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D..G. Bebb
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OA06 - Prognostic & Predictive Biomarkers (ID 452)
- Event: WCLC 2016
- Type: Oral Session
- Track: Biology/Pathology
- Presentations: 1
- Moderators:F. Shepherd, Y. Yatabe
- Coordinates: 12/05/2016, 14:20 - 15:50, Strauss 1
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OA06.03 - Transcriptome Analysis of ATM-Deficient NSCLC (ID 6196)
14:20 - 15:50 | Author(s): D..G. Bebb
- Abstract
- Presentation
Background:
Current targeted therapy options in lung cancer, such as EGFR and ALK inhibitors, are effective, though limited in use by the low percentage of patients that carry targetable mutations for these biomarkers. Targeting a broader biological process like DNA damage response (DDR), as with recent synthetic lethality exploits in BRCA-deficient tumours, may offer a form of precision therapy for a larger number of patients. We have shown that NSCLC cells deficient in the DDR protein ATM, exhibit similar synthetic lethality when treated with a PARP1 inhibitor, and that NSCLC patients lacking detectable ATM have poorer overall survival. In vitro, ATM deficient, or “ATMic” cells show increased sensitivity to chemotherapeutics at much lower levels when given in combination with PARP inhibitor. This data suggests that ATM status may be an important determinant for treatment modalities including low dose radiation or platin therapy, or novel synthetic lethality therapies. Here, we seek to determine the cause of ATM loss in NSCLC patients through targeted sequencing, and thorough transcriptomic and epigenetic analysis.
Methods:
We perform whole-transcriptome analysis on NSCLC patient samples previously characterized as normal or ATMic, to detect differences in intracellular pathway activation in these tumours. Additional analysis using OncoFinder software identifies possible effective therapies based on which signalling pathways are most active in the normal or ATMic patients. We also perform targeted NGS on these samples. To our knowledge, no sequencing of ATM has been performed on samples that have also been characterized through other methods (i.e. quantitative IHC) to be ATM deficient.
Results:
We have generated a substantial body of evidence showing that ATM loss has significant impact on the cell sensitivity to several therapeutic modalites. As such ATMic tumours may be treated more effectively using specific treatment strategies than their ATM competent counterparts. Initial analysis of NSCLC cell lines using the outlined methodologies distinguishes ATM status and identifies different therapeutic agents based on inherent molecular differences. A complete analysis of the transcriptome profiles of ATMic NSCLC patients will be presented and discussed.
Conclusion:
This research helps complete the overall picture of what the therapeutic implications of ATM loss in NSCLC actually are and how ATMic tumours can best be identified in the clinic. Together, these analyses will give us a stronger understanding of the mechanism for ATM loss in NSCLC, as well as allow us to develop an ATMic “signature” for reliably determining ATM status in patients for directing their treatment options.
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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)
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P1.02-086 - ATM Mutations in Lung Cancer Correlate to Higher Mutation Rates (ID 6339)
14:30 - 15:45 | Author(s): D..G. Bebb
- Abstract
Background:
Ataxia telangiectasia-mutated (ATM) is a critical first responder to DNA damage in the cell, but despite being one of the most mutated genes in lung cancer, no specific mutation hotspots have been linked with disease development. Our own quantitative analysis of ATM protein levels in patient samples suggests that ATM is lost in 20-25% of cases and that this loss correlates with poor overall survival and increased response to adjuvant chemotherapy treatments. We believe that this may be the result of increased genomic instability within the cancer cells caused by a lack of adequate DNA repair. Given that ATM-deficient cancers may have higher genetic instability, and that ATM is so highly mutated in lung cancer, we sought to quantify the relationship between ATM mutations and genomic instability, as measured by total somatic mutations.
Methods:
Using genomic and sequencing data available from the Broad Institute Cancer Cell Line Encyclopedia (CCLE) and the NIH Cancer Genome Atlas (TCGA), we correlated mutations in ATM and other genes involved with the DNA damage response with the total number of mutations annotated in ~900 cancer cell lines and ~500 lung adenocarcinomas.
Results:
We show that in cell lines across all cancer types, and particularly in lung, breast, and esophageal cancers, mutations in ATM correlate with a significantly higher number of total mutations. Only mutations in the direct damage response genes appeared to associate with total mutations, whereas p53 – while more commonly mutated – did not correlate with higher mutations in cell lines or patients. In lung cancer patients, ATM mutations were similarly correlated with high somatic mutations.
Conclusion:
We have identified a potential relationship between ATM mutation and total somatic mutations in cancer cell line and patient tumour genomes, which may be indicative of overall genetic instability. Analysis of the ATM mutations in cell lines and patient samples clearly shows that there are no specific hotspots for mutation in ATM that correlate with increased total mutations. Thus screening for ATM mutations alone may not be sufficient to indicate loss of function or instability. However, this data may prove useful in developing panels of targets to screen as mutation hotspots of instability, and ultimately to help identify patients that may benefit from targeted or modified therapy options based on ATM-deficiency or higher genetic instability.
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P1.06 - Poster Session with Presenters Present (ID 458)
- Event: WCLC 2016
- Type: Poster Presenters Present
- Track: Advanced NSCLC
- Presentations: 1
- Moderators:
- Coordinates: 12/05/2016, 14:30 - 15:45, Hall B (Poster Area)
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P1.06-022 - Clinical Characteristics of Survival Outliers in Stage IV Adenocarcinoma Lung Cancer Patients (ID 4304)
14:30 - 15:45 | Author(s): D..G. Bebb
- Abstract
Background:
Lung cancer is the leading cause of cancer deaths among men and women in Canada. Many lung cancer patients are diagnosed at advanced stages of disease, which is associated with poor survival outcomes. The mean survival of stage IV non-small cell lung cancer (NSCLC) patients is typically less than 12 months; however, there appears to be a small subset of patients with advanced disease that live substantially longer than the norm. Our study aims to determine whether certain clinical characteristics correlate with longer survival in stage IV NSCLC patients.
Methods:
Data on 1803 stage IV NSCLC patients (1291 adenocarcinoma, 512 squamous cell carcinoma) from 1999-2011 were extracted from the Glans Look Lung Cancer database. Adenocarcinoma data is presented here; squamous cell carcinoma data analysis is ongoing. Clinical characteristics such as age, gender, ethnicity, smoking history, histology, molecular testing, metastatic disease, treatments, and socioeconomic factors were compared between survival outliers and patients with average survival. Survival outliers were defined as those patients who lived > 5 years, or greater than 2 standard deviations from mean survival (42.1 months).
Results:
In the survival outlier group, there were 25 patients who lived >5 years, and 59 who lived >42.1 months. Survival outliers included a higher percentage of females, had a smaller smoking history, smaller tumour size at diagnosis, received more treatment lines, and had lower metastatic disease burden at diagnosis (P<0.05 in the outlier group with survival >42.1 months). Upon further characterization of metastatic disease, there appears to be survival outliers associated with no liver metastases and less sites of metastases at diagnosis, as well as with stage M1a disease compared to stage M1b.
Conclusion:
Adenocarcinoma patients with localized and lower metastatic disease burden and no liver metastases at the time of diagnosis appeared to live longer than their counterparts. Further statistical analysis is ongoing to determine the significance of other clinical characteristics with respect to survival. The present study will help us better understand the importance of various clinical parameters and their association with survival, in hopes of improving outcomes for lung cancer patients in the future.
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P2.01 - Poster Session with Presenters Present (ID 461)
- Event: WCLC 2016
- Type: Poster Presenters Present
- Track: Biology/Pathology
- Presentations: 1
- Moderators:
- Coordinates: 12/06/2016, 14:30 - 15:45, Hall B (Poster Area)
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P2.01-090 - Platin Induced Phosphorylation of ATM and ATM-Deficiency as a Predictive Marker of Platin Sensitivity in Non-Small Cell Lung Cancer (ID 5742)
14:30 - 15:45 | Author(s): D..G. Bebb
- Abstract
Background:
Platinum-based antineoplastic therapies (platins) are a first line treatment for non-small cell lung cancer (NSCLC) that generate DNA adducts leading to the formation of both single and double stranded DNA breaks. Effective DNA damage response pathways contribute to cell survival and resistance to these agents. Ataxia telangiectasia mutated (ATM) is an important mediator of the DNA damage response involved in the activation of key components of DNA repair, cell cycle arrest, and apoptosis. Our lab has demonstrated that cell lines lacking ATM show increased sensitivity to platins. We hypothesize that platin exposure will activate ATM and that cells deficient in ATM will be innately sensitivity to platins due to an impaired DNA damage response. Here we assess the molecular action of ATM in response to platins to determine if ATM-deficiency is predictive of platin sensitivity.
Methods:
ATM status was determined in five NSCLC cell lines using western blotting and rt-qPCR. Cell lines were treated with varying concentrations cisplatin, carboplatin and oxaliplatin for 18 hours and assessed for ATM phosphorylation by western blot. Additionally, downstream targets of ATM (KAP-1, p53, and g-H2AX) were investigated to determine ATM pathway activation. Finally, transient and stable ATM knockdowns were generated using siATM and shATM. These cells were then tested for platin sensitivity by trypan blue viability or clonogenic assays.
Results:
NSCLC cell lines NCI-H226, NCI-H460, and NCI-H522 were found to be ATM-proficient whereas cell lines NCI-H23 and NCI-H1373 were found to be ATM-deficient. ATM-proficient cell lines demonstrated an increased level of phosphorylated-ATM in response to platins. In addition, KAP-1, a downstream target of ATM showed increased phosphorylation in response to these treatments when compared to non-treated controls. In contrast, ATM-deficient cell lines showed no increased levels of phosphorylated ATM or KAP-1 in response to platins. Preliminary analysis of siATM transient knockdowns in NCI-H226 shows an increased sensitivity to cisplatin.
Conclusion:
It is clear that platin exposure activated an ATM mediated signalling response and that cells lacking ATM showed deficiencies in the phosphorylation of key downstream targets of this pathway. Cells deficient in ATM may therefore be more susceptible to platin therapy due to an impaired DNA damage response. This data suggests that individuals with low or non-functioning ATM may be candidates for precision low does therapies that exploit this deficiency.
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P3.01 - Poster Session with Presenters Present (ID 469)
- Event: WCLC 2016
- Type: Poster Presenters Present
- Track: Biology/Pathology
- Presentations: 1
- Moderators:
- Coordinates: 12/07/2016, 14:30 - 15:45, Hall B (Poster Area)
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P3.01-045 - Sex Differences in CXCR4-Dependent Motility of NSCLC Cells (ID 4932)
14:30 - 15:45 | Author(s): D..G. Bebb
- Abstract
Background:
The overwhelming majority of deaths due to lung cancer result from metastatic progression of the disease. Cytokines, a group of proteins involved in cell signaling, play an important role in activating the migratory and invasive capabilities of cancer cells, and studies have implicated the stromal-derived factor 1 (SDF-1/CXCL12)-CXCR4 cytokine signaling axis in the progression of several metastatic cancers, including that of the lung. Our previous investigations have shown that survival outcomes of female stage IV non-small cell lung cancer (NSCLC) patients with high CXCR4 levels are significantly worse compared to those of patients with low CXCR4, whereas male patients show no difference in survival. Studies in NSCLC cell lines have observed a link between CXCR4/SDF-1 and estrogen receptor (ER) function, as well as proliferation in response to treatment with estradiol (an estrogen) specifically in female cell lines. These previous results form the rationale for this project, which explores potential sex differences in the motility of NSCLC cell lines in response to cytokine and estrogen stimulation.
Methods:
Western blotting and PCR methodologies were used to assess the downstream activation of CXCR4 and estrogen receptor signaling as a means to confirm their activity in the cell lines studied. The migratory potential of NSCLC cells was measured using wound healing migration assays (scratch tests). Cells were incubated in phenol red-free RPMI 1640 media with or without the reagents of interest (SDF-1, beta-estradiol, estrogen and CXCR4 antagonists, among others) and the migration of cells into the wound was quantified to approximate the metastatic behavior of NSCLC cells in the presence or absence of the aforementioned stimuli.
Results:
All NSCLC cells studied showed high levels of CXCR4, but ER expression varied within our cell line panel, largely by gender of origin. Our preliminary data show a tentative but observable difference in how male and female NSCLC cells respond to both stimulation and inhibition of the CXCR4 axis. In addition, estrogen and SDF-1 co-stimulation induces a greater increase in cell motility of female NSCLC cells.
Conclusion:
The results observed may suggest a possible mechanism, through interactions between CXCR4 and estrogen receptor signalling pathways, to explain the extreme survival differences between male and female stage IV NSCLC patients with high CXCR4 expression.
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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)
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P3.02b-020 - Gender and Ethnicity Influence on Outcome in EGFRmut+ NSCLC Patients Treated at a Single Canadian Institution (ID 5096)
14:30 - 15:45 | Author(s): D..G. Bebb
- Abstract
Background:
EGFR-mutations are associated with adenocarcinoma, female, never/light smoking and East-Asian ethnicity. Several clinical studies have shown that EGFR-TKIs are superior to platin-based chemotherapy in EGFRmut[+] NSCLC patients; however there is insufficient literature that examines differences in survival based on East-Asian ethnicity, independent of the EGFR mutation type, gender, smoking history and disease recurrence in an all EGFRmut[+ ]NSCLC cohort.
Methods:
A retrospective study of EGFRmut[+] NSCLC patients treated with EGFR-TKIs was conducted at the Tom Baker Cancer Centre in Calgary, Canada between March 2010 and December 2014. Ethnicity was defined by place of birth. Comparison of smoking history was made using Pearson χ[2] statistic. Survival analyses according to gender, smoking history and EGFR mutation type were estimated using the Kaplan-Meier method, with comparisons made between groups by the log-rank test. All statistical analyses were conducted using STATA. Statistical significance was assumed for a two-tailed p value < 0.05.
Results:
A total of 138 patients were identified (61 [44.2%] Asians; 77 [55.8%] non-Asians). Of these, 92 died, eight were lost to follow up and 38 were alive as of June 1, 2016. In the Asian subgroup, the median age was 64.4 years, 67% were female, and 71% were never/non-smokers. Meanwhile, in the non-Asian subgroup, the median age was 66.5 years, 61% were female and only 39% were never/non-smokers. The median PFS for Asians vs non-Asians was 8.7m and 8.2m, while the median OS was 25.6m and 16.1m, respectively. The log rank test demonstrated a statistically significant difference in survival between Asians vs non-Asians (p= 0.026). KM curves showed that: (i) smoking was an important demographic feature as non-Asians with a smoking history had the worst median survival (17.5m); Pearson χ[2] showed that the overall relationship between ethnicity and smoking history was significant, p < 0.001; (ii) Asian-females/males had longer survival (23.4m; 21.5m, respectively) than non-Asian females/males (19.7m; 14.5m, respectively) and (iii) regardless of EGFR-mutation type, Asians (exon 19 deletions: 25.9m, exon 21 (L858R): 20.4m) outlived non-Asians (exon 19 deletions: 19.6m and exon 21: 14.9m).
Conclusion:
The demographics from our study are consistent with findings from IPASS and EURTAC. Our current survival estimates suggest that even in a cohort of EGFRmut[+]NSCLC treated with an EGFR-TKI, there seems to be a survival benefit for Asian-ethnicity, even after stratifying by gender, smoking history and EGFR-mutation type. *This study was supported by AstraZeneca.
