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G. Bebb



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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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      P1.02-002 - ATM deficiency increases radiation sensitivity in NSCLC cell lines in vitro and confers a poor outcome in early resected NSCLC. (ID 3468)

      09:30 - 16:30  |  Author(s): G. Bebb

      • Abstract

      Background
      ATM is a nuclear protein that plays a central role in the cellular response to DNA double stand breaks (DSBs), the DNA damage response (DDR). In mammalian cells, the DDR consists of co-ordinated multi-facet cellular processes, including DSB repair, cell cycle checkpoints and apoptosis which together act as barriers to tumor-genesis. Germ-line mutations in ATM result in the cancer-predisposing human disorder A-T (Ataxia telangiectasia). Inactivation of ATM due to gene mutations, epigenetic silencing and altered protein expression is found in many cancers including NSCLC. The role of ATM loss in NSCLC is incompletely defined. Early loss of ATM would be expected to cause an increased predisposition to mutation accumulation and the generation of neo-antigens that increase a tumour’s immunogenicity and lead to an increased sensitivity to PARP inhibition and radiation. We set out to determine the prevalence and significance of ATM loss in NSCLC cell lines and in resected NSCLC samples.

      Methods
      A panel of 7 NSCLC cell lines was screened for ATM protein expression levels using western blot. ATM signaling was investigated using specific antibodies to determine p-S1981 ATM, p-S824 KAP1 and p-S15 p53 following treatment of the cells with IR. ATM functionality was assessed by clonogenic survival assay to assess cellular viability after IR treatment. Clinical data was collected retrospectively through chart review of NSCLC patients diagnosed at the Tom Baker Cancer Centre from 2003 to 2006 and entered into the Glans-Look Lung Cancer Database. Archived formalin-fixed paraffin-embedded (FFPE) resected NSCLC tumour samples were retrieved and tissue microarrays constructed. Automated image acquisition was performed using a HistoRx PM-2000™. The ATM expression index was defined as the minimum ratio of the malignant cell specific AQUA score as compared with the non-malignant tumour-associated stromal cell specific AQUA score. Differences in the survival were compared using the log-rank test for low versus high ATM expression index groups and ATM expression groups as well as the stage subgroups. Cox proportional hazards regression was used to assess the prognostic effect of both ATM expression index.

      Results
      ATM deficiency was found in two NSCLC cell lines. The ATM deficient cell line, H23 demonstrates increased sensitivity to IR, disrupted ATM signaling and has the least surviving fraction of cells after treatment with single agent PARP inhibitor and topotecan. Our methodology identified an ATM-EI cutpoint of 0.716, defining 36/165 (21.8%) of patients as being ATM-deficient, many with stage I disease. After adjusting for histology, gender, age, and adjuvant treatment, the ATM-EI had no impact on stage I disease, but was a significant adverse prognostic factor for disease free survival (HR: 4.75, 95% CI: 2.02 to 11.17, p<0.001) and overall survival (HR: 5.09, 95% CI: 2.07 to 12.52, p<0.001) among those with stage II/III disease.

      Conclusion
      This study confirms ATM loss occurs in NSCLC cell lines and has therapeutic implications. It also demonstrates that ATM loss is seen in early stage NSCLC and is the first to show the prognostic consequences of this molecular deficiency. ATM status should be considered in designing new NSCLC clinical trials.

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    P2.02 - Poster Session 2 - Novel Cancer Genes and Pathways (ID 148)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P2.02-021 - Implications of CXCR4 and Estrogen/Progesterone Receptor (ER/PR) pathway activity in Non Small Cell Lung Cancer (NSCLC) (ID 3431)

      09:30 - 16:30  |  Author(s): G. Bebb

      • Abstract

      Background
      CXCR4 is a chemokine receptor that activates signaling pathways responsible for trafficking of cells to sites of inflammation, retention of stem cells in the bone marrow and plays a role in migration and metastatic processes in tumour cells. Previous work by the Bebb lab demonstrated that female stage IV NSCLC patients with high CXCR4 expression have significantly worse survival compared to their low CXCR4 expressing counterparts. Recently, a positive regulatory loop was discovered linking CXCR4 and ER signaling pathways leading to increased gene expression of SDF-1 and other ER dependent genes. We explored the relationship between CXCR4 and ER/PR signaling in NSCLC cell lines, and assessed the expression of ER/PR in stage IV tissue samples to determine if ER/PR could be contributing to the observed gender dependent difference in clinical outcome seen in CXCR4 high expressers.

      Methods
      Two male and two female NSCLC cell lines were characterized for ER and CXCR4 by western blot. Activity of the CXCR4 and ER pathways in each cell line after stimulation with SDF-1 and estradiol (E2), respectively, were assessed by western blot of downstream signaling targets ERK, AKT, phospho-ER and PR. Cross activity of the CXCR4 and ER pathways was examined by stimulating cells with E2 and quantifying SDF-1 and PR mRNA by rtPCR. Tumor specimens from stage IV NSCLC patients (Glans-Look Lung Cancer Database) previously assessed for CXCR4 were analyzed for ERα and PR expression by quantitative immunohistochemistry (IHC) using the HistoRx AQUA® platform. Correlation between CXCR4 and ER/PR expression was assessed using Pearson’s rank correlation, and associations between marker expression and clinical factors/overall survival were assessed using a Cox proportional hazards model.

      Results
      All cell lines expressed varying levels of CXCR4, ERα and ERβ, and were responsive to both SDF-1 and E2 stimulation. Preliminary data suggests that activation of AKT and ERK after stimulation with SDF-1 occurs more rapidly in the male cell lines than in the female cell lines, and that there were differences in SDF-1 gene expression after E2 stimulation between the male and female lines. Other experiments are ongoing and full data will be presented. ER and PR AQUA scores were obtained for 131 patients (63 females and 68 males). There were weak positive correlations between both ER/CXCR4 (r = 0.153) and PR/CXCR4 (r = 0.278), however, only the correlation between CXCR4 and PR was significant (p = 0.002). Similarly, only PR expression was significantly associated with overall survival in the model (p = 0.03)(HR = 1.46). More detailed sub-group analyses further exploring the association with gender is ongoing, and will be presented.

      Conclusion
      The CXCR4 and ER pathways are active in NSCLC cell lines. There appears to be differences in the responsiveness of male and female NSCLC cell lines to CXCR4 and ER pathway activation. ERs and PRs are present in stage IV NSCLC tissue samples, and are associated with both CXCR4 expression and overall survival in our patient cohort. The full implication of these observations is still being investigated and further analyses will be presented.

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    P3.10 - Poster Session 3 - Chemotherapy (ID 210)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Medical Oncology
    • Presentations: 1
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      P3.10-054 - Metastatic NSCLC Outcomes at a Single Canadian Institution Over a Decade (ID 3415)

      09:30 - 16:30  |  Author(s): G. Bebb

      • Abstract

      Background
      In the past 10 years, the standard of care in non small cell lung cancer has seen the adoption of several less toxic and better tolerated therapies, allowing a greater proportion of metastatic patients the opportunity to receive 2[nd] and even 3[rd] line treatment. We investigated whether this improvement in the number of available therapies for metastatic NSCLC had any bearing on overall patient survival, by retrospectively analyzing patients diagnosed in 1999/2000, 2004/2005 and 2009/2010 at our centre.

      Methods
      After ethical approval was obtained demographic details, clinical variables and outcome data were gathered retrospectively via chart review, on NSCLC patients diagnosed at the Tom Baker Cancer Centre (TBCC) in 1999/2000, 2004/2005 and 2009/2010 (Glans-Look Lung Cancer Database). All patients were restaged according to the new 7th Edition of the American Joint Committee on Cancer TNM system for NSCLC staging. Stage IV patients and early stage patients with subsequent metastatic recurrence were included in the analysis. Survival was analyzed using the Kaplan-Meier method and differences measured by a log rank test.

      Results
      1290 patients were included in the preliminary analysis (data only from 1999, 2004/2005 and 2009/2010), 1018 of which were stage IV at diagnosis, 272 who recurred with metastatic disease. The median overall survival (MOS) of patients increased slightly from 1999 to 2009/2010, from 4.5 months in 1999, to 5.7 months in 2004/2005 to 4.6 months in 2009/2010, as did the proportion of patients who received systemic treatment (21.3% in 1999, 28.9% in 2004/2005 and 28.1% in 2009/2010). However, the proportion of patients who received 2 or more lines of chemo doubled from 1999 to 2009/2010 (7.3% in 1999, 12.1% in 2004/2005 and 14.6% in 2009/2010)(p = 0.04). In addition, there was a trend towards increasing median overall survival (MOS) of systemically treated patients over 1999-2009, from 9.6 months (95% CI: 7.6-11.6) in 1999 to 12.7 months (95%CI: 11.0-14.4) in 2009/2010 (p=0.25).

      Conclusion
      Our analysis suggests that there are an increasing proportion of metastatic NSCLC patients being treated systemically at our centre, specifically, the proportion being treated with two or more lines of systemic therapy has significantly increased over the decade from 1999-2009/2010. This study also suggests a trend toward an increased MOS for systemically treated patients diagnosed in 2009/2010 compared to those diagnosed in 1999. However, the vast majority of patients (>3/4) are still not being treated with systemic therapy, despite the increase in available therapies now compared to a decade ago. The reasons for this are not clear but may include poor ECOG performance status, rapid decline, sub-optimal referral pathways and rural residence. Further analyses will be presented.