t(9;12)(q22;p13) ETV6::SYK: A new recurrent cytogenetic aberration and tyrosine kinase gene fusion in myeloid or lymphoid neoplasms associated with eosinophilia

Spleen tyrosine kinase (SYK), a 72 kDa cytoplasmic non-receptor protein tyrosine kinase, is expressed primarily in haematopoietic cells like B-cells, monocytes, macrophages, mast cells, and neutrophils. SYK is critical for B-cell receptor (BCR) signalling, but also has a role in the signal transduction of other receptors like Fc receptors and adhesion receptors.1 Based on this, targeted therapy with entospletinib (GS-9973) and TAK-659, oral SYK inhibitors, is being assessed in B- and T-cell malignancies, while fostamatinib (R788), and cerdulatinib (PRT062070) are being investigated in autoimmune/inflammatory disorders as rheumatoid arthritis, autoimmune haemolytic anaemia and immune thrombocytopenia.2 In addition, SYK gene fusions occur rarely in haematological malignancies. The t(5;9)(q33;q22) encoding the kinase fusion ITK::SYK is recurrent in follicular T-cell lymphoma.3, 4 A t(9;12)(q22;p13) encoding an ETV6::SYK tyrosine kinase fusion gene was reported twice so far, once in a case with an atypical myelodysplastic syndrome (MDS)5 and once in a B-lymphoblastic leukaemia.6 We here report a third case with an ETV6::SYK tyrosine kinases fusion gene. This case, originally diagnosed as a myelodysplastic/myeloproliferative neoplasm, unclassifiable7 with eosinophilia, bears remarkable resemblance to the case described by Kuno et al.5 We document sensitivity of ETV6::SYK-transformed cells in vitro to SYK inhibitors. We therefore propose to classify haematological neoplasms with ETV6::SYK tyrosine kinase gene fusion under the myeloid/lymphoid neoplasms with eosinophilia and tyrosine kinase gene fusions, as defined in the 5th edition of the World Health Organization (WHO) Classification of Haematolymphoid Tumours8 and the International Consensus Classification (ICC) of Myeloid Neoplasms and Acute Leukaemias.9 A male with a history of inflammatory bowel disease, presented at age 51 with weight loss, pruritic skin lesions, mild anaemia, and leucocytosis (28.8 × 109/L) with 40% neutrophils, 33% eosinophils, and 3% myeloid precursors. Bone marrow cytology revealed a hypercellular marrow with myeloid hyperplasia, prominent eosinophilia and marked megakaryocytic dysplasia (micromegakaryocytes, nuclear hypolobation, and separated nuclei) (Figure 1A). Examination of the bone marrow biopsy showed increased myelopoiesis, in the absence of reticulin fibrosis. The bone marrow karyotype was 45,X,-Y,t(9;12)(q22;p13)[9]/46,XY[1] and a breakpoint in the SYK gene was demonstrated by fluorescence in situ hybridization (FISH) (Figure 1C–E). The combination of anaemia, peripheral leucocytosis with megakaryocytic dysplasia, peripheral and central eosinophilia, and a clonal abnormality led to the diagnosis of a myelodysplastic/myeloproliferative neoplasm, unclassifiable7 with eosinophilia. Hydroxyurea (hydroxycarbamide) and steroids induced a partial haematological remission without effect on the cutaneous lesions. One year after diagnosis, the skin lesions were diagnosed as eruptive xanthogranuloma on the basis of clinicopathological examination.10 A second bone marrow examination confirmed the initial findings. In the following months, pegylated interferon (PEG-IFN), psoralen and ultraviolet A therapy, and thalidomide were added, leading to a complete resolution of the skin lesions and the eosinophilia after eight months. Due to toxicities, therapy was eventually reduced to PEG-IFN. Five years after diagnosis, he has extensive and abundant skin lesions (Figure 1B), anaemia, and normal leucocyte and eosinophil counts. Targeted Locus Amplification (TLA-technology) identified an in-frame ETV6::SYK tyrosine kinase gene fusion in bone marrow cells of the patient that was confirmed by Sanger sequencing (Figure 2A). This fusion leads to a transcript joining ETV6 exon 5 to SYK exon 6 (not shown), the same fusion transcript as in the two previous reports.5, 6 Expression of the ETV6::SYK fusion in BaF/3 cells induced interleukin-3-independent growth, as previously demonstrated.5 Both fostamatinib and entospletinib inhibited the growth of transformed Ba/F3 cells, with an IC50 of 333 nM and 141 nM, respectively (Figure 2B), which could be rescued by exogenous interleukin-3. We also explored the effect of fostamatinib and entospletinib on the phosphorylation of ETV6::SYK and its downstream targets STAT5, ERK1/2 and AKT by western blotting. Entosplenotinib inhibited phosphorylation of ETV6::SYK and its downstream targets in the expected range. For fostamatinib, unexpectedly, only a minor effect on ETV6::SYK phosphorylation was detected, even at concentrations three times as high as the calculated IC50 (Figure 2C). Kuno et al. were the first to report the t(9;12)(q22;p12), in a female with an atypical myelodysplastic syndrome, constitutional symptoms and facial nodules. She had mild neutrophilic leucocytosis, an absolute eosinophil count of 1.8 × 109/L, marked megakaryocytic dysplasia and marrow fibrosis, and evolved rapidly to leukaemia.5 The skin lesions, neutrophilic and eosinophilic leucocytosis and marked megakaryocytic dysplasia in the case reported here, are very reminiscent of the case described by Kuno et al.5 Based on the myeloproliferative features and the marked megakaryocytic dysplasia in our case, the diagnosis was a myelodysplastic/myeloproliferative neoplasm, unclassifiable with eosinophilia, but chronic eosinophilic leukaemia (CEL), not otherwise specified, could have been an alternative diagnosis.7 In the latest WHO and ICC classification, the favoured diagnosis would be CEL, as these recent classifications now emphasize abnormal bone marrow morphology as a criterion for this diagnosis. Zhou et al. described one case of B-cell acute lymphoblastic leukaemia (B-ALL) with t(9;12)(q22;p13).6 Mice transplanted with ETV6::SYK-transduced haematopoietic stem cells develop an aggressive myelodysplasia/panmyelosis with marked dysplastic features in the megakaryocytic lineage11, 12 that was only partially sensitive to the SYK inhibitor fostamatinib (R788).12 In summary, this is a second case of t(9;12)(q22;p13) ETV6::SYK with skin lesions, and a haematological neoplasm labelled as atypical MDS5 and as myelodysplastic/myeloproliferative neoplasm (unclassifiable), CEL NOS or CEL in this report, depending on the classification used.7-9 We confirm that the ETV6::SYK fusion gene conveys growth factor independence in BaF/3 cells that was sensitive to fostamatinib and entospletinib. Entospletinib (GS-9973) is an orally bioavailable selective SYK inhibitor with an IC50 of 7.7 nM in a cell-free assay. It is more active against and more selective for SYK than fostamatinib (R788), the prodrug of the active metabolite R406.13 To the best of our knowledge, we are the first to show the specific inhibitory potential of entospletinib on oncogenic SYK fusions: the inhibition of growth factor-independent growth correlated nicely with inhibition of phosphorylation of ETV6::SYK and its downstream targets, while fostamatinib failed to inhibit this phosphorylation. Entospletinib is also able to induce cell death in vitro in ETV6::RUNX1-derived cell lines, and is again stronger in this respect than fostamatinib.14 Together, the data strongly support that the ETV6::SYK tyrosine kinase gene fusion is the driver underlying this clinicobiological entity. Unfortunately, we have not been able to obtain permission to treat the patient with entospletinib. Lastly, t(9;12)(q22;p13) ETV6::SYK has also been reported in one case of B-ALL with t(9;12)(q22;p13). The report of a B-lineage lymphoid neoplasm with the t(9;12)(q22;p13) ETV6::SYK suggests that the mutation occurs in a pluripotent lymphoid/myeloid stem cell. We therefore propose to classify haematological neoplasms with t(9;12)(q22;p13) ETV6::SYK among the myeloid/lymphoid neoplasms with eosinophilia and tyrosine kinase gene fusions.8, 9 Els Lierman designed the in vitro study, analysed the data and wrote the paper; Sanne Smits and Koen Debackere performed research and analysed the data; Marc André provided patient data and revised the article for intellectual content; Lucienne Michaux and Peter Vandenberghe performed cytogenetic and FISH analysis, designed the study, analysed the data, and wrote the paper. The authors declare no competing financial interests. This research was supported by grant G090815N from Fonds voor Wetenschappelijk Onderzoek-Vlaanderen to Peter Vandenberghe. The authors thank Prof. dr. C Graux (CHU UCL-Namur) for providing images.