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In the scenario of systemic treatment for advanced non-small cell lung cancer (NSCLC) patients, one of the most relevant breakthroughs is represented by targeted therapies. Throughout the last years, inhibitors of the epidermal growth factor receptor (EGFR), anaplastic lymphoma kinase (ALK), c-Ros oncogene 1 (ROS1), and V-raf murine sarcoma viral oncogene homolog B (BRAF) have been approved and are currently used in clinical practice. However, other promising molecular drivers are rapidly emerging as therapeutic targets. This review aims to cover the molecular alterations with a potential clinical impact in NSCLC, including amplifications or mutations of the mesenchymal-epithelial transition factor (MET), fusions of rearranged during transfection (RET), rearrangements of the neurotrophic tyrosine kinase (NTRK) genes, mutations of the Kirsten rat sarcoma viral oncogene (KRAS) and phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA), as well as amplifications or mutations of human epidermal growth factor receptor 2 (HER2). Additionally, we summarized the current status of targeted agents under investigation for such alterations. This revision of the current literature on emerging molecular targets is needed as the evolving knowledge on novel actionable oncogenic drivers and targeted agents is expected to increase the proportion of patients who will benefit from tailored therapeutic approaches.

Cell-free DNA (cfDNA) and circulating tumor cells (CTCs) are promising prognostic and predictive biomarkers in non-small cell lung cancer (NSCLC). In this study, we examined the prognostic role of cfDNA and CTCs, in separate and joint analyses, in NSCLC patients receiving first line chemotherapy. Seventy-three patients with advanced NSCLC were enrolled in this study. CfDNA and CTC were analyzed at baseline and after two cycles of chemotherapy. Plasma cfDNA quantification was performed by quantitative PCR (qPCR) whereas CTCs were isolated by the ScreenCell Cyto (ScreenCell, Paris, France) device and enumerated according to malignant features. Patients with baseline cfDNA higher than the median value (96.3 hTERT copy number) had a significantly worse overall survival (OS) and double the risk of death (hazard ratio (HR): 2.14; 95% confidence limits (CL) = 1.24-3.68; p -value = 0.006). Conversely, an inverse relationship between CTC median baseline number (6 CTC/3 mL of blood) and OS was observed. In addition, we found that in patients reporting stable disease (SD), the baseline cfDNA and CTCs were able to discriminate patients at high risk of poor survival. cfDNA demonstrated a more reliable biomarker than CTCs in the overall population. In the subgroup of SD patients, both biomarkers identified patients at high risk of poor prognosis who might deserve additional/alternative therapeutic interventions.

Next-generation sequencing (NGS) is a cost-effective technology capable of screening several genes simultaneously; however, its application in a clinical context requires an established workflow to acquire reliable sequencing results. Here, we report an optimized NGS workflow analyzing 22 lung cancer-related genes to sequence critical samples such as DNA from formalin-fixed paraffin-embedded (FFPE) blocks and circulating free DNA (cfDNA). Snap frozen and matched FFPE gDNA from 12 non-small cell lung cancer (NSCLC) patients, whose gDNA fragmentation status was previously evaluated using a multiplex PCR-based quality control, were successfully sequenced with Ion Torrent PGM™. The robust bioinformatic pipeline allowed us to correctly call both Single Nucleotide Variants (SNVs) and indels with a detection limit of 5%, achieving 100% specificity and 96% sensitivity. This workflow was also validated in 13 FFPE NSCLC biopsies. Furthermore, a specific protocol for low input gDNA capable of producing good sequencing data with high coverage, high uniformity, and a low error rate was also optimized. In conclusion, we demonstrate the feasibility of obtaining gDNA from FFPE samples suitable for NGS by performing appropriate quality controls. The optimized workflow, capable of screening low input gDNA, highlights NGS as a potential tool in the detection, disease monitoring, and treatment of NSCLC.