TESTICULAR TUMORS-

Biomarkers and staging

Dr Rajesh Kumar

ATRCTRI

BIKANER

DICTUM FOR ANY SOLID SCROTAL SWELLINGS

All patients with a solid, Firm Intratesticular Mass that cannot be Transilluminated should be regarded as Malignant unless

otherwise proved.

Important facts about testicular tumors in Asian countries

Uncommon, incidence: 1/100,000 men

1% of all malignancies in men

Peak: 30-40 years, rare in prepubertal children & elderly

>95% are of germ cell origin.

Serum tumor markers found in 50% of patients. Eg: AFP, hCG

In 2013 our institute, 43 new case of testicular tumors registered.

Total Male cancer patients: 4118, thus 1.04% of all male patients.

I. Primary Neoplasma of Testis.

A. Germ Cell Tumour

B. Non-Germ Cell Tumour

II. Secondary Neoplasms.

III. Paratesticular Tumours.

A. Germinal Neoplasms : (90 - 95 %)

1. Seminomas - 40%

(a) Classic Typical Seminoma

(b) Anaplastic Seminoma

(c) Spermatocytic Seminoma

2. Embryonal Carcinoma - 20 - 25%

3. Teratoma - 25 - 35%

(a) Mature

(b) Immature

4. Choriocarcinoma - 1%

5. Yolk Sac Tumour

B. Nongerminal Neoplasms : ( 5 to 10% )

1. Specialized gonadal stromal tumor

(a) Leydig cell tumor

(b) Other gonadal stromal tumor

2. Gonadoblastoma

3. Miscellaneous Neoplasms

(a) Adenocarcinoma of the rete testis

(b) Mesenchymal neoplasms

(c) Carcinoid

(d) Adrenal rest “tumor”

A. Adenomatoid

B. Cystadenoma of Epididymis

C. Mesenchymal Neoplasms

D. Mesothelioma

E. Metastases

II. SECONDARY NEOPLASMS OF TESTIS

A. Reticuloendothelial Neoplasms

B. Metastases

III. PARATESTICULAR NEOPLASMS

Staging A or I - Tumour confined to testis.

Staging B or II - Spread to Regional nodes.

IIA - Nodes <2 cm in size or ≤ 5 Positive Nodes

IIB - 2 to 5 cm in size or > 5 Positive Nodes

IIC - Large, Bulky, abdominal mass usually > 5 to 10 cm

Staging C or III - Spread beyond retroperitoneal Nodes or Above Diaphragm or visceral disease

Diagnostic Work-up For Tumors of the Testis

A complete history should be taken, including information about previous

inguinal or scrotal surgery for cryptorchidism, retractile testes, and

orchiopexy.

Specifically, the abdomen should be examined to rule out the presence of

large abdominal masses, and both supraclavicular regions should be

palpated to rule out supraclavicular metastases.

The contra lateral testis should be examined clinically.

The presence or absence of gynecomastia is an important observation

Presentation Painless swelling/mass with or without hydrocele (5-10%)

30-40% report dull/aching sensation

10% present with metastatic symptoms

More common on the right Neck Mass / Cough / Anorexia / Vomiting / Back Ache/ Lower limb swelling

Gynecomastia

5% germ cell

30-50% Sertoli/Leydig

1-2% have bilateral disease at diagnosis

Cutaneous atypical nevi. It has been claimed that multiple cutaneous atypical nevi occur with increased frequency in patients with testicular germ cell tumors and that they could represent a marker for this disease.

Bilaterality Bilateral testicular involvement by germ cell tumors is seen

in 1.0–2.7% of the cases according to the different series.

The risk of bilaterality rises to 15% if both testes are undescended.

The most common situation is bilateral spermatocytic or classic seminoma.

In the presence of bilateral testicular tumors in an elderly individual, the most likely diagnosis is malignant lymphoma

Telomerase activity is present in all types of testicular germ cell tumors except for mature teratomas.

Spermatocytic seminoma shows completely different genetic features. Isochromosome 12p is not found.

Numerical chromosomal aberrations are common, and gain of chromosome 9 is characteristic.

Symptoms a painless enlargement of

the testis

usually gradual, and a sensation of testicular heaviness .

typical delay in treatment from initial recognition ofthe lesion by the patient to definitive therapy (orchiectomy) ranges from 3 to 6 months.

Signs testicular mass or diffuse

enlargement

typically firm and nontender and theepididymis should be easily separable from it

Hydrocele may accompany the testicular tumor and help to camouflage it

1. Ultrasound - Hypoechoic area

2. Chest X-Ray - PA and lateral views

3. CT Scan

4. MRI

5. PET CT

6. Tumour Markers

- AFP

- HCG

- LDH

- PLAP

Laboratory

Anemia may be detected in

advanced disease.

Liver function-may be elevated

Renal function may be diminished

(elevated serum creatinine) if

ureteral obstruction secondary to

bulky retroperitonealdisease is

present.

The assessment of renal function

(creatinine clearance) is mandatory

in patients with advanced disease

who require chemotherapy.

AFP Found in several NSGCT

Rarely in Seminomas

hCG Elevated in NSGCT

7% Seminomas

LDH

Elevation of total serum LDH and in particular isoenzyme-I

was shown to correlate with tumor burden in NSGCTs.

LDH may also be elevated in seminoma.

Imaging Ultrasonography scrotum-

1.Scrotal swelling

2. without s swelling-Young male with RPLN or visceral mass, increased AFP/HCG, infertility.

determine whether the mass

is truly intratesticular, can be

used to distinguish the tumor

from epididymal pathology,

and may also facilitate

testicular examination in the

presence of a hydrocele.

Testicular microlithiasis Associated with increased risk of testicular in situ

carcinoma(TIN).

Most common in GCT & their family members.

Need further investigations( biopsy or monitoring

) if microlithiasis is-

in b/l testes,

in subfertile male,

in c/l of GCT testes

Atrophy( vol≤ 12 ml)

inhomogeneous parenchyma

Once the diagnosis of testicular cancer has been established by inguinal orchidectomy, careful clinical staging of disease is mandatory.

Chest radiographs (postero-anterior and lateral) and computed tomography (CT scan) of the abdomen and pelvis are used to assess the 2 most common sites of metastatic spread, namely, the lungs and retroperitoneum.

Diagnostic Radiology

Computed tomography (CT) scan of

abdomen and pelvis :- to identify

metastatic involvement above and below

the diaphragm.

Sensitivity-40%, specificcity-95%

CT scan of chest for nonseminomas and

stage II seminomas.

lymph node SIZE- 1 to 2 cm in a

primary landing zone are involved by

GCT -70%

If 0.4 to 1 cm positive in -50%

Magnetic resonance imaging appears equivalent to CT in determining the size and location of retroperitoneal adenopathy. MRI of the scrotum offers a sensitivity of 100% and a specificity of 95-100%, but its high

cost does not justify its use for diagnosis.

Positron emission tomography (PET) has been shown to improve on the diagnostic accuracy of CT in early stage testicular cancer. It has a higher sensitivity (70%) and specificity (up to 100%) than CT . It is unable to detect lesions <5 mm in size or teratomas of any size due to their very low metabolic activity. PET is useful in the detection of residual viable seminoma in patients with masses greater than 3 cm in diameter after chemotherapy.

Special Studies:-

Bipedal lymphangiography has a greater sensitivity than

CT (71%) but a lower specificity (60%). It does not add to the

diagnostic accuracy of a positive CT, but is able to

demonstrate architectural abnormalities within normal-size

lymph nodes.

Pulmonary function test-

Semen analysis and banking of sperm

Differential DiagnosisTorsion

Epididymitis

Epididimo-orchitis

Hydrocele

Hernia

Haematoma

Spermatocele

Syphilitic gumma

Classification of Germ-Cell Tumors of the Testis

Intratubular germ-cell neoplasia

Seminoma :a) Classic type b) Spermatocytic type

Nonseminomatous germ-cell tumorsa) Embryonal carcinomab) Yolk sac (endodermal sinus) tumorc) Teratoma: Mature, Immature, Teratoma with malignant transformation, d) Choriocarcinomae) Mixed germ-cell tumors

Other than GCT :- Sex-Cord Stromal Tumors

Classification of Sex-Cord Stromal Tumors

a) Leydig cell tumorb) Sertoli cell tumorc) Granulosa cell tumord) Fibroma-thecoma stromal tumore) Sex cord-stromal tumor with annular tubulesf) Gonadoblastomag) Sex cord-stromal tumor unclassified type

95% germ cell tumours – malignant, but curable

Seminomas (40%)

Nonseminomatous germ cell tumors (NSGCT) (40%)

15% of tumors have both seminomatous & nonseminomatous elements.

Nonseminomatous elements are more malignant, therefore such tumors are clinically treated as NSGCT.

5% non-germ cell (aka sex cord stromal) tumours –usually benign, sometimes presenting hormonally.

Diagram showing relationships between various types of germ cell tumors

Seminoma Nonseminoma Age of presentation

• 3rd and 4th decade

Presentation

• present with uniform testicular swelling

Serum markers are rarely elevated. AFP never rises.

Lymphatic spread

• Good prognosis

• 2nd and 3rd decade

• present with multinodular testicular swelling

• Serum markers are commonly elevated.

• Lymphatic as well as hematogenous

• Worse prognosis

Dixon and Moore classification

Based on histology & has prognostic importance &

described in 1952

Group-1 seminoma pure.

Group-2 embryonal carcinoma pure, or with seminoma.

Group-3 teratoma pure, or with seminoma.

Group-4 teratoma with embryonal ca. and/ or

choriocarcinoma with or without seminoma.

Group-5 choriocarcinoma pure, or with embryonal

carcinoma and or seminoma.

For the purpose of treatment planning GCT may be

divided into 2 categories.

1 -pure seminoma

2 -all other histologic types(group 2 to 5), thereafter

referred to as

Mixed tumors

Immunochemical markers CD117 :- Seminoma, negative in

embryonal carcinoma

Loss of cKit asso. With more aggressive

phenotype.

SOX2 and CD30 :- Embryonal

carcinoma, negative in seminoma.

OCT ¾- positive in seminoma &

embryonal carcinoma

Placental alkaline phosphatase (PLAP) is

positive in a membranous pattern in up to

98% of seminomas.

SEMINOMA IMMUNOHISTO CHEMISTRY

Cells are OCT4+ve,

PLAP +ve, &

c-kit +ve

Contains cytokeratins, although only

36 % cases are +ve

EMA -ve

SPERMATOCYTIC SEMINOMA IMMUNOHISTO CHEMISTRY

Cells are PLAP –ve,

vimentin –ve,

muscle marker –ve,

cytokeratin –ve, AFP –ve,

HCG –ve,

EMA –ve

NY-ESO 1 +ve

SCP-1 +ve

EMBRYONAL CARCINOMA

IMMUNOHISTO CHEMISTRY

Tumor cells are CD 30 +ve, a finding unique to Embryonal carcinoma, and useful in ruling out solid pattern of Embryonal carcinoma, which can simulate Seminoma .

OCT 4 +ve,

PLAP +ve,

cytokeratin +ve,

c-kit –ve, and EMA -ve

YOLK SAC TUMOR IMMUNOHISTO CHEMISTRY

AFP + ( focal or patchy ),

cytokeratin +ve,

PLAP variable,

EMA –ve, CD 30 -ve

SERTOLI CELL TUMOR IMMUNOHISTO CHEMISTRY

Inhibin –+ve, but less consistently than in leydig cell

tumor & can be +ve with chromogranin,

S-100 proteins, synaptophysin, and cytokeratin AE1/3 &

CAM 5.2 in 64-100 % cases

MIS & CD99 +ve

IHC OF TESTICULAR GERM CELL TUMORSSeminoma Spermato.

SeminomaEmbryonalcarcinoma

Yolk sac tumor

Teratoma Choriocarcinoma

OCT-4 + - + - - -CD117 + -/+ - - - -CK -/+ - + + + +VIMENTIN + - - + + -PLAP + - + + + +AFP - - + + + +HCG + - + - + +CD30 + - + - - -PAS + - - + - -

Testicular Tumour & Molecular Biology

Seminoma & Embryonal - N-myc expressionCarcinoma

Seminoma - c-ras expression

ImmatureTeratomas - c-erb expression

PROTO-ONCOGENES in Germ Cell Tumours (Shuin et al)

Testicular Tumor & Molecular Biology (Recent Advances)

Testicular germ cell tumor show consistent expression of both:

Parental alleles of H19

IGF-2 genes.

Definition:

Biomarker is a substance used as an indicator of a biologic state

Existence of living organisms or biological process. A particular disease stateA fragment of DNA sequence

Proteins

Nucleic acids

Carbohydrate

Lipids

Detection of biomarker

Detection of biomarkerQuantitative

a link between quantity of the marker and disease

Qualitativea link between exist of a marker and disease

Detection of biomarker – diagnosise.g enzymatic activitiesAntibodies, IHC, ELISA

Enzymes –PSA - prostate specific antigen (serine protease), NSE (neurospecific isoenzyme of enolase), TK (thymidine kinase), LDH

Hormones - GH, prolactin, calcitonin, parathormon (PTH), gastrin, hCG

Imunoglobulines – IgG, IgM, IgA, IgD, IgE, 2-microglobulin

Glycoproteines, :- AFP, beta-hCG, CA 125

Glycolipides:- CA 19-9

Tumor markers are

Testicular tumorsTWO MAIN CLASSES

Onco-fetal Substances : AFP & HCG

Cellular Enzymes : LDH & PLAP

Hormones : Testosterone, estradiol,

Androstenidione, Inhibin

( AFP and b-HCG Trophoblastic Cells)

Common Serum Markers for Cancer Diagnosis/prognosis

AFP CEA CA15-3 CA19-9 CA125 PSA PLAP hTG HCGb NSE B2M LDH

Lung x x x x x x

Pancreas x x x x x

Kidney x x x x

Breast x x x

Ovarian x x x x x x

Cervical x x

Uterine x x x x

Prostate x x

Liver x x x x x

Gastro x x x

Colon x x x x

Bladder x

Lymphom

a

x

Myeloma x

Thyroid x x

Testicular x x x x

Common Biomarker for testicular CancerAFP (10-15 ng/ml)

Alpha-fetoprotein (AFP) is the major protein of fetal serum but falls to

an undetectable level after birth.

A glycoprotein, MW 70.000, discovered in 1956 in foetal serum

described as a tumour-associated protein in 1964 synthesised in the liver

and yolk sac of the foetus.

Because of the association of the rapid cell growth, this fetal protein is

also used as a tumor marker.

AFP is secreted into serum, reaching maximum levels at week 13 of

pregnancy. (Particularly very high if the pregnancy is complicated by a spinal cord defect or other

abnormality)

Normal adult concentration less than 15 ng/ml. Normal half life is 5 to 7 days.

The primary malignancies associated with AFP elevations are

Hepatocellular carcinoma

Nonseminomatous germ cell tumor:-

Pure embryonal carcinoma, Teratocarcinoma ,Yolk sac Tumor

Other gastrointestinal cancers occasionally (but rarely to greater than 1,000 ng

per mL)

AFP levels are abnormal in 80 percent of patients with hepatocellular carcinoma

and exceed 1,000 ng / mL in 40 percent of patients with this cancer.

An AFP level above 500 ng /mL is often used in lieu of biopsy to diagnose HCC.

Seminomas and Dysgerminomas are always AFP-negative

AFP level, (together with hCG level), is established regimen for monitoring

patients with non-seminomatous.

Non cancerous diseases Cirrhosis Viral hepatitis Marijuana abuser(although usually less than 500 ng/mL)

AFP rise in: Non seminomatous germ cell tumor . Direct relationship exists between AFP level and disease stage of non-seminomatous testicular carcinoma.

Stage I - 10-20%

Stage II – 20% - 40%

Stage III – 40 – 60%

AFP and b-HCG Levels in Germ Cell Tumors and Gestational TrophoblasticDisease

Tumor AFP elevation hCG elevation

Seminoma and dysgerminoma Never Occasional, minimal

Embryonal cell carcinoma Yes Yes

Choriocarcinoma No Yes

Yolk sac tumors Yes No

Teratoma No No

Gestational trophoblastic disease No Yes

AFP values in excess of 10,000 ng/mL or hCG levels above 50,000 mIU per mLat initial diagnosis portend a poor prognosis, with a five-year survival rate of 50percent. Similarly staged patients with lower AFP and -hCG levels have a cure rate higher than 90 percent.

Use of AFP or beta-hCG elevation

Recurrence and assess response to treatment.

HCG (10 mIU/ml ) The hormone HCG is secreted at the time of implantation by the syncytiotrophoblast cells (which later becomes the placenta), when the embryo attaches to the uterine wall.

Main role : to maintain the corpus luteum (whose role is to secrete progesterone) which is necessary for implantation of the embryo.

The protein can be detected in serum or urine.

Half life of HCG is 18 - 36 hours.

beta HCG promotes the maintenance of the corpus luteum during the beginning of pregnancy, causing it to secrete the hormone progesterone. Progesterone enriches the uterus with a thick lining of blood vessels and capillaries so that it can sustain the growing foetus.

This is first hormone that detects pregnancy as it is present in the blood about ten days after fertilization, and in the urine a few days later.

The most sensitive, accurate and reliable pregnancy test is a blood test for the presence of beta HCG .

Beta HCG levels vary according to the gestational age.

Non-malignant elevations may be observed in pregnancy and cirrhosis. Levels of

HCG are useful in monitoring the effectiveness of treatment.

Beta-HCG are typically about 100 mIU/ml 14 days after ovulation in a healthy

singleton pregnancy. They should double every 48- 72 hours in a healthy pregnancy.

The test for HCG to diagnose pregnancy, as well as other problems of pregnancy is

done by determining the levels of β-subunit of hCG.

The HCG hormone has 2 sub units- namely the ALPHA AND BETA sub-units.

The alpha sub-unit has components identical to that of luteinizing hormone

(LH), follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH)

The beta subunit is unique to only to hCG.

Importance : Confirmation of pregnancy

Tumor marker

Human chorionic gonadotropin can be used as a tumor marker, as its β subunit is secreted by some cancers.

The normal range for men is between 0-5 mIU/mL. Combined with alpha-fetoprotein, β-HCG is an excellent tumor marker for the monitoring of germ cell tumors.

The -hCG level is used to diagnose gestational trophoblastic disease, a rare neoplastic complication of pregnancy.

Choriocarcinoma may produce high levels of βhCG (due to the presence of syncytialtrophoblasts- part of the villi that make up the placenta) despite the absence of an embryo.

Non seminomatous germ cell tumor

Stage I - 10-20%

Stage II – 20% - 30%

Stage III – 40%

Also increase in 15 – 20% of advanced pure seminoma.

Non cancerous conditions

abnormal pregnancies i.e. ectopic pregnancies and potential miscarriages.

It is also used as part of a screening test for Down Syndrome. Where it is decrease.

150,000 mIU/ml is strongly indicative of an ectopic pregnancy..

Multiple Pregnancy

Gestational trophoblastic disease like Hydatidiform moles ("molar pregnancy")

Lactate Dehydrogenase Increased in 60% of advanced NSGCT and in 80% of

advanced seminoma.

LDH level reflects tumor burden, growth rate and cellular proliferation.

Yolk Sac Tumor is almost invariably associated with production of large amounts of alpha-fetoprotein (AFP) and also alpha-1 antitrypsin.

To properly Stage Testicular Tumours following

are pre-requisites:

(a) Pathology of Tumour Specimen

(b) History

(c) Clinical Examination

(d) Radiological procedure - USG / CT /

MRI / Bone Scan

(e) Tumour Markers - HCG, AFP

Requirements for staging

Serum tumor markersLDH HCG

Miu/ml

AFP

Ng/ml

S0 N N N

S1 <1.5 x N < 5000 < 1000

S2 1.5-10x N 5000 to

50000

1000 to

10000

S3 >10x N > 50000 >10000

Prognostic factors in testicular cancer(EAU)

For seminoma For non-seminoma

Pathological (for stage I)

Histopathological type •

Tumour size (> 4 cm)

• Invasion of the rete testis

• Vascular/lymphatic in or peri-tumoural invasion

• Proliferation rate > 70%

• Percentage of embryonal carcinoma > 50%

Clinical (for metastatic disease)

• Primary location

• Elevation of tumour marker levels

• Presence of non-pulmonary visceral metastasis

According to CMR invasion of testicular veins or lymphatics,

absence of yolk sack elements, and presence of embryonal cell

carcinoma.

Q 1. With respect to clinical staging of germ cell tumors of the testis, which of the following statements is incorrect?

A. Modern staging techniques have reduced the false-negative staging error in clinical staging of T1N0M0 to approximately 20%

B. Approximately 10-15% of patients with clinical stage T1N0M0 seminoma harbor occult retroperitoneal metastases

C. In general, 5% of patients with clinical stage I germ cell tumors harbor occult disease in extragonadal sites

D. Abdominal and pelvic MRI scans have a significant advantage over CT scans with respect to diagnosing micrometastatic disease.

E. Spermatic cord involvement increased the likelihood of metastatic involvement.

Answer: D

MRI offers no advantage over CT for imaging and staging the retroperitoneum in patients with testis cancer.

Q 2. In NSGCTs, all of the following prognostic factors

are used to determine risk of metastatic disease except

which one?

A. T stage

B. Embryonal cell carcinoma (>40%)

C. Teratoma (>50%)

D. Vascular invasion

E. Absence of yolk sac elements

Answer: CSix factors have been analyzed in many of these studies and include stage of the primary tumor (pT</= 2); vascular (including lymphatic) invasion; presence of embryonal carcinoma; absence of yolk sac elements; and elevated preorchiectomymarkers. In the Medical Research Council series, four were independently predictive of relapse; invasion of testicular veins or lymphatics, absence of yolk sack elements, and presence of embryonalcell carcinoma. Of the 259 patients, 55 patients had three or four factors and a relapse rate of 58%; 89 had two factors and a relapse rate of 24%; 81 had one factor and a relapse rate of 10%; and 8 patients had no factors and no relapses.

Q. 3. With respect to lymphatic drainage of the testis, which one of the following statements is correct?

A. The primary drainage of the right testis is usually located within the group of lymph nodes in the left para-aortic region.

B. The spermatic cord contains four to eight lymphatic channels that traverse the inguinal canal and peritoneal space.

C. The spermatic vessels cross dorsal to the ureter, whereas the testicular lymphatics cross ventrally.

D. Lymphatic drainage has been shown to cross over from right to left and therefore cross-metastasis occur more commonly in patients with right sided tumors.

E. Suprahilar lymph node spread is invariable in stage N1 disease.

Answer: D

Cross-metastases were reported to occur more commonly in patients with right sided tumors, because of lymphatic drainage from right to left. These observations have been important for the surgical management of testis cancer.

Remember: