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1.  Prognostic significance of RNA-dependent protein kinase (PKR) on non-small cell lung cancer patients 
Purpose
The role of RNA-dependent protein kinase (PKR) in antiviral defence mechanisms and in cellular differentiation, growth, and apoptosis is well known, but the role of PKR in human lung cancer remains poorly understood. To explore the role of PKR in human lung cancer, we evaluated PKR’s expression in tissue microarray specimens from both non-small cell lung cancer (NSCLC) and normal human bronchial epithelium tissue.
Experimental Design
Tissue microarray samples (TMA-1) from 231 lung cancers were stained with PKR antibody and validated on TMA-2 from 224 lung cancers. Immunohistochemical expression score was quantified by three pathologists independently. Survival probability was computed by the Kaplan-Meier method.
Results
The NSCLC cells showed lower levels of PKR expression than normal bronchial epithelium cells did. We also found a significant association between lower levels of PKR expression and lymph node metastasis. We found that loss of PKR expression is correlated with a more aggressive behavior, and that a high PKR expression predicts a subgroup of patients with a favorable outcome. Univariate and multivariate Cox proportional hazards regression models showed that a lower level of PKR expression was significantly associated with shorter survival in NSCLC patients. We further validated and confirmed that PKR to be a powerful prognostic factor in TMA-2 lung cancer (HR=0.22, P<0.0001).
Conclusions
Our findings first indicate that PKR expression is an independent prognostic variable in NSCLC patients.
doi:10.1158/1078-0432.CCR-10-0753
PMCID: PMC3070287  PMID: 20930042
PKR; Biomarker; Lung cancer
2.  Collagen XXIII—a potential biomarker for the detection of primary and recurrent non-small cell lung cancer 
Background
Collagen XXIII is a transmembrane collagen previously shown to be upregulated in metastatic prostate cancer. This study’s purpose was to determine the protein expression of collagen XXIII in tumor tissues from a variety of cancers and to assess collagen XXIII’s utility as a biomarker for non small-cell lung cancer (NSCLC).
Methods
A multi-cancer tissue microarray (TMA) was used for immunohistochemical examination of collagen XXIII protein expression in a variety of cancers. Subsequently, collagen XXIII expression was analyzed in three separate cohorts using TMAs with representative tumor and control lung tissues from NSCLC patients. In addition, NSCLC patient urine samples were analyzed for the presence of collagen XXIII via Western blot.
Results
Collagen XXIII was present in tissue samples from a variety of cancers. Within lung cancer tissues, collagen XXIII staining was enriched in NSCLC subtypes. Collagen XXIII was present in 294 of 333 (88%) lung adenocarcinomas and 97 of 133 (73%) squamous cell carcinomas (SqCC). In urine, collagen XXIII was present in 23 of 29 (79%) NSCLC patient samples but only in 15 of 54 (28%) control samples. High collagen XXIII staining intensity correlated with shorter recurrence-free survival in NSCLC patients.
Conclusions
We demonstrate the capability of collagen XXIII as a tissue and urinary biomarker for NSCLC, where positivity in tissue or urine significantly correlates with presence of NSCLC and high staining intensity is a significant recurrence predictor.
Impact
Inclusion of collagen XXIII in a tissue or urine-based cancer biomarker panel could inform NSCLC patient treatment decisions.
doi:10.1158/1055-9965.EPI-09-1095
PMCID: PMC2880394  PMID: 20447926
tissue microarray; fluid biomarker; cancer surveillance and screening
3.  High expression of a disintegrin and metalloproteinase-9 predicts a shortened survival time in completely resected stage I non-small cell lung cancer 
Oncology Letters  2013;5(5):1461-1466.
The aim of this study was to investigate the abnormal expression of a disintegrin and metalloproteinase-9 (ADAM9) in human resected non-small cell lung cancer (NSCLC) tissue, in order to evaluate the significance of ADAM9 expression in surgically resected NSCLC. Sixty-four cases of completely resected stage I NSCLC with mediastinal N2 lymph node dissection were immunohistochemically analyzed for ADAM9 protein expression. Survival, univariate and multivariate analyses were conducted to assess the significance of ADAM9 expression and its correlation with other clinicopathological characteristics. ADAM9 was observed to be significantly more highly expressed in NSCLC tissue compared with normal control lung tissue (P=0.001). The 5-year survival rate for patients with NSCLC tissues highly expressing ADAM9 was significantly lower when compared with NSCLC tissues of patients exhibiting low expression of ADAM9 (56.9 vs. 88.9%, P= 0.012). Multivariate analysis identified that high expression of ADAM9 is an independent factor of shortened survival time in resected stage I NSCLC (HR, 3.385; 95% CI, 1.224–9.360; P=0.019). These results clearly demonstrate that ADAM9 is highly expressed in NSCLC and highly expressed ADAM9 correlates with shortened survival time, suggesting that ADAM9 is a novel biomarker for predicting prognosis in resected stage I NSCLC. ADAM9 may also become a useful predictive biomarker for the selection of adjuvant chemotherapy treatment of NSCLC.
doi:10.3892/ol.2013.1209
PMCID: PMC3678878  PMID: 23761811
ADAM9; lung neoplasm; immunohistochemistry; prognosis; lobectomy
4.  Wnt Pathway Activation Predicts Increased Risk of Tumor Recurrence in Patients with Stage I Non-Small Cell Lung Cancer 
Annals of surgery  2013;257(3):548-554.
Objective
To determine the prevalence of Wnt pathway activation in patients with stage I NSCLC and its influence on lung cancer recurrence.
Background
Despite resection, the 5 year recurrence with localized stage I non-small cell lung cancer (NSCLC) is 18.4–24%. Aberrant Wnt signaling activation plays an important role in a wide variety of tumor types. However, there is not much known about the role Wnt pathway plays in patients with stage I lung cancer
Methods
Tumor and normal lung tissues from 55 patients following resection for stage I NSCLC were subjected to glutathione-S-transferase (GST) E-cadherin pull-down and immunoblot analysis to assess levels of uncomplexed β-catenin, a reliable measure of Wnt signaling activation. The β-catenin gene was also screened for oncogenic mutations in tumors with activated Wnt signaling. Cancer recurrence rates were correlated in a blinded manner in patients with Wnt pathway positive and negative tumors.
Results
Tumors in twenty patients (36.4%) scored as Wnt positive with only one exhibiting a β-catenin oncogenic mutation. Patients with Wnt positive tumors experienced a significantly higher rate of overall cancer recurrence than those with Wnt negative tumors (30.0% vs. 5.7%, p=0.02), with 25.0% exhibiting distal tumor recurrence compared to 2.9% in the Wnt negative group (p=0.02).
Conclusions
Wnt pathway activation was present in a substantial fraction of Stage I NSCLCs, which was rarely due to mutations. Moreover, Wnt pathway activation was associated with a significantly higher rate of tumor recurrence. These findings suggest that Wnt activation reflects a more aggressive tumor phenotype and identifies patients who may benefit from more aggressive therapy in addition to resection.
doi:10.1097/SLA.0b013e31826d81fd
PMCID: PMC3546156  PMID: 23011390
5.  FER overexpression is associated with poor postoperative prognosis and cancer-cell survival in non-small cell lung cancer 
Here, we show that overexpression of fer tyrosine kinase (FER), a non-receptor tyrosine kinase, predicts poor postoperative outcome and might be involved in cancer-cell survival in non-small cell lung cancer (NSCLC). Systematic screening using in silico analyses and quantitative RT-PCR revealed that FER was overexpressed in about 10% of NSCLC patients. Evaluation of FER expression using immunohistochemistry (IHC) on tissue microarrays was consistent with the mRNA level detected using quantitative RT-PCR. In analyses of 135 NSCLC patients who had undergone potential curative resection, we found that FER overexpression detected using IHC had no association with clinicopathological features such as age, sex, smoking history, histological type, disease stage, T factor, N factor, adjuvant chemotherapy history, or EGFR mutation, but was correlated with poor postoperative survival periods. A multivariate Cox regression analysis showed that this prognostic impact was independent of other clinicopathological features. In functional analyses of FER in vitro, FER exhibited a transforming activity, suggesting that it possesses oncogenic functions. We also found that human lung cancer NCI-H661 cells, which exhibited FER-outlier expression, were led to apoptosis by the knockdown of FER using RNA interference. FER overexpression might serve as a prognostic biomarker and be involved in cancer-cell survival in NSCLC.
PMCID: PMC3606849  PMID: 23573306
Non-small cell lung cancer; FER overexpression; prognostic factor
6.  Snail promotes CXCR2 ligand dependent tumor progression in NSCLC 
Purpose
As a transcriptional repressor of E-cadherin, Snail has predominantly been associated with epithelial-mesenchymal transition (EMT), invasion, and metastasis. However, other important Snail-dependent malignant phenotypes have not been fully explored. Here, we investigate the contributions of Snail to the progression of non-small cell lung cancer (NSCLC).
Experimental Design
Immunohistochemistry was performed to quantify and localize Snail in human lung cancer tissues, and tissue microarray analysis (TMA) was utilized to correlate these findings with survival. NSCLC cell lines gene-modified to stably over-express Snail were evaluated in vivo in two severe combined immunodeficiency (SCID) murine tumor models. Differential gene expression between Snail over-expressing and control cell lines was evaluated using gene expression microarray analysis.
Results
Snail is up-regulated in human NSCLC tissue, and high levels of Snail expression correlate with decreased survival (p<0.026). In a heterotopic model, mice bearing Snail over-expressing tumors developed increased primary tumor burden (p=0.008). In an orthotopic model, mice bearing Snail over-expressing tumors also demonstrated a trend toward increased metastases. In addition, Snail over-expression led to increased angiogenesis in primary tumors as measured by MECA-32 (p<0.05) positivity and CXCL8 (p=0.002) and CXCL5 (p=0.0003) concentrations in tumor homogenates. Demonstrating the importance of these pro-angiogenic chemokines, the Snail-mediated increase in tumor burden was abrogated with CXCR2 blockade. Gene expression analysis also revealed Snail-associated differential gene expression with the potential to affect angiogenesis and diverse aspects of lung cancer progression.
Conclusion
Snail up-regulation plays a role in human NSCLC by promoting tumor progression mediated by CXCR2 ligands.
doi:10.1158/1078-0432.CCR-09-1558
PMCID: PMC2783274  PMID: 19887480
Snail; lung cancer; angiogenesis; CXCL8; CXCL5
7.  Targeting Aromatase and Estrogen Signaling in Human Non-Small Cell Lung Cancer 
Lung cancer has become increasingly common in women, and gender differences in the physiology and pathogenesis of the disease have suggested a role for estrogens. In the lung recent data have shown local production of estrogens from androgens via the action of aromatase enzyme and higher levels of estrogen in tumor tissue as compared with surrounding normal lung tissue. High levels of aromatase expression are also maintained in metastases as compared with primary tumors. Consistent with these findings, clinical studies suggest that aromatase expression may be a useful predictive biomarker for prognosis in the management of non-small cell lung cancer (NSCLC), the most common form of lung malignancy. Low levels of aromatase associate with a higher probability of long-term survival in older women with early stage NSCLC. Treatment of lung NSCLC xenografts in vivo with an aromatase inhibitor (exemestane) alone or combined with standard cisplatin chemotherapy elicits a significant reduction in tumor progression as compared to paired controls. Further, lung cancer progression is also governed by complex interactions between estrogen and growth factor signaling pathways to stimulate the growth of NSCLC as well as tumor-associated angiogenesis. We find that combination therapy with the multitargeted growth factor receptor inhibitor vandetanib and the estrogen receptor antagonist fulvestrant inhibit tumor growth more effectively than either treatment administered alone. Thus, incorporation of antiestrogen treatment strategies in standard antitumor therapies for NSCLC may contribute to improved patient outcome, an approach that deserves to be tested in clinical trials.
doi:10.1111/j.1749-6632.2009.04116.x
PMCID: PMC2782616  PMID: 19250205
non-small cell lung cancer (NSCLC); aromatase; CYP19; estrogen receptor (ER); epidermal growth factor receptor (EGFR); vascular endothelial growth factor (VEGF) receptor; anastrazole; exemestane; fulvestrant; vandetanib
8.  Prognostic Impact of MiR-155 in Non-Small Cell Lung Cancer Evaluated by in Situ Hybridization 
Background
In recent years, microRNAs (miRNAs) have been found to play an essential role in tumor development. In lung tumorigenesis, targets and pathways of miRNAs are being revealed, and further translational research in this field is warranted. MiR-155 is one of the miRNAs most consistently involved in various neoplastic diseases. We aimed to investigate the prognostic impact of the multifunctional miR-155 in non-small cell lung cancer (NSCLC) patients.
Methods
Tumor tissue samples from 335 resected stage I to IIIA NSCLC patients were obtained and tissue microarrays (TMAs) were constructed with four cores from each tumor specimen. In situ hybridization (ISH) was used to evaluate the expression of miR-155.
Results
There were 191 squamous cell carcinomas (SCCs), 95 adenocarcinomas (ACs), 31 large cell carcinomas and 18 bronchioalveolar carcinomas. MiR-155 expression did not have a significant prognostic impact in the total cohort (P = 0.43). In ACs, high miR-155 expression tended to a significant negative prognostic effect on survival in univariate analysis (P = 0.086) and was an independent prognostic factor in multivariate analysis (HR 1.87, CI 95% 1.01 - 3.48, P = 0.047). In SCC patients with lymph node metastasis, however, miR-155 had a positive prognostic impact on survival in univariate (P = 0.034) as well as in multivariate (HR 0.45, CI 95% 0.21-0.96, P = 0.039) analysis.
Conclusions
The prognostic impact of miR-155 depends on histological subtype and nodal status in NSCLC.
doi:10.1186/1479-5876-9-6
PMCID: PMC3023703  PMID: 21219656
9.  Identifying and Targeting ROS1 Gene Fusions in Non-Small Cell Lung Cancer 
Purpose
Oncogenic gene fusions involving the 3’ region of ROS1 kinase have been identified in various human cancers. In this study, we sought to characterize ROS1 fusion genes in non-small cell lung cancer (NSCLC) and establish the fusion proteins as drug targets.
Experimental Design
A NSCLC tissue microarray (TMA) panel containing 447 samples was screened for ROS1 rearrangement by fluorescence in-situ hybridization (FISH). This assay was also used to screen NSCLC patients. In positive samples, the identity of the fusion partner was determined through inverse-PCR and RT-PCR. In addition, the clinical utility of ROS1 inhibition was assessed by treating a ROS1-positive patient with crizotinib. The HCC78 cell line, which expresses the SLC34A2-ROS1 fusion, was treated with kinase inhibitors that have activity against ROS1. The effects of ROS1 inhibition on proliferation, cell-cycle progression, and cell signaling pathways were analyzed by MTS assay, flow cytometry, and western blotting.
Results
In the TMA panel, 5/428 (1.2%) evaluable samples were found to be positive for ROS1 rearrangement. Additionally, 1/48 patients tested positive for rearrangement, and this patient demonstrated tumor shrinkage upon treatment with crizotinib. The patient and one TMA sample displayed expression of the recently identified SDC4-ROS1 fusion, while two TMA samples expressed the CD74-ROS1 fusion and two others expressed the SLC34A2-ROS1 fusion. In HCC78 cells, treatment with ROS1 inhibitors was anti-proliferative and down-regulated signaling pathways that are critical for growth and survival.
Conclusions
ROS1 inhibition may be an effective treatment strategy for the subset of NSCLC patients whose tumors express ROS1 fusion genes.
doi:10.1158/1078-0432.CCR-12-0550
PMCID: PMC3703205  PMID: 22919003
10.  The clinical significance of Psoriasin for non-small cell lung cancer patients and its biological impact on lung cancer cell functions 
BMC Cancer  2012;12:588.
Background
Psoriasin (S100A7) is a member of the S100 gene family. Alteration of Psoriasin expression has previously been reported to play an important role in cancer aggressive behaviour. The current study sought to investigate the level of Psoriasin expression at the mRNA level in a cohort of patients with non-small cell lung cancer (NSCLC), the association with clinical implication and outcomes, and the molecular and cellular impact of the protein on lung cancer cells.
Methods
Fresh frozen NSCLC cell carcinoma tissues, along with matched normal tissues were obtained from 83 NSCLC patients who received curative resection from January 2003 to December 2011. The expression of Psoriasin in the NSCLC specimens was assessed using both quantitative real time PCR (QPCR) and immunochemical staining. Knockdown and forced expression of Psoriasin in NSCLC cell lines were carried out using constructed plasmid vectors carrying either ribozyme transgenes targeting human Psoriasin or full-length coding sequence, respectively. The effect of Psoriasin on the functions of NSCLC cells was determined using a variety of in vitro cell function assays.
Results
Higher mRNA levels of Psoriasin were observed in tumour tissues when compared to both the paired normal background tissues and none paired normal tissues (p = 0.0251 and 0.0195). The mRNA level of Psoriasin was found to be higher in the squamous carcinoma (P=0.035). Higher Psoriasin expression is associated with poor prognosis. The cell function tests had supportive results to the clinical findings. Over-expression of Posriasin in lung cancer cells (SK-MES-1) resulted in an increase in in vitro growth and invasiveness. In contrast, Psoriasin knockdown suppressed cell growth and invasion (P<0.05), but increased cell adhesion (P<0.05).
Conclusions
Psoriasin expression is increased in lung cancer, more specifically in lung squamous carcinoma compared with adenocarcinoma, and is associated with poor prognosis. Psoriasin plays crucial roles in regulating the growth and invasion of lung cancer cells.
doi:10.1186/1471-2407-12-588
PMCID: PMC3537740  PMID: 23228205
Psoriasin; S100A7; Lung cancer; Adhesion and invasion
11.  Prognostic impact of Insulin Receptor Expression on Survival of Patients with Nonf-Small Cell Lung Cancer 
Cancer  2011;118(9):2454-2465.
Background
The purpose of this study was to characterize insulin receptor (IR) and insulin-like growth factor-1 receptor (IGF-1R) expression in patients with non-small cell lung cancer (NSCLC).
Methods
A total of 459 patients who underwent curative resection of NSCLC were studied (median follow-up duration, 4.01 years). Expression of the IR and IGF-1R protein in tumor specimens was assessed immunohistochemically using tissue microarrays.
Results
The cytoplasmic IR score was higher in patients with adenocarcinoma (ADC) than in those with squamous cell carcinoma (SCC) whereas cytoplasmic IGF-1R score was higher in patients with SCC than those with ADC. Neither IR nor IGF-1R expression was associated with sex, smoking history, or clinical stage. Patients with positive IR or IGF-1R expression levels had poor recurrence-free (RFS) (3.8 vs. 3.3 years; 3.8 vs. 2.0 years, respectively), but similar overall survival (OS). Patients with high expression levels of IR and IGF-1R had shorter RFS and OS compared to those with low levels of IR and/or IGF-1R expression. Finally, a multivariate analysis revealed the impact of IR, but not of IGF-1R, as an independent predictive marker of NSCLC survival: hazard ratio (HR) for OS, 1.005 (95% confidence interval [CI], 1.001 – 1.010], HR for RFS, 1.005 (95% CI, 1.001 – 1.009), when IR score was tested as a continuous variable.
Conclusions
Overexpression of IR predicts a poor survival among patients with NSCLC, especially those with SCC. These results might serve as future guidance to the clinical trials involving IR or IGR-1R targeting agents.
doi:10.1002/cncr.26492
PMCID: PMC3298843  PMID: 21952750
Carcinoma; Non-Small-Cell Lung; Receptor; Insulin; Receptor; IGF Type 1; Prognosis; Survival
12.  Analysis of GAGE, NY-ESO-1 and SP17 cancer/testis antigen expression in early stage non-small cell lung carcinoma 
BMC Cancer  2013;13:466.
Background
The unique expression pattern and immunogenic properties of cancer/testis antigens make them ideal targets for immunotherapy of cancer. The MAGE-A3 cancer/testis antigen is frequently expressed in non-small cell lung cancer (NSCLC) and vaccination with MAGE-A3 in patients with MAGE-A3-positive NSCLC has shown promising results. However, little is known about the expression of other cancer/testis antigens in NSCLC. In the present study the expression of cancer/testis antigens GAGE, NY-ESO-1 and SP17 was investigated in patients with completely resected, early stage, primary NSCLC.
Methods
Tumor biopsies from normal lung tissue and from a large cohort (n = 169) of NSCLC patients were examined for GAGE, NY-ESO-1 and SP17 protein expression by immunohistochemical analysis. The expression of these antigens was further matched to clinical and pathological features using univariate cox regression analysis.
Results
GAGE and NY-ESO-1 cancer/testis antigens were not expressed in normal lung tissue, while SP17 was expressed in ciliated lung epithelia. The frequency of GAGE, NY-ESO-1 and SP17 expression in NSCLC tumors were 26.0% (44/169), 11.8% (20/169) and 4.7% (8/169), respectively, and 33.1% (56/169) of the tumors expressed at least one of these antigens. In general, the expression of GAGE, NY-ESO-1 and SP17 was not significantly associated with a specific histotype (adenocarcinoma vs. squamous cell carcinoma), but high-level GAGE expression (>50%) was more frequent in squamous cell carcinoma (p = 0.02). Furthermore, the frequency of GAGE expression was demonstrated to be significantly higher in stage II-IIIa than stage I NSCLC (17.0% vs. 35.8%; p = 0.02). Analysis of the relation between tumor expression of GAGE and NY-ESO-1 and survival endpoints revealed no significant associations.
Conclusion
Our study demonstrates that GAGE, NY-ESO-1 and SP17 cancer/testis antigens are candidate targets for immunotherapy of NSCLC and further suggest that multi-antigen vaccines may be beneficial.
doi:10.1186/1471-2407-13-466
PMCID: PMC3851761  PMID: 24103781
Cancer/testis antigen; Immunotherapy; GAGE; NY-ESO-1; SP17; Lung cancer
13.  A five-gene and corresponding-protein signature for stage-I lung adenocarcinoma prognosis 
PURPOSE
Identification of effective markers for outcome is expected to improve the clinical management of non-small cell lung cancer (NSCLC). Here, we assessed in NSCLC the prognostic efficacy of genes, which we had previously found to be differentially expressed in an in vitro model of human lung carcinogenesis.
EXPERIMENTAL DESIGN
Prediction algorithms and risk-score models were applied to the expression of the genes in publicly available NSCLC expression datasets. The prognostic capacity of the immunohistochemical expression of proteins encoded by these genes was also tested using formalin-fixed paraffin-embedded (FFPE) tissue specimens from 156 lung adenocarcinomas and 79 squamous cell carcinomas (SCCs).
RESULTS
The survival of all-stages (p<0.001, HR=2.0) or stage-I (p<0.001, HR=2.84) adenocarcinoma patients that expressed the five-gene in vitro lung carcinogenesis model (FILM) signature was significantly poorer than that of patients who did not. No survival differences were observed between SCCs predicted to express or lack FILM signature. Moreover, all stages (p<0.001, HR=1.95) or stage-I (p=0.001, HR=2.6) adenocarcinoma patients predicted to be at high risk by FILM transcript exhibited significantly worse survival than patients at low risk. Furthermore, the corresponding protein signature was associated with poor survival (all stages, p<0.001, HR=3.6; stage-I, p<0.001, HR=3.5; stage-IB, p<0.001, HR=4.6) and mortality risk (all stages, p=0.001, HR=4.0; stage-I, p=0.01, HR=3.4; stage-IB, p<0.001, HR=7.2) in lung adenocarcinoma patients.
CONCLUSIONS
Our findings highlight a gene and corresponding protein signature with effective capacity for identification of stage-I lung adenocarcinoma patients with poor prognosis that are likely to benefit from adjuvant therapy.
doi:10.1158/1078-0432.CCR-10-2703
PMCID: PMC3079395  PMID: 21163870
Lung adenocarcinoma; NSCLC; gene signature; prognosis
14.  Prognostic potential of ERCC1 protein expression and clinicopathologic factors in stage III/N2 non-small cell lung cancer 
Background
Pathological stage III/N2 non-small cell lung cancer (NSCLC) is heterogeneous, and the optimal prognostic marker for survival remains unclear in Chinese patients. The aim of the present study was to assess the prognostic value of the clinicopathologic features and excision repair cross-complementing group-1 (ERCC1) in resected p-stage III/N2 NSCLC patients that received cisplatin-based adjuvant chemotherapy.
Methods
Clinical data concerning 115 patients with histopathologically confirmed stage III/N2 NSCLC who underwent a complete resection were reviewed retrospectively. All patients received cisplatin-based adjuvant chemotherapy. The protein expression levels for ERCC1 were immunohistochemically examined in 115 patients. The relationship between the ERCC1 protein expression level and the clinical outcomes of the patients was then observed.
Results
The 5-year survival rate and median survival time of patients with pathological stage III/N2 NSCLC after surgery and postoperative chemotherapy was 27.0% and 28.0 months, respectively. Survival of patients with ERCC1 negative tumors was significantly longer than those with ERCC1 positive tumors (p = 0.004). However, it was not entirely clear whether adjuvant chemotherapy with cisplatin-based agents was beneficial for ERCC1-negative patients with p-stage III/N2. A multivariate analysis of survival in patients with stage III/N2 NSCLC showed that surgical procedure (pneumonectomy vs. lobectomy; p = 0.001), number of involved lymph nodes (≤5 vs. >5; p = 0.001) and ERCC1 protein expression (negative vs. positive; p = 0.012) were significant prognostic factors. In addition, the prognosis of patients with skip mediastinal lymph node metastasis showed a tendency for improved survival, but this was no significant (p = 0.432).
Conclusions
Findings from this retrospective study suggested that the number of involved lymph nodes and the type of pulmonary resection are significant and independent prognosis factors in patients with p-stage III/N2 NSCLC. In addition, it was found that ERCC1 protein expression might play an important role in the prognosis of p-stage III/N2 NSCLC patients treated with cisplatin-based adjuvant chemotherapy.
doi:10.1186/1749-8090-8-149
PMCID: PMC3681661  PMID: 23759026
Excision repair cross complementation 1; Non-small lung cancer; Number of involved lymph nodes; Prognosis; Skip metastasis
15.  Simultaneous Multi-Antibody Staining in Non-Small Cell Lung Cancer Strengthens Diagnostic Accuracy Especially in Small Tissue Samples 
PLoS ONE  2013;8(2):e56333.
Histological subclassification of non-small cell lung cancer (NSCLC) has growing therapeutic impact. In advanced cancer stages tissue specimens are usually bioptically collected. These small samples are of extraordinary value since molecular analyses are gaining importance for targeted therapies. We therefore studied the feasibility, diagnostic accuracy, economic and prognostic effects of a tissue sparing simultaneous multi-antibody assay for subclassification of NSCLC. Of 265 NSCLC patients tissue multi arrays (TMA) were constructed to simulate biopsy samples. TMAs were stained by a simultaneous bi-color multi-antibody assay consisting of TTF1, Vimentin, p63 and neuroendocrine markers (CD56, chromogranin A, synaptophysin). Classification was based mainly on the current proposal of the IASLC with a hierarchical decision tree for subclassification into adenocarcinoma (LAC), squamous cell carcinoma (SCC), large cell neuroendocrine carcinoma (LCNEC) and NSCLC not otherwise specified. Investigation of tumor heterogeneity showed an explicit lower variation for immunohistochemical analyses compared to conventional classification. Furthermore, survival analysis of our combined immunohistochemical classification revealed distinct separation of each entity's survival curve. This was statistically significant for therapeutically important subgroups (p = 0.045). As morphological and molecular cancer testing is emerging, our multi-antibody assay in combination with standardized classification delivers accurate and reliable separation of histomorphological diagnoses. Additionally, it permits clinically relevant subtyping of NSCLC including LCNEC. Our multi-antibody assay may therefore be of special value, especially in diagnosing small biopsies. It futher delivers substantial prognostic information with therapeutic consequences. Integration of immunohistochemical subtyping including investigation of neuroendocrine differentiation into standard histopathological classification of NSCLC must, therefore, be considered.
doi:10.1371/journal.pone.0056333
PMCID: PMC3572034  PMID: 23418554
16.  An Integrated Analysis of miRNA and mRNA Expressions in Non-Small Cell Lung Cancers 
PLoS ONE  2011;6(10):e26502.
Using DNA microarrays, we generated both mRNA and miRNA expression data from 6 non-small cell lung cancer (NSCLC) tissues and their matching normal control from adjacent tissues to identify potential miRNA markers for diagnostics. We demonstrated that hsa-miR-96 is significantly and consistently up-regulated in all 6 NSCLCs. We validated this result in an independent set of 35 paired tumors and their adjacent normal tissues, as well as their sera that are collected before surgical resection or chemotherapy, and the results suggested that hsa-miR-96 may play an important role in NSCLC development and has great potential to be used as a noninvasive marker for diagnosing NSCLC. We predicted potential miRNA target mRNAs based on different methods (TargetScan and miRanda). Further classification of miRNA regulated genes based on their relationship with miRNAs revealed that hsa-miR-96 and certain other miRNAs tend to down-regulate their target mRNAs in NSCLC development, which have expression levels permissive to direct interaction between miRNAs and their target mRNAs. In addition, we identified a significant correlation of miRNA regulation with genes coincide with high density of CpG islands, which suggests that miRNA may represent a primary regulatory mechanism governing basic cellular functions and cell differentiations, and such mechanism may be complementary to DNA methylation in repressing or activating gene expression.
doi:10.1371/journal.pone.0026502
PMCID: PMC3203153  PMID: 22046296
17.  Integrative Proteomics and Tissue Microarray Profiling Indicate the Association between Overexpressed Serum Proteins and Non-Small Cell Lung Cancer 
PLoS ONE  2012;7(12):e51748.
Lung cancer is the leading cause of cancer deaths worldwide. Clinically, the treatment of non-small cell lung cancer (NSCLC) can be improved by the early detection and risk screening among population. To meet this need, here we describe the application of extensive peptide level fractionation coupled with label free quantitative proteomics for the discovery of potential serum biomarkers for lung cancer, and the usage of Tissue microarray analysis (TMA) and Multiple reaction monitoring (MRM) assays for the following up validations in the verification phase. Using these state-of-art, currently available clinical proteomic approaches, in the discovery phase we confidently identified 647 serum proteins, and 101 proteins showed a statistically significant association with NSCLC in our 18 discovery samples. This serum proteomic dataset allowed us to discern the differential patterns and abnormal biological processes in the lung cancer blood. Of these proteins, Alpha-1B-glycoprotein (A1BG) and Leucine-rich alpha-2-glycoprotein (LRG1), two plasma glycoproteins with previously unknown function were selected as examples for which TMA and MRM verification were performed in a large sample set consisting about 100 patients. We revealed that A1BG and LRG1 were overexpressed in both the blood level and tumor sections, which can be referred to separate lung cancer patients from healthy cases.
doi:10.1371/journal.pone.0051748
PMCID: PMC3526638  PMID: 23284758
18.  MALDI-MS derived prognostic protein markers for resected non-small cell lung cancer 
Proteomics. Clinical applications  2008;2(0):1508-1517.
Protein signals obtained directly from frozen lung tissue sections using MALDI-MS were used to predict nodal involvement and survival in resected non-small cell lung cancer (NSCLC). We have identified a list of these protein signals and further evaluated their prognostic values for NSCLC using immunohistochemistry (IHC). Kaplan-Meier analysis was used to assess the mortality risk associated with the prognostic protein IHC-staining intensities. The combined IHC scores of calmodulin, thymosin β4, and thymosin β10 were found to be correlated with NSCLC patient survival (p = 0.004). Furthermore, low cofilin-1 IHC-staining intensity was found to be correlated with a better outcome for patients with negative lymph node status (p = 0.006) while high cofilin-1 IHC-staining intensity was found to be correlated with a better outcome for patients with positive node status (p = 0.034). In conclusion, the prognostic protein signals selected using MALDI-MS can be identified and tested by IHC in formalin-fixed tissue samples. MALDI-MS-derived protein signals can be potentially translated to a conventional clinical setting to aid in the prognosis of patients with NSCLC at the molecular level.
doi:10.1002/prca.200800094
PMCID: PMC3728564  PMID: 21136798
Biomarkers; Immunohistochemistry; MALDI-MS; Non-small cell lung cancer; Prognosis
19.  Comparison of outcomes in patients with stage III versus limited stage IV non-small cell lung cancer 
Background
Standard therapy for metastatic non small cell lung cancer (NSCLC) includes palliative systemic chemotherapy and/or radiotherapy. Recent studies of patients with limited metastases treated with curative-intent stereotactic body radiation therapy (SBRT) have shown encouraging survival. We hypothesized that patients treated with SBRT for limited metastases have comparable outcomes with those treated with curative-intent radiation for Stage III NSCLC.
Methods
We retrospectively reviewed the records of NSCLC patients treated with curative-intent radiotherapy at the University of Rochester from 2000-2008. We identified 3 groups of patients with NSCLC: stage III, stage IV, and recurrent stage IV (initial stage I-II). All stage IV NSCLC patients treated with SBRT had ≤ 8 lesions.
Results
Of 146 patients, 88% had KPS ≥ 80%, 30% had > 5% weight loss, and 95% were smokers. The 5-year OS from date of NSCLC diagnosis for stage III, initial stage IV and recurrent stage IV was 7%, 14%, and 27% respectively. The 5-year OS from date of metastatic diagnosis was significantly (p < 0.00001) superior among those with limited metastases (≤ 8 lesions) versus stage III patients who developed extensive metastases not amenable to SBRT (14% vs. 0%).
Conclusion
Stage IV NSCLC is a heterogeneous patient population, with a selected cohort apparently faring better than Stage III patients. Though patients with limited metastases are favorably selected by virtue of more indolent disease and/or less bulky disease burden, perhaps staging these patients differently is appropriate for prognostic and treatment characterization. Aggressive local therapy may be indicated in these patients, though prospective clinical studies are needed.
doi:10.1186/1748-717X-6-80
PMCID: PMC3141527  PMID: 21718501
Stereotactic Body Radiotherapy; Oligometastases; Non-Small Cell Lung Cancer
20.  Fluorodeoxyglucose positron emission tomography and tumor marker expression in non–small cell lung cancer 
Objective
The best current noninvasive surrogate for tumor biology is fluorodeoxyglucose positron emission tomography (FDG–PET). Both FDG–PET maximal standardized uptake values and selected tumor markers have been shown to correlate with stage, nodal disease, and survival in non–small cell lung cancer (NSCLC). However, there are limited data correlating FDG–PET with tumor markers. The purpose of this study was to determine the correlation of tumor marker expression with FDG–PET maximal standardized uptake values in NSCLC.
Methods
FDG–PET maximal standardized uptake values were calculated in patients with NSCLC (n = 149). No patient had induction chemoradiotherapy. Intraoperative NSCLC tissue was obtained and tissue microarrays were created. Immunohistochemical analysis was performed for 5 known NSCLC tumor markers (glucose transporter 1, p53, cyclin D1, epidermal growth factor receptor, and vascular endothelial growth factor). Each tumor marker was assessed independently by two pathologists using common grading criteria. Subgroup analysis based on histologic characteristics and regional nodal status was performed.
Results
FDG–PET correlated with T classification (P<.0001), N stage (P = .002), and greatest tumor dimension (P<.0001). In addition, increasing maximal standardized uptake values correlated with increased expression of glucose transporter 1 (P<.0001) and p53 (P =.04) in adenocarcinoma. Epidermal growth factor receptor expression correlated with maximal standardized uptake values without predilection for histologic subtype (P = .004).
Conclusion
FDG–PET maximal standardized uptake values correlate with an increased expression of glucose transporter 1 and p53 in lung adenocarcinoma, but not squamous cell cancer. Future studies attempting to correlate FDG–PET with tumor biology will need to consider the effect of different tumor histologic types.
doi:10.1016/j.jtcvs.2008.10.014
PMCID: PMC3632077  PMID: 19154901
21.  High-level expression of Rad51 is an independent prognostic marker of survival in non-small-cell lung cancer patients 
British Journal of Cancer  2005;93(1):137-143.
High-level expression of Rad51, a key factor in homologous recombination, has been observed in a variety of human malignancies. This study was aimed to evaluate Rad51 expression to serve as prognostic marker in non-small-cell lung cancer (NSCLC). A total of 383 non-small-cell lung tumours were analysed immunohistochemically on NSCLC tissue microarrays. High-level Rad51 expression was observed in 29.4% (100 out of 340) of cases. Patients whose tumours displayed high-level Rad51 expression showed a significantly shorter median survival time of 19 vs 68 months (P<0.0001, log-rank test). Similarly T status, N status, M status, clinical stage and histological tumour grade were significant prognostic markers in univariate Cox survival analysis. Importantly, Rad51 expression (P<0.0001) together with tumour differentiation (P<0.009), clinical stage (P=0.004) and N status (P=0.0001) proved to be independent prognostic parameters in multivariate analysis. Rad51 expression predicted the outcome of squamous cell cancer as well as adenocarcinoma of the lung. Our results suggest that Rad51 expression provides additional prognostic information for surgically treated NSCLC patients. We hypothesise that the decreased survival of NSCLC patients with high-level expression of Rad51 is related to an enhanced propensity of tumour cells for survival, antiapoptosis and chemo-/radioresistance.
doi:10.1038/sj.bjc.6602665
PMCID: PMC2361489  PMID: 15956972
non-small-cell lung carcinoma; prognosis; tissue microarray; Rad51
22.  Methylation of the Candidate Biomarker TCF21 Is Very Frequent Across A Spectrum of Early Stage Non-Small Cell Lung Cancers 
Cancer  2010;117(3):606-617.
Background
The transcription factor TCF21 is involved in mesenchymal-to-epithelial differentiation and was shown to be aberrantly hypermethylated in lung and head and neck cancers. Because of its reported high frequency of hypermethylation in lung cancer, we sought to characterize the stages and types of non-small cell lung cancer (NSCLC) that are hypermethylated and to define the frequency of hypermethylation and associated “second hits”.
Methods
We determined TCF21 promoter hypermethylation in 105 NSCLC including various stages and histologies in smokers and nonsmokers. Additionally, we examined TCF21 loss-of-heterozygosity and mutational status. We also assayed 22 cancer cell lines from varied tissue origins. We validated and expanded our NSCLC results by examining TCF21 immunohistochemical expression on a tissue microarray containing 300 NSCLC cases.
Results
Overall, 81% of NSCLC samples showed TCF21 promoter hypermethylation and 84% showed decreased TCF21 protein expression. Multivariate analysis showed that TCF21 expression, although below normal in both histologies, was lower in adenocarcinoma than squamous cell carcinoma, and was not independently correlated with gender, smoking and EGFR mutation status, or clinical outcome. Cell lines from other cancer types also showed frequent TCF21 promoter hypermethylation.
Conclusions
Hypermethylation and decreased expression of TCF21 were tumor-specific and very frequent in all NSCLC, even early-stage disease, thus making TCF21 a potential candidate methylation biomarker for early-stage NSCLC screening. TCF21 hypermethylation in a variety of tumor cell lines suggests it may also be a valuable methylation biomarker in other tumor types.
doi:10.1002/cncr.25472
PMCID: PMC3023841  PMID: 20945327
TCF21; methylation; biomarker; lung cancer; screening
23.  Clinical significance of GADD153 expression in stage I non-small cell lung cancer 
Oncology Letters  2012;4(3):408-412.
The transcription factor growth arrest and DNA damage-inducible gene 153 (GADD153), also known as CHOP, is considered to function as a proapoptotic molecule. Overexpression of GADD153 leads to cell cycle arrest and/or apoptosis. However, its clinical implications in non-small cell lung cancer (NSCLC) remain controversial. Therefore, we investigated the expression of GADD153 in stage I NSCLC using immunohistochemistry. Paraffin-embedded tissue sections from 76 patients, who were diagnosed with primary stage I NSCLC and had undergone a curative lung resection, were stained using an anti-GADD153 antibody. The intensity of GADD153 immunostaining was evaluated within the cell membrane and cytoplasm of invasive cancer components. The correlation between the intratumoral expression of GADD153 and various clinical parameters were explored. GADD153 was detected in 29 (38.2%) cases. No statistically significant difference in expression was demonstrated between stage IA and stage IB tumors (35.0 vs. 39.3%; P=0.735). The expression of GADD153 was not affected by histological subtypes or histological grades of differentiation. The intratumoral expression of GADD153 did not influence the overall survival rate (53.29 vs. 52.18 months; P=0.743) or disease-free survival rate (46.97 vs. 54.19 months; P=0.084) of stage I NSCLC patients. However, patients with GADD153 expression demonstrated an improved disease-specific survival rate (28.80 vs. 53.85 months; P=0.020). No patients with GADD153 expression demonstrated distant metastasis (P=0.029). These data suggest that GADD153 expression may be a valuable prognostic factor of early-stage NSCLC in patients who have undergone curative lung resection.
doi:10.3892/ol.2012.768
PMCID: PMC3439019  PMID: 22970039
non-small cell lung cancer; growth arrest and DNA damage-inducible gene; immunohistochemistry; prognosis
24.  Clinicopathologic Implication of Ezrin Expression in Non-small Cell Lung Cancer 
Korean Journal of Pathology  2012;46(5):470-477.
Background
Ezrin, a member of the ezrin-radixin-moesin family, is implicated in tumor progression, metastatic dissemination, and adverse outcomes, in several cancer types. In this study, we explored the clinicopathological significance of ezrin expression in non-small cell lung carcinomas (NSCLCs).
Methods
Immunohistochemical analysis of tissue microarray with 112 surgically resected NSCLC specimens, was performed to examine the ezrin expression. We also correlated ezrin expression with other clinicopathological features and prognosis.
Results
The ezrin-positive group revealed significantly higher correlation with pleural invasion (p=0.016) and pathologic stage (p=0.050). Univariate survival analysis showed that ezrin-positive group had a significantly shorter cancer-specific survival than ezrin-negative group (p=0.016). Meanwhile, female (p=0.030), no pleural invasion (p=0.023), no lymphatic invasion (p=0.026), and early pathologic stage (p=0.008) significantly correlated with longer survival. Multivariate survival analysis showed that variables such as ezrin positivity (p=0.032), female (p=0.035), and early pathologic stage (p=0.001) were independent prognostic factors for NSCLC.
Conclusions
Ezrin might be a molecular marker to predict poor prognosis of NSCLC.
doi:10.4132/KoreanJPathol.2012.46.5.470
PMCID: PMC3490123  PMID: 23136574
Carcinoma, non-small cell lung; Ezrin; Prognosis
25.  Angiopoietin-like protein ANGPTL2 gene expression is correlated with lymph node metastasis in lung cancer 
Oncology Letters  2012;4(6):1325-1328.
Inflammation plays key roles at various stages of tumor development, including invasion and metastasis. In mice, the angiopoietin-like protein (ANGPTL2) gene has been implicated in inflammatory carcinogenesis. ANGPTL2 mRNA expression was investigated by real-time polymerase chain reaction (RT-PCR) assay using LightCycler in surgically treated non-small cell lung cancer (NSCLC) cases. In total, 110 surgically resected NSCLC cases were used for mRNA level analyses. The ANGPTL2/β-actin mRNA levels were not significantly different between lung cancer (1598.481±6465.781) and adjacent normal lung tissues (2116.639±8337.331, P=0.5453). The tumor/normal (T/N) ratio of ANGPTL2/β-actin mRNA levels was not different between gender, age, smoking status and pathological stages. The T/N ratio of ANGPTL2/β-actin mRNA levels was significantly higher in lymph node metastasis-positive cases (2.173±3.151) compared with lymph node metastasis-negative cases (1.212±1.778, P=0.0464). However, ANGPTL2 mRNA status was not correlated with tumor invasion status. Thus, ANGPTL2 may drive metastasis and provide a candidate for blockade of its function as a strategy to antagonize the metastatic process in NSCLC.
doi:10.3892/ol.2012.924
PMCID: PMC3506759  PMID: 23205131
ANGTL2; angiopoietin; lung cancer; metastasis; LightCycler

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