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

Abstract

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.

Figure 1. Schematic diagram of BRCAplus assay workflow.

Figure 2. Redundant primer tiling design limits allele drop-out.

A) A typical primer design with one to two amplicons covering region of interest. A polymorphism under the primer of amplicon one would result in allele drop-out and a false negative. B) BRCAplus tiling primer design has overlapping amplicons designed over region of interest. The same polymorphism in amplicon one would not result in false negative due to amplicon redundancy of amplicon two.

Figure 3. Primer sequence trimming increases detection sensitivity.

Two heterozygous causative variants A) BRCA1 c.3671_3672insCTTC and B) BRCA2 c.2918C>A that were missed without primer trimming were detected with the function enabled in the pipeline. Both calls were confirmed with Sanger sequencing.

Figure 4. Microarray has exon level resolution for deletion and duplication variant detection.

A sample harboring a 2 Kb exon 13 deletion in BRCA2 was detected using the custom designed microarray.

Figure 5. Average depth of coverage for each exon on the test from 3,000 representative samples.

The red line indicates the 50X coverage threshold in which any region with insufficient coverage would be Sanger sequenced.

Figure 6. Heterozygous read ratio vs. read coverage.

To identify the profile of false positives, sequencing coverage was plotted against the heterozygous read ratios using Sanger sequencing confirmed NGS variants. Green circle, Sanger confirmed variants. Red triangle, Sanger cleared false positive.

Figure 7. Distribution of pathogenic mutations reported out in first 3,000 patient samples referred for BRCAplus testing.

Figure 8. Causative heterozygous mutations, which had previously gone undetected, identified with BRCAplus assay.

A) Mosaic pathogenic mutation c.5583delA in BRCA2 was detected at allele ratio of 14.9% and confirmed by Sanger sequencing. B) A heterozygous splice site mutation c.1909+1 G>A in BRCA2, was detected by BRCAplus assay.