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K. O'Byrne



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    MO04 - Lung Cancer Biology I (ID 86)

    • Event: WCLC 2013
    • Type: Mini Oral Abstract Session
    • Track: Biology
    • Presentations: 1
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      MO04.03 - Chromosomal and mutational analysis of the cisplatin resistant phenotype in NSCLC cells (ID 3313)

      16:25 - 16:30  |  Author(s): 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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    MO07 - NSCLC - Targeted Therapies II (ID 114)

    • Event: WCLC 2013
    • Type: Mini Oral Abstract Session
    • Track: Medical Oncology
    • Presentations: 1
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      MO07.13 - Efficacy of afatinib vs. chemotherapy in treatment-naïve patients with non-small cell lung cancer (NSCLC) harbouring activating EGFR mutations with or without metastatic brain disease (ID 1923)

      17:25 - 17:30  |  Author(s): K. O'Byrne

      • Abstract
      • Presentation
      • Slides

      Background
      Afatinib, an irreversible ErbB Family Blocker, was superior to pemetrexed/cisplatin in previously untreated patients with epidermal growth factor receptor (EGFR) mutation-positive non-small cell lung cancer (NSCLC) in a global Phase III trial, LUX-Lung 3. In patients with the two most common EGFR mutations (Del19, L858R) median progression-free survival (PFS) was 13.6 vs. 6.9 months (HR=0.47, 95% CI: 0.34–0.65; p<0.0001). Here we present the results for subgroups of patients with or without brain metastases (BM) with NSCLC harbouring common EGFR mutations.

      Methods
      In LUX-Lung 3 EGFR mutation-positive patients were randomized 2:1 to afatinib 40 mg daily or up to 6 cycles of pemetrexed/cisplatin at standard doses. Patients with stable BM (asymptomatic, stable >4 weeks with no treatment required) were allowed. Presence of BM was documented by the investigator during screening. Tumour assessments were performed every 6 weeks until 48 weeks and every 12 weeks thereafter until progression, and reviewed independently and by the investigator.

      Results
      308 patients with common EGFR mutations were randomized (afatinib: 204, pemetrexed/cisplatin: 104), including 35 with baseline BM (afatinib: 20, pemetrexed/cisplatin: 15). Of these, Del19 mutation was detected in 11 (afatinib) and 8 (pemetrexed/cisplatin) patients and L858R in 9 (afatinib) and 7 (pemetrexed/cisplatin) patients. The baseline characteristics of patients with or without BM were comparable (females: 74% vs. 66%, median age: 61 vs. 62 years, ECOG 0: 31% vs. 41%, median time since diagnosis: 1.2 vs. 1.1 months, respectively). Within the BM group, baseline characteristics were balanced between treatment arms with the exception of ECOG 1; 80% of afatinib-treated patients had ECOG 1 compared with 53% of those treated with pemetrexed/cisplatin. Median PFS by independent review was 13.7 (afatinib) vs. 8.1 (pemetrexed/cisplatin) months in patients without BM (HR=0.47, 95% CI: 0.33–0.68; p<0.0001), and 11.1 (afatinib) vs. 5.4 (pemetrexed/cisplatin) months in patients with BM (HR=0.52, 95% CI: 0.22–1.23; p=0.13). Objective response in patients without BM was 59% (afatinib) vs. 23% (pemetrexed/cisplatin), odds ratio=4.8, p<0.0001, and 70% (afatinib) vs. 20% (pemetrexed/cisplatin), odds ratio=11.0, p=0.007, in patients with BM. Investigator review showed a median PFS of 13.6 (afatinib) vs. 6.9 (pemetrexed/cisplatin) months in patients without BM (HR=0.38, 95% CI: 0.27–0.53; p<0.0001), and 6.7 (afatinib) vs. 5.4 (pemetrexed/cisplatin) months in those with BM (HR=0.67, 95% CI: 0.29–1.57; p=0.36). By investigator review, progressive disease in the brain was observed for 4.2% (7/167) and 3.7% (3/82) of patients without BM at baseline for afatinib and pemetrexed/cisplatin, respectively. All but one of these patients (on afatinib) had intracranial progression only. The median (range) time to progression in the brain in this small group was 11.6 (1.3, 20.2) months (afatinib) and 5.5 (2.6, 8.2) months (pemetrexed/cisplatin).

      Conclusion
      In patients with previously untreated NSCLC harbouring common EGFR mutations afatinib remains efficacious regardless of the presence or absence of BM. Control of synchronous asymptomatic BM with afatinib compares favourably with existing data for cranial radiation therapy.

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    MO09 - Mesothelioma I (ID 120)

    • Event: WCLC 2013
    • Type: Mini Oral Abstract Session
    • Track:
    • Presentations: 1
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      MO09.08 - NF-kB in cisplatin resistance and as a prognostic marker in Malignant pleural mesothelioma (ID 3338)

      16:55 - 17:00  |  Author(s): K. O'Byrne

      • Abstract
      • Presentation
      • Slides

      Background
      Malignant pleural mesothelioma (MPM) is an aggressive inflammatory cancer associated with exposure to asbestos. Currently rates of MPM are rising and estimates indicate that the incidence of MPM will peak in western world within the next 10-15 years. Untreated, MPM has a median survival time of 6 months, with poor survival rates for most patients after 24 months of diagnosis. Nuclear Factor kappa B (NF-kB) is a pro-inflammatory transcription factor which is activated in many cancer types, including MPM. The NF-kB pathway regulates important cellular processes including survival and proliferation signals, which are often found to be dysregulated in cancer. Furthermore, we and others have shown that increased NF-kB activation is linked to development of cisplatin resistance. We aim to outline the potential role of NF-kB as a mediator of cisplatin resistance in MPM and determine its value as a potential candidate for therapeutic intervention.

      Methods
      NF-kB expression was examined in a cohort of MPM patients (n=200) by IHC, and correlated with clinicopathological variables and survival. NF-kB expression was examined in both a panel of MPM cell lines and isogenic parent/cisplatin resistant cell lines by Western blot analysis. The effect of NF-kB inhibition on cellular proliferation was measured by BrdU assay, in a panel of MPM and isogenic parent/cisplatin resistant cell lines, using the novel NF-kB inhibitor Dehydroxymethylepoxyquinomicin (DHMEQ). In addition, the effect of DHMEQ on nuclear translocation of NF-kB was examined by high content screening (HCS).

      Results
      Cytoplasmic or membranous immunostaining was seen in the majority of tumour samples (96.5%), but nuclear localisation of NF-kB was seen in only 11% cases. Kaplan-Meier survival analysis showed that nuclear NF-kB expression correlated with reduced survival (p=0.05). There was no significant correlation between the level of expression of NF-kB and standard clinicopathological parameters. NF-kB was expressed in all MPM cell lines tested to a varying extent (n=20), with no associations to histology. NF-kB levels were shown to be elevated in cisplatin resistant cell lines when compared to the isogenic parent from which they were derived. DHMEQ was shown to reduce nuclear translocation of NF-kB, inhibiting cell proliferation in all cell lines but to a lesser extent in NCI 2596 cells which have low NFkB expression.

      Conclusion
      Nuclear NFkB expression is a poor prognostic factor in MPM. DHMEQ, which inhibits nuclear translocation of NF-kB, inhibits cell proliferation in MPM cell lines. Furthermore, increased NF-kB expression in resistant cells suggests this pathway may play a role in development of cisplatin resistance in MPM. Inhibition of NF-kB may therefore prove to be of potential therapeutic benefit in MPM treatment and re-sensitisation of resistant MPM to cisplatin.

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    MO10 - Molecular Pathology II (ID 127)

    • Event: WCLC 2013
    • Type: Mini Oral Abstract Session
    • Track: Pathology
    • Presentations: 1
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      MO10.07 - ALK immunohistochemistry and fluorescence in-situ hybridization in Lung adenocarcinomas from the ETOP Lungscape tumour cohort (ID 2267)

      16:50 - 16:55  |  Author(s): K. O'Byrne

      • Abstract
      • Presentation
      • Slides

      Background
      The European Thoracic Oncology Platform LungScape database contains 2614 cases of primary resected lung carcinoma from 16 centres with patient demographics, pathological tumour data and detailed clinical follow-up. A total of 1281 cases of adenocarcinoma with >2 years clinical follow-up were selected for analysis of ALK status by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH). Test positive cases were matched, in order of importance at ratio 1:2, by stage, gender, smoking status, study centre, year of surgery and age with test negative cases -both for IHC and for FISH testing.

      Methods
      Testing was performed in all centres using the same protocol (IHC: Novocastra 5A4 clone antibody at 1:10 dilution, Novolink detection system. FISH: Abbott Vysis ALK break-apart probe). Each centre passed an external QA test using unknown cases in a tissue microarray before conducting the LungScape tumour testing. IHC was scored according to three intensity scores (1+, 2+, 3+) using ‘objective’ methodology previously described [1]. Maximum staining intensity was recorded. Any IHC staining was defined as IHC positive result. FISH preparations were assessed according to the Vysis protocol on all 82 IHCpositive cases plus their 164 IHCnegative matches.

      Results

      IHC cases, n=1281 FISH positive(264 tested)
      IHC negative 1199 (93.6%) 0 (0.0% of 164 controls) FISH specificity: 100%
      IHC 1+ 43 (3.35%) 2 (4.6% of IHC 1+)
      IHC 2+ 16 (1.25%) 6 (37.5% of IHC 2+)
      IHC 3+ 23 (1.8%) 20 (87% of IHC 3+)
      IHC any positive 82 (6.4%) 28 (34.1% of IHC+) FISH sensitivity: 34.1%
      FISH sensitivity was 87% for IHC 3+. IHCpositive/FISHnegative cases (n=54) were mostly IHC 1+ (75.9%), sometimes IHC 2+ (18.5%) and rarely IHC 3+ (5.5%). The frequency of never smokers was higher in the ALK IHCpositive group (29.3%) versus IHCnegative group (18.3%) {p=0.011}. Age, gender and tumour stage did not differ between IHC groups. The hazard of an event for IHCpositive cases decreases by 32% in relapse-free survival {RFS; p=0.03} and by 38% in either time-to-relapse {TTR; p=0.02} or overall survival {OS; p=0.016}. Multivariate models -adjusted for patient and tumour characteristics- indicated that IHC-ALK was a significant predictor for all three time-to-event outcomes (RFS, TTR, OS). In stratified Cox analysis, significantly higher OS was retained in the IHCpositive (HR=0.59, p=0.04) and FISHpositive (HR=0.34, p=0.03) cases in the matched cohorts, while conditional logistic regression yielded non-significant associations with 3-year survival status.

      Conclusion
      In this large cohort of surgically resected primary lung adenocarcinoma: ALK IHC positivity was 6.4%. IHC 3+ staining (prevalence 1.8%) showed 87% probability of ALK FISH positivity ALK IHC positivity was higher in never smokers and related to better clinical outcome ALK testing can be reliably implemented across multiple laboratories {1} Ruschoff et al. Virchows Arch. 2010;457(299-307).

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    P1.01 - Poster Session 1 - Cancer Biology (ID 143)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 4
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      P1.01-001 - VEGF is an autocrine survival factor in non-small cell lung cancer, mediating its effects via the Neuropilin-1 receptor. (ID 3344)

      09:30 - 09:44  |  Author(s): K. O'Byrne

      • Abstract

      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.

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      P1.01-011 - Targeting the Urokinase Plasminogen Activator (uPA) System to overcome cisplatin resistance in NSCLC (ID 3347)

      11:50 - 12:04  |  Author(s): K. O'Byrne

      • Abstract

      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.

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      P1.01-012 - The KDM6 Lysine Demethylases are candidate therapeutic targets in Malignant Pleural Mesothelioma (ID 3282)

      12:04 - 12:18  |  Author(s): K. O'Byrne

      • Abstract

      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.

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      P1.01-016 - Targeting NF-κB regulated pathways to overcome cisplatin resistance in non small cell lung cancer (ID 3270)

      13:00 - 13:14  |  Author(s): K. O'Byrne

      • Abstract

      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.

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    P1.02 - Poster Session 1 - Novel Cancer Genes and Pathways (ID 144)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 3
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      P1.02-007 - Validation and Function of a Novel miRNA signature in Cisplatin Resistant Non-Small Cell Lung Cancer Cells (ID 1410)

      09:30 - 09:30  |  Author(s): K. O'Byrne

      • Abstract

      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.

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      P1.02-008 - hSSB1: an essential regulator of genomic integrity in lung cancer (ID 2045)

      09:30 - 09:30  |  Author(s): K. O'Byrne

      • Abstract

      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.

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      P1.02-013 - SDHB is overexpressed and may be a candidate target for therapeutic intervention in Malignant Pleural Mesothelioma (ID 3287)

      09:30 - 09:30  |  Author(s): K. O'Byrne

      • Abstract

      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.

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    P1.05 - Poster Session 1 - Preclinical Models of Therapeutics/Imaging (ID 156)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 3
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      P1.05-022 - BET Bromodomains: Are they a potential therapeutic targets in Malignant Pleural Mesothelioma? (ID 3269)

      09:30 - 09:30  |  Author(s): K. O'Byrne

      • Abstract

      Background
      Malignant pleural mesothelioma (MPM) is an aggressive cancer affecting the pleura. Treatment options are limited and most patients die within 24 months of diagnosis. The recommended first line chemotherapy for MPM is a combination of cisplatin/pemetrexed (or alternatively Raltitrexed), and there is no recommeneded second-line therapy. As such new therapeutic approaches are required for the management of MPM. Bromodomain and extra terminal domain (BET) proteins function as epigenetic signaling factors that associate with acetylated histones to facilitate the transcription of target genes. Various inhibitors targeting the activity of BET proteins have been developed and have shown potent antiproliferative effects in hematological cancers, and more recently been studied for in vitro efficacy in lung adenocarcinoma cell lines (1). We examined the expression of various members of the BET in MPM and assessed the effects of one of these inhibitors (JQ-1) to determine if this family could represent a novel candidate target(s) for therapeutic intervention in MPM.

      Methods
      A panel of MPM cell lines inluding the normal pleural cells LP9 & Met5A were screened for expression of BRD2 and BRD4 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 expression of a known target of the BET inhibitor JQ1, oncogenic transcription factor FOSL1 was also examined. The effects of a small molecule inhibitor of BET proteins (JQ-1) on cellular proliferation was examined (BrdU ELISA).

      Results
      We show that the expression of the BRD2 and BRD4 variants are detectable in all cell lines across our panel of cell lines. In primary tumours however, the expression of BRD2 was very significantly downregulated (p=0.0006). BRD4 comprises 2 transcript variants, a long variant (BRD4L – Refseq NM_058243.2), and a short variant (BRD4S – Refseq NM_014299.2). BRD4L was not significantly affected in malignant MPM compared to benign pleura, whereas BRD4S was significantly elevated in the tumours compared to the benign pleura (p<0.05). When separated across histological subtype BRD2 was significantly decreased across all histological subtypes (p=0.0009). FOSL1 a candidate target of JQ1 (1) was found to be significantly elevated in malignant MPM compared to benign pleura (p<0.05). Treatment of REN/ NCI-H226 cells with JQ1 caused significant inhibition of cellular proliferation, with NCI-H226 being more sensitive than REN to this compound.

      Conclusion
      The BET domain proteins are altered in MPM, and a small molecule inhibitor of this protein shows significant anti-proliferative effects in MPM cell lines. We continue to asess the effects of this compound on gene expression and cellular health by other methodologies to confirm its potential utility in the treatment of MPM. Reference: 1. Lockwood WW et al., (2012). Proc Natl Acad Sci U S A. 109(47):19408-13.

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      P1.05-023 - The KDM4/JMJD2 Lysine Demethylases are candidate therapeutic targets in Malignant Pleural Mesothelioma (ID 3278)

      09:30 - 09:30  |  Author(s): K. O'Byrne

      • Abstract

      Background
      Malignant pleural mesothelioma (MPM) is a rare aggressive cancer of the pleura associated with exposure to asbestos. Treatment options are limited, and the current standard of care for MPM patients is a combination of cisplatin and pemetrexed (or alternatively cisplatin and raltitrexed). Despite this treatment option, almost all patients die within 24 months of diagnosis. Therefore, new therapeutic options are urgently required for the treatment of MPM. Lysine Demethylases (KDMs) represent novel targets for the treatment of cancer. Overexpression of KDMs are common in many cancers, and play important roles in tumorigenesis. The jumonji (JMJ) family of lysine demethylases are Fe2+- and α-ketoglutarate-dependent oxygenases that are essential components of regulatory transcriptional chromatin complexes. One such family, the KDM4/JMJD2 family, may therefore be altered in MPM and could represent a novel candidate target for intervention

      Methods
      A panel of MPM cell lines inluding the normal pleural cells LP9 & Met5A were screened for expression of KDM4 family members 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 KDM4A/JMJD2A, 3,4-dihydroxybenzaldehyde (protocatechuic aldehyde or PA) on cellular proliferation and gene expression were examined.

      Results
      We show that the expression of the KDM4 family is ubiquitously expressed across our panel of cell lines. In primary tumours however, the expression of KDM4 members KDM4A, KDM4B and KDM4C were significantly elevated in malignant MPM compared to benign pleura. Treatment of REN/ NCI-H226 cells with the small molecule PA caused significant inhibition of cellular proliferation (p<0.0001). We continue to asess the effects of this compound on gene expression and cellular health by other methodologies to confirm its potential utility in the treatment of MPM.

      Conclusion
      The KDM4/JMJD2 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. Targeting this protein may have important future implications for the management of MPM.

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      P1.05-024 - PARP inhibition increases sensitivity of NSCLC cells to cisplatin (ID 3300)

      09:30 - 09:30  |  Author(s): K. O'Byrne

      • Abstract

      Background
      Primary and acquired resistance to platinum agents is a serious clinical problem in lung cancer. Its mechanisms are probably multifactorial and remain poorly understood. Enhanced DNA repair can lead to increased cell viability in the face of DNA damage and has been proposed to be important in mediating platinum resistance. PARPs (poly(ADP-ribose) polymerases) are a family of nuclear enzymes that regulate the repair of DNA single-strand breaks (SSBs). Cisplatin sensitivity and DNA repair mechanisms following treatment with the PARP inhibitor, PJ34, was investigated in this study.

      Methods
      A panel of isogenic cisplatin resistant (CisR) NSCLC cells lines (MOR, SKMES-1, H1299) previously generated in our laboratory were used. The cisplatin resistant phenotype was initially assessed by treating CisR and parental (PT) cells with increasing doses of cisplatin (0-80uM) for 72h, after which time, cell proliferation was measured (BrdU). The effects of PJ34 on cell survival were also examined in a similar dose-response study. IC~25~ concentrations were calculated for each cell line using GraphPad statistical software. Cells were treated with PJ34 (IC~25~) alone, or in combination with cisplatin and cell survival/proliferation measured after 72h. Under similar experimental conditions, RNA was isolated from cells from which cDNA was reverse transcribed. All cell lines were screened for PARP1, PARP2, BRCA1, BRCA2 and ERCC1 mRNA at basal levels, and in response to treatment (RT-PCR). To investigate DNA double strand break (DSB) repair capacity in our panel of cell lines in response to PARP inhibition and cisplatin, phosphorylated γH2AX foci was examined by High Content Analysis (HCA) following treatment of cell lines for 24h. Cisplatin-DNA adduct formation (Pt-GpG) was studied following treatment of cells for 24h. Cells (1x10[6]/ml) were spotted on Superfrost® Gold glass slides. Immunofluorescence staining of specific DNA platination products, and quantification of adducts, was performed using an antibody that specifically recognises cisplatin-GpG DNA adducts.

      Results
      MOR and H1299 CisR cells were significantly more resistant to cisplatin (10µM and 20µM) compared to PT cells. SKMES-1 CisR cells were also significantly more resistant at 10µM, 20µM and 40µM cisplatin. While PJ34 had no effect on NSCLC cells when treated as a single agent, cell proliferation was significantly inhibited in MOR and H1299 cells when used in combination with cisplatin. No effect however was observed in our panel of CisR cell lines. While baseline expression levels of PARP1/2, BRCA1/2 and ERCC1 mRNA levels were similar in PT and CisR cell lines, BRAC1/2 mRNA expression was increased in cells treated with cisplatin alone, and in combination with PJ34 in PT cells but not in CisR cells. The formation of γH2AX foci and measurement of cisplatin-GpG DNA adducts in response to PARP inhibition and cisplatin are currently being investigated.

      Conclusion
      Data from this study show that inhibition of NSCLC cells with the PARP inhibitor, PJ34, sensitises lung cancer cells to the cytotoxic effects of the platinum drug, cisplatin. Further studies are warranted to investigate the role of PARP inhibitors in cisplatin resistant NSCLC cells.

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    P1.06 - Poster Session 1 - Prognostic and Predictive Biomarkers (ID 161)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 2
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      P1.06-055 - The RON (MST1R)/MSP pathway is a potential therapeutic target in malignant plural mesothelioma (ID 3250)

      09:30 - 09:30  |  Author(s): K. O'Byrne

      • Abstract

      Background
      Malignant pleural mesothelioma (MPM) is an aggressive inflammatory cancer. Treatment options are limited and drug resistance is common. Thus, there is a need to identify novel therapeutic targets in this disease in order to improve treatment options and survival times. Macrophage stimulating protein (MSP) is the only ligand recognised to bind to the RON receptor (MST1R). RON is a member of the MET proto-oncogene family. The MSP-RON signalling pathway has been implicated in a variety of cellular functions such as macrophage morphogenesis and phagocytosis. De-regulation of this pathway has been linked to tumour progression and metastasis in a number of cancers. We have previously identified RON as frequently activated in MPM and high positivity for RON by IHC was an independent predictor of favourable prognosis.

      Methods
      A panel of mesothelioma cell lines were screened for the expression of MSP and RON at the mRNA (RT-PCR) and protein (Western blot) level. The effect of MSP, IMC-RON8 (a humanised IgG1 monoclonal antibody), LCRF004 (a small molecule inhibitor) and NRWHE (a small peptide) was examined in the H226 cell line using proliferation (BrdU ELISA), apoptosis (Multi-parameter apoptosis assay) and migration assays (xCELLigence). A phospho-kinase proteome profiler array was utilised to detect the downstream signalling pathways activated upon MSP stimulation. The expression of MSP and the macrophage marker, CD-68, was examined by IHC using MPM TMAs. Studies are ongoing to determine the effect of the LCRF004 compound in vivo using a xenograft murine model with the H226 cells.

      Results
      The mRNA and protein levels of RON and MSP were differentially expressed in a panel of MPM cell lines. Treatment with LCRF004 resulted in significantly decreased proliferation and increased apoptosis in the H226 cells. MSP was unable to rescue the cells from the effects of LCRF004. NRWHE and RON8 had little effect on either proliferation or apoptosis. All of the compounds examined inhibited the migration capacity of the H226 cells. The combination of LCRF004 and MSP produced a synergistic effect, showing greater inhibition of migration than either compound alone. However, MSP treatment resulted in the up-regulation of a number of phosphor-kinases including Akt, ERK and the Src family. Currently, a number of proteins identified in the array studies are undergoing validation. Results of an in vivo H226 murine model using the LCRF004 compound will be presented at the meeting.

      Conclusion
      From previous work performed in this laboratory, we have determined that high expression of RON in MPM is an independent predictor of favourable prognosis. IHC was performed on a TMA of MPM patient samples and high expression levels of MSP correlated with better survival. There was no association between CD68 staining and MSP, nor correlation of CD68 expression with survival. Targeting the RTK domain of the RON receptor with a small molecule inhibitor is an effective interventional strategy in MPM. The seemingly counter intuitive results obtained from the MPM TMA studies and the in vitro experimental data, may be RON isoform dependant. Additional studies are ongoing to further delineate the RON-MSP axis in MPM.

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      P1.06-056 - Isolation & enumeration of Circulating Tumor Cells in Non-small Cell Lung Cancer, using Screencell & VitaAssay techniques. (ID 3318)

      09:30 - 09:30  |  Author(s): K. O'Byrne

      • Abstract

      Background
      Circulating Tumour Cells (CTCs) have been the subject of much interest as a potential biomarker however methods for isolating CTCs are still in their infancy. A promising method of CTC detection is ScreenCell. This technique uses polycarbonate filtration membranes containing multiple tiny pores. When blood is made to flow across the membrane, tumour cells are captured due to their greater size. Another such method is the use of the modified invasion assay, VitaAssay. This technique uses CAM (Collagen Adhesion Matrix) coated plates to capture CTCs with an invasive phenotype.

      Methods
      Peripheral blood samples were obtained from patients with advanced NSCLC using both Screencell & VitaAssay. In addition healthy blood samples spiked with NSCLC cells were also analysed. ScreenCell: Peripheral blood is diluted with specified buffer and drawn across the Screencell filter using a vacuum tube. The filters with captured fixed cells are then stained with H&E and/or immunocytochemistry. VitaAssay: Peripheral blood mononuclear cells (PBMCs) were obtained by Ficoll density centrifugation. PBMCs were seeded onto VitaAssay plates and cultured for 12-18 hrs. The supernatant is removed and the remaining captured cells are enriched for CTCs due to their invasive phenotype. Captured cells are fixed and stained using immunocytochemistry.

      Results
      Using the ScreenCell technique CTCs were identified by size & morphology using H&E staining. CTCs were detected in 70% of patient samples with. (n=10) Numbers of CTCs detected ranged from 6-82 per ml of blood. In addition, clumps of tumour cells or Circulating Tumour Microemboli (CTM) were detected in 50% of patient samples. (An example of CTM is illustrated in Fig. 1) Cells captured from NSCLC patients using VitaAssay were stained for EpCAM/pan-Cytokeratin and CD45. EpCAM/Pan-CK positive, CD45 negative cells were classed as CTCs. In healthy blood samples spiked with A549 & H2228 cells, approximately 20% (range 9%-26.4%) of spiked cells were recovered using VitaAssay. In NSCLC patients an average of 30.67 CTCs per ml of blood were identified. (range 14-52, n = 6) (An example of CTCs detected by immunocytochemistry is illustrated in Figs. 2 & 3) Figure 1

      Conclusion
      ScreenCell & VitaAssay techniques both appear to be viable methods of isolating & enumerating CTCs, in both model cell-spiking experiments and in NSCLC patient samples, as determined by morphology and antigen expression detected with immunocytochemistry. Of particular interest many of the CTCs isolated using Screencell, were detected as clusters or microemboli. Additional samples are being taken to compare CTC & CTM numbers with clinical outcomes.

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    P1.21 - Poster Session 1 - Diagnosis and Staging (ID 169)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Prevention & Epidemiology
    • Presentations: 1
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      P1.21-004 - Profiling the clinical and diagnostic pathway of Epidermal Growth Factor Receptor (EGFR) mutant Non-Small Cell Lung Cancer (NSCLC) in Ireland (ID 1455)

      09:30 - 09:30  |  Author(s): K. O'Byrne

      • Abstract

      Background
      The presence of an EGFR mutation in NSCLC provides prognostic and therapeutic information for patients and clinicians. This study investigates the clinical behaviour of EGFR mutant (MT) versus EGFR wild-type (WT) NSCLC in an Irish cohort of patients. Differences in the pattern of presentation, metastasis and diagnostic methods between patients with EGFR-MT and WT tumours are poorly characterised. In this retrospective study, we investigated these parameters, variations in EGFR mutation type and resultant impact on overall survival (OS).

      Methods
      Patients with EGFR-MT NSCLC were identified from a National Multi-Institutional database. Patient demographics, diagnostic and clinical data were collected by review of medical records. From the database, EGFR-WT controls matched for age, gender and stage were identified and used as a comparator group. Fisher’s exact and Mann-Whitney tests were used to compare variables between groups. Cox model was used to examine the effect of mutation type on OS.

      Results
      We identified 416 patients with NSCLC. Forty (10%) patients had EGFR-MT positive tumours, of which data were available on 35 (87%) patients. Among patients with EGFR-MT tumours, median age was 64 (range 35-89), 29 (83%) were female, 34 (97%) patients had adenocarcinoma, and 1 (3%) patient had adenosquamous carcinoma. Twelve (34%) patients had resected disease, and 23 (66%) had metastatic disease. At median follow up of 12.8 months, 3 (25%) patients with localised EGFR-MT disease recurred, 0 (0%) of EGFR-WT recurred. There were no significant differences in the pattern of disease between EGFR MT and WT in terms of central/peripheral localisation of primary lesion, or sites of metastasis such as the lung, liver, adrenal gland, bone or brain (p=1.0). Patients with EGFR-MT disease were more likely to be diagnosed via transbronchial biopsy (n=16, 47%) than EGFR-WT (n= 4, 11% p<0.01.) Patients with EGFR-WT disease were more likely to be diagnosed via endobronchial ultrasound/fine needle aspiration (FNA) (n= 21, 58%. p<0.01.) Among those with EGFR-MT disease, 19 (54%) patients had tumours which harboured Exon 19 deletions, and 6 (17%) harboured L858R mutations. The remaining mutations comprised L861Q, V689M and deletions in Exons 18, 20 and 21. Among patients with stage IV disease at diagnosis, the median OS was 20.9 months and 7.3 months for EGFR-MT and EGFR-WT disease respectively (p=0.16.) The median OS for patients who underwent resection was not reached in either group.

      Conclusion
      There were no significant differences in patterns of presentation and metastasis between patients with EGFR-MT and WT tumours in this cohort. Patients with EGFR mutations were more likely to be detected by transbronchial biopsy compared to patients with WT disease, who were diagnosed more commonly by FNA. Possible explanations for this include institutional preferences or ease of tissue acquisition. In this cohort, the most common mutations in EGFR were Exon 19 deletion or L858R. The likelihood of mutation detection might be improved with the inclusion of a full EGFR mutational analysis.

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    P2.01 - Poster Session 2 - Cancer Biology (ID 145)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 3
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      P2.01-010 - A model of chronic inflammation affects the invasive capacity and induces several hallmarks of cancer in a normal bronchial epithelial cell line (ID 3260)

      09:30 - 09:30  |  Author(s): K. O'Byrne

      • Abstract

      Background
      Acute inflammation is usually a rapid and self-limiting process, however it does not always resolve. This leads to the establishment of a chronic inflammatory state and provides the perfect environment for the transformation process. It has been estimated that approximately 25% of all malignancies are initiated or exacerbated by inflammation caused by infectious agents. Furthermore, inflammation is linked to all of the six hallmarks of cancer (evasion of apoptosis, insensitivity to anti-growth signals, unlimited replicative potential, angiogenesis, increase in survival factors and invasion and metastasis). It is thought that inflammation may play a critical role in lung carcinogenesis given that individuals with inflammatory lung conditions have an increased risk of lung cancer development. This study focuses on inflammation as a contributory factor in non small cell lung cancer (NSCLC), concentrating primarily on the pathological involvement of the pro-inflammatory cytokines, TNF-α, IL-1β, and hypoxia.

      Methods
      A normal bronchial epithelial cell line (HBEC4) was modified to stably and functionally over-express TNF-α and IL-1β (alone or in combination) and were continuously cultured for three months under normoxic or hypoxic (Invivo~2~ Hypoxia Workstation - 0.5% oxygen) conditions. Subsequently a range of experimental assays were carried out to assess functional cell change. These included: transformation assay (soft agar), proliferation (BrdU ELISA), invasion (Cell Invasion assay), migration (Scratch assay) and angiogenesis (Endothelial tube formation). Gene expression changes were also assessed using qPCR Cancer PathwayFinder arrays.

      Results
      Although transformation was not evident by soft agar, the adhesion potential (ICAM and VCAM levels) of normoxic and hypoxic clones has amplified and the growth rate has increased over time. Under normoxia the IL-1β and the TNF-α/IL-1β clones displayed an increased invasive capacity compared with the empty vector control (p<0.05). Differences were also detected in the gene expression profile implicated in the pathways involved in the hallmarks of cancer - cell signalling (FOS, JUN), apoptosis (BAD, BAX, BCL2), angiogenesis (CXCL8), adhesion (ITGB3), invasion (S100A4, TIMP1) and cell cycle regulation (p53, c-myc). A number of these targets are currently undergoing validation.

      Conclusion
      This study provides a valuable isogenic cell line model in which to study the effect of prolonged chronic inflammation. Although the cells have not developed anchorage independent growth as assessed by soft agar, there are distinct indications that phenotypic changes occurred within the three month time frame. As pro-longed chronic exposure to inflammation is a pre-requisite for many disease states, these results warrant extended growth studies to further delineate the complex roles of TNF-α, IL-1β and hypoxia in the process of carcinogenesis. This will assist in the development of novel cancer target therapeutics and chemo-preventive agents in lung cancer.

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      P2.01-011 - The IL-23 family may be a novel therapeutic target in non small cell lung cancer (ID 3304)

      09:30 - 09:30  |  Author(s): K. O'Byrne

      • Abstract

      Background
      IL-23A is a member of the IL-6 super-family and is composed of covalently bound IL-12p40 and IL-23p19 (IL-23A) subunits. The functional IL-23R is composed of the IL-23R and IL-12Rβ1 subunits and once activated can promote the STAT3 signalling pathway. IL-23A plays a key role in chronic inflammation through the promotion of IL-17 production by T helper 17 cells (Th 17). Stimulation of IL-23A associated pathways produces pro and anti oncogenic effects which are cell type dependant. We have previously identified IL-23A axis as deregulated in NSCLC and sought to examine the family in greater detail.

      Methods
      The expression of IL-23A/IL-23R was examined in a series of resected fresh frozen NSCLC tumours and in a panel of normal (HBEC) and NSCLC cell lines. Epigenetic regulation of IL-23A/IL-23R was determined in NSCLC cell lines using histone deacetylase (HDi) and DNA methyltransferase (DNMTi) inhibitors. A ChIP assay was performed to investigate the direct effect of Trichostatin A (TSA) on the IL-23A/IL-23R promoter regions. Additionally, the effect of Gemcitabine on IL-23A/IL-23R expression was examined. Finally, the effect of recombinant IL-23 treatment and Apilimod (STA 5326) (a small molecule which blocks the expression of IL-23 and IL-12) on NSCLC cell line proliferation was examined. IL-23 expression was also studied in a panel of NSCLC cisplatin sensitive and resistant cell lines. Furthermore due to its role in immune regulation, we are currently studying the effect of IL-23 on innate immune cell function.

      Results
      IL-23A expression was significantly elevated in a cohort of NSCLC patient tumour samples (p<0.05). Differential IL-23R expression was evident in a panel of NSCLC normal/tumour matched pairs (n=20), with no expression in 25%, reduced levels in 20% and increased levels in 55% of tumour samples compared with matched normal. In addition, IL-23A/IL-23R was found to be epigenetically regulated through histone post-translational modifications and DNA CpG residue methylation in the A549 cell line (p<0.05), with associated chromatin remodelling at both promoters. Gemcitabine, also induced IL-23A/IL-23R expression in this cell line (p<0.05). Furthermore, treatment with recombinant IL-23 resulted in increased cellular proliferation in the A549 cell line, while Apilimod reduced cellular proliferation. Preliminary results indicate that IL-23 can potentially affect the function of innate γδ T cells. In a panel of cisplatin resistant and sensitive cell lines, IL-23A was up-regulated in 3/5 resistant cell lines. Work is ongoing to assess the IL-23R expression in CisR and Parent cells lines and the effect of co-treating cells with HDi and Apilimod and to determine whether these compounds can re-sensitise cells to cisplatin therapy.

      Conclusion
      Based on our results the IL-23A/IL-23R axis is dysregulated in NSCLC. The IL-23 family is subject to epigenetic regulation through HDAC modifications and CpG island methylation. Gemcitabine treatment also affected the expression of this family. Recombinant IL-23 was pro-proliferative in NSCLC and blocking IL-23 with Apilimod reduced the proliferative capacity of the cells. Levels of IL-23A are differentially expressed between cisplatin sensitive and resistant cell lines. Apilimod may be a novel therapeutic drug in the treatment of NSCLC.

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      P2.01-016 - Targeting the PI3K-mTOR-NFκB pathway to overcome cisplatin resistance in NSCLC. (ID 2788)

      09:30 - 09:30  |  Author(s): K. O'Byrne

      • Abstract

      Background
      Non-small cell lung cancer (NSCLC) is the leading cause of cancer morbidity and mortality in the Western world with a poor overall 5 year survival of <15%. The most effective systemic chemotherapy for NSCLC is cisplatin-based combination treatment. However, chemoresistance is a major therapeutic problem and understanding the mechanisms involved is critical to the development of new therapeutic intervention strategies. The PI3K pathway plays an important role in NSCLC and we and others have shown increased PI3K signaling to be associated with a more aggressive disease with poor prognosis. Several proteins in this pathway have been indicated as potential mediators of cisplatin resistance in other cancers, and our group has previously identified the PI3K-activated transcription factor NFκB as a key player in this setting. In this study, targeted inhibition of three strategic points of the PI3K pathway was carried out with the aim of overcoming acquired resistance to cisplatin in these cell lines.

      Methods
      A panel of cisplatin resistant cell lines was previously generated in our laboratory through prolonged exposure to the drug. Expression of PI3K pathway related genes was compared between H460 parent (H460PT) and H460 cisplatin resistant (H460CR) cells using a PI3K pathway SABiosciences RTPCR array. Identified genes of interested were further investigated via PCR and Western blot in these cells as well as A549 parent (A549PT) and A549 cisplatin resistant (A549CR) cells. Three strategic points of the pathway were inhibited using GDC-0980, a dual PI3K-mTOR inhibitor currently in Phase II clinical trials in NSCLC, and DHMEQ, an inhibitor of NFkB translocation which has been used extensively both in vitro and in vivo. Effects of the two inhibitors on the parent & cisplatin resistant cell lines both with and without cisplatin were assessed by BrdU proliferation assay and multiparameter apoptosis assay (High Content Analysis).

      Results
      One of the most notable targets to emerge from the PI3K pathway RTPCR array screen was NFKBIA; the gene which codes for NFκB inhibitor IκBα. This gene was shown to be 12 fold overexpressed in H460CR compared to H460PT. This finding was validated at both the RNA and protein level by PCR and Western blot. NFκB was also found to be overexpressed in cisplatin resistant cells compared to their respective parent cells. Inhibition of NFκB by DHMEQ led to significantly improved inhibition of proliferation and induction of apoptosis in cisplatin resistant cells compared to parent cells. Preliminary data indicates that inhibition of PI3K and mTOR by GDC-0980 did not offer as significant a benefit as inhibition of NFκB in the cisplatin resistance setting, though further data from combination studies will be presented.

      Conclusion
      We conclude that the PI3K pathway plays an important role in resistance to cisplatin in NSCLC, particularly when signaling proceeds through the transcription factor NFκB. Targeting this pathway may be of benefit in re-sensitizing cisplatin resistant tumours to the drug.

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    P3.01 - Poster Session 3 - Cancer Biology (ID 147)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 2
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      P3.01-013 - KAT5 may be a candidate therapeutic target in Malignant Pleural Mesothelioma (ID 3273)

      09:30 - 09:30  |  Author(s): K. O'Byrne

      • Abstract

      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. Lysine acetyltransferases (KATs) including KAT5 have been linked with the development of cisplatin resistance. This gene may therefore be altered in MPM and could represent a novel candidate target for intervention

      Methods
      A panel of MPM cell lines inluding the normal pleural cells LP9 & Met5A were screened for expression of KAT5 variants 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. The effects of a small molecule inhibitor of KAT5 (MG-149) on cellular proliferation were examined.

      Results
      We show that the expression of KAT5A is dramatically increased in MPM. When separated according to histological subtype, KAT5A was significantly overexpressed in both the the sarcomatoid and biphasic subgroups for all transcript variants. Treatment of cells with the small molecule MG-149 caused significant inhibition of cellular proliferation (p<0.0001). We continue to asess this compound by other methodologies to confirm its potential utility in the treatment of MPM.

      Conclusion
      KAT5, a lysine acetyltransferase associated with cisplatin resistance in cancer is significantly altered in MPM. A small molecule inhibitor of this protein shows significant anti-proliferative effects in MPM cell lines. Targeting this protein may have important future implications for the management of MPM.

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      P3.01-014 - Expression and post-translational modifications of AKT isoforms in Malignant pleural Mesothelioma cells (ID 3252)

      09:30 - 09:30  |  Author(s): K. O'Byrne

      • Abstract

      Background
      Background: The PI3K/AKT signaling pathway is aberrantly active and has an important biologic impact in malignant pleural mesothelioma (MMe) cell cycle progression and chemo-resistance. Akt consists of three isoforms, Akt1, Akt2, and Akt3. Despite the growing amount of research demonstrating the existence of isoform-specific regulation, many papers still draw generalized conclusions about AKT without focusing on functional specificity of each isoforms. Recent data have clearly demonstrated a role of SIRT1 in the modulation of AKT1 activation and a role of PARP1 as a gatekeeper for SIRT1 activity by limiting NAD+ availability.

      Methods
      Methods: We explored the expression of AKT isoforms in MMe in vivo and in vitro and the balancing between their acetylation and phosphorylation status in human MMe derived cell lines in vitro.

      Results
      Results: We firstly described that MMe tumors in vivo and MMe derived cell lines express both AKT1 and AKT3 isoforms but not AKT2, and their expression results significantly increased in the biphasic histotype. Furthermore, we demonstrated an inverse correlation between AKTs acetylation and phosphorylation modulated by PARP1/SIRT1 activation status. By immunoprecipitation experiments, we evidenced that in basal conditions AKT1 is in part acetylated and in part phosphorylated and became highly phosphorylated and completely de-acetylated upon PARP1 inhibition. Interestingly, AKT1 activation, related to PARP1 inhibition, is unable to modulate pro-survival signals, because the downstream pathway is interrupted at the level of its effector mTOR. Conversely, SIRT1 inhibition or silencing result in a more evident AKT1 and its interactors acetylation.

      Conclusion
      Conclusions: In conclusion, our results clearly show how both PARP1 and SIRT1 affect critical cellular pathways involved in MMe progression and offer a model of a regulatory inter-relationship between these proteins. These data could be helpful for designing new effective therapeutic strategies.

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    P3.05 - Poster Session 3 - Preclinical Models of Therapeutics/Imaging (ID 159)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 2
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      P3.05-021 - Targeting HDACs to overcome cisplatin resistance in malignant plural mesothelioma (ID 3207)

      09:30 - 09:30  |  Author(s): K. O'Byrne

      • Abstract

      Background
      Malignant pleural mesothelioma (MPM) is an aggressive cancer of the mesothelial cells and is becoming a worldwide health burden. Progress in the treatment of MPM remains difficult, underscored by the fact that no single treatment option has proven particularly effective and chemo-resistance is a common problem. However, epigenetic modifiers, such as histone deacetylase inhibitors (HDi) have emerged as a novel class of anti-cancer agent and are currently undergoing clinical trials in numerous cancers. There is also evidence to suggest that HDAC expression may play a role in the development of chemo-resistance. We investigated the levels of HDACs in MPM cell lines, patient samples and determined the effect of HDi on MPM cisplatin sensitive and resistant cell lines.

      Methods
      A panel of MPM cell lines (n=16) was screened for the expression of HDAC11, Class I (HDAC1/2/3/8) and Class II (HDAC4/5/6/7/9/10) HDACs at the mRNA level (RT-PCR) and at the protein level (Western Blot). The HDAC expression profile was determined in an isogenic cell line model consisting of a cisplatin sensitive (Parent) and resistant (CisR) MPM cell line, P31. In addition, HDAC expression was examined in panel of twenty patient samples (benign/biphasic/sarcomatoid/epithelial). Also, MPM TMAs were stained by imummo-histochemistry for HDAC5 expression. Furthermore proliferation assays (BrdU ELISA) were performed to determine the effect of two HDi on the p31 cell lines. The HDi used were Vorinostat (pan HDAC inhibitor) and 19i (selective HDAC5 inhibitor).

      Results
      HDAC11, Class I and II HDACs were detected to varying degrees at the mRNA and protein level within our cell line panel. In the P31 isogenic parent/cisplatin resistant, HDAC protein expression was decreased (HDAC2/3/4/5/7) in the CisR compared with the Parent. HDAC5 was significantly reduced at both the protein and the mRNA level (p<0.05). The expression of HDACs 1/2/3/5 were increased in the MPM patient samples (n=15) compared with benign (n=5) (HDAC2/3/5, p<0.05). HDAC5 staining in a cohort of MPM samples, demonstrated no significant association between survival and a low HDAC5 score. However females had a greater median survival than their male counterparts. Vorinostat and 19i significantly reduced the proliferative capacity of the p31 Parent and CisR. SAHA was more effective in the Parent cells compared with CisR. The effect of 19i was similar in both cell lines.

      Conclusion
      Altered HDAC expression was observed in an isogenic Parent and CisR MPM cell model. Work ongoing in non-small cell lung cancer (NSCLC) has also demonstrated significantly reduced HDAC5 levels in CisR compared with Parent. This may suggest that a reduction in HDAC expression is involved in cisplatin resistance. Reduced levels of HDAC5, in an MPM TMA, were not associated with survival. It should be noted, however that patient numbers were small and biphasic and sarcomatoid sub-types had the lowest expression levels of HDAC5. We are currently increasing our patient numbers for an additional IHC study. HDi significantly reduced the proliferative capacity of CisR and Parent MPM cells. HDAC levels may represent an important biomarker in stratifying patients for future epigenetic targeted therapies in MPM.

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      P3.05-022 - BMS-777607 as a candidate therapeutic agent in Malignant Pleural Mesothelioma (ID 3272)

      09:30 - 09:30  |  Author(s): K. O'Byrne

      • Abstract

      Background
      Malignant pleural mesothelioma (MPM) is an aggressive inflammatory cancer associated with exposure to asbestos. Untreated, MPM has a median survival time of 6 months, and most patients die within 24 months of diagnosis. There is an urgent unmet need to identify new therapies for this disease. Receptor tyrosine kinases (RTK) are an area of active research in cancer therapy. The identification of “addicted” signalling networks could rapidly lead to candidate inhibitors (RTKi) that could potentially be uused to target MPM. We have previously identified RON/MST1R as a candidate therapeutic target in MPM. Additional RTKs identified by other studies include Axl and c-MET. The agent BMS-777607 is an orally bioavailable small molecule with the capacity to inhibit c-MET, RON/MST1R, Axl and Tyro3 (at nanomolar concentrations) and has just completed a Phase I/II trial (ClinicalTrials.gov Identifier: NCT00605618). This drug may therefore have applicability in MPM.

      Methods
      A panel of MPM cell lines inluding the normal pleural cells LP9 & Met5A were screened for expression of Tyro3, c-MET, RON/MST1R and Axl by RT-PCR. A cohort of snap-frozen patient samples isolated at surgery comprising benign, epithelial, biphasic, and sarcomatoid histologies were subsequently examined for the expression of these RTKs. The effects of BMS-777607 on MPM cellular proliferation and viability are being assessed.

      Results
      Expression of various RON/MST1R isoforms, c-MET, Tyro3 and Axl were observed in all cell lines. Significantly higher expression of all genes were found in the malignant tumour material versus benign pleura. The effects of BMS-777607 on cellular proliferation/viability are ongoing and the results will be presented at the meeting.

      Conclusion
      BMS-777607 represents a unique compound capable of targeting four RTKs whose expression are significantly elevated in maligant MPM. Given the many indications that these RTKs are associated with “addicted” RTK networks, this compound may therefore have potential clinical utility in the clinical management of MPM.

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    P3.11 - Poster Session 3 - NSCLC Novel Therapies (ID 211)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Medical Oncology
    • Presentations: 1
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      P3.11-042 - Molecular Inequality in the Treatment of Non-Small Cell Lung Cancer (NSCLC) and Implications for Clinical Trials (ID 2831)

      09:30 - 09:30  |  Author(s): K. O'Byrne

      • Abstract

      Background
      Activating mutations (MT) in the epidermal growth factor receptor (EGFR) gene are found in approximately 10-20% of patients with NSCLC. Guidelines recommend therapy with EGFR tyrosine kinase inhibitors (TKI’s) in these patients, and in patients with EGFR Wild type (WT) tumours beyond second line. Clinical trials have focussed on optimising the management of patients with an actionable target. The real-world management of patients with EGFR MT’s and clinical trial recruitment has yet to be explored. This retrospective study investigated treatment patterns in an Irish cohort of patients with non-squamous NSCLC, stratified by EGFR-MT status.

      Methods
      Patients with EGFR-MT positive tumours were identified from a National Multi-Institutional database. Patients with EGFR-WT tumours matched for age, stage and gender were identified. Treatment data including receipt of chemotherapy, EGFR TKI, and clinical trial participation were collected. Fisher’s exact and Mann-Whitney tests were used to compare variables. Cox model was used to examine the influence of treatment variables on overall survival (OS.) To ascertain the milieu of clinical trials applicable to this cohort, www.clinicaltrials.gov was searched for all phase III interventional studies in NSCLC between 1/1/2010 and 31/5/2013. Trial characteristics were summarized.

      Results
      We identified 416 patients with NSCLC. Forty (10%) patients had tumours with EGFR MT’s, of which data were available on 35 (87%) patients. Twelve (34%) patients had resected disease, and 23 (66%) had metastatic disease. Nineteen (82%) EGFR-MT positive patients with metastatic disease received first line systemic therapy, 12 (63%) receiving EGFR TKI (p=0.52.) Fifteen (65%) patients with EGFR-WT tumours received first line chemotherapy. The median number of lines of treatment was 1 (range: 0 – 4; 30% >1 line) for patients with EGFR-MT’s and 1 (range: 0 – 3; 13% >1 line) for EGFR-WT (p<0.01.) Receipt of second, third and fourth line therapy was 26%, 13% and 4.3% for EGFR-MT positive patients respectively, and 8.6%, 4.3% and 0% respectively in EGFR-WT (p<0.01.) Six (24%) patients with an EGFR MT and 0 (0%) with EGFR-WT participated in clinical trials (p<0.01.) Significant benefits were seen for 1) receipt of 1 line of treatment vs. 0 (HR=0.2, 95% CI=0.08 – 0.18, p=0.03) or 2) >1 line of treatment vs. 0 (HR=0.10, 95% CI= 0.01- 0.46, p< 0.01) Twenty-four phase III trials in advanced NSCLC were identified over the study period. The most commonly investigated agents were TKI's - 10 (42%) and monoclonal antibodies – 6 (25%). Ten (42%) trials required the presence of a driver mutation for eligibility, and 13 (54%) trials were in second line or beyond.

      Conclusion
      In Irish patients with NSCLC the incidence of EGFR MT’s is comparable to other European populations. Our real-world experience demonstrates that patients with EGFR MT’s tend to receive more lines of therapy and have a higher rate of clinical trials participation, reflecting the portfolio of currently available clinical trials. While trials should strive to optimise treatment for EGFR-MT positive NSCLC, the thoracic oncology community should consider that biological heterogeneity can lead to inequalities in clinical trial development and subsequent treatment.