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1.  Current Concepts on the Molecular Pathology of Non-small Cell Lung Carcinoma 
Seminars in diagnostic pathology  2014;31(4):306-313.
Recent advances in the understanding of the complex biology of non-small cell lung carcinoma (NSCLC), particularly activation of oncogenes by mutation, translocation and amplification, have provided new treatment targets for this disease, and allowed the identification of subsets of NSCLC tumors, mostly with adenocarcinoma histology, having unique molecular profiles that can predict response to targeted therapy. The identification of a specific genetic and molecular targetable abnormalities using tumor tissue and cytology specimens followed by the administration of a specific inhibitor to the target, are the basis of personalized lung cancer treatment. In this new paradigm, the role of a precise pathology diagnosis of lung cancer and the proper handling of tissue and cytology samples for molecular testing is becoming increasingly important. These changes have posed multiple new challenges for pathologists to adequately integrate routine histopathology analysis and molecular testing into the clinical pathology practice for tumor diagnosis and subsequent selection of the most appropriate therapy.
PMCID: PMC4190584  PMID: 25239274
adenocarcinoma; squamous cell carcinoma; targeted therapy; molecular testing; next-generation of sequencing
2.  Frequent co-amplification and co-operation between C-MYC and PVT1 oncogenes promote malignant pleural mesothelioma 
Malignant pleural mesothelioma (MPM) is a deadly disease with poor prognosis and few treatment options. We characterized and elucidate the roles of C-MYC and PVT1 involved in the pathogenesis of MPM.
We used siRNA-mediated knockdown in MPM cell lines to determine the effect of C-MYC and PVT1 abrogation on MPM cells undergoing apoptosis, proliferation, and cisplatin sensitivity. We also characterized the expression of microRNAs (miRNAs) spanning the PVT1 region in MPM cell lines. Copy number analysis was measured by quantitative PCR and fluorescence in situ hybridization.
Copy number analysis revealed copy number gains (CNGs) in chromosomal region 8q24 in six of twelve MPM cell lines. MicroRNA analysis showed high miR-1204 expression in MSTO-211H cell lines with ≥4 copies of PVT1. Knockdown by siRNA showed increased PARP-C levels in MSTO-211H transfected with siPVT1 but not in cells transfected with siC-MYC. C-MYC and PVT1 knockdown reduced cell proliferation and increased sensitivity to cisplatin. Analysis of the expression of apoptosis-related genes in the MSTO-211H cell line suggested that C-MYC maintains a balance between pro-apoptotic and anti-apoptotic gene expression, whereas PVT1 and to a lesser extent miR-1204, upregulate pro-apoptotic genes and downregulate anti-apoptotic genes. FISH analysis of MPM tumor specimens showed a high frequency of both CNGs (11/75) and trisomy (three copies; 11/75) for the C-MYC locus.
Our results suggest that C-MYC and PVT1 copy number gain promotes a malignant phenotype of MPM, with C-MYC CNG stimulating cell proliferation and PVT1 both stimulating proliferation and inhibiting apoptosis.
PMCID: PMC4287384  PMID: 24926545
C-MYC; PVT1; 8q24 chromosomal region; Malignant pleural mesothelioma
3.  Transcriptomic Architecture of the Adjacent Airway Field Cancerization in Non–Small Cell Lung Cancer 
Earlier work identified specific tumor-promoting abnormalities that are shared between lung cancers and adjacent normal bronchial epithelia. We sought to characterize the yet unknown global molecular and adjacent airway field cancerization (FC) in early-stage non–small cell lung cancer (NSCLC).
Whole-transcriptome expression profiling of resected early-stage (I–IIIA) NSCLC specimens (n = 20) with matched tumors, multiple cytologically controlled normal airways with varying distances from tumors, and uninvolved normal lung tissues (n = 194 samples) was performed using the Affymetrix Human Gene 1.0 ST platform. Mixed-effects models were used to identify differentially expressed genes among groups. Ordinal regression analysis was performed to characterize site-dependent airway expression profiles. All statistical tests were two-sided, except where noted.
We identified differentially expressed gene features (n = 1661) between NSCLCs and airways compared with normal lung tissues, a subset of which (n = 299), after gene set enrichment analysis, statistically significantly (P < .001) distinguished large airways in lung cancer patients from airways in cancer-free smokers. In addition, we identified genes (n = 422) statistically significantly and progressively differentially expressed in airways by distance from tumors that were found to be congruently modulated between NSCLCs and normal lung tissues. Furthermore, LAPTM4B, with statistically significantly increased expression (P < .05) in airways with shorter distance from tumors, was upregulated in human immortalized cells compared with normal bronchial epithelial cells (P < .001) and promoted anchorage-dependent and -independent lung cancer cell growth.
The adjacent airway FC comprises both site-independent profiles as well as gradient and localized airway expression patterns. Profiling of the airway FC may provide new insights into NSCLC oncogenesis and molecular tools for detection of the disease.
PMCID: PMC3982778  PMID: 24563515
4.  Validation of a Proliferation-based Expression Signature as Prognostic Marker in Early Stage Lung Adenocarcinoma 
New prognostic markers to guide treatment decisions in early stage non-small cell lung cancer are necessary to improve patient outcomes. In this report, we assess the utility of a pre-defined mRNA expression signature of cell cycle progression genes (CCP score) to define 5-year risk of lung cancer related death in patients with early stage lung adenocarcinoma.
A CCP score was calculated from the mRNA expression levels of 31 proliferation genes in stage I and II tumor samples from two public microarray data sets (Director’s Consortium (DC) and GSE31210). The same gene set was tested by quantitative PCR in 381 formalin-fixed paraffin-embedded (FFPE) primary tumors. Association of the CCP score with outcome was assessed by Cox proportional hazards analysis.
In univariate analysis the CCP score was a strong predictor of cancer-specific survival in both the DC cohort (p=0.00014, HR 2.08, 95%CI 1.43–3.02) and GSE31210 (p=0.0010, HR 2.25, 95%CI 1.42–3.56). In multivariate analysis the CCP score remained the dominant prognostic marker in the presence of clinical variables (p=0.0022, HR 2.02, 95%CI 1.29–3.17 in DC, p=0.0026, HR 2.16, 95%CI 1.32–3.53 in GSE31210). On a quantitative PCR platform the CCP score maintained highly significant prognostic value in FFPE derived mRNA from clinical samples in both univariate (p=0.00033, HR 2.10, 95%CI 1.39–3.17) and multivariate analyses (p=0.0071, HR 1.92, 95%CI 1.18–3.10).
The CCP score is a significant predictor of lung cancer death in early stage lung adenocarcinoma treated with surgery and may be a valuable tool in selecting patients for adjuvant treatment.
PMCID: PMC3834029  PMID: 24048333
non-small cell lung cancer; adenocarcinoma; prognosis; expression signature
5.  VEGF/VEGFR-2 upregulates EZH2 expression in lung adenocarcinoma cells and EZH2 depletion enhances the response to platinum-based and VEGFR-2–targeted therapy 
Investigate the mechanisms of regulation and role associated with EZH2 expression in lung cancer cells.
Experimental Design
We investigated the mechanisms of EZH2 expression associated with the vascular endothelial growth factor (VEGF)/VEGF receptor 2 (VEGFR-2) pathway. Furthermore, we sought to determine the role of EZH2 in response of lung adenocarcinoma to platinum-based chemotherapy, as well as the effect of EZH2 depletion on VEGFR-2–targeted therapy in lung adenocarcinoma cell lines. Additionally, we characterized EZH2 expression in lung adenocarcinoma specimens and correlated it with patients’ clinical characteristics.
In this study, we demonstrate that VEGF/VEGFR-2 activation induces expression of EZH2 through the upregulation of E2F3 and HIF-1α, and downregulated expression of miR-101. EZH2 depletion by treatment with 3-deazaneplanocin A and knockdown by siRNA decreased the expression of EZH2 and H3K27me3, increased PARP-C level, reduced cell proliferation and migration, and increased sensitivity of the cells to treatment with cisplatin and carboplatin. Additionally, high EZH2 expression was associated with poor overall survival in patients who received platinum-based adjuvant therapy, but not in patients who did not receive this therapy. Furthermore, we demonstrated for the first time that the inhibition of EZH2 greatly increased the sensitivity of lung adenocarcinoma cells to the anti-VEGFR-2 drug AZD2171.
Our results suggest that VEGF/VEGFR-2 pathway plays a role in regulation of EZH2 expression via E2F3, HIF-1α and miR-101. EZH2 depletion decreases the malignant potential of lung adenocarcinoma and sensitivity of the cells to both platinum-based and VEGFR-2–targeted therapy.
PMCID: PMC4190586  PMID: 24850841
EZH2; NSCLC; VEGF/VEGFR-2 pathway; DZNep
6.  ETS2 mediated tumor suppressive function and MET oncogene inhibition in human non-small cell lung cancer 
The ETS2 transcription factor is an evolutionarily conserved gene that is deregulated in cancer. We analyzed the transcriptome of lung adenocarcinomas and normal lung tissue by expression profiling and found that ETS2 was significantly down-regulated in adenocarcinomas. In this study, we probed the yet unknown functional role of ETS2 in lung cancer pathogenesis.
Lung adenocarcinomas (n=80) and normal lung tissues (n=30) were profiled using the Affymetrix Human Gene 1.0 ST platform. Immunohistochemical (IHC) analysis was performed to determine ETS2 protein expression in NSCLC histological tissue specimens (n=201). Patient clinical outcome, based on ETS2 IHC expression, was statistically assessed using the log-rank and Kaplan-Meier tests. RNA interference and over-expression strategies were employed to assess effects of ETS2 expression on the transcriptome and on various malignant phenotypes.
ETS2 expression was significantly reduced in lung adenocarcinomas compared to normal lung (p<0.001). Low ETS2 IHC expression was a significant predictor of shorter time to recurrence in NSCLC (p=0.009, HR=1.89) and adenocarcinoma (p=0.03, HR=1.86). Moreover, ETS2 was found to significantly inhibit lung cancer cell growth, migration and invasion (p<0.05), and microarray and pathways analysis revealed significant (p<0.001) activation of the HGF pathway following ETS2 knockdown. In addition, ETS2 was found to suppress MET phosphorylation and knockdown of MET expression significantly attenuated (p<0.05) cell invasion mediated by ETS2-specific siRNA. Furthermore, knockdown of ETS2 augmented HGF-induced MET phosphorylation, cell migration and invasion.
Our findings point to a tumor suppressor role for ETS2 in human NSCLC pathogenesis through inhibition of the MET proto-oncogene.
PMCID: PMC3846434  PMID: 23659968
NSCLC; ETS2; tumor suppressor; MET; HGF
7.  Characterizing the molecular spatial and temporal field of injury in early stage smoker non-small cell lung cancer patients after definitive surgery by expression profiling 
Gene expression alterations in response to cigarette smoke have been characterized in normal-appearing bronchial epithelium of healthy smokers and it has been suggested that adjacent histologically normal tissue display tumor-associated molecular abnormalities. We sought to delineate the spatial and temporal molecular lung field of injury in smoker early stage non-small cell lung cancer (NSCLC) patients (n=19) who were accrued into a surveillance clinical trial for annual follow-up and bronchoscopies within one year after definitive surgery. Bronchial brushings and biopsies were obtained from six different sites in the lung at the time of inclusion in the study and at 12, 24 and 36 months after the first time point. Affymetrix Human Gene 1.0 ST arrays were used for whole-transcript expression profiling of airways (n=391). Microarray analysis identified gene features (n=1165) that were non-uniform by site and differentially expressed between airways adjacent to tumors relative to more distant samples as well as those (n=1395) that were significantly altered with time up to three years. In addition, gene-interaction networks mediated by PI3K and ERK1/2 were modulated in adjacent compared to contralateral airways and the latter network with time. Furthermore, phosphorylated AKT and ERK1/2 immunohistochemical expression were significantly increased with time (nuclear pAKT, p=0.03; cytoplasmic pAKT, p<0.0001; pERK1/2, p=0.02) and elevated in adjacent compared to more distant airways (nuclear pAKT, p=0.04; pERK1/2, p=0.03). This study highlights spatial and temporal cancer-associated expression alterations in the molecular field of injury of early stage NSCLC patients after definitive surgery that warrant further validation in independent studies.
PMCID: PMC3774536  PMID: 23087048
Early stage NSCLC; gene expression profiling; lung airway epithelium; chemoprevention
8.  High Expression of Folate Receptor Alpha in Lung Cancer Correlates with Adenocarcinoma Histology and EGFR Mutation 
Journal of Thoracic Oncology  2012;7(5):833-840.
Folate receptor alpha (FRα) and reduced folate carrier-1 (RFC1) regulate uptake of folate molecules inside the cell. FRα is a potential biomarker of tumors response to antifolate chemotherapy and a target for therapy using humanized monocloncal antibody. Information on the protein expression of these receptors in non–small cell lung carcinoma (NSCLC) is limited.
Material and Methods
Expressions of FRα and RFC1 were examined by IHC in 320 surgically resected NSCLC (202 adenocarcinomas and 118 squamous cell carcinomas) tissue specimens and correlated with patients’ clinicopathologic characteristics. FOLR1 mRNA expression was examined using publicly available microarray datasets. FRα expression was correlated with thymidylate synthase (TS) and p53 expression in NSCLCs, and with EGFR and KRAS mutations in adenocarcinomas.
NSCLC overexpressed FRα and RFC1. In a multivariate analysis, lung adenocarcinomas were more likely to express FRα in the cytoplasm (odds ratio [OR] = 4.39; P<0.0001) and membrane (OR = 5.34; P<0.0001) of malignant cells than squamous cell carcinomas. Tumors from never-smokers were more likely to express cytoplasmic (OR = 3.35; P<0.03) and membrane (OR = 3.60; P=0.0005) FRα than those from smokers. In adenocarcinoma, EGFR mutations correlated with higher expression of membrane FRα and FOLR1 gene expressions. High levels of FRα expression was detected in 42 NSCLC advanced metastatic tumor tissues.
FRα and RFC1 proteins are overexpressed in NSCLC tumor tissues. The high levels of FRα in lung adenocarcinomas may be associated to these tumors’ better responses to antifolate chemotherapy and represents a potential novel target for this tumor type.
PMCID: PMC3383601  PMID: 22729036
non–small cell lung carcinoma; EGFR; membrane transporter; FRα; FRC1
9.  Robust Gene Expression Signature from Formalin-Fixed Paraffin-Embedded Samples Predicts Prognosis of Non-Small-Cell Lung Cancer Patients 
The requirement of frozen tissues for microarray experiments limits the clinical usage of genome-wide expression profiling using microarray technology. The goal of this study is to test the feasibility of developing lung cancer prognosis gene signatures using genome-wide expression profiling of formalin-fixed paraffin-embedded (FFPE) samples, which are widely available and provide a valuable rich source for studying the association of molecular changes in cancer and associated clinical outcomes.
Experimental Design
We randomly selected 100 Non-Small-Cell lung cancer (NSCLC) FFPE samples with annotated clinical information from the UT-Lung SPORE Tissue Bank. We micro dissected tumor area from FFPE specimens, and used Affymetrix U133 plus 2.0 arrays to attain gene expression data. After strict quality control and analysis procedures, a supervised principal component analysis was used to develop a robust prognosis signature for NSCLC. Three independent published microarray data sets were used to validate the prognosis model.
This study demonstrated that the robust gene signature derived from genome-wide expression profiling of FFPE samples is strongly associated with lung cancer clinical outcomes, can be used to refine the prognosis for stage I lung cancer patients and the prognostic signature is independent of clinical variables. This signature was validated in several independent studies and was refined to a 59-gene lung cancer prognosis signature.
We conclude that genome-wide profiling of FFPE lung cancer samples can identify a set of genes whose expression level provides prognostic information across different platforms and studies, which will allow its application in clinical settings.
PMCID: PMC3166982  PMID: 21742808
Lung Cancer Prognosis; Gene Expression Signature; Formalin Fixed Paraffin Embedded Samples
10.  Increased VEGFR-2 Gene Copy Is Associated With Chemoresistance and Shorter Survival in Patients with Non-small Cell Lung Carcinoma Who Receive Adjuvant Chemotherapy 
Cancer research  2011;71(16):5512-5521.
Vascular endothelial growth factor-2 (VEGFR-2 or KDR) is a known endothelial target also expressed in NSCLC tumor cells. We investigated the association between alterations in the KDR gene and clinical outcome in patients with resected NSCLC (n=248). KDR copy number gains (CNGs), measured by quantitative PCR and fluorescence in situ hybridization, were detected in 32% of tumors and associated with significantly higher KDR protein and higher microvessel density than tumors without CNGs. KDR CNGs were also associated with significantly increased risk of death (HR=5.16; P=0.003) in patients receiving adjuvant platinum-based chemotherapy, but no differences were observed in patients not receiving adjuvant therapy. To investigate potential mechanisms for these associations we assessed NSCLC cell lines and found that KDR CNGs were significantly associated with in vitro resistance to platinum chemotherapy as well as increased levels of nuclear HIF-1α in both NSCLC tumor specimens and cell lines. Furthermore, KDR knockdown experiments using small interfering RNA reduced platinum resistance, cell migration, and HIF-1α levels in cells bearing KDR CNGs, providing evidence for direct involvement of KDR. No KDR mutations were detected in exons 7, 11 and 21 by PCR-based sequencing; however, two variant SNP genotypes were associated with favorable overall survival in adenocarcinoma patients. Our findings suggest that tumor cell KDR CNGs may promote a more malignant phenotype including increased chemoresistance, angiogenesis, and HIF-1α levels, and that KDR CNGs may be a useful biomarker for identifying patients at high risk for recurrence after adjuvant therapy, a group that may benefit from VEGFR-2 blockade.
PMCID: PMC3159530  PMID: 21724587
11.  HER Family Receptor Abnormalities in Lung Cancer Brain Metastases and Corresponding Primary Tumors 
Clinical Cancer Research  2009;15(15):4829-4837.
To compare the characteristics of HER receptors and their ligands deregulation between primary tumor and corresponding brain metastases of non-small cell lung carcinoma (NSCLC).
Experimental design
Fifty five NSCLC primary tumors (PT) and corresponding brain metastases (BM) specimens were examined for the immunohistochemical expression of EGFR, phosphorylated (p)–EGFR, Her2, Her3, and p-Her3, and their ligands EGF, TGF-α, amphiregulin, epiregulin, betacellulin, heparin-binding EGFR-like growth factor, and neuregulins-1 and -2. Analysis of EGFR copy number using fluorescent in situ hybridization and mutation by PCR-based sequencing was also performed.
Metastases showed significantly higher immunohistochemical expression of EGF (membrane, BM 66.0 vs. PT 48.5; P=0.027; and nucleus, BM 92.2 vs. 67.4; P=0.008), amphiregulin (nucleus, BM 53.7 vs. PT 33.7; P=0.019), p-EGFR (membrane, BM 161.5 vs. PT 76.0; P<0.0001; and cytoplasm, BM 101.5 vs. PT 55.9; P=0.014), and p-Her3 (membrane, BM 25.0 vs. PT 3.7; P=0.001) than primary tumors (PT) did. Primary tumors showed significantly higher expression of cytoplasmic TGF–α (PT 149.8 vs. BM 111.3; P=0.008) and neuregulin-1 (PT 158.5 vs. BM 122.8; P=0.006). In adenocarcinomas, a similar high frequency of EGFR copy number gain (high polysomy and amplification) was detected in primary (65%) and brain metastasis (63%) sites. However, adenocarcinoma metastases (30%) showed higher frequency of EGFR amplification than corresponding primary tumors (10%). Patients whose primary tumors showed EGFR amplification tended to develop brain metastases at an earlier time points.
Our findings suggest that NSCLC brain metastases have some significant differences in HER family receptors-related abnormalities from primary lung tumors.
PMCID: PMC3372920  PMID: 19622585
12.  Histologic Patterns and Molecular Characteristics of Lung Adenocarcinoma Associated With Clinical Outcome 
Cancer  2011;118(11):2889-2899.
Lung adenocarcinoma is histologically heterogeneous and has 5 distinct histologic growth patterns: lepidic, acinar, papillary, micropapillary, and solid. To date, there is no consensus regarding the clinical utility of these patterns.
The authors performed a detailed semiquantitative assessment of histologic patterns of 240 lung adenocarcinomas and determined the association with patients’ clinicopathologic features, including recurrence-free survival (RFS) and overall survival (OS) rates. In a subset of tumors, expression levels of 2 prognostic molecular markers were evaluated: thyroid transcription factor-1 (TTF-1) (n = 218) and a panel of 5 proteins (referred as the FILM signature index) (n = 185).
Four mutually exclusive tumor histology pattern groups were identified: 1) any solid (38%), 2) any papillary but no solid (14%), 3) lepidic and acinar but no solid or papillary (30%), and 4) acinar only (18%). Patients in group 3 had a higher RFS rate than patients in group 1 (hazard ratio [HR], 0.4510; P = .0165) and group 2 (HR, 0.4253; P = .0425). Solid pattern tumors (group 1) were associated with a lower OS rate than nonsolid pattern tumors (all stages: HR; 1.665; P = .0144; stages I and II: HR, 2.157; P = .008). In the patients who had tumors with a nonsolid pattern, high TTF-1 expression was associated significantly with higher RFS (HR, 0.994; P = .0017) and OS (HR, 0.996; P = .0276) rates in all stages, and a high FILM signature index score was associated with lower RFS and OS rates in all stages (RFS: HR, 1.343; P = .0192; OS: HR, 1.371; P = .0156) and in stages I and II (RFS: HR, 1.419; P = .0095; OS: HR, 1.315; P = .0422).
The presence of a solid histologic pattern was identified as a marker of unfavorable prognosis in patients with primary lung adenocarcinoma. High TTF-1 expression and low FILM signature index scores were associated with a better prognosis for patients who had tumors with a nonsolid pattern.
PMCID: PMC3369269  PMID: 22020674
histologic patterns; lung adenocarcinoma; thyroid transcription factor 1; prognostic signature
13.  EGFR Abnormalities in the Pathogenesis and Progression of Lung Adenocarcinomas 
To identify the characteristics and sequence of epidermal growth factor receptor (EGFR) abnormalities relevant to the pathogenesis and progression of lung adenocarcinoma, we performed a precise mapping analysis of EGFR mutation, gene copy number and total and phosphorylated EGFR (pEGFR) protein expression for the same tissue sites. We examined normal bronchial and bronchiolar epithelium (NBE) and tumor tissues obtained from 50 formalin-fixed lung adenocarcinomas, including 24 EGFR-mutant primary tumors with nine corresponding lymph node metastases and 26 wild-type primary tumors. NBE in 12/24 (50%) mutant and 3/26 (12%) wild-type tumors harbored EGFR mutation; these NBE also showed lack of EGFR copy number increase and frequent EGFR (69%) and pEGFR (33%) overexpression. EGFR mutation and protein overexpression were more frequent in NBE sites within tumors than in NBE sites adjacent to and distant from tumors, suggesting a localized field effect. Sites with high and low EGFR copy numbers were heterogeneously distributed in six of nine primary tumors and one of eight metastases. EGFR protein overexpression was significantly higher in metastasis sites than in primary tumors. We conclude from our findings that EGFR mutations and protein overexpression are early phenomena in the pathogenesis of lung adenocarcinoma and that EGFR mutation precedes an increase in gene copy number. In EGFR-mutant adenocarcinoma metastases, the higher levels of EGFR overexpression and more homogeneously distributed high gene copy numbers suggest tumor progression. Our findings have important implications for the development of new strategies for targeted chemoprevention and therapy in lung adenocarcinoma using EGFR inhibitors.
PMCID: PMC3369271  PMID: 19138956
14.  Abnormalities of the TITF-1 lineage-specific oncogene in NSCLC: Implications in lung cancer pathogenesis and prognosis 
Emerging evidence suggests that aberrant expression of oncogenes contributes to development of lung malignancy. The thyroid transcription factor 1 (TITF-1) gene functions as a lineage survival gene abnormally expressed in a significant fraction of NSCLCs, in particular lung adenocarcinomas.
To better characterize TITF-1 abnormality: patterns in NSCLC, we studied TITF-1’s gene copy number using fluorescent in situ hybridization (FISH) and quantitative PCR, as well as its protein expression by immunohistochemistry analysis in a tissue microarray comprised of surgically resected NSCLC (N=321) including 204 adenocarcinomas and 117 squamous cell carcinomas (SCCs). TITF-1 copy number and protein expression were correlated with patients’ clinicopathologic characteristics, and in a subset of adenocarcinomas with EGFR and KRAS mutation status.
We found that increased TITF-1 protein expression was prevalent in lung adenocarcinomas only and was significantly associated with female gender (p<0.001), never smokers (p=0.004), presence of EGFR mutations (p=0.05) and better overall survival (all stages, p=0.0478. stages I and II, p=0.002). TITF-1 copy number gain (CBG) was detected by FISH analysis in both adenocarcinomas (18.9%; high CNG, 8.3%) and SCCs (20.1%; high CNG, 3.0%), and correlated significantly with the protein product (p=0.004) and presence of KRAS mutations (p=0.008) in lung adenocarcinomas. Moreover, multivariate analysis revealed that TITF-1 copy number gain was an independent predictor of poor survival of NSCLC (p=0.039).
Our integrative study demonstrates that the protein versus genomic expression patterns of TITF-1 have opposing roles in lung cancer prognosis and may occur preferentially in different subsets of NSCLC patients with distinct oncogene mutations.
PMCID: PMC3078948  PMID: 21257719
NSCLC; TITF-1; gene copy gain; lineage-specific oncogenes
15.  A five-gene and corresponding-protein signature for stage-I lung adenocarcinoma prognosis 
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.
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).
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.
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.
PMCID: PMC3079395  PMID: 21163870
Lung adenocarcinoma; NSCLC; gene signature; prognosis
16.  Nrf2 and Keap1 Abnormalities in Non-Small Cell Lung Carcinoma and Association with Clinicopathologic Features 
To understand the role of Nrf2 and Keap1 in NSCLC, we studied their expression in a large series of tumors with annotated clinicopathologic data, including response to platinum-based adjuvant chemotherapy.
Experimental design
We determined the immunohistochemical expression of nuclear Nrf2 and cytoplasmic Keap1 in 304 NSCLCs and its association with patients’ clinicopathologic characteristics, and in 89 tumors from patients who received neoadjuvant (n=26) or adjuvant platinum-based chemotherapy (n=63). We evaluated NFE2L2 and KEAP1 mutations in 31 tumor specimens.
We detected nuclear Nrf2 expression in 26% of NSCLCs; it was significantly more common in squamous cell carcinomas (38%) than in adenocarcinomas (18%; P<0.0001). Low or absent Keap1 expression was detected in 56% of NSCLCs; it was significantly more common in adenocarcinomas (62%) than in squamous cell carcinomas (46%; P=0.0057). In NSCLC, mutations of NFE2L2 and KEAP1 were very uncommon (2 of 29 and 1 of 31 cases, respectively). In multivariate analysis, Nrf2 expression was associated with worse overall survival (P=0.0139; HR=1.75) in NSCLC patients, and low or absent Keap1 expression was associated with worse overall survival (P=0.0181; HR=2.09) in squamous cell carcinoma. In univariate analysis, nuclear Nrf2 expression was associated with worse recurrence-free survival in squamous cell carcinoma patients who received adjuvant treatment (P=0.0410; HR=3.37).
Increased expression of Nrf2 and decreased expression of Keap1 are common abnormalities in NSCLC and are associated with a poor outcome. Nuclear expression of Nrf2 in malignant lung cancer cells may play a role in resistance to platinum-based treatment in squamous cell carcinoma.
PMCID: PMC2920733  PMID: 20534738
Nrf2; Keap1; NSCLC
17.  Expression of Interleukin-1 Receptor-Associated Kinase-1 (IRAK-1) in Non-Small Cell Lung Carcinoma and Preneoplastic Lesions 
To identify the pattern of IRAK-1 protein expression in non-small cell lung carcinoma (NSCLC) and corresponding preneoplastic lesions.
Experimental Design
Archived tissue from NSCLC (adenocarcinoma and squamous cell carcinoma; n = 306) and adjacent bronchial epithelial specimens (n = 315) were analyzed for the immunohistochemical expression of IRAK-1, and the findings were correlated with patients’ clinicopathologic features. Furthermore, we investigated the correlation between IRAK-1 expression and expression of NF-κB and IL-1α in tumor specimens.
NSCLC tumors demonstrated significantly higher cytoplasmic and lower nuclear IRAK-1 expression than normal epithelium. Squamous dysplasias had significantly higher cytoplasmic IRAK-1 expression that normal epithelium. In tumors, a significant positive correlation was detected between IRAK-1 expression (nuclear and cytoplasmic; P = 0.011) and IL-1α cytoplasmic expression (P < 0.0001). The correlation between the expression of the markers and patients’ clinicopathologic features varied according to tumor histologic type and sex. High IRAK-1 cytoplasmic expression correlated with worse recurrence-free survival in women with NSCLC (HR, 2.204; P = 0.033), but not in men. In adenocarcinoma, combined low level of expression of nuclear IRAK-1 and NF-κB correlated significantly with worse overall (HR, 2.485; P = 0.007) and recurrence-free (HR, 3.058; P = 0.006) survivals in stage I/II patients.
IRAK-1 is frequently expressed in NSCLC tissue specimens, and this expression is an early phenomenon in the sequential development of lung cancer. IRAK-1 is a novel inflammation-related marker and a potential target for lung cancer chemopreventive strategies.
PMCID: PMC2811365  PMID: 20028769
lung cancer; inflammation; preneoplastic lesions; dysplasia
18.  Immunohistochemical Expression of Estrogen and Progesterone Receptors Identifies a Subset of NSCLCs and Correlates with EGFR Mutation 
To determine the frequency of estrogen receptor α and β and progesterone receptor protein immunohistochemical expression in a large set of non–small cell lungcarcinoma (NSCLC) specimens and to compare our results with those for some of the same antibodies that have provided inconsistent results in previously published reports.
Experimental Design
Using multiple antibodies, we investigated the immunohistochemical expression of estrogen receptors α and β and progesterone receptor in 317 NSCLCs placed in tissue microarrays and correlated their expression with patients’ clinicopathologic characteristics and in adenocarcinomas with EGFR mutation status.
Estrogen receptors α and β were detected in the nucleus and cytoplasm of NSCLC cells; however, the frequency of expression (nucleus, 5-36% for α and 42-56% for β; cytoplasm: <1-42% for α and 20-98% for β) varied among the different antibodies tested. Progesterone receptor was expressed in the nuclei of malignant cells in 63% of the tumors. Estrogen receptor α nuclear expression significantly correlated with adenocarcinoma histology, female gender, and history of never smoking (P = 0.0048 to <0.0001). In NSCLC, higher cytoplasmic estrogen receptor α expression significantly correlated with worse recurrence-free survival (hazard ratio, 1.77; 95% confidence interval, 1.12, 2.82; P = 0.015) in multivariate analysis. In adenocarcinomas, estrogen receptor α expression correlated with EGFR mutation (P = 0.0029 to <0.0001). Estrogen receptor β and progesterone receptor but not estrogen receptor α expressed in the normal epithelium adjacent to lung adenocarcinomas.
Estrogen receptor α and β expression distinguishes a subset of NSCLC that has defined clinicopathologic and genetic features. In lung adenocarcinoma, estrogen receptor α expression correlates with EGFR mutations.
PMCID: PMC2893045  PMID: 19706809
19.  Loss and Reduction of Fus1 Protein Expression is a Frequent Phenomenon in the Pathogenesis of Lung Cancer 
FUS1, a novel tumor-suppressor gene located in the chromosome 3p21.3 region, may play an important role in lung cancer development. Currently, FUS1-expressing nanoparticles have been developed for treating patients with lung cancer. However, the expression of Fus1 protein has not been examined in a large series of lung cancers and their sequential preneoplastic lesions.
Experimental Design
Using tissue microarrays, we examined Fus1 immunohistochemical expression in 281 non – small cell lung carcinoma (NSCLC) and 22 small cell lung carcinoma tissue specimens and correlated the findings with patients’ clinicopathologic features. To investigate the expression of Fus1 in the early sequential pathogenesis of NSCLC, we studied Fus1 expression in 211 histologically normal and mildly abnormal bronchial epithelia, and 118 bronchial and alveolar preneoplastic lesions obtained from patients with lung cancer.
Loss and reduction of expression was detected in 82% of NSCLCs and 100% of small cell lung carcinomas. In NSCLCs, loss of Fus1 immunohistochemical expression was associated with significantly worse overall survival. Bronchial squamous metaplastic and dysplastic lesions expressed significantly lower levels of Fus1 compared with normal (P = 0.014 and 0.047, respectively) and hyperplastic (P = 0.013 and 0.028, respectively) epithelia.
Our findings show a high frequency of Fus1 protein loss and reduction of expression in lung cancer, and suggests that this reduction may play an important role in the early pathogenesis of lung squamous cell carcinoma. These findings support the concept that FUS1 gene and Fus1 protein abnormalities could be used to develop new strategies for molecular cancer therapy for a significant subset of lung tumors.
PMCID: PMC2833352  PMID: 18172250
20.  Epithelial-to-Mesenchymal Transition in the Development and Progression of Adenocarcinoma and Squamous Cell Carcinoma of the Lung 
Epithelial-to-mesenchymal transition is a process in which cells undergo a developmental switch from an epithelial to a mesenchymal phenotype. We investigated the role of this phenomenon in the pathogenesis and progression of adenocarcinoma and squamous cell carcinoma of the lung. Archived tissue from primary tumors (n=325) and brain metastases (n=48) and adjacent bronchial epithelial specimens (n=192) were analyzed for immunohistochemical expression by image analysis of E-cadherin, N-cadherin, integrin-αvβ6, vimentin, and matrix metalloproteinase-9. The findings were compared with patients’ clinicopathologic features. High expression of the epithelial-to-mesenchymal transition phenotype (low E-cadherin and high N-cadherin, integrin-αvβ6, vimentin, and matrix metalloproteinase-9) was found in most lung tumors examined, and the expression pattern varied according to the tumor histologic type. Low E-cadherin membrane and high N-cadherin cytoplasmic expression were significantly more common in squamous cell carcinoma than in adenocarcinoma (P=0.002 and 0.005, respectively). Dysplastic lesions had significantly lower expression of the epithelial-to-mesenchymal transition phenotype than did squamous cell carcinomas, and integrin-αvβ6 membrane expression increased stepwise according to the histopathologic severity. Brain metastases had decreased epithelial-to-mesenchymal transition expression compared with primary tumors. Brain metastases had significantly lower integrin-αvβ6 membrane (P=0.04) and N-cadherin membrane and cytoplasm (P<0.0002) expression than did primary tumors. The epithelial-to-mesenchymal transition phenotype is commonly expressed in primary squamous cell carcinoma and adenocarcinoma of the lung; this expression occurs early in the pathogenesis of squamous cell carcinoma. Brain metastases showed characteristics of reversed mesenchymal-to-epithelial transition. Our findings suggest that epithelial-to-mesenchymal transition is a potential target for lung cancer chemoprevention and therapy.
PMCID: PMC2675657  PMID: 19270647
epithelial-to-mesenchymal transition; tissue microarray; immunohistochemical analysis; lung cancer; preneoplasia; brain metastases
21.  Combined clinical and genomic signatures for the prognosis of early stage non-small cell lung cancer based on gene copy number alterations 
BMC Genomics  2015;16:752.
The development of a more refined prognostic methodology for early non-small cell lung cancer (NSCLC) is an unmet clinical need. An accurate prognostic tool might help to select patients at early stages for adjuvant therapies.
A new integrated bioinformatics searching strategy, that combines gene copy number alterations and expression, together with clinical parameters was applied to derive two prognostic genomic signatures. The proposed methodology combines data from patients with and without clinical data with a priori information on the ability of a gene to be a prognostic marker. Two initial candidate sets of 513 and 150 genes for lung adenocarcinoma (ADC) and squamous cell carcinoma (SCC), respectively, were generated by identifying genes which have both: a) significant correlation between copy number and gene expression, and b) significant prognostic value at the gene expression level in external databases. From these candidates, two panels of 7 (ADC) and 5 (SCC) genes were further identified via semi-supervised learning. These panels, together with clinical data (stage, age and sex), were used to construct the ADC and SCC hazard scores combining clinical and genomic data. The signatures were validated in two independent datasets (n = 73 for ADC, n = 97 for SCC), confirming that the prognostic value of both clinical-genomic models is robust, statistically significant (P = 0.008 for ADC and P = 0.019 for SCC) and outperforms both the clinical models (P = 0.060 for ADC and P = 0.121 for SCC) and the genomic models applied separately (P = 0.350 for ADC and P = 0.269 for SCC).
The present work provides a methodology to generate a robust signature using copy number data that can be potentially used to any cancer. Using it, we found new prognostic scores based on tumor DNA that, jointly with clinical information, are able to predict overall survival (OS) in patients with early-stage ADC and SCC.
Electronic supplementary material
The online version of this article (doi:10.1186/s12864-015-1935-0) contains supplementary material, which is available to authorized users.
PMCID: PMC4595201  PMID: 26444668
Early stage lung cancer; Prognosis; Copy number profiling; Gene filtering; Semi-supervised learning
22.  Essential role of aldehyde dehydrogenase 1A3 (ALDH1A3) for the maintenance of non-small cell lung cancer stem cells is associated with the STAT3 pathway 
Lung cancer stem cells (CSCs) with elevated aldehyde dehydrogenase (ALDH) activity are self-renewing, clonogenic and tumorigenic. The purpose of our study is to elucidate the mechanisms by which lung CSCs are regulated.
Experimental Design
A genome-wide gene expression analysis was performed to identify genes differentially expressed in the ALDH+ vs. ALDH− cells. RT-PCR, western blot and Aldefluor assay were used to validate identified genes. To explore the function in CSCs we manipulated their expression followed by colony and tumor formation assays.
We identified a subset of genes that were differentially expressed in common in ALDH+ cells, among which ALDH1A3 was the most upregulated gene in ALDH+ vs. ALDH− cells. ShRNA-mediated knockdown of ALDH1A3 in NSCLCs resulted in a dramatic reduction in ALDH activity, clonogenicity and tumorigenicity, indicating that ALDH1A3 is required for tumorigenic properties. By contrast, overexpression of ALDH1A3 by itself it was not sufficient to increase tumorigenicity. The ALDH+ cells also expressed more activated Signal Transducers and Activators of Transcription 3 (STAT3) than ALDH− cells. Inhibition of STAT3 or its activator EZH2 genetically or pharmacologically diminished the level of ALDH+ cells and clonogenicity. Unexpectedly, ALDH1A3 was highly expressed in female, never smokers, well differentiated tumors, or adenocarcinoma. ALDH1A3 low expression was associated with poor overall survival.
Our data show that ALDH1A3 is the predominant ALDH isozyme responsible for ALDH activity and tumorigenicity in most NSCLCs, and that inhibiting either ALDH1A3 or the STAT3 pathway are potential therapeutic strategies to eliminate the ALDH+ subpopulation in NSCLCs.
PMCID: PMC4438754  PMID: 24907115
Lung cancer; cancer stem cells; ALDH1A3; STAT3; Stattic
24.  Identification of a Subset of Human Non–Small Cell Lung Cancer Patients with High PI3Kβ and Low PTEN Expression, More Prevalent in Squamous Cell Carcinoma 
The phosphoinositide 3-kinase (PI3K) pathway is a major oncogenic signaling pathway and an attractive target for therapeutic intervention. Signaling through the PI3K pathway is moderated by the tumor suppressor PTEN, which is deficient or mutated in many human cancers. Molecular characterization of the PI3K signaling network has not been well defined in lung cancer; in particular, the role of PI3Kβ and its relation to PTEN in non–small cell lung cancer NSCLC remain unclear.
Experimental Design
Antibodies directed against PI3Kβ and PTEN were validated and used to examine, by immunohistochemistry, expression in 240 NSCLC resection tissues [tissue microarray (TMA) set 1]. Preliminary observations were extended to an independent set of tissues (TMA set 2) comprising 820 NSCLC patient samples analyzed in a separate laboratory applying the same validated antibodies and staining protocols. The staining intensities for PI3Kβ and PTEN were explored and colocalization of these markers in individual tumor cores were correlated.
PI3Kβ expression was elevated significantly in squamous cell carcinomas (SCC) compared with adenocarcinomas. In contrast, PTEN loss was greater in SCC than in adenocarcinoma. Detailed correlative analyses of individual patient samples revealed a significantly greater proportion of SCC in TMA set 1 with higher PI3Kβ and lower PTEN expression when compared with adenocarcinoma. These findings were reinforced following independent analyses of TMA set 2.
We identify for the first time a subset of NSCLC more prevalent in SCC, with elevated expression of PI3Kβ accompanied by a reduction/loss of PTEN, for whom selective PI3Kβ inhibitors may be predicted to achieve greater clinical benefit.
PMCID: PMC4503252  PMID: 24284056
25.  Copper Transporter CTR1 Expression and Tissue Platinum Concentration in Non-Small Cell Lung Cancer 
Platinum resistance is a major limitation in the treatment of advanced non-small cell lung cancer (NSCLC). We previously demonstrated that low tissue platinum concentration in NSCLC specimens was significantly associated with reduced tumor response. Furthermore, low expression of the copper transporter CTR1, a transporter of platinum uptake was associated with poor clinical outcome following platinum-based therapy in NSCLC patients. We investigated the relationship between tissue platinum concentrations and CTR1 expression in NSCLC specimens.
We identified paraffin-embedded NSCLC tissue blocks of known tissue platinum concentrations from 30 patients who underwent neoadjuvant platinum-based chemotherapy at MD Anderson Cancer Center. Expression of CTR1 in tumors and normal adjacent lung specimens was determined by immunohistochemistry with adequate controls.
Tissue platinum concentration significantly correlated with tumor response in 30 patients who received neoadjuvant platinum-based chemotherapy (P<0.001). CTR1 was differentially expressed in NSCLC tumors. A subset of patients with undetectable CTR1 expression in their tumors had reduced platinum concentrations (P=0.058) and tumor response (P=0.016) compared to those with any level of CTR1 expression. We also observed that African Americans had significantly reduced CTR1 expression scores (P=0.001), tissue platinum concentrations (P=0.009) and tumor shrinkage (P=0.016) compared to Caucasians.
To our best knowledge this is the first study investigating the function of CTR1 in clinical specimens. CTR1 expression may be necessary for therapeutic efficacy of platinum drugs, consistent with previous preclinical studies. A prospective clinical trial is necessary to develop CTR1 into a potential biomarker for platinum drugs.
PMCID: PMC4090351  PMID: 24792335

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