Liquid biopsy has emerged as an alternative source of nucleic acids for the management of Epidermal Growth Factor Receptor (EGFR)-mutant non-Small Cell Lung Cancer (NSCLC). The use of circulating cell-free DNA (cfDNA) has been recently introduced in clinical practice, resulting in the improvement of the identification of druggable EGFR mutations for the diagnosis and monitoring of response to targeted therapy. EGFR-dependent (T790M and C797S mutations) and independent (Mesenchymal Epithelial Transition [MET] gene amplification, Kirsten Rat Sarcoma [KRAS], Phosphatidyl-Inositol 4,5-bisphosphate 3-Kinase Catalytic subunit Alpha isoform [PI3KCA], and RAF murine sarcoma viral oncogene homolog B1 [BRAF] gene mutations) mechanisms of resistance to EGFR tyrosine kinase inhibitors (TKIs) have been evaluated in plasma samples from NSCLC patients using highly sensitive methods (i.e., digital droplet PCR, Next Generation Sequencing), allowing for the switch to other therapies. Therefore, liquid biopsy is a non-invasive method able to detect the molecular dynamic changes that occur under the pressure of treatment, and to capture tumor heterogeneity more efficiently than is allowed by tissue biopsy. This review addresses how liquid biopsy may be used to guide the choice of treatment strategy in EGFR-mutant NSCLC.

Systemic treatment of advanced non-small cell lung cancer (NSCLC) has changed dramatically since the introduction of targeted therapies. The analysis of circulating tumor DNA (ctDNA) is a valuable approach to monitor the clonal evolution of tumors during treatment with EGFR-tyrosine kinase inhibitors (TKIs) and to detect resistance mutations. A NSCLC patient with exon 19 deletion (ex19del) of EGFR was treated with osimertinib after multiple lines of treatment and obtained a partial response that lasted over 26 months. Blood was collected at each visit and ctDNA was extracted to monitor ex19del by digital droplet PCR. Within a few weeks from the beginning of osimertinib, ex19del disappeared from plasma but appeared again and steadily increased a few months later anticipating tumor progression. Interestingly, the change in ex19del was much more pronounced than other mutations, since T790M appeared 3 months after the increase of ex19del, and C797S was detectable a few weeks before clinical disease progression. Then the patient received cytotoxic chemotherapy, which was associated with a decrease in ex19del and disappearance of T790M and C797S; however, at disease progression, all EGFR mutations increased again in plasma together with MET amplification which was detected by NGS. The measurement of ex19del changes in ctDNA is a simple and sensitive approach to monitor clinical outcome to osimertinib and, potentially, to other therapeutic interventions.

Early-stage (ES) non-small cell lung cancer (NSCLC) is diagnosed in about 30% of cases. The preferred treatment is surgery, but a significant proportion of patients experience recurrence. Neoadjuvant and adjuvant chemotherapy has a limited clinical benefit. EGFR tyrosine kinase inhibitors and immunotherapy have recently opened new therapeutic scenarios. However, only a few data are available about the ES-NSCLC molecular landscape and the impact of oncogene addiction on therapy definition. Here, we determined the prevalence of the main lung cancer driver alterations in a monocentric consecutive cohort. Molecular analysis was performed on 1122 cases, including 368 ES and 754 advanced NSCLC. The prevalence of actionable alterations was similar between early and advanced stages. ES-NSCLC was significantly enriched for MET exon-14 skipping alterations and presented a lower prevalence of BRAF p.(V600E) mutation. PD-L1 expression levels, evaluated according to actionable alterations, were higher in advanced than early tumors harboring EGFR, KRAS, MET alterations and gene fusions. Taken together, these results confirm the value of biomarker testing in ES-NSCLC. Although approved targeted therapies for ES-NSCLC are still limited, the identification of actionable alterations could improve patients’ selection for immunotherapy, favoring the enrollment in clinical trials and allowing a faster treatment start at disease recurrence.

Pembrolizumab has been approved as first-line treatment for advanced Non-small cell lung cancer (NSCLC) patients with tumors expressing PD-L1 and in the absence of other targetable alterations. However, not all patients that meet these criteria have a durable benefit. In this monocentric study, we aimed at refining the selection of patients based on the expression of immune genes. Forty-six consecutive advanced NSCLC patients treated with pembrolizumab in first-line setting were enrolled. The expression levels of 770 genes involved in the regulation of the immune system was analysed by the nanoString system. PD-L1 expression was evaluated by immunohistochemistry. Patients with durable clinical benefit had a greater infiltration of cytotoxic cells, exhausted CD8, B-cells, CD45, T-cells, CD8 T-cells and NK cells. Immune cell scores such as CD8 T-cell and NK cell were good predictors of durable response with an AUC of 0.82. Among the immune cell markers, XCL1/2 showed the better performance in predicting durable benefit to pembrolizumab, with an AUC of 0.85. Additionally, CD8A, CD8B and EOMES showed a high specificity (>0.86) in identifying patients with a good response to treatment. In the same series, PD-L1 expression levels had an AUC of 0.61. The characterization of tumor microenvironment, even with the use of single markers, can improve patients’ selection for pembrolizumab treatment.