A novel 3-way translocation involving ETV6::IL3 drives AML with eosinophilia

TO THE EDITOR:

Eosinophilia is seen in both reactive and clonal processes. In acute myeloid leukemia (AML), the presence of eosinophilia often represents underlying gene rearrangements.^1-3 Underlying cytogenetic aberrations inform prognostic and therapeutic decision-making. ETV6 rearrangements are associated with different partner genes, including PDGFRA and NTRK3, and when involving ABL1 can present with eosinophilia and a chemotherapy resistant disease course.^4,5 Here, we investigate an unusual case of AML with profound eosinophilia involving a 3-way translocation of IL3, ETV6, and RP11-815J21.3 [t(5;12;15)].

A 48-year-old man presented with fevers, odynophagia, and cervical lymphadenopathy for several weeks. Initial workup demonstrated a white blood cell count of 243 × 10³/μL (77% blasts). Bone marrow (BM) biopsy and aspirate confirmed AML with myelomonocytic differentiation, with 90% blasts (Figure 1A-C). Cytogenetics revealed a NUP98::NSD1 [t(5;11)] translocation in 91% of cells and next generation sequencing (NGS) revealed FLT3^ITD and RUNX1^Q397fs∗¹⁹⁷ mutations (Figure 1D). He was treated with 7+3 and midostaurin. Day 21 BM biopsy showed residual disease and he was transitioned to azacitidine, venetoclax, and gilteritinib with subsequent remission, loss of FLT3^ITD, and undetectable NUP98::NSD1 by NGS and fluorescence in situ hybridization (FISH) (Figure 1D). Seven months after diagnosis, while receiving cycle 5 of therapy, he developed profound peripheral eosinophilia, reaching an absolute eosinophil (Eos) count of 61.5 × 10³/μL (Figure 1E-F). Strongyloides immunoglobulin G was positive and he was treated with ivermectin; however, eosinophilia was unresolved. BM biopsy showed AML relapse, with 5% to 8% myeloblasts and 19% eosinophils (Figure 1G-H). The NUP98::NSD1 translocation and RUNX1 mutation were again detected but notably there was no evidence of FLT3^ITD mutation (Figure 1D). Instead, a new rearrangement was identified in 45% of cells, a 3-way translocation of chromosomes 5, 12, and 15, respectively involving IL3, ETV6, and RP11-815J21.3, evolving from the NUP98::NSD1 clone (Figure 1I; supplemental Table 1). NGS identified fusion transcripts between ETV6 from exon 2 and RP11-815J21.3 (a lnc RNA gene) without an in-frame fusion product (supplemental Table 2), and the remaining 3’ ETV6 was juxtaposed to IL3 on der(12) (supplemental Figure 1).

Over the next 4 months, the patient was treated with various regimens including FLAG-Ida (fludarabine, cytarabine, idarubicin, and granulocyte colony-stimulating factor) and CLAG (cladribine, cytarabine, and granulocyte colony-stimulating factor). Higher eosinophilia levels corresponded to a higher abundance of t(5;12;15), with peak eosinophilia (52% in BM) corresponding to 57% FISH⁺ (gray arrow, Figure 1D-E). At a subsequent relapse when this translocation was reduced to 2.5%, the eosinophilia improved (green arrow, Figure 1D-E). Instead, this relapse, with 40% to 50% myeloblasts, was driven by recurrent and new NUP98::NSD1; RUNX1; FLT3^ITD clones.

During his course, the patient suffered and recovered from a ventricular arrhythmia and cardiac arrest. QTc prolongation was considered an etiology, though he was not receiving QTc prolongating agents at the time. Systolic dysfunction was also noted, which was primarily attributed to cumulative anthracycline. However, hypereosinophilia remained on the differential and may have contributed to cardiac dysfunction, and he was treated with a course of prednisone.⁶ 

Because of their correlation, we hypothesized that the translocation may drive dysregulated IL3, leading to eosinophilia. We measured serial plasma samples. At time of relapse with eosinophilia, peripheral blood, and BM aspirate interleukin-3 (IL-3) levels were elevated (54 pg/mL, 123 pg/mL) compared to normal and noneosinophilic AML samples (<0.3-1.3 pg/mL) (Figure 2A). After treatment, when eosinophilia had largely resolved, plasma IL-3 levels declined (8 pg/mL). IL-5 and IL-18, cytokines that regulate eosinophil activation, were elevated but did not correlate with the degree of eosinophilia (Figure 2A).^7,8 Other cytokines uninvolved in eosinophil expansion demonstrated levels similar to those in AMLs without eosinophilia (supplemental Figure 2). Elevated IL3 messenger RNA at relapse correlated with higher IL-3 levels (Figure 2B). To test whether the eosinophils were derived from the mutant clone, eosinophils were purified using the marker Siglec-8. ETV6::IL3 translocation was positive in 39% of these cells (Figure 2C). However, the percentage of peripheral blood eosinophils was greater than the percentage positive for ETV6 rearrangement (71% Eos vs 32% FISH⁺). This suggests both IL-3 autocrine and paracrine effects.

It is intriguing to speculate whether IL-3 also supported outgrowth of the NUP98::NSD1; RUNX1^Q397fs∗¹⁹⁷ clone as RUNX1 loss-of-function may confer IL-3 dependence.⁹ The initial relapse corresponded with elevated IL-3 without FLT3^ITD (Figure 1D, red arrow). In addition, the IL-3 receptor, Interleukin 3 Receptor Subunit alpha (IL3RA) or CD123, was negative at diagnosis but upregulated at relapse (Figure 2D).

To assess the frequency of ETV6::IL3 translocation, we reviewed our tumor cytogenomic database between October 2017 and August 2024. Three other t(5;12) cases were identified, but none involved the 5q31 and 12p13 breakpoints. Next, we searched for translocations involving the IL3 locus (5q31). A case of B-cell acute lymphoblastic leukemia (ALL) was identified with t(5;14)(q31;q32) involving the IGH and IL3 loci (Figure 2E). Notably, peripheral eosinophilia (80% Eos) was identified at presentation (Figure 2E). This patient was diagnosed with heart failure with an ejection fraction of 24% and an infiltrative process on echocardiogram, attributed to the eosinophilia. Pretreatment with mepolizumab was followed by chemotherapy for ALL, with subsequent improvement in peripheral eosinophilia (5% Eos) and an increase in ejection fraction 1 month later.

To our knowledge, this variant t(5;12;15) has not been previously reported. Rare t(5;12)(q31,p13) case reports have been described in hematologic malignancies, including AML, ALL, chronic eosinophilic leukemia, and myelodysplastic syndrome.^10,IL3 is in close genomic proximity to ACSL6, and some cases identified as ACSL6::ETV6 rearrangements have similar presentations with dysregulated IL-3 and eosinophilia.¹¹ Mechanistically, it has been shown that the translocation results in a super-enhancer (SE) in 3’ ETV6 being brought into cis-regulatory configuration, leading to upregulated IL3 expression.¹² 

In leukemia, ETV6 can be heavily H3K27 acetylated, a mark associated with SEs (supplemental Figure 3).¹³ Bromodomain and extraterminal (BET)-family members mediate these enhancers by binding acetylated histones.¹⁴ Given a similar 3’ ETV6 configuration in this case, we treated the patient’s leukemic BM cells with BET-inhibitors to test whether SEs were responsible for IL3 expression. Both JQ1 and ABBV 744 led to decreased expression of IL3 and ETV6, whereas JAK1/2 inhibitor, ruxolitnib, which blocks IL3 signaling, did not (Figure 2F). Expression of other genes not thought to be regulated by SEs, including CD123, were not affected.

Eosinophilia can readily be detected, but its etiology is not always apparent. Here, we describe a case of a 3-way translocation involving the IL3 locus. This results in elevated IL-3 production, with likely autocrine and paracrine induction of eosinophilia and leukemic blast adaptation. Other eosinophil-associated cytokines also became elevated. A similar mechanism of translocation-induced enhancer proximity is observed in IL3::IGH B-cell ALLs with eosinophilia.^15,16 This ETV6 enhancer-specific etiology is important to distinguish from the t(5;12)(q33;p13) PDGFRB::ETV6 rearrangement that is cell intrinsic and is tyrosine kinase inhibitor-responsive. Instead, eosinophilia involving t(5;12)(q31,p13) is not tyrosine kinase inhibitor-responsive and more likely responds to chemotherapy. Clinicians need to be vigilant for hypereosinophilia, as its treatment may mitigate end organ damage.¹⁷ The elevated IL-5 level observed here suggests that agents such as mepolizumab, used in hypereosinophilic syndrome, may be useful in symptom management.¹⁸ Targeting IL-3 signaling through JAK1/2 inhibition or directly blocking SEs through BET-inhibition may also have activity.^9,19 CD123-targeting agents have shown efficacy in CD123-positive AML, and should be explored as treatment in these patients.^18,19 Ultimately, this patient received further chemotherapy with BM blasts reduced to 5% and underwent allogeneic transplantation. He achieved a brief remission with NGS negative for mutations, but unfortunately later relapsed, first with eosinophilia then with increased blasts and succumbed to his AML.

Acknowledgments: A.H.S. is supported by an American Society of Hematology Scholars Award and a Leukemia Research Foundation Grant.

Contribution: J.M., V.N., and A.H.S. were responsible for study conception, design, and oversight of analysis; A.S., J.T., D.H., B.K. were responsible for experimental execution and data analysis; B.P., C.S., and A.S.D. were responsible for pathology interpretation; A.S., D.H., S.E.G.-B., M.B., B.F., K.B., D.T., J.F., H.L., M.K., A.K., B.K.M. were responsible for provision of study materials and/or patients; A.S. and A.H.S. wrote the manuscript; and all authors provided additional reviews, edits, and approval of the manuscript.

Conflict-of-interest disclosure: J.M. receives research funding paid to the institution from Incyte, Novartis, Celgene, Bristol Myers Squibb (BMS), Kartos, Karyopharm, PharmaEssentia, AbbVie, Geron, CTI BioPharma; and consulting fees from Incyte, Kartos, Karyopharm, Geron, Roche, AbbVie, CTIBiotech, GlaxoSmithKline (GSK), Pfizer, PharmaEssentia, Galecto, Celgene, BMS, and Novartis. D.T. receives contracted research funding paid to his institution from CTI BioPharma, Astellas Pharma, and Gilead; and consulting fees from CTI BioPharma, Novartis, AbbVie, Sierra Oncology, GSK, and Cogent Biosciences. J.F. receives contracted research funding paid to his institution from Syros Pharmaceuticals and ORYZON. M.K. receives research funding paid to the institution from Incyte, Celgene, BMS, MorphoSys, Protagonist, Ionis, Silence Therapeutics, Kura Oncology; and consulting fees from Incyte, AbbVie, MorphoSys, and Protagonist. The remaining authors declare no competing financial interests.

Correspondence: Alan Shih, Icahn School of Medicine at Mount Sinai, Box 1079, 1 Gustave L Levy Place, New York, NY 10029; email: [email protected].