Genetic testing for germline mutations in breast cancer predisposition genes can potentially identify individuals at a high risk of developing breast and/or ovarian cancer. There is a paucity of such mutational information for Asians. Panel testing of 25 cancer susceptibility genes and BRCA1/2 deletion/duplication analysis was performed for 220 Asian breast cancer patients or their family members referred for genetics risk assessment. All 220 participants had at least one high-risk feature: having a family history of breast and/or ovarian cancer in first- and/or second-degree relatives; having breast and ovarian cancer in the same individual or bilateral breast cancer; having early-onset breast cancer or ovarian cancer (⩽40 years of age). We identified 67 pathogenic variants in 66 (30.0%) patients. Of these, 19 (28.3%) occurred in BRCA1, 16 (23.9%) in BRCA2, 7 (10.4%) in PALB2, 6 (9.0%) in TP53, 2 (3.0%) in PTEN, 2 (3.0%) in CDH1 and 15 (22.4%) in other predisposition genes. Notably, 47.8% of pathogenic variants were in non-BRCA1/2 genes. Of the 66 patients with pathogenic mutations, 63.6% (42/66) were under the age of 40 years. Family history of breast and/or ovarian cancer is enriched in patients with BRCA1/2 pathogenic variants but less predictive for non-BRCA1/2 related pathogenic variations. We detected a median of three variants of unknown significance (VUS) per gene (range 0–21). Custom gene panel testing is feasible and useful for the detection of pathogenic mutations and should be done in the setting of a formal clinical cancer genetics service given the rate of VUS.

PurposeThe National Comprehensive Cancer Network (NCCN) has proposed guidelines for the genetic testing of the BRCA1 and BRCA2 genes, based on studies in western populations. This current study assessed potential predictive factors for BRCA mutation probability, in an Asian population.MethodsA total of 359 breast cancer patients, who presented with either a family history (FH) of breast and/or ovarian cancer or early onset breast cancer, were accrued at the National Cancer Center Singapore (NCCS). The relationships between clinico-pathological features and mutational status were calculated using the Chi-squared test and binary logistic regression analysis.ResultsOf 359 patients, 45 (12.5%) had deleterious or damaging missense mutations in BRCA1 and/or BRCA2. BRCA1 mutations were more likely to be found in ER-negative than ER-positive breast cancer patients (P=0.01). Moreover, ER-negative patients with BRCA mutations were diagnosed at an earlier age (40 vs. 48 years, P=0.008). Similarly, triple-negative breast cancer (TNBC) patients were more likely to have BRCA1 mutations (P=0.001) and that these patients were diagnosed at a relatively younger age than non-TNBC patients (38 vs. 46 years, P=0.028). Our analysis has confirmed that ER-negative status, TNBC status and a FH of hereditary breast and ovarian cancer (HBOC) are strong factors predicting the likelihood of having BRCA mutations.ConclusionsOur study provides evidence that TNBC or ER-negative patients may benefit from BRCA genetic testing, particularly younger patients (<40 years) or those with a strong FH of HBOC, in Asian patients.

It has been estimated that >1,000 genetic loci have yet to be identified for breast cancer risk. Here we report the first study utilizing targeted next-generation sequencing to identify single-nucleotide polymorphisms (SNP) associated with breast cancer risk. Targeted sequencing of 283 genes was performed in 240 women with early-onset breast cancer (≤40 years) or a family history of breast and/or ovarian cancer. Common coding variants with minor allele frequencies (MAF) >1% that were identified were presumed initially to be SNPs, but further database inspections revealed variants had MAF of ≤1% in the general population. Through prioritization and stringent selection criteria, we selected 24 SNPs for further genotyping in 1,516 breast cancer cases and 1,189 noncancer controls. Overall, we identified the SNP rs56118985 to be significantly associated with overall breast cancer risk. Subtype analysis performed for patient subgroups defined by ER, PR, and HER2 status suggested additional associations of the SNP rs200504060 and the SNP rs142179458 with breast cancer risk. analysis indicated that coding amino acids encoded at these three SNP sites were conserved evolutionarily and associated with decreased protein stability, suggesting a likely impact on protein function. Our results offer proof of concept for identifying novel cancer risk loci from next-generation sequencing data, with iterative data analysis from targeted, whole-exome, or whole-genome sequencing a wellspring to identify new SNPs associated with cancer risk. .

INTRODUCTION Although mutations in BRCA1 and BRCA2 are associated with familial hereditary breast and ovarian cancer, mutations are only found in 20 to 25% of these patients. This suggests that additional genes may be mutated. The Casitas B-lineage lymphoma (CBL) gene encodes a RING finger E3 ubiquitin ligase. Recent studies have shown that mutations in the CBL gene occurs in human acute myeloid leukemia and lung cancers. The relationship of the CBL gene with breast cancer, however, is not well characterized. The aim of this study was to identify mutations in the CBL gene in Asian patients with familial or early-onset breast cancer. METHODS All patients (n=75) were accrued at the risk assessment clinic at the National Cancer Centre Singapore, and presented with a family history of breast and/or ovarian cancer or early-onset breast cancer. Screening for mutations in the BRCA1 and BRCA2 genes was performed using Sanger sequencing and Multiplex Ligation-dependent Probe Amplification (MLPA). Next generation sequencing was done using the Agilent SureSelect target-enrichment technology on the Illumina MiSeq platform, followed by validation using Sanger sequencing. RESULTS Two mis-sense variants in the CBL gene were identified. They are CBL c. 560C&gt;T (p. A187V) and c. 1858C&gt;T (p.L620F) occurring in 2/75 (3%) and 8/75 (11%) of the patients, respectively. Both mis-sense mutations were predicted to be damaging using Polyphen-2 and SIFT. The p.A187V alteration was predicted to interrupt the N-terminal tyrosine kinase binding domain (TKB) that is important for ligand-induced ubiquitination of receptor tyrosine kinases (RTKs). The CBL c. 1858C&gt;T variant has previously been reported in the NCBI database (rs2227988). Three individuals, who carried this mutation, had early-onset breast cancer. In addition, the p.L620F alteration within the proline-rich region, may disrupt the interactions of binding partners to the c-CBL protein, which are required to recruit c-CBL to RTKs. All patients, except one, with germline CBL mutations were negative for BRCA1 and BRCA2 mutations. CONCLUSION This preliminary study has identified damaging CBL mutations in an Asian population with breast cancer and warrants further investigations in other breast cancer populations. Citation Format: Edward Wong, Yoon Sim Yap, Ying Ying Cheng, Delia Chua, Lewis Zuocheng Hong, William F. Burkholder, Gay Hui Ho, Min Han Tan, Peter Ang, Ann S.G. Lee. Mutations in the CBL gene among breast cancer patients in an Asian clinic-based population. [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 1287. doi:10.1158/1538-7445.AM2014-1287

<div>Abstract<p>It has been estimated that >1,000 genetic loci have yet to be identified for breast cancer risk. Here we report the first study utilizing targeted next-generation sequencing to identify single-nucleotide polymorphisms (SNP) associated with breast cancer risk. Targeted sequencing of 283 genes was performed in 240 women with early-onset breast cancer (≤40 years) or a family history of breast and/or ovarian cancer. Common coding variants with minor allele frequencies (MAF) >1% that were identified were presumed initially to be SNPs, but further database inspections revealed variants had MAF of ≤1% in the general population. Through prioritization and stringent selection criteria, we selected 24 SNPs for further genotyping in 1,516 breast cancer cases and 1,189 noncancer controls. Overall, we identified the <i>JAK2</i> SNP rs56118985 to be significantly associated with overall breast cancer risk. Subtype analysis performed for patient subgroups defined by ER, PR, and HER2 status suggested additional associations of the <i>NOTCH3</i> SNP rs200504060 and the <i>HIF1A</i> SNP rs142179458 with breast cancer risk. <i>In silico</i> analysis indicated that coding amino acids encoded at these three SNP sites were conserved evolutionarily and associated with decreased protein stability, suggesting a likely impact on protein function. Our results offer proof of concept for identifying novel cancer risk loci from next-generation sequencing data, with iterative data analysis from targeted, whole-exome, or whole-genome sequencing a wellspring to identify new SNPs associated with cancer risk. <i>Cancer Res; 77(19); 5428–37. ©2017 AACR</i>.</p></div>