title: A combination of immunohistochemistry and molecular approaches improves highly sensitive detection of BRAF mutations in papillary thyroid cancer.
abstract: The optimal method for BRAF mutation detection remains to be determined despite advances in molecular detection techniques. The aim of this study was to compare, against classical Sanger sequencing, the diagnostic performance of two of the most recently developed, highly sensitive methods: BRAF V600E immunohistochemistry (IHC) and peptide nucleic-acid (PNA)-clamp qPCR. BRAF exon 15 mutations were searched in formalin-fixed paraffin-embedded tissues from 86 papillary thyroid carcinoma using the three methods. The limits of detection of Sanger sequencing in borderline or discordant cases were quantified by next generation sequencing. BRAF mutations were found in 74.4 % of cases by PNA, in 71 % of cases by IHC, and in 64 % of cases by Sanger sequencing. Complete concordance for the three methods was observed in 80 % of samples. Better concordance was observed with the combination of two methods, particularly PNA and IHC (59/64) (92 %), while the combination of PNA and Sanger was concordant in 55 cases (86 %). Sensitivity of the three methods was 99 % for PNA, 94.2 % for IHC, and 89.5 % for Sanger. Our data show that IHC could be used as a cost-effective, first-line method for BRAF V600E detection in daily practice, followed by PNA analysis in negative or uninterpretable cases, as the most efficient method. PNA-clamp quantitative PCR is highly sensitive and complementary to IHC as it also recognizes other mutations besides V600E and it is suitable for diagnostic purposes.
authors: Department of Internal Medicine and Medical Specialties (DiMI), University of Genoa, V.le Benedetto XV 6, 16132, Genoa, Italy.|Department of Surgical Sciences (DISC), University of Genoa, Genoa, Italy.
authors: Claudia
authors: C
authors: Martinuzzi
authors: Department of Internal Medicine and Medical Specialties (DiMI), University of Genoa, V.le Benedetto XV 6, 16132, Genoa, Italy.
authors: Lorenza
authors: L
authors: Pastorino
authors: Department of Internal Medicine and Medical Specialties (DiMI), University of Genoa, V.le Benedetto XV 6, 16132, Genoa, Italy.
authors: Virginia
authors: V
authors: Andreotti
authors: Department of Internal Medicine and Medical Specialties (DiMI), University of Genoa, V.le Benedetto XV 6, 16132, Genoa, Italy.
authors: Anna
authors: A
authors: Garuti
authors: Department of Surgical Sciences (DISC), University of Genoa, Genoa, Italy.|Surgery 1 Unit, IRCCS AOU San Martino-IST, Genoa, Italy.
authors: Michele
authors: M
authors: Minuto
authors: Department of Surgical Sciences (DISC), University of Genoa, Genoa, Italy.|Pathology Unit, IRCCS AOU San Martino-IST, Genoa, Italy.
authors: Roberto
authors: R
authors: Fiocca
authors: Department of Internal Medicine and Medical Specialties (DiMI), University of Genoa, V.le Benedetto XV 6, 16132, Genoa, Italy.|Genetics of Rare Tumors, IRCCS AOU San Martino-IST, University of Genoa, Genoa, Italy.
authors: Giovanna
authors: G
authors: Bianchi-Scarrà
authors: Department of Internal Medicine and Medical Specialties (DiMI), University of Genoa, V.le Benedetto XV 6, 16132, Genoa, Italy. paola.ghiorzo@unige.it.|Genetics of Rare Tumors, IRCCS AOU San Martino-IST, University of Genoa, Genoa, Italy. paola.ghiorzo@unige.it.
authors: Paola
authors: P
authors: Ghiorzo
authors: Department of Surgical Sciences (DISC), University of Genoa, Genoa, Italy.|Pathology Unit, IRCCS AOU San Martino-IST, Genoa, Italy.
authors: Federica
authors: F
authors: Grillo
authors: Department of Surgical Sciences (DISC), University of Genoa, Genoa, Italy.|Pathology Unit, IRCCS AOU San Martino-IST, Genoa, Italy.
authors: Luca
authors: L
authors: Mastracci
mesh_terms: D000231:Adenocarcinoma, Papillary / Q000235:genetics* / Q000378:metabolism; D004252:DNA Mutational Analysis / Q000379:methods; D005260:Female; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods*; D008297:Male; D008875:Middle Aged; D009154:Mutation; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics* / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013964:Thyroid Neoplasms / Q000235:genetics* / Q000378:metabolism
chemical_list: C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf
keywords: BRAF; Immunohistochemistry; Molecular methods; PNA-clamp qPCR; Papillary thyroid cancer; Sanger sequencing