3045 – MOLECULAR EVOLUTION OF MEASURABLE RESIDUAL DISEASE IN MYELODYSPLASTIC SYNDROMES

Stem cell transplantation (SCT) is the sole curative approach for MDS. However, MDS relapse post-SCT, driven by MRD progression, presents the major obstacle to cure. We hypothesize that post-SCT MRD comprises a uniform population resistant to both graft-versus-leukemia effects and chemotherapy. To identify MRD molecular signatures, measure intra-patient phenotypic diversity, and elucidate pathways of MRD progression, we developed a novel single-cell (sc) workflow, “CARAMEL-seq”, to simultaneously capture RNA, ATAC, surface protein, and genotypic data, enabling identification and longitudinal tracking of (non)malignant hematopoietic stem and progenitor cells (HSPCs). We obtained multi-modal sc measurements from 160,858 CD34+ HSPCs and 135,666 CD34- marrow cells from 10 higher-risk MDS patients who relapsed after SCT across 30 timepoints. We identified malignant MRD cells at frequencies < 10-6 with uniquely defining features (FDR< 0.001). Recipient HSPCs at MRD comprised malignant (median 94%; range: 40-97%) and non-malignant residual cells based on aneuploidy, aberrant immunophenotype, and mitochondrial mutations. Unexpectedly, despite the narrow bottleneck of SCT, MRD cells exhibited diverse phenotypic states, sharing the majority with MDS cells at relapse and preSCT timepoints. MRD cells were enriched in G0/G1 phase compared to donor HSPCs (88% vs. 60%), indicating increased quiescence. Finally, by using mitochondrial/somatic mutations to track MRD clonal output to relapse, we found significant variability in outgrowth potentials across MRD cells with clear identification of ‘expanding’ and ‘contracting’ clones. These data suggest that MRD progression involves composition shifts between competing clones that drive immense phenotypic shifts from pre-SCT to post-SCT relapse. Our results reveal unexpected plasticity in MRD phenotypic states and outgrowth potentials.