title: Detection of BRAF V600E mutation in colorectal cancer: comparison of automatic sequencing and real-time chemistry methodology.
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.
authors.affiliation: Unidad de Investigación, Hospital General Universitario de Alicante, C/ Maestro Alonso 109, 03010 Alicante, Spain. benlloch_sus@gva.es
authors.forename: Susana
authors.initials: S
authors.lastname: Benlloch
authors.forename: Artemio
authors.initials: A
authors.lastname: Payá
authors.forename: Cristina
authors.initials: C
authors.lastname: Alenda
authors.forename: Xavier
authors.initials: X
authors.lastname: Bessa
authors.forename: Montserrat
authors.initials: M
authors.lastname: Andreu
authors.forename: Rodrigo
authors.initials: R
authors.lastname: Jover
authors.forename: Antoni
authors.initials: A
authors.lastname: Castells
authors.forename: Xavier
authors.initials: X
authors.lastname: Llor
authors.forename: F Ignacio
authors.initials: FI
authors.lastname: Aranda
authors.forename: Bartomeu
authors.initials: B
authors.lastname: Massutí
mesh_terms: D015179:Colorectal Neoplasms / Q000175:diagnosis* / Q000235:genetics; D003123:Colorectal Neoplasms, Hereditary Nonpolyposis / Q000191:economics* / Q000235:genetics; D004252:DNA Mutational Analysis / Q000191:economics*; D006801:Humans; D009154:Mutation; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000191:economics*; D017422:Sequence Analysis, DNA / Q000191:economics*
chemical_list: D048493:Proto-Oncogene Proteins B-raf