Quantitative DNA methylation analysis identifies a single CpG dinucleotide important for ZAP-70 expression and predictive of prognosis in chronic lymphocytic leukemia

Abstract

Purpose: Increased ZAP-70 expression predicts poor prognosis in chronic lymphocytic leukemia (CLL). Current methods for accurately measuring ZAP-70 expression are problematic, preventing widespread application of these tests in clinical decision making. We therefore used comprehensive DNA methylation profiling of the ZAP-70 regulatory region to identify sites important for transcriptional control.

Patients and methods: High-resolution quantitative DNA methylation analysis of the entire ZAP-70 gene regulatory regions was conducted on 247 samples from patients with CLL from four independent clinical studies.

Results: Through this comprehensive analysis, we identified a small area in the 5' regulatory region of ZAP-70 that showed large variability in methylation in CLL samples but was universally methylated in normal B cells. High correlation with mRNA and protein expression, as well as activity in promoter reporter assays, revealed that within this differentially methylated region, a single CpG dinucleotide and neighboring nucleotides are particularly important in ZAP-70 transcriptional regulation. Furthermore, by using clustering approaches, we identified a prognostic role for this site in four independent data sets of patients with CLL using time to treatment, progression-free survival, and overall survival as clinical end points.

Conclusion: Comprehensive quantitative DNA methylation analysis of the ZAP-70 gene in CLL identified important regions responsible for transcriptional regulation. In addition, loss of methylation at a specific single CpG dinucleotide in the ZAP-70 5' regulatory sequence is a highly predictive and reproducible biomarker of poor prognosis in this disease. This work demonstrates the feasibility of using quantitative specific ZAP-70 methylation analysis as a relevant clinically applicable prognostic test in CLL.

Fig 1.

Comprehensive quantitative DNA methylation profiling of CpG-rich regions at the ZAP-70 gene locus. (A) Schematic representation of the ZAP-70 gene locus. Black boxes represent exons, arrows indicate transcriptional start sites (TSSs), and AUG indicates the TSS of the main transcript. Gold bars represent CpG islands (CGIs). (B) Regions of analysis (Ulm University [ULM] set) covering the three major alternative TSSs—TSS1 to TSS3—are marked A1 to A6. Separate samples are organized in rows (B cells from 69 patients with chronic lymphocytic leukemia [CLL] and nine healthy donors; T cells from five healthy donors). Columns represent single CpG units. High methylation levels are depicted in dark blue, low methylation levels in light green, and missing data in gray. The unsupervised clustering refers to amplicon A2 (exon1 to intron 2 region). IGHV-mutated samples are marked with black boxes in the clustering tree, and unmutated samples with gray. White squares indicate missing data. Clusters are separated by colored bars (red, mostly methylated; black, CpG unit 1 unmethylated and other CpG units methylated; green, CpG unit 1 unmethylated and other CpG units with mixed methylation levels; blue, CpG units mostly unmethylated). (*) Informative CpG unit 1 located at position +223 relative to TSS1. (C) Correlation between DNA methylation levels and ZAP-70 mRNA expression assessed by TaqMan assay (upper panel) and ZAP-70 protein expression assessed by flow cytometry (Flow cyt; lower panel). Bar graphs display Spearman's rho (Spearman's rank correlation coefficient) between DNA methylation and expression data; (*) significance level of P < .05 (Spearman's rho P value). (D-F) Heat maps and unsupervised clustering displaying methylation levels across amplicon A2 in the Chronic Lymphocytic Leukemia Research Consortium (CRC), Mayo Clinic and Ohio State University (MAYO), and Cancer and Leukemia Group B 9712 trial (CALGB) sample sets. Cluster color coding corresponds to 1B, with the addition of orange (mostly mixed methylation). (*) CpG unit 1.

Fig 2.

Kaplan-Meier plots of DNA methylation-based clusters of ZAP-70 amplicon 2 show differential clinical outcome. (A) Treatment-free survival and (B) overall survival probabilities for the Ulm University set; (C) treatment-free survival probabilities for the Chronic Lymphocytic Leukemia Research Consortium set; (D) progression-free survival probabilities for the Mayo Clinic and Ohio State University set; (E) progression-free survival and (F) overall survival probabilities for the Cancer and Leukemia Group B 9712 trial (CALGB) set.

Fig 3.

Levels of DNA methylation at CpG unit 1 (+223) separate patient subgroups of different clinical outcome. DNA methylation measurements of CpG unit 1 were dichotomized into high and low methylation groups at a calculated threshold of 15%. Survival probabilities for each group were calculated and displayed using Kaplan-Meier plots. (A) Treatment-free and (B) overall survival probabilities in the Ulm University set; (C) treatment-free survival probability in the Chronic Lymphocytic Leukemia Research Consortium set; (D) progression-free survival probability in the Mayo Clinic and Ohio State University set; (E) progression-free and (F) overall survival probability in the Cancer and Leukemia Group B 9712 trial set.