AMP case report: ETV6/FLT3 fusion gene detected in a patient with T-cell lymphoblastic lymphoma

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Khadija Belhassan, MD; Abdulrahman Saadalla, MB, Bch
Nicole L. Hoppman, PhD; Yi-Shan Lee, MD, PhD
Camille N. Abboud, MD; Matt Webley, CG(ASCP)
Sarah Koon, CG(ASCP); Julie Neidich, MD
Yang Cao, PhD

April 2022—Genetic alterations of the gene FLT3, especially internal tandem duplications in the juxtamembrane domain and point mutations in the tyrosine kinase domain, are commonly seen in patients with newly diagnosed myeloid leukemias. However, chromosome rearrangements involving the FLT3 gene are extremely rare in hematologic malignancies. The FLT3 gene has only a few known partner genes, including the gene ETV6, which encodes a transcriptional repressor. ETV6 has a wide variety of translocation partner genes, several of which are tyrosine kinase genes. When ETV6 is fused to a tyrosine kinase gene, the N-terminal helix-loop-helix domain of ETV6 functions as a homodimerization motif that activates the tyrosine kinase domain of its partner gene. ETV6/FLT3 fusions are extremely rare and have been reported in a small number of cases with myeloid/lymphoid mixed neoplasms in association with eosinophilia.

We present a patient with T-lymphoblastic lymphoma-associated eosinophilia. Chromosome analysis revealed translocation t(12;13)(p13;q12) in the bone marrow specimen. The follow-up mate-pair sequencing analysis refined the translocation breakpoints and identified the ETV6/FLT3 gene fusion, which is potentially targetable by FLT3-specific tyrosine kinase inhibitors for treatment.

Case. A 22-year-old male patient presented to the clinic with rapid enlargement of the cervical lymph nodes. A complete blood count showed a normal white blood cell count with marked eosinophilia and slightly elevated absolute monocyte count. Computed tomography scan of the neck showed significant bilateral cervical lymphadenopathy at all anatomical levels. An excisional biopsy of a right enlarged deep cervical node was obtained. The lymph node specimen was extensively involved by T lymphoblastic lymphoma histologically characterized by immature-appearing CD3+ lymphoid cells with coexpression of Tdt, CD1a, CD2, CD4, CD5, and CD7. A significant subset of these cells showed coexpression of CD4 and CD8. A bone marrow aspirate/biopsy was obtained for histopathological evaluation. There was limited morphologic or immunophenotypic evidence of T-cell malignancy, with only scattered CD3+ T cells noticed in this bone marrow specimen. Instead, an increase of immature B cells were noticed in this bone marrow specimen. The patient was subsequently started on chemotherapy.

A follow-up positron emission tomography (PET)-CT scan at three months post-treatment initiation showed interval resolution of hypermetabolic activity. However, a later restaging PET-CT detected a widespread increase in fluorodeoxyglucose uptake involving multiple lymph node groups, and also an overall increased uptake in the bone marrow. Concurrent CBC also showed recurring eosinophilia indicative of disease relapse. A core needle axillary lymph node biopsy and a bone marrow aspirate/biopsy were then obtained and showed histopathologic features of disease persistence/relapse.

Chromosome analysis performed at diagnosis on bone marrow aspirate revealed a translocation involving chromosomes 12 and 13, with breakpoints at 12p13 and 13q12 (Fig. 1A) in five of 20 metaphases. The same translocation was identified at six months post-chemotherapy initiation, detected in 14 of 20 metaphases. Fluorescence in situ hybridization analysis using probe panels targeting common cytogenetic abnormalities in T-cell and B-cell neoplasms were negative: no aneuploidies of chromosomes 4, 10, or 17; no rearrangements of BCR/ABL1, MLL, IGH, ETV6/RUNX1, E2A, TRA/D, or TRB; and no CDKN2A deletion were detected.

To further characterize this t(12;13), post-chemotherapy bone marrow aspirate specimen was sent to the Mayo Clinic clinical genomics laboratory for DNA-based next-generation sequencing mate-pair sequencing (MPseq) for breakpoint detection and characterization. The t(12;13) translocation was confirmed by MPseq with breakpoints located within the ETV6 and the FLT3 genes on chromosomes 12 and 13, respectively. This rearrangement is predicted to create an in-frame ETV6/FLT3 fusion gene consisting of 5’ ETV6 (exons 1–6, transcript NM_001987) and 3’ FLT3 (exons 14–24, transcript NM_004119), seq[GRCh38]t(12;13) 12qter>12p13.2(11,896,468)::13q12.2(28,034,123)->13qter;12pter->12p13.2(11,884,677)::13q12.2(28,034,219)->13pter) (Fig. 1B). The fused 5’ end of ETV6 encodes the helix-loop-helix domain (exons 3 and 4), the internal domain (exon 5), and part of the ETS domain (exons 6–8), whereas the fused 3’ end of FLT3 includes the transmembrane domain (TMD) and the tyrosine kinase domain (TKD) (Fig. 1C).

Discussion. ETV6/FLT3 fusions are very rare in hematologic malignancies, with only eight cases reported to date since 2006 (Table 1). This gene fusion is oncogenic and has been shown to induce interleukin-3-independent transformation of Ba/F3 murine hematopoietic cells in vitro and development of myeloproliferative neoplasms phenotype in transgenic mice.^1,2 Structural rearrangements involving ETV6 are common in leukemias with more than 30 characterized partner genes.³ Several of these identified ETV6 partners encode tyrosine kinases, which are homo-dimerized when fused to the functional helix-loop-helix domain of ETV6, resulting in ligand-independent constitutive activation of the partner kinase domain receptors. Furthermore, ETV6 fusions could also contribute to leukemogenesis by modifying the original functions of fused transcription factors, including the loss of transcriptional repression mediated by wild-type ETV6.^3,4

Rearrangements of ETV6 with other tyrosine kinase encoding genes such as ABL1 and FGFR1 are among described drivers of clonal eosinophilia.^5-7 With the growing list of recurrent rearrangements associated with eosinophilia, rearrangements involving PDGFRA/B and FGFR1, or the PCM1-JAK2 rearrangement, are now recognized by the 2016 WHO classification of hematopoietic tumors as part of the diagnostic criteria for myeloid/lymphoid neoplasms with eosinophilia.⁸ The importance of the timely identification of these rearrangements lies in the potential response to tyrosine kinase inhibitor therapy. Although TKI monotherapy or with chemotherapy does not typically achieve long-term disease control, its early use for disease cytoreduction allows bridging to allogeneic stem cell transplant and seems to be associated with better outcomes.^9,10

In this case, the FLT3-specific TKI, midostaurin, was initiated as an adjuvant to hyper-CVAD chemotherapy after the identification of ETV6/FLT3 fusions. Following completion of chemotherapy, PET-CT scan showed resolution of hypermetabolic activity, and bone marrow biopsy was negative for disease involvement. The patient then proceeded to haploidentical bone marrow transplant. He continues to be in remission to date, over two years post-transplant. Although the genetic finding of an ETV6/FLT3 fusion may lead to effective treatment using FLT3-specific TKIs, it is still unclear how this rearrangement correlates with the patient’s hematopathologic findings.

In all previously reported cases (summarized in Table 1), the t(12;13)(12p13;13q12) rearrangements were not cryptic and were evident by karyotyping. Although these breakpoints are molecularly heterogeneous, the presence of eosinophilia, which is a constant feature (along with T-cell lymphoproliferative phenotypes or less specifically myeloid proliferative neoplasm), should raise suspicion of an ETV6/FLT3 rearrangement.^5,11 T-cell phenotypes associated with this rearrangement seem to commonly resemble T-cell lymphoblastic lymphoma, as reported in this case and other previous reports.^10,12 Employing an ETV6 break-apart FISH probe could be useful to focus testing on identifying partner-fused genes. Subsequent testing using conventional FISH probes or nested PCR primers could also be pursued. However, this strategy would entail clinical validations of multiple gene-specific fluorescent probes or primer sets. Testing workups to determine eligibility for TKIs based on gene rearrangements can hence become extensive and ultimately uninformative in some cases.¹³ With the availability and reduced cost of NGS-based sequencing methods capable of detecting structural rearrangements, there is potential for providing timely, cost-effective testing results that could be actionable for treating clinicians. This case is a perfect example to demonstrate superior performance of MPseq when identifying cryptic structural abnormalities across the entire genome and guiding treatment.

In summary, ETV6/FLT3 rearrangement falls in the spectrum of oncogenic tyrosine kinase fusions causing eosinophilia and are potentially targetable by FLT3 inhibitors. However, it remains unclear how this rearrangement leads to neoplastic lymphoid and/or myeloid phenotypes. The adoption of novel NGS-based methods such as MPseq will hopefully facilitate the characterization of more cases and increase our understanding of the underlying pathogeneses and presentations associated with ETV6/FLT3 rearrangements.

1. Baldwin BR, Li L, Tse KF, et al. Transgenic mice expressing Tel-FLT3, a constitutively activated form of FLT3, develop myeloproliferative disease. Leukemia. 2007;21(4):764–771.
2. Vu HA, Xinh PT, Masuda M, et al. FLT3 is fused to ETV6 in a myeloproliferative disorder with hypereosinophilia and a t(12;13)(p13;q12) translocation. Leukemia. 2006;20(8):1414–1421.
3. De Braekeleer E, Douet-Guilbert N, Morel F, Le Bris MJ, Basinko A, De Braekeleer M. ETV6 fusion genes in hematological malignancies: a review. Leuk Res. 2012;36(8):945–961.
4. Rasighaemi P, Ward AC. ETV6 and ETV7: siblings in hematopoiesis and its disruption in disease. Crit Rev Oncol Hematol. 2017;116:106–115.
5. Reiter A, Gotlib J. Myeloid neoplasms with eosinophilia. Blood. 2017;129(6):704–714.
6. Carll T, Patel A, Derman B, et al. Diagnosis and treatment of mixed phenotype (T-myeloid/lymphoid) acute leukemia with novel ETV6-FGFR2 rearrangement. Blood Adv. 2020;4(19):4924–4928.
7. Keene P, Mendelow B, Pinto MR, et al. Abnormalities of chromosome 12p13 and malignant proliferation of eosinophils: a nonrandom association. Br J Haematol. 1987;67(1):25–31.
8. Arber DA, Orazi A, Hasserjian R, et al. The 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia. Blood. 2016;127(20):2391–2405.
9. Gerds AT, Gotlib J, Bose P, et al. Myeloid/lymphoid neoplasms with eosinophilia and TK fusion genes, Version 3.2021, NCCN Clinical Practice Guidelines in Oncology. J Natl Compr Canc Netw. 2020;18(9):1248–1269.
10. Chonabayashi K, Hishizawa M, Matsui M, et al. Successful allogeneic stem cell transplantation with long-term remission of ETV6/FLT3-positive myeloid/lymphoid neoplasm with eosinophilia. Ann Hematol. 2014;93(3):535–537.
11. De Braekeleer E, Douet-Guilbert NDB, De Braekeleer M. t(12;13)(p13;q12) ETV6/FLT3. Atlas Genet Cytogenet Oncol Haematol. 2015;19(6):410–413.
12. Walz C, Erben P, Ritter M, et al. Response of ETV6-FLT3-positive myeloid/lymphoid neoplasm with eosinophilia to inhibitors of FMS-like tyrosine kinase 3. Blood. 2011;118(8):2239–2242.
13. Yang LH, Zhao Y, Maule J, Rapisardo S, Wang E. T-lymphoblastic lymphoma and acute myeloid leukaemia transformed from myeloid neoplasm with eosinophilia: a divergent evolution of myeloid neoplasm with monosomy 7 but no detectable tyrosine kinase gene rearrangements designated by the WHO Classification. Br J Haematol. 2020;190(5):e307–e312.
14. Falchi L, Mehrotra M, Newberry KJ, et al. ETV6-FLT3 fusion gene-positive, eosinophilia-associated myeloproliferative neoplasm successfully treated with sorafenib and allogeneic stem cell transplant. Leukemia. 2014;28(10):2090–2092.
15. Hosseini N, Craddock K, Salehi-rad S, et al. ETV6/FLT3 fusion in a mixed-phenotype acute leukemia arising in lymph nodes in a patient with myeloproliferative neoplasm with eosinophilia. J Hematopathol. 2014;7(2):71–77.
16. Zhang H, Paliga A, Hobbs E, et al. Two myeloid leukemia cases with rare FLT3 fusions. Cold Spring Harb Mol Case Stud. 2018;4(6):a003079.

Drs. Belhassan, Saadalla, Lee, Neidich, and Cao are in the Department of Pathology and Immunology, and Dr. Abboud is in the Division of Oncology, Department of Medicine—all at Washington University School of Medicine in St. Louis. Dr. Hoppman, Matt Webley, and Sarah Koon are in the Division of Laboratory Genetics and Genomics, Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minn.

Test yourself

Here are three questions taken from the case report.

1. Which of the following statements is true about FLT3?
a. FLT3 mutations are identified in about five percent of patients with newly diagnosed myeloid leukemias.
b. FLT3-ITD indicates the internal tandem duplications in the tyrosine kinase domain.
c. FLT3 activators have been approved for clinical use, leading to therapeutic paradigms for AML with FLT3 mutations.
d. It is recommended to screen for FLT3 mutations by the National Comprehensive Cancer Network guidelines for new acute leukemia cases.

2. Which of the following tests could potentially refine the breakpoints of a translocation detected in chromosomal analysis?
a. Chromosome microarray.
b. NGS-based mate-pair sequencing.
c. MLPA.
d. STR analysis.

3. Which of the following tyrosine kinase fusion genes is not recognized by the 2016 WHO classification of hematopoietic tumors as part of the diagnostic criteria for myeloid/lymphoid neoplasms with eosinophilia?
a. ETV6-RUNX1.
b. ETV6-PDGFRB.
c. PCM1-JAK2.
d. ZMYM2-FGFR1.

Answers are online now at www.amp.org/casereports.