Page 126 - 2019_08-Haematologica-web

J. Bond et al.
Introduction
Mutations in the DNA methyltransferase 3 alpha gene (DNMT3A) have been reported in a range of hematologic malignancies, most frequently in myeloid neoplasia, including acute myeloid leukemia (AML),1-5 myelodysplas- tic syndromes,6 myeloproliferative neoplasms7 and myelo- proliferative neoplasm/myelodysplastic overlap syn- dromes.8,9 DNMT3A alterations in lymphoid malignancies are less common, and reports to date are confined to T-lin- eage disease.10-16 In all cases, DNMT3A mutations increase in frequency with age, and are extremely rare in children and adolescents.17-19
Multiple studies have reported that DNMT3A alter- ations correlate with poor outcome in AML.1,2,4,20-22 In com- parison, the prognostic influence of DNMT3A mutation in T-cell acute lymphoblastic leukemia (T-ALL) is poorly characterized. Patients with DNMT3A alterations were reported to have shorter survival in three moderately sized (55 to 93 patients) T-ALL cohorts.9,11,13 DNMT3A sta- tus did not however independently predict prognosis in the only series for which multivariate analyses were doc- umented, as survival effects were linked to increased rates of DNMT3A mutation in poor-risk, phenotypically imma- ture disease.11 While that study did document a correlation between DNMT3A alteration and survival within the immature T-ALL subgroup, this finding was not corrobo- rated in an independent cohort of early thymic precursor (ETP) ALL cases.12
The issue of whether DNMT3A mutation truly alters the biology of T-ALL is therefore only partially addressed by the currently available evidence. In particular, it is unclear whether the associated poor survival simply reflects the prosaic fact that patients with DNMT3A alter- ations are older,11,12 and therefore do not tolerate intensive ALL treatment as well as their younger counterparts.
In order to address this question, we used next-genera- tion sequencing (NGS) to evaluate the DNMT3A genotype of a large cohort of 198 adult T-ALL patients treated as part of the multinational GRAALL-2003 and -2005 studies. We found that DNMT3A mutation strongly correlated with dis- ease relapse and shorter survival, and that these prognostic effects were independent of patients’ age. Furthermore, we report the presence of DNMT3A mutations in non- leukemic cells in a subset of patients, providing the first evi- dence of age-related clonal hematopoiesis in T-ALL.
Methods
Patients
Details of the GRAALL-2003 and -2005 studies are provided in the Online Supplementary Methods. Informed consent was obtained from all patients before inclusion into the trials. Both studies were conducted in accordance with the Declaration of Helsinki and approved by local and multicenter research ethical committees. The complete study protocols are detailed in the Online Data Supplement. Both trials were registered at http:/www.ClinicalTrials.gov (NCT00222027, NCT00327678). The criteria for inclusion in the current project were a diagnosis of T- ALL and the availability of diagnostic material for NGS analysis of DNMT3A genotype. Survival outcomes of the 198 patients (36 from GRAALL-2003 and 162 from GRAALL-2005) who ful- filled these criteria did not differ from those of the remaining 139 T-ALL patients of the study cohorts. As expected in retro-
spective studies, initial white blood cell count (WBC) was higher in the study cohort. However, no differences in allogeneic stem cell transplant rate, disease-free survival, event-free survival, or overall survival were found. A full comparison of the clinical fea- tures of each group is shown in Online Supplementary Table S1.
Next-generation sequencing
Nextera XT (Illumina) DNA Libraries were prepared accord- ing to the manufacturer’s instructions and sequenced using the Illumina MiSeq sequencing system. The custom NGS panel comprised genes coding for factors involved in molecular path- ways known to be mutated in T-ALL, namely cytokine receptor and RAS signaling (NRAS, KRAS, JAK1, JAK3, STAT3, STAT5B, IL7R, BRAF, NF1, SH2B3, PTPN11), hematopoietic development (RUNX1, ETV6, GATA3, IKZF1, EP300), chemical modification of histones (SUZ12, EED, EZH2, KMT2A, KMT2D, SETD2) and DNA methylation (DNMT3A, IDH1, IDH2, TET2, TET3). This panel was originally inspired by the repertoire of genes found to be preferentially altered in pediatric ETP-ALL,23 and we have reported a subset of the results described in the current paper in a previous clinico-biological and genetic analysis of adult ETP- ALL.24 Sequencing reads were analyzed using in-house software (Polyweb, Institut Imagine, Paris, France), and additional in- house custom filtering criteria (comprising minimum read counts and variant allele frequencies, and reference to external reference databases) were applied to minimize false-positive rates. Primers used to confirm mutations by direct sequencing are listed in Online Supplementary Table S2.
Outcome analyses
Comparisons between groups were performed with the Fisher exact and Mann-Whitney tests for categorical and contin- uous variables respectively. Corticosteroid sensitivity was defined as clearance of peripheral blood circulating blasts (<1 x 109/L) following steroid prophase treatment. Complete remis- sion was defined as clearance of bone marrow blasts (<5%) fol- lowing induction treatment. Overall survival was calculated from the date of inclusion in the trial to the last follow-up date, censoring patients alive at that date. Event-free survival was cal- culated from date of inclusion in the trial to the date of induction failure, relapse, or death, censoring patients alive in first com- plete remission without relapse at the last follow-up date. Cumulative incidence of relapse was calculated in patients who attained complete remission, from the date of achieving the complete remission to the date of relapse, with death in first complete remission being considered as a competing event. Univariate and bivariate analyses assessing the impact of DNMT3A mutations and age were performed with a Cox model. Variables that were significantly associated with out- come in univariate analysis were considered as covariates in multivariate Cox models. The proportional-hazards assumption was checked before conducting multivariate analyses. Statistical analyses were performed with STATA software (STATA 12.0 Corporation, College Station, TX, USA). All P values were two- sided, with P<0.05 denoting statistical significance.
Results
Analysis of DNMT3A genotype in patients with T-cell acute lymphoblastic leukemia in the GRAALL studies
We performed targeted NGS of a panel of genes, includ- ing DNMT3A, which have been described to be recurrent- ly mutated in T-ALL. This panel included all exons of DNMT3A, thereby providing a comprehensive picture of
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haematologica | 2019; 104(8)