Size-Exclusion Chromatography for Profiling Plasma Extracellular Vesicles in Chronic Myeloid Leukemia

Chronic myeloid leukemia (CML) is driven by the BCR::ABL1 fusion gene. While the use of tyrosine kinase inhibitors (TKIs) have transformed outcomes, resistance to treatment persists. Plasma extracellular vesicles (EVs), however, reflect their cell of origin, and they may serve as stable biomarkers. To characterize the plasma EV proteome in CML patients with distinct treatment responses and T315I mutation status, a joint study between researchers at the Carlos Chagas Institute, the Federal University of Paraná, and the University of São Paulo (all in Brazil) isolated EVs from the plasma of healthy controls (HC) and CML patients classified as good (GTR) or poor (PTR) treatment responders, treatment-free remission (TFR), and T315I or pre-T315I mutation carriers. The EVs were purified by size-exclusion chromatography (SEC), characterized by nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM), and analyzed by label-free mass spectrometry (LFQ), followed by differential expression, enrichment, and protein-protein interaction analyses. A paper based on this research was published in Frontiers in Genetics.¹

Leukemia accounted for approximately 486,777 new cases and 305,033 deaths globally in 2022, according to estimates from the World Health Organization, positioning the disease as the 13th most prevalent cancer and the 10th leading cause of cancer-related mortality.² CML is notably characterized among subtypes of the disease by a reciprocal translocation between chromosomes 9 and 22, known as t (9; 22) (q34; q11); this chromosomal rearrangement generates the Philadelphia chromosome, containing the fusion of the ABL1 gene on chromosome 9 (Murine Abelson) with the BCR gene (Breakpoint Cluster Region) on chromosome 22.^3,4 The consequent BCR::ABL1 fusion transcript encodes a chimeric oncoprotein with constitutive tyrosine kinase activity, which strangely results in the promotion of signaling pathways that lead to leukemogenesis and uncontrolled proliferation of myeloid cells.⁵

The researchers report that a total of 598 proteins were identified through this process, with 257 retained after quality and abundance filtering. Forty-two proteins were differentially expressed among HC, GTR, and PTR groups (p < 0.01), with PTR 27 samples showing marked downregulation of cytoskeletal and chaperone proteins (such as MYH9, 28 HSP90AB1, FERMT3). TFR patients exhibited distinct enrichment in complement and coagulation cascades (C3, C4B, F9, F11) and metabolic pathways.¹

“Plasma EV proteomes,” write the authors of the study,¹ “reflect CML clinical status, revealing immune and cytoskeletal alterations associated with treatment response, remission, and resistance, suggesting potential biomarkers for disease monitoring.”

“To our knowledge,” the authors continue,¹ “this is the only report showing differentially expressed proteins in plasma EVs from CML clinical samples, isolated from patients in distinct levels of response to therapy. The majority of reports are based on EVs from CML cell lines, making this a strength of the current report. As a weakness, a limited number of samples was evaluated. Nevertheless, a relevant number of DEPs were described in each group of patients, with special emphasis to those DEPS found in T315I patients and for the presence of complement components and coagulation factors.”

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References

1. Kusma Wosniaki, D.; Korte de Azevedo, A. L.; Marin, A. M. et al. Differentially Expressed Proteins in Plasma-Derived Extracellular Vesicles from Chronic Myeloid Leukemia Patients. Front Genet. 2026, 17, 1762244. DOI: 10.3389/fgene.2026.1762244
2. International Agency for Research on Cancer WHO (2022). Global Cancer Observatory, Globocan. Globocan 2022. https://gco.iarc.fr/today/fact-sheets-cancer/about
3. Wosniaki, D. K.; Marin, A. M.; Oliveira, R. N. et al. The Screening of microRNAs in Chronic Myeloid Leukemia: A Clinical Evaluation. Int J Mol Sci. 2024, 25 (6), 3363. DOI: 10.3390/ijms25063363
4. Luo, J.; Gao, Y.; Lin, X. et al. Systematic Analysis Reveals a lncRNA-miRNA-mRNA Network Associated with Dasatinib Resistance in Chronic Myeloid Leukemia. Ann Palliat Med. 2021, 10 (2), 1727-1738. DOI: 10.21037/apm-20-343
5. Cumbo, C.; Anelli, L.; Specchia, G. et al. Monitoring of Minimal Residual Disease (MRD) in Chronic Myeloid Leukemia: Recent Advances. Cancer Manag Res. 2020, 12, 3175-3189. DOI: 10.2147/CMAR.S232752