Single‐base LOH can be used as Specific Marker to Classify BRCAx Familial Breast Cancer into More Homogenous Subtypes

To the Editor: BRCAx familial breast cancer refers to the familial breast cancer without known germline predispositions; they account for 60–65% of familial breast cancer cases (1). BRCAx familial breast cancer is not a homologous disease entity but composed of more homogenous subtypes, as reflected by the differences in histologic grade, morphology, status of ER, PR and HER2/neu, basal phenotype (CK5/6, CK14, SMA, EGFR), and genetic sequences (2–12). Precise classification of BRCAx familial breast cancer into more homogeneous subtypes can allow better understanding genetic causes and developing targeted clinical treatment for the disease. Due to the lack of specific genetic markers, however, it is difficult to classify the disease further. Clinically, BRCAx breast cancer is diagnosed and treated as a single disease (13). LOH has high specificity. However, its potential as specific markers for disease classification is limited due to its small numbers identifiable by traditional cytogenetic method (14). In order to use LOH as the marker, the number of LOH candidates needs to be substantially increased. Molecular classification of complex disease is considered as promising (15), as it provides the repertoire of much larger number of potential candidates than conventional approaches do. Previous studies showed that single-base LOH can be used to classify highly heterogeneous diseases (16). We tested the possibility of using single-base LOH to classify BRCAx familial breast cancer. We performed the analysis in three steps: (a) Applying exome sequencing method to comprehensively identify the genetic changes enriched in the coding regions in the genome. We performed exome sequencing in paired blood and tumor of nine BRCAx familial breast cancer cases. All cases were tested with no pathogenic mutation in BRCA1 and BRCA2. The cancer tissues were diagnosed as invasive ductal carcinoma and ductal carcinoma in situ, with different TNM stages, grade, and different status of ER, PR, and HER2/Neu (Table 1A); (b) Identifying single-base LOHs. Through comprehensive bioinformatics data analysis, we identified the germline variants in blood DNA and somatic mutations in cancer tissues located at the same position between blood cells and cancer tissues. From them, we identified the single-base LOHs on the condition of heterozygote in blood DNA but homozygote in the paired tumor at the same position (Table 1B); and (c) Identifying the single-base LOHs shared between multiple BRCAx familial breast cancer cases. Those LOHs were distributed in exon, intron, 50 UTR and 30 UTR, and intergenic locations. They were highly reliable as 95% (774/812) are listed in dbSNP data base with assigned SNP ID and 95% (98/103) randomly selected candidates were validated as true single-base LOH by Sanger sequencing. From the identified single-base LOHs, we identified 812 shared LOHs, including 2 shared between 4, 12 shared between 12, and 798 shared between 2 cases (Table 1C). Our study demonstrates the presence of rich single-base LOHs shared between multiple cases, and the promising potential of using single-base LOHs as markers to further classify BRCAx familial breast cancer. While our study provides a proof-of-principle in using nine cases as the model, more cases are needed to determine specific single-base LOH markers to classify the disease into more homogeneous subtypes. Address correspondence and reprint requests to: San Ming Wang, MD, Department of Genetics, Cell Biology and Anatomy, University of Nebraska Medical Center, Omaha, NE, USA, or e-mails: sanming.wang@unmc.edu and sanwang78@gmail.com Bradley Downs and Fengxia Xiao are contributed equally.