The avoiding late diagnosis of ovarian cancer (ALDO) project; a pilot national surveillance programme for women with pathogenic germline variants in BRCA1 and BRCA2.

BACKGROUND: Our study aimed to establish 'real-world' performance and cost-effectiveness of ovarian cancer (OC) surveillance in women with pathogenic germline BRCA1/2 variants who defer risk-reducing bilateral salpingo-oophorectomy (RRSO). METHODS: Our study recruited 875 female BRCA1/2-heterozygotes at 13 UK centres and via an online media campaign, with 767 undergoing at least one 4-monthly surveillance test with the Risk of Ovarian Cancer Algorithm (ROCA) test. Surveillance performance was calculated with modelling of occult cancers detected at RRSO. The incremental cost-effectiveness ratio (ICER) was calculated using Markov population cohort simulation. RESULTS: Our study identified 8 OCs during 1277 women screen years: 2 occult OCs at RRSO (both stage 1a), and 6 screen-detected; 3 of 6 (50%) were ≤stage 3a and 5 of 6 (83%) were completely surgically cytoreduced. Modelled sensitivity, specificity, Positive Predictive Value (PPV) and Negative Predictive Value (NPV) for OC were 87.5% (95% CI, 47.3 to 99.7), 99.9% (99.9-100), 75% (34.9-96.8) and 99.9% (99.9-100), respectively. The predicted number of quality-adjusted life years (QALY) gained by surveillance was 0.179 with an ICER cost-saving of -£102,496/QALY. CONCLUSION: OC surveillance for women deferring RRSO in a 'real-world' setting is feasible and demonstrates similar performance to research trials; it down-stages OC, leading to a high complete cytoreduction rate and is cost-saving in the UK National Health Service (NHS) setting. While RRSO remains recommended management, ROCA-based surveillance may be considered for female BRCA-heterozygotes who are deferring such surgery.

Evaluation of tumour surveillance protocols and outcomes in von Hippel-Lindau disease in a national health service.

BACKGROUND: Von Hippel-Lindau (VHL) disease is an inherited tumour predisposition syndrome and a paradigm for the importance of early diagnosis and surveillance. However, there is limited information on the "real world" management of VHL disease. METHODS: A national audit of VHL disease in the United Kingdom. RESULTS: VHL disease was managed mostly via specialist clinics coordinated through regional clinical genetics services (but frequently involving additional specialties). Over the study period, 19 genetic centres saw 842 individuals (393 males, 449 females) with a clinical and/or molecular diagnosis of VHL disease and 74 individuals (35 male, 39 female) with a prior risk of 50% (affected parent). All centres offered retinal, central nervous system and abdominal surveillance to affected individuals and at-risk relatives though surveillance details differed between centres (but complied with international recommendations). Renal lesions detected on the first surveillance scan were, on average, larger than those detected during subsequent scans and the larger the diameter at detection the greater the likelihood of early intervention. CONCLUSIONS: In a state-funded health care system individuals with a rare inherited cancer predisposition syndrome are generally able to access appropriate surveillance and patient management is improved compared to historical data. The "real world" data from this study will inform the future development of VHL management protocols.

SDHC phaeochromocytoma and paraganglioma: A UK-wide case series.

OBJECTIVE: Phaeochromocytomas and paragangliomas (PPGL) are rare, but strongly heritable tumours. Variants in succinate dehydrogenase (SDH) subunits are identified in approximately 25% of cases. However, clinical and genetic information of patients with SDHC variants are underreported. DESIGN: This retrospective case series collated data from 18 UK Genetics and Endocrinology departments. PATIENTS: Both asymptomatic and disease-affected patients with confirmed SDHC germline variants are included. MEASUREMENTS: Clinical data including tumour type and location, surveillance outcomes and interventions, SDHC genetic variant assessment, interpretation, and tumour risk calculation. RESULTS: We report 91 SDHC cases, 46 probands and 45 non-probands. Fifty-one cases were disease-affected. Median age at genetic diagnosis was 43 years (range: 11-79). Twenty-four SDHC germline variants were identified including six novel variants. Head and neck paraganglioma (HNPGL, n = 30, 65.2%), extra-adrenal paraganglioma (EAPGL, n = 13, 28.2%) and phaeochromocytomas (PCC) (n = 3, 6.5%) were present. One case had multiple PPGLs. Malignant disease was reported in 19.6% (9/46). Eight cases had non-PPGL SDHC-associated tumours, six gastrointestinal stromal tumours (GIST) and two renal cell cancers (RCC). Cumulative tumour risk (95% CI) at age 60 years was 0.94 (CI: 0.79-0.99) in probands, and 0.16 (CI: 0-0.31) in non-probands, respectively. CONCLUSIONS: This study describes the largest cohort of 91 SDHC patients worldwide. We confirm disease-affected SDHC variant cases develop isolated HNPGL disease in nearly 2/3 of patients, EAPGL and PCC in 1/3, with an increased risk of GIST and RCC. One fifth developed malignant disease, requiring comprehensive lifelong tumour screening and surveillance.

Comprehensive Cancer-Predisposition Gene Testing in an Adult Multiple Primary Tumor Series Shows a Broad Range of Deleterious Variants and Atypical Tumor Phenotypes.

Multiple primary tumors (MPTs) affect a substantial proportion of cancer survivors and can result from various causes, including inherited predisposition. Currently, germline genetic testing of MPT-affected individuals for variants in cancer-predisposition genes (CPGs) is mostly targeted by tumor type. We ascertained pre-assessed MPT individuals (with at least two primary tumors by age 60 years or at least three by 70 years) from genetics centers and performed whole-genome sequencing (WGS) on 460 individuals from 440 families. Despite previous negative genetic assessment and molecular investigations, pathogenic variants in moderate- and high-risk CPGs were detected in 67/440 (15.2%) probands. WGS detected variants that would not be (or were not) detected by targeted resequencing strategies, including low-frequency structural variants (6/440 [1.4%] probands). In most individuals with a germline variant assessed as pathogenic or likely pathogenic (P/LP), at least one of their tumor types was characteristic of variants in the relevant CPG. However, in 29 probands (42.2% of those with a P/LP variant), the tumor phenotype appeared discordant. The frequency of individuals with truncating or splice-site CPG variants and at least one discordant tumor type was significantly higher than in a control population (χ2 = 43.642; p ≤ 0.0001). 2/67 (3%) probands with P/LP variants had evidence of multiple inherited neoplasia allele syndrome (MINAS) with deleterious variants in two CPGs. Together with variant detection rates from a previous series of similarly ascertained MPT-affected individuals, the present results suggest that first-line comprehensive CPG analysis in an MPT cohort referred to clinical genetics services would detect a deleterious variant in about a third of individuals.

The predictive ability of the 313 variant-based polygenic risk score for contralateral breast cancer risk prediction in women of European ancestry with a heterozygous BRCA1 or BRCA2 pathogenic variant.

PURPOSE: To evaluate the association between a previously published 313 variant-based breast cancer (BC) polygenic risk score (PRS313) and contralateral breast cancer (CBC) risk, in BRCA1 and BRCA2 pathogenic variant heterozygotes. METHODS: We included women of European ancestry with a prevalent first primary invasive BC (BRCA1 = 6,591 with 1,402 prevalent CBC cases; BRCA2 = 4,208 with 647 prevalent CBC cases) from the Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA), a large international retrospective series. Cox regression analysis was performed to assess the association between overall and ER-specific PRS313 and CBC risk. RESULTS: For BRCA1 heterozygotes the estrogen receptor (ER)-negative PRS313 showed the largest association with CBC risk, hazard ratio (HR) per SD = 1.12, 95% confidence interval (CI) (1.06-1.18), C-index = 0.53; for BRCA2 heterozygotes, this was the ER-positive PRS313, HR = 1.15, 95% CI (1.07-1.25), C-index = 0.57. Adjusting for family history, age at diagnosis, treatment, or pathological characteristics for the first BC did not change association effect sizes. For women developing first BC < age 40 years, the cumulative PRS313 5th and 95th percentile 10-year CBC risks were 22% and 32% for BRCA1 and 13% and 23% for BRCA2 heterozygotes, respectively. CONCLUSION: The PRS313 can be used to refine individual CBC risks for BRCA1/2 heterozygotes of European ancestry, however the PRS313 needs to be considered in the context of a multifactorial risk model to evaluate whether it might influence clinical decision-making.

Oral contraceptive use and ovarian cancer risk for BRCA1/2 mutation carriers: an international cohort study.

BACKGROUND: Ovarian cancer risk in BRCA1 and BRCA2 mutation carriers has been shown to decrease with longer duration of oral contraceptive use. Although the effects of using oral contraceptives in the general population are well established (approximately 50% risk reduction in ovarian cancer), the estimated risk reduction in mutation carriers is much less precise because of potential bias and small sample sizes. In addition, only a few studies on oral contraceptive use have examined the associations of duration of use, time since last use, starting age, and calendar year of start with risk of ovarian cancer. OBJECTIVE: This study aimed to investigate in more detail the associations of various characteristics of oral contraceptive use and risk of ovarian cancer, to provide healthcare providers and carriers with better risk estimates. STUDY DESIGN: In this international retrospective study, ovarian cancer risk associations were assessed using oral contraceptives data on 3989 BRCA1 and 2445 BRCA2 mutation carriers. Age-dependent-weighted Cox regression analyses were stratified by study and birth cohort and included breast cancer diagnosis as a covariate. To minimize survival bias, analyses were left truncated at 5 years before baseline questionnaire. Separate analyses were conducted for each aspect of oral contraceptive use and in a multivariate analysis, including all these aspects. In addition, the analysis of duration of oral contraceptive use was stratified by recency of use. RESULTS: Oral contraceptives were less often used by mutation carriers who were diagnosed with ovarian cancer (ever use: 58.6% for BRCA1 and 53.5% BRCA2) than by unaffected carriers (ever use: 88.9% for BRCA1 and 80.7% for BRCA2). The median duration of use was 7 years for both BRCA1 and BRCA2 carriers who developed ovarian cancer and 9 and 8 years for unaffected BRCA1 and BRCA2 carriers with ovarian cancer, respectively. For BRCA1 mutation carriers, univariate analyses have shown that both a longer duration of oral contraceptive use and more recent oral contraceptive use were associated with a reduction in the risk of ovarian cancer. However, in multivariate analyses, including duration of use, age at first use, and time since last use, duration of oral contraceptive use proved to be the prominent protective factor (compared with <5 years: 5-9 years [hazard ratio, 0.67; 95% confidence interval, 0.40-1.12]; >10 years [hazard ratio, 0.37; 95% confidence interval, 0.19-0.73]; Ptrend=.008). The inverse association between duration of use and ovarian cancer risk persisted for more than 15 years (duration of ≥10 years; BRCA1 <15 years since last use [hazard ratio, 0.24; 95% confidence interval, 0.14-0.43]; BRCA1 >15 years since last use [hazard ratio, 0.56; 95% confidence interval, 0.18-0.59]). Univariate results for BRCA2 mutation carriers were similar but were inconclusive because of limited sample size. CONCLUSION: For BRCA1 mutation carriers, longer duration of oral contraceptive use is associated with a greater reduction in ovarian cancer risk, and the protection is long term.

Cas9-based enrichment and single-molecule sequencing for precise characterization of genomic duplications.

The widespread use of genome-wide diagnostic screening methods has greatly increased the frequency with which incidental (but possibly pathogenic) copy number changes affecting single genes are detected. These findings require validation to allow appropriate clinical management. Deletion variants can usually be readily validated using a range of short-read next-generation sequencing (NGS) strategies, but the characterization of duplication variants at nucleotide resolution remains challenging. This presents diagnostic problems, since pathogenicity cannot generally be assessed without knowing the structure of the variant. We have used a novel Cas9 enrichment strategy, in combination with long-read single-molecule nanopore sequencing, to address this need. We describe the nucleotide-level resolution of two problematic cases, both of whom presented with neurodevelopmental problems and were initially investigated by array CGH. In the first case, an incidental 1.7-kb imbalance involving a partial duplication of VHL exon 3 was detected. This variant was inherited from the patient's father, who had a history of renal cancer at 38 years. In the second case, an incidental ~200-kb de novo duplication that included DMD exons 30-44 was resolved. In both cases, the long-read data yielded sufficient information to enable Sanger sequencing to define the rearrangement breakpoints, and creation of breakpoint-spanning PCR assays suitable for testing of relatives. Our Cas9 enrichment and nanopore sequencing approach can be readily adopted by molecular diagnostic laboratories for cost-effective and rapid characterization of challenging duplication-containing alleles. We also anticipate that in future this method may prove useful for characterizing acquired translocations in tumor cells, and for precisely identifying transgene integration sites in mouse models.

Correction to: Risk-reducing salpingo-oophorectomy, natural menopause, and breast cancer risk: an international prospective cohort of BRCA1 and BRCA2 mutation carriers.

After publication of the original article [1], we were notified that columns in Table 2 were erroneously displayed.

Risk-reducing salpingo-oophorectomy, natural menopause, and breast cancer risk: an international prospective cohort of BRCA1 and BRCA2 mutation carriers.

BACKGROUND: The effect of risk-reducing salpingo-oophorectomy (RRSO) on breast cancer risk for BRCA1 and BRCA2 mutation carriers is uncertain. Retrospective analyses have suggested a protective effect but may be substantially biased. Prospective studies have had limited power, particularly for BRCA2 mutation carriers. Further, previous studies have not considered the effect of RRSO in the context of natural menopause. METHODS: A multi-centre prospective cohort of 2272 BRCA1 and 1605 BRCA2 mutation carriers was followed for a mean of 5.4 and 4.9 years, respectively; 426 women developed incident breast cancer. RRSO was modelled as a time-dependent covariate in Cox regression, and its effect assessed in premenopausal and postmenopausal women. RESULTS: There was no association between RRSO and breast cancer for BRCA1 (HR = 1.23; 95% CI 0.94-1.61) or BRCA2 (HR = 0.88; 95% CI 0.62-1.24) mutation carriers. For BRCA2 mutation carriers, HRs were 0.68 (95% CI 0.40-1.15) and 1.07 (95% CI 0.69-1.64) for RRSO carried out before or after age 45 years, respectively. The HR for BRCA2 mutation carriers decreased with increasing time since RRSO (HR = 0.51; 95% CI 0.26-0.99 for 5 years or longer after RRSO). Estimates for premenopausal women were similar. CONCLUSION: We found no evidence that RRSO reduces breast cancer risk for BRCA1 mutation carriers. A potentially beneficial effect for BRCA2 mutation carriers was observed, particularly after 5 years following RRSO. These results may inform counselling and management of carriers with respect to RRSO.

Polygenic risk scores and breast and epithelial ovarian cancer risks for carriers of BRCA1 and BRCA2 pathogenic variants.

PURPOSE: We assessed the associations between population-based polygenic risk scores (PRS) for breast (BC) or epithelial ovarian cancer (EOC) with cancer risks for BRCA1 and BRCA2 pathogenic variant carriers. METHODS: Retrospective cohort data on 18,935 BRCA1 and 12,339 BRCA2 female pathogenic variant carriers of European ancestry were available. Three versions of a 313 single-nucleotide polymorphism (SNP) BC PRS were evaluated based on whether they predict overall, estrogen receptor (ER)-negative, or ER-positive BC, and two PRS for overall or high-grade serous EOC. Associations were validated in a prospective cohort. RESULTS: The ER-negative PRS showed the strongest association with BC risk for BRCA1 carriers (hazard ratio [HR] per standard deviation = 1.29 [95% CI 1.25-1.33], P = 3×10-72). For BRCA2, the strongest association was with overall BC PRS (HR = 1.31 [95% CI 1.27-1.36], P = 7×10-50). HR estimates decreased significantly with age and there was evidence for differences in associations by predicted variant effects on protein expression. The HR estimates were smaller than general population estimates. The high-grade serous PRS yielded the strongest associations with EOC risk for BRCA1 (HR = 1.32 [95% CI 1.25-1.40], P = 3×10-22) and BRCA2 (HR = 1.44 [95% CI 1.30-1.60], P = 4×10-12) carriers. The associations in the prospective cohort were similar. CONCLUSION: Population-based PRS are strongly associated with BC and EOC risks for BRCA1/2 carriers and predict substantial absolute risk differences for women at PRS distribution extremes.

Mendelian randomisation study of height and body mass index as modifiers of ovarian cancer risk in 22,588 BRCA1 and BRCA2 mutation carriers.

BACKGROUND: Height and body mass index (BMI) are associated with higher ovarian cancer risk in the general population, but whether such associations exist among BRCA1/2 mutation carriers is unknown. METHODS: We applied a Mendelian randomisation approach to examine height/BMI with ovarian cancer risk using the Consortium of Investigators for the Modifiers of BRCA1/2 (CIMBA) data set, comprising 14,676 BRCA1 and 7912 BRCA2 mutation carriers, with 2923 ovarian cancer cases. We created a height genetic score (height-GS) using 586 height-associated variants and a BMI genetic score (BMI-GS) using 93 BMI-associated variants. Associations were assessed using weighted Cox models. RESULTS: Observed height was not associated with ovarian cancer risk (hazard ratio [HR]: 1.07 per 10-cm increase in height, 95% confidence interval [CI]: 0.94-1.23). Height-GS showed similar results (HR = 1.02, 95% CI: 0.85-1.23). Higher BMI was significantly associated with increased risk in premenopausal women with HR = 1.25 (95% CI: 1.06-1.48) and HR = 1.59 (95% CI: 1.08-2.33) per 5-kg/m2 increase in observed and genetically determined BMI, respectively. No association was found for postmenopausal women. Interaction between menopausal status and BMI was significant (Pinteraction < 0.05). CONCLUSION: Our observation of a positive association between BMI and ovarian cancer risk in premenopausal BRCA1/2 mutation carriers is consistent with findings in the general population.

Mosaic PPM1D mutations are associated with predisposition to breast and ovarian cancer.

Improved sequencing technologies offer unprecedented opportunities for investigating the role of rare genetic variation in common disease. However, there are considerable challenges with respect to study design, data analysis and replication. Using pooled next-generation sequencing of 507 genes implicated in the repair of DNA in 1,150 samples, an analytical strategy focused on protein-truncating variants (PTVs) and a large-scale sequencing case-control replication experiment in 13,642 individuals, here we show that rare PTVs in the p53-inducible protein phosphatase PPM1D are associated with predisposition to breast cancer and ovarian cancer. PPM1D PTV mutations were present in 25 out of 7,781 cases versus 1 out of 5,861 controls (P = 1.12 × 10(-5)), including 18 mutations in 6,912 individuals with breast cancer (P = 2.42 × 10(-4)) and 12 mutations in 1,121 individuals with ovarian cancer (P = 3.10 × 10(-9)). Notably, all of the identified PPM1D PTVs were mosaic in lymphocyte DNA and clustered within a 370-base-pair region in the final exon of the gene, carboxy-terminal to the phosphatase catalytic domain. Functional studies demonstrate that the mutations result in enhanced suppression of p53 in response to ionizing radiation exposure, suggesting that the mutant alleles encode hyperactive PPM1D isoforms. Thus, although the mutations cause premature protein truncation, they do not result in the simple loss-of-function effect typically associated with this class of variant, but instead probably have a gain-of-function effect. Our results have implications for the detection and management of breast and ovarian cancer risk. More generally, these data provide new insights into the role of rare and of mosaic genetic variants in common conditions, and the use of sequencing in their identification.

Tumour risks and genotype-phenotype correlations associated with germline variants in succinate dehydrogenase subunit genes SDHB, SDHC and SDHD.

BACKGROUND: Germline pathogenic variants in SDHB/SDHC/SDHD are the most frequent causes of inherited phaeochromocytomas/paragangliomas. Insufficient information regarding penetrance and phenotypic variability hinders optimum management of mutation carriers. We estimate penetrance for symptomatic tumours and elucidate genotype-phenotype correlations in a large cohort of SDHB/SDHC/SDHD mutation carriers. METHODS: A retrospective survey of 1832 individuals referred for genetic testing due to a personal or family history of phaeochromocytoma/paraganglioma. 876 patients (401 previously reported) had a germline mutation in SDHB/SDHC/SDHD (n=673/43/160). Tumour risks were correlated with in silico structural prediction analyses. RESULTS: Tumour risks analysis provided novel penetrance estimates and genotype-phenotype correlations. In addition to tumour type susceptibility differences for individual genes, we confirmed that the SDHD:p.Pro81Leu mutation has a distinct phenotype and identified increased age-related tumour risks with highly destabilising SDHB missense mutations. By Kaplan-Meier analysis, the penetrance (cumulative risk of clinically apparent tumours) in SDHB and (paternally inherited) SDHD mutation-positive non-probands (n=371/67 with detailed clinical information) by age 60 years was 21.8% (95% CI 15.2% to 27.9%) and 43.2% (95% CI 25.4% to 56.7%), respectively. Risk of malignant disease at age 60 years in non-proband SDHB mutation carriers was 4.2%(95% CI 1.1% to 7.2%). With retrospective cohort analysis to adjust for ascertainment, cumulative tumour risks for SDHB mutation carriers at ages 60 years and 80 years were 23.9% (95% CI 20.9% to 27.4%) and 30.6% (95% CI 26.8% to 34.7%). CONCLUSIONS: Overall risks of clinically apparent tumours for SDHB mutation carriers are substantially lower than initially estimated and will improve counselling of affected families. Specific genotype-tumour risk associations provides a basis for novel investigative strategies into succinate dehydrogenase-related mechanisms of tumourigenesis and the development of personalised management for SDHB/SDHC/SDHD mutation carriers.

Mutational spectrum in a worldwide study of 29,700 families with BRCA1 or BRCA2 mutations.

The prevalence and spectrum of germline mutations in BRCA1 and BRCA2 have been reported in single populations, with the majority of reports focused on White in Europe and North America. The Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA) has assembled data on 18,435 families with BRCA1 mutations and 11,351 families with BRCA2 mutations ascertained from 69 centers in 49 countries on six continents. This study comprehensively describes the characteristics of the 1,650 unique BRCA1 and 1,731 unique BRCA2 deleterious (disease-associated) mutations identified in the CIMBA database. We observed substantial variation in mutation type and frequency by geographical region and race/ethnicity. In addition to known founder mutations, mutations of relatively high frequency were identified in specific racial/ethnic or geographic groups that may reflect founder mutations and which could be used in targeted (panel) first pass genotyping for specific populations. Knowledge of the population-specific mutational spectrum in BRCA1 and BRCA2 could inform efficient strategies for genetic testing and may justify a more broad-based oncogenetic testing in some populations.

Risks of breast or ovarian cancer in BRCA1 or BRCA2 predictive test negatives: findings from the EMBRACE study.

PURPOSE: BRCA1/BRCA2 predictive test negatives are proven noncarriers of a BRCA1/BRCA2 mutation that is carried by their relatives. The risk of developing breast cancer (BC) or epithelial ovarian cancer (EOC) in these women is uncertain. The study aimed to estimate risks of invasive BC and EOC in a large cohort of BRCA1/BRCA2 predictive test negatives. METHODS: We used cohort analysis to estimate incidences, cumulative risks, and standardized incidence ratios (SIRs). RESULTS: A total of 1,895 unaffected women were eligible for inclusion in the BC risk analysis and 1,736 in the EOC risk analysis. There were 23 incident invasive BCs and 2 EOCs. The cumulative risk of invasive BC was 9.4% (95% confidence interval (CI) 5.9-15%) by age 85 years and the corresponding risk of EOC was 0.6% (95% CI 0.2-2.6%). The SIR for invasive BC was 0.93 (95% CI 0.62-1.40) in the overall cohort, 0.85 (95% CI 0.48-1.50) in noncarriers from BRCA1 families, and 1.03 (95% CI 0.57-1.87) in noncarriers from BRCA2 families. The SIR for EOC was 0.79 (95% CI 0.20-3.17) in the overall cohort. CONCLUSION: Our results did not provide evidence for elevated risks of invasive BC or EOC in BRCA1/BRCA2 predictive test negatives.

Association of breast cancer risk in BRCA1 and BRCA2 mutation carriers with genetic variants showing differential allelic expression: identification of a modifier of breast cancer risk at locus 11q22.3.

PURPOSE: Cis-acting regulatory SNPs resulting in differential allelic expression (DAE) may, in part, explain the underlying phenotypic variation associated with many complex diseases. To investigate whether common variants associated with DAE were involved in breast cancer susceptibility among BRCA1 and BRCA2 mutation carriers, a list of 175 genes was developed based of their involvement in cancer-related pathways. METHODS: Using data from a genome-wide map of SNPs associated with allelic expression, we assessed the association of ~320 SNPs located in the vicinity of these genes with breast and ovarian cancer risks in 15,252 BRCA1 and 8211 BRCA2 mutation carriers ascertained from 54 studies participating in the Consortium of Investigators of Modifiers of BRCA1/2. RESULTS: We identified a region on 11q22.3 that is significantly associated with breast cancer risk in BRCA1 mutation carriers (most significant SNP rs228595 p = 7 × 10-6). This association was absent in BRCA2 carriers (p = 0.57). The 11q22.3 region notably encompasses genes such as ACAT1, NPAT, and ATM. Expression quantitative trait loci associations were observed in both normal breast and tumors across this region, namely for ACAT1, ATM, and other genes. In silico analysis revealed some overlap between top risk-associated SNPs and relevant biological features in mammary cell data, which suggests potential functional significance. CONCLUSION: We identified 11q22.3 as a new modifier locus in BRCA1 carriers. Replication in larger studies using estrogen receptor (ER)-negative or triple-negative (i.e., ER-, progesterone receptor-, and HER2-negative) cases could therefore be helpful to confirm the association of this locus with breast cancer risk.

Risks of Breast, Ovarian, and Contralateral Breast Cancer for BRCA1 and BRCA2 Mutation Carriers.

Importance: The clinical management of BRCA1 and BRCA2 mutation carriers requires accurate, prospective cancer risk estimates. Objectives: To estimate age-specific risks of breast, ovarian, and contralateral breast cancer for mutation carriers and to evaluate risk modification by family cancer history and mutation location. Design, Setting, and Participants: Prospective cohort study of 6036 BRCA1 and 3820 BRCA2 female carriers (5046 unaffected and 4810 with breast or ovarian cancer or both at baseline) recruited in 1997-2011 through the International BRCA1/2 Carrier Cohort Study, the Breast Cancer Family Registry and the Kathleen Cuningham Foundation Consortium for Research into Familial Breast Cancer, with ascertainment through family clinics (94%) and population-based studies (6%). The majority were from large national studies in the United Kingdom (EMBRACE), the Netherlands (HEBON), and France (GENEPSO). Follow-up ended December 2013; median follow-up was 5 years. Exposures: BRCA1/2 mutations, family cancer history, and mutation location. Main Outcomes and Measures: Annual incidences, standardized incidence ratios, and cumulative risks of breast, ovarian, and contralateral breast cancer. Results: Among 3886 women (median age, 38 years; interquartile range [IQR], 30-46 years) eligible for the breast cancer analysis, 5066 women (median age, 38 years; IQR, 31-47 years) eligible for the ovarian cancer analysis, and 2213 women (median age, 47 years; IQR, 40-55 years) eligible for the contralateral breast cancer analysis, 426 were diagnosed with breast cancer, 109 with ovarian cancer, and 245 with contralateral breast cancer during follow-up. The cumulative breast cancer risk to age 80 years was 72% (95% CI, 65%-79%) for BRCA1 and 69% (95% CI, 61%-77%) for BRCA2 carriers. Breast cancer incidences increased rapidly in early adulthood until ages 30 to 40 years for BRCA1 and until ages 40 to 50 years for BRCA2 carriers, then remained at a similar, constant incidence (20-30 per 1000 person-years) until age 80 years. The cumulative ovarian cancer risk to age 80 years was 44% (95% CI, 36%-53%) for BRCA1 and 17% (95% CI, 11%-25%) for BRCA2 carriers. For contralateral breast cancer, the cumulative risk 20 years after breast cancer diagnosis was 40% (95% CI, 35%-45%) for BRCA1 and 26% (95% CI, 20%-33%) for BRCA2 carriers (hazard ratio [HR] for comparing BRCA2 vs BRCA1, 0.62; 95% CI, 0.47-0.82; P=.001 for difference). Breast cancer risk increased with increasing number of first- and second-degree relatives diagnosed as having breast cancer for both BRCA1 (HR for ≥2 vs 0 affected relatives, 1.99; 95% CI, 1.41-2.82; P<.001 for trend) and BRCA2 carriers (HR, 1.91; 95% CI, 1.08-3.37; P=.02 for trend). Breast cancer risk was higher if mutations were located outside vs within the regions bounded by positions c.2282-c.4071 in BRCA1 (HR, 1.46; 95% CI, 1.11-1.93; P=.007) and c.2831-c.6401 in BRCA2 (HR, 1.93; 95% CI, 1.36-2.74; P<.001). Conclusions and Relevance: These findings provide estimates of cancer risk based on BRCA1 and BRCA2 mutation carrier status using prospective data collection and demonstrate the potential importance of family history and mutation location in risk assessment.

Robust diagnostic genetic testing using solution capture enrichment and a novel variant-filtering interface.

Targeted hybridization enrichment prior to next-generation sequencing is a widespread method for characterizing sequence variation in a research setting, and is being adopted by diagnostic laboratories. However, the number of variants identified can overwhelm clinical laboratories with strict time constraints, the final interpretation of likely pathogenicity being a particular bottleneck. To address this, we have developed an approach in which, after automatic variant calling on a standard unix pipeline, subsequent variant filtering is performed interactively, using AgileExomeFilter and AgilePindelFilter (http://dna.leeds.ac.uk/agile), tools designed for clinical scientists with standard desktop computers. To demonstrate the method's diagnostic efficacy, we tested 128 patients using (1) a targeted capture of 36 cancer-predisposing genes or (2) whole-exome capture for diagnosis of the genetically heterogeneous disorder primary ciliary dyskinesia (PCD). In the cancer cohort, complete concordance with previous diagnostic data was achieved across 793 variant genotypes. A high yield (42%) was also achieved for exome-based PCD diagnosis, underscoring the scalability of our method. Simple adjustments to the variant filtering parameters further allowed the identification of a homozygous truncating mutation in a presumptive new PCD gene, DNAH8. These tools should allow diagnostic laboratories to expand their testing portfolios flexibly, using a standard set of reagents and techniques.

Refined histopathological predictors of BRCA1 and BRCA2 mutation status: a large-scale analysis of breast cancer characteristics from the BCAC, CIMBA, and ENIGMA consortia.

INTRODUCTION: The distribution of histopathological features of invasive breast tumors in BRCA1 or BRCA2 germline mutation carriers differs from that of individuals with no known mutation. Histopathological features thus have utility for mutation prediction, including statistical modeling to assess pathogenicity of BRCA1 or BRCA2 variants of uncertain clinical significance. We analyzed large pathology datasets accrued by the Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA) and the Breast Cancer Association Consortium (BCAC) to reassess histopathological predictors of BRCA1 and BRCA2 mutation status, and provide robust likelihood ratio (LR) estimates for statistical modeling. METHODS: Selection criteria for study/center inclusion were estrogen receptor (ER) status or grade data available for invasive breast cancer diagnosed younger than 70 years. The dataset included 4,477 BRCA1 mutation carriers, 2,565 BRCA2 mutation carriers, and 47,565 BCAC breast cancer cases. Country-stratified estimates of the likelihood of mutation status by histopathological markers were derived using a Mantel-Haenszel approach. RESULTS: ER-positive phenotype negatively predicted BRCA1 mutation status, irrespective of grade (LRs from 0.08 to 0.90). ER-negative grade 3 histopathology was more predictive of positive BRCA1 mutation status in women 50 years or older (LR = 4.13 (3.70 to 4.62)) versus younger than 50 years (LR = 3.16 (2.96 to 3.37)). For BRCA2, ER-positive grade 3 phenotype modestly predicted positive mutation status irrespective of age (LR = 1.7-fold), whereas ER-negative grade 3 features modestly predicted positive mutation status at 50 years or older (LR = 1.54 (1.27 to 1.88)). Triple-negative tumor status was highly predictive of BRCA1 mutation status for women younger than 50 years (LR = 3.73 (3.43 to 4.05)) and 50 years or older (LR = 4.41 (3.86 to 5.04)), and modestly predictive of positive BRCA2 mutation status in women 50 years or older (LR = 1.79 (1.42 to 2.24)). CONCLUSIONS: These results refine likelihood-ratio estimates for predicting BRCA1 and BRCA2 mutation status by using commonly measured histopathological features. Age at diagnosis is an important variable for most analyses, and grade is more informative than ER status for BRCA2 mutation carrier prediction. The estimates will improve BRCA1 and BRCA2 variant classification and inform patient mutation testing and clinical management.

Gene-gene interactions in breast cancer susceptibility.

There have been few definitive examples of gene-gene interactions in humans. Through mutational analyses in 7325 individuals, we report four interactions (defined as departures from a multiplicative model) between mutations in the breast cancer susceptibility genes ATM and CHEK2 with BRCA1 and BRCA2 (case-only interaction between ATM and BRCA1/BRCA2 combined, P = 5.9 × 10(-4); ATM and BRCA1, P= 0.01; ATM and BRCA2, P= 0.02; CHEK2 and BRCA1/BRCA2 combined, P = 2.1 × 10(-4); CHEK2 and BRCA1, P= 0.01; CHEK2 and BRCA2, P= 0.01). The interactions are such that the resultant risk of breast cancer is lower than the multiplicative product of the constituent risks, and plausibly reflect the functional relationships of the encoded proteins in DNA repair. These findings have important implications for models of disease predisposition and clinical translation.