Branched evolution is a frequent route of clonal progression following treatment with LEN. (A) Mutational profiling of primary BM from P45 before and during LEN treatment reveals loss of subclonal lesions (DNMT3A/ATM/del(5q)) but stable maintenance of a mutation in TET2. (B) Schematic outline of an integrative NGS screening approach for unbiased evaluation of molecular disease evolution. (C) For 2 samples from P10 obtained before and after LEN treatment, validated mutations that have been requantified via UDS were bio-informatically separated into significant mutational subclusters with distinct MDS-specific driver lesions using the sciClone tool. (D-H) UDS-based profiling of somatically acquired mutations in P10 under LEN treatment and subsequent disease progression (D), and during long-term response under LEN therapy for P27 (E) and P31 (F) revealed complex branching evolution as illustrated in the graphical sketches. (G) Characterization of serial follow up BM aspirations from P13 during and after LEN treatment demonstrated linear acquisition of subclonal del(5q) and SF3B1 lesions during disease progression and emergence of an early PML/TET2-only “founder” clone after relapse from allo-SCT. (H) During 12 years of follow up, BM of P15 exhibited sequential acquisition of subclonal lesions including del(5q), which originated from an ASXL1-mutated “founder” clone that eventually gave rise to an independent branched subclone followed by clinical transformation into sAML and death of the patient. (Please note that loss of chromosome 7 carrying the EZH2 N130T-mutated allele results in decreasing VAF of this particular mutation and should not be mistaken with the disappearance of an early EZH2 N130T-mutated clone.) Mutations significantly associated with distinct clusters are equally colored. Depicted clinical parameters are WBC, Hb, PLT count, and proportion of CD34⁺ cells in BM quantified by FACS analysis. Samples that have been subjected to WES are underlined in red. Cytogenetic lesions throughout (D-H) were quantified via UDS-based SNP-skewing analysis. (I-J) Copy number and B-allele frequency profiling from WES data of P15 before (2010) and after (2014) LEN treatment (I), illustrates the transition of del(5q) into a copy number neutral UPD by duplication of the remaining nondeleted long arm of chromosome 5 (J). “+” indicates that mutational VAF was corrected for copy number bias. allo-SCT, allogeneic stem cell transplantation; chrom, chromosome; Hb, hemoglobin; LILRA5, leukocyte immunoglobulin-like receptor A5; PLT, platelet; vs, versus; WBC, white blood cell; WT, wild-type.