¹Division of Dermatology, Department of Medicine, Harbor – UCLA Medical Center, Torrance, California, USA

Correspondence: Dr Michael S. Kolodney, Division of Dermatology, Department of Medicine, Harbor – UCLA Medical Center and Los Angeles Biomedical Research Institute, 1000 W Carson St, Box 459, Torrance, California 90509, USA. E-mail: mkolodney@labiomed.org

Received 25 January 2007; Revised 9 April 2007; Accepted 12 April 2007; Published online 21 June 2007.

Abstract

Detection of mutated genomic DNA from cancer cells circulating in blood may improve tumor staging and patient selection for targeted therapy. However, the task of detecting a few mutated cells in the presence of a large excess of wild-type cells requires a sensitive and selective assay. We describe a novel approach to detect circulating melanoma cells harboring a common point mutation in the BRAF kinase. In the first step, primer binding to wild-type BRAF is competitively blocked by a locked nucleic acid (LNA) oligonucleotide. In the second step, the LNA-blocking approach is combined with a mutant-specific forward primer. This two-step approach easily detected 10 BRAF g[1799T>A]-mutated melanoma cells mixed with 10⁵ wild-type cells. To determine the clinical utility of this method, we tested its ability to detect human blood spiked with a defined number of BRAF1799T>A-mutated melanoma cells. Blood was first enriched for melanoma cells using an antibody-mediated negative selection procedure before whole genome amplification (WGA). Mutant BRAF in the WGA-amplified genomic DNA was further amplified by a two-step real-time PCR protocol. Using this approach, we could readily identify mutant DNA from as few as 10 melanoma cells in 1 ml of human blood.