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JMD 2006, Vol. 8, No. 5
Copyright © 2006 American Society for Investigative Pathology & Association for Molecular Pathology

Technical Advance

Detection of BRAF V600E Mutation in Colorectal Cancer

Comparison of Automatic Sequencing and Real-Time Chemistry Methodology

Susana Benlloch^*, Artemio Payá, Cristina Alenda, Xavier Bessa, Montserrat Andreu, Rodrigo Jover, Antoni Castells^¶, Xavier Llor^||, F. Ignacio Aranda and Bartomeu Massutí^**

From the Departments of Investigation, * Pathology, Gastroenterology, and Oncology, ** Hospital General Universitario de Alicante, Alicante; Department of Gastroenterology, Hospital del Mar, Barcelona; Department of Gastroenterology, ^|| Hospital Universitario Germans Trias i Pujol, Badalona; and Department of Gastroenterology, ^¶ Institut de Malalties Digestives, Hospital Clinic, Institut d’Investigacions Biomèdiques August Pi i Sunyer, University of Barcelona, Barcelona, Spain

Abstract

Mutation V600E of BRAF, a kinase-encoding gene from the RAS/RAF/MAPK pathway, in colorectal carcinoma (CRC) suggests a sporadic origin of the disease, providing an exclusion criterion for hereditary nonpolyposis colorectal cancer. Here we describe detection of this mutation by real-time chemistry TaqMan MGB probes, confirmed by direct DNA sequencing as the gold standard. DNA was extracted from paraffin-embedded tissue from 112 tumors obtained from the EPICOLON study. Seventy-two tumors were CRC with defective DNA mismatch repair (MMR; microsatellite instability and/or loss of protein expression by immunohistochemical analysis), and 40 were proficient MMR controls. BRAF mutation was detected in 20/72 (27.8%) CRC with defective MMR and in 3/40 (7.5%) proficient MMR controls (P = 0.011). BRAF mutation was detected in 19/51 (37.3%) tumors with loss of MLH1 expression and in none of the tumors with loss of MSH2 expression (0/13). BRAF mutation was not found in cases with germline mutation of MLH1 (4/112) or MSH2 (3/112) genes. The sensitivity and specificity of our real-time chemistry were both 100% for detecting the V600E mutation. Because real-time chemistry methodology has advantages in cost, time, and labor, we consider it a valuable alternative to automatic direct sequencing, particularly for serial measurements.