Association of Matrix Metalloproteinase-9 and p53 Gene Polymorphisms with Genetic Susceptibility to No-small-cell Lung Cancer

Matrix metalloproteinase-9(MMP-9)and p53 genes play an essential role in the multi-step process of tumorigenesis in lung cancer.Single nucleotide polymorphisms(SNPs)of MMP-9 and p53 genes are associated with the risk and progression of many cancers.In this study,we evaluated the association of the R279Q polymorphism of MMP-9 or the A1/A2 polymorphism of p53 gene with the risk of no-small-cell lung cancer(NSCLC)in Hart population of Northeast China.We examined the frequency of SNPs in the two kinds of genes of 50 patients with NSCLC and 50 cancer-free controls frequency-matched by age and sex.Polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP)technique was used to determine the genotypes.The results indicate that the 279RR genotype in MMP-9 gene and the A1/A2 genotype in p53 gene show a significantly increased risk of NSCLC.Therefore,the MMP-9 279RR and p53 A1/A2 genotypes may be used as markers for susceptibility to NSCLC in Han population of Northeast China.     

非小细胞肺癌患者ERCC1表达与铂类药物化疗敏感性的相关性的系统分析

目的: 分析非小细胞肺癌患者切除修复交叉互补基因1(ERCC1)表达与对铂类药物治疗敏感性的相关性。方法: 电子检索Medline(1991.1-2009.12)、Pubmed、CBMDisc等数据库,对回顾性病例研究和随机对照临床试验进行总结分析。结果: 共纳入10篇文献,包括9篇回顾性病例研究和1篇随机对照临床试验。9篇回顾性病例研究资料中,7篇结果表明ERCC1表达阴性患者对铂类药物的联合化疗方案的反应率高于阳性患者(2篇结果具有统计学意义),另2篇ERCC1表达阴性患者对铂类药物的联合化疗方案的反应率低于阳性患者;1篇文献报道化疗后肿瘤进展时间ERCC1阴性组患者显著长于ERCC1阳性组患者(P<0.05);化疗后中位生存期具有统计学差异的2篇报道均为ERCC1阴性组高于ERCC1阳性组 (P<0.05)。RCT研究结果表明辅助化疗可以明显延长ERCC1阴性患者的生存期,但不能延长ERCC1阳性患者的生存期。结论: 非小细胞肺癌患者中ERCC1低表达者可以从铂类化疗方案中受益,高表达者对铂类药物的化疗敏感性差,需要更好的辅助治疗方案。ERCC1作为预测NSCLC对铂类药物化疗方案敏感性的指标并指导临床个体化治疗具有临床意义。     

Disruption of Cytosolic Folate Integrity Aggravates Resistance to Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors and Modulates Metastatic Properties in Non-Small-Cell Lung Cancer Cells

Patients with advanced-stage non-small-cell lung cancer (NSCLC) are susceptible to malnutrition and develop folate deficiency (FD). We previously found that folate deprivation induces drug resistance in hepatocellular carcinoma; here, we assessed whether disrupted cytoplasmic folate metabolism could mimic FD-induced metastasis and affect the sensitivity of NSCLC cells to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs). We examined whether cytosolic folate metabolism in NSCLC cells was disrupted by FD or the folate metabolism blocker pemetrexed for 1–4 weeks. Our results revealed an increase in NF-κB overexpression–mediated epithelial-mesenchymal transition biomarkers: N-cadherin, vimentin, matrix metalloproteinases (MMPs), SOX9, and SLUG. This finding suggests that the disruption of folate metabolism can drastically enhance the metastatic properties of NSCLC cells. Cytosolic FD also affected EGFR-TKI cytotoxicity toward NSCLC cells. Because SLUG and N-cadherin are resistance effectors against gefitinib, the effects of SLUG knockdown in folate antagonist–treated CL1-0 cells were evaluated. SLUG knockdown prevented SLUG/NF-κB/SOX9-mediated invasiveness and erlotinib resistance acquisition and significantly reduced pemetrexed-induced gelatinase activity and MMP gene expression. To summarize, our data reveal two unprecedented adverse effects of folate metabolism disruption in NSCLC cells. Thus, the folic acid status of patients with NSCLC under treatment can considerably influence their prognosis.     

鼠抗人间变性淋巴瘤激酶单抗及其试剂盒的制备及鉴定

目的制备并鉴定鼠抗人间变性淋巴瘤激酶(anaplastic lymphoma kinase,ALK)单抗及其试剂盒。方法以人ALK重组蛋白免疫BALB/c小鼠,常规杂交瘤技术制备人ALK单抗,ELISA及免疫细胞化学(immunocytochemical,ICC)法筛选稳定分泌ALK单抗的杂交瘤细胞株;SDS-PAGE检测单抗纯度;蛋白定量测试检测单抗浓度;ELISA方法检测单抗效价、亲和力;单抗亚类检测试剂盒鉴定单抗亚型;Western blot及免疫组织化学(immunohistochemical,IHC)法鉴定单抗的特异性。IHC方法筛选ALK单抗检测试剂盒配套的相关试剂,应用制备的ALK单抗试剂盒及抗ALK(D5F3)兔单抗试剂(IHC法)分别检测1 162份临床样本。结果共筛选出3株稳定分泌ALK单抗的杂交瘤细胞株,其中细胞株1A4在IHC检测非小细胞肺癌(non small cell lung cancer,NSCLC)中ALK蛋白时表现强阳性。单抗1A4的浓度、效价及亲和常数分别为6.791 mg/mL、1∶51 200和2.75×109 L/moL,抗体亚型为IgG2b。在不同肺癌细胞系及过表达ALK和其他基因的细胞裂解液中,单抗1A4仅识别ALK蛋白,在多种NSCLC病理组织样本中可鉴定出ALK阳性的NSCLC,特异性高。选择EDTA修复液(pH 9.0)、DAB染色液(增强聚合物法)及中杉金桥生物科技有限公司的抗体稀释液作为试剂盒配套试剂。1 162份临床样本经ALK单抗试剂盒与抗ALK(D5F3)兔单抗试剂(IHC法)检测的ALK阳性及阴性结果一致性良好。结论成功制备了特异性强的鼠抗人ALK单抗(1A4),该ALK单抗试剂盒与抗ALK(D5F3)兔单抗试剂(IHC法)检测结果一致性良好,可用于NSCLC中ALK蛋白的检测。     

Glycoproteomics using fluid-based specimens in the discovery of lung cancer protein biomarkers: Promise and challenge

Lung cancer is the leading cancer in the United States and worldwide. In spite of the rapid progression in personalized treatments, the overall survival rate of lung cancer patients is still suboptimal. Over the past decade, tremendous efforts have been focused on the discovery of protein biomarkers to facilitate the early detection and monitoring of lung cancer progression during treatment. In addition to tumor tissues and cancer cell lines, a variety of biological material has been studied. Particularly in recent years, studies using fluid-based specimen or so-called “fluid-biopsy” specimens have progressed rapidly. Fluid specimens are relatively easier to collect than tumor tissue, and they can be repeatedly sampled during the disease progression. Glycoproteins are the major content of fluid specimens and have long been recognized to play fundamental roles in many physiological and pathological processes. In this review, we focus the discussion on recent advances of glycoproteomics, particularly in the identification of potential glyco protein biomarkers using fluid-based specimens in lung cancer. The purpose of this review is to summarize current strategies, achievements, and perspectives in the field. This insight will highlight the discovery of tumor-associated glycoprotein biomarkers in lung cancer and their potential clinical applications.     

多西他赛与吉西他滨分别顺泊治疗晚期非小细胞肺癌的临床研究

Objective: The aim of this study was to evaluate the clinical efficacy and side effects of docetaxel/cisplatin regiment and gemcitabine/cisplatin regiment in the patients with advanced non-small-cell lung cancer (NSCLC). Methods: Seventy six patients with advanced NSCLC who were chemotherapy-naive were enrolled in two groups. In docetaxel group (DP group) the patients received docetaxel 75 mg/m2 and cisplatin 60 mg/m2 on day 1. In gemcitabine group (GP group) the patients received gemcitabine 1000 mg/m2 on day 1 and day 8. The dosage of cisplatin was the same as DP group. The two regiments were administrated intravenously every 21 days as a cycle, each patient received 2-4 cycles. All patients were followed up until disease progressed or patients died. Results: The overall response rates were 43.5% in DP group and 45.9% in GP group. The response rate was significantly different between the initial treated group and retreated group in both two groups (53.8% vs 23.0% in DP group and 56% vs 25% in GP group, P < 0.05, respectively). The main side effects were bone marrow suppression and thrombocytopenia. Conclusion: Docetaxel/cisplatin regiment and gemcitabine/cisplatin regiment for the patients with advanced NSCLC were efficient and well-tolerated chemotherapeutic approachs with low toxicity levels. The efficacy and major toxicity in two groups were similar.     

MMP-7 and fcDNA Serum Levels in Early NSCLC and Idiopathic Interstitial Pneumonia: Preliminary Study

A non-invasive test to facilitate the diagnosis of non-small cell lung cancer (NSCLC) and idiopathic pulmonary fibrosis (IPF) is still not available and represents an important goal. Forty-eight patients with stage I NSCLC, 45 with IPF, 30 with other idiopathic interstitial pneumonias (IIPs) including idiopathic non-specific interstitial pneumonia (NSIP) and chronic hypersensitivity pneumonitis (HP), 35 with diffuse non-malignant disease and 30 healthy donors were enrolled onto the study. Free circulating (fc)DNA and MMP-7 levels were evaluated by Real Time PCR and ELISA, respectively. Median fcDNA levels were similar in NSCLC (127 ng/mL, range 23.6–345 ng/mL) and IPF (106 ng/mL, range 22–224 ng/mL) patients, and significantly lower in IIPs patients, in individuals with other diseases and in healthy donors (p < 0.05). Conversely, median MMP-7 values were significantly higher in IPF patients (9.10 ng/mL, range 3.88–19.72 ng/mL) than in those with NSCLC (6.31 ng/mL, range 3.38–16.36 ng/mL; p < 0.0001), NSIP (6.50 ng/mL, range 1.50–22.47 ng/mL; p = 0.007), other diseases (5.41 ng/mL, range 1.78–15.91, p < 0.0001) or healthy donors (4.35 ng/mL, range 2.45–7.23; p < 0.0001). Serum MMP-7 levels seem to be capable of distinguishing IPF patients from those with any other lung disease. fcDNA levels were similar in NSCLC and IPF patients, confirming its potential role as a biomarker, albeit non-specific, for the differential diagnosis of NSCLC.     

Gene Mutation Analysis in EGFR Wild Type NSCLC Responsive to Erlotinib: Are There Features to Guide Patient Selection?

Tyrosine kinase inhibitors (TKIs) are very efficacious in non-small-cell lung cancer (NSCLC) patients harboring activating Epidermal Growth Factor Receptor (EGFR) mutations. However, about 10% of EGFR wild type (wt) patients respond to TKI, with unknown molecular mechanisms of sensitivity. We considered a case series of 34 EGFR wt NSCLC patients responsive to erlotinib after at least one line of therapy. Responsive patients were matched with an equal number of non-responsive EGFR wt patients. A panel of 26 genes, for a total of 214 somatic mutations, was analyzed by MassARRAY^® System (Sequenom, San Diego, CA, USA). A 15% KRAS mutation was observed in both groups, with a prevalence of G12C in non-responders (80% vs. 40% in responders). NOTCH1, p53 and EGFR-resistance-related mutations were found more frequently in non-responders, whereas EGFR-sensitizing mutations and alterations in genes involved in proliferation pathways were more frequent in responders. In conclusion, our findings indicate that p53, NOTCH1 and exon 20 EGFR mutations seem to be related to TKI resistance. KRAS mutations do not appear to influence the TKI response, although G12C mutation is more frequent in non-responders. Finally, the use of highly sensitive methodologies could lead to the identification of under-represented EGFR mutations potentially associated with TKI sensitivity.