Single cell proteogenomic sequencing identifies a relapse-fated AML subclone carrying -ITD with CN-LOH at chr13q.

Internal tandem duplication of the Feline McDonough Sarcoma (FMS)-like tyrosine kinase 3 (-ITD) is one of the most clinically relevant mutations in acute myeloid leukemia (AML), with a high -ITD allelic ratio (AR) (≥0.5) being strongly associated with poor prognosis. -ITDs are heterogeneous, varying in size and location, with some patients having multiple -ITDs. Bulk cell-based approaches are limited in their ability to reveal the clonal structure in such cases. Using single-cell proteogenomic sequencing (ScPGseq), we attempted to identify a relapse-fated subclone in an AML case with mutations in , , and tyrosine kinase domain and two -ITDs (21 bp and 39 bp) (low AR) at presentation, then relapsed only with and mutations and one -ITD (high AR). This relapse-fated subclone at presentation (∼2.1% of sequenced cells) was characterized by the presence of a homozygous 21 bp -ITD resulting from copy neutral loss of heterozygosity (CN-LOH) of chr13q and an aberrant, immature myeloid cell surface signature, contrast to the cell surface phenotype at presentation. In contrast to results from multicolor flow-cytometry, ScPGseq not only enabled the early detection of rare relapse-fated subclone showing immature myeloid signature but also highlighted the presence of homozygous 21 bp -ITDs in the clone at presentation.