Abstract 7492: “Baseline-informed” unsupervised multiplexed CyTOF analysis enhances detection of minimal/measurable residual disease in acute myeloid leukemia

Abstract Multiparameter flow cytometry (MFC) and molecular techniques are the cornerstone for detecting acute myeloid leukemia (AML) minimal/measurable residual disease (MRD). However, some MRD(-) patients relapse, suggesting current methods miss persistent leukemia due to suboptimal detection limits or distinct leukemia-associated phenotypes(LAP). This highlights the need for improved MRD detection methods. Challenges in MFC-based MRD detection include reliance on limited markers, incomplete LAP detection, clonal evolution, surface-marker restrictions, and the need for specialized expertise. These limitations underscore the need for methodologies capable of capturing diverse and dynamic AML proteomic landscape. Single-cell proteomics via CyTOF uniquely dissects high-dimensional features, enabling comprehensive and unbiased leukemic cell characterization. We hypothesized that CyTOF, combined with unsupervised analysis, enhances MRD detection by identifying diverse proteomic features in a feature-agnostic manner. To explore this, we conducted integrated CyTOF analysis of 60 AML patients and 12 bone marrow (BM) samples. This revealed diverse AML proteomic profiles distinct from HSCs/HSPCs indicating unsupervised methods capture aberrant leukemia profiles in a feature-agnostic manner. Next, AML MRD was modeled by mixing barcoded primary AML cells (n=5) and cell lines (Kasumi, Molm13, OCI-AML3) with healthy BM at dilution ratios from 1:10 to 1:100, 000. A 50-parameter CyTOF panel and UMAP analysis assessed MRD detection sensitivity. UMAP analysis showed that primary AML cells and cell lines were well-segregated up to a 1:1, 000 dilution, with progressive loss of resolution at higher dilutions. At a 1:100, 000 dilution, AML cells became indistinguishable from healthy HSCs/HSPCs, highlighting the limitations of UMAP in detecting rare populations. Baseline-informed unsupervised analysis—leveraging phenotypically similar baseline AML cells—restored clustering sensitivity at a 1:100, 000 dilution (23 AML cells in 2.3 million BM cells). Furthermore, we extended these findings by conducting integrated CyTOF analysis on pre- and post-therapy primary AML samples (n=5). This "informed" analysis improved AML MRD detection and demonstrated its potential for clinical trials. This approach enabled detailed characterization of metabolic, signaling, apoptotic, and phenotypic features of AML MRD, providing deeper insights into its complexity and surpassing the limited insights of current MRD detection tools. In conclusion, multiplexed CyTOF analysis combined with an "informed" unsupervised analytic approach significantly enhances AML MRD detection and characterization. In addition to enumerating MRD cells with high sensitivity, CyTOF enables detailed proteomic profiling, offering insights that could guide AML MRD therapy. Citation Format: Mahesh Basyal, Muharrem Muftuoglu, Li Li, Jiangxing Lv, Yukun Tan, Ken Chen, Michael Andreeff. "Baseline-informed” unsupervised multiplexed CyTOF analysis enhances detection of minimal/measurable residual disease in acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 7492.