Circulating Cell Free Mitochondrial DNA As a Diagnostic and Prognostic Biomarker in Chronic and Late Acute Graft-Versus-Host Disease in Children

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. 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 validate this in children, we evaluated mtDNA plasma expression in a large pediatric cohort (ABLE /PBMTC 1202 study).

Methods:

Plasma cell-free mtDNA (cf-mtDNA) copy number was measured in 202 pediatric patients. cf-mtDNA was isolated from plasma and quantified using digital PCR by amplification of MT-CO1 and MT-ND1 human mitochondrial genes. To increase accuracy and efficiency for measuring levels of plasma cell-free mitochondrial DNA, two assays probe with FAM, VIC fluorescent dyes was used. Plasma cell-free mitochondrial DNA values as copies/μL separately and total number of wells were reported. Two evaluations were performed: a) before the onset of cGvHD or late aGvHD at day 100 +/- 14 days; and b) at the time of onset cGvHD, time-matched to non-cGvHD controls at 3, 6, and 12 months post-BMT. We also evaluated the impact of various clinical types of cGvHD, such as de novo, progressive, and pulmonary cGvHD on mtDNA at the onset sample of cGvHD. We evaluated potential clinical application using our previous definition of biological significance for a cGvHD biomarker which defines a potential biomarker as having: a ROC AUC ≥0.60; p-value ≤0.05; and effect ratio ≥1.3 or ≤0.75.

Results:

Comparing day 100 samples in patients developing late acute GvHD after day 100 (n=44) 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 (5810 ± 4103 copies/μL, [mean ± SD], vs 4352 ± 2881 copies/μL; p = 0.02). Most significant was that plasma mtDNA concentrations were elevated in

We found post HSCT immune reconstitution did not impact mtDNA copy numbers at 3, 6, and 12-months post-transplant in patients without cGvHD or late acute GvHD and was not impacted by previous aGvHD. There was no significant elevation 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 (N = 25, 5707 ± 4100 copies/μL, [mean ± SD], N = 103; 4352 ± 2881 copies/μL; p = 0.08).

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, confirming our previous observations in adults. Measurement of mt-DNA may have the potential as a diagnostic biomarker to guide therapy.

No relevant conflicts of interest to declare.