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M. Barr



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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)

      16:15 - 17:45  |  Author(s): M. Barr

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

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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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 - 16:30  |  Author(s): M. Barr

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

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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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 - 16:30  |  Author(s): M. Barr

      • 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.