Abstract
The objective of this study was to design and validate a next-generation sequencing assay (NGS) to detect BRCA1 and BRCA2 mutations. We developed an assay using random shearing of genomic DNA followed by RNA bait tile hybridization and NGS sequencing on both the Illumina MiSeq and Ion Personal Gene Machine (PGM). We determined that the MiSeq Reporter software supplied with the instrument could not detect deletions greater than 9 base pairs. Therefore, we developed an alternative alignment and variant calling software, Quest Sequencing Analysis Pipeline (QSAP), that was capable of detecting large deletions and insertions. In validation studies, we used DNA from 27 stem cell lines, all with known deleterious BRCA1 or BRCA2 mutations, and DNA from 67 consented control individuals who had a total of 352 benign variants. Both the MiSeq/QSAP combination and PGM/Torrent Suite combination had 100% sensitivity for the 379 known variants in the validation series. However, the PGM/Torrent Suite combination had a lower intra- and inter-assay precision of 96.2% and 96.7%, respectively when compared to the MiSeq/QSAP combination of 100% and 99.4%, respectively. All PGM/Torrent Suite inconsistencies were false-positive variant assignments. We began commercial testing using both platforms and in the first 521 clinical samples MiSeq/QSAP had 100% sensitivity for BRCA1/2 variants, including a 64-bp deletion and a 10-bp insertion not identified by PGM/Torrent Suite, which also suffered from a high false-positive rate. Neither the MiSeq nor PGM platform with their supplied alignment and variant calling software are appropriate for a clinical laboratory BRCA sequencing test. We have developed an NGS BRCA1/2 sequencing assay, MiSeq/QSAP, with 100% analytic sensitivity and specificity in the validation set consisting of 379 variants. The MiSeq/QSAP combination has sufficient performance for use in a clinical laboratory.
Highlights
Every year, more than 200,000 new cases of breast cancer are diagnosed in the United States [1]
Determining whether a woman with breast cancer is BRCA1/2 positive can assist in appropriate counseling regarding increased surveillance and the risks and benefits of undergoing contralateral mastectomy and/or salpingo-oophorectomy, both of which have been shown to be protective against breast cancer [13]
The American Society of Breast Surgeons recommends BRCA1/2 testing for individuals from high-risk populations, including those with early onset breast cancer; two primary breast cancers, either bilateral or ipsilateral; family history of early onset breast cancer; male breast cancer; personal or family history of ovarian cancer; Ashkenazi (Eastern European) Jewish heritage in the setting of a newly diagnosed breast cancer or family history of breast cancer; previously identified BRCA1 or BRCA2 mutation in the family; triple-negative breast cancer at 60 years of age; or pancreatic cancer associated with a family history of hereditary breast and ovarian related cancer [14]
Summary
More than 200,000 new cases of breast cancer are diagnosed in the United States [1]. Identifying a deleterious BRCA1/2 variant in a patient can be helpful to family members, who may need access to genetic counseling and testing to assess their cancer risk and identify appropriate management. The American Society of Breast Surgeons recommends BRCA1/2 testing for individuals from high-risk populations, including those with early onset breast cancer (diagnosed before age 50); two primary breast cancers, either bilateral or ipsilateral; family history of early onset breast cancer; male breast cancer; personal or family history of ovarian cancer ( nonmucinous types); Ashkenazi (Eastern European) Jewish heritage in the setting of a newly diagnosed breast cancer or family history of breast cancer; previously identified BRCA1 or BRCA2 mutation in the family; triple-negative breast cancer at 60 years of age; or pancreatic cancer associated with a family history of hereditary breast and ovarian related cancer [14]
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