The validation and clinical implementation of BRCAplus: a comprehensive high-risk breast cancer diagnostic assay

• Published in: PloS one Vol. 9; no. 5; p. e97408
• Main Authors: Chong, Hansook Kim, Wang, Tao, Lu, Hsiao-Mei, Seidler, Sara, Lu, Hong, Keiles, Steven, Chao, Elizabeth C, Stuenkel, A J, Li, Xiang, Elliott, Aaron M
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 15.05.2014; Public Library of Science (PLoS)
• Subjects: Algorithms; Alleles; Assaying; Bioinformatics; Biology and Life Sciences; BRCA1 protein; BRCA2 protein; BRCA2 Protein - genetics; Breast cancer; Breast Neoplasms - diagnosis; Breast Neoplasms - genetics; Cancer; Cancer genetics; Comparative Genomic Hybridization; Computational Biology - methods; Cytogenetics; Deoxyribonucleic acid; Design; Diagnostic systems; DNA; DNA Primers; E-cadherin; Early Detection of Cancer; Family medical history; Female; Gastric cancer; Gene sequencing; Genes; Genes, BRCA2; Genetic counseling; Genetic Predisposition to Disease; Genetics; Health risks; Humans; Hybridization; Laboratories; Low level; Medical diagnosis; Medical personnel; Medicine and Health Sciences; Mosaicism; Mutation; p53 Protein; Physicians; Polymerase Chain Reaction; PTEN protein; R&D; Reproducibility of Results; Research & development; Research and Analysis Methods; Risk; Risk factors; Sensitivity and Specificity; Stomach cancer; Surveillance; Tumor proteins; United States > US; California
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0097408

Breast cancer is the most commonly diagnosed cancer in women, with 10% of disease attributed to hereditary factors. Although BRCA1 and BRCA2 account for a high percentage of hereditary cases, there are more than 25 susceptibility genes that differentially impact the risk for breast cancer. Traditionally, germline testing for breast cancer was performed by Sanger dideoxy terminator sequencing in a reflexive manner, beginning with BRCA1 and BRCA2. The introduction of next-generation sequencing (NGS) has enabled the simultaneous testing of all genes implicated in breast cancer resulting in diagnostic labs offering large, comprehensive gene panels. However, some physicians prefer to only test for those genes in which established surveillance and treatment protocol exists. The NGS based BRCAplus test utilizes a custom tiled PCR based target enrichment design and bioinformatics pipeline coupled with array comparative genomic hybridization (aCGH) to identify mutations in the six high-risk genes: BRCA1, BRCA2, PTEN, TP53, CDH1, and STK11. Validation of the assay with 250 previously characterized samples resulted in 100% detection of 3,025 known variants and analytical specificity of 99.99%. Analysis of the clinical performance of the first 3,000 BRCAplus samples referred for testing revealed an average coverage greater than 9,000X per target base pair resulting in excellent specificity and the sensitivity to detect low level mosaicism and allele-drop out. The unique design of the assay enabled the detection of pathogenic mutations missed by previous testing. With the abundance of NGS diagnostic tests being released, it is essential that clinicians understand the advantages and limitations of different test designs.