ROLE OF PERITONEAL FLUID CYTOLOGY AND BIOLOGICAL (2).pptx

Role of p53,bcl2 and mib1 expression in malignant ovarian tumors

1. Title of the paper: “ROLE OF PERITONEAL FLUID CYTOLOGY AND BIOLOGICAL SIGNIFICANCE OF P53,BCL2 AND MIB1 IMMUNOEXPRESSION IN OVARIAN NEOPLASMS.” PRESENTER: Dr Abhishek Jaiswal CO- AUTHORS: Dr Nidhi Verma , Dr Deepali Mittal PRESENTER’S AFFILIATION: LLRM MEDICAL COLLEGE , MEERUT

2. INTRODUCTION

3. Ovarian cancer accounts for 2.5 % of all malignancies among female and 5% of female cancer death (1) . R isk factor (1) :- 1 Age, 2 F amily history of ovarian or breast cancer 3 I nheritance of BRCA-1 , BRCA-2 gene mutation. Wide histological spectrum.

4. Peritoneal dissemination of ovarian tumors is a major prognostic parameter in ovarian malignancies ( 2) . Analysis of peritoneal fluid cytology serves as a useful predictor of ovarian surface involvement and peritoneal metastasis even in the absence of clinical omental spread ( 2) .

5. Immunohistochemistry is a valuable adjunct. P53, BCL2(anti-apoptotic marker) and MIB1 serve as useful biomarkers for malignant ovarian neoplasm Programmed cell death or Apoptosis in maintenance of tissue homeostasis and pathogenesis of tumors .

6. Genetic Factors , important contributor to the regulation of both cell proliferation and apoptosis. Aberrant regulation of cell proliferation and apoptosis play an important role in tumor growth and development.

7. P53 protein produced in response to DNA damage and restrains cellular proliferation by binding to specific regions of DNA. P53 gene also regulates expression of the anti-apoptotic BCL2 gene (3) . Mutation of the P53 gene constitute the most common molecular genetics change associated with many cancers including ovarian cancer.

8. BCL2 (anti-apoptotic gene) is a proto-oncogenes (4) . I t is located on the mitochondrial membranes (4) . I nterferes with programmed cell death independently of its ability to promote cell division (4 ) .

9. Ki-67( MIB 1 ) is an axillary protein of DNA polymerase. DNA polymerase is a n enzyme involved in the catalysis of DNA synthesis and repair. The total proliferative compartment of the tumor can be determined by the expression of this protein ( 5) .

10. The present study examined 1 . Role of peritoneal fluid cytology in malignant ovarian neoplasm. 2 . The extent of apoptosis, apoptosis regulatory proteins P53 and BCL2 as well as the extent of tumor cell proliferation reflected by MIB1 expression in malignant ovarian tumors.

11. AIMS AND OBJECTIVES

12. To study cytological findings of peritoneal fluid in cases of ovarian neoplasms. To study immuno-expression of P53, BCL2 and MIB1 in malignant ovarian neoplasms.

13. MATERIAL AND METHODS

14. Study was done in Department of Pathology LLRM Medical college , Meerut in collaboration with the Department of Gynecology , SVBP hospital Study design : Prospective study Study period : from June 2022 to J une 2024 Sample size : 50

15. METHOD : Cytology of peritoneal fluid : The cytospin preparation was done. S tained with 1. May Grunewald Giemsa (MGG), 2. Papanicolaou stain 3. Hematoxylin and Eosin .

16. Histological examination : 3–5 μ thick H and E stained sections of 50 cases of ovarian neoplasms in 10% neutral buffered formalin. F ollowed by immunohistochemical staining for P53, BCL2 and MIB1 using primary antibodies

17. For immunohistochemistry:- extra 2-4 μ thick sections on Poly-L lysine coated slides. A ntigen retrieval by digital pressure cooker method. I ncubated with the HRP conjugated secondary antibody for 45 min. Reaction products were revealed with diaminobenzidine as the chromogen. C ounterstained with Harris's hematoxylin .

18. Postive control- tonsil( For BCL2). Negative control- Primary antibody substituted with phosphate buffer saline. Nuclear positivity for P53 and MIB1- dark brown color on bluish background. Cytoplasmic positivity for BCL2 – brown color.

19. RESULTS INTERPRETATION : IHC scoring for P 53 : I ntensity scoring system. Nuclear staining was interpreted as positive reaction (6) . NEGATIVE - (0) : no expression of nuclear protein, WEAK STAINING - (1) : 0–25% of the total cells shows positive staining in the nucleus MODERATE STAINING - (2) : >25– 75% of the total cells show positive nuclear staining, STRONG STAINING - (3) : >75–100% cells show positive nuclear staining.

20. IHC scoring for BCL2 : Cell proportion scoring system. C ytoplasmic membrane staining was interpreted as positive reaction (6) . SCORE ‘0’ or NEGATIVE - < 5% stained neoplastic cells SCORE ‘1’ or WEAK POSITIVE - 5-19% stained neoplastic cells SCORE ‘2’ or MODERATE POSITIVE - 20-50% neoplastic cells stained SCORE ‘3’ or STRONG POSITIVE - >50% neoplastic cells stained. 

21. MIB 1 Labeling Index (MIB 1 LI) : Percentage of cells with nuclear positivity. C alculated by counting 500 cells for nuclear staining in the highest proliferating area at 400 x magnification 5 . Low proliferative index (0%-10%) Moderate proliferative index (more than 10% and up to 25%) High proliferative index (25% and more)

22. INCLUSION CRITERIA : All malignant ovarian tumors along with peritoneal fluid in patients presenting with ascites will be included in the study. EXCLUSION CRITERIA : All cases with poorly preserved specimen and / or inadequate fluid for cytological examination. Patients presenting with ascites due to causes other than ovarian neoplasms.

23. OBSERVATION AND RESULT

24. S.No . Histological diagnosis No. Of cases Percentage (%) 1) surface epithelial tumors a) serous carcinoma – high grade low grade b) Mucinous carcinoma- c) Endometrioid carcinoma- d) Seromucinous carcinoma- 46 92 36 72 04 08 03 06 02 04 01 02 2) germ cell tumor a) Dyegerminoma b) Yolk sac tumor c) Choriocarcinoma 03 06 01 02 01 02 01 02 3) metastatic carcinoma 01 2 TOTAL 50 100 Table: 1 Morphological Spectrum of cases according to final histological diagnosis (n= 50 )

25. Table : 2 Age wise Distribution of total cases (n=50) AGE DISTRIBUTION NUMBER OF CASES PERCENTAGE (%) 31-40 5 10 41-50 10 20 51-60 20 40 61-70 13 26 71-80 2 4 Total 50 100

26. Sr. no Histological diagnosis Peritoneal fuide for malignant cells + ve -ve 1 surface epithelial tumors a) serous carcinoma – high grade (23) low grade(02) b) Mucinous carcinoma(2)- c) Endometrioid carcinoma- d) Seromucinous carcinoma(1)-  23(76.7%)  5(16.7%) 20(86.9%) 3(13.1%) 1(50%) 1(50%) 1(50%) 1(50%) - - 1(100%) 2 germ cell tumor a) Dyegerminoma b) Yolk sac tumor c) Choriocarcinoma(1) 1(3.3%) - - - - 1(100%) O 3 metastatic carcinoma(1)  1(3.3%)  0 TOTAL 25(83.3%) 5(16.7%) Table 4: P eritoneal fluid cytology for malignant cells in histological variants of Ovarian neoplasms(n= 30 )

27. Sr. no Histological diagnosis IHC P53 + ve -ve 1 surface epithelial tumors a) serous carcinoma – high grade(36) low grade(4) b) Mucinous carcinoma(3)- c) Endometrioid carcinoma(2)- d) Seromucinous carcinoma(1)-  37(74%)  09(18%) 30(83.3%) 6(16.7%) 2(50%) 2(50%) 2(66.7%) 1(33.3%) 2(50%) 1(100%) 2 germ cell tumor a) Dyegerminoma (1) b) Yolk sac tumor (1) c) Choriocarcinoma(1) 2(4%) 1(2%) 1(100%) 1(100%) 1(100%) o 3 metastatic carcinoma(1)  1(2%)  0 TOTAL 40(80%) 10(20%) Table 4: Expression of P53 in histological variants of Ovarian neoplasms (n=50)

28. Sr. no Histological diagnosis IHC BCL2 + ve -ve 1 surface epithelial tumors a) serous carcinoma – high grade(36) low grade(4) b) Mucinous carcinoma(3)- c) Endometrioid carcinoma(2)- d) Seromucinous carcinoma(1)-  24(48%)  22(44%) 15(41.7%) 21(58.3%) 4(100%) 2(66.7%) 1(33.3%) 2(100%) 1(100%) 2 germ cell tumor a) Dyegerminoma (1) b) Yolk sac tumor (1) c) Choriocarcinoma(1) 0(0%) 3(6%) 1(100%) 1(100%) 1(100%) 3 metastatic carcinoma(1)  1(2%)  0 TOTAL 26(52%) 24(48%) Table 4: Expression of BCL2 in histological variants of Ovarian neoplasms (n=50)

29. Sr. no Histological diagnosis IHC MIB1 + ve -ve 1 surface epithelial tumors a) serous carcinoma – high grade(36) low grade(4) b) Mucinous carcinoma(3)- c) Endometrioid carcinoma(2)- d) Seromucinous carcinoma(1)-  32(64%)  14(28%) 26(72.2) 10(27.8%) 2(50%) 2(50%) 2(66.7%) 1(33.3%) 2(100%) 1(100%) 2 germ cell tumor a) Dyegerminoma (1) b) Yolk sac tumor (1) c) Choriocarcinoma(1) 2(4%) 1(2%) 1(100%) 1(100%) 1(100%) o 3 metastatic carcinoma(1)  1(2%)  0 TOTAL 36(72%) 14(28%) Table 4: Expression of MIB1 in histological variants of Ovarian neoplasms (n=50)

30. RESULTS :- Present study was done on 50 cas es of ovarian neoplasms. Out of total 50 cases studied, 92% were surface epithelial tumors , 06% germ cell tumor and 02% metastatic carcinoma. Peritoneal fluid for malignant cells was seen positive in 83.3% of cases High grade carcinoma was found to have a significantly higher frequency of positive peritoneal fluid cytology i.e. in 86.9% of the high grade serous carcinoma cases.

31. Out of the 50 cases p53 nuclear positivity was seen in 74% cases . P53 accumulation was positively correlated with grade i.e. low grade serous carcinoma 50% positivity and high grade serous carcinoma 83.3 % positivity. BCL2 cytoplasmic positivity was seen in 54% of total cases. An inverse correlation was found with grade i.e. low grade serous carcinoma (100%) show increased positivity than high grade serous carcinoma (41.7%).

32. MIB1 expression was seen positive in 72% of the total cases. MIB1 lebelling index was positively correlated with grade i.e. high grade serous carcinoma (72.2%) show increased positivity than low grade seous carcinoma (50%). There was a positive correlation between P53 and MIB1 expression Whereas no significant correlation was found between P53 and BCL2 or MIB1 and BCL2 expression.

33. P53 expression in High Grade Serous Carcinoma (magnification 40x) b)

34. BCL2 expression in Low Grade Serous Carcinoma (magnification 40x)

35. MIB1 expression in High Grade Serous Carcinoma (magnification 40x)

36. Peritoneal wash cytology in High Grade Serous Carcinoma ( May- Grünwald Giemsa stain, magnification 100x)

37. DISCUSSION

38. Positive peritoneal fluid cytological findings represent an important predictive factor for prognosis. The sensitivity of peritoneal cytology stated in a study by Runyon et al (1994) can be upto 97%, depending on the study, disease stage and peritoneal inclusion (12) . According to a study by Zuna et al(1996), the sensitivity of peritoneal cytology was 82.9%. (13)

39. As per a study by Cheng et al(1998), sensitivity of peritoneal fluid cytology was 94%. (14) An another recent study by Cha ndralekha Janagam et al. (2017), sensitivity of peritoneal fluid was 90%. (18) In our study, the sensitivity was 86.7% and correlated well with the above studies. The result of cytology of peritoneal fluid is an important parameter in the diagnosis, staging, therapeutic approach and disease prognosis.

40. Several studies have been devoted to identifying novel prognostic indicators for ovarian carcinomas. (6,7,8,9) In a study J.E. PALMER*, et.al.(2008) found p53 positivity and overexpression to correlate with advancing tumor grade and bcl-2 was not found to correlate with p53 parameter. (6) In an a nother study Nesrin Gursan et.al. (2009) found a statistically significant correlation between the expression of p53 and Ki-67 LI and a significantly negative relationship between bcl-2 and p53 expression. (7)

41. Alexandra Kalogeraki et al (2015), found a statistically significant correlation between P53 and MIB1 expression correlated with tumor grade and a statistically significant correlation between BCL2 expression and tumor grade. (9) In above study A. Kalogeraki et.al. also found a positive correlation between P53 and MIB1 but no significant association was found between P53 and BCL2 expression or MIB1 labeling index. (9) Our results were also in accord with previous reports.

42. N uclear P53 accumulation was ascertained in more than 50 % of ovarian carcinomas and was found to be statistical correlated with the tumor grade. In our study BCL 2 expression was found in 52% of our cases and in more than 10% of neoplastic cells was significantly correlated with the grade of the tumors. This study clearly showed that MIB1 was positively correlated with grade of the tumors expressed in 72% of our cases and in more than 30% of the neoplastic cells.

43. A positive correlation between MIB 1 labeling index and P53 accumulation was ascertained. W hereas no significant association was found between BCL2 immunoreactivity and P53 accumulation or MIB 1labeling index in accord with previous study. (17)

44. CONCLUSION

45. Our data showed peritoneal cytology of ascitic fluid is highly sensitive for detection of ovarian malignancies. In addition to being an indicator of peritoneal metastasis, positive cytology also correlates with capsular invasion and histologic type in ovarian tumors. Therefore, it should always be used as an adjunctive tool in the surgical management of ovarian tumors.

46. Our data also showed significant correlation between tumor grade and expression of P53, BCL 2, and MIB1 in malignant ovarian neoplasms. Our study showed a possible role of P53, BCL2 and MIB1 as supplemental tool in prognosis as well as helps in future appropriate treatment.

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