PF782 MNPSEQ AS A HIGHLY SENSITIVE METHOD FOR DETECTING CHIMERISM AFTER ALLOGENEIC HEMATOPOIETIC STEM CELL TRANSPLANTATION

Background: Chimerism testing after allogeneic hematopoietic stem cell transplantation (Allo‐HSCT) can be used to monitor donor engraftment or to diagnose relapse. At present, short tandem repeats (STR) analysis is the most popular method for monitoring chimerism following Allo‐HSCT. However, this method suffers from a large number of technical and quality issues such as low detection sensitivity (3∼5%), stutter interference, and the need for genetic information of donor and recipient. Multiple Nucleotide Polymorphism (MNP) refers to the combination of multiple SNPs within <200 bp DNA, and the polymorphisms of different SNPs are MNP. As MNP contains multiple SNPs, it is a multi‐allele genetic marker, which contains more abundant genetic information. Aims: In this study, we carried out a clinical validation study of using MNPseq to detect chimerism based on next‐generation sequencing that enable us to analyze MNPseq's sensitivity and accuracy, and to assess the possibility of MNPseq used in the detection of chimerism without genetic information of donor and recipient. Methods: A total of 106 peripheral blood or bone marrow samples were collected from donors and patients undergoing Allo‐HSCT in Hematology Department of Henan Cancer Hospital, among which these samples were simultaneously detected fusion gene by real‐time PCR (RT‐PCR) and minimal residual disease (MRD) by flow cytometry (FC). In addition, linearity and detection sensitivity were tested by serially diluting white blood cells from two unrelated normal donors with varying dilution factors by 50, 25, 12.5, 6.25, 3.12, 1, 0.5, 0.1 and 0.01%. MNPseq was detected based on next‐generation sequencing (Ion Torrent PGM), which containing 100 amplicons and 646 SNPs. Mean read depth ranged from 3,427 × to 9,524 × , and the coverage of 96.8% of SNPs was over 2,000 × . Chimerism was calculated according to the polymorphism and frequency of MNP. Results: In a comparison study using serially diluting mixed samples, chimerism measured by MNPseq showed an excellent correlation and detection sensitivity with that measured by STR analysis (correlation: r 2  = 0.9992 vs. r 2  = 0.9968; sensitivity threshold: 0.01% vs. 1%). Among 57 samples with no recipient chimerism detected by STR, 32 samples were positive (0.04∼2.13%) by MNPseq analysis, all were positive detected by RT‐PCR, and only 2 samples were FC positive. MNPseq, RT‐PCR and FC results were all positive in 9 samples with recipient chimerism ranged from 6% to 71%. And MNPseq analysis was also performed in the case of incomplete donor and recipient genetic information. When separate genetic information was availiable or none, MNPseq could not only be used for chimerism detection, but also for detection sensitivity could reach 0.01% or 1%. Furthermore, MNPseq results were 2.13∼1.21% in 13 of 57 samples with no recipient chimerism when genetic information of donor and recipient was absent. Summary/Conclusion: MNPseq is not only a highly sensitive chimerism detection method, but also suitable for chimerism detection without genetic information of donor and recipient.