An optimized BRCA1/2 next-generation sequencing for different clinical sample types

Abstract

Objective: A simultaneous detection of germline and somatic mutations in ovarian cancer (OC) using tumor materials is considered to be cost-effective for BRCA1/2 testing. However, there are limited studies of the analytical performances according to various sample types. The aim of this study is to propose a strategy for routine BRCA1/2 next-generation sequencing (NGS) screening based on analytical performance according to different sample types.

Methods: We compared BRCA1/2 NGS screening assay using buffy coat, fresh-frozen (FF) and formalin-fixed paraffin-embedded (FFPE) from 130 samples.

Results: The rate of repeated tests in a total of buffy coat, FF and FFPE was 0%, 8%, and 34%, respectively. The accuracy of BRCA1/2 NGS testing was 100.0%, 99.9% and 99.9% in buffy coat, FFPE and FF, respectively. However, due to the presence of variant allele frequency (VAF) shifted heterozygous variants, tumor materials (FFPE and FF) showed lower sensitivity (95.5%-99.0%) than buffy coat (100%). Furthermore, FFPE showed 51.4% of the positive predictive value (PPV) on account of sequence artifacts. When performed in the post-filtration process, PPV was increased by approximately 20% in FFPE. Buffy coat showed 100% of sensitivity, specificity and accuracy in BRCA1/2 NGS test.

Conclusions: On the comparison of the analytical performance according to different sample types, the buffy coat was not affected by sequencing artifacts and VAF shifted variants. Therefore, the blood test should be given priority in detecting germline BRCA1/2 mutation, and tumor materials could be suitable to detect somatic mutations in OC patients without identifying germline BRCA1/2 mutation.

Keywords: BRCA1; BRCA2; Blood Buffy Coat; High-Throughput Nucleotide Sequencing; Tissue Preservation.

Fig. 1. Summary of quality metrics in the different form of specimens.

FF, fresh-frozen; FFPE, formalin-fixed paraffin-embedded; QC, quality control. ^*Final results excluded the results of the 21 first replicates that were reexamined because they did not meet the criteria of quality metrics and included the re-tested results from these 21 replicates. Fail sequencing QC metrics rate included replicates that did not meet the requirements of quality metrics 1) a coverage of 100% at a minimum depth of 20× and 2) an average depth of on-target regions>500×.

Fig. 2. Somatic mutations detected by NGS and validated by Sanger sequencing.

On the left, the results of NGS where the reads are aligned to the reference genome as provided by NextGENe software (SoftGenetics, LLC). On the right, we performed sequencing to validate the somatic nature of the mutation (c.3309dup, p.Lys1104^*) by its absence in the matching buffy coat DNA. This somatic nonsense mutation was shown in FF (B and E) and FFPE (C). Wild-type was demonstrated in buffy coat (A and D). FF, fresh-frozen; FFPE, formalin-fixed paraffin-embedded; NGS, next-generation sequencing.

Fig. 3. Comparison of VAF (%) among 186 SNVs detected as “heterozygous variants” from buffy coat and other forms of samples including FFPE and FF in subset A. The variant allele frequency (%, VAF) of SNVs from buffy coat, FFPE, and FF are depicted as a square dots, triangle dots and diamond dots, respectively. The black arrow indicates the direction of change in variant allele frequency of the 186 VAF shifted heterozygous variants in FFPE and FF.

FF, fresh-frozen; FFPE, formalin-fixed paraffin-embedded; SNV, single-nucleotide variant; VAF, variant allele frequency.