Rochelle L. Garcia

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.

Unexpected gynecologic neoplasms in patients with proven or suspected BRCA-1 or -2 mutations: implications for gross examination, cytology, and clinical follow-up.

Identification of inheritable mutations associated with the development of malignancy has led to prophylactic surgeries to remove tissues at risk. We report seven unrelated patients with family histories of breast and/or ovarian cancer, five of whom underwent prophylactic salpingo-oophorectomy with hysterectomy. Four had proven BRCA-1 or -2 mutations. Malignant cells were found unexpectedly in the peritoneal washings of two patients, leading to the discovery of early-stage fallopian tube carcinoma. After changing the sampling technique at our institution, two more cases of unexpected fallopian tube carcinoma in situ were discovered. Another patient had a significant family history and underwent hysterectomy for uterine fibroids, leading to the discovery of fallopian tube carcinoma. Another patient with BRCA-1 mutation had unexpected widespread primary peritoneal papillary serous adenocarcinoma. The final patient had a borderline malignant clear cell adenofibroma. These cases underscore the importance of peritoneal cytology and thorough sampling in the management of patients undergoing hysterectomy with a family history of breast/ovarian cancer and/or known BRCA-1/BRCA-2 mutations. As prophylactic surgeries are becoming more common secondary to advances in molecular diagnostics, pathologists need to be aware that surgical specimens from these patients may require more rigorous examination to uncover early neoplastic changes.

C-erbB-2 oncogene protein in in situ and invasive lobular breast neoplasia.

Lobular carcinoma in situ (LCIS) has uncertain malignant potential; biologic markers that will identify patients at risk for a poor clinical outcome have been sought actively. Amplification of the c‐erbB‐2 protooncogene has been correlated with poor prognosis in invasive mammary carcinoma, and immunohistochemical evaluation for expression of the oncogene protein has been correlated with gene amplification. The authors retrospectively evaluated 62 cases of lobular neoplasia for expression of the c‐erbB‐2 gene product on formalin‐fixed, deparaffinized sections, using two monoclonal anti‐erbB‐2 (p185) antibodies (c‐neu Ab3 and m‐erb) and one polyclonal anti‐erbB‐2 antibody (pAb 1) by the avidin‐biotin‐peroxidase method. All 62 cases were negative with the pAb 1 antibody; one of 62 cases was weakly positive with the c‐neu Ab3 in a membranous pattern. Expression of c‐erbB‐2 gene product was identified on adjacent invasive ductal carcinoma in one case and in adjacent ductal carcinoma in situ in another. None of 15 cases if infiltrating lobular carcinoma was positive with either of the two anti‐c‐erbB‐2 antibodies. Strong positivity was found on benign epithelium in one case, demonstrating epitheliosis. In summary, evidence of expression of the c‐erbB‐2 gene product was found in one of 57 cases of LCIS and none of 15 cases of invasive lobular carcinoma. This suggests that, in contrast to reported data concerning intraductal and invasive ductal carcinoma, c‐erbB‐2 oncogene amplification and/or overexpression does not play a significant role in the progression of lobular breast neoplasia.

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.

Methylation and protein expression of DNA repair genes: association with chemotherapy exposure and survival in sporadic ovarian and peritoneal carcinomas

BackgroundDNA 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.ResultsOf 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.ConclusionLow 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.

INCIDENCE OF ENDOMETRIAL HYPERPLASIA

OBJECTIVE The objective of the study was to estimate the age-specific incidence of endometrial hyperplasia: simple, complex, and atypical, in order of increasing likelihood of progression to carcinoma. STUDY DESIGN Women aged 18-90 years with endometrial pathology specimens (1985-2003) at a large integrated health plan were identified using automated data. Incidence rates were obtained by dividing the number of cases by the estimated number of female health plan enrollees who retained a uterus. RESULTS Endometrial hyperplasia peak incidence was: simple, 142 per 100,000 woman-years, complex, 213 per 100,000 woman-years, both in the early 50s; and atypical, 56 per 100,000 woman-years in the early 60s. Age-adjusted incidence decreased over the study period, especially for atypical hyperplasia. CONCLUSION Endometrial hyperplasia incidence without and with atypia peaks in the early postmenopausal years and in the early 60s, respectively. Given that some cases of endometrial hyperplasia likely go undiagnosed, the figures provided should be viewed as minimum estimates of the true incidence.

Colonization of the upper genital tract by vaginal bacterial species in nonpregnant women

OBJECTIVE The objective of the study was to evaluate the upper genital tract (UGT) presence of vaginal bacterial species using sensitive molecular methods capable of detecting fastidious bacterial vaginosis (BV)-associated bacteria. STUDY DESIGN Vaginal swabs were collected prior to hysterectomy. The excised uterus was sterilely opened and swabs collected from the endometrium and upper endocervix. DNA was tested in 11 quantitative polymerase chain reaction (PCR) assays for 12 bacterial species: Lactobacillus iners, L crispatus, L jensenii, Gardnerella vaginalis, Atopobium vaginae, Megasphaera spp, Prevotella spp, Leptotrichia/Sneathia, BVAB1, BVAB2, BVAB3, and a broad-range16S ribosomal ribonucleic acid gene assay. Endometrial fluid was tested with Luminex and an enzyme-linked immunosorbent assay for cytokines and defensins and tissue for gene expression of defensins and cathelicidin. RESULTS We enrolled 58 women: mean aged 43±7 years, mostly white (n=46; 79%) and BV negative (n=43; 74%). By species-specific quantitative PCR, 55 (95%) had UGT colonization with at least 1 species (n=52) or were positive by 16S PCR (n=3). The most common species were L iners (45% UGT, 61% vagina), Prevotella spp (33% UGT, 76% vagina) and L crispatus (33% UGT, 56% vagina). Median quantities of bacteria in the UGT were lower than vaginal levels by 2-4 log10 ribosomal ribonucleic acid gene copies per swab. There were no differences in the endometrial inflammatory markers between women with no bacteria, Lactobacillus only, or any BV-associated species in the UGT. CONCLUSION Our data suggest that the endometrial cavity is not sterile in most women undergoing hysterectomy and that the presence of low levels of bacteria in the uterus is not associated with significant inflammation.

Intratumoral T cells, tumor-associated macrophages, and regulatory T cells: Association with p53 mutations, circulating tumor DNA and survival in women with ovarian cancer

OBJECTIVES Forty percent of women with ovarian cancer have circulating free tumor DNA. We sought to determine if the tumor immune infiltrate varied based on tumor p53 mutation status or presence of circulating tumor DNA. METHODS We performed immunohistochemistry on 119 ovarian cancer specimens with CD3 and CD8 (Intratumoral T cells (TILs)), CD68 (tumor-associated macrophages (TAMs)), and FoxP3 (T regulatory cells (Tregs)). Tumors had been previously sequenced for mutations in exons 4-10 of p53, and plasma from women characterized for free tumor DNA. RESULTS TIL and TAM levels were positively correlated (P<0.0001). High levels of TILs were identified in 54 of 119 tumors (45.4%). No survival difference was identified according to the presence of TILs or TAMs. Patients with greater TILs were more likely to be optimally cytoreduced (P=0.005). p53 mutations were associated with more TILs (P=0.008). The presence of circulating tumor DNA did not correlate with TILs, TAMs, or Tregs. In the subgroup with a low host antitumor immune response, the intermediate response Tregs group did have a survival advantage (P=0.049). CONCLUSIONS p53 mutations are associated with higher levels of TILs. The ratio of Tregs to TILS may be more important than absolute levels. A brisk T cell response within the tumor predicts adequacy of cytoreduction, suggesting successful cytoreduction may be partially due to underlying tumor biology and host response.