MDS is an oligoclonal disease consisting of a founding clone and one or more subclones derived from the founding clone. Relapsed disease in myeloid malignancies is often characterized by the emergence of a subclone, presumably from selection pressure, following chemotherapy. The contribution of subclone evolution to MDS disease progression following an allogeneic hematopoietic cell transplant (alloHCT) has not been comprehensively studied.We used a combination of exome sequencing, ultra-deep error-corrected targeted sequencing, and copy number analysis to comprehensively describe the clonal dynamics of MDS in response to pretransplant and transplant therapy using 58 serially acquired tumors harvested from 9 patients who all progressed post-alloHCT after receiving either a myeloablative (n=3) or reduced-intensity (n=6) transplant (median time to progression 309 days, range 98-881). We performed enhanced exome sequencing (EES) to define the clonal architecture of 23 tumor samples at the following clinical landmarks (with matched skin as a source of normal DNA): first sampling (usually diagnosis), <2 months pre-alloHCT (where available), and progression post-alloHCT. Somatic mutations were validated in the 23 discovery samples and genotyped in 35 additional samples post-alloHCT, using capture probes targeting all putative single-nucleotide variants (SNV) and short insertions and deletions (indels) identified by enhanced exome sequencing. In addition, ultra-deep, error-corrected sequencing (i.e., barcoded sequencing) was performed on 49 of the 58 tumor samples to provide increased sensitivity to detect low-level mutations. Structural variants (SVs) were identified using exome and FISH data.We found that a subclone expanded or emerged at progression post-alloHCT in all 9 patients. We also observed that mutations (SNVs and/or indels) detected in the founding clone at first sampling always persisted at progression post-alloHCT. In 7 of 9 patients, the expanding/emerging subclone became the most abundant clone at relapse, underscoring the importance of subclonal expansion for driving disease progression. In 6 of these 7 patients (and 7 of 9 overall), the expanding/emerging subclone contained mutations and/or SVs known to be associated with myeloid malignancies. Furthermore, in 4 cases, a rare or minor pretransplant subclone escaped eradication, acquired new mutations or structural variants, and emerged as the most abundant clone at progression. This suggests that minor pre-existing, treatment-resistant subclones can undergo further evolution and drive progression post-alloHCT. In three of these patients, the emergent subclone at progression also gained SVs. In the other patient, the minor subclone, not detected by ultra-deep sequencing at first sampling, emerged during pre-transplant therapy with azacitidine and was characterized by the acquisition of C to G transversions, including coding C to G transversions in the recurrently mutated AML/MDS genes SF3A1 and EPPK1 ; thus the pretransplant therapy shaped the clonal evolution at progression. Overall, SVs were present in the emergent/expanding subclone in 8 of 9 patients. Structural variants were the only new somatic alteration in an expanding/emerging subclone in 5 patients at progression, highlighting the importance of monitoring SVs in post-alloHCT samples, and implicating SVs as late subclonal progression events.We interrogated 35 post-alloHCT specimens to determine if mutations were detectable prior to clinically-defined progression in the 9 patients. In 6 of the 8 patients with available specimens at days 30 and 100 post-alloHCT, mutations were detectable at either or both of these time points. In five instances, standard capture-based sequencing and/or error-corrected sequencing detected low-level mutation burden prior to clinical progression. In all other cases, sequencing results provided independent confirmation of relapse.In conclusion, MDS progression post allo-HCT is characterized by subclonal expansion and mutational evolution, which can be influenced by pretransplant therapy with hypomethylating agents. Although progression is characterized by increasing genetic diversity (including acquisition of SVs) the founding clone mutations are always present at progression, suggesting that tracking founding clone mutations may be useful to detect impending progression/relapse. DisclosuresNo relevant conflicts of interest to declare.