Chimerism Testing by Next Generation Sequencing for Detection of Engraftment and Early Disease Relapse in Allogeneic Hematopoietic Cell Transplantation and an Overview of NGS Chimerism Studies

Abstract

Chimerism monitoring after allogenic Hematopoietic Cell Transplantation (allo-HCT) is critical to determine how well donor cells have engrafted and to detect relapse for early therapeutic intervention. The aim of this study was to establish and detect mixed chimerism and minimal residual disease using Next Generation Sequencing (NGS) testing for the evaluation of engraftment and the detection of early relapse after allo-HCT. Our secondary aim was to compare the data with the existing laboratory method based on Short Tandem Repeat (STR) analysis. One hundred and seventy-four DNA specimens from 46 individuals were assessed using a commercially available kit for NGS, AlloSeq HCT NGS (CareDx), and the STR-PCR assay. The sensitivity, precision, and quantitative accuracy of the assay were determined using artificially created chimeric constructs. The accuracy and linearity of the assays were evaluated in 46 post-transplant HCT samples consisting of 28 levels of mixed chimerism, which ranged from 0.3–99.7%. There was a 100% correlation between NGS and STR-PCR chimerism methods. In addition, 100% accuracy was attained for the two external proficiency testing surveys (ASHI EMO). The limit of detection or sensitivity of the NGS assay in artificially made chimerism mixtures was 0.3%. We conducted a review of all NGS chimerism studies published online, including ours, and concluded that NGS-based chimerism analysis using the AlloSeq HCT assay is a sensitive and accurate method for donor-recipient chimerism quantification and minimal residual disease relapse detection in patients after allo-HCT compared to STR-PCR assay.

Utility of Next-Generation Sequencing-Based Chimerism Analysis for Early Relapse Prediction following Allogenic Hematopoietic Cell Transplantation

“…It is essential to note that artificial samples cannot fully substitute for the clinical post-HCT samples from patients. Liacini et al [ 2 ] calculated the overall CV as 5.08% (ranging from 0% to 11.21% at the expected chimerism value of 0.1–10%), and Janakiraman et al [ 13 ] demonstrated within- and across-run variability of 4.7% and 4.1%, respectively. Additionally, Picard et al documented a lower LOQ (0.22%) than that reported by the manufacturer ( Table 5 ) [ 8 ].…”

“…Additional benefits of AlloSeq HCT include its automated and user-friendly chimerism calculations [ 2 ]. The presented results encompass the percentage of DNA from each genetic contributor, QC status with reasons, the number of informative markers, and average marker coverage.…”

“…Simultaneously, the assessment of the patient-to-donor cell ratio, known as chimerism monitoring, serves to confirm donor cell engraftment. Moreover, chimerism monitoring is suggested for detecting potential relapses, especially in cases lacking suitable MRD markers [ 2 , 3 ]. Amanda et al [ 4 ] reported that 69% of the members of the Center for International Blood and Marrow Transplant Research use chimerism testing for detecting impending relapse, with most of them emphasizing engraftment monitoring as the primary indication.…”

“…Various methods can be employed for chimerism analysis utilizing polymorphic markers to differentiate donor cells from recipient cells. These methods encompass chromosome analysis, fluorescence in situ hybridization (FISH), short tandem repeat (STR) PCR, quantitative PCR (qPCR), digital PCR (dPCR), and next-generation sequencing (NGS) [ 2 , 7 , 8 ]. Each method has its strengths and weaknesses in terms of sensitivity, precision, applicability, and cost-effectiveness.…”