Monica Sorbini1, Gabriele Maria Togliatto1, Erika Simonato2, Massimo Boffini3, Margherita Cappuccio1, Alessandro Gambella1, Francesca Arruga1, Nicola Mora1, Matteo Marro2, Cristiana Caorsi4, Morteza Mansouri4, Paola Magistroni4, Luisa Delsedime5, Mauro Giulio Papotti5, Antonio Amoroso6, Mauro Rinaldi2, Tiziana Vaisitti1, Silvia Deaglio6. 1. Department of Medical Sciences, University of Turin, Turin, Italy. 2. Cardiac Surgery Division, Surgical Sciences Department, Heart and Lung Transplant Center, Città della Salute e della Scienza University Hospital of Torino, Torino, Italy. 3. Cardiac Surgery Division, Surgical Sciences Department, Heart and Lung Transplant Center, Città della Salute e della Scienza University Hospital of Torino, Torino, Italy. Electronic address: massimo.boffini@unito.it. 4. Immunogenetics and Transplant Biology Service, Città della Salute e della Scienza University Hospital, Turin, Italy. 5. Department of Oncology, University of Turin and Pathology Unit, Città della Salute e della Scienza Hospital, Turin, Italy. 6. Department of Medical Sciences, University of Turin, Turin, Italy; Immunogenetics and Transplant Biology Service, Città della Salute e della Scienza University Hospital, Turin, Italy.
Abstract
BACKGROUND: Donor-derived cell-free DNA (dd-cfDNA) is considered a reliable marker of organ damage with potential applications in the follow-up of transplant recipients. METHODS: In this work we present an assay based on the donor-recipient HLA-mismatch (human leukocyte antigen) at the HLA-DRB1 locus to monitor rejection by quantifying the percentage of dd-cfDNA using a droplet digital PCR (polymerase chain reaction) technique. A panel of probes targeting the HLA-DRB1 locus and covering >85% genetic variability was validated and used to assess dd-cfDNA levels in a prospective cohort of 19 adult heart transplant recipients (mean age 50.9±14.8 years). The assay was carried out on a total of 232 liquid biopsies collected at the same time as endomyocardial biopsy (EMB) during routine post-transplant follow-up. RESULTS: Results show a significant increase of dd-cfDNA related to ischemia-reperfusion injury (2.22±2.09%) and to acute cellular rejection (1.71±3.10%) compared to stable conditions (0.43±1.04%, p < 0.0001). On the contrary, no increase was observed during infections or vascular complications, underlining the potential role of this biomarker for rejection monitoring. With a cut-off of 0.11%, the test showed 70.8% specificity (95% CI, 58.17% - 81.40%) and 64.2% sensitivity (95% CI, 49.80% - 76.86%) in discriminating acute rejection from no rejection. CONCLUSIONS: These data demonstrate that this HLA mismatch-based droplet digital PCR method is effective for monitoring rejection in heart transplant recipients. Compared to next generation sequencing approaches, it is far more flexible, less expensive and provides faster results.
BACKGROUND: Donor-derived cell-free DNA (dd-cfDNA) is considered a reliable marker of organ damage with potential applications in the follow-up of transplant recipients. METHODS: In this work we present an assay based on the donor-recipient HLA-mismatch (human leukocyte antigen) at the HLA-DRB1 locus to monitor rejection by quantifying the percentage of dd-cfDNA using a droplet digital PCR (polymerase chain reaction) technique. A panel of probes targeting the HLA-DRB1 locus and covering >85% genetic variability was validated and used to assess dd-cfDNA levels in a prospective cohort of 19 adult heart transplant recipients (mean age 50.9±14.8 years). The assay was carried out on a total of 232 liquid biopsies collected at the same time as endomyocardial biopsy (EMB) during routine post-transplant follow-up. RESULTS: Results show a significant increase of dd-cfDNA related to ischemia-reperfusion injury (2.22±2.09%) and to acute cellular rejection (1.71±3.10%) compared to stable conditions (0.43±1.04%, p < 0.0001). On the contrary, no increase was observed during infections or vascular complications, underlining the potential role of this biomarker for rejection monitoring. With a cut-off of 0.11%, the test showed 70.8% specificity (95% CI, 58.17% - 81.40%) and 64.2% sensitivity (95% CI, 49.80% - 76.86%) in discriminating acute rejection from no rejection. CONCLUSIONS: These data demonstrate that this HLA mismatch-based droplet digital PCR method is effective for monitoring rejection in heart transplant recipients. Compared to next generation sequencing approaches, it is far more flexible, less expensive and provides faster results.