## Abstract

DNMT3A mutations are associated with poor prognosis in acute myeloid leukemia (AML), but the stability of this mutation during the clinical course remains unclear. In the present study of 500 patients with de novo AML, DNMT3A mutations were identified in 14% of total patients and in 22.9% of AML patients with normal karyotype. DNMT3A mutations were positively associated with older age, higher WBC and platelet counts, intermediate-risk and normal cytogenetics, FLT3 internal tandem duplication, and NPM1, PTPN11, and IDH2 mutations, but were negatively associated with CEBPA mutations. Multivariate analysis demonstrated that the DNMT3A mutation was an independent poor prognostic factor for overall survival and relapse-free survival in total patients and also in normokaryotype group. A scoring system incorporating the DNMT3A mutation and 8 other prognostic factors, including age, WBC count, cytogenetics, and gene mutations, into survival analysis was very useful in stratifying AML patients into different prognostic groups (P < .001). Sequential study of 138 patients during the clinical course showed that DNMT3A mutations were stable during AML evolution. In conclusion, DNMT3A mutations are associated with distinct clinical and biologic features and poor prognosis in de novo AML patients. Furthermore, the DNMT3A mutation may be a potential biomarker for monitoring of minimal residual disease.

## Introduction

DNMT3A encodes the enzyme DNA methyltransferase (DNMT) 3A, which catalyzes the addition of methyl groups to the cytosine residue of CpG dinucleotides in DNA.1,2  DNMT3A contains 3 main structure domains: an ATRX, DNMT3, and DNMT3L–type zinc finger domain, a proline-tryptophan-tryptophan-proline domain, and the methyltransferase (MTase) domain.1  The proline-tryptophan-tryptophan-proline domain is responsible for targeting the enzyme to nucleic acid, whereas the zinc finger domain mediates protein-protein interactions with the transcription factors Myc and RP58, the heterochromatin protein HP1, histone deacetylases, and the histone methyltransferase Suv39h1.2  Recently, mutations in DNMT3A were identified in patients with AML, myelodysplastic syndromes, and myeloproliferative neoplasms.3–7  The incidences of this mutation in AML varied: 4.1% in a Japanese study,8  9% (among all AML, including M4/M5 and other subtypes) in a Chinese study,9  and approximately 20% in 2 Western studies.3,4  Whether there is a geographic difference in the incidence of DNMT3A mutations needs to be determined. Furthermore, sequential analyses to evaluate the stability of DNMT3A mutations during the clinical course were limited to a small number of patients. In the present study, we investigated the DNMT3A mutation in 506 patients with de novo AML and analyzed its interactions with 16 other gene alterations. Sequential analysis of the DNMT3A mutation during the clinical course was also performed on 138 patients to investigate the stability and pathogenic role of this mutation in AML. Further, to better stratify AML patients into different risk groups, a scoring system integrating DNMT3A mutations with 8 other prognostic factors, including age, WBC count, cytogenetics, NPM1/FLT3 internal tandem duplication (NPM1/FLT3-ITD), CEBPA, AML1/RUNX1, WT1, and IDH2 mutations, into survival analysis was proposed.

## Methods

### Subjects

This study was approved by the institutional review board of the National Taiwan University Hospital (NTUH), and written informed consent was obtained from all participants in accordance with the Declaration of Helsinki. From March 1995 to December 2008, a total of 506 adult patients who were newly diagnosed as having de novo AML at NTUH and had enough cryopreserved cells for analysis were enrolled consecutively. Patients with antecedent hematologic diseases or therapy-related AML were excluded. Diagnosis and classification of AML were made according to the French-American-British (FAB) Cooperative Group Criteria.

In total, 363 (71.7%) patients received standard induction chemotherapy (idarubicin 12 mg/m2/d on days 1-3 and cytarabine 100 mg/m2/d on days 1-7) and then consolidation chemotherapy with 2-4 courses of high-dose cytarabine (2000 mg/m2 every 12 hours on days 1-4 for a total of 8 doses), with or without an anthracycline (idarubicin or Novantrone), after achieving complete remission (CR).10,11  The patients with acute promyelocytic leukemia (M3 subtype) received concurrent all-trans retinoic acid and chemotherapy. The remaining 143 patients received palliative therapy with supportive care and/or low-dose chemotherapy because of underlying comorbidities or based on patient decision. Forty-five patients received allogeneic hematopoietic stem cell transplantation (HSCT) in first CR.

### Cytogenetics

BM cells were harvested directly or after 1-3 days of unstimulated culture, as described previously.12  Metaphase chromosomes were banded with the trypsin-Giemsa technique and karyotyped according to the International System for Human Cytogenetic Nomenclature.

### Immunophenotype analysis

A panel of mAbs to myeloid-associated antigens, including CD13, CD33, CD11b, CD15, CD14, and CD41a, as well as lymphoid-associated antigens, including CD2, CD5, CD7, CD19, CD10, and CD20, and lineage-nonspecific antigens HLA-DR, CD34, and CD56, were used to characterize the phenotypes of the leukemia cells, as described previously.13

### Mutation analysis

Mutation analysis of DNMT3A exons 2-23 was performed by PCR and direct sequencing as described previously.4  Abnormal sequencing results were confirmed by at least 2 repeated analyses. Sequential analysis of the DNMT3A mutation during the clinical course was performed in 316 samples from 138 patients. Mutation analyses of 16 other relevant molecular marker genes, including class I mutations, such as FLT3-ITD and FLT3-TKD,13 N-RAS,14 K-RAS,14 JAK2,14 KIT,15  and PTPN1116  mutations, and class II mutations, such as MLL-PTD,17 CEBPA,18  and AML1/RUNX111  mutations, as well as NPM1,19 WT1,10 ASXL1,20 IDH1,21 IDH222  (including R140 and R172 mutations), and TET223  mutations, were performed as described previously. To detect DNMT3A mutation at diagnosis, we used DNA amplified in vitro from BM cells with the Illustra GenomiPhi V2 DNA-amplification kit as described by the manufacturer (GE Healthcare). All mutations detected were verified in the original nonamplified samples. All nucleotide alterations causing premature truncation of the DNMT3A proteins (nonsense or frame-shift mutations) were regarded as true mutations. Missense mutations were regarded as true only if they were documented in other studies or could be verified by sequencing of normal somatic tissues or matched remission BM samples.

### TA cloning analysis

For patients with double mutations, Taq polymerase-amplified (TA) cloning was performed to determine whether the 2 mutations were in the same or different alleles, as described previously.15  Briefly, the cDNA was amplified to cover both mutations and the PCR products were then cloned into the TA-cloning vector pGEM-T Easy (Promega) and more than 10 clones were selected for sequencing.

### Statistical analysis

The discrete variables of patients with and without gene mutation were compared using the χ2 tests, but if the expected values of contingency tables were smaller than 5, the Fisher exact test was used. If the continuous data were not normally distributed, Mann-Whitney U tests were used to compare continuous variables and medians of distributions. To evaluate the impact of the DNMT3 mutation on clinical outcome, only the patients who received conventional standard chemotherapy were included in the analysis.10,11  Overall survival (OS) was measured from the date of first diagnosis to the date of last follow-up or death from any cause, whereas relapse was defined as a reappearance of at least 5% leukemic blasts in a BM aspirate or new extramedullary leukemia in patients with a previously documented CR.24  Relapse-free survival (RFS) was measured from the date of attaining the leukemia-free state until the date of AML relapse or death from any cause, whichever occurred first. Cox regression survival estimation was used to plot survival curves and to test the differences between groups. Multivariate Cox proportional hazard regression analysis was used to investigate independent prognostic factors for OS and RFS. The proportional hazards assumption (constant hazards assumption) was examined using time-dependent covariate Cox regression before conducting multivariate Cox proportional hazard regression. The variables including age, WBC counts, karyotype, NPM1/FLT3-ITD, WT1, CEBPA, AML1/RUNX1, TET2, ASXL1, IDH2, and DNMT3A mutations were used as covariates. Those patients who received HSCT were censored at the time of transplantation in survival analysis to ameliorate the influence of the treatment.10,11 P < .05 was considered statistically significant. All statistical analyses were performed with SPSS Version 18 software and Statsdirect (2.7.8b, 2011).

## Results

### DNMT3A mutations in patients with de novo AML

Excluding the 8 single-nucleotide polymorphisms (P9P, S267S, G291G, A398A, P385P, L422L, V435V, and V563V) that were detected in 316 patients but did not alter the amino acid residues, and the 7 missense mutations (C586W, P896L, G543C, Y735C, A644T, G699D, and G707D) that were found in 6 patients but had uncertain biologic significance (because they were not reported previously and could not be verified because of lack of matched BM samples at CR), DNMT3A mutations at 30 different positions were identified in 70 patients (Table 1 and Figure 1). Twelve were missense mutations, 8 were nonsense mutations, 9 were frame-shift mutations, and 1 was an in-frame mutation. The most common mutation was R882H (n = 26), followed by R882C (n = 15), R882S (n = 3), R736H (n = 3), and R320X (n = 2). All other mutations were detected in only 1 patient each. Mutations at exon 23 occurred in 47 patients, including the 44 patients with R882 mutations. Four patients had double heterozygous mutations (patients 43, 64, 65, and 68); in 1 of them (patient 64), the 2 mutations were confirmed to be biallelic by DNA PCR and TA cloning, and for the other 3, the nature of the double mutations was not verified by this method because the 2 mutations were located in different exons too far apart to be amplified by a single-DNA PCR reaction. The remaining 66 patients showed only one mutation; all were heterozygous.

Table 1

Mutation patterns in 70 patients with DNMT3A mutations at diagnosis

UPN Age, y/Sex FAB Karyotype Location DNMT3 mutation

Other accompanying gene mutations
DNA change Protein change
79/M M5 46,XY 23 c.2646G > A p.R882H FLT3/ITD, MLL/PTD, IDH2
77/M M1 46,XY 23 c.2646G > A p.R882H AML1/RUNX1
64/M M5 NM 23 c.2646G > A p.R882H PTPN11, NPM1
73/M M4 46,XY 23 c.2646G > A p.R882H IDH2
16/M M4 46,XY 23 c.2645C > T p.R882C FLT3/TKD, NPM1
41/F M4 46,XX 23 c.2646G > A p.R882H FLT3/ITD, NPM1
80/F M4 46,XX 23 c.2646G > A p.R882H PTPN11, NPM1
61/F M5 46,XX t(5;17)(q33;q21) 23 c.2645C > T p.R882C FLT3/TKD, NPM1
46/M M4 46,XY 23 c.2646G > A p.R882H FLT3/ITD, NPM1
10 35/F M1 46,XX 18 c.2120delG p.G707AfsX72 NRAS, IDH1
11 82/M M0 ND 23 c.2645C > T p.R882C FLT3/ITD, MLL/PTD, AML1/RUNX1
12 79/F M4 46,XX 23 c.2646G > A p.R882H FLT3/ITD, FLT3/TKD, MLL/PTD, TET2
13 51/M M4 46,XY 23 c.2646G > A p.R882H FLT3/TKD, NPM1
14 55/M M4 46,XY 16 c.1865_1866 insGT p.Y623FfsX29 NPM1
15 54/M M4 46,XY c.890G > A p.W297X PTPN11, ASXL1
16 68/M M2 46,XY 23 c.2645C > A p.R882S FLT3/ITD, NPM1
17 45/F M5 46,XX 23 c.2645C > T p.R882C FLT3/TKD, AML1/RUNX1, IDH2
18 54/F M2 46,XX 23 c.2646G > A p.R882H NRAS, NPM1
19 87/M M4 46,XY 23 c.2606delG p.G869VfsX12 FLT3/TKD, NPM1
20 51/F M4 47,XX,+i(11)(q10) 20 c.2389A > T p.N797Y‡ ASXL1, IDH2
21 78/M M4 46,XY 23 c.2646G > A p.R882H FLT3/ITD, NPM1
22 38/F M5 46,XX 23 c.2645C > T p.R882C NRAS, NPM1, IDH1
23 72/F M2 46,XX,del(20)(q11q13) 13 c.1477delA p.I493SfsX158 FLT3/ITD, NPM1
24 65/F M5 46,XX 23 c.2646G > A p.R882H FLT3/ITD
25 42/F M4 46,XX 23 c.2646G > A p.R882H FLT3/ITD, AML1/RUNX1, ASXL1
26 78/M M2 46,X,−Y,+4 19 c.2246_2249del p.R749PfsX29 FLT3/TKD, NPM1
27 75/F M1 46,XX 23 c.2645C > T p.R882C FLT3/ITD, NPM1
28 51/M M4 46,XY 23 c.2646G > A p.R882H FLT3/ITD, NPM1
29 60/F M1 46,XX,t(9;22)(q34;q11) 18 c.2113A > T p.I705F§ IDH1
30 73/F M1 46,XX 23 c.2646G > A p.R882H CEBPA, TET2
31 22/F M4 46,XX 23 c.2646G > A p.R882H FLT3/ITD, NPM1
32 38/M M4 46,XY 23 c.2645C > T p.R882C FLT3/ITD, NPM1
33 31/F M5 46,XX 23 c.2646G > A p.R882H FLT3/ITD, NPM1
34 46/M M4 45X,−Y 23 c.2645C > A p.R882S NRAS, FLT3/ITD, NPM1
35 80/M M4 46,XY 23 c.2645C > T p.R882C FLT3/ITD, MLL/PTD
36 52/M M1 46,XY 23 c.2645C > T p.R882C FLT3/ITD, IDH2
37 44/M M2 46,XY 23 c.2645C > T p.R882C FLT3/ITD, NPM1
38 33/F M1 46,XX c.958C > T p.R320X FLT3/TKD, NPM1
39 42/M M4 45,X,−Y 15 c.1816C > T p.Q606X NPM1
40 78/F M2 46,XX 19 c.2255_2257del p.F752del FLT3/ITD, NPM1
41 75/F M2 47,XX,del(5)(q22q35),+8 c.958C > T p.R320X IDH2
42 49/M M1 46,XY 23 c.2646G > A p.R882H FLT3/ITD, NPM1
43 78/M M4 46,XY c.315C > A p.S105R PTPN11
44 64/M M5 46,XY 23 c.2645C > A p.R882S PTPN11, MLL/PTD
45 40/M M5 46,XY 23 c.2646G > A p.R882H FLT3/ITD, NPM1
46 75/M M4 46,XY 23 c.2646G > A p.R882H NRAS, NPM1, TET2
47 58/F M2 46,XX 23 c.2645C > T p.R882C NPM1
48 48/F M1 47,XX,+8 c.1001delG p.G334AfsX11 CEBPA, IDH2
49 67/M M5 47,XY,+8 23 c.2645C > T p.R882C PTPN11, KRAS, AML1/RUNX1, IDH2
50 51/M M2 46XY 23 c.2646G > A p.R882H IDH2
51 85/M M5 46,XY c.767_770del P256LfsX59 FLT3/ITD, NPM1, WT1
52 85/M M1 45,XY,−7 c.327_328insG Q110AfsX14
53 67/M M8 47,XY,+8 23 c.2646G > A p.R882H NRAS, IDH2
54 35/M M4 46,XY 23 c.2645C > T p.R882C FLT3/ITD, NPM1
55 47/F M2 46,XX 23 c.2646G > A p.R882H ASXL1, IDH2
56 50/F M1 46,XX 23 c.2645C > T p.R882C FLT3/ITD, MLL/PTD
57 86/M M5 46,XY c.866delG p.G289AfsX26 FLT3/ITD
58 69/M M1 NM 23 c.2645C > T p.R882C FLT3/ITD, NPM1, CEBPA
59 75/M M4 46,XY 23 c.2646G > A p.R882H AML1/RUNX1
60 79/F M4 Cplx* 22 c.2510C > G p.S837X
61 61/M M1 46,XY 23 c.2646G > A p.R882H NPM1, WT1, TET2
62 37/F M2 46,XX 19 c.2312G > A p.R771Q‡ NPM1, TET2
63 70/M M2 46,XY 23 c.2646G > A p.R882H FLT3/ITD, NPM1, IDH2
64 46/M M4 46,XY 19 c.2182G > C, c.2191T > C p.G728R‡, p.F731L‡ FLT3/ITD
65 69/M M4 47,XY,+X c.941G > A p.W314X NRAS, FLT3/TKD, AML1/RUNX1, IDH2
19 c.2207G > A p.R736H†
66 38/F M2 46,XX 19 c.2207G > A p.R736H† FLT3/ITD, NPM1, IDH1
67 66 M1 47,XY,del(5)(q31q35), der(7)t(5;7)(q13;q11),+8 15 c.1792C > T p.R598X IDH2
68 81 M4 46,XY 17 c.2032C > T p.Q678X NRAS, TET2
19 c.2210T > A p.L737H‡
69 50 M4 46,XX 15 c.1903C > T p.R635W† PTPN11, NPM1, IDH2
70 84 M0 ND 19 c.2207G > A p.R736H† AML1/RUNX1, IDH2
UPN Age, y/Sex FAB Karyotype Location DNMT3 mutation

Other accompanying gene mutations
DNA change Protein change
79/M M5 46,XY 23 c.2646G > A p.R882H FLT3/ITD, MLL/PTD, IDH2
77/M M1 46,XY 23 c.2646G > A p.R882H AML1/RUNX1
64/M M5 NM 23 c.2646G > A p.R882H PTPN11, NPM1
73/M M4 46,XY 23 c.2646G > A p.R882H IDH2
16/M M4 46,XY 23 c.2645C > T p.R882C FLT3/TKD, NPM1
41/F M4 46,XX 23 c.2646G > A p.R882H FLT3/ITD, NPM1
80/F M4 46,XX 23 c.2646G > A p.R882H PTPN11, NPM1
61/F M5 46,XX t(5;17)(q33;q21) 23 c.2645C > T p.R882C FLT3/TKD, NPM1
46/M M4 46,XY 23 c.2646G > A p.R882H FLT3/ITD, NPM1
10 35/F M1 46,XX 18 c.2120delG p.G707AfsX72 NRAS, IDH1
11 82/M M0 ND 23 c.2645C > T p.R882C FLT3/ITD, MLL/PTD, AML1/RUNX1
12 79/F M4 46,XX 23 c.2646G > A p.R882H FLT3/ITD, FLT3/TKD, MLL/PTD, TET2
13 51/M M4 46,XY 23 c.2646G > A p.R882H FLT3/TKD, NPM1
14 55/M M4 46,XY 16 c.1865_1866 insGT p.Y623FfsX29 NPM1
15 54/M M4 46,XY c.890G > A p.W297X PTPN11, ASXL1
16 68/M M2 46,XY 23 c.2645C > A p.R882S FLT3/ITD, NPM1
17 45/F M5 46,XX 23 c.2645C > T p.R882C FLT3/TKD, AML1/RUNX1, IDH2
18 54/F M2 46,XX 23 c.2646G > A p.R882H NRAS, NPM1
19 87/M M4 46,XY 23 c.2606delG p.G869VfsX12 FLT3/TKD, NPM1
20 51/F M4 47,XX,+i(11)(q10) 20 c.2389A > T p.N797Y‡ ASXL1, IDH2
21 78/M M4 46,XY 23 c.2646G > A p.R882H FLT3/ITD, NPM1
22 38/F M5 46,XX 23 c.2645C > T p.R882C NRAS, NPM1, IDH1
23 72/F M2 46,XX,del(20)(q11q13) 13 c.1477delA p.I493SfsX158 FLT3/ITD, NPM1
24 65/F M5 46,XX 23 c.2646G > A p.R882H FLT3/ITD
25 42/F M4 46,XX 23 c.2646G > A p.R882H FLT3/ITD, AML1/RUNX1, ASXL1
26 78/M M2 46,X,−Y,+4 19 c.2246_2249del p.R749PfsX29 FLT3/TKD, NPM1
27 75/F M1 46,XX 23 c.2645C > T p.R882C FLT3/ITD, NPM1
28 51/M M4 46,XY 23 c.2646G > A p.R882H FLT3/ITD, NPM1
29 60/F M1 46,XX,t(9;22)(q34;q11) 18 c.2113A > T p.I705F§ IDH1
30 73/F M1 46,XX 23 c.2646G > A p.R882H CEBPA, TET2
31 22/F M4 46,XX 23 c.2646G > A p.R882H FLT3/ITD, NPM1
32 38/M M4 46,XY 23 c.2645C > T p.R882C FLT3/ITD, NPM1
33 31/F M5 46,XX 23 c.2646G > A p.R882H FLT3/ITD, NPM1
34 46/M M4 45X,−Y 23 c.2645C > A p.R882S NRAS, FLT3/ITD, NPM1
35 80/M M4 46,XY 23 c.2645C > T p.R882C FLT3/ITD, MLL/PTD
36 52/M M1 46,XY 23 c.2645C > T p.R882C FLT3/ITD, IDH2
37 44/M M2 46,XY 23 c.2645C > T p.R882C FLT3/ITD, NPM1
38 33/F M1 46,XX c.958C > T p.R320X FLT3/TKD, NPM1
39 42/M M4 45,X,−Y 15 c.1816C > T p.Q606X NPM1
40 78/F M2 46,XX 19 c.2255_2257del p.F752del FLT3/ITD, NPM1
41 75/F M2 47,XX,del(5)(q22q35),+8 c.958C > T p.R320X IDH2
42 49/M M1 46,XY 23 c.2646G > A p.R882H FLT3/ITD, NPM1
43 78/M M4 46,XY c.315C > A p.S105R PTPN11
44 64/M M5 46,XY 23 c.2645C > A p.R882S PTPN11, MLL/PTD
45 40/M M5 46,XY 23 c.2646G > A p.R882H FLT3/ITD, NPM1
46 75/M M4 46,XY 23 c.2646G > A p.R882H NRAS, NPM1, TET2
47 58/F M2 46,XX 23 c.2645C > T p.R882C NPM1
48 48/F M1 47,XX,+8 c.1001delG p.G334AfsX11 CEBPA, IDH2
49 67/M M5 47,XY,+8 23 c.2645C > T p.R882C PTPN11, KRAS, AML1/RUNX1, IDH2
50 51/M M2 46XY 23 c.2646G > A p.R882H IDH2
51 85/M M5 46,XY c.767_770del P256LfsX59 FLT3/ITD, NPM1, WT1
52 85/M M1 45,XY,−7 c.327_328insG Q110AfsX14
53 67/M M8 47,XY,+8 23 c.2646G > A p.R882H NRAS, IDH2
54 35/M M4 46,XY 23 c.2645C > T p.R882C FLT3/ITD, NPM1
55 47/F M2 46,XX 23 c.2646G > A p.R882H ASXL1, IDH2
56 50/F M1 46,XX 23 c.2645C > T p.R882C FLT3/ITD, MLL/PTD
57 86/M M5 46,XY c.866delG p.G289AfsX26 FLT3/ITD
58 69/M M1 NM 23 c.2645C > T p.R882C FLT3/ITD, NPM1, CEBPA
59 75/M M4 46,XY 23 c.2646G > A p.R882H AML1/RUNX1
60 79/F M4 Cplx* 22 c.2510C > G p.S837X
61 61/M M1 46,XY 23 c.2646G > A p.R882H NPM1, WT1, TET2
62 37/F M2 46,XX 19 c.2312G > A p.R771Q‡ NPM1, TET2
63 70/M M2 46,XY 23 c.2646G > A p.R882H FLT3/ITD, NPM1, IDH2
64 46/M M4 46,XY 19 c.2182G > C, c.2191T > C p.G728R‡, p.F731L‡ FLT3/ITD
65 69/M M4 47,XY,+X c.941G > A p.W314X NRAS, FLT3/TKD, AML1/RUNX1, IDH2
19 c.2207G > A p.R736H†
66 38/F M2 46,XX 19 c.2207G > A p.R736H† FLT3/ITD, NPM1, IDH1
67 66 M1 47,XY,del(5)(q31q35), der(7)t(5;7)(q13;q11),+8 15 c.1792C > T p.R598X IDH2
68 81 M4 46,XY 17 c.2032C > T p.Q678X NRAS, TET2
19 c.2210T > A p.L737H‡
69 50 M4 46,XX 15 c.1903C > T p.R635W† PTPN11, NPM1, IDH2
70 84 M0 ND 19 c.2207G > A p.R736H† AML1/RUNX1, IDH2

Nucleotide numberings are according to the National Center for Biotechnology Information reference sequence NM_024426.

UPN indicates unique patient number; NM, no mitosis; and ND, not done.

*

In addition to R882 mutations, missense mutations in patients 65, 66, 69, and 70 have been reported in previous studies.4,7

Missense mutations in patients 20, 29, 62, 64, and 68 were confirmed to be significant by the analysis of remission BM samples.

Figure 1

Patterns and locations of the 30 different positions of mutations. The positions and predicted translational consequences of DNMT3A mutations detected in 500 AML samples are shown. The number of patients with the mutation is indicated in the parentheses behind each mutation. #, %, &, and $indicate that the patient has 2 mutations. Figure 1 Patterns and locations of the 30 different positions of mutations. The positions and predicted translational consequences of DNMT3A mutations detected in 500 AML samples are shown. The number of patients with the mutation is indicated in the parentheses behind each mutation. #, %, &, and$ indicate that the patient has 2 mutations.

### Correlation of DNMT3A mutations with clinical and laboratory features

In total, 500 de novo AML patients, including 70 (14%) DNMT3A-mutated and 430 DNMT3A-wild patients were enrolled into the study. The 6 patients with missense mutations of unknown significance were censored and were not included in the following analyses. A comparison of clinical characteristics of patients with and without distinct DNMT3A mutations is given in Table 2. DNMT3A-mutated patients were older (median, 61 vs 49 years, P < .0001) and had higher WBC, blast, and platelet counts than DNMT3A-wild patients (P = .0018, .0012, and .0001, respectively). Patients with the FAB M5 subtype of AML had the highest incidence (50%, P < .0001) of DNMT3A mutation, followed by those with the FAB M4 subtype (22.6%, P = .0026). DNMT3A mutations were positively associated with the expression of CD13 (P = .022) and CD14 (P = .0015), but inversely associated with the expression of CD34 (P = .0039) on leukemic cells (supplemental Table 1, available on the Blood Web site; see the Supplemental Materials link at the top of the online article). There was no difference in the expression of other antigens between the patients with and without the DNMT3A mutation.

Table 2

Comparison of clinical and laboratory features between AML patients with and without the DNMT3A mutation

Variable Total (n = 500) DNMT3A-mutated (n = 70, 14%) DNMT3A-wild (n = 430, 86%) P
Sex, n    .7961
Male 285 41 244
Female 215 29 186
Median age, y (range) 51 (15-90) 61 (16-87) 49 (15-90) < .0001
Laboratory data, median (range)
WBCs, /μL 19 075 (120-627 800) 32 490 (650-340 400) 15 940 (120-627 800) .0018
Hemoglobin, g/dL 8 (2.9-16.2) 8.7 (3.2-12) 7.9 (2.9-16.2) .0675
Platelets, × 1000/μL 42 (2-802) 57 (10-436) 39 (2-802) .0001
Blasts, /μL 7401 (0-456 725) 19 030 (111-283 212) 6263 (0-456 725) .0012
Lactase dehydrogenase, U/L 889 (206-15 000) 1064 (250-8116) 832 (206-15 000) .0883
FAB, n (%)    < .0001
M0 10 2 (20) 8 (80) .6375
M1 112 14 (12.5) 98 (87.5) .7572
M2 171 13 (7.6) 158 (92.4) .0027
M3 38 0 (0) 38 (100) .0056
M4 124 28 (22.6) 96 (77.4) .0026
M5 24 12 (50) 12 (50) < .0001
M6 12 0 (0) 12 (100) .3889
Undetermined 1 (11.1) 8 (88.9) > .9999
Variable Total (n = 500) DNMT3A-mutated (n = 70, 14%) DNMT3A-wild (n = 430, 86%) P
Sex, n    .7961
Male 285 41 244
Female 215 29 186
Median age, y (range) 51 (15-90) 61 (16-87) 49 (15-90) < .0001
Laboratory data, median (range)
WBCs, /μL 19 075 (120-627 800) 32 490 (650-340 400) 15 940 (120-627 800) .0018
Hemoglobin, g/dL 8 (2.9-16.2) 8.7 (3.2-12) 7.9 (2.9-16.2) .0675
Platelets, × 1000/μL 42 (2-802) 57 (10-436) 39 (2-802) .0001
Blasts, /μL 7401 (0-456 725) 19 030 (111-283 212) 6263 (0-456 725) .0012
Lactase dehydrogenase, U/L 889 (206-15 000) 1064 (250-8116) 832 (206-15 000) .0883
FAB, n (%)    < .0001
M0 10 2 (20) 8 (80) .6375
M1 112 14 (12.5) 98 (87.5) .7572
M2 171 13 (7.6) 158 (92.4) .0027
M3 38 0 (0) 38 (100) .0056
M4 124 28 (22.6) 96 (77.4) .0026
M5 24 12 (50) 12 (50) < .0001
M6 12 0 (0) 12 (100) .3889
Undetermined 1 (11.1) 8 (88.9) > .9999

### Association of DNMT3A mutations with cytogenetic abnormalities

Chromosome data were available for 482 patients at diagnosis, including 66 DNMT3A-mutated and 416 DNMT3A-wild patients (Table 3). DNMT3A mutations occurred more frequently in patients with intermediate-risk cytogenetics (19.5%) than in those with favorable karyotype or unfavorable cytogenetics (2.4%, P = .0069). There was a significant difference in the incidence of the DNMT3A mutation among patients with normal karyotype (22.9%), simple abnormalities with 1 or 2 changes (6.2%), and complex cytogenetics with 3 or more abnormalities (3.9%, P < .0001). None of the patients with t(8;21), t(15;17) inv(16), or 11q23 translocation had a DNMT3A mutation. There was no association of the DNMT3A mutation with other chromosomal abnormalities, including +8, +11, +13, +21, −5/del(5q), and −7/del(7q).

Table 3

Association of DNMT3A mutation with chromosomal abnormalities

Total DNMT3A-mutated DNMT3A-wild P
Karyotype   .0069
Favorable 99 0 (0) 99 (100) < .0001
Intermediate 318 62 (19.5) 256 (80.5) < .0001
Unfavorable 65 4 (6.2) 61 (93.8) .0783
Unknown 18 4 (22.2) 14 (77.7)
Normal 223 51 (22.9) 172 (77.1) < .0001
Simple 208 13 (6.2) 195 (93.8) < .0001
Complex 51 2 (3.9) 49 (96.1) .0303
t(8;21) 42 0 (0) 42 (100) .0034
t(15;17) 38 0 (0) 38 (100) .0053
inv(16) 19 0 (0) 19 (100) .0909
t(11q23) 16 0 (0) 16 (100) .145
t(7;11) 10 0 (0) 10 (100) .371
−5/5q−† 1 (50) 1 (50) .2554
−7/7q−† 10 1 (10) 9 (90) > .9999
+8‡ 27 4 (14.8) 23 (85.2) .7765
+11† 1 (33.3) 2 (66.7) .3577
+13† 0 (0) 1 (100) > .9999
+21† 0 (0) 9 (100) .6178
Total DNMT3A-mutated DNMT3A-wild P
Karyotype   .0069
Favorable 99 0 (0) 99 (100) < .0001
Intermediate 318 62 (19.5) 256 (80.5) < .0001
Unfavorable 65 4 (6.2) 61 (93.8) .0783
Unknown 18 4 (22.2) 14 (77.7)
Normal 223 51 (22.9) 172 (77.1) < .0001
Simple 208 13 (6.2) 195 (93.8) < .0001
Complex 51 2 (3.9) 49 (96.1) .0303
t(8;21) 42 0 (0) 42 (100) .0034
t(15;17) 38 0 (0) 38 (100) .0053
inv(16) 19 0 (0) 19 (100) .0909
t(11q23) 16 0 (0) 16 (100) .145
t(7;11) 10 0 (0) 10 (100) .371
−5/5q−† 1 (50) 1 (50) .2554
−7/7q−† 10 1 (10) 9 (90) > .9999
+8‡ 27 4 (14.8) 23 (85.2) .7765
+11† 1 (33.3) 2 (66.7) .3577
+13† 0 (0) 1 (100) > .9999
+21† 0 (0) 9 (100) .6178

Four hundred eighty-two patients, including 66 DNMT3A-mutated and 416 DNMT3A-wild patients, had chromosome data at diagnosis.

*

Favorable, t(15;17), t(8;21), inv(16); unfavorable, −7, del(7q), −5, del(5q), 3q abnormality, complex abnormalities; and intermediate, normal karyotype and other abnormalities.

Only including simple chromosomal abnormalities with ≤ 2 changes, but not those with complex abnormalities with ≥ 3 aberrations.

### Association of DNMT3A mutation with other molecular abnormalities

Table 4

Association of the DNMT3 mutation with other gene mutations

Variable Patients with alteration, n (%)

P
Whole cohort (n = 500) DNMT3A-mutated patients (n = 70) DNMT3A-wild patients (n = 430)
FLT3/ITD 113 (22.6) 30 (42.9) 83 (19.3) < .0001
FLT3/TKD 39 (7.8) 9 (12.9) 29 (6.7) .087
N-RAS 61 (12.2) 8 (11.4) 53 (12.3) > .9999
K-RAS 16 (3.2) 1 (1.4) 15 (3.5) .7112
PTPN11 18 (3.6) 7 (10) 11 (2.6) .007
KIT 15 (3.0) 0 (0) 15 (3.5) .2451
JAK2 3 (0.6) 0 (0) 3 (0.7) > .9999
WTI 33 (6.6) 2 (2.9) 31 (7.2) .2946
NPM1 104 (20.8) 38 (54.3) 66 (15.3) < .0001
CEBPA 66 (13.2) 3 (4.3) 63 (14.7) .0134
AML1/RUNX1 62 (12.4) 8 (11.4) 54 (12.6) > .9999
MLL/PTD 27 (5.4) 6 (8.6) 21 (4.9) .2475
ASXL1 51 (10.2) 4 (5.7) 46 (10.7) .2812
IDH1 27 (5.4) 4 (5.7) 23 (5.3) .7812
IDH2 55 (11) 16 (22.9) 39 (9.1) .0016
TET2 65 (13.0) 6 (8.6) 59 (13.7) .3365
Variable Patients with alteration, n (%)

P
Whole cohort (n = 500) DNMT3A-mutated patients (n = 70) DNMT3A-wild patients (n = 430)
FLT3/ITD 113 (22.6) 30 (42.9) 83 (19.3) < .0001
FLT3/TKD 39 (7.8) 9 (12.9) 29 (6.7) .087
N-RAS 61 (12.2) 8 (11.4) 53 (12.3) > .9999
K-RAS 16 (3.2) 1 (1.4) 15 (3.5) .7112
PTPN11 18 (3.6) 7 (10) 11 (2.6) .007
KIT 15 (3.0) 0 (0) 15 (3.5) .2451
JAK2 3 (0.6) 0 (0) 3 (0.7) > .9999
WTI 33 (6.6) 2 (2.9) 31 (7.2) .2946
NPM1 104 (20.8) 38 (54.3) 66 (15.3) < .0001
CEBPA 66 (13.2) 3 (4.3) 63 (14.7) .0134
AML1/RUNX1 62 (12.4) 8 (11.4) 54 (12.6) > .9999
MLL/PTD 27 (5.4) 6 (8.6) 21 (4.9) .2475
ASXL1 51 (10.2) 4 (5.7) 46 (10.7) .2812
IDH1 27 (5.4) 4 (5.7) 23 (5.3) .7812
IDH2 55 (11) 16 (22.9) 39 (9.1) .0016
TET2 65 (13.0) 6 (8.6) 59 (13.7) .3365

### Impact of DNMT3A mutation on response to therapy and clinical outcome

Of the 363 AML patients undergoing conventional intensive induction chemotherapy, 284 (78.5%) patients achieved a CR. The probability of achieving a CR was similar between patients with and without DNMT3A mutations (74.4% vs 79%, P = .5531). However, the patients with DNMT3A mutations had a trend of higher relapse rate than those without (65.6% vs 48.8%, P = .0911). With a median follow-up of 55 months (range, 1.0-160), patients with the DNMT3A mutation had significantly poorer OS and RFS than those without the DNMT3A mutation (median, 14.5 months vs 38 months, P = .013, and median, 7.5 months vs 15 months, P = .012, respectively, Figure 2A-C). The same was true among patients with non-M3 AML (P = .04 and P = .036, respectively). In the subgroup of 130 younger patients (< 60 years) with normal karyotype AML (CN-AML), the differences between patients with and without the DNMT3A mutation in OS (median, 15.5 months vs not reached, P = .018, Figure 2B) and RFS (median, 6 months vs 21 months, P = .004, Figure 2D) were still significant. We also observed that the prognostic impact of the DNMT3A mutation could only be demonstrated in the patients with a poor prognostic genotype (NPM1-mutated (NPM1+)/FLT3-ITD+, NPM1-wild (NPM1)/FLT3-ITD+ or FLT3/ITD), but not in those with favorable genotype (NPM1+/FLT3-ITD) among total AML patients (P < .001 and P = .823, respectively) or in CN-AML patients (P < .001 and P = .970, respectively). There was no significant difference in survival between patients with mutations of R882 and those with other mutations (P = .612).

Figure 2

OS and RFS in total patients and in younger patients with CN-AML. Kaplan-Meier survival curves for OS and RFS in 363 AML patients (A and C) and 130 younger patients (< 60 years) with CN-AML (B and D) who received standard intensive chemotherapy.

Figure 2

OS and RFS in total patients and in younger patients with CN-AML. Kaplan-Meier survival curves for OS and RFS in 363 AML patients (A and C) and 130 younger patients (< 60 years) with CN-AML (B and D) who received standard intensive chemotherapy.

In multivariate analysis (Table 5), the independent poor risk factors for OS were older age (> 50 years), high WBC count (> 50 000/μL), unfavorable karyotype, DNMT3A mutation, AML1/RUNX1 mutation, and WT1 mutation. Conversely, CEBPAdouble-mutation and NPM1+/FLT3ITD were independent favorable prognostic factors. There was a trend of better OS in patients with the IDH2 mutation (hazard ratio [HR], 0.573; 95% confidence interval [95% CI], 0.296-1.110, P = .099). The independent poor risk factors for RFS included high WBC count (> 50 000/μL), unfavorable karyotype, DNMT3A mutation, and WT1 mutation. NPM1+/FLT3-ITD was an independent favorable factor for RFS. In 130 CN-AML patients younger than 60 years, the DNMT3A mutation was still an independent poor prognosis for OS and RFS (HR, 2.303; 95% CI, 1.088-4.876, P = .029 and HR, 3.496; 95% CI, 1.773-6.896, P < .001, respectively, supplemental Table 3).

Table 5

Multivariate analysis (Cox regression) for relapse-free and overall survival

Variable Relapse-free survival

Overall survival

HR 95% CI

P HR 95% CI

P
Lower Upper Lower Upper
Age* 1.150 0.803 1.648 .446 2.531 1.790 3.580 < .001†
WBC‡ 1.649 1.120 2.428 .011† 1.970 1.358 2.857 < .001†
Karyotype§ 2.577 1.433 4.633 .002† 3.078 1.849 5.123 < .001†
NPM1/FLT3-ITD¶ 0.268 0.124 0.581 .001† 0.261 0.121 0.564 .001†
CEBPA0.629 0.362 1.093 .100 0.423 0.211 0.847 .015†
IDH2** 0.775 0.420 1.430 .415 0.573 0.296 1.110 .099
WT1 2.823 1.680 4.743 <.001† 2.576 1.490 4.454 .001†
AML1/RUNX1 1.448 0.718 2.918 .301 1.963 1.129 3.414 .017†
ASXL1 0.739 0.293 1.863 .521 1.439 0.798 2.597 .227
TET2 1.125 0.625 2.026 .694 1.033 0.601 1.777 .906
DNMT3A 2.898 1.673 5.022 <.001† 2.218 1.333 3.692 .002†
Variable Relapse-free survival

Overall survival

HR 95% CI

P HR 95% CI

P
Lower Upper Lower Upper
Age* 1.150 0.803 1.648 .446 2.531 1.790 3.580 < .001†
WBC‡ 1.649 1.120 2.428 .011† 1.970 1.358 2.857 < .001†
Karyotype§ 2.577 1.433 4.633 .002† 3.078 1.849 5.123 < .001†
NPM1/FLT3-ITD¶ 0.268 0.124 0.581 .001† 0.261 0.121 0.564 .001†
CEBPA0.629 0.362 1.093 .100 0.423 0.211 0.847 .015†
IDH2** 0.775 0.420 1.430 .415 0.573 0.296 1.110 .099
WT1 2.823 1.680 4.743 <.001† 2.576 1.490 4.454 .001†
AML1/RUNX1 1.448 0.718 2.918 .301 1.963 1.129 3.414 .017†
ASXL1 0.739 0.293 1.863 .521 1.439 0.798 2.597 .227
TET2 1.125 0.625 2.026 .694 1.033 0.601 1.777 .906
DNMT3A 2.898 1.673 5.022 <.001† 2.218 1.333 3.692 .002†

HR indicates hazard ratio; and 95% CI, 95% confidence interval.

*

Age > 50 relative to age ≤ 50 (the reference age).

Statistically significant (P < .05).

WBCs > 50 000/μL versus < 50 000/μL.

§

Unfavorable cytogenetics versus others.

NPM1mut/FLT3-ITDneg versus other subtypes.

#

**

IDH2 mutations included R140 and R172 mutations.

To better stratify the AML patients into different risk groups, a scoring system incorporating 9 prognostic markers—age, WBC count, cytogenetics at diagnosis, NPM1/FLT3-ITD, and mutations of CEBPA, DNMT3A, AML1/RUNX1, IDH2, and WT1 mutation—into the survival analysis was formulated based on the results of our Cox proportional hazards model. A score of −1 was assigned for each parameter associated with a favorable outcome (ie, CEBPAdouble-mutation, IDH2 mutation, and NPM1+/FLT3-ITD), whereas a score of +1 for each factor associated with an adverse outcome (ie, DNMT3A, WT1, and AML1/RUNX1 mutations, older age, and higher WBC counts at diagnosis). The karyotypes were stratified into 3 groups (+2, unfavorable; +1, intermediate; and 0, favorable). The algebraic summation of these scores for each patient was the final score. This score system divided the AML patients into 5 groups with different clinical outcomes (P < .001 for both OS and RFS, Figure 3).

Figure 3

OS and RFS stratified by proposed scoring system. Kaplan-Meier survival curves for OS (A) and RFS (B) in AML patients based on our new scoring system (P < .001 for both OS and RFS). AML patients were grouped according to our scoring system based on the DNMT3A mutation and 8 other prognostic markers (ie, age, WBC count at diagnosis, and CEBPAdouble-mutation, NPM1/FLT3-ITD, IDH2, DNMT3A, WT1, and AML1/RUNX1 mutations). A score of −1 was assigned for each parameter associated with a favorable outcome (ie, CEBPAdouble-mutation, IDH2 mutation, and NPM1+/FLT3-ITD); a score of +1 was assigned for each factor associated with an adverse outcome (ie, older age, higher WBC counts at diagnosis, and DNMT3A, WT1, and AML1/RUNX1 mutations). The karyotypes were stratified into 3 groups (+2, unfavorable; +1, intermediate; and 0, favorable). The algebraic summation of these scores for each patient was the final score. The 12 patients without chromosome data were not included in the analysis.

Figure 3

OS and RFS stratified by proposed scoring system. Kaplan-Meier survival curves for OS (A) and RFS (B) in AML patients based on our new scoring system (P < .001 for both OS and RFS). AML patients were grouped according to our scoring system based on the DNMT3A mutation and 8 other prognostic markers (ie, age, WBC count at diagnosis, and CEBPAdouble-mutation, NPM1/FLT3-ITD, IDH2, DNMT3A, WT1, and AML1/RUNX1 mutations). A score of −1 was assigned for each parameter associated with a favorable outcome (ie, CEBPAdouble-mutation, IDH2 mutation, and NPM1+/FLT3-ITD); a score of +1 was assigned for each factor associated with an adverse outcome (ie, older age, higher WBC counts at diagnosis, and DNMT3A, WT1, and AML1/RUNX1 mutations). The karyotypes were stratified into 3 groups (+2, unfavorable; +1, intermediate; and 0, favorable). The algebraic summation of these scores for each patient was the final score. The 12 patients without chromosome data were not included in the analysis.

### Sequential studies of DNMT3A mutations in AML patients

DNMT3A mutations were studied sequentially in 316 samples from 138 patients, including 35 patients with distinct DNMT3A mutations and 103 patients without mutations at diagnosis (Table 6). Among the 34 patients with DNMT3A mutations who had ever obtained a CR and had available samples for study, 29 lost the original mutation at remission status, but 5 (patients 5, 8, 28, 32, and 33) retained it (Table 6); all 5 patients relapsed within a median of 3.5 months and died of disease progression, suggesting the presence of leukemic cells. In the 13 patients who had available samples for serial study at relapse, 12 patients regained the original mutations, but 1 (patient 9) lost the mutation at relapse. Because direct sequencing might not be sensitive enough to detect low levels of DNMT3A mutation signal, we sequenced TA clones of the PCR product from patient 9 and 1 mutant clone of 17 was detected. Among the 103 patients who had no DNMT3A mutation at diagnosis, none acquired the DNMT3A mutation at relapse, whereas karyotypic evolution was noted at relapse in 39% of these patients (data not shown).

Table 6

Sequential studies in AML patients with DNMT3A mutations

UPN Date Status Karyotype DNMT3A mutation Other mutations
10/31/2006 Initial 46, XY p.R882H IDH2
11/29/2006   −
7/27/2000 Initial 46,XY p.R882C NPM1, FLT3/TKD
8/24/2000 CR1  p.R882C −
7/17/2001 Relapse 1 46,XY p.R882C NPM1, FLT3/TKD, WT1
10/23/2001 CR2  p.R882C −
5/7/2002 Relapse 2 46,XY,del(6)(p21) p.R882C NPM1, FLT3/TKD, WT1
8/31/2004 Initial 46,XX p.R882H NPM1, FLT3/ITD
9/14/2006 CR  − −
9/16/2005 Initial 46,XX,t(5;17)(q33;q21) p.R882C NPM1, FLT3/TKD
11/4/2005 CR 46,XX p.R882C −
5/27/1997 Initial 46,XY p.R882H FLT3/ITD, NPM1
6/23/1997 CR  − −
7/30/1997 Relapse 46,XY −* FLT3/ITD
10 5/16/2000 Initial 46,XX p.G707AfsX72 NRAS, IDH1
6/7/2000 CR  − −
13 7/26/2002 Initial 46,XY p.R882H FLT3/TKD, NPM1
9/2/2002 CR  − −
14 12/22/2003 Initial 46,XY p.Y623FfsX29 NPM1
3/5/2004 CR  − −
15 11/21/2006 Initial 46,XY p.W297X PTPN11, ASXL1
5/3/2007 CR  − −
17 4/24/2007 Initial 46,XX p.R882C FLT3/TKD, AML1/RUNX1, IDH2
6/28/2007 CR  − −
18 10/15/1999 Initial 46,XX p.R882H NRAS, NPM1
11/30/1999 CR  − −
1/18/2001 Relapse 46,XX p.R882H NPM1
20 12/28/2007 Initial 47,XX,+i(11)(q10) p.N797Y ASXL1, IDH2
6/20/2008 CR 46,XX − −
10/21/2008 Relapse 46,XX p.N797Y ASXL1, IDH2
22 9/16/2004 Initial 46,XX p.R882C NRAS, NPM1, IDH1
10/28/2004 CR  − −
28 8/7/2006 Initial 46,XY p.R882H FLT3/ITD, NPM1
9/26/2006 CR  p.R882H −
1/18/2007 Relapse ND p.R882H FLT3/ITD, NPM1
29 1/27/2004 Initial 46,XX,t(9;22)(q34;q11) p.I705F IDH1
3/1/2004 CR 46,XX − −
6/9/2005 Relapse 46,XX,t(9;22)(q34;q11) p.I705F IDH1
31 4/2/2001 Initial 46,XX p.R882H FLT3/ITD, NPM1
5/11/2001 CR  − −
8/20/2001 Relapse 44-46,XX,del(20)(q11q13)[cp6]/46,XX[7] p.R882H FLT3/ITD, NPM1
32 4/12/2000 Initial 46,XY p.R882C FLT3/ITD, NPM1
7/13/2000 CR  p.R882C −
10/5/2000 Relapse 46,XY p.R882C FLT3/ITD, NPM1
33 10/29/2007 Initial 46,XX p.R882H FLT3/ITD, NPM1
3/18/2008 CR  p.R882H −
5/8/2008 Relapse ND p.R882H FLT3/ITD, NPM1
34 6/25/1998 Initial 45,X,-Y p.R882S NRAS, FLT3/ITD, NPM1
7/7/2000 Relapse 45,X,-Y p.R882S FLT3/ITD, NPM1
8/11/2000 CR2 46,XY − −
37 2/3/2006 Initial 46,XY p.R882C FLT3/ITD, NPM1
4/19/2006 CR  − −
5/3/2006 Relapse ND p.R882C FLT3/ITD
38 8/15/2002 Initial 46,XX p.R320X FLT3/TKD, NPM1
1/28/2003 CR  − −
39 2/15/2002 Initial 45,X,-Y p.Q606X NPM1
4/8/2002 CR 46,XY − −
45 2/1/2005 Initial 46,XY p.R882H FLT3/ITD, NPM1
3/1/2005 CR  − −
11/24/2005 Relapse 46,XY p.R882H FLT3/ITD, NPM1
47 6/14/2000 Initial 46,XX p.R882C NPM1
10/19/2000 CR  − −
48 12/13/2006 Initial 47,XX,+8 p.G334AfsX11 CEBPA, IDH2
2/9/2007 CR  − −
50 9/25/2003 Initial 46,XY R882H IDH2
6/10/2005 CR  − −
51 5/29/2003 Initial 46,XY p.P256LfsX59 FLT3/ITD, NPM1, WT1
7/17/2003 CR  − −
12/26/2003 Relapse ND p.P256LfsX59 FLT3/ITD, NPM1, WT1
54 9/5/2002 Initial 46,XY p.R882C FLT3/ITD, NPM1
5/28/2003 CR  − −
55 2/21/2006 Initial 46,XX p.R882H ASXL1, IDH2
9/14/2006 CR  − −
56 3/24/2003 Initial 46,XX p.R882C FLT3/ITD, MLL/PTD
5/21/2003 CR  − −
10/1/2003 Relapse 46,XX p.R882C FLT3/ITD, MLL/PTD
61 10/30/1995 Initial 46, XY p.R882H NPM1, WT1, TET2
1/15/1996 CR  − −
10/22/1996 Relapse ND p.R882H NPM1, TET2
62 9/8/1995 Initial 46,XX p.R771Q NPM1, TET2
12/19/1995 CR  − −
9/23/1996 Relapse 46,XX p.R771Q NPM1, TET2
64 11/2/1999 Initial 46,XY p.G728R, p.F731L FLT3/ITD
3/16/2000 CR1  − −
6/12/2000 Relapse 1 ND p.G728R, p.F731L FLT3/ITD
7/14/2000 CR2 ND − −
1/11/2001 Relapse 2 ND p.G728R, p.F731L FLT3/ITD
3/13/2001 CR3  − −
66 3/25/2003 Initial 46,XX p.R736H FLT3/ITD, NPM1, IDH1
12/30/2003 CR  − −
69 4/2/2001 Initial 46,XX p.R635W PTPN11, NPM1, IDH2
5/17/2001 CR  − −
UPN Date Status Karyotype DNMT3A mutation Other mutations
10/31/2006 Initial 46, XY p.R882H IDH2
11/29/2006   −
7/27/2000 Initial 46,XY p.R882C NPM1, FLT3/TKD
8/24/2000 CR1  p.R882C −
7/17/2001 Relapse 1 46,XY p.R882C NPM1, FLT3/TKD, WT1
10/23/2001 CR2  p.R882C −
5/7/2002 Relapse 2 46,XY,del(6)(p21) p.R882C NPM1, FLT3/TKD, WT1
8/31/2004 Initial 46,XX p.R882H NPM1, FLT3/ITD
9/14/2006 CR  − −
9/16/2005 Initial 46,XX,t(5;17)(q33;q21) p.R882C NPM1, FLT3/TKD
11/4/2005 CR 46,XX p.R882C −
5/27/1997 Initial 46,XY p.R882H FLT3/ITD, NPM1
6/23/1997 CR  − −
7/30/1997 Relapse 46,XY −* FLT3/ITD
10 5/16/2000 Initial 46,XX p.G707AfsX72 NRAS, IDH1
6/7/2000 CR  − −
13 7/26/2002 Initial 46,XY p.R882H FLT3/TKD, NPM1
9/2/2002 CR  − −
14 12/22/2003 Initial 46,XY p.Y623FfsX29 NPM1
3/5/2004 CR  − −
15 11/21/2006 Initial 46,XY p.W297X PTPN11, ASXL1
5/3/2007 CR  − −
17 4/24/2007 Initial 46,XX p.R882C FLT3/TKD, AML1/RUNX1, IDH2
6/28/2007 CR  − −
18 10/15/1999 Initial 46,XX p.R882H NRAS, NPM1
11/30/1999 CR  − −
1/18/2001 Relapse 46,XX p.R882H NPM1
20 12/28/2007 Initial 47,XX,+i(11)(q10) p.N797Y ASXL1, IDH2
6/20/2008 CR 46,XX − −
10/21/2008 Relapse 46,XX p.N797Y ASXL1, IDH2
22 9/16/2004 Initial 46,XX p.R882C NRAS, NPM1, IDH1
10/28/2004 CR  − −
28 8/7/2006 Initial 46,XY p.R882H FLT3/ITD, NPM1
9/26/2006 CR  p.R882H −
1/18/2007 Relapse ND p.R882H FLT3/ITD, NPM1
29 1/27/2004 Initial 46,XX,t(9;22)(q34;q11) p.I705F IDH1
3/1/2004 CR 46,XX − −
6/9/2005 Relapse 46,XX,t(9;22)(q34;q11) p.I705F IDH1
31 4/2/2001 Initial 46,XX p.R882H FLT3/ITD, NPM1
5/11/2001 CR  − −
8/20/2001 Relapse 44-46,XX,del(20)(q11q13)[cp6]/46,XX[7] p.R882H FLT3/ITD, NPM1
32 4/12/2000 Initial 46,XY p.R882C FLT3/ITD, NPM1
7/13/2000 CR  p.R882C −
10/5/2000 Relapse 46,XY p.R882C FLT3/ITD, NPM1
33 10/29/2007 Initial 46,XX p.R882H FLT3/ITD, NPM1
3/18/2008 CR  p.R882H −
5/8/2008 Relapse ND p.R882H FLT3/ITD, NPM1
34 6/25/1998 Initial 45,X,-Y p.R882S NRAS, FLT3/ITD, NPM1
7/7/2000 Relapse 45,X,-Y p.R882S FLT3/ITD, NPM1
8/11/2000 CR2 46,XY − −
37 2/3/2006 Initial 46,XY p.R882C FLT3/ITD, NPM1
4/19/2006 CR  − −
5/3/2006 Relapse ND p.R882C FLT3/ITD
38 8/15/2002 Initial 46,XX p.R320X FLT3/TKD, NPM1
1/28/2003 CR  − −
39 2/15/2002 Initial 45,X,-Y p.Q606X NPM1
4/8/2002 CR 46,XY − −
45 2/1/2005 Initial 46,XY p.R882H FLT3/ITD, NPM1
3/1/2005 CR  − −
11/24/2005 Relapse 46,XY p.R882H FLT3/ITD, NPM1
47 6/14/2000 Initial 46,XX p.R882C NPM1
10/19/2000 CR  − −
48 12/13/2006 Initial 47,XX,+8 p.G334AfsX11 CEBPA, IDH2
2/9/2007 CR  − −
50 9/25/2003 Initial 46,XY R882H IDH2
6/10/2005 CR  − −
51 5/29/2003 Initial 46,XY p.P256LfsX59 FLT3/ITD, NPM1, WT1
7/17/2003 CR  − −
12/26/2003 Relapse ND p.P256LfsX59 FLT3/ITD, NPM1, WT1
54 9/5/2002 Initial 46,XY p.R882C FLT3/ITD, NPM1
5/28/2003 CR  − −
55 2/21/2006 Initial 46,XX p.R882H ASXL1, IDH2
9/14/2006 CR  − −
56 3/24/2003 Initial 46,XX p.R882C FLT3/ITD, MLL/PTD
5/21/2003 CR  − −
10/1/2003 Relapse 46,XX p.R882C FLT3/ITD, MLL/PTD
61 10/30/1995 Initial 46, XY p.R882H NPM1, WT1, TET2
1/15/1996 CR  − −
10/22/1996 Relapse ND p.R882H NPM1, TET2
62 9/8/1995 Initial 46,XX p.R771Q NPM1, TET2
12/19/1995 CR  − −
9/23/1996 Relapse 46,XX p.R771Q NPM1, TET2
64 11/2/1999 Initial 46,XY p.G728R, p.F731L FLT3/ITD
3/16/2000 CR1  − −
6/12/2000 Relapse 1 ND p.G728R, p.F731L FLT3/ITD
7/14/2000 CR2 ND − −
1/11/2001 Relapse 2 ND p.G728R, p.F731L FLT3/ITD
3/13/2001 CR3  − −
66 3/25/2003 Initial 46,XX p.R736H FLT3/ITD, NPM1, IDH1
12/30/2003 CR  − −
69 4/2/2001 Initial 46,XX p.R635W PTPN11, NPM1, IDH2
5/17/2001 CR  − −

The results of serial studies in 103 patients without DNMT3A mutation at diagnosis are not shown in this table. None of these 103 patients acquired DNMT3A mutation at relapse.

UPN indicates unique patient number; CR, complete remission; –, negative; ND not done; and NM, no mitosis.

*

Using the more sensitive TA cloning technique, 1 of 17 clones showed DNMT3A mutation.

## Discussion

In the present study, we found that the DNMT3A mutation was associated with distinct clinical and biologic features and was a poor prognostic factor in AML patients independent of age, WBC counts, karyotype, and other genetic markers.

DNMT3A mutations at 30 different positions, most commonly in the MTase domain, were demonstrated (Figure 1). All of the nonsense, frame-shift, and in-frame mutations generated truncated peptide with complete or partial deletion of the MTase domain and were thought to abolish the catalytic activity of this enzyme. The missense R882 mutations, the most common DNMT3A mutations, resulted in impaired enzyme activity,4,9  but the influence of other missense mutations on the enzyme remains unclear. These missense mutations all involved amino acid residues well conserved through evolution. We censored 6 patients with missense variants of unknown significance and did not include them in the analyses because there were no available remission BM samples or normal tissues to verify that their DNMT3A variants were true somatic mutations. In contrast to the report of Thol et al,3  who only found mutations between exons 15 and 23, 10 mutations in our patients were located outside of this region (Figure 1). Nine of these mutations were frame-shift or nonsense mutations and were expected to impair enzyme activity. Similar to our finding, Ley et al also detected mutations outside of exons 15 to 23.4  In the study by Thol et al, all 23 exons of DNMT3 were initially sequenced in 40 patients.3  Because only mutations between exons 15 and 23 were found in these patients, they subsequently sequenced exons 15 to 23, but not other exons, in other patients. Because all but one mutation outside of exons 15 to 23 in our study were detected in only one patient each (an incidence of 1 in 500 for each mutation), the absence of mutation in this area in 40 patients might not mean that it would not happen in other patients.

In this study, DNMT3A mutations were found in 14%, 15.2%, 19.5%, and 22.9%, respectively, in whole cohort, non-M3 AML, intermediate-risk cytogenetics, and CN-AML groups, lower than the reports of Ley et al (22.1% for total patients, 33.7% for those with intermediate-risk cytogenetics, and 36.7% for CN-AML patients)4  and Thol et al (17.8% in non-M3 and 27.2% in CN-AML patients).3  In a study of Chinese AML patients by Yan et al, DNMT3A mutations were detected in 20.5% and 13.6%, respectively, of patients with the FAB M5 and M4 subtypes of AML, but none of the patients with FAB M1 or M2 subtypes had the mutation, leading to an overall incidence of 9% for the DNMT3A mutation in the entire group of AML patients.9  Yamashita et al also reported a low incidence (4.1%) of DNMT3A mutations in Japanese AML patients.8  The reason for the variability in the incidence of DNMT3A mutations in different studies is unknown, but may be because of the differences in ethnic background, patient populations recruited, and methods used. Whether DNMT3A mutations occur less frequently in Asian than in Western AML patients needs to be determined by further studies.

In our comprehensive analysis of the 17 gene mutations in 500 patients, we found that the DNMT3A mutation was the third commonest recurrent genetic alteration, followed by FLT3-ITD and NPM1 mutations, in AML patients. In addition to its close association with NPM1 mutations and FLT3-ITD, which has been shown previously,3,4  we demonstrated herein that DNMT3A mutations were also positively associated with PTPN11 and IDH2 mutations and negatively associated with the CEBPA mutation. More intriguingly, we found that the DNMT3A mutation rarely occurred alone; all but 2 patients with DNMT3A mutations showed concurrent mutations of other genes, more frequently class I (51 of 68, 75%), but also class II mutations (16 of 68, 23.5%) and NPM1 mutations (38 of 68, 54.3%, supplemental Table 2), which behave more like class II mutations.13  In short, the development of AML may require concerted interaction among different genetic alterations.

The stability of DNMT3A mutations in the evolution of AML remains unclear. In a serial study of 5 patients with DNMT3A mutations at diagnosis, Thol et al found that the mutations disappeared at CR and reappeared at relapse in one patient tested.3  To the best of our knowledge, the present study recruited the largest number of AML patients for sequential analysis of DNMT3A mutations during the clinical course. In contrast to the instability of FLT3-ITD during disease evolution, we found that the DNMT3A mutation seemed to be stable, analogous to NPM1 and IDH1/2 mutations.13,21,22  At relapse, all DNMT3A-mutated patients who had available samples for serial study regained the same mutations, including the one in whom the mutation could be detected by a sensitive gene-cloning technique, but not by direct sequencing. Conversely, all 103 patients without DNMT3A mutation at diagnosis remained DNMT3A-wild at relapse. These results suggested that although DNMT3A mutations are important for the development of AML, they may play little role in disease progression. Given the stability of the DNMT3A mutation during AML evolution, it may be a potential biomarker for monitoring minimal residual disease.

We found that AML patients with DNMT3A mutations had distinct clinical and laboratory characteristics and a poor prognosis. Recently, many gene mutations have been detected in AML and some found to be independent prognostic factors. In the present study, to better stratify AML patients into different risk groups, a survival scoring system incorporating the DNMT3A mutation and 8 other prognostic factors, including age, WBC count, cytogenetics, and NPM1/FLT3-ITD, CEBPA, AML1/RUNX1, WT1, and IDH2 mutations, into the survival analysis was formulated. This scoring system was found to be more powerful than any single marker at separating patients into different prognostic groups. However, further study with an independent cohort will be needed to validate the proposed scoring system.

In summary, this study demonstrated that DNMT3A mutations could be detected in a substantial number of patients with de novo AML and were closely associated with older age and FAB M4/M5 subtypes. DNMT3A mutations occurred more frequently in patients with intermediate-risk cytogenetics and normal karyotype. They were mutually exclusive with CEBPA mutation, but were closely associated with FLT3/ITD, NPM1, PTPN11, and IDH2 mutations. Furthermore, the DNMT3A mutation was an independent poor risk factor for OS and RFS among total cohort and CN-AML patients. Sequential study during the clinical course showed that the DNMT3A mutation was stable during AML evolution. We conclude that the incorporation of the DNMT3A mutation with 8 other prognostic factors into survival analyses can better stratify AML patients into different risk groups.

## Acknowledgments

This work was partially sponsored by grants from the National Science Council (NSC 97-2314-B002-015-MY3, 99-2314-B-002-143, 100-2325-B-002-032, and 100-2628-B-002-003-MY3) and the Department of Health (DOH100-TD-C-111-001), Taiwan, Republic of China, and the Department of Medical Research (NTUH.99P14 and 100P07), National Taiwan University Hospital, Taipei, Taiwan.

## Authorship

Contribution: H.-A.H. collected the literature, managed and interpreted the data, performed the statistical analysis, and wrote the manuscript; Y.-Y.K. collected the literature, managed and interpreted the data, and wrote the manuscript; C.-Y.L. performed and interpreted the statistical analysis; L.-I.L. performed and interpreted the mutation analysis; C.-Y.C., W.-C.C., M.Y., S.-Y.H., J.-L.T., B.-S.K., S.-C.H., S.-J.W., W.T., and Y.-C.C. contributed patient samples and clinical data; M.C.L., M.-H.T., C.-F.H., Y.-C.C., C.-Y. L., F.-Y.L., and M.-C.L. performed the gene mutation and chromosomal studies; and H.-F.T. planned, designed, and coordinated the study and wrote the manuscript.

Conflict-of-interest disclosure: The authors declare no competing financial interests.

Correspondence: Hwei-Fang Tien, MD, PhD, Department of Internal Medicine, National Taiwan University Hospital, 7 Chung-Shan Street, Taipei, Taiwan; e-mail: hftien@ntu.edu.tw.

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## Author notes

*

H.-A.H. and Y.-Y.K. contributed equally to this work.