Plasma microRNA signature as a noninvasive biomarker for acute graft-versus-host disease

Abstract

Acute graft-versus-host disease (aGVHD) is the leading cause of morbidity and mortality after allogeneic hematopoietic cell transplantation (HCT). Approximately 35% to 50% of HCT recipients develop aGVHD; however, there are no validated diagnostic and predictive blood biomarkers for aGVHD in clinical use. Here, we show that plasma samples from aGVHD patients have a distinct microRNA (miRNA) expression profile. We found that 6 miRNAs (miR-423, miR-199a-3p, miR-93*, miR-377, miR-155, and miR-30a) were significantly upregulated in the plasma of aGVHD patients (n = 116) when compared with non-GVHD patients (n = 52) in training and validation phases. We have developed a model including 4 miRNAs (miR-423, miR-199a-3p, miR-93*, and miR-377) that can predict the probability of aGVHD with an area under the curve of 0.80. Moreover, these elevated miRNAs were detected before the onset of aGVHD (median = 16 days before diagnosis). In addition, the levels of these miRNAs were positively associated with aGVHD severity, and high expression of the miRNA panel was associated with poor overall survival. Furthermore, the miRNA signature for aGVHD was not detected in the plasma of lung transplant or nontransplant sepsis patients. Our results have identified a specific plasma miRNA signature that may serve as an independent biomarker for the prediction, diagnosis, and prognosis of aGVHD.

Figures

Figure 1

Expression of plasma miRNA signature for aGVHD diagnosis in a training set. (A) Expression of miR-423, miR-199a-3p, miR-93*, miR-377, miR-155, and miR-30a in plasma of aGVHD (n = 59) and non-GVHD patients (n = 19) 6 weeks after HCT. P values were calculated using the Mann-Whitney test. (B) ROC analysis for individual miRNAs and combined miRNA panel. Logistic regression demonstrated that a linear combination of values for miR-423, miR-199a-3p, miR-93*, and miR-377 produced the best model for aGVHD diagnosis; the equation was “Combined miRNA panel = 5.514 + 0.255 × miR-423 − 0.427 × miR-199a-3p + 0.522 × miR-93* − 1.161 × miR-377.” ROC curve of miRNA panel was generated based on the predicted probability (P) for each patient. Predicted probability = Exp (combined miRNA panel)/ [1+Exp (combined miRNA panel)]. (C) Comparison of sIL-2Rα protein levels in plasma between aGVHD and non-GVHD patients 6 weeks after HCT. P values were calculated using the Mann-Whitney test.

Figure 2

Expression of plasma miRNA signature for aGVHD diagnosis in a validation set. (A) Expression of miR-423, miR-199a-3p, miR-93*, miR-377, miR-155, and miR-30a in plasma of aGVHD (n = 57) and non-GVHD patients (n = 33) 6 weeks after HCT. (B) ROC analysis for individual miRNAs and combined miRNA signature including miR-423, miR-199a-3p, miR-93*, and miR-377 as described in the legend of Figure 1B. The equation of logistic regression in validation set was “Combined miRNA panel = 11.059 − 0.19 × miR-423 + 0.175 × miR-199a-3p-0.04 × miR-93* − 0.89 × miR-377.” (C) Comparison of sIL-2Rα protein levels in plasma between aGVHD and non-GVHD patients 6 weeks after HCT. (D) ROC curve of the 4-miRNA panel generated by analyzing all 168 samples from training and validation sets.

Figure 3

Predictive value of the miRNA signature for aGVHD. (A) The expression of miR-423, miR-199a-3p, miR-93*, miR-377, miR-155, and miR-30a in the plasma of aGVHD patients prior to aGVHD diagnosis (median = 16 days before diagnosis). (B) ROC curve of the 4-miRNA panel (miR-423, miR-199a-3p, miR-93*, miR-377) 6 weeks after HCT for prediction of aGVHD before onset. (C) Plasma concentration of sIL-2Rα 6 weeks after HCT in non-GVHD and aGVHD patients before aGVHD diagnosis. (D) The expression of the 4-miRNA panel 2 weeks after HCT in plasma of aGVHD and non-GVHD patients. (E) ROC curve of the 4-miRNA panel 2 weeks after HCT. (F) Plasma concentration of sIL-2Rα 2 weeks after HCT. (G) ROC curve of sIL-2Rα 2 weeks after HCT.

Figure 4

Association of the miRNA signature with aGVHD severity. (A) Expression levels of miR-423, miR-199a-3p, miR-93*, miR-377, miR-155, and miR-30a in aGVHD patients with different grades of disease (grade 1-2 vs grade 3-4). P values were calculated using the Mann-Whitney test. *P < .05; **P < .01; ***P < .001. (B) The kinetic expression of the miRNA signature in a set of 9 patients before disease onset (2 weeks after HCT) and after onset (6 weeks after HCT). We selected 9 aGVHD patients in the analysis according to the following criteria: they had similar diagnosis time (median day 20, range: 18 to 23) and didn’t develop aGVHD 2 weeks after HCT but had symptoms 6 weeks after HCT. P values were calculated using paired t tests.

Figure 5

Expression of the aGVHD miRNA signature in sepsis or lung transplant patients. (A) Expression levels of the aGVHD miRNA signature including miR-423, miR-199a-3p, miR-93*, miR-377, miR-155, and miR-30a in plasma of lung transplant patients with (n = 17) or without (n = 28) acute rejection. (B) Expression levels of the aGVHD miRNA signature in plasma of sepsis patients (n = 38) and healthy donors (n = 10). HD, healthy donors; NS, nonsignificant.