Circulating Tumor Extracellular Vesicles for Non-Invasive Monitoring of Metastatic Colorectal Cancer

Observational, ambispective, multicenter, non-interventional study without drugs or medical devices, aimed at the identification, validation, and clinical evaluation of extracellular vesicle (EV)-derived biomarkers in patients with metastatic colorectal cancer (mCRC).

The study will include patients diagnosed with metastatic colorectal cancer (mCRC), divided into a retrospective cohort and a prospective cohort.

This study aims to identify and validate circulating extracellular vesicle (EV)-derived biomarkers, including EV-RNA and EV-protein (EV-PROT) profiles, in patients with metastatic colorectal cancer (mCRC). The goal is to develop an integrated non-invasive biomarker panel to support patient stratification, treatment response prediction, and longitudinal disease monitoring.

The study will be conducted through three main phases:

Discovery phase: identification of novel EV-RNA and EV-PROT biomarkers from plasma samples of mCRC patients, integrated with spatial transcriptomic and proteomic analyses of tumor tissues and functional evaluation in patient-derived colorectal cancer organoids (CRC-org).

Retrospective validation: validation of selected biomarkers in an independent mCRC cohort using droplet digital PCR (ddPCR) for EV-RNA and nano-flow cytometry for EV-PROT, combined with clinical outcome analyses and functional studies using CRC-org-derived 3D tumor models (assembloids).

Prospective validation: assessment of biomarker clinical value in an independent prospective mCRC cohort through longitudinal plasma analyses at different treatment time points, evaluating associations with treatment response, progression-free survival (PFS), and overall survival (OS).

The integration of molecular, clinical, and experimental data will support the biological characterization of EV-derived biomarkers and their potential role in tumor progression, therapeutic response, and resistance mechanisms.

The statistical analysis will be conducted to evaluate the association between EV-RNA and EV-PROT biomarkers and clinical disease outcomes. Data will be analyzed using descriptive methods and appropriate statistical tests to compare groups and assess changes over time.

Retrospective Cohort Inclusion Criteria:

• Age ≥ 18 years;
• Histologically confirmed diagnosis of colorectal adenocarcinoma;
• Radiological evidence of metastatic or locally advanced unresectable disease;
• First-line treatment for metastatic disease already completed or ongoing according to standard clinical practice;
• Availability of stored biological samples collected at least at one of the protocol-defined time points (baseline, post-treatment, disease progression);
• Informed consent acquisition will be performed in accordance with Article 110-bis of the Italian Privacy Code.

Retrospective Cohort Exclusion Criteria:

• Absence of suitable biological samples or samples unsuitable for molecular analyses;
• Incomplete clinical data preventing the performance of the planned analyses;
• Insufficient quality of stored biological samples for the assessment of EV-RNA and EV-PROT analyses.

Prospective Cohort Inclusion Criteria:

• Age ≥ 18 years;
• Histologically confirmed diagnosis of colorectal adenocarcinoma;
• Radiological evidence of unresectable metastatic disease;
• Patients not previously treated for metastatic disease (prior completed adjuvant/neoadjuvant therapy is allowed if completed ≥ 6 months before study enrollment);
• Indication for first-line treatment with chemotherapy ± biological therapy according to standard clinical practice;
• Ability to understand the study procedures and provide written informed consent.

Prospective Cohort Exclusion Criteria:

• Previous systemic treatment for metastatic disease (except prior adjuvant/neoadjuvant therapy as specified above);
• Ongoing pregnancy or breastfeeding;
• Legal incapacity or limitation in the ability to provide informed consent.