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Figure 3. IDH1/2 mutant allele fraction assessed by droplet digital polymerase chain reaction at diagnosis of acute myeloid leukemia and during follow-up (A) in the whole cohort and (B) for the seven patients with persistent clonal hematopoiesis with IDH1/2 mutations. The plain lines in the dot plot indicate the median values.
IDH1/2 mutant allele burden in AML
PCR in this subgroup (data not shown). The association between IDH2R172 mutation and trisomy 11 observed in our cohort is consistent with results from a previous study,19 and suggests a potential cooperation between these two genetic alterations in leukemogenesis.
IDH1/2 mutation level at diagnosis of acute myeloid leukemia and during follow-up
At AML diagnosis, IDH1/2-VAF could be assessed by next-generation sequencing in 80/103 patients (Online Supplementary Table S5). The median IDH1/2-VAF value was 41% (range, 16-53%) in bone marrow and 39.5% (range, 6-50%) in peripheral blood samples. In the subset of NPM1-mutated AML, IDH1/2-VAF was systematically higher than NPM1-VAF, except in one patient with similar VAF for both mutations [n=34 comparisons; median dif- ference IDH1/2-VAF - NPM1-VAF, 10.5% (range, 0-25%); P<0.001] (Online Supplementary Figure S2). This finding supports the notion that IDH1/2 mutations were present in pre-existing clones that subsequently acquired NPM1 mutations.
We also performed ddPCR assays in diagnostic and fol- low-up samples to quantify the IDH1/2-VAF. A total of 322 samples from 103 patients with IDH1/2 mutations were analyzed by ddPCR at diagnosis (n=97, of which 69 were bone marrow and 28 peripheral blood samples), dur- ing hematologic remission (n=211 bone marrow samples), and at relapse (n=14 bone marrow samples). At AML diag- nosis, the median IDH1/2-VAF assessed by ddPCR was 42.3% (range, 8.2-49.9%) in bone marrow and 40.6% (range, 5.5-53%) in peripheral blood samples, consistent with our next-generation sequencing data. After induction therapy, the IDH1/2 mutant allele fraction in bone marrow samples decreased significantly compared to the pretreat- ment levels (P<0.001) with a median value below 0.2% (range, <0.2-39.3%). At AML relapse, the median IDH1/2- VAF was 21.3% (range, 0.2-38.5%). Among the 14 patients for whom a bone marrow sample was available for molecular analysis at AML relapse, only one lost the mutation during disease evolution (Figure 3A).
Persistent clonal hematopoiesis with IDH1/2 mutations
IDH1/2 mutations persisted at high levels during hema- tologic remission in 7/103 (7%) patients, including four
with an IDH2R140 mutation and three with an IDH1R132 mutation, but none with an IDH2R172 mutation. The main characteristics of these seven patients are summa- rized in Table 2 and their IDH1/2-VAF profiles are shown in Figure 3B. The only common characteristic identified in these patients was age over 50 years. In this subgroup, the median IDH1/2-VAF was 8% (range, 0.8-28.5%) after induction and 40% (range, 26-43.5%) after consolidation therapy. Of these seven patients, only one is still alive in first complete remission, one died from transplant-related mortality, three relapsed, and two developed overt myelodysplastic syndrome. Altogether, 5/7 (71%) patients with persistent clonal hematopoiesis with IDH1/2 muta- tions relapsed or progressed toward myelodysplastic syn- drome within 1 to 4 years after AML diagnosis.
Univariate and multivariate prognostic analyses
The prognostic impact of IDH1/2 mutations in AML remains controversial.9 In the present cohort composed exclusively of IDH1/2-mutated AML, the presence of an IDH2R172 mutation was associated with a shorter dis- ease-free survival compared to other IDH1/2 mutation types, but without the difference reaching statistical sig- nificance (P=0.088). No difference according to the type of IDH1/2 mutation was observed regarding overall survival (Table 3; Figure 4).
The prognostic impact of IDH1/2-VAF was evaluated in complete remission after induction therapy in a subset of 95 patients for whom a post-induction bone marrow sam- ple was available for IDH1/2-VAF assessment (Figure 1). We were not able to perform statistical analysis at later follow-up time-points, such as post-consolidation, because of the lack of available DNA samples for many patients. Variables considered for univariate and multivari- ate analyses were age, white blood cell count, cytogenet- ics, mutational status of five genes, and IDH1/2-VAF after induction therapy. In univariate analysis for disease-free survival, the presence of a normal karyotype, a NPM1 mutation, and a IDH1/2-VAF <0.2% were significantly associated with a longer disease-free survival. In multivari- ate analysis, these three variables remained significantly predictive of disease-free survival. Factors significantly associated with overall survival were age, the presence of a normal karyotype, the presence of a NPM1 mutation or a TET2 mutation. Other molecular abnormalities studied,
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