Abstract 1312: AXL mRNA overexpression in CTCs as a new potential liquid biopsy biomarker in NSCLC.

Abstract Background: AXL is a receptor tyrosine kinase that is over-expressed in NSCLC. It is involved in epithelial-to-mesenchymal transition (EMT), cell survival, invasion, metastasis, and resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) and immune checkpoint inhibitors (ICIs). Nowadays, clinical trials are investigating the efficacy of AXL-inhibitors such as bemcentinib, that has already received US FDA fast-track designation for the treatment of advanced or metastatic NSCLC combined with a PD-L1 inhibitor. In this study we evaluated AXL mRNA expression in circulating tumor cells (CTCs) isolated at different time points from two groups of NSCLC patients: a) under osimertinib treatment and b) under immunotherapy. Materials and Methods: Group A: Peripheral blood (PB) (15mL) was collected from 39 EGFR-mutant NSCLC patients before osimertinib (n=39), after one cycle of treatment (n=31), every 3 months during therapy (n=81) and at progression of disease (PD) (n=30). Group B: PB (10mL) was collected from 116 NSCLC patients before immunotherapy (n=116), after 3 or 4 cycles of treatment with ICIs (n=71) and at PD (n=10). CTCs were enriched using the ParsortixTM system (CelLBxHealth plc, UK) and further harvested in Trizol reagent, followed by extraction of total RNA and cDNA synthesis. An RT-qPCR assay was developed and analytically validated for the detection of AXL mRNA expression in CTCs using the COBAS z480 system (Roche). PB from healthy donors (n=46) was processed in the same way and used as control group for the estimation of overexpression, based on the 2-ΔΔCt approach. Results: In Group A (EGFR-mutant NSCLC), AXL mRNA overexpression in CTCs was detected in 5/39 (12.8%) patients’ samples before osimertinib, in 5/31 (16.1%) after one cycle of treatment, in 7/81 (8.6%) samples during treatment and in 4/30 (13.3%) samples at PD. In Group B (treatment with ICIs), AXL mRNA overexpression in CTCs was detected in 7/116 (6.0 %) patients prior to treatment, in 8/71 (11.3%) after 3 or 4 cycles of treatment, while no AXL transcripts were detected in CTCs at PD. Conclusions: This is the first time that AXL overexpression is detected in CTCs of NSCLC patients undergoing immunotherapy. Our results indicate that NSCLC patients with overexpression of AXL in CTCs could benefit from combination therapies with AXL-inhibitors to either overcome resistance to targeted therapies or enhance immune responses. The role of AXL as a potential biomarker and therapeutic target in advanced NSCLC needs to be further confirmed through larger clinical studies including liquid biopsy approach. Citation Format: Aliki Ntzifa, Elena Themistokli, Areti Strati, Martha Zavridou, Emilia Tsaroucha, Aggeliki Sfika, Amanda Psyrri, Athanasios Kotsakis, Vassilis Georgoulias, Evi Lianidou. AXL mRNA overexpression in CTCs as a new potential liquid biopsy biomarker in NSCLC abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1312.