The Role of DNA and RNA in NGS Analyses for Advaced Stage NSCLC Patients

Patients with advanced non-small cell lung cancer (NSCLC) usually undergo biopsies to obtain cytological material on which to perform Next Generation Sequencing (NGS) analysis, with the aim of identifying driver gene mutations that may be targeted by specific therapies. With the development of drugs with specific therapeutic targets, the clinical need for re-biopsy or even repeated biopsies is increasing; these biopsies are necessary to identify the mechanisms of drug resistance in the target lesions. Very often, lung cancer presents with small lesions and/or lesions located in areas that are difficult to reach with traditional biopsy techniques. An alternative way to obtain genetic material is to isolate extracellular vesicles (EVs). These are secreted by almost all cell types, transport bioactive molecules, including nucleic acids (RNA and DNA), enclosed in a double lipid layer, and act as essential mediators in cell-cell communication. EVs are an ideal biomarker for cancer, as the content of EVs originating from tumor cells reflects the molecular and genetic composition of the parent cells. Long-stranded, concentrated EV-DNA is easy to amplify, making it a suitable candidate for NGS analysis. EVs are widely distributed in various body fluids, making them easier to sample using less invasive methods than tumor cells. Recent studies have shown that EVs successfully isolated from bronchoalveolar lavage (BAL) fluid of lung cancer patients contain abundant amounts of dsDNA. In a study of patients with anatomopathologically confirmed NSCLC, the sensitivity and specificity of EGFR genotyping based on BAL EVs were high, and this test showed an even better mutation detection rate than tissue/cytology-based typing. Considering the high positive predictive value of EV genotyping in bronchoalveolar lavage, this study aims to evaluate its feasibility in NGS analyses. The primary objective of the study is to determine the technical feasibility of NGS analysis on EV-DNA/RNA