title: Requisite analytic and diagnostic performance characteristics for the clinical detection of BRAF V600E in hairy cell leukemia: a comparison of 2 allele-specific PCR assays.
abstract: Detection of high-frequency BRAF V600E mutations in hairy cell leukemia (HCL) has important diagnostic utility. However, the requisite analytic performance for a clinical assay to routinely detect BRAF V600E mutations in HCL has not been clearly defined. In this study, we sought to determine the level of analytic sensitivity needed for formalin-fixed, paraffin-embedded (FFPE) and frozen samples and to compare the performance of 2 allele-specific polymerase chain reaction (PCR) assays. Twenty-nine cases of classic HCL, including 22 FFPE bone marrow aspirates and 7 frozen specimens from blood or bone marrow were evaluated using a laboratory-developed allele-specific PCR assay and a commercially available allele-specific quantitative PCR assay-myT BRAF Ultra. Also included were 6 HCL variant and 40 non-HCL B-cell lymphomas. Two cases of classic HCL, 1 showing CD5 expression, were truly BRAF V600E-negative based on negative results by PCR and sequencing despite high-level leukemic involvement. Among the remaining 27 specimens, V600E mutations were detected in 88.9% (17/20 FFPE; 7/7 frozen) and 81.5% (15/20 FFPE; 7/7 frozen), for the laboratory-developed and commercial assays, respectively. No mutations were detected among the 46 non-HCL lymphomas. Both assays showed an analytic sensitivity of 0.3% involvement in frozen specimens and 5% in FFPE tissue. On the basis of these results, an assay with high analytic sensitivity is required for the clinical detection of V600E mutations in HCL specimens. Two allele-specific PCR assays performed well in both frozen and FFPE bone marrow aspirates, although detection in FFPE tissue required 5% or more involvement.
authors: *Molecular Diagnostics Laboratory, Department of Pathology, University of Michigan Health System †Swift Biosciences, Ann Arbor, MI.
authors: Noah A
authors: NA
authors: Brown
authors: Helmut C
authors: HC
authors: Weigelin
authors: Nathanael
authors: N
authors: Bailey
authors: Julie
authors: J
authors: Laliberte
authors: Kojo S J
authors: KS
authors: Elenitoba-Johnson
authors: Megan S
authors: MS
authors: Lim
authors: Bryan L
authors: BL
authors: Betz
mesh_terms: D000483:Alleles; D014408:Biomarkers, Tumor / Q000235:genetics*; D006801:Humans; D016130:Immunophenotyping; D007943:Leukemia, Hairy Cell / Q000175:diagnosis* / Q000235:genetics / Q000276:immunology; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D012680:Sensitivity and Specificity
chemical_list: D014408:Biomarkers, Tumor; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf