High frequencies of SF3B1 and JAK2 mutations in refractory anemia with ring sideroblasts associated with marked thrombocytosis strengthen the assignment to the category of myelodysplastic/myeloproliferative neoplasms.

Mutations of spliceosome genes were shown to occur frequently in different entities. Remarkably, mutations in SF3B1 (splicing factor 3b, subunit 1) were associated with the morphological feature of ring sideroblasts and were also found in refractory anemia with ring sideroblasts and marked thrombocytosis (RARS-T). This malignancy has been assigned as a provisional entity in the chapter “Myelodyplastic/myeloproliferative neoplasms, unclassifiable” of the WHO classification. Patients have anemia, clinical and morphological features of myelodysplastic syndromes (MDS), but also show marked thrombocytosis associated with abnormal megakaryocytes resembling BCR-ABL1 negative myeloproliferative neoplasms (MPN). In line with the myeloproliferative character of this disease, RARS-T patients often show JAK2V617F mutations and, much less common, MPLW515 mutations (MPLW515mut). To further characterize this entity, we analyzed 47 RARS-T patients for the occurrence of mutations in SF3B1 (SF3B1mut) and its association with JAK2V617F and MPLW515mut. To our knowledge, this is the largest cohort studied so far. All patients strictly met the criteria for RARS-T according to the WHO classification 2008. Twentyseven of 47 (57.4%) were female and 20 of 47 (42.6%) were male. The median age was 76 years (range 44-89 years), white blood cell count (WBC) 7.9x10/L (range 2.9-60.0x10/L), hemoglobin level (Hb) 9.7 g/dL (range 6.9-13.1 g/dL), platelet count 691x10/L (range 4661,500x10/L), and percentage of ring sideroblasts 60% (range 18-97%). Most patients had a normal karyotype (39 of 47, 83.0%) and 8 of 47 (17.0%) had various chromosomal aberrations consistent with MDS/MPN. Screening for SF3B1mut was performed by direct Sanger sequencing of exons 11-16. Using cDNA, a 1kb amplicon was generated with primers 5`-TGACCAGCCATCTGGAAATC-3` (forward, exon 10) and 5`-CACCATCTGTCCCACAACAC-3` (reverse, exon 17). Sequencing was performed with previous primers and 5`GCTTGCCAGGACTTCTTGCT-3` (reverse, exon 14), 5`-AGCTTTTGCTGTTGTAGCCTCTG-3` (forward, exon 14). Mutation load was determined according to the ratio of mutated/wild-type in the electropherograms of forward and reverse reaction. Cases being SF3B1 unmutated by Sanger sequencing were reanalyzed with a more sensitive 454 deep-sequencing approach (NGS) on genomic DNA. The lowest detectable mutation load with NGS was about 3% (1,013 reads) and, for Sanger sequencing, 5-10% as confirmed by NGS. JAK2V617F, JAK2exon12 and MPLW515 mutations were analyzed as published previously. Sensitivities of the assays were approximately 1%, 10%, and 5%, respectively. The frequency of SF3B1mut was 41 of 47 (87.2%). The 6 patients without SF3B1mut were reanalyzed by NGS but, despite higher sensitivity, no SF3B1mut was found. In the 41 SF3B1mut patients, 43 mutations with median mutation load of 40% (range 15-70%) were detected. The most frequent mutation was Lys700Glu (22 of 43, 51.2%), followed by Lys666Arg/Asn/Thr (7 of 43, 16.3%), Glu622Asp (4 of 43, 9.3%), His662Gln (4 of 43, 9.3%), Arg625Lys/Cys (3 of 43, 7.0%), Thr627Pro, Met784_Lys785delinsIle and a synonymous Leu575Leu (1 of 43, 2.3% each). The two double mutated cases had: i) Arg625Cys (load 45%) and Thr627Pro (load 45%); or ii) Lys700Glu (load 30%) and the synonymous Leu575Leu (load 70%) mutation. JAK2V617F was also very frequent (36 of 47, 76.6%). Twenty-five of 36 (69.4%) revealed a mutation load of 10-50%, 6 of 36