Study Of Activation Of BTAK Expression In Primary Ovarian Surface Epithelial Cells Carrying Heterozygous BRCA Mutation And Tumorigenesis Of Ovarian Carcinoma

Purpose:This study was to determine the morphologic changes in ovariansurface epithelial cells, and BTAK & p53 expression patterns inprophylactically removed ovaries with heterozygous BRCA1/2 mutationas compared with normal ovaries and ovarian cancer controls. We soughtto determine the relationship between BTAK and p53 so as to understandthe tumorigenesis of ovarian carcinoma.Methods:1. Morphologie features in ovarian surface epithelial cells withheterozygous BRCA1/2 mutation:Two pathologists (ZZ and DGR) observed the morphologic changesunder microscopy independently in a blinded manner in ovaries of 32patients with known BRCA1/2 mutation that underwent prophylacticoophorectomy and 27 normal ovaries from patients without any knownmutation. The evaluation of the following morphologic features in H&Estained slides includes deep surface epithelial invaginations, epithelialinclusion cysts, cortical stromal hyperplasia, psammoma bodies,endometriosis, surface papillations, papillomatosis, epithelial hyperplasia (defined by epithelial nuclear stratification), epithelial dysplasia (definedby epithelial hyperplasia with atypia, pleomorphism, and loss of polarity).2. Expression of BTAK in primary ovarian epithelial cells with orwithout BRCA1 heterozygous mutation:We performed immunohistochemical staining of morphologically normalovaries in a cohort of 32 patients who had prophylactic surgery andcompared these with 27 normal ovaries and 194 ovarian cancer controls.The BTAK intensity of staining was analyzed by computerized imageanalysis. The mean relative optical density was expressed as arbitraryunits of intensity and used for analysis. For statistical purposes opticaldensity values were grouped in a 4 score grading system as the meanoptical density±SD (177.46±15.10). Absence of staining was defined asnegative and given a score of 0, weak expression a score of 1, moderateexpression a score of 2 and strong expression a score of 3. Western blotanalysis of BTAK was performed in a primary cell culture carryingheterozygous BRCA1 mutation (OSE76) and three normal ovariansurface epithelial cell cultures (OSE72, OSE103, OSE137).3. Expression of p53 in primary ovarian epithelial cells with orwithout BRCA1 heterozygous mutation and study oftumorigenesis on ovarian carcinoma:We performed immunohistochemical staining in 32 patients with BRCAmutation compared these with 27 normal ovaries and 194 ovarian cancercontrols. Evaluation of the average expression for p53 expression wasperformed visually by two pathologists (ZZ and DGR) as follows: 0, lessthan 10% nuclear staining; 1, more than10%, less than25% nuclear staining; 2, more than 25%, less than 50% nuclear staining; 3, more than50% nuclear staining, discrepancy was resolved by third pathologist (JL).The relationship between expression of BTAK and p53 was analyzedwith Spearman Rank Order Correlation test. We developed aretrovirus-based system for stably expressing siRNA against p53 geneand then analyzed the BTAK expression by Western Blot. Withunderstanding of the relationship between BTAK expression and p53expression, the carcinogenesis of ovarian carcinoma was analyzed.Results:1. Morphologic Change:There was significantly difference in histological alterations betweencases and controls, especially including hyperplastic or dysplastic lesions(p=0.0418 and p=0.0289) on hematoxylin and eosin-stained sections.Normal ovaries have less frequency of pathologic lesions than BRCAmutation group (p=0.0341). The two groups both have deep surfaceepithelial invaginations, cortical stromal hyperplasia, surface papillations,but there were no significance.2. Expression of BTAK:The expression of BTAK was increased in 21 of 32 (66%) ovariescarrying a heterozygous BRCA mutation as compared with 27 normalcontrol ovaries which 4 of 27 (15%) positive for BTAK expression(p＜0.05; Mann-Whitney test). While ovarian cancer showed furtherincrease in BTAK as compared with BRCA heterozygous ovaries ina11-194 cases (p＜0.001; Mann-whitney test). The BTAK expression was significantly increased in primary culture carrying a heterozygousBRCA1 mutation (OSE76) as compared to those with no knownBRCA1/2 mutation (OSE72, OSE103 and OSE137) by Western blotanalysis. A specific band of BTAK protein (46 kDa) was detectedstrongly in OSE76 cell line, but no clear bands were found in OSE72,OSE 103 and OSE137.3. Expression of p53:The expression of p53 was positive in 1 of 27 cases (4%) in normal ovary,weakly increased in 10 of 32 (31%) positive in ovaries with knownBRCA mutation and highly expressed in 139 of 194 (72%) positive inovarian carcinomas. There were significantly difference expressionbetween ovarian carcinomas and ovaries with BRCA mutation (p＜0.05;Mann-Whitney test). Using Spearman correlation rank order test, weobserved BTAK was correlated with p53 (r=0.306, p＜0.001).4. Study of tumorigenesis:We developed a retrovirus-based system for stably expressing siRNAagainst p53 gene. SiRNA targeting p53 reduces expression of BTAK inOSE76 Cell Line. High expression of BTAK was in OSE76 cell line(46kDa) and no expression of BTAK was detected in OSE76 after p53siRNA introduction.Conclusions:1. The current study suggests pathologic evidence for the existence ofpreneoplastic changes in ovarian surface epithelium and support thepreviously proposed concept of ovarian dysplasia. The specimen of prophylactic oophorectomy is the best sample for findingpremalignant alterations in histology and early change intumorigenesis of ovarian carcinoma.2. Increased expression of BTAK is directly correlated with mutationstatus of BRCA1/2 genes, suggesting that mutation in a single alleleof either BRCA1 or 2 may be responsible for the activation of BTAK.This activation may be a key early genetic event in the developmentof hereditary ovarian cancer. Increased expression of BTAK may bean early signal in ovarian cancer and can be a potential diagnosticmarker and offer a novel target for chemoprevention.3. Increased expression of p53 is directly correlated with mutation statusof BRCA1/2 genes, suggesting that mutation in a single allele ofeither BRCA1 or 2 may be responsible for the activation of p53.BTAK expression correlates with p53 and their expressions may be anearly genetic event in the development of human ovarian cancer,especially in BRCA mutant patient. The interaction of BRCA, BTAKand p53 regulates the initiation of tumorigenesis in ovariancarcinoma.