Detection of EGFR mutation in supernatant,cell pellets of pleural effusion and tumor tissues from non-small cell lung cancer patients by high resolution melting analysis and sequencing

To determine epidermal growth factor receptor (EGFR) mutation in advanced non-small cell lung cancer (NSCLC) patients and compare the detection efficiency between different sample resources, both high resolution melting (HRM) analysis and direct sequencing method were used to analyze 36 pleural effusion samples and 22 matched biopsy tumor tissues collected from NSCLC patients. For each pleural effusion sample, the supernatant and the cell pellets were examined separately. Among all the 36 cases of pleural effusion samples, 18 mutations of EGFR were found in cell-free supernatant while 13 mutations were found in the cell pellets as detected by HRM analysis. In the 22 matched samples, 13 cases of EGFR mutations were identified in paraffin-embedded biopsy tissue samples, 12 cases in the cell-free supernatant and 9 cases in the cell pellets of pleural effusion. EGFR mutations in 15 cases out of the total 36 pleural effusion samples detected by direct sequencing were also identified by HRM analysis, giving 100% efficiency for HRM method. The results established the important role of HRM as a reliable and efficient method to determine EGFR mutation status and indicated the feasibility of using pleural effusion in replacement of biopsy tissues in particular clinical cases. Furthermore, the cell-free supernatant of pleural effusion might be a better resource for mutation detection than cell pellets.     

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。     

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.     

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.     

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.     

Microvessel count predicts metastatic disease and survival in non-small cell lung cancer

The growth of newly formed vessels, or neoangiogenesis, represents an important step in both physiological and pathological situations: in particular, tumour growth and metastasis require angiogenesis. Microvessel count (MC), which represents a measure of tumour angiogenesis, has been associated with metastatic spread in cutaneous, mammary, prostatic, head and neck, and early-stage lung cancer. In this study, the role of tumour angiogenesis as a prognostic indicator was examined in 253 primary non-small cell lung cancer (NSCLC) patients. Microvessels were counted by highlighting endothelial cells with anti-Factor VIII monoclonal antibody (MAb) in methacarn-fixed tumour samples. In univariat analysis, MC (P<0·000001), sex (P=0·0036), histotype (P<0·014), tumour status (P<0·007), and vessel invasion (P<0·019) were significantly related to hilar and/or mediastinal nodal involvement. However, in the stepwise logistic regression analysis, MC (P<0·000003) retained the most important influence on nodal metastasis. The overall survival analysis calculated by the Kaplan-Meier method revealed that tumours with high MC (>25 vessels/field) were significantly associated with increased death risk (log-rank test P=0·00067; Cox's test P=0·00046; Gehan's Wilcoxon test P=0·00108). In 94 patients, the development of metastatic disease during follow-up was significantly related to MC. Indeed, patients who developed metastasis during follow-up showed a higher MC, either as a dichotomous (P=0·01) or as a continuous (P=0·003) variable, than patients who had developed no metastasis at the time of the analysis. Moreover, in the stepwise logistic regression analysis, MC retained the most important influence on distant metastases. Microvessel count, as a method for the quantitation of tumour angiogenesis, has an important prognostic role in non-small cell lung carcinomas.     

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.     

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。     

MicroRNA-153 expression and prognosis in non-small cell lung cancer

Background: miR-153 has been found to be significantly decreased in non-small cell lung cancer (NSCLC) tissues; however, its clinical significance has not been investigated. Methods: The expression patterns of miR-153 in 137 pairs of human lung cancer tissues and adjacent normal lung tissues were analyzed using qRT-PCR. The relationships between miR-153 expression and clinicopathological parameters were examined by chi-square test. Kaplan-Meier method and the log-rank test were used to determine the difference in overall survival (OS) rates between two groups. Results: The expression of miR-153 was reduced significantly, compared with adjacent normal lung tissues (P<0.05). We observed that the expression level of miR-153 was positively correlated with the clinical stage (P=0.005), lymph node status (P=0.014), distant metastasis (P=0.004), and differentiated degree (P<0.001) in NSCLC patients. According to the Kaplan-Meier survival analysis, the patients with low miR-153 expression exhibited evidently poorer overall survival rates than those with high miR-153 expression (P=0.003). Multivariate analysis showed that the expression of miR-153 was an independent and significant factor associated with poor OS rates (P=0.002). Conclusion: Decreased expression of miR-153 might be a potential unfavorable prognostic factor for patients with NSCLC, and further studies would be needed to prove our findings.     

Reduced circulating exosomal miR-382 predicts unfavorable outcome in non-small cell lung cancer

Circulating microRNAs (miRNAs) have been demonstrated as robust and promising biomarkers for non-small cell lung cancer (NSCLC). Our aim was to determine the significance of serum exosomal miR-382 in NSCLC. Circulating exosomes were collected from 126 patients with NSCLC and 60 normal controls before treatment and one month after surgery. The circulating exosomal miR-382 expression was measured with quantitative RT-PCR (qRT-PCR) in all the participants. Our findings demonstrated that circulating exosomal miR-382 was very reduced in NSCLC. In addition, it showed high accuracy for discriminating NSCLC patients from healthy subjects. Interestingly, serum exosomal miR-382 improved the diagnostic accuracy of carcinoembryonic antigen (CEA). Moreover, its level increased significantly one month following surgical resection. Reduced circulating exosomal miR-382 was positively associated with poor clinical variables. NSCLC cases with lower serum exosomal miR-382 suffered worse overall survival (OS) and serum exosomal miR-382 was independently associated with OS. Taken together, circulating exosomal miR-382 is a robust biomarker for evaluating the progression of NSCLC.     

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。     

非小细胞肺癌中Naked1的表达及意义

目的探讨Naked1(NKD1)蛋白在非小细胞肺癌(non-small cell lung cancer,NSCLC)组织中的表达与预后的关系。方法采用RT-PCR、Western blot法对35例新鲜肺癌组织进行检测;采用免疫组化SP法对100例NSCLC石蜡标本进行检测。结果在79%的新鲜肺癌标本中,NKD1的蛋白水平明显低于相对应的癌旁正常肺组织,而mRNA水平明显高于正常肺组织(P<0.01)。在78%的NSCLC免疫组化标本中,NKD1的表达明显低于正常肺组织(P<0.01)。在肺鳞癌中NKD1的表达低于腺癌,并与分化程度呈正相关(P<0.01),与淋巴结转移(P<0.05)和TNM分期(P<0.01)呈负相关,NKD1低表达者的术后生存时间明显低于正常表达者(P=0.01)。结论在NSCLC中,NKD1 mRNA上调,而蛋白水平下调,其蛋白的下调程度与临床病理因素和不良预后相关。     

非小细胞肺癌中Frat1与Axin表达的相关性及意义

目的近年来肺癌的发病率及死亡率逐年升高,寻找新的靶向治疗方向的要求日益迫切。作为Wnt通路中的一个新成员,Frat1在肺癌组织中的表达情况以及Frat1与Axin在肺癌组织中表达相关性的研究甚少。方法用免疫组化SP法对120例原发性非小细胞肺癌(NSCLC)患者手术标本中Frat1和Axin的表达进行检测,使用SPSS 16.0统计数据。结果在NSCLC中Frat1的阳性表达与肺癌组织的低分化(P=0.004)、淋巴结转移(P=0.033)、TNM分期相关(P=0.039);Axin的异常表达与肺癌组织的低分化(P=0.001)、淋巴结转移(P=0.036)、TNM分期相关(P=0.043);Frat1的阳性表达与Axin的异常表达有相关性(P=0.025)。结论 Frat1和Axin在NSCLC中与肺癌的低分化、转移、高级别TNM分期等恶性表型有关,Frat1和Axin在癌组织中的表达有相关性,Frat1可能成为非小细胞肺癌患者靶向治疗的新靶点。     

Expression and prognostic significance of cyclin B1 and cyclin A in non-small cell lung cancer

Aims:  Aberrant expression of cell cycle regulators has been implicated in the pathogenesis of many neoplasms, including non-small cell lung cancer (NSCLC). The aim was to examine the expression and prognostic value of cyclin B1 and cyclin A, key regulators of the G₂/M checkpoint of the cell cycle, in NSCLC and bronchial precursor lesions.
Methods and results:  Immunohistochemical expression of cyclin B1 and A was examined in 90 cases of stage I–II primary NSCLC and bronchial precursor lesions using tissue microarrays. Increased cyclin B1 and A expression was found in 40.9 and 58.9% of NSCLC cases, respectively, and was significantly higher in primary NSCLC, lymph node metastases and some bronchial precursor lesions compared with normal bronchial epithelium. Increased expression of cyclin A and cyclin B1 correlated with tumour type, poorly differentiated tumours and male gender. A significant association was found between increased cyclin B1 expression and reduced survival using Kaplan–Meier survival analysis. On multivariate analysis, cyclin B1 was not an independent prognostic factor ( P  = 0.067). Cyclin A expression was not associated with survival.
Conclusions:  Cyclin B1 and cyclin A are aberrantly expressed in NSCLC and some precursor lesions. Cyclin B1, but not cyclin A, shows some promise as a potential prognostic marker in NSCLC.     

Clinicopathological significance of PTEN and PI3K/AKT signal transduction pathway in non-small cell lung cancer

A high frequency of mutations at the PTEN locus has been noticed in carcinoma of lung. However, the role of PTEN alternations and its association with outcome variables in the genesis of lung carcinoma are not understood fully. The purpose of our study was to examine the impact of EGFR, TGF-α, P-AKT and PTEN in the genesis of non-small cell lung cancer (NSCLC). Total numbers of 66 histopathologically confirmed cases of NSCLC and 10 cases of benign control samples embedded with wax were studied. We assessed EGFR, TGF-α and P-AKT by the use of specific antibody through immunohistochemistry as directed by the manufacturer, and detected PTEN expression by in situ hybridization. There were progressive loss of PTEN expression and significant increasing in EGFR, TGF-α, P-AKT expression from benign samples to NSCLC (p<0.05). The overexpression of EGFR, TGF-α, P-AKT and loss of PTEN expression were correlated to differentiation extent of cancer tissue, metastasis of lymph nodes and histological classification. Thus, alteration of EGFR, TGF-α, P-AKT and PTEN are likely important molecular events in pathogenesis and carcinogenesis of NSCLC.     

Prognostic assessment of apoptotic gene polymorphisms in non-small cell lung cancer in Chinese

Apoptosis plays a key role in inhibiting tumor growth, progression and resistance to anti-tumor therapy. We hypothesized that genetic variants in apoptotic genes may affect the prognosis of lung cancer. To test this hypothesis, we selected 38 potentially functional single nucleotide polymorphisms (SNPs) from 12 genes (BAX, BCL2, BID, CASP3, CASP6, CASP7, CASP8, CASP9, CASP10, FAS, FASLG and MCL1) involved in apoptosis to assess their prognostic significance in lung cancer in a Chinese case cohort with 568 non-small cell lung cancer (NSCLC) patients. Thirty-five SNPs passing quality control underwent association analyses, 11 of which were shown to be significantly associated with NSCLC survival (P < 0.05). After Cox stepwise regression analyses, 3 SNPs were independently associated with the outcome of NSCLC (BID rs8190315: P = 0.003; CASP9 rs4645981: P = 0.007 and FAS rs1800682: P = 0.016). A favorable survival of NSCLC was significantly associated with the genotypes of BID rs8190315 AG/GG (adjusted HR = 0.65, 95% CI: 0.49-0.88), CASP9 rs4645981 AA (HR = 0.22, 95% CI: 0.07-0.69) and FAS rs1800682 GG (adjusted HR = 0.67, 95% CI: 0.46-0.97). Time-dependent receptor operation curve (ROC) analysis revealed that the area under curve (AUC) at year 5 was significantly increased from 0.762 to 0.819 after adding the risk score of these 3 SNPs to the clinical risk score. The remaining 32 SNPs were not significantly associated with NSCLC prognosis after adjustment for these 3 SNPs. These findings indicate that BID rs8190315, CASP9 rs4645981 and FAS rs1800682 polymorphisms in the apoptotic pathway may be involved in the prognosis of NSCLC in the Chinese population.