Comparison of methodologies for the detection of BRAF mutations in bone marrow trephine specimens.

AIMS: BRAF V600E detection assists in the diagnosis of hairy cell leukaemia (HCL); however, testing practices vary. We evaluated the clinical utility of 5 BRAF mutation testing strategies for use on bone marrow trephines (BMT).

METHODS: 11 HCL, 5 HCL 'mimic', 2 treated HCL and 10 normal BMT specimens were tested for mutant BRAF, comparing Sanger sequencing, pyrosequencing, amplicon-based next generation sequencing (NGS), automated (Idylla) PCR and immunohistochemistry (IHC).

RESULTS: PCR and IHC were cheaper and identified V600E in 100 % of HCL cases. Pyrosequencing detected the mutation in 91%, NGS in 55% of cases and Sanger sequencing in 27%. All assays gave wild-type BRAF results in HCL mimics and normal BMT samples.

CONCLUSIONS: PCR and IHC were most sensitive and cost-effective, but these have limited scope for multiplexing and are likely to be replaced by NGS gene panels or whole genome sequencing in the medium to long term.