Analysis of Circulating Microrna Expression Profile In Patients Transplanted From Matched Unrelated Donors (MUD) Identifies mir203 as a Putative Marker of aGVHD

Allogeneic haemopoietic-stem-cell transplantation (HSCT) is the treatment of choice for many malignant and non-malignant disorders. Despite the recent advances in post-transplant immunosuppressive therapy, Graft-versus-Host Disease (GVHD) still represents the major life-threatening complication, developing in a substantial number of HSCT patients and resulting in poor outcome. Recent studies have indicated that microRNAs (miRNAs) circulate in a stable, cell-free form in the bloodstream and that the abundance of specific miRNAs in plasma or serum can serve as biomarkers of cancer and other diseases. We examined plasma microRNA (miRNA) expression levels from patients transplanted from matched unrelated donor (MUD) to assess their clinical application for diagnosing and monitoring GVHD.

Having obtained an informed consent, we collected plasma samples from 11 patients who received unmanipulated HSCT from MUDs. After HSCT, 5 of 11 patients developed acute GVHD. Blood samples were collected serially at day +30, +60, +90, +150 after HSCT. MicroRNAs were isolated from the peripheral blood (PB) plasma using a modified mirVana™ miRNA Isolation Kit (Ambion Inc). The miRNAs expression profile was examined using a quantitative PCR-method (TaqMan ® Human microRNA cards, Applied Biosystems) that allows the analysis of 384 human miRNAs by low density array technology. Plasma samples of normal subjects have been included in the study. Relative quantification of miRNA expression was calculated with the 2^-ΔΔCt method. The data were normalized respect to hsa-mir-122 and relative to a calibrator sample (average of normal subjects plasma samples).

Initial analysis showed that miRNAs are stable and detectable in all plasma samples from MUD transplanted patients. Among the 384 mirRNAs analyzed we identified a panel of mirRNAs that may have a predictive role for GVHD. Among these, we have identified mir203 which is upregulated in patients prior to the onset of GVHD and its expression decreases upon therapy. Mir203 acts downregulating SOCS3, the endogenous regulator of cytokine signaling through JAK-STAT3 which is involved in the activation of the immune response. The importance of SOCS3 in the pathogenesis of GVHD has been recenly demonstrated (Hill et al. Blood, 2010) confirming the potential regulatory role of mir203 in this disease. Of interest, the evaluation of mir203 expression levels at day +30 after HSCT is able to clearly classify the patients who will develop GVHD from those who will not (p-value<0.01).

Profiling of mir203 in a wider and heterogeneous group of patients receiving allo-HSCT is ongoing to extend and confirm the present findings. Although preliminary, these results indicate that the detection of circulating miRNAs might provide new complementary markers of GVHD. In particular the elevated plasma level of mir203 at day +30 after HSCT may be used to predict the risk of developing GVHD.

No relevant conflicts of interest to declare.