Thomas Carla, Amanuel Benhur, Finlayson Jill, Grieu-Iacopetta Fabienne, Spagnolo Dominic V, Erber Wendy N
1Department of Anatomical Pathology, PathWest Laboratory Medicine 2School of Pathology and Laboratory Medicine, The University of WA 3Department of Haematology, PathWest Laboratory Medicine, WA, Australia.
Pathology. 2015 Jun;47(4):349-54. doi: 10.1097/PAT.0000000000000245.
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 BRAF p.Val600Glu (V600E) mutation in HCL makes it a valuable molecular diagnostic marker. We compared the ability of Sanger sequencing, fluorescent single-strand conformational polymorphism (F-SSCP) and high resolution melting (HRM) analysis to detect BRAF mutations in 20 cases of HCL consisting of four archival Romanowsky stained air-dried peripheral blood and bone marrow aspirate smears, 12 mercury fixed decalcified bone marrow trephine biopsies, three formalin fixed, paraffin embedded (FFPE) splenectomy samples and one fresh peripheral blood sample. DNA was amplified and BRAF mutation status determined by the three methods above. V600E mutation was identified in 94%, 89% and 72% of HCL cases by F-SSCP, HRM and Sanger sequencing, respectively. In one case, in addition to the p.Val600Glu mutation, a p.Lys601Thr (K601T) mutation was identified. DNA from archival slide scrapings, mercury-fixed and FFPE tissue can be used to identify BRAF mutations with high sensitivity, especially using HRM/F-SSCP. The V600E mutation can be used as a supplementary molecular marker to aid in the diagnosis of HCL and the presence of the mutation may provide a target for therapy.
毛细胞白血病（HCL）是一种罕见的惰性慢性B细胞白血病，约占所有成人白血病的2%。HCL中最近发现的BRAF p.Val600Glu（V600E）突变使其成为一种有价值的分子诊断标志物。我们比较了桑格测序、荧光单链构象多态性（F-SSCP）和高分辨率熔解（HRM）分析检测20例HCL中BRAF突变的能力，这些病例包括4份存档的罗曼诺夫斯基染色空气干燥外周血和骨髓穿刺涂片、12份汞固定脱钙骨髓活检组织、3份福尔马林固定石蜡包埋（FFPE）脾切除样本和1份新鲜外周血样本。通过上述三种方法扩增DNA并确定BRAF突变状态。通过F-SSCP、HRM和桑格测序分别在94%、89%和72%的HCL病例中鉴定出V600E突变。在1例病例中，除了p.Val600Glu突变外，还鉴定出p.Lys601Thr（K601T）突变。存档玻片刮屑、汞固定组织和FFPE组织中的DNA可用于高灵敏度地鉴定BRAF突变，尤其是使用HRM/F-SSCP时。V600E突变可作为辅助诊断HCL的补充分子标志物，该突变的存在可能为治疗提供靶点。
