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G. Sozzi

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    MO04 - Lung Cancer Biology I (ID 86)

    • Event: WCLC 2013
    • Type: Mini Oral Abstract Session
    • Track: Biology
    • Presentations: 12
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      MO04.01 - Molecular mechanisms of cigarette smoke induced hyper-proliferation of human lung cells and its prevention. (ID 2636)

      16:15 - 17:45  |  Author(s): N. Dey, D. Chattopadhyay, I.B. Chatterjee

      • Abstract
      • Presentation
      • Slides

      Background
      Lung cancer is the leading cause of cancer-deaths throughout the World. Cigarette smoke (CS) is the strongest risk factor of lung cancer. Cigarette smoking and exposure to environmental tobacco smoke account for 90% of lung cancer cases, and smokers have a 20-fold increased risk of death from lung cancer compared to non-smokers. Unregulated cell proliferation together with suppressed apoptosis is a contributory factor for lung-carcinogenesis. However, the carcinogenic mechanism of cigarette smoking is not well understood. This is particularly because CS is a complex mixture of about 4000 compounds. We consider that identifying the risk factor(s) in CS and its prevention might be a novel way to prevent lung cancer.

      Methods
      Cytotoxicity was evaluated by the MTT assay. Cell cycle analysis was performed by propidium iodide (PI) staining followed by flow-cytometry. Apoptosis was assessed by AnnexinV- PI staining followed by flow-cytometry, phosphorylation of p53 and activation (cleavage) of caspase 3. Reactive Oxygen Species (ROS) production was detected by 2’, 7’-dichlorodihydrofluorescein diacetate (H~2~DCFDA) using confocal microscope. Cell proliferation was assessed using “In Situ Cell Proliferation Kit, FLUOS” (Roche Applied Science, Germany). DNA double-strand break was detected using “OxiSelect DNA Double Strand Break (DSB) Staining Kit” (CELL BIOLABS, INC.).

      Results
      We have identified p-benzoquinone (p-BQ) as a major risk factor that is derived from CS. p-BQ has a biphasic-nature as evidenced by MTT assay, AnnexinV-PI staining, cell cycle analysis and BrdU-incorporation assay. Low concentrations of p-BQ mimicked CS-induced proliferation of cultured lung adenocarcinoma cells (A549) as well as normal human primary small airway cells (ATCC[®] PCS-301-010[™]). On the contrary, high concentrations of CS/p-BQ resulted in cell death caused by oxidative stress and apoptosis. No such cell death was observed with low concentrations of CS/p-BQ. Coimmunoprecipitation and immunoblot experiments indicated that p-BQ-induced proliferation was mediated via aberrant phosphorylation of EGFR that lacked c-Cbl mediated ubiquitination and degradation. This resulted in prolonged EGFR signaling leading to persistent activation of Ras (a potent oncoprotein), the downstream survival and proliferative signaling molecules Akt and ERK1/2, as well as the transcription factors c-Myc and c-Fos. It is known that patients with lung cancer generally have a smoking history of more than 30-40 years. We therefore exposed A549 cells repeatedly to CS/p-BQ for 2 months. Repeated exposure of AECS/p-BQ generated high levels of DNA double-strand breaks in A549 cells that might lead to genomic instability as well as mutation of different proto-oncogenes and tumor suppressor genes – the hallmark events in cancer. In addition, CS/p-BQ altered the acetylation pattern of different histone epigenetic marks, thereby regulating the transcription of several candidate genes responsible for proliferation and apoptosis. Both anti-p-BQ antibody and vitamin C (a strong reductant of p-BQ) prevented CS/p-BQ-induced proliferation of lung cells.

      Conclusion
      Despite major advances in the treatment and management of lung cancer, most patients eventually die. Consequently, newer approaches such as chemoprevention(s) are necessary. We consider that prevention of CS-induced proliferation of lung cells by vitamin C and/or anti-p-BQ antibody may provide a novel intervention for preventing initiation of CS-induced lung cancer.

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      MO04.02 - Paclitaxel resistance is associated with drug accumulation in intracellular compartments and paclitaxel-binding proteins in human lung cancer cell lines (ID 75)

      16:15 - 17:45  |  Author(s): M. Shimomura, T. Yaoi, D. Kato, J. Shimada, S. Fushiki

      • Abstract
      • Presentation
      • Slides

      Background
      Several mechanisms have been suggested for paclitaxel resistance in cancer cells, including overexpression of the multidrug transporter gene, ATP-binding cassette, sub-family B, member 1 (ABCB1), and the presence of a point mutation in the β-tubulin gene at the paclitaxel-binding site. However, the mechanisms underlying resistance to this agent have not yet been completely elucidated.

      Methods
      Three human lung cancer cell lines, II18, A549, and RERF-LC-KJ, were analyzed; their 50% inhibitory concentrations of paclitaxel were -8.33, -7.69, and -4.51 logM, respectively. The cell lines did not have any β-tubulin mutation. We evaluated the expression levels of ABCB1, intracellular accumulation of paclitaxel, paclitaxel-induced stabilization of microtubules, and intracellular localization of Oregon Green[®] 488-conjugated paclitaxel in these cell lines. Moreover, we prepared paclitaxel conjugated ferriteglycidyl metacrylate (FG) beads to purify paclitaxel-binding proteins from whole cell lysates of these cells.

      Results
      The ABCB1 expression level was strongly correlated to intracellular [[3]H]-paclitaxel accumulation (r[2] = -0.804) but was not related with paclitaxel resistance. The changes in the quantities of polymerized tubulin and acetylated tubulin after paclitaxel exposure were not related to paclitaxel resistance. Differences were observed between the intracellular localization of paclitaxel in RERF-LC-KJ, the most resistant cell line, and in the other 2 cell lines. The use of Oregon Green[®] 488-conjugated paclitaxel enabled visualization of not only the normal microtubule formation in the partial cells but also the aggregated vesicle formation in RERF-LC-KJ cells; aggregated vesicle formation was not remarkable in the other cell lines. Affinity purification by paclitaxel immobilized beads revealed several specific bands in RERF-LC-KJ; these bands were not revealed in the other cell lines.

      Conclusion
      We propose that paclitaxel resistance is associated with intracellular compartments in which paclitaxel accumulates and paclitaxel-binding proteins expressed specifically in resistant cell line.

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      MO04.03 - Chromosomal and mutational analysis of the cisplatin resistant phenotype in NSCLC cells (ID 3313)

      16:15 - 17:45  |  Author(s): M.P. Barr, L.C. Alcock, R.L. Stallings, S. Toomey, A. Carr, B.T. Hennessy, D. Richard, K. O'Byrne

      • Abstract
      • Presentation
      • Slides

      Background
      Primary and acquired resistance to platinum agents such as cisplatin have become a major obstacle in the management of lung cancer patients, in particular non-small cell lung cancer (NSCLC). The availability of comprehensive genomic data on DNA copy number changes in cisplatin resistant NSCLC is limited, and little is known about the genes driving this chemoresistant phenotype. Detailed molecular portraits through high density genomic DNA arrays and genome wide mutation profiles will aid in understanding the molecular basis of individual responses to new molecular therapies.

      Methods
      A panel of cisplatin resistant (CisR) NSCLC cell lines were recently generated and characterised in our laboratory. In this study, high resolution array-based comparative genome hybridization (aCGH) was performed on a panel of five CisR NSCLC cell lines to examine DNA copy number gains, losses and amplifications. Cellular DNA (500ng) and control DNA was differentially labelled with Cy3 and Cy5, respectively. Labelled test (4µg) and reference DNA were hybridised to a 12-plex 135,000 probe array (Roche NimbleGen) for 18 hours in a MAUI hybridisation station (BioMicro Systems) at 42°C. Fluorescent intensities were extracted and log 2 ratios calculated and normalized using NimbleScan Software (version 2.4). Chromosomal aberrations were identified using the CGH-segMNT algorithm (NimbleScan 2.4). A significance log 2 ratio threshold of <−0.25 for loss and >0.25 for gain was used to identify DNA copy number imbalances. For mutational analysis, Sequenom®, a mass-spectrometry-based SNP genotyping technology, was used to identify mutations in our panel of resistant cell lines. Using a literature search and the Catalogue of Somatic Mutations in Cancer (COSMIC) database, a mutation panel was identified for the detection of 547 frequently occurring and potentially clinically relevant mutations in 49 cancer-related genes. Some of these include KRAS, NRAS, BRAF, PIK3CA, MET, CTNNB1, STK11, AKT, and EGFR. Matrix chips were analysed on a Sequenom® MassArray MALDI-TOF system. Visual inspection and Sequenom® typer software were used to perform genotyping based on mass spectra.

      Results
      Using aCGH arrays, a number of gains, losses and amplifications of various chromosomes were found across a panel of CisR cell lines, relative to corresponding PT cells. The most frequently occurring of these chromosomal imbalances included gains, losses and homozygous deletions on chromosome 3 (MOR, A549, H1299), deletions and amplifications on chromosome 7 (H460, A549, H1299) and deletions and gains on chromosome 15 (MOR, A549, H1299) and chromosome X (MOR, H460, SKMES-1). Deletions on chromosomes 4, 6, 11, 12, 14, and amplification of chromosome 5, were also identified among the different CisR cell lines. The collation and analysis of data arising from mutation analysis of CisR cells using the Sequenom® platform are currently being completed.

      Conclusion
      High-resolution mapping of chromosomal imbalances may offer potential in the identification of genes, including oncogenes and tumour suppressor genes, affected by these imbalances. These findings may further contribute to the delineation of the genomic profile of cisplatin resistant lung cancer, and offer perspectives for the identification of genes contributing to this disease phenotype and in assessing the response to new molecular treatments.

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      MO04.04 - Aerosol Azacitidine Reduces Methylation Levels of Tumor Suppressor Genes and Prolongs Survival in the Orthotopic Lung Cancer Models in Mice. (ID 1621)

      16:15 - 17:45  |  Author(s): X. Qiu, Y. Liang, B. Piperdi, R. Perez-Soler, Y. Zou

      • Abstract
      • Presentation
      • Slides

      Background
      Promoter hypermethylation plays an important role in lung cancer carcinogenesis by silencing key tumor suppressor genes (TSG). Reversing the hypermethylation of TSG by direct aerosolized administration of a demethylating agent may have a potential to inhibit lung cancer growth and development. This study examines the therapeutic potential of aerosolized azacytidine (Aza) in the orthotopic mice model of human NSCLC.

      Methods
      Vidaza® (the clinical grade Aza formulation for IV administration) was dissolved into sterile water for injection and aerosolized in a clinical standard aerosol system. The aerodynamic size, the lung deposition, and the Aza level in the lung and circulation of mice were measured by previously described methods. The demethylation and gene reactivation functions of aerosolized Aza were detected by qPCR methylation array and western blotting assay in the human non-small cell lung cancer (NSCLC) tumor tissues from the orthotopic intratracheally inoculated xenograft models in nude mice. The therapeutic efficacy and toxicity of the aerosolized Aza were also evaluated in mice. Mice treated with an intravenously administered Aza with clinically equivalent dose and schedules were used as a comparison.

      Results
      The aerosolized Aza is an appropriate pharmaceutical inhalation formulation with size distribution of about 80% of droplets from 0.1 to 5 micron. This dynamic size range ensures the aerosol droplets depositing in the lower airway as indicated by the recovery of approximately 80% Aza from the lung tissue 20 min after the aerosol administration. In efficacy and toxicity studies, aerosol administration of Aza significantly prolonged the survival of nude mice with intratracheally inoculated human NSCLC tumors. The %-increased in life span was 5- to 10-fold higher than that of a systemic treatment of Aza at a clinically equivalent dose. The aerosolized Aza, at a potentially therapeutic dose, did not cause any detectable lung toxicity or systemic toxicity (myelosuppression). After the aerosolized Aza treatment, the lung tumors were resected and the methylation levels of the 24 promoters driving the lung cancer related tumor suppressor genes (TSGs) in the lung tumors were examined by qPCR methylation array. The aerosolized Aza significantly reduced the methylation level in 9 of the 24 promoters examined. This demethylating effect also resulted in the gene reactivation at the protein level in several tested genes.

      Conclusion
      In our orthotopic NSCLC model, aerosolized Aza was well tolerated with good drug delivery to bronchial epithelium at non-cytotoxic doses. The pharmacodynamic effect of the TSG demethylation and reactivation was observed. Interesting therapeutic efficacy of prolonging survival was observed in our model from aerosolized 5-Aza over intravenous administration in mice. The phase I clinical trial of aerosolized Aza with pharmacodynamic endpoints will be conducted. Acknowledgement: Supported by a NIH (NCI) grant 5R01CA154755-02

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      MO04.05 - Tracheal basal cells in the lung squamous dysplasia: moving proximal. (ID 1700)

      16:15 - 17:45  |  Author(s): M. Ghosh, L.D. Nield, J. Kwon, D.T. Merrick, R.L. Keith

      • Abstract
      • Presentation
      • Slides

      Background
      Squamous cell carcinoma (SCC) is the second most common form of lung cancer, a disease primarily observed in smokers. Studies have shown that preneoplastic dysplasias are the precursors for SCC. However, only a subset of these lesions progress to invasive carcinoma and predicting the fate of individual lesions is difficult. Understanding the processes associated with the development of dysplasia would therefore have significant impact on preventative therapy for high-risk lesions. To this goal we have studied N-nitroso-tris chloroethyl urea (NTCU) model of premalignant murine squamous dysplasia. Bi-weekly topical application of NTCU for 32-weeks caused endobronchial dysplasias that were pathologically similar to dysplasias encountered in human smokers. In the current study we used NTCU model to identify the events that precede dysplastic changes in murine airways.

      Methods
      Immunofluorescence analysis of NTCU treated airways showed abundant expression of keratin 5 positive (K5+) basal cells. In normal mouse respiratory epithelium K5+ cells are confined only in the trachea and are absent in the bronchial epithelium. Ectopic expressions of K5+ cells in the mouse bronchial epithelium therefore suggested a role for tracheal basal cells in the pathogenesis of NTCU-induced dysplasia. To explore this possibility we analyzed tracheal and bronchial histology after vehicle and 20mM NTCU treatment at 4, 8, 12, 16, 25 and 32 weeks.

      Results
      Low-grade dysplasia of the tracheal epithelium was observed as early as 4 weeks of NTCU exposure. On the contrary, squamous metaplasia or low-grade dysplasia of the bronchial epithelium was not seen until 25 weeks. Morphometric analysis of immunostained tracheal tissue showed a time-dependent increase in the numbers of K5+ cells and a concomitant loss of cells expressing Clara cell secretory protein (CCSP) and ciliated cells. We have shown previously that injury to the trachea upregulates expression of keratin 14 (K14) in basal cells. Consistent with this phenomenon, NTCU exposure showed a time-dependent increase in the number of K5/K14 dual-positive basal cells. To understand the mechanism by which NTCU treatment mediates ectopic expression of basal cells in the lung, we used flow-cytometry to analyze cell-surface markers expressed by tracheal basal cells. These studies revealed a time-dependent decrease in the level of integrin a6b4 (CD49f). CD49f is a hemidesmosomal protein that facilitates attachment of basal cells to the basement membrane. These findings suggested that NTCU treatment might promote migration of tracheal basal cells by destabilizing their adherence to the basement membrane.

      Conclusion
      Collectively, our results showed an early involvement of tracheal basal cells in the generation of dysplastic lung lesions in a murine model of SCC. By moving the focus proximal, we have discovered a realm of biological developments in the trachea that had not been previously examined. Further research of these processes will aid in the elucidation of dysplastic development in SCC.

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      MO04.06 - DISCUSSANT (ID 3894)

      16:15 - 17:45  |  Author(s): G.R. Simon

      • Abstract
      • Presentation
      • Slides

      Abstract not provided

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      MO04.07 - Chronic lung injury by constitutive expression of AID leads to focal alveolar regeneration and cancer (ID 116)

      16:15 - 17:45  |  Author(s): J. Kitamura, K. Kinoshita, M. Uemura, M. Sonobe, H. Hiai, H. Date

      • Abstract
      • Presentation
      • Slides

      Background
      Activation-induced cytidine deaminase, AID, is an enzyme required for somatic hypermutation and class-switch recombination which diversify immunoglobulin genes and causes DNA mutations and strand breaks. Uncontrolled expression of AID is cytotoxic. AID transgenic mice invariably develop lung lesions morphologically similar to human atypical adenomatous hyperplasia (AAH), which can be a precursor of bronchioloalveolar carcinoma. About 10 % of these mice develop visible lung tumor including adenocarcinoma. However, the relationship between this mouse AAH-like lesion (MALL) and lung cancer is unclear. In the present study, we examined MALLs to elucidate their characteristics and involvement in lung cancer.

      Methods
      p53, KRAS, and EGFR mutation status in each laser-microdissected MALL were analyzed. The expression of airway epithelial cell markers and lung alveolar regeneration markers in MALLs were investigated by immunohistochemistry. Apoptosis assay were performed in murine lungs. For cell proliferation assay, AID Tg mice were received a daily intraperitoneal injection of 1 mg 5-ethynyl-2’-deoxyuridine (EdU) for 7 days. Then, mice were studied 1 day (day 1) or 3 weeks (day 20) after the last injection.

      Results
      We found mutations of p53 in 10.5% of MALLs (4/38), but no mutations of KRAS and EGFR. In immunohistochemistry, MALLs were partially positive for SP-C (lung alveolar type II cell-specific marker), but negative for CC-10 (clara cell-specific marker) and podoplanin (lung alveolar type I cell-specific marker). Frequency of apoptotic cells among lung alveolar wall cells was significantly higher in AID transgenic mice than in wild type mice. Moreover, frequency of Edu-positive MALL decreased significantly at day 20 compared to that at day 1. The expressions of p63, cytokeratin 5/14, and E-cadherin/Lgr6, the recently described markers of lung alveolar regeneration, were observed in MALLs.

      Conclusion
      Based on these observations, we speculate that MALL is a regenerating tissue compensating for alveolar epithelial cell loss caused by AID-induced genotoxic stress. AID expression in such regenerating tissue should predispose cells to malignant transformation by its mutagenic activity. AID transgenic mice could be a mouse model that may provide the link between lung regeneration after injury and the development of lung cancer.

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      MO04.08 - Activation of liver X receptor induces interferon-gamma production and inhibits tumor growth (ID 705)

      16:15 - 17:45  |  Author(s): Q. Wang, X. Ma, Y. Duan

      • Abstract
      • Presentation
      • Slides

      Background
      Interferon-γ (IFN-γ) has been well documented to have multiple functions including anti-tumorigenesis. Liver X receptors α and β (LXR) are members of ligand- activated nuclear receptor superfamily. LXR can be ac- tivated by natural ligands of some oxysterols and by numerous synthetic ligands (e.g. T317).The LXR-induced ABCA1 expression promotes free cholesterol efflux from macrophages thereby inhibiting the development of atherosclerosis. LXR has also been demonstrated the important functions in immune system . Here we tested the hypothesis that IFN-γ is a target for LXR activation, and the induction of IFN-γ expression by LXR can lead to inhibition of tumor growth.

      Methods
      C57 wild type or IFN-γ-/- mice (same background) in two groups were s.c. injected with2×10 5 LLC1 cells (ATCC) in the right flank to establish a carcinoma tumor model. For carcinogen-induced tumor: at the first week, C57 mice were i.p. injected with MCA (15 mg/kg body); from the second week, the mice were i.p. weekly injected with BHT for 6 weeks. The doses of BHT were: 1st injection, 150 mg/kg body weight; 2nd to 6th injections, 200 mg/kg.

      Results
      In this study, we observed that LXR ligand (T317) induced IFN-γ protein expression which was associated with increased mRNA and secreted protein levels in culture medium. In vivo,T317 increased wild type mouse serum IFN-γ levels and IFN-γexpression in tissues. T317 inhibited the inoculated LLC1 tumor growth in wild type mice but not in IFN-γ knockout (IFN-γ-/-) mice. In addition, T317 displayed inhibitory and therapeutic effects on 3-methylcholanthrene/butylated hydroxytoluene (MCA/BHT)-induced pulmonary carcinomas. T317 inhibits the growth of inoculated LLC1 tumor in wild type mice but not in IFN-γ-/- mice Wild type or IFN-γ-/- mice. Figure 1 T1317 inhibits MCA/BHT-induced pulmonary tumors Figure 2

      Conclusion
      Our study demonstrates IFN-γ is a target gene of LXR activation. LXR-induced IFN-γ expression can be attributed, at least in part, to the anti-tumorigenic properties of LXR.

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      MO04.09 - Exercise Induced Lung Cancer Tumor Growth Suppression via Activation of p53: Mechanistic Findings from a Mouse Model (ID 182)

      16:15 - 17:45  |  Author(s): K.A. Higgins, D. Park, W.J. Curran, X. Deng

      • Abstract
      • Presentation
      • Slides

      Background
      Regular exercise has been shown to improve the quality of life in patients undergoing treatment for lung cancer and has been associated with reductions in cancer-specific mortality in patients with colon and breast cancer. The direct effects of cardiovascular exercise on lung cancer tumor biology, however, remain unknown. This study evaluated cardiovascular exercise in a mouse model of lung adenocarcinoma, including clinically relevant endpoints such as lung tumor growth and distant metastasis. Furthermore, biologic mechanisms of action underlying clinical findings were also explored.

      Methods
      Luciferase-tagged A549 lung adenocarcinoma cells were injected through the tail vein of nude male mice. Mice underwent weekly bioluminescent imaging until lung tumors were clearly identified. After lung tumors were identified, mice were randomized to daily wheel-running versus no wheel-running. Mice were imaged weekly. After 4 weeks, all mice were euthanized and lung tumors were harvested. Western blots and immunohistochemistry (IHC) studies were undertaken on tumor tissue to identify potential differences in protein expression levels in exercise versus sedentary mice.

      Results
      Exercising mice tumors grew significantly more slowly relative to sedentary mice (figure 1). There was no change in development of metastatic lesions between the two groups. Protein analysis by Western blot or IHC demonstrated increased p53 protein levels in exercising mice relative to sedentary mice, as well as increased mediators of apoptosis including Bax, Bak and active caspase 3 in tumor tissues (figure 2 and data not shown). No normal tissue toxicity in other organs was observed in the two groups of mice. Figure 1. Figure 1 Figure 2. Figure 2

      Conclusion
      Daily cardiovascular exercise appears to mitigate growth of lung adenocarcinoma tumors by activation of p53 tumor suppressor function and increased apoptosis.

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      MO04.10 - Identification of biological properties of intralymphatic tumor related to the development of lymph node metastasis in lung adenocarcinoma (ID 1724)

      16:15 - 17:45  |  Author(s): K. Kirita, G. Ishii, R. Matsuwaki, Y. Matsumura, S. Umemura, S. Matsumoto, K. Yoh, S. Niho, K. Goto, H. Ohmatsu, Y. Ohe, K. Nagai, A. Ochiai

      • Abstract
      • Presentation
      • Slides

      Background
      Intralymphatic tumors in the extratumoral area are considered to represent the preceding phase of lymph node (LN) metastasis. The aim of this study was to clarify the biological properties of intralymphatic tumors susceptible to the development of LN metastasis, with special reference to the expression of cancer initiating/stem cell (CIC/CSC) markers in cancer cells and the number of infiltrating stromal cells.

      Methods
      A total of 2087 consecutive adenocarcinoma patients underwent complete resections and systematic LN dissections between May 1998 and December 2012 were identified. Among these cases, we selected those that had been diagnosed as having lymphatic permeation in the extratumoral area (n = 107). We examined the expression levels of CIC/CSC related markers including ALDH1, OCT4, NANOG, SOX2 and Caveolin-1 in the intralymphatic and primary tumor cells to evaluate their relationship to LN metastasis. The number of infiltrating stromal cells expressing CD34, α-smooth muscle actin, and CD204 were also evaluated. Moreover, we measured E-cadherin expression to identify a correlation between CIC/CSC related molecules and epithelial - mesenchymal transition (EMT) process.

      Results
      Intrathoracic LN metastases were detected in specimens from 86 patients (80%). Among the intralymphatic tissues, low ALDH1 expression in cancer cells, high SOX2 expression in cancer cells, and a high number of CD204(+) macrophages were independent predictive factors for LN metastasis (odds ratio [95%CI] = 3.25 [1.11 – 9.82], P = 0.031 for ALDH1; 4.09 [1.38 – 13.4], P = 0.011 for SOX2; and 3.45 [1.16 – 11.4], P = 0.026 for CD204(+) macrophages). However, in the primary tumors, only a high SOX2 expression level in the cancer cells within the primary tumor was significantly correlated with LN metastasis (p=0.008); ALDH1 expression in the cancer cells and the number of CD204(+) macrophages were not correlated with LN metastasis (P = 0.230 and P = 0.088, respectively). Among these factors, only low ALDH1 expression in intralymphatic cancer cells was significantly correlated with the farther spreading of LN metastasis (mediastinal LN, pN2) (P = 0.046) and higher metastatic LN ratio (metastatic/resected) (P = 0.028). Intralymphatic cancer cells expressing low ALDH1 levels exhibited lower E-cadherin expression levels than cancer cells with high levels of ALDH1 expression (P = 0.015). The expressions of other CIC/CSC related markers, including OCT4, NANOG, SOX2, and Caveolin-1, were not correlated with the E-cadherin expression.

      Conclusion
      Intralymphatic cancer cells expressing low levels of ALDH1 and infiltrating macrophages expressing CD204 have a critical impact on LN metastasis. Especially, intralymphatic cancer cells expressing low levels of ALDH1 might acquire a metastatic aggressiveness by the EMT process. Our study highlighted the significance of evaluating the biological properties of intralymphatic tumors for tumor metastasis and suggested the possibility of usefulness as a new molecular target, especially as an adjuvant therapy.

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      MO04.11 - The tumor microenvironment expression of the Inhibitor of Differentiation-1 (Id1) is determinant of the development of liver metastasis from lung cancer through gene regulation of tumor cells (ID 2429)

      16:15 - 17:45  |  Author(s): I. Gil-Bazo, E. Castañón, I. López, M. Ponz-Sarvisé, J.M. López-Picazo, M. Collantes, M. Ecay, I. Gil-Aldea, A. Calvo

      • Abstract
      • Presentation
      • Slides

      Background
      Liver metastases appear in about 30% of non-small cell lung cancer (NSCLC) patients during the disease course with a dramatic impact on clinical outcome and quality of life. TME gene expression might be crucial for allowing tumor cells to migrate and spread to the liver. However, no candidate genes have ever been proposed as responsible for this process. Id1, a member of the gene signature that facilitates breast cancer cells to disseminate to the lungs, might be determinant for NSCLC LM development.

      Methods
      For the first time, a mouse model of LM from lung cancer was developed. Two cohorts of mice were compared; C57BL/6 females vs. Id1-knockout (KO) females (gently provided by Dr. Benezra, MSKCC, New York). Lewis lung carcinoma cells (500,000) were selected for intrasplenic injection. Five minutes after tumor cells injection all mice were splenectomized to avoid bulky flank tumor formation. Mice were closely followed after tumor cells inoculation. Weekly FDG-micro-positron emission tomography (PET) scans were performed to study liver metastasis formation in both groups of mice. Animals were sacrificed at the time of LM development. Liver metastatic lesions were obtained for RNA extraction (Qiagen kit). A microarray gene expression analysis (Affymetrix) with the support of Ingenuity Pathways Analysis (IPA) was performed to evaluate the potential impact of Id1 genotype on the regulation of genes mediating proliferation, invasion, migration, angiogenesis and metastasis in LM.

      Results
      The first week after tumor cells intrasplenic injection, FDG-PET scans showed no liver metabolic uptake in any of the mice. By week 2 however, 70% of C57BL/6 mice and 10% of Id1-KO mice showed clear LM by FDG-PET (p=0.02). Three weeks after intrasplenic tumor cells injection, 100% of C57BL/6 animals showed LM compared to 30% of Id1-KO mice (p=0.03). In addition, 50% of Id1-KO mice remained LM-free 4 weeks after tumor cells injection. No other metastatic sites were indentified at the time of necropsy. In the mircroarray gene expression analysis, only a set of 50 out of nearly 900 genes appeared upregulated in the LM of Id1-KO mice compared to C57BL/6 animals whereas the rest of the genes were downregulated. Interestingly, amphiregulin, caveolin-1, aurora kinase B, MMP3, Hsp90aa1, Cdk1, Hif1a, Cyclin D2 were among the significantly downregulated genes in the Id1-KO LM.

      Conclusion
      A novel mouse model for liver metastasis from lung cancer has been developed allowing the study of this complex and unexplored process. Id1 gene expression seems to be a key mediator of the development of liver metastasis from lung cancer in this in vivo model. The absence of Id1 expression in the tumor microenvironment of Id1-KO mice was sufficient to significantly delay and impair the metastatic process of lung cancer tumor cells to the liver. Id1 might be able to modulate LM through a direct downregulation of genes involved in proliferation, invasion, migration, angiogenesis and metastasis. This study has been partially funded by "UTE project CIMA" and an ISCIII-FIS grant 2011.

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      MO04.12 - DISCUSSANT (ID 3895)

      16:15 - 17:45  |  Author(s): N. Watkins

      • Abstract
      • Presentation
      • Slides

      Abstract not provided

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    MS02 - Stem Cells and Epigenetics in Lung Cancer (ID 19)

    • Event: WCLC 2013
    • Type: Mini Symposia
    • Track: Biology
    • Presentations: 4
    • +

      MS02.1 - Stem Cells & the Cell of Origin of Lung Cancer (ID 462)

      14:00 - 15:30  |  Author(s): K. Sutherland

      • Abstract
      • Presentation
      • Slides

      Abstract
      The cellular hierarchy of the lung is quite complex and it is believed that different progenitor cells and stem cell populations residing within distinct spatial regions of the lung are responsible for orchestrating lung development, regeneration and repair. These different stem cell populations are also likely to be instrumental in the development of the various cancers in lung as particular lung tumour subtypes are almost exclusively found in distinct compartments within the lung. Squamous cell carcinomas are thought to arise from the proximal airways, small cell lung cancer are predominantly located in the bronchioles while adenocarcinomas are more frequently detected the in the distal part of the lung. To investigate the cellular origin of lung cancer, we utilized Cre-loxP recombination technology, which is an effective method for expressing or deleting a target gene in Cre-expressing cells. We generated a series of recombinant adenoviruses expressing Cre recombinase from specific lung epithelial gene promoters. For these studies we chose to target Clara cells, alveolar type 2 (AT2) cells and neuroendocrine (NE) cells. Comprehensive experiments performed in Rosa26R-lacZ and mT/mG reporter animals showed that these viruses exhibit a high level of cell selectivity in the adult mouse lung. To address the cellular origins of small cell lung cancer (SCLC) we have utilised a sporadic mouse model of small cell lung cancer based on the conditional inactivation of the tumour suppressor genes Tp53 and Rb1. We infected Tp53F/F;Rb1F/F animals with our cell type-restricted Adeno-Cre viruses: Ad5-CC10-Cre, Ad5-SPC-Cre and Ad5-CGRP-Cre. Results from these studies show that inactivation of Trp53 and Rb1 can efficiently transform neuroendocrine (CGRP-positive) and to a lesser extent, alveolar type 2 (SPC-positive) cells leading to SCLC. In contrast, CC10-expressing cells were largely resistant to transformation. The results clearly indicate that neuroendocrine cells serve as the predominant cell-of-origin of SCLC in this model. Interestingly a different, cell type specificity was observed when a K-rasG12D oncogene-driven non-small cell lung cancer (NSCLC) model was used to reveal the cell of origin of NSCLC (mostly of adenomas and adenocarcinomas). In this case we noted a difference between K-rasLSL-G12D/+ and K-rasLSL-G12D/+;Trp53F/F animals following infection with our cell type-restricted adenoviruses. In K-rasLSL-G12D/+ mice alveolar type 2 cells appeared to be the most effective target cell for inducing adenomas, whereas in K-rasLSL-G12D/+;Trp53F/F mice multiple cell types had the capacity to give rise to adenomas and adenocarcinomas. Moreover, preliminary data from these experiments indicates that the cell-of-origin may also influence the characteristics and behaviour of the resulting tumours. Taken together, our data show that both cell specific features and the nature of the genetic lesion(s) are critical factors in determining the tumour initiating capacity of lung (progenitor) cells to give rise to various lung cancer subtypes.

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      MS02.2 - Targeting Hedgehog Signaling in Small Cell Lung Cancer (ID 463)

      14:00 - 15:30  |  Author(s): N. Watkins

      • Abstract
      • Presentation
      • Slides

      Abstract
      Small cell lung cancer (SCLC) is a malignant neuroendocrine tumour responsible for 20% of all lung cancer deaths. Despite the effectiveness of platinum-based chemotherapy, the overwhelming majority of patients succumb to a chemoresistant recurrence within 2 years of diagnosis. Therefore, the discovery of novel strategies to prevent disease recurrence may have a significant impact on outcome. Many groups, including our own, have identified the importance of embryonic signaling pathways in promoting tumor-regeneration through the regulation of self-renewal. Activation of self-renewal pathways such as Notch, Hedgehog (Hh) and WNT is also thought to contribute to the survival of cancer cells with innate resistance to chemotherapeutic agents. Since the naturally occurring Hh inhibitor cyclopamine can block self-renewal in SCLC cells in vitro, we postulated that this pathway might be targetable following platinum-based chemotherapy. The Hh pathway is a highly conserved signaling system that specifies cell fate and self-renewal in development and homeostasis. The Hh ligand Sonic Hh (Shh) binds to, and inactivates, the receptor Patched (Ptch). This prevents Ptch from inhibiting Smoothened (Smo), the molecular target of the small molecule Hh inhibitors. Smo activation requires translocation to the tip of the primary cilium, a single, immotile, tubulin-based organelle present on most vertebrate cells. The ciliary motor protein Kif3a is essential for Smo translocation to the ciliary tip, and is required for Smo signaling in development. The importance of primary cilia in cancer is poorly understood. In addition, the formation of cilia is normally restricted to cells in the G0 of G1 phases of the cell cycle. In order to better understand how Hh signaling is regulated in SCLC, we investigated pathway activation in the context of cilia formation, and the expression of Smo protein in these cilia. Using a genetic mouse model of SCLC, we observed that approximately 25% of tumour cells express primary cilia, with a variable number expressing Smo at the cilia tip. Clonal growth of these tumour cells could be inhibited by blocking the Shh ligand with a monoclonal antibody, or by inactivating Smo with the small molecule LDE225 (Novartis). These data suggest that activation of Smo by Hh ligand at the level of the primary cilium is a crucial step in the initiation and self-renewal of SCLC. By contrast, cilia were rarely observed in human SCLC cells, both in vitro and in vivo. Based on our hypothesis that Hh signaling may be important in the regeneration of SCLC stem-like cells following chemotherapy, we employed a primary xenograft model in which we could identify minimal residual disease following treatment with single-agent carboplatin. Following chemotherapy, more than 50% of the residual tumour cells expressed primary cilia, and in the majority of these, Smo could be detected in the tip. In addition, marked upregulation of Shh ligand expression was observed. However, when these tumours were allowed to regrow to their original size, expression of Shh and cilia returned to the same pattern as seen in the treatment naive tumour, once again supporting a role for cilia-dependent activation of Smo by Hh ligand in SCLC stem-like cells. Furthermore, treatment of mice following chemotherapy with several small molecule inhibitors of Smo, including LDE225, delayed the regeneration of these tumours in vivo. One major controversy surrounding the role Hh signaling in cancer relates to the role of tumour stroma as a target of Shh signaling. To exclude this as a potential mechanism, we used two approaches. First, we crossed the mouse genetic model of SCLC referred to above with a reporter mouse in which LacZ activity can be used to measure Hh pathway activation. In this model, heterogeneous LacZ expression was clearly seen in tumour cells, but not stromal cells. Second, we recreated the carboplatin regeneration model of human SCLC in vitro using a three dimensional, stroma-free clonogenic assay. Transient manipulation of Hh signaling at all levels of the pathway by antibody blockade, siRNA, transfection or small molecule treatment dramatically affected long term cloning capacity in SCLC cells. Moreover, SCLC cells that survived an LD~95~ treatment with carboplatin in vitro demonstrated a marked increase in clonal capacity that was even more sensitive to Hh pathway blockade. Confocal immunofluoresence imaging of these cells revealed expression of Smo in the tips of numerous primary cilia, demonstrating that cell autonomous Hh pathway activation could be observed in a subset of innately chemoresistant, stem-like cells. In the last 5 years, targeted deletion of different components of the primary cilium in mice has dramatically expanded our understanding of the role of these structures in cell signaling. Once considered vestigial, cilia are now recognised as key signaling nodes for Hh, WNT, Notch, PDGF and mTOR signaling. In addition, we have identified heterogeneous expression of primary cilia in several other tumour models, most strikingly in osteosarcoma. More recently, we have shown that knockdown of KIF3a, an essential component of cilia assembly, causes a more dramatic loss of cloning capacity than inhibition of Hh signaling alone. These data suggest that in a subset of self-renewing tumour cells, cilia-dependent signaling pathways in addition to Hh are of importance, and may represent novel therapeutic targets. We are currently using genetically modified mouse models of cancer in which we can conditionally knockout Kif3a to address this question.

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      MS02.3 - Micro-RNA in Lung Cancer (ID 464)

      14:00 - 15:30  |  Author(s): G. Sozzi, M. Boeri, U. Pastorino

      • Abstract
      • Presentation
      • Slides

      Abstract
      Lung cancer, for its high incidence and mortality, is the most common cause of death from cancer in many developed countries. In contrast to other cancers, there has been almost no improvement in the 5-year survival rates of lung cancer in the past 30 years, rate just above 10% in Europe, primarily because lung cancer is detected in most cases in an advanced stage. Detecting lung cancer at an earlier stage and, ideally, predicting who will develop the disease and particularly the most aggressive forms of cancer are the biggest challenge. Imaging via low-dose computed tomography (LDCT) scanning is being actively evaluated as a screening tool for early detection of lung cancer in high risk patients but, although the positive results in mortality reduction reported in the large NLST trial (1) were very promising, at present, the real efficacy of LDCT lung cancer screening in heavy smokers remains a controversial issue (2). Nonetheless, the high false positive rates of LDCT, leading to multiple screening rounds, the issue of over-diagnosis, the unnecessary and sometimes harmful diagnostic follow-up and the costs underscore the need for non-invasive complementary biomarkers for standardized use. MicroRNAs are small, non coding, endogenous single–stranded ribonucleic acids with regulatory functions that are involved in tuning of many important pathways, including developmental and oncogenic pathways. Because of their fundamental role in development and differentiation, their involvement in the biological mechanisms underlying tumorigenesis, as well as their low complexity, stability and easily detection, they represent a promising class of tissue and blood-based biomarkers of cancer (3). We explored miRNA expression profiles of lung tumors and normal lung tissues from cases with variable prognosis identified in a completed spiral-CT screening trial with extensive follow-up (4). We found a panel of deregulated miRNAs discriminating normal lung tissue versus lung cancer and significant association of miRNA expression profiles in both tumor and non-involved lung tissue with clinical-pathological characteristics of the patients such as tumor histotype, tumor growth rate, disease free survival. miRNA expression profile in tumor and normal lung tissues from patients identifed in the first two years of the screening, including mainly Stage Ia ADC with excellent survival, was found to be significantly different from the profile of subjects with more aggressive tumors appearing in later years of screening, independently from tumor Stage. Overall these results indicate that, both in tumors and in non involved lung tissues, miRNA signatures are able to discriminate patients according to tumor aggressiveness, independently from Stage and type. We have then investigated mirRNA profiles in plasma samples from cases and controls belonging to two independent LDCT screening trials with extensive follow-up where multiple plasma samples, collected before and at time of disease detection were available. We reported that miRNA profiling in plasma samples collected 1–2 yrs before the onset of disease, at the time of lung cancer detection by LDCT and in disease-free smokers, resulted in the generation of four miRNA signatures with strong predictive, diagnostic, and prognostic potential (4). Overall, these results suggest that plasma miRNA profiles might be helpful in pinpointing those early stage tumors at high risk of aggressive evolution that would need additional treatments. We recently completed a large validation study where the diagnostic performance of the plasma-based miRNA test was retrospectively evaluated in samples prospectively collected from smoker subjects within the MILD trial. In this study, 1,000 consecutive MILD plasma samples collected from June 2009 to July 2010 among lung cancer-free individuals enrolled in the trial and all patients with lung cancer diagnosed by September 2012 (n=85) were obtained. In patients we analyzed plasma samples collected both pre-disease (four to 35 months before lung cancer detection, median lag time of 15 months) and at the time of diagnosis. Custom-made microfluidic cards containing the 24 microRNAs composing the signatures identified in the exploratory study were created, and on each card eight plasma samples were analyzed per time. Since the goal of this study was to combine the plasma miRNA assay with LDCT results, in order to have a clinical useful tool to classify plasma samples, we developed a three-level miRNA signature classifier (MSC) of Low, Intermediate, or High risk of disease with subject categorization to one of these three risk groups based on pre-defined cut-points of positivity for the four different expression signatures of the 24 miRNAs previously identified. The results of this large validation study indicates that MSC is a significant diagnostic instrument for lung cancer detection with prognostic performance and support the combined use of MSC and LDCT to improve the efficacy of lung cancer screening (5). References 1. Kramer BS, Berg CD, Aberle DR et al. Lung cancer screening with low-dose helical CT: results from the National Lung Screening Trial (NLST). J Med Screen. 2011;18:109-111. 2. Pastorino U, Rossi M, Rosato V, Marchianò A, Sverzellati N, Morosi C, Fabbri A, Galeone C, Negri E, Sozzi G, Pelosi G, La Vecchia C. Annual or biennial CT screening versus observation in heavy smokers: 5-year results of the MILD trial. Eur J Cancer Prev. 2012 May;21(3):308-15 3. Boeri M., Pastorino U. and Sozzi G. Role of MicroRNAs in Lung Cancer: MicroRNA Signatures in Cancer Prognosis. Cancer J. 2012 May;18(3):268-74 4. Boeri M, Verri C, Conte D, Roz L, Modena P, Facchinetti F, Calabrò E, Croce CM, Pastorino U, Sozzi G. MicroRNA signatures in tissues and plasma predict development and prognosis of computed tomography detected lung cancer. Proc Natl Acad Sci U S A. 2011 Mar 1;108(9):3713-8. 5. Sozzi G, Boeri M, Rossi M, Verri C, Suatoni P, Bravi F, Roz L, Conte D, Grassi M, Sverzellati N, Marchiano’ A, Negri, La Vecchia C, Pastorino U. Clinical Utility of a Plasma-based microRNA Signature Classifier within Computed Tomography Lung Cancer Screening: A Correlative MILD Trial Study. Submitted

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      MS02.4 - Targeting Epigenetic Changes in Lung Cancer (ID 465)

      14:00 - 15:30  |  Author(s): C.M. Rudin

      • Abstract
      • Presentation
      • Slides

      Abstract
      The process of carcinogenesis is driven by clonally maintained genetic and epigenetic events that lead to aberrant cell proliferation, inhibit cell death, promote cell dissemination, and affect other key pathways. Research in the past decade has led to new insights into the epigenetic mechanisms controlling gene expression, and into the multiple ways in which these mechanisms are specifically disrupted in cancer. Epigenetic control of gene expression is dependent on modifications of the DNA itself, primarily methylation at CpG dinucleotides, and also by a host of site-specific protein modifications of histones, histone modifiers, and transcriptional machinery. Progress in understanding the multiple layers of epigenetic control is leading to the development and clinical testing of anti-cancer agents specifically targeting these aberrant pathways. DNA methylation and histone acetylation are two well established epigenetic control mechanisms that are known to be aberrantly regulated in essentially all cancers, including lung cancer. We conducted an exploratory phase I/II trial combining an inhibitor of DNA methyltransferase, azacitidine, and an inhibitor of histone deacetylase, entinostat, in patients with recurrent metastatic non-small cell lung cancer. DNA methylation of gene promoters, and loss of histone acetylation, are coordinately regulated processes that can lead to selective silencing of gene expression: this mechanism has been implicated in silencing key tumor suppressor genes in cancer. Treatment with the combination of azacitidine and entinostat led to rare but impressive objective responses, including a complete response in a patient with extensively pretreated disease. In addition, a surprising fraction of patients experienced objective responses to the immediate subsequent therapy, including standard cytotoxic agents and investigational agents targeting the PD-1/PD-L1 immune checkpoint pathway. Preclinical data offer some potential explanations for this observation: many relevant immunoregulatory pathways in both tumor cells and immune effectors are markedly affected by azacitidine. We are now following up on the priming hypotheses suggested by these data, in randomized phase II studies assessing whether limited duration epigenetic therapy can enhance subsequent chemotherapy or immunotherapy efficacy in patients with advanced non-small cell lung cancer. This study represents an initial foray into combinatorial epigenetic strategy in lung cancer: many other strategies are now possible and are being pursued. “Second generation” agents targeting DNA methyltransferase, including an oral formulation of azacitidine and a prodrug, SG-110, are in early phase clinical development. So too are newer histone deacetylase inhibitors differing in specificity, selectivity, route of administration, and pharmacokinetics. Among the exciting new horizons in epigenetic therapy are new agents targeting more recently defined modifiers of epigenetic control, including many of the readers, writers, and erasers of histone modification. The recent remarkable expansion in knowledge about epigenetic regulatory pathways, and how they become dysregulated in cancer, is opening new therapeutic opportunities in lung cancer and other diseases.

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Author of

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    MS02 - Stem Cells and Epigenetics in Lung Cancer (ID 19)

    • Event: WCLC 2013
    • Type: Mini Symposia
    • Track: Biology
    • Presentations: 1
    • +

      MS02.3 - Micro-RNA in Lung Cancer (ID 464)

      14:00 - 15:30  |  Author(s): G. Sozzi

      • Abstract
      • Presentation
      • Slides

      Abstract
      Lung cancer, for its high incidence and mortality, is the most common cause of death from cancer in many developed countries. In contrast to other cancers, there has been almost no improvement in the 5-year survival rates of lung cancer in the past 30 years, rate just above 10% in Europe, primarily because lung cancer is detected in most cases in an advanced stage. Detecting lung cancer at an earlier stage and, ideally, predicting who will develop the disease and particularly the most aggressive forms of cancer are the biggest challenge. Imaging via low-dose computed tomography (LDCT) scanning is being actively evaluated as a screening tool for early detection of lung cancer in high risk patients but, although the positive results in mortality reduction reported in the large NLST trial (1) were very promising, at present, the real efficacy of LDCT lung cancer screening in heavy smokers remains a controversial issue (2). Nonetheless, the high false positive rates of LDCT, leading to multiple screening rounds, the issue of over-diagnosis, the unnecessary and sometimes harmful diagnostic follow-up and the costs underscore the need for non-invasive complementary biomarkers for standardized use. MicroRNAs are small, non coding, endogenous single–stranded ribonucleic acids with regulatory functions that are involved in tuning of many important pathways, including developmental and oncogenic pathways. Because of their fundamental role in development and differentiation, their involvement in the biological mechanisms underlying tumorigenesis, as well as their low complexity, stability and easily detection, they represent a promising class of tissue and blood-based biomarkers of cancer (3). We explored miRNA expression profiles of lung tumors and normal lung tissues from cases with variable prognosis identified in a completed spiral-CT screening trial with extensive follow-up (4). We found a panel of deregulated miRNAs discriminating normal lung tissue versus lung cancer and significant association of miRNA expression profiles in both tumor and non-involved lung tissue with clinical-pathological characteristics of the patients such as tumor histotype, tumor growth rate, disease free survival. miRNA expression profile in tumor and normal lung tissues from patients identifed in the first two years of the screening, including mainly Stage Ia ADC with excellent survival, was found to be significantly different from the profile of subjects with more aggressive tumors appearing in later years of screening, independently from tumor Stage. Overall these results indicate that, both in tumors and in non involved lung tissues, miRNA signatures are able to discriminate patients according to tumor aggressiveness, independently from Stage and type. We have then investigated mirRNA profiles in plasma samples from cases and controls belonging to two independent LDCT screening trials with extensive follow-up where multiple plasma samples, collected before and at time of disease detection were available. We reported that miRNA profiling in plasma samples collected 1–2 yrs before the onset of disease, at the time of lung cancer detection by LDCT and in disease-free smokers, resulted in the generation of four miRNA signatures with strong predictive, diagnostic, and prognostic potential (4). Overall, these results suggest that plasma miRNA profiles might be helpful in pinpointing those early stage tumors at high risk of aggressive evolution that would need additional treatments. We recently completed a large validation study where the diagnostic performance of the plasma-based miRNA test was retrospectively evaluated in samples prospectively collected from smoker subjects within the MILD trial. In this study, 1,000 consecutive MILD plasma samples collected from June 2009 to July 2010 among lung cancer-free individuals enrolled in the trial and all patients with lung cancer diagnosed by September 2012 (n=85) were obtained. In patients we analyzed plasma samples collected both pre-disease (four to 35 months before lung cancer detection, median lag time of 15 months) and at the time of diagnosis. Custom-made microfluidic cards containing the 24 microRNAs composing the signatures identified in the exploratory study were created, and on each card eight plasma samples were analyzed per time. Since the goal of this study was to combine the plasma miRNA assay with LDCT results, in order to have a clinical useful tool to classify plasma samples, we developed a three-level miRNA signature classifier (MSC) of Low, Intermediate, or High risk of disease with subject categorization to one of these three risk groups based on pre-defined cut-points of positivity for the four different expression signatures of the 24 miRNAs previously identified. The results of this large validation study indicates that MSC is a significant diagnostic instrument for lung cancer detection with prognostic performance and support the combined use of MSC and LDCT to improve the efficacy of lung cancer screening (5). References 1. Kramer BS, Berg CD, Aberle DR et al. Lung cancer screening with low-dose helical CT: results from the National Lung Screening Trial (NLST). J Med Screen. 2011;18:109-111. 2. Pastorino U, Rossi M, Rosato V, Marchianò A, Sverzellati N, Morosi C, Fabbri A, Galeone C, Negri E, Sozzi G, Pelosi G, La Vecchia C. Annual or biennial CT screening versus observation in heavy smokers: 5-year results of the MILD trial. Eur J Cancer Prev. 2012 May;21(3):308-15 3. Boeri M., Pastorino U. and Sozzi G. Role of MicroRNAs in Lung Cancer: MicroRNA Signatures in Cancer Prognosis. Cancer J. 2012 May;18(3):268-74 4. Boeri M, Verri C, Conte D, Roz L, Modena P, Facchinetti F, Calabrò E, Croce CM, Pastorino U, Sozzi G. MicroRNA signatures in tissues and plasma predict development and prognosis of computed tomography detected lung cancer. Proc Natl Acad Sci U S A. 2011 Mar 1;108(9):3713-8. 5. Sozzi G, Boeri M, Rossi M, Verri C, Suatoni P, Bravi F, Roz L, Conte D, Grassi M, Sverzellati N, Marchiano’ A, Negri, La Vecchia C, Pastorino U. Clinical Utility of a Plasma-based microRNA Signature Classifier within Computed Tomography Lung Cancer Screening: A Correlative MILD Trial Study. Submitted

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    O01 - Prognostic and Predictive Biomarkers I (ID 94)

    • Event: WCLC 2013
    • Type: Oral Abstract Session
    • Track: Medical Oncology
    • Presentations: 1
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      O01.04 - DISCUSSANT (ID 3909)

      10:30 - 12:00  |  Author(s): G. Sozzi

      • Abstract
      • Presentation
      • Slides

      Abstract not provided

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