Vaccine-elicited CD8+ T cells overcome immune suppressive environment to cure malignant mesothelioma in mice

Title Vaccine-elicited CD8+ T cells overcome immune suppressive environment to cure malignant mesothelioma in mice Author(s) Tan, Zhiwu; 譚 志 武 Citation Issued Date 2014 URL http://hdl.handle.net/10722/206430 Rights Creative Commons: Attribution 3.0 Hong Kong License

Vaccine-elicited CD8 + T cells overcome immune suppressive environment to cure malignant mesothelioma in mice by TAN Zhiwu Ph.D. THESIS The University of Hong Kong 2014

Abstract of thesis entitled Vaccine-elicited CD8 + T cells overcome immune suppressive environment to cure malignant mesothelioma in mice Submitted by TAN Zhiwu For the Degree of Doctor of Philosophy at the University of Hong Kong March 2014 Malignant mesothelioma is an aggressive cancer with increasing incidence worldwide. Exposure to asbestos is believed to be the main mechanistic basis of malignant transformation of mesothelial cells. Despite decades of efforts, treatment options for this malignancy are still limited to traditional surgery and chemotherapy, which do not provide significant survival benefits, highlighting the importance of finding novel therapeutic and preventive approaches to fight mesothelioma. For this reason, we aimed to examine the efficacy of immunotherapy strategy using DNA vaccines targeting tumor-expressing antigens. Immunotherapy targeting tumor associated self-antigen WT1 with conventional and PD1-based DNA vaccines was unable to induce tumor regression or improved survival in a quantitative mouse malignant mesothelioma model due to insufficient levels of antigen-specific immune responses being elicited. While why PD1-based DNA vaccine does not improve self-antigen WT1-specific immune responses remains to be investigated, it becomes important to define the level of vaccine-elicited immune responses for protection.

To date, the immune correlates of vaccine-elicited immunity remains poorly understood for the prevention and eradication of malignant mesothelioma. With the development of a malignant mesothelioma mouse model stably expressing HIV-1 GAG model antigen, we utilized the remarkably enhanced antigen-specific T cell responses elicited from our PD1-based HIV-1 GAG p24 vaccine to define antitumor responses. It has been demonstrated in this study that vaccineelicited host immunity not only achieved complete and long-lasting protection against murine mesothelioma cell challenges but also resulted in therapeutic eradication of pre-existing mesothelioma after four consecutive DNA vaccinations. Vaccine-elicited CD8 + T cells attributed primarily and dose-dependently to the protective efficacy in both preventive and therapeutic settings. Moreover, the consecutive vaccinations activated polyfunctional CD8 + T effector cells via T-bet and Eomes-mediated pathways, leading to the rejection of mesothelioma by releasing inflammatory IFN-γ and TNF-α in the vicinity of target cells and by triggering the TRAIL induced apoptosis. Importantly, the vaccination not only activated CD8 + T cells and maintained their effector function but also overcame immunosuppressive networks by downregulating inhibitory PD1 and Tim-3 molecule expression on CD8 + T cells and reducing suppressor cells such as myeloid-derived suppressor cells (MDSCs) and Treg, leading to the shift of tumor immune oediting from progression to elimination. Taken together, the generation of malignant mesothelioma mouse models in our study can enable targeting immunotherapy strategies to be evaluated in a quantitative way. Our data suggested that high frequency of vaccine-elicited CD8 + T cells could prevent and eradicate malignant mesothelioma. The activation of quantitatively and qualitatively enhanced CD8 + T cells caneliminate theimmune suppressive network contributing to the complete tumor rejection. (415 words)

Vaccine-elicited CD8 + T cells overcome immune suppressive environment to cure malignant mesothelioma in mice by TAN Zhiwu A thesis submitted in partial fulfillment of the requirements for the Degree of Doctor of Philosophy at The University of Hong Kong March 2014

DECLARATION I declare that this thesis represents my own work, except where due acknowledgement is made, and that it has not been previously included in a thesis, dissertation or report submitted to this University or to any other institution for a degree, diploma or other qualifications. Signed TAN Zhiwu I

Acknowledgement Over the past four years of study I have been grateful to work with such a group of people who are always enthusiastic to provide help with my experiments and discussion. Without their help, my thesis project would not have been possible. First and foremost, I would like to thank my primary supervisor, Dr. Zhiwei CHEN, for his guidance and great mentorship. The journey of a thousand miles begins with one step. His critical thinking and hard-working impress me a lot and lead me on the road to research. I would also like to thank my co-supervisor, Dr. Li LIU, for her warm assistant and suggestions in this project. And also thanks to Prof. Bojian Zheng for his support and admission of my study in the University of Hong Kong. Thanks to all involved in the AIDS Institute that helped me collecting, processing and running samples. And many thanks to Dr. Jingying ZHOU, Dr. Allen K.L CHEUNG and Dr. Faye CHEUNG for their critical discussion, heartful helping and contribution in this project. I would also like to thank Dr. Yuanxi KANG, Dr. Haibo WANG, Dr. Xiaofan LU, Dr Wenbo YU and Dr. Xian TANG for their patience teaching me various laboratory techniques. Much thanks to all the members of the core facility of the faculty of medicine in particular Dr. Andrew C.Y CHU, for giving me the opportunity to learn living imaging techniques from you. I would also like to thank Mr. Jianguo LIANG for sharing work bench and apartment with me and helping my experiments, Dr. Yanhua DU for providing convenient access of laboratory equipment and utilities. And great thanks to Mr. Boon Kiat LEE for his kind assistant in animal experiments. Special thanks are due to all the mice that participated in my research and sacrificed for it. The road to research can be full of potholes and frustration. The overwhelming feeling of failure and disappointment can really affect emotion and personality. Here, I would like to II

express my heartfelt gratitude to my fiancée, Moxi. Without your patience, love, support and buoyant personality to make me feel warm when I was in low mood and depression, the past four years might have been possible but definitely not as much fun. These streets we walked along, sunlight on the sea, soporific air of two in the afternoon, soft breath of the sea wind and the moist air in the summer, a great experience I will cherish for the rest of my life. Finally, I want to give my thanks to the members of my examining committee, Prof. Dongyan JIN, Prof. Liwei LU, Prof. Stephen K.W TSUI and Dr. Kwan MAN. It s my great honor to share my thesis work with you. This project is supported by Pneumoconiosis Compensation Fund Board (PCFB) Research Fund, Hong Kong Research Grant Council RGC762209 and RGC762712, RGC762811, and HKU-UDF/HKU-LKSFM matching fund to AIDS Institute. III

Contents Declaration... I Acknowledgement... II Table of Contents... V List of Figures... X Abbreviations... XIII IV

Table of Contents Chapter I Introduction 1.1 Introduction... 1 1.1.1 Malignant Mesothelioma... 1 1.1.2 Epidemiology of Malignant Mesothelioma... 4 1.1.3 Mouse Model of Malignant Mesothelioma... 7 1.1.4 Small Animal Imaging in malignant mesothelioma... 14 1.1.5 Malignant Mesothelioma Therapy... 18 1.1.5.1 Surgical treatment... 19 1.1.5.2 Radiotherapy and chemotherapy... 20 1.1.5.3 Gene therapy... 22 1.1.6 Immunotherapy of Malignant Mesothelioma... 25 1.1.6.1 DNA vaccines... 29 1.1.6.2 Methods of delivering DNA vaccines... 30 1.1.6.3 Existing DNA vaccines against mesothelioma... 31 1.1.6.4 Viral-vector cancer vaccines... 33 1.1.6.5 DC-targeting... 38 1.2 Relevance and focus of my project... 44 V

Chapter II Materials and Methods 2.1 Materials... 48 2.1.1 Cell culture... 48 2.1.2 Plasmids... 48 2.1.3 Mice... 49 2.1.4 Antibodies and tetramer for flow cytometry... 49 2.2 Methods... 50 2.2.1 Bioluminescence Imaging... 50 2.2.2 Design and generation of WT1 and luciferase expressing cell lines... 51 2.2.3 Construction of WT1 vaccines... 52 2.2.4 Detection of WT1 by western blotting and nested RT-PCR... 52 2.2.5 Mouse immunization... 53 2.2.6 WT1 Serum ELISA... 54 2.2.7 Evaluation of WT1-specific T cell responses... 54 2.2.8 Tumor challenge... 55 2.2.9 Generation of GAG-expressing AB1 cells... 55 2.2.10 Verification of GAG expression by western blot, flow cytometry and immunofluorescence staining... 55 2.2.11 Administration of p24 vaccines... 56 2.2.12 Accessing p24 specific immune responses by tetramer staining and serum ELISA.. 57 2.2.13 In vivo T cell depletion study... 57 2.2.14 T cell purification and adoptive transfer... 58 2.2.15 Cytotoxicity assay... 58 2.2.16 Cytokine production assay... 59 2.2.17 Cell cycle analysis... 59 2.2.18 Receptor blocking... 60 2.2.19 Separation of spleen cells and tumor cells... 60 2.2.20 Flow cytometry staining and analysis... 61 2.2.21 Statistical analysis... 62 VI

Chapter III WILMS TUMOR PROTEIN 1 (WT1) TARGETED IMMUNOTHERAPY FOR MALIGNANT MESOTHELIOMA 3.1 Introduction... 63 3.1.1 WT1 gene and protein... 63 3.1.2 WT1-targeted immunotherapy for various cancers... 67 3.2 Results... 72 3.2.1 Establishment of AB1-WT1 tumor cells... 72 3.2.2 Immunogenicity of conventional WT1 DNA vaccines in Balb/c mice... 81 3.2.3 Conventional WT1 DNA vaccines failed to protect mice from mesothelioma challenge... 85 3.2.4 Soluble PD-1 based WT1 vaccine failed to improve antigen specific immunity in Balb/c mice... 89 3.2.5 Dosage and mouse strain affected WT1 DNA vaccines immunogenicity... 95 3.3 Discussion... 101 VII

Chapter IV ASSESSING SOLUBLE PD-1 BASED p24 VACCINES IMMUNOTHERAPY IN A GAG EXPRESSION MALIGNANT MESOTHELIOMA MOUSE MODEL 4.1 Introduction... 107 4.2 Results... 109 4.2.1 Generation of HIV-1 GAG expressing AB1 malignant mesothelioma cell line... 109 4.2.2 The p24-specific immune responses protected mice from AB1-GAG challenge... 114 4.2.3 Cytotoxic ability of splenocytes from spd1-p24 fc vaccinated mice... 120 4.2.4 Anti-tumor effect was specific to GAG expressing AB1 malignant mesothelioma. 121 4.2.5 spd1-p24 fc immunization showed therapeutic effect on AB1-GAG growth in vivo...... 122 4.2.6 Increased p24 specific immune responses accompanied with AB1-GAG tumor rejection... 127 4.2.7 Dose-dependent efficacy of therapeutic vaccination... 130 4.2.8 CD8 + T cells from spd1-p24 fc vaccinated mice showed increased cytotoxicity and cytokine release in vitro... 132 4.2.9 Tumor cell cycle arrest and direct cytolysis induced by CD8 + T cells... 136 4.2.10 Functional p24-specific CD8 + T cells play a major role in tumor elimination in vivo...... 140 4.3 Discussion... 146 VIII

Chapter V VACCINE-ELICITED CD8 + T CELLS CAN OVERCOME TUMOR SUPPRESSIVE ENVIRONMENT TO ACHIEVE TUMOR CLEARANCE 5.1 Introduction... 151 5.1.1 Inhibitory signal pathways... 152 5.1.2 Inhibitory immune cells in the tumor... 155 5.2 Results... 160 5.2.1 Consecutive vaccination allowed tumor-specific CD8 + T cells to maintain effector function... 160 5.2.2 Consecutive vaccination leads to the elimination of suppressive environment... 173 5.2.3 Functional CD8 + T cells switched the tumor environment from suppression to tumor rejection... 181 5.2.4 Directing effector T cells to attack AB1 mesothelioma by genetic modification... 186 5.3 Discussion... 191 Chapter VI FINAL SUMMARY and FUTURE DIRECTIONS 6.1 Summary and future directions... 197 IX

List of Figures Figure 3.1.AB1 cell line does not have detectable AB1 protein expression. 73 Figure 3.2.Genetic representation of transfer vector. 75 Figure 3.3.Expression of constructed WT1-luc vectors. 76 Figure 3.4.WT1 expression in transduced-ab1 single clones. 78 Figure 3.5.WT1 expression pattern in AB1-WT1 cells. 79 Figure 3.6.Comparison of BLI signal intensity with cell number in vitro or tumor volume in vivo. 80 Figure 3.7.Conventional WT1 DNA vaccine constructs. 82 Figure 3.8.Immunogenicity of conventional WT1 DNA vaccines. 84 Figure 3.9.Representative bioluminescence images of mice on the indicated days after conventional WT1 DNA vaccination. 86 Figure 3.10.Conventional WT1 DNA vaccines failed to show benefits against AB-WT1 malignant mesothelioma. 87 Figure 3.11.Cytotoxic T lymphocyte (CTL) activity. 88 Figure 3.12.Counstrunction of soluble PD-1 based WT1 vaccine. 90 Figure 3.13.Analysis of WT1-specific antibody and T cell responses in vaccinated Balb/c mice. 92 Figure 3.14.Soluble PD-1 based WT1 vaccine failed to protect mice from AB1-WT1 tumor challenge. 94 Figure 3.15.20 µg plasmid DNA vaccinated Balb/c mice. 96 Figure 3.16.Incorporation of WT1 antigen did not affect soluble PD-1 antigen delivery. 98 Figure 3.17.Antibody and T cell responses of the two WT1 constructs generated in immunized C57BL/6N mouse. 100 Figure 4.1.Construction of HIV-1 GAG expressing transfer vector. 110 Figure 4.2.Verification of HIV-1 GAG protein expression in AB1-GAG cell line. 111 Figure 4.3.In vivo growth dynamics of AB1-GAG cells. 113 Figure 4.4.Vaccination of spd1-p24 fc protected Balb/c mice from AB1-GAG malignant mesothelioma challenge in a prophylactic setting. 115 X

Figure 4.5.Complete rejection of implanted tumor was observed only in spd1-p24 fc vaccinated mice. 116 Figure 4.6.Mice with tumor ablation rejected the second AB1-GAG tumor challenge. 118 Figure 4.7.Complete rejection and enhancement of survival were only observed in mice with tumor ablation. 119 Figure 4.8.Cytotoxic effect of splenocytes collected from spd1-p24 fc, p24 fc or PBS control mice. 120 Figure 4.9.Anti-tumor effect was specific to GAG-expressing AB1 cells. 121 Figure 4.10.sPD1-p24 fc vaccination rejected established AB1-GAG malignant mesothelioma. 124 Figure 4.11.Complete elimination of established AB1-GAG tumor was only observed in mice receiving spd1-p24 fc vaccination. 126 Figure 4.12.Significant higher levels of p24 specific immune responses were detected in spd1- p24 fc vaccinated mice. 129 Figure 4.13.Dose-dependent response was observed from spd1-p24 fc DNA vaccination. 131 Figure 4.14.CD8 + T cells from spd1-p24 fc vaccinated mice displayed cytotoxic effect and IFN-γ production against AB1-GAG cells. 133 Figure 4.15.CD4 + T cells from spd1-p24 fc vaccinated mice showed comparably low cytotoxicity and Th17 cytokine production. 135 Figure 4.16.Secreted cytokines from the CD8 + T cells of p24 vaccinated mice arrested AB1- GAG tumor cells. 137 Figure 4.17.Direct cytolytic effect from the cytotoxic CD8 + T cells by binding of TRAIL with its receptor on tumor cell surface. 139 Figure 4.18.Distinguishing anti-tumor effect of CD4 + and CD8 + T cells by T cells depletion study. 141 Figure 4.19.Passive transfer of anti-p24 serum revealed that serum antibodies did not contribute to the elimination of AB1-GAG tumor. 142 Figure 4.20.CD8 + T cells from spd1-p24 fc vaccinated mice showed potent anti-tumor effect in adoptive transfer. 144 Figure 4.21.CD4 + T cells from spd1-p24 fc vaccinated mice showed partial anti-tumor effect in adoptive transfer. 145 Figure 5.1.Schematic representation of treatment schedule. 162 Figure 5.2.Pictures of resected subcutaneous tumors. 163 Figure 5.3.Gating strategy for flow cytometric scatter plots. 164 XI

Figure 5.4.Population of p24 specific CD8 + T cells detected in the spleen and tumor. 165 Figure 5.5.sPD1-p24fc vaccination increased IFN-γ and TNF-α production CD8 + T cells population in both of the spleen and tumor site. 167 Figure 5.6.Significantly higher level of T-bet and Eomes expression were detected in CD8 + T cells of spd1-p24fc vaccinated mice. 168 Figure 5.7.Adoptive transfer of CD8 + T cells retarded established tumor. 170 Figure 5.8.The transferred CD8 + T cells from spd1-p24 fc vaccinated mice showed activated T- bet and Eomes production. 171 Figure 5.8.Alternation of the frequencies of IFN-γ and TNF-α production CD8 + T cell population.172 Figure 5.9.Vaccination of spd1-p24 fc maintained CD8 + T cells with lower level of inhibitory molecules expression including PD-1 and Tim-3. 174 Figure 5.10.PD-L2 expression on the whole cells from tumor was largely reduced in spd1-p24 fc vaccinated mice. 175 Figure 5.11.MDSCs and Treg population were reduced after repeated vaccination of spd1-p24 fc in both of the spleen and tumor. 177 Figure 5.12.Adoptive transfer of CD8 + T cells to tumor bearing SCID mice. 179 Figure 5.13.Fraction of MDSCs was depressed in SCIDmice of both of the spleen and tumor. 180 Figure 5.14.Correlations between frequency of antigen specific CD8 + T cells and tumor mass182 Figure 5.15.Correlations between the ratio of T effector cells /suppressor cells with tumor volume in Balb/c mouse model. 184 Figure 5.16.Correlations between the ratio of T effector cells /suppressor cells with tumor volume in SCID mouse model. 185 Figure 5.17.Efficacy of in vitro infection in wild-type AB1 cells. 187 Figure 5.18.MVTT-gag infection led to expression of target antigen, HIV-GAG, in the wild-type AB1 cells and also resulted in cell death. 188 Figure 5.19.In vivo infection of AB1 solid tumor with MVTT-gag. 190 Figure 5.20.Significant correlations of the frequencies of Tim3 + CD8 + T cells and Th1 cytokines production were observed in the tumor infiltrating CD8 + T cells from SCID mouse model.195 XII

Abbreviations ADCC antibody-dependent cell cytoxicity ADM antigen delivery method APC antigen presenting cell BLI bioluminescence imaging CAR chimeric T cell antigen receptor CCL2 chemokine C-C motif ligand 2 CMV strong human cytomegalovirus CT computed tomographic CTL cytotoxicity T lymphocyte CTLA-4 cytotoxic T lymphocyte associated antigen 4 Cy cyclophosphamide DC dendritic cell dsdna double-stranded DNA EF1α human elongation factor-1 alpha EGFR epidermal growth factor receptor EMA mucin-1 Eomes eomesodermin EPP extrapleural pneumonectomy EP eletroporation FDG fluoro-2-deoxy-d-glucose FLT fluoro-3 -doxythymidine FoxP3 forkhead transcription factor 3 HIV-1 human immunodeficiency virus type 1 HLA human leukocyte antigen HRP horseradish peroxidase HSV-TK herpes simplex virus thymidine kinase IDO indoleamine-2, 3-dioxygenase IFN interferon IL interleukin IMRT intensity-modulated radiotherapy i.p. intraperitoneally KLH keyhole limpet hemocyanin KTS lysine-threonine-serine LAG-3 lymphocyte activation gene 3 mab monoclonal antibody MDSC myeloid derived suppressive cell MHC major histocompatibility complex MLV murine leukemia virus MOI multiplicity of infection MRI magnetic resonance imaging MSLN mesothelin MVTT modified vaccinia Tiantan strain OVA ovalbumin XIII

P/D pleurectomy/decortication PBMC peripheral blood mononuclear cell PD-1 programmed cell death protein 1 PET positron-emission tomography PEI polyethylenimine PFA paraformaldehyde qrt-pcr quantitative reverse transcriptase-polymerase chain reaction ROI region of interest s.c. subcutaneously SCID severe combined immunodeficiency SPECT single photon emission computed tomography STAT3 signal transducer and activator of transcription 3 TAA tumor associated antigen TADC tumor associated DC TAM tumor associated macrophage TAN tumor associated neutrophil TCR T cell receptor TIL tumor infiltrating lymphocytes Tim-3 immune regulator T cell immunoglobulin mucin 3 TLR Toll-like receptors TMB tetramethylbenzidine TNF tumor necrosis factor TNM tumor-node-metastasis tpa tissue plasminogen activator Treg regulatory T cell TSA tumor specific antigen WT1 wilms tumor protein 1 VEGF vascular endothelial factor XIV

Chapter I Introduction

Chapter I 1.1 Introduction 1.1.1 Malignant Mesothelioma Malignant mesothelioma is an aggressive tumor that originates from the serosal surfaces of the pleura and the peritoneum, the tunica vaginalis, or the pericardium (Robinson and Lake 2005; Hansen and European Society for Medical Oncology. 2008). Before the 1950s, malignant mesothelioma was quite rare. However, since 1970s the annual incidence of this deadly tumor has been on the rise worldwide (Peto, Decarli et al. 1999; Le Stang, Belot et al. 2010; Linton, Kao et al. 2012). Long-term inhalational exposure to asbestos is considered to be the predominant cause of malignant mesothelioma (Wagner 1979; Mcdonald 2010). Epidemiology studies suggested that approximately 80% of mesothelioma cases are associated with asbestos exposure and up to sixty years of latent period has made the incidence rate peak in the coming decades in 1970s to 1990s (Chang, Leung et al. 2006). It is estimated that approximately 3300 cases of malignant mesothelioma occurred in the United States every year (Teta, Mink et al. 2008). Although the incidence of malignant mesothelioma has been declining since 2000 in the United States due to the control of asbestos exposure (Price 1997), other countries or regions of the world will definitely experience the incidence peaking in the near future, including Hong Kong (Pisani, Colby et al. 1988; Antman 1993; Hodgson, McElvenny et al. 2005; Chang, Leung et al. 2006; Nishikawa, Takahashi et al. 2008; Tse, Yu et al. 2010). The consumption level of asbestos in Hong Kong reached its highest point in the early 1960s (Lam, Kung et al. 1983). As a result of the consumption pattern, it is predicted that the number of cases will peak in 2014 locally (Tse, Yu et al. 2010). 1

Chapter I Asbestos is a group of hydrated magnesium-silicate fibrous minerals. It is intensively used in construction work because this material is heat/fire resistant and chemical degradation. There are two types of asbestos, serpentine or amphibole, which are found in brake pads, shipbuilding, cement, ceiling and pool titles. Although it remains to be debated whether the serpentine fibers can cause mesothelioma (Browne 2001), amphibole fibers are obviously more carcinogenic, especially the two types called blue and brown asbestos being the most harmful. Once inhaled, the asbestos fibers stick to the cells of lung surface, cause repeated scratching and local inflammation. Phagocytosis of these fibers by mesothelial cells triggers an oncogenic cascade of activation of oncogenes such as c-fos and c-jun, of which the expression product is the binding factor of epidermal growth factor receptors (EGFRs) (Goodglick, Vaslet et al. 1997; Heintz, Janssen-Heininger et al. 2010). These factors eventually lead to the onset of mesothelioma. With eighty percent of male patients suffering from malignant mesothelioma, this lethal disease seems to affect men more often than women (Goldberg, Imbernon et al. 2006). The strong male dominance of this type of tumor can be explained by the role that men mainly play in asbestos-related work (Bourgouin, Blais et al. 2012; La Vecchia and Boffetta 2012). The commonly known cancer syndrome, such as weight loss, fever, night sweats and cachexia, is not presented on mesothelioma patients at diagnosis but can develop in the advanced stage of malignant mesothelioma (West and Lee 2006). Therefore, it is suggested to physicians to investigate the possibility of malignant mesothelioma when unexplained pleural effusion and chest wall pain are observed in patients to reduce the time loss for treatment to occur. However, mesothelioma usually develops extensively when patients seek medical care because of the inherent characteristics of the tumor that it occurs covertly inside the body cavities. In recent 2

Chapter I years the development of immunohistochemistry has improved the accuracy and speed of diagnosis of malignant mesothelioma. This process is now an essential part in the clinical diagnosis (Husain, Colby et al. 2009). A panel of positive protein markers, such as EMA (mucin- 1), calretinin, wilms tumor protein 1 (WT1) and mesothelin (MSLN), are employed together with panel of negative markers such as MOC-31, B72.3 and Ber-EP4 (which are typically positive in pulmonary adenocarcinoma) to characterize and distinguish malignant mesothelioma from adenocarcinoma (Marchevsky and Wick 2007; Galateausalle, Le Stang et al. 2008). Currently no therapeutic cure is available for malignant mesothelioma, unless the tumor is detected at extremely early stage and surgical treatment is performed to remove the tumor completely (Delourme, Dhalluin et al. 2013; Haas and Sterman 2013). Given the hidden characteristic, this aggressive tumor has normally advanced into its later stages when it can be diagnosed for clinical treatment. Although the combined implication of treatment chemotherapy and radiotherapy with surgery prolongs the lifetime of patients with malignant mesothelioma, the medium survival is still expected to be quite short, with less than 12 months from diagnosis (Hansen and European Society for Medical Oncology. 2008). In order to predict prognosis and guide the choice of therapy, malignant mesothelioma is graded based on the degree of its differentiation and aggressiveness. The tumor-node-metastasis (TNM) staging system is widely used and adopted by both of the International Union Against Cancer and the International Mesothelioma Interest Group, which involves the evaluation of three main factors: tumor size, affection to the lymph node and degree of metastasis(rusch 1996). 3

Chapter I Under the TNM staging system, malignant mesothelioma is graded into four stages, stage I is the stage of localized tumor and stage IV the most advanced stage with intensive and distant tumor spreading which cannot be removed by surgery. In stage II surgical resection may be still impactful although tumor has evaded the lung. A much broader spreading of the tumor is visible in a single region including the chest wall, esophagus or lymph nodes, ruling out the surgery as an effective treatment method. Conventional chest radiography is useful in gathering information for the stage of mesothelioma progression. Computed tomographic (CT), magnetic resonance imaging (MRI), positron-emission tomography (PET) and ultrasound with thoracoscopy are commonly used in the assessment, although it varies among different geographical centers. 1.1.2 Epidemiology of Malignant Mesothelioma Since the first report published in 1960 by J.C. Wagner et al. (Wagner 1979), the convincing evidence of a link between asbestos exposure and pleural mesothelioma in North Western Cape Province of South Africa was described, other correlation studies of these two variables were conducted in various countries (Tossavainen 2004; Bianchi and Bianchi 2007). When the two curves of asbestos consumption and mesothelioma incidence are put together, a double-curve pattern can be always observed, that is, a direct relationship between mesothelioma incidence and the pattern of asbestos consumption. Nowadays it is commonly accepted that asbestos is the most important risk factor associated with malignant mesothelioma. Globally various countries are classified into three groups based on the level of mesothelioma incidence rates (Bianchi and Bianchi 2007). The highest incidence rates are 4

Chapter I occurring in Australia, Belgium, and Great Britain, with approximately 30 cases per million people annually. Australia experienced the most dramatic rise in incidence rate in recent years, because of its long history of asbestos mining, as early as 1939 in the town of Wittenoom, and intensive asbestos use in construction of approximately 70,000 tons in the peak year of 1975 (Leigh, Davidson et al. 2002). Despite rising concerns for asbestos-related diseases, asbestos mining continued until 1983, however, the use of asbestos as building product was finally banned in 1989. It is estimated that the incidence of mesothelioma in Australia will peak somewhere between 2014 and 2021 (Leigh, Corvalan et al. 1991). In Great Britain, the highest mortality rates are observed among workers involved in shipbuilding activities with the use of asbestos in the shipyard and the major role of the shipbuilding industry historically, especially around the time of World War II (McElvenny, Darnton et al. 2005). Although very few data of the incidence rate are available in Belgium, it has been estimated that a crude incidence rate of 29 cases per million is corresponding to its annual number of deaths from mesothelioma (Bianchi, Ramani et al. 2002). The second group of countries with comparable lower incidence rates includes a large portion of Europe (France, Germany, Italy, Scandinavian countries), and New Zealand (Bianchi and Bianchi 2007). 11 to 20 cases of crude incidence rates are reported annually among these countries. The third group of countries and regions has annual crude incidence rates below 11 cases per million. This group includes different countries in Europe, Asia, America, South America, and some parts of Africa where the incidence is accessible such as Morocco and Tunisia (Bianchi and Bianchi 2007). Hong Kong is not an area known for asbestos production and mining. However, asbestos has been extensively used since 1950s in construction industries and shipyard, especially during its booming economic development and explosive population growth. In response to the asbestos consumption, the Pneumoconiosis and 5

Chapter I Mesothelioma Board, which is the major source of local data on mesothelioma, confirmed only 22 cases of mesothelioma from 1988 to 2002, which is a rare incidence of only 0.3 cases per million (Chang, Leung et al. 2006). However, to parallel the peak of asbestos consumption in Hong Kong in early 1960s, given an average latency period of 40 years in Hong Kong, it is predicted that the number of cases will peak around 2014 (Tse, Yu et al. 2010). At that time, approximately 15 cases of mesothelioma will be diagnosed each year, giving rise to a predicted annual incidence rate of 2 cases per million. The category of high risk of asbestos exposure and mesothelioma incidence includes three principle cohorts. First, mesothelioma was initially unveiled and diagnosed in people who were directly exposed in asbestos-heavy environment, mainly asbestos mining and processing. Second, many people are exposed later in their occupations that involve the industrial manufacture and commercial use of asbestos products. Besides these two main sources of asbestos exposure, the third group of people can still be affected by their family members who have occupational exposure or by occasional environmental exposure that they may not notice in the myriad of situations (Robinson and Lake 2005). It was also found that family members could be exposed to clothing or equipment contaminated with asbestos brought home from the other member that works in the above two industries. However, epidemiology studies of malignant mesothelioma suggested that not all the people exposed to asbestos developed mesothelioma in their later life; moreover, non-exposed individuals can still develop mesothelioma. Thus, other factors other than asbestos may play a 6