Profiling Epidermal Growth Factor Receptor DNA Mutations In Circulating Rare Cells Using A Cell Isolation System And...

Mutations found within the epidermal growth factor gene (EGFR) are critical for determining the most effective treatments for patients with lung cancer. Routine testing for EGFR mutations can take the form of tumor biopsies or analysis of ctDNA from plasma of patients with lung cancer. Although testing blood samples from patients with lung cancer is performed routinely to provide insight into the presence or recurrence of lung cancer, via ctDNA detection or CTC detection, it typically does not involve the subsequent molecular analysis of the captured cells. Detection of individual mutations within the EGFR gene present within rare circulating tumor cells (CTCs) can offer additional insights into the potential efficacy of targeted therapies and beyond that, patient prognosis, especially in non-small cell lung cancer (NSCLC). Unfortunately, the rarity of such circulating tumor cells and lack of streamlined procedures to interrogate their genomic information present a significant detection challenge. The goal of this protocol is to illustrate the realistic potential of capturing CTCs from whole blood and assessing their molecular content using ddPCR. A semi-automated, size-based cell isolation platform was used to capture circulating tumor cells, which were subsequently recovered for downstream molecular analysis. To determine the presence of specific EGFR mutations within the genomic DNA of captured cells, ddPCR was performed using assays for EGFR pT790M and EGFR pL858R. Two separate lung cancer cell lines (A549 and H1975) were actively enriched from whole blood and accurate quantification of mutations present within the cell lines H1975 was performed using ddPCR.