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Y. Ferret et al.
IDH1/2 wild-type controls and IDH1/2 mutant samples are shown in Online Supplementary Figure S1. The mutant allele frequency was then estimated using a Poisson distribution model as the fraction of positive droplets divided by total droplets containing a target. The limit of detection was defined for each mutation as the mean value of IDH1/2 wild-type controls plus three standard deviations (Online Supplementary Table S1). The upper detection limit of these ddPCR assays (rounded to 0.2% of mutant allele frequency) was further considered as the threshold for statistical analysis. An IDH1/2-VAF level below 0.2% was hereafter considered as nega- tive MRD. Gene mutation analysis and next-generation sequenc- ing assays are described in the Online Supplementary Methods and Online Supplementary Tables S2-S4.
Statistical analysis
Group comparison for categorical and continuous variables was performed with the Fisher exact and Mann-Whitney test, respec- tively. Overall survival was calculated from the date of AML diag- nosis to the last follow-up date by censoring patients alive at that date. Disease-free survival was calculated from the date of com- plete remission to the date of relapse or death, censoring patients alive without an event at the last follow-up date. In some analyses, data were censored at the time of allogeneic stem cell transplanta- tion. Univariate and multivariate analyses assessing the impact of categorical and continuous variables were performed with a Cox model.15 The proportional-hazards assumption was checked before conducting multivariate analyses.16 Covariates with a P-value <0.1 in univariate analysis were included in the multivariable models. Statistical analyses were performed with STATA software (STATA 12.0 Corporation, College Station, TX, USA). P-values were two- sided, with P<0.05 denoting statistical significance.
Results
Baseline characteristics of the patients and acute myeloid leukemias
The patients’ median age was 54 years (range, 22-70). The median follow-up was 2.7 years (95% CI: 2.3-3.0). Results of conventional cytogenetic studies were available for 98/103 (95%) patients, of whom 72% had normal karyotype AML. A concomitant NPM1 mutation was found in 50/103 (48%) patients. Only 4/103 (4%) patients harbored a concomitant TET2 mutation (Table 1), in accor- dance with the fact that IDH1/2 and TET2 mutations tend to be mutually exclusive.10 As opposed to IDH1R132 and IDH2R140 mutations, IDH2R172 mutations are less likely to be accompanied by additional frequently recurring
mutations in AML.9,17 In our cohort, IDH2R172K muta- tions were mutually exclusive with NPM1 and FLT3 muta- tions, but co-occurred with DNMT3A mutations. An iso- lated trisomy 11 was identified in 5/21 (24%) patients with the IDH2R172K mutation, while this cytogenetic abnormality was not found in any patient with other types of IDH1/2 mutations (24% versus 0%; P<0.001) (Figure 2). In the subgroup of IDH2R172K mutant AML (n=21), single-nucleotide polymorphism array analysis revealed an additional genomic lesion involving chromo- some 11, consisting of a 11p11.2-q12.1 uniparental dis- omy, in one patient with normal karyotype AML. No MLL partial tandem duplication, known to be strongly associat- ed with trisomy 11,18 was found by reverse transcriptase
Table 1. Baseline characteristics of the patients and acute myeloid leukemias.
Gender Male Female
Median age (range), years
Median white blood cell count (range), x 109/L
Number of patients (%) ALFA-0701 ALFA-0702 Total
10 38 48(47)
16
62 (51-70) 18 (1-157)
39
50 (22-60) 5 (1-377)
50
23
55 (53)
54 (22-70) 7 (1-377)
71 (69)
27 (26)
Cytogenetics
Normal 21
Abnormal
Failure
IDH1/2 mutation IDH1 p.R132H/C/G IDH2 p.R140Q IDH2 p.R172K
4
1 4 5(5)
10 10 6
26 36 15
34
16
7
23
2
3 (2 sm, 1 dm)
20 52 5
36 (35) 46 (45) 21 (20)
50 (48) 19 (19) 9 (9) 29 (35) 4 (4) 4 (4)
33 (32) 64 (62) 6 (6)
Other gene mutations
NPM1 mutation
FLT3 internal tandem duplication FLT3-tyrosine kinase domain mutation2
DNMT3A mutation TET2 mutation CEBPA mutation
16 3
6
2
1 (1 sm) European LeukemiaNet 2008 risk-group
Favorable Non-favorable Not defined
sm: single mutation; dm: double mutation.
13 12 1
Figure 2. Barcoding representing the co-occurrence of gene mutations and cytogenetic alterations in our cohort of 103 patients with IDH1/2 mutant acute myeloid leukemia. ITD: internal tandem duplication; TKD: tyrosine kinase domain.
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