Barbara M. Norquist

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.

Secondary Somatic Mutations Restoring BRCA1/2 Predict Chemotherapy Resistance in Hereditary Ovarian Carcinomas

PURPOSE 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. METHODS 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. RESULTS 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, Fishers 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, Fishers 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, Fishers exact test). CONCLUSION 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.

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.

Contribution of Inherited Mutations in the BRCA2-Interacting Protein PALB2 to Familial Breast Cancer

Inherited mutations in the BRCA2-interacting protein PALB2 are known to be associated with increased risks of developing breast cancer. To evaluate the contribution of PALB2 to familial breast cancer in the United States, we sequenced the coding sequences and flanking regulatory regions of the gene from constitutional genomic DNA of 1,144 familial breast cancer patients with wild-type sequences at BRCA1 and BRCA2. Overall, 3.4% (33/972) of patients not selected by ancestry and 0% (0/172) of patients specifically of Ashkenazi Jewish ancestry were heterozygous for a nonsense, frameshift, or frameshift-associated splice mutation in PALB2. Mutations were detected in both male and female breast cancer patients. All mutations were individually rare: the 33 heterozygotes harbored 13 different mutations, 5 previously reported and 8 novel mutations. PALB2 heterozygotes were 4-fold more likely to have a male relative with breast cancer (P = 0.0003), 6-fold more likely to have a relative with pancreatic cancer (P = 0.002), and 1.3-fold more likely to have a relative with ovarian cancer (P = 0.18). Compared with their female relatives without mutations, increased risk of developing breast cancer for female PALB2 heterozygotes was 2.3-fold (95% CI: 1.5-4.2) by age 55 and 3.4-fold (95% CI: 2.4-5.9) by age 85. Loss of the wild-type PALB2 allele was observed in laser-dissected tumor specimens from heterozygous patients. Given this mutation prevalence and risk, consideration might be given to clinical testing of PALB2 by complete genomic sequencing for familial breast cancer patients with wild-type sequences at BRCA1 and BRCA2.

Inherited Mutations in Women With Ovarian Carcinoma

IMPORTANCE Germline mutations in BRCA1 and BRCA2 are relatively common in women with ovarian, fallopian tube, and peritoneal carcinoma (OC) causing a greatly increased lifetime risk of these cancers, but the frequency and relevance of inherited mutations in other genes is less well characterized. OBJECTIVE To determine the frequency and importance of germline mutations in cancer-associated genes in OC. DESIGN, SETTING, AND PARTICIPANTS A study population of 1915 woman with OC and available germline DNA were identified from the University of Washington (UW) gynecologic tissue bank (n = 570) and from Gynecologic Oncology Group (GOG) phase III clinical trials 218 (n = 788) and 262 (n = 557). Patients were enrolled at diagnosis and were not selected for age or family history. Germline DNA was sequenced from women with OC using a targeted capture and multiplex sequencing assay. MAIN OUTCOMES AND MEASURES Mutation frequencies in OC were compared with the National Heart, Lung, and Blood Institute GO Exome Sequencing Project (ESP) and the Exome Aggregation Consortium (ExAC). Clinical characteristics and survival were assessed by mutation status. RESULTS Overall, the median (range) age at diagnosis was 60 (28-91) years in patients recruited from UW and 61 (23-87) years in patients recruited from the GOG trials. A higher number of black women were recruited from the GOG trials (4.3% vs 1.4%; P = .009); but in patients recruited from UW, there was a higher proportion of fallopian tube carcinomas (13.3% vs 5.7%; P < .001); stage I and II disease (14.6% vs 0% [GOG trials were restricted to advanced-stage cancer]); and nonserous carcinomas (29.9% vs 13.1%, P < .001). Of 1915 patients, 280 (15%) had mutations in BRCA1 (n = 182), or BRCA2 (n = 98), and 8 (0.4%) had mutations in DNA mismatch repair genes. Mutations in BRIP1 (n = 26), RAD51C (n = 11), RAD51D (n = 11), PALB2 (n = 12), and BARD1 (n = 4) were significantly more common in patients with OC than in the ESP or ExAC, present in 3.3%. Race, histologic subtype, and disease site were not predictive of mutation frequency. Patients with a BRCA2 mutation from the GOG trials had longer progression-free survival (hazard ratio [HR], 0.60; 95% CI, 0.45-0.79; P < .001) and overall survival (HR, 0.39; 95% CI, 0.25-0.60; P < .001) compared with those without mutations. CONCLUSIONS AND RELEVANCE Of 1915 patients with OC, 347 (18%) carried pathogenic germline mutations in genes associated with OC risk. PALB2 and BARD1 are suspected OC genes and together with established OC genes (BRCA1, BRCA2, BRIP1, RAD51C, RAD51D, MSH2, MLH1, PMS2, and MSH6) bring the total number of genes suspected to cause hereditary OC to 11.

The Molecular Pathogenesis of Hereditary Ovarian Carcinoma: Alterations in the Tubal Epithelium of Women with BRCA1 and BRCA2 Mutations

BRCA1 or BRCA2 (BRCA1/2)‐mutated ovarian carcinomas may originate in the fallopian tube. The authors of this report investigated alterations in BRCA1/2 tubal epithelium to define the molecular pathogenesis of these carcinomas.

Challenges in Evaluating Patients Lost to Follow-up in Clinical Studies of Rotator Cuff Tears*

Background: Long-term follow-up studies are necessary to critically evaluate the outcome of a treatment intervention for a specific disorder. However, patients may cease participating in a long-term study and become lost to follow-up; thus, their current condition is unknown. The underlying characteristics that predispose a patient to become lost to follow-up are difficult to identify and control. Patients who are lost to follow-up may be contacted by telephone; however, the effect of administering a functional assessment questionnaire by telephone compared with that of mailing a questionnaire is unknown. The purpose of this study was to compare patients who continued to respond to requests for follow-up with those who did not. A second purpose was to compare responses obtained by mail with those obtained by telephone interview. Methods: Two hundred and twenty-four patients with a rotator cuff tear were enrolled in an ongoing study of shoulder function and general health. Self-assessment questionnaires were mailed to every patient at six-month intervals. Sixty-seven patients (30 percent) regularly responded to mailings (identified as responders in this study), fifty-five patients (25 percent) responded occasionally (these patients were not included in the analysis), and 102 patients (46 percent) ceased to respond and became lost to follow-up (identified as nonresponders in this study). This investigation was performed to determine: (1) the characteristics of nonresponders compared with those of responders, (2) the functional status of nonresponders as assessed with a questionnaire over the telephone, and (3) the effect of administering a self-assessment functional questionnaire by telephone compared with that of sending the same questionnaire by mail. Results: Nonresponders tended to have lower initial scores for the mental health summary (p = 0.03) and for social function (p = 0.01), were less likely to have had surgery (p = 0.009), and were less likely to consume alcohol (p = 0.03). At the last known time when they completed the mailed questionnaire, nonresponders reported significantly worse shoulder function than responders (p = 0.0001). However, on telephone questioning the mean number of shoulder functions that the nonresponders indicated that they could perform was greater than the mean number documented on their last mailed questionnaire (p < 0.0001). In a random subgroup of responders, the mean number of functions that the patients indicated that they could perform when interviewed by telephone was significantly greater than the number indicated on their most recent mailed questionnaire (p < 0.01). The results obtained by telephone from this random subgroup of responders were similar to those obtained by telephone from the nonresponders. Conclusions: There are differences between patients who continue to participate in a study and those who become lost to follow-up. Functional assessment questionnaires administered by telephone yield different results than the same questionnaires sent by mail. These considerations are relevant to the design, implementation, and interpretation of clinical studies in which functional questionnaires are used.

Long term follow up of BRCA1 and BRCA2 mutation carriers with unsuspected neoplasia identified at risk reducing salpingo-oophorectomy.

OBJECTIVES The reported incidence of neoplasia identified at the time of risk-reducing salpingo-oophorectomy (RRSO) in germline BRCA1/2 mutation carriers ranges from 4 to 12% but long-term outcomes have not been described. We evaluated recurrence and survival outcomes of mutation carriers with neoplastic lesions identified at RRSO. METHODS We identified BRCA1/2 mutation carriers with neoplasia at RRSO at three institutions. Data was collected on clinical variables, adjuvant treatment and follow-up. RESULTS We identified 32 mutation carriers with invasive carcinomas (n=15) or high-grade intraepithelial neoplasia (n=17) that were not suspected prior to surgery. 26 occurred in BRCA1 and 6 in BRCA2 mutation carriers. Median and mean age for carcinomas were 50 years and 49.3 respectively, significantly younger than for intraepithelial neoplasm, median 53 years, and mean 55 years (p=0.04). For the 15 invasive carcinomas, median follow up was 88 months (range 45-172 months), 7 recurred (47%), median time to recurrence was 32.5 months and 3 have died of disease; 1 additional patient died of breast cancer. Overall survival was 73%, disease specific overall survival was 80% and disease free survival was 66%. For the 17 high-grade intraepithelial neoplasms, median follow up was 80 months (range 40-150), 4 were treated with chemotherapy. One recurred at 43 months and is currently not on therapy with a normal CA125, 16 months later. All patients with noninvasive neoplasia are alive. CONCLUSIONS BRCA1 and BRCA2 mutation carriers with unsuspected invasive carcinoma at RRSO have a relatively high rate of recurrence despite predominantly early stage, small volume disease. High-grade intraepithelial neoplasms rarely recur as carcinoma and may not require adjuvant chemotherapy.

Somatic Mosaic Mutations in PPM1D and TP53 in the Blood of Women With Ovarian Carcinoma

IMPORTANCE Somatic mosaic mutations in PPM1D have been reported in patients with breast cancer, lung cancer, and ovarian cancer (OC), but cause or effect has not been established. OBSERVATIONS To test the hypothesis that somatic mosaic mutations are associated with chemotherapy exposure, we used massively parallel sequencing to quantitate mutations in peripheral blood mononuclear cells (PBMCs) of 686 women with primary OC (n = 412) or relapsed OC (n = 274). The frequency of somatic mosaic PPM1D mutations in PBMCs was significantly associated with prior chemotherapy (P < .001), and, in patients exposed to chemotherapy, with older age at blood draw (recurrent OC odds ratio [OR], 17.24; 95% CI, 6.80-43.69; and primary OC postchemotherapy OR, 4.82; 95% CI, 1.43-16.18). In contrast, somatic mosaic mutations in TP53 were not significantly associated with chemotherapy or age. In sequential PBMC samples harvested from 13 patients with OC near diagnosis and after a median of 2 different chemotherapy regimens, somatic mosaic PPM1D mutations increased in 11 individuals (84.6%) and TP53 mutations appeared in 2 (15.4%). CONCLUSIONS AND RELEVANCE Chemotherapy exposure and age influence the accumulation of PPM1D-mutated PBMC clones. Care should be taken to control for chemotherapy exposure and age at blood draw when testing the association of somatic mosaic mutations in PBMCs with cancer risk.

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.