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O.T. Brustugun



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    MO16 - Prognostic and Predictive Biomarkers IV (ID 97)

    • Event: WCLC 2013
    • Type: Mini Oral Abstract Session
    • Track: Medical Oncology
    • Presentations: 1
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      MO16.07 - Higher frequency of genetic aberrations in KRAS- than in EGFR-mutated NSCLCs. A next-generation sequencing study on 96 samples. (ID 1094)

      16:55 - 17:00  |  Author(s): O.T. Brustugun

      • Abstract
      • Presentation
      • Slides

      Background
      Genetic subtyping is increasingly being clinically relevant in NSCLC, and the search for novel targetable driver mutations is warranted. We intended to study the frequency and types of a vast number of potential druggable genetic aberrations in a large cohort of non-small cell lung cancers of all major histological subtypes. Herein we report the first findings.

      Methods
      Blood samples and tumor tissue was obtained from 96 operated early stage lung cancer patients admitted to Oslo University Hospital-Rikshospitalet in the period 2006-2011. Tissue was taken from the excised tumours, snap frozen in liquid nitrogen in the operation room, and stored at -80[o]C until DNA isolation. The tumor cell content in the specimens was found to be more than 70% in most samples. DNA was isolated from both tumor and corresponding blood sample according to standard procedures. High-throughput sequencing was performed using the SureSelect Human Kinome kit (Agilent Technologies), with capture probes that target 3.2 Mb of the human genome and include exons for all known kinases, select cancer-specific genes and their associated UTRs, in total 612 genes. The derived sequence reads were analyzed based on a pipeline including calling variations, somatic mutations, DNA copy number changes, indels and genomic rearrangements, as well as functional annotations.

      Results
      Tissues from 48 females and 48 males were analyzed; 73 adenocarcinomas, 21 squamous cell carcinomas and 2 large cell carcinomas. 55 patients were in stage I, 27 in II and 14 in stage III. 13 patients were never-smokers. 25 samples harbored a KRAS-mutation and 10 an EGFR mutation. The number of mutated genes per sample varied from 1 to 81. The median number of mutated genes was 14 in the overall cohort, 15 in the EGFR wildtype/KRAS wildtype tumors, 17 in KRAS- mutated patients, 5 in the EGFR-mutated group and 6 in the never-smoking patients (of whom 4 patients were EGFR-mutated).Figure 1

      Conclusion
      KRAS-mutated tumors contain the same amount of genetic aberrations as in wild-type tumors, whereas EGFR-mutated tumors show a much lower number of mutations per tumor. Never-smokers harbor a low number of mutations independent of EGFR-mutation status. Novel driver mutations are probably found in samples with low numbers of mutations.

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    MO19 - Lung Cancer Immunobiology (ID 91)

    • Event: WCLC 2013
    • Type: Mini Oral Abstract Session
    • Track: Biology
    • Presentations: 1
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      MO19.10 - Prevalence and prognostic association of PD-L1 protein and immune gene expression in NSCLC (ID 2437)

      11:25 - 11:30  |  Author(s): O.T. Brustugun

      • Abstract
      • Presentation
      • Slides

      Background
      Programmed Death Ligand 1 (PD-L1, CD274, B7-H1) is an immune checkpoint molecule that binds to the receptors PD-1 and B7.1 on activated T cells. Binding negatively regulates T-cell function in both physiological and pathological conditions. Recent clinical studies have suggested that numerous cancers, including NSCLC, may utilize PD-L1 expression to escape T-cell mediated cytotoxic activity. Inhibition of PD-L1 can restore anti-tumor immunity, leading to clinical responses. A better understanding of PD-L1 expression patterns, co-expression with other immune markers and actionable disease associated biomarkers may provide insight into the future design of cancer immunotherapy trials in NSCLC.

      Methods
      Expression of PD-L1 was measured by immunohistochemistry (IHC) in archival tumors and, in some cases, in paired metastases in 2 FFPE NSCLC tumor tissue collections. Set 1 (N=561) was collected from patients who were eligible for surgery with curative intent from 2003 to 2005 at MD Anderson Cancer Center. The samples from Set 2 (N=300) contained surgically resected NSCLC tissue collected between 2006 and 2011 (UCCC and Norwegian Radium Hospital). PD-L1 expression was analyzed in both malignant and non-malignant cells (e.g., infiltrating immune cells). In addition, a multiplex qPCR assay that measures ≈90 immune-related genes was used to characterize the tumor immune microenvironment in the NSCLC tumor samples. Disease associated biomarkers, including the mutation status of EGFR and KRAS, as well as expression of MET (by IHC) were also evaluated.

      Results
      Prevalence of PD-L1 was comparable between adenocarcinoma and squamous cell carcinoma (≈30% in tumor cells; ≈45% and ≈50%, respectively, in immune cells). PD-L1 prevalence varied depending on the pathological stage, and was higher in Stages I-IIIA than in Stages IIIB-IV. Similarly, the prognostic value of PD-L1 varied by both stage and histology. In adenocarcinoma, tumors with PD-L1–positive tumor cells had a higher frequency of KRAS mutation and high Met expression, and a lower frequency of EGFR mutation compared with PD-L1–negative tumors. In contrast, tumors with PD-L1–positive and PD-L1–negative immune cells had a comparable frequency of high Met expression. Expression of PD-L1 was frequently co-localized with CD8+ T-cell infiltrates. Gene expression profiling revealed differences in the tumor immune environment, including genes associated with cytotoxic T-cells, between adenocarcinomas and squamous cell carcinomas. PD-L1 protein and immune gene expression associations with patient characteristics will be described in further detail.

      Conclusion
      These data provide a comprehensive description of PD-L1 expression in the context of disease biology utilizing large independent cohorts of well-characterized lung cancer tissues. The results highlight the complexity of the tumor immune environment in NSCLC with particular emphasis on the association with factors such as pathological stage, histology and oncogenic mutational status. These analyses may help guide future development of immunotherapy trials in NSCLC.

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    MS01 - Radiation as a Systemic Therapy (ID 18)

    • Event: WCLC 2013
    • Type: Mini Symposia
    • Track: Radiation Oncology + Radiotherapy
    • Presentations: 1
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      MS01.3 - Hypoxia as a Cause of Treatment Failure in NSCLC (ID 459)

      14:45 - 15:05  |  Author(s): O.T. Brustugun

      • Abstract
      • Slides

      Abstract
      Hypoxia as a cause of treatment failure in NSCLC Odd Terje Brustugun, MD PhD Senior Consultant, The Norwegian Radium Hospital, Oslo, Norway & Assoc. professor, Faculty of Medicine, University of Oslo, Oslo, Norway Well-oxygenated tumors respond better to various therapies than hypoxic tumors. Hypoxia is therefore a predictive factor. However, emerging knowledge has underscored that hypoxia is also a prognostic factor, independently of therapy, and that hypoxia in a tumor’s microenvironment induces a more aggressive tumor phenotype. Here, factors involved in hypoxia-mediated therapeutic failures will be discussed both in the context of therapy resistance and as a tumor biology phenomenon per se. Most tumors (including lung cancer) have a low pO~2~ of 0-7.5 mmHg which can be measured indirectly using tracers as 18F-FAZA PET imaging, or via MR-based techniques. However, due to the heterogenous distribution and temporal instability of hypoxia, such methods are limited by lack of resolution. Oxygen molecules diffuse freely in normal tissues, with a diffusion range of ca 200 um. However, all solid tumors over 1 cm[3] contain hypoxic regions due to several factors: abnormal microvessel structure and function leading to increased diffusion distance from vessel to cell, increased oxygen demand due to increased cellular proliferation, reduced oxygen supply due to vascular constriction and increased interstitial pressure. Anemia, frequently observed in cancer patients, adds to the reduced oxygen supply (1). Radiation kills cells mainly via production of free radicals that bind to DNA and induce strand breaks. Oxygen stabilizes the chemical bond breaks in DNA, and makes the damage permanent. Therefore, in oxygen absence, DNA is less vulnerable to permanent damage, leading to relative radioresistance, and the dose has to be increased substantially to induce the same cell kill. Hypoxia-inducible factor-1 (HIF-1) is an intracellular protein whose transcriptional activity is increased as a response to cellular stresses, including hypoxia (2). HIF-1 consists of a labile unit (HIF-1α) and a stable unit (HIF-1β), which heterodimerize to become transcriptionally active. In normoxia, HIF-1α undergoes proteolysis, resulting in a very low level of HIF-hetereodimers. In hypoxia, degradation of the α-unit is reduced leading to an increased level of the functional heterodimer which via binding to hypoxia response elements (HRE) induces expression of genes. Notably, HIF-1 is also regulated by other factors apart from, or in concert with molecular oxygen. HRE-elements are found in promoter or enhancement regions of various tumor-promoting families of genes, involved in anaerobic metabolism (3), angiogenesis (4), anti-apoptosis (5) and invasion and metastasizing (6). Lysyl oxidase, LOX, is upregulated in hypoxia via HIF-1 and is shown to be an independent prognostic marker also in lung cancer (7). LOX exerts its effect both locally by stimulating migration and invasive behavior, and far away from its secretory origin, preparing the metastatic niche (8). Blockade of LOX is shown experimentally to reduce the metastatic propensity of tumors. HIF-1-mediated signaling regulates virtually every step of the metastatic cascade, from migration towards blood vessels and intravasation through HIF-induced leaky endothelial cells. Further, HIF-1 inhibits anoiokis of circulating tumor cells, and hypoxic primary tumors secrete factors that permeabilize the endothelium at distant premetastatic sites (9). Every element of the stromal compartment is also influenced by hypoxia, including fibroblasts, immune, lymph and blood cells, each playing important roles in tumor progression (10) Of special interest in lung cancer, epidermal growth factor receptor (EGFR) is involved in several aspects of hypoxia. Recently, hypoxia was shown to stimulate invasion via EGFR-activation (11). EGFR is also shown to suppress specific tumor-suppressing microRNAs in response to hypoxic stress through post-translational regulation of a Dicer-regulator, AGO2 (12). A number of HIF-1-upregulated genes contribute to radioresistance, perhaps most important is the shift from glucose metabolism to a glycolytic phenotype (13). This effect increases the cell’s antioxidant capacity via accumulation of redox-buffers, thereby reducing the level of free oxygen radicals produced by radiation and thus protects the DNA from damage. Hypoxic tumors reoxygenate after radiation therapy, as a result of reduced demand because of cell death, and due to increased perfusion in tissues (14). Based on this, one would expect HIF-1α levels to decline after radiation, but the opposite is observed. This phenomenon is primarily caused by 1) increased level of free radicals, and 2) liberation of “stress granula” content, both leading to stabilization of the HIF-1α subunit (15). The initial HIF-1-increase occurs within hours of radiation. A few days thereafter, increased NO produced by infiltrating macrophages induces a second peak of HIF-1 stabilization, via NO-mediated prevention of HIF-degradation (16). Both the initial and the later increase of HIF-levels may contribute to a more aggressive phenotype and ultimately to treatment failure as cells become more prone to invade and metastasize. Several hypoxia sensitizers are in clinical trials, but so far, none are in routine use in lung cancer (17). Both HIF-1-inhbitors, as well as drugs targeting glucose metabolism should be further examined in the context of radiation therapy (13). These studies should not only be confined to fractionated therapy, but may likely also have positive impact on stereotactic ablative radiotherapy. In conclusion, tumor hypoxia is a major cause of therapy failure and tumor aggressiveness, involving a multitude of factors. As knowledge emerges, the opportunities of therapeutic interventions should be ample. References 1. Brown JM et al. Nat Rev Cancer. 2004;4:437-47. 2. Greer SN et al. EMBO J. 2012;31:2448-60. 3. Semenza GL. Semin Cancer Biol. 2009;19:12-6. 4. Jackson AL et al. Expert Opin Ther Targets. 2010;14:1047-57. 5. Lenihan CR et al. Biochem Soc Trans. 2013;41:657-63. 6. Sullivan R et al. Cancer Metastasis Rev. 2007;26:319-31. 7. Wilgus ML et al. Cancer. 2011;117:2186-91. 8. Erler JT et al. Cancer Cell. 2009;15:35-44. 9. De Bock K et al. Nat Rev Clin Oncol. 2011;8:393-404. 10. Casazza A et al. Oncogene. 2013. 11. Arsenault D et al. PLoS One. 2013;8:e55529. 12. Shen J et al. Nature. 2013;497:383-7. 13. Meijer TW et al. Clin Cancer Res. 2012;18:5585-94. 14. Rubin P et al. Clin Radiol. 1966;17:346-55. 15. Moeller BJ et al. Cancer Cell. 2004;5:429-41. 16. Li F et al. Mol Cell. 2007;26:63-74. 17. Harada H. J Radiat Res. 2011;52:545-56.

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

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P1.06-011 - Next generation sequencing in lung cancers - focusing on the kinome (ID 1224)

      09:30 - 09:30  |  Author(s): O.T. Brustugun

      • Abstract

      Background
      The majority of newly diagnosed patients with lung cancer are at an advanced stage, implying small chances of cure. However, lung cancer treatment has in the last years taken advantage of newly developed targeted therapies. EGFR-mutations and ALK-translocations are druggable alterations used in treatment decisions. There are several additional druggable mutations in cancers, specially among kinases, but their frequency in lung cancer is not fully elucidated.

      Methods
      Blood samples and tumour tissue were obtained from 96 operated early stage lung cancer patients admitted to Oslo University Hospital-Rikshospitalet in the period 2006-2011. 48 were women, 21 squamous cell carcinomas, 73 adenocarcinomas and two large cell carcinomas. Tissue was taken from the excised tumours, snap frozen in liquid nitrogen in the operation room, and stored at -80[o]C until DNA isolation. The tumour cell content in the specimens was found to be more than 70% in most samples. DNA was isolated from both tumour and corresponding blood sample according to standard procedures. Targeted resequencing was performed using the SureSelect Human Kinome kit (Agilent Technologies), with capture probes targeting 3.2 Mb of the human genome, including exons for all known kinases, and selected cancer related genes and their associated UTRs, in total 612 genes. Targeted regions were sequenced at 50-60x coverage, allowing the detection of subpopulations down to 20%. The derived sequence was analysed based on a pipeline including calling variations, somatic mutations, DNA copy number changes, indels and genomic rearrangements, as well as functional annotations.

      Results
      There were significant differences in the number of somatic mutations detected within each tumour, ranging from 1 to 81, with a median of 14 mutations. Each mutation was supported by at least 20% mutant reads in the tumour, and the great majority corresponded to missense mutations. Over 1000 mutations were identified among all the samples analysed, but recurrent mutations were identified in specific pathways like the PI3K- and CHEK2-pathways. The TP53-gene was the most frequent mutated gene, in almost 50% of the samples, and these mutations have been validated by Sanger sequencing. Of the samples with more than 30 mutations, 55% revealed a mutation in the ATM-gene, whereas the frequency among the other samples was 14%, indicating a deregulation in DNA repair. Using the exon data from tumour and normal samples, we estimated DNA copy number changes, detecting gains and amplifications in cancer relevant genes i.e. KIT, ERK, EGFR.

      Conclusion
      In this pilot study, we have analysed 96 lung carcinomas by next generation sequencing, focusing on the kinome. We have identified several interesting mutational events, and analyses on different clinical subgroups are ongoing.