# Resolving intra-tumor heterogeneity and clonal evolution of core-binding factor acute myeloid leukemia patients with single-cell resolution

## TL;DR

This study uses single-cell DNA sequencing to understand the genetic diversity and evolution of core-binding factor acute myeloid leukemia tumors, revealing insights into therapy resistance and tumor development.

## Contribution

The study introduces a novel approach to integrate subclonal somatic copy number alterations into phylogenetic analysis of CBF AML, achieving high-resolution insights into tumor evolution.

## Key findings

- The fusion gene is among the earliest events in leukemogenesis at the single-cell level.
- Remaining tumor clones were identified in patients with complete remission, indicating incomplete eradication.
- The order of mutation acquisition provides insights into evolutionary history and can guide drug selection.

## Abstract

Reconstructing and understanding intra-tumor heterogeneity, the coexistence of multiple genetically distinct subclones within the tumor of a patient, and tumor development is essential for resolving carcinogenesis and for identifying mechanisms of therapy resistance. While bulk sequencing can provide a broad view on tumoral complexity/heterogeneity of a patient, single-cell analysis remains essential to identify rare subclones that might drive chemotherapy resistance. In this study, we performed an integrated analysis of bulk and single-cell DNA sequencing data of core-binding factor acute myeloid leukemia patients, defined by the presence of a RUNX1::RUNX1T1 or CBFB::MYH11 fusion gene. By single-cell sequencing, we inferred tumor phylogenies for 8 patients at diagnosis including patient-specific somatic variants, somatic copy-number alterations and fusion genes, and studied clonal evolution under the pressure of chemotherapy for 3 patients. As a result, we developed an approach to reliably integrate subclonal somatic copy number alterations into phylogenetic trees and clonal evolution analysis, obtaining unprecedented resolution of intra-tumor heterogeneity in CBF AML. We were able to show that the fusion gene is among the earliest events of leukemogenesis at single-cell level. We identified remaining tumor clones in 6 patients with complete remission samples indicating incomplete eradication of the tumor clones. Here, we show that identifying the order of mutation acquisition can provide valuable insights into evolutionary history, offering a framework to improve drug selection in the era of targeted therapies.

The online version contains supplementary material available at 10.1186/s40164-025-00718-4.