<i>JAK2</i> exon 12 mutation in myelodysplastic/myeloproliferative neoplasm with ring sideroblasts and thrombocytosis: Not an exclusive mutation to polycythaemia vera

Abstract

In myelodysplastic/myeloproliferative neoplasms (MDS/MPN) with ring sideroblasts and thrombocytosis (MDS/MPN-RS-T), somatic mutation in the Splicing Factor 3B subunit 1 gene (SF3B1) at the rate of approximately 85% was concurrently found with one of the MPN driver mutations, such as JAK2 V617F (50%), calreticulin (CALR) exon 9 (1–3%) and MPL W515 (0–3%) mutations (Patnaik and Tefferi 2017). Impaired erythropoiesis induced by mutant SF3B1 (Mupo et al, 2017, Obeng et al, 2016), and an increase in one or more lineages of blood cells induced by MPN driver mutations (Vainchenker and Kralovics 2017) have led to the notion that the overlapping phenotypes of MDS/MPN are at least partly, if not totally, due to overlapping disease-defining mutations (Cazzola et al, 2013). The MPN driver mutation, JAK2 exon 12, is rare in MPN and has been exclusively found in polycythaemia vera (PV) (Scott 2011), with no reports of this mutation in MDS/MPN-RS-T to date. A bone marrow biopsy of a patient, who was originally followed for Raynaud disease in our hospital and presented with anaemia associated with persistent leucocytosis and thrombocytosis (Fig S1 and Table SI), displayed MDS/MPN features (Fig S2) and lacked any chromosomal abnormality, including Philadelphia chromosome; the patient was thus suspected to have MDS/MPN-RS-T. Sequence analysis (see Data S1) of genomic DNA obtained from a peripheral blood sample revealed the allelic frequencies of SF3B1 E622D, JAK2 H538_K539delinsL (hereinafter called JAK2 exon 12, one of the typical mutations of JAK2 exon 12; Scott 2011), and TET2 I1873T (Fig S3) to be 44%, 75% and 14%, respectively. Based on this evidence, the patient was diagnosed with MDS/MPN-RS-T. As no report on the identification of JAK2 exon 12 mutation in MDS/MPN-RS-T was available, and only limited assessment has been performed for the capacity of haematopoietic cell differentiation in MDS/MPN-RS-T harbouring SF3B1 and MPN driver mutations, we examined the colony-forming ability of the patient's bone marrow mononuclear cells (BM-MNCs). Unlike BM-MNCs obtained from a patient with PV harbouring JAK2 exon 12 mutation and a patient with malignant lymphoma without bone marrow infiltration (hereinafter called control), those from the patient with MDS/MPN-RS-T formed smaller colonies of burst-forming unit-erythroid (BFU-E), colony-forming unit-granulocyte (CFU-G), CFU-macrophage (CFU-M), and CFU–granulocyte, macrophage (CFU-GM) (Fig 1A). Significantly lower number of BFU-Es was formed from BM-MNCs of the MDS/MPN-RS-T samples than from the PV and control samples, even in the presence of erythropoietin (EPO) (Fig 1B). EPO-independent BFU-Es were formed only in those with PV but not in those with MDS/MPN-RS-T harbouring JAK2 exon 12 mutation (Fig 1B), confirming that the patient had neither PV nor post-PV myelofibrosis. Based on the mutation status in individual colonies (Fig 1C and S3) determined by Sanger sequencing (see Data S1), diagrams were created to represent a hypothetical clonal evolution of haematopoietic progenitor cells (Fig 2A,2B). As expected from the patient's anaemic phenotype, an acquisition of SF3B1 mutation occurred earlier than any other mutation. JAK2 exon 12 mutation was always concurrently detected with the SF3B1 mutation, implying that BFU-E formation was blocked by mutant SF3B1, leading to anaemia in the patient, regardless of JAK2 exon 12 mutation. In contrast, in MPN, acquisition of SF3B1 mutation seems to be a later event (Boiocchi et al, 2019). Therefore, we hypothesized that the order of acquisition of SF3B1 mutation and an MPN driver mutation is likely to define the disease type (Fig 2C). Seventeen out of 28 BFU-Es (60.7%) were defined as wild-type (Fig 2A), which is contradictory to the anaemic phenotype of our patient. This discrepancy is presumably owing to the weakened capacity of erythroid cell differentiation in progenitor cells, considering the smaller size of BFU-Es, regardless of SF3B1 and JAK2 exon 12 mutations (Fig 1A). In contrast to the BFU-E formation, the ratio of wild-type (n = 4, 16.0%) versus SF3B1 mutant (n = 21, 84.0%) in CFU-G/M/GMs was significantly reduced compared to that in BFU-Es (P < 0.001, Table SII). This suggests skewing of the progenitor cells harbouring the SF3B1 mutation toward the myeloid lineage. Although the leucocytosis-like phenotype was not recapitulated in the colony assay, these genetic analyses implied that SF3B1 mutation, in combination with JAK2 exon 12 mutation, contributed to the leucocytosis in the patient by skewing the haematopoietic lineage towards myeloid (Fig 1C). Although this needs to be confirmed with bone marrow cells from multiple patients, it definitely suggests the possibility of cell extrinsic factor(s), such as cytokines and the bone marrow niche, playing a key role in the induction of leucocytosis in the patient with MDS/MPN-RS-T. Screening for JAK2 exon 12 mutations may not be routinely performed in daily clinical practice. The identification of concurrent SF3B1 mutation and MPN driver mutation supports the diagnosis of MDS/MPN-RS-T, the prognosis of which is different and treatment options vary across MDS or MPN. We would like to emphasize the importance of analysing JAK2 exon 12 mutation in patients with suspected MDS/MPN-RS-T, but who are negative for JAK2 V617F, CALR exon 9 and MPL W515 mutations. Furthermore, we would like to note that MDS/MPN-RS-T might often be misdiagnosed as essential thrombocythaemia (ET) or prefibrotic/early primary myelofibrosis (pre-PMF), especially when the bone marrow assessment, such as iron staining, is not properly performed. Detection of SF3B1 mutation does not support diagnosis, because this occurs in 5% of ET and 10% of PMF cases (Tefferi et al, 2016a, Tefferi et al, 2016b). A mild anaemia and megakaryocytic dysplasia are associated with pre-PMF. Therefore, we would like to emphasize that the absence or small number, if any, of ring sideroblasts and that of erythroid dysplasia should be confirmed in patients suspected with ET and pre-PMF, especially when they harbour SF3B1 mutation. In summary, we identified a patient with MDS/MPN-RS-T harboring SF3B1 and JAK2 exon 12 mutations. Considering the possibility that the order of acquisition of SF3B1 mutation and an MPN driver mutation defines the disease type, this study highlighted the importance of a comprehensive assessment of gene alterations, as well as pathology, for the diagnosis of MPN and MDS/MPN-RS-T. We thank the members of the Department of Haematology, Juntendo University Graduate School of Medicine for encouraging this study. TI designed the study, carried out the experiment, and wrote the initial draft of the manuscript. MA, YH and NK contributed to manuscript preparation, analysis and interpretation of data. MI performed histological diagnosis of clinical samples. SJ, HN, TO, KM, YF contributed to data collection. MI, YE, AO, NK supervised the study. This work was funded in part by the MEXT's Promotion Plan for the Platform of Human Resource Development for Cancer Project; the JSPS KAKENHI Grant (#17K16195); and Grant for Cross-disciplinary Collaboration in Juntendo University (#30-8). The funders had no role in manuscript preparation. Data S1. Patients and methods. Figure S1. Progressive leukocytosis, thrombocytosis, and anemia observed in the patient. Figure S2. Bone marrow morphologies of the patient harboring JAK2 exon 12 mutation, representing features associated with MDS/MPN-RS-T. Figure S3. Detection of SF3B1, JAK2 exon 12, and TET2 mutations in peripheral blood and colonies by Sanger sequencing. Table SI. Hematological and biochemical data for the patient with MDS/MPN-RS-T harboring JAK2 exon 12 mutation at initial diagnosis. Table SII. Enrichment of mutant cells in myeloid lineages. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. 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