Research Group
- Assist. Prof. Howard Slater, Principal Investigator
- Prof David Power, Co-Investigator
- Assist. Prof. Frank Ierino, Co-Investigator
- Dr Bruno Damien, Co-Investigator
- Ms Devika Ganesamoorthy, Res. Associate
- Assist. Prof. Nigel Toussaint, Collaborator
- Assist. Prof. John Kanellis, Collaborator
Location
- Murdoch Childrens Research Institute, Parkville, Australia
Title
- A Novel, Simple, Universal Blood Test Based on Copy Number Variants for Organ Transplant Monitoring
To date, 119 renal transplant patients have been recruited, representing 60% of the total planned within the first 21 months of the study (Milestone 1).
The protocol for the blood test, consisting of 10 copy number deletion assays, was finalised for plasma circulating free DNA (cfDNA) measurement by Month 3. We have shown each of the assays to be highly specific and sensitive; replicate measurements have been shown to be precise with a standard error of 0.01 and linear slope of 1 up to 16,000 GE/ml; the lower limit of detection was 4-16 GE/ml. This in essence achieves Aim 1 (To develop a non-invasive, sensitive blood test consisting of copy-number variation (CNV)-deletion assays to quantify ‘donor-derived’ DNA released into the recipient’s plasma from the transplant organ).
To date, 117 samples have been genotyped (1170 genotype assays) to identify informative markers. At least one informative marker was found in 90% of recipients with an average of 2 per recipient. This is in line with our predictions using a panel of 10 markers; for universal application to any donor-recipient pair (identical twins excepted) we estimate that a panel of 30 CND markers will be needed. Measurements of non-self, transplant-derived cfDNA have been made on 148 samples (an average of 2 per sample) including 69 samples from ‘stable’ patients indicating that the ‘stable’ level of non-self cfDNA is below 50 GE/ml. This is an important step towards Aim 2 (To determine inter-individual variation in baseline levels of ‘donor-derived’ DNA in the plasma of ‘stable’ transplant recipients).
Aim 3: To measure ‘donor-derived’ DNA levels in the plasma of patients with graft damage: Measurements of ccfDNA have been made in 30 patients with biopsy-proven graft damage, i.e. antibody-mediated rejection (AMR) (13), cell-mediated rejection (CMR) (6), tubulopathies (including glomerulonephritis), vasculitis, infections (renal and systemic), renal cancer (11). So far, cfDNA data from 81 patients have been reviewed and correlated with the clinical/biopsy data (Fig. 1). These results suggest there are four groups within the distribution that appear to be statistically separate. The AMR groups show significantly higher plasma cfDNA levels than either the ‘stable’ or CMR groups; the intermediate group may be a tail of the ‘stable’ group or potentially a transition group with low level rejection undetected by biopsy. The elevated levels in the AMR group are consistent with our hypothesis of ‘damage-induced cfDNA release’; however, the low levels in the CMR group are not. Testing a larger number of ‘rejecting’ patients (to be recruited from the Royal Melbourne Hospital and Monash Medical Centre renal units in addition to that at Austin Health) will confirm (or not) these findings.
Figure 1. Box plot on the measurements of donor-derived plasma cfDNA levels in ‘stable’ recipients and those experiencing antibody-mediated rejection (AMR), cell-mediated rejection (CMR) and an intermediate group.
