A minimally invasive and repeatable approach for real-time
epidermal growth factor receptor (EGFR) mutation surveillance would be highly beneficial for individualized
therapy of late stage
lung cancer patients whose surgical specimens are often not available. We aim to develop a viable method to detect EGFR mutations in single
circulating tumor cells (CTCs). Using a model CTC system of spiked
tumor cells in whole blood, we evaluated EGFR mutation determination in single
tumor cells enriched from blood. We used magnetic beads labeled with antibody against leukocyte
surface antigens to deplete leukocytes and enrich native CTCs independent of epithelial marker expression level. We then used
laser cell microdissection (LCM) to isolate individual CTCs, followed by whole-genome amplification of the
DNA for exon 19 microdeletion, L858R and T790M mutation detection by PCR sequencing. EGFR mutations were successfully measured in individual spiked
tumor cells enriched from 7.5 ml whole blood. Whole-genome amplification provided sufficient
DNA for mutation determination at multiple sites. Ninety-five percent of the single CTCs microdissected by LCM (19/20) yielded PCR amplicons for at least one of the three mutation sites. The amplification success rates were 55 % (11/20) for exon 19 deletion, 45 % (9/20) for T790M, and 85 % (17/20) for L858R. Sequencing of the amplicons showed allele dropout in the amplification reactions, but mutations were correctly identified in 80 % of the amplicons. EGFR mutation determination from single captured
tumor cells from blood is feasible with the approach described here. However, to overcome allele dropout and to obtain reliable information about the
tumor's EGFR status, multiple individual
tumor cells should be assayed.