Non-small Cell Lung Carcinoma Tumors Research Articles

Integrated 18 F-Fluorodeoxyglucose–Positron Emission Tomography/Dynamic Contrast-Enhanced Computed Tomography to Phenotype Non–Small Cell Lung Carcinoma

We applied modern molecular and functional imaging to the pretreatment assessment of lung cancer using combined dynamic contrast-enhanced computed tomography (DCE-CT) and (18)F-fluorodeoxyglucose-positron emission tomography ((18)F-FDG-PET) to phenotype tumors. Seventy-four lung cancer patients were prospectively recruited for (18)F-FDG-PET/DCE-CT using PET/64-detector CT. After technical failures, there were 64 patients (35 males, 29 females; mean age [± SD] 67.5 ± 7.9 years). DCE-CT yielded tumor peak enhancement (PE) and standardized perfusion value (SPV). The uptake of (18)F-FDG quantified on PET as the standardized uptake value (SUV(max)) assessed tumor metabolism. The median values for SUV(max) and SPV were used to define four vascular-metabolic phenotypes. There were associations (Spearman rank correlation [rs]) between tumor size and vascular-metabolic parameters: SUV(max) versus size (rs = .40, p = .001) and SUV/PE versus size (r = .43, p < .001). Patients with earlier-stage (I-IIA, n = 30) disease had mean (± SD) SUV/PE 0.36 ± 0.28 versus 0.56 ± 0.32 in later-stage (stage IIB-IV, n = 34) disease (p = .007). The low metabolism with high vascularity phenotype was significantly more common among adenocarcinomas (p = .018), whereas the high metabolism with high vascularity phenotype was more common among squamous cell carcinomas (p = .024). Other non-small cell lung carcinoma tumor types demonstrated a high prevalence of the high metabolism with low vascularity phenotype (p = .028). We show that tumor subtypes have different vascular-metabolic associations, which can be helpful clinically in managing lung cancer patients to hone targeted therapy.

Concurrent alteration of p16 and PTEN tumor suppressor genes could be considered as potential molecular marker for specific subgroups of NSCLC patients

p16 and PTEN are tumor suppressors that are commonly inactivated in human cancers. Loss of each of these molecules is widely studied in lung cancer, including non-small cell lung carcinoma (NSCLC), its most common clinical form. However, the importance of their mutual alterations for NSCLC pathogenesis has been barely examined so far. In this study we tested hypothesis that aberrant p16 might cooperate with inactive PTEN during pathogenesis of NSCLC, particularly in promoting tumor aggressiveness and invasiveness. Initially, we screened NSCLC tumor samples from patients for the presence of the most common genetic and epigenetic alterations of p16 and further correlated them with previously detected aberrations in PTEN gene. Statistical analyses showed that aberrant p16 directly correlated with altered PTEN. Such significant correlation was also observed in groups of patients with high genomic instability, with squamocellular histological subtype, with disease grade 2 and with lymph node invasion. Finally, survival analyses revealed dramatic decrease in survival rate of patients with mutual alterations of p16 and PTEN, but without prognostic significance. Overall results implicate cooperation between aberrant p16 and PTEN in pathogenesis of NSCLC and suggest that their combination might be considered as potential molecular marker for specific subgroups of NSCLC patients.

High Expression of Folate Receptor Alpha in Lung Cancer Correlates with Adenocarcinoma Histology and Mutation

Folate receptor alpha (FRα) and reduced folate carrier-1 (RFC1) regulate uptake of folate molecules inside the cell. FRα is a potential biomarker of tumors response to antifolate chemotherapy, and a target for therapies using humanized monocloncal antibody. Information on the protein expression of these receptors in non-small-cell lung carcinoma (NSCLC) is limited. Expressions of FRα and RFC1 were examined by immunohistochemistry (IHC) in 320 surgically resected NSCLC (202 adenocarcinomas and 118 squamous cell carcinomas) tissue specimens and correlated with patients' clinico-pathologic characteristics. Folate receptor α gene (FOLR1) mRNA expression was examined using publicly available microarray datasets. FRα expression was correlated with thymidylate synthase and p53 expression in NSCLCs, and with epidermal growth factor receptor (EGFR) and V-Ki-ras2 Kirsten rat sarcoma viral (KRAS) gene mutations in adenocarcinomas. NSCLC overexpressed FRα and RFC1. In a multivariate analysis, lung adenocarcinomas were more likely to express FRα in the cytoplasm (OR = 4.39; p < 0.0001) and membrane (OR = 5.34; p < 0.0001) of malignant cells than squamous cell carcinomas. Tumors from never-smokers were more likely to express cytoplasmic (OR = 3.35; p<0.03) and membrane (OR = 3.60; p=0.0005) FRα than those from smokers. In adenocarcinoma, EGFR mutations correlated with higher expression of membrane FRα and FOLR1 gene expressions. High levels of FRα expression was detected in 42 NSCLC advanced metastatic tumor tissues. FRα and RFC1 proteins are overexpressed in NSCLC tumor tissues. The high levels of FRα in lung adenocarcinomas may be associated to these tumors' better responses to antifolate chemotherapy and represents a potential novel target for this tumor type.

Down-regulated expression of apoptosis-associated genes APIP and UACA in non-small cell lung carcinoma

The Apaf-1 interacting protein (APIP) and the uveal autoantigen with coiled coil domains and ankyrin repeats (UACA) belong to endogenous regulators of the apoptosome apparatus, but their role in tumourigenesis and progression of non-small cell lung carcinoma (NSCLC) is not known. Previous studies demonstrated that APIP inhibits the apoptosome-mediated procaspase-9 activation while UACA induces translocation of Apaf-1 from the cytoplasm into the nucleus. Here, we report for the first time that the expression of APIP and UACA genes is down-regulated on the level of both mRNA and protein in NSCLC cells and tumours. In particular, the expression of APIP protein was strikingly decreased and the expression of UACA mRNA and protein was frequently down-regulated in NSCLC tumours of different histopathological types. Moreover, stage IA NSCLC tumours showed significantly lower expression of UACA mRNA compared to higher stage tumours. The weak increase of both APIP and UACA mRNA levels in the 5-aza-2'-deoxycytidine-treated NSCLC cells indicates that mechanisms other than DNA methylation are involved in the regulation of APIP and UACA gene expression in these cancer cells. Taken together, the down-regulation of APIP and UACA expression suggests that the threshold to activate the apoptosome apparatus may be decreased in NSCLC cells due to the lack of APIP-mediated suppression and UACA-assisted Apaf-1 nuclear entry. Moreover, the loss of UACA-assisted Apaf-1 nuclear translocation may underlie the failure of DNA damage checkpoint activation in NSCLC cells leading to their genomic instability.

Abstract B1: Targeting KRAS mutant NSCLC with the Hsp90 inhibitor ganetespib

Abstract Background: Mutant KRAS is detected in 20–25% of non-small cell lung carcinomas (NSCLC) and represents one of the most common oncogenic drivers of this disease. NSCLC tumors with oncogenic KRAS respond poorly to currently available therapies necessitating the pursuit of new treatment strategies. Heat shock protein 90 (Hsp90) is a molecular chaperone required for the maturation and stability of hundreds of client proteins, many of which are known oncogenic drivers or effectors of such proteins. Inactivation of Hsp90 results in the simultaneous inhibition of multiple oncogenic signaling pathways, making Hsp90 a highly attractive therapeutic target. Ganetespib is a second generation, small molecule Hsp90 inhibitor currently being evaluated in multiple clinical trials. Recent results from a Phase 2 trial with ganetespib revealed that &amp;gt;60% of patients with KRAS mutant NSCLC exhibited tumor shrinkage at 8 weeks, indicating that ganetespib has promising potential to benefit this disease. Aims: To further understand the actions of ganetespib in mutant KRAS NSCLC tumors, preclinical studies were executed in a diverse panel of KRAS mutant NSCLC cell lines to: (1) Investigate whether ganetespib is effective in suppressing critical cell signaling nodes responsible for KRAS-driven NSCLC cell survival and (2) Assess whether ganetespib can synergize with both clinical agents targeted against these signaling nodes and standard of care chemotherapies. Results: Ganetespib displayed potent anticancer activity across 15 KRAS mutant NSCLC cell lines assayed in vitro, with an average IC50 of 24 nM. Combining ganetespib with anti-mitotics, alkylating agents or topoisomerase inhibitors resulted in an increase in cell death of up to 44, 61 and 26%, respectively, versus monotherapy. At the molecular level, ganetespib induced the destabilization of several KRAS substrates, including BRAF and CRAF, leading to inactivation of their downstream effectors followed by programmed cell death. Ganetespib effectively suppressed the growth of human KRAS mutant NSCLC tumor xenografts in vivo; however, ganetespib did not induce tumor regression. In light of this, we sought to investigate whether inhibitors targeting KRAS driven signaling nodes would confer greater sensitivity to ganetespib. In vitro, combinations of low dose ganetespib with either MEK or PI3K/mTOR inhibitors consistently resulted in greater activity than monotherapy, up to 77% and 42%, respectively. Furthermore, ganetespib suppressed activating feedback loops that occur in response to MEK and PI3K/mTOR inhibition, providing a rationale for the enhanced combinatorial activity. To validate these results, in vivo combinations were performed with ganetespib and a PI3K/mTOR inhibitor in KRAS mutant NSCLC xenografts. While both agents promoted tumor shrinkage on their own, considerable improvement in tumor growth inhibition was observed in the combination arm. Conclusions: Ganetespib, a potent inhibitor of Hsp90, has shown encouraging evidence of clinical activity, including tumor shrinkage in patients with KRAS mutant NSCLC. In vitro, ganetespib exhibited potent anticancer activity in NSCLC cells harboring a diverse spectrum of KRAS mutations due in part to degradation and inactivation of critical KRAS signaling effectors. Combination with targeted therapies that overlap with these signaling nodes led to enhanced anticancer activity in vitro and in mouse models of KRAS mutant NSCLC. Taken together, these results could have interesting clinical utility in patients with KRAS mutant NSCLC.

Molecular Pathogenesis of Non-Small Cell Lung Carcinomas

Non-small cell lung carcinoma (NSCLC) from histological and biological perspectives is a complex neoplasm. The sequential preneoplastic changes have been defined for centrally arising squamous cell carcinomas (SCCs) of the lung, and they are less documented for peripherally arising adenocarcinomas. The main morphologic forms of preneoplastic lesions recognized in the lung are squamous dysplasias for SCC, and atypical adenomatous hyperplasia for adenocarcinoma. Several studies have provided information regarding the molecular characterization of lung preneoplastic changes, especially for SCC. These molecular changes have been detected in the histologically normal and abnormal respiratory epithelium of smokers and patients with lung cancer, phenomenon known as field of cancerization. Our improved understanding of the changes and origins of the field of cancerization can be applied clinically to improve early detection of lung cancer. In the last decade, significant progress has been made in the characterization of molecular abnormalities in NSCLC tumors that are being used as molecular targets and predictive biomarkers for patients’ selection for targeted therapy. As our understanding of the biology of the molecular pathogenesis of lung cancer evolves, there is an opportunity to use this knowledge for the development of novel chemoprevention strategies using those molecularly targeted agents used to treat advanced lung cancer, a concept coined as reverse migration. The rapid development of technologies for large-scale molecular analysis, includeing microarrays and next-generation sequencing will facilitate high-throughput molecular analysis of lung cancer preneoplastic lesions and the field of cancerization.

Critical Role of VCP/p97 in the Pathogenesis and Progression of Non-Small Cell Lung Carcinoma

BackgroundValosin-containing protein (VCP)/p97 is an AAA ATPase molecular chaperone that regulates vital cellular functions and protein-processing. A recent study indicated that VCP expression levels are correlated with prognosis and progression of non-small cell lung carcinoma (NSCLC). We not only verified these findings but also identified the specific role of VCP in NSCLC pathogenesis and progression.Methodology/Principal FindingsOur results show that VCP is significantly overexpressed in non-small cell lung carcinoma (NSCLC) as compared to normal tissues and cell lines (p<0.001). Moreover, we observed the corresponding accumulation of ubiquitinated-proteins in NSCLC cell lines and tissues as compared to the normal controls. VCP inhibition by si/shRNA or small-molecule (Eeyarestatin I, EerI) significantly (p<0.05, p<0.00007) suppressed H1299 proliferation and migration but induced (p<0.00001) apoptosis. Cell cycle analysis by flow cytometry verified this data and shows that VCP inhibition significantly (p<0.001, p<0.003) induced cell cycle arrest in the G0/G1 phases. We also found that VCP directly regulates p53 and NFκB protein levels as a potential mechanism to control tumor cell proliferation and progression. Finally, we evaluated the therapeutic potential of VCP inhibition and observed significantly reduced NSCLC tumor growth in both in vitro and xenograft murine (athymic-nude) models after EerI treatment (p<0.05).Conclusions/SignificanceThus, targeting VCP in NSCLC may provide a novel strategy to restore p53 and NFκB levels and ameliorate the growth and tumorigenicity, leading to improved clinical outcomes.

Proteins kinase Cɛ is required for non-small cell lung carcinoma growth and regulates the expression of apoptotic genes

Protein kinase C (PKC)ɛ, a member of the novel PKC family, has key roles in mitogenesis and survival in normal and cancer cells. PKCɛ is frequently overexpressed in epithelial cancers, particularly in lung cancer. Using a short-hairpin RNA approach, here we established that PKCɛ is required for non-small cell lung carcinoma (NSCLC) growth in vitro as well as tumor growth when inoculated into athymic mice. Moreover, sustained delivery of a PKCɛ-selective inhibitor peptide, ɛV1-2, reduced xenograft growth in mice. Both RNA interference depletion and pharmacological inhibition of PKCɛ caused a marked elevation in the number of apoptotic cells in NSCLC tumors. PKCɛ-depleted NSCLC cells show elevated expression of pro-apoptotic proteins of the Bcl-2 family, caspase recruitment domain-containing proteins and tumor necrosis factor ligands/receptor superfamily members. Moreover, a Gene Set Enrichment Analysis revealed that a vast majority of the genes changed in PKCɛ-depleted cells were also deregulated in human NSCLC. Our results strongly suggest that PKCɛ is required for NSCLC cell survival and maintenance of NSCLC tumor growth. Therefore, PKCɛ may represent an attractive therapeutic target for NSCLC.

Profound Coordinated Alterations of Intratumoral NK Cell Phenotype and Function in Lung Carcinoma

Both the innate and adaptive immune systems contribute to tumor immunosurveillance in mice and humans; however, there is a paucity of direct evidence of a role for natural killer (NK) cells in this important process. In this study, we investigated the intratumoral phenotypic profile and functions of NK cells in primary human tumor specimens of non-small cell lung carcinoma (NSCLC). We used in situ methods to quantify and localize NK cells using the NKp46 marker and we characterized their phenotype in blood, tumoral, and nontumoral samples of NSCLC patients. Intratumoral NK cells displayed a profound and coordinated alteration of their phenotype, with a drastic reduction of NK cell receptor expression specifically detected in the tumoral region. According to their altered phenotype, intratumoral NK cells exhibited profound defects in the ability to activate degranulation and IFN-γ production. We found that the presence of NK cells did not impact the clinical outcome of patients with NSCLC. Finally, we showed that tumor cells heterogeneously express ligands for both activating and inhibitory NK receptors. Taken together, our results suggest that the NSCLC tumor microenvironment locally impairs NK cells, rendering them less tumorcidal and thereby supportive to cancer progression.

Abstract 2252: ALDH7A1 expression is associated with recurrence in patients with resected non-small cell lung carcinoma

Abstract The aldehyde dehydrogenase (ALDH) enzymes are important for detoxification of endogenous and exogenous aldehydes. Expression of ALDH family members has recently been described as a potential marker for tumor-initiating cancer stem cells in a variety of human malignancies, including lung cancer. We were interested to determine whether expression of ALDH7A1, a member of the ALDH family, has prognostic significance in resected non-small cell lung carcinoma (NSCLC). Tumor specimens were obtained for 107 patients with completely resected stage I through stage III NSCLC from paraffin-embedded tissue microarrays and stained with an antibody specific for ALDH7A1. Staining patterns were graded by a pathologist based on the intensity of staining and the percentage of cells stained. A staining score index was calculated by multiplying intensity score by the percentage area with positive staining. An index of ≥100 was considered positive, while an index &amp;lt;100 was deemed negative, as determined by visually determining a clear positive stain supported by histograms of the range of scores. Staining index was subsequently correlated with clinical data. Positive ALDH7A1 staining was identified in 46 patients, and negative staining was noted in 48 patients, with 13 tumor sections missing. Multivariate analysis demonstrated that NSCLC patients with positive ALDH7A1 expression had decreased overall survival relative to ALDH7A1-negative tumors, although this did not reach significance (hazard ratio 1.42, 95% confidence interval 0.84 to 2.389; p=0.192). However, patients with ALDH7A1-expressing NSCLC tumors had a significantly higher incidence of lung cancer recurrence compared to patients with ALDH7A1-negative tumors (hazard ratio 6.24, 95% confidence interval 2.06 to 18.86; p=0.001). These data indicate that ALDH7A1 staining is present in a substantial number of NSCLC tumors and may be a biomarker predictive for increased incidence of cancer recurrence in patients with surgically resected NSCLC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2252. doi:10.1158/1538-7445.AM2011-2252

Abstract 4919: Multiplexed EGFR signaling pathway analysis in FFPE tissue using quantitative mass spectrometry

Abstract The epidermal growth factor receptor (EGFR) is a drug target for both small molecule and antibody therapeutics and has been approved in non small-cell lung carcinoma (NSCLC) and colorectal carcinoma (CRC) among other indications. These drugs block receptor signaling though blockade of the tyrosine kinase domain, or through inhibition of ligand binding. Current genomic tests measure receptor amplification, RNA levels, the mutation status of receptor or pathway molecules (EGFR or kRAS mutations) but no current assay can directly assess the activation state of the EGFR or its downstream signaling pathway components. Indeed, the EGFR mutation positive NSCLC tumors (thought to be constitutively active) show a high response rate to TKI therapy, but the many non responders (50% or more) demonstrate the limitation of genomic analysis. Since activation of EGFR is necessary for the response to these targeted agents, it is critical to measure what levels of receptor activation and downstream signaling determines tumor responsiveness to EGFR targeted therapies in these patients. For this reason, we have developed a panel of new diagnostic assays which measure the activation of the EGFR and key downstream signaling proteins through quantitation of the phosphorylation state of these proteins. These assays are based on the Liquid Tissue®-SRM technology platform. This approach enables relative and absolute quantification of proteins and their phosphorylation status directly in formalin fixed paraffin embedded (FFPE) tissue. We preclinically validated the multiplexed Liquid Tissue® phospho-SRM assay on formalin fixed EGF stimulated A431 cells. We followed up these in vitro studies with phospho-SRM analysis of FFPE NSCLC xenograft explants where extensive independent histopathologic and molecular characterization had been performed, allowing us to benchmark our phospho-SRM analysis with standard diagnostic analyses. We have now extended these quantitation studies by measuring the expression of EGFR and phospho-EGFR in FFPE tissues obtained from relevant human clinical trial cohorts – Gefitinib treated NSCLC and Cetuximab treated CRC. It is hoped that we will be able to correlate EGFR expression, activation and signaling in these tumors with responsiveness to EGFR targeted therapy, and to validate this assay for use as a companion diagnostic to guide therapy in both NSCLC and CRC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4919. doi:10.1158/1538-7445.AM2011-4919

Abstract 4113: Kinase activity based biomarkers: Identification of prognostic and erlotinib response prediction markers in NSCLC patients

Abstract Background: Reliable diagnostic tests are needed to identify early stage non-small cell lung carcinoma (NSCLC) patients with poor prognosis. Concomitantly there is a clear need for tests that enable the selection of patients who will benefit from targeted therapy with kinase inhibitors. We evaluated kinase activity profiles in two groups of early stage NSCLC patients, either for prognosis of long- or short-term survival, or for predicting erlotinib drug response. Method: Retrospective studies were performed on fresh frozen resection material of two groups of early stage NSCLC patients. The first group consisted of 48 short- and long-term survivors who underwent a complete surgical resection (5+ years follow-up). The second group consisted of 14 NSCLC patients who received 3 weeks of neo-adjuvant treatment with erlotinib prior to complete surgical resection. Response evaluation to neo-adjuvant treatment was based on histopathological examination of the surgical specimens. For both studies, kinase activity profiles of lysed cryosections of tumour tissues were generated in the presence and absence of protein tyrosine kinase inhibitors on PamChip® peptide micro-arrays, comprising 144 tyrosine containing peptides, derived from known phosphorylation sites of human proteins. Partial least square discriminant analysis was used to construct prediction models. ClustalW alignment algorithms were used to investigate the most informative phosphorylation sites. Results: Kinase activity profiles obtained in the absence of inhibitor did not distinguish between subgroups (long- versus short-term survival, responder or non-responder to TKI), whereas ratios of inhibited versus non-inhibited signals resulted in distinct classifiers predicting survival for the first group, and response for the second group. Multivariate unsupervised analysis with leave-one-out cross-validation resulted in an error rate for survival prediction of 29%. In the drug response prediction 13 out of 14 patients were correctly classified. Conclusion: This is the first study to show that kinase activity profiles of tumour tissue exposed to a kinase inhibitor can be used to identify NSCLC patients likely to respond to erlotinib treatment. Furthermore, based on kinase activity profiles of early stage NSCLC tumours, a prognostic classifier, for a set of 48 patients, was obtained. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4113. doi:10.1158/1538-7445.AM2011-4113

An in vitro chemoresponse assay defines a subset of colorectal and lung carcinomas responsive to cetuximab

Cetuximab is a chimeric monoclonal antibody for the epidermal growth factor receptor (EGFR) that may provide benefit to select cancer patients; however, identification of the characteristics of those patients who may benefit from its use is not complete. The ChemoFx® drug response marker (DRM) is an in vitro assay that can provide drug response data on tumor specimens before any patient treatment is initiated. We determined the feasibility of using the ChemoFx DRM to test tumor samples for sensitivity to cetuximab. We exposed four non-small cell lung carcinoma (NSCLC) cell lines (H358, H520, HCC827, and H1666) to cetuximab and determined their sensitivity using the ChemoFx DRM and, in parallel, EGFR status using immunocytochemistry, Western blotting, and In-Cell WesternTM analysis. We used the ChemoFx DRM to determine cetuximab sensitivity of primary NSCLC and colorectal tumor samples. The ChemoFx DRM distinguished between cetuximab-sensitive and -resistant cell lines. Cetuximab sensitivity was not dependent on EGFR mutational status; H358 cells were non-responsive to cetuximab yet contain wild-type EGFR, whereas H1666 cells were intermediately responsive to cetuximab and contain wild-type EGFR. HCC827 (EGFR-mutant) cells were intermediately responsive and, as expected, H520 cells (EGFR-null) were non-responsive to cetuximab. ChemoFx-determined cetuximab sensitivity of primary NSCLC and colorectal tumor samples was 9.0% and 7.5%, respectively. Use of the ChemoFx DRM is feasible for determining cetuximab sensitivity. The ChemoFx-determined cetuximab responses of primary NSCLC and colorectal tumor specimens were similar to published response rates of patients to treatment with cetuximab monotherapy.

Comparative analysis of the human urinary proteome by 1D SDS-PAGE and chip-HPLC-MS/MS identification of the AACT putative urinary biomarker

Urine is one of the most attractive analyte used for clinical diagnosis. NSCLC (non-small cell lung carcinoma), which includes adenocarcinoma, squamous cell carcinoma and large-cell carcinoma, is a leading cause of cancer-related deaths. In the present study, urinary proteomes of normal individuals and NSCLC patients were compared using 1D SDS-PAGE. From the distinctly differentially expressed bands in SDS-PAGE gel, 40 proteins were identified by chip-HPLC-MS/MS, including five proteins relevant to NSCLC. One of the selected proteins, alpha-1-antichymotrypsin (AACT), was further validated in urine by western blot and in lung tissue by immunohistochemistry staining. Higher expression level of AACT in NSCLC patients was observed by western blot when compared with normal urine samples. Significantly, the NSCLC tumor tissue (18 out of 20 cases, 90%) showed a significantly higher expression level of AACT compared to adjacent non-tumor lung tissue (3 out of 20 cases, 15%). These results establish AACT as a potential biomarker for objective and non-invasive diagnosis of NSCLC in urine and the other four NSCLC-related proteins were also listed.

The role of the c-Jun N-terminal kinase 2-α-isoform in non-small cell lung carcinoma tumorigenesis

The c-Jun N-terminal kinases (JNKs) are members of the mitogen-activated protein kinase family and have been implicated in tumorigenesis. One isoform in particular, JNK2α, has been shown to be frequently activated in primary brain tumors, to enhance several tumorigenic phenotypes and to increase tumor formation in mice. As JNK is frequently activated in non-small cell lung carcinoma (NSCLC), we investigated the role of the JNK2α isoform in NSCLC formation by examining its expression in primary tumors and by modulating its expression in cultured cell lines. We discovered that 60% of the tested primary NSCLC tumors had three-fold higher JNK2 protein and two- to three-fold higher JNK2α mRNA expression than normal lung control tissue. To determine the importance of JNK2α in NSCLC progression, we reduced JNK2α expression in multiple NSCLC cell lines using short hairpin RNA. Cell lines deficient in JNK2α had decreased cellular growth and anchorage-independent growth, and the tumors were four-fold smaller in mass. To elucidate the mechanism by which JNK2α induces NSCLC growth, we analyzed the JNK substrate, signal transducer and activator of transcription 3 (STAT3). Our data demonstrates for the first time that JNK2α can regulate the transcriptional activity of STAT3 by phosphorylating the Ser727 residue, thereby regulating the expression of oncogenic genes, such as c-Myc. Furthermore, reintroduction of JNK2α2 or STAT3 restored the tumorigenicity of the NSCLC cells, demonstrating that JNK2α is important for NSCLC progression. Our studies reveal a novel mechanism in which phosphorylation of STAT3 is mediated by a constitutively active JNK2 isoform, JNK2α.

The combination of stem cell markers CD133 and ABCG2 predicts relapse in stage I non-small cell lung carcinomas

The aim of the study was to investigate the expression of two cancer stem cell markers CD133 and ATP-binding cassette superfamily G member 2 (ABCG2) in non-small cell lung carcinomas (NSCLC) and evaluate their prognostic values for postoperative relapse. The expression levels of CD133 and ABCG2 in 145 stage I NSCLC tumors were detected by immunohistochemistry. Positive CD133 and ABCG2 expression was defined in 31.7 and 37.9% of the NSCLC tumors, respectively. Both stem markers alone did not correlate with any of the clinicopathological characteristics and were insufficient to predict recurrence after surgery. However, our results showed that the dual expression of CD133 and ABCG2 (CD133+/ABCG2+) status was an independent predictor of postoperative recurrence for patients with stage I NSCLC. Furthermore, CD133+/ABCG2+ NSCLC tumors (33 cases, 22.8%) had a significantly higher microvessel density and higher expression levels of angiogenic factors than the other subgroups. In conclusion, this study suggests that NSCLC patients with the dual expression of CD133 and ABCG2 have a high risk of early relapse and might benefit from anti-angiogenesis therapy.

Molecular Diagnosis of Activating EGFR Mutations in Non–Small Cell Lung Cancer Using Mutation-Specific Antibodies for Immunohistochemical Analysis

Therapeutic responses of non-small cell lung carcinoma (NSCLC) to epidermal growth factor receptor (EGFR)-targeted drugs, such as gefitinib and erlotinib, are closely associated with activating EGFR mutations. The most common mutations are delE746-A750 in exon 19 and L858R in exon 21, accounting for approximately 90% of all EGFR mutations. Recently, EGFR mutation-specific antibodies were developed and did well in immunohistochemical analysis, giving a sensitivity of approximately 90%. We have investigated whether this method detects activating EGFR mutations with sensitivity comparable with direct DNA sequencing, which is used to detect these mutations in NSCLC. We used antibodies specific for the E746-A750 deletion mutation in exon 19 and the L858R point mutation in exon 21 in Western blot analysis and immunohistochemistry to determine the presence of these mutations in NSCLC cell lines. We also examined these EGFR mutations in NSCLC tumor samples from 60 patients by immunohistochemically and direct DNA sequencing. We were able to identify EGFR mutations in NSCLC tumor samples immunohistochemically with a sensitivity of 79% using the anti-delE746-A750 antibody and 83% using the anti-L858R antibody. Additional DNA sequencing markedly improved the sensitivity obtained by immunohistochemistry. This simple and rapid assay for detecting EGFR mutations, even in the small bronchial biopsies obtained in stage IV NSCLC patients, will be useful for diagnosing responsiveness to EGFR-targeted drugs in patients with NSCLC. Combining this with DNA sequencing is recommended for the development of improved personalized EGFR-targeted therapeutics.

Abstract 374: VEGFR2/KDR polymorphisms and gene copy gain with outcome in non-small cell lung cancer

Abstract Background. The VEGF receptor 2 (VEGFR2) is the predominant mediator of VEGF-stimulated endothelial cell function. Recently, VEGFR2/KDR copy number gain and mutation, and VEGF copy number gain, have been described in lung adenocarcinoma tumor specimens. To better characterize the molecular changes of these two genes in non-small cell lung carcinoma (NSCLC), we investigated their abnormalities in NSCLC tumor tissue specimens and correlated with patients clinico-pathological characteristics. Methods. We extracted DNA from microdissected tissue obtained from 200 surgically resected NSCLCs. KDR single nucleotide polymorphism (SNP) 889G/A (rs2305948), SNP 1416A/T (rs1870377) and SNP −37A/G (rs2219471) were genotyped by PCR-based sequencing. KDR and VEGF copy number were examined by quantitative (q)-PCR. Protein expressions of VEGF and VEGFR2, and CD34 for microvascular density (MVD) analysis were studied by immunohistochemistry. SNP genotypes, genes copy number and protein expression were correlated with NSCLC clinico-pathological features, including overall survival (OS) and recurrence-free survival (RFS). Results. KDR 1416 AT/TT genotypes had significantly improved OS (HR = 0.56, 95% CI 0.33 to 0.96, P = 0.035) compared with KDR 1416 AA in all NSCLC patients in the multivariate analysis with adjustment of histology and tumor stage. In lung adenocarcinomas, KDR 1416 AT/TT genotypes and KDR −37AG/GG were associated with a favorable OS (HR = 0.43, 95% CI 0.20 to 0.92, P = 0.029; HR = 0.47, 95% CI 0.23 to 0.96, P = 0.039, respectively). Strikingly, KDR −37 AG/GG genotypes predicted a superior OS compared with KDR −37 AA genotype in the NSCLC patients treated with adjuvant therapy (HR = 2.45, 95% CI 1.06 to 5.66, P = 0.036). No genotype in KDR SNPs was associated with RFS in NSCLC patients. Gene copy gain of KDR and VEGF were detected respectively in 34/91 (37.4%) and 2/91 (2.2%) NSCLC tumors. Gene copy gain of KDR was associated with a poor overall survival in NSCLC patients (HR= 2.96, 95% CI 1.41 to 6.24, P = 0.004). Furthermore, tumors with gene copy gain of KDR showed significantly higher cytoplasmic (P = 0.013) and membrane (P = 0.009) VEGFR2 protein expression, lower cytoplasmic VEGF expression (P = 0.044), and higher MVD (P = 0.018) and larger vessel areas (P = 0.033) compared with tumors lacking KDR gene copy gain. Conclusion. Our findings suggest an association between KDR SNP genotypes and survival in NSCLC patients receiving adjuvant therapy. KDR copy number gain was frequently identified in NSCLC and was associated with worse survival in NSCLC patients. (Supported by grant US DoD W81XWH-07-1-0306). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 374.

Abstract 310: The role of c-Jun N-terminal kinase 2alpha in non-small cell lung carcinoma tumorigenesis

Abstract c-Jun N-terminal kinases (JNKs) are members of the mitogen activated protein kinase (MAPK) family and have been implicated in tumorigenesis. One isoform in particular, JNK2alpha, has been shown to be frequently activated in primary brain tumors, to enhance several tumorigenic phenotypes, and to increase tumor formation in mice. It was reported that there is frequent activation of JNK in non-small cell lung carcinoma (NSCLC). In this study, we investigated the role of the JNK2alpha isoform in NSCLC formation by examining its expression in primary tumors and by modulating its expression in cultured cell lines. We discovered 70% of the tested primary NSCLC tumors had 2 to 3-fold higher JNK2alpha protein and mRNA expression compared to normal lung tissue indicating a possible role of JNK2alpha in NSCLC tumorigenesis. To determine the importance of JNK2alpha in NSCLC progression, we reduced JNK2α in multiple NSCLC cell lines using short hairpin RNA. Cell lines deficient in JNK2alpha had decreased cellular growth and anchorage-independent growth, and the tumors were 4-fold smaller in mass. To elucidate the mechanism by which JNK2alpha induces NSCLC growth, we analyzed the JNK substrate, STAT3. Our data show that JNK2alpha can regulate the transcriptional activity of STAT3 by phosphorylating the Ser727 residue of STAT3 thereby regulating expression of oncogenic genes, such as c-Myc protein. Our studies revealed a novel mechanism in which phosphorylation of STAT3 is mediated by a constitutively active JNK2 isoform, JNK2alpha. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 310.

The EPHB6 Receptor Tyrosine Kinase Is a Metastasis Suppressor That Is Frequently Silenced by Promoter DNA Hypermethylation in Non–Small Cell Lung Cancer

Loss of EPHB6 receptor tyrosine kinase expression in early-stage non-small cell lung carcinoma (NSCLC) is associated with the subsequent development of distant metastasis. Here, we analyzed the regulation and function of EPHB6 in lung cancer metastasis. The expression levels of EPHB6 were compared among normal lung tissue (n = 9), NSCLC without metastasis (n = 39), and NSCLC with metastasis (n = 39) according to the history of the patients. In addition, EPHB6 expression levels of matched tumor-normal pairs from 24 NSCLC patients were analyzed. The promoter DNA methylation status and its association with the expression levels of EPHB6 were determined among 14 pairs of tumor-normal samples. Metastatic potential of EPHB6 was assessed in vitro and in vivo in a metastasis mouse model. Overexpression and RNA interference (RNAi) approaches were used for analysis of the biological functions of EPHB6. EPHB6 mRNA and protein levels were significantly reduced in NSCLC tumors compared with matched normal lung tissue. Decreased EPHB6 expression levels were associated with an increased risk for metastasis development in NSCLC patients. Loss of expression correlated with EPHB6 hypermethylation. EPHB6 expression was induced by 5-aza-2'-deoxycytidine treatment in an NSCLC cell line. Restoration of EPHB6 expression in lung adenocarcinoma cells increased adhesion and decreased migration. Reexpression of EPHB6 in lung cancer cells almost entirely abolished metastasis formation in non obese diabetic (NOD)/severe combined immunodeficient mice. Taken together, these analyses show that EPHB6 is a metastasis inhibitory gene that is frequently silenced by hypermethylation of its promoter in NSCLC.