Pre Operative nursing care

Assess patient. The health history and the physical and pelvic examinations are completed and the laboratory tests are performed.

Encourage patient to share details of her menstrual history, the date of her last menstrual period, the events leading up to admission and the current degree of vaginal blood loss or discharge.

Assess client’s knowledge of her condition and the surgery.

Perform skin operation: The lower half of the abdomen and the pubic and perineal area may be shaved and these areas may be cleaned with soap and water.

To prevent contamination and injury to the bladder or intestinal tract, the bladder and intestinal tract need to be empty before the patient is taken into the OR.

The patient who has previously been prescribed with oral contraceptive drug will have to stop taking the drug 6 weeks prior to operation.

Preoperative medications may be administered before surgery to help the patient relax.

The patient must be allowed time to talk and ask questions.

The nurse must know what information the physician has given the patient about the surgery.

Encourage patient to practice foot and leg exercises before operation to understand how to carry out the exercises while in bed after surgery.

Let the patient will wear anti-embolism socks to prevent venous stasis during the operation.

Provide education: Loss of fertility if ovaries are to be removed in conjunction with the operation. Discuss surgical menopause. Discuss how sexual intercourse may change. Client whose ovaries are removed may complain of a decrease in libido. Tell the client that once healing has occurred, intercourse should be pain free.

Let the patient relax on bed until she leaves the ward escorted by her nurse who completes a safe transfer to the operating theater staff.

Intra Operative Nursing Care

Prepare and assist for anesthesia.

Maintain homeostasis and asepsis.

Assist the surgeon and the whole team

Assist in transferring the patient to the Operating table in a supine position.

Ask patient to remove any jewelry or other objects that may interfere with the procedure.

Ask patient to remove clothing and be given a gown to wear.

Check for patency of the IV system.

Monitor client’s HR, BP and breathing and report abnormalities.

The skin over the surgical cite will be cleansed with an antiseptic solution

Post Op

Perform usual post operative assessments.

Evaluate psychological manifestations

Monitor proximity of the bladder to the reproductive organ.

Monitor Foley catheter to prevent susceptibility to UTI and temporary urinary retention

Assist GI functions by listening to bowel sounds.

Note distention and palpate whether abdomen is soft or firm

Assess abdominal incision for bleeding and intactness.

Assess vaginal bleeding.

There is no distinct diet. Simple, strong, distinct flavors rather than complicated and multi-flavored dishes seem to be preferred with anything with smaller-than-usual portions. It’s best to avoid gassy foods like beans, broccoli and cabbage and/or foods that typically cause gas for you. Many suggest avoiding extra-spicy foods. Remember that all pos top surgical patients need protein to aid in healing. Include fiber in your post op diet, drink lots of water, and consume caffeinated drinks sparingly.

If pain is experienced during sexual intercourse let the patient manipulate the penetration.

Avoid heavy lifting for about 6 weeks to prevent straining the abdominal muscles and surgical sites.

Avoid activities that increase pelvic congestion such as aerobics activity, horseback riding and prolonged standing.

Report any fresh bleeding and any abnormal vaginal discharge to surgeon.

Return for follow-up care as requested by the surgeon.

Post op pain and discomfort are common, therefore the nurse should assess it’s intensity and administer analgesics as prescribed.

If the patient has abdominal distention or flatus, rectal tube and application of heat to the abdomen may be prescribed

Encourage patient to contact nurse or surgeon when bleeding is excessive.

Encourage early ambulation o facilitate the return of normal peristalsis

Montior and manage potential complications such as: Hemorrhage: Count perineal pads used, assess the extent of saturation with blood and monitor vital

signs. Guidelines for activity restriction are given above to promote healing and prevent post operative bleeding.

Deep Vein Thrombosis: Encourage and assist patient to change position frequently and exercise leg and feet while in bed. Instruct patient to avoid prolonged sitting in the chair with pressure on the knees, sitting cross legs and inactivity.

Hysterectomy and Risk of Cardiovascular DiseaseA Population-Based Cohort StudyErik Ingelsson; Cecilia Lundholm; Anna L.V. Johansson; Daniel Altman

Authors and Disclosures

Posted: 04/29/2011; European Heart Journal. 2011;32(6):745-750. © 2011 Oxford University Press

Abstract and Introduction

Abstract

Aims Hysterectomy for benign indications is one of the commonest surgical procedures in women, but the association between the procedure and cardiovascular disease (CVD) is not fully understood. In this population-based cohort study, we studied the effects of hysterectomy, with or without oopherectomy, on the risk of later life CVD.Methods and results Using nationwide healthcare registers, we identified all Swedish women having a hysterectomy on benign indications between 1973 and 2003 (n = 184 441), and non-hysterectomized controls (n= 640 043). Main outcome measure was the first hospitalization or death of incident CVD (coronary heart disease, stroke, or heart failure). Occurrence of CVD was determined by individual linkage to the Inpatient Register. In women below age 50 at study entry, hysterectomy was associated with a significantly increased risk of CVD during follow-up [hazard ratio (HR), 1.18, 95% confidence interval (CI), 1.13–1.23; HR, 2.22, 95% CI, 1.01–4.83; and HR, 1.25, 95% CI, 1.06–1.48; in women without oopherectomy, with oopherectomy before or at study entry, respectively, using women without hysterectomy or oopherectomy as reference]. In women aged 50 or above at study entry, there were no significant associations between hysterectomy and incident CVD.Conclusions Hysterectomy in women aged 50 years or younger substantially increases the risk for CVD later in life and oopherectomy further adds to the risk of both coronary heart disease and stroke.

Introduction

Hysterectomy has traditionally been considered the method of choice for treating a variety of benign gynaecological disorders due to the low perioperative morbidity and definite cure of these diseases.[1] Incidence rates of hysterectomy in the USA and in western European countries have remained relatively stable,[2–6] despite recent years introduction of minimally invasive treatment options for conditions, such as menorrhagia and leiomyoma.[7]

Studies have shown that hysterectomy might be associated with an increased risk for cardiovascular disease (CVD),[8–11] but the epidemiological evidence is inconclusive. Previous studies have typically been too small to study associations of hysterectomy with separate types of CVD, such as coronary heart disease and stroke, with adequate statistical strength. Furthermore, temporal relations, such as time elapsed from hysterectomy and oopherectomy (concomitant with the hysterectomy or at a later time point), have been indicated to be of importance for CVD risks but are poorly understood.[10]

Bilateral salpingo-oopherectomy is the only unambiguous preventive measure for ovarian cancer and results in an abrupt transition to menopause in premenopausal patients. Considering that the majority of hysterectomies is performed in perimenopausal women on relative indications,[5,6] increased knowledge about the long-term effects of hysterectomy and/or oopherectomy is critical. Even a moderate risk increase in future disease would be important, as the attributed risk still could be large since hysterectomy is such a common surgical procedure. This is particularly important in the case of CVD, the major cause of death in women globally.[12] The aim of this nationwide, population-based cohort study using prospectively recorded data from the Swedish national health registers was to assess the risk of CVD after hysterectomy for benign indications, with or without oopherectomy, throughout the lifespan.

 

Methods

Ascertainment of Data

The Swedish Inpatient Register contains data on individual hospital discharges and contains (i) the dates of hospital admission and discharge; (ii) up to eight discharge diagnoses, coded according to the International Classification of Diseases (ICD) versions 7–10; and (iii) up to 12 operation codes from the Swedish Classification of Operations and Major Procedures. Correct coding for surgical procedures is achieved in 98% of the records.[13] The Inpatient Register also includes information about the unique national registration number (NRN), assigned to all Swedish residents, allowing unambiguous record linkage across all nationwide registers in Sweden.

The Cause of Death Register includes information about date and cause of death on all Swedish residents with a completeness exceeding 99%. The Register of Population and Population Changes includes information about dates of birth, death, emigration, and immigration of all Swedish residents. Information on socioeconomic status was retrieved from the Census 1990.

Study Population

We identified all records in the Inpatient Register indicating hysterectomy (study entry) from 1 January 1973, through 31 December 2003. To each patient with hysterectomy (n = 227 389), we randomly selected a control (n= 682 167) from the Register of the Total Population, who were individually matched by year of birth and county of residence at the year of hysterectomy. Using NRNs, our exposed and unexposed cohorts (i.e. women with and without hysterectomy) were linked to the Birth Register, Cancer Register, Emigration Register, and Cause of Death Register.

Within the Inpatient Register, we identified all women in the exposed and unexposed cohorts with a recorded CVD. In order to avoid bias introduced by CVD events occurring in relation to the surgical procedure, hysterectomized women contributed person-time to the study from 30 days after hysterectomy until first occurrence of a CVD, heart failure, cervical, corpus or ovarian cancer, death, emigration, or the end of the observation period (31 December 2003). Control women contributed person-time by the same criteria, but with start of follow-up 30 days after the date of matching, i.e. date of hysterectomy of the corresponding hysterectomized woman. We excluded from the analysis women: with incorrect information in the registers (n = 963); with CVD before entry (n = 17 889), with heart failure before start of follow-up (n = 2119), with hysterectomy on malignant indications or cervical, corpus or ovarian cancer registered within 365 days prior to hysterectomy/matching date (n = 37 642); with hysterectomy at age less than 18 years (n = 140); who died at/before start of follow-up (n = 165); who emigrated at/before start of follow-up (n = 12); or with start of follow-up after the end of the observation period (n = 2260). Women with the same values for matching variables were aggregated in order to achieve higher power, by combining the 1:3 matching strata into n:m matching strata. Thereafter, n:m strata containing either only hysterectomized or only non-hysterectomized women were excluded (n = 14 878), because they were deemed non-informative in the analysis. Thus, our final eligible study sample included 184 441 women with hysterectomy and 649 043 individually matched controls.

Classification of Exposures and Outcomes

The Swedish Classification of Operations and Major Procedures for 1973–1996 and 1997–2003 were used to identify: (i) hysterectomy in the Swedish Inpatient Register (operation codes 7210, 7211, 7261, 7262, 7467 for 1973–1996, and LCD00, LCC10, LCD10, LCD11,

LCD01-LCD04, LEF13 for 1997–2003) categorized as yes or no; and 2) oopherectomy performed concurrent with the hysterectomy or by itself, either bilateral once or unilateral twice (operation codes 7020, 7021, 7030, 7031, 7032, LAE10, LAE11, LAE20, LAE21, LAF00, LAF01, LAF10, LAF11, LAF20).

Incidence of CVD was defined as the first hospitalization (assessed from the Hospital Discharge Register) or death (assessed from the Cause of Death Register) caused by coronary heart disease, stroke, or heart failure. Coronary heart disease was defined as unstable angina (ICD-8 code 411, ICD-9 code 411B, ICD-10 code I20.0) or acute myocardial infarction (ICD-8 and ICD-9 code 410, ICD-10 codes I21–I22). Stroke was defined as cerebral infarction (ICD-8 codes 432–434, ICD-9 codes 433–434, ICD-10 code I63), cerebral haemorrhage (ICD-8 code 431, ICD-9 codes 431–432, ICD-10 codes I61–I62), subarachnoidal haemorrhage (ICD-8 and ICD-9 code 430, ICD-10 code I60), transient ischaemic attack (ICD-8 and ICD-9 code 435, ICD-10 code G45), or other acute stroke (ICD-8 and ICD-9 code 436, ICD-10 code I64). Heart failure was defined by ICD-8 codes 427.00 and 427.10, ICD-9 code 428, ICD-10 code I50. We only considered hospitalizations or deaths with the above diagnoses as primary diagnosis of the hospitalization or the primary cause of death. The validity of the stroke,[14] myocardial infarction,[14,15] and heart failure,[16] diagnoses in the Swedish hospital discharge register have been demonstrated to be ~95% when primary diagnoses are considered.

Statistical Analysis

We calculated crude incidence rates for CVD as the number of CVD events per 1000 person-years, with 95% confidence intervals (CIs) based on the Poisson distribution. Only the first occurrence of CVD in each individual was counted as an event. Rates were calculated separately by age (categorized as 18–49 years or ≥50 years), by hysterectomy (categorized as yes or no) and oopherectomy (categorized as never; before hysterectomy; at hysterectomy; or after hysterectomy). Categorization of age as 18–49 years or ≥50 years was based on 50 years being the median age of onset of menopause in Sweden.[17] Person time at risk was calculated from 30 days after the date of hysterectomy (exposed cohort) or from the date of matching (unexposed cohort), until the first occurrence of CVD or censoring.

Because we did not have access to information on smoking from the registers we investigated whether women with a hysterectomy were at increased risk for lung cancer as a proxy measure of smoking exposure. If women having hysterectomy are more inclined to smoke they would not only be at increased risk for CVD, but also for lung cancer, compared with women not having a hysterectomy.

Measures of association between exposures and subsequent CVD were investigated using conditional Cox proportional hazards models to retain and take the subject-level matching into account in the analysis. Data were presented as hazard ratios (HRs) with 95% CIs. Women with the same combination of matching variables (age, county, and calendar time) were collapsed into the same strata to gain statistical efficiency. We present HRs separately categorized by age, as well as, hysterectomy, and oopherectomy status (reference group 'no hysterectomy, never oopherectomy'). We performed two multivariate models: first, adjusting for time since start of follow-up, age, year, and county (model A) and second, further adjusting for socioeconomic status (data available for 71% of the cohort) as a covariate (model B). Oopherectomy was treated as a time-varying covariate in these analyses, i.e. a woman was considered as exposed to oopherectomy only from the time of operation. We used likelihood ratio tests (two-sided, 5% significance level) to assess the effect of exposure. Statistical analyses were performed using Intercooled Stata™ release 9 (StataCorp LP, College Station, TX, USA).

The study was approved by the regional ethical review board at Karolinska Institutet, Stockholm, Sweden and reporting conform to the STROBE guidelines for observational studies (http://www.strobe-statement.org).

Results

During the observation period of our study, 184 441 women having a hysterectomy fulfilled the inclusion criteria (exposed cohort) and were

individually matched to 649 043 women not undergoing hysterectomy (unexposed cohort). The median follow-up time was 10.4 years (range 0–31.6; 25–75th percentile, 5.0–17.1) contributing to 9 635 123 person-years at risk and a total of 51 181 CVD events.

To be able to separate effects of hysterectomy from those of oopherectomy, we took the occurrence and timing of oopherectomy (when present) into account in our analyses. During the observation period, 28 136 of the hysterectomized and 5118 of the non-hysterectomized women had an oopherectomy before, at or after entry in the study (i.e. date of hysterectomy or matching). Table 1 shows the distribution of women with or without hysterectomy and oopherectomy, respectively, along with person-years, number of CVD events and rates (composite or separately for coronary heart disease and stroke) by hysterectomy/oopherectomy category. As expected, the CVD rates were generally higher in the stratum of women over age 50 at entry than in the younger age group. Among younger women, the CVD incidence rates were higher in those that underwent surgical procedures, whereas a reverse pattern was observed in women over age 50 at study entry (Figure 1 and Table 1). There was no excess risk of lung cancer among women with hysterectomy compared with those without (HR 1.0, 95% CI 1.0–1.1) suggesting that the association between hysterectomy and CVD was not confounded by smoking.

(Enlarge Image)

Figure 1.

Incidence rates over time of cardiovascular disease (composite of coronary heart disease and stroke) in women below or above age 50 at baseline by mode of surgical procedure.

In women below age 50 at study entry, hysterectomy was associated with a significantly higher risk of CVD during follow-up (Table 2, upper panel). In models taking age, calendar time, county, and socioeconomic status into account, this risk increase was ~20% in women that did not go through an oopherectomy at all, or had an oopherectomy at the same time as the hysterectomy. In women who had an oopherectomy before study entry, and then a hysterectomy, the point estimate was higher, but this analysis was based on very few CVD events (n= 7); hence, the CI was very wide and the result borderline significant.

In women aged 50 or above at study entry, having a hysterectomy was associated with a slightly lower risk of CVD during follow-up. This risk decrease was borderline significant in women without oopherectomy, but non-significant in women with oopherectomy in addition to their hysterectomy (before, at or after study entry; Table 2, lower panel). Further, in this older age group, among women that did not have a hysterectomy, but that went through an oopherectomy after study entry, there was a >40% lower risk of CVD during follow-up.

Using models with oopherectomy as a time-varying covariate, the same patterns with differences across the two age categories emerged (Table 3). In women under age 50 at study entry, both hysterectomy and oopherectomy were independently associated with an increased risk of CVD during follow-up. This risk increase was rather substantial; almost 20% and >40% higher CVD risk, respectively. Further, the risk increase was consistent for incidence in coronary heart disease and stroke. In women aged 50 or above at study entry, hysterectomy was borderline significantly associated with a lower risk of CVD and coronary heart disease during follow-up, whereas there were no association of oopherectomy and CVD in this age category.

Discussion

In the present nationwide cohort study encompassing >800 000 women at risk, we report that having a hysterectomy and/or an oopherectomy before age 50 (corresponding to the median age of

onset of menopause in Sweden),[17] was associated with a substantially increased risk of later life CVD. This risk increase was consistent for incidence in coronary heart disease, as well as stroke, and it was evident also after taking socioeconomic status into account. In contrast, in women above age 50, hysterectomy as well as oopherectomy was associated with lower risk of CVD during follow-up. The most plausible explanation for this observation is a selection bias where women undergoing elective surgery for benign indications at older ages generally are healthier and at a lower risk for future CVD than those that do not undergo surgery.

The association between hysterectomy and CVD has been suggested to rely on three factors: age at surgery; disruption of ovarian circulation at hysterectomy; and the effects of concomitant bilateral oopherectomy.[18]Previous studies are largely in agreement that early surgical menopause increases the risk for CVD,[10,19,20] but prospective cohort studies are scarce and results are inconclusive with regard to the effects of simple hysterectomy without oopherectomy. In premenopausal women, bilateral oopherectomy results in an abrupt fall in circulating oestrogens and testosterone levels, and the patient enters menopause immediately. To our knowledge, there are no data to support that surgical menopause leads to hypertension, insulin resistance, or type 2 diabetes, whereas it has been shown to increase total cholesterol and LDL levels.[21] Hence, long-standing hypercholesterolaemia may be a mechanism linking surgical menopause with atherosclerosis and clinical CVD.

In women under the age of 50 years, hysterectomy by itself was associated with an increased risk of CVD compared with women with an intact uterus. This finding corroborates the notion that hysterectomy interferes with ovarian blood flow and may result in premature ovarian failure and hormone-related effects on the vascular bed. Having an oopherectomy at the time of the hysterectomy, and in particular before, added further to the risk also after adjusting for socioeconomic status. No previous studies have been stratified for the relation of having a bilateral oopherectomy before, concurrent with, or after the hysterectomy. The adverse cardiovascular effects of hysterectomy, and oopherectomy, were only notable in younger women, which give further corroboration to studies suggesting that it may be the surgical menopause itself that is associated with an increased risk for CVD.[11] Thus, the underlying biological rationale for an association between hysterectomy and CVD is likely to be found in ovarian failure. For this reason, information on age at surgery and the timeline of events is important to fully understand the relation, yet data on the intricate relation between hysterectomy, oopherectomy, and cardiovascular morbidity in the general population have been lacking.

Our data are in agreement with a meta-analysis suggesting that bilateral oopherectomy before the age of 50 years increases the overall CVD risk.[22] Risk estimates of coronary heart disease of a similar magnitude as those found in the present study were observed

in the Nurses Health Study.[20] However, in contrast to the Nurses Health Study, we also found a significantly increased risk of stroke. The reason for this difference could be attributed to differences in methodology, where the Nurses' Health Study relies on voluntary participation and self-reported measures of outcomes, which to some extent may involve selection, ascertainment, and classification bias. If the risk for CVD after hysterectomy and/or oopherectomy is attributed to early surgical menopause and precipitated atherosclerosis, it seems biologically plausible that the risk also applies to cerebral vascularization and stroke.

Data from high-quality nationwide healthcare registers, such as the Swedish Hospital Discharge Register, have proved highly accurate for diagnoses of CVD,[14–16] and the register has a negligible loss to registration.[13]Furthermore, our data are representative for the general population and controls are drawn from the same population as where cases arose. The register also allows for establishing a temporal chain of events given the prospective data collection. Despite the strengths of our study, use of register data is at the same time a weakness compared with other prospective cohorts, since information on life events not requiring hospitalization goes unregistered. Thus, we may have missed milder cases of CVD that only were treated as outpatients. On the other hand, this would be expected to increase the validity of the endpoints, limiting the inclusion of false positive cases. Another inherent limitation of using nationwide registry data is that we lack information about other CVD risk factors than age and socioeconomic status. We recognize that our study cannot adjust for some factors, such as diabetes and obesity, known to influence the risk of CVD. However, these factors would make women less likely to have abdominal surgery due to co-morbidity and underestimate the risk for CVD attributed to hysterectomy and oopherectomy in our study. Further, there was no indication that smoking confounded the observed associations since the risk for lung cancer was the same for women with and without the surgical procedures. The relatively homogenous population of women living in Sweden is expected to increase the internal validity and reliability of the findings but at the same time the ability to generalize our findings to other populations is unknown and the observational study design cannot infer causality.

Hysterectomy rates of ~5.5 hysterectomies per 1000 female US resident and 2.1 per 1000 in Sweden[6] make hysterectomy the second most common abdominal surgical procedure in women after cesarean delivery. The low perioperative morbidity and cost-effectiveness of the procedure make hysterectomy an attractive and definitive treatment option for a number of gynaecological disorders. Current trends in hysterectomy for benign disorders indicate both a broadening of indications, as well as, a shift towards providing younger women of reproductive and perimenopausal age a definitive surgical solution to gynaecological disorders.[6] Thus, our findings of an increased risk of CVD in women having a hysterectomy and/or bilateral oopherectomy in premenopausal age may have global and important implications for women's health.

Circulating Free DNA and p53 Antibodies in Plasma of Patients with Ovarian Epithelial CancersB. Dobrzycka; S. J. Terlikowski; M. Kinalski; O. Kowalczuk; W. Niklinska; L. Chyczewski

Authors and Disclosures

Posted: 06/21/2011; Annals of Oncology. 2011;22(5):1133-1140. © 2011 Oxford University Press

Abstract and Introduction

Abstract

Background: This study was conducted in order to evaluate the significance of circulating free DNA (CFDNA), blood plasma p53 antibodies (p53-Ab) and mutations of KRAS gene in the prognosis of ovarian epithelial cancers.Patients and methods: A total of 126 patients were included in this study. KRAS mutations and CFDNA were detected by means of the PCR–restriction fragment length polymorphism (PCR-RFLP) and enriched by the PCR-RFLP method. Enzyme-linked immunosorbent assay was used to analyze plasma p53-Ab.Results: KRAS mutations were detected in 27 (21.4%) of examined tumors. The frequency of KRAS mutations was especially high in mucinous cancers (P < 0.001). CFDNA and p53-Ab were frequently detected in patients with serous cancers in high grade (P < 0.001). The overall survival rate was significantly lower for patients with serous tumors and CFDNA and p53-Ab-positive than negative tumors (P = 0.022 and P < 0.001, respectively). In mucinous ovarian cancer, a worse overall survival was correlated with the KRAS mutations (P = 0.03).Conclusions: The results of the present study suggested that a presence of KRAS mutations in mucinous ovarian cancer and CFDNA and p53-Ab in serous tumors was correlated with the highest risk of cancer progression.

Introduction

Epithelial ovarian cancer comprises the majority of malignant ovarian tumors in adult women. About 190 000 new cases and 114 000 deaths from ovarian cancer are estimated to occur annually. The highest rates are reported in Scandinavia and Eastern Europe, the United States and Canada. The age-adjusted incidence rate in the United States is 12.48 per 100 000 women per year.[1] Low rates are found in Africa and Asia.[2] In Poland, the age-adjusted incidence is 10.8 per 100 000 women per year.[3] The overall 5-year survival rate for all stages combined range from 30%/INS< to 50%. Most women, however, present with late-stage disease, which is associated with a rate of about 20%.[4]

Activation of protooncogenes is a feature of many malignancies and, not surprisingly, there have been numerous searches for oncogene mutations as well as for specific genes dysregulation involved in apoptotic, neoangiogenetic and transduction signal pathways.[5] In ovarian cancer, KRAS mutations are seen in 4%–30% of cases.[6–

10] TP53 mutations have been found in 50% of cases.[11–13] The frequencies of p53 antibodies (p53-Ab) in serum vary from 8% to 46%.[14–17] Several methods have been used for circulating free DNA (CFDNA) quantification, but none have been evaluated in terms of reproducibility and therefore, results from different studies are not comparable.[18–22]

This study was conducted in order to evaluate the significance of CFDNA, blood plasma p53-Ab and mutations ofKRAS gene in the prognosis of ovarian epithelial cancers.

Materials and Methods

Patient and Clinical Samples

A total of 126 patients with ovarian cancers (aged 18–79 years; median value = 58.3 years), all treated in the Department of Gynecology at District Hospital in Bialystok between 2002 and 2005, were included in this study. The protocol was previously approved by the Bioethical Committee of the Medical University of Bialystok. Patients were informed and gave their consent for the study. Follow-up data was completed until April 2010. At a median follow-up of 38 months (range 1–96), 85 patients had died as a consequence of cancer progression.

Primary treatment generally consisted of surgery, which entails total abdominal hysterectomy, omentectomy, multiple peritoneal and lymph node samplings as well as peritoneal washings for cytology. Adjuvant chemotherapy consisted of different platinum-based treatment regimens. Bilateral tumors were resected from 19 patients and remaining tumors were found unilaterally. Tumor stage and histological diagnosis of each case were determined according to the criteria of the International Federation of Gynecology and Obstetrics and the histological typing system of the World Health Organization, respectively. Patients were categorized as having limited disease (stage I or II) and advanced disease (stage III or IV). Tumors were graded as well (G1), moderately (G2), or poorly (G3) differentiated. Histologically, 64 (50.8%) of the patients had a serous cystadenocarcinoma, 18 (14.3%) a mucinous cystadenocarcinoma, 26 (20.6%) an endometrioid carcinoma and 18 (14.3%) was other (therein 10 cases of clear-cell carcinomas and 8 of different histological types). Two experienced pathologists evaluated the histological appearance of all tissue samples in a blinded fashion.

KRAS Mutation Detection

Tissue specimens of ovarian cancer were obtained during surgery and immediately frozen in liquid nitrogen and then stored at a temperature of −80°C until analyzed. Before DNA extraction, it was microscopically confirmed that the tumor specimens consisted mainly of carcinoma tissue (80%). Tumor samples (30–50 mg) were minced with a sterile scalpel and then digested overnight in 180 μl of tissue lysis buffer, containing 20 μl of proteinase K solution (10 mg/ml) at 37°C. DNA extraction from tissue lysates was carried out with the GeneElute Mammalian Genomic DNA Miniprep Kit (Sigma-Aldrich, St Louis, MO), according to the manufacture's instructions. Isolated DNA was stored at a temperature of −20°C before further assays.

The detection of KRAS mutations at codon 12 was carried out using PCR–restriction fragment length polymorphism (PCR-RFLP) method as described previously.[7, 23] PCR products were sequenced with the use of the sense primer K1, the ABI PRISM BigDye Terminator v3.0 Cycle Sequencing Ready Reaction (Applied Biosystems, Foster City, CA) and the automated ABI PRISM 377 DNA sequencer (Applied Biosystems). Mutations were confirmed by sequencing reaction with the use of the antisense primer DD5P. Antisense strain of PCR products was used for sequencing.

Circulating Free DNA

Three-milliliter sample of peripheral blood was collected in vials containing EDTA on the day before surgery. Plasma was immediately separated from the cellular fraction by centrifugation at 3000 g for 20 min at 4°C and then stored at −80°C. DNA isolation from 200 μl blood plasma was carried out with commercial kit for blood DNA isolation of Sigma-Aldrich according to manufacturer's specifications. DNA solutions were stored at a temperature of −80°C before mutation detection. KRAS codon 12 mutation was detected by enriched PCR-RFLP method with some modification.[23] The method consists of two DNA amplification and two digestion reactions.

Assay of p53-Ab in Plasma

A three-milliliter sample of peripheral blood was collected in vials containing EDTA on the day before surgery. Plasma was immediately separated from the cellular fraction by centrifugation at 3000 g during the period of 20 min at a temperature of 4°C and the supernatant was stored at a temperature of −80°C until use.

The level of p53-Ab in plasma was measured with the use of anti-p53 ELISA II Kit (Pharmacell, Paris, France) according to the manufacture's instruction. The value over 0.85 IU/ml indicates a probable presence of the antibody.

Statistical Analysis

Statistical analysis was carried out using Statistica software version 8.0 (StatSoft Inc., StatSoft Polska Sp. z o.o., Poland). A chi-square test was used to evaluate the relationship between categorical variables. Fisher's exact test was used to determine significance between the two groups. A P value of <0.05 was considered as statistically significant. In addition, survival time was calculated from the date of surgery to the date of death and survival analysis was carried out using the Kaplan–Meier method.

Results

KRAS Gene Mutations

Our results show that mutations of the KRAS gene in codon 12 were present in 27 of 126 cases (21.4%) examined with epithelial ovarian cancer (Figure 1). We found this frequency to be lower in serous tumors (12.5%), with a slight increase to 23.1% in endometrioid tumors and an increase to 61.1% in mucinous carcinomas. These differences were statistically significant (P < 0.001) (Table 1). KRAS mutations were present in all analyzed types of the ovarian tumors, the most commonly, in grade 1 of histopathological differentiation. These differences are not statistically significant (Table 2).

(Enlarge Image)

Figure 1.

(A) PCR-RFLP analysis of codon 12 KRAS mutations: 1, 4, 7, 8, 9, 10 – samples with normal (wild type) codon 12 KRAS gene sequence; 2, 3, 5, 6 – samples with mutated codon 12 KRASsequence (M1 - size standard, DNA, Gdansk,

Poland). (B) Automated sequence analysis of the DNA product from PCR amplification of codon 12 KRAS from two samples with mutated (M) sequence (1, 2) in the PCR-RFLP analysis. The arrows indicate the site of mutation. In the first mutated sample (1) there is a G to T substitution in the second position of the codon 12, and in the second mutated sequence (2) there is a G to A substitution in the second position of the codon (antysense strain of the KRAS gene has been sequenced in this assay).

Among patients with KRAS-mutated mucinous cystadenocarcinomas, the Kaplan–Meier survival estimates of the 1-year and 5-year survival rates were 97.4% and 90.8%, respectively, while the respective rates among patients with lack of KRAS mutations were 97.4% and 93.4%. Statistically significant difference was observed between survival rates over time (P = 0.03) (Figure 2). Presence of KRAS gene mutation was not associated with survival outcome in serous and endometrioid tumors (data not shown).

(Enlarge Image)

Figure 2.

Kaplan-Meier survival analysis concerning KRAS mutation and cumulative survival in mucinous ovarian cancer patients. The prognosis of patients was significantly worse in KRAS-mutated tumors (thin line) as compared to nonmutated tumors (thick line) (P = 0.03).

Circulating Free DNA

CFDNA was detectable in 55 of treated patients (43.7%). Among them, there were 39 patients with serous tumors (60.9%), 2 patients with mucinous tumors (11.1%) and 8 with endometrioid tumors (30.8%). The difference between the groups was statistically significant (P < 0.001). A positive correlation was found between the presence of CFDNA and the grade (mostly in G2 and G3; P < 0.001) at late tumor stage (III/IV; P < 0.001) and only in serous cancers (Table 2 and Table 3 and Figure 3).

(Enlarge Image)

Figure 3.

Semi-nested PCR products of KRAS gene in DNA extracted from plasma samples. Samples 1, 2, 3, 5, 6, 7, 9, 10 – blood plasmas with detectable CFDNA, samples 4 and 8 – blood plasmas without

detectable CFDNA (M1 - size standard, DNA, Gdansk, Poland).

The Kaplan–Meier survival estimates of the 1-year and 5-year survival rates for patients with serous cystadenocarcinomas and the presence of CFDNA were 97.4% and 90.8%, respectively, while the respective rates for patients with absence of CFDNA were 97.4% and 93.4%. The median overall survival was 21 months for patient with CFDNA presence and 52 months for patient with CFDNA absence. The differences were statistically significant (P = 0.022) (Figure 4). The survival rate was unaffected by presence of CFDNA in mucinous and endometrioid tumors (data not shown).

(Enlarge Image)

Figure 4.

Kaplan-Meier survival analysis concerning CFDNA and cumulative survival in serous ovarian cancer patients. The prognosis of patients was significantly worse in CFDNA-positive patients (thin line) as compared to patients who were CFDNA-negative (thick line) (P = 0.022).

Positivity of Plasma p53-Ab

Using the cutoff value, 42 (33.3%) of 126 patients were p53-Ab positive. The increased presence of Ab to p53 in plasma of women with serous ovarian cancer (57.8%) was statistically significant as compared with other examined ovarian cancer (P < 0.001) (Table 1). Plasma of women with advanced stage (III/IV) of serous ovarian cancer was more likely to have detectable antibodies specific for p53 (96.4%) than samples taken from patients with early-stage (I/II) disease (27.8%) (P < 0.001) (Table 3). There was a tendency toward a higher presence of Ab to p53 in plasma of the serous cystadenocarcinomas than remaining histological subtypes of epithelial ovarian cancer. The p53-Ab was especially high in the G2 and G3 serous ovarian cancer (81.8% and 100%, respectively) (P < 0.001) (Table 2).

The Kaplan–Meier survival estimates of the 1-year and 5-year survival rates in p53-Ab-positive patients with serous cystadenocarcinomas were 86.4% and 72.7%, respectively, while the respective rates among p53-Ab-negative patients were 100% and 84.8%. The median overall survival was 11 months for p53-Ab-positive patient and 57 months for p53-Ab-negative patient. The differences were statistically significant (P < 0.001) (Figure 5). Presence of p53-Ab in plasma of patients with mucinous and endometrioid tumors did not influence survival significantly (data not shown).

(Enlarge Image)

Figure 5.

Kaplan-Meier survival analysis concerning p53-Ab and cumulative survival in serous ovarian cancer patients. The prognosis of patients was significantly worse in p53-Ab-positive patients (thin line) as compared to patients who were p53-Ab-negative (thick line) (P <

0.001).

 

Discussion

Recently, a dualistic model of ovarian serous carcinogenesis has been proposed.[24–26] In this model, ovarian cancers are divided into two types. Type I tumors include low-grade serous, mucinous, endometrioid and clear-cell carcinomas and malignant Brenner tumors. These are characterized by a high frequency of mutations ofKRAS, low proliferation and a 5-year survival of ~55%.[27–29] Type II tumors are composed of what are currently classified as moderately and poorly differentiated serous carcinoma, malignant mixed mesodermal tumors and undifferentiated carcinoma. They are characterized by a high frequency of mutations in TP53 and a high proliferative index.[29, 30] Type I tumors evolve slowly and are associated with distinct molecular changes that are rarely found in type II tumors. In contrast, type II tumors grow up rapidly, arising directly from the surface epithelium or inclusion cysts and metastasize early in their course.[8, 27, 31] Thus, there is a need to discover novel biomarkers for early diagnosis and prediction of prognosis.

The unexpected high frequency of KRAS mutations in type I tumors supports the hypothesis that KRASmutations are genetic events closely related to the development of a mucinous phenotype in ovarian cancer.[10, 28, 32] As it is shown in Table 4, KRAS mutations are quite frequent in ovarian mucinous tumors when compared with other histological types. However, the lack of a statistically significant correlation between KRAS mutations and stage or grade in our study indicates that we cannot rely on this molecular genetic abnormality for the identification of cancer patients for whom more aggressive treatment is necessary.

Serum p53-Ab is predominantly associated with P53 gene missense mutations and P53 accumulation in the tumor. They have been found in human cancer patients with a specificity of 96%, but the sensitivity of such detection is only 30%.[16] In our study, plasma p53-Ab was detected in 33.3% patients, which is similar to the previous findings presented in Table 4.[33–37] The clinical value of these antibodies remains a subject to debate, but consistent results have been observed in other cancers in which they have been associated with high-grade tumors and poor survival.[15, 23, 38]

Point mutations in KRAS oncogene have been detected in the plasma DNA of patients with colorectal and pancreatic cancer, even preceding clinical diagnosis of pancreatic cancer by 5–14 months before clinical diagnosis.[39, 40] We also detected the same KRAS point mutation in the plasma DNA of patients with epithelial ovarian cancer. The fact that early-stage tumors of low-grade or in situ carcinomas could present DNA alterations in the plasma/serum/ascites suggests that CFDNA may become a useful diagnostic tool for early and potentially curable cancers.[41–43]

Our study showed a significant difference between the presence of CFDNA in patients with serous cancers in high grade compared with other histological subtypes. This result is in accordance with the finding of Kamat et al.,[18, 21] who reported an increase in the median levels of CFDNA in serum patients with high-grade advanced stage (III or IV) serous ovarian cancer.

Mechanisms regulating appearance and distribution of CFDNA in blood are under pressure of factors that influence its circulation and clearance including hydrolyzing enzymes. The invading cells have the ability of shedding DNA into the circulation and this may explain the high level of CFDNA in the plasma of patients with cancer.[43] Thus, elevated levels of CFDNA before diagnosis may be a promising independent DNA-based prognostic plasma marker for epithelial ovarian cancer. However, at the moment, the true origin of CFDNA is not fully understood.

In early stage of ovarian cancer, the relapse rate ranges between 10% and 40%. More than 50% of patients with advanced disease will eventually progress. Only 10%–30% of such patients attain long-term survival with a median progression-free survival of 16 months.[1–3, 44] In our study, the presence in plasma CFDNA and p53-Ab in patients with

serous cystadenocarcinoma was correlated with the highest risk of cancer progression and short survival time. In mucinous cystadenocarcinomas, mutation of the KRAS gene in codon 12 may predict improved overall survival.

The results of our study suggest that the detection of KRAS mutation, CFDNA and p53-Ab in plasma of patients with type I ovarian tumors offers a possibility to individualize treatment regimen. These findings suggest that a biologically less aggressive tumor may be more amenable to optimal cytoreduction. Probably, tumor biology and other mechanisms may also influence patient's outcome. It is believed that a combined measurement of currently used tumor biomarkers will improve the sensitivity and specificity for ovarian cancer management. However, the values of longitudinal measurements of the used markers are yet to be determined.