Background RAS assessment is mandatory for therapy decision in metastatic colorectal cancer (mCRC) patients. This determination is based on tumor tissue, however, genotyping of circulating tumor (ct)DNA offers clear advantages as a minimally invasive method that represents tumor heterogeneity. Our study aims to evaluate the use of ctDNA as an alternative for determining baseline RAS status and subsequent monitoring of RAS mutations during therapy as a component of routine clinical practice.Patients and methods RAS mutational status in plasma was evaluated in mCRC patients by OncoBEAM™ RAS CRC assay. Concordance of results in plasma and tissue was retrospectively evaluated. RAS mutations were also prospectively monitored in longitudinal plasma samples from selected patients.ResultsAnalysis of RAS in tissue and plasma samples from 115 mCRC patients showed a 93% overall agreement. Plasma/tissue RAS discrepancies were mainly explained by spatial and temporal tumor heterogeneity. Analysis of clinico-pathological features showed that the site of metastasis (i.e. peritoneal, lung), the histology of the tumor (i.e. mucinous) and administration of treatment previous to blood collection negatively impacted the detection of RAS in ctDNA. In patients with baseline mutant RAS tumors treated with chemotherapy/antiangiogenic, longitudinal analysis of RAS ctDNA mirrored response to treatment, being an early predictor of response. In patients RAS wt, longitudinal monitoring of RAS ctDNA revealed that OncoBEAM was useful to detect emergence of RAS mutations during anti-EGFR treatment.ConclusionThe high overall agreement in RAS mutational assessment between plasma and tissue supports blood-based testing with OncoBEAM™ as a viable alternative for genotyping RAS of mCRC patients in routine clinical practice. Our study describes practical clinico-pathological specifications to optimize RAS ctDNA determination. Moreover, OncoBEAM™ is useful to monitor RAS in patients undergoing systemic therapy to detect resistance and evaluate the efficacy of particular treatments.

Metastatic colorectal cancer (mCRC) relies on the detachment of aggressive malignant cells from the primary tumor into the bloodstream and, concordantly, the presence of these Circulating Tumor Cells (CTC) is associated with a poor prognosis. In this work, the molecular characterization of CTC from mCRC patients was approached, with the aim of understanding their biology and improving their clinical utility in the management of colorectal cancer patients. For this, EpCAM-based immunoisolation of CTC was combined with whole transcriptome amplification and hybridization onto cDNA microarrays. Gene expression data from mCRC patients, once the background of unspecific immunoisolation from a group of controls had been subtracted, resulted in 410 genes that characterized the CTC population. Bioinformatics were used for the biological interpretation of the data, revealing that CTC are characterized by genes related to cell movement and adhesion, cell death and proliferation, and cell signalling and interaction. RTqPCR on an independent series of mCRC patients and controls was used for the validation of a number of genes related to the main cellular functions characterizing the CTC population. Comparison between primary carcinomas and lung and liver metastases further involved the CTC-genes in the promotion of metastasis. Moreover, the correlation of CTC-gene expression with clinical parameters demonstrated detection and prognosis significance. In conclusion, the molecular characterization of CTC from mCRC patients and the identification of diagnostic and prognostic biomarkers represent an innovative and promising approach in the clinical management of this type of patients.