Virtual Library
Start Your Search
K. O’byrne
Author of
-
+
MA02 - RNA in Lung Cancer (ID 377)
- Event: WCLC 2016
- Type: Mini Oral Session
- Track: Biology/Pathology
- Presentations: 1
- Moderators:E. Brambilla, M. Noguchi
- Coordinates: 12/05/2016, 14:20 - 15:50, Stolz 2
-
+
MA02.02 - A Novel 5-miR Signature Shows Promise as a Diagnostic Tool and as a Predictor of Cisplatin Response in NSCLC (ID 5948)
14:20 - 15:50 | Author(s): K. O’byrne
- Abstract
Background:
MicroRNAs are a class of small non-coding RNAs that range in size from 19-25 nucleotides. They have been shown to regulate a number of processes within tumour biology, including metastasis, invasion and angiogenesis. More recently, miRNAs have been linked to chemoresistance in solid tumours, including lung cancer.
Methods:
MicroRNA expression within an isogenic panel of age-matched parent (PT) and cisplatin resistant (CisR) NSCLC cell lines was profiled using the 7[th] generation miRCURY LNA arrays (Exiqon). Significantly altered miRNAs within the CisR sublines were manipulated using antagomirs (Exiqon) and Pre-miRs (Ambion) and functional studies were carried out in the presence and absence of cisplatin. To examine the translational relevance of these miRNAs, their expression was examined in a cohort of chemo-naïve patient-matched normal and lung tumour tissue and serum from NSCLC patients of different histologies. To create a xenograft model of cisplatin resistance 1x10[3 ]cells H460 PT or CisR cells were injected into 5-7week old NOD/SCID mice. Tumour volume was measured over time and harvested once the tumour mass measured 500mm[3] and formalin-fixed and paraffin embedded (FFPE). Expression of the 5-miR signature was analysed within FFPE murine tumours and cisplatin resistance was investigated relative to cisplatin sensitive controls.
Results:
Profiling and subsequent validation revealed a 5-miR signature associated with our model of cisplatin resistance (miR-30a-3p, miR-30b-5p, miR-30c-5p, miR-34a-5p, miR-4286). Inhibition of the miR-30 family and miR-34a-5p reduced clonogenic survival of CisR cells when treated cisplatin. Expression of the miRNA signature was significantly altered in both adenocarcinoma (AD) and squamous cell carcinoma (SCC) relative to matched normal lung tissue and between SCC and AD tissue. miR-4286 was significantly up-regulated in SCC sera compared to normal control and AD sera. Similarly to the cell line expression of the miRNAs, the miR-30 family members and miR-34a-5p were up-regulated in the CisR xenograft FFPE tissue relative to PT.
Conclusion:
A novel miRNA signature associated with cisplatin resistance was identified in vitro, genetic manipulation of which altered clonogenic response to cisplatin. The 5-miR signature shows both diagnostic and prognostic biomarker potential across a number of diagnostically relevant biological mediums.
-
+
OA23 - EGFR Targeted Therapies in Advanced NSCLC (ID 410)
- Event: WCLC 2016
- Type: Oral Session
- Track: Advanced NSCLC
- Presentations: 1
- Moderators:O.T. Brustugun, S. Lu
- Coordinates: 12/07/2016, 14:20 - 15:50, Stolz 2
-
+
OA23.05 - First-Line Afatinib versus Gefitinib in EGFRm+ Advanced NSCLC: Updated Overall Survival Analysis of LUX-Lung 7 (ID 5347)
14:20 - 15:50 | Author(s): K. O’byrne
- Abstract
- Presentation
Background:
The irreversible ErbB family blocker afatinib and the reversible EGFR TKI gefitinib are approved for first-line treatment of advanced EGFRm+ NSCLC. This Phase IIb trial prospectively compared afatinib versus gefitinib in this setting.
Methods:
LUX-Lung 7 assessed afatinib (40 mg/day) versus gefitinib (250 mg/day) in treatment-naïve patients with stage IIIb/IV NSCLC harbouring a common EGFR mutation (Del19/L858R). Co-primary endpoints were PFS (independent review), time to treatment failure (TTF) and OS. Other endpoints included ORR and AEs. In case of grade ≥3/selected grade 2 drug-related AEs the afatinib dose could be reduced to 30 mg or 20 mg (minimum). The primary analysis of PFS/TTF was undertaken after ~250 PFS events. The primary OS analysis was planned after ~213 OS events and a follow-up period of ≥32 months.
Results:
319 patients were randomised (afatinib: 160; gefitinib: 159). At the time of primary analysis, PFS (HR [95% CI] 0.73 [0.57‒0.95], p=0.017), TTF (0.73 [0.58‒0.92], p=0.007) and ORR (70 vs 56%, p=0.008) were significantly improved with afatinib versus gefitinib. The most common grade ≥3 AEs were diarrhoea (13%) and rash/acne (9%) with afatinib and elevated ALT/AST (9%) with gefitinib. 42% of patients treated with afatinib had ≥1 dose reduction due to AEs; dose reductions were more common in females than males (77%/23%) and non-Asians than Asians (64%/36%). Dose reduction of afatinib did not negatively impact PFS (<40mg vs ≥40mg; HR [95% CI]: 1.34 [0.90‒2.00]) but reduced incidence and severity of drug-related grade ≥3 AEs. Treatment discontinuation due to drug-related AEs was the same in each arm (6%). The data cut-off for primary OS analysis occurred on 8 April 2016. At this time, median treatment duration (range) was 13.7 (0‒46.4) versus 11.5 (0.5‒48.7) months with afatinib and gefitinib. 25% (afatinib) and 13% (gefitinib) of patients received treatment for >24 months. 73% and 77% of patients in the afatinib and gefitinib arms had ≥1 subsequent systemic anti-cancer treatment, with 46% and 56% receiving a subsequent EGFR-TKI including osimertinib (14%)/olmutinib (14%). OS data, including subgroup analysis with respect to subsequent therapy will be presented at the meeting.
Conclusion:
Afatinib significantly improved PFS, TTF and ORR versus gefitinib in EGFRm+ NSCLC patients, with a manageable AE profile and few drug-related discontinuations. Dose adjustment of afatinib reduced drug-related AEs without compromising efficacy. Primary OS analysis will be reported.
Only Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.
-
+
P1.02 - Poster Session with Presenters Present (ID 454)
- Event: WCLC 2016
- Type: Poster Presenters Present
- Track: Biology/Pathology
- Presentations: 1
- Moderators:
- Coordinates: 12/05/2016, 14:30 - 15:45, Hall B (Poster Area)
-
+
P1.02-065 - Elucidating the Role of PIM Kinase and Its Therapeutic Potential in NSCLC (ID 4328)
14:30 - 15:45 | Author(s): K. O’byrne
- Abstract
Background:
PIM kinases are a family of three serine/threonine kinases: PIM1, PIM2 and PIM3 that have been shown to play a role in tumorigenesis. PIM1 is a downstream effector of oncoproteins ABL and JAK/STAT and regulator of BCL2/BAD and CXCR4. PIM activity is synergistic with the PI3K/Akt/mTOR pro-survival pathway and PIM2 has been shown to phosphorylate translational repressor 4E-BP1 and p70S6 independently of the PI3K pathway. Furthermore a synergism between PIM kinases and c-Myc has been reported. Here we investigate the expression of PIM1/PIM2/PIM3 in NSCLC cell lines and patient matched normal/tissue samples. The effect of a novel combined inhibitor of PI3K/mTOR/PIM kinases (IBL-301) on cell signalling, cell death and proliferation is also examined.
Methods:
PIM1/PIM2/PIM3 expression were examined by Western blot analyses in NSCLC cells (H1975 and H1838). Additionally, the frequencies of PIM1/PIM2/PIM3 expression in NSCLC patient tumour and matched normal adjacent samples (n=31) were investigated. The effectiveness of IBL-301 on cell signalling, cell viability and proliferation were examined by Western blot analysis, cell titre blue and BrdU assay respectively.
Results:
All three PIM isoforms were detected in the lung cancer cell lines tested. Similarly, all three PIM isoforms were expressed across the 31 NSCLC patient tumour and match normal adjacent tissue samples. To investigate this further PIM1 staining of FFPE tumour and match normal tissue from this cohort is currently underway. In two lung cancer cell lines, H1975 and H1838, IBL-301 was found to have a dose dependent effect on proliferation/viability with IC~50~ values in the nanomolar range. Additionally, western blot analyses have indicated that these novel drugs can suppress the phosphorylation of key players in cell signalling pathways linked to tumorigenesis including pAkt, p4E-BP1 and peIF4B.
Conclusion:
This is the first study to investigate the expression of all 3 isoforms of PIM in lung cancer specifically. All 3 isoforms were abundantly expressed across cells lines and patient tumour samples. Observed PIM expression in the immune cells of normal adjacent tissue may indicate a role in inflammation. This finding coupled with the promising in vitro data demonstrate the therapeutic potential of targeting PIM in NSCLC.
-
+
P2.01 - Poster Session with Presenters Present (ID 461)
- Event: WCLC 2016
- Type: Poster Presenters Present
- Track: Biology/Pathology
- Presentations: 2
- Moderators:
- Coordinates: 12/06/2016, 14:30 - 15:45, Hall B (Poster Area)
-
+
P2.01-031 - CCL Chemokines May Play an Important Role in Cisplatin Resistance (ID 4861)
14:30 - 15:45 | Author(s): K. O’byrne
- Abstract
Background:
In the absence of a targetable mutation, cisplatin based chemotherapy is the backbone of NSCLC treatment. However, a diverse patient population combined with complex tumour heterogeneity is hampering its’ clinical utility. Although intrinsic and acquired resistance to cisplatin is common, the mechanisms have not yet been fully elucidated. However, some studies have suggested that inflammatory pathways may play a key role in chemo-resistance. The aim of this project is to increase our understanding of inflammatory mediated cisplatin resistance in NSCLC.
Methods:
A number of isogenic cell line models of NSCLC (adenocarcinoma, squamous cell carcinoma, large cell carcinoma) cisplatin resistance were utilised to assess the role of inflammation in chemo-resistance. These included a sensitive parental cell line (PT) and a matched resistant subtype (CisR). The cell lines were screened for NFKB and a number of inflammatory mediators including chemokines and TLRs at the mRNA (RT-PCR/qPCR) and protein level (Western Blot/ELISA). A specific NFKB inhibitor, DHMEQ, and recombinant chemokines were employed to further characterise inflammatory pathways in PT and CisR cells in terms of cisplatin sensitivity, proliferation (BrdU ELISA), cellular viability (Cytell Cell Imaging System) and DNA damage response (Comet). An in vivo study was also completed using DHMEQ alone and in combination with cisplatin.
Results:
A number of NFKB targets and responsive pathways are deregulated in CisR cells compared with their matched sensitive PT cell line. Amongst others, CCL2 and CCL5 were altered across all NSCLC subtypes. Preliminary data suggests that DHMEQ enhances cisplatin sensitivity in both PT and CisR cells, conversely recombinant chemokines elicit a protective effect. Additionally, DHMEQ treatment resulted in opposite affects on CCL2 and CCL5 mRNA levels in the PT and CisR cell lines. This may reflect an alternative pathway hierarchy within the cells. Further characterisation is ongoing assessing chemokine specific inhibitors. Although, in vivo data suggests a trend of decreased tumour growth in the DHMEQ cohorts compared with vehicle control, the data was not significant. However, tumour samples appeared more necrotic with DHMEQ and are currently being characterised using IHC for necrosis and proliferation.
Conclusion:
Targeting chemokines downstream of NFKB may provide a means to overcome inflammatory mediated acquired and intrinsic NSCLC chemo-resistance. Given the increased significance of immuno-oncology agents to harness the body’s own immune system in the fight against cancer, these agents may also prove fruitful in re-sensitising patients to chemotherapy.
-
+
P2.01-081 - CDCA3 is a Novel Prognostic Cell Cycle Protein and Target for Therapy in Non-Small Cell Lung Cancer (ID 5823)
14:30 - 15:45 | Author(s): K. O’byrne
- Abstract
Background:
Lung cancer is the leading cause of cancer-related mortality worldwide with a 5 year survival rate of 15%. Non-small cell lung cancer (NSCLC) is the most commonly diagnosed form of lung cancer. Cisplatin-based regimens are currently the most effective chemotherapy for NSCLC, however, chemoresistance poses a major therapeutic problem. New and reliable strategies are required to avoid drug resistance in NSCLC. Cell division cycle associated 3 (CDCA3) is a key regulator of the cell cycle. CDCA3 modulates this process by enabling cell entry into mitosis through degradation of the mitosis-inhibitory factor WEE1. CDCA3 itself is also degraded in G1 yet re-expressed in G2/M phase, to allow successful progression through the cell cycle. Herein, we describe CDCA3 as a novel prognostic factor in NSCLC and target to delay or prevent cisplatin resistance in NSCLC.
Methods:
CDCA3 expression was investigated in squamous and non-squamous NSCLC using several approaches including bioinformatic analysis of publicly available datasets, immunohistochemistry of a tissue microarray and western blot analysis of matched tumour and normal tissue and NSCLC cell lines. CDCA3 function in NSCLC was determined using several in vitro assays by siRNA depleting CDCA3 in a panel of three immortalized bronchial epithelial cell lines (HBEC) and seven NSCLC cell lines.
Results:
CDCA3 transcripts and protein levels are elevated in NSCLC patient tissue and highly expressed in tumour cells relative to proximal normal cells. High mRNA levels are associated with poor survival in resected NSCLC. Depletion of CDCA3 in vitro markedly impairs proliferation in seven NSCLC cell lines by inducing a mitotic cell cycle arrest, ultimately resulting in p21-dependent cellular senescence. Importantly, silencing of CDCA3 also greatly sensitises NSCLC cell lines to cisplatin. In line with these in vitro data, NSCLC patients that have elevated levels of CDCA3 and are treated with cisplatin have a poorer outcome than patients with reduced levels of the protein. To improve patient response to cisplatin, we are exploring novel strategies to suppress CDCA3 expression in tumour cells.
Conclusion:
Our data highlight CDCA3 as a novel factor in mediating NSCLC. We propose that evaluating novel strategies to target CDCA3 may prove a useful strategy is enhancing the anti-tumour activity of platinum-based chemotherapy and may ultimately benefit patient outcomes by preventing cisplatin resistance.
-
+
P2.03a - Poster Session with Presenters Present (ID 464)
- Event: WCLC 2016
- Type: Poster Presenters Present
- Track: Advanced NSCLC
- Presentations: 3
- Moderators:
- Coordinates: 12/06/2016, 14:30 - 15:45, Hall B (Poster Area)
-
+
P2.03a-062 - Characterisation and Targeting of the DNA Repair Gene, XRCC6BP1, in Cisplatin Resistant NSCLC (ID 5198)
14:30 - 15:45 | Author(s): K. O’byrne
- Abstract
Background:
In the absence of specific treatable mutations, platinum-based doublet chemotherapy remains the gold standard treatment for NSCLC patients. However, its clinical efficacy is hindered in many patients due to intrinsic and acquired resistance to these agents, in particular cisplatin. Alterations in the DNA repair capacity of damaged cells is now recognised as an important factor in mediating this phenomenon.
Methods:
DNA Repair Pathway RT[2 ]Profiler Arrays were used to elucidate key DNA repair genes implicated in chemoresistant NSCLC cells using cisplatin resistant (CisR) and corresponding parental (PT) H460 cells previously established in our laboratory. DNA repair genes significantly altered in CisR cells were validated at the mRNA and protein level, using RT-PCR and Western blot analysis, respectively. The translational relevance of differentially expressed genes was examined in a cohort of chemo-naïve matched normal and tumour lung tissues from NSCLC patients. Loss of function studies were carried out using siRNA technology. The effect of XRCC6BP1 gene knockdown on apoptosis was assessed by FACS using Annexin-V/PI staining. Cellular expression and localisation of XRCC6BP1 protein and γH2AX foci in response to cisplatin were examined by immunofluorescence using the Cytell™ Imaging System. To investigate a role for XRCC6BP1 in lung cancer stem cells, Side Population (SP) studies were used to characterise stem-like cells in chemoresistant cells. XRCC6BP1 mRNA analysis was also examined in ALDH1[+] and ALDH1[- ]subpopulations.
Results:
We identified a number of critical DNA repair genes that were differentially regulated between H460 PT and CisR NSCLC cells, where XRCC6BP1 mRNA and protein expression was significantly increased (mRNA; 19.4-fold) in H460 CisR cells relative to their PT counterparts. Relative to matched normal lung tissues, XRCC6BP1 mRNA was significantly increased in lung adenocarcinoma patients. Gene silencing of XRCC6BP1 induced significant apoptosis of CisR cells and reduced the DNA repair capacity of these cells relative to scrambled (negative) controls. Immunofluorescence studies showed an increase in XRCC6BP1 protein expression and γH2AX foci in CisR cells relative to their PT counterparts. SP analysis revealed a significantly higher stem cell population in CisR cells, while XRCC6BP1 mRNA expression was considerably increased in SKMES-1, H460 and H1299 ALDH1[+] CisR cells compared to ALDH1[-] cells.
Conclusion:
We identified XRCC6BP1 as key DNA repair gene implicated in cisplatin resistant NSCLC. Our data highlight the potential of targeting components of the DNA repair pathway in chemoresistant lung cancer, in particular, XRCC6BP1, either alone or in combination with conventional cytotoxic therapies.
-
+
P2.03a-063 - Small Molecule Cancer Stemness Inhibitor, BBI608, Restores Cisplatin Sensitivity in Resistant NSCLC (ID 5962)
14:30 - 15:45 | Author(s): K. O’byrne
- Abstract
Background:
The cancer stem cell (CSC) hypothesis is now a well-established and widely investigated field within oncology. It hypothesizes that there is a robustly resistant stem-like population of cells that survive initial chemotherapeutic treatment. These surviving CSCs contribute to the recapitulation of a heterogeneous tumour via a combination of asymmetric and symmetric cell division, subsequently resulting in relapse and therapeutic resistance. BBI608 is a small molecule inhibitor of cancer stemness; it targets STAT3, leading to the inhibition of critical genes required for the maintenance of cancer stemness. To date, preclinical studies investigating BBI608 in in vitro and in vivo models of pancreatic and prostate cancer have shown promise.
Methods:
Aldefluor (Stemcell Technologies) staining and flow cytometry analysis of an isogenic panel of matched parent (PT) and cisplatin resistant (CisR) NSCLC cell lines identified the ALDH1-positive (ALDH1+ve) subpopulation of cells as a key CSC subset across cisplatin resistant NSCLC cell lines. PT and CisR cell lines were treated with BBI608 (1μM) and stemness factors investigated, the presence of the ALDH1+ve CSC population was reassessed by flow cytometry and expression of stemness factors (Nanog, Oct-4, Sox-2, Klf4 and cMyc) were examined by reverse transcriptase PCR. The functional parameters of proliferation, clonogenic survival and apoptosis were investigated with increasing concentrations of cisplatin (0-100μM) in the presence and absence of 1μM BBI608.
Results:
The NSCLC CisR sublines showed a significantly greater ALDH1+ve CSC population relative to their PT counterparts. Treatment of the CisR sublines with 1μM BBI608 significantly depleted the ALDH1+ve CSC population and decreased gene expression of stemness markers. BBI608 significantly decreased the proliferative capacity and clonogenic survival of the CisR sublines when in combination with cisplatin relative to cisplatin alone. Cisplatin in combination with BBI608 significantly increased cisplatin-induced apoptosis in the CisR sublines indicating restoration of cisplatin sensitivity.
Conclusion:
To date, BBI608 has not been investigated in terms of a cisplatin resistant CSC population in lung cancer. BBI608, via the inhibition of STAT3, pharmacologically depleted the CSC subpopulation and stemness expression while simultaneously restoring cisplatin sensitivity. There are currently a number of clinical trials recruiting patients to further investigate BBI608. These data suggest a promising role for BBI608 in the treatment of non-responsive or recurrent NSCLC.
-
+
P2.03a-064 - Inhibition and Exploitation of Aldehyde Dehydrogenase 1 as a Cancer Stem Cell Marker to Overcome Cisplatin Resistant NSCLC (ID 5954)
14:30 - 15:45 | Author(s): K. O’byrne
- Abstract
Background:
The root of therapeutic resistance is hypothesized to be the presence a rare CSC population within the tumour population which survives chemotherapeutic treatment and has the potential to recapitulate a heterogenic tumour. Aldehyde dehydrogenase 1 (ALDH1) is involved the catalytic conversion of vitamin A (retinol) to retinoic acid and has been identified as a CSC marker in a number of solid malignancies.
Methods:
FACS analysis of an isogenic panel of matched parent (PT) and cisplatin resistant (CisR) NSCLC cell lines identified ALDH1 as a promising CSC-marker associated with cisplatin resistance across NSCLC histologies. The H460 CisR subline was separated by FACS into ALDH1-positive and negative subpopulations and subcutaneously injected into NOD/SCID mice to assess tumour initiation and growth. ALDH1 was inhibited in vitro within the cell lines using two pharmacological ALDH1 inhibitors, DEAB and disulfiram, alone and in combination with cisplatin. Cell lines were treated in vitro with retinol and all-trans retinoic acid (ATRA) to exploit the vitamin A/retinoic acid axis in which ALDH1 is involved.
Results:
The CisR sublines showed significantly greater ALDH1 activity relative to their PT counterparts. In vivo subcutaneous injection of ALDH1-positive and negative subpopulations revealed no significant difference in tumour initiation or growth rate. ALDH1 inhibition in combination with cisplatin significantly decreased clonogenic and proliferative competencies and increased apoptosis cell death compared to cisplatin alone. Vitamin A supplementation and ATRA treatment in combination with cisplatin showed similar re-sensitising effects.
Conclusion:
This pharmacological CSC depletion in conjunction with cisplatin treatment resulted in re-sensitisation of cisplatin resistant cells to the cytotoxic effects of cisplatin. These data suggest vitamin A supplementation or the addition of ATRA or an ALDH1 inhibitor to the cisplatin-based chemotherapeutic regimen may be of clinical benefit in overcoming tumour recurrence and cisplatin resistance.
-
+
P2.03b - Poster Session with Presenters Present (ID 465)
- Event: WCLC 2016
- Type: Poster Presenters Present
- Track: Advanced NSCLC
- Presentations: 1
- Moderators:
- Coordinates: 12/06/2016, 14:30 - 15:45, Hall B (Poster Area)
-
+
P2.03b-096 - Utilisation of a Novel 3D Culture Technology for the Assessment of Chemo-Resistance in Non-Small Cell Lung Cancer (ID 4954)
14:30 - 15:45 | Author(s): K. O’byrne
- Abstract
Background:
Elucidation of the key mechanisms underlying resistance to chemotherapy is an on-going and complex process. Owing to its suggested increased biological relevance, many are now transitioning from two-dimensional (2D) to three-dimensional (3D) cellular-based assay systems. These systems permit the formation of 3D multicellular structures (MCS). The internal micro-environment of these structures mimics closely those found in vivo. In addition they are considered to provide a more biologically relevant model of chemo-resistance. This study focuses on the utilisation of a novel 3D culture technology to compare chemo-resistant models of non-small cell lung cancer (NSCLC) in 3D culture with those cultured in 2D monolayers. Critically, this will provide a valuable tool to determine the biological discrepancies which exist between the two culture methods with the aim to identify novel mechanisms of chemo-resistance in NSCLC.
Methods:
Isogenic NSCLC models of cisplatin resistance, which encompassed sensitive parent (PT) and matched cisplatin resistant (CisR) cell lines, were used for this study. The 3D MCS were cultured in Happy Cell Advanced Suspension Medium™ and 2D monolayers as standard. Both 2D and 3D cultures were exposed to a range of cisplatin concentrations (0 – 100 µM) for a period of 72 h. Subsequently, cellular viability, hypoxia, proteomic and morphological assays were conducted in order to compare the response of both PT and CisR cells in 2D and 3D.
Results:
High content imaging has identified a central necrotic core within the 3D MCS, which is a feature of the asymmetric growth patterns observed in vivo; that being a decrease in viable cells as you move inwards from the periphery of the MCS. Preliminary data suggests that at equivalent cisplatin concentrations, H460 3D MCS exhibit increased resistance to cisplatin compared with 2D monolayers in both PT and CisR cell lines. Proteomic analysis has also identified diverse pathway modifications in 3D compared with 2D culture, with a number of proteins expressed exclusively in each. Additional cellular characterisation and further bioinformatic analysis is on-going.
Conclusion:
Chemotherapeutic intervention is most frequently employed in the treatment of NSCLC, however many patients exhibit intrinsic and acquired resistance to common chemotherapy drugs, such as cisplatin and targeted agents. As it has been argued that 3D models and their micro-environment are more reflective of the in vivo situation, 3D culture may provide a more accurate in vitro model to elucidate mechanisms of chemo-resistance and possibly aid in the identification of novel targets to re-sensitise and stratify patients for therapy.
-
+
P3.01 - Poster Session with Presenters Present (ID 469)
- Event: WCLC 2016
- Type: Poster Presenters Present
- Track: Biology/Pathology
- Presentations: 2
- Moderators:
- Coordinates: 12/07/2016, 14:30 - 15:45, Hall B (Poster Area)
-
+
P3.01-042 - Lung Cancer Cells Can Stimulate Functional and Genotypic Modifications in Normal Bronchial Epithelial Cells (ID 4852)
14:30 - 15:45 | Author(s): K. O’byrne
- Abstract
Background:
Normal lung epithelium cells may act in concert with tumour cells, given that bystander effects may exist between the two. This interaction may lead to inappropriate activation of pro-oncogenic signalling pathways, which may result in high mutational load and tumour heterogeneity. The aim of this project is to evaluate the effects of non-small cell lung cancer (NSCLC) cells on an immortalised normal bronchial epithelial cell line.
Methods:
A normal bronchial epithelial cell line (HBEC4) was exposed to A549 (adenocarcinoma), H460 (large cell carcinoma) and SK-MES-1 (squamous cell carcinoma) NSCLC cell lines in a trans-well co-culture system. Cellular characteristics were examined using a Cytell Cell Imaging System (cell number, viability, apoptosis, cell cycle). The gene expression profile was also determined in terms of inflammatory mediators, stem cell markers (RT-PCR) and miRNA profiling (Nanostring). The proliferative effect of NSCLC cancer exosomes was also examined (BrdU ELISA) on the HBEC4 cell line.
Results:
A number of functional and gene modifications were observed in the HBEC4 cell line after seven days of co-culture. While patterns were similar amongst all NSCLC subtypes, SK-MES-1 elicited the most significant effects in terms of cell number, viability, cell cycle progression and proliferative potential of isolated cancer exosome fraction. Promotion of both inflammatory mediators and stem cell marker expression was evident at the mRNA level. There was no apparent consensus between NSCLC subtypes and miRNA expression, as exposure to each cell line resulted in distinct profiles of miRNAs in HBEC4 cells. Bioinformatic analysis of miRNA target genes, demonstrated that pathways such as p53, MAPK, VEGF, TLR and Wnt were amongst those altered.
Conclusion:
Cancer cells may promote significant genotypic and phenotypic alterations within the normal lung epithelium though multiple mechanisms. These modifications may, in part, contribute to the heterogeneity of lung cancer tumours and influence response to both chemotherapeutics and targeted agents.
-
+
P3.01-064 - The Overexpression and Cleavage of SASH1 by Caspase-3 Stimulates Cell Death in Lung Cancer Cells (ID 5811)
14:30 - 15:45 | Author(s): K. O’byrne
- Abstract
Background:
SASH1 (SAM and SH3 domain-containing protein 1) is a recently identified gene with tumour suppressor properties and has a role in induction of apoptosis. Previous work has shown that 90 % of lung cancer cell lines have a decrease in SASH1 mRNA levels (Zeller et al., 2003), however little characterisation of SASH1 function in lung cancer has been undertaken.
Methods:
We evaluated SASH1 expression in transformed normal and malignant non-small cell lung cancer cell lines. We also utilised cell based assays to study the effects of altered SASH1 levels on cell survival and proliferation. Identification of a novel SASH1 targeting drug was performed through connectivity mapping.
Results:
SASH1 protein expression was down regulated in two of the five lung cancer cell lines compared to immortalized normal bronchial epithelial cells. Prognoscan assessment identified decreased SASH1 mRNA expression reduced patient survival. The depletion of SASH1 in lung cells resulted in a significant increase in cellular proliferation in cancer lung cells. Connectivity mapping predicted the drug Chloropyramine would lead to an increase in SASH1 expression. We demonstrated that Chloropyramine upregulates SASH1 in malignant cell lines. In keeping with this we have demonstrated the Chloropyramine inhibited lung cancer proliferation in vitro. We also explored the role of SASH1 in apoptosis. Following ultraviolet light exposure SASH1 is cleaved by Caspase-3. The C-terminal fragment of SASH1 then translocates from the cytoplasm to the nucleus where it associates with chromatin. The overexpression of wild type SASH1 or cleaved SASH1 amino acids 231-1247 leads to an increase in apoptosis, however loss of the SASH1 cleavage site and/or nuclear translocation prevents this initiation of apoptosis. Mechanistically SASH1 cleavage is required for the translocation of the transcription factor NF-κB to the nucleus. The use of the NF-κB inhibitor DHMEQ demonstrated that the effect of SASH1 on apoptosis was dependent on NF-κB, indicating a co-dependence between SASH1 and NF-κB for this process.
Conclusion:
We have shown that SASH1 contributes to apoptosis via a NF-κB-dependent mechanism. Agents that upregulate SASH1, such as chloropyramine or SASH1 gene therapy, are potential novel approaches to the management of NSCLC in the future.
-
+
P3.02b - Poster Session with Presenters Present (ID 494)
- Event: WCLC 2016
- Type: Poster Presenters Present
- Track: Advanced NSCLC
- Presentations: 1
- Moderators:
- Coordinates: 12/07/2016, 14:30 - 15:45, Hall B (Poster Area)
-
+
P3.02b-044 - Afatinib versus Gefitinib as First-Line Treatment for EGFR Mutation-Positive NSCLC Patients Aged ≥75 Years: Subgroup Analysis of LUX-Lung 7 (ID 5327)
14:30 - 15:45 | Author(s): K. O’byrne
- Abstract
Background:
The irreversible ErbB family blocker afatinib and the reversible EGFR tyrosine kinase inhibitor gefitinib are approved for first-line treatment of advanced EGFRm+ NSCLC. In the Phase IIb LUX-Lung 7 trial, afatinib significantly improved median progression-free survival (PFS; HR=0.73 [95% CI, 0.57–0.95], p=0.017), objective response rate (70% vs 56%, p=0.008) and time to treatment failure (TTF; HR=0.73 [95% CI, 0.58–0.92], p=0.007) versus gefitinib in this setting (Park et al. Lancet Oncol 2016). Here we evaluated the efficacy and safety of afatinib versus gefitinib in patients aged ≥75 years in a subgroup analysis of LUX-Lung 7 (NCT01466660).
Methods:
Treatment-naïve patients with stage IIIB/IV EGFRm+ NSCLC were randomized (1:1) to oral afatinib (40 mg/day) or gefitinib (250 mg/day), stratified by EGFR mutation type (Del19/L858R) and presence of brain metastases (Yes/No). Co-primary endpoints were PFS, TTF, and overall survival. Subgroup analyses of PFS and adverse events (AEs) by age (≥75/<75 years) were exploratory.
Results:
Of 319 patients randomised in LL7, 40 (13%) were aged ≥75 years (afatinib n=19, gefitinib n=21). Median PFS for both age groups was in line with the overall population and favoured afatinib versus gefitinib (patients ≥75 years: 14.7 vs 10.8 months, HR=0.69 [95% CI, 0.33–1.44]; patients <75 years: 11.0 vs 10.9 months, HR=0.76 [95% CI, 0.58–1.00]). The incidence of treatment-related AEs (grade 3/4) was slightly higher in the older subgroup (afatinib: 42%/0%; gefitinib: 24%/5%) than in the younger subgroup (afatinib: 28%/2%; gefitinib: 15%/<1%). There were no unexpected safety findings. The most common treatment-related AEs (all grade [grade 3]) with afatinib in the older patient subgroup were diarrhoea (89% [21%]), rash (63% [5%]), dry skin (37% [0%]), and decreased appetite (32% [0%]). Dose reduction/discontinuation of afatinib due to treatment-related AEs was required in 53%/16% and 40%/5% of the older and younger subgroup, respectively.
Conclusion:
A small subgroup of patients in the LUX-Lung 7 trial were ≥75 years old (13%). In exploratory subgroup analyses of patients aged ≥75 and <75 years old, advancing age did not adversely affect the PFS benefit and tolerability observed with afatinib versus gefitinib in treatment-naïve EGFRm+ NSCLC patients. These findings suggest that afatinib can provide an effective and tolerable treatment for older patients with EGFRm+ NSCLC.
-
+
P3.02c - Poster Session with Presenters Present (ID 472)
- Event: WCLC 2016
- Type: Poster Presenters Present
- Track: Advanced NSCLC
- Presentations: 1
- Moderators:
- Coordinates: 12/07/2016, 14:30 - 15:45, Hall B (Poster Area)
-
+
P3.02c-010 - Resistance Mechanisms to PI3K-mTOR Inhibition in NSCLC (ID 5355)
14:30 - 15:45 | Author(s): K. O’byrne
- Abstract
Background:
Non-small cell lung cancer (NSCLC) is a leading cause of cancer mortality globally, having a 5 year survival rate of less than 15%. PI3K-mTOR signalling has been implicated in various hallmarks of cancer and this pathway is often dysregulated in NSCLC. Efforts to therapeutically target the PI3K-mTOR pathway have been hindered by emerging drug resistance. In this study, mechanisms of drug resistance were investigated in a H1975 cell line model of acquired resistance, following chronic exposure to a phase II PI3K-mTOR inhibitor (GDC-0980), Additionally, short term exposure of BEZ235 (another phase II PI3K-mTOR inhibitor) and IBL-301 (a novel PIM/PI3K/mTOR inhibitor) were investigated for effects on cell viability/proliferation and downstream signalling pathways.
Methods:
Alterations to the mRNA expression profile of GDC-0980 acquired resistant H1975 cells versus matched parent cells were examined using an 84-gene IL-6/STAT3 signalling-specific profiler array. Subsequently, selected genes were validated by qPCR. The effectiveness of BEZ235 and IBL-301 on cell viability of two lung cancer cell lines (H1975 and H1838) following 72 hour treatment were investigated by Cell Titre Blue. pAkt and p4E-BP1 expression were examined by Western blot analyses following treatment with BEZ235 and IBL-301 at 3, 6 and 24 hours.
Results:
Thirty candidate gene alterations were identified from the array profile and six genes were chosen for validation by qPCR (n=3). The pro-proliferative and pro-metabolic regulator mTOR and the anti-apoptotic protein BCL-2 were increased in GDC-0980 resistant cells (p<0.05 and p<0.001). Similarly, TNF-α and its receptor co-stimulatory molecule CD40 were increased (p<0.05 and p<0.01). Furthermore, the cell cycle inhibitor, CDKN1, and JAK-signalling blocker, SOCS1 were downregulated (both p<0.01) in GDC-0980 resistant cells. BEZ235 and IBL-301 had a dose-dependent effect on the viability of NSCLC cell lines with respective IC~50~ values of 9.42nM and 136.55nM in H1975 cells and 103.35nM and 159.27nM in H1838 cells. Treatments of 250nM BEZ235 or IBL-301 inhibited pAKt at all time points in the lung cancer cell lines. BEZ235 blocked translation repressor protein (p4E-BP1) across all 3 cell lines and time points while IBL-301 treatment resulted in a reduction in p4E-BP1 at 24 hours.
Conclusion:
This study highlights a number of genes involved in IL-6/STAT3 signalling that may contribute to PI3K-mTOR inhibitor resistance. BEZ235 and IBL-301 both decrease cell viability and inhibit PI3K pathway signalling and cap-dependent translation in NSCLC cell lines that warrant further investigation.
-
+
P3.03 - Poster Session with Presenters Present (ID 473)
- Event: WCLC 2016
- Type: Poster Presenters Present
- Track: Mesothelioma/Thymic Malignancies/Esophageal Cancer/Other Thoracic Malignancies
- Presentations: 1
- Moderators:
- Coordinates: 12/07/2016, 14:30 - 15:45, Hall B (Poster Area)
-
+
P3.03-021 - When RON MET TAM in Mesothelioma: All Druggable for One, and One Drug for All? (ID 5025)
14:30 - 15:45 | 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. Therefore an urgent need exists to identify new therapies for treating MPM patients. The potential for therapeutically targeting receptor tyrosine kinase (RTK) signalling networks is emerging as a critical mechanism in ‘oncogene addicted’ cancer, with RTK inhibitors evolving as areas of considerable importance in cancer therapy. Furthermore, RTK hetero-dimerization has emerged as a key element in the development of resistance to cancer therapy. As such TKIs which target several RTKs may have superior efficacy compared with TKIs targeting individual RTKs. We and others have identified c-MET, RON, Axl and Tyro3 as RTKs frequently overexpressed and activated in MPM, making these attractive candidate therapeutic targets. A number of orally bioavailable small molecule inhibitors have been developed which can target these receptors. LCRF0004 specifically targets RON, whereas ASLAN002 (BMS-777607) or Merestinib (LY2801653) are orally bioavailable small molecule inhibitors which inhibit c-MET, RON, Axl and Tyro3 at nanomolar concentrations. These drugs may therefore have applicability in the treatment/management of MPM.
Methods:
A panel of MPM and normal pleural cell lines were screened for expression of Tyro3, c-MET, RON and Axl by RT-PCR, and subsequently examined in a cohort of patient samples comprising benign, epithelial, biphasic, and sarcomatoid histologies by qPCR. The effects of two small molecule inhibitors LCRF0004, ASLAN002 on MPM cellular health were assessed in vitro. The effects of LCRF0004 and ASLAN002 were subsequently examined in an in vivo SQ xenograft tumour model.
Results:
Expression of various RON 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 and this was validated in other datasets. Both LCRF0004 and ASLAN002 demonstrated significant anti-tumour efficacy in vitro. In xenograft models ASLAN002 was far superior to LCRF0004.
Conclusion:
Our results suggest that a multi-TKI, targeting the RON/MET/TAM signalling pathways, may be a more effective therapeutic strategy for the treatment of MPM as opposed to targeting RON alone.
-
+
SC27 - P53 and KRAS Mutations in NSCLC (ID 351)
- Event: WCLC 2016
- Type: Science Session
- Track: Biology/Pathology
- Presentations: 1
- Moderators:F. De Marinis, H. Kunitoh
- Coordinates: 12/07/2016, 11:00 - 12:30, Strauss 3
-
+
SC27.03 - Transforming KRAS into a Clinically Relevant Biomarker (ID 6715)
11:00 - 12:30 | Author(s): K. O’byrne
- Abstract
- Presentation
Abstract:
TP53 mutations represent the commonest mutation seen in NSCLC. Over 50% of NSCLC tumours harbour a TP53 mutations. TP53 mutations in tobacco smokers are predominantly G-to-T transversions and deletions. In non-smokers, however, these alterations are rare. p53 is a stress response protein/transcription factor and is involved in cellular response to DNA damage induced by oxidative stress and external factors such as sunlight and radiation. Expression of p53 protein is largely controlled through degradation of the protein by the mouse double minute 2 (MDM2) E3 ligase and MDM4. Post-translational modification by various kinases/phosphatases and acetylases/deacetylases also regulates p53 activity. Amongst the various type of TP53 mutation that can occur, conformational TP53 mutations may contribute to the emergence of new functions leading to genomic instability, inhibition of apoptosis, cell migration, and drug resistance. These mutations may result in the binding and inactivation of p53-related proteins such as p63 and p73, or with other transcriptional factors resulting in modification of their activity and, hence, altered gene expression. Apart from the loss of tumour-suppressor functions, TP53 mutations may result in gain of function favouring cellular proliferation, inhibition of apoptosis, and genomic instability. As a result not all P53 mutations are the same with some more likely to affect the pathogenesis of NSCLC than others. Broadly speaking TP53 mutations may be divided into disruptive and non-disruptive cohorts. Many non-disruptive p53 mutations result in gain of function. In a recent series of 318 patients that included 125 with EGFR-mutations, non-disruptive TP53 mutations were associated with a poor prognosis. A recent study using an ELISA demonstrated p53 antibodies in 20.4% of lung cancer patients. A significant correlation between serum p53 antibodies and high levels of p53 expression in the corresponding tumour samples was seen. In NSCLC, the presence of p53 antibodies were significantly associated with poorly differentiated. High levels of p53 antibodies were also associated with high grade tumours, with squamous cell histology and with a poor prognosis in squamous cell carcinoma. Oncogenic KRAS mutations have been reported in up to 40% of adenocarcinomas and 5% of squamous cell carcinomas of the lung. These mutations are found largely in lung adenocarcinomas with solid growth patterns. While KRAS mutations are classically associated with a significant smoking history, they are also identified in a substantial proportion of never-smokers. KRAS mutations are relatively mutually exclusive from EGFR, BRAF and ALK mutations/rearrangements but have considerable overlap with both P53 and PIK3CA mutations. The commonest mutation is in codon 12 but mutations in codon 13 and 61 are also been described. Substitutions in these residues result in constitutively elevated levels of Ras-GTP due to reduced intrinsic GTP hydrolysis and resistance to GTPase-activating proteins and hence activation of the Raf and phosphatidylinositol 3-kinase. Although controversy on the prognostic and predictive value of the presence of a KRAS mutation in a tumour exists, recent studies indicate that patients with KRAS mutations are resistant re chemotherapy and radiotherapy, with a lower objective response rate and worse progression free and overall survival rates. A number of recent studies have allowed us to gain novel insights into the role of KRAS in the pathogenesis of lung cancer, in particular adenocarcinoma of the lung. Increasing evidence indicates a role for chronic inflammation in the pathogenesis of lung cancer, an observation being exploited clinically through the use of immune checkpoint inhibitors such as pembrolizumab. A proportion of KRAS mutation positive tumours have been found to have a high tumour mutation burden that may indicate sensitivity to such agents. KRAS mutations have also been associated with tumour-infiltrating lymphocytes. Recent work has demonstrated that KRAS mutation in lung epithelial cells preferentially leads to recruitment of Th17 positive immune cells that produce IL-17, a cytokine that promotes inflammation. IL-17 induces tumorigenesis by recruiting GR1C CD11bC immune cells. Recent work has demonstrated that miRs may play a role in the regulation of KRAS. For example miR-31 has recently been reported to be over-expressed in lung adenocarcinoma and to correlate with worse survival. Using a transgenic mouse model that allows for lung-specific expression, induction of miR-31 results in lung hyperplasia, followed by adenoma formation and later the development of adenocarcinoma. Induced expression of miR-31 acts with mutant KRAS to accelerate lung tumourigenesis by down-regulating a number of negative regulators of RAS/MAPK signaling. The expression of mesothelin, a cell surface glycoprotein that may have a role in cell adhesion and metastases, is seen in several epithelial cancers and has recently been assessed in adenocarcinoma of the lung using immunohistochemistry. The intensity of staining and the percentage of cells expressing mesothelin in the report was blinded for molecular data and outcome. Mutations of EGFR, KRAS, BRAF, AKT1, PIK3CA and HER2 were assessed by pyrosequencing; HER2 amplification and ALK translocation were assessed by fluorescence in situ hybridization. Of the advanced lung adenocarcinomas, 53% expressed mesothelin to some degree. High mesothelin expression, defined as mesothelin positivity in more than 25% of cells, was found in 24% of patients. High mesothelin expression was associated with a worse survival (median 18.2 months vs. 32.9 months; P = 0.014) and with wild-type EGFR, and was strongly associated with mutant KRAS. The increased understanding of the tumour promoting activities of KRAS mutations, and the association with biomarkers, provides novel insights that will facilitate targeting of these tumours with novel agents in the future.
Only Members that have purchased this event or have registered via an access code will be able to view this content. To view this presentation, please login, select "Add to Cart" and proceed to checkout. If you would like to become a member of IASLC, please click here.
