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. 2012;7(2):e31723.
doi: 10.1371/journal.pone.0031723. Epub 2012 Feb 16.

Specific and sensitive hydrolysis probe-based real-time PCR detection of epidermal growth factor receptor variant III in oral squamous cell carcinoma

Affiliations

Specific and sensitive hydrolysis probe-based real-time PCR detection of epidermal growth factor receptor variant III in oral squamous cell carcinoma

John B McIntyre et al. PLoS One. 2012.

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.

Methodology: 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.

Principal findings: 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.

Conclusion and significance: 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.

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Conflict of interest statement

Competing Interests: Yes, we have the following competing interest. This study was funded by the Ohlson Research Initiative, which functions within the University of Calgary - Faculty of Medicine as well as the Southern Alberta Cancer Research Institute (SACRI). The corresponding author (JCD) is the Director of the Ohlson Research Initiative. This does not alter our adherence to all the PLoS ONE policies on sharing data and materials, as detailed online in the guide for authors.

Figures

Figure 1
Figure 1. CONSORT Diagram outlining patient selection criteria and the different stages at which specific analyses were undertaken for the OSCC study cohort.
Figure 2
Figure 2. Specific detection of EGFRvIII mRNA by real-time RT-PCR.
A. Real-time amplification plot of EGFRvIII transcript as detected in the U87MGvIII cell-line. U87MGvIII cells were used as a positive control, U87MG cells were used as negative control: water was used for no template control. B. Direct sequencing of U87MG and U87MGvIII cDNA confirms the presence of EGFRvIII transcript in U87MGvIII cells only. The broken arrow indicates the EGFRvIII-specific exon 1 and exon 8 junction. C. Linear dynamic range of the EGFRvIII real-time RT-PCR assay (100, 10, 1, 0.1 and 0.01 ng cDNA). D. EGFRvIII real-time RT-PCR assay amplification efficiency.
Figure 3
Figure 3. Conventional RT-PCR and direct cDNA sequencing confirm the presence of EGFRvIII in glioblastoma FFPE tissue.
A. Conventional RT-PCR failed for GBM 11, GBM 23 and GBM 19. GBM 9 is positive for EGFRvIII. EGFRvIII positive control: U87MGvIII (384 bp), negative control: U87MG (1184 bp) and no template control (NTC): water. B. Sequencing of GBM 9 cDNA confirms the presence of EGFRvIII. C. Real-time amplification plot showing EGFRvIII-positive OSCC patient (13A) and the positive control (U87MGvIII).
Figure 4
Figure 4. Immunofluorescent staining and quantitative analysis of EGFR protein expression in OSCC samples using the HistoRx AQUA® platform.
A. Representative examples of normal oral cavity squamous epithelium in the top panel, a low expressing tumor from an EGFRvIII-negative patient in the top-mid panel, and the EGFRvIII negative and EGFRvIIIitive samples from patient 13 in the bottom two panels. DAPI (blue) = nuclei, pan-cytokeratin (green) = epithelial/tumor cells, and EGFR (red). B. Histogram distribution of the EGFR AQUA scores for the entire OSCC patient cohort. The red and green lines indicate the EGFR scores for patient 13 who had both an EGFRvIII-positive (red, Sample ID 13A) and EGFRvIII-negative (green, Sample ID 13B) tumor sample. The solid blue line indicates the median EGFR AQUA score for normal oral cavity squamous epithelium, surrounded by the hashed blue lines representing one standard deviation above and below the median.

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