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1.  Comprehensive biomarker analysis and final efficacy results of sorafenib in the BATTLE (Biomarker-Integrated Approaches of Targeted Therapy for Lung Cancer Elimination) trial 
Purpose
To report the clinical efficacy of sorafenib and to evaluate biomarkers associated with sorafenib clinical benefit in the BATTLE program.
Patients and Methods
Patients with previously treated non-small–cell lung cancer (NSCLC) received sorafenib until progression or unacceptable toxicity. Eight-week disease control rate (DCR), progression-free survival (PFS), and overall survival (OS) were assessed. Prespecified biomarkers included K-RAS, EGFR, and B-RAF mutations, and EGFR gene copy number. Gene expression profiles from NSCLC cell lines and patient tumor biopsies with wild-type EGFR were used to develop a sorafenib sensitivity signature (SSS).
Results
105 patients were eligible and randomized to receive sorafenib. Among 98 patients evaluable for 8-week DCR, the observed DCR was 58.2%. The median PFS and OS were 2.83 (95% confidence interval [CI], 2.04-3.58) and 8.48 months (95% CI, 5.78-10.97), respectively. Eight-week DCR was higher in patients with wt-EGFR than patients with EGFR mutation (P=0.012), and in patients with EGFR gene copy number gain (FISH positive) versus patients FISH negative (P=0.048). In wt-EGFR tumors, the SSS was associated with improved PFS (median PFS 3.61 months in high SSS versus 1.84 months in low SSS, P=0.026) but not with 8-week DCR. Increased expression of fibroblast growth factor-1, NF-kB and hypoxia pathways were identified potential drivers of sorafenib resistance.
Conclusion
Sorafenib demonstrates clinical activity in NSCLC, especially with wt-EGFR. SSS was associated with improved PFS. These data identify subgroups that may derive clinical benefit from sorafenib and merit investigation in future trials. ClinicalTrials.gov: NCT00411671.
doi:10.1158/1078-0432.CCR-12-1818
PMCID: PMC3905243  PMID: 24166906
multikinase inhibitor; non–small cell lung cancer; sorafenib; biomarkers; targeted treatment
2.  EZH2 Protein Expression Associates With the Early Pathogenesis, Tumor Progression and Prognosis of Non-small Cell Lung Carcinoma 
Purpose
Enhancer of zeste homolog 2 (EZH2) promotes carcinogenesis by epigenetically silencing tumor suppressor genes. We studied EZH2 expression by immunohistochemistry in a large series of non-small cell lung carcinomas (NSCLC) in association with tumor characteristics and patient outcomes.
Experimental Design
EZH2 immunohistochemistry expression was analyzed in 265 normal and premalignant bronchial epithelia, 541 primary NSCLCs [221 squamous cell carcinomas (SCCs) and 320 adenocarcinomas] and 36 NSCLCs with paired brain metastases. An independent set of 91 adenocarcinomas was also examined. EZH2 expression was statistically correlated with clinico-pathological information, and EGFR/KRAS mutation status.
Results
EZH2 expression was significantly (P<0.0001) higher in SCCs compared to adenocarcinomas and in brain metastasis relative to matched primary tumors (P=0.0013). EZH2 expression was significantly (P<0.0001) elevated in bronchial preneoplastic lesions with increasing severity. In adenocarcinomas, higher EZH2 expression significantly correlated with younger age, cigarette smoking and higher TNM stage (P=0.02 to P<0.0001). Higher EZH2 expression in adenocarcinoma was associated with worse recurrence-free survival (RFS; P=0.025; HR 1.54) and overall survival (OS; P=0.0002; HR 1.96). Furthermore, lung adenocarcinomas with low EZH2 levels and high expression of the lineage-specific transcription factor, TTF-1, exhibited significantly improved RFS (P=0.009; HR 0.51) and OS (P=0.0011; HR 0.45) which was confirmed in the independent set of 91 adenocarcinomas.
Conclusion
In lung, EZH2 expression is involved in early pathogenesis of SCC and correlates with a more aggressive tumor behavior of adenocarcinoma. When EZH2 and TTF-1 expressions are considered together, they serve as a prognostic marker in patients with surgically resected lung adenocarcinomas.
doi:10.1158/1078-0432.CCR-12-3946
PMCID: PMC3890101  PMID: 24097870
EZH2; NSCLC; lung adenocarcinoma; lung squamous cell carcinoma; bronchial preneoplasia; brain metastasis; KRAS mutations; EGFR mutations
3.  The BATTLE Trial: Personalizing Therapy for Lung Cancer 
Cancer discovery  2011;1(1):44-53.
Background
Lung cancer is the leading cancer cause of mortality worldwide; large-scale trials have failed to improve clinical outcomes of patients with chemorefractory non-small-cell lung cancer (NSCLC).
Methods
Following an initial equal randomization period, BATTLE adaptively randomized patients with chemorefractory NSCLC to erlotinib, vandetanib, erlotinib plus bexarotene, or sorafenib based on molecular biomarkers of NSCLC pathogenesis in fresh core needle biopsy specimens. The primary end point was disease control rate (DCR) at 8 weeks.
Results
Of 255 patients randomly assigned to erlotinib (59 patients), vandetanib (54), erlotinib plus bexarotene (37), and sorafenib (105), 244 were eligible for the DCR analysis. Pneumothorax after lung biopsy occurred in 11.5% and treatment-related toxicities grade 3–4 in 6.5% of patients. Overall results were a 46% 8-week DCR, 1.9-month median progression-free survival, 9-month median overall survival, and 35% 1-year survival. Individual markers predicting a significantly superior DCR for a treatment included: epidermal growth factor receptor (EGFR) mutation (P=0.04) for erlotinib; cyclin D1 positivity (P=0.01) or EGFR amplification (P=0.006) for erlotinib plus bexarotene; vascular endothelial growth factor receptor 2 positivity (P=0.05) for vandetanib; and absence of EGFR mutation (P=0.01) or of EGFR high polysomy (P=0.05) for sorafenib. A better 8-week DCR occurred with sorafenib versus all other regimens (64% versus 33%; P<0.001) among EGFR wild-type patients and versus all other regimens (61% versus 32%; P=0.11) among mutant-KRAS patients. The prespecified biomarker groups were less predictive than the individual biomarkers analyzed in this study.
Conclusions
The first completed biopsy-mandated study in pretreated NSCLC, BATTLE confirmed our pre-specified hypotheses regarding biomarker and targeted treatment interactions, establishing a new paradigm for personalizing therapy for patients with NSCLC. (ClinicalTrials.gov numbers, NCT00409968, NCT00411671, NCT00411632, NCT00410059, NCT00410189.)
doi:10.1158/2159-8274.CD-10-0010
PMCID: PMC4211116  PMID: 22586319
4.  A Phase I/II Study Combining Erlotinib and Dasatinib for Non-Small Cell Lung Cancer 
The Oncologist  2014;19(10):1040-1041.
Background.
EGFR and Src are frequently activated in non-small cell lung cancer (NSCLC). In preclinical models, combining EGFR and Src inhibition has additive synergistic effects. We conducted a phase I/II trial of the combination of Src inhibitor dasatinib with EGFR inhibitor erlotinib to determine the maximum tolerated dose (MTD), pharmacokinetic drug interactions, biomarkers, and efficacy in NSCLC.
Methods.
The phase I 3+3 dose-escalation study enrolled patients with solid tumors to determine the MTD. The phase II trial enrolled patients with advanced NSCLC who had undergone no previous treatments to determine progression-free survival (PFS) and response. Pharmacokinetic and tissue biomarker analyses were performed.
Results.
MTD was 150 mg of erlotinib and 70 mg of dasatinib daily based on 12 patients treated in the phase I portion. No responses were observed in phase I. The 35 NSCLC patients treated in phase II had an overall disease control rate of 59% at 6 weeks. Five patients (15%) had partial responses; all had activating EGFR mutations. Median PFS was 3.3 months. Epithelial-mesenchymal transition markers did not correlate with outcomes.
Conclusion.
The combination of erlotinib and dasatinib is safe and feasible in NSCLC. The results of this study do not support use of this combination in molecularly unselected NSCLC.
doi:10.1634/theoncologist.2014-0228
PMCID: PMC4201000  PMID: 25170013
5.  Biological Activity of Celecoxib in the Bronchial Epithelium of Current and Former Smokers 
Purpose
Non-small-cell lung cancer (NSCLC) is the primary cause of cancer-related death in Western countries. One important approach taken to address this problem is the development of effective chemoprevention strategies. In this study, we examined whether the cyclooxygenase-2 (COX-2) inhibitor celecoxib, as evidenced by decreased cell proliferation, is biologically active in the bronchial epithelium of current and former smokers.
Patients and Methods
Current or former smokers with at least a 20 pack-year (pack-year = number of packs of cigarettes per day times number of years smoked) smoking history were randomized into one of four treatment arms (3-month intervals of celecoxib then placebo, celecoxib then celecoxib, placebo then celecoxib, or placebo then placebo) and underwent bronchoscopies with biopsies at baseline, 3 months, and 6 months. The 204 patients were primarily (79.4%) current smokers; 81 received either low-dose celecoxib or placebo and 123 received either high-dose celecoxib or placebo. Celecoxib was originally administered orally at 200 mg twice daily and the protocol subsequently increased the dose to 400 mg twice daily. The primary endpoint was change in Ki-67 labeling (from baseline to 3 months) in bronchial epithelium.
Results
No cardiac toxicities were observed in the participants. Although the effect of low-dose treatment was not significant, high-dose celecoxib decreased Ki-67 labeling by 3.85% in former smokers and by 1.10% in current smokers—a significantly greater reduction (P = 0.02) than that seen with placebo after adjusting for metaplasia and smoking status.
Conclusion
A 3–6-month celecoxib regimen proved safe to administer. Celecoxib 400 mg bid was biologically active in the bronchial epithelium of current and former smokers; additional studies on the efficacy of celecoxib in NSCLC chemoprevention may be warranted.
doi:10.1158/1940-6207.CAPR-09-0233
PMCID: PMC4028718  PMID: 20103722
6.  Feasibility of Image-Guided Transthoracic Core Needle Biopsy in the BATTLE Lung Trial 
Purpose
As therapy for non-small cell lung cancer (NSCLC) patients becomes more personalized, additional tissue in the form of core needle biopsies (CNBs) for biomarker analysis is increasingly required for determining appropriate treatment and for enrollment into clinical trials. We report our experience with small-caliber percutaneous transthoracic (PT) CNBs for the evaluation of multiple molecular biomarkers in BATTLE (Biomarker-integrated Approaches of Targeted Therapy for Lung Cancer Elimination), a personalized, targeted therapy NSCLC clinical trial.
Methods
The medical records of patients who underwent PTCNB for consideration of enrollment in BATTLE, were reviewed for diagnostic yield of 11 predetermined molecular markers, and procedural complications. Univariate and multivariate analyses of factors related to patient and lesion characteristics were performed to determine possible influences on diagnostic yield.
Results
One hundred and seventy PTCNBs were performed using 20-gauge biopsy needles in 151 NSCLC patients screened for the trial. 82.9% of the biopsy specimens were found to have adequate tumor tissue for analysis of the required biomarkers. On multivariate analysis, metastatic lesions were 5.4 times more likely to yield diagnostic tissue as compared to primary tumors (p = 0.0079). Pneumothorax and chest tube insertion rates were 15.3% and 9.4%, respectively.
Conclusions
Image-guided 20-gauge PTCNB is safe and provides adequate tissue for analysis of multiple biomarkers in the majority of patients being considered for enrollment into a personalized, targeted therapy NSCLC clinical trial. Metastatic lesions are more likely to yield diagnostic tissue as compared to primary tumors.
doi:10.1097/JTO.0b013e318287c91e
PMCID: PMC3879952  PMID: 23442309
research biopsy; biomarker analysis; percutaneous transthoracic biopsy
7.  Kinase Impaired BRAF Mutations Confer Lung Cancer Sensitivity to Dasatinib 
Science translational medicine  2012;4(136):10.1126/scitranslmed.3003513.
During a clinical trial of the tyrosine kinase inhibitor dasatinib for advanced non–small cell lung cancer (NSCLC) one patient responded dramatically and remains cancer-free 4 years later. A comprehensive analysis of his tumor revealed a previously undescribed, kinase inactivating BRAF mutation (Y472CBRAF); no inactivating BRAF mutations were found in the non-responding tumors taken from other patients. Cells transfected with Y472CBRAF exhibited CRAF, MEK, and ERK activation – characteristics identical to signaling changes that occur with previously known kinase inactivating BRAF mutants. Dasatinib selectively induced senescence in NSCLC cells with inactivating BRAF mutations. Transfection of other NSCLC cells with these BRAF mutations also increased these cells’ dasatinib sensitivity, whereas transfection with an activating BRAF mutation led to their increased dasatinib resistance. The sensitivity induced by Y472CBRAF was reversed by the introduction of a BRAF mutation that impairs RAF dimerization. Dasatinib inhibited CRAF modestly, but concurrently induced RAF dimerization resulting in ERK activation in NSCLC cells with kinase inactivating BRAF mutations. The sensitivity of NSCLC with kinase impaired BRAF to dasatinib suggested synthetic lethality of BRAF and a dasatinib target. Inhibiting BRAF in NSCLC cells expressing wild-type BRAF likewise enhanced these cells’ dasatinib sensitivity. Thus, the patient’s BRAF mutation was likely responsible for his tumor’s marked response to dasatinib, suggesting that tumors bearing kinase impaired BRAF mutations may be exquisitely sensitive to dasatinib. Moreover, the potential synthetic lethality of combination therapy including dasatinib and BRAF inhibitors may lead to additional therapeutic options against cancers with wild-type BRAF.
doi:10.1126/scitranslmed.3003513
PMCID: PMC3836384  PMID: 22649091
8.  Effect of KRAS Oncogene Substitutions on Protein Behavior: Implications for Signaling and Clinical Outcome 
Background
Mutations in the v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) play a critical role in cancer cell growth and resistance to therapy. Most mutations occur at codons 12 and 13. In colorectal cancer, the presence of any mutant KRas amino acid substitution is a negative predictor of patient response to targeted therapy. However, in non–small cell lung cancer (NSCLC), the evidence that KRAS mutation is a predictive factor is conflicting.
Methods
We used data from a molecularly targeted clinical trial for 215 patients with tissues available out of 268 evaluable patients with refractory NSCLC to examine associations between specific mutant KRas proteins and progression-free survival and tumor gene expression. Transcriptome microarray studies of patient tumor samples and reverse-phase protein array studies of a panel of 67 NSCLC cell lines with known substitutions in KRas and in immortalized human bronchial epithelial cells stably expressing different mutant KRas proteins were used to investigate signaling pathway activation. Molecular modeling was used to study the conformations of wild-type and mutant KRas proteins. Kaplan–Meier curves and Cox regression were used to analyze survival data. All statistical tests were two-sided.
Results
Patients whose tumors had either mutant KRas-Gly12Cys or mutant KRas-Gly12Val had worse progression-free survival compared with patients whose tumors had other mutant KRas proteins or wild-type KRas (P = .046, median survival = 1.84 months) compared with all other mutant KRas (median survival = 3.35 months) or wild-type KRas (median survival = 1.95 months). NSCLC cell lines with mutant KRas-Gly12Asp had activated phosphatidylinositol 3-kinase (PI-3-K) and mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) signaling, whereas those with mutant KRas-Gly12Cys or mutant KRas-Gly12Val had activated Ral signaling and decreased growth factor–dependent Akt activation. Molecular modeling studies showed that different conformations imposed by mutant KRas may lead to altered association with downstream signaling transducers.
Conclusions
Not all mutant KRas proteins affect patient survival or downstream signaling in a similar way. The heterogeneous behavior of mutant KRas proteins implies that therapeutic interventions may need to take into account the specific mutant KRas expressed by the tumor.
doi:10.1093/jnci/djr523
PMCID: PMC3274509  PMID: 22247021
9.  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.
doi:10.1158/0008-5472.CAN-10-2614
PMCID: PMC3159530  PMID: 21724587
10.  HER Family Receptor Abnormalities in Lung Cancer Brain Metastases and Corresponding Primary Tumors 
Clinical Cancer Research  2009;15(15):4829-4837.
Purpose
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.
Results
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.
Conclusions
Our findings suggest that NSCLC brain metastases have some significant differences in HER family receptors-related abnormalities from primary lung tumors.
doi:10.1158/1078-0432.CCR-08-2921
PMCID: PMC3372920  PMID: 19622585
11.  Histologic Patterns and Molecular Characteristics of Lung Adenocarcinoma Associated With Clinical Outcome 
Cancer  2011;118(11):2889-2899.
BACKGROUND
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.
METHODS
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).
RESULTS
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).
CONCLUSIONS
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.
doi:10.1002/cncr.26584
PMCID: PMC3369269  PMID: 22020674
histologic patterns; lung adenocarcinoma; thyroid transcription factor 1; prognostic signature
12.  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.
doi:10.1158/1940-6207.CAPR-08-0032
PMCID: PMC3369271  PMID: 19138956
13.  Abnormalities of the TITF-1 lineage-specific oncogene in NSCLC: Implications in lung cancer pathogenesis and prognosis 
PURPOSE
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.
EXPERIMENTAL DESIGN
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.
RESULTS
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).
CONCLUSIONS
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.
doi:10.1158/1078-0432.CCR-10-1412
PMCID: PMC3078948  PMID: 21257719
NSCLC; TITF-1; gene copy gain; lineage-specific oncogenes
14.  Immunohistochemical Expression of Estrogen and Progesterone Receptors Identifies a Subset of NSCLCs and Correlates with EGFR Mutation 
Purpose
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.
Results
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.
Conclusions
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.
doi:10.1158/1078-0432.CCR-09-0033
PMCID: PMC2893045  PMID: 19706809
15.  Sex Determining Region Y-Box 2 (SOX2) Is a Potential Cell-Lineage Gene Highly Expressed in the Pathogenesis of Squamous Cell Carcinomas of the Lung 
PLoS ONE  2010;5(2):e9112.
Background
Non-small cell lung cancer (NSCLC) represents the majority (85%) of lung cancers and is comprised mainly of adenocarcinomas and squamous cell carcinomas (SCCs). The sequential pathogenesis of lung adenocarcinomas and SCCs occurs through dissimilar phases as the former tumors typically arise in the lung periphery whereas the latter normally arise near the central airway.
Methodology/Principal Findings
We assessed the expression of SOX2, an embryonic stem cell transcriptional factor that also plays important roles in the proliferation of basal tracheal cells and whose expression is restricted to the main and central airways and bronchioles of the developing and adult mouse lung, in NSCLC by various methodologies. Here, we found that SOX2 mRNA levels, from various published datasets, were significantly elevated in lung SCCs compared to adenocarcinomas (all p<0.001). Moreover, a previously characterized OCT4/SOX2/NANOG signature effectively separated lung SCCs from adenocarcinomas in two independent publicly available datasets which correlated with increased SOX2 mRNA in SCCs. Immunohistochemical analysis of various histological lung tissue specimens demonstrated marked nuclear SOX2 protein expression in all normal bronchial epithelia, alveolar bronchiolization structures and premalignant lesions in SCC development (hyperplasia, dysplasia and carcinoma in situ) and absence of expression in all normal alveoli and atypical adenomatous hyperplasias. Moreover, SOX2 protein expression was greatly higher in lung SCCs compared to adenocarcinomas following analyses in two independent large TMA sets (TMA set I, n = 287; TMA set II, n = 511 both p<0.001). Furthermore, amplification of SOX2 DNA was detected in 20% of lung SCCs tested (n = 40) and in none of the adenocarcinomas (n = 17).
Conclusions/Significance
Our findings highlight a cell-lineage gene expression pattern for the stem cell transcriptional factor SOX2 in the pathogenesis of lung SCCs and suggest a differential activation of stem cell-related pathways between squamous cell carcinomas and adenocarcinomas of the lung.
doi:10.1371/journal.pone.0009112
PMCID: PMC2817751  PMID: 20161759
16.  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.
doi:10.1038/modpathol.2009.19
PMCID: PMC2675657  PMID: 19270647
epithelial-to-mesenchymal transition; tissue microarray; immunohistochemical analysis; lung cancer; preneoplasia; brain metastases
17.  Oncogene Mutations, Copy Number Gains and Mutant Allele Specific Imbalance (MASI) Frequently Occur Together in Tumor Cells 
PLoS ONE  2009;4(10):e7464.
Background
Activating mutations in one allele of an oncogene (heterozygous mutations) are widely believed to be sufficient for tumorigenesis. However, mutant allele specific imbalance (MASI) has been observed in tumors and cell lines harboring mutations of oncogenes.
Methodology/Principal Findings
We determined 1) mutational status, 2) copy number gains (CNGs) and 3) relative ratio between mutant and wild type alleles of KRAS, BRAF, PIK3CA and EGFR genes by direct sequencing and quantitative PCR assay in over 400 human tumors, cell lines, and xenografts of lung, colorectal, and pancreatic cancers. Examination of a public database indicated that homozygous mutations of five oncogenes were frequent (20%) in 833 cell lines of 12 tumor types. Our data indicated two major forms of MASI: 1) MASI with CNG, either complete or partial; and 2) MASI without CNG (uniparental disomy; UPD), due to complete loss of wild type allele. MASI was a frequent event in mutant EGFR (75%) and was due mainly to CNGs, while MASI, also frequent in mutant KRAS (58%), was mainly due to UPD. Mutant: wild type allelic ratios at the genomic level were precisely maintained after transcription. KRAS mutations or CNGs were significantly associated with increased ras GTPase activity, as measured by ELISA, and the two molecular changes were synergistic. Of 237 lung adenocarcinoma tumors, the small number with both KRAS mutation and CNG were associated with shortened survival.
Conclusions
MASI is frequently present in mutant EGFR and KRAS tumor cells, and is associated with increased mutant allele transcription and gene activity. The frequent finding of mutations, CNGs and MASI occurring together in tumor cells indicates that these three genetic alterations, acting together, may have a greater role in the development or maintenance of the malignant phenotype than any individual alteration.
doi:10.1371/journal.pone.0007464
PMCID: PMC2757721  PMID: 19826477

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