Abstract 172

Somatic mutations of the RNA splicing machinery have been recently identified in patients with myelodysplastic syndrome (MDS). In particular, a strong association has been found between SF3B1 mutation and the MDS subtype defined as refractory anemia with ring sideroblasts (RARS). Similarly, within myelodysplastic/myeloproliferative neoplasms (MDS/MPN) a high prevalence of SF3B1 mutations has been reported in the provisional entity defined as refractory anemia with ring sideroblasts associated with marked thrombocytosis (RARS-T). These findings strongly support a causal relationship between SF3B1 mutations and ring sideroblasts. Interestingly, a high proportion of RARS-T patients also harbor JAK2 and/or MPL mutations. The available evidence suggests that somatic mutations of SF3B1 might be an early pathogenetic event determining myelodysplastic features, and that subsequent occurrence of JAK2 and/or MPL mutations may cause the myeloproliferative phenotype.

In this work, we studied the mutation status of SF3B1, JAK2 and MPL in circulating granulocytes and bone marrow cells from RARS-T patients. We also studied the in vitro growth of hematopoietic progenitors (BFU-E, CFU-GM), and genotyped individual colonies to examine the mutation status of the above genes. The coding exons of SF3B1 were screened using massively parallel pyrosequencing. A real time PCR-based allelic discrimination assay was used for the detection of JAK2 (V617F), while Sanger sequencing was employed for JAK2 exon 12 and MPL exon 10 mutation analysis.

Twenty-eight patients affected with RARS-T were assessed for SF3B1, JAK2 and MPL exon 10 mutation status. Eighteen patients (64%) showed somatically acquired mutation of SF3B1. The median mutant allele burden was 43%, consistent with the presence in the majority of patients of clonal hematopoiesis characterized by a dominant clone carrying a heterozygous SF3B1 mutation. Fourteen patients carried the JAK2 (V617F) mutation (median allele burden 6.5%, range 0.4–29.5%), while one had a JAK2 exon 12 mutation. In 13 cases, the JAK2 mutation was detected at the time of diagnosis, whereas in 2 patients, who had a typical RARS phenotype and were negative for JAK2 mutations at clinical onset, JAK2 (V617F) was detected 18 and 32 months after diagnosis, respectively, and concomitantly with a progressive increase in platelet count. Four patients, two of whom were JAK2 (V617F)-positive, carried the MPL (W515L) mutation (median allele burden 27.5%, range 25–50%).

Concomitant mutations of SF3B1 and JAK2 or MPL were observed in 8 cases. Seven patients carried an SF3B1 mutation and JAK2 (V617F), while one carried SF3B1 (K700E), JAK2 (V617F), and MPL (W515L). In all these cases, the SF3B1 mutant allele burden was higher than that of JAK2 or MPL, indicating the existence of an SF3B1-mutated dominant clone with minority JAK2- or MPL-mutated clones.

We genotyped individual colonies from peripheral blood in 2 patients with concomitant mutations. In a patient with granulocyte SF3B1 and JAK2 mutant allele burdens equal to 45% and 8%, respectively, SF3B1 (H662Q) was detected in 9 of 11 colonies, three of which also carried JAK2 (V617F); the remaining two colonies had wild type SF3B1 and JAK2. These data are consistent with the existence of a dominant hematopoietic clone carrying the SF3B1 mutation and the subsequent emergence of a JAK2-mutated subclone. The other patient, who was initially SF3B1- mutated and JAK2 wild type, at the time of colony assay had a mutant allele burden equal to 50% and 1% for SF3B1 (K700E) and JAK2 (V617F), respectively. Forty-three of 45 colonies were heterozygous for SF3B1 (K700E) and wild type for JAK2. The opposite pattern was observed in the remaining 2 colonies, which carried just JAK2 (V617F). These data indicate the coexistence of two distinct clones, a dominant one carrying the SF3B1 mutation and a minority one carrying JAK2 (V617F).

In summary, these observations suggest that the occurrence of an SF3B1 mutation represents an early event in patients with RARS-T, likely causing mitochondrial iron overload, ring sideroblasts, ineffective erythropoiesis and anemia, typical myelodysplastic features. The subsequent occurrence of a somatic mutation of JAK2 or MPL involves the emergence of minority clones and the acquisition of myeloproliferative features. JAK2- mutated clones may emerge as subclones of the dominant SF3B1-mutated clone or as independent clones.


No relevant conflicts of interest to declare.

Author notes


Asterisk with author names denotes non-ASH members.