Changes in clonal composition in BM are reflected in clinical alterations and course of the disease. UDS-based VAF quantification of mutational evolution during clinical follow up: (A) For P03, LEN treatment induced hemotoxicity without a profound reduction in MDS clonal burden resulting in transfusion dependence after treatment discontinuation and outgrowth of a novel EZH2-mutated subclone shortly after temsirolimus therapy. (B) P20 acquires 3 distinct subclonal mutation clusters during a follow-up of 11 years, prior to treatment with LEN. This induces a massive molecular remission, accompanied by a hematotoxic reaction, which is reversed after withdrawal of LEN and followed by rapid reconstitution of the pretreatment clone. (C) P37 received 5-aza in a high-risk disease stage and achieved transfusion independence, which was accompanied by overall clonal reduction. However, upon treatment discontinuation, his MDS clone acquired a novel subclonal LZTR1 mutation coinciding with prolonged transfusion dependence. Reinitiating the 5-aza treatment resulted only in a minor molecular response of the new subclone and was quickly followed by the patient’s death. (D) P19 shows a stable disease with fully clonal BM and novel appearance of a branched del(ETV6) clone following treatment with panobinostat, which remains stable on a subclonal level. Without any specific treatment applied, a rapid, diverse subclonal expansion can be observed in P29 (E) and P30 (F) leading to disease progression and rapid death. Completely independent clones were found in P02 (G) and P40 (H). (G) P02 showed a highly stable coexistence of a CHRM2/del(5q) clone and a DNMT3A-mutated clone for 16 years. Upon LEN treatment, the CHRM2/del(5q) clone completely disappeared, whereas the DNMT3A-mutant clones immediately fully clonal expanded, which changed the clinical phenotype of the patient, currently receiving phlebotomy. (H) In P40, a SF3B1/TET2/DNMT3A/del(5q) clone was likewise completely outcompeted by an independent DNMT3A-mutant clone after LEN treatment, ultimately resulting in a fatal outcome. Cytogenetic lesions throughout (A-H) were quantified via UDS-based SNP-skewing analysis unless stated otherwise. “+” indicates that mutational VAF was corrected for copy number bias.