title: Comparison of high resolution melting analysis, pyrosequencing, next generation sequencing and immunohistochemistry to conventional Sanger sequencing for the detection of p.V600E and non-p.V600E BRAF mutations.
abstract: BACKGROUND
  The approval of vemurafenib in the US 2011 and in Europe 2012 improved the therapy of not resectable or metastatic melanoma. Patients carrying a substitution of valine to glutamic acid at codon 600 (p.V600E) or a substitution of valine to leucine (p.V600K) in BRAF show complete or partial response. Therefore, the precise identification of the underlying somatic mutations is essential. Herein, we evaluate the sensitivity, specificity and feasibility of six different methods for the detection of BRAF mutations.
  
  
  METHODS
  Samples harboring p.V600E mutations as well as rare mutations in BRAF exon 15 were compared to wildtype samples. DNA was extracted from formalin-fixed paraffin-embedded tissues by manual micro-dissection and automated extraction. BRAF mutational analysis was carried out by high resolution melting (HRM) analysis, pyrosequencing, allele specific PCR, next generation sequencing (NGS) and immunohistochemistry (IHC). All mutations were independently reassessed by Sanger sequencing. Due to the limited tumor tissue available different numbers of samples were analyzed with each method (82, 72, 60, 72, 49 and 82 respectively).
  
  
  RESULTS
  There was no difference in sensitivity between the HRM analysis and Sanger sequencing (98%). All mutations down to 6.6% allele frequency could be detected with 100% specificity. In contrast, pyrosequencing detected 100% of the mutations down to 5% allele frequency but exhibited only 90% specificity. The allele specific PCR failed to detect 16.3% of the mutations eligible for therapy with vemurafenib. NGS could analyze 100% of the cases with 100% specificity but exhibited 97.5% sensitivity. IHC showed once cross-reactivity with p.V600R but was a good amendment to HRM.
  
  
  CONCLUSIONS
  Therefore, at present, a combination of HRM and IHC is recommended to increase sensitivity and specificity for routine diagnostic to fulfill the European requirements concerning vemurafenib therapy of melanoma patients.
authors.affiliation: Institute of Pathology, University of Cologne, Medical Centre, Cologne, Germany. michaela.ihle@uk-koeln.de.
authors.forename: Michaela Angelika
authors.initials: MA
authors.lastname: Ihle
authors.forename: Jana
authors.initials: J
authors.lastname: Fassunke
authors.forename: Katharina
authors.initials: K
authors.lastname: König
authors.forename: Inga
authors.initials: I
authors.lastname: Grünewald
authors.forename: Max
authors.initials: M
authors.lastname: Schlaak
authors.forename: Nicole
authors.initials: N
authors.lastname: Kreuzberg
authors.forename: Lothar
authors.initials: L
authors.lastname: Tietze
authors.forename: Hans-Ulrich
authors.initials: HU
authors.lastname: Schildhaus
authors.forename: Reinhard
authors.initials: R
authors.lastname: Büttner
authors.forename: Sabine
authors.initials: S
authors.lastname: Merkelbach-Bruse
mesh_terms: D000970:Antineoplastic Agents / Q000627:therapeutic use; D014408:Biomarkers, Tumor / Q000032:analysis / Q000235:genetics; D004252:DNA Mutational Analysis / Q000379:methods*; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D007150:Immunohistochemistry; D007211:Indoles / Q000627:therapeutic use; D008545:Melanoma / Q000188:drug therapy / Q000201:enzymology / Q000235:genetics; D058990:Molecular Targeted Therapy; D009154:Mutation; D016612:Paraffin Embedding; D018579:Patient Selection; D016133:Polymerase Chain Reaction; D057285:Precision Medicine; D011237:Predictive Value of Tests; D047428:Protein Kinase Inhibitors / Q000627:therapeutic use; D048493:Proto-Oncogene Proteins B-raf / Q000032:analysis / Q000037:antagonists & inhibitors / Q000235:genetics; D012878:Skin Neoplasms / Q000188:drug therapy / Q000201:enzymology / Q000235:genetics; D013449:Sulfonamides / Q000627:therapeutic use; D016707:Tissue Fixation; D000077484:Vemurafenib
chemical_list: D000970:Antineoplastic Agents; D014408:Biomarkers, Tumor; D007211:Indoles; D047428:Protein Kinase Inhibitors; D013449:Sulfonamides; D000077484:Vemurafenib; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf