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K. Gately



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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): K. Gately

      • 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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    MO20 - Preclinical Therapeutic Models II (ID 93)

    • Event: WCLC 2013
    • Type: Mini Oral Abstract Session
    • Track: Biology
    • Presentations: 1
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      MO20.03 - Development and characterization of a panel of GDC-0980 resistant NSCLC cell lines (ID 2798)

      10:30 - 12:00  |  Author(s): K. Gately

      • Abstract
      • Presentation
      • Slides

      Background
      The PI3K-Akt- mTOR pathway regulates cell growth and proliferation and is often dysregulated in cancer due to mutation, amplification, deletion, methylation and post-translational modifications. PI3K pathway activation in NSCLC has been shown by us and others to lead to a more aggressive disease correlating to poor prognosis for patients. Multiple novel agents, targeting different regulators within the pathway are currently under development. GDC-0980 is a selective dual inhibitor of PI3K and mTOR, which demonstrated excellent downstream inhibition of the PI3K pathway in vitro, with the strongest effects being observed in lung, breast and prostate cancer cell lines. There are 12 clinical trials ongoing for this drug, with Phase I studies in solid tumours and Phase II studies in endometrial carcinoma, renal cell carcinoma, prostate cancer and breast cancer. As with all targeted therapies, acquired resistance to GDC-0980 is anticipated to be a major hurdle in the success of this drug. Multiple mechanisms of resistance to GDC-0980 may develop while a patient is being treated with this drug. The aim of this project is to develop four cell line models of resistance to GDC-0980, each representing a different molecular subtype of NSCLC, in order to predict which mechanisms of resistance may occur in patients. This will allow us to identify biomarkers of response/resistance to the drug that may dictate beneficial treatment strategies.

      Methods
      H460, A549, H1975 and SKMES-1 cells were treated with a dose response curve of GDC-0980 and BrdU proliferation assays determined IC50 values for each cell line. Each cell line was then cultured in GDC-0980 at IC50 concentrations over a period of several months, along with matched ‘parent’ cell lines. Each month, BrdU proliferation assay were carried out in order to track the development of resistance to the drug. When a log fold difference between the parent and resistant IC50s was observed, the cells were deemed to be resistant. Matched parent and resistant cells were then screened for a panel of mutations. Cells lines were also screened for gene alterations using a human cancer drug resistance PCR array. Identified genes of interest were validated at the RNA and protein level by PCR and Western blot, respectively.

      Results
      All four cell lines exhibited a dose-dependent decrease in proliferation when treated with GDC-0980. H1975 cells (adenocarcinoma; PIK3CA mutant) were most sensitive to GDC-0980, however they developed resistance to the drug more rapidly than the other 3 cell lines. Results from mutational analysis and investigation of the gene and protein expression of each of the 4 pairs of parent and resistant cell lines will be presented.

      Conclusion
      While the panel of four NSCLC cell lines all responded well to GDC-0980 treatment initially, resistance to the drug developed rapidly. As such, understanding the mechanisms involved in the development of resistance to this drug will be crucial so that we may design optimal treatment strategies. Specific conclusions regarding the mechanisms of resistance in this panel of cell lines will be drawn based on identified genes and proteins of interest.

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

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 2
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      P1.01-011 - Targeting the Urokinase Plasminogen Activator (uPA) System to overcome cisplatin resistance in NSCLC (ID 3347)

      09:30 - 16:30  |  Author(s): K. Gately

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

      09:30 - 16:30  |  Author(s): K. Gately

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

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 2
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      P1.06-054 - Targeting MCL1 amplification in NSCLC through anthracycline-mediated transcriptional suppression (ID 3213)

      09:30 - 16:30  |  Author(s): K. Gately

      • Abstract

      Background
      Targeting oncogene dependency for effective therapy has been one of the most successful strategies for managing metastatic non-small cell lung cancer (NSCLC). Although validating therapeutically tractable oncogenic driver mutations are a major focus, non-driver mutations may also confer dependencies that may also be exploitable. The prosurvival BCL2 protein, MCL1 prevents mitochondrial apoptosis by blocking interaction of proapoptotic BH3 only proteins with their multidomain proapototic counterparts, BAX and BAK. MCL1 is often mutated in cancers, and ranks as one of the most frequently amplified loci at 1q21.2. MCL1 amplified tumours exhibit addiction to this oncogene. Anthracyclines have been shown to transcriptionally suppress MCL1. Phase IIA studies in NSCLC have shown that epirubicin has useful single agent activity in unselected patients, with a significantly greater response rate than that achieved with standard chemotherapy. We therefore set out to evaluate MCL1 addiction in NSCLC, its correlation with anthracyline induced apoptosis and the prevalence of 1q21.2amplification to support a planned 1q21.2 stratified phase II trial in NSCLC, (EORTC-1303-LCG).

      Methods
      RNAi targeting MCL1was conducted in NCI-H460, NCI-H1299, NCI-H28 and NCI-H23 cell lines. Doxorubicin activity was measured by viability assay and apoptosis was assessed by western blot. gDNA from cell lines was obtained by Phenol-Chloroform extraction. The QIAamp DNA FFPE Tissue Kit was used to extract gDNA from FFPE tissues. MCL1 amplification was quantified by real-time PCR with a set of two primers and one probe (minor groove-binding (MGB) hydrolysis probe assay) for the gene of interest MCL1 and the two reference genes CCT3 and H6PD. Tonsil samples were used as a control diploid population.

      Results
      MCL1 silencing efficiently induced apoptosis in a subset of NSCLC cells, however we identified two cell lines that were resistant to MCL1 knockdown (NCI-H1299 and NCI-H28). Doxorubicin efficiently induced apoptosis in MCL1 addicted cells but exhibited significantly less activity in cells that were not addicted. We developed a genomic DNA based quantitative real time PCR assay to evaluate copy number variation (CNV) at the 1q21.2 locus. A clear correlation r[2] >0.91 was observed for 1q21.2 CNV compared with reference Conan Copy Number Analysis Tool (Cancer genome project, Sanger). Increased 1q21.2 copy number was consistently associated with MCL1addiction; however addiction also occurred in cells lacking 1q21.2 CNV, suggesting that MCL1 amplification represents a subset of MCL1 dependence. The concentration of doxorubicin was titrated against MCL1 protein downregulation into therapeutically sub-micromolar concentration range and we observed that MCL1 downregulation occurred coincidently with cleavage of poly-ADP ribose polymerase. We then screened DNA isolated from 19 adenocarcinomas, and identified 1q21.2 CNVs in 36.8%, with high level amplification (CNV >5) in 1q21.2 in 10.5%.

      Conclusion
      Targeting MCL1 addiction in 1q21.2 amplified NSCLC induces apoptosis and this dependence can be exploited by anthracyclines at therapeutically relevant concentrations. Given its significant prevalence in NSCLC, our data suggests that 1q21.2 amplification could be a novel non-driver mutation predictive for anthracycline response.

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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): K. Gately

      • 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 - 16:30  |  Author(s): K. Gately

      • 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: 1
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      P2.01-016 - Targeting the PI3K-mTOR-NFκB pathway to overcome cisplatin resistance in NSCLC. (ID 2788)

      09:30 - 16:30  |  Author(s): K. Gately

      • 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: 1
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      P3.01-012 - Co-targeting the PI3K and MEK pathways in NSCLC: an in vitro evaluation and mutation prevalence in an Irish patient cohort. (ID 2794)

      09:30 - 16:30  |  Author(s): K. Gately

      • Abstract

      Background
      PI3K pathway activation in NSCLC has been shown by us and others to lead to a more aggressive disease correlating to poor prognosis for patients. Unfortunately, the success of PI3K targeted inhibition has been hampered by a high rate of innate and acquired resistance. Mutations in KRAS and B-RAF, ERK hyperactivation as well as extensive PI3K-MEK pathway cross-talk allow the MEK pathway to provide a bypass track. Preclinical studies demonstrate a rationale for a PI3K-MEK co-targeted treatment strategy which may provide a more effective response. A Phase I clinical trial is underway investigating the combination of GDC-0941, a pan-PI3K inhibitor, with GDC-0973, a MEK inhibitor. GDC-0980 is a dual PI3K-mTOR inhibitor which may offer improved pathway inhibition compared to GDC-0941. No data has been published to date on the combination of GDC-0980 and GDC-0973, which we believe may offer improved overall inhibition of survival signaling in NSCLC cells. We aim to elucidate the role of mutation status in response to this co-targeted inhibition approach in vitro, as well as investigating the frequency of PI3K and MEK pathway mutations in a well characterized Irish NSCLC patient cohort.

      Methods
      The effects of GDC-0941, GDC-0980 and GDC-0973 on proliferation and apoptosis in a panel of four NSCLC cell lines were analysed by BrdU Assay and HCA Apoptosis Assay, respectively. The four cell lines investigated were H460 (adenocarcinoma, PIK3CA mutant & KRAS mutant), A549 (adenocarcinoma, PIK3CA wild type & KRAS mutant), H1975 (adenocarcinoma, PIK3CA mutant, KRAS wild type & EGFR TKI resistant) and SKMES-1 (squamous cell carcinoma, PIK3CA wild type & KRAS mutant). Further investigation involved expression analysis of pAkt, pGSK-3β, pp70S6K, pS6RP, ERK and pERK in cell lines treated with each inhibitor alone or in combination using Mesoscale technology and Western blot. DNA was extracted from 120 NSCLC patient tissue samples, and screened for 547 mutations in 59 genes (including PI3K and MEK pathway members) using the Sequenom.

      Results
      GDC-0941 and GDC-0980 treatment induced dose-dependent anti-proliferative and pro-apoptotic responses across all four NSCLC cell lines, while GDC-0973 treatment induced only anti-proliferative responses. Protein expression analysis showed that GDC-0980 & GDC-0973 combination treatment induced significantly improved phosphoprotein inhibition compared to treatment with either inhibitor alone in cell lines harbouring PIK3CA mutations, while in one cell line bearing WT PIK3CA (SKMES-1), combination treatment actually increased pathway signalling. NSCLC patient mutational profiling data will be presented.

      Conclusion
      This research underpins the importance of mutation status in sensitivity to targeted therapies. While combination treatment approaches may be beneficial in certain molecular subtypes, in others they may be detrimental. In the era of personalised medicine, patient genotyping is crucial to improve patient survival and reduce toxicities.

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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 - 16:30  |  Author(s): K. Gately

      • 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.