Title: Role of mitochondrial DNA biomarker in chronic and late acute graft-versus-host disease in children

Abstract

Background: Chronic graft-vs.-host disease (cGvHD) is a major long-term complication of hematopoietic stem cell transplantation (HSCT) resulting in high levels of morbidity and mortality post-HSCT. Few biomarkers for prediction and early diagnosis of cGvHD are available. Previously, we found that mitochondrial DNA (mtDNA) is elevated in adults with chronic graft-versus-host disease (cGvHD), acting as an endogenous source of TLR9 agonists to augment B cell responses. To further evaluate the role of mtDNA in cGvHD, we evaluated mtDNA plasma expression in a large pediatric cohort from the ABLE study. Methods: Plasma cell-free mtDNA (cf-mtDNA) copy number was isolated from plasma and quantified using digital PCR by amplification of MT-CO1 and MT-ND1 human mitochondrial genes. Two assays probe with FAM and VIC fluorescent dyes were used to increase accuracy and efficiency. Two evaluations were performed: a) before the onset of cGvHD or late aGvHD at day and b) at the time of onset cGvHD. Results: Comparing day 100 samples in patients developing late acute GvHD after day 100 to the 3-month samples in controls with no cGvHD/no late acute GvHD, higher levels of cell-free mtDNA were found in the late acute GvHD cohort associated with VIC - MT-CO1 assay. Most significant was that plasma mtDNA concentrations were elevated in patients at the onset of cGvHD. There was no significant elevation of plasma cell-free mtDNA at day 100 after HSCT in patients who later developed cGvHD compared with no cGvHD controls with mtDNA measured by the VIC - MT-CO1 assay. Conclusions: Our study shows that cf-mtDNA level is increased at day 100 after HSCT in patients later developing late acute GVHD after day 100 (before the onset of cGvHD) and at the onset cGvHD. Measurement of mt-DNA may have the potential as a diagnostic biomarker to guide therapy.