An evaluation of the challenges to developing tumor BRCA1 and BRCA2 testing methodologies for clinical practice

Area of Science:

• Oncology
• Genetics
• Molecular Diagnostics

Background:

• Poly ADP ribose polymerase (PARP) inhibitors offer benefits for ovarian cancer patients with specific germline or somatic pathogenic variants.
• Testing for these variants in tumors presents challenges due to formalin-fixed paraffin-embedded (FFPE) sample limitations, complex tumor genomes, and low allelic fractions of somatic variants.

Purpose of the Study:

• To compare different next-generation sequencing (NGS) approaches for identifying clinically significant BRCA1/2 variants in FFPE tumor DNA.
• To identify common issues and challenges in tumor BRCA (tBRCA) testing across multiple laboratories.

Main Methods:

• An inter-laboratory comparison study involving 10 laboratories performing their preferred NGS methods on identical FFPE tumor DNA samples.
• Samples varied in genotype, quantity, quality, and variant allele frequency (VAF) to simulate real-world testing conditions.

Main Results:

• No false positive results were reported across the participating laboratories.
• Most methods successfully detected variants with low VAF, but some variants were missed due to bioinformatics analysis, variant classification, or insufficient DNA.
• Hybridization capture or short amplicon methods were recommended based on bioinformatic data assessment.

Conclusions:

• Standardization of tBRCA testing is crucial, especially when results guide life-extending therapeutic decisions.
• Addressing challenges in bioinformatics, variant classification, and DNA quality is essential for accurate tBRCA testing in FFPE samples.