Clonal involvement of stem and progenitor cell compartments in patients with MDS in CR after allo-HSCT. (A-B) Representative FACS profiles of BM cells from an aged-matched healthy control (A) and from a patient during CR who had later relapsed (B; patient 8). Numbers in left panels indicate mean (± SEM) percentages of BM MNCs; in the second and third columns, percentages of total CD34⁺ cells for 12 age-matched normal controls (A) and 15 patients who relapsed (B). Also indicated are the quadrants from which investigated HSPC populations were sorted for mutational MRD analysis (for individual FACS profiles at diagnosis, CR, and relapse for all patients who experienced relapse, see supplemental Figure 3). (C-D) ddPCR screening for patient-specific mutations identified in BM MNCs before transplantation and still present at relapse was used to assess clonal involvement in remission BM MNCs and purified HSPCs. For each patient the number of months (mth) before relapse are shown, and in parenthesis months after transplantation. (C) Ten patients in whom MRD-positivity of HSPCs preceded MRD-positivity of BM MNCs and (D) 5 additional patients in whom MRD-positivity of distinct HSPCs was higher than MRD-positivity of BM MNC. For all patients (C-D), with the exception of patient 7, the earliest time point after allo-HSCT at which MNCs and/or HSPCs were found to be confidently MRD-positive, is shown. For patient 1, in whom the dominating diagnostic and relapse clones were mutually exclusive (see supplemental Figure 2), data are shown for the earliest time point after allo-HSCT when both the dominant diagnostic (D) and dominant relapse (R) mutation was detected in HSPCs. Error bars represent the 95% confidence interval of VAFs calculated according to Poisson distribution, as further specified in “Methods.” Blue color indicates highly confident clonal involvement, red indicates negative, and green indicates inconclusive data based on the LOD established in normal BM samples for each ddPCR probe (supplemental Table 6) as well as the number of cells analyzed as described in supplemental Methods. ∗Analysis negative for clonal involvement (red) but based on analysis of <50 purified cells. Hashtag (#): mutation of “unknown” significance as specified in supplemental Methods. Raw data including cell numbers analyzed can be found in source data file 1. HSC, hematopoietic stem cell (LIN⁻CD34⁺CD38^low/−CD90⁺CD45RA⁻); MPP, multipotent progenitor (LIN⁻CD34⁺CD38^low/−CD90⁻CD45RA⁻); LMPP, lymphoid-primed MPP (LIN⁻CD34⁺CD38^low/−CD90⁻CD45RA⁺); CMP, common myeloid progenitor (LIN⁻CD34⁺CD38⁺CD90⁻CD123⁺CD45RA⁻); GMP, granulocyte-monocyte progenitor (LIN⁻CD34⁺CD38⁺CD90⁻CD123⁺CD45RA⁺); and MEP, megakaryocyte-erythroid progenitor (LIN⁻CD34⁺CD38⁺CD90⁻CD123⁻CD45RA⁻).