BRCA1基因复杂甲基化模式

应用体外甲基化酶M-SssI处理和甲基化敏感单链构象分析法(Methylation-sensitivesingle-strand conformation     

卵巢癌细胞中OY-TES-1基因启动子CpG位点甲基化状态分析

目的:了解卵巢癌SKOV3细胞中OY-TES-1启动子区域CpG位点的甲基化状态.方法:运用免疫细胞化学法检测SKOV3细胞中OY-TES-1蛋白表达;通过硫化测序PCR法,分析SKOV3细胞中OY-TES-1启动子区域CpG位点的甲基化状态,并与睾丸组织相比较.结果:免疫细胞化学法检测结果显示,SKOV3细胞中OY-TES-1蛋白呈阳性反应;在检测的OY-TES-1启动子区域(转录起始点-127 bp～+110 bp,含24个CpG位点)中,CpG位点的甲基化频率为80.21％,其甲基化状态明显高于睾丸组织.结论:SKOV3细胞中OY-TES-1启动子CpG位点处于高甲基化状态,进一步可探讨甲基化抑制剂氮杂脱氧胞嘧啶对OY-TES-1甲基化状态及其表达水平的影响.     

Recurrent copy number alterations in BRCA1-mutated ovarian tumors alter biological pathways

Array CGH was used to identify recurrent copy number alterations (RCNA) characteristic of either BRCA1-related or sporadic ovarian cancer. After preprocessing, both groups of patients were modeled using a recurrent Hidden Markov Model to detect RCNA. RCNA with a probability higher than 80% were called. After removing RCNA present in both groups, the genes present in the remaining RCNA were investigated for enrichment of pathways from external databases. More RCNA were observed in the BRCA1 group, and they display more losses than gains compared to the sporadic group. When focusing on the type of RCNA, no significant difference in length was seen for the gains, but there was a statistically significant difference for the losses. In the sporadic group, a great proportion of the altered regions contain genes known to have a function in cell adhesion and complement activation, whereas the BRCA1 samples are characterized by alterations in the HOX genes, metalloproteinases, tumor suppressor genes, and the estrogen-signaling pathways. We conclude that BRCA1 ovarian tumors present a different type, number, and length of RCNA; a huge amount of the genome is lost, resulting in important genomic instability. Moreover, important biological pathways are altered differentially when compared to the sporadic group. Hum Mutat 30:1–10, 2009. © 2009 Wiley-Liss, Inc.     

Mutation analysis of BRCA1, TP53, and KRAS2 in ovarian and related pelvic tumors

Cancer may be viewed as a genetic disease resulting from critical mutations that disrupt normal cell growth. To characterize the involvement of the BRCA1 and TP53 tumor suppressor genes and of the KRAS2 protooncogene in gynecologic cancer, mutation analysis of these genes was conducted in pelvic tumors of 85 patients that included 49 epithelial ovarian carcinoma cases. The 85 pelvic tumors contained 5 tumors with BRCA1 mutations, 33 with TP53 mutations, and 1 with a KRAS2 mutation. Each of the BRCA1 and KRAS2 mutations, and 25 of the TP53 mutations, were in ovarian carcinomas. Four of the BRCA1 mutations were germline and 1 was somatic. The 4 patients with germline BRCA1 mutations had an early age of disease onset (33-48 years) relative to the mean age of onset (58 years) of all 49 ovarian carcinoma patients, and 3 of these 4 patients had a family history of ovarian or breast cancer. None of the 4 tumors with germline BRCA1 mutations had a KRAS2 mutation or a TP53 mutation, despite a 51% frequency of TP53 mutations in the 49 ovarian carcinomas. Three of the 4 tumors with germline BRCA1 mutations retained a wild-type BRCA1 allele. The tumor with the somatic BRCA1 mutation contained a TP53 mutation and had no evidence for wild-type BRCA1 and TP53 alleles. These data suggest that both BRCA1 and TP53 were inactivated in 1 of 49 ovarian carcinomas. Moreover, mutational inactivation of both BRCA1 and TP53 did not occur in 4 tumors with a germline BRCA1 mutation. It has been proposed that tumorigenesis in cells with a heterozygous BRCA1 mutation requires inactivation of the wild-type BRCA1 and TP53 alleles, which results in genomic instability and acquisition of mutations in protooncogenes. Clearly, mutational inactivation of TP53 and the wild-type BRCA1 allele in ovarian tumors with a heterozygous, germline BRCA1 mutation is not an absolute requirement for tumor formation. It is possible that these alleles may be inactivated by nonmutational mechanisms or that other tumor formation pathways exist.     

Aberrant hypermethylation of RASSF1A promoter in ovarian borderline tumors and carcinomas

The newly identified 3p21.3 tumor suppressor gene RASSF1A is inactivated by hypermethylation in variable solid tumors, including those of the lung, breast, prostate, kidney, and ovary. The purpose of this study was to evaluate the methylation status of RASSF1A in various types and stages of ovarian epithelial tumors. We analyzed the DNA methylation status of ovarian tumors using methylation-specific polymerase chain reaction in 54 frozen ovarian tumor tissues and in 97 cases of archival ovarian serous epithelial tumors using a microdissection procedure. Hypermethylation statuses were examined vs clinicopathologic findings. RASSF1A promoter methylation rates in the various types of fresh ovarian tissues were as follows: serous cystadenoma (1/5), serous tumor of borderline malignancy (2/7), serous adenocarcinoma (4/10), mucinous cystadenoma (0/5), mucinous tumor of borderline malignancy (2/7), mucinous adenocarcinoma (3/6), transitional-cell carcinoma (1/3), clear-cell carcinoma (3/3), and malignant müllerian mixed tumor (3/3). In archived serous tumor tissues, RASSF1A promoter hypermethylation was detected in serous cystadenoma (1/6, 16.6%), serous tumor of borderline malignancy (20/41, 48.8%), and in serous adenocarcinoma (25/50, 50%). The status of RASSF1A hypermethylation in borderline tumors was found to correlate statistically with the presence of microinvasion (p=0.002), peritoneal implant (p<0.001), and bilaterality (p=0.019). The RASSF1A promoter hypermethylation was frequently found in borderline tumors and carcinomas, suggesting that RASSF1A promoter hypermethylation may be a useful molecular marker for the early detection of ovarian tumors.     

BRCA1 mutation analysis in breast/ovarian cancer families from Greece

Germline mutations in BRCA1 gene account for varying proportions of breast/ovarian cancer families, and demonstrate considerable variation in mutational spectra coincident with ethnic and geographical diversity. We have screened for mutations the entire coding sequence of BRCA1 in 30 breast/ovarian cancer women with family history of two or more cases of breast cancer under age 50 and/or ovarian cancer at any age. Genomic DNA from patient was initially analyzed for truncating mutations in exon 11 with PTT followed by DNA sequencing. In the cases where no frameshift mutation was observed in exon 11, all other exons were screened with direct sequencing. Two novel (3099delT, 3277insG) and three already described (3741insA, 1623del5-TTAAA, 5382insC-twice) truncating mutations were identified. In addition, 6 point mutations (L771L, P871L, E1038G, K1183R, S1436S, S1613G) which are already classified as polymorphisms were identified. Three unclassified intronic variants (IVS16-68 G>A, IVS16-92 G>A, IVS18+65G>A) were also detected. These results show that BRCA1 deleterious mutations are present in a fraction (20%) of Greek breast/ovarian cancer families similar to other European countries. Mutations were detected in high- (>/=3 members) as well as in moderate-risk (2 members) families. This is the first report of BRCA1 mutation analysis in Greece.     

Expression of BRCA1 protein in benign, borderline, and malignant epithelial ovarian neoplasms and its relationship to methylation and allelic loss of the BRCA1 gene

BRCA1 is a putative tumour suppressor gene responsible for a hereditary ovarian cancer syndrome. To clarify the possible involvement of BRCA1 in the development of sporadic ovarian neoplasms, this study analysed the immunohistochemical expression of BRCA1 protein in normal ovarian surface epithelium and 119 epithelial ovarian tumours (19 benign, 24 borderline, and 76 malignant tumours). Loss of heterozygosity (LOH) of BRCA1 was examined using three microsatellite markers to analyse the relationship between BRCA1 expression and alterations of the BRCA1 gene. Methylation of the BRCA1 promoter was also analysed by methylation-specific PCR. In ovarian carcinomas showing heterogeneous expression of BRCA1 protein in the same tumour, LOH and methylation status were analysed using microdissection techniques. Finally, the relationship of BRCA1 expression or its genetic alteration to clinicopathological parameters and patient survival was analysed. Ovarian surface epithelial cells expressed BRCA1 protein. Decreased expression of BRCA1 was found in 16% of benign tumours, 38% of borderline tumours, and 72% of carcinomas. LOH of BRCA1 was demonstrated in no benign tumours, 15% of borderline tumours, and 66% of carcinomas. Methylation of BRCA1 was not detected in benign or borderline tumours, but was present in 31% of carcinomas. Reduced expression of BRCA1 correlated with the presence of gene methylation. The frequency of BRCA1 methylation and LOH was higher in serous carcinomas than in other types. In one of the three serous carcinomas that showed heterogeneous expression of BRCA1, BRCA1-positive borderline-like tumour cells were LOH-positive and methylation-negative, whereas adjacent BRCA1-negative carcinoma cells were LOH-positive and methylation-positive. The prognosis of carcinoma patients did not correlate with BRCA1 expression or genetic status. These findings suggest that reduced expression of BRCA1 protein along with genetic and epigenetic changes of the BRCA1 gene play an important role in the development of sporadic ovarian carcinomas, particularly those of serous histology.     

Genetic analysis of the BRCA1 region in a large breast/ovarian family: refinement of the minimal region containing BRCA1

We have analyzed a single multi-affected breast/ovarian cancerpedigree (BOV3) and have shown consistent inheritance of markerson chromosome 17q with the disease confirming that this familyis due to the BRCA1 gene. Analysis of 17q hapiotypes shows arecombination event in a bilateral breast cancer case whichsuggests that the BRCA1 gene lies distal to D17S857; D17S857is thus the new proximal boundary for the region containingBRCA1. Combining this information with previously publishedmapping information suggests that BRCA1 is contained in a regionestimated at 1 – 1 .5 Mb in length. All seven breast tumour/bloodpairs examined from this family show loss of heterozygosityin the tumours. The allele retained in each tumour was fromthe disease-bearing chromosome implicating BRCA1 as a tumoursuppressor gene. We have sequenced the 17ß-oestradioldehydrogenase genes (EDH17B1 and EDH17B2) which have been suggestedas candidate genes for BRCA1 in four members of this family.No germline mutations were detected.     

BRCA1 and BRCA2 mutation analysis in 86 early onset breast/ovarian cancer patients.

Eighty-six women fulfilling specific selection criteria were studied for germline mutations in two breast cancer susceptibility genes, BRCA1 and BRCA2, using the protein truncation test (PTT). Nine germline mutations were identified, six in BRCA1 and three in BRCA2. Of the six BRCA1 mutations, three have previously been described and three are new, and for BRCA2, one is a new mutation and the other two appear to occur at a site that has been described several times. Four kindreds were breast cancer families, one a breast/ovarian cancer family, and the sixth an ovarian cancer family. The three kindreds with BRCA2 mutations were classified as one breast/ovarian cancer family, one breast cancer family, and one family which harboured one early onset breast cancer patient and two melanoma patients. The mutations in BRCA1 were either insertions, deletions, or transitions which all resulted in a premature stop codon. Mutations in BRCA2 were all frameshift mutations as a result of either 2 or 4 bp deletions. Two BRCA2 mutations were identical, suggesting a Swiss founder effect which was confirmed by haplotype sharing. The 10% mutation detection rate is compatible with the relaxed criteria used for patient selection. Considering the relative ease with which coding sequences can be screened by PTT, this assay is useful as a first screen for BRCA1 and BRCA2 mutations.     

Histopathology of familial ovarian tumors in women from families with and without germline BRCA1 mutations

Breast cancers from patients with germline BRCA1 mutations show characteristic histopathologic features. However, similar studies of BRCA1-associated ovarian cancers have reported inconsistent findings. Interobserver differences in histopathologic classification are a significant source of variation, and most studies have obtained histopathologic information from pathology reports rather than from review of histopathology slides. We therefore reviewed the histopathology slides and pathology reports to determine histologic type, grade, and stage for cancers of the ovary or peritoneum in 217 women from 126 families enrolled in the Gilda Radner Familial Ovarian Cancer Registry. Peripheral blood DNA from at least 1 affected member of each family was analyzed for BRCA1 mutations, and tumors from BRCA1 mutation-positive families were compared with those from BRCA1-negative families. Of 66 patients from 36 BRCA1-positive families, 64 had ovarian carcinoma, 1 had an ovarian carcinoma in situ, and 1 had a dysgerminoma. Of 151 patients from 90 BRCA1-negative families, 135 had ovarian carcinoma, 10 had ovarian borderline tumors, 3 had ovarian sex cord/stromal tumors, and 3 had primary peritoneal carcinoma. There were fewer grade 1 (P <.001) and stage I (P =.10) cancers in patients from BRCA1-positive families than in patients from BRCA1-negative families. Neither mucinous nor borderline tumors were found in the BRCA1-positive families. Ovarian cancers arising in women from BRCA1-positive families are more likely to be high grade and nonmucinous than cancers arising in women from BRCA1-negative families. The absence of borderline tumors in patients from BRCA1-positive families adds to accumulating evidence that BRCA1 mutations do not play a role in the development of these tumors. HUM PATHOL 31:1420-1424.     

Mutational analysis of BRCA1 and BRCA2 genes in Chinese ovarian cancer identifies 6 novel germline mutations

Germline mutations in the BRCA1 and BRCA2 genes predispose women to breast and ovarian cancer. An incidence of 5% and 3.3% respectively has been reported of BRCA1 and BRCA2 mutations in women with ovarian cancer unselected for family history. The contribution of BRCA1 and BRCA2 mutations to ovarian cancer in Chinese women is unknown. A total of 60 samples of ovarian cancer diagnosed in Chinese unselected for age or family history were analyzed for BRCA mutations using the protein truncation test. The entire coding exon of BRCA1 of 53 cases and that of exon 11 of BRCA2 of 43 cases were successfully screened. Six germline (11.3%) mutations (633C>T, 1080delT, 1129delA, 2371-2372delTG, 3976-3979delGTGA, and IVS 22+7 A>G) were detected in BRCA1. One germline mutation (3337C>T) (2.1%) was detected in BRCA2. None of these seven cases were associated with strong family history of breast and/or ovarian cancer. Five out of our six BRCA1 mutations and the one BRCA2 mutation identified are novel. Our 11.3% incidence of BRCA1 mutations in ovarian cancer found amongst Chinese with insignificant family history is apparently higher than that previously reported in other populations. It suggests that BRCA1 mutation may play a significant role in the development of sporadic ovarian cancer in Chinese women.     

FISH analysis of BRCA1 copy number in paraffin-embedded ovarian cancer tissue samples

We investigated the BRCA1 gene copy number in unselected ovarian malignancies. Both additional genes (amplification) as well as deletion (loss of heterozygosity, LOH) are often thought to have a role in the initiation or progression of cancer. In addition, if there were little change, deletion studies might help identify BRCA1 mutation carriers. Forty-seven paraffin-embedded ovarian tissue blocks obtained between 1984 and 1997 were used for this study. A sample was "deletion-positive" when BRCA1-deleted cells in the tumor area were significantly different from the benign area. Twenty-five (53%) cases were found to be "deletion-positive". The average age of onset of "deletion-positive" patients was 50.8 years and of "deletion-negative" 57.8 years (P < 0.05). There was no statistical difference between groups in the staging, histology, or prognosis. A Kaplan-Meier study did show a trend towards poorer survival for "deletion-positive" patients. FISH permits unique molecular characterization of malignancies at a cellular level. Double amplification of HER-2 and c-myc predicts poor ovarian cancer survival. There appears to be a definite role for BRCA1 deletion in reducing the age of ovarian cancer onset and possibly in overall survival. Further FISH studies of this and other patient sets using additional molecular markers are needed.     

Microarray analysis of promoter methylation in lung cancers

Aberrant DNA methylation is an important event in carcinogenesis. Of the various regions of a gene that can be methylated in cancers, the promoter is the most important for the regulation of gene expression. Here, we describe a microarray analysis of DNA methylation in the promoter regions of genes using a newly developed promoter-associated methylated DNA amplification DNA chip (PMAD). For each sample, methylated Hpa II-resistant DNA fragments and Msp I-cleaved (unmethylated + methylated) DNA fragments were amplified and labeled with Cy3 and Cy5 respectively, then hybridized to a microarray containing the promoters of 288 cancer-related genes. Signals from Hpa II-resistant (methylated) DNA (Cy3) were normalized to signals from Msp I-cleaved (unmethylated + methylated) DNA fragments (Cy5). Normalized signals from lung cancer cell lines were compared to signals from normal lung cells. About 10.9% of the cancer-related genes were hypermethylated in lung cancer cell lines. Notably, HIC1, IRF7, ASC, RIPK3, RASSF1A, FABP3, PRKCDBP, and PAX3 genes were hypermethylated in most lung cancer cell lines examined. The expression profiles of these genes correlated to the methylation profiles of the genes, indicating that the microarray analysis of DNA methylation in the promoter region of the genes is convenient for epigenetic study. Further analysis of primary tumors indicated that the frequency of hypermethylation was high for ASC (82%) and PAX3 (86%) in all tumor types, and high for RIPK3 in small cell carcinoma (57%). This demonstrates that our PMAD method is effective at finding epigenetic changes during cancer.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

CDH1基因启动子甲基化在上皮性卵巢癌转移方面的研究

目的探讨CDH1基因启动子甲基化对上皮性卵巢癌转移的影响。方法采用免疫组织化学方法检测38例正常卵巢上皮和80例上皮性卵巢癌组织中E-钙黏附素（E-cadherin）表达;应用甲基化特异的PCR（MSP）检测上述组织中CDH1基因启动子区甲基化;应用5-氮-2＇-脱氧胞苷（5-aza-CdR）使SKOV3细胞去甲基化,观察SKOV3细胞体外侵袭性的改变,并通过RT-PCR检测CDH1基因的改变。结果E-cadherin在正常卵巢组织中表达明显高于上皮性卵巢癌（P〈0．05）。34例CDH1基因启动子区甲基化全部出现在卵巢癌组织中,有淋巴结转移组织中甲基化明显高于无淋巴结转移者（P〈0．05）,而CDH1基因启动子区有甲基化的卵巢癌组织中E-cadherin表达明显降低（P〈0．05）。经5-Aza-CdR处理后的SKOV3细胞体外侵袭性降低（P〈0．01）,CDH1基因的表达明显上调（P〈0．01）。结论E-cadherin表达降低与上皮性卵巢癌转移关系密切,CDH1基因启动子区甲基化可能是导致E-cadherin蛋白表达减低的重要原因之一,因此启动子区甲基化与上皮性卵巢癌转移有关。     

Borderline and malignant phyllodes tumors display similar promoter methylation profiles

Mammary phyllodes tumors (PTs) are uncommon fibroepithelial neoplasms. On the basis of histologic criteria, PTs can be divided into benign, borderline, and malignant groups; however, the histologic distinction of PTs is often difficult and arbitrary. In breast cancer, promoter hypermethylation is a common phenomenon, but there are no data available concerning methylation status in PTs. The aim of this study was to assess whether the methylation profiles support the classification of PTs into three subgroups. A multiplex, nested, methylation-specific polymerase chain reaction approach was used to examine promoter methylation of five genes (GSTP1, HIN-1, RAR-β, RASSF1A, and Twist) in 87 PTs (54 benign, 23 borderline, and 10 malignant). Immunohistochemical staining for GSTP1 was performed using tissue microarray blocks to determine whether GSTP1 promoter hypermethylation correlated with loss of GSTP1 expression. There was a trend of increasing methylation frequency with increasing grade of PTs. The methylation frequency of all genes and the mean number of methylated genes in borderline and malignant PTs were higher than those in benign PTs; however, there were no statistically significant differences between borderline and malignant PTs. GSTP1 promoter hypermethylation was associated with loss of GSTP1 expression (p < 0.001). These results suggest that PTs segregate into only two groups on the basis of their methylation profiles: the benign group and the combined borderline/malignant group.     

Recurrent BRCA1 and BRCA2 germline mutations in ovarian cancer: a founder mutation of BRCA1 identified in the Chinese population

Previous mutational analysis for BRCA gene mutations in sporadic ovarian cancer occurring in Chinese patients in Hong Kong identified six germline BRCA1 mutations and one germline BRCA2 mutation, six of which were novel (Khoo et al., 2000). Knowledge of BRCA gene mutations in the Chinese population is relatively scant. In this study, we focussed on whether any of these mutations could be recurrent in our Chinese population, making use of archival paraffin embedded tissue. A consecutive series of 214 ovarian cancer cases, half of Southern Chinese origin from Hong Kong whilst the other half of Northern Chinese origin from Beijing were used for the study. We identified one further novel mutation, 1081delG, in BRCA1. This was found to occur in two unrelated individuals with shared haplotype as revealed by allelotype analysis, thus demonstrating founder effect. Two other recurrent mutations were also identified, the 2371-2372delTG mutation in BRCA1 and the 3337C>T mutation in BRCA2 recurring in two and three unrelated individuals respectively, giving an overall prevalence 4.7% of recurrent BRCA mutations in ovarian cancer in the Southern Chinese population. Most importantly, all our recurrent mutation carriers were identified from Southern Chinese patients from Hong Kong whilst such mutations were absent in samples from the Northern Chinese. Our findings indicate possible heterogeneity in the BRCA genotype between Northern and Southern Chinese. The identification of a founder mutation and two recurrent mutations moreover, has important implications towards screening strategies for breast and ovarian cancer among Chinese of southern ancestral origin who are now dispersed throughout the world.     

Contribution of BRCA1 and BRCA2 mutations to inherited ovarian cancer

A total of 283 epithelial ovarian cancer families from the United Kingdom (UK) and the United States (US) were screened for coding sequence changes and large genomic alterations (rearrangements and deletions) in the BRCA1 and BRCA2 genes. Deleterious BRCA1 mutations were identified in 104 families (37%) and BRCA2 mutations in 25 families (9%). Of the 104 BRCA1 mutations, 12 were large genomic alterations; thus this type of change represented 12% of all BRCA1 mutations. Six families carried a previously described exon 13 duplication, known to be a UK founder mutation. The remaining six BRCA1 genomic alterations were previously unreported and comprised five deletions and an amplification of exon 15. One of the 25 BRCA2 mutations identified was a large genomic deletion of exons 19-20. The prevalence of BRCA1/2 mutations correlated with the extent of ovarian and breast cancer in families. Of 37 families containing more than two ovarian cancer cases and at least one breast cancer case with diagnosis at less than 60 years of age, 30 (81%) had a BRCA1/2 mutation. The mutation prevalence was appreciably less in families without breast cancer; mutations were found in only 38 out of 141 families (27%) containing two ovarian cancer cases only, and in 37 out of 59 families (63%) containing three or more ovarian cancer cases. These data indicate that BRCA1 and BRCA2 are the major susceptibility genes for ovarian cancer but that other susceptibility genes may exist. Finally, it is likely that these data will be of clinical importance for individuals in families with a history of epithelial ovarian cancer, in providing accurate estimates of their disease risks.     

Morphologic patterns associated with BRCA1 and BRCA2 genotype in ovarian carcinoma

This study was undertaken with the hypothesis that certain common morphologic features of ovarian carcinomas are predictably associated with BRCA1 and BRCA2 deficiencies. We selected 43 high-grade serous carcinomas diagnosed at Memorial Sloan-Kettering Cancer Center that were studied as part of The Cancer Genome Atlas pilot project. In addition to 12 randomly selected nonfamilial BRCA-unassociated cases, all 31 Memorial Sloan-Kettering Cancer Center cases with BRCA1 or BRCA2 abnormality were included (n=43). Slides were examined to assess tumor architecture, mitotic index, tumor-infiltrating lymphocytes (TILs), nuclear pleomorphism, necrosis, and involvement of fallopian tube epithelium. Comparing BRCA1-associated cases (BRCA1 germline mutation, n=4, BRCA1 somatic mutation, n=6, BRCA1 promoter methylation, n=13) with unassociated cases (n=12) identified statistically significant differences in morphology. BRCA1-associated high-grade serous carcinomas had more frequent Solid, pseudoEndometrioid, and Transitional cell carcinoma-like morphology (SET features) (P=0.0045), higher mitotic indexes (P=0.012), more TILs (P=0.034), and either geographic or comedo necrosis (P=0.034). BRCA2-associated cases (germline mutation, n=4 and somatic mutation, n=4) tended to show SET features, but they were relatively deficient in TILs and necrosis. Two algorithms incorporating tumor architecture, necrosis, and either mitotic indexes or TILs separated cases that showed 2 of 3 features (BRCA1 associated) from those with 0 of 3 features (BRCA unassociated; P=0.0016 and P=0.0033). A test set comprising 9 BRCA1 germline mutants and 14 high-grade serous carcinoma controls lacking BRCA1 and BRCA2 germline mutation was used to validate the algorithms, with specific emphasis on the ability to detect cases with BRCA1 germline mutation. Best results were obtained with the algorithm that incorporated SET features, necrosis, and mitotic index (P=0.0072; sensitivity of 1.0 (95% CI, 0.66-1.0); specificity of 0.57 (95% CI, 0.29-0.82); positive predictive value of 0.60 (95% CI, 0.32-0.84) and a negative predictive value of 1.0 (95% CI, 0.63-1.0)). These preliminary data indicate potential strong associations between morphology and genotype in high-grade serous carcinomas.