A novel three-way rearrangement involving ETV6 (12p13) and ABL1 (9q34) with an unknown partner on 3p25 resulting in a possible ETV6-ABL1 fusion in a patient with acute myeloid leukemia: a case report and a review of the literature

Acute myeloid leukemia (AML) is commonly characterized by several chromosomal abnormalities resulting in the formation of chimeric genes that play various roles in leukemogenesis. Translocations resulting in the ETV6-ABL1 fusion gene are rare in AML and other hematologic malignancies with only thirty-two previously reported cases in the literature, five of which were AML. Herein, we report the case of a 73-year-old male with acute myeloid leukemia arising from MDS, negative for PDGFRA and PDGFRB, positive for bone marrow eosinophilia, rash, and marked fluid retention, which improved dramatically with imatinib therapy. Conventional cytogenetics revealed a t(3;9)(p25;q34), t(5;18)(q13;p11.2), and additional material of unknown origin at 12p11.2 in 2 out of 10 metaphases analyzed. Interphase FISH studies showed evidence of ETV6 (12p13) and ABL1 (9q34) rearrangements in 41.3 % and 5.7 % of the cells respectively. FISH studies on previously G-banded metaphases showed colocalization of ABL1 and ETV6 signals to the short arm of chromosome 3 at 3p25 suggesting a possible ETV6-ABL1 fusion. Subtelomeric metaphase FISH studies also showed the presence of a subtelomere 3p signal on the long arm of the derivative 9, and no subtelomere 3p signal on the derivative chromosome 12. These findings suggest a complex rearrangement involving an insertion of ETV6 into 3p25 followed by a reciprocal translocation involving 3p25 and 9q34, resulting in a possible ETV6-ABL1 fusion. This case highlights the importance of FISH to characterize complex rearrangements in myeloid malignancies, particularly those resulting in clinically significant chimeric genes.


Introduction
The ETV6-ABL1 fusion gene is uncommon in hematological malignancies, including chronic myeloid leukemia (CML), acute myeloid leukemia (AML), and acute lymphoblastic leukemia (ALL), and has been reported in only thirty-two patients [1]. Six reported cases of hematological malignancies bearing ETV6-ABL1 in the context of complex rearrangements involving additional translocation partners have been reported, resulting in the translocation of the fusion gene to a third derivative chromosome [2][3][4][5][6][7]. The rarity of this event is due in part to the opposite transcriptional orientation of ETV6 and ABL1 relative to the centromere, which requires at least three separate chromosomal breaks to form a functional fusion gene [8]. The structure and function of the ETV6-ABL1 oncoprotein is very similar to that of the BCR-ABL1 protein with the ETV6 helix loop helix domain (HLH) deregulating the kinase activity of ABL1 leading to activation of a non-receptor tyrosine kinase that initiates downstream pathways affecting growth rate, cellular survival, and independence as well as transforming capacity [1,3]. Because of the common functional activity with the BCR-ABL1 fusion protein, ETV6-ABL1 positive patients have been observed to respond to therapy with tyrosine kinase inhibitors, albeit at varying degrees and with high likelihood of relapse [1].
Herein, we present the case of a 73-year-old male diagnosed with acute myeloid leukemia. Cytogenetic analysis revealed a t(5;18) and a t(3;9), as well as additional material of unknown origin on the short arm of chromosome 12. Additional interphase and metaphase FISH studies revealed an insertion of ETV6 into 3p and translocation of ABL1 to the same locus on 3p, resulting in a possible ETV6-ABL1 fusion gene. The patient responded transiently to imatinib therapy, but eventually relapsed and expired.

Case presentation
The patient was a 73-year-old male with acute myeloid leukemia (AML) and hypereosinophilia, arising from antecedent myelodysplastic syndrome (MDS). He was initially found to have thrombocytopenia fifteen months prior to transfer during a pre-surgical workup for surgery to treat carpal tunnel syndrome. A bone marrow biopsy performed six months later had findings consistent with myelodysplastic syndrome with fewer than 5 % blasts in the bone marrow. He subsequently received three cycles of decitabine: the first dose was given in February 2015, the second dose was given in May 2015, and the third dose was given in July 2015. Eight months after bone marrow biopsy, he presented to an outside hospital with a fever and was found to have leukocytosis with circulating blasts, and a repeat bone marrow biopsy identified AML, possibly acute eosinophilic leukemia, with 20 % blasts identified in the bone marrow. Broadspectrum antibiotics were started and the patient was transferred to UCLA for escalation of care. Shortly after transfer, he developed progressive renal failure requiring dialysis. Persistent blasts were treated with azacytidine, but he developed severe pancytopenia. In addition, eosinophilia, rash and marked fluid retention led his clinical team to consider therapy with imatinib, which promptly led to resolution of those findings. A follow-up bone marrow aspiration and biopsy one month later identified a hypercellular marrow showing marked eosinophilia with increased atypical immature forms, markedly reduced myeloid precursors other than the eosinophilic series including increased atypical immature eosinophils, reduced erythropoiesis and megakaryopoiesis, and increased blasts (10-11 % of the marrow elements). The overall marrow histology was consistent with acute myeloid leukemia possibly, acute myelocytic leukemia.

Conventional cytogenetics
Chromosome analysis was performed using standard cytogenetic techniques on the bone marrow samples from this patient. The karyotypes were prepared using the Applied Imaging CytoVision software (Applied Imaging, Genetix, Santa Clara, CA) and described according to the ISCN 2013 nomenclature [9].

FISH
Fluorescence in situ hybridization (FISH) was performed on interphase nuclei using the Vysis BCR/ABL1/ASS1 Tri-color DF FISH Probe Kit, Vysis LSI BCR/ABL Dual Color, Dual Fusion Probe Kit, and Vysis ETV6 Break Apart FISH Probe Kit from Abbott Molecular (Des Plaines, Illinois 60018) on interphase nuclei. Additionally, metaphase FISH was performed with the TotelVysion 3p, Spectrum Green, TotelVysion 3q Spectrum Orange probes, as well as the previously mentioned probes on previously G-banded metaphases.

Discussion
This case highlights the formation of a potential ETV6-ABL1 fusion gene as a result of a complex, three-way rearrangement. The conventional and molecular cytogenetic findings in this case suggest an insertion of ETV6 in the short arm of the derivative chromosome 3 followed by a reciprocal translocation involving the same derivative chromosome 3 and ABL1 (9q34), resulting in the potential fusion gene. The breakpoint on chromosome 3 at which the aforementioned rearrangements occurred -3p25harbors ANKRD28 (3p25.1), which has been implicated in AML in the context of t(3;11)(p25;p15) involving NUP98 (11p15) [10]. In other studies, 3p25 was found to be the most frequently deleted chromosomal band on 3p in AML [11]. Given this information, the involvement of 3p25 in this three-way rearrangement may result in deregulation of particular target genes relevant to leukemogenesis in this region. The translocation that occurred concomitant to the three-way rearrangement -t(5;18)(q13;p11.2) -has not been observed in AML and has not been associated with any clinical or hematopathologic features.
In total, there have been thirty-two reported cases of ETV6-ABL1 fusion gene in numerous hematologic malignancies including eleven cases of acute lymphoblastic leukemia, five cases of acute myeloid leukemia, and sixteen cases of myeloproliferative neoplasms (including CML) ( Table 1). The rarity of this chromosomal rearrangement is thought to be due in part to the opposite transcriptional orientation of two genes relative to the centromere, which requires at least three break-and-join events for an in-frame fusion transcript to be formed. The rearrangement is often not detected using conventional cytogenetic techniques because of its cryptic nature due to the similar G-banding pattern of the distal long arm of chromosome 9 and the distal short arm of chromosome 12 [12]. Additionally, it has been observed that commercially available ABL1 FISH probes may not detect aberrations in the gene in this context, suggesting that the abnormality may remain undetected in a number of cases. In interphase cells, for example, the resulting ABL1 signal can be disproportionately small and can potentially be considered as noise and disregarded [1]. Thus, the rarity of the ETV6-ABL1 fusion is not only due to the multi-step mechanism required for its formation, but also because of technological limitations of FISH probes and molecular cytogenetic analysis.
To date, five cases of AML bearing a possible ETV6-ABL1 fusion have been reported ( Table 1). Two of the cases were designated M1, two were M6, and one was not reported. One of the cases showed a straightforward t(9;12)(q34;p13) with concurrent FISH studies showing the 3' ABL1 signal on the 12p, albeit in the context of a complex karyotype [4]. Two of the cases showed     [3,4]. The two remaining cases showed normal karyotypes by conventional cytogenetics, but showed 3' ABL1 signals on 12p [13,14]. Among all of these cases, reported secondary abnormalities observed included gains of chromosomes 8, 9, 11, 12, 14, 17, and 19, as well as a t(1;22) [4,14]. The ETV6-ABL1 fusion includes a helix loop helix (HLH) domain of ETV6 and tyrosine kinase domain of ABL1, and each domain is necessary for constitutive phosphorylation to occur [4]. Millon et al. found that mice transplanted with ETV6-ABL1-positive hematopoietic stem cells developed CML-like myeloproliferative disease, and that the TEL pointed homology oligomerization domain was essential to ETV6-ABL1-driven leukemogenesis [15]. It is well known that the ABL1 kinase has altered catalytic specificity in human leukemia. Coimmunoprecipitation studies show that the ETV6-ABL1 fusion protein tends to form complexes with CrkL in Ba/F3 cells, and this interaction phosphorylates CrkL and possibly CrkII [16]. However, it is known that the in vitro studies tend to have a wider range of substrates than the cellular forms [16]. Further analysis of Crk and CrkL adaptor proteins show that they play an essential role in integrating signals from a wide variety of sources such as apoptotic cells, extracellular matrix molecules, and growth factors, and there is mounting evidence to indicate that these proteins are associated with human diseases including susceptibility to pathogens and cancer [17].
ETV6 along with five other genes, BCR, ZMIZ, EML, and Nup21 form chimeric transcripts with ABL1. There must be a joining of the 3' sequence of ABL1 with the 5' end of the partner genes, and most of these genes are associated with a wide spectrum hematologic malignancies. Despite this heterogeneity, there is likely a common pluripotent stem cell that gives rise to similar transduction pathways and transforming activity [1]. Due to diagnostic, prognostic, and treatment-related implications, these cases further underscore the use of FISH along with routine chromosome analysis to properly characterize rare, albeit clinically significant fusion genes.
Eosinophilia is a recurrent morphologic finding in ETV6-ABL1-positive myeloid malignancies [4]. Of the five known AML ETV6-ABL1 positive cases, three out of the five were reported to have an increased abnormal eosinophil count, consistent with the findings in the present case. Another finding common to most of the patients was leukocytosis and out of those three cases, two had both leukocytosis and eosinophilia [3,4,13,14].
Each patient was treated with chemotherapy including, cytosine-arabinoside, idarubicin, etoposide, mitoxantrone, and cytarabine. The two patients treated with imatinib responded transiently with resolved fluid retention, eosinophilia, and leukocytosis; however, full remission was not achieved [14]. There was only one patient who achieved full cytogenetic and hematological remission 20 months after undergoing allogeneic hematopoietic stem cell transplantation, which suggests its effectiveness in the treatment of ETV6-ABL1-positive AML patients with eosinophilia and leukocytosis [4].
Although there is limited information about the pathogenesis of myeloid neoplasms positive for ETV6-ABL1, chronic myeloid leukemia (CML) positive for BCR-ABL1 has been well studied and the molecular mechanisms of leukemogenesis and courses of clinical management are established. Tyrosine kinase inhibitors (TKI) such as imatinib are effective agents for inhibiting the constitutively activated BCR-ABL1 tyrosine kinase in CML [18]. Similarly, ETV6-ABL1 is also known to constitutively activate the ABL1 tyrosine kinase, leading to cell cycle deregulation and leukemogenesis [19]. Due to the similar molecular pathogenesis of BCR-ABL1 and ETV6-ABL1 driven leukemogenesis, TKIs have also been considered in the treatment of patients bearing the ETV6-ABL1 fusion. Six out of eleven CML patients positive for ETV6-ABL1 reported in the literature were treated with imatinib: three patients showed a transient favorable response followed by relapse, one patient showed significantly decreased levels of leukemic clones, and two patients treated with 400 mg/day during the chronic phase achieved complete remission [1,6,18,[20][21][22]. Of the three that relapsed, Gancheva et al. reported a case in which the patient was administered an additional TKI, nilotinib, and the patient was able to sustain a positive response following the relapse [1]. Perna et al. did further analysis on another patient who achieved complete remission posttreatment which showed that the ETV6-ABL1 transcript became undetectable, the white blood count normalized, and expression of C-MYC, ID1, BCL-XL, and NUP-98 had decreased significantly [22]. All in all, the molecular targets of ETV6-ABL1 and BCR-ABL1 have significant overlaps that warrant further investigation to elucidate the effectiveness of TKIs on ETV6-ABL1 positive hematologic malignancies.
All in all, this is the sixth reported case of AML bearing the ETV6-ABL1 fusion gene and provides additional insight into the pathogenesis of this subset of malignancies. It is particularly important to utilize complimentary cytogenetic methodologies -namely conventional cytogenetics and FISH -in order to elucidate cryptic abnormalities, which occur more frequently in this context, and to properly characterize karyotypic changes. Additionally, screening using RT-PCR as well as other methodologies has proven useful when cytogenetic analysis is unavailable or yields negative results and in the context of broad molecular screening to identify previously unreported cases. Finally, the consideration of tyrosine kinase inhibitors, particularly second-generation ones, in the treatment of ETV6-ABL1-positive hematological malignancies has shown varying responses, and further investigation of its utility and clinical efficacy is warranted.