Discrepant dynamics of TP53 mutations during follow-up compared with IG/TR MRD. (A) Longitudinal DNA samples of 5 patients with ALL selected based on TP53 mutation positivity in an MRD-negative follow-up sample and availability of archived left-over viable cells were analyzed for the presence of specific TP53 mutations using mutation-specific digital droplet PCR. In all 5 cases, the respective TP53 mutations (red lines) were sustained despite MRD-negativity (blue lines) during remission (top panel). Pat32 relapsed with a clone harboring an entirely unrelated IG/TR (dotted blue line) rearrangement and gained a second TP53 mutation (dotted red line) while sustaining the diagnostic TP53 mutation. In patients 14, 19, and 29, false MRD negativity owing to potential clonal evolution of the MRD marker was excluded by IGH amplicon next-generation sequencing analysis that neither showed the diagnostic IGH clonotype nor a related IGH or any other unrelated highly abundant IGH clonotype in the remission sample (data not shown). Distinct hematopoietic cell populations (early hematopoietic cells; hematopoietic stem cells/multipotent progenitors [HSCs/MPPs]), common myeloid/lymphoid progenitors (L/M prog), myeloid progenitors (M prog), and B-cell precursors (B-cell prec), as well as the mature immune cell populations such as T cells, monocytes, and granulocytes of an MRD-negative follow-up sample, were isolated using the FACS Aria sorter. Sorted subpopulations were analyzed for the presence of specific TP53 mutations by mutation-specific digital droplet PCR. Values in percentages indicate the TP53 mutant-allele burden in the isolated populations. (B) TP53 mutations in ALL originate in a preleukemic compartment that can self-renew and cause clonal expansions in hematopoietic compartments; thus, they persist in tumor-free cells during remission and increase at the (re)occurrence of the disease. IGH, immunoglobulin heavy locus; TP53^mt, mutated TP53; TP53^wt, wild-type TP53. Panel B was created with BioRender.