Extracellular Vesicles as Biomarkers of Acute Graft-vs.-Host Disease After Haploidentical Stem Cell Transplantation and Post-Transplant Cyclophosphamide

Abstract

Even with high-dose post-transplant cyclophosphamide (PT-Cy) which was initially introduced for graft-versus-host disease (GvHD) prevention in the setting of HLA-haploidentical transplantation, both acute and chronic GvHDs remain a major clinical challenge. Despite improvements in the understanding of the pathogenesis of both acute and chronic GvHDs, reliable biomarkers that predict their onset have yet to be identified. We recently studied the potential correlation between extracellular vesicles (EVs) and the onset of acute (a)GvHD in transplant recipients from related and unrelated donors. In the present study, we further investigated the role of the expression profile of membrane proteins and their microRNA (miRNA) cargo (miRNA100, miRNA155, and miRNA194) in predicting the onset of aGvHD in haploidentical transplant recipients with PT-Cy. Thirty-two consecutive patients were included. We evaluated the expression profile of EVs, by flow cytometry, and their miRNA cargo, by real-time PCR, at baseline, prior, and at different time points following transplant. Using logistic regression and Cox proportional hazard models, a significant association between expression profiles of antigens such as CD146, CD31, CD140a, CD120a, CD26, CD144, and CD30 on EVs, and their miRNA cargo with the onset of aGvHD was observed. Moreover, we also investigated a potential correlation between EV expression profile and cargo with plasma biomarkers (e.g., ST2, sTNFR1, and REG3a) that had been associated with aGVHD previously. This analysis showed that the combination of CD146, sTNFR1, and miR100 or miR194 strongly correlated with the onset of aGvHD (AUROC >0.975). A large prospective multicenter study is currently in progress to validate our findings.

Keywords: acute GvHD; biomarkers; correlation; extracellular vesicles; haploidentical; miRNA.

Figure 1

Extracellular vesicle (EV) characterization by light scattering and fluorescence. (A) Forward and side scatter dot plots of EVs analyzed after incubation with non-immune isotypic FITC and PE-IgG (iso-FITC/iso-PE, negative controls, blue dots). Red dots represent debris. (B) Representative fluorescence dot plots showing EV fluorescence after incubation with non-immune isotypic FITC and PE-IgG (negative controls, red dots), and after incubation with anti-CD146-FITC and anti-CD31-PE (blue dots). The red line marks the threshold to discriminate the positive FITC (green fluorescence) and the positive PE fluorescence (yellow fluorescence) signal from the background. (C) Representative histograms showing the shift in fluorescence after incubation of EVs with the indicated antibodies (blue peaks) with respect to isotypic control (FMO, red peaks). (D) Donor EV dimension histograms by nanoparticle tracking analysis. Inset: representative image of EVs detected by transmission electron microscopy (magnification ×60,000; scale bar 50 nm). (E, F) Plasma concentration of total EVs (E) and concentration of CD120+ EVs (F) in donors (blue) and in patients prior to transplant (preTX, red). (G) Representative flow cytometry histograms showing the shift in fluorescence after incubation of donor EVs with anti-CD9-PE and anti-CD81-PE (exosomes biomarkers). EVs = gated region; red line marks threshold to discriminate positive florescence signal from background.

Figure 2

Impact of acute GvHD onset on the kinetics of EV membrane protein expression. Proportional change of fluorescence levels of CD146, total EV concentration, CD140a⁺ EV concentration and CD31⁺ EV concentration (left side, from top to bottom), CD120a⁺ EV concentration, CD144⁺ EV concentration, CD26⁺ EV concentration, and fluorescence levels of CD30 from precipitated EVs at different time points before and after aGvHD onset and compared to the pre-transplant baseline values. Dashed black line: pre-transplant levels; dashed red line: time of aGvHD onset; circle and star dots represent outliers (>1.5 box length from median) and extreme values (>3 box length from median), respectively. Significant mean differences before the onset (p ≤ 0.05) between patients with aGvHD (red) and without (blue) are indicated.

Figure 3

Impact of acute GvHD onset on the kinetics of EV miRNA. Proportional change of miR100, miR155, and miR194 (from top to bottom) quantified by real-rime PCR from precipitated EVs at different time points before and after aGvHD onset and compared to the pre-transplant baseline values. Dash black line: pre-transplant levels; dashed red line: time of aGvHD onset; circle and star dots represent outliers (>1.5 box length from median) and extreme values (>3 box length from median), respectively. Significant mean differences before the onset (p ≤ 0.05) between patients with aGvHD (red) and without (blue) are indicated.

Figure 4

Impact of aGvHD onset on the circulating levels of ST2, sTNFR1, and REG3a. Variations of (A) absolute ST2 plasma level concentrations (ng/ml), (B) sTNFR1 relative plasma concentrations, and (C) absolute REG3a plasma level concentrations (ng/ml) from pre-transplant baseline levels, in patients with (red) and without (blue) aGvHD at different time points before and after aGvHD onset. Dashed black line: pre-transplant levels; dashed red line: time of aGvHD onset; circle and star-shaped dots represent outliers (>1.5 box length from median) and extreme values (>3 box length from median), respectively. Significant mean differences before the onset (p ≤ 0.05) between patients with aGvHD (red) and without (blue) are indicated.

Figure 5

Biomarker combination improves aGvHD prediction. Individual AUROC curve analysis of aGVHD diagnostic performance of (A) EV membrane protein and (B) EV miRNA biomarkers. Individual and multivariate AUROC curve analysis of aGvHD diagnostic performance of (C) CD146 and CD144; (D) miR100 and miR194; (E) CD146, miR100, and sTNFR1; and (F) CD146, miR194, and sTNFR1. AUROC, area under the receiver operating characteristics; dot line, reference line; Fl., fluorescence; conc., concentration; cha., proportional change from basal; abs., absolute value. Three-D scatter plot of standardized proportional change from baseline of (G) CD146 fluorescence, miR100 expression, and sTNFR1 plasma concentration, and of (H) CD146 fluorescence, miR194 expression, and sTNFR1 plasma concentration, in patients with (red) and without (blue) aGvHD.