Array-comparative genomic hybridization reveals loss of SOCS6 is associated with poor prognosis in primary lung squamous cell carcinoma

Background

Primary tumor recurrence commonly occurs after surgical resection of lung squamous cell carcinoma (SCC). Little is known about the genes driving SCC recurrence.

Methods

We used array comparative genomic hybridization (aCGH) to identify genes affected by copy number alterations that may be involved in SCC recurrence. Training and test sets of resected primary lung SCC were assembled. aCGH was used to determine genomic copy number in a training set of 62 primary lung SCCs (28 with recurrence and 34 with no evidence of recurrence) and the altered copy number of candidate genes was confirmed by quantitative PCR (qPCR). An independent test set of 72 primary lung SCCs (20 with recurrence and 52 with no evidence of recurrence) was used for biological validation. mRNA expression of candidate genes was studied using qRT-PCR. Candidate gene promoter methylation was evaluated using methylation microarrays and Sequenom EpiTYPER analysis.

Results

18q22.3 loss was identified by aCGH as being significantly associated with recurrence (p = 0.038). Seven genes within 18q22.3 had aCGH copy number loss associated with recurrence but only SOCS6 copy number was both technically replicated by qPCR and biologically validated in the test set. SOCS6 copy number loss correlated with reduced mRNA expression in the study samples and in the samples with copy number loss, there was a trend for increased methylation, albeit non-significant. Overall survival was significantly poorer in patients with SOCS6 loss compared to patients without SOCS6 loss in both the training (30 vs. 43 months, p = 0.023) and test set (27 vs. 43 months, p = 0.010).

Conclusion

Reduced copy number and mRNA expression of SOCS6 are associated with disease recurrence in primary lung SCC and may be useful prognostic biomarkers.     

miR-141 and miR-200c as Markers of Overall Survival in Early Stage Non-Small Cell Lung Cancer Adenocarcinoma

Background

Several treatments in non-small cell lung cancer (NSCLC) are histology-dependent, and the need for histology-related markers is increasing. MicroRNAs (miRNAs) are promising molecular markers in multiple cancers and show differences in expression depending on histological subtype. The miRNA family miR-200 has been associated with the regulation of epithelial-mesenchymal (EMT)/mesenchymal-epithelial transition (MET). EMT involves profound phenotypic changes that include the loss of cell-cell adhesion, the loss of cell polarity, and the acquisition of migratory and invasive properties that facilitates metastasis. A dual role for the miR-200 family in the prognosis of several tumors has been related to tumor cell origin. However, the prognostic role and function of miR-200 family in early-stage NSCLC adenocarcinoma and squamous cell carcinoma (SCC) have not been well established.

Methods

miRNA expression was determined using TaqMan assays in 155 tumors from resected NSCLC patients. Functional studies were conducted in three NSCLC cell lines: H23, A-549 and HCC-44.

Results

High miR-200c expression was associated with shorter overall survival (OS) in the entire cohort (p = 0.024). High miR-200c (p = 0.0004) and miR-141 (p = 0.009) expression correlated with shorter OS in adenocarcinoma – but not in SCC. In the multivariate analysis, a risk score based on miR-141 and miR-200c expression emerged as an independent prognostic factor for OS in the entire cohort (OR, 2.787; p = 0.033) and in adenocarcinoma patients (OR, 10.649; p = 0.002). Functional analyses showed that miR-200c, was related to mesenchymal-epithelial transition (MET) and affected cell migration and E-cadherin levels, while overexpression of miR-141 reduced KLF6 protein levels and produced an increase of secretion of VEGFA in vitro (H23, p = 0.04; A-549, p = 0.03; HCC-44, p = 0.02) and was associated with higher blood microvessel density in patient tumor samples (p<0.001).

Conclusion

High miR-141 and miR-200c expression are associated with shorter OS in NSCLC patients with adenocarcinoma through MET and angiogenesis.     

Over-expression of LSD1 promotes proliferation, migration and invasion in non-small cell lung cancer

Background

Lysine specific demethylase 1 (LSD1) has been identified and biochemically characterized in epigenetics, but the pathological roles of its dysfunction in lung cancer remain to be elucidated. The aim of this study was to evaluate the prognostic significance of LSD1 expression in patients with non-small cell lung cancer (NSCLC) and to define its exact role in lung cancer proliferation, migration and invasion.

Methods

The protein levels of LSD1 in surgically resected samples from NSCLC patients were detected by immunohistochemistry or Western blotting. The mRNA levels of LSD1 were detected by qRT-PCR. The correlation of LSD1 expression with clinical characteristics and prognosis was determined by statistical analysis. Cell proliferation rate was assessed by MTS assay and immunofluorescence. Cell migration and invasion were detected by scratch test, matrigel assay and transwell invasion assay.

Results

LSD1 expression was higher in lung cancer tissue more than in normal lung tissue. Our results showed that over-expression of LSD1 protein were associated with shorter overall survival of NSCLC patients. LSD1 was localized mainly to the cancer cell nucleus. Interruption of LSD1 using siRNA or a chemical inhibitor, pargyline, suppressed proliferation, migration and invasion of A549, H460 and 293T cells. Meanwhile, over-expression of LSD1 enhanced cell growth. Finally, LSD1 was shown to regulate epithelial-to-mesenchymal transition in lung cancer cells.

Conclusions

Over-expression of LSD1 was associated with poor prognosis in NSCLC, and promoted tumor cell proliferation, migration and invasion. These results suggest that LSD1 is a tumor-promoting factor with promising therapeutic potential for NSCLC.     

Gene Expression-Based Classification of Non-Small Cell Lung Carcinomas and Survival Prediction

Background

Current clinical therapy of non-small cell lung cancer depends on histo-pathological classification. This approach poorly predicts clinical outcome for individual patients. Gene expression profiling holds promise to improve clinical stratification, thus paving the way for individualized therapy.

Methodology and Principal Findings

A genome-wide gene expression analysis was performed on a cohort of 91 patients. We used 91 tumor- and 65 adjacent normal lung tissue samples. We defined sets of predictor genes (probe sets) with the expression profiles. The power of predictor genes was evaluated using an independent cohort of 96 non-small cell lung cancer- and 6 normal lung samples. We identified a tumor signature of 5 genes that aggregates the 156 tumor and normal samples into the expected groups. We also identified a histology signature of 75 genes, which classifies the samples in the major histological subtypes of non-small cell lung cancer. Correlation analysis identified 17 genes which showed the best association with post-surgery survival time. This signature was used for stratification of all patients in two risk groups. Kaplan-Meier survival curves show that the two groups display a significant difference in post-surgery survival time (p = 5.6E-6). The performance of the signatures was validated using a patient cohort of similar size (Duke University, n = 96). Compared to previously published prognostic signatures for NSCLC, the 17 gene signature performed well on these two cohorts.

Conclusions

The gene signatures identified are promising tools for histo-pathological classification of non-small cell lung cancer, and may improve the prediction of clinical outcome.     

EZH2 Is Associated with Malignant Behavior in Pancreatic IPMN via p27Kip1 Downregulation

Background

The epigenetic mechanism of tumorigenesis in pancreatic intraductal papillary mucinous neoplasm (IPMN) remains largely unknown. The aim of this study is to examine the role of enhancer of zeste homologue 2 (EZH2) alteration in pancreatic IPMN progression.

Methods

Fifty-four surgically resected pancreatic IPMN specimens, including a total of 181 lesions (normal duct in 48, adenoma in 50, borderline atypia in 53, carcinoma in situ (CIS) in 19, and invasive carcinoma in 11) were analyzed by immunohistochemical staining (EZH2, Ki-67, p27^Kip1). Using paraffin embedded sections, total RNA was successfully extracted from 20 IPMN lesions (borderline IPMN in 9, CIS in 6, invasive carcinoma in 5) and 7 pancreatic normal ducts, and then levels of EZH2 and p27^Kip1 mRNA were analyzed by real time PCR.

Results

In immunohistochemical analysis, cell proliferative activity revealed by Ki-67 positive nuclei was increased during IPMN progression (normal duct<adenoma<borderline atypia<CIS ≈ invasive carcinoma). EZH2 expression displayed a similar pattern (normal duct<adenoma<borderline atypia<CIS ≈ invasive carcinoma) with cell proliferative activity. EZH2 expression in malignant (CIS and invasive carcinoma) IPMNs was significantly higher than that in adenoma and borderline-atypia IPMNs. EZH2 expression level in IPMN lesions was positively correlated with the Ki-67 positive nuclear ratio (p<0.0001). EZH2-positive cells in malignant IPMN did not express p27^Kip1. EZH2 mRNA expressions in malignant lesions were significantly higher than those in benign lesions (p<0.0001). In contrast, p27^Kip1 mRNA in malignant lesions was significantly decreased compared to those in benign lesion (p<0.05), and there was an inverse correlation between EZH2 and p27^Kip1 mRNA levels (p = 0.0109).

Conclusion

EZH2 is associated with the accelerated cell proliferation and malignant step in pancreatic IPMN via the downregulation of p27^Kip1.     

Integration of gene dosage and gene expression in non-small cell lung cancer, identification of HSP90 as potential target

Background

Lung cancer causes approximately 1.2 million deaths per year worldwide, and non-small cell lung cancer (NSCLC) represents 85% of all lung cancers. Understanding the molecular events in non-small cell lung cancer (NSCLC) is essential to improve early diagnosis and treatment for this disease.

Methodology and Principal Findings

In an attempt to identify novel NSCLC related genes, we performed a genome-wide screening of chromosomal copy number changes affecting gene expression using microarray based comparative genomic hybridization and gene expression arrays on 32 radically resected tumor samples from stage I and II NSCLC patients. An integrative analysis tool was applied to determine whether chromosomal copy number affects gene expression. We identified a deletion on 14q32.2-33 as a common alteration in NSCLC (44%), which significantly influenced gene expression for HSP90, residing on 14q32. This deletion was correlated with better overall survival (P = 0.008), survival was also longer in patients whose tumors had low expression levels of HSP90. We extended the analysis to three independent validation sets of NSCLC patients, and confirmed low HSP90 expression to be related with longer overall survival (P = 0.003, P = 0.07 and P = 0.04). Furthermore, in vitro treatment with an HSP90 inhibitor had potent antiproliferative activity in NSCLC cell lines.

Conclusions

We suggest that targeting HSP90 will have clinical impact for NSCLC patients.     

Visualization-Aided Classification Ensembles Discriminate Lung Adenocarcinoma and Squamous Cell Carcinoma Samples Using Their Gene Expression Profiles

Introduction

The widespread application of microarray experiments to cancer research is astounding including lung cancer, one of the most common fatal human tumors. Among non-small cell lung carcinoma (NSCLC), there are two major histological types of NSCLC, adenocarcinoma (AC) and squamous cell carcinoma (SCC).

Results

In this paper, we proposed to integrate a visualization method called Radial Coordinate Visualization (Radviz) with a suitable classifier, aiming at discriminating two NSCLC subtypes using patients'' gene expression profiles. Our analyses on simulated data and a real microarray dataset show that combining with a classification method, Radviz may play a role in selecting relevant features and ameliorating parsimony, while the final model suffers no or least loss of accuracy. Most importantly, a graphic representation is more easily understandable and implementable for a clinician than statistical methods and/or mathematic equations.

Conclusion

To conclude, using the NSCLC microarray data presented here as a benchmark, the comprehensive understanding of the underlying mechanism associated with NSCLC and of the mechanisms with its subtypes and respective stages will become reality in the near future.     

Concomitant active tuberculosis prolongs survival in non-small cell lung cancer: a study in a tuberculosis-endemic country

Background

Adjuvant tumor cell vaccine with chemotherapy against non-small cell lung cancer (NSCLC) shows limited clinical response. Whether it provokes effective cellular immunity in tumor microenvironment is questionable. Concomitant active tuberculosis in NSCLC (TBLC) resembles locoregional immunotherapy of tumor cell vaccine; thus, maximally enriches effective anti-tumor immunity. This study compares the survival and immunological cell profile in TBLC over NSCLC alone.

Methods

Retrospective review of NSCLC patients within 1-year-period of 2007 and follow-up till 2010.

Results

A total 276 NSCLC patients were included. The median survival of TBLC is longer than those of NSCLC alone (11.6 vs. 8.8 month, p<0.01). Active tuberculosis is an independent predictor of better survival with HR of 0.68 (95% CI, 0.48∼0.97). Squamous cell carcinoma (SCC) (55.8 vs. 31.7%, p<0.01) is a significant risk factor for NSCLC with active TB. The median survival of SCC with active tuberculosis is significantly longer than adenocarcinoma or undetermined NSCLC with TB (14.2 vs. 6.6 and 2.8 months, p<0.05). Active tuberculosis in SCC increases the expression of CD3 (46.4±24.8 vs. 24.0±16.0, p<0.05), CXCR3 (35.1±16.4 vs. 19.2±13.3, p<0.01) and IP-10 (63.5±21.9 vs. 35.5±21.0, p<0.01), while expression of FOXP3 is decreased (3.5±0.5 vs. 13.3±3.7 p<0.05, p<0.05). Survival of SCC with high expression of CD3 (12.1 vs. 3.6 month, p<0.05) and CXCR3 (12.1 vs. 4.4 month, p<0.05) is longer than that with low expression.

Conclusions

Active tuberculosis in NSCLC shows better survival outcome. The effective T lymphocyte infiltration in tumor possibly underlies the mechanism. Locoregional immunotherapy of tumor cell vaccine may deserve further researches.     

Cell-Free MicroRNA Expression Profiles in Malignant Effusion Associated with Patient Survival in Non-Small Cell Lung Cancer

Objective

MicroRNAs (miRNAs) expression is altered in cancer cells, and miRNAs could serve as diagnostic and prognostic biomarker for cancer patients. This study was designed to analyze circulating miRNAs expression in the malignant pleural effusion (MPE) and their association with patient survival in non-small cell lung cancer (NSCLC).

Methods

Pleural effusion from 184 patients with NSCLC and MPE were collected. MiRNA microarray and bioinformatics interpretation were used to evaluate miRNA expression profiles in 10 NSCLC patients with different survival prognosis. Associations were validated in 184 patients (randomly classified into training and validation set with equal number in each group) using quantitative RT-PCR. Risk scores were formulated based on the expression signature of miRNAs. Clinical data, such as patient survival, were collected for correlation analysis.

Results

Thirty-three miRNAs were found to be altered more than two-fold by microarray in malignant effusions between longer-survival and shorter-survival groups, and levels of five miRNAs (miRNA-93, miRNA-100, miRNA-134, miRNA-151 and miRNA-345) were significantly associated with overall survival. High expression of miR-100 and low expression of miRNA-93, miRNA-134, miRNA-151 and miRNA-345 were associated with poor survival in both the training and validation cohort. Patients with high risk scores had overall poor survival compared to the patients with low risk scores. Risk score was an independent predictor of patient survival.

Conclusions

Expression patterns of miRNAs are systematically altered in MPE of patient with NSCLC. The five miRNA signature from the effusion may serve as a predictor for the overall survival of patients with lung cancers.     

Performance of Epigenetic Markers SEPT9 and ALX4 in Plasma for Detection of Colorectal Precancerous Lesions

Background

Screening for colorectal cancer (CRC) has shown to reduce cancer-related mortality, however, acceptance and compliance to current programmes are poor. Developing new, more acceptable non-invasive tests for the detection of cancerous and precancerous colorectal lesions would not only allow preselection of individuals for colonoscopy, but may also prevent cancer by removal of precancerous lesions.

Methods

Plasma from 128 individuals (cohort I – exploratory study: 73 cases / 55 controls ) was used to test the performance of a single marker, SEPT9, using a real-time quantitative PCR assay. To validate performance of SEPT9, plasma of 76 individuals (cohort II – validation study: 54 cases / 22 controls) was assessed. Additionally, improvement of predictive capability considering SEPT9 and additionally ALX4 methylation was investigated within these patients.

Results

In both cohorts combined, methylation of SEPT9 was observed in 9% of controls (3/33), 29% of patients with colorectal precancerous lesions (27/94) and 73% of colorectal cancer patients (24/33). The presence of both SEPT9 and ALX4 markers was analysed in cohort II and was observed in 5% of controls (1/22) and 37% of patients with polyps (18/49). Interestingly, also 3/5 (60%) patients with colorectal cancer were tested positive by the two marker panel in plasma.

Conclusions

While these data confirm the detection rate of SEPT9 as a biomarker for colorectal cancer, they also show that methylated DNA from advanced precancerous colorectal lesions can be detected using a panel of two DNA methylation markers, ALX4 and SEPT9. If confirmed in larger studies these data indicate that screening for colorectal precancerous lesions with a blood-based test may be as feasible as screening for invasive cancer.     

PD-1 and PD-L1 Expression in NSCLC Indicate a Favorable Prognosis in Defined Subgroups

Background

Immunotherapy can become a crucial therapeutic option to improve prognosis for lung cancer patients. First clinical trials with therapies targeting the programmed cell death receptor PD-1 and its ligand PD-L1 have shown promising results in several solid tumors. However, in lung cancer the diagnostic, prognostic and predictive value of these immunologic factors remains unclear.

Method

The impact of both factors was evaluated in a study collective of 321 clinically well-annotated patients with non-small lung cancer (NSCLC) using immunohistochemistry.

Results

PD-1 expression by tumor infiltrating lymphocytes (TILs) was found in 22%, whereas tumor cell associated PD-L1 expression was observed in 24% of the NSCLC tumors. In Fisher’s exact test a positive correlation was found for PD-L1 and Bcl-xl protein expression (p = 0.013). Interestingly, PD-L1 expression on tumor cells was associated with improved overall survival in pulmonary squamous cell carcinomas (SCC, p = 0.042, log rank test), with adjuvant therapy (p = 0.017), with increased tumor size (pT2-4, p = 0.039) and with positive lymph node status (pN1-3, p = 0.010). These observations were confirmed by multivariate cox regression models.

Conclusion

One major finding of our study is the identification of a prognostic implication of PD-L1 in subsets of NSCLC patients with pulmonary SCC, with increased tumor size, with a positive lymph node status and NSCLC patients who received adjuvant therapies. This study provides first data for immune-context related risk stratification of NSCLC patients. Further studies are necessary both to confirm this observation and to evaluate the predictive value of PD-1 and PD-L1 in NSCLC in the context of PD-1 inhibition.     

Integrated analyses of copy number variations and gene differential expression in lung squamous-cell carcinoma

Background

Although numerous efforts have been made, the pathogenesis underlying lung squamous-cell carcinoma (SCC) remains unclear. This study aimed to identify the CNV-driven genes by an integrated analysis of both the gene differential expression and copy number variation (CNV).

Results

A higher burden of the CNVs was found in 10–50 kb length. The 16 CNV-driven genes mainly located in chr 1 and chr 3 were enriched in immune response [e.g. complement factor H (CFH) and Fc fragment of IgG, low affinity IIIa, receptor (FCGR3A)], starch and sucrose metabolism [e.g. amylase alpha 2A (AMY2A)]. Furthermore, 38 TFs were screened for the 9 CNV-driven genes and then the regulatory network was constructed, in which the GATA-binding factor 1, 2, and 3 (GATA1, GATA2, GATA3) jointly regulated the expression of TP63.

Conclusions

The above CNV-driven genes might be potential contributors to the development of lung SCC.     

The Prognostic Value of SOX2 Expression in Non-Small Cell Lung Cancer: A Meta-Analysis

Objective

To investigate the association of SOX2 expression in tumor with clinicopathological features and survival of non-small-cell lung carcinoma (NSCLC) patients.

Methods

Publications assessing the clinicopathological characteristics and prognostic significance of SOX2 in NSCLC were identified up to May 2013. A meta-analysis of eligible studies was performed using standard statistical methods to clarify the association between SOX2 expression and these clinical parameters.

Results

A total of eight studies met the inclusion criteria. Analysis of these data showed that SOX2 expression was positively associated with squamous histology, (pooled OR = 5.26, 95% CI: 1.08–25.6, P = 0.040). Simultaneously, we also found that SOX2 expression was positively associated with overall survival (pooled HR = 0.65, 95% CI: 0.47–0.89, P = 0.007, random-effect).

Conclusions

SOX2 expression in tumor is a candidate positive prognostic biomarker for NSCLC patients.     

Expression analysis of asthma candidate genes during human and murine lung development

Background

Little is known about the role of most asthma susceptibility genes during human lung development. Genetic determinants for normal lung development are not only important early in life, but also for later lung function.

Objective

To investigate the role of expression patterns of well-defined asthma susceptibility genes during human and murine lung development. We hypothesized that genes influencing normal airways development would be over-represented by genes associated with asthma.

Methods

Asthma genes were first identified via comprehensive search of the current literature. Next, we analyzed their expression patterns in the developing human lung during the pseudoglandular (gestational age, 7-16 weeks) and canalicular (17-26 weeks) stages of development, and in the complete developing lung time series of 3 mouse strains: A/J, SW, C57BL6.

Results

In total, 96 genes with association to asthma in at least two human populations were identified in the literature. Overall, there was no significant over-representation of the asthma genes among genes differentially expressed during lung development, although trends were seen in the human (Odds ratio, OR 1.22, confidence interval, CI 0.90-1.62) and C57BL6 mouse (OR 1.41, CI 0.92-2.11) data. However, differential expression of some asthma genes was consistent in both developing human and murine lung, e.g. NOD1, EDN1, CCL5, RORA and HLA-G. Among the asthma genes identified in genome wide association studies, ROBO1, RORA, HLA-DQB1, IL2RB and PDE10A were differentially expressed during human lung development.

Conclusions

Our data provide insight about the role of asthma susceptibility genes during lung development and suggest common mechanisms underlying lung morphogenesis and pathogenesis of respiratory diseases.     

Environmental Factors Associated with Disease Progression after the First Demyelinating Event: Results from the Multi-Center SET Study

Objectives

To investigate the associations of environmental MS risk factors with clinical and MRI measures of progression in high-risk clinically isolated syndromes (CIS) after the first demyelinating event.

Methods

We analyzed 211 CIS patients (age: 28.9±7.8 years) enrolled in the SET study, a multi-center study of high-risk CIS patients. Pre-treatment samples were analyzed for IgG antibodies against cytomegalovirus (anti-CMV), Epstein Barr virus (EBV) early nuclear antigen-1 (EBNA-1), viral capsid antigen (VCA), early antigen-diffuse (EA-D), 25 hydroxy-vitamin D3 and cotinine levels and HLA DRB1*1501 status. The inclusion criteria required evaluation within 4 months of the initial demyelinating event, 2 or more brain MRI lesions and the presence of two or more oligoclonal bands in cerebrospinal fluid. All patients were treated with interferon-beta. Clinical and MRI assessments were obtained at baseline, 6, 12, and 24 months.

Results

The time to first relapse decreased and the number of relapses increased with anti-CMV IgG positivity. Smoking was associated with increased number and volume of contrast-enhancing lesions (CEL) during the 2-year period. The cumulative number of CEL and T2 lesions during the 2-year period was greater for individuals in the highest quartile of anti-EBV VCA IgG antibodies. The percent loss of brain volume was increased for those in the highest quartile of with anti-EBV VCA IgG antibodies.

Conclusions

Relapses in CIS patients were associated with CMV positivity whereas anti-EBV VCA positivity was associated with progression on MRI measures, including accumulation of CEL and T2 lesions and development of brain atrophy.     

GWAS Meets Microarray: Are the Results of Genome-Wide Association Studies and Gene-Expression Profiling Consistent? Prostate Cancer as an Example

Background

Genome-wide association studies (GWASs) and global profiling of gene expression (microarrays) are two major technological breakthroughs that allow hypothesis-free identification of candidate genes associated with tumorigenesis. It is not obvious whether there is a consistency between the candidate genes identified by GWAS (GWAS genes) and those identified by profiling gene expression (microarray genes).

Methodology/Principal Findings

We used the Cancer Genetic Markers Susceptibility database to retrieve single nucleotide polymorphisms from candidate genes for prostate cancer. In addition, we conducted a large meta-analysis of gene expression data in normal prostate and prostate tumor tissue. We identified 13,905 genes that were interrogated by both GWASs and microarrays. On the basis of P values from GWASs, we selected 1,649 most significantly associated genes for functional annotation by the Database for Annotation, Visualization and Integrated Discovery. We also conducted functional annotation analysis using same number of the top genes identified in the meta-analysis of the gene expression data. We found that genes involved in cell adhesion were overrepresented among both the GWAS and microarray genes.

Conclusions/Significance

We conclude that the results of these analyses suggest that combining GWAS and microarray data would be a more effective approach than analyzing individual datasets and can help to refine the identification of candidate genes and functions associated with tumor development.