Mutations in the RNA splicing machinery genes in myelofibrotic transformation of essential thrombocythaemia and polycythaemia vera

Mutations in mRNA splicing machinery genes, including SF3B1, SRSF2 and U2AF1, have been recently described in lymphoid and myeloid haematopoietic malignancies (Maciejewski & Padgett, 2012; Cazzola et al, 2013). Regarding the myeloproliferative neoplasms (MPN), alterations in the SF3B1 gene have been reported in essential thrombocythaemia (ET, 3%) and primary myelofibrosis (PMF, 4-6%) (Brecqueville et al, 2012; Lasho et al, 2012a; Maciejewski & Padgett, 2012; Cazzola et al, 2013). SRSF2 mutations, which have been associated with adverse outcome, have been detected in 17% of PMF and in 19% of MPN that evolve to acute leukaemia (Lasho et al, 2012b; Zhang et al, 2012; Vannucchi et al, 2013). Concerning U2AF1, mutations have been reported in 3% of PMF (Zhang et al, 2012). Although mutations in these genes have been described in PMF and, in some cases, associated with adverse outcome, there is limited information about their incidence in patients with ET or polycythaemia vera (PV) who develop myelofibrotic transformation during the evolution of the disease. SF3B1 (exons 14 and 15), SRSF2 (exon 1) and U2AF1 (exons 2, 6 and 7) were analysed in 22 PV and 14 ET patients who transformed to myelofibrosis and 27 PMF, 30 ET and 30 PV patients in chronic phase. Diagnosis of ET, PV and PMF was determined according to the 2008 World Health Organization criteria (Vardiman et al, 2009). Prefibrotic MF was excluded by histomorphological criteria. Post-ET and post-PV MF patients were diagnosed according to the International Working Group for Myelofibrosis Research and Treatment (IWG-MRT) guidelines (Barosi et al, 2008). Screening for mutations was performed on granulocyte DNA at the myelofibrotic phase of the disease using Next Generation Sequencing (NGS; 454GSJunior platform, Roche, Mannheim, Germany). Mutations were confirmed by independent polymerase chain reaction amplification and Sanger sequencing. The study was approved by the local Ethics Committee and patients provided written informed consent according to the Declaration of Helsinki. From the 36 PV or ET patients who evolved to myelofibrosis, we identified 1 SRSF2 (3%), 3 SF3B1 (8%) and no U2AF1 (0%) mutations (Fig 1, Table I). Two post-ET MF patients harboured the SF3B1 p.K666N mutation and one post-PV MF patient simultaneously carried a SRSF2 mutation (p.P95H) and a SF3B1 mutation (p.I671T) (Fig 1, Table I). As the SF3B1 p.I671T mutation has not been previously described, a prediction of its possible impact on the structure and function of the protein was analysed using the PolyPhen, Align GVGD and SIFT bioinformatic tools. All three analyses predicted that this change probably results in protein function damage. A diagnostic sample from one of the two post-ET MF patients harbouring the p.K666N mutation, 8 years before transformation, showed the presence of the p.K666N mutation by NGS in 2 4% of sequences, in contrast to the 45% observed in the sample at the time of transformation, suggesting expansion of the clone during the evolution of the disease. On the other hand, a diagnostic sample from the post-PV MF patient, 2 years before transformation, showed a similar percentage of both SF3B1 and SRSF2 mutations. The prevalence of mutations in 30 PV and 30 ET patients at chronic phase was very low with only 1/30 ET patients (3%) showing two simultaneous SRSF2 mutations (p.R94P and p.P95H). No mutations in these genes were detected in PV patients (Table I). However, the presence of a small clone with the SF3B1 mutation at diagnosis that expanded during the transformation of the disease in one post-ET MF patient, suggests that, at least in some cases, the presence of mutations in splicing machinery genes at chronic phase might play a role in myelofibrotic transformation. Nevertheless, larger studies including sequential mutational studies at diagnosis and during follow-up are needed to confirm the frequency and the role of splicing machinery gene mutations in myelofibrotic transformation of PV and ET patients. To determine whether the prevalence of mutations in myelofibrotic transformation was comparable to PMF, we analysed these genes in 27 PMF patients. In PMF, we detected mutations in SRSF2 in 6 cases (22%), SF3B1 in 2 patients (7%) and U2AF1 in 4 patients (15%) (Table I). All SRSF2 mutations affected the hotspot P95 amino acid position (p.P95H (n = 3), p.P95R (n = 2) and p.P95_R103delinsR (n = 1). The two PMF patients positive for SF3B1 mutations presented the p.K666N mutation, and all mutations affecting the U2AF1 gene were detected at amino acid position Q157 (p.Q157P (n = 2) and p.Q157R (n = 2)) (Fig 1, Table I). Our results identified SRSF2 mutations in 1/36 patients (3%) with myelofibrotic transformation, in contrast to 6/27 correspondence