Pyrosequencing of BRAF V600E in Routine Samples of Hairy Cell Leukemia identifies CD5+ Variant Hairy Cell Leukemia That Lacks V600E
Jochen K Lennerz, Beate Klaus, Ralf Marienfeld, Peter Moeller. University Ulm, Ulm, Germany
Background: Tiacci et al. identified that 100% of hairy-cell leukemias (HCL) carry the BRAF-V600E mutation (NEJM 2011; 364). Based on the frequency and availability of BRAF inhibitors there is an implicit understanding that BRAF-V600E testing could become a component of the laboratory diagnosis of HCL. During the process of implementing this new HCL test in our lab, we performed validation experiments using pyrosequencing. Here, we report the diagnostic performance measures in synopsis with reported data as well as the unexpected finding of a variant HCL that was BRAF wild-type.
Design: We reviewed our files, microdissected tissues, extracted DNA and performed pyrosequencing of BRAF V600E. Mutant peak heights and density of the leukemic infiltrate were assessed and correlated (Spearman with P<0.05 considered significant). The overall diagnostic performance of BRAF V600E in HCL in our series was reviewed in conjunction with published reports and cases in the COSMIC database (www.sanger.ac.uk/genetics/CGP/cosmic; last accessioned Sept 19, 2011).
Results: We identified a consecutive series of 18 HCL-patients with a median age of 56 (range: 40-74 years; 16 males). Immunolabeling showed CD20+, DBA-44+, CD11c+ whereas CD23 or cyclin-D1 was negative. Using formalin-fixed, decalcified and paraffin-embedded bone marrow biopsies, in one case the DNA-quality was insufficient and 16 of the remaining 17 informative cases were BRAF V600E mutant (analytical and diagnostic sensitivity 94%). Mutant peak heights and infiltrate density correlated significantly (r=0.66; P=0.006). The one case without BRAF V600E was that of a 74-year old Caucasian man with HCL, which, in addition to CD20/CD11+ and cyclin-D1-, showed strong CD5+, diagnostic of so-called variant HCL; all other BRAF V600E cases were CD5-negative. Currently, a total of 2019 lymphoid/myeloid neoplasms have been reported and the Youden index for BRAF V600E as diagnostic of HCL is 0.97 with specificity and sensitivity of 97.65% and 99.11%, respectively.
Conclusions: Pyrosequencing for BRAF V600E in routine samples offers a quick, reliable and cost-effective option for clinical testing of HCL. One case without BRAF V600E suggests that CD5+ variant HCL may represent an outlier with marginal influence on the exceptional diagnostic performance of BRAF V600E in HCL. Comparison of other methods for detection in clinical samples is paramount; however, given the high efficacy of current therapies, ultimately cost-benefit analysis will determine whether BRAF testing will become part of the laboratory diagnosis of HCL.
Monday, March 19, 2012 1:00 PM
Poster Session II # 224, Monday Afternoon