Promising role of extracellular vesicles as biomarker of acute graft-versus-host-disease

Reliable biomarkers could be crucial to define patients at high-risk of developing acute Graft-vs.-Host Disease (aGVHD) after allogeneic hematopoietic stem cell transplant (HSCT). We recently investigated the potential role of serum Extracellular Vesicles (EVs) as biomarkers of aGVHD (Lia G. et.al. Leukemia 2017). In a retrospective study, we correlated three EVs membrane antigens (CD146, Cd31, CD140alpha) with the onset of aGVHD. To confirm our preliminary findings, a study where serum samples are prospectively collected from patients undergoing an allograft at different time-points (pre-transplant, on day 0, +3, +7, +14,+21,+28, +45 and then monthly up to 1 year) has been designed. Moreover, EVs content in MiRNAs (miR100, miR155, miR92b, and miR194) is also evaluated. EVs are extracted using a protamine-based precipitation method and analyzed for the expression of membrane proteins (CD146, CD31, CD140a, CD120, CD44, KRT18, CD106) by flow-cytometry (Guava EasyCyte Flow Cytometer). Total EVs concentration, fluorescence distribution and percentage of positive EVs are evaluated for all biomarkers. MiRNAs are extracted from EVs by miRNeasy mini kit Qiagen.MiRNAs are then quantified by real time PCR and expressed as relative levels compared to healthy donors. By logistic regression analysis, odds ratio (OR) is calculated as proportional change from pre-transplant levels for each marker. Thirty-one patients have so far been enrolled and 17/31 have developed grade II-IV aGVHD. A preliminary analysis on a patient subset has confirmed a correlation of the expression of CD146, CD31 and CD140alpha with the onset of aGVHD. These potential biomarkers have shown a significant proportional change in signal levels from pre-transplant up to the onset of aGVHD onset (Figure 1). By logistic regression analysis (Table 1), higher expression of CD146 and lower expression of CD31 and CD140alpha have significantly been associated with increased risk of developing aGVHD. CD146, and CD31 (also known as MCAM-1, and PECAM-1, respectively) belong to the Cell Adhesion Molecule family and are crucial for endothelium and immune-cells interactions. CD140-alpha, also known as Platelets Derived Growth Factor Receptor alpha (PDGFRa), plays a role in fibroblast migration and tissue wound healing. Moreover, preliminary results on miRNAs expression levels have shown their upregulation at day +14 in patients who have later developed aGVHD (Figure 2). EVs antigen expression and their content (MiRNAs) appear promising biomarkers of aGVHD. Further analyses on a larger patient cohort with longer follow-up will be presented at the meeting.Table 1MarkerTypeOR (95%CI)PCD146Fluorescence4.11.04Percentage2.99.18CD31Fluorescence.36.01Percentage.48.14CD140alphaFluorescence.48.21Percentage.22.03 Open table in a new tab Figure 2MiR100, miR155, miR92b and miR194 relative expression respect donors mean (PreTx: before HSCT).View Large Image Figure ViewerDownload Hi-res image Download (PPT)