Refractory anemia with ringed sideroblasts and thrombocytosis (RARS-T) has been considered a provisional subtype within the diagnostic entity of myelodysplastic/myeloproliferative diseases (MDS/MPD). Since JAK2 V617F mutation is present in a significant proportion of RARS-T patients (Szpurka et al. Blood, 2006), many investigators consider this entity to be more closely related to classical MPD. However, a significant minority of patients with RARS-T do not display a JAK2 V617F mutation. We have studied a cohort of patients with RARS-T (N=18) characterized by the presence of ringed sideroblasts, reticulin fibrosis and thrombocytosis (>600×109/L), that lack obvious causes of secondary thrombocytosis. While 8/18 patients harbored a JAK2 V617F mutation, a molecular pathogenesis for the remaining patients was unexplained. The successful application of SNP-A to characterize the genomic lesions in MDS prompted us to use this technology to study RARS-T. SNP-A allows detection of copy neutral loss of heterozygosity such as UPD9p which is associated with JAK2 V617F mutation. SNP-A facilitated detection of previously cryptic lesions; 9/18 patients showed an abnormal SNP-A-based karyotype often involving multiple lesions (only 5 of these defects were detected by metaphase cytogenetics). The new lesions seen by SNP-A included gains of chromosome 11p, 20q and 21q; deletion of 2p and various areas of UPD including 1p, 9p, 6p, 2p and 8p. SNP-A allowed identification of seemingly invariant UPD1p in 4/18 patients. As this region includes the Mpl gene, we analyzed patients for the presence of MPL W515L/K mutations which have been described in MPD. We did not find any patients with MPL W515K, however MPL W515L mutation was present in 2/4 RARS-T patients with UPD1p; another patient showed monoallelic MPL W515L variant. In addition, 1 patient with UPD1p harbored both JAK2 V617F and MPL W515L mutations. To further delineate the molecular lesion we analyzed all patients for the presence of abnormal STAT5 activation. An aberrant phospho-STAT5 staining pattern was present in all cases that were positive for either JAK2 V617F or MPL W515L mutations (N=10); unexplained STAT5 activation was found in only 4 cases, pointing towards a molecular defect involving this pathway. In these 4 patients, and in 1 additional with UPD1p who did not harbor MPL W515L mutation, we searched for other genes which might explain the pathogenesis of this disease by potentially causing aberrant activation of STAT5. We sequenced Jak1T478S, Jak1V623A and Ntrk1S677N as well as the transmembrane, juxtamembrane and kinase domain of Tie1, Epha2 and Ephb2 genes, but no mutation was found. In addition, we found a group of phospho-STAT5-negative patients (N=4) that showed typical genetic features of myelodysplasia e.g. del(5), +8 and partial loss of chromosome X; these cases are probably best considered to be of MDS origin rather than MPD. To our knowledge, our work is the first description of biallelic MPL W515L mutation and UPD1p found in RARS-T patients. This data is important for understanding the clonal selection process and pathophysiology of activating mutations in MDS/MPD. Overall, our studies demonstrate that somatic UPD1p is associated with homozygous MPL W515L mutation in MDS/MPD cases. Localizing areas of somatic UPD by SNP-A may help identify candidate genes within the shared regions that are likely targets for mutations.

Disclosures: No relevant conflicts of interest to declare.

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