Epidermal growth factor receptor (EGFR) high gene copy number and activating mutations in lung adenocarcinomas are not consistently accompanied by positivity for EGFR protein by standard immunohistochemistry

The purpose of this study was to investigate whether detectable protein biomarker overexpression is a prerequisite for the presence of increased gene copy number or activating mutations and responsiveness to the epidermal growth factor receptor (EGFR) inhibitors gefitinib and erlotinib in patients with lung adenocarcinomas. EGFR status was prospectively analyzed in tumor biopsy samples by three methods: protein expression (n = 117) by standardized immunohistochemistry (IHC), gene copy number (n = 97) by fluorescent in situ hybridization (FISH), and mutation analysis by sequencing (n = 126). Fifty-nine percent of the samples were positive by IHC, 40% were positive by FISH, and 13.5% contained activating kinase domain mutations. Thirty-four percent of the FISH-positive and 27% of the mutant samples were also IHC-negative. All EGFR mutant patients had major clinical responses (five complete response and five partial response) to gefitinib or erlotinib treatment, although three of these tumors were IHC-negative and four were FISH-negative. In a retrospective analysis of samples from nine patients with excellent therapeutic responses (three complete response, five partial response, one stable disease) to erlotinib or gefitinib, mutations were identified in eight cases, but IHC was negative in four of these tumors. These results indicate that molecular diagnostic methods appear to be most important for the identification of lung adenocarcinoma patients who may benefit from EGFR inhibitor treatments.     

肺癌EGFR的表达及意义

目的　探讨EGFR在肺癌中的表达及与癌组织分型、淋巴结转移等方面的关系。方法　应用免疫组化S P法分别对 86例肺癌组织、35例正常肺组织EGFR的表达进行检测。结果　肺癌组织EGFR的表达明显高于正常肺组织。EGFR在鳞癌与腺癌中的表达无显著差异。淋巴结转移阳性癌组织EGFR表达与淋巴结转移阴性癌组织比较 ,无明显差别。结论　EGFR参与了肺癌的发生发展。EGFR表达与鳞癌、腺癌分型无关 ,与淋巴结转移与否无关。     

表皮生长因子受体基因在非小细胞肺癌中的突变情况及蛋白表达情况

目的本研究旨在探讨非小细胞肺癌(NSCLC)患者中表皮生长因子受体(EGFR)基因突变的情况,分析EGFR蛋白表达情况对吉非替尼治疗的指导意义。方法收集140例非小细胞肺癌患者新鲜组织,采用荧光定量PCR法检测EGFR基因突变的状态;同时采用免疫组化SP法检测EGFR蛋白的表达。结果 140例患者中,46例患者发生了EGFR基因突变,其基因突变率为33%,主要发生在19号外显子(20例,43.5%)和21号外显子(23例,50.0%)。腺癌突变发生率为50.6%,腺鳞癌突变发生率为87.5%,鳞癌突变发生率为2.5%,大细胞癌突变发生率为5.3%(P=0.000),无吸烟史患者突变率为50%,有吸烟史患者的突变率为20.0%(P=0.005),女性患者突变率为50.0%,男性患者突变率为23.3%(P=0.001)。EGFR蛋白表达阳性组患者的中位无疾病进展生存时间为9个月(95%CI:7.3¹0.6个月),EGFR蛋白表达阴性组患者的中位无疾病进展生存时间为5个月(95%CI:3.510.6个月),EGFR蛋白表达阴性组患者的中位无疾病进展生存时间为5个月(95%CI:3.5⁶.4个月)。结论 EGFR基因突变和EGFR蛋白表达情况联合检测对于筛选EGFR酪氨酸激酶抑制剂(TKI)治疗敏感人群有帮助,并且也能够帮助预测吉非替尼对于晚期NSCLC的疗效及预后。     

非小细胞肺癌表皮生长因子受体基因突变和EML4-ALK融合基因表达的研究

目的研究广西地区非小细胞肺癌(NSCLC)表皮生长因子受体(EGFR)基因突变和棘皮动物微管相关类蛋白4与间变性淋巴瘤激酶(EML4-ALK)融合基因表达的情况。方法分别采用扩增耐突变系统(ARMS-PCR)法和实时荧光定量PCR扩增检测119例NSCLC患者EGFR基因外显子18~21的突变和9种EML4-ALK融合突变体,同时分析其与临床病理特征的关系。结果 119例NSCLC患者共检出44例EGFR基因突变,检出率为37.0%,检出7例EML4-ALK融合基因阳性表达,检出率为5.9%。其中EGFR基因突变主要见于腺癌、女性、不吸烟患者;EML4-ALK融合基因表达可能与不吸烟有关,而在性别、年龄、肿瘤病理类型、淋巴转移方面比较,差异无统计学意义(P0.05);EGFR基因突变与EML4-ALK融合基因不共存。结论广西地区NSCLC患者的EGFR基因突变率与EML4-ALK融合基因阳性表达率与中国其他地区总体水平接近,均具有一定临床病理特征。     

EGFR mutations and EGFR tyrosine kinase inhibition in non-small cell lung cancer

OBJECTIVES: To discuss selected molecular targets and new clinical variables that can serve as both predictive and prognostic markers for outcome in patients with non-small cell lung cancer (NSCLC) treated with EGFR-TKIs. DATA SOURCES: Research and journal articles. CONCLUSION: In the near future, treatment for NSCLC will rely ever increasingly on molecular targets rather than empirically chosen cytotoxic chemotherapy for some patients. This will improve outcomes for patients with NSCLC. IMPLICATIONS FOR NURSING PRACTICE: An understanding of the molecular targets and clinical variables that are predictive and prognostic of outcome in NSCLC will help nurses better care for these patients.     

非小细胞肺癌EGFR和KRAS基因突变和临床病理特征分析

目的 探讨非小细胞肺癌(NSCLC)中表皮生长因子受体(EGFR)和鼠类肉瘤病毒癌基因(KRAS)突变状态及其与临床病理特征的关系.方法 回顾性收集2019年1月至2021年6月首都医科大学大兴教学医院183例病理确诊NSCLC患者临床病理资料,采用扩增阻滞突变系统(ARMS)法检测肿瘤组织中EGFR和KRAS基因的突...     

非小细胞肺癌PD-1PD-L1表达与EGFR基因突变临床特征及预后的相关性

目的探讨非小细胞肺癌患者肺组织中程序性死亡受体-1、程序性死亡配体-1蛋白表达与表皮生长因子受体基因突变、临床特征及预后的相关性,为临床治疗提供参考.方法将117例非小细胞肺癌患者设为研究组,60例良性病例设为对照组,采用免疫组化法检测两组肺组织中程序性死亡受体-1、程序性死亡配体-1蛋白表达,采用荧光聚合酶链式反应法检测研究组表皮生长因子受体基因突变情况,对研究组不同临床特征患者的表皮生长因子受体基因突变及程序性死亡受体-1、程序性死亡配体-1蛋白表达进行分析.结果研究组肺组织中程序性死亡受体-1、程序性死亡配体-1蛋白阳性表达率(35.0%、54.7%)显著高于对照组(19.0%、15.5%)(P<0.05或0.01);研究组女性、高分化患者表皮生长因子受体基因突变率高,肿瘤分期Ⅲ期、Ⅳ期患者程序性死亡配体-1蛋白表达阳性率高,程序性死亡配体-1蛋白表达与表皮生长因子受体基因突变呈显著正相关,程序性死亡配体-1蛋白表达阳性较阴性患者的无进展生存期显著缩短(P<0.05或0.01).结论非小细胞肺癌患者肺组织中程序性死亡受体-1、程序性死亡配体-1蛋白表达阳性率高,程序性死亡配体-1蛋白表达与表皮生长因子受体基因突变显著相关;对表皮生长因子受体基因突变型非小细胞肺癌患者使用表皮生长因子受体的酪氨酸激酶抑制剂治疗,程序性死亡配体-1蛋白表达阳性患者可能预后较差.     

Rapid polymerase chain reaction-based detection of epidermal growth factor receptor gene mutations in lung adenocarcinomas

Somatic mutations in the tyrosine kinase domain of the epidermal growth factor receptor (EGFR) gene are present in lung adenocarcinomas that respond to the EGFR inhibitors gefitinib and erlotinib. Two types of mutations account for approximately 90% of mutated cases: short in-frame deletions in exon 19 and a specific point mutation in exon 21 at codon 858 (L858R). Screening for these mutations has been based mainly on direct sequencing. We report here the development and validation of polymerase chain reaction-based assays for these two predominant types of EGFR mutations. The assay for exon 19 mutations is based on length analysis of fluorescently labeled polymerase chain reaction products, and the assay for the exon 21 L858R mutation is based on a new Sau96I restriction site created by this mutation. Using serial dilutions of DNAs from lung cancer cell lines harboring either exon 19 or 21 mutations, we detected these mutations in the presence of up to approximately 90% normal DNA. In a test set of 39 lung cancer samples, direct sequencing detected mutations in 25 cases whereas our assays were positive in 29 cases, including 4 cases in which mutations were not apparent by sequencing. These assays offer higher sensitivity and ease of scoring and eliminate the need for sequencing, providing a robust and accessible approach to the rapid identification of most lung cancer patients likely to respond to EGFR inhibitors.     

HER-2/neu gene copy number quantified by real-time PCR: comparison of gene amplification,heterozygosity, and immunohistochemical status in breast cancer tissue

BACKGROUND: Amplification of the oncogene HER-2/neu influences breast cancer pathogenesis, and therapy and prognosis may be affected by the degree of amplification. The extent of amplification or protein overexpression typically is analyzed by fluorescence in situ hybridization or immunohistochemistry (IHC), but quantitative PCR techniques have been described that may provide alternatives to these methods. METHODS: We developed a rapid-cycle, real-time PCR assay for quantification of HER-2/neu gene status. We compared results obtained with this assay with short tandem repeat findings by capillary electrophoresis (CE) and with protein overexpression assessments by IHC. Accuracy and linearity were tested on cell lines and with simulation experiments. We analyzed the amplification of HER-2/neu in 51 clinical tissue samples from patients with suspected breast cancer. RESULTS: The intra- and interrun CVs for HER-2/neu quantification by real-time PCR were 12% and 18%, and the CV for different simulated amplification and deletion experiments was <7%. The results for HER-2/neu gene status in cell lines matched the values reported in literature. We detected HER-2/neu amplification by real-time PCR in 11 samples, all from patients with invasive ductal carcinoma. Allelic imbalances were found by CE analyses in three samples and by protein overexpression in six samples; five of these were also detected by real-time PCR. Comparison of the quantification results with known prognostic indices yielded results similar to those reported in several other published studies. CONCLUSIONS: The assay is suitable for accurate and precise quantification of HER-2/neu copy numbers in tumor tissue samples obtained in routine clinical practice.     

肺癌中EGFR、VEGF与NET-1的表达及与临床病理因素的关系

目的探讨表皮生长因子受体(EGFR)、血管内皮生长因子(VEGF)、NET-1在肺癌中的表达及其与临床病理因素的关系。方法采用免疫组织化学SP法,检测120例肺癌组织及10例正常肺组织(对照组)中EGFR、VEGF、NET-1的表达。结果 EGFR、VEGF、NET-1在肺癌中的阳性表达率(69.16%、55.83%、85.00%)明显高于正常肺组织(10.00%、0.00%、40.00%)。EGFR、VEGF、NET-1蛋白在肺癌中的表达差异与患者性别、年龄、病变部位无关(P均>0.05),但与肺癌组织的分化程度、临床分期、有无淋巴结转移相关(P<0.05)。结论 EGFR、VEGF、NET-1的阳性表达与肺癌的分化程度、临床分期、淋巴结转移有关,且在癌组织中表达强度显著高于正常组织,提示三者具有促进肿瘤细胞转化、增殖、迁移的作用,可作为临床判定肺癌恶性程度及评估预后的指标。     

Elevated epidermal growth factor receptor gene copy number and expression in a squamous carcinoma cell line.

The human epidermal growth factor (EGF) receptor is known to be homologous to the v-erb B oncogene protein of the avian erythroblastosis virus. Overexpression of the EGF receptor gene in A431 epidermoid carcinoma cells is due to gene amplification. In this study, a variety of squamous cell carcinomas were examined and one, SCC-15, contained high levels of the EGF receptor as determined by immunoprecipitation via an EGF receptor-specific polyclonal antibody. Using a cloned EGF receptor complementary DNA as a probe, the level of EGF receptor RNA was found to be elevated four-fold in SCC-15 relative to normal cultured keratinocytes. When the same probe was used to identify EGF receptor gene fragments on a genomic DNA blot, the SCC-15 cell line was shown to possess an EGF receptor gene copy number amplified four to five times. Gene amplification results in the enhancement in the level of the EGF receptor in several carcinomas and could be responsible for the appearance of the transformed phenotype in these cells.     

非小细胞肺癌中EGFR和K-ras基因突变检测分析与病理特征的关系

目的探讨不同分型非小细胞肺癌的EGFR与K-ras基因突变及其相关病理特征的关系。方法基因测序方法检测260例非小细胞肺肿瘤患者肿瘤组织中的EGFR(18,19,20,21外显子)和K-ras(12,13,61密码子)基因的突变。结果 260例样本中,EGFR基因突变检出率为48.8%(127/260),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。K-ras基因突变检出率为10%(26/260),其中12、13和61密码子均发现突变。受检的所有样本中没有发现EGFR与K-ras基因同时出现突变的情况。腺癌的EGFR突变检出率明显高于鳞状细胞癌等其他组织类型。在分化程度上,高分化与中分化癌基因突变检出率和中分化与低分化突变检出率比较差异显著(P<0.01)。EGFR突变与有无淋巴结转移无关(P>0.05)。K-ras基因突变与组织类型、患者性别、分化程度、有无淋巴结转移间无相关性。结论非小细胞肺癌EGFR基因突变检出率较高,K-ras基因突变检出率较低,且两种突变并不同时出现。EGFR基因突变与性别、肺癌组织类型、分化程度有关,存在多种突变。     

Limited lentiviral transgene expression with increasing copy number in an MGMT selection model: lack of copy number selection by drug treatment.

Retroviral vector integration into the human genome carries increased risk of oncogenesis with increasing integrations. To boost transgene expression for gene therapy, multiple integrations are often sought. We studied the relationship between the number of vector integrations and transgene expression and the effect that drug selection in an MGMT-selection model would have on vector copy number. K562 cells were transduced using a lentiviral vector and a library of clones was generated. Median proviral copy number was 4 and a positive correlation with transgene expression was observed. Transgene expression increased at a linear rate between 1 and 4 vector copies/cell, but was unpredictable at >4 integrations/cell. When lentivirus MGMT(P140K)-transduced K562 cells were treated with O(6)-benzylguanine (BG)/BCNU, there was no selection for increased median copy number in colony-forming units, despite strong selection pressure and an increase in transgene expression and activity. These data show a direct and linear correlation between MGMT(P140K) transgene expression and vector copy number. Strong BG/BCNU selective pressure does not result in preferential survival of high-copy-number clones but does select for strong transgene expression. Thus drug selection would not be expected to increase the risk of oncogenesis due to exaggerated selection in favor of high-copy-number vector integration.     

EGFR mutations in non-small cell lung cancer: an audit from West China Hospital

Objectives: To discover the incidence and characteristics of EGFR mutations in non-small cell lung cancer (NSCLC) in a single, large cohort as a part of routine diagnostic investigations.

Methods: We reviewed EGFR mutations investigated by Amplification Refractory Mutation System (ARMS) PCR (covering 29 known mutations) using DNA samples from FFPE tissue or cell clot specimens in a total of 3894 cases of NSCLC analysed between 2012-2014.

Results: EGFR mutations are preferentially associated with adenocarcinomaand adenosquamous histology, particularly those well to moderately differentiated, and were significantly more common in female than male patients irrespective of histological subtypes. Exon 19 deletion (45.7%) and exon 21 L858R (45.6%) accounted for the vast majority of the EGFR mutations detected, with the remaining mutations being infrequent (<2%). Compound mutations were seen in 51 (3%) of the mutant cases, the combination of these compound mutations could be classified into three subgroups according to the potential impact of individual mutations on EGFR TKI therapy. Accordingly, 7 cases had both sensitive mutations, 4 cases harboured one sensitive and one less responsive /uncertain mutation, 19 cases contained one sensitive and one resistant change, and a further 21 cases had two less responsive /uncertain mutations.

Conclusion: Our data represents the largest EGFR mutation survey based on routine clinical diagnostic laboratory data from a single institution, it confirms the incidence and characteristics of EGFR mutations in NSCLC seen in Asian patients, and also unravels the combinatorial nature of rare compound EGFR mutations.     

非小细胞肺癌患者p53基因突变检测的临床意义

目的探讨非小细胞肺癌（NSCLC）患者呼出气冷凝液（EBC）中p53基因突变检测的临床意义。方法采用巢式PCR结合DNA测序法,检测53例NSCLC患者EBC中p53基因第5～8外显子的突变情况,并以32例健康体检者EBC标本作为对照。结果肺癌组53例EBC标本中扩增到1）53基因26例,其中10例检测到p53基因突变,突变率为38．5％（10／26）;正常对照组32例EBC标本中扩增到p53基因15例,但未检测到p53基因突变。结论p53基因突变可能参与NSCLC的发生发展,检测EBC中p53基因的突变对肺癌的早期诊断有一定的价值。     

非小细胞肺癌组织及胸水中EGFR基因TKD结构域突变的临床病理研究

目的检测非小细胞肺癌（NSCLC）EGFR基因酪氨酸蛋白激酶结构域（TKD）18—21外显子突变情况;比较常见突变（19号外显子的缺失突变及21号外显子错义突变-L858R）和非常见突变患者的临床病理改变。方法从91例NSCLC组织及胸水标本中提取基因组DNA;巢式PCR方法扩增EGFR基因18～21外显子;应用PCR-LIS-SSCP及直接测序方法检测EGFR基因18～21外显子突变情况;对存在常见及非常见突变患者的组织病理、酪氨酸蛋白激酶小分子抑制剂（TKIs）的治疗反应和预后等的临床病理改变进行分析。结果91例NSCLC患者中27例存在EGFR基因TKD突变,其中常见突变19例,突变率为20．9％（19／91）;非常见突变8例（其中4例为20号外显子的短片段插入突变）,突变率为8．8％（8／91）。两组患者组织学类型均为腺癌,且对TKIs治疗均有较好反应。2例非常见突变患者化疗时出现肺间质纤维化（ILD）。结论NSCLC患者EGFR基因TKD存在多种非常见突变;具有非常见突变的患者可能存在独特的临床病理改变。