TET2 gene is not deleted in chronic myelomonocytic leukemia: a FISH retrospective study

We read with interest the paper TET2 gene mutation is a frequent and adverse event in chronic myelomonocytic leukemia, published in this Journal in December 2009. This study contributes significantly to the study of chronic myelomonocytic leukemia (CMML), providing important molecular and survival information. It reported the incidence and importance of TET2 mutations in this pathology.1 Taking this into account, the aim of the present study was to determine the copy number status of the TET2 gene by FISH in patients with CMML and acute myeloid leukemia (AML) secondary to CMML. As well as to evaluate the usefulness of fluorescence in situ hybridization (FISH) to analyze the status of TET2 in this cohort of patients.

Table 1.Cases with abnormal karyotype.

Regarding TET2 (4q24), acquired somatic mutations in the coding sequence of this gene, as well as loss of heterozygosity or uniparental disomy of chromosome 4, were identified in hematopoietic cells from patients with myeloproliferative disorders (MPD) or myelodysplastic syndromes (MDS).2 Kosmider et al.1 detected TET2 mutations in 44 of 88 (50%) patients with CMML; TET2 deletion was not observed by cytogenetics in any of the cases. Array comparative genomic hybridization (CGH-A) was also performed and they compared the leukemic cell profile to normal DNA in 28 of the 88, detecting TET2 deletion in one of 10 studied patients with a mutated TET2 copy. Thus, copy number alterations and deletion of the wild-type TET2 copy in TET2-mutant CMML cases seem uncommon.

Besides, Delhommeau et al.,3 using a combination of molecular, cytogenetic, CGH and single-nucleotide-polymorphism (SNP) arrays analyses, obtained the coding sequence of TET2 gene in 320 patients with myeloid malignancies. They found 3 patients diagnosed of AML that presented 4q24 deletion, corroborating the results by FISH. Also, bone marrow cells from 3 patients with MDS and one with MPD had a similar deletion. However, they detected TET2 defects in 15 of 81 patients with MDS (19%), in 24 of 198 patients with MPD (12%), in 5 of 21 patients with secondary AML (sAML) (24%), and in 2 of 9 patients with CMML (22%); suggesting that deletions or mutations in TET2 are early events. Nevertheless, CGH/SNP arrays and sequencing of TET2 are costly and time consuming.

The study cohort included 79 patients diagnosed with CMML and 4 with AML transformed from CMML. Patients were selected from 1990 until 2010. Conventional routine cytogenetic study was performed at the moment of diagnosis from bone marrow samples in a 24-hour culture without mitogens. FISH analysis was applied using BAC clones from a 32K library (http://bac-pac.chori.org/) following the standard procedures in the bone marrow fixed cells.4 Two labeled probes, RP11-542F11 (corresponding to the TET2 gene) and RP11-1377H10 (control probe for chromosome 4) were used to identify possible alterations in the status of TET2. To establish the cut-offs, 10 peripheral blood normal samples (5 male and 5 female) were used as controls. The cutoff value to consider one sample as positive was defined as average plus three standard deviations, and was 3.88% for deletions. Both controls and samples were analyzed counting 200 nuclei by 2 independent observers. Additionally, a positive control was used to make sure that the probe detected TET2 deletions. The case used was a patient diagnosed with AML with a translocation involving a loss of 4q24 band [t(4;15(q24;q26)] resulting in a deletion of the TET2 gene that was corroborated by FISH. The study was conducted with the approval of the ethical committee from our institution and in keeping with the guidelines of the Declaration of Helsinki.

Cytogenetic results revealed that 25.3% (21/83) presented an abnormal karyotype; trisomy 8 was the most frequent alteration (6/21) followed by loss of Y chromosome (4 cases) and –7/7q- and del(5q) (3 cases) (Table 1). No chromosomal structural and numerical alterations were found in chromosome 4 for any of the cases included in the series. FISH results revealed that no cases with the diagnosis of CMML or AML transformed from CMML presented a deletion of TET2.

Other authors have used SNP arrays to study the status of TET2 showing that the deletions are present in few cases. Langemeijer et al. examined 102 patients and found 2 cases with TET2 deletion, both with a diagnosis of RAEB-2.5 Also, Jankowska et al. used SNP arrays and sequencing in 396 patients. Eight of the patients studied had deletion of the gene and their diagnosis corresponded to MDS and sAML.6 A similar study to ours was published by Viguié et al. in 2005, who, before the discovery of the involvement of the TET2 gene in hematologic malignancies, studied 4 cases of AML with the 4q24 deletion suggesting the implication of a tumor suppressor gene.7

Taking into account our results and those previously reported, we can conclude that TET2 is not deleted in CMML patients, although it is mutated in a high proportion of cases, as had been previously reported.1,3,5–6 In addition, FISH is not a useful technique for analyzing the status of TET2 in CMML.

Acknowledgments

we would like to thank Blanca Espinet and Marta Salido for the cytogenetic analysis, and Carme Melero and María Rodríguez-Rivera for their expert technical assistance.

Footnotes

• Funding: this work has been partially supported by grants from Instituto de Salud Carlos III, Ministerio de Sanidad y Consumo, Spain (FI07/00107 and PI07/1009) and Ministerio de Ciencia e Innovación, Red Temática de Investigación Cooperativa en Cáncer (RTICC): RD07/0020/2004 FEDER.
• The information provided by the authors about contributions from persons listed as authors and in acknowledgments is available with the full text of this paper at www.haematologica.org.
• Financial and other disclosures provided by the authors using the ICMJE (www.icmje.org) Uniform Format for Disclosure of Competing Interests are also available at www.haematologica.org.