The circulating free tumor DNA (ctDNA) represents an alternative, minimally invasive source of tumor DNA for molecular profiling. Targeted sequencing with next generation sequencing (NGS) can assess hundred mutations starting from a low DNA input. We performed NGS analysis of ctDNA from 44 patients with metastatic non-small-cell lung carcinoma (NSCLC) and 35 patients with metastatic colorectal carcinoma (CRC). NGS detected EGFR mutations in 17/22 plasma samples from EGFR-mutant NSCLC patients (sensitivity 77.3%). The concordance rate between tissue and plasma in NSCLC was much lower for other mutations such as KRAS that, based on the allelic frequency and the fraction of neoplastic cells, were likely to be sub-clonal. NGS also identified EGFR mutations in plasma samples from two patients with EGFR wild type tumor tissue. Both mutations were confirmed by droplet digital PCR (ddPCR) in both plasma and tissue samples. In CRC, the sensitivity of the NGS plasma analysis for RAS mutations was 100% (6/6) in patients that had not resection of the primary tumor before blood drawing, and 46.2% (6/13) in patients with primary tumor resected before enrollment. Our study showed that NGS is a suitable method for plasma testing. However, its clinical sensitivity is significantly affected by the presence of the primary tumor and by the heterogeneity of driver mutations.

The presence of EGFR mutations predicts the sensitivity to EGF receptor (EGFR)-tyrosine kinase inhibitors in a molecularly defined subset of non-small-cell lung carcinoma (NSCLC) patients. For this reason, EGFR testing of NSCLC is required to provide personalized treatment options and better outcomes for NSCLC patients. As surgery specimens are not available in the majority of NSCLC, other currently available DNA sources are small biopsies and cytological samples, providing however limited and low-quality material. In order to address this issue, the use of surrogate sources of DNA, such as blood, serum and plasma samples, which often contains circulating free tumor DNA or circulating tumor cells, is emerging as a new strategy for tumor genotyping.

No abstract

The introduction in the clinic of immune checkpoint inhibitors (IOs) has represented an important improvement for the treatment of patients with advanced non-small cell lung cancer (NSCLC). These drugs have shown a higher activity as compared with chemotherapy in both first-and second-line of treatment, with some patients experiencing a long-lasting response. More recently, combinations of IOs have entered clinical trials in different tumor types including NSCLC. Nevertheless, IOs are active only in a subgroup of patients and biomarkers for appropriate patients' selection are urgently needed to offer the patients an effective therapy, and also to manage the costs. Tumor mutation burden (TMB) has powerfully emerged as a potential biomarker for immunotherapy and might enter the clinic in the next months, although different challenges are still unsolved. Different methods exist to evaluate TMB in tissue, ranging from whole exome sequencing (WES) to targeted sequencing of smaller sets of genes, which need to be fully standardized to ensure that patients receive an appropriate TMB test with clear clinical interpretation. In addition, as already happened for the implementation of liquid biopsy testing from NSCLC patients to identify targetable alterations, researchers are also evaluating the possibility to calculate TMB in blood, to further enlarge the number of NSCLC patients who may benefit from immunotherapy. Preliminary data highlight the difficulty to develop targeted sequencing panels for the assessment of TMB starting from the circulating cell free DNA (cfDNA). The applicability of TMB testing on liquid biopsy needs further investigation and may be clarified within the ongoing clinical trials.