ZAP-70 mRNA quantification in B-cell chronic lymphocytic leukaemia.
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.
Catherwood
Mark A
MA
Department of Haematology, Belfast City Hospital, Belfast, Northern Ireland, UK. mark.catherwood@bll.n-i.nhs.uk
Matthews
Christine
C
Niblock
Roberta
R
Dobbin
Edwina
E
Morris
T C M
TC
Alexander
H Denis
HD
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*
D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D012333:RNA, Messenger; D051746:ZAP-70 Protein-Tyrosine Kinase