【作者】 尹光浩；

【导师】 刘国津；

【作者基本信息】 吉林大学 ， 外科学， 2009， 博士

【摘要】 肺癌已成为导致死亡的最主要的恶性肿瘤,尽管治疗方法有所改进,但是肺癌的5年生存率并没有得到明显的改善。肺癌中非小细胞肺癌(non-small cell lung cancer NSCLC)比率为80%,大部分NSCLC很早就有远处转移,平均生存期比较短。表皮生长因子受体(epidermal growth factor receptor,EGFR)是分子量为170kDa的酪氨酸激酶,含有一个胞外配体结合结构,一个跨膜结构和一个具有酪氨酸激酶活性的胞浆结构。EGFR信号转导途径在肿瘤细胞的增殖、细胞凋亡、新生血管形成、细胞粘附力和活性有关,而且对肿瘤进展起到非常重要的作用。EGFR酪氨酸激酶抑制剂(tyrosine kinase inhibitor TKI)具有显著的抗肿瘤作用。现已证实EGFR的基因突变可作为预测对EGFR-TKI疗效敏感的指标,已经发现EGFR-TKI的治疗疗效与性别、病理类型及吸烟史相关。EGFR基因突变的检测是通过PCR和基因测序方法,该方法操作复杂,无法作为日常临床中的常规检查。有研究发现在EGFR-TKI敏感的细胞株发生EGFR磷酸化,EGFR的磷酸化水平在野生型和突变型中的比较具有差异。目前关于EGFR基因突变与磷酸化EGFR关系的研究较少,国内尚无研究报道。本研究目的检测NSCLC磷酸化EGFR与EGFR基因突变及临床特征的关系。在130例手术切除的NSCLC中用免疫组化方法检测EGFR和EGFR 2个酪氨酸磷酸化位点(pEGFR-tyr1045和pEGFR-tyr1068)的表达,并采用PCR扩增和基因测序检测EGFR基因突变情况,研究磷酸化EGFR与EGFR基因突变及临床特征的关系。结果发现EGFR基因突变与性别、病理类型和吸烟史有关,而且pEGFR-tyr1045和pEGFR-tyr1068与EGFR基因突变有关,pEGFR-tyr1045和pEGFR-tyr1068能够预测EGFR基因突变情况,可以预测EGFR-TKI治疗疗效。更多还原

【Abstract】 Background: Lung cancer has become the most common cause of cancer death. Despite strong therapeutic efforts, the overall 5-year survival rate of patients with lung cancer is only 15% without having improved over the last 30 years. Non-small cell lung cancer (NSCLC) accounts for approximately 80% of lung cancers, NSCLC is thought to originate in lung epithelial cells, and comprises diverse histological subtypes including adenocarcinoma, bronchioloalveolar, squamous and large-cell carcinomas. Most patients with advanced NSCLC present with metastatic disease and, if left untreated, have a median survival after diagnosis of 4-5 months and a year survival of less than 10%.EGFR is a 170 kDa tyrosine kinase receptor consisting of an extracellular domain, a transmembrane domain, and a cytoplasmic domain comprised of the tyrosine kinase domain. Ligand binding results in receptor dimerization, autophosphorylation of specific tyrosine residues, and activation of downstream signaling. The EGFR signal regulates proliferation, apoptosis, angiogenesis, cell adhesion, and motility, and therefore the receptor has a great impact on tumour growth and progression.EGFR is an important target for cancer therapy, these mutations endow the receptor with ligand-independent catalytic activity and are oncogenic in a number of model systems. Most tumors harboring EGFR kinase domain mutations are dependent on them as EGFR tyrosine kinase inhibitors induce a marked antitumor effect. Gefitinib has been widely used in the treatment of NSCLC as tyrosine kinase inhibitor (EGFR-TKI). To date, EGFR mutation has been thought to be the most sensitive predictor of gefitinib response and is correlated with gender, histology and smoking history. The observation of higher response rates with EGFR-TKI in selected groups of patients, as well as the disappointing results with EGFR-TKI in unselected patients, it is very important to study the potential predictive value in patients treated with EGFR-TKI.Studies have showed that gefitinib was effective in 80-90% of patients with EGFR mutations, and in 10-20% of patients with wild-type EGFR. If we rely solely on gene mutation in gefitinib treatment as predictor, will make part of patients who respond to gefitinib missed opportunities. So screening for the treatment of EGFR-TKI-sensitive patients is particularly important, although the gene mutation as the current predictor of EGFR-TKI treatment, but still need to improve. In a recent study, found that about half of the tumor of lung cancer have expression of EGFR phosphorylation, EGFR phosphorylation, not only in the expression of EGFR mutations in lung cancer, but also exist in the wild-type EGFR of lung tumor. Some studies found that the level of phosphorylation of the EGFR mutant cell line was comparable with the level of phosphorylation in wild-type cells.Objective: To study the correlation between phosphorylated EGFR and EGFR mutation in resected non-small cell lung cancers.Methods: DNAs were extracted from 130 cases paraffin embedded NSCLC tissues and the exon 18, 19, 21 of EGFR were sequenced to detect the EGFR mutation status; the expression of EGFR and phosphorylated EGFR at tyrosine 1045 and 1068 (pEGFR-tyr1045 and pEGFR-tyr1068) were also studied in total 130 cases NSCLC by immuohistochemical method. Examined the correlations between EGFR mutation and phosphorylated EGFR..Results: EGFR mutation (Exon 18,19,21) were seen in 29.2% (38/130) of 130 NSCLCs, In adenocarcinoma, 38.7% (29/75) of patients had EGFR mutation. 21.3% (17/80) of males and 42.0% (21/50) of females had EGFR mutation. 40.3% (25/62) of patients who had never smoked and 19.1% (13/68) of smokers had the EGFR mutation. Furthermore, adenocarcinoma, female and non-smoking were significantly correlated to the EGFR mutations (P<0.05). There is no correlations were found between EGFR mutations and patient’s age, tumor size, nodal status, and clinical stage (P>0.05).Expression of EGFR were positive in 67.7% (88/130) of all patients, 78.9% (30/38) of patients had EGFR mutation , 76.0% (38/50) of females ,72.0% (54/75) of patients with adenocarcinoma , and 74.2%(46/62)of patients who had never smoked were positive of EGFR expression. No correlations were found between EGFR expression and these characteristics(P>0.05), But EGFR expression was correlated to pEGFR-tyr1045 and pEGFR-tyr1068 (P<0.05).Expression of pEGFR-tyr1045 were positive in 51.5% (67/130) of patients, 73.7%(28/38) of patients had EGFR mutation, 64.0%(32/50)of females, and 64.0% (48/75) of patients with adenocarcinoma were pEGFR-tyr1045 positive, the correlations between pEGFR- tyr1045 and these characteristics were significant(P<0.05).Expression of pEGFR-tyr1068 were positive in 55.4% (72/130) of patients, 76.3%(29/38) of patients had EGFR mutation, 70.0%(35/50)of females, and 66.7%(50/75)of patients with adenocarcinoma, 64.5%(40/62)of patients who had never smoked were pEGFR-tyr1068 positive, pEGFR-tyr1068 was significantly correlated with EGFR mutation, gender, histology and smoking history (P<0.05).Conclusions: Statistical analysis of these results revealed that the presence of EGFR mutations was significantly correlated with gender, histology and smoking history, pEGFR-tyr1045 and pEGFR-tyr1068 were significantly correlated with EGFR mutation, pEGFR-tyr1045 and pEGFR-tyr1068 can predict EGFR mutation, and they may be a sensitive predictor of EGFR-TKI response.It has been reported that some patients develop adverse effect of gefitinib treatment, therefore, careful judgment in balancing the potential benefits and toxicity is required, and a useful method for predicting the responsiveness to EGFR-TKI has been awaited. Our immunohistochemical diagnosis of EGFR phosphorylation status at tyrosine residue 1045 and 1068 appears to have a number of advantages over mutation analysis of the EGFR gene. First, immunohistochemistry with anti–pEGFR-tyr1045 and 1068 antibody is easier to perform than mutation analysis of the EGFR gene. Second, immunohistochemistry facilitates direct identification of the tumor cells in which EGFR is phosphorylated, whereas in mutation analysis, contamination with the genomic DNA of normal cells cannot be ruled out. Third, our method is less invasive for patients, as paraffin-embedded tissue samples taken from patients for histopathologic diagnosis can be reused for immunohistochemical analysis, obviating the need for another biopsy sample for mutation analysis.10-20% of wild-type EGFR in NSCLC cancer patients sensitive to TKI treatment, but these patients with gene mutation as an indicator of prediction gefitinib treatment, may be excluded from the treatment of TKI-sensitive patients. Although phospho-EGFR analysis might not be superior to sequence analysis as a predictive marker of TKI response, however, detection of phosphorylated EGFR can supplement the omission of screening, and immunohistochemistry of EGFR is a conveninent tool for predicting the presence of EGFR gene mutation, and can be applied to daily clinical practice examination.更多还原