Multi-Walled Carbon Nanotube-Induced Gene Expression Biomarkers for Medical and Occupational Surveillance.

As the demand for multi-walled carbon nanotube (MWCNT) incorporation into industrial and biomedical applications increases, so does the potential for unintentional pulmonary MWCNT exposure, particularly among workers during manufacturing. Pulmonary exposure to MWCNTs raises the potential for development of lung inflammation, fibrosis, and cancer among those exposed; however, there are currently no effective biomarkers for detecting lung fibrosis or predicting the risk of lung cancer resulting from MWCNT exposure. To uncover potential mRNAs and miRNAs that could be used as markers of exposure, this study compared in vivo mRNA and miRNA expression in lung tissue and blood of mice exposed to MWCNTs with in vitro mRNA and miRNA expression from a co-culture model of human lung epithelial and microvascular cells, a system previously shown to have a higher overall genome-scale correlation with mRNA expression in mouse lungs than either cell type grown separately. Concordant mRNAs and miRNAs identified by this study could be used to drive future studies confirming human biomarkers of MWCNT exposure. These potential biomarkers could be used to assess overall worker health and predict the occurrence of MWCNT-induced diseases.

Similar and Differential Canonical Pathways and Biological Processes Associated With Multiwalled Carbon Nanotube and Asbestos-Induced Pulmonary Fibrosis: A 1-Year Postexposure Study.

Respiratory exposure to multiwalled carbon nanotubes (MWCNT) or asbestos results in fibrosis; however, the mechanisms to reach this end point may be different. A previous study by our group identified pulmonary effects and significantly altered messenger RNA (mRNA) signaling pathways following exposure to 1, 10, 40, and 80 µg MWCNT and 120 µg crocidolite asbestos on mouse lungs over time at 1-month, 6-month, and 1-year postexposure following pulmonary aspiration. As a continuation to the above study, this current study took an in-depth look at the signaling pathways involved in fibrosis development at a single time point, 1 year, and exposure, 40 µg MWCNT, the lowest exposure at which fibrosis was pathologically evident. The 120 µg asbestos exposure was included to compare MWCNT-induced fibrosis with asbestos-induced fibrosis. A previously validated computational model was used to identify mRNAs with expression profiles matching the fibrosis pathology patterns from exposed mouse lungs. mRNAs that matched the pathology patterns were then input into ingenuity pathway analysis to determine potential signaling pathways and physiological disease functions inherent to MWCNT and asbestos exposure. Both MWCNT and asbestos exposure induced changes in mouse lungs regarding gene expression, cell proliferation, and survival, while MWCNT uniquely induced alterations in pathways involved in oxidative phosphorylation, mitochondrial dysfunction, and transcription. Asbestos exposure produced unique alterations in pathways involved in sustained inflammation. Although typically considered similar due to scale and fiber-like appearance, the different compositional properties inherent to either MWCNT or asbestos may play a role in their ability to induce fibrosis after pulmonary exposure.

Multiwalled carbon nanotube-induced pulmonary inflammatory and fibrotic responses and genomic changes following aspiration exposure in mice: A 1-year postexposure study.

Pulmonary exposure to multiwalled carbon nanotubes (MWCNT) induces an inflammatory and rapid fibrotic response, although the long-term signaling mechanisms are unknown. The aim of this study was to examine the effects of 1, 10, 40, or 80 µg MWCNT administered by pharyngeal aspiration on bronchoalveolar lavage (BAL) fluid for polymorphonuclear cell (PMN) infiltration, lactate dehydrogenase (LDH) activity, and lung histopathology for inflammatory and fibrotic responses in mouse lungs 1 mo, 6 mo, and 1 yr postexposure. Further, a 120-µg crocidolite asbestos group was incorporated as a positive control for comparative purposes. Results showed that MWCNT increased BAL fluid LDH activity and PMN infiltration in a dose-dependent manner at all three postexposure times. Asbestos exposure elevated LDH activity at all 3 postexposure times and PMN infiltration at 1 mo and 6 mo postexposure. Pathological changes in the lung, the presence of MWCNT or asbestos, and fibrosis were noted at 40 and 80 µg MWCNT and in asbestos-exposed mice at 1 yr postexposure. To determine potential signaling pathways involved with MWCNT-associated pathological changes in comparison to asbestos, up- and down-regulated gene expression was determined in lung tissue at 1 yr postexposure. Exposure to MWCNT tended to favor those pathways involved in immune responses, specifically T-cell responses, whereas exposure to asbestos tended to favor pathways involved in oxygen species production, electron transport, and cancer. Data indicate that MWCNT are biopersistent in the lung and induce inflammatory and fibrotic pathological alterations similar to those of crocidolite asbestos, but may reach these endpoints by different mechanisms.

mRNAs and miRNAs in whole blood associated with lung hyperplasia, fibrosis, and bronchiolo-alveolar adenoma and adenocarcinoma after multi-walled carbon nanotube inhalation exposure in mice.

Inhalation exposure to multi-walled carbon nanotubes (MWCNT) in mice results in inflammation, fibrosis and the promotion of lung adenocarcinoma; however, the molecular basis behind these pathologies is unknown. This study determined global mRNA and miRNA profiles in whole blood from mice exposed by inhalation to MWCNT that correlated with the presence of lung hyperplasia, fibrosis, and bronchiolo-alveolar adenoma and adenocarcinoma. Six-week-old, male, B6C3F1 mice received a single intraperitoneal injection of either the DNA-damaging agent methylcholanthrene (MCA, 10 µg g(-1) body weight) or vehicle (corn oil). One week after injections, mice were exposed by inhalation to MWCNT (5 mg m(-3), 5 hours per day, 5 days per week) or filtered air (control) for a total of 15 days. At 17 months post-exposure, mice were euthanized and examined for the development of pathological changes in the lung, and whole blood was collected and analyzed using microarray analysis for global mRNA and miRNA expression. Numerous mRNAs and miRNAs in the blood were significantly up- or down-regulated in animals developing pathological changes in the lung after MCA/corn oil administration followed by MWCNT/air inhalation, including fcrl5 and miR-122-5p in the presence of hyperplasia, mthfd2 and miR-206-3p in the presence of fibrosis, fam178a and miR-130a-3p in the presence of bronchiolo-alveolar adenoma, and il7r and miR-210-3p in the presence of bronchiolo-alveolar adenocarcinoma, among others. The changes in miRNA and mRNA expression, and their respective regulatory networks, identified in this study may potentially serve as blood biomarkers for MWCNT-induced lung pathological changes.

System-based identification of toxicity pathways associated with multi-walled carbon nanotube-induced pathological responses.

The fibrous shape and biopersistence of multi-walled carbon nanotubes (MWCNT) have raised concern over their potential toxicity after pulmonary exposure. As in vivo exposure to MWCNT produced a transient inflammatory and progressive fibrotic response, this study sought to identify significant biological processes associated with lung inflammation and fibrosis pathology data, based upon whole genome mRNA expression, bronchoaveolar lavage scores, and morphometric analysis from C57BL/6J mice exposed by pharyngeal aspiration to 0, 10, 20, 40, or 80 µg MWCNT at 1, 7, 28, or 56 days post-exposure. Using a novel computational model employing non-negative matrix factorization and Monte Carlo Markov Chain simulation, significant biological processes with expression similar to MWCNT-induced lung inflammation and fibrosis pathology data in mice were identified. A subset of genes in these processes was determined to be functionally related to either fibrosis or inflammation by Ingenuity Pathway Analysis and was used to determine potential significant signaling cascades. Two genes determined to be functionally related to inflammation and fibrosis, vascular endothelial growth factor A (vegfa) and C-C motif chemokine 2 (ccl2), were confirmed by in vitro studies of mRNA and protein expression in small airway epithelial cells exposed to MWCNT as concordant with in vivo expression. This study identified that the novel computational model was sufficient to determine biological processes strongly associated with the pathology of lung inflammation and fibrosis and could identify potential toxicity signaling pathways and mechanisms of MWCNT exposure which could be used for future animal studies to support human risk assessment and intervention efforts.

Multi-walled carbon nanotubes induce human microvascular endothelial cellular effects in an alveolar-capillary co-culture with small airway epithelial cells.

BACKGROUND: Nanotechnology, particularly the use of multi-walled carbon nanotubes (MWCNT), is a rapidly growing discipline with implications for advancement in a variety of fields. A major route of exposure to MWCNT during both occupational and environmental contact is inhalation. While many studies showed adverse effects to the vascular endothelium upon MWCNT exposure, in vitro results often do not correlate with in vivo effects. This study aimed to determine if an alveolar-capillary co-culture model could determine changes in the vascular endothelium after epithelial exposure to MWCNT. METHODS: A co-culture system in which both human small airway epithelial cells and human microvascular endothelial cells were separated by a Transwell membrane so as to resemble an alveolar-capillary interaction was used. Following exposure of the epithelial layer to MWCNT, the effects to the endothelial barrier were determined. RESULTS: Exposure of the epithelial layer to MWCNT induced multiple changes in the endothelial cell barrier, including an increase in reactive oxygen species, actin rearrangement, loss of VE-cadherin at the cell surface, and an increase in endothelial angiogenic ability. Overall increases in secreted VEGFA, sICAM-1, and sVCAM-1 protein levels, as well as increases in intracellular phospho-NF-κB, phospho-Stat3, and phospho-p38 MAPK, were also noted in HMVEC after epithelial exposure. CONCLUSION: The co-culture system identified that alveolar-capillary exposure to MWCNT induced multiple changes to the underlying endothelium, potentially through cell signaling mediators derived from MWCNT-exposed epithelial cells. Therefore, the co-culture system appears to be a relevant in vitro method to study the pulmonary toxicity of MWCNT.

New perspectives for in vitro risk assessment of multiwalled carbon nanotubes: application of coculture and bioinformatics.

Nanotechnology is a rapidly expanding field with wide application for industrial and medical use; therefore, understanding the toxicity of engineered nanomaterials is critical for their commercialization. While short-term in vivo studies have been performed to understand the toxicity profile of various nanomaterials, there is a current effort to shift toxicological testing from in vivo observational models to predictive and high-throughput in vitro models. However, conventional monoculture results of nanoparticle exposure are often disparate and not predictive of in vivo toxic effects. A coculture system of multiple cell types allows for cross-talk between cells and better mimics the in vivo environment. This review proposes that advanced coculture models, combined with integrated analysis of genome-wide in vivo and in vitro toxicogenomic data, may lead to development of predictive multigene expression-based models to better determine toxicity profiles of nanomaterials and consequent potential human health risk due to exposure to these compounds.

Multiwalled carbon nanotube-induced gene signatures in the mouse lung: potential predictive value for human lung cancer risk and prognosis.

Concerns over the potential for multiwalled carbon nanotubes (MWCNT) to induce lung carcinogenesis have emerged. This study sought to (1) identify gene expression signatures in the mouse lungs following pharyngeal aspiration of well-dispersed MWCNT and (2) determine if these genes were associated with human lung cancer risk and progression. Genome-wide mRNA expression profiles were analyzed in mouse lungs (n = 160) exposed to 0, 10, 20, 40, or 80 µg of MWCNT by pharyngeal aspiration at 1, 7, 28, and 56 d postexposure. By using pairwise statistical analysis of microarray (SAM) and linear modeling, 24 genes were selected, which have significant changes in at least two time points, have a more than 1.5-fold change at all doses, and are significant in the linear model for the dose or the interaction of time and dose. Additionally, a 38-gene set was identified as related to cancer from 330 genes differentially expressed at d 56 postexposure in functional pathway analysis. Using the expression profiles of the cancer-related gene set in 8 mice at d 56 postexposure to 10 µg of MWCNT, a nearest centroid classification accurately predicts human lung cancer survival with a significant hazard ratio in training set (n = 256) and test set (n = 186). Furthermore, both gene signatures were associated with human lung cancer risk (n = 164) with significant odds ratios. These results may lead to development of a surveillance approach for early detection of lung cancer and prognosis associated with MWCNT in the workplace.

The gene expression signature of genomic instability in breast cancer is an independent predictor of clinical outcome.

Recently, expression profiling of breast carcinomas has revealed gene signatures that predict clinical outcome, and discerned prognostically relevant breast cancer subtypes. Measurement of the degree of genomic instability provides a very similar stratification of prognostic groups. We therefore hypothesized that these features are linked. We used gene expression profiling of 48 breast cancer specimens that profoundly differed in their degree of genomic instability and identified a set of 12 genes that defines the 2 groups. The biological and prognostic significance of this gene set was established through survival prediction in published datasets from patients with breast cancer. Of note, the gene expression signatures that define specific prognostic subtypes in other breast cancer datasets, such as luminal A and B, basal, normal-like, and ERBB2+, and prognostic signatures including MammaPrint and Oncotype DX, predicted genomic instability in our samples. This remarkable congruence suggests a biological interdependence of poor-prognosis gene signatures, breast cancer subtypes, genomic instability, and clinical outcome.

An integrative genomic and proteomic approach to chemosensitivity prediction.

New computational approaches are needed to integrate both protein expression and gene expression profiles, extending beyond the correlation analyses of gene and protein expression profiles in the current practices. Here, we developed an algorithm to classify cell line chemosensitivity based on integrated transcriptional and proteomic profiles. We sought to determine whether a combination of gene and protein expression profiles of untreated cells was able to enhance the performance of chemosensitivity prediction. An integrative feature selection scheme was employed to identify chemosensitivity determinants from genome-wide transcriptional profiles and 52 protein expression levels in 60 human cancer cell lines (the NCI-60). A set of 118 anti-cancer drugs whose mechanisms of action were putatively understood was evaluated. Classifiers of the complete range of drug response (sensitive, intermediate, or resistant) were generated for the evaluated anti-cancer drugs, one for each agent. The classifiers were designed to be independent of the cells' tissue origins. The classification accuracy of all the evaluated 118 agents was remarkably better (P<0.001) than that would be achieved by chance. Furthermore, 76 out of the 118 classifiers identified from integrated genomic and protein profiles significantly (P<0.05) improved the accuracy of protein expression-based classifiers identified previously. These results demonstrate that our integrated genomic and proteomic approach enhances the performance of chemosensitivity prediction. This study presents a new analytical framework to identify integrated gene and protein expression signatures for predicting cellular behavior and clinical outcome in general.

Confirmation of gene expression-based prediction of survival in non-small cell lung cancer.

PURPOSE: It is a critical challenge to determine the risk of recurrence in early stage non-small cell lung cancer (NSCLC) patients. Accurate gene expression signatures are needed to classify patients into high- and low-risk groups to improve the selection of patients for adjuvant therapy. EXPERIMENTAL DESIGN: Multiple published microarray data sets were used to evaluate our previously identified lung cancer prognostic gene signature. Expression of the signature genes was further validated with real-time reverse transcription-PCR and Western blot assays of snap-frozen lung cancer tumor tissues. RESULTS: Our previously identified 35-gene signature stratified 264 patients with NSCLC into high- and low-risk groups with distinct overall survival rates (P < 0.05, Kaplan-Meier analysis, log-rank tests). The 35-gene signature further stratified patients with clinical stage 1A diseases into poor prognostic and good prognostic subgroups (P = 0.0007, Kaplan-Meier analysis, log-rank tests). This signature is independent of other prognostic factors for NSCLC, including age, sex, tumor differentiation, tumor grade, and tumor stage. The expression of the signature genes was validated with real-time reverse transcription-PCR analysis of lung cancer tumor specimens. Protein expression of two signature genes, TAL2 and ILF3, was confirmed in lung adenocarcinoma tumors by using Western blot analysis. These two biomarkers showed correlated mRNA and protein overexpression in lung cancer development and progression. CONCLUSIONS: The results indicate that the identified 35-gene signature is an accurate predictor of survival in NSCLC. It provides independent prognostic information in addition to traditional clinicopathologic criteria.

Differential gene regulation in human small airway epithelial cells grown in monoculture versus coculture with human microvascular endothelial cells following multiwalled carbon nanotube exposure.

Concurrent with rising production of carbon-based engineered nanomaterials is a potential increase in respiratory and cardiovascular diseases due to exposure to nanomaterials in the workplace atmosphere. While single-cell models of pulmonary exposure are often used to determine the potential toxicity of nanomaterials in vitro, previous studies have shown that coculture cell models better represent the cellular response and crosstalk that occurs in vivo. This study identified differential gene regulation in human small airway epithelial cells (SAECs) grown either in monoculture or in coculture with human microvascular endothelial cells following exposure of the SAECs to multiwalled carbon nanotubes (MWCNTs). SAEC genes that either changed their regulation direction from upregulated in monoculture to downregulated in coculture (or vice versa) or had a more than a two-fold changed in the same regulation direction were identified. Genes that changed regulation direction were most often involved in the processes of cellular growth and proliferation and cellular immune response and inflammation. Genes that had a more than a two-fold change in regulation in the same direction were most often involved in the inflammatory response. The direction and fold-change of this differential gene regulation suggests that toxicity testing in monoculture may exaggerate cellular responses to MWCNTs, and coculture of cells may provide a more in-depth assessment of toxicological responses.

Multi-walled carbon nanotube-induced gene expression in vitro: concordance with in vivo studies.

There is a current interest in reducing the in vivo toxicity testing of nanomaterials in animals by increasing toxicity testing using in vitro cellular assays; however, toxicological results are seldom concordant between in vivo and in vitro models. This study compared global multi-walled carbon nanotube (MWCNT)-induced gene expression from human lung epithelial and microvascular endothelial cells in monoculture and coculture with gene expression from mouse lungs exposed to MWCNT. Using a cutoff of 10% false discovery rate and 1.5 fold change, we determined that there were more concordant genes (gene expression both up- or downregulated in vivo and in vitro) expressed in both cell types in coculture than in monoculture. When reduced to only those genes involved in inflammation and fibrosis, known outcomes of in vivo MWCNT exposure, there were more disease-related concordant genes expressed in coculture than monoculture. Additionally, different cellular signaling pathways are activated in response to MWCNT dependent upon culturing conditions. As coculture gene expression better correlated with in vivo gene expression, we suggest that cellular cocultures may offer enhanced in vitro models for nanoparticle risk assessment and the reduction of in vivo toxicological testing.

mRNA and miRNA regulatory networks reflective of multi-walled carbon nanotube-induced lung inflammatory and fibrotic pathologies in mice.

Multi-walled carbon nanotubes (MWCNTs) are known for their transient inflammatory and progressive fibrotic pulmonary effects; however, the mechanisms underlying these pathologies are unknown. In this study, we used time-series microarray data of global lung mRNA and miRNA expression isolated from C57BL/6J mice exposed by pharyngeal aspiration to vehicle or 10, 20, 40, or 80 µg MWCNT at 1, 7, 28, or 56 days post-exposure to determine miRNA and mRNA regulatory networks that are potentially involved in MWCNT-induced inflammatory and fibrotic lung etiology. Using a non-negative matrix factorization method, we determined mRNAs and miRNAs with expression profiles associated with pathology patterns of MWCNT-induced inflammation (based on bronchoalveolar lavage score) and fibrosis (based on Sirius Red staining measured with quantitative morphometric analysis). Potential binding targets between pathology-related mRNAs and miRNAs were identified using Ingenuity Pathway Analysis and the miRTarBase, miRecords, and TargetScan databases. Using these experimentally validated and predicted binding targets, we were able to build molecular signaling networks that are potentially reflective of and play a role in MWCNT-induced lung inflammatory and fibrotic pathology. As understanding the regulatory networks between mRNAs and miRNAs in different disease states would be beneficial for understanding the complex mechanisms of pathogenesis, these identified genes and pathways may be useful for determining biomarkers of MWCNT-induced lung inflammation and fibrosis for early detection of disease.

Integrated DNA Copy Number and Gene Expression Regulatory Network Analysis of Non-small Cell Lung Cancer Metastasis.

Integrative analysis of multi-level molecular profiles can distinguish interactions that cannot be revealed based on one kind of data in the analysis of cancer susceptibility and metastasis. DNA copy number variations (CNVs) are common in cancer cells, and their role in cell behaviors and relationship to gene expression (GE) is poorly understood. An integrative analysis of CNV and genome-wide mRNA expression can discover copy number alterations and their possible regulatory effects on GE. This study presents a novel framework to identify important genes and construct potential regulatory networks based on these genes. Using this approach, DNA copy number aberrations and their effects on GE in lung cancer progression were revealed. Specifically, this approach contains the following steps: (1) select a pool of candidate driver genes, which have significant CNV in lung cancer patient tumors or have a significant association with the clinical outcome at the transcriptional level; (2) rank important driver genes in lung cancer patients with good prognosis and poor prognosis, respectively, and use top-ranked driver genes to construct regulatory networks with the COpy Number and EXpression In Cancer (CONEXIC) method; (3) identify experimentally confirmed molecular interactions in the constructed regulatory networks using Ingenuity Pathway Analysis (IPA); and (4) visualize the refined regulatory networks with the software package Genatomy. The constructed CNV/mRNA regulatory networks provide important insights into potential CNV-regulated transcriptional mechanisms in lung cancer metastasis.

Systematic analysis of multiwalled carbon nanotube-induced cellular signaling and gene expression in human small airway epithelial cells.

Multiwalled carbon nanotubes (MWCNT) are one of the most commonly produced nanomaterials, and pulmonary exposure during production, use, and disposal is a concern for the developing nanotechnology field. The airway epithelium is the first line of defense against inhaled particles. In a mouse model, MWCNT were reported to reach the alveolar space of the lung after in vivo exposure, penetrate the epithelial lining, and result in inflammation and progressive fibrosis. This study sought to determine the cellular and gene expression changes in small airway epithelial cells (SAEC) after in vitro exposure to MWCNT in an effort to elucidate potential toxicity mechanisms and signaling pathways. A direct interaction between SAEC and MWCNT was confirmed by both internalization of MWCNT and interaction at the cell periphery. Following exposure, SAEC showed time-dependent increases in reactive oxygen species production, total protein phosphotyrosine and phosphothreonine levels, and migratory behavior. Analysis of gene and protein expression suggested altered regulation of multiple biomarkers of lung damage, carcinogenesis, and tumor progression, as well as genes involved in related signaling pathways. These results demonstrate that MWCNT exposure resulted in the activation of SAEC. Gene expression data derived from MWCNT exposure provide information that may be used to elucidate the underlying mode of action of MWCNT in the small airway and suggest potential prognostic gene signatures for risk assessment.

A population-based gene signature is predictive of breast cancer survival and chemoresponse.

It remains a critical issue to improve the survival rate in patients with recurrent or metastatic breast cancer. This study sought to develop a prognostic scheme based on a 28-gene signature in a broad patient population, including those with advanced disease. Clinically annotated transcriptional profiles of 1,734 breast cancer patients were obtained to validate the 28-gene signature in prognostic categorization. The 28-gene signature generated significant patient stratification with regard to breast cancer disease-free survival (log-rank P<0.0001; n=1,337) and overall survival (log-rank P<0.0001; n=806) in Kaplan-Meier analyses. The gene expression signature provides refined prognosis of disease-free survival (log-rank P<0.006; Kaplan-Meier analysis) within each classic clinicopathologic factor-defined subgroup, including LN-, LN+, ER-, ER+ and tumor grade II. Furthermore, it was investigated whether this gene signature predicts chemoresponse to drugs commonly used to treat breast cancer. The mRNA expression levels of this gene signature in NCI-60 cell lines were used to predict chemoresponse to CMF, tamoxifen, paclitaxel, docetaxel, and doxorubicin (adriamycin). The 28-gene prognostic signature accurately (P<0.02) predicted chemotherapeutic response to the studied drugs. This study confirmed the prognostic applicability of the breast cancer gene signature in a broad clinical setting. This prognostic signature is also predictive of drug response in cancer cell lines.

Impact and interactions between smoking and traditional prognostic factors in lung cancer progression.

BACKGROUND: Cigarette smoking is a well-known risk factor of lung carcinogenesis. The clinical impact of smoking on lung cancer metastases and survival remains unclear. We sought to investigate the effect of smoking intensity on lung cancer treatment failure (represented by overall survival), and the interactions between smoking and clinicopathological factors in lung cancer progression. METHODS: Clinical information was obtained from four non-small cell lung cancer patient cohorts (n=347). Twenty patients were excluded from the analysis because their smoking history was not available. The distribution of smoking intensity on patient age (> or =60 years or <60 years), gender, tumor differentiation (poor, moderate and well differentiated), and clinical stage (1, 2, or 3) was assessed with Kruskal-Wallis rank sum tests. The effect of smoking on cause-specific lung cancer mortality was estimated by using Cox proportional hazard models and Kaplan-Meier analysis. The interactions between clinicopathological factors and smoking intensity with regard to lung cancer overall survival were evaluated with analysis of variance (ANOVA) for Cox modeling. RESULTS: Greater smoking intensity at diagnosis was found in older patients (> or =60 years; p=0.022), male (p=1.35e-7), poorly differentiated tumors (p=8.51e-5), patients with tumor stage 2 (p=0.031), and squamous cell lung cancer patients (p=2.2e-16). Patients who smoked more than 61 packs/year had an increased risk for lung cancer recurrence (hazard ratio=1.41, 95% CI: [1.03, 1.94], log-rank p=0.032) and shorter overall survival period (log-rank p=0.033, Kaplan-Meier analysis) than those who smoked less than 61 packs/year. ANOVA analysis showed that smoking intensity (p=0.03) and tumor stage (p=1.2e-6) are the only significant prognostic factors of lung cancer, whereas patient age, gender, and tumor differentiation were not significant in lung cancer prognostication. There were significant interactions between smoking and clinical stage (p=0.02) as well as patient age and tumor differentiation (p=0.03) in lung cancer progression. CONCLUSION: Smoking intensity at diagnosis is an independent, significant prognostic factor of non-small cell lung cancer. This factor could be used in patient selection for chemoprevention of tumor metastases and relapse. Additionally, the information may be used for clinically relevant tobacco prevention and intervention messages.