ETV6::ABL1-Positive Myeloid Neoplasm: A Case of a Durable Response to Imatinib Mesylate without Additional or Previous Treatment

ETV6::ABL1 rearranged neoplasms are rare hematological diseases. To date, about 80 cases have been reported, including myeloid and lymphoid leukemias. The ETV6 gene codes for an ETS family transcription factor and several fusion partners have been described. When translocated, ETV6 causes the constitutive activation of the partner genes. Here, we report the case of a 54-year-old woman with a cryptic insertion of the 3′ region of ABL1 in the ETV6 gene. The patient was first diagnosed with idiopathic hypereosinophilic syndrome, according to the clinical history, conventional cytogenetics, standard molecular analyses and pathologist description. Next generation sequencing of diagnosis samples unexpectedly detected both ETV6::ABL1 type A and B fusion transcripts, which were then confirmed by FISH. The diagnosis was Myeloid/Lymphoid neoplasm with ETV6::ABL1 fusion, and the patient received imatinib mesylate treatment. In a follow-up after more than one year, the patient still maintained the molecular and complete hematological responses. This case highlights the importance of timely and proper diagnostics and prompt tyrosine kinase inhibitor treatment.


Introduction
ETV6::ABL1 (also known as TEL::ABL1) is a rare fusion that has been found in different types of hematological diseases.To date, about 80 cases of hematological neoplasms (more frequently, acute lymphoblastic leukemia (ALL), myeloproliferative neoplasms (MPNs), Philadelphia-negative chronic myeloid leukemia (CML) [1], atypical (a)CML and chronic myelomonocytic leukemia (CMML)) carrying ETV6::ABL1 translocation have been reported [2][3][4][5][6].ETV6::ABL1 chimeric protein has been found in less than 1% of ALL cases; however, due to the lack of a systematic screening for this fusion transcript, its overall incidence in hematological neoplasms cannot be precisely defined [2].The ETV6 (ETS variant 6) gene encodes for an ETS family transcription factor containing two functional domains: the N-terminus PNT (exons 3-4) and the C-terminus ETS (exons 6-7) domains, flanking the central domain coded by the exon 5 [7].ETV6 is involved in the maintenance of the vascular network, hematogenesis, embryogenesis and development of different tissues [7].The ABL1 gene encodes for a non-receptor tyrosine kinase containing different structural domains, including SRC-homology domains (SH1, SH2 and SH3) responsible for the regulation of their own activity, DNA-binding (DB) domains and actin-binding (AB) domains, in addition to a nuclear translocation signal (NTS) sequence, sites for phosphorylation by protein kinase C (PKC) and a proline-rich sequence [8].Two types of ETV6::ABL1 in-frame fusion isoforms have been described: the so-called "type A", involving exon 4 of ETV6 and exon 2 of ABL1, and the "type B" translocation, involving exon 5 of ETV6 and exon 2 of ABL1.The ETV6::ABL1 fusion protein retains both the SH domains and the tyrosine kinase domain of ABL1 [9], leading to the loss of ABL1 autoinhibitory activity, thus resulting in a constitutive active enzyme.Both transcripts encode for a chimeric non-receptor tyrosine kinase resembling the BCR::ABL1 structure.Moreover, in vitro studies have demonstrated that ETV6::ABL1 phosphorylates the same substrates activated by BCR::ABL1 chimeric proteins, suggesting that ETV6 may replace the BCR role and activate ABL1 [10], and explaining why patients harboring ETV6::ABL1 translocation are sensitive to TKI treatment [9].Here, we present the case of a patient who received a diagnosis of Myeloid/Lymphoid neoplasm with ETV6::ABL1 fusion and reached a durable response by imatinib mesylate treatment.

Case Description
In January 2019, a 54-year-old Caucasian woman was referred to our institution for leukocytosis.She had a mild increase in white blood cell (WBC) count over one year (mean WBC 12 × 10 9 /L, mean neutrophils 8 × 10 9 /L), basophilia (5%) and eosinophilia (14%).JAK2, CALR and MPL mutations and BCR::ABL1 rearrangements were negative (peripheral blood).The patient was asymptomatic and was not receiving any chronic treatment.She had no significant medical history, no history of smoking, no ongoing infections, negative inflammation markers, a normal chest and abdomen examination, and a normal abdomen ultrasonography (US).The patient underwent a regular follow-up (every 4 months) and did not receive any treatment.In the following 18 months, the WBC counts fluctuated around 11-13 × 10 9 /L and the clinical patients' conditions were stable.In June 2020, the WBC count was raised to 32 × 10 9 /L (basophilia 4%, eosinophilia 11%), while hemoglobin and platelets count were within the normal range, spleen was not palpable and the abdomen US results were normal.BCR-ABL1 translocation and JAK2, CALR, and MPL mutations were confirmed to be negative (peripheral blood).Furthermore, no PDGFRA, PDGFRB or FGFR1 rearrangements were detected.A trephine biopsy showed hypercellular bone marrow (95%) with a diffuse eosinophilic infiltration, slightly reduced erythropoiesis, normal CD34 + cells and mastocytes (Figure 1).Cytogenetic examination showed a unique clone characterized by 47, XX, +12 (on 20 metaphases, Figure 2A).Chest X-ray and heart US excluded any significant organ involvement.Therefore, the patient was disagosed with idiopathic hypereosinophilic syndrome.
Next generation sequencing (NGS) was performed on the diagnosis peripheral blood sample, on both DNA and RNA, using the Oncomine Myeloid Assay (Thermo Fisher Scientific, Waltham, MA, USA), surprisingly revealing the presence of both ETV6::ABL1 type A and B fusion transcripts (Figure 2B and Table S1), while confirming the absence of the Philadelphia chromosome.The ETV6::ABL1 fusions were confirmed by RT-PCR (Figure 2C) and Sanger sequencing (Figure 2D,E).No additional DNA variants were found.Further information on the sample collection, nucleic acids isolation, library preparation and RT-PCR are reported in the Supplementary Methods.The variants and fusions tested are reported in Tables S2-S4.In order to confirm the fusion transcripts revealed by NGS, we performed fluorescent in situ hybridization (FISH).FISH analysis using an ETV6 break-apart probe revealed three copies of the ETV6 gene without evidence of ETV6 rearrangement (Figure 3A).On the contrary, FISH analysis with a BCR::ABL1 Tricolor Color Dual Fusion (TCDF) probe confirmed the presence of the ABL1 rearrangement with ABL located on one chromosome 12 (Figure 3B).In order to confirm the ETV6::ABL1 fusion, we performed FISH analysis combining ETV6::RUNX1 ES Dual Color Dual Fusion and BCR::ABL1 TCDF.FISH analysis showed the presence of the ETV6::ABL1 fusion on chromosome 12 (Figure 3C).Therefore, we concluded that the ETV6::ABL1 fusion was the result of a cryptic insertion of the 3 of ABL1 (q34) into the ETV6 locus (12p13).Details on chromosome banding analysis (CBA) and FISH are described in the Supplementary Methods.Based on the results, the diagnosis was modified to Myeloid/Lymphoid neoplasm with ETV6::ABL1 fusion, and the patient started imatinib mesylate treatment at the dose of 200 mg QD (8 September 2020).
NGS and involving exon 5 of ETV6 and exon 2 of ABL1, or exon 4 of ETV6 and exon 2 of ABL1, respectively.(C) RT-PCR revealing the 706 bp ETV6exon5::ABL1exon2 fusion transcript (upper band) and the 160 bp ETV6exon4::ABL1exon2 fusion transcript (lower band).A 100 bp molecular weight marker was used.(D,E) Electropherograms of the sequences spanning the breakpoint confirming in-frame fusions involving ETV6 exon 5 or ETV6 exon 4 and ABL1 exon 2. The dashed lines indicate the breakpoint regions.Each peak represents a single nucleotide in the DNA sequence, and each nucleotide has a different colour; A is green, T is red, C is blue and G is black.MM: Molecular Marker; A: diagnosis; B: 3 months follow-up; C: 6 months follow-up; D: 12 months follow-up; E: RT negative; F: PCR negative.
Next generation sequencing (NGS) was performed on the diagnosis peripheral blood sample, on both DNA and RNA, using the Oncomine Myeloid Assay (Thermo Fisher Scientific, Waltham, MA, USA), surprisingly revealing the presence of both ETV6::ABL1 type A and B fusion transcripts (Figure 2B and Table S1), while confirming the absence of the Philadelphia chromosome.The ETV6::ABL1 fusions were confirmed by RT-PCR (Figure 2C) and Sanger sequencing (Figure 2D,E).No additional DNA variants were found.Further information on the sample collection, nucleic acids isolation, library preparation and RT-PCR are reported in the Supplementary Methods.The variants and fusions tested are reported in Tables S2-S4.
In order to confirm the fusion transcripts revealed by NGS, we performed fluorescent in situ hybridization (FISH).FISH analysis using an ETV6 break-apart probe revealed three copies of the ETV6 gene without evidence of ETV6 rearrangement (Figure 3A).On the contrary, FISH analysis with a BCR::ABL1 Tricolor Color Dual Fusion (TCDF) probe confirmed the presence of the ABL1 rearrangement with ABL located on one chromosome 12 (Figure 3B).In order to confirm the ETV6::ABL1 fusion, we performed FISH analysis combining ETV6::RUNX1 ES Dual Color Dual Fusion and BCR::ABL1 TCDF.FISH analysis showed the presence of the ETV6::ABL1 fusion on chromosome 12 (Figure 3C).Therefore, we concluded that the ETV6::ABL1 fusion was the result of a cryptic insertion of the 3′ of ABL1 (q34) into the ETV6 locus (12p13).Details on chromosome banding analysis (CBA) and FISH are described in the Supplementary Methods.Based on the results, the diagnosis was modified to Myeloid/Lymphoid neoplasm with ETV6::ABL1 fusion, and the patient started imatinib mesylate treatment at the dose of 200 mg QD (8 September 2020).We monitored the most abundant fusion transcript, ETV6exon5::ABL1 exon2, using both RT-PCR and Nested PCR.Follow-up peripheral blood samples were collected and analyzed at 3 months (time point B), at 6 months (time point C), at 12 months (time point D) and at 18 months (time point E) of therapy, according to clinical practice.RT-PCR results were negative in all the follow-up samples (Figure 4A).Negativity was confirmed by NGS at time points B and C using the Myeloid Plus Solution panel (Sophia Genetics).Nested-PCR negativity was reached at time point D, while samples at time points B and C were still weakly positive (1/2 replicates, Figure 4B).The complete hematological response was assessed by blood count, revealing all the parameters to be within the normal range.
The last evaluation-after 3 years of imatinib mesylate treatment-showed a WBC count of around 5 × 10 9 /L and a neutrophils count of 3.70 × 10 9 /L (basophilia 0.6%, eosinophilia 2.4%).The main clinical and laboratory information are summarized in Figure 5.
analyzed at 3 months (time point B), at 6 months (time point C), at 12 months (time point D) and at 18 months (time point E) of therapy, according to clinical practice.RT-PCR results were negative in all the follow-up samples (Figure 4A).Negativity was confirmed by NGS at time points B and C using the Myeloid Plus Solution panel (Sophia Genetics).Nested-PCR negativity was reached at time point D, while samples at time points B and C were still weakly positive (1/2 replicates, Figure 4B).The complete hematological response was assessed by blood count, revealing all the parameters to be within the normal range.The last evaluation-after 3 years of imatinib mesylate treatment-showed a WBC count of around 5 × 10 9 /L and a neutrophils count of 3.70 × 10 9 /L (basophilia 0.6%, eosinophilia 2.4%).The main clinical and laboratory information are summarized in Figure 5.

Discussion
In this study, we report the case of an ETV6::ABL1 rearranged patient with a diagnosis of Myeloid/Lymphoid neoplasm, who received imatinib mesylate treatment and achieved a durable response.
The formation of an in-frame ETV6::ABL1 fusion gene involves complex genomic rearrangements because ETV6 and ABL1 genes have opposite chromosome orientations.Conventional diagnostic techniques (such as conventional cytogenetics) sometimes fail to detect this rearrangement 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.Moreover, no ready-to-use ETV6::ABL1 FISH probes are commercially available, suggesting that the ETV6-ABL1 fusion may remain undetected in a number of patients [5,11].
In the case we present, the use of next generation sequencing allowed us to overcome the above-mentioned limitations and to detect this fusion transcript, which changed the initial diagnosis of hypereosinophilic syndrome into the proper diagnosis of Myeloid/Lymphoid neoplasm with ETV6::ABL1 fusion.
The patient showed the presence of both type "A" and "B" fusion transcripts, first detected by NGS and then confirmed by RT-PCR and Sanger sequencing.FISH analysis combining ETV6::RUNX1 DCDF and BCR-ABL1 TCDF probes performed on metaphases showed a cryptic insertion of the 3′ region of ABL1 in the ETV6 gene on one chromosome 12, in addition to a signal consistent with trisomy 12.
ETV6::ABL1 rearrangements have been reported in different hematological malignancies and, in particular, in ALL, followed by MPNs and acute myeloid leukemia (AML) [11,12].Eosinophilia represents a common clinical feature and a hallmark of all ETV6::ABL1 rearranged MPNs cases reported in the literature [2], while the most frequent molecular alterations observed in ALL or in lymphoid blast crisis (LCB) patients are deletions of CDKN2A/CDKN2B, IKZF1 or PAX5 [2].

Discussion
In this study, we report the case of an ETV6::ABL1 rearranged patient with a diagnosis of Myeloid/Lymphoid neoplasm, who received imatinib mesylate treatment and achieved a durable response.
The formation of an in-frame ETV6::ABL1 fusion gene involves complex genomic rearrangements because ETV6 and ABL1 genes have opposite chromosome orientations.Conventional diagnostic techniques (such as conventional cytogenetics) sometimes fail to detect this rearrangement 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.Moreover, no ready-to-use ETV6::ABL1 FISH probes are commercially available, suggesting that the ETV6-ABL1 fusion may remain undetected in a number of patients [5,11].
In the case we present, the use of next generation sequencing allowed us to overcome the above-mentioned limitations and to detect this fusion transcript, which changed the initial diagnosis of hypereosinophilic syndrome into the proper diagnosis of Myeloid/Lymphoid neoplasm with ETV6::ABL1 fusion.
The patient showed the presence of both type "A" and "B" fusion transcripts, first detected by NGS and then confirmed by RT-PCR and Sanger sequencing.FISH analysis combining ETV6::RUNX1 DCDF and BCR-ABL1 TCDF probes performed on metaphases showed a cryptic insertion of the 3 region of ABL1 in the ETV6 gene on one chromosome 12, in addition to a signal consistent with trisomy 12.
ETV6::ABL1 rearrangements have been reported in different hematological malignancies and, in particular, in ALL, followed by MPNs and acute myeloid leukemia (AML) [11,12].Eosinophilia represents a common clinical feature and a hallmark of all ETV6::ABL1 rearranged MPNs cases reported in the literature [2], while the most frequent molecular alterations observed in ALL or in lymphoid blast crisis (LCB) patients are deletions of CDKN2A/CDKN2B, IKZF1 or PAX5 [2].
ETV6::ABL1 rearranged hematological neoplasms share many clinical features with CML.Indeed, the ETV6::ABL1 fusion protein functionally resembles the BCR::ABL1 ones, being characterized by a constitutive activation of the chimeric transcript [13], and sensitivity to both first- [14] and second-generation tyrosine kinase inhibitor (TKI) treatments [15].For these reasons, the patient received imatinib therapy.Imatinib competitively binds the ABL1 ATP binding site in the ETV6::ABL1 fusion protein by the same mechanism of action described for the BCR::ABL1 protein.
Despite this, in the literature, few patients have received TKI treatment at first manifestation of the disease and/or at the first progression, and most of them died or relapsed/evolved [2].Schwaab et al. presented data on ETV6::ABL1 rearranged MPN patients that received imatinib, nilotinib or dasatinib after a prior treatment with hydroxyurea and/or cytarabine or intensive chemotherapy.Patients receiving imatinib did not achieve a complete cytogenetic (CCR) or molecular (CMR) response, which was instead obtained by patients under nilotinib or dasatinib treatment [16].Accordingly, previous reports showed that imatinib allowed only an initial reduction of disease followed by a mild neutrophilia, basophilia and eosinophilia after 6 months, with persistent ETV6::ABL1 positivity in FISH and nested PCR [13], or a transient response followed by transformation into ALL [17].Moreover, most patients reported in the literature were diagnosed as atypical AML or Ph-like ALL.
Conversely, our patient presented without any blast excess at the diagnosis, and the clinical features were consistent with chronic diseases.She started TKI treatment as a frontline therapy after receiving the correct diagnosis and is still maintaining complete hematological and molecular responses after 36 months of imatinib mesylate.To our knowledge, this is the first case reporting a follow-up longer than one year without any additional [13] or previous chemotherapy treatment [18].Our data likely rule out the co-occurrence of genomic events accounting for resistance in our patient and suggest a potential driver role for the ETV6::ABL1 rearrangement.Studies accounting for different sensitivity profiles, gene expressions and BCR-ABL1-like signatures are warranted in this case; however, an accurate and multicenter sample collection is needed to meet this endpoint.

Conclusions
The detection of the ETV6::ABL1 rearrangement remains difficult, due to its cryptic nature.
A deeper genomic characterization of patients with ETV6::ABL1 fusion transcripts may improve our understanding of the biological complexity behind this disease.Although this could represent a limitation of our study, overall, our results underline the importance of timely and proper diagnostics, and the need to revise the current screening algorithms-for example, by recommending NGS RNA panels-in order to detect and monitor ETV6::ABL1 rearrangements.In this case, molecular biology was instrumental in the diagnosis and, consequently, in the selection of an appropriate therapy.Notably, in the absence of molecular biology results, supportive therapies would have been the only ones administered to the patient.

Figure 3 .
Figure 3. FISH analyses (100× magnification).(A) FISH analysis with ETV6 break-apart on previously G-banded metaphase showing 3 fusion signals on 3 chromosomes 12, indicating 3 copies of ETV6.(B) FISH analysis with BCR-ABL1 TCDF probes on previously G-banded metaphase showing 2 green signals on chromosome 22, two blue/red signals on chromosome 9 and an extra red signal on the short arm of chromosome 12, indicating ABL1 rearrangement.(C) FISH analysis combining ETV6-RUNX1 DCDF and BCR-ABL1 TCDF probes on metaphase showing a fusion between ETV6 marked in spectrum green and ABL1 marked in spectrum orange on derivative chromosome 12, confirming ETV6::ABL1 rearrangement.The arrows indicate the derivatives chromosome.

Figure 3 .
Figure 3. FISH analyses (100× magnification).(A) FISH analysis with ETV6 break-apart on previously G-banded metaphase showing 3 fusion signals on 3 chromosomes 12, indicating 3 copies of ETV6.(B) FISH analysis with BCR-ABL1 TCDF probes on previously G-banded metaphase showing 2 green signals on chromosome 22, two blue/red signals on chromosome 9 and an extra red signal on the short arm of chromosome 12, indicating ABL1 rearrangement.(C) FISH analysis combining ETV6-RUNX1 DCDF and BCR-ABL1 TCDF probes on metaphase showing a fusion between ETV6 marked in spectrum green and ABL1 marked in spectrum orange on derivative chromosome 12, confirming ETV6::ABL1 rearrangement.The arrows indicate the derivatives chromosome.

Figure 4 .
Figure 4. Monitoring of ETV6exon5::ABL1exon2 fusion overtime on peripheral blood samples.(A) RT-PCR revealing the 421-bp ETV6exon5-ABL1exon2 fusion transcript.(B) Nested PCR revealing the 321-bp ETV6exon5::ABL1exon2 fusion transcript.A 100 bp molecular weight marker was used.(C) Electropherogram of the sequence spanning the breakpoint that confirmed in-frame fusion involving ETV6 exon 5 and ABL1 exon 2. The dashed lines indicate the breakpoint regions.Each peak represents a single nucleotide in the DNA sequence, and each nucleotide has a different colour; A is green, T is red, C is blue and G is black.MM: Molecular Marker; A: diagnosis; B: 3 months followup; C: 6 months follow-up; D: 12 months follow-up; E: 18 months follow-up; F: RT negative; G: PCR negative; H: Nested-PCR negative.

Figure 4 .
Figure 4. Monitoring of ETV6exon5::ABL1exon2 fusion overtime on peripheral blood samples.(A) RT-PCR revealing the 421-bp ETV6exon5-ABL1exon2 fusion transcript.(B) Nested PCR revealing the 321-bp ETV6exon5::ABL1exon2 fusion transcript.A 100 bp molecular weight marker was used.(C) Electropherogram of the sequence spanning the breakpoint that confirmed in-frame fusion involving ETV6 exon 5 and ABL1 exon 2. The dashed lines indicate the breakpoint regions.Each peak represents a single nucleotide in the DNA sequence, and each nucleotide has a different colour; A is green, T is red, C is blue and G is black.MM: Molecular Marker; A: diagnosis; B: 3 months follow-up; C: 6 months follow-up; D: 12 months follow-up; E: 18 months follow-up; F: RT negative; G: PCR negative; H: Nested-PCR negative.

Figure 5 .
Figure 5. Timeline displaying clinical and therapy schedule (upper part) and laboratory data (bottom part).

Figure 5 .
Figure 5. Timeline displaying clinical and therapy schedule (upper part) and laboratory data (bottom part).