Analyses of TET2 mutations in post-myeloproliferative neoplasm acute myeloid leukemias

Classic myeloproliferative neoplasms (MPN) include essential thrombocythemia, polycythemia vera and primitive myelofibrosis.1 These diseases are characterized by an over production of myeloid cells and may transform into acute myeloblastic leukemia (AML). An acquired point mutation that leads to the expression of a constitutively active form of the Janus kinase 2 (JAK2) tyrosine kinase, JAK2V617F, is observed in almost all polycythemia vera cases, and in 50–60% of essential thrombocythemia and primitive myelofibrosis. In experimental models, JAK2V617F recapitulates most of the features of MPN, establishing its essential role in the clinical phenotype of the diseases. It is, however, unclear how a single mutation gives rise to three related but distinct disorders. Moreover, when AML develops in a patient with a JAK2V617F-positive myeloproliferative neoplasm, the fully transformed clone is often JAK2V617F negative.2 These and other observations suggested that, in some patients, another unknown mutation has preceded the JAK2V617F mutation in the development of MPN.3 Recent publications have reported acquired mutations of the TET2 gene in various hematopoietic malignancies.4 The most frequent type of variation affecting TET2 interrupts the open reading frame of the gene. In MPN, TET2 mutations were observed in both JAK2V617F-positive and -negative patients and in both JAK2V617F-positive and -negative clones in individual patients.5

To investigate whether TET2 mutations could represent a preJAK2V617F event predisposing to the development of AML, we recruited a series of secondary AMLs (sAMLs) derived from MPN patients on behalf of the Groupe Francophone de Cytogénétique Hématologique. All patients gave informed consent according to the Helsinki declaration. DNA was available for 19 samples, and it was analyzed for the presence of TET2 and JAK2V617F mutations as described.5 JAK2V617F mutations were observed in the majority of sAMLs (10 of 19; Table 1). An investigation of the coding sequence of the TET2 gene identified seven variations in six samples. These were frame shift (n=3), nonsense (n=2) and missense (n=2) variations. The results are summarized Table 1 and Figure 1a. It is not known whether the Arg1366His variation is acquired, but it affects a conserved amino acid in an evolutionarily conserved region of the protein (data not shown), and is therefore expected to have functional consequences.5, 6 An Ile1873Thr change has already been reported as acquired. It is noteworthy that all possible combinations between the wild-type and mutated form of the JAK2 and TET2 genes were observed in sAML samples. These results show that sAML may arise from MPNs in the presence or absence of TET2 mutations.

Table 1 Clinical, cytogenetic and genetic data
Figure 1

Molecular and cytogenetic analyses of patients. (a) Consequences of TET2 variations in secondary AML (sAML) patients. (b) Traces of TET2 sequences obtained from the cDNA of patient 4 at chronic and blastic phases. The same variation is observed at both phases and is indicated by an arrow. Wild-type sequences are shown for comparison purposes. (c) Fluorescence in situ hybridization (FISH) analyses of the t(10;16)(q22;q22) translocation observed in blast metaphasic chromosome using the Vysis CBFB break apart probe (Abbott, Rungis, France). The red (centromeric) signal remains on the derivative chromosome 16 and the green (telomeric) signal is translocated to the derivative chromosome 10. The probes closely bracket the CBFB gene.

We were particularly interested in patient 4, for whom chronic phase cDNA was available. Molecular analyses of this patient have already been reported as patient BO-04.7 She suffered from essential thrombocythemia and the JAK2V617F mutation was present only in a fraction of neutrophils. At the transformation phase, the blasts were devoid of the JAK2V617F mutation,7 but a C → T variation was observed resulting in the creation of a stop codon (p.Gln803X, Figures 1a and b). The identified TET2 mutation was searched in the cDNA obtained from chronic phase peripheral blood cells. As shown in Figure 1b, the TET2 mutation was already present in the chronic phase sample, and the ratio between wild-type and mutated nucleotide was roughly similar between the two phases. These data indicated that, although JAK2V617F had been observed in a minority of myeloid cells, TET2 mutations were present in the majority of blood cells. Unfortunately, no material from neutrophils at the chronic phase was available, precluding any experimental confirmation that TET2 and JAK2 mutations had been present in the same cells. Nevertheless, our data strongly suggest that in patient 4, TET2-mutated AML derived from TET2-mutated JAK2 wild-type cells was present during the chronic phase.

The karyotype was not determined during the chronic phase, but an isolated t(10;16)(q22;q22) was observed at the blastic phase. The location of the break point on chromosome 16 suggested the involvement of the CBFB gene in the translocation. CBFB is located on human chromosome 16, band q22, and is fused to MYH11 in inv(16)(p13q22) or t(16;16)(p13;q22) observed in AML.8 Fluorescence in situ hybridization on metaphase chromosomes from blasts of patient 4 demonstrated the direct involvement of CBFB in t(10;16) (Figure 1c), whereas reverse transcription PCR analyses aimed at detecting that the CBFB–MYH11 fusion was negative (data not shown). These results suggest that a novel CBFB fusion might have cooperated with the TET2 mutation to drive the development of AML in patient 4.

Taken together, our data indicate that TET2 mutations are not observed in all cases of JAK2V617F-negative AML that develops from MPN, but cooperate with other mutations to induce AML independently of JAK2V617F. A detailed analysis of a larger series is required to determine whether the example of patient 4 is paradigmatic.

Conflict of interest

The authors declare no conflict of interest.


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This work was supported by INSERM, INCa, the Fondation de France and the Ligue Nationale Contre le Cancer. We thank William Vainchenker for critical reading of the manuscript.

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Correspondence to F Nguyen-Khac.

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Couronné, L., Lippert, E., Andrieux, J. et al. Analyses of TET2 mutations in post-myeloproliferative neoplasm acute myeloid leukemias. Leukemia 24, 201–203 (2010).

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