Comprehensive Characterization of Oncogene-Addicted Non-Small Cell Lung Cancer and its Immune Microenvironment

Lung cancer remains the leading cause of cancer-related mortality worldwide, with non-small cell lung cancer (NSCLC) accounting for approximately 85% of cases. A relevant molecular subset is represented by oncogeneaddicted (OA) NSCLC, characterized by tumor dependence on specific driver alterations. This PhD thesis, comprising multiple studies, provides a comprehensive analysis of OA NSCLC, integrating tumor-intrinsic features, immune microenvironment characterization, and liquid biopsy applications to improve understanding of the disease, its monitoring, and therapeutic strategies. The first study established a free-of-charge molecular profiling platform for advanced NSCLC across Europe to increase detection of targetable drivers and access to matched therapies and clinical trials. A robust NGS workflow enabled the identification of 570 molecular alterations across 59 genes in 92% of samples. Although only 50% were potentially targetable, patients receiving matched therapies showed improved survival, supporting the implementation of comprehensive genomic profiling in routine clinical practice. The second study evaluated the correlation between circulating tumor DNA (ctDNA) KRAS G12C dynamics and clinical outcomes in advanced NSCLC patients treated with sotorasib in a real-world setting. Early clearance of KRAS G12C-mutant ctDNA predicted improved tumor response and longer survival. Moreover, rising KRAS G12C variant allele frequency (VAF) anticipated radiologic progression in 70% of evaluable patients at resistance, highlighting the clinical value of dynamic ctDNA monitoring. The third study assessed the diagnostic accuracy of plasma-based KRAS mutation testing compared with tissue genotyping in treatment-naïve advanced NSCLC patients from six Italian centers. Among 186 baseline plasma samples analyzed for KRAS, EGFR, and BRAF mutations, the assay showed high specificity (0.98), acceptable sensitivity (0.60), and 71.5% overall concordance. Longitudinal VAF assessment in KRAS-positive patients demonstrated heterogeneous molecular dynamics, supporting liquid biopsy as a complementary diagnostic and monitoring tool. The fourth study focused on young NSCLC patients (<50 years) in the Piedmont region, aiming to refine molecular classification through wide-panel tissue NGS combined with liquid biopsy analysis. Among 57 patients, 81.1% harbored oncogene-addicted tumors, consistent with enrichment of targetable drivers in this population. Fresh tumor samples were also collected to generate preclinical in vitro models for drug testing and personalized therapeutic prediction. Another study investigated the immune microenvironment of NSCLC using immunohistochemistry and flow cytometry on surgical samples. KRAS-mutated tumors showed increased expression of immune checkpoint markers and regulatory T cells, suggesting chronic antigen exposure and T-cell exhaustion. Additional analyses revealed broader immune remodeling, including predominance of proliferative over cytotoxic NK cells, indicating that oncogenic signaling shapes immune composition and function. The final study, performed as a visiting PhD student, was aimed at evaluating RNA-based splice-switching oligonucleotides (SSOs) targeting TYMS in NSCLC cell lines as a potential strategy against KRAS-driven disease. SSOs reduced thymidylate synthase expression, impaired proliferation, and induced apoptosis. The most effective sequence, SSO e4.3, showed synergistic activity with 5-fluorouracil and pemetrexed. Mechanistically, efficacy appeared independent of canonical enzymatic inhibition or exon skipping, suggesting interference with alternative splicing products such as circular RNAs. Overall, this work integrates molecular profiling, liquid biopsy, immune characterization, and RNA-based therapeutic strategies to advance the biological understanding and clinical management of oncogene-addicted NSCLC.