Comparison of EGFR mutation status between plasma and tumor tissue in non-small cell lung cancer using the Scorpion ARMS method and the possible prognostic significance of plasma EGFR mutation status

Background: The aims were to compare the consistency of epidermal growth factor receptor (EGFR) mutations in the plasma and tumor tissue of NSCLC patients, and to explore the prognostic significance of plasma EGFR mutation status in tyrosine kinase inhibitors (TKIs)-treated patients with tumor EGFR mutation. Methods: We evaluated EGFR gene (exons 18, 19, 20 and 21) mutation status in paired plasma and tumor tissue from 94 NSCLC patients before EGFR-TKIs treatments using the Scorpion amplification refractory mutation system (Scorpion-ARMS) method. Results: Our results demonstrated that the rates for EGFR mutations in 94 NSCLC patients were 20% (19/94, plasma samples) and 40% (38/94, tumor tissue samples), respectively. The consistency of EGFR mutations between plasma and tissue reached 80% (75/94, P<0.001). The sensitivity of tests using plasma samples was 50% (19/38) and the specificity was 100% (49/49) compared with tissue samples. 29 of the 38 patients were treated with TKIs. Among the 29 patients, 14 patients had EGFR mutations in both plasma and tumor tissue, and these patients had a significantly shorter overall survival (OS) than those with EGFR mutations in tumor tissue only by univariate analysis (P=0.019). Conclusions: Our data demonstrated the feasibility and potential utility of plasma cell-free DNA (cfDNA) as a source of specimens for EGFR mutation detection using the Scorpion ARMS method. Moreover, plasma EGFR mutation status before TKIs therapy might be of prognostic significance for TKIs-treated NSCLC patients with tumor tissue EGFR mutation.     

非小细胞肺癌患者肿瘤组织中EGFR和KARS基因突变的分子病理检测分析

目的：探讨非小细胞肺癌患者肿瘤组织中EGFR和KRAS基因各亚型突变情况。方法：应用直接测序方法检测非小细胞肺癌石蜡组织中1273例EGFR基因和1062例KRAS基因突变情况。结果：非小细胞肺癌肿瘤组织中EGFR基因总突变率为36.68%(467/1273),外显子18、19、20和21的突变率分别为1.02%(13/1273)、18.93%(241/1273)、2.59%(33/1273)和15.95%(203/1273);EGFR基因各外显子之间双重突变共17例(1.34%),其中18外显子与20外显子双重突变3例(0.24%),19外显子与20外显子双重突变7例(0.55%),19外显子与21外显子双重突变4例(0.31%)和20外显子与21外显子双重突变3例(0.24%);EGFR基因各外显子内双重突变共2例(2.18%),均为21外显子双重突变。KRAS基因总突变率为3.01%(32/1062),外显子2的密码子5、12、13和25的突变率分别为0.09%(1/1062)、2.64%(28/1062)、0.18%(2/1062)和0.09%(1/1062),外显子3密码子61的突变率为0.09%(1/1062)。结论：非小细胞肺癌患者中EGFR基因存在较高的突变率,尤其为19和21外显子突变,其基因突变亚型分类能指导EGFR-TKI的肿瘤靶向治疗,KRAS基因突变率虽低但不容忽视,其基因突变预示着EGFR-TKI原发耐药。     

恩度联合含铂类化疗方案治疗晚期非小细胞肺癌胸腔积液急性发作的临床疗效观察

目的：观察恩度联合含铂类化疗方案治疗晚期非小细胞肺癌胸腔积液急性发作的临床疗效。方法：选择2014年3月至2016年1月在我院接受治疗的晚期非小细胞肺癌胸腔积液急性发作患者100例,按照随机抽签的方式将其分为观察组和对照组,对照组患者给予单纯的含铂类化疗,观察组则给予恩度联合含铂类化疗,治疗结束后,观察两组的临床疗效。结果：治疗后,观察组的总缓解率为88%、生活质量总改善率为82%,明显的高于对照组的总缓解率56%、总改善率44%,两组比较差异具有统计学意义(P<0.05);治疗后,观察组胸水VEGF、HIF-1α水平分别为(334.7±81.4) pg/mL、(42.7±3.9) ng/L明显的低于对照组胸水VEGF水平(451.7±93.2) pg/mL、HIF-1α水平(48.9±3.2) ng/L,且观察组的肿瘤标记物水平明显的低于对照组,两组比较差异具有统计学意义(P<0.05);观察组在治疗期间总共发生69人次不良反应,对照组总共发生68人次不良反应,两组比较无显著差异(P>0.05)。结论：恩度联合含铂类化疗方案治疗晚期非小细胞肺癌胸腔积液急性发作具有显著的临床效果,有效地降低患者胸水VEGF、HIF-1α水平,且不会增加不良反应的发生。     

非小细胞肺癌胸水细胞块c-M ET基因扩增检测分析

目的：探讨非小细胞肺癌胸水细胞块在间充质上皮转化因子受体(c-mesenchymal-epithelial transition,c-MET)基因扩增检测中的临床价值。方法：采用RT-PCR法检测215例非小细胞肺癌细胞块和404例非小细胞肺癌组织块中c-MET基因扩增,并检测细胞块同时送检组织块的患者74例的一致性。结果：细胞块c-MET基因扩增31例,扩增率14.42%(31/215);组织块c-MET基因扩增35例,扩增率8.66%(35/404);74例有组织块对照的细胞块c-MET结果一致性有68例,一致率达91.89%(68/74),其中细胞块c-MET基因扩增率12.16%(9/74),组织块扩增率17.57%(13/74)。结论：非小细胞肺癌胸水细胞块c-MET的扩增率略高于组织块;有恶性胸水的非小细胞肺癌患者原发灶组织发生c-MET扩增的概率较高。     

肺腺癌胸水细胞块EGFR基因突变检测分析

目的探讨肺腺癌胸水细胞块中表皮生长因子受体(epidermal growth factor receptor,EGFR)基因突变检测的临床价值。方法采用ARMS-PCR法检测143例肺腺癌细胞块和269例肺腺癌组织块中EGFR的29种突变类型,并检测细胞块同时送检组织块的患者49例的一致性。结果细胞块EGFR突变型71例,阳性率49.65%(71/143);组织块EGFR突变型131例,阳性率44.26%(131/269);49例有组织块对照的细胞块EGFR结果一致性有36例,一致率达73.47%(36/49),其中细胞块EGFR的阳性率44.90%(22/49),组织块阳性率67.34%(33/49)。结论肺腺癌胸水细胞块EGFR的阳性率略高于组织块;有恶性胸水的肺腺癌患者原发灶组织发生EGFR突变的概率较高。     

Mig-6 overcomes gefitinib resistance by inhibiting EGFR/ERK pathway in non-small cell lung cancer cell lines

Non small cell lung cancer (NSCLC) accounts for 85% of all lung cancers and is the most common cause of lung cancer death. Currently, the epidermal growth factor receptor inhibitor gefitinib is widely used for patients with advanced NSCLC. However, drug resistance is a major obstacle. Mig-6 is a feedback inhibitor of EGFR and its down-stream pathway; it has been shown to play a role in gefitinib sensitivity. There is neither systematical research on the relationship between Mig-6 expression and gefitinib sensitivity, nor has the contribution of up-regulated Mig-6 on the gefitinib-resistant cell lines. In the present work, four NSCLC cell lines (H1299, A549, PC-9, and PC-9/AB11) with different sensitivities to gefitinib were subjected to analysis of the expression of Mig-6. We found that Mig-6 is over-expressed in gefitinib-sensitive NSCLC cell lines, but is low in gefitinib-resistant NSCLC cell lines. Further analysis revealed that over-expression of Mig-6 increased cell apoptosis and inhibited proliferation of gefitinib-resistant NSCLC cells treated with gefitinib, whereas lowering the expression of Mig-6 decreased cell apoptosis and promoted cell proliferation after treatment with gefitinib in gefitinib-sensitive NSCLC cell lines. These results suggest that Mig-6 is involved in mediating the response to gefitinib in NSCLC cell lines. Additionally we demonstrated that Mig-6 could reverse gefitinib resistance through inhibition of EGFR/ERK pathway in NSCLC cells. Our work uncovered that Mig-6 may be an effective therapeutic target in gefitinib-resistant lung cancer patients.     

Distinct profile of cell-free DNA in malignant pleural effusion of non-small cell lung cancer and its impact on clinical genetic testing

Cell-free DNA (cfDNA) in supernatant of pleural effusion from advanced NSCLC patients has been proved as surrogate sample detecting therapeutic targets as well as tumor mutation burden (TMB). As recently reported, cfDNA in pleural effusion supernatant is superior to plasma in TMB evaluation. It is reasonable to hypothesize that cfDNA profile in pleural effusion (PE) and plasma might be different. It remains to be elucidated why cfDNA in PE supernatant impacts on genetic analysis. Consequently, the approach dealing with cfDNA from PE supernatant might need to be different from that for plasma cfDNA in order to obtain accurate clinical genetic testing result.Methods: Pleural effusion samples from 32 patients with stage IV lung adenocarcinoma were collected. Supernatant and sediment were processed separately to extract Cell-free DNA as well as sediment DNA (PE-S). cfDNA from pleural effusion was analyzed by Agilent 2100 bioanalyzer. Libraries were prepared by 1) direct use of the total cfDNA without fragmentation step (PE-FL) or 2) use of full-length cfDNA fragmented to 150-250bp (PE-F), 3) use of cfDNA fragments enriched to ~167bp (PE-E167) as well as 4) use of cfDNA fragments larger than 500bp enriched (PE-E500). All samples were subjected to targeted next-generation sequencing (NGS) with a panel of 448 cancer-related genes as well as a panel of 10 NSCLC driver genes.Results: cfDNA were successfully extracted from 30 MPE samples. cfDNA displayed distinct profile in supernatant of malignant pleural effusion from that of plasma cfDNA. No statistical difference in detection of hotspot variations between PE-E167 and PE-F by 448-gene or 10-gene panel. While TMB from PE-F samples was significantly higher than that from PE-E167 and PE-FL. Higher TMB from PE-F was resulted from cancer-unspecific variants with low allele frequency (0.1%-1%) which were mainly introduced by long-fragment cfDNA. Similar genetic profile was observed between paired cfDNA of PE-FL and cfDNA of PE-E167.Conclusion: Long-fragment cfDNA in the PE supernatant will introduce low abundant cancer unrelated variants which leads overestimation of TMB. Paired PE-FL and PE-E167 gave comparable outcomes. Direct use of the total cfDNA without fragmentation step (PE-FL) is recommended for library preparation of NGS testing in clinical practice to exclude interference from long fragments of the cfDNA.     

非小细胞肺癌患者ALK,EGFR及KRAS基因的检测及临床病理特征

目的:研究汉族非小细胞肺癌患者中间变性淋巴瘤激酶(anaplastic lymphoma kinase,ALK)、表皮生长因子受体(epidermal growth factor receptor,EGFR)及Kirsten鼠肉瘤基因(Kirstenrat sarcoma,KRAS)突变的阳性率及其与临床病理特征的关系.方法:采用免疫组织化学(immunohistochemistry,IHC)的方法检测ALK融合基因异常表达,采用PCR检测EGFR基因和KRAS基因突变,采用x2检验及Fisher精确概率法进行数据分析.结果:共2 267例进行了ALK融合基因检测,其中1 655例同时进行了EGFR突变检测,951例同时进行了KRAS检测.ALK融合基因、EGFR基因及KRAS基因突变阳性率分别为7.28％(165/2 267)、48.58％(804/1 655)、11.40％(108/947).ALK基因突变多见于年轻、腺癌患者;EGFR基因突变多见于女性、腺癌患者;KRAS基因突变多见于老年、男性、腺癌患者.1 655例同时进行了ALK与EGFR检测的病例中,共6例存在双基因突变(0.36％);947例同时进行了ALK与KRAS检测,共4例存在双基因突变(0.42％);943例同时进行了EGFR与KRAS突变检测,未发现双突变病例.结论:非小细胞肺癌患者ALK,EGFR和KRAS基因的突变与患者的年龄、性别、组织学类型均存在相应的联系,个别病例可以出现ALK融合基因与EGFR或KRAS突变共存.     

Detection of low-level EGFR T790M mutation in lung cancer tissues

Epidermal growth factor receptor (EGFR) gene mutation status is critical to predicting responsiveness to EGFR tyrosine kinase inhibitor (TKI) therapies in non-small cell lung cancer (NSCLC) patients. However, a vast majority of the patients experience recurrence of the cancers by a secondary mutation of EGFR (T790M). Earlier studies suggested evidence that subclones bearing EGFR T790M mutation pre-exist in NSCLCs even prior to the therapies. However, to date, the status of T790M mutation in primary NSCLC is largely known. In this study, we developed an assay using peptide nucleic acid (PNA)-clamping PCR for detection of low-level EGFR T790M mutation. We found that the assay showed the highest sensitivity (0.01% mutation detection) in the clamping condition. We analyzed 147 NSCLC tissues [70 adenocarcinomas (AD), 62 squamous cell carcinomas (SQ), 12 large cell carcinomas (LC), and three adenosquamous carcinomas] that had not been exposed to the TKI therapies, and found 12 (8.2%; 12/147) EGFR T790M mutation in eight AD (11.4%), three SQ (4.8%), and one LC (8.3%) by the PNA-clamping PCR. However, this mutation was not detected by conventional DNA sequencing. Our data indicate that EGFR T790M exists in pretreatment NSCLC at low levels irrespective of histologic types. This study provides a basis for developing an applicable protocol for detecting low-level EGFR T790M mutation in primary NSCLC, which might contribute to predicting recurrence of the tumor in response to the TKI therapies.     

A comparison of ARMS and mutation specific IHC for common activating EGFR mutations analysis in small biopsy and cytology specimens of advanced non small cell lung cancer

We have compared mutation analysis by Amplification Refractory Mutation System (ARMS) and epidermal growth factor receptor (EGFR) mutant-specific antibodies for their ability to detect two common activating EGFR mutations in a cohort of 115 advanced non-small cell lung cancer (NSCLC), including cytology material, core biopsy, and bronchoscopic biopsies. Assessment of EGFR mutation status was performed by using antibodies and ARMS assay specific to the two major forms of mutant EGFR, exon 19 deletion E746-A750 (c.2235_2249del15 or c.2236_2250del15, p. Glu746_Ala750 del) and exon 21 L858R point mutation (c.2573T>G, p.Leu858Arg). In this study the optimal buffer for antigen retrieval was sodium citrate (pH 6.0). Q score was used to evaluate the specific mutant EGFR proteins expression. Validation using clinical material showed deletions in exon 19 were detected in 19.1% and L858R mutation in 20% of all cases by ARMS assay. A cutoff value of score 1 was used as positive by IHC. No wild type cases were immuno-reactive. The antibodies performed well in cytology, core biopsies and bronchoscopic biopsies. There were only one false positive case using L858R IHC (sensitivity 100%, specificity 98.5%, positive predictive value 96%, negative predictive value 100%). All 23 E746-A750 exon 19 deletions identified by mutation analysis were positive by IHC. The sensitivity of exon 19 IHC for E746-A750 was 100%, specificity 100%, positive predictive value 100% and negative predictive value 100%. The result of the IHC stains was finely correlated with mutations status determined by ARMS assay. Although inferior to molecular genetic analysis of the EGFR gene, IHC is highly specific and sensitive for the targeted EGFR mutations. The antibodies are likely to be of clinical value in cases especially where limited tumor material is available, or in situations where molecular genetic analysis is not readily available.     

Kinase switching in mesenchymal-like non-small cell lung cancer lines contributes to EGFR inhibitor resistance through pathway redundancy

NSCLC cells with a mesenchymal phenotype have shown a marked reduction in sensitivity to EGFR inhibitors, though the molecular rationale has remained obscure. Here we find that in mesenchymal-like tumor cells both tyrosine phosphorylation of EGFR, ErbB2, and ErbB3 signaling networks and expression of EGFR family ligands were decreased. While chronic activation of EGFR can promote an EMT-like transition, once having occurred EGFR family signaling was attenuated. We investigated the mechanisms by which mesenchymal-like cells bypass EGFR signaling and acquire alternative routes of proliferative and survival signaling. Mesenchymal-like NSCLC cells exhibit aberrant PDGFR and FGFR expression and autocrine signaling through these receptors can activate the MEK-ERK and PI3K pathways. Selective pharmacological inhibition of PDGFR or FGFR receptor tyrosine kinases reduced cell proliferation in mesenchymal-like but not epithelial NSCLC cell lines. A metastable, reversible EMT-like transition in the NSCLC line H358 was achieved by exogenous TGFβ, which served as a model EMT system. The H358/TGFβ cells showed many of the attributes of established mesenchymal-like NSCLC cells including a loss of cell-cell junctions, a loss of EGF-family ligand expression, a loss of ErbB3 expression, increased EGFR-independent Mek-Erk pathway activation and reduced sensitivity to EGFR inhibition. Notably an EMT-dependent acquisition of PDGFR, FGFR and TGFβ receptors in H358/TGFbeta cells was also observed. In H358/TGFbeta cells both PDGFR and FGFR showed functional ligand stimulation of their intrinsic tyrosine kinase activities. The findings of kinase switching and acquired PDGFR and FGFR signaling suggest investigation of new inhibitor combinations to target NSCLC metastases.     

Detection of EGFR mutations in archived cytologic specimens of non-small cell lung cancer using high-resolution melting analysis

Mutations of the epidermal growth factor receptor (EGFR), particularly deletional mutations (DEL) in exon 19 and L858R in exon 21, are reportedly correlated with clinical outcome in patients with non-small cell lung cancer (NSCLC) receiving the EGFR tyrosine kinase inhibitors gefitinib and erlotinib, suggesting that detection of EGFR mutations would have an important role in clinical decision making. We established and validated an easy, inexpensive, and rapid method for detecting DEL and L858R from cytologic material by high-resolution melting analysis (HRMA). Dilution for sensitivity studies revealed that DEL and L858R were detectable in the presence of at least 10% and 0.1% EGFR-mutant cells, respectively. We analyzed 37 archived cytological slides of specimens from 29 patients with advanced NSCLC and compared the results with direct sequencing data obtained previously. Of 37 samples, 34 (92%) yielded consistent results with direct sequencing, 2 were false negative, and 1 was indeterminate. The sensitivity of this analysis was 90% (19/21) and specificity, 100% (15/15). These results suggest that HRMA of archived cytologic specimens of advanced NSCLC is useful for detecting EGFR mutations in clinical practice.     

Antigenic specificity and subset analysis of T cells isolated from the bronchoalveolar lavage and pleural effusion of patients with lung disease.

Cellular infiltrates of bronchoalveolar lavage (BAL) and pleural effusion from patients with tuberculosis (TB) and lung cancer were characterized for the presence of different T cell subsets by phenotypic analysis. The specificity of the T cells for mycobacterial antigens was then compared for the two disease compartments. The composition of T cell subsets within the BAL, in contrast to pleural effusion cells (PEC), revealed evidence of sequestration of CD8+ cells. BAL T cells were found to be a predominantly CD29+ DR+ memory population of activated cells. Although polyclonal populations of BAL T cells proliferated poorly to Mycobacterium tuberculosis antigens, mycobacterial antigen-reactive monoclonal T cell populations could be derived from the alveolar compartment. Two clones were shown to recognize the 65-kD heat shock protein of mycobacteria, and one of these clones recognized a conserved sequence of the molecule. Several BAL-derived clones, responding to a mycobacterial soluble extract, did not, however, recognize purified mycobacterial antigens, previously identified as highly stimulatory for PEC-derived T cells. T cell clones, derived from PEC of two TB patients, responded to the 38-kD and 71-kD, as well as the 65-kD mycobacterial antigens. Examination of the activation requirements of BAL-derived T cell clones, specific for mycobacterial antigens, revealed that exogenous IL-2 was necessary for the T cells to sustain proliferation. This was in contrast to the mycobacterial antigen-reactive T cells cloned from PEC. These results suggest that T cell populations with distinct antigen specificities and activation requirements are present in BAL and PEC.     

EGFR 19-Del上调Fn14及JAK/STAT的表达促进非小细胞肺癌的发生发展

目的：本研究检测Fn14、p-JAK1、p-STAT1在EGFR 19-Del的非小细胞肺癌(non-small cell lung cancer,NSCLC)细胞系中的表达,并探讨EGFR 19-Del对Fn14及JAK1/STAT1表达的调控作用,以期为EGFR 19-Del之NSCLC的发展机制研究奠定基础。方法：采用EGFR TKI(吉非替尼)处理HCC827细胞系(EGFR 19-Del)、H1975(L858R)及H292细胞系(正常肺上皮细胞),Western blot法检测Fn14、p-JAK1、p-STAT1蛋白表达。结果：相比于H1975和H292细胞系,Fn14、p-JAK1及p-STAT1基因和蛋白在HCC827细胞系中有较高的表达水平。对比未经吉非替尼处理的HCC827细胞系,经抑制剂处理后的HCC827细胞系中Fn14、p-JAK1、p-STAT1蛋白表达明显受到抑制,而在H292细胞系中无此现象。由此说明,EGFR 19-Del对Fn14及JAK/STAT信号分子的表达具有调控作用。结论：EGFR 19-Del可能通过上调Fn14及JAK/STAT信号分子的表达促进NSCLC的发生发展,而Fn14可能是EGFR 19-Del之NSCLC潜在的治疗靶点。     

Cytological features of lung adenocarcinoma with micropapillary pattern in the pleural or pericardial effusion: analysis of 5 cases

Micropapillary pattern is a distinct histopathological pattern, and usually shows a high frequency of lymphatic invasion and lymph node metastases. This pattern is also reported in lung adenocarcinoma, however, only one cytological report of lung adenocarcinoma with micropapillary pattern has been reported. In this study, we analyzed the cytological features of this type of carcinoma in the pleural or pericardial effusion. This study was comprised of 5 consecutive cases of lung adenocarcinoma with micropapillary pattern, in which the tumor cells were present in the pleural or pericardial effusion and whose diagnoses were histopathologically confirmed. The characteristic cytological findings in the pleural or pericardial effusion were as follows: i) tightly cohesive small nests of tumor cells showing papillary structure without fibrovascular core, ii) these nests were comprised of approximately 5-20 tumor cells, iii) cauliflower-like and acinar-like structures were also observed, iv) intracytoplasmic vacuoles were observed in 40% of the cases, and v) the neoplastic cells had large round to oval nuclei containing coarse chromatin and occasional conspicuous nucleoli. It has been reported that the presence of micropapillary structure and intracytoplasmic vacuolation are also characteristic cytological features of micropapillary carcinoma of the urinary bladder, therefore, they are thought to be common cytological features of carcinomas with micropapillary pattern. Consequently, detection of these features can lead to a cytodiagnosis of lung adenocarcinoma with micropapillary pattern in the pleural or pericardial effusion. Recognition of these features is important because this type of tumor shows an aggressive clinical course.     

非小细胞肺癌中EGFR基因突变与ERCC1、Ki-67表达及临床病理特征的关系

目的：探讨非小细胞肺癌(non-small cell lung cancer, NSCLC)中EGFR基因突变与切除修复交叉互补基因1(excision re-pair cross-complementation group 1, ERCC1)、Ki-67蛋白表达及其临床意义。方法采用DNA测序法检测EGFR基因突变,免疫组化EnVision法检测ERCC1、Ki-67蛋白表达,分析与临床病理特征的关系。结果 EGFR 基因突变率为49．1%(143/291),多见于女性、不吸烟、腺癌患者。 EGFR基因突变在腺癌的不同亚型中差异有显著性(P=0．008);EGFR基因突变组肿瘤直径的中位数小于野生型组(P=0．020);EGFR基因突变与患者年龄、淋巴结有无癌转移无相关性(P>0．050);鳞状细胞癌中ERCC1阳性率高于腺癌(P=0．039)。 Ki-67表达与肺癌分化程度有关,低分化高于高、中分化(P=0．010);ERCC1、Ki-67表达与EGFR基因突变无相关性(P>0．050)。结论 EGFR基因突变与NSCLC患者性别、组织学类型、分化程度等相关,存在多种突变。 EGFR基因突变与ERCC1、Ki-67表达无相关性。     

非小细胞肺癌EGFR基因突变与ERCC1、RRM1、TUBB3 mRNA表达相关性研究

目的：探讨非小细胞肺癌(non-small cell lung cancer,NSCLC)患者表皮生长因子受体(epidermal growth factor receptor,EGFR)基因突变与切除修复交叉互补蛋白1(excision repair cross-complementation 1,ERCC1)、核苷酸还原酶亚单位M1(ribonucleotide reductase subunit M1,RRM1)及3型β-微管蛋白基因(classⅢβ-tubulin,TUBB3)表达的关系。方法：回顾性分析我院经病理诊断的69例NSCLC患者的手术切除的肿瘤标本,利用扩增受阻突变系统(amplification refractory mutation system,ARMS)进行EGFR基因突变检测,应用实时荧光定量PCR检测ERCC1、RRM1及TUBB3 mRNA的表达水平。结果：69例患者中,EGFR突变率为33.33%(23/69),在性别、吸烟史、病理类型组间突变率有显著性差异。ERCC1低、高表达率分别为59.42%(41/69)、40.58%(28/69),EGFR突变与ERCC1表达呈负相关性,EGFR无突变时ERCC1趋向于高表达(P=0.024);RRM1低、高表达率分别为31.88%(22/69)、68.12%(47/69),EGFR突变与RRM1表达无相关性(P>0.05);TUBB3低、高表达率分别为8.70%(6/69)、91.30%(63/69),EGFR突变与TUBB3表达无相关性(P>0.05)。ERCC1、RRM1、TUBB3三者间的基因表达均无相关性。结论：NSCLC患者肿瘤组织中EGFR突变患者ERCC1倾向低表达,这类患者可能更能从铂类化疗和靶向药物中受益。     

Next‐generation sequencing of Chinese stage IV lung cancer patients reveals an association between EGFR mutation status and survival outcome

Large‐scale genomic characterization of non‐small cell lung cancer (NSCLC) has revealed several putative oncogenic driver mutations that may constitute druggable therapeutic targets. However, there are little data to suggest that such gene alterations have clinical relevance. Over 12 consecutive months, tumor biopsy samples from 80 patients with stage IV NSCLC were analyzed for mutations in selected exons of 508 cancer‐related genes using next‐generation sequencing. From 85 specimens referred for genomic characterization, 80 (94%) specimens were successfully genotyped, and all had identifiable somatic alterations. Epidermal growth factor receptor (EGFR) and TP53 genes contained the highest frequency of observed mutations (65% and 40%, respectively) in the stage IV NSCLC cases. Notably, patients with EGFR mutations showed a significantly shorter survival time compared with patients expressing wild‐type EGFR (p = 0.0053). Moreover, of the 32 patients harboring EGFR mutations, EGFR‐L858R mutant patients showed a significantly shorter survival time compared with patients with other EGFR mutations (p = 0.036). In conclusion, tumors from stage IV NSCLC patients harbor characteristic gene alterations, of which EGFR L858R in particular appears to be a poor prognostic factor for overall survival.