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J. Bond et al.
The prevalence of other mutations detected by NGS is shown in Figure 1B. DNMT3A-altered cases had an increased frequency of alterations in other genes included in the NGS panel, compared with the rest of the cohort (88.9% DNMT3A mutated vs. 64.4% DNMT3A WT, P=0.036). There were no statistically significant differences in the prevalence of mutations in any specific functional gene category, namely factors involved in cytokine receptor and RAS signaling (61.1% DNMT3A mutated vs. 41.7% DNMT3A WT, P=0.113), hematopoiesis (38.5% DNMT3A mutated vs. 12.8% DNMT3A WT, P=0.082) and chemical modification of histones (50.0% DNMT3A mutated vs. 30.0% DNMT3A WT, P=0.082). However, we did observe significant co-occurrence of DNMT3A alterations and IDH2 mutations (27.8% DNMT3A mutated vs. 2.2% DNMT3A WT, P<0.001). This association has been described previ- ously in both AML25 and myelodysplastic syndromes.26
Evidence of possible clonal hematopoiesis in DNMT3A- mutated T-cell acute lymphoblastic leukemia
DNMT3A is the most commonly altered gene in age- related clonal hematopoiesis,27-29 and DNMT3A mutations have been detected in non-malignant cells in AML30,31 and peripheral T-cell lymphoma.14 We therefore tested whether DNMT3A mutations were present in non- leukemic hematopoietic cells in T-ALL patients.
DNA from remission bone marrow was available for only three of the 18 patients with DNMT3A mutations. While two of these samples had a DNMT3A WT geno- type (Online Supplementary Figure S1), one interesting case had evidence of DNMT3A alterations in non-leukemic bone marrow cells. At diagnosis, this patient (case 6 in Online Supplementary Table S3) had mutations in exons 14 and 15 of DNMT3A, a NOTCH1 PEST domain insertion, and an NRAS G12D substitution. Sequencing of remission DNA revealed mutation of DNMT3A exon 14 in non- leukemic cells, while NOTCH1, NRAS and DNMT3A exon 15 all presented wild-type genotypes (Figure 2A). We confirmed that the exon 14 mutation has never been reported as a polymorphic variant, while SIFT analysis (http://sift.jcvi.org/) predicted this M548T substitution to be highly deleterious to protein function, with a SIFT score of 0. These results suggest that this T-ALL may have devel- oped on a background of DNMT3A-mutated clonal hematopoiesis, and that the other genetic alterations, including the second DNMT3A mutation, were acquired at leukemic transformation.
In order to extend this analysis of non-leukemic DNMT3A mutation, we performed immunophenotypic sorting of two further diagnostic bone marrow samples, and extracted DNA from both the leukemic and the minor residual non-leukemic fractions. We detected a mutation in non-leukemic DNA in one patient (case 4 in Online Supplementary Table S3). Again, we confirmed that this mutation has not been reported as a polymorphism, and that the resultant P385L substitution is predicted to dam- age protein function, with a SIFT score of 0.02. Similar to the case with mutated remission DNA, this sample was negative for two NOTCH1 alterations detected at T-ALL diagnosis, confirming the specificity of DNMT3A muta- tion persistence (Figure 2B). The other tested non- leukemic DNA had a DNMT3A WT genotype (Online Supplementary Figure S2), giving an overall rate of non- leukemic DNMT3A mutant positivity of 2/5 samples from the GRAALL-2003 and -2005 studies. We also tested a fur-
ther three T-ALL cases not included in this cohort, but found no evidence of non-leukemic DNMT3A mutation. This gives an overall incidence of possible clonal hematopoiesis in 2/8 T-ALL samples assessed in our labo- ratory. It was unfortunately not possible to obtain non- hematopoietic tissue from either of these patients, in order to exclude that these alterations were not constitu- tional, and to confirm definitively that these results reflect the persistence of a DNMT3A-mutated clonal hematopoi- etic population in these cases.
DNMT3A mutations are associated with older age and treatment resistance
A clinico-biological comparison of cases with and with- out DNMT3A mutations is shown in Table 1. In keeping with previous reports,11,12 patients with mutations were considerably older than the rest of the T-ALL cohort (median age 43.9 years mutated vs. 29.4 years non-mutat- ed, P<0.001). In addition, DNMT3A-mutated leukemias were more likely to have an immature T-receptor geno- type32 (53.3% mutated vs. 24.4% non-mutated, P=0.016), although this did not correspond to a significantly higher incidence of an ETP-ALL immunophenotype33 (35.7% mutated vs. 20.3% non-mutated, P=0.184).
DNMT3A mutation was notably associated with poor initial treatment response. We observed trends towards early corticosteroid resistance (66.7% mutated vs. 43.3% non-mutated, P=0.081) and induction failure (13.3% vs. 2.9%, P=0.096), and patients with DNMT3A mutations had significantly higher rates of death during induction (16.7% vs. 2.8%, P=0.027), and lower attainment of com- plete remission (72.2% mutated vs. 94.4% non-mutated, P=0.006). As only four patients with mutations were eval- uated for minimal residual disease, we could not verify that molecular remission was similarly compromised.
We found that the type of DNMT3A mutation did not significantly correlate with any individual clinico-biologi- cal parameter, suggesting that the alterations detected in this study are likely to have broadly similar biological con- sequences.
DNMT3A mutation correlates with poor outcome in T-cell acute lymphoblastic leukemia
The median follow-up of the cohort was 5.5 years. Prognostic analyses revealed that DNMT3A mutation was associated with an increased 5-year cumulative incidence of relapse (53.9% mutated vs. 28.7% non-mutated, P=0.037) (Figure 3A) and with 5-year event-free survival [27.8% mutated vs. 61.0% non-mutated; hazard ratio (HR) 3.22, (95% confidence interval (95% CI): 1.81-5.72, P<0.001] (Figure 3B). Patients with DNMT3A mutations also had a markedly inferior 5-year overall survival (38.8% mutated vs. 68.7% non-mutated, HR 2.91, 95% CI: 1.56- 5.43, P=0.001) (Figure 3C).
The poor prognosis of DNMT3A-mutated T-cell acute lymphoblastic leukemia is age-independent
Our and others’ data11,12 have shown that the incidence of DNMT3A mutation in T-ALL increases with age, but previous reports have not documented whether this factor contributes to prognosis. As older patients treated during the GRAALL studies had worse outcomes due to impaired tolerance of intensive chemotherapy,34 we considered it critical to determine to what extent age was a confound- ing prognostic variable.
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haematologica | 2019; 104(8)