Cell-free DNA and exoDNA analysis in metastatic colorectal cancer patients (mCRC).

Abstract

e16093 Background: Liquid biopsy is a growing field in translational cancer research. Two of the most studied liquid biopsy biomarkers are cell-free DNA (cfDNA) and exosomes, nano-sized vesicles that transport protein and nucleic acids including DNA (exoDNA). Therefore, both cfDNA and exoDNA are potentially useful to investigate the molecular landscape of tumor with a minimally invasive approach. Here we investigate the prognostic and predictive role of both cfDNA and exoDNA in mCRC using Next Generation Sequencing (NGS) analysis. Methods: From July 2017 to September 2018, samples of 40 mCRC patients were collected at the Medical Oncology of the AOUP Paolo Giaccone of Palermo. Blood samples were collected in EDTA-tubes before chemotherapy infusion (T1), at the first instrumental evaluation (T2) and every 2-3 months (T3). Plasma was used to isolate cfDNA and exosomes using the QIAamp Circulating Nucleic Acid and ExoEasy Maxi Kit respectively. ExoDNA was isolated through the QIAamp DNA micro kit. NGS analysis was conducted on IonS5 with AmpliSeq Cancer Hotspot Panel v2, which includes hotspot regions of 50 cancer-related genes. Results: Patients with cfDNA concentration > 0.47ng/μl have worse PFS compared with those with < 0.47 ng/μl cfDNA level. NGS analysis from 16 cfDNA samples showed a total of 292 mutations, with a median of 5 non-synonymous mutations in T1 vs. 8 non-synonymous mutations in T2 sample. We report an inverse and significant correlation between non-synonymous mutations count and disease-specific survival (DSS). Only 2 cfDNA samples carried KRAS mutations, despite it were previously reported in 5 paired tissue samples. An interesting result emerged from exoDNA sequencing of CRC20. KRAS mutation was identified in T1 exoDNA but not in the paired cfDNA samples, suggesting that exoDNA is able to intercept KRAS mutation earlier than cfDNA. Conclusions: cfDNA analysis could be a useful tool for better patients’ stratification. exoDNA could allow an early interception of disease recurrence that could be explained by the ability of exosomes to protect their cargo from degradation.