LOCAL IMMUNOPOTENTIATION OF THE BLADDER IN RESPONSE TO BCG IMMUNOTHERAPY by Dr Kesavan Esuvaranathan MBBS (Singapore), FRCSEd, FRCSG, FAMS(Urol) A thesis submitted for the degree of Doctor of Medicine National University of Singapore 2003 Acknowledgements I am deeply grateful to my wonderful wife, Roshni for her unfailing support and encouragement in all my activities. I thank my parents, Mr. & Mrs. P.E. Nathan, my father-in-law, Dr V.K. Pillay and my children, Arshvin, Nisha and Sharmera for providing substance to my life. I am grateful to my sister, Vijaya, for her help in the UK. I am also grateful to my dear friend and colleague, Dr Shabbir Moochhala, for his encouragement and advice. I thank Dr Ratha Mahendran, Professor Keith James, Dr Revathi Kamaraj, my graduate students and my fellow doctors and scientists for making work as enjoyable as play. I thank our patients with bladder cancer for inspiring this research and making it worthwhile. I am grateful to God for the opportunity of this experience and its fulfillment. I thank the National Medical Research Council, Faculty of Medicine Cancer Research Fund, Cancer Research Campaign U.K., the European Community and the Singapore Cancer Society for funding this research. i Table of Contents Acknowledgement i Table of Contents ii List of Publications v List of Figures viii List of Tables xii List of Abbreviations xiv Summary xvi Chapter 1. Introduction 1.1 Epidemiology of bladder cancer 1.2 Etiology of bladder cancer 1.3 Genetic basis of bladder cancer 1.4 Clinical issues 1.4.1 Histology 1.4.2 Morphology 1.4.3 Grade 1.4.4 Stage 1.4.5 Low risk and high risk tumours 1.5 Clinical management 1.5.1 Presenting symptoms 1.5.2 Investigation 1.6 Primary treatment and follow-up 1.6.1 Transurethral resection 1.6.2 Follow-up 1.7 Intravesical chemotherapy 1.8 Bacillus Calmette-Guérin immunotherapy 1.8.1 History of BCG as an antineoplastic agent 1.8.2 BCG and bladder cancer 1.8.3 Efficacy of BCG Immunotherapy 1.8.3.1 BCG Strains 1.8.3.2 Efficacy in clinical trials 1.8.3.3 Toxicity of BCG Immunotherapy 1.8.4 Mechanism of action of BCG 1.8.4.1 Immune surveillance 1.8.4.2 Tumour cell antigens 1.8.4.3 Tumour cell detection and killing 1.8.4.4 Cytokines 1.8.4.5 Cell adhesion molecules 1.9 Current problems in BCG immunotherapy for superficial bladder cancer 12 12 12 13 14 15 16 16 16 17 17 17 18 19 19 21 21 21 22 24 26 26 27 27 29 33 34 ii 1.9.1 Schedule 1.9.2 Dose 1.9.2.1 BCG dose reduction – when more isn’t better! 1.10 BCG and Interferon alpha 1.11 Aims of this study 35 36 36 37 38 Chapter 2. Methods 39 Part I. Section A. Development of a Protocol for the Assay of Cytokines in the Urine of BCG-treated Patients 40 Section B. Study of Changes in Urinary Cytokines and soluble Intercellular Adhesion Molecule-1 in Patients with Carcinoma-in-situ after bacillus Calmette-Guérin immunotherapy and their Functions 43 Part II. A multicentre, double blind randomized clinical trial of intravesical bacillus Calmette-Guerin and interferon alpha-2b in the treatment of superficial bladder cancer 55 Chapter 3. Results 56 Part I. Section A. Development of a protocol for the assay of cytokines in the urine of BCG-treated patients 57 Section B. (I) Changes in Urinary Cytokines and soluble Intercellular Adhesion Molecule-1 in Patients with Carcinoma-in-situ after bacillus Calmette-Guérin immunotherapy 65 Section B. (II) Interleukin-6 Studies 68 Section B. (III) ICAM-1 Studies 82 Part II. A multicentre, double blind randomized clinical trial of intravesical bacillus Calmette-Guerin and IFNα -2b in the treatment of superficial bladder cancer 93 Section A. A comparison of urinary cytokines in low and standard dose BCG therapy and low dose BCG with IFNα in a “6+3” schedule for bladder cancer 93 Section B. Toxicity Analysis Serious adverse events BCG cystitis Previous exposure to tuberculosis or BCG vaccination Comparison of toxicities between the treatment arms Local side effects vs. systemic side effects Fever 100 100 101 101 101 103 104 iii Dysuria Frequency Burning sensation Hematuria Malaise Urgency Nocturia Incontinence Lassitude Work and other activities 105 107 109 111 114 117 120 123 123 126 Section C. Efficacy Analysis 1. Recurrence 2. Progression 3. Maintenance therapy 4. Mortality 5. Extravesical recurrence 129 129 133 133 134 134 Chapter 4. Discussion 135 Part I. Section A. Development of a protocol for the assay of cytokines in the urine of BCG-treated patients 136 Section B. (I) Changes in Urinary Cytokines and soluble Intercellular Adhesion Molecule-1 in Patients with Carcinoma-in-situ after BCG immunotherapy 141 Section B. (II) Interleukin-6 Studies 146 Section B. (III) ICAM-1 Studies 150 Part II. A multicentre, double blind randomized clinical trial of intravesical BCG and IFNα-2b in the treatment of superficial bladder cancer 153 Section A. A comparison of urinary cytokines in low and standard dose BCG therapy and low dose BCG with IFNα in a “6+3” schedule for bladder cancer 153 Section B. Toxicity Analysis 155 Section C. Efficacy Analysis 156 Conclusion 157 Future Studies 159 References 160 iv Publications arising from this thesis 1. Young Surgeon's Award, 27th Annual Combined Surgical Meeting, Academy of Medicine, Singapore 1993. Refereed Journals 1. Esuvaranathan K, Jackson AM, Alexandroff AB, James K. The expression of ICAM-1 and ICAM-2 in bladder cells. J Urol 1993; 149:271A. 2. Jackson AM, Alexandroff AB, Gribben SC, Esuvaranathan K, James K. Expression and shedding of ICAM-1 in bladder cancer and its immunotherapy. Int J Cancer 1993; 55: 9215. 3. Jackson AM, Alexandroff AB, McIntyre M, , James K, Esuvaranathan K, Chisholm GD. Induction of ICAM-1 expression on bladder tumours by BCG immunotherapy. J Clin Path 1994; 47:309-312. 4. Jackson AM, Alexandroff AB, Lappin MB, Esuvaranathan K, James K, Chisholm GD. Control of LFA-1 dependent cellular conjugation by divalent cations. Immunology 1994; 81:120-3. 5. Alexandroff AB, Jackson AM, Esuvaranathan K, Prescott S, James K. Autocrine regulation of ICAM-1 expression on bladder cancer cell lines: evidence for the role of IL1. Immunol Letters 1994; 40:117-124. 6. Jackson AM, Alexandroff AB, Kelly RW, Skibinska A, Esuvaranathan K, Prescott S, Chisholm GD, James K. Changes in urinary cytokines and soluble intercellular adhesion molecule-1 (ICAM-1) in bladder cancer patients after bacillus Calmette-Guérin (BCG) immunotherapy. Clin Experimental Immunol 1995; 99(3):369-375. 7. Esuvaranathan K, Alexandroff AB, McIntyre M, Jackson AM, Prescott S, Chisholm GD, James K. Interleukin-6 production by bladder cancers is upregulated by BCG immunotherapy. J Urol 1995; 154:572. 8. Esuvaranathan K, Moochhala S, Png D, Kour NW, Cheng C, Tung KH. Diurnal urinary IL-10 and IL-6 production in patients with urinary tract infections, bladder cancers and during BCG immunotherapy. J Urol 1995; 53: 486A. 9. Esuvaranathan K, Tey HB, Mohan RS, , Tung KH, Tan PK, Moochhala SM. Maintenance BCG induces a secondary immune response. J Urol 1996; 155(4):A1417. 10. C Lawrencia, RS Mohan, R Mahendran, K Esuvaranathan et al. Comparison of the Immune Response during Induction and Booster BCG Immunotherapy for Bladder cancer. Br J Urol 1997; 80 (Supple 2): 46:177A 11. Zhang Y, Khoo HE & Esuvaranathan K. Effects of Bacillus Calmette-Guérin and Interferon-2b on Cytokine Production in Human Bladder Cancer Cell Lines. J Urol March 1999; 161(3):977-83 12. Gan YH, Zhang Y & Esuvaranathan K. Antitumour immunity of Bacillus Calmette-Guérin and interferon alpha in murine bladder cancer. Eur J Cancer. 1999; 35(7):1123-9. 13. Esuvaranathan K, Lawrencia C, Kamaraj R, Mahendran R. Both standard and low dose BCG elicit similar urinary cytokine production in patients with bladder cancer. Br J Urol 2000; supple 3:252 14. Esuvaranathan K, Lawrencia C, Moochhala S, Mahendran R. Urinary cytokines in BCG immunotherapy. (Submitted) 15. Esuvaranathan K, Kamaraj R, Mohan RS, Cheng C, Chia SJ, Ng FC, Mahendran R & Bladder Cancer Workgroup. Report of a multicentre, double blind randomized clinical trial of intravesical interferon alpha-2b and bacillus Calmette-Guérin in the treatment of superficial bladder cancer. (In preparation) v Conference papers 1. Esuvaranathan K. Local immunological markers in intravesical BCG therapy for bladder cancer. In Proceedings of 27th Annual Combined Surgical Meeting, 5-7 November 1993, Academy of Medicine, Singapore 1993. Young Surgeon’s Award 2. Esuvaranathan K. Interleukin-6 production by bladder tumours is upregulated by BCG immunotherapy. Societe Internationale d’Urologie 23rd World Congress, 18-22 September 1994, Sydney, Australia. 3. Esuvaranathan K, S Moochhala, D Png, NW Kour, CWS Cheng, Tung KH. Diurnal Urinary Interleukin-10 and Interleukin-6 Production in Patients with Urinary Tract Infections, Bladder Cancers and during BCG Immunotherapy. 90th Annual Meeting of the American Urological Association, Las Vegas, USA, April 1995. 4. Esuvaranathan K. Interleukin-6 production by bladder tumours is upregulated by BCG immunotherapy. In University Surgeons of Southeast Asia – 1st Scientific Congress, 1-3 August 1994, National University Hospital, Singapore, p.65. National University of Singapore, Singapore. Best paper prize 5. Esuvaranathan K, Tung KH, Png D, Kour NW, Cheng WS, Moochhala SM. IL-10: The probable braking mechanism in the Th1-mediated response to intravesical BCG. In 28th Annual Combined Surgical Meeting, 18-20 November 1994, pp.43. Singapore: Academy of Medicine, 1994. 6. Esuvaranathan K, Moochhala S, Png D, Kour NW, Cheng C, Tung KH. Diurnal urinary IL10 and IL-6 production in patients with urinary tract infections, bladder cancers and during BCG immunotherapy. At 90th Annual Meeting, American Urological Association, April 1995, San Francisco, USA. 7. Esuvaranathan K, Tey HB, RS Mohan, Tung KH, S Moochhala. Urinary IL-10 secretion influences the response of patients with bladder cancers to intravesical BCG immunotherapy. Proceedings 12th Asia Pacific Cancer Conference, October 1995, Singapore. 8. Esuvaranathan K. Mode of action of intravesical BCG therapy. Presented at Focus on Prostate and Bladder Cancer, Singapore Urological Association, October 1995, Singapore. (Invited Speaker) 9. Esuvaranathan K. Optimization of BCG therapy for superficial bladder cancer. 5th Congress of the Malaysian Urological Association, November 1995, Awana, Malaysia. (Invited Speaker) 10. Esuvaranathan K. Maintenance intravesical BCG therapy for bladder cancer induces a secondary immune response. 5th Congress of the Malaysian Urological Association, November 1995, Awana, Malaysia. (Invited Speaker) 11. Esuvaranathan K, RS Mohan, Tey HB, Tung KH, PK Tan, S Moochhala. Maintenance BCG induces a secondary immune response. 91st Annual Meeting of the American Urological Association, May 1996, Orlando, USA. 12. Esuvaranathan K, Tey HB, RS Mohan, S Moochhala. IL-10 and IL-6 exert differential growth effects on human bladder cancer. 91st Annual Meeting of the American Urological Association, May 1996, Orlando, USA. 13. Esuvaranathan K. The remarkable success of BCG immunotherapy for superficial bladder cancer. University Surgeons of Asia – 2nd Scientific Congress, 10-12 August 1996, National University of Singapore, Singapore. 14. Esuvaranathan K. BCG Immunotherapy – Lessons for gene therapy. 2nd Nordic Conference on BCG Immunotherapy, Sep 1996, Oslo, Norway. (Invited Speaker) 15. Esuvaranathan K. Ying-yang states in Bladder Cancer & BCG immunotherapy. 30th Annual Combined Surgical Meeting, 30 October – November 1996, Academy of Medicine Singapore, 1996, Singapore. (Invited speaker) 16. Esuvaranathan K. Bladder cancer: Prospects for immunotherapy. 30th Annual Combined Surgical Meeting, 30 October – November 1996, Academy of Medicine Singapore, 1996, Singapore. vi 17. C Lawrencia, RS Mohan, R Mahendran, K Esuvaranathan et al. Comparison of the Immune Response during Induction and Booster BCG Immunotherapy for Bladder cancer. SIU - World Congress, 1997, Montreal, Canada. 18. Lawrencia, C, Mohan RS, Zhang Y & Esuvaranathan K. Responders to BCG immunotherapy display a strong Th1-like immune response to maintenance immunotherapy. In University Surgeons of Asia – 3rd Scientific Congress, 5-7 August 1998 Singapore, p. 20. 19. Mohan RS, Lawrencia C, Begum S, Kamaraj R, Chia SJ, Ng FC, Tay KP, Cheng C & Esuvaranathan K. Interim analysis of a multicentre, double-blind randomized clinical trial of intravesical bacillus Calmette-Guérin and interferon alpha-2b in the treatment of superficial bladder cancer. In Proceedings of the 4th Asian Congress of Urology, 17-20 September 1998, Singapore, p.241. 20. Esuvaranathan K. Novel therapies for superficial bladder cancer. Uro-oncology Workshop, 4th Asian Congress of Urology, 17-20 September 1998, Singapore. (Invited Speaker) 21. Esuvaranathan K. Approaches for gene therapy in bladder cancer. International Symposium on Human Genetics and Gene Therapy, 5-6 February 1999, Singapore. (Invited speaker) 22. Esuvaranathan K. Update on BCG Immunotherapy. Winter Meeting, Finnish Urological Association, 10-11 March 1999, Rovaniemi, Finland. (Invited Speaker) 23. Esuvaranathan K. Immunotherapy for bladder cancer. Malaysian Urological Association Annual Meeting, December 1999, Penang, Malaysia. (SUA Lecturer) 24. Esuvaranathan K. 12th Annual Scientific Meeting, Singapore, Jan 2000. From bench to bedside – A priori. (Invited Speaker) 25. Esuvaranathan K. Combined use of BCG and interferon alpha for high risk superficial bladder cancer. Presented at National BCG & Interferon Bladder Cancer Trial Investigators Meeting at the AUA Annual Meeting, May 2000, Hyatt Regency Hotel, Atlanta, USA. 26. Esuvaranathan K. Interim analysis of a multicentre, double-blind randomized clinical trial of intravesical bacillus Calmette-Guérin and interferon alpha-2b in the treatment of superficial bladder cancer. 95th Annual Meeting of the American Urological Association, May 2000, Atlanta, USA. 27. Esuvaranathan K, Cheng WS, Chia JS, Chin CM, Mohan RS, Revathi P. Report of a double-blind, randomized, controlled trial of the combined use of bacillus CalmetteGuérin and interferon alpha for superficial bladder cancer. The 5th Asian Congress on Urology, 27-30 August 2000, Beijing, China. 28. Esuvaranathan K. Superficial bladder cancer. The 5th Asian Congress on Urology, 27-30 August 2000, Beijing, China. 29. Esuvaranathan K. An update on the management of bladder cancer. NHG Annual Scientific Congress 2003, 4-5 October 2003, Raffles City Convention Centre, Singapore. (Invited Speaker) 30. Esuvaranathan K, Cheng WS, Chia SJ, Ng FC, R Mahendran. Combined use of Interferon alpha and BCG for superficial bladder cancer. 2nd Asia-Pacific Uro-oncology Meeting, 911 December 2003, Hong Kong. (Invited Speaker) 31. Esuvaranathan K. Principles in Bladder cancer – Time for a Change? 2nd Asia-Pacific Urooncology Meeting, 9-11 December 2003, Hong Kong. (Invited Speaker) vii LIST OF FIGURES Fig. 1. Age-standardized and age-specific incidences of bladder cancer in Singapore Fig. 2. Hypothetical model of carcinogen activation and detoxification Fig. 3(A). Schematic representation of cell cycle phases Fig. 3(B). Schematic representation of changes in cyclins and cdks through the cell cycle 10 Fig. 4. Pathways of development and progression in bladder cancer 10 Fig. 5. Stability of IL-2 in urine from BCG-treated Patients 59 Fig. 6. Measurement of Urinary IL-2 & IL-2R 60 Fig. 7(A). Stability of IFNγ in urine of pH 7.2 before dialysis 62 Fig. 7(B). Stability of IFNγ in acidic urine before dialysis 63 Fig. 8. Constitutive and IFNγ induced IL-6 in vitro 69 Fig. 9. Constitutive and BCG induced IL-6 in vitro 70 Fig. 10(A). IL-6 production in multicellular spheroids 70 Fig. 10(B). An SD human bladder cancer MCS shows increased IL-6 positivity after exposure to IFNγ 71 Fig. 10(C). SD MCS showing increased IL-6 positivity after exposure to BCG 71 Fig. 11. Urinary IL-6 in 13 patients with CIS bladder treated with BCG 72 Fig. 12. Cytospin preparation from urine of patient after BCG therapy 73 Fig. 13. Positive IL-6 staining in bladder tumour before BCG therapy 74 Fig. 14. IL-6 positive staining in a post-BCG persistent bladder tumour 75 viii Fig. 15. Diurnal pattern of urinary IL-6 secretion in normal subjects 76 Fig. 16. Diurnal pattern of urinary IL-6 secretion in normal subjects 76 Fig. 17. Diurnal pattern of urinary IL-10 secretion in patients with TCC bladder 77 Fig. 18. Diurnal pattern of urinary IL-10 secretion in patients with TCC bladder 77 Fig. 19. Diurnal pattern of urinary IL-6 secretion in patients with UTI 78 Fig. 20. Diurnal pattern of urinary IL-10 secretion in patients with UTI 78 Fig. 21. Ratio of urinary IL-10 to IL-6 in bladder cancer, UTI and normal subjects 79 Fig. 22. IL-6 positive bladder tumour biopsy 80 Fig. 23. IL-10 positive bladder tumour biopsy 80 Fig. 24. IL-10 positivity in a biopsy from pre-BCG treated TCC bladder 81 Fig. 25. High power magnification of an IL-10 positive bladder tumour biopsy 81 Fig. 26. Urinary cytospins after BCG Immunotherapy 83 Fig. 27. Post-BCG therapy urinary cytospin showing LFA-1 positive leucocytes 84 Fig. 28. Post-BCG urinary cytospin of HLA class II expressing urothelial cells 84 Fig. 29. A series of urinary cytospins from post-BCG therapy patients showing ICAM-1 positive urothelial cells in apposition to polymorphonuclear cells 85 Fig. 30. HLA-DR expression in RT4 (1), RT112 (2) and SD (3) spheroids 86 Fig. 31. ICAM-1 expression in RT4 (1), RT112 (2) and SD (3) spheroids 86 Fig. 32. ICAM-1 expression in the RT4 multicellular spheroids 87 Fig. 33(A). RT112 MCS, control (magnification 300X) 88 ix References Alexandroff, A. B., Jackson, A. M., Chisholm, G. 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Fig 45 The secondary immune response 154 Fig 46 Model of Th1-Th2 cytokine interactions 155 Fig 47 Schema of how IFNα may modulate the effects of BCG 157 x Fig 48 Schema of mechanism of action of BCG immunotherapy 158 xi LIST OF TABLES Table 1 1997 AJCC/TNM Staging of Bladder Cancer 15 Table 2 Comparison of BCG strains in the treatment of CIS of the bladder 23 Table 3 Effect of intravesical BCG on recurrence... clinical trial using a novel combination of interferon alpha and BCG, to explore the mechanisms of action of BCG immunotherapy We have developed a reliable protocol for measuring cytokines in the urine after intravesical BCG instillation because cytokines are very unstable in this environment We discovered that in addition to immune cells, bladder cancer cells and normal urothelium also produce cytokines... the toxicity of BCG immunotherapy IFNα in combination with low dose BCG has superior efficacy to standard dose BCG immunotherapy The combination appears to have amongst the best results reported to date for BCG immunotherapy, even when compared with more intensive schedules of BCG alone, but these results need to be confirmed in a larger trial It is possible that the combination of IFNα and low dose BCG. .. markers to distinguish between these two groups We found that BCG induces a mixed Th1 and Th2 cytokine response, with a predomination of TH1 cytokines The addition of interferon alpha to BCG induces xvi more urinary interferon gamma production, a cytokine known to increase the TH1 cellular immune response and possibly contribute to better anti-tumour efficacy As BCG invokes an immunological response, dose... will not be discussed further in this thesis Urothelial carcinomas may arise anywhere along the collecting system of the genitourinary tract with more than 90% occurring in the bladder, 8% in the renal pelvis and 2% in the ureter or rarely, the proximal two-thirds of the urethra (National Board of Health and Welfare: Cancer Incidence in Sweden in 1985 Stockholm: National Board of Health and Welfare,... flexible cystoscopy (bladder and urethra) In my own experience, neither contrast-enhanced computer tomography nor ultrasonography has sufficient definition to exclude small lesions in the renal pelvis or urinary bladder conclusively Urine cytology may be useful in the specific settings of the presence of abnormal bladder mucosa in the absence of gross tumour or a normal bladder in the presence of persistent... carcinomas, similar to findings in the West Fig 1 Age–standardized and age–specific incidences of bladder cancer in Singapore Age and incidence Bladder cancer is uncommon in men aged less than 30 years and women aged less than 40 The age–specific incidence increases from the age of 50 in Singapore, similar to elsewhere (Fig 1) (Chia et al 1996) Gender differences The 2-4 times lower incidence of bladder. .. inhalation, age of initiating smoking and type of tobacco have all been shown to influence the risk of developing bladder cancer (Steineck et al 2001) Several studies have shown an increased incidence of p17 and p53 genetic mutations in bladder cancers of smokers (Zhang et al 1994; Uchida et al 1995) Compounds associated with an increased risk of bladder cancer include aromatic amines (arylamines) and polycyclic... populations in the US, the Basque region in Spain and in Denmark The high incidence of bladder cancer seen in the Middle East and Africa are associated with Schistosoma hematobium infections and are usually squamous carcinomas rather than the transitional cell carcinomas seen elsewhere (Bedwani et al 1998) 2 In Singapore, bladder cancer is the 7th most common cancer in males with an agestandardized incidence... mechanisms of action – the clinical regimens used are empirical and frequently cause morbidity This work investigated the local immunopotentiation in the bladder to intravesical BCG, using a variety of immunological techniques, including urinary cytokine assays, immunohistochemistry, in vitro bladder cancer cell line monolayer and multicellular microspheroid culture and LAK cell experiments, scanning electron . BCG, to explore the mechanisms of action of BCG immunotherapy. We have developed a reliable protocol for measuring cytokines in the urine after intravesical BCG instillation because cytokines. work investigated the local immunopotentiation in the bladder to intravesical BCG, using a variety of immunological techniques, including urinary cytokine assays, immunohistochemistry, in vitro. Urinary cytospins after BCG Immunotherapy 83 Fig. 27. Post -BCG therapy urinary cytospin showing LFA-1 positive leucocytes 84 Fig. 28. Post -BCG urinary cytospin of HLA class II expressing urothelial