Background The myelodysplastic syndromes and myeloproliferativedisorders are associated with deregulated production of myeloidcells. The mechanisms underlying these disorders are not welldefined.
Methods We conducted a combination of molecular, cytogenetic,comparative-genomic-hybridization, and single-nucleotide–polymorphismanalyses to identify a candidate tumor-suppressor gene commonto patients with myelodysplastic syndromes, myeloproliferativedisorders, and acute myeloid leukemia (AML). The coding sequenceof this gene, TET2, was determined in 320 patients. We analyzedthe consequences of deletions or mutations in TET2 with theuse of in vitro clonal assays and transplantation of human tumorcells into mice.
Results We initially identified deletions or mutations in TET2in three patients with myelodysplastic syndromes, in three offive patients with myeloproliferative disorders, in two patientswith primary AML, and in one patient with secondary AML. Weselected the six patients with myelodysplastic syndromes orAML because they carried acquired rearrangements on chromosome4q24; we selected the five patients with myeloproliferativedisorders because they carried a dominant clone in hematopoieticprogenitor cells that was positive for the V617F mutation inthe Janus kinase 2 (JAK2) gene. TET2 defects were observed in15 of 81 patients with myelodysplastic syndromes (19%), in 24of 198 patients with myeloproliferative disorders (12%) (withor without the JAK2 V617F mutation), in 5 of 21 patients withsecondary AML (24%), and in 2 of 9 patients with chronic myelomonocyticleukemia (22%). TET2 defects were present in hematopoietic stemcells and preceded the JAK2 V617F mutation in the five samplesfrom patients with myeloproliferative disorders that we analyzed.
Conclusions Somatic mutations in TET2 occur in about 15% ofpatients with various myeloid cancers.
The myelodysplastic syndromes and myeloproliferative disordersare clonal disorders that arise in hematopoietic stem cells,1whereas acute myeloid leukemia (AML) can arise from stem cellsor more mature progenitor cells.2 The main feature of the heterogeneoussubtypes of the myelodysplastic syndromes is ineffective hematopoiesisthat affects one or several lineages.3 In about a third of patients,the disorder progresses to AML. Some of the molecular abnormalitiesthat are associated with specific subtypes of myelodysplasticsyndromes4 or the transformation of myelodysplastic syndromesto AML5 have been identified, but the basis of these relatedsyndromes remains poorly understood.6
The myeloproliferative disorders, especially polycythemia veraand essential thrombocythemia, are frequently associated witha somatic mutation, V617F, in the Janus kinase 2 (JAK2) gene,which results in a constitutively active Janus protein kinase.7,8,9In mouse models, the presence of JAK2 V617F alone can causemyeloproliferative disorders but does not give hematopoieticstem cells a proliferative advantage.10,11,12 Recent evidenceindicates that JAK2 V617F does not account for all the pathophysiologicaleffects of the myeloproliferative disorders.13,14,15,16 Forexample, JAK2 V617F was absent in some patients in whom a JAK2V617F–positive myeloproliferative disorder progressedto AML.17
We hypothesized that an early genetic event in a hematopoieticstem cell could be common to the myelodysplastic syndromes,myeloproliferative disorders, and AML. By using various geneticapproaches, we identified a tumor-suppressor gene, TET2, thatis deleted or mutated in patients with various myeloid cancers.
Methods
Patients
Table 1 in the Supplementary Appendix (available with the fulltext of this article at NEJM.org) lists the clinical featuresof the patients, whose conditions were diagnosed with the useof standard international criteria.18,19,20 Samples of bonemarrow or blood were obtained from 320 patients (84 with myelodysplasticsyndromes, 203 with myeloproliferative disorders, 2 with primaryAML, 22 with secondary AML, and 9 with chronic myelomonocyticleukemia) after the patients provided written informed consentand the local research ethics committees (at Hôtel-Dieuand Cochin hospitals) gave approval.
Genetic Analyses
A detailed description of the materials and methods used inthese experiments is provided in the Supplementary Appendix.Briefly, bone marrow mononuclear cells, lymphocytes, or granulocyteswere isolated and stored in liquid nitrogen. Cellular experimentswere performed as described previously.10,11 Purified CD34+cells (1x105 to 10x105 cells) were injected intravenously intomice that had undergone sublethal irradiation.14
Polymerase-chain-reaction (PCR) assays and direct sequencingreactions were performed with primers that are listed in Table2 of the Supplementary Appendix. JAK2 mutational status wasdetermined as described previously.10
In comparative-genomic-hybridization and single-nucleotide–polymorphism(SNP) analyses, paired DNA samples from malignant and nonmalignantcells were analyzed with the use of 244K oligonucleotide arrays(Agilent Technologies) and Affymetrix 250K SNP (Affymetrix)arrays.
No commercial entity was involved in the conduct of the study,the analysis or storage of the data, or the preparation of themanuscript. The authors vouch for the completeness and accuracyof the data and the analysis.
Results
Deleted Region on Chromosome 4q24
We previously reported on six patients with myelodysplasticsyndromes or AML who had acquired rearrangements on chromosome4q24.21 In two of these patients, the chromosomal abnormalitywas found in both myeloid and lymphoid cells, indicating theinvolvement of a lymphomyeloid progenitor. In this study, wediscovered a deleted region on chromosome 4q24 containing asingle gene, TET2, in cells from the three patients with AML,by means of fluorescence in situ hybridization (Figure 1A).Bone marrow cells from the three patients with myelodysplasticsyndromes (Patients MDS01, MDS02, and MDS03) had a similar deletion.Taken together, five patients had a heterozygous loss, and onehad a deletion of both copies.
Figure 1. Structural DNA Rearrangements and Mutations in TET2 on Chromosome 4q24 in Subgroups of Patients.
In Panel A, a minimally deleted region on chromosome 4q24 is seen in subgroups of patients with myelodysplastic syndromes and acute myeloid leukemia (AML). This region was defined with the use of RP11-1061M121, RP11-356L5, and RP11-16G16 bacterial artificial chromosomes (represented by horizontal lines with double arrows) in fluorescence in situ hybridization. The region encompasses a unique gene, TET2. In Panel B, acquired abnormalities on chromosome 4q24 are shown for three of five patients with myeloproliferative disorders who carried the JAK2 V617F mutation. The abnormalities were detected with the use of high-resolution single-nucleotide-polymorphism (SNP) and comparative-genomic-hybridization (CGH) arrays. The blue bars indicate the regions affected by loss of heterozygosity (LOH). Patient MPD05 had a smaller LOH because of an acquired 325-kb deletion. TET2 was the only open reading frame observed in this region. In Panel C, nucleotide sequences of DNA from bone marrow (BM) samples from patients with AML and myelodysplastic syndromes are compared with wild-type and nontumor samples from the same patients (when available). The presence of wild-type nucleotide sequences in the samples was attributed to residual normal hematopoietic cells. Matched samples consisted of bone marrow cells obtained after transplantation, during a period of clinical health (Patient nAML1); B type lymphoid cells transformed by Epstein–Barr virus (Patient nAML2); and peripheral-blood mononuclear cells stimulated by a phorbol ester (Patient MDS03). In Panel D, wild-type TET2 sequences are compared with TET2 sequences in tumor cells and matched mononuclear cells from Patients MPD01 and MPD04. The dashed vertical lines frame the nine-nucleotide deletion shown for Patient MPD04. The arrow indicates the presence of a small, residual peak of wild-type allele in mononuclear cells from Patient MPD01. In Panels C and D, asterisks indicate nucleotide changes. In Panel E, alignment of the conserved region spanning amino acids 1842 through 1921 of human TET2 protein is shown. Invariant amino acids (identical in all species) are marked by a star, with conserved amino acids indicated by a double dot under the alignment. An arrow points from the amino acids targeted by mutational events in myeloid cancers to the resulting amino acid. In Panel F, the TET protein family is shown with the location of acquired missense mutations. The arrowhead indicates the fusion point of mixed-lineage leukemia (MLL) protein on TET1. The conserved regions that are shared by all TET family proteins appear as shaded boxes. DM denotes Drosophila melanogaster, Fugu Takifugu rubripes, Hs Homo sapiens, and Mm Mus musculus.
We discovered involvement of the same region on chromosome 4q24using a different method in patients with myeloproliferativedisorders. Two subgroups of patients with myeloproliferativedisorders and different profiles of JAK2 V617F clonal expansionhave been described previously.10 The first subgroup had a lowproportion of CD34+ cells bearing the JAK2 V617F mutation anda high proportion of granulocytes with the mutation, whereasthe second subgroup had a high proportion of CD34+ cells bearingJAK2 V617F. We analyzed samples from five patients from thesecond subgroup (Patients MPD01 through MPD05) using comparativegenomic hybridization and SNP arrays to compare DNA from cellsthat were presumed to be affected (granulocytes) with DNA fromcells that were presumed to be normal (mononuclear cells orlymphocytes) (Figure 1B). In this subgroup, one patient withprimary myelofibrosis (Patient MPD01) and one with polycythemiavera (Patient MPD04) had loss of heterozygosity (LOH) withoutcopy-number modification in chromosome 4, ranging from q22 tothe end of the long arm of the chromosome, which was not foundin presumably normal cells.22 A third patient (Patient MPD05)had an acquired 325-kb deletion in the region on chromosome4q24 containing TET2 as a single candidate gene. This regionwas normal in Patients MPD02 and MPD03.
Acquired Mutations in TET2
The TET2 gene comprises 11 exons spread over 150 kb and is widelyexpressed.23,24 In humans, the predicted TET2 protein belongsto a three-member family containing two highly conserved regions(Figure 1, and Fig. 1 and 2 in the Supplementary Appendix).TET2 has not previously been implicated in human cancers, butTET1 is fused to the mixed-lineage leukemia (MLL) gene in thechromosomal translocation t(10;11)(p12;q23), which occurs rarelyin patients with acute leukemia.23,24 The function of the TETproteins is unknown, but the presence of the conserved carboxy-terminaldomain in the MLL–TET1 fusion protein suggests its importancein cellular transformation.
The loss of both copies of TET2 in Patient MDS01 and the lossof one copy in eight other patients suggested that TET2 is atumor-suppressor gene. We therefore searched for nucleotidesequence variation in the coding region of the remaining copyof TET2 in the eight patients with either a heterozygous deletionor a copy-neutral loss of heterozygosity. A comparison of theseeight sequences with human sequences in the databases identifiednucleotide changes in the remaining TET2 genes in six patients.One change caused a frame shift, two generated stop codons,one was an in-frame deletion, and two resulted in amino acidsubstitutions of extremely conserved residues (Table 1 and Figure 1C, 1D, and 1E,and Fig. 2 in the Supplementary Appendix). No variations werefound in the coding sequence of TET2 in Patients sAML1 and MPD05.TET2 coding sequences were wild type in Patients MPD02 and MPD03,who retained both copies of TET2.
Table 1. Clinical and Molecular Data for Patients with TET2 Mutations.
To investigate whether these variations were somatically acquiredin myeloid cells, we studied DNA obtained from lymphocytes fromfour patients. In analyses of the coding sequences of TET2 inthese DNA samples, we found no mutations in samples from PatientsMDS03, MPD04, and nAML2 (Figure 1C and 1D) and no deletion ina sample from Patient MPD05 (data not shown). In addition, wild-typesequence was detected in mononuclear cells from Patient MPD01(Figure 1D). In Patient nAML1, the wild-type sequence predominated,as compared with the mutated sequence, in a sample obtainedwhen the patient was in remission. Together, these data indicatethat the TET2 defects were somatically acquired.
To determine the prevalence of TET2 mutations in malignant myeloiddisorders, we analyzed the TET2 coding sequence in 309 availablesamples from 81 patients with various subtypes of myelodysplasticsyndromes, 21 patients with secondary AML, 9 patients with chronicmyelomonocytic leukemia, 181 patients with JAK2 V617F–positivemyeloproliferative disorders, and 17 patients with JAK2 V617F–negativemyeloproliferative disorders. Sequence variants of TET2, resultingin conserved amino acid substitution, stop codons, or frameshifts, were detected in 45 of 309 patients (15%) (Table 1 andFigure 1E and 1F, and Fig. 3 in the Supplementary Appendix).In Patient MDS07, amplification of the 5' region of exon 11on PCR failed, suggesting an uncharacterized structural genomicrearrangement in this region. TET2 defects were found in 22of 111 patients with various types of myelodysplastic syndromes,in 21 of 181 patients with myeloproliferative disorders associatedwith JAK2 V617F, in 1 of 6 patients with disease associatedwith a W515L/K mutation in the myeloproliferative leukemia virusoncogene (MPL), and in 2 of 11 patients who did not carry eitheran MPL W515L/K or JAK2 V617F mutation.
Overall, we found TET2 defects in diverse myeloid disorders,with a prevalence of 15% (46 of 309 patients). Because mostof such mutations are predicted to truncate the protein, theycould result in partial or total loss of function of the TET2protein. In 25 of 55 patients with TET2 defects, two differentmutations were detected, which probably targeted both copiesof TET2 (Table 1). This inference was confirmed in Patient MDS42by subcloning and analysis of individual DNA molecules (Fig.4 in the Supplementary Appendix). A single defect was observedin 30 of 55 samples, suggesting that haploinsufficiency of TET2has a role in these cancers.
Early Progenitor Cells with TET2 Mutations
We sought TET2 defects in CD34+ cells, which include hematopoieticstem cells and hematopoietic progenitors, from four patientswith myelodysplastic syndromes (Patients MDS03, MDS09, MDS28,and MDS35). In all four patients, a mutated TET2 sequence wasdetected (Figure 2, and Fig. 5 and 6 in the Supplementary Appendix).In one patient (Patient MDS03), CD34+ cells were analyzed inthe first phase of refractory anemia with excess blasts (RAEB1,5 to 9% blasts) and in the second phase (RAEB2, 10 to 19% blasts).The wild-type sequence was detected in the RAEB1 phase, butonly a mutated sequence was found in the RAEB2 phase, suggestingthe presence of expanded clones of progenitors bearing the mutationduring disease progression (Figure 2A).
Figure 2.TET2 Mutations in Fractionated CD34+ Cells from Patients with Myelodysplastic Syndromes.
In samples from patients with myelodysplastic syndromes, mutated TET2 sequence is observed in immature CD34+ cells and is associated with in vivo expansion of the mutated clone. In Panel A, sequencing chromatograms of sorted CD34+ cells from Patient MDS03 are shown in samples obtained during phases of refractory anemia with excess blasts of 5 to 9% (RAEB1) and with excess blasts of 10 to 19% (RAEB2). Nucleotide sequences that were observed in an unsorted bone marrow sample from the patients and in a wild-type control sample are shown for comparison purposes. Asterisks indicate the mutated nucleotide. In Panel B, polymerase-chain-reaction (PCR) assay followed by restriction-fragment–length polymorphism (RFLP) analysis shows DNA that was isolated from sorted CD34+CD38– and CD34+CD38+ cells from Patient MDS03 at the RAEB1 phase. Amplified fragments were digested with the use of the Taq endonuclease (Tas1) and underwent size fractionation with the use of agarose-gel electrophoresis. The proportion of TET2 that was mutated was evaluated by measuring the intensity of the mutated or wild-type signal relative to that of the signal generated by both alleles (wild type plus mutated), as compared with a control sample. MW denotes molecular weight (see Fig. 5 in the Supplementary Appendix for details). In Panel C, the results of PCR–RFLP analysis of TET2 that was directly performed from sorted CD34+CD38– and CD34+CD38+ cells from Patient MDS09 with the use of the Bacillus stearothermophilus endonuclease (BseL1) are shown. In Panel D, PCR-RFLP genotyping analysis with the use of BseL1 endonuclease shows sorted CD34+CD38– and CD34+CD38+ clones from Patient MDS09; the clones were grown at one cell per well. The proportion of mutated clones differs significantly between the two cell populations (P=0.01 by Fisher's exact test). The histograms represent the fraction of clones with wild-type or mutated TET2. The absence of wild-type fragment in CD34+CD38+ clones is indicated by asterisks.
We fractionated the CD34+ cells from these four patients intoCD34+CD38– populations (corresponding to stem cells andmultipotent progenitors) and into CD34+CD38+ populations (correspondingto more mature progenitors). In Patient MDS03, mutations inTET2 were found in 16% of cells in the CD34+CD38– populationand in 54% of cells in the CD34+CD38+ population (Figure 2B).The corresponding proportions for Patient MDS09 were 26% and48%, respectively (Figure 2C). Further analysis was performedby seeding single hematopoietic progenitors from Patient MDS09on a stromal-cell layer or in a methylcellulose medium. A TET2mutation was identified in 8 of 32 clones (25%) derived fromCD34+CD38– cells and in 18 of 30 clones (60%) derivedfrom CD34+CD38+ cells (Figure 2D). Wild-type TET2 was not alwaysdetected in clones bearing a mutated TET2, suggesting its lossin a minority of the cells.
In Patients MDS28 and MDS35, the difference in the occurrenceof TET2 mutations in CD34+CD38– and CD34+CD38+ cells wasevaluated from the respective peak intensities and confirmedby subcloning and sequencing of bacterial clones (Fig. 6 inthe Supplementary Appendix). In Patient MDS28, the mutated TET2sequence was barely detectable in CD34+CD38– cells, whereasit represented one third of the TET2 sequences from the CD34+CD38+cells. The data for these four patients with myelodysplasticsyndromes indicate that TET2 mutations were present in a smallnumber of immature CD34+CD38– cells and increased in thepopulation of mature progenitors.
Timing of TET2 and JAK2 V617F Mutations
We analyzed hematopoietic progenitors from five patients whohad myeloproliferative disorders with mutations in TET2 andJAK2. Immature CD34+CD38– cells were seeded at one cellper well in culture conditions that supported lymphomyeloiddifferentiation, whereas CD34+CD38+ cells were grown in erythroid–granulocyticmethylcellulose assays. In all cells that were tested, bothTET2 and JAK2 defects were present in clones containing lymphoidand myeloid cells together (Figure 3A and 3B). Overall, aftertesting of 232 clones from immature progenitors, the JAK2 V617Fmutation was not observed in the absence of the TET2 mutation.These results demonstrate that in patients with myeloproliferativedisorders (as in patients with myelodysplastic syndromes), theTET2 mutation is present in immature progenitors. In addition,the findings indicate that in the five patients with myeloproliferativedisorders, TET2 defects preceded JAK2 mutations during the evolutionof the disease. Of course, this finding does not formally ruleout an alternative sequence of mutations in other patients withmyeloproliferative disorders.
Figure 3.TET2 and JAK2 Defects in Clones Containing Lymphoid and Myeloid Cells.
The TET2 mutation is a primary event in myeloproliferative disorders and is associated with in vivo expansion of the mutated clone. Sequence analyses of TET2 and JAK2 mutations in patients with myeloproliferative disorders were performed in CD34+CD38– cells grown in culture derived from clonal B cells, myeloid cells, and natural killer cells (BMNK) and in CD34+CD38+ cells in methylcellulose culture for differentiation of burst-forming unit–erythroid (BFU-E) or colony-forming unit–granulocyte–macrophage (CFU-GM) activity. In Panel A, the sequencing chromatograms show representative results from three clones per patient. Asterisks indicate the positions of nucleotide changes. For Patient MPD05, sequence analysis was performed with the use of a single-nucleotide polymorphism to detect the loss of heterozygosity (LOH) resulting from the deletion in TET2. In Panel B, the histograms show the fractions of immature progenitors (BMNK) and mature progenitors (BFU-E and CFU-GM) carrying defects in both TET2 and JAK2, clones with mutated TET2, and nonmutated clones. Panel C shows the fractions of immature progenitors (BMNK) and mature myeloid progenitors (BFU-E and CFU-GM) carrying TET2 defects in JAK2 wild-type progenitor cells from Patients MPD05 and MPD20. In Panels B and C, the numbers of analyzed clones are shown on the bars.
In Patients MPD01, MPD04, and MPD35, almost all the coloniesderived from immature and mature progenitors carried a TET2mutation, indicating the dominance of the TET2-mutated cloneat early stages of hematopoiesis (Figure 3B). In Patients MPD05and MPD20, most immature progenitor cells were wild type, whereasmore mature progenitor cells had a mutated TET2. Within JAK2wild-type progenitors from these two patients, the proportionof clones with TET2 defects in the immature population was 2of 37 cells in Patient MPD05 and none of 34 cells in PatientMPD20; in the mature population, the proportion was 10 of 23cells in Patient MPD05 and 9 of 54 cells in Patient MPD20 (Figure 3C).The proportions of mutated clones differed significantly betweenthe two populations (P<0.001 for Patient MPD05 and P=0.01for Patient MPD20, by Fisher's exact test), indicating thatthe increase in cells with TET2 mutations does not require thepresence of the JAK2 V617F mutation.
Studies of TET2 Mutation in Mice
To add to the evidence that the TET2 mutation occurs initiallyin hematopoietic stem cells, we grafted CD34+ cells from patientswith polycythemia vera or myelofibrosis in nonobese diabeticmice with severe combined immunodeficiency (NOD–SCID)and depletion of natural killer cells, independently of theproportion of JAK2 V617F mutations.14 Purified CD34+ cells isolatedfrom patients with myeloproliferative disorders and the JAK2V617F mutation either with the TET2 mutation (Patients MPD01and MPD04) or without the TET2 mutation (Patients MPD09, MPD11,and MPD27) were injected into NOD–SCID mice (Fig. 7 inthe Supplementary Appendix).
The percentage of human cells from the three samples withouta TET2 mutation decreased with time, whereas the percentageof human cells from the two TET2-mutated samples increased withtime (Figure 4A). Furthermore, human hematopoietic reconstitutionfrom TET2-mutated samples was skewed toward myeloid progenitors,at the expense of lymphoid progenitors, as judged from CD33and CD19 antigen expression, unlike the predominantly lymphoidreconstitution observed with wild-type hematopoietic stem cells25(Figure 4B).
Figure 4. Repopulating Capacities of Stem Cells from Patients with Myeloproliferative Disorders, with and without TET2 Mutations, Transplanted into Mice.
Hematopoietic stem cells with the JAK2 V617F mutation from patients with myeloproliferative disorders with TET2 defects show enhanced repopulating capacities when the cells are grafted into nonobese diabetic mice with severe combined immunodeficiency (NOD–SCID). CD34+ cells from patients with myeloproliferative disorders were injected intravenously into NOD–SCID mice that had undergone sublethal irradiation. The percentage of human CD45+ cells in mouse bone marrow was monitored at 3, 6, 12, and 15 weeks after transplantation. As shown in Panel A, Patients MPD01 and MPD04 had TET2 defects, whereas Patients MPD09, MPD11, and MPD27 had normal TET2 and served as controls. Panel B shows the results of flow-cytometric analysis of human cells present in the bone marrow of NOD–SCID mice 15 weeks after transplantation with 3x105 CD34+ cells from Patients MPD04 and MPD09. The percentages of human CD45+ myeloid and lymphoid cells were determined with the use of anti–CD45-PC7, anti–CD33-APC, and anti–CD19-PE antibodies. Panel C shows the results of clonogenic methylcellulose assays performed on CD34+ cells before transplantation. The number of colony-forming units (D0 CFU) is shown. Fifteen weeks after transplantation, human cells were sorted from mouse bone marrow and analyzed in methylcellulose assays (W15 CFU) and long-term-culture–initiating cell (W15 LTC-IC) assays. JAK2 and TET2 sequence analyses were performed on progenitor-derived colonies. Bar graphs show the fraction of individual clones with both JAK2 and TET2 defects or with wild-type JAK2 and a TET2 defect. No clone with both wild-type JAK2 and wild-type TET2 was observed. Sequence traces show the presence of TET2 defects in pooled W15 CFU cells. The number of clones in each category is indicated on the bars. Asterisks indicate the positions of nucleotide changes.
Mouse bone marrow samples that contained human cells 15 weeksafter transplantation were tested in vitro for their contentof mature progenitors and long-term-culture–initiatingcells, a surrogate assay for hematopoietic stem cells, and wereanalyzed for the presence of TET2 and JAK2 mutations. TET2 defectswere found in all human long-term-culture–initiating cellsand progenitors in the mice (Figure 4C).
Because only hematopoietic stem cells can sustain long-termhematopoietic reconstitution in NOD–SCID mice, these resultsare consistent with the occurrence of a TET2 mutation in a truehematopoietic stem cell. The proportion of progenitor cellscarrying only the TET2 mutation increased at the expense ofcells carrying both TET2 and JAK2 V617F mutations from baselineto 15 weeks after transplantation, which suggests that cellswith a mutated TET2 can proliferate in vivo independently ofthe JAK2 V617F mutation (Figure 4C).
Discussion
We report that deletions or mutations in TET2 are early eventsin some patients with myelodysplastic syndromes, myeloproliferativedisorders, or secondary AML. The frequency of TET2 mutationsin unselected patients was 19% (15 of 81 patients) with myelodysplasticsyndromes, 12% (24 of 198 patients) with myeloproliferativedisorders, 24% (5 of 21 patients) with secondary AML, and 22%(2 of 9 patients) with chronic myelomonocytic leukemia. Thedetection of acquired genetic defects targeting both TET2 copiesin 24 of 55 patients indicates that TET2 is a bona fide tumor-suppressorgene in some myeloid cancers. In samples from patients withmyeloproliferative disorders who had both TET2 and JAK2 mutations,TET2 mutations occurred first in the course of the disease.TET2 mutations were observed in various subtypes of myelodysplasticsyndromes, in keeping with reported loss of heterozygosity andinterstitial deletions on chromosome 4q24 in these subtypes.26,27Similar events affecting chromosome 4q24 have been reportedin patients with myeloproliferative disorders28 and those withrelapsed AML.29
TET2 mutations were observed in primitive CD34+CD38– cells,and xenograft transplantation demonstrated the presence of mutantTET2 in hematopoietic stem cells in samples from patients withmyeloproliferative disorders. In both myelodysplastic syndromesand myeloproliferative disorders, the TET2 mutation appearedto be associated with the amplification of the mutated cloneat the early stages of hematopoietic differentiation.
TET2 defects were observed in both myelodysplastic syndromesand myeloproliferative disorders, which are two distinct myeloiddiseases. The presence of clinical and biologic phenotypes characteristicof these diseases probably requires at least one additionalcontributing event, as exemplified by our finding that the JAK2V617F or MPL W515L mutations, which are responsible for somebiologic features of myeloproliferative disorders, were associatedwith TET2 mutations.
Our results support a role for wild-type TET2 in the controlof balance between survival, growth, and differentiation innormal hematopoiesis. Elucidation of the biochemical pathwayunderlying the function of TET2 will require additional workand may help to define novel mechanisms of renewal and differentiationof hematopoietic stem cells.
Supported by grants from INSERM, La Ligue Nationale contre leCancer, La Fondation de France, Institut National du Cancer,Canceropole Ile de France (Cellules Souches Cancéreusesand Apoptosis 2007), L'association Française contre lesMyopathies, GIS-Institut des Maladies Rares, la Direction Régionalede la Recherche Clinique AP–HP, Ministère de laRecherche et de la Technologie (to Dr. Dupont), and the EuropeanHematology Association (to Dr. Delhommeau).
No potential conflict of interest relevant to this article wasreported.
We thank J. Prchal, S. Constantinescu, T. Mercher, F. Wendling,and E. Solary for their critical reading of the manuscript;D. Bouscary and I. Callebaut for their help and support; O.Beyne-Rauzy, C. Delacroix, C. Preudhomme, B. Quesnel, and E.Solary for the recruitment of patients with myelodysplasticsyndromes; P.J. Weiller, L. Roy, J.-A. Ribeil, and E. Lippertfor the recruitment of patients with myeloproliferative disorders;and I. Teyssandier, H. Bouamar, and C. Pierre-Eugene for thepreparation of samples.
Source Information
From INSERM U790, Institut Gustave Roussy (F.D., S.D., C.J., A.M., J.-P.L.C., Y.L., I.P., N.C., P.D., W.V.) and Université Paris XI (F.D., S.D., A.M., J.-.P.L.C., N.C., P.D., W.V.), Villejuif; Hôpital St. Antoine, Paris (F.D., C.M., N.C.); Université Paris VI Pierre et Marie Curie, Paris (F.D., N.C.); INSERM E0210, Hôpital Necker, Paris (V.D.V., S.T., F.R., S.P.R., O.A.B.); Université René Descartes, Paris (V.D.V., O.K., F.J.D., C.L., S.P.R., F.V., M.F., O.A.B.); and Hopital Cochin, Paris; INSERM Unité 567, Centre National de la Recherche Scientifique UMR8104, Institut Cochin, Hôpital Cochin, Paris (O.K., F.J.D., C.L., M.F.); Assistance Publique–Hôpitaux de Paris, Laboratoire d'Hématologie (O.K., C.L., M.F., O.A.B.) and Unité Fonctionnelle d'Hématologie (F.J.D.), Hôpital Cochin, Paris; INSERM Unité 876, Université Bordeaux 2, Bordeaux (C.J.); and Université Paris Diderot, INSERM Unité MR944, Hôpital Saint-Louis, Paris (A.A., J.S.); — all in France. Drs. Delhommeau, Dupont, Della Valle, and James contributed equally to this article.
Address reprint requests to Dr. Bernard at INSERM E0210, Hôpital Necker, 149 rue de Vaugirard, Paris 75015, or at olivier.bernard{at}inserm.fr.
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