Detection of the BRAF V600E mutation in colon carcinoma: critical evaluation of the imunohistochemical approach.
Recently BRAF V600E mutant-specific antibody (clone VE1) became available to immunohistochemically pinpoint the occurrence of these BRAF-mutant proteins in different tumors, such as colon carcinoma. Detection of BRAF mutations is important for the accurate application of targeted therapy against BRAF serine-threonine kinase activation. In this study, we evaluated 113 colon carcinomas including 95 primary and 27 metastatic tumors with the VE1 antibody using Leica Bond-Max automated immunohistochemistry. To ensure comprehensive BRAF V600E mutation detection, all cases were evaluated using 4 molecular methods (Sanger sequencing, the Cobas 4800 BRAF V600 Mutation Test, BRAF V600 allele-specific polymerase chain reaction, and BRAF V600 quantitative polymerase chain reaction) with nearly 100% concordance. Molecular and immunohistochemical studies were blinded. Furthermore, all cases were evaluated for KRAS and NRAS mutations as parameters mutually exclusive with BRAF mutations offering parallel evidence for BRAF mutation status. Strong to moderate VE1 positivity was seen in 34 tumors. Twelve colon carcinomas showed weak VE1 immunohistochemical staining, and 67 were entirely negative. An identical c.1799T>A single nucleotide substitution leading to the BRAF V600E mutation was identified in 27 of 113 (24%) colon carcinomas. A majority of BRAF-mutant tumors were located in the right side of the colon and had mismatch-repair deficiency. V600E mutation-negative carcinomas were more often sigmoid tumors and usually showed intact mismatch-repair proteins and KRAS or NRAS mutations. The sensitivity and specificity of positive results (strong to moderate staining) of VE1 immunohistochemistry were 85% and 68%, respectively. If any positivity would be considered, then the specificity declined to 51% with no significant improvement of sensitivity. Therefore, only strong positivity should be considered when using the VE1 antibody and Leica Bond-Max automated immunohistochemistry with these parameters. Although VE1 antibody can be useful in the screening of colon carcinomas for BRAF V600E-mutant proteins, molecular genetic confirmation is always necessary for mutation diagnosis.
*Laboratory of Pathology, National Cancer Institute (NCI), Bethesda, MD Departments of †Molecular Diagnostics §Surgical Pathology ∥Department of Clinical Oncology, Holycross Cancer Center ¶Faculty of Health Sciences, The Jan Kochanowski University, Kielce ‡Department of Biology and Genetics, Medical University of Gdansk, Gdansk, Poland.
Jerzy
J
Lasota
Artur
A
Kowalik
Bartosz
B
Wasag
Zeng-Feng
ZF
Wang
Anna
A
Felisiak-Golabek
Tiffany
T
Coates
Janusz
J
Kopczynski
Stanislaw
S
Gozdz
Markku
M
Miettinen
D000230:Adenocarcinoma / Q000201:enzymology* / Q000235:genetics* / Q000556:secondary; D000328:Adult; D000368:Aged; D057205:Automation, Laboratory; D014408:Biomarkers, Tumor / Q000235:genetics*; D001706:Biopsy; D003110:Colonic Neoplasms / Q000201:enzymology* / Q000235:genetics* / Q000473:pathology; D053843:DNA Mismatch Repair; D004252:DNA Mutational Analysis; D005260:Female; D020558:GTP Phosphohydrolases / Q000235:genetics; D020022:Genetic Predisposition to Disease; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D008565:Membrane Proteins / Q000235:genetics; D008875:Middle Aged; D009154:Mutation; D010641:Phenotype; D011044:Poland; D016133:Polymerase Chain Reaction; D011237:Predictive Value of Tests; D011518:Proto-Oncogene Proteins / Q000235:genetics; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D016283:Proto-Oncogene Proteins p21(ras); D015203:Reproducibility of Results; D014481:United States; D018631:ras Proteins / Q000235:genetics
D014408:Biomarkers, Tumor; C117307:KRAS protein, human; D008565:Membrane Proteins; D011518:Proto-Oncogene Proteins; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D020558:GTP Phosphohydrolases; C579846:NRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins