ZAP70 Methylation Is An Independent Prognostic Biomarker For Front Line Therapy Of Chronic Lymphocytic Leukemia : Results From The UK LRF CLL4 Trial
Authors: Parker Anton , Gregory Ben , Davis Zadie , Else Monica , Rose-Zerilli Matthew JJ , Strefford Jonathan C , Catovsky Daniel , Oscier David 
Published: October 2019

Abstract
In CLL, over-expression of ZAP70 is well established as a prognostic biomarker for unfavourable disease course. However, technical challenges limit the application of flow cytometry for ZAP70 detection and measurement of ZAP70 mRNA expression is complicated by the need for T cell depletion. Having identified a correlation between DNA methylation of a 5' regulatory region of ZAP70 and expression of the gene, we and others have proposed the measurement of ZAP70 methylation as an alternative biomarker. Of note, recent work by Claus et al showed ZAP70 methylation to be prognostic for progression free survival (PFS) after therapy in two small, phase II, trial cohorts, treated with regimes incorporating Rituximab. However, as no comprehensive assessment of the prognostic value of ZAP70 methylation has been reported, we analysed ZAP70 methylation in 416 samples from patients entered into the phase III UK LRF CLL4 trial (comparing Chlorambucil(C) with Fludarabine alone (F) or in combination with Cyclophosphamide(FC)).
The methylation of a single CpG site 334bp downstream of the transcription start site (C334) was interrogated in peripheral blood mononuclear cell DNA samples, using a quantitative bisulfite pyrosequencing assay. All samples were bisulfite converted, amplified and pyrosequenced in triplicate with a 6 point standard curve in each run. Standard curves for each of the 20 runs were linear (r2 > 0.98) and sensitivity of pyrosequencing was ±5%.
C334 methylation ranged from 0-100% and showed a significant inverse correlation with percentage of ZAP70 positive cells, IGHV identity to germline and percentage of CD38 positive cells (all p≤0.001). Methylation levels showed a trimodal, W-shaped, distribution with major modes at 5% and 95% and a minor mode at 50%. Based on this distribution, samples were classified as having Low (C334L<33.3%), Intermediate (C334Int 33.3-66.6%) or High (C334H >66.6%) methylation (Figure 1).Figure 1Distribution of methylation levelsFigure 1. Distribution of methylation levels
In univariate Kaplan Meier (KM) analysis, no significant difference was seen between C334L and C334Int groups in terms of PFS or OS (median PFS 1.68 versus 1.76 years, p=0.36 and median OS 4.71 years versus 5.41 years, p=0.18). However, C334H cases had a significantly longer PFS and OS than either group (median PFS=2.86 years, median OS=8.60 years, all p<0.001). C334H cases were significantly enriched with good risk features such as the presence of a 13q deletion, absence of 11q deletion, absence of NOTCH1 mutation, ZAP70 negativity, CD38 negativity and IGHV mutated status (all p<0.02). However, C334H status retained significant prognostic value for PFS and OS in KM analysis when cases were stratified by each of these good risk features. Furthermore, in multivariate Cox regression analysis, C334H cases showed a significantly reduced risk of progression compared to all other cases (HR=0.67 (95%CI 0.48-0.95), p=0.023), independent of treatment received, IGHV status and 11q and 17p deletion status (Table 1).Table 1Multivariate Analysis of Progression Free SurvivalVariable95% CI for HRpHRLowerUpperTreatment<0.001 F v C0.690.510.930.015 FC v C0.420.300.58<0.001IGHV unmutated or IGHV 3-211.861.322.62<0.00117p deletion5.713.549.21<0.00111q deletion1.461.081.970.015C334H0.670.480.950.023NOTE. Variables selected as being significantly associated with hazard of progression are shown. Variables not selected but included in the model were age, sex, Binet stage, deletion 13q, trisomy 12, ZAP70 (>10%) and CD38 (>7%).Abbreviations: HR, hazard ratio; IGHV, immunoglobulin heavy chain variable gene
By identifying a group of patients with hypermethylation of ZAP70, who display a superior outcome with first line chemotherapy, our results confirm the prognostic value of ZAP70 methylation and show it to be superior to ZAP70 expression as measured by flow cytometry. The independent prognostic value of this, compared with established biomarkers, together with the ease and low cost of the assay, warrant its validation in other large trial cohorts.

Disclosures:
No relevant conflicts of interest to declare.