A Broad Distribution of ZAP-70 Expression Is Demonstrated in Adult Acute Lymphoblastic Leukaemia by Quantitative PCR Analysis.
Authors: Kochethu Geothy , Bell Andy , Griffiths Mike , Begum Gulnaz , Wandroo Farooq , Moss Paul A.H. 
Published: October 2019

Abstract
The ZAP-70 tyrosine kinase plays a critical role in signal transduction in T cells and NK cells but has limited expression in primary human B cells. However ZAP-70 is expressed in many cases of B cell chronic lymphocytic leukemia and correlates with a poor prognosis. Microarray analysis has recently identified ZAP-70 expression in a range of other B cell malignancies, including acute lymphoblastic leukaemia (ALL), and ZAP-70 expression may carry prognostic value in these diseases.
Microarray analysis may not offer the most accurate method for quantitation of small changes in mRNA expression and this technology may therefore limit the potential information to be derived from ZAP-70 expression. We developed a real time quantitative PCR assay for ZAP-70 and used this to determine ZAP-70 expression in 76 adult patients with ALL (64 B lineage and 12 T lineage).
RNA was extracted from diagnostic bone marrow specimens taken from 76 patients aged over 18 years presenting with ALL. Quantitative PCR was optimised using Jurkat T cells and utilized GAPDH as the internal control. ZAP-70 and GAPDH cDNA were co-amplified in a multiplex PCR reaction with five-fold serial dilutions of Jurkat cDNA ranging from 25ng to 40pg, and this data was used to construct the calibration curve.
ZAP-70 expression within tumours was determined relative to expression within the T cell line Jurkat, defined as having an arbitrary value of 1.0. A wide distribution of ZAP-70 expression was seen in individual ALL cases with a range between 0.002 and 5.3. The average ZAP-70 expression level within ALL was below that of Jurkat with a mean of 0.332 and median of 0.185. Expression of ZAP-70 showed a relatively continuous pattern of expression across the cohort apart from six samples (8%) with a level of ZAP-70 expression above that of the Jurkat T cell line.
Statistical analysis of a potential association between ZAP-70 expression and cytogenetic subgroup was performed by group comparison using either a two-sample test or ANOVA. No significant association between ZAP-70 expression and cytogenetic subgroup was found. Only 4 cases of t(1,19) translocation encoding the E2A/PBX1 gene fusion were seen in the cohort and all had ZAP-70 levels above the median level. Within the T-ALL subgroup, 11 of the 12 patients showed ZAP-70 expression above the median with a mean of 0.26. Low expression was observed in 4 of the 5 cases in the b3a2/b2a2 p210 BCR/ABL subgroup. Study of a potential correlation between ZAP-70 expression and clinical prognosis is continuing but the three patients with the highest levels of ZAP-70 failed to achieve remission with induction therapy and died of refractory disease.
These data confirm that ZAP-70 expression is seen in the majority of cases of ALL and demonstrate the wide range of expression between individual cases. Large cohort studies are likely to be required to confirm an association between ZAP-70 expression and specific cytogenetic subgroup and to confirm the potential prognostic value of ZAP 70 expression.