Nathaly Poveda-Garavito ^1,2* Carlos A Orozco ¹ Yulieth Torres-Llanos ^1,2,3 Nataly Cruz-Rodriguez ⁴ Rafael Parra-Medina ^1,5 Sandra Quijano ⁶ Jovanny Zabaleta ⁷ Alba Lucia Combita ^1,2*

• ¹ National Cancer Institute (Colombia), Bogotá, Colombia
• ² National University of Colombia, Bogotá, Bogota, Colombia
• ³ San Ignacio University Hospital, Bogotá, Cundinamarca, Colombia
• ⁴ Versiti Blood Research Institute, Milwaukee, Wisconsin, United States
• ⁵ Foundation University of Health Sciences, Bogotá, Cundinamarca, Colombia
• ⁶ Pontifical Javeriana University, Bogotá, Cundinamarca, Colombia
• ⁷ LSU Health Sciences Center New Orleans, Louisiana State University, New Orleans, Louisiana, United States

The diagnosis of B-Cell acute lymphoblastic leukaemia (B-ALL) in adults often carries a poor prognosis. ID1 and ID3 genes have been identified as predictors of poor response in Colombian adult B-ALL patients, playing roles in cancer development. In different cancer models, these genes have been associated with immune regulator populations within the tumour immune microenvironment (TIME). B-ALL development alters the immune cell composition and the bone marrow (BM) tumour microenvironment, affecting disease progression and response to therapy. This study analyses gene expression levels of ID1 and ID3 in relation to TIME and immune evasion. This exploratory study analysed BM samples from 10 B-ALL adult patients diagnosed at the National Cancer Institute of Colombia. First, RT-qPCR was used to assess ID1 and ID3 expression in BM tumor cells. Flow cytometry characterized immune populations in the TIME. RNA-seq evaluated immune genes associated with B-ALL immune response, while xCell and CytoSig analyzed TIME cell profiles and cytokines. Pathway analysis, gene ontology, and differential gene expression (DEGs) were examined, with functional enrichment analysis performed using KEGG ontology. Patients were divided into two groups based on ID1 and ID3 expression, namely basal and overexpression. A total of 94 differentially expressed genes were identified between these groups, with top overexpressed genes associated with neutrophil pathways. Gene set enrichment analysis revealed increased expression of genes associated with neutrophil degranulation, immune response-related neutrophil activation, and neutrophilmediated immunity. These findings correlated with xCell data. Overexpression group showed significant differences in neutrophils (p=0.0008), monocytes (<0.0001) and CD4+ naive T cells (p=0.0240) compared to basal group patients. Microenvironment and immune scores were also significantly different (p=0.0016 and p=0.0017, respectively), consistent with the flow cytometry results. Elevated cytokine levels associated with neutrophil activation supported these findings. Validation was performed using the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) TCGA B-ALL cohorts. Our results show important differences between the expression level of ID1 and ID3 in cancer cells and the populations of TIME, suggesting a role in evading the immune response of ID1 and ID3 in B-ALL, mainly related to neutrophil pathways.