title: Heterogeneity and frequency of BRAF mutations in primary melanoma: Comparison between molecular methods and immunohistochemistry.
abstract: Finding the best technique to identify BRAF mutations with a high sensitivity and specificity is mandatory for accurate patient selection for target therapy. BRAF mutation frequency ranges from 40 to 60% depending on melanoma clinical characteristics and detection technique used.Intertumoral heterogeneity could lead to misinterpretation of BRAF mutational status; this is especially important if testing is performed on primary specimens, when metastatic lesions are unavailable.Aim of this study was to identify the best combination of methods for detecting BRAF mutations (among peptide nucleic acid - PNA-clamping real-time PCR, immunohistochemistry and capillary sequencing) and investigate BRAF mutation heterogeneity in a series of 100 primary melanomas and a subset of 25 matched metastatic samples.Overall, we obtained a BRAF mutation frequency of 62%, based on the combination of at least two techniques. Concordance between mutation status in primary and metastatic tumor was good but not complete (67%), when agreement of at least two techniques were considered. Next generation sequencing was used to quantify the threshold of detected mutant alleles in discordant samples. Combining different methods excludes that the observed heterogeneity is technique-based. We propose an algorithm for BRAF mutation testing based on agreement between immunohistochemistry and PNA; a third molecular method could be added in case of discordance of the results. Testing the primary tumor when the metastatic sample is unavailable is a good option if at least two methods of detection are used, however the presence of intertumoral heterogeneity or the occurrence of additional primaries should be carefully considered.
authors.affiliation: Department of Internal Medicine and Medical Specialties (DiMI), University of Genoa and IRCCS AOU San Martino-IST, Genoa, Italy.
authors.forename: William
authors.initials: W
authors.lastname: Bruno
authors.affiliation: Department of Internal Medicine and Medical Specialties (DiMI), University of Genoa and IRCCS AOU San Martino-IST, Genoa, Italy.
authors.forename: Claudia
authors.initials: C
authors.lastname: Martinuzzi
authors.affiliation: Department of Internal Medicine and Medical Specialties (DiMI), University of Genoa and IRCCS AOU San Martino-IST, Genoa, Italy.
authors.forename: Virginia
authors.initials: V
authors.lastname: Andreotti
authors.affiliation: Department of Internal Medicine and Medical Specialties (DiMI), University of Genoa and IRCCS AOU San Martino-IST, Genoa, Italy.
authors.forename: Lorenza
authors.initials: L
authors.lastname: Pastorino
authors.affiliation: Department of Medical Oncology, IRCCS AOU San Martino-IST, Genoa, Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Spagnolo
authors.affiliation: Department of Internal Medicine and Medical Specialties (DiMI), University of Genoa and IRCCS AOU San Martino-IST, Genoa, Italy.
authors.forename: Bruna
authors.initials: B
authors.lastname: Dalmasso
authors.affiliation: Department of Pathology, IRCCS AOU San Martino-IST, Genoa, Italy.
authors.forename: Francesco
authors.initials: F
authors.lastname: Cabiddu
authors.affiliation: Department of Pathology, IRCCS AOU San Martino-IST, Genoa, Italy.
authors.forename: Marina
authors.initials: M
authors.lastname: Gualco
authors.affiliation: Department of Internal Medicine and Medical Specialties (DiMI), University of Genoa and IRCCS AOU San Martino-IST, Genoa, Italy.
authors.forename: Alberto
authors.initials: A
authors.lastname: Ballestrero
authors.affiliation: Department of Internal Medicine and Medical Specialties (DiMI), University of Genoa and IRCCS AOU San Martino-IST, Genoa, Italy.
authors.forename: Giovanna
authors.initials: G
authors.lastname: Bianchi-Scarrà
authors.affiliation: Department of Medical Oncology, IRCCS AOU San Martino-IST, Genoa, Italy.
authors.forename: Paola
authors.initials: P
authors.lastname: Queirolo
authors.affiliation: Department of Surgical and Diagnostic Sciences, Pathology Unit, University of Genoa and IRCCS AOU San Martino-IST, Genoa, Italy.
authors.forename: Federica
authors.initials: F
authors.lastname: Grillo
authors.affiliation: Department of Surgical and Diagnostic Sciences, Pathology Unit, University of Genoa and IRCCS AOU San Martino-IST, Genoa, Italy.
authors.forename: Luca
authors.initials: L
authors.lastname: Mastracci
authors.affiliation: Department of Internal Medicine and Medical Specialties (DiMI), University of Genoa and IRCCS AOU San Martino-IST, Genoa, Italy.
authors.forename: Paola
authors.initials: P
authors.lastname: Ghiorzo
mesh_terms: D014408:Biomarkers, Tumor / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods*; D005260:Female; D005787:Gene Frequency; D020022:Genetic Predisposition to Disease; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D008545:Melanoma / Q000201:enzymology / Q000235:genetics* / Q000556:secondary; D008875:Middle Aged; D009154:Mutation; D015588:Observer Variation; D010641:Phenotype; D011237:Predictive Value of Tests; D011379:Prognosis; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction; D015203:Reproducibility of Results; D012878:Skin Neoplasms / Q000201:enzymology / Q000235:genetics* / Q000473:pathology
chemical_list: D014408:Biomarkers, Tumor; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf
keywords: BRAF; PNA-clamping; heterogeneity; immunohistochemistry; primary melanoma