Comparison of ZAP-70/Syk mRNA levels with immunoglobulin heavy-chain gene mutation status and disease progression in chronic lymphocytic leukemia.
OBJECTIVE
The protein tyrosine kinase ZAP-70 has recently emerged as a major prognostic indicator in chronic lymphocytic leukemia (CLL). ZAP-70 is structurally and functionally homologous to Syk, a key mediator of B-cell receptor signaling. We therefore evaluated ZAP-70 expression in CLL B cells using Syk as an intracellular standard.


METHODS
The relative amounts of ZAP-70 and Syk were determined in purified B cells from 92 CLL patients using a novel reverse transcriptase/polymerase chain reaction (RT-PCR) procedure that co-amplifies both transcripts with equal efficiency. The ZAP-70/Syk mRNA ratio was correlated with VH gene mutation status, median treatment-free survival and FACS analysis of ZAP-70 expression.


RESULTS
ZAP-70 was expressed in the majority of cases with unmutated VH genes (88%), but also at lower levels in a substantial fraction of cases with mutated VH genes (44%). High levels of ZAP-70, defined as ZAP-70/Syk mRNA ratios above 0.25, were observed mainly in cases with unmutated VH genes and correlated with short treatment-free survival. In contrast, no difference was observed in the median treatment-free survival between patients with low ZAP-70/Syk ratios (0.05-0.25) and patients with no or negligible ZAP-70 expression (ZAP-70/Syk<0.05). In 73 cases ZAP-70 expression was investigated by RT/PCR and FACS analysis; concordance with VH gene mutation status was 86% and 71%, respectively.


CONCLUSIONS
ZAP-70 is frequently expressed in CLL B cells, but only high levels correlate with unmutated VH gene status and progressive disease. Expression of ZAP-70 can be accurately assessed by analysis of the ZAP-70/Syk mRNA ratio, thus providing an alternative to FACS analysis.
Laurenti
Luca
L
Department of Hematology, Catholic University Hospial A. Gemelli, Rome, Italy. laurenti@rm.unicatt.it
Petlickovski
Aleksandar
A
Rumi
Carlo
C
Gobessi
Stefania
S
Piccioni
Paola
P
Tarnani
Michela
M
Puggioni
Pierluigi
P
Marietti
Sara
S
Sica
Simona
S
Leone
Giuseppe
G
Efremov
Dimitar G
DG
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001483:Base Sequence; D018450:Disease Progression; D005260:Female; D050438:Genes, Immunoglobulin Heavy Chain / Q000235:genetics*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics*; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000201:enzymology / Q000235:genetics*; D008297:Male; D008875:Middle Aged; D008969:Molecular Sequence Data; D009154:Mutation; D011505:Protein-Tyrosine Kinases / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics*; D000072377:Syk Kinase; D051746:ZAP-70 Protein-Tyrosine Kinase / Q000235:genetics*
D047908:Intracellular Signaling Peptides and Proteins; D012333:RNA, Messenger; D011505:Protein-Tyrosine Kinases; C000606896:SYK protein, human; D000072377:Syk Kinase; D051746:ZAP-70 Protein-Tyrosine Kinase