A donor-specific epigenetic classifier for acute graft-versus-host disease severity in hematopoietic stem cell transplantation

Abstract

Background: Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative treatment for many hematological conditions. Acute graft-versus-host disease (aGVHD) is a prevalent immune-mediated complication following HSCT. Current diagnostic biomarkers that correlate with aGVHD severity, progression, and therapy response in graft recipients are insufficient. Here, we investigated whether epigenetic marks measured in peripheral blood of healthy graft donors stratify aGVHD severity in human leukocyte antigen (HLA)-matched sibling recipients prior to T cell-depleted HSCT.

Methods: We measured DNA methylation levels genome-wide at single-nucleotide resolution in peripheral blood of 85 HSCT donors, matched to recipients with various transplant outcomes, with Illumina Infinium HumanMethylation450 BeadChips.

Results: Using genome-wide DNA methylation profiling, we showed that epigenetic signatures underlying aGVHD severity in recipients correspond to immune pathways relevant to aGVHD etiology. We discovered 31 DNA methylation marks in donors that associated with aGVHD severity status in recipients, and demonstrated strong predictive performance of these markers in internal cross-validation experiments (AUC = 0.98, 95% CI = 0.96-0.99). We replicated the top-ranked CpG classifier using an alternative, clinical DNA methylation assay (P = 0.039). In an independent cohort of 32 HSCT donors, we demonstrated the utility of the epigenetic classifier in the context of a T cell-replete conditioning regimen (P = 0.050).

Conclusions: Our findings suggest that epigenetic typing of HSCT donors in a clinical setting may be used in conjunction with HLA genotyping to inform both donor selection and transplantation strategy, with the ultimate aim of improving patient outcome.

Fig. 1

Overview of the study design. We aimed to identify specific epigenetic marks in peripheral blood of healthy graft donors that delineate aGVHD severity in HLA-matched sibling recipients prior to HSCT. At the discovery stage, we assessed genome-wide DNA methylation levels in peripheral blood of 85 HSCT donors, matched to recipients with various transplant outcomes, that is, ‘severe’ aGVHD (grades III + IV; n = 9) and ‘no/mild’ aGVHD (grades 0, I + II; n = 76). HSCT recipients received reduced-intensity (non-myeloablative) T cell-depleted conditioning using in vivo alemtuzumab. At the replication stage, we used a semi-quantitative DNA methylation assay, MethyLight, which can be easily used in a clinical setting. We validated the top-ranked differentially methylated positions associated with aGVHD severity status in donors in the context of both T cell-depleted and T cell-replete conditioning regimens for HSCT

Fig. 2

Identification of DMPs associated with aGVHD severity. a Genomic locus on chromosome 14q24.2 harboring four top-ranked DMPs associated with aGVHD severity. A DNA hypomethylation phenotype was observed in HSCT donors matched to recipients with severe aGVHD (red points) compared to donors paired with recipients with no/mild aGVHD (blue points). Lines represent the means of the measured DNA methylation levels (β-values) across HSCT donors. Statistically significant DMPs are indicated with a black triangle. Annotation of all significant DMPs is provided in Table 2. b ROC measures evaluating the epigenetic classifier performance. LOOCV was used to assess the classifier performance (Methods). Over 85 iterations of the LOOCV, the mean AUC was 0.98 (95 % confidence interval = 0.96–0.99), with a maximal specificity and sensitivity of 0.93 and 0.93, respectively

Fig. 3

Validation of top-ranked DMP cg20475486 using a clinical biomarker assay. Replication of the top-ranked DMP associated with aGVHD severity, cg20475486, using a semi-quantitative DNA methylation assay. a Box-and-whisker plot of DNA methylation values in graft donors in T cell-depleted HSCT (initial discovery cohort). We replicated the DNA hypomethylation phenotype in HSCT donors matched to recipients with severe aGVHD compared to no/mild aGVHD (P = 0.039, Wilcoxon rank-sum test). b At a relative DNA methylation threshold of 8.295 (dotted line), the AUC was 0.74 with a maximal specificity and sensitivity of 0.75 and 0.71, respectively. c Box-and-whisker plot of DNA methylation values in graft donors in T cell-replete HSCT (that is, without the application of in vivo alemtuzumab). In an independent sample cohort, we confirmed the observed DNA methylation phenotype, suggesting the epigenetic classifier is also effective in the context of a T cell-replete conditioning regimen (P = 0.050). For two samples, C_t-values could not be detected in the MethyLight experiments. d At a threshold of PMR = 17.73 (dotted line), the area under the ROC curve was 0.73 with a maximal specificity and sensitivity of 0.71 and 0.78, respectively