Zap70 Methylation Correlates with Zap70 Expression in Chronic Lymphocytic Leukemia.
Authors: Oscier David Graham , Parker Anton , Davis Zadie , Michaela Wirtz , Oliver Schmitz , Orchard Jenny , Corcoran Martin 
Published: October 2019

Abstract
ZAP70 expression in chronic lymphocytic leukemia (CLL) correlates with the presence of unmutated immunoglobulin chain (IgVH) genes and with poor clinical outcome. We hypothesised that expression of ZAP70 may be related to the methylation status of the ZAP70 gene. Bisulphite sequencing of a 5′ region of the ZAP70 gene showed a significant difference in CpG methylation between ZAP70 positive and ZAP70 negative cells. We developed a bisulfite restriction PCR assay, insensitive to 10% T cell contamination, to determine the methylation status of a CpG dinucleotide 332 base pairs downstream of the transcription start site, which represented methylation across the sequenced region. This assay was used to detect ZAP70 methylation in 87 patients with CLL, 12 patients with mantle cell lymphoma (MCL) and 12 patients with splenic marginal zone lymphoma (SMZL). ZAP70 protein expression and IgVH gene mutation status had been previously established in all cases. Global cytosine methylation was also analysed in a subset of 15 of the CLL patients by the capillary electrophoresis with laser-induced fluorescence method. Considering the CLL cases, the ZAP70 gene was methylated in 53 patients, unmethylated in 32 patients and an equivocal result was obtained for 2 patients. Correlating methylation status with ZAP70 expression, 51 of 53 (96%) ZAP70 positive patients were methylated and 30 of 32 (94%) ZAP70 negative patients were unmethylated (p<0.0001). Similarly, 49 of 54 (91%) patients with mutated IgVH genes were methylated and 27 of 31 (87%) unmutated patients were unmethylated (p<0.001). The median survivals of methylated and unmethylated CLL patients were 211 and 85 months respectively (p<0.0001) which was very similar to that found for ZAP70 expression or IgVH gene mutational status. The mean global cytosine methylation level was 3.9±0.15% in the ZAP70 methylated CLL patients and 4.1±0.12% in the ZAP70 unmethylated CLL patients; the difference between the two patient groups was significant (p=<0.001) which implies that if ZAP70 methylation is malignancy-related, then hypomethylation of ZAP70 in a subset of CLL arises as a consequence of gene specific rather than global hypomethylation. Among the 24 MCL and SMZL patients an absolute correlation was seen between ZAP70 expression and methylation. However, in marked contrast to the correlation seen in CLL, all 7 non-CLL patients with unmutated IgVH-genes and 16 of 17 non-CLL patients with mutated IgVH-genes showed methylated ZAP70. Therefore, measurement of ZAP70 gene methylation is a simple and reproducible method for predicting prognosis in CLL, which correlates closely with ZAP70 expression and IgVH gene mutational status. The assay is suitable for stored DNA and histological samples as well as for fresh or DMSO-frozen cells. In addition, the methodology of our assay presents none of the standardisation problems, which are relevant to the current flow cytometric assays for ZAP70.