Fiorini Claudia, Massari Francesco, Pedron Serena, Sanavio Sara, Ciccarese Chiara, Porcaro Antonio Benito, Artibani Walter, Bertoldo Francesco, Zampini Claudia, Sava Teodoro, Ficial Miriam, Caliò Anna, Chilosi Marco, D'Amuri Alessandro, Sanguedolce Francesca, Tortora Giampaolo, Scarpa Aldo, Delahunt Brett, Porta Camillo, Martignoni Guido, Brunelli Matteo
Departments of Pathology and Diagnostic, University of Verona Italy.
Medical Oncology, Azienda Ospedaliera Universitaria Integrata di Verona AOUI, University of Verona Italy.
Am J Cancer Res. 2014 Nov 19;4(6):907-15. eCollection 2014.
Since target therapy with mTOR inhibitors plays an important role in the current management of clear cell renal cell carcinoma (RCC), there is an increasing demand for predictive biomarkers, which may help to select patients that are most likely to benefit from personalized treatment. When dealing with formalin-fixed paraffin-embedded (FFPE) cancer tissue specimens, several techniques may be used to identify potential molecular markers, yielding different outcome in terms of accuracy. We sought to investigate and compare the capability of three main techniques to detect molecules performing an active function in mTOR pathway in RCC. Immunohistochemistry (IHC), Western blot (WB) and immunofluorescence (IF) analyses were performed on FFPE RCC tissue specimens from 16 patients by using the following mTOR pathway-related: mTOR (Ser235/236), phospho-mTOR (p-mTOR/Ser2448), phospho-p70S6k (p-p70S6k/Thr389), both monoclonal and polyclonal, phospho-S6Rb (p-S6Rb) and phospho-4EBP1 (p-4EBP1/Thr37/46). No single molecule was simultaneously revealed by all three techniques. Only p-p70S6k was detected by two methods (IHC and IF) using a monoclonal antibody. The other molecules were detected exclusively by one technique, as follows: p-mTOR and polyclonal p-p70S6K by IHC, p70S6K, p-S6Rb and p-4EBP1 by WB, and, finally, mTOR by IF. We found significant differences in detecting mTOR pathway-related active biomarkers by using three common techniques such as IHC, WB and IF on RCC samples. Such results have important implications in terms of predictive biomarker testing, and need to be related to clinical end-points such as responsiveness to targeted drugs by prospective studies.
由于mTOR抑制剂的靶向治疗在目前透明细胞肾细胞癌（RCC）的治疗中发挥着重要作用，对预测性生物标志物的需求日益增加，这些生物标志物可能有助于选择最有可能从个性化治疗中获益的患者。在处理福尔马林固定石蜡包埋（FFPE）的癌症组织标本时，可以使用几种技术来识别潜在的分子标志物，在准确性方面会产生不同的结果。我们试图研究和比较三种主要技术检测RCC中mTOR通路中发挥活性功能分子的能力。通过使用以下与mTOR通路相关的物质：mTOR（Ser235/236）、磷酸化mTOR（p-mTOR/Ser2448）、磷酸化p70S6k（p-p70S6k/Thr389）（单克隆和多克隆）、磷酸化S6Rb（p-S6Rb）和磷酸化4EBP1（p-4EBP1/Thr37/46），对16例患者的FFPE RCC组织标本进行免疫组织化学（IHC）、蛋白质免疫印迹（WB）和免疫荧光（IF）分析。没有一个分子能被所有三种技术同时检测到。只有p-p70S6k通过使用单克隆抗体的两种方法（IHC和IF）检测到。其他分子仅通过一种技术检测到，如下所示：p-mTOR和多克隆p-p70S6K通过IHC检测，p70S6K、p-S6Rb和p-4EBP1通过WB检测，最后，mTOR通过IF检测。我们发现在RCC样本上使用IHC、WB和IF等三种常用技术检测mTOR通路相关活性生物标志物存在显著差异。这些结果在预测性生物标志物检测方面具有重要意义，并且需要通过前瞻性研究与临床终点（如对靶向药物的反应性）相关联。
