Abstract 3159: BRAFV600E mutation analysis in circulating tumor cells
Authors: Pace Margaret , Melnikova Vladislava O. , Brandl Kerry , Kahn Larry , Woo Jacky , Davis Darren W. 
Published: June 2012

Abstract
Background: The concept of “liquid biopsy” refers to analysis of rare cells in the blood such as Circulating Tumor Cells (CTC) and represents a powerful tool for molecular characterization of tumors for which biopsies are not available. Because of a low frequency of CTCs, assay sensitivity for mutational analysis of CTCs needs to be below 1%. An activating mutation in the serine/threonine kinase BRAF (V600E) is present in up to 70% of melanomas and 10% of colon carcinomas. Herein, we developed and validated a highly-sensitive allele-specific PCR (AS-PCR) assay to detect the presence of the BRAFV600E mutation in CTCs.
Methods: Genomic DNA isolation was optimized for rare cell analysis. AS-PCR for detection of BRAFV600E mutation was developed and tested, using cell spiking experiments to define assay sensitivity, limit of detection, assay precision and specificity. Use of the CTC BRAFV600E AS-PCR assay was demonstrated in melanoma and colon cancer patient blood.
Results: Performance of the BRAFV600E AS-PCR was tested using gDNA extracted from HT-29 colon cancer cells heterozygous for BRAFV600E, and BRAFwild-type MDA-MB-231 breast cancer cells. Assay sensitivity and limit of detection for determination of BRAFV600E in a background of BRAFwt was tested by spiking HT-29 cells into 50,000 MDA-MB-231 cells at following ratios: 5000 (5% mutant DNA copies), 500 (0.5%), 50 (0.05%), 10 (0.01%), 5 (0.005%) and 0 (0%). The assay limit of detection was found to be 0.01% or 5 BRAFV600E mutant copies in a wt background. Precision was demonstrated by repeating assay sensitivity testing at the limit of detection on three different days. The performance and specificity of the BRAF AS-PCR assay in enriched CTCs was further verified using HT-29 and MDA-MB-231 cells spiked into donor blood and isolated by EpCAM-based CellSearch® CTC Profile kit, which we have previously shown to yield a higher number of CTCs than the standard CellSearch® CTC enumeration kit. Finally, CTC BRAFV600E AS-PCR assay performance was demonstrated in patients with colon cancer as well as melanoma, in which CTCs were isolated using a previously developed melanoma immunomagnetic enrichment panel.
Conclusions: We have developed an AS-PCR assay for detection of the BRAFV600E mutation in CTCs. Validation testing demonstrated that the assay detects 0.01% or 5 copies of BRAFV600E DNA in a wild-type background. Testing melanoma and colon cancer patient blood specimens demonstrated feasibility of the BRAFV600E mutation analysis using liquid biopsy. Further concordance testing between tumor biopsies and blood specimens is ongoing and will establish whether liquid biopsies are a suitable alternative for identifying patients for whom a particular targeted therapy may or may not be effective based on genetic status.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3159. doi:10.1158/1538-7445.AM2011-3159