[show abstract][hide abstract] ABSTRACT: The founder mutations in BRCA (BRCA1*185delAG, BRCA1*5382insC, and BRCA2*6174delT) account for 95% of the detectable BRCA mutations in breast and ovarian cancer families of Ashkenazi Jewish ancestry. Optimal clinical management of individuals from these high-risk families relies on the identification of BRCA founder mutations in the laboratory. We have therefore developed a rapid and reliable approach using pyrosequencing, which allows for the detection of these frequent frameshift mutations on different types of specimens. We were able to correctly identify all mutants in a blinded analysis of 177 DNA samples, including 120 DNA samples extracted from paraffin tissues, 30 samples derived from blood specimens, and 27 samples derived from saliva. The mutation detection rate of pyrosequencing was 100% for all of the DNA samples tested with neither false-positive nor false-negative results. The assay also demonstrated both high accuracy and high precision for the detection of these common mutations in paraffin tissues. Furthermore, saliva collection is a noninvasive alternative for DNA isolation in both clinical and research settings. We show that pyrosequencing is a rapid and reliable method that serves as an excellent platform for BRCA founder mutation analysis, especially when only paraffin-embedded tissues are available.
The Journal of molecular diagnostics: JMD 04/2009; 11(3):176-81. · 3.48 Impact Factor
[show abstract][hide abstract] ABSTRACT: For individuals genetically predisposed to breast and ovarian cancer through inheritance of a mutant BRCA allele, somatic loss of heterozygosity affecting the wild-type allele is considered obligatory for cancer initiation and/or progression. However, several lines of evidence suggest that phenotypic effects may result from BRCA haploinsufficiency.
Archival fixed and embedded tissue specimens from women with germ line deleterious mutations in BRCA1 or BRCA2 were identified. After pathologic review, focal areas of normal breast epithelium, atypical ductal hyperplasia, ductal carcinoma-in-situ, and invasive ductal carcinoma were identified from 14 BRCA1-linked and 9 BRCA2-linked breast cancers. Ten BRCA-linked prophylactic mastectomy specimens and 12 BRCA-linked invasive ovarian carcinomas were also studied. Laser catapult microdissection was used to isolate cells from the various pathologic lesions and corresponding normal tissues. After DNA isolation, real-time polymerase chain reaction assays were used to quantitate the proportion of wild-type to mutant BRCA alleles in each tissue sample.
Quantitative allelotyping of microdissected cells revealed a high level of heterogeneity in loss of heterozygosity within and between preinvasive lesions and invasive cancers from BRCA1 and BRCA2 heterozygotes with breast cancer. In contrast, all BRCA-associated ovarian cancers displayed complete loss of the wild-type BRCA allele.
These data suggest that loss of the wild-type BRCA allele is not required for BRCA-linked breast tumorigenesis, which would have important implications for the genetic mechanism of BRCA tumor suppression and for the clinical management of this patient population.
Annals of Surgical Oncology 10/2007; 14(9):2510-8. · 4.12 Impact Factor
[show abstract][hide abstract] ABSTRACT: A major limitation in counseling unaffected women from families with inherited breast and ovarian cancer is that a "true-negative" interpretation of wild type BRCA analysis of the proband cannot be inferred in the absence of demonstration of a BRCA mutation segregating in the kindred. Documentation of familial BRCA mutations from paraffin-derived DNA of deceased patients has been limited due to reports of technical complications leading to lack of reproducibility of BRCA testing of archival material.
DNA was extracted from formalin-fixed paraffin-embedded (FFPE) morphologically normal tissue of 161 blinded, coded samples from women previously genotyped for the three Ashkenazi Jewish BRCA founder mutations from lymphocyte-derived DNA. Multiplex PCR followed by denaturing polyacrylamide gel electrophoresis was performed for the three founder mutations to determine if analysis on FFPE tissue could produce results concordant with those of the lymphocyte-derived DNA.
After disclosure of the sample codes, the results were compared with the original lymphocyte-derived DNA genotypes. Excluding one sample unevaluable due to PCR failure, there was 100% concordance of 160 genotypes (120 mutation samples) derived from DNA from archival FFPE tissue compared to peripheral lymphocytes.
The method described reliably detected BRCA founder mutations in archival DNA derived from FFPE tissue. These results suggests that this technique may be useful in clinical settings to inform wild type BRCA results of unaffected probands, leading to avoidance of unnecessary intensified surveillance or risk-reducing surgery. With further validation this approach can also be applied to other populations where founder mutations are observed.
Familial Cancer 02/2006; 5(4):337-42. · 1.94 Impact Factor
[show abstract][hide abstract] ABSTRACT: The goal of this study was to determine whether distinct gene expression profiles are associated with intrinsic and/or acquired chemoresistance in epithelial ovarian carcinoma.
Gene expression profiles were generated from 21 primary chemosensitive tumors and 24 primary chemo-resistant tumors using cDNA-based microarrays. Gene expression profiles of both groups of primary tumors were then compared with those of 15 ovarian carcinomas obtained following platinum-based chemotherapy ("post-chemotherapy" tumors). A theme discovery tool was used to identify functional categories of genes involved in drug resistance.
Comparison of primary chemosensitive and chemo-resistant tumors revealed differential expression of 85 genes (P < 0.001). Comparison of gene expression profiles of primary chemosensitive tumors and post-chemotherapy tumors revealed more robust differences with 760 genes differentiating the two groups (P < 0.001). In contrast, only 230 genes were differentially expressed between primary chemo-resistant and post-chemotherapy groups (P < 0.001). Common to both gene lists were 178 genes representing transcripts differentially expressed between post-chemotherapy tumors and all primary tumors irrespective of intrinsic chemosensitivity. The gene expression profile of post-chemotherapy tumors compared with that of primary tumors revealed statistically significant overrepresentation of genes encoding extracellular matrix-related proteins.
These data show that gene expression profiling can discriminate primary chemo-resistant from primary chemosensitive ovarian cancers. Gene expression profiles were also identified that correlate with states of intrinsic and acquired chemoresistance and that represent targets for future investigation and potential therapeutic interventions.
Clinical Cancer Research 10/2005; 11(17):6300-10. · 7.84 Impact Factor
[show abstract][hide abstract] ABSTRACT: Activation of the phosphatidylinositol 3-kinase (PI3K)-AKT pathway, resulting in increased cell proliferation, survival, and motility, is believed to play an oncogenic role in many cancer types. The PIK3CA gene encodes the p110alpha catalytic subunit of PI3K, and is amplified in some ovarian cancers, whereas the AKT2 gene is amplified in some ovarian, breast, and pancreatic cancers. Recently, in a mutational screen of eight PI3K genes and eight PI3K-like genes, PIK3CA was found to be the only gene affected by somatic mutations, which were observed frequently in gastrointestinal and brain cancers. Here, we test whether PIK3CA is subject to mutation in ovarian and breast cancers.
Exons 9 and 20, encoding the highly conserved helical and kinase domains of PIK3CA, were subjected to sequence analysis in 198 advanced stage epithelial ovarian carcinomas and 72 invasive breast carcinomas (48 of ductal histology and 24 of lobular histology).
Somatic missense mutations were observed in 24 of 198 (12%) ovarian carcinomas, and in 13 of 72 (18%) breast carcinomas.
These data indicate that mutations of PIK3CA play an oncogenic role in substantial fractions of ovarian and breast carcinomas, and in consideration of mutation of other components of the PI3K-AKT pathway in both tumor types, confirm the major oncogenic role of this pathway in ovarian and breast carcinomas.
Clinical Cancer Research 05/2005; 11(8):2875-8. · 7.84 Impact Factor
[show abstract][hide abstract] ABSTRACT: Recently BRCA1 has been implicated in the regulation of gene expression from the X chromosome. In this study the influence of BRCA1 on expression of X chromosome genes was investigated. Complementary DNA microarrays were used to compare the expression levels of X chromosome genes in 18 BRCA1-associated ovarian cancers to those of the 13 "BRCA1-like" and 14 "BRCA2-like" sporadic tumors (as defined by previously reported expression profiling). Significance was determined using parametric statistics with P < 0.005 as a cutoff. Forty of 178 total X-chromosome transcripts were differentially expressed between the BRCA1-associated tumors and sporadic cancers with a BRCA2-like molecular profile. Thirty of these 40 genes showed higher mean expression in the BRCA1-associated samples including all 11 transcripts that mapped to Xp11. In contrast, four of 178 total X chromosome transcripts showed significant differential expression between BRCA1-associated and sporadic tumors with a BRCA1-like molecular profile. All four mapped to Xp11 and showed higher mean expression in BRCA1-associated tumors. Re-expression of BRCA1 in HCC1937 BRCA1-deficient breast cancer cell resulted in the repression of 21 transcripts. Eleven of the 21 (54.5%) transcripts mapped to Xp11. However, there was no significant overlap between these Xp11 genes and those found to be differentially expressed between BRCA1-associated and sporadic ovarian cancer samples. These results demonstrate that BRCA1 mediates the repression of several X chromosome genes, many of which map to the Xp11 locus.
Journal of Translational Medicine 10/2004; · 3.46 Impact Factor
[show abstract][hide abstract] ABSTRACT: The aims of this study were to determine the incidence of BRCA mutations among Ashkenazi Jewish patients with fallopian tube carcinoma (FTC) or primary peritoneal carcinoma (PPC), to study the clinicopathologic features of these cancers, and to estimate the risks of these cancers in association with a BRCA mutation.
A retrospective review at two institutions identified 29 Jewish patients with FTC and 22 Jewish patients with PPC. These patients were genotyped for the three Ashkenazi Jewish BRCA founder mutations (185delAG and 5382insC in BRCA1 and 6174delT in BRCA2). Surgical and pathologic information, family history, and survival data were obtained from hospital records. All statistical tests were two sided.
Germline BRCA mutations were identified in five of 29 patients with FTC (17%) and nine of 22 patients with PPC (41%). Mutation carriers had a younger mean age at diagnosis than patients without a mutation (60 v 70 years; P =.002). The overall median survival was 148 months for mutation carriers and 41 months for patients without a mutation (P =.04). For BRCA mutation carriers, the lifetime risks of FTC and PPC were 0.6% and 1.3%, respectively.
Substantial proportions of Ashkenazi Jewish patients with FTC or PPC are BRCA mutation carriers. Patients with BRCA-associated FTC or PPC are younger at diagnosis and have improved survival compared with patients without a BRCA mutation. Although the lifetime risks of FTC or PPC for patients with BRCA heterozygotes are greater than those for the general population, the absolute risks seem relatively low.
Journal of Clinical Oncology 12/2003; 21(22):4222-7. · 18.04 Impact Factor
[show abstract][hide abstract] ABSTRACT: Germline mutations in BRCA1 and BRCA2 are responsible for 5%-10% of epithelial ovarian cancers, but the molecular pathways affected by these mutations are unknown. We used complementary DNA (cDNA) microarrays to compare gene expression patterns in ovarian cancers associated with BRCA1 or BRCA2 mutations with gene expression patterns in sporadic epithelial ovarian cancers and to identify patterns common to both hereditary and sporadic tumors.
Tumor samples from 61 patients with pathologically confirmed epithelial ovarian adenocarcinoma with matched clinicopathologic features were studied, including 18 with BRCA1 founder mutations, 16 with BRCA2 founder mutations, and 27 without either founder mutation (termed sporadic cancers). The cDNA microarrays contained 7651 sequence-verified features. Gene expression data were analyzed with a modified two-sided F test, with P<.0001 considered statistically significant. The expression level of six genes was also studied with reverse transcription-polymerase chain reaction.
The greatest contrast in gene expression was observed between tumors with BRCA1 mutations and those with BRCA2 mutations; 110 genes showed statistically significantly different expression levels (P<.0001). This group of genes could segregate sporadic tumors into two subgroups, "BRCA1-like" and "BRCA2-like," suggesting that BRCA1-related and BRCA2-related pathways are also involved in sporadic ovarian cancers. Fifty-three genes were differentially expressed between tumors with BRCA1 mutations and sporadic tumors; six of the 53 mapped to Xp11.23 and were expressed at higher levels in tumors with BRCA1 mutations than in sporadic tumors. Compared with the immortalized ovarian surface epithelial cells used as reference, several interferon-inducible genes were overexpressed in the majority of tumors with a BRCA mutation and in sporadic tumors.
Mutations in BRCA1 and BRCA2 may lead to carcinogenesis through distinct molecular pathways that also appear to be involved in sporadic cancers. Sporadic carcinogenic pathways may result from epigenetic aberrations of BRCA1 and BRCA2 or their downstream effectors.
JNCI Journal of the National Cancer Institute 07/2002; 94(13):990-1000. · 14.34 Impact Factor