Researchers developed a method, called CombiFlow, for processing expression data from plasma membrane-related protein markers examined in patients with acute myeloid leukemia (AML). The researchers described utilization of CombiFlow to monitor clonal evolution in patients with AML in a study published in the journal Blood Advances.

As the researchers explained in their report, AML frequently presents with multiple subclones related to the disease that have differing genetic profiles. Subclones may also possess varying degrees of sensitivity to AML treatments, and relapse frequently occurs in patients with AML. The researchers conducting this study stressed the importance of devising a method to detect measurable residual disease (MRD) that could be used for early identification of relapse-initiating clones.

In this study, the researchers focused on gene expression data from 7 AML-specific protein markers, which included CD82, CD97, FLT3, IL1RAP, TIM3, CD25, and CD123, and all of which are plasma membrane-related proteins. Patients with AML or myelodysplastic syndrome (MDS) were evaluated for these markers obtained from bone marrow during diagnostic testing. Expression was assessed through flow cytometry.

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CombiFlow was used as a pipeline for combining multiple of these markers for antibody-based labeling of individual markers in sample tubes. Data were used in principal components analyses (PCAs) and, ultimately, to follow clonal evolution at the single-cell level.

The study population consisted of 256 patients with AML and 33 patients with MDS. Clustering of expression signals from markers suggested FLT3, IL1RAP, CD97, and TIM3 were often expressed together, while another expression cluster included CD123 and CD25. Expression of CD82 was not found to be associated with expression of other markers. CD82 was also the only assessed marker for which positive expression detected at diagnosis was a predictor of overall survival (hazard ratio [HR], 0.52; 95% CI, 0.31-0.86).

With data longitudinal data from 72 patients with AML, the researchers were able to track 5 markers over time, including CD82, CD97, FLT3, IL1RAP, and TIM3. The researchers found that increased expression of at least 1 of these markers following 2 intensive chemotherapy courses was a predictor of shorter relapse-free survival (HR, 5.004; 95% CI, 1.663-15.06; P =.004). The researchers considered this observation to be in line with a role for these markers in detection of MRD.

Additionally, in a set of samples obtained at times of diagnosis and relapse from 12 patients, the researchers were able to follow clonal evolution using CombiFlow. The research team also expanded the set of AML-specific markers used with CombiFlow to 36 markers, which they indicated led to improved refinement of the system. They also suggested the next step for research with this system involves analysis of a larger number of longitudinal samples from patients with AML.

“Here, we created an analysis pipeline termed CombiFlow, that allows tracking of disease progression based on previously defined aberrant AML PM marker expression, which may serve a clinical tool to identify relapse-inducing cell populations,” the researchers wrote in their report.


Houtsma R, van der Meer NK, Meijer K, et al. CombiFlow: combinatorial AML-specific plasma membrane expression profiles allow longitudinal tracking of clones. Blood Adv. Published online September 20, 2021. doi:10.1182/bloodadvances.2021005018