International Survey and Next-Generation Sequencing-Based Indel Chimerism Detection of Human Leukocyte Antigens Loss in Relapsed Haploidentical and Mismatched Unrelated Donor Transplant Recipients.

Human leukocyte antigen (HLA) loss-mediated relapse is an important mechanism of relapse after haploidentical or mismatched unrelated donor (MMUD) allogeneic hematopoietic stem cell transplantation (allo-HCT), impacting the efficacy of donor lymphocyte infusion (DLI) and second transplants. Real-world use of HLA loss testing is poorly characterized. We conducted an international survey of 62 transplant physicians from 11 countries across North America, Europe, Asia, and Central and South America to assess practices surrounding HLA loss testing after haploidentical or MMUD allo-HCT. We also applied a next-generation sequencing (NGS)-based indel chimerism HLA loss assay to 58 samples from 36 patients who relapsed post-transplant, of whom 29 samples with recipient chimerism greater than 5% were evaluable for HLA loss assessment. Most respondents practiced in academic centers (76%). For molecular relapse, 74.5% used DLI alone; for hematologic relapse, 72.5% favored a second allo-HCT. Most (85.7%) preferred a different donor for second transplants, but HLA loss testing was infrequent prior to second allo-HCT (21%) or DLI (17.7%), with no differences by center type or geography. Testing barriers included cost (88.1%), limited assay availability (87.8%), and long turnaround times (44.5%). At our center, application of an NGS-based indel chimerism assay to 29 evaluable samples (recipient chimerism >5%) identified HLA loss in 3 patients (10.3%), including both complete and partial haplotype loss. Confirmatory NGS HLA typing in one patient validated the indel chimerism findings. The assay was rapid, sensitive, did not require patient-specific markers, and could reduce both cost and turnaround time compared to conventional methods, directly addressing the major barriers to HLA loss testing identified in the international survey. HLA loss testing remains underutilized worldwide. Increased awareness and broader access to rapid, cost-effective assays are needed, alongside prospective studies to establish optimal testing strategies.