Neuhausen, Susan L.

Abstract Part of the substantial unexplained familial aggregation of breast cancer may be due to interactions between common variants, but few studies have had adequate statistical power to detect interactions of realistic magnitude. We aimed to assess all two-way interactions in breast cancer susceptibility between 70 917 single nucleotide polymorphisms (SNPs) selected primarily based on prior evidence of a marginal effect. Thirty-eight international studies contributed data for 46 450 breast cancer cases and 42 461 controls of European origin as part of a multi-consortium project (COGS). First, SNPs were preselected based on evidence (P < 0.01) of a per-allele main effect, and all two-way combinations of those were evaluated by a per-allele (1 d.f.) test for interaction using logistic regression. Second, all 2.5 billion possible two-SNP combinations were evaluated using Boolean operation-based screening and testing, and SNP pairs with the strongest evidence of interaction (P < 10−4) were selected for more careful assessment by logistic regression. Under the first approach, 3277 SNPs were preselected, but an evaluation of all possible two-SNP combinations (1 d.f.) identified no interactions at P < 10−8. Results from the second analytic approach were consistent with those from the first (P > 10−10). In summary, we observed little evidence of two-way SNP interactions in breast cancer susceptibility, despite the large number of SNPs with potential marginal effects considered and the very large sample size. This finding may have important implications for risk prediction, simplifying the modelling required. Further comprehensive, large-scale genome-wide interaction studies may identify novel interacting loci if the inherent logistic and computational challenges can be overcome.

Abstract BACKGROUND Accurate evaluation of unclassified sequence variants in cancer predisposition genes is essential for clinical management and depends on a multifactorial analysis of clinical, genetic, pathologic, and bioinformatic variables and assays of transcript length and abundance. The integrity of assay data in turn relies on appropriate assay design, interpretation, and reporting. METHODS We conducted a multicenter investigation to compare mRNA splicing assay protocols used by members of the ENIGMA (Evidence-Based Network for the Interpretation of Germline Mutant Alleles) consortium. We compared similarities and differences in results derived from analysis of a panel of breast cancer 1, early onset (BRCA1) and breast cancer 2, early onset (BRCA2) gene variants known to alter splicing (BRCA1: c.135-1G>T, c.591C>T, c.594-2A>C, c.671-2A>G, and c.5467+5G>C and BRCA2: c.426-12_8delGTTTT, c.7988A>T, c.8632+1G>A, and c.9501+3A>T). Differences in protocols were then assessed to determine which elements were critical in reliable assay design. RESULTS PCR primer design strategies, PCR conditions, and product detection methods, combined with a prior knowledge of expected alternative transcripts, were the key factors for accurate splicing assay results. For example, because of the position of primers and PCR extension times, several isoforms associated with BRCA1, c.594-2A>C and c.671-2A>G, were not detected by many sites. Variation was most evident for the detection of low-abundance transcripts (e.g., BRCA2 c.8632+1G>A Δ19,20 and BRCA1 c.135-1G>T Δ5q and Δ3). Detection of low-abundance transcripts was sometimes addressed by using more analytically sensitive detection methods (e.g., BRCA2 c.426-12_8delGTTTT ins18bp). CONCLUSIONS We provide recommendations for best practice and raise key issues to consider when designing mRNA assays for evaluation of unclassified sequence variants.

Abstract Rare germline mutations in the breast and ovarian cancer predisposition genes BRCA1 and BRCA2 are present in roughly 5 percent of ovarian cancer patients. Both genes play key roles in DNA damage repair but appear to have distinct, although often complementary, functions. The risks of breast and ovarian cancer have been shown to differ between BRCA1 and BRCA2 carriers, and mutation-specific effects have also been suggested for both groups. Published reports comparing the survival of ovarian cancer patients with and without BRCA1/2 mutations generally show a survival advantage among mutation carriers. This is thought to be related to an improved response of carriers to platinum-based therapies. Since it is not known whether BRCA1 and BRCA2 carriers show similar survival patterns, we have performed a large, multicenter study to investigate the impact of germline BRCA1 and BRCA2 mutations on ovarian cancer survival. 3,531 invasive epithelial ovarian cancer cases (1,178 BRCA1, 367 BRCA2 and 1,986 BRCA-negative) with survival time data from twenty-four studies in the U.S.A, Europe, Israel and Asia were included. In our main analysis, we excluded patients who were known or who were likely to have not received platinum-based therapy. Compared to non-carriers, BRCA1 and BRCA2 carriers were more likely to present with advanced stage (BRCA1; p=2×10−4, BRCA2; p=4×10−6), high grade (BRCA1; p=4×10−9, BRCA2; p=1×10−4), serous disease (BRCA1; p=3×10−6, BRCA2; p=0.003). BRCA1 carriers were younger at diagnosis (p=2×10−9) and BRCA2 slightly older at diagnosis (p=0.002) than non-carriers. BRCA1 and BRCA2 carriers did not differ in terms of tumor stage, grade or histology but did show differences in the age at diagnosis (p=2×10−14). In an unadjusted analysis, neither BRCA1 nor BRCA2 carriers differed from non-carriers in overall survival (BRCA1: HR=1.02, p=0.77, BRCA2: HR=0.89, p=0.36). After adjusting for stage, grade, histology and age at diagnosis, BRCA1 carriers showed a modest, non-significant, survival advantage (HR=0.84, p=0.12) while BRCA2 carriers showed a marked improvement (HR=0.57, p=6×10−4) compared to non-carriers. This survival advantage was also seen when comparing BRCA2 carriers to BRCA1 carriers after adjustment for age at diagnosis (HR=0.69, p=5×10−4). Similar results were obtained when we restricted the analysis to high grade, advanced stage, serous cases. The survival differences we observed between BRCA1 and BRCA2 carriers could be related to differences in tumor biology or chemo-sensitivity. Additional analyses investigating the later will be presented in addition to mutation-specific effects within BRCA1 and BRCA2. In this study, we provide strong evidence for genetic heterogeneity of survival effects in ovarian cancer patients with BRCA mutations. This could be applied to both clinical prediction and to aid our understanding of the complex biology of BRCA mutations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2752. doi:10.1158/1538-7445.AM2011-2752

Abstract Background: Genome-wide association studies (GWAS) identified variants at 19p13.1 and ZNF365 (10q21.2) as risk factors for breast cancer among BRCA1 and BRCA2 mutation carriers, respectively. We explored associations with ovarian cancer and with breast cancer by tumor histopathology for these variants in mutation carriers from the Consortium of Investigators of Modifiers of BRCA1/2 (CIMBA). Methods: Genotyping data for 12,599 BRCA1 and 7,132 BRCA2 mutation carriers from 40 studies were combined. Results: We confirmed associations between rs8170 at 19p13.1 and breast cancer risk for BRCA1 mutation carriers [HR, 1.17; 95% confidence interval (CI), 1.07–1.27; P = 7.42 × 10−4] and between rs16917302 at ZNF365 (HR, 0.84; 95% CI, 0.73–0.97; P = 0.017) but not rs311499 at 20q13.3 (HR, 1.11; 95% CI, 0.94–1.31; P = 0.22) and breast cancer risk for BRCA2 mutation carriers. Analyses based on tumor histopathology showed that 19p13 variants were predominantly associated with estrogen receptor (ER)-negative breast cancer for both BRCA1 and BRCA2 mutation carriers, whereas rs16917302 at ZNF365 was mainly associated with ER-positive breast cancer for both BRCA1 and BRCA2 mutation carriers. We also found for the first time that rs67397200 at 19p13.1 was associated with an increased risk of ovarian cancer for BRCA1 (HR, 1.16; 95% CI, 1.05–1.29; P = 3.8 × 10−4) and BRCA2 mutation carriers (HR, 1.30; 95% CI, 1.10–1.52; P = 1.8 × 10−3). Conclusions: 19p13.1 and ZNF365 are susceptibility loci for ovarian cancer and ER subtypes of breast cancer among BRCA1 and BRCA2 mutation carriers. Impact: These findings can lead to an improved understanding of tumor development and may prove useful for breast and ovarian cancer risk prediction for BRCA1 and BRCA2 mutation carriers. Cancer Epidemiol Biomarkers Prev; 21(4); 645–57. ©2012 AACR.

Abstract Background: BRCA1 and BRCA2 mutation carriers have a lifetime breast cancer risk of 40% to 80%, suggesting the presence of risk modifiers. We previously identified significant associations in genetic variants in the insulin-like growth factor (IGF) signaling pathway. Here, we investigate additional IGF signaling genes as risk modifiers for breast cancer development in BRCA carriers. Methods: A cohort of 1,019 BRCA1 and 500 BRCA2 mutation carriers were genotyped for 99 single-nucleotide polymorphisms (SNP) in 13 genes. Proportional hazards regression was used to model time from birth to diagnosis of breast cancer for BRCA1 and BRCA2 carriers separately. For linkage disequilibrium (LD) blocks with multiple SNPs, an additive genetic model was used. For an SNP analysis, no additivity assumptions were made. Results: Significant associations were found between risk of breast cancer and LD blocks in IGF2 for BRCA1 and BRCA2 mutation carriers (global P values of 0.009 for BRCA1 and 0.007 for BRCA2), HTRA1 for BRCA1 carriers (global P value of 0.005), and MMP3 for BRCA2 carriers (global P = 0.0000007 for BRCA2). Conclusions: We identified significant associations of genetic variants involved in IGF signaling. With the known interaction of BRCA1 and IGF signaling and the loss of PTEN in a majority of BRCA1 tumors, this suggests that signaling through AKT may modify breast cancer risk in BRCA1 carriers. Impact: These results suggest potential avenues for future research targeting the IGF signaling pathway in modifying risk in BRCA1and BRCA2 mutation carriers. Cancer Epidemiol Biomarkers Prev; 20(8); 1690–702. ©2011 AACR.

Abstract Inherited BRCA1/2 mutations confer elevated ovarian cancer risk. Knowledge of factors that can improve ovarian cancer risk assessment in BRCA1/2 mutation carriers is important because no effective early detection for ovarian cancers exists. A cohort of 1,575 BRCA1 and 856 BRCA2 mutation carriers was used to evaluate haplotypes at ATM, BARD1, BRIP1, CTIP, MRE11, NBS1, RAD50, RAD51, and TOPBP1 in ovarian cancer risk. In BRCA1 carriers, no associations were observed with ATM, BARD1, CTIP, RAD50, RAD51, or TOPBP1. At BRIP1, an association was observed for one haplotype with a multiple testing corrected P (Pcorr) = 0.012, although no individual haplotype was significant. At MRE11, statistically significant associations were observed for one haplotype (Pcorr = 0.007). At NBS1, we observed a Pcorr = 0.024 for haplotypes. In BRCA2 carriers, no associations were observed with CTIP, NBS1, RAD50, or TOPBP1. Rare haplotypes at ATM (Pcorr = 0.044) and BARD1 (Pcorr = 0.012) were associated with ovarian cancer risk. At BRIP1, two common haplotypes were significantly associated with ovarian cancer risk (Pcorr = 0.011). At MRE11, we observed a significant haplotype association (Pcorr = 0.012), and at RAD51, one common haplotype was significantly associated with ovarian cancer risk (Pcorr = 0.026). Variants in genes that interact biologically withBRCA1 and/or BRCA2 may be associated with modified ovarian cancer risk in women who carry BRCA1/2 mutations. [Cancer Res 2009;69(14):5801–10]

AbstractThe AURKA oncogene is associated with abnormal chromosome segregation and aneuploidy and predisposition to cancer. Amplification of AURKA has been detected at higher frequency in tumors from BRCA1 and BRCA2 mutation carriers than in sporadic breast tumors, suggesting that overexpression of AURKA and inactivation of BRCA1 and BRCA2 cooperate during tumor development and progression. The F31I polymorphism in AURKA has been associated with breast cancer risk in the homozygous state in prior studies. We evaluated whether the AURKA F31I polymorphism modifies breast cancer risk in BRCA1 and BRCA2 mutation carriers from the Consortium of Investigators of Modifiers of BRCA1/2. Consortium of Investigators of Modifiers of BRCA1/2 was established to provide sufficient statistical power through increased numbers of mutation carriers to identify polymorphisms that act as modifiers of cancer risk and can refine breast cancer risk estimates in BRCA1 and BRCA2 mutation carriers. A total of 4,935 BRCA1 and 2,241 BRCA2 mutation carriers and 11 individuals carrying both BRCA1 and BRCA2 mutations was genotyped for F31I. Overall, homozygosity for the 31I allele was not significantly associated with breast cancer risk in BRCA1 and BRCA2 carriers combined [hazard ratio (HR), 0.91; 95% confidence interval (95% CI), 0.77-1.06]. Similarly, no significant association was seen in BRCA1 (HR, 0.90; 95% CI, 0.75-1.08) or BRCA2 carriers (HR, 0.93; 95% CI, 0.67-1.29) or when assessing the modifying effects of either bilateral prophylactic oophorectomy or menopausal status of BRCA1 and BRCA2 carriers. In summary, the F31I polymorphism in AURKA is not associated with a modified risk of breast cancer in BRCA1 and BRCA2 carriers. (Cancer Epidemiol Biomarkers Prev 2007;16(7):1416–21)