Liquid biopsy in clinical oncology

Circulating tumor DNA (ctDNA), commonly known as liquid biopsy, is shed from tumor cells into the bloodstream of the patient. ctDNA containing somatic mutations which occur in tumor tissues may represent valid biomarkers to follow disease progression. Despite the major challenge remains assay sensitivity and specificity for ctDNA analysis, advancements represented by the use of new technical approaches such as NGS and droplet digital PCR assays - able to detect and quantify small amounts of mutated DNA molecules - have enabled the use of mutations as predictive/prognostic tool in the clinical practice. Several studies in melanoma, breast, lung and colorectal cancers demonstrated the potential clinical application of ctDNA analysis at each stage of cancer management: early diagnosis, molecular profiling for prognosis, detection of residual disease, monitoring response or resistance to treatment. In malignant melanoma and breast cancer, recent studies reveal that ctDNA could potentially be an excellent marker of residual disease, guiding therapeutic strategies. Moreover, occurrence of circulating KRAS mutations may predict failure of anti-EGFR therapies or, on the contrary, their disappearance from blood may address the rechallenge with EGFR inhibitors in patients with colorectal cancer (CRC). In our preliminary experience within the ongoing clinical trial DISTINCTIVE (Second-D line folfiri/aflIbercept in proSpecTIvely stratified, anti-EGFR resistaNt, metastatic coloreCTal cancer patients wIth RAS Validated wild typE status), Sardinian patients (N=44) with RAS wild-type advanced CRC who received first-line anti-EGFR treatment were investigated for circulating RAS or BRAF mutations at disease progression in order to determine whether the status of tumor biomarkers changes during tumor evolution. We found that about one third of patients presented a circulating oncogene mutation (29.5% in KRAS, 4.5% in NRAS, and 2.3% in BRAF), whereas in the remaining larger fraction (>60%) of the series the lack of mutated ctDNA addressed cases to the rechallenge therapy with anti-EGFR drugs. Efforts to improve the outcome of the oncological treatments for non-small cell lung cancer (NSCLC), together with the technological advances in DNA sequencing, led to the development of new therapeutic strategies. Subsets of patients with adenocarcinoma and activating mutations within the kinase domain of the epidermal growth factor receptor (EGFR) gene have been successfully treated with selective tyrosine kinase inhibitors (TKI). We previously described the molecular epidemiology of EGFR, KRAS, BRAF, ALK and MET genetic alterations and their correlations with the demographic and clinical characteristics of 1,440 Sardinian patients with lung adenocarcinoma. In NSCLC patients treated with the EGFR TKIs, the T790M mutation in EGFR gene represents the most common mechanism of acquired resistance. We started the analysis of 165 consecutive liquid biopsies from advanced NSCLC patients treated with TKIs, detecting the T790M in 11.5% of cases. EGFR MutationPositive (%)Negative (%) T790M 19 (11.5%) 146 (88.5%) Exon 19 Deletions 35 (21.2%) 130 (78.8%) L858R 18 (10.9%) 147 (89.1%) In addition to complete such clinical trials, our group will extend the analysis of ctDNAs in other neoplastic pathologies through the participation to further studies by using NGS-based strategies.