Wang, Xianshu

Abstract Distinct subtypes of triple negative (TN) breast cancer have currently been identified by tumor expression profiling and are thought to be influenced by the tumor microenvironment. However, little is known about the relationship between distinguishing histopathologic features of TN tumors, which reflect aspects of both tumor behavior and tumor microenvironment, and molecular TN subtypes. We analyzed whole genome expression profiles of 593 TN tumors from the Triple Negative Breast Cancer Consortium (TNBCC) using the Illumina Whole Genome DASL array. Histopathologic review of mitotic index, grade, trabecular pattern, growth pattern, involution, presence of ductal carcinoma in situ, and degree of lymphoid infiltrate, necrosis, fibrosis, and lobulitis was completed for 310 of these tumors. Clusters were identified both agnostically and using genes associated with these histopathologic features, and subtypes were evaluated for enriched pathways and associations with histopathologic features. Agnostic clustering identified four stable clusters which represented a basal-like (BL) signature, a luminal signature with overexpression of androgen receptor and targets, an immune signature, and a stromal (STR) signature. Overall, the TN tumors tended to have little or no necrosis (91%), high grade (88%), a high mitotic index (80%), fibrosis (77%), an epicentric growth pattern (62%), incomplete lobular involution (55%), trabecular structures (37%), lymphocytic infiltration (25%), adjacent ductal carcinoma in situ (24%), and variable amounts of lobulitis. Using a combined total of 2,776 unique probes individually associated with the ten histopathologic features measured, we identified six distinct TN subtypes, with an additional BL and STR signature compared to the four agnostic subtypes. Our findings suggest that histopathologic features that reflect heterogeneity in tumor cell structure and behavior as well as the tumor microenvironment, particularly the presence of lymphocytic infiltrate, are critical for TN subtype identification. Further work is needed to validate these signatures and to better understand the immunologic pathways associated with each subtype. Citation Format: Kristen Purrington, Daniel Visscher, Drakoulis Yannoukakos, Jane Carpenter, Heli Nevanlinna, Arto Mannerma, Xianshu Wang, Graham Giles, Wei Zheng, Angela Cox, Hiltrud Brauch, Ute Hamann, Diana Eccles, Celine M. Vachon, Fergus J. Couch. Genes associated with histopathologic features of triple negative breast tumors predict molecular subtypes. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1924. doi:10.1158/1538-7445.AM2015-1924

Abstract Recent studies have shown that associations between common genetic variation and gene expression in breast tumors provide insight into the functional mechanisms underlying breast cancer risk loci. However, the relationship between genetic variation, gene expression, and risk of triple negative (TN) breast cancer remains largely unexplored. We performed a genome-wide expression quantitative trait locus (eQTL) analysis of TN breast cancer using 668 formalin-fixed paraffin embedded TN tumors from the Triple Negative Breast Cancer Consortium (TNBCC) to identify novel genes relevant to TN breast cancer risk and to further explore the biology underlying known TN risk loci. Cis-eQTLs were defined as correlations between single nucleotide polymorphisms (SNP) and expression of genes within 1Mb, and trans-eQTLs were defined as the remainder of all SNP-gene correlations. In a genome-wide analysis, we identified 68,012 cis-eQTLs, representing correlations between 42,193 SNPs and 2,092 genes that were significant at a 10% false discovery rate (FDR). We also identified 70 trans-eQTLs across two unique genes at the same FDR threshold. Five cis-eQTLs involved SNPs previously associated with TN breast cancer risk (p<5.0x10-5) in 3,700 TN cases and 4,700 controls from the TNBCC. Three of these, located on chromosomes 5p (cis-eQTL p=4.8x10-34; risk p=1.1x10-7), 7p (cis-eQTL p=6.1x10-10; risk p=1.3x10-7), and 17q (cis-eQTL p=1.8x10-15; risk p=2.9x10-7), have previously been reported in public databases of normal tissue. The remaining two TN breast cancer cis-eQTLs, located on chromosomes 8q (cis-eQTL p=6.3x10-6, risk p=8.3x10-6) and 14q (cis-eQTL p=3.1x10-6, risk p=1.2x10-5), have not been reported as eQTLs in other tissues. In addition, 12 of 25 known TN breast cancer risk loci (PEX14, MDM4, 2q31.1, ESR1, 11q13.1, 11q24.3, 12p13.1, PTHLH, NTN4, 12q24, 19p13.1, MLK1), contained at least one significant cis-eQTL. In summary, we have identified five cis-eQTLs that may identify novel risk loci for TN breast cancer. Citation Format: Kristen S. Purrington, Drakoulis Yannoukakos, Jane Carpenter, Heli Nevanlinna, Angela Cox, Gianluca Severi, Christine Ambrosone, Amanda Ewart Toland, Andrew K. Godwin, Hiltrud Brauch, Peter A. Fasching, Penelope Miron, Jenny Chang-Claude, Nicholas G. Martin, Grant W. Montgomery, Vessela Kristensen, Hoda Anton-Culver, Paul Goodfellow, Janet E. Olson, Hugues Sicotte, Naresh Prodduturi, Daniel W. Visscher, Jeanette E. Eckel-Passow, S. Keith Anderson, Seth Slettedahl, Curtis Olswold, Xianshu Wang, V. Shane Pankratz, Susan Slager, Wei Zheng, Arto Mannermaa, Ute Hamann, Diana M. Eccles, Celine M. Vachon, Fergus J. Couch. Expression quantitative trait locus analysis of triple negative breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3266. doi:10.1158/1538-7445.AM2014-3266

Abstract BRCA1 and BRCA2 (BRCA) testing is uninformative in approximately 80% of families with clinical features of inherited susceptibility to breast cancer. Early identification of the individuals at risk in BRCAx families may lead to enhanced screening and prevention strategies, and potentially improved overall survival as has been seen for carriers of BRCA1/2 mutations. A proportion of these families are likely explained by mutations in other high penetrance and moderate penetrance breast cancer susceptibility genes. Although multi-gene panels are now commercially available for clinical testing, only limited information is available on the spectrum of mutations in breast cancer susceptibility genes in specific subgroups of high-risk breast cancer patients. We performed whole exome sequencing in 278 BRCA1/2 negative individuals with high-risk familial breast cancer, defined as a proband and at least two first or second degree relatives with breast cancer. Samples included 232 individuals from 97 families and 49 unrelated individuals, for a total of 146 independent cases. Data were analyzed for identification of all variants in 18 known high and moderate penetrance breast cancer susceptibility genes, in addition to BRCA1 and BRCA2. Two of the 97 families and one of the 49 singletons (2% of independent cases) were found to have deleterious mutations in high penetrance genes, namely TP53 (2) and CDH1 (1). In addition, nine of 146 independent cases (6%) were found to have deleterious mutations in the other predisposition genes. Mutations in ATM and CHEK2 accounted for seven of the ten moderate penetrance mutations identified; additionally one family had a mutation in BLM, one in MRE11A, and one in PALB2. Furthermore, 15 of 146 independent cases (10%) were found to have variants of unknown significance (VUS) in one of eight genes. There were no deleterious mutations or VUS in eight of the 18 genes. Our data show that significant genetic heterogeneity exists in BRCAx families. Large-scale collaborative efforts will be required to attain sufficient power to understand how to appropriately apply these results clinically in cancer risk evaluation. Citation Format: Kara N. Maxwell, Lucia Guidugli, Kasmintan Schrader, Steven Hart, Vijai Joseph, Tinu Thomas, Xianshu Wang, Bradley Wubbenhorst, Robert Klein, Susan M. Domchek, Csilla Szabo, Susan Neuhausen, Jeffrey Weitzel, Katherine L. Nathanson, Kenneth Offit, Fergus Couch. High and moderate penetrance germline mutations in a number of genes are responsible for a small proportion of familial breast cancer risk in BRCAx families. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1291. doi:10.1158/1538-7445.AM2014-1291

Purpose Recent advances in DNA sequencing have led to the development of breast cancer susceptibility gene panels for germline genetic testing of patients. We assessed the frequency of mutations in 17 predisposition genes, including BRCA1 and BRCA2, in a large cohort of patients with triple-negative breast cancer (TNBC) unselected for family history of breast or ovarian cancer to determine the utility of germline genetic testing for those with TNBC. Patients and Methods Patients with TNBC (N = 1,824) unselected for family history of breast or ovarian cancer were recruited through 12 studies, and germline DNA was sequenced to identify mutations. Results Deleterious mutations were identified in 14.6% of all patients. Of these, 11.2% had mutations in the BRCA1 (8.5%) and BRCA2 (2.7%) genes. Deleterious mutations in 15 other predisposition genes were detected in 3.7% of patients, with the majority observed in genes involved in homologous recombination, including PALB2 (1.2%) and BARD1, RAD51D, RAD51C, and BRIP1 (0.3% to 0.5%). Patients with TNBC with mutations were diagnosed at an earlier age (P < .001) and had higher-grade tumors (P = .01) than those without mutations. Conclusion Deleterious mutations in predisposition genes are present at high frequency in patients with TNBC unselected for family history of cancer. Mutation prevalence estimates suggest that patients with TNBC, regardless of age at diagnosis or family history of cancer, should be considered for germline genetic testing of BRCA1 and BRCA2. Although mutations in other predisposition genes are observed among patients with TNBC, better cancer risk estimates are needed before these mutations are used for clinical risk assessment in relatives.

Abstract Background:BRCA1 and BRCA2 mutation carriers are at substantially increased risk for developing breast and ovarian cancer. The incomplete penetrance coupled with the variable age at diagnosis in carriers of the same mutation suggests the existence of genetic and nongenetic modifying factors. In this study, we evaluated the putative role of variants in many candidate modifier genes. Methods: Genotyping data from 15,252 BRCA1 and 8,211 BRCA2 mutation carriers, for known variants (n = 3,248) located within or around 445 candidate genes, were available through the iCOGS custom-designed array. Breast and ovarian cancer association analysis was performed within a retrospective cohort approach. Results: The observed P values of association ranged between 0.005 and 1.000. None of the variants was significantly associated with breast or ovarian cancer risk in either BRCA1 or BRCA2 mutation carriers, after multiple testing adjustments. Conclusion: There is little evidence that any of the evaluated candidate variants act as modifiers of breast and/or ovarian cancer risk in BRCA1 or BRCA2 mutation carriers. Impact: Genome-wide association studies have been more successful at identifying genetic modifiers of BRCA1/2 penetrance than candidate gene studies. Cancer Epidemiol Biomarkers Prev; 24(1); 308–16. ©2014 AACR.

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 &lt; 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 &lt; 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 &lt; 10−8. Results from the second analytic approach were consistent with those from the first (P &gt; 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.

Background Recently, rare germline variants in XRCC2 were detected in non-BRCA1/2 familial breast cancer cases, and a significant association with breast cancer was reported. However, the breast cancer risk associated with these variants needs further evaluation. Methods The coding regions and exon–intron boundaries of XRCC2 were scanned for mutations in an international cohort of 3548 non-BRCA1/2 familial breast cancer cases and 1435 healthy controls using various mutation scanning methods. Predictions on functional relevance of detected missense variants were obtained from three different prediction algorithms. Results The only protein-truncating variant detected was found in a control. Rare non-protein-truncating variants were detected in 20 familial cases (0.6%) and nine healthy controls (0.6%). Although the number of variants predicted to be damaging or neutral differed between prediction algorithms, in all instances these categories were evenly represented among cases and controls. Conclusions Our data do not confirm an association between XRCC2 variants and breast cancer risk, although a relative risk smaller than two could not be excluded. Variants in XRCC2 are unlikely to explain a substantial proportion of familial breast cancer.