Purpose

This prospective study aimed to develop a robust and clinically-applicable method to identify high-risk early stage lung cancer patients and then to validate this method for use in future translational studies.

Patients and Methods

Three published Affymetrix microarray data sets representing 680 primary tumors were used in the survival-related gene selection procedure using clustering, Cox model and random survival forest (RSF) analysis. A final set of 91 genes was selected and tested as a predictor of survival using a qRT-PCR-based assay utilizing an independent cohort of 101 lung adenocarcinomas.

Results

The RSF model built from 91 genes in the training set predicted patient survival in an independent cohort of 101 lung adenocarcinomas, with a prediction error rate of 26.6%. The mortality risk index (MRI) was significantly related to survival (Cox model p < 0.00001) and separated all patients into low, medium, and high-risk groups (HR = 1.00, 2.82, 4.42). The MRI was also related to survival in stage 1 patients (Cox model p = 0.001), separating patients into low, medium, and high-risk groups (HR = 1.00, 3.29, 3.77).

Conclusions

The development and validation of this robust qRT-PCR platform allows prediction of patient survival with early stage lung cancer. Utilization will now allow investigators to evaluate it prospectively by incorporation into new clinical trials with the goal of personalized treatment of lung cancer patients and improving patient survival.

Background

Esophageal cancer consists of two major histologic types: esophageal squamous cell carcinoma (ESCC) predominant globally and esophageal adenocarcinoma (EAC) with a higher incidence in westernized countries. Five-year overall survival is 15%. Clinical trials frequently combine histologies although they are different diseases with distinct origins. In the evolving era of personalized medicine and targeted therapies, we hypothesized that ESCC and EAC have genomic differences important for developing new therapeutic strategies for esophageal cancer.

Methods

We explored DNA copy number abnormalities (CNAs) in 70 ESCCs with publicly available array data and 189 EAC from our group. All data was from Affymetrix single nucleotide polymorphism (SNP) arrays. Analysis was performed with Nexus 5.0 Copy number software using a SNPRank segmentation algorithm. Log ratio thresholds for copy number gain and loss were set at +/− 0.2 (approximately 2.3 and 1.7 copies respectively).

Results

ESCC and EAC genomes showed some CNAs with similar frequencies (e.g., CDKN2A, EGFR, KRAS, MYC, CDK6, MET) but also many CNAs with different frequencies between histologies, most of which were amplification events. Some of these regions harbor genes to which targeted therapies are currently available (VEGFA, ERBB2) or where agents are in clinical trials (PIK3CA, FGFR1). Other regions contain putative oncogenes that may be targeted in the future.

Conclusions

Using SNP arrays we compared genomic abnormalities in a large cohort of EAC and ESCC. We report here the similar and different frequencies of CNAs in ESCC and EAC. These results may allow development of histology-specific therapeutic agents for esophageal cancer.

Purpose

LRP1 is a broadly-expressed receptor that binds multiple extracellular ligands and participates in protein clearance. LRP1 is expressed numerous cancers, but its role in lung cancer has not been characterized. Here, we investigate the relationship between LRP1 and lung cancer.

Experimental Design

LRP1 mRNA levels were determined in lung tumors from several large, multicenter studies. LRP1 protein localization was determined by immunohistochemical analysis of lung tumor microarrays. Normal fibroblasts, fibroblasts treated with the LRP1 inhibitor RAP, and LRP1 null fibroblasts were co-cultured with three independent lung cancer cell lines to investigate the role of LRP1 on tumor cell proliferation.

Results

LRP1 mRNA levels are significantly decreased in lung tumors relative to non-tumorous lung tissue. Lower expression of LRP1 in lung adenocarcinomas correlates with less favorable clinical outcome in a cohort of 439 patients. Immunohistochemical analysis demonstrates that LRP1 is primarily expressed in stromal cells in 94/111 lung cancers, with very little protein found in cancer cells. A growth suppressive function of mouse embryonic fibroblast cells (MEF) was observed in three lung cancer cell lines tested (H460, H2347, and HCC4006 cells); growth suppression was blocked by the LRP1 inhibitor, RAP. LRP1 deletion in fibroblasts reduced the ability of MEF cells to suppress tumor cell mitosis. In a validation set of adenocarcinomas, we confirmed a significant positive correlation between both LRP1 mRNA and protein levels and favorable clinical outcomes.

Conclusions

LRP1 expression is associated with improved lung cancer outcomes. Mechanistically, stromal LRP1 may non-cell autonomously suppress lung tumor cell proliferation.

Background & Aims

Dysplasia is a pre-malignant condition in Barrett's esophagus that is difficult to detect on screening endoscopy because of its flat architecture and patchy distribution. Peptides are promising for use as novel molecular probes that identify cell surface targets unique to disease, and can be fluorescence-labeled for detection. We aim to select and validate an affinity peptide that binds to esophageal dysplasia for future clinical studies.

Methods

Peptide selection was performed using phage display by removing non-specific binders using Q-hTERT (intestinal metaplasia) cells and achieving specific binding against OE33 (esophageal adenocarcinoma) cells. Selective binding was confirmed on bound phage counts, ELISA, flow cytometry, competitive inhibition, and fluorescence microscopy. On stereomicroscopy, specific peptide binding to dysplasia on endoscopically resected specimens was assessed by rigorous registration of fluorescence intensity to histology in 1 mm intervals.

Results

The peptide sequence SNFYMPL was selected and demonstrated preferential binding to target cells on bound phage counts, ELISA, and flow cytometry. Reducing binding was observed on competition with unlabeled peptide in a dose dependent manner, an affinity of Kd = 164 nM was measured, and peptide binding to the surface of OE33 cells was validated on fluorescence microscopy. On esophageal specimens (n=12), the fluorescence intensity (mean±SEM) in 1 mm intervals classified histologically as squamous (n=145), intestinal metaplasia (n=83), dysplasia (n=61) and gastric mucosa (n=69) was 46.5±1.6, 62.3±5.8, 100.0±9.0, and 42.4±3.0 arb units, respectively.

Conclusions

The peptide sequence SNFYMPL binds specifically to dysplasia in Barrett's esophagus, and can be fluorescence-labeled to target pre-malignant mucosa on imaging.

Purpose

The active form of vitamin D, 1α,25-dihydroxyvitamin D3 (1,25-D3) exerts antiproliferative effects in cancers, including lung adenocarcinoma (AC). CYP24A1 is overexpressed in many cancers and catabolizes 1,25-D3. The purpose of our study was to assess CYP24A1 as a prognostic marker and to study its relevance to antiproliferative activity of 1,25-D3 in lung AC cells.

Experimental Design

Tumors and corresponding normal specimens from 86 patients with lung AC (stages I–III) were available. AffymetrixR array data and subsequent confirmation by quantitative real time-PCR were used to determine CYP24A1 mRNA expression. A subsequent validation set of 101 lung AC was used to confirm CYP24A1 mRNA expression and its associations with clinical variables. The antiproliferative effects of 1,25-D3 were examined using lung cancer cell lines with high as well as low expression of CYP24A1 mRNA.

Results

CYP24A1 mRNA was elevated 8–50 fold in lung AC (compared to normal nonneoplastic lung) and significantly higher in poorly-differentiated cancers. At 5 years of follow-up, the probability of survival was 42% (high CYP24A1, n = 29) versus 81% (low CYP24A1, n = 57) (P = 0.007). The validation set of 101 tumors showed that CYP24A1 was independently prognostic of survival (multivariate Cox model adjusted for age, gender and stage, P = 0.001). A549 cells (high CYP24A1) were more resistant to antiproliferative effects of 1,25-D3 compared with SKLU-1 cells (low CYP24A1).

Conclusions

CYP24A1 overexpression is associated with poorer survival in lung AC. This may relate to abrogation of antiproliferative effects of 1,25-D3 in high CYP24A1 expressing lung AC.

We profiled receptor tyrosine kinase pathway activation and key gene mutations in eight human lung tumor cell lines and 50 human lung tumor tissue samples to define molecular pathways. A panel of eight kinase inhibitors was used to determine whether blocking pathway activation affected the tumor cell growth. The HER1 pathway in HER1 mutant cell lines HCC827 and H1975 were found to be highly activated and sensitive to HER1 inhibition. H1993 is a c-MET amplified cell line showing c-MET and HER1 pathway activation and responsiveness to c-MET inhibitor treatment. IGF-1R pathway activated H358 and A549 cells are sensitive to IGF-1R inhibition. The downstream PI3K inhibitor, BEZ-235, effectively inhibited tumor cell growth in most of the cell lines tested, except the H1993 and H1650 cells, while the MEK inhibitor PD-325901 was effective in blocking the growth of KRAS mutated cell line H1734 but not H358, A549 and H460. Hierarchical clustering of primary tumor samples with the corresponding tumor cell lines based on their pathway signatures revealed similar profiles for HER1, c-MET and IGF-1R pathway activation and predict potential treatment options for the primary tumors based on the tumor cell lines response to the panel of kinase inhibitors.

Epidermal growth factor receptor (EGFR) is overexpressed in a variety of epithelial tumors and is considered to be an important therapeutic target. Although gene amplification is responsible for EGFR overexpression in certain human malignancies including lung and head and neck cancers, additional molecular mechanisms are likely. Here, we report a novel interaction of EGFR with an HECT-type ubiquitin ligase SMURF2, which can ubiquitinate, but stabilize EGFR by protecting it from c-Cbl-mediated degradation. Conversely, small interfering RNA (siRNA)-mediated knockdown of SMURF2 destabilized EGFR, induced an autophagic response and reduced the clonogenic survival of EGFR-expressing cancer cell lines, with minimal effects on EGFR-negative cancer cells, normal fibroblasts, and normal epithelial cells. UMSCC74B head and neck squamous cancer cells, which form aggressive tumors in nudemice, significantly lost in vivo tumor-forming ability on siRNA-mediated SMURF2 knockdown. Gene expressionmicroarray data from 443 lung adenocarcinoma patients, and tissue microarray data from 67 such patients, showed a strong correlation of expression between EGFR and SMURF2 at the messenger RNA and protein levels, respectively. Our findings suggest that SMURF2-mediated protective ubiquitination of EGFR may be responsible for EGFR overexpression in certain tumors and support targeting SMURF2-EGFR interaction as a novel therapeutic approach in treating EGFR-addicted tumors.

Cancer genomes contain many aberrant gene fusions—a few that drive disease and many more that are nonspecific passengers. We developed an algorithm (the concept signature or ‘ConSig’ score) that nominates biologically important fusions from high-throughput data by assessing their association with ‘molecular concepts’ characteristic of cancer genes, including molecular interactions, pathways and functional annotations. Copy number data supported candidate fusions and suggested a breakpoint principle for intragenic copy number aberrations in fusion partners. By analyzing lung cancer transcriptome sequencing and genomic data, we identified a novel R3HDM2-NFE2 fusion in the H1792 cell line. Lung tissue microarrays revealed 2 of 76 lung cancer patients with genomic rearrangement at the NFE2 locus, suggesting recurrence. Knockdown of NFE2 decreased proliferation and invasion of H1792 cells. Together, these results present a systematic analysis of gene fusions in cancer and describe key characteristics that assist in new fusion discovery.

Purpose

The chemopreventive effects of selenium have been extensively examined but its role in cancer development or as a chemotherapeutic agent have only recently been explored. Because Selenium Binding Protein 1 (SELENBP1, SBP1, hSP56) has been shown to bind selenium covalently and selenium deficiency has been associated with esophageal adenocarcinoma (EAC), we examined its role in EAC development and its potential effect on chemosensitivity in the presence of selenium.

Experimental Design

SELENBP1 expression level and copy number variation were determined by oligonucleotide microarrays, real-time RT-PCR, tissue microarrays, immunoblotting and SNP arrays. Bisulfite sequencing and sequence analysis of RT-PCR-amplified products explored epigenetic and post-transcriptional regulation of SELENBP1 expression, respectively. WST-1 cell proliferation assays, senescence-associated β-galactosidase staining, immunoblotting, and flow cytometry were performed to evaluate the biological significance of SELENBP1 overexpression in selenium-supplemented EAC cells.

Results

SELENBP1 expression decreased significantly in Barrett's esophagus to adenocarcinoma progression. Both epigenetic and post-transcriptional mechanisms appeared to modulate SELENBP1 expression. Stable overexpression of SELENBP1 in methylseleninic acid-supplemented Flo-1 cells resulted in enhanced apoptosis, increased cellular senescence, and enhanced cisplatin cytotoxicity. Although inorganic sodium selenite similarly enhanced cisplatin cytotoxicity, these 2 forms of selenium elicited different cellular responses.

Conclusions

SELENBP1 expression may be an important predictor of response to chemoprevention or chemosensitization with certain forms of selenium in esophageal tissues.

For decades, hundreds of different human tumor type–specific cell lines have been used in experimental cancer research as models for their respective tumors. The veracity of experimental results for a specific tumor type relies on the correct derivation of the cell line. In a worldwide effort, we verified the authenticity of all available esophageal adenocarcinoma (EAC) cell lines. We proved that the frequently used cell lines SEG-1 and BIC-1 and the SK-GT-5 cell line are in fact cell lines from other tumor types. Experimental results based on these contaminated cell lines have led to ongoing clinical trials recruiting EAC patients, to more than 100 scientific publications, and to at least three National Institutes of Health cancer research grants and 11 US patents, which emphasizes the importance of our findings. Widespread use of contaminated cell lines threatens the development of treatment strategies for EAC.

Abstract

The L-type amino acid transporter-1 (LAT-1) has been associated with tumor growth. Using cDNA microarrays, overexpression of LAT-1 was found in 87.5% (7/8) of esophageal adenocarcinomas relative to 12 Barrett's samples (33% metaplasia and 66% dysplasia) and was confirmed in 100% (28/28) of Barrett's adenocarcinomas by quantitative reverse transcription polymerase chain reaction. Immunohistochemistry revealed LAT-1 staining in 37.5% (24/64) of esophageal adenocarcinomas on tissue microarray. LAT-1 also transports the amino acid-related chemotherapeutic agent, melphalan. Two esophageal adenocarcinoma and one esophageal squamous cell line, expressing LAT-1 on Western blot analysis, were sensitive to therapeutic doses of melphalan (P < .001). Simultaneous treatment with the competitive inhibitor, BCH [2-aminobicyclo-(2,1,1)-heptane-2-carboxylic acid], decreased sensitivity to melphalan (P < .05). In addition, confluent esophageal squamous cultures were less sensitive to melphalan (P < .001) and had a decrease in LAT-1 protein expression. Tumors from two esophageal adenocarcinoma cell lines grown in nude mice retained LAT-1 mRNA expression. These results demonstrate that LAT-1 is highly expressed in a subset of esophageal adenocarcinomas and that Barrett's adenocarcinoma cell lines expressing LAT-1 are sensitive to melphalan. LAT-1 expression is also retained in cell lines grown in nude mice providing a model to evaluate melphalan as a chemotherapeutic agent against esophageal adenocarcinomas expressing LAT-1.

INTRODUCTION: The incidence of Barrett esophageal adenocarcinoma (BEAC) has been increasing at an alarming rate in western countries. In this study, we have evaluated the therapeutic potential of sulforaphane (SFN), an antioxidant derived from broccoli, in BEAC. METHODS: BEAC cells were treated with SFN, alone or in combination with chemotherapeutic, paclitaxel, or telomerase-inhibiting agents (MST-312, GRN163L), and live cell number determined at various time points. The effect on drug resistance/chemosensitivity was evaluated by rhodamine efflux assay. Apoptosis was detected by annexin V labeling and Western blot analysis of poly(ADP-ribose) polymerase cleavage. Effects on genes implicated in cell cycle and apoptosis were determined by Western blot analyses. To evaluate the efficacy in vivo, BEAC cells were injected subcutaneously in severe combined immunodeficient mice, and after the appearance of palpable tumors, mice were treated with SFN. RESULTS: SFN induced both time- and dose-dependent decline in cell survival, cell cycle arrest, and apoptosis. The treatment with SFN also suppressed the expression of multidrug resistance protein, reduced drug efflux, and increased anticancer activity of other antiproliferative agents including paclitaxel. A significant reduction in tumor volume was also observed by SFN in a subcutaneous tumor model of BEAC. Anticancer activity could be attributed to the induction of caspase 8 and p21 and down-regulation of hsp90, a molecular chaperon required for activity of several proliferation-associated proteins. CONCLUSIONS: These data indicate that a natural product with antioxidant properties from broccoli has great potential to be used in chemoprevention and treatment of BEAC.

Abstract

The prolonged use of nonsteroidal anti-inflammatory drugs (NSAIDs) has been shown to exert a chemopreventive effect in esophageal and other gastrointestinal tumors. The precise mechanism by which this occurs, however, is unknown. While the inhibition of COX-2 as a potential explanation for this chemopreventive effect has gained a great deal of support, there also exists evidence supporting the presence of cyclooxygenase-independent pathways through which NSAIDs may exert their effects. In this study, immunohistochemical analysis of 29 Barrett's epithelial samples and 60 esophageal adenocarcinomas demonstrated abundant expression of the COX-2 protein in Barrett's epithelium, but marked heterogeneity of expression in esophageal adenocarcinomas. The three esophageal adenocarcinoma cell lines, Flo-1, Bic-1, and Seg-1, also demonstrated varying expression patterns for COX-1 and COX-2. Indomethacin induced apoptosis in all three cell lines, however, in both a time- and dose-dependent manner. In Flo-1 cells, which expressed almost undetectable levels of COX-1 and COX-2, and in Seg-1, which expressed significant levels of COX-1 and COX-2, indomethacin caused upregulation of the pro-apoptotic protein Bax. The upregulation of Bax was accompanied by the translocation of mitochondrial cytochrome c to the cytoplasm, and activation of caspase 9. Pre-treatment of both cell lines with the specific caspase 9 inhibitor, z-LEHD-FMK, as well as the broad-spectrum caspase inhibitor, z-VAD-FMK, blocked the effect of indomethacin-induced apoptosis. These data demonstrate that induction of apoptosis by indomethacin in esophageal adenocarcinoma cells is associated with the upregulation of Bax expression and mitochondrial cytochrome c translocation, and does not correlate with the expression of COX-2. This may have important implications for identifying new therapeutic targets in this deadly disease.

Purpose

An embryonic stem cell profile correlates with poorly differentiated breast, bladder and glioma cancers. In this manuscript, we assess the correlation between the embryonic stem cell profile and clinical variables in lung cancer.

Experimental Design

Microarray gene expression analysis was done using Affymetrix Human Genome U133A on 443 samples of human lung adenocarcinoma and 130 samples of squamous cell carcinoma. To identify gene-set enrichment patterns we used the Genomica software.

Results

Our analysis showed that an increased expression of the embryonic stem cell gene set and decreased expression of Polycomb target gene set identified poorly-differentiated lung adenocarcinoma. In addition, this gene expression signature was associated with markers of poor prognosis and worse overall survival in lung adenocarcinoma. However, there was no correlation between this embryonic stem cell gene signature and any histological or clinical variable assessed in lung squamous cell carcinoma.

Conclusions

This work suggests that not all poorly-differentiated non-small cell lung cancers exhibit a gene expression profile similar to ESC, and that other characteristics may play a more important role in the determination of differentiation and survival in squamous cell carcinoma of the lung.

Lung adenocarcinoma (AD) represents a predominant type of lung cancer demonstrating significant morphologic and molecular heterogeneity. We sought to understand this heterogeneity by utilizing gene expression analyses of 432 AD samples and examining associations between 27 known cancer-related pathways and the AD subtype, clinical characteristics and patient survival. Unsupervised clustering of AD and gene expression enrichment analysis reveals that cell proliferation is the most important pathway separating tumors into subgroups. Further, AD with increased cell proliferation demonstrate significantly poorer outcome and an increased solid AD subtype component. Additionally, we find that tumors with any solid component have decreased survival as compared to tumors without a solid component. These results lead to the potential to use a relatively simple pathological examination of a tumor in order to determine its aggressiveness and the patient's prognosis. Additional results suggest the ability to use a similar approach to determine a patient's sensitivity to targeted treatment. We then demonstrated the consistency of these findings using two independent AD cohorts from Asia (N = 87) and Europe (N = 89) using the identical analytic procedures.

The transcription factor, nuclear factor κB (NF-κB), plays a central role as a key mediator of cell survival and proliferation, and its activation may confer increased tumor chemoresistance. Curcumin, an orally available naturally occurring compound, has been shown to inhibit NF-κB and has a potential role in cancer chemoprevention. We investigated the effects of curcumin on NF-κB activity, on cell viability, and as a chemosensitizing agent with 5-fluorouracil (5-FU) or cisplatin (CDDP) in esophageal adenocarcinoma (EAC). Oligonucleotide microarray analysis of 46 cases, consisting of Barrett metaplasia, low-grade dysplasia, high-grade dysplasia and EAC, showed increased expression of NF-κB and IκB kinase subunits and decreased effector caspase expression in EAC compared with Barrett metaplasia. Stromal expression of both IκB and phospho-IκB was detected in several EAC samples by tissue microarray analysis. Curcumin alone inhibited NF-κB activity and induced apoptosis in both Flo-1 and OE33 EAC cell lines as determined by Western blot analysis, NF-κB reporter assays, and Caspase-Glo 3/7 assays. It also increased 5-FU- and CDDP-induced apoptosis in both cell lines. These data suggest that activation of NF-κB and inhibition of apoptosis may play a role in the progression from Barrett metaplasia to EAC. In addition, curcumin, a well-known inhibitor of NF-κB activity, was shown to increase apoptosis and enhance both 5-FU- and CDDP-mediated chemosensitivity, suggesting that it may have potential application in the therapy of patients with EAC.

Kruppel-like factor 6 (KLF6) is a tumor suppressor gene that is functionally inactivated in human cancer by loss of heterozygosity, somatic mutation, decreased expression, and increased alternative splicing into an oncogenic splice variant, KLF6-SV1. Here we show that increased expression of KLF6-SV1 is associated with decreased survival in patients with lung adenocarcinoma. In addition, KLF6-SV1 is a novel antiapoptotic protein in lung cancer cell lines, and targeted reduction of KLF6-SV1 using siRNA induces apoptosis both alone and in combination with the chemotherapeutic drug cisplatin. Together, these findings highlight a critical role for KLF6-SV1 in lung cancer, and show a potential novel therapeutic strategy for the treatment of lung cancer.

Introduction

The expression, mechanisms of regulation, and functional impact of INHBA (activin A) in lung adenocarcinoma (AD) have not been fully elucidated.

Methods

INHBA expression was examined in 96 lung samples (86 ADs, 10 normal lung) using oligonucleotide microarrays and 187 lung samples (164 ADs, 6 bronchioalveolar carcinomas, and 17 normal lung) using immunohistochemistry. The proliferation of AD cell lines H460 and SKLU1 was examined with WST-1 assays after treatment with recombinant activin A, follistatin, and INHBA-targeting small-interfering RNA. Cells were also treated with 5-aza-2′ deoxycytidine and trichostatin A to investigate the role of epigenetic regulation in INHBA expression.

Results

Primary ADs expressed 3.1 times more INHBA mRNA than normal lung. In stage I AD patients, high levels of primary tumor INHBA transcripts were associated with worse prognosis. Immunohistochemistry confirmed higher inhibin βA protein expression in ADs (78.7%) and bronchioalveolar carcinomas (66.7%) compared with normal lung (11.8%). H460 and SKLU1 demonstrated increased proliferation when treated with exogenous activin A and reduced proliferation when treated with follistatin or INHBA-targeting small-interfering RNA. INHBA mRNA expression in H460 cells was upregulated after treatment with trichostatin A and 5-aza-2′ deoxycytidine.

Conclusions

INHBA is overexpressed in AD relative to controls. Inhibin βA may promote cell proliferation, and its overexpression is associated with worse survival in stage I AD patients. In addition, overexpression of INHBA may be affected by promoter methylation and histone acetylation in a subset of lung ADs.

Clinical Relevance

It remains a critical issue to reliably identify specific patients at high risk for recurrence and metastasis of lung cancer. To date, there has been no clinically applied gene test for predicting lung cancer recurrence. This study validated a 35-gene prognostic signature in various cell types of non-small cell lung cancer. The analysis showed that the 35-gene signature could further stratify patients at stage 1A into distinct prognostic subgroups. This lung cancer prognostic signature is independent of traditional clinicopathological factors, including patient age, clinical stage, tumor differentiation, and tumor grade. This signature had better prognostic performance than other lung cancer signatures, including the 5-gene signature and the 133-gene signature in the studied cohorts. The gene expression and protein expression of the identified biomarkers were validated in real-time RT-PCR and Western blots analysis of clinical specimens. This study indicates that the 35-gene signature could be applied in clinics for patient stratification.

Purpose

It remains a critical challenge to determine the risk for recurrence in early stage non-small cell lung cancer (NSCLC) patients. Accurate gene expression signatures are needed to classify patients into high- and low-risk groups to improve the selection of patients for adjuvant therapy.

Experimental Design

Multiple published microarray datasets were used to evaluate our previously identified lung cancer prognostic gene signature. Expression of the signature genes was further validated with real-time RT-PCR and Western blot assays of snap frozen lung cancer tumor tissues.

Results

Our previously identified 35-gene signature stratified 264 patients with non-small cell lung cancer into high- and low-risk groups with distinct overall survival rates (P < 0.05, Kaplan-Meier analysis, log-rank tests). The 35-gene signature further stratified patients with clinical stage 1A diseases into poor prognostic and good prognostic subgroups (P = 0.0007, Kaplan-Meier analysis, log-rank tests). This signature is independent of other prognostic factors for non-small cell lung cancer, including age, sex, tumor differentiation, tumor grade, and tumor stage. The expression of the signature genes was validated with real-time RT-PCR analysis of lung cancer tumor specimens. Protein expression of two signature genes, TAL2 and ILF3, was confirmed in lung adenocarcinoma tumors by using Western blot analysis. These two biomarkers showed correlated mRNA and protein over-expression in lung cancer development and progression.

Conclusions

The results indicate that the identified 35-gene signature is an accurate predictor of survival in non-small cell lung cancer. It provides independent prognostic information in addition to traditional clinicopathological criteria.

Purpose:

The aims of this study were to investigate telomere function in normal and Barrett's esophageal adenocarcinoma (BEAC) cells purified by laser capture microdissection (LCM) and to evaluate the impact of telomerase inhibition in cancer cells in vitro and in vivo.

Experimental Design:

Epithelial cells were purified from surgically resected esophagi. Telomerase activity was measured by modified “Telomeric Repeat Amplification Protocol” and telomere length determined by Real-Time PCR assay. To evaluate the impact of telomerase inhibition, adenocarcinoma cell lines were continuously treated with a specific telomerase inhibitor (GRN163L) and live cell number determined weekly. Apoptosis was evaluated by annexin labeling and senescence by beta-galactosidase staining. For in vivo studies, SCID-mice were subcutaneously inoculated with adenocarcinoma cells and following appearance of palpable tumors, injected intraperitoneally with saline or GRN163L.

Results:

Telomerase activity was significantly elevated whereas telomeres were shorter in BEAC cells relative to normal esophageal epithelial cells. The treatment of adenocarcinoma cells with telomerase inhibitor, GRN163L, led to loss of telomerase activity, reduction in telomere length, and growth arrest through induction of both the senescence and apoptosis. GRN163L induced cell death could also be expedited by addition of chemotherapeutic agents, doxorubicin and ritonavir. Finally, the treatment with GRN163L led to a significant reduction in tumor volume in a subcutaneous tumor model.

Conclusions:

We show that telomerase activity is significantly elevated whereas telomeres are shorter in BEAC and suppression of telomerase inhibits proliferation of adenocarcinoma cells both in vitro and in vivo.

Although prognostic gene expression signatures for survival in early stage lung cancer have been proposed, for clinical application it is critical to establish their performance across different subject populations and in different laboratories. Here we report a large, training-testing, multi-site blinded validation study to characterize the performance of several prognostic models based on gene expression for 442 lung adenocarcinomas. The hypotheses proposed examined whether microarray measurements of gene expression either alone or combined with basic clinical covariates (stage, age, sex) can be used to predict overall survival in lung cancer subjects. Several models examined produced risk scores that substantially correlated with actual subject outcome. Most methods performed better with clinical data, supporting the combined use of clinical and molecular information when building prognostic models for early stage lung cancer. This study also provides the largest available set of microarray data with extensive pathological and clinical annotation for lung adenocarcinomas.

A recurrent gene fusion between EML4 and ALK in 6.7% of non-small cell lung cancers (NSCLCs) and NKX2-1 (TTF1, TITF1) high-level amplifications in 12% of adenocarcinomas of the lung were independently reported recently. Because the EML4-ALK fusion was only shown by a reverse transcription-polymerase chain reaction approach, we developed fluorescent in situ hybridization assays to interrogate more than 600 NSCLCs using break-apart probes for EML4 and ALK. We found that EML4-ALK fusions occur in less than 3% of NSCLC samples and that EML4 and/or ALK amplifications also occur. We also observed that, in most cases in which an EML4/ALK alteration is detected, not all of the tumor cells harbor the lesion. By using a detailed multi-fluorescent in situ hybridization probe assay and reverse transcription-polymerase chain reaction, we have evidence that other, more common mechanisms besides gene inversion exist including the possibility of other fusion partners for ALK and EML4. Furthermore, we confirmed the NKX2-1 high-level amplification in a significant subset of NSCLC and found this amplification to be mutually exclusive to ALK and EML4 rearrangements.

Angiogenesis is crucial for tumor biology. There are many mechanisms by which tumors induce angiogenesis. We hypothesize that each individual tumor develops a unique mechanism to induce angiogenesis, and that activation of a particular angiogenic pathway suppresses the evolution of alternative pathways. We characterized 168 human non–small cell lung cancer (NSCLC) specimens for levels of angiogenic factors (angiogenic CXC chemokines, basic fibroblast growth factor, and vascular endothelial growth factor). We also induced lung tumor formation in A/J mice by injecting the tobacco carcinogen NNK. We dissected individual lung tumors and measured expression of angiogenic factors from three distinct families using real-time PCR. Finally, we controlled the angiogenic milieu using in vivo models to determine the resultant phenotype of the angiogenic factors expressed by NSCLC cells. Human tumors displayed marked variation in the expression of angiogenic factors. Individual mouse tumors, even from within the same mouse, displayed variability in their pattern of expression of angiogenic factors. In a sponge model of angiogenesis using murine lung cancer cells, implanting LLC cells with an angiogenic factor suppressed the expression of other angiogenic factors in implanted sponges. This suppressive effect was not seen in vitro. We conclude that lung cancer tumors evolve a unique and dominant angiogenic phenotype. Once an angiogenic pathway is activated, it may allow for tumor growth to proceed in the absence of a selection pressure to activate a second pathway.

Abstract

Ubiquitin-dependent proteolysis of cyclins plays a critical role in cell cycle progression and tumorigenesis. We examined the expression of ubiquitin-conjugating enzyme E2C (UBE2C) during progression from Barrett's metaplasia to esophageal adenocarcinoma (EA) and the effects of targeting this enzyme on EA-derived cell lines. Using oligonucleotide microarrays UBE2C expression was elevated in 73% (11 of 15) of EAs relative to Barrett's metaplasia. Tissue microarray showed elevated UBE2C in 70% (7 of 10) of dysplastic samples and in 87% (58 of 67) of tumors relative to metaplastic samples. Transfection of dominant-negative UBE2C into Seg-1 cells decreased proliferation (P = .04) and increased mitotic arrest compared to vector controls (63.5% vs 6.8%; P < .001). Transfection of UBE2C small interfering RNA also caused inhibiton of cell proliferation and distortion of the cell cycle, with maximal increase of G2 cells (155% of mock cells) at 72 hours and of S-phase cells (308% of mock cells) at 24 hours. Treatment of Seg-1 cells with the proteasome inhibitor MG-262 (1 nM-1 µM) showed decreased proliferation (P = .02). EA-derived cells expressing UBE2C are sensitive to treatment with MG-262 and to silencing of UBE2C, suggesting that patients with EAs overexpressing UBE2C may benefit from agents targeting this ubiquitin-conjugating enzyme.

Purpose

To identify stage I lung adenocarcinoma patients with a poor prognosis who will benefit from adjuvant therapy.

Patients and Methods

Whole gene expression profiles were obtained at 19 time points over a 48-hour time course from human primary lung epithelial cells that were stimulated with epidermal growth factor (EGF) in the presence or absence of a clinically used EGF receptor tyrosine kinase (RTK)-specific inhibitor, gefitinib. The data were subjected to a mathematical simulation using the State Space Model (SSM). “Gefitinib-sensitive” genes, the expressional dynamics of which were altered by addition of gefitinib, were identified. A risk scoring model was constructed to classify high- or low-risk patients based on expression signatures of 139 gefitinib-sensitive genes in lung cancer using a training data set of 253 lung adenocarcinomas of North American cohort. The predictive ability of the risk scoring model was examined in independent cohorts of surgical specimens of lung cancer.

Results

The risk scoring model enabled the identification of high-risk stage IA and IB cases in another North American cohort for overall survival (OS) with a hazard ratio (HR) of 7.16 (P = 0.029) and 3.26 (P = 0.0072), respectively. It also enabled the identification of high-risk stage I cases without bronchioalveolar carcinoma (BAC) histology in a Japanese cohort for OS and recurrence-free survival (RFS) with HRs of 8.79 (P = 0.001) and 3.72 (P = 0.0049), respectively.

Conclusion

The set of 139 gefitinib-sensitive genes includes many genes known to be involved in biological aspects of cancer phenotypes, but not known to be involved in EGF signaling. The present result strongly re-emphasizes that EGF signaling status in cancer cells underlies an aggressive phenotype of cancer cells, which is useful for the selection of early-stage lung adenocarcinoma patients with a poor prognosis.

Trial Registration

The Gene Expression Omnibus (GEO) GSE31210