Citations

Formalin-fixed paraffin-embedded (FFPE) specimens, when used in conjunction with patient clinical data history, represent an invaluable resource for molecular studies of cancer. Even though nucleic acids extracted from archived FFPE tissues are degraded, their molecular analysis has become possible. In this study, we optimized a laboratory-based next-generation sequencing barcoded cDNA library preparation protocol for analysis of small RNAs recovered from archived FFPE tissues. Using matched fresh and FFPE specimens, we evaluated the robustness and reproducibility of our optimized approach, as well as its applicability to archived clinical specimens stored for up to 35 years. We then evaluated this cDNA library preparation protocol by performing a miRNA expression analysis of archived breast ductal carcinoma in situ (DCIS) specimens, selected for their relation to the risk of subsequent breast cancer development and obtained from six different institutions. Our analyses identified six miRNAs (miR-29a, miR-221, miR-375, miR-184, miR-363, miR-455-5p) differentially expressed between DCIS lesions from women who subsequently developed an invasive breast cancer (cases) and women who did not develop invasive breast cancer within the same time interval (control). Our thorough evaluation and application of this laboratory-based miRNA sequencing analysis indicates that the preparation of small RNA cDNA libraries can reliably be performed on older, archived, clinically-classified specimens.

Abstract Breast cancer risks conferred by many germline missense variants in the BRCA1 and BRCA2 genes, often referred to as variants of uncertain significance (VUS), have not been established. In this study, associations between 19 BRCA1 and 33 BRCA2 missense substitution variants and breast cancer risk were investigated through a breast cancer case–control study using genotyping data from 38 studies of predominantly European ancestry (41,890 cases and 41,607 controls) and nine studies of Asian ancestry (6,269 cases and 6,624 controls). The BRCA2 c.9104A>C, p.Tyr3035Ser (OR = 2.52; P = 0.04), and BRCA1 c.5096G>A, p.Arg1699Gln (OR = 4.29; P = 0.009) variant were associated with moderately increased risks of breast cancer among Europeans, whereas BRCA2 c.7522G>A, p.Gly2508Ser (OR = 2.68; P = 0.004), and c.8187G>T, p.Lys2729Asn (OR = 1.4; P = 0.004) were associated with moderate and low risks of breast cancer among Asians. Functional characterization of the BRCA2 variants using four quantitative assays showed reduced BRCA2 activity for p.Tyr3035Ser compared with wild-type. Overall, our results show how BRCA2 missense variants that influence protein function can confer clinically relevant, moderately increased risks of breast cancer, with potential implications for risk management guidelines in women with these specific variants. Cancer Res; 77(11); 2789–99. ©2017 AACR.

Abstract Clinical genetic testing panels are broadly used to gather information about cancer predisposition in individuals with personal and/or family history of breast cancer. However, the involvement of several of the genes on clinical testing panels in predisposition to breast cancer, such as MRE11A and RAD50, has recently come into question. In addition, accurate risk estimates for breast and other cancer are not well defined for the majority of genes on testing panels. We studied 60,000 women diagnosed with breast cancer who were tested for germline cancer predisposing mutations using hereditary cancer gene panels. Information on personal and family cancer history, age of diagnosis, and ethnicity of patients was obtained from test requisition forms. Greater than 90% met National Comprehensive Cancer Network HBOC testing criteria. To estimate gene-specific risks for breast cancer, case-control analyses were performed comparing the frequencies of pathogenic mutations from Caucasian cancer cases with frequencies from Caucasian, non-Finnish, non-TCGA controls from the Exome Aggregation Consortium (ExAC) database. Mutations were detected in 9% of breast cancer patients. Twelve genes displayed a significant association (p<0.05) with breast cancer. Nine of these genes, including ATM, RAD51D, NF1, and MSH6, were associated with moderate risk (RR>2.0) of breast cancer and three genes (BRCA1, BRCA2, PALB2) were associated with high risk (RR>5.0) of breast cancer. Cumulative age-dependent risk models were developed for each gene. This large clinical testing dataset of 60,000 women with breast cancer provides useful data for many predisposition genes previously lacking risk estimates, and should prove useful for clinical risk management of patients with inherited mutations in these genes. Citation Format: Couch FJ, Hu C, Lilyquist J, Shimelis H, Akinhanmi M, Na J, Polley EC, Hart SN, McFarland R, LaDuca H, Huether R, Goldgar DE, Dolinsky JS. Breast cancer risks associated with mutations in cancer predisposition genes identified by clinical genetic testing of 60,000 breast cancer patients [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr S2-01.

Abstract Clinical genetic testing of individuals with a personal or family history of breast and ovarian cancer using panels for BRCA1/2 and other candidate cancer predisposition genes has become routine clinical practice. Several of the genes on hereditary cancer testing panels have been strongly associated with specific subtypes of breast cancer. In particular, individuals with germline mutations in BRCA1 predominantly develop estrogen receptor (ER)-negative and triple negative (TN) (estrogen receptor negative, progesterone receptor negative, HER2 negative) breast tumors. In contrast, CHEK2 and ATM mutations have been associated with ER-positive breast cancer. In this study, associations between mutations in panel genes and breast cancer subtypes were evaluated. A cohort of 60,000 breast cancer patients tested for germline cancer predisposing mutations using hereditary cancer gene panels was utilized. Information on personal and family cancer history, age of diagnosis, tumor pathology, and ethnicity of patients was obtained from test requisition forms or by follow up with ordering health care providers. Mutations in each gene were combined into four histological subtypes (triple negative; HER2 positive; ER-positive,HER2-positive; and ER-positive,HER2 negative). Associations for each subtype were estimated by case-control analyses comparing the frequencies of pathogenic mutations in each subtype with frequencies from non-TCGA controls from the Exome Aggregation Consortium (ExAC) database. In addition, case-case analyses were conducted to assess enrichment of gene mutations in specific breast cancer subtypes. Among the observed associations between genes and breast cancer subtypes, mutations in CHEK2 and ATM were highly enriched in luminal breast cancers and BARD1 was specifically associated with TN breast cancer. Refining the spectrum of pathological correlates with mutations in hereditary breast cancer genes will aid gene specific cancer risk management, and may accelerate the development of novel gene-specific therapeutic interventions. Citation Format: Couch FJ, Lilyquist J, Na J, Hu C, Polley EC, Shimelis H, Akinhanmi M, McFarland R, LaDuca H, Goldgar DE, Dolinsky JS. Associations between breast cancer subtypes and mutations in cancer predisposition genes identified by clinical genetic testing of breast cancer patients [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P5-09-03.

Abstract Approximately 15% of genetic screens for mutations in BRCA1 and BRCA2 identify Variants of Uncertain Significance (VUS). Primarily missense mutations, VUS are often difficult to interpret, leading to either uncertainty in how to properly counsel a patient or an unnecessary prophylactic surgery. Given the paucity of data for which missenses are classified as truly pathogenic, computational deleterious missense prediction (DMP) algorithms are used to predict whether a mutation is likely deleterious or neutral. Accuracy of DMPs can vary considerably and have only been calibrated on a relatively small number of missense mutations of demonstrable effect on protein function. In this study, the performance of 41 different DMPs was compared to functional data from 455 functionally characterized missense variants in BRCA1 and BRCA2. New optimized thresholds for classifying missense mutations as deleterious are presented for several existing models as well as a newly derived naïve voting method (NVM). The areas under the curve estimates for the NVM approach are between 0.889-0.922, much higher than previous methods. We estimate that the overall pathogenic potential of missense variants to be 6.8% for BRCA1 and 3.2% of BRCA2, but can be as high as 50% depending on protein location. Overall these results provide key insights into how to predict deleterious missense mutations in BRCA1 and BRCA2. Citation Format: Hart SN, Hoskin T, Shimelis H, Feng B, Lindor NM, Monteiro A, Iversen E, Goldgar DE, Suman V, Couch FJ. Optimized prediction of deleterious missense mutations in BRCA1 and BRCA2 genes [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-02-03.

Abstract Women with one or more first-degree female relatives with a history of breast cancer have a two-fold increased risk of developing breast cancer. This risk increases with the number of affected family members, particularly for those diagnosed at a young age or with bilateral disease. Segregation analyses of breast cancer families led to the identification of inactivating mutations in the BRCA1 and BRCA2 breast cancer susceptibility genes. Pathogenic variants resulting in truncation of these proteins, along with a small number of pathogenic missense variants, have been associated with 55% to 65% lifetime risk of breast cancer in the general population and up to 85% lifetime risks among women with a strong family history of the disease. In contrast, the influence on cancer risk of many rare variants of uncertain significance (VUS) is not known. Mutations in several genes in addition to BRCA1 and BRCA2 have been implicated in predisposition to breast cancer through family segregation studies. These include moderate risk mutations (relative risk between 2.0 and 5.0) in CHEK2 and ATM, high-risk mutations (relative risk greater than 5.0) in PALB2, and high-risk mutations in CDH1, PTEN, STK11, and TP53 that are associated with hereditary diffuse gastric cancer, Cowden, Peutz-Jeghers, and Li-Fraumeni syndromes. However, risks of breast and other cancers associated with mutations in these and other genes that have been suggested to predispose to cancer have not been established or have very broad confidence intervals. Despite the absence of accurate risk estimates clinical testing with gene panels for hereditary cancer gene mutations has become an integral component of medical management for high-risk families and individuals with triple negative breast cancer. To estimate breast cancer risks several large studies of breast cancer patients from the general population and from high-risk families are underway. These studies are expected to identify the subset of panel genes that are associated with increased risk of breast cancer or breast cancer subtypes and to establish more accurate risks of breast and other cancers. In addition, classification of the clinical relevance of VUS in BRCA1, BRCA2, and other predisposition genes remains a significant problem, with patients unable to benefit from enhanced risk assessment, prevention, and clinical management. Thousands of VUS in BRCA1 and BRCA2 have already been identified through clinical testing and many more in BRCA1, BRCA2, and other predisposition genes are being identified through panel-based genetic testing and tumor sequencing studies. Efforts to classify VUS as neutral or pathogenic have focused on large case-control studies and quantitative multifactorial models based on family history of cancer and pathology. However, more recent studies have established the utility of validated functional assays for VUS assessment. Hundreds of BRCA1 and BRCA2 VUS have been reclassified by commercial testing laboratories or by the ENIGMA consortium that serves as the expert review panel for variants in these genes for the ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/) and BRCA Exchange (http://brcaexchange.org) publically accessible websites. Common genetic variants associated with breast cancer risk have also been identified through genome-wide association studies (GWAS). The magnitude of these associations have ranged from 1.03 to 1.29 in women of European ancestry. While the majority of the 179 known breast cancer variants are associated with estrogen receptor (ER) positive breast cancer, 21 of these show specific associations with ER negative or triple negative (ER negative, PR negative, HER2 negative) breast cancer. The variants in risk loci account for approximately 19% of the familial relative risk of breast cancer. Several studies have recently established that polygenic risk scores derived from these risk variants improve personalized risk assessment by identifying women at increased or reduced risk of breast cancer in the general population and by modifying risks of breast cancer among BRCA1 and BRCA2 mutation carriers. In summary, germline genetic studies have substantially changed the landscape of breast cancer, identifying mutations associated with moderate and high risks of breast cancer in up to 5% of population-based breast cancer cases and 35% of high-risk families and providing personalization of underlying breast cancer risk for all women. Citation Format: Couch FJ. OI-1 Decoding breast cancer predisposition genes [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr OI-1.

Abstract Background: Common cancers develop through a multistep process often including inherited susceptibility. Collaboration among multiple institutions, and funding from multiple sources, has allowed the development of an inexpensive genotyping microarray, the OncoArray. The array includes a genome-wide backbone, comprising 230,000 SNPs tagging most common genetic variants, together with dense mapping of known susceptibility regions, rare variants from sequencing experiments, pharmacogenetic markers, and cancer-related traits. Methods: The OncoArray can be genotyped using a novel technology developed by Illumina to facilitate efficient genotyping. The consortium developed standard approaches for selecting SNPs for study, for quality control of markers, and for ancestry analysis. The array was genotyped at selected sites and with prespecified replicate samples to permit evaluation of genotyping accuracy among centers and by ethnic background. Results: The OncoArray consortium genotyped 447,705 samples. A total of 494,763 SNPs passed quality control steps with a sample success rate of 97% of the samples. Participating sites performed ancestry analysis using a common set of markers and a scoring algorithm based on principal components analysis. Conclusions: Results from these analyses will enable researchers to identify new susceptibility loci, perform fine-mapping of new or known loci associated with either single or multiple cancers, assess the degree of overlap in cancer causation and pleiotropic effects of loci that have been identified for disease-specific risk, and jointly model genetic, environmental, and lifestyle-related exposures. Impact: Ongoing analyses will shed light on etiology and risk assessment for many types of cancer. Cancer Epidemiol Biomarkers Prev; 26(1); 126–35. ©2016 AACR.