TY - JOUR
T1 - Cheaper and Faster Analysis of Donor-Derived Cell-Free DNA in Children after Heart and Lung Transplantation; Validation of a New Assay
AU - Ellershaw, D.
AU - Preka, E.
AU - Chandler, N.
AU - Ahlfors, H.
AU - Spencer, H.
AU - Marks, S.
AU - Fenton, M. J.
PY - 2020/4/1
Y1 - 2020/4/1
N2 - PURPOSE: Donor-derived cell-free cell free DNA (ddcfDNA) is a noninvasive biomarker undergoing investigation in adult heart and lung allograft recipients. Elevated ddcfDNA levels have been correlated with active rejection, most robustly for antibody-mediated rejection (ABMR). However, complex methodology limits clinical implementation of this promising biomarker. There are no data in the paediatric population. METHODS: Blood from 96 heart and 19 lung pediatric transplant recipients were taken at various time points, e.g. as a baseline, before/after biopsies and the clinical status was recorded. Donor allograft biopsies were available from 7 heart patients. A multiplex PCR assay consisting of a panel of 61 highly heterogeneous single nucleotide polymorphic markers (SNPs), HLA exon 3 and X/Y-markers was designed and validated by using genomic DNA (gDNA). This assay was then used to look for evidence of the donor's unique genotype in the recipient's cell free DNA (cfDNA). A statistical model using R was developed to perform donor genotype-free analysis which was validated against the known donor genotype from donor allograft biopsies. Total cell free DNA concentration was also measured. RESULTS: The ddcfDNA assay showed a limit of blank (LoB) of 0.12% (based on SNPs homozygous for the same allele from each genomic contribution) and a limit of detection/quantification (LoD/LoQ) of 0.28%. The accuracy of the genotype free analysis was greatest for donor fractions above 0.5%. For patients without clinical evidence of allograft damage a stable low baseline of ddcfDNA was observed. A single heart patient in this series died approximately 2 years post-transplantation. The assay demonstrated a stable baseline initially but increased to 2.03% five weeks prior to death and further increased to 12.16% one week prior to death due to poor function and cardiac allograft rejection. CONCLUSION: We present the first study in pediatric heart and lung transplantation validating the performance of a SNP-based PCR assay, rather than a whole genome assay, to detect recipient ddcfDNA fraction. This offers the potential for a rapid and cheaper noninvasive biomarker for monitoring allograft damage.
AB - PURPOSE: Donor-derived cell-free cell free DNA (ddcfDNA) is a noninvasive biomarker undergoing investigation in adult heart and lung allograft recipients. Elevated ddcfDNA levels have been correlated with active rejection, most robustly for antibody-mediated rejection (ABMR). However, complex methodology limits clinical implementation of this promising biomarker. There are no data in the paediatric population. METHODS: Blood from 96 heart and 19 lung pediatric transplant recipients were taken at various time points, e.g. as a baseline, before/after biopsies and the clinical status was recorded. Donor allograft biopsies were available from 7 heart patients. A multiplex PCR assay consisting of a panel of 61 highly heterogeneous single nucleotide polymorphic markers (SNPs), HLA exon 3 and X/Y-markers was designed and validated by using genomic DNA (gDNA). This assay was then used to look for evidence of the donor's unique genotype in the recipient's cell free DNA (cfDNA). A statistical model using R was developed to perform donor genotype-free analysis which was validated against the known donor genotype from donor allograft biopsies. Total cell free DNA concentration was also measured. RESULTS: The ddcfDNA assay showed a limit of blank (LoB) of 0.12% (based on SNPs homozygous for the same allele from each genomic contribution) and a limit of detection/quantification (LoD/LoQ) of 0.28%. The accuracy of the genotype free analysis was greatest for donor fractions above 0.5%. For patients without clinical evidence of allograft damage a stable low baseline of ddcfDNA was observed. A single heart patient in this series died approximately 2 years post-transplantation. The assay demonstrated a stable baseline initially but increased to 2.03% five weeks prior to death and further increased to 12.16% one week prior to death due to poor function and cardiac allograft rejection. CONCLUSION: We present the first study in pediatric heart and lung transplantation validating the performance of a SNP-based PCR assay, rather than a whole genome assay, to detect recipient ddcfDNA fraction. This offers the potential for a rapid and cheaper noninvasive biomarker for monitoring allograft damage.
UR - http://www.scopus.com/inward/record.url?scp=85085636476&partnerID=8YFLogxK
U2 - https://doi.org/10.1016/j.healun.2020.01.1272
DO - https://doi.org/10.1016/j.healun.2020.01.1272
M3 - Article
C2 - 32466006
SN - 1053-2498
VL - 39
SP - S67-S68
JO - Journal of Heart and Lung Transplantation
JF - Journal of Heart and Lung Transplantation
IS - 4
ER -