Page 118 - Haematologica - Vol. 105 n. 6 - June 2020

A. Touzart et al.
ture/early thymic precursor (ETP) (LyL1, MEF2C), late cor- tical (TAL1), early cortical (TLX1/3 and NKX2.1) and HOXA clusters. Although cancer is typically considered a genetic disease, epigenetic aberrations also play important roles in tumor potentiation, initiation, and progression.7 Epigenetics is defined as changes in gene expression that are not due to changes in gene sequence, and include DNA methylation, histone modifications, microRNA (miRNA) and nucleosome positioning. Unlike genetic alterations, epigenetic changes are reversible by enzymatic activity and pharmacological treatment with small molecule inhibitors, like those targeting enzymes involved in DNA methylation or chromatin modifications. Altered epige- netic states are a common feature of all cancer types and the most studied epigenetic modification in primary can- cer samples is DNA methylation, which is known to dis- play characteristic changes in malignant cells compared to normal tissue. These include diffuse hypomethylation and focal hypermethylation changes at discrete loci potentially associated with repression of specific genes related to can- cer pathogenesis.
In the field of ALL, DNA methylation studies have mostly focused on pediatric B-cell precursor ALL (BCP- ALL) describing promoter hypermethylation and specific methylation signatures according to the cytogenetic sub- group.8 In pediatric T-ALL, DNA methylation was ana- lyzed by Infinium 27 K and 450 K arrays and two distinct CpG island methylator phenotype (CIMP) groups were identified. Patients with a CIMP-negative profile dis- played a significantly higher cumulative incidence of relapse (CIR) compared to CIMP-positive patients sug- gesting a prognostic relevance of aberrant DNA methyla- tion profiles in T-ALL.9,10 Furthermore, it has more recently been shown in a pediatric series that CIMP status corre- lates with known oncogenic subgroups, for instance, with higher expression of TAL1 in a CIMP-negative subgroup (11). However such data for adult T-ALL are still lacking. In this work, we report genome-wide promoter methyla- tion profiling by methylation-dependent immunoprecipi- tation (MeDIP) in a cohort of adult T-ALL. Subsequently, a nine-promoter classifier was applied to a large series of 168 adult T-ALL included in the GRAALL 03/05 trial that distinguished two subgroups with highly significant dif- ferences in the clinical outcome. Thus, MeDIP profiling is a potential candidate for risk stratification of adult T-ALL and could provide important information in treatment decision making and therapeutic targeting.
Methods
Patients and treatments
Adult patients (15-60 years old) included in two successive French ALL cooperative group trials (GRAALL-2003 and GRAALL-2005) with T-ALL, and defined according to the 2008 World Health Organisation classification, were analyzed. The GRAALL-2003 protocol was a multicenter phase 2 trial, which enrolled 76 adults with T-ALL between November 2003 and November 2005 of whom 50 had sufficient diagnostic tumor material available.12 The multicenter randomized GRAALL-2005 phase 3 trial was very similar to the GRAALL-2003 trial, with the addition of a randomized evaluation of an intensified sequence of hyperfractionated cyclophosphamide during induction and late intensification.13 Between May 2006 and May 2010, 337 adults with T-ALL were randomized in the GRAALL-2005, of which 185
had available diagnostic material. All samples contained >80% blasts. Phenotypic and oncogenetic characteristics were as described.14-16 Informed consent was obtained from all patients at enrollment. All trials were conducted in accordance with the Declaration of Helsinki and approved by local and multicenter research ethical committees.
MeDIP-assay
Global DNA methylation was assessed by a MeDIP assay on an initial series of 24 T-ALL and three human thymi and a second (confirmatory series) of 17 T-lymphoblastic lymphomas (T-LBL) and three human thymi. Briefly, methylated DNA was immuno- precipitated as described previously17 using 2 μg of sonicated genomic DNA. MeDIP samples were directly subjected to labeling and hybridization to previously described custom human promot- er arrays (Agilent, Santa Clara, CA, USA) covering either 17,970 promoters17 (T-ALL series) or 25,490 promoters18 (T-LBL series), following the manufacturer’s instructions. The median-normal- ized log2 enrichment ratios (MeDIP/Input) were calculated for each probe using the CoCAS software19 and visualized using the IGB tool (http://bioviz.org/igb). Finally, a methylation score was computed for each promoter by calculating the median enrich- ment ratio of overlapping probes. A summary of the methylation scores per promoter in T-ALL and T-LBL samples is provided in the Online Supplementary Tables S1-2, respectively.
Clustering of methylation profiles
Hierarchical clustering analysis (Average Linkage) based on the methylation signal of the top 5% genes with highest variance was performed with the TIGR MeV v. 4.9.0 program,20 using the -1 Spearman rank correlation method. Analysis of the differential methylation signal between the groups was performed using the significant analysis of microarrays (SAM) algorithm (threshold value: FDR<0.121 and delta=2.144). The graphical clustering rep- resentation of the clustering was done with the GenePattern soft- ware.21 The list of differentially methylated promoters in T-ALL and T-LBL is provided in Online Supplementary Table S3.
Validation of DNA methylation signature
Direct methylation levels were analyzed by methylation-specif- ic multiplex ligation-dependent probe amplification (MS-MLPA) with custom probes (Online Supplementary Table S4) and, SALSA® MLPA® P200 Reference-1 probemix and EK1 reagent kits from MRC-Holland (Amsterdam, the Netherlands), according to manu- facturer’s recommendations. Data were analyzed with the Coffalyser software (MRC-Holland, Amsterdam, the Netherlands). In addition, the promoter methylation patterns were verified by quantitative PCR (qPCR) analysis of MeDIP sam- ples and by bisulfite sequencing using specific primers for the MEIS1 gene promoter.
Results
DNA methylation signatures in T-ALL/T-LBL
Global promoter regions DNA methylation by MeDIP- array was performed in a training series of 24 adult T-ALL. Unsupervised hierarchical clustering defined two major groups (group 1 and group 2) with distinct methylation profiles (Figure 1A). The supervised signature of differen- tial methylation (FDR<0.121) between these two groups resulted in 300 unique differentially methylated gene pro- moters with a vast majority of hypermethylated (hyperM) promoters (297 of 300) in the so-called hyperM group. The second group displayed an intermediate methylation
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