Ovarian cancer is the leading cause of mortality of female gynecological cancers and ranks seventh as the most common cancer world wide1. It is one of the most common malignancies affecting women in India and has shown an increase in the incidence rates over the years .2 Immunohistochemical staining for P53 is used with mutational analysis in the diagnostic workup of carcinomas at various sites including ovarian cancer. Strong and diffuse immunoexpression of P53 is generally interpreted as likely indicating P53 gene mutation 3. P53 is one of the human tumor suppressor genes. Mutations in the Tp53 gene are the commonest and frequent in epithelial ovarian neoplasm's 3. P53 is altered in 30-80% of cases 4. Advances in screening and molecular pathogenesis of ovarian neoplasms, development of novel targeted therapies, and practical intra peritoneal techniques for drug delivery are most likely to improve patient outcome 5,6..The present study is to know about the association of P53 expression by immunohistochemistry and P53 mutations by DNA analysis with in the epithelial ovarian neoplasms. The incidence of P53 mutations in epithelial ovarian neoplasms will be calculated and compared with age, clinical presentation, histological grading of tumor. These findings will be correlated, evaluating the P53 gene mutations in epithelial ovarian neoplasms suggesting its role in prognosis. AIMS AND OBJECTIVES 1. To Study the immunohistochemical expression of P53 in epithelial ovarian neoplasms 2. To study p53 mutations in (5-8 exons) by PCR in different types of epithelial ovarian neoplasms, and to understand its role in predicting prognosis. 3. To study the variation of P53 mutations in benign, borderline and malignant ovarian neoplasms

This study is conducted for a period of 2 years (August 2017 to July 2019). The cases were retrieved from the archives of Department of Pathology at MNJ Institute of Oncology and Regional Cancer Centre - Osmania Medical College, Hyderabad. Immunohistochemistry: Immunohistochemical staining of P53 was done using peroxidase–antiperoxidase method according to the protocol described by DAKO with P53 Monoclonal Mouse, Clone-DO-7. PCR: Formalin fixed paraffin embedded blocks of Surface epithelilal ovarian tumors were collected and DNA extraction done for all the 50 patients using QIAamp DNA FFPE tissue Kit.

The present study included the morphological, immunohistochemical, and genetic analysis of 50 cases of ovarian neoplasms in the department of pathology, Osmania Medical College, Hyderabad over a period of 2 years from August 2017 to July 2019. Out of 50 Surface epithelial ovarian tumors, 12(24%) cases were benign, 10(20%) borderline, and 28 (56%) were malignant. Age Distribution The present study shows age ranging from 33 to 66 years, with a peak incidence in 5th decade of life [Chart 1]. Majority of benign cases were seen between 30-40 years. Maximum number of malignant tumors were seen between 40-60 years. Laterality Left sided tumors of ovary 27 (54%) were more common than the right sided tumors 16 (32%) whereas bilateral cases were 07, i.e., constituting14% [Chart 2]. Clinical Presentation The most common clinical presentation was mass per abdomen seen in 37 cases (74%), followed by ascites in 21(42%) cases [Table 1]. Specimen Type In the present study, TAH with BSO was the most common procedure opted in 35 cases (70%) followed by unilateral oophorectomy in 07 cases (14%), bilateral oophorectomy in 08 cases (16%). Size There was a wide range of size in ovarian neoplasms, i.e., from 4x5cm to 30x16 cm. Majority of them 20(40%) were between 10-20cm [Table 2]. Consistency The present study includes cystic, solid and tumors with solid and cystic consistency. Tumors were cystic in 13 cases while the malignant tumors were predominantly solid and cystic in 30 (60%) cases. 7 (14%) of ovarian tumors included both were solid in consistency. HISTOPATHOLOGICAL SPECTRUM The tumors were classified according to WHO classification of ovarian tumors and the incidence of various histopathological types were noted. Surface Epithelial Tumors The commonest surface epithelial tumors were serous type, 30 cases (60%), followed by mucinous type 16(32%) cases and seromucinous type 4 cases (8%). Mucinous Tumors These tumors constituted the second most common ovarian neoplasms and included 16 cases (32%) of all ovarian tumors. Gross picture of ovarian mass with cut section showing solid and cystic areas with multiple cysts filled with mucinous material. The benign tumors were cystic while borderline and malignant ones were partly solid and partly cystic containing mucinous fluid. Six cases of borderline mucinous were diagnosed in tumor with papillary projections lined by atypical cells up to one- two cell thickness and no stromal invasion was seen. Malignant tumors showed complex papillary pattern with pleomorphic mucinous cells. Areas of necrosis and stromal invasion were seen. Seromucinous Tumors The Present study shows only 04 cases of Borderline seromucinous tumor, constituting 8% of all surface epithelial ovarian neoplasms. IMMUNOHISTOCHEMISTRY Immunoreactivity for P53 was assessed in each case by estimating the Percentage of cells showing nuclear staining, the case was considered positive, when at least more than 5% of the cells showed nuclear positivity. The reaction for p53 was recorded as either positive or negative. P53 expression was seen in majority of malignant tumors, 21/28 i.e., (75%) and in one case of borderline tumor, 1/10, i.e., (10%) and negative in all benign cases, 0/12, i.e.,(0%). P53 MUTATIONS IN OVARIAN NEOPLASMS BY PCR TECHNIQUE In the present study, 5 mutations were detected, and all of them were missense mutations in exon 5. Among them, 4 mutations were seen in serous cystadenocarcinoma and 1 mutation in borderline mucinous tumor. Total of 5 mutations altering the TP53 protein in 50 different patients of benign, borderline, and malignant ovarian neoplasms.

50 cases of surface epithelial ovarian tumors were included in the study. The tumors are categorized according to WHO classification of ovarian tumors (2014). In the present study, we analyzed P53 immunoexpression in SEOTs and in different grades of these neoplasms, and see the variation of P53 mutations in exons 5-8 and to correlate with clinicopathological parameters. Peak incidence of SEOTs was seen in the age group of 40 to 50 years and is comparable with the study done by Purti etal7. In the study done by Chinedu etal (2018)8 most of the cases were in the age range of 50 to 60 years and T.T Sreeja etal (2016)9 majority were in 31 to 40 age group. Many of the studies correlates with increase in age increases the risk of developing EOC, hence the age of developing EOC is variable, mostly in 3rd to 6th decade. Majority of benign tumors were unilateral (86%) and only 14% cases show bilaterality. This is comparable to findings of study done by Jha etal10 (Unilateral - 93.34%) and in contrast to the study of Swamy etal11 , who reported bilaterality rate of 29% in benign tumors. The present study shows malignant tumors with bilaterality rate of 8%, whereas Swamy etal11 study reported bilaterality rate of 50% and Jha etal10 study, 42%. HISTOPATHOLOGY The current study showed a slightly higher percentage of malignant ovarian tumors (56%) but this was similar to the study done by Nisha etal 12 (44%). The study done by Jha etal10 recorded the least percentage of malignant cases (16.1%). Borderline tumors were constituting 26 % and less common were benign, 24%. This study is discordant with studies of Verma and Bhatia13 (66.99%), Swamy etal11(71.6%) and Pradhan A etal14(79.5%). This variation could be because the hospital from which data was collected for this study (MNJ) is a tertiary level specialised hospital for cancer and hence may be majority were malignant. This selection bias may have inflated the number of malignant cases. It was found that surface epithelial tumors to be the most common tumors ranging from 56% (Jha et al) 10 to 71% (Pilli etal) 15in the present and also other studies. The relative percentage of various histological subtypes of ovarian tumors in this study was comparable to most of the studies (Jha etal10, Swamy et al11, Ganga Pilli et al515 etc. Maximum number of cases in the current study were Serous cystadenocarcinoma comprising of 25 cases (50%) similar with the study of Jha et al 10 (46.2%) IMMUNOHISTOCHEMISTRY P53 expression was seen in 57.8% (22/28) of both malignant and borderline tumors. p53 expression was negative (0/12) 0% in benign, (1/10)10% in borderline tumors and 75% in malignant cases (21/28). In the present study, P53 expression is more in malignant tumors (75%) followed by borderline tumors(10%) but negative in all benign tumors (0%) which are in concordance with the studies of Sylvia MT etal16, Kumar A etal17.The study done by Pooja S etal 18 also showed p53 positivity in 75% of the borderline and malignant tumors.P53 expression is more common in malignant serous ovarian tumors19/25 (76%) followed by borderline(10%) and all the benign cases were p53 negative i.e., (0%), which is in concordance with other studies like YWang etal, and Leanne M etal19. Among the 19 malignant serous tumors which showed positive p53 expression, only 3 cases showed mutations (3/19, i.e.,16%). This variation could be due to the fact that some mutations create a stop codon and hence no protein is encoded, or some mutations do not lead to stable proteins that can be detected by IHC. TP53 MUTATION BY DNA ANALYSIS All the mutations in the present study are misssense mutations, with G>A nucleotide change at codon 550 in 4 cases and G>T nucleotide change at codon 417 in 1 case.All of the mutations are in Exon 5 in the present study. This study shows most of the mutations in ovarian tumors were seen in serous tumors i.e., (16%) and was in concordance with studies done by YWANG etal44, and LEANNE M etal19 [Table -9].In comparison to mutations with IHC in this study, the percentage of mutations (exons 5-8) in ovarian tumors , were less than the p53 immunoexpression, but this does not rule out the other mutations in the ovarian tumors and also mutations in other exons. The positive immunoexpression by p53 may also be due to increased normal protein but not due to any mutation. Limitation of the present study include small number of sample size, selection of cases where equal proportion of cases were not included. Large collaborative studies across the spectrum of various tumor types are required.

Histomorphological, immunohistochemical and genetic analysis of 50 cases of ovarian neoplasms was done in correlation with clinicopathological parameters. Among them, 56% were malignant, 20% borderline and 24% were benign tumors. The serous tumors were the commonest histomorphological type, constituting 60% (83.4% were Malignant and 16.6% were benign) followed by mucinous tumors. P53 immunoexpression was more commonly seen in malignant serous ovarian neoplasms, followed by mucinous cystadenocarcinomas and in only one case of borderline tumor out of 6. All the Benign tumors were p53 negative. The role of P53 mutation is evident by increased immunoexpression in malignant and borderline tumors as compared to benign tumors. However, IHC has its limitations. As IHC analysis of TP53 protein accumulation is a poor predictor of Tp53 mutations, accurate assessment of TP53 mutations requires DNA analyses. P53 mutational study done in Exons 5-8 by PCR, only 5 cases out of 50, showed missense mutation in Exon 5. Among them, 4 cases were serous cystadenocarcinoma and the other case is a borderline mucinous cystadenoma. In comparison of mutational study with IHC, the percentage of mutations (exons 5-8) in ovarian tumors were less than the p53 immunoexpression, but this does not rule out the other mutations, and mutations in the other exons in ovarian tumors. Presence of P53 mutation indicates poor prognosis as they are chemo-resistant and might require specific targeted therapy to make them more susceptible to chemotherapy and to improve the outcome. Hence identification of P53 mutation by DNA analysis in ovarian neoplasms is essential.

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