表皮生长因子受体变体 III 在口腔鳞状细胞癌中的特异性和灵敏性基于水解探针的实时 PCR 检测。

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

机构信息

Department of Pathology and Laboratory Medicine, Tom Baker Cancer Centre, University of Calgary, Calgary, Canada.

出版信息

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

DOI:10.1371/journal.pone.0031723

PMID:22359620

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC3280998/

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.

摘要

背景

肿瘤特异性 EGFR 缺失突变体 EGFRvIII 的特征是配体非依赖性组成型信号转导。表达 EGFRvIII 的肿瘤对目前的 EGFR 靶向治疗具有抗性。头颈部鳞状细胞癌 (HNSCC) 中 EGFRvIII 的频率存在争议，并且可能因特定的亚部位而异。本研究的目的是使用新型实时 PCR 检测评估特定 HNSCC 亚部位口腔鳞状细胞癌 (OSCC) 中 EGFRvIII 突变的发生。

方法

使用新型水解探针基于实时 PCR 检测评估了 50 例 OSCC 患者的预处理福尔马林固定石蜡包埋 (FFPE) 癌标本中 EGFRvIII 的存在。使用荧光免疫组化 (IHC) 和 AQUA®技术定量肿瘤样本中的 EGFR 蛋白表达。

主要发现

我们在我们的队列中检测到单个 OSCC 患者中存在 EGFRvIII（2%）。我们在独立的胶质母细胞瘤患者队列中证实了我们的检测技术的有效性。我们还比较了我们新型实时 EGFRvIII 检测与传统 RT-PCR 和直接测序的敏感性和特异性。我们的检测方法可以特异性检测 EGFRvIII，并能在 FFPE 肿瘤样本中区分野生型 EGFR。AQUAnalysis®显示 EGFRvIII 转录本的存在与非常高的 EGFR 蛋白表达（第 98 百分位数）相关。与之前的报告相反，在我们的研究中只有 44%的 OSCC 过度表达 EGFR。

结论和意义

我们的结果表明 EGFRvIII 突变在 OSCC 中很少见，并证实了之前关于 EGFRvIII 仅在 EGFR 过度表达的肿瘤中表达的报告。我们得出结论，EGFRvIII 特异性治疗可能不适合作为 OSCC 的一线治疗。此外，如这里描述的实时 RT-PCR 检测和 AQUAnalysis®等高度特异性和敏感的方法将提供 EGFR 突变频率和 EGFR 表达的准确评估，并将促进为 OSCC 患者选择最佳的靶向治疗。