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.
|Publication status||Published - 2019|
|Event||24th Congress of the European Hematology Association - RAI Amsterdam at Europaplein 24, Amsterdam, Netherlands|
Duration: 13 Jun 2019 → 16 Jun 2019
|Conference||24th Congress of the European Hematology Association|
|Location||RAI Amsterdam at Europaplein 24|
|Period||13/06/2019 → 16/06/2019|