Development Of Transcriptional Amplification Systems
To Target and Characterize Cancer Cells Based On
Gene expression Altered During Prostate Cancer
Development and Treatment
Thèse
Pallavi Jain
Doctorate en biologie cellulaire et moléculaire
Philosophiae Doctor (Ph. D.)
Québec, Canada
Development Of Transcriptional Amplification Systems
To Target and Characterize Cancer Cells Based On
Gene expression Altered During Prostate Cancer
Development and Treatment
Thèse
Pallavi Jain
Sous la direction de:
Résumé
Le cancer de la prostate (CaP) est le cancer dont l’incidence augmente le plus vite parmi les hommes. Selon la Société Canadienne du Cancer, en 2015, 24 000 nouveaux cas de cancer de la prostate seront diagnostiqués et 4 100 patients en décèderont. Bien que des techniques cliniques pour la détection, le diagnostique et le traitement du CaP soient disponibles et importantes dans le traitement actuel de la maladie, elles sont cependant limitées. L’exploitation de plusieurs promoteurs dont l’activité est altérée au cours du développement du cancer est un moyen pour surmonter ces limitations. L’ARN non codant PCA3 est un biomarqueur unique du CaP qui a été largement étudié et dont l’expression est 60 fois plus forte dans les cellules de CaP que dans les cellules bégnines de prostate. Le gène de l’APS (PSEBC) est un marqueur important en clinique, il reflète la réponse au traitement par privation androgénique. Ces études ont pour objectif de développer des systèmes d’amplification transcriptionnel avec les promoteurs PCA3 et PSEBC pour non seulement cibler mais aussi caractériser les cellules cancéreuses de prostate lors de la progression de la maladie. Nous avons générés plusieurs systèmes dans des adénovirus contenant différentes constructions avec le promoteur proximal PCA3 de 152 pb, le système d’amplification TSTA et le gène rapporteur de la luciférase. Nous avons testé leur spécificité pour les cellules du CaP par infection transitoire. Nous avons amélioré le système TSTA et généré le PCA3-3STA. Nous avons ensuite intégré le promoteur PCA3 avec le promoteur PSA pour générer un autre nouveau système d’amplification transcriptionnelle qui se nomme le système «Multiple Promoter Integrated Transcriptional Amplification (MP-ITSTA)». Ces systèmes ont ensuite été exploités avec un microscope à bioluminescence pour cibler des cellules de CaP provenant de biopsies liquides de patients. Dans le chapitre deux, nous avons montré que l’activité de PCA3-3STA était hautement spécifique pour les cellules de CaP. Son activité était de 98,7 à 108 fois plus fortes dans les cellules de CaP que dans les cellules primaires bégnines de prostate ou dans les cellules cancéreuses non-prostatiques. Dans des modèles murins de xénogreffes de lignées cellulaires de CaP, nous avons montré que PCA3-3STA pouvait imager de manière très sensible l’activité du promoteur PCA3. De plus, sur des modèles de cultures primaires de biopsies, nous avons montré que le système PCA3-3STA ciblait spécifiquement les cellules épithéliales de CaP sans affecter les cellules stromales. Dans le chapitre trois, nous avons ensuite développé une technique en combinat la microscopie à bioluminescence avec le système TSTA et le promoteur PSA pour cibler les cellules de CaP purifiées de sang de patients et évaluer, cellule par cellule, l’hétérogénéité de leur réponse aux anti-androgènes. Cette technique a aussi montré que la microscopie à bioluminescence est hautement quantitative et a la capacité de détecter les changements moléculaires à l’échelle de la cellule. Le quatrième chapitre présente le système MP-ITSTA. Le système intègre l’activation combinée de deux promoteurs qui contrôlent l’expression d’un seul gène rapporteur. La combinaison du promoteur PCA3 avec celui de l’APS permet d’évaluer, cellule par cellule, la réponse aux anti-androgènes de
cellules de CaP prélevés à partir d’urine de patients. C’est pourquoi, les systèmes PCA3-3STA et MP-ITSTA sont des systèmes d’expression spécifiques au cancer de la prostate avec le potentiel de cibler et détecter avec précision les cellules épithéliales de CaP ainsi que leur réponse aux traitements thérapeutiques in vivo et ex vivo. Ces systèmes peuvent jouer un rôle important pour l’imagerie moléculaire, l’immunothérapie et la thérapie génique.
Abstract
Development Of Transcriptional Amplification Systems To Target and Interrogate Cancer Cells Based On Gene Expression Altered During Cancer Development and Treatment
Prostate cancer (PCa) is the fastest rising cancer among the males. According to the Canadian Cancer Society in 2015 it was estimated that 24 000 new cases will be diagnosed with prostate cancer and 4100 patients will die from the disease. Although already available clinical techniques for the detection, prognosis and treatment of PCa play an important role in decision making, they are limited in terms of the ability of detecting PCa cells, prognosis and increasing over all survival of patients. Exploitation of several gene promoters altered during cancer development act as important tool to overcome these limitations. PCA3 non-coding long RNA is a unique PCa biomarker that has been widely studied for its sixty-fold overexpression in PCa cells, compared to benign prostate cells. PSA (PSEBC) gene is of high clinical significance as it can give an account of response to androgen deprivation treatments. These studies aim to develop Transcriptional Amplification Systems that can target as well as characterise cancer cells during disease progression using PCA3 and PSA gene promoters. Various adenovirus constructs incorporating the proximal 152 bp PCA3 promoter, the TSTA system and the Firefly luciferase reporter gene were generated and the specificity of the promoter was tested in PCa cells by transient infection. We have improved the TSTA system and generated the (PCA3-3STA). We further integrated the PCA3 promoter along with the PSA promoter to generate a new transcriptional amplification system that we named the Multiple Promoter Integrated Transcriptional Amplification (MP-ITSTA) system. These systems were further applied to target PCa cells from body fluids of patients using bioluminescence microscopy. In chapter two we show that PCA3-3STA activity was highly specific for PCa cells, ranging between 98.7 and 108.0-fold higher, respectively, than that for benign prostate or non-PCa cells. In PCa cell line mouse xenografts, PCA3-3STA was shown to image PCA3 promoter activity with high sensitivity. Moreover, when primary PCa biopsies were infected with PCA3-3STA, it managed to image PCa epithelial cells but not stromal cells. In chapter three we further developed a bioluminescence microscopy technique using the TSTA system with PSA promoter to target PCa cells from blood of patients and assess heterogeneous single cell response to antiandrogens. This technique also shows that bioluminescence microscopy is highly quantitative and has the ability to detect molecular changes at the cellular level. The fourth chapter presents the MP-ITSTA system. This system integrates the combined activation of two promoters giving a single reporter gene expression. PCA3 when combined with the PSA promoter could assess single cell response to antiandrogens in cells isolated from urine of patients. Hence, PCA3-3STA and MP-ITSTA utilizing the bioluminescence microscopy represent a prostate- and PCa-specific expression systems with the potential to target, with high accuracy, PCa epithelial cells, assess their response to therapy in vivo and ex vivo. This can play an important role for imaging, immunotherapy, or gene therapy.
Table of contents
Résumé ... III
Abstract ... V
Table of contents ... VII
List of figures ... XIII
List of tables ... XV
List of abbreviations ... XVI
Acknowledgement ... XXI
Chapter 1 ... 23
Introduction ... 23
1.1) Prostate ... 23
1.1.2) Anatomy of the prostate ... 24
1.2) Prostate carcinoma ... 25
1.2.1) Epidemiology of prostate cancer ... 25
1.2.2) Incidence ... 26
1.2.3) Risk factor for prostate cancer ... 26
1.2.3.1) Endogenous risk factors ... 26
1.2.3.1.1) Familial history ... 26
1.2.3.1.2) Hormones ... 26
1.2.3.1.3) Race ... 26
1.2.3.1.4) Age ... 26
1.2.3.2) Exogenous risk factors ... 27
1.2.3.2.1) Diet ... 27
1.2.3.2.1) Environmental agents ... 27
1.2.3.2.1) Occupation and other factors ... 28
1.2.4) Classification and staging of Prostate cancer ... 28
1.2.4.1) Classification ... 28
1.2.5) Diagnostic evaluation ... 29
1.2.5.1) Screening and early detection ... 29
1.2.5.2) Clinical Diagnosis ... 30
1.2.5.2.1) Digital rectal Examination (DRE) ... 30
1.2.5.2.2) Prostate Specific Antigen (PSA) ... 31
1.2.5.2.2.1) PSA density ... 31
1.2.5.2.2.2) PSA velocity and doubling time ... 31
1.2.5.2.2.3) Free/total PSA ratio ... 32
1.2.5.2.2.4) Prostate Health Index (PHI) test ... 32
1.2.5.2.3) PCA3 marker ... 32
1.2.5.2.4) Prostate biopsy ... 33
1.2.5.2.4.1) Baseline biopsy ... 33
1.2.5.2.5) Role of imaging ... 33
1.2.5.2.5.1) Trans rectal ultrasound (TRUS) ... 33
1.2.5.2.5.2) Multi-parametric magnetic resonance imaging (MRI) ... 33
1.2.5.2.5.4) Positron emission tomography (PET scan) ... 35
1.2.5.2.6) Lymphadenectomy ... 35
1.2.5.2.7) Bone scan ... 35
1.2.6) Treatment for Prostate cancer ... 36
1.2.6.1) Active surveillance / watchful waiting ... 36
1.2.6.2) Surgery ... 36
1.2.6.3) Radiotherapy ... 37
1.2.6.4) Cryosurgery ... 37
1.2.6.5) High intensity focused ultrasound of the prostate (HIFU) ... 38
1.2.6.6) Focal therapy of prostate cancer ... 38
1.2.6.7) Hormonal therapy ... 38
1.2.6.7.1) Castration ... 38
1.2.6.7.2) Bilateral orchiectomy ... 39
1.2.6.7.3) Luteinising-hormone-releasing hormone agonists ... 39
1.2.6.7.4) Castrated levels ... 39
1.2.6.7.5) Luteinising-hormone-releasing hormone antagonists ... 39
1.2.6.7.5.1) Abarelix ... 39
1.2.6.7.5.2) Degarelix ... 39
1.2.6.7.6) Anti-androgens ... 40
1.2.6.7.6.1) Steroidal anti-androgens ... 40
1.2.6.7.6.1.1) Cyproterone acetate (CPA) ... 40
1.2.6.7.6.1.2) Megestrol acetate and medroxyprogesterone acetate ... 40
1.2.6.7.6.2) Non-steroidal anti-androgens ... 40
1.2.6.7.6.2.1) Nilutamide ... 41
1.2.6.7.6.2.2) Flutamide ... 41
1.2.6.7.6.2.3) Bicalutamide ... 41
1.2.6.7.6.3) Drugs post-castration resistance ... 41
1.2.6.7.6.3.1) Abiraterone acetate ... 41
1.2.6.7.6.3.2) Enzalutamide ... 41
1.2.6.8) Non-hormonal therapy ... 42
1.2.6.8.1) Docetaxel ... 42
1.2.6.8.2) Vaccine ... 42
1.2.6.8.3) Cabazitaxel ... 42
1.2.6.8.4) Radium 223 ... 42
1.2.6.8.5) Biphosphonates ... 43
1.2.7) Molecular changes associated with Prostate cancer ... 43
1.2.7.1) Inherited genetic changes ... 43
1.2.7.2) Somatic genetic changes ... 44
1.2.7.2.1) NKX3.1 down-regulation ... 44
1.2.7.2.2) Myc up-regulation ... 44
1.2.7.2.3) TMPRSS2-ERG translocations ... 45
1.2.7.2.4) PTEN ... 46
1.2.7.3) Signalling pathways—Akt/mTOR and MAPK signalling ... 46
1.2.7.5) Developmental signalling pathways ... 47
1.2.7.5.1) Genomic modifications ... 48
1.2.7.6) Invasion and metastasis suppressor genes ... 48
1.2.7.7) Telomere shortening ... 49
1.2.7.8) Other genetic changes ... 49
1.2.7.9) Overexpressed genes with clinical potential ... 49
1.2.7.10) Micro RNAs (miRNAs) ... 50
1.2.7.11) Androgen receptors ... 50
1.2.7.11.1) Genomic Androgen receptor pathway ... 51
1.2.7.11.2) Non-genomic Androgen receptor pathway ... 52
1.2.7.11.3) Mechanism involved in development of castration resistant prostate cancer ... 54
1.2.7.11.3.1) AR overexpression ... 54
1.2.7.11.3.2) Recruitment of adrenal gland hormones ... 54
1.2.7.11.3.3) Change in balance of co-activators and co-repressors ... 54
1.2.7.11.3.4) Gain of functional mutations ... 55
1.2.7.11.3.5) Alternative activation through signalling cross-talk ... 55
1.2.7.11.3.6) Expression of constitutive active receptor variants ... 56
1.2.7.11.3.7) Intra-tumoural de-novo synthesis as alternative androgen hormone source ... 57
1.2.7.11.3.8) Neuroendocrine differentiation ... 58
1.2.8) Models for Biomarker study in prostate cancer patients ... 59
1.2.8.1) Tissue based approach ... 60
1.2.8.2) Fluid based approach ... 61
1.2.8.2.1) Blood based molecular characterization of prostate cancer ... 62
1.2.8.2.1.1) Circulating tumour cells (CTCs) ... 62
1.2.8.2.1.1.1) Isolation of circulating tumour cells ... 63
1.2.8.2.1.1.2) Identification and characterization of CTCs ... 65
1.2.8.2.1.1.3) Biological implications of circulating tumour cells ... 67
1.2.8.2.1.1.4) Clinical utility of CTCs ... 67
1.2.8.2.1.1.4.2) Potential predictive biomarkers ... 68
1.2.8.2.1.1.4.3) CTC characterisation for novel therapeutics ... 70
1.2.8.2.1.2) Circulating tumour nucleic acid ... 70
1.2.8.2.1.2.1) Circulating tumour DNA ... 70
1.2.8.2.1.3) Exosomes ... 72
1.2.8.2.2) Urine tumour cells (UTC) ... 73
1.2.8.2.2.1) Targeting cancer cells ... 74
1.2.8.2.2.1.1) Two –Step Transcriptional Amplification System ... 75
1.2.9) Research Questions ... 76
Chapter 2 ... 77
A PCA3 Gene-Based Transcriptional Amplification System Targeting Primary Prostate Cancer ... 77
Foreword ... 79
Résumé ... 80
Abstract ... 81
2.1) Introduction ... 82
2.2.1) Plasmid construction and adenovirus production ... 83
2.2.2) Cell cultures ... 83
2.2.3) Adenoviral infection and androgen sensitivity ... 84
2.2.4) Time course experiments and Western blot analysis ... 84
2.2.5) In vivo experiments ... 84
2.2.6) Ex vivo primary PCa detection ... 84
2.2.7) Statistical analysis ... 85
2.3) Results ... 85
2.3.1) PCA3-3STA is a highly amplified expression system ... 85
2.3.2) PCA3-3STA directs a robust expression that is specific to prostate cancer ... 86
2.3.3) PCA3-3STA directs a robust expression in vivo in PCa tumour models ... 87
2.3.4) PCA3-3STA can detect primary PCa ... 87
2.4) Discussion ... 88
2.5) Conclusion ... 90
References ... 91
Figures and legends ... 95
Supplementary figures and legend: ... 100
Chapter 3 ... 104
Bioluminescence Microscopy as a Method to Measure Single Cell Androgen Receptor Activity Heterogeneous Responses to Antiandrogens. ... 104
Foreword ... 106
Résumé ... 107
Abstract ... 108
3.1) Introduction ... 109
3.2) Results ... 110
3.2.1) Single cell bioluminescence microscopy imaging optimization after reporter system delivery ... 110
3.2.2) Bioluminescense microscopy is highly quantitative. ... 111
3.2.3) Bioluminescence microscopy is able to quantify single cell heterogeneous response to antiandrogens. ... 112
3.3) Discussion ... 113
3.4) Material and Methods ... 115
3.4.1) Plasmid construction and adenoviral production ... 115
3.4.2) Cell cultures ... 115
3.4.3) Production of stable transduced cell lines ... 116
3.4.4) Bioluminescence microscopy ... 116
3.4.5) Adenoviral infection and viability assay ... 116
3.4.6) D-luciferin concentration optimization ... 117
3.4.7) Exposure time optimization ... 117
3.4.8) Androgen responsiveness assessment by luciferase assay ... 117
3.4.9) Single cell treatment response ... 118
3.4.10) Single cell treatment response from spiked cancer cells isolated from blood ... 118
3.4.11) Statistical analysis ... 119
Author contributions ... 119
References ... 120
Figure and Legends ... 123
Supplementary information ... 127
Chapter 4 ... 134
Development of a multigenic bioluminescence imaging system to Detect Prostate Cancer Cells and Assess their Response to Therapy ... 134
Foreword ... 134
Résumé ... 135
Abstract ... 137
4.1) Introduction ... 138
4.2) Results ... 139
4.2.1) Integration of PCA3 and PSEBC promotor’s specificities as a single output to image PCa cell antiandrogen response. ... 139
4.2.2) PCA3-CRE-PSEBC-ITSTA system is specific for PCa cells and can measure the transcriptional activity of the androgen receptor. ... 141
4.2.3) PCA3-CRE-PSEBC-fl and bioluminescence microscopy can detect PCa cells from urine and quantify single cell response to anti-androgen therapy. ... 141
4.2.4) PCA3-CRE-PSEBC-ITSTA can detect primary PCa cells from urine and assess their response to antiandrogen therapy. ... 143
4.2.5) PCA3-CRE-PSEBC-ITSTA allows dynamic primary PCa cell imaging and can assess single cell antiandrogen sensitivity. ... 143
4.3) Discussion ... 144
4.4) Material and methods ... 147
4.4.1) Plasmid and adenoviral construction ... 147
4.4.1.1) Generation of stop cassette plasmids ... 147
4.4.1.2) Generation of modified cre recombinase ... 147
4.4.1.3) Construction of plasmid with LoxP sites ... 148
4.4.1.4) Adenoviral construction ... 148
4.4.2) Cell culture ... 148
4.4.3) Transfection experiments ... 149
4.4.4) Adenoviral infection and treatment experiments ... 149
4.4.5) RT-qPCR technique ... 149
4.4.6) Isolation and infection of PCa cells isolated from urine sample ... 149
4.4.7) Immunoflourescence ... 150
4.4.8) Statistical analysis ... 151
References ... 151
Figures and Legends ... 153
Supplementary Information ... 162
Chapter 5 ... 166
Discussion ... 166
5.1) Current tools for Disease management of prostate cancer ... 166
5.2) Molecular-tools for tissue specific targeting ... 168
5.3) Bioluminescence: a tool to study molecular changes in cancer cells ... 170
Conclusion ... 175
References ... 175
List of figures
Figure 1: Position of the prostate. ... 23Figure 2: Zonal anatomy of the prostate the prostate is divided into four distinct zones. ... 24
Figure 3: Clinician performing a DRE examination. ... 31
Figure 4: The molcular structure of the androgen receptor gene and protein, showing the domain regions. ... 51
Figure 5: Genomic AR pathway. ... 52
Figure 6: Non-genomic AR pathway. ... 53
Figure 7: Schematic structures of human AR splice variants reported in Genbank. ... 57
Figure 8: Scheme showing all the resistome mechanisms leading to a single output causing castration resistance. ... 58
Figure 9: Scheme showing advances in prostate cancer biomarker uses. ... 59
Figure 10: Metastasis forming cascade. ... 63
Figure 11: Scheme depicting the tissue specific promoter driven reporter gene expression having the ability to target tissue-specific cancer cells. ... 75
Figure 12: The Three Step Transcriptional Amplification System provides strong amplification of the PCA3 promoter activity. ... 95
Figure 13: PCA3-3STA: A prostate- and prostate cancer-specific expression system that is not androgen-dependent. ... 96
Figure 14: PCA3-3STA provided reporter expression levels in vivo comparable to those of the PSEBC-TSTA system. ... 97
Figure 15: PCA3-3STA detected primary prostate cancer cells from radical prostatectomy specimens ex vivo ... 98
Figure 16: PCA3-3STA activity can discriminate benign from primary cancerous prostate glands. ... 99
Figure 17: (supplementary) Characterisation of the best PCA3-3STA conformations for prostate cancer-specific expression. ... 100
Figure 18: (Supplementary) PCA3-3STA is prostate cancer and not induced by androgen but PSEBC-TSTA is highly induced by androgens. ... 101
Figure 19: (Supplementary) The enhanced amplification provided by PCA3-3STA compared to that of the TSTA was after 3 days and was secondary to increased GAL4VP16 protein expression. ... 102
Figure 20: Optimization of a bioluminescence micorscopy method for single cell imaging after adenoviral system transduction. ... 123
Figure 21: Imaging single cell heterogeneous response to AR agonist and antagonist by using bioluminescence microscopy. ... 126
Figure 22: (Supplementary) optimization of D-luciferin concentration for bioluminescence microscopy. 127
Figure 23: (Supplementary) Optimization of viral transduction conditions for PCa cell lines. ... 128
Figure 24: (Supplementary) Optimization of exposure time to be used for Bioluminescence imaging. .... 129
Figure 25: (Supplementary) Growth curves of PCa treated with either bicalutamide (Bic) or enzalutamide (Enz). ... 130
Figure 26: (Supplementary) Comaprison of bioluminescence microscopy and conventional luciferase assay to measure AR-transcription activity changes associated with DHT or Enz treatments in PCa cells. 131
Figure 27: (Supplementary) single cell bioluminescence microscopy using PSEBC-TSTA detected heterogeneous response to treatment. ... 133
Figure 28: (Supplementary) LNCaP cells display greater single cell cell AR-transcriptional heterogeneity upon treatment when comapred to LAPC4 cells. ... 133
Figure 29: PCA3 promoter is PCa specific while PSEBC promoter is androgen responsive. ... 153
Figure 30: Characterization of the MP-ITSTA system for prostate cancer-specific expression. ... 154
Figure 31: PCA3-CRE-PSEBC-ITSTA shows activity specifically in AR responsive PCa cells giving an induction comparable to PSEBC-TSTA with DHT. ... 155
Figure 32: PCA3-CRE-PSEBC-ITSTA could specifically detect and assess response to treatment of spiked PCa cells in urine of patients. ... 156
Figure 33: PCA3-CRE-PSEBC-ITSTA could specifically detect PCa cells in urine of patients. ... 157
Figure 34: PCA3-CRE-PSEBC-ITSTA could specifically detect and assess response to treatment of PCa cells in urine of patients. ... 159
Figure 35: Scheme for single cell dynamic imaging. ... 160
Figure 36: (Supplementary) Bovine growth hormone poly A (BHGstop) could efficiently inhibit the expression of fl and gives better reactivation in the presence of cre comapred to SV40 polyA. ... 162
Figure 37: (Supplementary) Insertion of the chimeric human intron within cre recombinase could inhibit leakage of cre in bacteria and enhanced the expression of firefly luciferase. ... 163
Figure 38: (Supplementary) multiple marker immunofluorescence in cell lines. ... 164
Figure 39: (Supplementary) Viability assay for PCa cells either on treatment with enzalutamide or bicalutamide. ... 164
List of tables
Tableau 1: TNM staging of prostate cancer. The table is adapted from Duckward Black et al. ... 29
Tableau 2: Summary of techniques used to isolate prostate cancer CTCs. ... 65
Tableau 3: Clinical and histopathological information of patients from which prostate tissue was sampled.
List of abbreviations
AA- Abiraterone Acetate
ADT- Androgen Deprivation Therapy AF- Activation Factors
AFP- Alpha-fetoprotein ALP- Alkaline Phosphatase
AMACR- Alph-methylacyl-CoA racemase AR- Androgen Receptor
ASAP- Atypic Small Acinar Proliferation AS- Active Survillance
Bic- Bicalutamide
BPE- Benign Prostatic enlargement BPH- Benign Prostatic Hyperplasia BS- Bone Scan
CAB- Complete Androgen Blockage CAM- Cell Adhesion Matrix
CCP- Cell Cycle Progression cDNA- Complementary DNA cfDNA- Cell free DNA CMV- Cytomegalovirus COX- Cyclooxygenase
CPA- Cyproterone Acetate CRT- Conformal Radiotherapy CTC- Circulating Tumour cell ctDNA- Circulating Tumour DNA
CT Scan- Computerised Tomography Scan CYP17- Cytochrome P450 17alpha hydroxylase DBD- DNA Binding Domian
DHEA- Dehydroepiandrosterone DHT- Dihydrotestosterone DRE- Digital Rectal Examination EDCs- Endocrine Disrupting Chemicals EMT- Epithelial Mesynchymal Transition Enz- Enzalutamide
EpCAM- Epithelial Cell Adhesion Molecule ER- Estrogen Receptor
ERSPC- European Randomised Study of Screeing for Prostate Cancer ETS- Erythroblast Transformation Specific
Evs- Extracellular Vesicles FAS- Fatty Acid Synthase FDG- Fludeoxyglucose
FHBG- Fluoro-3-(Hydroxymethyl) Butyl Guanine FISH- Fluorescence In Situ Hybridization Fl- Firefly Luciferase
FSH- Follicular Stimulating Hormone GAL4RE- GAL4 Response Elements GATA3- GATA binding protein 3 GFP- Green Fluorescent Protein GSTP1- Glutathione-S-Transferase Pi 1
HGPIN- High Grade Prostatic Epithelial Neoplasia HIFU- High Intensity Focused Ultrasound
HPC1- Hereditory Prostate Cancer 1 HSP- Heat Shock Protein
hTERT- Human Telomerase Reverse Transcriptase ICC- Immunocytochemistry
IF- Immunofluorescence IHC- Immunohistochemistry
IMRT- Intensity Modulated Radiotherapy LDR- Low Dose rate
LHRH- Luteinizing Hormone- Releasing Hormone LND- Lymph Node Dissection
mCRPC- Metastatic Castration Resistant Prostate Cancer miRNA- MicroRNA
MRI- Magnetic Resonance Imaging ncRNA- Non-Coding RNA
NTD- N-Terminal Domain OS- Overall Survival PCa- Prostate Cancer PCA3- Prostate Cancer gene 3
PET scan- Positron Emission tomography scan PFS- Progression Free Survival
PIA- Proliferative inflammatory atrophy PIN- Prostatic Epethelial Neoplasia PSA- Prostate Specific Antigen
PSMA- Prostate Specific Membrane Antigen RBC- Red Blood Cells.
RCTs- Randomised Controlled Trials ROS- Reactive Oxygen Species RP- Radical Prostatectomy
RTAs- Recombinant Transcriptional Activators RT-PCR- Real-Time Polymerase Chain reaction
SR39tk- Herpes Simplex Virus type 1 Thymidine Kinase SV40- Simian Virus 40
TNM- Tumour, Node, Metastasis Staging TSTA- Two Step transcriptional Amplification TURP- Transurethral Resection
Acknowledgement
I am very proud to present my doctoral work, which provides a clear idea of what I have done for last four years during my doctoral study. The work of this thesis would not have been possible without the generous help and support of my supervisor, colleagues, teachers, friends and family. This part of my thesis is to acknowledge all those excellent people around me for making this thesis possible.
First of all, I would like to thank my supervisor, mentor, guide and “philosopher” Dr Frédéric Pouliot. He has been a great teacher for me and guided me to be a better scientist. Every scientific interaction with him makes me more delightful and enthusiastic about science. I truly admire his passion for science, which has been a great encouragement and inspiration for me during my doctoral studies. During all those years of my hard work, he supported me during the tough time and appreciative when I had success. It has been a great honor for me to work with him and I am grateful to him for believing in me and giving me the best opportunity. Secondly, I would like to thank Dr Bertrand Neveu for all the support provided by him during my thesis. He has been a mentor for all the technical difficulties that I faced. He has always supported me not only within the laboratory but also for problems I faced being a foreign student outside the laboratory. It was an honour to work with him and I am grateful to him for making my thesis and stay in Québec easier.
I am grateful to my earlier supervisor Dr Ritu Gaur who gave me the opportunity to learn basic molecular biological techniques and for giving me the first exposure to science and research experience.
I would also like to thank Dr Yves Fradet for providing the laboratory space and all the technical support that I received from his team members. I would also like to thank Dr Vincent Fradet, Dr Louis Lacombe, Michele Lodde and Dr Paul Toren for their constant inputs and suggestions in making my work better. I would also like to thank Dr Lily Wu and Dr Bernard Têtu for their expertise while writing my articles. I wish to thank Dr Alain Bergeron for his knowledge. He has been very generous for sharing his wisdom and expertise with me always. I would like to thank my pre-doctoral committee members Dr Jacque Landry, Dr Éric Lévesque and Dr. Claude Labrie for their valuable comments helped me to improve my doctoral study. I would also like to thank Dr Dr Éric Lévesque, Dr Amina Zoubeidi, and Dr Steve Bilodeau for agreeing to evaluate this thesis.
I am lucky enough to have such excellent people as colleagues first of all I like to thank Dre Mélanie Rouleau for being there for every problem in the laboratory and also supporting me in my work. I learned at lot of techniques from her. I am thankful to Ms Lauriane Velot and Ms Audrey champagne, for their valuable suggestion and also helping me in carrying out experiments.
Further I would like to thank all my other lab members Ms Valérie Picard, Mr Oscar Eduardo Molina, Ms Marjorie Besançon, Ms Denise St-Onge, Mr Nikunj Gevariya, Mr Sébastien Le Batteux, Ms Claire Ménard, Mr Jean-francois Pelletier, Dre Fanny Gaignier, Dre Hélène Hovington, Dr Molière Nguile, Vanessa Bussieres and Michèle Orain for supporting me in my thesis and making my stay in quebec enjoyable. I would like to thank the research nurse for the clinical help and collection of patient samples.
I owe my gratitude to my past lab members. I was lucky to work with Dre Hélène LaRue, an interesting person and I learned lot from her. I would also like to thank Dre Marie Josée Beaudet, Annie-Claude Blouin, Fannie Morin, Goran Rimac, Francis Desmules and Vanessa Collin for taking interest in my work and supporting me. I would also like to thank Dr Darren Richard for providing the luminometer, Dr Jean Charron for providing the lab materials, Anne Lorange and Carl St-Pierre for providing the imaging platform and technical help.
Also I would like to thank Ms Katia Mercier and Ms Lucie Marcoux for being such good people and amazing friends.
I would like to thank Priyanka Patel, Minty Thomas, Ranjan Maity, Wajid Bhat, Prakash Mishra, Irfan Bukhari, Jayesh Patel, Kallol Dutta, Sai Sampath, Arojit Mitra, Hemanta Adhikary, Shubhendu Tripathi, Rishabh
Kataria, Salar Ahmed and Pavan Kumar Kakumani, all my Indian friends in Quebec city, because of you I never felt I am half a world away from our country.
Last but not the least, I am lucky enough to have unconditional love, support and encouragement from my parents and family, without their encouragements it would never have been possible.
Chapter 1
Introduction
1.1) Prostate
Prostate is an accessory gland of the male reproductive and urinary system. It can vary in size from one person to another. It is located at the base of the bladder. Urethra, the tube carrying the urine from the bladder to the penis passes through the prostate dividing it into two: right and left lobes.
This exocrine gland is encapsulated in a layer of connective tissues. It consists of 3 different types of cells namely: gland cells, muscle cells and fibrous cells. Function of these cells is to produce the fluid portion of the semen, control ejaculation and provide support to the glands respectively. There are various other organs around the prostate that aid in its efficient functioning. These include the seminal vesicles, vas deferens and nerve bundles. The seminal vesicle surrounds the prostate on both the sides and is involved in producing semen. The Vas deferens is the duct that carries the spermatozoids from the testicles to the seminal vesicles. The nerve bundles around the prostate control the bladder and erectile function (Figure 1).
Figure 1: Position of the prostate. The figure is adapted from WebMD (2008)
1.1.1) Function of prostate
The main function of the prostate is to secrete seminal fluid and help in ejaculation during sexual activity. It produces a part an alkaline fluid that mixes with the seminal fluid and maintains the viability and mobility of the sperms. The muscular layer present around the prostate helps to expel the semen during ejaculation. The
major component of the fluid secreted by the prostate is prostate specific antigen (PSA), along with citrate, spermine, zinc and cholesterol (Livermore et al. 2016)
1.1.2) Anatomy of the prostate
The prostate gland is mainly composed of 70% glandular cells and 30% of stroma. It is divided into 3 major zones: the peripheral zone (PZ) is the external most regions and constitutes 70% of glandular tissue (Figure 2). It is also the site for the development of prostate cancer in 80% of the cases. The zone beneath the PZ is the central zone that usually forms 25% of the prostate mass. Adenocarcinoma is less likely to develop in this zone and once it occurs it is mostly more aggressive. The third zone is the transitional zone that directly surrounds the urethra. This zone enlarges with age in males and after an age of 40 extends to cover a large part of the gland. It is also the main site for the development of benign prostatic hyperplasia (BPH) (Livermore et al. 2016).
Figure 2: Zonal anatomy of the prostate the prostate is divided into four distinct zones.
Consisting of three glandular zones (peripheral zone, central zone and transition zone) and a fibromuscular stroma. Figure is adapted from Livermore et al AIMS mol. sci. (2016)
Human prostate consists of two tissue compartments namely epithelial and stromal. These two compartments are separated by the basement membrane, a packaged structure of collagen fibers containing various extracellular matrix proteins produced by both epithelial and stromal cells (Bonkhoff et al; 1991). The epithelial compartment further consists of two layes the luminal cell and the basal cells. The luminal cellular layer is composed of polarised columnar laminal epithelial cells and seperates the luminal cells from the stromal
(Brawer et al; 1985, Schlaken et al; 2003). Luminal cells form majority of the prostatic adenocarcinoma. Basal and luminal cells express different cell-type specific proteins, which are used to detect glandular pathologies. In addition to the epithelial cells, the epithelial compartment also contains rare post-mitotic neuroendocrine cells. These cells secreate various neuro-peptides and growth factors necessary for growth of luminal cells (Bankhoff et al; 1995).
1.2) Prostate carcinoma
Cancer is referred to a disease state when cells attain the ability of unregulated growth and subsequently spread to other parts of the body. Various types of cells undergo changes and become malignant but only epithelial cells transform into adenocarcinoma. The normal course of cell cycle is disrupted forming the tumour, that spreads locally and then to other body parts forming metastatic sites by invading into the blood stream. Several reasons can cause a normal cell to become cancerous. Some of these reasons include mutations in the genetic make that alter the DNA repair mechanism, and in turn hampers the balance between proliferation and cell death though the normal cell cycle. This may lead to an over grown mass of cells which might be benign or malignant.
A possible precursor of prostate carcinoma is prostatic epithelial neoplasia (PIN). PIN is a state that involves abnormal growth of the epithelial cells surrounding the prostate glands. It has been shown that presence of PIN is not directly correlated with prostate cancer but may involve several other factors. (Epstein JL et al. 2006)
Another condition involved in prostate cancer development is Atypical small acinar proliferation (ASAP) which is a not a confirmatory pathological, but is an evidence of possible cancer development. Histologically, it can be detected within a benign tissue to a marginally cancerous tissue. ASAP is more closer to being a proliferation marker suggestive of cancer, but not for diagnostic purpose (Bostwick D.G.et al. 2006). While the predictive value of HGPIN is reduced, ASAP remains constant making it an important tool for staging (Schlesinger et al. 2005).
1.2.1) Epidemiology of prostate cancer
The epidemiology of prostate cancer has been extensively studied. There is an increasing burden to public health and the benefit of screening has been debated for sometime. PSA screening has been a constant cause of debate prostate cancer screening (Neal et al. 2003). However it could be efficiently used to identify the disease in high-risk groups.
1.2.2) Incidence
According to prostate cancer Canada statistics, it has been seen that prostate cancer is the most commonly diagnosed cancer in Canadian men with 24% of all new cases. In 2015 it was estimated that 24 000 new cases will be diagnosed with prostate cancer and 4100 patients will die from the disease.
1.2.3) Risk factor for prostate cancer
Three major risk factors associated with prostate cancer are: age, race and positive familial history of prostate cancer (Bostwick et al. 2004).
1.2.3.1) Endogenous risk factors
Endogenous risk factors mainly consist of the following factors:
1.2.3.1.1) Familial history
Familial history is one of the most significantly associated factors with prostate cancer. But no clinical evidence is present that could discriminate familial and non-familial prostate cancer (Gronberg et al. 2003). A positive family history has been consistently associated with 2 to 4-fold increase in the risk of the disease. Family history further reflects an association of both genetic and environmental factor that are shared by the family members (Hemminki et al. 2012)
1.2.3.1.2) Hormones
Androgens play an important role in prostate cancer growth and progression. Increase in the levels of testosterone and its metabolites can increase the risk of prostate cancer development. Variability in hormone concentration can be caused by both endogenous (e.g.; genetic) and exogenous (e.g.; exposure to chemicals) factors (Roddam et al. 2008).
1.2.3.1.3) Race
Incidence of prostate cancer based on race is mainly a result of effects caused to difference in diet and genetic difference. It mainly reflects the difference in the provided medical facility among the race, difference in clinical decision-making (whether or not to obtain medical attention and follow up) and genetic variation (Bostwick et al. 2004).
1.2.3.1.4) Age
Prostate cancer is significantly associated with age. Development and progression of prostate cancer is very slow, therefore localised cancer can remain undetected for many years before it develops into a clinically significant disease. Development of PCa at an early state (upto 50 years) shows an increased risk of 42% but associated mortality risk at this age is only 3% (Pienta et al. 1993).
1.2.3.2) Exogenous risk factors
Exogenous risk factors consist of the following factors:
1.2.3.2.1) Diet
Several Epidemiological studies have shown multiple dietary factors to be associated with the development of PCa. High fat containing diet, especially polyunsaturated fat shows a strong positive correlation with prostate cancer incidence and mortality. This can be mainly caused due to a change in hormones, the effect of fat metabolites as protein or DNA-reactive intermediates or increase in oxidative stress. Vitamin A belonging to the retinoid family has been seen to play an important role in cell proliferation and differentiation. It was seen that vitamin A positively correlated with PCa incidence. Vitamin C even though is a scavenger of reactive oxygen species (ROS) and free radicals. Studies have not proved its positive association with PCa incidence. Vitamin D deficiency has been seen to decrease the incidence of PCa. Its hormonal form 1-25-dihydroxyvitamin D has anti-proliferative functions thereby preventing PCa. Vitamin E (tocopherol) is an antioxidant that inhibits prostate cancer cell growth through apoptosis. Selenium is an essential tracer element that inhibits viral and chemical, carcinogen-induced tumours in animals, but its role in humans has not yet been identified. Selenium and vitamin E cancer prevention trial (SELECT) study has shown that supplementation of diet with vitamin E has a 17% increases in the risk of the disease (Klein et al. 2011). Alcohol was not seen to have positive association with PCa. Intake of cruciferous vegetables is associated with a decreased incidence of several cancers, but their role in PCa has not yet been identified. Instead, lycopene, an abundant constituent of tomato with antioxidant properties, has a significant protective effect (Giovannucci et al. 1993).
1.2.3.2.1) Environmental agents
Endocrine disrupting chemicals (EDCs) is an important category of chemicals studied in association to PCa. An EDC can be defined as an environmental agent that has the ability to alter hormonal regulation and leads to effects on reproduction, development, and/or carcinogenesis. Majority of the EDCs studied work as estrogen agonists that by binding to the estrogen receptor (ER) increase its activity. Some of them however are believed to affect other hormone activities as well. Taking example of the active metabolite of the pesticide vinclozolin, an androgen antagonist that binds to AR and decreases the expression of androgen-regulated genes. Several pesticide residues on foods, chemicals used in plastics production, and phytoestrogens in dietary plant products (e.g. soy) are believed to behave as EDCs. Some epidemiological studies have shown cadmium as a significant environmental contaminant associated with PCa. Carcinogenic potential of cadmium can be reduced by incorporation of zinc (Prins et al. 2008).
1.2.3.2.1) Occupation and other factors
Association of occupational exposure with PCa has not yet been concretely proved and the studies are inconclusive. Some of the major occupations associated with the risk are farming and rubber industry.
Apart from the factors enlisted above there are several other factors which are believed to be associated with PCa, but have shortcoming such as inconsistent results, negative associations, or have very limited data. These factors include smoking, energy intake, sexual activity, marital status, vasectomy, social factors (lifestyle, socioeconomic factors, and education), physical activity, and anthropometry (Pienta et al. 1993).
1.2.4) Classification and staging of Prostate cancer
1.2.4.1) Classification
Classification plays an important role in staging of the disease by clustering together patients predicted to show a similar outcome. This allows the determination of a more homogeneous patient population for studying the clinical and pathological aspects of patients around the world and make efficient decision over their treatment. The most appropriately studied classification system for PCa is TNM staging (Table 1) (Sobin et al. 2009).
Tableau 1: TNM staging of prostate cancer. The table is adapted from Duckward Black et al.
1.2.5) Diagnostic evaluation
1.2.5.1) Screening and early detection
There are two types of screening existing in a population. Initial screening is the process that involves examination of asymptomatic men (at risk) in a population by health authorities. Another type of screening is the one that involves early diagnosis undertaken by a patient on recommendation of his physician. Both the screening aims at reducing the mortality caused by PCa and improving the QoL for a patient. To evaluate the efficacy of PCa screening, two large randomized trials have been published: the Prostate, Lung, Colorectal and Ovary (PLCO) trial in the United States and the European Randomized Study of Screening for Prostate Cancer (ERSPC) in Europe.
PCa mortality varies around the globe. Even though the mortality rate has decreased in the western population its magnitude is still variable. Rigorous PCa screening is believed to be the major reasons for this reduction in incidence and mortality.
Early diagnosis should be done using a PSA test and DRE. The optimal intervals for PSA testing and DRE follow-up are unknown, and it has varied between several trials. Initial PSA level can help in determining an early strategy which could help in determining a timeline for follow-up, e.g.: 2 years for those initially at risk, or postponed up to 8 years in those not at risk.
Current tools involving individualised screening are believed to detect several insignificant lesions (above 50% in some trials), most of which do not require active treatment. Breaking the link between diagnosis and active treatment is the only way to decrease overtreatment, while still maintaining the potential benefit of individual early diagnosis.
Mass screening of PCa is not recommended, but early diagnosis on an individual basis is possible based on DRE and PSA testing. Individual patient screening requires informed consent from the patient after discussion with a physician taking into account the patient’s risk factors, age and life expectancy. The interval for follow-up of the screening depends on age and baseline PSA level.
1.2.5.2) Clinical Diagnosis
Initial diagnosis of prostate cancer involves recognition of abnormal digital rectal examination DRE and/or prostate-specific antigen (PSA) levels. Confirmative diagnosis is further done based on adenocarcinoma in prostate biopsy cores or specimens from TURP or prostatectomy for benign prostatic enlargement (BPE).
1.2.5.2.1) Digital rectal Examination (DRE)
As seen earlier, peripheral zone is the major site for development of PCa and can be screened by an abnormal digital rectal examination (DRE) (Figure 3). DRE alone was seen to screen PCa in 18% of the cases irrespective of the serum PSA level (Richie 1993). A patient with suspicious DRE and serum PSA level less than 2 ng/mL has a positive predictive value of 5-30%. Abnormal DRE also acts as an indication for biopsy (Okotie et al. 2007).
Figure 3: Clinician performing a DRE examination.
The image was released by the National Cancer Institute, NIH, USA.
1.2.5.2.2) Prostate Specific Antigen (PSA)
The most revolutionary discovery made for the screening of prostate cancer was the detection of PSA level in serum as a marker (Stamey et al. 1987). PSA is organ but not cancer-specific. Therefore, it may be elevated in benign prostatic hypertrophy (BPH), prostatitis and other non-malignant conditions. As an independent variable, PSA is a better predictor of cancer than DRE or trans rectal ultrasound (TRUS). PSA is a continuous parameter, with higher levels indicating greater likelihood of PCa. Several men may develop PCa despite low serum PSA (Catalona et al. 1994).
1.2.5.2.2.1) PSA density
PSA density is the level of serum PSA divided by the TRUS-determined prostate volume. Higher the PSA density, the more clinically significant is the PCa.
1.2.5.2.2.2) PSA velocity and doubling time
There are two methods for measuring a change in PSA: 1) PSA velocity (PSAV) which is calculated as the absolute annual increase in serum PSA (ng/mL/year). 2) PSA doubling time (PSA-DT) which measures the exponential increase in serum PSA over time.
PSAV and PSA-DT may have a prognostic role in treated PCa, but not useful for diagnosis because of background noise, different intervals between PSA screening, and acceleration/deceleration of PSAV and PSA-DT over time. These measurements do not provide additional information compared with PSA alone (Carter et al. 1992; Schmid et al. 1993).
1.2.5.2.2.3) Free/total PSA ratio
Free/total (f/t) PSA ratio is used to differentiate BPH from PCa. It stratifies the risk of PCa in men with 4-10 ng/mL total PSA and negative DRE. f/t PSA is not of clinical use if total serum PSA is higher than 10 ng/mL or during follow-up of known PCa. f/t PSA must be used cautiously because it may be adversely affected by several preanalytical and clinical factors (e.g., instability of free PSA at 4°C and room temperature, variable assay characteristics, and concomitant BPH in large prostates) (Stephen et al. 1997).
1.2.5.2.2.4) Prostate Health Index (PHI) test
The Prostate Health Index (PHI) test is a recently approved diagnostic blood test, combining free and total PSA and the PSA isoform (p2PSA). Studies have shown that PHI test has better prediction of clinically significant PCa, both in men with a PSA between 4-10 ng /mL and between 2-10 ng /mL compared to free and total PSA. The PHI test may therefore also have a role in monitoring men under active surveillance (Loeb et al. 2014). Its clinical impact is undetermined due to limited benefit in clinical decision-making (Fossati et al. 2014). However, PSA screening has raised controversial questions due to its harms that were identified as clinically relevant included over diagnosis (detection of cancers that if left untreated would not result in symptoms or death), false positives, and harms associated with biopsy. The quality of the evidence on harms of screening identified in the observational studies was assessed as very low. The results were reported descriptively using proportions (%) with 95% confidence intervals, since the data were primarily obtained from uncontrolled or modelling studies. Over diagnosis was estimated in modeling studies and was from 40.45% and 42% of men screened for screening every four years using a PSA threshold of 3 ng/m and was 54% with a threshold of 4ng/mL. An over diagnosis rate for screening every year with a PSA threshold of 4 ng/mL was estimated at 42% and 56%.
1.2.5.2.3) PCA3 marker
PCA3 (Prostate cancer antigen 3) earlier known as Differential Digital Code 3 (DD3), it is a prostate cancer specific marker. PCA3 long non-coding RNA is 66 fold over expressed in PCa compared to benign or normal prostatic tissue. PCA3 test is performed by detection of RNA is the urine sediments after three strokes of prostatic massage during DRE. The test kit is available with progensa and the PCA3 score is calculated as (PCA3 mRNA)/(PSA mRNA) × 1,000 (Hessels et al. 2003).
A meta-analysis of literature in 2014 showed that PCA3 score at first biopsy shows excellent value. Some studies showed that during the first biopsy, when a PCA3 cut-off score of 35 was used, the sensitivity and specificity were up to 82.3% and 89.0%, respectively, with little differences between these studies. The results were much better than those using PSA. The best PSA cut-off value showed only 57.4% and 53.8% sensitivity and specificity, respectively. Also when PCA3 cut-off score of 20 and 35 were compared it was seen that
specificity for cut-off was higher with a score of 35 but sensitivity was higher with a score of 20. 75% of the patients could be diagnosed with a cut-off score of 20 concluding that a PCA3 cut-off score of 20 is better than a cut-off score of 35 and that PCA3 is a much better diagnostic marker than PSA (Marks et al. 2007).
1.2.5.2.4) Prostate biopsy
1.2.5.2.4.1) Baseline biopsy
The need for prostate biopsy is based on PSA level and/or suspicious DRE. Before taking a biopsy age, potential comorbidity, and therapeutic consequences should be considered. Risk stratification is a potential tool for reducing biopsies (Roobol et al. 2010). Ultrasound-guided biopsy is now the most common procedure. A trans rectal approach is used for most prostate biopsies, although some urologists prefer a perineal approach. Cancer detection rates are comparable with both approaches (Hara et al. 2008).
Repeated biopsies in certain cases might be undertaken after a negative biopsy. Indications that suggest a repeat biopsy are: rising or persistently elevated PSA, suspicious DRE, presence of atypical small acinar proliferation, extensive high-grade prostatic intraepithelial neoplasia (HGPIN) or a few atypical glands near HGPIN. Isolated high-grade PIN in one or two biopsy is not considered as an indicator for repeat biopsy (Kronz et al. 2001).
1.2.5.2.5) Role of imaging
1.2.5.2.5.1) Trans rectal ultrasound (TRUS)
Trans rectal ultrasound is an important technique involved in diagnosis of PCa. Only 60% of tumours are visible with TRUS, and 40% are undetectable due to isoechogenicity. TRUS is not considered accurate for predicting localised disease when compared to DRE. Combined DRE and TRUS can detect T3a PCa more accurately than each method alone (Borlaza et al. 1985). 3D-TRUS is claimed to have better staging accuracy than 2D-TRUS. Greater sensitivity for cancer detection is achieved by the addition of coloured Doppler and contrast agents. All TRUS techniques are largely operator dependent and cannot differentiate between T2 and T3 tumours with sufficient accuracy (Yang et al. 2012).
1.2.5.2.5.2) Multi-parametric magnetic resonance imaging (MRI)
MRI is considered an efficient system for detection of local staging of PCa. At 1.5T (Tesla), MRI has low sensitivity for detecting extra prostatic extension of carcinoma (22-82%), but shows high specificity (61-100%). MRI accuracy for distinguishing T1/T2 stages from T3 stage is 50-85%. These disappointing results are because MRI cannot detect microscopic extra-prostatic extension. Its sensitivity increases with the radius of extension within peri-prostatic fat. MRI sensitivity, specificity and accuracy for detecting pT3 stage were, 40, 95 and 76%, respectively, for focal extra-prostatic extension, and 62, 95 and 88% for extensive extra-prostatic extension (Turbey et al. 2010).
Staging accuracy at 1.5T can be improved using an endorectal coil. Accuracy of detection was improved by 20 % on combining endorectal and external coils when compared to external coil alone. Dynamic contrast enhanced imaging combined with T2-WI (weighted imaging) may also improve local staging. The high field strength allows high resolution T2-WI and results at 3T seem better than at 1.5T, but vary based on the experience of individual image analyser. MRI accuracy at 3T varies between 67% and 93% depending on the experience of the analyser. Even if MRI is not perfect for local staging, it may improve prediction of the pathological stage when combined with clinical data.
Because of low sensitivity for focal extra-prostatic extension, mpMRI (Multi-parametric MRI) is not recommended for local staging in low-risk patients. However, mpMRI can still be useful for treatment planning in selected low-risk patients (e.g. candidates for brachytherapy). MRI can detect assess nodal invasion by measuring lymph node diameter but the sensitivity is very low. MRI is not preferred for nodal staging in low risk patients.
Diffusion-weighted whole-body and axial MRI are more sensitive than bone scanning and targeted radiography in detecting bone metastasis in high-risk PCa. Whole-body MRI is also more sensitive and specific than combined bone scan, targeted radiography and abdominopelvic CT. A recent meta-analysis found iron superoxide combined MRI to have higher sensitivity than choline PET/CT and bone scan for detecting bone metastasis, although PET/CT had the highest specificity.
1.2.5.2.5.3) Computerised tomography (CT scan)
Computed tomography (CT) scan (also known as a computed axial tomography scan, or CAT scan) is one of the most commonly used tools for the screening, diagnosis and treatment of cancer. A CT scan is an X-ray procedure that uses a computer to produce three-dimensional, cross-sectional images of inside the body. Unlike conventional X-rays, CT scans provide exceptionally detailed images of the bones, organs and tissues. A CT scan may be used to pinpoint the location of a tumour, evaluate the extent of cancer in the body, and assess whether the disease is responding to treatment. In some cases, CT technology is used to accurately guide cancer treatment during a procedure.
According to EUA guidelines abdominal CT can assess nodal invasion similar to MRI by measuring lymph node diameter. Using a 10-mm threshold, CT sensitivity is greater than 40%. Median estimated CT sensitivity, specificity, NPV and PPV were 40%, 80%, 85% and 80%, respectively. For CT or MRI, detection of microscopic lymph node invasion is < 1% in patients with a Gleason score < 8, PSA < 20 ng/mL, or localised disease. CT and MRI should not be used for nodal staging in low-risk patients and reserved for high-risk cancer.
1.2.5.2.5.4) Positron emission tomography (PET scan)
Positron emission tomography (PET) is a non-invasive technique used to follow cellular and molecular events in small animals as well as patients. Radiolabelled molecular probes (tracers) are injected into the patient and used to map out the underlying biochemistry. Both small-animal and clinical PET is being used to study cancer in living subjects. 2-18F-fluoro-2-deoxy-D-glucose (FDG) is actively taken up and accumulates in cancer cells. Many tracers already exist for PET that measure cell proliferation, bone remodelling, perfusion, oxygen metabolism, tumour-receptor density and reporter-gene expression. 11C- or 18F-choline positron emission tomography (PET)/CT has good specificity for lymph node metastasis, but sensitivity of 10-73%.
In a meta-analysis of 609 patients pooled sensitivity and specificity of choline PET/CT for pelvic lymph node metastasis were 62% and 92%, respectively. Currently, psmaPET-CT (prostate-specific membrane antigen-PET CT) remains experimental (Eyben et al. 2014).
Ultra-small particles of iron oxide (USPIOs), improves the detection of microscopic lymph node metastasis using MRI and shows high sensitivity, comparable to 11C-choline PET/CT. This approach is cost-effective, but is limited by a lack of availability (Choi et al. 2007). 18F-fluoride PET or PET/CT shows superior sensitivity to bone scanning. It remains unclear whether 11C-choline PET/CT is more sensitive than conventional bone scanning, but it has higher specificity, with fewer indeterminate lesions.
1.2.5.2.6) Lymphadenectomy
The gold standard for N-staging is open or laparoscopic lymphadenectomy. Pelvic lymph node dissection (LND) does not detect 50% of metastasis. When decisions are based on pelvic LND, extended lymphadenectomy should be considered. Removal of sentinel lymph nodes aims to improve the accuracy of detecting tumourous nodes while reducing morbidity associated with extended pelvic LND. Image guidance allows intra-operative sentinel node (SN) detection visually. Difficulty in accessing the SN and the lack of large multicenter cohorts are major limitations of this technique. Therefore, this technique is limited for clinical application (winter et al. 2008).
1.2.5.2.7) Bone scan
Bone scan (BS) has been the most widely used method for evaluating bone metastasis of PCa. However, it suffers from relatively low specificity. Thus, in patients with equivocal findings or a small number of hot spots, the metastatic nature of the lesions needs to be checked by other imaging modalities. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of planar BS were 70%, 57%, 64%, and 55%, respectively, and of SPECT (single photon emission computerised tomography) were 92%, 82%, 86%, and 90%, respectively (Tombal et al. 2012)
1.2.6) Treatment for Prostate cancer
1.2.6.1) Active surveillance / watchful waiting
During active surveillance the patient remains under surveillance and treatment is started based on pre-defined threshold of potential life threatening disease state. Where as watchful waiting involves waiting till local or systemic progression related symptoms appear. The patient is then treated with palliative treatment. As PCa progresses slowly and is diagnosed mainly in older men watchful waiting is often believed to to efficient. Watchful waiting is possible in patients with localised PCa and limited life expectancy, or older patients with less aggressive cancer (Welty et al. 2014).
Active surveillance mostly involves no treatment for patients at an age above 70 years, while in younger patients treatment may be delayed for several years. AS is mainly adapted to reduce overtreatment in patients with very-low-risk PCa. Active surveillance is only proposed for highly selected low-risk patients. Current data are from ongoing prospective or retrospective cohorts, without any available randomised clinical trials and the results of active surveillance (AS) are consistent throughout the published cohorts for survival.
During AS imaging with mpMRI, biological markers, include urine markers such as PCA3, the TMPRSS2: ERG fusion gene or PSA isoforms such as the Phi index act as important tools equally efficient as genomics on the tissue sample itself. However, further study data will be needed before such markers can be used in standard clinical practice.
1.2.6.2) Surgery
The surgical treatment of prostate cancer involves removal of the gland between the membranous urethra and bladder, and resection of both seminal vesicles, along with sufficient surrounding tissue to obtain a negative margin. This procedure is called the radical prostatectomy. This is usually a good treatment option for patients whose cancer has not yet spread outside the prostate (stages I and II). It can be achieved by either open surgery or laparoscopic surgery. In open surgery, the surgeon makes a small incision either in the perineum (perineal approach) or in the lower abdomen (retro pubic approach). The retropubic approach is the most common method for treating prostate cancer; however the recovery time is longer compared with the perineal approach.
In laparoscopic surgery, several small incisions are made in the abdomen and a laparoscope is inserted to allow the tumour to be viewed. The surgeon will then remove the prostate. Men undergoing laparoscopic surgery will lose less blood compared to open surgery and also have a shorter recovery time.
Radical prostatectomy is very effective in the treatment of early stage cancer. With the prostate removed and if the cancer has not spread, PSA levels can drop to zero. Radical prostatectomy reduces the rate of death from
PCa as well as a reduced risk of metastasis compared to the watchful waiting or active surveillance. However in some cases the tumour cannot be completely removed and disease can reoccur. Adverse effects of radical prostatectomy usually occur within 30 days of surgery and include erectile dysfunction and urinary incontinence. These effects can either be short (resolved within 90 days) or long term (continuing for up to 12 months after surgery). Additional general surgery risks exist such as blood clot; reactions to anaesthesia, blood loss and infection of the wound are also seen to be involved (Bill-Axelson et al. 2014).
1.2.6.3) Radiotherapy
The 2 main types of radiation therapy are used for prostate cancer: External beam radiation or Brachytherapy (internal radiation). The combination of radiation therapy with LHRH androgen deprivation therapy has been proved to have better response compared to radiation therapy alone.
Intensity-modulated radiotherapy (IMRT) with or without image-guided radiotherapy (IGRT) is the gold standard for external beam radiation therapy. Intensity modulated radiotherapy (IMRT) allows radiation to be adjusted around the target to protect adjacent organs.
Conformal radiotherapy (CRT) is an external beam radiotherapy technique were the high energy x-rays are carefully shaped to match the shape of the prostate gland, focussing only on the affected area and protecting surrounding tissue. Three-dimensional CRT (3D-CRT) uses special computers to precisely map the location of the prostate and improves local control.
Brachytherapy on the other hand with low dose rate (LDR) was identified to be safe and effective technique for patients with low risk of PCa. No randomised trials have compared brachytherapy with other curative treatment modalities.
Short-term adverse effects of radiotherapy include bowel disturbances and urinary symptoms such as irritative voiding, incontinence and urinary retention. Long-term erectile dysfunction can often occur for up to two years following surgery (Kieler et al. 2007).
1.2.6.4) Cryosurgery
Cryosurgery uses freezing techniques to induce cell death by dehydration resulting in protein denaturation, direct rupture of cellular membranes by ice crystals or vascular stasis and micro thrombi leading to apoptosis. The procedure is carried out with freezing the prostate by TRUS guided placement of 12-15 x 17 gauge cryo-needles, thermosensors at the level of the external sphincter and bladder neck, and insertion of a urethral warmer. Two freeze-thaw cycles are used under TRUS guidance, resulting in a temperature of -40°C in the mid-gland and at the neurovascular bundle. Currently, the third generation cryosurgery devices are mainly used. According to a recent meta-analysis of 566 cryosurgery related publications, there were no controlled
trials, survival data or validated biochemical surrogate end-points available for analysis. Cryosurgery showed progression-free survival (PFS) of 36-92%, depending on risk groups and the definition of failure. Erectile dysfunction occurs in about 80% of patients. The complication rates described in third-generation cryosurgery include tissue sloughing in about 3%, incontinence in 4.4%, pelvic pain in 1.4% and urinary retention in about 2% (Long J.P. et al. 2001).
1.2.6.5) High intensity focused ultrasound of the prostate (HIFU)
HIFU consists of focused ultrasound waves, emitted from a transducer, that cause tissue damage by mechanical and thermal effects as well as by cavitation. The goal of HIFU is to heat malignant tissues above 65°C so that they are destroyed by coagulative necrosis. The procedure is performed under spinal anaesthesia (Madersbacher et al. 2003).
1.2.6.6) Focal therapy of prostate cancer
The main aim of focal therapy is to limit treatment toxicity in patients that could benefit from local disease control. It is achieved with ablative technologies: cryotherapy, HIFU or photodynamic therapy, electroporation, focal radiotherapy by brachytherapy, or CyberKnife Robotic Radiosurgery System technology. Focal therapy should be limited to patients with a low to moderate risk in investigational settings, retrospective data have shown the presence of grade I-III toxicity in 13% of case. Focal therapy lacks data on functional and oncological outcomes (Barret et al. 2013).
1.2.6.7) Hormonal therapy
Hormone therapy involves suppression of secretion of testicular androgens, or inhibiting the circulating androgens at the level that their receptor uses competing compounds (anti-androgens). These two methods can also be combined to achieve complete androgen blockade (CAB) (Pagliarulo et al. 2012).
1.2.6.7.1) Castration
Surgical castration is still considered the ‘gold standard’ for ADT. It leads to a considerable decline in testosterone levels and the patient attains a hyogonadal state, known as the castrated level. The standard castrate level was less than 50 ng/dL (1.7 nmol/L). Although this was defined more than 40 years ago, when testosterone level testing was limited. Current testing methods have found that the mean value of testosterone after surgical castration to be 15 ng/dL. This new definition is important as better results are repeatedly observed with levels around or below 1 nmol/l compared to 1.7 nmol/L. However, the castrate level considered by the regulatory authorities is still 50 ng/dL (1.7 mmol/L), which is also the threshold that has been used in clinical trials addressing castration in PCa patients (Pickels et al. 2012).
