Lack of evidence for clonal involvement of HSPCs in patients in long-term continuous-CR after HSCT. (A) Kinetic assessment of MRD by ddPCR for patient-specific mutations identified at diagnosis, in BM MNCs and HSPCs in patients in continuous-CR (≥66 months); diamond-shaped symbol indicates time point of last clinical assessment without evidence of relapse after allo-HSCT, from time of transplantation (0). For complete ddPCR data for each patient and time point see supplemental Figure 6. MNC MRD⁺, at least 1 mutation confidently detected in BM MNCs by ddPCR; MNC MRD⁻; no mutation confidently detected in BM MNCs by ddPCR; HSPC MRD⁺, at least 1 mutation confidently detected in ≥1 stem and progenitor cell populations by ddPCR; HSPC MRD⁻, no mutation confidently detected by ddPCR in any investigated stem and progenitor cell populations (see “Methods”). (B) ddPCR-based VAF analysis of CR samples from 4 patients who relapsed for patient-specific SNPs (left; see supplemental Methods for selection of SNPs) as compared with mutational VAF (right) in BM MNCs, HSCs, and the most clonally involved HSPC population (in patient 5, HSCs were most clonally involved). (C) Patient-specific SNP analysis in all HSPC populations from CR samples from 6 patients in continuous-CR, at the earliest and latest available time point after allo-HSCT at which remission BM was analyzed, and for which mutational MRD was negative in all cell populations and cases. Error bars represent the 95% confidence interval of VAFs calculated according to Poisson distribution as further specified in “Methods.” Blue and red indicate confidently positive and negative data, respectively, and green inconclusive, as defined in the supplemental Methods. Raw data can be found in source data file 1 (mutational HSPC ddPCR) and source data file 3 (SNP ddPCR).