EVI1 overexpression in t(3;17) positive myeloid malignancies results from juxtaposition of EVI1 to the MSI2 locus at 17q22

Abstract

Chromosomal translocations involving the EVI1 locus are a recurrent finding in myeloid leukemia and are associated with poor prognosis. In this study, we performed a detailed molecular characterization of the recurrent translocation t(3;17)(q26;q22) in 13 hematologic malignancies. The EVI1 gene locus was rearranged in all 13 patients and was associated with EVI1 overexpression. In 9 out of 13 patients, the 17q breakpoints clustered in a 250 kb region on band 17q22 encompassing the MSI2 (musashi homologue 2) gene. Expression analyses failed to demonstrate ectopic MSI2 expression or the presence of an MSI2/EVI1 fusion gene. In conclusion, we show for the first time that the t(3;17) is indeed a recurrent chromosomal aberration in myeloid malignancies. In keeping with findings in other recurrent 3q26 rearrangements, overexpression of the EVI1 gene appears to be the major contributor to leukemogenesis in patients with a t(3;17).

Introduction

The Ecotropic Viral Integration site 1 ( EVI1) gene is located at chromosome band 3q26.2 and was identified as a proto-oncogene by retroviral integration assays in mice.1 Rearrangements of the EVI1 locus are found in acute myeloid leukemias (AML), myelodysplastic syndromes (MDS) and chronic myeloid leukemias (CML). EVI1 gene rearrangements account for approximately 5% of cytogenetic abnormalities in these disease entities.2 Patients with an EVI1 rearrangement have distinct clinical features, such as marked hyperplasia with dysplasia of the megakaryocytes3 and, in some cases, hyperthrombocytosis.4

These 3q26 chromosomal aberrations confer an adverse prognosis and contribute to ectopic expression of either full length or truncated EVI1 transcripts, or to the formation of EVI1 fusion genes.2 Common recurrent rearrangements affecting the 3q26 locus include the inv(3)(q21q26) and the translocation t(3;3)(q21;q26), in which EVI1 overexpression is caused by juxtaposition of the EVI1 gene to enhancer elements of the Ribophorin (RPN) gene at 3q21 (5). EVI1 activation in the translocations t(3;12)(q26;p13) and t(3;21)(q26;q22) is due to generation of the fusion genes ETV6/EVI1 and RUNX1/EVI1 respectively.6,7

In addition to these well-characterized rearrangements, the EVI1 locus is also involved in rare 3q26 aberrations such as the t(3;17)(q26;q22),8 the t(2;3)(p21~22;q26)9,10 and the t(3;6)(q26;q25).11 For these EVI1 rearrangements, the true recurrent nature and the partner chromosomes involved, have not been analyzed in detail. Therefore, we performed an in depth characterization of the 17q breakpoints in 13 hematologic malignancies with a t(3;17).

Design and Methods

Patients and cell lines

In this multicenter retrospective study 13 leukemias were included according to the following criteria; presence of a hematologic malignancy with a t(3;17) and 3q26 rearrangement. Karyotyping of diagnostic samples was performed according to standard procedures. Several myeloid leukemia cell lines (K562 and U937) and EVI1 rearranged cell lines (Kasumi-3 and UCSD-AML1) were included as positive controls for EVI1 overexpression.12–15 The study was approved by the ethics committee of the Ghent University Hospital (2003/273). Patients’ characteristics and karyotypes are described in Table 1.

Fluorescence in situ hybridization

Dual-color fluorescence in situ hybridization (FISH) with a dedicated EVI1 probe set (RP11-362K14, RP11-82C9 and RP11-694D5) was performed on fixed nuclei and/or metaphases of patients and cell lines.8 To characterize the 17q breakpoints, eight different 17q BAC/PAC probes over a region of 34.3 Mb on 17q were selected from the UCSC (http://genome.ucsc.edu) or Ensembl (http://ensembl.org) databases. The 17q and EVI1 probes were all obtained from the Sanger Wellcome Trust Institute, Hinxton, Cambridge (United Kingdom). Position of the probes used and location of translocation breakpoints is described in Figures 1 A–B.

Real-time quantitative RT-PCR

Total RNA was extracted from total bone marrow samples or bone marrow leukocytes from 9 of 13 t(3;17) samples, from 4 myeloid cell lines, from bone marrow of 3 patients without a hematologic disorder and from 2 stem cell (CD34) fractions, using the miRNeasy kit (Qiagen, Belgium) according to the manufacturer's recommendations. The cell lines and CD34 cells served as positive controls for the study of EVI1 expression as well as for MSI2 expression. cDNA was prepared from 2 μg of total RNA with the iScript cDNA Synthesis Kit (Bio-Rad, Belgium) according to the manufacturer’s descriptions.

Real-time quantitative RT-PCR (qRT-PCR) for the EVI1 (exon 1b-2), cEVI1 (exon 8–9, primers located at the 3’ end of the EVI1 gene to detect possible 5’ transcript variants),16 MDS1/EVI1 (data not shown) and MSI2 (primer pairs for exon 3 and 10) transcripts was performed as previously described.8,17 For normalization three housekeeping genes (RPL13A, YWHAZ and HPRT1) were selected in view of their stability in bone marrow samples as analyzed using the Genorm software.17 The EVI1 overexpressing cell line Kasumi-3 carrying a t(3;7)(q26;q22) translocation12 was used as a positive control.

Table 1.Patients’ characteristics, diagnosis, sex, age at diagnosis, G-banded karyotype.

Figure 1.Schematic overview of the location of the 17q and 3q probes and breakpoints. (A). Physical map of the 3q26.2 probes used. The locations of the EVI1 breakpoints in the 13 t(3;17) patients are indicated by shaded boxes. (B). Location of the 17q22 breakpoint for 9 out of 13 patients. Vertical arrows indicate location of the breakpoints within the MSI2 gene.

RT-PCR

To investigate the possibility of MSI2/EVI1 fusion gene formation, RT-PCR was performed. Based on the known EVI1 breakpoint position in fusion genes and the location of the MSI2 17q breakpoints, primers for the second exon of EVI1 and the second exon of MSI2 were selected. For PCR analysis, the following touch-down program was used: an initial denaturation step at 94°C for 2 min, 12 cycles of 20 sec at 94°C, 15 sec at the initial annealing temperature (Ta) 62°C (Ta-1°C for each cycle ), 1 min at 72°C followed by 24 cycles of 40 sec at 94°C, 40 sec at 50°C, 30 sec at 72 °C and a final extension step of 4 min at 72°C.

Results and Discussion

FISH analysis demonstrated a rearrangement of the EVI1 locus in all samples. For 9 out of 13 leukemic samples, the 3q26 breakpoint was located 5’ (telomeric) of the EVI1 gene which is in keeping with previous observations of 3q26 translocation breakpoint positions.8 In 3 out of 13 samples, the EVI1 breakpoint was located 3’ (centromeric) of the EVI1 gene and in case 5 the EVI1 breakpoint was located within the region corresponding to the RP11-82C9 probe and could thus be located either 5’, 3’ or within the EVI1 gene (Figure 1A).

In 9 of 13 patients the 17q breakpoint clustered in a 250 kb region on 17q22 harboring the MSI2 gene (Figure 1B). For the remaining 3 cases, unique break-points were detected in BAC clones RP11-386F9 (patient #5, 17q11.2), RP11-1094H24 (patient #3, 17q21.33) and in the PAC clone RP5-1171I10 (patient #6, 17q22), respectively (data not shown). For patient #13, the 17q breakpoint overlapping clone could not be determined.

MSI2 plays a role in post-transcriptional gene regulation and in maintaining stem cell status,18 and involvement of MSI2 in myeloid leukemias has previously been reported in 2 CML patients, one of which carried a MSI2/HOXA9 fusion gene.19 In our patient series however, no MSI2/EVI1 fusion gene could be detected in RT-PCR analysis. Given the opposite orientation of MSI2 and EVI1 and the lack of karyotypic evidence for complex rearrangements, this result was indeed anticipated.20 Further study of a possible MSI2/EVI1 fusion transcript was complicated by limited availability of patient sample. MSI2 rearrangements have also been linked to CML and myeloproliferative disorder disease progression.19,21,22 Interestingly, 2 out of 9 patients with a MSI2 breakpoint were CML-BC patients.

Frequently observed secondary karyotypic changes in CML include +8, i(17q), +19 and +Ph, but in some occasions recurrent translocations and 3q26 rearrangements are reported.23 We postulate that the t(3;17) involving MSI2 is associated with CML as a secondary aberration and could serve as a progression marker.

Real-time quantitative PCR with EVI1 and cEVI1 primers indicated that 5 out of 9 patients displayed ectopic EVI1 expression (data not shown). In the remaining 4 patients, no ectopic EVI1 expression could be detected. In a previous study, we showed that the majority of hematologic malignancies displaying a rearrangement in the vicinity of EVI1 show ectopic EVI1 expression.8 Therefore, we hypothesize that in these 4 patients EVI1 expression could not be detected because of the low blast counts in these samples. Overexpression of the EVI1 oncogene conveys a poor prognosis in AML and CML-BC.24 In our cohort, patients without detectable EVI1 overexpression had prolonged survival compared to the patients with EVI1 overexpression (Table 1). This enhanced prognosis could in part be explained by the absence of a high blast count which is indicative for an early stage of the disease. Therefore the patient’s blast count might have to be taken into account when addressing prognosis.

The exact cause of EVI1 overexpression is not known, but in the inv(3)(q21q26) and the t(3;3)(q21;q26) EVI1 is juxtaposed to the enhancer elements of the Ribophorin gene on chromosome band 3q21.5 Possibly, EVI1 overexpression in t(3;17) leukemias is driven by enhancer elements located in the vicinity of the MSI2 locus.

We found no significant differences in MSI2 expression between MSI2 rearranged and MSI2 non-rearranged t(3;13) hematologic malignancies (data not shown). Expression of MSI2 in patients with a 17q breakpoint in MSI2 is not straightforward. Due to the translocation, a part of the MSI2 gene is separated from its promoter. Therefore, we postulate that the observed expression is due to residual expression from the normal allele.

No MSI2/EVI1 fusion gene could be detected, and no significant MSI2 expression differences were found between MSI2 rearranged and MSI2 non-rearranged patients. Therefore, we have no evidence of a transforming role of MSI2. Given the expression of MSI2 in CD34 cells (data not shown) it is feasible that the gene is located in a region with open chromatin within the genome. As these regions are more susceptible to double strand DNA breaks, this could lead to increased vulnerability of the MSI2 locus in chromosomal aberrations. Rearrangement and consecutive overexpression of EVI1 seem to be the main contributor to tumorigenesis in t(3;17) patients.

In conclusion, we confirm the t(3;17) as a recurrent chromosomal aberration in myeloid malignancies, consistently involving EVI1. We found that a distinct subgroup (9 out of 13) shares a recurrent 17q breakpoint region implicating the MSI2 gene, and postulate that overexpression of the EVI1 gene is the major contributor to leukemogenesis in patients with a t(3;17).

Footnotes

• Authorship and Disclosures ADW: performed FISH and expression analysis, drafted the manuscript; FS: helped drafting the manuscript; BC: helped with the RT-PCR; NVR: helped drafting the manuscript; NY: helped with FISH experiments; BV: helped with drafting the manuscript; BDM, YB, LN, DS, EL, SS, CB, PV, AH, ND: supplied patient samples; ADP: helped drafting the manuscript; BP: helped drafting the manuscript. This text presents research results of the Belgian Program of Interuniversity Poles of Attraction initiated by the Belgian State, Prime Minister’s Office, Science Policy Programming. The scientific responsibility is assumed by the authors.
• Funding: ADW is the recipient of a BOF grant (Bijzonder Onderzoeksfonds UGent, grant n. 01D28905). BP, PV and BV are senior clinical investigators of FWO-Vlaanderen. This study was supported by the FWO-Vlaanderen, grant n. G.0106.05 and by GOA-UGent, grant n. 12051203.

• Received April 7, 2008.
• Revision received July 16, 2008.
• Accepted August 4, 2008.