Virtual Library

Abstract
Neuroendocrine Tumours of the Lung encompass low grade tumors (typical and atypical carcinoids) and high grade carcinomas (large cell neuroendocrine lung carcinoma and small cell lung carcinoma). Typical carcinoids (TC) and atypical carcinoids (AC) are usually diagnosed in front of symptoms (bleeding or pneumonia) or abnormal chest Xray or CT scan. Patients with TC are younger. Association with MEN1 syndrom is rare. Bronchial endoscopy often shows the tumor and biopsies made with caution usually confirms the diagnosis of carcinoid. However, the pathological analysis of the whole tumour is necessary to distinguish TC and AC, according to WHO criteria based on mitotic count (less or more than 2 per 2mm[2]) and necrosis. Staging differs from that of other lung cancers, including somatostatin receptor scintigraphy (SRS), hepatic MRI and bone MRI. Treatment of typical or atypical bronchial carcinoid is implemented in the multidisciplinary setting after an in depth characterization of host and tumor presentations. Goals of the treatment are both to control hormone-related symptoms if they exist, and tumor growth. Surgery remains the gold standard of treatment of bronchial carcinoids. Thoracic surgery should not differ from that of lung cancer, while it should be conservative for young patients, with complete resection and lymph node dissection. In case of lymph node involvement, there is no consensus about adjuvant treatment, which is not recommended by ENETS. Prognostic heterogeneity and absence of cure at the metastatic stage make quality of life a key issue of advanced bronchial carcinoid management. Advanced atypical carcinoids are more aggressive than typical carcinoids. The spontaneous slope without treatment, the number of tumor organs including the primary, SRS grade of uptake and quality of control of hormone-related symptoms are key parameters to be characterized before any therapeutic decision. Based on the limited number of studies available in patients with bronchial primary, recommendations of treatment also take into account data established from the field of digestive NET. Treatment is based on locoregional therapies for bone and liver metastasis, and/or systemic treatment (somatostatin analogues, everolimus, chemotherapy, Peptide receptor radionuclide therapy). Large cell neuroendocrine carcinomas (LCNEC) are high grade malignant tumours. Their behavior is very different from that of bronchial carcinoids. Patients are usually male and smokers. Locally advanced or metastatic stages are usual at presentation. Brain metastases are frequent. Surgery is possible only in one third of patients. Adjuvant chemotherapy with platinum-etoposide based chemotherapy is recommended. For more advanced LCNEC, prognosis is poor and treatment does not differ from that of SCLC, without any significant progress made these last years.

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Abstract
The purpose of this session is to allow delegates to bring cases for discussion in relation to unusual thoracic tumours, primarily related to the pathology but also in relation to clinical management of complex pathological cases. Respiratory medicine does not provide a huge volume of material for the diagnostic pathologist, but much of this is related to thoracic cancers and their differential diagnoses. This sometimes leads to a lack of experience in managing rare and complex cases and most pathologists will have an, albeit informal, link for specialist opinion to help in this instance. Indeed, it is now a recommendation in the United Kingdom that a pathway of referral is part of the multidisciplinary management of lung cancer patients. In the speaker’s experience, around 60% of referrals for complex cases relate to tumour pathology, and this is perhaps increasing with the relative decrease in biopsy for non-neoplastic diseases and increased interest in the subtyping of more common tumours. Cases referred for specialist opinion tend to breakdown into small lung biopsies, in particular asking the question, "is there enough evidence to call the biopsy definitively malignant?" This is, in particular, in relation to adenocarcinoma. Another common area is the differential diagnosis of mesothelioma, in particular in relation to spindle cell rather than epithelioid proliferations in the pleura. The third common area for referral is the "rare" tumour, where cases will often be sent because the referring pathologist has been no previous experience of the particular neoplasm. This process is not only of benefit to the referrer but also for the specialist as it allows accrual of potential new entities, an example being primary pulmonary myxoid sarcomas with EWSR1-CREB1 fusion where cases were collected over a period of 12 years from first sighting. Even within these groups, the pattern of referral has changed over the last two decades. Between 1995 and 2000, there were frequent referrals in relation to the differential diagnosis of pulmonary marginal zone lymphoma of MALT origin and reactive pulmonary lymphoid hyperplasia. Now that distinguishing criteria are much more established, these referrals are much rarer. However the last two years has seen an increase in referrals of non-small cell carcinoma, in relation to the need for further subtyping, with many of the cases being those with diverging immunophenotype. Little has been done to assess the value of specialist referral. However a review of personal experience of 50 cases affirmed diagnoses in 26/50 (52%) cases, revised in 16/50 (32%) and favoured diagnoses in 8/50. 80% of reports were deemed to have major clinical significance. Average (laboratory and pathologist) coast was £130.47 respectively (in 2009), excluding lymphoma investigations and corporate costs and current charges in the UK vary between £150 and £250 per case for slide/block reviews. The author would argue that the cost of referral appears is offset by a high level of definitive diagnosis, negating further investigation. Average turnaround time was 5 days, excluding those over holidays, with 80% reported within 7 days. Prolonged reporting times were mainly due to those referring not sending blocks or unstained slides, necessitating further despatch of material. There is also additional delay when cases require molecular analysis. With the advance of digital pathology, this has potential to facilitate and speed up external opinions, although is still in its infancy. This is because referrals often comprise numerous slides which are still quicker to send and review via the microscope than scan and then go through remotely. However, for single or small numbers of slides, the technology has greater potential. Finally, the question is often asked what happens when the putative specialists are unable to make a diagnosis. In the author’s experience, some are undiagnosable for practical reasons, such as poor fixation, whilst some are sufficiently complex and unusual that a further collective opinion from fellow specialists is obtained and fed back to the referring pathologist. This is often after much emailing and debate!

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Abstract
Tobacco use is the leading preventable cause of death worldwide, responsible for over 5 million deaths per year. By 2030 there will be more than 8 million tobacco-attributable deaths annually, if present trends continue, and 80% of them will occur in low and middle-income countries. Approximately half of regular smokers will die of a tobacco-related disease, losing an average of ten years of life compared to never smokers. Tobacco use is the major cause of lung cancer, increasing the risk 15-20 fold, and accounts a substantial fraction of the burden of tobacco-attributable mortality. In the U.S., for example, lung cancer is responsible for 29% of tobacco-attributable deaths. Lung cancer risk extends beyond tobacco users to nonsmokers who are exposed to secondhand smoke, who have a 30% higher risk of lung cancer than other nonsmokers. Reducing tobacco smoking is central to the prevention and treatment of lung cancer. The risk of lung cancer declines progressively with time after a smoker quits, reaching an asymptote at 15-20 years after quitting. Most cancer prevention efforts focus on preventing lung cancer by promotion smoking cessation among smokers and preventing smoking initiation by youths. A strong evidence base exists to guide efforts to reduce the harms of tobacco use. Offering tobacco cessation treatment is a core component of the World Health Organization’s Framework Convention on Tobacco Control, the world’s first global health treaty. It complements policy efforts such as increasing tobacco excise taxes, expanding smoke-free areas, conducting public education campaigns, and restricting tobacco promotion. However, there is an important gap. Reducing tobacco use among smokers with diagnosed lung cancer has received far less attention in cancer care than it deserves, despite a small but growing body of evidence to indicate that continuing to smoke after a lung cancer diagnosis increases all-cause mortality, impairs the response to treatment and worsens the toxicity of treatment, whether surgery, radiation, or chemotherapy. Stopping smoking also improves lung cancer patients’ quality of life by reducing symptoms such as dyspnea and fatigue. A majority of patients diagnosed with lung cancer try to quit after the diagnosis, and effective treatments are available. However, clinicians caring for smokers with lung cancer often neglect to address this issue and to connect smokers to treatment to help them sustain tobacco abstinence. The randomized controlled clinical trial evidence supporting the efficacy of treatments for tobacco use is strong. Systematic reviews agree that effective smoking cessation treatments exist and help many smokers to quit. Proven treatment methods fall into two major categories: psychosocial counseling (also called behavioral support) and pharmacotherapy. Each of these modalities is effective, but combining behavioral support and pharmacotherapy enhances success because the treatments are complementary. Pharmacotherapy primarily relieves nicotine withdrawal symptoms, while counseling aims to improve a smoker’s motivation and confidence to quit and teaches practical coping skills for quitting. Counseling can be delivered in person or by telephone (either voice or text messaging). The evidence base for the efficacy of delivery via the web or smart phone applications is being developed. Smoking cessation pharmacotherapy approximately doubles the success rate of a quit attempt. Three categories of first-line treatment are approved in the U.S. and many other countries: nicotine replacement therapy (NRT), bupropion (an atypical antidepressant), and varenicline (a selective nicotine receptor partial agonist). NRT, the most widely used product, is available in multiple forms including skin patch, gum, lozenge or sublingual tablet, oral inhaler, mouth spray and nasal spray. Combining NRT products (patch plus a shorter acting form) is the most effective way to administer NRT and is recommended by U.S. and U.K. clinical guidelines. Varenicline, the newest product to market, is effective but enthusiasm has been tempered by post-marketing concerns about psychiatric side effects and a possible increased risk of cardiovascular events. Current evidence does not suggest a large adverse effect but studies are ongoing. Cytisine, a partial nicotinic receptor agonist that is less selective for the α4β2 nicotinic receptor than varenicline, is inexpensive and sold in some Eastern European countries. It showed efficacy for smoking cessation in a recent double-blind randomized controlled trial although quit rates were low. If replicated, its low cost could make pharmacotherapy affordable in settings where cost limits medication availability. Nortriptyline, a tricyclic antidepressant, has evidence of efficacy in systematic reviews but is supported by a smaller body of evidence. No other antidepressant and no anti-anxiety medication has demonstrated efficacy for smoking cessation. To date there are few trials of smoking cessation interventions specifically targeted to patients with cancer. However, there is substantial evidence about what works in ambulatory and inpatient medical practice, and this is likely to be generalizable to lung cancer patients. Physicians who routinely deliver brief advice to quit to all smokers increase smokers’ odds of quitting. Providing a brief counselling intervention during an office visit is more effective than advice alone at promoting smoking cessation. Clinicians in ambulatory practice can promote smoking cessation by encouraging a smoker to make a quit attempt and connecting the smoker to evidence-based medication and behavioural treatments available in the health care system or community. Caring for hospitalized smokers provides another opportunity to encourage smoking cessation. Hospital-initiated smoking cessation counselling interventions increase cessation rates after discharge. A system-wide approach to promoting and supporting smoking cessation should be part of standard care in cancer centers and other sites that deliver health care to cancer patients. This would include the systematic identification and documentation of smoking status throughout cancer treatment, routine and repeated offers of tobacco cessation treatment during cancer treatment, and coordination of care among various types of specialists who deliver the cancer care. In our institution, we conducted a pilot trial in newly diagnosed patients with early stage lung cancer to identify smoking status and routinely offer treatment. We compared 12 weeks of varenicline plus counselling with usual care. At end of treatment, quit rates were higher with treatment (34% vs. 14%), and a full-scale randomized trial is underway.

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Abstract
The single most important advance in the approach to lung cancer treatment in the past decade has been the iterative identification and targeting of subsets of lung cancer defined by distinct oncogenic driver mutations. This has changed the way we think about these diseases, and is accelerating the development of driver-targeted therapies that are improving multiple measures of clinical outcome, most dramatically documented by waterfall plots of tumor response, for patients with advanced lung cancer. Despite the clinically meaningful benefits provided by selective inhibition of mutated oncogenic drivers, however, none of these treatments have changed the fundamental incurability of metastatic lung cancer. Hence the impetus for this series of talks: we have markedly improved the short-term prognosis for select subsets of our patients, but the survival curves even for these subsets go to ground. This observation, entirely consistent with the clinical experience of targeted inhibitor development in other malignancies, has prompted focused research in a number of directions, including inhibiting secondary mutations and escape pathways implicated in acquired resistance, priming the immune system to effectively respond to advanced cancer, and defining and targeting non-mutational (epigenetic) mechanisms contributing to oncogenesis and disease persistence. Epigenetics refers to the somatically heritable differences in gene expression not attributable to intrinsic alterations in the primary sequence of DNA. In general, the cells that comprise an individual have identical genomes, but have many entirely different epigenomes that dictate tissue specificity and cellular function. The past decade has seen remarkable expansion of our understanding of how epigenetic control influences the patterns of gene expression, how these controls can be manipulated, and how such manipulation can influence cell fate. These emerging insights have clear translational implications for therapeutic targeting epigenetic abnormalities in cancer. Cancers of all types demonstrate extensive and biologically significant changes in the epigenetic code; these changes collectively can be termed the “cancer epigenome.” Clonally heritable alterations in the cancer epigenome include important oncogenic drivers, entirely analogous to somatic mutations in the cancer genome. One nice example demonstrating the complementary nature of genetic and epigenetic alterations in cancer has emerged from The Cancer Genome Atlas sequencing efforts in lung cancer, revealing key tumor suppressors affected by mutational inactivation or epigenetic silencing in non-overlapping sets of cases. There is a key difference between genetic and epigenetic changes in cancer-causing genes. Mutation is in general irreversible, while epigenetic changes are not: these could be reversed with appropriately directed therapy. This plasticity may represent both an opportunity and a limitation for epigenetic therapy approaches. The existence of intermediate states of gene silencing or activation can further complicate identification of epigenetically dysregulated “driver” and “passenger” genes in cancer. I will review our recent progress in epigenetic profiling of small cell lung cancer. The development of epigenetically targeted anti-cancer drugs has lagged behind the recent explosive expansion of mutant kinase inhibitors. In part, this may be because many key components of epigenetic dysregulation in cancer are only now being described. Currently available epigenetically targeted drugs include the DNA methyltransferase inhibitors azacitidine and decitabine and the histone deacetylase inhibitors romidepsin and vorinostat; a much larger portfolio of both of these classes of drugs, particularly the HDAC inhibitors, are now in development. These classes of drugs both induce broad changes in gene expression. Our experience in combining azacitidine with the HDAC inhibitor entinostat in patients with advanced lung cancer will be discussed in another presentation at this meeting. An additional development with translational implications for drug development that has emerged from recent genomic sequencing efforts is the recognition that virtually all tumors harbor mutations in critical regulators of the epigenome. Novel agents targeting other epigenetic factors, including multiple chromatin modifiers, are now in development. Bromodomain inhibitors targeting the BET family proteins may be of particular interest in blocking activation of MYC target genes, of central relevance in small cell lung cancer. If cancer-specific epigenetic alterations were just an alternative to mutation in heritably affecting specific oncogenic drivers, one might not expect epigenetically directed therapy to lead to durable responses. However, several preclinical observations that suggest that effective epigenetic therapy could have very different, and potentially complementary, effects. One set of observations, first noted by the Settleman laboratory, suggests a strategy to convert excellent responses to targeted therapy to durable responses. These investigators define drug-tolerant “persisters” among driver-oncogene dependent tumor cells treated with supratherapeutic doses of tyrosine kinase inhibitors – these persisters are clonogenic but are dependent on a particular histone demethylase (KDM5A) and can be eliminated by exposure to any of multiple HDAC inhibitors. A second set of observations, from the Baylin laboratory and others, demonstrates that low dose exposure to demethylating agents alone, below the level of substantial cytotoxicity, can alter the long-term clonogenic and tumorigenic potential of cancer cells. A final set of observations concerns the extensive array of cancer-relevant pathways and processes that can be concomitantly affected by epigenetically targeted therapy; of particular note are multiple immunologically relevant pathways in both tumor cells and immunologic effectors. Induced re-expression of silenced tumor antigens together with stimulation of immune response pathways may enhance tumor susceptibility to immunotherapy. These recent observations define clinically testable hypotheses using currently available investigational agents that could affect the long-term survival of patients with lung cancer.

Abstract
Despite advances in cancer genomics, most advanced solid tumors remain incurable and drug resistance is almost inevitable. Two important lessons have emerged, which may provide an explanation for difficulties that have been encountered in improving cancer survival outcomes. First, each tumor contains an individual assortment of multiple genomic aberrations, few of which are shared between patients with the same histopathological tumor subtype. Second, evidence suggests that these anomalies appear to vary within individual tumors, both spatially and temporally during the disease course, indicating substantial intratumor heterogeneity. Branched evolutionary growth and intratumor heterogeneity results in coexisting cancer cell subclones with variegated genotypes and phenotypes that may be regionally separated within the same tumor and alter in dominance over time. Variegated phenotypes, resulting from intratumoral genetic heterogeneity and the emergence of new subclones at relapse, are likely to have important implications for developing novel targeted therapies and for preventing the emergence of drug resistance. Intratumor heterogeneity and sampling bias, resulting from single biopsy-driven biomarker discovery and validation approaches, may also contribute to the recently reported failures in implementation of robust biomarkers in the clinical setting. In this talk, the two fundamental principles of Darwinian Evolution, diversity and selection, will be discussed in relation to achieving better cancer survival outcomes.

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Background
The VEGF pathway has become an important therapeutic target in lung cancer. In Phase III trials, blocking VEGF using Bevacizumab (Avastin[®]) has yielded improved progression-free and overall survival in NSCLC patients when combined with standard chemotherapy, demonstrating the use of VEGF as a target for therapy. In this study, we examined the mechanisms of VEGF-mediated survival in NSCLC.

Methods
NSCLC cells (H460, H647, A549 and SKMES-1) were screened for expression of VEGF and its receptors at the mRNA and protein levels. The effect of recombinant VEGF (100ng/ml) and its blockade using neutralising antibodies (1µg/ml) on lung tumour cell proliferation (BrdU) and cell cycle (FACS) was examined. Phosphorylation of Akt and Erk1/2 proteins was examined by High Content Analysis (HCA) and confocal microscopy. The effects of silencing VEGF (100nM siVEGF)) on cell proliferation and survival signalling were assessed using BrdU and Western blot analysis, respectively. The role of NP1 in cell proliferation (BrdU), apoptosis (HCA) and cell survival signalling was also examined. A NP1 stable transfected H460 cell line (NP1-negative) was generated and NP1 overexpression verified at the mRNA (RT-PCR) and protein (Western Blot) levels relative to empty vector controls (EVC). Cell proliferation, pAkt and pErk1/2 levels were assessed in response to recombinant VEGF and VEGF antibodies. Tumour growth studies were carried out in female Balb/c nude mice following subcutaneous injection of NP1-overexpressing cells (n=8) and EVC control cells (n=8). The role of epigenetic modifications in the regulation of VEGF and its receptors was also examined using the histone deacetylase (HDAC) inhibitors, Trichostatin-A (TSA) and Virinostat (SAHA).

Results
VEGF increased proliferation of NP1-expressing NSCLC cells (H647, A549 and SKMES-1). Blocking VEGF inhibited VEGF-mediated proliferation and induced growth arrest in the G0/G1 phase of the cell cycle. Phosphorylation of Akt and Erk1/2 proteins was significantly upregulated in response to VEGF, while antibodies to VEGF inhibited this effect. VEGF siRNA significantly inhibited lung tumour cell proliferation and decreased phosphorylation of pAkt and pErk1/2. NP1 blockade significantly inhibited proliferation and apoptosis of NP1-expressing NSCLC cells, with no effect on the NP1-negative cell line, H460. Cell proliferation was significantly decreased in response to NP1 siRNA. Stable transfection of H460 cells with NP1 pcDNA significantly increased proliferation relative to EVC cells. This effect was further increased in NP1 stable transfectants upon the addition of VEGF, while neutralising antibodies to VEGF inhibited this effect. Expression levels of pAkt protein was significantly increased following treatment of cells with VEGF, with little or no effect on the pErk1/2 pathway. Tumour growth was significantly increased in mice injected with NP1-overexpressing cells (p=0.0052). HDAC inhibitors increased VEGFR1 and VEGFR2 and downregulated NP1 and NP2 expression. Significant inhibition in proliferation of NP1-positive lung cancer cells was associated with a decrease in the NP1 receptor at the mRNA and protein levels.

Conclusion
We demonstrate that VEGF is an autocrine survival factor for NSCLC cells, mediating its effects through the Neuropilin-1 receptor. Combining anti-VEGF therapies with HDAC inhibitors may offer potential as a promising avenue for future clinical research in the treatment of NSCLC.

Background
KRAS mutations are one of the most common driver mutations in non-small cell lung cancer (NSCLC) and efficient therapeutic stratergies for oncogenic KRAS-driven NSCLC are urgently needed. We recently identified epiregulin (EREG) as one of several putative transcriptional targets of oncogenic KRAS signaling in KRAS-mutant NSCLC cells and immortalized bronchial epithelial cells expressing ectopic mutant KRAS. In the present study, we assessed clinicopathological and biological significance of EREG expression in NSCLC.

Methods
Seventy-eight lung cancer cell lines (23 small cell lung cancers [SCLCs] and 35 NSCLCs), five noncancerous bronchial epithelial cell lines and 174 surgical specimens from NSCLC patients (136 adenocarcinomas and 38 squamous cell carcinomas) were used for EREG expression analysis by real-time RT-PCR methods. In vitro cell growth was evaluated by MTT assay, colony-formation assay in liquid culture and soft agar assay. Apoptosis was evaluated by the DNA fragment detection method and the annexin-V-fluorescein staining method. The Kaplan-Meier method was used for analysis of disease-free survival (DFS) and overall survival (OS) and log-rank test was used for survival differences. Cox proportional hazards model was used to identify independent prognostic factors for PFS and OS.

Results
EREG is predominantly expressed in NSCLC lines harboring KRAS, BRAF or EGFR mutations whereas most SCLC lines lack EREG expression. Small interfering RNAs (siRNAs) targeting against these mutations resulted in down-regulation of EREG expression in NSCLC cells. EREG expression was significantly reduced by treatments with the inhibitors of MEK or ERK in EREG-overexpressing NSCLC cell lines, irrespective of mutation status of KRAS, BRAF and EGFR. EREG expression significantly correlated with KRAS copy number in KRAS-mutant NSCLC cell lines whereas EREG expression significantly correlated with EGFR copy number in NSCLC cell lines with wild-type KRAS/BRAF/EGFR. In the analysis of surgical specimens from NSCLC patients, EREG was predominantly expressed in lung adenocarcinomas. In a subgroup of adenocarcinomas, EREG expression was significantly higher in the tumors from elderly patients (≥70-year-old), males and smokers and was higher in the tumors with pleural involvement-, lymphatic permeation- or vascular invasion-positive. EREG was highly expressed in lung adenocarcinomas with KRAS mutation compared to those with EGFR mutation or wild-type EGFR/KRAS. Lung adenocarcinoma patients with high EREG expression had significantly shorter DFS and OS compared to those with low EREG expression. When the patients were divided into four groups according to EREG expression levels and KRAS mutation status, DFS and OS were significantly shorter in the patients with KRAS-mutant/EREG-high than those with wild-type KRAS/EREG-low. Cox regression analysis demonstrated that elevated EREG expression was an unfavorable prognostic factor. siRNA-mediated EREG silencing inhibited anchorage-dependent and -independent growth and induced apoptosis in KRAS-mutant and EREG-overexpressed lung adenocarcinoma cells.

Conclusion
Our findings suggest that oncogenic KRAS-induced EREG overexpression contributes to an aggressive phenotype and unfavorable prognosis in lung adenocarcinoma patients, and EREG could be a promising therapeutic target in oncogenic KRAS-driven NSCLC.

Background
We previously developed a 76-gene signature of epithelial-to-mesenchymal transition (EMT) that predicted resistance to EGFR and PI3K inhibition in non-small cell lung cancer (NSCLC). This analysis also identified Axl, a receptor tyrosine kinase, as a novel target for mesenchymal lung cancers. Here, we conducted an integrated molecular analysis of EMT in resected, treatment-naïve tumors from three clinical cohorts, including the Cancer Genome Atlas (TCGA) lung adenocarcinomas (LUAD) and squamous cell carcinomas (LUSC), with particular focus on Axl as a potential target in mesenchymal NSCLC.

Methods
Using our 76-gene EMT signature, TCGA patient tumors (230 LUAD, 178 LUSC) and a large MDACC cohort of resected tumors (n=279) were assigned an “EMT score.” Expression of >160 total and phosphoproteins were measured in the tumors by reverse phase protein array (RPPA). Proteomic profiles and other molecular markers (including mutation status, miRNA expression, and copy number) were correlated with EMT scores and Axl expression levels.

Results
The EMT score, derived from our EMT signature, identified NSCLC tumors with mesenchymal gene expression signatures (average 23% of tumors across all cohorts, range 14-34%). In both LUAD and LUSC, EMT scores were highly correlated with (1) expression levels of the miR200 family, a group of miRNAs previously known to regulate EMT (p-values <0.001 by Pearson correlation) and (2) levels of proteins central to EMT (e.g., E-cadherin, alpha-catenin, beta-catenin, claudin-7, fibronectin; p<0.001 for all). Mesenchymal tumors also had lower expression of TTF1 in LUAD (p=0.0002) and lower p63 in LUSC (p=0.003). Although pEGFR levels were higher in epithelial LUAD tumors (p=0.01), the frequency of EGFR mutations was not significantly higher in this group. EMT score was not associated with smoking status. Consistent with our previous findings in cell lines and patients with advanced NSCLC (BATTLE trial), protein expression of the receptor tyrosine kinase Axl was significantly higher in tumors with mesenchymal signatures (high EMT scores) and with low E-cadherin protein expression (p<0.005 for both). The inverse correlation between tumor E-cadherin and Axl expression was confirmed in an independent group of NSCLC cases by immunohistochemistry. Although a small number of Axl mutations were observed (<3% of tumors), few occurred in the kinase domain and their biological significance is unknown. Other potential therapeutic targets expressed at higher levels in mesenchymal lung cancers included PKC-alpha, NFKB, and FGFR1.

Conclusion
The EMT gene expression signature performed well in the TCGA LUAD, TCGA LUSC, and MDACC cohorts, correlating strongly with established markers of EMT on other data platforms (miRNA and protein). We observed strong protein expression of the receptor tyrosine kinase Axl (as well as other targets) among mesenchymal tumors, supporting further investigation of AXL as a potential EMT target and into the mechanism of its overexpression in NSCLC.

Background
Among the most common features of highly invasive tumors, such as lung adenocarcinomas (AD) and squamous cell carcinomas(SqCC), is the massive degradation of the extracellular matrix (ECM); and the remarkable qualitative and quantitative modifications of hyaluronidases (HAases), hyaluronan sinthases (HAS), E-cadherin adhesion molecules, and the transformer growth factor β (TGF-β) may favor invasion, cellular motility, and proliferation.

Methods
We examined HAases proteins (Hyal), HAS, E-cadherin, and TGF-β profiles in lung AD subtypes and SqCC obtained from smokers and nonsmokers.Fifty-six patients, mean age 64 years, who underwent lobectomy for AD (n = 31) and SqCC (n = 25) were included in the study.

Results
HAS1, 2 and 3, and Hyal 1 and 3 were significantly more expressed by tumor cells than normal and stroma cells (P<0.01) . E-cadherin and TGF-β immunostaining indices were significantly increased in the tumour cells compared to stroma cells (p<0.01). When stratified according to histologic types, HAS3 and Hyal 1 immunoreactivity was significantly increased in tumour cells of AD (p = 0.01) and stroma of SqCC (p = 0.002), respectively. Tobacco history in patients with AD was significantly associated with increased HAS 3 immunoreactivity in tumour cells (p<0.01). Stroma cells of SqCC from non smokers patients presented a significant association with HAS3 (p<0.01). A positive association was found between TGF-β in tumor cells and HAS2 in stroma cells (R = 0.40, p= 0.03), Hyal 3 and HAS1 in stroma cells (R = 0.58 p = 0.02), Hyal 1 in tumor cells and HAS1 in stroma cells (R: 0.44, p = 0.01), Hyal 3 and HAS1 in tumor cells (R: 0.35, p = 0.01). A negative association was found between TGF-β and HAS3 (R = -0.54, p = 0.004), and TGF- β and Hyal 1 (R = -0.40, p = 0.03). A Cox model analysis show low risk of death associated with female patients (R=0.11) age < 69 yrs (R=0.02), I and II stage (R=0.18 and 0.00, respectively), and high risk of death associated to HAS2 < 14.6% (R=8.69) and HAS3 > 7.4%..

Conclusion
HAase,HAS, E-cadherin, and TGF-β modulate a different tumor-induced invasive pathway in lung AD subgroups and SqCC. An inverse relationship between epithelial and stroma biomarker expression provides a different spectrum of aggressiveness. While an overexpression of HAS1 and HAS3 indicates aggressive subtypes of SqCC and AD, an overexpression of TGF-β and E-cadherin, indicates a protective role of the ECM in avoiding invasion by tumor cells in both histological types. HAasesin resected AD and SqCC were strongly related to the prognosis,Therefore, our findings suggest that strategies aimed at preventing high HAS3 and Hyal1 synthesis, or local responses to low TGF-β and E-cadherin, may have a greater impact in lung cancer prognosis. Proof of this idea will require further study in a randomized, prospective clinical trial.

Background
Many studies have reported the presence of human papilloma virus (HPV) primary oncoproteins in lung cancer patients. Their detection depends on histological and geographical patterns and seems to be associated with the response obtained to EGFR inhibitors.

Methods
Information regarding 84 patients suffering lung adenocarcinomas and EGFR mutations and another 48 patients lacking them (including 7 KRAS carriers) was explored for the presence of HPV16 in paraffin-embedded tumour tissue using INNO-LiPA PCR-based assays. The results were correlated with clinical characteristics and multiple outcomes, including response rate, progression-free survival (PFS) and overall survival (OS).

Results
Mean age was 59.9 years (+/- 12.2) and HPV16 infection positivity was 39% (N=52). HPV was predominant in females (N=42; p=0.032), no differences being found regarding histological pattern (p=0.72) or having a background of smoking (p=0.54). 62% of the patients had EGFR exon 19 deletions and 22.6% the L858R mutation. Changes in exon 19 were positively related to the presence of HPV16 (p=0.043), differently to the exon 21 mutation (p=0.3). Overall response rate to tyrosine -kinase inhibitors in EGFR mutation carriers’ was 65%, stable disease was 31% and clinical benefit 86.5%. Positive differences were found for response according to HPV virus status (p=0.03). PFS rate was greater in patients who were EGFR+/HPV+ compared to the EGFR+/HPV- population (p=0.014). Likewise, OS was longer for the EGFR+/HPV+ population compared to the EGFR+/HPV- population (34 months versus 24 months; p=0.0001). OS was also longer for HPV+ patients in the absence of EGFR mutations (p=0.001). The presence of HPV also discriminated OS in the small cohort of KRAS+ patients.

Conclusion
The present study has documented a high HPV positivity rate in Hispanic patients suffering lung adenocarcinoma. The presence of viral DNA can thus be presumed to be a positive prognostic factor for EGFR and KRAS mutated patients, thereby leading to considering infection as a dominant part of carcinogenesis amongst non-smokers in Latin America.

Background
Intra-tumor pressure (ITP) was reported to be one of prognostic factors for head and neck cancer and hepatocellular carcinoma. It was reported that ITP of rectal cancer fell after Bevacizumab treatment. We measured ITP of lung cancer and investigated relationship between ITP and clinicopathological factors of lung cancer.

Methods
After institutional review board approval, we measured ITP in the center of the tumor after resection of lung cancer using Ultra-Miniature Mikro-Tip Pressure Transducer Catheters (SPR1000, Millar). Lung cancer showing pure ground glass opacity (GGO) appearance in CT was excluded from this study. Between September 2009 and January 2013, 219 lung cancer patients, 142 male and 77 female, entered the study. Mean age of patients was 70 ± 10 year old (range: 39 to 87). The histologic types of lung cancer were adenocarcinoma (AD) in 148 patients, squamous cell carcinoma (SqCC) in 52 patients, adenosquamous carcinoma (AdSq) in 10 patients, and others. The clinical (c) T stages were T1a in 69 patients, T1b in 64 patients, T2a in 60 patients, T2b in 11 patients, T3 in 14 patients, and T4 in one patient. The cN stages were N0 in 187 patients, N1 in 22 patients, N2 in 8 patients, and N3 in 2 patients. The cStages were IA in 122 patients, IB-IIIB in 97 patients. The pathological (p) T stages were T1a in 84 patients, T1b in 45 patients, T2a in 67 patients, T2b in 9 patients, T3 in 9 patients, and T4 in 4 patients. The pN stages were N0 in 183 patients, N1 in 21 patients, N2 in 14 patients. The pStages were IA in 117 patients, IB in 53 patients, IIA in 19 patients, IIB in 6 patients, IIIA in 21 patients, and IIIB in 2 patients. Mean maximal diameter of the tumors on CT was 2.7±1.4cm (range: 0.8 to 7.9). Of these tumors, 59 showed part-solid and 147 showed solid appearance on CT.

Results
Mean ITP was 8.7 ± 6.5 mmHg（range: 0 to 37.2）. Mean ITPs according to histological type were as follows: 8mmHg in AD, 10mmHg in SqCC, 7mmHg in AdSq. Mean maximal standardized uptake value (SUVmax) in fluoro-deoxy glucose-positron emission tomography was 4.6±3.5 (range: 0.3 to 15.1). ITP correlated to maximal diameter of tumor on CT (r=0.312) and pT factor (r=0.336). The ITP in pN0 (8mmHg) was significantly lower than that in pN(+) (12mmHg) (p=0.005). The ITP in pStage IA patients (6mmHg) was significantly lower than that in other stages patients (11mmHg) (p=0.001). The ITP of tumors showing solid appearance (10mmHg) was significantly higher than that of tumors showing part-solid (5mmHg). While ITP correlated to SUVmax (r=0.402), lymphatic vessel invasion (r=0.269), blood vessel invasion (r=0.293), pleural invasion (r=0.287), tumor grade (r=0.238), MIB-1 index (r=0.292), did not correlate to microvessel density (CD34) .

Conclusion
ITP showed correlation to tumor aggressiveness factors of lung cancer.

Background
Heat shock protein 90 (Hsp90) maintains the stability and activity of numerous signaling proteins involved in cancer metastasis. Interim results of the GALAXY-1 trial (NCT01348126) showed an improvement in overall survival in patients with non-small cell lung cancer (NSCLC) treated with an Hsp90 inhibitor, ganetespib (G), plus docetaxel (D) compared to D alone. We evaluated the antimetastatic activity of ganetespib in preclinical models, and compared time to appearance of new lesions in patients with radiologic progression in the two arms of the GALAXY-1 trial.

Methods
Preclinical effects of ganetespib were investigated on the: (1) migration and invasion of cancer cells via scratch and Boyden chamber assays, (2) expression of metastatic factors using protein array and gene profiling, (3) architecture of NSCLC xenografts by IHC, and (4) metastasis to the lung in tail vein and orthotopic breast cancer mouse models assessed by bioluminescence and histology. GALAXY-1 is a Synta sponsored randomized, international open-label study of D with or without G in patients with advanced lung adenocarcinoma, one prior systemic therapy, and ECOG PS 0/1. D was given at 75 mg/m2 on Day 1 of a three-week cycle in both arms. In the combination arm, G was given at 150 mg/m2 on days 1 and 15. Time to appearance of new lesions (TTNL) was measured using serial computed tomography scans, starting from randomization and until a new metastatic lesion was reported. Patient enrollment completed in November 2012.

Results
Ganetespib blocked the directional migration of NSCLC cells in a monolayer of cancer cells, significantly reduced their invasion into type I collagen and induced the degradation of metastasis drivers including HIF-1α, activated FAK and MET. In mouse models, ganetespib significantly reduced tumor angiogenesis and proliferation in NSCLC xenografts; significantly blocked (>8X, p<0.005) the development of lung cancer metastases in a tail vein model; and significantly reduced multi-organ metastasis in an orthotopic model and blocked extramedullary hematopoiesis induced by the primary tumor. In GALAXY-1 trial patients, the population that exhibited the strongest survival improvement (diagnosis of advanced disease >6 months, N=175), median TTNL increased from 6.9 months (D) to 11.3 months (G+D), with hazard ratio 0.5 (p=0.0053) at time of abstract submission.

Conclusion
Ganetespib induces the degradation of key drivers of metastasis, resulting in the reduction of cancer cell migration and invasion in vitro and tumor angiogenesis and new lesion formation in vivo. Preliminary data from the GALAXY-1 study suggests that ganetespib treatment may reduce the risk of emergence of new metastatic lesions by 50% in advanced NSCLC patients.

Background
Osteopontin (OPN) is a ubiquitous extracellular protein associated with a wide range of normal and pathologic functions. It is a central regulator of the malignant phenotype in non-small cell lung cancer (NSCLC) through binding of cell surface receptors, but underlying mechanisms are poorly understood. Among OPN’s critical roles in NSCLC progression, is preventing apoptosis related to oxidative stress by activating alternative survival pathways. While OPN’s central RGD domain is considered important to this function, we hypothesize that exon 4, in the amino terminus, which is present in OPN’s A and B isoforms, but not in the C isoform, is essential to this process. We sought to determine the impact of inibition of binding between NSCLC cells and OPN exon 4 on apoptosis and apoptotic protein expression in NSCLC.

Methods
A 16 amino acid peptide mimicking the central sequence of OPN exon 4 and a scrambled sham were constructed. Competitive binding assays had previously determined that the OPN exon 4 peptide binds the NSCLC cell surface and inhibits OPN binding. Two NSCLC cell lines with wt p53, A549 (moderate endogenous OPN) and H460 (high endogenous OPN), were placed in serum-free media with OPN exon 4 peptide or scrambled peptide for 48 hours. Cells were then evaluated by TUNEL assay or harvested, lysed and protein expression measured using Human Apoptosis Array (R&D, Minneapolis, MN).

Results
Exon 4 peptide treatment resulted in significant increases in apoptosis in both cell lines (Fig). A similar pattern of change in apoptotic protein expression was seen in both cell lines, with significant increases in Bax, cleaved Caspase-3, CytC, Hsp32, Pon2, Cdnk1, and p53-pS15, p53-pS46, and p53-pS392, while Survivin and Claspin expression were significantly decreased (Fig). Notably no significant change was seen in Bclx, Bcl2, and pro-Caspase 3 expression. Scrambled peptide had no effect on apoptosis or protein expression. Figure 1

Conclusion
Inhibition of binding between OPN exon 4 and NSCLC cells significantly increased the rate of apoptosis and expression of proteins which modulate apoptosis associated with oxidative stress, including several key phosphorylated p53 variants. These data implicate OPN exon 4 interactions in NSCLC progression and resistance mechanisms and may explain the importance of OPN’s A and B isoforms as opposed to isoform C in NSCLC pathogenesis.

Background
Background and Aim of Study: Non-small cell lung cancer (NSCLC) is one of the leading causes of cancer deaths. Erlotinib (tyrosine kinase inhibitor, TKI) is widely used as a specific treatment targeting epidermal growth factor receptor (EGFR), while crizotinib serves as a c-MET and anaplastic lymphoma kinase (ALK) inhibitor. Autophagy is a regulated cellular catabolic process in response to stress. This study aimed to investigate whether autophagy could confer acquired resistance to TKI in NSCLC.

Methods
Methods: Two NSCLC cell lines (HCC827 and HCC4006) with EGFR mutations (exon 19 deletions) and two NSCLC cell lines (H1993 and H2228) with c-MET amplification or ALK rearrangement were selected. Cell proliferation (MTT) and annexin-V binding assays were performed to determine cell proliferation and apoptotic cell death upon TKI treatment respectively. Autophagy was determined by conversion of LC3I to LC3II and p62 degradation using Western blot. Acidic vesicular organelle (AVO) formation was shown by acridine orange staining. Autophagy inhibitor (chloroquine) was used to study the functional significance of TKI-induced autophagy.

Results
Results: MTT assay confirmed that all cell lines were sensitive to corresponding TKI after 72 hours incubation (IC~50~ <0.5 μM). Erlotinib or crizotinib increased LC3II expression, p62 degradation and formation of AVO, compatible with induction of autophagy. Combination of 10 μM chloroquine (autophagy inhibitor) with 0.2 μM erlotinib or crizotinib for 48 hours increased apoptotic events compared to single treatment (Table 1)

Conclusion
Conclusions: TKI induced both apoptosis and autophagy in EGFR-mutated, c-MET-amplified and ALK-rearranged NSCLC cell lines. Inhibition of autophagy with chloroquine enhanced TKI-induced cell death. Autophagy may serve as a protective mechanism in NSCLC upon treatment with TKI.

Background
Recently, the personalized treatments for non-small cell lung cancer (NSCLC) have progressed with the understanding of the biology and the molecular mechanisms of the tumor. Treatment strategy is determined based on the biomarker profiles examined with the primary tumor tissue or the metastatic lymph nodes. However, because tumor cells acquire the metastatic phenotype through the process of clonal evolution occurring during the tumor progression, there could be the heterogeneity of the biomarker profiles between the primary tumor and the metastatic lesions. The aim of this study was to investigate the heterogeneity of the biomarkers between the primary tumor and the metastatic lymph nodes, and to evaluate the clinical significance of its discordancy.

Methods
Seventy-two patients of NSCLC with lymph node metastases who underwent complete resection with systematic lymph node dissection between 2004 and 2010 were studied. The study was conducted with the approval of the institutional ethics committee. Immunohistochemical analysis of ERCC1 and VEGF and the mutation analysis of EGFR were performed with the paraffin-embedded tissue of both the primary tumor (PT) and the metastatic lymph nodes (LN).

Results
There were 47 male and 25 female, with the median age of 69.4 years. Thirty-eight patients had adenocarcinoma, 23 had squamous cell carcinoma, and 11 had other histologic types of NSCLC. Twenty-nine patients had N1 disease, and 43 had N2. Median observation period was 35.7 months. The 5-year overall survival rate of 72 patients was 38.0%, with 53.2% in N1 cases and 28.8% in N2 cases. ERCC1 was positive in 41.7% of PT and 58.3% of LN. There were 34 cases (47.2%) with the discordant expression of ERCC1. VEGF was positive in 66.7% of both PT and LN, whereas 32 cases (44.4%) were discordant. EGFR mutation was detected in 31.9% of PT and 18.1% of LN. There were 10 cases (13.9%) with discordant mutation status. All 10 cases had mutant EGFR in PT, whereas EGFR mutation was not found in LN. Of the 49 patients with EGFR mutation-negative in PT, none showed mutations in LN. There were no differences in relapse-free survival and overall survival among the biomarker profile groups. Among patients who had negative ERCC1 in PT or LN, overall survival was significantly better in patients who received platinum-based chemotherapy perioperatively compared with that in patients who did not. However, there were no differences in patients with positive ERCC1 in PT or LN. Seventeen patients who had mutant EGFR in PT were treated with gefitinib. The responses were CR in 4, PR in 7, SD in 2, and PD in 4, with the response rate of 64.7% and the disease control rate of 76.5%. All the nine patients who had mutant EGFR in both PT and LN showed disease control, whereas 4 out of 8 patients who had mutant-negative EGFR in LN showed PD.

Conclusion
It should be noted that discordancy of biomarker profiles between PT and LN was not a few. Understanding the heterogeneity of biomarkers would be essential for the establishing the personalized treatments for NSCLC.

Background
The urokinase plasminogen activator (uPA) system (uPAS) has been shown to play a significant multifunctional role in tumour progression including angiogenesis, adhesion and migration. Increased levels of urokinase plasminogen activator (uPA) and its receptor uPAR (CD87) strongly correlate with poor prognosis and a poor clinical outcome. It has been shown previously that a subpopulation of uPAR-positive cells in Small Cell Lung Cancer (SCLC) cell lines demonstrate significant drug resistance to traditional chemotherapeutic agents such as cisplatin, 5-fluorouracil (5-FU) and etoposide. The uPAS is regulated by NF-κB which has been shown to be constitutively activated in several cancer types including non-small cell lung cancer (NSCLC). Furthermore, we have shown NF-κB to be involved in the development of resistance to cisplatin in NSCLC. This project focuses on determining the role of the uPA system in the invasive phenotype of cisplatin resistant NSCLC cells.

Methods
Expression of NF-κB (p65) in parent and resistant NSCLC cell lines was quantified by qPCR, western blot and high content screening (HCS). The expression profiles of NFκB target genes were quantified using a Roche custom NFκB RTPCR array. Gene “hits” with a fold change >2 between parent and cisplatin resistant cells were validated by qPCR analysis. The upregulation of the urokinase-type plasminogen activator (uPA) in cisplatin resistant cells was determined by western blot. The effect of uPA inhibition on cell migration and invasion, using the monoclonal anti-uPAR antibody ATN-658, is being determined using the novel impedance-based xCELLigence Real-Time Cell Analysis detection platform.

Results
Gene expression data, from the NFκB target gene array identified a panel of genes including; PLAU (gene for uPA), RIPK and NLRP12 amongst others that were over-expressed in H460 cisplatin resistant cell lines compared to the isogenic parent cell line. uPA overexpression at the protein level was confirmed in a panel of cisplatin resistant cells compared to parent cell lines. The effect of ATN-658 on the inhibition of cell migration and invasion in cisplatin sensitive and resistant cell lines will be presented.

Conclusion
Overexpression of uPA across a panel of cisplatin resistant NSCLC cell lines highlights its significance as a marker of resistance. Targeting the uPA system may be exploited in cisplatin resistant NSCLC to inhibit cell migration and invasion.

Background
Malignant pleural mesothelioma (MPM) is a rare cancer affecting the pleura and is commonly caused by prior exposure to asbestos. Treatment of MPM is difficult with limited options. The current standard of care for MPM patients is a combination of cisplatin and pemetrexed (or alternatively cisplatin and raltitrexed), yet most patients die within 24 months of diagnosis. There is therfore an unmet need to identify new therapeutic approaches for the treatment of MPM. Lysine Demethylases (KDMs) represent novel targets for the treatment of cancer. Overexpression of many KDMs occurs in many cancers, and these proteins play important roles in tumorigenesis. One such family, the KDM6/JMJD3 family, was investigated for changes in expression in MPM and to determine if this family could represent a novel candidate target(s) for intervention in MPM.

Methods
A panel of MPM cell lines inluding the normal pleural cells LP9 & Met5A were screened for expression of KDM6 family members (KDM6A/UTX and KDM6B/JMJD3) by RT-PCR. mRNA levels were subsequently examined by RT-PCR in a cohort of snap-frozen patient samples isolated at surgery comprising benign, epithelial, biphasic, and sarcomatoid histologies. The effects of a small molecule inhibitor of KDM6B/JMJD3, (GSK-J4) on cellular proliferation and gene expression were examined.

Results
We show that the expression of the KDM6 family is detectable in all cell lines across our panel of cell lines. In primary tumours however, the expression of KDM6A/UTX was very significantly (p<0.001) and KDM6B/JMJD3 was significantly elevated (p<0.05) in malignant MPM compared to benign pleura. When separated across histological subtype KDM6A/UTX was most significantly elevated in the Sarcomatoid subtype (p<0.001), while only KDM6B/JMJD3 was significantly elevated (p<0.05) in the Biphasic subset. Treatment of REN/ NCI-H226 cells with the KDM6B/JMJD3 inhibitor GSK-J4 caused significant inhibition of cellular proliferation, with the REN cell line being more sensitive than NCI-H226. The effects of GSK-J4 on gene expression were examined on a panel of genes associated with Tumor-Invasion/Metastasis and on pro-inflammatory cytokines.

Conclusion
The KDM6 family of lysine demethylases are significantly altered in MPM. A small molecule inhibitor of this protein shows significant anti-proliferative effects in MPM cell lines. We continue to assess the effects of this compound on gene expression and cellular health by other methodologies to confirm its potential utility in the treatment of MPM.

Background
Cisplatin-based chemotherapy is the standard therapy used to many cancers. However, its efficacy is largely limited by the acquired drug resistance. So far, little is known about the RNA expression changes in the cisplatin resistant cancers. Identification the RNAs related to the cisplatin resistance may provide precise clues for cancer therapy.

Methods
Expression profiling of 7 cancer cell lines that performed using oligonucleotide microarray analysis got from GEO database. Bioinformatic analyses such as the Gene Oncology and KEGG pathway were used to identify genes and pathways specifically associated with cisplatin resistance. Signaling transduction network was established to identify the core genes in regulating cancer cell cisplatin resistance.

Results
A number of genes were differentially expressed in 7 groups of cancer cell lines. They mainly participated in 85 GO terms and 11 pathways in common. All differential gene interactions in the Signal-Net were analyzed. CTNNB1, PLCG2 and SRC were the most significantly altered.Figure 1

Conclusion
Bioinformatics may help excavate and analyze large amounts of data in microarrays by means of rigorous experimental planning, scientific statistical analysis and collection of complete data about cancer cell cisplatin resistance. In the present study, a novel differential gene expression pattern was constructed and advanced study will provide new targets for diagnosis and mechanism of cancer cisplatin resistance.

Background
EZH2, a polycomb group protein, has histone methyltransferase activity, and is involved in malignant transformation of several cancers. We have previously shown that an EZH2 inhibitor, DZNep, inhibits growth of non-small cell lung cancer (NSCLC) cell lines via G1 arrest and apoptosis. Recent studies have shown that EZH2-mediated gene silencing requires a recruitment of the histone deacetylase (HDAC) activity. Co-treatment with DZNep and an HDAC inhibitor has been shown to induce apoptosis synergistically in AML, ovarian cancers and renal cancers. However, the effect of combined therapy with DZNep and an HDAC inhibitor on NSCLC cells has not been reported.

Methods
We evaluated the effect of combined therapy with DZNep and an HDAC inhibitor, SAHA, on four human NSCLC cell lines, H1299, H1975, A549 and PC-3. Cell proliferation of untreated or drug-treated cells was measured by MTT assay. Percentage of apoptotic cells was measured using FITC-conjugated annexin V with a flow cytometer. Western blot analysis was performed on total cell lysates.

Results
Co-treatment with DZNep and SAHA inhibited cell proliferation synergistically, and reduced EZH2 expression and histone H3 lysine 27 trimethylation more effectively compared with each agent alone. The co-treatment greatly induced accumulation of p27[ Kip1] and decrease in cyclin A expression. Flow cytometry analysis demonstrated that the apoptotic fraction was increased in an additive or synergistic manner by the combination therapy. These effects were more evident in H1975 and PC-3 cells with EGFR mutation, in which expression of EGFR and phosphorylation of EGFR, AKT and ERK1/2 were markedly decreased by the co-treatment.

Conclusion
Combined epigenetic therapy with an EZH2 inhibitor and an HDAC inhibitor may represent an effective strategy for NSCLCs.

Background
Breathing demands our lungs to be soft and elastic. In contrast, lung tumors exhibit an abnormal tissue hardening concomitantly with an altered lung architecture and function, which brings the mechanical microenvironment of the tumor closer to that of muscle or bone. Carcinoma-associated fibroblasts (TAFs) within the tumor stroma are known to be conspirators of tumor progression. It remains unknown whether tissue hardening is sufficient to drive abnormal abundance of activated fibroblasts in the stroma of the different NSCLC subtypes. We aimed to determine the role of abnormal tumor tissue hardening in the overabundance of TAFs in different NSCLC subtypes

Methods
To address this question, we cultured the human lung fibroblast cell line CCD-19Lu or primary human lung fibroblasts on collagen-coated polyacrylamide gels exhibiting normal (soft) or tumour (stiff)-like substrates in the absence (0%) or presence (0.1%) of serum for 5 days. Primary cells were isolated from both tumor-free regions (referred to as control fibroblasts) or tumors of NSCLC patients diagnosed with either Adenocarcinoma (ADC, n=5) or Squamous Cell Carcinoma (SCC, n=5) histological subtypes.

Results
We observed a significantly higher density of CCD-19Lu fibroblasts in tumor-like gels compared to normal-like gels regardless the presence of serum. Similar results were obtained in both primary control fibroblasts and SCC-TAFs. In contrast, tumor-like gels induced a weaker density increase in primary ADC-TAFs. The increased fibroblast density in the tumor-like gels was associated with increased survival rather than proliferation, as revealed by a downregulation of caspase-3 and a rise in FAK and AKT phosphorylation. To further understand the apoptotic effect on stiffer substrates, we examined the expression of the mechanoreceptor integrin beta-1, responsible of sensing stiffness signals from the ECM. We found a significantly higher expression of integrin beta-1 in SCC compared to control and ADC primary fibroblasts .

Conclusion
Our results indicate that tumor extracellular hardening alone is sufficient to induce an increased density in activated lung fibroblasts, a well known conspirators of tumor progression, by activating pro-survival FAK-AKT pathways. Collectively these findings underline the major role of tissue hardening in the abnormal abundance of TAFs in NSCLC, particularly in the SCC subtype. These data warrants the development of novel targeted therapies against the pro-survival effects of tissue hardening.

Background
Cisplatin based doublet chemotherapy is the mainstay of non small cell lung cancer (NSCLC) treatment with an initial objective response rate of approximately 40-50%. However, intrinsic and acquired resistance to cisplatin constitutes a major clinical obstacle in lung cancer management and has yet to be fully understood. Inflammatory mediators may play an important role in the development of cisplatin resistance, such as those regulated by NF-κB. We have previously demonstrated that levels of NF-κB are increased in cisplatin resistant cells compared with sensitive Parent cells. We are currently assessing a number of NF-κB regulated targets in cisplatin resistant cell line models, using DHMEQ, a specific NF-κB inhibitor. DHMEQ treatment results in greater cell death in the cisplatin resistant cells compared with Parent. This study will elucidate the efficacy of DHMEQ to overcome cisplatin resistance and identify novel targets within the NF-κB pathway that may improve therapeutic strategies for NSCLC patients.

Methods
NF-κB downstream targets and signalling mediators were examined using NF-κB signalling and target pathway qPCR arrays (168 genes) in the H460 CisR and Parent cell line model. Targets identified are currently undergoing validation using qPCR and western blot. Biological and functional relevance of these targets in the development of cisplatin resistance will be examined further using DHMEQ and siRNA knockdown strategies. In addition, a xenograft murine model will be utilised to assess the effect of DHMEQ alone and in combination with cisplatin on tumour growth in vivo.

Results
Data from qPCR arrays have demonstrated that a number of genes are differentially regulated between the CisR and Parent cell lines. These include genes which activate the NF-κB signalling cascade (TLR3, TLR4), regulators of the pathway (BIRC3, CASP1), transcription factors (Myc) and NF-κB responsive genes (TNF, CXCL8). A number of these genes will be modulated to determine their involvement in cisplatin resistance. In addition, DHMEQ is being used in combination studies to determine, whether it can re-sensitise cells to cisplatin therapy. At present a dosing study is ongoing to establish the effect of DHMEQ on xenograft tumours derived from Parent and CisR cells. The results of which will be presented.

Conclusion
Preliminary data indicates that NF-κB and a number of its downstream targets are deregulated in cisplatin resistant cells. This project aims to validate the role of these NF-κB regulated genes in cisplatin resistant NSCLC. It will also determine whether DHMEQ may be a novel targeted agent for the treatment of NSCLC. The data obtained in this study will ultimately benefit patients by providing insights into novel druggable targets and new clinical strategies to re-sensitise patients to cisplatin therapy.

Background
Model of metastatic spreading of extrapulmonary malignancy into mediastinal lymhnodes.

Methods
Subcutaneous administration of 0,4 ml of the ink (compound from black carbon dispersed on colloid casein solution i.e. colloidal carbon / KOH-I-NOOR Hardtmuth Ltd., Praha, CZ) diluted with sterile saline solution 1:3 was performed under i.m. general anesthesia (diazepam, xylazine, ketamine) in 42 outbred Wistar rats (An-Lab Praha, CZ) divided into 7 groups according the application sites: retroperitoneum, testis / ovary, hind leg, front leg, thoracic wall, pleural cavity. On seventh day after the administration all rats were euthanatized, the thoracic wall was opend and mediastinal lymphnodes were detected macroscopically, taken out, and preserved in 18% formalin. Paraplast fixation and the hematoxyline-and-eosine staining were used for histological examination by 40-times and 100-times magnification. The samples with monocyte- macrophage system cells containing colloidal carbon (CC) in the mediastinal lymphnodes (MLNs) were considered positive. Two statistical methods were used to determine the probability of model cell transport and retention. First is based on random error theory and consists of mean values, standard deviations and confidence intervals. Second analysis calculated the confidence intervals of probabilities using F-distribution. Programs Matlab 2012 (MathWorks) and Microsoft Office 2010 Excel (Microsoft Corp.) were used for the statistical analysis. FIG 1 The sites of ink administration 1 2 3 4 5 6 7 1 - thoracic wall, 2 - front leg, 3 - pleural cavity, 4 - retroperitoneum, 5 – testis, 6 - hind leg, 7- ovary

Results
Macrophages containing carbon particles were found in MLNs of all groups of animals, at least in one animal of each tested group. Positive lymphnodes were found in 4/6 (66.7%) of animals after paraovarial, paratesticular and pleural cavity application and in 2/6 (33.3%) with retroperitoneal and distal part of hind leg application. In animals injected in distal part of front leg and in thoracic wall it was 1/6 (16.7%) with positive nodes. Statistical analysis showed significant retain of CC administered into different parts of the periphery of the body on the 5% significance level.

Conclusion
The transport of CC with the monocyte macrophage system cells from very different parts of the body to the mediastinal lymph nodes of Wistar rats and their persistence there after seven days has been proved on the 5% significance level. That indicates the relation between mediastinal lymphnodes and alien loading of the body.

Background
CD151 is a member of the tetraspanin superfamily. It is expressed on the surface of a variety of cell types and is involved in cellular processes such as cell motility, adhesion, proliferation, differentiation and signal transduction. CD151 is a positive regulator of tumor progression; metastasis, tumor cell growth and survival. It is over-expressed in various cancers, including non-small cell lung cancers (NSCLCs). The 5-year survival rate for NSCLC patients with CD151 gene-positive tumors has been shown to be lower than that of those with CD151 gene-negative tumors. Most CD151 studies to date have compared the cellular functions in the presence or absence of CD151 via over-expression and knockdown techniques respectively. However, the endogenous mechanisms by which CD151 protein is up-regulated in cancer cells remain unknown. The epidermal growth factor (EGF) is a distinct ligand for EGF receptors (EGFRs). Over-expression of EGFRs has been observed in more than 60% of NSCLCs. EGF is secreted by NSCLCs, causing the hyperactivity of EGFRs and the activation of its downstream signaling pathways to promote cell proliferation. In this study, we investigated the impact of EGF stimulation on CD151 protein expression in NSCLC cell proliferation.

Methods
A549 cells (a NSCLC cell line) were stimulated with increasing concentrations of EGF for 48hr and the CD151 protein abundance was measured via immunoblotting. To examine whether the effect of EGF on CD151 expression was a class-effect of growth factors, we also stimulated A549 cells with various stimulants (IL-1b, TNF-a, TGF-b1 and VEGF). To determine whether the effect of EGF on CD151 protein expression in NSCLC cells was cell-type selective, we examined the effects of EGF stimulation on CD151 protein abundance in other NSCLCs (H358, H1975), colon cancer (SW620) and breast cancer (MDA-MB-231) cell lines. Subsequently, silencing of CD151 via siRNA was carried out to investigate the effect of CD151 on EGF-stimulated A549 cell proliferation. Cell number was measured via trypan blue exclusion after 72hr of EGF stimulation, and phospho-ERK1/2 levels were examined via immunoblotting after 6hr of EGF stimulation.

Results
In A549 cells, EGF induced significant up-regulation of CD151 protein expression and this effect was not a class effect of growth factors. The up-regulation of CD151 protein by EGF was observed in more than one type of NSCLCs and in other cancer cell types. Silencing of CD151 down-regulated EGF-stimulated increase in A549 cell number and this effect was dependent on ERK1/2 phosphorylation.

Conclusion
This is the first study to show that EGF up-regulates CD151 protein expression to promote NSCLC cell proliferation, possibly via the MAPK (ERK1/2) pathway.

Background
MLPA® is a robust method that detects aberrant copy numbers in one simple PCR reaction by using one primer-pair. The SALSA® MLPA® kits P171, P172-B1 and P173 (MRC-Holland, Amsterdam, Netherlands) containing 42, 42 and 43 probes respectively for genes that often have an increased copy number in one or more types of tumours were used.

Methods
Tumor samples from 42 lung adenocarcinoma of never-smokers were studied (17 with EGFR L858R mutation, 9 with EGFR exon 19 deletions, and 16 with wild type EGFR). Normal lung parenchyma from the same individual was used as internal control. The normalization with reference controls and data analysis was done according to the MLPA® protocol and Coffalyser developed at MRC-Holland.

Results
Significant copy number gains in > 15% cases (incidence rates indicated in brackets) were reported here. For tumours with EGFR L858R mutation, significant gains were detected in EGFR (60%), RNF139 (50%), TERT (28%), NFKBIE (22%), UCKL1 (23%), MYC (18%), NTRK1 (17%), RUNX1 (17%) and SERPINB9 (17%). For tumours with EGFR exon 19 mutations, significant gains were detected in PSMB4 (50%), EGFR (45%), UCKL1 (23%), MYC (23%), MET (23%), CENPF (23%), CYP27B1 (23%), CDK4 (23%) and ERBB4 (23%). For tumours with wild-type EGFR, significant gains were detected in STK6 (25%), IRS2 (20%) and MYC (20%). Therefore, gains in MYC copy numbers were common in all 3 groups. Gains in EGFR and UCKL1 were common in the 2 groups with EGFR mutations. Gains in STK6 were common to L858R mutation and wild-type EGFR groups.

Conclusion
The 3 groups of adenocarcinoma in never-smokers with respect to the absence, presence and types of EGFR mutation showed quite different patterns of aberrant copy numbers in other cancer-associated genes. This in turn reflected the distinctively different patterns of genetic instability.

Background
Recently developed molecular targeting drugs are highly effective against various cancers, however the duration of response is limited. To overcome this problem, researchers have sought to find the resistance mechanism and identify novel targets. The majority of the drugs are related to receptors tyrosine kinase (RTKs), and recently, G-protein coupled receptor (GPCR) has also been reported to play a critical role in cancer biology. We thus attempted to identify the target GPCR for the treatment of lung cancer cells.

Methods
We analyzed 124 patients with non-small-cell-lung cancer who had undergone surgery from January 2008 through November 2011. Expression levels of GPCR mRNA in the tumor tissues and the adjacent normal tissues were examined by comparative genomic analysis. We then sought to find the relationship between clinical features and the GPCRs that showed differential expression levels between the two types of tissues. In addition, we examined the protein levels of the GPCRs by immunohistochemistry.

Results
We identified 3 GPCRs and 1 related molecule. Of the 4 molecules, serotonin receptor 2B (HTR2B) was expressed higher in tumor tissues than in normal tissues. HTR2B was expressed statistically higher in the tumor tissues of female, adenocarcinoma, and non-smokers (p=0.012. 0.001, 0.045, respectively), and tended to be expressed higher in patients who harbored EGFR mutation (p=0.086). No statistically significant differences were observed in relapse-free survival. Immunohistochemistry demonstrated that HTR2B was expressed especially in the invasive front of the tumor.

Conclusion
Differential expression of HTR2B between cancer cells and normal tissues and its invasive potential suggest that further investigation into this molecule is needed.

Background
Paclitaxel is one of the key drugs used in chemotherapy for the non-small cell lung cancer (NSCLC). Anti-microtubule agents, such as paclitaxel, stabilizes the microtubule polymer and prevents their breakdown. Data from the metastatic study suggest high tumor class III beta-tubulin (TUBB3) expression is a determinant of insensitivity to paclitaxel. To clarify whether TUBB3 is a true predictive marker for chemotherapy with paclitaxel, chemosensitivity was examined using an in vitro drug sensitivity assay.

Methods
Initially, 12 specimens were obtained to analyze the dose-response curve and to measure the median effective dose 50 (ED50) in the histoculture drug response assay (HDRA). The HDRA was perfomed for paclitaxel at several concentrations (minimum 0 μg/ml ,maximal 256μg/ml), in order to analyze the dose-response curves for individual patients. The value that was obtained from HDRA were directly applied for non-linear least square analysis using a formula of simplified dose-response curve. Subsequently, 41 surgically resected NSCLC specimens were applied to the HDRA and inhibition ratio at the concentration of 25μg/ml paclitaxel (IR25) was measured. H-scores were calculated by immunohistochemical staining. The patients comprised 26 male patients and 15 female patients. Mean age was 72±6.8. The specimens examined were 24 adenocarcinomas 17 squamous cell carcinomas.

Results
The mean (±SD) slope factor, ED50 and maximal response was 11.7±6.5, 24.6±7.73 μg/ml and 87.4±6.15% respectively. And the mean H-score was 50; (20-140). Among 12 specimens whose dose-response curve was obtained, the ED50 was higher than 25μg/ml in 5 (Resistant group) and lower in 7 (Sensitive group). The median H-score was significantly (p=0.0076) higher in Resistant group (240) than in Sensitive group (10). The mean IR25 was 53.8±26.6%. The median H-score in the specimens with IR25 above 50% (60, n=15) was significantly (p=0.0337) higher than that in specimens with IR25 under 50% (35, n=26).

Conclusion
Tumors with high TUBB3 levels exhibited chemoresistance to paclitaxel than tumors with low TUBB3 levels. HDRA revealed that TUBB3 expression is a true predictive factor for the response of paclitaxel in NSCLC.

Background
Lung cancer easily metastasizes to multiple organs, such as bone, lung, brain, liver, and adrenal gland. The frequency of the distant metastasis usually appears to be dependent on the volume of each target organ. However, metastasis to the adrenal gland is frequently observed in spite of its small volume. We hypothesized that some organotropic mechanisms exist in lung cancer adrenal metastasis. We applied microarray analysis to find the gene expression influencing organotropism of lung cancer adrenal metastasis.

Methods
Human lung adenocarcinoma cell lines PC-14, A549, and VMRC-LCD were used. Cell were cultured for 7 days on the fresh tissue slice of athymic mouse (Balb/c-nu/nu) adrenal gland. After that, proliferated cancer cells were collected from the surface of adrenal gland and cultured in the flask again. This process was repeated up to 5 times. This procedure was also applied to the other organs, which were lung, kidney, bone, and muscle, at the same time. Then the conditioned cells from each organ were obtained. Microarray analysis was applied to these cells including original cells in order to detect specific gene alteration in each organ. Expressions of 28869 genes were evaluated in each cell using microarray analysis. Gene expressions of conditioned cells obtained from each organ were compared with original cells. The genes with expression altered 1.5 fold or more were regarded as significant. Adrenal gland specific alterations were checked using unpaired t-test. The values P<0.05 were regarded as significant.

Results
Adrenal gland metastasis specific alteration was observed in 76 genes. There were 59 genes with increased expressions and 17 genes with decreased expressions. The same statistical analysis was applied to lung, liver, kidney, bone, and muscle, too. We detected 22 genes as lung metastasis specific alterations, 212 with liver, 25 with kidney, 141 with bone, and 27 with muscle. The most change in adrenal grand was increased expression of calbindin-D28k. Calbindin-D28k has anti-apoptotic properties. These properties may suppress steroid induced apoptosis and promote adrenal metastasis in lung cancer. In vitro evaluation revealed that proliferation of original PC-14 cells was inhibited by 1,4, 16, 64 µg/ml of dexamethasone in the dose-dependent manner. On the other hand, proliferation of PC-14 cells obtained from adrenal grand was not inhibited by dexamethasone in all concentrations.

Conclusion
Microarray analysis was applied to find the gene expression influencing organotropism of lung cancer adrenal metastasis. Our study results detected 76 genes as the candidates. Calbindin-D28k seemed to regulate lung cancer adrenal metastasis.

Background
Sex difference is an important factor to differentiate the clinical characteristics of lung cancers. Sex hormones derived signaling should be involved in the etiology of lung cancers. The most important factor may be estrogen that is suspected carcinogen, since strong epidemiological evidence associates the hormone to breast, endometrial, and uterine cancers. We have been studied the involvement of ATBF1 that is a large transcription factor playing an important role as a tumor suppressor in various cancers. Intriguing fact is that estrogen at lower levels increases the expression of ATBF1, but at higher levels decreases ATBF1. The response of ATBF1 is explained by the negative feedback through the estrogen-responsive proteasome system. This is the first study to reveal the expression of ATBF1 in lung cancers and explain the clinical characteristic of lung cancer differentiated by sex.

Methods
We prepared a cell line array from 17 lung cancer cell lines, which was consisted of twelve adenocarcinomas, three squamous cell carcinomas, one large cell carcinoma and one undifferentiated cancer. We generated five antibodies against ATBF1 (MB34-2, MB39-1, D1-120, MB44-2, MB47-2) at distinct part of the protein from N-terminal to C-terminal. We also analyzed the expression of p53, p21, ATM, psATM, estrogen receptors (ER), progesterone receptor (PR) , thyroid transcription factor 1 (TTF-1) and CEA by immunohistochemical analysis. The intensity of each factor was graded (score: 0-3) from weak expression (score: 0) to strong expression (score: 3). The intensity was scored in comparison with the intensity of ATB1 for other factors.

Results
Positive rates of ATBF1 with each antibody (MB34-2, MB39-1, D1-120, MB44-2, MB47-2) were 88%, 100%, 100%, 76% and 71%. Positive rates of p53, p21, ATM, psATM, ER, PR, TTF-1 and CEA were 53%, 41%, 47%, 41%, 65%, 42%, 29% and 76%, respectively. There was no significant difference in the expression pattern of each factor between adenocarcinoma and squamous cell carcinoma but TTF-1. We divided lung cancer cells into two groups by expression pattern of ATBF1. Wide range expression (W) group is characterized by the positive expression with all antibody whereas the limited expression (L) group that is characterized by limited number of positive with these antibodies. The expression rate of ER was significantly low in W group (45%, 5/11) in contrast to L group (100%, 6/6) (p=0.025).

Conclusion
The higher expression of ER, the lower and limited expression of ATBF1. The observation may be relevant to the breaking down mechanism of ATBF1 through estrogen-ER signaling discovered in breast cancer. The protein stability of ATBF1 should be an important factor for the prognosis of lung cancer distinguished by the sex.

Background
Microvasculature and microenvironment play important roles in proliferation, metastasis and prognosis in human lung adenocarcinoma, which might be altered by many anti-angiogenic drugs and cause “vessel normalization”. Epigallocatechin-3-gallate (EGCG), a natural anti-angiogenesis agent refined from green tea, was defined to have multiple effects on angiogenesis factors. So we hypothesizing that EGCG might cause “vessel normalization”, and in addition combined chemotherapy exert a synergistic effect in the tumor vessel normalization window caused by EGCG.

Methods
Build nude mice xenograft tumor model(A549 cell line). Randomly divided them into three groups (treated with saline, EGCG, bevacizumab). Test following indexes at day of 0, 2, 4, 6, 9, 12: Vessel structure: MVD, MPI; vessel GBM; Transmission-electron-microscope of microvessles; Vessel functional: perfusion function, vessel permeability; Microenvironment effect: IFP, PO2. Test cisplatin concentration in tumor tissues with different combination of EGCG and cisplatin. Treated mice with saline, cisplatin, EGCG, EGCG+cisplatin on day0 and EGCG+cisplatin on day5 and record growth delay.

Results
EGCG treated group undergoing a persisting decrease of MVD, a gradual decrease of MPI, a transient elevation of vessel perfusion function, permeability and PO2, transient decrease of IFP in tumor tissue. Full-dose cisplatin at day5 had a concentration significantly higher than Full-dose at day0 and half-dose at d5. Statistical analysis shows EGCG and cisplatin had synergistic effect as a combined anti-tumor chemotherapy. Combined treatment groups had significantly lower xenograft tumor growth rates than other three groups, and tumor growth rate in combining cisplatin on day5 was significantly lower than on day0.

Conclusion
EGCG causes vessel normalization in human lung adenocarcinoma tumor, the window is between Day 4 to Day 9. Combined therapy in this window period can escalate drug concentration in local tumor tissue, and leads to anti-tumor synergistic effect, providing a new strategy for EGCG applying as a complementary chemotherapy drug.

Background
Extracellular adenosine induces apoptosis in a variety of cancer cells via diverse signaling pathways. The present study investigated the mechanism underlying adenosine-induced apoptosis in A549 human lung cancer cells.

Methods
MTT assay, TUNEL staining, flow cytometry using propidium iodide and annexin V-FITC, real-time RTPCR, Western blotting, monitoring of mitochondrial membrane potentials, and assay of caspase-3, -8, and -9 activities were carried out in A549 cells, and the siRNA to silence the A~3 ~adenosine receptortargeted gene was constructed.

Results
Extracellular adenosine induces A549 cell apoptosis in a concentration(0.01-10 mM)-dependent manner, and the effect was inhibited by the A~3~ adenosine receptor inhibitor MRS1191 or knocking-down A~3~ adenosine receptor. Like adenosine, the A~3~ adenosine receptor agonist 2-Cl-IB-MECA also induced A549 cell apoptosis. Adenosine increased expression of mRNAs for Puma, Bax, and Bad, disrupted mitochondrial membrane potentials, and activated caspase-3 and -9 in A549 cells, and those adenosine effects were also suppressed by knocking-down A~3~ adenosine receptor.

Conclusion
Adenosine induces A549 cell apoptosis by upregulating expression of Bax, Bad, and Puma, to disrupt mitochondrial membrane potentials and to activate caspase-9 followed by the effector caspase-3, via A~3~ adenosine receptor.

Background
Malignant mesothelioma (MM) is an aggressive, fatal tumour of the pleura or peritoneum and strongly related to asbestos exposure. Malignant pleural mesothelioma (MPM) is the most common and occurs with a latency of up to 40 years. The mechanism of MM carcinogenesis is not well understood and the heterogeneity of the tumour is considered to be a major barrier to successful therapy. Several studies have identified changes in the expression and activities of defined cell signalling pathways in mesothelial and stromal cells, but the relationship between different cell types in the process of tumorigenesis has not been studied.

Methods
To examine the pro-oncogenic role(s) of different cell populations, the effect of primary fibroblasts from human mesotheliomas and activated macrophages on cellular signalling in normal untransformed mesothelial cells was monitored using imaging and immunoblotting techniques.

Results
Although the subcellular location of damage-associated molecular pattern (DAMP) protein HMGB1 remained nuclear in normal mesothelial cells co-cultured or treated with conditioned medium, the activation levels of growth modulating signalling pathways were altered in these cells. The proliferation and migration rates of normal mesothelial cells were also affected by paracrine signalling from activated fibroblasts and macrophages.

Conclusion
Thus, non-mesothelial cells instigate alterations in cellular signalling in mesothelial cells. Further integral examination of the aberrant signalling pathways, especially at early stages of neoplasia, will provide new insights into the mechanisms underlying malignant mesothelioma carcinogenesis.

Background
With the development of whole genome and transcriptome sequencing technologies, long noncoding RNAs (lncRNAs) have received increased attention. Multiple studies indicate that lncRNAs act not only as the intermediary between DNA and protein but also as important protagonists of cellular functions. LncRNAs can regulate gene expression in many ways, including chromosome remodeling, transcription and post-transcriptional processing. Moreover, the dysregulation of lncRNAs has increasingly been linked to many human diseases, especially in cancers. Here, we reviewed the rapidly advancing field of lncRNAs and described the relationship between the dysregulation of lncRNAs and human diseases, highlighting the specific roles of lncRNAs in human diseases.

Methods
not applicable

Results
not applicable

Conclusion
Continuing advances in transcriptomics indicate that lncRNAs fulfill important functions in the regulation of gene expression. With this updated view of molecular biology, the central dogma may be re-written. In this review, we have described some examples of lncRNAs involved in disease-associated processes (such as cancer initiation and progression), as well as highlighted the diverse mechanisms of lncRNAs. As with the dysregulation of miRNAs, dysregulation of lncRNAs is becoming recognized as a hallmark feature of many types of diseases. Importantly, cancer-associated lncRNAs may serve as diagnostic or predictive biomarkers of cancer and also provide a new therapeutic strategy of selectively silencing cancer-associated lncRNAs. However, compared with coding RNA and miRNA, there are still significant gaps in our current understanding of lncRNA function. Further studies are needed to elucidate the networks of proteins, miRNAs and lncRNAs.

Background
MicroRNAs (miRs) play important roles in the development and progression of human cancers. MiR-146a down-regulates epidermal growth factor receptor and the nuclear factor-κB regulatory kinase interleukin-1 receptor-associated kinase 1 genes that play important roles in lung carcinogenesis. This study was conducted to evaluate the association between rs2910164C>G, a functional polymorphism in the pre-miR-146a, and lung cancer risk.

Methods
The rs2910164C>G genotypes were determined in 1,094 patients with lung cancer and 1,100 healthy controls who were frequency matched for age and gender.

Results
The rs2910164 CG or GG genotype was associated with a significantly decreased risk for lung cancer compared to that of the CC genotype (adjusted odds ratio = 0.80, 95% confidence interval = 0.66-0.96, P = 0.02). When subjects were stratified according to smoking exposure (never, light and heavy smokers), the effect of the rs2910164C>G genotype on lung cancer risk was significant only in never smokers (adjusted odds ratio = 0.66, 95% confidence interval = 0.45-0.96, P = 0.03, under a dominant model for the C allele) and decreased as smoking exposure level increased (P~trend~ < 0.001).

Conclusion
These findings suggest that the rs2910164C>G in pre-miR-146a may contribute to genetic susceptibility to lung cancer, and that miR-146a might be involved in lung cancer development.

Background
ATM is a nuclear protein that plays a central role in the cellular response to DNA double stand breaks (DSBs), the DNA damage response (DDR). In mammalian cells, the DDR consists of co-ordinated multi-facet cellular processes, including DSB repair, cell cycle checkpoints and apoptosis which together act as barriers to tumor-genesis. Germ-line mutations in ATM result in the cancer-predisposing human disorder A-T (Ataxia telangiectasia). Inactivation of ATM due to gene mutations, epigenetic silencing and altered protein expression is found in many cancers including NSCLC. The role of ATM loss in NSCLC is incompletely defined. Early loss of ATM would be expected to cause an increased predisposition to mutation accumulation and the generation of neo-antigens that increase a tumour’s immunogenicity and lead to an increased sensitivity to PARP inhibition and radiation. We set out to determine the prevalence and significance of ATM loss in NSCLC cell lines and in resected NSCLC samples.

Methods
A panel of 7 NSCLC cell lines was screened for ATM protein expression levels using western blot. ATM signaling was investigated using specific antibodies to determine p-S1981 ATM, p-S824 KAP1 and p-S15 p53 following treatment of the cells with IR. ATM functionality was assessed by clonogenic survival assay to assess cellular viability after IR treatment. Clinical data was collected retrospectively through chart review of NSCLC patients diagnosed at the Tom Baker Cancer Centre from 2003 to 2006 and entered into the Glans-Look Lung Cancer Database. Archived formalin-fixed paraffin-embedded (FFPE) resected NSCLC tumour samples were retrieved and tissue microarrays constructed. Automated image acquisition was performed using a HistoRx PM-2000™. The ATM expression index was defined as the minimum ratio of the malignant cell specific AQUA score as compared with the non-malignant tumour-associated stromal cell specific AQUA score. Differences in the survival were compared using the log-rank test for low versus high ATM expression index groups and ATM expression groups as well as the stage subgroups. Cox proportional hazards regression was used to assess the prognostic effect of both ATM expression index.

Results
ATM deficiency was found in two NSCLC cell lines. The ATM deficient cell line, H23 demonstrates increased sensitivity to IR, disrupted ATM signaling and has the least surviving fraction of cells after treatment with single agent PARP inhibitor and topotecan. Our methodology identified an ATM-EI cutpoint of 0.716, defining 36/165 (21.8%) of patients as being ATM-deficient, many with stage I disease. After adjusting for histology, gender, age, and adjuvant treatment, the ATM-EI had no impact on stage I disease, but was a significant adverse prognostic factor for disease free survival (HR: 4.75, 95% CI: 2.02 to 11.17, p<0.001) and overall survival (HR: 5.09, 95% CI: 2.07 to 12.52, p<0.001) among those with stage II/III disease.

Conclusion
This study confirms ATM loss occurs in NSCLC cell lines and has therapeutic implications. It also demonstrates that ATM loss is seen in early stage NSCLC and is the first to show the prognostic consequences of this molecular deficiency. ATM status should be considered in designing new NSCLC clinical trials.

Background
The ataxia telangiectasia mutated (ATM) gene produces a protein essential in mechanisms responsible for regulating the controlled growth and proliferation of cells, as well as the DNA damage response. The lack of ATM expression can lead to a predisposition to cancer due to the inability for the protein to actively respond to DNA damage. Previous work in the Bebb lab has shown that the lack of ATM gene expression in non-small cell lung cancer (NSCLC) cells increases sensitivity to ionizing radiation, however little is known about whether ATM status can influence sensitivity to commonly used chemotherapeutic agents. In this study we will explore the chemosensitivity of NSCLC cells in relation to their ATM status, as well as the synergistic effects of combining multiple agents to help develop new treatment strategies for patients with low or absent ATM expression.

Methods
Several NSCLC cell lines were screened to identify the ATM and p53 status. Of the cell lines screened, four NSCLC cell lines: NCI-H460, NCI-H226, NCI-H23, and NCI-H1395 were chosen to treat with various chemotherapeutic agents. Optimization of cell proliferation for each cell line was conducted to determine the appropriate concentration of cells that allows for continued proliferation after a 72 hour period. Each of the agents used for cytotoxicity assays target a different mechanism of the cell, including topoisomerase I inhibitors, DNA damaging and alkylating agents, and mitotic and cell cycle inhibitors. The Chou-Talalay method for drug combinations was utilized as the combination index theorem provides a quantitative definition for examining additive, synergistic and antagonistic effects with our primary focus of observing synergy.

Results
Several NSCLC cell lines were characterized for their ATM status, and four were selected for cytotoxicity assays: H460 and H226 (ATM normal), and H23 and H1395 (ATM deficient). We observed a noticeable difference in growth rate between cell lines in addition to the differences in ATM and p53 status. Similarly, the cytotoxic response to the chemotherapeutic agents varied greatly, and the relationship of drug sensitivity to ATM status in the cells, as well as the synergistic response to combinational therapy will be presented and discussed.

Conclusion
The results of this study have several important implications. Our previous work has shown that resected, early stage NSCLC patients with low ATM expression have worse overall survival, however our demonstration that ATM deficient cell lines are more sensitive to cancer therapy could indicate that these same patients would respond better to certain lines of treatment. Similarly, increased sensitivity to combinational therapy will help mitigate detrimental side effects, leading to better quality of life during treatment periods.

Background
Lung cancers in smokers and never smokers (NS) are distinct clinical diseases. Specific molecular differences identified in these two groups include: EGFR and KRAS mutation, DNA methylation levels at specific loci, and most recently, global mutation spectra. However, much remains to be understood about the biology driving lung tumourigenesis in smokers and NS in order to improve treatment outcome. To date, no multi-dimensional integrative genomics (i.e. multi-omics) analysis designed to specifically compare current (CS) and NS lung tumours has been performed. We hypothesize that a multi-omics analysis which considers each tumour as its own unique perturbed system (as opposed to a grouped approach) will reveal molecular mechanisms of lung adenocarcinoma (AC) biology that are common or different in CS and NS.

Methods
Copy number, DNA methylation, and gene expression profiles were generated for lung AC and matched non-malignant lung tissues from 34 CS and 30 NS. PCR was performed to determine EGFR and KRAS mutation status. Copy number, methylation and expression alterations were integrated for 14,000 genes on an individual tumour basis. Disrupted genes were ranked according to the magnitude of alterations they exhibited using a novel algorithm we developed denoted MITRA. Of the genes scored by MITRA, those ranking in the 99th and 1st (top) percentiles for up- and downregulation, respectively, were subjected to Ingenuity Pathway Analysis (IPA). IPA was performed separately on all 64 lung tumours and pathway results for CS and NS were compared.

Results
We identified 361 genes that ranked in the top percentiles for up- or downregulation in at least 20% of the lung ACs we assessed. Identification of recurrent RASSF1A downregulation, and EGFR upregulation predominantly in NS demonstrates the ability of our ranking algorithm to prioritize genes known to be involved in lung tumour biology using multi-dimensional genomics data. To determine cellular pathways and functions likely deregulated as a consequence of gene disruption, we performed IPA on each tumour and determined the frequency of individual pathway disruption across tumours. This analysis revealed 88 annotated pathways with a minimum disruption frequency of 15% in either or both CS and NS. Commonly affected pathways involved: adhesion and extravasation implicating tumour invasion and migration; various catabolic and anabolic processes implicating cell metabolism; and several specific signaling pathways including atherosclerosis and Wnt/β-catenin signaling implicating inflammation and cell proliferation. Comparison of the pathways identified in CS and NS revealed 13 differentially disrupted pathways (Fisher's Exact test p < 0.05 and disruption frequency difference > 15%). Eleven pathways were preferentially disrupted in CS and affected metabolic, immune response, and inflammatory pathways. Anandamine degradation and ephrin receptor signaling were preferential to NS.

Conclusion
Our novel, multi-omics tumour system based approach revealed genes prominently disrupted in CS and NS lung AC which were associated with several cellular pathways commonly or differentially disrupted in these two groups. Pathways affected by genes disrupted at both the DNA and RNA level may contribute to the distinct clinical characteristics associated with CS and NS lung cancer and may serve as targets for intervention.

Background
In resected lung cancer, no reliable clinical or molecular predictors are currently available for identifying patients with high risk for developing recurrent disease. In a previous study, we compared gene expression profiling from adenocarcinoma specimens and normal lung tissue with non relapse (NR) and early relapse (ER), using Affimetrix human microarray HG-U133Plus 2.0. We selected 5 genes up-regulated and 4 genes down-regulated were predictive for clustering patients in ER and NR (Siggillino et al. ECCO 2011). Here, we validate our results using an independent cohort of patients with lung adenocarcinoma stage I to identify novel genes involved in the risk of ER compared to NR disease.

Methods
From tissue banking of 180 consecutive resected NSCLC stage I patients at two Italian institutions, we selected 58 frozen specimens of lung adenocarcinoma tissue with corresponding normal lung. Total RNA was isolated from tumor and normal lung specimens using RNA Universal Tissue Kit and automatically purified by Biorobot-EZ1 instrument (Qiagen). Quantification of mRNA expression levels of 9 genes (5 up-regulated: CLCA2, FABP3, H19, TFPI2, AKR1B10 and 4 down-regulated: CYP3A5, ALDH3A1, SCGB3A2, SCGB1A1, were analyzed by real-time one-step RT-PCR using QuantiFast technology by RotorGeneQ instrument (Qiagen), and the results were compared considering β-actin as the internal reference gene and as calibrator the pool of normal tissues of analyzed patients. The gene expression of all evaluated genes and their association with relapse disease measures were assessed by t-test and logistic regression model was used for multivariate analysis.

Results
Fifteen-eight adenocarcinoma stage I patients were evaluable, 17% of which had an ER. Patients characteristics were as follows: median age was 65.8 years (38.7-81.5), 67.2% were male, 91.3% were PS 0, 70.7% were ever-smokers. The expression median values of 9 genes: CLCA2, FABP3, H19, TFPI2, AKR1B10, CYP3A5, ALDH3A1, SCGB3A2, SCGB1A1 were 0.30, 0.71, 0.34, 1.10, 0.26, 0.24, 0.42, 0.53, 0.09, respectively. Among all genes evaluated, the NR vs ER mean expression levels of two genes down-regulated (CYP3A5, 1.09 vs 0.30; SCGB3A2, 2.28 vs 0.98) and two genes up-regulated (AKR1B10, 4.53 vs 34.20; FABP3 1.25 vs 1.55) were superimposable respect to the results of previous microarray analysis. The median disease free survival (DFS) and overall survival (OS) were 21 and 23 months, respectively. In the logistic multivariate analysis the mean expression levels of all genes showed a tendency to predict the ER in the overall population (p=0.07). Nevertheless considering only the expression levels of genes (FABP3, H19, TFPI2, AKR1B10, CYP3A5, SCGB3A2) identified as significant with t-test, the covariates in multivariate analysis increased their capacity of ER prediction (p= 0.028).

Conclusion
Our results indicate that it is possible to define, through gene expression, a characteristic gene profiling of early relapse tumor patients with an increased risk of relapse disease. The contemporary expression levels of 6 genes (FABP3, H19, TFPI2, AKR1B10, CYP3A5, SCGB3A2) predicted a worse DFS. Such features may have important implications for future targeted therapies. We thank Italian Association for Cancer Research (AIRC) for supporting the study.

Background
Approximately 25% of lung cancers occur in lifelong never smokers. Although no dominant risk factor has been identified yet, the discover of molecular drivers potentially targetable with biological agents, makes lung cancer in never smokers a unique disease, candidate for a personalized therapy. Through the FISH test, we performed a screening for ALK, ROS1, and RET rearrangements, in a highly selected population of lung adenocarcinoma never smoker patients, previously demonstrated to be wild-type for EGFR and K-RAS mutations.

Methods
We collected archived histological material of 28 EGFR and K-RAS wild-type patients (pts), from a 200 never-smoker advanced lung adenocarcinomas database, to be analyzed for the presence of rearrangements in ALK, ROS1 and RET genes. All pts were treated at the Division of Medical Oncology of the S Maria della Misericordia Hospital in Perugia from October 2003 to February 2013. 20 specimens were included in a tissue microarray (TMA) analysis, whereas 8 were screened in separate subset, due to the scarce samples. FISH test was performed using a combination of commercial reagents and custom designed probes. Median overall survival (OS) of mutated pts compared to the pan-negative ones, was evaluated by Cox multivariate analysis.

Results
Clinicopathological characteristics: among the 28 patients, 27 were never smokers and 1 former light smoker, with a good performance status; 20 (72%) presented with a metastatic disease at diagnosis, 8 (28%) were locally advanced; median age was 56 years-old, with a predominance of female sex (18/28, 64%). All cases were invasive adenocarcinomas and classified into 18 (64%) solid predominant type, 1 (3.5%) mixed acinar/lepidic pattern, 1 (3.5%) papillary, no predominant subtype for 8 (28%) patients, because of unsufficient histological material available. Of the 28 never smoker cases, we identified 7 gene fusions (25%), including 2 pts ALK+ (7.1%), 3 pts ROS1+ (10.7%) and 2 RET+ cases (7.1%), one compatible with KIF5B:RET and other with CCDC6:RET fusion. Median OS for the entire cohort was 24.5 months (mo), 61.2 mo for mutated pts (any rearrangement) vs 24.1 mo for not-mutated, respectively (P = .292).

Conclusion
Molecularly selected never smoker lung adenorcinomas associates with a high incidence of driver genes mutations and further investigations to confirm our frequencies in larger cohorts are needed. In line with literature data, our findings suggest a different survival outcome among genotypes, and identification of specific subsets in this special population can lead to successful treatment with target therapies.

Background
Lung cancer is the leading cause of cancer-related deaths worldwide, where non-small cell lung cancer (NSCLC) accounts for 85% of cases. While cisplatin-based chemotherapy remains the gold standard treatment for lung cancer, response rates are low due to increasing development of resistance to cisplatin. Circumventing cisplatin resistance, therefore, remains a critical goal for anti-cancer therapy. The aim of this study is to examine the role of miRNA’s in the regulation of cisplatin resistance in NSCLC using a panel of isogenic cisplatin resistant NSCLC cell lines developed in our laboratory, and to examine the putative cancer stem cell markers within this chemoresistant phenotype.

Methods
MicroRNA profiling of a panel of isogenic cisplatin resistant (CisR) NSCLC cell lines, and age-matched parent cells (PT) was carried out using a 749 miRNA in-situ hybridisation array platform (Nanostring Technologies). The miRNA signature obtained was validated by qPCR using miRCURY LNA[TM ]Universal RT miRNA PCR technology (Exiqon). In order to determine the role of these miRNA’s in conferring cisplatin resistance, transfection of cell lines using miR-specific antagomirs and pre-miR’s will be carried out to examine this effect on sensitising lung cancer cells to cisplatin using clonogenic survival assays, apoptosis (APC), proliferation (BrdU) and DNA damage repair (γH2AX) assays. Furthermore, the characterisation and potential role of exosomes and exosomal-derived miRNA in modulating the cellular response to cisplatin chemotherapy will also be examined in this model of resistance. Putative stem cell markers (Oct-4, Sox-2, Nanog, SSEA4, Klf-4 and c-Myc) will be assessed in holoclones derived from resistant sublines. An in vivo model will be used to The tumourigenic potential of putative cancer stem-like cells within the cisplatin resistant population will be investigated in vivo using NOD/SCID mice. In parallel, asymmetric division assays will also be used.

Results
MicroRNA profiling of MOR, H460, A549, SKMES-1 and H1299 cisplatin resistant cell lines deduced a 3-miR signature across all five chemoresistant cell lines. Validation of this miR signature by qPCR showed differential expression of only one miRNA, miR30-c. miR-30c was significantly up-regulated (15-40 fold) in MOR, H460, SKMES-1 and H1299 CisR NSCLC cell lines and significantly down-regulated (5-fold) in A549 CisR cells relative to PT cells. The effects of miR-30c antagomirs and pre-miR’s in reversing the cisplatin resistant phenotype of NSCLC cells are currently being examined.

Conclusion
Differential expression of 3 specific miRNA’s was demonstrated in CisR lung cancer cells, relative to their parent counterparts, using an in-situ miRNA profiling hybridisation platform. While validation of this panel of miRNA’s identified only one differentially regulated miRNA species, miR30-c, further studies are warranted to explore the role of miR-30c in the cisplatin resistance phenotype of NSCLC. Exosome and cancer stem cell studies are currently under investigation.

Background
Lung cancer remains a leading cause for cancer mortality worldwide. A key feature of lung cancer development is genomic instability resulting from an accumulation of DNA lesions. In normal settings, these DNA lesions, such as double strand breaks and oxidised DNA, are rapidly repaired to prevent cytotoxicity and loss of genetic information. However, the molecular basis for the loss of genome integrity during cancer development remains to be determined. Herein, we have examined the involvement of hSSB1 in maintenance of genome stability and its potential role in lung cancer progression. hSSB1 is a critical component of the repair of DNA double strand breaks. As part of this study, we examined if hSSB1 is also involved in the repair of oxidative stress-induced DNA modifications. The most common nucleotide modification, 8-oxo-7,8-dihydro-guanine (8-oxoG), is repaired by the enzyme OGG1. Failure to repair these modifications results in mismatch mutations which are common in cancer. Therefore, understanding the molecular basis for genomic instability is key to the development of future therapeutics with clinical relevance.

Methods
To determine if hSSB1 expression is associated with lung cancer progression, a tissue microarray (TMA) with cores from 550 patients was stained with an anti-hSSB1 antibody. Kaplan-Meier survival curves were generated with the clinical data and patient prognosis was correlated with hSSB1 expression levels. To determine an in vitro association with lung cancer, A549 lung cancer cells were treated with hSSB1 specific siRNA. Cell survival was determined by microscopy and MTT assay. To examine the role of hSSB1 in repair of oxidised DNA, U2OS cells were treated with 500 µM H~2~O~2~. Cells treated with H~2~O~2~ were fixed, stained with antibodies for hSSB1 and 8-oxoG and examined by deconvolution microscopy. Lysates were collected from H~2~O~2~ treated U2OS cells and subjected to immunoprecipitation and Western blot analysis. Genomic DNA isolated from scrambled or hSSB1 siRNA U2OS cells treated with H~2~O~2~ were immobilised on nylon membrane and stained with antibodies for 8-oxoG.

Results
Significantly, TMA staining of lung cancer tissues indicated universal overexpression of hSSB1. Survival curves generated from the patient data indicated a poorer prognosis for patients with increased hSSB1 expression versus lower expressing tumours. Interestingly, in vitro inhibition of hSSB1 using siRNA significantly reduced cell survival. These data highlight the potential prognostic value of hSSB1 expression. As oxidative stress is prevalent in lung cancers, we also tested whether hSSB1 is capable of repairing 8-oxoG DNA lesions. Following oxidative DNA damage, hSSB1 localises rapidly to chromatin. hSSB1 also directly interacts with OGG1 to facilitate OGG1 recruitment to chromatin and repair of DNA damage. Importantly, cells lacking hSSB1 display ineffective repair of 8-oxoGs.

Conclusion
Our data highlight a potential role for hSSB1 in lung cancer progression and a novel role in maintenance of genome integrity. As tumours have increased genomic instability, elevated hSSB1 expression in lung cancer may enable tumours to cope with genomic instability. Taken together, these data present hSSB1 both as a prognostic marker and a novel therapeutic target.

Background
Lung cancer is the leading cause of cancer-related death worldwide. Nowdays, the histological subtypes of lung cancer and corresponding genetic polymorphisms play an important role in deciding the treatment options. KCTD11, a novel tumor suppressor, acts on antagonizing Hedgehog (Hh) signaling pathway . The deregulation of KCTD11 might activate glioma-associated oncogene homolog 1(Gli) transcription factors and lead to tumorigenesis. The present study was designed to analyze expression of the KCTD11 in different pathological lung cancer cell lines and lung cancer tissues, also the mechanism of KCTD11 inactivation and the possible affected downstream signal pathway.

Methods
Lung cancer cell lines and tissues were used to detect the expression of KCTD11. The clinical significance was analysed.Methylation detection was completed. KCTD11 vectors were transfected to squamous cell lung cancer cell lines to detect the Gli1 expression.

Results
The expression of KCTD11 was detected in Small airway epithelial cell (SAEC) and 14 lung cancer cell lines. RT-PCR showed reduced expression in lung cancer cell lines, especially in small cell lung cancer cell (SCLC) lines and squamous cell lung cancer cell (SCC) lines compared with SAEC. In all 14 tested cell lines (5 adenocarcinoma, 2 large cell, 3 SCC and 4 SCLC), we found these CpG islands were densely methylated in 3 of 4 SCLC cell lines, 2 of 3 SCC cell lines and 1 of 2 large cell lung cancer (LCC) cell linesbut none of the 5 adenomacarcinoma cell lines and SAEC. TMA staining showed significantly lower expression of KCTD11 in SCC lung cancer tissues than in normal tissues (24.2% vs 87.5%, p=0.000) .Also 5 of 10 SCC tissues compared with 1 of 12 adenocarcinoma showed methylation status. IHC analysis showed that there was 44.1% (55/118) positive Gli1 expression in group of negative KCTD11 expression. Correlation analysis showed they might exist marginally negative correlation (r=-0.165, p=0.052). H2170 cell lines with KCTD11 plasmid transfection showed significantly reduced Gli-1 expression compared with control plasmid transfection. (picture 1) Figure 1

Conclusion
In summary, we found the reduced KCTD11 expression in lung cancer. Hypermethylation was the important mechanism of KCTD11 epigenetic change in non-adenocarcinoma lung cancer cell lines and tissues. KCTD11 silencing resulted in the high expression of Gli1, a downstream mediator of Hh signaling pathway, and might affected the tumorigenesis and development in lung cancer. Targeting the KCTD11 treatment and thus controlling the Hh pathway might a new treatment target in non-adenocarcinoma lung cancer.

Background
Lung cancer is a highly lethal disease, and is believed to develop through a multistep carcinogenic process, which involves numerous genetic and epigenetic alterations. Among these alterations, mutations in “driver genes” such as KRAS and EGFR are found in non-small cell lung cancer (NSCLC) and they are demonstrated to contribute to a phenomenon, oncogene addiction. Recently, the EML4-ALK (echinoderm microtubule-associated protein–like 4 anaplastic lymphoma kinase) fusion gene has been discovered as a novel driver gene in a subset of NSCLC. We evaluated the oncogenic transformation ability of EML4-ALK by using an hTERT/CDK4-immortalized normal human bronchial epithelial cell (HBEC) model.

Methods
We used two HBEC lines, HBEC3 and HBEC4. Mutant KRAS[V12]-expressing HBEC was used as a positive control for oncogenic transformation. A lentiviral vector system was used to generate HBECs stably expressing EML4-ALK. EML4-ALK protein expression was confirmed by westernblotting, and downstream pathways were analyzed by westernblotting with phospho-specific antibodies. Malignant phenotypes of EML4-ALK-expressing HBECs were examined by WST-1 proliferation assay and liquid and soft agar colony formation assays.

Results
Westernblotting analysis showed that EML4-ALK was expressed in HBECs. Analysis of downstream pathways did not show significant differences between EML4-ALK-expressing and control HBECs. Introduction of EML4-ALK in HBECs increased the number of soft agar colonies but its effect was not as strong as KRAS[V12].Figure 1 A. Soft agar colony formation assay showing that EML4-ALK increased the number of colonies compared to control cells to a lesser extent than did KRAS[V12]. B. Cell proliferation assay (MTS-1) showing no significant difference between EML4-ALK-expressing and control HBECs.

Conclusion
EML4-ALK alone did not induce dramatic oncogenic changes in HBECs. To acquire more malignant phenotype, additional genomic alterations may be required and this is now under investigation.

Background
RANKL is an essential mediator of osteoclast differentiation, function, and survival. Tumor cells can induce RANKL expression in the bone stroma, causing activation of osteoclasts, leading to bone breakdown and subsequent tumor growth. In mouse models of lung cancer bone metastasis, RANKL inhibition by OPG-Fc can prevent tumor-induced osteolysis, decrease skeletal tumor burden, and increase survival. The effects on disease progression and survival may be explained by direct effects on the tumor in addition to indirect effects via osteoclast suppression. Recent clinical studies in patients with advanced cancer, including lung cancer, showed that RANKL inhibition with denosumab reduced the risk of skeletal-related events. Denosumab also improved overall survival compared with zoledronic acid in patients with lung cancer. In addition to the well-defined expression in the bone compartment, RANK and RANKL expression has also been demonstrated in human lung cancer cell lines and the functional expression of RANK has been confirmed through the demonstration of RANKL-dependent responses. The current study assessed the expression of human RANK and RANKL in human primary lung cancer samples in order to gain a potential mechanistic understanding of these clinical observations.

Methods
RANK and RANKL expression was analyzed in a panel of human primary lung cancer samples. Specific, monoclonal antibodies against human RANK (N-1H8, N-2B10; Amgen) and human RANKL (M366, AMG161; Amgen) were validated and optimized for immunohistochemistry (IHC) and used for expression analysis. mRNA was quantified using RT-PCR. Expression of RANK and RANKL was determined by IHC in the carcinoma element, tumor adjacent normal lung, and infiltrating cells. Incidence was scored as a positive IHC signal (any intensity). The specificity of the antibodies was substantiated by concordant signals observed using multiple independent analyses, including IHC, flow cytometry, and Western blots of positive and negative control cells and xenograft samples.

Results
Analysis of primary human lung cancer using IHC demonstrated RANK staining in the tumor epithelium of 9/16 (56%) non-small cell lung cancer (NSCLC) adenocarcinomas, 9/26 (34%) squamous cell carcinomas (SCC), and 5/10 (50%) small cell carcinomas (SCLC). RANKL staining was observed in the tumor epithelium of 12/16 (75%) adenocarcinomas, 5/26 (19%) SCC, and 3/10 (30%) SCLC. RANK and RANKL were also observed in infiltrating macrophages and lymphocytes, respectively, within the majority of tumors examined. In addition, RANKL expression was frequently observed in type II pneumocytes of the alveoli, Clara cells of the terminal bronchioles, and in lymphocytes within the bronchus-associated lymphoid tissue (BALT); RANK expression was observed in alveolar macrophages proximal to the tumor and in M-cells of BALT. Analysis of mRNA levels indicated significantly greater levels of both RANK and RANKL in primary human NSCLC as compared with normal lung.

Conclusion
RANK and RANKL expression was observed in the epithelial carcinoma element in human primary lung cancer. Whether the expression of RANK and/or RANKL on lung cancer will directly contribute to tumor progression and/or metastatic activity and whether RANKL inhibition has a potential direct anti-tumor effect in lung cancer beyond the well established bone-targeted mechanism remains an objective of ongoing research.

Background
Rearrangement of the proto-oncogene RET is a newly identified potential driver mutation in lung adenocarcinoma. Clinically available tyrosine kinase inhibitors (TKIs) such as sunitinib, sorafenib, and vandetanib target RET kinase activity, suggesting that the patients with RET fusion genes may be treatable with a kinase inhibitor. However, the mechanisms of resistance to these agents remain largely unknown. Cancer cell microenvironments can critically affect cancer cell behaviors, including drug sensitivity. We determine whether microenvironmental factors trigger RET inhibitor resistance in LC-2/ad cell with CCDC6-RET fusion gene.

Methods
We investigated the effects of epidermal growth factor (EGF) and hepatocyte growth factor (HGF) on the susceptibility of CCDC6-RET lung cancer cell line to RET inhibitors (sunitinib, sorafenib, vandetanib and E7080)

Results
CCDC6-RET lung cancer cell was highly sensitive to RET inhibitors. EGF receptor (EGFR) ligand, EGF, activated EGFR and triggered resistance to sunitinib, E7080, sorafenib and vandetanib by transducing bypass survival signaling through Erk1/2 and Akt. The resistance to RET inhibitors was not induced by EGF in EGFR siRNA-treated cells. EGFR-TKI (gefitinib) resensitized cancer cells to RET inhibitors even in the presence of EGF. Endothelial cells, which were known to produce EGF, decreased the sensitivity of CCDC6-RET lung cancer cell to RET inhibitors, an effect inhibited by anti-EGFR antibody (cetuximab). HGF, MET receptor ligand, affected a drug response of sunitinib, not sorafenib, vandetanib and E7080.

Conclusion
Paracrine receptor activation by ligand from the microenvironment may trigger resistance to RET inhibition in CCDC6-RET lung cancer cells, suggesting that receptor ligands from microenvironment may be additional targets during treatment with RET inhibitors. LC-2/ad cell line was sensitive to E7080, which inhibited RET and its downstream targets. E7080 and other RET inhibitors may provide therapeutic benefits in the treatment of RET-positive lung cancer patients.

Background
Malignant pleural mesothelioma (MPM) is a rare aggressive cancer of the pleura. Asbestos exposure (through inhalation) is the most well established risk factor for mesothelioma. The current standard of care for patients suffering from MPM is a combination of cisplatin and pemetrexed (or alternatively cisplatin and raltitrexed). Most patients however, die within 24 months of diagnosis. New therapies are therefore urgently required for this disease. The Succinate Dehydrogenase Complex, Subunit B, IronSulfur Protein (SDHB), is a subunit of the Succinate dehydrogenase or succinate-coenzyme Q reductase (SQR) or respiratory Complex II, and has recently been identified by us as an important element in MPM.

Methods
A panel of MPM cell lines inluding the normal pleural cells LP9 & Met5A were screened for expression of SDHB by RT-PCR. Levels were subsequently examined in a cohort of snap-frozen patient samples isolated at surgery comprising benign, epithelial, biphasic, and sarcomatoid histologies by RT-PCR and western blot. Finally the expression of SDHB in a large cohort of MPM specimens with clinical data was asessed by IHC.

Results
Expression of SDHB occurs in all cell lines. Significantly higher expression of SDHB is observed in the malignant tumour material versus benign pleura. There was a trend towards better survival for patients expressing higher levels of SDHB, but this was not statistically significant.

Conclusion
SDHB, a key member of oxidative energy metabolism is significantly altered in MPM. This may have important future implications for the management of MPM.

Background
Environmental factors contributed toward lung carcinogenesis. Tobacco-smoking is the major etiological factor related with lung cancer in 90% in the world compared with Mexico with 66% of cases. In previous works we have demonstrated that other factors such as chronic exposure to wood smoke (WSE) are related to non-small-cell lung cancer (NSCLC) and it´s clinically and pathologically different from lung cancer arisen from tobacco-exposure, regarding on tumor histology, mutation profiles, brain metastasis incidence, response rate and overall survival. This work is aimed to estimate expression profiles related WSE on adenocarcinoma histology associated to pathological mechanisms that differ from other carcinogenic factors, such as tobacco exposure.

Methods
This study used clinical, longitudinal, prospective and observational. From January 2008 to June 2011, patients were admitted in Cancerology Institute with lung adenocarcinoma in stage IV, eligible for the inclusion. Clinical variables, WSE, gender and age. WSE was defined to being exposed to fumes by burning wood in fireplaces and wood stoves for five years for at least 4 hours per day. The WSE index was calculated multiplying the number of daily hours exposed by years of exposure. Collaboration agreements with the Institute of Medical Genomics and approved within the Health Research Sectorial Fund, CONACyT-México (SALUD-2009-01-115552). Primary biopsies were taken by guided tru-cut needle by tomography. Samples were analyzed by the Pathology for histological/quantification of neoplasic celularity, and stored at -80°C. RNA was extracted from tumor biopsies. RNA integrity (RIN>6) was analyzed using the Agilent 6000. We used an Affymetrix Human GeneChip® 1.0 ST. Two-Cycle Target Labeling was followed for microarrays. Analysis was done by Affymetrix console and “R” software language. Matrices employ 29 microarrays with experimental contrasts. Differential expressed genes were analyzed by linear model that analyze contrasts between experimental contrasts. The Partek Genomic Software 4 and SAM (Significant Analysis) was used for microarray comparisons and integration of genomic data.

Results
Using computational genomics using fold changes and confiability data we found differences in genomic expression. In brain metastasis (BM) patients the Fold Change values >1 shows 6 upregulated and 11 downregulated genes compared with or without WSE. Patients without BM the Fold Change values >1 shows 90 downregulated and 7 upregulated genes with or without WSE. Prior both analysis were confirmed with B statistics data (significant value ≥0). No difference were found using computational Genomics SAMS (False discovery rate), neither PARTEK (p-value, fold change), on gene expression evaluation.

Conclusion
Our results demonstrate that gene expression profiles are different in advanced lung adenocarcinoma patients with brain metastasis with or without WSE. These results could be used for predictive models related with BM in advanced lung cancer. The best genomic statistical value was obtained with Affymetrix console and “R” software language in this work. These results must be confirmed by increasing the experimental samples and validating in an independent cohort by PCR.

Background
Acquisition of ability to uncontrolled migration is one of the fundamental properties of cancer cells, enabling them to infiltrate tissues and metastasize. PAI-1 is the major physiological inhibitor of urokinase (uPA), which plays a key role in migration and invasion of tumor cells.

Methods
The aim of present study was to analyze the impact of increasing concentrations of PAI-1 mutated forms: VLHL PAI-1 with very long half-life time, Vn neg PAI-1 - devoid of affinity towards vitronectin and wPAI -1 on lung (A549, NCI-H1299) and prostate (LNCaP, DU145) cancer cells invasive activity. Selected cell lines are characterized by different (normal and high) urokinase production.

Results
No effect of PAI-1 proteins on invasiveness of lung cancer cells was observed, while dose-dependent significant inhibition was demonstrated in both prostate cancer lines (DU145 and LNCaP) cultured with VLHL PAI-1 (respectively p<0,05 and p<0,01) and Vn PAI-1 neg (p<0,05). Not surprisingly wPAI-1 significantly stimulated prostate cancer cells invasiveness in all concentrations.

Conclusion
PAI-1 inhibitory effect on prostate cancer invasive activity is associated with anti-proteinase activity. Lung cancer cells invasiveness regulation seems not to be PAI-1-urokinase regulated.

Background
Lung cancer is the first cause of death among all cancers in the world and every year more than 1.6 million patients die from lung cancer. In Polish population morbidity and mortality from lung cancer persist in the first location and amount respectively 21.4% and 31.2% of all cancers. Over the last 30 years the development of targeted therapy significantly influenced the routine practice in pathology. The treatment based on identification of the type of tumor is still crucial information however the importance of predictive or prognostic molecular markers has become significant step in the choice of treatment. Identification of mutations in the EGFR gene in non-small cell lung cancer (NSCLC) fully illustrates the impact of molecular biology in treatment decisions. The use of one of the small molecule tyrosine kinase inhibitors, gefitinib or erlotinib depends on confirmation presence of activating somatic EGFR mutation.

Methods
The aim of the study was to evaluate the EGFR mutations in two types of material cytological and histological. 189 histological, paraffin-embedded materials as well as 12 fresh and 72 fixed cytology specimens obtained by trans-thoracic, computed tomography supported biopsy or during bronchofiberoscopy were included. Material and methods. 273 patients with confirmed NSCLC were entered into the study. Each specimen contained at least 50% of tumor cells. The macrodissecion was performed on histological specimens to maximize tumor cells content. DNA was extracted from both types of material and the EGFR mutation was analyzed in exons 18, 19 20 and 21 using the direct sequencing method

Results
The mean age of patients was 61.5 years (range 25 – 84 years) with males predominance (151 males vs. 122 females). The histological types included: adenocarcinoma 186 (69%), squamous cell carcinoma 44 (16%), NSCLC, not-otherwise specified 30 (11%), large cell carcinoma 9 (3%) and adenosquamous carcinoma 4 (1%). Both types of material were equally sufficient to evaluate EGFR mutations. The ratio of molecularly non-diagnostic material due to DNA degradation or too scant DNA probe was respectively for cytological and histological material 1.2% vs. 4.75% (p<0.01). The percentage of EGFR somatic mutations were 10.62%. Females suffered more frequently from adenocarcinoma (females 70.49% vs. males 66.23%, p <0.01) and had significantly higher rate of EGFR mutations (females 17.21% vs. males 3.97%, p<0.01). Mutations in exons 21 and 19 together accounted for 87% of all types of mutations of the EGFR gene. Apart from these, two patients had co-existence of activating/inhibiting mutations: L858R (exon 21) with T790M (exon 20) and G719C (exon 18) with S768I (exon 20).

Conclusion
This study allows for the first time to estimate an EGFR mutation frequency in Polish population. Moreover the results have strengthened the importance of reusing cytological material in molecular evaluation. Cytological material recovered from fixed preparations and stained with hematoxylin and eosin showed comparable DNA quality to fresh tumor cells. The cytology especially fine needle aspirates as well as small biopsy material provide molecular biological information which is a key issue for the targeted treatment options.

Background
NLBP (novel-LZAP binding protein, also known as KIAA0776 or Maxer) was originally indentified as a reciprocal binding partner of LZAP. LZAP possesses dual functionality as a tumor suppressor and an oncogene in human cancers. Despite its strong association with LZAP, the biological roles and underlying molecular mechanisms of NLBP remain unknown. Recent studies indicating a possible link between p120 catenin (p120ctn) and NLBP in tumorigenesis.

Methods
We examined NLBP expression in 29 non-small cell lung cancers using immunohistochemical staining and immunoblotting, as well as an in vitro analysis of NLBP activity during cell proliferation in lung cancer cell lines. We next examined whether p120ctn plays a role in the molecular mechanism underlying NLBP activity

Results
In this study, we found that NLBP expression was increased in human lung adenocarcinomas, particularly early-stage adenocarcinomas, compared to squamous cell carcinomas. Furthermore, the overexpression of NLBP in H1299 cells resulted in an increase in cell proliferation. We next identified p120ctn as a novel NLBP-binding protein, and identified the reciprocal binding regions of these proteins in lung cancer cell lines. We also demonstrated that NLBP promotes cell proliferation by regulating the stability of p120ctn. This effect is mediated through the inhibition of ubiquitination, which occurs as a result of NLBP binding to p120ctn, leading to an increase in protein stability. Figure 1

Conclusion
In conclusion, the data presented here show that NLBP may act as a novel oncogene in the early stages of lung adenocarcinoma, and may promote cell proliferation in lung adenocarcinoma through interactions with p120ctn. The interaction between NLBP and p120ctn provides new implications for interplay between signaling pathways and protein networks in tumor development

Background
Among many molecular markers associated with tumor progression, the Wnt family has been shown to encode the multifunctional signaling glycoproteins that are involved in the regulation of a wide variety of normal and pathological processes in lung epithelium. Defects in Wnt signaling are associated with several tumor types, including lung cancer. Numerous reports have demonstrated aberrant Wnt (wnt-1, 2, 5a, and 7) activation in lung cancer. The Wnt signaling pathway has been extensively investigated in NSCLC, but not in lung carcinoid tumors.

Methods
Sixty formalin-fixed paraffin embedded typical (TC) and atypical (AC) human lung carcinoid tumor samples were analyzed by qRT-PCR for Wnt-1, 5a, 7a, 10b, 13 and Fz2 and Fz5 gene expression as potential tumor-associated markers, using SYBR Green-based qRT-PCR.

Results
The heatmap (Figure) shows low expression of Wnt genes (Wnt-1, 5a, 7a, 10b and 13) in almost all samples analyzed. Otherwise, Wnt-ligands are frequently positive, strongly positive for Fz2 and with lower levels for Fz5 in both carcinoid types (AC and TC). Wnt-1 and 13 expression was found negative in all TC and AC samples; Wnt-7a was expressed in 0% AC and 4.55% TC; Wnt -10b was expressed in 0% AC and 11.36% TC; and Wnt-5a was expressed in 18.18% AC and 20.45% TC. Regarding Wnt receptors, Fz2 was positive in 90.9% AC and 95.45% TC, and Fz5 was positive in 45.45% AC and 20.45% TC. Figure 1

Conclusion
In the current study, we assessed the clinical-pathological implications of changes in Wnt expression across a serie of lung carcinoids. Our data indicate that Wnt gene family is scarcely expressed both in TC and AT lung carcinoids. Conversely, ligands (Fz2, Fz5) are positively expressed in both types of lung carcinoids. Whereas the Wnt pathway has been shown to have a role in NSCLC, there are limits on the contribution of this signaling mechanism in lung carcinoids (TC and AC).

Background
EGFR mutation and EML4/ALK rearrangement have become standard genetic tests for patients with advanced adenocarcinoma of the lung. Other driven mutations and genetic abnormalities are still investigational including c-Met expression. c-Met overexpression has shown to confer resistance to EGFR TKI. By targeting c-Met, studies suggest that lung cancer cells may respond again to EGFR TKI therapy. The incidence of c-Met in NSCLC is not clear yet as well as how it does interact with other biomarkers.

Methods
A retrospective study of 32 consecutive biomarker profile tests from patients with either unresectable or stage IV chemonaive adenocarcinoma of the lung were analyzed. We adopted a comprehensive biomarker panel as part of a common decision made from a lung cancer consortium. All patients’ samples were sent to Response Genetics Inc with an order to perform a panel of 9 biomarkers. We report the incidence of c-Met expression and its co-existence with other biomarkers.

Results
The tumor sample of 32 patients with unresectable or stage IV chemonaive adenocarcinoma of the lung were analyzed. All patients were Caucasian; gender: 18 females/14 males; median age: 76 (range, 58-89); distribution of staging was: stage I: n=3, stage II: n=1, stage III: n=7 and stage IV: n= 21 patients. c-Met was ordered in 26 patients: 9 patients had c-Met overexpressed (34%), 5 had low-expression (19%); 12 had no enough tissue for the test. None of the c-Met high expression patients had EGFR mutation; in this cohort, 4/27 patients had EML4/ALK (15%) rearrangement; 2 of them had high c-Met expression; the other 2 did not have enough tissue. The presence of K-ras (25 tumor samples tested), PI3K (25 tumor sample tested), and ROS-1 (n=26 tumor samples tested) mutations were 12/25 (48%), 1/25 (4%), and 0/26, respectively.

Conclusion
Our cohort is small, and this may explain the high EML4/ALK and low EGFR incidence (sample size and patient selection). However, it is crucial to increase our knowledge on how these biomarkers interact themselves and what kind of role do they play in lung carcinogenesis. Some novel trials propose a double target approach in patients who have two pathways overexpressed. Hence, it is imperative that we develop a molecular phenotype data to understand these co-existence genetic phenomenon. Our cohort confirmed the high incidence of K-ras mutations in adenocarcinoma of the lung and also showed that c-Met high expression may be common in chemonaive patients without prior exposure to EGFR TKI. Interestingly, our 2 EML4/ALK patients also had c-Met highly expressed. PI3K and ROS-1 mutations seem to be rare genetic abnormalities.

Background
Squamous cell carcinoma is the most commonly diagnosed lung cancer type in Turkey; however lung adenocarcinoma diagnosis has risen among women and nonsmokers. MPM is common in Turkey because of the high asbestos exposition rates. Mostly, lung adenocarcinoma cannot be clearly differentiated from malignant pleural mesothelioma (MPM). Because of The limitations in the immunohistochemical methods there has been a growing interest in the use of gene expression profiling for diagnosis in many cancers. So we aimed to evaluate the gene expression profiles in tumor cells by using RT-PCR array, between two separate groups of lung adenocarcinoma and MPM patients.

Methods
Ten newly diagnosed patients with adenocarcinoma and paraffin-embedded tissues of 12 patients with MPM were included in this study without considering gender differences. Eight healthy individual were recruited as a control group. After processing the fresh samples of lung adenocarcinoma stored at -80°C for RNA isolation, cDNA synthesis and the expression of 84 genes were associated with DNA repair were analyzed with RT-PCR Array. Paraffin tissues of patients with MPM were deparaffinized and the same procedure was applied. Fold change values of gene expressions in each group are calculated in “SA Bioscience” data analysis expression page.

Results
ACBT, B2N, GADPH AND RPLPO genes were identified as housekeeping genes. Table 1 and 2 shows the comparisons of fold change values ​​of the gene expression differences between lung adenocarcinoma, MPM and control groups.

Conclusion
We showed that adenocarcinoma and MPM tumor cells have different expression profiles of DNA repair genes. Our study suggests that TREX1, PRKDC, PMS1 genes can be significant in support of differential diagnosis between MPM and lung adenocarcinoma.

Background
αB-Crystallin has been shown to correlate with the invasion of several tumors. In this study we investigate the role and mechanism of αB-Crystallin in non-small cell lung cancer (NSCLC).

Methods
Twelve cases of NSCLC and matched nontumorous samples were used to analyze αB-Crystallin expression at the level of protein. Then, we up- and down-regulated the expression of αB-Crystallin in NSCLC cells with specific vshRNA and αB-Crystallin cDNA , and assessed the role of αB-Crystallin in the proliferation, invasion of NSCLC cell line, Furthermore, The molecular mechanism of αB-Crystallin in NSCLC cells were determined by genetic and functional screens. Finally, expression of αB-Crystallin was further examined by Immunohistochemistry (IHC) in tissue microarray (TMA) consisting of 208 cases of NSCLC, and the prognostic role of αB-Crystallin in NSCLC was evaluated by Kaplan-Meier and Cox regression analysis.

Results
The expression of αB-Crystallin in NSCLC tissues was much higher than those in nontumorous samples, and forced loss of αB-Crystallin expression suppressed the invasive potential of lung cancer cell lines, whereas up-regulated αB-Crystallin expression enhanced the cells invasion both in vitro and in vivo. Furthermore, we demonstrated overexpression of αB-Crystallin enhanced the invasion ability of lung cancer cells by resulting in lung cancer cells epithelial-mesenchymal transition (EMT) through ERK1/2/slug pathway. Clinically, we showed that αB-Crystallin overexpression is associated with lymph node metastasis and poor prognosis of NSCLC. Together, our findings indicate that αB-Crystallin may represent a potential therapeutic target and a novel prognostic marker of NSCLC.

Conclusion
Our findings indicate that αB-Crystallin may represent a potential therapeutic target and a novel prognostic marker of NSCLC.

Background
The transcription factor PAX6 is primarily expressed in embryos. PAX6 is also expressed in several tumors and plays an oncogenic role. However, little is known about the role of PAX6 in lung cancer.

Methods
In this study, the function of PAX6 in lung cancer cells was evaluated by small interfering RNA-mediated depletion of the protein followed by analyses of cell proliferation, anchorage-independent growth, and cell cycle arrest. The PAX6 mRNA level in 53 pairs of tumors and corresponding matched adjacent normal tissues from non-small cell lung cancer patients and lung cancer cell lines was detected by real-time PCR.

Results
Suppression of PAX6 expression inhibited cell growth and colony formation by A549 and H1299 cells. The percentage of cells in G1-phase increased when PAX6 expression was suppressed. The cyclin D1 protein level, as well as the pRB phosphorylation level, decreased as a result of PAX6 down-regulation. PAX6 mRNA was highly expressed in lung cancer tissue and lung cancer cell lines.

Conclusion
Our data support the hypothesis that PAX6 accelerates cell cycle progression by potentiating G1-S progression.

Background
NSCLC is a heterogeneous neoplasm comprising several histologic types, etiology, genetics, survival and response to therapy. Accurate analysis of these subtypes has increased sample requirements, which is challenging in the era of minimally invasive procedures. A recent survey of 90 US pathologists presented at ASCO 2013 meeting, concluded insufficient sample availability in 6% of all NSCLC samples recently handled by these pathologists. Moreover, subcellular localization of marker expression linked to tumor pathobiology necessitates methodological advancement. With the development of a new platform that allows in situ, multiplexed sub-cellular analysis of over 60 proteins, this project aims to demonstrate the feasibility of detailed in situ molecular profiling and perform comparative analysis of known cancer pathways and prognostic markers on the same serial section.

Methods
Multiplex immunofluorescence staining and imaging of over 30 biomarkers, including several RTKs, cell adhesion molecules, select members of PI3K, MAPK/ERK, JAK/STAT pathways, angiogenesis, hypoxia, proliferation and chemotherapy resistance markers were performed on replicate FFPE tissue microarrays (TMA) from 382 samples. Cell-level and subcellular-level marker expressions were quantified using image analysis algorithms and compared between serial sections. Associations between marker expressions and histological subtypes and survival were investigated in European male smokers. Multivariate analysis was performed using logistic regression and Cox proportional hazard models on over 300 quantitated features of marker expression. All models controlled for age. Serial sections were modeled separately and combined to improve confidence in associations. EGFR and cMET positivity was evaluated using whole cohort median expression values to define positive cells and the summary statistics are reported using 10% positive cells as cutoff for characterizing positive samples.

Results
In concordance with previous reports, differential expression of RRM1, CK5 and CK7 was observed in SCC vs AD in the high grade, early stage male smokers (N=86). With a 10% cell positivity threshold, 72.7% (76.3%, serial section (SS)) of all male smokers (N=183 (190, SS)) were positive for EGFR. EGFR positivity was higher in SCC, 83.9% (86.0%, SS) compared to AD 54.9% (61.8%, SS). Opposite was observed for cMET with 81.7% (78.9%, SS) of AD characterized as positive compared to only 58.0% (57.0%, SS) SCC. Among the several previously reported prognostic markers evaluated in this study, only CA9 expression was associated with overall patient survival with a hazard ratio of 1.47, p-value 0.0005 (N=278). Again, analysis of serial section produced a similar result confirming the robustness of the platform.

Conclusion
The study demonstrates the capabilities of multiplexing technology (MultiOmyx[TM]) for assessment of limited lung samples, encompassing topographic expression features and the ability to observe relationships between markers through in situ pathway profiling. Additionally, by evaluating markers on exactly the same sample set (same section), a direct comparison of their relative significance in predicting course of disease is now feasible.

Background
Lung cancer is a leading cause of cancer related deaths, 80% of which are classified as non-small cell carcinomas (NSCLC). Differentiating the two main sub-types of NSCLC, adenocarcinoma (AD) from squamous cell carcinoma (SCC) is crucial for therapeutic decision-making. Current methods for characterizing subtypes may involve DAB stains on up to 7 tissue sections, depending on complexity of diagnosis. This process may deplete precious tissue required for molecular studies sequencing and other predictive markers. The goal of the current study was to measure 11 proteins on a single section using a novel multiplexed immunofluorescence (IF) technology (MultiOmyx[TM]) and evaluate performance of analytical workflows in automatic biomarker scoring and in AD, SCC discrimination, with reference to the Pulmotype® test of 5markers

Methods
The protein markers included in the study were comprised of the five antibodies from the Pulmotype® test - Muc1, CK5/6, TRIM29, CEACAM5 and SLC7A5. Six additional markers TTF1, p40, CK7, CK20, p63, NapsinA were selected based on literature reports. These markers were applied to two separate cohorts of NSCLC cases. The entire set of 11 markers was multiplexed on a 378 core tissue microarray (TMA) containing 213 cases of AD or SCC diagnosis (cohort 1). A second 74 core TMA with 50 cases of AD or SCC was stained with the Pulmotype® markers. Manual scores were generated for the immunofluorescence protein images and DAB stained serial tissue sections were used to generate manual ground truth protein expression scores. The first cohort was used to model diagnosis of AD or SCC using an implementation of Breiman and Cutler’s Random Forest and compared to the performance of a previously published lung classifier using manual DAB scores. Image and data analysis algorithms were developed to aid automated biomarker scoring. These algorithms segment the immunofluorescence images into tumor and stromal areas and compute a large number of biomarker-related metrics. Linear regression modeling was used on a down-selected set of metrics to generate automated protein expression scores per biomarker for the second cohort.

Results
Manual scoring of all 11 targets demonstrated excellent concordance between fluorescence and DAB. Concordance was also demonstrated between manual DAB scores and automated IF metrics for the five Pulmotype® markers with an overall sensitivity and specificity of 95% and 87%, respectively. Statistical modeling indicated that 9 (of the 11) multiplexed markers provided 97% specificity and 90% sensitivity in classifying AD versus SCC. The observed 7% indeterminate rate measures well against the existing published indeterminate rates for the Pulmotype® test (11%) and the classic IHC marker combination TTF-1/p63 (29%).

Conclusion
Multiplexed analysis of a single tissue section allows maximum use of limited sample and enhanced protein profiling in context of tissue histology. We have shown that differential diagnosis of AD and SCC may be achieved using a multiplexed panel of markers in a single tissue section, when compared to the Pulmotype® test panel. Concordance between fluorescence and DAB shows transferability of the two detection methods. Furthermore, we demonstrated that image and data analysis tools can be applied for consistent automatic biomarker scoring.

Background
Figure 1Epidermal growth factor receptor (EGFR) mutation status is the primary issue on the appropriate therapy of EGFR-tyrosine kinase inhibitor (EGFR-TKI) in non-small lung cancer. The point mutation of EGFR T790M (C→T) is known to be an acquired and the most common resistance against EGFR-TKI. Highly sensitive mutation detection system has been desired considering the difficulty in　obtaining tissue specimens during disease progression. Eprobe is new fluorescence labeled probe with high affinity toward target ssDNA and works as competitive probe (Figure 1). Here we describe a novel method to identify T790M mutation using Eprobe on real-time monitoring of PCR and melting curve analysis.

Methods
Figure 1Eprobe was designed to bind wild type allele including T790M region with competing primer, which enriches mutant allele amplification (Figure 2). The T790M mutation was detected by melting curve analysis following real-time monitoring of PCR. We verified detection ability by genomic DNA containing wild type and mutated EGFR T790M (cell lines H1975) gene. For clinical evaluation, 338 tumor tissues from the patients with lung adenocarcinoma, of which EGFR gene mutation status had been revealed by the nucleic acid-locked nucleic acid PCR clamp (PNA-LNA PCR clamp), were assayed by Eprobe method. We compared the two methods on T790M mutation assay.

Results
The T790M mutant genome could be detected when it accounted for as little as 0.5% of a mixture of wild type genome by enrichment of mutation amplicon. The activating EGFR mutation (exon19 deletion, L858R, and L861Q) had been detected in 143 out of 338 samples (42.3%) but no T790M mutation was identified by PNA-LNA PCR clamp. Among 143 samples harboring activating EGFR mutation, T790M mutation was identified in 2 samples (1.4%) by Eprobe method.

Conclusion
The Eprobe method is sensitive for detecting EGFR T790M. Since Eprobe works in simple manner, current method is expected to apply to other gene detections.

Background
MicroRNAs (miRNA) are endogenous, small regulatory nucleotides that negatively regulate gene expression post-transcriptionally. They are involved in a wide range of cellular functions, including growth, development, and apoptosis. Given their widespread roles in biological processes, changes in their expression are likely to be associated with the development and progression of diseases. Understanding their patterns of expression could provide new insights into complex biological processes and the possible clinical implications of miRNA dysfunction. As such, global miRNA expression profiling of human malignancies is increasingly performed, but to date, the majority of such analyses have used microarrays and quantitative real-time PCR (qRT-PCR). With the introduction of digital count technologies, such as next-generation sequencing (NGS) and the NanoString nCounter System, we have at our disposal, many more options.

Methods
To compare the attributes of different profiling methodologies, five pairs of non-small cell lung cancer cell lines and their corresponding xenograft models were analysed using a microarray platform (Illumina Human microRNA Expression Profiling v2), NGS (Applied Biosystems SOLiD™ 3 Plus and 4 Systems and Illumina HiSeq2500), and the NanoString nCounter System (Human miRNA Expression Assay v1). The platforms were evaluated according to the following criteria: (i) inter-platform concordance, (ii) concordance with an independent validation method, qRT-PCR, and (iii) detection of differentially expressed miRNAs in a biologically relevant setting.

Results
Inter-platform correlations ranged from 0.62 – 0.80, while correlations with qRT-PCR, the current gold standard for validating expression profiling studies, was highly statistically significant for all platforms, with Spearman’s ρ ranging from 0.79 – 0.86. The accuracy in detecting differential expression was the highest for NGS (88%). Overall, sequencing technologies had the greatest detection sensitivity, along with the largest dynamic range of detection, and highest concordance with qRT-PCR. To assess the technical reproducibility of NGS, the same set of samples was profiled in duplicates. Using unsupervised hierarchical clustering, technical replicates for each biological sample clustered together, with Spearman’s ρ > 0.93 in all cases. miRNA analysis of formalin-fixed paraffin-embedded tissue (FFPE) was also evaluated. FFPE samples represent a rich source of archived specimen for retrospective studies of human disease. The feasibility of miRNA analysis with FFPE tissues would offer many opportunities to evaluate such large banks of archival materials. Three pairs of matched frozen and FFPE xenografts tumors were profiled using the Illumina HiSeq2000 platform. Hierarchical clustering showed similarity between expression profiles of paired frozen and FFPE samples (Spearman’s ρ > 0.88); whereas, samples of different biological origin were less correlated (Spearman’s ρ < 0.81).

Conclusion
These results show the superior sensitivity, accuracy and robustness of NGS for global miRNA profiling in both frozen and FFPE tissue. Although microarrays and qRT-PCR have been used more extensively for expression profiling and are highly reproducible, they are limited to the detection of only known targets identified at the time of assay development and manufacturing. With the rapid increase in miRNAs being discovered and deposited in public databases, sequencing will offer a more comprehensive view of the miRNA transcriptome.

Background
The personalized therapies against specific genetic targets in tumoral cells like EGFR mutations or EML-ALK translocation, or the development of minimized invasive procedures for diagnosis and cancer staging have dramatically improved the outcome of patients with lung cancer. The combination of the wide use of Endobronchial Ultrasound-Guided Transbronchial Needle Aspiration (EBUS-TBNA) and genetic testing to individually tailor chemotherapy should become the standard of care. However, the rapid evolution of personalized medicine is toward the routine testing of high number of biomarkers per patient. Therefore, we have tested whether the recently developed DNA arrays and multiplex technologies could increase the suitability of very small size biopsies performed by EBUS-TBNA for the high-throughput molecular diagnosis

Methods
Fourty consecutive EBUS TBNAs with histologically diagnosed lung adenocarcinoma were tested using the high throughput array and sensitive mass spectrometry technology (Massarray, Sequenom) for the detection of 216 mutations in 26 cancer genes (Lungcarta).Each of the 40 DNAs extracted from only one 19- to 22-gauge needle aspiration spread on a glass slide per patient has been successfully amplified and analyzed within one-day experiment for all of the 238 mutations

Results
Five patients (12.5 %) exhibited EGFR mutations and received either gefitinib or erlotinib, 1 of them exhibiting the T790M resistance mutation. One patient had the BRAFV600E mutation confirmed 1-y later on a skin metastasis and thus entered a phase II trial to receive anti-BRAF therapy. There were EBUS-TBNA exhibiting mutations in KRAS (n=7), PI3K (n=1) and cMET (n=1), and insertion in HER2 (n=2).

Conclusion
In conclusion, only one 19-22 gauge needle aspiration is suitable in routine care for the detection of at least 238 mutations, to guide treatment decisions and offer patients to benefit from current and future individually targeted drugs

Background
Circulating tumour cells (CTCs) are present in the blood of a proportion of patients with lung cancer. However, it is currently unclear how suitable CTCs are for use in the detection of predictive genetic mutations. We sought to determine the utility of DNA extracted from CTCs to screen for the underlying primary tumour mutation.

Methods
Using ScreenCell™ MB devices, from 20/01/12 to 25/01/2013, CTCs were captured in peripheral blood of 100 patients who underwent surgery for lung cancer at The Royal Brompton Hospital. DNA was extracted using QIAamp DNA Micro kit (QIAGEN) followed by whole-genome amplification using GenomePlex® SingleCell WGA kit (Sigma). DNA from matched primary tumours was used as reference. Mutation detection in EGFR and KRAS genes was undertaken using cobas®4800 (Roche) and single-strand conformation analysis for BRAF gene. Sensitivity and specificity analyses were undertaken to measure predictive performance of mutation testing in CTCs.

Results
The DNA extracted from CTCs, were of sufficient quality to allow mutation analyses to be successfully performed in 100%, 99%, and 98% of samples for EGFR, KRAS, and BRAF genes, respectively. In CTC DNA, the KRAS mutation rate (codons 12/13 and 61) was 9.1% and concordance with the primary tumour was 78.8%. Six mutations were detected in CTCs, but not in primary tumours, and 13 mutations in primary tumours were not detected in corresponding CTC samples. Three mutations were detected in matched CTC and primary tumour specimens. One mutation in EGFR was detected in CTC DNA and 3 mutations were detected in primary tumours. In all cases, the mutations were detected in discordant specimens. The concordance between mutations detection in CTCs and primary tumours was 95.8%. BRAF V600E mutation was not detected in any sample. In general, the results suggested low sensitivity but high specificity (Table). Due to low number of EGFR mutations detected, test performance results require further validation.

Conclusion
The result of our study indicates that the DNA extracted from CTCs can be used to screen for primary tumour mutations with reasonable concordance. Differences in the mutation results from the CTC and primary tumours needs to be explored in more detail and may be due to issues related to processing and / or tumour versus CTC heterogeneity.