Germline mutations in Black patients with ovarian, fallopian tube and primary peritoneal carcinomas.

OBJECTIVE: Routine genetic testing for ovarian cancer and identification of germline mutations can help improve early detection of cancer as well as guide treatment. Knowledge of genetic counseling and referral rates for genetic testing has been lower for Black patients, compared to White patients. We aimed to describe the demographics and presence of germline mutations in Black individuals with ovarian, fallopian tube or peritoneal carcinoma at two large academic institutions. METHODS: Fifty-one Black patients with invasive epithelial ovarian, fallopian tube, or primary peritoneal carcinoma were identified via institutional tissue banks over a 20-year time-period. Germline DNA was sequenced using BROCA, a targeted capture and parallel sequencing assay that identified pathogenic germline mutations in ovarian carcinoma susceptibility genes. RESULTS: Germline mutations in ovarian cancer susceptibility genes were found in 25.5% of women, most commonly BRCA1 and BRCA2. This mutation frequency mirrors those previously described among predominantly White populations. Our data suggests there may be an advantage in survival among those with germline mutations, although this was not statistically significant. CONCLUSIONS: Given similar frequencies of germline mutations between Black and White patients with ovarian cancer, we conclude that there are not major differences in the genetic predisposition to ovarian carcinoma. Equitable access to genomic advancements including germline and tumor sequencing would facilitate equal access to PARP inhibitors, the standard of care for patients with BRCA mutated advanced ovarian cancer.

Cryptosporidium infecting wild cricetid rodents from the subfamilies Arvicolinae and Neotominae.

We undertook a study on Cryptosporidium spp. in wild cricetid rodents. Fecal samples were collected from meadow voles (Microtus pennsylvanicus), southern red-backed voles (Myodes gapperi), woodland voles (Microtus pinetorum), muskrats (Ondatra zibethicus) and Peromyscus spp. mice in North America, and from bank voles (Myodes glareolus) and common voles (Microtus arvalis) in Europe. Isolates were characterized by sequence and phylogenetic analyses of the small subunit ribosomal RNA (SSU) and actin genes. Overall, 33·2% (362/1089) of cricetids tested positive for Cryptosporidium, with a greater prevalence in cricetids from North America (50·7%; 302/596) than Europe (12·1%; 60/493). Principal Coordinate analysis separated SSU sequences into three major groups (G1-G3), each represented by sequences from North American and European cricetids. A maximum likelihood tree of SSU sequences had low bootstrap support and showed G1 to be more heterogeneous than G2 or G3. Actin and concatenated actin-SSU trees, which were better resolved and had higher bootstrap support than the SSU phylogeny, showed that closely related cricetid hosts in Europe and North America are infected with closely related Cryptosporidium genotypes. Cricetids were not major reservoirs of human pathogenic Cryptosporidium spp.

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.

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

BACKGROUND: 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. METHODS: Using targeted capture and massively parallel genomic sequencing, 151 subjects with USC were assessed for germline mutations in 30 tumor suppressor genes, including BRCA1 (breast cancer 1, early onset), BRCA2, the DNA mismatch repair genes (MLH1 [mutL homolog 1], MSH2 [mutS homolog 2], MSH6, PMS2 [postmeiotic segregation increased 2]), TP53 (tumor protein p53), and 10 other genes in the Fanconi anemia-BRCA pathway. Ten cases with < 10% serous histology were also assessed. RESULTS: Seven subjects (4.6%) carried germline loss-of-function mutations: 3 subjects (2.0%) with mutations in BRCA1, 2 subjects (1.3%) with mutations in TP53, and 2 subjects (1.3%) with mutations in CHEK2 (checkpoint kinase 2). One subject with < 10% serous histology had an MSH6 mutation. Subjects with MSH6 and TP53 mutations had neither personal nor family histories suggestive of Lynch or Li-Fraumeni syndromes. Of the 22 women with USC and a personal history of breast carcinoma, the frequency of BRCA1 mutations was 9%, compared to 0.9% in 119 women with no such history. CONCLUSIONS: Approximately 5% of women with USC have germline mutations in 3 different tumor suppressor genes: BRCA1, CHEK2, and TP53. Mutations in DNA mismatch repair genes that cause Lynch syndrome are rare in USC. The germline BRCA1 mutation rate in USC subjects of 2% is higher than expected in a nonfounder population, suggesting that USC is associated with hereditary breast and ovarian carcinoma in a small proportion of cases. Women with USC and breast cancer should be offered genetic testing for BRCA1 and BRCA2 mutations.

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, (Fisher's 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.

Detection of inherited mutations for breast and ovarian cancer using genomic capture and massively parallel sequencing.

Inherited loss-of-function mutations in the tumor suppressor genes BRCA1, BRCA2, and multiple other genes predispose to high risks of breast and/or ovarian cancer. Cancer-associated inherited mutations in these genes are collectively quite common, but individually rare or even private. Genetic testing for BRCA1 and BRCA2 mutations has become an integral part of clinical practice, but testing is generally limited to these two genes and to women with severe family histories of breast or ovarian cancer. To determine whether massively parallel, "next-generation" sequencing would enable accurate, thorough, and cost-effective identification of inherited mutations for breast and ovarian cancer, we developed a genomic assay to capture, sequence, and detect all mutations in 21 genes, including BRCA1 and BRCA2, with inherited mutations that predispose to breast or ovarian cancer. Constitutional genomic DNA from subjects with known inherited mutations, ranging in size from 1 to >100,000 bp, was hybridized to custom oligonucleotides and then sequenced using a genome analyzer. Analysis was carried out blind to the mutation in each sample. Average coverage was >1200 reads per base pair. After filtering sequences for quality and number of reads, all single-nucleotide substitutions, small insertion and deletion mutations, and large genomic duplications and deletions were detected. There were zero false-positive calls of nonsense mutations, frameshift mutations, or genomic rearrangements for any gene in any of the test samples. This approach enables widespread genetic testing and personalized risk assessment for breast and ovarian cancer.

Diagnosis of Ovarian Carcinoma Homologous Recombination DNA Repair Deficiency From Targeted Gene Capture Oncology Assays.

PURPOSE: Homologous recombination DNA repair deficiency (HRD) is a therapeutic biomarker for sensitivity to platinum and poly(ADP-ribose) polymerase inhibitor therapies in breast and ovarian cancers. Several molecular phenotypes and diagnostic strategies have been developed to assess HRD; however, their clinical implementation remains both technically challenging and methodologically unstandardized. METHODS: We developed and validated an efficient and cost-effective strategy for HRD determination on the basis of calculation of a genome-wide loss of heterozygosity (LOH) score through targeted, hybridization capture and next-generation DNA sequencing augmented with 3,000 common, polymorphic single-nucleotide polymorphism (SNP) sites distributed genome-wide. This approach requires minimal sequence reads and can be readily integrated into targeted gene capture workflows already in use for molecular oncology. We interrogated 99 ovarian neoplasm-normal pairs using this method and compared results with patient mutational genotypes and orthologous predictors of HRD derived from whole-genome mutational signatures. RESULTS: LOH scores of ≥11% had >86% sensitivity for identifying tumors with HRD-causing mutations in an independent validation set (90.6% sensitivity for all specimens). We found strong agreement of our analytic approach with genome-wide mutational signature assays for determining HRD, yielding an estimated 96.7% sensitivity and 50% specificity. We observed poor concordance with mutational signatures inferred using only mutations detected by the targeted gene capture panel, suggesting inadequacy of the latter approach. LOH score did not significantly correlate with treatment outcomes. CONCLUSION: Targeted sequencing of genome-wide polymorphic SNP sites can be used to infer LOH events and subsequently diagnose HRD in ovarian tumors. The methods presented here are readily generalizable to other targeted gene oncology assays and could be adapted for HRD diagnosis in other tumor types.

Loss of function germline mutations in RAD51D in women with ovarian carcinoma.

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Microparticles from ovarian carcinomas are shed into ascites and promote cell migration.

OBJECTIVE: Microparticles are cellular-derived vesicles (0.5-1.0 µm) composed of cell membrane components, which are actively shed from the surface of various cells, including epithelial cells. We compared microparticles in ascites between women with ovarian carcinoma and women with benign ovarian pathology, and isolated tumor-derived (epithelial cell adhesion molecule [EpCAM]-positive) microparticles for functional analysis and proteomics. MATERIALS AND METHODS: Cases included 8 patients with benign ovarian neoplasms and 41 with ovarian carcinoma. Ascites from a high-grade stage III serous carcinoma was used for functional and proteomic analysis. Cancer cells were isolated using EpCAM-coated beads, microparticles were isolated by ultracentrifugation/flow cytometry, and sorting was achieved using markers (eg, EpCAM). Binding and migrations assays were performed with 3 ovarian cancer cell lines. Proteomic analysis of EpCAM-positive microparticles and ascites cancer cells was performed by mass spectrometry. RESULTS: Microparticles in benign pelvic fluid were similar to early and advanced-stage ascites (2.4 vs 2.8 vs 2.0 × 106 microparticles/mL). Advanced stage had a greater proportion of EpCAM-positive microparticles than early or benign disease (13.3% vs 2.5% vs 2.1%; P = 0.001), and serous histology had more than endometrioid (13.2% vs 1.8%; P = 0.01). Microparticles bound to the surface of 3 cultured cell lines, and were internalized into the EpCAM-positive microparticles, resulting in more cell migration than buffer alone or EpCAM-negative microparticles (P = 0.007). A dose-dependent increase was seen with increasing numbers of EpCAM-positive microparticles. Proteomics revealed that most proteins in EPCAM-positive microparticles were shared with cancer cells, and many are associated with cell motility and invasion, such as fibronectin, filamin A, vimentin, myosin-9, and fibrinogen. CONCLUSIONS: Ascites from advanced-stage and serous ovarian carcinomas contain large numbers of tumor-derived microparticles. In vitro, these microparticles bind to cancer cells and stimulate migration. Tumor-derived microparticles in ascites could mediate the predilection for peritoneal spread in serous ovarian carcinomas.

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.

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, 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). 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.

Identification of a preneoplastic gene expression profile in tubal epithelium of BRCA1 mutation carriers.

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.