Introduction

We evaluated the ability of histopathologic response criteria to predict overall survival (OS) and disease-free survival (DFS) in patients with surgically resected non-small cell lung cancer (NSCLC) treated with or without neoadjuvant chemotherapy.

Methods

Tissue specimens from 358 patients with NSCLC were evaluated by pathologists blinded to the patient treatment and outcome. The surgical specimens were reviewed for various histopathologic features in the tumor including percentage of residual viable tumor cells, necrosis, and fibrosis. The relationship between the histopathologic findings and OS was assessed.

Results

The percentage of residual viable tumor cells and surgical pathologic stage were associated with OS and DFS in 192 patients with NSCLC receiving neoadjuvant chemotherapy in multivariate analysis (p = 0.005 and p = 0.01, respectively). There was no association of OS or DFS with percentage of viable tumor cells in 166 patients with NSCLC who did not receive neoadjuvant chemotherapy (p = 0.31 and p = 0.45, respectively). Long-term OS and DFS were significantly prolonged in patients who had ≤10% viable tumor compared with patients with >10% viable tumor cells (5 years OS, 85% versus 40%, p < 0.0001 and 5 years DFS, 78% versus 35%, p < 0.001).

Conclusion

The percentages of residual viable tumor cells predict OS and DFS in patients with resected NSCLC after neoadjuvant chemotherapy even when controlled for pathologic stage. Histopathologic assessment of resected specimens after neoadjuvant chemotherapy could potentially have a role in addition to pathologic stage in assessing prognosis, chemotherapy response, and the need for additional adjuvant therapies.

Purpose

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.

Results

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.

Conclusions

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.

Background

Activating enhancer-binding protein-2β (AP2β) is a transcription factor involved in apoptosis. The purpose of the current study was to assess the cellular location and level of AP2β in Non-Small Cell Lung Cancer (NSCLC) and normal lung tissue and investigate whether the level and localization of AP2β expression is predictive of overall survival in patients with stage I NSCLC.

Methods

We performed immunohistochemical analysis of tissue microarrays (TMAs) prepared from stage I NSCLC specimens with adjacent normal lung tissue from two independent sets of patients who underwent lung resection with curative intent at our institution. AP2β intensity was assessed in TMAs, and AP2β staining patterns were classified as either diffuseor nucleolar in the TMAs. AP2β intensity and localization were analyzed for correlation with patients' survival.

Results

Immunohistochemical analysis of TMAs showed that the intensity of AP2β immunohistochemical staining did not correlate with overall survival. When location of AP2β was analyzed in TMAs, all of the normal lung tissue had diffuse pattern of AP2β. In the first set of NSCLC, patients with nucleolar pattern had a significantly lower 5-year survival rate than patients with diffuse pattern (67% vs. 100%; P = 0.004); this finding was confirmed in the second set (64% vs. 91%; P = 0.02). Multivariate analysis revealed that nucleolar pattern was an independent predictor of poor overall survival in both sets.

Conclusions

The AP2β which is located in the nucleoplasm in normal lung tissue is found in either nucleoplasm or nucleoli in NSCLC. The patients with AP2β in the nucleoli had poor survival compared to patients with AP2β in the cytoplasm.

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.

Lung cancer continues to be a major deadly malignancy. The mortality of this disease could be reduced by improving the ability to predict cancer patients' survival. We hypothesized that genes differentially expressed among cells constituting an in vitro human lung carcinogenesis model consisting of normal, immortalized, transformed, and tumorigenic bronchial epithelial cells are relevant to the clinical outcome of non–small cell lung cancer (NSCLC). Multidimensional scaling, microarray, and functional pathways analyses of the transcriptomes of the above cells were done and combined with integrative genomics to incorporate the microarray data with published NSCLC data sets. Up-regulated (n = 301) and down-regulated genes (n = 358) displayed expression level variation across the in vitro model with progressive changes in cancer-related molecular functions. A subset of these genes (n = 584) separated lung adenocarcinoma clinical samples (n = 361) into two clusters with significant survival differences. Six genes, UBE2C, TPX2, MCM2, MCM6, FEN1, and SFN, selected by functional array analysis, were also effective in prognosis. The mRNA and protein levels of one these genes—UBE2C—were significantly up-regulated in NSCLC tissue relative to normal lung and increased progressively in lung lesions. Moreover, stage I NSCLC patients with positive UBE2C expression exhibited significantly poorer overall and progression-free survival than patients with negative expression. Our studies with this in vitro model have lead to the identification of a robust six-gene signature, which may be valuable for predicting the survival of lung adenocarcinoma patients. Moreover, one of those genes, UBE2C, seems to be a powerful biomarker for NSCLC survival prediction.

Lung cancer is the leading cause of cancer-related death. Despite a number of studies that have provided prognostic biomarkers for lung cancer, a paucity of reliable markers and therapeutic targets exist to diagnose and treat this aggressive disease. In this study we investigated the potential of nuclear receptors (NRs), many of which are well-established drug targets, as therapeutic markers in lung cancer. Using quantitative real-time PCR, we analyzed the expression of the 48 members of the NR superfamily in a human panel of 55 normal and lung cancer cell lines. Unsupervised cluster analysis of the NR expression profile segregated normal from tumor cell lines and grouped lung cancers according to type (i.e. small vs. non-small cell lung cancers). Moreover, we found that the NR signature was 79% accurate in diagnosing lung cancer incidence in smokers (n = 129). Finally, the evaluation of a subset of NRs (androgen receptor, estrogen receptor, vitamin D receptor, and peroxisome proliferator-activated receptor-γ) demonstrated the therapeutic potential of using NR expression to predict ligand-dependent growth responses in individual lung cancer cells. Preclinical evaluation of one of these receptors (peroxisome proliferator activated receptor-γ) in mouse xenografts confirmed that ligand-dependent inhibitory growth responses in lung cancer can be predicted based on a tumor's receptor expression status. Taken together, this study establishes NRs as theragnostic markers for predicting lung cancer incidence and further strengthens their potential as therapeutic targets for individualized treatment.

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.

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.

For therapeutic purposes, non-small cell lung cancer (NSCLC) has traditionally been regarded as a single disease. However, recent evidence suggest that the two major subtypes of NSCLC, adenocarcinoma (AC) and squamous cell carcinoma (SqCC) respond differently to both molecular targeted and new generation chemotherapies. Therefore, identifying the molecular differences between these tumor types may impact novel treatment strategy. We performed the first large-scale analysis of 261 primary NSCLC tumors (169 AC and 92 SqCC), integrating genome-wide DNA copy number, methylation and gene expression profiles to identify subtype-specific molecular alterations relevant to new agent design and choice of therapy. Comparison of AC and SqCC genomic and epigenomic landscapes revealed 778 altered genes with corresponding expression changes that are selected during tumor development in a subtype-specific manner. Analysis of >200 additional NSCLCs confirmed that these genes are responsible for driving the differential development and resulting phenotypes of AC and SqCC. Importantly, we identified key oncogenic pathways disrupted in each subtype that likely serve as the basis for their differential tumor biology and clinical outcomes. Downregulation of HNF4α target genes was the most common pathway specific to AC, while SqCC demonstrated disruption of numerous histone modifying enzymes as well as the transcription factor E2F1. In silico screening of candidate therapeutic compounds using subtype-specific pathway components identified HDAC and PI3K inhibitors as potential treatments tailored to lung SqCC. Together, our findings suggest that AC and SqCC develop through distinct pathogenetic pathways that have significant implication in our approach to the clinical management of NSCLC.

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.

PURPOSE

Identification of effective markers for outcome is expected to improve the clinical management of non-small cell lung cancer (NSCLC). Here, we assessed in NSCLC the prognostic efficacy of genes, which we had previously found to be differentially expressed in an in vitro model of human lung carcinogenesis.

EXPERIMENTAL DESIGN

Prediction algorithms and risk-score models were applied to the expression of the genes in publicly available NSCLC expression datasets. The prognostic capacity of the immunohistochemical expression of proteins encoded by these genes was also tested using formalin-fixed paraffin-embedded (FFPE) tissue specimens from 156 lung adenocarcinomas and 79 squamous cell carcinomas (SCCs).

RESULTS

The survival of all-stages (p<0.001, HR=2.0) or stage-I (p<0.001, HR=2.84) adenocarcinoma patients that expressed the five-gene in vitro lung carcinogenesis model (FILM) signature was significantly poorer than that of patients who did not. No survival differences were observed between SCCs predicted to express or lack FILM signature. Moreover, all stages (p<0.001, HR=1.95) or stage-I (p=0.001, HR=2.6) adenocarcinoma patients predicted to be at high risk by FILM transcript exhibited significantly worse survival than patients at low risk. Furthermore, the corresponding protein signature was associated with poor survival (all stages, p<0.001, HR=3.6; stage-I, p<0.001, HR=3.5; stage-IB, p<0.001, HR=4.6) and mortality risk (all stages, p=0.001, HR=4.0; stage-I, p=0.01, HR=3.4; stage-IB, p<0.001, HR=7.2) in lung adenocarcinoma patients.

CONCLUSIONS

Our findings highlight a gene and corresponding protein signature with effective capacity for identification of stage-I lung adenocarcinoma patients with poor prognosis that are likely to benefit from adjuvant therapy.

Background

For processes that follow first order kinetics, exponential decay nonlinear regression analysis (EDNRA) may delineate curve characteristics and suggest processes affecting curve shape. We conducted a preliminary feasibility assessment of EDNRA of patient survival curves.

Methods

EDNRA was performed on Kaplan-Meier overall survival (OS) and time-to-relapse (TTR) curves for 323 patients with resected NSCLC and on OS and progression-free survival (PFS) curves from selected publications.

Results and Conclusions

In our resected patients, TTR curves were triphasic with a “cured” fraction of 60.7% (half-life [t1/2] >100,000 months), a rapidly-relapsing group (7.4%, t1/2=5.9 months) and a slowly-relapsing group (31.9%, t1/2=23.6 months). OS was uniphasic (t1/2=74.3 months), suggesting an impact of co-morbidities; hence, tumor molecular characteristics would more likely predict TTR than OS. Of 172 published curves analyzed, 72 (42%) were uniphasic, 92 (53%) were biphasic, 8 (5%) were triphasic. With first-line chemotherapy in advanced NSCLC, 87.5% of curves from 2-3 drug regimens were uniphasic vs only 20% of those with best supportive care or 1 drug (p<0.001). 54% of curves from 2-3 drug regimens had convex rapid-decay phases vs 0% with fewer agents (p<0.001). Curve convexities suggest that discontinuing chemotherapy after 3-6 cycles “synchronizes” patient progression and death. With postoperative adjuvant chemotherapy, the PFS rapid-decay phase accounted for a smaller proportion of the population than in controls (p=0.02) with no significant difference in rapid-decay t1/2, suggesting adjuvant chemotherapy may move a subpopulation of patients with sensitive tumors from the relapsing group to the cured group, with minimal impact on time to relapse for a larger group of patients with resistant tumors. In untreated patients, the proportion of patients in the rapid-decay phase increased (p=0.04) while rapid-decay t1/2 decreased (p=0.0004) with increasing stage, suggesting that higher stage may be associated with tumor cells that both grow more rapidly and have a higher probability of surviving metastatic processes than in early stage tumors.

This preliminary assessment of EDNRA suggests that it may be worth exploring this approach further using more sophisticated, statistically rigorous nonlinear modelling approaches. Using such approaches to supplement standard survival analyses could suggest or support specific testable hypotheses.

Aldehyde dehydrogenase (ALDH) is a candidate marker for lung cancer cells with stem cell-like properties. Immunohistochemical staining of a large panel of primary non-small cell lung cancer (NSCLC) samples for the ALDH1A1, ALDH3A1 and CD133 revealed a significant correlation between ALDH1A1 (but not ALDH3A1 or CD133) expression and poor prognosis in patients including those with stage I and N0 disease. Flow cytometric analysis of a panel of lung cancer cell lines and patient tumors revealed most NSCLCs contain a subpopulation of cells with elevated ALDH activity, and that this activity is associated with ALDH1A1 expression. Isolated ALDH+ lung cancer cells were observed to be highly tumorigenic and clonogenic as well as capable of self-renewal compared to their ALDH− counterparts. Expression analysis of sorted cells revealed elevated Notch pathway transcript expression in ALDH+ cells. Suppression of the Notch pathway by treatment with either a gamma-secretase inhibitor or stable expression of shRNA against NOTCH3 resulted in a significant decrease in ALDH+ lung cancer cells, commensurate with a reduction in tumor cell proliferation and clonogenicity. Taken together, these findings indicate that ALDH selects for a subpopulation of self-renewing NSCLC stem-like cells with increased tumorigenic potential, that NSCLCs harboring tumor cells with ALDH1A1 expression have inferior prognosis, and that ALDH1A1 and CD133 identify different tumor subpopulations. Therapeutic targeting of the Notch pathway reduces this ALDH+ component, implicating Notch signaling in lung cancer stem cell maintenance.

Purpose

The role of RNA-dependent protein kinase (PKR) in antiviral defence mechanisms and in cellular differentiation, growth, and apoptosis is well known, but the role of PKR in human lung cancer remains poorly understood. To explore the role of PKR in human lung cancer, we evaluated PKR’s expression in tissue microarray specimens from both non-small cell lung cancer (NSCLC) and normal human bronchial epithelium tissue.

Experimental Design

Tissue microarray samples (TMA-1) from 231 lung cancers were stained with PKR antibody and validated on TMA-2 from 224 lung cancers. Immunohistochemical expression score was quantified by three pathologists independently. Survival probability was computed by the Kaplan-Meier method.

Results

The NSCLC cells showed lower levels of PKR expression than normal bronchial epithelium cells did. We also found a significant association between lower levels of PKR expression and lymph node metastasis. We found that loss of PKR expression is correlated with a more aggressive behavior, and that a high PKR expression predicts a subgroup of patients with a favorable outcome. Univariate and multivariate Cox proportional hazards regression models showed that a lower level of PKR expression was significantly associated with shorter survival in NSCLC patients. We further validated and confirmed that PKR to be a powerful prognostic factor in TMA-2 lung cancer (HR=0.22, P<0.0001).

Conclusions

Our findings first indicate that PKR expression is an independent prognostic variable in NSCLC patients.

Background

In this study, we investigated whether PKR protein expression is correlated with mRNA levels and also evaluated molecular biomarkers that are associated with PKR, such as phosphorylated PKR (p-PKR) and phosphorylated eIF2α (p-eIF2α).

Methodology and Findings

We determined the levels of PKR protein expression and mRNA in 36 fresh primary lung tumor tissues by using Western blot analysis and real-time reverse-transcriptase PCR (RT-PCR), respectively. We used tissue microarrays for immunohistochemical evaluation of the expression of p-PKR and p-eIF2α proteins. We demonstrated that PKR mRNA levels are significantly correlated with PKR protein levels (Spearman's rho = 0.55, p<0.001), suggesting that PKR protein levels in tumor samples are regulated by PKR mRNA. We also observed that the patients with high p-PKR or p-eIF2α expression had a significantly longer median survival than those with little or no p-PKR or p-eIF2α expression (p = 0.03 and p = 0.032, respectively). We further evaluated the prognostic effect of combined expression of p-PKR plus PKR and p-eIF2α plus PKR and found that both combinations were strong independent prognostic markers for overall patient survival on stage I and all stage patients.

Conclusions

Our findings suggest that PKR protein expression may controlled by transcription level. Combined expression levels of PKR and p-PKR or p-eIF2α can be new markers for predicting the prognosis of patients with NSCLC.

Purpose

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.

Results

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).

Conclusions

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.

Purpose

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.

Results

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.

Conclusions

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.

David Mangelsdorf and colleagues show that nuclear receptor expression is strongly associated with clinical outcomes of lung cancer patients, and this expression profile is a potential prognostic signature for lung cancer patient survival time, particularly for individuals with early stage disease.

Background

The identification of prognostic tumor biomarkers that also would have potential as therapeutic targets, particularly in patients with early stage disease, has been a long sought-after goal in the management and treatment of lung cancer. The nuclear receptor (NR) superfamily, which is composed of 48 transcription factors that govern complex physiologic and pathophysiologic processes, could represent a unique subset of these biomarkers. In fact, many members of this family are the targets of already identified selective receptor modulators, providing a direct link between individual tumor NR quantitation and selection of therapy. The goal of this study, which begins this overall strategy, was to investigate the association between mRNA expression of the NR superfamily and the clinical outcome for patients with lung cancer, and to test whether a tumor NR gene signature provided useful information (over available clinical data) for patients with lung cancer.

Methods and Findings

Using quantitative real-time PCR to study NR expression in 30 microdissected non-small-cell lung cancers (NSCLCs) and their pair-matched normal lung epithelium, we found great variability in NR expression among patients' tumor and non-involved lung epithelium, found a strong association between NR expression and clinical outcome, and identified an NR gene signature from both normal and tumor tissues that predicted patient survival time and disease recurrence. The NR signature derived from the initial 30 NSCLC samples was validated in two independent microarray datasets derived from 442 and 117 resected lung adenocarcinomas. The NR gene signature was also validated in 130 squamous cell carcinomas. The prognostic signature in tumors could be distilled to expression of two NRs, short heterodimer partner and progesterone receptor, as single gene predictors of NSCLC patient survival time, including for patients with stage I disease. Of equal interest, the studies of microdissected histologically normal epithelium and matched tumors identified expression in normal (but not tumor) epithelium of NGFIB3 and mineralocorticoid receptor as single gene predictors of good prognosis.

Conclusions

NR expression is strongly associated with clinical outcomes for patients with lung cancer, and this expression profile provides a unique prognostic signature for lung cancer patient survival time, particularly for those with early stage disease. This study highlights the potential use of NRs as a rational set of therapeutically tractable genes as theragnostic biomarkers, and specifically identifies short heterodimer partner and progesterone receptor in tumors, and NGFIB3 and MR in non-neoplastic lung epithelium, for future detailed translational study in lung cancer.

Please see later in the article for the Editors' Summary

Editors' Summary

Background

Lung cancer, the most common cause of cancer-related death, kills 1.3 million people annually. Most lung cancers are “non-small-cell lung cancers” (NSCLCs), and most are caused by smoking. Exposure to chemicals in smoke causes changes in the genes of the cells lining the lungs that allow the cells to grow uncontrollably and to move around the body. How NSCLC is treated and responds to treatment depends on its “stage.” Stage I tumors, which are small and confined to the lung, are removed surgically, although chemotherapy is also sometimes given. Stage II tumors have spread to nearby lymph nodes and are treated with surgery and chemotherapy, as are some stage III tumors. However, because cancer cells in stage III tumors can be present throughout the chest, surgery is not always possible. For such cases, and for stage IV NSCLC, where the tumor has spread around the body, patients are treated with chemotherapy alone. About 70% of patients with stage I and II NSCLC but only 2% of patients with stage IV NSCLC survive for five years after diagnosis; more than 50% of patients have stage IV NSCLC at diagnosis.

Why Was This Study Done?

Patient responses to treatment vary considerably. Oncologists (doctors who treat cancer) would like to know which patients have a good prognosis (are likely to do well) to help them individualize their treatment. Consequently, the search is on for “prognostic tumor biomarkers,” molecules made by cancer cells that can be used to predict likely clinical outcomes. Such biomarkers, which may also be potential therapeutic targets, can be identified by analyzing the overall pattern of gene expression in a panel of tumors using a technique called microarray analysis and looking for associations between the expression of sets of genes and clinical outcomes. In this study, the researchers take a more directed approach to identifying prognostic biomarkers by investigating the association between the expression of the genes encoding nuclear receptors (NRs) and clinical outcome in patients with lung cancer. The NR superfamily contains 48 transcription factors (proteins that control the expression of other genes) that respond to several hormones and to diet-derived fats. NRs control many biological processes and are targets for several successful drugs, including some used to treat cancer.

What Did the Researchers Do and Find?

The researchers analyzed the expression of NR mRNAs using “quantitative real-time PCR” in 30 microdissected NSCLCs and in matched normal lung tissue samples (mRNA is the blueprint for protein production). They then used an approach called standard classification and regression tree analysis to build a prognostic model for NSCLC based on the expression data. This model predicted both survival time and disease recurrence among the patients from whom the tumors had been taken. The researchers validated their prognostic model in two large independent lung adenocarcinoma microarray datasets and in a squamous cell carcinoma dataset (adenocarcinomas and squamous cell carcinomas are two major NSCLC subtypes). Finally, they explored the roles of specific NRs in the prediction model. This analysis revealed that the ability of the NR signature in tumors to predict outcomes was mainly due to the expression of two NRs—the short heterodimer partner (SHP) and the progesterone receptor (PR). Expression of either gene could be used as a single gene predictor of the survival time of patients, including those with stage I disease. Similarly, the expression of either nerve growth factor induced gene B3 (NGFIB3) or mineralocorticoid receptor (MR) in normal tissue was a single gene predictor of a good prognosis.

What Do These Findings Mean?

These findings indicate that the expression of NR mRNA is strongly associated with clinical outcomes in patients with NSCLC. Furthermore, they identify a prognostic NR expression signature that provides information on the survival time of patients, including those with early stage disease. The signature needs to be confirmed in more patients before it can be used clinically, and researchers would like to establish whether changes in mRNA expression are reflected in changes in protein expression if NRs are to be targeted therapeutically. Nevertheless, these findings highlight the potential use of NRs as prognostic tumor biomarkers. Furthermore, they identify SHP and PR in tumors and two NRs in normal lung tissue as molecules that might provide new targets for the treatment of lung cancer and new insights into the early diagnosis, pathogenesis, and chemoprevention of lung cancer.

Additional Information

Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1000378.

The Nuclear Receptor Signaling Atlas (NURSA) is consortium of scientists sponsored by the US National Institutes of Health that provides scientific reagents, datasets, and educational material on nuclear receptors and their co-regulators to the scientific community through a Web-based portal

The Cancer Prevention and Research Institute of Texas (CPRIT) provides information and resources to anyone interested in the prevention and treatment of lung and other cancers

The US National Cancer Institute provides detailed information for patients and professionals about all aspects of lung cancer, including information on non-small-cell carcinoma and on tumor markers (in English and Spanish)

Cancer Research UK also provides information about lung cancer and information on how cancer starts

MedlinePlus has links to other resources about lung cancer (in English and Spanish)

Wikipedia has a page on nuclear receptors (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)

Insulin-like growth factor 1 receptor (IGF-1R) signaling has been implicated in several human neoplasms. However, the role of serum levels of insulin-like growth factors (IGFs) in lung cancer risk is controversial. We assessed the role of tissue-derived IGFs in lung carcinogenesis. We found that IGF-1 and IGF-2 levels in bronchial tissue specimens containing high-grade dysplasia were significantly higher than in those containing normal epithelium, hyperplasia, and squamous metaplasia. Derivatives of human bronchial epithelial cell lines with activation mutation in KRAS (V12) or loss of p53 overexpressed IGF-1 and IGF-2. Transformed characteristics of these cells were significantly suppressed by inactivation of IGF-1R or inhibition of IGF-1 or IGF-2 expression but enhanced by ovrexpresion of insulin-like growth factor receptor (IGF-1R) or exposure to the tobacco carcinogens (TC) 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and benzo[a]pyrene (BaP). We further determined the role of IGF-1R signaling in lung tumorigenesis by determining antitumor activities of the selective IGF-1R tyrosine kinase inhibitor cis-3-[3-(4-methyl-piperazin-l-yl)-cyclobutyl] -1-(2-phenyl-quinolin-7-yl) –imidazo [1,5-a]pyrazin-8-ylamine (PQIP) using an in vitro progressive cell system and an in vivo mouse model with a lung-specific IGF-1 transgene after exposure to TCs, including NNK plus BaP. Our results demonstrate that airway epithelial cells produce IGFs in an autocrine or paracrine manner, and these IGFs act jointly with TCs to enhance lung carcinogenesis. Further, the use of selective IGF-1R inhibitors may be a rational approach to controlling lung cancer.

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.

Purpose

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.

Results

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.

Conclusions

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.

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.

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.

Background

Data, data everywhere. The diversity and magnitude of the data generated in the Life Sciences defies automated articulation among complementary efforts. The additional need in this field for managing property and access permissions compounds the difficulty very significantly. This is particularly the case when the integration involves multiple domains and disciplines, even more so when it includes clinical and high throughput molecular data.

Methodology/Principal Findings

The emergence of Semantic Web technologies brings the promise of meaningful interoperation between data and analysis resources. In this report we identify a core model for biomedical Knowledge Engineering applications and demonstrate how this new technology can be used to weave a management model where multiple intertwined data structures can be hosted and managed by multiple authorities in a distributed management infrastructure. Specifically, the demonstration is performed by linking data sources associated with the Lung Cancer SPORE awarded to The University of Texas MDAnderson Cancer Center at Houston and the Southwestern Medical Center at Dallas. A software prototype, available with open source at www.s3db.org, was developed and its proposed design has been made publicly available as an open source instrument for shared, distributed data management.

Conclusions/Significance

The Semantic Web technologies have the potential to addresses the need for distributed and evolvable representations that are critical for systems Biology and translational biomedical research. As this technology is incorporated into application development we can expect that both general purpose productivity software and domain specific software installed on our personal computers will become increasingly integrated with the relevant remote resources. In this scenario, the acquisition of a new dataset should automatically trigger the delegation of its analysis.