Department of Pathology, Chi-Mei Medical Center, Tainan, Taiwan.
Ann Surg Oncol. 2012 Feb;19(2):443-54. doi: 10.1245/s10434-011-1942-6. Epub 2011 Jul 15.
The understanding of epidermal growth factor receptor (EGFR) deregulation in carcinogenesis remains incomplete. We investigated the implications of EGFR gene status and EGFR nuclear translocation in gallbladder carcinoma (GBCA).
Subcellular localization of EGFR and phosphorylated EGFR (pEGFR) was analyzed by fractional immunoblotting and confocal immunofluorescence in GBCA cell lines. pEGFR binding to iNOS promoter was assessed by chromatin immunoprecipitation with iNOS promoter activity evaluated by luciferase assay. EGFR, pEGFR, and iNOS were immunohistochemically assessable for localization and level in the training set of 104 GBCAs on tissue microarrays, with 76 cases analyzed for EGFR gene by chromogenic in situ hybridization (CISH) and mutant-enriched PCR targeting exons 19 and 21. The prognostic impact of nuclear pEGFR (N-pEGFR) immunoexpression was reaffirmed on whole sections of 58 GBCAs in the test set.
Nuclear expression of EGFR and pEGFR was substantiated in vitro with augmented activity of iNOS promoter elicited by pEGFR binding upon EGF treatment. Despite no mutation, EGFR amplification, identified in 11 cases (15%) by CISH, strongly correlated with cytoplasmic EGFR expression (P < 0.001) but not with disease-specific survival (DSS). Immunoexpression of nuclear EGFR (N-EGFR), cytoplasmic pEGFR, and N-pEGFR was strongly related to that of iNOS (all ≤0.005). N-pEGFR independently predicted worse DSS in both training (P = 0.0468, HR = 2.024) and test sets (P = 0.0223, HR = 5.573).
N-EGFR and N-pEGFR express in GBCA, conferring clinical aggressiveness partly through iNOS transactivation. Lacking response-predicting mutation, EGFR gene status, albeit amplified in 15% of GBCA, is neither related to nuclear EGFR translocation nor prognostically useful.
表皮生长因子受体（EGFR）失调在癌变中的作用仍不完全清楚。本研究旨在探讨 EGFR 基因状态和 EGFR 核转位在胆囊癌（GBCA）中的意义。
通过免疫印迹和免疫荧光共聚焦技术分析 EGFR 和磷酸化 EGFR（pEGFR）在 GBCA 细胞系中的亚细胞定位。通过染色质免疫沉淀法检测 pEGFR 与 iNOS 启动子的结合，通过荧光素酶报告基因检测 iNOS 启动子活性。应用组织微阵列免疫组化法检测 104 例 GBCA 中 EGFR、pEGFR 和 iNOS 的定位和水平，76 例采用显色原位杂交（CISH）检测 EGFR 基因，11 例采用针对外显子 19 和 21 的突变富集 PCR 检测 EGFR 基因扩增。采用免疫组化法检测 58 例 GBCA 全切片核 pEGFR（N-pEGFR）的表达，验证 N-pEGFR 免疫表达的预后意义。
体外实验证实 EGFR 和 pEGFR 核表达，且 pEGFR 与 EGF 结合后可增强 iNOS 启动子的活性。尽管没有发现突变，但 CISH 检测到 11 例（15%）GBCA 存在 EGFR 扩增，与细胞质 EGFR 表达显著相关（P<0.001），但与疾病特异性生存无关（DSS）。核 EGFR（N-EGFR）、细胞质 pEGFR 和 N-pEGFR 的免疫表达与 iNOS 显著相关（均 P<0.005）。在训练集和测试集中，N-pEGFR 独立预测 DSS 更差（训练集 P=0.0468，HR=2.024；测试集 P=0.0223，HR=5.573）。
N-EGFR 和 N-pEGFR 在 GBCA 中表达，通过 iNOS 反式激活赋予其临床侵袭性。尽管 EGFR 基因状态在 15%的 GBCA 中扩增，但缺乏预测反应的突变，与核 EGFR 易位无关，也无预后意义。
