ETV6 (TEL) gene amplification in a myelodysplastic syndrome with excess of blasts


Oncogene amplifications are frequent events in solid tumors, observed either as large chromosomes in which the amplified DNA appears as a homogeneous stained region (hsr) or as small chromosomal structures described as double minute chromosomes (dmin). In malignant hemopathies, such chromosomal alterations are rather rare, concerning a limited number of genes often implicated in recurrent translocations. Amplifications, now more easily detected by extensive use of FISH and/or comparative genomic hybridization, involve in majority c-MYC, MLL1 and RUNX1 (AML1)2 genes, more rarely other sequences. We report here the amplification of ETV6 gene, diagnosed with FISH and quantitative PCR, in a myelodysplastic syndrome with excess of blasts.

LR, a 66-year-old man, was admitted in Hôpital Hautepierre, Strasbourg, France, for fatigue and weight loss. Laboratory data were as follows: hemoglobin level, 7.3 g/dl; mean corpuscular volume, 69.9 fl; platelet count, 53 × 109/l; white blood cell count, 12.3 × 109/l with 30% neutrophils, 8% eosinophils, 3% blasts, 27% monocytes (3.32 × 109/l), 9% lymphocytes, and 3% metamyelocytes. Bone marrow smears examination revealed dyserythropoiesis, severe dysgranulopoiesis with excess of blasts (13%), abnormal eosinophils (8%) and monocytosis (21%). The patient was classified as refractory anemia with excess blasts-2 using WHO classification. The evolution was fatal in 5 weeks.

Conventional cytogenetics (RHG banding) showed a structural abnormality interpreted as a t(5;12) translocation (Figure 1a). Breakpoints were difficult to precise because of the variable length and the lack of RHG banding contrast of the der(12) in most of the metaphases, suggesting a hsr (Figure 1b). All metaphases carried a small marker (mar1). If some metaphases showed a der(12)t(5;12) with variable length, some other showed a der(12)t(5;12) with a constant length (without hsr), carrying the small mar1 and also 3–7 small other markers (mar2). Modal karyotype was: 46,X,-Y,-5,del(7)(q?12q?32), +8,der(12)t(5;12)(?;p13?)hsr(12)(p13?),+mar1[26] /4953,X, -Y,-5,del(7)(q?12q?32),+8,der(12)t(5;12)(?;p13?),+mar1,+3 7mar2[4]. FISH studies with painting probe wcp5 and wcp12 confirmed the t(5;12) translocation and revealed alternation of chromosome 5 and 12 material in the hsr (Figure 2a), suggesting a possible coamplification. Mar1, exclusively composed of chromosome 5 material and carrier of a chromosome 5 centromere, was identified as a del(5)(?q). Specific chromosome 5 unique sequence probes (LSI EGR1 (5q31)/D5S721,D5S23 (5p15.2), LSI CSF1R (5q33)/D5S721,D5S23 (5p15.2) and YAC probe 885A6 (5q35)) demonstrated the presence of a 5p15.2 signal at the extremity of the der(12)t(5;12) and the absence of 5q31, 5q33 and 5q35 signals on this der(12). Surprisingly, specific ETV6 probe (YAC 936E2) demonstrated two distinct locations of ETV6 amplification: in hsr for the clone with the der(12) of variable length and in small markers (mar2) for the second clone, each one being mutually exclusive. M-FISH results confirmed the global structural abnormalities and did not show additional abnormalities. We next tried to demonstrate the specific amplification of ETV6 and quantify the mean number of ETV6 copies in each bone marrow cell. As no frozen cells were available, thus preventing Southern blot analysis, real-time quantitative DNA PCR was used in order to compare the copy number of ETV6 gene to the reference albumin (ALB) gene, located on chromosome 4q11, in which no cytogenetic abnormality was detected. Reactions containing the different primer sets (ETV6 exons 4 and 8, ALB exon 11) were amplified side by side using the same protocol, and amplifications were compared. Relative quantification as well as standard curve quantification determined a 10-fold increase of ETV6 gene copy number, for exon 4 (coding for part of the HLH protein interaction motif) as well as exon 8 (immediately downstream the DNA binding ETS domain), suggesting that the whole gene (that is composed of at least 8 exons, spanning 241 kb) may be amplified. Accordingly, dual-color FISH of ETV6 exon 2 and exons 6–8 (PACs was kindly provided by M Rocchi, Bari, Italy) confirmed the integrity of the gene within the hsr (Figure 2b). Sizing of the amplicon was not possible because of the lack of material.

Figure 1

R-banded karyotype of a metaphase cell showing a der(12)t(5;12)(?;p13?)hsr(12)(p13?) (a) and examples of R-banded derivative chromosomes 12 showing length variability and lack of contrast of the der(12) (b).

Figure 2

FISH analysis with painting probes wcp5 (red) and wcp12 (green) showing alternation of chromosome 5 and 12 material in the hsr (a) and hybridization with dJ852F10 (exon 2) SpectrumOrange and dJ846M9 (exons 6, 7, 8) SpectrumGreen (b) showing the integrity of the of ETV6 in the hsr.

As the patient harbored an excess of eosinophils and monocytes in a myelodysplasic syndrome with excess of blasts, we nevertheless suspected a variant form of the t(5;12)(q31–32;p13) implicating ETV6 and PDGFβ-R genes.3 Specific PDGFβ-R FISH probe (BAC CTB-5M9) was not splitted, arguing against the hypothesis of a t(5;12)(q31–32;p13). Accordingly, our attempts to detect the ETV6-PDGFβ-R fusion transcript corresponding to the previously described t(5;12), in which the HLH domain of ETV6 is fused with transmembrane and kinase domains of PDGFβ-R, were unsuccessful. Using cDNA obtained from 25 μl of the cytogenetic sample, three different ETV6 primers located 5′ to the different breakpoints were tested, in combination with a PDGFβ-R primer known to detect the truncated form of PDGFβ-R.2 The β-globin gene was used for control of RNA quality. In our hands, all three PCRs failed to detect an aberrant ETV6-PDGFβ-R transcript whereas controls, harboring the classical t(5;12), were positive. Accordingly, quantification of the copies number of PDGFβ-R (realized with the most 5′ (exon 1) and 3′(exon 23) of PDGFβ-R) showed no gene amplification. Taken together, these data strongly suggested that PDGFβ-R gene was not implicated in the chromosome 5 sequences detected by FISH in the altered chromosome 12.

If the t(5;12)(q31;q33) was not present, another fusion transcript involving ETV6 may also have resulted from the translocation described here. Indeed, in rare cases, HLH and ETS domains of ETV6 were both included in a fusion transcript generated by another balanced t(5;12)(q31;p13) translocation,4 in which exons 2–8 of ETV6 were joined to the exons 2–19 of FACL6 (ACS2) gene on 5q31. It was not the case here, as on one hand, chromosome 5p15 was detected on the der12 but not EGR1 (5q31) sequences, and on the other, we did not detect the ETV6-FACL6 transcript by RT-PCR using published primers.4

Our results indicated that fusion transcripts described previously in t(5;12) were not present in our case. So, how ETV6 could be implicated here? Three main hypotheses remain to be explored. First, a new fusion transcript involving ETV6 and an unknown gene may be expressed, even if in most of cases, no fusion transcript was detected in malignant hemopathies with gene amplification, as reported for MLL1 and RUNX1 (AML1)2: rearranged chromosomes were not amplified and only structurally normal genes were amplified.1, 2 A frequent exception for this general rule is, to our knowledge, the amplification of the BCR-ABL chimeric sequences, described previously in chronic myeloid leukemia cases treated by imatinib mesylate.5 Second, as hypothesized for RUNX1 (AML1) and MLL, the isolated presence of amplified copies of ETV6 may be oncogenic by gene dosage effect,6 even if deletions of ETV6 have also been implicated in leukemogenesis.7 Third, ETV6 position may activate critically important genes in chromosome 5, as described for a t(4;12)(q11–q12;p13) and a t(5;12)(q31;p13) translocation in acute myeloid leukemia (AML) cases.8 In the two cases studied, no fusion transcript explaining their pathogenic character was generated by these translocations. However, the homeobox gene GSH2 at 4q11–q12 and the IL-3/CSF2 locus at 5q31 were found to be located close to the respective breakpoints, and ectopically expressed in the leukemic cells, probably underlying leukemogenic mechanisms of these translocations. Also, in one AML case with eosinophilia and one patient with acute eosinophilic leukemia,4 ETV6-ACS2 transcripts were out of frame and could not lead to a fusion protein, suggesting a similar mechanism. Remarkably, in our case, ETV6 amplification harbored two different presentations. In the 3–7 small markers (mar 2), only chromosome 12 was detected, but in hsr, FISH showed interspersed material from chromosomes 5 and 12. These results suggest that two different mechanisms may have driven the amplification of ETV6. It can be hypothesized that the first abnormality may have been a breakage into ETV6 locus, followed by two different types of secondary events: either a recombination with chromosome 5 sequences or a recombination within ETV6 sequences, each of the mechanisms appearing in different clones.

Although ETV6 is implicated in many translocations, leading to the generation of fusion genes, the present case indicated that amplification might be a possible new mechanism for leukemogenesis involving ETV6, as it was described for MLL and RUNX1 (AML1). It is not presently possible to further investigate this case, and confirmation of the mechanism proposed here still waits for other similar cases.


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Correspondence to M Lessard.

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Mauvieux, L., Helias, C., Perrusson, N. et al. ETV6 (TEL) gene amplification in a myelodysplastic syndrome with excess of blasts. Leukemia 18, 1436–1438 (2004).

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