Chimerism Monitoring with Highly Sensitive and Precise Next-Generation Sequencing Assay in Patients Post-Allogeneic Hematopoietic Stem Cell Transplantation

Abstract

<h3>Introduction</h3> Chimerism testing of peripheral blood and bone marrow aspirates following allogeneic hematopoietic cell transplantation (HCT) is important to evaluate engraftment kinetics and potentially early detection of disease relapse¹. NGS-based chimerism assays use SNP panels and provide standardized workflow with automated data analyses. <h3>Objectives</h3> We aim to evaluate performance of an NGS-based chimerism assay and compare it to existing laboratory method based on Short Tandem Repeat (STR) analysis. <h3>Methods</h3> Genomic DNA (gDNA) derived from PBMCs of unrelated donors were mixed at fractions ranging from 0.1% - 50% to evaluate sensitivity and specificity of the NGS-based chimerism assay in artificial genomic chimeras. 3-10 ng gDNA was used to prepare libraries with the assay kit and sequenced on Illumina MiSeq. <h3>Results</h3> Linear regression analyses of gDNA fractions (expected, observed) detected a linear signal at a minimum of 0.1% of minor gDNA in a two-gDNA chimeric mix (R²=1, average CV = 4.5%, range = 0.2-12.4). Chimeric samples with three distinct gDNAs gave linear signal as low as 0.5% of minor gDNA fraction (CV < 2%, range = 0.01-5.5%), a scenario that is clinically relevant in double-cord HCT or second HCT from different donors. The NGS-based chimerism assay was also tested on post-transplant samples derived from three HCT recipients. Results were compared longitudinally and between the two test methods at various time points (days) post transplantation. While majority results were consistent with previous data from STR, some discrepancies were observed. Patient 1, day+128, showed microchimerism (0.1%) that was undetected by STR (0%). Patient 2, day+287, showed low level (2.42%) of recipient DNA while STR reported full donor engraftment. Such clinical cases underscore the need for high sensitivity assays to more accurately quantitate donor-recipient fractions in post-HCT samples. <h3>Conclusions</h3> Using the test NGS-based chimerism assay on two and three gDNA mixes, we observe 10X greater sensitivity of minor fraction quantitation compared to current STR method. In our experience, high sensitivity, precision over a wide range of detection and robust performance with low input DNA may qualify this NGS-based chimerism assay as a potential method of choice for routine engraftment monitoring and potential early detection of disease relapse.