K. Pennington

Mutations in 12 genes for inherited ovarian, fallopian tube, and peritoneal carcinoma identified by massively parallel sequencing

Inherited loss-of-function mutations in BRCA1 and BRCA2 and other tumor suppressor genes predispose to ovarian carcinomas, but the overall burden of disease due to inherited mutations is not known. Using targeted capture and massively parallel genomic sequencing, we screened for germ-line mutations in 21 tumor suppressor genes in genomic DNA from women with primary ovarian, peritoneal, or fallopian tube carcinoma. Subjects were consecutively enrolled at diagnosis and not selected for age or family history. All classes of mutations, including point mutations and large genomic deletions and insertions, were detected. Of 360 subjects, 24% carried germ-line loss-of-function mutations: 18% in BRCA1 or BRCA2 and 6% in BARD1, BRIP1, CHEK2, MRE11A, MSH6, NBN, PALB2, RAD50, RAD51C, or TP53. Six of these genes were not previously implicated in inherited ovarian carcinoma. Primary carcinomas were generally characterized by genomic loss of normal alleles of the mutant genes. Of women with inherited mutations, >30% had no family history of breast or ovarian carcinoma, and >35% were 60 y or older at diagnosis. More patients with ovarian carcinoma carry cancer-predisposing mutations and in more genes than previously appreciated. Comprehensive genetic testing for inherited carcinoma is warranted for all women with ovarian, peritoneal, or fallopian tube carcinoma, regardless of age or family history. Clinical genetic testing is currently done gene by gene, with each test costing thousands of dollars. In contrast, massively parallel sequencing allows such testing for many genes simultaneously at low cost.

Germline and Somatic Mutations in Homologous Recombination Genes Predict Platinum Response and Survival in Ovarian, Fallopian Tube, and Peritoneal Carcinomas

Purpose: Hallmarks of germline BRCA1/2-associated ovarian carcinomas include chemosensitivity and improved survival. The therapeutic impact of somatic BRCA1/2 mutations and mutations in other homologous recombination DNA repair genes is uncertain. Experimental Design: Using targeted capture and massively parallel genomic sequencing, we assessed 390 ovarian carcinomas for germline and somatic loss-of-function mutations in 30 genes, including BRCA1, BRCA2, and 11 other genes in the homologous recombination pathway. Results: Thirty-one percent of ovarian carcinomas had a deleterious germline (24%) and/or somatic (9%) mutation in one or more of the 13 homologous recombination genes: BRCA1, BRCA2, ATM, BARD1, BRIP1, CHEK1, CHEK2, FAM175A, MRE11A, NBN, PALB2, RAD51C, and RAD51D. Nonserous ovarian carcinomas had similar rates of homologous recombination mutations to serous carcinomas (28% vs. 31%, P = 0.6), including clear cell, endometrioid, and carcinosarcoma. The presence of germline and somatic homologous recombination mutations was highly predictive of primary platinum sensitivity (P = 0.0002) and improved overall survival (P = 0.0006), with a median overall survival of 66 months in germline homologous recombination mutation carriers, 59 months in cases with a somatic homologous recombination mutation, and 41 months for cases without a homologous recombination mutation. Conclusions: Germline or somatic mutations in homologous recombination genes are present in almost one third of ovarian carcinomas, including both serous and nonserous histologies. Somatic BRCA1/2 mutations and mutations in other homologous recombination genes have a similar positive impact on overall survival and platinum responsiveness as germline BRCA1/2 mutations. The similar rate of homologous recombination mutations in nonserous carcinomas supports their inclusion in PARP inhibitor clinical trials. Clin Cancer Res; 20(3); 764–75. ©2013 AACR.

Hereditary ovarian cancer: Beyond the usual suspects

In the past, hereditary ovarian carcinoma was attributed almost entirely to mutations in BRCA1 and BRCA2, with a much smaller contribution from mutations in DNA mismatch repair genes. Recently, three new ovarian cancer susceptibility genes have been identified: RAD51C, RAD51D, and BRIP1. In addition, germline mutations in women with ovarian carcinoma have been recently identified in many of the previously identified breast cancer genes in the Fanconi anemia (FA)-BRCA pathway. While mutations in genes other than BRCA1 and BRCA2 are each individually rare, together they make up a significant proportion of cases. With at least 16 genes implicated in hereditary ovarian cancer to date, comprehensive testing for ovarian cancer risk will require assessment of many genes. As the cost of genomic sequencing continues to fall, the practice of evaluating cancer susceptibility one gene at a time is rapidly becoming obsolete. New advances in genomic technologies will likely accelerate the discovery of additional cancer susceptibility genes and increase the feasibility of comprehensive evaluation of multiple genes simultaneously at low cost. Improved recognition of inherited risk will identify individuals who are candidates for targeted prevention. In addition, identifying inherited mutations in a variety of FA-BRCA pathway genes may aid in identifying individuals who will selectively benefit from PARP inhibitors.

BRCA1, TP53, and CHEK2 germline mutations in uterine serous carcinoma

Uterine serous carcinoma (USC) is not recognized as part of any defined hereditary cancer syndrome, and its association with hereditary breast and ovarian carcinoma and Lynch syndrome are uncertain.

The role of the endogenous opioid system in polycystic ovary syndrome

OBJECTIVE To review the complex role of the opioid system in reproduction and carbohydrate metabolism, abnormalities in the opioid system in women with polycystic ovary syndrome (PCOS), and the role of opioid antagonists in the management of PCOS-related infertility. DESIGN Pertinent articles were identified through a computer PubMed search. References of selected articles were hand searched for additional citations. CONCLUSION(S) Endogenous opioids are generally considered inhibitory central neurotransmitters. Peripherally, opioids are involved in the regulation of pancreatic islet function, hepatic insulin clearance, and glucose metabolism, potentially contributing to the pathogenesis of hyperinsulinemia and insulin resistance in PCOS. The presence of sex steroids is required for normal function of the opioid system in both GnRH secretion and carbohydrate metabolism. In women with PCOS, growing evidence suggests dysregulation of the opioid system both centrally and peripherally, with complex interactions. The opioid system effects on carbohydrate metabolism appear to be modulated by obesity. Finally, naltrexone has been demonstrated to successfully augment traditional ovulation induction regimens, but has limited support as a single ovulation induction agent for PCOS.

53BP1 expression in sporadic and inherited ovarian carcinoma: Relationship to genetic status and clinical outcomes.

OBJECTIVES 53BP1, a critical mediator of the DNA damage response, functions by regulating the balance between homologous recombination (HR) and the more error-prone non-homologous endjoining (NHEJ). Deletion of 53BP1 in brca1 (but not brca2) null cells partially restores HR and reverses sensitivity to poly-ADP-ribose polymerase inhibitors (PARPi). We characterized 53BP1 and BRCA1 expression and their association with clinical outcomes in sporadic and inherited ovarian carcinomas. METHODS We evaluated 53BP1 and BRCA1 protein expression using immunohistochemistry in 248 ovarian carcinomas and mRNA expression in 89 cases with quantitative reverse transcriptase PCR. All subjects were comprehensively characterized for germline mutations in BRCA1 and BRCA2. RESULTS BRCA1-mutated (but not BRCA2-mutated) ovarian carcinomas had significantly higher 53BP1 protein expression than wildtype carcinomas. 53BP1 message levels were significantly associated with BRCA1 message levels in wildtype and BRCA1-mutated but not BRCA2-mutated carcinomas. In wildtype carcinomas, lower 53BP1 message predicted improved survival (p=0.02, median survival 74 vs. 41months, HR 0.49, 95% CI 0.27-0.88). Survival was not impacted by BRCA1 message level. 53BP1 expression was not associated with primary platinum resistance. In 54 paired primary and recurrent cases, 53BP1 protein expression was equally likely to decrease or increase, and there was no association between decreased 53BP1 at recurrence and the development of platinum resistance. CONCLUSIONS BRCA1-mutated ovarian carcinomas have higher 53BP1 protein expression than wildtype or BRCA2-mutated carcinomas, in opposition to previous findings in breast carcinomas. Higher 53BP1 protein, which promotes NHEJ, could explain the frequent chromosomal aberrations that are characteristic of BRCA1-mutated ovarian carcinomas. In wildtype ovarian carcinomas, decreased 53BP1 message predicts improved survival, but message and protein expression were not associated.

Characteristics of women with ovarian carcinoma who have BRCA1 and BRCA2 mutations not identified by clinical testing

OBJECTIVES Few studies have comprehensively tested all ovarian cancer patients for BRCA1 and BRCA2 (BRCA1/2) mutations. We sought to determine if clinically identified mutation carriers differed in clinical characteristics and outcomes from mutation carriers not identified during routine clinical care. METHODS We included women with ovarian, tubal or peritoneal carcinoma. BROCA, an assay using targeted capture and massively parallel sequencing was used to identify mutations in BRCA1/2 and 19 other tumor suppressor genes. We identified subjects with BRCA1/2 mutations using BROCA that had not previously received standard genetic testing (BROCA, n=37) and compared them to subjects with BRCA1/2 mutations identified during routine clinical care (known, n=70), and to those wildtype for 21 genes using BROCA (wildtype, n=291). RESULTS BROCA mutation carriers were older than known carriers, median age of 58 (range 41-77), vs. 51 (range 33-76, p=0.003, Mann-Whitney). 58/70 (82.9%) of known carriers had a strong family history, compared with 15/37 (40.5%) of BROCA carriers, p<0.0001, (Fishers Exact). Median overall survival was significantly worse for BROCA mutation carriers compared to known mutation carriers, (45 vs. 93months, p<0.0001, HR 3.47 (1.79-6.72), Log-rank test). The improved survival for BRCA1/2 mutation carriers (known and BROCA) compared with wildtype cases (69 vs. 44months, p=0.0001, HR 0.58 (0.43-0.77), Log-rank test) was driven by known mutation carriers. CONCLUSIONS Older age, absence of a strong family history, and poor survival are all associated with decreased clinical identification of inherited BRCA1/2 mutations in women with ovarian cancer. Using age and family history to direct genetic testing will miss a significant percentage of mutation carriers. Testing should be initiated at the time of diagnosis to maximize identification of mutations and minimize survival bias.

Too much of a good thing: suicide prevention promotes chemoresistance in ovarian carcinoma.

Ovarian cancer is the most lethal of gynecologic malignancies. Currently, standard treatment for epithelial ovarian cancer consists of surgical debulking followed by adjuvant chemotherapy with a platinum-based drug coupled with paclitaxel. While initial response to chemotherapy is high, the majority of patients develop recurrent disease which is characterized by chemoresistance. The primary cytotoxic effect of many chemotherapy drugs is mediated by apoptotic response in tumor cells. Recent data indicates that cross talk between the tumor microenvironment and malignant epithelial cells can influence apoptotic response as well. The identification of molecules involved in the regulation and execution of apoptosis, and their alterations in ovarian carcinoma have provided new insights into the mechanism behind the development of chemoresistance in this disease. Our challenge is now to devise strategies to circumvent cell death defects and ultimately improve response to treatment in ovarian carcinoma patients.

Clinical characteristics and outcomes of patients with BRCA1 or RAD51C methylated versus mutated ovarian carcinoma

OBJECTIVE In ovarian carcinoma, mutations in homologous recombination DNA repair (HRR) genes, including BRCA1 and RAD51C, are associated with increased survival and specific clinical features. Promoter hypermethylation is another mechanism of reducing gene expression. We assessed whether BRCA1 and RAD51C promoter hypermethylation is associated with similar survival and clinical characteristics. METHODS Promoter methylation of BRCA1 and RAD51C was evaluated using methylation-sensitive PCR in 332 primary ovarian carcinomas. Damaging germline and somatic mutations in 16 HRR genes were identified using BROCA sequencing. RESULTS BRCA1 methylation was detected in 22 carcinomas (6.6%) and RAD51C methylation in 9 carcinomas (2.7%). These small numbers limited the power to detect differences in survival and platinum sensitivity. Mutations in one or more HRR genes were found in 95 carcinomas (29%). Methylation of BRCA1 or RAD51C was mutually exclusive with mutations in these genes (P=0.001). Patients whose carcinomas had BRCA1 methylation (57.7years±2.5) or BRCA1 mutations (54.1years±1.4) were younger than those without (63.3years±0.8; P=0.029, P<0.0001). BRCA1 methylation and germline BRCA1 mutation were associated with high grade serous (HGS) histology (P=0.045, P=0.001). BRCA1 mutations were associated with increased sensitivity to platinum chemotherapy while BRCA1 methylation was not (P=0.034, P=0.803). Unlike HRR mutations, methylation was not associated with improved overall survival compared to cases without methylation or mutation. CONCLUSIONS Patients with BRCA1-methylated carcinomas share clinical characteristics with patients with BRCA1-mutated carcinomas including younger age and predominantly HGS histology. However, unlike mutation, RAD51C and BRCA1 methylation were not associated with improved survival or greater sensitivity to platinum chemotherapy.

Abstract AS09: Germline mutations in cancer susceptibility genes in BRCA1 and BRCA2 negative families with ovarian and breast cancer

Abstracts: 10th Biennial Ovarian Cancer Research Symposium; September 8-9, 2014; Seattle, WA Objectives: Germline mutations in cancer susceptibility genes other than BRCA1 and BRCA2 ( BRCA1/2 ) are found in approximately 6% of women with ovarian, fallopian tube, or primary peritoneal cancer. Our objective was to sequence BRCA1/2 -negative ovarian cancer patients with a family history of ovarian or breast cancer to identify inherited mutations that may explain the familial risk. Methods: We used a targeted capture, massively parallel sequencing test called BROCA on ovarian cancer probands with a family history of ovarian or breast cancer, or a personal history of breast cancer. BROCA testing included all known breast and ovarian cancer genes. Only clear loss of function mutations were included. 118 probands were ascertained from a gynecologic oncology tissue bank or outside referrals and provided informed consent. A family history of ovarian cancer was defined as having a first or second degree relative with ovarian cancer. A family history of breast cancer was defined as having a first or second degree relative with pre-menopausal breast cancer, or 2 or more regardless of menopausal status. Subjects were only included in one category. Results: Of 118 ovarian cancer probands, 22 (18.6%) were found to carry deleterious mutations in non- BRCA1/2 cancer susceptibility genes. 8/29 (27.6%) ovarian cancer patients with a personal history of breast cancer had mutations in 7 genes (2 CHEK2 , 2 RAD51D , 1 BRIP1 , 1 TP53 , 1 ATM , and one with both PALB2 and PMS2 ). This included mutations found in 2/5 (40%) who also had a family history of ovarian cancer and 4/10 (40%) who also had a family history of breast cancer. 38 patients had a family history of ovarian cancer with no personal history of breast cancer; 9/38 (23.7%) had mutations in 5 genes (3 BRIP1 , 3 RAD51C , 1 RAD51D , 1 TP53 , and 1 ATM ). Finally, 5/51 (9.8%) ovarian cancer patients with a family history of breast cancer and no personal history of breast cancer had mutations in 5 genes (1 MSH6 , 1 FAM175A , 1 NBN , 1 PALB2 , and 1 CHEK2 ). Conclusions: Germline mutations in DNA-repair genes are present in a substantial fraction of BRCA1/ 2-negative ovarian cancer patients with a personal or family history suggestive of inherited disease. These women may benefit from multiplex gene testing. The detection of inherited mutations in these women may be useful to identify the risk of other cancers, to inform family members of possible risk, and to direct therapy by suggesting candidates for PARP inhibitor therapy. Citation Format: B. Norquist, M. Harrell, T. Walsh, J. Mandell, S. Bernards, K. Agnew, M. Lee, K. Pennington, M.C. King, E. Swisher. Germline mutations in cancer susceptibility genes in BRCA1 and BRCA2 negative families with ovarian and breast cancer [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr AS09.