Generation of mRNA and miRNA gene expression profiles in circulating tumor cells of metastatic colorectal cancer patients

Abstract

Patients with solitary resectable liver metastases might benefit from curative liver resection, but unfortunately, half of the patients relapse within one year after this major surgery. Reliable prognostic factors are urgently needed to identify those patients benefitting from liver metastasis resection. Circulating tumor cells (CTCs) have been identified as a powerful prognostic marker in metastatic colorectal cancer. In addition to enumeration, CTCs can be enriched for subsequent molecular characterization. In this sense, CTCs are a very promising diagnostic and prognostic tool enabling the repeated and non-invasive evaluation of the expression of drug targets, predictive and prognostic factors at the time of metastasis resection. Here, we describe the identification of a CTC-specific mRNA and miRNA profile generated on CTC-enriched fractions isolated from blood drawn from metastatic CRC patients at the time of liver metastasis resection, and their correlation with clinical parameters. In this study we included 161 metastatic colorectal cancer patients who were about to undergo liver metastasis resection and 30 healthy donors (HD). CTCs were isolated from 30 mL blood using the CellSearch Profile Kit (Veridex LLC) after a modified Ficoll enrichment, and HD blood was subjected to the same procedure. Total RNA was isolated from the enriched CTC fraction and a panel of mRNAs and miRNAs with potential clinical relevance was measured in CTCs and in enriched HD blood to identify CTC-specific genes. Gene expression levels of CTC-specific mRNAs and miRNAs were correlated with clinical parameters. From 100 of these patients, the liver resection indeed showed pathology-confirmed metastasis of colorectal origin and both mRNA and miRNA was of sufficient quality. These 100 patients (mean age 63, range 40 - 85) had a median CTC count of 1 (range 0 - 35) per 30 mL blood. Most patients (63%) presented with synchronous metastatic disease and median time between primary tumor resection and metastasis was 313 days (range 0 - 2486). Just over half of patients (52%) had received induction chemotherapy before liver surgery. Out of 96 mRNAs and 45 miRNAs, we were able to identify a panel of CTC-specific mRNAs and miRNAs, of which the transcripts were more abundantly expressed in patients with β2 CTCs compared to 30 healthy donors (Mann-Whitney U-test P<0.05). Among the differentially expressed were, besides epithelial-specific genes, genes involved in cell differentiation, proliferation and inflammation. Associations between CTC gene expression and clinicopathologic characteristics will be presented at the meeting. In this study, we show that it is feasible to molecularly characterize rare CTCs from colorectal cancer patients in a background of leukocytes, a promising application which will greatly increase the amount of information that can be obtained from the CTCs of colorectal cancer patients.