Intrapatient functional clonality deconvoluted by coupling intracellular flow cytometry and next-generation...

Area of Science:

• Hematology
• Molecular Biology
• Cancer Research

Background:

• Leukemia progression is driven by tumor heterogeneity and microenvironmental factors, leading to clonal selection.
• Intrapatient genetic architecture may reflect functional clonal differences, but detection methods are limited.
• Understanding functional clonal heterogeneity is key to deciphering leukemia's complex biology.

Purpose of the Study:

• To develop and validate a scalable method for directly measuring functional properties of genetically defined clones within leukemia patients.
• To investigate the interplay between tumor heterogeneity and microenvironmental factors, specifically using granulocyte-macrophage colony stimulating factor (GM-CSF).
• To establish a proof of concept for resolving functional clonal architecture in primary leukemia samples.

Main Methods:

• Coupling of intracellular flow cytometry and next-generation sequencing (NGS) to analyze clonal function.
• Validation using mixtures of human leukemia cell lines with known GM-CSF response.
• Application to primary chronic myelomonocytic leukemia (CMML) samples.

Main Results:

• The method accurately discriminated between GM-CSF sensitive and insensitive cell lines.
• Somatic variants were identified in primary leukemia samples.
• Functional clonal architecture was resolved in an illustrative patient sample.
• Proof of concept for detecting intrapatient functional clonal heterogeneity was established.

Conclusions:

• A novel, scalable method enables direct measurement of intrapatient functional clonal heterogeneity in leukemia.
• This approach provides a powerful tool for investigating the clinical relevance of functional clonal differences.
• Future research can leverage this method to explore leukemia's complex clonal dynamics and therapeutic strategies.