New challenges for BRCA testing: a view from the diagnostic laboratory

Abstract

Increased demand for BRCA testing is placing pressures on diagnostic laboratories to raise their mutation screening capacity and handle the challenges associated with classifying BRCA sequence variants for clinical significance, for example interpretation of pathogenic mutations or variants of unknown significance, accurate determination of large genomic rearrangements and detection of somatic mutations in DNA extracted from formalin-fixed, paraffin-embedded tumour samples. Many diagnostic laboratories are adopting next-generation sequencing (NGS) technology to increase their screening capacity and reduce processing time and unit costs. However, migration to NGS introduces complexities arising from choice of components of the BRCA testing workflow, such as NGS platform, enrichment method and bioinformatics analysis process. An efficient, cost-effective accurate mutation detection strategy and a standardised, systematic approach to the reporting of BRCA test results is imperative for diagnostic laboratories. This review covers the challenges of BRCA testing from the perspective of a diagnostics laboratory.

Figure 1

Components/complexities to consider in the NGS workflow, including the NGS platform, enrichment methods, sequencing chemistries and analytical procedures.

Figure 2

Enrichment methods. (a) PCR-based approach. Multiplex PCR kits enrich for a specific gene or panel of genes in a small number of PCR amplifications. (b) Molecular inversion probes consist of amplicons containing a universal spacer region flanked by target-specific sequences. Genomic DNA is digested, and the target DNA is PCR-amplified and sequenced. (c) Hybridisation enrichment methods work on the principle of selection using probes complementary to DNA in the genomic area of interest either by surface microarray or in solution with labelled beads. Reprinted with permission from Mamanova et al.

Figure 3

(a) NGS percentage 100 × coverage vs DNA input from 98 breast and ovarian cancer FFPE DNA samples. (b) Comparison of DNA concentration measurements using a hgDNA Quantification and QC Kit (KapaBiosystems, Anachem, Luton, UK) and three different amplicon sizes. The ovarian DNA samples were also quantified using a Nanodrop (Thermo Fisher). The 129 bp product was selected to determine the amount of DNA to add into the BRCA panel, as it was the closest measure to the mean amplicon size of all methods being evaluated (GeneRead (Qiagen) V.1: 155 bp (estimated), V.2: 153 bp, Ion AmpliSeq (Life Technologies) ~197 bp). Figure reproduced under the terms of the Creative Commons Attribution License from Ellison et al.

Figure 4

VUS classification for BRCA: a complex task involving many elements.