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Updated: Jun 5, 2026
Intracellular Phosphoflow Cytometry of Acute Myeloid Leukemia Patient-Derived Xenotransplants
Published on: June 6, 2025
Normal bone marrow signal-transduction profiles: a requisite for enhanced detection of signaling dysregulations in
James Marvin1, Suchitra Swaminathan, Geoffrey Kraker
1Robert H. Lurie Comprehensive Cancer Center, Northwestern University, Chicago, IL, USA.
Abstract:
Molecular and cytogenetic alterations are involved in virtually every facet of acute myeloid leukemia (AML), including dysregulation of major signal-transduction pathways. The present study examines 5 phosphoproteins (pErk, pAkt, pS6, pStat3, and pStat5) in response to 5 cytokine/growth factors (stem cell factor [SCF], Flt-3/Flk-2 ligand [FL], granulocyte/macrophage-colony stimulating factor [GM-CSF], interleukin-3 [IL-3], and granulocyte-CSF [G-CSF]) within 7 immunophenotypically defined populations, spanning progenitor to mature myeloid/myelomonocytic cells in normal bone marrows with further comparison to AML samples. The normal cohort showed pathway-specific responses related to lineage, maturation, and stimulus. Heterogeneous-signaling responses were seen in homogeneous immunophenotypic subsets emphasizing the additive information of signaling. These profiles provided a critical baseline for detection of dysregulated signaling in AML falling into 4 broad categories, viz lack of response, increased activation, altered constitutive expression, and dysregulated response kinetics, easily identified in 10 of 12 AMLs. These studies clearly show robust and reproducible flow cytometry phosphoprotein analyses capable of detecting abnormal signal-transduction responses in AML potentially contributing to definitive reliable identification of abnormal cells. As functional correlates of underlying genetic abnormalities, signal-transduction abnormalities may provide more stable indicators of abnormal cells than immunophenotyping which frequently changes after therapy and disease recurrence.
Insights
This study reveals that analyzing phosphoprotein signaling pathways using flow cytometry can effectively detect abnormal signaling in acute myeloid leukemia (AML). This method offers a stable way to identify abnormal AML cells, even when immunophenotyping changes after treatment.
Area of Science:
- Hematology
- Molecular Biology
- Cell Signaling
Background:
- Acute myeloid leukemia (AML) is characterized by molecular and cytogenetic alterations affecting signal-transduction pathways.
- Understanding signaling pathway dysregulation is crucial for AML diagnosis and treatment.
Purpose of the Study:
- To examine phosphoprotein responses to cytokines in normal bone marrow cells and compare them to acute myeloid leukemia (AML) samples.
- To establish a baseline for normal signaling to identify aberrant signaling in AML.
Main Methods:
- Flow cytometry was used to analyze 5 phosphoproteins (pErk, pAkt, pS6, pStat3, pStat5) in response to 5 cytokines (SCF, FL, GM-CSF, IL-3, G-CSF).
- Analyses were performed on 7 immunophenotypically defined cell populations from normal bone marrow and AML samples.
Main Results:
- Normal bone marrow cells exhibited pathway-specific signaling responses related to lineage, maturation, and stimulus.
- AML samples showed heterogeneous signaling responses, categorized as lack of response, increased activation, altered constitutive expression, or dysregulated kinetics.
- Aberrant signaling was identified in 10 out of 12 AML cases.
Conclusions:
- Robust and reproducible flow cytometry phosphoprotein analyses can detect abnormal signal-transduction responses in AML.
- Signal-transduction abnormalities serve as stable indicators of abnormal AML cells, potentially more reliable than immunophenotyping, which can change after therapy.
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