Published online before print August 9, 2007

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Copyright © 2007 American Society for Investigative Pathology
Journal of Molecular Diagnostics, doi:10.2353/jmoldx.2007.060191

Accepted for publication March 23, 2007.

Article

Application of a BRAF Pyrosequencing Assay for Mutation Detection and Copy Number Analysis in Malignant Melanoma

Cynthia Spittle^*, M. Renee Ward, Katherine L. Nathanson, Phyllis A. Gimotty, Eric Rappaport^¶, Marcia S. Brose^||, Angelica Medina, Richard Letrero, Meenhard Herlyn^**, and Robin H. Edwards^@

From the Fox Chase Cancer Center,* Philadelphia, Pennsylvania; the Departments of Medicine, Biostatistics and Epidemiology, Otorhinolaryngology,^|| and Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania; The Joseph Stokes Jr. Research Institute,^¶ Children's Hospital of Philadelphia, Philadelphia, Pennsylvania; The Wistar Institute,** Philadelphia, Pennsylvania; and Pharmion Corporation, San Francisco, California

^@ To whom correspondence should be addressed. E-mail: edwardsr{at}mail.med.upenn.edu.

	Abstract

Mutations in the BRAF gene are found in the majority of cutaneous malignant melanomas and subsets of other tumors. These mutations lead to constitutive activation of BRAF with increased downstream ERK (extracellular signal-regulated kinase) signaling; therefore, the development of RAF kinase inhibitors for targeted therapy is being actively pursued. A methodology that allows sensitive, cost-effective, high-throughput analysis of BRAF mutations will be needed to triage patients for specific molecular-based therapies. Pyrosequencing is a high-throughput, sequencing-by-synthesis method that is particularly useful for analysis of single nucleotide polymorphisms or hotspot mutations. Mutational analysis of BRAF is highly amenable to pyrosequencing because the majority of mutations in this gene localize to codons 600 and 601 and consist of single or dinucleotide substitutions. In this study, DNAs from a panel of melanocyte cell lines, melanoma cell lines, and melanoma tumors were used to validate a pyrosequencing assay to detect BRAF mutations. The assay demonstrates high accuracy and precision for detecting common and variant exon 15 BRAF mutations. Further, comparison of pyrosequencing data with 100K single nucleotide polymorphism microarray data allows characterization of BRAF amplification events that may accompany BRAF mutation. Pyro-sequencing serves as an excellent platform for BRAF genotyping of tumors from patients entering clinical trial.

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