Immunophenotype signatures in acute leukemias unveiled by integrative systems immunology

Acute leukemias (ALs) are complex hematological disorders, and accurate diagnosis is crucial for guiding treatment decisions and predicting patient outcomes. While changes in cell marker levels are well documented, the impact of these changes on marker relationships through an integrative systems approach remains uncharacterized. To address this gap, we conducted a 12-year study investigating 41 markers, including ontogenic markers and those used to diagnose both common and rare leukemia types, using immunophenotyping flow cytometry (IFC) data from 1,069 leukocyte samples obtained from peripheral blood (PB) or bone marrow (BM) aspirates of patients with suspected ALs. Machine learning techniques, such as principal component analysis (PCA) and random forest (RF) classification, demonstrated the stratification power of the cellular markers. Hierarchical clustering analysis of leukocyte ontogenetic markers revealed disease-specific clusters, irrespective of sex or sample type (PB or BM). Additionally, we found that patients with acute myeloid leukemia (AML) showed mild disruption in cell marker correlations, whereas the most significant dysregulation was observed in patients with T-cell acute lymphoblastic leukemia (T-ALL). Importantly, we identified ontogenic correlation changes indicating clusters of immature versus mature leukocyte markers, as well as cell lineage-specific markers influencing cellular relationships. These findings underscore the value of integrating systems strategies into conventional IFC analyses to enhance synthetic diagnosis and deepen our understanding of ALs pathophysiology. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This study was funded by the Coordination of Superior Level Staff Improvement under Academic Excellence Program (CAPES/PROEX grants 88887.954341/2024-00 to IAFB, 88887.917898/2023-00 to JNU and 88887.801068/2023-00 to ALN). We thank the National Council for Scientific and Technological Development (CNPq) Brazil (grants: 309482/2022-4 to OCM). We acknowledge the Sao Paulo Research Foundation (FAPESP grants 2018/18886-9, 2020/01688-0, and 2020/07069-0 to OCM, 2020/16246-2 and 2023/06086-6 to PMB, 2023/07806-2 to ISF, 2020/16246-2 and 2023/13356-0 to DLMF, 2023/12268-0 to ASA, 2023/14417-2 to JNU and 2018/14933-2 to HIN) for financial support. We recognize and thank the infrastructure support and raw data for the Hematology Center Dalton Cunha and Flowmentor. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: This project was approved by the Research Ethics Committee of the Onofre Lopes University Hospital, Federal University of Rio Grande do Norte (CEP/HUOL.UFRN, n 4.378.690). The Hemocenter Dalton Cunha, the institution responsible for the immunophenotypic analyses, granted permission for data use, ensuring informed consent and compliance with ethical and legal standards. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data used in this study are provided in the Supplementary Data. Raw data (FCS files) are available upon reasonable request.