Noninvasive Prediction of Acute Graft-Verus-Host Disease Following Allogeneic Hematopoietic Stem Cell Transplantation by Circulating miRNA Profiling

Acute graft-versus-host disease (aGVHD) results in significant morbidity and mortality and remains the main complication of alloHSCT. Noninvasive, diagnostic and prognostic tests for aGVHD are currently lacking but essential to predict GVHD and to improve the safety and accessibility of alloHSCT. We hypothesized that the prospective analysis of miRNA expression profile in the plasma of allografted patients could allow for the detection of specific miRNAs with predictive role for aGVHD.

After informed consent, we collected plasma samples from 10 healthy donors and 22 patients (median age: 59 and 41 years) who received unmanipulated alloHSCT (18 from Matched Unrelated Donors and 4 from HLA-matched siblings). Blood samples were collected weekly after HSCT and patients were monitored to assess aGVHD onset. MicroRNAs were isolated from the plasma and the miRNA expression profile examined using a quantitative PCR-method (TaqMan® Human microRNA Cards, Applied Biosystems). The results obtained were subsequently validated with specific miRNA Single Assays (Applied Biosystems). To verify whether the miRNAs emerged from the human studies represent markers of aGVHD and provide information regarding the involvement of specific target organs, a major histocompatibility complex (MHC) mismatched HSCT mouse model was used. Recipient BALB/c mice were lethally irradiated and treated either with spleen and bone marrow (BM) cells from C57BL/6 (B6) donors (GVHD cohort, n=22) or with BM cells only (negative control, n=18). Syngeneic transplants (B6àB6, n=6 were also included. Mice were characterized for GVHD onset by monitoring overall survival and weight loss. Recipient mice were sacrificed and tissues harvested on day 9, 14 and 18 post transplant and GVHD confirmed by histology and scored according to Foley et al, 2008. MiRNAs expression profile have been characterized in the plasma, skin, liver, colon and lymphocytes of GVHD and non-GVHD control cohorts

Three of 22 patients developed intestinal GVHD (grade 2) and 9 of 22 patients developed cutaneous GVHD (grade 2–3). By comparing the circulating miRNAs expression profiles of GVHD patients and non GVHD patients, we identified a group of 8 differentially expressed miRNAs (miR-136, 194, 203, 367, 148b, 196b, 26a, 340) (p<0.05). On day 14 post transplant mice in the GVHD cohort developed GVHD as confirmed by organ evaluation (GVHD score: 14–15 out of 20) and weight loss while the control group had no GVHD (GVHD score 0–1 out of 20). The analysis of circulating miRNAs in mice supported the results in humans: the 8 circulating microRNA signature could clearly predict those subjects developing GVHD (area under the ROC curve ≥ 0.75). Of note, pathway enrichment analysis performed using DIANA-mirPath software on the gene targets predicted by microT-4.0, indicate that these miRNAs regulate critical pathways of GVHD pathogenesis (TGFbeta and cytokine signaling, T and B cell differentiation and proliferation, immune response). The analysis of the miRNAs expression profiles of the lymphocytes and of histologically confirmed skin, liver and colon biopsies of GVHD mice highlighted the presence of several deregulated miRNAs when compared to control samples. Pathway enrichment analysis indicated that the differentially expressed miRNAs mainly target genes involved in the TGFbeta and Wnt signaling pathways as well as in the cytoskeleton rearrangements. Of interest, when analysing the expression of the 8 circulating miRNAs able to discriminate GVHD samples from controls, in the organs of mice developing GVHD, miR-203 is also abundant in the skin and colon, miR-367 in the colon and miR-136 in the lymphocytes. These findings strengthen a role for these miRNAs in the modulation of aGVHD and suggest that the presence of plasma miRNAs is linked to the specific organs target of this pathological process.

Considering the noninvasive characteristics of plasma sampling and the reproducible and easy detection of miRNAs, our results indicate that circulating miRNAs might represent a promising tool for the early diagnosis of aGVHD thus enhancing therapeutic success and increasing life expectancy of allografted patients. In addition the miRNA profiling of the target organs and lymphocytes of GVHD mice, allowed the identification of several deregulated genes that might play a role in the modulation of aGVHD and warrant further investigations.

No relevant conflicts of interest to declare.