title: Specific and sensitive hydrolysis probe-based real-time PCR detection of epidermal growth factor receptor variant III in oral squamous cell carcinoma.
abstract: BACKGROUND
  The tumor-specific EGFR deletion mutant, EGFRvIII, is characterised by ligand-independent constitutive signalling. Tumors expressing EGFRvIII are resistant to current EGFR-targeted therapy. The frequency of EGFRvIII in head and neck squamous cell carcinoma (HNSCC) is disputed and may vary by specific sub-site. The purpose of this study was to measure the occurrence of EGFRvIII mutations in a specific HNSCC subsite, oral squamous cell carcinoma (OSCC), using a novel real-time PCR assay.
  
  
  METHODS
  Pre-treatment Formalin Fixed Paraffin Embedded (FFPE) cancer specimens from 50 OSCC patients were evaluated for the presence of EGFRvIII using a novel hydrolysis probe-based real-time PCR assay. EGFR protein expression in tumor samples was quantified using fluorescent immunohistochemistry (IHC) and AQUA® technology.
  
  
  RESULTS
  We detected EGFRvIII in a single OSCC patient in our cohort (2%). We confirmed the validity of our detection technique in an independent cohort of glioblastoma patients. We also compared the sensitivity and specificity of our novel real-time EGFRvIII detection assay to conventional RT-PCR and direct sequencing. Our assay can specifically detect EGFRvIII and can discriminate against wild-type EGFR in FFPE tumor samples. AQUAnalysis® revealed that the presence of EGFRvIII transcript is associated with very high EGFR protein expression (98(th) percentile). Contrary to previous reports, only 44% of OSCC over-expressed EGFR in our study.
  
  
  CONCLUSIONS
  Our results suggest that the EGFRvIII mutation is rare in OSCC and corroborate previous reports of EGFRvIII expression only in tumors with extreme over-expression of EGFR. We conclude that EGFRvIII-specific therapies may not be ideally suited as first-line treatment in OSCC. Furthermore, highly specific and sensitive methods, such as the real-time RT-PCR assay and AQUAnalysis® described here, will provide accurate assessment of EGFR mutation frequency and EGFR expression, and will facilitate the selection of optimal tailored therapies for OSCC patients.
authors.affiliation: Department of Pathology and Laboratory Medicine, Tom Baker Cancer Centre, University of Calgary, Calgary, Canada.
authors.forename: John B
authors.initials: JB
authors.lastname: McIntyre
authors.forename: Pinaki
authors.initials: P
authors.lastname: Bose
authors.forename: Alexander C
authors.initials: AC
authors.lastname: Klimowicz
authors.forename: Nigel T
authors.initials: NT
authors.lastname: Brockton
authors.forename: Stephanie
authors.initials: S
authors.lastname: Petrillo
authors.forename: Wayne
authors.initials: W
authors.lastname: Matthews
authors.forename: Jay
authors.initials: J
authors.lastname: Easaw
authors.forename: Anthony
authors.initials: A
authors.lastname: Magliocco
authors.forename: Joseph C
authors.initials: JC
authors.lastname: Dort
mesh_terms: D002294:Carcinoma, Squamous Cell / Q000737:chemistry*; D066246:ErbB Receptors / Q000032:analysis / Q000235:genetics*; D006801:Humans; D006868:Hydrolysis; D009062:Mouth Neoplasms / Q000737:chemistry*; D050505:Mutant Proteins / Q000032:analysis; D009363:Neoplasm Proteins / Q000032:analysis / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012680:Sensitivity and Specificity
chemical_list: D050505:Mutant Proteins; D009363:Neoplasm Proteins; D012333:RNA, Messenger; D066246:ErbB Receptors