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A.F. Gazdar



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    CALC - Chinese Alliance Against Lung Cancer Session (ID 79)

    • Event: WCLC 2013
    • Type: Other Sessions
    • Track: Other Topics
    • Presentations: 2
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      CALC.03 - Pathogenesis and Pathology of Never Smoking Lung Cancer (ID 3867)

      A.F. Gazdar

      • Abstract
      • Slides

      Abstract
      Lung cancer (LC) is the leading cause of cancer deaths in the world. While smoking is universally accepted as the major cause of lung cancer in tobacco users, lung cancer in lifetime never smokers (LCNS) is among the 10 major causes of cancer deaths. LCNS is a very different disease than LC arising in ever smokers (LCES), and these differences are discussed in this Abstract. Because LCNS is highly influenced by gender and ethnicity, we put special emphasis on LCNS arising in East Asians. The reader is referred to several recent review articles on this subject [1-5] Etiology: Unlike LCES, the etiology of LCNS is not fully elucidated. The suspected factors include exposure to environmental tobacco smoke (ETS), exposure to industrial or domestic carcinogens including coal smoke and volatile cooking oils, radon exposure, viruses including HPV, and genetic factors. While these factors may individually or in combination contribute to the pathogenesis of LCNS, none of them is likely to be the major causative factor. Further investigation of causation is required. Clinico-patholgoical differences. While adenocarcinoma is the predominant form of NSCLC, the vast majority of LCNS are of adenocarcinoma histology, or large cell carcinomas (which may represent poorly differentiated adenocarcinomas). Squamous cell histology is rare and small cell carcinomas almost never occur. A retrospective study from Singapore identified significantly better performance status, younger age at diagnosis, and higher proportion of females (68.5% vs. 12-13%) and more advanced stage at diagnosis in never smokers compared with current and former smokers [6]. The disease stage variation at diagnosis might be explained by late presentation of symptoms and delayed diagnosis by physicians. The survival outcome of never smokers was significantly better than smokers, with the 5-year overall survival rate of LCES, respectively [6]. The differences in the treatment response and survival outcome between never smokers and smokers with lung cancer may be attributed to the differences in molecular pathogenesis and tumor biology (see below). Genetics: While lung cancer prone families have been well described, the risk of affected subjects is greatly increased after smoke exposure. Recently the interest has focused on single nucleotide polymorphisms (SNPs). Genome wide association studies (GWAS) identified a locus in chromosome region 15q25 that was strongly associated with lung cancer. The association region contains several genes, including three that encode nicotinic acetylcholine receptor subunits [7]. Such subunits are expressed in neurons and other tissues, including alveolar epithelial cells, pulmonary neuroendocrine cells and lung cancer cell lines, and they bind to potential lung carcinogens. Thus variants in this region undoubtedly code for increased susceptibility to smoke and are unlikely to be associated with LCNS. Not unexpectedly, GWAS studies indicate different patterns of susceptibility for Asians and never smokers and possibly related to gender [8, 9]. Molecular differences: The molecular differences between LCES and LCNS show marked differences and characteristic patterns. While TP53 mutations are common to all types of lung cancer, the mutational spectra are very different [10]. KRAS mutations are largely limited to LCES, and, along with TP53, show the typical smoking associated characteristic G to T transversions. In addition, the total numbers of non-synonymous and synonymous mutations in LCES tumors are much higher than that in LCNS, indicating that tobacco exposure results in in widespread genomic instability. Paradoxically, some of the most responsive currently available or potential molecular targets for precision medicine are more frequent in never smokers, including EGFR, BRAF and HER2 mutations and ALK translocations. Therapeutic options and precision medicine: While the overall treatment strategy is the same for LCES and LCNS, the differences in molecular profiles dictate differences in precision medicine and, response to targeted agents and overall survival. These factors are also influenced by gender and ethnicity. For instance, one study found that the frequency of driver mutations (EGFR, HER2, ALK, KRAS, or BRAF) in lung adenocarcinoma from female never-smokers in China was over 87% [11]. Summary: The differences between LCES and LCNS are major, and cover etiologic factors, clinic-pathological changes, genetic susceptibility genes, mutational and molecular changes and precision medicine. These differences are vast enough so that we can regard lung cancers arising in ever and never smokers as two different diseases. References: 1. Rudin CM, Avila-Tang E, Harris CC, et al. Lung cancer in never smokers: molecular profiles and therapeutic implications. Clin Cancer Res 2009;15(18):5646-61. 2. Sun S, Schiller JH, Gazdar AF. Lung cancer in never smokers - a different disease. Nat Rev Cancer 2007;7(10):778-90. 3. StatBite lung adenocarcinoma in smoker vs. never smokers. J Natl Cancer Inst 2010;102(10):674. 4. Lee YJ, Kim JH, Kim SK, et al. Lung cancer in never smokers: change of a mindset in the molecular era. Lung Cancer 2011;72(1):9-15. 5. Subramanian J, Govindan R. Lung cancer in never smokers: a review. J Clin Oncol 2007;25(5):561-70. 6. Toh CK, Gao F, Lim WT, et al. Never-smokers with lung cancer: epidemiologic evidence of a distinct disease entity. J Clin Oncol 2006;24(15):2245-51. 7. Hung RJ, McKay JD, Gaborieau V, et al. A susceptibility locus for lung cancer maps to nicotinic acetylcholine receptor subunit genes on 15q25. Nature 2008;452(7187):633-7. 8. Shiraishi K, Kunitoh H, Daigo Y, et al. A genome-wide association study identifies two new susceptibility loci for lung adenocarcinoma in the Japanese population. Nat Genet 2012;44(8):900-3. 9. Lan Q, Hsiung CA, Matsuo K, et al. Genome-wide association analysis identifies new lung cancer susceptibility loci in never-smoking women in Asia. Nat Genet 2012;44(12):1330-5. 10. Pfeifer GP, Besaratinia A. Mutational spectra of human cancer. Hum Genet 2009;125(5-6):493-506. 11. Zhang Y, Sun Y, Pan Y, et al. Frequency of driver mutations in lung adenocarcinoma from female never-smokers varies with histologic subtypes and age at diagnosis. Clin Cancer Res 2012;18(7):1947-53

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      CALC.07 - Molecular Signatures for the Accurate Classification of NSCLC and Neuroendocrine Tumors and Cell Lines (ID 3874)

      A.F. Gazdar

      • Abstract
      • Slides

      Abstract
      Until recently the oncologist was only interested to know whether a lung cancer was SCLC or NSCLC. However, recent changes, particularly during this century, require more precise classification of lung cancer, and, in some cases, for subclassification [1]. The different classes of lung cancer respond differently to conventional therapy and to precision medicine. The patterns of driver mutations are highly tumor type dependent, with very little overlap between classes. Thus mutation testing depends of accurate classification. Other reasons for accurate classification include: 1) Adenocarcinoma histology is a strong predictor of response to pemetrexed therapy in patients with advanced disease; and 2) Serious hemorrhagic complications after bevacizumab therapy have been reported in patients with squamous histologies. With the development and application of newer agents for precision medicine, the need for accurate classification will only increase. Complicating the increased need for accurate classification is the fact that currently 70% of lung cancers are diagnosed from small biopsies or cytological samples. Thus more accurate diagnoses are demanded from smaller amounts of materials [1, 2]. A further complication is that large international clinical trials often require that tumor materials be reserved for entry requirements or various tests. In routine pathology practice, immunostains are often used to classify poorly differentiated lung carcinomas. While many immunostains have been proposed, a simple algorithm utilizing TTF1 and Napsin A for adenocarcinoma and p63 (or its isoform p40) and high molecular weight keratins have is effective [3]. However, even with excellent pathology practices, over 10% of cases will be incorrectly classified or be unclassified (undifferentiated large cell carcinoma or NSCLC-not other wise specified (NSCLC-NOS). Pathology practices and quality may vary from institution to institution or country to country. The SEER data on cancer incidence indicates that over 20% of lung cancer cases in the USA are not further classified. For these reasons we developed highly specific and sensitive RNA expression signatures as an adjunct test for routine pathological classification. The signatures not only classify the smaples, but provide a numeric scor ranging from 0-1.0, indicating the degree of differentiation. We utilized expression arrays from multiple public and private sources including The Tumor Cell Genome Atlas (TCGA), which used several platforms including Illumina, Affymetrix and RNA-Seq. For complete identification, four signatures had to be developed and validated and can be utilized independently or in combination. These signatures are: 1) Adenocarcinoma-squamous cell carcinoma discrimination, 2) lung specific neuroendocrine (NE)-non neuroendocrine lung cancer discrimination, 3) Non-malignant lung- lung carcinoma discrimination and 4) lung respiratory cell-lung carcinoma cell discrimination. The adenocarcinoma signature includesTTF1, the squamous cell carcinoma signature includes p63 and several high molecular weight keratins, and the NE cell signature includes chromgranin A, synaptophysin and dopa decarboxyase, adding credence to the signatures. These signatures have <10% discrepancy rates with expert pathology review and have helped n the correct classification of NSCLC, large cell and NSCLC-NOS carcinomas, NE lung tumors and lung cancer cell lines. John Minna and Alex Augustyn, in collaboration with us, have utilized their modification of the NE cell signature, and have identified two potential major clinical applications. These include identification of the full NE expression signature in a subset (5-10%) of NSCLC. While some of these may represent misclassified large cell neuroendocrine carcinomas, others appear to be typical adenocarcinomas. In addition, they have identified that BCL2 is one of the downstream targets of ASCL1, the driving force for NE differentiation in the lung, and that inhibition of BCL2 results in apoptosis of SCLC and NSCLC-NE tumors. Practical application of our signatures requires modification to a more practical platform such as Nanostring technology, and application to formalin fixed paraffin embedded small biopsies. These are currently in development. We are grateful to Drs. William Travis and Natasha Rehktman, members of the TCGA pathology panel and Ignacio Wistuba, John Minna and Alex Augustyn for their invaluable assistance. References 1. Gazdar AF. The evolving role of the pathologist in the management of lung cancer. Lung Cancer Management 2012;1(4):1-9. 2. Travis WD, Brambilla E, Noguchi M, et al. Diagnosis of Lung Cancer in Small Biopsies and Cytology: Implications of the 2011 International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society Classification. Arch Pathol Lab Med 2012. 3. Travis WD, Rekhtman N. Pathological diagnosis and classification of lung cancer in small biopsies and cytology: strategic management of tissue for molecular testing. Semin Respir Crit Care Med 2011;32(1):22-31.

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    O12 - Lung Cancer Biology II (ID 87)

    • Event: WCLC 2013
    • Type: Oral Abstract Session
    • Track: Biology
    • Presentations: 1
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      O12.05 - Defining the role of ZEB1 in the pathogenesis of non-small cell lung cancer (NSCLC) using immortalized human bronchial epithelial cells (HBECs) (ID 1139)

      A.F. Gazdar

      • Abstract
      • Presentation
      • Slides

      Background
      To study the role of common lung cancer mutations in transforming lung epithelial cells in an appropriate cellular context we used cdk4/hTERT-immortalized normal HBECs. We developed an isogenic series of HBECs by introducing genetic manipulations representing common lung cancer mutations (such as p53, KRAS[V12], cMYC, and LKB1). This defined in vitro system allows characterization of specific tumorigenic contributions as well as identification of acquired changes, likely representing tumor acquired vulnerabilities and novel therapeutic targets (Mol Cancer Res 2013). One acquired change observed with oncogenic transformation of HBECs is a spontaneous epithelial-to-mesenchymal transition (EMT), an important biologic process in cancer. This study sought to characterize the role of EMT in driving tumorigenesis in HBECs and, in turn, lung cancer to identify novel therapeutic targets.

      Methods
      Genetic manipulations were introduced into cell lines using siRNA/shRNA or over-expression constructs. Tumorigenicity was measured using in vitro (anchorage-dependent and -independent colony formation, proliferation, migration and transwell Matrigel invasion assays) and in vivo (subcutaneous or intravenous injection into NOD/SCID mice) methods. Genome-wide mRNA expression data from five independent datasets was obtained either in-house using Illumina HumanHT-12v4 BeadChips or from publicly available databases.

      Results
      Analysis of EMT-promoting transcription factors in our isogenic series of oncogenically-manipulated HBECs found ZEB1 expression highly correlated with mesenchymal-like HBECs. Functional studies confirmed ZEB1 was a significant driver of tumorigenic phenotypes in both oncogenic HBECs and human lung cancer cell lines where loss of ZEB1 resulted in decreased colony formation, migration and invasion in vitro and subcutaneous tumor growth and intravenous colonization in vivo. A set of ZEB1-associated genes was identified from analyzing five independent mRNA microarray datasets comprising both cell lines and lung adenocarcinomas. From this gene set we found ZEB1 directly represses ESRP1 by binding to its promoter, which leads to increased mesenchymal splicing of the ESRP1 target CD44. The mesenchymal isoform of CD44, CD44s, conferred a CD44[hi] flow cytometry profile which, in turn, could be used to select for a highly tumorigenic subpopulation in partially transformed HBECs. To identify candidate ZEB1-activated targets we screened ZEB1-upregulated genes in a siRNA invasion assay. Several genes including PMP22 and CD70 could phenocopy ZEB1 where siRNA-mediated loss of expression led to decreased invasiveness in multiple NSCLC cell lines. CD70 (also called TNFSF7, tumor necrosis factor ligand superfamily member 7) may represent a prime therapeutic target for anti-metastatic growth in lung cancer. The ligand for CD27, it is involved in immune regulation, upregulated in some cancers and is being studied as a potential target for antibody therapeutics. Importantly, an anti-CD70 monoclonal antibody inhibited invasion of NSCLC cell lines comparably to siCD70 and siZEB1.

      Conclusion
      We demonstrate in vitro models of defined oncogenic HBEC transformation provide an invaluable tool to study lung cancer progression where EMT is an important mediator. ZEB1 is spontaneously expressed with malignant transformation of HBECs and is a significant driver of oncogenic progression in both HBECs and NSCLC cells. Identification of CD70 and PMP22 as downstream targets of ZEB1 may represent novel therapeutic targets for lung cancer.

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