Abstract

Mutations of the ten eleven translocation 2 gene (TET2) have recently been reported in myelodysplastic syndrome and myeloproliferative neoplasms. We analyzed the incidence and prognostic value of TET2 point mutations and other genomic alterations by direct sequencing and single nucleotide polymorphism microarray analysis in 111 de novo acute myeloid leukemia, who had all achieved complete remission (CR). Mutations were observed in 19 (17%) of the 111 patients compared with 10 (27%) of 36 patients who had failed to achieve CR (P = .2). In the 111 patients who had achieved CR, TET2 alterations were only significantly associated with NPM1 mutations but not with other pretreatment characteristics. TET2 gene status was not significantly correlated with disease-free survival and overall survival, both in the entire cohort and in patients with normal karyotype.

Introduction

In acute myeloid leukemia (AML), both cytogenetic and molecular abnormalities are strongly associated with prognosis. In particular, in cytogenetically normal AML, FLT3-ITD (internal tandem duplication) carries adverse prognostic factor, whereas NPM1 or CEBPA mutations are associated with favorable outcome.1-3 

Recently, mutations of the ten eleven translocation 2 gene (TET2) have been reported in 20% to 26% of myelodysplastic syndromes (MDSs) or secondary AML,4,5  in 14% of myeloproliferative neoplasms (MPNs),4,6,7  and in 12% of de novo AML.8  We evaluated the frequency and type of TET2 alterations in 111 de novo AML patients who had achieved complete remission (CR) with intensive chemotherapy. The incidence of mutations was compared with that observed in 36 AML patients who failed to achieve CR with intensive chemotherapy. In patients who achieved CR, the presence of TET2 mutation was correlated with other molecular abnormalities and outcomes.

Methods

A total of 147 patients 15 to 69 years of age with previously untreated de novo AML from 7 French centers (Avicenne Hospital, Cochin Hospital, Dunkerque Hospital, Lille Hospital, Saint Louis Hospital, Roubaix Hospital, and Versailles Hospital) were analyzed. Among them, 111 had reached CR using anthracycline-cytosine arabinoside induction treatment, followed by high-dose cytosine arabinoside consolidation courses (most of them had been enrolled in the French ALFA 9801 and 9802 cooperative group trials: 28 received an allogeneic bone marrow transplantation in first CR). The remaining 36 patients had failed to achieve CR using the same induction chemotherapy.

In all patients who achieved CR, bone marrow DNA was available both at diagnosis and after CR achievement, so that diagnostic and CR samples could both be analyzed, separating acquired mutations from polymorphisms, whereas in patients who failed to achieve CR, only diagnostic samples were studied, and no germline DNA was available. Blast percentage, after Ficoll density gradient centrifugation, was evaluated by May-Grunewald-Giemsa staining: all diagnosis samples contained more than 80% of blast cells complying with single nucleotide polymorphism (SNP) array analysis sensitivity criteria. Informed consent was obtained from all patients or their parents in accordance with the Declaration of Helsinki. This project was approved by the Institutional Review Board of Centre Hospitalier Regional Universitaire de Lille. Main characteristics of the patients studied are listed in supplemental Table 1 (available on the Blood Web site; see the Supplemental Materials link at the top of the online article). The median follow-up in patients who achieved CR was 2.5 years.

Analysis of TET2 sequence variations was performed as described previously4  by direct sequencing of polymerase chain reaction products at diagnosis and, in patients who reached CR, after CR achievement. Frameshift and nonsense variations were all scored as mutations, whereas missense variations were considered as mutations only when observed at diagnostic but absent in the paired sample obtained after CR achievement. Previously identified SNPs were not considered. TET2 anomalies were numbered according to GenBank reference FM992369. TET2 variations were also researched by direct sequencing in 36 DNA samples obtained at diagnosis from patients with refractory AML.

Paired diagnosis and CR genomic DNAs were analyzed using Affymetrix Genome-Wide Human SNP Array Version 6.0 (Affymetrix). Data were analyzed using Gene Chip Genotyping Console Version 3.0.2 (GTC, Affymetrix), DChip (www.biosun1.harvard.edu/complab/dchip), and Partek Genomics Suite (www.partek.com). Array data had been uploaded in the European Genome-Phenom archive at the European Bioinformatics Institute (www.EBI.ac.uk/ega). All microarray data have been deposited in the European Molecular Biology Laboratory-European Bioinformatics Institute under accession no. E-MTAB-278.

Comparisons were made by Fisher exact test for binary variables and Mann-Whitney test for continuous variables. In patients who achieved CR, disease-free survival (DFS) and overall survival (OS) were calculated according to the Kaplan-Meier method, respectively, from the date of CR achievement to the date of relapse or last follow-up, censoring patients alive in first CR and from the day of first diagnosis to the day of death or last follow-up censoring patients alive at last follow-up. Patients were not censored at the allogenic transplantation time. Comparisons regarding DFS and OS were performed with the log-rank test. A P value less than or equal to .05 was considered to indicate statistical significance.

Results and discussion

Analysis of TET2 mutations in the 111 patients who reached CR and correlation with other mutations and outcome

In the 111 patients who had achieved CR, comparison between diagnosis and germline samples allowed us to distinguish between SNP and acquired alterations. We found 11 different SNPs, including 8 SNPs already present in National Center for Biotechnology Information Single Nucleotide Polymorphism database (www.ncbi.nlm.nih.gov/projects/SNP) and the 3 other (L34F, Y867H, and Q1084P) previously reported by Abdel-Wahab et al.8  Twenty-five acquired mutations of the TET2 coding sequence were observed in 19 of the 111 (17%), suggesting alteration of the 2 TET2 alleles in 6 patients (Table 1). They included 24 different events: 6 frameshift mutations, 7 nonsense mutations, and 11 missense mutations. Six of the missense mutations were located in conserved regions and 5 outside of those regions. All those mutations were heterozygous and detected in the diagnostic sample but absent in the paired remission sample. Except for 1 missense mutation (S282F) detected in 2 patients, no recurrent TET2 mutation was observed (Table 1).

Table 1

Clinical and biologic characteristics of the 111 patients with de novo AML

Patient no.SexTET2 alterationsKaryotypeNPM1FLT3-ITDOS, moDFS, mo
Male p.Gln417X Not informative Mutation Negative Not done Not done 
Male p.Cys784X 46,XY Wild-type Positive 22+ 21+ 
Female p.Gly1313X 46,XX,del(15)(q1?4;q2?2) Mutation Positive 31+ 
Male p.Arg1516X 46,XY Mutation Negative 23+ 22 
Male p.Tyr2001X 46,XY Wild-type Negative 18+ 17+ 
Female p.[Ser1292Arg]+[Ala1355X] 46,XX,t(3;5)(q25;q34) Wild-type Negative 49+ 21+ 
7* Male p.[Pro101His]+[Gln690X] 46,XY Mutation Positive 22+ 21+ 
Male p.Leu1637fs 46,XY Wild-type Negative 14+ 
Female p.Asn1156fs 46,XX Mutation Negative 18 14 
10 Female p.[Val1199Ile]+[Glu1437fs] 46,XX,t(4;11)(q?;q1?),del(7)(q2?1) Mutation Negative 
11 Male p.G613fs 46,XY Mutation Negative 53+ 17 
12 Female p.[Cys1271Trp]+[Asp1830fs] 46,XX Mutation Negative 13 
13 Female p.[L226Rfs]+[Gly1370Val] Not informative Mutation Positive 
14 Male p.Gly1282Asp 46,XY Mutation Positive 10+ 9+ 
15* Male p.Thr492Ser 46,XY Mutation Negative 11 
16* Female p.Ser282Phe 46,XX Wild-type Negative 
17* Male p.Ser282Phe 47,XY,t(8;21)(q22;q22),+der(8)t(8;21) Wild-type Negative 6+ 4+ 
18* Female p.[Gln548Leu]+[Phe1368Tyr] 46,XX Wild-type Positive 11 
19* Male p.Gly773Val 46,XY,inv(16)(p13q22) Wild-type Negative 38+ 37+ 
20 Female Deletion Not informative Wild-type Negative 34+ 34+ 
Patient no.SexTET2 alterationsKaryotypeNPM1FLT3-ITDOS, moDFS, mo
Male p.Gln417X Not informative Mutation Negative Not done Not done 
Male p.Cys784X 46,XY Wild-type Positive 22+ 21+ 
Female p.Gly1313X 46,XX,del(15)(q1?4;q2?2) Mutation Positive 31+ 
Male p.Arg1516X 46,XY Mutation Negative 23+ 22 
Male p.Tyr2001X 46,XY Wild-type Negative 18+ 17+ 
Female p.[Ser1292Arg]+[Ala1355X] 46,XX,t(3;5)(q25;q34) Wild-type Negative 49+ 21+ 
7* Male p.[Pro101His]+[Gln690X] 46,XY Mutation Positive 22+ 21+ 
Male p.Leu1637fs 46,XY Wild-type Negative 14+ 
Female p.Asn1156fs 46,XX Mutation Negative 18 14 
10 Female p.[Val1199Ile]+[Glu1437fs] 46,XX,t(4;11)(q?;q1?),del(7)(q2?1) Mutation Negative 
11 Male p.G613fs 46,XY Mutation Negative 53+ 17 
12 Female p.[Cys1271Trp]+[Asp1830fs] 46,XX Mutation Negative 13 
13 Female p.[L226Rfs]+[Gly1370Val] Not informative Mutation Positive 
14 Male p.Gly1282Asp 46,XY Mutation Positive 10+ 9+ 
15* Male p.Thr492Ser 46,XY Mutation Negative 11 
16* Female p.Ser282Phe 46,XX Wild-type Negative 
17* Male p.Ser282Phe 47,XY,t(8;21)(q22;q22),+der(8)t(8;21) Wild-type Negative 6+ 4+ 
18* Female p.[Gln548Leu]+[Phe1368Tyr] 46,XX Wild-type Positive 11 
19* Male p.Gly773Val 46,XY,inv(16)(p13q22) Wild-type Negative 38+ 37+ 
20 Female Deletion Not informative Wild-type Negative 34+ 34+ 

AML indicates acute myeloid leukemia; OS, overall survival; and DFS, disease-free survival.

*

Missense mutations located outside the conserved domains.

Recurrent mutation.

As observed in previous studies in myeloid malignancies, acquired mutations were spread over all exons (Figure 1A). However, missense mutations targeting amino acids located outside the 2 conserved domains detected in our study were mainly found in the N-terminal part of the protein. Similar results were reported in AML by Abdel-Wahab et al.8  Interestingly, the missense mutations seem to be less frequent in MPNs and preferentially located in the C-terminal part of the protein, which could point to different pathogenetic mechanisms. In contrast to previous reports in MDS and MPN,9,10  no case of uniparental disomy (UPD) was observed in accordance with Gupta et al who reported the presence of UPD in 17% of AMLs11  but very rare UPD on chromosome 4. Only 1 patient (patient 20) presented a small deletion of 60 kb in the TET2 gene locus without TET2 mutation

Figure 1

TET2 alterations and clinical outcome. (A) Schematic representation of the TET2 protein. The 2 conserved domains are colored in grey. TET2 mutations reported in the study by Abdel-Wahab et al8  and found in our cohort of de novo AML are represented, respectively, with broken and straight lines (dark gray background represents frameshift and nonsense mutations; light gray background, missense mutations; and white background, polymorphisms). Kaplan-Meier estimates of disease-free survival (B) and overall survival (C) according to TET2 mutational status in cytogenetically normal AML patients (N = 54, 3-year DFS, 0% for patients with TET2 mutations, 95% CI, 1%-66% vs 57%, 95% CI, 41%-73%, P = .17; 3-year OS, 51% for patients with TET2 mutations, 95% CI, 19%-83% vs 54%, 95% CI, 37%-72%, P = .63).

Figure 1

TET2 alterations and clinical outcome. (A) Schematic representation of the TET2 protein. The 2 conserved domains are colored in grey. TET2 mutations reported in the study by Abdel-Wahab et al8  and found in our cohort of de novo AML are represented, respectively, with broken and straight lines (dark gray background represents frameshift and nonsense mutations; light gray background, missense mutations; and white background, polymorphisms). Kaplan-Meier estimates of disease-free survival (B) and overall survival (C) according to TET2 mutational status in cytogenetically normal AML patients (N = 54, 3-year DFS, 0% for patients with TET2 mutations, 95% CI, 1%-66% vs 57%, 95% CI, 41%-73%, P = .17; 3-year OS, 51% for patients with TET2 mutations, 95% CI, 19%-83% vs 54%, 95% CI, 37%-72%, P = .63).

No significant difference was observed between patients with or without TET2 alterations for most pretreatment characteristics, including gender, age, hemoglobin level, white blood cell count, platelet count, French-American-British subtype distribution, and cytogenetics according to Medical Research Council classification (supplemental Table 1). No significant association was observed between TET2 mutations and FLT3 or CEBPA alterations. On the other hand, TET2 alterations were significantly associated with NPM1 mutations (P = .032; supplemental Table 1). This association between TET2 mutations and NPM1 mutations had not been found by Abdel-Wahab et al8  in 91 AML patients. This difference was possibly because of the AML population analyzed. Our study included only de novo AML patients who had all achieved CR to clearly distinguish TET2 mutations from potential polymorphisms, whereas 50% of the patients in the cohort reported by Abdel-Wahab et al8  had a secondary AML.

A negative impact of TET2 alterations was reported in MPN but was not in MDS. Contrary to Abdel-Wahab et al8  who found an overall negative prognostic impact of TET2 alterations in de novo AML, there was no difference in DFS or OS between patients with and without TET2 alteration in our entire cohort of patients with de novo AML who achieved CR.

We also analyzed the influence of TET2 mutations in patients with normal karyotype (NK)–AML. No significant difference was observed in OS or DFS in NK-AML between patients with and without TET2 mutations (3-year DFS, 0%, 95% confidence interval [CI], 1%-66% vs 57%, 95% CI, 41%-73%, respectively, P = .17; 3-year OS, 51%, 95% CI, 19%-83% vs 54%, 95% CI, 37%-72%, respectively P = .63; Figure 1B-C).

Analysis of TET2 mutations in 36 patients who did not achieve CR

Of the 36 AML patients who failed to achieve CR with intensive chemotherapy, we found 16 mutations in 10 patients (27%): 5 nonsense, 1 frameshift, and 10 missense mutations. Five of these missense mutations were located in the conserved domain and 5 outside. Because of the absence of germline DNA in these last cases, we could not verify the exact frequency of TET2 mutations in refractory de novo AML. However, this maximum incidence of 27% mutations in patients who failed to achieve CR was not different from that observed in patients who achieved CR (P = .2).

In conclusion, in de novo AML, point mutations of TET2 appear to be as frequent as in MDSs or MPNs, whereas TET2 deletion or UPD is very rare. In our study, the incidence of TET2 mutations appeared similar in patients who achieved or did not achieve CR with intensive chemotherapy. In patients who achieved CR, where TET2 mutations could be clearly distinguished from polymorphisms, TET2 mutations were associated with NPM1 mutations. In our study, the presence of TET2 mutations did not affect OS and DFS in the overall population and in NK-AML patients. However, the patient number remained relatively low, and those data will have to be confirmed prospectively on a larger number of patients.

The online version of this article contains a data supplement.

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 USC section 1734.

Acknowledgments

The authors thank all patients, families, and clinicians for their participation.

This work was supported by the Association Laurette Fugain, the Fondation de France (Leukemia Committee), and North-West Canceropole (Onco-Hematology axis), France.

Authorship

Contribution: O.N. analyzed data and wrote the paper; O.K. and M.F. provided samples and data and contributed to writing the paper; M.C. and C.R. analyzed data and wrote the paper; N.B. performed statistical analysis and wrote the paper; A.R., C.R.-L., and J.-M.C. performed the research and contributed to writing the paper; N.P. and S.G. performed the research; S.Q. and J.S. performed the research, analyzed data, and contributed to writing the paper; H.D., F.D., B.Q., P.F., W.V., and O.A.B. contributed to writing the paper; and C.P. conceptualized the idea, designed research, analyzed data, and wrote the paper.

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

Correspondence: Claude Preudhomme, Centre Hospitalier Regional Universitaire de Lille, Laboratoire d'hématologie, Bd du Professeur Leclercq, 59037 Lille Cedex, France; e-mail: cpreudhomme@chru-lille.fr.

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