Background: Targeted therapies directed against somatic mutations in genes involved in signalling pathways have been shown to improve outcome compared with chemotherapies in patients with tumors carrying the respective mutations.

Purpose/Objectives: Identification of such mutations is performed from the primary tumor. However, such material is not always available but cells with metastatic potential must be released  to reach their distant loci. Using maintrac® CETC can be detected and individually isolated in almost all patients with lung and colon cancer and melanoma and can provide a liquid biopsy to monitor the course of disease. We, here, report on the successful analysis of such isolated cells for gene mutations in tumor driver genes EGFR, KRAS and BRAF.

Materials and Methods: Blood from patients with NSCLC, colon cancer and malignant melanoma was analyzed for cells positive for epithelial antigen (EpCAM) using the maintrac® approach. Between 8-20 EpCAM positive cells from each patient were isolated individually. The DNA was subsequently amplified by whole genome amplification and assayed using either the cobas® EGFR, the cobas® KRAS or the cobas® BRAF V600 Test.

Results: DNA could be amplified from all indiviually isolated cells. EGFR mutation was detected in 12% of isolated tumor cells from a patient with NSCLC, KRAS  mutation was detectable in 28% of cells from a patient with colon cancer and the BRAF mutation in 100% of cells from a BRAF cell line and in 50% of cells from a melanoma patient.

Conclusions: Individually isolating epithelial tumor cells from the peripheral blood allows not only detection of driver mutations in circulating tumor cells but also to determine the frequency of mutated cells. The results were confirmed by single cell analysis of a BRAF mutated cell line. This proves that at least part of the CETC from the tumor. They can, in the future, be used as markers of response to the action of drugs and contribute insight into how resistance may be acquired.