title: ZAP-70 mRNA quantification in B-cell chronic lymphocytic leukaemia.
abstract: OBJECTIVE
  The mutational status of the immunoglobulin (Ig) V(H) gene in B-cell chronic lymphocytic leukaemia (B-CLL) identifies two subgroups of patients with significantly different outcomes. We investigated the association of ZAP-70 expression with IgVH mutational status in B-CLL by quantifying ZAP-70 mRNA, to evaluate its use as a surrogate marker for mutational status. The aim of this study was to develop a quantitative reverse transcriptase-polymerase chain reaction (RQ-PCR) assay for the detection of ZAP-70 expression in a group of patients whose mutational status and cytogenetics had been determined previously.
  
  
  METHODS
  RQ-PCR was used to analyse ZAP-70 expression from 42 B-CLL patients. B cells were purified using CD19 magnetic bead system and total RNA was isolated. RQ-PCR was performed using Taqman PCR.
  
  
  RESULTS
  Twenty-five patients (60%) had mutated and 17 (40%) had unmutated IgVH genes; 94% (16/17) of patients with unmutated IgVH gene were ZAP-70 positive as assessed by RQ-PCR and 92% (23/25) of patients with mutated IgVH gene were ZAP-70 negative. In three patients, ZAP-70 expression and IgVH mutational status were discordant.
  
  
  CONCLUSIONS
  This paper describes an RQ-PCR assay for the detection of ZAP-70 expression and confirms that IgV(H) unmutated CLL cells have a high expression of ZAP-70 in comparison with IgVH mutated CLL. This robust method acts as a surrogate marker for IgVH mutational status albeit with <100% concordance. However, it does provide better concordance with mutational status than that reported using flow cytometry.
authors.affiliation: Department of Haematology, Belfast City Hospital, Belfast, Northern Ireland, UK. mark.catherwood@bll.n-i.nhs.uk
authors.forename: Mark A
authors.initials: MA
authors.lastname: Catherwood
authors.forename: Christine
authors.initials: C
authors.lastname: Matthews
authors.forename: Roberta
authors.initials: R
authors.lastname: Niblock
authors.forename: Edwina
authors.initials: E
authors.lastname: Dobbin
authors.forename: T C M
authors.initials: TC
authors.lastname: Morris
authors.forename: H Denis
authors.initials: HD
authors.lastname: Alexander
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D005260:Female; D005434:Flow Cytometry / Q000379:methods; D015973:Gene Expression Regulation, Leukemic / Q000235:genetics*; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000096:biosynthesis / Q000235:genetics*; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis / Q000235:genetics*; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000235:genetics* / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D009154:Mutation; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D051746:ZAP-70 Protein-Tyrosine Kinase / Q000096:biosynthesis / Q000235:genetics*
chemical_list: D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D012333:RNA, Messenger; D051746:ZAP-70 Protein-Tyrosine Kinase