Specific and Sensitive Hydrolysis Probe-Based Real-Time PCR Detection of Epidermal Growth Factor Receptor Variant III in Oral Squamous Cell Carcinoma

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

Polymorphisms in the methylenetetrahydrofolate reductase (MTHFR) gene, intakes of folate and related B vitamins and colorectal cancer : a case-control study in a population with relatively low folate intake

Peer reviewedPublisher PD

Operationalizing the 2018 World Cancer Research Fund/American Institute for Cancer Research (WCRF/AICR) Cancer Prevention Recommendations: A Standardized Scoring System

Background: Following the publication of the 2018 World Cancer Research Fund (WCRF) and American Institute for Cancer Research (AICR) Third Expert Report, a collaborative group was formed to develop a standardized scoring system and provide guidance for research applications. Methods: The 2018 WCRF/AICR Cancer Prevention Recommendations, goals, and statements of advice were examined to define components of the new Score. Cut-points for scoring were based on quantitative guidance in the 2018 Recommendations and other guidelines, past research that operationalized 2007 WCRF/AICR Recommendations, and advice from the Continuous Update Project Expert Panel. Results: Eight of the ten 2018 WCRF/AICR Recommendations concerning weight, physical activity, diet, and breastfeeding (optional), were selected for inclusion. Each component is worth one point: 1, 0.5, and 0 points for fully, partially, and not meeting each recommendation, respectively (Score: 0 to 7–8 points). Two recommendations on dietary supplement use and for cancer survivors are not included due to operational redundancy. Additional guidance stresses the importance of accounting for other risk factors (e.g., smoking) in relevant models. Conclusions: The proposed 2018 WCRF/AICR Score is a practical tool for researchers to examine how adherence to the 2018 WCRF/AICR Recommendations relates to cancer risk and mortality in various adult populations.

Operationalizing the 2018 World Cancer Research Fund/American Institute for Cancer Research (WCRF/AICR) Cancer Prevention Recommendations: A Standardized Scoring System

Background: Following the publication of the 2018 World Cancer Research Fund (WCRF) and American Institute for Cancer Research (AICR) Third Expert Report, a collaborative group was formed to develop a standardized scoring system and provide guidance for research applications. Methods: The 2018 WCRF/AICR Cancer Prevention Recommendations, goals, and statements of advice were examined to define components of the new Score. Cut-points for scoring were based on quantitative guidance in the 2018 Recommendations and other guidelines, past research that operationalized 2007 WCRF/AICR Recommendations, and advice from the Continuous Update Project Expert Panel. Results: Eight of the ten 2018 WCRF/AICR Recommendations concerning weight, physical activity, diet, and breastfeeding (optional), were selected for inclusion. Each component is worth one point: 1, 0.5, and 0 points for fully, partially, and not meeting each recommendation, respectively (Score: 0 to 7–8 points). Two recommendations on dietary supplement use and for cancer survivors are not included due to operational redundancy. Additional guidance stresses the importance of accounting for other risk factors (e.g., smoking) in relevant models. Conclusions: The proposed 2018 WCRF/AICR Score is a practical tool for researchers to examine how adherence to the 2018 WCRF/AICR Recommendations relates to cancer risk and mortality in various adult populations.

Specific detection of <i>EGFRvIII</i> mRNA by real-time RT-PCR.

<p>A. Real-time amplification plot of <i>EGFRvIII</i> 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 <i>EGFRvIII</i> transcript in U87MGvIII cells only. The broken arrow indicates the <i>EGFRvIII</i>-specific exon 1 and exon 8 junction. C. Linear dynamic range of the <i>EGFRvIII</i> real-time RT-PCR assay (100, 10, 1, 0.1 and 0.01 ng cDNA). D. <i>EGFRvIII</i> real-time RT-PCR assay amplification efficiency.</p

Conventional RT-PCR and direct cDNA sequencing confirm the presence of <i>EGFRvIII</i> in glioblastoma FFPE tissue.

<p>A. Conventional RT-PCR failed for GBM 11, GBM 23 and GBM 19. GBM 9 is positive for <i>EGFRvIII</i>. 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 <i>EGFRvIII</i>. C. Real-time amplification plot showing EGFRvIII-positive OSCC patient (13A) and the positive control (U87MGvIII).</p