The Philadelphia chromosome-negative chronic myeloproliferative neoplasms (MPNs) encompass essential thrombocythemia (ET), polycythemia vera (PV) and primary myelofibrosis (PMF), including early prefibrotic myelofibrosis. These neoplasms arise due to an acquired stem cell insult with ensuing clonal myeloproliferation in the biological continuum from the early cancer stages (ET and PV) to the advanced myelofibrosis stage and ultimately leukemic transformation. Molecular markers for MPNs include JAK2V617F, CALR -and MPL-mutations. Interferon-alpha2 (IFN) has convincingly demonstrated to be safe and highly efficacious in normalizing elevated cell counts. Indeed, prolonged treatment (about 5 years) may be followed by normalization of the bone marrow and low-burden JAK2V617F in a subset of patients, even being sustained for 2–3 years after discontinuation of IFN. Several studies have described aberrant gene expression profiling in MPNs but only a few have focused upon the miRNA signatures in patients with MPNs and no studies have so far been reported dealing with the impact of IFN upon the miRNAs in MPNs.
The aims of the present study are: 1) to find and describe putative miRNAs that can be used to discriminate ET, PV and PMF 2) to describe the differences in miRNA expression profiles at study entry and after 3 month's therapy with IFN.
In total 32 MPN patients: ET (n = 8), PV (n = 20), PMF (n = 4) were enrolled in the study. Whole blood was obtained at study entry and during a three-month follow-up while treated with IFN. JAK2V617F allele burden, total leukocyte count, thrombocytes and haemoglobin were analysed during study entry and follow-up. miRNA was extracted from PAXgene tubes using the Paxgene RNA kit and RNeasy minElute Cleanup kit. miRNA quantity and integrity were assessed by Qubit miRNA assay and Bioanalyzer-2100. 4 patients from each MPN group were selected based on miRNA concentration for subsequent small RNA sequencing. Small RNA library synthesis was performed using the Ion Total RNA-Seq Kit v2 for small RNA libraries. The NGS Ion Torrent platform was used for sequencing. Reads Per Kilobase Million (RPKM) was applied as the normalization strategy. Likewise, a RPKM cut-off was established for enabling statistical analysis.
Time to follow-up was median 3 months (range: 2–8 months). A significant total leukocyte reduction from study entry was evident in all MPN groups after 3-months of therapy, while the molecular response in JAK2V617F allele burden was insignificant. Using the NGS Ion Torrent Platform, 930 miRNAs were detected of which 139 had an RPKM count ≥5 among the entire MPN cohort (N = 12). Among the 139 miRNAs, 30 (21.6%) were significantly differentially expressed during IFN therapy compared to baseline. Specifically, hsa-miR-378a-3p was observed to be positively correlated with both total leukocyte count and the JAK2V617F allele burden. Moreover, a significant reduction in RPKM read count was recorded (P = 0.02).
In this study, we present the preliminary results of a miRNA-sequencing project. Interestingly, hsa-miR-378–30 was positively correlated with JAK2V617F allele burden and the leukocyte count. The RNA-sequencing data permits us to design a validation study using RT-qPCR on a number of selected miRNAs including hsa-miR-378a-3p. These miRNAs are selected not only by the level of significance but also in their ability to correlate to known and daily used clinical parameters.