Local immunopotentiation of the bladder in response to BCG immunotherapy

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


i
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.

ii
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

1.1 Epidemiology of bladder cancer 2
1.2 Etiology of bladder cancer 4
1.3 Genetic basis of bladder cancer 7
1.4 Clinical issues 12
1.4.1 Histology 12
1.4.2 Morphology 12
1.4.3 Grade 13
1.4.4 Stage 14
1.4.5 Low risk and high risk tumours 15
1.5 Clinical management 16
1.5.1 Presenting symptoms 16

1.5.2 Investigation 16
1.6 Primary treatment and follow-up 17
1.6.1 Transurethral resection 17
1.6.2 Follow-up 17
1.7 Intravesical chemotherapy 18
1.8 Bacillus Calmette-Guérin immunotherapy 19
1.8.1 History of BCG as an antineoplastic agent 19
1.8.2 BCG and bladder cancer 21
1.8.3 Efficacy of BCG Immunotherapy 21
1.8.3.1 BCG Strains 21
1.8.3.2 Efficacy in clinical trials 22
1.8.3.3 Toxicity of BCG Immunotherapy 24
1.8.4 Mechanism of action of BCG 26
1.8.4.1 Immune surveillance 26
1.8.4.2 Tumour cell antigens 27
1.8.4.3 Tumour cell detection and killing 27
1.8.4.4 Cytokines 29
1.8.4.5 Cell adhesion molecules 33
1.9 Current problems in BCG immunotherapy for superficial bladder cancer 34

iii
1.9.1 Schedule 35
1.9.2 Dose 36
1.9.2.1 BCG dose reduction – when more isn’t better! 36
1.10 BCG and Interferon alpha 37
1.11 Aims of this study 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 100
Serious adverse events 100
BCG cystitis 101
Previous exposure to tuberculosis or BCG vaccination 101
Comparison of toxicities between the treatment arms 101
Local side effects vs. systemic side effects 103
Fever 104

iv
Dysuria 105
Frequency 107
Burning sensation 109
Hematuria 111
Malaise 114
Urgency 117
Nocturia 120
Incontinence 123
Lassitude 123
Work and other activities 126

Section C. Efficacy Analysis 129
1. Recurrence 129
2. Progression 133
3. Maintenance therapy 133
4. Mortality 134
5. Extravesical recurrence 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

v
Publications arising from this thesis

1. Young Surgeon's Award, 27
th
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: 921-
5.
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 IL-
1. 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)


vi
Conference papers
1. Esuvaranathan K. Local immunological markers in intravesical BCG therapy for bladder
cancer. In Proceedings of 27

th
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 23
rd
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 – 1
st
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 28
th

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 IL-
10 and IL-6 production in patients with urinary tract infections, bladder cancers and during
BCG immunotherapy. At 90
th

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 – 2
nd
Scientific Congress, 10-12 August 1996,
National University of Singapore, Singapore.
14. Esuvaranathan K. BCG Immunotherapy – Lessons for gene therapy. 2
nd
Nordic
Conference on BCG Immunotherapy, Sep 1996, Oslo, Norway. (Invited Speaker)

15. Esuvaranathan K. Ying-yang states in Bladder Cancer & BCG immunotherapy. 30
th

Annual Combined Surgical Meeting, 30 October – 2 November 1996, Academy of
Medicine Singapore, 1996, Singapore. (Invited speaker)
16. Esuvaranathan K. Bladder cancer: Prospects for immunotherapy. 30
th
Annual Combined
Surgical Meeting, 30 October – 2 November 1996, Academy of Medicine Singapore,
1996, Singapore.

vii
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 – 3
rd
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 4
th
Asian Congress of Urology, 17-20
September 1998, Singapore, p.241.
20. Esuvaranathan K. Novel therapies for superficial bladder cancer. Uro-oncology Workshop,
4

th
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. 12
th
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, 1 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. 95
th
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 Calmette-
Guérin and interferon alpha for superficial bladder cancer. The 5
th
Asian Congress on
Urology, 27-30 August 2000, Beijing, China.
28. Esuvaranathan K. Superficial bladder cancer. The 5

th
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. 2
nd
Asia-Pacific Uro-oncology Meeting, 9-
11 December 2003, Hong Kong. (Invited Speaker)
31. Esuvaranathan K. Principles in Bladder cancer – Time for a Change? 2
nd
Asia-Pacific Uro-
oncology Meeting, 9-11 December 2003, Hong Kong. (Invited Speaker)


viii
LIST OF FIGURES

Fig. 1. Age-standardized and age-specific incidences of bladder cancer in Singapore 3
Fig. 2. Hypothetical model of carcinogen activation and detoxification 6
Fig. 3(A). Schematic representation of cell cycle phases 9
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

ix
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

x
Fig. 33(B). RT112 MCS exposed to IFNγ 100 IU for 24h (magnification 300X) 88
Fig. 33(C). RT112 MCS, control (magnification 625X) 89
Fig. 33(D). RT112 MCS exposed to IFNγ 100 IU for 24h (magnification 625X) 89
Fig. 33(E). RT112 MCS exposed to IFNγ 100 IU & LAK cells for 24h 90
Fig. 33(F). RT112 MCS exposed to IFNγ 100 IU & LAK cells for after 48h 90
Fig. 33(G). RT112 MCS, control (magnification 2500X) 91
Fig. 33(H). RT112 MCS exposed to IFNγ 100 IU for 24h (magnification 2500X) 91
Fig. 33(I). SD MCS exposed to IFNγ and LAK cells (magnification 2500X) 92
Fig. 34. Post-BCG urinary IL-2 in patients on a 6+3 schedule of instillations 93
Fig. 35. Post-BCG urinary IL-6 in patients on a 6+3 schedule of instillations 94
Fig. 36. Post-BCG urinary IL-10 in patients on a 6+3 schedule of instillations 95
Fig. 37. Post-BCG urinary TNFα in patients on a 6+3 schedule of instillations 95
Fig. 38. Post-BCG urinary IFNγ in patients on a 6+3 schedule of instillations 96
Fig. 39. Urinary cytokine profile during maintenance therapy 97
Fig. 40. Urinary IL-2 after the 1
st
, 6
th
, 7
th
& 9
th
instillations in responders and non-
responders 98
Fig. 41. Scattergram of urinary IL-2 in responders and non-responders 98
Fig. 42. Scattergram of urinary IFNγ in BCG responders and non-responders 99
Fig. 43. Scattergram of urinary IL-10 in BCG responders and non-responders 99

Fig. 44. Recurrence-free functions (KM curve) by treatments 133
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

xi
Fig. 48. Schema of mechanism of action of BCG immunotherapy 158


xii
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 in controlled studies 23
Table 4. Incidence of Common Adverse Events Associated with any Instillation During
Induction versus Maintenance Therapy 26
Table 5. Procedure for the processing of the urine 41
Table 6. ELISA kits used 42
Table 7. Details of the antibodies used in these studies 51
Table 8. Stability of cytokines in acidic & neutral urine at 4
o
C and -20
o
C 58
Table 9. Comparison of pre-freezing and post-thawing dialysis on urinary cytokines 64
Table 10. Modal week of first detection of each cytokine (n=11) 66
Table 11. Mean Total Post-instillation Cytokine Production (12 h) 67
Table 12. Severe adverse events occurring in this study 100
Table 13. Patients with prior exposure to tuberculosis had a higher incidence of toxicity 101
Table 14. Local side effects vs. systemic side effects 103

Table15. Fever 104
Table 16. Dysuria 105
Table 17. Frequency 107
Table 18. Burning sensation 109

xiii
Table 19. Hematuria 111
Table 20. Malaise 114
Table 21. Urgency 117
Table 22. Nocturia 120
Table 23. Incontinence 123
Table 24. Lassitude 123
Table 25. Work and activities 126
Table 26. Interim analysis: Recurrence rate 129
Table 27. Profiles of patients by treatment categories 130
Table 28. Mean time to recurrence 131
Table 29. Recurrence rates and hazard ratios by treatments 132


xiv
Abbreviations
ADI Adoptive cellular immunotherapy
ADCC Antibody dependent cellular cytotoxicity
AFP Alpha-foetoprotein
Ag Antigen
APC Antigen presenting cell
BCG bacillus Calmette-Guérin
BRM Biological response modifiers
CD Cluster designation
cDNA Complementary Deoxyribonucleic acid

CEA Carcinoembryonic antigen
CFU Colony-forming units
CIS Carcinoma in situ
CNS Central nervous system
CTL Cytotoxic T lymphocyte
DAB Diaminobenzidine
DAG Diacylglycerol
DTH Delayed type hypersensitivity
DMSO Dimethylsulphoxide
DNA Deoxyribonucleic acid
EDTA Ethylenediaminetetraacetic acid
EGF Epidermal growth factor
ELISA Enzyme linked immunosorbent assay
Fc Complement binding fragment
FCS Foetal calf serum
FITC Fluorescein isothiocyanate
GMCSF Granulocyte macrophage colony stimulating factor
HBSS Hanks balanced salt solution
HLA Human leucocyte antigen
HIV Human immunodeficiency virus
HRP Horse radish peroxidase
ICAM Intercellular adhesion molecule
IL Interleukin
IL-2R Interleukin-2 receptor
IFNα Interferon-alpha
IFNβ Interferon-beta
IFNγ Interferon-gamma
LAK Lymphokine-activated killer cell

xv

LFA-1 Leucocyte function-associated antigen-l
MHC Major histocompatibility antigen
mRNA Messenger ribonucleic acid
M-VAC Methotrexate, vinblastine, adriamycin, cisplatin
NK Natural killer cell
n.s. Not statistically significant
OD Optical density
OR Odds Ratio
PBL Peripheral blood lymphocyte
PBMC Peripheral blood mononuclear cells
PBS Phosphate buffered saline
PE Phycoerythrin
PGE Prostaglandin E
PHA Phytohaemagglutinin
PKC Protein kinase C
PMA Phorbol myristate acetate
PMN Polymorphonuclear cell
RA Rheumatoid arthritis
RNA Ribonucleic acid
TAA Tumour associated antigen
TB Tuberculosis
TBS Tris buffered saline
TCC Transitional cell carcinoma
TcR T cell receptor
TGFβ Transforming growth factor beta
TIL Tumour infiltrating lymphocyte
TNFα Tumour necrosis factor alpha
TNFβ Tumour necrosis factor beta
TUR Transurethral resection
UK United Kingdom

UTI Urinary tract infection
UV Ultraviolet

xvi
Summary

Bacillus Calmette-Guérin (BCG) immunotherapy for superficial bladder cancer is the
most successful example of immunotherapy for human malignancies. However much
is still unknown about its 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 microscopy and a 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 and adhesion molecules, constitutively and on
stimulation with BCG, both in vitro and in vivo. Activation of the cellular immune
response responsible for the anti-tumour effects are enhanced and possibly even
dependent on these immunomodulators. The pattern of cytokine production also varies
between responders and nonresponders; the TH1 cytokines, IL-2 and IFNγ are
potential prognostic 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

xvii

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 and schedule are important in
preventing overstimulation, which may lead to tolerance to BCG antigen. Indeed, non-
responders did not display immunological memory for BCG when rechallenged.
Booster instillations invoke a secondary immune response and are probably a better
method for inducing persistent immunoactivation than a prolonged induction course of
BCG.

Dose reduction reduces the toxicity of BCG. IFNα does not increase 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 in the “6+3”
schedule used in this study could become the new standard in immunotherapy for
superficial bladder cancer.

1




Chapter 1


INTRODUCTION

2

Bladder Cancer
Introduction
Bladder cancer is a spectrum of superficial, invasive and metastatic disease. Each differs
in natural history and prognosis and consequently, in primary management. For
superficial tumours, the goals of management are prevention of recurrence and
progression; for muscle-invasive disease, the goals become survival and improvement in
quality of life.
1.1 Epidemiology
Incidence
Cancer of the urinary bladder is the 9
th
most common cancer worldwide representing
3.2% of the total cancer burden (Parkin et al 1999), with an estimated total of 261,000
incident cases and 114,000 deaths in 1990 (Pisani et al 1999). As median survival for
bladder cancer exceeds 10 years, its prevalence is approximately 10 times its incidence.
This, compounded by its recurrent nature, makes bladder cancer one of the costliest
cancers today (Riley et al 1995).
Geographical distribution
There are wide geographical differences in incidence rates with the highest rate in males
of 34.0 per 100,000 seen in Trieste, Italy. High incidence rates are also seen in white
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).

3
In Singapore, bladder cancer is the 7
th
most common cancer in males with an age-
standardized incidence rate per 100,000 (ASR) of 7.2 compared with 1.9 in females.

These rates are relatively low compared with the incidences in some other parts of Asia,
notably Hong Kong (ASR 14.5 per 100 000 per year). The ASR has increased by 2.3%
over a 22-year period between 1968 and 1992 (Chia et al 1996). The ASR is highest in
Chinese males (7.7) compared with Malay males (7.1) and Indian males (4.2). 87% of
the cases were transitional cell 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 cancer in women relative to men is consistent
worldwide. Even in the absence of known carcinogenic factors, the risk of bladder

4
cancer for men is almost 3 times higher in men than in women (Hartge et al 1990). Also,
parous women are at decreased risk relative to nulliparous women (OR = 0.67, 95% CI =
0.44-1.00), after adjustment for age, tobacco use and previous bladder infection (Cantor
et al, 1992). Why this is so, is unknown.
1.2 Etiology
Tobacco smoking
There is a strong and well-established association between bladder cancer and smoking.
In different regions of the world, smoking accounts for 33% to 50% of cancers among
men and about 25% of that among women (Silverman et al 1992; Parkin et al 1994).
The relative risk in smokers is from 2 to 5 (Howe et al 1980; Cole et al 1971). The dose

and duration of smoking, depth of 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 aromatic hydrocarbons (e.g. 2-naphthyline, 4-
aminobiphenyl, benzene, benzidine, benzopyrene etc.) commonly found in tobacco
smoke or exhaust gases from combustion engines (Steineck, 2001)
179
.
Carcinogens may be genotoxic, non-genotoxic or cause epigenetic changes (MacLeod et
al 1996)
126
. These carcinogens or their metabolites, are electrophilic and bind to
electron-rich nucleic acids or proteins to form adducts or cause mutagenesis (Talaska et
al 1991; Wormhoudt et al 1999). Interestingly, neither arylamines nor polycyclic

5
aromatic hydrocarbons are direct carcinogens and it is their metabolites which are
carcinogenic (Theodorescu & See, 2001). Foreign lipophilic compounds are converted to
hydrophilic compounds, which are more readily excreted. In the case of arylamines, the
first step is N-oxidation catalysed by hepatic cytochrome P450 1A2 isoenzyme (Butler et
al 1989). The resulting metabolites, hydroxylamines, are active compounds, which bind
to hemoglobin or form glucuronide conjugates and are excreted in the urine.
Hydroxylamines are hydrolysed in the acidic urinary environment, allowing formation of
adducts to nucleophilic sites in transitional bladder epithelium. Subjects with mutations
in the promoter region of the CYP1A gene have increased transcription rate leading to
increased production of these highly reactive metabolites. This mutation occurs more
frequently in patients with bladder cancer (OR 1.54, CI 2.99-4.11) (Horn et al 2003).
Fortunately, arylamines can be detoxified more safely by N-acetylation. Two

isoenzymes of N-acetyltransferase, NAT 1 and NAT 2 catalyze this reaction. A single
polymorphic gene encodes NAT2, with individuals having any two of several mutant
alleles displaying a slow acetylator phenotype. Slow acetylators are also at increased
risk of developing bladder cancer (OR 1.88) (Johansson and Cohen, 1997).
Metabolites of polycyclic aromatic hydrocarbons and some arylamines may undergo
detoxification by conjugation to glutathione (Bell et al). Heterologous carriers of the
null phenotype for GSTM1 have an increased risk of developing bladder cancer (OR
3.54, 95%CI, 2.99-4.11) (Horn et al 2003) (Fig. 1).

6
Fig. 2. Hypothetical model of carcinogen activation and detoxification. Note the
resulting cellular consequences in (A) patients with normal detoxification, and (B) in
individuals with abnormal detoxification mechanisms. Abbreviations: CYP1A2, hepatic
cytochrome P450 1A2; NAT 2, N-acetyltransferase 2; GST-M1, glutathione S
transferase M1. (From: Theodorescu & See, 2001)

Environmental Exposure
Smoking Industrial Pollution
Carcinogen Carcinogen
DNA Adduct DNA Adduct
DNA Adduct
DNA Repair
Cell Death
Tumor Formation
DNA Repair
Cell Death
Tumor Formation
Metabolic
Detoxification
Pro-Carcinogen
Metabolic
Activation
CYP1A2
Inert Compounds
NAT2 GSTM1
A
Environmental Exposure
Smoking Industrial Pollution
Carcinogen Carcinogen
DNA Adduct DNA Adduct
DNA Adduct
DNA Repair
Cell Death
Tumor Formation
DNA Repair
Cell Death

Tumor Formation
Metabolic
Detoxification
Pro-Carcinogen
Metabolic
Activation
CYP1A2
Inert Compounds
NAT2 GSTM1
B

7
Interestingly, lifestyle studies appear to support this model. Many carcinogens are
lipophilic and may be stored in animal fat. Dietary factors associated with greater risk
include fried meats and animal fat (Steineck et al 1990). Also, drinking more fluids (2l
or more) decreases risk of bladder cancer, perhaps by decreasing concentration of
carcinogenic metabolites and increasing transit time for urine (Michaud et al 1999a). A
high intake of cruciferous vegetables may also decrease risk of developing bladder
cancer (Michaud et al 1999b). Also, ingestion of live lactic acid bacteria (lactobacillus),
which replaces carcinogen-producing colonic bacteria and may additionally stimulate
interferon production, has been shown to reduce recurrence rates in some patients with
superficial bladder cancer (Aso et al 1992).
1.3 Genetic basis of bladder cancer
The exact genetic events leading to bladder cancer are unknown but they appear to be
multiple and involve activation of oncogenes or loss of tumour suppressor genes, giving
rise to abnormal gene expression (Olumi et al 1990). A number of specific genes are
known to be mutated by chemical carcinogens. Two of these genes, HRAS and p53 have
been implicated in bladder cancer. Approximately 40% of bladder tumours have HRAS
codon 12 mutations (Czerniak et al 1992). Mutations in p53 occur in up to 40% of
bladder tumours (Sidransky et al 1991). p53 has important regulatory roles in cell

division (see below) and p53 mutations may be important in both transformation and
progression (Simoneau et al 1994; Esrig et al 1994).
In bladder cancer, alterations have been observed in chromosomes 1, 5, 8, 11 (Tyrkus et
al 1992; Vanni et al 1988; Atkin et al 1986), 7 (Babu et al 1987; Atkin et al 1993), 9
(Gibas et al 1984; Atkin et al 1985), 13 (Gibas et al 1984), 17 (Chaturvedi et al 1997;