BRAF mutation detection in hairy cell leukaemia from archival haematolymphoid specimen

Abstract

Introduction Hairy cell leukaemia (HCL) is a rare, indolent chronic B-cell leukaemia accounting for approximately 2% of all adult leukaemias. The recent association of the V600E BRAF mutation in HCL makes it a valuable molecular diagnostic marker. Objective To compare the ability of Sanger sequencing (SS), fluorescent single-strand conformational analysis (F-SSCA) and high resolution melting (HRM) analysis to detect BRAF mutations in HCL with DNA extracted predominantly from archival material. Methodology 20 cases of HCL consisting of four archival Roma-novsky stained air-dried peripheral blood and bone marrow aspirate smears, 12 mercury fixed decalcified bone marrow trephine biopsies, three formalin fixed, paraffin embedded (FFPE) sple-nectomy samples and one fresh peripheral blood sample were reviewed. DNA was amplified by PCR and BRAF mutation status determined by the three methods above. Results V600E mutation was identified in 95%, 87% and 76% of HCL cases by F-SSCA, HRM and SS, respectively. In one HCL case, in addition to the V600E mutation, a K601T mutation was identified. One of the Romanowsky stained samples was negative for mutation by all three methods. Three mercury fixed cases were negative for mutation by sequencing but positive for the mutation using F-SSCA and HRM. This discrepancy could be due to mutations below the limit of sensitivity of SS. Conclusion DNA from archival slide scrapings, mercury-fixed and FFPE tissue can be used to identify BRAF mutations with high sensitivity especially using HRM/F-SSCA. The V600E mutation can be used as a supplementary molecular marker to aid in the diagnosis of HCL. The presence of the mutation may provide a target for therapy. Hairy cell leukaemia (HCL) is a rare, indolent chronic B-cell leukaemia accounting for approximately 2% of all adult leukaemias. The recent association of the V600E BRAF mutation in HCL makes it a valuable molecular diagnostic marker. To compare the ability of Sanger sequencing (SS), fluorescent single-strand conformational analysis (F-SSCA) and high resolution melting (HRM) analysis to detect BRAF mutations in HCL with DNA extracted predominantly from archival material. 20 cases of HCL consisting of four archival Roma-novsky stained air-dried peripheral blood and bone marrow aspirate smears, 12 mercury fixed decalcified bone marrow trephine biopsies, three formalin fixed, paraffin embedded (FFPE) sple-nectomy samples and one fresh peripheral blood sample were reviewed. DNA was amplified by PCR and BRAF mutation status determined by the three methods above. V600E mutation was identified in 95%, 87% and 76% of HCL cases by F-SSCA, HRM and SS, respectively. In one HCL case, in addition to the V600E mutation, a K601T mutation was identified. One of the Romanowsky stained samples was negative for mutation by all three methods. Three mercury fixed cases were negative for mutation by sequencing but positive for the mutation using F-SSCA and HRM. This discrepancy could be due to mutations below the limit of sensitivity of SS. DNA from archival slide scrapings, mercury-fixed and FFPE tissue can be used to identify BRAF mutations with high sensitivity especially using HRM/F-SSCA. The V600E mutation can be used as a supplementary molecular marker to aid in the diagnosis of HCL. The presence of the mutation may provide a target for therapy.