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

Area of Science:

• Oncology
• Genetics
• Molecular Biology

Background:

• Simultaneous germline and somatic mutation detection in ovarian cancer (OC) using tumor tissue is cost-effective for BRCA1/2 testing.
• Limited studies exist on the analytical performance of BRCA1/2 next-generation sequencing (NGS) across various sample types.

Purpose of the Study:

• To propose a routine BRCA1/2 NGS screening strategy based on analytical performance across different sample types.
• To evaluate the performance of BRCA1/2 NGS assays using buffy coat, fresh-frozen (FF), and formalin-fixed paraffin-embedded (FFPE) samples.

Main Methods:

• Comparison of BRCA1/2 NGS screening assays.
• Utilized 130 samples including buffy coat, FF, and FFPE.
• Assessed analytical performances: accuracy, sensitivity, specificity, and positive predictive value (PPV).

Main Results:

• Buffy coat showed 0% repeat tests, 100% accuracy, sensitivity, and specificity for BRCA1/2 NGS.
• FFPE samples had a 34% repeat rate and 99.9% accuracy, but lower sensitivity (95.5%-99.0%) due to VAF shifts.
• FFPE showed a 51.4% PPV, improved by ~20% post-filtration; FF had an 8% repeat rate.

Conclusions:

• Buffy coat is unaffected by sequencing artifacts and VAF shifts, making it ideal for germline BRCA1/2 mutation detection.
• Tumor materials (FFPE, FF) are suitable for detecting somatic mutations in OC patients when germline mutations are not the primary focus.