Tumour Markers in Peritoneal Washing Fluid-Contribution to Cytology

Gynaecologic cancers are a group of disease including vulvar, vaginal, cervical, endometrial and ovarian cancers as well as gestational trophoblastic neoplasm. Gynaecologic cancers are still an important problem of public health in developing countries and constitute 19% of new cancer cases (Sankaranarayanan et al., 2004). Intra peritoneal spread of gynaecologic cancers is an important reason of mortality and morbidity and presents as malignant ascites. Microscopic tumour spread may be detected by PWC before formation of malignant ascites and detection of these malignant cells gives information about the prognosis of the disease (Shiel et al., 2004). Low rates of positive PWC may be seen in occult spread of benign diseases. In many studies, positive PWC has been shown in the progress of benign diseases (Sharifi et al., 1999).


Introduction
Gynaecologic cancers are a group of disease including vulvar, vaginal, cervical, endometrial and ovarian cancers as well as gestational trophoblastic neoplasm.Gynaecologic cancers are still an important problem of public health in developing countries and constitute 19% of new cancer cases (Sankaranarayanan et al., 2004).Intra peritoneal spread of gynaecologic cancers is an important reason of mortality and morbidity and presents as malignant ascites.Microscopic tumour spread may be detected by PWC before formation of malignant ascites and detection of these malignant cells gives information about the prognosis of the disease (Shiel et al., 2004).Low rates of positive PWC may be seen in occult spread of benign diseases.In many studies, positive PWC has been shown in the progress of benign diseases (Sharifi et al., 1999).

Tumour Markers in Peritoneal Washing Fluid -Contribution to Cytology
Mustafa Yıldırım 1 , Dınc Suren 2 , Mustafa Yıldız 3 , Arsenal Sezgın Alıkanoglu 2 , Vildan Kaya 4 *, Suleyman Gunhan Doluoglu 5 , Ozgur Aydın 6 , Necat Yılmaz 6 , Cem Sezer 2 , Mehmet Karaca 5 Tumour markers are soluble glycoproteins.Levels of these markers can be evaluated in blood, urine and other body fluid samples of the patients.The ideal tumour marker of gynaecologic cancers has still not been determined.However many tumour-related antigens are determined frequently by monoclonal antibody technique in serum samples of the patients.The tumour markers used most frequently in gynaecologic cancers are CEA, CA 125 and CA 19-9.
The limitations of PWC examination are the possibility of false positivity in benign diseases and false negativity in the early stage of disease.Although positive PWC does not seem to change the stage of the disease in the latest guidelines, it is a poor prognostic factor and effective in determining the intensity of the treatment (Selvaggi et al., 2003).In this study, CEA, CA 125 and CA 19-9 in peritoneal washing fluid were evaluated in patients operated due to a gynaecologic cancer.

Patients
This study includes the patients who were operated due to either a malignant or a benign gynaecologic pathology in Obstetrics and Gynaecology Clinic of Antalya Education and Research Hospital between 2010 and 2012.Inoperable patients with advanced stage disease or metastasis and patients with inadequate sample for evaluation and clinically detected ascites were excluded from the study.A standard preoperative evaluation for staging the disease and determining the prognosis was carried out for all of the patients.The local ethic committee gave approval for the study.Patients' files were analysed and information on demographic data such as age, gender and disease stage were obtained.

Obtaining the samples
Preoperative tumour markers were studied in serum of blood samples obtained from the patients for preoperative evaluation of a gynaecologic operation.PWC was obtained by aspiration after injecting 100 mL saline solution to the peritoneal cavity just after penetration to peritoneum surgically.Cytopathological examination was performed by two pathologists (DS, ASA).After completing the cytopathological examination, remaining samples were saved in -80 0 C. Peritoneal washing fluid tumour markers were quantified in these samples.

Quantification of tumour markers
Levels of CEA, CA-125 and CA 19,9 were quantified in preoperative serum samples and peritoneal washing fluid samples obtained intraoperative by chemiluminescence method using Bechman DX-800.Reference values for CEA, CA-125 and CA 19-9 were as follows respectively; <2.5ng/ml, <35 U/ml and <39 U/ml.

Statistical analysis
Statistical analyses were performed using the SPSS ("Statistical software system for Windows") software version 15.Variables either normal or not, were detected by visual (histograms and probability calculations) and analytical methods (Kolmogorov-Simirnov/Shapiro-Wilk's test).Since CEA, CA-125, CA 19-9 and CA 15-3 are not present in preoperative serum samples and peritoneal washing fluid of patients ordinarily, Mann-Whitney "rank sum test" was used for comparison of malignant and benign groups.A p value of <0.05 was considered significant for all of the tests.

Results
This study includes 94 patients operated for a gynaecologic pathology.Median age of the patients was 48.5±12.3(range 14-78).Histopathological examination revealed malignancy in 62 patients (66%), and a benign disease in 32 (34%) patients.Median age of the patients with a benign disease was significantly lower (median value: 6 years) than the patients with malignancy.Benign diseases originated most frequently from ovary (54.8%) whereas malignant ones originated from the uterus (59.4).
The most frequent benign ovarian disease was simple ovarian cyst and the most frequent malignant uterus disease was endometrioid carcinoma.
In patients with a histopathological diagnosis of malignancy, T1, T2 and T3 lesions were determined in 74.2%, 3.2% and 22.6% of patients, respectively.Patients with a cytopathological diagnosis of malignancy had a tumour with a more advanced stage (p<0.001).
Histopathological findings of the patients with a cytopathological diagnosis of malignancy were all consistent with malignancy.Sensitivity and specificity of the cytological examination was found as 21% and 100% respectively.The patient group with a diagnosis of malignancy after cytopathological examination included patients with ovarian and uterine malignancies.Malignancies with an origin of cervix constituted 5% of all the patients.When all the cytological examination was evaluated with pathological level of CEA in peritoneal washing fluid, the sensitivity of the cytological examination increased to 37%.
PWC was found negative in all patients (3 malignant and 2 benign) with a cervix disease.Many of the patients with a malignant cytology had a malignancy of ovary and uterus.Among patients with cervical malignancy, three of the preoperative tumour markers were found positive in one patient whereas only CEA was found high in the peritoneal washing fluid.
No significant difference was found between benign and malignant groups with respect to CEA, CA 125 and CA 19-9 levels quantified in preoperative serum.While no difference was found between the two groups in terms of CA125 and CA19-9 levels in peritoneal washing fluid, levels of CEA was found significantly higher (p:0.009)(Table 1).

Discussion
PWC is used for detecting the microscopic tumour foci that cannot be observed in intraoperative gross examination.Although the presence of malignant cells in the peritoneal cavity does not change the stage of the disease, it has a prognostic significance (Anastasiadis et al., 2011).In our study, we found that CEA, quantified as a tumour marker in the peritoneal fluid has an additional  diagnostic benefit to the diagnostic value of PWC.Specificity and sensitivity of PWC in gynaecological pathologies were searched in many studies.Zuna et al. (1996) determined the role of PWC in gynaecological malignancies.They found a positive cytology result in 80.4% of 112 ovarian carcinoma, 31.2% of 16 borderline ovarian tumour, 12.6% of 135 endometrial carcinoma and 8.7% of 92 cervical carcinoma.They suggested that PWC is highly specific (98.1%)but less sensitive (82.9%) (Zuna et al., 1996).In our study we found a less sensitivity rate in comparison with these rates.We think that the reason of this finding is including the patients with a benign disease in our study.
Tumour markers can be determined in peritoneal washing fluid in progress of both malignant and benign diseases (Ismail et al., 1994).Levels of tumour markers quantified in peritoneal fluid and serum samples may be different (Barbati et al., 1992).Studies performed in patients with ascites, showed that tumour markers quantified in ascites fluid does not play a role in distinction of benign and malignant diseases (Sari et al., 2001).Tüzün et al. (2009) showed just the contrary in a study determining the levels of tumour markers including CA125, CEA and CA19.9 in samples of serum and ascites fluid.In this study, they found the levels of tumour markers correlated in serum and ascites fluid and revealed that the tumour markers were high in patients with a malignancy (Tuzun et al., 2009).In our study patients with ascites were excluded from the study and the study group was different from the study mentioned above in which only patients with a gynaecological pathology were included.CEA was found to be the only tumour marker providing an additional benefit to PWC Positive PWC is found rarely in vulvar cancers.It ranges between 2.5% and 11% in cervix cancer (Takeshima et al., 1997).CEA and CA125 can be used in diagnosis and follow-up of patients with cervix cancer (Yoon et al., 2007;Gadducci et al., 2008).Positive PWC was not determined in patients with a cervical pathology in our study.We found a high level of CEA in peritoneal washing fluid of a patient with high preoperative tumour markers and this indicates that quantifying CEA in peritoneal washing fluid may be useful in case of negative PWC.
Positive PWC is determined in 7-22% of patients with endometrial cancer (Mlyneek et al., 2005).Positive cytology rate is lower in early-stage patients when compared with advanced stage patients.Kashimura et al. found this rate as 9% in stage I and 25% in stage II endometrial cancer (Kashimura et al., 1997).
Positive cytology is found in 45-60% of patients with ovarian cancer.Presence of peritoneal implants in ovarian cancers with a low malignant potential indicates a high risk of recurrence.PWC may be used to establish this risk (Sneige et al., 2012).In our study there was no patient with an ovarian cancer of low malignant potential.While high levels of serum CA-125 are found in 50% of patients with ovarian cancer, the rate increases to 90% in patients with an advanced stage (Gadducci et al., 1992).High levels of CA125 are determined not only in peritoneal fluid but also in pleural fluid in ovarian cancer (Topalak et al., 2002).In a study searching the levels of CA125 and CEA in ascites fluid and serum samples in ovarian pathologies, levels of CA125 and CEA in ascites fluid were found statistically higher than serum levels independent of the histology of the ovarian cancer.Levels of CA125 and CEA are found higher in malignant diseases when compared with benign diseases (Harłozińska et al., 1991).In patients with ovarian cancer, high levels of CA19-9 are found in advanced stage more frequently (Yurkovetsky et al., 2010).
In conclusion, CEA, one of the tumour markers quantified in peritoneal washing fluid has a diagnostic contribution to PWC.Quantification of CEA in peritoneal washing fluids seems to be useful in management of gynaecologic cancers.