Embed Size (px)
Citation preview
1
2
Scientific Chair of Prof. Abdullah Hussain Basalamah
for Gynecological Cancer and Gynecology Oncology Unit
MEMBER OF
Prof. Khalid Hussain Sait
Professor and Consultant Gynecology Oncology.
King Abdulaziz University Hospital, Jeddah KSA.
Dr. Nisreen Mohammed Omer Anfinan
Assistant Professor and Consultant Gynecology Oncology.
King Abdulaziz University Hospital, Jeddah KSA.
Prof. Abdullah Hussain Basalamah
First Dean of Faculty of Medicine, King Abdulaziz University
Professor and sponsor of Scientific Chair of
Prof. Abdullah Hussain Basalamah for Gynecological Cancer.
Prof. James Bentley
Professor and Consultant Gynecology Oncology
Dalhousie University, Halifax, Canada
3
Scientific Chair of Prof. Abdullah Hussain Basalamah for Gynecological Cancer and Gynecology Oncology Unit
MEMBER OF
Dr. Hussain Abdullah Basalamah
Resident of Obstetrics and Gynecology
King Abdulaziz University Hospital, Jeddah KSA.
Hesham Khalid Sait
Medical Student
Faculty of Medicine, King Abdulaziz University, Jeddah KSA.
Dr. Bassem Salama El-Deek
Associate Professor Community Medicine, Joint master of health profession education (JMHPE) Maastricht
KAU FOM, Medical education department.
Dr. Jawaher Ahmad Al Ahmadi
Family and Community Medicine
King Abdulaziz University Hospital, Jeddah KSA.
Dr. Faten SALAH Gazaz
Consultant in Medical Virology - Director of Virology Laboratory
King Abdulaziz University Hospital, Jeddah KSA.
4
Treatment of cancer become one of the
fundamental pillars of the strategical plan in the
health sector in Saudi Arabia, represented by the
Ministry
of Health and other health sectors under the care of our government of
Saudi Arabia and the leadership of his Majesty King Salman bin
Abdulaziz Al Saud.
According to the latest statistics of Saudi Cancer registry, the number
of cancer cases has been increased over the last few years, It reached
more than 12,000 cases in 2007 in Comparison to 7,000 cases in 2005.
The female patients represent 51% of those cases.
The global development in the world over the last 10 years in the field
of gynecological cancer in addition to gestational trophoblastic
neoplasia showed imprecise development, implication of cancer and how
to contain it.
This Journal is one step further to promote awareness and education
among physicians, nurses and health care providers in Saudi Arabia and
hence more researches in the failed of gynecological Cancer.
I would like to thanks professor Abdullah Hussain Basalama for his
support in establishing this Scientific Chair of Gynecological Cancer and
many thanks for all members of the Chair for the great effort in
achieving the goals.
Prof. khalid Sait
Director of Scientific Chair of Prof. Abdullah Hussein Basalamah For
Gynecological Cancer
EDITORIAL
5
In Gynecological Cancer
in Saudi Arabia 1989 – 2012
PREVIOUSLY PUBLISHED ARTICLE
6
PREVIOUSLY PUBLISHED ARTICLE
In Gynecological Cancer in Saudi Arabia 1989-2012
Cancer. 1989 Dec 1;64(11):2309-12.
Dysgerminoma of the ovary with rhabdomyosarcoma. Report of a case.
Akhtar M1, Bakri Y, Rank F.
Department of Pathology and Laboratory Medicine, King Faisal Specialist Hospital, Riyadh, Saudi
Arabia.
1
Acta Obstet Gynecol Scand. 1990;69(7-8):657-8.
Carcinoma of the cervix in a pregnant woman with negative Pap smears and colposcopic examination.
Bakri YN1, Akhtar M, al-Amri A.
Department of Obstetrics and Gynecology, King Faisal Specialist Hospital and Research Centre,
Riyadh, Saudi Arabia.
2
3
Aust N Z J Obstet Gynaecol. 1991 Nov;31(4):327-30.
Ovarian tumours in pregnancy.
el-Yahia AR1, Rahman J, Rahman MS, al-Suleiman SA.
Department of Obstetrics and Gynaecology, College of Medicine and Medical Sciences, King Faisal
University, Dammam, Saudi Arabia.
Int Orthop. 1991;15(4):393-5.
Ollier's disease with secondary chondrosarcoma associated with ovarian tumour. A case report.
Asirvatham R1, Rooney RJ, Watts HG.
Department of Orthopaedic Surgery, King Faisal Specialist Hospital and Research Centre,
Riyadh, Saudi Arabia.
4
7
PREVIOUSLY PUBLISHED ARTICLE
In Gynecological Cancer in Saudi Arabia 1989-2012
Int J Gynaecol Obstet. 1992 Apr;37(4):289-91.
Bilateral and synchronous cervical carcinoma in situ in a didelphic uterus. Bakri Y
1, Salem H, Sadi AR, Mansour M.
Department of Obstetrics and Gynaecology, King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia.
5
J Egypt Public Health Assoc. 1992;67(3-4):465-77.
An epidemiologic study of ovarian cancer. Part 1: Reproductive and social factors. Badawy YA
1, Bayoumi DM.
Department of Community Medicine, Faculty of Medicine, University of Alexandria, Egypt.
6
7
Gynecol Oncol. 1992 Sep;46(3):384-90.
Vulvar sarcoma: a report of four cases.
Bakri YN1, Akhtar M, el-Senoussi M, Wierzbicki R.
1Department of Obstetrics and Gynecology, King Faisal Specialist Hospital
and Research Centre, Riyadh, Saudi Arabia.
Acta Obstet Gynecol Scand. 1992 Jan;71(1):67-8.
Gestational choriocarcinoma in a tubal ectopic pregnancy.
Bakri YN1, Amri A, Mulla J.
1Department of Obstetrics and Gynecology, King Faisal Specialist Hospital and
Research Centre, Riyadh, Saudi Arabia.
8
8
PREVIOUSLY PUBLISHED ARTICLE
In Gynecological Cancer in Saudi Arabia 1989-2012
Eur J Obstet Gynecol Reprod Biol. 1994 Jun 30;55(3):205-8.
Struma ovarii with pseudo-Meigs' syndrome: report of a case and review of the literature.
Amr SS1, Hassan AA.
1Dhahran Health Center, Saudi Aramco, Saudi Arabia.
9
J Reprod Med. 1994 Mar;39(3):175-8.
Pulmonary metastases of gestational trophoblastic tumor. Risk factors for early respiratory failure. Bakri YN
1, Berkowitz RS, Khan J, Goldstein DP, von Sinner W, Jabbar FA.
1King Faisal Gestational Trophoblastic
Center, King Faisal Specialist Hospital and Research Centre, Riyadh, Kingdom of Saudi Arabia.
10
11
J Clin Pharmacol. 1995 Oct;35(10):1003-7.
Pharmacokinetics of carboplatin in a patient with cervical cancer with ureteric obstruction before, during, and after hemodialysis.
el-Yazigi A1, Alfurayh O, Amer M.
1Department of Biological and Medical Research, King Faisal Specialist Hospital and Research Centre, Riyadh,
Saudi Arabia.
Gynecol Oncol. 1996 Jun;61(3):451-3.
Pericardial metastasis in carcinoma of the uterine cervix.
Jamshed A1, Khafaga Y, El-Husseiny G, Gray AJ, Manji M.
1Oncology Department,King Faisal Specialist Hospital and Research Centre, Riyadh,
Saudi Arabia.
12
9
PREVIOUSLY PUBLISHED ARTICLE
In Gynecological Cancer in Saudi Arabia 1989-2012
J Reprod Med. 1998 Jan;43(1):11-3.
Complete molar pregnancy. Clinical trends at King Fahad
Hospital, Riyadh, Kingdom of Saudi Arabia.
Felemban AA1, Bakri YN, Alkharif HA, Altuwaijri SM, Shalhoub J, Berkowitz RS.
1Department of Obstetrics and Gynaecology, King Fahad Hospital, Riyadh, Kingdom of Saudi Arabia.
13
Int J Radiat Oncol Biol Phys. 1998 Aug 1;42(1):91-100.
Carcinoma of the uterine cervix in Saudi Arabia: experience in the management of 164 patients with stage-I & -II disease.
El-Senoussi M1, Bakri Y, Amer MH, DeVol EB.
1Department of Oncology, King Faisal Specialist Hospital and Research Centre, Riyadh, Kingdom
of Saudi Arabia.
14
15
Pediatr Surg Int. 1998 Nov;14(1-2):62-5.
Laparoscopy for ovarian pathology in infancy and childhood.
Jawad AJ1, Al-Meshari A.
1Division of Paediatric Surgery, Department of Surgery, King Khalid University Hospital,
P.O. Box 7805, Riyadh 11472, Saudi Arabia.
Scand J Urol Nephrol. 1998 Feb;32(1):73-6.
Metastatic transitional cell carcinoma of the ovary from superficial bladder tumour. Kardar AH
1, Lindstedt EM, Tulbah AM, Bazarbashi SN, al Suhaibani HS.
1Department of Urology, King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia.
16
10
PREVIOUSLY PUBLISHED ARTICLE
In Gynecological Cancer in Saudi Arabia 1989-2012
Ann Saudi Med. 1999 Sep-Oct;19(5):438-9.
Ovarian cancer recurrence at the laparoscopic port for cholecystectomy. Al-Sobhi S
1, Shirah HA, Subhi J, Amin T, Martan A, Al Deery M.
1Departments of Surgery and Obstetrics and Gynecology, King Faisal Specialist Hospital and Research
Centre, Riyadh, Saudi Arabia.
17
Acta Oncol. 1999;38(4):455-60.
Malignant ovarian germ cell tumours -- a survival and prognostic analysis. Ezzat A
1, Raja M, Bakri Y, Subhi J, Memon M, Schwartz P, Stuart R.
1Department of Oncology, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia.
18
19
Eur J Gynaecol Oncol. 1999;20(5-6):412-5.
Carcinoma of cervix, the King Faisal Specialist Hospital & Research Center experience--the need for screening forcervical cancer in developing countries.
Manji MF1, Pradhan D, El-Senoussi M, Bakri Y, Subhi J, Ezzat A, Zwan F, Ramirez C.
1Department of Oncology, King Faisal Specialist Hospital & Research Center, Riyadh,
Kingdom of Saudi Arabia.
Eur J Pediatr Surg. 2000 Aug;10(4):252-7.
Congenital cervical teratoma in neonates. Case report and review. Elmasalme F
1, Giacomantonio M, Clarke KD, Othman E, Matbouli S.
1Department of Pediatric Surgery, Maternity and Children's Hospital, Jeddah, Saudi Arabia.
20
11
PREVIOUSLY PUBLISHED ARTICLE
In Gynecological Cancer in Saudi Arabia 1989-2012
Eur J Obstet Gynecol Reprod Biol. 2000 Jan;88(1):103-6.
Polypoid endocervical stromal sarcoma with heterologous elements. Report of a case with review of the literature.
Amr SS1, Sheikh SM.
1Pathology Services Division, Saudi Aramco-Dhahran Health Center, Saudi Aramco Medical Services
Organization, Dhahran, Kingdom of Saudi Arabia. [email protected]
21
APMIS. 2000 Nov;108(11):756-60.
p53 and Bcl-2 oncoprotein expression in placentas with hydropic changes and partial and complete moles.
Al-Bozom IA1.
1Department of Pathology, King Khalid University Hospital, Riyadh, Kingdom of Saudi Arabia.
22
23
J Reprod Med. 2000 Feb;45(2):94-6.
CSF/serum beta-hCG ratio in patients with brain metastases of gestational trophoblastic tumor.
Bakri Y1, al-Hawashim N, Berkowitz R.
1Department of Obstetrics and Gynecology, King Faisal Specialist Hospital and Research Center,
Riyadh, Saudi Arabia.
Eur J Obstet Gynecol Reprod Biol. 2000 May;90(1):87-91.
Malignant germ cell tumors of the ovary. Pregnancy considerations.
Bakri YN1, Ezzat A, Akhtar, Dohami, Zahrani.
1Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, King Fahad
National Guard Hospital, Riyadh, Saudi Arabia
24
12
PREVIOUSLY PUBLISHED ARTICLE
In Gynecological Cancer in Saudi Arabia 1989-2012
Eur J Obstet Gynecol Reprod Biol. 2001 Aug;97(2):245-8.
Uterine artery aneurysm mimicking pelvic sarcoma. A case report and review of literature. [email protected].
Raslan WF1, Marier RR.
1Pathology Services Division, Saudi Aramco, Dhahran Health Center, Room D-205-2, Box 76, Saudi Aramco
Medical Services Organization, 31311, Dhahran, Saudi Arabia
25
Saudi Med J. 2001 Oct;22(10):914-6.
Synchronous endometrioid carcinoma of the ovary and endometrium associated with ovulation induction. Ghourab S
1.
1Department of Obstetrics and Gynecology, King Khalid University Hospital, King Saud University, PO Box 2925,
Riyadh 11461, Kingdom of Saudi Arabia. [email protected]
26
27
Gynecol Oncol. 2001 Jun;81(3):477-80.
Positron emission tomography for the evaluation of metastases in patients with carcinoma of the cervix: a retrospective review.
Kerr IG1, Manji MF, Powe J, Bakheet S, Al Suhaibani H, Subhi J.
1Department of Oncology, King Faisal Specialist Hospital and Research Centre, Riyadh, 11211, Saudi Arabia.
Gynecol Oncol. 2001 Apr;81(1):105-9.
Scalp and cranial bone metastasis of endometrial carcinoma: a case report and literature review. Mustafa MS
1, Al-Nuaim L, Inayat-Ur-Rahman N.
1Department of Obstetrics and Gynaecology, King Khalid University Hospital, Riyadh 11472, Saudi Arabia.
28
13
PREVIOUSLY PUBLISHED ARTICLE
In Gynecological Cancer in Saudi Arabia 1989-2012
Histopathology. 2002 Nov;41(5):446-9.
Extrauterine placental site trophoblastic tumour in association with a lithopedion.
El Hag IA1, Ramesh K, Kollur SM, Salem M.
1Department of Pathology, PARAS Central Hospital, Sakaka, Al Jouf, Kingdom of Saudi Arabia.
29
Am J Clin Oncol. 2002 Jun;25(3):256-60.
Prognostic factors and treatment modalities in uterine sarcoma.
El Husseiny G1, Al Bareedy N, Mourad WA, Mohamed G, Shoukri M, Subhi J, Ezzat A.
1Department of Radiation Oncology, King Faisal Specialist Hospital and Research
Centre, Riyadh, Saudi Arabia.
30
31
J Ayub Med Coll Abbottabad. 2003 Apr-Jun;15(2):65-8.
Cancer ovary, present and future of management.
Parvez T1.
1King Fahad Hospital, Al Madma Al Munawra, Kingdom of Saudi Arabia.
Saudi Med J. 2003 Dec;24(12):1329-33.
Prevalence of gestational trophoblastic disease. A single institution experience. Khashoggi TY
1.
1Department of Obstetrics and Gynecology, King Khalid University Hospital, King Saud University, Riyadh, Kingdom of Saudi
Arabia.
32
14
PREVIOUSLY PUBLISHED ARTICLE
In Gynecological Cancer in Saudi Arabia 1989-2012
Saudi Med J. 2004 Oct;25(10):1492-4.
Uterine lipoma.
Al-Maghrabi JA1, Sait KH, Lingawi SS.
1Department of Pathology, King Faisal Specialist Hospital and Research Centre,
Jeddah, Kingdom of Saudi Arabia.
33
Saudi Med J. 2004 Jul;25(7):857-61.
Assessment of risk factors of uterine cancer in Saudi patients
With postmenopausal bleeding.
Al-Kadri HM1, Al-Awami SH, Madkhali AM.
1Department of Obstetrics and Gynecology, King Fahad National Guard Hospital, PO Box 57374, Riyadh
11574, Kingdom of Saudi Arabia. [email protected]
34
35
Appl Immunohistochem Mol Morphol. 2004 Mar;12(1):79-82.
Common blue nevus of the uterine cervix: case report and review.
Zevallos-Giampietri EA1, Barrionuevo C.
1Department of Pathology and Laboratory Medicine, King Faisal Specialist Hospital &
Research Center at Jeddah, Jeddah, Saudi Arabia.
Saudi Med J. 2004 Sep;25(9):1270-3.
Ovarian mucinous cystadenoma in a female with Turner syndrome. Sait KH
1, Alkhattabi MA, Alkushi AO, Alqahtani MH.
1Department of Obstetrics and Gynecology, King Abdul-Aziz University Hospital, PO Box
80215, Jeddah 21589, Kingdom of Saudi Arabia.
36
15
PREVIOUSLY PUBLISHED ARTICLE
In Gynecological Cancer in Saudi Arabia 1989-2012
J Obstet Gynaecol Can. 2004 Feb;26(2):137-42.
Ovarian teratoma diagnosis and management: case presentations.
Sait K1, Simpson C.
1Department of Obstetrics and Gynecology, King Abdulaziz University Hospital, Jeddah, Saudi
Arabia.
37
Saudi Med J. 2004 May;25(5):615-20.
Potential regulation of PTH/PTHrP receptor expression in choriocarcinoma cells. Alokail MS
1.
1Department of Biochemistry, College of Science, King Saud University, Riyadh, Kingdom of Saudi Arabia.
38
39
Saudi Med J. 2004 May;25(5):552-6.
Genito-urinary cancer in Saudi Arabia.
Abomelha MS1.
1Department of Urology, Armed Forces Hospital, Riyadh, Kingdom of Saudi Arabia.
Arch Gynecol Obstet. 2005 Apr;271(4):346-9. Epub 2004 Jun 2.
Pregnancy outcome in non-gynecologic cancer.
Sait KH1, Ashour A, Rajabi M.
1Departments of Obstetrics and Gynecology, King Abdulaziz University Hospital, P.O. Box
80215, 21589 Jeddah, Saudi Arabia. [email protected]
40
16
PREVIOUSLY PUBLISHED ARTICLE
In Gynecological Cancer in Saudi Arabia 1989-2012
Saudi Med J. 2005 Apr;26(4):588-92.
Cytopathological pattern of cervical Pap smear according to the Bethesda system in Southwestern Saudi Arabia.
Elhakeem HA1, Al-Ghamdi AS, Al-Maghrabi JA.
1Department of Pathology, King Fahad Hospital, Al-Baha, Kingdom of Saudi Arabia.
41
West Afr J Med. 2005 Jan-Mar;24(1):31-5.
Elective hysterectomy: a clinicopathological review from Abha catchment area of Saudi Arabia.
Sobande AA1, Eskandar M, Archibong EI, Damole IO.
1Department of Obstetrics & Gynaecology, College of Medicine, King Khalid University, Abha, Saudi Arabia.
42
43
Int J Radiat Oncol Biol Phys. 2005 Jan 1;61(1):257-66.
Minimizing urinary bladder radiation dose during brachytherapy
for carcinoma of the cervix using balloon inflation technique.
Malaker K1, Shukla V, D'Souza H, Weatherburn H.
1Radiation Oncology Section, Princess Norah Oncology Centre, Jeddah, Saudi Arabia.
Saudi Med J. 2006 Sep;27(9):1412-4.
Ovarian mucinous cystadenocarcinoma of low malignant potential associated with a mature cystic teratoma.
Al-Ghamdi FA1, Al-Khattabi MA.
1Department of Pathology, King Abdul-Aziz University Hospital, PO Box 21342, Jeddah 124743, Kingdom of
Saudi Arabia.
44
17
PREVIOUSLY PUBLISHED ARTICLE
In Gynecological Cancer in Saudi Arabia 1989-2012
Saudi Med J. 2006 Oct;27(10):1498-502.
Cervical cancer screening with pattern of pap smear. Review of multicenter studies.
Altaf FJ1.
1Department of Pathology, Faculty of Medicine, King Abdul-Aziz University Hospital, PO Box
51241, Jeddah 21543, Kingdom of Saudi Arabia.
45
Int J Gynecol Pathol. 2006 Jul;25(3):262-7.
Cotyledonoid dissecting leiomyoma of the uterus: a case report of a benign
uterine tumor with sarcomalike gross appearance and review of literature. Saeed AS
1, Hanaa B, Faisal AS, Najla AM.
1Department of Pathology and Laboratory Medicine, King Fahad National Guard Hospital, King Abdulaziz
Medical City, Riyadh, Saudi Arabia.
46
47
Int J Gynecol Cancer. 2006 Mar-Apr;16(2):675-80.
Female circumcision (female genital mutilation): a problem for brachytherapy in cervical cancer. Manji MF
1, Al Badawi I, El Enbaby A, Al Bareedy N.
Departments of Oncology, King Faisal Specialist Hospital & Research Center, Riyadh, Kingdom of Saudi Arabia.
Ann Saudi Med. 2007 Jul-Aug;27(4):268-72.
Pattern of abnormal Pap smears in developing countries: a report from a large referral hospital in Saudi Arabiausing the revised 2001 Bethesda System.
Abdullah LS1.
1Department of Pathology, King Abdulaziz University Hospital, Jeddah, Saudi Arabia.
48
18
PREVIOUSLY PUBLISHED ARTICLE
In Gynecological Cancer in Saudi Arabia 1989-2012
Saudi Med J. 2007 Dec;28(12):1810-8.
Molecular testing of human papillomavirus in cervical specimens.
Gazzaz FB1.1Virology Laboratory, Faculty of Medicine, King Abdul-Aziz University Hospital,
PO Box 80215, Jeddah 21589, Kingdom of Saudi Arabia.
49
nt J Gynaecol Obstet. 2007 Dec;99(3):201-5. Epub 2007 Apr 24.
Three-dimensional ultrasound and three-dimensional power Doppler in the assessment of ovarian tumors.
Laban M1, Metawee H, Elyan A, Kamal M, Kamel M, Mansour G.
1Department of Gynecology and Obstetrics, Taibah University, Saudi Arabia.
50
51
Ann Saudi Med. 2007 Jan-Feb;27(1):1-5.
Human papilloma virus-16/18 cervical infection among women attending a family medical clinic in Riyadh.
Al-Muammar T1, Al-Ahdal MN, Hassan A, Kessie G, Dela Cruz DM, Mohamed GE.
1Department of Family Medicine, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi
Arabia.
Ann Saudi Med. 2008 Jul-Aug;28(4):282-6.
Hysterectomy for benign conditions in a university hospital in Saudi Arabia.
Sait K1, Alkhattabi M, Boker A, Alhashemi J.
1Department of Obstetrics and Gynecology, King Abdulaziz University, Jeddah, Saudi Arabia.
52
19
PREVIOUSLY PUBLISHED ARTICLE
In Gynecological Cancer in Saudi Arabia 1989-2012
J Egypt Natl Canc Inst. 2008 Mar;20(1):1-9.
Using the computed tomography in comparison to the orthogonal radiography based treatment planning in high dose rate (HDR) brachytherapy in cervical uteri cancer patients; a single institution feasibility study.
Bahadur YA1, El-Sayed ME, El-Taher ZH, Zaza KO, Moftah BA, Hassouna AH, Ghassal NM.
1The Department of Radiation Oncology, King Abdul- Aziz University Hospital, Kingdom of Saudi
Arabia. [email protected]
53
Saudi Med J. 2008 Jan;29(1):126-8.
Mucinous cystadenoma of the ovary in a 12-year-old girl.
Alobaid AS1.
1Department of Obstetrics and Gynecology, College of Medicine, King Saud University,
PO Box 7805, Riyadh 11472, Kingdom of Saudi Arabia.
54
55
J Obstet Gynaecol Res. 2008 Aug;34(4):538-42. doi: 10.1111/j.1447-0756.2008.00735.x.
Gynecological cancer incidence in a hospital population in Saudi Arabia: the effect of foreign immigration over two decades.
Makoha FW1, Raheem MA.
1Maternity and Children's Hospital, Jeddah, Saudi Arabia.
Fertil Steril. 2008 Nov;90(5):2015.e17-9. doi: 10.1016/j.fertnstert.2008.07.021. Epub 2008 Sep 7.
Massive ascites as a presentation in a young woman with endometriosis: a case report. Sait KH
1.1Department of Obstetrics and Gynecology, King Abdulaziz University Hospital,
Jeddah, Saudi Arabia.
56
20
PREVIOUSLY PUBLISHED ARTICLE
In Gynecological Cancer in Saudi Arabia 1989-2012
J Family Community Med. 2008 Sep;15(3):127-31.
Ovarian dysgerminoma in two sisters.
Aldhafery BF1.
1Department of Radiology, King Fahd Hospital of the University, Al-Khobar, Saudi Arabia.
57
Saudi Med J. 2008 Mar;29(3):444-6.
Primitive neuroectodermal tumor of the ovary.
Anfinan NM1, Sait KH, Al-Maghrabi JA.
1Department of Obstetrics & Gynecology, King Abdulaziz University Hospital, Jeddah,
Kingdom of Saudi Arabia.
58
59
Saudi Med J. 2009 Sep;30(9):1208-12.
Attitudes, knowledge, and practices in relation to cervical cancer and its screening among women in Saudi Arabia.
Sait KH1.
1Department of Obstetrics & Gynecology, King Abdul-Aziz University Hospital, Jeddah 21589, PO Box
80215, Kingdom of Saudi Arabia.
Arch Gynecol Obstet. 2009 Nov;280(5):823-5. doi: 10.1007/s00404-009-0992-5. Epub 2009 Feb 26.
Cisplatinum and docetaxel for ovarian cancer in pregnancy. Rouzi AA
1, Sahly NN, Sahly NF, Alahwal MS.
1Department of Obstetrics and Gynecology, King Abdulaziz University, PO Box 80215, Jeddah
21589, Saudi Arabia.
60
21
PREVIOUSLY PUBLISHED ARTICLE
In Gynecological Cancer in Saudi Arabia 1989-2012
Lab Invest. 2009 Oct;89(10):1115-27. doi: 10.1038/labinvest.2009.75. Epub 2009 Jul 27.
Bortezomib-mediated expression of p27Kip1 through S-phase kinase protein 2 degradation in epithelial ovarian cancer. Uddin S
1, Ahmed M, Hussain AR, Jehan Z, Al-Dayel F, Munkarah A, Bavi P, Al-Kuraya KS.
1Department of Human Cancer Genomic Research, King Fahad National Centre for Children's Cancer and
Research, King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia.
61
Mol Cancer. 2009 Sep 18;8:74. doi: 10.1186/1476-4598-8-74.
Overexpression of leptin receptor predicts an unfavorable outcome in Middle Eastern ovarian cancer. Uddin S
1, Bu R, Ahmed M, Abubaker J, Al-Dayel F, Bavi P, Al-Kuraya KS.
1Department of Human Cancer Genomic Research, Research Center, Department of Pathology, King Faisal
Specialist Hospital and Research Center, Riyadh, Saudi Arabia.
62
63
Mol Cancer. 2009 Jul 28;8:51. doi: 10.1186/1476-4598-8-51.
PIK3CA alterations in Middle Eastern ovarian cancers.
Abubaker J1, Bavi P, Al-Haqawi W, Jehan Z, Munkarah A, Uddin S, Al-Kuraya KS.
1Department of Human Cancer Genomic Research, Research Center, King Faisal Specialist
Hospital and Research Center, Riyadh, Saudi Arabia.
Cutis. 2009 Jul;84(1):33-8.
Cutaneous metastasis of uterine adenocarcinoma: a case report and review of the literature.
Selim AA1, Shaheen S, Lockshin N, Khachemoune A.
1Biotechnology Center, King Fahd University of Petroleum & Minerals, Dhahran, Saudi Arabia
64
22
PREVIOUSLY PUBLISHED ARTICLE
In Gynecological Cancer in Saudi Arabia 1989-2012
Surg Technol Int. 2010 Apr;19:141-3.
Robotic-assisted ovarian transposition before radiation.
Al-Badawi I1, Al-Aker M, Tulandi T.
1King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia.
65
Ann Saudi Med. 2010 Sep-Oct;30(5):397-400. doi: 10.4103/0256-4947.68550.
Prevalence of abnormal cervical cytology among subfertile Saudi women.
Al-Jaroudi D1, Hussain TZ.
1Women's Specialized Hospital, King Fahad Medical City, Riyadh, Saudi Arabia.
66
67
Hematol Oncol Stem Cell Ther. 2010;3(3):161-2.
Carcinosarcoma of the uterus in a woman with Down
syndrome.
Al-Badawi IA1, AlOmar O, Kornfeld I.
1King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia.
Int J Cancer. 2010 Jan 15;126(2):382-94. doi: 10.1002/ijc.24757.
Cyclooxygenase-2 inhibition inhibits PI3K/AKT kinase activity in epithelial ovarian cancer.
Uddin S1, Ahmed M, Hussain A, Assad L, Al-Dayel F, Bavi P, Al-Kuraya KS, Munkarah A.
1King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia.
68
23
PREVIOUSLY PUBLISHED ARTICLE
In Gynecological Cancer in Saudi Arabia 1989-2012
Int J Gynecol Cancer. 2010 Aug;20(6):1082-6. doi: 10.1111/IGC.0b013e3181e2ace5.
Laparoscopic ovarian transposition before pelvic irradiation: a Saudi tertiary center experience.
Al-Badawi IA1, Al-Aker M, AlSubhi J, Salem H, Abduljabbar A, Balaraj K, Munkarah A.
1King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia.
69
Arch Gynecol Obstet. 2010 Nov;282(5):529-33. doi: 10.1007/s00404-009-1346-z. Epub 2010 Jan 5.
Ovarian tumors associated with pregnancy: a 20-year experience in a teaching hospital. Gasim T
1, Al Dakhiel SA, Al Ghamdi AA, Al Ali M, Al Jama F, Rahman J, Al Suleiman SA, Rahman MS.
1Department of Obstetrics and Gynecology, College of Medicine, King Faisal University, Dammam, Saudi Arabia.
70
71
Int J Gynecol Pathol. 2010 Jul;29(4):343-50. doi: 10.1097/PGP.0b013e3181cd6552.
High- grade endometrial carcinoma: serous and grade 3 endometrioid carcinomas have different immunophenotypes and outcomes.
Alkushi A1, Köbel M, Kalloger SE, Gilks CB.
1Department of Pathology, King Fahad National Guard Hospital, Riyadh, Saudi Arabia.
Reprod Biol Endocrinol. 2010 Mar 11;8:24. doi: 10.1186/1477-7827-8-24.
A massive ovarian mucinous cystadenoma: a case report. Kamel RM
1.
1Department of Obstetrics and Gynaecology, Faculty of Medicine, Jazan University, Saudi
Arabia.
72
24
PREVIOUSLY PUBLISHED ARTICLE
In Gynecological Cancer in Saudi Arabia 1989-2012
Int J Womens Health. 2011;3:219-26. doi: 10.2147/IJWH.S23046. Epub 2011 Jul 26.
Early experience with the da Vinci surgical system robot in gynecological surgery at King Abdulaziz University Hospital.
Sait KH1.
1Obstetrics and Gynecology Department, Faculty of Medicine, Gynecology Oncology Unit,
King Abdulaziz University Hospital, Jeddah, Saudi Arabia.
73
Saudi Med J. 2011 May;32(5):495-503.
Treatment planning for high dose rate brachytherapy of cervical cancer based on total dose constraints.
Bahadur YA1, Constantinescu CT, Hassouna AH, El-Sayed ME.
1Department of Radiology, King Abdulaziz University Hospital, PO Box 80215, Jeddah 21589, Kingdom of
Saudi Arabia.
74
75
Int J Gynecol Cancer. 2011 Jul;21(5):907-10. doi: 10.1097/IGC.0b013e318214219f.
Detection and genotyping of human papilloma virus in cervical cancer specimens from Saudi patients.
Al-Badawi IA1, Al-Suwaine A, Al-Aker M, Asaad L, Alaidan A, Tulbah A, Fe Bohol M, Munkarah AR.
1King Faisal Specialist Hospital and Research Center, Riyadh, Saudi Arabia.
Lab Invest. 2011 Jan;91(1):124-37. doi: 10.1038/labinvest.2010.136. Epub 2010 Jul 26.
HGF/c-Met pathway has a prominent role in mediating antiapoptotic signals through AKT in epithelial ovariancarcinoma.
Bu R1, Uddin S, Bavi P, Hussain AR, Al-Dayel F, Ghourab S, Ahmed M, Al-Kuraya KS.
1Human Cancer Genomic Research, Research Center, King Faisal Specialist Hospital and Research
Center, Riyadh, Saudi Arabia.
76
25
PREVIOUSLY PUBLISHED ARTICLE
In Gynecological Cancer in Saudi Arabia 1989-2012
Eur J Obstet Gynecol Reprod Biol. 2011 Dec;159(2):388-93. doi: 10.1016/j.ejogrb.2011.06.018. Epub 2011 Jul 7.
Antibiotic use and risk of gynecological cancer.
Tamim HM1, Musallam KM, Al Kadri HM, Boivin JF, Collet JP.
1King Abdullah International Medical Research Center, King Saud bin Abdulaziz University for Health
Sciences, Riyadh, Saudi Arabia.
77
Saudi Med J. 2011 Sep;32(9):913-8.
Conservative treatment of ovarian cancer. Safety, ovarian function preservation, reproductive ability, and emotional attitude of the patients in Saudi Arabia.
Sait KH1.
1Gynecology Oncology Unit, Obstetrics and Gynecology Department, Faculty of Medicine, King Abdulaziz
University and King Abdulaziz University Hospital, PO Box 80215, Jeddah, Kingdom of Saudi Arabia.
78
79
Food Chem Toxicol. 2011 Dec;49(12):3281-6. doi: 10.1016/j.fct.2011.09.023. Epub 2011 Sep 24.
Catechin hydrate inhibits proliferation and mediates apoptosis of SiHa human cervical cancer cells.
Al-Hazzani AA1, Alshatwi AA.
1Dept. of Botany and Microbiology, King Saud University, Saudi Arabia.
BMC Syst Biol. 2011 Nov 3;5:183. doi: 10.1186/1752-0509-5-183.
Network analysis of microRNAs and their regulation in human
ovarian cancer.
Schmeier S1, Schaefer U, Essack M, Bajic VB.
1Computational Bioscience Research Center (CBRC), 4700 King Abdullah University of Science and Technology
(KAUST), Thuwal 23955-6900, Kingdom of Saudi Arabia.
80
26
PREVIOUSLY PUBLISHED ARTICLE
In Gynecological Cancer in Saudi Arabia 1989-2012
Mol Med. 2011;17(7-8):635-45. doi: 10.2119/molmed.2011.00046. Epub 2011 Mar 21.
Overexpression of fatty acid synthase in Middle Eastern epithelial ovarian
Carcinoma activates AKT and Its inhibition potentiates cisplatin-induced apoptosis. Uddin S
1, Jehan Z, Ahmed M, Alyan A, Al-Dayel F, Hussain A, Bavi P, Al-Kuraya KS.
1Human Cancer Genomic Research, Research Center, Riyadh, Saudi Arabia.
81
Gynecol Oncol. 2011 Jun 1;121(3):522-6. doi: 10.1016/j.ygyno.2011.01.033. Epub 2011 Feb 24.
Prevalence and genotypes' distribution of human papillomavirus in
invasive cervical cancer in Saudi Arabia.
Alsbeih G1, Ahmed R, Al-Harbi N, Venturina LA, Tulbah A, Balaraj K.
1Radiation Biology Laboratory, Biomedical Physics Department, King Faisal Specialist Hospital and Research
Centre, Riyadh, Saudi Arabia.
82
83
Saudi Med J. 2011 Nov;32(11):1155-60.
Knowledge, attitudes, and practices regarding cervical cancer screening among physicians in the Western Region of Saudi Arabia. Sait KH
1.
1Department of Obstetrics & Gynecology, Faculty of Medicine, King Abdulaziz University, Kingdom of Saudi
Arabia.
BMC Syst Biol. 2011 Sep 19;5:144. doi: 10.1186/1752-0509-5-144.
In Silico discovery of transcription factors as potential diagnostic biomarkers of ovarian cancer.
Kaur M1, MacPherson CR, Schmeier S, Narasimhan K, Choolani M, Bajic VB.
1Computational Bioscience Research Center, King Abdullah University of Science and Technology,
Thuwal 23955-6900, Kingdom of Saudi Arabia.
84
27
PREVIOUSLY PUBLISHED ARTICLE
In Gynecological Cancer in Saudi Arabia 1989-2012
Brachytherapy. 2011 Nov-Dec;10(6):498-502. doi: 10.1016/j.brachy.2011.03.004. Epub 2011 Jun 2. In vivo diode dosimetry vs. computerized tomography and digitally reconstructed radiographs for critical organ dose calculation in high-dose-rate brachytherapy of cervical cancer.
Hassouna AH1, Bahadur YA, Constantinescu C, El Sayed ME, Naseem H, Naga AF.
1Department of Oncology, King Faisal Specialist Hospital & Research Center, Jeddah, Saudi Arabia.
85
J Pediatr Adolesc Gynecol. 2011 Feb;24(1):25-8. doi: 10.1016/j.jpag.2010.06.005. Epub 2010 Aug 14.
Ovarian tumors in children and adolescents--a clinical study of 52 patients in a university hospital. Al Jama FE
1, Al Ghamdi AA, Gasim T, Al Dakhiel SA, Rahman J, Rahman MS.
1Department of Obstetrics and Gynecology, College of Medicine, University of Dammam and King Fahad
University Hospital, Al-Khobar, Saudi Arabia.
86
87
J Cytol. 2011 Oct;28(4):173-7. doi: 10.4103/0970-9371.86343.
Cytological pattern of cervical Papanicolaou smear in eastern region of Saudi Arabia. Balaha MH
1, Al Moghannum MS, Al Ghowinem N, Al Omran S.
1Department of Obstetrics and Gynecology, King Faisal University, Al Ahsa, Kingdom of Saudi Arabia.
Arq Bras Endocrinol Metabol. 2011 Oct;55(7):490-3.
Incidental finding of ovarian teratoma on post-therapy scan for papillary thyroid cancer and impact of SPECT/CT imaging.
Jammah AA1, Driedger A, Rachinsky I.
1Department of Medicine, King Saud University, Riyadh, Saudi Arabia.
88
28
PREVIOUSLY PUBLISHED ARTICLE
In Gynecological Cancer in Saudi Arabia 1989-2012
J Obstet Gynaecol. 2011 Aug;31(6):555. doi: 10.3109/01443615.2011.587052.
Cervical angioleiomyoma. Al-Sannaa GA
1, Al-Manea M.
1Department of Histopathology, Regional Lab and Blood Bank, Dammam, Saudi Arabia.
89
Eur J Obstet Gynecol Reprod Biol. 2011 Dec;159(2):388-93. doi: 10.1016/j.ejogrb.2011.06.018. Epub 2011 Jul 7.
Antibiotic use and risk of gynecological cancer.
Tamim HM1, Musallam KM, Al Kadri HM, Boivin JF, Collet JP.
1King Abdullah International Medical Research Center, King Saud bin Abdulaziz University for Health
Sciences, Riyadh, Saudi Arabia.
90
Ann Saudi Med. 2012 Sep-Oct;32(5):541-3. doi: 10.5144/0256-4947.2012.26.5.1110.
Sarcomatoid carcinoma of the cervix. Nageeti TH
1, Jastania RA.
1Department of Oncology, King Abdul-Aziz Medical City, Jeddah, Saudi Arabia.
92
91
Medicine International 2011:3 25–29
Molecular tests to detect human papillomavirus infection in patients with cervical dysplasia and invasive cervical cancer in Saudi Arabia
Sait K, Gazzaz FS Department of Obstetrics & Gynecology, Faculty of Medicine, King Abdulaziz University, Kingdom of
Saudi Arabia.
29
PREVIOUSLY PUBLISHED ARTICLE
In Gynecological Cancer in Saudi Arabia 1989-2012
J Cancer Res Clin Oncol. 2012 Jul;138(7):1173-86. doi: 10.1007/s00432-012-1182-6. Epub 2012 Mar 15.
Analysis of molecular cytogenetic alterations in uterine leiomyosarcoma by array-based comparative genomic hybridization.
Raish M1, Khurshid M, Ansari MA, Chaturvedi PK, Bae SM, Kim JH, Park EK, Park DC, Ahn WS.
1Department of Pharmaceutics, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia.
93
Saudi Med J. 2012 Jan;33(1):61-5.
Histopathological pattern of ovarian neoplasms and their age distribution in the western region of Saudi Arabia.
Abdullah LS1, Bondagji NS.
1Pathology Department, King Abdul-Aziz University, College of Medicine, PO Box 80215, Jeddah 21589,
Kingdom of Saudi Arabia.
94
95
Med Oncol. 2012 Dec;29(4):2944-8. doi: 10.1007/s12032-012-0194-z. Epub 2012 Mar 10.
Pure dysgerminoma of the ovary: a single institutional experience of 65 patients.
A L Husaini H1, Soudy H, El Din Darwish A, Ahmed M, Eltigani A, A L Mubarak M, Sabaa AA, Edesa W, A L-
Tweigeri T, Al-Badawi IA. 1Department of Oncology, King Faisal Specialist Hospital, Riyadh, Saudi Arabia.
Ann Saudi Med. 2012 Nov-Dec;32(6):588-92. doi: 10.5144/0256-4947.2012.588.
Ovarian volume assessment in relation to histologic findings and sex hormone levels in women with postmenopausal bleeding and thickened endometrium.
Elfayomy AK1, El Tarhouny SA.
1Department of Obstetrics and Gynecology, Faculty of Medicine, Taibah University, Al Madinah 30001, Saudi
Arabia.
96
30
PREVIOUSLY PUBLISHED ARTICLE
In Gynecological Cancer in Saudi Arabia 1989-2012
Saudi Med J. 2012 Jun;33(6):634-9.
Pattern of cervical smear abnormalities using the revised Bethesda
system in a tertiary care hospital in Western Saudi Arabia. Altaf FJ
1, Mufti ST.
1Department of Anatomic Pathology, Faculty of Medicine, King Abdulaziz University Hospital, PO Box 51241,
Jeddah 21543, Kingdom of Saudi Arabia.
97
J Immunoassay Immunochem. 2012;33(3):223-33. doi: 10.1080/15321819.201
.634472.
Plexin D1: new potential biomarker for cervical cancer. Shalaby MA
1, Hampson L, Oliver A, Hampson I.
1Biochemistry Department, King Saud University Girls Department, Kingdom of Saudi Arabia-Riyadh.
98
99
J Biomed Opt. 2012 Sep;17(9):98001-1. doi: 10.1117/1.JBO.17.9.098001.
Fluorescence spectra of blood and urine for cervical cancer detectio. Masilamani V
1, Alsalhi MS, Vijmasi T, Govindarajan K, Rathan Rai R, Atif M, Prasad S, Aldwayyan AS.
1King Saud University, College Of Science, Department of Physics and Astronomy, P.O. Box 2455,
Riyadh, Kingdom of Saudi Arabia
Ann Saudi Med. 2012 Jan-Feb;32(1):27-31.
Evaluation of adjuvant therapy in women with uterine papillary serous cancer. Al Husaini H
1, Soudy H, Darwish A, Ahmed M, Eltigani A, Edesa W, Abdelsalam M.
1Department of Medical Oncology, King Faisal Specialist Hospital and Research Center, Riyadh, Saudi
100
31
PREVIOUSLY PUBLISHED ARTICLE
In Gynecological Cancer in Saudi Arabia 1989-2012
The Open Women’s Health Journal, 2012, 6, 1-5
Cervical Cancer Prevention in Saudi Arabia: It is Time to Call for Action! Khalid Sait1, James Bentley2, Nisrin Anfinan*, 1and Patti Power3
1Gynecology Oncology Unit, King Abdulaziz University, Jeddah, Saudi Arabia 2Gynecology Oncology Unit, Dalhousie University, Halifax, Nova Scotia, Canada
3Gynecology Oncology Unit, Memorial University of Newfoundland, St. John’s, Newfoundland, Canada
101
32
In Gynecological Cancer
in Saudi Arabia 2013 – 2015
33
PREVIOUSLY PUBLISHED ARTICLE
In Gynecological Cancer in Saudi Arabia 2013 - 2015
Prevalence of high-risk human papillomavirus infections in healthy Saudi women attending gynecologic clinics in the western region of Saudi Arabia.
Bondagji NS1, Gazzaz FS, Sait K, Abdullah L.
Cervical Cancer Prevalence at King Abdulaziz University Hospital. Fadwa J. Altaf1, Ghadeer A. Mokhtar2 and Faris M. Altaf3
HPV prevalence and genetic predisposition to cervical cancer in
Saudi Arabia. Alsbeih G1, Al-Harbi N, El-Sebaie M, Al-Badawi I.
Prevalence of human papillomavirus in women from Saudi
Arabia. Turki R1, Sait K, Anfinan N, Sohrab SS, Abuzenadah AM.
Five-year outcome of concurrent radiotherapy and chemotherapy in Saudi women
with locally advanced cervicalcancer: single-institution experience.
Al Asiri M1, Tunio M, Al Hadab A, Mohamed R, Bayoumi Y, Al Saeed E, Al Arifi M, Al Amro A.
A detailed study of patients and tumor characteristics of epithelial ovarian cancer in Saudi women.
Al-Badawi IA1, Munkarah AR, Tulbah A, Babic II, Al Husaini H, Ahmad S.
Methanolic extract of Nigella sativa seed inhibits SiHa human cervical cancer Cell proliferation through apoptosis.
Hasan TN1, Shafi G, Syed NA, Alfawaz MA, Alsaif MA, Munshi A, Lei KY, Alshatwi AA.
40
45
50
57
62
69
74
34
PREVIOUSLY PUBLISHED ARTICLE
In Gynecological Cancer in Saudi Arabia 2013 - 2015
Naringin induces death receptor and mitochondria-mediated apoptosis in human cervical cancer (SiHa) cells.
Ramesh E1, Alshatwi AA.
Hypermethylation of P15, P16, and E-cadherin genes in ovarian cancer.
Moselhy SS1, Kumosani TA, Kamal IH, Jalal JA, Abdul Jabaar HS, Dalol A.
Comprehensive laparoscopic surgical staging of ovarian dysgerminoma in a 10-year-old girl—A case report Anfinan N
Supraclavicular lymphadenopathy: initial manifestation of metastasis in
carcinoma of cervix. Tunio MA1, Al Asiri M, Mohamed R, Al-Dandan S.
Poorly differentiated ovarian sertoli-leydig cell tumor in a 16year - old single woman: a case report and literaturereview.
Abu-Zaid A1, Azzam A, Alghuneim LA, Metawee MT, Amin T, Al-Hussain TO.
HPV DNA And Liquid Base Cervical Cytology Co-Testing For Cervical Cancer Screening In Saudi Females Of Different Age Groups
Abdullah Layla S, Gazzaz Faten S, Sait Khalid H, Bondagji Nabeel S
Gestational trophoblastic disease in the western region of Saudi Arabia (single-institute experience).
Anfinan N1, Sait K
2, Sait H
1.
78
88
95
98
101
107
115
35
PREVIOUSLY PUBLISHED ARTICLE
In Gynecological Cancer in Saudi Arabia 2013 - 2015
Uterine sarcoma. Clinico-pathological characteristics and outcome.
Sait HK1, Anfinan NM, El Sayed ME, Alkhayyat SS, Ghanem AT, Abayazid RM, Sait KH.
Uterine leiomyosarcoma metastasizing to the heart. Tunio MA, Al-Asiri M, Fareed MM.
Expression of Thyroid Transcription Factor-1 (TTF-1) in Endometrial Carcinoma.
Jaudah Al-Maghrabi1(MD, FRCPC), Nisrin Anfinan2,3 (MD, FRCSC), Khalid Sait 2,3 (MD, FRCSC), Hesham
Sait2,3(MD), Mahmou Al-Ahwal4 (MD, FRCPC), Hussain Basalamah2,3 (MD, FRCSC).
HPV Infection in Cervical and Other Cancers in Saudi Arabia: Implication for Prevention and Vaccination.
Alsbeih G1.
Coronary sinus metastasis from cervical carcinoma.
Al-Ebrahim KE1.
Human papillomavirus prevalence and type distribution among
women attending routine gynecologicalexaminations in Saudi Arabia. AlObaid A, Al-Badawi IA, Al-Kadri H, Gopala K, Kandeil W, Quint W, Al-Aker M, DeAntonio R.
The Trend of Prophylactic Oophorectomy During Hysterectomy for Benign Disease at Different age Groups Single Institute Experience
Anfinan N,
119
127
129
133
6
139
142
151
36
PREVIOUSLY PUBLISHED ARTICLE
In Gynecological Cancer in Saudi Arabia 2013 - 2015
Incidence of malignant ovarian germ cell tumors (MOGCTs)in Saudi Arabia.
Abu-Zaid A1, Nazer A
2, Alomar O
3, Azzam A
4, Al-Eid HS
5, Elhassan TA
6, Al-Badawi IA
7.A
Is extended-field concurrent chemoradiation an option for radiologic negative paraaortic lymph node, locally advanced cervical cancer?
Asiri MA1, Tunio MA
1, Mohamed R
2, Bayoumi Y
2, Alhadab A
1, Saleh RM
3, AlArifi MS
1, Alobaid A
4.
Five years treatment outcomes of postoperative radiotherapy in Saudi women with
uterine cancers: singleinstitutional experience.
Al Asiri M1, Tunio MA
2, Bayoumi Y
3, Mohamed R
3, Al Saeed E
4, Al Hadab A
1, Al Amro A
1.
Gestational trophoblastic neoplasia: treatment outcomes from a single
institutional experience.
Al-Husaini H1, Soudy H, Darwish A, Ahmed M, Eltigani A, Edesa W, Elhassan T, Omar A,
Elghamry W, Al-Hashem H, Al-Hayli S, Madkhali I, Ahmad S, Al-Badawi IA.
Cytoreductive surgery and hyperthermic intraperitoneal chemotherapy for management of recurrent/relapsedovarian granulosa cell tumor: a single-center experience.
Al-Badawi IA1, Abu-Zaid A, Azzam A, AlOmar O, AlHusaini H, Amin T.
Ovarian surface epithelium receptors during pregnancy and estrus cycle of rats with
emphasis on steroids andgonadotropin fluctuation.
Saddick SY1.
Single versus multichannel applicator in high-dose-rate vaginal brachytherapy optimized by inverse treatment planning Yasir A. Bahadur, MD1, Camelia Constantinescu, PhD2, Ashraf H.
Hassouna, MD3,4, Maha M. Eltaher, MD1,4, Noor M. Ghassal, BSc2, Nesreen A. Awad, MD1,4
161
164
174
182
189
199
205
37
PREVIOUSLY PUBLISHED ARTICLE
In Gynecological Cancer in Saudi Arabia 2013 - 2015
Incidence rate of ovarian cancer cases in Saudi Arabia: an observational descriptive epidemiological analysis ofdata from Saudi Cancer Registry 2001-2008.
Alghamdi IG1, Hussain II
2, Alghamdi MS
3, Alghamdi MM
4, Dohal AA
4, El-Sheemy MA
5.
Knowledge of Saudi female university students regarding cervical cancer and acceptance of the human papillomavirus vaccine.
Al-Shaikh GK1, Almussaed EM, Fayed AA, Khan FH, Syed SB, Al-Tamimi TN, Elmorshedy HN.
Cervical cancer detection by time resolved spectra of blood components.
Kalaivani R1, Masilamani V
2, AlSalhi MS
2, Devanesan S
3, Ramamurthy P
4, Palled SR
5, Ganesh KM
5.
Evaluating the association between p53 codon 72 Arg>pro polymorphism and risk of ovary cancer: a meta-analysis.
Alqumber MA1, Akhter N
1, Haque S
2, Panda AK
3, Mandal RK
4.
Comparative Study of Alterations in Tri-iodothyronine (T3) and Thyroxine (T4) Hormone Levels in Breast and Ovarian Cancer Mahmood Rasool,
1
Muhammad Imran Naseer,2
Kalsoom Zaigham,3
Arif Malik,4
Naila Riaz,5
Rabail
Alam,6
Abdul Manan,7
Ishfaq Ahmed Sheikh,8
and Muhammad Asif9
Knowledge, perception, and attitudes about cancer and its treatment among healthy
relatives of cancer patients:single institution hospital-based study in Saudi Arabia. Eldeek B1, Alahmadi J, Al-Attas M, Sait K, Anfinan N, Aljahdali E, Ajaj H, Sait H.
Lumbosacral plexus delineation, dose distribution, and its correlation with radiation induced lumbosacralplexopathy in cervical cancer patients.
Tunio M1, Al Asiri M
1, Bayoumi Y
2, Abdullah O Balbaid A
1, AlHameed M
3, Gabriela SL
1, Amir O Ali A
1.
214
221
229
235
242
247
258
38
PREVIOUSLY PUBLISHED ARTICLE
In Gynecological Cancer in Saudi Arabia 2013 - 2015
Assessment of air pockets in high-dose-rate vaginal cuff brachytherapy using cylindrical applicators
Ashraf Hassouna, MD1,2, Prof. Yasir Abdulaziz Bahadur3, Camelia Constantinescu, PhD1
Perception of patients with cancer towards support management services and use of complementary alternative medicine – a single institution hospital based study in Saudi Arabia.
Sait KH1
, Anfinan NM, Eldeek B, Al-Ahmadi J, Al-Attas M, Sait HK, Basalamah HA, Al-Ama N, El-Sayed ME.
Vulvar extra uterine endometrial stromal sarcoma: A case report and literature review.
Zaza KJ1, Arafah MA2, Al-Badawi IA
3.
Human Papilloma Virus Early Proteins E6 (HPV16/18-E6) and the Cell Cycle Marker P16 (INK4a)are Useful Prognostic Markers in Uterine Cervical Carcinomas in Qassim Region-
Saudi Arabia. Omran OM1, AlSheeha M.
Uterine perforation and its dosimetric implications in cervical cancer high-dose-rate brachytherapy Yasir A. Bahadur, MD1, Maha M. Eltaher, MD1,2, Ashraf H. Hassouna, MD2,3, Mohammad
A. Attar, MD1, Camelia Constantinescu, PhD4
Human papillomavirus genotyping and integration in ovarian cancer Saudi
patients Al-Shabanah OA, Hafez MM1, Hassan ZK, Sayed-Ahmed MM, Abozeed WN, Al-Rejaie SS, Alsheikh AA.
265
270
278
283
293
301
39
PREVIOUSLY PUBLISHED ARTICLE
In Gynecological Cancer in Saudi Arabia 2013 - 2015
Chemo sensitizing effect of aqueous extract of sweet fennel in Cisplatin treated HeLa cells Abstract presented in Chicago on 28-31 March 2015,Annual Meeting on Women's Cancer
Wafaa S.Ramadan a, Khalid H. Saitb , Nisreen M. Anfinanb and Hesham Sait c a Department of Anatomy, Faculty of Medicine, King Abdulaziz University bGynecology Oncology Unit, Faculty of Medicine, King Abdulaziz University Hospital c Medical student, Faculty of Medicine, King Abdulaziz University
1
Outcome of Cervix Uteri Cancer Patients Treated With or Without Concurrent Chemotherapy and radiotherapy Incorporating High Dose Rate Brachytherapy: A Retrospective Single Institution Study In Saudi Arabia Abstract presenting in Quebec on 09-12 July 2015,Annual Clinical and Scientific Conference
Mohamed E. El Sayed1,6, Yasir A. Bahadur1,3, Ehab E. Fawzy 1,7,Bakr Ben Sadiq2, Azza M. Nasr 5,6,Rayed Dada8 , Khalid H. Sait 4and Nisreen
M.Anfian4
2
Assessment of knowledge, awareness and attitudes towards cervical
cancer and its screening among women in Makkah – Saudi Arabia
Abstract presented in 24th Saudi Obstetrics & Gynecology Society Annual Meeting Intercontinental Hotal,Jeddah31st Mars – 2nd April,2015 Jeddah ,KSAHani A. Aziz Jokhdar, Mohammed A. Garout, Enas H. AlFalogy, Rahma A. ALZahrany, Weaam S. Al-Harbi, Wafaa I. Bahwaini, Hibah S.Alharbi
3
original article
Ann Saudi Med 2013 January-February www.annsaudimed.net 13
Cervical cancer is the third most common cancer a!ecting females and the fourth leading cause of cancer death in females worldwide, account-
ing for 9% (529 800) of the total newly diagnosed can-cer cases and 8% (275 100) of the total cancer deaths among females in the year 2008. More than 85% of these cases and deaths occur in developing countries.1
"e incidence of cervical cancer is low in Saudi women. According to the 2007 Saudi cancer registry report, cervical cancer is the thirteenth most frequent cancer in Saudi women. "e incidence rate in Saudi Arabia is one of the lowest in the world at 1.9 cases per 100 000 women, accounting for 2.2% of diagnosed cas-es of cancer in Saudi women.2 Although cervical cancer is both preventable and curable, most women in Saudi Arabia present at advanced stages that require extensive
Prevalence of high-risk human papillomavirus infections in healthy Saudi women attending gynecologic clinics in the western region of Saudi ArabiaNabeel Salem Bondagji,a Faten Salah Gazzaz,b Khalid Sait,a Layla Abdullahc
From the aFaculty of Medicine King Abdulaziz University, Obstetrics and Gynecology, Jeddah, Saudi Arabia, bKing Abdulaziz University, Laboratory Medicine, Jeddah, Saudi Arabia, cKing Abdulaziz University, Pathology, Jeddah, Saudi Arabia
Correspondence: Dr. Nabeel Salem Bondagji · Faculty of Medicine King Abdulaziz University, Obstetrics and Gynecology, P.O. Box 80215 Jeddah 21589 Saudi Arabia · T:+966(2)6408310, F:+966(2)6408316 · [email protected]
Ann Saudi Med 2013; 33(1): 13-17
DOI: 10.5144/0256-4947.2013.13
BACKGROUND AND OBJECTIVES: Infection with human papillomavirus (HPV) is the major cause of cervical cancer. There is little published data on the prevalence of HPV infection among Saudi women. The aim of this study was to determine the prevalence of HPV in a group of women in the western region of Saudi Arabia.DESIGN AND SETTING: A prospective study of Saudi women seeking gynecologic care at King Abdulaziz University Hospital from March 2010 to January 2011.PATIENTS AND METHODS: Four hundred eighty-five Saudi women of different age groups attending gynecol-ogy clinic were tested for high-risk HPV DNA. HPV DNA was detected in cervical scrapes using Hybrid Capture 2 (HC2) high-risk HPV DNA test. The prevalence of HPV DNA positivity in different age groups was calculated. RESULTS: Out of the 485 specimens, 27 (5.6%) were positive for the high-risk HPV. The highest percentage was among women aged 60 years and older. Patients in the age group 40-49 years were more likely to accept HPV testing with a total of 188 patients.CONCLUSION: The prevalence of HPV in this group of Saudi women is similar to what was reported in some Arab countries and lower than that reported in developed countries. This information could be used to help in establishing a primary screening program using HPV DNA testing in Saudi Arabia.
chemoradiation therapy.3,4 "is is due to the lack of a proper screening program.5 Cervical cancer is caused by sexual exposure to an oncogenic type of the human pap-illomavirus (HPV), usually types 16 and 18.6-9
"e FDA has approved the Digene Hybrid Capture 2 High-Risk HPV DNA Test as a cervical screening test for HPV infection.10 "ere are clear benefits for the use of HPV DNA testing in the triage of equivocal smears, low-grade smears in older women and in the post-treatment surveillance of women after treatment for cervical intraepithelial neoplasia. However, there are still issues regarding how best to test in primary screen-ing.11 "e most resourceful and cost-e!ective screening techniques include visual inspection of the cervix after applying acetic acid or Lugol iodine and DNA testing for human HPV DNA in cervical cell samples.12 A
original article PAPILLOMAVIRUS IN SAUDI WOMEN
Ann Saudi Med 2013 January-February www.annsaudimed.net14
recent clinical trial from India in a low-resource area concluded that a single set of HPV DNA testing was associated with a 50% reduction in the risk of devel-oping advanced cervical cancer and associated deaths.13 Currently, little is known about the prevalence and type of distribution of HPV in Saudi Arabia. Introduction of appropriate screening for cervical cancer in Saudi Arabia requires extensive work to find whether HPV infection is a significant healthcare problem. In addition, baseline information on HPV prevalence and genotype distribution is highly desirable to evaluate the impact of prophylactic HPV vaccines in the near future. "is study aimed to evaluate the feasibility of using HPV testing as a primary screening for cervical cancer by de-termining the prevalence in a group of Saudi women and assessing the prevalence among di!erent age groups to evaluate the feasibility of using HPV DNA testing as a primary screening test for cervical cancer.
PATIENTS AND METHODS"is was a prospective cohort hospital-based study of all Saudi women attending gynecology clinics at King Abdulaziz University Hospital from March 2010 to January 2011 who fulfilled the inclusion criteria of be-ing sexually active and of childbearing or postmeno-pausal age. Exclusion criteria included virginity, preg-nancy, known cases of HPV, cervical precancerous le-sions or cervical cancer, positive cytology on Pap smear, patient refusal to participate in the study and nonSaudi nationality. Women who were eligible for the study consented to participation after being counseled by one of the gynecologist in the clinics and given the liberty to participate or decline; only women who signed the consent form were included in the study. "e women then had the traditional Pap smear using the wet mount technique and the Hybrid Capture 2 (HC2) reagents and materials for HPV detection (Digene Corporation, USA). Pap smear results were reported according to the Bethesda system for reporting of cervical cytol-ogy.14 Smears with no abnormalities or one with reac-tive changes were considered normal while all smears of atypical squamous cell of undetermined significance (ASC-US) or higher were considered as abnormal.
"e specimen collection was done via cells taken from the cervix with the Digene cervical sampler kit, then placed into the Digene liquid collection medium. "e specimen collection was performed by a gynecolo-gist after taking a detailed history and performing a physical examination including pelvic examination. "e Digene HPV HC2 test used in the study detects the high/intermediate risk HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68). "e target DNA
hybridizes with a specific high-risk HPV RNA probe-forming RNA/DNA hybrids which are captured onto the surface of a microplate well coated with antibod-ies specific for RNA/DNA hybrids. Fixed hybrids are then reacted with alkaline phosphatase conjugated antibodies specific for the RNA/DNA hybrids, which are then identified with a chemiluminescent substrate, where several alkaline phosphatase molecules unite to each antibody. Multiple united antibodies bind to each captured hybrid give considerable signal amplification. Light is released when the substrate is broken by the bound alkaline phosphatase, then measured as relative light units (RLUs) on a luminometer and its strength indicates the presence or absence of target DNA in the specimen. "e interpretations of the test results were carried out according to the manufacturer’s instruc-tion. "e women were classified into groups based on their age (19-29 years), (30-39), (40-49), (50-59) and (60 years and older). Data were collected and analyzed using SPSS statistical package version 16. "e insti-tutional human ethics committee for King AbdulAziz University Hospital approved the study protocol based on the international recommendations on hu-man subject research and according to principles of the Helsinki declaration.
RESULTSDuring the study period, 6585 women were seen in the gynecology clinics for di!erent clinical complaints. "e majority of cases had menstrual cycle abnormalities fol-lowed by pelvic pain, vaginal discharge, urinary inconti-nence and dyspareunia in order of frequency. Applying the inclusion criteria, 1649 patients were eligible. Out of 1649 eligible women, 1164 were excluded from the study for the following reasons: 704 refused to partici-pate in the study after counseling, 429 were pregnant or seen for pregnancy complications, and 16 had tech-nical di#culties in collecting or processing the sample according to the study protocol, 15 for previous posi-tive HPV, cervical carcinoma or cervical precancerous lesions. "e commonest cause for refusal to participate was the psychological fear of the impact of positive test on the patient’s physical and social life.
Four hundred and eighty-five women participated in the study. "e age range was 19 to 91 years with a mean age of 44.7 years. "e majority of women were multiparous, 403 (83%). "e parity ranged from 1 to 9 with a mean of 3.5. Four hundred and seventeen women (86%) were married, 44 (9%) divorced and 24 (5%) were widows. "e Pap smear was abnormal in 118 (24.3%) women and normal in 367 (75.7%).
Of 485 patients, 458 (94.4%) were negative for
original articlePAPILLOMAVIRUS IN SAUDI WOMEN
Ann Saudi Med 2013 January-February www.annsaudimed.net 15
HPV infection while 27 (5.6%) were positive (Table 1). "e detection rate of HPV, DNA varied according to age showing the highest rate among women age 60 years and older. Furthermore the study showed that 16 women of 334 patients in the reproductive age group (19-49 years) tested positive for HPV DNA with a de-tection rate of 4.8% compared to 11 of 151 postmeno-pausal women (50 years and older) with a detection rate of 7.3%. "e highest number of tested patient was reported in the group 40-49 years old with 188 women (32% of the collected samples).
DISCUSSION"e unduly high burden of cervical cancer in develop-ing countries is mostly due to a lack of screening pro-grams that allows detection of precancerous and early stage cervical cancer.15,16 Out of the 30 to 40 known HPV genotypes that infect the mucosa of the female genital tract, eight types (16, 18, 45, 31, 33, 52, 58, and 35) are accountable for 95% of cervical cancers and two genotypes (16 and 18) are responsible for 70 percent of the cervical cancer cases.17 "e HPV vaccine pro-tects against the most common strains of HPV infec-tions (HPV types 16 and 18). E!ective utilization of the available vaccine depends on the prevalence and the genotype of HPV in the targeted population. "e estimated global HPV prevalence was 11.7%. It was estimated to be 24.0% in Sub-Saharan Africa, 21.4% in Eastern Europe, and 16.1% in Latin America. "e age-specific HPV prevalence distribution showed a first peak at younger ages (<25 years) in Latin America and older ages ($45 years) in North America and Africa.18
In two recent studies from Saudi Arabia on the HPV genotype associated with cervical cancer, Alsbeih et al19 showed that 81% of cervical cancers specimens tested in their institution in the central part Saudi Arabia were associated with HPV infection, the ma-jority 78.7% (70/89) of HPV-positive tumors were infected with HPV-16/18. Al-Badawi et al20 reported similar finding with 95.5% detection of HPV in cervi-cal cancer specimens, the most common HPV geno-type detected being HPV-16 (63.4%), followed by HPV-18. "ese two studies clearly show that the most prevalent HPV genotype in Saudi women with cervi-cal cancer were 16 and 18 which is no di!erent than was reported globally.17
"e role of high-risk HPV DNA testing is grow-ing and HPV DNA testing, either alone or in combi-nation with cervical cytology, has been shown in many studies to be more sensitive than cervical cytology alone in detecting low- or high-grade cervical lesions.21-23 In addition, HPV DNA testing has been proposed both
as a primary screening method (either as an adjunct or instead of Pap smear) and as a method to triage Pap smear results that are equivocal.24-26
Many studies have documented the use of HPV DNA testing as a primary screening tool. In a Canadian randomized control trial on 10 154 women, Mayrand et al27 compared HPV DNA testing with conventional Pap smear and concluded that HPV testing has greater sensitivity for the detection of cer-vical intraepithelial neoplasia than the conventional Pap smear. Ronco et al,28 in a large randomized con-trolled Italian trial that included two groups of wom-en, 47,001 were assigned to the cytology group and 47,369 to the HPV testing group. "ey concluded that HPV-based screening is more e!ective than cy-tology in preventing invasive cervical cancer, by detect-ing persistent high-grade lesions earlier.
In a low-resource setting, a single round of HPV DNA testing was associated with a significant reduc-tion in the numbers of advanced cervical cancers and deaths from cervical cancer. "is was clearly demon-strated in a randomized trial of 131 746 women aged 30 to 59 years in rural India that compared a single lifetime screening with one of three screening modalities with standard care; the screening modalities were HPV test-ing using the Hybrid Capture HC2, cervical cytology, or visual inspection of the cervix with acetic acid.29
"e current study reported a prevalence of 5.6% of the high/intermediate-risk HPV types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 68) in Saudi women attending gynecology clinics for di!erent complains. "e only other reports from Saudi Arabia identified through a PubMed search were by Al-Muammar et al30 and Gazzaz.31 In a small number of patients attend-ing family medicine clinics in Riyadh, Saudi Arabia, Muammar et al30 reported a high prevalence of HPV infection, reaching 31.6% with the majority of cases be-
Table 1. Result of negative and positive HPV DNA in cervical specimens by Hybrid Capture 2.
Age group (years) n
Number of negative
patients (%)
Number of positive
patients (%)
19-29 54 53 (98.1) 1 (1.9)
30-39 92 87 (94.6) 5 (5.4)
40-49 188 178 (94.7) 10 (5.3)
50-59 108 101 (93.5) 7 (6.5)
!60 43 39 (90.7) 4 (9.3)
19-91 485 458 (94.4) 27 (5.6)
original article PAPILLOMAVIRUS IN SAUDI WOMEN
Ann Saudi Med 2013 January-February www.annsaudimed.net16
ing HPV-16 followed by 18. "e age distribution was not mentioned in that study.
"e prevalence in the current study (5.6%) is similar to that reported by Gazzaz31 (5%) and much lower than the one reported by Al-Muammar et al30 (31.6%). "e current study showed a prevalence peak on relatively older women (>60 years), which might be explained by the lack of screening program and earlier testing in those women. A report from Egypt on 5453 women32 showed a prevalence of 4.0% for HPV among Egyptian women included in the study. "ese figures are in ac-cordance with our figure, but the age distribution for HPV positivity was younger in the Egyptian women compared to our results. "e prevalence of HPV IN 1026 Lebanese women aged 18-76 years33 was 4.9% with 3% for high risk HPV type 16 DNA with peak at 60-69 years of age, which was similar to our results.
Comparing our positive results (5.6%) with those from other countries such as the United States34 (26.8%) and China21,35 (13.5%-17.6%) show that the prevalence of cervical HPV infection among females in Saudi Arabia is relatively low. In addition, the age dis-tribution shows a marked di!erence. "e current study shows a prevalence peak in women in the age group 60 years and older compared to a prevalence peak in the age group of 20 to 24 years in the United States.34
"e current study shows a low detection rate (1 case, 1.9%) of HPV DNA positivity in women un-der the age of 30 years. Our findings concurs with the findings of Kjaer et al36 in their study to determine the absolute risk of cervical abnormalities in women with normal cytology and a positive high-risk Hybrid Capture 2 (HC2) test, which showed that the rate of development of a cervical lesion to be 17.7% in younger women compared to 24.5% in older women. In another
study by Khan et al37 from the United States to explore the risk of cervical precancerous lesions in women with normal cytology and positive testing for HPV DNA type 16 and 18 showed that women 30 years of age and older had a higher risk of developing cervical lesions than younger women.
Datta et al38 measured the Pap test results and high-risk HPV prevalence by Hybrid Capture 2 assay in 9657 women age 14 to 65 years receiving routine cervi-cal screening and concluded that high-risk HPV was widespread among women receiving cervical screening in the United States. "ey suggested that many women 30 years of age or older with normal Pap tests would need follow-up if Hybrid Capture 2 testing is added to cytology screening.
In the present study HPV infection among females in a Saudi community was done on a larger number than that was done by Gazzaz31 and Muammar et al.30 "e current study provides a unique opportunity to gather an idea about baseline data on cervical HPV prevalence among females in the western region of Saudi Arabia. "e protocol and methodology applied in the current study was successful and could be used in a larger nationwide research.
"e prevalence of HPV in this group of Saudi women in the western region of Saudi Arabia is similar to what is reported in some other Arab countries and lower than what is reported in developed countries and some parts of Asia. "is information can be used in es-tablishing a proposal for using HPV testing by hybrid capture as a primary screening for cervical cancer in Saudi Arabia. Multicenter population prevalence data for HPV on a larger scale in women in Saudi Arabia is required before the implementation of routine HPV vaccination in this country.
original articlePAPILLOMAVIRUS IN SAUDI WOMEN
Ann Saudi Med 2013 January-February www.annsaudimed.net 17
1. Jemal A,Bray F, Center M,Ferlay J, Ward E,Forman D.Global cancer statistics. CA CANCER J CLIN 2011; 61:69– 90.2. Saudi Cancer Registry cancer incidence and survival reports Saudi Arabia 2007. National Saudi Cancer Registry. Riyadh (KSA): Ministry of Health. Available at: http://www.scr.org.sa/reports/SCR2007.pdf. 3. El Dosoky M, Ismail N, Dagastani M. Preinvasive cervical carcinoma in Saudi Arabia. Lancet 1995 Mar 11; 345(8950): 650.4. Manji M. Cervical cancer screening program in Saudi Arabia: action is overdue. Ann Saudi Med 2000 Sep-Nov; 20(5-6): 355–357.5. Kitchener HC, Symonds P. Detection of cervical intraepithelial neoplasia in developing countries. Lancet 1999 Mar 13; 353(9156): 856-857.6. Zur Hausen H. Human papillomaviruses in the pathogenesis of anogenital cancer. Virology 1991 Sep; 184(1): 9–13.7. Bosch FX, Manos MM, Munoz N, et al. Preva-lence of human papillomavirus in cervical cancer: a worldwide perspective. International biological study on cervical cancer (IBSCC) Study Group. J Natl Cancer Inst 1995 June 7; 87(11): 796–802.8. Walboomers JM, Jacobs MV, Manos MM, et al. Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. J Pathol 1999 Sep; 189(1): 12–19.9. Munoz N, Bosch FX, de Sanjose S, et al. Epide-miologic classification of human papillomavirus types associated with cervical cancer. N Engl J Med 2003 Feb 6; 348(6): 518–527.10. US Food and Drug Administration. FDA News. FDA approves expanded use of HPV test. www.fda.gov/bbs/topics/news/2003/new00890.html. Ac-cessed July 14, 2003.11. Cuzick J, Arbyn M, Sankaranarayanan R, Tsu V, Ronco G, Mayrand MH, Dillner J,et al. Overview of human papillomavirus-based and other novel op-tions for cervical cancer screening in developed and developing countries. Vaccine. 2008 Aug 19; 26 Suppl 10:K29-41.12. Sherris J, Wittet S, Kleine A, et al. Evidence-based, alternative cervical cancer screening ap-proaches in low-resource settings. Int Perspect Sex Reprod Health. 2009; 35:14 7-154.13. Sankarana rayanan R, Nene BM, Shastri SS, et al. HPV screening for cervical cancer in rural India. N Engl J Med. 2009; 360: 1385-13 94.14. Solomon D, Davey D, Kurman R, Moriarty A, O’Connor D, Prey M et al. The 2001 Bethesda Sys-tem: terminology for reporting results of cervical cytology. JAMA 2002; 287:2114-2119.
15. Parkin DM, Almonte M, Bruni L, Clifford G, Cu-rado MP, Pineros M. Burden and trends of type-specific human papil lomavirus infections and re-lated diseases in the Latin America and Caribbean an region. Vaccine. 2008; 26(suppl 11):L1-L 15.16. Mathew A, Geoge PS. Trends in incidence and mortality rates of squamous cell carcinoma and adenocarcinoma of cervix– worldwide. Asia n Pac J Cancer Prev. 2009; 10:645-6 50.17. Kahn JA. HPV vaccination for the prevention of cervical intraepithelial neoplasia. N Engl J Med 2009; 361:271. 18. Bruni L, Diaz M, Castellsagué X, Ferrer E, Bosch FX, de Sanjosé S.Cervical human papillo-mavirus prevalence in 5 continents: meta-analysis of 1 million women with normal cytological find-ings. J Infect Dis. 2010 Dec 15; 202(12):1789-99. 19. Alsbeih G, Ahmed R, Al-Harbi N, Venturina LA, Tulbah A, Balaraj K. Prevalence and genotypes’ distribution of human papillomavirus in invasive cervical cancer in Saudi Arabia. Gynecol Oncol. 2011 Jun 1; 121(3):522-6. 20. Al-Badawi I, Al-Suwaine A, Al-Aker M, Asaad L, Alaidan A. Tulbah A,et al. Detection and Geno-typing of Human Papilloma Virus in Cervical Can-cer Specimens from Saudi Patients. Int J Gynae-col Cancer: 2011; 21: 907-910.21. Wu RF, Dai M, Qiao YL, Clifford GM, Liu ZH, Arslan A, et al. Human Papillomavirus infection in women in Shenzhen City, People’s Republic of China, a population typical of recent Chinese ur-banisation. Int J Cancer 2007, 121:1306-1311.22. ASCUS-LSIL Traige Study (ALTS) Group. A randomized trial on the management of low-grade squamous intraepithelial lesion cytology interpre-tations. Am J Obstet Gynecol 2003; 188:1393. 23. Cuzick J, Clavel C, Petry KU, et al. Overview of the European and North American studies on HPV testing in primary cervical cancer screening. Int J Cancer 2006; 119:1095. 24. Kim JJ, Wright TC, Goldie SJ. Cost-effective-ness of human papillomavirus DNA testing in the United Kingdom, The Netherlands, France, and Italy. J Natl Cancer Inst 2005; 97:888. 25. Denny LA, Wright TC Jr. Human papillomavirus testing and screening. Best Pract Res Clin Obstet Gynaecol 2005; 19:501. 26. Koliopoulos G, Arbyn M, Martin-Hirsch P, et al. Diagnostic accuracy of human papillomavirus testing in primary cervical screening: a system-atic review and meta-analysis of non-randomized studies. Gynecol Oncol 2007; 104:232.27. Mayrand MH, Duarte-Franco E, Rodrigues I, et al. Human papillomavirus DNA versus Papanico-
laou screening tests for cervical cancer. N Engl J Med 2007; 357:1579.28. Ronco G, Giorgi-Rossi P, Carozzi F, Confortini M, Dalla Palma P, Del Mistro A et al. Efficacy of human papillomavirus testing for the detection of invasive cervical cancers and cervical intraepithelial neo-plasia: a randomized controlled trial. Lancet Oncol. 2010 Mar; 11(3):249-57.29. Sankaranarayanan R, Nene BM, Shastri SS, Jayant K, Muwonge R, Budukh AM et al. HPV screening for cervical cancer in rural India. N Engl J Med. 2009 Apr 2;360(14):1385-94.30. Al-Muammar T, Al-Ahdal MN, Hassan A, Kes-sie G, Dela Cruz DM, Mohamed GE. Human papil-loma virus-16/18 cervical infection among women attending a family medical clinic in Riyadh. Ann Saudi Med. 2007 Jan-Feb; 27(1):1-5.31. Gazzaz FS. Molecular Testing of Human Pap-illomavirus (HPV) in Cervical Specimens. Saudi Med J 2007; Vol. 28 (12): 1810-1818.32. El-All HS, Refaat A, Dandash K.Prevalence of cervical neoplastic lesions and Human Papilloma Virus infection in Egypt: National Cervical Cancer Screening. Infect Agent Cancer. 2007 Jul 4; 2:12. 33. Mroueh AM, Seoud MA, Kaspar HG, Zalloua PA.Prevalence of genital human papillomavirus among Lebanese women Eur J Gynaecol On-col. 2002; 23(5):429-32.34. Dunne EF, Sternberg M, McQuillan G, Swan DC, Patel SS, Markowitz LE. Prevalence of HPV in-fection among females in the United States. JAMA 2007 Feb 28; 297(8):813-819.35. Li LK, Dai M, Clifford GM, Yao WQ, Arslan A, Li N, Shi JF,et al. Human Papillomavirus infection in Shenyang City, People’s Republic of China: A population-based study. Br J Cancer 2006, 95:1593-1597.36. Kjaer S, Høgdall E, Frederiksen K, Munk C, van den Brule A, Svare E, et al. The absolute risk of cervical abnormalities in high-risk human papil-lomavirus-positive, cytologically normal women over a 10-year period. Cancer Res 2006; 66:10630.37. Khan MJ, Castle PE, Lorincz AT, Wacholder S, Sherman M, Scott DR et al. The elevated 10-year risk of cervical precancerous and cancer in women with human papillomavirus (HPV) type 16 or 18 and the possible utility of type-specific HPV testing in clinical practice. J Natl Cancer Inst 2005; 97:1072.38. Datta SD, Koutsky LA, Ratelle S, Unger ER, Shlay J, McClain T et al. Human papillomavirus in-fection and cervical cytology in women screened for cervical cancer in the United States, 2003-2005. Ann Intern Med. 2008 Apr 1; 148(7):493-500.
REFERENCES
Journal of American Science 2013;9(5) http://www.jofamericanscience.org
375
Cervical Cancer Prevalence at King Abdulaziz University Hospital
Fadwa J. Altaf1 Ghadeer A. Mokhtar2 and Faris M. Altaf3
1Professor of Pathology and Consultant Pathologist, Department of Pathology, King Abdulaziz University. Principle investigator of "Cervical cancer research"[email protected]
2 Ghadeer A. Mokhtar Associate Professor and Consultant Pathologist, Department of Pathology King Abdulaziz University.
3 Faris MS. Altaf. PhD. Medical School, Umm Al-Qura University Abstract: Introduction: Cervical cancer (CC) is the tenth most frequent cancer in females in Saudi Arabia. Squamous intra epithelial changes are increasing in the recent years in many reports from different regions of the Kingdom. However, no information on the frequent histopathological types of CC. Objective: The objective of this study is to identify the most frequent histopathological types of CC diagnosed at King Abdulaziz University Hospital (KAUH) and to compare it with literature. Methods: A retrospective study was designed to reclassify all cases that were diagnosed as CC by using the World Health Organization (WHO) classification system at the Department of Pathology of KAUH from January 1990- September 2012. We identified and reclassified 167 cases. Results: The most frequent type was Squamous cell carcinoma (83%) followed by adenocarcinoma (7.7%) and then adenosquamous (3.6%). The Non keratinizing and keratinizing squamous cells are of nearly equal distribution in frequency and age (32%) versus (30%) with mean age of 50 years versus 54 years. Adenocarcinoma tends to be presented at slightly earlier age. Conclusion: Cervical carcinoma occurs in matching frequency with major histological type and age group in literature although the keratinizing and non keratinizing are almost similar in prevalence which is different than other reports. Lack of CC screening program and research in this area obscure a lot of information of the natural history of this health problem in this part of the world. [Fadwa J. Altaf Ghadeer A. Mokhtarand Faris M. Altaf. Cervical Cancer Prevalence at King Abdulaziz University Hospital. J Am Sci 2013;9(5):375-379]. (ISSN: 1545-1003). http://www.jofamericanscience.org. 47 Key words: Cervical cancer, Saudi Arabia, Human Papilloma virus. Squamous intraepithelial lesions 1. Introduction:
Our understanding of CC has changed a lot after series of publications in this aspect. It is well documented in the literature that CC has many risk factors; top of the list is Human Papilloma Virus infection (HPV) (1). The viral infection will cause series of cytological changes in cervical epithelium that can be detected by cytological examination of the cervix by Pap smear. Its incidence was the second cancer of the females in Western Societies in the early sixties, and nowadays it drops down to eight (2-6). In the United State of America, according to the National Cancer Institute (NCI), it is estimated that 11,270 women were diagnosed with cancer of the cervix uteri in 2009; about 4,070 women died of it (7). Its incidence is higher in Hispanics and black in comparison to Asian/Pacific Islander and Whites. The mortality rate was higher in Black American in comparison to the other ethnic groups. The median age of diagnosis of cervical cancer was 48 years.
In Canada, Incidence rates of cervical cancer have declined dramatically over the last 3 decades, from 19.4 per 100 000 women in 1971 to 8.4 per 100 000 women as estimated in 2000 (8). Dr Liu et al., reported that the overall age-adjusted incidence rates of cervical squamous cell carcinoma declined by 51%, from 13.39 per 100 000 women in 1970–1972
to 6.56 per 100 000 women in 1994–1996. Conversely, incidence rates of adenocarcinoma and adenosquamous carcinoma increased considerably, from 1.30 and 0.15 per 100 000 women respectively, in 1970–1972, to 1.83 and 0.41 per 100 000 women respectively, in 1994–1996. Further analysis showed that the observed increase in overall incidence rates of adenocarcinoma and adenosquamous carcinoma was mainly in women aged 20–49 years (9).
In Saudi Arabia (SA), the World Health Organization ( WHO) currently estimates that women population of 6.5 million are at age 15 years and older, and they are at risk of developing cervical cancer, which ranks as the 11th most frequent cancer among females in SA in general.
It is the 8th most frequent cancer among women between 15 and 44 years of age (10). According to Saudi cancer registry of 2001(NCR) report, carcinoma of cervix rank number tenth preceded by cancer of breast, thyroid, leukemia, lymphoma, brain, colon, oral cavity, ovary and Hodgkin lymphoma. Its prevalence is 3% and Age Specific Rate (ASR) is 2/100,000 female population (11). No recent data from NCR of cervical cancer prevalence in their 2006 report.
WHO recognize more than 30 histological types of CC (12). In this study we tried to reevaluate all the
Journal of American Science 2013;9(5) http://www.jofamericanscience.org
376
cases of carcinoma of cervix by reclassifying them based on WHO histopathology classification (12&13)
2.Material and Methods:
We used the data base of Phoenix System which is available in Anatomical pathology laboratory, to identify the cases with cervical biopsies that had cervical cancer diagnosis or carcinoma in situ in the archives of the Department of Pathology King Abdulaziz University Hospital. We also used the manual search of the old cases (from 1995-1990). The list of the cases and the slides were prepared for the pathologist (authors) to examine them. We excluded cervical biopsies with the diagnosis of cervical intraepithelial neoplasia( CIN I& CIN II), endometrioid carcinoma of endometrial type that presented as cervical mass, or even adenocarcinoma
of cervix (endometrioid ) type that cannot be differentiated histopathologically from endometrial type based on the material submitted.
The hematoxcilin and eosin stained (H&E) slides of each case were reviewed and reclassified according to modified World Health Organization (WHO) of invasive carcinoma of the uterine cervix by two of the authors (FA &GM).
3.Results:
A total of 167 cases were found with the diagnosis of carcinoma of cervix. Some cases had single histopathology diagnosis (n=147), others had multiple specimen over a period of time (n=20), but the diagnosis was based on one specimen and they are counted as one case. The different histological types that we identified were tabulated in table II.
Table I: Modified World Health Organization (WHO) histological classification of invasive carcinomas of the
uterine cervix 1- Squamous cell carcinoma – Keratinizing / Non-keratinizing Microinvasive squamous cell carcinoma Invasive squamous cell carcinoma Verrucous carcinoma Warty (condylomatous) carcinoma Papillary squamous cell (transitional) carcinoma Lymphoepithelioma-like carcinoma 2- Adenocarcinoma Mucinous adenocarcinoma Endocervical type Intestinal type Signet-ring type Endometrioid adenocarcinoma Endometrioid adenocarcinoma with squamous metaplasia Clear cell adenocarcinoma Minimal deviation adenocarcinoma Endocervical type (adenoma malignum) Endometrioid type Well-differentiated villoglandular adenocarcinoma Serous adenocarcinoma Mesonephric carcinoma 3- Other epithelial tumors Adenosquamous carcinoma Glassy cell carcinoma Clear cell adenosquamous carcinoma Mucoepidermoid carcinoma Adenoid cystic carcinoma Adenoid basal carcinoma Typical carcinoid tumor Atypical carcinoid tumor Large cell neuroendocrine carcinoma Small cell carcinoma Undifferentiated carcinoma
Journal of American Science 2013;9(5) http://www.jofamericanscience.org
377
Table II: KAU cases Histolopathological type identified, mean age & Frequency Std. Deviation Mean Age % N Histological Types 14.54937 50.7037 32.1 54 Squamous cell carcinoma, non-keratinizing 13.51531 54.2353 30.1 51 Squamous cell carcinoma, keratinizing 30.0000 0.6 1 Microinvasive squamous cell carcinoma 29.0000 0.6 1 Invasive squamous cell carcinoma 11.98655 44.2500 19.6 32 Carcinoma in-situ / CIN III . 40.0000 0.6 1 Invasive papillary squamous cell carcinoma . 58.0000 0.6 1 In-situ adenocarcinoma 10.35725 48.0000 7.1 12 Invasive adenocarcinoma endocervical type 11.90658 47.8333 3.6 6 Adenosquamous carcinoma 6.36396 56.5000 1.2 2 Papillary serous adenocarcinoma 16.26346 51.5000 1.2 2 Mucoepidermoid carcinoma .00000 70.0000 1.2 2 Invasive adenosarcoma 38.0000 0.6 1 Small cell carcinoma . 46.000 0.6 1 Undifferentiated carcinoma 13.73053 50.2048 99.4 167 Total
4.Discussion:
The most common histological types of Cervical Carcinoma in the literature is the squamous cell carcinoma (SCC) accounted for 75-80% of CC with a mean age 55 years. The next common type is adenocarcinoma 20-25%, followed by adenosquamous and small cell carcinoma (12, 13). Majority of SCC are non- keratinizing, which means it lakes squamous pearls but individual cell keratinization is evident in most of the cases. The keratinized SCC contains squamous pearls and may also show individual cell keratinization. The risk factors of CC are as for squamous intraepithelial changes (SILs). WHO recognizes three major types of Cervical Carcinoma (CC) and 31 histological subtypes, as demonstrated in table I (12).
There are many reports from different provinces of the Kingdom mainly from Western region, and they are all hospital based studies (10&16-19). These reports indicate there is a definite increase in the prevalence of cervical epithelial changes in PS from (1.6 % to 7.9% and recently 17.3%). Which reflect an increase in the incidence of HPV infection.
Study from the Eastern province also indicates an increase of squamous epithelial changes (SIL) up to 4.9% seen in PS (20).
Few studies had looked at the presence of HPV in cytology by molecular methods (21) and found 5% of the cervical smears have oncogenic HPV.
Al-Muammar et al., looked also at HPV DNA in cytology specimen and correlates it with PS. Of the 120 samples used in their study, 38 (31.6%) were found positive by PCR for HPV-16 or HPV-18 or both (22). When they correlated that finding with the cytology they found 10 cases (8.3%) showed minor cytological changes (reactive and reparative changes, inflammatory cellular changes, and low-grade squa-
mous epithelial lesions [LGSL]) in the Pap smear test. Of these 10, only 6 were HPV-infected (1 HPV-16, 1 HPV-18, and 4 HPV-16/18), making the prevalence of HPV 60% (6/10) in those with cytological changes and 5% (6/120) in all subjects. Very much similar result to Gazaz study in the Western region 6% HPV DNA detected in cervical cytology (21).
Al Hebishi et al., looked at the prevalence of HPV virus DNA in 100 cases that have the diagnosis of cervical cancer. They concluded that eighty-nine percent of cervical cancers in Saudi Arabia were associated with HPV infection, and 78.7% (70/89) of HPV-positive tumors were infected with HPV-16/18, which caused the cancer to appear 5 years earlier than the combined HPV-negative and other HPV genotypes (P=0.013). (23)
We did not find a single study in Saudi Arabia that looked at the frequency of the histological types of cervical cancer and to compare it to literature.
We found no much difference in the prevalence between the non Keratinizing and keratinizing squamous cell carcinoma 32&31%. In addition no big differences in their mean age as well 51 versus 54 years. The literature review reveal there is difference in the prognosis in the treatment of the 2 groups when radiotherapy is a modality of treatment. Keratinizing SCC is less sensitive to radiotherapy in comparison to non keratinizing SCC and subsequently to survival (24).
The in situ cancer of squamous component in our study represent 20% (32/167) with mean age of 44 years, which is younger than the invasive SCC and still older than literature, the reason of this could be due lack of detection earlier as a result of absence of cervical cancer screening program or it could be
Journal of American Science 2013;9(5) http://www.jofamericanscience.org
378
related to the disease natural history in this part of the world.
Invasive adenocarcinoma represented 7% of our cases. It is presenting few years earlier than invasive SCC (48 years) but no big age difference as reported in literature
Liu et al., (9) reported in their study that several reports from Sweden, United States, and Australia have reported an increase of incidence of invasive cervical adenocarcinoma, although the overall incidence of cervical cancer has declined. It accounts for 10%–15% of all cervical cancers and it has been increasing in young women (age range 29-49yrs.). The cause of the increase is unclear, but the possibly of Changes in sexual habits and increased transmission of HPV, but it are of concern because some studies have shown a poorer prognosis for patients with cervical adenocarcinoma than for those with squamous cell carcinoma. At the time of diagnosis, adenocarcinoma tends to be larger and exhibits a propensity for early lymphatic and hematogenous metastasis (25).
We identified 2 cases of serous carcinoma (1.2%) with average age of 56 years and both of them present with stage IV disease. It is a rare type of endocervical adenocarcinoma. Serous carcinoma of the cervix occurs much less frequently than serous carcinoma of the endometrium. Only one series with a significant number of patients has been reported and it contained only 17 cases (26). In one study, serous carcinoma did not appear to be associated with HPV infection. (27)
Serous carcinoma of the cervix occurs over a wide age range, but there appears to be a bimodal distribution, with a peak <40 years and another >65 years. (26) The typical clinical presentation is with abnormal vaginal bleeding or an abnormal Pap smear. Treatment has been the same as for other types of cervical carcinoma. In the largest series 6 of 15 patients died of carcinoma, an outcome similar to that observed in adenocarcinoma of the cervix overall. Serous carcinoma can occur as a pure type or a second type of cervical adenocarcinoma can be admixed.
Since serous carcinoma of the endometrium is much more common than serous carcinoma of the cervix, endocervical spread of an endometrial serous carcinoma must be excluded before a diagnosis of serous carcinoma of the cervix is made (26).
Papillary squamous cell carcinoma is a type of squamous cell carcinoma that grow in thin and thick papillae with fibrovascular core and the epithelium mimicking that of high grade squamous intraepithelial lesion. (28). typically this tumor occurs in women in the late reproductive period or postmenopausal age group. Mirhashemi et al reported
presence of HPV antibodies in 50% of their cases. (29) Declaration:
The authors declare that the study and manuscript have no conflict of interest and is not supported/funded by any Drug Company. Acknowledgement:
This project was funded by the Deanship of Scientific Research (DSR), King Abdulaziz University, Jeddah, under grant no. (259/140/1431). The authors acknowledge with thanks DSR technical and financial support. Corresponding Author: Fadwa Jameel Altaf Professor of pathology and consultant pathologist King Abdulaziz University. Jeddah Saudi Arabia. P.O. Box 51241, SA 21543. Email. [email protected] References: 1. Papanicolaou GN, Traut HF. The diagnostic value
of vaginal Smears in carcinoma of the uterus. 1941. Arch Pathol Lab Med 1997 ; 121: 211-224.
2. Pettersson F, Bjorkholm E, Naslund I. Evaluation of screening for cervical cancer in Sweden: trends in incidence and mortality 1958–1980. Int J Epidemiol 1985; 14:521-7
3. Anderson GH, Boyes DA, Benedet JL, Le Riche JC, Matisie JP, Suen KC, et al., Organisation and results of the cervical cytology screening program in British Columbia, 1955–85. BMJ 1988; 296:975-8.
4. Quinn M, Babb P, Jones J, Allen E, on behalf of the United Kingdom Association of Cancer Registries. Effect of screening on incidence of and mortality from cancer of the cervix in England: evaluation based on routinely collected statistics. BMJ 1999; 318:904-7.
5. Bergstrom R, Sparen P, Adami HO. Trends in cancer of the cervix uteri in Sweden following cytological screening. Br J Cancer 1999; 81:159-66.
6. Miller AB, Lindsay J, Hill GB. Mortality from cancer of the uterus in Canada and its relationship to screening for cancer of the cervix. Int J Cancer 1976; 17:602-12.
7. Howlader N, Noone AM, Krapcho M, Neyman N, Aminou R, Altekruse SF, Kosary CL, Ruhl J, Tatalovich Z, Cho H, Mariotto A, Eisner MP, Lewis DR, Chen HS, Feuer EJ, Cronin KA (eds). SEER Cancer Statistics Review, 1975-2009 (Vintage 2009 Populations), National Cancer Institute. Bethesda, MD, http://seer.cancer.gov/csr/1975_2009_pops09/, based on November 2011 SEER data submission,
Journal of American Science 2013;9(5) http://www.jofamericanscience.org
379
posted to the SEER web site, 2012. Cervical Cancer Incidence
8. National Cancer Institute of Canada. Canadian Cancer Statistics 2000. Toronto. The Institute 2000.p.17-38
9. Shiliang Liu, Robert Semenciw, Yang Mao Cervical cancer: the increasing incidence of adenocarcinoma and adenosquamous carcinoma in younger women. JAMA 2001.17 AVR: 164(8).
10. Altaf F.J, Mufti ST, Pattern of cervical smear abnormalities using the revised Bethesda system in a tertiary care hospital in Western Saudi Arabia. Saudi Med J 2012; Vol. 33 (6)
11. Altaf- FJ, Cervical Cancer Screening with Pattern of Pap Smears Review of Multicenter Studies. Saudi Med J 2006; Vol 27 (10)
12. Wright TC, A. Frenczy and RJ Kurman. Chapter 8. Carcinoma and Other Tumors of the Cervix. Blaustein Gynecological pathology 2002
13. Clement PB, Young RH. Atlas of Gynecologic Surgical Pathology. Saunders Elsevier. Second Edition 2008.Chapter 5&6, pages, 96-138.
14.Benedet JL, Bender H, Jones H III et al., FIGO staging classification and clinical practice guidelines in the management of gynecological Oncology. Int J. Gynaecol Obstet 70:209-262. 2000
15. Coutlee F, Ratnam S, Ramanakumar AV, Insinga RR, Bentley J, Escott N, Ghatage P, Koushik A, Ferenczy A, Franco EL. Distribution of human Papillomavirus genotype in cervical intraepithelial neoplasia and invasive cancer in Canada. J Med Virol.2011; 83(6):1034-41
16. Altaf FJ. Pattern of cervical smear cytology in the Western Region of Saudi Arabia. Ann Saudi Med. 2001; 21:92–6
17. Jamal A, Al-Maghrabi JA. Profile of Pap smear cytology in the Western region of Saudi Arabia. Saudi Med J. 2003; 24:1225–9.
18. Elhakeem HA, Al-Ghamdi AS, Al-Maghrabi JA. Cytopathological pattern of cervical Pap smear according to the Bethesda system in Southwestern Saudi Arabia. Saudi Med J. 2005; 26:588–92.
19. Abdullah LS. Pattern of abnormal Pap smears in developing countries: a report from a large
referral hospital in Saudi Arabia using the revised 2001 Bethesda System. Ann Saudi Med. 2007; 27:268–72.
20. Balaha M H , M S Al Moghannum, NAl Ghowinem, S. Al Omran Cytological pattern of cervical Papanicolaou smear in eastern region of Saudi Arabia J Cytol. 2011; 28(4): 173–177.
21. Gazaz FS. Molecular testing of human Papilloma in cervical specimen. Saudi Med J 2007; 28:1810-1818.
22. Al-Muammar T. , M N Al-Ahdal, A. Hassan, G. Kessie, D.MD.Cruz, G. E Mohamed. Human papilloma virus-16/18 cervical infection among women attending a family medical clinic in Riyadh. Ann Saudi Med .2007;27(1):1-5.
23. Alsbeih G. , R. Ahmed, N. Al-Harbi, L. A.Venturina, A. Tulbah, K.Balaraj Prevalence and genotypes' distribution of human papillomavirus in invasive cervical cancer in Saudi Arabia. Gynecologic Oncology. Volume 121, Issue 3, 1 June 2011, Pages 522–526.
24. Kumar S, Shah JP, Bryant SC, Media AN· Rouba Ali-Fehmi ·John M. Malone Jr · Robert T. Morris Prognostic significance of keratinization in squamous cell cancer of uterine cervix: a population based study. Arch Gynecol Obstet (2009) 280:25–32
25. DiSaia. Adenocarcinoma of the cervix: Clinical Gynecologic Oncology, 6th ed., 2002
26. Gilks, C. B. & P. B. Clement. "Papillary serous adenocarcinoma of the uterine cervix: a report of three cases." Modern Pathology 5: 426-431. 1992.
27. Pirog EC, Kleter B, (2000). "Prevalence of human papillomavirus DNA in different histological subtypes of cervical adenocarcinoma." American Journal of Pathology 157(4): 1055-1062.
28. Randall ME, Andersen WA, Mills SE, Kim JC, Papillary squamous cell of uterine cervix. A clincopathological study of nine cases. Int J Gynecol Pathol 1986; 5: 1-10.
29. Mirhashemi R. Ganjel-Azar P. Nadji M. Papillary squamous cell carcinoma of uterine cervix: an immunohistochemical appraisal of 12 cases. Gynecol Oncol 2003; 90:657- 661
3/12/2013
Alsbeih et al. Infectious Agents and Cancer 2013, 8:15http://www.infectagentscancer.com/content/8/1/15
RESEARCH ARTICLE Open Access
HPV prevalence and genetic predisposition tocervical cancer in Saudi ArabiaGhazi Alsbeih1,4*, Najla Al-Harbi1, Medhat El-Sebaie2 and Ismail Al-Badawi3
Abstract
Background: Cervical cancer incidence is low in Saudi Arabian women, suggesting low prevalence to HPVinfection due to environmental, cultural and genetic differences. Therefore, we investigated HPV prevalence andgenotype distribution in cervical cancer as well as the association with 9 genetic single nucleotide polymorphisms(SNPs): CDKN1A (p21) C31A, TP53 C72G, ATM G1853A, HDM2 promoter T309G, HDM2 A110G, LIG4 A591G, XRCC1G399A, XRCC3 C241T and TGFβ1 T10C, presumed to predispose to cancer.
Methods: One hundred cervical cancer patients (90 squamous cell carcinoma and 10 adenocarcinoma) and 100age/sex-matched controls were enrolled. SNPs were genotyped by direct sequencing and HPV was detected andtyped in tumors using the HPV Linear Array Test.
Results: Eighty-two cases (82%) were positive for HPV sequences. Seven HPV genotypes were present as singleinfections (16, 18, 31, 45, 56, 59, 73) and five double infections (16/18, 16/39, 16/70, 35/52, 45/59) were detected.Most common genotypes were HPV-16 (71%), 31 (7%), and 18, 45, 73 (4% each). Only XRCC1 SNP was significantlyassociated with cervical cancer (P=0.02, OD=1.69; 95% CI= 1.06–2.66). However, nested analysis revealed apreponderance of HPV-positivity in patients harboring the presumed risk allele TP53 G (P=0.06). Both XRCC1 andTP53 SNPs tended to deviate from Hardy-Weinberg equilibrium (HWE; P=0.03-0.07).
Conclusions: HPV prevalence (82%) in cervical cancer is at the lower range of the worldwide estimation (85 - 99%).While XRCC1 G399A was significantly associated with cervical cancer, TP53 G72C showed borderline association onlyin HPV-positive patients. Deviation from HWE in HPV-positive patients indicates co-selection, hence implicating thecombination of HPV and SNPs in cancer predisposition. Thus, SNPs could be more relevant biomarkers ofsusceptibility to cervical cancer when associated with HPV infection.
Keywords: Cervical cancer, Human papillomavirus (HPV), Predisposition, Single nucleotide polymorphism (SNP)
BackgroundCancer of the uterine cervix is the 3rd most frequentmalignancy affecting women worldwide and the seventhoverall, with an estimated 530,000 new cases in 2008[1,2]. Among all the known risk factors, human papillo-mavirus (HPV) stands as a main cause, and high-riskHPV infections play a major role in the pathogenesis ofcervical cancer with an estimated prevalence between85% to 99% [3-7]. More than 85% of the global burdenoccurs in developing countries, where it accounts for
* Correspondence: [email protected] Physics, King Faisal Specialist Hospital & Research Centre, Riyadh,Saudi Arabia4Research Centre, Biomedical Physics Department, KFSHRC, MBC-03, P.O. Box3354, Riyadh 11211, Saudi ArabiaFull list of author information is available at the end of the article
© 2013 Alsbeih et al.; BioMed Central Ltd. ThisAttribution License (http://creativecommons.oreproduction in any medium, provided the or
13% of all female cancers. This is due to the lack ofproper screening program that has helped reducingcervical cancer incidence and mortality rates by 70% indeveloped countries [8,9].In contrast to the global view, the incidence of cervical
cancer is very low in Saudi Arabia, ranking number 11between all cancers in females and accounts only for2.4% of all new cases [10], despite the lack of nationalscreening programs. The actual reason for this low inci-dence is unknown. The closed society and standards ofmores could reduce women exposure to HPV infection[11-14]. In addition, male circumcision is associated witha reduced risk of penile HPV infection and a reducedrisk of cervical cancer in their female partners [15]. Theprevalence of HPV infection among women and its
is an Open Access article distributed under the terms of the Creative Commonsrg/licenses/by/2.0), which permits unrestricted use, distribution, andiginal work is properly cited.
Alsbeih et al. Infectious Agents and Cancer 2013, 8:15 Page 2 of 8http://www.infectagentscancer.com/content/8/1/15
association with cervical cancer in Saudi Arabia and insimilar socio-cultural societies is scanty [14,16-20]. In alimited study performed on 120 women attending routinegynecological examination, Al-Muammar et al. reported aprevalence of 31.6% infection with HPV-16/18 [21]. Inaddition, early reports are discordant [22,23], particularlythat some show high incidence, such as in Indonesia,where cervical cancer ranks number 3 after breast andcolorectal tumors [2].Inherited genetic predisposition may contribute to the
risk of cervical cancer. Genetic polymorphisms in tumorsuppressor genes might be related to HPV persistenceand progression to cancer. The gene encoding the tumorsuppressor TP53 is one example of a candidate gene thathas been suggested to affect the oncogenic potential ofthe HPV E6 protein. A common polymorphism in thep53 amino acid sequence is the arginine or proline atposition 72 (G/C) (rs1042522). Storey et al. found anassociation between the majority allele, arginine (G) formof p53, and cervical cancer development and proposedthat this genotype is more susceptible to HPV E6-mediated degradation [24]. Since then, there have beenmany reports on this TP53 polymorphism and risk forcervical cancer and the results are largely contradictory[25,26]. The frequency of TP53 codon 72 polymorphism
Cell cycle arrest
Genotodama
HR
TP53
CDKNTGFB1
ATM
TissuesMitochondria
RAD52,54
XRCC3
Cyclin E/
NBS1 M
RAD5
C-JUNC-ABL
JNKMKP5
NBS1
E2FRB
CDKN1B
H2AX
CHK1
POL CHK2
BRCA1
LIG53BP1
DSB
BCL2BAX
BRCA2(FANCD1)RAD51
Cross-links
SMAD2,3,4
Maintain Genomic Stability: Cell R
FA Complex:A C D E F G
Figure 1 Schematic representation of main pathways involved in proDNA single-strand breaks (SSBs) and double-strand breaks (DSBs). BDmechanisms. DSBs are repaired by non-homologous end joining (NHEJ) aninteracting proteins in tissues, cells and mitochondria that lead to the exprcycle arrest to allow for accurate DNA healing to prevent the cells from engenomic integrity which enables recovery or otherwise triggers cell death.respectively interact with TP53 and RB tumor suppressor proteins and inhibinteractions. Arrows indicate activation and blunt ends indicate inhibition. Tdesignates encoding genes selected for polymorphic variations predisposin
and its relationship with HPV infection and cervicalcancer is still unknown among Saudi women. In addition,TP53 is a central node in cell cycle control and DNArepair and orchestrates multiple pathways to maintaingenomic integrity that can be compromised by HPV infec-tion (Figure 1). The following SNPs: CDKN1A C31A Ser/Arg (rs1801270), ATM G1853A Asp/Asn (rs1801516),HDM2 T309G promoter (rs2279744), HDM2 A110G Ile/Val (rs11177386), LIG4 A591G Ile/Val (rs2232641),XRCC1 G399A Arg/Gln (rs25487), XRCC3 C241T Thr/Met (rs861539) and TGFβ1 T10C Lue/Pro (rs1982073)selected from various pathways could also alter proteinfunction and contribute to p53-mediated cell cycle dere-gulation and genomic instability [27-29]. Therefore, theaims of this study were to investigate HPV prevalence andgenotype in our cervical cancer patients and the potentialassociation with these 9 genetic SNPs presumed to predis-pose to cancer.
MethodsStudy populationOne hundred patients with histopathologically proven,locally advanced, cervical cancer were enrolled in thisstudy out of 218 patients followed at King Faisal SpecialistHospital and Research Centre (KFSHRC) from 2009 to
DNA synthesis,
Cell division
xicge
MDC1
SSBR, BERBases
HDM2
PRKDC
LIG4
XRCC4
1A
XRCC5
XRCC1
NHEJ
CDK2
RE11
0
s
LIG3
APE1
PNKPARP
POL
ARTEMIS
POL
XRCC6
SSBs
ecovery, Death, Transformation
HPV: E6, E7
RB E2F
cessing of genotoxic DNA damage including base damages (BDs),s and SSBs are efficiently repaired by base-excision (BER) and SSBRd homologous recombination (HR). These activate panoply ofession and inhibition of multiple genes. These normally results in celltering DNA synthesis with damaged DNA. The aim is to maintainThe E6 and E7 oncoproteins produced by high risk HPV infections willit their functions leading to genomic instability. Lines representhickness represents the strength of the actions. Underlined textg to cervical cancer (See text for details).
Alsbeih et al. Infectious Agents and Cancer 2013, 8:15 Page 3 of 8http://www.infectagentscancer.com/content/8/1/15
2012. There was no restriction on patients’ age or histo-logical type of cervix cancer (squamous cell carcinoma,adenocarcinoma or other). The cervix tumor sampleswere obtained during routine procedure for regular biopsyor from paraffin embedded tissues. One hundred age-matched women without history of cancer were enrolledand served as normal controls. Upon signing an institu-tionally approved informed consent, 5-ml blood sampleswere withdrawn for the genetic study. The KFSHRCResearch Ethics Committee has approved the study (RAC #2060 029).
DNA extraction, PCR amplification, DNA sequencing andSNP genotypingDNA was extracted using Puregene DNA PurificationKit (Gentra System). The PCR primers used for amplifi-cation were published previously [28]. Relevant segmentsof DNA were amplified by thermal cycling (95°C for 15min, 39 rounds of 95°C for 1 min, 56°C for 1 min and72°C for 1 min and final extension at 72°C for 7 min)using HotStarTaq DNA polymerase (Qiagen), and 50 ngtemplate DNA in 25 micro-litter volume with standardreaction conditions. The amplified fragment was directlysequenced using the DYEnamic ET Dye Terminator CycleSequencing Kit (Amersham Biosciences) and were run onthe MegaBase 1000 sequencer (Applied Biosystems). Se-quencing results were aligned to the corresponding refe-rence sequence and the SNPs were genotyped usingSeqManII sequence analysis software (DNASTAR Inc.).
HPV detection and genotypingWe used the Linear Array HPV Genotyping Test (LAHPV GT; Roche Diagnostics). The LA HPV GT is based
Figure 2 The distribution of squamous cell carcinoma and adenocarc
on four major processes including DNA extraction, PCRamplification of target DNA, hybridization of amplifiedproducts to oligonucleotide probes and finally, the colori-metric determination using the Linear Array DetectionKit (LA DK). It enables the concurrent detection andgenotyping of 37 most common anogenital HPV DNAgenotypes [6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 45, 51,52, 53, 54, 55, 56, 58, 59, 61, 62, 64, 66, 67, 68, 69, 70, 71,72, 73 (MM9), 81, 82 (MM4), 83 (MM7), 84 (MM8), 89(CP6108) and IS39]. The test has the betaglobin gene asan internal control to show adequacy of the sample. Themanufacturer states a sensitivity of 96% (95% CI: 92-98%)and a specificity of 99% (95% CI: 98-100%) and hasincluded 3 controls, 2 negatives and 1 positive for HPV-16.The kit also enables detection of multiple infections. Theprimers and PCR reaction conditions were provided withthe test kit. The manufacturer’s recommended metho-dology was strictly followed as also described previously[17]. Positive reactions appear as blue bands on the teststrip. The strips were interpreted using the HPV referenceguide provided with the kit. Results were considered nega-tive if no HPV band was detected after at least two repeatedtesting.
Statistical analysis and ethical considerationsA total of 100 patients and 100 controls subjects wereincluded in the project following signing an informedconsent. Samples were coded with no identifiable per-sonal data. HPV status was compared between thepatients in 5-year age groups. The association betweenSNPs and cervical cancer was evaluated by the odd ratio(OR) with its confidence interval. The degree of signifi-cance was calculated using the Chi-Squares method. A
inoma by 5-year age group in 100 cervical cancer patients.
Table 1 Detection and Prevalence of different HPVgenotypes in 100 cervical cancer patients
HPV genotypes Classification Number ofpatients
Prevalence(%)
HPV-positive 82 82%
HPV-negative 18 18%
Single infection:
HPV-16 HR 58 70.73
HPV-18 HR 3 3.66
HPV-31 HR 6 7.32
HPV-45 HR 3 3.66
HPV-56 HR 1 1.22
HPV-59 HR 1 1.22
HPV-73 HR 3 3.66
Co-infections:
HPV-16/18 HR/HR 3 3.66
HPV-16/39 HR/HR 1 1.22
HPV-16/70 HR/LR 1 1.22
HPV-35/52 HR/HR 1 1.22
HPV-45/59 HR/HR 1 1.22
LR: low risk; HR: high risk.
Alsbeih et al. Infectious Agents and Cancer 2013, 8:15 Page 4 of 8http://www.infectagentscancer.com/content/8/1/15
p-value of 0.05 or less was considered statistically signi-ficant. Statistical analysis was carried out using theSigmaPlot platform (Version 12.0, SPSS Science, IL, USA)and the free online software: Case Control Studies, Testsfor Association, Institute of Human Genetics, HelmholtzCenter Munich, Germany (http://ihg.gsf.de/cgi-bin/hw/hwa1.pl).
Figure 3 The distribution of HPV detection and genotypes by 5-year
Results and discussionSubjects and clinical dataNormal controls had similar socio-economic and demo-graphic distribution and age range (30 to 73, median = 48)as the patients. Age of patients at diagnosis of cervicalcancer ranged between 30 and 76 years, with a median of46 years. The FIGO stage of the cancer ranged betweenIA2 and IVA but most patients (n=76) had stage II/IIIdisease. By histology, 90 patients had squamous cell car-cinoma while 10 had adenocarcinoma of the cervix. Thedistribution of these two histopathological types by 5-yearage groups is given in Figure 2. Taking into considerationtwo previously published data [17,18], adenocarcinomaforms only 12% of cervical cancers compared to 88% ofsquamous cell carcinoma.
HPV detection and genotypingHPV detection and genotyping showed that 82 patients(82%) were positive for HPV infection while 18 speci-mens proved to be negative (18%) after at least two inde-pendent testing. This prevalence of HPV infection inSaudi cervical cancer patients is at the lower range of theestimated 85% to 99% worldwide [5,7,30], and alsocontrasting with the high burden estimated from previ-ously published data from the extended Middle East andNorth Africa that showed up to 98% positivity in womenwith preinvasive and invasive lesions [31]. Khorasanizadehet al. has reported slightly lower prevalence (76%) in anearby country [19].By histology, 60% of adenocarcinomas and 84% of
squamous cell carcinomas were HPV-positive. LinearArray HPV genotyping test had detected seven differentsingle HPV genotypes and five double infections in this
age group in 100 cervical cancer patients.
Table 2 Genotypes’ and alleles’ frequencies of 7 assessedpolymorphisms in 100 cervix carcinoma patients inaddition to 100 age-and-gender matched controlvolunteers without cancers
Gene,genotypeand allele
Cancerpatients’frequency
Normalvolunteers’frequency
Odds ratio P- Value
(95% CI)
(%, n = 100) (%, n = 100)
CDKN1A (p21) codon 31 C>A Ser/Arg rs1801270
C/C 64 (64) 62 (62)
C/A 32 (32) 35 (35) 0.89 (0.49-1.60) 0.69
A/A 4 (4) 3 (3) 1.29 (0.28-6.01) 0.74
C 160 (80) 159 (79)
A 40 (20) 41 (21) 0.97 (0.59-1.58) 0.90
TP53 (p53) codon 72 G>C Arg/Pro rs1042522
G/G 20 (20) 22 (22)
G/C 58 (58) 52 (52) 1.23 (0.60-2.50) 0.57
C/C 22 (22) 26 (26) 0.93 (0.41-2.14) 0.87
G 98 (49) 96 (48)
C 102 (51) 104 (52) 0.96 (0.65-1.42) 0.84
ATM codon 1853 G>A Asp/Asn rs1801516
G/G 90 (90) 88 (88)
G/A 8 (8) 12 (12) 0.65 (0.25-1.67) 0.37
A/A 2 (2) 0 (0) 4.89 (0.23-103.2) 0.16
G 188 (94) 188 (94)
A 12 (6) 12 (6) 1.00 (0.44-2.28) 1
HDM2 promoter 309 T>C rs2279744
T/T 30 (30) 27 (27)
T/C 46 (46) 44 (44) 0.94 (0.48-1.83) 0.86
C/C 24 (24) 29 (29) 0.75 (0.35-1.58) 0.44
T 106 (53) 98 (49)
C 94 (47) 102 (51) 0.85 (0.58-126) 0.42
TGFβ1 codon 10 T>C Leu/Pro rs1982073
T/T 48 (48) 39 (39)
T/C 31 (31) 38 (38) 0.89 (0.42-1.91) 0.77
C/C 21 (21) 23 (23) 1.35 (0.65-2.79) 0.42
T 127 (64) 116 (58)
C 73 (37) 84 (42) 1.26 (0.84-1.88) 0.26
XRCC1 codon 399 G>A Arg/Gln rs25487
G/G 52 (52) 59 (59)
G/A 34 (34) 40 (40) 0.96 (0.54-1.74) 0.90
A/A 14 (14) 1 (1) 15.88 (2.0-124.9)) 0.0007
G 138 (69) 158 (79)
A 62 (31) 42 (21) 1.69 (1.07-2.66) 0.02
XRCC3 codon 241 C>T Thr/Met rs861539
C/C 45 (45) 41 (41)
C/T 44 (44) 37 (37) 1.08 (0.59-1.99) 0.79
Table 2 Genotypes’ and alleles’ frequencies of 7 assessedpolymorphisms in 100 cervix carcinoma patients inaddition to 100 age-and-gender matched controlvolunteers without cancers (Continued)
T/T 11 (11) 22 (22) 0.46 (0.20-1.05) 0.06
C 134 (67) 119 (60)
T 66 (33) 81 (41) 0.72 (0.48-1.09) 0.12
Alsbeih et al. Infectious Agents and Cancer 2013, 8:15 Page 5 of 8http://www.infectagentscancer.com/content/8/1/15
cohort. Results are summarized in Table 1. The agedistribution of HPV detection and genotypes is shown inFigure 3. Furthermore, age-specific HPV distribution inthe Saudi cervical cancer patients showed a bimodalcurve with a first peak at younger ages (41–50 years)and a relative rebound at older ages (56–60 years) as ithas been described in other population [32].The most common HPV genotypes were HPV-16 (71%),
followed by HPV-31 (7%), HPV-18, 45, 73 (4% each).Seven patients had double infections involving HPV-16/18(4%), HPV-16/39, 16/70, 35/52, and 45/59 (1% each). Inagreement with other studies, the most common HPVgenotype was HPV-16 [33] with an overall prevalence,including co-infections, of 77% compared to 54% in theworld [30]. In addition, HPV-16 and/or HPV-18 werepresent in 66% (66/100) of all patients and formedtogether 80% (66/82) of all HPV positive patients. This isslightly higher than the prevalence observed in Europe(74.5%), North America (76.5%) and in the whole world(70.9%). These results are close to those reported inanother neighboring country where HPV-16 (54%), HPV-18 (14%), and HPV-31 (6%) were the most commonlydetected in cervical cancer patients [19]. However, ourresults seem to be different from those obtained in otherMiddle Eastern country where the most common HPVgenotype was HPV-33, which was not detected in ourpatients, followed by HPV-16 and HPV-18 [34].
Genetic polymorphic variationsGenetic polymorphic variation in candidate SNPs weredetermined for all 100 cervical cancer patients inaddition to the 100 age-matched female volunteers with-out cancer (controls). The two SNPs, LIG4 rs2232641and HDM2 rs11177386 were all majority alleles, andtherefore, were omitted from the analysis. Cancer predis-position study showed significant association betweenXRCC1 rs25487 and having cervical cancer (Table 2).Patients harboring the variant allele (A, Gln) have about2-fold increased risk to develop cervical cancer (P = 0.02).These results suggest that the variant (or minority) allelecould confer higher susceptibility to cervical cancer andprobably the HPV-related transformation.Indeed, the analysis of this nested case–control study
shows that 93% (13/14) of patients with homozygousvariant alleles (A/A) are HPV-positive compared to 82%
Alsbeih et al. Infectious Agents and Cancer 2013, 8:15 Page 6 of 8http://www.infectagentscancer.com/content/8/1/15
(27/33) in heterozygous and 79% (42/53) in majorityallele (G/G), suggesting a trend toward an associationbetween the HPV-positivity and XRCC1 G399A geno-type; however, it did not reach statistical significance(P = 0.28; Table 3). To check for skewness in the distri-bution of XRCC1 G399A genotypes, we tested for devi-ation from Hardy-Weinberg equilibrium (HWE), bycomparing observed-to-expected distributions in HPV-positive (cases) and HPV-negative (controls) patients(Table 3). Results showed statistically significant devi-ation from HWE for cases (P = 0.03) but not for thecontrols. Therefore, in HPV+ cervical cancer patients,the null hypothesis that the population is in Hardy–Weinberg frequencies is rejected, which put forward thehypothesis of probable selection. This suggests that theco-occurrence of XRCC1 G399A genotypes and HPV-positive cancer is not random, thus, implicating thisSNP as a susceptibility locus to develop cervical cancer.At the molecular level, XRCC1 protein is required forefficient DNA single-strand breaks repair to maintaingenomic stability in human cells (Figure 1) and its
Table 3 Genotypes’ association with HPV status and test for d
Gene,genotype and allele
HPV+/− n (%)
Cases Controls
(HPV+) (n = 82) (HPV-) (n = 18)
XRCC1 codon 399 G>A
Genotype
G/G 42 (51) 11 (61)
G/A 27 (33) 6 (33)
A/A 13 (16) 1 (6)
G/A+A/A 40 (49) 7 (39)
Allele
G 111 (68) 28 (78)
A 53 (32) 8 (22)
Armitages’ trend test
Significance level (P-value) for deviation from HWE
TP53 (p53) codon 72 G>C
Genotype
G/G 18 (22) 2 (11)
G/C 49 (60) 9 (50)
C/C 15 (18) 7 (39)
G/C+C/C 64 (78) 9 (89)
Allele
G 85 (52) 13 (36)
C 79 (48) 23 (64)
Armitages’ trend test:
Significance level (P-value) for deviation from HWE
HWE: Hardy-Weinberg Equilibrium. OR: Odds Ratio.Computed online at: http://ihg.gsf.de/cgi-bin/hw/hwa1.pl.
reduction leads to increased sensitivity to cell killing byionizing radiation [35]. The codon 399 is situated in theBRCT I active domain of the protein and could possiblyaffect its function [36]. In addition, this SNP was asso-ciated with cellular and clinical sensitivity to cancertreatment [37] and has recently been implicated in suscep-tibility to cervical cancer among Asian women [38].In contrast, no association was found for TP53 G72C
where cancer patients and controls without cancer haveshowed similar frequencies (Table 3). Nonetheless, nestedanalysis showed that 90% of patients with majority (G/G)allele were HPV-positive compared to 74% of heterozy-gous (G/C), and 68% of homozygous (C/C) variant allele,revealing a preponderance of HPV-positivity in patientsharboring the majority (G) allele. In fact, this allele hasbeen suggested to be more susceptible to high-risk HPVE6 degradation [24]. In addition, statistical analysisshowed a trend towards an association between TP53G72C SNP genotype and HPV infection (P = 0.06;Table 3). Furthermore, testing for deviation from HWEalso showed a borderline significant deviation (P = 0.07)
eviation from Hardy-Weinberg Equilibrium
Odds ratio P-Value Deviation from HWE
(95% CI) Expected
Cases Controls
Arg/Gln (rs25487)
37.56 10.89
1.18 (0.39-3.56) 0.77 35.87 6.22
3.41 (0.40-28.93) 0.24 8.56 0.89
1.49 (0.53-4.24) 0.45
1.67 (0.71-3.91) 0.23
Common OR= 1.67 0.28
0.03 0.88
Arg/Pro (rs1042522)
22.03 2.35
0.61 (0.12-3.07) 0.54 40.95 8.31
0.24 (0.04-1.32) 0.08 19.03 7.35
0.44 (0.09-2.12) 0.29
0.53 (0.25-1.11) 0.09
Common OR= 0.48 0.06
0.07 0.72
Alsbeih et al. Infectious Agents and Cancer 2013, 8:15 Page 7 of 8http://www.infectagentscancer.com/content/8/1/15
for HPV-positive cases, meanwhile no significant devi-ation was observed for HPV-negative controls. Again,these results suggest that cervical cancer occurrence is notrandom in the population and that certain factors such asgenetic SNPs, for instance having the XRCC1 A-allele orthe TP53 G-allele in connection with HPV infection,favors its development. To answer the question whetherthese 2 SNPs exhibit cumulative effect towards HPVmediated cervical cancer, , we have computed the numberof risk alleles for XRCC1 and TP53 in patients andcontrols. Although the patients had higher median num-ber of risk alleles (2 compared to 1), the difference wasnot statistically significant (Mann–Whitney rank sum test,P = 0.12) suggesting independent effects.Since the first identification of the potential role of Arg/
Arg genotype as a risk marker for uterine cervix neoplasia[24], there have been many studies that investigated theassociation between the TP53 codon 72 polymorphismand cervical cancer in various populations; however,results were inconsistent [29]. Although several factorswere proposed as contributing factors to the discrepan-cies, the deviation from the Hardy-Weinberg equilibriumwas identified as a principal source of divergent results[39]. Nonetheless, there is sufficient evidence to support apositive association particularly when HPV status and/orhistopathology is known. Thus, while Sousa et al. failed toconfirm the association in most European countries,except Italy and United Kingdom [40], two other meta-analyses confirmed the association of homozygous Argwith invasive cervical cancer [39,41], In line with ourresults, a recent family-based association study whereHPVs status was also determined, Hu et al. confirmed thatthe TP53 codon 72 G (arginine) is significantly overtran-smitted in Caucasian cervical cancer subjects, especially incases infected with HPV16- and/or HPV-18 [29].
ConclusionsThe prevalence of HPV infection in invasive cervicalcancer in Saudi Arabia (82%) is at the lower range ofthat observed in the world (85%-99%), the most com-mon HPV genotype was by far HPV-16 (71%), followedby HPV-31 (7%), HPV-18, 45, and 73 (4% each) withdouble infections were present in 8.5% of HPV-positivepatients. Genetic predisposition showed that among thenine SNPs studied, only XRCC1 G399A was significantly(P = 0.02) associated with cervical cancer, while TP53G72C showed borderline association (P = 0.06) only inHPV-positive patients. In addition, both SNPs showeddegrees of deviation from Hardy-Weinberg equilibriumonly in HPV-infected tumors, indicative of non-randomdistribution, hence implicating the combination of HPVand SNPs in cancer predisposition. Thus, SNPs could bemore relevant biomarkers of susceptibility to cervicalcancer when associated with HPV infection. Further
studies with larger cohort are needed to confirm theseresults and better postulate the use of SNPs as bio-markers of susceptibility to cervical cancer.
AbbreviationsSNP: Single nucleotide polymorphism; HPV: Human papilloma virus;CDKN1A: cyclin-dependent kinase inhibitor 1A; TP53: Tumor protein p53;ATM: Ataxia-telangiectasia mutated; HDM2: Human double minutes 2 (alsoknown as MDM2); LIG4: DNA ligase IV; XRCC1: X-ray repair cross-complementing 1; XRCC3: X-ray repair cross-complementing 3;TGFB1: Transforming Growth Factor Beta 1; HWE’: Hardy-Weinbergequilibrium; OD: Odd Ratio; CI: Confidence interval.
Competing interestsThe authors declare that there are no conflicts of interest related to thisresearch manuscript.
Authors’ contributionsGA designed the study, analyzed results and drafted manuscript. NAHprocessed and genotyped samples. MES and IA selected study population,gynecological sampling and patient care. All authors read and approved thefinal manuscript.
AcknowledgmentsWe would like to thank: Asma Tulbah (Pathology & Laboratory Medicine),Hani Salem, Osama AlOmar, Murad Al-Aker (Obstetrics & Gynecology), KhalidBalaraj, Raef Ahmad, Rana Mahmood, Mohamed Al-Dehaim, Ehab Khalil(Radiation Oncology), Belal Moftah, Khaled Al-Hadyan, Muneera Al-Buhairi,Sarah Al-Qahtani (Radiation Biology) for helping in collecting samples andclinical data; Brian Meyer (Genetics) for DNA sequencing and MohamedShoukri for statistical advice. This project was supported by King AbdulazizCity for Science and Technology (KACST) grant # ARP-27-12 (RAC# 2060 029).
Author details1Biomedical Physics, King Faisal Specialist Hospital & Research Centre, Riyadh,Saudi Arabia. 2Radiation Oncology, King Faisal Specialist Hospital & ResearchCentre, Riyadh, Saudi Arabia. 3Obstetrics & Gynecology, King Faisal SpecialistHospital & Research Centre, Riyadh, Saudi Arabia. 4Research Centre,Biomedical Physics Department, KFSHRC, MBC-03, P.O. Box 3354, Riyadh11211, Saudi Arabia.
Received: 31 December 2012 Accepted: 17 April 2013Published: 4 May 2013
References1. Parkin DM, Bray F, Ferlay J, Pisani P: Global cancer statistics, 2002. CA
Cancer J Clin 2005, 55:74–108.2. Cancer Incidence and Mortality Worldwide: IARC CancerBase No. 10
[Internet]. Available from: http://globocan.iarc.fr.3. Zur Hausen H: Human papillomaviruses in the pathogenesis of
anogenital cancer. Virology 1991, 184:9–13.4. Bosch FX, Manos MM, Munoz N, Sherman M, Jansen AM, Peto J, Schiffman
MH, Moreno V, Kurman R, Shah KV: Prevalence of human papillomavirusin cervical cancer: a worldwide perspective. International biologicalstudy on cervical cancer (IBSCC) Study Group. J Natl Cancer Inst 1995,87:796–802.
5. Walboomers JM, Jacobs MV, Manos MM, Bosch FX, Kummer JA, Shah KV,Snijders PJ, Peto J, Meijer CJ, Munoz N: Human papillomavirus is anecessary cause of invasive cervical cancer worldwide. J Pathol 1999,189:12–19.
6. Munoz N, Bosch FX, de Sanjose S, Herrero R, Castellsague X, Shah KV,Snijders PJ, Meijer CJ: Epidemiologic classification of humanpapillomavirus types associated with cervical cancer. N Engl J Med 2003,348:518–527.
7. de Sanjose S, Quint WG, Alemany L, Geraets DT, Klaustermeier JE, Lloveras B,Tous S, Felix A, Bravo LE, Shin HR, et al: Human papillomavirus genotypeattribution in invasive cervical cancer: a retrospective cross-sectionalworldwide study. Lancet Oncol 2010, 11:1048–1056.
8. Kurman RJ, Henson DE, Herbst AL, Noller KL, Schiffman MH: Interimguidelines for management of abnormal cervical cytology. The 1992National Cancer Institute Workshop. Jama 1994, 271:1866–1869.
Alsbeih et al. Infectious Agents and Cancer 2013, 8:15 Page 8 of 8http://www.infectagentscancer.com/content/8/1/15
9. Kitchener HC, Symonds P: Detection of cervical intraepithelial neoplasia indeveloping countries. Lancet 1999, 353:856–857.
10. SCR Report 2007: Cancer Incidence and Survival Report: Saudi Arabia2007. In Special Edition. Saudi Cancer Registry. Riyadh, Kingdom of SaudiArabia: Ministry of Health; 2011 (URL: www.scr.org.sa).
11. Bhurgri Y, Bhurgri A, Rahim A, Bhutto K, Pinjani PK, Usman A, Hasan SH: Thepattern of malignancies in Karachi (1995 to 1996). J Pak Med Assoc 1999,49:157–161.
12. Altaf F: Pattern of cervical smear cytology in western region of SaudiArabia. Ann Saudi Med 2001, 21:94–96.
13. Jamal A, Al-Maghrabi JA: Profile of Pap smear cytology in the Westernregion of Saudi Arabia. Saudi Med J 2003, 24:1225–1229.
14. Raza SA, Franceschi S, Pallardy S, Malik FR, Avan BI, Zafar A, Ali SH, Pervez S,Serajuddaula S, Snijders PJ, et al: Human papillomavirus infection inwomen with and without cervical cancer in Karachi, Pakistan. Br J Cancer2010, 102:1657–1660.
15. Castellsague X, Bosch FX, Munoz N, Meijer CJ, Shah KV, de Sanjose S,Eluf-Neto J, Ngelangel CA, Chichareon S, Smith JS, et al: Male circumcision,penile human papillomavirus infection, and cervical cancer in femalepartners. N Engl J Med 2002, 346:1105–1112.
16. Hammouda D, Clifford GM, Pallardy S, Ayyach G, Chekiri A, Boudrich A,Snijders PJ, van Kemenade FJ, Meijer CJ, Bouhadef A, et al: Humanpapillomavirus infection in a population-based sample of women inAlgiers, Algeria. Int J Cancer 2011, 128:2224–2229.
17. Alsbeih G, Ahmed R, Al-Harbi N, Venturina LA, Tulbah A, Balaraj K:Prevalence and genotypes' distribution of human papillomavirus ininvasive cervical cancer in Saudi Arabia. Gynecol Oncol 2011, 121:522–526.
18. Al-Badawi IA, Al-Suwaine A, Al-Aker M, Asaad L, Alaidan A, Tulbah A, FeBohol M, Munkarah AR: Detection and genotyping of human papillomavirus in cervical cancer specimens from Saudi patients. Int J GynecolCancer 2011, 21:907–910.
19. Khorasanizadeh F, Hassanloo J, Khaksar N, Taheri SM, Marzaban M, RashidiBH, Sari AA, Zendehdel K: Epidemiology of cervical cancer and humanpapilloma virus infection among Iranian women - Analyses of nationaldata and systematic review of the literature. Gynecol Oncol 2012,128:277–281.
20. Hussain S, Bharadwaj M, Nasare V, Kumari M, Sharma S, Hedau S, Das BC:Human papillomavirus infection among young adolescents in India:impact of vaccination. J Med Virol 2012, 84:298–305.
21. Al-Muammar T, Al-Ahdal MN, Hassan A, Kessie G, Dela Cruz DM, MohamedGE: Human papilloma virus-16/18 cervical infection among womenattending a family medical clinic in Riyadh. Ann Saudi Med 2007, 27:1–5.
22. Duttagupta C, Sengupta S, Roy M, Sengupta D, Bhattacharya P, LaikangbamP, Roy S, Ghosh S, Das R: Are Muslim women less susceptible tooncogenic human papillomavirus infection? A study from rural easternIndia. Int J Gynecol Cancer 2004, 14:293–303.
23. de Boer MA, Vet JN, Aziz MF, Cornain S, Purwoto G, van den Akker BE,Dijkman A, Peters AA, Fleuren GJ: Human papillomavirus type 18 andother risk factors for cervical cancer in Jakarta, Indonesia. Int J GynecolCancer 2006, 16:1809–1814.
24. Storey A, Thomas M, Kalita A, Harwood C, Gardiol D, Mantovani F, Breuer J, LeighIM, Matlashewski G, Banks L: Role of a p53 polymorphism in the developmentof human papillomavirus-associated cancer. Nature 1998, 393:229–234.
25. Makni H, Franco EL, Kaiano J, Villa LL, Labrecque S, Dudley R, Storey A,Matlashewski G: P53 polymorphism in codon 72 and risk of humanpapillomavirus-induced cervical cancer: effect of inter-laboratoryvariation. Int J Cancer 2000, 87:528–533.
26. Klug SJ, Ressing M, Koenig J, Abba MC, Agorastos T, Brenna SM, Ciotti M,Das BR, Del Mistro A, Dybikowska A, et al: TP53 codon 72 polymorphismand cervical cancer: a pooled analysis of individual data from 49 studies.Lancet Oncol 2009, 10:772–784.
27. Bond GL, Hu W, Bond EE, Robins H, Lutzker SG, Arva NC, Bargonetti J, BartelF, Taubert H, Wuerl P, et al: A single nucleotide polymorphism in theMDM2 promoter attenuates the p53 tumor suppressor pathway andaccelerates tumor formation in humans. Cell 2004, 119:591–602.
28. Al-Hadyan KS, Al-Harbi NM, Al-Qahtani SS, Alsbeih GA: Involvement ofsingle-nucleotide polymorphisms in predisposition to head and neckcancer in Saudi Arabia. Genet Test Mol Biomarkers 2012, 16:95–101.
29. Hu X, Zhang Z, Ma D, Huettner PC, Massad LS, Nguyen L, Borecki I,Rader JS: TP53, MDM2, NQO1, and susceptibility to cervical cancer.Cancer Epidemiol Biomarkers Prev 2010, 19:755–761.
30. WHO/ICO Information Centre on HPV and Cervical Cancer (HPV InformationCentre): Human Papilomavirus and Related Cancers in the World. SummaryReport; 2010. Available at: : www.who.int/hpvcentre. Accessed 2012.
31. Seoud M: Burden of human papillomavirus-related cervical disease in theextended middle East and north Africa-a comprehensive literaturereview. J Low Genit Tract Dis 2012, 16:106–120.
32. Bruni L, Diaz M, Castellsague X, Ferrer E, Bosch FX, de Sanjose S: Cervicalhuman papillomavirus prevalence in 5 continents: meta-analysis of 1million women with normal cytological findings. J Infect Dis 2010,202:1789–1799.
33. Sjoeborg KD, Trope A, Lie AK, Jonassen CM, Steinbakk M, Hansen M,Jacobsen MB, Cuschieri K, Eskild A: HPV genotype distribution accordingto severity of cervical neoplasia. Gynecol Oncol 2010, 118:29–34.
34. Darnel AD, Wang D, Ghabreau L, Yasmeen A, Sami S, Akil N, Al Moustafa AE:Correlation between the presence of high-risk human papillomavirusesand Id gene expression in Syrian women with cervical cancer.Clin Microbiol Infect 2010, 16:262–266.
35. Brem R, Hall J: XRCC1 is required for DNA single-strand break repair inhuman cells. Nucleic Acids Res 2005, 33:2512–2520.
36. Taylor RM, Thistlethwaite A, Caldecott KW: Central role for the XRCC1 BRCTI domain in mammalian DNA single-strand break repair. Mol Cell Biol2002, 22:2556–2563.
37. Brem R, Cox DG, Chapot B, Moullan N, Romestaing P, Gerard JP, Pisani P,Hall J: The XRCC1–77T->C variant: haplotypes, breast cancer risk,response to radiotherapy and the cellular response to DNA damage.Carcinogenesis 2006, 27:2469–2474.
38. Li Y, Liu F, Tan SQ, Wang Y, Li SW: X-ray repair cross-complementinggroup 1 (XRCC1) genetic polymorphisms and cervical cancer risk: a hugesystematic review and meta-analysis. PLoS One 2012, 7:e44441.
39. Koushik A, Platt RW, Franco EL: p53 codon 72 polymorphism and cervicalneoplasia: a meta-analysis review. Cancer Epidemiol Biomarkers Prev 2004,13:11–22.
40. Sousa H, Santos AM, Pinto D, Medeiros R: Is the p53 codon 72polymorphism a key biomarker for cervical cancer development? Ameta-analysis review within European populations. Int J Mol Med 2007,20:731–741.
41. Jee SH, Won SY, Yun JE, Lee JE, Park JS, Ji SS: Polymorphism p53 codon-72and invasive cervical cancer: a meta-analysis. Int J Gynaecol Obstet 2004,85:301–308.
doi:10.1186/1750-9378-8-15Cite this article as: Alsbeih et al.: HPV prevalence and geneticpredisposition to cervical cancer in Saudi Arabia. Infectious Agents andCancer 2013 8:15.
Submit your next manuscript to BioMed Centraland take full advantage of:
• Convenient online submission
• Thorough peer review
• No space constraints or color figure charges
• Immediate publication on acceptance
• Inclusion in PubMed, CAS, Scopus and Google Scholar
• Research which is freely available for redistribution
Submit your manuscript at www.biomedcentral.com/submit
3177
DOI:http://dx.doi.org/10.7314/APJCP.2013.14.5.3177Prevalence of Human Papillomavirus in Women from Saudi Arabia
14 (5), 3177-3181
Introduction
Cervical cancer is the second most prevalent disease worldwide occurrence with new cases every year and approximately 80% of them reported in developing countries (Munoz et al., 2003; Parkin and Bray, 2006; Lin et al., 2008; Chen et al., 2011 and 2012). Such type of cases appears mainly in between the age of 30 and 50. HPV belongs to family Papillomavirus which is non enveloped virus with 52-55 nm in size with circular double stranded circular DNA and a genome with 7,900 base pairs followed by eight overlapping open reading frames. The late genes (L1 and L2) and early genes (E6, E7, E1, E2, E4 and E5) that are expressed in more than 100 different HPV
variations. They are classified according to various criteria such as their tissue tropism, potential of oncogenic and phylogenetic relationship using molecular biology
(HPV) plays a very important role in cervical cancer
carcinomas (Walboomers et al., 1999; Munoz et al., 2006). The prevalence of HPV infection varies substantially among countries and according to lifestyle, age, food and nutrition. The majority of HPV infections are
1 3
2
Abstract
Background: Human papillomavirus (HPV) infection is the main causes of cervical cancer in women worldwide. The goal of the present study was to determine the prevalence and distribution of HPV genotypes in women from Saudi Arabia. Recently, several HPV detection methods have been developed, each with different
Methods: In this study, total forty cervical samples were subjected to polymerase chain reaction and hybridization to BioFilmChip microarray assessment. Results: Human papillomavirus (HPV) infections were found in 43% of the specimens. The most prevalent genotypes were HPV 16 (30%) HPV 18 (8.0%) followed by type HPV 45, occurring at 5.0%. Conclusionprevalence is increasing at alarming rate in women of Saudi Arabia. There was no low risk infection detected in the tested samples. The BioFilmChip microarray detection system is highly accurate and suitable for detection of single and multiple infections, allowing rapid detection with less time-consumption and easier performance as compared with other methods. Keywords: Human papillomavirus (HPV) - cervical cancer - prevalence - diagnosis - Saudi Arabia
RESEARCH ARTICLE
Prevalence of Human Papillomavirus in Women from Saudi Arabia Rola Turki1, Khalid Sait1, Nisreen Anfinan1, Sayed Sartaj Sohrab2*, Adel Mohammed Abuzenadah3
asymptomatic and temporary in the adolescent population. It has been observed that up to 98% of cervical cancers in females are associated with HPV infection and more than 90% of new infections appear to induce high grade cervical neoplasia (Moscicki et al., 2006; Woodman et al., 2007). The most important risk factors include, drinking, smoking, education system, many partners, food, nutrition, use of long-term oral contraceptive and immune-suppression. Less involvement for testing is a probable cofactor that should be considered in an analysis of HPV in cervical female cancer. More than 100 HPV genotypes have been characterized at molecular level, and
of genital tract infection. The World Health Organization
agents. According to earlier information, HPV-16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, -69, -73, -59
2003; Bouvard et al., 2009), and associated with cervical
in cervical lesions but associated with anogenital warts. Female individuals infected with low risk HPV types have a minimal possibility of developing cervical cancer.
Rola Turki et al
3178
However, 10-20% of positive cases with HPV infection by several genotypes have been reported (Hwang et al., 2004; Meijer et al., 2006; Matsumoto 2007; Lie et al., 2008). HPV 16 is detectable in 21% of women with low
than 50% of women with cervical intraepithelial neoplasia grade 3 (CIN3). HPV 18 causes 10 15% of CIN 3 and also causes more than 35% of cervical adenocarcinomas, and it is complex to identify by current diagnostic tools (Bosch et al., 1995). The relationship between HPV infection and cervical cancer had given great impetus to the development of prophylactic vaccines against the most common HR-HPV types. Worldwide, HPV16 and 18 resulted for more thn 70% of cervical cancer cases (Munoz et al., 2003). Currently very few HPV vaccines; Gardasil (HPV6/11/16/18) (Villa et al., 2005) and Cervarix (HPV16/18) (Harper et al., 2004) were developed against these two major cancer-causing HPV types (Yoshikawa, 2009). Based on the meta-analysis reported worldwide, HPV type 16 and 18 were the most common while in (de Sanjose et al., 2007; Bruni et al., 2010), HPV type 45, 31 and 33, were in Europe and Africa. HPV 52 and 58
2008; Sun et al., 2010; Chen et al., 2012).
infections since HPV is asymptomatic. Various diagnostic
countries such as by using the traditional detection based on the Papanicolaou smear discovered in 1943. However, false negatives (between 15-50% for cervical premalignant lesions and cervical cancer) and false positives (30%) should be considered during screening by using that
al., 2003).Cytology examinations have limitations with
sensitive methods have been developed to detect HPV such as: polymerase chain reaction (PCR) method; and
worldwide (Young et al., 1989). PCR is based on the use of primers such as the MY09/MY11 primer set (MY PCR),
for the detection of the virus in clinical samples. This was synthesized with multiple nucleotides in each primer and mixed with 25 primers including HMB01M, which detect a large spectrum of HPV types? This has been used in North and South America as well as in Asia (Wheeler et al., 1993; Hildesheim et al., 1994; Ley et al., 1994; Liaw et al., 1995; Lazcano et al., 2001; Thomas et al., 2004). On the basis of published information, this study was design and conducted to detect and determine the
Saudi Arabian population suffering from cervical cancer at KAUH and to establish a correlation between HPV genotypes involved in malignancies and the high risk type
Materials and Methods
A woman was eligible for study subject if she was a gynecological outpatient with genital tract disease related symptom, was not presently pregnant; had not undergone a total uterus or cervix resection; and was willing to undergo HPV testing and also consent to participate in the present study. The study was carried out with the approval of the hospital ethical committee, and patients consent was obtained for the collection of cervical cells. Total forty tissue biopsies were freshly collected from cervical cancer cases. Examination performed by gynaecological practitioners in above-mentioned hospital. The ages of the patients enrolled in this study were between 36-80 years. The control group comprised specimens with normal cytology and HPV DNA negative by PCR.
DNA extraction was performed by using by Qiamp DNA mini kit (QIAGEN, Valencia, CA) with following manufacturer’s instructions. After extraction, all
at –20oC until tested.
Total DNA was isolated by using Kit (QIAGEN, Valencia, CA) followed by manufacturer instructions and multiplex PCR was performed of the targeted DNA followed by primer extension detection. Positive samples for HPV DNA by multiplex PCR were selected for
detection were performed using autogenomics INFINITI Analyzer (Vista, CA) according to the manufacturer’s
probes of 26 HPVs (6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52,
targets was chosen.
Results
were selected for testing. Applying the assay, total three
covering high-risk.
The most common HR-HPV types observed in this study were HPV 16, HPV 18 and HPV 45. Detection
showed that the highest prevalence HPV 16 infection was found in the 35-76 year old age group and decreased slowly in older age groups in Saudi Arabian population and no other genotypes like HPV 31, 33, 35/68, 39/56, 51/59, 52/58, 6/11 were detected in all the screened samples (Table 1).
HPV genotypes (Figure 1). Outpatients the age of 35-76
3179
DOI:http://dx.doi.org/10.7314/APJCP.2013.14.5.3177Prevalence of Human Papillomavirus in Women from Saudi Arabia
years had the highest HPV prevalence (30.0%), which
Very less HPV infection (8%) was found in women 45-64
middle aged groups age ranged from 41 to 45 years. The LR-HPV genotypes were not detected in any samples. Discussion
This study reports the detection and prevalence of HPV
hybridization assay. The HPV 16 genotype was detected at the highest prevalence rate (30%) in Saudi Arabian population followed by HPV 18 and HPV 45. The Low risk genotype was not detected in any samples. In women,
intraepithelial lesions may disappear spontaneously within 12 months whereas the clearance rate in older women is lower (Chumworathayi et al., 2010). Approximately 1%
to invasive cervical cancer (Clifford et al., 2005) and more than 90% of HR-HPV genotypes pose an increased risk for progression to cervical cancer. Thus, HR-HPV genotypes
cancer development and is important for developing suitable HPV vaccines.
According to other studies, diagnosis based on cytology showed various multiple infections in 7% to 23% of patient samples (Rousseau et al., 2003). The infection shows a peak in young women aged below 25 and decreases slightly to less than 4% above the age of 40 (Peto et al., 2004). The variations depend on
characteristics of the population studied and detection methods.Geographical, clinical factors and demographics contribute to various extents to multiple HPV infections (Trottier et al., 2006). Variable results have been achieved by PCR detection for different genotypes. For multiple
typing methods and cytology is more suitable for risk assessment but expensive for routine diagnosis (Galan-Sanchez et al., 2009). Multiple infections, which increase the risk of invasive cervical cancer compared with single
intraepithelial lesions patients (Tucker et al., 1993). In this study, highly sensitive and time saving
BiofilmChip hybridization assay was used for HPV detection. Upon testing with DNA chip, the highest prevalence of HPV infection was found in the 35-76 year age groups and decreased slightly. Single HPV infection was more prevalent than multiple HPV infections. HPV 16 is the most prevalent genotype in cervical cancer (30.0%) and the second most predominant genotypes HPV 18 (8.0%) respectively which correlated with other
HPV 18 is the second in the CA group, nearly the world average (15.0%) (Munoz et al., 2003; Clifford et al., 2005). In addition, the third most prevalent genotype HPV45 (5.0%) was detected in Saudi Arabian followed by Yemini and Somalian population. These results have shown the diversity of HPV genotypes which provide information with regard to the design of multivalent prophylactic vaccines suitable for each geographical area.
Interestingly, a high incidence of HPV58 has been found in Asia, Africa and other areas while it is not common worldwide especially in Europe and North America. According to a study conducted in Korea and
(Miura et al., 2006). The evolution and spread of HPV 58 have been spread earlier. The original source of ancient HPV58 may have been in West Africa and Southeast Asia
the capacity to detect these genotypes and others which are uncommon such as HPV 67, HPV 70 and HPV 84. HR-HPV testing can be applied to primary screening and management. For this reason, clinical laboratories should determine advantages and disadvantages of each method for HPV typing. All methods produced highly similar results. In the previous study, the prevalence of
and normal cytology such as HPV 66, HPV 11 and HPV 71, respectively (Chansaenroj et al., 2010). Thus, multiple
the other commercial kits. Finally, the operation of HPV genotyping assays depends on their primer sets. The differences in HPV prevalence depend on many factors such as sample size, measurement and detection method. Combinations of highly concordant HPV genotyping methods for primary screening are recommended.
Table 1. HPV Genotypes in Women from Saudi ArabiaHPV genotypes Total No. of Age Prevalence HR-HPV LR-HPV positive groups (%) (%) (%) cases
beta-globin 35/40 35-82 87.50% HPV16 12/40 35-76 30% 30% 0HPV18 3/40 45-64 7.50% 7.50% 0HPV31 0/40 0 0 0 0HPV33 0/40 0 0 0 0HPV45 2/40 41-45 5% 5% 0HPV35/68 0/40 0 0 0 0HPV39/56 0/40 0 0 0 0HPV51/59 0/40 0 0 0 0HPV52/58 0/40 0 0 0 0HPV6/11 40 0 0 0 0
Figure 1. Prevalence of HPV Genotypes in Women from Saudi Arabia
Rola Turki et al
3180
considered for vaccination program.
is more suitable to identify both single and multiple
of viral genotypes of HPV and others should be considered for vaccination programs. Finally, combinations of highly concordant HPV genotyping methods are recommended for primary screening. It is reported that cervical cancer
in recent years. Our study showed that HPV infection is one of the major causes of cervical cancer in the screened group of patients. HPV type 16, 18 and 45 were detected
about involvement of HPV in cervical cancer. Managing costs associated with increased detection of positive HPV patients is needed in our population. Autogenomics
reliable method for detection of many genetic diseases. The load and go capability of the machine, makes it one of the few technologies available today that are considered to be the new generation of analyzers with full automation
infection and prevalence is increasing at alarming rate in Saudi Arabia. There was no any low risk infection was detected in tested samples.
Acknowledgements
Authors gratefully acknowledge the research facility provided by King Abdulaziz University Hospital and King Fahd Medical Research Center (KFMRC), King Abdulaziz
Abdulaziz University for providing grant, bearing number: 5-17/430 for the establishment of state of the art research facilities at King Fahd Medical Research Center.
References
type distribution in women from Asia: a meta-analysis. , 18, 71-9.
Bosch FX, Manos MM, Munoz N, et al (1995). Prevalence of human papillomavirus in cervical cancer: a worldwide perspective. International biological study on cervical cancer (IBSCC) study group. , 87, 796-802.
Bouvard V, Baan R, Straif K, et al (2009). A review of human carcinogens--Part B: biological agents. , 10, 321-2.
Bruni L, Diaz M, Castellsague X, et al (2010). Cervical human papillomavirus prevalence in 5 continents: meta-analysis of
Dis, 202, 1789-99.
(2010). Prevalence and genotypes of human papillomavirus among Thai women. , 11, 117-22.
Chen HC, Schiffman M, Lin CY, et al (2011). Persistence of
long-term risk of cervical cancer. , 103, 1387-96.
Chen Q, Xie LX, Qing ZR, et al (2012). Epidemiologic characterization of human papillomavirus infection in rural chaozhou, eastern guangdong province of china. , 7, 32149.
Chen Q, Zhao Y L, Min L et al (2012). Prevalence and Genotype Distribution of Human Papillomavirus Infections in Women Attending Hospitals in Chaozhou of Guangdong Province.
, 13, 1519-24.
Cryotherapy for HPV clearance in women with biopsy-
lesions. , 108, 119-22. Clifford GM, Gallus S, Herrero R, et al (2005). Worldwide
distribution of human papillomavirus types in cytologically normal women in the International Agency for Research on Cancer HPV prevalence surveys: a pooled analysis. , 366, 991-8.
Clifford GM, Rana RK, Franceschi S, et al (2005). Human papillomavirus genotype distribution in low-grade cervical lesions: comparison by geographic region and with cervical cancer. , 14, 1157-64.
of the role of human papillomavirus (HPV) testing within a cervical screening programme: summary and conclusions.
, 83, 561-65.de Sanjose S, Diaz M, Castellsague X, et al (2007). Worldwide
prevalence and genotype distribution of cervical human papillomavirus DNA in women with normal cytology: a meta-analysis. , 7, 453-9.
Galan-Sanchez F, Rodriguez-Iglesias MA (2009). Comparison of human papillomavirus genotyping using commercial assays based on PCR and reverse hybridization methods.
, 117, 708-15.
a bivalent L1 virus-like particle vaccine in prevention of infection with human papillomavirus types 16 and 18 in young women: a randomised controlled trial. , 364, 1757-65.
Hildesheim A, Schiffman MH, Gravitt P, et al (1994). Persistence
cytologically normal women. , 169, 235-240. Hwang HS, Park M, Lee SY, et al (2004). Distribution and
prevalence of human papillomavirus genotypes in routine pap smear of 2.470 Korean women determined by DNA chip.
, 13, 2153-6.
risk HPV DNA testing by polymerase chain reaction: an inter method and intra method comparison. , 52, 498-503.
Lazcano Ponce E, Herrero R, Munoz N, et al (2001). Epidemiology of HPV infection among Mexican women with normal cervical cytology. , 91, 412-20.
Ley C, Bauer H, Reingold A, et al (1991). Determinants of genital human papillomavirus infection in young women.
, 83, 997-1003.
and cervical neoplasia: a case control study in Taiwan. , 62, 565-71.
Lie AK, Kristensen G (2008). Human papillomavirus E6/E7 mRNA testing as a predictive marker for cervical carcinoma.
, 8, 405-415.
screening by gene chip in Chinese women of Guangdong province. , 48, 189-94.
Matsumoto K (2007). Human papillomavirus and cervical
3181
DOI:http://dx.doi.org/10.7314/APJCP.2013.14.5.3177Prevalence of Human Papillomavirus in Women from Saudi Arabia
cancer. , 65, 2113-124.
genotyping. , 103, 12-17.Middleton K, Peh W, Southern S, et al (2003). Organization of
human papillomavirus productive cycle during neoplastic progression provides a basis for selection of diagnostic markers. , 77, 10186-201.
Miura S, Matsumoto K, Oki A, et al (2006). Do we need a different strategy for HPV screening and vaccination in East Asia? , 119, 2713-5.
Moscicki AB, Schiffman M, Kjaer S et al (2006). Updating the natural history of HPV and anogenital cancer. Chapter 5.
, 24, 42-51.
cervical cancer. , 348, 518-27. Munoz N, Castellsague X, de Gonzalez AB et al (2006). HPV in
the etiology of human cancer. Chapter 1. , 24, 1-10. Parkin DM, Bray F (2006). The burden of HPV related cancers.
, 24, 11-25.
and neoplasia in a large population-based prospective study: the Manchester cohort. , 91, 942-53.
abnormal cervical smear: what are we screening for? , 345, 1469-73.
Rousseau MC, Villa LL, Costa MC, et al (2003). Occurrence of cervical infection with multiple human papillomavirus types is associated with age and cytologic abnormalities.
, 30, 581-7. Shadrina MI, Semenova EV, Slominsky PA, et al (2007).
analysis of the parkin gene (PARK2) exon 1-12 dosage. , 26, 8-6.
Cytological surveillance avoids treatment. , 309, 590-1.
prevalence among women in Liaoning province, China. , 109, 105-9.
Szostek S, Klimek M, Zawilinska B et al (2008). Genotype
, 55, 687-92.
of Papillomavirus in women in Idaban, Nigeria: a population based study. , 90, 638-45.
Trottier H, Burchell AN (2009). Epidemiology of mucosal human papillomavirus infection and associated diseases.
, 12, 291-307.Trottier H, Mahmud S, Costa MC, et al (2006). Human
papillomavirus infections with multiple types and risk of cervical neoplasia. , 15, 1274-80.
polymerase chain reaction to genotype human papillomavirus DNAs in samples containing multiple HPVs may produce inaccurate results. , 43, 321-33.
Villa LL, Costa RL, Petta CA, et al (2005). Prophylactic
18) L1 virus-like particle vaccine in young women: a randomised double-blind placebo-controlled multicentre
, 6, 271-8.
papillomavirus is a necessary cause of invasive cervical cancer worldwide. , 189, 12-19.
Wheeler CM, Parmenter CA, Hunt WC, et al (1993). Determinants of genital human papillomavirus infection
among cytologically normal women attending the University of New Mexico Student Health Center. , 20, 286-89.
Woodman CB, Collins SI, Young LS (2007). The natural history of cervical HPV infection: unresolved issues.
, 7, 11-22. Yoshikawa H (2009). Progress and challenges on HPV
vaccination. , 59, 243-8.
chain reaction. , 298, 14-18.
original article
Ann Saudi Med 2013 July-August www.annsaudimed.net 327
Cervical cancer ranks as the 11th most frequent cancer among women in Saudi Arabia, and the 8th most frequent cancer among women in the
age group of 15 to -44 years, and every year 152 new cases are diagnosed, of whom 55 (36.1%) die from the disease.1 Because of inadequate cytological screening program and poor acceptance by women population, the majority of women are diagnosed at locally advanced stages—International Federation of Gynecologists and Obstetrics (FIGO) stage IIB-IVA—for which surgery is inadequate due to parametrial invasion.2 The stan-dard treatment for locally advanced cervical cancer is
Five-year outcome of concurrent radiotherapy and chemotherapy in Saudi women with locally advanced cervical cancer: single-institution experienceMushabbab Al Asiri,a Mutahir Tunio,ab Abdulrehman Al Hadab,a Reham Mohamed,a Yasser Bayoumi,a Eyad Al Saeed,b Mohannad Al Arifi,a Abdulllah Al Amroa
From the aRadiation Oncololgy, King Fahad Medical City, Riyadh, Saudi Arabia; bRadiation Oncology, King Khalid University Hospital, Riyadh, Saudi Arabia
Correspondence: Dr. Mutahir Tunio · Radiation Oncology, King Fahad Medical City, Riyadh, Saudi Arabia · T: +966112889999 F: +96611461 4006 · [email protected]
Ann Saudi Med 2013; 33(4): 327-333
DOI: 10.5144/0256-4947.2013.327
BACKGROUND AND OBJECTIVES: We aimed to evaluate long-term treatment outcomes and toxicity profile of combined radiotherapy and chemotherapy in Saudi women with locally advanced cervical cancer.DESIGN AND SETTINGS: Retrospective study in a tertiary care cancer center in Riyadh, Saudi Arabia.METHODS AND MATERIALS: The medical records of patients with histopathologically proven, locally ad-vanced cervical cancer were analyzed. These patients received three-dimensional conformal radiotherapy with concurrent chemotherapy followed by high dose rate brachytherapy in our center between July 2007 and April 2012. The data regarding the safety profile, response rates, occurrence of locoregional or distant failure, disease-free survival, and overall survival rates were recorded.RESULTS: The median follow-up period was 60 months (range, 8-66) for 74 patients. The median age of study population was 52.3 years (32-78), and the stage IIB was the predominant stage (49 patients [66.2%]). A total of 45 patients (60.9%) had radiologic-positive pelvic ± para-aortic lymphadenopathy. The 5-year locoregional and distant control rates were 84.3% and 78.5%, respectively. The 5-year disease-free and overall survival rates were 75.7% and 64.5%, respectively. Stage, nodal status, and hemoglobin levels were found to be important prog-nostic factors for locoregional and distant control. Acute grade 3 hematological and nonhematological toxicities were seen in 4 (5.4%) and 4 (5.4%) patients. Late toxicities were mild, and only 1 (1.4%) patient presented with subacute intestinal obstruction.CONCLUSION: Concurrent chemoradiation in Saudi women with locally advanced cervical cancer showed better locoregional and distant control and survival rates with minimal toxicity.
prescribed as combined chemotherapy and pelvic irra-diation, based on the results of 5 Western and North American randomized trials showing survival benefit by 10% to 15% and local and distant recurrence re-duction rates by 30% to 40%.3-5 However, disease-free survival (DFS) and overall survival (OS) benefit of combined chemotherapy and pelvic irradiation in Saudi women with locally advanced cervical cancer is not well known. El-Senoussi, et al have reported 5-year DFS and OS rates of 68.3% and 57.9%, respectively, in 164 Saudi women with FIGO stage I and II cervical cancer.6 However, this study incorporated conventional EBRT
original article rAdiotherApy And cheMotherApy in SAudi woMen
Ann Saudi Med 2013 July-August www.annsaudimed.net328
followed by low dose rate brachytherapy techniques. However, over the last decade the advent of novel radia-tion therapy techniques (three-imensional conformal radiation therapy [3DCRT] and intensity modulated radiation therapy [IMRT]) have shown improvements in locoregional control and minimal toxicity.
We aimed to evaluate the efficacy, toxicity, and treat-ment outcomes of 3DCRT pelvic irradiation with che-motherapy followed by high dose rate brachytherapy in Saudi women with locally advanced cervical cancer and also to evaluate the prognostic factors affecting locore-gional and distant control.
METHODS AND MATERIALS
EligibilityAfter getting approval from institutional Review Committee, the medical records of patients with locally advanced cervical cancers were looked for demographic, symptomatology, tumor characteristics, radiologic im-aging, pathologic examination, radiation therapy tech-niques, concurrent chemotherapy regimen, acute and chronic side effects, and response rates. Patients with locally advanced cervical cancer between July 2007 and May 2012 were studied when they met the following eligibility criteria:
(1) histologically proven squamous cell carcinoma, adenocarcinoma, and adenosquamous carcinoma of cervix uteri; (2) clinical and radiologic FIGO stage IIB-IVA with no other evidence for distant metastasis outside the pelvis; (3) Eastern Co-operative Oncology Group performance status 0 to 2; and (4) patients who received radical concurrent chemoradiation. Patients with a previous history of hysterectomy, retroperitoneal surgery, abdominal or pelvic radiotherapy, prior chemo-therapy, and pregnancy were excluded.
Treatment Protocol
External beam irradiation All patients were scanned for simulation on a CT simu-lator from epigastrium to mid thighs, and 3DCRT planning was performed. After data acquisition, the gross tumor volume (GTV; clinical target volume [CTV-1] (GTV+uterus+1 cm margins+common, internal, and external iliac lymph nodes±para-aortic nodes), planning target volume (PTV-1; CTV-1 + 0.5 to 1 cm margins), CTV-2 (parametrium + 1 cm mar-gin with 4 cm midline shielding), PTV-2 (CTV+ 0.5 cm margins), and organs at risks (kidneys, small bowel, bladder, rectum, and femoral heads) were delineated. Four equally spaced, coplanar 3DCRT field plans for
pelvis and anteroposterior and posteroanterior field for para-aortic regions were generated. The prescribed ra-diation doses were 45 to 50.4 Gy/25 to 28 fractions to PTV-1 and 54 to 59.4Gy/30 to 33 fractions to PTV-2, 5 days per week, and up to 7% variation was considered acceptable. During planning, the mean dose to the rec-tum was constrained to <50 Gy, and the total doses to the small bowel, kidneys and bladder were constrained to <45 Gy, <20 Gy, <60 Gy, respectively (Figures 1 and 2).
Concurrent chemotherapyAll patients received weekly cisplatin 40 mg/m2 before the administration of radiotherapy for 6 doses. During concurrent chemoradiation (CCRT), the doses modifi-cations were done on weekly basis. If the WBC count was below 2000/mm3 or the platelet count was below 50 000/mm3, all chemotherapy for that week was omit-ted. If the white blood cell count was below 1000/mm3
or the platelet count was below 25 000/mm3, CCRT was withheld until the white blood cell count and the platelet count recovered to 1000/mm3 or greater and 25 000/mm3 or greater, respectively. If the serum cre-atinine level was above 1.8 mg/dL, the cisplatin was withheld. Patients were deemed unsuitable for further chemotherapy if the delay to resume treatment was lon-ger than 2 weeks.
High dose rate brachytherapyFletcher-Suit tandem and ovoids afterloading applica-tors were used for high dose rate (HDR)-brachytherapy with iridium-192 sources once a week under conscious sedation after 45 Gy of external beam irradiation therapy (EBRT). A dose of 7 Gy per fraction using 4-6 insertions to point A with a total dose of 21 Gy was delivered, based on the dose limit derived from the treatment plan for the rectum and bladder. The dose constraints were 75 Gy and 80 Gy for the rectum and bladder, respectively (Figure 2).
Toxicity scoring The National Cancer Institute Common Toxicity Criteria version 2.0 were used to score acute radiation and chemotherapy toxicity (<90 days from the start of radiation therapy). During CCRT, weekly weight, performance status, pelvic examination findings, hema-tologic, and blood chemistry determinations were also recorded.
The Radiation Therapy Oncology Group Late Radiation Morbidity Scoring Criteria were used to score radiation toxicity persisting beyond 90 days from the completion of radiotherapy. After completion of the
original articlerAdiotherApy And cheMotherApy in SAudi woMen
Ann Saudi Med 2013 July-August www.annsaudimed.net 329
Figure 1. initial phase radiotherapy (A) extended field and (B) pelvic field three-dimensional conformal radiation therapy (3dcrt) plans showing 45 Gy to ptV-1 in coronal and sagittal views.
CCRT, patients were seen every 3 months for the first 2 years, and every 6 months thereafter at radiation on-cology and gynecology oncology clinics. The response evaluation consisted of a physical and pelvic examina-tion; a Pap smear; hematology, hepatic, renal function tests; computed tomography (CT) chest and abdomen; and pelvic magnetic resonance imaging (MRI) every 6 months for the first 2 years.
Statistical analysis The primary endpoints were toxicity, locoregional and systemic control, and DFS and OS rates. DFS was de-fined as the duration between the completion of CCRT and the date of documented disease recurrence, death resulting from the cancer, and/or last follow-up visit (censored). OS was defined as the duration between the completion of CCRT and the date of patient death or last follow-up visit (censored). The probabilities of local, para-aortic, and distant control, and DFS and OS rates were determined with the Kaplan-Meier method. The comparisons for various endpoints were performed using the log-rank test. The Student unpaired t test was used to determine the significance of the difference be-tween the 2 groups. A P value of .05 was considered statistically significant. Cox regression model was used to evaluate the effect of the potential prognostic fac-tors on locoregiona controll, distant control, and DFS. Bonferroni correction was applied to overcome multi-plicity problem. Statistical analyses were performed us-ing the computer program SPSS, version 17.0 (SPSS Inc., Chicago, IL, USA).
RESULTS
Characteristics of the patients and treatment compliance and toxicity profileCommon presenting symptoms were vaginal bleeding in 45 patients (60.8%), followed by vaginal discharge in 16 patients (21.6%), and low backache in 13 pa-tients (17.6%). The median duration of symptoms was 9 months (3-15). The median age of cohort was 52.3 years (32-78). FIGO stage IIB was the predominant stage in 49 patients (66.2%) followed by IIIA in 12 pa-tients (16.2%). Twenty-nine patients (39.1%) had nega-tive pelvic nodal at the time of CCRT. The common site of gross pelvic lymph nodes were as follows: iliac in 21 patients (28.4%), common iliac in 16 patients (21.6%), and gross para-aortic in 8 patients (10.8%). Thirty-six patients (48.7%) were treated with pelvic irradiation, and pelvic and para-aortic irradiation was used in 38 patients (51.3%) (Table 1).
The median follow-up time was 5 years (range, 3-5.5
years). Among all 74 patients who received CCRT, the treatment protocol completion rate was 90 % (95% confidence interval [CI], 85-100). The weekly concur-rent cycles of cisplatin in both treatment groups were completed in all 74 (100%) patients with no interrup-tion. The median duration of radiation therapy in both groups was 55.5 days (48-58).
The overall grade 3 or 4 acute hematological and nonhematological toxicities were seen in 8 patients (10.8%). Grade 3 or 4 acute gastrointestinal toxicity was seen in 4 patients (5.4%) (Table 2).
During the follow-up time of 5 years in the EF-CCRT group, 1 patient (1.4%) experienced sub-acute intestinal obstruction. No patient in either group un-derwent surgery for the radiation-induced damage or died because of the treatment-related side effects.
Response rates, locoregional and distant control, and sur-vival ratesAt the time of the last follow-up, 13 patients (17.6%)
original article rAdiotherApy And cheMotherApy in SAudi woMen
Ann Saudi Med 2013 July-August www.annsaudimed.net330
Table 1. patient characteristics.
Variables ccrt (n=74)
Age 52.3 y (32-78)
Comorbiditieshypertension yes nodiabetes yes no
33 (44.6%)41 (55.4%)
30 (40.6%)44 (59.4%)
Histopathology Squamous cell carcinoma Adenocarcinoma Adenosquamous cell carcinoma
67 (90.5%) 5 (6.7%) 2 (2.7%)
FIGO staging iiB iiiA iiiB iVA
49 (66.2%) 12 (16.2%)
7 (9.5%) 6 (8.1%)
Radiological primary tumor size <5 cm >5 cm
27 (36.5%) 47 (63.5%)
MRI-based nodal involvement negative iliac common iliac para-aortic
29 (39.1%)21 (28.4%) 16 (21.6%) 8 (10.8%)
Pretreatment hemoglobin >10 gm/dL <10 gm/dL
69 (93.3%) 5 (6.7%)
Treatment eBrt: whole pelvis para-aortic parametrial boost
hdr-Bt: dose/Fraction total dose/fraction point A Bed icru 38 rectal point Bed icru 38 bladder point Bed
concurrent weekly cisplatin cycle: dose/wk Mean cycles
45 Gy (42-50.4)45 Gy (45-50.4)
9 Gy (0-9)
7 Gy/fraction21 Gy/3
86.4 Gy (80.5-102.7)85 Gy (80.5-100)86 Gy (80.5-102)
30 mg/m2 5 (4-7)
FiGo: international Federation of Gynecologists and obstetrics, ccrt: concurrent chemoradiation, Mri: magnetic resonance imaging, eBrt: external beam radiation therapy, Bed: biologic effective dose, icru: international commission of radiation units, hdr-Bt: high dose rate brachytherapy.had locoregional recurrences (Figure 3A). Among
those, 6 patients (46.1%) had pelvic recurrences, 5 patients (38.5%) had para-aortic recurrences, and 2 patients (15.4%) had isolated vaginal recurrences that were treated with surgery. Eight patients (72.7%) with pelvic/para-aortic nodal recurrences were treated with salvage chemotherapy and 2 patients (18.1%) received additional para-aortic irradiation. Salvage chemothera-py was generally poorly tolerated.
Distant failures were seen in 9 patients (12.1%). The
Figure 2. (A) parametrial boost of 9 Gy with midline shielding followed by high dose rate brachytherapy 21 Gy in 3 sessions.
common sites of distant failure were as follows: lungs in 4 patients (44.6%), mediastinal in 3 patients (33.3%), supraclavicular nodes in 1 patient (11.1%), and bone in 1 patient (11.1%) (Figure 3B). The median time to initial locoregional and distant failure was 20 months (range 19–24 months). Combined distant and locore-gional failures were observed in 4 patients (18.2%). All
original articlerAdiotherApy And cheMotherApy in SAudi woMen
Ann Saudi Med 2013 July-August www.annsaudimed.net 331
Table 2. Acute and late toxicity profile.
Toxicity CCRT (n=74)
Acute:hematologicneutropeniathrombocytopeniaAnemia
nonhematologicnausea/Vomitingdiarrheacystitisderanged renal functionsderanged liver functions
G3
4 (5.4%) 0 0
4 (5.4%) 0000
G4
000
00000
Late:chronic cystitisintestinal obstructionproctitisneuropathy/plexopathyhearing lossrenal
1 (1.35%)
2 (2.7%)
0
0
0
ccrt: concurrent chemoradiation, G: grade.
Figure 3. Five-year (A) locoregional control, (B) distant control, (c) disease-free survival, and (d) overall survival rates in concurrent chemoradiation.
Table 3. cox regression model analysis of variables on locoregional, distant control, and disease-free survival rates.
Overall SurvivalP value HR (95% CI)
Disease-Free SurvivalP value HR (95% CI)
Distant Metastasis Control
P value HR (95% CI) Locoregional ControlP value HR (95% CI) Variable
0.80 0.80 (0.77-0.98) 0.70 0.50 (0.10-2.41) 0.60 1.10 (0.89-2.00) 0.80 1.80 (0.79-2.10) Age (<50 y vs > 50 y)
0.04 2.20 (1.60-4.11) 1.0 1.80 (0.79-2.10) 0.90 1.80 (0.79-2.10) 0.90 0.88 (0.67-0.97) comorbids (yes vs no)
0.01 2.56 (1.70-6.50) 0.01 3.95 (1.91-10.35) 0.02 4.65 (1.81-9.65) 0.001 7.21 (3.22-16.30) FiGo stage (<iiB vs >iiB)
0.03 3.0 (2.40-7.25) 0.01 4.01 (2.21-11.59) 0.03 3.66 (1.75-9.36) 0.001 6.34 (4.52-11.34) n stage (n0 vs n1)
0.50 1.10 (0.89-2.00) 0.40 0.78 (0.23-2.38) 0.50 1.10 (0.89-2.00) 0.05 1.10 (0.96-1.20)hemoglobin level
(<10 gm/dL vs >10 gm/dL)
0.40 1.21 (1.10-2.10) 0.70 1.21 (1.10-2.10) 0.60 1.10 (0.89-2.00) 0.40 1.21 (1.10-2.10)cell type
(squamous vs nonsquamous)
0.50 1.10 (0.98-1.20) 0.60 1.10 (0.89-2.00) 0.90 0.88 (0.67-0.97) 0.03 3.21 (2.45-7.85) treatment duration (<54 vs >54 d)
FiGo: international Federation of Gynecologists and obstetrics, or: odds ratio, ci: confidence intervals, n: nodal status.
distant failures were treated with salvage chemotherapy and radiotherapy for bone and supraclavicular nodes. At the time of analysis, 51 patients were found without disease with overall DFS of 75.7%. Forty-seven patients were alive at the time of analysis with OS of 64.5% (Figure 3C and D).
Cox regression model analysis showed that nodal
status, FIGO stage, and CCRT duration were impor-tant prognostic factors for locoregional and distant con-trol (Figure 4 and Table 3).
DISCUSSIONPelvic irradiation with concurrent chemotherapy is considered the standard treatment by many North
original article rAdiotherApy And cheMotherApy in SAudi woMen
Ann Saudi Med 2013 July-August www.annsaudimed.net332
Figure 4. Five-year locoregional control according to (A) FiGo stage, (B) nodal status, and (c) pretreatment hemoglobin levels.
American and European teams for the treatment of locally advanced cervical cancer, which has resulted in survival gain of 10% to 20%.7 The 10-year results of a Radiation Therapy Oncology Group (RTOG-97-20) trial, which compared pelvic irradiation with para-aor-tic and pelvic irradiation, have shown an improvement in the survival gain of 11% in the prophylactic para-
aortic and pelvic irradiation group, but no difference in locoregional control.8,9 A subsequent another RTOG trial (RTOG 90-01) compared prophylactic extended-field radiotherapy versus pelvic irradiation with che-motherapy in locally advanced cervical cancer patients. This showed 5-year OS rates for patients treated with pelvic irradiation and concurrent chemotherapy, which was significantly greater than for patients treated with the extended-field radiation alone (67% vs41% at 8 years), and pelvic irradiation with concurrent chemo-therapy resulted in a 51% reduction in the risk of re-currence and a 52% reduction in the risk of death.10
To the best of our knowledge, our single institu-tional study is the first one to report the 5-year treat-ment outcomes of 3DCRT and HDR brachytherapy–based concurrent chemoradiation in FIGO IIB-IVA locally advanced cervical cancer in Saudi Arabia. The 5-year locoregional control, distant control, and DFS and OS rates in our study were 84.3%, 78.5%, 75.7%, and 64.5%, respectively, which are similar or better in some outcomes than the previously reported tri-als.11,12 The course of treatment was well tolerated in our patients, and hematological and nonhematological toxicities were minimal and better than those reported in other trials of using CCRT. The possible explana-tion for lower toxicity profile and better outcomes in our study is as follows: (a) majority of our patients had FIGO Stage IIB, pelvic nodes negative, (b) surgi-cal staging for para-aortic lymph nodes was not carried out as reported in other trials that enhances the gas-trointestinal toxicity, instead, (c) optional FDG/PET was performed, (d) moderate doses of weekly cisplatin, and (e) use of better radiation techniques (3DCRT and HDR brachytherapy). We used radiologic (CT/MRI) staging along with FIGO staging, as the tumor size and lymph node involvement are also important prognostic factors that influence the locoregional con-trol and survival as seen in our study and mentioned by other related studies.13,14 The reason for the relatively lower OS rate is that the majority of our cohort was hypertensive (44.6%) and diabetic (59.4%), which have been known as important prognostic factors for OS in cervical cancer.15
Further, our results have shown that in addition to the FIGO stage, nodal status and treatment duration are also significant prognostic factors for locoregional control; however prognostic factors for distant control and DFS in our study were FIGO stage and nodal sta-tus. Pretreatment hemoglobin <10 gm/dL has been considered an important prognostic factor for OS,16 but we could not see its impact on locoregional con-troland OS in our patients. The total duration of con-
original articlerAdiotherApy And cheMotherApy in SAudi woMen
Ann Saudi Med 2013 July-August www.annsaudimed.net 333
comitant chemoradiation has been suggested to be re-stricted within 56 days to increase locoregional control and OS;17 however, the majority (90%) of our patients finished treatment within 55 days, which reflects the better locoregional control and DFS in our patients.
The strengths of our study were as follwos: reason-able sample size of Saudi women with locally advanced cervical cancer, use of modern radiation therapy tech-niques (3DCRT and HDR brachytherapy), and lon-ger follow-up period. The limitations of our study were as follows: retrospective data and the majority of
cohort was with comorbidities and gross pelvic lymph-adenopathy, which warrants the need of diabetic and hypertension awareness campaigns along with routine cytological screening in Saudi Arabia.
In conclusion, the combined chemotherapy and ra-diation therapy in Saudi women with locally advanced cervical cancer results into better locoregional control, distant control, and DFS, which is consistent with in-ternational data. However, in future, trials incorporat-ing IMRT in locally advanced cervical shall be done to see additional benefit of combined chemoradiation.
REfERENCES1. Makoha Fw, raheem MA. Gynecological cancer incidence in a hospital population in Saudi Arabia: the effect of foreign immigration over two decades. J obstet Gynaecol res. 2008;34:538–42.2. Jamal A, Al-Maghrabi JA. profile of pap smear cytology in the western region of Saudi Arabia. Saudi Med J. 2003;24:1225–9.3. Keys hM, Bundy Bn, Stehman FB, Muders-pach Li, chafe we, Suggs cL 3rd, et al. cisplatin, radiation and adjuvant hysterectomy compared with radiation and adjuvant hysterectomy for bulky stage iB cervical cancer. n eng J Med. 1999;340:1154-61.4. rose pG, Bundy Bn, watkins eB, thigpen Jt, deppe G, Maiman MA, et al. concurrent cis-platin based radiotherapy and chemotherapy for locally advanced cervical cancer. n eng J Med. 1999;340:1144-53.5. whitney cw, Sause w, Bundy Bn, Malfetano Jh, hannigan eV, Fowler wc Jr, et al. randomized comparison of fluorouracil plus cisplatin versus hydroxyurea as an adjunct to radiation therapy in stages iiB-iV carcinoma of the cervix with negative para-aortic lymph nodes: a Gynecologic oncology Group and Southwest oncology Group Study. J clin oncol. 1999;17:1339-48.6. el-Senoussi M, Bakri y, Amer Mh, deVol eB. carcinoma of the uterine cervix in Saudi Arabia: experience in the management of 164 patients with stage-i & -ii disease. int J radiat oncol Biol phys. 1998;42:91-100.
7. Morris M, eifel pJ, Lu J, Grigsby pw, Leven-back c, Stevens re, et al. pelvic radiation with concurrent chemotherapy compared with pelvic and para-aortic radiation for high-risk cervical cancer. n eng J Med. 1999;340:1137-43.8. rotman M, choi K, Guse c, Marcial V, horn-back n, John M. prophylactic irradiation of the pa-ra-aortic lymph node chain in stage iiB and bulky stage iB carcinoma of the cervix, initial treatment results of rtoG 7920. int J radiat oncol Biol phys. 1990;19:513-21.9. rotman M, pajak tF, choi K, clery M, Marcial V, Grigsby pw, et al. prophylactic extended-field irradiation of para-aortic lymph nodes in stages iiB and bulky iB and iiA cervical carcinomas. ten-year treatment results of rtoG 79-20. JAMA. 1995;274:387-93.10. eifel pJ, winter K, Morris M, Levenback c, Grigsby pw, cooper J, et al. pelvic irradiation with concurrent chemotherapy versus pelvic and para-aortic irradiation for high-risk cervical cancer: an update of radiation therapy oncology group trial (rtoG) 90-01. J clin oncol. 2004;22:872-80.11. peters wA 3rd, Liu py, Barrett rJ 2nd, Stock rJ, Monk BJ, Berek JS, et al. concurrent che-motherapy and pelvic radiation therapy com-pared with pelvic radiation therapy alone as ad-juvant therapy after radical surgery in high-risk early-stage cancer of the cervix. J clin oncol. 2000;18:1606-13.12. Small w Jr, winter K, Levenback c, iyer r,
Gaffney d, Asbell S, et al. extended-field irra-diation and intracavitary brachytherapy combined with cisplatin chemotherapy for cervical cancer with positive para-aortic or high common iliac lymph nodes: results of Arm 1 of rtoG 0116. int J radiat oncol Biol phys. 2007;68:1081-7.13. Kodaira t, Fuwa n, toita t, nomoto y, Kuzuya K, tachibana h, et al. comparison of prognostic value of Mri and FiGo stage among patients with cervical carcinoma treated with radiotherapy. int J radiat oncol Biol phys. 2003;56:769– 77. 14. Scheidler J, hricak h, yu KK, Subak L, Segal Mr. radiological evaluation of lymph node me-tastases in patients with cervical cancer. A meta-analysis. JAMA. 1997;278:1096– 101.15. cetina L, Garcia-Arias A, uribe Mde J, can-delaria M, rivera L, oñate-ocaña L, et al. concur-rent chemoradiation with carboplatin for elderly, diabetic and hypertensive patients with locally advanced cervical cancer. eur J Gynaecol oncol. 2008;29:608-12.16. demirci S, ozsaran Z, ozsaran A, yavas F, demircioglu B, hanhan M, et al. evaluation of treatment results and prognostic factors in early-stage cervical carcinoma patients treated with postoperative radiotherapy or radiochemothera-py. eur J Gynaecol oncol. 2012;33:62-7.17. petereit dG, Sarkaria Jn, chappell r, Fowler JF, hartmann tJ, Kinsella tJ, et al. the adverse effect of treatment prolongation in cervical carci-noma. int J radiat oncol Biol phys. 1995;32:1301-7.
A Detailed Study of Patients and Tumor Characteristics ofEpithelial Ovarian Cancer in Saudi Women
Ismail A. Al-Badawi, MD, MBChB, FRCSC,* Adnan R. Munkarah, MD,Þ Asmaa Tulbah, MD,þInas I. Babic, MD,* Hamed Al Husaini, MD,§ and Sarfraz Ahmad, PhD||
Background: The Saudi population is characterized by high parity and intermarriages thatmay impact ovarian carcinogenesis. Herein, we analyzed the tumor characteristics andoutcomes in Saudi patients with epithelial ovarian cancer (EOC).Methods: Patients with EOC treated at King Faisal Specialist Hospital and ResearchCenter during 1995Y2007 were identified retrospectively through a review of their medicalrecords. Patients’ and tumor characteristics were collected including age at diagnosis,marital status, parity, histology, stage, treatment rendered, and follow-up data.Results: One hundred-ninety-three patients with EOC were identified in this cohort. Themean age of the patients was 55 T 15 years, the mean T SD body mass index was 27.0 T5.6 kg/m2, and the median parity was approximately 7.0. Whereas 4 patients reported afamily history of cancer, 164 women reported negative family history; and it was unknownin 27 cases. Tumor distribution by International Federation of Gynecology and Obstetricsstage was the following: 12 patients (6.2%) had stage I disease at diagnosis, 1 patient (0.5%)stage II disease, 130 patients (67.4%) stage III disease, 39 patients (20.2 %) stage IV disease,and that of 11 patients (5.7%) was unknown. Information on residual disease after surgerywas available on 98 patients with optimal debulking (G1 cm) achieved in 61 cases. Medianprogression-free survival from end of chemotherapy to recurrence/progression was 11.9 months(95% confidence interval, 9.4Y15.2). Tumor histology, size of residual disease, and stage atdiagnosis were significant prognostic factors. The patients’ age, body mass index, tumorhistology, and grade had no impact on survival.Conclusions: Patients presenting with advanced-stage disease are higher among Saudisthan those reported in global literature. Despite high intermarriage rates, reported familyhistory for related cancers was quite low in this cohort. Notably, this is the first studyevaluating EOC in Saudi patients.
Key Words: Epithelial ovarian cancer in Saudi patients, Tumor characteristics, Treatmentpatterns, Clinical outcomes
Received August 2, 2012, and in revised form December 6, 2012.Accepted for publication December 24, 2012.
(Int J Gynecol Cancer 2013;23: 456Y460)
ORIGINAL STUDY
456 International Journal of Gynecological Cancer & Volume 23, Number 3, March 2013
*Department of Obstetrics and Gynecology, King Faisal SpecialistHospital and Research Center, Riyadh, Saudi Arabia; †Department ofWomen’s Health Services, Henry Ford Hospital, Detroit, MI48202; ‡Department of Anatomical Pathology, and §OncologyCenter, King Faisal Specialist Hospital and Research Center, Riyadh,
Saudi Arabia; and ||Department of Gynecologic Oncology, FloridaHospital Cancer Institute, Orlando, FL.Address correspondence and reprint requests to Ismail
A. Al-Badawi, MD, MBChB, FRCSC, Department of Obstetricsand Gynecology, King Faisal Specialist Hospital & ResearchCenter, PO Box 3354, Riyadh 11211, MBC-52, Saudi Arabia.E-mail: [email protected].
The authors declare no conflicts of interest.
Copyright * 2013 by IGCS and ESGOISSN: 1048-891XDOI: 10.1097/IGC.0b013e318284aafe
Copyright © 2013 by IGCS and ESGO. Unauthorized reproduction of this article is prohibited.
Ovarian cancer is the leading cause of death among gyne-cologic malignancies, being the sixth most common
cancer in the world and the seventh cause of cancer deaths inwomen.1 Worldwide, more than 200,000 cases are diagnosedwith this disease,1,2 and higher rates are observed in industri-alized and northern European populations.3 The incidencevaries between different regions of the globe, with rates rangingfrom 2.6 cases per 100,000 populations in North Africa to11.5 cases per 100,000 populations in Northern Europe. TheAmerican Cancer Society recently estimates that there will be22,280 new cases of ovarian cancer and 15,500 deathswith thisdisease in the United States alone in the year 2012.4
It is established that age is an important prognosticfactor in this disease. Younger patients tend to do better thantheir older counterparts even when matched for the diseasetype, stage, and treatment(s).5 Published literature furthersuggests that ethnicity and parity are also important prog-nostic factors in this disease.6 Extensive research is currentlybeing done to advance our understanding of the cellularchanges leading to the development/progression of ovariancancer. Whereas several groups around the world are inves-tigating newer/sensitive methods for screening and early de-tection of epithelial ovarian cancer (EOC), many large clinicaltrials are also being conducted with novel regimens includingnewer chemotherapeutic agents and small-molecule ‘‘designerdrugs’’ that target specific molecular sites in the cancer cells.
According to the National Cancer Registry in theKingdom of Saudi Arabia, ovarian cancer is the fifth mostcommon cancer diagnosed in women representing 4.9% of allcancer cases diagnosed during 2010.7 Detailed descriptivedata regarding the clinical course of this disease among Saudiwomen is largely unknown.8 Although there are numerouspublications addressing the course of this disease, its treat-ment, and molecular characteristics from different parts of theworld, there is no substantial study reported in the peer-reviewed literature on Arab women. Therefore, we decided toconduct this study of Saudi women diagnosed with EOCincluding a detailed analysis of their tumor characteristics,evaluation of their disease course, and the prognostic factorsthat may influence survival.
MATERIALS AND METHODSA retrospective review of medical records of all con-
secutive patients diagnosed with EOC and managed atthe King Faisal Specialist Hospital and Research Center(KFSHRC) in Riyadh was performed during the period1995Y2007. The institutional Research Advisory Councilapproval was obtained for this study. Patients were identifiedthrough the institutional tumor registry, the pathology data-base, and medical records using the International Classifi-cation of Diseases, Ninth Revision (ICD-9) codes for ovariancancer. A comprehensive review of the charts was conductedfor data abstraction, including patients’ demographics at thetime of diagnosis, reproductive history, and personal/familyhistory of other cancers (if any). Tumor characteristics in-cluding histology, International Federation of Gynecologyand Obstetrics (FIGO) stage 1988, and grade were recorded.Treatments rendered were also reviewed, including the type of
surgery performed, size of residual disease, the type(s) andduration of primary chemotherapy, and survival outcomes.
It is to be noted that approximately 25% of all the pa-tients with EOC that were treated at the KFSHRC could notbecome part of the survival analysis because of inherentlimitations encountered as (1) lack of regular follow-up in ourgynecologic oncology clinics, and (2) inability to contact thepatients from different districts around Saudi Arabia. Theoverall survival could not be calculated/reported in this studyowing to the lack of accurate information available about thepatients’ date of death (if it occurred).
TABLE 1. Patients’ demographics and histopathologiccharacteristics (n = 193)
Factors Results
Age, mean T SD (range), yrs 54 T 15 (19Y92)Body mass index,mean T SD (range), kg/m2
27 T 5.6 (14Y45)
No. pregnancies (n = 148),mean T SD (range)
6.4 T 3.7 (1Y15)
Marital status, n (%)Married 145 (75.1)Widowed 29 (15.0)Single 12 (6.2)Divorced 7 (3.6)
FIGO stage, n (%)I 12 (6.2)II 1 (0.5)III 130 (67.4)IV 39 (20.2)Unknown 11 (5.7)
Tumor grade, n (%)1 42 (21.8)2 80 (41.5)3 71 (36.8)
Tumor histology, n (%)Serous 125 (64.8)Mucinous 37 (19.2)Endometrioid 22 (11.4)Clear cell 4 (2.1)Undifferentiated 4 (2.1)Mixed epithelial 1 (0.5)
Residual tumor size afterprimary surgery, n (%)G1 cm 7 (3.6)1Y2 cm 6 (3.1)92 cm 31 (16.1)None 54 (28.0)Unknown 95 (49.2)FIGO, International Federation of Gynecology and Obstetrics.
International Journal of Gynecological Cancer & Volume 23, Number 3, March 2013 Epithelial Ovarian Cancer in Saudi Women
* 2013 IGCS and ESGO 457
Copyright © 2013 by IGCS and ESGO. Unauthorized reproduction of this article is prohibited.
Data are summarized as mean T standard deviation (SD)for continuous variables and as proportions for categoricalvariables. The impact of prognostic factors (such as patients’demographics, molecular, clinical, and pathological factors)on progression-free survival (PFS) outcomes was analyzed bythe Kaplan-Meier method. Log-rank test was used to compareoutcomes by specific prognostic variables. Two separateanalyses were run for PFS, one from the time of diagnosis toprogression or recurrence and another one from the end ofprimary therapy to progression or recurrence. Overall survivaldata could not be obtained because most patients are lost tofollow-up when their disease progressed after the second orthird chemotherapy regimen. Furthermore, there is no na-tional or regional registry for death and its causes. Stepwise,Cox proportional hazards regression was used to select themost important predictors of PFS.
RESULTSAlthough initially, this study comprised of a total of
256 patients with ovarian cancer, 63 cases were excludedowing to incorrect histology or incomplete data. Hence, 193cases (75.4%) formed the basis of this study cohort analysis.Table 1 summarizes the patients’ demographics and tumorcharacteristics. The mean T SD age of the patients with EOCwas 54 T 15 years with a mean T SD body mass index of27 T 5.6 kg/m2. A total of 148 cases (76.7%) reportedpregnancies with a mean T SD pregnancy rate of 6.4 T 3.7.Whereas most [128/148 (86.5%)] of these women have hadless than 10 pregnancies, 20 cases (13.5%) experienced morethan 10 pregnancies. Most of the patients with EOC in thiscohort were married women and living with their spouse.Four patients (2.1%) only reported family history of cancerincluding breast cancer (n = 2), lymphoma (n = 1), and ovariancancer (n = 1). Most of the patients (87.6%) had advanced-stage disease (stages III and IV), and high-grade (92) histology.More than 60% of cases were of serous histology followed bymucinous, endometrioid, and other histologic findings.
Almost all cases (99.5%) of the study cohort under-went surgery as part of their treatment for ovarian cancer(Table 2). Whereas 152 EOC cases (79.2%) received surgery
at KFSHRC, 40 cases (20.8%) had their surgery at an outsideinstitution. The surgical procedures included hysterectomywith adnexectomy and omentectomy in 169 patients,adnexectomy with or without omentectomy in 15 patients,and laparotomy with biopsies in the rest of the cases. Otherperformed procedures included colon resection in 10 patients,small bowel resection in 7 patients, and splenectomy in 3cases. Although 163 patients were reported to have ‘‘tumordebulking’’, almost half of the cases lacked sufficient docu-mentation in the chart to either assess the extent of disease atthe beginning of surgery or the size of residual tumor at itscompletion (Table 1)
Most patients (73%) received primary chemotherapy.The chemotherapeutic regimens are described in Table 2, withpaclitaxel plus carboplatinum being the most commonly usedregimens. We recognize the fact that approximately 88% of ourpatients had stage III/IV disease and only 73%of cases receivedprimary chemotherapy; this happened as some of the patientsdeclined treatment with chemotherapy, and somewanted to usealternative medicine against our medical advice. The responserates to chemotherapy are described in Table 3. One hundredtwenty-seven patients (66%) experienced disease progressionor recurrence after primary therapy.
The median follow-up was 60 months. The median PFSwas 17.0 months (95% confidence interval, 14.0Y19.2) fromthe time of diagnosis and 11.9 months (95% confidence inter-val, 9.4Y15.2) from the end of primary therapy. Figures 1AYDdepicts the Kaplan-Meier PFS curves from the end of pri-mary therapy with respect to various prognostic factors. In theunivariate analyses, the size of residual disease (no gross re-sidual, e, or 91 cm), tumor histology (serous vs nonserous),and FIGO stage had significant impact on the PFS. This effectwas retained for all the 3 factors in a Cox regression analysis.Interestingly, parity did not have an impact on prognosis ex-cept in women who had more than 10 deliveries. The PFS ofthat highly multiparous group of women was 3 months shorter(P = 0.005).
DISCUSSIONSaudi Arabia is one of the major Gulf countries in
which intermarriages and multiparity are fairly common.Furthermore, the routine use of oral contraceptive pills andhormone replacement therapy is not common among Saudipopulation. Data on the ovarian cancer prevalence and theimpact of known prognostic factors in this population arelargely unknown. The only available information is from the
TABLE 2. Surgical treatment strategy andchemotherapeutic regimens for patients with EOC
Surgical Treatment (n = 193) No. Cases (%)
Debulking surgery only 43 (22.2)Surgery followed by chemotherapy 131 (67.8)Chemotherapy followed by surgery 18 (9.3)Chemotherapy only 1 (0.5)Chemotherapeutic regimens (n = 150)*Platinum + taxol 115 (59.6)Platinum + cyclophosphamide 24 (12.4)Platinum only 9 (4.7)Others 2 (1)
*Median no. cycles, 6 months; range, 1Y12 months.
TABLE 3. Response on primary treatment of patientswith EOC (n = 193)
Treatment Response No. Cases (%)
Complete 111 (57.1)Partial 16 (8.3)Progression 15 (7.8)Stable 5 (2.6)Not clinically evaluable 46 (23.8)
Al-Badawi et al International Journal of Gynecological Cancer & Volume 23, Number 3, March 2013
458 * 2013 IGCS and ESGO
Copyright © 2013 by IGCS and ESGO. Unauthorized reproduction of this article is prohibited.
Saudi National Tumor Registry, in which distribution by thegeographic districts, age group, American Joint Committeeon Cancer tumor stage, and histopathologic classification areavailable based on review of hospital records. Most recently, apreliminary assessment from the Saudi Cancer Registry be-tween the years 1999 and 2008 suggested that the genital tractmalignancies in the region were, in order of frequency, asfollows: ovarian cancer (35.2%), endometrial cancer (34.2%),cervical cancer (27.6%), vulvar cancer (2%), and vaginalcancer (1%).8 Other details about the clinicopathologic andsurgical outcomes of patients with EOC are lacking seriously.Therefore, we conducted this retrospective review to evaluatethe tumor and the patients’ characteristics that are pertinent toovarian cancer in Saudi women and also analyzed the prog-nostic impact of factors described in the literature.
Eighty percent of younger patients (younger than age40) in this study were free of disease at follow-up of 60 months.On the other hand, elderly patients (970 years) had 100%recurrence rate within 55 months. Most of the patients in our
study were slightly overweight (with a mean body mass indexof 27 kg/m2), which is rather similar to the globally publishedliterature.9 Interestingly, the family history for breast, ovary,or colon cancer was very low in this study population. Wewere expecting to find a higher proportion of familial cancersyndromes in view of the high occurrence of intermarriages inthe society as well as local data showing high prevalence ofbreast cancer in younger Saudi women.10
Multiparity is known to reduce the risk of EOC, but weare not aware of any study about its effect on the PFS oroverall survival. Parity in our study was not an independentprognostic factor except at a cutoff of 10 pregnancies. It ishard to speculate as to the cause of worse PFS in these grandmultiparous women.
Most (87%) of the patients in this cohort had theircondition diagnosed at an advanced stage of the disease(FIGO stage III or IV). This is probably due to limited accessto medical care as well as awareness regarding ovarian cancer.Until recently, cancer care in Saudi Arabia was centralized
FIGURE 1. Kaplan-Meier curves showing the progression-free survival (PFS) computed from the completion ofprimary therapy. A, Progression-free survival for the study population; B, PFS by FIGO stage; C, PFS by residual disease;D, PFS by parity.
International Journal of Gynecological Cancer & Volume 23, Number 3, March 2013 Epithelial Ovarian Cancer in Saudi Women
* 2013 IGCS and ESGO 459
Copyright © 2013 by IGCS and ESGO. Unauthorized reproduction of this article is prohibited.
with most patients in the country being referred to and caredfor in a couple of tertiary centers in 2 large cities. Further-more, the symptoms and signs of ovarian cancer are nonspe-cific and frequently confused with gastrointestinal diseases.Delays in referring the patients to specialized centers mayhave contributed to this problem. Fortunately, this scenario haschanged over the past decade. A number of physicians havereceived cancer training specifically in gynecologic oncologyin large international and Western centers. Many cancer cen-ters are now available around the nation and help improvepatients’ access.
Analysis of treatments rendered showed that most of thepatients had cytoreductive surgery and chemotherapy. Pacli-taxel and carboplatinum was the most commonly used regi-men. Response rates to chemotherapy were similar to thosereported in the literature. The PFS noted in the current study isclose to what is reported in the literature for women withadvanced-stage disease. Unfortunately, the lack of informa-tion on residual disease after cytoreductive surgery in 50% ofthe population limited our survival analysis. However, theprognostic impact of minimal residual disease was confirmedin the subgroup of patients where such information wasavailable (P G 0.001).
Some of the limitations of this study include its retro-spective nature, its time span over a decade, and lack ofsurvival data due to limited follow-up. Its strengths includecentral pathologic review of all cases and a relatively largecohort of patients. Furthermore, this is the first study of itssort in this patient population. It confirmed that the histologicdistribution and natural course of the disease is comparable towhat is described in the literature. The reproductive factorsthat are specific to this population did not show any signifi-cant impact on the biology of the disease or the patients’prognosis. Furthermore, the family history of cancer was
surprisingly low despite the high rate of intermarriages.Molecular studies on the malignant tissues are being com-pleted to help characterize the molecular phenotype of thesecancers.
REFERENCES1. Jemal A, Bray F, Center MM, et al. Global cancer statistics.
CA Cancer J Clin. 2011;61:69Y90.2. Benerjee S, Gore M. The future of targeted therapies in ovarian
cancer. Oncologist. 2009;14:706Y716.3. Farley J, Shin HR, Bray F, et al. GLOBOCON 2008 v1.2,
cancer incidence and mortality worldwide: IARC cancer baseno. 10. Lyon (France). International Agency for Researchon Cancer; 2010. Available at: http://globocon.iarc.fr.Accessed January 17, 2012.
4. Siegel R, Naishadham D, Jemal A. Cancer statistics, 2012.CA Cancer J Clin. 2012;62:10Y29.
5. Chan JK, Loizzi V, Lin YG, et al. Stages III and IV invasiveepithelial ovarian carcinoma in younger versus older women:what prognostic factors are important? Obstet Gynecol.2003;102:156Y161.
6. Barnholtz-Sloan JS, Schwartz AG, Qureshi F, et al. Ovariancancer: changes in patterns at diagnosis and relative survivalover the last three decades. Am J Obstet Gynecol.2003;189:1120Y1127.
7. King Faisal Specialist Hospital, Oncology Centre(Tumor Registry), Annual Report-2010. 2010; p. 26.
8. Nazer A, Al-Badawi IA. Incidence of gynecological malignancyamong the Saudi population. Hematol Oncol Stem Cell Ther.2012;5:69Y70.
9. Olsen CM, Green AC, Whiteman DC, et al. Obesity and therisk of epithelial ovarian cancer: a systematic review andmeta-analysis. Eur J Cancer. 2007;43:690Y709.
10. Auranen A, Pukkala E, Makinen J, et al. Cancer incidence in thefirst-degree relatives of ovarian cancer patients. Br J Cancer.1996;74:280Y284.
Al-Badawi et al International Journal of Gynecological Cancer & Volume 23, Number 3, March 2013
460 * 2013 IGCS and ESGO
Copyright © 2013 by IGCS and ESGO. Unauthorized reproduction of this article is prohibited.
Methanolic Extract of Nigella sativa Seed Inhibits SiHa Human Cervical Cancer Cell Proliferation Through Apoptosis Tarique N. Hasan1, Gowhar Shafi1, Naveed A Syed1, Muhammad A Alfawaz1, Mohammed A. Alsaif2, Anjana Munshi1,3, Kai Y. Lei1,4 and Ali A. Alshatwi1*
1Molecular Cancer Biology Research Lab, Dept. of Food Science and Nutrition, College of Food and Agricultural Sciences, King Saud University, Saudi Arabia
2Department of Clinical Nutrition, College of Applied Medical Sciences, King Saud University, Riyadh Saudi Arabia
3Department of Molecular Biology, Institute of Genetics and Hospital for Genetic Diseases, Hyderabad, India
4Department of Nutrition and Food Science, University of Maryland, College Park, MD 20742, USA [email protected]
Received: January 4th, 2011; Accepted: December 12th, 2012
Nigella sativa (NS), also known as black cumin, has long been used in traditional medicine for treating various cancer conditions. In this study, we sought to investigate the potential anti-cancer effects of NS extract using SiHa human cervical cancer cells. NS showed an 88.3% inhibition of proliferation ofSiHa human cervical cancer cells at a concentration of 125 µL/mL methanolic extract at 24 h, and an IC50 value 93.2 µL/mL. NS exposure increased the expression of caspase-3, -8 and -9 several-fold. The analysis of apoptosis by DeadEnd terminaltransferase-mediated dUTP-digoxigeninend labeling (TUNEL) assay was used to further confirm that NS induced apoptosis. Thus, NS was concluded to induce apoptosis in SiHa cell through bothp53and caspases activation. NS could potentially be an alternative source of medicine for cervical cancer therapy. Keywords: Nigella sativa seeds, SiHa cells, Anticancer, Apotosis, p53, Caspases-3, Caspases-8, Caspases-9. Nigella sativaL.(NS), commonly known as black cumin, has been used in many Middle Eastern countries for thousands of years, as a spice, food preservative, and curative remedy for numerous disorders [1a-1c]. The extract of NS seeds has been shown to exert in vitro antitumor effects on carcinoma, lymphoma and sarcoma [2a, b]. Methanolic extract of NS seeds has been reported to induce in vitro cytotoxicity in human prostate cancer PC3 cell line [2c], whereas the chloroform fraction of NS was found to be able to induce apoptosis in HeLa cells [2d]. In addition to these direct anti-tumor effects, black seed preparations may have potential for cancer chemoprevention, as well as for reducing the toxicity of standard antineoplastic drugs [2a,3a]. Most of these effects have been attributed to the volatile oil obtained from the black seed preparation. Thymoquinone and dithymoquinone, the main active components of the volatile oil, were observed to be cytotoxic to both parental and multidrug resistant tumor cells in vitro [3b, c]. NS seeds are well known for their medicinal property [3d] and no side effect and health hazard has been reported [3e]. Cervical cancer is the fifth most common type of cancer among women. Considering the significance of this cancer sub-type, we used SiHa cell lines to analyze the anticancer activity of the NS seed extract as well as to investigate the ability of NS to induce apoptosis and to determine the responsible molecular mechanisms underlying apoptosis in SiHA cells. To determine its antiproliferative activity, we studied the effect of NS on cell viability by Cell Titer Blue®viability assay. Induction of apoptosis was assessed by DeadEnd TUNEL assay and real-time quantitative PCR for the expression of the pro-apoptotic genes like caspase-3, caspase-8 and caspase-9 and p53. The results led us to conclude that certain bioactive compounds in NS seeds are capable of effectively causing death of cancer cells by up-regulating the expression of certain pro-apoptotic genes.
The cytotoxic effect of NS from 0 µL/mL to 125 µL/mL on SiHa cells was examined using the Cell Titer Blue®viability assay. A dose-dependent reduction in color was observed after 24 hours of treatment with NS, and 88.3% of the cells were dead at the highest concentration tested (125 µL/mL), whereas the IC50value of NS for SiHa cell was achieved at 93.2 µL/mL. To determine whether the inhibition of cell proliferation by NS was due to the induction of apoptosis, a TUNEL assay was used.Figures 1-3 summarize the effect of NS on SiHa cells. A dose-and time-dependent increase in theinduction of apoptosis was observed when SiHa cells were treated with NS. At 24 h, 16.7 and 26.2% of the cells treated with 50 µL/mL and 100 µL/mL NS, respectively, underwent apoptosis when normalized with control. Similarly, 29.6 and 38.8% of the treated cells for 48 hours, and 36.4 and 60.1% of the treated cells for 72 hours were apoptotic when treated with 50 µL/mL and 100 µL/mL, respectively. To investigate the molecular mechanism of NS-induced apoptosis in SiHa cells, the expression levels of several apoptosis-related genes were examined. The relative quantification of caspase-3, -8, and -9 and p53 mRNA expression levels was performed by SYBR Green–based quantitative real-time PCR using a 7500 Fast Real Time PCR System. NS increased the transcripts of caspase 3, -8, and -9, and p53by several fold. The expression levels of these genes in SiHa cells treated with 50 µL/mL NS for 24 hours increased by 4.34, 2.23, 7.41 and 4.69 fold, respectively, as compared with the levels in untreated control cells. Similarly, the expression levels of caspase-3, -8, and -9 and p53 in SiHa cells treated with 100 µL/mL NS for 24 hours increased by 8.12, 2.42, 8.15 and 8.14 fold, respectively, as compared with levels in untreated control cells. In a time-dependent manner, the expression levels of the apoptosis-related genes in SiHa cells treated with 50 or 100 µL/mL NS for 48 hours increased when compared with the levels in untreated control cells.
NPC Natural Product Communications 2013 Vol. 8 No. 2
213 - 216
214 Natural Product Communications Vol. 8 (2) 2013 Hasan et al.
Figure 1: TUNEL assay (microscopic, resolution=200 X) after 24 h incubation of SiHa cells with NS. The red fluorescence is due to propidium iodide staining with observations made under a green filter. The green fluorescence is due to FITC staining with observations made under a blue filter.
Figure 2: TUNEL assay (microscopic, resolution=200 X) after 48 h incubation of SiHa cells with NS. The red fluorescence is due to propidium iodide staining with observations made under a green filter. The green fluorescence is due to FITC staining with observations made under a blue filter.
Figure 3: TUNEL assay (microscopic, Resolution=200 X) after a 72-h incubation of SiHa cells with NS treatment. The red fluorescence is due to propidium iodide staining with observations made under a green filter. The green fluorescence is due to FITC staining with observations made under a blue filter. However, the expression levels of caspase-3, -8, and -9 and p53in SiHa cells treated with 100 µL/mL NS for 48 hours markedly increased by 30.7, 23.9, 15.2 and 26.1 fold, as compared with control, untreated cells. Together, these data suggest that these caspases and p53 were induced by NS in a dose- and time-dependent manner. Agents suppressing the proliferation of malignant cells by enhancing apoptosis may constitute a useful mechanistic approach to both cancer chemoprevention andchemotherapy. Hence, apoptosis was hypothesized to be the possible mode of cell death in
SiHa cancer cell by following either nuclear (p53) or mitochondrial (caspase) or both pathways. To test this hypothesis, we sought to determine the role of NS in inhibiting cell growth and modulating the expression of crucial apoptotic genes likecaspases-3, -8, and -9 and p53. The data presented in this paper demonstrate a time-and dose-dependent inhibition of SiHa human cancer cell proliferation by NS exposure. There are many mechanisms through which apoptosis can be enhanced in cells. Caspases have a central role in the apoptotic process in that they trigger a cascade of apoptotic pathways. The release of cytochrome-c from mitochondria leads to the activation of procaspase-9 and then caspase-3 [3h]. The activation of caspase-3 is an important downstream step in the apoptotic pathway [3i]. In addition, the effector caspase, caspase-3, and the initiator caspases, caspase-8 and -9, are the main executors of apoptosis [4a]. Caspase-8 is in the death receptor pathway, whereas caspase-9 is in the mitochondrial pathway, and both pathways share caspase-3 [4b]. The possibility that p53-mediated apoptosis may be associated with the activation of caspase-3, -8 and -9 was suggested by the evidence, which established the ability of p53 to activate both the extrinsic and intrinsic apoptotic pathways [4c, 4d, 5a]. p53 enhances cancer cell apoptosis, and it prevents cell replication by stopping the cell cycle at G1 or interphase [5b]. By inducing the release of mitochondrial cytochrome c, p53 might be able to activate effector caspases, including caspase -3 and other caspases. Furthermore, caspases-3, -8, and -9 may be part of the apoptotic effector machinery engaged by p53 to mediate teratogen-induced apoptotic pathways [5c]. The data presented in this study indicate that the expression levels of p53 and caspase-3, -8 and -9 weremarkedly increased after NS treatment in a concentration-dependent manner. Taken together, the data presented in this study suggest that the NS induced apoptosis is mediated by the death receptor andmitochondrial apoptotic pathways as demonstrated by increasedexpression levels of caspase-3, -8 and -9 after NS treatment. In addition, this study suggests that NS activates the extrinsic death pathway, as demonstrated by increased expression levels of caspase-8.Therefore, these data suggested that NS may induce apoptosis through various mechanisms, and the NS-induced apoptosis occurred partly through its ability to increase the expression of pro-apoptotic genes such as p53 and caspase-3, 8, and 9 in SiHa cervical cancer cells. In conclusion, to the best of our knowledge, the results presented in this study show for the first time that NS exhibits anticancer effects by blocking the proliferation of cervical cancer SiHa cells and inducing apoptosis in part by modulating p53 in addition to caspase-3, 8, and 9 expression. The present data shown here confirm our earlier reports and suggest that the induction of apoptosis by NS is partly attributed to its ability to increase the expression of the pro-apoptotic genes such as p53 and caspase-3, 8, and 9. Taken together with our previous observations, the results described here suggest that NS induced apoptosis in SiHa cells, in part by mediating the induction of p53 and caspase-3, -8, and -9. Most importantly, findings of the present study support the development of dietetic plant product based drug(s) for treatment of cervical cancer. However, further research will be required to characterize and analyze different components of the NS seed before the development of a promising chemo-therapeutic agent for cervical cancer treatment from NS. Experimental
Preparation of NS extract: NS seeds, purchased from a local market, were powdered mechanically. The extract was obtained by
Cytotoxic activity of Nigella sativa Natural Product Communications Vol. 8 (2) 2013 215
cold soaking of the powdered seeds in 1 L of methanol thrice for 24 h. The solvent was removed under reduced pressure. Maintenance of SiHa cells: The SiHa cancer cell line was kindly gifted by Dr MAAkbarshah, Mahatma Gandhi-Doerenkamp Center (MGDC), Bharathidasan University, India. The cell line was maintained and propagated in 90% Dulbecco's Modified Eagle's Medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Cells were cultured as adherent monolayers (i.e., cultured at ~70% to 80% confluence) and maintained at 37°C in a humidified atmosphere of 5% CO2. Cells were harvested after brief trypsinization. Toxicity and cell proliferation assays: To assess the toxicity of different concentrations of NS on SiHa cells, the Cell Titer Blue®
viability assay (Promega Madison, WI) was performed following the manufacturer’s instructions. Briefly, SiHa cells (2 x 104
cells/well) were plated in 96-well plates and treated with a range of doses (0 µL/mL NS to 125 µL/mL NS for 24 h). Then, 40 µL of the Cell Titer Blue solution was directly added to the wells and incubated at 37°C for 6 h. The fluorescence was recorded with a 560 nm/590 nm (excitation/emission) filter set using a Bio-Tekmicroplate fluorescence reader (FLx800TM), and the IC50 was calculated. Quadruplet samples were run for each concentration of NS in 3 independent experiments. NS treatment for a dose- and time-dependent study: For a dose -and time-dependent study, SiHa cells were treated with either 50 µL/mL or 100 µg/mL NS for 24, 48, and 72 h for the terminal deoxynucleotidyltransferase–mediated dUTP nick end labeling (TUNEL) assay. The cells were incubated with the same NS concentrations for 24 and 48 h for real-time quantitative PCR analysis. TUNEL assay: The DeadEnd® TUNEL assay kit (Promega, Madison, WI) was used for studying apoptosis in a time-and dose-dependent manner. Briefly, SiHa cells (1.5 x 106 cells/well) were cultured in 6-well plates to study apoptosis in adherent cells. Cells were treated with 50 µL/mL and 100 µL/mL NS for 24, 48 and 72 h. After the incubation period, the culture medium was removed by aspiration, and the cell layers were trypsinized. The treated cells were reattached on 0.01% polylysine-coated slides, fixed with 4% methanol-free formaldehyde solution, and stained according to the DeadEndfluorometric TUNEL system protocol [5d]. The stained
cells were observed using a Carl-Zeiss (Axiovert) epifluorescence microscope using a triple band-pass filter. To determine the percentage of cells demonstrating apoptosis, 1000 cells were counted in each experiment [2d]. Real-time quantitative PCR analysis: Real Time-PCR (ABI 7500 Fast, Foster City, CA) was used to analyze the expression of apoptotic genes by using a real-time SYBR Green/ROX gene expression assay kit (QIAGEN, Germany). A Fastlane® Cell cDNA kit (QIAGEN, Germany) was used to prepare cDNA directly from cultured cells., and the mRNA levels of caspase-3, -8, and -9 and p53, as well as the reference gene, GAPDH, were assayed using gene-specific SYBR Green–based QuantiTect® Primer assays. Briefly, 12.5 µL of master mix, 2.5 µL of primer assay (10x) and 10 µL of template cDNA (100 µg) were added to each well. After brief centrifugation, the PCR plate was subjected to 35 cycles of the following conditions: (i) PCRactivation at 95°C for 5 min, (ii) denaturation at 95°C for 5 sec and (iii) annealing/extension at 60°C for 10 sec. All samples and controls were run in triplicate on an ABI 7500 Fast Real-time PCR system. The quantitative RT-PCR data were analyzed by a comparative threshold (Ct) method, and the fold inductions of samples were compared with the untreated samples. GAPDH was used as an internal reference gene to normalize the expression of the apoptotic genes. The Ct cycle was used to determine the expression level in control cells and SiHa cells treated with NS for 24 and 48 h. The gene expression level was then calculated as described by Yuan et al. [5e]. The results were expressed as the ratio of reference gene to target gene by using the following formula: ΔCt = Ct (apoptotic genes) – Ct (GAPDH). To determine the relative expression levels, the following formula was used: ΔΔCt = ΔCt (Treated) -ΔCt (Control). Thus, the expression levels were expressed as n-fold differences relative to the calibrator. The value was used to plot the expression of apoptotic genes using the expression of 2 -ΔΔCt. Statistical analysis: The data were expressed as the mean (± SD). Analyses were performed using Microsoft excel statistical software. Acknowledgments – This work was supported by grants from the Research Center, Research Center, Deanship of Scientific Research, College of Food and Agriculture Science, King Saud University, Saudi Arabia.
.
References
[1] (a) Chopra RN, Nayar SL, Chopra IC. (1956) Glossary of Indian Medicinal Plants. CSIR, New Delhi, India, 175; (b) Nadakarni KM. (1976) Crocus sativus, Nigella sativa. In Indian Materia Medica.Nadkarni KM. (Ed.). Popular Prakashan, Bombay, India, 386–411; (c) Yesilada E, Honda G, Sezik E, Tabata M, Fujita T, Tanaka T, Takeda Y, Takaishi Y. (1995) Traditional medicine in Turkey. V. Folk medicine in the inner Taurus Mountains. Journal of Ethnopharmacology, 46, 133-152.
[2] (a) Salomi NJ, Nair SC, Panikkar KR. (1991) Inhibitory effects of Nigella sativa and saffron (Crocus sativus) on chemical carcinogenesis in mice. Nutrition and Cancer, 16, 67–72; (b) Salomi NJ, Nair SC, Jayawardhanan KK, Vargghese CD, Pantikkar KR. (1992) Anti-tumor principles from Nigella sativa seeds. Cancer Letters, 63, 41–46; (c) Shafi G, Hasan TN, Sayed NA. (2008) Methanolic extract of Nigella sativa seeds is potent clonogenic inhibitor of PC3 Cells. International Journal of Pharmacology, 4, 477-481; (d) Shafi G, Munshi A, Hasan TN, Alshatwi AA, Jyothi A, Lei DKY. (2009) Induction of apoptosis in HeLa cells by chloroform fraction of seed extracts of Nigella sativa. Cancer Cell International, 9, 29-36, doi:10.1186/1475-2867-9-29
[3] (a) Nair SC, Salomi, MJ, Panikkar B, Panikkar KR. (1991) Modulatory effects of Crocus sativus and Nigella sativa extracts on cisplatin-induced toxicity in mice. Journal of Ethnopharmacology, 31, 75–83; (b) Worthen DR, Ghosheh OA, Peter AC. (1998) The in-vitro anti-tumor activity of some crude and purified components of black seed, Nigella sativa L. Anticancer Research, 18, 1527–1532; (c) Gali-Muhtasib H, Diab-Assaf M, Boltze C, Al-Hmaira J, Hartig R, Roessner A, Schneider-Stock R. (2004) Thymoquinone extracted from black seed triggers apoptotic cell death in human colorectal cancer cells via a p53dependent mechanism. International Journal of Oncology, 25, 857-866; (d) Hasan TN, Ahmed SN, Aalam SMM, Kumar C, Shafi G. (2007) Evaluation of Cichorium extract for the growth supporting property in rat hepatocyte primary culture. Asian Journal of Plant Science, 6,431-434; (e) Sharma NK, Ahirwar D, Jhade D, Gupta S. (2009) Medicinal and pharmacological potential of Nigella sativa: Areview. Ethnobotanical Review, 13,946-955; (f) Thabrew MI, Mitry RR, Morsy MA, Hughes RD. (2005) Cytotoxic effects of a decoction of Nigella sativa, Hemidesmus indicus and Smilax glabra on human hepatoma HepG2 cells. Life Science, 77, 1319-1330; (g) Ivankovic S, Stojkovic R, Jukic M, Milos M, Jurin M. (2006) The antitumor activity of thymoquinone and thymohydoquinone in vitro and in vivo. Experimental Oncology, 28, 220-224; (i) Hengartner MO. (2000) The biochemistry of apoptosis. Nature, 407, 770–776.
216 Natural Product Communications Vol. 8 (2) 2013 Hasan et al.
[4] (a) Earnshaw WC, Martins LM, Kaufmann SH. (1999) Mammalian caspases Structure, activation, substrates, and functions during apoptosis. Annual Review Biochemistry, 68, 383-424; (b) Riedl SJ, Shi Y. (2004) Molecular mechanisms of caspase regulation during apoptosis. Nature Review. Molecular Cell Biology, 5, 897–907; (c) Malik F, Kumar A, Bhushan S, Khan S, Bhatia A, Suri KA, Qazi GN, Singh J. (2008) Reactive oxygen species generation and mitochondrial dysfunction in the apoptoticcell death of human myeloid leukemia HL-60 cells by a dietary compoundwithaferin A with concomitant protection by N-acetyl-cysteine. Apoptosis, 12, 2115–2133; (d) Fridman JS, Lowe SW. (2003) Control of apoptosis by p53. Oncogene, 22, 9030–9040.
[5] (a) Michalak E, Villunger A, Erlacher M, Strasser A (2005) Death squads enlisted by the tumor suppressor p53. Biophysical Biochemical Research Communications, 331, 786-798; (b) Takaoka A, Hayakawa S, Yanai H, Stoiber D, Negishi H, Kikuchi H, Sasaki S, Imai K, Shibue T, Honda K, Taniguchi T. (2003) Integration of interferon-alpha/beta signalling to p53 responses in tumour suppression and antiviral defence. Nature, 424, 516–523; (c) Pekar O, Molotski N, Savion S, Fein A, Toder V, Torchinsky A.(2007) p53 regulates cyclophosphamide teratogenesis by controlling caspases 3, 8, 9 activation and NFkappaBDNA binding. Reproduction, 134, 379–388; (d) Wang W, Sun W, Wang X. (2004) Intramuscular gene transfer of CGRP inhibits neointimal hyperplasia after balloon injury in the rat abdominal aorta. American Journal of Physiology: Heart Circulation Physiology, 287, H1582-H1589; (e) Yuan JS, Reed A, Chen F, Stewart Jr CN. (2006) Statistical analysis of real-time PCR data. BMC Bioinformatics, 7, 85. doi:10.1186/1471-2105-7-85.
(This is a sample cover image for this issue. The actual cover is not yet available at this time.)
This article appeared in a journal published by Elsevier. The attachedcopy is furnished to the author for internal non-commercial researchand education use, including for instruction at the authors institution
and sharing with colleagues.
Other uses, including reproduction and distribution, or selling orlicensing copies, or posting to personal, institutional or third party
websites are prohibited.
In most cases authors are permitted to post their version of thearticle (e.g. in Word or Tex form) to their personal website orinstitutional repository. Authors requiring further information
regarding Elsevier’s archiving and manuscript policies areencouraged to visit:
http://www.elsevier.com/copyright
Author's personal copy
Naringin induces death receptor and mitochondria-mediated apoptosisin human cervical cancer (SiHa) cells
E. Ramesh, Ali A. Alshatwi ⇑Department of Food Science and Nutrition, King Saud University, Riyadh 11451, Saudi Arabia
a r t i c l e i n f o
Article history:Received 8 December 2011Accepted 19 July 2012Available online 27 July 2012
Keywords:NaringinApoptosisSiHa cellsCancer
a b s t r a c t
Cervical cancer is the second most common female cancer worldwide, and it remains a challenge to man-age preinvasive and invasive lesions. Fruit-based cancer prevention entities, such as flavonoid and theirderivatives, have demonstrated a marked ability to inhibit preclinical models of epithelial cancer cellgrowth and tumor formation. Here, we extend the role of naringin-mediated chemoprevention to thatof cervical carcinogenesis. The present study sought to investigate the therapeutic potential effect ofnaringin on apoptosis in human cervical SiHa cancer cells. Viability of SiHa cells was evaluated by theMTT assay, apoptosis and mitochondrial transmembrane potential by flow cytometry, and pro-apoptoticrelated genes by Real-time quantitative PCR. Naringin showed a 50% inhibition of SiHa human cervicalcancer cells at a concentration of 750 lM. SiHa cells exhibited apoptotic cell death, intranucleosomalDNA fragmentation, morphological changes and decline in the mitochondrial transmembrane potential.In addition, administration of naringin increased the expression of caspases, p53 and Bax, Fas deathreceptor and its adaptor protein FADD. These results suggest that the induction of apoptosis by naringinis through both death-receptor and mitochondrial pathways. Taken together, our results suggest thatnaringin might be an effective agent to treat human cervical cancer.
� 2012 Elsevier Ltd. All rights reserved.
1. Introduction
Cervical carcinoma is the second most common female cancerworldwide. It is estimated that 12,200 new cases and about 4210deaths will be attributed to cervical cancer in the United Statesin 2010 (American Cancer Society, 2010). Cervical cancer, the sev-enth most frequent women cancer in Saudi Arabia and the eighthmost common cancer among women aged between 15 and44 years (WHO, 2007). It is generally accepted that radical surgeryor radiotherapy can be curative for the majority of patients withearly-stage cervical cancer, while chemotherapy or neoadjuvantchemotherapy is always the first choice for those patients with ad-vanced cervical cancer, where the prognosis remains very poor(Thomas, 1999; Ren et al., 2008). Therefore, many researchers havebeen trying to find more effective chemotherapeutics to treat cer-vical cancer cell. Apoptosis is an active form of cell suicide con-trolled by a network of genes and it is an essential process, as
well as a key role, in the pathogenesis of diseases including cancer(Li et al., 2009). During the past decade, numerous reports haveproven that many cancer chemotherapeutic agents kill the cancercell by inducing apoptosis (Mandal et al., 2010; Kim et al., 2010.Thus, clarification of the induction mechanisms of cell apoptosisis useful to approach in cancer therapies.
Epidemiological evidence also indicates a high intake of fruitsand vegetables leads to a reduction in cancer incidence, and it isthe phytochemicals within fruits and vegetables that have beenproposed as responsible for their protective effects (Kroon and Wil-liamson, 2005). Consequently, in the past few decades chemopre-vention through dietary phytochemicals has become anincreasingly active area of research and entails using non-toxicsubstances to interfere with carcinogenesis (Surh, 2003; Johnsonet al., 2010). Flavonoids, a family of natural polyphenolic com-pounds, are commonly found in fruits and vegetables, regularlyconsumed by humans. Flavonoids are composed of several classesincluding flavonols, flavonones, flavones, flavanols, iso-flavonoidsand antho-cyanidins. They have been demonstrated to possessanti-cancer and chemopreventive property in numerous epidemio-logical studies (Park and Surh, 2004; Ramos, 2007; Arts, 2008;Miller et al., 2008). At the cellular and molecular level, flavonoidshave a broad range of effects and interactions that contribute totheir chemopreventive and anti-carcinogenic activities. Themodulation of cellular processes such as cell cycle and apoptosiscontributes to their anti-proliferative effects (Kuntz et al., 1999;
0278-6915/$ - see front matter � 2012 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.fct.2012.07.033
Abbreviations: DMSO, dimethylsulfoxide; DMEM, Dulbecco’s Modified Eagle’sMedium; FBS, fetal bovine serum; wM, mitochondrial transmembrane potential; JC-1, 5,50 ,6,60-tetrachloro-1,10 ,3,30-tetraethylbenzimidazolylcarbocyanineiodide; MTT,3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PI, propidiumiodide.⇑ Corresponding author. Address: Department of Food Science and Nutrition,
College of Food Sciences and Agriculture, King Saud University, P.O. Box 2460Riyadh 11451, Saudi Arabia. Tel.: +966 1 467 7122; fax: +966 1 467 8394.
E-mail address: [email protected] (A.A. Alshatwi).
Food and Chemical Toxicology 51 (2013) 97–105
Contents lists available at SciVerse ScienceDirect
Food and Chemical Toxicology
journal homepage: www.elsevier .com/locate / foodchemtox
Author's personal copy
Agarwal, 2000). In addition, flavonoids interact with receptors, en-zymes, and kinases in their molecular levels (Gamet-Payrastreet al., 1999; Collins-Burow et al., 2000; Manthey et al., 2001; Jacob-son et al., 2002).
Naringin is a kind of bioflavonoid derived from grapefruit andrelated citrus species (Jagetia and Reddy, 2002). Naringin or itsmetabolite naringenin has been reported to possess diverse biolog-ical and pharmacological properties including anticarcinogenic (Soet al., 1996), lipid-lowering (Jeon et al., 2004), superoxide scaveng-ing (Rajadurai et al., 2009), anti-apoptotic (Kim et al., 2009), anti-atherogenic (Choe et al., 2001), metal chelating (Jagetia et al.,2003) and antioxidant activities (Jagetia and Reddy, 2005). Re-cently, growing evidence has indicated that naringin or naringenindisplays anti-inflammatory effects both in in vitro and in vivo sys-tems (Kanno et al., 2006; Amaro et al., 2009; Lee et al., 2009; Ribe-iro et al., 2009; Shi et al., 2009). Fig. 1 depicted the structure ofnaringin. In recent years, experiments have been performed onsupplementation with natural flavanones, especially compoundsfound in vegetables, fruits and medicinal plants, have receivedincreasing attention for their potential role in prevention of cancer(Szliszka and Krol, 2011). Therefore, this study will be designed toinvestigate the effects of flavanones on the cell viability, oxidativestress, and expression of caspases, and p53 in SiHa cancer cells. Inthe present study, naringin was shown to kill human cervical can-cer cells preferentially. Apart from inhibiting cell proliferation,naringin was also found to induce apoptosis in human cancer cellsthrough both death receptor and mitochondrial pathways. The re-sults are significant as they provide new insights in understandingthe molecular mechanism of naringin that might be a potent che-motherapeutic agent for the treatment of human cervical cancer.
2. Materials and methods
2.1. Materials
Naringin, propidium iodide, (PI), acridine orange and ethidium bromide werepurchased from Sigma–Aldrich chemical co. (St. Louis, MO, USA). 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 5,50 ,6,60-tetrachloro-1,10 ,3,30-tetraethylbenzimidazolylcarbocyanineiodide (JC-1) and Dimethylsulfoxide(DMSO), Dulbecco’s Modified Eagle’s Medium (DMEM) and fetal bovine serum (FBS)were purchased from Invitrogen (Carlsbad, CA, USA). The DeadEnd TUNEL assay kitwas purchased from Promega (Madison, USA). Annexin V-Cy3 apoptosis detectionkit was procured from Abcam (Cambridge, MA, USA). QuantiTect Primer assay, Fast-Lane Cell cDNA Kit, and QuantiFast SYBR Green PCR Kit were obtained from QIAGEN(Germany). All the other chemicals used were of research grade.
2.2. Maintenance of SiHa cells
The SiHa cervical cancer cell line was provided by Prof. M.A. Akbarsha at theMahatma Gandhi-Doerenkamp Center (MGDC) for Alternatives to Use of Animalsin Life Science Education, Bharthidasan University, India. The cell line was main-tained and propagated in 90% Dulbecco’s Modified Eagle’s Medium (DMEM) con-taining 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Cells were
cultured as an adherent monolayer at approximately 70–80% confluence and at37 �C in a humidified atmosphere of 5% CO2. Cells were harvested after brief tryp-sinization. Cells in the exponential growth phase were used in all experiments.
2.3. Preparation of naringin
A stock solution of 20 mg/ml was prepared in DMSO (Sigma, USA). The solutionwas stored in aliquots at �20 �C. Further dilutions were made in DMEM to requiredconcentrations between 250 and 2000 lM for the treatment of SiHa cells.
2.4. Measurement of cell viability
Cell viability was assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltet-razolium bromide (MTT) assay based on the reduction of MTT by mitochondrialdehydrogenases of viable cells to a purple formazon product (Mosmann, 1983).Briefly, SiHa cells were plated at a density of 2.5 � 103 cells/ml in 96-well plates.After overnight growth, cells were treated with a 250–2000 lM of naringin for24/48 h. Subsequently, the cells were washed with 200 lL of PBS, and incubatedwith 100 ll of 500 lg/ml MTT in PBS at 37 �C for 3 h. The MTT-formazon productdissolved in 200 ll of DMSO was estimated by measuring the absorbance at570 nm in a Bio-Rad multiwell plate reader and the IC50 was calculated. Quadrupli-cate samples were run for each concentration of naringin in three independentexperiments. The cell viability of SiHa cell line was expressed as the percent viabil-ity of treated cells compared with the untreated control.
2.5. Analysis of mitochondrial transmembrane potential
Changes in the mitochondrial transmembrane potential (DwM) were deter-mined using JC-1, a fluorescent carbocyanine dye, which accumulates in the mito-chondrial membrane as a monomer or dimer depending on the mitochondrialmembrane potential (Smiley et al., 1991). Briefly, cells were plated at a seeding den-sity of 5 � 104 cells/well in a 24-well plate. After 24 h of treatment with IC50
(750 lM) of naringin, cells were incubated with 2.5 lM JC-1 for 15 min at 37 �Cin darkness. Subsequently, stained cells were washed with PBS, followed by FACSanalysis (BD FACSCantoTM II, San Jose, CA, USA). The presence of JC-1 monomersor dimers was examined under a fluorescent microscope using filter pairs of530 nm/590 nm (dimers) and 485 nm/538 nm (monomers).
2.6. Detection of apoptosis by fluorescence microscopy
The nuclear morphology was analyzed by treatment of SiHa cells with (750 lM)naringin for 24 h. Control cells were grown in the same manner in absence of narin-gin. Cells were trypsinized and fixed with methanol. Then, cell nuclei were stainedby treatment with 1 mg/mL Propidium Iodide (Sigma) at 37 �C for 15 min in thedark. Characteristic apoptotic morphological changes were determined by acridineorange/ethidium bromide (AO/EB) staining as described by Leite et al. (1999).Stained cells were examined under a fluorescence inverted microscope (Carl Zeiss,Jena, Germany).
2.7. Analysis of the cell cycle distribution
SiHa cells were plated at 2 � 105 cells/ml in a six-well plate. After a 24 h incu-bation (37 �C, 5% CO2), the cells were treated with naringin prepared in serum-freemedia and incubated for an additional 24 h. The IC50 determined from the cytotox-icity assay for each treatment was used in the cell cycle assay. The cell cycle phaseevaluation was performed as described by Grassi et al. (2007). Following trypsiniza-tion, cells were centrifuged at 1000 � g for 10 min, and the pellet was resuspendedin 0.5 ml of PBS. Fixation was completed by adding 4.5 ml of 70% cold ethanol for atleast 2 h. The fixed cells were centrifuged at 1000g for 10 min, and the pellet sus-pended in 5 ml of PBS. After 60 s, the cells were centrifuged as before, and the pelletresuspended in 1 ml of propidium iodide (PI) staining solution. Immediately after15 min incubation at 37 �C, the cells were analyzed to determine the cell cycle stageusing flow cytometry (BD FACSCantoTM II, San Jose, CA, USA) with an excitationwavelength of 488 nm and an emission at 670 nm. The data presented are represen-tative of those obtained in at least three independent experiments conducted intriplicate.
2.8. Annexin V-Cy3 assay
The phosphatidylserine-binding protein Annexin V was conjugated to Cy3 usingthe commercially available Annexin V-Cy3 apoptosis detection kit with SYTOX (Ab-cam, Cambridge, MA, USA) according to the manufacturer’s protocol. To examinewhether cell death occurred via apoptosis or necrosis, SYTOX was used, which beinga non permeable stain having affinity towards nucleic acids, selectively enters ne-crotic or late apoptotic cells. Therefore, double-staining of Ann-Cy3 and SYTOXhelps discriminate between live cells (SYTOX and Annexin V negative), cells in earlyapoptosis (Annexin V positive, SYTOX negative), cells undergoing late apoptosis(both Annexin V and SYTOX positive) and necrotic cells (SYTOX positive, AnnexinV negative). SiHa cells were plated in 25-cm2 tissue culture flasks at 2 � 105 cells
Fig. 1. Molecular structure of naringin. Naringin [7-(2-O-(6-deoxy-alpha-L-man-nopyranosyl)-beta-D-glucopyranosyloxy)-2,3-dihydro-40 ,5,7-trihydroxyflavone]CAS: 10236–47-2. Molecular formula: C27H32O14; molecular weight: 580.50.
98 E. Ramesh, A.A. Alshatwi / Food and Chemical Toxicology 51 (2013) 97–105
Author's personal copy
per flask, incubated overnight, and treated with naringin (750 lM) or DMSO controlfor 24 h. At the end of incubation, treatment and control media were collected sincethey could contain dead or dying cells that had detached from the flasks. Adherentcells were collected by trypsinization and added to the non-adherent population.Briefly, the cells were washed and pellet was suspended in binding buffer with dou-ble-label staining solution (Ann-Cy3 and SYTOX) and then incubated at room tem-perature in the dark for 5 min. After the incubation, excess label was removed bywashing the cells with PBS buffer. Cells were subject to FACS analysis (BD FACSCan-toTM II, San Jose, CA, USA) within 30 min of Ann-Cy3 and SYTOX staining. A mini-mum of 10,000 events was collected for each sample. Quantification of cytotoxicitywas determined by the percentage of pre-apoptotic, apoptotic and necrotic or deadtarget cells scored by FACS analysis.
2.9. Terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling assay
Terminal deoxynucleotidyl transferase–mediated dUTP end labeling (TUNEL)assay was performed to visualize apoptotic cells. Ten thousand cells were seededinto the wells of permanox chamber slides (Thermo Scientific Nunc, Rochester,NY, USA). The next day, the media were exchanged for fresh DMEM medium. Thecells were treated with naringin for 24 h and untreated cells as negative controls.Cells were fixed with 4% methanol-free paraformaldehyde in PBS for 10 min atroom temperature. After fixation, wells were washed with PBS and underwent per-meabilization with a 0.2% Triton X-100 solution for 5 min, washed twice in phos-phate-buffered saline and then added with 100 ll of equilibration buffer at roomtemperature for 5–10 min. Samples were washed with PBS and then incubated withTerminal Deoxynucleotidyl Transferase, Recombinant (rTdT) buffer at 37 �C for60 min inside the humidified chamber according to the manufacturer’s protocol(Promega, Madison, USA). Termination of the reaction was done by adding 2XSSC for 15 min. The wells were washed thrice, by using PBS, for 5 min to removeunincorporated fluorescein-12-dUTP nucleotides. Detection of fragmented DNAwas examined under a fluorescence inverted microscope (Carl Zeiss, Jena, Ger-many). For each sample, the total number of cells and the number of TUNEL-posi-tive cells were quantified in 10 representative fields. The results are presented as arepresentation from a series of three separate experiments.
2.10. Real-time quantitative PCR analysis
The expression of apoptotic genes was analyzed by reverse transcription-PCR(RT-PCR; Applied Biosystems 7500 Fast, Foster City, CA) using a real-time SYBRGreen/ROX gene expression assay kit (QIAGEN). The cDNA was directly preparedfrom cultured cells using a Fastlane� Cell cDNA kit (QIAGEN, Germany), and themRNA levels of Caspase -3, -8, and - 9, p53, Bax, Fas and FADD as well as the refer-ence gene, GAPDH, were assayed using gene-specific SYBR Green-based Quanti-Tect� Primer assays (QIAGEN, Germany). Quantitative real-time RT-PCR wasperformed in a reaction volume of 25 lL according to the manufacturer’s instruc-tions. Briefly, 12.5 lL of master mix, 2.5 lL of primer assay (10�) and 10 lL of tem-plate cDNA (100 lg) were added to each well. After a brief centrifugation, the PCRplate was subjected to 35 cycles under the following conditions: (i) PCR activationat 95 �C for 5 min, (ii) denaturation at 95 �C for 5 s and (iii) annealing/extension at60 �C for 10 s. All samples and controls were run in triplicates on an ABI 7500 FastReal-time PCR system. The quantitative RT-PCR data was analyzed by a comparativethreshold (Ct) method, and the fold inductions of samples were compared with theuntreated samples. GAPDH was used as an internal reference gene to normalize theexpression of the apoptotic genes. The Ct cycle was used to determine the expres-sion level in control cells and SiHa cells treated with naringin for 24 and 48 h. Thegene expression level was then calculated as described by Yuan et al. (2006). Theresults were expressed as the ratio of reference gene to target gene by using the fol-lowing formula: DCt = Ct (apoptotic genes)-Ct (GAPDH). To determine the relativeexpression levels, the following formula was used: DDCt = DCt (treated) � DCt(control). Thus, the expression levels were expressed as n-fold differences relativeto the calibrator. The value was used to plot the expression of apoptotic genes usingthe expression of 2�DDCt.
2.11. Statistical analysis
Results are expressed as mean ± standard deviation (SD). All data were derivedfrom at least three independent experiments with a similar pattern. The data wereanalyzed by one-way ANOVA using the SPSS (Version 11.5; SPSS Inc., Chicago, IL,USA) followed by Dunnett’s multiple comparison test. For all comparisons, differ-ences were considered statistically significant at p < 0.05.
3. Results
3.1. Naringin inhibited cell profileration
The effects of naringin on cell proliferation were measured withthe MTT assay, using human cervical cancer SiHa cells were ex-
posed to different concentrations of naringin (0–2000 lM) for24 h. Naringin did not affect cell proliferation at low concentra-tions, but the cell proliferation were first exhibited as inhibited cellproliferation at 250 lM after 24 h of naringin treatment. At highconcentrations of 2000 lM, naringin markedly decreased cell pro-liferation in a dose-dependent manner, with an IC50 value of750 lM (Fig. 2). Thus, the IC50 value was used in subsequentexperiments.
3.2. Role of mitochondria in naringin-induced cell death
Analysis of DwM using JC-1 dye revealed a shift in fluorescencefrom reddish-orange to greenish-yellow in SiHa cells treated withnaringin indicating depolarization of the mitochondrial membranepotential (Fig. 3A).
3.3. Naringin disruption of mitochondrial membrane potential
Collapses in DwM are the critical steps in the mitochondrialapoptotic pathway. In this study, the effects of naringin on DwMwere explored using a mitochondrial specific dye, JC-1. Using flowcytometric analysis, a shift in the cell population with JC-1 aggre-gates to that with JC-1 monomers was observed in cells undernaringin treatment, indicating mitochondrial membrane depolar-ization in cells with naringin treatment (Fig. 3B).
3.4. Naringin induces apoptosis in SiHa cells
Treatment of SiHa cells with naringin for 24 h resulted in signif-icant chromosomal condensation, internucleosomal fragmentationand morphological changes, indicating that the cytotoxic action ofnaringin induces apoptosis (Fig. 4).
3.5. Naringin arrest SiHa cells at G2/M phase of cell cycle
To test whether naringin could affect the cell cycle of SiHa cells,cells treated with DMSO or naringin (IC50) for 24 h were subjectedto flow cytometric analysis after DNA staining. As shown in Fig. 5Aand B, the exposure of SiHa cells to the growth-suppressive con-centrations of naringin resulted in evidently increase in the pro-portion of cells in the G2/M and Sub-G0 phases, which wasaccompanied by a significant decrease in cells in the G0/G1 phase.However, there was slight increase in the proportion of cells in Sphase on SiHa cells treated with naringin for 24 h when comparedto control.
Fig. 2. The IC50 determination for naringin against the SiHa human cervical cancercell line. Effect of naringin treatment on SiHa cell viability determined by MTTassay. Data are presented as mean ± SD of three determinations each performed intriplicate.
E. Ramesh, A.A. Alshatwi / Food and Chemical Toxicology 51 (2013) 97–105 99
Author's personal copy
3.6. Apoptosis induction by Naringin in SiHa cells
TUNEL assay confirmed the presence of SiHa cells in terminalapoptotic stages in naringin treated cells (Fig. 6A and B). Increasedstaining intensity indicates the degree of DNA damage induced bynaringin. The results confirm naringin treated cancer cells areundergoing programmed cell death by apoptosis. Quantificationof apoptotic cell death by annexin V-SYTOX assay indicated in-crease in the proportion of early and late apoptotic cells in naringintreated cells (8.74% and 37.15%) compared to control (1.2% and4.8%) respectively (Fig. 6C).
3.7. Quantization of mRNA levels of apoptosis-related genes
To investigate the molecular mechanism of naringin-inducedapoptosis in SiHa cells, the expression levels of several apoptosis-related genes were examined. The relative quantitation of cas-pase-3, -8, and -9, p53, Bax, Fas and FADD mRNA expression wasperformed by SYBR Green-based quantitative real-time PCR (RT–PCR) using a 7500 Fast Real Time System (Applied Biosystem).Fig. 7 summarizes the gene expression changes of caspase-3, -8,and -9. p53, Bax, Fas and FADD. A significant increase (p < 0.05)in the expression of caspase-3, and -9, p53, Bax, Fas and FADD inSiHa cells treated with naringin 24 or 48 h compared to untreatedcontrol; however, there is no significant difference between theexpression of caspase-3,-8 and -9, Bax, Fas and FADD in cells trea-
ted with naringin (IC50) for 24 and 48 h. In addition, the expressionof p53 was significant increase (p < 0.05) in SiHa cells treated withnaringin for 24 and 48 h, however, no significant difference wasobserved between the expression of caspase-8 in cells treated withnaringin for 24 or 48 h when compared to control. These resultssuggested that naringin is an efficient inducer of apoptosis, maybe involved in both extrinsic and intrinsic pathways.
4. Discussion
Cell death occurs via two major mechanisms: apoptosis ornecrosis. Necrosis involves the death of cells via external damage,mediated by destruction of the plasma membrane or the biochem-ical supports of its integrity. Apoptosis, a form of programmed celldeath, is characterized by cell shrinkage, chromatin condensation;inter nucleosomal DNA fragmentation, and the formation of apop-totic bodies (D’Agostini et al., 2005). Apoptosis is arguably one ofthe most potent forms of defense against cancer (Ghavami et al.,2009). Apoptosis has emerged as an important mechanism bywhich dietary compounds may exhibit chemopreventive potential.
Flavonoids markedly differ from one another in their bioavail-ability and intestinal metabolism. The bioavailability of naringinwas reported 8.8% (based on urinary excretion) at an intake levelof 50 mg aglycone equivalents, reaching plasma concentrations of0.5 lmol/l, which are absorbed after release of the aglycones bythe microflora, reached Cmax at �5.5 h (Manach et al., 2005). Bio-
Fig. 3. (A) Fluorescent microscopy images of control cells and cells treated with naringin. Reddish-orange fluorescence of JC-1 dimers is seen in cell areas with highmitochondrial membrane potential, while green fluorescence of JC-monomer is prevalent in cell areas with low mitochondrial membrane potential. (B) Naringin inducedmitochondrial disorganization in SiHa cells. Depolarization of mitochondrial membrane in naringin-treated SiHa cells. Cells were incubated with vehicle only (control) or IC50
of naringin for 24 h. The cells were then incubated with JC-1 dye for staining at 37 �C in dark. Fluorescence emission was measured by flow cytometry. The number at thecorner indicates the percentage of cells with depolarized mitochondrial membrane. The experiments have been repeated at least three times with results similar to the oneshown. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
100 E. Ramesh, A.A. Alshatwi / Food and Chemical Toxicology 51 (2013) 97–105
Author's personal copy
availability is determined by different factors, including the sugarmoiety of the compound and its further metabolism by the gutmicroflora (Manach et al., 2004). The dietary intake of flavonoidsand cancer has been assessed by Linseisen and Rohrmann (2008).Recently, fruits and vegetable consumption have been shown to
be inversely related to cancer mortality (Willett, 2002), and pro-vides the foundation for food based approaches to cancer preven-tion. It has been observed that diet rich in plant-based nutrientsis important in reducing the risk of cervical cancer (Ghosh et al.,2008). In the present study, fruit flavonoid naringin exerted potent
Fig. 4. Apoptotic and nuclear morphological changes in cervical cancer cells treated with narginin evaluated with acridine orange/ethidium bromide (AO/EB) and propidiumIodide (PI) staining. (A) Viable cells stained only by AO were bright green with intact structure; apoptotic cells stained by AOLOW and EB were red-orange with condensation ofchromatin as dense orange areas and reduced cells. (B) PI staining shows apoptotic cells with chromosomal condensation and nuclear fragmentation and viable cells withintact nucleus. (C) Bar-diagram showing the manual morphological counts of viable cells (normal) and nuclear fragmentation (abnormal). Data are represented as mean ± SDof decuplicate. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
E. Ramesh, A.A. Alshatwi / Food and Chemical Toxicology 51 (2013) 97–105 101
Author's personal copy
anti-proliferative effects on SiHa cells in a dose-dependent mannerwith an IC50 value of 750 lM. In addition to growth inhibition,administration of naringin induced morphological features charac-teristic of apoptotic cell death such as increased number of sub-diploid cells, chromatin condensation, intranucleosomal DNA frag-mentation and decline in the mitochondrial transmembrane po-tential. Furthermore, TUNEL confirmed that naringin treatedcancer cells are undergoing programmed cell death by apoptosis.The dead cells as evidenced by microscopic inspection presentedmorphological changes are well-characterized features of celldeath due to apoptosis (Rodriguez and Schaper, 2005). Severalresearchers have documented the cytotoxic and anti-proliferativeeffects of naringin in human neuroblastoma and mouse p388 cells(Kanno et al., 2003; Newhouse et al., 2004; Kim et al., 2009)
In the present study, the exposure of SiHa cells to growth-sup-pressive concentrations of naringin resulted in a significant in-crease in the proportion of cells in the G2/M and Sub-G0 phases,which was accompanied by a decrease of cells in the G0/G1 phase.However, no significant difference was observed in the proportionof cells in the S phase for treated and untreated cells; these find-ings are consistent with a recent report that quercetin can inducecell cycle arrest (Shan et al., 2009). Similarly, cell cycle arrest inthe G2/M phase in colon cancer cells following the administrationof apigenin has also been reported (Wang et al., 2000). The datapresented herein show that naringin induces the G2/M phase ofcell cycle arrest.
Our previous studies shown that catechin hydrate possessesthe induction of apoptosis and increased the expression levels ofpro-apoptotic genes such as caspase-3, -8, and -9 and p53(Alshatwi, 2010; Al-Hazzani and Alshatwi, 2011). Similarly, wealso observed that naringin-induced apoptosis increases theexpression levels of caspase-3, -8, and -9, p53, Bax, Fas and FADDin SiHa cancer cells. Earlier studies have shown that epigallocate-chin gallate (EGCG) increases the mitochondrial membrane poten-tial loss through cytochrome c release and caspase-3, and -9 andalso activated an extrinsic pathway as evident by the time-depen-dent increases in Fas expression and caspase-8 activity (Ahmeda
et al., 2010). There are many mechanisms to induce apoptosis incells. The activation of caspase-3 is an important downstreameffect in the apoptotic pathway (Earnshaw et al., 1999). In addi-tion, the effector caspase-3 and the initiators caspase-8, and -9are the main executors of apoptosis (Riedl and Shi, 2004).Caspase-8 operates in the death-receptor pathway, whereas cas-pase-9 acts in the mitochondrial pathway and both pathwaysshare caspase-3 (Pommier et al., 2004). Once caspase-8 is acti-vated, two distinct downstream pathways have been identifiedfor the activation of apoptosis. In one pathway, caspase-8 directlyprocesses downstream effectors caspase-3, -6, and -7, and in analternative pathway, caspase-8 can activate a cross-talk pathwaybetween the death receptor and the mitochondrial pathways bythe cleavage of Bid to tBid, a pro-apoptotic member of the Bcl-2family. The cleavage of Bid, which is an important pro-apoptoticmember of the Bcl-2 family of proteins, has been shown to beassociated with caspase-8 activation (Malik et al., 2008). Taken to-gether, the data presented in this study suggested that naringininduced apoptosis through the mitochondrial apoptotic pathwaysas evident by increased expression of caspase-3, and -9. Further-more, data presented in this study suggested that the naringin-activated extrinsic pathway was related to the increased expres-sion of caspase-8.
The role of the death receptor-dependent pathway in naringin-induced apoptosis is further supported as both Fas and its adaptorprotein FADD, which were up-regulated by naringin (Fig. 7). Inaddition to that administration of naringin upregulated the expres-sion of pro-apoptotic Bax, and p53 in SiHa cells. Bernard et al. haveshown that HPV status and p53 functionality can influence the sus-ceptibility of staurosporine-induced apoptosis in various cervicalcancer cell lines (Bernard et al., 2001). The dose-dependentapoptotic responses by the human cervical cancer cells are mir-rored by the growth inhibition induced by naringin in SiHa cellline. Therefore, these data suggested that naringin may induceapoptosis through various mechanisms, and naringin-inducedapoptosis occurred partly through its ability to increase the expres-sion of pro-apoptotic genes.
Fig. 5. The effects of naringin on cell cycle distribution. (A) Histogram representing the cellular DNA was stained with PI, and the distribution of the cells in the Sub-G0, G0/G1, S and G2/M phases was assessed with flow cytometry. The experiments have been repeated at least three times with results similar those shown. (B) Bar diagram showingthe distribution of the cells in the Sub-G0, G0/G1, S and G2/M phases in SiHa cells treated with naringin or vehicle control. The data represent the mean ± SD of triplicates.
102 E. Ramesh, A.A. Alshatwi / Food and Chemical Toxicology 51 (2013) 97–105
Author's personal copy
In conclusion, results of the present study provided clearevidence that naringin inhibits the proliferation through cell cyclearrest at the G2/M phase of human cervical SiHa cells and apopto-
sis induction through the disruption of DwM, and the activation ofboth intrinsic and extrinsic pathway. Thus, our findings provide amechanistic framework for further exploration of the use of
Fig. 6. Induction of apoptosis in SiHa cells by naringin. (A and B) Cells were incubated with vehicle (control) or the IC50 of naringin for 24 h. The blue fluorescence is due toDAPI staining were considered as counterstain, while the green fluorescence is due to FITC staining in nuclei were considered as TUNEL-positive cells. (C) Scatter plots ofAnnexin V-FITC/ SYTOX staining was analyzed using flow cytometry. The numbers represents the percentage of cells in each quadrant. FL1-SYTOX green; FL2-Annexin V-Cy3;LR-early apoptotic cells; UR-late apoptotic cells. The experiments have been repeated at least three times with results similar to the one shown. (For interpretation of thereferences to color in this figure legend, the reader is referred to the web version of this article.)
E. Ramesh, A.A. Alshatwi / Food and Chemical Toxicology 51 (2013) 97–105 103
Author's personal copy
naringin as a promising chemotherapeutic agent for human cervi-cal cancer.
Conflict of Interest
Authors declare that there are no conflicts of interest.
Acknowledgments
We gratefully acknowledge the help of Mr. R. Vishnu Balaji, Col-lege of Medicine, King Saud University, for his technical supportduring flow cytometry. The authors thank the Research Center,Deanship of Scientific Research, College of Food and AgricultureScience, King Saud University, Saudi Arabia, for their generousfunding in support of this research. We also acknowledge theexcellent technical support and critical discussions of Dr. V.S.Periasamy, Department of Food Science and Nutrition, King SaudUniversity.
References
Agarwal, R., 2000. Cell signaling and regulators of cell cycle as molecular targets forprostate cancer prevention by dietary agents. Biochem. Pharmacol. 60, 1051–1059.
Ahmeda, K., Weia, Z., Zhaoa, Q., Nakajimab, N., Matsunagac, T., Ogasawarac, M.,Kondoa, T., 2010. Role of fatty acid chain length on the induction of apoptosis bynewly synthesized catechin derivatives. Chem. Biol. Interact. 185, 182–188.
Al-Hazzani, A.A., Alshatwi, A.A., 2011. Catechin hydrate inhibits proliferation andmediates apoptosis of SiHa human cervical cancer cells. Food Chem. Toxicol. 49,3281–3286.
Alshatwi, A.A., 2010. Catechin hydrate suppresses MCF-7 proliferation throughTP53/caspase-mediated apoptosis. J. Exp. Clin. Cancer Res. 29, 167.
Amaro, M.I., Rocha, J., Vila-Real, H., Eduardo-Figueira, M., Mota-Filipe, H., Sepodes,B., Ribeiro, M.H., 2009. Anti-inflammatory activity of naringin and thebiosynthesized naringenin by naringinase immobilized in microstructuredmaterials in a model of DSS-induced colitis in mice. Food Res. Inter. 42, 1010–1017.
American Cancer Society. 2010. Cancer facts & figures 2010. Atlanta (GA): AmericanCancer Society.
Arts, I.C., 2008. A review of the epidemiological evidence on tea, flavonoids, andlung cancer. J. Nutr. 138, 1561S–1566S.
Bernard, B., Fest, T., Prétet, J.L., Mougin, C., 2001. Staurosporine-induced apoptosis ofHPV positive and negative human cervical cancer cells from different points inthe cell cycle. Cell Death Differ. 8, 234–244.
Choe, S.C., Kim, H.S., Jeong, T.S., Bok, S.H., Park, Y.B., 2001. Naringin has an anti-atherogenic effect with the inhibition of intercellular adhesion molecule-1 inhypercholesterolemic rabbits. J. Cardiovasc. Pharmacol. 38, 947–955.
Collins-Burow, B.M., Burow, M.E., Duong, B.N., McLachlan, J.A., 2000. Estrogenicand antiestrogenic activities of flavonoid phytochemicals through estrogenreceptor binding-dependent and -independent mechanisms. Nutr. Cancer 38,229–244.
D’Agostini, F., Izzotti, A., Balansky, R.M., Bennicelli, C., de Flora, S., 2005. Modulationof apoptosis by cancer chemopreventive agents. Mutat. Res. 591, 173–186.
Earnshaw, W.C., Martins, L.M., Kaufmann, S.H., 1999. Mammalian caspasesstructure, activation, substrates, and functions during apoptosis. Annu. Rev.Biochem. 68, 383–424.
Gamet-Payrastre, L., Manenti, S., Gratacap, M.P., Tulliez, J., Chap, H., Payrastre, B.,1999. Flavonoids and the inhibition of Pkc and Pi 3-kinase. Gen. Pharmacol. 32,279–286.
Ghavami, S., Hashemi, M., Ande, S.R., Yeganeh, B., Xiao, W., Eshraghi, M., Bus, C.J.,Kadkhoda, K., Wiechec, E., Halayko, A.J., Los, M., 2009. Apoptosis and cancer:mutations within caspase genes. Br. Med. J. 46, 497–510.
Ghosh, C., Baker, J.A., Moysich, K.B., Rivera, R., Brasure, J.R., McCann, S.E., 2008.Dietary intakes of selected nutrients and food groups and risk of cervical cancer.Nutr. Cancer 60, 331–341.
Grassi, G., Scaggiante, B., Farra, R., Dapas, B., Agostini, F., Baiz, D., 2007. Theexpression levels of the translational factors eEF1A ½ correlate with cell growthbut not apoptosis in hepatocellular carcinoma cell lines with differentdifferentiation grade. Biochimie 89, 1544–1552.
Jacobson, K.A., Moro, S., Manthey, J.A., West, P.L., Ji, X.D., 2002. Interactions offlavones and other phytochemicals with adenosine receptors. Adv. Exp. Med.Biol. 505, 163–171.
Jagetia, G.C., Reddy, T.K., 2002. The grapefruit flavonone naringin protects againstthe radiation-induced genomic instability in the mice bone marrow: amicronucleus study. Mutat. Res. 519, 37–48.
Jagetia, G.C., Reddy, T.K., 2005. Modulation of radiation induced alteration in theantioxidant status of mice by naringin. Life Sci. 77, 780–794.
Jagetia, G.C., Venkatesha, V.A., Reddy, T.K., 2003. Naringin, a citrus flavonone,protects against radiation-induced chromosome damage in mouse bonemarrow. Mutagenesis 18, 337–343.
Jeon, S.M., Park, Y.B., Choi, M.S., 2004. Antihypercholesterolaemic property ofnaringin alters plasma and tissue lipids, cholesterol-regulating enzymes, fecalsterol and tissue morphology in rabbits. Clin. Nutr. 23, 1025–1034.
Johnson, J.J., Bailey, H.H., Mukhtar, H., 2010. Green tea polyphenols for prostatecancer chemoprevention: a translational perspective. Phytomedicine 17, 3–13.
Kanno, S., Shouji, A., Asou, K., Ishikawa, M., 2003. Effects of naringin on hydrogenperoxide-induced cytotoxicity and apoptosis in P388 cells. J. Pharmacol. Sci. 92,166–170.
Kanno, S., Shouji, A., Tomizawa, A., Hiura, T., Osanai, Y., Ujibe, M., Obara, Y.,Nakahata, N., Ishikawa, M., 2006. Inhibitory effect of naringin onlipopolysaccharide-induced endotoxin shock in mice and nitric oxideproduction in RAW264 7 macrophages. Life Sci. 78, 673–681.
Kim, H.G., Song, H., Yoon, D.H., Song, B.W., Park, S.M., Sung, G.H., Cho, J.Y., Park, H.I.,Choi, S., Song, W.O., Hwang, K.C., Kim, T.W., 2010. Cordyceps pruinosa extractsinduce apoptosis of HeLa cells by a caspase dependent pathway. J.Ethnopharmacol. 128, 342–351.
Kim, H.J., Song, J.Y., Park, H.J., Park, H.K., Yun, D.H., Chung, J.H., 2009. Naringinprotects against rotenone-induced apoptosis in human neuroblastoma SH-SY5YCells. Korean. J. Physiol. Pharmacol. 13, 281–285.
Kroon, P., Williamson, G., 2005. Polyphenols: dietary components with establishedbenefits to health? J. Sci. Food Agric. 85, 1239–1240.
Kuntz, S., Wenzel, U., Daniel, H., 1999. Comparative analysis of the effects offlavonoids on proliferation, cytotoxicity, and apoptosis in human colon cancercell lines. Eur. J. Nutr. 38, 133–142.
Lee, E.J., Kim, D.I., Kim, W.J., Moon, S.K., 2009. Naringin inhibits matrixmetalloproteinase-9 expression and AKT phosphorylation in tumor necrosisfactor-alpha-induced vascular smooth muscle cells. Mol. Nutr. Food Res. 53,1582–1591.
Leite, M., Quinta-Costa, M., Leite, P.S., Guimarães, J.E., 1999. Critical evaluation oftechniques to detect and measure cell death–study in a model of UV radiationof the leukaemic cell line HL60. Anal. Cell. Pathol. 19, 139–151.
Li, H.N., Nie, F.F., Liu, W., Dai, Q.S., Lu, N., Qi, Q., Li, Z.Y., You, Q.D., Guo, Q.L., 2009.Apoptosis induction of oroxylin A in human cervical cancer HeLa cell linein vitro and in vivo. Toxicology 257, 80–85.
Fig. 7. Effect of naringin on markers of apoptosis related genes in SiHa cells. Comparison of expression level changes are expressed as fold changes (in ratio of target:reference gene, GAPDH) in SiHa cells after 24/48 h exposure of naringin (IC50) Statistical analyses (⁄ and # represents significance at p < 0.05). ⁄, control vs 24/48 h; #, 24 vs48 h.
104 E. Ramesh, A.A. Alshatwi / Food and Chemical Toxicology 51 (2013) 97–105
Author's personal copy
Linseisen, J., Rohrmann, S., 2008. Biomarkers of dietary intake of flavonoids andphenolic acids for studying diet-cancer relationship in humans. Eur. J. Nutr. 47,60–68.
Malik, F., Kumar, A., Bhushan, S., Khan, S., Bhatia, A., Suri, K.A., Qazi, G.N., Singh, J.,2008. Reactive oxygen species generation and mitochondrial dysfunction in theapoptotic cell death of human myeloid leukemia HL-60 cells by a dietarycompound withaferin A with concomitant protection by N-acetyl-cysteine.Apoptosis 12, 2115–2133.
Manach, C., Scalbert, A., Morand, C., Remesy, C., Jimenez, L., 2004. Polyphenols: foodsources and bioavailability. Am. J. Clin. Nutr. 79, 727–747.
Manach, C., Williamson, G., Morand, C., Scalbert, A., Remesy, C., 2005. Bioavailabilityand bioefficacy of polyphenols in humans. I. Review of 97 bioavailabilitystudies. Am. J. Clin. Nutr. 81, 230S–242S.
Mandal, S.K., Biswas, R., Bhattacharyya, S.S., Paul, S., Dutta, S., Pathak, S., Khuda-Bukhsh, A.R., 2010. Lycopodine from Lycopodium clavatum extract inhibitsproliferation of HeLa cells through induction of apoptosis via caspase-3activation. Eur. J. Pharmacol. 626, 115–122.
Manthey, J.A., Grohmann, K., Guthrie, N., 2001. Biological properties of citrusflavonoids pertaining to cancer and inflammation. Curr. Med. Chem. 8, 135–153.
Miller, E.G., Peacock, J.J., Bourland, T.C., Taylor, S.E., Wright, J.M., Patil, B.S., Miller,E.G., 2008. Inhibition of oral carcinogenesis by citrus flavonoids. Nutr. Cancer.60, 69–74.
Mosmann, T., 1983. Rapid colorimetric assay for cellular growth and survival:application to proliferation and cytotoxicity assays. J. Immunol. Methods 65,55–63.
Newhouse, K., Hsuan, S.L., Chang, S.H., Cai, B., Wang, Y., Xia, Z., 2004. Rotenone-induced apoptosis is mediated by p38 and JNK MAP kinases in humandopaminergic SH-SY5Y cells. Toxicol. Sci. 79, 137–146.
Park, O.J., Surh, Y.J., 2004. Chemopreventive potential of epigallocatechin gallateand genistein: evidence from epidemiological and laboratory studies. Toxicol.Lett. 150, 43–56.
Pommier, Y., Sordet, O., Antony, S., Haywrd, R.L., Kohn, K.W., 2004. Apoptosis defectsand chemotherapy resistance. molecular interaction maps and networks.Oncogene 23, 2934–2949.
Rajadurai, M., Stanely, P., Prince, M., 2009. Naringin ameliorates mitochondrial lipidperoxides, antioxidants and lipids in isoproterenol-induced myocardialinfarction in Wistar rats. Phytother. Res. 23, 358–362.
Ramos, S., 2007. Effects of dietary flavonoids on apoptotic pathways related tocancer chemoprevention. J. Nutr. Biochem. 18, 427–442.
Ren, G., Zhao, Y.P., Yang, L., Fu, C.X., 2008. Anti-proliferative effect of clitocine fromthe mushroom Leucopaxillus giganteus on human cervical cancer HeLa cells byinducing apoptosis. Cancer Lett. 262, 190–200.
Ribeiro, M., Barateiro, A., Vila-Real, H., Fernandes, A., Brites, D., 2009. Anti-inflammatory effect of naringin and naringenin on TNF-a secretion incultured cortical astrocytes after stimulation with LPS. New Biotech. 25, S10–S11.
Riedl, S.J., Shi, Y., 2004. Molecular mechanisms of caspase regulation duringapoptosis. Nat. Rev. Mol. Cell Biol. 5, 897–907.
Rodriguez, M., Schaper, J., 2005. Apoptosis: measurement and technical issues. J.Mol. Cell. Cardiol. 38, 15–20.
Shan, B.E., Wang, M.X., Li, R.G., 2009. Quercetin inhibit human sw480 colon cancergrowth in association with inhibition of cyclin d1 and surviving expressionthrough wnt/b-catenin signaling pathway. Cancer Invest. 27, 604–612.
Shi, Y., Dai, J., Liu, H., Li, R.R., Sun, P.L., Du, Q., Pang, L.L., Chen, Z., Yin, K.S., 2009.Naringenin inhibits allergen-induced airway inflammation and airwayresponsiveness and inhibits NF-jB activity in a murine model of asthma. Can.J. Physiol. Pharmacol. 87, 729–735.
Smiley, S.T., Reers, M., Mollola-Hartsorn, C., Lin, M., Chen, A., Smith, T.W., 1991.Intracellular heterogeneity in mitochondrial membrane potentials revealed by aJ-aggregate-forming lipophilic cation JC-1. Proc. Natl. Acad. Sci. 88, 3671–3675.
So, F.V., Guthrie, N., Chambers, A.F., Moussa, M., Carroll, K.K., 1996. Inhibition ofhuman breast cancer cell proliferation and delay of mammary tumorigenesis byflavonoids and citrus juices. Nutr. Cancer 26, 167–181.
Surh, Y.J., 2003. Cancer chemoprevention with dietary phytochemicals. Nat. Rev.Cancer 3, 768–780.
Szliszka, E., Krol, W., 2011. The role of dietary polyphenols in tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-induced apoptosis for cancerchemoprevention. Eur. J. Cancer Prev. 20, 63–69.
Thomas, G.M., 1999. Improved treatment for cervical cancer-concurrentchemotherapy and radiotherapy. N. Engl. J. Med. 340, 1198–1200.
Wang, W., Heideman, L., Chung, C.S., Pelling, J.C., Koehler, K.J., Birt, D.F., 2000. Cell-cycle arrest at G2/M and growth inhibition by apigenin in human coloncarcinoma cell lines. Mol. Carcinog. 28, 102–110.
WHO/ICO, 2007. Summary report on HPV and cervical cancer statistics in SaudiArabia.4, 3–12.
Willett, W.C., 2002. Balancing life-style and genomics research for diseaseprevention. Science 296, 695–698.
Yuan, J.S., Reed, A., Chen, F., Stewart Jr., C.N., 2006. Statistical analysis of real-timePCR data. BMC Bioinformatics 7, 85.
E. Ramesh, A.A. Alshatwi / Food and Chemical Toxicology 51 (2013) 97–105 105
Article
Hypermethylation of P15, P16, andE-cadherin genes in ovarian cancer
Said S. Moselhy1,2, Taha A. Kumosani1,3, I.H. Kamal1,2,J.A. Jalal1,4, Hassan S. Abdul Jabaar5 and Ashraf Dalol6
AbstractBoth p16 and p15 proteins are inhibitors of cyclin-dependent kinases that prevent the cell going through theG1/S phase transaction. E-cadherin is a transmembrane glycoprotein that mediates calcium-dependentinteractions between adjacent epithelial cells. Two groups of patients were selected: the first group sufferedfrom epithelial serous ovarian tumors and the second group suffered from benign ovarian lesions; ovariantissue samples from all the subjects (benign and malignant) were subjected to methylation-specific polymerasechain reaction for methylated and unmethylated alleles of the genes (E-cadherin, p15, and p16). Resultsobtained showed that aberrant methylation of p15 and p16 genes were detected in 64.29 and 50% of ovariancancer patients, while E-cadherin hypermethylation was detected in 78.57% of ovarian cancer patients. Methy-lation of E-cadherin was significantly correlated with different stage of disease (p < 0.05). It was found that therisk of E-cadherin hypermethylation was 1.347-fold, while risk of p15 hypermethylation was 1.543-fold and p16was 1.2-fold among patients with ovarian cancer than that among patients with benign ovarian lesions. In con-clusion, Dysfunction of the cell cycle and/or the cell–cell adhesion molecule plays a role in the pathogenesis ofovarian cancer and that the analysis of the methylation of p15 and E-cadherin genes can provide clinicallyimportant evidence on which to base the treatment.
KeywordsHypermethylation, P15, E-cadherin, ovarian cancer
Introduction
Epithelial ovarian cancer causes more deaths than any
other female reproductive organ cancer, with an
estimated 23,100 new cases of ovarian cancer and
about 14,000 deaths (Bishop, 1987). Because this
cancer strikes silently, revealing no obvious
symptoms until late in its course, most ovarian cancer
cases are diagnosed in the late stages. A better under-
standing of advanced ovarian cancer may help
improve the long-term outcome for patients diag-
nosed in the late stages (Fearon, 1997). DNA
methylation plays an important role in tissue- and
stage-specific gene regulation, genomic imprinting,
and X-chromosome inactivation (Look, 1997). Previ-
ous studies revealed that the silencing of tumor
suppressor genes by promoter hypermethylation is a
common feature in human cancer (Stanbridge,
1990). Changes in methylation levels as well as mod-
ifications in methylation patterns have been observed
in cancer cells (Eshleman and Markowitz, 1996).
CpG island (CGI) hypermethylation is a frequent epi-
genetic event in cancer and is known to be associated
with gene silencing in cancer (Bird, 1986).
1 Biochemistry Department, Faculty of Science, King AbdulazizUniversity, Jeddah, Kingdom of Saudi Arabia2 Biochemistry Department, Faculty of Science, Ain ShamsUniversity, Cairo, Egypt3 Experimental Biochemistry Unit, King Fahad Medical ResearchCenter, King Abdulaziz University, Jeddah, Kingdom of Saudi Arabia4 Pharmaceutical Chemistry Unit, King Fahad Medical ResearchCenter, King Abdulaziz University, Jeddah, Kingdom of Saudi Arabia5 Gynecology Department, Faculty of Medicine, King AbdulazizUniversity, Jeddah, Kingdom of Saudi Arabia6 Excellence center of human Genome, King Fahad MedicalResearch Center, King Abdulaziz University, Jeddah, Kingdom ofSaudi Arabia
Corresponding author:Said S. Moselhy, Biochemistry department, Faculty of Science, AinShams University, Cairo, Egypt.Email: [email protected]
Toxicology and Industrial Health1–7© The Author(s) 2013Reprints and permissions:sagepub.co.uk/journalsPermissions.navDOI: 10.1177/0748233713484657tih.sagepub.com
at King Abdul Aziz University on March 23, 2015tih.sagepub.comDownloaded from
Tumor suppressor genes p15 (CDKN2B and
INK4b) is located at 9p21, which encodes p15
cyclin-dependent kinase inhibitor (Kass et al., 1997).
It was found that inhibition of p15 expression by hyper-
methylation appears to be a common event in human
lymphoid tumors (Razin and Shemer, 1995) and in
mouse T-cell lymphomas (Razin and Cedar, 1994).
E-cadherin is a transmembrane glycoprotein that med-
iates calcium-dependent interactions between adjacent
epithelial cells (Laird and Jaenisch, 1996). Decreased
or loss of E-cadherin expression is a common finding
in many human epithelial cancers including colon and
prostate cancer especially in poorly differentiated,
advanced-stage carcinomas (Herman et al., 1994). It
has been reported that aberrant hypermethylation of
CGIs in the E-cadherin promoter region (Lengauer
et al., 1997), together with alterations in chromatin
structure and transcription factor activity may conspire
to suppress E-cadherin expression. However, in most
cancers with reduced or absent E-cadherin gene and
protein expression, mutations in E-cadherin are rarely
detected (Lee et al., 1994).
Both p16 and p15 proteins are inhibitors of cyclin-
dependent kinases that prevent the cell going through
the G1/S phase transaction. Inactivation of p16 and
p15 genes is the important step in cancer development
(Ushijima, 2005). Downregulation of p16 and p15
gene expression is found in many cancers (Fodde and
Brabletz, 2007). We have previously reported a high
frequency (48%) of p16 downregulation in a study
of 225 head and neck squamous cell carcinomas.
Transcriptional silencing of these inhibitors is
frequently associated with the methylation of 50 CGIs
(Mirza et al., 2007). Methylation of p16 has been
found in precancerous oral dysplastic tissues, adjacent
non-neoplastic areas of gastric carcinoma, and ulcera-
tive colitis (Clevers, 2006). The role of p15 is less
well documented. Methylated p16 is present in bron-
chial epithelia before the clinical evidence of lung
cancer in chronic smokers (Gil and Peters, 2006). It
therefore suggests the potential of using methylated
DNA as a tumor marker in cancer screening, monitor-
ing chemoprevention, and the treatment of cancer.
Apart from examining cells, the other possible source
of detection of these tumor markers is in the absorbed
tumor DNA in peripheral blood. Elevation of cell-free
circulating DNA is observed in cancer patients (Kim
and Sharpless, 2006). The circulating DNA shares
many properties of the cancerous DNA, and it is
suggested that it originates mostly from the cancerous
tissues (Yeh et al., 2003). By using the methylation-
specific polymerase chain reaction (PCR; MSP),
several methylated genes have been detected in the
peripheral blood of patients with head and neck, liver,
lung, oesophageal, gastric and colorectal cancers (Lin
et al., 2004).
DNA methylation plays an important role in the
gene expression, and it occurs almost exclusively in the
context of CpG dinucleotides in the form of a covalent
attachment of a methyl residue to the cytosine residue.
The DNA methylation is an important epigenetic bio-
marker to cancer. CGIs are the regions with an elevated
GC content and a high frequency of CpG dinuleotides,
which overlap the promoter region of 60–70% of all
human genes. Hypermethylation of CGIs at gene
promoters is mostly associated with gene silencing.
The aim of this work is to use MSP to identify
promoter region hypermethylation changes in the
tumor suppressor genes P15 and P16 and a cell adhe-
sion molecule gene (E-cadherin) in ovarian cancer
in comparison with benign tumors and to examine the
correlation between methylation of these three genes
and the increased risk of ovarian cancer. This may
help in understanding the mechanism of carcinogen-
esis and the rapid distant spread of this cancer.
Subjects and methods
Subjects
The protocol was approved by the university ethical
committee after obtaining fully informed consent
from all subjects studied. This study was conducted
on 50 formalin-impeded paraffin-embedded ovarian
tissue samples obtained from two groups of patients;
the first group includes 18 female patients whose age
ranged from 25 to 70 years with mean + SD (52.30
+ 12.10) and diagnosed as having epithelial serous
ovarian tumor (cystadenocarcinoma). Pathological
classification was assessed according to the World
Health Organization criteria, and the tumor stage was
established according to the Federation International
Gynecological Oncologists (FIGO) classification, and
the second group includes 32 female patients whose
age ranged from 18 to 55 years with mean + SD
(35.7 + 12.0) and diagnosed as having benign ovarian
lesions (fibroma, mucinous, teratoma and serous cyst).
Reagents
The following reagents were used during the experi-
ment: TaqMan 2X Universal PCR Master Mix, No
EmpErase1; TaqMan Probe with TAMRA quencher
2 Toxicology and Industrial Health
at King Abdul Aziz University on March 23, 2015tih.sagepub.comDownloaded from
(Bioline Reagents Ltd London,UK) for a gene of choice;
sequence detection primers for the gene of choice.
Methods
DNA extraction. DNA was extracted from 100 mg of
frozen tissue specimens and placental tissue control
by overnight digestion with 20 mg/ml proteinase K
at 37�C, followed by phenol/chloroform/isoamyl
alcohol (25:24:1) extraction and ethanol precipitation
according to the method of Muller et al. (2003).
Bisulfite modification. It was carried out according to the
method of Niwa et al. (2000) with few modifications.
Briefly, DNA (1 mg) in a volume of 50 ml was
denatured by NaOH (final concentration, 0.3 M) for
30 min at 50�C. To this mixture, sodium bisulfite
(3.9 M; 600 ml) and hydroquinone mixture (10 mM;
pH 5) were added and the samples were incubated
at 95�C for 2 h. Modified DNA was purified using the
Wizard DNA purification resin according to the man-
ufacturer’s protocol (Promega, Ireland) and eluted
into 50 ml of water. Modification was completed by
NaOH (final concentration, 0.3 M) treatment for 5 min
at room temperature, followed by ethanol precipita-
tion. DNA was resuspended in water and used imme-
diately or stored at 20�C.
Methylation-specific PCR. DNA methylation of CGIs
was determined by PCR using specific primers for
both methylated and unmethylated DNA. Two sets
of primers designed according to Osborne et al.
(1980) were used to amplify each region of interest:
one pair recognized a sequence in which CpG sites are
unmethylated (bisulfite modified to UpG) and the
other recognized a sequence in which CpG sites are
methylated (unmodified by bisulfite treatment).
Negative control samples without DNA were
included for each set of PCR. The final volume of
50 ml of PCR mixture contained l� PCR buffer
(50 mM KCl, 2 mM MgCl2, and 10 mM Tris-HCl;
pH 9.0 at room temperature), dNTPs mixture
(10 mM), primers (300 ng each per reaction), and
bisulfite-modified DNA (50 ng) or unmodified DNA
(50–100 ng). Amplification was carried out in Biome-
tra Uno II thermocycler (Biometra GmbH, Goettin-
gen, Germany) for 35 cycles (1 min at 95�C, 1 min
at 60�C for p15 and at 55�C for E-cadherin, and 1 min
at 72�C), followed by a final 7 min extension at 72�C.
For negative control, placental DNA was modified
using sodium bisulfite and then one PCR reaction was
preformed using the sets of primers for unmethylated
E-cadherin and another reaction was performed using
the sets of primers for unmethylated p15.
Methylation of placental DNA using S1s1 methyltransferase.For positive control, placental DNA was treated by
CpG methylase enzyme (New England biolabs Inc.,
Beverly, Massachusetts, USA), which methylates all
cytosine residues (C5) within the double-stranded
dinucleotide recognition sequence 50 . . . .CG . . . .30.Treatment was carried out by the addition of 1 mg of
placental DNA to 1 ml of CpG methylase enzyme, 2
ml of S-adenosyl methionine (diluted five times), and
8 ml of NE buffer 2, and then the volume was made-up
to 20 ml with double-distilled water and incubated at
37�C for 2 h. One PCR reaction was preformed using
the sets of primers for methylated E-cadherin and
another reaction was performed using the sets of
primers for methylated p15. PCR products were ana-
lyzed by electrophoresis on mini-gel submarine using
2% agarose gel stained with ethidium bromide and
directly visualized under UV illumination using DNA
documentation system; 100 bp DNA ladder was used.
Statistical analysis
Multiregression analysis was used to search for a
panel (independent parameters) that can predict the
target parameter (dependant variable). Using stepwise
multiregression analysis, the panels can be stored
according to their sensitivity to discriminate; Chi-
square test is used for comparison between two or
more groups regarding proportions or correlations
between two or more variables among a group; and
calculated relative risk ratio measures how many
times the risk is present among the target or diseased
group relative to that of nondiseased group.
Results
The malignant group was classified according to their
clinicopathological data (clinical stages and histologi-
cal grades): eight of 18 (28.57%) were of stages Ι and
ΙΙ, while six of 18 (42.85%) were of stage ΙΙΙ, and four
of 18 (28.57%) were of stage ΙV; also, it was found
that six of 18 (33%) were of grade Ι, six of 18
(33%) were of grade ΙΙ, and six of 18 (33%) belong
to grade ΙΙΙ (Table 1). It was found that promoter
methylation in E-cadherin gene was 18% (eight of
18) and 82% (12 of 32) of malignant and benign tis-
sues, respectively, while promoter methylation in
P15 gene was 26% (10 of 18) and 82% (nine of 14)
Moselhy et al. 3
at King Abdul Aziz University on March 23, 2015tih.sagepub.comDownloaded from
of malignant and benign tissues, respectively. How-
ever, promoter methylation in P16 gene was 87%(14 of 18) and 30% (12 of 32) of benign and malignant
tissues, respectively (Table 2). Association of E-
cadherin methylation with p15 methylation in malig-
nant group showed that eight cases (18%) of serous
epithelial ovarian cancer had methylation in at least
one of the two gene promoters, while eight cases
(57.14%) had methylation in both the gene promoters
and only 24 cases (14.29%) had no methylation in any
of the two gene promoters; while association of E-
cadherin methylation with p15 methylation in benign
group showed that four cases (33.33%) of benign
ovarian lesion had methylation at one of the two gene
promoters, while four cases (33.33%) had methyla-
tion in both the gene promoters, and four cases
(33.33%) had no methylation in any of the two gene
promoters. Association of E-cadherin methylation
with disease progression (stage of disease) showed
that only one case (7.14%) belongs to stage (Ι, ΙΙ),while six cases (42.86%) belonging to stage ΙΙΙ and
four cases (28.57%) belonging to stage ΙV had methy-
lation in E-cadherin promoter. On the other hand, two
cases (14.29%) belong to stage (Ι, ΙΙ), while five cases
(35.71%) belonging to stage ΙΙΙ and two cases
(14.29%) belonging to stage ΙV had methylation in
p15 promoter.
Association of E-cadherin with tumor differentia-
tion (grade of tumor) showed that two cases
(14.29%) belonging to grade Ι, three cases (21.43%)
belonging to grade ΙΙ, and six cases (42.86%) belong-
ing to grade ΙΙΙ had methylation in E-cadherin promo-
ter, while two cases (14.29%) belonging to grade Ι,two cases (14.29%) belonging to grade ΙΙ, and five
cases (35.71%) belonging to grade ΙΙΙ had methylation
of p15 promoter. It was observed that there was a sig-
nificant correlation (p < 0.05) between E-cadherin
methylation and disease progression, while there was
no correlation between E-cadherin methylation and
tumor differentiation (Table 3). It was also found that
p15 methylation showed no significant correlation with
any of E-cadherin methylation, disease progression, or
tumor differentiation. Multiregression analysis showed
significant association between age, E-cadherin and
p15 methylation, and increased risk of ovarian cancer
(p < 0.05), while there was a high significant associa-
tion between age and E-cadherin methylation and inci-
dence of ovarian cancer (p < 0.01; Table 4).
The risk of E-cadherin hypermethylation increases
among patients with ovarian cancer by 1.347-fold
than that among patients with benign ovarian lesions,
while the risk of p15 hypermethylation increases
among patients with ovarian cancer by 1.543-fold
than that among patients with benign ovarian lesions
(Table 5).
Discussion
It seems likely that most ovarian tumors will have
many genes targeted by methylation, a proportion of
which will likely be important in the development
of the tumor. As these genes are not normally methy-
lated in adult tissue (Parkin et al., 2005), this repre-
sents one of the most prevalent tumor-specific
markers yet identified. In addition, numerous studies
have demonstrated that reversal of CGI methylation
can result in reactivation of the associated gene
(Raman et al., 2000a). Consequently, reversal of pro-
moter hypermethylation and resultant re-expression
of tumor suppressor genes represents a very promis-
ing molecular target for developing novel therapies.
In this study, the mean age + SD in malignant
group was 52.30 + 12.10 years, and there was a high
significant correlation between aging and increases
the risk of epithelial ovarian cancer (z ¼ �2.880,
p < 0.01). In the current study, tumor progression
(stage) and pathological classification of tumor
(grade) of malignant group were assessed. With
respect to grading, grades Ι, ΙΙ, and ΙΙΙ tumors consti-
tuted 28.57%, 21.43%, and 50%, respectively, while
stages (Ι, ΙΙ), ΙΙΙ, and ΙV tumors constituted 28.57%,
42.86%, and 28.57%, respectively.
It is known that pregnancy decreases the risk of
ovarian cancer, and multiple pregnancies have an
increasing protective effect against ovarian cancer
(Raman et al., 2000b). In this study, it was found that
women who had not been pregnant before, those who
had been pregnant for one to three times, and those
who had been pregnant more than three times
Table 1. Frequency of clinicopathological data in malignantgroup.
Clinicopathological No (%)
Clinical stagesI, II 8 (28.5%)III 6 (42%)IV 4 (28%)
Histological gradesI 6 (28.5%)II 6 (42%)III 6 (28%)
4 Toxicology and Industrial Health
at King Abdul Aziz University on March 23, 2015tih.sagepub.comDownloaded from
represented 0%, 35.71%, and 64.29%, respectively,
and there was a positive significant correlation
between parity (times of pregnancy) and incidence
of ovarian cancer (p < 0.05).
Previous study reported that the frequent methyla-
tion of E-cadherin gene promoter in ovarian cancer
was 29% (14 of 49) (Rice and Futscher, 2000), while
in the current study, a high incidence of E-cadherin
methylation 78.57% (11 of 14) was observed and only
one case of four (25%) that showed E-cadherin
methylation was found in early stages Ι or ΙΙ of dis-
ease, while 10 of 10 cases (100%) that showed
E-cadherin methylation were in advanced disease
stages ΙΙΙ or ΙV. This data are in line with those of
Sharma et al. (2007) who reported that 26.1% (six
of 23) of invasive ductal breast cancer from Chinese
women demonstrated E-cadherin promoter hyper-
methylation. Hypermethylated promoters of E-
cadherin gene preferentially occurred in the invasive
ductal breast cancers at an advanced stage, three of 18
(16.7%) cases with stage I or II disease showed fewer
molecular alterations in primary tumors than three of
five (60%) cases with stage III or IV disease. Also, a
reduction in E-cadherin expression was observed in
approximately 50% of cervical carcinomas, especially
in more advanced cervical cancers (Shukla et al.,
2006). In this study, nine of 14 cases (64.29%) of ovar-
ian cancer had p15 promoter methylation, and only two
of four cases (50%) showed p15 methylation that were
found in early stage Ι or ΙΙ of disease, while seven of
10 cases (70%) showed p15 methylation that were in
advanced disease stage ΙΙΙ or ΙV.
It was found that p15 and p16 was rarely methy-
lated (1%) in tumor samples and no methylation was
detected in any of normal ovarian tissues, and in
contrast, promoter hypermethylation of p15 (INK4b)
was more common in ovarian cancer (30.8%) than
in normal (5%), but in this study, p15 methylation
showed a relatively high incidence of 64.29% (nine
of 14) among the malignant group and 41.67% (five
of 12) in benign group. In the current study, 28.57%of malignant cases had methylation in one locus,
while 57.14% had methylation of both loci, this
means that methylation may occur in more than one
gene simultaneously and there was no correlation
between E-cadherin, p15 methylation, and increase
risk of ovarian cancer (Singletary et al., 2003).
In spite of this high simultaneous methylation in
these two loci, there was no significant correlation
between E-cadherin methylation and p15 methylation.
Similar results in acute myeloid leukemia (AML)
suggest that these methylation abnormalities may
occur independently. The relationship between p15 and
p16 methylation and E-cadherin methylation seems to
Table 2. Frequency of E-cadherin, P15, and P16 methylation in benign ovarian lesion and ovarian cancer.
Groups
E-cadherin methylation P15 methylation P16 methylation
positive negative positive negative positive negative
Ovarian cancer (n ¼ 32) 8 (18%) 24 (82%) 10 (26%) 22 (74%) 12 (30%) 20 (70%)Benign ovarian lesion (n ¼ 18) 12 (82%) 6 (18%) 12 (82%) 6 (18%) 14 (87%) 4 (13%)
Table 4. Results of stepwise multiregression analysisparameters.
ParametersF ratio p ValueVariables
Parity 1.89 NSP15 1.56 NSP16 8.97 <0.05Disease progression 9.9 <0.05Tumor differentiation 6.8 NS
Table 5. Results of relative risk ratio gene odd ratio amongodd ratio among relative risk ratio.
GenesOdd ratio
among benignOdd ratio among
malignantRelativerisk ratio
P15 0.543 0.543 1.34P16 0.642 0.642 1.54E-cadherin 0.71 0.71 1.61
Table 3. Correlation between E-cadherin methylation andother parameters in malignant group.
Parametersw2 p ValueVariables
Age, Parity, E-cadherine methylation 1.89 NSAge, Parity, P15 methylation 1.56 NSAge, Parity, P16 methylation 8.97 <0.05
Moselhy et al. 5
at King Abdul Aziz University on March 23, 2015tih.sagepub.comDownloaded from
be independent since these two tumor-related genes are
functionally different. In the current study, there was a
significant correlation (p < 0.05) between E-cadherin
methylation and disease progression, while there was
no correlation between E-cadherin methylation and
tumor differentiation. These data indicated that epige-
netic inactivation of E-cadherin may occur early, prior
to invasion but increases as cells acquire invasiveness
and metastatic potential.
In this study, it was found that p15 and p16 methy-
lation shows neither significant correlation with
disease progression nor tumor differentiation. It was
reported that p15 and p16 methylation did not have
prognostic significance in acute leukemia patients
(Rice and Futscher, 2000).
The median survival time of AML with p15
methylation was notably shortened. This minimizes
the role of p15 methylation in solid tumors. Our
results revealed a significant association between age,
E-cadherin and p15 methylation, and increase risk of
ovarian cancer (p < 0.05), while there was a high sig-
nificant association between age and E-cadherin
methylation and incidence of ovarian cancer
(p < 0.01). We observed in this study that the risk
of E-cadherin hypermethylation increases among
patients with ovarian cancer by 1.347-fold than that
among patients with benign ovarian lesions, while
risk of p15 hypermethylation increases among
patients with ovarian cancer by 1.543-fold than that
among patients with benign ovarian lesions; so we
demonstrate here for the first time that methylation
in suppressor genes occur in benign conditions.
This means that benign tumor methylation showed
a very important indication to the fact that the process
of ovarian carcinogenesis occur through methylation
than hyper methylation. This assumption is supported
by the fact that certain precancerous conditions may
undergo methylation in their suppressor genes fol-
lowed by hypermethylation status that leads to malig-
nant transformation (Shukla et al., 2006).
Conclusion
The low frequency of genes methylation in the group
of nonmalignant ovarian samples is an important find-
ing with respect to developing these markers as early
detection aids for ovarian cancer arising in women at
increased risk. However, significant methylation of E-
cadherin gene loci in invasive tumors compared with
control tissues indicates that methylation of that gene
may play a role in ovarian carcinogenesis. This gene
may be a suitable candidate for exploring ovarian
tumor progression, and may represent additional tar-
get for molecular detection of invasive ovarian tumors
and for development of antimethylation therapeutic
strategies in future studies.
Acknowledgment
The authors acknowledge Center of Excellence in Genomic
Medicine Research support for Scientific Research.
Funding
This study was funded by the Center of Excellence in
Genomic Medicine Research (CEGMR), King Abdulaziz
University, Jeddah, , Kingdom of Saudi Arabia, under grant
no. CEGMR 08-17.
References
Bird AP (1986) CpG-rich islands and the function of DNA
methylation. Nature 321: 209–213.
Bishop JM (1987) The molecular genetics of cancer.
Science 235: 305–311.
Clevers H (2006) Wnt/beta-catenin signaling in develop-
ment and disease. Cell 127: 469–480.
Eshleman JR, Markowitz SD (1996) Mismatch repair
defects in human carcinogenesis. Human Molecular
Genetics 5: 1489–1494.
Fearon ER (1997) Human cancer syndromes: clues to the
origin and nature of cancer. Science 278: 1043–1050.
Fodde R, Brabletz T (2007) Wnt/beta-catenin signaling in
cancer stemness and malignant behavior. Current
Opinion in Cell Biology 19: 150–158.
Gil J, Peters G (2006) Regulation of the INK4b-ARF-
INK4a tumour suppressor locus: all for one or one for
all. Nature Reviews Molecular Cell Biology 7: 667–677.
Herman JG, Latif F, Weng Y, et al. (1994) Silencing of
VHL tumor-suppressor gene by DNA methylation in
renal carcinoma. Proceedings of the National Academy
of Sciences United States of America 91: 9700–9704.
Kass SU, Pruss D and Wolffe AP (1997) How does DNA
methylation repress transcription? Trends in Genetics
13: 444–449.
Kim WY, Sharpless NE (2006) The regulation of INK4/
ARF in cancer and aging. Cell 127: 265–275.
Laird PW, Jaenisch R (1996) The role of DNA methylation
in cancer genetics and epigenetics. Annual Review of
Genetics 30: 441–464.
Lee WH, Morton RA, Epstein JI, et al. (1994) Cystidine
methylation of regulatory sequences near the pi-class glu-
tathione S-transferase gene accompanies human prostatic
carcinogenesis. Proceedings of the National Academy of
Sciences United States of America 91: 11733–11737.
6 Toxicology and Industrial Health
at King Abdul Aziz University on March 23, 2015tih.sagepub.comDownloaded from
Lengauer C, Kinzler KW and Vogelstein B. DNA methyla-
tion and genetic instability in colorectal cancer cells.
Proceedings of the National Academy of Sciences
United States of America 94: 2545–2550.
Lin SY, Yeh KT, Chen WT, et al. (2004) Promoter CpG
methylation of tumor suppressor genes in colorectal
cancer and its relationship to clinical features. Oncology
Reports 11: 341–348.
Look AT (1997) Oncogenic transcription factors in the
human acute leukemia. Science 278: 1059–1064.
Mirza S, Sharma G, Prasad CP, et al. (2007) Promoter
hypermethylation of TMS1, BRCA1, ERalpha and PRB
in serum and tumor DNA of invasive ductal breast
carcinoma patients. Life Sciences 81(4): 280–287.
Muller HM, Widschwendter A, Fiegl H, et al. (2003) DNA
methylation in serum of breast cancer patients: an inde-
pendent prognostic marker. Cancer Research 63(22):
7641–7645.
Niwa Y, Oyama T and Nakajima T (2000) BRCA1 expres-
sion status in relation to DNA methylation of the BRCA1
promoter region in sporadic breast cancers. Japanese
Journal of Cancer Research 91(5): 519–526.
Osborne CK, Yochmowitz MG, Knight WA 3rd, et al.
(1980) The value of estrogen and progesterone receptors
in the treatment of breast cancer. Cancer 46(12 Suppl):
2884–2888.
Parkin DM, Bray F, Ferlay J, et al. (2005) Global cancer
statistics, 2002. Cancer Journal for Clinicians 55(2):
74–108.
Raman V, Martensen SA, Reisman D, et al. (2000a)
CompromisedHOXA5 function can limit p53 expression
in human breast tumours. Nature 405(6789): 974–978.
Raman V, Tamori A, Vali M, et al. (2000b) HOXA5 regu-
lates expression of the progesterone receptor. Journal of
Biological Chemistry 275(34): 26551–26555.
Razin A, Cedar H (1994) DNA methylation and genomic
imprinting. Cell 77: 473–476.
Razin A, Shemer R (1995) DNA methylation in early devel-
opment. Human Molecular Genetics 4: 1751–1755.
Rice JC, Futscher BW (2000) Transcriptional repression of
BRCA1 by aberrant cytosinemethylation, histone
hypoacetylation and chromatin condensation of the
BRCA1 promoter. Nucleic Acids Research 28(17):
3233–3239.
Sharma G, Mirza S, Prasad CP, et al. (2007) Promoter
hypermethylation of p16(INK4A), p14(ARF), CyclinD2
and Slit2 in serum and tumor DNA from breast cancer
patients. Life Sciences 81(4): 280–287.
Shukla S, Mirza S, Sharma G, et al. (2006) Detection of
RASSF1A and RAR beta hypermethylation in Serum
DNA from breast cancer patients. Epigenetics 1: 88–93.
Singletary SE, Allred C, Ashley P, et al. (2003) Staging
system for breast cancer: revisions for the 6th edition
of the AJCC cancer staging manual. Surgicals of Clini-
cians of North America 83(4): 803–819.
Stanbridge EJ (1990) Identifying tumor suppressor genes in
human colorectal cancer. Science 247: 12–13.
Ushijima T (2005) Detection and interpretation of altered
methylation patterns in cancer cells. Nature Review
Cancer 5: 223–231.
Yeh KT, Chang JG, Lin TH, et al. (2003) Correlation
between protein expression and epigenetic and mutation
changes of Wnt pathway-related genes in oral cancer.
International Journal of Oncology 23: 1001–1007.
Moselhy et al. 7
at King Abdul Aziz University on March 23, 2015tih.sagepub.comDownloaded from
Vol.2, No.7, 402-404 (2013) Case Reports in Clinical Medicine http://dx.doi.org/10.4236/crcm.2013.27107
Copyright © 2013 SciRes. OPEN ACCESS
Comprehensive laparoscopic surgical staging of ovarian dysgerminoma in a 10-year-old girl—A case report
Nisrin Anfinan*
Gynecology Oncology Unit, Department of Obstetric and Gynaecology, Faculty of Medicine, King Abdulaziz University, Jeddah, KSA; *Corresponding Author: [email protected] Received 4 August 2013; revised 5 September 2013; accepted 20 September 2013 Copyright © 2013 Nisrin Anfinan. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
Minimal access surgery to stage early ovarian cancer (EOC) is still regarded as an investiga- tion among many gynecologic oncologists. This is a case of comprehensive laparoscopic surgi-cal stage of an ovarian dysgerminoma in a 10- year-old girl described. This patient was re- ferred to the gynecology oncology unit status post left salpingo-oophorectomy through a mid- line incision when the histopathology showed pure dysgerminoma. We then performed the laparoscopic stage including peritoneal washing; resection of the left infundibulopelvic ligament; systematic pelvic, common iliac, and infrarenal bilateral paraaortic lymphadenectomy; and omen- tectomy. The uterus and right adnexum were spared to preserve future fertility. The final his- topathology showed no metastatic disease (stage ovarian dysgerminoma), and patient has no evi- dence of recurrence after 52 months follow up. Conclusion: This is the youngest patient report- ed in the literature with a comprehensive laparo- scopic surgical stage for ovarian neoplasm. A full laparoscopic staging for ovarian cancer in a 10-year-old girl is safe and might be considered as an alternative to the stander of care. Keywords: Ovarian Dysgerminoma; Laparoscopy; Comprehensive Surgical Staging; Child
1. INTRODUCTION
In recent years, minimally invasive surgery (MIS) has been widely used to establish a diagnosis, for staging
purposes, and evaluation of recurrent or metastatic dis-ease in children [1,2]. The use of MIS techniques in children is growing with the availability of smaller in- struments and equipment more suitable for the pediatric patients [3]. The main indications for use of MIS tech- niques are diagnostic biopsies (mediastinal or lung tu- mors, and retroperitoneal extrarenal masses), resection of the primary tumor in patients with thoracic and abdomi- nal neuroblastic tumors, and post-treatment of the resid- ual mass in patients with lymphoma [4]. The role of MIS for surgical staging of the early stage ovarian cancer is feasible [5,6].
This is a case of comprehensive surgical restaging per- formed in a 10-year-old girl affected by a pure ovarian dysgerminoma apparently confined to one ovary.
2. CASE REPORT
A 10-year-old premenarchal girl was referred to our gynecology oncology unit at King Abdulaziz University Hospital in January 2011 after she had undergone a la- parotomy through a midline incision performed by a ge- neral gynecologist and left salpingo-oophorectomy was done. At that time, histological analysis of a surgical specimen revealed a pure dysgerminoma, but unfortu- nately she was not staged. The options were discussed with her parents who elected for her to undergo a restag- ing procedure.
Her medical history was unremarkable; physical ex- amination revealed a well-healed incision that extended from the midline up to the umbilicus. The patient’s weight and height were 25 kg and 140 cm, respectively. Laboratory tests performed revealed preserved renal function and a normal hemoglobin level. The LDH was elevated 4461 U/L, other tumor markers CA 125, CEA, and α-FP were normal. She was prepared and admitted for surgery, and the informed consent form was signed
*MD, ABOG. Scientific Chair of Professor Abdullah Hussain Ba-salamah for Gynecological Cancer.
N. Anfinan / Case Reports in Clinical Medicine 2 (2013) 402-404
Copyright © 2013 SciRes. OPEN ACCESS
403
by her parents. Considering the young age of the patient, we per-
formed comprehensive surgical staging via laparoscopy. Intra-abdominal inspection revealed an adhesion of the omentum over the left uterine cornua with no evidence of disseminated disease in the pelvis and upper abdomen. The right adnexum and uterus were carefully examined and appeared normal. Peritoneal washing was performed before starting the procedure. The procedure consisted of systematic bilateral pelvic (including external iliac, in- ternal iliac, and obturator), bilateral common iliac, and infrarenal bilateral paraaortic lymph node dissection (Figure 1); omentectomy; and obtaining peritoneal bi- opsy specimens. The staging procedure was successfully completed in 249 minutes with an estimated blood loss of 200 mL, using a 10-mm umbilical trocar, 3 suprapubic 5-mm ancillary trocars, and one 3-mm ancillary trocar placed in the left hypochondrium at the level of the mid- clavicular line. Abdominal drainage was placed and re- moved the next day. The decrease in the patient’s hemo- globin (Hb) level was 1 g/dL (Hb before 11 g/Dl-Hb after 10 g/dL), and the postoperative hospital stay was not complicated. The postoperative period was unevent- ful, and the girl was discharged from the hospital 2 days after the surgery. All other surgical specimens including total of 24 pelvic and para aortic lymph nodes and the peritoneal washing revealed no evidence of neoplastic lesions, so the final diagnosis was a pure ovarian dys- germinoma at Ia stage (FIGO classification).
Follow-up visits with physical examination and eval- uation of tumor markers were planned 1 month and 3 months after the surgery and then every 3 months. Her lactate dehydrogenase level soon returned to the normal range with no evidence of recurrence after 52 months follow up.
Figure 1. Laparoscopic view of the retroperitoneal space after complete para-aortic lymphadenctomy (A) inferior vena cava; (B) inferior mesenteric artery; (C) aorta.
3. DISCUSSION
Ovarian germ cell tumors are rare neoplasms, repre- senting about 3% to 5% of all ovarian malignancies. Dysgerminomas are commonly diagnosed in the 2nd and 3rd decades of life, approximately 85% - 90% are con- fined to one ovary [7].
As for other ovarian tumors, the initial management of a dysgerminoma is surgery, and the traditional approach to this condition is staging. Comprehensive surgical stag- ing allows to identify patients with a more advanced stage disease that should receive adjuvant chemotherapy. For unstaged IA ovarian dysgerminoma, the management is still controversial. Vicus reported 4 cases of relapsed patients with unstaged IA ovarian dysgerminoma [8], Kasenda also reported 2 cases of recurrence with appar- ent IA, surgically unstaged patients. The authors con- cluded that comprehensive initial surgical staging is man- datory to minimize treatment burden [9].
Traditionally surgical staging performed through a ge- nerous midline laparotomy incision. However, the role of MIS for early stage ovarian cancer is feasible [5,6], opening new perspectives for the management of ovarian germ cell malignant tumors. Laparoscopy now allows performance of all procedures required by International Federation of Gynecology and Obstetrics (FIGO) guide-lines for surgical staging of ovarian malignancies (i.e., complete removal of ovarian neoplasm, pelvic and pa- raaortic lymph nodes, and omentum).
Uccella et al. reported the first case of comprehensive laparoscopic surgical staging of an ovarian dysgermi- noma in a 13-year-old girl [10]. For a 20-cm pelvic mass rising from the right adnexum, she underwent laparo- scopic right salpingo-oophorectomy. Pathologic findings were consistent with a diagnosis of pure dysgerminoma. Laparoscopic staging included peritoneal washing; re- section of the right infundibulopelvic ligament; system- atic pelvic, common iliac, and infrarenal paraaortic lym- phadenectomy; appendectomy; and infracolic omentec- tomy. The uterus and left adnexum were spared to pre- serve future fertility [10].
Our report of performing comprehensive surgical re- staging of an apparent early-stage ovarian dysgerminoma via laparoscopy in a 10-year-old child is of great interest for a number of reasons. This type of neoplasm is largely curable [11], and its chemosensitivity has been proven to be preserved also in cases of recurrent disease. In addi- tion, the postoperative recovery is much faster when the operation is performed laparoscopically compared with via laparotomy. Our patients were able to go home next day with less pain which is certainly much less psycho- logical and physical consequences of having another major abdominal incision. In addition, the lower rate of postoperative adhesions after laparoscopic procedures (when compared with laparotomy) is of utmost impor-
N. Anfinan / Case Reports in Clinical Medicine 2 (2013) 402-404
Copyright © 2013 SciRes. OPEN ACCESS
404
tance in these patients, because it is well known that ad- hesions can be responsible for infertility [12]. In addition, she was spared the unnecessary adjuvant chemotherapy considering negative staging after the second surgery.
Written informed consent was obtained from her par- ents for publication of this case report and accompanying images.
4. CONCLUSION
This is the youngest patient reported in the literature with a comprehensive laparoscopic surgical stage for ovarian neoplasm. A full laparoscopic stage for ovarian cancer in 10 year old girl is safe and might be considered as an alternative to the stander of care.
5. ACKNOWLEDGEMENTS
I would like to thank professor Khalid Sait, my team, the anesthesia
department and the operating room staff for the their help and assistant.
REFERENCES [1] Spurbeck, W.W., Davidoff, A.M., Lobe, T.E., Rao, B.N.,
Schropp, K.P. and Shochat, S.J. ( 2004) Minimally inva- sive surgery in pediatric cancer patients. Annals of Surgi- cal Oncology, 3, 340-343. http://dx.doi.org/10.1245/ASO.2004.04.021
[2] Sandoval, C., Strom, K. and Stringel, G. (2004) Laparo- scopy in the management of pediatric intraabdominal tu- mors. JSLS, 2, 115-118.
[3] Cribbs, R.K., Wulkan, M.L., Heiss, K.F. and Gow, K.W. (2007) Minimally invasive surgery and childhood cancer. Surgical Oncology, 3, 221-228. http://dx.doi.org/10.1016/j.suronc.2007.09.002
[4] Leclair, M.D., Sarnacki, S., Varlet, F. and Heloury, Y.
(2007) Minimally-invasive surgery in cancer children. Bull Cancer, 12, 1087-1090.
[5] Tozzi, R., Köhler, C., Ferrara, A. and Schneider A. (2004) Laparoscopic treatment of early ovarian cancer: Surgical and survival outcomes. Gynecologic Oncology, 1, 199- 203. http://dx.doi.org/10.1016/j.ygyno.2004.01.004
[6] Leblanc, E., Querleu, D., Narducci, F., Occelli, B., Papa- georgiou, T. and Sonoda, Y. (2004) Laparoscopic restag- ing of early invasive adnexal tumors: A 10-year experi- ence. Gynecologic Oncology, 3, 624-629. http://dx.doi.org/10.1016/j.ygyno.2004.05.052
[7] Williams, S.D. (1998) Ovarian germ cell tumors. Semi- nars in Oncology, 25, 407-413.
[8] Vicus, D., Beiner, M.E., Klachook, S., Le, L.W., La- framboise, S. and Mackay, H. (2010) Pure dysgerminoma of the ovary 35 years on: A single institutional experi- ence. Gynecologic Oncology, 1, 23-26. http://dx.doi.org/10.1016/j.ygyno.2009.12.024
[9] Kasenda, B., Harter, P., Hirsch, T., Ast, A., Buhrmann, C., Glaser, F. and Du Bois A. (2009) Para-aortic lymph node metastasis in malignant dysgerminoma of the ovary. Acta Obstetricia et Gynecologica Scandinavica, 11, 1288- 1290. http://dx.doi.org/10.3109/00016340903242461
[10] Uccella, S., Cromi, A., Seveso, A., Siesto, G. and Ghezzi, F. (2008) Comprehensive laparoscopic surgical staging of ovarian dysgerminoma in a 13-year-old girl: A case report. Journal of Minimally Invasive Gynecology, 1, 110-112. http://dx.doi.org/10.1016/j.jmig.2007.08.599
[11] Smith, H.O., Berwick, M., Verschraegen, C.F., Wiggins, C., Lansing, L., Muller, C.Y. and Qualls, C.R. (2006) In- cidence and survival rates for female malignant germ cell tumors. Obstetrics & Gynecology, 5, 1075-1085. http://dx.doi.org/10.1097/01.AOG.0000216004.22588.ce
[12] Tulandi, T. and Al-Shahrani, A. (2005) Adhesion preven- tion in gynecologic surgery. Current Opinion in Obstet- rics and Gynecology, 4, 395-398. http://dx.doi.org/10.1097/01.gco.0000175357.25932.89
Hindawi Publishing CorporationCase Reports in Obstetrics and GynecologyVolume 2013, Article ID 409196, 3 pageshttp://dx.doi.org/10.1155/2013/409196
Case ReportSupraclavicular Lymphadenopathy: Initial Manifestationof Metastasis in Carcinoma of Cervix
Mutahir A. Tunio,1 Mushabbab Al Asiri,1 Reham Mohamed,1 and Sadeq Al-Dandan2
1 Radiation Oncology, Comprehensive Cancer Center, King Fahad Medical City, Riyadh 59046, Saudi Arabia2 Comprehensive Cancer Center, King Fahad Medical City, Riyadh 59046, Saudi Arabia
Correspondence should be addressed to Mutahir A. Tunio; [email protected]
Received 20 December 2012; Accepted 28 January 2013
Academic Editors: P. De Franciscis, C.-C. Liang, and E. F. C. Murta
Copyright © 2013 Mutahir A. Tunio et al. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.
Introduction. Carcinoma of cervix rarely metastasizes to cervical lymph nodes and is associated with poor prognosis. To date, onlyfew case reports have been reported in the medical literature. Here, we report a case of this unusual manifestation of carcinoma ofcervix. Case Presentation. A 39-year-old Saudi woman who was treated three years ago for bulky IB stage carcinoma of cervixwith total abdominal hysterectomy and bilateral salpingo-oophorectomy and adjuvant chemoradiation presented to us duringher routine follow-up visit with left supraclavicular lymphadenopathy. Staging workup revealed additional para-aortic nodal andosseous metastases. The biopsy of left supraclavicular mass confirmed the diagnosis of carcinoma of the cervix. Patient was startedon chemotherapy and bisphosphonates.Conclusion. Supraclavicular lymph nodes are a rare site ofmetastasis in carcinoma of cervix,and this can be explained by outlining the drainage of the lymphatic system from the cervix. Supraclavicular lymphadenopathy isassociated with variable prognosis.
1. Introduction
The incidence of invasive cervical cancer is decreasing inthe United States [1]. However, cervical cancer continues tobe a major women’s health issue in many countries becauseof inadequate cytological screening programs, and manypatients present locally advanced or metastatic FIGO stagesIIB-IVB.
The pattern of metastasis in carcinoma of cervix initiallyinvolves pelvic lymph nodes, followed by para-aortic nodesand distant sites. The most frequent metastatic sites are thelungs, extrapelvic nodes, liver, and bones [2]. However, due tomultidisciplinary treatment and increased survival, differentmetastatic sites have been reported, including the cervicallymph nodes. However, supraclavicular lymphadenopathyfrom carcinoma of cervix is extremely rare. Only few casereports have been published so far in the medical literature[3, 4]. The presence of supraclavicular lymphadenopathyis associated with grave prognosis for survival outcome inpatients with carcinoma of cervix.
Here, we present a case report of 39-year-old Saudiwoman, who presented with left supraclavicular lymphad-
enopathy as initial metastatic site after 3 years of treatmentfor FIGO IB bulky carcinoma of cervix.
2. Case Presentation
A 39-year-old Saudi woman presented in our oncology clinicfor her routine visit with left lower neck swelling. She hadnoticed this swelling for 2 months, and it had been rapidlyincreasing in size over a month causing pain, for which shewas taking nonsteroidal anti-inflammatory drugs (NSAIDs),but no benefit. Her previous medical history revealed thatthree years ago shewas treatedwith total abdominal hysterec-tomy and bilateral salpingo-oophorectomy (TAH and BSO)followed by adjuvant chemoradiation for bulky FIGO stageIB. She had no history comorbid conditions and no historyof smoking, and her weight was stable. On physical examina-tion, her vitals were stable. A fixed, solitary, hard neckmass ofsize 4 × 4 cm was palpable in the left supraclavicular region.There was no other palpable cervical lymphadenopathy, andthe examination of chest, heart, nervous system, abdomen,
2 Case Reports in Obstetrics and Gynecology
Figure 1: Computed tomography (CT) axial images of neck showingleft supraclavicular solid mass of size 3.2 × 2.5 cm encasing thevessels.
Figure 2: Histopathology of left supraclavicular node showingmetastatic squamous cells with abundant eosinophilic cytoplasmand cell keratinization.
and pelvis was normal. Clinical differential diagnosis wastuberculosis or carcinoma of breast or lung.
Computed tomography (CT) neck showed left supraclav-icular solid mass of size 3.2 × 2.5 cm encasing the vessels(Figure 1). The mammogram of both breasts was normalwith no solid or cystic lesion. Hematological, renal, and liverfunction tests, tuberculin, and serum electrolytes were withinnormal limits.The core biopsy of mass was performed, whichrevealed metastatic squamous cell carcinoma consisting withcervix primary (Figure 2). CT of chest, abdomen, and pelvisrevealed para-aortic lymphadenopathy and lytic lesion indorsal spine however, no local recurrence. Bone scintigraphyconfirmed bone metastasis in skull, dorsal spine, left femur,distal right femur, and proximal right tibia (Figure 3).
Patient was started on duplet chemotherapy (cisplatinand paclitaxel). After four cycles, she has responded well tothe treatment with a reported decrease in the size of thesupraclavicular nodes (Figure 4).
3. Discussion
The reported incidence of metastasis of carcinoma of thecervix to left supraclavicular nodes is 0.1%–1.5% [5]. To
Figure 3: Bone scan showing bone metastasis in skull, dorsal spine,left femur, distal right femur, and proximal right tibia.
Figure 4: Postchemotherapy CT scan axial images showing adecrease in the size of the left supraclavicular nodes.
date, only few case reports have been published [6, 7]. Themanifestation of supraclavicular lymphadenopathy indicateshigh tumor burden and poor prognosis in patients withcarcinoma of cervix. Henriksen [5], in his retrospectivereview of 18 cases of cervical cancer with supraclavicularlymphadenopathy, has reported a survival time of between 1and 16 months after the appearance of metastases.
Possible pattern of spread of tumor cells carcinomaof cervix to the supraclavicular region is best understoodthrough its lymphatic drainage. The carcinoma of the cervixspreads by internal and external iliac lymph nodes from pri-mary lesion, then common iliac andpara-aortic lymphnodes,then into the thoracic duct. The thoracic duct communicateswith the systemic venous system in the neck at the junctionof the left subclavian and internal jugular vein. The left-sidedsupraclavicular nodes represent the final common path of thebody’s infradiaphragmatic lymphatic drainage [8].
Our patient has responded well to cisplatin and paclitaxelbased palliative chemotherapy with partial response and
Case Reports in Obstetrics and Gynecology 3
is alive at 6 months after the initial presentation of supr-aclavicular lymphadenopathy. However, recent trials havedocumented response rates of 27% achieved by cisplatin incombination with paclitaxel [9, 10].
A previous study of 33 cases with cervical cancerand supraclavicular metastasis reported that the SCC-Ag <15 ng/mL at initial diagnosis and staging/restaging includ-ing 18-flouro-deoxyglucose positron emission tomography(FDG-PET) to be associated with a better prognosis [11–13].However, these tests were not carried out in our patient butare highly recommended for prompt treatment. Further, ourpatient’s TAH+BSO is an inadequate therapeutic model forcervical cancer IB, and there was no complete staging at thetime of the first operation, which possibly resulted in cancerrecurrence later.
In conclusion, supraclavicular lymphadenopathy sec-ondary to carcinoma of cervix is rare, and prognosis in suchpatients is usually poor but not incurable, and treatment ismainly palliative. However, the incorporation of FDG-PETand SCC-Ag markers can be helpful.
Abbreviations
CT: Computed tomographyNSAID: Nonsteroidal anti-inflammatory drugsTAH: Total abdominal hysterectomyBSO: Bilateral salpingo-oophorectomyFIGO: International Federation of Gynecologists
and ObstetricsFDG-PET: Flouro-deoxyglucose positron emission
tomography.
Consent
A written permission from the patient was taken for thepublication of this paper.
Conflict of Interests
There is no competing interest among authors, and nofinancial or nonfinancial support was taken from anywhere.
Authors’ Contribution
The paper writing was done by M. Tunio, data collection byR. Mohamed, and pathological part of the paper by S. Al-Dandan; the final review and approval were done by M. AlAsiri.
References
[1] R. Siegel, D. Naishadham, and A. Jemal, “Cancer statistics 2012,”CA: A Cancer Journal for Clinicians, vol. 62, pp. 10–29, 2012.
[2] S. E.Waggoner, “Cervical cancer,”TheLancet, vol. 361, pp. 2217–2225, 2003.
[3] A.W. Diddle, “Carcinoma of the cervix uteri with metastases tothe neck,” Cancer, vol. 29, no. 2, pp. 453–455, 1972.
[4] M.Manoharan,D. Satyanarayana, andA.R. Jeyarajah, “Cervicallymphadenopathy—an unusual presentation of carcinoma of
the cervix: a case report,” Journal of Medical Case Reports, vol.2, article 252, 2008.
[5] E. Henriksen, “The lymphatic spread of carcinoma of the cervixand of the body of the uterus. A study of 420 necropsies,”American Journal of Obstetrics and Gynecology, vol. 58, no. 5,pp. 924–942, 1949.
[6] M. S. Shin, H. M. Shingleton, E. E. Partridge, V. M. Nicolson,and K. J. Ho, “Squamous cell carcinoma of the uterine cervix:patterns of thoracic metastases,” Investigative Radiology, vol. 30,no. 12, pp. 724–729, 1995.
[7] I. Scott, C. J. Bergin, and N. L. Muller, “Mediastinal andhilar lymphadenopathy as the only manifestation of metastaticcarcinoma of the cervix,” Canadian Association of RadiologistsJournal, vol. 37, no. 1, pp. 52–53, 1986.
[8] E. Ellison, P. LaPuerta, and S. E. Martin, “Supraclavicularmasses: results of a series of 309 cases biopsied by fine needleaspiration,” Head and Neck, vol. 21, pp. 239–246, 1999.
[9] D. H. Moore, J. A. Blessing, R. P. McQuellon et al., “PhaseIII study of cisplatin with or without paclitaxel in stage IVB,recurrent, or persistent squamous cell carcinoma of the cervix:a Gynecologic Oncology Group study,” Journal of ClinicalOncology, vol. 22, no. 15, pp. 3113–3119, 2004.
[10] J. Y. Kim, J. Y. Kim, and J. H. Kim, “Curative chemoradiotherapyin patients with stage IVB cervical cancer presenting withparaaortic and left supraclavicular lymph node metastases,”International Journal of Radiation,Oncology, Biology andPhysics, vol. 84, pp. 741–747, 2012.
[11] J. T. Qiu, K. C. Ho, C. H. Lai et al., “Supraclavicular lymph nodemetastases in cervical cancer,” European Journal of Gynaecolog-ical Oncology, vol. 28, no. 1, pp. 33–38, 2007.
[12] K.-C.Ho,C.-C.Wang, J.-T.Qiu et al., “Identification of prognos-tic factors in patients with cervical cancer and supraclavicularlymph node recurrence,” Gynecologic Oncology, vol. 123, no. 2,pp. 253–256, 2011.
[13] B. N. Tran, P. W. Grigsby, F. Dehdashti, T. J. Herzog, andB. A. Siegel, “Occult supraclavicular lymph node metastasisidentified by FDG-PET in patients with carcinoma of theuterine cervix,” Gynecologic Oncology, vol. 90, no. 3, pp. 572–576, 2003.
Hindawi Publishing CorporationCase Reports in Obstetrics and GynecologyVolume 2013, Article ID 858501, 6 pageshttp://dx.doi.org/10.1155/2013/858501
Case ReportPoorly Differentiated Ovarian Sertoli-LeydigCell Tumor in a 16-Year-Old Single Woman: A Case Report andLiterature Review
Ahmed Abu-Zaid,1 Ayman Azzam,2 Lama Abdulhamid Alghuneim,1
Mona Tarek Metawee,1 Tarek Amin,3 and Turki Omar Al-Hussain4
1 College of Medicine, Alfaisal University, P.O. Box 50927, Riyadh 11533, Saudi Arabia2Department of General Surgery, Faculty of Medicine, Alexandria University, Alexandria 21526, Egypt3 Department of Surgical Oncology, King Faisal Specialist Hospital and Research Center (KFSH&RC),P.O. Box 3354, Riyadh 11211, Saudi Arabia
4Department of Pathology and Laboratory Medicine, King Faisal Specialist Hospital and Research Center (KFSH&RC),P.O. Box 3354, Riyadh 11211, Saudi Arabia
Correspondence should be addressed to Ahmed Abu-Zaid; [email protected]
Received 14 May 2013; Accepted 18 June 2013
Academic Editors: S. Z. A. Badawy, X. Deffieux, L. Nilas, and M. G. Porpora
Copyright © 2013 Ahmed Abu-Zaid et al. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properlycited.
Sertoli-Leydig cell tumor (SLCT) of ovary is an exceedingly unusual neoplasm that belongs to a group of sex cord-stromal tumorsof ovary and accounts for less than 0.5% of all primary ovarian neoplasms. Very few case reports have been documented in theliterature so far. Herein, we report a case of primary poorly differentiated ovarian Sertoli-Leydig cell tumor (SLCT) involving theleft ovary in a 16-year-old single woman who presented with a 3-month history of a pelviabdominal mass, acne, hirsutism, andmenstrual irregularities. In addition, a literature review on ovarian SLCTs is provided.
1. Introduction
Sertoli-Leydig cell tumor (SLCT) of ovary is an exceedinglyunusual neoplasm that belongs to a group of sex cord-stromaltumor of ovary and accounts for less than 0.5% of all primaryovarian neoplasms [1]. The vast majority of SLCTs are largelydiagnosed during reproductive age group (second and thirddecades of life) [1–4], frequently unilateral, mostly confinedto ovary and nearly 90% classified as stage I at the time ofclinical diagnosis [5]. Clinical signs and symptoms can berelated to either hormonal production (mostly androgen andrarely estrogen) [6] or presence of mass-occupying lesion(mostly pelviabdominal mass and/or pain) [1–3]. Elevatedserum levels of testosterone and androstenedione can beidentified in approximately 80% of patients with ovarianSLCTs and virilizing manifestations [7, 8]. SLCTs are by farthe most common virlizing tumors of ovary [1].
Ultrasound (particularly transvaginal sonography) re-mains the best imaging modality of preference for initial
assessment of adnexal masses [6, 9]. Microscopically, SLCTsare classically made up of uncontrolled proliferation ofvarying degrees of differentiation of tubules lined by Sertolicells and intervening nests of Leydig cells [10]. Immuno-histochemically, almost all SLCTs, including moderately andpoorly differentiated variants, stain positive for inhibin andcalretinin, and negative for epithelial membrane antigen(EMA) [11]. A collective profile of hematoxylin and eosin(H&E) stains in addition to immunohistochemical studiesare expected to yield the most accurate definitive diagnosisof ovarian SLCTs [4].
Prognosis of ovarian SLCTs is significantly correlatedwith degree of tumor grading and staging [1]. Managementof ovarian SLCTs remains challenging owing to lack of stan-dardized management protocol guidelines [12]. Surgicalresection represents the mainstay of management of ovarianSLCTs [4]. The role of postoperative chemotherapy remainsquestionable and is only indicated in patients with poorprognostic factors [1–3].
2 Case Reports in Obstetrics and Gynecology
Herein, we report a case of primary poorly differentiatedovarian Sertoli-Leydig cell tumor (SLCT) involving the leftovary in a 16-year-old single woman who presented with a3-month history of a pelvi-abdominal mass, acne, hirsutism,and menstrual irregularities. In addition, a literature reviewon ovarian SLCTs is provided.
2. Case Report
A 16-year-old single woman presented to King Faisal Spe-cialist Hospital and Research Center (KFSH&RC) with a3-month history of a pelvi-abdominal mass, acne, hir-sutism, and menstrual irregularities. Past medical historyand surgical history were unremarkable. Systemic review wasremarkable for decreased appetite, shortness of breath, andconstipation. Physical examination revealed acne, hirsutism,abdominal swelling, and a diffuse, tender, and palpable massextending from lower pelvis/abdomen to umbilicus.
Laboratory tests showed increased total serum levels oftestosterone (T) of 7.8 ng/mL (normal range: 0.2–0.7 ng/mL),dehydroepiandrosterone (DHEA) of 7.2 ng/mL (normalrange: 0.8–3.2 ng/mL), and CA-125 of 272U/mL (normalrange <35U/mL). All other laboratory tests including com-plete blood count (CBC), renal, bone, hepatic and coag-ulation profiles, alkaline phosphatase, carcino-embryonicantigen (CEA), alfa-feto protein (AFP), CA 15-3, CA 19-9,estradiol (17𝛽-estradiol), luteinizing hormone (LH), follicle-stimulating hormone (FSH), sex hormone-binding globulin(SHBG), adrenocorticotropic hormone (ACTH), prolactin,and cortisol levels were within normal ranges.
A chest radiograph (X-ray) showed no evidence of pul-monary nodules. Ultrasound (US) showed intraperitonealascites and extremely huge well-vascularizedmass with cysticand solid components, mostly arising from left ovary. Achest, abdominal, and pelvic contrast-enhanced computedtomography (CT) scan at the portal venous phase with rectalcontrast showed a huge, intraperitoneal, complex, cystic,and multilocular lesion, extending from pelvis up to mid-abdomen just above umbilicus, collectively measuring 13.5× 23.3 × 21.5 cm. The source of lesion was more likely to beleft ovary which was probably ruptured and leaked into theabove-mentioned cystic lesion. In addition, large ascites wasidentified. No significant lymphadenopathies in abdomenand pelvis were noticed (Figure 1). In view of a possible neo-plastic process involving the left ovary with androgen-excessmanifestations (i.e., virilizing ovarian neoplasm), patient wassubjected to laparotomy.
During laparotomy, frozen section biopsy from theleft ovarian mass was consistent with poorly differentiatedSertoli-Leydig cell tumor. Subsequently, the patient under-went left unilateral salpingoophorectomy, omentectomy, andappendectomy. Resected specimens were sent for histopatho-logical evaluation. Macroscopic and microscopic examina-tion of omentum, appendix, and left fallopian tube revealedno significant pathology and were negative for neoplasm.
Macroscopically, the ovarian mass weighed 1945 g andmeasured 24 × 21 × 7 cm. The mass was well circumscribedand had whitish nodular outer surface. A break of ovarian
Figure 1: A chest, abdominal, and pelvic contrast-enhanced com-puted tomography (CT) scan at the portal venous phase with rectalcontrast showed a huge, intraperitoneal, complex, cystic, and multi-locular lesion, extending from pelvis up to midabdomen just aboveumbilicus, collectivelymeasuring 13.5× 23.3× 21.5 cm.The source oflesion was more likely to be left ovary which was probably rupturedand leaked into the above-mentioned cystic lesion. In addition,large ascites was identified. No significant lymphadenopathies inabdomen and pelvis were noticed.
# SP12-5668
Figure 2: Macroscopic examination of the left ovarian mass. Themass weighed 1945 g and measured 24 × 21 × 7 cm. The masswas well circumscribed and had whitish nodular outer surface. Abreak of ovarian capsule was noted for a distance measuring 5.5 cm.Cut-surface showed multiple nodules separated by fibrous septa.Some of nodules were necrotic and others had tan-yellow nodularappearance.
capsule was noted for a distance measuring 5.5 cm. Cut-surface showed multiple nodules separated by fibrous septa.Some of the nodules were necrotic and others had tan-yellownodular appearance (Figure 2).
Microscopically, the ovarian tumor mass was composedof immature, poorly differentiated, spindle-shaped Sertolicells forming cords, and ill-defined tubules (Figure 3(a)).Theneoplastic Sertoli cells exhibited nuclear atypia and highmitotic index (Figure 3(b)). Focal myxoid area containingspindle-shaped cells was identified (Figure 3(c)). Infrequent
Case Reports in Obstetrics and Gynecology 3
(a) (b)
(c) (d)
Figure 3: Microscopic examination of the left ovarian mass. (a) The ovarian mass was composed of immature, poorly differentiated, andspindle-shaped Sertoli cells forming cords and ill-defined tubules (H&E stain, magnification power: 20x). (b) The neoplastic Sertoli cellsexhibited nuclear atypia and high mitotic indices (H&E stain, magnification power: 40x). (c) Focal myxoid area containing spindle-shapedcells was identified (H&E stain, magnification power: 20x). (d) Infrequent Leydig cells with eosinophilic cytoplasm were identified focally(H&E stain, magnification power: 20x).
Leydig cells with eosinophilic cytoplasm were identifiedfocally (Figure 3(d)).
Immunohistochemically, the neoplastic cells stained pos-itive for calretinin, inhibin, CD56, WT-1, and CK 8/18(Figures 4(a)–4(e). Conversely, the neoplastic cells stainednegative for EMA, PLAP, chromogranin A, and synapto-physin. Based on the clinical, histopathological and immuno-histochemical findings, a diagnosis of poorly differentiatedovarian Sertoli-Leydig cell tumor (SLCT) was established.
In consideration of poorly differentiated ovarian SLCT,the patient was considered for adjuvant chemotherapy reg-imen of bleomycin, etoposide, and cisplatin (BEP). A post-operative 3-month followup failed to show any evidence ofrecurrence.
3. Discussion
Sertoli-Leydig cell tumor (SLCT) of ovary is an exceedinglyunusual neoplasm that belongs to a group of sex cord-stromal tumors of ovary and accounts for less than 0.5%of all primary ovarian neoplasms [1]. It is characterized byuncontrolled proliferation of naturally occurring testicularstructures (Sertoli and Leydig cells) of varying degrees ofdifferentiation in ovary. The neoplastic Sertoli and Leydig
cells exhibit varying degrees of differentiation (grading)which include well differentiated, moderately differentiated,poorly differentiated, and with heterologous elements [13].
SLCTs can affect any age group ranging from 2 to 75 yearsof age. However, 75% of SLCTs take place during second andthird decades of life. The average age at the time of clinicaldiagnosis is roughly 25 years of age. Around less than 10% ofSLCTs take place prior to menarche or following menopause[1–3]. Degree of tumor differentiation (grading) seems to beage linked. Patients exhibiting poorly differentiated SLCTsappear to be on average 10 years younger than patientsexhibiting well-differentiated SLCTs [1, 3]. The vast majorityof SLCTs are frequently unilateral, mostly confined to ovary,andnearly 90%classified as stage I at time of clinical diagnosis[5]. At the time of clinical diagnosis, occurrence of extraovarian spread of SLCTs is extremely uncommon accountingfor roughly 2-3% [1–3]. Moreover, bilateral ovarian SLCTs areexceptionally rare accounting for approximately 1.5–2.0% ofall SLCT cases [5].
Clinical signs and symptoms of SLCT can be related toeither hormonal production [6] or presence of mass-occu-pying lesion [1–3]. While SLCTs can be functionally inac-tive, abnormal hormonal production (mostly androgen orrarely estrogen excess) can be identified in more than half of
4 Case Reports in Obstetrics and Gynecology
(a) (b)
(c) (d)
(e)
Figure 4: Immunohistochemical examination of the left ovarian mass. (a) The neoplastic cells stained positive for calretinin (magnificationpower: 40x). (b)The neoplastic cells stained positive for inhibin (magnification power: 40x). (c)The neoplastic cells stained positive for CD56(magnification power: 40x). (d) The neoplastic cells stained positive for WT-1 (magnification power: 40x). (e) The neoplastic cells stainedpositive for CK 8/18 (magnification power: 40x).
patients [6]. Clinical expression of virilization is recog-nized in more than one-third (33–38%) of patients [1, 2,4]. Androgen-excess manifestations with varying degreesinclude virilism, hirsutism, hyperseborrhea, acne, recedinghairline, alopecia, hoarseness of voice, loss of subcutaneoustissue deposits, breast atrophy, clitoromegaly, oligomenor-rhea and amenorrhea. Conversely, although rare, estrogen-excess manifestations include: precocious puberty, abnor-mal uterine bleeding, abnormal vaginal bleeding, menstrualirregularities, generalized edema, weight gain, breast hyper-trophy, endometrial hyperplasia, endometrial polyps andendometrial carcinoma.
Elevated serum levels of testosterone and androstene-dione can be often identified in approximately 80%of patients
with ovarian SLCTs and virlizing manifestations [7, 8].Testosterone serum levels greater than 200 ng/dL (7 nmol/L)are generally associated with an androgen-secreting neo-plasm from ovaries, adrenals, or elsewhere [14]. Urinary 17-ketosteroid levels are often normal or a bit elevated in patientswith SLCTs as opposed to patients with virilizing adrenaltumors who often express extremely elevated levels of urinary17-ketosteroid levels [1–3].
Nearly half of SLCTpatients experience symptoms relatedto growing space-occupying lesions of ovary [1–3]. Thesesymptoms frequently manifest as abdominal/pelvic mass orpain [1–3]. Mass is often adnexal, unilateral, and mobile[1–3]. Mass can be detected by self-examination or clinical(abdominal, vaginal, or rectovaginal) examination [6]. Pain is
Case Reports in Obstetrics and Gynecology 5
typically chronic and dull in nature, and occurs secondarily tocapsular expansion and possible subsequent compression ofnearby visceral structures [6]. Acute abdominal pain requir-ing prompt emergency intervention happens in roughly lessthan one-fifth (15%) of SLCT cases and can be attributable toovarian torsion, capsular rupture, or bleeding [6].
Imaging studies can be utilized in the diagnosis of ovarianSLCTs. Sonography (ultrasound) remains the best imagingmodality of preference for initial assessment of adnexalmasses, due to its high sensitivity, suitability, and cost-effectiveness [6, 9]. Transvaginal sonography, as opposed toabdominal sonography, appears to yield better morphologicfeatures of adnexal masses [15]. SLCTs typically exhibit solidsonographic appearance [6, 9] and mostly unilateral tumors[1–3, 5]; bilateral tumors are exceptionally uncommon [5].Components of SLCTs can be purely solid, purely cystic, ormixed [1]. Mixed (solid and cystic) components are mostcommonly encountered in roughly 60% of all ovarian SLCTs[1]; pure cystic ovarian SLCTs are extremely unusual [1, 6].Average SLCT diameter is 13.5 cm and can reach as hugeas 50 cm in poorly differentiated histological variants [1]. Inthe settings of clinical and laboratory proofs of androgenor estrogen excess, a normal sonographic study does notrule out diagnosis of ovarian SLCT, as the size of tumor cansometimes be undetectable by sonography [6]. ColorDopplersonography offers further categorization and evaluation ofneoplastic masses. Moderate-to-rich ovarian vascular masseswith low-resistance indices highly suggest malignant ratherthan benign lesions [16]. Other imaging modalities such ascomputed tomography (CT), magnetic resonance imaging(MRI), and positron imaging tomography (PET) scans can beused for better characterization of ovarian SLCTs, detectionof extraovarian disease/metastasis and identification of otherpossible primary neoplasms (e.g., a functional androgen-producing adrenal gland tumor).
Macroscopically [10], SLCTs are frequently unilateral,well-encapsulated, solid, firm, lobulated, and yellow-graymasses of roughly 7 cm in diameter on average. Cut-sectionsurface exhibits varying degrees of greasy/fleshy consistency,straw-colored fluid, necrosis, hemorrhage, and cystic spacesseparated by fibrous septae.
Microscopically [10], SLCTs are classically made up ofuncontrolled proliferation of varying degrees of differentia-tion of tubules lined by Sertoli cells and intervening nests ofLeydig cells. Well-and moderately differentiated SLCTs arethemost frequently encountered histological variants. Leydigcells are typically found in clusters in interstitial stroma andtypically exhibit polygonal cells with well-defined margins,centric nuclei, prominent nucleoli, and eosinophilic cyto-plasm. Sertoli cells typically form tubular structures lined bysingle ormultiple layers of cuboidal-columnar cells withwell-bounded margins, oval dark (basal) nuclei, inconspicuousnucleoli and eosinophilic or vacuolated cytoplasm. Mitoticfigures are extremely rare. Poorly differentiated SLCTs, as inour case report, represent a considerable diagnostic challengeowing to the huge range of microscopic/histopathologicaldiversity. The classical arrangement of tubules lined bySertoli cells and intervening nests of Leydig cells is greatlyminimal, very occasional and most often difficult to identify.
The sex-cord neoplastic cells exhibit immature (poor) dif-ferentiation with high nuclear atypia, increased nuclear-to-cytoplasmic ratio, coarse chromatin and extremely abundantmitotic figures. For histopathologists with low index ofsuspicion of ovarian SLCTs, all these microscopic featurescan be misleading and easily mistaken for a diagnosis ofundifferentiated sarcoma.
Immunohistochemically, almost all SLCTs, includingmoderately and poorly differentiated variants, stain positivefor inhibin and calretinin, and negative for epithelial mem-brane antigen (EMA) [11]. In addition, it has been shown thatSLCTs stain positive forWT-1 [17] andCD56 [18]. A collectiveprofile of hematoxylin and eosin (H&E) stains in addition toimmunohistochemical studies are expected to yield the mostaccurate definitive diagnosis of SLCTs [4].
Management of ovarian SLCTs remains challengingowing to lack of standardized management protocol guide-lines [12]. Surgical resection represents the mainstay of man-agement of ovarian SLCTs [4]. Fortunately, the vast majorityof SLCTs are largely diagnosed during reproductive age[1–4], frequently unilateral, mostly confined to ovary andnearly 90% classified as stage I at time of clinical diagnosis[5]. Therefore, fertility-sparing surgery (unilateral salpin-gooophorectomy) can be considered in all patients with well-differentiated ovarian SLCTs. Patients desiring fertility andexhibiting moderately or poorly differentiated ovarian SLCTscan be considered for unilateral salpingo-oophorectomyplus standard staging surgery (omentectomy, appendectomy,and pelvic lymphadenectomy) [12, 19]. The need for pelviclymphadenectomy is still debatable. However, the widelyaccepted conclusion is that pelvic lymph node metastasis isextremely rare in ovarian SLCTs and therefore, pelvic lym-phadenectomy may be excluded during the staging surgery[20]. Patients who are elderly, not desiring fertility or withprogressively advanced SLCTs should be considered for totalhysterectomy, bilateral salpingooophorectomy in addition tocomplete standard staging surgery [18].
Owing to rarity of ovarian SLCTs, limited number ofdocumented case reports/series and lack of randomizedclinical trials, effectiveness of post-operative chemotherapyremains questionable and requires further evaluation [4,12, 19, 21]. Generally, postoperative chemotherapy is con-sidered for patients with poor prognostic factors such as:advanced disease staging, moderate-to-poor tumor grading,high mitotic profile, existence of heterologous elements andtumor rupture [1–3]. The first-line and most frequently usedchemotherapeutical regimen is bleomycin, etoposide, andcisplatin (BEP) [22, 23]. Other regimens also exist such as (1)cisplatin, Adriamycin, and cyclophosphamide (CAP), and (2)cisplatin, vinblastine, and bleomycin (PVB) [24].
Prognosis of ovarian SLCTs is significantly correlatedwith degree of tumor differentiation (grading) and tumorextent (staging) [1]. Well-differentiated (grade 1) SLCTs areassociatedwith zeromalignant potential, whereasmoderately(grade 2) and poorly (grade 3) differentiated SLCTs areassociated with 11% and 59%malignant potential respectively[1]. The overall 5-year survival rate for well-differentiated(grade 1) SLCTs is 100%, whereas for moderately (grade 2)and poorly (grade 3) differentiated SLCTs is collectively 80%
6 Case Reports in Obstetrics and Gynecology
[21].With respect to tumor staging, the overall 5-year survivalrate for stage I is 95% [2]whereas for stages III and IV is nearlyzero percent [1]. Long-term followup is highly advised in allpatients.
Acknowledgment
The authors sincerely acknowledge the editorial assistanceof Ms. Ranim Chamseddin, College of Medicine, AlfaisalUniversity, Riyadh, Saudi Arabia.
References
[1] R. H. Young and R. E. Scully, “Ovarian Sertoli-Leydig celltumors. A clinicopathological analysis of 207 cases,” AmericanJournal of Surgical Pathology, vol. 9, no. 8, pp. 543–569, 1985.
[2] C. Zaloudek and H. J. Norris, “Sertoli-Leydig tumors of theovary. A clinicopathologic study of 64 intermediate and poorlydifferentiated neoplasms,” American Journal of Surgical Pathol-ogy, vol. 8, no. 6, pp. 405–418, 1984.
[3] L.M.Roth,M.C.Anderson, andA.D. T.Govan, “Sertoli-Leydigcell tumors: a clinicopathologic study of 34 cases,” Cancer, vol.48, no. 1, pp. 187–197, 1981.
[4] C. S. Weng, M. Y. Chen, T. Y. Wang et al., “Sertoli-Leydig celltumors of the ovary: a Taiwanese gynecologic oncology groupstudy,”Taiwanese Journal of Obstetrics &Gynecology, vol. 52, no.1, pp. 66–70, 2013.
[5] R. H. Young andR. E. Scully, “Sex cord-stromal, steroid cell, andother ovarian tumors,” inBlaustein’s Pathology of Female GenitalTract, R. J. Kurman, Ed., p. 929, Springer, New York, NY, USA,5th edition, 2002.
[6] K. M. Zanotti, “The clinical manifestations and diagnosisof Sertoli-Leydig cell tumors of the ovary,” CME Journal ofGynecologic Oncology, vol. 7, no. 2, pp. 129–133, 2002.
[7] R. H. Osborn and M. E. Yannone, “Plasma androgens in thenormal and androgenic female: a review,” Obstetrical & Gyne-cological Survey, vol. 26, no. 3, pp. 195–228, 1971.
[8] F. T. Prunty, “Hirsutism, virilism and apparent virilism and theirgonadal relationship. II,” Journal of Endocrinology, vol. 38, no. 2,pp. 203–227, 1967.
[9] C. M. de Oliveira Franzin, M. L. Kraft, D. Faundes, L. C.Zeferino,M.Alvarenga, andE. F.Marussi, “Detection of ovarianSertoli-Leydig cell tumors exclusively by color Doppler sonog-raphy,” Journal of Ultrasound in Medicine, vol. 25, no. 10, pp.1327–1330, 2006.
[10] M. Nouriani, J. C. Felix, and L. Dubeau, “Histogenesis andhistopathological characteristics of Sertoli-Leydig cell tumors,”CME Journal of Gynecologic Oncology, vol. 7, no. 2, pp. 114–120,2002.
[11] W. G. McCluggage and R. H. Young, “Immunohistochemistryas a diagnostic aid in the evaluation of ovarian tumors,” Semi-nars in Diagnostic Pathology, vol. 22, no. 1, pp. 3–32, 2005.
[12] R. A. Bhat, Y. K. Lim, Y. N. Chia, and K. L. Yam, “Sertoli-Leydigcell tumor of the ovary: analysis of a single institution database,”Journal of Obstetrics and Gynaecology Research, vol. 39, no. 1, pp.305–310, 2013.
[13] V.W. Chen, B. Ruiz, J. L. Killeen, T. R. Cote, X. C.Wu, and C. N.Correa, “Pathology and classification of ovarian tumors,” Can-cer, vol. 97, supplement 10, pp. 2631–2642, 2003.
[14] D. R. Meldrum and G. E. Abraham, “Peripheral and ovarianvenous concentrations of various steroid hormones in virilizing
ovarian tumors,” Obstetrics and Gynecology, vol. 53, no. 1, pp.36–43, 1979.
[15] E. B. Mendelson, M. Bohm-Velez, N. Joseph, and H. L. Neiman,“Gynecologic imaging: comparison of transabdominal andtransvaginal sonography,”Radiology, vol. 166, no. 2, pp. 321–324,1988.
[16] E. H. Yanushpolsky, D. L. Brown, and B. L. Smith, “Localizationof small ovarian Sertoli-Leydig cell tumors by transvaginalsonography with color Doppler,” Ultrasound in Obstetrics &Gynecology, vol. 5, no. 2, pp. 133–135, 1995.
[17] H. P. Cathro andM. H. Stoler, “The utility of calretinin, inhibin,and WT1 immunohistochemical staining in the differentialdiagnosis of ovarian tumors,” Human Pathology, vol. 36, no. 2,pp. 195–201, 2005.
[18] W. G. McCluggage, M. McKenna, and H. A. McBride, “CD56is a sensitive and diagnostically useful immunohistochemicalmarker of ovarian sex cord-stromal tumors,” InternationalJournal of Gynecological Pathology, vol. 26, no. 3, pp. 322–327,2007.
[19] T. Gui, D. Cao, K. Shen et al., “A clinicopathological analysisof 40 cases of ovarian Sertoli-Leydig cell tumors,” GynecologicOncology, vol. 127, no. 2, pp. 384–389, 2012.
[20] J. Brown,A.K. Sood,M. T.Deavers, L.Milojevic, andD.M.Ger-shenson, “Patterns of metastasis in sex cord-stromal tumors ofthe ovary: can routine staging lymphadenectomy be omitted?”Gynecologic Oncology, vol. 113, no. 1, pp. 86–90, 2009.
[21] C. Sigismondi, A. Gadducci, D. Lorusso et al., “Ovarian Sertoli-Leydig cell tumors. A retrospective MITO study,” GynecologicOncology, vol. 125, no. 3, pp. 673–676, 2012.
[22] F. Y. Chen, B. C. Sheu, M. C. Lin, S. N. Chow, and H. H. Lin,“Sertoli-Leydig cell tumor of the ovary,” Journal of the FormosanMedical Association, vol. 103, no. 5, pp. 388–391, 2004.
[23] P. Sachdeva, R. Arora, C. Dubey, A. Sukhija, M. Daga, and D. K.Singh, “Sertoli-Leydig cell tumor: a rare ovarian neoplasm.Casereport and review of literature,” Gynecological Endocrinology,vol. 24, no. 4, pp. 230–234, 2008.
[24] B. J. Roth, A. Greist, P. S. Kubilis, S. D. Williams, and L. H.Einhorn, “Cisplatin-based combination chemotherapy for dis-seminated germ cell tumors: long-term follow-up,” Journal ofClinical Oncology, vol. 6, no. 8, pp. 1239–1247, 1988.
Gestational trophoblastic disease in the western region of Saudi Arabia(single-institute experience)
Nisrin Anfinan, Khalid Sait *, Hesham SaitScientific chair of prof. Abdullah Hussain Basalamah for Gynecological Cancer, Faculty of Medicine, King Abdulaziz University Hospital, Jeddah, Saudi Arabia
A R T I C L E I N F O
Article history:Received 21 December 2013Received in revised form 11 April 2014Accepted 4 June 2014
Keywords:Trophoblastic diseaseIncidenceSurvival rate
A B S T R A C T
Objective: To estimate the prevalence of gestational trophoblastic disease (GTD) in the western region ofSaudi Arabia, and to evaluate the success of treatment and the effect of age and risk group on survival.Methods: Between January 2001 and December 2010, all patients treated for GTD were identified from theKing Abdulaziz University Hospital database. Patients with persistent disease were evaluated accordingto their clinical treatment outcomes.Results: In total, 122 cases of GTD were identified in the database. Of these, 77 (63%) cases were diagnosedand received initial treatment at the study centre, resulting in an incidence of 1.26 cases per 1000deliveries. The mean (!standard deviation) age of the study participants was 31.52 ! 10.8 years, meangestational age at diagnosis was 12.42 ! 3.2 weeks, and mean follow-up for each patient was 24 months.There were 20 cases (26%) of persistent GTD after treatment. The majority of patients with low-riskdisease were treated with single-agent methotrexate, with an overall success rate of 83%. The overall 5-year survival rate for all patients was 98%. Using the Wilcoxon (Gehan) test, risk group and age (cut-off 40years) were not found to be significantly associated with survival (p = 0.69).Conclusions: This single-institute study reports the first survival data for GTD for Saudi Arabia. However,the overall incidence of GTD in Saudi Arabia will be defined by establishment of a GTD registry.
ã 2014 Elsevier Ireland Ltd. All rights reserved.
Introduction
Gestational trophoblastic disease (GTD) represents a heteroge-neous group of disorders characterized by abnormally formedplacentas and neoplasms, elevated human chorionic gonadotropin(hCG), and a constellation of clinical symptoms. The World HealthOrganization (WHO) classification of GTD includes hydatidiformmole (HM), complete mole, partial mole, invasive mole, chorio-carcinoma, placental site trophoblastic tumour (PSTT) andepithelioid trophoblastic tumour [1,2].
The classic presenting symptoms and clinical findings of HMinclude vaginal bleeding, passage of vesicles, anaemia, excessiveuterine enlargement, toxaemia of pregnancy, hyperemesis gravida-rum, hyperthyroidism, trophoblastic emboli and theca lutein cystsassociated with an extremely high level of hCG [3]. Occasionally, theclinical presentation is incomplete or missed abortion [4]. Factorsthat have been associated with increased risk for HM includematernal age, previous molar pregnancy, parity, ethnicity, socio-economic status and environmental exposure [1,5,6].
Gestational trophoblastic neoplasia (GTN) (which may repre-sent invasive mole, choriocarcinoma and PSTT) may be diagnosedafter evacuation of a complete or partial molar pregnancy. It canalso be diagnosed following abortion, ectopic pregnancy or normalpregnancy, typically based on plateauing of hCG level over at least3 weeks, an increase of "10% in hCG level for three or more valuesover at least 2 weeks or persistent hCG 6 months after molarevacuation [7].
According to the prognostic scoring index of the modified WHOscoring system [8], the majority of women diagnosed with low-riskGTN can be treated with single-agent methotrexate (MTX) andfolinic acid. Women with high-risk GTN require multi-agentchemotherapy with etoposide, methotrexate, actinomycin D,cyclophosphamide and vincristine (EMA-CO) [5,9–11].
The reported incidence of GTD varies widely in different regionsof the world at different times. In North America and Europe, HMoccurs in approximately 0.5–1 per 1000 pregnancies [1]. Higherfrequencies have been reported in some areas of Asia and theMiddle East, with rates ranging from 1 to 12 per 1000 pregnancies[12,13].
In Saudi Arabia, studies have reported prevalence rates of HM of0.94 per 1000 deliveries [14] and 2.2 per 1000 deliveries [15].Felemban et al. reported that the prevalence of complete HM is 1
* Corresponding author.E-mail address: [email protected] (K. Sait).
http://dx.doi.org/10.1016/j.ejogrb.2014.06.0050301-2115/ã 2014 Elsevier Ireland Ltd. All rights reserved.
European Journal of Obstetrics & Gynecology and Reproductive Biology 180 (2014) 8–11
Contents lists available at ScienceDirect
European Journal of Obstetrics & Gynecology andReproductive Biology
journal homepage: www.else vie r .com/ locat e/e jogrb
per 676 live births [16]. A worldwide GTD registry would help todefine the incidence of GTD accurately, and provide active follow-up for all patients and early detection and treatment of patientswith persistent disease.
This hospital-based study evaluated temporal trends in the rateof GTD, treatment success and patient survival in the westernregion (Makkah) of Saudi Arabia in order to establish theinfrastructure for a Saudi GTD registry.
Methods
Between January 2001 and December 2010, all patients withGTD treated at the King Abdulaziz University Hospital (KAUH),Jeddah, Saudi Arabia were identified using the hospital’s database.The study was approved by the Unit of Biomedical Ethics, Faculty ofMedicine, King Abdulaziz University. The total number ofdeliveries at KAUH ranges from 2761 to 4950 per year. KAUH isa referral centre for the western region of Saudi Arabia, so GTN andmolar pregnancies diagnosed at KAUH, and cases of persistent GTDthat require chemotherapy and have been referred from elsewhereare treated at the centre. Therefore, referred cases were excludedfrom calculations regarding the prevalence of molar pregnancy andthe risk of persistent disease after molar pregnancy. However, themedical records of all patients with GTD were reviewedretrospectively with regard to maternal age, parity, clinicalpresentation, gestational age, initial treatment and follow-updetails. Patients with persistent disease were evaluated for clinicaltreatment outcome, type of chemotherapy, response rate andsurvival. The toxicity of chemotherapy was evaluated according tothe common toxicity criteria of the National Cancer Institute(Version 2.0; http://ctep.cancer.gov/forms/CTCv20-4_30-992.pdf).
The effect of maternal age was evaluated, and a cut-off of 40 yearswas selected for group comparison. Data were entered intoMicrosoft Excel (Microsoft Corp., Redmond, WA, USA) for analysis,which was performed using Statistical Package for Social SciencesVersion 17 (SPSS Inc., Chicago, IL, USA). Qualitative data arereported as numbers and percentages. Chi-squared test with Yates’correction was used to compare qualitative data. Odds ratios (OR)and 95% confidence intervals (CI) were calculated to estimate risk.Quantitative data are reported as mean, standard deviation andrange. Student’s t-test was used to compare quantitative data. Theoverall survival functions of the study groups were constructedusing the log-rank test to compare survival rates. p < 0.05 wasconsidered to indicate statistical significance.
Results
In total, 122 cases of GTD were identified in the database; 77(63%) of these cases were diagnosed as molar pregnancies andtreated at the study centre, and 45 (37%) cases had persistent GTDand had been referred from other hospitals in the western region ofSaudi Arabia. Therefore, the prevalence of molar pregnancy at thestudy centre was 1.26 per 1000 deliveries. Table 1 shows thedemographic characteristics of the study population.
The main clinical presentations were vaginal bleeding (n = 90,74%), large size for date (n = 54, 44%) and abdominal pain (n = 46,38%). Five patients (4%) presented with associated pre-eclampsia.Only three patients (3%) had been diagnosed previously with amolar pregnancy.
Ninety-one (75%) of the 122 cases had a pre-evacuationultrasound to confirm the diagnosis of HM. Only 31 (26%) caseswere diagnosed after evacuation for incomplete or missedabortion, based on the histopathological results.
Table 1Demographic data of patients (n = 122).
Variable n
Age (years)Mean ! SD 31.52 ! 10.8Range 15–56
ParityMedian 3Range 0–7
Gestational age (weeks)Mean ! SD 12.42 ! 3.2Range 4–22
Pre-evacuation level of hCG<100,000 u/ml 30 (25%)>100,000 u/ml 92 (75%)
NationalitySaudi 53 (43%)Non-Saudi 69 (57%)
Median follow-up (months) 24Range 12–72
hCG: human chorionic gonadotropin; SD: standard deviation.
Table 2Summary of patients who underwent hysterectomy.
Case Age (years) Parity (n) Indication Pathology
1 52 10 Initial treatment Complete mole2 45 7 Initial treatment Complete mole3 54 10 Initial treatment Complete mole4 49 7 Initial treatment Complete mole5 47 7 Initial treatment Complete mole6 54 9 Initial treatment Complete mole7 50 7 Failure of initial treatment Choriocarcinoma
Table 3Characteristics of patients with persistent and non-persistent gestational tropho-blastic disease.
Odds ratio Persistent(n = 65)n (%)
Non-persistent(n = 57)n (%)
p-value
Age (years)"40
15 (23) 15 (26) 0.38
<40 50 (77) 42 (74) 0.48 (OR 1.08, 95% CI0.46–2.53)
Pre-evacuationlevel of hCG<100,000 u/ml>100,000 u/ml
11 (16.9)54 (83.1)
19 (33.3)38 (66.7)
0.103 (OR 4.36, 95% CI1.76–25.8)
hCG: human chorionic gonadotropin; OR: odds ratio; CI: confidence interval.
Table 4Summary of high-risk cases of gestational trophoblastic neoplasia.
Case Age(years)
Parity(n)
Antecedentpregnancy
Site ofmetastases
WHOscore
Alive
1a 54 7 Complete mole Abdomen/lung
9 Yes
2 27 1 Partial mole Lung 7 Yes3 29 12 Complete mole Lung 7 Yes4 37 3 Complete mole Lung/liver 10 Yes5 35 6 Complete mole Lung/
abdominalwall
9 Yes
6 25 2 Complete mole Lung 7 Yes7 27 9 Complete mole Lung 7 Yes8 26 2 Complete mole Lung 7 Yes9 30 9 Term pregnancy Vagina/lung 8 Yes
10 21 1 Complete mole Lung 7 Yes11b 35 3 Unknown Liver/lung 10 No
WHO: World Health Organization.a Patient elected to have hysterectomy prior to chemotherapy.b Patient did not receive chemotherapy, as died 24 h after arrival.
N. Anfinan et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 180 (2014) 8–11 9
At the study centre, 52 (68%) cases were complete molarpregnancies and 25 (33%) cases were partial molar pregnancies.Suction dilatation and curettage was performed as the initialtreatment in 71 cases (92%), and six (8%) patients elected toundergo hysterectomy (Table 2). The mean age of the patients whounderwent hysterectomy as an initial treatment was 50 years(range 47–54 years), and average parity was 8 (range 7–12).
The diagnostic criteria used in most US centres were used todiagnose persistent GTD in this study [7]. At the study centre,patients diagnosed with persistent GTD undergo chest X ray, pelvicand abdominal ultrasound, and computer tomography of the brainas initial work up.
Nineteen (25%) cases of complete mole, one (1%) case of partialmole and 20 (26%) cases of both complete and partial mole werediagnosed at the study centre. In total, 65 patients (including 45patients referred from other hospitals) were treated for persistentGTD (one followed a term pregnancy). No cases of PSTT were seenat the study centre.
Forty-eight (81%) cases were categorized as low risk accordingto the prognostic scoring index of the modified WHO scoringsystem [8]. Forty-seven of these cases were treated with single-agent MTX according to the protocol at the study centre, consistingof an 8-day regimen of MTX (1 mg/kg Days 1, 3, 5 and 7) alternatingwith folinic acid (0.1 mg/kg Days 2, 4, 6 and 8). The remaining low-risk case had a contraindication for MTX, and was treated withsingle-agent actinomycin D 1.25 mg/m2 every 2 weeks. All low-riskpatients received one additional cycle after their hCG levelreturned to normal. One patient, who was treated with MAC(methotrexate, actinomycin D and cyclophosphamide) because shewas thought to be high risk at her initial presentation,unfortunately died of septic shock due to severe febrile neutrope-nia. One patient treated with MTX experienced Grade 2 mucositis.
The success rate of single-agent MTX was 81%. Nine patientswere not treated successfully with single-agent MTX, and wereswitched to single-agent actinomycin D; this treatment wassuccessful in all cases. Table 3 compares patients with persistentGTD with patients with non-persistent GTD. There was nosignificant difference in persistent GTD rates between patientsaged <40 years and "40 years (p = 0.3, OR 1.08, 95% CI 0.46–2.52),and the pre-evacuation level of hCG was not associated with therisk of persistent GTD (p = 0.1, OR 4.36, 95% CI 1.76–25.8).
Table 4 shows the characteristics of the high-risk group. At thestudy centre, high-risk patients receive EMA-CO [Days 1 and 2:etoposide 100 mg/m2, methotrexate 300 mg/m2 (Day 1 only) andactinomycin D 0.3 mg/m2; Days 7 and 8: cyclophosphamide600 mg/m2 and vincristine 1 mg/m2]. All high-risk patientsreceived two additional cycles of EMA-CO after their hCG levelreturned to normal.
There were three deaths from GTN. The first patient died beforetreatment started; she presented with respiratory failure and diedin the intensive care unit on the first day of admission. The secondpatient died a few years after treatment from a road trafficaccident; she was in the low-risk group. The third patient, also inthe low-risk group, was treated with MAC and died due tochemotherapy toxicity. The overall 5-year survival rate in thisstudy was 98% (Fig. 1; 91% for high-risk patients and 96% for low-risk patients). A comparison using the Wilcoxon (Gehan) testindicated that this difference was not statistically significant(p = 0.69).
The survival rates of patients aged "40 years and <40 yearswere 93% and 98%, respectively. A comparison using the Wilcoxon(Gehan) test revealed that this difference was not statisticallysignificant (p = 0.49) (Fig. 2).
Discussion
The reported incidence of HM is 1.26 per 1000 deliveries inSouth Africa [17] and 0.87–4.88 per 1000 deliveries in Nigeria inWest Africa [18]. Higher frequencies have been reported for someareas of Asia and the Middle East, with rates ranging from 1 to 12per 1000 pregnancies [1,19]. However, as many of these countrieslack a tumour registry, the true incidence of GTD is unknown.
The few studies undertaken at different centres in Saudi Arabiareported variable prevalence rates and used small study popula-tions [14–16]. The population for the present study had the largestnumber of molar pregnancies in a single centre, with an incidenceof 1.26 per 1000 deliveries. This prevalance was higher than thatreported by Khashoggi for the eastern region of Saudi Arabia [14].This difference may be explained by the fact that the westernregion of Saudi Arabia is considered a sea coast, unlike the easternregion of Saudi Arabia, and the western region welcomes manypilgrims every year, some of whom remain. These pilgrims may beof different ethnic backgrounds, and 57% of the study populationincluded expatriates (Table 5).
Complete mole was the most common histological type of HMin this study, and accounted for 68% of cases, which is consistentwith previously published data [19,20]. This study demonstrated
Fig. 1. Overall 5-year survival rate (98%).
Fig. 2. Survival rate for patient aged "40 years was 93%, compared with 98% forpatients aged <40 years. According to a Wilcoxon (Gehan test) comparison, thisdifference was not significant (p = 0.49).
Table 5Worldwide incidence of molar pregnancy.
Regions Incidence/1000 pregnancies
North America and Europe [1] 0.5–1Asia and Middle East [12,13] 1–12Saudi Arabia [14–16] 0.94–2.2South Africa [17] 0.87–4.88West Africa and Nigeria [18] 1.26
10 N. Anfinan et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 180 (2014) 8–11
that persistent GTD occurred in 26% of cases, which is highcompared with the literature (20%) [1,21].
Reported predictors of GTN in women with a complete moleinclude a pre-evacuation serum level of b-hCG >100,000 mIU/ml,clinical and/or pathological evidence of marked trophoblasticovergrowth (including excessive uterine size), and maternal age>40 years [6,20]. However, this study did not find that maternal ageand b-hCG level affected the risk of developing persistent disease.
Chemotherapy is a major component of curative therapy for themajority of women diagnosed with malignant GTD. As a group,malignantgestational trophoblastic tumoursare extremelysensitiveto chemotherapy, and represent one of the only cancers for whichsingle-agent therapy is still in use. The choice of single-agent vscombination chemotherapy depends on the patient's risk category.
Different regimens of MTX may be similarly effective but lesstoxic than the 8-day regimen in patients with non-metastaticmalignant GTD (low risk) [21–23]; however, to the authors'knowledge, no randomized controlled trials have been conducted.
The most commonly used MTX regimen in Europe, and possiblyin the rest of the world other than North America, is an 8-dayalternating regimen of intramuscular MTX and folinic acid,repeated every 14–16 days [21]. In North America, many centresuse the weekly low-dose MTX protocol, which was first tested andreported by the Gynecologic Oncology Group. This parenteralregimen is initiated at 30 mg/m2 per week, and the dose isescalated by 5 mg/m2 at 3-week intervals to 50 mg/m2 [23]. Todate, there is no consensus on the optimum regimen. This studyused the 8-day alternating regimen of MTX with folinic acid in themajority of low-risk patients. The overall success of this group ofpatients was 88%, which is similar to reports in the literature [21–23]. This study included 10 high-risk patients with modified WHOscores >7; these patients received the EMA-CO regimen andachieved complete remission. This regimen has been the standardof care in managing high-risk GTN, and success rates up to 78%have been reported [24].
GTD carries a good prognosis, even in the presence ofmetastasis. With correct management, survival rates of 94% and60% for low- and high-risk cases, respectively, have been reported[24,25]. The present study observed an overall survival rate of 98%.
Limitations of this study include the inability to assessabortions and ectopic pregnancies, and the fact that it was asingle-centre study and may not be representative of the incidenceof GTD throughout Saudi Arabia. However, this study confirms thepreviously reported low incidence of GTD in Saudi Arabia.
This study reports survival data for GTD in Saudi Arabia;however, the overall incidence of GTD in Saudi Arabia will be betterdefined by establishment of a GTD registry.
Conflict of interest
None declared.
Acknowledgements
The authors would like to thank the Deanship of ScientificResearch, King Abdulaziz University, Jeddah (399/140/1431) fortheir research support, and the Facility of the Scientific Chair ofProf. Abdullah Hussain Basalamah for Gynaecological Cancer.
References
[1] Altieri A, Francheschi S, Ferlay J, Smith J, La Vecchia C. Epidemiology andetiology of gestational trophoblastic diseases. Lancet Oncol 2003;4:670–8.
[2] Shih LM, Kurman RJ. Pathogenesis of gestational trophoblastic lesions. In:Giordano A, Bovicelli A, Kurman R, editors. Current clinical oncology:molecular pathology of gynecologic cancer. Totowa, NJ: Humana Press;2007. p. 157–66.
[3] Berkowitz RS, Goldstein DP. Molar pregnancy. N Engl J Med 2009;360:1639–45.
[4] Lindholm H, Flam F. The diagnosis of molar pregnancy by sonography andgross morphology. Acta Obstet Gynecol Scand 1999;78:6–9.
[5] Drake RD, Rao GG, McIntire DD, Miller DS, Schorge JO. Gestationaltrophoblastic disease among Hispanic women: a 21-year hospital-basedstudy. Gynecol Oncol 2006;103:81–6.
[6] Altman AD, Bentley B, Murray S, Bentley JR. Maternal age-related rates ofgestational trophoblastic disease. Obstet Gynecol 2008;112:244–50.
[7] Kohorn EI. Negotiating a staging and risk factor scoring system for gestationaltrophoblastic neoplasia: a progress report. J Reprod Med 2002;47:445–50.
[8] Goldstein DP, Zanten- IV, Przybysz, Bernstein MR, Berkowitz RS. Revised FIGOstaging system for gestational trophoblastic tumors. Recommendationregarding therapy. J Reprod Med 1998;43:37–43.
[9] Lurain JR, Singh DK, Schink JC. Primary treatment of metastatic high-riskgestational trophoblastic neoplasia with EMA-CO chemotherapy. J ReprodMed 2006;51:767–72.
[10] Turan T, Karacay O, Tulunay G, et al. Results with EMA/CO (etoposide,methotrexate, actinomycin D, cyclophosphamide, vincristine) chemotherapyin gestational trophoblastic neoplasia. Int J Gynecol Cancer 2006;16:1432–8.
[11] Escobar PF, Lurain JR, Singh DK, Bozorgi K, Fishman DA. Treatment of high-riskgestational trophoblastic neoplasia with etoposide, methotrexate, actinomy-cin D, cyclophosphamide, and vincristine chemotherapy. Gynecol Oncol2003;91:552–7.
[12] Aziz MF, Kampono N, Moegni EM, Sjamsuddin S, Barnas B, Samil RS.Epidemiology of gestational trophoblastic neoplasm at the Dr. CiptoMangunkusumo Hospital, Jakarta, Indonesia. Adv Exp Med Biol1984;176:165–75.
[13] Bracken MB, Brinton LA, Hayashi K. Epidemiology of hydatidiform mole andchoriocarcinoma. Epidemiol Rev 1984;6:52–75.
[14] Khashoggi TY. Prevalence of gestational trophoblastic disease. A singleinstitution experience. Saudi Med J 2003;24:1329–33.
[15] Chattopadhyay SK, Senupta BS, Chattopadhyay C, Hassounah MH. Changingpattern of maternal mortality in Riyadh, Saudi Arabia. Saudi Med J1985;6:441–53.
[16] Felemban AA, Bakri YN, Alkharif HA, Altuwaijri SM, Shalhoub J, Berkowitz RS.Complete molar pregnancy: clinical trends of King Fahad Hospital, Riyadh,Kingdom of Saudi Arabia. Reprod Med J 1998;43:11–3.
[17] Osamor JO, Olowasola AO, Adewole IF. A clinic-pathological study of completeand partial hydatidiform moles in Nigeria. J Obstet Gynaecol 2002;22:423–5.
[18] Steigrad SJ. Epidemiology of gestational trophoblastic diseases. Best Pract ResClin Obstet Gynaecol 2003;17:837–47.
[19] Sakumoto K, Higashi M, Kanazawa K. Hydatidiform mole in Okinawa islandsand mainland Japan. Int J Gynaecol Obstet 1999;64:10–30.
[20] Murad TM, Longley JV, Lurain JR, Brewer JI. Hydatidiform mole: clinicopatho-logic associations with the development of postevacuation trophoblasticdisease. Int J Gynaecol Obstet 1990;32:359–67.
[21] McNeish IA, Strickland S, Holden L, et al. Low-risk persistent gestationaltrophoblastic disease: outcome after initial treatment with low-dosemethotrexate and folinic acid from 1992 to 2000. J Clin Oncol2002;20:1838–44.
[22] Homesley HD, Blessing JA, Rettenmaier M, Capizzi RL, Major FJ, Twiggs LB.Weekly intramuscular methotrexate for nonmetastatic disease. ObstetGynecol 1988;72:413–8.
[23] Osborne RJ, Filiaci V, Schink JC, et al. Phase III trial of weekly methotrexate orpulsed dactinomycin for low-risk gestational trophoblastic neoplasia: aGynecologic Oncology Group study. J Clin Oncol 2011;29:825–31.
[24] Curry SL, Blessing JA, DiSaia PJ, Soper JT, Twiggs LB. A prospective randomizedcomparison of methotrexate, dactinomycin, and chlorambucil versus metho-trexate, dactinomycin, cyclophosphamide, doxorubicin, melphalan, hydroxy-urea, and vincristine in “poor prognosis” metastatic gestational trophoblasticdisease: a Gynecologic Oncology Group study. Obstet Gynecol 1989;73:357–62.
[25] Gilani MM, Fariba B, Behtash N, Ghaemmaghami F, Moosavi AS, Rezayof E. TheWHO score predicts treatment outcome in low risk gestational trophoblasticneoplasia patients treated with weekly intramuscular methotrexate. J CancerRes Ther 2013;9:38–43.
N. Anfinan et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 180 (2014) 8–11 11
Uterine sarcoma
Clinico-pathological characteristics and outcome
Hesham K. Sait, MS, Nisrin M. Anfinan, MBChB, SBOG, Mohamed E. El Sayed, MBBCh, PhD, Shadi S. Alkhayyat, MBBS, FRCPC, Ahmad T. Ghanem, MD, PhD, Reem M. Abayazid, MBBS, SBOG, Khalid H. Sait, MBChB, FRCS(C).
1215
ABSTRACT
األهداف: أجريت هذه الدراسة االستعادية للتحقيق في اخلصائص املؤثرة، العوامل إلى جنب مع املرضية، جنب السريرية والتشريحية
نتائج العالج، ومنط االنتكاس وحتليل وبقاء مرض ساركوما الرحم.
ساركوما من يعانون اللذين املرضى جميع حتديد مت الطريقة: عبدالعزيز، امللك جامعة ملستشفى بيانات قاعدة واستخدم الرحم جدة، اململكة العربية السعودية خالل الفترة ما بني2000م وديسمبر
2012م.
الرحم ساركوما من يعانون 36مريضا استعراض مت الطريقة: وجد وقد ، عاما 57 للمجموعة العمر متوسط أن التقارير أفادت 21 (%58) أن احلاالت carcinosarcomaمن 7 و (19%), ( leiomyosarcoma (LMS)ساركوما اللحمية الرحم بطانة ،
undifferentiated endometrial sarcoma 6 في شخصت .rhabdomyosarcoma (RMS) مرضى (%17) و حالتني (6%) و قد وجد أن ما يقارب نصف احلاالت شخصت في املرحلة الثالثة، %28)والرابعة (15)و(25% كانت حني في . التوالي على (%6) وحالتني فقط حالة أي (%41) من في املرحلة األولى، الرحم كامل باستئصال اجلراحي العالج وكان الثانية. املرحلة في واملبايض وقنوات فالوب باإلضافة إلى استئصال الغدد اللمفاوية لدى 18 مريضة %50، أما استئصال كامل الرحم واملبايض وقنوات فالوب اجريت على 4 مريضات (%19 ) .العالجdebulkingوكامل الورم 24 مريضة %69(الكيميائي والعالج اإلشعاعي املصاحب اعطي ( مريضات %14(و5 التوالي) على املتابعة. فترة متوسط وفي
. وفي الدراسة أصيبت 8مرضيات (%22) بانتكاسه 13.5 شهرا.ان5-2 التوالي(DFS) وجدنا على و14% 22% هو للمرضى
وغزو اللمفاوية الدموية األوعية أن املتقدمة املرحل DFSارتبطت p=0.015 وp=0.0001في حني استخدم العالج الكيماوي في
.DFS (p=0.027) حتسن في
اخلامتة: في هذه السلسلة الصغيرة من املرضى مت حتديد العوامل التياملرضى على %30 من فقط الرحم تزيد من سوء مرض ساركوما قيد احلياة ملدة سنتني. و يدعو ذلك للضرورة امللحة إلى أدوات أكثر
عدوانية للقضاء على هذا املرض.
Objectives: To investigate the clinical and histopathological characteristics, with the prognostic factors, treatment outcome, pattern of relapse, and survival analysis of uterine sarcoma patients.
Methods: All patients with histologically proven uterine sarcoma were identified using the database at King Abdulaziz University Hospital, Jeddah, Saudi Arabia between January 2000 and December 2012.
Results: A total of 36 patients with uterine sarcoma were reviewed. The median age of all patients was 57 years, and the mean age was 57.72±13.17 years. Carcinosarcoma was reported in 21 patients (58%), leiomyosarcoma in 7 (19%), undifferentiated endometrial sarcoma in 6 (17%), and rhabdomyosarcoma in 2 (6%). Approximately half of the patients were stages III and IV (28% and 25%), while 15 patients (41%) were stage I; only 2 patients (6%) were stage II. The surgical treatment was hysterectomy and bilateral salpingoophorectomy (H+BSO) plus staging in 18 patients (50%), while in 4 patients (19%), H+BSO plus debulking was performed. Adjuvant chemotherapy was given in 24 (69%) and adjuvant radiotherapy in 5 (14%) cases, At a median follow-up period of 13.5 months, 8 patients (22%) relapsed. The 2-year disease-free survival (DFS) rate was 22% and the 5-year was 14%. In the multivariate analysis, the advanced stages (p=0.015) and lymph vascular invasion (p=0.0001) were associated with poor DFS , while the use of chemotherapy significantly improved the DFS (p=0.027).
Conclusions: The poor outcome of high-grade uterine sarcoma patients was identified, and only one third of patients (30%) survived for 2 years. This finding necessitates the need for more aggressive tools to fight this disease.
Saudi Med J 2014; Vol. 35 (10): 1215-1222
From the Scientific Chair of Prof. Abdullah Hussein Basalamah for Gynecological Cancer (Sait H, Abayazid, Sait K), Gynecology Oncology Unit (Anfinan), Department of Medicine (Alkhayyat), Department of pathology (Ghanem); Faculty of Medicine, King Abdulaziz University, Jeddah, Kingdom of Saudi Arabia, and the National Cancer Institute, Radiotherapy and Nuclear Medicine Department (El Sayed), Cairo University, Cairo, Egypt.
Received 10th April 2014. Accepted 5th June 2014.
Address correspondence and reprint request to: Dr. Khalid H. Sait, Gynecology Oncology Unit, Faculty of Medicine, King Abdulaziz University, Jeddah, Saudi Arabia. E-mail: [email protected]
www.smj.org.sa Saudi Med J 2014; Vol. 35 (10)OPEN ACCESS
1216
Outcome of uterine sarcoma in Saudi Arabia ... Sait et al
Saudi Med J 2014; Vol. 35 (10) www.smj.org.sa
Uterine sarcomas are rare tumors that account for 1-3% of all female genital tract malignancies and 3-8%
of all uterine malignancies.1-3 This heterogeneous group of tumors originates from uterine mesodermal tissue. The major uterine sarcomas consist of leiomyosarcoma (LMS) and endometrial stromal tumors (EST), the latter of which are sub-classified into 3 categories by the World Health Organization (WHO): endometrial stromal nodules (ESNs), endometrial stromal sarcoma (ESS, historically referred to as low-grade sarcoma), and undifferentiated endometrial sarcoma (UES, historically called high-grade sarcoma). Carcinosarcoma, previously called malignant mixed mullerian tumor, is considered a deviant of carcinoma, and its behavior, and treatment resembles those of high-grade carcinoma. However, there are still some centers that treat this as sarcoma.4 Uterine sarcomas occur primarily in women who are 40-60 years old.5,6 A history of pelvic irradiation was also considered a risk factor in 5-10% of cases.7 Compared with the more common types of endometrial cancer, women with uterine sarcoma have a poor prognosis due to the aggressiveness of the disease.5-7
The most frequent prognostic factors include the stage, histological subtype, grade, lymph vascular invasion, and menopausal status.8-10 Standard treatment of early stage patients are hysterectomy and surgical staging, and approximately half of these patients develop recurrent disease.10 Post-operative radiotherapy reduces local recurrence and improves local disease but does not affect the overall survival.11,12 Adjuvant chemotherapy with a single agent, isofosamide or doxorubicin, has been used,11 and combination chemotherapy (which did not show any superiority) has non-proven value over a single agent.13 Due to its rarity, heterogeneity, and aggressiveness, there is no consensus regarding the optional therapeutic approaches with considerable variation in the type of surgery and choice of adjuvant treatment. The purpose of this study is to investigate the clinical and histopathological characteristics, with the prognostic factors, treatment outcome, and pattern of relapse and survival analysis of uterine sarcoma patients.
Methods. All patients with histologically proven uterine sarcoma were identified using the database at King Abdulaziz University Hospital, Jeddah, Saudi Arabia between January 2000 and December 2012.
The study inclusion criteria required the pathological diagnosis of uterine sarcoma at the time of the study. The patients’ medical records were reviewed, and information regarding the patients’ characteristics, medical history, tumor characteristics, treatment modalities, follow-up, and survival data was reviewed and recorded. A single pathologist reviewed all the cases, and the stages of the disease were determined and adjusted retrospectively according to the FIGO staging 2009.14 The surgical procedures included one of the following procedures: hysterectomy and bilateral salpingoophorectomy (H+BSO), H+BSO+staging (pelvic and para-aortic lymphadenectomy ± omentectomy), or H+BSO+debulking (cytoreductive surgery). Patients were offered adjuvant therapy chemotherapy, radiotherapy, or hormonal therapy based on the patients’ performance status, histological type, and tumor board decision. Study approval was obtained from the Unit of Biomedical Ethics, Faculty of Medicine, King Abdulaziz University.
Statistical analysis. The Statistical Science for Social Package Version 20 (IBM SPSS Statistics for Windows, Version 20.0. Armonk, NY, USA) was used for data analysis. Data were expressed as median (minimum-maximum) or number (percentage) as appropriate. The overall survival (OS) time was calculated as the number of months from the date of surgery to either the date of death or the date of last follow up. The disease-free survival (DFS) time was calculated as the number of months from the date of surgery to either the date of recurrence or the date of last follow up. The endpoints were 2 and 5 years OS and DFS. Survival curves were calculated with the Kaplan-Meir estimator. The statistical significance of each variable was first tested by the log rank test (univariate analysis). A Cox regression model was used for multivariate analysis. For all tests, p<0.05 was considered significant.
Results. A total of 36 patients with uterine sarcoma were reviewed. The median age of all patients was 57 years, and the mean age was 57.72±13.17 years. The median parity was 4 (minimum-maximum, 0.00-15.00). Carcinosarcoma was the most common presenting histopathological type (58%), followed by LMS (20%). No patients with endometrial stromal sarcoma (previously called a low-grade stromal sarcoma) were found during the study period. The stage of the tumors was mostly stage I (41%), followed by stage III (28%), stage IV (25%) and stage II (6%). Cervical involvement was found in 8 (38.1%) in carcinosarcoma and 2 (33%) in undifferentiated endometrial sarcoma. Adnexal involvement was present in 3 (50%) and 7 (33.3%) in undifferentiated endometrial sarcoma and carcinosarcoma (Table 1).
Disclosure. Authors have no conflict of interests, and the work was not supported or funded by any drug company.
1217www.smj.org.sa Saudi Med J 2014; Vol. 35 (10)
Outcome of uterine sarcoma in Saudi Arabia ... Sait et al
Table 2 shows the lines of treatment and adjuvant therapy of the uterine sarcoma patients. In all patients, the surgical procedure was mostly H+BSO+staging (50% of patients), followed by H+BSO (39%), and H+BSO+debulking (11%). Thirty-three (91.7%) underwent surgery using an open approach. Two patients had subtotal abdominal hysterectomy for clinical diagnosis of uterine fibroids; the final pathology showed LMS, and the cases were referred to us and had complete surgery with removal of the cervix and pelvic and para-aortic lymphadenectomy using
the Robot DeVinci method. Both patients received adjuvant chemotherapy. One patient had initial laparoscopic total hysterectomy and BSO for uterine fibroids. The uterus was delivered vaginally and was found in the final pathology to have LMS; the patient was given adjuvant chemotherapy and is alive and well. Approximately two-thirds (24/36, 69%) of the patients received adjuvant chemotherapy. Adjuvant chemotherapy was given in 81% of patients with carcinosarcoma and 72% of LMS patients (72%). The type of adjuvant chemotherapy in all carcinosarcoma
Table 1 - Demographic, clinical characteristics and tumor characteristics of the patients with uterine sarcoma.
Parameters Carcinosarcoma(n=21)
Leiomyosarcoma
(n=7)
Undifferentiated endometrial
sarcoma (n=6)
Rhabdomyo sarcoma
(n=2)
Total (n=36)
Age (years) 65.05±10.40 45.43±10.00 46.00±7.67 59.00±0.00 57.72±13.17Body mass index (kg/m2) 26.24±6.09 31.07±9.79 27.53±6.97 21.05±2.19 27.11±7.10Parity: median(range) 4 (0.00-12.00) 1 (0.00-6.00) 5.50 (3.00-15.00) 0.50 (0.00-1.00) 4 (0.00-15.00)Medical illness
Diabetes mellitus 2 (9.5) 1 (14.3) 1 (16.7) - - 4 (11.1)Hypertension 2 (9.5) 1 (14.3) - - - - 3 (8.3)
Preoperative biopsyYes 11 (52.4) 3 (42.9) 2 (33.3) 1 (50) 17 (47.2)No 10 (47.6) 4 (57.1) 4 (66.7) 1 (50) 19 (52.9)
StagingI 7 (33.3) 5 (71.4) 3 (50.0) - - 15 (41.7)II 2 (9.5) - - - - - - 2 (5.6)III 7 (33.3) 1 (14.3) 1 (16.7) 1 (50) 10 (27.8)IV 5 (23.8) 1 (14.3) 2 (33.3) 1 (50) 9 (25.0)
Myometrium invasionNone 5 (23.8) 7 (100.0) 3 (50.0) 1 (50) 16 (44.4)<50 5 (23.8) - - 1 (16.7) - - 6 (16.7)≥50 11 (52.4) - - 2 (33.3) 1 (50) 14 (38.9)
Cervical involvement Yes 8 (38.1) - - 2 (33.3) - - 10 (27.8)No 13 (61.9) 7 (100.0) 4 (66.7) 2 (100) 26 (72.2)
Lymph nodes Negative 9 (42.9) 2 (28.6) 1 (16.7) - - 12 (33.3)Positive 5 (23.8) - - - - 2 (100) 7 (19.4)Not done 7 (33.3) 5 (71.4) 5 (83.3) - - 17 (47.2)
Lymph vascular involvementNegative 10 (47.6) 7 (100.0) 3 (50.0) 1 (50) 21 (58.3)Positive 11 (52.4) - - 3 (50.0) 1 (50) 15 (41.7)
Adnexa involvementNegative 14 (66.6) 6 (85.7) 3 (50.0) 2 (100) 25 (69.4)Positive 7 (33.3) 1 (14.3) 3 (50.0) - - 11 (30.6)
Pelvic washingNegative 12 (57.1) 3 (42.9) 1 (16.7) 1 (50) 17 (47.2)Positive 7 (33.3) 1 (14.3) 2 (33.3) - - 10 (27.8)Not done 2 (9.5) 3 (42.9) 3 (50.0) 1 (50) 9 (25.0)
Omentum involvementNegative 12 (57.1) - - - - - - 12 (33.0)Positive 6 (28.6) 1 (14.3) 3 (50.0) 1 (50) 11 (30.6)Not done 3 (14.3) 6 (85.7) 3 (50.0) 1 (50) 13 (36.1)Data are expressed as the median (minimum-maximum) and number and percentage (%) as appropriate.
1218
Outcome of uterine sarcoma in Saudi Arabia ... Sait et al
Saudi Med J 2014; Vol. 35 (10) www.smj.org.sa
cases was carboplatin (AUC 5) + docetaxel at 75 mg/m2. The LMS patients were treated with doxorubicin at a dose of 65 mg/m2 + cisplatian at dose of 65 mg/m2. For rhabdomyosarcoma, we administered VAC (vincristine, 65 mg/m2, actinomycin 1.5 mg/m2, and 500 mg/m2 of cyclophosphamide). Adjuvant radiotherapy was given to only 5 out of the 36 patients (14%), including 3 patients with carcinosarcoma, one with LMS and one with undifferentiated endometrial sarcoma. The adjuvant radiotherapy consisted of pelvis radiotherapy with a dose of 45-50 Gy/25-28 fractions/5 weeks. Vaginal brachytherapy was added if there was a cervical invasion, a positive vaginal margin or parametrial infiltration, and was performed with high dose-rate brachytherapy using a vaginal cylinder applicator with dose 12 Gy/3F and a dose calculated at a depth of 0.50 CM.
At a mean follow-up period of 24.00±0.00 months, 8 patients (22%) relapsed (Table 3). The median time of relapse was 7.50 months. The initial clinical stages of the relapsed patients were stages III, IV, and I. Most relapses occurred in patients who did not receive
adjuvant chemotherapy (62.5% versus 37.5%) or adjuvant radiotherapy (75% versus 25%). Relapse was reported in the peritoneum, vaginal vault, abdominal wall, inguinal region, omentum and lung. Six out of 8 (75%) with recurrence were in advanced stages III and IV. The treatment of recurrence was mostly palliative, surgery, radiotherapy, and surgery or chemotherapy and radiotherapy (Table 3). Thirteen patients (36.1%) died during follow-up; 10 cases had carcinosarcoma, 2 cases had undifferentiated endometrial sarcoma, and one case had rhabdomyosarcoma.
The 2-year DFS rates were 22% and 5-year was 14% (Figure 1). The advanced stage (p=0.015) and lymph vascular invasion (p=0.0001) were significantly associated with poor DFS in the multivariate analysis. The use of chemotherapy significantly improved the DFS (p=0.027). The 2-year OS rate was 30%, and the 5-year was 18% (Figure 2). Older age (>71 years of age) and lymph node involvement were significantly associated with poor OS (p=0.005, p=0.029). Adjuvant chemotherapy (p=0.033) and radiotherapy were significantly associated with a better OS (p=0.015).
Table 2 - Treatment characteristics and outcomes of the patients with uterine sarcoma.
Parameters Carcinosarcoma(n=21)
Leiomyosarcoma(n=7)
Undifferentiated endometrial
sarcoma (n=6)
Rhabdomyosarcoma
(n=2)
Total (n=36)
Surgical procedure (%)H+BSO 5 (23.8) 4 (57.1) 4 (66.7) 1 (50) 14 (39)H+BSO+staging 12 (57.1) 3 (42.9) 2 (33.3) 1 (50) 18 (50)H+BSO+debulking 4 (19.0) 0 (0) 0 (0) 0 (0) 4 (11)
Adjuvant chemotherapy (%)Yes 17 (80.9) 5 (71.4) 0 (0) 2 (100) 24 (69)No 4 (19.0) 2 (28.6) 6 (100) 0 (0) 12 (31)Carboplatin + taxoter 17 (80.9) - - - - 1 (50) 18 (75)Doxorubicin + cisplatian - - 5 (71.4) - - - - 5 (20)Vincristine + actinomycin + cyclophosphamide - - - - - - 1 (50) 1 (5)
Adjuvant radiotherapy (%)Yes 3 (14.3) 1 (14.3) 1 (16.7) (0) (0) 5 (14)No 18 (85.7) 6 (85.7) 5 (83.3) 2 (100) 31 (86)
Recurrence (%)No 18 (85.7) 6 (85.7) 2 (33.3) 2 (100) 28 (78)Yes 3 (14.3) 1 (14.3) 4 (66.7) 0 (0) 8 (22)
Follow-up (months) (median)(Minimum-maximum)
13(1.00-88.00)
14(12.00-118.00)
23.5(2.00-136.00)
9.5(7.00-12.00)
13.5(1.00-136.00)
Duration free from diseases (months) (median)(Minimum-maximum)
12.00(1.00-88.00)
14.00(11.00-118.00)
13.50(1.00-108.00)
9.50(7.00-12.00)
13.50(1.00-118.00)
Duration till recurrence (months) (median)(Minimum-maximum)
8.00(7.00-11.00)
11.00(11.00-11.00)
3.00(1.00-10.8.00)
- 7.50(1.00-108.00)
Alive (%)Yes 1 (4.7) 7 (100) 4 (66.7) 1 (50) 23 (64)No 10 (47.6) 0 (0) 2 (33.3) 1 (50) 13 (36)
Duration until death (months) (median)(Minimum-maximum)
9.50(1.00-36.00)
- 2(2.00-2.00)
7(7.00-7.00)
8(1.00-36.00)
H+BSO - hysterectomy + bilateral salpingo-opherectomy
1219www.smj.org.sa Saudi Med J 2014; Vol. 35 (10)
Outcome of uterine sarcoma in Saudi Arabia ... Sait et al
Discussion. Uterine sarcomas are relatively uncommon cancers, accounting for between 3% and 8% of all uterine malignancies.1-3 The age at presentation varies with the different histologic subtypes; LMS often occurs in women of peri-menopausal age, but carcinosarcoma and EST tend to be associated with women who are beyond menopause.1,15 In our center, we treated only 36 cases with uterine sarcoma over a 13-year period. The mean age for LMS was 45, whereas for carcinosarcoma and EST, the ages were 65 and 46 years old, Although rabdomyosarcoma is usually present in the pediatric age group,16 we had 2 patients who presented at the age of 59 years; they were in the advanced stages. Carcinosarcoma was the most common presenting histopathological type (58%), followed by LMS (20%), and EST in 6 (17%) patients. However, Nassar et al15 reported in their series that LMS was the
most commonly reported (42% of patients), followed by carcinosarcoma in 35.5%, and EST in 18.6%.15 In our study, 10 (28%) of the patients were stage III and 9 (25%) were stage IV, at presentation, while 15 patients (41%) were stage I and only 2 patients (6%) were stage II. This finding is different from other series that reported that stages I and II disease comprise approximately 70% of the patients at presentation, while stages III and IV comprise 30% of the group.15,17
Hysterectomy and BSO are the standard treatment for uterine sarcomas.18 Recent reports insist that patients who undergo extended or radical hysterectomy have a more favorable outcome than those who undergo hysterectomy.19 The role of BSO in uterine sarcoma is controversial. Some studies have found that adnexectomy is associated with improved prognosis in patients with LMS and decreased recurrence in
Table 3 - Characteristics of the recurrence among patients with uterine sarcoma.
Parameters Patients with recurrence(n=8)
Surgical procedureH+BSO 4 (50.0)H+BSO+staging 3 (38.0)H+BSO+debulking 1 (12.0)
Final histopathologyUndifferentiated endometrial sarcoma 4 (50.0)Carcinosarcoma 3 (38.0)Leiomyosarcoma 1 (12.0)
Adjuvant chemotherapyNo 5 (63.0)Yes 3 (37.0)
Adjuvant radiotherapyNo 6 (75.0)Yes 2 (25.0)
Initial stage Stage I 2 (25.0)Stage III 3 (37.5)Stage IV 3 (37.5)
Site of recurrencePeritoneum 2 (25.0)Vault 2 (25.0)Abdominal 1 (12.5)Abdominal wall + inguinal 1 (12.5)Omental 1 (12.5)Lung 1 (12.5)
Treatment Palliative care 5 (62.5)Surgery 1 (12.5)Radiotherapy 1 (12.5)Surgery, chemotherapy and radiotherapy 1 (12.5)
AliveNo 4 (50.0)Yes 4 (50.0)
H+BSO - hysterectomy + bilateral salpingo-opherectomyData are expressed as number and percentage (%)
Figure 1 - Disease-free survival for patients with uterine sarcomas.
Figure 2 - Overall survival for patients with uterine sarcomas.
1220
Outcome of uterine sarcoma in Saudi Arabia ... Sait et al
Saudi Med J 2014; Vol. 35 (10) www.smj.org.sa
patients with EST.20,21 However, others have reported contradictory results.22,23 In our series, the surgical procedure was mostly H+BSO+staging (50%), followed by H+BSO (39%) and H+BSO+debulking (11%). The role of lymphadenectomy in uterine sarcoma is controversial;24,25 in a cohort study performed by Hoellen et al25 involving 52 patients diagnosed with uterine sarcoma, pelvic lymphadenectomy was performed in 48%, while 10% had both pelvic and para-aortic lymphadenectomy. These authors25 found that patients who underwent lymphadenectomy had better survival.
In our study, 50% of our patients underwent pelvic and para-aortic lymphadenectomy.
Figure 3 shows that there are no significant differences among the different surgical procedures performed for uterine sarcoma (log rank, p=0.786). Park et al17 reported in their series that complete surgical resection is an important factor affecting patient outcome. Complete surgical resection may be the best option for uterine sarcomas because the role of effective adjuvant treatment remains undetermined. Approximately two-thirds of the patients (69%) received adjuvant chemotherapy in this series. The adjuvant chemotherapy was given in 81% and 71% of carcinosarcoma and LMS patients. Adjuvant radiotherapy was given in 5/36 (14%). Other series15,17,21 reported a variable percentage of patients who received adjuvant chemotherapy or radiotherapy (50-60% for adjuvant chemotherapy and 5-30% for adjuvant radiotherapy).
In this study, 8 patients (22%) had confirmed relapse during the follow-up period; 75% of relapses occurred in patients who were initially stages III and IV, whereas other series reported an incidence of relapse between 30-37%.4,14 Although relapse occurred in 63% and 75% of patients who did not receive adjuvant chemotherapy or radiotherapy versus 38% and 25% of patients who did, respectively, this difference was not statistically significant due to the small number of patients (p<0.480 and p<0.157).
Our 2-year DFS rate was 22% and the 5-year was 14% (Figure 1). Ghaemmaghami et al26 reported that the median survival for their patients was 2.8 years. Moskovic et al27 reported a median survival of 22 months. Advanced stage and lymph vascular invasion were significantly associated with poor DFS (p=0.015 and p=0.0001). The use of chemotherapy significantly improved the DFS (p=0.027). Park et al17 reported a 10-year DFS of 30%; adjuvant chemotherapy and radiotherapy have limited impact on the outcome of early stage disease. However, patients with advanced disease that received adjuvant chemotherapy had significantly longer OS times. Multivariate analysis revealed that the
FIGO stage (p=0.025), depth of myometrium invasion (p=0.004), and complete cytoreduction (p=0.030) significantly affected the DFS. Nassar et al15 also reported that the DFS was significantly affected by the stage, adjuvant radiotherapy, tumor size, depth of invasion, and cervical and lymph vascular invasion, while the histological type had no significant value.15 In their series of 127 patients, which had a median follow-up period of 38 months, Park et al17 reported a 10-year OS of 48%; the menopausal status (p=0.044), FIGO stage (p=0.016), depth of myometrium invasion (p=0.029), and lymph vascular invasion (p=0.020) were significantly associated with the OS. In our series, the 2-year overall survival (OS) rate was 30% and in the 5-year was 18%. Older age (>71 years) and lymph node involvement were significantly associated with poor OS (p=0.005 and p=0.029). The use of adjuvant chemotherapy (p=0.033) and radiotherapy (p=0.015) was significantly associated with a better OS. Nassar et al15 reported that patients who received adjuvant chemotherapy showed a poorer median survival time, with a 2-year survival of 40% versus 53% for those who did not receive chemotherapy; the difference was not statistically significant. The role of adjuvant chemotherapy is poorly defined, although chemotherapy has been used because of the high risk of systemic relapse for stage I uterine LMS and undifferentiated sarcoma with complete resection of the tumor. However, for stages II and III, because of the increase risk profile for systemic relapse, it is appropriate to consider adjuvant chemotherapy.28 Adjuvant chemotherapy is still performed with controversial results.11-13 The value of
Figure 3 - Overall survival for patients with uterine sarcomas per surgical procedure. Log rank, p=0.786.
1221www.smj.org.sa Saudi Med J 2014; Vol. 35 (10)
Outcome of uterine sarcoma in Saudi Arabia ... Sait et al
post-operative radiotherapy, if any, was shown to reduce local recurrence and improve local disease, but it had no effect on the overall survival.11,12 An ongoing phase III randomized trial (GOG 277) is currently assessing the role of postoperative chemotherapy versus observation in patients with uterine LMS and undifferentiated sarcoma. If chemotherapy is needed, docetaxel and gemcitabine have been the most commonly used regimen based on data evaluating their used in LMS.29 For undifferentiated endometrial sarcoma, some data suggest that docetaxel plus gemcitabine and a doxorubicin-containing regimen exhibit activity.30
Although radiotherapy was given in only 5 patients in our series, these patients had better outcomes than patients who did not receive radiotherapy. The role of adjuvant radiotherapy in non-metastatic disease is controversial. Most of the retrospective studies of adjuvant radiation with LMS and undifferentiated sarcoma suggest an improvement in the local pelvic control but not in the overall survival.31 However, in a phase III randomized trial performed by Reed et al32
on 224 patients diagnosed with uterine sarcoma that underwent radiation versus observation, the initial analysis showed a reduction in the local relapse (14 versus 24; p=0.004), but there was no effect on either the OS or PFS.
In conclusion, we report the first survival data for uterine sarcoma in Saudi Arabia. The poor outcome of high grade uterine sarcoma patients was identified and one third of patients survived for 2 years. The management of uterine sarcomas is challenging due to the lack of consensus or guidelines. Adjuvant treatment should be individualized.
References 1. Chauveinc L, Deniaud E, Plancher C, Saastre X, Amesani F, de
La Rochefordiere A, et al. Uterine sarcomas: the Curie institute experiences. Prognosis factors and adjuvant treatment. Gynecol Oncl 1999; 72: 232-237.
2. Tropé CG, Abeler VM, Kristensen GB. Diagnosis and treatment of sarcoma of the uterus. A review. Acta Oncol 2012; 51: 694-695.
3. Brooks SE, Zhan M, Cote T, Baquet CR. Surveillance epidemiology and end results analysis of 2677 cases of uterine sarcoma 1989-1999. Gynecol Oncol 2004; 93: 204-208.
4. Shi Y, Liu Z, Peng Z, Liu H, Yang K, Yao X. The diagnosis and treatment of Mullerian adenosarcoma of the uterus. Aust N Z J Obstet Gynaecol 2008; 48: 596-600.
5. Dinh TA, Oliva EA, Fuller AF Jr, Lee H, Goodman A. The treatment of uterine leiomyosarcoma. Results from a 10-year experience (1990-1999) at the Massachusetts General Hospital. Gynecol Oncol 2004; 92: 648-652.
6. Kelly KL, Craighead PS. Characteristics and management of uterine sarcoma patients treated at the Tom Baker Cancer Centre. Int J Gynecol Cancer 2005; 15: 132-239.
7. Meredith RF, Eisert DR, Kaka Z, Hodgson SE, Johnston GA Jr, Boutselis JG. An excess of uterine sarcoma after pelvic irradiation. Cancer 1986; 58: 2003-2007.
8. Schick U, Bolukbasi Y, Thariat J, Abdah-Bortnyak R, Kuten A, Igdem S. Outcome and prognostic factors in endometrial stromal tumors: a rare cancer network study. Int J Radiat Oncol Biol Phys 2012; 82: e757-e763.
9. Durnali A, Tokluoğlu S, Özdemir N, Inanç M, Alkiş N, Zengin N. Prognostic factors and treatment outcomes in 93 patients with uterine sarcoma from 4 centers in Turkey. Asian Pac J Cancer Prev 2012; 13: 1935-1941.
10. Iwasa Y, Haga H, Konishi Y, Kobashi Y, Higuchi K, Katsuyama. Prognostic factors in uterine carcinosaromas. Cancer 1998; 83: 512-519.
11. Sutton GP, Brunetto V, Kilgore L, Soper JT, McGehee R, Olt G. A phase III trial of isofsamide with and without cisplatian in carcniosarcoma of the uterus. a Gynecologic Oncology Group study. Gynecol Oncol 2000; 79: 147-153.
12. Giuntoli RL 2nd, Metzinger DS, DiMarco CS, Cha SS, Sloan JA, Keeney GL. Retrospective review of 208 patients with Leiomyosarcoma of the uterus: prognostic indicators, surgical mamgement and adjuvant therapy. Gynecol Oncol 2003; 89: 460-469.
13. Pautier P, Floquet A, Penel N, Piperno-Neumann S, Isambert N, Rey A . Randomized multicenter and stratified phase II study of gemcitabine alone versus gemcitabine and docetaxel in patients with metastatic or relapsed Leiomyosarcoma: a Federation Nationale des Centers de LutteContre le Cancer (FNCLCC) French Sarcoma Group Study (TAXOGEM study). Oncologist 2012; 17: 1213-1220.
14. Prat J. FIGO staging for uterine sarcomas. Int J Gynaecol Obstet 2009; 104: 177-178.
15. Nassar OA, Abdul Moaty SB, Khalil el-SA, El-Taher MM, El Najjar M. Outcome and prognostic factors of uterine sarcoma in 59 patients: single institutional results. J Egypt Natl Canc Inst 2010; 22: 113-122.
16. Hagiyama Y, Hashimoto H, Hamada K, Matsubara K, Fujioka T, Nawa A. Embryonal rhabdomyosarcoma of the uterus in a 35-year-old woman: case report and review of the literature. Eur J GynaecolOncol 2013; 34: 332-335.
17. Park JY, Kim DY, Suh DS, Kim JH, Kim YM, Kim YT. Prognostic factors and treatment outcomes of patients with uterine sarcomas: analysis of 127 patients at single institution, 1989-2007. J Cancer Res Clin Oncol 2008; 134: 1277-1287.
18. Gadducci A, Cosio S, Romanaine A, Genazzani AR. The management of patients with uterine sarcoma: A debated clinical challenge. Crit Rev Oncol Hematol 2008; 65: 129-142.
19. Kokawa K, Nishiyama K, Ikeuchi M, Ihara Y, Akamatsu N, Enomoto T. Clinical outcome of uterine sarcomas; results from 14 years’ worth of experience in Kinki district in Japan (1990-2003). Int J Gynecol Cancer 2006; 16: 1358-1363.
20. Gadducci A, Sartori E, Landoni F, Zola P, Maggino T, Urgesi A, et al. Endometrial stoma sarcoma: analysis in treatment failure and survival. Gynecol Oncol 1996; 63: 247-253.
21. Morice P, Rodrigues A, Pautier P, Rey A, Camatte S, Atallah D. Surgery for uterine sarcoma: review of literature and recommendation for the standard surgical procedure. Gynecol Obstet Fertil 2003; 32: 147-150.
22. Giuntoli RL 2nd, Metzinger DS, DiMarco CS, Cha SS, Sloan JA, Keeney GL, et al. Retrospective review of 2008 patients with Leiomyosarcoma of the uterus; prognostic indicators, surgical management and adjuvant therapy. Gynecol Oncol 2003; 89: 460-469.
1222
Outcome of uterine sarcoma in Saudi Arabia ... Sait et al
Saudi Med J 2014; Vol. 35 (10) www.smj.org.sa
23. Li AJ, Giuntoli RL 2nd, Drake R, Byun SY, Rojas F, Barbuto D, et al. Ovarian preservation in stage I low-grade endometrial stromal sarcomas. Obstet Gynecol 2005; 106: 1304-1308.
24. Rothmund R, Hartkopf A, Joachim C, Walter CB, Wallwiener M, Kraemer B, et al. Clinical characteristics, pathological reevaluation, surgical management and adjuvant therapy of patients with endometrial stromal tumors. Arch Gynecol Obstet 2014; 1. [Epub ahead of print]
25. Hoellen F, Waldmann A, Benthin S, Hanker L, Rody A, Fischer D. The role of lymphadenectomy in uterine sarcoma: a clinical practical approach based on retrospective analysis. Anticancer Res 2014; 34: 985-993.
26. Ghaemmaghami F, Karimi-Zarchi M, Gilani MM, Mousavi A, Behtash N, Ghasemi M. Uterine sarcoma: clinicopathological characteristics, treatment and outcome in Iran. Asian Pac J Cancer Prev 2008; 9: 421-426.
27. Moskovic E, MacSweeney E, Law M, Price A. Survival, patterns of spread and prognostic factors in uterine sarcoma: a study of 76 patients. Br J Radiol 1993; 66: 1009-1015.
28. Ricci S, Giuntoli RL 2nd, Eisenhauer E, Lopez MA, Krill L, Tanner EJ 3rd, et al. Does adjuvant chemotherapy improve survival for women with early-stage uterine leiomyosarcoma? Gynecol Oncol 2013; 131: 629-633.
29. Hensley ML, Ishill N, Soslow R, Larkin J, Abu-Rustum N, Sabbatini P, et al. Adjuvant gemcitabine plus docetaxel for completely resected stages I-IV high grade uterine leiomyosarcoma: Results of a prospective study. Gynecol Oncol 2009; 112: 563-567.
30. Tanner EJ, Garg K, Leitao MM Jr, Soslow RA, Hensley ML. High grade undifferentiated uterine sarcoma: surgery, treatment, and survival outcomes. Gynecol Oncol 2012; 127: 27-31.
31. Brenner DJ, Hall EJ. Computed tomography-an increasing source of radiation exposure. N Engl J Med 2007; 357: 2277-2284.
32. Reed NS, Mangioni C, Malmström H, Scarfone G, Poveda A, Pecorelli S, et al. Phase III randomised study to evaluate the role of adjuvant pelvic radiotherapy in the treatment of uterine sarcomas stages I and II: an European Organisation for Research and Treatment of Cancer Gynaecological Cancer Group Study (protocol 55874). Eur J Cancer 2008; 44: 808-818.
Related Articles
Saraydaroglu O, Ozuysal S, Kasap M, Ozerkan K. The importance of CD10 and h-Caldesmon in the distinction of smooth muscle tumors of the uterus and endometrial stromal sarcoma. Saudi Med J 2008; 29: 1349-1350.
Abu-Rafea BF, Vilos GA, Al Jasser RS, Al Anazy RM, Javaid K, Al-Mandeel HM. Linguistic and clinical validation of the Arabic-translated Aberdeen Menorrhagia Severity Scale as an indicator of quality of life for women with abnormal uterine bleeding. Saudi Med J 2012; 33: 869-874.
Ulkumen B, Pala HG, Baytur Y. Acute abdomen and massive hemorrhage due to placenta percreta leading to spontaneous uterine rupture in the second trimester. Saudi Med J 2014; 35: 1131-1132.
S20 Journal of the College of Physicians and Surgeons Pakistan 2014, Vol. 24 (Special Supplement 1): S20-S21
INTRODUCTIONUterine leiomyosarcoma accounts for 3% of all uterinemalignancies; it metastasizes through hematogenousroute to the lungs, liver, bones and the brain.1 Heart is anextremely rare site of metastasis from uterine leiomyo-sarcoma. Common malignancies which metastasize toheart are lung, gastrointestinal tract, genitourinary tract,breast and pancreas.2
Cardiac metastasis in uterine leiomyosarcoma is usuallya sign of extensive disease and poor prognosis. Firstsymptomatic case of cardiac metastasis secondary touterine leiomyosarcoma was reported by Rosenblattand Featherston in 1960.3 Since then only a few casereports of uterine leiomyosarcoma associated cardiacmetastasis have been published.4
Herein we present the case of 57 years female withpreviously operated uterine leiomyosarcoma havinginterventricular cardiac metastasis.
CASE REPORTA 57 years old Saudi woman presented with symptomsof progressive dyspnea, dizziness, fever off and on, andtachycardia for one month. Her medical history andfamily history were unremarkable. Past surgical historyrevealed a total abdominal hysterectomy and bilateralsalpingo-oophorectomy followed by adjuvant pelvicirradiation for uterine leiomyosarcoma 10 years back.On physical examination, she was well-nourishedwith mild pallor, no cyanosis and no palpable
lymphadenopathy. A 5 x 5 cm soft tissue mobile non-tender mass on back of right shoulder was noticed. Oncardiovascular system (CVS) examination, she wasnormotensive with heart rate of 80/minute, sinus rhythmlow volume and non-paradoxical pulse. Jugular venouspressure (JVP) was not raised. The apex beat was notpalpable and auscultation revealed faint normal first (S1)and second (S2) heart sounds with no murmur or rubs.Percussion of the cardiac outline suggested cardiacenlargement. Rest of the examination was unremark-able. Hematological, biochemical, hepatic and renalfunction tests were found to be within normal limits.Echocardiography revealed 4 x 4 cm solid massadherent to the interventricular septum and mitral valvewith flow impedance with left ventricular ejection fractionof 55% (Figure IA and IB). Differential diagnosis wascardiac myxoma, infective endocarditis or metastasis.Computed tomography (CT) of chest showed cardiacmass around the interventricular septum and mitralvalve. Further CT chest, abdomen and pelvis findingswere recurrent soft tissue mass in pelvis invading theupper vagina and base of the urinary bladder causingbilateral hydronephrosis, metastases into lungs, liver,subcutaneous right upper back, pelvic and retro-peritoneal lymph nodes (Figure IC). Biopsies of rightback mass and recurrent pelvic mass were consistentwith metastatic uterine leiomyosarcoma (Figure ID).After discussing the case in multi-disciplinary board,patient was started on palliative radiotherapy to heartfollowed by systemic chemotherapy. At two months,patient's symptoms were improved and on her last visitshe was receiving chemotherapy.
DISCUSSIONCardiac metastases are uncommon seen usually inautopsy series. Since last decade, symptomatic cardiacmetastasis have been reported frequently possibly dueto prolonged survival of cancer patients with treatment
CASE REPORT
Uterine Leiomyosarcoma Metastasizing to the HeartMutahir Ali Tunio, Mushabbab Al-Asiri and Muhammad Mohsin Fareed
ABSTRACTUterine leiomyosarcoma is an uncommon and aggressive gynecologic malignancy with an overall poor prognosis. Lungs,bones and brain are common sites of metastases of uterine leiomyosarcoma. Metastases of uterine leiomyosarcoma tothe heart are extremely rare, and only a few case reports have been published so far. Herein we present the case of57 years old female, who had undergone total abdominal hysterectomy with salpingo-oophorectomy 10 years back foruterine leiomyosarcoma, she presented with dyspnea, dizziness, fever, and tachycardia, subsequently was found to haveinterventricular septum cardiac as well extensive system metastasis and recurrent pelvic mass. Patient’s symptomsresponded to palliative radiotherapy followed by chemotherapy.
Key Words: Uterine leiomyosarcoma. Cardiac metastasis. Echocardiography. Radiotherapy. Chemotherapy. Palliation recurrence.Interventricular septum.
Department of Radiation Oncology, Comprehensive CancerCentre, King Fahad Medical City, Riyadh-59046, Saudi Arabia.
Correspondence: Dr. Mutahir Ali Tunio, Assistant Consultant,Radiation Oncology, Comprehensive Cancer Center, King FahadMedical City, Riyadh-59046, Saudi Arabia.E-mail: [email protected]
Received: January 03, 2013; Accepted: August 03, 2013.
advancements. Uterine leiomyosarcoma metastatic tothe heart is extremely rare and only few cases havebeen reported so far in world literature.4-7
The mechanism of metastasis to the heart is not wellknown. Possible routes that have been postulated arehematogenous spread through the coronary arteries,direct contiguous extension, and retrograde dissemi-nation through lymphatics. Common site of cardiacmetastasis is the pericardium, followed by myocardium,epicardium, endocardium, and interventricular septum.7
Most cardiac metastases can be difficult to diagnoseantemortem unless the patients are symptomatic.Echocardiography and CT imaging are important tools inthe antemortem diagnosis of cardiac metastasis inasymptomatic cancer patients.
This patient had symptomatic cardiac metastasis ininterventricular septum and mitral valve, which areextremely rare sites.8 Such patients are not considered
candidates for surgical resection. This patient wastreated with palliative radiotherapy followed bychemotherapy due to metastatic location and extent ofmetastatic disease. Her symptoms responded to thepalliative regime. In addition, she had multiple othermetastatic foci along with a local recurrence whichnecessitated further chemotherapy.
In conclusion, cardiac metastasis secondary to uterineleiomyosarcoma is extremely rare entity and patientswith uterine malignancies must undergo early echo-cardiographic evaluation with imaging if patients developcardiac symptoms for prompt diagnosis and treatment.
REFERENCES1. D'Angelo E, Prat J. Uterine sarcomas: a review. Gynecol Oncol
2010; 116:131-9.
2. Klatt EC, Heitz DR. Cardiac metastases. Cancer 1990;65:1456-9.
3. Rosenblatt WH, Featherston WP. Metastatic leiomyosarcomaof the heart. Am J Cardiol 1960; 6:672-5.
4. Moreno AF, Casado HA, Puente VJ, Gomez DR, Aragoncillo P,Diaz-Rubio GE. Cardiac metastasis from uterine leiomyo-sarcoma. Clin Transl Oncol 2006; 8:375-8.
5. Peng YJ, Hueng GG, Lee HS. Acute heart failure asmanifestation of metastatic uterine leiomyosarcoma to theheart and lung. Heart Lung 2004; 33:46-9.
6. Calleja AM, Wellnitz CV, Alharthi MS, Khandheria BK, ChalikiHP. Extensive cardiac metastases secondary to uterineleiomyosarcoma. J Am Soc Echocardiogr 2009; 22:1419.5-7.
7. Nguyen SK, Wong F. Right atrial metastasis of uterineleiomyosarcoma causing obstructive shock. Curr Oncol 2012;19:292-4.
8. Neragi-Miandoab S, Kim J, Vlahakes GJ. Malignant tumoursof the heart: a review of tumour type, diagnosis and therapy.Clin Oncol 2007; 19:748-56.
Uterine leiomyosarcoma metastasizing to the heart
Figure 1: (A) Echocardiography, (B) computed tomography (CT) chestshowing 4x 4 cm solid mass adherent to interventricular septum and mitralvalve with flow impedance, (C) recurrent pelvic mass and (D) a section of therecurrent pelvic mass biopsy which showed the uterine leiomyosarcoma x 40magnification and (E) Immunohistochemistry of primary leiomyosarcomashowing positivity for SMA.
Journal of the College of Physicians and Surgeons Pakistan 2014, Vol. 24 (Special Supplement 1): S20-S21 S21
Life Science Journal 2014;11(4) http://www.lifesciencesite.com
317
Expression of Thyroid Transcription Factor-1 (TTF-1) in Endometrial Carcinoma.
Jaudah Al-Maghrabi1 (MD, FRCPC), Nisrin Anfinan2,3 (MD, FRCSC), Khalid Sait 2,3 (MD, FRCSC), Hesham Sait2,3
(MD), Mahmou Al-Ahwal4 (MD, FRCPC), Hussain Basalamah2,3 (MD, FRCSC).
Department of Pathology1, Gynecology Oncology Unit2, Scientific Chair of Prof. Abdullah Hussain Basalamah for Gynecological Cancer3, Department of Medicine4, King Abdulaziz University, Jeddah, Saudi Arabia
Abstract: Introduction: TTF-1 is a helpful marker for primary lung adenocarcinoma and is considered as a reliable marker to distinguish between primary and secondary lung carcinoma. However, some studies showed that TTF-1 also can be expressed in extrapulmonary adenocarcinomas. The data on TTF-1 expression in endometrial cancer is limited and conflicting. The aim of this study is to evaluate the immunoexpression of TTF-1 in primary endometrial adenocarcinoma. Material and methods: Tissue microarrays were prepared from archival of endometrial carcinoma obtained from the Department of Pathology at King Abdulaziz University Jeddah, Saudi Arabia. Tissue sections were immunostained using monoclonal antibodies to TTF-1. The immunohistochemical stains were scored semiquantitativly from 0 to 5+. Results: The categories of endometrial adenocarcinoma include 78 grade I endometrioid, 17 grade II endometrioid, 12 grade III endometrioid, 7 serous, 2 clear cell. TTF-1 immunoexpression was detected only in 2 carcinoma (1 serous and one endometrioid type) and in both cases the staining score was 1+. Conclusion: TTF-1 is a reliable marker for lung carcinomas; however, in patient with focal TTF-1 immunoexpression, endometrial carcinomas should be considered when evaluating patients with adenocarcinoma of unknown origin and in patients with a history of endometrial adenocarcinoma. [Jaudah Al-Maghrabi, Nisrin Anfinan, Khalid Sait, Hesham Sait, Mahmou Al-Ahwal, Hussain Basalamah. Expression of Thyroid Transcription Factor-1 (TTF-1) in Endometrial Carcinoma. Life Sci J 2014;11(4):317-320]. (ISSN:1097-8135). http://www.lifesciencesite.com. 43 Key Words: Immunoexpression, thyroid transcription factor-1, TTF-1, endometrial carcinoma, metastasis. 1. Introduction
Thyroid transcription factor-1 (TTF-1) is DNA-binding protein that is encoded by a gene located on chromosome 14q13. It belongs to a family of homeodomain transcription factors. It is selectively expressed in the thyroid and lung. In the thyroid, TTF-1 is expressed in C-cells and follicular cells and it activates thyroglobulin and thyroperoxidase gene transcription (1-4). TTF-1 is useful marker for primary adenocarcinoma of the lung (1). Some studies have shown that TTF-1 also can be expressed in extrapulmonary adenocarcinomas (2-7). However the results of the expression of TTF-1 in endometrial adenocarcinoma are conflicting. The aim of this study is to provide data regarding the incidence and distribution of TTF-1 expression in endometrial carcinoma. 2. Material and methods:
Archival paraffin-embedded tissue samples from patients with endometrial carcinoma were used to construct tissue microarrays. Cases were retrieved by diagnosis search from the Department of Pathology at King Abdulaziz University Jeddah, Saudi Arabia, covering the period from January 1995 to December 2012. The study was performed in accordance with the ethics committee of Faculty of Medicine, King Abdulaziz University, Saudi Arabia,
and according to the ethical guidelines of the 1975 Declaration of Helsinki. Tissue sections from microarray blocks were immunostained using monoclonal antibodies to TTF-1, clones 8G7G3/1 (dilution 1:100, Dako North America, Inc. Carpinteria, CA). Staining was carried out by an automatic immunostainer (Ventana Bench Mark XT, Ventana Inc., Tucson, AZ). The immunohistochemical stains were scored according to the distribution of TTF-1 nuclear staining as follows: 0 = negative, 1+ = <5%, 2+ = 5% to 25%, 3+ = 26% to 50%, 4+ = 51% to 75%, and 5+ = >75%). 3. Result:
A total of 116 endometrial carcinomas were retrieved from the authors’ institutions. The patient had abdominal hysterectomy with bilateral salpingo-oophorectomy. The age of the patients ranged between 25 and 80 years. The categories of endometrial adenocarcinoma include endometrioid endometrial adenocarcinomas 107 (78 grade I, 17 grade II, and 12 grade III), and 7 uterine serous carcinomas and 2 clear cell carcinoma. TTF-1 immunoexpression was detected only in 2 carcinomas (1 serous and one endometrioid type) and in both cases the staining score was 1+. Our data
Life Science Journal 2014;11(4) http://www.lifesciencesite.com
318
demonstrated that all the other cases were completely negative.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 1: Tissue microarray reveals endometrial carcinoma cases. Each core represents a different cancer (hematoxylin-eosin, original magnification X100) Figure 2: Immunohistochemistry stain for TTF-1 using tissue microarray reveals negative staining (original magnification X200). Figure 3 Immunohistochemistry stain for TTF-1 using tissue microarray reveals negative staining in another case (original magnification X200). Figure 4: higher power on another case reveal few cells that reveal nuclear staining for TTF-1 (arrow) (original magnification X400). 4. Discussion:
In normal tissue, TTF ‑ 1 is reported to be expressed in epithelial cells of thyroid and type II pneumocytes and Clara cells in lung (8). It is unreactive with other tissues examined including prostate, pituitary, testes, adrenal gland, skin, mammary gland, kidney, colon, liver, pancreas, small intestine, brain, and stomach.
In cancerous tissue, TTF‑1 has been detected in pulmonary adenocarcinoma (3;8), large-cell carcinoma (8), small cell carcinoma of lung (9) and thyroid carcinoma (10). Carcinomas arising from
lung show frequent TTF ‑ 1expression. TTF-1 is expressed in about 90% of bronchogenic adenocarcinoma. Lung also is a common site for metastases from extrapulmonary carcinomas (11). Nearly half of lung cancers are adenocarcinomas (11). Differential diagnoses of primary lung adenocarcinomas from metastatic carcinomas, particularly those with poor differentiation are challenges for practicing pathologists.
TTF-1 immunoreactivity is a very sensitive and highly specific marker in the differential diagnosis of lung adenocarcinoma and other non-pulmonary carcinoma and highly recommended to be used in
Life Science Journal 2014;11(4) http://www.lifesciencesite.com
319
regular clinical practice for this purpose (3;12). Apart from thyroid carcinoma, all non-pulmonary adenocarcinomas classically lacked TTF-1 staining
Endometrial carcinoma is the most common cancer of the female genital cancer. Metastatic carcinomas to the lung from the endometrium, endocervix, ovary and colon have been reported to be positive for TTF-1 in rare occasion (2-7). Ye et al (14) found that fourteen (13.6%) of 103 metastatic carcinomas to the lung showed positive TTF-1 immunostaining including carcinomas from the endometrium (2), thyroid (5), colon (3), kidney (2), ovary (1), prostate (1), and salivary gland (1). Although the reported frequency of positivity is low, its misinterpretation can lead to an incorrect diagnosis. Lung metastases were found at the time of diagnosis of the primary endometrial cancer in 22%, so pulmonary metastases represent a common site of metastasis of endometrial carcinoma (13). However the results of TTF-1 expression in these tumors are conflicting.
Ervine et al. demonstrated that TTF-1 is expressed in a small subset of all categories of endometrial adenocarcinoma as follows: 2% low grade endometrioid, 11% grade 3 endometrioid, 9% serous and 7% clear cell and they showed that TTF-1 positivity in low grade endometrioid adenocarcinomas is a poor prognostic factor (14). Zhang et al. showed that TTF-1 is frequently detected in uterine malignant mixed Mullerian tumor (82%), more common in uterine tumors than ovarian tumors. When present, tumor cells can be rarely positive or diffusely positive for TTF-1 reactivity (7).
TTF-1 positivity was identified in 1 out of 48 endometrial adenocarcinoma by Turner et al. (15). TTF-1 has been reported in 1 of 8 endometrial adenocarcinomas by others (16). At M. D. Anderson, Deavers et al. studied TTF-1 expression in 31 endometrial endometrioid adenocarcinomas (11 grade I, 8 grade II, and 12 grade III), TTF-1 immunoreactivity was identified in 5 cases (16%), and ranged from focal to diffuse in distribution (17). There was no correlation between TTF-1 expression and the degree of differentiation, and no distinguishing histologic features of the positive tumors were noted (17). Recently it has been demonstrated that all primary lung adenocarcinomas were negative for PAX8, whereas all endometrial carcinoma (5/5) were positive for PAX8 and they suggested that combined use of PAX8, TTF-1 and napsin A is reliable to separate reliably lung primary from metastatic tumors including endometrial carcinoma (18). PAX 8 is expressed in the vast majority of endometrial carcinomas both of endometrioid and non-endometrioid type (19).
In this study we demonstrated that the vast majority of endometrial carcinomas are negative for TTF-1 immunoexpression. The rare cases that expressed TTF-1 showed only focal weak expression of TTF-1. So, diffuse expression is not seen in any of the cases in this study and we think that this pattern of expression is against endometrial carcinoma origin of metastatic carcinoma. However, in patient with focal TTF-1 immunoexpression, endometrial carcinomas should be considered when evaluating patients with adenocarcinoma of unknown origin and in patients with a history of endometrial adenocarcinoma. Additional markers such as PAX-8 to help in differentiating lung and endometrial carcinomas are recommended. Acknowledgment:
This study was supported by a grant from the Scientific Chair of Prof. Abdullah Hussain Basalamah for Gynecological Cancer3, King Abdulaziz University, Jeddah, Saudi Arabia
Reference 1. Ye J, Findeis-Hosey JJ, Yang Q, McMahon LA,
Yao JL, Li F et al. Combination of napsin A and TTF-1 immunohistochemistry helps in differentiating primary lung adenocarcinoma from metastatic carcinoma in the lung. Appl Immunohistochem Mol Morphol 2011; 19(4):313-317.
2. Bishop JA, Sharma R, Illei PB. Napsin A and thyroid transcription factor-1 expression in carcinomas of the lung, breast, pancreas, colon, kidney, thyroid, and malignant mesothelioma. Hum Pathol 2010; 41(1):20-25.
3. Comperat E, Zhang F, Perrotin C, Molina T, Magdeleinat P, Marmey B et al. Variable sensitivity and specificity of TTF-1 antibodies in lung metastatic adenocarcinoma of colorectal origin. Mod Pathol 2005; 18(10):1371-1376.
4. Klingen TA, Chen Y, Gundersen MD, Aas H, Westre B, Sauer T. Thyroid transcription factor-1 positive primary breast cancer: a case report with review of the literature. Diagn Pathol 2010; 5:37.
5. Kubba LA, McCluggage WG, Liu J, Malpica A, Euscher ED, Silva EG et al. Thyroid transcription factor-1 expression in ovarian epithelial neoplasms. Mod Pathol 2008; 21(4):485-490.
6. Siami K, McCluggage WG, Ordonez NG, Euscher ED, Malpica A, Sneige N et al. Thyroid transcription factor-1 expression in endometrial and endocervical adenocarcinomas. Am J Surg Pathol 2007; 31(11):1759-1763.
Life Science Journal 2014;11(4) http://www.lifesciencesite.com
320
7. Zhang PJ, Gao HG, Pasha TL, Litzky L, Livolsi VA. TTF-1 expression in ovarian and uterine epithelial neoplasia and its potential significance, an immunohistochemical assessment with multiple monoclonal antibodies and different secondary detection systems. Int J Gynecol Pathol 2009; 28(1):10-18.
8. Nakamura N, Miyagi E, Murata S, Kawaoi A, Katoh R. Expression of thyroid transcription factor-1 in normal and neoplastic lung tissues. Mod Pathol 2002; 15(10):1058-1067.
9. Ordonez NG. Value of thyroid transcription factor-1 immunostaining in distinguishing small cell lung carcinomas from other small cell carcinomas. Am J Surg Pathol 2000; 24(9):1217-1223.
10. Katoh R, Miyagi E, Nakamura N, Li X, Suzuki K, Kakudo K et al. Expression of thyroid transcription factor-1 (TTF-1) in human C cells and medullary thyroid carcinomas. Hum Pathol 2000; 31(3):386-393.
11. Travis WD. Pathology of lung cancer. Clin Chest Med 2002; 23(1):65-81, viii.
12. Moldvay J, Jackel M, Bogos K, Soltesz I, Agocs L, Kovacs G et al. The role of TTF-1 in differentiating primary and metastatic lung adenocarcinomas. Pathol Oncol Res 2004; 10(2):85-88.
13. Bouros D, Papadakis K, Siafakas N, Fuller AF, Jr. Patterns of pulmonary metastasis from uterine cancer. Oncology 1996; 53(5):360-363.
14. Ervine A, Leung S, Gilks CB, McCluggage WG. Thyroid transcription factor-1 (ttf-1) immunoreactivity is an adverse prognostic factor in endometrioid adenocarcinoma of the uterine corpus. Histopathology 2013.
15. Turner BM, Cagle PT, Sainz IM, Fukuoka J, Shen SS, Jagirdar J. Napsin A, a new marker for lung adenocarcinoma, is complementary and more sensitive and specific than thyroid transcription factor 1 in the differential diagnosis of primary pulmonary carcinoma: evaluation of 1674 cases by tissue microarray. Arch Pathol Lab Med 2012; 136(2):163-171.
16. Bejarano PA, Baughman RP, Biddinger PW, Miller MA, Fenoglio-Preiser C, al-Kafaji B et al. Surfactant proteins and thyroid transcription factor-1 in pulmonary and breast carcinomas. Mod Pathol 1996; 9(4):445-452.
17. Deavers MT. Immunohistochemistry in gynecologic pathology. Arch Pathol Lab Med 2008; 132(2):175-180.
18. Ye J, Hameed O, Findeis-Hosey JJ, Fan L, Li F, McMahon LA et al. Diagnostic utility of PAX8, TTF-1 and napsin A for discriminating metastatic carcinoma from primary adenocarcinoma of the lung. Biotech Histochem 2012; 87(1):30-34.
19. Brunner AH, Riss P, Heinze G, Meltzow E, Brustmann H. Immunoexpression of PAX 8 in endometrial cancer: relation to high-grade carcinoma and p53. Int J Gynecol Pathol 2011; 30(6):569-575.
3/1/2014
REVIEW ARTICLEpublished: 31 March 2014
doi: 10.3389/fonc.2014.00065
HPV infection in cervical and other cancers in Saudi Arabia:implication for prevention and vaccinationGhazi Alsbeih*
Research Centre, King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia
Edited by:Silvia De Sanjose, Catalan Institute ofOncology, Spain
Reviewed by:Mahboobeh Safaeian, NationalCancer Institute, USASophia S. Wang, City of Hope, USA
*Correspondence:Ghazi Alsbeih, Radiation BiologySection, Biomedical PhysicsDepartment, KFSHRC, P.O. Box 3354,MBC-03, Riyadh 11211, Saudi Arabiae-mail: [email protected]
Human papillomavirus (HPV) is closely associated with cervical cancer that the incidenceof this tumor is regarded as a surrogate marker for HPV infection in countries lacking epi-demiological studies. HPV is also implicated in subsets of anogenital and oropharyngealcancers. Although cervical cancer is the third most common cancer in women worldwide,its reported incidence is low in Saudi Arabia, ranking number 12 between all cancers infemales and accounts only for 2.4% of all new cases, despite the lack of national screen-ing programs. However, the limited available studies from Saudi Arabia indicate that HPVprevalence and genotypes’ distribution in invasive cervical cancer show similar pattern asin the world. Cytology screening (Pap smear) and HPV vaccinations are the two preven-tive measures against cervical cancer. The two available vaccines are effective against thetwo most common HPV genotypes (HPV-16 and -18). Since 92% of cervical tumors in theKingdom are infected with HPV of which 78% are HPV-16 and -18 genotypes, vaccinationis expected to protect against more than two-third of cervical cancers in Saudi Arabia.Nevertheless, due to its low incidence (2.1/100,000 women), a proper cost-effectivenessanalysis is required to justify the implementation of a costly vaccine bearing in mind thatHPV could potentially be associated with about 3% of all cancers. However, further studiesare needed to ascertain the real prevalence of HPV at the population level at large, its asso-ciation with various types of cancers, and also the impact of local tradition and emergingbehavioral trends that could affect HPV transmission and consequently the effectivenessof applying national vaccination program.
Keywords: human papillomavirus, HPV genotype, HPV-16, cervical cancer, Saudi Arabia
INTRODUCTIONHuman papillomavirus (HPV) has been overwhelmingly associ-ated with cervical cancer that the incidence of this malignancy isdeemed as surrogate indicator of HPV infection in countries lack-ing epidemiological studies. This can easily be justified because theworldwide HPV prevalence in cervical cancer has been estimatedbetween 85 and 99% (1, 2). According to the International Agencyfor Research on Cancer, report GLOBOCAN 2008, cervical can-cer is the third most common cancer in women worldwide, andthe seventh overall, with an estimated 530,000 new cases in 2008(3). More than 85% of the global burden occurs in developingcountries, where it accounts for 13% of all female cancers. Agestandardized rates show that cervical cancer is the second mostcommon cancer in women in less developed countries while itranks number 7 in developed countries. This is due to the lack ofproper screening program (4). Pap smear screening, which identi-fies cytological abnormalities of the cervical transformation zone,has helped reducing cervical cancer incidence and mortality ratesby 70% in developed countries (5).
HPV INFECTION AND RELATED CANCERSHuman papillomavirus infection is common particularly in sex-ually active period of age. Reported estimates of incident HPVinfection among initially negative women have reached as highas 60% over a 5-year follow-up period (6). The population-wide
prevalence of HPV in women varies among studies and countriesfrom 1.5 to 39% and closely reflects age and sexual activity (7–9).In addition, HPV infection has also been implicated in other can-cers such as 90% of anal cancers and a smaller subset (<50%) oforopharyngeal, penile, vaginal, and vulvar cancers. In total, HPVmay account for 5.2% of the worldwide cancer burden (10).
In contrast to the global view, the incidence of cervical canceris very low in Saudi Arabia, ranking number 12 between all can-cers in females and accounts only for 2.4% of all new cases (11),despite the lack of national screening programs. The incidencerate is extracted from the Saudi Cancer Registry (SCR), which isa population-based registry developed in 1992. It was establishedunder the jurisdiction of the Ministry of Health and commencedreporting cancer cases from 01 January 1994. Although it relies onthe collaboration of 500 governmental and private hospitals,physi-cian’s offices, cancer treatment centers, and pathology laboratorieslocated throughout the country, full coverage of all cancer casescannot be ascertained. Nevertheless, in view of the lack of nationalscreening programs, the actual reason for this low incidence isunknown. The closed society and standards of mores could reducewomen’s exposure to HPV infection (12–15). Although cervicalcancer is both preventable and curable, due to the lack of accessiblescreening in Saudi Arabia, most cases are presented at advancedstages (16, 17), that require extensive chemo-radiation therapy.This is due to the lack of proper screening program (4).
www.frontiersin.org March 2014 | Volume 4 | Article 65 | 1
Alsbeih HPV and related cancers in KSA
Data concerning the prevalence of HPV infection, HPV geno-types, and its relationship with cervical cancer are globally scarcein Saudi Arabia. Studies profiling Pap smears examined at Uni-versity Hospitals have revealed precursor lesions of cervical cancer(14, 18–20). A study combining HPV detection with Pap test in100 women undergoing voluntarily cervical cytological screeninghave found 6% HPV-positive cases that consisted of 5% high-risk and 1% low risk HPV (21). This percentage has further beenconfirmed in a larger, more recent study including 485 womenseeking general gynecologic care at King Abdulaziz University Hos-pital in western region of Saudi Arabia (22). In contrast to theselow risk settings, a study performed on 120 women attendingroutine gynecological examination in a hospital-based commu-nity, with dominant human components that has internationalacquaintances, has reported a prevalence of 31.6% infection withHPV-16/18 (23). In this study, none of the seven subjects withabnormal cytology had progressed to cervical intraepithelial neo-plasia (CIN3) after 4 years of follow-up; suggesting non-persistinginfection (24, 25). However, these studies were all confined tocommunities revolving around major hospitals and had includedlimited number of subjects. In addition, as local tradition lim-its sexual activities to marriage with no studies of the impact oftravel on infection transmission considering the large portion ofSaudi young generation traveling abroad for studies, business, andleisure, the real epidemiological prevalence of HPV infection innative Saudi population is still unknown.
The magnitude of the association between HPV infections andgenotypes as causative agent of cervical cancer has been recentlyevaluated in limited number of patients in two independent stud-ies from the same institute (26, 27). The first study had included100 paraffin-embedded cervical biopsies with histopathologicallyproven cervical cancer. By histology,82% were squamous cell carci-noma and 18% were adenocarcinoma of the cervix. Eleven patientshad reported other unidentified past cervical infections and onlysix had prior screening. HPV detection and genotyping was car-ried out using the Linear Array kit (Roche Diagnostic) that enablesthe concomitant detection of 37 mucosal HPVs including 13 mostcommon high-risk viruses. Results showed that 89% were positivefor HPV infection. By histopathology, 93% of squamous cell car-cinomas and 72% of adenocarcinomas are HPV positive. In total,11 different HPV genotypes were detected, 8 of which (16, 18,31, 39, 45, 51, 59, 73) are commonly classified as high-risk (87%)and 3 (6, 64, and 70) are classified as low risk (2%) HPVs. Thus,the prevalence of high-risk genotypes was 97.8% of HPV-positivetumors, which is comparable to the regional results obtained byDarnel et al. involving 44 Syrian women with invasive cervical can-cer (28). The frequencies of the different HPV genotypes detectedare summarized in (Table 1).
In agreement with other studies, the most common HPV geno-type was HPV-16 (29) with a prevalence of 65.2% compared to54.4% in the world (30). The following most common genotypesby decreasing prevalence were: HPV-31 (7.9%), HPV-45 (6.7%),HPV-18 (3.4%), and HPV-73 (2.3%). The HPV genotypes (6, 39,51, 59, 64, and 70) had an estimated prevalence of 1.1% each.Co-infections implicated HPV-16 with HPV-18 (6.7%), HPV-39,HPV-51, and HPV-70 (1.1% each), and HPV-45/59 (1.1%). Withdouble infections, the two most common HPV genotypes were 16
Table 1 | Prevalence of different HPV genotypes in two studies
including 190 (92% HPV positive) cervical cancer patients in Saudi
Arabia.
HPV
genotypes
Classification Prevalence (%)
Alsbeih et al. (26) Al-Badawi et al. (27)
SINGLE INFECTION
HPV-6 LR 1.1
HPV-16 HR 65.2 63.4
HPV-18 HR 3.4 11.1
HPV-31 HR 7.9 2.2
HPV-33 3.3
HPV-45 HR 6.7 4.5
HPV-52 2.2
HPV-53 2.2
HPV-58 2.2
HPV-59 HR 1.1 2.2
HPV-64 LR 1.1
HPV-66 2.2
HPV-73 HR 2.3
CO-INFECTIONS
HPV-16/18 HR/HR 6.7
HPV-16/39 HR/HR 1.1
HPV-16/51 HR/HR 1.1
HPV-16/70 HR/LR 1.1
HPV-45/59 HR/HR 1.1
LR, low risk; HR, high-risk.
and 18 with an estimated overall prevalence of 70% of all patientsand 78.7% of HPV-positive tumors. This is comparable to theprevalence observed in Europe (74.5%), North America (76.5%),and in the whole-world (70.9%). However, these results seem tobe different from those obtained in another Middle Eastern coun-try where the most common HPV genotype was 33, which wasdetected at low prevalence in one of the two referenced studyin Table 1, followed by 16 and 18 (28). Interestingly, as it hadbeen described in the literature (25, 31), the two most commonHPV genotypes (16 and 18) were more frequent in younger agegroup, and caused cervical cancer to occur 5 years earlier thanother HPV infected patients. Furthermore, age-specific HPV dis-tribution in Saudi cervical cancer patients showed a bimodal curvewith a first peak at younger ages (41–45 years) and a relativerebound at older ages (56–60 years) as it has been described inother population (32).
The second study included 90 patients with cervical can-cer and had essentially reached similar results using polymerasechain reaction amplification methods with two common primers,MY09, MY11 and GP5+, GP6+ that amplify a wide range ofHPVs of which isolates were genotyped using DNA sequencingand reverse line blot hybridization assay to identify the high-riskHPV genotypes (27). Results showed that 95.5% were HPV pos-itive. The most common HPV genotype detected was HPV-16(63.4%), HPV-18 (11.1%), HPV-45 (4.5%), HPV-33 (3.3%), and
Frontiers in Oncology | Cancer Epidemiology and Prevention March 2014 | Volume 4 | Article 65 | 2
Alsbeih HPV and related cancers in KSA
HPV-31, HPV-52, HPV-53, HPV-58, HPV-59, and HPV-66 with2.2% prevalence rate each (Table 1). Both studies concluded thatthe results obtained in Saudi cervical cancer patients are compa-rable to international rates, namely: (1) the prevalence of HPVinfection (89–96%) is in range of the published worldwide esti-mates of 85–99% (1, 2); and (2) the most common genotypes arethe high-risk HPV-16 and -18 that affect together 74.5–78.7% ofall HPV-positive patients (Figure 1).
In North America, it was noted that, although there has beensignificant reduction of the incidence of head and neck cancers asa result of the anti-smoking campaigns since the late 80s, there wasa significant increase in the incidence of oropharyngeal squamouscell carcinomas in young (40–55 years) specifically in the tonsilsand the base of the tongue where most of these patients are notalcohol or tobacco consumers (33). About 60% of these tumorswere found positive for HPV-16, the same type that leads to HPV-associated anogenital cancers. Interestingly, the prevalence of HPVinfections in the oral cavity is significantly higher among men thanwomen. In a systematic meta-analysis, Kreimer et al. have reviewed60 eligible studies that included 5,046 cases of squamous cell carci-nomas of the head and neck (34). HPV was prevalent in 35.6% oforopharyngeal, in 23.5% of oral, and in 24% of laryngeal cancers.HPV-16 was by far the commonest subtype in all HPV-positivecancers (87% of oropharyngeal, 68% of oral, and 69% of laryngealcancers). HPV-18 was the next most common subtype.
So far, the prevalence of HPV infection in anal, penile, vagi-nal, vulvar, and oropharyngeal cancers has not yet been exploredin Saudi Arabia. Nevertheless, it is important to include theseforecasted potential when discussing the eventual impact of HPVvaccines in a society. In fact, the extrapolation of these rough esti-mated percentages of HPV-positive anogenital (96%) and headand neck (30%) cancers in Saudi Arabia is given in Figure 2. Intotal, potential HPV-related cancers would represent about 3% ofall cancers in both genders. In addition, HPV has more recentlybeen suspected to be implicated in subgroups of colorectal andbreast malignancies, however; these studies remain inconclusive,
particularly that some experts in the field still do not support sucha role in view of the inadequate evidence.
Acknowledging that detection of HPV infection in head andneck cancers remains lacking with no published report to datefrom Saudi Arabia, few studies had looked at this potential insome other types of tumors such as ocular and prostatic cancers.Karcioglu and Issa had examined the presence of HPV-16/18 in96 paraffin-embedded external ocular tissues with neoplastic andnon-neoplastic lesions and 19 conjunctiva samples free from overtdisease (35). They reported HPV positivity in 57% of in situ squa-mous cell carcinoma, in 55% of invasive squamous cell carcinoma,in 20% of climatic droplet keratopathy, in 35% of scarred corneas,and in 32% of normal conjunctival tissue obtained during routinecataract extractions. They concluded that HPV is detectable notonly in epithelial neoplasms of the ocular mucous membrane butalso in non-neoplastic lesions as well as in apparently healthy con-junctiva. Gazzaz and Mosli had explored the possibility of findingHPV infection in prostatic tissues of 56 patients presenting withbenign prostatic hyperplasia (BPH) or prostate cancer (36). Theresults showed that all the prostatic biopsies were negative for HPVDNA as assessed by the hybrid capture 2 technology that can detect13 carcinogenic types of HPV infection, and differentiate betweenthe 2 HPV groups, the low and the high/intermediate risk types.The authors concluded that it is unlikely that HPV enhance therisk of prostate cancer.
PREVENTION AND HPV VACCINESPrevention of cervical cancer is provided by HPV screening andvaccination, which is an effective measure in many infectious dis-eases (24, 25). Vaccines were developed against HPV infection toprevent cervical cancer and probably other HPV-related diseases(37). Two types, a bivalent (Cervarix) vaccine that protect againstHPV-16 and -18 and a quadrivalent (Gardasil) that is effectiveagainst HPV-6, -11, -16, and -18 are being widely introduced inwestern countries (38, 39), and promising new broad-spectrumHPV vaccines are in development (40). The short term results
FIGURE 1 |The prevalence of HPV infection and genotypes distribution in cervical cancer in Saudi Arabia compiled from two published studies (26, 27).
www.frontiersin.org March 2014 | Volume 4 | Article 65 | 3
Alsbeih HPV and related cancers in KSA
FIGURE 2 | Burden of potentially HPV-mediated cancers in SaudiArabia.
showed nearly complete efficacy against cervical cytological abnor-malities, precancerous lesions, and even genital warts in the caseof the quadrivalent vaccine (37, 38).
In principle, the vaccines could be applicable in Saudi Arabiasince the incidence of HPV infection in invasive cervical cancer isvery high (89–96%) and comparable to the whole-world (85–99%)and that about 75% are HPV-16/18 genotypes (Figure 1), whichare covered by both currently available vaccines. Therefore, intheory vaccination is expected to protect against more than three-quarters of cervical cancers in Saudi Arabia as it has been estimatedworldwide (38). In contrast, the incidence of cervical cancer is verylow in Saudi Arabia, forming only 2.4% of all females’ cancers.From an expenditure point of view, an expert cost-effectivenessanalysis that takes into consideration the particular incidence ofcervical cancer in the country, is required to justify the implemen-tation of a costly national vaccination program (41). The currentinformation from the SCR and the WHO/ICO indicates that theincidence of cervical cancer in Saudi Arabia is 2.1/100,000 women.This rate is at the threshold of the best performance that cytol-ogy screening (Pap smear) can offer and is much lower than thetwo available vaccines against HPVs can achieve at 9/100,000 forCervarix and 14/100,000 for Gardasil (37, 38).
Although cost-effectiveness criteria may vary between coun-tries, a preliminary evaluation would suggest that there is no reasonfor public health to institute either screening or vaccination ifSaudi Arabia truly has such a low incidence of cervical cancer.Therefore, theoretically, some might argue that implementing anational vaccination program may not sensibly decrease the inci-dence of cervical cancer at the population level because it is alreadyvery low. In addition, if all 10–14 years old girls are to be vacci-nated in Saudi Arabia (1,006,745 girls× 3 shots/girl), this wouldbe a burden on health system and may not be cost-effective incomparison with other health priorities. Nevertheless, there maybe specific individual benefits that screening or vaccination mayoffer for selected women, who would be identified upon carefulanalysis of the frequency of cervical cancer in groups at risk for
HPV infection or women well-informed about the risk and vol-untarily wish to be vaccinated. However, taking into considerationthe projection of all potentially HPV-associated tumors outlinedin Figure 2, in theory, the vaccination is expected to protect about3% of cancer patients in Saudi Arabia, which would be, froman expenditure standpoint, still require proper cost-effectivenessevaluation in view of the incidence of head and neck [excludingnasopharyngeal carcinoma that would rather be associated withEpstein–Barr virus (EBV) infection] and anogenital cancers in thecountry. In addition, many DNA vaccines are being developedfor the treatment of HPV-16 induced malignancies (42). Mostof these vaccines consist of a fusion of E6 or E7 with a “carrier-protein” to generate highly immunogenic E6- or E7-directed DNAvaccines. These vaccines can be used to treat HPV-positive cancersto improve outcome.
Currently, the vaccines are available in major hospitals in SaudiArabia and are offered with or without fee to requesting girls uponphysician’s prescription. Family physicians should also be pro-vided with objective information regarding the HPV vaccine sothey would recommend the vaccine to their patients (43). Parents’involvement is a significant factor in decision making since cur-rent practice recommends vaccination to be carried out on minorsand young age (9–26 years old) for best efficacy. Hence, educatingfamilies and medical staff on the vaccines is important to recon-cile with religious values and beliefs bearing in mind that vaccinesat large have saved more lives than any medical treatment everdeveloped.
CONCLUSIONBeside the salient difference of having very low incidence of cer-vical cancer in Saudi Arabia, the involvement of HPV infection inthis malignancy is comparable to the rest of the world with HPV-16 and -18 being the two most common genotypes and accounttogether for three-quarters of HPV infection. Although vaccina-tion against HPV would protect three-quarters of cervical cancerpatients, the currently reported low incidence and the high costof the vaccine would make it not-cost-effective in the Kingdomof Saudi Arabia. However, to ascertain or refute these conclusions,further studies are needed to find out the real prevalence of HPVat the population level at large, its association with various typesof cancers, and also the impact of local tradition and emergingbehavioral trends that could affect HPV transmission.
ACKNOWLEDGMENTSI would like to thank: Drs Medhat El-Sebaie, Nasser Alrajhi, AsmaTulbah, Hadeel Almanea, Anas Dababo, Nabil Alzaher, HatimKhoja for their collaboration, Najla Al-Harbi, Khaled Al-Hadyan,Sarah Al-Qahtani, Nikki Venturina for assistance. This work issupported by the National Science, Technology and InnovationPlan (NSTIP), King Abdulaziz City for Science and Technology(KACST), grant # 12-MED2945-20 (KFSHRC, RAC# 2130 025).
REFERENCES1. Walboomers JM, Jacobs MV, Manos MM, Bosch FX, Kummer JA, Shah KV, et al.
Human papillomavirus is a necessary cause of invasive cervical cancer world-wide. J Pathol (1999) 189(1):12–9. doi:10.1002/(SICI)1096-9896(199909)189:1<12::AID-PATH431>3.0.CO;2-F
Frontiers in Oncology | Cancer Epidemiology and Prevention March 2014 | Volume 4 | Article 65 | 4
Alsbeih HPV and related cancers in KSA
2. de Sanjose S, Quint WG, Alemany L, Geraets DT, Klaustermeier JE, LloverasB, et al. Human papillomavirus genotype attribution in invasive cervical can-cer: a retrospective cross-sectional worldwide study. Lancet Oncol (2010)11(11):1048–56. doi:10.1016/S1470-2045(10)70230-8
3. Ferlay J, Shin HR, Bray F, Forman D, Mathers C, Parkin DM. Cancer Incidenceand Mortality Worldwide: IARC CancerBase No. 10 [Internet]. Lyon: Interna-tional Agency for Research on Cancer (2010). [cited 2012 Apr 29]. Availablefrom: http://globocan.iarc.fr
4. Kitchener HC, Symonds P. Detection of cervical intraepithelial neoplasia indeveloping countries. Lancet (1999) 353(9156):856–7.
5. Kurman RJ, Henson DE, Herbst AL, Noller KL, Schiffman MH. Interim guide-lines for management of abnormal cervical cytology. The 1992 National Can-cer Institute Workshop. JAMA (1994) 271(23):1866–9. doi:10.1001/jama.271.23.1866
6. Baseman JG, Koutsky LA. The epidemiology of human papillomavirus infec-tions. J Clin Virol (2005) 32(Suppl 1):S16–24. doi:10.1016/j.jcv.2004.12.008
7. Cogliano V, Baan R, Straif K, Grosse Y, Secretan B, Ghissassi FE. Carcinogenicityof human papillomaviruses. Lancet Oncol (2005) 6(4):204. doi:10.1016/S1470-2045(05)70086-3
8. Castellsague X, Iftner T, Roura E, Vidart JA, Kjaer SK, Bosch FX, et al. Preva-lence and genotype distribution of human papillomavirus infection of thecervix in Spain: the CLEOPATRE study. J Med Virol (2012) 84(6):947–56.doi:10.1002/jmv.23282
9. Howell-Jones R, de Silva N, Akpan M, Oakeshott P, Carder C, Coupland L, et al.Prevalence of human papillomavirus (HPV) infections in sexually active adoles-cents and young women in England, prior to widespread HPV immunisation.Vaccine (2012) 30:3867–75. doi:10.1016/j.vaccine.2012.04.006
10. Tota JE, Chevarie-Davis M, Richardson LA, Devries M, Franco EL. Epidemi-ology and burden of HPV infection and related diseases: implications forprevention strategies. Prev Med (2011) 53(Suppl 1):S12–21. doi:10.1016/j.ypmed.2011.08.017
11. SCR Report 2007. Special edition. In: Bazarbashi S,Al-Zahrani A, editors. CancerIncidence and Survival Report 2007. Riyadh: Ministry of Health (2011). p. 1–119.
12. Bhurgri Y, Bhurgri A, Rahim A, Bhutto K, Pinjani PK, Usman A, et al. The patternof malignancies in Karachi (1995 to 1996). J Pak Med Assoc (1999) 49(7):157–61.
13. Altaf F. Pattern of cervical smear cytology in western region of Saudi Arabia.Ann Saudi Med (2001) 21:94–6.
14. Jamal A, Al-Maghrabi JA. Profile of Pap smear cytology in the western region ofSaudi Arabia. Saudi Med J (2003) 24(11):1225–9.
15. Raza SA, Franceschi S, Pallardy S, Malik FR, Avan BI, Zafar A, et al.Human papillomavirus infection in women with and without cervical cancerin Karachi, Pakistan. Br J Cancer (2010) 102(11):1657–60. doi:10.1038/sj.bjc.6605664
16. el Dosoky M, Ismail N, Dagastani M. Preinvasive cervical carcinoma in SaudiArabia. Lancet (1995) 345(8950):650. doi:10.1016/S0140-6736(95)90551-0
17. Manji M. Cervical cancer screening program in Saudi Arabia: action is overdue.Ann Saudi Med (2000) 20(5):355–7.
18. Mansoor I. Experience with cervical Pap smears at the University Hospital inJeddah. Ann Saudi Med (2003) 23(3–4):229–30.
19. Altaf FJ. Cervical cancer screening with pattern of Pap smear. Review of multi-center studies. Saudi Med J (2006) 27(10):1498–502.
20. Abdullah LS. Pattern of abnormal Pap smears in developing countries: areport from a large referral hospital in Saudi Arabia using the revised 2001Bethesda System. Ann Saudi Med (2007) 27(4):268–72. doi:10.4103/0256-4947.51488
21. Gazzaz FB. Molecular testing of human papillomavirus in cervical specimens.Saudi Med J (2007) 28(12):1810–8.
22. Bondagji NS, Gazzaz FS, Sait K, Abdullah L. Prevalence of high-risk humanpapillomavirus infections in healthy Saudi women attending gynecologic clin-ics in the western region of Saudi Arabia. Ann Saudi Med (2013) 33(1):13–7.doi:10.5144/0256-4947.2013.13
23. Al-Muammar T, Al-Ahdal MN, Hassan A, Kessie G, Dela Cruz DM, MohamedGE. Human papilloma virus-16/18 cervical infection among women attend-ing a family medical clinic in Riyadh. Ann Saudi Med (2007) 27(1):1–5.doi:10.4103/0256-4947.51540
24. McCredie MR, Sharples KJ, Paul C, Baranyai J, Medley G, Jones RW, et al. Nat-ural history of cervical neoplasia and risk of invasive cancer in women withcervical intraepithelial neoplasia 3: a retrospective cohort study. Lancet Oncol(2008) 9(5):425–34. doi:10.1016/S1470-2045(08)70103-7
25. Schiffman M, Rodriguez AC. Heterogeneity in CIN3 diagnosis. Lancet Oncol(2008) 9(5):404–6. doi:10.1016/S1470-2045(08)70110-4
26. Alsbeih G, Ahmed R, Al-Harbi N, Venturina LA, Tulbah A, Balaraj K. Prevalenceand genotypes’ distribution of human papillomavirus in invasive cervical cancerin Saudi Arabia. Gynecol Oncol (2011) 121(3):522–6. doi:10.1016/j.ygyno.2011.01.033
27. Al-Badawi IA, Al-Suwaine A, Al-Aker M, Asaad L, Alaidan A, Tulbah A,et al. Detection and genotyping of human papilloma virus in cervical can-cer specimens from Saudi patients. Int J Gynecol Cancer (2011) 21(5):907–10.doi:10.1097/IGC.0b013e318214219f
28. Darnel AD, Wang D, Ghabreau L, Yasmeen A, Sami S, Akil N, et al. Correla-tion between the presence of high-risk human papillomaviruses and Id geneexpression in Syrian women with cervical cancer. Clin Microbiol Infect (2010)16(3):262–6. doi:10.1111/j.1469-0691.2009.02774.x
29. Sjoeborg KD, Trope A, Lie AK, Jonassen CM, Steinbakk M, Hansen M, et al.HPV genotype distribution according to severity of cervical neoplasia. GynecolOncol (2010) 118(1):29–34. doi:10.1016/j.ygyno.2010.03.007
30. Human Papillomavirus and Related Cancers in the World [Internet]. (2010) [cited2010 Sep 8]. Available from: http://www.who.int/hpvcentre
31. Wheeler CM, Hunt WC, Joste NE, Key CR, Quint WG, Castle PE. Humanpapillomavirus genotype distributions: implications for vaccination and can-cer screening in the United States. J Natl Cancer Inst (2009) 101(7):475–87.doi:10.1093/jnci/djn510
32. Bruni L, Diaz M, Castellsague X, Ferrer E, Bosch FX, de Sanjose S. Cervicalhuman papillomavirus prevalence in 5 continents: meta-analysis of 1 millionwomen with normal cytological findings. J Infect Dis (2010) 202(12):1789–99.doi:10.1086/657321
33. Marur S, D’Souza G, Westra WH, Forastiere AA. HPV-associated head andneck cancer: a virus-related cancer epidemic. Lancet Oncol (2010) 11(8):781–9.doi:10.1016/S1470-2045(10)70017-6
34. Kreimer AR, Clifford GM, Boyle P, Franceschi S. Human papillomavirus types inhead and neck squamous cell carcinomas worldwide: a systematic review. CancerEpidemiol Biomarkers Prev (2005) 14(2):467–75. doi:10.1158/1055-9965.EPI-04-0551
35. Karcioglu ZA, Issa TM. Human papilloma virus in neoplastic and non-neoplastic conditions of the external eye. Br J Ophthalmol (1997) 81(7):595–8.doi:10.1136/bjo.81.7.595
36. Gazzaz FS, Mosli HA. Lack of detection of human papillomavirus infection byhybridization test in prostatic biopsies. Saudi Med J (2009) 30(5):633–7.
37. Harper DM, Williams KB. Prophylactic HPV vaccines: current knowledge ofimpact on gynecologic premalignancies. Discov Med (2010) 10(50):7–17.
38. Harper DM. Currently approved prophylactic HPV vaccines. Expert Rev Vaccines(2009) 8(12):1663–79. doi:10.1586/erv.09.123
39. Mahdavi A, Monk BJ. Vaccines against human papillomavirus and cervicalcancer: promises and challenges. Oncologist (2005) 10(7):528–38. doi:10.1634/theoncologist.10-7-528
40. Jagu S, Karanam B, Gambhira R, Chivukula SV, Chaganti RJ, Lowy DR, et al.Concatenated multitype L2 fusion proteins as candidate prophylactic pan-human papillomavirus vaccines. J Natl Cancer Inst (2009) 101(11):782–92.doi:10.1093/jnci/djp106
41. Techakehakij W, Feldman RD. Cost-effectiveness of HPV vaccination comparedwith Pap smear screening on a national scale: a literature review. Vaccine (2008)26(49):6258–65. doi:10.1016/j.vaccine.2008.09.036
42. Oosterhuis K, Aleyd E, Vrijland K, Schumacher T, Haanen JB. Rational designof DNA vaccines for the induction of HPV16 E6 and E7 specific cytotoxic T cellresponses. Hum Gene Ther (2012) 23:1301–12. doi:10.1089/hum.2012.101
43. Sait KH. Knowledge, attitudes, and practices regarding cervical cancer screeningamong physicians in the western region of Saudi Arabia. Saudi Med J (2011)32(11):1155–60.
Conflict of Interest Statement: The author declares that the research was conductedin the absence of any commercial or financial relationships that could be construedas a potential conflict of interest.
Received: 28 November 2013; accepted: 17 March 2014; published online: 31 March2014.Citation: Alsbeih G (2014) HPV infection in cervical and other cancers inSaudi Arabia: implication for prevention and vaccination. Front. Oncol. 4:65. doi:10.3389/fonc.2014.00065
www.frontiersin.org March 2014 | Volume 4 | Article 65 | 5
Alsbeih HPV and related cancers in KSA
This article was submitted to Cancer Epidemiology and Prevention, a section of thejournal Frontiers in Oncology.Copyright © 2014 Alsbeih. This is an open-access article distributed under the termsof the Creative Commons Attribution License (CC BY). The use, distribution or
reproduction in other forums is permitted, provided the original author(s) or licen-sor are credited and that the original publication in this journal is cited, in accordancewith accepted academic practice. No use, distribution or reproduction is permittedwhich does not comply with these terms.
Frontiers in Oncology | Cancer Epidemiology and Prevention March 2014 | Volume 4 | Article 65 | 6
In the presence of UCS with PLSVC, either simpleligation of PLSVC or intracardiac or extracardiac rerout-ing to right atrium can be performed.8–10
REFERENCES
1. Raghib G, Ruttenberg HD, Anderson RC, et al: Termina-
tion of left superior vena cava in left atrium, atrial septal
defect and absence of the coronary sinus. A developmen-
tal complex. Circulation 1965;31:906–918.
2. Joffe DC, Rivo J, Oxorn DC: Coronary sinus atrial septal
defect. Anesth Analg 2008;107:1163–1165.
3. Kwon SH, Oh JH, Kim SJ: Partially unroofed coronary
sinus found incidentally in adult patient. Arch Cardiovasc
Dis 2011;104(3):202–203.
4. Ootaki Y, Yamaguchi M, Yoshimura N, et al: Unroofed
coronary sinus syndrome: Diagnosis, classification, and
surgical treatment. J Thorac Cardiovasc Surg 2003;126:
1655–1656.
5. Vimal R, Sanjiv J, Yuen CH, et al: Case report: Completely
unroofed coronary sinus with a left superior vena cava
draining into the left atrium studied by cardiovascular
magnetic resonance. Indian J Radiol Imaging 2010;20
(3):215–217.
6. ThangaroopanM, TruongQA, KalraMK, et al: Rare case of
an unroofed coronary sinus: Diagnosis by multidetector
computed tomography. Images Cardiovasc Med 2009;
119(16):518–520.
7. Ngee T, LimMC,DeLarrazabal C, et al: Unroofed coronary
sinus defect. J Comput Assist Tomogr 2011;35(2):246–
247.
8. Chihara S, Yasunaga H, Todo K: Anastomosis of left to
right superior vena cava for repair of unroofed coronary
sinus. Gen Thorac Cardiovasc Surg 2012;60(4):244–
246.
9. Jian Z, Li J, Xiao Y: Rare association of tetralogy of
Fallot with partially unroofed coronary sinus and PLSVC:
Case report. Thorac Cardiovasc Surg 2010;58(2):117–
119.
10. Hayase S, Yano Y, Ogawa K, et al: A case of completely
unroofed coronary sinuswith persistent left superior vena
cava. Kyobu Geka 1991;44(2):172–175.
Coronary Sinus Metastasis fromCervical Carcinoma
Khaled E. Al-Ebrahim, MBBCh, FRCSC
Department of Cardiothoracic Surgery,
King Abdulaziz University Hospital,
Jeddah, Saudi Arabia
ABSTRACT In this case report, we present a patientwith metastatic squamous cell cervical carcinomato the coronary sinus. The management of thisunusual metastasis is discussed. doi: 10.1111/jocs.12041 (J Card Surg 2013;28:22–24)
1
Metastases to the heart are very rare.1,2 The rightventricle is the most common metastatic site withinthe cardiac chambers. In this report, we describea metastatic lesion from a cervical squamous cellcarcinoma to the coronary sinus.
CASE REPORT
IRB permission was obtained in King AbdulazizUniversity hospital.
A 22-year-old female, one-year post-radical hysterec-tomy for stage IV B cervical squamous cell carcinomafollowed by radiation and chemotherapy, presentedwith dyspnea and palpitations. Echocardiographyshowed a large right atrial mass, protruding acrossthe tricuspid valve, resembling a myxoma (Fig. 1, videoin Supporting Information). Intraoperatively, undercardiopulmonary bypass and cardioplegic arrest, themass was found to be filling the right atrium (Fig. 2A).Excision of the mass revealed that its origin was thecoronary sinus (Fig. 2B). It was arising deep in thecoronary sinus, extending to the triangle of Koch (Fig. 3).The mass was soft, and very fragile. The postoperativecoursewas uneventful. The histopathology of themassand cytology of the pericardial fluid confirmed ametastatic squamous cell carcinoma of uterine origin.Metastatic workup including chest X-ray, liver ultra-sound, CT brain and bone scan were all negative. Thepatientwas referred back to oncology for further chemoand radiotherapy cycles. A repeat echocardiogramsix months later showed no recurrence.
COMMENT
Cervical carcinoma commonly metastasizes to theliver, bone brain, and lung.1–3 Cardiac metastasis fromuterine cancer is very rare with a frequency rangingfrom 1.6% to 8.0%.4–6 Secondary heart tumors usually
Disclosure: The authors report no conflict of interest.
Address for correspondence: Khaled E. Al-Ebrahim, Department of
Surgery, King Abdulaziz University Hospital, P. O. Box 80215, Jeddah
21589, Saudi Arabia. Fax: þþ6401238; e-mail: [email protected]
22 AL-EBRAHIM J CARD SURGCORONARY SINUS METASTASIS 2013;28:19–32
remain silent and are discovered only at autopsy.4–6
Echocardiography should be performed as soon assymptoms of heart failure, angina pectoris, embolism,or rhythm disturbances develop or heart size increasesradiologically.4–6 In this case, the mechanism of spreadwas most likely hematogenous through the coronariesand residing in the coronary sinus. Bhatia et al.7
described an intracardiac recurrent thymoma arisingfrom the coronary sinus, via infiltration from the lateralLV wall by pleural metastases. In selected patients,palliative resectionmay be necessary to improve qualityof life. Advanced cervical carcinoma is not consideredcurable and the prognosis is very poor with less than20% five-year survival.3
Figure 1. Echocardiography static view showing the mass protruding across the tricuspid valve into the right ventricle.
Figure 3. Mass arising deep in the coronary sinus (blackarrow).
Figure 2. (A) Huge right atrial mass almost filling the rightatrium. (B) The mass postexcision showing the coronarysinus origin at the tapering end (black arrow).
J CARD SURG AL-EBRAHIM 232013;28:19–32 CORONARY SINUS METASTASIS
REFERENCES
1. Lam KY, Dickens P, Chan ACL: Tumors of the heart. A 20-
year experience with a review of 12485 consecutive
autopsies. Arch Pathol Lab Med 1993;117:1027–1031.
2. Thurber DL, Edwards JE, Achor RWP: Secondary malig-
nant tumors of the pericardium. Circulation 1962;26:228–
224.
3. Wright TC Jr, Massad LS, Dunton CJ, et al: 2006
Consensus guidelines for the management of women
with cervical intraepithelial neoplasia or adenocarcinoma in
situ. Am J Obstet Gynecol 2007;197(4):340–345.
4. Hanfling SM:Metastatic cancer to the heart: Review of the
literature and report of 127 cases. Circulation 1960;22:474–
448.
5. Itoh K, Matsubara T, Yanagisawa K, et al: Right ventricular
metastasis of cervical squamous cell carcinoma. Am Heart
J 1984;108:1369–1371.
6. Klatt EC, Heitz DR: Cardiac metastases. Cancer 1990;65:
1456–1459.
7. Bhatia L, Lee-Elliot C, Grieves K: Intracardiac recurrent
thymoma arising from the coronary sinus. Can J Cardiol
2009;25(6):e203–e204.
Lymphangioma of the LeftVentricle
Zhixiong Huang, M.D.
Department of Cardiovascular Surgery,
Cardiovascular Institute, Fu Wai Hospital,
Chinese Academy of Medical Science,
Peking Union Medical College, Beijing,
China
ABSTRACT Cardiac lymphangiomas are one of therarest types of cardiac tumors.Wedescribe the caseof a 31-year-old male with a large left ventricularlymphangioma which was totally excised oncardiopulmonary bypass. The surgical manage-ment of this rare cardiac tumor is reviewed. doi:10.1111/jocs.12037 (J Card Surg 2013;28:24–26)2
Primary cardiac lymphangioma is among the raresttumors of the heart.1 We present a case of a patient
with lymphangioma of the left ventricle which wastotally resected on cardiopulmonary bypass and discussthe management of these tumors.
CASE REPORT
A 31-year-old male was referred with substernalchest pain on exercise. The physical examination wasunremarkable. Electrocardiogram showed a sinusrhythm with occasional premature ventricular com-plexes, and T wave inversion in leadI, aVL, V5, V6.Echocardiography revealed a large mass occupyingalmost the entire lateral wall of left ventricle, anda moderate pericardial effusion (Fig. 1A). Contrast-enhanced computed tomography showed a largeintramuralmass in the anterolateralwall of left ventricle.Magnetic resonance imaging (MRI) showed a largeintramural mass in the left ventricle, compressing theleft ventricular cavity (Fig. 2). Surgery was performedvia a median sternotomy under cardiopulmonarybypass and cardiac arrest. Two-hundred milliliters ofstraw yellow pericardial effusion was removed. A gray-white, firm mass was deeply embedded in the antero-lateral wall of the left ventricle between the diagonal
The authors acknowledge no conflict of interest in the submission.
Address for correspondence: Zhixiong Huang, M.D., Department of
Cardiovascular Surgery, Cardiovascular Institute, Fu Wai Hospital, A
167 Beilishi Road, Fuchengmenwai, Beijing 100037, China. e-mail:
Figure 1. Echocardiography. (A) A large mass occupyingalmost the entire lateral wall of left ventricle, and a moderatepericardial effusion. (B) The tumor being completely excised.
SUPPORTING INFORMATION
Additional supporting information may be found inthe online version of this article.
Supporting Video Metastatic mass protrudingacross the tricuspid valve.
24 HUANG J CARD SURGLYMPHANGIOMA OF THE LEFT VENTRICLE 2013;28:19–32
RESEARCH ARTICLE Open Access
Human papillomavirus prevalence and typedistribution among women attending routinegynecological examinations in Saudi ArabiaAbdulaziz AlObaid1, Ismail A Al-Badawi2*, Hanan Al-Kadri3, Kusuma Gopala4, Walid Kandeil5, Wim Quint6,Murad Al-Aker2,7 and Rodrigo DeAntonio5
Abstract
Background: Cervical cancer (CC) is caused by persistent infection with high-risk (HR) human papillomavirus (HPV)types. In Saudi Arabia which has a population of 6.5 million women over the age of 15 years, approximately 152new cases of CC are diagnosed and 55 women die from the disease annually. Nevertheless current epidemiologicaldata for HPV in this population are limited. This study evaluated the prevalence and type distribution of HPV anddocumented the awareness of HPV infection and health-related behavior among Saudi and non-Saudi womenattending routine examination.
Methods: This was an observational, epidemiological cross-sectional study conducted between April 2010 andDecember 2011 at three hospitals in Saudi Arabia. Cervical samples from women aged ≥15 years, who were attendingroutine gynecological examinations were collected and tested for HPV-DNA by polymerase chain reaction and typedusing the SPF10 DEIA/LiPA25 system. Two questionnaires on health-related behavior and awareness of HPV infectionwere completed.
Results: A total of 417 women, mean age (standard deviation) 41.9 (±10.4) years, were included in the final analysis, ofwhom 77% (321/417) were Saudi nationals. HPV-DNA was detected in 9.8% women (41/417, 95% confidence interval[CI]: 7.1-13.1). The prevalence of any HR-HPV by age was: 25–34 years: 3.0%; 35–44 years: 4.5%; 45–54 years: 3.2%;>55 years: 10.9%. The most prevalent HR-HPV-types were: HPV-68/73 (5 cases); HPV-18 (4 cases); HPV-16 (3 cases). Themost prevalent low risk (LR) types were HPV-6 (4 cases); HPV-42, HPV-53 and HPV-54 (2 cases each). The prevalence ofHPV was higher among non-Saudi nationals vs. Saudi nationals (16.7% vs. 7.8%, P = 0.0234). No statistically significant riskfactors were identified: 32.2% (101/314) women were aware of HPV and 89.9% (285/317) showed an interest in HPVvaccination.
Conclusion: The overall prevalence of HPV was 9.8% in Saudi Arabia, but was higher in women over 55 years, as wellas in non-Saudi nationals. These data provide a reference for public health authorities and may also help in determiningfuture policies for the prevention of CC.
Clinical trial registration: NCT01213459
Keywords: Human papillomavirus, Saudi Arabia, Epidemiology
* Correspondence: [email protected] Faisal Specialist Hospital & Research Centre, P.O. Box 3354, Riyadh11211, MBC-52, Saudi ArabiaFull list of author information is available at the end of the article
© 2014 AlObaid et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the CreativeCommons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, andreproduction in any medium, provided the original work is properly credited. The Creative Commons Public DomainDedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article,unless otherwise stated.
AlObaid et al. BMC Infectious Diseases 2014, 14:643http://www.biomedcentral.com/1471-2334/14/643
BackgroundCervical cancer (CC) is the third most frequent cancerin women throughout the world and was associated withan estimated 530,000 new cases and 275,000 deaths in2008 [1]. The global age standardized incidence rate forCC is 15.2 per 100,000 population [2]. Saudi Arabia hasa population of 6.5 million women over the age of15 years [3]. Based on the available data, around 152women are diagnosed with CC and 55 die from the dis-ease annually, corresponding to the 11th most frequentcancer among women of all ages in this population [3].However, as not all CC cases are reported in SaudiArabia, there is a concern that the real incidence of CCmay be somewhat higher.It is known that CC is caused by persistent infection
with high-risk (HR) human papillomavirus (HPV) types[4,5], of which HPV-16 and HPV-18 are responsible forapproximately 70% of the overall cases [6]. Two HPVvaccines are currently licensed in many countries: a bi-valent vaccine (Cervarix®, GlaxoSmithKline, Belgium)and a quadrivalent vaccine (Gardasil®, Merck and Co.,Inc., Whitehouse Station, New Jersey), both of which arewell-tolerated and have good efficacy profiles [7-14]. Theintroduction of these vaccines provides an opportunityto reduce CC, but the introduction of such a preventivemeasure, requires baseline data on national epidemi-ology and prevalent circulating HPV strains.The epidemiology of HPV amongst women in Saudi
Arabia is not fully understood and only limited publica-tions about the prevalence, detection and genotyping ofHPV [15-17] and attitudes towards screening are avail-able in this population [18,19]. In order to bridge thisgap and provide baseline data, this study was undertakento evaluate the prevalence and type distribution of HPV,including HR and low risk (LR)-types, among Saudi andnon-Saudi women. The study also documented the levelof awareness of HPV infection, health-related behavior,and potential risk factors for HPV infection amongwomen attending routine gynecological screening.
MethodsStudy design and study populationThis multicenter, observational, cross-sectional, epidemio-logical study (NCT01213459) was conducted betweenApril 2010 and December 2011 at three large hospitals:King Fahd Medical City (KFMC), King Faisal SpecialistHospital and Research Centre (KFSH and RC) and KingAbdulaziz Medical City-National Guard Health Affairs(KAMC-NGHA) in Riyadh, Saudi Arabia. Women aged≥15 years undergoing routine gynecological examinationand willing to provide a cervical sample were enrolled.Pregnant women above 25 years or women with a knowndiagnosis of immunosuppression, or those who hadundergone hysterectomy were excluded from the study.
Cytological examination of the collected cervical sampleswas undertaken locally in the laboratories at each hos-pital. The investigator issued two questionnaires for com-pletion by all women; these assessed health-relatedbehavior and their awareness of HPV. The responses tothese questionnaires were anonymous and confidentialitywas maintained.
Sample collection and laboratory proceduresEndocervical samples were also collected during the firstvisit by a trained practitioner/gynecologist using a cyto-brush and placed in a liquid-based cytology transportmedium (PreservCyt®, ThinPrep Pap Test; Cytyc Corpor-ation, Boxborough, Massachusetts). Samples were storedat room temperature at the sites for four weeks and thenat −20°C until shipment to the DDL Diagnostic Labora-tory (Rijswijk, The Netherlands).DNA was isolated from 500 μl of the cervix-vagina on
a MagNA Pure Robot (Roche Diagnostics, Almere, TheNetherlands) using the MagNA Pure LC Total NAILVkit and eluted in 50 μl of elution buffer [20]. Sampleswere tested for HPV-DNA at DDL by broad-spectrumpolymerase chain reaction (PCR) using HPV short PCRfragment 10 (SPF-10) and PCR DNA enzyme immuno-assay (PCR-DEIA) to amplify and recognize at least 57HPV genotypes by hybridization with a cocktail of nineconservative probes. If positive by SPF10-DEIA theamplimers were further analyzed by Line probe assay 25(LiPA25) version 1 system (Labo Biomedical Products,Rijswijk, The Netherlands). This Line probe assay 25(LiPA25) version 1 system (Labo Biomedical Products,Rijswijk, The Netherlands) was used to genotype 25 HRand LR HPV types [21]. (The sequence variation withinthe SPF10 inter-primer region did not allow HPV type 68and 73 to be distinguished [22,23]). DEIA positive-LiPAnegative samples were denoted as non-typeable.
Sample size calculationThe primary objective of the study was to describe theprevalence and types of HPV (including multiple infec-tions) among women ≥15 years of age. To meet this ob-jective, an estimated HPV prevalence ranging from 10 to30% as previously reported [17,24,25], was considered.Given a precision level of 0.045, the required number ofsubjects ranged from 188 subjects for a 10% HPV preva-lence to 450 subjects for a 30% prevalence, including anassumption of 10% of subjects non-evaluable.
Statistical analysesThe percentage of women in each category who wereHPV positive was tabulated with corresponding 95%confidence intervals (CI). Descriptive analyses regardingHPV prevalence, HPV-types, age distribution, potentialrisk factors (education level, life-time marital partners,
AlObaid et al. BMC Infectious Diseases 2014, 14:643 Page 2 of 8http://www.biomedcentral.com/1471-2334/14/643
parity and smoking status) and HPV status were per-formed. An exploratory analysis was performed to assessthe association between the HPV status and nationality(two sided Fisher’s exact test) and the adjusted odds ra-tio (adjusted for factors which are associated with therisk of HPV infection) was calculated using multivariatelogistic regression model. All statistical analyses wereperformed using the statistical analysis software (SAS®)version 9.2.
Ethical considerationsThe study was approved by the following local ethics re-view bodies: Institutional Review Board at KAMC-NGHA;Institutional Review Board at KFMC; Research EthicsCommittee of Office of Research Affairs at KFSH and RC.The study was conducted in accordance with the Declar-ation of Helsinki, good clinical practice guidelines andlocal rules and regulations of the country. A written in-formed consent was obtained from all eligible women be-fore entering the study. The investigator communicatedresults as appropriate to the subjects, including the needfor additional testing or treatment.
ResultsStudy populationOf 420 enrolled women, 417 were included in the finalanalysis (three were excluded: two due to pregnancy andone due to hysterectomy). A total of 151, 152 and 117women were enrolled at the KFMC, KFSH and RC, andKAMC-NGHA hospitals, respectively. Overall, 319 womencompleted the health-related behavior questionnaire and317 completed the HPV awareness questionnaire. Themean age (standard deviation) of the population was 41.9(±10.45) years and 77% (321/417) were Saudi nationals.Most women (93.1%, 297/319) were married and 63.0%(201/319) had studied up to post-secondary/universitylevel.
Overall HPV prevalence and type distributionHPV-DNA was detected in 41 out of 417 women (9.8%),of whom 25 had single HPV-type infection, 4 had multipleHPV-type infection and at least12 women were infectedwith non-typeable HPV-types. Overall, the most prevalentHR-HPV-types were HPV-68/73 (5 cases); HPV-18 (4cases); HPV-16 (3 cases) and the most prevalent LR typeswere HPV-6 (4 cases); HPV-42, HPV-53 and HPV-54 (2cases each) (Table 1).
HPV prevalence and type distribution by ageThe prevalence of any HR-HPV was highest (10.9%)among women over 55 years; LR-HPV-types were alsofound in 6.5% of this age group (Figure 1). However, nostatistical significance was noted by age group.
HPV prevalence and type distribution by nationalityThe prevalence of HPV was higher (16.7% vs. 7.8%, P =0.0234) among non-Saudi nationals (n = 96) as comparedwith Saudi nationals (n = 321) respectively (Figure 2).
HPV co-infectionMultiple infections were observed in four women; three ofwhom were infected with HR-HPV-68/73 (−68/73 with−52, −39, −53; −68/73 with −40; and −68/73 with −54).One woman had a co-infection of HR-HPV-16 with −31.No HR-HPV-18 positive women were co-infected withany other HR-HPV-types.
Table 1 HPV prevalence and type distribution (N = 417)
N = 417 n % 95% CI
HPV negative 376 90.2 86.9–92.9
HPV positive 41 9.8 7.1–13.1
Single infection 25 61.0 44.5–75.8
Multiple infection 4 9.8 2.7–23.1
Non-typeable# 12 29.3 16.1–45.5
HPV-types (n = 41)*
Any high-risk HPV** 18 43.9 28.5–60.3
HPV-68/73 5 12.2 4.1–26.2
HPV-18 4 9.8 2.7–23.1
HPV-16 3 7.3 1.5–19.9
HPV-31 2 4.9 0.6–16.5
HPV-51 2 4.9 0.6–16.5
HPV-52 2 4.9 0.6–16.5
HPV-39 1 2.4 0.1–12.9
HPV-56 1 2.4 0.1–12.9
HPV-58 1 2.4 0.1–12.9
Any low-risk HPV*** 14 34.1 20.1–50.6
HPV-6 4 9.8 2.7–23.1
HPV-42 2 4.9 0.6–16.5
HPV-53 2 4.9 0.6–16.5
HPV-54 2 4.9 0.6–16.5
HPV-11 1 2.4 0.1–12.9
HPV-40 1 2.4 0.1–12.9
HPV-70 1 2.4 0.1–12.9
HPV-74 1 2.4 0.1–12.9
N: number of women whose cervical samples were tested; n: number ofwomen in a given category; 95% CI: exact 95% confidence interval.#DEIA positive-LiPA negative.*Some women were infected with multiple HR/LR HPV types.**Includes 14 women with single HR-HPV infection and 4 women with multipleinfections and at least one HR-HPV type.***Includes 11 women with single LR-HPV infection and 3 women withmultiple infections and at least one LR-HPV type.Note: Single infection = 25/417 = 6.0% (95% CI: 3.9–8.7); Multiple infection = 4/417 = 0.9% (95% CI: 0.3–2.4); non-typeable = 12/417 = 2.9% (95% CI: 1.5–5.0).
AlObaid et al. BMC Infectious Diseases 2014, 14:643 Page 3 of 8http://www.biomedcentral.com/1471-2334/14/643
Awareness and health related behavior questionnaireresultsThe potential risk factors of educational level, number oflife-time partners, parity and smoking status assessedusing univariate analyses showed no statistical associationswith any HPV infection (Table 2). Of 317 women complet-ing the HPV awareness questionnaire, 101 (32.2%) wereaware of HPV and 285 (89.9%) expressed an interest invaccination (Table 3).
DiscussionThis study estimated the prevalence and type distributionof HPV in 417 women above 15 years of age attendingroutine gynecological screening at three large hospitals inRiyadh, Saudi Arabia. We reported a 9.8% prevalence forHPV, which is much lower than the 31.6% overall
prevalence of HPV-16/18 reported previously in a smallstudy involving subjects from Riyadh [24]. An earlier re-port from Jeddah reported a 5.6% prevalence of HR-types[17]. These differences could be due to many factors in-cluding differences in HPV testing technology, study size,age groups or geographical variations. The study whichwas undertaken in Riyadh [24] included a limited numberof subjects (75 Saudi nationals and 45 from other coun-tries) from just one hospital (which was also included inour study). The higher proportion of non-Saudi nationalsin this study might help to explain the observed higherHPV prevalence [24]. Conversely, although the samplesize in the study conducted in Jeddah [17] was similar tothat in our present study, the exclusion of non-Saudinationals might explain the lower reported HPV preva-lence. According to available data, our study included a
Figure 1 HPV prevalence and type distribution by age (N = 417*). *Note: For 2 women, dates were not available; hence age could not beestimated. Note: The error bars represent 95% confidence intervals.
Figure 2 HPV prevalence and type distribution by nationality (N = 417). Note: The error bars represent 95% confidence intervals.
AlObaid et al. BMC Infectious Diseases 2014, 14:643 Page 4 of 8http://www.biomedcentral.com/1471-2334/14/643
representative number of Saudi and non-Saudi nationals,which reflects the current demography of Saudi Arabia[25]. This might explain higher HPV prevalence in ourstudy compared to the Jeddah study. Our study mighttherefore be more representative of the current situationin Saudi Arabia given the larger sample size as well as theinclusion of non-Saudi nationals.Indeed, data from the Saudi Cancer registry suggest
that there are regional differences in the percentage dis-tribution of CC with the northern region having thehighest (6.4%) percentage distribution compared withthe other regions [26].According to the United Nations classification, Saudi
Arabia belongs to the Western Asia region [27], whichhas a lower HPV prevalence rate (2.2% [95% CI, 1.5–3.1]) compared with global rates (11.4% [95% CI, 11.3–11.5]) in women with normal cytology [2]. The crudeincidence rate of CC per 100,000 women per year inSaudi Arabia is 1.3, which is lower than that in WesternAsia (3.6) and the world (15.8) [2]. The prevalence ofHPV in women with CC in Saudi Arabia has a broadrange (43–89%) [28,29] compared with the global preva-lence (85–99%) [6]. The low rates of CC in Saudi Arabiaas compared with other countries could be due to differ-ences in sexual practices and attitudes. For example, thepopulation in Saudi Arabia is more conservative than
western countries where most of the data derive [30].The exact reasons for these low rates in Saudi Arabiaare not known, but highlight the need for recent data tobetter understand the disease burden of HPV and theprevalent circulating types.The most prevalent HR-HPV-types reported in our
study were HPV-68/73, 18 and 16; the most commonLR-HPV-types were HPV-6, 42, 53 and 54. HPV-types16 and 18 have been predominantly reported in HPVinfections globally and the results of our study weretherefore consistent [24,31]. We did however note ahigh prevalence of HPV-68/73 for the first time, espe-cially among non-Saudi nationals. HPV-68 type hasbeen reported throughout the world, with the exceptionof North America, albeit at a lower prevalence [32].However, our results should be considered with cautionas only five women were positive for HPV-68/73. Fu-ture studies to substantiate this finding are thereforeindicated.Our study also found that HR-HPV infection was high-
est (10.9%) in the oldest age group (>55 years). These re-sults are consistent with the general worldwide trend ofhigher HPV burden in older women. However, the com-parator study from Bruni et al. [31] only included womenwith normal cytology whereas the cytology status was notknown in the present study [32].
Table 2 Prevalence of any HPV by risk factors (N = 319)
Risk factors Categories N HPV+ % Adj. OR LL–UL P value
Age at sample collection (years) <30* 56 3 5.36 . . .
30-39 124 12 9.68 3.310 0.391–28.052 0.2723
40-49 132 12 9.09 2.097 0.230–19.085 0.5110
50-60 89 12 13.48 4.136 0.454–37.681 0.2079
>60 14 2 14.29 9.570 0.601–152.366 0.1097
Nationality Non-Saudi* 96 16 16.67 . . .
Saudi 321 25 7.79 0.315 0.116–0.855 0.0234
Education level No formal education* 21 2 9.52 . . .
Primary 47 3 6.38 1.039 0.144–7.489 0.9693
Secondary 50 4 8.00 1.515 0.200–11.492 0.6879
Post-secondary/University 201 24 11.94 1.158 0.180–7.447 0.8770
Number of marital partners 1* 286 29 10.14 . . .
2-5 32 4 12.50 1.129 0.324–3.941 0.8488
Parity 0* 8 0 0.00 . . .
1-2 100 11 11.00 >999.999 <0.001– > 999.999 0.9516
3-5 112 14 12.50 >999.999 <0.001– > 999.999 0.9502
≥ 6 71 5 7.04 >999.999 <0.001– > 999.999 0.9518
Smoking status No* 273 30 10.99 . . .
Yes 44 3 6.82 0.502 0.134–1.875 0.3053
N: number of subjects in a given cohort; %: HPV+/number of subjects with available results × 100; Adj. OR: Adjusted odds ration from simple logistic regressionmodel Odds ratio adjusted for the other variables; 95% CI: Wald 95% confidence interval; LL: lower limit; UL: upper limit.*Reference category.
AlObaid et al. BMC Infectious Diseases 2014, 14:643 Page 5 of 8http://www.biomedcentral.com/1471-2334/14/643
We estimated a higher proportion of infection in non-Saudi nationals compared with the Saudi nationals. Thisdifference could be due to many reasons including dif-ferent cultural behaviors such as male circumcision[32,33], sexual behavior or prevalence in the nativecountries. Further studies are therefore warranted. Al-though, it appears that non-Saudi nationals are at agreater risk of contracting infection, prevalent HPVtypes indeed pose a risk of infecting Saudi nationals.Our study did not find educational level, number of life-time partners, parity or smoking status to be signifi-cantly associated risk factors for HPV-16, HPV-18 orany HR-HPV infection.Our study results should be interpreted with caution
as the study design was cross sectional, i.e., we obtainedsingle point estimates of women with HPV infections.These infections could have been transient and resolvedon their own rather than leading to CC. In addition,since the overall number of women positive for HPV it-self was low, the prevalence of HPV types should beinterpreted with caution. Another limitation lies withinthe recruitment process: women with higher levels ofeducation are more likely to opt for cervical cancerscreening and therefore would be more likely to partici-pate in our study. Nevertheless, our study did also in-clude women who did not have formal education.A major strength of this study was the high quality of
HPV-DNA testing across the three hospitals in all agegroups which helps to provide a representative sampleof the population. The study also met the required sam-ple size to calculate an overall prevalence of 9.8% with
Table 3 Awareness of HPV infection among women(N = 317)
Characteristics Categories n %
How common is cervical cancerin women?
Very common 48 15.1
Common 157 49.5
Rare 56 17.7
Not sure 56 17.7
What do you think is/are themain reasons for cervical cancer?*
It develops from inside 95 30.0
Bacterial infection 49 15.5
Viral infection 80 25.2
None 14 4.4
Not sure 85 26.8
Which among these cancause cervical cancer?*
Persistent infectionwith HPV
82 25.9
Rous sarcoma virus 23 7.3
Hereditary/genetic factors 115 36.3
None 18 5.7
Not sure 88 27.8
What do you think can turnin to cervical cancer*
Genital warts 109 34.4
Bacterial infection 56 17.7
Fungal infection 26 8.2
None 23 7.3
Not sure 108 34.1
Apart from avoiding unwantedpregnancy, what would youthink can happen with usingcontraceptive pills*
Protects againstcervical cancer
31 9.8
Increases risk ofcervical cancer
123 38.8
No ill effect at all 77 24.3
Not sure 86 27.1
Did you hear about HPV before? Yes 101 32.2
No 213 67.8
Missing 3 -
If yes*, General physician 28 8.8
Friend or family member 20 6.3
Internet 22 6.9
TV/Magazine/Newspaper 46 14.5
Other 14 4.4
How is HPV transmitted?* Contaminatedfood/Water
10 3.2
Mosquito bite 3 0.9
Sexually 159 50.2
None 20 6.3
Not sure 127 40.1
How is cervical cancerdiagnosed?*
Pap smear test 77 24.3
Colposcopy 24 7.6
Biopsy sample testing(histological)
122 38.5
Table 3 Awareness of HPV infection among women(N = 317) (Continued)
All above 82 25.9
None 2 0.6
Not sure 30 9.5
Is it possible to prevent cancer? Yes 243 78.9
No 26 8.4
Not sure 39 12.7
Missing 9 -
If yes*, Through cancer vaccine 58 18.3
Through responsiblesexual behavior
46 14.5
Through cervicalscreening
173 54.6
Through condom use 14 4.4
If the vaccine against cervicalcancer is available, would you beinterested in getting vaccinated?
Yes 285 89.9
No 32 10.1
N: number of women in a specified category for whom questionnaire datawere collected.*Women could have selected more than one option.
AlObaid et al. BMC Infectious Diseases 2014, 14:643 Page 6 of 8http://www.biomedcentral.com/1471-2334/14/643
good precision. Furthermore, the questionnaires werecompleted by most enrolled women giving an importantinsight into behavior and attitudes, and suggesting thatthe introduction of a preventive measure such as vaccin-ation would be accepted. Reports indicate that the pro-portion of non-Saudi nationals represent 20%–30% ofthe entire population in Saudi Arabia [25,34] which isconsistent with our results (23%). In addition, the pro-portion of women completing up to post-secondary/uni-versity level education in our study (63%) is comparableto the Organization for Economic Co-operation andDevelopment (OECD) reports, where at least 50% ofwomen were educated and at least 54% of womenamong all OECD countries completed post-secondaryeducation [35]. These comparisons therefore indicatethe representativeness of our study population.The study results emphasize the need for a future up-
dated policy for HPV and CC prevention in Saudi Arabia.A World Health Organization document on cancer con-trol in the Eastern Mediterranean region describes thatonly 35% of CC cases present at early stage; as in other de-veloping countries, the rest are reported at later stageswhen cure is unlikely, even with the best treatment [34]. Ithas also been reported that when women are double nega-tive in the conventional cytological screening test and thehighly sensitive HPV molecular test, then screening canbe performed at longer intervals [36]. Our study findingstogether with these data will help determine the best strat-egy for targeting preventive interventions, and designingpublic health measures for Saudi Arabia.
ConclusionThe overall results from this study emphasize that the HPVburden in Saudi Arabia is a cause for concern and prevent-ive strategies such as screening, HPV-DNA testing of cer-vical samples and vaccination might reduce the burden ofthe disease. Our data can raise the awareness of local au-thorities and public health officials and help to guide policyin Saudi Arabia to implement strategies to prevent CC.
TrademarkCervarix is a trademark of the GlaxoSmithKline groupof companiesGardasil is a trademark of Merck & Co. Inc.PreservCyt is a trademark of Cytyc Corporation,
Massachusetts, United States of AmericaLabo Biomedical Products, Rijswijk, The Netherlands
AbbreviationsCC: Cervical cancer; CI: Confidence interval; DEIA: DNA enzymeimmunoassay; HPV: Human papillomavirus; HR: High-risk; KAMC-NGHA: KingAbdulaziz Medical City-National Guard Health Affairs; KFMC: King FahdMedical City; KFSH and RC: King Faisal Specialist Hospital and ResearchCentre; LiPA25: Line probe assay 25; LR: Low-risk; OECD: Organization forEconomic Co-operation and Development; PCR: Polymerase chain reaction;SAS: Statistical analysis software; SPF-10: Short PCR fragment 10.
Competing interestsMAA and his institution received a grant from GlaxoSmithKline group ofcompanies to cover costs of samples collection and initial processing; IABand HAK and their respective institutions received financial assistance fromGlaxoSmithKline group of companies for study conduct; RD, WK and KG areemployed by the GlaxoSmithKline group of companies and RD has stockoptions; AAO and WQ have no conflict of interest to declare.
Authors’ contributionsAAO, IAB and HAK were the coordinating investigators and in collaborationwith MAA were responsible for the conduct of the study at the three sites,AAO, IAB, HAK, MAA and WK were involved in the design of the study,analysis and interpretation of the results. KG contributed to the study designand performed the statistical analysis. WQ was responsible for HPVgenotyping, and contributed to the analysis of the results. RD managed thestudy at GlaxoSmithKline Vaccines and contributed to the analysis,interpretation and critically reviewed the study report. All authors had accessto the data and participated in the drafting, review and approval of themanuscript. The corresponding author took final responsibility for submittingthe manuscript.
AcknowledgementsThe authors would like to thank Dr. Mamoun ElAwad for his participation inthe conduct of the study, Karin Hallez and Mohammed Bassyouni formonitoring the study sites (both employed by GlaxoSmithKline group ofcompanies). The authors also thank Shruti Priya Bapna, Harshith Bhat andPreethi Govindarajan (all employed by GlaxoSmithKline group of companies)for preparation of the manuscript, Julia Donnelly (on behalf of GlaxoSmithKlineVaccines) for language editing and Abdelilah Ibrahimi (XPE Pharma and Scienceon behalf of GlaxoSmithKline Vaccines) for publication coordination.
Funding sourceGlaxoSmithKline Biologicals SA sponsored and funded the study conduct,analyses of data and the development and publication of the manuscript.
Author details1King Fahd Medical City, P.O. Box 59046, Riyadh 11525, Saudi Arabia. 2KingFaisal Specialist Hospital & Research Centre, P.O. Box 3354, Riyadh 11211,MBC-52, Saudi Arabia. 3Department of Obstetrics and Gynecology, KingAbdulaziz Medical City, College of Medicine, King Saud bin AbdulazizUniversity for Health Sciences, P.O Box 22490, Riyadh 11426, Saudi Arabia.4GlaxoSmithKline Pharmaceuticals Ltd, Bangalore, India. 5GlaxoSmithKlineVaccines, Wavre, Belgium. 6DDL Diagnostic Laboratory, Rijswijk, theNetherlands. 7Sydney Gynecologic Oncology Group, Royal Prince AlfredHospital, Sydney, Australia.
Received: 22 April 2014 Accepted: 19 November 2014
References1. Ferlay J, Shin HR, Bray F, Forman D, Mathers C, Parkin DM: Estimates of
worldwide burden of cancer in 2008: GLOBOCAN 2008. Int J Cancer 2010,127(12):2893–2917.
2. Rana MM, Huhtala H, Apter D, Eriksson T, Luostarinen T, Natunen K,Paavonen J, Pukkala E, Lehtinen M: Understanding long-term protection ofhuman papillomavirus vaccination against cervical carcinoma: Cancerregistry-based follow-up. Int J Cancer 2013, 132(12):2833–2838.
3. WHO/ICO Information Centre on HPV and CC Human Papillomavirus andRelated Cancers in Saudi Arabia. [http://www.who.int/immunization/topics/hpv/en/]. Last accessed: 06 Jan 2014.
4. Walboomers JM, Jacobs MV, Manos MM, Bosch FX, Kummer JA, Shah KV,Snijders PJ, Peto J, Meijer CJ, Munoz N: Human papillomavirus is anecessary cause of invasive cervical cancer worldwide. J Pathol 1999,189(1):12–19.
5. Munoz N, Bosch FX, de Sanjose S, Herrero R, Castellsague X, Shah KV,Snijders PJ, Meijer CJ, International Agency for Research on CancerMulticenter Cervical Cancer Study G: Epidemiologic classification ofhuman papillomavirus types associated with cervical cancer. N Engl JMed 2003, 348(6):518–527.
6. de Sanjose S, Quint WG, Alemany L, Geraets DT, Klaustermeier JE, Lloveras B,Tous S, Felix A, Bravo LE, Shin HR, Vallejos CS, de Ruiz PA, Lima MA, Guimera
AlObaid et al. BMC Infectious Diseases 2014, 14:643 Page 7 of 8http://www.biomedcentral.com/1471-2334/14/643
N, Clavero O, Alejo M, Llombart-Bosch A, Cheng-Yang C, Tatti SA, KasamatsuE, Iljazovic E, Odida M, Prado R, Seoud M, Grce M, Usubutun A, Jain A,Suarez GA, Lombardi LE, Banjo A, et al: Human papillomavirus genotypeattribution in invasive cervical cancer: a retrospective cross-sectionalworldwide study. Lancet Oncol 2010, 11(11):1048–1056.
7. Descamps D, Hardt K, Spiessens B, Izurieta P, Verstraeten T, Breuer T, DubinG: Safety of human papillomavirus (HPV)-16/18 AS04-adjuvanted vaccinefor cervical cancer prevention: a pooled analysis of 11 clinical trials. HumVaccin 2009, 5(5):332–340.
8. Paavonen J, Naud P, Salmeron J, Wheeler CM, Chow SN, Apter D, KitchenerH, Castellsague X, Teixeira JC, Skinner SR, Hedrick J, Jaisamrarn U, Limson G,Garland S, Szarewski A, Romanowski B, Aoki FY, Schwarz TF, Poppe WA,Bosch FX, Jenkins D, Hardt K, Zahaf T, Descamps D, Struyf F, Lehtinen M,Dubin G, Group HPS: Efficacy of human papillomavirus (HPV)-16/18 AS04-adjuvanted vaccine against cervical infection and precancer caused byoncogenic HPV types (PATRICIA): final analysis of a double-blind,randomised study in young women. Lancet 2009, 374(9686):301–314.
9. Einstein MH, Baron M, Levin MJ, Chatterjee A, Fox B, Scholar S, Rosen J,Chakhtoura N, Meric D, Dessy FJ, Datta SK, Descamps D, Dubin G, GroupHPVS: Comparative immunogenicity and safety of human papillomavirus(HPV)-16/18 vaccine and HPV-6/11/16/18 vaccine: follow-up frommonths 12–24 in a Phase III randomized study of healthy women aged18–45 years. Hum Vaccin 2011, 7(12):1343–1358.
10. Verstraeten T, Descamps D, David MP, Zahaf T, Hardt K, Izurieta P, Dubin G,Breuer T: Analysis of adverse events of potential autoimmune aetiologyin a large integrated safety database of AS04 adjuvanted vaccines.Vaccine 2008, 26(51):6630–6638.
11. Castellsague X, Munoz N, Pitisuttithum P, Ferris D, Monsonego J, Ault K,Luna J, Myers E, Mallary S, Bautista OM, Bryan J, Vuocolo S, Haupt RM, SaahA: End-of-study safety, immunogenicity, and efficacy of quadrivalentHPV (types 6, 11, 16, 18) recombinant vaccine in adult women 24–45years of age. Br J Cancer 2011, 105(1):28–37.
12. Lehtinen M, Paavonen J, Wheeler CM, Jaisamrarn U, Garland SM, CastellsagueX, Skinner SR, Apter D, Naud P, Salmeron J, Chow SN, Kitchener H, Teixeira JC,Hedrick J, Limson G, Szarewski A, Romanowski B, Aoki FY, Schwarz TF, PoppeWA, De Carvalho NS, Germar MJ, Peters K, Mindel A, De Sutter P, Bosch FX,David MP, Descamps D, Struyf F, Dubin G, et al: Overall efficacy of HPV-16/18AS04-adjuvanted vaccine against grade 3 or greater cervical intraepithelialneoplasia: 4-year end-of-study analysis of the randomised, double-blindPATRICIA trial. Lancet Oncol 2012, 13(1):89–99.
13. Roteli-Martins CM, Naud P, De Borba P, Teixeira JC, De Carvalho NS, Zahaf T,Sanchez N, Geeraerts B, Descamps D: Sustained immunogenicity andefficacy of the HPV-16/18 AS04-adjuvanted vaccine: up to 8.4 years offollow-up. Hum Vaccin Immunother 2012, 8(3):390–397.
14. Wheeler CM, Castellsague X, Garland SM, Szarewski A, Paavonen J, Naud P,Salmeron J, Chow SN, Apter D, Kitchener H, Teixeira JC, Skinner SR, JaisamrarnU, Limson G, Romanowski B, Aoki FY, Schwarz TF, Poppe WA, Bosch FX, HarperDM, Huh W, Hardt K, Zahaf T, Descamps D, Struyf F, Dubin G, Lehtinen M,Group HPS: Cross-protective efficacy of HPV-16/18 AS04-adjuvanted vaccineagainst cervical infection and precancer caused by non-vaccine oncogenicHPV types: 4-year end-of-study analysis of the randomised, double-blindPATRICIA trial. Lancet Oncol 2012, 13(1):100–110.
15. Al-Badawi IA, Al-Suwaine A, Al-Aker M, Asaad L, Alaidan A, Tulbah A, FeBohol M, Munkarah AR: Detection and genotyping of human papillomavirus in cervical cancer specimens from Saudi patients. Int J Gynecol Cancer2011, 21(5):907–910.
16. Alsbeih G, Ahmed R, Al-Harbi N, Venturina LA, Tulbah A, Balaraj K:Prevalence and genotypes' distribution of human papillomavirus in invasivecervical cancer in Saudi Arabia. Gynecol Oncol 2011, 121(3):522–526.
17. Bondagji NS, Gazzaz FS, Sait K, Abdullah L: Prevalence of high-risk humanpapillomavirus infections in healthy Saudi women attending gynecologicclinics in the western region of Saudi Arabia. Ann Saudi Med 2013,33(1):13–17.
18. Sait KH: Attitudes, knowledge, and practices in relation to cervical cancerand its screening among women in Saudi Arabia. Saudi Med J 2009,30(9):1208–1212.
19. Sait KH: Knowledge, attitudes, and practices regarding cervical cancerscreening among physicians in the Western Region of Saudi Arabia.Saudi Med J 2011, 32(11):1155–1160.
20. Geraets DT, van Baars R, Alonso I, Ordi J, Torne A, Melchers WJ, Meijer CJ,Quint WG: Clinical evaluation of high-risk HPV detection on self-samples
using the indicating FTA-elute solid-carrier cartridge. J Clin Virol 2013,57(2):125–129.
21. van Doorn LJ, Molijn A, Kleter B, Quint W, Colau B: Highly effectivedetection of human papillomavirus 16 and 18 DNA by a testingalgorithm combining broad-spectrum and type-specific PCR. J ClinMicrobiol 2006, 44(9):3292–3298.
22. Kleter B, van Doorn LJ, Schrauwen L, Molijn A, Sastrowijoto S, ter ScheggetJ, Lindeman J, ter Harmsel B, Burger M, Quint W: Development and clinicalevaluation of a highly sensitive PCR-reverse hybridization line probeassay for detection and identification of anogenital human papillomavirus.J Clin Microbiol 1999, 37(8):2508–2517.
23. de Sanjose S, Diaz M, Castellsague X, Clifford G, Bruni L, Munoz N, Bosch FX:Worldwide prevalence and genotype distribution of cervical humanpapillomavirus DNA in women with normal cytology: a meta-analysis.Lancet Infect Dis 2007, 7(7):453–459.
24. Al-Muammar T, Al-Ahdal MN, Hassan A, Kessie G, Dela Cruz DM, MohamedGE: Human papilloma virus-16/18 cervical infection among womenattending a family medical clinic in Riyadh. Ann Saudi Med 2007, 27(1):1–5.
25. Central Intelligence Agency: The World factbook: Middle East: SaudiArabia. [https://www.cia.gov/library/publications/the-world-factbook/geos/sa.html]. Last accessed: 06 Jan 2014.
26. Cancer Incidence Report Saudi Arabia 2004. National Cancer Registry,Ministry of Health. [http://www.kfshrc.edu.sa/wps/wcm/connect/40dcba804a8d741fb731f7e404c39865/SCR2004W.pdf?MOD=AJPERES&lmod=1265914960&CACHEID=40dcba804a8d741fb731f7e404c39865]. Last accessed:06 Jan 2014.
27. United Nations Statistics Division: Standard country and area codesclassifications (M49). [http://unstats.un.org/unsd/methods/m49/m49regin.htm].Last accessed: 06 Jan 2014.
28. Turki R, Sait K, Anfinan N, Sohrab SS, Abuzenadah AM: Prevalence ofhuman papillomavirus in women from Saudi Arabia. Asian Pac J CancerPrev 2013, 14(5):3177–3181.
29. Alsbeih G, Al-Harbi N, El-Sebaie M, Al-Badawi I: HPV prevalence and geneticpredisposition to cervical cancer in Saudi Arabia. Infect Agent Cancer 2013,8(1):15.
30. Mackay J: Global sex: sexuality and sexual practices around the world.Sex Relation Ther 2001, 16(1):71–82.
31. Bruni L, Diaz M, Castellsague X, Ferrer E, Bosch FX, de Sanjose S: Cervicalhuman papillomavirus prevalence in 5 continents: meta-analysis of 1million women with normal cytological findings. J Infect Dis 2010,202(12):1789–1799.
32. Davis MA, Gray RH, Grabowski MK, Serwadda D, Kigozi G, Gravitt PE,Nalugoda F, Watya S, Wawer MJ, Quinn TC, Tobian AA: Male circumcisiondecreases high-risk human papillomavirus viral load in female partners:A randomized trial in Rakai. Uganda Int J Cancer 2013, 133(5):1247–1252.
33. Morris BJ, Mindel A, Tobian AA, Hankins CA, Gray RH, Bailey RC, Bosch X,Wodak AD: Should male circumcision be advocated for genital cancerprevention? Asian Pac J Cancer Prev 2012, 13(9):4839–4842. Demographicsof Saudi Arabia [http://www.princeton.edu/~achaney/tmve/wiki100k/docs/Demographics_of_Saudi_Arabia.html]. Last accessed: 06 Jan 2014.
34. Towards a Stratergy for Cancer Control in the Eastern MediterraneanRegion. [http://applications.emro.who.int/dsaf/dsa1002.pdf]. Last accessed:06 Jan 2014.
35. Education at a Glance 2012. [http://www.oecd.org/edu/highlights.pdf]. Lastaccessed: 06 Jan 2014.
36. Gazzaz FB: Molecular testing of human papillomavirus in cervicalspecimens. Saudi Med J 2007, 28(12):1810–1818.
doi:10.1186/s12879-014-0643-8Cite this article as: AlObaid et al.: Human papillomavirus prevalence andtype distribution among women attending routine gynecologicalexaminations in Saudi Arabia. BMC Infectious Diseases 2014 14:643.
AlObaid et al. BMC Infectious Diseases 2014, 14:643 Page 8 of 8http://www.biomedcentral.com/1471-2334/14/643
brief communication
Incidence of malignant ovarian germ celltumors (MOGCTs) in Saudi ArabiaAhmed Abu-Zaid a,b, Ahmed Nazer b, Osama AlOmar b, Ayman Azzam c,d, Haya S Al-Eid e,Tusneem Ahmed Elhassan c, Ismail A Al-Badawi a,b,*
a College of Medicine, Alfaisal University, Riyadh, Saudi Arabia, b Department of Obstetrics and Gynecology, King Faisal Specialist
Hospital and Research Center, Riyadh, Saudi Arabia, c Oncology Center, King Faisal Specialist Hospital and Research Center, Riyadh,
Saudi Arabia, d Faculty of Medicine, Alexandria University, Alexandria, Egypt, e Saudi Cancer Registry, Ministry of Health, Riyadh, Saudi Arabia
* Corresponding author at: Department of Obstetrics and Gynecology, King Faisal Specialist Hospital and Research Centre (MBC-52), P.O.
Box 3354, Riyadh 11211, Saudi Arabia. Tel.: +966 11 442 4885; fax: +966 11 442 7397 Æ [email protected] Æ [email protected] Æ[email protected] Æ [email protected] Æ [email protected] Æ [email protected] Æ [email protected] Æ Accepted for
publication 26 December 2013
Hematol Oncol Stem Cell Ther 2014; xx(xx): xxx–xxx
ª 2013 King Faisal Specialist Hospital & Research Centre. Published by Elsevier Ltd. All rights reserved.DOI: http://dx.doi.org/10.1016/j.hemonc.2013.12.001
Abstract Incidence of malignant ovarian germ cell tumors (MOGCTs) in the Saudi Arabian population has not
been studied before. Therefore, the primary objective of this study was to define the population-based incidence
rates and histopathological types of MOGCTs in the Saudi Arabian population from 1999 to 2008. Our study
showed that MOGCTs are a common type of ovarian tumors in the Saudi Arabian population, and the incidence
rates and histopathological types are relatively comparable to the international populations with few differences.
Ovarian germ cell tumors (OGCTs) are rapidlygrowing neoplasms thought to originate fromprimitive germ cells of the embryonic gonad
ovary.1 OGCTs account for roughly 20–25% of all ovar-ian tumors overall and can be benign or malignant.1
Malignant ovarian germ cell tumors (MOGCTs) areexceedingly uncommon neoplasms accounting forroughly 5% of all malignant ovarian tumors.1 Histopa-thological classification of MOGCTs include: dysgermi-noma, germinoma, yolk sac (endodermal sinus) tumor,malignant (immature) teratoma, embryonal carcinoma,polyembryoma, primary ovarian (nongestational) chorio-carcinoma and mixed germ cell tumor.2
Incidence of MOGCTs in the Saudi Arabian pop-ulation has not been studied before. Therefore, theprimary objective of this study is to define thepopulation-based incidence and histopathologicaltypes of MOGCTs in the Saudi Arabian populationfrom 1999 to 2008. Comprehensive details of MOG-CTs are beyond the scope of research report and willbe addressed in a couple of future research projects.
MATERIAL AND METHODS
1Using the Saudi Cancer Registry (SCR), we con-ducted a retrospective cross-sectional study fromJanuary 1999 to December 2008 to explore the
following epidemiological data: histopathologicaltypes, frequency of cases, crude incidence rates(CIRs), and age-standardized incidence rates (ASIRs)of MOGCTs among the Saudi Arabian population.
RESULTS
Among all female tumors diagnosed over the ten yearsexamined (n = 30,720), incidence of ovarian tumorswas 4.1% (n = 1271/30,720). MOGCTs specificallyaccounted for 13.8% (n = 175/1271) of all ovariantumors overall.
Table 1 displays the distribution of MOGCT his-topathological types in the Saudi Arabian populationfrom 1999 to 2008. Dysgerminoma was the mostcommon histopathological type of MOGCTsaccounting for approximately 41.1% (n = 72/175),followed by malignant teratoma 25.1% (n = 44/175),yolk sac tumor 18.3% (n = 32/175) and mixed germ celltumor 14.3% (n = 25/175).
Table 2 shows the total number of cases, CIRs andworld ASIRs of MOGCTs in the Saudi Arabian pop-ulation from 1999 to 2008 adjusted by age. Around60.5% of all MOGCT cases occurred in childrenand young adolescents aged between 10 and 24 yearsof age. Occurrence of MOGCTs in patients beyond40 years of age was extremely low accounting for up
Hematol Oncol Stem Cell Ther xx(xx) xxx Quarter 2014 hemoncstem.edmgr.com 1
to 6.8%. CIRs and world ASIRs were almost similarand did not differ significantly.
DISCUSSION
In our study, incidence of ovarian tumors (4.1%) wasfairly comparable to the worldwide incidence in Uni-ted States and most developed countries (3%).3 More-over, among ovarian tumors overall, incidence ofMOGCTs in the Saudi Arabian population (13.8%)was approximately 3 times the incidence of Westernpopulations (5%) and roughly matched the incidenceof Asian and African populations (15%).4
Dysgerminoma was the most frequent histopathol-ogical type comprising 41.1% of all MOGCTs in theSaudi Arabian population. Our finding greatlymirrored the worldwide incidence of dysgerminoma(35–50%) as the most frequently diagnosed histopa-thological type of MOGCTs.2 While dysgerminomaaccounts for less than 2% of all ovarian tumorsinternationally,5 it accounted for roughly 5.7%(n = 72/1271) of all ovarian tumors in the SaudiArabian population (around 3 folds higher).
Moreover, while ovarian malignant teratomasaccount for less than 1% of all ovarian tumors androughly 20% of MOGCTs internationally,6 they
Table 2. The total number of cases, crude incidence rates (CIRs), and world age-standardized incidence rates (ASIRs) of malignant ovarian germ cell tumors (MOGCTs) in theSaudi Arabian population from 1999 to 2008 adjusted by age.
Yr All ages Un-known
age
0–4
Yr
5–9 Yr 10–14
Yr
15–19
Yr
20–24
Yr
25–29
Yr
30–34
Yr
35–39
Yr
40–44
Yr
45–49
Yr
50–54
Yr
55–59
Yr
60–64
Yr
65–69
Yr
70+
Yr
CIR ASIR
world
1999 21 0 0 1 5 3 5 4 1 1 0 1 0 0 0 0 0 0.3 0.3
2000 18 0 0 3 3 4 3 0 2 1 1 0 0 0 0 0 1 0.2 0.2
2001 14 0 0 0 4 4 1 2 1 0 1 0 0 1 0 0 0 0.2 0.2
2002 19 1 0 3 4 3 3 1 1 0 0 0 0 0 0 0 3 0.2 0.2
2003 22 0 1 3 3 5 5 2 1 2 0 0 0 0 0 0 0 0.3 0.2
2004 13 0 0 1 4 2 2 1 1 0 1 1 0 0 0 0 0 0.2 0.1
2005 16 0 0 4 4 4 2 2 0 0 0 0 0 0 0 0 0 0.2 0.1
2006 15 0 1 0 2 3 4 1 1 2 0 0 0 0 0 0 0 0.2 0.2
2007 19 0 0 3 5 6 3 1 0 0 0 0 0 0 0 0 1 0.2 0.2
2008 18 0 1 2 4 3 3 2 3 0 0 0 0 0 0 0 0 0.2 0.2
Total
(%)
175
(100)
1 (0.6) 3 (1.7) 20
(11.4)
38 (21.7)37 (21.1)31 (17.7) 16 (9.1) 11 (6.3) 6 (3.4) 3 (1.7) 3 (1.7) 0 (0) 1 (0.6) 0 (0) 0 (0) 5 (2.8) 0.2 0.2
Abbreviations: yr: year; CIR: crude incidence rate; ASIR: age-standardized incidence rate.
Table 1. The distribution of malignant ovarian germ cell tumor (MOGCT) histopathological types in the Saudi Arabian population from 1999 to 2008.
Year 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 Total (%)
Histological type
Dysgerminoma 10 7 5 5 8 4 9 8 10 6 72 (41.1)
Embryonal carcinoma 0 0 0 2 0 0 0 0 0 0 2 (1.1)
Yolk sac tumor 2 2 3 6 4 4 2 1 2 6 32 (18.3)
Malignant teratoma 5 7 5 5 6 3 1 2 6 4 44 (25.1)
Mixed germ cell tumor 4 2 1 1 4 2 4 4 1 2 25 (14.3)
Total (%) 21 (12) 18 (10.3) 14 (8) 19 (10.9) 22 (12.6) 13 (7.4) 16 (9.1) 15 (8.6) 19 (10.9) 18 (10.3) 175 (100)
2 Hematol Oncol Stem Cell Ther xx(xx) xxx Quarter 2014 hemoncstem.edmgr.com
brief communication INCIDENCE OF MOGCTS IN SAUDI ARABIA
accounted for roughly 3.5% (n = 44/1271) of all ovar-ian tumors (3.5 folds higher) and 25.1% of MOGCTsin the Saudi Arabian population.
Furthermore, the estimated international incidenceof ovarian yolk sac tumor (20%) was comparativelyequivalent to our study findings of 18.3%.2 In addi-tion, mixed germ cell tumors account for roughly10% of all MOGCTs worldwide,2 and this percentagewas slightly higher in our study finding of 14.3%.
Polyembryoma and primary ovarian (nongesta-tional) choriocarcinoma are extremely rare neoplasmsaccounting for less than 1% of all MOGCTs interna-tionally.2 In our study, no single case of ovarian poly-embryoma or nongestational choriocarcinoma wasidentified. They almost always exist in admixed formsand hardly ever exist in solitary pure forms.7
Looking into Western literature, the reportedmedian age at time of clinical diagnosis of MOGCTsis 18 years.8–10 This figure greatly paralleled our find-ing in which the vast majority of MOGCT cases(60.6%) in the Saudi Arabian population occurredin patients between 10 and 25 years of age. Thiscan be greatly attributed to the younger average agegroup of the Saudi Arabian population. AsMOGCTs happen predominately in early children,young adolescents, early-to-late adulthood, andchild-bearing age group women, the issues of fertilitypreservation, tumor curability and long-term survivalrates are of great significance to patients, and mark-edly present difficult management challenges to theattending gynecologic oncologists.4
CONCLUSION
This is the first study published documenting theincidence and histopathological types of MOGCTs
in the Saudi Arabian population. This study confirmsMOGCTs are a common type of ovarian tumors inthe Saudi Arabian population and the incidence ratesand histopathological types are relatively comparableto the international populations with few differences.Future research directions include exploring furtherepidemiological facts, risk factors, clinical presenta-tions (signs and symptoms), tumor biological behav-iors, diagnostic blood markers and radiologicalinvestigations, surgical management and fertility pres-ervation, chemotherapy responses and overall progno-sis of MOGCTs in Saudi Arabia. Additional researchobjectives include MOGCT screening and earlydetection programs. These studies are anticipated tofurther our understanding of MOGCTs in SaudiArabia and accordingly optimize our practice towardsbest approach of MOGCTs.
ROLE OF FUNDING SOURCE
No funding source was received to conduct this re-search study.
CONFLICTS OF INTERESTS
Authors report no conflicts of interests to regardingthe publication of this research study.
AcknowledgmentsAuthors sincerely acknowledge Saudi Cancer Registry(SCA) and King Faisal Specialist Hospital & ResearchCenter (KFSH&RC), Riyadh, Saudi Arabia, for provid-ing the epidemiological data.
REFERENCES
1. Koshy M, Vijayananthan A, Vadiveloo V. Malig-nant ovarian mixed germ cell tumour: a rarecombination. Biomed Imaging Interv J 2005;1:e10.2. Gershenson DM. Update on malignant ovariangerm cell tumors. Cancer 1993;71(Suppl. 4):1581–90.3. Siegel R, Naishadham D, Jemal A. Cancerstatistics, 2013. CA Cancer J Clin 2013;63:11–30.4. Low JJ, Ilancheran A, Ng JS. Malignant ovariangerm-cell tumours. Best Pract Res Clin ObstetGynaecol 2012;26:347–55.
5. Chen VW, Ruiz B, Killeen JL, Cot� TR,Wu XC, Correa CN. Pathology and classifi-cation of ovarian tumors. Cancer 2003;97:2631–42.6. Deodhar KK, Suryawanshi P, Shah M, Rekhi B,Chinoy RF. Immature teratoma of the ovary: aclinicopathological study of 28 cases. Indian J PatholMicrobiol 2011;54:730–5.7. Tewari K, Cappuccini F, Disaia PJ, Berman ML,Manetta A, Kohler MF. Malignant germ celltumors of the ovary. Obstet Gynecol 2000;95:128–33.
8. Talerman A. Germ cell tumors of the ovary. In:Kurman RJ, editor. Blaustein's pathology of thefemale genital tract. New York: Springer-Verlag;1994. p. 849–914.9. Williams SD, Gershenson DM, Horowitz CJ,Scully RE. Ovarian germ-cell tumors. In: HoskinsWJ, Perez CA, Young RC, editors. Principles andpractice of gynecologic oncology. Pennsylva-nia: Lippincott-Raven; 1997. p. 987–1002.10. Gershenson DM. Management of early ovariancancer: germ cell and sex cord-stromal tumors.Gynecol Oncol 1994;55:S62–72.
Hematol Oncol Stem Cell Ther xx(xx) xxx Quarter 2014 hemoncstem.edmgr.com 3
INCIDENCE OF MOGCTS IN SAUDI ARABIA brief communication
© 2014 Asiri et al. This work is published by Dove Medical Press Limited, and licensed under Creative Commons Attribution – Non Commercial (unported, v3.0) License. The full terms of the License are available at http://creativecommons.org/licenses/by-nc/3.0/. Non-commercial uses of the work are permitted without any further
permission from Dove Medical Press Limited, provided the work is properly attributed. Permissions beyond the scope of the License are administered by Dove Medical Press Limited. Information on how to request permission may be found at: http://www.dovepress.com/permissions.php
Cancer Management and Research 2014:6 339–348
Cancer Management and Research Dovepress
submit your manuscript | www.dovepress.com
Dovepress 339
O R i g i n a l R e s e a R C h
open access to scientific and medical research
Open access Full Text article
http://dx.doi.org/10.2147/CMAR.S68262
Is extended-field concurrent chemoradiation an option for radiologic negative paraaortic lymph node, locally advanced cervical cancer?
Mushabbab al asiri1
Mutahir a Tunio1
Reham Mohamed2
Yasser Bayoumi2
abdulrehman alhadab1
Rasha M saleh3
Muhannad saud alarifi1
abdelaziz alobaid4
1Radiation Oncology, Comprehensive Cancer Center, King Fahad Medical City, Riyadh, saudi arabia; 2Radiation Oncology, national Cancer institute, Cairo University, Cairo, egypt; 3Medical Oncology, Comprehensive Cancer Center, King Fahad Medical City, Riyadh, saudi arabia; 4Women’s specialized hospital, King Fahad Medical City, Riyadh, saudi arabia
Correspondence: Mutahir Tunio Radiation Oncology, Comprehensive Cancer Center, King Fahad Medical City, Riyadh 59046, saudi arabia Tel +966 1 288 9999 Fax +966 1 461 4006 email [email protected]
Background: The aim was to evaluate whether extended-field concurrent chemoradiation
(EF-CCRT) leads to results better than those obtained by standard whole-pelvis concurrent
chemoradiation (WP-CCRT) in locally advanced cervical cancer with radiologic negative
paraaortic lymph nodes (PALNs).
Patients and methods: A total of 102 patients with histopathologically proven squamous
cell carcinoma, adenocarcinoma, or adenosquamous cell carcinoma, and radiologic negative
PALN locally advanced cervical cancer, stage IIB-IVA, were accrued between July 2007 and
April 2008 and were randomly assigned to WP-CCRT (50 patients) or EF-CCRT (52 patients),
followed by high-dose rate brachytherapy. Data regarding the safety profile, response rates, and
occurrence of local, PALN, or distant failure were recorded.
Results: During a median follow-up time of 60 months (18–66), 74/102 patients completed
the treatment protocol and were analyzed. Overall PALN, distant-metastasis control, disease-
free survival, and overall survival rates were 97.1%, 86.9%, 80.3%, and 72.4% in EF-CCRT
respectively in comparison with WP-CCRT (82.1%,74.7%, 69.1%, and 60.4%), with P-values of
0.02, 0.03, 0.03 and 0.04 respectively. No difference in acute toxicity profile was seen between
the groups, and late toxicities were mild and minimal.
Conclusion: Prophylactic EF-CCRT can be a reasonable option in patients with locally advanced
cervical cancer with radiologic positive pelvic lymph nodes and radiologic negative PALN.
Keywords: prophylactic extended field radiation therapy, concurrent chemotherapy
IntroductionThe incidence of invasive cervical cancer is decreasing in United States.1 However,
cervical cancer continues to be a major women’s health issue in many countries
because of inadequate cytological screening programs, and many patients present
as locally advanced International Federation of Gynecology and Obstetrics (FIGO)
stage IIB-IVA, for which surgery is inadequate. The standard treatment for locally
advanced cervical cancer is combined chemotherapy and pelvic irradiation, based on
results of four randomized trials showing survival benefit of 10%–15% and local and
distant recurrence reduction rates of 30%–40%.2–5 However, 15%–25% of patients
experience paraaortic lymph nodes (PALN) failures after receiving combined pelvic
chemoradiation, in long-term follow up.6 Data suggests that patients with locally
advanced cervical cancer harbor 17%–37% paraaortic nodal micrometastases, at the
time of diagnosis, just outside of standard radiation fields; therefore, the paraaortic
nodal area is a common extrapelvic site of failure, which certainly compromises the
distant control and survival.7
Cancer Management and Research 2014:6submit your manuscript | www.dovepress.com
Dovepress
Dovepress
340
asiri et al
Since the last three decades, clinical trials of radiotherapy
alone have failed to document the efficacy of prophylactic
paraaortic irradiation or extended-field irradiation for
biopsy-proven PALNs metastases, PALN failures with 5-year
survival rates as low as 29% to 50%.8 Further observations
of chemotherapy incorporated with extended-field irradia-
tion for biopsy-proven PALN metastases showed significant
gastrointestinal and hematological toxicities and no survival
benefit.9
We aimed to evaluate whether prophylactic extended-field
irradiation with concurrent chemotherapy leads to results
better than those obtained by standard pelvic irradiation with
concurrent chemotherapy in patients with locally advanced
cervical cancer with radiologic negative PALN status.
Materials and methodseligibilityAfter getting the approval from Institutional (King Fahad
Medical City, Riyadh Saudi Arabia) and National ethical
review committees (King Abdulaziz City for Science and
Technology) and obtaining informed consents from patients
for participation in the study in March 2007, consecutive
patients with locally advanced cervical cancer, between July
2007 and April 2008, were accrued when they met the fol-
lowing eligibility criteria: 1) histologically proven squamous
cell carcinoma, adenocarcinoma or adenosquamous carci-
noma of the cervix uteri; 2) clinical and radiologic FIGO
stage IIB-IVA, with no other evidence of distant metasta-
sis outside the pelvis; 3) Eastern Cooperative Oncology
Group (ECOG) performance status 0–2; 4) negative
PALNs on imaging (computed tomography [CT], mag-
netic resonance imaging [MRI]), or flourodeoxyglucose
positron emission tomography (FDG-PET); and 5) normal
hematologic, hepatic, and renal functions tests (white
blood cell count [WBC] $4,000/mm3, absolute neutrophil
count [ANC] $1,500/mm3, platelets $100,000/mm3, total
bilirubin #1.5 mg/dL, alanine transaminase #2 of normal
and creatinine #1.5 mg/dL).
Patients with a previous history of hysterectomy, retro-
peritoneal surgery, abdominal or pelvic radiotherapy, prior
chemotherapy, pregnancy, or positive paraaortic nodes on
imaging or biopsy proven were excluded.
The pretreatment workup consisted of a complete history
and physical examination; bimanual examination; CT and
MRI of chest, abdomen, and pelvis; complete hematology,
hepatic, renal, and electrolytes studies; bone scan; and cys-
toscopy and sigmoidoscopy when necessary.
Treatment protocolexternal beam radiation therapy (eBRT)All patients were scanned, for simulation, on a CT simula-
tor, from epigastrium to midthighs, and noncontrast and
contrast images were obtained. After the data acquisition,
all patients were randomized into two groups according to
radiation therapy techniques. In the first group, whole-pelvis
concurrent chemoradiation (WP-CCRT), the following were
delineated: gross tumor volume (GTV); uterus; the presacral,
common iliac, internal iliac, and external iliac lymph nodes;
initial clinical target volume (CTV-1), covering GTV and the
uterus, with 1 cm margins, and the presacral, common iliac,
internal iliac, and external iliac lymph nodes; initial planning
target volume (PTV)-1, with 0.5 to 1 cm margins around
CTV-1; boost clinical target volume (CTV-2), covering the
positive lymph nodes and parametria; PTV-2 (4 cm midline
block and 0.5 cm margins to CTV2); and organs at risk,
including kidneys, small bowel, bladder, rectum, and femoral
heads. For the majority of patients; equally spaced, coplanar
three-dimensional chemoradiation (3D-CRT) field plans
(box-field) were generated; however, intensity-modulated
radiation therapy (IMRT) was also generated for some cases,
to achieve better dose distribution. Radiation (3D-CRT) was
delivered with anteroposterior, posteroanterior, and opposed
lateral beams of 18-MV photons. The borders of (antero-
posterior/posteroanterior) fields were kept, cranially, at the
junction of L3/L4; caudally, 3 cm below the most inferior
vaginal involvement (as marked by golden seeds); and later-
ally, 2 cm lateral to the pelvic brim. The borders of lateral
fields were kept, anteriorly, 1 cm anterior to the symphysis
pubis, and posteriorly, were extended to the sacrum hollow.
The prescribed radiation doses were 45–50.4 Gy/25–28 frac-
tions to PTV-1, and 54– 59.4 Gy/30–33 fractions to PTV-2, 5
days per week, and up to 7% variation was considered accept-
able (Figures 1 and 2A). Radiologic positive lymph nodes
were boosted to a dose of 50.4–54 Gy. During planning, the
mean dose to the rectum was constrained to ,50 Gy, and
the total doses to the small bowel, kidneys, and bladder were
constrained to ,45 Gy, ,20 Gy, and ,60 Gy respectively.
In the second group, receiving prophylactic extended-field
concurrent chemoradiation (EF-CCRT), the pelvis was
treated similarly as for the WP-CCRT group, and additional
paraaortic fields were added as a continuous area or with a
half-beam block, with a superior field border at the junction
of T12/L1, to cover PALNs up to the level of the renal hila.
In no cases were anteroposterior or posteroanterior fields
allowed, to minimize the toxicity. The prescribed radiation
Cancer Management and Research 2014:6 submit your manuscript | www.dovepress.com
Dovepress
Dovepress
341
Extended-field or pelvic field chemoradiation in cervical cancer
Figure 1 initial-phase radiotherapy: (A,B) extended-field and (C,D) three-dimensional conformal radiation therapy (3D-CRT) plans showing 45 gy to planning target volume (PTV)-1, in coronal (A,C) and sagittal views (B,D).
dose to PALNs was 45 Gy (up to 7% variation was considered
acceptable). All patients in the EF-CCRT arm were given
routine antiemetics before planned radiotherapy sessions.
Concurrent chemotherapyBoth groups of patients received weekly cisplatin 40 mg/m2
before the administration of radiotherapy for six doses.
During CCRT, the doses were modified on a weekly basis.
If the WBC count was below 2,000/mm3 or the platelet count
was below 50,000/mm3, all chemotherapy for that week was
omitted. If the WBC count was below 1,000/mm3 or the plate-
let count was below 25,000/mm3, CCRT was withheld until
the WBC count and the platelet count recovered to 1,000/mm3
or greater and 25,000/mm3 or greater, respectively. If the
serum creatinine level was above 1.8 mg/dL, the cisplatin
was withheld. Patients were deemed unsuitable for further
chemotherapy if the delay to resume treatment was longer
than 2 weeks.
high-dose rate (hDR) brachytherapyFletcher–Suit tandem and ovoids afterloading applicators
were used for HDR-brachytherapy with iridium-192 sources,
once a week under conscious sedation, after 45 Gy of EBRT.
A dose of 7 Gy per fraction, using three insertions to point
A with a total dose of 21 Gy, was delivered, based on the
dose limit derived from the treatment plan for the rectum and
bladder. The dose constraints were 75 Gy and 80 Gy for the
rectum and bladder, respectively (Figure 2B).
Cancer Management and Research 2014:6submit your manuscript | www.dovepress.com
Dovepress
Dovepress
342
asiri et al
Toxicity scoringDuring CCRT, patients were evaluated every week for
weight, performance status, pelvic examination, hematologic
and blood chemistry determinations, and other related
characteristics. The National Cancer Institute Common
Toxicity Criteria (NCI-CTC) version 2.0 were used to score
acute radiation and chemotherapy toxicity (,90 days from
the start of radiation therapy).
After completion of the CCRT, patients were seen every
3 months for the first 2 years, and every 6 months thereaf-
ter, at radiation oncology and gynecology oncology clin-
ics. Response evaluation consisted of physical and pelvic
examination; Pap smear; hematology, hepatic, and renal
function tests; and CT chest, and abdomen and pelvic MRI
every 6 months for the first 2 years. The Radiation Therapy
Oncology Group (RTOG) Late Radiation Morbidity Scoring
Criteria were used to score radiation toxicity persisting
beyond 90 days from the completion of radiotherapy.
statistical analysisThe study was initially designed to be able to detect an
improvement of approximately 10% in the 5-year locore-
gional control (LRC), with a statistical power of 80% and a
two-sided significance level of 0.05. To detect such a differ-
ence, we predicted that we would need to enroll 163 women
in the study over a 2-year period and then follow them for an
additional 4 years. We estimated that 50% (86) women would
have died by the time of the analysis. The primary end point
was the LRC (pelvic and paraaortic control). Secondary end
points were distant metastatic control, disease-free survival
(DFS), and the overall survival (OS) for each group. DFS was
defined as the duration between the completion of CCRT and
the date of documented disease recurrence, death resulting
from the cancer, and/or last follow-up visit (censored). OS
was defined as the duration between the completion of CCRT
and the date of patient death or last follow-up visit (censored).
The probabilities of LRC, distant metastatic control, DFS,
Figure 2 (A) Parametrial boost (planning target volume [PTV]-2) of 5.4-9 gy with midline shielding, followed by (B) high-dose rate brachytherapy of 21 gy in three sessions, in the two treatment groups (rectum on the left and bladder on the right).
Cancer Management and Research 2014:6 submit your manuscript | www.dovepress.com
Dovepress
Dovepress
343
Extended-field or pelvic field chemoradiation in cervical cancer
and OS were determined with the Kaplan–Meier method.
The comparisons for various end points were performed
using the log-rank test. The Student’s unpaired t test was
used to determine the significance of the difference between
two groups. A P-value of 0.05 was considered statistically
significant. Statistical analyses were performed using the
computer program SPSS (Statistical Package for the Social
Sciences, Version 17.0; IBM Corp, Chicago, IL, USA).
ResultsCharacteristics of the patientsAfter accrual, 83 patients with locally advanced cervical can-
cer were randomly assigned to WP-CCRT (50 patients) and
EF-CCRT (52 patients), as shown in Figure 3. The characteris-
tics of remaining 74 patients are given in Table 1. The majority
of the cohort was presented with FIGO stage IIB (66.2%) and
with radiologic positive pelvic lymph nodes (51.4%). FIGO
stage IIIB with hydronephrosis/ nonfunctioning kidneys was
seen in three cases (4.05%), and for these patients, percu-
taneous nephrostomy or JJ stenting was performed prior to
starting CCRT. The median follow-up time was 60 months
(range, 18–66). The histopathologic characteristics of patients
with positive pelvic lymph nodes are shown in Table 2.
Among all 74 patients who received CCRT, the treat-
ment protocol completion rate was 90% (95% confidence
interval [CI]: 85–100) in the WP-CCRT group and 88.4%
(95% CI: 90–100) in the EF-CCRT group (P=0.8). Weekly
concurrent cycles of cisplatin, in both treatment arms, were
completed in all 74 (100%) patients, with no interruption. The
median duration of radiation therapy (including EBRT and
Estimated sample size =163 patients
Accrued =102 patients
WP-CCRT =50 patients
WP-CCRT =36 patients
EF-CCRT =52 patients
EF-CCRT =38 patients
Withdrawal
Randomization
Final analysis
• Histopathologically confirmed squamous cell /adenocarcinoma/ adenosquamous carcinoma
• FIGO stage IIB–IVA
Incomplete treatment protocol =eleven patients
Incomplete staging =seven patientsIrregular follow up =six patients
Social issues=four patients
• Radiological negative paraaortic lymph nodes
Figure 3 Treatment algorithm.Abbreviations: EF-CCRT, extended-field concurrent chemoradiation; FIGO, international Federation of gynecology and Obstetrics; WP-CCRT, whole-pelvis concurrent chemoradiation.
HDR-brachytherapy) in both arms was 55.5 days (7.8 weeks)
with 95% CI: 48–58.
Toxicity profileThe overall incidence of the grade 3 or 4 acute hematological
and nonhematological toxicities was 5.2% and 2.6%, respec-
tively, in the EF-CCRT arm and 5.4% and 2.7%, respectively,
in the WP-CCRT arm (P=0.7). Both treatment arms had simi-
lar grade 3 or 4 acute gastrointestinal toxicity (Table 3).
During the follow-up time of 60 months, in the EF-CCRT
group, one patient (2.6%) experienced subacute intestinal
obstruction. No patient in either arm underwent surgery
for radiation-induced damage or died because of treatment-
related side effects.
Pelvic, paraaortic, distant control, and survival ratesAt the time of the last follow-up visit, three (7.9%) patients
had pelvic recurrences (one vaginal and two pelvic nodal)
in the EF-CCRT arm, and three (8.3%) patients had local
recurrences (pelvic nodal) in the WP-CCRT arm (P=0.8).
Isolated vaginal recurrence in the EF-CCRT group was
successfully salvaged with surgery, and pelvic nodal recur-
rences were treated with salvage chemotherapy. First pelvic
recurrence was manifested within 24 months of completion
of CCRT in both arms.
Paraaortic nodal failures were seen in five (13.9%)
patients in the WP-CCRT arm and one (2.6%) patient in EF-
CCRT arm (P=0.02) (Figure 3A). In the WP-CCRT arm, three
(60%) of five paraaortic nodal failures were seen at the level
of junction L2 and L3 vertebrae and two (40%) at the level
of L1 vertebrae; and in the EF-CCRT arm, one paraaortic
nodal failure was seen at the junction of T12 and L1 vertebrae.
In both arms, paraaortic nodal failures were seen within 20
months of completion of CCRT. Distant failures were seen in
seven (19.4%) patients in the WP-CCRT and in four (10.5%)
patients from the EF-CCRT arm (P=0.02) (Figure 4B). The
common sites of distant failure were: lungs (four patients),
mediastinal (three patients), and supraclavicular nodes (one
patient), and thoracic spine (one patient). Combined distant
and paraaortic nodal failures were observed in four (11.4%)
in the WP-CCRT and one (2.6%) in the EF-CCRT group. All
distant and PALN were treated with salvage chemotherapy
± radiotherapy for bony lesions. Important prognostic fac-
tors for PALN were positive pelvic nodes on imaging (CT/
MRI), the FIGO stage .IIB, and prolonged treatment time
.55 days (P=0.001, 0.001 and 0.03, respectively) (Table 4).
At the time of analysis, 32 patients in the EF-CCRT and 27
Cancer Management and Research 2014:6submit your manuscript | www.dovepress.com
Dovepress
Dovepress
344
asiri et al
Table 1 Patients characteristics
Variables Arm A Extended-field CCRT (n=38)
Arm B Pelvic-field CCRT (n=36)
P-value
Mean age 52.3 years (32–78) 51.6 years (34–76) 0.9eCOg performance scale 0–2 0–2 1.0histopathology squamous cell carcinoma adenocarcinoma adenosquamous cell carcinoma
34 (89.5%)3 (7.9%)1 (2.6%)
33 (91.7%)2 (5.6%)1 (2.8%)
0.7
FigO staging iiB iiia iiiB iVa
24 (63.1%)6 (15.8%)4 (10.5%)4 (10.5%)
25 (69.4%)6 (16.6%)3 (8.3%)2 (5.6%)
0.8
Radiological primary tumor size ,5 cm .5 cm
13 (34.2%)25 (65.8%)
14 (38.9%)22 (61.1%)
0.6
MRi-based nodal involvement negative iliac Common iliac Paraaortic
15 (39.5%)9 (23.7%)14 (36.8%)–
21 (58.4%)13 (36.1%)2 (5.6%)–
0.05
Pretreatment hemoglobin .10 gm/dl ,10 gm/dl
35 (92.1%)3 (7.9%)
34 (94.4%)2 (5.6%)
0.9
Treatment 3D-CRT iMRTeBRT Whole pelvis Paraaortic Parametrial/positive ln boosthDR-BT Dose/fraction Total dose/fraction Point a BeD iCRU 38 rectal point BeD iCRU 38 bladder point BeDConcurrent weekly cisplatin cycle Dose/week Mean cycles
30 (78.9%)8 (21.1%)
45 gy (42–50.4)45 gy (45–50.4)9 gy (5–9)
7 gy/fraction21 gy/386.4 gy (80.5–102.7)85 gy (80.5–100)86 gy (80.5–102)
30 mg/m2
5 (4–7)
36 (100.0%)–
45 gy (42–50.4)–9 gy (5–9)
7 gy/fraction21 gy/386.4 gy (80.5–102.7)85 gy (80.5–100)86 gy (80.5–102)
30 mg/m2
5 (4–7)
0.9
Abbreviations: 3D-CRT, three-dimensional conformal radiation therapy; BeD, biologic effective dose; CCRT, concurrent chemoradiation; eBRT, external beam radiation therapy; eCOg, european Cooperative Oncology group; FigO, international Federation of gynecologists and Obstetricians; hDR-BT, high-dose rate brachytherapy; iCRU, international commission of radiation units; iMRT, intensity modulated radiation therapy; ln, lymph nodes; MRi, magnetic resonance imaging.
patients in the WP-CCRT group were found without evidence
of disease (Figure 4C and D).
DiscussionWP-CCRT is deemed the standard treatment, by many North
American and European teams, for treatment of locally
advanced cervical cancer, which has resulted in survival gain
but also, 10%–25% paraaortic nodal failures.10 The 10-year
results of a RTOG (RTOG-97-20) trial, which compared
pelvic irradiation with paraaortic plus pelvic radiotherapy
alone, have shown an improvement in survival gain of 11%
in the prophylactic paraaortic plus pelvic irradiation group
but no difference in LRC.11,12 A subsequent other RTOG
trial (RTOG 90-01) compared prophylactic extended-field
radiotherapy versus pelvic irradiation with chemotherapy in
locally advanced cervical cancer patients; the study showed
that 5-year OS rates for patients treated with pelvic irradia-
tion and concurrent chemotherapy was significantly greater
than for patients treated with extended-field radiation alone
(67% vs 41% at 8 years), and pelvic irradiation with concur-
rent chemotherapy resulted in a 51% reduction in the risk
of recurrence and a 52% reduction in the risk of death but
Cancer Management and Research 2014:6 submit your manuscript | www.dovepress.com
Dovepress
Dovepress
345
Extended-field or pelvic field chemoradiation in cervical cancer
Table 2 Clinicopathological findings variation in cervical patients with positive lymph nodes
Variables Negative lymph nodes (n=36)
Metastatic lymph nodes (n=38)
P-value
age (range) 51.6 years (44–76)
47.3 years (32–57)
0.04
eCOg performance scale 0–2 0–2 1.0histopathology squamous cell carcinoma Keratinizing nonkeratinizing adenocarcinoma adenosquamous
carcinoma
33 (91.7%)8 (24.3%)25 (75.7%)4 (11.1%)0
34 (89.5%)10 (29.4%)24 (70.6%)1 (2.6%)2 (5.3%)
0.6 0.0030.04
FigO staging iiB iiia iiiB iVa
34 (94.4%)1 (2.8%)1 (2.8%)0 (0%)
15 (39.4%)11 (28.9%)6 (15.8%)6 (15.8%)
0.003
Radiological primary tumor size ,5 cm .5 cm
17 (47.2%)19 (52.8%)
10 (26.3%)28 (73.7%)
0.04
Uterine corpus invasion no Yes
29 (80.6%)7 (19.4%)
13 (34.2%)25 (65.8%)
0.003
Vaginal invasion no Yes
27 (75.0%)9 (25.0%)
12 (31.6%)26 (68.4%)
0.004
Abbreviations: eCOg, european Cooperative Oncology group; FigO, international Federation of gynecologists and Obstetricians.
no difference in the incidence of paraaortic failures (7% in
pelvic irradiation with concurrent chemotherapy vs 4% in
extended-field irradiation [P-value 0.15]).6
The role of incorporating concurrent chemotherapy
with prophylactic extended-field irradiation to reduce the
paraaortic nodal failures and promote a survival gain is not
well known; due to lack of Phase III trials to pursue this ques-
tion, Malfetano et al used weekly cisplatin concomitant with
prophylactic extended-field radiation, followed by low-dose
rate brachytherapy, in a Phase II study in which 67 women
were enrolled, of whom 54 had radiologic negative paraaortic
nodes. Treatment was well tolerated, with high efficacy, and
at a median follow up of 47.5 months, 75% of patients were
alive without evidence of disease and with no paraaortic
failures.13 However, this trial was criticized for nonrandom-
ization, selection bias, and use of older conventional exter-
nal beam and low-dose rate radiation techniques. Another
Phase II study by Chung et al treated 63 locally advanced
cervical cancers with concurrent cisplatin during the first
and fifth weeks of extended-field irradiation, followed by
two adjuvant cycles of cisplatin- and 5-flouro-uracil-based
chemotherapy; results showed that toxicity was minimal,
with paraaortic nodal failures in only 4.5% and an OS rate of
81% at 3 years.14 Advanced FIGO stage, parametrial invasion,
elevated CEA, and SCC-Ag are all associated with increased
risk of PALN failure.
To the best of our knowledge, our single institutional
study is the first randomized, prospective study to compare
prophylactic EF-CCRT plus HDR brachytherapy with stan-
dard WP-CCRT plus HDR brachytherapy, in patients with
locally advanced cervical cancer with radiologic negative
PALNs. We used radiologic (CT/MRI) staging along with
clinical FIGO staging, as tumor size and lymph node involve-
ment are also important prognostic factors that influence the
LRC and survival.17,18 For such patients, CT/PET imaging
would be superior to other imaging modalities to assess the
lymph node status and distant metastasis;19 however, our study
can be criticized for not performing baseline CT/PET in all
patients. Possible explanation was the nonavailability of PET
imaging in our institute at the time of accrual of patients (and
later on, only 16% of patients had routine follow-up CT/PET
imaging). The course of treatment was well tolerated in our
patients, and hematological and nonhematological toxicities
were minimal or even better than those reported in other
trials of EF-CCRT.4,5 Further, EF-CCRT resulted in excel-
lent 5-year paraaortic distant control rates and better DFS
and OS rates, in comparison with WP-CCRT, in the present
study, especially in patients with radiologic gross pelvic
lymphadenopathy; however, we did not see any difference
in pelvic control between the two groups.
Possible explanation for the lower toxicity profile and
better outcomes (LRC and distant control) in the EF-CCRT
group may be that: a) the majority of our patients (90%)
had gross pelvic nodes .1 cm on imaging, which enhances
the risk of paraaortic nodal micrometastasis, and these were
eradicated by prophylactic EF-CCRT; b) surgical staging
causes more intestinal fibrosis which results in more gastro-
intestinal toxicity; c) moderate doses of weekly cisplatin were
used than elsewhere; d) use of better radiation techniques
in our study (CT simulation, 3D-CRT/IMRT and HDR
brachytherapy); and e) treatment duration was 55 days for
the majority of patients.
Although our study showed encouraging treatment
outcomes and low morbidity, limitations of the present
study were: a) the low sample size; b) poor randomization
to study treatment and selection bias as many patients with
common iliac lymphadenopathy were included in the EF-
CCRT arm; c) lack of baseline FDG-PET-based staging;
and e) the lack of baseline carcinoembryonic antigen and
SCC-Ag levels.
Cancer Management and Research 2014:6submit your manuscript | www.dovepress.com
Dovepress
Dovepress
346
asiri et al
Table 3 Acute and late treatment related toxicity profile
Toxicity Arm A Extended-field CCRT (n=38)
Arm B WP CCRT (n=36)
P-value
G3 G4 G3 G4
Acutehematologic neutropenia Thrombocytopenia anemianonhematologic nausea/vomiting Diarrhea Cystitis Deranged renal functions Deranged liver functions
1 (2.6%)00
01 (2.6%)000
1 (2.6%)00
00000
1 (2.7%)00
001 (2.7%)00
1 (2.7%)00
00000
0.7
LateChronic cystitisintestinal obstructionProctitisneuropathy/plexopathyhearing lossRenal
01 (2.6%)0000
1 (2.8%)0
0.8
Abbreviations: CCRT, concurrent chemoradiation; WP-CCRT, whole-pelvis concurrent chemoradiation; g, grade.
Log-rank P-value 0.02
EF-CCRT 80.3%
Follow-up, in months Follow-up, in months
Follow-up, in monthsFollow-up, in months
Pelvic-CCRT 69.1%
EF-CCRT 97.1%
1.0
0.8
0.6
0.4
0.2
0.0
0 10 20 30 40 50 60 70
Par
aao
rtic
co
ntr
ol (
%)
Dis
tan
t co
ntr
ol (
%)
Ove
rall
surv
ival
(%
)
Dis
ease
-fre
e su
rviv
al (
%)
A B
DC
Pelvic-CCRT 82.1%
EF-CCRT 86.9%
Pelvic-CCRT 74.7%
EF-CCRT 72.4%
Pelvic-CCRT 60.4
Log-rank P-value 0.03
Log-rank P-value 0.04Log-rank P-value 0.03
0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70
0 10 20 30 40 50 60 700 10 20 30 40 50 60 70
1.0
0.8
0.6
0.4
0.2
0.0
1.0
0.8
0.6
0.4
0.2
0.0
1.0
0.8
0.6
0.4
0.2
0.0
Figure 4 Five year (A) paraaortic control, (B) distant control, (C) disease-free survival, and (D) overall survival, after extended-field chemoradiation and WP-CCRT.Abbreviations: CCRT, concurrent chemoradiation; EF-CCRT, extended-field concurrent chemoradiation; WP-CCRT, whole-pelvis concurrent chemoradiation.
Cancer Management and Research 2014:6 submit your manuscript | www.dovepress.com
Dovepress
Dovepress
347
Extended-field or pelvic field chemoradiation in cervical cancer
Table 4 Multivariate analysis of variables on paraaortic, distant control, and disease-free survival rates
Variable Paraaortic control OR (95% CI) P-value
Distant metastasis control OR (95% CI) P-value
Disease-free survival OR (95% CI) P-value
age (,50 vs .50 years) 1.80 (0.79–2.10) 0.8 1.10 (0.89–2.00) 0.6 0.71 (0.10–2.41) 0.5Comorbidities (yes vs no) 0.88 (0.67–0.97) 0.9 1.80 (0.79–2.10) 0.7 1.80 (0.79–2.10) 0.7FigO stage (,iiB vs .iiB) 6.11 (2.22–16.30) 0.001 3.65 (1.81–9.65) 0.02 3.85 (1.91–10.35) 0.01n stage (n0 vs n1) 4.34 (3.52–11.34) 0.001 4.01 (2.21–11.59) 0.01 2.76 (1.75–9.36) 0.03hemoglobin level (,10 vs .10) 2.24 (1.65–4.40) 0.03 1.10 (0.89–2.00) 0.5 0.78 (0.23–2.38) 0.4Cell type ( sCC vs non-sCC) 1.21 (1.10–2.10) 0.4 1.10 (0.89–2.00) 0.6 1.21 (1.10–2.10) 0.7eF-CCRT vs WP-CCRT 3.45 (1.61–9.45) 0.02 2.97 (1.95–10.5) 0.04 3.65 (1.81–9.65) 0.02Treatment duration (,55 vs .55 days) 2.21 (1.45–7.85) 0.03 1.1 (0.67–1.97) 0.9 1.10 (0.89–2.00) 0.6
Abbreviations: CI, confidence interval; EF-CCRT, extended-field concurrent chemoradiation; FigO, international Federation of gynecologists and Obstetricians; OR, odds ratio; sCC, squamous cell carcinoma; WP-CCRT, whole-pelvis concurrent chemoradiation.
However, the results of our study recommend use
prophylactic EF-CCRT in patients with locally advanced cervi-
cal cancer with radiologic positive pelvic lymph nodes. Further,
our results warrant a multicenter, Phase III trial using IMRT
with prophylactic EF-CCRT in FDG-PET negative PALN
locally advanced cervical patients, to evaluate whether the out-
comes of EF-CCRT are truly better than those of WP-CCRT.
AcknowledgmentWe thank Laura Stanciu Gabriella, medical physicist, who did
manuscript language editing on behalf of the authors.
DisclosureThe authors report no conflicts of interest in this work.
References1. Siegel R, Naishadham D, Jemal A. Cancer statistics, 2012. CA Cancer
J Clin. 2012;62(1):10–29.2. Keys HM, Bundy BN, Stehman FB, et al. Cisplatin, radiation, and
adjuvant hysterectomy compared with radiation and adjuvant hys-terectomy for bulky stage IB cervical carcinoma. N Engl J Med. 1999;340(15):1154–1161.
3. Rose PG, Bundy BN, Watkins EB, et al. Concurrent cisplatin based radiotherapy and chemotherapy for locally advanced cervical cancer. N Eng J Med. 1999;340(15):1144–1153.
4. Whitney CW, Sause W, Bundy BN, et al. Randomized comparison of fluorouracil plus cisplatin versus hydroxyurea as an adjunct to radiation therapy in stage IIB-IVA carcinoma of the cervix with negative para-aortic lymph nodes: a Gynecologic Oncology Group and Southwest Oncology Group study. J Clin Oncol. 1999;17(5):1339–1348.
5. Peters WA III, Liu PY, Barrett RJ, et al. Cisplatin and 5-Flourouracil plus radiation therapy are superior to radiation therapy as adjunctive in high risk early stage carcinoma of the cervix after radical hysterectomy and pelvic lymphadenectomy: report of a phase III intergroup study. J Clin Oncol. 2000;18:1606–1613.
6. Eifel PJ, Winter K, Morris M, et al. Pelvic irradiation with concurrent chemotherapy versus pelvic and para-aortic irradiation for high-risk cervical cancer: an update of radiation therapy oncology group trial (RTOG) 90-01. J Clin Oncol. 2004;22(5):872–880.
7. Berman ML, Keys H, Creasman W, DiSaia P, Bundy B, Blessing J. Sur-vival and patterns of recurrence in cervical cancer metastatic to periaortic lymph nodes (a Gynecologic Oncology Group study). Gynecol Oncol. 1984;19(1):8–16.
8. Morris M, Eifel PJ, Lu J, et al. Pelvic radiation with concurrent chemo-therapy compared with pelvic and para-aortic radiation for high-risk cervical cancer. N Engl J Med. 1999;340(15):1137–1143.
9. Small W, Winter K, Levenback C, et al. Extended-field irradiation and intracavitary brachytherapy combined with cisplatin chemotherapy for cervical cancer with positive para-aortic or high common iliac lymph nodes: results of ARM 1 of RTOG 0116. Int J Radiat Oncol Biol Phys. 2007;68(4):1081–1087.
10. Gouy S, Morice P, Narducci F, et al. Nodal-staging surgery for locally advanced cervical cancer in the era of PET. Lancet Oncol. 2012;13(5):e212–e220.
11. Rotman M, Choi K, Guse C, et al. Prophylactic irradiation of the para-aortic lymph node chain in stage IIB and bulky stage IB carcinoma of the cervix, initial treatment results of RTOG 7920. Int J Radiat Oncol Biol Phys. 1990;19(3):513–521.
12. Rotman M, Pajak TF, Choi K, et al. Prophylactic extended-field irra-diation of para-aortic lymph nodes in stages IIB and bulky IB and IIA cervical carcinomas. Ten-year treatment results of RTOG 79-20. JAMA. 1995;274(5):387–393.
13. Malfetano JH, Keys H, Cunningham MJ, Gibbons S, Ambros R. Extended field radiation and cisplatin for stage IIB and IIIB cervical carcinoma. Gynecol Oncol. 1997;67(2):203–207.
14. Chung YL, Jian JJ, Cheng SH, et al. Extended-field radiotherapy and high-dose-rate brachytherapy with concurrent and adjuvant cisplatin-based chemotherapy for locally advanced cervical cancer: a phase I/II study. Gynecol Oncol. 2005;97(1):126–135.
15. Huang EY, Huang YJ, Chanchien CC, et al. Pretreatment carcino-embryonic antigen level is a risk factor for para-aortic lymph node recurrence in addition to squamous cell carcinoma antigen following definitive concurrent chemoradiotherapy for squamous cell carcinoma of the uterine cervix. Radiat Oncol. 2012;7:13.
16. Jeong BK, Choi DH, Huh SJ, Park W, Bae DS, Kim BG. The role of squamous cell carcinoma antigen as a prognostic and predictive factor in carcinoma of uterine cervix. Radiat Oncol J. 2011;29(3):191–198.
17. Kodaira T, Fuwa N, Toita T, et al. Comparison of prognostic value of MRI and FIGO stage among patients with cervical carcinoma treated with radiotherapy. Int J Radiat Oncol Biol Phys. 2003;56(3):769–777.
18. Scheidler J, Hricak H, Yu KK, Subak L, Segal MR. Radiological evaluation of lymph node metastases in patients with cervical cancer. A meta-analysis. JAMA. 1997;278(13):1096–1101.
19. Herrera FG, Prior JO. The role of PET/CT in cervical cancer. Front Oncol. 2013;3:34.
Cancer Management and Research
Publish your work in this journal
Submit your manuscript here: http://www.dovepress.com/cancer-management-and-research-journal
Cancer Management and Research is an international, peer-reviewed open access journal focusing on cancer research and the optimal use of preventative and integrated treatment interventions to achieve improved outcomes, enhanced survival and quality of life for the cancer patient. The journal welcomes original research, clinical & epidemiological
studies, reviews & evaluations, guidelines, expert opinion & commen-tary, case reports & extended reports. The manuscript management system is completely online and includes a very quick and fair peer-review system, which is all easy to use. Visit http://www.dovepress.com/ testimonials.php to read real quotes from published authors.
Cancer Management and Research 2014:6submit your manuscript | www.dovepress.com
Dovepress
Dovepress
DovepressDovepress
348
asiri et al
3232
Five years treatment outcome of Postoperative radiotherapy in uterine cancers, M.A. Tunio, et. al.
Five Years Treatment Outcomes of Postoperative Radiotherapy in Saudi Women with Uterine Cancers:
Single Institutional ExperienceM. Al Asiri1, M.A. Tunio1, Y. Bayoumi2, R. Mohamed2, E.Al Saeed3, A. Al Hadab1, A. Al Amro1
1Radiation oncology, Comprehensive Cancer Center, King fahad Medical City, Riyadh 59046, Saudi Arabia. 2Radiation oncology, national Cancer Institute, Cairo University, Cairo-
Egypt. 3Radiation oncology, King Khalid University Hospital, Riyadh, Saudi Arabia
Corresponding Author: Mutahir Tunio, MBBS, FCPS (Radiation Oncology), Assistant Consultant, Radiation Oncology, Comprehensive Cancer Center, King Fahad Medical City, Riyadh 59046, Saudi Arabia, Tel: +966 1 2889999 Fax: 966 1 4614006. Email: [email protected]
Abstract
Purpose:We aimed to evaluate long-term treatment
outcomes and toxicity profile of postoperative radiotherapy (PORT) in Saudi women with uterine cancers.
Methods and Materials:Medical records of patients with
histopathologically proven uterine cancers were reviewed and identified those who received PORT (45-50.4 Gy in 25-28 fractions) followed by vaginal brachytherapy (15-20 Gy in 3 to 4 sessions) after total abdominal hystrectomy and bilateral salpingo-oophorectomy (TAH-BSO) in our center between August 2007 and April 2012. Data regarding the safety profile, locoregional control (LRC) or distant metastases control (DMC) and overall survival (OS) rates were analyzed.
Results: Median follow-up period was 60 months
(range, 12-70) for 89 patients. Predominant histological type was endometrial (59 patients), followed by carcinosarcoma (17 patients) and leiomyosarcoma (13 patients). Median age
at time of diagnosis was 57.6, 56 and 51.1 years for endometrial, carcinosarcoma and leiomyosarcoma respectively. LRC rates were 80.9%, 87.1% and 100% for leiomyosarcoma, carcinosarcoma and endometrial carcinoma respectively (p 0.4). DMC rates were 69.3%, 45% and 16.3% for endometrial, leiomyosarcoma and carcinosarcoma respectively (p 0.0001). Five-year OS rates were 71.1%, 60% and 16.3% for endometrial, leiomyosarcoma and carcinosarcoma respectively (p 0.001). Cox-proportional hazard ratio model showed body mass index, FIGO stage, lymphovascular invasion in endometrial carcinoma, tumor size in leiomyosarcoma and histology in carcinosarcoma important prognostic factors for LRC. Acute grade 3 and 4 proctitis/enteritis seen only in 4 patients (4.5%) and late toxicities were minimal.
Conclusion: PORT in Saudi women with uterine cancers
showed better LRC, DMC and OS rates with minimal toxicity.
Keywords: Postoperative radiotherapy, uterine
carcinomas, Saudi women
IntroductionUterine cancer is the tenth most common
cancer in Saudi Arabia; it is also the second most common gynecologic malignancies in women
and endometrial carcinoma is the most common histologic type (1). For endometrial carcinoma, surgery is the primary treatment involving a total abdominal hysterectomy, bilateral salpingo-oophorectomy (TAH+BSO), pelvic and para-aortic lymphadenectomy, and pelvic washings, with five year survival rates of 78% (2, 3). Randomized trials by Post-operative Radiation therapy in endometrial carcinoma (PORTEC)
33
G. J. O. Issue 16, 2014Five years treatment outcome of Postoperative radiotherapy in uterine cancers, M.A. Tunio, et. al.
and Gynecological Oncology Group 99 (GOG-99) have shown significant reduction of the risk of pelvic and vaginal recurrence by adjuvant radiotherapy, although a survival benefit is not yet proven (4, 5). Thus radiotherapy remains mainstay of the adjuvant treatment.
Uterine sarcomas are rare and account for 3-7% of all uterine malignancies and are associated with dismal prognosis. Histologically, it is divided into three parts: (a) leiomyosarcoma, (b) carcinosarcoma or mixed Mullerian tumors and (c) endometrial stromal sarcoma (6). Uterine sarcomas are treated in similar way as to endometrial carcinoma with TAH+ BSO excluding lymph node dissection followed by adjuvant radiotherapy and systemic chemotherapy due to increased tendency to metastasize (7).
We aimed to evaluate the efficacy, toxicity and treatment outcomes of postoperative pelvic radiotherapy by three dimensional conformal (3DCRT) followed by high dose rate vaginal brachytherapy in Saudi women with all types of uterine cancers and also to evaluate the prognostic factors affecting locoregional and distant control.
Materials and Methods
EligibilityAfter getting approval from institutional
Review Committee, medical records of patients with uterine cancers were looked for demographic, symptomatology, tumor characteristics, radiologic imaging, pathology, radiation therapy techniques, additional systemic chemotherapy regimen, acute and chronic side effects and response rates. Patients with uterine cancers between August 2007 and April 2012 were studied when they met the following eligibility criteria:
(1) Histologically proven endometrial carcinoma, leiomyosarcoma and carcinosarcoma of corpus uteri;
(2) Clinico-pathologic FIGO stage IB-IVA with no other evidence for distant metastasis outside the pelvis;
(3) Eastern Co-operative Oncology Group (ECOG) performance status 0-2; and
(4) Patients received postoperative pelvic radiation therapy followed by high dose rate vaginal brachytherapy
Patients with a previous history of abdominal or pelvic radiotherapy and prior chemotherapy were excluded.
Treatment techniques
SurgeryAll endometrial cancer and carcinosarcoma
patients underwent TAH and BSO with pelvic and para-aortic lymphadenectomy and leiomyosarcoma patients underwent TAH and BSO without lymph node dissection. After 6-8 weeks of surgery all patients were referred to radiotherapy department for postoperative radiation therapy (PORT).
Postoperative radiation therapy techniquesAll patients were simulated using Siemens
Emotions 6 computed tomography (CT) simulator. Contrast enhanced axial images of 5 mm slice thickness were obtained from the top of fourth lumbar (L4) vertebra to 5 cm below ischial tuberosities. After the acquisition of CT data, delineation of contouring of CTV (vaginal cuff, parametrial soft tissue, external, internal iliac, presacral and common iliac lymph nodes), planning target volume (PTV) = CTV + 1 cm margin and critical structures (urinary bladder, rectum, small bowel) was performed using Varian Eclipse Contouring software by two radiation oncologists according to Radiation Therapy Oncology Group (RTOG) guidelines (8). After contouring treatment planning for conformal therapy (3DCRT) was carried out by two medical physicists. Treatment plans were made using box field technique for 3DCRT. The PTV was prescribed to 45-50.4 Gy in 25-28 fractions, 1.8 Gy per fraction, one fraction per day. Efforts were made to receive 45-50.4 Gy to 95% of PTV and to reduce hot spots less than 120%. During planning, total doses to the small bowel, rectum and bladder were constrained to < 40 Gy, < 45Gy and 45 Gy respectively. After completion of pelvic external beam irradiation, high dose rate (HDR) vaginal brachytherapy (VBT) was given. Total VBT dose was 15-20 Gy
34
Five years treatment outcome of Postoperative radiotherapy in uterine cancers, M.A. Tunio, et. al.Five years treatment outcome of Postoperative radiotherapy in uterine cancers, M.A. Tunio, et. al.
delivered in 3-4 sessions (each session three days apart). The reference point for dose prescription was 0.5 cm from surface of vaginal applicators.
adjuvant chemotherapyAdditional adjuvant chemotherapy was given
to high risk FIGO II (G3, papillary serous and clear cell types), FIGO III, IVA endometrial cancers and all stages carcinosarcoma patients initially 6 weeks after surgery, before starting radiation therapy. Adjuvant chemotherapy consisted of four cycles of Paclitaxal (175mg/m2) and Carboplatin (350 mg/m2) every 21 days. Dose modifications were done according to side effects and tolerance of patients.
toxicity and response evaluation During radiation therapy, patients were
evaluated every week for weight, performance status, hematology/chemistry and side effects. The National Cancer Institute Common Toxicity Criteria (NCI-CTC) version 2.0, were used to score acute radiation toxicity (≤ 90 days from start of radiation therapy). The Radiation Therapy Oncology Group (RTOG) Late Radiation Morbidity Scoring Criteria were used to score radiation toxicity persisting beyond 90 days from the completion of radiotherapy. After completion of therapy, periodic follow ups were carried out every 3 months for first two years and every 6 months subsequent 3rd to 5th year with physical and per vaginal examination, Pap smear and CT chest, abdomen and pelvis.
Statistical analysis The primary endpoints were toxicity,
locoregional control (LRC) and distant metastases control (DMC), disease free survival (DFS) and the overall survival (OS) rates. DFS was defined as the duration between the completion of PORT and the date of documented disease recurrence, death resulting from the cancer, and/or last follow-up visit (censored). OS was defined as the duration between the completion of PORT and the date of patient death or last follow-up visit (censored). The probabilities of locoregional and distant control, DFS and OS were determined with the Kaplan-Meier method. The comparisons for various endpoints were performed using
the log-rank test. The Student’s unpaired t-test was used to determine the significance of the difference between two groups. A p value of 0.05 was considered statistically significant. Cox-proportional hazard model was used to calculate the key prognostic factors affecting LRC, DMC, DFS and OS. Statistical analyses were performed using the computer program SPSS (Statistical Package for the Social Sciences, version 17.0, SPSS Inc, Chicago, IL).
ResultsA total of 89 patients with uterine cancers
were given PORT and were followed for a period of 60 months (range: 12-70). Characteristics of patients are shown in Table 1. Endometrial carcinoma was predominant histology (66.3%) followed by carcinosarcoma (19.0%) and leiomyosarcoma (14.7%). Median time between surgery and radiotherapy was 7.1 weeks (range: 5-24). For patients who were given adjuvant chemotherapy, the mean time between surgery and radiotherapy was 13 weeks (11-18). The median dose to PTV was 47.5 Gy (range: 45-50.4) and VBT dose was 15 Gy at 0.5 cm from surface of applicators and mean radiotherapy duration was 6.5 weeks (range: 6- 8).
Toxicity profileRadiotherapy treatment was generally well
tolerated by all patients with grade 1 and 2 acute side effects and only four patients (3.42%) had grade 3 enteritis (Table. 2). Patients who received additional systemic chemotherapy, febrile neutropenia was seen in two patients (5.7%), which required hospitalization and Granulocyte- colony stimulating factor (GCSF) support. Late toxicity was seen only in one patient who presented with sub-acute intestinal obstruction which was managed conservatively.Locoregional control, distant control and overall survival rates
endometrial carcinomaThe Kaplan-Meier estimates of LRC, DMC,
DFS and OS were 80.9%, 69.3%, 70.6% and 71.1% respectively (Fig. 1 - A, B, C and D). Five patients developed locoregional recurrences (in-field). One patient had vaginal recurrences (1.7%)
Five years treatment outcome of Postoperative radiotherapy in uterine cancers, M.A. Tunio, et. al.
35
G. J. O. Issue 16, 2014Five years treatment outcome of Postoperative radiotherapy in uterine cancers, M.A. Tunio, et. al.
Table 1. Patients characteristics Abbreviations: n= number, HTN= hypertension, DM= diabetes mellitus, BMI= body mass index, FIGO= International Federation of Gynecologists and Obstetricians, PALN= para-aortic lymph nodes, LVSI= lymphovascular invasion, G= grade, ER= estrogen receptors, PR= progesterone receptors, EBRT= external beam radiation therapy, Gy= Gray
and three patients have pelvic nodal recurrence (5.1%). Distant metastases were seen in eight patients (13.6%) of whom two patients (3.4%) have simultaneous locoregional failures. First event was seen at 13 months of completion of treatment. Lungs (4 patients), liver (one patient) and para-aortic lymph nodes (3 patients) were common sites of metastasis. All patients received
salvage chemotherapy. At 60 months of follow-up, seven patients (11.9%) were dead; of whom one patient (1.7%) died of non-endometrial cancer related causes. Further subgroup analysis showed DFS was superior in FIGO stage I and II (93.8% and 71.4%) as compared to III and IV (22.7% and 0%) with p value < 0.001 (Fig. 2A). Presence of lymphovascular stromal invasion
36
Five years treatment outcome of Postoperative radiotherapy in uterine cancers, M.A. Tunio, et. al.Five years treatment outcome of Postoperative radiotherapy in uterine cancers, M.A. Tunio, et. al.
(LVSI) resulted in 14.7% reduction in DFS rates (Fig.2B).
Cox-proportional hazard ratio model showed body mass index, FIGO stage, lymphovascular invasion and adjuvant chemotherapy as significant prognostic factors for LRC, DMC and OS (Table. 3).
CarcinosarcomaThe Kaplan-Meier estimates of LRC, DMC,
DFS and OS were 87.1%, 16.3%, 17.9% and 16.3% respectively (Fig. 1 - A, B, C and D). Despite excellent LRC rates, majority of patients (59%) developed metastases in lungs (6 patients), bones (3 patients) and brain (one patient). Cox-proportional hazard ratio model showed carcinosarcoma itself and absence of chemotherapy poor prognostic factors for DMC (Table 3).
Table 2. Incidence of grade 2 and 3 acute and late toxicities
Table 3. Cox Proportional Hazard analysis of prognostic factors on Locoregional, distant control and overall survival‡ factors for endometrial carcinoma
† factors for leiomyosarcoma
Abbreviations: OR= odds ratio, CI= confidence intervals, BMI= body mass index, FIGO= International Federation of Gynecologists and Obstetricians, LVSI= lymphovascular stromal invasion, ER= estrogen receptors, PR= progesterone receptors, CT= chemotherapy, RT= radiation therapy, Gy= Gray
Five years treatment outcome of Postoperative radiotherapy in uterine cancers, M.A. Tunio, et. al.
37
G. J. O. Issue 16, 2014Five years treatment outcome of Postoperative radiotherapy in uterine cancers, M.A. Tunio, et. al.
figure 1.A
figure 1.C
figure 1.B
figure 1.D
figure 1: Kaplan Meier Curves of (A) locoregional Control, (B) distant metastases control, (C) disease free survival and (D) overall survival according to histologic types of uterine cancer
figure 2: Kaplan Meier Curves of disease free survival in endometrial carcinoma according to (A) fIGo stage and (B) lymphovascular stromal invasion
figure 2.A figure 2.B
38
Five years treatment outcome of Postoperative radiotherapy in uterine cancers, M.A. Tunio, et. al.Five years treatment outcome of Postoperative radiotherapy in uterine cancers, M.A. Tunio, et. al.
figure 3. Kaplan Meier Curves of disease free survival according to tumor size in leiomyosarcoma
leiomyosarcomaThe Kaplan-Meier estimates of LRC, DMC,
DFS and OS were 100%, 45%, 64.3% and 60% respectively (Fig. 1 - A, B, C and D). Four patients developed distant metastasis in lungs (3 patients and bones (one patient). Tumor size above 5 cm was found as an important prognostic factor for DMC (Fig. 3) and (Table 3).
DiscussionPostoperative pelvic radiation therapy
(PORT) in uterine malignancies have shown to be associated with improved LRC and DFS by various trials, but treatment outcomes of PORT have not been studied in Saudi population previously. Our study revealed several interesting and different characteristics and outcomes which are different from similar previously published western data. First, our cohort of endometrial carcinoma and carcinosarcoma were obese with median BMI > 30 Kg/m2 with significant co-morbidities, which is associated with poor LRC and OS as we reported previously (9). Further in our study, the patients with obesity and elevated BMI > 30 kg/m2 were mainly with good prognostic features, i.e., early stage disease and less lymphovascular invasion and ER and PR receptor positivity. Second, frequency of uterine sarcoma (leiomyosarcoma and carcinosarcoma) is significantly high as compared to reported western literature (10). Third, positive lymph nodes were not found prognostic factors for LRC, DMC and OS contrary to reported in other
studies (11). Fourth, LRC, DMC, DFS and OS for endometrial carcinoma were equal or far better than other studies (12); however, LRC, DMC, DFS and OS for uterine sarcoma were similar to other studies (13). Fifth, carcinosarcoma were seen at fifth decade of age and have elevated baseline serum CA-125 levels and with advanced stages which were associated with dismal survival similar to other studies (14). Sixth, tumor size above 5 cm was found as an important prognostic factor for DMC and OS in leiomyosarcoma but grade and stage were not found significant factors for DMC and OS (15).
Further, adjuvant systemic chemotherapy prior to adjuvant radiotherapy has been employed in FIGO stages high risk II, III and IVA endometrial carcinoma and all stages of carcinosarcoma after surgery, with minimal improvement in DFS but with high hematological toxicity (16). However our study showed that incorporation of adjuvant chemotherapy prior to adjuvant radiotherapy was associated with reduced LRC (p value 0.003) in patients with high risk II and III, IVA stage endometrial carcinoma and all stages of carcinosarcoma, yet there was minimal impact of additional adjuvant chemotherapy on distant control, DFS and OS. The possible explanation for high locoregional recurrences in adjuvant sequential chemotherapy and radiotherapy patients can be explained by delayed pelvic radiotherapy, i.e. mean time between surgery and radiotherapy was 13 weeks (11-18) as described in our previous publication (17).
Strengths of our study were the long follow up period of our cohort and use of 3DCRT in PORT and the limitations of our study were retrospective review, low sample size and heterogeneous cohort.
ConclusionOur first retrospective review of uterine cancer
Saudi patients who received PORT concludes following key points;
(a) Saudi women with endometrial cancers were found more obese with BMI > 30 k/m2 and are associated with co-morbidities, which warrant a national level obesity awareness campaign.
Five years treatment outcome of Postoperative radiotherapy in uterine cancers, M.A. Tunio, et. al.
39
G. J. O. Issue 16, 2014Five years treatment outcome of Postoperative radiotherapy in uterine cancers, M.A. Tunio, et. al.
(b) Frequency of uterine sarcoma is more in Saudi women as compared to other parts of world. These are far aggressive tumor than endometrial variant and associated with high distant metastasis even after pelvic irradiation.
(c) Incorporation of better radiation therapy techniques; 3DCRT or intensity modulated radiation therapy will enhance LRC, DMC and DFS rates
(d) FIGO stage and LVSI are important prognostic factors for LRC rates in endometrial carcinoma
(e) Tumor size above 5 cm is an important prognostic factor for DMC and DFS in leiomyosarcoma and histology itself an important prognostic factor for LRC, DMC and DFS in carcinosarcoma.
(f) Our results did not support the cincorporation of sequential chemotherapy in adjuvant care of early endometrial carcinoma due to poor LRC rates, however future large trials should consider the appropriate sequencing of chemotherapy.
References
1. Cancer incidence report Saudi Arabia 1999-2000.
2. Berrino F, De Angelis R, Sant M, Rosso S, Bielska-Lasota M et al. Survival for eight major cancers and all cancers combined for European adults diagnosed in 1995–99: results of the EUROCARE-4 study. Lancet Oncol 2007; 8:773–783.
3. Obermair A, Manolitsas TP, Leung Y, Hammond IG, McCartney AJ. Total laparoscopic hysterectomy for endometrial cancer: patterns of recurrence and survival. Gynecol Oncol 2004; 92: 789-793.
4. Creutzberg CL, Nout RA, Lybeert ML, Wárlám-Rodenhuis CC, Jobsen JJ, et al. Fifteen-year radiotherapy outcomes of the randomized PORTEC-1 trial for endometrial carcinoma. Int J Radiat Oncol Biol Phys 2011; 81: e631-638.
5. Keys HM, Roberts JA, Brunetto VL, Zaino RJ, Spirtos NM, et al. A phase III trial of surgery with or without adjunctive external pelvic radiation therapy in intermediate risk endometrial adenocarcinoma: a Gynecologic Oncology Group Study. Gynecol Oncol 2004; 92: 744-751.
6. Brooks SE, Zhan M, Cote T. Surveillance, epidemiology, and end results analysis of 2677 cases of uterine sarcoma 1989–1999. Gynecol Oncol 2004; 93: 204–208.
7. Kahanpaa KV, Wahlstrom T, Grohn P et al. Sarcomas of the uterus: a clinicopathologic study of 119 patients. Obstet Gynecol 1986; 67: 417–424.
8. Small W Jr. Mell LK, Anderson P. Consensus guidelines for delineation of clinical target volume for intensity-modulated pelvic radiotherapy in postoperative treatment of endometrial and cervical cancer. Int J Radiat Oncol Biol Phys 2008; 71:428–434.
9. Asiri MA, Tunio MA, AlHadab AR, Mohamed R, Bayoumi Y, Alsaeed E, et al. Impact of Body Mass Index on Treatment Outcomes of Adjuvant Radiation Therapy in Saudi Females with Endometrial Carcinoma. World J Surg Med Rad Onc 2012; 2:119-129.
10. Sherman ME & Devesa SS. Analysis of racial differences in incidence, survival, and mortality for malignant tumors of the uterine corpus. Cancer 2003; 98:176–186.
11. Chia VM, Newcomb PA, Trentham-Dietz A, Hampton JM. Obesity, diabetes and other factors in relation to survival after endometrial cancer diagnosis. Int J Gynecol Cancer 2007; 17: 441-446.
12. Trovik J, Mauland KK, Werner HM, Wik E, Helland H, Salvesen HB. Improved survival related to changes in endometrial cancer treatment, a 30-year population based perspective. Gynecol Oncol 2012; 125:381-7.
13. D’Angelo E & Prat J. Uterine sarcomas: a review. Gynecol Oncol 2010; 116: 131–139.
14. Sleijfer S, Seynaeve C & Verweij J. Gynaecological sarcomas. Curr Opin Oncol 2007; 19: 492–496.
15. Reed NS. The management of uterine sarcomas. Clin Oncol (R Coll Radiol) 2008; 20: 470–478.
16. Randall ME, Spirtos NM, Dvoretsky P. Whole abdominal radiotherapy versus combination chemotherapy with doxorubicin and cisplatin in advanced endometrial carcinoma (phase III): Gynecologic Oncology Group Study No.122. J Natl Cancer Inst Monogr 2003; 19: 13-15.
17. Secord AA, Havrilesky LJ, O’Malley DM, Bae-Jump V, Fleming ND, et al. A multicenter evaluation of sequential multimodality therapy and clinical outcome for the treatment of advanced endometrial cancer. Gynecol Oncol 2009; 114: 442-447.
RESEARCH ARTICLE
Gestational trophoblastic neoplasia: treatment outcomesfrom a single institutional experience
H. Al-Husaini • H. Soudy • A. Darwish • M. Ahmed • A. Eltigani •
W. Edesa • T. Elhassan • A. Omar • W. Elghamry • H. Al-Hashem •
S. Al-Hayli • I. Madkhali • S. Ahmad • I. A. Al-Badawi
Received: 15 July 2014 / Accepted: 17 October 2014! Federacion de Sociedades Espanolas de Oncologıa (FESEO) 2014
AbstractPurpose To report the outcomes of gestational tropho-
blastic neoplasia (GTN) at a single institution and to
determine the factors affecting response to chemotherapyand survival.
Methods/Patients From 1979–2010, we retrospectively
reviewed the data of 221 patients treated at our center.GTN Patients were assigned to low-risk (score B6) or
high-risk (score C7) based on the WHO risk factor scoring
system. Overall survival (OS) probabilities were estimatedusing Kaplan–Meier method. Logistic regression was
applied to study the impact of different factors on theresponse to initial therapy.
Results Patients’ OS rate was 97 %. Median age at
diagnosis was 37 year. 131 (59 %) patients had low-riskand 88 (40 %) cases had high-risk GTN. Complete
remission rates to initial chemotherapy in low-risk group
were 53 % and 87 % for single-agent methotrexate ordactinomycin, respectively. In high-risk group, 94 %
achieved complete remission to initial chemotherapy with
etoposide, methotrexate, dactinomycin, cyclophosphamide,and vincristine (EMA-CO). Etoposide, cisplatin, and dac-
tinomycin as primary therapy in high-risk patients was
successful in 70 %, while bleomycin, etoposide, and cis-platin (BEP) was successful in 53 % of cases. Salvage
chemotherapy, surgical intervention or radiation therapy
resulted in overall complete remission of 90 % in low-riskand 73 % in high-risk groups. Factors associated with
resistance to initial chemotherapy were advanced-stage III/
IV (p = 0.005), metastatic site other than lung or vagina(p = 0.005) and high-risk prognostic score (p = 0.05). OS
was significantly influenced by the type of antecedentpregnancy (molar 98 % vs. others 93 %; p = 0.04), FIGO
stage (I, II 100 % vs. III, IV 94 %; p = 0.02), score (low-
risk 100 % vs. high-risk 92 %; p = 0.01), and site ofmetastasis (lung/vagina 98 % vs. others 85 %; p = 0.002).
Conclusions GTNs have excellent prognosis if properly
treated at experienced centers. Single-agent dactinomycinseems more effective for low-risk GTN. EMA-CO remains
the preferred primary treatment regimen for high-risk
group. The excellent outcome reflects the success of sal-vage therapy.
Keywords Gestational trophoblastic disease !Chemotherapy, treatment ! Survival ! Clinical outcomes !Salvage therapy
H. Al-Husaini ! H. Soudy ! M. Ahmed ! T. Elhassan !A. Omar ! S. Al-Hayli ! I. Madkhali ! I. A. Al-BadawiMedical Oncology Consultant, King Faisal Cancer Center, KingFaisal Specialist Hospital & Research Center, P.O. Box 3354,MBC 64, Riyadh 11211, Saudi Arabiae-mail: [email protected]
A. DarwishOncology Department, Dr. Soliman Fakeeh Hospital, Jeddah,Saudi Arabia
M. Ahmed ! W. EdesaCairo University, Cairo, Egypt
A. EltiganiNational Guard Hospital, Riyadh, Saudi Arabia
W. ElghamryAin Shams University, Cairo, Egypt
H. Al-HashemPrincess Margaret Hospital, Toronto, Canada
S. Ahmad (&)Florida Hospital Cancer Institute, Orlando, FL, USAe-mail: [email protected]
123
Clin Transl Oncol
DOI 10.1007/s12094-014-1251-1
Introduction
The proliferative process arising from an aberrant fertil-
ization event that has potential to develop into invasive
malignant neoplasm is known as gestational trophoblasticdisease (GTD). This includes hydatidiform mole (complete
and partial) and gestational trophoblastic neoplasia (GTN),
which encompasses persistent/invasive mole, choriocarci-noma, placental site trophoblastic tumors, and epithelioid
trophoblastic tumor [1].
Most commonly, GTN is diagnosed following molarpregnancy, but it can also occur after any gestation
including miscarriages and term pregnancies. Any form of
GTN can metastasize and the most common metastatic siteis lung (80 %) followed by vagina (30 %), brain and liver
(10 %). GTN is highly sensitive to chemotherapy and most
curable cancer with success rate exceeding 90 % [2]. Itrepresents one of the only cancers for which single-agent
chemotherapy is still in wider use.
Therapeutic decision is based on the International Fed-eration of Gynecology and Obstetrics (FIGO) anatomic
staging and prognostic scoring index. The low-risk group
(prognostic score B6) can be treated with single-agentchemotherapy resulting in the survival rate approaching
100 %. The high-risk group (prognostic score C7) requires
initial multi-agent chemotherapy with or without adjuvantradiation therapy and surgery to achieve a survival rate of
80–90 % [3, 4].The King Faisal Specialist Hospital & Research Center
(KFSHRC) has been a natural referral center and many
patients were directed to our institution from all around theKingdom of Saudi Arabia. The aim of this study was to
review our clinical experience in the treatment of malig-
nant GTN over the past 30 years at KFSHRC, to evaluatecomplete response rate to chemotherapy, and to analyze the
risk factors affecting patients’ response and overall
survival.
Materials and methods
From 1979 to 2010, a total of 221 patients with GTN
(invasive mole and choriocarcinoma, excluding placentalsite and epithelioid trophoblastic tumor) were treated at the
primary institution. The diagnosis of GTN was made by
both clinical and histopathologic criteria. Post-molar GTNwas diagnosed using the following criteria: 1) plateau of
human chorionic gonadotropin (hCG) levels for four con-
secutive tests over at least 3 weeks, 2) a rise in hCG levelsby C10 % for three or more values over at least 2 weeks,
3) persistent of hCG 6 months after the molar evacuation,
4) histologic identification of choriocarcinoma, or 5) pre-sence of metastatic disease. Invasive mole was diagnosed
by pathologic confirmation or, if tissue was not available,
after the molar pregnancy when there was rising or plateauin the hCG level or detection of metastatic disease in the
absence of histologic diagnosis of choriocarcinoma.
Patients were diagnosed with choriocarcinoma based onhistologic findings of hyperplastic and dysplastic tropho-
blast in the absence of villi or if GTN developed following
a non-molar pregnancy, thus excluding the possibility of aninvasive mole.
The inclusion criteria were patients diagnosed to haveGTN post-molar, abortion or full-term pregnancy using the
FIGO anatomic staging and prognostic scoring index.
Exclusion criteria were cases with the diagnosis of pla-cental site trophoblastic tumor or epithelioid trophoblastic
tumor.
After the diagnosis of GTN was made, staging work-upwas performed. Along with complete history and physical
examination, patients underwent laboratory tests: complete
blood counts, quantitative serum hCG level, renal and liverfunction tests. Chest X-ray with computed tomography
(CT) scan of the chest, abdomen, and pelvis were regularly
performed, as well as either CT scan or magnetic resonanceimaging (MRI) of the brain if other scans showed meta-
static disease.
Based upon the staging work-up, patients with GTNwere categorized according to the FIGO anatomic staging
and the World Health Organization (WHO) scoring system
called the prognostic scoring index. The WHO risk factorscoring system was determined by age, antecedent preg-
nancy, interval from antecedent pregnancy, pre-treatment
hCG level, largest tumor size, site of metastasis, number ofmetastasis, and previous failed chemotherapy. Patients
with score \7 were considered low-risk, whereas patients
with score 7 or more were considered high-risk group.At our primary institution, the first-line chemotherapy
for patients with low-risk, including metastatic and non-
metastatic disease, was usually started with single-agentchemotherapy consisting of methotrexate 1 mg/kg intra-
muscular (IM) weekly (n = 59) or an 8-day regimen of
methotrexate (1 mg/kg IM every other day for four doses;n = 13). An alternative regimen of dactinomycin 1.25 mg/
m2 intra-venous (IV) every other week was given as initial
therapy to 19 patients; and three patients received dacti-nomycin 0.5 mg IV daily for 5 days. Dactinomycin was
given to 11 patients who developed methotrexate resistance
or toxicity. Patients who failed single-agent chemotherapyreceived combination chemotherapy, including bleomycin,
etoposide, and cisplatin (BEP) in 12 patients; etoposide,
methotrexate, dactinomycin, cyclophosphamide, and vin-cristine (EMA-CO) in eight patients; and methotrexate,
dactinomycin, and either chlorambucil or cyclophospha-
mide (MAC) in three patients. Other combination regimensused were etoposide, cisplatin, dactinomycin in two
Clin Transl Oncol
123
patients, and vinblastine, ifosfamide, and cisplatin (VeIP)
in two patients.Patients with high-risk GTN were initially managed
with combination chemotherapy, including EMA-CO in 16
patients, BEP in 19 patients (between 1994 and 2003), andetoposide, cisplatin, and dactinomycin in 20 patients
(between 1987 and 1993). Seven patients were treated with
MAC (from 1982 to 1983). Patients who developed resis-tant or relapsed disease were treated with salvage combi-
nation chemotherapy included EMA-EP (EMA-CO, butcyclophosphamide and vincristine were replaced by eto-
poside and cisplatin) in two patients, VeIP in eight patients,
and etoposide, ifosfamide, and cisplatin (VIP) in fourpatients. Other salvage therapy used was taxane/platinum
in two patients.
During the therapies, history and physical examination,complete blood counts, chemistry profiles, and hCG were
done at first day of each course of the treatment. Patients
were considered to be responding by the decrease in hCGlevels. Complete remission was defined when three con-
secutive weekly hCG levels were within the normal range
(\5 mIU/mL). Additional two courses of chemotherapywere usually given after the first normal hCG level. After
the complete remission was achieved, serum hCG level
was measured monthly for one year with the use of oral orbarrier contraception to prevent pregnancy. Relapse was
defined when the hCG level rose after an initial complete
remission. Surgery was considered to either excise sites ofbulky and/or resistant tumor or less frequently to treat
complications such as tumor hemorrhage or infection.
The clinical data about the patient and disease charac-teristics, including age, antecedent pregnancy, interval
from pregnancy, clinico-pathologic diagnosis, pre-treat-
ment hCG, extent of tumor and treatment were descripted.Descriptive statistics included medians and ranges for
continuous variables and frequencies for categorical vari-
ables. Overall survival probabilities were calculated usingKaplan–Meier method. Survival curves were compared
using log-rank test. A univariate analysis was utilized using
logistic regression to study the impact of each risk factor onthe response to initial therapy. Significant level was set to
0.05. Statistical analysis was done using SPSS version 20
(IBM, Chicago, IL).
Results
A total of 221 patients with GTN were treated at our pri-
mary institution from the year 1979 through 2010. Thepatient and disease characteristics are presented in Table 1.
The median age of patients at the time of diagnosis was
37 years (range 14–55). The overall survival rate for thepatients was 97 % (Fig. 1). Most patients developed GTN
Table 1 Patients and disease characteristics
Demographics Number = 221
Median age (range) 37 years (14–55)
\40 years 135 (61 %)
C40 years 86 (39 %)
Antecedent pregnancy
Hydatidiform mole 157 (71 %)
Abortion 27 (12 %)
Term pregnancy 30 (14 %)
Other 7 (3 %)
Interval
\4 months 130 (59 %)
4–6 months 39 (18 %)
7–12 months 14 (6 %)
12 months 31 (14 %)
Unknown 7 (3 %)
Pre-treatment hCG level
\1,000 29 (13 %)
1,000–\10,000 43 (20 %)
10,000–\100,000 72 (33 %)
C100,000 69 (31 %)
Unknown 8 (4 %)
Clinico-pathologic type
Persistent/invasive GTN 103 (46.6 %)
Choriocarcinoma 117 (53 %)
Unknown 1 (0.4 %)
Previous chemotherapy outside
No 175 (79 %)
Yes 46 (21 %)
FIGO stage
I 91 (41 %)
II 17 (8 %)
III 86 (39 %)
IV 27 (12 %)
Prognostic score
Low-risk 131 (59 %)
High-risk 88 (40 %)
Unknown 2 (1 %)
Radiation therapy
Yes 17 (8 %)
No 203 (92 %)
Unknown 1 (0.4 %)
Surgery
No 16 (7 %)
Yes 203 (92 %)
Uterine evacuation 158 (71 %)
Hysterectomy 69 (31 %)
Metastatectomy 8 (4 %)
hCG human chorionic gonadotropin, GTN gestational trophoblasticneoplasia, FIGO International Federation of Gynecology andObstetrics
Clin Transl Oncol
123
following the molar pregnancy (71 %), had disease dura-
tion from antecedent pregnancy of less than 4 months(59 %), and had the pre-treatment hCG level
of B100,000 IU/L (65 %). The lung (n = 112, 51 %
cases) was the most common site of metastasis, followedby brain (n = 15, 7 %), vagina (n = 14, 6 %), and liver
(n = 11, 5 %). The diagnosis was based on histopathologic
evidence in 86 (39 %) cases and elevated hCG levels withhistory consistent with GTN in 135 (61 %) patients. The
median follow-up period was 39 months.Treatment outcomes of low-risk group of GTN patients
are shown in Table 2 [131 (59 %) patients falling into the
low-risk (0-6 FIGO prognostic score)]. The overall com-plete remission rate to the initial chemotherapy regimen
(CRI) was 69.5 % (91/131). We observed a complete
remission in 53 % of patients treated with single-agentmethotrexate (39/73), while the remission rate was 87 % in
patients treated with single-agent dactinomycin (20/23).
Thirty-five patients received combination chemotherapyregimen as first-line therapy because nine cases had failed
the single-agent chemotherapy outside the KFSHRC, four
had hCG level of more than 100,000 IU/L and theremaining cases with no clear reason(s). The group sub-
jected to combination regimen had remission rate of 91 %
(32/35).Thirty-three (25 %) patients required second-line sal-
vage therapy. The complete remission rate to sequential
single-agent dactinomycin for the low-risk group was 82 %(9/11). Two patients with resistant to first-line dactino-
mycin were treated with single-agent methotrexate and one
patient achieved complete remission. Twenty patientsreceived a multi-agent chemotherapy as second-line
therapy. Finally, eight patients required third-line treat-
ment, four patients required fourth-line treatment, and onlyone patient required fifth-line treatment. The overall com-
plete remission to sequential chemotherapy, initial and
salvage chemotherapy, for low-risk disease was 90 % (118/131) as summarized in Table 2.
Thirty-six (27.5 %) low-risk patients underwent hys-
terectomy. Hysterectomy was performed either as prior toinitial chemotherapy in 13 patients, due to persistent dis-
ease in 15 patients, because of acute bleeding in 5 patients,or unknown reason in 3 patients. Seven of 15 patients
(47 %) who had hysterectomy for persistent disease
achieved complete remission without further salvage che-motherapy. Two patients underwent thoracotomy for
resistant focus. One patient received radiation therapy to
pelvis. The overall survival rate of the low-risk patientswas 100 %.
Treatment outcomes of high-risk group of GTN patients
are shown in Table 3 [88 (40 %) patients of the studypopulation falling into the high-risk group]. The overall
complete remission rate to the initial multi-agent chemo-
therapy was 57 % (50/88). We observed a completeremission in 94 % of patients treated with EMA-CO regi-
men (15/16), while the remission rate was 70 % in patients
treated with etoposide, cisplatin, and dactinomycin (14/20).Of the 19 high-risk patients who received BEP regimen, 10
(53 %) patients achieved the complete remission. The
MAC regimen was administered to seven patients withremission rate of 28.5 % (2/7).
Thirty (34 %) patient required second-line therapy. Only
47 % (14/30) of patients achieved complete remission tosecondary chemotherapy resulting in an overall complete
Fig. 1 Overall survival inpatients with gestationaltrophoblastic neoplasia (GTN)as determined by Kaplan–Meieranalysis
Clin Transl Oncol
123
remission rate to sequential chemotherapy, initial and sal-
vage chemotherapy, in high-risk patients of 73 % (64/88)as summarized in Table 3.
Thirty-two (36 %) high-risk patients underwent hyster-
ectomy, 14 of whom had hysterectomy prior to or with theinitiation of chemotherapy. The others had this procedure
either to persistent disease in 11 patients or due to acute
bleeding in six patients. Two high-risk patients had lungresection of resistant focus and got complete remission
without any further chemotherapy. Two patients underwentcraniotomy and one patient had bowel resection for the
persistent disease. Radiation therapy was given to 16
(18 %) patients, 13 of whom received whole brain radia-tion therapy for brain metastasis. Three patients were
required pelvic radiation and one patient was given radia-
tion to the lung. Most of the patients who received radia-tion therapy were earlier cases before the year 2002. The
overall survival for the high-risk patients was 92 %.
As shown in Table 4, the factors that were significantlyassociated with resistant to the initial chemotherapy include
advanced-stage disease (III and IV; odd ratio ‘‘OR’’ 0.4) as
compared to the early-stage disease (I and II; OR 1.0)(p = 0.005). Patients with metastatic site other than lung or
vagina had increased risk of resistance to initial chemo-
therapy when compared to metastatic disease localized to
vagina and/or lung (OR 1.0 vs. 0.5; p = 0.005). Patients
with high-risk prognostic score compared to the low-risk
group showed an increased risk of resistant to the initialchemotherapy (OR 1.0 vs. 1.7; p = 0.05). Age, pre-treat-
ment hCG level, antecedent pregnancy, and duration of
disease from pregnancy did not significantly affect theresistance to initial chemotherapy in this study cohort.
The overall survival (OS) was also significantly influ-
enced by the type of antecedent pregnancy (molar OS 98 %vs. other OS 93 %; p = 0.04). Patients with advanced-
stage disease had poor survival when compared to the
early-stage group (III and IV OS 94 % vs. I and II OS100 %; p = 0.02). Patients with high-risk prognostic score
compared to low-risk group showed poor outcome (OS
92 vs. 100 %; p = 0.01). Patients with metastatic site otherthan lung or vagina were associated with poor survival
when compared to metastatic disease localized to vagina
and/or lung (OS 85 vs. 98 %; p = 0.002).There were six relapses that occurred after complete
remission to the initial chemotherapy from the entire 221
patients giving an overall 3 % risk of relapse. The mediantime to relapse was 4 months. Three of the relapsed
patients had low-risk prognostic score and were success-
fully cured with salvage chemotherapy. In contrast, onlyone of the three high-risk patients who relapsed was suc-
cessfully cured with salvage therapy.
Table 2 The overall treatment results for the low-risk group ofpatients with GTN
Disease category/chemotherapy Number ofpatients
Completeremission (%)
Low-risk 131 CRI: 91 (69.5 %)
Single-agent methotrexate 73 39 (53 %)
Single-agent dactinomycin 23 20 (87 %)
Combination chemotherapy(EMA-CO or BEP or MAC)
35 32 (91 %)
Low-risk: salvage chemotherapy
Single-agent dactinomycin 11 9 (82 %)
Single-agent methotrexate 2 1 (50 %)
EMA-CO 8 6 (75 %)
MAC 3 1 (33 %)
BEP or EP 12 7 (58 %)
VeIP 2 2 (100 %)
VIP 2 1 (50 %)
Overall CR:118 (90 %)
GTN gestational trophoblastic neoplasia, CRI complete remission toinitial chemotherapy, EMA-CO etoposide, methotrexate, dactinomy-cin, cyclophosphamide, and vincristine, MAC methotrexate, dactino-mycin, and either chlorambucil or cyclophosphamide, BEPbleomycin, etoposide, and cisplatin, EP etoposide and cisplatin, VeIPvinblastine, ifosfamide, and cisplatin, VIP etoposide, ifosfamide, andcisplatin, CR complete remission
Table 3 The overall treatment for high-risk group for patients withGTN
Disease Category/Chemotherapy Number ofPatients
CompleteRemission (%)
High-risk 88 CR1: 50 (57 %)
EMA-CO 16 15 (94 %)
BEP 19 10 (53 %)
Etoposide/cisplatin/actinomycin 20 14 (70 %)
MAC 7 2 (28.5 %)
High-risk: salvage chemotherapy
EMA-CO 6 3 (50 %)
EMA-EP 2 2 (100 %)
BEP or EP 7 5 (71 %)
MAC 8 1 (12.5 %)
VeIP 8 1 (12.5 %)
VIP 4 2 (50 %)
Overall CR:64 (73 %)
GTN gestational trophoblastic neoplasia, CRI complete remission toinitial chemotherapy, EMA-CO etoposide, methotrexate, dactinomy-cin, cyclophosphamide and vincristine, MAC methotrexate, dacti-nomycin, and either chlorambucil or cyclophosphamide, BEPbleomycin, etoposide, and cisplatin, EP etoposide and cisplatin, VeIPvinblastine, ifosfamide, and cisplatin, VIP etoposide, ifosfamide, andcisplatin, CR complete remission
Clin Transl Oncol
123
With regard to fertility outcomes, 38 (17 %) patients
became pregnant after the completion of chemotherapy.Thirteen of these pregnant women were of the high-risk
group. Healthy babies without congenital malformation
were delivered in 24 (63 %) pregnant patients. Abnormalpregnancies occurred in seven (18 %) patients that include
miscarriage (n = 5), stillbirth (n = 2), and molar preg-
nancy (n = 1) patient. The remaining pregnant women hadunknown outcome, if any.
Discussion
The majority of women diagnosed with GTN can be cured
with overall worldwide survival rate of low-risk group
approaching 100 %, and 80–90 % for high-risk group [5–9].However, these tumors are rare in any individual hospital and
most treatment recommendations are based on the
observational studies from larger series. Our KFSHRC has
become a referral center and many patients were directed toour center from all across the Kingdom of Saudi Arabia. In this
large series of 221 patients spanning over 30 years of period,
we confirm the previously reported highly curable rates ofGTN when therapeutic decisions are based on FIGO anatomic
stage and prognostic scoring index. The overall survival rate
for patients with GTN treated at our center approached 97 %.The median age of women needing treatment in our study was
37 years which seem quite older than what is published in thepeer-reviewed English literature (usually under the age of
35 years) [15]. This is presumably due to the greater number
of cases getting pregnant in older age.Patients with low-risk GTN can usually be treated suc-
cessfully with single-agent chemotherapy. In our series of 131
low-risk patients, we observed a complete remission rate of53 % to single-agent methotrexate and 87 % to single-agent
dactinomycin. In keeping with our results, other peer-
Table 4 Univariable logistic regression analysis of prognostic factors for complete response to initial chemotherapy and overall survival
Prognostic factors Number (%) OR for response to initialchemotherapy (95 % CI)
p value Overallsurvival (%)
p value
Type of pregnancy 0.04
Molar 157 (71 %) 0.9 (0.5-1.8) 0.9 98
Non-molar 64 (29 %) 1.0 93
Metastatic sites 0.002
Lung or vagina 91 (41 %) 1.0 0.02 98
Other sites 35 (16 %) 0.38 (0.87-1.74) 85
Prognostic score 0.01
Low 131 (59 %) 1.7 (0.9-3.0) 0.05 100
High 88 (40 %) 1.0 92
FIGO stage 0.02
I–II 108 (49 %) 1.0 0.005 100
III–IV 113 (51 %) 0.4 (0.2-0.7) 94
Age (years) 0.7
\40 135 (61 %) 1.0 0.8 96
C40 86 (39 %) 0.9 (0.5-1.5) 97
Pre-treatment hCG 0.6
\1,000 29 (13 %) 1.0 0.08 100
1,000–\10,000 43 (20 %) 1.1 (0.3-3.2) 0.8 97
10,000–\100,000 72 (33 %) 0.4 (0.1-1.08) 0.07 98
C100,000 69 (31 %) 0.67 (0.2-1.7) 0.4 95
Interval 0.3
\4 months 130 (59 %) 1.0 0.2 98
4–6 months 39 (18 %) 2 (0.8-4.6) 0.09 97
7–12 months 14 (6 %) 0.6 (0.2-1.8) 0.3 92
[12 months 31 (14 %) 1.1 (0.48-2.4) 0.8 90
Chemotherapy outside 0.75
No 175 (79 %) 1 0.5 96.3
Yes 46 (21 %) 1.1 (0.5-2.1) 97.5
OR odds ratio, CI confidence interval, FIGO International Federation of Gynecology and Obstetrics, hCG human chorionic gonadotropin
Clin Transl Oncol
123
reviewed studies have also reported the superiority of single-
agent dactinomycin over methotrexate as a frontline therapyin low-risk patients. Randomized clinical trial comparing
biweekly dactinomycin to weekly IM methotrexate has
demonstrated a superior response rate for dactinomycin overmethotrexate (69 vs. 53 %; p = 0.015) [10]. Retrospective
review of 247 patients with low-risk GTN treated with single-
agent therapy showed significantly higher primary remissionrate with dactinomycin than with methotrexate regimen [11].
For patients who developed evidence of methotrexateresistant, we observed a successful salvage with single-
agent dactinomycin (in 82 % of cases). Due to lower tox-
icity, decreased cost, and advantage of convenience ofsingle weekly IM methotrexate and also successful salvage
therapy with dactinomycin, we preferred to start treatment
with methotrexate and keeping dactinomycin as secondarytherapy in the presence of methotrexate resistance or as
primary therapy when there is contraindication to metho-
trexate (effusion, renal or hepatic impairment). In ourseries, the overall complete remission rate to sequential
chemotherapy and overall survival rate of low-risk GTN
patients was 90 and 100 %, respectively.The proportion of women with high-risk disease in our
study was high due to the tertiary nature of the referrals.
Among the patients with high-risk GTN, our data supportthe effectiveness of the combination chemotherapy. A large
majority of patients (94 %) achieved complete remission
with the first-line use of the EMA-CO regimen. Despite theabsence of randomized trials to prove its superiority, EMA-
CO has emerged as the current optimal primary treatment
for the patients with the high-risk GTN [12–14]. The overallsurvival rate was 92 %, reflecting successful salvage of
some non-responders with cisplatin-based chemotherapy.
Following the successful therapy of GTN, the overallrelapse rate in our study is low (3 %), which is similar to
other studies reported in the peer-reviewed literature [15,
16]. The median time from remission to relapse was4 months. Most of the relapsed patients were successfully
cured with additional salvage chemotherapy. Patients can
anticipate subsequent pregnancy outcomes similar to thegeneral population [17, 18].
Treatment of patients with GTN in specialized centers
optimizes the opportunity for cure and minimizes formorbidity. Factors that are known to increase resistance to
initial chemotherapy include high-risk prognostic score,
advanced-stage (III and IV), metastatic disease other thanlung or vagina, prior unsuccessful chemotherapy, had
higher pre-treatment hCG level, antecedent term gestation,
and clinico-pathologic diagnosis of choriocarcinoma [19,20]. Our study confirms the lower outcomes in patients
with advanced-stage disease, metastatic site other than the
lung and vagina, high-risk prognostic score, and antecedentnon-molar pregnancy.
In summary, our data confirm the previously reported
high rate of remission and survival in both low-risk andhigh-risk GTN patients when using the FIGO staging cri-
teria and the WHO prognostic scoring system. Referral of
such patients to relatively more experienced centers ishighly recommended for appropriate risk stratification and
subsequent therapeutic management of patients with GTN.
Conflict of interest None.
References
1. Seckl MJ, Sebire NJ, Berkowitz RS. Gestational trophoblastic disease. Lancet.2010;376(9742):717–29.
2. Soper JT. Gestational trophoblastic disease. Obstet Gynecol. 2006;108:176–87.3. Lurain JR, Casanova LA, Miller DS, Rademaker AW. Prognostic factors in
gestational trophoblastic tumors: A proposed new scoring system based onmultivariate analysis. Am J Obstet Gynecol. 1991;164:611–6.
4. Ngan HY, Bender H, Benedet JL, Jones H, Montruccoli GC, Pecorelli S. FIGOCommittee on Gynecologic Oncology. Gestational trophoblastic neoplasia,FIGO 2000 staging and classification. Int J Gynaecol Obstet. 2003;83(Suppl1):175–7.
5. Berkowitz RS, Goldstein DP, Bernstein MR. Ten years’ experience withmethotrexate and folinic acid as primary therapy for gestational trophoblasticdisease. Gynecol Oncol. 1986;23:111–8.
6. Ngan S, Seckl MJ. Gestational trophoblastic neoplasia management: An update.Curr Opin Oncol. 2007;19:486–91.
7. Chakrabarti BK, Mondal NR, Chatterjee T. Gestational trophoblastic tumor at atertiary level cancer center: A retrospective study. J Reprod Med.2006;51:875–8.
8. Lok CA, Ansink AC, Grootfaam D, van der Velden J, Verheijen RH, ten Kate-Booij MJ. Treatment and prognosis of post term choriocarcinoma in TheNetherlands. Gynecol Oncol. 2006;103:698–702.
9. El-Lamie IK, El Sayed HM, Badawie AG, Bayomi WA, El-Ghazaly HA,Khalaf-Allah AE, et al. Evolution of treatment of high-risk metastatic gesta-tional trophoblastic tumors: Ain Shams University experience. Int J GynecolCancer. 2006;16:866–74.
10. Osborne RJ, Filiaci V, Schink JC, Mannel RS, Alvarez Secord A, Kelley JL,et al. Phase III trial of weekly methotrexate or pulsed dactinomycin for low-riskgestational trophoblastic neoplasia: A Gynecologic Oncology Group study.J Clin Oncol. 2011;29:825-31.
11. Matsui H, Iitsuka Y, Seki K, Sekiya S. Comparison of chemotherapies withmethotrexate, VP-16 and actinomycin-D in low-risk gestational trophoblasticdisease: Remission rates and drug toxicities. Gynecol Obstet Invest.1998;46:5–8.
12. Newlands ES, Bagshawe KD, Begent RH, Rustin GJ, Holden L. Results with theEMA-CO (etoposide, methotrexate, actinomycin D, cyclophosphamide, vin-cristine) regimen in high-risk gestational trophoblastic tumours, 1979 to 1989.Br J Obstet Gynaecol. 1991;98:550–7.
13. Lurain JR, Singh DK, Schink JC. Primary treatment of metastatic high-riskgestational trophoblastic neoplasia with EMA-CO chemotherapy. J ReprodMed. 2006;51:767–72.
14. Escobar PF, Lurain JR, Singh DK, Bozorgi K, Fishman DA. Treatment of high-risk gestational trophoblastic neoplasia with etoposide, methotrexate, actino-mycin D, cyclophosphamide, and vincristine chemotherapy. Gynecol Oncol.2003;91:552–7.
15. Sita-Lumsden A, Short D, Lindsay I, Sebire NJ, Adjogatse D, Seckl MJ, et al.Treatment outcomes for 618 women with gestational trophoblastic tumoursfollowing a molar pregnancy at the Charing Cross Hospital, 2000-2009. Br JCancer. 2012;107:1810–4.
16. Matsui H, Iitsuka Y, Suzuka K, Yamazawa K, Mitsuhashi A, Sekiya S. Salvagechemotherapy for high-risk gestational trophoblastic tumor. J Reprod Med.2004;49:438–42.
17. Loret de Mola JR. Goldfarb JM. Reproductive performance of patients aftergestational trophoblastic disease. Semin Oncol. 1995;22:193–7.
18. Berkowitz RS, Goldstein DP, Bernstein MR. Reproductive experience aftercomplete and partial molar pregnancy and gestational trophoblastic tumors.J Reprod Med. 1991;36:3–8.
19. Roberts JP, Lurain JR. Treatment of low-risk metastatic gestational tropho-blastic tumors with single-agent chemotherapy. Am J Obstet Gynecol.1996;174:1917–23; Discussion 1923–4.
20. Hoekstra AV, Lurain JR, Rademaker AW, Schink JC. Gestational trophoblasticneoplasia: Treatment outcomes. Obstet Gynecol. 2008;112:251–8.
Clin Transl Oncol
123
Cytoreductive surgery and hyperthermic intraperitonealchemotherapy for management of recurrent/relapsedovarian granulosa cell tumor: A single-center experience
Ismail A. Al-Badawi1,2, Ahmed Abu-Zaid1,2, Ayman Azzam3,4, Osama AlOmar1,Hamed AlHusaini3 and Tarek Amin3
1Department of Obstetrics and Gynecology, 3Oncology Center, King Faisal Specialist Hospital and Research Center, 2College ofMedicine, Alfaisal University, Riyadh, Saudi Arabia; and 4Faculty of Medicine, Alexandria University, Alexandria, Egypt
Abstract
Aim: The aim of this study was to retrospectively report our experience (efficacy/morbidity) withcytoreductive surgery+hyperthermic intraperitoneal chemotherapy (CRS+HIPEC) for the management ofrecurrent/relapsed ovarian granulosa cell tumors (OGCT).Material and Methods: From 2010 to 2013, six patients underwent CRS+HIPEC. CRS was performed withstandard peritonectomy procedures and visceral resections directed towards complete elimination of tumorsfrom the abdominopelvic cavity. HIPEC was performed with cisplatin (50 mg/m2) and doxorubicin (15 mg/m2) and allowed to circulate in the abdominopelvic cavity for 90 min at 41.0–42.2°C.Results: Cytoreduction completeness (CC-0) was achieved in all except one patient (CC-1). Five patients hadOGCT recurrences in abdomen+pelvis and one patient in abdomen only. No grade V morbidity (Clavien–Dindo classification) occurred. Two patients developed lung atelectasis, which was managed by mere chestphysiotherapy (grade I). One patient developed urinary tract infection (grade II) and another patient devel-oped pneumonia (grade II) – both of which were managed by antibiotics. One patient developed splenic bedand anterior abdominal wall collections requiring ultrasound-guided aspiration without general anesthesia(grade III). One patient developed pulmonary embolism requiring intensive care-unit management (grade IV).Four chemo-naïve patients received adjuvant chemotherapy whereas the remaining two previously chemo-exposed patients received no adjuvant therapy. All patients were alive and disease-free without proof ofrecurrence/relapse at 40, 32, 27, 24, 20 and 16 months. The average interval of follow-up after CRS+HIPEC wasroughly 27 months (range: 16–40 months).Conclusion: CRS+HIPEC appears to be an efficacious and morbidly well-tolerated therapeutic modality forrecurrent/relapsed OGCT. Long-term follow-up data and further research are needed.Key words: cytoreductive surgery, hyperthermic intraperitoneal chemotherapy, ovarian granulosa celltumor.
Introduction
Ovarian granulosa cell tumors (OGCT) are quiteinfrequent neoplasms accounting for roughly 70%of ovarian sex cord-stromal tumors and 2–5% of all
ovarian neoplasms.1 The natural biological behaviorof OGCT is unique. OGCT are characterized by clini-cally indolent course, slow-paced tumor growth andincreased propensity for late recurrence.2–4 Recurrencesof OGCT most commonly happen between 5 and 30
Received: January 28 2014.Accepted: March 31 2014.Reprint request to: Dr Ismail A. Al-Badawi, Gynecologic Oncology, Department of Obstetrics and Gynecology, MBC-52, King FaisalSpecialist Hospital and Research Centre, P.O. Box 3354, Riyadh 11211, Saudi Arabia. Email: [email protected]
bs_bs_banner
doi:10.1111/jog.12460 J. Obstet. Gynaecol. Res. Vol. 40, No. 9: 2066–2075, September 2014
2066 © 2014 The AuthorsJournal of Obstetrics and Gynaecology Research © 2014 Japan Society of Obstetrics and Gynecology
years following the initial management.5 To date, thelongest time to recurrence of an OGCT documentedin the published work is 40 years following initialtreatment.6
Prognosis of patients with recurrent OGCT largelysignifies unfavorable outcomes.7 There are no univer-sally approved guidelines for the management ofrecurrent OGCT.7,8 Complete surgical debulking – withor without adjuvant chemotherapy – is the standard ofcare management, and has been shown to yield longersurvival rates.9 The benefit of adjuvant chemotherapy isstill questionable, and the bleomycin, etoposide andcisplatin (BEP) combination has emerged as the bestactive chemotherapeutic regimen.10
Cytoreductive surgery (CRS) and hyperthermicintraperitoneal chemotherapy (HIPEC) have revealedfortunate, favorable and promising outcomes in themanagement of advanced and recurrent epithelialovarian cancers.11,12 Use of CRS and HIPEC for the man-agement of recurrent OGCT is very limited. The objec-tive of this study was to report our experience (efficacyand morbidity) with CRS and HIPEC for the manage-ment of recurrent/relapsed OGCT.
Methods
The study took place at King Faisal Specialist Hos-pital and Research Center (KFSH&RC), Riyadh, SaudiArabia – a tertiary health care institute. From Novem-ber 2010 to September 2013, all patients with recurrentOGCT managed by CRS and HIPEC were retrospec-tively analyzed. Approval of the Research AdvisoryCouncil was obtained to publish this research work.
In our institute, intraoperative administration ofHIPEC is optional. Patients were informed in detailabout the current literature, benefits and risks ofundergoing the standard treatment (CRS plus/minuspostoperative chemotherapy) or the optional treatment(CRS plus HIPEC plus/minus postoperative chemo-therapy). Afterwards, patients were requested to signa written consent regarding the desired treatmentoption.
Inclusion criteria for considering CRS and HIPECincluded: (i) age below 75 years; (ii) Karnofsky per-formance status more than 50%; (iii) satisfactory hema-tological, hepatic, coagulation, renal and electrolyteprofiles; (iv) proven diagnosis of recurrent OGCT con-firmed by imaging modality and/or intraoperativebiopsy; (v) no evidence of distant OGCT metastaticfoci to brain, lungs or bones; and (vi) signed writteninformed consent by patients.
All operations were carried out by the same sur-geons from the departments of surgical oncologyand gynecologic oncology at KFSH&RC. Undergeneral anesthesia, a midline incision extending fromthe xiphoid process to the pubic tubercle was per-formed to completely explore the abdominopelviccavity for peritoneal carcinomatosis (PC). Foci ofrecurrent OGCT were confirmed intraoperatively byanalysis of frozen section biopsies. Extent of PC wasevaluated intraoperatively by utilizing the PeritonealCancer Index (PCI).13 CRS was performed as previ-ously portrayed by Sugarbaker14 and included multiplevisceral resections directed towards optimal eradica-tion of neoplastic foci from the abdominopelvic cavity.After completion of CRS, assessment of residualtumors was determined intraoperatively using thecytoreduction completeness (CC) scores.13 Only CC-0scores were regarded as complete cytoreduction.
HIPEC was performed at the end of CRS.Abdominopelvic cavity was lavaged 15 times with 1 Lof normal saline prior to HIPEC. Two inflow drainswere positioned below hemidiaphragms whereastwo outflow drains were positioned in the pouch ofDouglas. All drains were connected to an extracorpo-real closed sterile circuit in which a 2-L perfusate wascirculated by means of two peristaltic rollup pumps(one inflow and one outflow) at a flow rate of 2 L/min.Cisplatin (50 mg/m2) and doxorubicin (15 mg/m2)were supplemented to the perfusate and allowed tocirculate in the abdominopelvic cavity for 90 min at41.0–42.2°C. The heated perfusate plus chemotherapy(41.0–42.2°C) was achieved by means of a heatexchanger connected to the sterile circuit. Intrape-ritoneal temperature was continuously checked bythermometers situated in the abdominopelvic cavity toensure maintenance of 41.0–42.2°C.
During the HIPEC procedure, hemodynamic andcardiopulmonary parameters were continuously andcarefully monitored. At the end of the HIPEC proce-dure, the abdominopelvic cavity was again lavaged15 times with 1 L of normal saline. Moreover, the lefthemidiaphragmatic drain was kept for a couple ofdays to facilitate draining of the residual perfusate.All other drains were taken out intraoperatively. Allpatients were moved to the intensive care unit for1–3 days, and afterwards transferred to wards forrecovery.
Postoperative morbidity and mortality follow-ing CRS and HIPEC were evaluated according to theClavien–Dindo classification system for postoperativecomplications.15
CRS+HIPEC for recurrent/relapsed OGCT
© 2014 The Authors 2067Journal of Obstetrics and Gynaecology Research © 2014 Japan Society of Obstetrics and Gynecology
Following CRS and HIPEC, selected patients under-went adjuvant systemic chemotherapy. Such chemo-therapeutic drugs included the BEP regimen and thecarboplatin and paclitaxel (CP) regimen. The BEPchemotherapy regimen was administered at 4-weekintervals for a total of three cycles. Conversely, the CPchemotherapy regimen was administered at 3-weekintervals for a total of four cycles.
All patients were followed up regularly. No patientwas lost during follow-up visits. During the first yearfollowing HIPEC, patients were followed up every4 months. During the second year and afterwards,patients were followed up every 6 months. Thefollow-up work-up included: routine physical exami-nation, hematological profiles (complete blood count),biochemical profiles (electrolyte, renal, bone, hepaticand coagulation), serum tumor markers (cancerantigen [CA]-125, inhibin, lactate dehydrogenase,α-fetoprotein, β-human chorionic gonadotrophin),chest X-ray, whole-body computed tomography (CT)scan or positron emission tomography (PET)–CT scan(whenever deemed necessary).
Results
A total of six patients (n = 6) with recurrent/relapsedOGCT had undergone CRS and HIPEC. The character-istics of the patients are demonstrated in Table 1.
All patients (100%) had adult OGCT histology. Atinitial presentation, four patients and two patients hadInternational Federation of Gynecology and Obstetricsstage IB and IC, respectively. Moreover, at initial pre-sentation, four patients (66.7%) were managed withsurgery alone, whereas one patient (16.7%) wasmanaged with surgery plus the BEP regimen. Onepatient (33.3%) was managed with hormonal therapy(leuprolide), which failed and was replaced withsingle-agent paclitaxel chemotherapy and failed, too.No patient had previously received radiation therapyat initial treatment.
The average age at time of CRS and HIPEC was 51years (range: 26–64 years). CRS and HIPEC were per-formed for the first OGCT recurrence in two patients(33.3%), second recurrence in two patients (33.3%),third recurrence in one patient (16.7%) and fourthrecurrence in one patient (16.7%). The average periodfrom initial surgery to first recurrence was roughly 31months (range: 2–62 months). The average period frominitial diagnosis to CRS and HIPEC was roughly 101months (range: 46–150 months). Prior to HIPEC, the
Tab
le1
Cha
ract
eris
tics
ofpa
tien
ts
Pati
ent
Age
(yea
rs)
His
tolo
gyIn
itia
lFI
GO
stag
ing
Init
ialm
anag
emen
tN
umbe
rof
rela
pses
befo
reH
IPE
C
Num
ber
ofd
ebul
king
befo
reH
IPE
C
Num
ber
ofch
emo
lines
befo
reH
IPE
C
Tim
efr
omin
itia
ld
iagn
osis
tofir
stre
curr
ence
(mon
ths)
Tim
efr
omin
itia
ld
iagn
osis
toH
IPE
C(m
onth
s)
Kar
nofs
kype
rfor
man
cest
atus
scor
ing
(%)
Surg
ery
Che
mo
Rad
ioH
orm
onal
126
Adu
ltIB
Yes
Yes
No
Yes
22
228
7190
253
Adu
ltIB
Yes
No
No
No
11
—17
119
803
62A
dult
ICYe
sN
oN
oN
o2
2—
5084
704
43A
dult
IBYe
sYe
sN
oN
o1
11
2546
805
55A
dult
ICYe
sN
oN
oN
o4
4—
215
070
664
Adu
ltIB
Yes
No
No
No
33
—62
120
70
FIG
O,I
nter
nati
onal
Fed
erat
ion
ofG
ynec
olog
yan
dO
bste
tric
s;H
IPE
C,h
yper
ther
mic
intr
aper
iton
ealc
hem
othe
rapy
.
I. A. Al-Badawi et al.
2068 © 2014 The AuthorsJournal of Obstetrics and Gynaecology Research © 2014 Japan Society of Obstetrics and Gynecology
Karnofsky performance status scores were as follows:three patients with 70%, two patients with 80%, andone patient with 90%.
Details of CRS and HIPEC are shown in Table 2. Onepatient (16.7%) had restricted recurrence in theabdomen whereas the remaining five patients (83.3%)had recurrences in both the abdomen and the pelvis.Multifocal involvement of recurrence was noted inall patients. Further, all patients (100%) underwentperitonectomy and omentectomy surgical procedures.Three patients (50%) had anterior low resections andone patient (16.7%) had diaphragmatic stripping.
The average PCI, examined intraoperatively, was 9(range: 5–12). Five patients (83.3%) achieved completecytoreduction (CC-0). The remaining patient (16.7%)achieved near complete cytoreduction (CC-1). Theaverage duration of CRS and HIPEC was 9 h (range:8–12 h). The average postoperative hospital stay was 18days (range: 12–32 days).
Details and grading of postoperative complicationsaccording to the Clavien–Dindo classification systemare shown in Table 3. No intraoperative or grade Vmorbidity occurred. Two patients (33.3%) developedlung atelectasis, which was managed by mere chestphysiotherapy (grade I). One patient (16.7%) developedurinary tract infection (grade II) and another patient(16.7%) developed pneumonia (grade II) – both ofwhich were managed only by antibiotics. One patientdeveloped splenic bed and anterior abdominal wallcollections requiring ultrasound-guided aspirationwithout the need for general anesthesia (grade III). Onepatient (16.7%) developed pulmonary embolism requir-ing intensive care unit (ICU) management (grade IV).
Details of myelosuppression (immediately) andblood product transfusion (during hospital stay)post-HIPEC are presented in Table 4. The averagewhite blood cell count was 6.02 × 109/L (range:3.45–11.31 × 109/L), red blood cell (RBC) count was3.56 × 1012/L (range: 2.22–4.17 × 1012/L), platelet countwas 141 × 109/L (range: 105–173 × 109/L) and hemo-globin was 105 g/L (range: 85–121 g/L). During hospi-tal stay, no patient experienced severe chemotherapy-induced myelosuppression requiring administrationof granulocyte colony-stimulating factor to promoterecovery of bone marrow granulocytes. Only twopatients required 1 unit of packed RBC transfusion tocorrect anemia. No patient required platelet transfu-sion. Overall, myelosuppression was well tolerated.Moreover, with respect to chemotherapy-relatedtoxicity, no patient developed doxorubicin-inducedcardiotoxicity or cisplatin-induced nephrotoxicity.
Tab
le2
Det
ails
ofC
RS
and
HIP
EC
Pati
ent
Site
ofre
curr
ence
Surg
ical
rese
ctio
ns(p
roce
dure
s)Pe
rito
neal
canc
erin
dex
scor
e
Cyt
ored
ucti
onco
mpl
eten
ess
scor
e
Dur
atio
nof
CR
San
dH
IPE
C(h
)
Hos
pita
lst
ay(d
ays)
1A
bdom
en+
pelv
isO
men
tect
omy,
umbi
lical
rese
ctio
n,sm
allb
owel
rese
ctio
n,lo
wan
teri
orre
sect
ion
120
912
2A
bdom
en+
pelv
isC
hole
cyst
ecto
my,
omen
tect
omy,
rect
osig
moi
dre
sect
ion,
smal
lbow
elre
sect
ion
50
1214
3A
bdom
enal
one
Cho
lecy
stec
tom
y,sp
lene
ctom
y,om
ente
ctom
y,ri
ght
dia
phra
gmat
icst
ripp
ing
90
920
4A
bdom
en+
pelv
isO
men
tect
omy,
part
ialp
erit
onec
tom
y,re
ctos
igm
oid
rese
ctio
n,to
tala
bdom
inal
hyst
erec
tom
yw
ith
left
salp
ingo
-oop
hore
ctom
y
90
814
5A
bdom
en+
pelv
isC
hole
cyst
ecto
my,
appe
ndec
tom
y,om
ente
ctom
y,pa
rtia
lper
iton
ecto
my,
tota
labd
omin
alhy
ster
ecto
my,
pelv
icd
isse
ctio
n
111
1016
6A
bdom
en+
pelv
isC
hole
cyst
ecto
my,
appe
ndec
tom
y,sp
lene
ctom
y,om
ente
ctom
y,pa
rtia
lper
iton
ecto
my,
peri
hepa
tic
dis
sect
ion,
pelv
icd
isse
ctio
n
70
832
CR
S,cy
tore
duct
ive
surg
ery;
HIP
EC
,hyp
erth
erm
icin
trap
erit
onea
lche
mot
hera
py.
CRS+HIPEC for recurrent/relapsed OGCT
© 2014 The Authors 2069Journal of Obstetrics and Gynaecology Research © 2014 Japan Society of Obstetrics and Gynecology
Details of postoperative therapy, follow-up andoutcome following CRS and HIPEC are presentedin Table 5. Four patients (66.67%) received adjuvantchemotherapy, whereas the remaining two patients(33.33%) received no modality of adjuvant therapy. Allpatients are alive and disease-free without proof ofrecurrence/relapse at 40, 32, 27, 24, 20 and 16 months.The average interval of follow-up after CRS and HIPECis approximately 27 months (range: 16–40 months).
Discussion
There are no universally approved guidelines for themanagement of recurrent OGCT.7,8 As a consequence,
various therapeutic approaches, such as surgicaldebulking, chemotherapy, radiotherapy, hormonaltherapy, targeted therapy and novel therapy, havebeen suggested. Some of these are associated withprolonged disease-free survival rates in isolatednumbers of case reports and small-sized retrospec-tive series, and owing to relative rarity of OGCT,it has largely been impossible to conduct prospec-tive clinical randomized trials and draw concreteconclusions.16,17
The aim of this study was to explore the feasibility,efficacy and morbidity of CRS and HIPEC for the man-agement of a retrospective series of six patients withrecurrent OGCT.
Table 3 Postoperative complications following cytoreductive surgery+hyperthermic intraperitoneal chemotherapy(according to Clavien–Dindo classification)
Patient Postoperative complication, grade Management
1 Lung atelectasis, grade I Chest physiotherapy2 Lung atelectasis, grade I Chest physiotherapy3 Splenic bed collection, grade III
Anterior abdominal wall collection, grade IIIUltrasound-guided aspiration without general anesthesiaUltrasound-guided aspiration without general anesthesia
4 Urinary tract infection, grade II Antibiotics5 Pneumonia, grade II Antibiotics6 Pulmonary embolism, grade IV Intensive care unit management
Table 4 Myelosuppression (immediately) and blood product transfusion (during hospital stay) post HIPEC
Patient Whitebloodcount109/L
Redbloodcount1012/L
Hemoglobing/L
Platelet109/L
Bloodtransfusionduring hospitalstay post HIPEC
Platelettransfusionduring hospitalstay post HIPEC
Granulocyte colony-stimulating factoradministration duringhospital stay post HIPEC
1 4.75 4.15 115 145 — — —2 6.62 4.17 107 169 — — —3 11.31 4.00 121 139 — — —4 4.76 2.22 85 105 1 unit — —5 5.23 3.73 114 115 — — —6 3.45 3.09 86 173 1 unit — —
HIPEC, hyperthermic intraperitoneal chemotherapy.
Table 5 Postoperative therapy, follow-up and outcome following CRS+HIPEC
Patient Adjuvanttherapy
Recurrence afterCRS+HIPEC
Period from CRS+HIPEC tolast follow-up (months)
Current status
1 — No 40 Alive and disease-free2 BEP No 32 Alive and disease-free3 CP No 27 Alive and disease-free4 — No 24 Alive and disease-free5 BEP No 20 Alive and disease-free6 BEP No 16 Alive and disease-free
BEP, bleomycin, etoposide, cisplatin; CP, carboplatin and paclitaxel; CRS, cytoreductive surgery; HIPEC, hyperthermic intraperitonealchemotherapy.
I. A. Al-Badawi et al.
2070 © 2014 The AuthorsJournal of Obstetrics and Gynaecology Research © 2014 Japan Society of Obstetrics and Gynecology
There is a general consensus that complete surgicaldebulking of recurrent OGCT masses – whenever tech-nically possible and regardless of the number of recur-rences – should be carried out.8,9 In our study, completecytoreduction (CC-0) was achieved in 83.3% of patientswith recurrent OGCT. Our findings mirrored anotherstudy in which CC-0 was achieved in more than 80% ofpatients with recurrent OGCT.18
At present, complete surgical resection remains thestandard of care for primary and recurrent OGCT set-tings,8,9 as residual tumor disease considerably influ-ences disease-free survival and accelerates recurrencerate.4,9,19,20
In the Multicenter Italian Trials in Ovarian Cancer 9retrospective study about recurrent OGCT,8 35 patientswith recurrent OGCT were identified. At the time ofthe first recurrence, the median disease-free survivalwas 64.5 months in patients receiving completecytoreduction and 47 months in patients receivingincomplete cytoreduction. Moreover, at the time of thefirst recurrence, although CC-0 was achieved in 30patients, 11 patients developed a second relapse after amedian period of 38 months, and four patients devel-oped a third relapse after a median period of 41months from the time of the second recurrence. The5-year survival from the time of first recurrence wasroughly 60% and 87% for patients with and withoutresidual tumor foci at successive debulking surgery,respectively.
In another study by Fotopoulou et al.,18 45 patientswith OGCT were analyzed. Eighteen patients hadprimary OGCT and CC-0 was achieved in all patients.Alternatively, 27 patients had recurrent OGCT, andCC-0 was achieved in only 23 patients (85.2%). For allpatients, during a median follow-up period of 69months (range: 4–120 months), the median disease-freesurvival and overall survival were 58 months and 63months, respectively.
Optimal surgical debulking remains the standard ofcare for primary and recurrent OGCT settings,8,9 andcytoreduction completeness of residual disease sig-nificantly influences recurrence-free survival andoverall survival.4,9,19,20 However, with subsequentrelapses, optimal (complete) surgical debulking usingmultivisceral resections may be more challengingand unfeasible, and hence one should look for othereffective intraoperative therapeutic modalities, one ofwhich is intraoperative HIPEC.
The justification for using HIPEC is primarily basedon the thermal-improved efficacious penetration, cyto-toxicity and synergism of chemotherapeutic drugs on
the malignant cells.21,22 Moreover, administrationof perioperative intraperitoneal chemotherapy offershigher local drug concentrations, lower unwanted sys-temic side-effects23 and effectively bathes the surgicalfields7 for microscopic/macroscopic residual tumorimplants, which are the most significant factors influ-encing the recurrence rates.
The above-mentioned benefits of HIPEC allow com-bination of CRS and HIPEC to emerge as a therapeuticmodality that is potentially worth testing for the man-agement of recurrent OGCT. Moreover, in line with thepromising outcomes of HIPEC in management of PCarising from recurrent gynecological11,12,24–26 and non-gynecological27 malignancies, we highly anticipate thatutilization of optimal CRS and HIPEC will also beeffective in: (i) managing recurrent OGCT with PC;(ii) reducing the risk of future repetitive recurrences;and (iii) decreasing the need for subsequent debulkingsurgeries.
The selection of chemotherapeutic drugs for HIPECshould be rationalized according to proven scientificresearch-based improved heat-enhancing effects onthe chemotherapeutic drugs. Chemotherapeuticdrugs of such nature include cisplatin,28 doxorubicin(adriamycin),29 and mitomycin C.30
The combined platinum- and doxorubicin-basedchemotherapy regimen is widely used as a commonHIPEC cytotoxic regimen in the management ofwidespread PC arising from gynecological (epithelialovarian and endometrial)24–26 and non-gynecological(mesothelioma)27 malignancies with acceptable efficacyand morbidity results. Canbay et al.31 and Gouy et al.7
have previously used platinum-based HIPEC cytotoxicagents in the management of recurrent OGCT with PC.Therefore, the use of cisplatin in our study is well ratio-nalized based on the two previous studies. Conversely,doxorubicin-based HIPEC cytotoxic agents have notbeen previously used for management of recurrentOGCT with PC. However, in our study, we decided touse doxorubicin for two reasons: (i) doxorubicin iscommonly used as a standard HIPEC cytotoxic drug inthe management of PC arising from many gynecolo-gical and non-gynecological malignancies with veryacceptable morbidity and promising results; and (ii)this HIPEC chemotherapy combination of cisplatinplus doxorubicin is expected to maximize the cytotoxiceffects with tolerable toxicity and morbidity. However,further research is needed.
The morbidity of platinum-based versusdoxorubicin-based chemotherapy has been studiedand shown to be insignificant. A recent study by
CRS+HIPEC for recurrent/relapsed OGCT
© 2014 The Authors 2071Journal of Obstetrics and Gynaecology Research © 2014 Japan Society of Obstetrics and Gynecology
Glockzin et al.32 compared the treatment-related mor-bidity in patients receiving oxaliplatin versus doxoru-bicin plus mitomycin C as HIPEC cytotoxic agents formanagement of PC. A total of 307 patients were retro-spectively analyzed. Forty patients received oxaliplatinwhereas the remaining patients received doxorubicinplus mitomycin C. According to Common Terminol-ogy Criteria for Adverse Events (version 3.0), gradeIII/IV (severe adverse events) morbidity was relativelylower in the doxorubicin plus mitomycin C treatedgroups (37.5%) as compared to the oxaliplatin treatedgroups (42.5%) without significant statistical difference(P = 0.648). This study suggests that doxorubicin toxic-ity is generally well tolerated in selected patients andfairly comparable to oxaliplatin.
Multifocal pattern of recurrence is very commonamong OGCT. The most frequently reported regions ofrecurrence (descending order of frequency) are thepelvis, liver and bowel.18,20 In our study, recurrencemultifocality was present in all patients (100%) andreflected by the number of various surgical resectionsperformed (peritonectomy, omentectomy, splenec-tomy, low anterior resections, etc.). Furthermore,abdominopelvic recurrences occurred in five patients(83.3%) whereas only recurrences confined to theabdomen occurred in one patient (6.7%). Interestingly,no patient developed hepatic recurrences in our study,as opposed to the reported 14% (n = 4/27) and 10%(n = 9/82) of patients in other studies by Fotopoulouet al.18 and Lee et al.,4 respectively. Dissection of para-aortic or pelvic lymph nodes was not performed owingto the quite low rate of retroperitoneal disease involve-ment in recurrent OGCT settings (less than 6%).33
In our study, all patients are alive and disease-freewithout proof of recurrence/relapse at 40, 32, 27, 24, 20and 16 months. The average interval of follow-up afterCRS and HIPEC is 27 months (range: 16–40 months).Our study suggests that CRS and HIPEC seem to be afeasible modality for management of recurrent OGCT.
Morbidity of CRS and HIPEC was very tolerable andacceptable. Four patients developed grade I/II mor-bidities without the need for surgical, endoscopicor radiological interventions. One patient developedsplenic bed and anterior abdominal wall collectionsrequiring ultrasound-guided aspiration without theneed for general anesthesia (grade III). One patient(16.7%) developed pulmonary embolism requiringICU management (grade IV). All complications weremanaged successfully without problematic events.No intraoperative mortality or grade V morbidityoccurred. There is a learning curve associated with this
procedure; with accumulated experience in this proce-dure, an acceptable morbidity rate can be achieved.In our tertiary care center, up to this moment, a totalof 161 HIPEC cases (over the past 5 years) were treatedby surgeons from the departments of surgical oncologyand gynecologic oncology. In particular, a total of48 HIPEC cases were treated for various malignantovarian tumors, six cases of which were OGCT with PC– presented here in this study as a sub-group analysis.
Complete surgical debulking – despite repetitiverecurrences – with or without adjuvant chemotherapyis the standard of care management and has beenshown to yield longer survival rates.8,9 Currently, thereis no powerful proof that postoperative adjuvant che-motherapy prevents further recurrences of OGCT.9,34,35
However, adjuvant chemotherapy should be em-ployed in the management of widespread disease,inoperable/unresectable metastatic disease or diseasewith suboptimal cytoreduction.36,37 The BEP combina-tion emerges as the best active chemotherapeuticregimen.10 Other chemotherapeutic regimens withdocumented usefulness include doxorubicin38 and thecisplatin, vinblastine and bleomycin protocol.37 Earlierstudies showed promising results for taxane-basedchemotherapy in management of GCT and with lesstoxicity and fewer adverse side-effects when comparedto the BEP regimen.39–41 At present, the GynecologicOncology Group is carrying out a randomized phase IIclinical trial exploring efficacy of BEP regimen versusthe combination of carboplatin and paclitaxel for man-agement of patients with newly diagnosed and chemo-naïve recurrent metastatic ovarian sex-cord stromaltumors.42 The outcomes of this important trial areanticipated to provide high-quality and powerful evi-dences regarding the best active regimens for manage-ment of the uncommon recurrent OGCT. In our study,all patients who received prior chemotherapy did notreceive adjuvant chemotherapy following CRS andHIPEC. We anticipated they would be unlikely tobenefit from further adjuvant chemotherapeuticschedules and we did not want to expose them to addi-tional unnecessary chemotherapy-related toxicities.For chemo-naïve patients (n = 4), we elected to employadjuvant BEP regimen in three patients and CPregimen in one patient.
A genuine question arises: in chemo-naïve patientswith recurrent OGCT, is HIPEC needed? No studieshave been carried out to explore this question, andthis is indeed an interesting area for future research.However, the natural clinical behavior of OGCT iscommonly featured by slow growth and increased
I. A. Al-Badawi et al.
2072 © 2014 The AuthorsJournal of Obstetrics and Gynaecology Research © 2014 Japan Society of Obstetrics and Gynecology
predisposition for late recurrences, despite completedebulking.2–4 Complete surgical cytoreduction –regardless of multiple recurrences – with or withoutpostoperative chemotherapy remains the standard ofcare and has been demonstrated to result in prolongedoverall survival rates.9 At present, there is no strongevidence supporting that postoperative adjuvant che-motherapy in chemo-naïve patients prevents furtherrecurrences of OGCT.9,34,35 Considering the above-mentioned advantages of HIPEC, we anticipatedchemo-naïve patients with multiple OGCT recurrenceswould greatly benefit from a combination therapy ofintraoperative HIPEC (optional) plus postoperativechemotherapy. No studies have been conducted on thismatter and further research is needed – a study worthyof exploration.
Postoperative radiotherapy43,44 and hormonaltherapy45,46 – despite limited efficacy and variabledisease-free survival – can be offered to patients withrefractory high-risk recurrent OGCT, localized orinoperable metastatic disease, or patients who are notcandidates for surgical debulking or chemotherapeuticintervention.
To our best knowledge, use of CRS and HIPEC formanagement of recurrent/relapsed OGCT is verylimited to two studies only.7,31 Canbay et al.31 employedCRS and HIPEC for five patients with recurrentOGCT. HIPEC was performed with 100 mg cisplatinand allowed to circulate in the abdominopelvic cavityfor 40 min at 43.0°C. Median follow-up of patients was4 years (range: 1–6 years). Among the five patients, onepatient died and another three patients were alive withno disease progression. Recently, Gouy et al.7 exploredthe feasibility of CRS and HIPEC for managementof seven patients with recurrent OGCT. Completecytoreduction was achieved in all patients. HIPEC wasperformed with oxaliplatin (460 mg/m2) or oxaliplatin(360 mg/m2) plus irinotecan (360 mg/m2) and allowedto circulate in the abdominopelvic cavity for 30 minat 41.0–43.0°C. Median follow-up of patients was 32months (range: 25–56 months). Among the sevenpatients, two were disease-free, three had relapsedwith PC and two had relapsed with liver metastases.As opposed to our study, all patients were alive anddisease-free without proof of recurrence/relapse at40, 32, 27, 24, 20 and 16 months. The average interval offollow-up after CRS and HIPEC is approximately 27months (range: 16–40 months).
Limitations to this study include the retrospectivestudy design, comparatively small sample size (caseseries of six patients), relatively short period of follow-
up, and lack of consistent therapy and control group.Such limitations make it fairly uncertain to draw con-crete and definitive conclusions.
In summary, our study suggests that CRS andHIPEC appear to be a well-tolerated and feasible treat-ment modality in management of recurrent OGCT.However, long-term follow-up of patients is manda-tory to verify efficacy of CRS and HIEPC in reducingthe risk of further recurrences of OGCT. Moreover,a larger series of patients and possibly prospectiverandomized clinical trials, along with prolongedfollow-up data and control groups, are needed to drawdefinitive conclusions regarding CRS and HIPEC forthe management of patients with recurrent OGCT.
Acknowledgment
The authors sincerely acknowledge the editorial assis-tance of Ms Evelyn Dinio.
Disclosure
None declared.
References
1. Pectasides D, Pectasides E, Psyrri A. Granulosa cell tumor ofthe ovary. Cancer Treat Rev 2008; 34: 1–12.
2. Evans AT 3rd, Gaffey TA, Malkasian GD Jr, Annegers JF.Clinicopathologic review of 118 granulosa and 82 theca celltumors. Obstet Gynecol 1980; 55: 231–238.
3. Bjorkholm E, Silfversward C. Prognostic factors in granulosa-cell tumors. Gynecol Oncol 1981; 11: 261–274.
4. Lee YK, Park NH, Kim JW, Song YS, Kang SB, Lee HP. Char-acteristics of recurrence in adult-type granulosa cell tumor.Int J Gynecol Cancer 2008; 18: 642–647.
5. Malmström HH, Hogberg T, Risberg B, Simonsen E. Granu-losa cell tumors of the ovary: Prognostic factors and outcome.Gynecol Oncol 1994; 52: 50–55.
6. East N, Alobaid A, Goffin F, Ouallouche K, Gauthier P.Granulosa cell tumour: A recurrence 40 years after initialdiagnosis. J Obstet Gynaecol Can 2005; 27: 363–364.
7. Gouy S, Uzan C, Pautier P, Lhomme C, Duvillard P, Morice P.Results of oxaliplatin-based hyperthermic intraperitonealchemotherapy in recurrent ovarian granulosa cell tumors.Eur J Obstet Gynecol Reprod Biol 2013; 170: 464–467.
8. Mangili G, Sigismondi C, Frigerio L et al. Recurrent granulosacell tumors (GCTs) of the ovary: A MITO-9 retrospectivestudy. Gynecol Oncol 2013; 130: 38–42.
9. Al-Badawi IA, Brasher PM, Ghatage P, Nation JG, SchepanskyA, Stuart GC. Postoperative chemotherapy in advancedovarian granulosa cell tumors. Int J Gynecol Cancer 2002; 12:119–123.
CRS+HIPEC for recurrent/relapsed OGCT
© 2014 The Authors 2073Journal of Obstetrics and Gynaecology Research © 2014 Japan Society of Obstetrics and Gynecology
10. Pautier P, Gutierrez-Bonnaire M, Rey A et al. Combination ofbleomycin, etoposide, and cisplatin for the treatment ofadvanced ovarian granulosa cell tumors. Int J Gynecol Cancer2008; 18: 446–452.
11. Cotte E, Glehen O, Mohamed F et al. Cytoreductive surgeryand intraperitoneal chemo-hyperthermia for chemo-resistantand recurrent advanced epithelial ovarian cancer: Prospectivestudy of 81 patients. World J Surg 2007; 31: 1813–1820.
12. Jaaback K, Johnson N. Intraperitoneal chemotherapy for theinitial management of primary epithelial ovarian cancer.Cochrane Database Syst Rev 2006; (1): CD005340.
13. Sugarbaker PH. Management of peritoneal-surface malig-nancy: The surgeon’s role. Langenbecks Arch Surg 1999; 384:576–587.
14. Sugarbaker PH. Peritonectomy procedures. Ann Surg 1995;221: 29–42.
15. Dindo D, Demartines N, Clavien PA. Classification of surgicalcomplications: A new proposal with evaluation in a cohortof 6336 patients and results of a survey. Ann Surg 2004; 240:205–213.
16. Colombo N, Parma G, Zanagnolo V, Insinga A. Managementof ovarian stromal cell tumors. J Clin Oncol 2007; 25: 2944–2951.
17. Schumer ST, Cannistra SA. Granulosa cell tumor of the ovary.J Clin Oncol 2003; 21: 1180–1189.
18. Fotopoulou C, Savvatis K, Braicu EI et al. Adult granulosa celltumors of the ovary: Tumor dissemination pattern at primaryand recurrent situation, surgical outcome. Gynecol Oncol 2010;119: 285–290.
19. Sehouli J, Drescher FS, Mustea A et al. Granulosa cell tumorof the ovary: 10 years follow-up data of 65 patients. AnticancerRes 2004; 24: 1223–1229.
20. Sun HD, Lin H, Jao MS et al. A long-term follow-up study of176 cases with adult-type ovarian granulosa cell tumors.Gynecol Oncol 2012; 124: 244–249.
21. Witkamp AJ, de Bree E, Van Goethem R, Zoetmulder FA.Rationale and techniques of intra-operative hyperthermicintraperitoneal chemotherapy. Cancer Treat Rev 2001; 27: 365–374.
22. Mohamed F, Marchettini P, Stuart OA, Urano M, SugarbakerPH. Thermal enhancement of new chemotherapeutic agentsat moderate hyperthermia. Ann Surg Oncol 2003; 10: 463–468.
23. Glehen O, Beaujard AC, Arvieux C, Huber O, Gilly FN. Peri-toneal carcinomatosis. Surgical treatment, peritonectomy andintraperitoneal chemohyperthermia. Gastroenterol Clin Biol2002; 26: 210–215. (In French).
24. Ansaloni L, Agnoletti V, Amadori A et al. Evaluation of exten-sive cytoreductive surgery and hyperthermic intraperitonealchemotherapy (HIPEC) in patients with advanced epithelialovarian cancer. Int J Gynecol Cancer 2012; 22: 778–785.
25. Deraco M, Kusamura S, Virzì S et al. Cytoreductive surgeryand hyperthermic intraperitoneal chemotherapy as upfronttherapy for advanced epithelial ovarian cancer: Multi-institutional phase-II trial. Gynecol Oncol 2011; 122: 215–220.
26. Jérôme D, Mariangela D, Mélanie F et al. Cytoreductivesurgery with hyperthermic intraperitoneal chemotherapy forthe treatment of endometrial cancer with peritoneal carcino-matosis. Eur J Obstet Gynecol Reprod Biol 2014; 172: 111–114.
27. Baratti D, Kusamura S, Cabras AD, Bertulli R, Hutanu I,Deraco M. Diffuse malignant peritoneal mesothelioma: Long-
term survival with complete cytoreductive surgery followedby hyperthermic intraperitoneal chemotherapy (HIPEC). EurJ Cancer 2013; 49: 3140–3148.
28. Rietbroek RC, van de Vaart PJ, Haveman J et al. Hyperthermiaenhances the cytotoxicity and platinum-DNA adduct forma-tion of lobaplatin and oxaliplatin in cultured SW 1573 cells.J Cancer Res Clin Oncol 1997; 123: 6–12.
29. Herman TS, Henle KJ, Nagle WA, Moss AJ, Monson TP.Effect of step-down heating on the cytotoxicity of adriamycin,bleomycin, and cis-diamminedichloroplatinum. Cancer Res1984; 44: 1823–1826.
30. Teicher BA, Kowal CD, Kennedy KA, Sartorelli AC.Enhancement by hyperthermia of the in vitro cytotoxicity ofmitomycin C toward hypoxic tumor cells. Cancer Res 1981; 41:1096–1099.
31. Canbay E, Ishibashi H, Sako S, Miyata R, Nishino E,Yonemura Y. Management of peritoneal dissemination ofrecurrences granulosa cell tumor of the ovary. Gan To KagakuRyoho 2012; 39: 2435–2437.
32. Glockzin G, von Breitenbuch P, Schlitt HJ, Piso P. Treatment-related morbidity and toxicity of CRS and oxaliplatin-basedHIPEC compared to a mitomycin and doxorubicin-basedHIPEC protocol in patients with peritoneal carcinomatosis: Amatched-pair analysis. J Surg Oncol 2013; 107: 574–578.
33. Abu-Rustum NR, Restivo A, Ivy J et al. Retroperitoneal nodalmetastasis in primary and recurrent granulosa cell tumors ofthe ovary. Gynecol Oncol 2006; 103: 31–34.
34. Gershenson DM, Copeland LJ, Kavanagh JJ, Stringer CA,Saul PB, Wharton JT. Treatment of metastatic stromal tumorsof the ovary with cisplatin, doxorubicin, and cyclophospha-mide. Obstet Gynecol 1987; 70: 765–769.
35. Uygun K, Aydiner A, Saip P et al. Clinical parameters andtreatment results in recurrent granulosa cell tumor of theovary. Gynecol Oncol 2003; 88: 400–403.
36. Gershenson DM, Morris M, Burke TW, Levenback C,Matthews CM, Wharton JT. Treatment of poor-prognosis sexcord-stromal tumors of the ovary with the combination ofbleomycin, etoposide, and cisplatin. Obstet Gynecol 1996; 87:527–531.
37. Zambetti M, Escobedo A, Pilotti S, De Palo G. Cis-platinum/vinblastine/bleomycin combination chemotherapy inadvanced or recurrent granulosa cell tumors of the ovary.Gynecol Oncol 1990; 36: 317–320.
38. Disaia P, Saltz A, Kagan AR, Rich W. A temporary responseof recurrent granulosa cell tumor to adriamycin. ObstetGynecol 1978; 52: 355–358.
39. Brown J, Shvartsman HS, Deavers MT et al. The activity oftaxanes compared with bleomycin, etoposide, and cisplatin inthe treatment of sex cord-stromal ovarian tumors. GynecolOncol 2005; 97: 489–496.
40. Shavit T, Bruchim I, Ben-Harim Z, Fishman A. Successfulresponse to docetaxel treatment in recurrent ovarian granu-losa cell tumor: A case report. Eur J Gynaecol Oncol 2012; 33:419–420.
41. Tresukosol D, Kudelka AP, Edwards CL, Charnsangavej C,Narboni N, Kavanagh JJ. Recurrent ovarian granulosa celltumor: A case report of a dramatic response to Taxol. Int JGynecol Cancer 1995; 5: 156–159.
42. Gershenson DM. Current advances in the managementof malignant germ cell and sex cord-stromal tumors of theovary. Gynecol Oncol 2012; 125: 515–517.
I. A. Al-Badawi et al.
2074 © 2014 The AuthorsJournal of Obstetrics and Gynaecology Research © 2014 Japan Society of Obstetrics and Gynecology
43. Wolf JK, Mullen J, Eifel PJ, Burke TW, Levenback C,Gershenson DM. Radiation treatment of advanced or recur-rent granulosa cell tumor of the ovary. Gynecol Oncol 1999; 73:35–41.
44. Hauspy J, Beiner ME, Harley I et al. Role of adjuvant radio-therapy in granulosa cell tumors of the ovary. Int J RadiatOncol Biol Phys 2011; 79: 770–774.
45. Kim HJ, Lee SC, Bae SB et al. GnRH agonist therapy in apatient with recurrent ovarian granulosa cell tumors. J KoreanMed Sci 2009; 24: 535–538.
46. Ameryckx L, Fatemi HM, De Sutter P, Amy JJ. GnRH anta-gonist in the adjuvant treatment of a recurrent ovariangranulosa cell tumor: A case report. Gynecol Oncol 2005; 99:764–766.
CRS+HIPEC for recurrent/relapsed OGCT
© 2014 The Authors 2075Journal of Obstetrics and Gynaecology Research © 2014 Japan Society of Obstetrics and Gynecology
ORIGINAL ARTICLE
Ovarian surface epithelium receptors
during pregnancy and estrus cycle of rats
with emphasis on steroids and gonadotropin
fluctuation
Salina Y. Saddick *
Biological Studies Dept., Faculty of Science, King Abdulaziz University, PO Box 42671, Jeddah 21551, Saudi Arabia
Received 31 December 2013; revised 8 February 2014; accepted 20 February 2014Available online 13 March 2014
KEYWORD
OSE pregnancy rat steroid
receptors gonadotropins
Abstract The present study is designed to demonstrate the ovarian surface epithelial cells’ (OSE)
estrogen receptor a (ERa) and progesterone receptor (PR) during pregnancy and estrous cycle in
rat. Moreover, determination of the levels of plasma progesterone, estradiol, FSH and LH was also
made. The levels of plasma progesterone, estradiol, FSH and LH concentrations were determined on
days 7 (n= 5), 14 (n= 5), and 21 (n= 5) of pregnancy in three groups of rats and during the estrous
cycle (n = 5) using an ELISA kit. Immunohistochemical method for PR and ERa expressions was
also made on the ovary. During pregnancy, FSH and LH remained low except at term when LH lev-
els began to increase from 16 ng/ml to 47 ng/ml. Progesterone levels significantly exceeded estradiol
values in all pregnant rats with a peak value of 202 ng/ml on day 14. Elevated progesterone levels
were associated negatively with LH and estradiol levels during pregnancy. The levels of estradiol
surged significantly on day 21. Immunohistochemistry of the ovary showed low levels of OSE cells
staining positive for ERa expression. ERa positive cells were absent on day 7 and 14 of pregnancy,
only day 21 recorded a very low percentage of immunostaining (0.5%) within the nuclei of OSE cells.
On the contrary, immunostaining of PR was not observed within the nuclei of OSE cells in all groups
of study. In conclusion, these results may suggest that the progesterone effect during pregnancy
seems to be overriding the positive effect of estrogens on OSE cells. High progesterone levels may
have a direct negative effect on gonadotropin production and thereby it might inhibit events leading
to both follicular development and OSE proliferation. Understanding the factors affecting OSE pro-
liferation may help elucidating the mechanism(s) of assisted diseases such as ovarian cancer.ª 2014 Production and hosting by Elsevier B.V. on behalf of King Saud University.
1. Introduction
The mammalian ovary is a highly dynamic organ with dramat-ically changing environment, it is covered by a single layer of
epithelial tissue; the ovarian surface epithelium (OSE). OSE
* Tel.: +966 556942202; fax: +966 26861898.
E-mail address: [email protected]
Peer review under responsibility of King Saud University.
Production and hosting by Elsevier
Saudi Journal of Biological Sciences (2014) 21, 232–237
King Saud University
Saudi Journal of Biological Sciences
www.ksu.edu.sawww.sciencedirect.com
1319-562X ª 2014 Production and hosting by Elsevier B.V. on behalf of King Saud University.
http://dx.doi.org/10.1016/j.sjbs.2014.02.004
cells derived from mesodermal epithelium of gonadal ridges,are flattened mesothelium of peritoneum separated fromunderneath ovarian stroma by a basement membrane and a
connective tissue layer, tunica albuginea (Leung and Choi,2007). OSE occupies the entire ovarian lining, and varies inmorphology from simple squamous to cuboidal to low pseudo
stratified columnar (Auersperg et al., 2001). Despite theirinconspicuous appearance, OSE participates in transportingand exchanging nutrients and other bioactive metabolites from
the peritoneal cavity and ovary. At pre-ovulation, OSE inproximity to rupture site undergoes apoptotic cell death andthe wound caused by ovulation is repaired by highly prolifer-ating OSE cells from surroundings of the ruptured follicle
(Murdoch and Van Kirk, 2002). OSE cell proliferation also oc-curs at post-ovulatory phase especially in post-menopausalwoman due to ageing of ovary; epithelial line invaginates, pro-
ducing crypts and glands, which eventually develop into cystswithin the stromal compartment (Auersperg, 2013). It has beenhypothesized that repeated cycles of ovulation-induced trauma
and repair of the OSE at the site of ovulation contribute tomalignancy, it makes the OSE susceptible to mitogenic factorsand other genotoxic radicals. There are several studies that
suggested that menstrual cycle can affect tumor growththrough the high levels of reproductive hormones (Woodet al., 2005; Wood and Hrushesky, 2005). A host of tumorige-netic factors, viz. cytokeratin, desmoplakin, transforming
growth factor-a (TGF-a) and receptors for estrogen, proges-terone and epidermal growth factor (EGF) are expressed byOSE (Li et al., 2003).
Approximately 90% of human ovarian cancers (OCs) orig-inate from OSE. Epithelial ovarian cancer (EOC) is the fifthleading cause of cancer deaths and the third among gynecolog-
ical cancer worldwide (Ferlay et al., 2010). It is well knownthat pregnancy and use of oral contraceptives can reduce therisk of EOC development (Tiedemann, 2000; Clow et al.,
2002). Additionally, it has been reported that EOC is absentin animal species where seasonal ovulation and multiple preg-nancies take place. Suppression of ovulation and steroid pro-duction by the pre-ovulatory follicles are main significant
alterations that occur within the ovary during pregnancy.OC is highly associated with high exposure to gonadotropins.This is evident because a research on the use of ovulation
inducing treatments by infertile women showed a direct pro-portionality between the number of OC cases and the ovula-tion induction use (Rizzuto et al., 2013). At menopause, it is
evident that gonadotropins are released in high quantitiesand hence causing the high risk of cancer in post-menopausefemales (Leung and Choi, 2007). Both the follicle stimulating(FSH) and luteinizing hormones (LH) are very important tro-
phic factors especially for the survival and proliferation of thefollicular somatic cells and aiding in the cyclic recruitment ofthe antral follicles (Zhang et al., 2013). On the other hand, the-
ory states that OC is triggered by an increase of estrogen pro-duction that triggers OSE proliferation (Baby and Bartlewski,2011). Steroid hormones have been implicated in the etiology
of some EOC. Expression of estrogen (ER), progesterone(PR) and androgen (AR) receptors has been reported in hu-man EOC (Akahira et al., 2002; Hamilton et al., 1981).
According to Fortune (1994) senescent ovary, like the oneduring pregnancy or postnatal period, maintains oocytes in‘‘resting’’ or primordial follicles (Fortune, 1994). Growingfollicles either ovulate to release oocytes for fertilization, or
degenerate through into atresia. Differentiated follicles alsoprovide steroidal hormones to maintain the ovarian cycleand prepare uterus for implant, and corpus luteum (CL) pro-
duces hormones for establishing and maintaining pregnancy.Gonadotropins and estrogens are vital for maintaining ovariancycle until antral stage just before ovulation.
The objective of this study is to determine hormonal fluctu-ation namely steroids and gonadotropins during pregnancy. Asecond purpose of our investigations is to examine the expres-
sion of steroid receptors in OSE cells at different stages ofpregnancy.
2. Materials and methods
2.1. Animals
Adult female rats weighing 250–300 g were obtained from thecentral animal house at the King Fahd Medical and ResearchCentre (KFMRC) and maintained under controlled
conditions of temperature (23–27 �C) and lighting (lights on05:00–19:00 h). Vaginal smears were examined daily and onthe day of estrous. Pregnancy was induced in the animals by
transferring 1 female rat to the cage of a single male rat andleft overnight. Mating was verified the next morning by thepresence of vaginal plug in the vaginal smear; this was
designated as Day 1 of pregnancy. Rats were bled 1 ml byretro-orbital plexus puncture under light diethyl-etheranesthesia. Blood samples were centrifuged immediately after
collection. Plasma was separated and stored at �20 �C untilassayed for, estradiol, progesterone, LH and FSH hormones.All experimental procedures were approved by the animalethics committee.
2.2. Sample collection
Ovaries from estrus and pregnant animals were collected. The
ovaries were gently handled during collection to avoid cell lossby excessive handling as the OSE is a very fragile layer. Theovaries were classified according to the reproductive status of
the rats: ovaries from estrus rats (n= 5) and ovaries frompregnant rats (n = 15). This last group was further sub-dividedinto three groups based on the stage of pregnancy, the threegroups were as follows; ovaries in early gestation (day 7)
(n= 5) and ovaries in mid gestation (day 14) (n= 5) and lategestation (day 21) (n = 5). Pregnancy was confirmed byabdominal palpation. Full term pregnancies range from
21–22 days. Ovarian tissue samples from pregnant animalsand estrous animals were collected alongside material from.
2.3. Histological methods
Ovarian tissues were fixed in Bouin’s, dehydrated in gradedalcohols and embedded in paraffin, and sectioned serially at
6 lm and mounted on Super Frost plus microscope slides(VWR International Ltd., Leicestershire, UK) and left to dryovernight at 37 �C.
2.4. PR and ERa immunohisthemistry
The effect of the reproductive stage (estrous or pregnancy) onPR and ERa expressions within OSE cells was investigated
Ovarian surface epithelium receptors during pregnancy and estrus cycle of rats 233
using immunohisthemistry (IHC) methods. Sections werede-waxed in xylene, and hydrated in descending grades of alco-hol to distilled water, and followed by double washing with
phosphate buffer saline (PBS). A heat-induced epitope retrie-val method was performed (antigen retrieval) by microwaving(high power at 800 W) the sections in a 0.01 M citrate buffer
(pH 6.0). Endogenous peroxidase activity was inactivatedthrough incubation with 3 percent hydrogen peroxide in meth-anol. Sections were incubated with monoclonal mouse anti-hu-
man estrogen receptor a clone 1D5 (Dako Autostainer/Autostainer Plus-Code IS657) to detect ER and with monoclo-nal mouse anti-human progesterone receptor clone PgR 636(Dako/FLEX Code IR068) to detect PR. Sections were
incubated overnight with the primary antibody at 4 �C. TheNovoLink polymer detection system (RE7 150-K, NovastraLaboratories Ltd., UK) was used for visualization. Sections
were incubated with protein blk for 5 min, followed by anovernight incubation with the primary antibody (1:2000 at4 �C). After being washed with PBS, a post-primary blk was
performed for 30 min. Sections were incubated with the Nov-oLink polymer solution for 30 min, and then washed withPBS. Peroxidase activity was developed using a DAB solution
for 5 min. Sections were counterstained using hematoxylinstain. Optimization of antibody was necessary to enhance thepermeability to the tissue. Antibody incubation time and tem-perature were adjusted to overnight at 4 �C instead of 60 min
at 37 �C. As a negative control, the blking solution was lefton the sections instead of the primary antibody to insure thepurity of the staining procedure. Samples of breast cancer were
used as a positive control. At each run of IHC, a set of slidesincluding sections from all groups of study plus negative andpositive control were run together under the same experimen-
tal condition. Two sections from each ovary were selectedrandomly for the analysis.
2.5. Steroids and gonadotropins determination
Concentrations of progesterone, estradiol, FSH and LH inperipheral plasma were determined in all participants usingELISA kits. For estradiol determination, the Estradiol
ELISA kit (catalog no. 582251; Cayman Chemical Co.,Ann Arbor, MI) was used. For progesterone determination,the DHT ELISA kit form BioVendor (catalog no. RCAN-
DHT-280R) was used. For FSH determination, the RATFSH ELISA kit form BioVendor (catalog no. RSHAKRFS-010R) was used. For LH determination, the RAT LH ELISA
kit form BioVendor (catalog no. RSHAKRLH-010SR) wasused. For all hormones the standard protocols provided by themanufacturers were followed using undiluted, 1:50, or 1:150dilutions.
2.6. Statistics
Student’s t test was used to test the significance of difference
between two means. Analysis of variance was carried outwhen more than two means were compared and the signifi-cance of the difference between means was determined by
Duncan’s multiple range tests. Differences were consideredto be significant at P 6 0.05. Analysis was done usingMinitab version 15.
3. Results
3.1. Estradiol and Progesterone concentrations during theestrous cycle and pregnancy
During a rat’s estrous cycle and pregnancy period, levels of
estradiol and progesterone fluctuated as shown in Fig. 1(a and b). The concentrations of estradiol on day 7 of preg-nancy (62 pg/ml) were similar to the levels measured at estrous
(53 pg/ml). Then on day 14 of pregnancy a significant increasein the concentration of estradiol was observed (152 pg/ml).Further increased was noticed on day 21 (288 pg/ml), the levelof estradiol surged significantly and progressively up to term.
The estradiol peak during pregnancy was higher than its peakduring estrous (Fig. 1-a).
As measured by the radioimmunoassay technique, proges-
terone concentrations during all stages of pregnancy signifi-cantly exceeded estrous values. On day 7 of pregnancy,progesterone values increased sharply to 131 ng/ml, highest
levels were recorded during the second week of pregnancy onday 14 (202 ng/ml) followed by a gradual decrease in the levelson day 21 (124 ng/ml) but values remain above estrous values(5 ng/ml). The concentrations of progesterone decrease gradu-
ally until parturition (Fig. 1-b).
0
50
100
150
200
250
300
350
400
Estrous Day 7 Day 14 Day 21
Estr
adio
l pg
/ml)
Stage of ovarian cycle
(a)
Estrous
Day 7
Day 14
Day 21
0
50
100
150
200
250
Estrous Day 7 Day 14 Day 21
Prog
este
rone
(ng/
ml)
Stage of ovarian cycle
(b)
Estrous
Day 7
Day 14
Day 21
Figure 1 Histogram representing plasma concentrations of
estradiol (a) and progesterone (b) at estrous cycle (n= 5), day 7
pregnancy (n= 5), day 14 pregnancy (n= 5) and day 21
pregnancy (n= 5). Different letters above the bars indicate
statistically differences between groups among groups
(P 6 0.05). Values are means of concentrations ± MSE.
234 S.Y. Saddick
3.2. FSH and LH concentrations during the estrous cycle andpregnancy
FSH and LH levels remained low throughout pregnancy com-pared to the estrous values (117 and 86 ng/ml) respectively. On
day 7 of pregnancy, FSH concentration decreased significantly(78 ng/ml) onward, the levels remained steady low as recordedon day 14 (81 ng/ml). A slight increase in FSH concentrationwas recorded on day 21 (112 ng/ml) (Fig. 2-a).
During pregnancy, LH levels were significantly lower thanthe estrous levels (86 ng/ml). On day 7 of pregnancy, the valueof LH hormone was 46 ng/ml followed with sharp decreasing
on day 14 (16 ng/ml). An unexpected rise in LH value wasreported on day 21 (47 ng/ml) which was continuous untilparturition (Fig. 2-b).
3.3. Expression of Estrogen a receptor and progesterone
receptor
The ovary of estrous rat showed low levels of ERa expression,3.5% of OSE nuclei were stained with DAB brown color(Fig. 3). Generally the percentage of OSE cells staining positivefor ERa was relatively low in the pregnant rats. ERa positive
cells were absent on day 7 and 14 of pregnancy, only day 21recorded a very low percentage of immunostaining (0.5%)within the nuclei of OSE cells (Fig. 4). On the contrary,
immunostaining of PR was not observed within the nuclei ofOSE cells in all groups of study, PR was absent in the ratOSE cells (Fig. 5).
4. Discussion
Multiple pregnancies play a significant role in reducing
chances of OC. Recent research shows that breastfeedingand pregnancy reduces the risk of getting OC. Pregnancy isone of the factors which suppress ovulation process. Ceaseless
ovulation hypothesis insinuates that recurring monthlymenstrual sequence of epithelium trauma and successivereplacement of worn out tissues after ovulation increases the
exposure of the OSE to various risk factors including geneticinstabilities (Fathalla, 1971). On the other hand, theory statesthat OC is triggered by an increase of estrogen production and
the proliferation of ovarian surface epithelium (Baby andBartlewski, 2011).
The physiology of OSE cells is of interest since the majorityof female OCs originates in these cells (Vanderhyden et al.,
2003). Several studies have investigated the dynamic of follic-ular growth at pregnancy but none had focused on the roleof pregnancy as a protective factor against OC. The current
study was designed to determine hormonal fluctuation namelysteroids and gonadotropins during pregnancy and to examinethe expression of steroid receptors in OSE cells at different
stages of pregnancy.During pregnancy several factors, notably high steroidal
hormones produced in feto-placental unit and CL, may retardfollicle development, and suppress OSE proliferation. Accord-
ing to a recent ‘‘gonadotropin theory’’, the expression of LHand FSH receptors on the surface of OSE cells is a direct re-sponse of gonadotropins (Choi et al., 2007). Most likely the
hormone-stimulated receptors trigger the localized prolifera-tion events. Besides, these receptors also initiate the oncogenicpathways. OSE cell differentiation was investigated in the
presence of exogenously supplemented estrogens and proges-terone (Ho, 2003; Laviolette et al., 2010). In the current study,determination of the levels of steroids and gonadotropins in
the plasma of pregnant rats has been made. The pattern of
0
20
40
60
80
100
120
140
Estrous Day 7 Day 14 Day 21
FSH
(ng/
ml)
Stage of ovarian cycle
Estrous
Day 7
Day 14
Day 21
0
10
20
30
40
50
60
70
80
90
100
Estrus Day 7 Day 14 Day 21
LH (n
g/m
l)
Stage of ovarian cycle
Estrus
Day 7
Day 14
Day 21
(a)
(b)
Figure 2 Histogram representing plasma concentrations of FSH
(a) and LH (b) at estrous cycle (n= 5), day 7 pregnancy (n= 5),
day 14 pregnancy (n= 5) and day 21 pregnancy (n= 5). Different
letters above the bars indicate statistically differences between
groups among groups (P 6 0.05). Values are means of
concentrations ±MSE.
0
0.5
1
1.5
2
2.5
3
3.5
4
Estrous Day 7 Day 14 Day 21
Perc
enta
ge o
f ER α
expr
essi
on %
Stage of the ovarian cycle
Estrous
Day 7
Day 14
Day 21
Figure 3 Histogram representing percentage of ER at estrous
cycle (n= 5), day 7 pregnancy (n= 5), day 14 pregnancy (n= 5)
and day 21 pregnancy (n= 5). Different letters above the bars
indicate statistically differences between groups among groups
(P 6 0.05). Values are means of concentrations ± MSE.
Ovarian surface epithelium receptors during pregnancy and estrus cycle of rats 235
hormone fluctuation during pregnancy reported here is consis-
tent with previous studies (Kallen, 2004).In this study, estradiol values were generally low except at
the end of pregnancy. At the first week of pregnancy on day
7, the levels of estradiol rose then followed by a steady declineuntil the middle stage of pregnancy and then continued to risegradually toward the end of term.
In this study, FSH and LH levels remained low throughoutthe pregnancy period except for some instances like on day 21.Low concentrations of plasma LH during pregnancy may con-tribute to the suppression of follicular maturation during this
time. Gonadotropins like LH, FSH and human chorionicgonadotropin (hCG) are the other hormones regulating OSEproliferation and transformation into cancerous cells (Trabert
et al., 2013; Yang et al., 2013). The levels of FSH and LHwhich are required for ovulation may be suppressed by thehigh concentration of progesterone. FSH is considered to be
the most important hormone controlling folliculogenesis,especially at later stages of follicle growth (Chaffin andVandevoort, 2013). According to theory, high level of FSH
and LH is known to wear the epithelium, and hence responsi-ble for OC. Several studies revealed that gonadotropins treat-ment may increase OSE proliferation in different animalmodels (Hilliard et al., 2013; Stewart et al., 2004). The results
of this study suggest that pregnancy may protect from OC bydecreasing the levels of gonadotropins throughout pregnancyand therefore reduce the exposure of OSE cells to FSH and
LH. However, the role of gonadotropins on OSE cells is notfully characterized.
In conclusion, these results may suggest that progesterone
effect during pregnancy seems to be overriding the positiveeffect of estrogens on OSE cells. High progesterone levelsmay have a direct negative effect on gonadotropin production
and thereby it might inhibit events leading to both folliculardevelopment and OSE proliferation. Understanding the fac-tors affecting OSE proliferation may help elucidating the
mechanism(s) of assisted diseases such as ovarian cancer.
Acknowledgment
This project was funded by the Deanship of Scientific Research(DSR), King Abdulaziz University, Jeddah, under grant No.(504/247/1433). The authors, therefore, acknowledge with
thanks DSR technical and financial support.
References
Akahira, J., Suzuki, T., Ito, K., Kaneko, C., Darnel, A.D., Moriya, T.,
Okamura, K., Yaegashi, N., Sasano, H., 2002. Differential
expression of progesterone receptor isoforms A and B in the
normal ovary, and in benign, borderline, and malignant ovarian
tumors. Jpn. J. Cancer Res. 93, 807–815.
Auersperg, N., 2013. Ovarian surface epithelium as a source of ovarian
cancers: unwarranted speculation or evidence-based hypothesis?
Gynecol. Oncol. 130, 246–251.
Auersperg, N., Wong, A.S., Choi, K.C., Kang, S.K., Leung, P.C.,
2001. Ovarian surface epithelium: biology, endocrinology, and
pathology. Endocr. Rev. 22, 255–288.
m0 µ525 µm(a) (b)
Figure 4 Photomicrographs showing ERa expression within OSE cells at estrous (a) and on day 14 of pregnancy. (b) Red arrow shows a
positive staining (brown color).
m0 µ5m0 µ5(a) (b)
Figure 5 Photomicrographs showing PR expression within OSE cells at estrous (a) and on day 14 of pregnancy. (b) Red arrow shows a
negative staining (blue color).
236 S.Y. Saddick
Baby, T.E., Bartlewski, P.M., 2011. Circulating concentrations of
ovarian steroids and follicle-stimulating hormone (FSH) in ewes
with 3 or 4 waves of antral follicle emergence per estrous cycle.
Reprod. Biol. 11, 19–36.
Chaffin, C.L., Vandevoort, C.A., 2013. Follicle growth, ovulation, and
luteal formation in primates and rodents: a comparative perspec-
tive. Exp. Biol. Med. (Maywood.) 238, 539–548.
Choi, J.H., Wong, A.S., Huang, H.F., Leung, P.C., 2007. Gonado-
tropins and ovarian cancer. Endocr. Rev. 28, 440–461.
Clow, O.L., Hurst, P.R., Fleming, J.S., 2002. Changes in the mouse
ovarian surface epithelium with age and ovulation number. Mol.
Cell Endocrinol. 191, 105–111.
Fathalla, M.F., 1971. Incessant ovulation–a factor in ovarian neopla-
sia? Lancet 2, 163.
Ferlay, J., Shin, H.R., Bray, F., Forman, D., Mathers, C., Parkin,
D.M., 2010. Estimates of worldwide burden of cancer in 2008:
GLOBOCAN 2008. Int. J. Cancer 127, 2893–2917.
Fortune, J.E., 1994. Ovarian follicular growth and development in
mammals. Biol. Reprod. 50, 225–232.
Hamilton, T.C., Davies, P., Griffiths, K., 1981. Androgen and
oestrogen binding in cytosols of human ovarian tumours. J.
Endocrinol. 90, 421–431.
Hilliard, T.S., Modi, D.A., Burdette, J.E., 2013. Gonadotropins
activate oncogenic pathways to enhance proliferation in normal
mouse ovarian surface epithelium. Int. J. Mol. Sci. 14, 4762–4782.
Ho, S.M., 2003. Estrogen, progesterone and epithelial ovarian cancer.
Reprod. Biol. Endocrinol. 1, 73.
Kallen, C.B., 2004. Steroid hormone synthesis in pregnancy. Obstet.
Gynecol. Clin. North Am. 31, 795–816, x.
Laviolette, L.A., Garson, K., Macdonald, E.A., Senterman, M.K.,
Courville, K., Crane, C.A., Vanderhyden, B.C., 2010. 17beta-
estradiol accelerates tumor onset and decreases survival in a
transgenic mouse model of ovarian cancer. Endocrinology 151,
929–938.
Leung, P.C., Choi, J.H., 2007. Endocrine signaling in ovarian surface
epithelium and cancer. Hum. Reprod. Update 13, 143–162.
Li, A.J., Baldwin, R.L., Karlan, B.Y., 2003. Estrogen and progester-
one receptor subtype expression in normal and malignant ovarian
epithelial cell cultures. Am. J. Obstet. Gynecol. 189, 22–27.
Murdoch, W.J., Van Kirk, E.A., 2002. Steroid hormonal regulation of
proliferative, p53 tumor suppressor, and apoptotic responses of
sheep ovarian surface epithelial cells. Mol. Cell Endocrinol. 186,
61–67.
Rizzuto, I., Behrens, R.F., Smith, L.A., 2013. Risk of ovarian cancer
in women treated with ovarian stimulating drugs for infertility.
Cochrane Database Syst. Rev. 8, CD008215.
Stewart, S.L., Querec, T.D., Gruver, B.N., O’Hare, B., Babb, J.S.,
Patriotis, C., 2004. Gonadotropin and steroid hormones stimulate
proliferation of the rat ovarian surface epithelium. J. Cell Physiol.
198, 119–124.
Tiedemann, D., 2000. Oncology today: ovarian cancer. RN 63, 36–41.
Trabert, B., Lamb, E.J., Scoccia, B., Moghissi, K.S., Westhoff, C.L.,
Niwa, S., Brinton, L.A., 2013. Ovulation-inducing drugs and
ovarian cancer risk: results from an extended follow-up of a large
US infertility cohort. Fertil. Steril. 100, 1660–1666.
Vanderhyden, B.C., Shaw, T.J., Ethier, J.F., 2003. Animal models of
ovarian cancer. Reprod. Biol. Endocrinol. 1, 67.
Wood, P.A., Bove, K., You, S., Chambers, A., Hrushesky, W.J., 2005.
Cancer growth and spread are saltatory and phase-locked to the
reproductive cycle through mediators of angiogenesis. Mol. Cancer
Ther. 4, 10651075.
Wood, P.A., Hrushesky, W.J., 2005. Sex cycle modulates cancer
growth. Breast Cancer Res. Treat. 91, 95–102.
Yang, H.P., Anderson, W.F., Rosenberg, P.S., Trabert, B., Gierach,
G.L., Wentzensen, N., Cronin, K.A., Sherman, M.E., 2013.
Ovarian cancer incidence trends in relation to changing patterns
of menopausal hormone therapy use in the United States. J. Clin.
Oncol. 31, 2146–2151.
Zhang, C., Guo, L., Zhu, B., Feng, Y., Yu, S., An, N., Wang, X., 2013.
Effects of 3,5,30-Triiodothyronine (T3) and Follicle Stimulating
Hormone on Apoptosis and Proliferation of Rat Ovarian Gran-
ulosa Cells. Chin. J. Physiol. 56.
Ovarian surface epithelium receptors during pregnancy and estrus cycle of rats 237
Journal of Contemporary Brachytherapy (2014/volume 6/number 4)
Clinical InvestigationsOriginal paper
Single versus multichannel applicator in high-dose-rate vaginal brachytherapy optimized by inverse treatment planningYasir A. Bahadur, MD1, Camelia Constantinescu, PhD2, Ashraf H. Hassouna, MD3,4, Maha M. Eltaher, MD1,4, Noor M. Ghassal, BSc2, Nesreen A. Awad, MD1,4
1Department of Radiology, King Abdulaziz University Hospital, Jeddah, Saudi Arabia, 2Department of Bio-Medical Physics, King Faisal Specialist Hospital & Research Center, Jeddah, Saudi Arabia, 3Department of Oncology, King Faisal Specialist Hospital & Research Center, Jeddah, Saudi Arabia, 4Department of Radiation Oncology, National Cancer Institute, Cairo University, Egypt
AbstractPurpose: To retrospectively compare the potential dosimetric advantages of a multichannel vaginal applicator vs.
a single channel one in intracavitary vaginal high-dose-rate (HDR) brachytherapy after hysterectomy, and evaluate the dosimetric advantage of fractional re-planning.
Material and methods: We randomly selected 12 patients with endometrial carcinoma, who received adjuvant vag-inal cuff HDR brachytherapy using a multichannel applicator. For each brachytherapy fraction, two inverse treatment plans (for central channel and multichannel loadings) were performed and compared. The advantage of fractional re-planning was also investigated.
Results: Dose-volume-histogram (DVH) analysis showed limited, but statistically significant difference (p = 0.007) regarding clinical-target-volume dose coverage between single and multichannel approaches. For the organs-at-risk rectum and bladder, the use of multichannel applicator demonstrated a noticeable dose reduction, when compared to single channel, but statistically significant for rectum only (p = 0.0001). For D2cc of rectum, an average fractional dose of 6.1 ± 0.7 Gy resulted for single channel vs. 5.1 ± 0.6 Gy for multichannel. For D2cc of bladder, an average fractional dose of 5 ± 0.9 Gy occurred for single channel vs. 4.9 ± 0.8 Gy for multichannel. The dosimetric benefit of fractional re-plan-ning was demonstrated: DVH analysis showed large, but not statistically significant differences between first fraction plan and fractional re-planning, due to large inter-fraction variations for rectum and bladder positioning and filling.
Conclusions: Vaginal HDR brachytherapy using a multichannel vaginal applicator and inverse planning provides dosimetric advantages over single channel cylinder, by reducing the dose to organs at risk without compromising the target volume coverage, but at the expense of an increased vaginal mucosa dose. Due to large inter-fraction dose vari-ations, we recommend individual fraction treatment plan optimization.
J Contemp Brachytherapy 2014; 6, 4: 362–370 DOI: 10.5114/jcb.2014.47816
Key words: endometrial cancer, inverse planning, multichannel applicator, vaginal brachytherapy.
PurposeIntracavitary vaginal brachytherapy can be perform-
ed either alone or in combination with external beam ra-diation therapy (EBRT) for patients with endometrial or cervical carcinomas [1,2]. Late radiation reactions after high-dose-rate (HDR) brachytherapy are serious concerns, particularly when the patients are young and sexually ac-tive. The risk of late reaction depends on the dose received by the nearby critical organs, as bladder and rectum. Dose optimisation is an old concept in brachytherapy practice, but just recently became a topic for extensive study, due to technological advances in both treatment planning sys-tems and 3D imaging possibilities, as computerized to-mography (CT) and magnetic resonance (MR).
Recently, the American Brachytherapy Society (ABS) reported the recommendations for adjuvant vaginal HDR brachytherapy after hysterectomy, establishing dose prescription and optimization guidelines, as well as dose fractionation schedules [3]. Although there is a general consensus to treat the upper third of the vagina as a tar-get for this group of patients because the majority of the treatment failures were reported in this region [4,5], the choice of optimal applicator type is widely varying [3].
The most commonly used applicator for intracavitary vaginal brachytherapy is single channel vaginal cylinder. However, due to its radial symmetry of dose distribution, a single channel applicator offers limited possibilities to optimize the treatment plan according to the patient’s
Address for correspondence: Yasir A. Bahadur, MD, Department of Radiology, King Abdulaziz University Hospital, P.O. Box 80215, Jeddah 21589, Saudi Arabia, phone: +966 2 2401000, ext. 17444, mobile: +966 505528528, fax: +966 2 6408047, e-mail: [email protected]
Received: 30.07.2014Accepted: 11.11.2014Published: 30.12.2014
Journal of Contemporary Brachytherapy (2014/volume 6/number 4)
Single vs. multichannel applicator in inverse treatment planning 363
anatomy. Aiming to improve the capabilities of vaginal brachytherapy, multichannel applicators have been de-veloped. The additional channels at the periphery of the applicator support more conformal dosimetry and amend for the anisotropy generated by a single line source at the vaginal apex [3]. Differential loading of the channels can also potentially reduce the dose to the bladder and rec-tum, compared with the single channel cylinder.
The purpose of this study is to retrospectively com-pare, via 3D inverse planning optimization, the potential dosimetric advantages of a multichannel vaginal applica-tor versus a single channel one, in intracavitary vaginal HDR brachytherapy after hysterectomy. This comparison is made by analyzing dose distributions to target and or-gans at risk (OARs) volumes.
As the dosimetric and clinical benefits of optimized treatment plans for every fraction are still controversial, we also evaluated the dosimetric advantage of fractional re-planning.
Material and methodsWe are reporting the analysed data of 12 patients with
endometrial carcinoma, randomly selected and identified as candidates for vaginal vault brachytherapy, according to our current treatment practice. The patients were treat-ed in the Radiotherapy Unit of King Abdulaziz Universi-ty Hospital, Jeddah, Saudi Arabia, between January 2012 and November 2013.
The patients had either post-operative EBRT (45 Gy in 25 fractions, one fraction per day, five times per week) to the whole pelvis, using four-fields CT-based planning, followed by HDR brachytherapy (12 Gy in 3 fractions, two fractions per week), or HDR brachytherapy alone (21 Gy in 3 fractions, one fraction per week). However, for the purpose of this study we performed all brachythera-py plans with a prescription dose of 21 Gy in 3 fractions. Brachytherapy was performed with Miami 7-channels vaginal applicator (Varian Medical Systems, Palo Alto, CA, USA), of various diameters (3 and 3.5 cm); the ap-plicator has one central and 6 peripheral channels. The peripheral channels produce an asymmetric dose distri-bution, enabling the treatment plan optimization based on patient anatomy. Patients were requested to evacuate the rectum and bladder just before the applicator inser-tion. In order to help delineating the bladder and rectum, a 20 cc of Urographine contrast media was injected into the bladder (using Foley’s catheter) and 35 cc into the rec-tum (using a rectal catheter). Computerized tomography images without intravenous contrast were acquired using a Siemens Somatom Emotion CT scanner (Siemens Med-ical Systems, Erlangen, Germany), with 2 mm slice inter-vals from the iliac crest to the distal end of the applicator.
Target and organs at risk delineation
The clinical target volume (CTV) and the OARs vol-umes have been delineated on axial CT images for each patient and each brachytherapy fraction prospectively, at the time of each treatment planning, and reviewed ret-rospectively by one radiation oncologist, for the purpose
of this study. The CTV was a 5 mm expansion of the ap-plicator surface along 5 cm length measured from the tip of the applicator, and cropped from rectum and bladder. Organ at risk volumes included the rectum and bladder. The outer rectal wall was contoured from the recto-sig-moid junction till 1 cm above the anal verge, and the out-er bladder wall was contoured till the urethra.
Computed tomography image-based planning was per-formed in Varian Brachyvision planning system, version 10 (Varian Medical Systems, Palo Alto, CA, USA), for a Varian HDR VariSource iX remote afterloader (Varian Medical Sys-tems, Palo Alto, CA, USA). The dose calculation algorithm is based on the TG-43 formalism, as recommended by the American Association of Physicists in Medicine (AAPM) [6]. The inverse planning optimizer identifies the combina-tion of dwell times that best fulfill the dose constraints of target volume and critical organs. Two treatment plans (for both the central channel and multichannel loadings) were performed for each brachytherapy fraction, such that the dose distribution of the two plans could be compared. All plans were optimized by inverse planning, using multic-riterial objectives (dose and time), and the following con-straints: 1) for CTV coverage: 95% of the volume to receive 7 Gy and 100% of the volume to receive 6.65 Gy (95% of the prescribed dose, as a minimum); 2) for CTV dose homo-geneity: source dwell times to not exceed 300 s for single channel and 100 s for multichannel applicator; 3) for rectum: 2 cc of the volume to receive maximum 4.9 Gy (70% of the prescribed dose); 4) for bladder: 2 cc of the volume to receive maximum 5.6 Gy (80% of the prescribed dose).
All dose constraints were set to have the same pri-ority. Dose distributions and dose-volume histograms (DVHs) from these plans were analyzed, and all plans were evaluated and compared using the following indi-ces: the dose covering 100% of volume (D100%) and 95% of volume (D95%) for CTV. As a measure of high dose expo-sure of vaginal mucosa, the dose received by the most ex-posed volumes 2 cm3 (D2cc) of CTV and plan quality indi-ces, such as dose nonuniformity index (DNR, defined as the ratio of CTV receiving a dose equal to or greater than 1.5 times of the prescribed dose to the volume that re-ceives a dose equal to or greater than the prescribed dose; DNR = V150%/V100%) and the overdose index (OI, defined as the ratio of CTV receiving a dose equal to or greater than 2.0 times the prescribed dose to the volume that re-ceives a dose equal to or greater than the prescribed dose; OI = V200%/V100%). Ideally, both quality indices should be equal to zero. In addition, the dose at 5 mm from the ap-plicator tip was analysed.
For OARs, the doses received by the most exposed volumes of 1 cm3 (D1cc) and 2 cm3 (D2cc), the volume re-ceiving 70% of prescription dose (V70%) for rectum, and the volume receiving 80% of prescription dose (V80%) for bladder were considered for comparison. The dosimetric advantage of fractional re-planning was also investigat-ed, using inverse planning optimization and multichan-nel approach. For each patient, we applied the dwell-time values of the first fraction, corrected for the source decay, to the CT-data set of the subsequent fractions, and com-pared the dosimetric indices mentioned above to the ones generated by fractional re-planning.
Journal of Contemporary Brachytherapy (2014/volume 6/number 4)
Yasir A. Bahadur, Camelia Constantinescu, Ashraf H. Hassouna et al.364
The statistical analysis was performed using the Wilcoxon matched pairs test and an in-house software; a p value of < 0.05 was considered significant.
ResultsData of 12 patients who underwent CT-guided vag-
inal cuff HDR brachytherapy with multichannel ap-plicators and inverse treatment planning optimization were retrospectively reviewed. Due to relatively large size of available vaginal multichannel cylinder diame-ter (3 and 3.5 cm), not all patients could tolerate the in-sertion for each fraction, and their treatment was com-pleted using a single channel applicator with smaller diameter. Two patients started the treatment with sin-gle channel applicator and, due to unfavourable plan dosimetry of single channel, had subsequent fractions with multichannel applicator, attempting for a better plan optimization. Treatment characteristics are pre-sented in Table 1.
A total of 22 paired HDR brachytherapy inverse plans were performed and analyzed. Dose distributions and DVHs were generated for the CTV and OARs for both the central channel and multichannel approaches for all
Table 1. Treatment characteristics
No. of patients
n (%)
No. of fractions
n (%)
Multichannel in 3/3 fractions 3 (25%) 9
Multichannel in 2/3 fractions 4 (33%) 8
Multichannel in 1/3 fractions 5 (42%) 5
Total 12 22
Multichannel diameter 3 cm 10 (83%) 19 (86%)
Multichannel diameter 3.5 cm 2 (17%) 3 (14%)
Fig. 1. Dose distribution comparison between single channel (A) and multichannel (B), in axial and sagittal views. Isodose line of 7 Gy (prescribed dose) is displayed in yellow, 6.3 Gy (90% of prescribed dose) in green, 4.9 Gy (70% of prescribed dose) in pink and 14 Gy (200% of prescribed dose) in magenta. The capability of multichannel to correct for anisotropy at vaginal apex is clearly illustrated
A
B
Journal of Contemporary Brachytherapy (2014/volume 6/number 4)
Single vs. multichannel applicator in inverse treatment planning 365
patients. Figures 1 and 2 show a comparative example of single channel and multichannel approaches, for a rep-resentative patient. The comparison is performed with respect to the dose distribution (Fig. 1) and DVH (Fig. 2).
Out of 22 brachytherapy inverse plans, none met all dose constraints for single channel, while 6 (27%) fulfilled all dose constraints for multichannel. Clinical target vol-ume dose coverage was achieved in 2 (9%) single channel and 10 (45%) multichannel plans. Rectum dose limit was attained in 1 (4%) single channel and 10 (45%) multichan-nel plans, and bladder in 17 (77%) single channel and 21 (95%) multichannel plans.
Rectum and bladder doses are reduced by the mul-tichannel approach. However, limited improvement in the CTV dose coverage was achieved, but DVH analysis showed significant difference in D95% of CTV between single and multichannel approaches (p = 0.007).
For the OARs, the use of multichannel applicator de-monstrated a noticeable dose reduction, when compared to single channel, but statistically significant for rectum only (p = 0.0001). For D2cc of rectum, an average fractional dose of 6.1 ± 0.7 Gy (95% CI: 5.8-6.4 Gy) resulted for single channel vs. 5.1 ± 0.6 Gy (95% CI: 4.8-5.3 Gy) for multi-channel. For D2cc of bladder, an average fractional dose of 5 ± 0.9 Gy (95% CI: 4.6-5.4 Gy) occurred for single channel vs. 4.9 ± 0.8 Gy (95% CI: 4.5-5.2 Gy) for multichannel.
An increase of dose at 5 mm from the applicator tip of up to 41.3%, as well as an increase of the vaginal mucosa dose of up to 34.3%, was also noted in the multichannel setting. The vaginal dose at 5 mm from the applicator tip
was significantly increased (p = 0.001), from a fractional average of 7.8 ± 3.3 Gy for the single channel to about 9 ± 2.6 Gy for multichannel approach. This demonstrates the capability of multichannel applicator to compensate for anisotropy effect.
Table 2 summarizes the dosimetric comparison be-tween single and multichannel inverse plans. The dosim-etric benefit of fractional re-planning was also investigat-ed, using inverse planning optimization and multichannel applicator, for 7 patients (who had more than one mul-tichannel insertion) and 10 paired brachytherapy plans. The DVH analysis showed large, but not statistically significant differences between first fraction plan and fractional re-planning, for all dosimetric parameters an-alysed, and are revealed in Table 3 and Figure 3. Patient with fraction 4 presented outstanding inter-fraction vari-ation and was excluded from statistical analysis, because the large dose differences and small sample size would have been affected the statistical results. While the D2cc of rectum varied with up to 23.4%, the bladder appeared to be more stable to inter-fraction variations, with differenc-es of D2cc up to 11.3%. The differences were due to large inter-fraction variations for rectum and bladder position-ing, as well as marked inconsistencies in OARs filling, as shown in Figure 4.
DiscussionBrachytherapy has been a standard component of
endometrial carcinoma therapy for over 100 years. As
Fig. 2. Dose-volume histogram comparison between single channel (squares) and multichannel (triangles) approaches. Clinical target volume is displayed in red, rectum in green and bladder in blue
Ratio
of t
otal
str
uctu
re v
olum
e (%
)
100
80
60
40
20
0 0 200 400 600 800 1000 1200 1400
Dose (cGy)
Relative dose (%) 0 28.571 57.142 85.714 114.28 142.85 171.42 200
multichannel
multichannel
multichannel
single channel
single channel
single channel
Journal of Contemporary Brachytherapy (2014/volume 6/number 4)
Yasir A. Bahadur, Camelia Constantinescu, Ashraf H. Hassouna et al.366
the use of anatomy-based treatment planning for HDR brachytherapy becomes more widely used, systematic methods of dose optimization are important for qual-ity assurance, reproducibility, and respect of the clin-ical issues. Although the complications rate for vaginal brachytherapy is considerably diminished than for pelvis EBRT, published reports support a need for dose optimi-zation in vaginal brachytherapy [7-10].
Adapting a HDR vaginal brachytherapy plan to any patient specific anatomy and disease presentation using single channel cylindrical applicators is limited, as recog-nized by the latest ABS guidelines for adjuvant vaginal cuff brachytherapy after hysterectomy, which recom-mends custom applicators [3]. If a brachytherapy plan with a single channel applicator delivers unacceptable doses to the rectum and bladder, a multichannel applica-tor can be considered, as improves the plan dosimetry and compensates for the anisotropy at the vaginal apex [3].
Among the patients included in this study, 2 started the brachytherapy with single channel applicator and, due to unfavourable dosimetry, decision was made to complete their treatment with multichannel applicator, resulting in a better plan optimization.
The present study evaluated the magnitude of dose sparing to OARs that can be accomplished using a mul-tichannel applicator and 3D CT-based inverse planning. Our results showed a reduction of the rectum and blad-der doses, without compromising the CTV coverage. In order to maintain a uniform methodology and unbiased comparison between single and multichannel, we appli-ed the same constraints and priorities to all treatment plans, for the purpose of this dosimetric study. However, in clinical practice, these constraints can be adapted to in-dividual clinical circumstances.
Table 2. Dosimetric comparison between single and multichannel approaches, presented as mean and standard deviation for fractional prescribed dose of 7 Gy
Single channel (mean ± SD) Multi-channel (mean ± SD) p
CTV D100% (%) 73.7 ± 7 74.8 ± 6.8 0.15
D95% (%) 94.7 ± 6 95.6 ± 6.7 0.007
D2cc (Gy) 12.3 ± 2.2 14.1 ± 1.9 0.0002
DNR 0.14 ± 0.08 0.26 ± 0.05 0.0001
OI 0.04 ± 0.03 0.07 ± 0.03 0.0001
Rectum D1cc (Gy) 6.5 ± 0.7 5.5 ± 0.6 0.0001
D2cc (Gy) 6.1 ± 0.7 5.1 ± 0.6 0.0001
V70% (cc) 12.2 ± 8 6.3 ± 3.5 0.0001
Bladder D1cc (Gy) 5.4 ± 0.9 5.2 ± 0.8 0.081
D2cc (Gy) 5 ± 0.9 4.9 ± 0.8 0.053
V80% (cc) 1.2 ± 1.5 1 ± 1.2 0.77
CTV – clinical target volume; D100% – the dose covering 100% of volume; D95% – the dose covering 95% of volume; DNR – dose nonuniformity index DNR = V150%/V100%; OI – overdose index OI = V200%/V100%; D1cc – the dose received by the most exposed volume of 1 cm3; D2cc – the dose received by the most exposed volume of 2 cm3; V70% – the volume receiving 70% of prescription dose; V80% – the volume receiving 80% of prescription dose
Table 3. Dose percentage differences between first fraction plan and fractional re-planning, presented as mean and standard deviation for fractional pre-scribed dose of 7 Gy
Dose difference (%)(mean ± SD)
p
CTV D100% (%) –12.1 ± 26.9 0.12
D95% (%) –9.8 ± 18 0.09
Rectum D1cc (Gy) 3.9 ± 13.6 0.19
D2cc (Gy) 1.9 ± 12.6 0.29
Bladder D1cc (Gy) 1.4 ± 6.6 0.41
D2cc (Gy) 0.6 ± 6 0.45
CTV – clinical target volume; D100% – the dose covering 100% of volume; D95% – the dose covering 95% of volume; D1cc – the dose received by the most expos - ed volume of 1 cm3; D2cc – the dose received by the most exposed volume of 2 cm3
Fig. 3. Dose percentage differences between first fraction plan and fractional re-planning
Inte
r-fr
actio
n do
se d
iffer
ence
(%)
Fraction number
CTV D100% CTV D95% rectum 1cc rectum 2cc bladder 1cc bladder 2cc
0 1 2 3 4 5 6 7 8 9 10
40
20
0
–20
–40
–60
–80
–100
–120
Journal of Contemporary Brachytherapy (2014/volume 6/number 4)
Single vs. multichannel applicator in inverse treatment planning 367
With single channel, the inverse planning calculation could produce unacceptable high doses at the applicator tip and the normal tissue above, such as small bowel. The multichannel approach minimizes the effect of ani-sotropy and significantly improves CTV dose coverage at 5 mm from applicator tip by up to 40% (p = 0.001). Our data revealed also a significant increase of dose to the vaginal mucosa if using a multichannel applicator of about 15% (p = 0.0002), as estimated by D2cc of CTV. Be-cause the source channels of the multichannel applicator are placed close to the vaginal mucosa, the dose gradient in the radial direction is steep, when compared to a single channel applicator, raising therefore the caution that the mucosal dose is acceptably increased, as recommended by ABS guidelines [3].
Although defined in interstitial brachytherapy, some plan quality indices such as DNR and OI have been used by several studies reporting intracavitary treatments, in order to assess the high dose within CTV [11-13]. Gloi et al., analysing the potential of an inflatable multichan-nel vaginal applicator to optimize the dose distribution in 5 pa tients with endometrial carcinoma, found the values of
DNR in the range of 0.1-0.3, and OI of about 0.1, compara-ble to our data: DNR = 0.26 ± 0.05 and OI = 0.07 ± 0.03 [13]. This value of DNR will result in a homogeneity index (DHI = 1 – DNR) of about 0.74, a bit higher that the value of 0.6-0.7 stated by ABS guidelines for interstitial brachytherapy for vaginal cancer [11]. The result is not surprising: unlike an interstitial implant, where the catheters are in direct contact with vaginal mucosa, the multichannel applicator is able to provide a slightly better dose homogeneity. Nev-ertheless, DNR and OI values for multichannel are two times higher than for single channel applicator (p = 0.0001).
To date, there are no clear guidelines regarding the acceptable high doses in intracavitary brachytherapy, and inhomogeneous dose distributions are inherently generated. Rather than controlling the high dose inside CTV by dose constraints, that might affect its coverage during inverse planning optimization process, we de-cided on a multicriterial approach (dose and time con-straints), using the active dwell times as hard constraints forced to not exceed 300 s for single channel and 100 s for multichannel applicator, and thus eliminating possible hot spots [14].
Fig. 4. The inter-fraction variation in rectum and bladder positioning (A) and filling (B), in sagittal view, for two representative patients. Figure 4A presents the patient with fraction 4; outstanding inter-fraction variations in OARs positioning is observed
A
B
Journal of Contemporary Brachytherapy (2014/volume 6/number 4)
Yasir A. Bahadur, Camelia Constantinescu, Ashraf H. Hassouna et al.368
As shown by other authors, the multichannel vaginal cylinders may provide more conformal dosimetry, by de-creasing the dose to the rectum and bladder without a re-duction in the target dose, but with the liability of a larger mucosal dose [3,15-22]. Using the flexibility of selecting source dwell positions and adjusting dwell times would result in a better shaping of individual dose distributions, compared to a single channel cylinder. An important ad-vantage would be achieved in the specific case of treating vaginal cancer or vaginal recurrences, when the vaginal mucosa will be only partially defined as CTV [16,20]. Tanderup et al. have previously shown the dosimetric ad-vantage of a 13-channels vaginal cylinder in comparison to a single channel one, in a dosimetric study using point dose optimization [21]. They reported a reduction of the hot spot doses to rectum and bladder of 17% and 16%, re-spectively, while the vaginal mucosal dose remained the same, and recommended the use of CT-based 3D dose planning to fully exploit the capabilities of a multichan-nel vaginal cylinder.
The latest ABS report recognizes that, for fixed geo-metry applicators and assuming that the geometry of implant remains the same for each insertion, although in-dividualized treatment planning for every fraction most accurately documents the actual total dose delivered to the patient, this practice is time consuming, involves technical costs, and may not improve patient outcomes [3]. In the particular case of vaginal brachytherapy alone, the need for routine calculation of the rectal and bladder doses has been questioned; the relatively low dose to or-gans at risk and the overall low morbidity of brachyther-apy should be balanced against the possible risks of re-peated Foley catheter insertion [3,23,24].
Several dosimetric studies, describing attempts of an-atomically optimized vaginal cuff HDR brachytherapy, also investigated the benefit of fractional re-planning, but with contradicting results for both single and multi-channel approaches [16-20,25-31].
Yaparpalvi et al. analyzed the inter-fraction variations of single channel applicator insertion, as well as the fluc-tuations in bladder and rectal volumes, which have led to variations of bladder and rectal doses; they concluded that the dose to OARs should be assessed on individual fraction basis [27]. Consequently, each fraction of vagi-nal cuff brachytherapy should be image-based, in order to achieve an accurate and complete dosimetric assess-ment of the treatment. However, other investigators stat-ed that the small inter-fraction variation in doses to the bladder and rectum do not support treatment planning and reporting doses to the OARs beyond the first fraction [23,32,33]. Small concluded that the need for individual-ized fraction optimization for single channel vaginal cyl-inders is yet to be determined [26].
The OARs dose can vary considerably from frac-tion to fraction during the course of vaginal cuff HDR brachytherapy. A number of patient-based and tech-nique-based factors, as: changes in bladder and rectum filling, inconsistencies in patient orientation, and differ-ences in cylinder position within the vagina may contrib-ute to this variation [28,34]. Contouring of critical organs on CT images and 3D dosimetric analysis provide a reli-
able method to elucidate the nature of these daily geome-tric variations, despite a large inter-observer variations that has been reported [28,35,36]. Siddiqui et al. showed that over a series of patients, such variations result in an increased rectum volume receiving a percentage of the prescribed dose, but over the course of multiple frac-tions for an individual patient, this effect is dosimetrically averag ed out [28].
For multichannel approach, the available data are also inconsistent. While Symon et al., analysing 44 paired brachytherapy plans, showed that individual fraction optimization is important, in order to minimize doses to critical structures, Zhou et al. concluded that there is no advantage of re-planning for each fraction, analysing the ICRU 38 points dose for rectum and bladder, in a recent dosimetric study over 9 paired brachytherapy plans and employing manual optimization [17,25].
Our data demonstrate a large variation in the rec-tum and bladder doses, if fractional re-planning is not performed. Image analysis confirmed these findings, showing marked inter-fraction variation in OARs filling and positioning relative to the multichannel applicator. Unlike the single channel applicator, the steep dose gra-dient in the radial direction exhibited by a multichannel applicator seems to noticeably affect the dose to the near-by OARs, if inter-fraction variations occur. We noticed a reduction of mean values of doses to CTV, concomitant with an increase of rectum and bladder dose in the ab-sence of fractional re-planning. These findings reveal that inter-fraction variations could be detrimental for both the local control and OARs toxicity, and emphasize the im-portance of image guidance and fractional re-planning, if inverse planning optimization is performed. Neverthe-less, the bladder seems to be more stable to inter-fraction variations: the differences of D2cc being about half of the values for rectum. A possible explanation of this result could be the filling protocol. All patients were requested to empty the rectum and bladder just prior applicator in-sertion, but voluntarily evacuation of bladder is usually easier than of rectum. The current literature is still lacking studies investigating the effect of inter-fraction variation on 3D inverse planning optimization for a multichannel applicator; therefore a comparison of our results, show-ing the importance of fractional re-planning, is not cur-rently possible.
The outcome of HDR vaginal cuff brachytherapy re-lies on choosing the applicator size that would maintain a permanent contact with the vaginal mucosa and most comfortably fit the patient. However, the commercially available multichannel applicators have the minimum di-ameter of 3 cm, this making them unsuitable for patients with narrow vagina or post-radiotherapy vaginal steno-sis. In our clinical practice of HDR brachytherapy, the use of multichannel applicators is often challenging, despite of all our efforts (lubrication of the applicator, patient education, pain medication) to make the insertion and treatment tolerable. Out of 12 patients selected for this study, only 3 (25%) were able to tolerate the insertion for all 3 fractions of brachytherapy.
Pelvic and vaginal radiotherapy may damage the va-gina and produce stenosis, shortening and loss of elastici-
Journal of Contemporary Brachytherapy (2014/volume 6/number 4)
Single vs. multichannel applicator in inverse treatment planning 369
ty [37-40]. Although scarcely reported, vaginal stenosis is a common side effect of radiotherapy, occurring in 38% of patients, most often during the first year after treat-ment, and patients older than 50 years presents the high-est risk [37]. Research has shown that maximum dose to the vaginal vault correlates with stenosis grade, and ad-juvant chemotherapy before vaginal cuff brachytherapy is associated with subsequent stenosis [39]. In an effort to prevent vaginal stenosis, the use of vaginal dilators has been advocated [40].
An obvious limitation of the present study is the small number of patients data considered for statistical analysis. Such a small sample size could not be valid if generalized at patient population level, and future studies with large sample sizes are recommended. 3D CT-based vaginal HDR brachytherapy using a multichannel vaginal appli-cator and inverse planning optimization provides dosim-etric advantages over single channel cylinder, by reduc-ing the dose to organs at risk without compromising the coverage of target volume. However, this improvement comes at the expense of an increased vaginal mucosa dose.
For vaginal HDR inverse planning brachytherapy with multichannel applicator, our data show that large inter-fraction dose variations can occur, due to marked differences in rectum and bladder volumes and positions, therefore we recommend individual fraction optimization.
DisclosureAuthors report no conflict of interest.
References1. Horowitz NS, Peters WA 3rd, Smith MR et al. Adjuvant high
dose rate vaginal brachytherapy as treatment of stage I and II endometrial carcinoma. Obstet Gynecol 2002; 99: 235-240.
2. Pearcey RG, Petereit DG. Post-operative high dose rate bra-chytherapy in patients with low to intermediate risk endo-metrial cancer. Radiother Oncol 2000; 56: 17-22.
3. Small W Jr, Beriwal S, Demanes DJ et al. American Bra-chytherapy Society consensus guidelines for adjuvant vag-inal cuff brachytherapy after hysterectomy. Brachytherapy 2012; 11: 58-67.
4. Nag S, Erickson B, Parikh S et al. The American Brachythera-py Society recommen dations for high-dose-rate brachythera-py for carcinomas of the endometrium. Int J Radiat Oncol Biol Phys 2000; 48: 779-790.
5. Li Z, Liu C, Palta JR. Optimized dose distribution of a high dose rate vaginal cylinder. Int J Radiat Oncol Biol Phys 1998; 41: 239-244.
6. Rivard MJ, Coursey BM, DeWerd LA et al. Update of AAPM Task Group No. 43 report. A revised AAPM protocol for brachytherapy dose calculations. Med Phys 2004; 31: 663-674.
7. Dimopoulos JC, Schmid MP, Fidarova E et al. Treatment of locally advanced vaginal cancer with radiochemotherapy and magnetic resonance image-guided adaptive brachytherapy: dose-volume parameters and first clinical results. Int J Radiat Oncol Biol Phys 2012; 82: 1880-1888.
8. Nout RA, Smit VT, Putter H et al. Vaginal brachytherapy versus pelvic external beam radiotherapy for patients with endometrial cancer of high-intermediate risk (PORTEC-2): an open-label, non-inferiority, randomised trial. Lancet 2010; 375: 816-823.
9. Onsrud M, Strickert T, Marthinsen AB. Late reactions after postoperative high-dose-rate intravaginal brachytherapy for
endometrial cancer: a comparison of standardized and indi-vidualized target volumes. Int J Radiat Oncol Biol Phys 2001; 49: 749-755.
10. Libby B, Sen N. Retrospective analysis of toxicity from vag-inal cuff brachytherapy. American Brachytherapy Society Annual Meeting, Atlanta, GA, 2010.
11. Beriwal S, Demanes DJ, Erickson B et al. American Brachy-therapy Society consensus guidelines for interstitial brachy-therapy for vaginal cancer. Brachytherapy 2012; 11: 68-75.
12. Shwetha B, Ravikumar M, Katke A et al. Dosimetric compar-ison of various optimization techniques for high dose rate brachytherapy of interstitial cervix implants. J Appl Clin Med Phys 2010; 11: 3227.
13. Gloi AM. First clinical implementation of the Capri applica-tor. J Appl Clin Med Phys 2014; 15: 386-394.
14. De Boeck L, Beliën J, Egyed W. Dose optimization in high-dose-rate brachytherapy: A literature review of quantitative models from 1990-2010. ORHC 2014; 3: 80-90.
15. Demanes DJ, Rege S, Rodriquez RR et al. The use and advan-tages of a multichannel vaginal cylinder in high-dose-rate brachytherapy. Int J Radiat Oncol Biol Phys 1999; 44: 211-219.
16. Iftimia I, Cirino ET, Mower HW, McKee AB. Treatment planning methodology for the Miami multichannel applica-tor following the American Brachytherapy Society recently published guidelines: the Lahey Clinic experience. J Appl Clin Med Phys 2013; 14: 214-227.
17. Zhou J, Prisciandaro J, Lee C et al. Single or multi-channel vaginal cuff high-dose-rate brachytherapy: Is replanning nec-essary prior to each fraction? Pract Radiat Oncol 2014; 4: 20-26.
18. Peng J, Sinha R, Patel R et al. A comparison of 3D dose plan-ning regimens for vaginal cuff brachytherapy using a flexi-ble inflatable multichannel gynecologic applicator. Scientific Poster PD66, ABS 2011 Annual Meeting.
19. Kuo HC, Mehta KJ, Yaparpalvi R et al. Feasibility study and optimum loading pattern of a multi-ring inflatable intravagi-nal applicator. J Contemp Brachytherapy 2013; 5: 93-100.
20. Lapuz C, Dempsey C, Capp A, O’Brien PC. Dosimetric com-parison of optimization methods for multichannel intra-cavitary brachytherapy for superficial vaginal tumors. Brachytherapy 2013; 12: 637-644.
21. Tanderup K, Lindegaard JC. Multi-channel intracavitary vag-inal brachytherapy using three-dimensional optimization of source geometry. Radiother Oncol 2004; 70: 81-85.
22. Kim H, Rajagopalan MS, Houser C, Beriwal S. Dosimetric comparison of multi-channel vaginal cylinder high-dose-rate brachytherapy to one single channel vaginal cylinder for pa-tients with vaginal cancer. Brachytherapy 2013; 12: S59.
23. Holloway CL, Macklin EA, Cormack RA, Viswanathan AN. Should the organs at risk be contoured in vaginal cuff bra chy-therapy? Brachytherapy 2011; 10: 313-317.
24. Zhu L, Craciunescu O, Cai J et al. Dosimetric impact of CTs with each fraction on organs at risk for HDR vaginal cylinder brachytherapy. Radiother Oncol 2012; 103: S110.
25. Symon Z, Menhel J, Alezra D, Pfeffer MR. Individual fraction optimization vs. first fraction optimization for multichan-nel applicator vaginal cuff high-dose-rate brachytherapy. Brachytherapy 2006; 5: 211-215.
26. Small W Jr. To plan or not to plan? That is the question. Brachytherapy 2006; 5: 216-217.
27. Yaparpalvi R, Shah S, Sharma R et al. Variance in bladder and rectal doses in the course of fractionated cylinder high-dose-rate brachytherapy. Brachytherapy 2009; 8: 145-148.
28. Siddiqui MU, Bellon MR, Fraser C et al. The nature and do-simetric effect of daily variations in vaginal cuff high-dose-rate brachytherapy: a prospective study. Brachytherapy 2010; 9: S89.
Journal of Contemporary Brachytherapy (2014/volume 6/number 4)
Yasir A. Bahadur, Camelia Constantinescu, Ashraf H. Hassouna et al.370
29. Supe SS, Bijina TK, Varatharaj C et al. Optimized dose dis-tribution of Gammamed plus vaginal cylinders. Med Dosim 2009; 34: 20-25.
30. Bahadur YA, Hassouna AH, Constantinescu CT et al. Three- dimension anatomy-based planning optimization for high dose rate vaginal vault brachytherapy. Saudi Med J 2012; 33: 640-647.
31. Longo JM, Chen S, Thompson R et al. Measuring the inter-fraction variation in dose to the rectum and the bladder using CT-based treatment planning for postoperative high dose rate vaginal brachytherapy in women with endometrial can-cer. Int J Radiat Oncol Biol Phys 2011; 81: S478.
32. Nixon E, Modrick JM, Jacobson GM. Retrospective evaluation of single treatment plan per course vs. plan per fraction for high-dose-rate vaginal cylinders. Brachytherapy 2006; 5: 102.
33. Nunnery EW, Ali AN, Corso C et al. Fractional re-planning with three-dimensional CT for single channel vaginal cylin-der high-dose-rate brachytherapy: imaging technology over-kill? Int J Radiat Oncol Biol Phys 2011; 81: S474.
34. Bellon M, Fraser C, Sintay B et al. Interfraction geomet-ric and dosimetric variations in vaginal cuff high dose-rate brachytherapy using CT based planning: a prospective study. Int J Radiat Oncol Biol Phys 2009; 75: S381-S382.
35. Hung J, Shen S, De Los Santos JF et al. Image-based 3D treatment planning for postoperative vaginal cylinder bra-chytherapy: Dose-volume histograms of the bladder, rectum, and bowel. Brachytherapy 2008; 7: 148.
36. Craciunescu OI, Pepek JM, Steffey BA et al. Relevance of re-peating the planning CT for HDR vaginal cylinder treatments: a comparison of intra-patient variability and inter user con-touring variability. Int J Radiat Oncol Biol Phys 2009; 75: S379.
37. Brand AH, Bull CA, Cakir B. Vaginal stenosis in patients treated with radiotherapy for carcinoma of the cervix. Int J Gynecol Cancer 2006; 16: 288-293.
38. Wolf JK. Prevention and treatment of vaginal stenosis result-ing from pelvic radiation therapy. Commun Oncol 2006; 3: 665-671.
39. Vakilian S, Al-Suhaibani A, Bahoric B. What causes vagi-nal stenosis in patients undergoing adjuvant intracavitary brachytherapy for endometrial cancer? Int J Radiat Oncol Biol Phys 2011; 81: S475.
40. Johnson N, Miles TP, Cornes P. Dilating the vagina to pre-vent damage from radiotherapy: systematic review of the lite-rature. BJOG 2010; 117: 522-531.
© 2014 Alghamdi et al. This work is published by Dove Medical Press Limited, and licensed under Creative Commons Attribution – Non Commercial (unported, v3.0) License. The full terms of the License are available at http://creativecommons.org/licenses/by-nc/3.0/. Non-commercial uses of the work are permitted without any further
permission from Dove Medical Press Limited, provided the work is properly attributed. Permissions beyond the scope of the License are administered by Dove Medical Press Limited. Information on how to request permission may be found at: http://www.dovepress.com/permissions.php
International Journal of Women’s Health 2014:6 639–645
International Journal of Women’s Health Dovepress
submit your manuscript | www.dovepress.com
Dovepress 639
O r I g I n a l r e s e a r c H
open access to scientific and medical research
Open access Full Text article
http://dx.doi.org/10.2147/IJWH.S63636
Incidence rate of ovarian cancer cases in saudi arabia: an observational descriptive epidemiological analysis of data from saudi cancer registry 2001–2008
Ibrahim g alghamdi1,2
Issam I Hussain1
Mohamed s alghamdi3
Mansour M alghamdi4
ahlam a Dohal4
Mohammed a el-sheemy5
1school of life sciences, University of lincoln, Brayford Pool, lincoln, UK; 2al-Baha University, Kingdom of saudia arabia; 3Ministry of Health, general Directorate of Health affairs, al-Baha, Kingdom of saudi arabia; 4King Fahad specialist Hospital–Dammam, Kingdom of saudi arabia; 5Department of research and Development, lincoln Hospital, United lincolnshire Hospitals, national Health service Trust, lincoln, UK
correspondence: Ibrahim g alghamdi 15 copgrove close, Hamilton, leicester, le5 1aW, UK Tel +44 116 276 1913 email [email protected]
Purpose: This study provides descriptive epidemiological data, such as the percentage of cases
diagnosed, crude incidence rate (CIR), and age-standardized incidence rate (ASIR) of ovarian
cancer in Saudi Arabia from 2001–2008.
Patients and methods: A retrospective descriptive epidemiological analysis of all ovarian
cancer cases recorded in the Saudi Cancer Registry (SCR) from January 2001–December 2008
was performed. The data were analyzed using descriptive statistics, analysis of variance tests,
Poisson regression, and simple linear modeling.
Results: A total of 991 ovarian cancer cases were recorded in the SCR from January 2001–De-
cember 2008. The region of Riyadh had the highest overall ASIR at 3.3 cases per 100,000 women,
followed by the Jouf and Asir regions at 3.13 and 2.96 cases per 100,000 women. However, Hail
and Jazan had the lowest rates at 1.4 and 0.6 cases per 100,000 women, respectively. Compared
to Jazan, the incidence rate ratio for the number of ovarian cancer cases was significantly higher
(P,0.001) in the Makkah region at 6.4 (95% confidence interval [CI]: 4.13–9.83), followed
by Riyadh at 6.3 (95% CI: 4.10–9.82), and the eastern region of Saudi Arabia at 4.52 (95% CI:
2.93–6.98). The predicted annual CIR and ASIR for ovarian cancer in Saudi Arabia could be
defined by the equations 0.9 + (0.07× years) and 1.71 + (0.09× years), respectively.
Conclusion: We observed a slight increase in the CIRs and ASIRs for ovarian cancer in Saudi
Arabia from 2001–2008. Riyadh, Jouf, and Asir had the highest overall ASIR, while Jazan and
Hail had the lowest rates. Makkah, Riyadh, and the eastern region of Saudi Arabia had the high-
est incidence rate ratio for the number of ovarian cancer cases. Further analytical studies are
required to determine the potential risk factors of ovarian cancer among Saudi women.
Keywords: cancer epidemiology, ovarian cancer, Saudi Cancer Registry, incidence rate
IntroductionOvarian cancer is the eighth most common type of cancer among women in the world.
Furthermore, it is the seventh leading cause of cancer-related mortality with a severe
impact on socioeconomic and community health, globally.1–4 Compared to other types
of gynecologic malignancies, ovarian cancer has the highest mortality rate in developed
countries worldwide.5–7 The American Cancer Society estimated that 22,240 women
were going to be diagnosed with ovarian cancer in the US in 2013, while the number
of ovarian cancer-related deaths was estimated to be 14,230.8 Moreover, ovarian cancer
was estimated to account for 3% of all cancers among women. Currently, the lifetime
International Journal of Women’s Health 2014:6submit your manuscript | www.dovepress.com
Dovepress
Dovepress
640
alghamdi et al
risk of developing ovarian cancer is approximately 1 in
72, while the lifetime risk of dying from ovarian cancer is
approximately 1 in 100.8
The International Agency for Research on Cancer
estimated that the age-standardized incidence rate (ASIR)
and the age-standardized mortality rate for ovarian cancer
in Saudi Arabia in 2012 was 3.4 per 100,000 women and
2.5 per 100,000 women, respectively.9 Furthermore, the
registry of the King Faisal Specialist Hospital and Research
Centre (2011) recorded 1510 cases of ovarian cancer with
admission to the hospital from 1975–2011.10 In 2008, data
from the Saudi Cancer Registry (SCR) suggested that ovar-
ian cancer ranked seventh in cancer incidence among Saudi
women.11 The ASIR of ovarian cancer in Saudi Arabia
is low in comparison with other countries in the Arabian
Gulf. For example, in 2012, the reported ASIR of ovarian
cancer for Oman, United Arab Emirates, Kuwait, Qatar, and
Bahrain was 10.2, 6.4, 4.7, 4.6, and 4.4 per 100,000 women,
respectively.9
Despite a lack of data on the geographic distribution of
ovarian cancer among women in Saudi Arabia, we aimed to
investigate and describe the crude incidence rate (CIR) and
ASIR of ovarian cancer cases by providing an observational
descriptive epidemiological analysis of cancer of the ovary
while considering spatial/temporal distribution of recorded
cases in the SCR from 2001–2008.12
Materials and methodsA retrospective descriptive epidemiological study of the
ovarian cancer cases and diagnosed in Saudi Arabia from
January 2001–December 2008 was performed. The data of
reports regarding cancer incidence in Saudi Arabia are pub-
licly available and easily accessible to be downloaded from
the website of SCR. The main mission of SCR is to gather,
register, and provide high standard data for cancer incidence
stratified by region, age group, and year of diagnosis. Fur-
thermore, the published reports by SCR would be available
to policy makers, cancer researchers, treating physician,
cancer control, and prevention programs. Therefore, no ethi-
cal approval was required for this observational descriptive
epidemiological study. The data source for this study was
the SCR, a population-based registry established in 1992 by
the Ministry of Health of Saudi Arabia. The data cannot be
obtained directly from the SCR, but it is published in reports.
However, no data was available from 1994–2000 and the lat-
est published report of the SCR was available in 2008.
Since 2001, the SCR has been providing reports on
the patterns of cancers in Saudi Arabia with the primary
objective of defining the population-based incidence of the
disease. Currently, there are comprehensive reports for each
of the 13 administrative regions of Saudi Arabia. Each report
discusses the number and percentage of cases diagnosed, the
CIR, and the ASIR (adjusted by region and year of diagnosis)
of cancer from 2001–2008. The current study utilized the
SCR reports to gather all the information on ovarian cancer
with the aim of presenting the descriptive epidemiology of
ovarian cancer in Saudi Arabia.
Data were analyzed using the Statistical Package for the
Social Sciences version 20.0 (IBM Corporation, Armonk,
NY, USA). The descriptive analysis of the epidemiological
data of ovarian cancer was performed by calculating the
overall percentage of ovarian cancer cases, CIR, and the
ASIR adjusted by the age group, region, or year of diagnosis.
The analysis of variance (ANOVA) test was performed to
determine the effect of the geographical area and the year
of diagnosis on CIR and ASIR.13 Simple linear regression
was used separately with an independent variable (year of
diagnosis) to predict the annual CIR and the ASIR of ovar-
ian cancer among Saudi women.14 In addition, the Poisson
regression was conducted to calculate the incidence rate
ratio (IRR) of ovarian cancer cases for each region of Saudi
Arabia.
ResultsA total of 991 ovarian cancer cases were recorded in the
SCR from January 2001–December 2008. The percent-
age of women diagnosed with ovarian cancer increased
slightly from 2001–2008. Figure 1A and B shows that
there were 101 new ovarian cancer cases in 2001 (3.7%
of all cancer cases diagnosed in women; 95% confidence
interval [CI]: 1.7%–5.7%) and 96 in 2002. This indicates
a 0.4% decline in cases. By 2005, 136 ovarian cancer
cases were reported, corresponding to a 0.2% increase.
The number of ovarian cancer cases diagnosed annually
remained slightly high, but the percentages were stable
in 2005 and 2008 at 3.5%.
The average number and percentage of ovarian cancer
cases diagnosed from 2001–2008, adjusted by age group,
was calculated using the data from the SCR (Table 1 and
Figure 1C). Women aged 45–59 years were most fre-
quently diagnosed with ovarian cancer followed by those
aged 60–74 years, representing 31% (38 cases) and 26.0%
(33 cases) of the total number of ovarian cancer cases. In
contrast, the younger (0–44 years) and older (75 years and
over) groups recorded the lowest overall number and per-
centage of cases.
International Journal of Women’s Health 2014:6 submit your manuscript | www.dovepress.com
Dovepress
Dovepress
641
analysis of incidence rate of ovarian cancer cases in saudi arabia
The CIRs per 100,000 women, adjusted for the year of
diagnosis, indicated a steady increase in ovarian cancer cases
in Saudi Arabia from 2001–2008. As shown in Table 2 and
Figure 2A, a CIR of 1.3 (95% CI: 0.6–2.0) ovarian cancer
cases per 100,000 women was estimated in 2001; and a
CIR of 1.8 (95% CI: 0.9–2.7) cases per 100,000 women in
2008. The latter CIR was the highest rate observed, but it
was not statistically significantly different compared to the
other years (F[7, 96]=0.106; P.0.05). Linear regression
analysis suggested that the annual increase of CIR for ovar-
ian cancer in Saudi Arabia can be predicted by the equation
0.9 + (0.07× years), which indicates that the CIR of ovarian
cancer increased on average by 0.9 cases per 100,000 Saudi
women per year.
Table 3 and Figure 2B show the overall CIR of ovarian
cancer per 100,000 women, adjusted for the region of Saudi
Arabia from 2001–2008. The region of Riyadh had the highest
overall CIR for ovarian cancer at 1.9 (95% CI: 1.6–2.2) cases per
100,000 women, followed by the regions of Baha and Makkah
at 1.8 (95% CI: 1.0–2.6) and 1.7 (95% CI: 1.4–3.0) cases per
100,000 women, respectively. The ANOVA test revealed
that the incidence rates of ovarian cancer were significantly
180 3.8
3.7
3.6
3.5
3.4
3.3
3.2
3.1
% o
f ca
ses
3
2.9
2.8
A B
C
160
140
120
100
80
40
60Nu
mb
er o
f ca
ses
20
0
50
45
40
35
Mea
n
30
25
20
15
10
5
Number of cases
15–290–14
%
30–44
Age groups
45–59 60–74 ≥75
2001 2002 2003
Year of diagnosis2004 2005 2006 2007 2008 2001 2002 2003
Year of diagnosis
2004 2005 2006 2007 2008
Figure 1 number and percentage of ovarian cancer cases (A and B) in saudi arabia from 2001–2008. Overall number and percentage of ovarian cancer cases’ distribution by age group (C) in saudi arabia from 2001–2008.
Table 1 Ovarian cancer cases distribution by age group in saudi arabia from 2001–2008
Year Age group of ovarian cancer
0–14 15–29 30–44 45–59 60–74 $75 Total
# % # % # % # % # % # % Total % of total
2001 4 4 15 14.8 17 16.8 31 30.7 21 20.8 13 12.9 101 3.72002 6 6 15 16 13 14 29 30 24 25 9 9 96 3.32003 4 4 14 12.8 18 16.5 33 30 26 23.9 14 12.8 109 3.42004 6 6 11 10 22 20 32 30 27 25 10 9 108 3.12005 8 6 17 12.5 18 13 39 29 40 29.5 14 10 136 3.52006 2 2 15 11 22 17 44 33 38 29 11 8 132 3.22007 10 6.7 19 12.8 25 17 47 31.5 36 24 12 8 149 3.12008 6 3.7 17 10.6 22 13.7 51 32 50 31 14 9 160 3.5average 6 5 15 13 20 16 38 31 33 26 12 9.8 123.9 3.4
International Journal of Women’s Health 2014:6submit your manuscript | www.dovepress.com
Dovepress
Dovepress
642
alghamdi et al
higher for these three regions compared to the other regions
of Saudi Arabia (F[12, 91]=4.988; P,0.001). Furthermore,
Jazan (95% CI: 0.1–0.5), Najran (95% CI: 0.3–1.3), and the
northern region of Saudi Arabia (95% CI: 0.3–1.3) had the low-
est average CIRs for ovarian cancer at 0.3, 0.8, and 0.8 cases
per 100,000 women, respectively.
The overall ASIRs per 100,000 women were calculated
for all of the SCR reports on the basis of (Segi) world standard
for intermediate-age populations (Table 4). This standard can
be used in the direct method of standardization to calculate
the ASIR. It allows to control the confounding effects of
age when making a comparison between populations with
5
A
B
4.5
4
3.5
3
2.5
2
1.5
Rat
e p
er 1
00,0
00 w
om
enM
ean
rat
e p
er10
0,00
0 w
om
en
1
0.5
0
5
4.5
43.5
3
2.5
21.5
1
0.5
0Asir
CIR Mean ASIR Mean
Baha Jazan Madinah Hail Qassim
Regions
Riyadh Makkah Najran Jouf Tabuk Easternregion
Northernregion
2001
CIR ASIR Linear (ASIR)Linear (CIR)
2002 2003 2004
Year of diagnosis2005 2006 2007 2008
Figure 2 cIr and asIr of ovarian cancer cases (A) in saudi arabia from 2001–2008. Overall cIr and asIr of ovarian cancer cases’ distribution by region (B) in saudi arabia from 2001–2008.Abbreviations: cIr, crude incidence rate; asIr, age standardized incidence rate.
Table 2 cI for %, cIr, and asIr of ovarian cancer cases in saudi arabia from 2001–2008
Year Percentage CIR per 100,000
ASIR per 100,000
% 95% CI CIR 95% CI ASIR 95% CI
2001 3.7 1.7–5.7 1.3 0.6–2.0 2.3 1.3–3.32002 3.3 1.5–4.8 1.2 0.5–1.9 2.0 1.0–3.02003 3.4 1.6–5.0 1.4 0.7–2.2 2.0 0.9–3.12004 3.1 0.6–3.7 1.3 0.4–1.7 2.2 1.4–3.02005 3.5 0.8–4.3 1.6 0.7–2.5 2.8 2.0–3.62006 3.2 0.7–3.9 1.5 0.5–2.5 2.8 1.9–3.72007 3.1 0.8–3.9 1.7 1.0–2.4 2.6 1.8–3.42008 3.5 1.3–4.8 1.8 0.9–2.7 3.0 2.3–3.7Overall 3.4 1.1–4.5 1.5 0.6–2.2 2.5 1.5–3.3
Note: Values in bold denote there are no significant differences between years of diagnosis, P-value .0.05.Abbreviations: cIr, crude incidence rate; asIr, age-standardized incidence rate; CI, confidence interval.
Table 3 cI for overall percentage, cIr, and asIr of ovarian cancer cases in the regions of saudi arabia from 2001–2008
Regions Overall percentage
Overall CIR Overall ASIR
% 95% CI CIR 95% CI ASIR 95% CI
asir 3.8 2.6–5.0 1.3 0.8–1.8 2.96 1.25–2.66Baha 5.0 3.4–6.6 1.8 1.0–2.6 2.28 1.32–3.25Jazan 1.6 0.5–2.7 0.3 0.1–0.5 0.57 0.17–0.97Madinah 3.7 2.5–4.9 1.3 1.0–1.6 2.27 1.67–2.87Hail 3.4 1.8–5.0 1.0 0.5–1.5 1.35 0.70–1.99Qassim 2.3 1.2–3.4 0.9 0.3–1.5 1.46 0.69–2.23riyadh 3.4 3.1–3.7 1.9 1.6–2.2 3.30 2.68–3.91Makkah 3.6 3.1–4.1 1.7 1.4–2.0 2.81 2.20–3.42najran 3.1 1.0–5.2 0.8 0.3–1.3 1.92 0.52–3.32Jouf 4.8 1.4–8.2 1.4 0.7–2.1 3.13 1.50–4.76Tabuk 3.0 2.5–3.5 1.1 0.8–1.4 2.35 1.51–3.18eastern region 3.2 2.6–3.8 1.6 1.3–1.9 2.92 2.39–3.45northern region
2.6 0.7–4.5 0.8 0.3–1.3 1.55 0.24–2.85
Note: Values in bold denote for CIR, Riyadh, Baha and Makkah were significantly higher than other regions of saudi arabia, P-value ,0.05; for asIr, riyadh, Jouf and Asir were significantly higher than other regions of Saudi Arabia, P-value ,0.05.Abbreviations: CI, confidence interval; CIR, crude incidence rate; ASIR, age-standardized incidence rate.
International Journal of Women’s Health 2014:6 submit your manuscript | www.dovepress.com
Dovepress
Dovepress
643
analysis of incidence rate of ovarian cancer cases in saudi arabia
different age structures.15,16 Therefore, the ASIR of ovarian
cancer per 100,000 women, adjusted for the year of diag-
nosis in Saudi Arabia, from 2001–2008, is displayed in
Table 2 and Figure 2A. The SCR reports indicated a slight
increase in the number of cases of ovarian cancer diagnosed
from 2001–2008, with the highest ASIR of three cases per
100,000 women recorded in 2008 (95% CI: 2.3–3.7). Linear
regression showed that the annual increase in ASIR of ovar-
ian cancer in Saudi Arabia can be predicted by the equation
1.71 + (0.09× years), which suggests that the ASIR of ovarian
cancer increased by a predicted average of 0.09 cases per
100,000 women per year. Furthermore, Riyadh had the high-
est overall ASIR for ovarian cancer at 3.3 (95% CI: 2.68–3.91)
cases per 100,000 women, followed by Jouf and Asir at 3.13
(95% CI: 1.5–4.76) and 2.96 (95% CI: 1.25–2.66) cases per
100,000 women, respectively (Table 3 and Figure 2B). The
ANOVA test indicated that the ASIRs of ovarian cancer were
significantly higher for these three regions compared to other
provinces of Saudi Arabia (F[12, 91]=4.100; P,0.001). Fur-
thermore, Jazan, Najran, and the northern region of Saudi
Arabia had the lowest average ASIRs for ovarian cancer at:
0.3 (95% CI: 0.1–0.5); 0.8 (95% CI: 0.3–1.3); and 0.8 (95%
CI: 0.3–1.3) cases per 100,000 women, respectively.
The Poisson regression was conducted to calculate
the IRR of ovarian cancer cases for the different regions
of Saudi Arabia. The model was statistically significant
(LRχ2[15]=850.6; P,0.001). As shown in Table 5, Jazan
was used as the reference region for comparing the other
provinces of Saudi Arabia. The IRRs were significantly higher
(P,0.001) for Makkah at 6.4 (95% CI, 4.12–9.83), Riyadh at
6.3 (95% CI, 4.10–9.82), and the eastern region of Saudi Arabia
at 4.52 (95% CI, 2.93–6.98).
The differences in the CIRs and ASIRs between
2001–2008 were calculated to investigate the pattern of
ovarian cancer in the different regions of Saudi Arabia
(Table 6). The greatest changes in percentages and rates
were observed in the region of Baha (6.4%; CIR, 2.9;
ASIR, 3.6). However, the CIR and ASIR for all regions,
with the exception of Baha, were under the predicted values
based on the equations for the linear regression, ie, 0.9 + (0.07×8 years) = 1.46 per 100,000 women for the CIR and
2.43 + (0.09×8 years) = 2.43 per 100,000 women for the ASIR.
The smallest changes in the CIR and ASIR were observed
in the regions of Najran (−5.8%, −1.3 CIR, −4.1 ASIR), and
Hail (−2.9%; CIR, −0.5; ASIR, −1.1). The percentage of
ovarian cancer cases decreased by 5.8% and 2.9% in Najran
and Hail, respectively. Furthermore, in the Najran region the
CIR and ASIR decreased by 1.3 and 4.1 cases per 100,000
women, respectively; whereas, in the Hail region the CIR
decreased by 0.5 case and the ASIR decreased by 1.1 cases
per 100,000 women. However, these data indicate downtrend-
ing rates for both regions from 2001–2008.
DiscussionOvarian cancer is an important malignancy in women with
regards to its incidence and mortality. The current study
Table 5 Poisson regression model to calculate the Irr and check if there is an effect due to geographical area
# casesa IRR P-value 95% CI
Regions 1. asir 0.55 0.06 0.34–0.87 2. Baha 2.56 0.05* 1.56–3.98 3. Jazan 1 referenceb
4. Madinah 2.21 0.08 1.39–3.50 5. Hail 0.76 0.23 0.49–1.18 6. Qassim 1.21 0.40 0.77–1.91 7. riyadh 6.3 0.001* 4.10–9.82 8. Makkah 6.4 0.001* 4.12–9.83 9. najran 0.50 0.09 0.32–0.8110. Jouf 0.48 0.12 0.30–0.7611. Tabuk 0.98 0.60 0.64–1.5112. eastern region 4.52 0.001* 2.93–6.9813. northern region 0.44 0.10 0.26–0.62
Notes: anumber of cases occurring in regions numbered from 1 to 13. breference is the comparison region, and can be used for comparison with other regions and to calculate IRR. There are no significant differences between regions, P-value .0.05, *P-value ,0.05.Abbreviations: IRR, incidence rate ratio; CI, confidence interval.
Table 4 segi (world) standard for making comparisons with intermediate-age population (segi, 1960)
Age group Segi (world) standard
0–4 12,0005–9 10,00010–14 9,00015–19 9,00020–24 8,00025–29 8,00030–34 6,00035–39 6,00040–44 6,00045–49 6,00050–54 5,00055–59 4,00060–64 4,00065–69 3,00070–74 2,00075+ 2,000Total 100,000
Note: The segi (world) standard can be used in direct method of standardization to calculate the asIr and asMr. asIr is a technique used to compare the incidence rates among different populations when the confounding variable is age. asMr is a technique used to compare mortality rates among different populations when the confounding variable is age.Abbreviations: asIr, age-standardized incidence rate; asMr, age-standardized mortality rate.
International Journal of Women’s Health 2014:6submit your manuscript | www.dovepress.com
Dovepress
Dovepress
644
alghamdi et al
focused on the CIR and ASIR of ovarian cancer in different
regions of Saudi Arabia from 2001–2008. To the best of our
knowledge, this is the first geographical descriptive epide-
miological study of ovarian cancer cases in Saudi Arabia.
It presents the pattern and the trend of the disease from
2001–2008. In addition, this study provides an important
explanation for the observed trend, thereby highlighting the
significance of this disease.
The results indicated that the regions of Riyadh, Jouf,
and Asir had the highest overall ASIRs for ovarian cancer in
Saudi Arabia from 2001–2008; however, the overall ASIRs
of these regions did not increase above the expected levels. In
contrast, the regions of Jazan and Hail had the lowest overall
ASIRs of ovarian cancer in Saudi Arabia from 2001–2008.
Women .44 years of age were the most affected by ovarian
cancer in the different provinces of Saudi Arabia. Further-
more, the Poisson regression showed that the IRR of ovarian
cancer cases in the regions of Riyadh and Makkah was more
than six times higher than the reference region of Jazan.
This study also reported the changes in the percentage
of ovarian cancer cases diagnosed, CIR, and ASIR from
2001–2008. The Baha region had the largest differences in
CIR and ASIR, which were both higher than their predicted
values for the years 2001–2008. These differences in rates
suggest that, from 2001–2008, more women in the region
of Baha were affected by ovarian cancer than predicted. In
contrast, the rates of ovarian cancer decreased in the regions
of Najran and Hail. These regions reported the lowest changes
in the respective study outcomes, with a downtrend observed
in the CIR and ASIR from 2001–2008.
However, with the exception of the SCR reports, only
limited information was available on the incidence of ovarian
cancer in Saudi Arabia. This study intended to explore the
real pattern of ovarian cancer in different regions of Saudi
Arabia from 2001–2008. The findings of this study may be
useful for researchers and decision makers in the field of
medicine and health care administration in Saudi Arabia as
they can provide opportunities for generating hypotheses
about the potential risk factors of ovarian cancer in the highest
affected regions. Furthermore, the findings can be utilized
to conduct analytical epidemiological studies that aim to
identify the relationship between exposure and disease.13 The
availability of data from the SCR reports can also encourage
further descriptive epidemiological studies of other types of
cancers among the Saudi population.
ConclusionThe epidemiological analysis of the reports by the SCR, from
2001–2008, revealed that the CIR and ASIR of ovarian cancer
were increasing slightly. The regions of Riyadh, Jouf, and
Asir had the highest overall ASIR in Saudi Arabia, while
Jazan and Hail recorded the lowest rates. The IRR of ovarian
cancer cases indicated a concern in the number of ovarian
cancer cases diagnosed in certain regions, such as Makkah,
Riyadh, and the eastern region of Saudi Arabia. Moreover, the
region of Baha documented the greatest changes in CIR and
ASIR from 2001–2008, while the regions of Najran and Hail
reported the lowest. Nonetheless, further analytical studies
are required to determine and to explore the potential risk
factors of ovarian cancer in Saudi Arabia.
Table 6 Differences in the percentage, cIr, and asIr of ovarian cancer cases in the regions of saudi arabia from 2001–2008
Regions Percentage % CIR per 100,000 ASIR per 100,000
2008 2001 Difference 2008 2001 Difference 2008 2001 Difference
asir 4.7 4.0 0.7 1.9 0.9 0.9 3.0 1.7 1.3Baha 8.8 2.5 6.4 3.3 0.4 2.9a 4.2 0.6 3.6a
Jazan 2.0 2.5 −0.5 0.5 0.3 0.2 1.1 0.4 0.7Madinah 3.6 6.5 −2.9 1.6 1.7 −0.1 2.6 3.7 −1.1Hail 2.2 5.1 −2.9 0.8 1.3 −0.5 1.2 2.3 −1.1Qassim 2.6 1.4 1.2 1.1 0.5 0.6 2.0 0.9 1.1riyadh 4.0 3.6 0.4 2.5 1.8 0.7 4.5 3.8 0.7Makkah 3.8 3.1 0.7 2.1 1.0 1.1 3.5 1.5 1.5najran 1.5 6.8 −5.8 0.5 1.8 −1.3 1.1 5.2 −4.1Jouf 3.2 3.6 −0.4 1.2 0.5 0.7 2.8 3.3 −0.5Tabuk 2.4 2.5 −0.1 0.9 0.8 0.1 1.9 1.1 0.8eastern region 2.2 3.8 −1.6 1.3 1.6 −0.3 2.1 3.2 −1.1northern region 1.9 0.0 1.9 0.8 0.0 0.8 1.3 0.0 1.3
Note: Values in bold denote the differences in the percentages, cIrs and asIrs of ovarian cancer cases between 2001 and 2008 in different regions of saudi arabia. aFor cIr and asIr, the region of Baha showed the greatest changes compared to other provinces of saudi arabia, P-value ,0.05.Abbreviations: cIr, crude incidence rate; asIr, age-standardized incidence rate.
International Journal of Women’s Health
Publish your work in this journal
Submit your manuscript here: http://www.dovepress.com/international-journal-of-womens-health-journal
The International Journal of Women’s Health is an international, peer-reviewed open-access journal publishing original research, reports, editorials, reviews and commentaries on all aspects of women’s healthcare including gynecology, obstetrics, and breast cancer. The manuscript management system is completely online and includes
a very quick and fair peer-review system, which is all easy to use. Visit http://www.dovepress.com/testimonials.php to read real quotes from published authors.
International Journal of Women’s Health 2014:6 submit your manuscript | www.dovepress.com
Dovepress
Dovepress
Dovepress
645
analysis of incidence rate of ovarian cancer cases in saudi arabia
DisclosureThe authors report no conflicts of interest in this work.
References1. Ferlay J, Shine H, Bray F, Forman D, Mathers C, Parkin D. Estimates of
worldwide burden of cancer in 2008: GLOBOCAN 2008. Int J Cancer. 2010;127(12):2893–2917.
2. Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA Cancer J Clin. 2011;61(2):69–90.
3. Bast RC, Hennessy B, Mills GB. The biology of ovarian cancer: new opportunities for translation. Nat Rev Cancer. 2009;9(6):415–428.
4. Kusumbe AP, Bapat SA. Ovarian stem cell biology and the emergence of ovarian cancer stem cells. In: Cancer Stem Cells. 2008:95–110.
5. Jemal A, Siegel R, Xu J, Ward E. Cancer statistics, 2010. CA Cancer J Clin. 2010;60(5):277–300.
6. Holschneider CH, Berek JS. Ovarian cancer: epidemiology, biology, and prognostic factors. Semin Surg Oncol. 2000;19(1):3–10.
7. Klint A, Tryggvadóttir L, Bray F, et al. Trends in the survival of patients diagnosed with cancer in female genital organs in the Nordic countries 1964–2003 followed up to the end of 2006. Acta Oncol. 2010;49(5): 632–643.
8. American Cancer Society. Global Cancer Facts and Figures. 2nd ed. Atlanta: American Cancer Society; 2012. Available from: http://www.cancer.org/acs/groups/content/@epidemiologysurveilance/documents/document/acspc-027766.pdf. Accessed January 1, 2014.
9. GLOBOCAN (IARC). Section of Cancer Information; 2008. Available from: http://globocan.iarc.fr/ia/World/atlas.html. Accessed September 21, 2013.
10. King Faisal Specialist Hospital and Research Centre. Tumor Registry Annual Report; 2011. Available from: http://www.kfshrc.edu.sa/oncology/ Tumor%202011%20New%206%20Final.pdf. Accessed June 9, 2014.
11. Saudi Cancer Registry. Cancer Incidence Report in Saudi Arabia. Ministry of Health; 2008. Available from http://www.scr.org.sa/files/file/1eo1lhvt_compressPdf.pdf. Accessed June 9, 2014.
12. Friis RH, Sellers TA. Epidemiology for Public Health Practice. 4th ed. Sudbury, MA: Jones and Bartlett; 2009.
13. McHugh ML. Multiple comparison analysis testing in ANOVA. Biochem Med (Zagreb). 2011;21(3):203–209.
14. Tabatabai MA, Eby WM, Li H, Bae S, Singh KP. TELBS robust linear regression method. Open Access Medical Statistics. 2012;2:65–84.
15. Ahmad OB, Boschi-Pinto C, Lopez AD, Murray CJL, Lozano R, Inoue M. Age Standardization of Rates: a New WHO Standard. Geneva: World Health Organization; 2001. Available from: http://www.who.int/ healthinfo/paper31.pdf. Accessed January 20, 2014.
16. Doll R, Payne PM, Waterhouse JAH. Cancer incidence in five countries. International Union Against Cancer. Berlin: Springer-Verlag; 1966.
Knowledge of Saudi female university students regarding cervical cancer and acceptance of the human papilloma virus vaccineGhadeer K. Al-Shaikh, MD, FRCSC, Eman M. Almussaed, MD, Amel A. Fayed, MD, Dr PH, Farida H. Khan, MD, FCPS, Sadiqa B. Syed, MD, PhD, Tahani N. Al-Tamimi, MD, SBFM, Hala N. Elmorshedy, MD, Dr PH.
1223
ABSTRACT
األهداف: تقييم مستوى املعرفة عن سرطان عنق الرحم وتقييم مدي قبول لقاح فيروس الورم احلليمي البشري بني طالبات الكليات الصحية بجامعة
األميرة نورة بنت عبد الرحمن.
طالبات من متاحة لعينة الوصفية املقطعية الدراسة هذه الطريقة: الكليات الصحية بجامعة األميرة نورة بنت عبد الرحمن مكونة من 1400 فبراير إلى ديسمبر2013م من الفترة خالل الدراسة أجريت وقد طالبة، 2014م. مت توزيع استبيانات على الطالبات تضمنت البيانات االجتماعية والدميوجرافية واملعرفية حول سرطان عنق الرحم، مسحة عنق الرحم ودور الرحم كما تضمنت بسرطان عنق اإلصابة في البشري احلليمي الفيروس الثبات درجة اختبار مت . املرض من للوقاية للقاح الطالبات تقبل مدى .0.82 قيمته بلغت والتي ألفا كرونباخ اختبار باستخدام لالستبيان
تدني الدراسة أظهرت وقد ،89.9% املشاركات نسبة بلغت النتائج: على الطالبات من 95.7% واضح حيث حصل بشكل املعرفي املستوى مجموع نقاط معرفية أقل من املستوى املقبول ، كما بلغت نسبة الطالبات الالتي سمعن مبسحة عنق الرحم %46.7 ، وقد لوحظ أن املستوى املعرفي يتأثر إيجابيا باملستوى الدراسي للطالبات وبنوع الكلية حيث كان األعلى بني طالبات كلية الطب، وقد كان ذلك التأثر واضحا وذو داللة إحصائية. إلى لألسرة املرتفع الشهري والدخل لآلباء الصحية املهن أن وجد كما على تأثير له الرحم عنق بسرطان املعارف أو األقارب أحد إصابة جانب سعر أن الدراسة وأظهرت إحصائية. داللة بدون ولكن املعرفي املستوى اللقاح من العوامل الهامة لدى غالبية الطالبات حيث رأت حوالي 80% بينت كما ريال سعودي، 300 يتجاوز أال ينبغي اللقاح سعر أن منهن الدراسة أن اخلوف من احلقن واآلثار اجلانبية للقاح كانت من العوامل التي
تعوق قبول التطعيم.
املعلومات وشيوع املعرفي املستوى تدني الدراسة هذه أبرزت اخلامتة: فيروس وكذلك ، الرحم عنق و مسحة الرحم عنق اخلاطئة حول سرطان الورم احلليمي البشري باعتباره احد عوامل اخلطورة لإلصابة بسرطان عنق الرحم بني الغالبية العظمى من الطالبات. وهذه البيانات ميكن أن تستخدم
كقاعدة لصياغة برامج توعوية فعالة.
Objectives: To assess the level of knowledge regarding cervical cancer and the acceptance of the human papilloma virus (HPV) vaccine among Saudi female students in health colleges.
Methods: This cross-sectional study of a convenient sample encompassed 1400 students in Health Colleges at Princess Nora Bint Abdul Rahman University, Riyadh, Saudi Arabia was conducted between December 2013 and February 2014. A self-administrated questionnaire was distributed to all participants. Data collected included socio-demographic data, knowledge of cervical cancer risk factors and clinical presentation, Pap smear, and HPV vaccine acceptance. The questionnaire reliability as tested by Cronbach’s alpha was 0.82.
Results: The response rate was 89.9%, and data analysis revealed that 95.7% of students had poor knowledge level. The Pap smear was poorly recognized as a screening tool, with 46.7% of students having heard of the test. Senior and medical students had a significantly higher knowledge score. Father’s health profession, high monthly income, and presence of cervical cancer among family members or friends increased the level of knowledge. Vaccine acceptance is influenced by its price, approximately 80% of students thought that an affordable vaccine price should not exceed 300 Saudi Riyals. Perceived barriers to the vaccine were fear of injections and vaccine side effects.
Conclusion: There is a lack of knowledge and misinformation regarding cervical cancer, Pap smear, and HPV as a major risk factor for cancer of the cervix. These data can be used as a benchmark to formulate effective awareness programs.
Saudi Med J 2014; Vol. 35 (10): 1223-1230
From the Obstetrics and Gynecology Department (Al-Shaikh), College of Medicine, King Khalid University Hospital, King Saud University, College of Medicine (Almussaed, Khan, Syed, Elmorshedy), Princess Nora Bint Abdul Rahman University, Riyadh, Department of Family and Community Medicine (Al-Tamimi), College of Medicine, University of Hail, Hail, Kingdom of Saudi Arabia, and Department of Biostatistics (Fayed), High Institute of Public Health, Alexandria University, Alexandria, Egypt.
Received 30th January 2014. Accepted 22nd July 2014.
Address correspondence and reprint request to: Dr. Ghadeer Al-Shaikh, Associate Professor and Consultant, Department of Obstetrics & Gynecology, King Khalid University Hospital, King Saud University, Riyadh, Kingdom of Saudi Arabia. Tel. +966 (11) 4699339. Fax. +966 (11) 4679557. E-mail: [email protected]
www.smj.org.sa Saudi Med J 2014; Vol. 35 (10)OPEN ACCESS
1224
Knowledge regarding cervical cancer and the acceptance of HPV ... Al-Shaikh et al
Saudi Med J 2014; Vol. 35 (10) www.smj.org.sa
Cancer of the cervix uteri is a frequent cancer affecting women, and is a leading cause of
mortality worldwide.1 The highest incidence rates have been reported from sub-Saharan Africa, Central and South America, Southeast Asia, and Brazil. In contrast, the incidence rates were the lowest in the Middle East, particularly among Muslims and Jews, as compared to other religious groups.2,3 In the Kingdom of Saudi Arabia, carcinoma of the cervix uteri accounts for 2.6% of female cancers, and is ranked ninth among all carcinomas affecting Saudi females.4 The pathogenesis of cervical cancer in Muslim countries might be different from that of Western societies because of differences in cultural and religious factors that influence human behavior, and reduce the risk of exposure to cervical cancer.4-7 Among all known risk factors, persistent infection with high-risk human papillomavirus (HPV) plays a considerable role in the pathogenesis of cervical cancer.8-11 The worldwide HPV prevalence in cervical cancer was estimated to be between 85-99%.10,12
The HPVs are grouped according to their association with cervical cancer and their genomic sequence into oncogenic high, probable high, and non-oncogenic low-risk.11-14 The strong association of oncogenic HPV infection and the development of cervical cancer provides an opportunity for primary prevention through prophylactic vaccination. Human papillomavirus vaccines (bivalent and quadrivalent) have been shown to be immunogenic, safe, and highly effective in preventing chronic infection and precancerous lesions in women.15,16 The vaccine is available in the KSA market, but national campaigns to vaccinate females are not launched yet.
Cytological screening based on Pap smear plays a major role in reducing both the incidence and mortality of invasive cervical cancer. In the USA and Canada, the reduction in the incidence of cervical cancer and the subsequent reduction of female mortality rate was attributed to the widespread introduction of the Pap smear screening program as a secondary preventive measure for early detection of cases.17-19 However, in Saudi Arabia, most cases present at advanced stages that require extensive chemoradiation therapy. This might be due to lack of proper screening programs,20,21 and inadequate knowledge among the target population. Noteworthy, most female cancer awareness campaigns in KSA are mainly focused on breast cancer. Appropriate
level of knowledge, attitude, and beliefs are key elements for adopting a healthy lifestyle, influencing human behaviors, and accepting newly introduced preventive measures. Concerning cervical cancer, the gap of knowledge of clinical presentation, risk factors, primary and secondary prevention has been documented in several studies both in developed and developing countries.22-24 However, few studies have been reported from Saudi Arabia. The present study was designed to assess the level of knowledge and beliefs regarding cervical cancer, and the acceptance of the HPV vaccine among Saudi university students enrolled in health colleges because of their important role as health care providers to raise community awareness and to modify population behavior.
Methods. Study design and study sample. A descriptive cross-sectional study was conducted at the Health Colleges of Princes Nora Bint Abdul Rahman University (PNU) Riyadh, Saudi Arabia between December 2013 and February 2014. The study followed the principals of the Helsinki Declaration, and was approved by the Research and Ethical Committee at PNU. A convenient sample of students aged between 18-25 years were invited to participate in the study. The objectives of the study were explained to the students, and a verbal consent was obtained from each of the participants. The total sample size amounted to 1400 representing students enrolled in Medical, Dental, Nursing, Pharmacy and Health and Rehabilitation Colleges. Lack of consent to complete the questionnaire was the exclusion criteria.
Data collection. A self-administered close ended questionnaire consisting of 48 questions was developed by investigators, guided by study objectives and review of literature.22-24 The questionnaire included 4 sections: the first section comprises socio-demographic data such as age, college, year of study, profession of parents, and monthly income. The second section included 27 items for assessment of knowledge regarding risk factors and symptoms of cancer of the cervix. The responses to these questions included true, false, and don’t know. The third section was related to awareness of the Pap smear as a screening test. In the fourth section, questions related to participants’ perception of safety, efficacy, and acceptance of HPV vaccination were asked in a 5-point Likert scale format, where one indicated strongly disagree and 5 indicated strongly agree. Questions were prepared in the English language and translated into Arabic by 2 experts for better understanding of students. The questionnaire was tested for face and content validity as well as reliability by a pilot study
Disclosure. Authors have no conflict of interests, and the work was not supported or funded by any drug company.
1225www.smj.org.sa Saudi Med J 2014; Vol. 35 (10)
Knowledge regarding cervical cancer and the acceptance of HPV ... Al-Shaikh et al
carried out on 20 students, and Cronbach’s alpha was found to be 0.82.
Data analysis. The data were compiled, checked for completeness, and analyzed using the Statistical Package for Social Sciences (SPSS Inc., Chicago, IL, USA) version 20. Results of descriptive analysis of socioeconomic variables, knowledge of cervical cancer, Pap smear, attitude, and acceptance of HPV vaccine were tabulated. The knowledge score represents the sum of the correct answers of the 27 questions in the second section of the questionnaire. The value one was given for the correct answer, and zero for wrong answers, and for “don’t know” responses. The cutoff for a poor knowledge score was set at values below 60% of the total score. Testing the normality distribution of knowledge score revealed skewed distribution; hence, Mann-Whitney test and Kruskal-Wallis were used to compare knowledge scores among different subgroups. P-values of less than 0.05 were considered statistically significant. Multivariate linear regression analysis was adopted to adjust the effect of independent variables on the knowledge score.
Results. The response rate was 89.9%, and 1258 questionnaires were collected out of the total 1400 students representing the Health Colleges. Table 1 shows the distribution of students according to socio demographic characteristics; a total of 1258 students participated in the survey; their average age was 20.4±1.3 years. Students were distributed between different colleges; comparable percentages were from Pharmacy and Health Rehabilitation (33.1% and 34.8%), and the lowest percentage was from the Dentistry College (n=67, 5.3%). All year groups participated in the study with various percentages; the most common group was year 3 (n=380, 30.4%) whilst the least were from fifth or higher levels (n=131, 10.4%). Regarding parents work, most fathers were working in a non-health professions (81.6%) while most of the mothers were house wives (54.6%). Approximately 1% of students left these fields blank or wrote death as an answer. Only 6.1% of students reported low monthly income (<5,000 Saudi Riyals [SR]) and approximately 49% of students declared high income (more than 15,000 SR/month); moreover, approximately 10% of students did not answer this question.
The responses to different items for knowledge assessment are displayed in Table 2. Approximately 80% of students recognized breast cancer as the most common cancer among females in Saudi Arabia, and nearly half of them (51.1%) considered cancer cervix as a preventable disease. More than half of the students identified sexually transmitted disease as a risk factor for cancer cervix (59.6%), and a comparable proportion of them marked AIDS (48.9%) and other immunosuppressive disorders as risk factors (48.8%), whereas, nearly 25% of students thought that old age and sexual/perianal warts are risk factors for cancer cervix.
As for clinical presentation of cancer of the cervix, 54.8% knew that irregular vaginal bleeding was a symptom of cervical cancer, whereas 6% deemed that cervical cancer may be asymptomatic (Table 2). Fewer than half of students had heard of the pap smear (46.7%), and nearly 30% of them reported it as sensitive in early detection of cancer cervix. Concerning the frequency of the Pap smear, 38% of students thought that one Pap smear is enough, and 28.7% consider that it has serious complications (Table 2).
Knowledge of the HPV vaccine revealed that merely 10.9% could correctly recognize the viral component of the HPV vaccine, and 6.1% of students positively defined that the HPV vaccine is protective against genital warts, whilst 8% recognized the suitable age for vaccination (Table 2).
Table 1 - Distribution of students according to socio-demographic characteristics (1258 items).
Socio-demographic n (%)College
Medicine 122 (9.7)Dental 67 (5.3)Pharmacy 416 (33.1)Nursing 215 (17.1)Health and rehabilitation 438 (34.8)
Year group*Year 1 335 (26.8)Year 2 140 (11.2)Year 3 380 (30.4)Year 4 263 (20.9)Higher levels 131 (10.4)
Father’s occupationHealth professional 89 (7.1)Non health professional 1027 (81.6)Not working 127 (10.1)Death/blank 15 (1.2)
Mother’s occupationHealth professional 37 (2.9)Non health professional 524 (41.7)Not working 687 (54.6)Death/blank 10 (0.8)
Monthly income (Saudi Riyals [SR[)Up to 5,000 SR/month 77 (6.1)5,001-10,000 SR/month 197 (15.7)10,001-15,000 SR/month 252 (20.0)More than 15,000 SR/month 612 (48.6)Did not mention 120 (9.6)
*1249 completed the questionnaire, 9 had incomplete response
1226
Knowledge regarding cervical cancer and the acceptance of HPV ... Al-Shaikh et al
Saudi Med J 2014; Vol. 35 (10) www.smj.org.sa
Table 2 - Knowledge on cervical cancer: symptoms, risk factors, secondary and primary prevention.
Items for knowledge assessment n (%) 1. What is the most common type of cancer among females in Saudi Arabia?
1000 (79.5)
2. Is cervical cancer a preventable disease? 643 (51.1)Risk factors of cervical cancer
3. Perianal warts 298 (24.1) 4. Contraceptive pills 428 (34.6) 5. Human Immune deficiency virus (AIDS) 603 (48.9) 6. Smoking 418 (33.7) 7. Obesity 280 (22.6) 8. Human papilloma virus 326 (26.5) 9. Sexually transmitted diseases (STD) 742 (59.6)10. Immunosuppressive disorders 607 (48.8)11. Old age 321 (25.9)
Symptoms of cervical cancer12. Irregular vaginal bleeding 686 (54.8)13. Vaginal discharges 398 (31.9)14. Post coital bleeding 270 (21.8)15. Pelvic pains 488 (39.3)16. Weight loss 276 (22.3)17. Asymptomatic 76 (6.3)
Pap smear knowledge18. Did you hear about Pap smear? 286 (46.7)19. Is Pap smear a sensitive way to early diagnose cervical cancer?
384 (30.8)
20. Is Pap smear painful? 197 (15.7)21. Is one pap smear enough? 478 (38.0)22. Does pap smear have serious complications?
359 (28.7)
23. What is the most appropriate time to have pap smear?
224 (17.8)
24. What is the best frequency of pap smear? 227 (18.0)HPV vaccine knowledge
25. Vaccine against cervical cancer contains Human Papilloma Virus
137 (10.9)
26. The vaccine is protective against genital warts
76 (6.1)
27. Appropriate age to get the vaccine is 12-25 years
102 (8.1)
The total score of knowledge 8.33±4.58Poor knowledge 95.7%Acceptable knowledge 4.3%
Table 4 - Acceptance and barriers for human papillomavirus vaccine.
Questions n (%)What is the most appropriate place to get the vaccine*
Hospital 993 (78.9)Health center 83 (6.6)Private clinic 11 (0.9)
Acceptable vaccine cost* (Saudi Riyals)<100 481 (38.2)>100-300 511 (40.6)<500 114 (11.4)>500 57 (4.5)
Who is the best to advise for vaccination*Family doctor 812 (64.5)Friends 14 (1.1)Family 66 (5.2)Internet 64 (5.1)Television 48 (3.8)Own decision 170 (13.5)
Reasons for refusal to be vaccinatedBeing afraid of the vaccine injection 333 (26.5)Being worried about side effects 652 (51.9)The cervical cancer is rare and is not worth vaccination (1258)
116 (9.2)
The vaccine might be unaffordable 168 (13.3)She might have no time 257 (20.4)Family refusal 106 (8.4)She does not believe in the effect of vaccination
82 (6.5)
*Total number of respondents is not equal depending on the number of completed questionnaires in every questions.
Table 3 - Average knowledge scores, median, and interquartile range (IQR) according to various subgroups in the studied sample.
Variables Knowledge score P-valueMean±SD Median IQR
Father’s occupationHealth professional (n=82) 9.05±4.46 9 7.25 0.27Non health professional(n=919) 8.47±4.57 9 7
Mother’s occupationHealth professional(n=28) 8.50±3.56 8 3 0.94Non health professional (n=471) 8.43±4.62 8 7
Family income (Saudi Riyals)<5,000 (n=71) 8.02±4.59 8 6 0.29>5,000 and 10,000 (n=182) 8.49±4.27 8 6>10,00 and 15,000 (n=228) 7.89±4.45 8 7>15,000 (n=547) 8.53±4.66 8 7
Year groupYear 1 (n=301) 8.06±4.41 8 7 0.002Year 2 (n=32) 8.73±4.98 7 7Year 3 (n=340)* 7.72±4.59 9 6Year 4 (n=235)* 9.18±4.61 10 5Higher levels (n=114) 8.75±4.31 9 6
CollegeMedicine (n=86)* 8.9±4.9 8.5 8
0.001Dental (n=43) 7.9±4.3 8 6Pharmacy (n=338)* 9.1±4.4 9 6Nursing (n=211) 7.8±4.4 8 7Health and rehabilitation (n=354) 7.7±4.5 8 7
Having a friend or a family member with cancer cervixYes (n=85) 8.91±4.20 9 5.25 0.35No (n=908) 8.42±4.66 8 7
*incomplete response, thus, the total knowledge score was not calculated and they were excluded from the analysis.
Table 3 demonstrates the mean, median, and interquartile range of knowledge score according to the independent variables. For most of the items, the means, and the medians are closely related. The average total score of knowledge among students was 8.33±4.58, the minimum score was (0 out of 27) and the maximum was (24/27). Considering poor knowledge as correctly answering less than 60%, 1080 students (95.7%) were found to have poor knowledge. Further analyses of total knowledge score according to socioeconomic characteristics revealed that students whose parents work in the health professional field showed relatively higher scores (9.05±4.46 for health professional father, and 8.50±3.56 for health professional mother) when compared to those from non-health professions
1227www.smj.org.sa Saudi Med J 2014; Vol. 35 (10)
Knowledge regarding cervical cancer and the acceptance of HPV ... Al-Shaikh et al
fathers (8.47±4.57) and mothers (8.43±4.62); however, this difference was not conclusive, p>0.05. Average knowledge scores were not widely dissimilar among students of various family income groups (p=0.29); the top average score was 8.53 (monthly income more than 15,000 SR group), whilst the least average score was 7.72 among students whose family income was ranging from 10,001 SR to 15,000 SR per month.
Inequality of knowledge score among different year groups was evident (p=0.002), with the uppermost average score reported for year 4 students (9.18±4.61), and the least was obtained by third-year students (7.72±4.59). The Post-Hoc test (Mann-Whitney) identified that the score of students enrolled in year 3 is significantly lower than other groups, and year 4 students scored significantly higher than other groups (p<0.05). A significant difference among different colleges was also reported; students from colleges of medicine and pharmacy showed significantly higher scores than students from nursing and rehabilitation colleges (Table 3). The linear regression model was used to test the effect of year group on the knowledge score with adjustment of college. The mean knowledge score was found to increase by 0.4 points with each increase in the year group holding the colleges constant (beta coefficient =0.4, p<0.001). Validation of the model was tested via testing the distribution of the residuals, which were found normally distributed.
Table 4 demonstrates vaccine acceptance, the principal proportion of students preferred to have the vaccine in hospitals (n=993, 78.9%); however, less than 1% of them preferred the private clinics as a venue for vaccination. Approximately 80% of students thought that the affordable cost of vaccine should not exceed 300 Saudi Riyals (SR) and merely 4.5% will accept the cost to surpass 500 SR. Most students trust the family doctor as the main source of information regarding the vaccine, yet, 13.5% of students would accept to be vaccinated based merely on their own judgment. Regarding barriers for HPV vaccination, being worried regarding side effects was the first reason (51.9%) followed by being afraid of the injection (26.5%). Skepticism in the efficiency of the vaccine was the last reasons behind rejection of the vaccine (6.5%), whereas family refusal was reported by 8.5% of students as a barrier to obtaining the vaccine.
Discussion. It is becoming important to raise awareness of cervical cancer in KSA, and the level of knowledge and perception are key elements for adopting healthy behaviors and accepting newly introduced preventive measures. The HPV-related
cancer studies have reported the presence of HPV infection, according to the WHO, the HPV accounts for 99% of cases of cervical cancer.25 The vaccine against HPV cervical cancer-related strains has been available in the market since 2006;26 however, most of the available data generally portray a picture of widespread ignorance among the target population regarding HPV infection and vaccination.27-29 In KSA, although there has been increased attention to community health awareness programs, yet, most of the available research has focused on breast cancer, diabetes, and obesity. In contrast, very few studies with a limited sample size have been undertaken to assess the level of knowledge of cervical cancer, and the acceptance of HPV vaccine among Saudis.30,31
Results of the present study regarding knowledge of risk factors of cervical cancer demonstrate knowledge deficits in all items, with a percentage of less than 60% for correct answer in all the 9 questions. Moreover, student’s responses demonstrate confusion, while the percent of correct answers were 59.6% for sexually transmitted diseases, 48.9% for HIV, and 48.8% for immune suppression, only 26.9% considered HPV virus a risk factor for cervical cancer. In addition, students’ knowledge of the link between HPV and the primary prevention of cervical cancer by HPV vaccine is very poor. In 2014, a study was performed among 181 Saudi medical students at King Faisal University, and most of the students had poor knowledge of the early warning signs, symptoms, and risk factors of cervical cancer. On average, the percentages of correct answers ranged from 43.7-55%. In addition, most of the students (67%) were not aware of the availability of a vaccine against HPV.30 Moreover, among Saudi physicians at the Faculty of Medicine, King Abdulaziz University Hospital, Jeddah, only 60.5% of non-gynecological doctors knew that HPV is the major risk factor for cervical cancer, and only 33.8% believed that cervical cancer could be a curable disease.31 Similarly, in Western countries, general awareness of cervical cancer among 17-26 year old students in Poland is insufficient. The HPV infection is not considered to be the major etiological factor.22 Surprisingly, a report from a recent study in Greece among health caregivers demonstrated a significant gap in knowledge of HPV, where only 30% of the sample appears to be aware that HPV infection plays a considerable role in carcinogenesis.23
In accordance with this, findings from a recent survey in Thailand revealed that the level of knowledge regarding HPV is very low.24 However, it can be argued that this study sample represents the top academic level of university students, in addition, a good percentage of
1228
Knowledge regarding cervical cancer and the acceptance of HPV ... Al-Shaikh et al
Saudi Med J 2014; Vol. 35 (10) www.smj.org.sa
them (48.6%) belong to a high socioeconomic standard where the monthly income exceeds 15,000 SR. These 2 factors are expected to impose a positive influence on the level of knowledge both directly and indirectly with good opportunities for better education of family members, which is usually associated with passion to acquire knowledge, in addition to better chances of a high standard of healthcare facilities where health care providers have more time to raise the awareness of their customers.
Questions regarding clinical presentation of cervical cancer revealed lack of knowledge and misinformation, 54.8% consider irregular vaginal bleeding as the presenting symptom, around one third of students thought that vaginal discharge and pelvic pain are the presenting symptoms. The response to post-coital bleeding was 21.8%, while only 6% thought that cervical cancer could be asymptomatic. Lack of knowledge of disease symptomatology might have serious consequences on quality of life as well as on health expenditure. In Saudi Arabia, due to the lack of a national screening program, most cases of cervical cancer present at the advanced stages and require extensive treatment plans with more disabilities and hazards to individual health,20,21 in addition to the burden on the health system and tremendous expenditure of health budget. The influence of clinical presentation of diseases on human attitude and behavior should be considered while planning awareness intervention programs, several studies pointed out that absence of symptoms and vulnerability to the disease might influence the attitude towards preventive measures.32-34
In the present study, the value of the Pap smear as a screening tool for cervical cancer is poorly recognized. Only 46.7% of students have heard about the Pap smear. The percentages of correct answer concerning the test sensitivity to diagnose cervical cancer, frequency of performing Pap smear, and the appropriate time to have it carried out are all low. It is worth mentioning that misconceptions regarding the Pap smear were observed; the test was deemed to be painful by 15.7%, and cause serious complications by 28.7% of participants. Contrary to this, in the USA, and Poland, the data obtained on cytology and the need for regular testing demonstrated a good level of knowledge. The percentage of women who have heard about cytological screening was approximately 90%.22,35-38 The contributing factors for the observed difference are multifactorial including; the widespread introduction of Pap smear as a screening program, gender education programs in schools, an intensive public awareness campaign, and the onset of
HPV vaccine programs in Western countries started earlier than in the Middle East and the Gulf region.
In broad terms, the vast majority of students had a low level of knowledge, the mean knowledge score amounted to 8.33±4.58, and overall 95% of participants had poor knowledge. Although father’s health profession, monthly income >15,000 SR, and presence of cervical cancer among a family member or a friend increased the level of knowledge, yet, none of these factors imposed a conclusive effect on the knowledge score. A significantly higher knowledge score was reported for year 4 students, as well as for students enrolled in Medical and Pharmacy schools than for junior students or students from other colleges. This might be related to the content of curricula exposing students to more in-depth information in topics pertaining to viral infection and cancers. The association between age and knowledge of causes of cervical cancer and transmission of HPV is a reflection of the educational level as shown in studies carried out among nurses from Tanzania and Thailand.39-41 Assessment of knowledge of 289 adults recruited from a local university health service, and 2 family practice clinics in Michigan confirmed that knowledge score is positively correlated with years of education, female gender, and being married.42
Data pertaining to vaccine acceptance demonstrate that most students preferred to obtain the vaccine in hospitals. Despite the relatively high monthly income, around 80% of students thought that the affordable vaccine price should not exceed 300SR. Several studies have confirmed that the price of the vaccine is an important motivating factor to accept the HPV vaccine.43-46 Among Hungarian school children, 80% of respondents would get vaccinated if the vaccine were available free of charge.47 It is worth mentioning that vaccine side effects and fear of injections are perceived barriers for HPV vaccine among a considerable proportion of students enrolled in the present study. Similarly, concerns regarding the vaccine’s side effects and other factors related to vaccine efficacy, age of target population, religious consideration have been reported.48
The limitations of the present study were; the dissimilar proportions of the numbers of respondents in different colleges, also, males and students from non-health colleges are not represented. However, the results provide valuable information on the awareness of Saudi female university students pertaining to cervical cancer. This study is the first phase of a 2 phase project, the second intervention phase will be guided by the results of the present study. Also, this data will be useful for health caregivers in planning community education.
1229www.smj.org.sa Saudi Med J 2014; Vol. 35 (10)
Knowledge regarding cervical cancer and the acceptance of HPV ... Al-Shaikh et al
In conclusion, this study indicates a poor level of knowledge of cervical cancer and misinformation regarding primary and secondary preventive measures among Saudi female students enrolled in health colleges at Princess Nora University in Riyadh City. The data provide a benchmark on the level of knowledge, which can be used to formulate effective awareness program.
References 1. Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer statistics,
2002. CA Cancer J Clin 2005; 55: 74-108. 2. Christoperson WM, Lundin FE Jr, Mendez WM, Parker JE.
Cancer cervical control. Study of morbidity and mortality trends over a twenty-one-year period. Cancer 1976; 38: 1357-1366.
3. Konno R, Sagae S, Yoshikawa H, Basu PS, Hanley SJ, Tan JH, et al. Cervical Cancer Working Group Report. Jpn J Clin Oncol 2010; 40 Suppl 1: i44-i50.
4. Saudi Cancer Registry. Cancer Incidence and Survival Report: Saudi Arabia 2007. In Special Edition. Riyadh (KSA): Saudi Cancer Registry, Ministry of Health; 2011.
5. Jamal A, Al-Maghrabi JA. Profile of Pap smear cytology in the Western region of Saudi Arabia. Saudi Med J 2003; 24: 1225-1229.
6. Bhurgri Y, Bhurgri A, Rahim A, Bhutto K, Pinjani PK, Usman A, et al. The pattern of malignancies in Karachi (1995 to 1996). J Pak Med Assoc 1999; 49: 157-161.
7. Raza SA, Franceschi S, Pallardy S, Malik FR, Avan BI, Zafar A, et al. Human papillomavirus infection in women with and without cervical cancer in Karachi, Pakistan. Br J Cancer 2010; 102: 1657-1660.
8. zur Hausen H. Papillomaviruses in the causation of human cancers-a brief historical account. Virology 2009; 384: 260-265.
9. Bosch FX, Manos MM, Munoz N, Sherman M, Jansen AM, Peto J, et al. Prevalence of human papillomavirus in cervical cancer: a worldwide perspective. International biological study on cervical cancer (IBSCC) Study Group. J Natl Cancer Inst 1995; 87: 796-802.
10. Walboomers JM, Jacobs MV, Manos MM, Bosch FX, Kummer JA, Shah KV, et al. Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. J Pathol 1999; 189: 12-19.
11. Muñoz N, Bosch FX, de Sanjosé S, Herrero R, Castellsagué X, Shah K, Epidemiologic classification of human papillomavirus types associated with cervical cancer. N Engl J Med 2003; 348: 518-527.
12. de Sanjose S, Quint WG, Alemany L, Geraets DT, Klaustermeier JE, Lloveras B,et al. Human papillomavirus genotype attribution in invasive cervical cancer: a retrospective cross-sectional worldwide study. Lancet Oncol 2010; (11): 1048-1056.
13. Bernard HU, Burk RD, Chen Z, van Doorslaer K, Hausen H, de Villiers EM. Classification of papillomaviruses (PVs) based on 189 PV types and proposal of taxonomic amendments. Virology 2010; 401: 70-79.
14. IARC Working Group on the Evaluation of Carcinogenic Risks to Humans. Human papillomaviruses. IARC Monogr Eval Carcinog Risks Hum 2007; 90: 1-636.
15. FUTURE II Study Group. Quadrivalent vaccine against human papillomavirus to prevent high-grade cervical lesions. N Engl J Med 2007; 356: 1915-1927.
16. Paavonen J, Naud P, Salmerón J, Wheeler CM, Chow SN, Apter D, et al. Efficacy of human papillomavirus (HPV)-16/18 AS04-adjuvanted vaccine against cervical infection and precancer caused by oncogenic HPV types (PATRICIA): final analysis of a double-blind, randomised study in young women. Lancet 2009; 374: 301-314.
17. Wang SS, Sherman ME, Hildesheim A, Lacey JV Jr, Devesa S. Cervical adenocarcinoma and squamous cell carcinoma incidence trends among white women and black women in the United States for 1976-2000. Cancer 2004; 100: 1035-1044.
18. Sherman ME1, Wang SS, Carreon J, Devesa SS. Mortality trends for cervical squamous and adenocarcinoma in the United States. Relation to incidence and survival. Cancer 2005; 103: 1258-1264.
19. Mathew A, George PS. Trends in incidence and mortality rates of squamous cell carcinoma and adenocarcinoma of cervix--worldwide. Asian Pac J Cancer Prev 2009; 10: 645-650.
20. el Dosoky M, Ismail N, Dagastani M. Preinvasive cervical carcinoma in Saudi Arabia. Lancet 1995; 345: 650.
21. Manji M. Cervical cancer screening program in Saudi Arabia: action is overdue. Ann Saudi Med 2000; 20: 355-357.
22. Kamzol W, Jaglarz K, Tomaszewski KA, Puskulluoglu M, Krzysztof K. Assessment of knowledge about cervical cancer and its prevention among female students aged 17–26 years. Eur J Obstet Gynecol Reprod Biol 2013; 166: 196-203.
23. Notara V, Soultatou P, Tselika A. Lay knowledge of HPV infection and the vaccine against HPV in Greece. Health Science Journal 2012; 6: 270-279.
24. Kietpeerakool C, Phianmongkhol Y, Jitvatcharanun K, Siriratwatakul U, Srisomboon J. Knowledge, awareness, and attitudes of female sex workers toward HPV infection, cervical cancer, and cervical smears in Thailand. Int J Gynaecol Obstet 2009; 107: 216-219.
25. World Health Organization. Cervical cancer, human papillomavirus (HPV), and HPV vaccines. Geneva: WHO; 2008.
26. Markowitz LE, Dunne EF, Saraiya M, Lawson HW, Chesson H, Unger ER, et al. Quadrivalent Human Papillomavirus Vaccine: Recommendations of the Advisory Committee on Immunization Practices (ACIP). MMWR Recomm Rep 2007; 56 (RR-2): 1-24.
27. Das A, Madhwapathi V, Davies P, Brown G, Dearnley E, Spencer A, et al. Knowledge and acceptability of the HPV vaccine by school children and their parents in Birmingham. Vaccine 2010; 28:1440-1446.
28. Picerno I, Sturniolo G, Spadafora M, Nasso E, Di Pietro A, Spataro P. Secondary school students knowledge and awareness of HPV infections and vaccine. Prev Med 2010; 51: 427-428.
29. Agius PA, Pitts MK, Smith AM, Mitchell A. Human papillomavirus and cervical cancer: Gardasil vaccination status and knowledge amongst a nationally representative sample of Australian secondary school students. Vaccine 2010; 28: 4416-4422.
30. Al-Darwish AA, Al-Naim AF, Al-Mulhim KS, Al-Otaibi NK, Morsi MS, Aleem AM. Knowledge about Cervical Cancer Early Warning Signs and Symptoms, Risk Factors and Vaccination among Students at a Medical School in Al-Ahsa, Kingdom of Saudi Arabia. Asian Pac J Cancer Prev 2014; 15: 2529-2532.
1230
Knowledge regarding cervical cancer and the acceptance of HPV ... Al-Shaikh et al
Saudi Med J 2014; Vol. 35 (10) www.smj.org.sa
31. Sait KH. Knowledge, attitudes, and practices regarding cervical cancer screening among physicians in the Western Region of Saudi Arabia. Saudi Med J 2011; 32: 1155-1160.
32. Roy B, Tricia ST. Cervical cancer screening in Kolkata, India: Beliefs and predictors of cervical cancer screening among women attending a women’s Health Clinic in Kolkata. J Cancer Educ 2008; 23: 253-259.
33. Ackerson K, Gretebeck K. Factors influencing cancer screening practices of underserved women. J Am Acad Nurse Pract 2007; 19: 591-601.
34. Byrd TL, Petterson SK, Chavez R, Heckert A. Cervical cancer screening beliefs among young Hispanic women. Prev Med 2004; 38: 192-197.
35. Doescher MP, Jackson JE. Trends in cervical and breast cancer screening practices among women in rural and urban areas of the United States. Journal of Public Health Management and Practice 2009; 15: 200-209.
36. Nelson W, Moser RP, Gaffey A, Waldron W. Adherence to cervical cancer screening guidelines for U.S. women aged 25–64: data from the 2005 Health Information National Trends Survey (HINTS). Journal of Women’s Health 2009; 18: 1759-1768.
37. Hawkins NA, Cooper CP, Saraiya M, Gelb CA, Polonec L. Why the pap test? Awareness and use of the Pap test among women in the United States. Journal of Women’s Health 2011; 20: 511-515.
38. Smith RA, Cokkinides V, Brooks D, Saslow D, Shah M, Brawley OW. Cancer screening in the United States, 2011: a review of current American Cancer Society guidelines and issues in cancer screening. CA Cancer J Clin 2011; 61: 8-30.
39. Urasa M, Darj E. Knowledge of cervical cancer and screening practices of nurses at a regional hospital in Tanzania. Afr Health Sci 2011;11: 48-57.
40. Duval B, Gilca V, Boulianne N, Pielak K, Halperin B, Simpson M A, et al. Cervical cancer prevention by vaccination: nurses’ knowledge, attitudes and intentions. J Adv Nurs 2009; 65: 499-508.
41. Tessaro I, Herman C. Changes in public health nurses’ knowledge and perception of counselling and clinical skills for breast and cervical cancer control. Cancer Nurs 2000; 23: 401-405.
42. Holcomb B, Bailey JM, Crawford K, Ruffin MT. Adults’ Knowledge and Behaviors Related to Human Papillomavirus Infection. J Am Board Fam Pract 2004; 17: 26-31.
43. Dinas K, Nasioutziki M, Arvanitidou O, Mavromatidis G, Loufopoulos P, Pantazis K, et al. Awareness of human papillomavirus infection, testing and vaccination in midwives and midwifery students in Greece. J Obstet Gynaecol 2009; 29: 542-546.
44. Madhivanan P, Krupp K, Yashodha MN, Marlow L, Klausner JD, Reingold AL. Attitudes toward HPV vaccination among parents of adolescent girls in Mysore, India. Vaccine 2009; 27: 5203-5208.
45. Donders GG, Bellen G, Declerq A, Berger J, Van Den Bosch T, Riphagen I, et al. Change in knowledge of women about cervix cancer, human papillomavirus (HPV) and HPV vaccination due to introduction of HPV vaccines. Eur J Obstet Gynecol Reprod Biol 2009; 145: 93-95.
46. Sauvageau C, Duval B, Gilca V, Lavoie F, Ouakki M. Human papillomavirus vaccine and cervical cancer screening acceptability among adults in Quebec, Canada. BMC Public Health 2007; 7: 304.
47. Marek E, Dergez T, Rebek-Nagy G, Kricskovics A, Kovacs K, Bozsa S,et al. Adolescents’ awareness of HPV infections and attitudes towards HPV vaccination 3 years following the introduction of the HPV vaccine in Hungary. Vaccine 2011; 29: 8591-8598.
Related Articles
Wang YH, Chen DJ, Yi TN, Liu XH. The relationship among human papilloma virus infection, survivin, and p53 gene in lung squamous carcinoma tissue. Saudi Med J 2010; 31: 1331-1336.
Sait KH. Attitudes, knowledge, and practices in relation to cervical cancer and its screening among women in Saudi Arabia. Saudi Med J 2009; 30: 1208-1212.
Jamal A, Al-Maghrabi JA. Profile of Pap smear cytology in the Western region of Saudi Arabia. Saudi Med J 2003; 24: 1225-1229.
Cervical cancer detection by time-resolved spectra of blood components
Rudran KalaivaniVadivel MasilamaniMohamad Saleh AlSalhiSandhanasamy DevanesanP. RamamurthySiddanna R. PalledK. M. Ganesh
Downloaded From: http://biomedicaloptics.spiedigitallibrary.org/ on 03/18/2015 Terms of Use: http://spiedl.org/terms
Cervical cancer detection by time-resolvedspectra of blood components
Rudran Kalaivani,a,b Vadivel Masilamani,a,* Mohamad Saleh AlSalhi,a Sandhanasamy Devanesan,cP. Ramamurthy,d Siddanna R. Palled,e and K. M. Ganeshe
aKing Saud University, Department of Physics and Astronomy, Riyadh 11451, Kingdom of Saudi ArabiabUniversity of Madras, Department of Nuclear Physics, Chennai 600005, IndiacKing Saud University, Research Chair, Laser Diagnosis of Cancers, Kingdom of Saudi ArabiadUniversity of Madras, National Centre for Ultrafast Processes, Taramani Campus, Chennai 600113, IndiaeKidwai Memorial Institute of Oncology, Bangalore 560029, India
Abstract. Fluorescence spectral techniques are very sensitive, and hence they are gaining importance incancer detection. The biomarkers indicative of cancer could be identified and quantified by spectral or timedomain fluorescence spectroscopy. The results of an investigation of time-resolved spectra of cellular compo-nents of blood obtained from cervical cancer patients and normal controls are given. The cancer indicative bio-marker in this paper is porphyrin; it has a fluorescence decay time of 60% more in samples of cancer patientsthan those of normal controls. Based on suchmeasurements, a randomized set comprising samples from cancerpatients and controls (N ¼ 27 in total) could be classified with sensitivity (92%) and specificity (86%).© The Authors.
Published by SPIE under a Creative Commons Attribution 3.0 Unported License. Distribution or reproduction of this work in whole or in part requires full
attribution of the original publication, including its DOI. [DOI: 10.1117/1.JBO.19.5.057011]
Keywords: time-resolved spectra; picosecond laser excitation; cervical cancer detection; blood components; porphyrin biomarker.
Paper 140091R received Feb. 22, 2014; revised manuscript received Apr. 19, 2014; accepted for publication Apr. 23, 2014; publishedonline May 22, 2014.
1 IntroductionOptical biopsy of cancer is a novel, minimally invasive,real-time technique for cancer detection. From laboratory inves-tigations, it has gone to clinical settings in some cases, withspecific examples such as laser endoscopes.1–3
The concept is based on the proven principle that a certain setof fluorescence biomarkers grow out of proportion when normaltissue turns malignant. Fluorescence spectroscopy has twocomplementary approaches: steady-state spectral domain andtransient time-resolved spectral domain.
Steady-state fluorescence spectra are not sufficient to distin-guish between fluorophores that have highly overlapping spec-tral characteristics. Also, differences in the fluorescenceintensities due to quenching by other molecules, aggregationor energy transfer can be difficult to quantify or interpret.Therefore, fluorescence lifetime data are additional parametersto distinguish the spectrally overlapping fluorophores.4–7 Due tothis advantage, there is a considerable interest in time-resolvedmeasurements, and it is a new emerging technique to study thestructure and dynamics of biological molecules. The photophys-ical properties of the intrinsic biomolecules and biostructureshave been considered as a possible parameter that may be relatedto the morphofunctional state of biological substrate. It can alsobe used to identify cancer by using the intrinsic or extrinsicfluorophores.1–7
The commonly used fluorescence lifetime τ depends on theintrinsic characteristics of the fluorophore and also on localenvironments, such as viscosity, pH, refractive index, aggrega-tion as well as interactions with other molecules.4–6 The
time-resolved spectral information is obtained by exciting thesample with a short pulse laser and observing the decay ofthe fluorescence intensity with the time-correlated single photoncounting (TCSPC) technique. Employing this technique, a fewreports had shown significant differentiation between normaland tumor sites of the same organ, in terms of lifetime τ.4–6
In this brief paper, we investigated the possibility of usingtime-resolved fluorescence spectroscopy to differentiate bloodcomponents drawn from normal controls and patients with cer-vical cancer. We reported in our previous papers8–10 on theresults of steady-state measurements of blood components forcancer diagnosis using porphyrin as a biomarker. The resultsshowed a significant discrimination accuracy to classify thediseased blood from normal blood. Such results have motivatedus to study the time-resolved measurements of porphyrin inblood components.
2 Methods and Materials
2.1 Sample Processing
Exactly 5 ml of venous blood from each of fourteen healthywomen (age range 25 to 60 years) was collected in a violet ster-ile vial that contained the anticoagulant ethylenediamine tetra-acetic acid (EDTA). The vial was gently rocked five times toadequately mix the EDTAwith the whole blood, and the samplewas centrifuged at a rotational speed (rpm) of 3000 for 15 min.Clear, pale, greenish-yellow plasma supernatant was obtainedby this centrifugation. A total of 1.5 ml of supernatant plasma(containing mostly proteins and enzymes) and buffy coat (con-taining mostly white blood cells) were removed and discardedleaving the formed elements (FE) undisturbed.
Then, exactly 1 ml of the thick FE from the bottom layer,which contained mostly red blood cells (RBCs), was removed
*Address all correspondence to: Vadivel Masilamani, E-mail: [email protected]
Journal of Biomedical Optics 057011-1 May 2014 • Vol. 19(5)
Journal of Biomedical Optics 19(5), 057011 (May 2014)
Downloaded From: http://biomedicaloptics.spiedigitallibrary.org/ on 03/18/2015 Terms of Use: http://spiedl.org/terms
to a sterile vial and mixed with 2 ml of analytical grade acetone.Proper care was taken to ensure that the FE did not developlumps. After thorough mixing to enable the acetone extractfluorophores within and around the cells, the sample was cen-trifuged again (3000 rpm for 15 min). The resulting supernatantwas subjected to steady-state fluorescence emission spectra(FES) and time-resolved spectra.
The same protocol was used to process blood samples fromthe cervical cancer patients (N ¼ 13, age range from 25 to 60) atthe Government Hospital in Royapettah, Chennai. The classifi-cation of cervical cancer subjects was made by the histopatho-logical examination as early stages (Stage I and Stage II). All ofthe above said patients were confirmed cases of cancer, waitingfor the onset of treatment. They had been informed about theresearch investigation and consent from each had been obtained.Suitable permission had been given from the InternalReview Board.
2.2 Spectral Measurements
Steady-state FES for the acetone extract of samples (control andcervical cancer patients) were obtained by PerkinElmerSpectrofluorometer LS 55. Each sample, taken in a quartzcuvette of 1 cm × 1 cm × 4 cm, was excited at 400 nm, and thefluorescence signal from the sample was collected (transversely)and scanned from 425 to 700 nm. This was the spectral domainanalysis.
The same set of samples was used to obtain transient,excited-state laser-induced fluorescence spectra, employing theTCSPC technique. This was the time domain analysis. Ourattempt was to establish correspondence between these twotypes of spectral analyses.
The fluorescence decay measurements were carried out witha micro-channel plate photomultiplier tube (MCP-PMT) asdetector and a picosecond laser as the excitation source. Thelaser used was mode locked Ti-Sapphire Tsunami laser(Spectra Physics, 3960) capable of generating ps∕fs laser pulsesat 800 nm. Such pulses were passed through the pulse picker(Spectra Physics, 3980 2S) to generate 35 ps 4 MHz pulsesfrom the 82 MHz train of pulses. Following this, the second-harmonic laser (400 nm) output was obtained from the flexibleharmonic generator (Spectra Physics, Santa Clara, California).The vertically polarized 400 nm laser beam was used to excitethe sample.
The fluorescence emission at the magic angle (54.7 deg) wasacquired by an MCP-PMT (Hamamatsu R 3809U, Tokyo,Japan) after passing through the monochromator and processedthrough a constant fraction discriminator, a time to amplitudeconverter, and multichannel analyzer modules. The instrumentresponse function for this system was 50 ps. Since we are inter-ested in studying the porphyrin lifetime of blood components at630 nm, a band-pass filter was used before the PMT to obtainthe desired native fluorescence and to exclude undesired fluo-rescence from other sources.
The measured fluorescence decay was the convolution of thetrue fluorescence decay, the excitation function, and the instru-ment response function. The fluorescence kinetic parameters(lifetime, amplitude, etc.) were obtained by deconvoluting theexcitation function and the instrument response function fromthe measured fluorescence decay function. The data analysiswas carried out by the software provided by the fluorocubeof Horiba Jobin Yuvon (formerly IBH product) IBH (DAS-6),
which was based on the reconvolution technique using iterativenonlinear least-squares methods.11
The fluorescence decay curves obtained from the samplewere fitted to single, double, and triple exponential decay func-tions. The three component approaches improved the fitting, andthe quality of fit was judged by the reduced χ2 value. The threeexponential functions used to fit the data were
IðtÞ ¼ α1 exp
�−
tτ1
�þ α2 exp
�−
tτ2
�þ α3 exp
�−
tτ3
�;
where α1, α2, and α3 were the amplitudes of the componentsat t ¼ 0; τ1, τ2, and τ3 were the decay times.
The fractional contribution of each component to the fluores-cence intensity could be defined as
fi ¼αiτiPjαjτj
:
The average lifetime for the three exponential decay was
τ ¼ α1τ21 þ α2τ
22 þ α3τ
23
α1τ1 þ α2τ2 þ α3τ3:
2.3 Statistical Analysis
Statistical analyses were performed to evaluate the diagnosticpotential of the collected time-resolved spectra of blood compo-nents of normal and cancer patients using ANOVA discriminantanalysis commonly available in the SPSS +11 software. Thistechnique assigns a case to group K, if its score on the functionfor that group is greater than its score on the function for anyother group. This function called the Fisher classification func-tion will give n classification function coefficients and (n − 1)canonical discriminant functions.11 To check the classificationresults, we used “leave-one-out cross-validation” method. Thealgorithm of “leave-one-out cross-validation” involves classify-ing each case into a group according to the classification func-tions computed from all the data except the case beingclassified.12 Such an analysis gave a measure of reliability ofcollected time-resolved data.
3 Results
3.1 Steady-State FES
Figure 1(a) shows the typical FES of acetone extract of cellularcomponents in the normal control. This spectrum has threeimportant fluorescence peaks: one at 500 nm (due to Flavinadenine dinucleotide), next at 585 nm, and third at 630 nm(due to basic and neutral forms of porphyrin). Figure 1(b)shows the typical FES of acetone extract of cellular componentsof a cancer patient. This too has three peaks similar to that ofFig. 1(a), but porphyrin peaks are out of proportion. If we defineratio parameters R1 ¼ I635∕I585, intensities at 635 and 585 nm,it is 1.1 for the control group, but 2.4 for a cancer patient. Thatis, the normal porphyrin is twice elevated for the patient. This istrue for many other types of cancer, too.8–12
Journal of Biomedical Optics 057011-2 May 2014 • Vol. 19(5)
Kalaivani et al.: Cervical cancer detection by time-resolved spectra of blood components
Downloaded From: http://biomedicaloptics.spiedigitallibrary.org/ on 03/18/2015 Terms of Use: http://spiedl.org/terms
3.2 Fluorescence Lifetime Spectra
The native fluorescence lifetime spectra of porphyrin found inthe blood samples of normal patients and cervical cancerpatients were acquired at 630 nm emission under 400 nm exci-tation. Figure 2(a) shows the typical time-resolved spectra fornormal samples. A time decay curve, such as the one shownin Fig. 2(a) may be fitted with single, double, or triple exponen-tial decay functions. For us, it was the three component func-tions that gave the best fit, and the quality of such a fit couldbe judged by the residuals lying between −1.4 and þ1.4 asshown in Fig. 2(b). A similar set of fluorescent decay curvesand residuals for a typical cancer sample are shown inFigs. 3(a) and 3(b), respectively.
The three decay times and their amplitudes for normal andcancer samples are shown in Table 1.
4 DiscussionAs the steady-state fluorescence spectra for blood samples ofcancer patients have been studied extensively,8–12 the discussionwill focus only on one pertinent observation. In the field of fluo-rescence spectroscopy, the relative intensities of different peaksare more important than the actual, absolute intensities. Theyreduce the errors arising out of the quantity and possible turbid-ity of a sample in addition to the instrumental sensitivity varia-tion. The relative intensity between two peaks, say R1, as givenin Fig. 1, depends upon the concentration of the intrinsic natureof the fluorophores under observation and not on sample vol-ume, or RBC concentrations.
The time-resolved spectral parameters of FE of blood sam-ples indicated that the fluorescence at 630 nm emission origi-nated from three molecular species with three characteristiclifetimes. For normal FE, the fast component (component 1)lifetime was 0.035 ns and had the maximum amplitude; the
Fig. 1 (a) Steady-state fluorescence emission spectra (FES) ofacetone extract of formed elements (FE) of normal control.(b) Steady-state FES of acetone extract of FE of cervical cancerpatients. Both are typical spectra.
Fig. 2 (a) Fluorescence decay curve of FE of normal control and(b) with fitted function and residuals.
Fig. 3 (a) Fluorescence decay curve of FE of cervical cancer patientsand (b) fitted functions with residuals.
Table 1 Mean values of lifetimes and their amplitudes of formedelement.
Mean Normal FE Diseased FE
α1 (%) 56.53 31.69
τ1 (ns) 0.035� 0.006 0.091� 0.011
α2 (%) 18.58 20.41
τ2 (ns) 1.14� 0.09 1.6� 0.16
α3 (%) 24.88 47.89
τ3 (ns) 6.0� 0.6 9.31� 1.16
Journal of Biomedical Optics 057011-3 May 2014 • Vol. 19(5)
Kalaivani et al.: Cervical cancer detection by time-resolved spectra of blood components
Downloaded From: http://biomedicaloptics.spiedigitallibrary.org/ on 03/18/2015 Terms of Use: http://spiedl.org/terms
intermediate component (component 2) had the lowest ampli-tude and lifetime was 1.140 ns; and the third component hadthe longest decay time of 6.000 ns. In the cervical cancerFE, component 1 was 0.091 ns and the amplitude was lowwhen compared to normal; the component 2 was 1.6 ns; andthe component 3 was 9.31 ns and the amplitude was foundhigher than for the normal. One may ignore the fast componentvalues (0.035 and 0.091 ns), as the resolution of the instrumentwas only 50 ps. Yet, the other two components were about 1.6times higher for diseased FE than for normal FEs, and hence,such data did have the potential to distinguish the two setsunambiguously.
In an earlier report by Cubeddu et al., porphyrin in normaltissue had a fluorescence lifetime of 10 ns, but in tumor tissues itwas 18 ns.13,14 That is, for this tissue study, the tumor tissue hada 1.8 times longer lifetime than that of normal ones. In anothersimilar study on oral cancer, it was again reported that the con-centration of porphyrin was larger, and it had a longer lifetime.15
It is important to mention here that no other reports ontime-resolved spectra of blood components are available formore precise comparison.
Under normal conditions, the synthesis of protoporphyrin isunder a high feedback control; that is, cells produce it at a ratejust sufficient to match the heme, but in excessive abnormalcell proliferation the feedback mechanism loses its control andthe protoporphyrin that is produced in excess is left unutilizedand released into tissuewhich is carried through,by thecirculatingfluid,blood.Sotheporphyrinconcentrationismorein thediseasedblood components.8–10 The porphyrin which gets accumulatedabnormally on the tumor tissue experiences different local envi-ronment, such as aggregation, higher acidity and also bondingto the intercellular matrix. All these could lead to enhanced life-time,14,15 in tissueas shownbyothers and inblood, as shown in thispaper. It is important to draw attention to two important facts:
1. There is more than one porphyrin (such as copropor-phyrin I, coproporphyrin III, uroporhyrins, etc.) foundin blood and, more importantly, in urine.16 All haveoverlapping spectral profiles, and hence the three com-ponents in decay times could be due to three differenttypes of porphyrins.
2. In the spectral domain also, there are three overlappingprofiles for porphyrin: one at 585 nm, the next at630 nm, and a third at 685 nm attributed to thebasic, neutral, and acidic forms of porphyrins.8,9 Itmight be possible that all these different porphyrinsoccurring in different concentrations could lead tothree different lifetimes. However, an attempt hasnot been made to resolve them in this report, becauseour aim was to assess the diagnostic potential of thetechnique, which may have clinical value in thelong run.
In conclusion, the lifetime of FE porphyrin fit well for threecomponent models. The lifetime values of the porphyrinsavailable in the FE showed a significant difference betweenthe normal and cervical cancer subjects.
4.1 Statistical Results
The statistical analysis was performed as described in Sec. 2 toevaluate the diagnostic potential of the time-resolved spectra of
blood components using porphyrin as a biomarker. One of themultivariate analyses called linear stepwise discriminant analy-sis was used to distinguish the diseased blood components fromnormal blood components. Out of twenty-seven samples, knowncases of normal and cervical cancer (each four in number) wereselected for preliminary measurement of lifetimes; these aver-aged out values are in Table 1. With these values as classificationtools, three component lifetimes obtained from each samplewere given as inputs for the subsequent discriminant analysisfor classification into two separate sets.
4.2 Discriminant Analysis
Discriminant analysis gives the discriminant function, calledfirst canonical discriminant function, that is used to classifythe diseased FE from the normal control FE using discriminantscores. Figure 4 shows the scatter plot between the sample num-ber and the discriminant score obtained from the first canonicaldiscriminant function. It shows that normal and cervical cancerpatients are well separated.
Table 2 shows the classification results of the first discrimi-nant analysis. The classification accuracy for classifying normalsubjects and cervical diseased subjects is 88.9% for the originalcase and 81.5% for the cross-validation case. The discriminantanalysis results show that cervical malignant subjects arediscriminated with a sensitivity of 92.3% and a specificity of
Fig. 4 Plot between the sample number and values of discriminantfunction for normal and cervical cancer subjects.
Table 2 Classification results of discriminant analysis.
Group
Predicted groupmembership
TotalNormal 1 Diseased 2
Original
Count normal 1 12 2 14
Diseased 2 1 12 13
% 1 85.7 14.3 100.0
2 7.7 92.3 100.0
Cross-validated
Count 1 12 2 14
2 3 10 13
% 1 85.7 14.3 100.0
2 23.1 76.9 100.0
Journal of Biomedical Optics 057011-4 May 2014 • Vol. 19(5)
Kalaivani et al.: Cervical cancer detection by time-resolved spectra of blood components
Downloaded From: http://biomedicaloptics.spiedigitallibrary.org/ on 03/18/2015 Terms of Use: http://spiedl.org/terms
85.7% for the original case and sensitivity of 76.9% and speci-ficity of 85.7% for the cross-validated case. In other words,88.9% of the original grouped cases were correctly classified,and 81.5% of cross-validated grouped cases were correctlyclassified.
5 ConclusionIn this short paper on time-resolved spectra of porphyrin foundin the cellular component of blood obtained from cervical cancerpatients and normal controls are reported. It was found that thefluorescence decay could be fitted very well by three exponen-tial functions. The porphyrin of cervical cancer samples hasapproximately 1.6 times longer fluorescence time than that fromthe normal control. Based on the statistical analysis, discrimina-tion was done for a total in 27 samples. This technique hasshown a 92% sensitivity and an 86% specificity. It is importantto mention that this is the only time-resolved spectral study doneon blood components. Although this study was confined to cer-vical cancer, it may be extended to other cancers, too. Such astudy is in progress in our laboratory.
AcknowledgmentsThe authors would like to extend their sincere appreciation tothe Deanship of Scientific Research at King Saud Universityfor its funding of this research through the Research GroupProject No. “RGP-VPP-223.”
References1. T. Glanzmann et al., “Time-resolved spectrofluorometer for clinical
tissue characterization during endoscopy,” Rev. Sci. Instrum. 70(10),4067–4077 (1999).
2. D. W. Thomas and V. D. Jacques, “Optical biopsy: a new frontierin endoscopic detection and diagnosis,” Clin. Gastroenterol.Hepatol. 2(9), 744–753 (2004).
3. H. J. F. Herth, A. Ernst, and H. D. Becker, “Autofluorescence bronchos-copy: a comparison of two systems (LIFE and DLight),” Respiration70(4), 395–398 (2003).
4. Q. Fang et al., “Time-domain laser-induced fluorescence spectroscopyapparatus for clinical diagnostics,” Rev. Sci. Instrum. 75(1), 151–162(2004).
5. J. D. Pitts and M. A. Mycek, “Design and development of a rapidacquisition laser-based fluorometer with simultaneous spectral and tem-poral resolution,” Rev. Sci. Instrum. 72(9), 3061–3072 (2001).
6. M. M. Mycek, K. T. Schomacker, and N. S. Nishioka, “Colonic polypdifferentiation using time-resolved autofluorescence spectroscopy,”Gastrointest. Endosc. 48(4), 390–394 (1998).
7. H. Liu et al., “Raman, fluorescence, and time-resolved light scattering asoptical diagnostic techniques to separate diseased and normal biomedi-cal media,” J. Photochem. Photobiol. B. 16(2), 187–209 (1992).
8. V. Masilamani et al., “Cancer diagnosis by auto fluorescence of bloodcomponents,” J. Lumin. 109(3–4), 143–154 (2004).
9. R. Kalaivani et al., “Fluorescence spectra of blood components forbreast cancer diagnosis,” Photomed. Laser Surg. 26(3), 251–256(2008).
10. V. Masilamani et al., “Fluorescence spectra of blood and urine for cer-vical cancer detection,” J. Biomed. Opt. 17(9), 098001 (2012).
11. R. Kalaivani, “Optical diagnosis of cancer by spectra of body fluid,” Ph.D. Thesis, University of Madras, India (2011).
12. S. Madhuri et al., “Native fluorescence spectroscopy of blood plasma inthe characterization of oral malignancy,” Photochem. Photobiol. 78(2),197–204 (2003).
13. R. Cubeddu et al., “Noninvasive absorption and scattering spectroscopyof bulk diffusive media: an application to the optical characterization ofhuman breast,” Appl. Phys. Lett. 74(6),874–876 (1999).
14. R. Cubeddu et al., “Clinical system for skin tumour detection by fluo-rescence lifetime imaging,” Eng. Med. Biol. 3(3), 2295–2296 (2002).
15. H. M. Chen et al., “Time-resolved auto fluorescence spectroscopy forclassifying normal and premalignant oral tissues,” Lasers Surg. Med.37(1), 37–45 (2005).
16. K. Inoue et al., “Porphyrins as urinary biomarkers for bladder cancerafter 5-aminolevulinic acid (ALA) administration: the potential ofphotodynamic screening for tumors,” Photodiagn. Photodyn. Ther.10(4), 484–489 (2013).
Rudran Kalaivani did her MSc in physics and PhD in physics atUniversity of Madras.
Vadivel Masilamani, PhD, is working as a distinguished professor inlaser physics at KSU and professor in Research Chair in LaserDiagnosis of Cancer. He has over thirty-eight years of research expe-rience in the field of design of gas, liquid and solid-state lasers andtheir applications to medicine and material processing. He has pub-lished 70 research papers and six U.S. patents.
Mohamad Saleh AlSalhi, PhD, is head of the Chair of LaserDiagnosis of Cancer, former vice dean of King Abdullah Institutefor Nanotechnology (KAIN). He has published 96 research papersand six U.S. patents.
Sandhanasamy Devanesan, MPhil, is pursuing his final year PhD,working as a researcher at Research Chair in Laser Diagnosis ofCancer, College of Science, King Saud University. He has sixyears of research experience in the field of cancer biology, opticalbiopsy and hemoglobinopathies. He had published seven researchpapers in international journals.
Siddanna R. Palled, MD, is a radiation oncologist with 10 years ofexperience. He is working at HCG Bangalore Institute of Oncology,and also in Kidwai Memorial Institute of Oncology, Bangalore, India.
Biographies of the other authors are not available.
Journal of Biomedical Optics 057011-5 May 2014 • Vol. 19(5)
Kalaivani et al.: Cervical cancer detection by time-resolved spectra of blood components
Downloaded From: http://biomedicaloptics.spiedigitallibrary.org/ on 03/18/2015 Terms of Use: http://spiedl.org/terms
Evaluating the Association between p53 Codon 72Arg.Pro Polymorphism and Risk of Ovary Cancer: AMeta-AnalysisMohammed A. A. Alqumber1, Naseem Akhter1, Shafiul Haque2, Aditya K. Panda3, Raju K. Mandal4*
1 Department of Laboratory Medicine, Faculty of Applied Medical Sciences, Albaha University, Albaha, Saudi Arabia, 2 Department of Biosciences, Jamia Millia Islamia (A
Central University), New Delhi, India, 3 Department of Infectious Disease Biology, Institute of Life Sciences, Bhubaneswar, Odisha, India, 4 Department of Urology, Sanjay
Gandhi Post Graduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India
Abstract
Aim: Allelic polymorphism in codon 72 of the p53 tumor suppressor gene causes imbalance of p53 protein expression.Earlier studies have shown association between allelic polymorphism in codon 72 of the p53 gene with risk of ovary cancer(OC); however the results are inconclusive and conflicting. Therefore, we performed this meta-analysis to investigate therelation between p53 codon 72 Arg.Pro polymorphism and overall OC susceptibility.
Methods: We searched all eligible published studies based on the association between codon 72 of the p53 Arg.Propolymorphism and risk of OC. Data were pooled together from individual studies and meta-analysis was performed. Pooledodds ratios (ORs) and 95% CI were calculated for allele contrast, homozygous, heterozygous, dominant and recessivegenetic models.
Results: A total of twelve studies comprising of 993 OC cases and 1264 healthy controls were included in this meta-analysis.Overall, no significant association was detected for Pro allele carrier (Pro vs. Arg: p = 0.916; OR = 0.980, 95% CI = 0.677 to1.419), homozygous (Pro/Pro vs. Arg/Arg: p = 0.419; OR = 0.731, 95% CI = 0.341 to 1.564), heterozygous (Arg/Pro vs. Arg/Arg:p = 0.248; OR = 1.237, 95% CI = 0.862 to 1.773), dominant (Pro/Pro+Arg/Pro vsArg/Arg: p = 0.699; OR = 1.089, 95% CI = 0.706to 1.681), and recessive (Pro/Pro vs Arg/Arg+Arg/Pro: p = 0.329; OR = 0.754, 95% CI = 0.428 to 1.329) genetic models,respectively. Also, in the stratified analysis by ethnicity, no significant association of this polymorphism with risk of OC wasfound in the Caucasian population.
Conclusions: This meta-analysis suggested that codon 72 of the p53 Arg.Pro polymorphism may not significantlycontribute in ovary cancer susceptibility. However, future large studies with gene-gene and gene-environment interactionsare needed to validate these findings.
Citation: Alqumber MAA, Akhter N, Haque S, Panda AK, Mandal RK (2014) Evaluating the Association between p53 Codon 72 Arg.Pro Polymorphism and Risk ofOvary Cancer: A Meta-Analysis. PLoS ONE 9(4): e94874. doi:10.1371/journal.pone.0094874
Editor: Klaus Roemer, University of Saarland Medical School, Germany
Received January 2, 2014; Accepted March 20, 2014; Published April 18, 2014
Copyright: � 2014 Alqumber et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The authors have no support or funding to report.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: [email protected]
Introduction
Ovary cancer (OC) is the most common carcinoma among
females with poor prognosis. It is the sixth leading cause of death
among gynecological malignancies in females worldwide [1,2].
The etiology of OC is still unclear and epidemiological studies
have suggested that susceptibility to OC of an individual is
influenced by several genetic factors [3]. However, there is no
thorough screening technique for this malignancy, indicating that
the identification of a gene related to the risk of OC may improve
the early diagnosis and prevention of this deadly disease.
The p53 tumor suppressor gene (TP53 at 17p13), recognized as
‘‘the guardian of the genome’’, plays a significant role in the cell
cycle arrest, senescence, DNA damage repair, regulates the cell
cycle and requires loss of function mutations for tumor formation
[4].
The p53 protein functions by interfering with central regulators
of hypoxia which mediate angiogenesis, and eventually inhibit
production of pro-angiogenic factors and endogenous angiogenesis
inhibitors [5,6,7]. The ability of p53 to eliminate excess, damaged
or infected cells by apoptosis is essential for the proper regulation
of cell proliferation in multi-cellular organisms. Differential
expression of p53 in various cancers and association of serum
p53 levels with malignant tumors highlights the significance role of
p53 in malignancy [8,9]. The major modes of TP53 inactivation
are single-base substitutions and loss of alleles, with inactivation by
viral or cellular proteins [10].
Several polymorphisms have been detected in both coding and
non-coding region of this gene [11]. An important single
nucleotide germ line polymorphism in the proline- rich domain
of exon 4 of p53 gene induces an arginine to proline residue
change at amino acid position 72 [12]. The two polymorphic
PLOS ONE | www.plosone.org 1 April 2014 | Volume 9 | Issue 4 | e94874
forms (Pro72 and Arg72) of p53 gene have different primary
structures, electrophoretic migration and functional properties
[13]. The arginine (Arg72) allele increases the ability of p53 to
locate to mitochondria and induce cellular death, whereas proline
allele (Pro72) impart a lower apoptotic potential and an increased
cellular arrest in G1 phase of the cell cycle [14].
Considering the functional significance of p53 gene in
carcinogenesis, it is speculated that codon 72 Arg.Pro polymor-
phism may be a potential susceptibility factor for OC. Lately,
several epidemiological case-control studies have evaluated the
association between p53codon 72 Arg.Pro polymorphisms and
OC risk [15–26]. Despite several studies globally, the putative
association between p53 codon 72 Arg.Pro genetic polymor-
phism and OC risk remains uncertain and lacks consensus.
Therefore, to derive a more precise conclusion of the possible
association between p53 codon 72 Arg.Pro polymorphism and
OC risk, a meta-analysis was performed based on eligible
published studies.
Materials and Methods
Publication search strategyWe carried out a PubMed (Medline), EMBASE and Google
Scholar web database search covering all research articles
published with a combination of the following key words: ‘p53
gene (polymorphism OR mutation OR variant) AND ovarian
carcinoma or ovary cancer, tumor susceptibility (last updated on
November 2013). All the searched studies were retrieved and their
reference lists were checked as well for other relevant studies.
When, more than one of the same population was included in
several publications, only the most recent or complete study was
included in this meta-analysis. Since, this is a meta-analysis of
published articles based on the association of p53 codon 72
Arg.Pro polymorphism and OC risk, so ethical approval was not
required for this study.
Inclusion and exclusion criteriaIn order to minimize heterogeneity and ease the appropriate
interpretation of this study, published articles included in the
current meta-analysis had to meet all the following inclusion
criteria: a) studies should have a cross-sectional, case-control or
cohort design, b) must evaluated the association between p53
codon 72 Arg.Pro polymorphism and OC risk, c) recruited
pathologically or histologically confirmed OC patients and healthy
controls, d) have available genotype frequency in case and control,
e) and published in English language. Also, when the case-control
study was included by more than one research article using the
same case series, we selected the study that included the largest
number of individuals. On the other side, the major reasons for
study exclusion were, overlapping of the data, case-only studies,
review articles, and genotype frequencies or numbers are not
reported. The study selection procedure has been shown in the
form of flow-diagram as Figure S1 (PRISMA Flow Diagram).
Data extraction and quality assessmentFor each retrieved research publication, the methodological
quality assessment and data extraction were independently
abstracted in duplicate using a standard protocol by two
independent investigators. Data-collection form was used to
guarantee the accuracy of the collected data by stringently
following the inclusion-exclusion criteria mentioned above. The
main characteristics abstracted from the retrieved studies included
the name of the first author, publication year, the country of
origin, the number of cases and controls, study type, and genotype
frequencies. Cases associated with disagreement on any item of the
data from the collected research studies were fully debated with
investigators to achieve a final consensus.
Statistical analysisIn order to examine the relationship between p53 codon 72
Arg.Pro polymorphism and OC risk, pooled ORs and their
corresponding 95% CIs were estimated. Heterogeneity assump-
tion was examined by the chi-square-based Q-test [27]. Hetero-
geneity was considered significant when p-value,0.05. The data
from single comparison was pooled using fixed effects model [28]
when no heterogeneity presented. Otherwise, the random-effects
model [29] was used for pooling purpose. Additionally, I2 statistics
was employed to quantify inter-study variability and larger values
suggested an increasing degree of heterogeneity [30]. Hardy-
Weinberg equilibrium (HWE) in the controls was calculated via
chi-square test. Funnel plot asymmetry was estimated by Egger’s
linear regression test which is a type of linear regression
methodology to measure the funnel plot asymmetry on the natural
logarithm scale of the OR. The significance of the intercept was
determined by the t-test considering p-value,0.05 as representa-
tion of statistically significant publication bias [31]. Also, the
subgroup analysis was carried out by the ethnicity, and the
ethnicity was defined mainly as Caucasians. A comparative
examination of ‘meta-analysis’ softwares was performed by using
url address http://www.meta-analysis.com/pages/comparisons.
html. The Comprehensive Meta-Analysis (CMA) Version 2
software program (Biostat, USA) was chosen and utilized to
perform all statistical analysis involved in this study.
Results
Characteristics of included studiesAccording to our selection (inclusion-exclusion) criteria, a total
of twelve research articles were finally included through literature
search from the PubMed (Medline), EMBASE and Google
Scholar web databases in this meta-analysis. All retrieved research
publications were examined carefully by reading the titles and
abstracts, and the full texts for the potentially relevant research
articles were further checked for their aptness for the current meta-
analysis. Studies either showing p53 codon 72 Arg.Pro polymor-
phism to predict survival in OC patients or considering p53
variants as an indicators for response to therapy were excluded
straightaway. Similarly, studies investigating the levels of p53
mRNA or protein expression or relevant review articles were also
excluded. In the present meta-analysis, only case-control or cohort
design studies having frequency of all three genotypes were
included. Besides the database search, the reference lists present in
the retrieved articles were also checked for other potential research
publications (Table 1). Distribution of genotypes, minor allele
frequency (MAF) and HWE in the controls and cases have been
presented in Table 2.
Publication biasBegg’s funnel plot and Egger’s test were carried out to evaluate
the publication bias among the selected studies for the meta-
analysis. The appearance of the shape of funnel plots was seemed
to be symmetrical in all the genetic models. The Egger’s test was
performed to provide the statistical evidence of funnel plot. The
results showed lack of publication bias among all comparison
models (Table 3).
Association of p53 Variant with Ovary Cancer
PLOS ONE | www.plosone.org 2 April 2014 | Volume 9 | Issue 4 | e94874
Evaluation of heterogeneityIn order to analyze heterogeneity among the selected studies, Q-
test and I2 statistics were employed and heterogeneity was noticed
in all the five genetic models. Therefore, random effects model was
applied to synthesize the data (Table 3).
Association of p53 codon 72 Arg.Pro polymorphismand OC susceptibility
We pooled all the twelve studies together and it resulted into
1264 controls and 993 OC cases, to review the overall association
between p53 codon 72 Arg.Pro polymorphism and OC risk.
Overall pooled analysis did not suggest any correlation between
p53 codon 72 Arg.Pro polymorphism and OC risk in all the five
genetic comparison models, i.e., allele (Pro vs. Arg: p = 0.916;
OR = 0.980, 95% CI = 0.677 to 1.419), homozygous (Pro.Pro vs.
Arg.Arg: p = 0.419; OR = 0.731, 95% CI = 0.341 to 1.564),
heterozygous (Arg.Pro vs. Arg.Arg: p = 0.248; OR = 1.237, 95%
CI = 0.862 to 1.773), recessive (Pro.Pro vs. Arg.Arg+Arg.Pro:
p = 0.329; OR = 0.754, 95% CI = 0.428 to 1.329) and dominant
model (Pro.Pro+Arg.Pro vs. Arg.Arg: p = 0.699; OR = 1.089, 95%
CI = 0.706 to 1.681) (Figure 1).
Subgroup analysis of racial descentWe have analyzed only Caucasian population by study design
and participants. This meta-analysis included eight studies (717
cases and 797 controls), Heterogeneity was observed in all genetic
Table 1. Characteristics of studies included in this meta-analysis.
First Authors and year Country of origin Study Design Cases Controls Source of genotyping
Dholariya et al. 2013 [15] India HB 100 100 Blood
Malisic et al. 2013 [16] Serbia HB 47 70 Tissue
Matei et al. 2012 [17] Romania HB 21 21 Blood
Ueda et al. 2006 [18] Japan HB 68 95 Blood
Morari et al. 2006 [19] Brazil HB 69 222 Blood
Santos et al. 2006 [20] Portugal HB 99 188 Blood
Agorastos et al. 2004 [21] Greece HB 51 30 Cytobrush
Pegoraro et al. 2003 [22] South Africa HB 85 340 Blood
Hogdall et al. 2002 [23] Denmark HB 211 83 Blood
Li et al. 2002 [24] China HB 39 50 Tissue
Buller et al. 1997 [25] America HB 190 52 Blood
Peller et al. 1999 [26] Israel HB 13 13 Blood
doi:10.1371/journal.pone.0094874.t001
Table 2. Distribution of p53 polymorphism of twelve studies included in the meta-analysis.
Authors andyear Control Case HWE
GenotypeMinorallele Genotype
Minorallele
Arg/Arg Arg/Pro Pro/Pro MAF Arg/Arg Arg/Pro Pro/Pro MAF HWEF
Dholariya et al.2013
62 32 6 0.22 33 50 17 0.42 0.49
Malisic et al. 2013 45 22 3 0.2 22 22 3 0.29 0.88
Matei et al. 2012 7 7 7 0.5 9 6 6 0.42 0.12
Ueda et al. 2006 34 54 7 0.35 21 41 6 0.38 0.02
Morari et al. 2006 117 91 14 0.26 23 46 0 0.33 0.51
Santos et al. 2006 117 58 13 0.22 49 40 10 0.30 0.12
Agorastos et al.2004
6 19 25 0.69 26 22 3 0.27 0.42
Pegoraro et al.2003
32 147 161 0.68 14 41 30 0.59 0.85
Hogdall et al.2002
48 27 8 0.25 118 73 20 0.26 0.16
Li et al. 2002 10 26 14 0.54 14 20 5 0.38 0.74
Buller et al. 1997 30 18 4 0.25 98 79 13 0.27 0.57
Peller et al. 1999 8 5 0 0.19 7 6 0 0.23 0.39
doi:10.1371/journal.pone.0094874.t002
Association of p53 Variant with Ovary Cancer
PLOS ONE | www.plosone.org 3 April 2014 | Volume 9 | Issue 4 | e94874
models; thus, random effect model was applied to analyze the
data. In subgroup analysis publication bias did not exist (Table 4).
We did not observe any association of p53 codon 72 Arg.Pro
polymorphism with OC risk in Caucasian population in all genetic
comparison models, i.e., allele (Pro vs. Arg: p = 0.542;
OR = 0.862, 95% CI = 0.535 to 1.388), homozygous (Pro.Pro vs.
Arg.Arg: p = 0.307; OR = 0.630, 95% CI = 0.260 to 1.527),
heterozygous (Arg.Pro vs. Arg.Arg: p = 0.781; OR = 1.059, 95%
CI = 0.707 to 1.586), recessive (Pro.Pro vs. Arg.Arg+Arg.Pro:
p = 0.259; OR = 0.686, 95% CI = 0.356 to 1.320) and dominant
model (Pro.Pro+Arg.Pro vs. Arg.Arg: p = 0.710; OR = 0.905, 95%
CI = 0.534 to 1.532) (Figure 2)
Table 3. Statistics to test publication bias and heterogeneity in the present meta-analysis.
Comparisons Egger’s regression analysis Heterogeneity analysisModel used forthe meta-analysis
Intercept95% ConfidenceInterval p-value Q-value Pheterogeneity I2 (%)
Pro vs. Arg 21.56 27.40 to 4.26 0.56 70.75 ,0.0001 84.45 Random
Pro/Pro vs. Arg/Arg 21.61 26.48 to 3.26 0.47 45.35 ,0.0001 77.95 Random
Arg/Pro vs. Arg/Arg 22.72 25.95 to 0.51 0.09 30.12 0.002 63.48 Random
Pro/Pro+Arg/Pro vs. Arg/Arg 23.38 27.58 to 0.82 0.10 48.78 ,0.0001 77.45 Random
Pro/Pro vs. Arg/Arg+Arg/Pro 20.49 23.70 to 2.72 0.73 31.08 0.001 67.83 Random
doi:10.1371/journal.pone.0094874.t003
Figure 1. Forest plot of OR with 95% CI of ovary cancer associated with the p53 codon 72 Arg.Pro gene polymorphism. Black squarerepresent the value of OR and the size of the square indicates the inverse proportion relative to its variance. Horizontal line is the 95% CI of OR. Thestudies are listed by year of publication.doi:10.1371/journal.pone.0094874.g001
Association of p53 Variant with Ovary Cancer
PLOS ONE | www.plosone.org 4 April 2014 | Volume 9 | Issue 4 | e94874
Discussion
The p53 tumor suppressor gene, mutated in many human
cancer types, suggests its key role in the host’s defense against
malignancy [6]. The cellular level of the p53 protein is regulated in
a complex manner by a negative feedback loop involving ubiquitin
medicated degradation [32]. Based upon the nature of the genetic
insult, wild-type p53 induces either growth arrest or apoptosis.
Any alterations within p53 gene restrict these activities and allow
the continuous proliferation of cells, ultimately resulting in the
progression to malignancy [33].
Figure 2. Forest plot of OR with 95% CI of ovary cancer associated with the p53 codon 72 Arg.Pro gene polymorphism accordingto the ethnicity group by the random effect model. Black square represent the value of OR and the size of the square indicates the inverseproportion relative to its variance. Horizontal line is the 95% CI of OR. The studies are listed by year of publication.doi:10.1371/journal.pone.0094874.g002
Table 4. Statistics to test publication bias and heterogeneity in the present meta-analysis.
Comparisons Egger’s regression analysis Heterogeneity analysisModel used forthe meta-analysis
Intercept95% ConfidenceInterval p-value Q value Pheterogeneity I2 (%)
Pro vs. Arg 20.65 27.59 to 6.28 0.82 44.95 ,0.0001 84.43 Random
Pro/Pro vs. Arg/Arg 21.29 29.14 to 6.54 0.68 26.87 ,0.0001 77.67 Random
Arg/Pro vs. Arg/Arg 21.69 25.29 to 1.91 0.29 14.34 0.04 51.19 Random
Pro/Pro+Arg/Pro vs. Arg/Arg 22.34 27.37 to 2.68 0.29 27.67 ,0.0001 74.70 Random
Pro/Pro vs. Arg/Arg+Arg/Pro 20.17 24.97 to 4.63 0.93 18.46 0.005 67.51 Random
doi:10.1371/journal.pone.0094874.t004
Association of p53 Variant with Ovary Cancer
PLOS ONE | www.plosone.org 5 April 2014 | Volume 9 | Issue 4 | e94874
Despite remarkable progress in mechanistic understanding of
p53 structure and function, the contribution of specific p53 gene
polymorphisms to OC risk remains equivocal and proven to be
extremely complex biomarkers. Interest in the genetic susceptibil-
ity to OC has led to an emerging trend to the study of
polymorphisms of genes involved in OC risk. Due to the different
roles of p53 gene in human genome, it has been hypothesized that
codon 72 Arg.Pro polymorphism is associated with risk of OC.
As a result, a large number of studies have been performed to
evaluate the association between p53 codon 72 Arg.Pro
polymorphism and risk of OC, but the results from different
published studies lacks consensus. Inconsistency in results from
these studies can be attributed to low statistical power to evaluate
the overall effect of the p53 codon 72 Arg.Pro polymorphism
with OC risk. The answer to this limitation is a meta-analysis, a
powerful tool for investigating the risk factors associated with
genetic diseases, which employs quantitative technique to pool the
data from individual studies where individual sample sizes are
small with lower statistical power, and provides reliable conclusion
[34]. Hence, we have done the present meta-analysis from twelve
eligible published case-control studies to evaluate the said relation
of p53 codon 72 Arg.Pro polymorphism and risk of OC. This
study might help to explore a more robust estimate about the role
of this polymorphism with OC risk, as combining data from many
studies has the advantage of reduced random errors [35].
The overall pooled results of this meta-analysis revealed that
p53 codon 72 Arg.Pro polymorphism did not influence an
increased or decreased risk of OC in all the five genetic models as
per the eligible studies when compared with wild type allele. Even
in the stratified analysis by ethnicity, no statistically significant
relationship between p53 codon 72 Arg.Pro genotype and OC
risk was detected in Caucasian population. One possible
explanation is that several other SNPs have been reported in the
p53 gene and previous research showed that various other SNPs
are related to the susceptibility to OC. It is possible that the
analyzed variant does not act as primary susceptibility polymor-
phism and may be inhibiting p53 function by linking with other
functional polymorphism alleles found in linkage disequilibrium
(LD). Our results are in agreement with Shen et al. [36], whereas
Zhang et al. 2008 has reported a decreased risk [37]. Moreover,
pooled and meta-analyses for breast [38], lung [39] and
endometrial [40] cancers do not support a significant role for this
polymorphism in susceptibility. Susceptibility to OC is a multistep
process in which environmental and genetic factors interact closely
and a single genetic variant is usually insufficient to predict the risk
of this deadly disease.
Heterogeneity between studies is very common in the genetic
association studies of meta-analysis. In the present meta-analysis
we found inter-study heterogeneity in overall analysis. There are
several factors responsible for such heterogeneity, i.e., the genetic
backgrounds for cases and controls, diverse genotype distribution
of codon 72 Arg.Pro in different ethnic groups and uneven
selection criteria for the cases and controls in different studies.
Despite the important findings from our current analysis, we still
have to acknowledge some limitations of this study. First, we only
included studies published in English language, abstracted and
indexed by the selected electronic databases for the data analysis; it
is possible that some pertinent reports published in other languages
and indexed in other electronic databases may have missed.
Second, the result of this meta-analysis was based on unadjusted
ORs because not all eligible studies stated adjusted ORs. Third,
the role of gene-environment interactions were not considered
which may affect the risk of OC. Also, it is worthwhile to mention
several strengths of our study. First, we have included significantly
more number of cases and controls comparison to the previous
meta-analysis study by using effective and efficient search strategy
to increase the statistical power of the analysis. Second, the quality
of the case-control studies included in the present pooling analysis
was satisfactory and met with the pre-set inclusion criteria.
Conclusion
In conclusion, a meta-analysis is a rational approach of data-
analysis which pools both statistically significant and non-
significant findings from individual studies to improve the
statistical performance by increasing the sample size. Our meta-
analysis demonstrates that p53 codon 72 Arg.Pro polymorphism
might not significantly modulate the OC risk. However, future
well designed large studies, particularly stratified by gene-gene and
gene-environment interactions might be necessary to clarify the
possible role of the p53 codon 72 Arg.Pro polymorphism in the
susceptibility to OC.
Supporting Information
Figure S1 PRISMA 2009 Flow Diagram. Showing identifi-
cation and selection of studies for the meta-analysis.
(TIF)
Checklist S1 PRISMA 2009 Checklist.
(DOC)
Acknowledgments
We thank Institute of Life Sciences (Bhubaneswar, India) and Albaha
University (Albaha, Saudi Arabia) for providing the software related
support in data-analysis.
Author Contributions
Conceived and designed the experiments: MAAA NA SH RKM.
Performed the experiments: MAAA AKP RKM. Analyzed the data:
AKP RKM. Contributed reagents/materials/analysis tools: NA SH AKP.
Wrote the paper: RKM SH.
References
1. Clarke-Pearson DL (2009) Clinical practice. Screening for ovarian cancer.
N Engl J Med 361:170–177.
2. Jemal A, Bray F, Center MM, Ferlay J, Ward E, et al. (2011) Global cancer
statistics. CA Cancer J Clin 61(2):69–90.
3. Diaz-Padilla I, Amir E, Marsh S, Liu G, Mackay H (2012) Genetic
polymorphisms as predictive and prognostic biomarkers in gynecological
cancers: a systematic review. Gynecol Oncol 124(2):354–365.
4. Berchuck A, Kohler MF, Marks JR, Wiseman R, Boyd J, et al. (1994) The p53
tumor suppressor gene frequently is altered in gynecologic cancers. Am J Obstet
Gynecol 170(1 Pt 1):246–252.
5. Donehower LA, Harvey M, Slagle BL, McArthur MJ, Montgomery CA Jr, et al.
(1992) Mice deficient for p53 are developmentally normal but susceptible to
spontaneous tumours. Nature 356(6366):215–221.
6. Hollstein M, Sidransky D, Vogelstein B, Harris CC (1991) p53 mutations in
human cancers. Science 253(5015):49–53.
7. Greenblatt MS, Bennett WP, Hollstein M, Harris CC (1994) Mutations in the
p53 tumor suppressor gene: clues to cancer etiology and molecular pathogenesis.
Cancer Res 54(18):4855–4878.
8. Lane DP (1994) On the expression of the p53 protein in human cancer. Mol Biol
Rep 19(1):23–29.
9. Wu M, Mao C, Chen Q, Cu XW, Zhang WS (2010) Serum p53 protein and
anti-p53 antibodies are associated with increased cancer risk: a case-control
study of 569 patients and 879 healthy controls. Mol Biol Rep 37(1):339–343.
10. Tommasino M, Accardi R, Caldeira S, Dong W, Malanchi I, et al. (2003) The
role of TP53 in Cervical carcinogenesis. Hum Mutat 21(3):307–312.
Association of p53 Variant with Ovary Cancer
PLOS ONE | www.plosone.org 6 April 2014 | Volume 9 | Issue 4 | e94874
11. Olivier M, Eeles R, Hollstein M, Khan MA, Harris CC, et al. (2002) The IARC
TP53 database: new online mutation analysis and recommendations to users.
Hum Mutat 19(6):607–614.
12. Whibley C, Pharoah PD, Hollstein M (2009) p53 polymorphisms: cancer
implications. Nat Rev Cancer 9(2):95–107.
13. Thomas M, Kalita A, Labrecque S, Pim D, Banks L, et al. (1999) Two
polymorphic variants of wild-type p53 differ biochemically and biologically. Mol
Cell Biol 19(2):1092–1100.
14. Pim D, Banks L (2004) p53 polymorphic variants at codon 72 exert different
effects on cell cycle progression. Int J Cancer 108(2):196–199.
15. Dholariya S, Zubari M, Ray PC, Gandhi G, Khurana N, et al. (2013) TP53
Gene Polymorphism in Epithelial Ovarian Carcinoma Patients from North
Indian Population and its Pro/Pro Variant is Potentially Contributing to Cancer
Susceptibility. J Genet Syndr Gene Ther 4(5)1–6.
16. Malisic EJ, Jankovic RN, Jakovljevic KV, Radulovic SS (2013) Association of
TP53 codon 72 polymorphism with susceptibility to ovarian carcinomas in
Serbian women. Eur J Obstet Gynecol Reprod Biol 166(1):90–93.
17. Matei MC, Negura L, Liliac L, Negura A, Azoicai D (2012) Validation of PCR-
RFLP techniques for the evaluation of codon 72 of p53 and CYP1A1 gene’s
polymorphisms in relation with ovarian cancer in a Romanian population.
Rom J Morphol Embryol 53(1):47–54.
18. Ueda M, Terai Y, Kanda K, Kanemura M, Takehara M, et al. (2006) Germline
polymorphism of p53 codon 72 in gynecological cancer. Gynecol Oncol
100(1):173–178.
19. Morari EC, Lima AB, Bufalo NE, Leite JL, Granja F, et al. (2006) Role of
glutathione-S-transferase and codon 72 of P53 genotypes in epithelial ovarian
cancer patients. J Cancer Res Clin Oncol 132:521–528.
20. Santos AM, Sousa H, Pinto D, Portela C, Pereira D, et al. (2006) Linking TP53
codon 72 and P21 nt590 genotypes to the development of cervical and ovarian
cancer. Eur J Cancer 42(7):958–963.
21. Agorastos T, Masouridou S, Lambropoulos AF, Chrisafi S, Miliaras D, et al.
(2004) P53 codon 72 polymorphism and correlation with ovarian and
endometrial cancer in Greek women. Eur J Cancer Prev 13(4):277–280.
22. Pegoraro RJ, Moodley M, Rom L, Chetty R, Moodley J (2003) P53 codon 72
polymorphism and BRCA 1 and 2 mutations in ovarian epithelial malignancies
in black South Africans. Int J Gynecol Cancer 13(4):444–449.
23. Høgdall EV, Høgdall CK, Christensen L, Glud E, Blaakaer J, et al. (2002)
Distribution of p53 codon 72 polymorphisms in ovarian tumour patients and
their prognostic significance in ovarian cancer patients. Anticancer Res
22(3):1859–1864.
24. Li T, Lu ZM, Guo M, Wu QJ, Chen KN, et al. (2002) p53 codon 72
polymorphism (C/G) and the risk of human papillomavirus-associatedcarcinomas in China. Cancer 95(12):2571–2576.
25. Buller RE, Sood A, Fullenkamp C, Sorosky J, Powills K, et al. (1997) The
influence of the p53 codon 72 polymorphism on ovarian carcinogenesis andprognosis. Cancer Gene Ther 4(4):239–245.
26. Peller S, Halperin R, Schneider D, Kopilova Y, Rotter V (1999) Polymorphismsof the p53 gene in women with ovarian or endometrial carcinoma. Oncol Rep
6(1):193–197.
27. Wu R, Li B (1999) A multiplicative-epistatic model for analyzing interspecificdifferences in outcrossing species. Biometrics 2:355–365.
28. Mantel N, Haenszel W (1959) Statistical aspects of the analysis of data fromretrospective studies of disease. J Natl Cancer Inst 4:719–748.
29. DerSimonian R, Laird N (1986) Meta-analysis in clinical trials. Control ClinTrials 7:177–188.
30. Higgins JP, Thompson SG, Deeks JJ, Altman DG (2003) Measuring
inconsistency in meta-analyses. BMJ 7414:557–560.31. Egger M, Davey Smith G, Schneider M, Minder C (1997) Bias in meta-analysis
detected by a simple, graphical test. BMJ 7109:629–634.32. Vousden KH, Lu X (2002) Live or let die: the cell’s response to p53. Nat Rev
Cancer 2(8):594–604.
33. Vogelstein B, Kinzler KW (1992) p53 function and dysfunction. Cell 70(4):523–526.
34. Cohn LD, Becker BJ (2003) How meta-analysis increases statistical power.Psychol Methods 3:243–253.
35. Bouillon R, Carmeliet G, Verlinden L, van Etten E, Verstuyf A, et al. (2008)Vitamin D and Human Health: Lessons from Vitamin D Receptor Null Mice.
Endocrine Reviews 29(6):726–776.
36. Shen SQ, Jiang DK, Liu GY, Chen F, Yu L (2012) Meta-analysis showssignificant association of the TP53 Arg72Pro with ovarian cancer risk. Mol Biol
Rep 39(4):4683–4690.37. Zhang Z, Fu G, Wang M, Tong N, Wang S, et al. (2008) P53 codon 72
polymorphism and ovarian cancer risk: a meta-analysis. JNMU 22(5):279–285.
38. Schmidt MK, Reincke S, Broeks A, Braaf LM, Hogervorst FB, et al. (2007)Breast Cancer Association Consortium. Do MDM2 SNP309 and TP53 R72P
interact in breast cancer susceptibility? A large pooled series from the breastcancer association consortium. Cancer Res 67(19):9584–9590.
39. Matakidou A, Eisen T, Houlston RS (2003) TP53 polymorphisms and lungcancer risk: a systematic review and meta-analysis. Mutagenesis 18(4):377–385.
40. Tang W, He X, Chan Y, Luo Y (2012) Lack of association between p53 codon
72 polymorphism and endometrial cancer: a meta-analysis. Cancer Epidemiol36(3):e153–7.
Association of p53 Variant with Ovary Cancer
PLOS ONE | www.plosone.org 7 April 2014 | Volume 9 | Issue 4 | e94874
Open Access
1356 Pak J Med Sci 2014 Vol. 30 No. 6 www.pjms.com.pk
INTRODUCTION
Thyroid hormone plays an imperative role in the regulation of cellular metabolism, proliferation and differentiation.1 There are two basic forms of thyroid hormone: T4 (3,5,3’,5’-tetraiodothyronine) and T3
(3, 3’,5-triiodothyronine). The two forms of thyroid hormone (T3, T4) are produced and secreted by the follicular cells of the thyroid gland.2
Thyroid hormones mediate their action on different types of cells (plasma membrane, nucleus, cytoplasm and in the mitochondria) in different ways. For example, the biological activities of T3 are regulated by thyroid hormone nuclear receptors (TRs) via transcriptional regulation. There are two different TR genes in humans; α and β which encode for different T3-binding receptor isoforms (α1, β1, β2 and β3). Any mutations in the TRβ gene decrease the sensitivity of the target tissue of the thyroid hormone.3 However, the transcriptional activity of T3 can be upregulated by many factors which include host of nuclear co-regulatory proteins, the type of thyroid hormone response elements
Correspondence:
Dr. Muhammad Imran Naseer, Center of Excellence in Genomic Medicine Research (CEGMR), Post Box No. 80216, King Abdulaziz University, Jeddah-21589, Saudi Arabia. E-mail: [email protected]
* Received for Publication: March 20, 2014
* 1st Revision Received: April 2, 2014
* 2nd Revision Received: May 20, 2014
* Third Revision Received: August 28, 2014
* Final Revision Accepted: September 2, 2014
Original Article
Comparative study of alterations in Tri-iodothyronine (T3) and Thyroxine (T4) hormone levels in breast and ovarian cancer
Mahmood Rasool1, Muhammad Imran Naseer2, Kalsoom Zaigham3,Arif Malik4, Naila Riaz5, Rabail Alam6, Abdul Manan7,
Ishfaq Ahmed Sheikh8, Muhammad Asif9
ABSTRACTObjective: The present study was designed to investigate variations in the levels of thyroid hormones (T3, T4) in breast and ovarian cancers patients.Methods: A total 120 subjects were recruited (without thyroid history) divided into three groups; A, B and C. Group A as control with healthy individuals. While group B and group C were consisting of breast cancer and ovarian cancer patient respectively. Blood samples (5 ml) were taken and analyzed to estimate the levels of serum T3 (tri-iodothyronine) and T4 (thyroxin) hormones.Results: Statistically significant difference (P=0.000* and P=0.017*) was obtained among all groups. A significant increase in T3 (P=0.000*) and T4 (0.005*) levels was observed among breast cancer patients as compared to healthy controls. While for ovarian cancer patients conflicting results were found for T3 and T4 levels in the serum i.e. insignificant difference was found in T3 (P=0.209) and T4 (P=0.050) as compared to control. Our results showed that in the breast cancer and ovarian cancer patients the thyroid hormone (T3 and T4) level has been altered from the normal ranges as compared to the normal healthy individuals. Conclusion: We conclude that hyperthyroidism has profound effects on breast cancer and ovarian cancer cells proliferation.
KEYWORDS: Breast cancer, Ovarian cancer, Thyroid hormones, Tri-iodothyronine (T3), Thyroxine (T4).
doi: http://dx.doi.org/10.12669/pjms.306.5294How to cite this:Rasool M, Naseer MI, Zaigham K, Malik A, Riaz N, Alam R, et al. Comparative study of alterations in Tri-iodothyronine (T3) and Thyroxine (T4) hormone levels in breast and ovarian cancer. Pak J Med Sci 2014;30(6):1356-1360. doi: http://dx.doi.org/10.12669/pjms.306.5294
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Thyroid hormones (T3, T4) in breast and ovarian cancer
Pak J Med Sci 2014 Vol. 30 No. 6 www.pjms.com.pk 1357
situated on the promoter sites of the T3 target genes as well as the developmental- and tissue dependent expressions of TR isoforms. These co-regulatory proteins consist of corepressors and coactivators, which either repress or activate the transcription, respectively.3
Autoimmune thyroid diseases affect the thyroid hormone production that results in either hypothyroidism or hyperthyroidism. They cause two opposing clinical syndromes, Hashimoto’s thyroiditis (HT) and Graves’ disease (GD).4,5
Thyroid disease has been associated with many diseases including breast cancer.3,6 It is a well-known fact that breast cancer is a hormone-dependent neoplasm. There have been reports on the clinical association between breast cancer and thyroid diseases but at the same time there are many contradictory results reported in literature which do not verify the clinical association between these two diseases. Geographical distinction in the incidence of breast cancer has been ascribed due to differences in intake of iodine in diet, and an effect of iodine on breast was also suggested. Our results signify an increased occurrence of thyroid diseases (both autoimmune and nonautoimmune) in breast cancer patients.6
The concept that thyroid hormone may act as a growth factor for the breast cancer has been reported by various scientists.7 The thyroid hormone receptors and steroids belong to the same family with similar molecular structure, but having different transcriptional functions which define their genomic actions.1 Thyroid hormones, T3 and T4 play a role in tumor cell proliferation and angiogenesis via binding to αVβ3 receptor at plasma membrane. Tetraiodothyroacetic acid (tetrac), displays an anti-proliferative activity by blocking the proangiogenic and proliferative actions of T3 and T4. In nucleus many transactivator proteins like estrogen receptor-α (ERα), thyroid hormone receptor-β1 (TRβ1) and signal transducer and activator of transcription-1α (STAT1α) are phosphorylated by translocated pMAPK. These downstream phosphorylated transactivator proteins start gene transcription of fibroblast growth factor factor (bFGF), (that induces thyroid hormone-induced angiogenesis). These also start transcription of other proliferation factors which are important for cell division of tumor cells.8
On the other hand ovarian cancer is also a hormone-dependent neoplasm like breast cancer. Ovarian cancer develops when a mutation or genetic change occurs in the cells on the surface
the ovaries or in the fallopian tubes that leads to uncontrolled cell growth which may often metastasize.9 In hyperthyroidism where there is a higher concentration of thyroid hormones (T3 and T4), circulating in the blood and reaches to the ovarian tissue, causes its inflammation. The ovarian surface epithelial (OSE) cells exhibit the receptor for thyroid hormone and estrogen hormone. T3 exerts direct inflammatory effects on the ovarian epithelial cells. When T3 binds to its receptor at OSE, it increases the expression of ERα and mRNA which mimic the action of estrogen receptor and encodes the ER isoforms. These isoforms are strongly associated with ovarian cancer. It suggests a possible link between hyperthyroidism and ovarian cancer.10
The role of thyroid hormones in the development and differentiation of normal breast tissue is a well known fact. However, the clinical association between the breast cancer and thyroid diseases is controversial. Although some of the studies do not verify this association but there are significant number of studies which established the clinical association. In addition to this, there are some studies which suggested a possible link between ovarian cancer and thyroid diseases.6,7,10-12 This study is perhaps the first from Lahore, Pakistan to elucidate the role of thyroid hormone abnormalities (autoimmune or non-autoimmune disorders) and to correlate them with breast and ovarian cancers with respect to T3 and T4 (having genetic and/or environmental influences). Moreover, if it is established as expected, then the effect of interferon received by hepatitis C patients,13 particularly in females who suffer from thyroid disease (as side effect of interferon) may be linked to breast and ovarian tumor/cancer.
METHODS
The blood (5 ml) from breast and ovarian cancer patients were collected from pathology Laboratory at INMOL (Institute of Nuclear Medicine and Oncology) hospital Lahore. The samples of healthy individuals (control) were collected from different cities of the Punjab i.e. Lahore, Gujranwala and Faisalabad etc with age limits of 18 to 70 years old.Experimental design: A total 120 subjects were selected for the present study. They were divided into three groups as A, B and C (Table-I). Group A consisted of normal healthy individuals (control). Groups B and C consisted of histopathologically confirmed breast cancer and ovarian cancer
1358 Pak J Med Sci 2014 Vol. 30 No. 6 www.pjms.com.pk
patients respectively without their thyroid history (i.e. patients having symptomatic for thyroid disorder).Sample collection and processing: Blood samples (5 ml) from female subjects were collected in a clot activator vacutainer blood collection tube (BD®). After clot formation in the blood tubes the serum was separated out by centrifugation at 4000 rpmfor 10 min.Sample storage: The serum was then separated from the clotted blood and transferred into a sterile glass tubes/eppendorf tubes. It was properly labeled with patient’s identification number and was stored at 4°C.Estimation of serum thyroid hormone levels tri-iodothyroxine (T3) and thyroxine (T4): Serum thyroid hormones (T3, T4) level was estimated by competitive radioimmunoassay.14,15
Statistical Analysis: The data thus obtained was subjected to statistical analysis for the determination of significance by using ANOVA (Analysis of Variance).
RESULTS
Overall statistically significant difference (P<0.05) was observed in mean values of serum thyroid hormones (T3, T4) level in all groups, A (control), B (Breast cancer) and C (Ovarian cancer) as compared to control group. Multiple comparisons (Table-II) indicated that there was a significant difference (P<0.05) in serum T3 levels in CA breast cancer patients when compared to control and CA ovarian cancer patients while an insignificant difference (P>0.05) was observed in ovarian cancer patients as compared to control group. Significant difference (P<0.05) was observed in serum T4 levels in CA breast and CA ovarian patients when compared to control.
A positive significant (P<0.05) correlation was observed between T3 and T4 levels among breast and ovarian cancer patients (Table-III), but there was no significant correlation for the levels among the control group.
DISCUSSION
As mentioned in introduction, this study is the stepping stone to elucidate the role of thyroid hormone abnormalities and to correlate them with breast and ovarian cancers with respect to T3 and T4. Significant increase in T3 and T4 levels were seen among breast cancer patients as compared to control (P<0.05) which was in agreement with work of11,16 where it has been observed that hyperthyroidism is associated with breast cancer patients with significantly higher T3 and T4 values and lower TSH levels which is suggesting a possible tumor growth promoting effect caused by this imbalance. They evaluated that in iodine deficient women the administration of thyroid hormone seems to elevate the chance of developing breast cancer. The occurrence of breast cancer was twice higher among hypothyroid women who were on supplemental thyroid hormone when compared to women not on thyroid hormone supplements. It has been reported that the incidence of breast cancer was almost half in women taking thyroid hormone for 5 years (10%) in comparison to women who have been taking thyroid hormone more than 15 years (19.5%).17
Significant increase (P<0.05) in T4 level was observed in ovarian cancer patients as compared to control,10,12 where associations between
Muhammad Imran Naseer et al.
Table-I: Estimation of serum T3 and T4 levels Group A Group B Group C P value
T3 (nm/L) 2.01 ± 0.39 2.73 ± 0.82 2.25 ± 0.71 0.000*T4 (nm/L) 117.65 ± 25.35 146.91 ± 42.92 137.68 ± 37.41 0.017*
Table-II: Multiple comparisons of T3 and T4 levels among the groups.
Control Group Cancer Group P value
T3 Control CA Breast 0.000 CA Ovary 0.209T4 Control CA Breast 0.005 CA Ovary 0.050
Table-III: Pearson correlation (two-tailed) between t3 and t4 levels among breast and ovarian cancer patients.
T3 Control T3 Ovarian T3 Breast
T4 Control 0.373 0.008 0.138 r-value 0.105 0.974 0.563 p-valueT4 Ovarian -0.520 0.502 0.206 r-value 0.019* 0.000** 0.151 p-valueT4 Breast 0.596 0.047 0.533 r-value 0.006** 0.746 0.000** p-value*, **: Significant at 0.05 and 0.01 respectivelyr-value: correlation coefficient.
Pak J Med Sci 2014 Vol. 30 No. 6 www.pjms.com.pk 1359
hyperthyroidism and ovarian cancer has been observed which was a novel observation. Ovarian cancer is hormone dependent inflammation and the most frequent reason of hyperthyroidism is autoimmune inflammation of the thyroid gland. However, no significant difference was observed in T3 level in ovarian cancer patients when compared to controls which may be attributed to its lower concentrations in the circulation as well as due to its very short half life (5.3 days) as compared to T4 (5.7 days).18 It was inferred from the highly positive correlation between T4 and T3 levels in ovarian cancer as well as among breast cancer patients. This showed a definite relationship between these two hormones (T3 and T4) as T4 is transformed to T3 within the target cells with the help of deiodinases (types of deiodinases, D1, D2, D3).19
Geographical variations in the rates of endometrial, breast, and ovarian cancers are inversely correlated with iodine intake in the diet. Endocrinological studies20-22 have indicated that a low dietary iodine intake may result in a state of increased gonadotrophin stimulation which in turn leads to a hyperestrogenic state that gives rise to increased production of estradiol and estrone as well as a relatively low estriol to estrone plus estradiol ratio. The difference in the endocrine state may increase the chance of endometrial, breast and ovarian cancers while the increase in dietary intake of iodine may decrease the risk of the above mentioned cancers.23
Since significantly elevated levels of T3 and T4 were observed in breast cancer patient, it can be concluded that manifestation of breast cancer is associated with serum thyroid hormones (T3, T4) levels. However, for ovarian cancer, there was no significant difference in the level of T3 but not T4 levels when compared to control. A hyperthyroidism has profound effects on breast cancer and ovarian cancer cells proliferation since significant alterations in amount of thyroid hormone (both T3 and T4) in breast cancer and T4 in ovarian cancer patients has been reported. This study would be of significant importance in screening of these patients related to the risk of these cancers (breast and ovarian) with other molecular biomarkers of tumor growth.
CONCLUSION
This study has significant impact on screening of female patients for breast and ovarian cancers. Estimation of T3 and T4 levels along with the panel of other biomarkers may have profound effect in screening of these patients. Since there have been
contradictory reports regarding correlation of hyperthyroidism and breast cancer, this study is an addition in this regard and suggests the correlation between hyperthyroidism (higher T3 and T4 levels) and breast cancer. In addition this study also provides basis and opens new dimensions in screening of ovarian cancer patients using T4 level estimation along with other biomarkers. Since interferon therapy may lead to thyroid dysfunction, this study also suggests possibility of progression of breast and ovarian cancer in patients receiving interferon therapy. Hence patients receiving interferon therapy shall also be screened for these cancers. Over all this study has impeccable impact on screening of breast and ovarian cancer patients having thyroid gland dysfunction.
ACKNOWLEDGEMENT
This article was funded by the Deanship of Scientific Research (DSR), King Abdulaziz University, Jeddah. The authors, therefore, acknowledge with thanks DSR technical and financial support.
Conflict of interest: The authors declare that they have no competing interest
REFERENCES1. Meng R, Tang HY, Westfall J, London D, Cao JH, Mousa
SA, et al. Crosstalk between integrin αvβ3 and estrogen receptor-α is involved in thyroid hormone-induced proliferation in human lung carcinoma cells. PLoS One. 2011;6(11):27547. doi: 10.1371/journal.pone.0027547
2. Schwartz CE, Stevenson RE. The MCT8 thyroid hormone transporter and allan- Herndon- Dudley syndrome. Best pract Res Clin Endocrinol Metab. 2007;21(2):307-321. DOI: 10.1016/j.beem.2007.03.009
3. Cheng SY, Leonard JL, Davis PJ. Molecular aspects of thyroid hormone actions. Endocrinol Rev. 2010;31(2):139-170. doi: 10.1210/er.2009-0007
4. Tomer Y. Genetic susceptibility to autoimmune thyroid disease: past, present, and future. Thyroid. 2010;20(7):715-725. doi: 10.1089/thy.2010.1644
5. Cappa M, Bizzarri C, Crea F. Autoimmune thyroid diseases in children. J Thyroid Res. 2010;2011:675703. doi: 10.4061/2011/675703
6. Turken O, NarIn Y, DemIrbas S, Onde ME, Sayan O, KandemIr EG, et al. Breast cancer in association with thyroid disorders. Breast Cancer Res 2003;5(5):110-113. doi:10.1186/bcr609
7. Cristofanilli M, Yamamura Y, Bevers SW, Strom TS, Patangan M, Rishnamurthy S, et al. Thyroid hormone and breast carcinoma, primary hypothyroidism is associated with a reduced incidence of breast carcinoma. Cancer. 2005;103(6):1122-1128. doi: 10.1002/cncr.20881
8. Lin HY, Cody V, Davis FB, Hercbergs AA, Luidens MK, Mousa SA, et al. Identification and functions of the plasma membrane receptor for thyroid hormone analogues. Discov Med. 2011;11(59):337-347.
Thyroid hormones (T3, T4) in breast and ovarian cancer
9. Brown PO, Palmer C. The Preclinical Natural History of Serous Ovarian Cancer: Defining the Target for Early Detection. PLoS Med. 2009;6(7):1-14. doi: 10.1371/journal.pmed.1000114
10. Rae MT, Gubbay O, Kostogiannou A, Price D, Critchley HO, Hillier SG. Thyroid hormone signaling in human ovarian surface epithelial cells. J Clin Endocrinol Metab. 2007;92(1):322-327. doi:10.1210/jc.2006-1522
11. Ghandrakant C. Breast cancer relationship to thyroid supplements for hypothyroidism. JAMA. 1976;238(11):1124-1127.
12. Ness RB, Grisso JA, Cottreau C, Klapper J, Vergona R, Wheeler JE, et al. Factors related to inflammation of the ovarian epithelium and risk of ovarian cancer. Epidemiology. 2000;11(2):111-117.
13. Malik A, Qureshi MS, Manan A, Saleem S, Munir M, Fatima A, et al. Assessment of Oxidative Stress in Hepatitis C Patients Receiving Interferon Therapy. Pak J Med Dentist. 2013;2(4):10-15.
14. Kabat EA. Basic principle of antigen-antibody reaction. In: methods in enzymology, immunochemical techniques, part A. academic press, London. 1980:7.
15. Karush F, Oterness I. Principles of antibody reaction in: antibody as a tool (eds. Marchalonis J.J., Warr, G.W) John Wiley & sons, New York 1982.
16. Saraiva PP, Figueiredo NB, Padovani CR, Brentani MM, Nogueira CR. Profile of thyroid hormones in breast cancer patients. Braz J Med Biol Res. 2005;38(5):761-765. doi: 10.1590/S0100-879X2005000500014
17. Howenstine J. Thyroid hormone therapy appears to cause breast cancer. Epidemiology. 2006;11(2):111-117.
Authors:
1. Mahmood Rasool, Center of Excellence in Genomic Medicine Research (CEGMR),2. Muhammad Imran Naseer, Center of Excellence in Genomic Medicine Research (CEGMR),3. Kalsoom Zaigham,4. Arif Malik,5. Naila Riaz,6. Rabail Alam,7. Abdul Manan,8. Ishfaq Ahmed Sheikh, King Fahd Medical Research Center (KFMRC),9. Muhammad Asif1,2,8: King Abdulaziz University, Jeddah, Saudi Arabia.3-7: Institute of Molecular Biology and Biotechnology (IMBB), The University of Lahore, Lahore, Pakistan.9: Department of Biotechnology and Informatics, (BUITEMS), Quetta, Pakistan.
1360 Pak J Med Sci 2014 Vol. 30 No. 6 www.pjms.com.pk
18. Shilo L, Kovatz S, Hadari R, Weiss E, Nabriski D, Shenkman L. Massive Thyroid Hormone Overdose: Kinetics, Clinical Manifestations and Management. Isr Med Assoc J. 2004;4:298-299.
19. Bianco AC, Kim BW. Deiodinases: implications of the local control of thyroid hormone action. J. Clin. Invest. 2006;116:2571–2579. doi:10.1172/JCI29812
20. Burns R, Azizi F, Hedayati M, Miamian PO, Herlihy C, Smyth PPA. Does placental iodine storage compensate for low maternal iodine intake? Clin Endocrinol. 2011;75(2):261-264. doi: 10.1111/j.1365-2265.2011.04039.x.
21. Burns RO, Herlihy C, Smyth PPA. The placenta as a compensatory iodine storage organ. Thyroid. 2011;21(5):541-546. doi: 10.1089/thy.2010.0203
22. Gulaboglu M, Borekci B, Halici Z. Placental tissue iodine level and blood magnesium concentration in pre-eclamptic and normal pregnancy. Int J Gynaecol Obstet. 2007;98:100-104. doi:10.1016/j.ijgo.2007.03.047
23. Stadel BV. Dietary iodine and risk of breast, endometrial, and ovarian cancer. Lancet. 1976;24(7965):890-891.
Author Contribution:
MR, AM and KZ designed the study and prepared the manuscript.NR, RA, AM and MA collected the data and performed the laboratory work.IAS, MA and MIN analyzed the data and critically revised the manuscript.
Muhammad Imran Naseer et al.
1 23
1 23
Your article is protected by copyright and allrights are held exclusively by Springer Science+Business Media New York. This e-offprint isfor personal use only and shall not be self-archived in electronic repositories. If you wishto self-archive your article, please use theaccepted manuscript version for posting onyour own website. You may further depositthe accepted manuscript version in anyrepository, provided it is only made publiclyavailable 12 months after official publicationor later and provided acknowledgement isgiven to the original source of publicationand a link is inserted to the published articleon Springer's website. The link must beaccompanied by the following text: "The finalpublication is available at link.springer.com”.
Knowledge, Perception, and Attitudes About Cancerand its Treatment Among Healthy Relatives of Cancer Patients:Single Institution Hospital-Based Study in Saudi Arabia
Bassem Eldeek & Jawaher Alahmadi & Maha Al-Attas &Khalid Sait & Nisrin Anfinan & Ettedal Aljahdali &Hamzah Ajaj & Hesham Sait
# Springer Science+Business Media New York 2014
Abstract This study was conducted to assess knowledge,perception, and attitudes regarding cancer and treatmentamong healthy relatives of cancer patients who attended anoutpatient cancer clinic with their relatives who suffer fromcancers. The participants recruited in this cross-sectional,interview-based study were 846 (557 female and 289 malesubjects) healthy relatives of cancer patients from the outpa-tient cancer clinic at King Abdulaziz University Hospital,Jeddah, Saudi Arabia. Most of the participants answered thatthey believed the causes of cancer were genetic (44.90 %),followed by environmental factors (30.10 %), diet (26.90 %),other causes (26.90 %), envy (26.90 %), and black magic(17.60 %). Most of the healthy participants believed thatdoctors should tell patients the full truth about the diagnosis(83.57%). More than half of the healthy population stated thatcancer patients should accept all types of treatment (chemo-therapy and/or radiotherapy and/or surgery), with more malesubjects having this position than females (P=0.014). Most ofthe participants believed that cancer cannot be caught fromanother person who suffered from cancer (67.50 %). Most of
the participants believed that cancer education was sufficient(66.70 %), with a significant difference between male andfemale respondents (P=0.004). With regard to why cancerpatients hide their disease, most of the participants in the agegroup <25 years believed that the causes were fear of loss ofhealth insurance (56.20 %), followed by job loss (34.40 %),and then social stigma (9.40 %); in the age group between 25and 45 years, the causes were fear of loss of health insurance(76.50 %), followed by social stigma (14.70 %), and then jobloss (8.80 %); while in the age group >45 years, the reasonswere job loss (47.10 %), followed by health insurance loss(41.20%), and then social stigma (11.80%), with a significantdifference between groups (P=0.034). This study demonstrat-ed that still a large number of healthy participants had defi-cient perceptions and poor attitudes about important issuesconcerning cancers such as different mode of treatments,alternative treatment, biological causes, and prognosis, partic-ularly among male respondents. Prevention education strate-gies should be considered, including targeted approaches thataim to reduce disparities in cancer perception among thegeneral population.
Keywords Attitude . Behavior . Knowledge . Cancer .
Cross-sectional studies . Public education
Introduction
Cancer is a major public health problem, with significantlyassociated mortality and disability. It is the second leadingcause of death in developed countries and is one of the threeleading causes of death for adults in developing countries [1].In the whole world, cancer rates are set to double by 2030, asreported by the World Health Organization [1]. The estimatednumber of new cases could rise from 12.3 million in 2007 to
B. EldeekCommunity Medicine in the Medical Education Department, KingAbdulaziz University, Jeddah, Saudi Arabia
J. AlahmadiFamily and Community Medicine, King Abdulaziz University,Jeddah, Saudi Arabia
M. Al-Attas :K. Sait :N. Anfinan : E. Aljahdali :H. Ajaj :H. SaitObstetrics and Gynecology Department, King Abdulaziz University,Jeddah, Saudi Arabia
M. Al-Attas :K. Sait (*) :N. Anfinan :H. SaitScientific Chair of Professor Abdullah Hussain Basalamah forGynecological Cancer, Faculty of Medicine, King AbdulazizUniversity, Jeddah, Saudi Arabiae-mail: [email protected]
J Canc EducDOI 10.1007/s13187-014-0653-7
Author's personal copy
16 million in 2020, and approximately 60 % of these newcases are likely to occur in low- and middle-income countriesin the world [2]. However, approximately half of the 11 majorcancer types are potentially preventable [3–5].
Currently, the major problems facing low- and medium-income countries are the growth and increasing age of theirpopulations and the westernization of their lifestyles. For thesereasons, the role of cancer prevention programs in thesecountries is critical. However, to develop successful preven-tive programs, knowledge regarding cancer and awareness ofearly symptoms must be high. In addition, there should bepositive attitudes toward screening programs [1]. Several fac-tors, such as culture, false beliefs, and taboos, can affect earlydetection and proper treatment. Failure to identify these inter-nal obstacles can impair the success of any cancer care pro-gram, even with adequate resources [1]. Patients will com-monly turn to alternative healthcare strategies and traditionalhealers, believing them to have equal or superior ability toaddress difficult health problems [6].
In Saudi Arabia, few studies have been conducted to detectthe perception of cancer and attitudes toward conventionaland alternative therapies, and these studies have mostly fo-cused on knowledge of and attitudes toward breast cancer[7–12]. According to the Saudi Cancer Registry, which waspublished in 2007, the total number of adult cancer Saudicases reported was 11,437. Overall, the incidence of cancerwas slightly higher in women than in men, as cancer affected5,473 (47.9 %) men and 5,964 (52.1 %) women [13]. It is thuscrucial to assess the general population’s behaviors, opinions,and perceptions regarding cancer.
Therefore, this cross-sectional study was conducted toassess knowledge, perception, and attitudes regarding cancerand treatment among healthy relatives of cancer patients, whowere attendees of an outpatient cancer clinic with/for their sickrelatives at King Abdulaziz University Hospital in Jeddah,Saudi Arabia.
Subjects and Methods
After receiving ethical approval from the Unit of BiomedicalEthics and Research Committee, Faculty of Medicine, KingAbdulaziz University, Jeddah, Saudi Arabia, a descriptive,cross-sectional study was conducted among healthy relativesof cancer patients, who were attendees of an outpatient cancerclinic with/for their sick relatives at King Abdulaziz Univer-sity Hospital (KAUH) in Jeddah, Saudi Arabia. KAUH is thebiggest governmental teaching hospital in Jeddah. It has 790beds with a total number of 250,915 patients seen for the year2012, and total admissions in the period were 41,923. Thetotal number of cancer patients seen in 2012 was 1,453 pa-tients. The total number of surgical procedures done in 2012were 9,282, and 80 cases were done for cancer diagnosis. We
chose relatives of cancer patients who did not suffer fromcancer to measure their knowledge, perception, and attitudesregarding cancer. The study was conducted from November2012 to March 2013. The participants recruited for this studytotaled 846 (557 female and 289 male subjects), and their agesranged from 15 to 92 years old. It was noticed that the numberof female participants (65.80 %) was higher than male partic-ipants (34.20 %). The participants were apparently healthyand their medical records were completely free from anymedical or psychological illness. All of the participants wereinformed about the study and signed a written consent form,which was approved to be used by the ethical committee ofKing Abdulaziz University Hospital in Jeddah, Saudi Arabia.
Tools of the Study
Interviews were conducted face to face, using a standardizedquestionnaire. The questionnaire was designed to measure theknowledge, perception, and factors influencing the perceptionand attitudes of healthy individuals toward cancer. The ques-tionnaire was divided into four sections as follows:sociodemographic data, knowledge about the causes, knowl-edge about treatment, and support services for cancer.Sociodemographic variables included age, sex, marital status,and education. Knowledge about cancer causes was assessedwith the following four questions: “What do you think are thecauses of cancer?” The attitude of a healthy individual towardcancer was assessed with the following questions: “Shoulddoctors tell patients the full truth about the diagnosis?,” withresponse categories of “yes,” “no,” and “do not know”; “Whatkind of treatment should cancer patients accept?,” with re-sponse categories of “all types of treatments (chemotherapyand/or radiotherapy and/or surgery),” “surgery,” “chemother-apy,” and “radiotherapy”; “Should cancer patients receivealterative treatment (e.g., hormonal, biological, and trans-plant)?,” with response categories of “yes,” “no,” and “notsure,” and if the answer was “yes,” the response categorieswere “alone” and “with other treatments”; and “Is cancer aninfectious disease?,” with response categories of “yes,” “no,”and “do not know.” The factors influencing the perception ofand attitudes toward cancer were assessed with the followingseven questions: “Shouldn’t cancer patients tell anyone thatthey have been diagnosed?,” with response categories of“yes,” “no,” and “not sure”; “Shouldn’t genitalia cancer pa-tients tell anyone that they have been diagnosed?,” withresponse categories of “yes,” “no,” and “not sure”; “Why dopatients didn’t tell anyone that they have been diagnosed?,”with response categories of “loss of job,” “loss of healthinsurance,” and “social stigma”; “Is health education aboutcancer sufficient?,” with response categories of “yes,” “no,”and “not sure”; “Do you require more knowledge about can-cer?,” with response categories of “yes” and “no”; “Do youknow about alternative treatments (e.g., hormonal, biological,
J Canc Educ
Author's personal copy
transplant)?,”with response categories of “yes” and “no”; and“Do you require more information about alternative treat-ments (e.g., hormonal, biological, transplant)?,”with responsecategories of “yes” and “no.” Each question had a unique codeto make entering and processing of the data easier. The sam-pling units were the working days of the clinics. The workingdays were 4 days, we chose Sunday and Tuesday by simplerandom samples, and any relatives of patients with cancerattending the clinic on those days were chosen.
Statistical Analysis
The statistical analysis was performed using the StatisticalPackage of Social Sciences computer program (SPSS), ver-sion 20. The data are presented in the forms of numbers andpercentages or means±standard deviations (minimum–maxi-mum). The chi-square test was used for comparisons of thequantitative data. The data were considered significant whenthe P value was less than 0.05.
Results
Demographic Details of the Study Population
Table 1 shows the demographic characteristics of the patients.Most of the patients involved in this studywere in the age group<25 years, followed by 25–45 years, and finally >45 years witha significant difference between them (P<0.0001). There weresignificantly more Saudi citizens than non-Saudis (81.20 versus18.80%,P<0.0001) and significantlymore female respondentsthan male respondents (68.50 versus 31.40 %, P<0.0001).Significantly more of the participants were single (57.40 %),compared with married (37.70 %), divorced (1.80 %), orwidowed (1.70 %, P<0.0001). There were significantly moreuniversity level-educated participants (72.10 %), comparedwith the following education levels: secondary school(14.90 %), illiterate (6.90 %), primary school (4.02 %), andpreparatory school (2.10 %, P<0.0001).
Knowledge of the Causes of Cancer
Regarding the studied population’s knowledge of the causesof cancer, most of the participants answered genetic(44.90 %), followed by environmental causes (30.10 %), diet(26.90 %), others (26.90 %), envy (26.90 %), black magic(17.60 %), stress (17.00 %), inflammation (14.20 %), do notknow (14.20 %), and finally sadness (12.80 %) (Table 2).
Perception of Cancer
Regarding whether doctors should tell patients the full truthabout the diagnosis, most of the healthy participants answered
yes (83.57 %) versus no (12.06 %) or do not know (4.26 %),with a significant difference between male and female respon-dents (P=0.018). More than half of the healthy populationstated that cancer patients should accept chemotherapy and/orradiotherapy and/or surgery (59.10 %), followed by chemo-therapy (33.10 %), surgery (5.70 %), and finally radiotherapy(2.10 %), with a significant difference between male andfemale respondents (P=0.014). More than half of the healthyparticipants (57.57 %) believed that cancer patients should
Table 1 Demographic characteristics of the studied population (n=846)
Variables Data Significance
Age (years) 30.36±12.20 (15.00–92.00)
Age groups P<0.0001
<25 years 434 (51.30 %)
25–45 years 291 (34.30 %)
>45 years 121 (14.30 %)
Nationality P<0.0001
Saudi 687 (81.20 %)
Non-Saudi 159 (18.80 %)
Sex P<0.0001
Female 557 (65.80 %)
Male 289 (34.20 %)
Marital status P<0.0001
Single 485 (57.30 %)
Married 331 (39.10 %)
Divorced 16 (1.80 %)
Widowed 14 (1.70 %)
Education P<0.0001
University level 610 (72.10 %)
Secondary school 126 (14.90 %)
Illiterate 59 (7.00 %)
Primary school 34 (4.00 %)
Preparatory school 17 (2.00 %)
Data are expressed as the means±SDs (minimum–maximum) or aspercentages (%) as appropriate. Significance was determined using thechi-square test
Table 2 Questionnaireregarding the causes ofcancer
Causes of cancer Number (%)
Genetic 441 (44.90 %)
Environment 296 (30.10 %)
Diet 264 (26.90 %)
Other 264 (26.90 %)
Envy 264 (26.90 %)
Black magic 173 (17.60 %)
Stress 167 (17.00 %)
Inflammation 140 (14.20 %)
Do not know 140 (14.20 %)
Sadness 126 (12.80 %)
J Canc Educ
Author's personal copy
receive alternative treatments (e.g., hormonal, biological,transplant), while 32.74 % were not sure, and 9.70 % didnot believe in alternative treatments, with an insignificantdifference between male and female respondents (P=0.052).Most of the participants who agreed about alternative treat-ments said that these treatments must occur with other treat-ments (e.g., hormonal, biological, transplant) (88.30%), while11.70 % said the treatments must be performed alone, with aninsignificant difference between male and female respondents(P=0.167). In addition, most of the healthy participants be-lieved that cancer cannot be caught from another person asthey might catch flu (67.50 %), while 29.10 % did not know,and only 3.40 % believed that cancer can be caught, with asignificant difference between male and female respondents(P=0.002) (Table 3).
Regarding whether doctors should tell patients the full truthabout the diagnosis, most of the healthy participants in thedifferent age groups answered yes versus no or do not know,with a significant difference between volunteers in the differentage groups (P=0.798). More than half of the healthy popula-tion stated that cancer patients should accept chemotherapyand/or radiotherapy and/or followed by chemotherapy surgeryand finally radiotherapy, with an insignificant difference be-tween volunteers in the different age groups (P=0.059). Morethan half of the healthy participants believed that cancer
patients should receive alternative treatments followed by notsure then did not believe in alternative treatments, with asignificant difference between volunteers in the different agegroups (P=0.0001). Most of the participants who agreed aboutalternative treatments said that these treatments must occurwith other treatments compared with volunteers who said thattreatments must be performed alone, with a significant differ-ence between volunteers in the different age groups (P=0.003).In addition, most of the healthy participants believed thatcancer cannot be caught from another person as they mightcatch flu compared with those who answered did not know orthose who believed that cancer can be caught, with a signifi-cant difference between volunteers in the different age groups(P<0.0001) (Table 4).
Factors Influencing the Perception of and AttitudesToward Cancer
Most of the participants believed that patients should tellanyone at work that they have been diagnosed (76.80 %),while 13.70% believed that they shouldn’t tell anyone at workthat they have been diagnosed, and 9.50 % did not know, withan insignificant difference between male and female respon-dents (P=0.935). Regarding patients with genital cancer, mostof the participants believed that those patients should tell
Table 3 Perception of the healthy population regarding some concepts of cancer according to sex
Questionnumber
Perception questionnaire Total (n=846) Male (n=289) Female(n=557)
Significance
1 Should doctors tell patients the full truth about the diagnosis? 0.018
Yes 707 (83.57 %) 252 (87.20 %) 455 (81.70 %)
No 102 (12.06 %) 32 (11.10 %) 70 (12.60 %)
Do not know 37 (4.26 %) 5 (1.70 %) 32 (5.70 %)
2 What kind of treatment should cancer patients accept? 0.014
Chemotherapy and/or radiotherapy and/or surgery 500 (59.10 %) 172 (59.50 %) 328 (58.90 %)
Chemotherapy 280 (33.10 %) 105 (36.30 %) 175 (31.40 %)
Surgery 48 (5.70 %) 11 (3.80 %) 37 (6.60 %)
Radiotherapy 18 (2.10 %) 1 (0.30 %) 17 (3.10 %)
3 Should cancer patients receive alternative treatments(e.g., hormonal, biological, transplant)?
0.052
No 82 (9.70 %) 21 (7.30 %) 61 (11.00 %)
Yes 487 (57.57 %) 160 (55.40 %) 327 (58.70 %)
Not sure 277 (32.74 %) 108 (37.70 %) 169 (30.30 %)
3.1 If yes: 0.167
With other treatment (e.g., hormonal, biological, transplant) 430 (88.30 %) 145 (90.60 %) 285 (87.20 %)
Alone 57 (11.70 %) 15 (9.40 %) 42 (12.80 %)
4 Can a person catch cancer from another person as they might catch flu? 0.002
Yes 29 (3.40 %) 18 (6.20 %) 11 (2.00 %)
No 571 (67.50 %) 180 (62.30 %) 391 (70.20 %)
Do not know 246 (29.10 %) 91 (31.50 %) 155 (27.80 %)
Data are expressed as percentages (%). Significance was determined using the chi-square test
J Canc Educ
Author's personal copy
anyone at work that they have been diagnosed with genitalcancer (79.90 %), while 9.80 % believed that these patientsshouldn’t tell anyone at work about their disease, and 10.30 %were not sure, with an insignificant difference between maleand female respondents (P=0.767). With regard to why cancerpatients hide their disease, most of the participants believedthat the causes were fear of job loss (61.40 %), followed byloss of health insurance (26.50 %), and social stigma(12.00 %), with an insignificant difference between maleand female respondents (P=0.498). Most of the participantsbelieved that current cancer education was sufficient(66.70 %), while 31.80 % did not believe it was sufficient,and 1.50 % were not sure, with a significant difference be-tween male and female respondents (P=0.004). Most of theparticipants did not require more information about cancer(55.40 %), while 44.60 % needed more information, with asignificant difference between male and female respondents(P=0.032). Most of the participants know alternative treat-ments (e.g., hormonal, biological, transplant) (74.90%), while25.10 % did not know about these treatments, with a signifi-cant difference between male and female respondents(P=0.006). Most of the participants did not require moreinformation about alternative medicine (57.20 %), while42.80 % did need more, with an insignificant differencebetween male and female respondents (P=0.196) (Table 5).
In the different studied age groups, most of the participantsbelieved that patients should tell anyone at work that they havebeen diagnosed compared to those believed that they shouldn’ttell anyone at work that they have been diagnosed, and thosedid not know, with an insignificant difference between groups(P=0.214). Regarding patients with genital cancer, most of theparticipants in the different studied age groups believed thatthose patients should tell anyone at work that they have beendiagnosed with genital cancer compared with those who be-lieved that these patients shouldn’t tell anyone at work abouttheir disease, and those who were not sure, with an significantdifference between groups (P=0.043). With regard to whycancer patients hide their disease, most of the participants inthe age group <25 years believed that the causes were fear ofloss of health insurance (56.20 %), followed by job loss(34.40 %) and social stigma (9.40 %); in the age group be-tween 25 and 45 years, the causes were fear of loss of healthinsurance (76.50 %), followed by social stigma (14.70 %), andthen job loss (8.80 %); while in the age group >45 years, thereasons were job loss (47.10 %) followed by health insuranceloss (41.20 %), and then social stigma (11.80 %), with asignificant difference between groups (P=0.034). Most of theparticipants in the different age groups believed that currentcancer education was sufficient compared to those who did notbelieve it was sufficient, and those who were not sure, with a
Table 4 Perception of the healthy population regarding some concepts of cancer according to age groups
Questionnumber
Perception questionnaire <25 years(n=434)
25–45 years(n=291)
>45 years(n=121)
Significance
1 Should doctors tell patients the full truth about the diagnosis? 0.798
Yes 361 (83.20 %) 247 (84.90 %) 99 (81.80 %)
No 51 (11.80 %) 34 (11.70 %) 17 (14.00 %)
Do not know 22 (5.10 %) 10 (3.40 %) 5 (4.10 %)
2 What kind of treatment should cancer patients accept? 0.059
Chemotherapy and/or radiotherapy and/or surgery 250 (57.60 %) 176 (60.50 %) 74 (61.20 %)
Chemotherapy 160 (36.90 %) 82 (28.20 %) 38 (31.40 %)
Surgery 17 (3.90 %) 24 (8.20 %) 7 (5.80 %)
Radiotherapy 7 (1.60 %) 9 (3.10 %) 2 (1.70 %)
3 Should cancer patients receive alternative treatments(e.g., hormonal, biological, transplant)?
0.0001
No 49 (11.30 %) 22 (7.60 %) 11 (9.10 %)
Yes 203 (46.80 %) 203 (69.80 %) 81 (66.90 %)
Not sure 182 (41.90 %) 66 (22.70 %) 29 (24.00 %)
3.1 If yes: 0.003
With other treatment (e.g., hormonal, biological, transplant) 191 (94.10 %) 170 (83.70 %) 60 (85.20 %)
Alone 12 (5.90 %) 33 (16.30 %) 12 (14.80 %)
4 Can a person catch cancer from another person as they might catch flu? 0.034
Yes 12 (2.80 %) 11 (3.80 %) 6 (5.00 %)
No 276 (63.60 %) 212 (72.90 %) 83 (68.60 %)
Do not know 146 (33.60 %) 68 (23.40 %) 32 (26.40 %)
Data are expressed as percentages (%). Significance was determined using the chi-square test
J Canc Educ
Author's personal copy
significant difference between groups (P=0.001). Most of theparticipants did not require more information about cancercompared to those who need more information, with an insig-nificant difference between the different age groups(P=0.927). Most of the participants know alternative treat-ments compared to those did not know about these treatments,with a significant difference between the different age groups(P=0.006). Most of the participants did not require moreinformation about alternative medicine compared to thosewho need more information, with an insignificant differencebetween the different age groups (P=0.837) (Table 6).
Discussion
The majority of volunteers included in this study were Saudiwith high education levels, mostly university. There weremore female participants than male participants. Unfortunate-ly, the participants’ knowledge about the causes of cancer was
poor, and they did not know the proven risk factors for cancer.Most of the participants believed the cause of cancer might begenetic followed by environmental causes, diet, other causes,envy, black magic, stress, inflammation, do not know, andfinally sadness. These findings support previous studies thathave found low levels of awareness of cancer risk factors ingeneral population samples [14–22].
A higher level of knowledge regarding breast cancer wasobserved with older age in a study from Saudi Arabia, basedon female secondary school students in Jeddah, Saudi Arabia[7]. A study assessing the knowledge of doctors, nurses,patients, and the public regarding the causes of gastrointestinalcancers in Al-Madinah Al-Munawara, Saudi Arabia, foundthat the majority of doctors and nurses had a high level ofknowledge, while the knowledge of patients and the generalpublic was low [23]. A study assessing the knowledge, atti-tudes, and practices regarding cervical cancer screening, itsetiology, and preventive measures among women in Jeddah,Saudi Arabia, reported that awareness of cervical cancer was
Table 5 Factors influencing the perception and attitudes of the healthy population toward cancer
Questionnumber
Beliefs questionnaire Total (n=846) Male (n=289) Female(n=557)
Significance
1. Shouldn’t cancer patients tell anyone that they have been diagnosed? 0.935
Yes 116 (13.70 %) 38 (13.10 %) 78 (14.00 %)
No 650 (76.80 %) 224 (77.50 %) 426 (76.50 %)
Not sure 80 (9.50 %) 27 (9.30 %) 53 (9.50 %)
2. Shouldn’t genital cancer patients tell anyone that theyhave been diagnosed?
0.767
Yes 83 (9.80 %) 30 (10.40 %) 53 (9.50 %)
No 676 (79.90 %) 232 (80.30 %) 444 (79.40 %)
Not sure 87 (10.30 %) 27 (9.30 %) 60 (10.80 %)
3. Why should patients not disclose their disease? 0.498
Job loss 51 (61.40 %) 16 (53.30 %) 35 (66.00 %)
Health insurance loss 22 (26.50 %) 10 (33.30 %) 12 (22.60 %)
Social stigma 10 (12.00 %) 4 (13.30 %) 6 (11.30 %)
4. Is health education sufficient? 0.004
Yes 564 (66.70 %) 173 (59.90 %) 391 (70.20 %)
No 269 (31.80 %) 113 (39.10 %) 156 (28.00 %)
Not sure 13 (1.50 %) 3 (1.00 %) 10 (1.80 %)
5. Do you require more information about cancer? 0.032
Yes 377 (44.60 %) 142 (49.10 %) 235 (42.20 %)
No 469 (55.40 %) 147 (50.90 %) 322 (57.80 %)
6. Do you know about alternative treatments (e.g., hormonal,biological and transplant)?
0.006
Yes 634 (74.90 %) 201 (69.60 %) 433 (77.70 %)
No 212 (25.10 %) 88 (30.40 %) 124 (22.30 %)
7. Do you require more information about alternative treatments(e.g., hormonal, biological and transplant)?
0.196
Yes 362 (42.80 %) 130 (45.00 %) 232 (41.70 %)
No 484 (57.20 %) 159 (55.00 %) 325 (58.30 %)
Data are expressed as percentages (%). Significance was determined using the chi-square test
J Canc Educ
Author's personal copy
far behind that in developed countries [12]. A study from theUK suggested that public knowledge of cancer (based onawareness of risk factors, presenting symptoms, treatments,and support services) was poor, and greater attempts should bemade to raise awareness [24]. In a cross-sectional survey fromBirmingham and Melbourne, evaluating knowledge aboutcommon cancers and their risk factors among adults, theresearchers found that several protective lifestyle choices wereincorrectly identified by participants, and they concluded thatfurther health education is needed to clarify public awarenessabout the differences between proven and nonproven riskfactors and about protective lifestyle choices for major cancers[25]. However, the most likely way to improve cancer knowl-edge is via the popular media, particularly television [24].Ravichandran et al. [26] reported that 56.7 % of their respon-dents indicated that they would like to know more aboutcancer. Consequently, culturally sensitive health educationmessages should be tailored to fill the knowledge gaps in allpopulation strata.
There has been a significant debate regarding whether totell patients the full truth about their disease and the manage-ment options that should be offered to them. We asked ourrespondents about this dilemma, and 83.57 % of the healthyparticipants believed that doctors should tell patients the fulltruth about the diagnosis, with the male responses beingsignificantly more positive than the female responses. Regard-ing various types of treatments, more than half of the healthypopulation stated that cancer patients should accept chemo-therapy and/or radiotherapy and/or surgery (59.10 %), follow-ed by chemotherapy, surgery, and finally radiotherapy, and themale responses were significantly higher than female re-sponses. Unfortunately, 57.57 % of the healthy participantsbelieved that cancer patients should receive alternative treat-ments (e.g., hormonal, biological, transplant), while 32.74 %were not sure, and 9.70 % did not believe in alternativetreatments, with an insignificant difference between maleand female respondents. In addition, most of the healthyparticipants believed that persons cannot catch cancer from
Table 6 Factors influencing the perception and attitudes of the healthy population toward cancer according to age groups
Questionnumber
Beliefs questionnaire <25 years(n=434)
25–45 years(n=291)
>45 years(n=121)
Significance
1. Shouldn’t cancer patients tell anyone that they have been diagnosed? 0.214
Yes 51 (11.80 %) 41 (14.10 %) 24 (19.80 %)
No 344 (79.30 %) 221 (75.90 %) 85 (70.20 %)
Not sure 39 (9.00 %) 29 (10.00 %) 12 (9.90 %)
2. Shouldn’t genital cancer patients tell anyone that they havebeen diagnosed?
0.043
Yes 32 (7.40 %) 34 (11.70 %) 17 (14.00 %)
No 349 (80.40 %) 235 (80.80 %) 92 (76.00 %)
Not sure 53 (12.20 %) 22 (7.60 %) 12 (9.90 %)
3. Why should patients not disclose their disease? 0.034
Job loss 11 (34.4 %) 3 (8.80 %) 8 (47.10 %)
Health insurance loss 18 (56.20 %) 26 (76.50 %) 7 (41.20 %)
Social stigma 3 (9.40 %) 5 (14.70 %) 2 (11.80 %)
4. Is health education sufficient? 0.001
Yes 261 (60.10 %) 208 (71.50 %) 95 (78.50 %)
No 165 (38.00 %) 79 (27.10 %) 25 (20.70 %)
Not sure 8 (1.80 %) 4 (1.40 %) 1 (0.80 %)
5. Do you require more information about cancer? 0.927
Yes 194 (44.70 %) 131 (45.00 %) 52 (43.00 %)
No 240 (55.30 %) 160 (55.00 %) 69 (57.00 %)
6. Do you know about alternative treatments (e.g., hormonal,biological, and transplant)?
0.006
Yes 309 (71.20 %) 237 (81.40 %) 88 (72.70 %)
No 125 (28.80 %) 54 (18.60 %) 33 (27.30 %)
7. Do you require more information about alternative treatments(e.g., hormonal, biological, and transplant)?
0.837
Yes 187 (43.10 %) 121 (41.60 %) 54 (44.60 %)
No 247 (56.90 %) 170 (58.40 %) 67 (55.40 %)
Data are expressed as percentages (%). Significance was determined using the chi-square test
J Canc Educ
Author's personal copy
another person as they might catch flu (67.50 %), while29.10 % did not know, and only 3.40 % said cancer can becaught from others, with a significant difference between maleand female respondents. We believe that the remainder of theparticipants might have had an incorrect perception of cancerand a lack of knowledge about the consequences of cancer andabout the advanced therapies that are available today.
Most of the participants in this study believed that cancerpatients should tell anyone at work that they have been diag-nosed. With regard to why cancer patients shouldn’t tellanyone at work that they have been diagnosed, most of theparticipants believed that the causes were fear of losing theirjob, fear of losing their health insurance, and social stigma.With regard to why cancer patients hide their disease, most ofthe participants in the age group <25 years believed that thecauses were fear of loss of health insurance, followed by jobloss, and then social stigma; in the age group between 25 and45 years, the causes were mostly fear of loss of health insur-ance, followed by social stigma, and then job loss; while in theage group >45 years, the reasons were job loss followed byhealth insurance loss, and then social stigma. Most of theparticipants in this study believed that current cancer educa-tion was sufficient, with female subjects responding signifi-cantly more positively than male subjects. Most of the partic-ipants did not require more information about cancer(55.40 %), while 44.60 % needed more, with male subjectsresponding significantly more positively than female subjects.Most of the participants knew about alternative treatments(e.g., hormonal, biological, transplant) (74.90 %), while25.10 % did not know about these treatments, with femalerespondents knowing more than male respondents. It wasexpected that those with family histories of cancer would beparticularly likely to acknowledge the potential warning signsof cancer, as they might be more motivated to adopt preven-tive behaviors [27]. However, the findings of our study indi-cated that family history had little influence on increasing thelevel of awareness. The results of our research showed thatmen were significantly less likely than women to be aware ofthe early warning signs of cancer. These findings are consis-tent with those of other studies evaluating the effects of sex onknowledge of cancer [15, 18–20, 22, 28–33]. A possibleexplanation for this difference might be women’s greaterfamiliarity with and use of primary healthcare servicesthrough, for example, their participation in mass health pro-grams, being responsible for their children’s health care, andtheir interest in health within families [34].
Conclusions
This study demonstrated that the healthy relatives of cancerpatients, especially the male subjects, had deficient perceptionand poor attitudes about important issues concerning cancer
such as causes, treatment, and prognosis. These data can beconsidered the first step in the development of an interventionbased on empirical findings that will identify areas for publiceducation and intervention efforts as important components inpreventing the disease. Such educational and interventionprograms should be culture sensitive and accessible to allindividuals, with special attention placed on reaching thepopulations at the greatest risk, to increase awareness aboutthe disease in hopes of increasing early diagnosis. As a na-tional cancer strategy, public education focused on high-riskpopulations, combined with the use of cancer-screening tech-nologies, could be a cost-effective approach.
Recommendation
Further studies are necessary to assess cancer knowledge andbehavioral risk factors. Health education campaigns are need-ed to raise awareness about risk factors and the prevention ofcancer among the general population.
Limitations of the Study
An important limitation of our study was the possibility ofselection bias; relatives of cancer patients might have betterknowledge of cancer than the general population. Most of thepatients who participated in this study were female. Further-more, there is no international standardized questionnaire toassess cancer knowledge, and this study addressed only a fewissues regarding cancer knowledge; therefore, the possibilityof having neglected some issues cannot be ruled out. Qualita-tive studies are necessary in these areas to assess views of andconcepts about cancer.
Acknowledgments We would like to thank all the participants for theirsupport and agreement to participate in the survey.
Conflict of Interest None declared.
References
1. Boyle P, Levin B (2009) World cancer report 2008, 1st edn. WorldHealth Organization, Geneva
2. Omar S, Alieldin NH, Khatib OM (2007) Cancer magnitude, chal-lenges and control in the EasternMediterranean region. East MediterrHealth J 13:1486–1496
3. Kanavos P (2006) The rising burden of cancer in the developingworld. Ann Oncol 17(Suppl 8):15–23
4. Soerjomataram I, de Vries E, Pukkala E, Coebergh JW (2007) Excessof cancers in Europe: a study of eleven major cancers amenable tolifestyle change. Int J Cancer 120:1336–1343
5. GladeMJ (1999) Food, nutrition, physical activity and the preventionof cancer: a global perspective. American Institute for CancerResearch/World Cancer Research Fund, American Institute forCancer Research. Nutrition 15:523–526
J Canc Educ
Author's personal copy
6. Gansler T, Kaw C, Crammer C, Smith T (2008) A population-basedstudy of prevalence of complementary methods use by cancer survi-vors: a report from the American Cancer Society’s studies of cancersurvivors. Cancer 113:1048–1057
7. Milaat WA (2000) Knowledge of secondary-school female studentson breast cancer and breast self-examination in Jeddah, Saudi Arabia.East Mediterr Health J 6:338–344
8. Alsaif AA (2004) Breast self-examination among Saudi female nurs-ing students in Saudi Arabia. Saudi Med J 25:1574–1578
9. Alam AA (2006) Knowledge of breast cancer and its risk andprotective factors among women in Riyadh. Ann Saudi Med 26:272–277
10. Jahan S, Al-Saigul AM, Abdelgadir MH (2006) Breast cancer.Knowledge, attitudes and practices of breast self examination amongwomen in Qassim region of Saudi Arabia. SaudiMed J 27:1737–1741
11. Amin TT, Al Mulhim AR, Al Meqihwi A (2009) Breast cancerknowledge, risk factors and screening among adult Saudi women ina primary health care setting. Asian Pac J Cancer Prev 10:133–138
12. Sait KH (2009) Attitudes, knowledge, and practices in relation tocervical cancer and its screening among women in Saudi Arabia.Saudi Med J 30:1208–1212
13. Al-Attas SA (2005) Knowledge, attitude and behavior about oralcancer, among a group of adult Jeddah population. Saudi Dental J17:34–42
14. Brown ML, Potosky AL, Thompson GB, Kessler LG (1990) Theknowledge and use of screening tests for colorectal and prostatecancer: data from the 1987 National Health Interview Survey. PrevMed 19:562–574
15. Breslow RA, Sorkin JD, Frey CM, Kessler LG (1997) Americans’knowledge of cancer risk and survival. Prev Med 26:170–177
16. Fitch MI, Greenberg M, Levstein L, Muir M, Plante S et al (1997)Health promotion and early detection of cancer in older adults:assessing knowledge about cancer. Oncol Nurs Forum 24:1743–1748
17. Paul C, Barratt A, Redman S, Cockburn J, Lowe J (1999) Knowledgeand perceptions about breast cancer incidence, fatality and riskamong Australian women. Aust N Z J Public Health 23:396–400
18. Brunswick N, Wardle J, Jarvis MJ (2001) Public awareness ofwarning signs for cancer in Britain. Cancer Causes Control 12:33–37
19. Wardle J, Waller J, Brunswick N, Jarvis MJ (2001) Awareness of riskfactors for cancer among British adults. Public Health 115:173–174
20. McCaffery K, Wardle J, Waller J (2003) Knowledge, attitudes, andbehavioral intentions in relation to the early detection of colorectalcancer in the United Kingdom. Prev Med 36:525–535
21. Waller J, McCaffery K, Wardle J (2004) Beliefs about the risk factorsfor cervical cancer in a British population sample. Prev Med38:745–753
22. Feizi A, Kazemnejad A, Hosseini M, Parsa-Yekta Z, Jamali J (2011)Assessing awareness level about warning signs of cancer and itsdeterminants in an Iranian general population. J Health Popul Nutr29:656–659
23. Parvez T, Gumgumji AA, Anwar MS, Al-Ahmadi SS (2004)Awareness about causes of gastrointestinal tract (GIT) malignancies.J Coll Physicians Surg Pak 14:98–101
24. Adlard JW, HumeMJ (2003) Cancer knowledge of the general publicin the United Kingdom: survey in a primary care setting and reviewof the literature. Clin Oncol (R Coll Radiol) 15:174–180
25. Kaur J, Brown KT (2009) Public awareness of risk factors for majorcancers in the UK and Australia. Clin Oncol (R Coll Radiol) 21:426–427
26. RavichandranK,MohamedG,Al-Hamdan NA (2010) Public knowl-edge on cancer and its determinants among Saudis in the RiyadhRegion of Saudi Arabia. Asian Pac J Cancer Prev 11:1175–1180
27. Ryan EL, Skinner CS (1999) Risk beliefs and interest in counseling:focus-group interviews among first-degree relatives of breast cancerpatients. J Cancer Educ 14:99–103
28. Pearlman DN, Clark MA, Rakowski W, Ehrich B (1999) Screeningfor breast and cervical cancers: the importance of knowledge andperceived cancer survivability. Women Health 28:93–112
29. Yardley C, Glover C, Allen-Mersh TG (2000) Demographicfactors associated with knowledge of colorectal cancer symp-toms in a UK population-based survey. Ann R Coll Surg Engl82:205–209
30. Beier ME, Ackerman PL (2003) Determinants of health knowledge:an investigation of age, gender, abilities, personality, and interests. JPers Soc Psychol 84:439–448
31. Ma GX, Fleisher L (2003) Awareness of cancer information amongAsian Americans. J Commun Health 28:115–130
32. San Turgay A, Sari D, Türkistanli EC (2005) Knowledge, attitudes,risk factors, and early detection of cancer relevant to the school-teachers in Izmir, Turkey. Prev Med 40:636–641
33. Lykins EL, Graue LO, Brechting EH, Roach AR, Gochett CG et al(2008) Beliefs about cancer causation and prevention as a function ofpersonal and family history of cancer: a national, population-basedstudy. Psychooncology 17:967–974
34. Evans R, Brotherstone H, Miles A, Wardle J (2005) Gender differ-ences in early detection of cancer. J Mens Health Gend 2:209–217
J Canc Educ
Author's personal copy
© 2015 Tunio et al. This work is published by Dove Medical Press Limited, and licensed under Creative Commons Attribution – Non Commercial (unported, v3.0) License. The full terms of the License are available at http://creativecommons.org/licenses/by-nc/3.0/. Non-commercial uses of the work are permitted without any further
permission from Dove Medical Press Limited, provided the work is properly attributed. Permissions beyond the scope of the License are administered by Dove Medical Press Limited. Information on how to request permission may be found at: http://www.dovepress.com/permissions.php
OncoTargets and Therapy 2015:8 21–27
OncoTargets and Therapy Dovepress
submit your manuscript | www.dovepress.com
Dovepress 21
O r i g i n a l r e s e a r c h
open access to scientific and medical research
Open access Full Text article
http://dx.doi.org/10.2147/OTT.S71086
lumbosacral plexus delineation, dose distribution, and its correlation with radiation-induced lumbosacral plexopathy in cervical cancer patients
Mutahir Tunio1
Mushabbab al asiri1
Yasser Bayoumi2
ali abdullah O Balbaid1
Majid alhameed3
stanciu laura gabriela1
ahmad amir O ali1
1radiation Oncology, comprehensive cancer center, King Fahad Medical city, riyadh, saudi arabia; 2national cancer institute, cairo University, cairo, egypt; 3neurology, comprehensive cancer center, King Fahad Medical city, riyadh, saudi arabia
correspondence: Mutahir Tunio radiation Oncology, comprehensive cancer center, King Fahad Medical city, PO Box 59046, riyadh 59046, saudi arabia Tel +966 1 288 9999 Fax +966 1 461 4006 email [email protected]
Background: To evaluate the dose distribution to the lumbosacral plexus (LSP) and its
correlation with radiation-induced lumbosacral plexopathy (RILSP) in patients with cervical can-
cer treated with intensity-modulated radiotherapy (IMRT) and high-dose-rate brachytherapy.
Materials and methods: After meeting eligibility criteria, 50 patients with cervical cancer
were selected who were treated with IMRT and high-dose-rate brachytherapy, and the LSP was
contoured. Mean volume; percentages of LSP volume absorbing 40, 50, 55, and 60 Gy (V30,
V40, V50, V55, and V60) and point doses (P1, P2, P3, P4, P5, P6, P7, P8, P9, and P10); and
RILSP incidence were calculated.
Results: At 60 months of follow-up, four patients (8%) were found to have grade 2/3 RILSP. The
mean maximal LSP dose in patients with RILSP was 59.6 Gy compared with 53.9 Gy in patients
without RILSP (control; P=0.04). The mean values of V40, V50, V55, and V60 in patients with
RILSP versus control were 61.8% versus 52.8%, 44.4% versus 27.7%, 8.0% versus 0.3% and
1.8% versus 0%, respectively (P=0.01, 0.001, 0.001, and 0.001, respectively).
Conclusion: The delineation of the LSP during IMRT planning may reduce the risk for RILSP.
The mean values of V40, V50, V55, and V60 for LSP should be less than 55%, 30%, 5%, and
0.5%, respectively; however, further studies are warranted.
Keywords: cervical cancer, intensity-modulated radiation therapy, lumbosacral plexus
delineation, radiation-induced lumbosacral plexopathy, dosimetric analysis
IntroductionRadiation-induced lumbosacral plexopathy (RILSP) in gynecologic cancers is a rare
but extremely serious complication of pelvic irradiation. The reported frequency of
RILSP ranges from 1.3% to 6.67%, and the clinical course is associated with different
degrees of bilateral lower limb pain, numbness, weakness, paresis or paralysis, and
rarely, urinary or fecal incontinence leading to poor quality of life.1–3 However, the
true incidence of RILSP is underestimated because it is not commonly evaluated by
radiation oncologists, and symptoms are often overlooked, given the prevalence of
lower back pain.4 In addition, the lumbosacral plexus (LSP) is not routinely contoured
in practice for patients with gynecologic cancer receiving intensity-modulated radia-
tion therapy (IMRT), which may lead to dose dumping, with higher than expected
doses seen in the LSP because it is not specified as an organ at risk.5
A standardized method for LSP delineation was devised by Yi et al for 15 patients
treated with IMRT for rectal or anal cancers.6 However, their study was criticized for its
small cohort sample size and short follow-up period. Recently, Min et al found the Yi
et al LSP contouring protocol to be a useful and reproducible guideline tool; however,
OncoTargets and Therapy 2015:8submit your manuscript | www.dovepress.com
Dovepress
Dovepress
22
Tunio et al
they proposed the delineation of lumbosacral plexus regions
(LSPRs), the regions in which LSP is likely to be present if
it is radiologically invisible.7
In the present study, we aimed to delineate LSP and
evaluate both dose distribution within LSP and its correla-
tion with RILSP.
Materials and methodsAfter receiving approval from our institutional review
board, we chose our study participants from among
patients with cervical cancer who were treated at our
institute between August 2007 and July 2012, using a
whole-pelvis concurrent chemoradiation with IMRT
technique (using six or eight coplanar beams), followed
by image-guided conformal high-dose-rate brachytherapy
(BT). Only those patients who met the following eligibil-
ity criteria were selected: histologically proven cervical
cancer; FIGO stage IIB–IIIB, but with no evidence for
distant metastasis; had undergone radical whole-pelvis
concurrent chemoradiation, using the IMRT technique,
followed by high-dose-rate BT; no locoregional or distant
failure (disease-free); and a minimal follow-up period of at
least 24 months. Patients who underwent adjuvant hyster-
ectomy or had uncontrolled diabetes or dual-energy X-ray
absorptiometry-confirmed severe osteopenia/osteoporosis
were excluded.
lumbosacral plexus delineationFor the purpose of study, LSP was delineated in each patient
from the L4–L5 interspace (junction of L4 and L5 vertebrae)
to the level of the sciatic nerve on contrast-enhanced treat-
ment planning axial slices of 5 mm thickness by a radia-
tion oncologist with assistance of a neuroradiologist using
anatomy manuals and the Yi et al protocol.6 For radiologically
visible LSP, a 5 mm diameter paint tool was used, and to the
points of radiologically invisible LSP, a freehand tool was
used to contour only the regions in which LSP was likely to
be seen (LSP regions; Figure 1).
Dose–volume histograms dataFor the purpose of study, the combined mean equivalent
dose in 2 Gy/fraction (EQD2 IMRT+BT
) was calculated, and
Figure 1 (Continued)
OncoTargets and Therapy 2015:8 submit your manuscript | www.dovepress.com
Dovepress
Dovepress
23
lumbosacral plexus delineation, dose distribution in cervical cancers
based on each patient’s dose–volume histogram (DVH),
the total LSP volume, mean maximum dose to whole LSP,
and volume percentages of the LSP absorbing, respectively,
5, 10, 20, 30, 40, 50, 55, and 60 Gy (V5,V10,V20,V30,
V40,V50,V55,V60) were then estimated. The continuous
variables were dichotomized at their median values. In
addition, point doses were calculated on LSP as P1 and P2
(point doses at the right and left portion of LSP at the level
of the L4/L5 interspace), P3 and P4 (point doses at the right
and left portion of LSP at the level of interspace L5/S1), P5
and P6 (point doses at the right and left portion of LSP at the
level of the inferior part of the sacroiliac joint), P7 and P8
(point doses at the right and left portion of LSP at the level
of ischial spine/acetabulum), and P9 and 10 (point doses
at the right and left portion of LSP at the levels of femoral
neck; Figure 1H).
evaluation of rilsPAll patients were evaluated by a neurologist. A detailed his-
tory was obtained, and neurological examination was done to
exclude other potential etiologies of lumbosacral plexopathy,
along with magnetic resonance imaging (MRI). Additional
positron emission tomography and needle electromyography
were performed if suggested by the neurologist. The RILSP
was defined as the “occurrence of paresthesias, numbness,
dysesthesias, pain or lower extremity weakness confirmed on
T2-weighted MRI (diffuse marrow and perineural foramina
hyper-intensity)”. The time of onset of RILSP was defined
as the interval between the end of concurrent chemoradiation
and the occurrence of the first RILSP symptom. Grading
of RILSP was defined as follows: 1 is asymptomatic; 2 is
symptomatic, but not interfering with activities of daily life;
3 is symptomatic and interfering with activities of daily life;
and 4 is symptomatic with disability.
statistical analysisAssociations between RILSP with relevant clinical data
(age, comorbidities, FIGO stage, chemotherapy) and dosi-
metric data (total LSP volume, LSP mean dose [Dmean
], LSP
maximum dose [Dmax
], V5, V10, V20, V30, V40, V50, V55,
and V60, and point doses [P1, P2, P3, P4, P5, P6, P7, P8, P9,
and P10]) were tested by Fisher exact test. Dosimetric data
Figure 1 cranial to piriformis portion of lumbosacral plexus (l4, l5, s1, and s2).Notes: The areas shaded green indicate the lumbosacral plexus. (A–D) Prepiriformis portion of lumbosacral plexus (anterior to piriformis and posterior to internal obturator muscle). (E and F) Postpiriformis portion of lumbosacral plexus at level of femoral neck (between gluteus maximus and obturator internus muscles). (G and H) Point doses calculation on the right and left lumbosacral plexus on digitally reconstructed radiographs.
OncoTargets and Therapy 2015:8submit your manuscript | www.dovepress.com
Dovepress
Dovepress
24
Tunio et al
100
90
80
70
60
60
50
50
40
40
30
30
Dose (Gy)
Vo
lum
e (%
)
20
20
10
100
0
Figure 2 cumulative lumbosacral plexus dose–volume histograms of all patients (red lines) on dose–volume histogram, showing patients in whom radiation-induced lumbosacral plexopathy was observed.
comparison between patients with RILSP and without RILSP
(control patients) was done by using the Student’s unpaired
t-test. A P-value lower than 0.05 was considered statistically
significant. All analysis was done using SPSS version 17.0
and Mathematica 9 software systems.
ResultsClinicopathologic and treatment characteristics of the
50 patients are summarized in Table 1. Median follow-up
was 60 months (range, 24.1–65.4 months). All patients were
treated by IMRT to total prescribed doses covering 95% of the
planned target volume, ranging between 50.4 Gy and 59.0 Gy
(median, 54 Gy) in 1.8 Gy fractions, followed by image-
guided high-dose-rate BT 21 Gy in three sessions. The
EQD2 IMRT+BT
were 80.9 Gy (standard deviation, ±7.2 Gy).
All patients received concurrent chemotherapy consisting
of weekly cisplatin 40 mg/m2 (total dose not exceeding
70 mg). There was no significant interobserver or interop-
erational variation when the LSP was contoured.
lsP DVh dataMean LSP volume was 93.2 cm3 (range, 72.3–117.3 cm3).
The mean dose (Dmean
) and mean maximal dose (Dmax
) to
the LSP were 47.9 Gy (range, 30.9–56.9 Gy) and 56.7 Gy
(range, 54.6–62.7 Gy). The mean volume percentages of the
LSP absorbing, respectively, 5, 10, 20, 30, 40, 50, 55, and
60 Gy were then calculated (V5, V10, V20, V30, V40, V50,
V55, and V60), at 99.35%, 97.8%, 91.8%, 76.15%, 57.3%,
36.05%, 4.15%, and 1.7%, respectively. Forty-eight patients
(96%) and 44 patients (88%) received doses to the LSP in
excess of 40 Gy or more and 50 Gy, respectively. Twelve
(24%) and 5 (10%) patients received doses to LSP in excess
of 55 Gy, respectively. The cumulative LSP DVHs for all
patients are shown in Figure 2. The points P5, P6, P7, and
P8 absorbed the highest doses when compared with other
points, as shown in box plot Figure 3.
rilsP and correlation with DVh dataAmong 50 patients, four (8%) were found to have a clinicora-
diologically confirmed diagnosis of RILSP of grade 2 and 3
without any evidence of tumor recurrence. The initial time
for onset of RILSP from the completion of treatment was
20 months (Table 2). In patients with RILSP, the Dmean
, Dmax
,
Table 1 clinicopathological and treatment characteristics
Variables N (%)
age 46.2 years (range, 33–55)ecOg performance scale, range 0–2comorbidities hypertension Yes 5 (10.0%) no 45 (90.0%) Diabetes Yes 3 (6.0%) no 47 (94.0%)histopathology squamous cell carcinoma 41 (82.0%) adenocarcinoma 8 (16.0%) adenosquamous cell carcinoma 1 (2.0%)FigO staging iiB 38 (76.0%) iiia 6 (12.0%) iiiB 6 (12.0%)radiological primary tumor size ,5 cm 17 (34.0%)
.5 cm 33 (66.0%)Mri-based nodal involvement negative 19 (38.0%) iliac 18 (36.0%) common iliac 13 (26.0%)Pretreatment hemoglobin .10 g/dl 46 (92.0%)
,10 g/dl 4 (8.0%)Treatment eBrT Whole pelvis 45 gy Parametrial/positive ln boost 9 gy (range, 5–9) high-dose-rate brachytherapy Dose/fraction 7 gy/fraction Total dose/fraction 21 gy/3 concurrent weekly cisplatin Dose/week 40 mg/m2
Mean cycles 5 (range, 4–7)
Abbreviations: ecOg, european cooperative Oncology group; FigO, international Federation of gynecologists and Obstetricians; Mri, magnetic resonance imaging; eBrT, external beam radiation therapy; ln, lymph node.
OncoTargets and Therapy 2015:8 submit your manuscript | www.dovepress.com
Dovepress
Dovepress
25
lumbosacral plexus delineation, dose distribution in cervical cancers
Table 2 characteristics of patients with radiation-induced lumbosacral plexopathy
Patient Age, years
Stage Onset of RILSP, months
Grade Diagnostic tool Treatment
1 44 iiia 20 2 Mri, PeT nsaiDs, Tca, aeDs, PT2 52 iiB 43 2 Mri, PeT, eMg nsaiDs, Tca, aeDs, PT3 49 iiB 52 2 Mri, PeT nsaiDs, Tca, aeDs, PT4 55 iiB 52 3 Mri, PeT, eMg nsaiDs, Tca, aeDs,
epidural steroid injections, PT
Abbreviations: MRI, magnetic resonance imaging; PET, positron imaging tomography; NSAIDs, nonsteroidal anti-inflammatory drugs; TCA, tricyclic antidepressants; AEDs, antiepileptic drugs; PT, physical therapy; eMg, electromyography.
6,000
5,000
4,000
3,000
Do
se (
cGy)
2,000
1,000
0P1 P2 P3 P4
Point dose within lumbosacral plexus
P5 P6 P7 P8 P9 P10
Figure 3 Box plot of the absorbed doses at points 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10.
V40, V50, V55, and V60 were 52.9 Gy, 59.6 cGy, 61.8%
(range, 55%–64.3%), 44.4% (range, 36%–53%), 8% (range,
6.1%–10.2%), and 1.8% (range, 1.0%–2.3%), respectively.
Statistically significant associations are presented in Table 3,
where the Dmean
, Dmax
, V40, V50, V55, and V60 of LSP were
associated with a higher number of RILSP, with P-values of
0.03, 0.04, 0.04, 0.01, 0.001, and 0.001, respectively. Point
doses (P5, P6, P7, and P8) were also found to be significantly
associated with RILSP (P=0.02).
DiscussionRILSP is rare, but is one of the most disabling complications of
pelvic radiation therapy, as the leg weakness or incapacitating
pain immobilize the patient, leading to infection, deep venous
thrombosis, and poor quality of life.8 However, the true preva-
lence of RILSP in patients with cervical cancer treated with
IMRT is underestimated, as the LSP is not routinely sought
during target delineation in such cases. Differential diagnosis
of RILP is always not easy; however, some clinical features
may be useful. For example, in tumor/metastatic LSP, the pain
may be relieved to a certain extent by either lying on one side
with the knees flexed or flexing the affected extremity at the
hip in bed. In contrast, pain is not relieved with positioning in
patients with RILSP.9 Neurological findings are unilateral in
patients with tumor/metastatic LSP, and bilateral in RILSP.10
Autonomic involvement or sphincter disturbance is unusual
in patients with RILSP.11 There is also the negative reverse
straight leg raising test in RILSP.3 Finally, no definite relief of
pain is seen in patients with RILSP after an intravenous bolus
of 100 mg dexamethasone.9 Patients with RILSP have usually
unremarkable computed tomography scans, and MRI is con-
sidered the most accurate diagnostic tool for RILSP.12 Positron
emission tomography (rule out tumor/metastatic LSP) and
electromyography (low-amplitude motor unit potentials in
RILSP) are helpful adjunct tools, along with MRI.13,14 To the
best of our knowledge, this is the first study that has sought
LSP contouring and its correlation with RILSP in patients
with cervical cancer (Table 4).
OncoTargets and Therapy 2015:8submit your manuscript | www.dovepress.com
Dovepress
Dovepress
26
Tunio et al
Table 4 studies reporting the lumbosacral plexus delineation, dosimetric data, and its correlation with rilsP
Study Patients Malignancy LSP volume Mean maximal dose to the LSP, Gy
V40, % V50, % V55, % Incidence of RILSP and initial onset after treatment
Yi et al6 15 rectal/anal cancer 100±22 cm3 (71–138)
52.6 (44.5–58.6)
58±19 22±23 0.5±0.9 7%,13 months
Min et al7 10 cervical/endometrial/ rectal/anal cancer
40.9–58.4 cm3 52.2 57.83 (30–74.5)
16.86 (0–55.3)
1.23 (0–11.2)
none
Present study
50 cervical cancer 93.2 cm3 (72.3–117.3)
56.7 (54.6–62.7)
61.8 (55–64.3)
44.4 (36–53)
8 (6.1–10.2)
8%, 20 months
Abbreviations: lsP, lumbosacral plexus; rilsP, radiation-induced lumbosacral plexopathy.
In present study, the incidence of RILSP (8%) in cervical
cancer is much higher than that reported in the literature
(∼1.3%–6.67%).4,6 However, none of the patients in present
study had grade 3 (disabling) RILSP. The possible explanation
for high incidence of RILSP in our cohort can be explained
by the fact that previous reports were mainly retrospective,
case reports/series, with low sample size and nongynecologic
tumors, and were mostly from the radiotherapy-alone era. When
using radiotherapy alone with or without BT, the tolerance to
the LSP (tolerance dose; 5% probability of severe sequelae in 5
years) has been estimated at 47 and 60 Gy, respectively; RILSP
was estimated at 70–80 Gy for full-volume irradiation.15,16 How-
ever, the radiosensitivity of peripheral nerves is likely enhanced
by concomitant chemotherapy, and the RILSP has been reported
at much lower doses (50–60 Gy), as seen in the present study,
making LSP an organ at risk during IMRT planning in such
patients.17 In our study, LSP volumes (mean volume, 93.2 cm3)
were consistent with those described by Yi et al (mean volume,
100 cm3), which also validated this study; however, it is larger
than those described by Min et al (mean volume, 40.9–58.4
cm3).6,7 The reason for larger volumes can be explained by the
contouring of LSPRs when LSP was radiologically invisible
in our series. Further in our study, the mean V50, V55, and
V60 were slightly higher (ie, 36% versus 22%; 4.15% versus
0.5%; and 1.7% versus 0%), which can be explained by dif-
ferent IMRT techniques, different concurrent chemotherapy,
parametrial boost in patients with cervical cancer, and higher
point doses to P5, P6, P7, and P8. In the present study, we also
noticed that risk for RILSP is more in patients older than 50
years of age and in those who are diabetic, although this dif-
ference was not significantly high in our cohort. In addition,
the mean LSP dose (42.95 Gy) and mean V40 (52.8%), V50
(27.7%), V55 (0.31%), and V60 (0%) in our patients without
RILSP indicates that mean dose lower than 45 Gy, V40 lower
than 55%, V50 lower than 30%, V55 lower than 5%, and V60
lower than 0.5% for LSP during IMRT planning (especially
focusing at the levels of P5, P6, P7, and P8) can significantly
reduce the risk for RILSP,7 as treatment for RILSP is usually
symptomatic and often refractory.
Strengths of our study are that it was the first study to
mention RILSP and dosimetric data correlation in patients
with cervical cancer and its appropriate selection of patients,
Table 3 associations between age, lumbosacral volume, and its dose distribution and radiation-induced lumbosacral plexopathy (rilsP)
Variables Patients without LSP (n=46) Patients with LSP (n=4) P-value
age 46.2 years (range, 33–53) 50 years (range, 47–55) 0.06Diabetes 1 (2%) 2 (4%) 0.05lsP volume, cm3 92.97 (range, 72.3–115) 93.56 (range, 74.0–117.3) 0.9Mean doses to lsP 42.95 gy 52.90 gy 0.03Maximum doses to lsP, mean 53.9 cgy 59.6 cgy 0.04 V5 98.7% (range, 79.1–100) 100% 0.9 V10 95.6% (range, 71.8–100) 100% 0.8 V20 91.1% (range, 64.0–100) 92.5% (range, 70%–100%) 0.9 V30 75.1% (range, 33.2–97.6) 77.2% (range, 64.3%–91.6%) 0.7 V40 52.8% (range, 21.9–62.8) 61.8% (range, 55.0%–64.3%) 0.01 V50 27.7% (range, 10.6–49.0) 44.4% (range, 36.0%–53.0%) 0.001 V55 0.31% (range, 0.0–1.71) 8.0% (range, 6.1%–10.2%) 0.001 V60 0 1.8% (range, 1.0%–2.3%) 0.001
Abbreviation: lsP, lumbosacral plexus.
OncoTargets and Therapy
Publish your work in this journal
Submit your manuscript here: http://www.dovepress.com/oncotargets-and-therapy-journal
OncoTargets and Therapy is an international, peer-reviewed, open access journal focusing on the pathological basis of all cancers, potential targets for therapy and treatment protocols employed to improve the management of cancer patients. The journal also focuses on the impact of management programs and new therapeutic agents and protocols on
patient perspectives such as quality of life, adherence and satisfaction. The manuscript management system is completely online and includes a very quick and fair peer-review system, which is all easy to use. Visit http://www.dovepress.com/testimonials.php to read real quotes from published authors.
OncoTargets and Therapy 2015:8 submit your manuscript | www.dovepress.com
Dovepress
Dovepress
Dovepress
27
lumbosacral plexus delineation, dose distribution in cervical cancers
periodic neurological assessment, and long follow-up.
However, our study can be criticized for its low sample size
and shorter follow-up.
ConclusionRILSP in IMRT with concurrent chemotherapy in cervical
cancer still represents a clinically underestimated problem,
and LSP delineation is not yet performed routinely, prob-
ably because of limited literature data. The mean dose lower
than 45 Gy, V40 lower than 55%, V50 lower than 30%, V55
lower than 5%, and V60 lower than 0.5% for LSP during
IMRT planning (especially focusing at levels of P5, P6, P7,
and P8) can reduce the risk for RILSP. However, further large
prospective studies on dose–RILSP in patients with cervical
cancer are therefore warranted, and LSP should be considered
as an organ at risk in all patients with cervical cancer who
are receiving IMRT.
DisclosureThe authors report no conflicts of interest in this work.
References1. Dahele M, Davey P, Reingold S, Shun Wong C. Radiation-induced
lumbo-sacral plexopathy (RILSP): an important enigma. Clin Oncol (R Coll Radiol). 2006;18(5):427–428.
2. Topikan E, Onal HC, Yavuz AA, Yavuz MN. Pathophysiology and man-agement of radiation-induced lumbosacral plexopathy. Turk Onkoloji Dergisi. 2008;23:147–152.
3. Saphner T, Gallion HH, Van Nagell JR, Kryscio R, Patchell RA. Neurologic complications of cervical cancer. A review of 2261 cases. Cancer. 1989;64(5):1147–1151.
4. Georgiou A, Grigsby PW, Perez CA. Radiation induced lumbosacral plexopathy in gynecologic tumors: clinical findings and dosimetric analysis. Int J Radiat Oncol Biol Phys. 1993;26(3):479–482.
5. Galvin JM, Ezzell G, Eisbrauch A, et al; American Society for Therapeutic Radiology and Oncology; American Association of Physi-cists in Medicine. Implementing IMRT in clinical practice: a joint docu-ment of the American Society for Therapeutic Radiology and Oncology and the American Association of Physicists in Medicine. Int J Radiat Oncol Biol Phys. 2004;58(5):1616–1634.
6. Yi SK, Mak W, Yang CC, et al. Development of a standardized method for contouring the lumbosacral plexus: a preliminary dosimetric analysis of this organ at risk among 15 patients treated with intensity-modulated radiotherapy for lower gastrointestinal cancers and the incidence of radiation-induced lumbosacral plexopathy. Int J Radiat Oncol Biol Phys. 2012;84(2):376–382.
7. Min M, Roos D, Keating E, et al. External validation of the lumbosacral plexus-contouring protocol developed by Yi et al. (IJROBP 2012;84: 376–382) for pelvic malignancies. J Med Imaging Radiat Oncol. 2014;58(1):117–124.
8. Pettigrew LC, Glass JP, Maor M, Zornoza J. Diagnosis and treatment of lumbosacral plexopathies in patients with cancer. Arch Neurol. 1984;41(12):1282–1285.
9. Thomas JE, Cascino TL, Earle JD. Differential diagnosis between radia-tion and tumor plexopathy of the pelvis. Neurology. 1985;35(1):1–7.
10. Jaeckle KA, Young DF, Foley KM. The natural history of lumbosacral plexopathy in cancer. Neurology. 1985;35(1):8–15.
11. Jaeckle KA. Neurological manifestations of neoplastic and radiation-induced plexopathies. Semin Neurol. 2004;24(4):385–393.
12. Planner AC, Donaghy M, Moore NR. Causes of lumbosacral plexopathy. Clin Radiol. 2006;61(12):987–995.
13. Brejt N, Berry J, Nisbet A, Bloomfield D, Burkill G. Pelvic radiculopa-thies, lumbosacral plexopathies, and neuropathies in oncologic disease: a multidisciplinary approach to a diagnostic challenge. Cancer Imaging. 2013;13(4):591–601.
14. Tong HC. Specificity of needle electromyography for lumbar radicu-lopathy in 55- to 79-yr-old subjects with low back pain and sciatica without stenosis. Am J Phys Med Rehabil. 2011;90(3):233–238.
15. Emami B, Lyman J, Brown A, et al. Tolerance of normal tissue to thera-peutic irradiation. Int J Radiat Oncol Biol Phys. 1991;21(1):109–122.
16. Stryker JA, Sommerville K, Perez R, Velkley DE. Sacral plexus injury after radiotherapy for carcinoma of cervix. Cancer. 1990;66(7): 1488–1492.
17. Klimek M, Kosobucki R, Luczyńska E, Bieda T, Urbański K. Radiotherapy-induced lumbosacral plexopathy in a patient with cervi-cal cancer: a case report and literature review. Contemp Oncol (Pozn). 2012;16(2):194–196.
Journal of Contemporary Brachytherapy (2014/volume 6/number 3)
Clinical InvestigationsOriginal paper
Assessment of air pockets in high-dose-rate vaginal cuff brachytherapy using cylindrical applicatorsAshraf Hassouna, MD1,2, Prof. Yasir Abdulaziz Bahadur3, Camelia Constantinescu, PhD1
1King Faisal Specialist Hospital & Research Center, Jeddah, Saudi Arabia, 2National Cancer Institute, Cairo University, Egypt, 3King Abdulaziz University Hospital, Jeddah, Saudi Arabia
AbstractPurpose: To retrospectively assess the incidence and magnitude of air pockets around vaginal cylinders and its
impact on dose distribution in vaginal cuff image-guided high-dose-rate (HDR) brachytherapy. Material and methods: Fifty endometrial carcinoma patients treated by postoperative HDR vaginal cuff brachyther-
apy were included in the study. The average age of patients was 58.3 ± 11.8 years (range: 31-87 years). Brachytherapy was performed using cylindrical applicators, and the dose prescribed to 0.5 cm from the applicator’s surface, over a length of 5 cm from the applicator’s tip. Computed tomography (CT) simulation was used for each brachytherapy fraction. The incidence, vaginal mucosa displacement, volume, and dosimetric effect of air pockets around the vaginal cylinder were evaluated.
Results: A total of 78 air pockets were found in 29/50 patients (58%) and 45/135 (33%) brachytherapy plans. They were located at the apex: 16/78 (20%) and lateral to the applicator: 62/78 (80%). The volume of air pockets ranged be-tween 0.01 and 2.1 cm3 (mean: 0.15 cm3 ± 0.36 cm3), and the maximum displacement of vaginal mucosa from cylinder surface was between 0.1 and 1.09 cm (mean: 0.34 cm ± 0.2 cm). The dose reduction to the vaginal mucosa generated by the air pockets ranged from 0.5 to 66% (mean: 26.4% ± 13.9%).
Conclusions: The presence of air pockets around vaginal cylinder applicators is frequently noticed in post-operative vaginal cuff brachytherapy. The dose to the vaginal mucosa is reduced, as a result of displacement generated by air pock-ets. The effect on the clinical outcome of this dose reduction is yet to be determined.
J Contemp Brachytherapy 2014; 6, 3: 271–275 DOI: 10.5114/jcb.2014.45436
Key words: air pockets, brachytherapy cylinder, endometrial carcinoma, vaginal brachytherapy.
PurposePost-operative vaginal cuff brachytherapy is used to
reduce the risk of vault recurrence for patients with endo-metrial or cervical carcinomas [1,2]. Although the choice of applicator for high-dose-rate (HDR) brachytherapy of vagina is both institutional and patient dependent, the most commonly used applicator is a vaginal cylinder [1]. Recently, the American Brachytherapy Society (ABS) re-ported the recommendations for adjuvant vaginal HDR brachytherapy after hysterectomy, establishing dose pre-scription and optimization guidelines [3]. As the preva-lent location of the vaginal lymphatic channels is with-in 1 mm of tissue surrounding the vaginal cylinder, the ABS report recognizes that it is imperative for the vaginal mucosa to be in contact with the cylindrical applicator surface to achieve the optimal dose distribution, and rec-ommends computed tomography (CT) planning to con-firm the presence of significant air gaps [3,4]. Air pockets around vaginal cylinders have been often noticed in our brachytherapy practice. Their presence may result in the
failure to eradicate all microscopic malignant cells, there-fore increasing the risk of recurrence.
To date, there is not enough literature addressing this subject [5,6]. The purpose of this study is to retrospec-tively assess the incidence and magnitude of air pockets around vaginal cylinders, and its impact on dose distribu-tion in CT-image based HDR brachytherapy.
Material and methodsWe retrospectively reviewed the data of 50 patients
with endometrial carcinoma, treated between July 2010 and September 2013 by post-operative HDR vaginal cuff brachytherapy in the Radiotherapy Unit of King Abdu-laziz University Hospital Jeddah, Saudi Arabia. The pa-tients had either post-operative external beam radiothera-py (EBRT; 45 Gy in 25 fractions, one fraction per day, five times per week) to the whole pelvis, using four-fields CT-based planning, followed by HDR brachytherapy (12 Gy in 3 fractions, two fractions per week), or HDR brachyther-apy alone (21 Gy in 3 fractions, one fraction per week).
Address for correspondence: Yasir A. Bahadur, MD, Department of Radiology, King Abdulaziz University Hospital, Jeddah, Saudi Arabia, PO Box 80215, Jeddah 21589, Saudi Arabia, phone: +966 505528528, e-mail: [email protected]
Received: 06.05.2014Accepted: 09.08.2014Published: 30.09.2014
Journal of Contemporary Brachytherapy (2014/volume 6/number 3)
Ashraf Hassouna, Yasir Abdulaziz Bahadur, Camelia Constantinescu272
Clinical examination was performed before brachy-therapy, to assess the patient and estimate the applicator size to be used. The appropriate cylinder size was selected according to the clinical examination before brachyther-apy. Clinical judgement (inserting the cylinder accord-ing to the vaginal stump length and monitoring patient discomfort during insertion) was used to insure that the cylinder reaches the vaginal apex. Gold seeds were not used to mark the vaginal apex. Application was com-pleted without anesthesia, and the applicator was fixed in place by a perineal belt ensuring its immobilization. Brachytherapy was carried out with cylindrical vaginal applicators of various diameters (2, 2.6, 3, and 3.5 cm), and the dose prescribed to 0.5 cm from the applicator’s surface, over a length of 5 cm from the applicator’s tip.
Computed tomography images were acquired for each brachytherapy fraction using a Siemens Somatom Emo-tion CT scanner (Siemens AG, Erlangen, Germany), with 2 mm slice intervals from the iliac crest to the ischial tu-berosities, without intravenous contrast. Dose distribution was calculated by Varian Brachyvision planning system, version 8.10 (Varian Medical Systems, Inc., Palo Alto, CA), for a brachytherapy remote afterloader Varian HDR Var-iSource iX (Varian Medical Systems, Inc.). The dose cal-
culation algorithm is based on the TG-43 formalism, as recommended by the American Association of Physicists in Medicine (AAPM) [7]. All treatment plans were retro-spectively analyzed for the presence of air pockets in the proximal 5 cm of the vaginal vault, that were contoured, and the volumes calculated by the treatment planning sys-tem, as shown in Figure 1.
The incidence, vaginal mucosa displacement, volume, and dosimetric effect of the air pockets around the vagi-nal cylinder were evaluated.
ResultsFor 50 patients, a total number of 135 CT-based bra-
chy therapy plans were retrospectively reviewed. The av- erage age of patients was 58.3 ± 11.8 years (range: 31- 87 years). We could not find any correlation between the incidence of air pockets and patient’s age. A total of 78 air pockets were found in 29/50 patients (58%) and 45/135 (33%) brachytherapy plans. They were located at the apex: 16/78 (20%) and lateral to the applicator: 62/78 (80%). The apex air pockets occurred during first brachythera-py fraction for 4 patients and in subsequent fractions for 6 patients. One patient presented air pockets at applicator apex through all treatment.
Fig. 1. Computed tomography images of air pockets present around a vaginal cylinder for a representative patient, in axial and sagittal views
Journal of Contemporary Brachytherapy (2014/volume 6/number 3)
Assessment of air pockets in high-dose-rate vaginal cuff brachytherapy 273
The incidence of air pockets per patient during the course of 3 brachytherapy fractions is presented in Figure 2. Among the 29 patients presenting air pockets, 21 (72%) had them occurring during first fraction of treatment, while 8 (28%) patients developed air pockets later in their brachytherapy course: 6 (21%) of them during second fraction and 2 (7%) during third fraction. Three patients presented air pockets during all treatment. The maxi-mum number of air pockets for a single patient during the whole brachytherapy course was 8.
The correlation between the incidence of air pockets and vaginal applicator size was analysed and is shown in Figure 3. The highest incidence of air pockets occurred for 3 cm cylinder diameter, in 25/48 (52%) fractions, and the lowest for 2 and 2.6 cm diameter, in 7/9 (21%) and 7/11 (20%) fractions, respectively.
The volume of air pockets ranged between 0.01 and 2.1 cm3 (mean: 0.15 cm3 ± 0.36 cm3) and the maximum dis-placement of vaginal mucosa from cylinder surface was be-tween 0.1 and 1.09 cm (mean: 0.34 cm ± 0.2 cm). The dosim-etric impact of air pockets was the reduction of dose to the vaginal mucosa from 0.5% to 66% (mean: 26.4% ± 13.9%), as presented in Figure 4. Even for small displacements of vaginal wall (0.1-0.2 cm), the dose reduction exceeds 10%. The dose reduction is increasing with the air pockets dis-placement, this effect being more significant for cylinders with small diameters due to the inverse square factor.
DiscussionAdapting a HDR vaginal brachytherapy plan to pa-
tient specific anatomy is challenging, as recognized by the recent ABS guidelines for adjuvant vaginal cuff bra-chytherapy after hysterectomy [3]. The applicator selec-tion may be influenced by the post-operative shape of va-gina, but is mainly physician or institutional depending, and the most commonly used applicator is a properly sized vaginal cylinder [8]. Regardless the choice of appli-cator, and though the total dose required to eradicate the disease is unknown, it is essential for the vaginal mucosa to be in contact with the applicator surface to achieve the optimal dose distribution [3,9].
The vaginal cylinder should be fitted to the vagina in size and introduced to reach the vaginal vault. Rich-ardson et al. used gold seeds to mark the vaginal vault apex and CT-imaging to verify that the vaginal cylinder reached these seeds [5]. In our study, we have used the clinical sense and patient’s feeling to ensure that the cyl-inder reached the vaginal vault apex. We reported 58% incidence of air gaps in our patients and 16/78 (20%) be-ing at the apex, while Richardson et al. [5] reported 80% incidence of air gaps for all patients and 21/90 (23%) lo-cated at the apex. It seems that clinical judgement works well and gold seeds are not mandatory to verify the cyl-inder-vaginal vault relationship.
In some patients, the vagina may not be cylindrical af-ter surgery and have an enlargement in the lateral apices, due to surgical residues of the vaginal fornices. Parity, hormonal status, or normal anatomic irregularities (e.g., larger vaginal apex in relation to introitus) can make dif-ficult the adequate placement of a vaginal cylinder. Also,
Fig. 2. Number of air pockets per patient over the course of 3 brachytherapy fractions
40
30
20
10
0
Num
ber o
f pat
ient
s
0 1 2 3 4Number of air pockets
Fraction 1 Fraction 2 Fraction 3
Fig. 3. The distribution of air pockets according to vaginal cylinder diameter
80
60
40
20
0
Num
ber o
f fra
ctio
ns (%
)
2.0 2.6 3.0 3.5Cylinder diameter (cm)
With air pockets Without air pockets
Fig. 4. The percentage dose differences between cylinder lateral surface and vaginal mucosa surface at the point of maximum displacement created by the air pockets, for various diameters of vaginal cylinders. The solid trend lines represent the inverse square factor for each cylinder
0
–10
–20
–30
–40
–50
–60
–70
Dos
e re
duct
ion
(%)
2 cm 2.6 cm 3 cm 3.5 cm
Vaginal wall displacement (cm) 0 0.5 1
Journal of Contemporary Brachytherapy (2014/volume 6/number 3)
Ashraf Hassouna, Yasir Abdulaziz Bahadur, Camelia Constantinescu274
if the chosen cylinder is too small, air gaps or folds may be present, leading to under-dosage of the vaginal target tissue. Alternative approaches for a treatment individ-ualized to the patient’s anatomy are the use of ovoids for vaginas with ‘‘dog-ear’’ configuration, or a vaginal custom mold [10,11]. The use of anatomically conformal applicators, such as intra-vaginal balloon applicator or multi-channel applicator has been also suggested [12,13].
Vaginal relapse rates in post-operative vaginal cuff brachytherapy for endometrial carcinoma patients are reported as ranging from less than 5% to 18% [1,14-16]. Since the predominant location of the vaginal lymphatic channels is within 1 mm of tissue surrounding the vaginal cylinder [4], a possible cause of relapse is that the target volume does not receive the prescribed dose, due to the presence of air pockets. Besides, many institutions do not perform fractional imaging of brachytherapy treatments; therefore the size and significance of these air pockets are widely undetermined.
We found 78 air pockets in 29/50 patients (58%) and 45/135 (33%) brachytherapy plans. Out of 50 patients, 29 (58%) did not reveal air pockets during the first frac-tion of treatment, but had air pockets present in following fractions, and 43 (86%) patients had no air pockets during last fraction of treatment. This indicates that CT-imaging only the first fraction of brachytherapy is insufficient for assessing the conformity of vaginal mucosa to the cylin-der over the whole course of treatment, as suggested by other investigators [5,17] as well. Yaparpalvi et al. ana-lysed the inter-fraction variations of cylindrical applica-tor insertion, as well as the fluctuations in bladder and rectal volumes, which have led to variations of bladder and rectal doses; they concluded that each fraction of vaginal cuff brachytherapy should be image-based, in order to achieve an accurate and complete dosimetric as-sessment of the treatment [17].
Analysing the correlation between the incidence of air pockets and vaginal applicator size, we found that the highest incidence of air pockets occurred for 3 cm cylin-der diameter, and the lowest for 2 and 2.6 cm diameter. Although selecting the largest tolerated applicator was suggested to ensure an optimal contact with the vaginal mucosa, it is not necessary to be associated with fewer incidences of air pockets. A prospective study is neces-sary to explore the role of using larger applicator in the remaining fractions in patients having air pockets in the first brachytherapy application.
Our data show that, even for small displacements of vaginal wall (1-2 mm), the dose reduction exceeds 10%. The dose reduction is increasing with the displacement generated by air pockets, this effect being more signifi-cant for cylinders with small diameters due to the inverse square factor.
Similar data have been reported by Richardson et al. [5]. In a study analysing brachytherapy plans of 25 pa-tients with post-operative endometrial carcinoma, they found an average pocket volume of 0.34 cm3 (range: 0.01-1.32 cm3), and an average mucosa displacement of 0.37 mm (range: 0.13-0.8 mm), the dosimetric impact being a reduction of the dose to the vaginal mucosa of
about 27% (range: 9-58%). Cameron et al. compared the dose to 0.5 cm from the surface of the vaginal mucosa at air gaps location to the dose that mucosa would have received if there was no air gap, and found it ranging between 54.7% and 97.3% (average: 86.7%) [6]. Although barely reported, the presence of air pockets around vag-inal cylinders seems to be a frequent event in vaginal cuff brachytherapy. However, brachytherapy appears to reduce the risk of vaginal relapse despite the vaginal mucosa potentially being displaced from the applicator by air pockets [1,14-16,18].
Even if air pockets have a considerable dosimetric ef-fect, it seems that it may be of minor clinical consequence. The highest risk of microscopic malignant cells prevails for vaginal mucosa located at the apex of applicator. Our data show that only 20% of air pockets were located at the apex, this finding decreasing the risk of not treating the microscopic disease. Moreover, except for one patient, the position of air gaps was different for each brachyther-apy fraction, additionally attenuating their overall effect, as also noticed by Cameron et al. [6].
Furthermore, for the most treatment planning sys-tems currently available, the brachytherapy dose calcu-lation algorithms do not take in account the tissue inho-mogeneity, and the dose is calculated as if the sources are surrounded by water. Yet, if low density air pockets are present, the delivered doses are fairly increased by scat-tering effects from adjacent tissues. The literature indi-cates that post-hysterectomy vaginal HDR brachytherapy is an efficient treatment, resulting in good clinical out-come for many different dose and fractionation schedules [3]. However, the dose required to eradicate microsco-pic disease is still unknown. Randomizing 290 patients with low-risk endometrial carcinoma to 15 vs. 30 Gy in 6 fractions, prescribed to 0.5 cm vaginal tissue, Sorbe et al. reported no difference in local recurrence [9]. It is not yet established if this is valid for high-intermediate-risk disease too, but it is possible that a lower dose would be similarly efficient. Therefore, even if air pockets are pres-ent, the dose to vaginal mucosa may still be sufficient to treat microscopic malignant cells [6].
The magnitude of air pockets around vaginal cylinders and their effect on dose distribution was scarcely reported in the literature [5,6], and our report will add more evidence that this issue substantiate attention and more research. Further clinical trials are needed to estimate the uncertain-ties in delivering vaginal cuff HDR brachytherapy [18,19].
DisclosureAuthors report no conflict of interest.
References1. Horowitz NS, Peters WA 3rd, Smith MR et al. Adjuvant high
dose rate vaginal brachytherapy as treatment of stage I and II endometrial carcinoma. Obstet Gynecol 2002; 99: 235-240.
2. Pearcey RG, Petereit DG. Post-operative high dose rate bra-chytherapy in patients with low to intermediate risk endo-metrial cancer. Radiother Oncol 2000; 56: 17-22.
3. Small W, Beriwal S, Demanes DJ et al. American Brachyther-apy Society consensus guidelines for adjuvant vaginal cuff
Journal of Contemporary Brachytherapy (2014/volume 6/number 3)
Assessment of air pockets in high-dose-rate vaginal cuff brachytherapy 275
brachytherapy after hysterectomy. Brachytherapy 2012; 11: 58-67.
4. Choo JJ, Scudiere J, Bitterman P et al. Vaginal lymphatic chan-nel location and its implication for intracavitary brachythera-py radiation treatment. Brachytherapy 2005; 4: 236-240.
5. Richardson S, Palaniswaamy G, Grigsby PW. Dosimetric ef-fects of air pockets around high-dose-rate brachytherapy vag-inal cylinders. Int J Radiat Oncol Biol Phys 2010; 78: 276-279.
6. Cameron AL, Cornes P, Al-Booz H. Brachytherapy in endo-metrial cancer: Quantification of air gaps around a vaginal cylinder. Brachytherapy 2008; 7: 355-358.
7. Rivard MJ, Coursey BM, DeWerd LA et al. Update of AAPM Task Group No. 43 Report. A revised AAPM protocol for Bra-chytherapy dose calculations. Med Phys 2004; 31: 633-674.
8. Small JW, Erickson B, Kwakwa F. American Brachytherapy Society survey regarding practice patterns of postoperative irradiation for endometrial cancer: Current status of vaginal brachytherapy. Int J Radiat Oncol Biol Phys 2005; 63: 1502-1507.
9. Sorbe B, Straumits A, Karlsson L. Intravaginal high-dose-rate brachytherapy for stage I endometrial cancer: A randomized study of two dose-per-fraction levels. Int J Radiat Oncol Biol Phys 2005; 62: 1385-1389.
10. Tuncel N, Garipagaoglu M, Kizildag AU et al. Optimisation techniques in vaginal cuff brachytherapy. Br J Radiol 2009; 82: 936-940.
11. Pötter R, Gerbaulet A, Haie-Meder C. The GEC ESTRO hand-book of brachytherapy. Endometrial Cancer 2002; 15: 365-401.
12. Miller DA, Richardson S, Grigsby PW. A new method of anatomically conformal vaginal cuff HDR brachytherapy. Gynecol Oncol 2010; 116: 413-418.
13. Symon Z, Menhel J, Alezra D et al. Individual fraction optimization vs. first fraction optimization for multichan-nel applicator vaginal cuff high-dose-rate brachytherapy. Brachytherapy 2006; 5: 211-215.
14. Knocke TH, Kucera H, Weidinger B et al. Primary treatment of endometrial carcinoma with high-dose-rate brachythera-py: Results of 12 years of experience with 280 patients. Int J Radiat Oncol Biol Phys 1997; 37: 359-365.
15. MacLeod C, Fowler A, Duval P et al. High-dose-rate bra-chytherapy alone post-hysterectomy for endometrial cancer. Int J Radiat Oncol Biol Phys 1998; 42: 1033-1039.
16. Alektiar KM, Venkatraman E, Barakat RR. Intravaginal bra-chytherapy alone for intermediate-risk endometrial cancer. Int J Radiat Oncol Biol Phys 2005; 62: 111-117.
17. Yaparpalvi R, Mutyala S, Thawani N et al. Variance in blad-der and rectal doses in the course of fractionated cylinder high-dose-rate brachytherapy. Brachytherapy 2009; 8: 145.
18. Nout RA, Smit VT, Putter H et al. Vaginal brachytherapy versus pelvic external beam radiotherapy for patients with endometrial cancer of high-intermediate risk (PORTEC-2): an open-label, non-inferiority, randomised trial. Lancet 2010; 375: 816-823.
19. Reed NS, Harrand RL. Counterpoint: The role of adjuvant radiation in endometrial cancer. Inside, outside, or not at all? Brachytherapy 2011; 10: 4-6.
Asian Pacific Journal of Cancer Prevention, Vol 15, 2014 2547
DOI:http://dx.doi.org/10.7314/APJCP.2014.15.6.2547Perception of Patients Regarding Cancer Support Services in Saudi Arabia
Asian Pac J Cancer Prev, 15 (6), 2547-2554
Introduction
Cancer is a major public health problem and an emerging global problem, with significant associated death and disability. It is the second leading cause of death in developed countries and is one of the three leading causes of death for adults in developing countries. Worldwide cancer rates are set to double by 2030, according to the World Health Organization. Over half of newly diagnosed cases and two thirds of cancer deaths are in low and medium-income countries. Currently the most important problems facing low and medium resource countries in this
1Gynecological Oncology Unit, 2Medical Education Department, Community Medicine, 3Family and Community Medicine Department, 4Scentific chair of professsor Abdullah Hussain Basalamah for Gynecological Cancer, Faculty of Medicine, King Abdulaziz University, 5Radiation Oncology Section, Oncology Department, King Faisal Specialist Hospital and Research Centre, Jeddah, 6Radiation Oncology and Nuclear Medicine Department, National Cancer Institute, Cairo University, Cairo, Egypt *For correspondence: [email protected]
Abstract
Background: To evaluate the perception of cancer patients toward treatment services and influencing factors and to inquire about the use of complementary alternative medicine (CAM). Materials and Methods: Information was obtained through pre-tested structured questionnaires completed by cancer patients during treatment at King Abdulaziz University Hospital, Jeddah, Saudi Arabia. Results: Of 242 patients, 137 (64.6%) accepted to enter this study. Most were Saudi (n=93, 68%), female (n= 80, 58%), educated at university (n=71, 52%), married (n=97, 72%) and with breast cancer (n=36, 26%). One-hundred (73%) patients were satisfied with the services provided; 61% were Saudi. Ninety-four (68%) respondents were satisfied with the explanation of their cancer. Twenty-eight (21.6%) patients received CAM, of them 54.0% received herbal followed by rakia (21.0%), nutritional supplements/vitamins (7.0%) and Zamam water (18.0%), with significant differences among them (p =0.004). Seven (5%) patients believed this therapy could be used alone; 34 (25%) patients believed it could be used with other treatments, regardless of whether they themselves used this therapy. Fifty-three (53%) satisfied patients felt they received enough support; 31 (58%) patients received support from family and friends; 22 (41.6%) patients received support from the health-care team. Patients who received information about their disease from their physicians and those who felt they had enough support were more satisfied. The patients who took alternative treatment were older age, mostly female and highly educated but values did not reach significance. Conclusions: We stress enhancing the educational and supportive aspects of cancer-patient services to improve their treatment satisfaction and emphasize the need for increasing the educational and awareness programs offered to these patients. Keywords: Perception - cancer - services - support - complementary alternative medicine - Saudi Arabia
RESEARCH ARTICLE
Perception of Patients with Cancer towards Support Management Services and Use of Complementary Alternative Medicine - a Single Institution Hospital-Based Study in Saudi ArabiaKhalid Hussain Sait1,4*, Nisrin Mohammad Anfinan1,4, Basem Eldeek2,4, Jawher Al-Ahmadi3,4, Maha Al-Attas4, Hesham Khalid Sait4, Hussain Abdullah Basalamah4, Nabeel Al-Ama4, Mohamed Eid El-Sayed4,5,6
century are the growth and aging of the population and the westernization of their lifestyle, which is why the role of cancer prevention programs in these countries is essential. However, to develop successful preventive programs, there needs to be an increase in the understanding of cancer and the awareness of early symptoms. Positive attitudes toward screening programs are also necessary (Boyle, Levin, 2008; Mena et al., 2013). In Saudi Arabia the total number of cancer cases among Saudis, in 2006, as reported by the Saudi Cancer Registry (SCR), was 8,054. Cancer was more among women than men, with a female to male ratio of 1: 0.94. Cancer of breast, thyroid,
Khalid Hussain Sait et al
Asian Pacific Journal of Cancer Prevention, Vol 15, 20142548
colorectal, and Non-Hodgkin’s lymphoma, were the most common cancers among women, while colorectal, Non-Hodgkin’s lymphoma, leukemia, and liver cancers were common among men (Saudi Cancer Registry, 2010). Complementary and alternative medicine (CAM) can be defined as ‘a group of diverse medical and health care systems, practices, and products that are not presently considered to be part of conventional medicine’. Complementary medicine is typically used together with conventional medicine, whereas alternative medicine is used in place of conventional medicine. The CAM therapies are classified into five categories: whole medical systems, mind-body medicine, biologically-based therapies, manipulative and body-based methods, and energy therapies (NCCAM, 2008). Several recent studies with the general population have documented widespread use of CAM (Wade et al., 2008; Nazik et al., 2012; Pan et al., 2012). Consistent with this general trend, many people dealing with chronic illnesses, including cancer, use CAM during and after treatment (Barnes et al., 2002; American Association of Retired Persons, National Center for Complementary and Alternative Medicine, 2007; Boon et al., 2007). Reasons commonly cited by people with cancer for using CAM include attempting to improve one’s physical and emotional health (Humpel et al., 2006; Fox et al 2013; Suzuki etal 2013), the desire to exert a sense of personal control over one’s illness, dissatisfaction with the medical care system or health providers (Wanchai et al., 2010), and doubts concerning the effectiveness of conventional treatment (Ritvo et al., 1999). In Saudi Arabia, studies regarding the perceptions of cancer and attitudes toward conventional and CAM therapy were few and focused mostly on the knowledge of and attitudes toward breast cancer (Alam, 2006, Bismark et al., 2014). This cross section study aimed to evaluate the perception of cancer patients toward treatment services and the factors affecting this perception. Also this study aimed to use one-on-one interviews to gain a greater knowledge and in-depth understanding of the whole process of CAM use in survivors’ cancer patients. From the previous literature, identified domains in need of investigation included: CAM use, including types and timing during the cancer journey; motivations for use of CAM; perceived positive and negative effects incurred from CAM.
Materials and Methods
This cross-sectional study was conducted among patients with cancer attending for follow-up at King Abdulaziz University Hospital (KAUH), Jeddah, Saudi Arabia, during the period from December 2011 to December 2012. All participants gave informed consent. The study was approved by the University’s Human Research Ethics Committee at King Abdelaziz University hospital. A questionnaire was designed to measure the knowledge, attitudes, and practices of patients with cancer regarding treatment services. The questionnaire was divided into four variable sections: personal data, knowledge about cancer, satisfaction, and the use of alternative and complementary treatments. Each question
had a unique code for ease of data entry and processing. The research team met 212 cancer patients attending cancer unit on Saturday and Monday of every week to obtain a simple random sample for data analysis, only 137 patients accepted to participate in this study. To fulfill the inclusion criteria, patients had to be in or outpatients; to be at least 18 years old; to be aware of their cancer diagnosis; to be fit enough to complete the questionnaire according to their physician; and to be free of any cognitive dysfunction. The patients were excluded when they had just been diagnosed or when they were hospitalized in palliative care units. Patients were assessed individually by independent investigators. Completion of the questionnaire lasted about 1 h. All patients completed the first part of the questionnaire with the help of the investigator. When they were used to the way of completion, they could choose to continue with the investigator or to do it alone. As this could have led to bias, the way patients completed the questionnaire was recorded. Physicians completed the questionnaire concerning each included patient immediately after the consultation in the ambulatory setting and the same day as the patient in the hospitalization setting.
Statistics analysis The statistical analysis was performed using Statistical Package for the Social Sciences (SPSS) version 20, IBM Corporation. Data were expressed in the form of number (percentage) or mean (standard deviation) and minimum and maximum as appropriate. A Chi-square test was used for comparisons of non-parametric values and student “t” test for comparisons of parametric values. Significance was considered when the P value was less than 0.05.
Results
The Saudi patients were more than non-Saudi (68.0% versus 32.0%, p=0.0001); female were more than male (58.0% versus 42.0%, p=0.049). Education were mostly university (52.0%), followed by illiterate (17%), primary school (12.0%), secondary school (10.0%) and preparatory school (9.0%) with significant difference between them (p=0.0001). Most of participants were married (72.0%) followed by single (27.0%), widowed (7.0%) then divorced (4.0%) with significant difference between them (p=0.0001). The type of cancer was mostly breast cancer (26.0%) then gastrointestinal (23.0%), gynecological (18.0%), urological (12.0%), leukemia and lymphoma (10.0%), lung (7.0%), soft tissue (2.0%) and head and neck (2.0%) with significant difference between them (p=0.0001) (Table 1). Regarding attitude of the cancer patients towards treatment, 7.3% of patients did not received the prescribed treatment, mostly due to financial cause (60.0%) followed by believed that treatment led to death (20.0%), did not preferred chemotherapy (10.0%) and believed that treatment were useless (10.0%) with insignificant difference between them (p=0.079). Most of patients (40.0%) were satisfied regarding treatment of cancer, 32.0% were very satisfied, 18.0% were dissatisfied and 10.0% were very dissatisfied with significant difference
Asian Pacific Journal of Cancer Prevention, Vol 15, 2014 2549
DOI:http://dx.doi.org/10.7314/APJCP.2014.15.6.2547Perception of Patients Regarding Cancer Support Services in Saudi Arabia
between them (p=0.0001). About 20.6% received alternative treatment, of them 54.0% received herbal followed by rakia (21%), nutritional supplements/vitamins (7.0%) and Zamam water (18.0%) with significant difference between them (p=0.004). Most of patients (47.0%) received alternative treatment with cancer treatment, 43% before cancer treatment and 10.0% after cancer treatment with significant difference between them (p=0.0001). Most of patients (50.0%) received alternative treatment to improve general condition, 36.0% to increase response to cancer therapy and 14.0% in response to peer pressure with insignificant difference between them (p=0.066). Most of patients (61.3%) believed that
alternative medicine should not be used, while 25.0% believed that it should be taken with medical treatment, 8.1% were not sure and 5.1% believed that it should be used alone with significant difference between them (p=0.0001) (Table 2). Using a univariate analysis to detect the predictive factors that may influence the satisfaction and dissatisfaction among the population studied, the Saudi patients were dissatisfied with the service provided to them more than non-Saudis (p=0.003). In satisfied patients female were more than male (63.0% versus 37.0%), while in dissatisfied patients male were more than female (54.0% versus 46.0%) with insignificant difference between them (p=0.055). The received information regarding cancer were in satisfied patients mostly by doctors (65.0%) followed by nurses (17.0%), report (8.0%), relative (6.0%) then accidental (4.0%); while in dissatisfied patients mostly by doctors (45.0%) followed by nurses (43.0%) then accidental (12.0%) with significant difference between them (p=0.010). Regarding satisfaction regarding explanation of cancer diagnosis, in satisfied patients were mostly strongly satisfied (42.0%) followed by satisfied (27.0%), not satisfied (25.0%) then strongly dissatisfied (6.0%); while were in dissatisfied patients were mostly satisfied (54.0%), then not satisfied (24.0%), strongly satisfied (14.0%) then strong dissatisfied (8.0%) with significant difference between them (p=0.006). Regarding enough support, in satisfied patients were mostly yes than no (53.0% versus 47.0%); while in dissatisfied patients mostly no than yes (73.0% versus 27.0%) with significant difference between them (p=0.005) (Table 3). The patients who took alternative treatment were older, mostly females and highly educated than those did not take alternative medicine but these values did not reach significant level (p=0.750, p=0.523, 0.208). The marital status in patients who received alternative treatment were mostly married (75.0%) then widowed (18.0%)
Table 1. Demographic Characteristics of the Studied Patients with Cancer (n=137).Variables Number (%) Significance
Nationality Saudi 93 (68.0%) p=0.0001 Non-Saudi 44 (32.0%) Gender Male 57 (42.0%) p=0.049 Female 80 (58.0%) Age Mean ±SD 41.00± 17.00 Minimum-maximum (19.00-75.00) Education Illiterate 23 (17.0%) p=0.0001 Primary 17 (12.0%) Preparatory 12 (9.0%) Secondary 14 (10.0%) University 71 (52.0%) Marital status Married 97 (72.0%) p=0.0001 Single 37 (27.0%) Widowed 9 (7.0%) Divorced 5 (4.0%) Type of cancer Breast 36 (26.0%) p=0.0001 Gastrointestinal 32 (23.0%) Gynecological 25 (18.0%) Urological 17 (12.0%) Leukemia & lymphoma 12 (10.0%) Lung 10 (7.0%) Soft tissue 3 (2.0%) Head & neck 2 (2.0%)
Table 2. Attitude of the Studied Patients with Cancer Towards Treatment (n=137)Questions (number) Response No (%) SignificanceDid you receive the prescribed treatment? No 10 (7.3%) p=0.079If no, what was the cause? (n=10) Financial 6 (60.0%) Treatment lead to death 2 (20.0%) I do not prefer chemotherapy 1 (10.0%) Treatment useless 1 (10.0%) Are you satisfied regarding the treatment of cancer patients? (n=137) Very satisfied 45 (32.0%) p=0.0001 Satisfied 55 (40.0%) Dissatisfied 24 (18.0%) Very dissatisfied 13 (10.0%) Did you receive complementary alternative methods? (n=137) Yes 28 (20.6%) p=0.004If yes, what type of treatment did you receive? (n=28) Herbal 15 (54.0%) Religious/spirit practice (Rakia) 6 (21.0%) Zamzam water 5 (18.0%) Nutritional supplements/vitamins 2 (7.0%) When did you receive the alternative treatment? (n=28) Before treatment 12 (43.0%) p=0.0001 With treatment 13 (47.0%) After treatment 3 (10.0%) Why do you receive alternative treatment? (n=28) Improve general condition 14 (50.0%) p=0.066 Increase response 10 (36.0%) Peer pressure 4 (14.0%) How you believe that alternative methods should be used? (n=137) Alone 7 (5.1%) p=0.0001 With medical treatment 34 (25.0%) Never 84 (61.3%) I am not sure 12 (8.5%)
Khalid Hussain Sait et al
Asian Pacific Journal of Cancer Prevention, Vol 15, 20142550
and single (2.0%); while in patients who did not received alternative medicine were mostly married (59.0%) then single (32.0%), divorced (5.0%) then widowed (4.0%) with significant difference between the (p=0.003) (Table 4).
Discussion
According to the 2007 Saudi cancer registry (Bazerbashi, 2007), the total number of new adult cancer cases reported was 11,437. Overall, cancer was slightly more prevalent in females than in males (Bazerbashi, 2007). These numbers encouraged us to study the knowledge and attitudes of these patients to improve the role of the health team in dealing with this disease and those patients. During our study, we mainly explored patient satisfaction with their care and the factors influencing satisfaction. In addition, we inquired about the use of CAM among cancer patients. In this study, the Saudi patients were more than non-Saudi; female were more than male university education was more than other types of educations; married status were more than single, widowed and divorced. The patients included in this study had various cancer types, mostly breast cancer then gastrointestinal, gynecological, urological, leukemia & lymphoma, lung, soft tissue and head and neck. The National Cancer Registry Report in 2001 revealed that breast cancer represented 12.9% of all cancers among Saudi population (Amin et al., 2009).
Regarding attitude of the our cancer patients towards treatment, 7.3% of patients did not received the prescribed treatment, mostly due to financial cause followed by believed that treatment led to death, did not preferred chemotherapy and believed that treatment were useless. As could be seen, financial constraint was described as a major obstacle to accessing health care in cancer care in particular. In this study, most of patients (40.0%)
Table 3. Predictors of the Satisfaction of Patients with CancerParameters Dissatisfied (n=37) Satisfied (n=100) p value Number (%) Number (%)
Nationality Saudi 32 (86.0%) 61 (61.0%) p=0.003 Non-Saudi 5 (14.0%) 39 (39.0%) Gender Male 20 (54%) 37 (37.0%) p=0.055 Female 17 (46%) 63 (63.0%) Age Mean±SD 41.0±18.0 40.9±17.0 p=0.930Education Illiterate 4 (11.0%) 19 (19.0%) p=0.166 Primary 3 (8.0%) 14 (14.0%) Preparatory 2 (5.0%) 10 (10.0%) Secondary 7 (19.0%) 7 (7.0%) University 21 (57.0%) 50 (5.0%) Marital status Married 22 (60%) 63 (63%) p=0.470 Single 12 (32.0%) 26 (26.0%) Widowed 3 (8.0%) 6 (6.0%) Divorced - 5 (5.0%) Who informed you? Doctor 17 (45%) 65 (65%) p=0.010 Nurse 16 (43.0%) 17 (17.0%) Report - 8 (8.0%) Relative - 6 (6.0%) Accidental 5 (12.0%) 4 (4.0%) Satisfaction regarding explanation of cancer diagnosis Strongly dissatisfied 3 (8.0%) 6 (6.0%) p=0.006 Not satisfied 9 (24.0%) 25 (25.0%) Satisfied 20 (54.0%) 27 (27.0%) Strongly satisfied 5 (14.0%) 42 (42.0%) Did you receive enough support? Yes 10 (27.0%) 53 (53.0%) p=0.005 No 17 (73.0%) 37 (47.0%) Who provided you with support? Friends and family 6 (60.0%) 31 (58.0%) p=0.152 Health care team 4 (40.0%) 22 (42.0%)
Table 4. Comparison between Patients Who Received and Did not Receive Complementary Alternative MedicineParameters Received Did not receive alternative alternative treatment treatment (n=28) (n=109) p value
Nationality Saudi 17 (61.0%) 79 (69.0%) p=0.250 Non-Saudi 11 (39.0%) 33 (31.0%) Gender Male 12 (43.0%) 45 (42.0%) p=0.523 Female 16 (57.0%) 64 (58.0%) Age p=0.750 Mean±SD 49.76±20.10 36.92±15.35 Marital status Married 21 (75.0%) 65 (59.6%) p=0.003 Single 2 (7.0%) 35 (32.1%) Widowed 5 (18.0%) 4 (3.7%) Divorced - 5 (4.6%) Education p=0.208 Illiterate 7 (25.0%) 16 (14.7%) Primary 6 (21.4%) 11 (10.1%) Preparatory 1 (3.6%) 11 (10.1%) Secondary 3 (10.7%) 11 (10.1%) University 11 (39.3%) 60 (55.0%)
Asian Pacific Journal of Cancer Prevention, Vol 15, 2014 2551
DOI:http://dx.doi.org/10.7314/APJCP.2014.15.6.2547Perception of Patients Regarding Cancer Support Services in Saudi Arabia
were satisfied regarding treatment of cancer, 32.0% were very satisfied, 18.0% were dissatisfied and 10.0% were very dissatisfied. In our study, education level did not significantly affect satisfaction, contradicting other studies that showed that higher levels of education were associated with better appreciation of the cancer disease (Opoku et al., 2012). A study performed in Jeddah among a group of adults regarding oral cancer knowledge found that greater the awareness of oral cancer was observed most among highly educated individuals (Al-attas, 2005). The patient-physician relationship is a significant factor influencing patient medication adherence and, ultimately, positive outcomes. If patients are satisfied with the relationship, they are more likely to comply with the advice of the health-care provider. Improving patient-physician relationships can effectively promote patient medication adherence. In this study, the Saudi patients were dissatisfied with the service provided to them more than non-Saudis. In satisfied patients female were more than male (63.0% versus 37.0%), while in dissatisfied patients male were more than female (54.0% versus 46.0%) with insignificant difference between them. The received information regarding cancer were in satisfied patients mostly by doctors (65.0%) followed by nurses (17.0%), report (8.0%), relative (6.0%) then accidental (4.0%); while in dissatisfied patients mostly by doctors (45.0%) followed by nurses (43.0%) then accidental (12.0%) with significant difference between them. These numbers draw our attention to the importance of the patient-doctor relationship in increasing patient and public knowledge about cancer and its determinants among Saudi. In Riyadh Region of Saudi Arabia, a study showed that 67.6% of patients had no knowledge of any cancer warning signals, but 80.7% of patients believed some cancers can be cured if detected early. It also found that 65.1% of patients received information about cancer from television or radio (Ravichandran et al., 2010).
Cancer patients often experience distress. However, the majority of newly diagnosed patients gradually adapt to the crisis. When symptoms of distress and anxiety persist over months, patients require psychosocial support; the psychological status of cancer patients plays a major role in adherence and general condition. Fifty three (53%) satisfied patients felt they were receiving enough support, with 58% of the support provided by family and friends, while 42% of the support came from the health-care team. Many studies have shown the need for the cancer patient to receive psychological support (Ernstmann et al., 2009; Goldzweig et al., 2010). In a German study, 18.9% of patients indicated an unmet need for psychosocial support and 9.5% of patients were actually using psychosocial services; that need was higher in female patients (Merckaert et al., 2010).
There is a growing interest in the use of CAM among people diagnosed with cancer. In this study, about 20.6% of cancer patients received CAM therapies. The use of CAM therapies among patients with cancer ranged from 7-64% (1998; Richardson et al., 2000; Greenlee et al., 2009 ;Muhamad et al 2012 ; Marriam and Muhamad 2013). The large variability in CAM use between studies (from 7% to 64%) was likely to be due to the various
researchers’ and patients’ differing definition of CAM (Bismark et al 2014). There have been a limited number of studies that include patients with a wider variety of cancers. A US study of 453 patients attending a cancer centre found 83% had used at least one CAM (Richardson et al., 2000). A Finnish study found 30% of 216 breast cancer and 28% of 190 prostate cancer patients used CAM after diagnosis (Salmenpera, 2002).
Variations among the most commonly used CAMs are quite substantial and attributable to factors such as differences in study populations and inconsistency in the way CAMs are defined (Gansler et al 2008 ; Wang & Chung 2012; Saibul et al., 2012). In our study, the most commonly used CAM in cancer survivors are herbal (54.0%) then rokia (21.0%), which can be considered prayer/spiritual practices methods, Zamzam water (18.0%), and nutritional supplements/vitamins (7.0%). In 2002, Ni et al. reported that the three most commonly used therapies to be spiritual healing or prayer (13.7%), herbal medicine (9.6%), and chiropractic therapies (7.6%). The distinction between prevalence when spiritual and nonspiritual CAMs are included is important, as opinions and practices differ regarding whether spiritual practices should be considered as CAMs. For this reason, some studies exclude spiritual-based practices (American Association of Retired Persons, National Center for Complementary and Alternative Medicine, 2007; Matthews et al., 2007) whereas others report the results with and without spiritual-based practices (Barnes et al., 2002; Grzywacz et al., 2006). The analysis of the 2002 NHIS data for CAM use by US adults found that the most commonly used therapies were prayer for one’s own health (43.0%), prayer by others for one’s own health (24.4%), natural products (18.9%), deep breathing exercises (11.6%), participation in prayer group for one’s own health (9.6%), meditation (7.6%), and chiropractic care (7.5%) (Barnes et al., 2004). In the Middle East, traditional herbal medicine is the leading CAM modality in the context of cancer care (Yildirim et al., 2006; Afifi et al., 2010; Ulger and Yagli, 2010). The use of herbs for treating cancer is documented in various historical documents that include well-known Arab and Jewish physicians such as Ibn Sina (980-1037 AD) and Maimonides (1135-1204 AD). The contemporary prevalence of CAM use among patients with cancer has been studied in Jordan (Yildirim et al., 2006), and Turkey (Tarhan et al., 2009) and may reach as many as half of the patients. Accumulating evidence from epidemiological studies supports the anticancer properties of garlic and its organosulfur compounds (Nagini, 2008). The potential of phytochemicals present in spices such as turmeric, red chilli, cloves, ginger, fennel, fenugreek, and black cumin in cancer prevention has been established (Aggarwal et al., 2008). There is accumulating evidence that repeated daily brief cold stress by hydrotherapy over many months can increase both numbers and activity of peripheral cytotoxic T lymphocytes and natural killer cells, the major effectors of adaptive and innate tumour immunity (Shevchuk and Radoja, 2007).
In this study, twelve of these patients (43.0%) received these CAMs prior to treatment, which may affect the early treatment of the disease. Thirteen other patients
Khalid Hussain Sait et al
Asian Pacific Journal of Cancer Prevention, Vol 15, 20142552
(47.0%) reported their use of CAM in conjunction with standard treatment, which raises the possibility of drug-herb interaction with possible serious side effects that may interfere with delivery of the standard treatment or possibly reduce its efficacy. And three patients (10.0%) used CAM after treatment of disease. Additionally, of those who used and those who did not use CAM, 7 (5.1%) patients believed that it could be taken alone and 34 (25.0%) patients thought it could be used with other treatments, which points to the importance of patient education and the responsibility of the physician to meticulously explore patients’ ideas and behaviors regarding CAM. Salmenpera (2002) examined whether the expected benefits from CAM use were realized among a sample of cancer patients. Forty-three women (90%) and 29 men (62%) reported positive effects on physical coping and general health.
In this study, most of patients (50.0%) received alternative treatment to improve general condition, 36.0% to increase response to cancer therapy and 14.0% in response to peer pressure. In most cases, CAM users are not disappointed or dissatisfied with conventional medicine but want to do everything possible to regain their health and improve their quality of life (Rabow et al., 2004). Patients may use CAM to reduce side effects and toxicity, to protect and stimulate immunity, or to prevent second cancers or recurrences. Other expectations included treating some of the side-effects from conventional treatment and because they perceived there would be less side-effects from CAM products and therapies. The use of CAM to boost the immune system is not without risk, especially if the person is currently undergoing conventional treatment for cancer. For example, combined use of some herbal supplements with an anticancer therapy such as chemotherapy, may change the amount of the drugs absorbed (Sparreboom et al., 2004). An Australian study of 156 cancer patients found the expectations of benefits varied widely depending on the CAM used (Miller et al. 1998). For example, more than 50% of patients using shark cartilage believed that it would cure their cancer, whereas only 7-14% of those using meditation, change of diet or multivitamins believed that a cure would be the outcome. Shen et al. (2002) also found the primary reason for use depended on the CAM modality. For example, among the women with breast cancer using relaxation and meditation techniques, the main reason was to boost the immune system (40% of participants), followed by to treat the cancer (32%). Similarly, in a survey of 411 Canadian breast cancer survivors (Boon et al., 2000), 63% reported boosting the immune system was the main reason for CAM use.
In this study, married patients were significantly higher likely to use CAM. Meanwhile old age, female and higher education patients were more likely to use CAM than those who did not use CAM but levels did not reach significant level, that may be the small number of patients used CAM in this study. Previous studied reported that CAM use for cancer treatment is more prevalent among women than men and is associated with younger age, higher education, higher socioeconomic status, advanced disease, active coping behaviour, and a change in life outlook and beliefs
since the diagnosis of cancer (Edgar et al., 2000; Paltiel et al., 2001; Alferi et al., 2001; Wolsko et al., 2004; AARP, NCCAM, 2007; Gansler et al., 2008; Truant et al., 2013 ; Saghatchian et al., 2014 ). Some potential explanations why female used CAM than male are that women tend to pay more attention to their health (Berrigan et al., 2003); more frequent physician visits (Bertakis et al., 2000). A study of CAM use among persons aged ≥50 years found that use decreased with age. Persons aged 50 to 54 years (69%) and 55 to 64 years (70%) were more likely to have used a CAM practice than those aged ≥65 (54%) (AARP, NCCAM, 2007). An analysis of 2002 NHIS data found that age was positively correlated with CAM use if prayer was included (Barnes et al., 2002). If prayer (aside from praying for health for oneself) was excluded, all the CAM categories demonstrated “inverted U-shaped” relationships with age, with highest use levels among the middle-aged, whereas the youngest and oldest groups reported the least use of CAM (Barnes et al., 2002). Meanwhile, others reported negative associations between CM use and age (Barnes et al., 2002; Committee on the Use of Complementary and Alternative Medicine by the American Public IoM, 2005),
In conclusion, we stress the need for a change in cancer patient care to improve satisfaction. In this study, these changes demonstrated improved outcomes in the study-population. This change is centered upon improving the patient-doctor relationship, as that has been shown to greatly affect not only awareness but also behavior. This study provides additional evidence that CAM use among cancer survivors is common and may be determined by disease and demographic factors. In order to encourage open communication of CAM use by their patients, oncologists should be knowledgeable about the most commonly used remedies, or at least be able to direct patients to reliable sources of information. Establishment of support groups at KAUH for patients with cancer is essential for the improvement of health-care team relationships with cancer patients. Regular feedback will be obtained from those patients with the aim of detecting areas that need improvement. Further research should investigate the connection between CM use and particular long-term and late effects of treatment, such as fear of recurrence or fatigue.
Acknowledgements
We would like to thank our colleagues for helping us to complete the interview and to populate the questionnaires. We express great appreciation to all of the cancer patients for taking the time to complete the questionnaire. May Allah continue their cure from their cancer. Special thanks to the team of the scientific chair of Prof. Abdullah Hussain Basalamah for gynecological cancer at King Abdulaziz University for their support.
References
AARP, NCCAM (2007). Complementary and alternative medicine: what people 50 and older are using and discussing with their physicians. Washington, DC: AARP.
Asian Pacific Journal of Cancer Prevention, Vol 15, 2014 2553
DOI:http://dx.doi.org/10.7314/APJCP.2014.15.6.2547Perception of Patients Regarding Cancer Support Services in Saudi Arabia
Afifi FU, Wazaify M, Jabr M, Treish E (2010). The use of herbal preparations as complementary and alternative medicine (CAM) in a sample of patients with cancer in Jordan. Complement Ther Clin Pract, 16, 208-12.
Aggarwal BB, Kunnumakkara AB, Harikumar KB, et al (2008). Potential of spice-derived phytochemicals for cancer prevention. Planta Med, 74, 1560-9.
Alam AA (2006). Knowledge of breast cancer and its risk and protective factors among women in Riyadh. Ann Saudi Med, 26, 272-7.
Al-attas SA (2005). Knowledge, attitude and behavior about oral cancer, among a group of adult Jeddah population. Saudi Dental J, 17, 34-42.
Alferi SM, Antoni MH, Ironson G, Kilbourn KM, Carver CS (2001). Factors predicting the use of complementary therapies in a multi-ethnic sample of early-stage breast cancer patients. J Am Med Womens Assoc, 56, 120-3.
American Association of Retired Persons (2007). National Center for Complementary and Alternative Medicine. Complementary and Alternative Medicine: What People 50 and Older Are Using and Discussing With Their Physicians. Washington, DC: AARP.
Amin TT, Al Mulhim AR, Al Meqihwi A (2009). Breast cancer knowledge, risk factors and screening among adult Saudi women in a primary health care setting. Asian Pac J Cancer Prev, 10, 133-8.
Barnes PM, Powell-Griner E, McFann K, Nahin RL (2004). Complementary and alternative medicine use among adults: United States, 2002. Adv Data, 343, 1-19.
Bazerbashi S (2007). Cancer incidence for specific site. National Saudi Cancer Registry. Riyadh (KSA): Ministry of Health.
Bernstein BJ, Grasso T (2001). Prevalence of complementary and alternative medicine use in cancer patients. Oncology, 15, 1267-72.
Berrigan D, Dodd K, Troiano RP, Krebs-Smith SM, Barbash RB (2003). Patterns of health behavior in US adults. Prev Med, 36, 615-23.
Bertakis KD, Azari R, Helms LJ, Callahan EJ, Robbins JA (2000). Gender differences in the utilization of health care services. J Fam Pract, 49, 147-52.
Bismark RS, Chen H, Dy GK, Gage-Bouchard EA, Mahoney MC (2014). Complementary and alternative medicine use among patients with thoracic malignancies: Support Care Cancer.
Boon H, Stewart S, Kennard M, et al (2000). Use of complimentary/alternative medicine by breast cancer survivors in Ontario: prevalence and perceptions. J Clin Oncol, 18, 2515-21.
Boon HS, Olatunde F, Zick SM (2007). Trends in complementary/alternative medicine use by breast cancer survivors: comparing survey data from 1998 and 2005. BMC Womens Health, 7, 4.
Boyle P, Levin B. World cancer report 2008. Lyon: International Agency for Research on Cancer; 2008.
Committee on the Use of Complementary and Alternative Medicine by the American Public IoM. Complementary and alternative medicine in the United States. Washington, DC: National Academies Press; 2005.
Edgar L, Remmer J, Rosberger Z, Fournier MA (2000). Resource use in women completing treatment for breast cancer. Psychooncology, 9, 428-38.
Ernstmann N, Neumann M, Ommen O, et al (2009). Determinants and implications of cancer patients’ psychosocial needs. Support Care Cancer, 17, 1417-23.
Fox P Butler M, Coughlan B, Murray M, et al (2013). Using a mixed methods research design to investigate complementary alternative medicine (CAM) use among women with breast cancer in Ireland. Eur J Oncol Nurs, 17, 490-7.
Gansler T, Kaw C, Crammer C, Smith T (2008). A population-based study of prevalence of complementary methods use by cancer survivors: a report from the American Cancer Society’s studies of cancer survivors. Cancer, 113, 1048-57.
Goldzweig G, Meirovitz A, Hubert A, et al (2010). Meeting expectations of patients with cancer: relationship between patient satisfaction, depression, and coping. J Clin Oncol, 28 1560-5.
Greenlee H, Kwan ML, Ergas IJ, et al (2009). Complementary and alternative therapy use before and after breast cancer diagnosis: the Pathways Study. Breast Cancer Res Treat, 117, 653-65.
Grzywacz JG, Suerken CK, Quandt SA, et al (2006). Older adults’ use of complementary and alternative medicine for mental health: findings from the 2002 National Health Interview Survey. J Altern Complement Med, 12, 467-73.
Humpel N, Jones SC (2006). Gaining insight into the what, why and where of complementary and alternative medicine use by cancer patients and survivors. Eur J Cancer Care, 15, 362-8.
Matthews AK, Sellergren SA, Huo D, List M, Fleming G (2007). Complementary and alternative medicine use among breast cancer survivors. J Altern Complement Med, 13, 555-62
Mena M1, Wiafe-Addai B, Sauvaget C, et al (2014). Evaluation of the impact of a breast cancer awareness program in rural Ghana: a cross-sectional survey. Int J Cancer, 134, 913-24.
Meriam S, Merriam s ,Muhamad M (2013). Roles traditional healers play in cancer treatment in Malaysia: implications for health promotion and education. Asian Pac J Cancer Prev, 14, 3593-601.
Merckaert I, Libert Y, Messin S, et al (2010). Cancer patients’ desire for psychological support: prevalence and implications for screening patients’ psychological needs. Psychooncology, 19, 141-9.
Miller M, Boyer M, Butow P, Gattellari M, Dunn S, Childs A (1998). The use of unproven methods of treatment by cancer patients: frequency, expectations and cost. Supportive Care Cancer, 6, 337-47.
Muhamad M, Merriam S, Suhami N (2012). Why breast cancer patients seek traditional healers. Int J Breast Cancer, 2012, 689168.
Nagini S (2008). Cancer chemoprevention by garlic and its organosulfur compounds-panacea or promise. Anticancer Agents Med Chem, 8, 313-21.
Nazik E, Nazik H, Api M, Kale A, Aksu M (2012). Complementary and alternative medicine use by gynecologic oncology patients in Turkey. Asian Pac J Cancer Prev, 13, 21-5
NCCAM (National Center for Complementary and Alternative Medicine): http://nccam. nih.gov/health/whatiscam/ 2008.
Ni H, Simile C, Hardy AM (2002). Utilization of complementary and alternative medicine by United States adults: results from the 1999 national health interview survey. Med Care, 40, 353-8.
Opoku SY, Benwell M, Yarney J (2012). Knowledge, attitudes, beliefs, behavior and breast cancer screening practices in Ghana, West Africa. Pan Afr Med J, 11, 28.
Paltiel O, Avitzour M, Peretz T, et al (2001). Determinants of the use of complementary therapies by patients with cancer. J Clin Oncol, 19, 2439-48.
Pan SY, Gao SH, Zhou SF, et al (2012).New perspectives on complementary and alternative medicine: an overview and alternative therapy. Altern Ther Health Med, 18, 20-36.
Rabow MW, Dibble SL, Pantilat SZ, McPhee SJ (2004). The comprehensive care team: a controlled trial of outpatient palliative medicine consultation. Arch Intern Med, 164, 83-91.
Ravichandran K, Mohamed G, Al-Hamdan NA (2010). Public knowledge on cancer and its determinants among Saudis
Khalid Hussain Sait et al
Asian Pacific Journal of Cancer Prevention, Vol 15, 20142554
in the Riyadh Region of Saudi Arabia. Asian Pac J Cancer Prev, 11, 1175-80.
Richardson MA, Sanders T, Palmer JL, Greisinger A, Singletary SE (2000). Complementary/alternative medicine use in a comprehensive cancer center and the implications for oncology. J Clin Oncol, 18, 2505-14.
Ritvo P, Irvine J, Katz J, et al (1999). The patient’s motivation in seeking complementary therapies. Patient Educ Couns, 38, 161-5.
Saghatchian M, Bihan C, Chenailler C, et al (2014). Exploring frontiers:Use of complementary and alternative medicine among patients with early-stage breast cancer. Breast. [Epub ahead of print]
Saibul N, Shariff ZM, Rahmat A, Sulaiman S, Yaw YH (2012). Use of complementary and alternative medicine among breast cancer survivors. Asian Pac J Cancer Prev, 13, 4081-6
Salmenpera L (2002). The use of complementary therapies among breast and prostate cancer patients in Finland. Eur J Cancer Care, 11, 44-50.
Saudi Cancer Registry (2010). Cancer incidence report, Saudi Arabia-2006. Kingdom of Saudi Arabia: Ministry of Health.
Shen J, Andersen R, Albert P, et al (2002). Use of complementary/alternative therapies by women with advanced-stage breast cancer. BMC Complement Altern Med, 2, 1-7.
Shevchuk NA, Radoja S (2007). Possible stimulation of anti-tumor immunity using repeated cold stress: a hypothesis. Infect Agent Cancer, 2, 20-9.
Sparreboom A, Cox M, Acharya M, Figg W (2004). Herbal remedies in the United States: potential interactions with anticancer agents. J Clin Oncol, 22, 2489-503.
Suzuki N, N Takimoto Y, Suzuki R, et al (2013). Efficacy of oral administration of Lentinula eododes mycelia extract for breast cancer patients undergoing postoperative hormone therapy. Asian Pac J Cancer Prev, 14, 3469-72
Tarhan O, Alacacioglu A, Somali I, et al (2009). Complementary-alternative medicine among cancer patients in the western region of Turkey. J BUON, 14, 265-9
Truant TL, Porcino AJ, Ross BC, Wong ME, Hilario CT (2013). Complementary and alternative medicine (CAM) use in advanced cancer: a systematic review. J Support Oncol, 11, 105-13.
Ulger O, Yagli NV (2010). Effects of yoga on the quality of life in cancer patients. Complement Ther Clin Pract, 16, 60-3.
Wade C, Chao M, Kronenberg F, Cushman L, Kalmuss D (2008). Medical pluralism among American women: results of a national survey. J Womens Health (Larchmt), 17, 829-40.
Wanchai A, Armer JM, Stewart BR (2010). Complementary and alternative medicine use among women with breast cancer: a systematic review. Clin J Oncol Nurs, 14, 45-55
Wang HH1, Chung UL (2012). Use of complementary and alternative medicine among breast cancer survivors in Taiwan. Asian Pac J Cancer Prev, 13, 4789-92.
Wolsko PM, Eisenberg DM, Davis RB, Phillips RS (2004). Use of mind-body medical therapies. J Gen Intern Med, 19, 43-50.
Yildirim YK, Fadiloglu C, Uyar M (2006). Complementary therapies in palliative cancer care. Agri, 18, 26-32.
E
case report
Vulvar extrauterine endometrialstromal sarcoma: A case reportand literature review
Hematol Oncol Stem Cell Ther xx(xx) xxx Quarter 2015
Khaled J Zaza a, Maria A Arafah b, Ismail A Al-Badawi c,*
a College of Medicine, Alfaisal University, P.O. Box 50927, Riyadh 11533, Saudi Arabia, b Department of Pathology, College of Medicine,
King Saud University, P.O. Box 2925, Riyadh 11461, Saudi Arabia, c Department of Obstetrics and Gynecology, King Faisal Specialist Hospital
and Research Centre (KFSH & RC), P.O. Box 3354, Riyadh 11211, Saudi Arabia
* Corresponding author at: Department of Obstetrics and Gynecology, King Faisal Specialist Hospital and Research Centre (MBC-52), P.O.
Box 3354, Riyadh 11211, Saudi Arabia. Tel.: +966 11 442 4885; fax: +966 11 442 7397 Æ [email protected] Æ [email protected] Æ[email protected] Æ Received for publication 28 May 2014 Æ Accepted for publication 9 December 2014
Hematol Oncol Stem Cell Ther 2015; xx(xx): xxx–xxx
ª 2015 King Faisal Specialist Hospital & Research Centre. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-NDlicense (http://creativecommons.org/licenses/by-nc-nd/4.0/).DOI: http://dx.doi.org/10.1016/j.hemonc.2014.12.003
Endometrial stromal sarcoma (ESS) is an extremely rare neoplasm accounting for only 0.2% of all uterine
malignancies and for 15–26% of primary uterine sarcomas. The annual incidence of ESS is 1–2 per million
women. Herein, to the best of our knowledge, we present the first reported case of ESS of the vulva in a
50-year-old female presenting with per vaginal spotting over a period of three months. Her past surgical
history included a subtotal hysterectomy and left salpingo-oophorectomy for uterine fibroids ten years
previously. On examination, a 3.5 · 3 · 2 cm cystic mass was found in the right labia majora. The mass
was excised and the diagnosis of endometrial stromal sarcoma was made. Subsequent metastatic workup
was negative and the patient was started on megestrol acetate. She has remained disease free with no
signs or symptoms of recurrent or advanced disease for 28 months.
KEYWORDS: Endometrial stromal sarcoma; Vulva; Uterine sarcoma
INTRODUCTION
ndometrial stromal sarcoma (ESS) is an extre-mely rare neoplasm accounting for only 0.2%of all uterine malignancies and for 15–26% of
primary uterine sarcomas.1 The annual incidence ofESS is 1–2 per million women. Compared to otheruterine malignancies, ESS affects younger womenwith a mean age of 42–58 years.2 ESS resembles stro-mal cells in the proliferative stage of the normal endo-metrium and are often low-grade, indolent, butmetastatic exhibiting myometrial and/or vascularinvasion.3 The incidence of ESS in extrauterine loca-tions is exceedingly rare especially in the absence ofmetastasis or extension of a primary neoplasm. Prin-cipal extrauterine sites of ESS include the ovary,bowel wall, abdomen, peritoneum, pelvis, andvagina.4–6
Herein, to the best of our knowledge, we presentthe first reported case of ESS of the vulva in a 50-year-old female presenting with per vaginal spotting
of three months’ duration. A literature review ofESS is also presented.
CASE REPORT
A 50-year-old female, para 8+0 was referred to ourservice with per vaginal spotting. Her past medicalhistory was unremarkable; her previous surgicalhistory included a subtotal hysterectomy and left sal-pingo-oophorectomy for uterine fibroids ten yearspreviously. On clinical examination, a soft yellowishlesion with hemorrhagic foci, measuring3.5 · 3 · 2 cm, was found and a Bartholin gland cystwas suspected. On February 2012, an excisionalbiopsy was performed and the rendered histopatholo-gical diagnosis was low-grade endometrial stromalsarcoma. Microscopy showed a diffuse cellular infil-trate composed of monotonous bland looking ovalto spindle cells (simulating endometrial stromal cells)surrounding arterioles resembling endometrial spiral
1
Figure 1. Endometrial stromal sarcoma. (a) A cellular neoplasm composed of bland oval cells surrounding arterioles resembling endometrial spiralarterioles (hematoxylin and eosin stain, ·200 magnification). (b) A higher magnification microphotograph showing the spindle cells surrounding ablood vessel (hematoxylin and eosin stain, ·400 magnification).
Figure 2. Endometrial stromal sarcoma. (a) Plaques of hyalinized fibrosis (hematoxylin and eosin stain, ·200 magnification). (b) Areas of tumornecrosis on the left of the image in contrast to the viable tumor on the right (hematoxylin and eosin stain, ·100 magnification).
2
case report VULVAR EXTRAUTERINE ESS
arterioles (Figure 1). Few mitoses and large foci oftumor necrosis and hemorrhage were seen, as wellas plaques of hyaline fibrosis (Figure 2). The lesionwas infiltrative with poorly defined margins. The sur-gical resection margins were negative for tumor andthere was no evidence of associated endometriosis.Moreover, immunohistochemical staining wasstrongly positive for CD10, vimentin, estrogen recep-tor and progesterone receptor (ER and PR) (Figures3 and 4). Cytokeratin cocktail (CKAE1/AE3) wasmoderately positive, whereas h-caldesmon, desmin,CD34, SMA and S-100 were all negative (Figure5). The patient was started on megestrol acetate. InMay 2012, surveillance computed tomography of the
chest, abdomen, and pelvis displayed an unremarkablecervix and no signs of metastasis or recurrence. In Jan-uary 2013, the patient had a positron emissiontomography (PET) scan which was unremarkable;the patient was therefore kept on megestrol. In July2013, magnetic resonance imaging (MRI) revealedan ill-defined lesion in the right side of her perineumand lower vagina, encasing the urethra, with no lymphnode involvement (Figure 6). The patient was referredto urology, and a cystoscopy was performed withunremarkable results. An excisional biopsy wasperformed showing a benign polypoid piece ofendocervical mucosa with chronic inflammation.Subsequent cervical smears throughout the patient’s
Hematol Oncol Stem Cell Ther xx(xx) xxx Quarter 2015
Figure 3. CD10 immunostain showing a strong and diffuse membranous staining of the tumor cells (CD10, Dako, ·400 magnification).
Figure 4. Estrogen receptor immunostain showing a diffuse nuclear reactivity in the tumor cells (ER, Dako, ·400 magnification).
Figure 5. H-caldesmon immunostain showing negative tumor cells in contrast to the internal control smooth muscle cells in the walls of the bloodvessels. (H-caldesmon, Dako, ·200 magnification).
Hematol Oncol Stem Cell Ther xx(xx) xxx Quarter 2015 3
VULVAR EXTRAUTERINE ESS case report
Figure 6. T1-weighted axial magnetic resonance imaging (MRI) scan showing slight high signal intensity around an ill-defined lesion encasing theurethra from the right side.
4
case report VULVAR EXTRAUTERINE ESS
follow-up were all negative. The patient was main-tained on megestrol 80 mg once daily and hasremained disease free with no signs or symptoms ofrecurrent or advanced disease for the subsequent28 months of follow-up.
DISCUSSION
ESS is an extremely rare neoplasm accounting for only0.2% of all uterine malignancies and for 15–26% ofprimary uterine sarcomas.1 The annual incidence ofESS is 1–2 per million women. Compared to otheruterine malignancies, ESS affects younger women witha mean age of 42–58 years.2 The pathogenesis of ESSis still vague, but hormonal exposure through tamoxi-fen, estrogens, and conditions such as polycysticdisease of the ovary have been implicated.7 Clinically,symptoms of ESS are non-specific and usually includeirregular vaginal bleeding pelvic pain/pressure and anabdominal mass or discomfort. Diagnosis shouldtherefore be based on pathological examination.
ESS often forms distinctive finger-like projectionsthat invade the myometrium, veins, and lymphatics.Histologically, ESS is characterized by denselyuniform stromal cells with minimal cellular pleomor-phism, mild nuclear atypia, and variable mitoses. Thedifferential diagnosis of our lesion included: smoothmuscle tumors (leiomyosarcoma, cellular leiomyoma),adenosarcoma, hemangiopericytoma/solitary fibroustumor (HPC/SFT), and cellular angiofibroma.
Smooth muscle tumors tend to have larger cellswith typical cigar-shaped nuclei. Large thick-walled
blood vessels are commonly present, and the smallarterioles that are characteristic of ESS are usuallyabsent. Immunostains are also helpful in making thedistinction, as smooth muscle tumors are stronglypositive for SMA, desmin and h-caldesmon, andextensive staining for CD10 is unusual. HPC/SFTform well-circumscribed masses with neoplastic cellsintimately arranged around a myriad of thin-walledvessels and partially collapsed branching capillaries.The absence of large vessels, which often exhibit astaghorn-like appearance, and the immunostains didnot support this diagnosis. HPC/SFT are stronglypositive for CD34 and SMA. In comparison withHPC/SFT, ESS shows strong expression of CD10and PR. ESS is also ER positive and CD34 negative.We also excluded adenosarcoma with stromalovergrowth because of the lack of any epithelial ele-ments, and, subsequently, any periglandular stromalcondensations. Furthermore, the possibility of cellularangiofibroma was considered. However, cellularangiofibromas tend to be well-circumscribed withuniformly distributed small blood vessels, thick colla-gen bundles and variable amounts of fat. Cellularangiofibromas can show variable mitotic activity,though atypical forms, including tumor necrosis, areabsent. There are notable differences in the reportedimmunoprofile of this tumor. SMA and desminexpression are positive in <20% and <10% respec-tively; however, strong diffuse reactivity for CD34 isreported in about half of such cases.8
The origin of the ESS in our case is unclear.Despite an unknown route of metastasis and negative
Hematol Oncol Stem Cell Ther xx(xx) xxx Quarter 2015
VULVAR EXTRAUTERINE ESS case report
metastatic workup, a primary metastasizing ESSshould be considered because remote recurrencesoccurring after decades from the primary diagnosishave been previously documented in the literature.9In a series of 78 patients, Gloor et al. reported higherlocal and metastatic recurrence rates in cases whereone or both ovaries were preserved;10 such is the casein our patient. ESS has also been shown to arise fromovarian and extraovarian endometriosis.11 Yet ourpatient did not have any pathological or radiologicalevidence of endometriosis.
According to the hypothesis postulated byLauchlan et al., mesenchymal cells present in tis-sues derived from the coelomic epithelium havethe potential to differentiate into Müllerian-typeepithelium and stroma. This could explain theoccurrence of ESS in extrauterine sites lined bya coelomic-type of epithelium such as the ovaries,fallopian tube, and pelvic peritoneum, but not thevulva or vagina.12,13 However, four cases ofprimary ESS occurring in the vagina without evi-dence of endometriosis or metastasis have beenreported in the literature.14
Treatment of ESS has been challenging due to therarity of the disease. Performing prospective random-ized clinical trials to adjust treatment is difficult;management has therefore been mainly guided byretrospective case series and case reports. Treatmentinvolves various approaches including radiation,
Hematol Oncol Stem Cell Ther xx(xx) xxx Quarter 2015
surgical excision, chemotherapy, or a combination ofsurgery and chemotherapy. Hormonal therapyremains the most studied in the management ofESS. We therefore report the successful control ofthis neoplasm on megestrol acetate after local excisionwith negative margins.
ESS has a highly recurrent nature with around50% of patients presenting with recurrence mostlyafter long latency periods.15 They most commonlyrecur in the abdomen/pelvis (40–50%) followed bythe lung (25%). However, spine and hematologicrecurrences have also been described in the litera-ture.16–19
ESS has shown overall survival rates of 65% at fiveyears but of 32% in those with extrauterine disease.20
In conclusion, although extremely rare, the diagnosisof ESS presenting as a Bartholin gland cyst shouldbe kept in mind especially in patients between the agesof 40–50 presenting with symptoms of menorrhagia,dysmenorrhea, and pelvic pain or pressure.
DISCLOSURE OF CONFLICT OF INTEREST
None.
CONSENT
Informed consent was obtained from the patient.
REFERENCES
1. Asada Y, Isomoto H, Akama F, Nomura N, WenCY, Nakao H, et al. Metastatic low-grade endome-trial stromal sarcoma of the sigmoid colon threeyears after hysterectomy. World J Gastroenterol2005;11(15):2367–9.2. Tavassoli FA, Devilee P. (WHO) World HealthOrganization classification of tumours. In: TavassoliFA, Devilee P, editors. Pathology and Genetics ofTumours of the Breast and Female Genital Organs.Lyon: IARC Press; 2003. p. 233–6.3. Hendrickson MR, Tavassoli FA, Kempson RL,McCluggage WG, Haller U, Kubik-Huch RA. Mesen-chymal tumors and related lesions. In: Tavassoli FA,Devilee P, editors. World Health Organization Clas-sification of Tumours: Pathology and Genetics ofTumours of the Breast and Female Genital Organs.France.: IARC Press, Lyon; 2003. p. 233–44.4. Chang KL, Crabtree GS, Lim-Tan SK, Kempson RL,Hendrickson MR. Primary extrauterine endometrialstromal neoplasms: a clinicopathologic study of 20cases and a review of the literature. Int J GynecolPathol 1993;12(4):282–96.5. Kim L, Choi SJ, Park IS, Han JY, Kim JM, Chu YC,et al. Endometrial stromal sarcoma of the smallbowel. Ann Diagn Pathol 2008;12(2):128–33.6. Masand RP, Euscher ED, Deavers MT, Malpica A.Endometrioid stromal sarcoma: a clinicopathologicstudy of 63 cases. Am J Surg Pathol2013;37(11):1635–47.
7. Iwasa Y, Fletcher CD. Cellular angiofibroma:clinicopathologic and immunohistochemical analysisof 51 cases. Am J Surg Pathol 2004;28(11):1426–35.8. Cohen I. Endometrial pathologies associated withpostmenopausal tamoxifen treatment. GynecolOncol 2004;94(2):256–66.9. Ayuso A, Fadare O, Khabele D. A case ofextrauterine endometrial stromal sarcoma in thecolon diagnosed three decades after hysterectomyfor benign disease. Case Rep Obstet Gynecol2013;2013:202458.10. Gloor E, Schnyder P, Cikes M, Hofstetter J,Cordey R, Burnier F, et al. Endolymphatic stromalmyosis. Surgical and hormonal treatment of exten-sive abdominal recurrence 20 years after hysterec-tomy. Cancer 1982;50(9):1888–93.11. Baiocchi G, Kavanagh JJ, Wharton JT. Endo-metrioid stromal sarcomas arising from ovarian andextraovarian endometriosis: report of two cases andreview of the literature. Gynecol Oncol1990;36(1):147–51.12. Lauchlan SC. The secondary M�llerian system.Obstet Gynecol Surv 1972;27(3):133–46.13. Lauchlan SC. The secondary M�llerian systemrevisited. Int J Gynecol Pathol 1994;13(1):73–9.14. Liu Z, Ding J, Li X, Yu K. Endometrial stromalsarcoma arising in vagina. Int J Clin Exp Pathol2013;6(12):2997–3002.
15. Wood CL, Sederberg 2nd J, Russ P, Seres T.Cystic appearance of low-grade endometrial stromalsarcoma in the right atrium: case report. CardiovascUltrasound 2011;9:23.16. Cheng X, Yang G, Schmeler KM, Coleman RL, TuX, Liu J, et al. Recurrence patterns and prognosis ofendometrial stromal sarcoma and the potential oftyrosine kinase-inhibiting therapy. Gynecol Oncol2011;121(2):323–7.17. Batista LM, Carvalho CH, Acioly MA, Ghara-baghi A, Ramina KF, Schittenhelm J, et al. Spinalmetastasis of endometrial stromal sarcoma: clinico-pathological features and management. Surg Oncol2011;20(2):e78–83.18. Huang MI, Debernardo RL, Rodgers M, Hart DJ.Endometrial stromal sarcoma metastasis to thelumbar spine and sphenoid bone. Rare Tumors2011;3(3):e27.19. Thomas MB, Keeney GL, Podratz KC, Dowdy SC.Endometrial stromal sarcoma: treatment and pat-terns of recurrence. Int J Gynecol Cancer2009;19(2):253–6.20. Ramia JM, De la Plaza R, Garcia I, Perna C,Veguillas P, Garc�a-ParreÇo J. Liver metastasis ofendometrial stromal sarcoma. World J Hepatol2012;4(12):415–8.
5
RESEARCH
Human Papilloma Virus Early Proteins E6 (HPV16/18-E6)and the Cell Cycle Marker P16 (INK4a) are Useful PrognosticMarkers in Uterine Cervical Carcinomasin Qassim Region- Saudi Arabia
O. M. Omran & M. AlSheeha
Received: 17 December 2013 /Accepted: 16 May 2014 /Published online: 13 June 2014# Arányi Lajos Foundation 2014
Abstract Cervical cancer is a common and an importantpublic health problem for adult women in developing coun-tries. In contrast, cervical cancer incidence is low in SaudiArabia. High-risk types of human papilloma viruses (HPV16and HPV18) are the most significant risk factors for cervicalcancer. HPV16/18-E6 oncoprotein is associated with HPVetiology, viral persistence and epithelial transformation. Cellcycle protein p16 INK4a (p16) plays an important role in thepathophysiology of cervical carcinomas. The aims of thisstudy were to investigate the expression of HPV16/18-E6and p16 in uterine cervical carcinomas in Qassim Region -Saudi Arabia, and to relate the results to the establishedclinicopathological prognostic parameters (age of the patient,educational level, birth control methods, number of pregnan-cy, smoking status, degree of histological differentiation, clin-ical stage, and lymph node metastasis) The study included 40specimens of uterine cervical squamous cell carcinomas diag-nosed and confirmed by biopsy. Histopathological classifica-tion of cervical tumors cases was performed according to theInternational Federation of Gynecology and Obstetrics(FIGO). Immunohistochemical analysis for HPV16/18-E6and p16 were carried out on formalin-fixed paraffin-embedded sections of cervical tissues using avidin-biotin
peroxidase method. There was a significant statistical correla-tion between HPV16/18-E6 expression in cervical carcinomaand nationality, smoking status and size of the tumor. HPV16/18-E6 oncoprotein expression in normal lymphocytes andendothelial cells in the tumor tissues and the adjacent normalcervical tissues suggest the possibility that HPV infectionmight spread to other organs through blood circulation. P16expression has been correlated with high grade, stage of cer-vical SCC and HPV16/18-E6 expression. The current studysupports the critical function of p16 and HPV16/18-E6 asspecific markers for cervical carcinoma. However the potentialfor usage of p16 and HPV16/18-E6 as prognostic markers willrequire detailed follow data for a larger group of patients.
Keywords Immunohistochemistry . Cervical carcinoma .
HPV16/18E6 . P16 (INK4a)
Introduction
Worldwide, cervical cancer is an important public healthproblem for adult women [1, 2]. The International Agencyfor Research on Cancer (IARC) of the World HealthOrganization (WHO) estimated that nearly 80 % of cervicalcancer cases occur in developing countries and, in many suchregions, it is the most common cancer and cause of death fromcancer amongwomen [3]. The vast majority of cervical cancercases are caused by infection with certain subtypes of humanpapilloma virus (HPV), a sexually transmitted virus that in-fects cells and may result in precancerous lesions and invasivecancer [4, 5]. The worldwide prevalence of HPV is 10.4% [6].Women are generally infected with HPV in their teens; it cantake as long as 20 years after HPV infection for the cancer todevelop. The prevalence of cervical HPV infection decreasessharply in women after the age of 30 [7]. The American
O. M. OmranDepartment of Pathology, College of Medicine, Assiut University,Assiut, Egypt
M. AlSheehaDepartment of Obstetrics and Gynacology, College of Medicine,Qassim University, Buridah, Qassim Region,Kingdom of Saudi Arabia
Present Address:O. M. Omran (*)Department of Pathology, College of Medicine, Qassim University,Buridah, Qassim Region, Kingdom of Saudi Arabiae-mail: [email protected]
Pathol. Oncol. Res. (2015) 21:157–166DOI 10.1007/s12253-014-9801-y
Cancer Society provides the following list of risk factors forcervical cancer: Human Papillomavirus (HPV) infection,smoking, HIV infection, chlamydia infection, dietary factors,oral contraception, multiple pregnancies, exposure to the hor-monal drug diethylstilbestrol (DES) and a family history ofcervical cancer. These factors probably modify the risk inwomen infected with HPV [8, 9]. Malignant transformationof the cervical epithelial cells is associated with persistenthigh-risk HPV infections, such as HPV 16 and 18 whichencode E6 and E7 oncoproteins that bind to and inactivatethe tumor suppressor proteins p53 and pRB (retinoblastomaprotein), respectively [10].
In contrast, cervical cancer incidence is very low in SaudiArabia, ranking number 11 between all cancers in females [11].The closed society, standards of mores and male circumcisionmight reduce women exposure to HPV infection [12–16]. Theprevalence of HPV infection among women and its associationwith cervical cancer in Saudi Arabia and in similar socio-cultural societies is scanty [17–21]. Discordant reports showedhigh incidence in some countries, such as Indonesia [22, 23].Inherited genetic predisposition may contribute to the risk ofcervical cancer. Tumor suppressor gene TP53 has been sug-gested to affect the oncogenic potential of the HPV E6 protein[24]. Recent study reported that activation of interleukin-6/signal transducer and activator of transcription 3 by HPV16/18-E6 induces fibroblast senescence to promote cervicaltumourigenesis through autocrine and paracrine pathways intumormicroenvironment [25].Moreover, HPV16 oncoproteinspromote invasiveness of cervical cancer by upregulating spe-cific matrix metalloproteinases [26].
P16 is part of cascade cell cycle regulators, which controlthe transition cell from G1 to S phase [27]. Cyclin D andcyclin-dependent kinases (CDK) make complexes that phos-phorylate pRB and ensure cell proliferation to which p16 has anegative regulation role [28]. Many studies indicated thepotential of this biomarker for cervical carcinoma diagnosisand prognosis.
An association between HPV16/18-E6 and p16 expressionin human cervical squamous cell carcinoma in QassimRegion-Saudi Arabia has not been studied. Therefore, weanalyzed immunohistochemical expression of HPV16/18E6and P16 in cervical carcinoma to examine their relationshipand to determine any association with other existing clinico-pathological parameters.
Patients and Methods
This study was carried out with cooperation of PathologyDepartment, King Fahd Specialist Hospital, Buraidah, andother hospitals in Qassim region. The retrospective studywas conducted on cervix uteri specimens obtained from cer-vical carcinoma patients from December 2000 to March
2013. Patients’ clinical information including age, na-tionality, smoking status, number of pregnancies, themain complaints, the clinical presentations was obtainedfrom electronic medical records.
Histopathology
Specimens were examined, diagnosed, and classified accord-ing to the International Federation of Gynecology andObstetrics (FIGO) of cervical tumors. The normal cervicalepithelium was obtained from areas adjacent to the tumortissues. The tumor size, site and number were recorded andanalyzed. The tissue specimens were fixed in 10 % formalin,dehydrated and embedded in paraffin and stained with hema-toxylin and eosin (H&E). The slides were then examined forhistological diagnosis and assessment.
Immunohistochemistry
From the formalin fixed paraffin-embedded tissues, 4 μ sectionswere immunostained with monoclonal antibodies againstHPV16E6/18-E6 (C1P5) (Santa Cruz Biotechnology, Inc.USA, dilution 1:50) and p16 (F-12) (Santa CruzBiotechnology, Inc. USA, dilution 1:50) using labeledstreptavidin biotin (LSAB) method. Sections were cut and driedat 37 °C, dewaxed in xylene and rehydrated using serial con-centrations of ethanol and washed in running water. Sectionswere washed with Tris buffer and preincubated with normalrabbit serum (10 %) for 20 min. Slides were incubated withthe monoclonal primary antibodies for one hour and exposed toImmunoCruz™ mouse LSAB Staining (Santa CruzBiotechnology, Inc. USA). The slides were then counterstainedwith hematoxylin and reviewed. Known positive control sec-tions were included in each run to ensure proper immunostain-ing whereas the negative control consisted of the same sectionwhere the primary antibody was omitted. Sections of thep16INK4a-positive human uterine adenocarcinoma tissue andHPV16/18 E6-positive HPV-positive vulva carcinoma wereincluded to serve as the positive controls for p16INK4a andHPV16/18 E6 IHC, respectively. Dichotomic negative/positiveevaluation was adapted to determine E6 immunoreaction assuggested by Lin et al. with some modification [29]. Brownnuclear or cytoplasmic staining was considered as a positivereaction to E6 HPV 16/18 proteins. Immunohistochemicalexpression for p16INK4a was quantified according tostaining intensity (weak, moderate and strong) and dis-persion pattern (diffuse or focal) in each block [30].
Statistical Analysis
Statistical calculations were performed by using Statviewprogramme package. Data were compared using Chi-squaretest, and the differences between the means of continuous data
158 O. M. Omran, M. AlSheeha
will be compared by using paired t-test. The percentage ofexpression of HPV16 E6/18-E6 and p16 in malignant cervicalsquamous cell carcinoma, were used to assess the role of(FIGO)’s classification, differentiation, age, and tumorsize. A probability p value of <0.05 was considered statisti-cally significant.
Results
Clinical and Histopathological Data
This study included 40 cases of cervical carcinoma. Thestudy included 21 Saudi women and 19 non-Saudiwomen. The patient ages ranged from 28 years to76 years, (median for Saudi women=50 and for non-Saudi women=35). The non Saudi women showed de-velopment of carcinoma at younger ages compared toSaudi women (p <0.05). The clinical presentations weremostly vaginal bleeding for more than one month. Fortyeight percent of the patient were smokers and 35 %either take or having a history of contraceptive pillsadministration. Histologically, the specimen were classi-fied as 14 cases (35 %) well differentiated, 19 cases(47.5 %) moderately differentiated and 7 cases (17.5 %)poorly differentiated cervical squamous cell carcinoma.
HPV16/18-E6 Oncoprotein is Expressed in CervicalSquamous Cell Carcinoma
To explore whether HPV16 infection could be linked tocervical carcinoma development in Qassim region, SaudiArabia, HPV16/18-E6 oncoprotein expression was eval-uated by immunohistochemistry in cervical SCC. Ourdata showed HPV 16/18-E6 positivity in 21cases(52.5 %); 73.6 % of positive cases were non Saudi (p<0.05). The staining reaction was seen cytoplasmic andnuclear. Meanwhile, most of the mature squamous epi-thelial cells were negative. Most malignant squamouscells showed diffuse expression (Fig. 1). The accompa-nied squamous dysplasia in the studied sections alsoshowed positive immunoreactivity for the HPV16/18-E6; it was seen positive mainly in the superficial epi-thelial cells. There was statistically significant correlationbetween HPV16/18-E6 expression and smoking status,where more positive expression were associated withsmoking (p <0.011). There was statistically significantcorrelation between HPV16/18-E6 expression and tumorsize, where it was more commonly expressed in tumordiameter more than 2 cm. There was no statisticallysignificant correlation between HPV16/18-E6 expressionand age of the patient, educational level, contraceptivesadministration, number of pregnancies, grade and stage ofthe tumor and lymph node metastasis (p >0.05) (Tables 1and 2).
Fig. 1 Immunohistochemical analysis of human papilloma virus 16/18-E6 protein in cervical SCC, and adjacent normal tissues. A a negativeHPV16/18-E6 immunostaining in normal cervical epithelial cells, andpositive expression in lymphocytes, macrophages, fibroblast and endo-thelial cells (×200); B HPV16/18-E6 protein expressed in lymphocytesand in carcinoma in situ tumor cells (×200); C: HPV16/18-E6 expressed
in well differentiated SCC and lymphocytes in the stroma (×400); DHPV16/18-E6 expressed in moderately differentiated SCC (×400); EHPV16/18-E6 expressed in poorly differentiated SCC (×400); F a nega-tive HPV16/18-E6 immunostaining in tumor cells of moderately differ-entiated SCC sheets (×400) (L=lymphocyte, F=fibroblast, E=endothelialcell, M=macrophage)
HPV16/18-E6 and P16 are Useful Prognostic Markers in Uterine Cervical Carcinomas 159
Tab
le1
The
correlationbetweenHPV
16/18-E6andp16proteinexpression
incervicalsquamouscellcarcinom
aandtheclinicalparameters
HPV
16/18E6Expression
p16
Expression
Intensity
Dispersion
Sam
ples
Total
−(%
)+(%
)p
−(%
)weak(%
)moderate(%
)Strong(%
)p
Focal(%)
Diffuse
(%)
p
SCC
4019
(47.5)
21(52.5)
8(20)
10(25)
17(42.5)
5(12.5)
9(22.5)
23(57.5)
1.Age
(Years)
<45
22(55)
10(45.5)
12(54.5)
0.781
5(22.7)
5(22.7)
8(36.4)
4(18.2)
0.858
6(27.3)
11(50)
0.224
>45
18(45)
9(50)
9(50)
3(16.7)
5(27.7)
9(50)
1(56)
3(16.3)
12(66.7)
2.Nationality
Saudi
21(52.5)
14(66.4)
7(33.3)
0.024(S)
4(9)
7(33.3)
9(42.9)
1(4.8)
0.29
4(19.1)
13(61.9)
0.411
None-Saudi
19(47.9)
5(26.3)
14(73.7)
4(21.1)
3(15.8)
8(42.1)
4(21.1)
5(26.4)
10(52.6)
3.Educatio
nallevel
Noform
alEducatio
n18
(45)
10(55.6)
8(44.4)
0.796
3(16.7)
5(27.8)
8(44.4)
2(11.1)
0.454
5(27.7)
10(55.6)
0.893
BasicEducatio
n16
(40)
6(37.5)
10(62.5)
2(12.5)
4(25)
8(50)
2(12.5)
4(25)
10(62.5)
Highereducation
6(15)
3(50)
3(50)
3(50)
1(16.7)
1(16.7)
1(16.7)
0(0)
3(50)
4.Sm
okingstatus
No
21(52.5)
14(66.5)
7(33.5)
0.011(S)
3(14.3)
6(28.6)
11(52.4)
1(4.7)
0.978
5(23.8)
13(61.9)
0.677
Yes
19(47.5)
5(26.3)
14(73.7)
5(26.2)
4(21.1)
6(31.5)
4(21.1)
4(21.1)
10(52.6)
5.Contraceptiv
es
No
26(65)
15(57.9)
11(42.3)
0.132
6(23.1)
6(23.1)
12(46.2)
2(7.6)
0.424
6(23.1)
14(53.8)
0.479
Yes
14(35)
4(28.6)
10(71.4)
2(14.3)
4(28.6)
5(35.7)
3(21.4)
3(21.4)
9(64.3)
6.Num
berof
pregnancies
No
9(22.5)
4(44.4)
5(55.6)
0.659
0(0)
1(11.1)
5(%
)3(%
)0.235
3(33.3)
6(66.7)
0.648
1–2
11(27.5)
5(45.5)
6(54.5)
4(36.4)
3(27.2)
4(36.4)
0(0)
3(27.2)
4(36.4)
3–4
10(25)
6(60)
4(40)
3(30)
2(20)
4(40)
1(10)
1(10)
6(60)
>4
10(25)
4(40)
6(60)
1(10)
4(40)
4(40)
1(10)
2(20)
7(70)
160 O. M. Omran, M. AlSheeha
Tab
le2
The
correlationbetweenHPV16/18-E6andp16proteinexpression
incervicalsquamouscellcarcinom
aandtheclinicopathologicalp
aram
eters
HPV
16/18E
6Expression
p16Expression Intensity
Dispersion
Sam
ples
Total
−(%
)+(%
)p
−(%
)weak(%
)moderate(%
)Strong(%
)p
Focal(%)
Diffuse
(%)
p
SCC
4019
(47.5)
21(52.5)
8(20)
10(25)
17(42.5)
5(12.5)
9(22.5)
23(57.5)
1.Pathologictumor
size
<2cm
9(22.5)
8(88.9)
1(11.1)
0.041(S)
2(22.2)
4(44.4)
3(33.4)
0(0)
0.190
4(44.4)
3(33.4)
0.073
2–5cm
21(52.5)
8(38.1)
13(61.9)
5(23.8)
4(19)
8(38.2)
4(19)
4(19)
12(57.2)
>5cm
10(25)
3(30)
7(70)
1(10)
2(20)
6(60)
1(10)
1(10)
8(80)
2.HistologicGrade
WD
14(35)
8(57.1)
6(42.9)
0.416
5(35.7)
6(42.9)
2(14.3)
1(7.1)
0.029(S)
6(50)
3(21.4)
0.000(H
S)MD
19(47.5)
8(42.1)
11(57.9)
2(10.5)
2(10.5)
12(63.2)
3(15.8)
3(10.5)
14(79.8)
PD7(17.5)
3(42.9)
4(57.1)
1(14.3)
2(28.6)
3(42.8)
1(14.3)
0(0)
6(85.7)
3.Clin
icalstage
I17
(42.5)
10(58.8)
7(41.2)
0.521
4(23.5)
7(41.2)
6(35.3)
0(0)
0.035(S)
6(35.3)
7(41.2)
0.037(S)
II11
(27.5)
4(36.4)
7(63.6)
2(18.2)
3(27.3)
5(45.4)
1(9.1)
3(27.3)
6(54.5)
III/VI
12(30)
5(41.7)
7(58.3)
2(16.7)
0(0)
6(50)
4(33.3)
0(0)
10(83.3)
4.Lym
phnode
metastasis
No
28(70)
15(53.6)
13(46.4)
0.344
7(25)
9(32.1)
11(39.3)
1(3.6)
0.007(H
S)8(28.6)
13(46.4)
0.04
(S)
Yes
12(30)
4(33.3)
8(66.7)
1(8.3)
1(8.3)
6(50)
4(33.3)
1(8.3)
10(83.3)
HPV16/18-E6 and P16 are Useful Prognostic Markers in Uterine Cervical Carcinomas 161
HPV16/18-E6 Oncoprotein is Expressed in Lymphocytesand Endothelial Cells of Cervical Tumors and AdjacentNormal Tissues
To understand whether HPV-infected cervical tumorscould spread to other organs through blood circulation,HPV16/18-E6 oncoprotein expression in normal cervicaltissues adjacent to cervical carcinomas were examinedby IHC. Our data showed that HPV16/18-E6oncoprotein is indeed expressed in lymphocytes, macro-phages infiltrating cervical tumors and adjacent normalcervical tissue and in endothelial cells of blood vessels(Figs. 1A–C). These results seem to support the possi-bility that HPV16/18 infection may spread to otherorgans through blood circulation.
The Cell Cycle Marker P16INK4a is Expressed in CervicalSquamous Cell Carcinoma
The p16 reaction was evaluated as positive when nucle-ar or cytoplasmic immunostaining was clearly demon-strated. Different patterns of p16 signal were observedbased on staining intensity (weak, moderate and strong)and dispersion pattern (diffuse or focal). Thirty twocases (80 %) of the cervical cancer tissues were positive(Fig. 2). Moderate and diffuse pattern of p16 expressionwas statistically associated with histologic grade of thecervical cancer. Cases presenting a moderate p16 ex-pression were more common in the advanced FIGOstages III/IV (50 %) while weak expression was
associated with early stage I (41.2 %) (p=0.035)(Table 2). No association was observed between p16staining intensity or dispersion and age of the patient,educational level, contraceptives administration, smokingstatus, number of pregnancy or tumor size (p >0.05)(Tables 1 and 2).
The Correlation Between HPV16/18-E6 and p16 Expression
The correlation between the expression of HPV16/18-E6 withp16 proteins intensity and dispersion in the cervical SCC isshown in Tables 3 and 4 respectively. The negative expressionof p16 was significantly correlated with the negative expres-sion of HPV16/18-E6 oncoprotein and the positive expressionof p16 was significantly correlated with the positive expres-sion of HPV16/18-E6 oncoprotein. These data were statisti-cally significant (p <0.05).
Discussion
Cervical cancer remains the second most common malignan-cy in women in the world and accounts for 9.8 % of all cancercases [31]. Infection with Human Papillomavirus (HPV) isconsidered to be the principal causative agent in the develop-ment of cervical squamous cell carcinoma [9]. The objectivesof our study were to investigate the expression of HPV16/18-E6 and p16 in uterine cervical carcinomas in Qassim Region-Saudi Arabia, and to relate the results to the established
Fig. 2 Immunohistochemicalanalysis of p16 protein in cervicalSCC. A: moderate cytoplasmicimmunohistochemical staining ofp16 in carcinoma in situ; Bnuclear expression of p16 in welldifferentiated SCC; C nuclearexpression of p16 in moderatelydifferentiated SCC. D bothcytoplasmic and nuclearexpression of p16 in poorlydifferentiated SCC; thedistribution of P16 expressionwas focal in A, B and C anddiffuse in D (×400)
162 O. M. Omran, M. AlSheeha
clinicopathological prognostic parameters. A previous reportshowed the prevalence of HPV infection in invasive cervicalcancer in Saudi Arabia (82 %) at the lower range of thatobserved in the world (85–95 %) [24]. Similar studies report-ed low prevalence of HPV infection among women withcervical cancer in Saudi Arabia and similar socio-culturalsocieties [14, 16, 20, 24].
Our data showed HPV16/18-E6 positivity in 21 cases(52.5 %); 73.6 % of positive cases were non Saudi (p<0.05). The non Saudi women showed development ofcervical carcinoma at younger ages compared to Saudi women(p <0.05). A previous study demonstrated a trend for higherrates of HPV 16/18 infection leading to a greater proportion ofhigh grade cervical lesion in young women in New Zealand[32]. A recent study observed the distribution of HPV16variants worldwide and their relative risks for cervicalcancer appear to be population-dependant [33].
The high-risk HPV types encode two oncoproteins, E6 andE7. Protein E6 is a transcriptional transactivator, bindingdouble stranded DNA. It has transforming activityinactivating the human TP53/p53 tumor suppressor proteinby targeting it for degradation [34]. The HPV E6 oncoproteinis involved in cell cycle deregulation, where p53 is abrogated.Sonoporation delivery of monoclonal antibodies against hu-man papilloma virus 16 E6 restores p53 expression in trans-formed cervical carcinoma derived cell lines using chemical
transfection [35]. The immunohistochemical expression of E6has been proposed to be a useful maker for determining adiagnosis and/or prognosis of cancer [36]. The E7 oncoproteinbinds to the retinoblastoma protein (pRb) tumor suppressor,leading to continuous cell cycling without any repair check-points [37]. In an attempt to prevent this continuous cellcycling, p16, a pRb regulator, is overexpressed and accumu-lates inside the cells [38].
High-risk human papillomavirus E6 might contribute tosome form of immunosuppression as it inhibits monocytedifferentiation to Langerhans cells (competent antigen pre-senting cells) which in charge of the induction of T cell-dependent immunity [39, 40]. The vigilance of the immunesystem is readily exploited by HPV to escape immune de-struction, resulting in persistent infections and development ofHPV-positive cancers. HPV can evade host immune responsesthrough avoidance of killing the host cells during viral repli-cation and therefore neither presents viral antigen nor inducesinflammation and downregulates the expression of HLA-class1, facilitating evasion of cytotoxic T lymphocytes (CTL)attack [41]. Moreover, HPV16 E6 and E7 proteins reducetype-1 interferons (IFNs) expression in host cells thus induc-ing immune tolerance rather than the appropriate responses[41]. A recent study demonstrated that attenuation of IL-1β bythe HPV16 E6 oncoprotein in immortalized cells is ap-parently a crucial step in viral immune evasion andinitiation of malignancy [42].
Our data showed that HPV16/18-E6 oncoprotein isexpressed in lymphocytes, macrophages, infiltrating cervicaltumors and in endothelial cells of blood vessels. Previousstudies demonstrated that peripheral blood mononuclear cells(PBMCs) might be HPV reservoir, thus spreading the virusthrough blood, providing a potential new route of transmis-sion [43, 44]. Moreover a recent study demonstrated that HPVmarkers are not only found in PBMCs of peripheral blood butalso present in CD20+ B lymphocytes and CD56+ naturalkiller cells in the semen of infected patients with high riskHPV-16 raising a concerns about the risk of developing can-cers to distal organs and to the sexual partner [45]. On theother side, the competency of HPV to be uptaken by Blymphocytes cannot be excluded. In fact, this possibility issupported by the fact that heparan-sulfate proteoglycans,theorised to potentially be one of the primary receptors in-volved in HPVentry, are expressed by B lymphocytes and area requirement for normal cell maturation, differentiation andfunction [46]. Although, sexual activity has been consideredto be a major route of transmission for HPV resulting ingenital cancers and oropharyngeal cancers [47], HPV infec-tions in infants and female university students who are virgins,revealing that HPV transmission via other routes may exist[48, 49]. Furthermore, peripheral blood lymphocytes (PBLs)from healthy donors have been shown to be infected withHPV [44]. Therefore, we support the previous possibility that
Table 3 The correlation between the expression of HPV16/18-E6 andp16 intensity in cervical squamous cell carcinoma
HPV16/18-E6 Expression
Samples Total Negative (%) Positive (%) p
Cervical SCC 40 19 (47.5) 21 (52.5)
p16 Expression (intensity)
Negative 8 (20) 7 (87.5) 1 (12.5) 0.000 (HS)Weak 10 (25) 6 (60) 4 (40)
Moderate 17 (42.5 %) 6 (32.3) 11 (64.7)
Strong 5 (12.5) 0 (0) 5 (100)
HS highly significant statistical correlation
Table 4 The correlation between the expression of HPV16/18-E6 andp16 dispersion in cervical squamous cell carcinoma
HPV16/18-E6 Expression
Samples Total Negative (%) Positive (%) p
Cervical SCC 40 19 (47.5) 21 (52.5)
p16 Expression (dispersion)
Negative 8 (40) 7 (87.5) 1 (12.5) 0.014 (S)Focal 9 (25) 4 (44.4) 5 (55.6)
Diffuse 23 (35) 8 (34.8) 15 (65.2)
S significant statistical correlation
HPV16/18-E6 and P16 are Useful Prognostic Markers in Uterine Cervical Carcinomas 163
HPV infection may spread to other organs through bloodcirculation [50, 51].
In the present study, immunohistochemical staining of p16antibody in normal cervical epithelial tissues were negative.Meanwhile, 80% (32/40 cases) of the invasive cervical cancerwere positive for p16. A previous study observed p16 expres-sion in 95.4 % of cervical squamous cell carcinoma [30]. Fewstudies have observed p16 expression in normal tissue [52,53]. P16 is a protein that is expressed in low concentrations inhealthy cells, but is overexpressed in cervical cancer and high-grade precursor lesions. Still, it is well established that nondysplastic epithelial cells can express p16, for example, undercertain physiological conditions, such as the shortening oftelomeres in older tissues. Here, the expression of p16 imme-diately induces cell cycle arrest and may ultimately induceapoptosis with the end results being a focal pattern with weakintensity [54]. Our study confirms increased expression of p16in invasive cervical squamous cell carcinoma. Several studieshave documented overexpression, with a diffuse and strongpattern, not only in high-grade cervical intraepithelial le-sions but also with invasive cancer compared to the normalspecimens [55].
The results obtained in our study showed p16 expression aspredominantly in the nuclei of most of our tumor samples,while only a few of these tumors displayed both nuclear andcytoplasmic staining. Wang et al. and Murphy et al. found allthe invasive squamous cancers exhibited strong nuclear andcytoplasmic staining. Thus, the p16 overexpressions eithernuclear or cytoplasmic in affected cells are to be consideredpositive [56, 57]. The presence of p16 in the cytoplasm mayresults from a type of post transcriptional modification oroverproduction of p16 protein which force its transfer to thecytoplasm [56].
In this study, we demonstrated a significant correla-tion between a diffuse and moderate p16 immunoreac-tivity, and high grade, stage, and lymph node metastasisof cervical SCC (P <0.05). A recent study observed thatHPV16-E6 induces epithelial mesenchymal transition,thus may contribute to tumor progression and metastasis[58]. Previous studies observed strong and diffuse p16expression in all cases of invasive cervical carcinoma[56, 57]. Consequently, p16 overexpression is a signif-icant marker of cervical lesions and is considered to bea useful test that may facilitate an improved diagnosisof severe cervical lesions.
In our study, negative p16 and HPV16/18-E6 reactionswere observed in combination in 17.5 % of cases, but 50 %of positive reactions were identified in combination. A corre-lation between p16 and HPV16/18-E6 expression was pre-dicted, however, the reason for this discrepancy may be at-tributed to limitation of sensitivity of Immunohistochemicalreaction [59, 51]. However, other risk factors or other types ofhigh risk- HPV contribution such as HPV45 to the cervical
carcinoma development could not be excluded. Theoretically,p16 represent a promising biomarker, because its expressionreflects both that oncogenic HPV is present and that it hasdisrupted normal cell cycle function and might be usefuldiagnostic marker of oncogenic HPV infection. The increasedlevel of p16 expression by high risk-HPV points to the rec-ommendation of this protein as a marker of HPV infection[59–62]. However the detection of p16 per se, should not beconsidered as proof of high risk of HPV infection withoutconsideration of other markers such as HPV16/18-E6. Ourprimary limitation is the relatively few number of cases ofcervical carcinoma in Qassim region- Saudi Arabia, thuslimiting adequate analysis.
Conclusions
This is the first study to investigate the expression of HPV16/18-E6 and p16 in uterine cervical squamous cell carcinomasin Qassim Region - Saudi Arabia. The present study revealedstatistical correlation between HPV16/18-E6 expression incervical carcinoma and nationality, smoking status and sizeof the tumor. HPV16/18-E6 oncoprotein expression in normallymphocytes, macrophages, fibroblasts and endothelial cellsin the tumor tissues and adjacent normal cervical tissuessupport the possibility that HPV infection may spread to otherorgans through blood circulation. P16 expression has beencorrelated with high grade, stage of cervical SCC and withHPV16/18-E6 expression. The current study supports thecritical function of p16 and HPV16/18-E6 as specific markersfor cervical carcinoma. However the potential for usage of p16and HPV16/18-E6 as prognostic markers will require detailedfollow data for a larger group of patients.
Acknowledgments This work was funded by Qassim University
References
1. Parkin DM, Bray F, Ferlay J, Pisani P (2005) Global cancer statistics2002. CA Cancer J Clin 55:74–108
2. Sankaranarayanan R, Madhukar A, Rajkumar R (2001) Effectivescreening programmes for cervical cancer in low- and middle-income developing countries. Bull World Health Organ 79(10):954–962
3. GLOBOCAN 2000. Cancer incidence, mortality and prevalenceworldwide, version 10. IARC Cancer Base No 5 Lyon, IARCPress. 2001
4. Bosch FX, de Sanjose S (2003) Human papillomavirus and cervicalcancer – burden and assessment of causality. J Natl Cancer InstMonogr 31:3–13
5. Walboomers JM, Jacobs MV, Manos MM, Bosch FX, Kummer JA,Shah KV, Snijders PJ, Peto J, Meijer CJ, Muñoz N (1999) Humanpapillomavirus is a necessary cause of invasive cervical cancerworldwide. J Pathol 189(1):12–19
164 O. M. Omran, M. AlSheeha
6. de Sanjosé S, DiazM, Castellsagué X, Clifford G, Bruni L,MuñozN,Bosch FX (2007)Worldwide prevalence and genotype distribution ofcervical human papillomavirus DNA in women with normalcytology: a meta-analysis. Lancet Infect Dis 7(7):453–459
7. Wright TC Jr, Schiffman M (2003) Adding a test for human papillo-mavirus DNA to cervical- cancer screening. N Engl J Med 348:489
8. What Causes Cancer of the Cervix?. American Cancer Society(2014-01-31).
9. RashedMM, Bekele A (2011) The prevalence and pattern of HPV-16immunostaining in uterine cervical carcinomas in ethiopian women:a pilot study. Pan Afr Med J 8:21
10. Missaoui N, Hmissa S, Frappart L, Trabelsi A, Ben Abdelkader A,Traore C, Mokni M, Yaacoubi MT, Korbi S (2006) p16INK4Aoverexpression and HPV infection in uterine cervix adenocarcinoma.Virchows Arch 448(5):597–603
11. Bhurgri Y, Bhurgri A, Rahim A, Bhutto K, Pinjani PK, Usman A,Hasan SH (1999) The pattern of malignancies in Karachi (1995 to1996). J Pak Med Assoc 49:157–161
12. Altaf F (2001) Pattern of cervical smear cytology in western region ofSaudi Arabia. Ann Saudi Med 21:94–96
13. Jamal A, Al-Maghrabi JA (2003) Profile of pap smear cytology in theWestern region of Saudi Arabia. Saudi Med J 24:1225–1229
14. Raza SA, Franceschi S, Pallardy S, Malik FR, Avan BI, Zafar A, AliSH, Pervez S, Serajuddaula S, Snijders PJ et al (2010) Humanpapillomavirus infection in women with and without cervical cancerin Karachi, Pakistan. Br J Cancer 102:1657–1660
15. Castellsague X, Bosch FX, Munoz N, Meijer CJ, Shah KV, deSanjose S, Eluf-Neto J, Ngelangel CA, Chichareon S, Smith JSet al (2002) Male circumcision, penile human papillomavirus infec-tion, and cervical cancer in female partners. N Engl J Med 346:1105–1112
16. Hammouda D, Clifford GM, Pallardy S, Ayyach G, Chekiri A,Boudrich A, Snijders PJ, van Kemenade FJ, Meijer CJ, BouhadefA et al (2011) Human papillomavirus infection in a population-basedsample of women in Algiers, Algeria. Int J Cancer 128:2224–2229
17. Alsbeih G, Ahmed R, Al-Harbi N, Venturina LA, Tulbah A, BalarajK (2011) Prevalence and genotypes’ distribution of human papillo-mavirus in invasive cervical cancer in Saudi Arabia. Gynecol Oncol121:522–526
18. Al-Badawi IA, Al-Suwaine A, Al-Aker M, Asaad L, Alaidan A,TulbahA, FeBoholM,Munkarah AR (2011)Detection and genotyp-ing of human papiloma virus in cervical cancer specimens fromSaudipatients. Int J Gynecol Cancer 21:907–910
19. Khorasanizadeh F, Hassanloo J, Khaksar N, Taheri SM, MarzabanM, Rashidi BH, Sari AA, Zendehdel K (2012) Epidemiology ofcervical cancer and human papilloma virus infection among Iranianwomen - analyses of national data and systematic review of theliterature. Gynecol Oncol 128:277–281
20. Hussain S, Bharadwaj M, Nasare V, Kumari M, Sharma S, Hedau S,Das BC (2012) Human papillomavirus infection among young ado-lescents in India: impact of vaccination. J Med Virol 84:298–305
21. Al-Muammar T, Al-Ahdal MN, Hassan A, Kessie G, Dela Cruz DM,Mohamed GE (2007) Human papilloma virus-16/18 cervical infec-tion among women attending a family medical clinic in Riyadh. AnnSaudi Med 27:1–5
22. Duttagupta C, Sengupta S, Roy M, Sengupta D, Bhattacharya P,Laikangbam P, Roy S, Ghosh S, Das R (2004) Are Muslim womenless susceptible to oncogenic human papillomavirus infection? astudy from rural Eastern India. Int J Gynecol Cancer 14:293–303
23. de BoerMA,Vet JN, AzizMF, Cornain S, Purwoto G, van den AkkerBE, Dijkman A, Peters AA, Fleuren GJ (2006) Human papillomavi-rus type 18 and other risk factors for cervicacancer in Jakarta,Indonesia. Int J Gynecol Cancer 16:1809–1814
24. Alsbeih G, Al-Harbi N, El-Sebaie M, Al-Badawi I (2013) HPVprevalence and genetic predisposition to cervical cancer in SaudiArabia. Infect Agent Cancer 8(1):15
25. Ren C, Cheng X, Lu B, Yang G. Activation of interleukin-6/signal transducer and activator of transcription 3 by humanpapillomavirus early proteins 6 induces fibroblast senescenceto promote cervical tumourigenesis through autocrine and para-crine pathways in tumour microenvironment. Eur J Cancer. 2013Aug 14.
26. Kaewprag J, Umnajvijit W, Ngamkham J, Ponglikitmongkol M(2013) HPV16 oncoproteins promote cervical cancer invasivenessby upregulating specific matrix metalloproteinases. PLoS One 8(8):e71611
27. Sano T, Oyama T, Kashiwabara K, Fukuda T, Nakajima T (1998)Expression status of p16 protein is associated with human papillo-mavirus oncogenic potential in cervical and genital lesions. Am JPathol 153(6):1741–1748
28. Bringold F, Serrano M (2000) Tumor suppressors and oncogenes incellular senescence. Exp Gerontol 35(3):317–329
29. Lin HP, Wang YP, Chiang CP (2011) Expression of p53, MDM2,p21, heat shock protein 70, and HPV 16/18 E6 proteins in oralverrucous carcinoma and oral verrucous hyperplasia. Head Neck33:334–340
30. Amaro-Filho SM, Golub JE, Nuovo GJ, Cunha CB, Levi JE, VillaLL, Andrade CV, Russomano FB, Tristão A, Pires A, Nicol AF(2013) A comparative analysis of clinical and molecular factors withthe stage of cervical cancer in a Brazilian cohort. PLoS One 8(3):e57810
31. Mendoza L, Mongelos P, Paez M, Castro A, Rodriguez-Riveros I,Gimenez G, Araujo P, Echagüe G, Diaz V, Laspina F, Castro W,Jimenez R, Marecos R, Ever S, Deluca G, Picconi MA (2013)Human papillomavirus and other genital infections in indigenouswomen from Paraguay: a cross-sectional analytical study. BMCInfect Dis 13(1):531
32. Simonella LM, Lewis H, Smith M, Neal H, Bromhead C, Canfell K(2013) Type-specific oncogenic human papillomavirus infection inhigh grade cervical disease in New Zealand. BMC Infect Dis 13:114
33. Cornet I, Gheit T, Iannacone MR, Vignat J, Sylla BS, Del Mistro A,Franceschi S, Tommasino M, Clifford GM (2013) HPV16 geneticvariation and the development of cervical cancer worldwide. Br JCancer 108(1):240–244
34. Tungteakkhun SS, Duerksen-Hughes PJ (2008) Cellular bindingpartners of the human papillomavirus E6 protein. Arch Virol153(3):397–408
35. Togtema M, Pichardo S, Jackson R, Lambert PF, Curiel L, Zehbe I(2012) Sonoporation delivery of monoclonal antibodies against hu-man papillomavirus 16 E6 restores p53 expression in transformedcervical keratinocytes. PLoS One 7(11):e50730
36. Lin HP, Wang YP, Chiang CP (2011) Expression of p53, MDM2,p21, heat shock protein 70, and HPV 16/18 E6 proteins in oralverrucous carcinoma and oral verrucous hyperplasia. Head Neck33(3):334–340
37. Doorbar J, Quint W, Banks L, Bravo IG, Stoler M, Broker TR,Stanley MA (2012) The biology and life-cycle of human papilloma-viruses. Vaccine 30(Suppl 5):F55–F70
38. Cuschieri K, Wentzensen N (2008) Human papillomavirus mRNAand p16 detection as biomarkers for the improved diagnosis ofcervical neoplasia. Cancer Epidemiol Biomarkers Prev 17(10):2536–2545
39. Iijima N, Goodwin EC, Dimaio D, Iwasaki A (2013) High-riskhuman papillomavirus E6 inhibits monocyte differentiation toLangerhans cells. Virology 444(1-2):257–262
40. Offringa RI, de Jong A, Toes RE, van der Burg SH, Melief CJ (2003)Interplay between human papillomaviruses and dendritic cells. CurrTop Microbiol Immunol 276:215–240
41. Sasagawa T, Takagi H, Makinoda S (2012) Immune responsesagainst human papillomavirus (HPV) infection and evasion ofhost defense in cervical cancer. J Infect Chemother 18(6):807–815
HPV16/18-E6 and P16 are Useful Prognostic Markers in Uterine Cervical Carcinomas 165
42. Niebler M, Qian X, Höfler D, Kogosov V, Kaewprag J, KaufmannAM, Ly R, Böhmer G, Zawatzky R, Rösl F, Rincon-Orozco B (2013)Post-translational control of IL-1β via the human papillomavirustype 16 E6 oncoprotein: a novel mechanism of innate immune escapemediated by the E3-ubiquitin ligase E6-AP and p53. PLoS Pathog9(8):e1003536
43. Bodaghi S, Wood LV, Roby G, Ryder C, Steinberg SM, Zheng ZM(2005) Could human papilloma viruses be spread through blood? JClin Microbiol 43(11):5428–5434
44. Chen AC, Keleher A, Kedda MA, Spurdle AB, McMillan NA,Antonsson A (2009) Human papillomavirus DNA detected in pe-ripheral blood samples from healthy Australian male blood donors. JMed Virol 81(10):1792–1796
45. Foresta C, Bertoldo A, Garolla A, Pizzol D, Mason S, Lenzi A, DeToni L (2013)Human papillomavirus proteins are found in peripheralblood and semen Cd20+ and Cd56+ cells during Hpv-16 semeninfection. BMC Infect Dis 13:593
46. Reijmers RM, Spaargaren M, Pals ST (2013) Heparan sulfate pro-teoglycans in the control of B cell development and the pathogenesisof multiple myeloma. FEBS J 280(10):2180–2193
47. Leemans CR, Braakhuis BJ, Brakenhoff RH (2011) The molecularbiology of head and neck cancer. Nat Rev Cancer 11:9–22
48. Carozzi F, Gillio-Tos A, Confortini M, Del Mistro A, Sani C, DeMarco L, Girlando S, Rosso S, Naldoni C, Dalla Palma P, Zorzi M,Giorgi-Rossi P, Segnan N, Cuzick J, Ronco G (2013) NTCCworkinggroup. risk of high-grade cervical intraepithelial neoplasia duringfollow-up in HPV-positive women according to baseline p16-INK4A results: a prospective analysis of a nested substudy of theNTCC randomised controlled trial. Lancet Oncol 14(2):168–176
49. Chiou HL, Wu MF, Liaw YC, Cheng YWSchiffman M, Castle PE,Jeronimo J, Rodriguez AC, Wacholder S (2007) Human papilloma-virus and cervical cancer. Lancet 370(9590):890–907
50. Chen TH, Huang CC, Yeh KT, Chang SH, Chang SW, Sung WW,Cheng YW, Lee H (2012) Human papilloma virus 16 E6 oncoproteinassociated with p53 inactivation in colorectal cancer. World JGastroenterol 18(30):4051–8.14
51. Roncaglia MT, Fregnani JH, Tacla M, DE Campos SG, Caiaffa HH,Ab’saber A, DAMotta EV, Alves VA, Baracat EC, Longatto Filho A(2013) Characterization of p16 and E6 HPV-related proteins in uter-ine cervix high-grade lesions of patients treated by conization withlarge loop excision. Oncol Lett 6(1):63–68
52. Giarnieri E, Mancini R, Pisani T, Alderisio M, Vecchione A (2000)Msh2, Mlh1, Fhit, p53, Bcl-2, and Bax expression in invasive and in
situ squamous cell carcinoma of the uterine cervix. Clin Cancer Res6(9):3600–3606
53. Volgareva G, Zavalishina L, Andreeva Y, Frank G, Krutikova E,Golovina D, Bliev A, Spitkovsky D, Ermilova V, Kisseljov F(2004) Protein p16 as a marker of dysplastic and neoplastic alter-ations in cervical epithelial cells. BMC Cancer 4:58
54. Hampl M, Wentzensen N, Vinokurova S, von Knebel-Doeberitz M,Poremba C, Bender HG, Kueppers V (2007) Comprehensive analysisof 130 multicentric intraepithelial female lower genital tract lesionsby HPV typing and p16 expression profile. J Cancer Res Clin Oncol133(4):235–245
55. MimicaM, Tomić S, KardumG, Hofman ID, Kaliterna V, Pejković L(2010) Ki-67 quantitative evaluation as a marker of cervicalintraepithelial neoplasia and human papillomavirus infection. Int JGynecol Cancer 20(1):116–119
56. Wang JL, Zheng BY, Li XD, Nokelainen K, Angström T, LindströmMS, Wallin KL (2005) p16INK4A and p14ARF expression patternby immunohistochemistry in human papillomavirus-related cervicalneoplasia. Mod Pathol 18(5):629–637
57. Murphy N, RingM, Killalea AG et al (2003) p16INK4A as a markerfor cervical dyskaryosis: CIN and cGIN in cervical biopsies andThinPrep smears. J Clin Pathol 56:56–63
58. Jung YS, Kato I, Kim HR (2013) A novel function of HPV16-E6/E7in epithelial-mesenchymal transition. Biochem Biophys ResCommun 435(3):339–344
59. Qi ZL, Huo X, Xu XJ, Zhang B, DuMG, Yang HW, Zheng LK, Li J,Shen ZY (2006) Relationship between HPV16/18 E6 and 53,21WAF1, MDM2, Ki67 and cyclin D1 expression in esophagealsquamous cell carcinoma: comparative study by using tissue micro-array technology. Exp Oncol 28(3):235–240
60. Galgano MT, Castle PE, Atkins KA, Brix WK, Nassau SR, StolerMH (2010) Using biomarkers as objective standards in the diagnosisof cervical biopsies. Am J Surg Pathol 34(8):1077–1087
61. Nicol AF, Golub JE, e Silva JR, Cunha CB, Amaro-Filho SM,Oliveira NS, Menezes W, Andrade CV, Russomano F, Tristão A,Grinsztejn B, Friedman RK, Oliveira MP, Pires A, Nuovo GJ (2012)An evaluation of p16 (INK4a) expression in cervical intraepithelialneoplasia specimens, including women with HIV-1. Mem InstOswaldo Cruz 107(5):571–577
62. Cheah PL, Looi LM, Teoh KH, Mun KS, Nazarina AR (2012) p16(INK4a) is a useful marker of human papillomavirus integrationallowing risk stratification for cervical malignancies. Asian Pac JCancer Prev 13(2):469–472
166 O. M. Omran, M. AlSheeha
Journal of Contemporary Brachytherapy (2015/volume 7/number 1)
Clinical InvestigationsOriginal paper
Uterine perforation and its dosimetric implications in cervical cancer high-dose-rate brachytherapy Yasir A. Bahadur, MD1, Maha M. Eltaher, MD1,2, Ashraf H. Hassouna, MD2,3, Mohammad A. Attar, MD1, Camelia Constantinescu, PhD4
1Department of Radiology, King Abdulaziz University Hospital, Jeddah, Saudi Arabia, 2Department of Radiation Oncology, National Cancer Institute, Cairo University, Egypt, 3Department of Oncology, King Faisal Specialist Hospital & Research Center, Jeddah, Saudi Arabia, 4Department of Bio-Medical Physics, King Faisal Specialist Hospital & Research Center, Jeddah, Saudi Arabia
AbstractPurpose: To retrospectively assess the incidence of sub-serosal and uterine perforation of intra-uterine tandem in
intracavitary high-dose-rate (HDR) brachytherapy for cervical cancer, and to evaluate its dosimetric implications on computed tomography (CT)-based treatment planning.
Material and methods: Computed tomography images and brachytherapy plans of cervical cancer patients treated from February 2006 to December 2012 were reviewed for sub-optimal implants (sub-serosal and uterine perforation), and their correlation with cancer FIGO stage and patients’ age. For each patient, the plans showing sub-optimal in-sertion of intra-uterine tandem were analyzed and compared to plans with adequate insertion. The difference in dose coverage of clinical-target-volume (CTV) and variation of the dose delivered to organs-at-risk (OARs) rectum and bladder were evaluated.
Results: A total of 231 brachytherapy plans for 82 patients were reviewed. We identified 12 (14.6%) patients and 14 (6%) applications with uterine perforation, and 12 (14.6%) patients and 20 (8.6%) applications with sub-serosal in-sertion of tandem. Data analysis showed that advanced stage correlates with higher incidence of sub-optimal implants (p = 0.005) but not the age (p = 0.18). Dose-volume-histograms (DVHs) analysis showed large variations for CTV dose coverage: D90 significantly decreased with average of –115.7% ± 134.9% for uterine perforation and –65.2% ± 82.8% for sub-serosal insertion (p = 0.025). The rectum and bladder dose assessed by D2cc increased up to 70.3% and 43.8%, respectively, when sub-optimal insertion of uterine tandem occurred.
Conclusions: We report a low incidence of uterine perforation and sub-serosal insertion of uterine tandem in intra-cavitary HDR brachytherapy for cervical cancer. However, the effects on treatment plan dosimetry can be considerably detrimental. Therefore, we recommend image-guided insertion, at least for the challenging cases.
J Contemp Brachytherapy 2015; 7, 1: DOI: 10.5114/jcb.2015.48898
Key words: cervix brachytherapy, cervical cancer, uterine perforation.
Address for correspondence: Prof. Yasir Bahadur, MD, Department of Radiology, King Abdulaziz University Hospital, P.O. Box 80215, Jeddah 21589, Saudi Arabia, phone: +966 12 2401000, ext. 17444, fax: +966 12 6408047, mobile: +966 505528528, e-mail: [email protected]; [email protected]
Received: Accepted: Published:
PurposeLocally advanced cervical cancer is optimally treated
by concurrent chemo-radiotherapy [1-3]. Radiotherapy includes external beam radiotherapy (EBRT), followed by a brachytherapy boost. Several studies have shown that the outcome of patients treated for bulky and advanced stage disease is directly correlated with the combined dose of radiation delivered by EBRT and brachytherapy [4-6]. Intracavitary brachytherapy involves insertion of a uterine tandem into the cervix and uterus. Sub-optimal insertion of uterine tandem can perforate the uterus and can result in increased dose to nearby organs-at-risk (OARs). A per-foration rate of 8% has been reported in applicators inser-tions, even if the operator is confident of placement [7,8].
Uterine perforation is a potential complication of uterine tandem insertion, as a result of the tandem being inserted in a false passage outside the uterine canal. If perforation remains undetected and radiation delivered, this can sig-nificantly affect the treatment outcome [9-11].
The purpose of this study was to retrospectively assess the incidence of sub-serosal and uterine perforation in intracavitary high-dose-rate (HDR) brachytherapy for cervical cancer, and to evaluate its effects on computed to-mography (CT)-based treatment planning dosimetry.
Material and methods Cervical cancer patients treated with HDR brachyther-
apy from February 2006 to December 2012 at the Radio-
Journal of Contemporary Brachytherapy (2015/volume 7/number 1)
Yasir A. Bahadur, Maha M. Eltaher, Ashraf H. Hassouna et al.2
therapy Unit of King Abdulaziz University Hospital, Jeddah, Saudi Arabia, were retrospectively identified. The study was reviewed and approved by the institution-al Research Ethics Committee.
All brachytherapy applications were performed un-der general anesthesia, without radiological guidance for uterine tandem insertion, by a radiation oncologist. Computed tomography images were then acquired using a Siemens Somatom Emotion CT-scanner (Siemens Med-ical Systems, Erlangen, Germany), with 2 mm slice inter-vals from the iliac crest to the distal end of the applicator.
The clinical-target-volume (CTV) and OARs volumes were defined on axial CT images for each brachytherapy fraction at the time of treatment planning, and retrospec-tively reviewed by one radiation oncologist, for the pur-pose of this study. The gross-target-volume (GTV) was delineated based on the CT information at the time of the brachytherapy and clinical findings. Isotropic margin (usually 1 cm) was added to generate the CTV. Primarily, the cervix and the lower uterine segment were included. If there was an involvement of the fornices or proximal vagina, these were also encompassed. Organ-at-risk vol-umes were the rectum and bladder. The outer rectal wall
was contoured from the recto-sigmoid junction till 1 cm above the anal verge, and the outer bladder wall was con-toured till the urethra.
Demographic and clinical data such as: patients’ age, International Federation of Gynecology and Obstetrics (FIGO) stage, initial tumor volume and residual tumor volume after EBRT were collected.
Computed tomography-based treatment plans were reviewed for uterine perforation or near-perforation (sub-serosal insertion of tandem, i.e. within 5 mm from the uterine serosa), as presented in Figure 1. For each patient, the plans showing sub-optimal insertion of intra-uterine tandem were analyzed and compared to plans with opti-mal insertion for the same patient. The difference in dose coverage of CTV was evaluated by the doses delivered to 90% (D90) and 100% (D100) of CTV. The variation of the dose received by 1 cc (D1cc) and 2 cc (D2cc) of OARs was also determined and analyzed to assess any possible cor-relation with the position of the uterine tandem relative to the uterus. All CT-based brachytherapy plans were gener-ated using Varian Brachyvision planning system, versions 8.0 and 10.0, for Varian HDR VariSource 200 and iX (Vari-an Medical Systems Inc., Palo Alto, CA, USA).
Fig. 1. Examples of uterine perforation (A) and sub-serosal insertion of intra-uterine tandem (B), in sagittal and coronal views
A
B
Journal of Contemporary Brachytherapy (2015/volume 7/number 1)
Uterine perforation in brachytherapy for cervix cancer 3
The correlation between incidence of uterine perfo-ration and patients’ variables was assessed using logistic regression analysis. The statistical analysis of dosimetry data was performed using the Student’s t-test and a p val-ue of < 0.05 was considered significant.
ResultsA total of 231 brachytherapy plans for 82 patients
were reviewed. We identified 34 (14.7%) treatment plans with sub-optimal insertion of intra-uterine tandem in 20 (24.4%) patients: 14 (6%) applications with uterine perfo-ration and 20 (8.6%) applications with sub-serosal inser-tion of tandem. Four patients (4.8%) presented both uter-ine perforation and sub-serosal insertion. Patient clinical characteristics are summarized in Table 1.
Applicators were not removed in all sub-optimal insertions; the tip of the tandem (which was outside of the uterine canal) was not loaded and the planned radiotherapy dose was delivered. Patients were stable and managed conservatively, with no reported ma-jor complications such as excessive bleeding, sepsis or other organ injury. Although it has been noticed a cor-relation between patient’s age (median 55.5 years) and advanced disease stage (IIB and IIIB) and the incidence of a sub-optimal insertion of intra-uterine tandem, the logistic regression analysis showed that only the stage could be a predictor for a sub-optimal implant (p = 0.005) but not the age (p = 0.18). Due to limited data regarding the initial and residual tumor volume post EBRT, its relation to the incidence of sub-optimal implants could not be analyzed.
Table 1. Patient characteristics (n = 82)
Characteristics Optimal insertion (n = 62) Sub-optimal insertion (n = 20)
n % n %
Brachytherapy fractions 197 85.3 34 14.7
Age (years)
Range 22-76 38-79
Median 52 55.5
Stage
IB 2 3.2 1 5
IIA 3 4.8 0 0
IIB 48 77.4 10 50
IIIA 2 3.2 0 0
IIIB 4 6.5 9 45
IVA 1 1.6 0 0
Unknown 2 3.2 0 0
Initial tumor size (cc)
Range 1.1-510.1 4.4-189.8
Mean ± SD 74.7 ± 97.1 63.4 ± 58.3
Unknown 19 30.6 6 30
Residual tumor size (cc)
Range 0.5-90.8 0.5-20.7
Mean ± SD 14.4 ± 25.8 7.1 ± 8.7
CR 16 25.8 4 20
Unknown 30 48.4 8 40
Site of sub-optimal insertion
Fundus uteri 5 25
Posterior uterine wall 12 60
Anterior uterine wall 3 15
SD – standard deviation, CR – complete response
Journal of Contemporary Brachytherapy (2015/volume 7/number 1)
Yasir A. Bahadur, Maha M. Eltaher, Ashraf H. Hassouna et al.4
Among the 20 patients (34 plans) having sub-optimal implants, there were 6 patients (17 plans) in which all the brachytherapy plans revealed sub-optimal insertion of intra-uterine tandem, therefore they were excluded from the dosimetric analysis, as no individual reference plans were available for comparison. Dose distribution and dose-volume histograms (DVHs) analysis was performed for the remaining 14 patients (17 sub-optimal implant plans), and showed large variations when compared to the plans with optimal intra-uterine insertion. The per-cent differences in dose coverage of CTV (assessed by D90) and dose received by 1 cc and 2 cc of rectum and bladder are presented in Table 2.
The percent difference of D90 between sub-optimal and optimal implant plans showed a significant decrease: from 0.74% to –319% (average: –115.7% ± 134.9%) for uterine perforation and from 12% to –223.8% (average: –65.2% ± 82.8%) for sub-serosal insertion of uterine tan-dem (p = 0.025), as shown in Figure 2.
The variations of the dose received by the OARs rec-tum and bladder were further analyzed, in order to de-tect a possible correlation of the dose with the position of the uterine tandem (site of perforation) relative to the uterus. The results showed that the rectum dose, assessed by D2cc increased up to 70.3% if the sub-serosal insertion of uterine tandem occurred in the posterior uterine wall, and up to 39.7% if in the uterine fundus. The bladder dose appeared not to be so sensitive to the uterine tandem po-sition: the maximum variations of D2cc were 37.4% in the posterior uterine wall and 43.8% in the uterine fundus. The percent differences between sub-optimal and opti-mal intra-uterine tandem insertion for D2cc of rectum and bladder are presented in Figure 3.
Discussion Brachytherapy is an essential component of cervical
cancer treatment. The technical quality of brachytherapy implant has been shown to significantly affect the clin-ical outcome of the treatment [9-11]. In a recent study, Viswanathan et al. showed that inappropriate placement of brachytherapy applicators led to significant risk of local recurrence (2.5 times) and lower DFS rate (2.28 times) [11].
Few published reports addressed the uterine perfo-ration during brachytherapy, using either low-dose-rate (LDR) or HDR [7,12-14]. In the case of LDR intracavitary brachytherapy, Barnes et al. presented an incidence of CT detected uterine perforation of 13.7%, although in 8.2% of insertions the physician was confident of correct tandem placement [7]. An incidence of 2% was reported by Pete-reit et al., using ultrasound in only 6% of cases, therefore the true occurrence of uterine perforation was probably underestimated [14]. Granai et al. employed post-opera-tive B-mode ultrasound to evaluate final tandem position in 50 consecutive insertions in 28 patients. In 34%, the tan-dem was found to be sub-optimally positioned: in 24% it penetrated the myometrium, and in 10% completely per-forated the uterus [15]. In a recent study reporting one of the lowest incidence of uterine perforation in literature, Segedin et al. determined 3% incidence of uterine perfo-ration among 428 image-guided brachytherapy applica-tions [16]. The authors emphasized the importance of us-ing CT/MR imaging post insertion to accurately identify
Table 2. Dose comparison in 14 patients between plans for sub-optimal (sub-serosal and uterine perforation) and optimal insertion of intra-uterine tandem, reported as percentage dose difference
Location Constraints Sub-serosal insertion of tandem Uterine perforation
Difference (%)(mean ± SD)
p Difference (%)(mean ± SD)
p
CTV D90 –65.2 ± 82.8 0.025 –115.7 ± 134.9 0.042
D100 –103.4 ± 170.4 0.078 –102.8 ± 132.3 0.13
Rectum D1cc –12.2 ± 52.3 0.46 20.2 ± 32.7 0.065
D2cc –12.2 ± 48.2 0.49 21. ± 30.6 0.051
Bladder D1cc –16.3 ± 31.7 0.061 4.1 ± 36.7 0.23
D2cc –14.1 ± 29.6 0.085 2.1 ± 36.3 0.27
CTV – clinical-target-volume, D90 – the minimum dose to 90% of the CTV, D100 – the minimum dose to 100% of the CTV, D1cc – minimum dose to the most exposed 1 cm3, D2cc – minimum dose to the most exposed 2 cm3
Fig. 2. Percent differences between sub-optimal implant and optimal insertion of intra-uterine tandem in the same patient for 14 patients (17 plans), regarding D90 of CTV. The black markers show the sub-serosal insertion of tan-dem and the gray ones the uterine perforation
Dos
e di
ffere
nce
betw
een
sub-
optim
al
and
optim
al im
plan
t for
CTV
D90
(%)
50
0
–50
–100
–150
–200
–250
–300
–350Patient number
Fundus
Posterior wall
Anterior wall
1 2 3 4 5 6 7 8 9 10 11 12 13 14
Journal of Contemporary Brachytherapy (2015/volume 7/number 1)
Uterine perforation in brachytherapy for cervix cancer 5
uterine perforation and used ultrasound guidance in 40% of subsequent applications after perforation.
Our study shows comparable data: 14.7% of the ana-lyzed brachytherapy treatment plans presented sub-op-timal insertion of intra-uterine tandem: 6% with uter-ine perforation and 8.6% with sub-serosal insertion of tandem. At our institution, uterine tandem insertion is a blind technique, performed without image guidance that would directly visualize pelvic structures, and de-pends on physician’s clinical judgment and skills. This explains a higher incidence of perforation in our study, when compared to Segedin et al. (6% vs. 3%) [16]. Prior to tandem insertion, it may be difficult to predict which patients are at risk of perforation. Risk factors that have been reported include age over 60 and anatomical distor-tion of the cervix (due to advanced disease, cervical ste-nosis, radiation fibrosis, or previous cone biopsy), which can make it difficult to localize the cervical os or subse-quent dilation of the endocervical canal [7,12,17-19].
Barnes et al. demonstrated that significant predictors of uterine perforation are patient age greater than or equal to 60 (p = 0.0019) and tumor size (p = 0.0016) [7]. Our data showed stage to be a predictor for a sub-optimal insertion (p = 0.005) but not the age (p = 0.18). Advanced disease may be associated with distortion of the cervix, difficulty to identify or localize the os, friability of cervical stroma, and uterine wall (if involved), which could increase the risk of a false track and perforation.
Nevertheless, post-insertion imaging is currently used to confirm correct uterine tandem placement, as well as to enable advanced brachytherapy planning [20-24]. Given the fixed angulation of commercially available uterine tandems, perforation risk is increased in marked ante-verted or retro-verted uteri [25,26]. We found that 60% of the perforations occurred in the posterior uterine wall, 25% in the fundus, and 15% in the anterior wall, while Segedin et al. reported 70% in the posterior uterine wall, 15% in the fundus, and 15% in the anterior wall [16].
In addition to injuring the pelvic organs, sub-optimal in-sertion of intra-uterine tandem can lead to inadequate ra-diation delivery and can subsequently alter the expected clinical outcome.
Our study showed that sub-optimal uterine tan-dem insertion can considerably affect the quality of the brachytherapy plans. The dose coverage of CTV, assessed by D90, showed a significant decrease, with an average of 115.7% ± 134.9% for uterine perforation, and 65.2% ± 82.8% for sub-serosal insertion of uterine tandem (p = 0.025). The doses received by rectum and bladder, and evaluated by D2cc increased with an average of 21.0% ± 30.6% for rectum, and 2.1% ± 36.3% for bladder, in the case of uterine perforation. While the bladder dose ap-peared not to be sensitive to the position of the uterine tandem relative to the uterine cavity, the rectum dose markedly increased when the sub-optimal insertion of tandem occurred in the posterior uterine wall.
Several reports investigated the effect of applicator displacement on brachytherapy plan dosimetry, and emphasized the importance of image guidance and com-prehensive quality control in the practice of brachyther-apy [27-30]. However, published data on the effect of mal-placement of brachytherapy applicators on plan quality are scarce, hence a comparison with our data was not possible. Furthermore, the clinical significance of car-rying out brachytherapy with a sub-optimally inserted intra-uterine tandem is not yet determined.
Pelvic magnetic resonance (MR) imaging after EBRT and before brachytherapy is a useful modality to as-sess the residual tumor and the orientation of uterus. For patients predicted to be at risk of perforation, ultra-sound-guided insertion of applicators should be consid-ered to minimize patient morbidity [21]. Intracavitary brachytherapy procedures are gradually becoming im-age-guided. Various authors have used X-ray fluoros-copy, ultrasonography, CT scan, MRI, and laparoscopy for guiding brachytherapy procedures in gynecological
Dos
e di
ffere
nce
betw
een
sub-
optim
al a
nd o
ptim
al im
plan
t (%
)
Dos
e di
ffere
nce
betw
een
sub-
optim
al a
nd o
ptim
al im
plan
t (%
)
806040200
–20–40–60–80
–100–120
60
40
20
0
–20
–40
–60
–80
–100Patient number Patient number
Fundus
Posterior wall
Anterior wall
Fundus
Posterior wall
Anterior wall
Fig. 3. Percent differences between sub-optimal implant and optimal insertion of intra-uterine tandem in the same patient for 14 patients (17 plans), regarding D2cc of rectum (A) and D2cc of bladder (B). The black markers show the sub-serosal insertion of tandem and the gray ones the uterine perforation
A B
1 2 3 4 5 6 7 8 9 10 11 12 13 141 2 3 4 5 6 7 8 9 10 11 12 13 14
Journal of Contemporary Brachytherapy (2015/volume 7/number 1)
Yasir A. Bahadur, Maha M. Eltaher, Ashraf H. Hassouna et al.6
malignancies. Methods such as CT and MR scans have practical limitations and are rarely used. Transrectal ul-trasound (TRUS) can provide real time imagery of pelvic structures and movement of applicators, and thus poten-tially help in avoiding perforation and injury to OAR. It has many advantages in terms of easy availability, sim-ple, and cost effective equipment, free from ionizing ra-diation [31]. Even though ultrasound-guidance is known to reduce the risk of uterine perforation, it is not yet used routinely by many clinicians [32].
The use of traditional X-ray simulations may make the planner unaware of the true position of the central tandem, which if loaded in the intra-abdominal location could risk the small intestine to be exposed to serious ra-diation dosages [33]. Post-insertion imaging techniques such as ultrasound, CT or MR, have been shown to detect uterine perforation in up to 66% of cases when clinical assessment suggested proper tandem placement, and have been recommended by several authors [19-22,33,34]. Some authors have even suggested additional pelvic im-aging following tandem insertion for all patients [7,35]. Image-guided brachytherapy has a double advantage in that it not only allows the detection of inadequate place-ment of the applicators but also gives the opportunity of dose optimization during the planning process.
An obvious limitation of the present study is the small number of patients presenting uterine perforation. Statis-tical analysis of such a small sample size could not be val-id if generalized at patient population level, and future studies with large sample sizes are recommended.
Conclusions
We report relatively low incidence rates of uterine perforation and sub-serosal insertion of uterine tandem in CT-guided intracavitary HDR brachytherapy for cer-vix carcinoma, without image guidance during applica-tors insertion. The effects on CTV coverage and rectal dose were considerably detrimental. Based on our data, we advise image-guided tandem insertion, at least for challenging cases.
Disclosure
Authors report no conflict of interest.
References1. NCCN Guidelines Version 1.2015. Cervical Cancer, 2015.2. National Cancer Institute. Cervical Cancer Treatment, 2014. 3. Haie-Meder C, Morice P, Castiglione M. Cervical cancer:
ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol 2010; 21: 37-40.
4. Eifel PJ, Thomas WW, Smith TL. The relationship between brachytherapy dose and outcome in patients with bulky en-docervical tumors treated with radiation alone. Int J Radiat Oncol Biol Phys 1994; 28: 113-118.
5. Lanciano RM, Won M, Coia L. Pretreatment and treatment factors associated with improved outcome in squamous cell carcinoma of the uterine cervix: a final report of the 1973 and 1978 patterns of care studies. Int J Radiat Oncol Biol Phys 1991; 20: 667-676.
6. Perez CA, Kao MS. Radiation therapy alone or combined with surgery in the treatment of barrel-shaped carcinoma of the uterine cervix (stages IB, IIA, IIB). Int J Radiat Oncol Biol Phys 1985; 11: 1903-1909.
7. Barnes EA, Thomas G, Ackerman I et al. Prospective com-parison of clinical and computed tomography assessment in detecting uterine perforation with intracavitary brachyther-apy for carcinoma of the cervix. Int J Gynecol Cancer 2007; 17: 821-826.
8. Irvin W, Rice L, Taylor P et al. Uterine perforation at the time of brachytherapy for carcinoma of the cervix. Gynecol Oncol 2003; 90: 113-122.
9. Corn BW, Hanlon AL, Pajak TF et al. Technically accurate intracavitary insertions improve pelvic control and survival among patients with locally advanced carcinoma of the uter-ine cervix. Gynecol Oncol 1994; 53: 294-300.
10. Eifel PJ, Morris M, Wharton JT et al. The influence of tumor size and growth habit on outcome of patients with FIGO stage IB squamous cell carcinoma of the uterine cervix. Int J Radiat Oncol Biol Phys 1994; 29: 9-16.
11. Viswanathan AN, Moughan J, Small W et al. The quality of cervical cancer brachytherapy implantation and the impact on local recurrence and disease-free survival in radiation therapy oncology group prospective trials 0116 and 0128. Int J Gynecol Cancer 2012; 22: 123-131.
12. Kim RY, Levy DS, Brascho DJ et al. Uterine perforation during intracavitary application. Prognostic significance in carcinoma of the cervix. Radiology 1983; 147: 249-251.
13. Matsuyama T, Tsukamoto N, Matsukuma K et al. Uterine perforation at the time of brachytherapy for the carcinoma of the uterine cervix. Gynecol Oncol 1986; 23: 205-211.
14. Petereit DG, Sarkaria JN, Chappell RJ. Perioperative morbid-ity and mortality of high-dose-rate gynecologic brachythera-py. Int J Radiat Oncol Biol Phys 1998; 42: 1025-1031.
15. Granai CO, Allee P, Doherty F et al. Intraoperative real-time ultrasonography during intrauterine tandem placement. Obstet Gynecol 1986; 67: 112-114.
16. Segedin B, Gugic J, Petric P. Uterine perforation – 5-year ex-perience in 3-D image guided gynaecological brachytherapy at Institute of Oncology Ljubljana. Radiol Oncol 2013; 47: 154-160.
17. Jhingran A, Eifel PJ. Perioperative and postoperative com-plications of intracavitary radiation for FIGO stage I-III car-cinoma of the cervix. Int J Radiat Oncol Biol Phys 2000; 46: 1177-1183.
18. Corn BW, Shaktman BD, Lanciano RM et al. Intra- and perioperative complications associated with tandem and col-postat application for cervix cancer. Gynecol Oncol 1997; 64: 224-229.
19. Granai CO, Doherty F, Allee P et al. Ultrasound for diagnos-ing and preventing malplacement of intrauterine tandems. Obstet Gynecol 1990; 75: 110-113.
20. Beddy P, Rangarajan RD, Sala E. Role of MRI in intracavitary brachytherapy for cervical cancer: what the radiologist needs to know. AJR 2011; 196: 341-347.
21. Watkins JM, Kearney PM, Opfermann KJ et al. Ultra-sound-guided tandem placement for low-dose-rate brachy- therapy in advanced cervical cancer minimizes risk of intra-operative uterine perforation. Ultrasound Obstet Gynecol 2011; 37: 241-244.
22. Addley HC, Vargas HA, Moyle PL et al. Pelvic imaging fol-lowing chemotherapy and radiation therapy for gynecologic malignancies. Radiographics 2010; 30: 1843-1856.
23. Nag S, Erickson B, Thomadsen B et al. The American Bra- chytherapy Society recommendations for high-dose-rate brachytherapy for carcinoma of the cervix. Int J Radiat Oncol Biol Phys 2000; 48: 201-211.
Journal of Contemporary Brachytherapy (2015/volume 7/number 1)
Uterine perforation in brachytherapy for cervix cancer 7
24. Sahinler I, Cepni I, Oksuz DC et al. Tandem application with transvaginal ultrasound guidance. Int J Radiat Oncol Biol Phys 2004; 59: 190-196.
25. Prabhakar GS, Revannasiddaiah S, Susheela SP et al. Uterine perforation during intracavitary brachytherapy for carcino-ma of the cervix. BMJ Case Reports 2012; doi:10.1136/bcr-2012-007830.
26. Mayr NA, Montebello JF, Sorosky JI et al. Brachytherapy man-agement of the retroverted uterus using ultrasound-guided implant applicator placement. Brachytherapy 2005; 4: 24-29.
27. Garipagaoglu M, Tuncel N, Dalmaz MG et al. Changes in ap-plicator positions and dose distribution between high dose rate brachytherapy fractions in cervix carcinoma patients re-ceiving definitive radiotherapy. Br J Radiol 2006; 79: 504-509.
28. Yoshida K, Ueda M, Takenaka T et al. Daily CT measurement of needle applicator displacement during multifractionated high-dose-rate interstitial brachytherapy for postoperative recurrent uterine cancer. J Radiat Res 2012; 53: 295-300.
29. Koushik K, Bilimagga R, Rao N et al. Positional variation of applicators during low dose rate intracavitary brachytherapy for cervical cancer: a prospective study. J Contemp Brachyther-apy 2010; 2: 93-97.
30. Tanderup K, Hellebust TP, Lang S et al. Consequences of ran-dom and systematic reconstruction uncertainties in 3D image based brachytherapy in cervical cancer. Radiother Oncol 2008; 89: 156-163.
31. Sharma DN, Rath GK, Thulkar S et al. Use of transrectal ultrasound for high dose rate interstitial brachytherapy for patients of carcinoma of uterine cervix. J Gynecol Oncol 2010; 21: 12-17.
32. Davidson MT, Yuen J, D’Souza DP et al. Optimization of high-dose-rate cervix brachytherapy applicator place-ment: the benefits of intraoperative ultrasound guidance. Brachytherapy 2008; 7: 248-253.
33. Makin WP, Hunter RD. CT scanning in intracavitary ther-apy: unexpected findings in “straightforward” insertions. Radiother Oncol 1988; 13: 253-255.
34. Rotmensch J, Waggoner SE, Quiet C. Ultrasound guidance for placement of difficult intracavitary implants. Gynecol On-col 1994; 54: 159-162.
35. Small W Jr, Strauss HB, Hwang CS et al. Should uterine tan-dem applicators ever be placed without ultrasound guid-ance? No: a brief report and review of the literature. Int J Gynecol Cancer 2011; 21: 941-944.
RESEARCH Open Access
Human papillomavirus genotyping andintegration in ovarian cancer Saudi patientsOthman A Al-Shabanah1, Mohamed M Hafez1*, Zeinab K Hassan2, Mohamed M Sayed-Ahmed1,Waleed N Abozeed3, Salem S Al-Rejaie1 and Abdulmalik A Alsheikh4
Abstract
Background: Human papillomavirus (HPV) is associated with different malignancies but its role in the pathogenesisof ovarian cancer is controversial. This study investigated the prevalence, genotyping and physical state of HPV inovarian cancer Saudi patients.
Methods: Hundred formalin fixed paraffin embedded (FFPE) ovarian carcinoma tissues and their normal adjacenttissues (NAT) were included in the study. HPV was detected by nested polymerase chain reaction (PCR) usingdegenerated HPVL1 consensus primer pairs MY09/MY11 and GP5+/GP6 + to amplify a broad spectrum of HPVgenotypes in a single reaction. The HPV positive samples were further genotyped using DNA sequencing. Thephysical state of the virus was identified using Amplification of Papillomavirus Oncogene Transcripts (APOT) assay inthe samples positive for HPV16 and/or HPV18.
Results: High percentage of HPV (42%) was observed in ovarian carcinoma compared to 8% in the NAT. Thehigh-risk HPV types 16, 18 and 45 were highly associated with the advanced stages of tumor, while low-risk types 6and 11 were present in NAT. In malignant tissues, HPV-16 was the most predominant genotype followed byHPV-18 and −45. The percentage of viral integration into the host genome was significantly high (61.1%) comparedto 38.9% episomal in HPV positive tumors tissues. In HPV18 genotype the percentage of viral integration was 54.5%compared to 45.5% episomal.
Conclusion: The high risk HPV genotypes in ovarian cancer may indicate its role in ovarian carcinogenesis. The HPVvaccination is highly recommended to reduce this type of cancer.
Keyword: DNA sequencing, Formalin paraffin embedded tissue, Human papilloma virus, Ovarian cancer,Polymerase chain reaction
IntroductionHuman papillomavirus (HPV) belongs to Papillomaviridaefamily that consists of small double stranded DNA virusesassociated with cutaneous and mucosal squamous epithe-lial lesions [1]. HPV infection is detected in cancers of thefemale lower genital tract [2,3]. However, its role in thedevelopment of cancers in the upper genital tract, suchas endometrial and ovarian cancer, is less clear [4].More than 200 genotypes of HPV have been identifiedand were subdivided into two groups the oncogenic andnon-oncogenic group [5]. The oncogenic HPV genotypes
are 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68[6,7]. Of these, type 16 and 18 have been classified as“high-risk” (HR-HPV) because they are associated with themalignant progression of cervical tumors and with othergenital and head-neck malignancies [8]. The high-riskHPV types produced two oncogenes, designed E6 and E7proteins induce transformation by interference with en-dogenous cell cycle regulatory proteins, including P53,retinoblastoma (Rb) and breast cancer type 1 suscepti-bility protein (BRAC1) [9]. The L1 open reading frame(ORF) region is the most conserved gene within theHPV genome, and has been used for identification ofgenotyping and new HPV genotypes [10]. The HPVdetection in clinical samples is based on the DNA frag-ments amplification in the L1 region [11].
* Correspondence: [email protected] of pharmacology, College of pharmacy; King Saud University,P.O. Box 2457, Riyadh 11451, Kingdom of Saudi ArabiaFull list of author information is available at the end of the article
© 2013 Al-Shabanah et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of theCreative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use,distribution, and reproduction in any medium, provided the original work is properly cited.
Al-Shabanah et al. Virology Journal 2013, 10:343http://www.virologyj.com/content/10/1/343
Ovarian carcinoma is the most lethal gynecologicalmalignancy because it is detected in advanced stageswith 5-year survival rate is <40% [12,13]. The etiology ofovarian cancer remains unclear and may be multifactorial.Ovarian cancer is either epithelial carcinomas or malig-nant germ cell tumors [14]. Its incidence rates are thehighest among developed countries, with rates exceeding9/100,000 women per year. In Saudi Arabia, ovarian can-cer is the seventh most common malignancy among fe-males and accounting for 3.1% of all newly diagnosedcases with median age of 50 years [15]. Epithelial ovariancancer accounts for 85-90% of total ovarian tumor [16].The genetic alterations associated with ovarian carcin-
omas are well known [17,18]. The risk factors that lead toovarian carcinomas include positive family history of ovar-ian, breast or colon cancer; old age; number of ovulations;endocrine factors; endometriosis; pelvic inflammation; fatintake [19]. Other environmental factors as HPV infectionare recently under investigations. The participation ofHPV infection could be suspected to be involved in thedevelopment of ovarian cancer [20]. Several studies pro-vided highly controversial results [21-26].The integration site of HPV is extensively investigated
in cell lines and clinical samples of HPV related cancersat various sites of the body. The integration site for HPVis random throughout the genome but the Integrationmechanisms are not fully understood [27]. Integration ofHPV into host cell genome is found with a high per-centage in cervical cancer infected with HPV16 andHPV18 genotypes, and low in the precancerous lesionsand undetected in early HPV-induced lesion [28-30]. Inlate stage of cancers, viral integration into host genomeis important in the disease progression. Viral integrationoccurs downstream of the early genes E6 and E7 or inthe E1 or E2 region causing gene inactivation [31].Therefore, the integration of viral genomes may con-tribute to a large extent to the neoplastic transformationprocess and may trigger chromosomal instability. Thereare number of methods for the detection of HR-HPVintegrants in human genome such as Amplification ofPapillomavirus Oncogene Transcripts (APOT), RestrictionSite PCR (RS-PCR), Southern blot and Detection ofIntegrated Papillomavirus Sequences (DIPS). The APOTmethod is able to detect the integration of viral genome inclinical lesions even in the presence of a large excess ofnon-integrated, episomal, form of viral genomes [32]. Thecurrent study aimed to determine the prevalence, genotyp-ing and physical state of HPV in cancerous and normaladjacent tissues from ovarian cancer Saudi patients.
ResultsGenomic DNA was isolated from 100 FFPE ovariancarcinoma and their normal adjacent tissues. All sam-ples were positive for β-globin gene amplification. The
amplified HPV DNA with MY09/MY11 followed byGP5+/GP6+ were considered HPV positive and weresubjected to DNA sequencing. The negative samplesby nested PCR were subjected to HPV-type specificPCR to confirm that the samples were negative. Fortytwo out of 100 tumor samples and 8 out of 100 NATwere positive for HPV by nested PCR as shown inFigure 1.The patients’ mean age was 50 ± 11 years (range, 25–78
years). The patients’ age distribution was analyzed and theprevalence of HPV genotypes was detected among them.There was no significant difference (P > 0.5) observed inHPV infection among patients with age <45 comparedto >45 years old, in which 15/35 (42.8%) of the patientswith age <45 years old and 27/65 (41.5%) were positivefor HPV. The HPV infection in relation to histologicalgrade, HPV was detected in 25% (7/28) of cases withgrade I, 50% (21/42) of cases with grade II and 46.7%(14/30) of cases with grade III.By using sequencing technique, the most common de-
tected HPV genotype was HPV-16 in 18/42 (42.9%),followed by HPV-18 in 11/42 (26.2%), finally HPV-45 in7.1% as in Figures 2A and 2B. The overlapped sequenceswas seen in 10/42 (23.8%) cases that indicates the pres-ences of more than one HPV genotype.As the sequencing technique failed to identify the spe-
cific mixed HPV genotypes, therefore type specific PCRassay was used. Mixed infection with HPV-16/18, HPV-
Figure 1 PCR products were analyzed on a 2% agarose gelstained with ethidium bromide and visualized byUV-trans-illumination. Lane M is 50 PCR marker (Promega), Lanes1, 5 and 8 are weak positive sample, lanes 2, 3, 4, 6 and 7 arenegative samples, from lane 9-12 are positive samples and lane 13 ispositive control.
Al-Shabanah et al. Virology Journal 2013, 10:343 Page 2 of 9http://www.virologyj.com/content/10/1/343
16/45, HPV-18/45 or HPV = 16/18/45 were observed in7/42 (16.6%), 2.4%, 2.4%, 2.4% respectively as showed inTable 1. The prevalence of HPV-6 and HPV-11 (low riskHPV genotypes) were detected only in NAT HPV posi-tive samples with 50% of both genotypes (Table 1).The clinical stages were determined according to the
International Federation of Gynecology and Obstetricssystem (FIGO). The ovarian cancer samples were classifiedas followed: stage 1-23%, stage II-37%, stage III-29% andstage IV-11%. HPV was detected in 21.7% (5/23) stage 1,40.5% (15/37) stage II, 41% (16/39) stage III and 75% (9/11) stage IV as showed in Figure 3. The presence of HPVinfection was significantly higher in patients with ad-vanced stages (FIGO stage III and IV) 50% compared tolocalized disease (FIGO stage I and II) 33.3% (P < 0.05).
Figure 2 Sequencing data of HPV genotypes (A) A sequence excised from an electropherogram for HPV type 18 (B) Sequencealignment of HPV type 16, 18 and 45 using Basic Local Alignment Search (BLAST).
Table 1 The prevalence of different humanpapillomavirus genotypes in ovarian cancer and itsadjacent normal tissues
HPV infection Ovarian cancer Normal adjacent tumor
Total positive (42/100) 42% (8/100) 8%
HPV-16 (18/42) 42.9% 0 (0%)
HPV-18 (11/42) 26.2% 0 (0%)
HPV-16/18 (7/42) 16.6% 0 (0%)
HPV-45 3/42 (7.1%) 0 (0%)
HPV-16/45 1/42 (2.4%) 0 (0%)
HPV-18/45 1/42 (2.4%) 0 (0%)
HPV-16/18/45 1/42 (2.4%) 0 (%)
HPV-6 0 (0%) 4/8 (50%)
HPV-11 0 (0%) 4/8 (50%)
Al-Shabanah et al. Virology Journal 2013, 10:343 Page 3 of 9http://www.virologyj.com/content/10/1/343
The HPV genotypes were distributed among differentFIGO stages as followed: HPV-16 were (7/37) 18.9%, (9/39) 23% and (2/11) 18.2% in stage II, III and IV respect-ively as showed in Figure 4. HPV-18 was 2/23 (8.7%) instage I, 3/37 (8.1%) in stage II, 4/39 (10.%) in stage III and2/11 (18.2%) in stage IV (Figure 5). On the other handHPV-45 was observed in 2 cases ofstageI and in one casewith stage II.The APOT assay is based on the structural differences
among the 3′-ends of viral oncogene transcripts. The in-tegration of HR-HPV genomes into the host genome re-sults in both disruption of the E1 or E2 open readingframes and deletion of viral early-region from the viraloncogene-encoding sequences. Thirty six HPV-16, HPV-18 or HPV-16/-18 positive samples with good qualityRNA, were subjected to study the physical states byAPOT assay. The viral genome was found to be inte-grated in 22/36 (61.1%), whereas the episomal tran-scripts were found in 38.9%. Out of the 22 cases withintegrated viral genome the episomal form of HPV wasalso detected in 5/22 (22.7%). In HPV-18 genotype posi-tive samples the percentage of viral integration was54.5% compared to 45.5% episomal. On the other handin HPV-16 genotype positive samples the percentage ofviral integration was 65% compared to 35% episomal.The integrated state of the virus was significantly foundin 90% in advanced stages III and IV compared to 10%
FIGO stage
I
FIGO stage
II
FIGO stage
III
FIGO stage
IV
0
20
40
60
80 *#$
Perc
en
tag
e
Figure 3 The incidence of human papillomavirus in relation toovarian cancer stages. *,# and $ indicate significant difference fromStage I, stage II and stage III respectively.
FIGO stage
II
FIGO stage
III
FIGO stage
IV
0
5
10
15
20
25
Perc
en
tag
e
Figure 4 The incidence of human papillo mavirus type 16 inrelation to ovarian cancer stages.
FIGO stage
I
FIGO stage
II
FIGO stage
III
FIGO stage
IV
0
5
10
15
20
Perc
en
tag
e
*#$
Figure 5 The incidence of human papillomavirus type 18 inrelation to ovarian cancer stages. *, # and $ indicate significantdifference from stage I, satge II and stage III, respectively.
Al-Shabanah et al. Virology Journal 2013, 10:343 Page 4 of 9http://www.virologyj.com/content/10/1/343
in localized stages I and II (p < 0.05) showing that theintegrated form was associated with the advanced stagesof cancer.
DiscussionThe role of HPV infection in cervical cancer [33] andother types of cancer [34-37] has been studied. The role ofHPV in ovarian cancer development is debated [24,38-43]may be due to the different samples size or the techniqueused to detect HPV. Therefore, subsequent studies are inneed to confirm the potential impact of HPV in ovariancancer. In Saudi Arabia, ovarian cancer represents the sev-enth most common malignancy and cancer-related deathsamong females [15]. To the best of our knowledge, this isthe first study on the association between HPV infectionand ovarian cancer in Saudi Arabia. This study investi-gated the presence of HPV genotypes and its physicalstates in ovarian cancer Saudi women.HPVs are classified as high and low risk, according to
their relationship with benign or malignant proliferativelesions [44]. The oncogenic activity of high-risk HPVtypes occurred when they integrated in the host genome.In many studies, molecular assays were used to identify
different types of HPV in cells and tissues [45-47]. The useof MY09/MY11 followed by GP5+/GP6+ primers toenhance the detection sensitivity in samples containinglow viral copy numbers and to amplify a wide spectrum ofHPV genotypes [48]. In the present study, the MY09/MY11 and GP5+/GP6+ primers followed by DNA sequen-cing and type specific PCR were used to confirm the HPVgenotypes and to identify the mixed infection. In thecurrent study, the incidence of HPV was higher in cancer-ous tissues than in NAT by both the PCR and sequencingtechniques. Some studies detect the high incidence ofhigh-risk HPV DNA in both benign and ovarian malig-nant tumors [49,50]. Similar study indicates the import-ance of HPV in malignant development via the statisticaldifference of HPV distribution in benign compared to ma-lignant ovarian tissue of Chinese patients [4]. In contrast,other study didn’t find any association between ovariancancers and the presence of HPV infection by using PCRassay [51]. A study in India included 20 ovarian cancerbiopsies, demonstrated complete absence of HPV infec-tion in ovarian cancer [52]. Other studies have shownlower rate of HPV DNA in ovarian than cervical cancer[4,24]. Hence, the identification of HPV DNA in ovariantumors may provide an evidence of a metastatic cervicalcancer [53,54]. The geographical variation in HPV variantsshowed that the virus and the host has co-evolved overtime [55]. In our study, the identification of high-risk HPVin cancerous tissues reflects the HPV possible role in ovar-ian carcinogenesis and the viral type is probably an im-portant factor. Some host genetic factors is implicated inHPV persistence such as polymorphism or variation is
human major histocompatibility class II [56]. Other studyon the cervical cancer Saudi patients found high incidenceof HPV with HPV-16 and −18 the common genotypes.They concluded that the HPV prevalence in cervical can-cer patients is comparable to the international rates [8].In the current study, the high-risk HPV types 16, 18
and 45 were associated with advanced stages of the dis-ease, while low-risk types 6 and 11 were associated withnormal tissues adjacent to the tumor. This finding wasin agreement with other study found that the presenceof high-risk types of HPV was found in the serous histo-logical subtype and advanced stages of the disease (FIGOstages III and IV) [39]. Other studies showed no evi-dence of high-risk HPV association with histologicalsubtype and/or stage of disease [4,24]. In ovarian carcin-oma Serbia patients, HPV infections were more frequentin FIGO III/IV in relation to FIGO I/II stages [57].There is no statistical significant difference in HPV
infection in relation to age group more/or less than 45years. The current study are consistent with thosereports based the detection of HPV in relation to age,they found that the median age of diagnosis of ovariancancer patients infected with HPV was 57 years forpatients with HPV and 59 years for patients withoutHPV infection [4].HR-HPV DNA integration in cervical cancer genome
plays an important role in cancer pathogenesis [1].HPV-transformed cells growth rate depends on E6 andE7 oncogenes expression level [58,59], so the integrationis important in HR-HPV-induced cancer. Therefore, theviral genomes integration resulted in elevated expressionlevels of the E6 and E7 oncogenes [60].Integration of HPV is common in late stage cervical
cancers and considered as an important event in diseaseprogression. The molecular detection of integrated HR-HPV genomes may represent a suitable marker for theidentification of invasive carcinoma. Integration gener-ally occurs in E1 or E2 region downstream of the earlygenes E6 and E7. Viral E2 gene is well known to play arole in viral replication as well as negative regulation ofE6 and E7 genes [39] and transcriptionally inactivatedonce the virus gets integrated due to disruption of itsopen reading frame. Some studies showed that bothepisomal and integrated forms are able to transformnormal keratinocytes [61-63].The APOT assay allows distinguishing between
integrate- and episome-derived transcripts encompass-ing HR-HPV E7 sequences. To study the physical stateof HPV, APOT assay was used to detect the integrationof viral genome [32]. In the current study, the percent-age of viral integration into the host genome was de-tected in 61.1% of HPV positive tumors and was 38.9%episomal. The incidence of integration in HPV18 posi-tive samples was 54.5% compared to 45.% the virus was
Al-Shabanah et al. Virology Journal 2013, 10:343 Page 5 of 9http://www.virologyj.com/content/10/1/343
episomal. Also the virus integrated state was associatedwith the advanced stages of cancer. This was inconsist-ent with other study that was done on the relationshipbetween integration of HPV and cervical cancer [32].Similar study found, by applying APOT assay to samplesinfected with HR-HPV types 16 and 18, a strong correl-ation between the detection of integrate-derived tran-scripts and the progression stage of the cervical dysplasia.Some investigators detected integrated HPV genomes invarious preneoplastic lesions [64] or in invasive carcinomasamples [65]. This discrepancy was attributed to the differ-ent methodological approaches used for the detection ofintegrated HPV DNA [65].
ConclusionThis study supports the hypothesis that there was a correl-ation between HPV infection and ovarian cancer in SaudiArabia. The high percentage of HR-HPV associated withovarian cancer and its integrated form may reflect a pos-sible role of this virus in the carcinogenesis of ovariantumors or it may facilitate its progression. From this study,we recommended theadmission of HPV vaccination in thenational vaccination program.
Material and methodsHuman papillomavirus and its integration were investi-gated in ovarian cancer and its normal adjacent tissues(NAT). The study was conducted in compliance withHelsinki Declaration and was approved by the reviewboard of King Khalid Hospital, King Saud University. Itincluded 100 archival formalin-fixed paraffin embedded(FFPE) ovarian cancer and their normal adjacent tissueswere collected from Pathology Department, College ofMedicine, King Saud University and Riyadh RegionalLaboratory and blood bank. All the pathological datawere taken from the pathological reports. The mean agewere 50 ±11 years ranging from 25–78 years.
Nucleic acid extractionCervical cancers cell lines positive for HPV-16 (SiHa,CaSki) and HPV-18 (HeLa, C-4 I) were used as positivecontrol for genotypes and for viral integration. All FFPEsamples were thin sectioned at 8 μm thicknesses usingLeica Microtome (Manual Rotary Microtome RM2235).Tissue sections were floated in a DEPC-treated waterbath then picked up on glass slides then allowed to dry.Genomic DNA was used for HPV genotyping and
RNA was used to detect the integration of HPV. Threetissue sections were used for RNA and DNA extractionusing Recover All total Nucleic Acid Isolation Kit(Ambion, Life Technologies, USA) following the manu-facturer instructions. In brief the tissue sections weredeparaffinized then digested by proteases. The nucleicacid was isolated by preparing the isolation additive/
ethanol mixture followed by transfer to the column theneluted. The quantity and quality of the RNA and DNAwere characterized using a UV spectrophotometer(NanoDrop8000, Thermo scientific, USA).
Human papillomavirus detection by nested PCRThe specimens were amplified with beta-globin primersGH20 and GH21 in order to check the DNA quality.Nested PCR with consensus primers MY09/MY11 wasused to amplify a wide spectrum of HPV types with PCRproduct of 450 bp followed by GP5+/GP6+ with PCRproduct of approximately 150 bp as previously described[66]. Each sample was tested three times. The primerssequences are shown in Table 2. The PCR reaction wasdone in 50 μl, contained 500 ng of DNA, 1XPCR MasterMix (Promega, Madison, USA), 3 mMMgCl2, 300 nMof each primer. Amplifications using MY09/MY11wereperformed with the following cycling profile: incubationat 94°C for 5 min followed by 40 cycles of 1 min de-naturation at 95°C, 1 min annealing at 55°C, and 1 minelongation at 72°C. The last cycle was followed by afinal extension of 10 min at 72°C. The annealing step ofGP5+/GP6+ primers-based PCR was performed at 40°Cfor 2 min. During amplification positive and negativecontrol samples were included. PCR products were ana-lyzed on a 2% agarose gel stained with ethidium bromideand visualized by UV-transillumination Figure 1.
Type‐specific PCRMultiple infections and negative samples were subjectedto type-specific PCR to confirm the results. The amplifi-cation reactions were performed using 18 HPV primersas previously described [67] (13 for HR-HPV 16, 18, 31,33,45, 35, 39, 51, 52, 56, 58, 66, and 68 and 5 for Lowrisk HPV 6,11,42,43 and 44) in separate reactions. Eachreaction was performed in a final volume of 50 μL con-taining 500 ng of DNA, 1 × PCR Buffer 300 nM of eachprimer, and 1 U of Taq polymerase (KAPABIOSYSTEM,USA). The amplification conditions were 95°C for 10min followed by 40 cycle of 1 min denaturation at 95°C,1 min annealing temperature vary for each primers, and2 min extension at 72°C. The last cycle was followed bya final extension of 10 min at 72°C.
Amplification of papillomavirus oncogene transcripts(APOT)cDNA was synthesized from 1 μg of total RNA by re-verse transcription using an oligo(dT)17-primer coupledto a linker sequence p3 using a high capacity cDNAkit (Applied biosystem, life technology, USA), accord-ing to the manufacturer’s instructions. To controlRNA integrity and cDNA quality, PCR reactions usingglyceraldehyde-3-phosphate dehydrogenase–specific primerswere performed.
Al-Shabanah et al. Virology Journal 2013, 10:343 Page 6 of 9http://www.virologyj.com/content/10/1/343
cDNAs include viral oncogene sequences were subse-quently amplified by PCR using HPVE7–specific primer[P1-16] for HPV16 and [P1-18] for HPV18 as forwardprimers and linker p3 as the reverse primer. The PCRwas performed in 50 μL reaction volume containing5 μL of the RT reaction mixture (cDNA), 2.5 units Taqpolymerase (promega madison wisconsin, USA), 1×PCR buffer (500 mMKCl, 1.5 mM MgCl2), 200 μM eachof the deoxyribonucleotide triphosphate and 0.25 μM ofeach primer. The reaction mixture was subjected toinitial denaturation for 2 min, followed by 35 cycles ofdenaturation at 94°C for 45 s, annealing at 58°C for 45 s,elongation at 72°C for 2 min, and a final elongation stepat 72°C for 7 min. Five μl of the amplified products wereused as template for nested PCR under the same condi-tions at annealing temperature 65°C using forwardHPVE7-specific primer [p2-16] specific for HPV16 and
[P2-18] specific for HPV18 as forward primers and (dT)17-p3 as reverse primer [64].
DNA sequencingTo identify the HPV genotypes and the integration, allpositive PCR products were analyzed by direct DNA se-quencing. PCR products were purified using QIAquickPurification Kit according to manufacturer’s instructions(QIAGEN, Hilden, Germany). Purified PCR products werelabeled with fluorescent dyes using BigDye Terminatorv3.1 Cycle Sequencing Kit Applied Biosystem. Labeled oli-gonucleotides were purified using BigDye X TerminatorPurification Kit (Applied Biosystems, CA, USA).The samples were sequenced by automatic ABI 3500
genetic analyzer (Applied Biosystems, USA). Chromato-grams with sharp peaks and quality values ≥20 with littleor no background noise consider as single HPV infection.
Table 2 Primers sequence used in this study
Primers Forward primer Reverse primer
MY09/MY11 5′-CGTCC(AC)A(AG)(AG)GGA(T)ACTGATC-3′ 5′-GC(AC)CAGGG(AT)CATAA(CT)AATGG-3′
GP5+/GP6+ 5′-TTTGTTACTGTGGTAGATACTAC-3′ 5′-GAAAAATAAACTGTAAATCATATTC-3′
HPV-16 5′-CAC AGT TAT GCA CAG AGC TGC-3′ 5′-CAT ATA TTC ATG CAA TGT AGG TGTA-3′
HPV-18 5′-CAC TTC ACT GCA AGA CAT AGA-3′ 5′-GTT GTG AAA TCG TCG TTT TTC A-3′
HPV-31 5′-GAA ATT GCA TGA ACT AAG CTC G-3′ 5′-CAC ATA TAC CTT TGT TTG TCA A-3′
HPV-59 5′-CAA AGG GGA ACT GCA AGA AAG-3′ 5′-TAT AAC AGC GTA TCA GCA GC-3′
HPV-45 5′-GTG GAA AAG TGC ATT ACA GG-3′ 5′-ACC TCT GTG CGT TCC AAT GT-3′
HPV-33 5′-ACT ATA CAC AAC ATT GAA CTA-3′ 5′-GTT TTT ACA CGT CAC AGT GCA-3′
HPV 6/11 5′-TGC AAG AAT GCA CTG ACC AC-3′ 5′-TGC ATG TTG TCC AGC AGT GT-3′
HPV-58 5′-GTA AAG TGT GCT TAC GAT TGC-3′ 5′-GTT GTT ACA GGT TAC ACT TGT-3′
HPV-52 5′-TAA GGC TGC AGT GTG TGC AG-3′ 5′-CTA ATA GTT ATT TCA CTT AAT GGT-3′
HPV-56 5′-GTG TGC AGA GTA TGT TTA TTG-3′ 5′-TTT CTG TCA CAA TGC AAT TGC-3′
HPV-35 5′-CAA CGA GGT AGA AGA AAG CAT C-3′ 5′-CCG ACC TGT CCA CCG TCC ACCG-3′
HPV-42 5′-CCC AAA GTA GTG GTC CCA GTT A-3′ 5′-GAT CTT TCG TAG TGT CGC AGT G-3′
HPV-43 5′-GCA TAA TGT CTG CAC GTA GCT G-3′ 5′-CAT GAA ACT GTA GAC AGG CCA AG-3′
HPV-44 5′-TAA ACA GTT ATA TGT AGT GTA CCG-3′ 5′-TAT CAG CAC GTC CAG AAT TGA C-3′
HPV-68 5′-GCA GAA GGC AAC TAC AAC GG-3′ 5′-GTT TAC TGG TCC AGC AGT GG-3′
HPV-39 5′-GAC GAC CAC TAC AGC AAA CC-3′ 5′-TTA TGA AAT CTT CGT TTG CT-3′
HPV-51 5′-GAG TAT AGA CGT TAT AGC AGG-3′ 5′-TTT CGT TAC GTT GTC GTG TAC G-3′
HPV-66 5′-TTC AGT GTA TGG GGC AAC AT-3′ 5′-AAA CAT GAC CCG GTC CAT GC-3′
GAPDH 5′-CCACTCCTCCACCTTTGA-3′ 5′-ACCCTGTTGCTGTAGCCA-3′
GH20 and GH21 5′-GAA GAG CCA AGG ACA GGT AC-3′ 5′-CAA CTT CAT CCA CGT TCA CC-3′
P1-16 5′-CGGACAGAGCCCATTACAAT-3′
P1-18 5′-TAGAAAGCTCAGCAGACGACC-3′
P3 5′-GACTCGAGTCGACATCG-3′
P2-16 5′-CTTTTTGTTGCAAGTGTGACTCTACG-3′
P2-18 5′-ACGACCTTCGAGCATTCCAGCAG-3′
(dt)17-P3- 5′GACTCGAGTCGACATCGATTTTTTTTTTTTTTTTT-3′
Al-Shabanah et al. Virology Journal 2013, 10:343 Page 7 of 9http://www.virologyj.com/content/10/1/343
When the samples contain more than one HPV genotype,direct sequencing gave mixed chromatograms, with over-lapping peaks or two or more fluorescent signals. Sampleswith mixed chromatogram were subsequently subjected totype-specific PCR. The nucleotide sequences were sub-sequently subjected to Basic Local Alignment Search(BLAST) provided by the National Cancer Institute,USA.
StatisticsChi-square test used to test prevalence differences in HPVgenotypes and integration frequencies between differentHPV types in cancerous tissues and NAT in relation toage, tumor stage and tumor grade. A p value of < 0.05 wasconsidered statistically significant. SPSS, version 17.0 wasused for these analyses.
Competing interestsThe authors declare that they have no competing interests.
Authors’ contributionsMMH and ZK shared in molecular studies and their statistical analysis anddraft the manuscript. MMH, OA, MS, WN, SM and AA participated in studydesign, sample and data collection and shared in revising the manuscript. SSshared in sample and data collections. All authors read and approved thefinal manuscript.
AcknowledgmentThe present work was supported by operating grant from the KingAbdul-Aziz City for Science and Technology (Grand Number AT-30-26).
Author details1Department of pharmacology, College of pharmacy; King Saud University,P.O. Box 2457, Riyadh 11451, Kingdom of Saudi Arabia. 2Zoology department,Faculty of science, King Saud University, Riyadh, Kingdom of Saudi Arabia.3Medical Oncology Unit; King Khalid University Hospital; King SaudUniversity, Riyadh, Kingdom of Saudi Arabia. 4Pathology department, Collegeof Medicine, King Saud University, Riyadh, Kingdom of Saudi Arabia.
Received: 17 June 2013 Accepted: 8 November 2013Published: 20 November 2013
References1. zur Hausen H: Papillomavirus infections–a major cause of human cancers.
Biochim Biophys Acta 1996, 1288(2):F55–78.2. El-All HS, Refaat A, Dandash K: Prevalence of cervical neoplastic lesions
and human papilloma virus infection in egypt: national cervical cancerscreening project. Infect Agent Cancer 2007, 2:12.
3. Melo A, Montenegro S, Hooper T, Capurro I, Roa JC, Roa I: Humanpapilloma virus (HPV) typing in preneoplastic and neoplastic lesionsof the uterine cervix in the IX region-Chile. Revista Med Chil 2003,131(12):1382–1390.
4. Wu QJ, Guo M, Lu ZM, Li T, Qiao HZ, Ke Y: Detection of humanpapillomavirus-16 in ovarian malignancy. Br J Cancer 2003, 89(4):672–675.
5. Bosch FX, Lorincz A, Munoz N, Meijer CJ, Shah KV: The causal relationbetween human papillomavirus and cervical cancer. J Clin Pathol 2002,55(4):244–265.
6. Bell MC, Schmidt-Grimminger D, Patrick S, Ryschon T, Linz L, Chauhan SC:There is a high prevalence of human papillomavirus infection inAmerican Indian women of the northern plains. Gynecol Oncol 2007,107(2):236–241.
7. Vaccarella S, Franceschi S, Clifford GM, Touze A, Hsu CC, de Sanjose S, PhamTH, Nguyen TH, Matos E, Shin HR, et al: Seroprevalence of antibodiesagainst human papillomavirus (HPV) types 16 and 18 in four continents:the international agency for research on cancer HPV prevalence surveys.Cancer Epidemiol Biomarkers Prev 2010, 19(9):2379–2388.
8. Al-Badawi IA, Al-Suwaine A, Al-Aker M, Asaad L, Alaidan A, Tulbah A, FeBohol M, Munkarah AR: Detection and genotyping of human papillomavirus in cervical cancer specimens from Saudi patients. Int J GynecolCancer 2011, 21(5):907–910.
9. Chong PP, Asyikin N, Rusinahayati M, Halimatun S, Rozita R, Ng CK, Hamilton WH,Tan BC, Noraihan N, Rohani A, et al: High prevalence of human papillomavirusDNA detected in cervical swabs from women in southern Selangor, Malaysia.Asian Pac J Cancer Prev 2010, 11(6):1645–1651.
10. Depuydt CE, Boulet GA, Horvath CA, Benoy IH, Vereecken AJ, Bogers JJ:Comparison of MY09/11 consensus PCR and type-specific PCRs in thedetection of oncogenic HPV types. J Cell Mol Med 2007, 11(4):881–891.
11. Lee SH: Detection of human papillomavirus (HPV) L1 gene DNA possiblybound to particulate aluminum adjuvant in the HPV vaccine gardasil.J Inorg Biochem 2012, 117:85–92.
12. Devolder K, Amant F, Neven P, van Gorp T, Leunen K, Vergote I: Role ofdiaphragmatic surgery in 69 patients with ovarian carcinoma. Int JGynecol Cancer 2008, 18(2):363–368.
13. Fishman DA, Bozorgi K: The scientific basis of early detection of epithelialovarian cancer: the national ovarian cancer early detection program(NOCEDP). Cancer Treat Res 2002, 107:3–28.
14. Piek JM, van Diest PJ, Verheijen RH: Ovarian carcinogenesis: an alternativehypothesis. Adv Exp Med Biol 2008, 622:79–87.
15. Al-Eid MSM HS, Shouki B, Ali A-Z: Cancer incidence and survival reportSaudi Arabia. 2007. http://www.scr.org.sa/reports/SCR2007.pdf.
16. Dilek FH, Sahin O, Tokyol C, Mazlum M, Aycicek A: Expression ofcyclooxygenase-1 and 2 in chronic tonsillitis. Indian J Pathol Microbiol2010, 53(3):451–454.
17. Shih Ie M, Kurman RJ: Ovarian tumorigenesis: a proposed model basedon morphological and molecular genetic analysis. AmJ Pathol 2004,164(5):1511–1518.
18. Bell DA: Origins and molecular pathology of ovarian cancer. Mod Pathol2005, 18(2):S19–32.
19. Modugno F: Ovarian cancer and high-risk women-implications for prevention,screening, and early detection. Gynecol Oncol 2003, 91(1):15–31.
20. Falaki F, Amir Chaghmaghi M, Pakfetrat A, Delavarian Z, Mozaffari PM,Pazooki N: Detection of human papilloma virus DNA in seven cases offocal epithelial hyperplasia in Iran. J Oral Pathol Med 2009, 38(10):773–776.
21. Liao Y, Zhou Y, Guo Q, Xie X, Luo E, Li J, Li Q: Simultaneous detection,genotyping, and quantification of human papillomaviruses by multicolor real-time PCR and melting curve analysis. J Clin Microbiol 2013, 51(2):429–435.
22. Cherne S, Popov V, Feng Q: Protocol for the detection and genotyping ofhuman papillomaviruses using a liquid bead microarray assay. MethodsMol Biol 2012, 903:205–223.
23. Lee HP, Kim SO, Hwang TS, Bae JM, Kim SN, Kim JW, Hwang SY, Lee HS, Shin SK,Cho W, et al: Analytical and clinical performances of a restriction fragmentmass polymorphism assay for detection and genotyping of a wide spectrumof human papillomaviruses. J Med Virol 2011, 83(3):471–482.
24. Ip SM, Wong LC, Xu CM, Cheung AN, Tsang PC, Ngan HY: Detection of humanpapillomavirus DNA in malignant lesions from Chinese women withcarcinomas of the upper genital tract. Gynecol Oncol 2002, 87(1):104–111.
25. Kawano Y, Kypta R: Secreted antagonists of the Wnt signalling pathway.J Cell Sci 2003, 116(Pt 13):2627–2634.
26. Su HY, Lai HC, Lin YW, Chou YC, Liu CY, Yu MH: An epigenetic markerpanel for screening and prognostic prediction of ovarian cancer. Int JCancer 2009, 124(2):387–393.
27. Darwich L, Canadas MP, Sirera G, Alameda F, Forcada P, Delas J, Fernandez I,Llatjos M, Coll J, Clotet B, et al: Human papillomavirus genotypedistribution and human papillomavirus 16 and human papillomavirus 18genomic integration in invasive and in situ cervical carcinoma in humanimmunodeficiency virus-infected women. Int J Gynecol Cancer 2011,21(8):1486–1490.
28. Gauger KJ, Hugh JM, Troester MA, Schneider SS: Down-regulation of sfrp1in a mammary epithelial cell line promotes the development of acd44high/cd24low population which is invasive and resistant to anoikis.Cancer cell Int 2009, 9:11.
29. Smith JS, Herrero R, Bosetti C, Munoz N, Bosch FX, Eluf-Neto J, CastellsagueX, Meijer CJ, Van den Brule AJ, Franceschi S, et al: Herpes simplex virus-2 asa human papillomavirus cofactor in the etiology of invasive cervicalcancer. J Natl Cancer Inst 2002, 94(21):1604–1613.
30. Munoz N, Franceschi S, Bosetti C, Moreno V, Herrero R, Smith JS, Shah KV,Meijer CJ, Bosch FX: Role of parity and human papillomavirus in cervical
Al-Shabanah et al. Virology Journal 2013, 10:343 Page 8 of 9http://www.virologyj.com/content/10/1/343
cancer: the IARC multicentric case–control study. Lancet 2002,359(9312):1093–1101.
31. Xue Y, Bellanger S, Zhang W, Lim D, Low J, Lunny D, Thierry F: HPV16 E2 is animmediate early marker of viral infection, preceding E7 expression inprecursor structures of cervical carcinoma. Cancer Res 2010, 70(13):5316–5325.
32. Das P, Thomas A, Mahantshetty U, Shrivastava SK, Deodhar K, Mulherkar R:HPV genotyping and site of viral integration in cervical cancers in Indianwomen. PloS one 2012, 7(7):e41012.
33. zur Hausen H: Papillomaviruses in the causation of human cancers - abrief historical account. Virology 2009, 384(2):260–265.
34. Syrjanen KJ: HPV infections and oesophageal cancer. J Clin Pathol 2002,55(10):721–728.
35. Rezazadeh A, Laber DA, Ghim SJ, Jenson AB, Kloecker G: The role of humanpapilloma virus in lung cancer: a review of the evidence. Am J Med Sci2009, 338(1):64–67.
36. Lee YM, Leu SY, Chiang H, Fung CP, Liu WT: Human papillomavirus type18 in colorectal cancer. J Microbiol Immunol Infect 2001, 34(2):87–91.
37. Damin AP, Karam R, Zettler CG, Caleffi M, Alexandre CO: Evidence for anassociation of human papillomavirus and breast carcinomas. BreastCancer Res Treat 2004, 84(2):131–137.
38. Kaufman RH, Bornstein J, Gordon AN, Adam E, Kaplan AL, Adler-Storthz K:Detection of human papillomavirus DNA in advanced epithelial ovariancarcinoma. Gynecol Oncol 1987, 27(3):340–349.
39. Atalay F, Taskiran C, Taner MZ, Pak I, Or M, Tuncer S: Detection of humanpapillomavirus DNA and genotyping in patients with epithelial ovariancarcinoma. J Obstet Gynaecol Res 2007, 33(6):823–828.
40. Giordano G, D’Adda T, Gnetti L, Froio E, Merisio C, Melpignano M: Role ofhuman papillomavirus in the development of epithelial ovarianneoplasms in Italian women. J Obstet Gynaecol Res 2008, 34(2):210–217.
41. Kuscu E, Ozdemir BH, Erkanli S, Haberal A: HPV and p53 expression inepithelial ovarian carcinoma. Eur J Gynaecol Oncol 2005, 26(6):642–645.
42. Leake JF, Woodruff JD, Searle C, Daniel R, Shah KV, Currie JL: Humanpapillomavirus and epithelial ovarian neoplasia. Gynecol Oncol 1989,34(3):268–273.
43. Quirk JT, Kupinski JM, DiCioccio RA: Analysis of ovarian tumors for thepresence of human papillomavirus DNA. J Obstet Gynaecol Res 2006,32(2):202–205.
44. Varsani A, Williamson AL, de Villiers D, Becker I, Christensen ND, Rybicki EP:Chimeric human papillomavirus type 16 (HPV-16) L1 particles presentingthe common neutralizing epitope for the L2 minor capsid protein ofHPV-6 and HPV-16. J Virol 2003, 77(15):8386–8393.
45. Gravitt PE, Peyton CL, Alessi TQ, Wheeler CM, Coutlee F, Hildesheim A,Schiffman MH, Scott DR, Apple RJ: Improved amplification of genitalhuman papillomaviruses. J Clin Microbiol 2000, 38(1):357–361.
46. Hubbard RA: Human papillomavirus testing methods. Arch Pathol Lab Med2003, 127(8):940–945.
47. Kosel S, Burggraf S, Mommsen J, Engelhardt W, Olgemoller B: Type-specificdetection of human papillomaviruses in a routine laboratory setting–improved sensitivity and specificity of PCR and sequence analysiscompared to direct hybridisation. Clin Chem Lab Med 2003, 41(6):787–791.
48. Pannier-Stockman C, Segard C, Bennamar S, Gondry J, Boulanger JC,Sevestre H, Castelain S, Duverlie G: Prevalence of HPV genotypesdetermined by PCR and DNA sequencing in cervical specimens fromFrench women with or without abnormalities. J Clin Virol 2008,42(4):353–360.
49. Lai CH, Hsueh S, Lin CY, Huang MY, You GB, Chang HC, Pao CC: Humanpapillomavirus in benign and malignant ovarian and endometrialtissues. Int J Gynecol Pathol 1992, 11(3):210–215.
50. Sana'a MH Alizi FAM, Abdul-Majeed BA: Detection of humanpapillomavirus in surface epithelial ovarian carcinoma using in situhybridization technique. Fac Med Baghdad 2012, 54(1):54–62.
51. Giordano G, D’Adda T, Gnetti L, Merisio C, Melpignano M: Endometrialmucinous microglandular adenocarcinoma: morphologic,immunohistochemical features, and emphasis in the humanpapillomavirus status. Int J Gynecol Pathol 2006, 25(1):77–82.
52. Shukla S, Bharti AC, Mahata S, et al: Infection of human papillomavirusesin cancers of different human organ sites. Indian J Med Res 2009,130(3):222–233.
53. Powell JL, Bock KA, Gentry JK, White WC, Ronnett BM: Metastaticendocervical adenocarcinoma presenting as a virilizing ovarian massduring pregnancy. Obstet Gynecol 2002, 100(5 Pt 2):1129–1133.
54. Plaza JA, Ramirez NC, Nuovo GJ: Utility of HPV analysis for evaluation ofpossible metastatic disease in women with cervical cancer. Int J GynecolPathol 2004, 23(1):7–12.
55. Heinzel PA, Chan SY, Ho L, O’Connor M, Balaram P, Campo MS, Fujinaga K,Kiviat N, Kuypers J, Pfister H, et al: Variation of human papillomavirus type6 (HPV-6) and HPV-11 genomes sampled throughout the world. J ClinMicrobiol 1995, 33(7):1746–1754.
56. Scheurer ME, Tortolero-Luna G, Adler-Storthz K: Human papillomavirusinfection: biology, epidemiology, and prevention. Int J Gynecol Cancer2005, 15(5):727–746.
57. Malisic E, Jankovic R, Jakovljevic K: Detection and genotyping of humanpapillomaviruses and their role in the development of ovariancarcinomas. Arch Gynecol Obstet 2012, 286(3):723–728.
58. von Knebel DM, Oltersdorf T, Schwarz E, Gissmann L: Correlation ofmodified human papilloma virus early gene expression with alteredgrowth properties in C4-1 cervical carcinoma cells. Cancer Res 1988,48(13):3780–3786.
59. von Knebel DM, Rittmuller C, Aengeneyndt F, Jansen-Durr P, Spitkovsky D:Reversible repression of papillomavirus oncogene expression in cervicalcarcinoma cells: consequences for the phenotype and E6-p53 andE7-pRB interactions. J Virol 1994, 68(5):2811–2821.
60. von Knebel Doeberitz M, Bauknecht T, Bartsch D, Zur Hausen H: Influenceof chromosomal integration on glucocorticoid-regulated transcription ofgrowth-stimulating papillomavirus genes E6 and E7 in cervicalcarcinoma cells. Proc Natl Acad Sci U S A 1991, 88(4):1411–1415.
61. Cullen AP, Reid R, Campion M, Lorincz AT: Analysis of the physical state ofdifferent human papillomavirus DNAs in intraepithelial and invasivecervical neoplasm. J Virol 1991, 65(2):606–612.
62. Ronco G, Franceschi S, Segnan N: HPV16 and HPV18 genotyping incervical cancer screening. Lancet Oncol 2011, 12(9):831–832.
63. Botezatu A, Socolov D, Goia CD, Iancu IV, Ungureanu C, Huica I, Anton G:The relationship between HPV16 and HPV18 viral load and cervicallesions progression. Roum Arch Microbiol Immunol 2009, 68(3):175–182.
64. Klaes R, Woerner SM, Ridder R, Wentzensen N, Duerst M, Schneider A,Lotz B, Melsheimer P, von Knebel DM: Detection of high-risk cervicalintraepithelial neoplasia and cervical cancer by amplification oftranscripts derived from integrated papillomavirus oncogenes. Cancer Res1999, 59(24):6132–6136.
65. Arias-Pulido H, Peyton CL, Joste NE, Vargas H, Wheeler CM: Humanpapillomavirus type 16 integration in cervical carcinoma in situ and ininvasive cervical cancer. J Clin Microbiol 2006, 44(5):1755–1762.
66. Qu W, Jiang G, Cruz Y, Chang CJ, Ho GY, Klein RS, Burk RD: PCR detectionof human papillomavirus: comparison between MY09/MY11 and GP5+/GP6+ primer systems. J Clin Microbiol 1997, 35(6):1304–1310.
67. Sotlar K, Diemer D, Dethleffs A, Hack Y, Stubner A, Vollmer N, Menton S,Menton M, Dietz K, Wallwiener D, et al: Detection and typing of humanpapillomavirus by e6 nested multiplex PCR. J Clin Microbiol 2004,42(7):3176–3184.
doi:10.1186/1743-422X-10-343Cite this article as: Al-Shabanah et al.: Human papillomavirusgenotyping and integration in ovarian cancer Saudi patients. VirologyJournal 2013 10:343.
Submit your next manuscript to BioMed Centraland take full advantage of:
• Convenient online submission
• Thorough peer review
• No space constraints or color figure charges
• Immediate publication on acceptance
• Inclusion in PubMed, CAS, Scopus and Google Scholar
• Research which is freely available for redistribution
Submit your manuscript at www.biomedcentral.com/submit
Al-Shabanah et al. Virology Journal 2013, 10:343 Page 9 of 9http://www.virologyj.com/content/10/1/343
REGARD
