P e n n y N Penny Nymark y m a r k Molecular Alterations in Asbestos is still a serious problem all around the world, even though the devastating health effects of breathing in the M Asbestos-Related Lung Cancer microscopically tiny mineral fibres have been known for over a o le century. Asbestos exposure causes a variety of severe pulmonary c u diseases and unfortunately due to the long latency period la r between exposure and development of disease, this epidemic will A continue, even in countries where asbestos use has been banned lt e for many years. ra t io Asbestos-related lung cancer is one of the most common types n s of occupational cancer. It is clinically indistinguishable from lung in cancer in patients with no known history of asbestos exposure A and the treatment is the same for both etiologic types. Never- sb e theless, the molecular basis may be different and diagnosis as s t well as prognosis and treatment strategies may benefit from the o s identification of specific asbestos-related molecular alterations. In -R e addition, these kinds of molecular correlates could be of impor- la tance in resolving some of the medico-legal issues arising from te d occupational diseases. L u n This study sheds light on the molecular alterations related to g asbestos exposure in lung cancer and may point the way for the C a development of molecular-based clinical methods for asbestos- n c related lung cancer. e r Orders: Finnish Institute of Occupational Health Topeliuksenkatu 41 a A FI-00250 Helsinki Finland Fax +358-9 477 5071 E-mail [email protected] www.ttl.fi/bookstore ISBN 978-951-802-982-6 (paperback) 978-951-802-983-3 (PDF) ISSN-L 1237-6183 ISSN 1237-6183 Cover photograph: A bronchoalveolar lavage sample 90 with macrophages attempting to engulf an asbestos body, formed on a crocidolite asbestos fibre People and Work (courtesy of S. Anttila). Research Reports 90 Molecular Alterations in Asbestos-Related Lung Cancer Penny Nymark People and Work Research Reports 90 Finnish Institute of Occupational Health, Helsinki, Finland MOLECULAR ALTERATIONS IN ASBESTOS-RELATED LUNG CANCER PENNY NYMARK Health and Work Ability Finnish Institute of Occupational Health Helsinki, Finland Department of Pathology Haartman Institute and HUSLAB University of Helsinki and Helsinki University Central Hospital Helsinki, Finland Department of Biosciences Faculty of Biological and Environmental Sciences University of Helsinki, Finland ACADEMIC DISSERTATION To be presented for public examination with the permission of the Faculty of Biological and Environmental Sciences, University of Helsinki, in the Small Lecture Hall of the Haartman Institute, Haartmaninkatu 3, Helsinki, on June 11th, 2010, at 12 o´clock noon. Helsinki 2010 SUPERVISED BY Sisko Anttila, MD, Ph.D Health and Work Ability Finnish Institute of Occupational Health Helsinki, Finland Sakari Knuutila, PhD Department of Pathology Haartman Institute and HUSLAB University of Helsinki and Helsinki University Central Hospital Helsinki, Finland REVIEWED BY Janna Saarela, MD, PhD Institute for Molecular Medicine Finland (FIMM) Biomedicum Helsinki 2U Helsinki, Finland Veli-Matti Kosma, MD, PhD Department of Pathology and Forensic Medicine Institute of Clinical Medicine School of Medicine Faculty of Health Sciences University of Eastern Finland, Kuopio, Finland OFFICIAL OPPONENT Antti Sajantila, MD, PhD Department of Forensic Medicine Laboratory of Forensic Biology Helsinki University Helsinki, Finland 4 TABLE OF CONTENTS LIST OF PUBLICATIONS ......................................................8 ABBREVIATIONS................................................................9 ABSTRACT ................................................................... 11 ABSTRACT IN FINNISH – TIIVISTELMÄ ............................... 13 INTRODUCTION .............................................................. 15 REVIEW OF THE LITERATURE ............................................ 19 1. LUNG CANCER ............................................................. 19 1.1 Epidemiology ......................................................... 19 1.2 Histology .............................................................. 21 1.3 Genetics and epigenetics ......................................... 22 1.3.1 Methods used in the detection of genetic alterations and gene expression ...................... 26 1.3.2 Gene Ontology (GO) ...................................... 28 2. ASBESTOS.................................................................. 28 2.1 Characteristics ....................................................... 28 2.2 Use ................................................................... 29 2.3 Toxicity and carcinogenicity ..................................... 32 2.4 Asbestos and tobacco smoke as co-carcinogens.......... 35 3. ASBESTOS-RELATED LUNG CANCER ............................... 35 3.1 Lung cancer risk associated with asbestos exposure .... 36 3.2 Clinical features ..................................................... 37 3.3 Molecular alterations attributable to asbestos exposure in lung cancer .......................................... 38 3.3.1 Genetic alterations ......................................... 38 3.3.2 Signalling pathways ....................................... 41 AIMS OF THE STUDY ........................................................ 43 MATERIALS AND METHODS ............................................... 44 1. LUNG CANCER PATIENTS AND DNA SAMPLES (I, II and III) 44 2. CELL LINES (IV) .......................................................... 47 2.1 Asbestos exposure .................................................. 47 5 TABLE OF CONTENTS 3. ANALYSIS METHODS .................................................... 48 3.1 Chromosomal and array CGH (I) .............................. 48 3.2 Microsatellite analysis for detection of allelic imbalance (II and III) ............................................. 49 3.3 Fluorescence in situ hybridization (FISH) (II and III) ... 52 3.3.1 Tissue microarrays ......................................... 52 3.3.2 Locus-specifi c FISH ........................................ 52 3.3.3 Centromere FISH ........................................... 54 3.4 Gene expression microarrays (IV) ............................ 55 3.4.1 GO analysis .................................................. 56 3.4.2 Clustering ..................................................... 57 3.4.3 Enriched chromosomal regions ........................ 58 RESULTS ................................................................... 59 1. ASBESTOS-RELATED GENETIC ALTERATIONS IN LUNG CANCER ............................................................. 59 1.1 Genome-wide copy number alterations (I) ................. 59 1.2 Genetic alterations at 9q (II) ................................... 62 1.2.1 Allelic imbalance ............................................ 62 1.2.2 Copy number alterations ................................ 65 1.3 Genetic alterations at 2p (III) .................................. 66 1.3.1 Allelic imbalance ............................................ 66 1.3.2 Copy number alterations ................................ 66 1.4 Polyploidy (II) ........................................................ 69 2. ASBESTOS-RELATED GENE EXPRESSION CHANGES IN CELL LINES (IV) ....................................................... 70 2.1 Genes ................................................................... 71 2.2 Biological processes ................................................ 71 2.3 Enriched chromosomal regions ................................. 74 DISCUSSION .................................................................. 76 1. GENOMIC ALTERATIONS IN ASBESTOS-RELATED LUNG CANCER ............................................................. 76 1.1 Allelic imbalance and copy number alterations at 9q (II) .............................................................. 78 1.2 Allelic imbalance and loss at 2p (III) ......................... 80 1.3 Chromosomal regions enriched with asbestos exposure response genes (IV).................................. 83 1.4 Polyploidy and aneuploidy ....................................... 84 2. DYSREGULATED BIOLOGICAL PROCESSES IN ASBESTOS-RELATED LUNG CANCER ................................ 85 6 TABLE OF CONTENTS CONCLUSIONS AND FUTURE PROSPECTS ........................... 90 ACKNOWLEDGEMENTS ..................................................... 93 REFERENCES .................................................................. 96 7 LIST OF PUBLICATIONS This thesis is based on the following original publications, referred to in the text by the Roman numerals I–IV as indicated below. I Nymark P*, Wikman H*, Ruosaari S, Hollmén J, Vanhala E, Kar- jalainen A, Anttila S, Knuutila S. Identifi cation of specifi c gene copy number changes in asbestos-related lung cancer. Cancer Res. 2006; 66(11):5737–43. II Nymark P, Kettunen E, Aavikko M, Ruosaari S, Kuosma E, Vanhala E, Salmenkivi K, Pirinen R, Karjalainen A, Knuutila S, Wikman H, Anttila S. Molecular alterations at 9q33.1 and polyploidy in asbestos-related lung cancer. Clin Cancer Res. 2009; 15(2):468–75. III Kettunen E, Aavikko M, Nymark P, Ruosaari S, Wikman H, Vanhala E, Salmenkivi K, Pirinen R, Karjalainen A, Kuosma E, Anttila S. DNA copy number loss and allelic imbalance at 2p16 in lung cancer associated with asbestos exposure. Br J Cancer. 2009; 100(8):1336–42. doi 10:1038/sj.bjc. 6605012 IV Nymark P*, Lindholm PM*, Korpela MV, Lahti L, Ruosaari S, Kaski S, Hollmén J, Anttila S, Kinnula VL, Knuutila S. Gene ex- pression profi les in asbestos-exposed epithelial and mesothelial lung cell lines. BMC Genomics. 2007; 8:62. *Equal contribution The original publications are reprinted with the permission of their copyright holders. In addition, unpublished data is presented. 8 ABBREVIATIONS AC adenocarcinoma AI allelic imbalance AM alveolar macrophages aCGH array CGH BAC bacterial artifi cial chromosome BEGM bronchial epithelial cell growth medium CCA canonical correlation analysis CGH comparative genomic hybridization cCGH chromosomal CGH CIN chromosomal instability Cip1 CDK-interacting protein 1 CNA copy number alteration CNC Carney complex CNV copy number variation Cy3 cyanine3 Cy5 cyanine5 DNA deoxyribonucleic acid ER endoplasmic reticulum FBS fetal calf (or bovine) serum FFPE formalin-fi xed paraffi n-embedded FIOH Finnish Institute of Occupational Health FISH fl uorescence in situ hybridization GCOS GeneChip opertaing software GO Gene Ontology iGA iterative Group Analysis LCLC large cell lung cancer LCNEC large cell neuroendocrine carcinoma 9