Maxwell, Kara N.

Abstract A better understanding of gene-specific risks for development of breast cancer will lead to improved screening, prevention, and therapeutic strategies for individuals identified to carry germline mutations. We performed targeted massively-parallel sequencing to identify mutations and large genomic rearrangements in 26 known or proposed breast cancer susceptibility genes in 2134 BRCA-negative women with familial breast cancer (FBC). A case-control analysis was performed comparing the frequency of internally classified mutations identified in FBC cases to that in non-Finnish European controls from the Exome Aggregation Consortium (ExAC) excluding samples from The Cancer Genome Atlas. Including large genomic rearrangements, mutations were identified in 8.2% of FBC cases compared to 6.2% of ExAC controls, including mutations in high-penetrance genes (0.6% in cases vs. 0.1% in controls), moderate-penetrance genes (3.7% vs 1.7%), and seven cases with two mutations (0.3%). The remainder of FBC cases and ExAC controls had mutations in proposed breast cancer genes (1.6% of cases vs 2.4% of controls), Lynch syndrome genes (0.5% vs. 0.5%) or were heterozygous MUTYH carriers (1.5% vs. 1.5%). Case-control analysis demonstrated significant associations with FBC for ATM, PALB2, and TP53 mutations (OR>3.0, p<10-4), BARD1 mutations (OR=3.2, p=0.012), and CHEK2 truncating mutations (OR=1.6, p=0.041). Our results therefore demonstrate that only approximately 4% of BRCA1/2 negative FBC patients have mutations in genes definitively associated with breast cancer at this time. Large case-control studies are needed to fully evaluate the breast cancer risks associated with moderate penetrance and proposed breast cancer susceptibility genes. Citation Format: Kara N. Maxwell, Thomas Paul Slavin, Jenna M. Lilyquist, Joseph Vijai, Susan L. Neuhausen, Steven N. Hart, Vignesh Ravichandran, Tinu Thomas, Ann Maria, Kasmintan A. Schrader, Raymond Moore, Chunling Hu, Brad Wubbenhorst, Brandon M. Wenz, Kurt D'Andrea, Susan M. Domchek, Mark E. Robson, Paulo Peterlongo, Paolo Radice, James M. Ford, Judy E. Garber, Csilla Szabo, Kenneth Offit, Katherine L. Nathanson, Fergus J. Couch, Jeffrey N. Weitzel. Risks of familial breast cancer associated with known and proposed breast cancer susceptibility genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4265. doi:10.1158/1538-7445.AM2017-4265

Abstract Known gene mutations account for approximately 50% of the hereditary risk for breast cancer. Moderate and low penetrance variants, discovered by genomic approaches, account for an as-yet-unknown proportion of the remaining heritability. A truncating mutation c.325C>T:p.Arg109* (R109X) in the ATP-dependent helicase ERCC3 was observed recurrently among exomes sequenced in BRCA wild-type, breast cancer–affected individuals of Ashkenazi Jewish ancestry. Modeling of the mutation in ERCC3-deficient or CRISPR/Cas9-edited cell lines showed a consistent pattern of reduced expression of the protein and concomitant hypomorphic functionality when challenged with UVC exposure or treatment with the DNA alkylating agent IlludinS. Overexpressing the mutant protein in ERCC3-deficient cells only partially rescued their DNA repair–deficient phenotype. Comparison of frequency of this recurrent mutation in over 6,500 chromosomes of breast cancer cases and 6,800 Ashkenazi controls showed significant association with breast cancer risk (ORBC = 1.53, ORER+ = 1.73), particularly for the estrogen receptor–positive subset (P < 0.007). Significance: A functionally significant recurrent ERCC3 mutation increased the risk for breast cancer in a genetic isolate. Mutated cell lines showed lower survival after in vitro exposure to DNA-damaging agents. Thus, similar to tumors arising in the background of homologous repair defects, mutations in nucleotide excision repair genes such as ERCC3 could constitute potential therapeutic targets in a subset of hereditary breast cancers. Cancer Discov; 6(11); 1267–75. ©2016 AACR. This article is highlighted in the In This Issue feature, p. 1197

Abstract Background: Known gene mutations account for approximately 50% of the risk for breast cancer. However, a considerable fraction of heritable risk remains unexplained. Fifteen percent of the risk is accounted for by BRCA1/2 and another 3% by TP53, PTEN, LKB1 and CDH1. CHEK2, ATM, PALB2, BRIP1, RAD51C, RAD51D and BARD1 account for 4%, while SNPs discovered from large multicenter genome-wide association studies explain another 14% of the heritable risk. Founder mutations in the DNA repair pathway genes such as BRCA1 and BRCA2 account for the majority of AJ breast cancer mutations. Methods: We performed exome sequencing of 49 early onset (age <35) breast cancer cases, and 85 BRCA wild type familial breast cancer cases, all of Ashkenazi Jewish (AJ) from the New York City area. A recurrent truncating mutation was then analyzed in 3131 breast cancer cases and 2716 unaffected women of Ashkenazi ancestry from New York and Israel. Using CRISPR and overexpression systems on the human mammary epithelial cell line HMLE, transcript and protein levels were assayed for the mutant and compared to wild-type. Treatment with IlludinS and UVC were performed to assess DNA damage response. Finally, clonogenic survival assay was also performed. Results: Amongst the DNA repair pathway genes, exome sequencing revealed a heterozygous recurrent truncating mutation in ERCC3 (R109X) in 2 of 49 early onset breast cancer cases of AJ ancestry and 4 familial AJ probands. Taqman genotyping in a case control setting from New York and Israel revealed 54 mutation carriers in 3131 cases and 32 in the 2716 controls. In total, there were 60 heterozygotes detected in 3209 cases and 32 in 2716 controls [OR 1.59 (95% CI 1.01-2.50)]; p = 0.02 Fisher one- tailed). Functional studies using CRISPR and overexpression systems on human mammary epithelial cells, show that the mutation results in lower transcript levels and this reduction is effected by nonsense mediated decay of the mutant transcript. Western blotting showed that the mutation resulted in a smaller protein (∼12kDa). Clonogenic assays showed similar survival rate of mutant and wildtype under UVC exposure, however the mutant cell line showed significantly smaller colony size demonstrating a growth disadvantage that was further increased upon DNA damage. Treatment with fungal sesquiterpene IlludinS, a known sensitizer to mutant ERCC3 cell lines, showed drastically reduced survival when compared to the wild type human mammary epithelial cells. Conclusions: We demonstrate that ERCC3 is a moderate risk gene for breast cancer in individuals of Ashkenazi ancestry. ERCC3 is somatically mutated in multiple cancers including breast, ovarian and pancreatic cancers, however its role as a cancer susceptibility gene requires further elucidation. Additional functional and population genetic studies to further characterize this novel ERCC3 variant are underway. Citation Format: Joseph Vijai, Sabine Topka, Kara Maxwell, Vignesh Ravichandran, Tinu Thomas, Danylo Villano, Ann Maria, Pragna Gaddam, Anne Lincoln, Steven Hart, Susan Neuhausen, Mark Robson, Jeffrey Weitzel, Mark Daly, Katherine Nathanson, Fergus Couch, Gadi Rennert, Kenneth Offit. ERCC3 R109X is a moderate risk breast cancer risk variant in Ashkenazi Jews. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 796.

Abstract Sequencing tests assaying panels of genes or whole exomes are widely available for cancer risk evaluation. However, methods for classification of variants resulting from this testing are not well studied. We evaluated the ability of American College of Medical Genetics and Genomics (ACMG) guidelines to define the rate of mutations and variants of uncertain significance (VUS) in 180 medically relevant genes, including all ACMG designated reportable cancer and non-cancer genes, in individuals who met guidelines for hereditary cancer risk evaluation. We performed whole exome sequencing in 404 individuals in 257 families and classified 1640 variants from these genes. Potentially clinically actionable (likely-pathogenic/pathogenic, LP/P) versus nonactionable (VUS/likely-benign/benign) calls were 92% and 88% concordant with locus specific databases and Clinvar, respectively. LP/P mutations were identified in 11 of 25 breast cancer susceptibility genes in 27 BRCA1/2 negative families (11%). Evaluation of 84 additional autosomal dominant cancer susceptibility genes identified LP/P mutations in only four additional families (1.7%), suggesting they do not influence risk in this cohort. However, individuals from nine of 257 families (3.5%) had incidental LP/P mutations in 32 non-cancer disease genes, and 7% of individuals were monoallelic carriers of an LP/P mutation in 39 autosomal recessive cancer syndrome genes. Furthermore, 90% of individuals had at least one VUS. In summary, these data support the clinical utility of ACMG variant classification guidelines. In addition, evaluation of extended panels of cancer genes in breast/ovarian cancer families leads to only an incremental clinical benefit but substantially increases the complexity of the results. Citation Format: Kara N. Maxwell, Steven N. Hart, Joseph Vijai, Kasmintan A. Schrader, Tinu Thomas, Bradley Wubbenhorst, Vignesh Ravichandran, Raymond M. Moore, Chunling Hu, Lucia Guidugli, Brandon Wenz, Thomas P. Slavin, Susan M. Domchek, Mark E. Robson, Csilla Szabo, Susan L. Neuhausen, Jeffrey N. Weitzel, Kenneth Offit, Fergus J. Couch, Katherine L. Nathanson. Evaluation of ACMG guideline classified variants in 180 cancer and incidental non-cancer genes in families with breast/ovarian cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5220.

Abstract Germline mutations of BRCA1 and BRCA2 underlie joint susceptibility to breast and ovarian cancers, but do not account for all cases. Genome wide association studies show little overlap between common variants associated with breast and ovarian cancer susceptibility, whereas rare variants in genes initially identified as breast cancer susceptibility genes also confer susceptibility to ovarian cancer cases, unselected for breast cancer. Ovarian and breast tumors also are observed in Peutz-Jeghers and possibly in Lynch syndrome. To further elucidate rare germline susceptibilities for breast and ovarian cancer, we have ascertained a unique cohort of 90 BRCA1/2-negative probands with a personal history of breast cancer and ovarian/fallopian tube/peritoneal cancer, unselected for family history. Cases are from the clinical genetics services of Memorial Sloan-Kettering Cancer Center and University of Pennsylvania. Analysis is ongoing using both targeted and exome sequencing approaches. Germline DNA of 69 cases will be subjected to whole exome sequencing. A total of 21 additional cases will undergo targeted sequencing for 28 genes (8 cases) and an extended panel of 54 genes (13 cases). The majority of ovarian cancers are high-grade serous epithelial ovarian carcinoma (n=51), in combination with invasive ductal or DCIS (n=36), invasive lobular (n=4), or unspecified (n=11) breast cancers. The remaining ovarian subgroups comprise other epithelial subtypes; endometrioid (n=9), clear cell (n=3), mucinous and low-grade serous (n=3) and non-epithelial (n=4) or unclassified (n=20), in combination with invasive ductal or DCIS (n=21), invasive lobular (n=5) or unspecified (n=13) breast cancers. Preliminary analysis of the targeted set of genes in 69 cases, reveals 43 predicted pathogenic coding or splice site variants; 17 of which are within PALB2, MSH2, MSH6, BARD1, FANCE, CDKN2A, TP53, SETD2, SIRT1, BRIP1, RAD50, and RAD51D and are not seen in dbSNP138, 1000 Genomes or Exome Variant Server ESP6500 and 26 variants with reported frequencies in MUTYH, MSH6, MLH1, FAM175A, RAD50, UIMC1, JARID2, PHF3, SIRT1, MRE11A, ATM, BRCA2, TP53BP1, CDH1, RAD51D, BRCA1, BABAM1 of less than 1% allele frequency in public databases. Analysis of these variants across and within histologic subtypes is being undertaken to look for specific genotype-phenotype correlations. Analysis of novel candidate genes and pathways by co-segregation, functional analysis, as well as external replication is underway to determine whether shared rare variants, mutated genes, or altered pathways confer cancer susceptibility in this cohort. Germline susceptibility to breast and ovarian cancer is heterogenous and remains a critical research and clinical question. Improved understanding of the susceptibility genetics will allow further examination of the utility of targeted cancer prevention strategies in those who are found to be at risk. Citation Format: Kasmintan A. Schrader, Kara N. Maxwell, Joseph Vijai, Steven Hart, Tinu Thomas, Bradley Wubbenhorst, Lucia Guidugli, Robert Klein, Marina Corines, Liying Zhang, Susan Neuhausen, Jeffrey Weitzel, Namrata Gupta, Larry Norton, Clifford Hudis, Gad Getz, Mark Daly, Steven Lipkin, David Altshuler, Fergus Couch, Katherine Nathanson, Kenneth Offit. Determination of cancer susceptibility in probands with breast and ovarian 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 3282. doi:10.1158/1538-7445.AM2014-3282

Abstract Massively parallel next generation sequencing data (NGS) has proved to be useful in identification of rare Mendelian disorders. Efforts are on-going to use NGS for discovery of novel rare variants in common disease etiology. Distinguishing rare private mutations from causal variants remains a major challenge in complex disorders. With decreasing cost, investigators are generating NGS data for common cancers. However, power and other considerations suggest that a consortia approach of pooling data is more likely to succeed where individual efforts fail. Combining data within such consortia brings its own level of challenges. We enumerate these challenges and suggest recommendations based on our experience of combining and harmonizing NGS data on 358 breast cancer samples from four centers; (City of Hope n=8, Mayo Clinic n=221, MSKCC n=96 and University of Pennsylvania n=33). Major concerns were ability of individual datasets to discover known and novel variations, depth of coverage across the exome, and quality of variants. Using pre- and post-processed data, we demonstrate an increased power for detecting variants when using a joint-calling method, quality control (QC) and quality assurance pre- and post-merge, as well as the advantages of combined annotations and filtering procedures. To generate a filtered list of high confident calls, we subset the data on Depth per Allele (DP), Genotyping Quality (GQ), Allele balance, Phred likelihood score (PL) as well as using frequency filters on the alternate allele both within the data and comparing against convenience control summaries. We describe general principles and QC measures to be adopted when NGS data is used from diverse sources, specifically applicable to large consortia. Note: This abstract was not presented at the meeting. Citation Format: Joseph Vijai, Steven Hart, Tinu Thomas, Bradley Wubbenhorst, Lucia Guidugli, Kasmintan Schrader, Kara Maxwell, Lauren Jacobs, Danylo Villano, Robert Klein, Steven Lipkin, Susan Neuhausen, Jeffrey Weitzel, David Altshuler, Fergus Couch, Kenneth Offit, Katherine Nathanson. Harmonization of next generation sequencing data within consortia for gene discovery in familial 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 2378. doi:10.1158/1538-7445.AM2014-2378

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