Abstract A040-PR008: Characterization of cytotoxic cell abundance and genetic variants in immune related genes in pediatric acute lymphoblastic leukemia patients

Abstract Acute lymphoblastic leukemia (ALL) is the leading cause of cancer-related mortality in pediatrics. Between 15-30% of patients relapse, and current treatments are associated with acute and long-term toxicity. The immune component of the tumor microenvironment is an under-explored factor that could play a role in the establishment or progression of the disease, as well as modulate response to chemo- and immunotherapies. We characterized the molecular profile of leukemias from 32 patients with pediatric ALL treated under the ALLIC-GATLA 2010 clinical protocol in Argentina by transcriptome sequencing (Illumina). We estimated the immune component through digital cytometry (MIXTURE) and a cytolytic score based on the expression of five genes. We searched for single nucleotide variants (SNV) in the transcriptome (CTAT-Mutations) that could alter the interaction with immune cells in the tumor microenvironment. For this, we analyzed 843 genes from Reactome collections associated with the immune system. Variants were filtered and annotated using publicly available germline and somatic variant databases. The group of patients with higher abundance of cytotoxic cells (Z-score>1) was associated with an increased risk of relapse/death (HR=4.5; p=0.05), and higher levels of CX3CR1 (q-val<0.05) and TIM-3 (q-val=0.06). Analysis of an independent high-risk cohort (TARGET B-ALL phase II, N=88), confirmed that a higher cytolytic score was associated with the risk of relapse in an univariate (HR=2.66, p=0.001) and multivariate model including cytolytic score (p<0.006), protocol (p=0,07), ETV6::RUNX1 (p:>0.05) and TCF3::PBX1 status (p<0.001). Cytolytic score was not associated with molecular subtype, age, sex or early response to treatment (p>0.05). For SNV analysis, we developed a threshold value for the QUAL parameter to select for high-confidence variants, establishing a specificity of 95%. Eleven variants were found in nine patients, being 8/11 classified by Franklin as TIER3 in CEP290, UBE3B, CPNE3, DUSP4, DAPP1, RNF135, TBC1D10C and TLR1; 2/11 as TIER1 in TP53, KRAS; and 1/11 as TIER4 in BTN3A3. Patient harboring variants in these genes were significantly associated with a lower GSVA score in the Reactome dataset “MHC_Class_II_Antigen_Presentation” (q-val<0.01). We found a variant in UBE3B, located in the active site of the E3 ubiquitin ligase, and associated with a low GSVA score in the Reactome “Antigen presentation through MHC class I” pathway. A novel variant in CPNE3 gene was found, located in a highly conserved residue and predicted to be deleterious by multiple tools. Conclusion: the abundance of cytotoxic cells was independent of molecular subtype and associated with a worse prognosis in independent cohorts. Further analysis of the variants found in this study has the potential to identify novel mechanisms modulating the interaction between the immune system and leukemic cells in ALL. Citation Format: Ignacio Gomez Mercado, Daniel Avendaño, Sabrina Ledesma Bazan, Pablo Sanchis, Maria Cecilia Riccheri, Geraldine Gueron, Javier Cotignola, Maria Sol Ruiz. Characterization of cytotoxic cell abundance and genetic variants in immune related genes in pediatric acute lymphoblastic leukemia patients [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Discovery and Innovation in Pediatric Cancer— From Biology to Breakthrough Therapies; 2025 Sep 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl_2):Abstract nr A040-PR008.