Figure 1.
CONSORT Diagram outlining patient selection criteria and the different stages at which specific analyses were undertaken for the OSCC study cohort.
Table 1.
Clinicopathological characteristics of OSCC patients evaluated for EGFR protein expression and EGFRvIII frequency.
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.
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.
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.