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

Ellison G1, Ahdesmäki M2, Luke S3, Waring PM4, Wallace A5, Wright R5, Röthlisberger B6, Ludin K6, Merkelbach-Bruse S7, Heydt C7, Ligtenberg MJL8,9, Mensenkamp AR8, Castro DG10,11, Jones T11, Vivancos A12, Kondrashova O4, Pauwels P13, Weyn C13, Hahnen E14, Hauke J14, Soong R15, Lai Z16, Dougherty B17, Carr TH2, Johnson J17, Mills J1, Barrett JC17.

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Abstract

Ovarian cancer patients with germline or somatic pathogenic variants benefit from treatment with PARP inhibitors. Tumour BRCA1/2 testing is more challenging than germline testing as the majority of samples are formalin fixed paraffin embedded (FFPE), the tumour genome is complex and the allelic fraction of somatic variants can be low. We collaborated with 10 laboratories testing BRCA1/2 in tumours to compare different approaches to identify clinically important variants within FFPE tumour DNA samples. This was not a proficiency study but an inter-laboratory comparison to identify common issues. Each laboratory received the same tumour DNA samples ranging in genotype, quantity, quality and variant allele frequency (VAF). Each lab performed their preferred Next Generation Sequencing method to report on the variants. No false positive results were reported in this small study and the majority of methods detected the low VAF variants. A number of variants were not detected due to the bioinformatics analysis, variant classification or insufficient DNA. The use of hybridisation capture or short amplicon methods are recommended based on a bioinformatic assessment of the data. The study highlights the importance of establishing standards and standardisation for tBRCA testing particularly when the test results dictate clinical decisions regarding life extending therapies. This article is protected by copyright. All rights reserved.