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.     

Increased expression of the lncRNA PVT1 promotes tumorigenesis in non-small cell lung cancer

Introduction: Non-small cell lung cancer (NSCLC) is the major cause of cancer death worldwide. Increasing evidence shows that long non coding RNAs (lncRNAs) are widely involved in the development and progression of NSCLC. lncRNA PVT1 in several cancers has been studied, its role in lung cancer remains unknown. Our studies were designed to investigate the expression, biological role and clinical significance of PVT1 in lung cancer. Methods: lncRNA PVT1 expression in 82 NSCLC tissues and 3 lung cancer cell lines was measured by quantitative Real-time PCR (qRT-PCR). Its association with overall survival of patients was analyzed by statistical analysis. RNA interference (RNAi) approaches were used to investigate the biological functions of PVT1. The effect of PVT1 on proliferation was evaluated by MTT, cell migration and invasion ability was evaluated by cell migration and invasion assays. Results: lncRNA PVT1 expression was significantly upregulated in NSCLC tissues and lung cancer cells when compared with corresponding adjacent normal tissues and normal bronchial epithelial cells. Increased PVT1 expression was significantly correlated with histological grade and lymph node metastasis. In addition, NSCLC patients with PVT1 higher expression have shown significantly poorer overall survival than those with lower PVT1 expression. And PVT1 expression was an independent prognostic marker of overall survival in a multivariate analysis. In vitro assays our results indicated that knockdown of PVT1 inhibited cell proliferation, migration, and invasion. Conclusions: Our data indicated that lncRNA PVT1 is significantly upregulated in NSCLC tissues and may represent a new biomarker and a potential therapeutic target for NSCLC intervention.     

晚期非小细胞肺癌组织中ERCC1、RRM1表达水平与含铂治疗方案疗效及预后相关性分析

目的：探讨ERCC1及RRM1的表达在预测晚期非小细胞肺癌含铂治疗方案疗效及预后中的作用。方法：用免疫组织化学的方法检测124例ⅢB和Ⅳ期非小细胞肺癌患者的石蜡包埋活检组织标本中ERCC1和RRM1的表达水平,并分析其与临床病理特征以及疗效、预后的相关性。124例均为接受以铂类为基础的三代药物联合化疗的初治患者。结果：ERCC1、RRM1表达阳性者分别为43例（35％）和50例（40％）。ERCC1及RRM1的表达与疗效相关,ERCC1表达阴性者疗效达PR的患者（54％）明显高于阳性者（33％）,差异有统计学意义（P=0．022）。同样,RRM1表达阴性患者疗效为PR的明显高于RRM1阳性者（53％VS34％,P=0．042）。ERCC1和RRM1同时阴性表达者的中位生存时问明显长于同时阳性表达者（11．7个月VS9．2个月,P=0．025）,多因素分析表明,ERCC1为独立的预后预测因素（P=0．0066）。结论：ERCC1的表达与晚期非小细胞肺癌预后及含铂治疗方案疗效相关。     

Screening and identification of potential novel lipid biomarkers for non-small cell lung cancer using ultra-high performance liquid chromatography tandem mass spectrometry

Lung cancer is characterized by a high incidence rate and low survival rate. It is important to achieve early diagnosis of the disease. We applied ultra-high performance liquid chromatography tandem mass spectrometry to screen plasma lipid spectrum in non-small cell lung cancer (NSCLC) patients, healthy controls (HC), and community-acquired pneumonia (CAP) patients. Modeling employing orthogonal partial least squares-discriminant analysis combined with t-test was used to screen the differential lipids. Logistic regression analysis was used to establish the diagnostic model, while the accuracy was verified by 10-fold cross-validation. The results showed that the abnormal metabolism of lipid in NSCLC mainly comprised fatty acid metabolism, phospholipid metabolism, and glyceride metabolism. Four potential biomarkers, including LPC (14:0/0:0), LPI (14:1/0:0), DG (14:0/18:2/0:0), and LPC (16:1/0:0), were fitted by the receiver operating characteristic curve model with the area under curve (AUC) value of 0.856, and the specificity and sensitivity were 87.0 and 78.0%, respectively. The results of cross validation showed that the AUC value of the model was 0.812, the sensitivity was 72.9%, and the specificity was 82.6%. The positive rate of four potential lipid biomarkers in this study (>60.0%) was higher than that of existing tumor biomarkers in the clinical application. We investigated the plasma lipid profile of NSCLC patients and identified lipid biomarkers with potential diagnostic values. From the lipidomics perspective, our study may lay a foundation for the biomarker-based early diagnosis of lung cancer.     

LncRNA NCK1-AS1 promotes non-small cell lung cancer progression via regulating miR-512-5p/p21 axis

ObjectiveThis study aimed at probing into the effect of lncRNA NCK1-AS1 on proliferation, migration and invasion of non-small cell lung cancer (NSCLC) cells and its regulatory function on miR-512-5p/p21 molecular axis.MethodsQuantitative real-time polymerase chain reaction (qRT-PCR) was used to assess the expressions of NCK1-AS1 and miR-512-5p in NSCLC tissues and cell lines. The alterations of cell proliferation, migration, invasion and cell cycle were examined by cell counting kit-8 (CCK-8) assay, BrdU experiment, Transwell experiment and flow cytometry, respectively. The dual-luciferase reporter assay and RNA immunoprecipitation experiment were performed to validate the binding relationships between miR-512-5p and NCK1-AS1, and miR-512-5p the 3'UTR of p21 mRNA. Western blot was used to determine the effects of NCK1-AS1 and miR-512-5p on p21 protein expression.ResultsNCK1-AS1 expression was up-regulated in NSCLC tissues and cells, and its high expression was correlated with shorter overall survival time and faster progression of patients. Overexpression of NCK1-AS1 promoted NSCLC cell proliferation, migration and invasion, and accelerated the cell cycle, whereas NCK1-AS1 siRNA inhibited these malignant biological behaviors, and arrested cell cycle. NCK1-AS1 could bind to miR-512-5p, p21 was verified as a target gene of miR-512-5p, and NCK1-AS1 could up-regulate the expression of p21 in NSCLC cells via repressing miR-512-5p expression.ConclusionNCK1-AS1 promotes NSCLC progression by regulating miR-512-5p/p21 molecular axis.     

非小细胞肺癌影像学特征与基因表达间相关性的探索性研究

影像基因组学通过挖掘肿瘤的影像和基因组学间的关联性,将两者的优势互补,以此指导不同患者个体化治疗方案的制定、预后评估、疗效检测等。针对非小细胞肺癌(NSCLC),建立其CT影像定量特征与基因表达之间的映射。首先,将对应的CT影像中肿瘤区域进行分割和特征提取,选用66种三维定量特征作为肿瘤区域影像特征集;然后,利用基因组学数据分析流程,在原始基因数据经过预处理、聚类后,获取其第一主成分作为具有相似表达谱基因聚类结果的代表;最后,运用基因芯片显著性分析算法探寻两者之间的相关性,并进行基因集的富集分析和预测模型的建立。对癌症图像归档数据库(TCIA)中的26例NSCLC影像数据和基因表达综合数据库(GEO)中相对应的基因数据进行分析,共得到126组成对的显著关联(q<0.05)。将所得结果中的29组元基因建立预测模型,并通过TCIA中更新的211组数据,对其中10组预测准确率大于70%且预测的元基因有生物学意义的预测模型进行验证,最终预测准确率为35.48%～80.85%,10个预测模型中有6个的准确率在70%以上。实验结果表明,特定的影像特征或其组合可以作为基因表达的影像标记物。     

High expression of nitric oxide synthases is a favorable prognostic sign in non-small cell lung carcinoma

Immunohistochemical expression of neuronal (n), endothelial (e), and inducible (i) NOS and their association with the type, grade, apoptotic index, proliferation of tumors and the survival of patients were investigated in 89 biopsies of non-small cell lung carcinoma (NSCLC). In tumor cells, expression of iNOS was detected in 35/89 (40%) cases, while 79/89 (89%) and 72/89 (81%) cases showed weak to intense positivity for eNOS and nNOS, respectively. Strong eNOS staining was seen significantly more often in adenocarcinomas than in squamous cells carcinomas (p=0.016), and iNOS immunoreactivity was seen more often in grade I-II tumors than in grade III tumors (p=0.024). There was no significant difference between the low and high apoptotic indexes or between the low and high proliferation rates of tumors in any instance of NOS staining. The patients with tumors showing high nNOS expression tended to have better survival than the others (p=0.06, log-rank; p=0.04, Bresow; p=0.048, Tarone-Ware). Similarly, the patients with tumors showing high expression of iNOS, eNOS and nNOS, as determined by a combined sum index, had a better survival than those with a low sum index for these enzymes (p<0.05). The results show intense expression of eNOS and nNOS, and moderate expression of iNOS in tumor cells of non-small cell carcinoma. Intense NOSs expression seems to be a favorable prognostic sign in non-small cell lung carcinoma.     

Differential diagnosis between 18F-FDG-avid metastatic lymph nodes in non-small cell lung cancer and benign nodes on dual-time point PET/CT scan

Objective  To clarify the difference of ¹⁸F-FDG uptake kinetics between FDG-avid metastatic lymph nodes (LNs) in patients with non-small-cell lung cancer (NSCLC) and FDG-avid benign LNs associated with various etiologies on dual-time point PET/CT scan, and to determine the optimal parameter for differentiation. Methods  The subjects were 134 FDG-avid metastatic LNs in 67 patients with NSCLC and 62 FDG-avid benign LNs in 61 patients with various lung disorders including NSCLC. PET/CT scan was performed at 2 time points (at 60 min and at 120 min) after intravenous injection of 4.4 MBq/kg ¹⁸F-FDG. The maximum standardized uptake value (SUVmax) on early and delayed scans and the percent change of SUVmax (%ΔSUVmax) were measured at each FDG-avid LN. The optimal parameter for differentiation was determined by the receiver-operating characteristic analysis. Results  Delayed SUVmax was increased compared with early SUVmax in 114 (85.0%) FDG-avid metastatic LNs and 42 (67.7%) FDG-avid benign LNs, with significant higher delayed SUVmax than early values (7.0 ± 5.0 vs. 5.9 ± 3.4; P < 0.0001, and 3.0 ± 1.3 vs. 2.8 ± 1.0; P < 0.05, respectively). Early and delayed SUVmax and %ΔSUVmax in metastatic LNs were significantly higher than those in benign LNs (P < 0.0001). The optimal parameter for the differentiation was the combined use of early SUVmax > 3.0 or delayed SUVmax > 4.0, yielding sensitivity of 88.8%, specificity of 80.6%, accuracy of 86.2%, negative predictive value of 76.9%, and positive predictive value of 90.6%. It provided better results than the use of early SUVmax > 3.0 alone (P = 0.019) or the optimal parameter for %ΔSUVmax (>5%) (P = 0.012). However, 12 (19.3%) benign LNs were indistinguishable from metastatic LNs. Conclusions  Although dual-time point PET/CT scan enhances the difference of FDG uptake between FDG-avid metastatic and benign LNs and improves the differentiation when compared with a single scan, biopsy procedure may be still required for accurate assessment of LN status in patients with NSCLC and possible etiologies showing intensive FDG uptake in benign LNs.