Expression of ETV6 rearrangement in a subject with acute myeloid leukemia-M4Eo

Acute myeloid leukemia (AML) M4Eo type is a hematological malignancy with abnormal eosinophilia, which is often accompanied by inv(16). The Ets variant gene 6 (ETV6), mapped to 12p13, is an ETS family transcription factor that is essential for hematopoietic processes.1 The ETV6 gene-involved chromosomal translocations have been found in many hematological malignancies characterized by fusing to a number of different partner genes; mainly coding for tyrosine kinases or transcription factors which are important for the initiation, progress and prognosis of disease.2 In particular, the ETV6 gene has been reported to be fused to ABL in acute lymphocytic leukemias (ALL),3 and chronic myeloid leukemia (CML).4 However, there have been few domestic reports of ETV6 fusion genes, especially in cases of acute leukemia. We investigated 3 cases of AML-M4Eo patients using Split-signal Fluorescence in situ hybridization (FISH) and found one case with a translocation between 12p13 and 1q25 co-occurring with an inv(16). The ETV6/ARG (ABL-related gene) fusion transcript was confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR). This is the report of ARG involvement in a translocation in a human malignancy. METHODS Patient samples Three patients with AML-M4Eo (all males) were recently diagnosed from the Qilu Hospital, Shandong University according to the diagnostic criteria of AML. Bone marrow cell culture and chromosome preparation methods Bone marrow specimens were acquired from patients in the absence of stimulus conditions and cultivated for 16-24 hours before harvesting cells. Bone marrow cell chromosomes were prepared conventionally and analyzed by the R-banding technique. Karyotype abnormalities were identified and described according to an international system for human cytogenetic nomenclature ISCN (1995). Split-signal FISH analysis Split-signal FISH was applied on the chromosome samples from the three patients according to the standard manufacture procedures. Briefly, bacteria artificial chromosome (BAC) clones (RP11-434C1 and RP11-525I3) containing ETV6 gene were amplified and DNA was extracted using a plasmid DNA extraction kit (QIAGEN, German). Selected BAC sequences on either side of ETV6 were used as probes and labeled by a Dig-Nick Translation Mix (Roche, Switzerland) and Biotin-Nick Translation Mix (Roche). The probes were then purified by Quick Spin Columns (Roche). The labeled probes are DIG525I23 and Bio407P10. Hybridization procedures were previously described.2 RT-PCR ETV6/ARG fusion gene was detected by RT-PCR in patients using the TRIzol reagent (Invitrogen, USA) and an RT-PCR kit (Biological Engineering Limited, Dalian). RT-PCR was performed according to standard procedures, with the forward primers F1: (5′-ATCGGGAAGACCTG-GCTTACA-3′) and F2: (5′-TGAAGAGCACGCCATGC-CCATTG-3′) on ETV6 and the reverse primers R1: (5′-TGCCTGGGGTTCAACATCAC-3′) and R2: (5′-TA-CTGCCTCCAGTCTTGTCT-3′) on ARG. Electrophoresis, scanning and analysis of results were then conducted. RESULTS Characterization of ETV6-involved rearrangement The bone marrow cell’s R-banding chromosome karyotype analysis from 3 cases of AML-M4Eo patients showed inv(16); the traditional cytogenetic analysis had not found any other chromosome abnormities. We further probed the three samples by Split-signal FISH and found chromosome translocation between 12p13 and 1q25. The karyotype showed 46 XY, inv(16), t(1;12)(q25;p13). Red and green fluorescence signal separation was observed in cell nuclei under fluorescence microscopy (Figure 1). Both of the chromosomes 12p and 1q showed fluorescence signals, suggesting that there is translocation on homologous chromosomes between 12p and 1q. RT-PCR detected ETV6/ARG fusion gene products (Figure 2).Figure 1.: Split-signal FISH analysis of ETV6 rearrangement of M4Eo patient. The signals were found on the der (1) and the der (12), indicating that red fluorescence signals translocated to 1q25.Figure 2.: RT-PCR analysis of ETV6/ARG fusion products. Lane M: DNA marker DL2000; lane 1: negative control; lane 2: patient sample. Two special bands of ETV6/ARG fusion products were detected in the patient sample. The different products are the result of an alternative splicing event in the ARG gene.Clinical implication The patient is a 13-year-old boy, hospitalized for “neck lymphadenopathy, fever, and weakness” in March 2006. Blood assay showed markedly increased leukocytes with naïve neutrophils and monocytes, following thrombocytopenia. Morphological assay of bone marrow cells showed significantly active myelodysplasia with 26.8% myeolblasts, 17.8% eosinophil and 13.0% monoblasts. The eosinophils had a relatively large size, containing abnormal coarse eosinophilic particles. After treated with two courses of daunorubicin and cytarabine chemotherapy, the patient achieved complete hematological remission (CR). Five months later he suffered from bone marrow (BM) relapse with 30% blast invasion of the BM. CR was obtained after a three-courses of high-dose cytarabine chemotherapy treatment, followed by a second BM relapse and central nervous system involvement 4 months later. In the course of treatment this resistance phenomenon happened frequently. The patient abandoned the treatment and left the hospital. We could not detect the ETV6 rearrangement in the other two cases of M4Eo patients, who were sensitive to chemotherapy. Their CR periods were significantly longer than the first case. DISCUSSION ETV6 (also named TEL), has a total length of 300 kb and expressed as a 452 amino-acid protein with an ETS DNA-binding domain near its carboxyterminus and a helix-loop-lelix (HLH) domain near the aminoterminus. ETV6 was identified as a fusion partner gene of many other genes; including PDGFRB,5 AML1,6 JAK2,2 BAZ2A7 and MN1.8 The ETV6-related chimeric genes have been reported in many types of hematological malignancies. The repression of ETV6 appears to be important in the regulation of cell growth and differentiation. Moreover, ETV6 has been shown to stimulate erythroid differentiation of the murine erythroid leukemia cell line.9 Wang et al10 reported that chimeric mice with ETV6 knockout embryonic stem cells showed defective hematopoiesis within the first week after birth. So far it has been reported that there are more than 40 kinds of ETV6 gene-related chromosomal translocations. Different ETV6 gene rearrangements are involved in the occurrence of leukemia through different mechanisms. Inv(16) is a characteristic chromosomal abnormality of AML-M4Eo. AML-M4Eo patients with inv(16) have a higher CR rate after chemotherapy (78%) and a longer median survival period (> 66 weeks), which constitutes a special clinic pathologic entity. Most studies show that this subtype is sensitive to high-dose cytarabine. That central nervous system leukemia is rarely involved when patients relapse indicates better prognosis. In this study R-banding chromosome karyotype analysis of 3 cases of AML-M4Eo patients all showed inv(16). Split-signal FISH detected an ETV6 rearrangement in one case who had a shorter CR and was not sensitive to chemotherapy after relapse; indicating a worse prognosis than the other two M4Eo patients. RT-PCR further confirmed the existence of ETV6/ARG in this patient. This is the second report of an M4Eo patient with ETV6/ARG rearrangement after Cazzaniga et al.11 The ARG gene, also called the ABL2 gene, is a non-receptor tyrosine kinase that together with ABL1 is characterized by high homology in its protein tyrosine kinase (PTK), Src homology 2 (SH2) and Src homology 3 (SH3) domains. So far there have been very little in vivo data of ARG and the role of Arg in the hematological system remains unknown. The ETV6/ARG translocation is the first instance in which ARG has been implicated in human cancers. Previous in vitro studies showed that there were two ETV6-ARG fusion transcripts11 as the result of an alternative splicing event in the ARG gene. Our results are in agreement with the previous reports. The transforming potential of the ETV6/ABL1 fusion gene has been demonstrated to be dependent on the oncogenic activity of the chimeric PTK proteins by forming HLH domain-dependent homo-oligomers that result in ligand-independent tyrosine kinase activation.1 A similar mechanism may play a role in cases with ETV6/ARG fusion transcripts. The presence of the ETV6/ARG translocation raises the possibility that the resulting fusion protein functions as an oncogene. The ETV6/ARG fusion protein might activate a more myeloid differentiation process and push the cells towards a more myeloid phenotype.12 It may also prevent G-CSF stimulation of the myeloid hematopoietic process, blocking myeloid differentiation and maturation. Further experiments are needed to confirm the oncogenic mechanism of ETV6/ARG fusion transcripts. We have also detected ETV6 gene rearrangements in the other hematological malignancies such as ALL, CML and myelodysplastic syndrome (MDS) by Split-signal FISH as well as ETV6/ARG detected in a M4Eo patient.2 We found the ETV6 rearrangement in the M4Eo patient co-occurred with inv(16), suggesting that the ETV6/ARG fusion may not be the primary transforming event. The inv(16) was also found in the AML-M4 patient with ETV6/ARG rearrangement reported by Cazzaniga et al.11 In addition, this patient also showed BM relapse resistance to chemotherapy in the course of treatment which is similar to our case, suggesting ETV6/ARG in M4Eo patients may also be associated with poor prognosis. Domestic and foreign studies indicate that some chromosome abnormalities can be taken as important indicators for prognosis. Additional chromosomal abnormalities, for example, i(17q), +8, +19, in Ph-positive chronic myeloid leukemia predict a poor prognosis. In addition to the t (8; 21) abnormality, a 9q-in AML-M2 is a sign of a poor prognosis. Further studies on the ETV/ARG fusion product in M4Eo patients will help understand the role of the fusion gene in the pathogenesis, process or prognosis of the disease.

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