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R. Kratzke
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ORAL 06 - Next Generation Sequencing and Testing Implications (ID 90)
- Event: WCLC 2015
- Type: Oral Session
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:G. De Lima Lopes, V. Miller
- Coordinates: 9/07/2015, 10:45 - 12:15, Mile High Ballroom 1a-1f
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ORAL06.03 - Genome-Wide Gene Copy Number Analysis by OncoScan<sup>TM</sup> FFPE Assay in 976 Resected NSCLC From LACE-Bio2 (ID 1561)
10:45 - 12:15 | Author(s): R. Kratzke
- Abstract
- Presentation
Background:
Genome wide SNP array studies have identified systematic gene copy number aberrations (CNA) in non-small cell lung cancer (NSCLC), but their prognostic implication is unknown. This study aimed to investigate associations between CNAs and survival using the LACE-Bio bio-bank. The LACE-Bio consortium includes large clinical trials comparing adjuvant platinum-based chemotherapy to observation after complete resection of stage I-III NSCLC.
Methods:
DNA was extracted from FFPE tumor samples from 3 pivotal adjuvant chemotherapy trials (CALGB 9633, IALT, JBR.10); 1013 samples were profiled using Affymetrix OncoScan[TM] arrays with over 300,000 probes and normalized relative to a pool of normal tissues. Segmentation was performed using the CBS algorithm and minimally recurrent regions (MCR) across the series identified by CGHregions. All analyses were performed on the level of MCRs. CNAs were correlated with clinicopathological factors and adjusted for the False Discovery Rate (FDR). The primary endpoint, disease-free survival (DFS), was assessed via univariate Cox models stratified by trial and adjusted for treatment, age, sex, PS, histology, T, and N stage.
Results:
Among 976 successfully profiled samples, 414 (42%) were adenocarcinoma (ADC), 430 (44%) squamous cell carcinoma (SCC) and 132 (14%) other NSCLC; 710 (73%) were male. Across the 431 MCRs identified, patients had on average 94 (SD 69) CNAs: 51 gains and 43 losses. A gain or loss was observed in at least 10% of patients for 177 and 166 regions respectively. The most common gains (up to 48%) were on chromosomes 1p, 3q, 5p, 6p, and 22q. The most common losses (up to 40%) were on chromosomes 3p, 8p and 9p. The size of 253 of the 431 MCRs (59%) was smaller or equal to 3Mb (and 79% ≤10 Mb). Sensitivity analyses on the subset of samples with optimal quality (n=777, defined by MAPD<0.3) gave consistent results. The CNA frequency of 195 regions was significantly different with FDR≤0.05 between ADC and SCC (of which 49% regions of size ≤3Mb and 71% ≤10Mb); the most significant were more gains in 3q, 22q and 12 in SCC and more losses in 3p, 4, 5q in SCC. With a median follow-up of 5.3 years, 510 DFS events and 451 deaths were recorded. In univariate analyses for DFS, 13 regions in loci 19p11–13, 7p12, 9p21, 15q14 had a raw p-value <0.005 (FDR<0.13, the top 8 corresponded to FDR≤0.05); 9 of those 13 regions were of size ≤3Mb (12 regions ≤10Mb). In adjusted analyses, 10 of the 13 regions retained raw adjusted p-values ≤0.005 (FDR≤0.15). Losses of focal regions including CDKN2A/B and STK11 (≤3Mb) were associated with poorer DFS: the hazard ratio (HR) for a 2-fold copy number decrease in region 9p21.3 (including CDKN2A/B) was 1.50 (95% CI: 1.2–1.9, P<0.001, FDR=0.02), and the HR for a 2-fold copy number decrease in 19p13 (including STK11) was 2.4 (1.3–4.3, P=0.005, FDR=0.15). Similar results were obtained for overall survival and lung-cancer specific survival. Results of histology-specific analyses will be presented.
Conclusion:
These large-scale genome-wide analyses of gene CNA provide new candidate prognostic markers for stage I-III NSCLC.
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P1.04 - Poster Session/ Biology, Pathology, and Molecular Testing (ID 233)
- Event: WCLC 2015
- Type: Poster
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:
- Coordinates: 9/07/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P1.04-110 - Use of Blood Outgrowth Endothelial Cells as a Carrier of Oncolytic Vesicular Stomatitis Virus-Interferon Beta in Treating Metastatic NSCLC (ID 1008)
09:30 - 17:00 | Author(s): R. Kratzke
- Abstract
Background:
Oncolytic viruses have been extensively studies in the past two decades and are promising for cancer treatment. We have shown previously that vesicular stomatitis virus expressing interferon β (VSV-IFNβ) has oncolytic activity in vitro and in vivo in an immune competent mouse model of NSCLC. However, for treatment of metastatic NSCLC, intravenous delivery of VSV-IFNβ still faces several challenges, such as rapid clearance from bloodstream due to serum complement as well as sequestration in lymphoid tissue. In order to overcome these problems, we are exploring the potential role of blood outgrowth endothelial cells (BOECs) as carrier cells to deliver VSV-IFNβ to lung tumor sites.
Methods:
Efficacy of VSV-IFNβ-infected BOECs in transferring VSV-IFNβ to co-cultured human lung cancer cell lines in presence or absence of VSV antiserum were tested in vitro. A/J mice intravenously injected with LM2 non-small cell lung cancer cells were treated with PBS, VSV-IFNβ or VSV-IFNβ-infected BOECs (3 sequential treatments 3 weeks after tumor cell injection). Tumor growth, intratumor viral titer and survival were tested.
Results:
We demonstrated that VSV-IFNβ-infected BOECs can effectively transfer VSV-IFNβ to co-cultured human lung cancer cells and result in viral oncolysis even in the presence of VSV antiserum. In mice bearing metastatic lung cancer, BOECs injected via tail vein preferentially accumulated in lung tumor tissues, and were absent in either normal lung or liver tissues. Moreover, treatment with VSV-IFNβ-BOECs had higher and more sustained intra-tumoral viral titers comparing with those treated with either PBS or naked VSV-IFNβ. Furthermore, there was a trend (p=0.09) towards reduced tumor burden in the VSV-IFNβ-BOEC treated mice (n=5). Currently, we are testing the survival benefit of VSV virus in metastatic lung cancer model.
Conclusion:
In summary, the pre-clinical data showed promise to support developing a clinical protocol in the near future to assess the safety, response and efficacy of VSV-IFNβ-infected BOECs in treatment of metastatic lung cancer.
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P2.04 - Poster Session/ Biology, Pathology, and Molecular Testing (ID 234)
- Event: WCLC 2015
- Type: Poster
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:
- Coordinates: 9/08/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P2.04-052 - Targeting Oncogenic Eukaryotic Protein Translation in Thoracic Malignancies with Small-Molecule Inhibitors (ID 3094)
09:30 - 17:00 | Author(s): R. Kratzke
- Abstract
Background:
Hyperactivation of cap-mediated translation can induce oncogenic transformation by enhancing the translation of a subset of mRNAs involved in the genesis, maintenance and progression of cancer.In this investigation, disabling the eIF4F complex by disrupting the eIF4E-mRNA-cap interaction is evaluated as a therapy for mesothelioma and non-small cell lung cancer (NSCLC).
Methods:
Cell lines and culture. H513 and H2373 were from American Type Culture Collection (ATCC) and cultured in either RPMI 1640 containing 10% calf serum supplemented with 10% calf serum and maintained at 37[o]C. Cell proliferation assay. 5000 cells were seeded into wells of 96 well plates. Following overnight incubation cells were treated with varying doses of cpd 267, 272 or pemetrexed for 72 h. Viable cells were counted employing Cell Counting Kit 8 (Dojindo). Cap-affinity assay. Cell lysate was mixed with 50 mL of a 50% mixture of m[7]GTP-Sepharose resin with and without 400 mM of cpd 267 for 2 h at 4[o]C to capture eIF4E and eIF4G. The captured bound proteins were eluted and prepared for immunoblot analysis. Immunoblot analysis. Protein samples were separated by SDS-PAGE and transferred to Hybond PVDF membrane. Blots were probed for eIF4E and eIF4G (both from Cell Signaling and diluted 1:1000). Detection was carried out using ECL Plus Western Blotting System (Amersham) to visualize the bands of interest.
Results:
Mesothelioma and NSCLC cells were treated with small-molecule inhibitors [compounds 267 and 272] that mimic the cap structure that displace capped mRNAs from the eIF4F complex resulting in suppression of cap-dependent translation of malignancy-related proteins. Treatment with the compounds resulted in a dose dependent decrease in cell viability. Combination therapy of the compounds with cytotoxic agents further decreased cell survival. Binding to a synthetic cap-analogue was employed to assess the strength of eIF4F complex activation in lysates exposed to the compounds.
Conclusion:
These novel compounds reduce cancer cell proliferation,reduce eIF4F complex formation and sensitizes mesothelioma and NSCLC cells to cytotoxic agents.
