An Ultrasensitive Next Generation Sequencing Method for the Detection of Minimal Residual Disease and the Assessment of Chimerism Following Hematopoietic Cell Transplantation

Abstract

Introduction Accuracy and precision are critically important in the assessment of minimal residual disease (MRD) and chimerism following hematopoietic cell transplantation. Reliable and sensitive detection of changes in engraftment status can impact patient care by early detection of potential failure of engraftment or relapse. We evaluated a commercially available NGS technology for engraftment monitoring for sensitivity, accuracy and reproducibility. Objective Introduction of next generation sequencing technology for engraftment monitoring. Methods Samples analyzed by NGS at Baylor Transplant Immunology Laboratory included archived clinical specimens with historic results by STR, n=19, or qPCR, n=15, test results; 72 artificial mixtures of volunteer donors, and 5 ASHI external proficiency specimens. NGS analysis assessed 182 SNPs validated in the 1000 Genomes HapMap project and distributed across all 23 chromosomes, including the x chromosome. The rapid workflow allows a TAT of 2 days for up to 24 samples, with a less than 6 hour NGS library preparation protocol, followed by a 6 hour sequencing run time and 30 minute analysis. The low sample requirement, 1 ng DNA per PCR, facilitates chimerism assessment in multiple cell subsets including low frequency populations such as CD34-positive cells. Results NGS showed close concordance with historic results from STR and qPCR (figure 1), providing validation of NGS technology. Figure 2 compares NGS results versus the reference values of artificial mixtures and demonstrates NGS accuracy, while the low tech to tech variation data reveals assay precision. Limit of detection experiments showed reliable 0.1% sensitivity, as illustrated in Figure 3, with NGS analysis of 4 mixtures at 0.1%.. In addition, NGS reported results for the external proficiency samples correlated closely with the mean % donor reported throughout the full range of donor/recipient proportions including concordance with a sample of 100% donor. Conclusions Application of NGS technology to chimerism analysis in HCT provides accurate and precise test results. With the reliable 0.1% sensitivity and low input DNA that allows testing of multiple critical cell subsets, NGS is particularly relevant to early detection of MRD, graft failure, and relapse.