基因突变检测技术在非小细胞肺癌表皮生长因子受体 与临床病理特征的关系研究中的作用

摘要：目的 探讨非小细胞肺癌（NSCLC）表皮生长因子受体（EGFR）基因突变情况及其与临床病理的关系。方法 选取我院2010年1月至2014年1月收集的159份非小细胞肺癌手术切除标本为研究对象,利用基因测序方法检测标本中的EGFR基因突变情况,并分析其与临床病理的关系。结果 159例样本中,EGFR基因突变检出率为12.6%(20/159),突变主要集中在19号外显子的缺失和21号外显子的点突变。女性患者基因突变检出率明显高于男性患者（P﹤0.01）。腺癌及细支气管肺泡癌患者基因突变检出率明显高于其他组织学分型（P﹤0.01）。高分化患者基因突变检出率高于中－低分化检出率（P＜0.05）。EGFR基因突变与年龄及淋巴结转移与否无关（P＞0.05）。结论 非小细胞肺癌患者EGFR基因突变与性别、组织学分型及分化程度密切相关。     

肺癌的表皮生长因子受体分子靶向治疗与基因突变

肺癌分子靶向治疗近年来取得较大进展,特别是针对表皮生长因子受体（EGFR）分子靶向药物表现出确定的临床效果。临床应用表明,EGFR基因酪氨酸激酶域体细胞突变与非小细胞肺癌患者对酪氨酸激酶抑制剂吉非替尼的敏感性相关,本文就相关的研究进行了简述。     

ARMS法检测非小细胞肺癌表皮生长因子受体基因突变及临床意义

目的：探讨应用突变特异性扩增系统法（ARMS）检测非小细胞肺癌（NSCLC）表皮生长因子受体（EGFR）基因突变的特点。方法：收集220例浙江南部地区NSCLC患者肿瘤样本,分别采用ARMS法和测序法检测EGFR基因18、19、20、21号外显子突变情况,并分析EGFR突变与病理类型、突变种类、患者年龄和性别的关系。结果：两方法比较,相符率为86．81％（158/182,Kappa=0．732,P〈0．01）。总突变率为47．27％（104/220）,其中腺癌的突变率明显高于鳞癌（52．46％zJs17．14％;P〈0．01）。肺腺癌中,女性患者EGFR突变率明显高于男性（65．56％∞39．78％;P〈0．01）。突变样本中,21外显子错义突变（L858R）所占比例最高（62．5％,65/104）,19外显子缺失（19Del）其次（43．27％,45/104）,其中两者同时突变占5．77％（6/104）;但腺癌女性与男性患者L858R突变率（64．41％∞56．76％）不存在显著性差别。结论：采用ARMS法检测EGFR基因突变较测序法敏感,突变主要发生在女性肺腺癌患者,以L858R突变为主。     

肺腺癌组织中表皮生长因子受体基因突变的检测及其临床意义

目的:通过检测肺腺癌组织中表皮生长因子受体(EGFR)基因的突变情况,研究EGFR突变与患者临床特征(性别、年龄和吸烟史)的相关性。方法:收集160例肺腺癌患者术前的石蜡组织标本,提取DNA后用实时定量PCR方法对EGFR基因18~21外显子进行突变检测;对基因突变结果与患者的性别、年龄和吸烟史分别做χ2检验。结果:160例肺腺癌组织标本中,有57例检测到EGFR基因突变,突变率为35.6%,且突变与患者性别、年龄和吸烟史均无显著相关性(P0.05);57例EGFR基因突变标本中,27例为19外显子缺失,22例为21外显子L858R点突变,这2种突变占总突变类型的85.96%,并且与患者性别、年龄及吸烟史无显著相关性(P0.05)。结论:EGFR基因在女性不吸烟肺腺癌中有较高的突变率,突变主要集中在19外显子缺失和21外显子L858R点突变,但突变率及突变类型与患者性别、年龄和吸烟史均不相关。     

基因突变检测技术在非小细胞肺癌EGFR与临床病理特征的关系研究中的作用

目的探讨非小细胞肺癌(NSCLC)表皮生长因子受体(EGFR)基因突变情况及其与临床病理的关系。方法选取我院2010年1月至2014年1月收集的159份非小细胞肺癌手术切除标本为研究对象,利用基因测序方法检测标本中的EGFR基因突变情况,并分析其与临床病理的关系。结果 159例样本中,EGFR基因突变检出率为12.6%(20/159),突变主要集中在19号外显子的缺失和21号外显子的点突变。女性患者基因突变检出率明显高于男性患者(P﹤0.01)。腺癌及细支气管肺泡癌患者基因突变检出率明显高于其他组织学分型(P﹤0.01)。高分化患者基因突变检出率高于中-低分化检出率(P0.05)。EGFR基因突变与年龄及淋巴结转移与否无关(P0.05)。结论非小细胞肺癌患者EGFR基因突变与性别、组织学分型及分化程度密切相关。     

晚期NSCLC患者血液EGFR基因检测研究进展

目的:随着表皮生长因子受体酪氨酸酶抑制剂(epidermal growth factor receptor-tyrosine kinase inhibitor,EGFR-TKI)在非小细胞肺癌(Non-Small Cell Lung Cancer,NSCLC)治疗中的应用,患者的生活质量及生存期均有很大程度的提高,但是,组织表皮生长因子受体(epidermal growth factor receptor,EGFR)检测结果为能否接受EGFR-TKI治疗的先决条件,而晚期肺癌患者却因组织量少、质量不佳、组织异质性无法进行检测,血液EGFR检测便应运而生,本研究将综述晚期非小细胞肺癌(Non-Small Cell Lung Cancer,NSCLC)患者血液EGFR基因检测研究。方法:检索Pub-med、SCI、Medline及中国生物文献数据库中晚期非小细胞肺癌患者血液EGFR基因检测的相关研究。结果:对于晚期NSCLC患者,血液EGFR基因检测有较高的敏感度与特异度,并且能够较好的预测患者对EGFR-TKI的疗效以及进行耐药监测。结论:当组织获取困难及质量不佳时,血液可替代组织行EGFR基因检测。     

表皮生长因子及受体在甲状腺肿瘤中的表达

目的:研究表皮生长因子(Epidermal Growth Factor,EGF)及受体(Epidermal Growth Factor Receptor,EGFR)及在甲状腺肿瘤中的表达。方法:应用免疫组织化学法检测91例甲状腺病变组织中EGFR和EGF的表达情况。结果:结节性甲状腺肿、甲状腺腺瘤、分化型甲状腺癌标本中EGFR表达的阳性率分别为15%、25%、68.62%,EGF表达的阳性率分别为10%、15%、68.62%,其中EGFR、EGF在分化型甲状腺癌与其余两组间差异均有统计学意义(P<0.05)。EGFR和EGF在甲状腺乳头状癌中的表达与性别、年龄、肿瘤大小、淋巴结转移、临床分期等临床因素无明显相关。结论:EGF和EGFR的表达可作为鉴别甲状腺肿瘤良恶性的一个指标。     

陕西省南部地区非小细胞肺癌EGFR基因突变分析

目的:探讨陕西南部非小细胞肺癌表皮生长因子受体基因的突变状况。方法:采用测序方法检测陕西省南部地区233例非小细胞肺癌(non-small cell lung cancer,NSCLCs)患者表皮生长因子受体(epithelial growth factor receptor,EGFR)基因第18、19、20和21号外显子突变情况,并分析其基因突变与肺癌人口学分布及组织类型的关系。结果:233例非小细胞肺癌患者中,共检出82例含有EGFR基因突变,其中第18、19、21号外显子突变率分别为1.3%、16.3%和18.0%,第20号外显子无突变;男性EGFR基因突变率(31.2%,39/125)低于女性(39.8%,43/108);腺癌EGFR基因突变率(39.1%,75/192)高于鳞癌(22%,9/41)。结论:陕西南部NSCLC的EGFR基因突变率较高,以第19、21号外显子突变为主。EGFR基因变率与NSCLC患者性别和病理类型均无关。     

抗人表皮生长因子受体单克隆抗体的制备、鉴定及初步应用

 用人上皮癌细胞系A 431细胞作为抗原免疫BalB/c小鼠,制备七株抗人表皮生长因子受体的单克隆抗体的杂交瘤,这些杂交瘤经三次亚克隆后仍能稳定地分泌单克隆抗体。对其中四株杂交瘤分泌的单克隆抗体进行了鉴定。免疫沉淀放射自显影结果示单克隆抗体3、101和176均可识别A 431细胞膜抗原MW为170000的蛋白质即EGF受体。单克隆抗体59可以识别低分化鼻咽癌细胞膜上EGF受体。单抗3、176和59等可抑制EGF与受体的特异结合,而101和94则不能抑制EGF与受体的结合。 用Protein-A Sepharose CL4B纯化了单抗,纯化的单抗主要为IgG_1亚类。用SDS聚丙烯酰胺凝胶电泳对纯化的单抗进行了纯度测定。     

Mechanisms for Kinase-mediated Dimerization of the Epidermal Growth Factor Receptor

We study a mechanism by which dimerization of the EGF receptor (EGFR) cytoplasmic domain is transmitted to the ectodomain. Therapeutic and other small molecule antagonists to the kinase domain that stabilize its active conformation, but not those that stabilize an inactive conformation, stabilize ectodomain dimerization. Inhibitor-induced dimerization requires an asymmetric kinase domain interface associated with activation. EGF and kinase inhibitors stimulate formation of identical dimer interfaces in the EGFR transmembrane domain, as shown by disulfide cross-linking. Disulfide cross-linking at an interface in domain IV in the ectodomain was also stimulated similarly; however, EGF but not inhibitors stimulated cross-linking in domain II. Inhibitors similarly induced noncovalent dimerization in nearly full-length, detergent-solubilized EGFR as shown by gel filtration. EGFR ectodomain deletion resulted in spontaneous dimerization, whereas deletion of exons 2–7, in which extracellular domains III and IV are retained, did not. In EM, kinase inhibitor-induced dimers lacked any well defined orientation between the ectodomain monomers. Fab of the therapeutic antibody cetuximab to domain III confirmed a variable position and orientation of this domain in inhibitor-induced dimers but suggested that the C termini of domain IV of the two monomers were in close proximity, consistent with dimerization in the transmembrane domains. The results provide insights into the relative energetics of intracellular and extracellular dimerization in EGFR and have significance for physiologic dimerization through the asymmetric kinase interface, bidirectional signal transmission in EGFR, and mechanism of action of therapeutics.     

Epidermal Growth Factor Receptor (EGFR) Signaling Requires a Specific Endoplasmic Reticulum Thioredoxin for the Post-translational Control of Receptor Presentation to the Cell Surface

The epidermal growth factor receptor (EGFR) is a well characterized receptor-tyrosine kinase that functions in development and serves a vital role in many human cancers. Understanding EGFR regulatory mechanisms, and hence approaches for clinical intervention, has focused on ligand-receptor interactions and tyrosine kinase activity. Here, we show using the NCI-H460 lung and A431 epidermoid human cancer cell lines that EGFR binding to anterior gradient homolog 2 (AGR2) in the endoplasmic reticulum is required for receptor delivery to the plasma membrane and thus EGFR signaling. Reduced AGR2 protein levels or mutation of an essential cysteine in the active site result in decreased cell surface EGFR and a concomitant decrease in signaling as reflected by AREG, EGR1, and FOS expression. Similar to previously described EGFR nulls, an AGR2 null also resulted in embryonic lethality. Consistent with its role in regulating EGFR-mediated signaling, AGR2 expression is also enhanced in many human cancers and promotes the transformed phenotype. Furthermore, EGFR-mediated signaling in NCI-H460 cells, which are resistant to the tyrosine kinase inhibitor AG1478, is also disrupted with reduced AGR2 expression. The results provide insights into why cancer prognosis or response to therapy often does not correlate with EGFR protein or RNA levels because they do not reflect delivery to the cell surface where signaling is initiated. AGR2, therefore, represents a novel post-translational regulator of EGFR-mediated signaling and a promising target for treating human cancers.     

Growth Differentiation Factor (GDF)-15 Blocks Norepinephrine-induced Myocardial Hypertrophy via a Novel Pathway Involving Inhibition of Epidermal Growth Factor Receptor Transactivation

Accumulating evidence suggests that growth differentiation factor 15 (GDF-15) is associated with the severity and prognosis of various cardiovascular diseases. However, the effect of GDF-15 on the regulation of cardiac remodeling is still poorly understood. In this present study, we demonstrate that GDF-15 blocks norepinephrine (NE)-induced myocardial hypertrophy through a novel pathway involving inhibition of EGFR transactivation. Both in vivo and in vitro assay indicate that NE was able to stimulate the synthesis of GDF-15. The up-regulation of GDF-15 feedback inhibits NE-induced myocardial hypertrophy, including quantitation of [³H]leucine incorporation, protein/DNA ratio, cell surface area, and ANP mRNA level. Further research shows that GDF-15 could inhibit the phosphorylation of EGF receptor and downstream kinases (AKT and ERK1/2) induced by NE. Clinical research also shows that serum GDF-15 levels in hypertensive patients were significant higher than in healthy volunteers and were positively correlated with the thickness of the posterior wall of the left ventricle, interventricular septum, and left ventricular mass, as well as the serum level of norepinephrine. In conclusion, NE induces myocardial hypertrophy and up-regulates GDF-15, and this up-regulation of GDF-15 negatively regulates NE-induced myocardial hypertrophy by inhibiting EGF receptor transactivation following NE stimulation.     

The F-BAR Protein PACSIN2 Regulates Epidermal Growth Factor Receptor Internalization

Signaling via growth factor receptors, including the epidermal growth factor (EGF) receptor, is key to various cellular processes, such as proliferation, cell survival, and cell migration. In a variety of human diseases such as cancer, aberrant expression and activation of growth factor receptors can lead to disturbed signaling. Intracellular trafficking is crucial for proper signaling of growth factor receptors. As a result, the level of cell surface expression of growth factor receptors is an important determinant for the outcome of downstream signaling. BAR domain-containing proteins represent an important family of proteins that regulate membrane dynamics. In this study, we identify a novel role for the F-BAR protein PACSIN2 in the regulation of EGF receptor signaling. We show that internalized EGF as well as the (activated) EGF receptor translocated to PACSIN2-positive endosomes. Furthermore, loss of PACSIN2 increased plasma membrane expression of the EGF receptor in resting cells and increased EGF-induced phosphorylation of the EGF receptor. As a consequence, EGF-induced activation of Erk and Akt as well as cell proliferation were enhanced in PACSIN2-depleted cells. In conclusion, this study identifies a novel role for the F-BAR-domain protein PACSIN2 in regulating EGF receptor surface levels and EGF-induced downstream signaling.     

Epidermal Growth Factor Receptor (EGFR) Signaling Regulates Global Metabolic Pathways in EGFR-mutated Lung Adenocarcinoma

Genetic mutations in tumor cells cause several unique metabolic phenotypes that are critical for cancer cell proliferation. Mutations in the tyrosine kinase epidermal growth factor receptor (EGFR) induce oncogenic addiction in lung adenocarcinoma (LAD). However, the linkage between oncogenic mutated EGFR and cancer cell metabolism has not yet been clearly elucidated. Here we show that EGFR signaling plays an important role in aerobic glycolysis in EGFR-mutated LAD cells. EGFR-tyrosine kinase inhibitors (TKIs) decreased lactate production, glucose consumption, and the glucose-induced extracellular acidification rate (ECAR), indicating that EGFR signaling maintained aerobic glycolysis in LAD cells. Metabolomic analysis revealed that metabolites in the glycolysis, pentose phosphate pathway (PPP), pyrimidine biosynthesis, and redox metabolism were significantly decreased after treatment of LAD cells with EGFR-TKI. On a molecular basis, the glucose transport carried out by glucose transporter 3 (GLUT3) was downregulated in TKI-sensitive LAD cells. Moreover, EGFR signaling activated carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD), which catalyzes the first step in de novo pyrimidine synthesis. We conclude that EGFR signaling regulates the global metabolic pathway in EGFR-mutated LAD cells. Our data provide evidence that may link therapeutic response to the regulation of metabolism, which is an attractive target for the development of more effective targeted therapies to treat patients with EGFR-mutated LAD.     

A Novel Glycoengineered Bispecific Antibody Format for Targeted Inhibition of Epidermal Growth Factor Receptor (EGFR) and Insulin-like Growth Factor Receptor Type I (IGF-1R) Demonstrating Unique Molecular Properties

In the present study, we have developed a novel one-arm single chain Fab heterodimeric bispecific IgG (OAscFab-IgG) antibody format targeting the insulin-like growth factor receptor type I (IGF-1R) and the epidermal growth factor receptor (EGFR) with one binding site for each target antigen. The bispecific antibody XGFR is based on the “knob-into-hole” technology for heavy chain heterodimerization with one heavy chain consisting of a single chain Fab to prevent wrong pairing of light chains. XGFR was produced with high expression yields and showed simultaneous binding to IGF-1R and EGFR with high affinity. Due to monovalent binding of XGFR to IGF-1R, IGF-1R internalization was strongly reduced compared with the bivalent parental antibody, leading to enhanced Fc-mediated cellular cytotoxicity. To further increase immune effector functions triggered by XGFR, the Fc portion of the bispecific antibody was glycoengineered, which resulted in strong antibody-dependent cell-mediated cytotoxicity activity. XGFR-mediated inhibition of IGF-1R and EGFR phosphorylation as well as A549 tumor cell proliferation was highly effective and was comparable with a combined treatment with EGFR (GA201) and IGF-1R (R1507) antibodies. XGFR also demonstrated potent anti-tumor efficacy in multiple mouse xenograft tumor models with a complete growth inhibition of AsPC1 human pancreatic tumors and improved survival of SCID beige mice carrying A549 human lung tumors compared with treatment with antibodies targeting either IGF-1R or EGFR. In summary, we have applied rational antibody engineering technology to develop a heterodimeric OAscFab-IgG bispecific antibody, which combines potent signaling inhibition with antibody-dependent cell-mediated cytotoxicity induction and results in superior molecular properties over two established tetravalent bispecific formats.     

A Pathophysiologic Role for Epidermal Growth Factor Receptor in Pemphigus Acantholysis

The pemphigus family of autoimmune bullous disorders is characterized by autoantibody binding to desmoglein 1 and/or 3 (dsg1/dsg3). In this study we show that EGF receptor (EGFR) is activated following pemphigus vulgaris (PV) IgG treatment of primary human keratinocytes and that EGFR activation is downstream of p38 mitogen-activated protein kinase (p38). Inhibition of EGFR blocked PV IgG-triggered dsg3 endocytosis, keratin intermediate filament retraction, and loss of cell-cell adhesion in vitro. Significantly, inhibiting EGFR prevented PV IgG-induced blister formation in the passive transfer mouse model of pemphigus. These data demonstrate cross-talk between dsg3 and EGFR, that this cross-talk is regulated by p38, and that EGFR is a potential therapeutic target for pemphigus. Small-molecule inhibitors and monoclonal antibodies directed against EGFR are currently used to treat several types of solid tumors. This study provides the experimental rationale for investigating the use of EGFR inhibitors in pemphigus.     

Destabilization of the Epidermal Growth Factor Receptor (EGFR) by a Peptide That Inhibits EGFR Binding to Heat Shock Protein 90 and Receptor Dimerization

An eight-amino acid segment is known to be responsible for the marked difference in the rates of degradation of the EGF receptor (ErbB1) and ErbB2 upon treatment of cells with the Hsp90 inhibitor geldanamycin. We have scrambled the first six amino acids of this segment of the EGF receptor (EGFR), which lies in close association with the ATP binding cleft and the dimerization face. Scrambling these six amino acids markedly reduces EGFR stability, EGF-stimulated receptor dimerization, and autophosphorylation activity. Two peptides were synthesized as follows: one containing the wild-type sequence of the eight-amino acid segment, which we call Disruptin; and one with the scrambled sequence. Disruptin inhibits Hsp90 binding to the EGFR and causes slow degradation of the EGFR in two EGFR-dependent cancer cell lines, whereas the scrambled peptide is inactive. This effect is specific for EGFR versus other Hsp90 client proteins. In the presence of EGF, Disruptin, but not the scrambled peptide, inhibits EGFR dimerization and causes rapid degradation of the EGFR. In contrast to the Hsp90 inhibitor geldanamycin, Disruptin inhibits cancer cell growth by a nonapoptotic mechanism. Disruptin provides proof of concept for the development of a new class of anti-tumor drugs that specifically cause EGFR degradation.     

紫草素逆转埃克替尼对肺癌EGFR-TKI 耐药及其机制的研究

目的:观察紫草素联合埃克替尼对肺腺癌耐药细胞H1975增殖的影响,探讨克服耐药可能的作用机制。方法:应用MTT法检测紫草素(1.25~20μmo/L)、埃克替尼(5~100μmol/L)及两药联合干预对H1975细胞生长的抑制作用;流式细胞术观察紫草素(1.25μmol/L)、埃克替尼(10μmol/L)及联合使用对H1975凋亡作用;Western blot检测不同干预对H1975细胞EGFR、p-EGFR、AKT、p-AKT、ERK、p-ERK和凋亡相关蛋白PARP表达水平的影响。结果:MTT检测结果显示,与单药组相比,联合用药组细胞H1975增殖能力明显减弱,差异有统计学意义(P0.05);流式细胞术结果显示,联合用药组细胞的凋亡率达到(52.45±3.04)%,较紫草素组细胞凋亡率(22±1.17)%和埃克替尼处理组细胞凋亡率(15.35±5.85)%明显提高,差异有统计学意义(P0.05)。Western blot结果显示,单药组下调了p-EGFR、p-Akt蛋白水平的表达,而联合用药组显著抑制了p-ERK、PARP蛋白水平的表达,差异有统计学意义(P0.05),EGFR、AKT、ERK蛋白表达无差异(P0.05)。结论:紫草素联合埃克替尼能明显抑制H1975细胞增殖,促进肿瘤细胞凋亡;抗肿瘤机制可能与调节EGFR信号通路相关蛋白表达有关。     

Meprinα Transactivates the Epidermal Growth Factor Receptor (EGFR) via Ligand Shedding,thereby Enhancing Colorectal Cancer Cell Proliferation and Migration

Meprinα, an astacin-type metalloprotease is overexpressed in colorectal cancer cells and is secreted in a non-polarized fashion, leading to the accumulation of meprinα in the tumor stroma. The transition from normal colonocytes to colorectal cancer correlates with increased meprinα activity at primary tumor sites. A role for meprinα in invasion and metastatic dissemination is supported by its pro-angiogenic and pro-migratory activity. In the present study, we provide evidence for a meprinα-mediated transactivation of the EGFR signaling pathway and suggest that this mechanism is involved in colorectal cancer progression. Using alkaline phosphatase-tagged EGFR ligands and an ELISA assay, we demonstrate that meprinα is capable of shedding epidermal growth factor (EGF) and transforming growth factor-α (TGFα) from the plasma membrane. Shedding was abrogated using actinonin, an inhibitor for meprinα. The physiological effects of meprinα-mediated shedding of EGF and TGFα were investigated with human colorectal adenocarcinoma cells (Caco-2). Proteolytically active meprinα leads to an increase in EGFR and ERK1/2 phosphorylation and subsequently enhances cell proliferation and migration. In conclusion, the implication of meprinα in the EGFR/MAPK signaling pathway indicates a role of meprinα in colorectal cancer progression.