title: A comparison of high resolution melting, allele-specific priming and Sanger sequencing for the detection of BRAFV600E mutation in hairy cell leukaemia from different haematological specimens.
abstract: The BRAFV600E mutation is a highly sensitive and specific marker for the diagnosis of hairy cell leukaemia (HCL) and a potential therapeutic target. We assessed the performance of high resolution melting (HRM), allele-specific priming (ASP) and Sanger sequencing (SS) for BRAFV600E detection in 17 unenriched samples from 15 HCL patients: blood (n = 7), marrow aspirate (n = 3), ethylenediaminetetraacetic acid (EDTA)-decalcified trephine biopsy (n = 2), formic acid (FA)-decalcified trephine biopsy (n = 5). Our results showed that for blood and marrow aspirate samples, both HRM and ASP had a very high analytical sensitivity (1%) in clinical specimens and excellent diagnostic sensitivity (100%) and specificity (100%) in analysable samples. Sanger sequencing had a lower analytical sensitivity (4%), resulting in false-negative analysis in samples with a low tumour cell percentage. High resolution melting was technically the simplest and had the shortest turn-around time (2 hours). Analysis of decalcified trephine biopsies was more difficult because of suboptimal DNA preservation. Although Sanger sequencing was least demanding on sample DNA quality for a successful analysis, none of the three techniques showed satisfactory diagnostic performance on trephine biopsies. Therefore, careful selection of a suitable sample type and testing platform is important to optimise the detection of this important mutation in HCL.
authors.affiliation: 1Department of Pathology, Faculty of Medicine, University of Hong Kong 2Department of Pathology and Clinical Biochemistry, Queen Mary Hospital 3Department of Pathology, Queen Elizabeth Hospital, Hong Kong SAR, China.
authors.forename: Chi-Chiu
authors.initials: CC
authors.lastname: So
authors.forename: Amy
authors.initials: A
authors.lastname: Chan
authors.forename: Lap-Ping
authors.initials: LP
authors.lastname: Chung
authors.forename: Lisa
authors.initials: L
authors.lastname: Siu
authors.forename: Wai-Shan
authors.initials: WS
authors.lastname: Wong
authors.forename: Kit-Fai
authors.initials: KF
authors.lastname: Wong
mesh_terms: D000483:Alleles; D014408:Biomarkers, Tumor / Q000235:genetics*; D001707:Biopsy, Needle; D001853:Bone Marrow / Q000473:pathology; D004252:DNA Mutational Analysis / Q000379:methods*; D004273:DNA, Neoplasm / Q000097:blood / Q000235:genetics; D006801:Humans; D007943:Leukemia, Hairy Cell / Q000175:diagnosis / Q000235:genetics*; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D009154:Mutation; D009691:Nucleic Acid Denaturation; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA
chemical_list: D014408:Biomarkers, Tumor; D004273:DNA, Neoplasm; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf