MiRNAs’ characterization in metastatic colorectal cancer: a multicenter prospective translational study

Introduction: Colorectal cancer (CRC) is the second leading cause of cancer-related deaths worldwide. Biology and evolutive stage of the disease are responsible for prognosis, treatment and follow-up. Since in early stages 5y-OAS is close to 90% while in advanced CRC it is comprised between 12 and 17%, screening programs and preventative measures are widely adopted in an effort to anticipate the diagnosis; nevertheless, about 50-60 % of patients with CRC will develop liver metastases in the course of their disease, primary factor of long-term survival. At present, decision making depends on the TNM staging system of the American Joint Committee on Cancer, which allocates patients in different protocols of treatment and follow-up depending on their stage. Several limits of this system have been recently questioned and updates have been performed, nonetheless, current prognostic features don’t seem to be unfailingly efficient in identifying patients with early-stage disease at higher risk for relapse, likely to receive benefit from adjuvant chemotherapy. Since their aberrant expression showed to be connected with cancer development, progression and even with treatment response, micro-RNAs (miRNAs) have been widely and still are investigated as a new class of biomarkers, valuable in a number of neoplasms including CRC. This PhD project aims to investigate miRNAs expression in both liquid and solid biopsies of metastatic vs non-metastatic CRC patients candidate for surgery, in order to identify reliable biomarkers suitable for prognosis, treatment and patient’s monitoring. Materials and methods: Blood sampling from each patient was obtained at the time of admission and on fifth postoperative day. Circulating exosomal RNA was extracted from plasma, isolated and RNA profiles analyzed. RNA from solid tumors, sampled immediately after surgery, was extracted and quantified. Samples were then processed and subjected to microarray analysis. Results: Globally, 25 patients have been enrolled in the study, in between whom 10 non-metastatic and 15 metastatic. No significant differences in terms of demographics were observed between the two groups. Colorectal resections were conducted by laparoscopy and no conversion to open surgery occurred. Any significant difference in perioperative outcomes was noticed. Liquid biopsies estimate of small non-coding RNA concentration revealed a high percentage of miRNAs. Bioinformatic analysis of solid biopsies identified 16 dysregulated miRNAs differently expressed in metastatic vs non-metastatic patients, 11 upregulated and 5 downregulated. Bibliographic research focused on these 16 selected miRNAs was conducted. Three miRNAs overexpressed in our casuistic haven’t been associated with CRC before; all the remains have been already reported in the literature as linked to CRC, even if with discordant expression between different experiences and different queries, possibly misleading. Conclusion: Since the biology of the primary tumor is responsible for the disease aggressiveness, the knowledge of tumor’s genomic expression is the key to propose treatment and follow-up protocols tailored on individual cancer characteristics, overcoming staging systems and prognostic scores commonly used in clinical practice, which suffer from important limits bound to imaging discrimination rather than bias related to surgical sampling or anatomopathological analyses. This project leads to identify 16 miRNAs capable to distinguish metastatic from non-metastatic CRC. In the future, if real-time PCR of circulating exosomal miRNA isolated from blood samples will confirm miRNAs expression of solid biopsies, we would be able to add important prognostic information in the expanding field of liquid biopsies’ in the management of metastatic cancer.