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Real-world experience of BRAF V600E mutation testing in hairy cell leukaemia
  1. Stephen E Langabeer1,
  2. David O'Brien2,
  3. Sarah McCarron1,
  4. C Larry Bacon2,
  5. Elisabeth Vandenberghe1,2
  1. 1 Cancer Molecular Diagnostics, St James's Hospital, Dublin, Ireland
  2. 2 Department of Haematology, St James's Hospital, Dublin, Ireland
  1. Correspondence to Dr Stephen E Langabeer, Cancer Molecular Diagnostics, St James's Hospital, Dublin 8, Ireland; slangabeer{at}

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Hairy cell leukaemia (HCL) is a rare, mature, B-cell malignancy characterised clinically by fatigue, splenomegaly, pancytopenia and recurrent opportunistic infections. Morphologically, hairy cells have prominent cytoplasmic projections with classical HCL (HCLc) cells immunophenotypically expressing CD11c, CD19, CD25 and CD103. A variant form (HCLv), that lacks CD25 expression, exhibits a more aggressive clinical course and may not respond to standard HCLc therapies.1 The discovery of the acquired BRAF V600E mutation (c.1799T>A; p.Val600Glu) in nearly all cases of HCLc in 2011, but absent in those patients with HCLv, has added an additional level to the diagnostic algorithm with current guidelines recommending molecular testing for the BRAF V600E in the diagnostic work-up.1–4 In order to establish adherence to guidelines, a retrospective audit was performed on a series of patients with HCL identified at a haemato-oncology diagnostic referral centre.

From January 2012 to December 2019 inclusive, a consecutive series of 49 diagnostic cases of HCL were identified by immunophenotyping. This series consisted of 42 men and 7 women (M:W ratio 6:1) with a median age of 61 years (range 35–90 years). Forty-five (91.8%) patients possessed an HCLc immunophenotype with four (8.2%) patients with HCLv. BRAF V600E detection was not performed reflexively on a suggestive immunophenotype but on request only. A further 18 individual patient samples were received requesting BRAF V600E mutation analysis of which 11 were at HCLc presentation from external centres and seven were HCLc at relapse or who had refractory disease. During the audit period, detection methodologies transitioned from an allele-specific PCR for the V600E to a commercially available companion diagnostic for codon V600 (cobas 4800 BRAF V600 Mutation Test; Roche Diagnostics, Burgess Hill, UK) to a next-generation sequencing (NGS) approach covering BRAF exon 15.

Of the 49 individual patients identified with a diagnostic HCL immunophenotype BRAF V600E mutational analysis was requested and performed in only 20 (40.8%) comprising those with HCLc (n=18) and HCLv (n=2). Of these tested samples, the BRAF V600E was identified in 14 HCLc (77.8%) and not in those with HCLv. Of the external HCLc diagnostic samples, the BRAF V600E mutation was identified in 10 (90.9%) and in 5 (71.4%) of patients with HCLc at relapse or with refractory disease.

In an era of intensive genetic scrutiny of haematological malignancies, it is striking that more than half of those patients with an HCL immunophenotype did not proceed to molecular confirmation. Possible reasons for this include lack of awareness of the service, molecular analysis being performed elsewhere or the perceived lack of requirement for molecular confirmation given the pervasive existence of the CD19/CD11c/CD25/CD103 positive immunophenoptype of HCLc and the discrimatory value of lack of CD25 expression. Five diagnostic samples from patients with HCLc demonstrated no evidence of the BRAF V600E with possible explanations including rare cases of BRAF V600E-negative HCLc,5 mutations in alternative BRAF exons6 or lack of sensitivity of the NGS assay to detect low levels of the mutation: difficult aspiration due to disease-associated fibrosis can result in an unrepresentative degree of bone marrow infiltration.7

Given that frontline therapy for immunophenotypically characterised patients with HCLc with a purine nucleoside analogue with or without rituximab has historically resulted in favourable response rates, it may be understandable why BRAF mutation testing is deemed unnecessary in such patients. However, the increasing uptake of BRAF inhibitor therapy in patients who relapse or have refractory diseases8 mandates BRAF V600E testing in these clinical situations in order to guide therapeutic choice and assess disease response.

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  • Handling editor Mary Frances McMullin.

  • Contributors SEL, DO and SM performed laboratory studies. CLB and EV provided clinical oversight. All authors contributed to manuscript preparation and approved the submitted version.

  • Funding The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors.

  • Competing interests None declared.

  • Provenance and peer review Not commissioned; externally peer reviewed.