Microhaplotype-based chimerism analysis for sensitive monitoring following allogeneic hematopoietic stem cell transplantation

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BACKGROUND: Relapse after allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains a leading cause of treatment failure in patients with hematologic malignancies. Short tandem repeat (STR)-based chimerism analysis has limited analytical sensitivity, whereas minimal residual disease (MRD) assays are disease-specific and not universally applicable. We developed a microhaplotype (MH)-based next-generation sequencing assay for sensitive quantification of low-level recipient DNA after allo-HSCT. METHODS: Analytical performance was evaluated using artificial donor-recipient DNA mixtures containing 0.01% to 10% recipient DNA. Among 74 MH loci, highly informative loci were defined as those containing at least one recipient-specific allele that differed from both donor alleles by two or more single nucleotide polymorphisms. Recipient chimerism was estimated as the median value across highly informative loci. Analytical sensitivity was determined using unmixed donor-recipient samples across six donor-recipient pairs. Clinical performance was assessed in 10 allo-HSCT recipients and compared with STR-based chimerism and available MRD results. The limit of detection was 0.022%, with an estimated limit of quantitation of 0.1%. A median of 17 highly informative loci was identified per donor-recipient pair in 10 clinical samples. MH-based chimerism monitoring detected relapse-associated recipient chimerism changes, including cases not adequately resolved by STR-based analysis. CONCLUSIONS: Quantification of highly informative MH loci enables sensitive and reliable chimerism monitoring after allo-HSCT. This approach may complement existing methods for longitudinal assessment of recipient chimerism dynamics.