CombiFlow: Combinatorial AML-specific plasma membrane expression profiles allow longitudinal tracking of clones.

Acute myeloid leukemia (AML) often presents as an oligoclonal disease whereby multiple genetically distinct subclones can co-exist within patients. Differences in signaling and drug sensitivity of such subclones complicates treatment and warrants tools to identify them and track disease progression. We previously identified over 50 AML-specific plasma membrane (PM) proteins and seven of these (CD82, CD97, FLT3, IL1RAP, TIM3, CD25 and CD123) were implemented in routine diagnostics in patients with AML (n=256) and MDS (n=33). We developed a pipeline termed CombiFlow in which expression data of multiple PM markers is merged, allowing a Principle Component-based analyses to identify distinctive marker expression profiles and to generate single cell tSNE landscapes to longitudinally track clonal evolution. Positivity for one or more of the markers after 2 courses of intensive chemotherapy predicted a shorter relapse-free survival supporting a role of these markers in measurable residual disease (MRD) detection. CombiFlow also allowed the tracking of clonal evolution in paired diagnosis and relapse samples (n=12). Extending the panel to 36 AML-specific markers further refined the CombiFlow pipeline. In conclusion, CombiFlow provides a valuable tool in the diagnosis, MRD detection, clonal tracking, and the understanding of clonal heterogeneity in AML.