RAD51D, a gene in the Fanconi Anemia-BRCA homologous recombination pathway, has recently been shown to harbor germline mutations responsible for ovarian carcinoma in multiply affected families. We aimed to extend these results to ovarian carcinoma in the general population.
We sequenced RAD51D in germline DNA from 360 individuals with primary ovarian, peritoneal or fallopian tube carcinoma who were not selected for age of cancer onset or family history. We also sequenced RAD51D in 459 probands from 226 high risk breast cancer families who were wild type for 21 breast and ovarian cancer genes.
Of 360 cases, three (0.8%) carried loss-of-function mutations in RAD51D. All three subjects had ovarian carcinoma; one was also diagnosed with a synchronous endometrial carcinoma. Only one of the three subjects had a family history of breast or ovarian cancer. Combined with previous data for this series, 23.9% of women with unselected ovarian, fallopian tube, or peritoneal carcinoma carried a germline loss-of-function mutation in any of 13 tumor suppressor genes. Among the 449 women and 10 men with familial breast cancer, none carried a loss of function mutation in RAD51D.
These data support the previous observation that loss-of-function mutations in RAD51D predispose to ovarian carcinoma but not to breast carcinoma. We conclude that inherited ovarian cancer is highly heterogeneous genetically, and that approximately one in four ovarian carcinoma patients carry a germline mutation in a known tumor suppressor gene that confers high risk.
Secondary somatic BRCA1/2 mutations may restore BRCA1/2 protein in hereditary ovarian carcinomas. In cell lines, BRCA2 restoration mediates resistance to platinum chemotherapy and poly (ADP-ribose) polymerase (PARP) inhibitors. We assessed primary and recurrent BRCA1/2-mutated ovarian carcinomas to define the frequency of secondary mutations and correlate these changes with clinical outcomes.
Neoplastic cells were isolated with laser capture microdissection, and DNA was sequenced at the site of the known germline BRCA1/2 mutation. When secondary mutations were found that restored wild-type sequence, haplotyping was performed using single nucleotide polymorphisms in tumor and paired lymphocyte DNA to rule out retention of the wild-type allele.
There were 64 primary and 46 recurrent ovarian carcinomas assessed. Thirteen (28.3%) of 46 (95% CI, 17.3% to 42.6%) recurrent carcinomas had a secondary mutation compared with two (3.1%) of 64 (95% CI, 1.0% to 10.7%) primary carcinomas (P = .0003, Fisher's exact test). Twelve (46.2%) of 26 (95% CI, 28.7% to 64.7%) platinum-resistant recurrences had secondary mutations restoring BRCA1/2, compared with one (5.3%) of 19 (95% CI, 1.2% to 24.8%) platinum-sensitive recurrences (P = .003, Fisher's exact test). Six (66.7%) of nine (95% CI, 34.8% to 87.8%) women with prior breast carcinoma had a recurrent carcinoma with a secondary mutation, compared with six (17.1%) of 35 (95% CI, 8.2% to 32.8%) with no history of breast carcinoma (P = .007, Fisher's exact test).
Secondary somatic mutations that restore BRCA1/2 in carcinomas from women with germline BRCA1/2 mutations predict resistance to platinum chemotherapy and may also predict resistance to PARP inhibitors. These mutations were detectable only in ovarian carcinomas of women whom have had previous chemotherapy, either for ovarian or breast carcinoma.
OBJECTIVES: Forty percent of women with ovarian carcinoma have circulating free neoplastic DNA identified in plasma. Angiogenesis is critical in neoplastic growth and metastasis. We sought to determine whether circulating neoplastic DNA results from alterations in the balance of angiogenesis activators and inhibitors. METHODS: Sixty patients with invasive ovarian carcinomas with somatic TP53 mutations that had been characterized for circulating neoplastic DNA had carcinoma analyzed for microvessel density using immunohistochemistry with CD31 and for the expression of VEGF, ANGPT1, ANGPT2, PTGS2, PLAU, THBS1, CSF1, PIK3CA, HIF1A, IL8, MMP2, and MMP9 message by real-time quantitative polymerase chain reaction. The expression of each gene was calculated relative to GAPDH expression for each neoplasm. Patient plasma had been tested for circulating neoplastic DNA using a ligase detection reaction. RESULTS: MMP2 expression was significantly correlated with free plasma neoplastic DNA (P = .007). Microvessel density was not correlated with plasma neoplastic DNA or BRCA1/2 mutation status. The expression pattern of other angiogenic factors did not correlate with plasma neoplastic DNA but correlated with each other. BRCA1/2 mutated carcinomas had significantly different expression profiles of angiogenesis activators and inhibitors in comparison to sporadic carcinomas. CONCLUSIONS: MMP2 expression is associated with the presence of circulating neoplastic DNA in women with ovarian carcinoma. These data are consistent with the proinvasive properties of MMP2 and suggest that the presence of circulating neoplastic DNA indicates a more aggressive malignant phenotype. Carcinomas with germ line BRCA1/2 mutations had a lower angiogenic profile than those without mutations.
To identify biomarkers associated with progestin therapy resistance and persistence/progression of endometrial hyperplasia.
We performed a nested case-control study among women with complex (n=73) and atypical (n=41) hyperplasia treated with oral progestin, followed 2–6 months for persistence/progression. We evaluated index endometrial protein expression for progesterone receptors A (PRA) and B (PRB), PTEN, Pax-2 and Bcl-2. Odds ratios and 95% confidence intervals were estimated.
Among women with atypical hyperplasia, high PRB expression was associated with 90% decreased risk of persistence/progression (95% CI: 0.01–0.8). High expression of PRA and PRB suggested decreased risk of persistence/ progression (OR=0.1, 95% CI: 0.02–1.0). These findings were not observed among women with complex hyperplasia. No associations were found with PTEN, Pax-2, and Bcl-2 protein expression.
PRB expression shows promise as a biomarker of progestin response. Further research is warranted to understand how PRB expression may guide treatment decisions.
Biological Markers; Endometrial Hyperplasia; Immunohistochemistry; Progesterone Receptors; Progestin
To define patterns of aberrant DNA methylation, p53 mutation and Her-2/neu overexpression in tissues from benign (N=29), malignant (N=100), and border line malignant ovaries (N=10), as compared to normal (N=68) ovarian tissues. Further, to explore the relationship between the presence of genetic and epigenetic abnormalities in ovarian cancers, and assess the association between epigenetic changes and clinical stage of malignancy at presentation and response to therapy.
The methylation status of 23 genes that were previously reported associated with various epithelial malignancies was assessed in normal and abnormal ovarian tissues by methylation specific PCR. The presence of p53 mutation (N=82 cases) and Her-2/neu overexpression (N=51 cases) were assessed by DNA sequencing and immunohistochemistry, respectively.
Methylation of four genes (MINT31, HIC1, RASSF1, and CABIN1) was significantly associated with ovarian cancer but not other ovarian pathology. Her-2/neu overexpression was associated with aberrant methylation of three genes (MINT31, RASSF1, and CDH13), although aberrant methylation was not associated with p53 mutations. Methylation of RASSF1 and HIC1 was more frequent in early compared to late stage ovarian cancer, while methylation of CABIN1 and RASSF1 was associated with response to chemotherapy.
DNA methylation of tumor suppressor genes is a frequent event in ovarian cancer, and in some cases is associated with Her-2/neu overexpression. Methylation of CABIN1 and RASSF1 may have the utility to predict response to therapy.
hypermethylation; Her-2/neu overexpression; p53; ovarian cancer
BRCA1 or BRCA2 (BRCA1/2) mutated ovarian carcinomas may originate in the fallopian tube. We investigated alterations in BRCA1/2 tubal epithelium to define the molecular pathogenesis of these carcinomas.
Tubal epithelium was evaluated from 31 BRCA1/2 mutation carriers with gynecologic carcinomas (BRCA CA), 89 mutation carriers undergoing risk-reducing salpingo-oophorectomy (RRSO), and 87 controls. Ki-67 expression and p53 foci (≥10/12 consecutive staining cells) were scored by two investigators blinded to case designation. p27 and p21 expression was evaluated within p53 foci. Loss of heterozygosity at the BRCA1/2 mutation site was evaluated in microdissected p53 foci and tubal neoplasms.
Background tubal proliferation as measured by Ki-67 staining was increased in BRCA1 RRSO (p=0.005) compared with controls. Women with BRCA1/2 mutations were found to have more p53 foci per tubal segment than controls (p=0.02). p27 was decreased in 12/28 p53 foci from women with BRCA1 mutations and 0/16 from controls (p=0.002). There was no loss of the wildtype BRCA1/2 allele in 5 tested p53 foci. Tubal neoplasia lost the wildtype allele in 6/6 cases (p=0.002).
These observations suggest a model of tubal carcinogenesis in women with BRCA1/2 mutations. Increased proliferation occurs globally in at-risk tubal epithelium. A TP53 mutation with clonal proliferation and loss of p27 occurs prior to neoplastic proliferation. Loss of the wildtype BRCA1/2 allele occurs with neoplastic proliferation and prior to invasion.
fallopian tube; BRCA1; BRCA2; p53; p27; Ki-67; ovarian cancer
The HE4 protein is overexpressed in ovarian carcinomas and can be detected in serum by an ELISA with sensitivity similar to CA125 and higher specificity for malignant disease. We now demonstrate that HE4 can also be detected in the urine at a specificity level of 94.4%, including 13/15 (86.6%) with stage I/II and 57/64 (89.0%) with stage III/IV disease and including 90.5% of patients with serous ovarian carcinoma. Assaying serum and urine from the same patients showed similar sensitivity. Our data indicate that measuring HE4 in urine may aid diagnosis and the monitoring of response to therapy.
ELISA; ovarian cancer diagnostics; urine; HE4
The majority of tumors arising in BRCA1 mutation carriers exhibit inactivation of p53, a key effector of cell death following DNA damage. Despite the loss of p53, BRCA1-deficient tumor cells exhibit increased sensitivity to cisplatin, and patients with BRCA1-associated ovarian carcinomas experience improved outcomes with platinum-based chemotherapy compared to sporadic cases. While it is known that chemosensitivity in BRCA1-associated cancers is associated with unrepaired DNA damage, the specific effector pathway mediating the cellular response to platinum-induced damage in these tumors is poorly understood. Here we demonstrate that the p53-related gene p73, encoding a pro-apoptotic protein which is linked to chemosensitivity in many settings, is upregulated through a novel epigenetic mechanism in both human and murine models of BRCA1-associated ovarian carcinoma. BRCA1-deficient ovarian carcinoma cells exhibit hypermethylation within a p73 regulatory region which includes the binding site for the p73 transcriptional repressor ZEB1, leading to abrogation of ZEB1 binding and increased expression of transactivating p73 isoforms (TAp73). Cisplatin chemotherapy induces TAp73 target genes specifically in BRCA1-deficient cells, and knockdown of TAp73 in these cells causes chemoresistance while having little or no effect on BRCA1-expressing tumor cells. In primary ovarian carcinomas, ZEB1 binding site methylation and TAp73 expression correlate with BRCA1 status and with clinical response. Together, these findings uncover a novel regulatory mechanism that supports the contribution of TAp73 as an important mediator of the response to platinum chemotherapy in a subset of ovarian carcinomas. TAp73 may represent a response predictor and potential therapeutic target for enhancing chemosensitivity in this disease.
Ovarian Carcinoma; BRCA1; Cisplatin; TP73; Methylation; Chemosensitivity
Although microRNAs (miRNAs) are important regulators of gene expression, the transcriptional regulation of miRNAs themselves is not well understood. We employed an integrative computational pipeline to dissect the transcription factors (TFs) responsible for altered miRNA expression in ovarian carcinoma. Using experimental data and computational predictions to define miRNA promoters across the human genome, we identified TFs with binding sites significantly overrepresented among miRNA genes overexpressed in ovarian carcinoma. This pipeline nominated TFs of the p53/p63/p73 family as candidate drivers of miRNA overexpression. Analysis of data from an independent set of 253 ovarian carcinomas in The Cancer Genome Atlas showed that p73 and p63 expression is significantly correlated with expression of miRNAs whose promoters contain p53/p63/p73 family binding sites. In experimental validation of specific miRNAs predicted by the analysis to be regulated by p73 and p63, we found that p53/p63/p73 family binding sites modulate promoter activity of miRNAs of the miR-200 family, which are known regulators of cancer stem cells and epithelial–mesenchymal transitions. Furthermore, in chromatin immunoprecipitation studies both p73 and p63 directly associated with the miR-200b/a/429 promoter. This study delineates an integrative approach that can be applied to discover transcriptional regulatory mechanisms in other biological settings where analogous genomic data are available.
MicroRNAs are often aberrantly expressed in human neoplasms and are postulated to play a role in neoplastic initiation and progression. miR-221 and miR-222 negatively regulate expression of CDKN1B (p27)and CDKN1C (p57), two cell cycle regulators expressed in ovarian surface epithelium and down-regulated in ovarian carcinomas. We characterized miR-221 and miR-222 expression in 49 sporadic high grade ovarian carcinomas and determined whether somatic mutation or epigenetic alterations explained the differences in expression of these miRNAs. We correlated these findings with protein expression of CDKN1B and CDKN1C as assessed by immunohistochemistry. Expression of miR-221 and miR-222 were closely correlated with each other (P=0.0001). Interestingly, a lower ratio of miR-221 to miR-222 expression was significantly correlated with worse overall survival (P=0.01) and remained a significant predictor of overall survival in multivariate analysis using the co-variate adequacy of surgical cytoreduction (P=0.03). Higher miR-222 and miR-221 expression were significantly associated with decreased CDKN1C expression (P=0.009 and 0.01). In contrast, CDKN1B expression was not associated with miR-221 or miR-222 expression. Neither somatic mutations nor methylation of the studied region explained the alterations in miR-221 and miR-222 expression in most carcinomas.
Inherited mutations in the tumor suppressor genes BRCA1 and BRCA2 cause increased risk of developing various cancers, especially breast and ovarian cancers. Tumors that develop in patients with inherited BRCA1/2 mutations are generally believed to be BRCA1/2 deficient. Cancer cells with BRCA1/2 deficiency are defective in DNA repair by homologous recombination and sensitive to interstrand DNA crosslinking agents, such as cisplatin and carboplatin, and poly(ADP-ribose) polymerase (PARP) inhibitors. Therefore, these agents are logical choices for the treatment for BRCA1/2-deficient tumors and have shown to be clinically effective. However, BRCA1/2-mutated tumors often develop resistance to these drugs. Restoration of BRCA1/2 functions due to secondary BRCA1/2 mutations has been recognized as a mechanism of acquired resistance to cisplatin and PARP inhibitors in BRCA1/2-mutated cancer cells. This indicates that even disease-causing inherited mutations of tumor suppressor genes can be genetically reverted in cancer cells, if the genetic reversion is advantageous for the cells' survival. In this review, we will discuss this drug resistance mechanism.
Microinvasive carcinomas and high-grade intraepithelial neoplasms are commonly discovered within the fallopian tube of BRCA1 mutation carriers at the time of risk-reducing salpingo-oophorectomy, suggesting that many BRCA1-mutated ovarian carcinomas originate in tubal epithelium. We hypothesized that changes in gene expression profiles within the histologically normal fallopian tube epithelium of BRCA1 mutation carriers would overlap with the expression profiles in BRCA1-mutated ovarian carcinomas and represent a BRCA1 preneoplastic signature. Laser capture microdissection of frozen sections was used to isolate neoplastic cells or histologically normal fallopian tube epithelium, and expression profiles were generated on Affymetrix U133 Plus 2.0 gene expression arrays. Normal-risk controls were 11 women wild type for BRCA1 and BRCA2 (WT-FT). WT-FT were compared with histologically normal fallopian tube epithelium from seven women with deleterious BRCA1 mutations who had foci of at least intraepithelial neoplasm within their fallopian tube (B1-FTocc). WT-FT samples were also compared with 12 BRCA1 ovarian carcinomas (B1-CA). The comparison of WT-FT versus B1-FTocc resulted in 152 differentially expressed probe sets, and the comparison of WT-FT versus B1-CA resulted in 4079 differentially expressed probe sets. The BRCA1 preneoplastic signature was composed of the overlap between these two lists, which included 41 concordant probe sets. Genes in the BRCA1 preneoplastic signature included several known tumor suppressor genes such as CDKN1C and EFEMP1 and several thought to be important in invasion and metastasis such as E2F3. The expression of a subset of genes was validated with quantitative reverse transcription-polymerase chain reaction and immunohistochemistry.
Acquired platinum resistance is a serious problem in the treatment of ovarian carcinomas. However, the mechanism of the drug resistance has not been elucidated. Here, we show functional significance of restoration of BRCA2 protein by secondary BRCA2 mutations in acquired drug resistance of BRCA2-mutated ovarian carcinoma. Three ovarian cancer cell lines (PEO1, PEO4 and PEO6) were derived from a BRCA2 mutation (5193C>G (Y1655X)) carrier with ovarian carcinoma with acquired cisplatin resistance and a secondary BRCA2 mutation (5193C>T (Y1655Y)) that canceled the inherited mutation. PEO1 was BRCA2-deficient and sensitive to cisplatin and a poly(ADP-ribose) polymerase inhibitor, AG14361, while PEO4 was resistant. PEO4 and PEO6, derived from ascites at the time of relapse with cisplatin resistance, had the secondary mutation and were BRCA2-proficient. In vitro cisplatin/AG14361 selection of PEO1 led to restoration of BRCA2 due to another secondary BRCA2 mutation. BRCA2 depletion sensitized BRCA2-restored PEO1 clones and PEO4 to cisplatin/AG14361. Thus, restoration of BRCA2 due to secondary BRCA2 mutation is involved in acquired drug resistance of BRCA2-mutated ovarian carcinoma.
BRCA2; cisplatin; drug resistance; ovarian cancer; PARP inhibitor
DNA repair genes critically regulate the cellular response to chemotherapy and epigenetic regulation of these genes may be influenced by chemotherapy exposure. Restoration of BRCA1 and BRCA2 mediates resistance to platinum chemotherapy in recurrent BRCA1 and BRCA2 mutated hereditary ovarian carcinomas. We evaluated BRCA1, BRCA2, and MLH1 protein expression in 115 sporadic primary ovarian carcinomas, of which 31 had paired recurrent neoplasms collected after chemotherapy. Additionally, we assessed whether promoter methylation of BRCA1, MLH1 or FANCF influenced response to chemotherapy or explained alterations in protein expression after chemotherapy exposure.
Of 115 primary sporadic ovarian carcinomas, 39 (34%) had low BRCA1 protein and 49 (42%) had low BRCA2 expression. BRCA1 and BRCA2 protein expression were highly concordant (p < 0.0001). MLH1 protein loss occurred in 28/115 (24%) primary neoplasms. BRCA1 protein loss in primary neoplasms was associated with better survival (p = 0.02 Log Rank test) and remained significant after accounting for either stage or age in a multivariate model (p = 0.04, Cox proportional hazards). In paired specimens, BRCA1 protein expression increased in 13/21 (62%) and BRCA2 protein expression increased in 15/21 (71%) of recurrent carcinomas with low or intermediate protein in the paired primary. In contrast MLH1 expression was rarely decreased in recurrent carcinomas (1/33, 3%). Similar frequencies of MLH1, BRCA1, and FANCF promoter methylation occurred in primary carcinomas without previous chemotherapy, after neoadjuvant chemotherapy, or in recurrent neoplasms.
Low BRCA1 expression in primary sporadic ovarian carcinoma is associated with prolonged survival. Recurrent ovarian carcinomas commonly have increased BRCA1 and/or BRCA2 protein expression post chemotherapy exposure which could mediate resistance to platinum based therapies. However, alterations in expression of these proteins after chemotherapy are not commonly mediated by promoter methylation, and other regulatory mechanisms are likely to contribute to these alterations.
The aim of this study was to determine the genomic structure of the deletions on chromosome 17 in ovarian carcinomas from women with inherited BRCA1 mutations. Normal and tumor DNA from 14 ovarian tumors associated with inherited BRCA1 mutations were extracted and tested for loss of heterozygosity (LOH) at microsatellite markers along chromosome 17. Finer mapping using more microsatellite markers and single nucleotide polymorphisms (SNPs) helped further define the LOH margins. The genomic repeated elements within the LOH breakpoint regions were identified using the University of California Santa Cruz Genome Database and the frequencies were compared to regions of equal GC percentages across the genome. Of the 14 ovarian tumors, 12 showed LOH of the entire chromosome 17. The other two tumors lost the distal end of the 17q arm. The breakpoints of these two tumors occurred in regions with significantly high frequencies of SINE repeating elements, specifically Alu elements. Ovarian tumors of high grade and stage have large regions of LOH along chromosome 17, with most tumors showing loss of the entire chromosome. In those tumors with retention of part of chromosome 17, LOH margins suggest that a high Alu content may have a role in the deletions.
Although ovarian carcinomas with mutated BRCA1 or BRCA2 are sensitive to platinum compounds, such carcinomas eventually develop platinum resistance. Previously, we showed that acquired resistance to cisplatin in BRCA2-mutated tumors can be mediated by secondary intragenic mutations in BRCA2 that restore the wild-type BRCA2 reading frame. Here, we show that secondary mutations of BRCA1 also occur in BRCA1-mutated ovarian cancer with platinum resistance. We evaluated 9 recurrent BRCA1-mutated ovarian cancers previously treated with platinum compounds, including five with acquired platinum resistance, one with primary platinum resistance, and three with platinum sensitivity. Four of the 6 recurrent platinum-resistant tumors had developed secondary genetic changes in BRCA1 that restored the reading frame of the BRCA1 protein, while none of 3 platinum-sensitive recurrent tumors developed BRCA1 sequence alterations. We immunohistochemically confirmed restored expression of BRCA1 protein in 2 cases with secondary mutations. Intriguingly, the case with primary platinum resistance showed back mutation of BRCA1 in the primary tumor, and showed another secondary mutation in the recurrent tumor. Our results suggest that secondary mutations in BRCA1 can mediate resistance to platinum in BRCA1-mutated ovarian tumors.
DNA damage and repair mechanisms; BRCA1; BRCA2; cisplatin; drug resistance
Ovarian carcinomas with mutations in the tumor suppressor BRCA2 are particularly sensitive to platinum compounds 1. However, such carcinomas ultimately develop cisplatin resistance. The mechanism of that resistance is largely unknown 2. Here we show that acquired resistance to cisplatin can be mediated by secondary intragenic mutations in BRCA2 that restore the wild-type BRCA2 reading frame. First, in a cisplatin-resistant BRCA2-mutated breast cancer cell line, HCC1428, a secondary genetic change in BRCA2 rescued BRCA2 function. Second, cisplatin selection of a BRCA2-mutated pancreatic cancer cell line, Capan-1 3,4, led to 5 different secondary mutations that restored the wild-type BRCA2 reading frame. All clones with secondary mutations were resistant both to cisplatin and to a poly(ADP-ribose) polymerase (PARP) inhibitor (AG14361). Finally, we evaluated recurrent cancers from patients whose primary BRCA2-mutated ovarian carcinomas were treated with cisplatin. The recurrent tumor that acquired cisplatin resistance had undergone reversion of its BRCA2 mutation. Our results suggest that secondary mutations that restore the wild-type BRCA2 reading frame may be a major clinical mediator of acquired resistance to platinum-based chemotherapy.