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THE RELATIONSHIP OF HUMAN CHORIONIC GONADOTROPIN BETA (hCGβ) EXPRESSION AND LUTEINIZING HORMONE GENE MUTATION (vLH) WITH PROSTATE CANCER IN POPULATION GROUPS OF MULTI-ETHNIC ORIGIN Ali Thwaini Queen Mary School of Medicine and Dentistry, Academic Department of Urology, St Bartholomew’s and the Royal London Hospitals & Department of Biomedical Sciences, Middlesex University A THESIS SUBMITTED TO THE UNIVERSITY OF LONDON FOR THE DEGREE OF “DOCTOR OF MEDICINE” 2007 1 DECLARATION I declare that this thesis has been composed and written entirely by myself, and the work contained herein to have been principally conducted by myself. Ali Thwaini I declare that the conditions of ordinance and regulations (M.D.) have been fulfilled. Mr. Frank Chinegwundoh Supervisor 2 ABSTRACT Data have shown that Black men have the highest incidence and mortality from prostate cancer in the world. Even after adjusting for stage at diagnosis, black men have higher mortality rates than white men (Freedland, 2005). One of the postulated theories is the elevated androgen level in African-Caribbean men (Mohler, 2004, Abdelrahaman, 2005). In addition, it has been shown that the prevalence of prostatic carcinoma in the United Arab Emirates (UAE), like other Arabian Gulf and Asian countries, is very low compared to Western countries (Ghafoor, 2003). The beta subunit of the human chorionic gonadotropin (hCG) has been associated with aggressive cancers, including prostate cancer (Rao, 2004; Weissbach, 1999; Dirnhofer, 1998; Dirnhofer, 2000; Noeman, 1994; Crawford et al., 1998). It has been suggested that it may act as an autocrine growth factor by inhibiting apoptosis, mediated by inhibition of the transforming growth factor (TGF) receptor complex (Iles, 2007). 3 A common genetic variant of Luteinizing Hormone (LH) has been identified. The prevalence of this variant LH (vLH) allele has been estimated worldwide as a carrier frequency of 0% to 53% of Caucasians and is known to be more active than the wild-type protein (Elkins, 2003). In addition to its well-known physiological role in androgen metabolism, its role in prostate tissue is possibly through the conversion of testosterone into dihydrotestosterone (DHT), thus potentiating the androgen effect of prostatic tissue (Douglas, 2005). Archival prostate tissue samples –including benign prostate hyperplasia (BPH) and prostate cancer- were obtained from Caucasians (n=118, UK), AfricanCaribbean men (n=92, UK) and men from the UAE (Middle Eastern Caucasoids) (n=148, UAE). Of those with prostate cancer, seventy-one had organ-confined disease, thirty had locally advanced disease and seventeen had metastatic disease at presentation. hCG and vLH were studied using Immunohistochemistry (IHC) for the former and Nested Polymerase Chain Reaction (PCR) for the latter. It was found, using Kruskal-Wallis analysis, that the expression of hCG in the African-Caribbean group was significantly higher than the Caucasians and the Middle East group (African-Caribbean group versus Middle East: P = 0.0009, African-Caribbean group versus Caucasians: P = 0.0082, Middle East versus 4 Caucasians: P = 0.653). However, there was no significant association between the positive expression of hCG and survival (P = 0.8881). Regarding the vLH, it was found that the presence of the vLH in the three ethnic groups is extremely rare (0.011%) in BPH and prostate cancer patients. These findings show that there is a significant association of the hCG expression with the African-Caribbean men; a group known to have a higher baseline testosterone levels (Mohler, 2004; Abdelrahaman, 2005). This may be mediated through a possible role of hCG in cross-interaction with TGF beta receptor, leading to inhibition of apoptosis (Iles, 2007), or with LH beta receptor, increasing the conversion of testosterone into its active metabolite; dihydrotestosterone with ultimate increase in the risk of development of prostate cancer (Douglas, 2005). 5 ACKNOWLEDGEMENTS I would like to express my sincere gratitude to my supervisor Mr. Frank Chinegwundoh for giving me the opportunity to work on this project. His encouragement and support were pivotal to completing this project. I would also like to thank Dr. Baithun for his guidance and brilliant ideas for the research material. I am indebted to Prof. Ray Iles for his kindness, support and advice at all stages of my research, including his invaluable advice on the statistical analysis and writing up my thesis. Special thanks to Dr. Mahmoud Naase for his day-to-day supervision, help and guidance, without which, I would not have been able to accomplish this task. Many thanks to the laboratory staff at the Department of Biomedical Sciences, School of Health and Social Sciences, Middlesex University, for all their help and advice, and for making it such an enjoyable place to work, particularly to Dr. Lucy Ghali, Song, Ella, Peter and Myrl. 6 DEDICATION This thesis is dedicated to my parents, my wife Suhair and my sons Hassan and Mustafa. 7 TABLE OF CONTENTS PAGE TITLE 1 ABSTRACT 3 ACKNOWLEDGEMENTS 6 DEDI CATION 7 TABLE OF CONTENTS 8 LIST OF FIGURES 15 LIST OF TABLES 18 ABBREVIATIONS 19 PUBLISHED WORK AND PRESENTATIONS 21 CHAPTER 1 INTRODUCTION 23 1.1 Prostate cancer epidemiology: 24 1.2 Risk factors: 25 1.3 Pathology of prostate cancer: 28 1.3.1 Adenocarcinoma of the prostate: 29 1.3.1.1 Tumour grade 29 1.3.1.2 Spread of tumour 32 Prostate specific antigen 33 1.4 1.4.1 PSA and cancer screening 34 1.5 Clinical presentation 34 8 1.5.1 Localized prostate cancer 34 1.5.2 Locally advanced cancer 35 1.5.3 Metastatic disease 35 1.6 Staging of prostate cancer 35 1.7 Investigations for prostate cancer 37 1.7.1 TRUS 37 1.7.2 CT and MRI of prostate 38 1.7.3 Isotope bone scan 38 1.8 Management of prostate cancer 38 1.8.1 Localized prostate cancer 38 1.8.1.1 Active surveillance 38 1.8.1.2 Radical prostatectomy 39 1.8.1.3 Radical external beam radiotherapy and brachytherapy 39 1.8.1.4 Cryotherapy and high frequency focused ultracound 39 1.8.2 Management of locally advanced prostate cancer 40 1.8.2.1 External beam radiotherapy 40 1.8.2.2 Hormone therapy 40 1.8.2.3 Active surveillance 41 1.8.3 Management of advanced prostate cancer 9 41 1.9 Gonadotropins as tumour markers 41 1.9.1 Gonatotropin receptors 43 1.9.2 Gonadotropins and prostate cancer 45 1.9.3 Human Chorionic Gonadotropin (hCG): 45 1.9.3.1 Metabolism and excretion of hCG 46 1.9.3.2 Genetic encoding of hCG 46 1.9.3.3 hCGß and cancer 46 1.9.3.4 hCGß and prostate cancer 47 1.9.4 Luteinizing Hormone (LH): 48 1.9.4.1 Physiology of action 49 1.9.4.2 Genetic encoding of LH and its variant 50 1.9.4.3 vLH bioactivity 51 1.9.4.4 Luteinizing Hormone and Prostate cancer 52 1.10 HYPOTHESIS 54 1.11 AIM OF THESIS 56 CHAPTER II 57 PATIENTS DEMOGRAPHICS AND EQUIPMENT 2.1 Introduction 58 2.2 Case selection 58 2.3 The study (prostate cancer) population 60 2.4 The Controls population 62 10 2.5 Specimen collection and storage 64 2.6 Materials and equipment 65 2.7 Statistical analysis 67 2.8 Results 68 2.8.1 Age at presentation in the three groups 68 2.8.2 PSA in the cancer and control populations 70 2.8.3 Progression-free survival and Gleason scoring in the 72 three groups 2.9 Discussion 77 CHAPTER III 80 IMMUNOIHISTOCHEMICAL DETECTION OF THE hCG EXPRESSION IN PROSTATE CANCER AND CONTROLS IN THE THREE GROUPS 3.1 Introduction 81 3.2 Patients 82 3.3 Materials 83 3.3.1 Primary Antibody 83 3.3.2 Secondary antibody 83 3.3.3 Enzyme labelling 83 3.3.4 Detection Reagent 83 3.4 Methods 83 11 3.4.1 Deparaffinization 84 3.4.2 Peroxidase blocking 84 3.4.3 Epitope retrieval 84 3.4.4 Serum blocking 85 3.4.5 Primary antibody 85 3.4.6 Secondary antibody 85 3.4.7 Enzyme labelling 85 3.4.8 Homogen/Substrate 86 3.4.9 Controls 86 3.4.10 Scoring and evaluation of results 86 3.5 Results 93 3.5.1 hCG expression in the three groups 93 3.5.2 hCG expression in the controls of the three groups 95 3.5.3 hCG expression in the study cases (prostate cancer) 97 of the three groups. 3.5.4 PSA distribution in the three groups in relation to 101 hCGβ status 3.5.5 The relation between the progression-free survival 106 and hCG in all the ethnic groups 3.6 Discussion 108 12 CHAPTER IV 111 LH-BETA GENE ISOLATION 4.1 DNA extraction from paraffin-embedded prostate tissues 112 4.1.1 112 Introduction 4.1.1.1 Reagents 112 4.1.1.2 Preparation 113 4.1.1.3 Methods 113 4.2 PCR 115 4.2.1 Introduction 115 4.2.1.1 117 nested PCR 4.2.2 PCR using LH long primers 121 4.2.3 Methods 122 4.3 DNA extraction and purification from Agarose gel 124 4.4 PCR LH Short Primers (LH-SP) 125 4.4.1 Materials 125 4.4.2 Methods 126 4.5 DNA sequencing 128 4.5.1 Introduction 128 4.5.2 Methods 129 4.6 Results 131 4.7 Discussion 132 13 CHAPTER V GENERAL DISCUSSION 136 LIMITATIONS OF THE STUDY 142 SUMMARY AND CONCLUSIONS 143 RECOMMENDATIONS 144 REFERENCES 145 APPENDICES 164 14 LIST OF FIGURES Fig 1.1 Age standardised (world) incidence and mortality rates, prostate 25 cancer in selected countries, 2002 estimates. Fig 2.1 Age distribution (cases) at presentation in the three groups 68 Fig 2.2 Age distribution (controls) at presentation in the three groups 69 Fig 2.3 PSA distribution in the three ethnic groups 72 Fig 2.4 Gleason scoring in the three ethnic groups 73 Fig 2.5 Progression-free survival, Gleason score (6) in the three groups 75 Fig 2.6 Progression-free survival, Gleason score (7) in the three groups 76 Fig 2.7 Progression-free survival, Gleason score (8-10) in the three groups 76 Fig 3.1 Immunostaining of placenta tissue for hCG beta as positive control 88 Fig 3.2 Immunostaining of placenta tissue for hCG beta as positive control 88 Fig 3.3 Negative control of prostate using Immunostaining. 89 Fig 3.4 Negative control of prostate using immunostaining. 89 Fig 3.5 H&E staining of prostate cancer showing hCG beta positive (I) 90 Fig 3.6 hCGβ staining of prostate cancer showing hCG beta negative (II) 90 Fig 3.7 H&E staining of prostate cancer showing hCG beta positive (II) 91 Fig 3.8 hCGβ staining of prostate cancer showing hCG beta positive (II) 91 Fig 3.9 H&E staining of prostate tissue showing hCG beta positive (III) 92 Fig 3.10 hCGβ staining of prostate tissue showing hCG beta positive (III) 92 Fig 3.11 hCG expression in the three groups 94 Fig 3.12 Scoring distribution of hCGβ+ cells in the three ethnic groups 95 15 Fig 3.13 hCG expression in the controls of the three groups 96 Fig 3.14 Scoring distribution of the hCGβ+ cells in controls in the 97 three ethnic groups Fig 3.15 hCG expression in the study cases (prostate cancer) of the 98 three groups Fig 3.16 Scoring distribution of the hCGβ+ cells in cancer cases 99 in the three ethnic groups. Fig 3.17 Scoring distribution in relation to Gleason system 100 Fig 3.18 PSA distribution in hCG positive and negative cases in the 101 three ethnic groups. Fig 3.19 PSA in hCGβ positive and negative cancer cases in Group I 102 Fig 3.20 PSA in hCG beta positive controls in Group I 102 Fig 3.21 PSA in hCG beta positive cancer cases in Group II 103 Fig 3.22 PSA in hCG beta positive controls in Group II 104 Fig 3.23 PSA in hCG beta positive controls in Group III 105 Fig 3.24 PSA in hCG beta positive cancer cases in Group III 105 Fig 3.25 Progression-free survival between the hCG positive vs. 106 hCG negative patients. Fig 3.26 Progression-free survival in relation to the immunochemistry 107 grading system used in this study. Fig 4.1 Gel electrophoresis of the first PCR (LH-LP) 123 Fig 4.2 Gel electrophoresis of the second PCR (LH-SP) 127 16 Fig 4.3 Diagram showing DNA sequencing using Capillary Array 129 Electrophoresis Fig 4.4 vLH and Wild LH sequence 130 Fig 4.5 The prevalence of vLH in the three study groups 131 Fig 4.6 The prevalence of vLH in the three study groups 132 17 LIST OF TABLES Table 1.1 TNM staging of prostate cancer 36 Table 2.1 Age distribution of Study (prostate cancer) and 61 Control population. Table 2.2 Clinical tumour stage of patients with prostate cancer 63 Table 2.3 PSA distribution in the three groups 70 Table 3.1 The distribution of the hCGβ cases and controls among the 93 three ethnic groups. LIST OF DIAGRAMS Diagram 1.1 Gonadotropins and their receptors 44 Diagram 2.1 Consort diagram showing the steps involved in our study 59 Diagram 4.1 Nested PCR 119 18 ABBREVIATIONS BMI body mass index BPH benign prostate hyperplasia DHT dihydrotestosterone DHEAS dehydroepiandrosterone sulphate DRE digital rectal examination EAU European Association of Urology guidelines FSH follicle stimulating hormone GPH glycoprotein-hormones GnRH gonadotropin-releasing hormone hCGβ human chorionic gonadotropin beta hCG-cf hCG core fragment H&E Haematoxylin-Eosin HIFU high frequency focused ultracound IHC immunohistochemistry LH luteinizing hormone LH-LP luteinizing hormone long primers LHR luteinizing hormone receptors LHRH luteinizing hormone-releasing hormone LH-SP luteinizing hormone short primers LUTS lower urinary tract symptoms PBS phosphate-buffered saline PCR polymerase chain reaction 19 PSA prostate specific antigen RP radical prostatectomy SEER Surveillance, Epidemiology, and End Results SHBG sex hormone-binding globulin TCC transitional cell carcinoma TRUS trans rectal ultrasound TSH thyroid stimulating hormone TURP transurethral resection of prostate UAE United Arab Emirates vLH luteinizing hormone genetic variant wtLH wild-type of luteinizing hormone 20 PUBLISHED WORK AND PRESENTATIONS 1. The incidence of Luteinizing Hormone genetic mutation (vLH) and prostate cancer. Pathological Society of Great Britain & Ireland 189th meeting. Cambridge. Jan 2006. 2. The relation between Luteinizing Hormone genetic mutation (vLH), Human Chorionic Gonadotropin Beta (hCGβ) expression and ethnicity in prostate cancer patients. Urology Research Society Annual Meeting, London. Jan 2006. 3. Gonadotropins and Prostate Cancer- revisited. Thwaini A, Naase M, Chinegwundoh F, Baithun S, Ghali L, Shergill I, Iles R. Urol Int. 2006;77(4):28996. Published. 4. Gleason scoring, and ethnicity: its prognostic significance in prostate cancer patients of multi-ethnic origins. British Prostate Group Autumn Meeting, Cardiff. Sept 2006. 5. The genetic variant of Luteinizing Hormone, prostate cancer and ethnicity; the true story. 82nd Congress of the Western Section of the American Urology Association. Oct 2006. 6. hCG beta as a prognostic marker in prostate cancer in multi-ethnic population.” 82nd Congress of the Western Section of the American Urology Association. Oct 2006. 21 7. Does the genetic variant of Luteinizing hormone have any role in the incidence and prognosis of prostate cancer? The 28th Congress of the Société Internationale d'Urologie, Cape Town. Nov 2006. 8. hCG beta as a prognostic marker in adenocarcinoma of the prostate in men of multi-ethnic origins.The 28th Congress of the Société Internationale d'Urologie, Cape Town. Nov 2006. 22 CHAPTER 1 INTRODUCTION 23 1.1 Prostate cancer epidemiology: Prostate cancer accounts for around 13% of male deaths from cancer in the UK and is the second most common cause of cancer death in men after lung cancer. There are around 24,700 new cases per year, and with approximately 9,900 deaths per year (www.cancerresearchuk.org). Survival from prostate cancer has greatly improved since the 1970s, and rates now stand at 84%, 60% and 28% at one, five and ten years respectively. Part of the reason for the improved survival is the huge increase in patients presenting with early stage over the past 10 years, resulting from prostate-specific antigen (PSA) testing and increased health education (www.cancerresearchuk.org). The highest prostate cancer incidence rates are in the developed world and the lowest rates in Africa and Asia (Parkin, 2002). The extremely high rate in the USA (125 per 100,000), more than twice the reported rate in the UK (52 per 100,000), is likely to be due to the particularly high rates of PSA testing in the USA (Gann, 2002). Figure 1.1 shows the incidence and mortality rates in a number of countries in the world. 24 USA Sweden Canada Switzerland Australia France Netherlands Brazil UK Italy Denmark Zimbabwe Singapore Japan India China Incidence Mortality 0 20 40 60 80 100 120 140 Rate per 100,000 males Figure 1.1: Age standardised (world) incidence and mortality rates, prostate cancer in selected countries, 2002 estimates (www.cancerresearchuk.org) 1.2 Risk factors: Age is one of the most important factors in developing prostate cancer. The disease is very rare below the age of 40 years, but becomes increasingly common with increasing age. However, most prostate cancer does not achieve a clinically recognizable and aggressive state. Increasing age is associated with a higher risk of high grade prostate cancer in men of different ethnic origins (Reed et al.,2007) 25 Geographic variation: the incidence of prostate cancer is 10 times higher in the western world than in Asian countries (www.ncic.cancer.ca). This might be explained by genetic factors. However, the incidence of prostate cancer also tends to increase in Asian immigrants to western countries, mainly after one generation. This may suggest the involvement of environmental factors as well (Nasseri, 2007). Ethnicity: Black men have the highest incidence and mortality from prostate cancer in the world. In the UK the incidence of prostate cancer is approximately treble in men of African origin than in white men, and lowest in Asian and Oriental men, suggesting a strong ethnic influence. (Chinegwundoh, 2003; BenShlomo, 2008) Even after adjusting for stage at diagnosis, black men have higher mortality rates than white men. Multiple reasons have been postulated to explain these findings including access to care, attitudes about care, socio-economic and education differences (Shavers, 2004; Oakley-Girvan, 2004; Meyer, 2007), differences in type and aggressiveness of treatment, dietary, and genetic differences. While each reason may contribute to the higher incidence or higher mortality, likely combination of reasons will best explain all the findings. Also, with recent advances in the understanding of genetic variation in the human genome, in general, and in the genes involved in pathways relevant to prostate cancer biology, in particular, a number of genes with alleles which differ in frequency 26 between black and white men have been proposed as a genetic cause or contributor to the increased prostate cancer risk in black men. However, the clinical significance of these genetic differences is not fully known. It has been shown that the prevalence of prostate carcinoma in the UAE, like other Arabian Gulf and Asian countries, is very low compared to Western Countries despite the high intake of calories and consumption of animal fat. These are considered as contributing factors to the increased risk of development of prostate cancer (Segev, 2006). However, interaction of genetic and environmental factors believed to be involved in the complex aetiology of prostate cancer in UAE and else where await further investigations. Family history: It has been shown that there is a strong association between early onset prostate cancer and positive family history of the disease. This is mainly governed by the number of relatives with prostate cancer and their age at diagnosis. Dominant pattern of inheritance with high penetrance is responsible for 5% to 10% of all prostate cancer cases, and as much as 30% to 40% of early onset disease (Bratt, 2002). Chronic inflammation: There has been an increasing evidence of the association of chronic inflammation of the prostate, with the development of prostate cancer (Wagenlehner, 2007). 27 Hormones: Being a hormone dependant cancer, one of the postulated theories was the elevated androgen level in African-Caribbean men. While reports of racial differences in gonadal steroid hormone levels in middle-aged men have produced conflicting results, there is evidence that high sex hormone-binding globulin (SHBG) and androstenedione levels are more common among young adult African American men than white men (Mohler, 2004; Abdelrahaman, 2005). Furthermore, it has been proved that the concentrations of androstenedione, testosterone, and progesterone were notably higher in African-American compared with Caucasian or Hispanic women. These data are consistent with hypotheses that in-utero hormonal exposures may explain some of the ethnic group differences in cancer risk (Potischman, 2005). Furthermore, it has been shown that the androgen levels in the Arab population are significantly lower compared with Caucasian population (Kehinde, 2006). 1.3 Pathology of prostate cancer: The most common primary prostatic malignancy is the adenocarcinoma of the ductal or acinar epithelium. The basal layer is normally absent and thereafter there is infiltration into the prostatic stroma. Macroscopically, the gland tends to be hard and white, though a soft mucin-producing variety exists. Less commonly, the transitional cell carcinoma (TCC) might primarily present in the prostatic urethra, or the prostate may be invaded by a primary TCC of the bladder. 28 Rhabdomyosarcoma of the prostate is rare but may be seen in childhood. Secondary deposits (metastases) from other sites are also rare. 1.3.1 Adenocarcinoma of the prostate: About 75% of adenocarcinomas occur in the peripheral zone of the prostate and most (85%) are multifocal. 20% appear to arise from the transition zones and 5% from the embryologically distinct central zone. 1.3.1.1 Tumour grade: Although numerous grading systems exist for the evaluation of prostatic adenocarcinoma, the Gleason grading system is the most widely accepted. The Gleason system is based on the glandular pattern of the tumour as identified at relatively low magnification. Cytological features play no role in the grade of the tumour. Both the primary (predominant) and the secondary (second most prevalent) architectural patterns are identified and assigned a grade from 1 to 5, with 1 being the most differentiated and 5 being the least differentiated. When Gleason compared his grading system with survival rates, it was noted that in tumours with two distinct tumour patterns, the observed number of deaths generally fell between the number expected on the basis of the primary pattern and that based on the secondary pattern. Because both the primary and the secondary patterns were influential in predicting prognosis, there resulted a Gleason sum obtained by the addition of the primary and secondary grades. If a tumour had only one histological pattern, then for uniformity the primary and 29 secondary patterns were given the same grade. Gleason sums range from 2 (1 + 1=2), which represents tumours uniformly composed of Gleason pattern 1 tumour, to 10 (5 + 5=10), which represents totally undifferentiated tumours. Synonyms for Gleason sum are combined Gleason grade and Gleason score. Pathologists may assign only a Gleason pattern rather than a Gleason sum in cases with limited adenocarcinoma of the prostate on needle biopsy. However, it should be made perfectly clear by the pathologist that she or he is assigning only a pattern and not a sum. Cases in which the pathologist signs out a case as Gleason grade 4 (i.e., Gleason pattern 4) may be misconstrued as Gleason sum 4. Consequently, the author prefers to assign both a primary and a secondary pattern even when presented with limited cancer so as to not give rise to any confusion. The Gleason system does not account for the existence of a tertiary (third most prevalent) pattern. In radical prostatectomy specimens, it has been demonstrated that tertiary high-grade components adversely affect biologic behaviour. It is proposed that the Gleason system for radical prostatectomy specimens be modified, either by recording the Gleason grade as a combination of the most common pattern along with the highest grade pattern or by giving the routine Gleason grade (primary and secondary patterns) with a note stating that there is a tertiary high-grade pattern (Gleason et al., 1974). Gleason pattern 1 and pattern 2 tumours are composed of relatively circumscribed nodules of uniform, single, separate, closely packed, mediumsized glands. Gleason pattern 3 tumour infiltrates the non-neoplastic prostate, and the glands have marked variation in size and shape, with smaller glands 30 than seen in Gleason pattern 1 or 2. Gleason pattern 4 glands are no longer single and separate as seen in patterns 1 to 3. In Gleason pattern 4, one may also see large, irregular, cribriform glands as opposed to the smoothly circumscribed smaller nodules of cribriform Gleason pattern 3. Tumours with this pattern have a significantly worse prognosis than those with pure Gleason pattern 3. It has been demonstrated in radical prostatectomy specimens that tumours with Gleason score 4 + 3=7 have a worse prognosis than those with Gleason score 3 + 4=7. Gleason pattern 5 tumour shows no glandular differentiation and is composed of solid sheets, cords, single cells, or solid nests of tumour with central comedonecrosis (Chan et al., 2000). The Gleason grade on biopsy material has also been shown to correlate fairly well with that of subsequent prostatectomy. One of the most frequent causes of discordant grading is grading of tumours that straddle two grades. Undergrading of needle biopsy is more of a problem than overgrading and is unavoidable to some extent owing to sampling error. Gleason scoring can be improved by not assigning Gleason scores 2 to 4 for adenocarcinoma of the prostate on needle biopsy. The reasons for doing this are as follows: (1) The vast majority of tumours graded as Gleason score 2 to 4 on needle biopsy are graded as Gleason score 5 to 6 or higher when reviewed by experts in urologic pathology. In a study of men visiting The Johns Hopkins Hospital for radical prostatectomy, an outside needle biopsy grade of Gleason score 2 to 4 was matched in only 4 of 87 cases; (2) there is poor reproducibility in the diagnosis of Gleason score 2 to 4 on needle biopsy even among urologic pathology experts; and (3) most importantly, assigning Gleason 31 score 2 to 4 to adenocarcinoma on needle biopsies can adversely affect patient care because clinicians may assume that low-grade cancers on needle biopsy do not need definitive therapy. Of 87 needle biopsies with cancers graded as Gleason score 2 to 4 by outside institutions, 48 (55%) showed extraprostatic extension at radical prostatectomy, including four cases with invasion of either seminal vesicles or lymph nodes. Although incidentally found low-volume, Gleason score 2 to 4 adenocarcinoma of the prostate on trans urethral resection of prostate (TURP) has a relatively indolent course (T1a, T1b), low-grade cancer on needle biopsy does not (Steinberg et al., 1997). The ultimate value of any grading system is its prognostic ability. Both Gleason's data with 2911 patients and subsequent studies with long-term follow-up have demonstrated a good correlation between Gleason sum and prognosis. When stage of disease is factored in with grade, prognostication is enhanced. The Gleason grade of tumour at radical prostatectomy and the preoperative Gleason grade also correlate with final pathologic stage. 1.3.1.2 Spread of the tumour: The tumour spreads locally through the poorly formed prostatic capsule into surrounding tissue, at which time it is termed ‘locally advanced’. Hence, the disease may involve the urethral sphincter, corpora of the penis, seminal vesicles, and trigone of the bladder including the distal ureters. Local spread is often along the course of autonomic nerves (perineural invasion). The most 32 frequent sites of metastasis are lymph nodes and bone, although lung, liver, testis, and brain are not uncommon. Bone metastases are characteristically sclerotic, rarely lytic. The axial skeleton (spine and pelvis) are most commonly affected, followed by the proximal long bones, ribs, clavicles, and the skull. 1.4 Prostate specific antigen (PSA) PSA is a serine protease enzyme with a molecular weight of 34 kilo Daltons. It is produced by prostatic epithelial cells. Its function is to liquefy the ejaculate, enabling fertilization. Large amounts are secreted into the semen, and small quantities are found in the urine and blood. Seventy-five percent of circulating PSA is bound to plasma proteins (complexed PSA) and metabolized in the liver, while 25% is free and excreted in the urine. Complexed PSA is stable, bound to alpha-1 antichymotrypsin and alpha-2 macroglobulin. The half-life of serum PSA is 2.2 days. The normal range for the serum PSA assay in men is <4.0ng/ml, though this varies with age. In the absence of prostate cancer, serum PSA concentrations also vary physiologically, according to prostate volume and race. In an epidemiological study it was found that the mean values of total PSA were 0.87 for men 40-49 years, 1.36 for men 50-59 years, 1.81 for men 60-69 years, 2.32 for men 70-79 years and 2.36 for men 80-89 years in Saudi population, indicating a lower references range of the normal PSA values in the Middle East population (Kamal, 2003). 33 1.4.1 PSA and prostate cancer screening Screening for prostate cancer is normally carried out in men above the age of 50 years with PSA and DRE. However, until now there is no screening programme for the prostate cancer in the UK. Supporters of screening rely on the fact of acceptable and relatively inexpensive tests, which may detect clinically significant disease and offer a better therapeutic potential. However, opponents argue that many men would suffer unnecessary anxiety, especially with the fact of incompletely understood natural history of the disease. (www.cancerscreening.nhs.uk). 1.5 Clinical presentation 1.5.1 Localized prostate cancer (T1-2) Most patients are asymptomatic and are incidentally found to have the disease during screening with PSA or digital rectal examination (DRE). Some patients may present with lower urinary tract symptoms (LUTS) secondary to a coexisting benign hyperplasia of the prostate. 34 1.5.2 Locally advanced cancer (T3-4) Men could still be asymptomatic and discovered during routine checks. However, they may also present with LUTS, haematospermia, and renal failure due to ureteric obstruction. 1.5.3 Metastatic disease (N+, M+) In addition to the above symptoms men would present with bone pain, pathological fracture, anorexia, weight loss, anaemia, lower limbs swelling secondary to lymphatic obstruction. 1.6 Staging of prostate cancer: Prostate cancer is staged using the TNM system. This widely used system has been produced by the American Joint Committee on Cancer. It separately assesses the tumour (T), lymph nodes (N) and secondary cancer or metastases (M), according to table 1.1 (James, 1994). 35 TNM staging of adenocarcinoma of the prostate T0 No tumour (pT0 if no cancer found by histological examination) Tx T stage uncertain T1a Cancer non-palpable on digital rectal examination (DRE), present in <5% of TURP specimens (in up to 18% of TURPs) T1b Cancer non-palpable on DRE, present in >5% of TURP specimens T1c Cancer non-palpable on DRE, present in needle biopsy taken because of elevated PSA T2a Palpable tumour, feels confined, in <half of one lobe on DRE T2b Palpable tumour, feels confined, in >half of one lobe on DRE T2c Palpable tumour, feels confined, in both lobes on DRE T3a Palpable tumour, locally advanced into periprostatic fat, uni- or bi-lateral and mobile on DRE T3b Palpable tumour, locally advanced into seminal vesicle(s) on DRE T4a Palpable tumour, locally advanced into adjacent structures, feels fixed on DRE T4b Palpable tumour, locally advanced into pelvic side-wall, feels fixed on DRE Nx Regional lymph not assessed N0 No regional lymph node metastasis N1 Tumour involves regional (pelvic) lymph nodes Mx Distant metastases not assessed M0 No distant metastasis M1a Non-regional lymph node metastasis M1b Tumour metastasis in bone M1c Tumour metastasis in other sites Table 1.1 TNM staging of prostate cancer (James, 1994) 36 1.7 Investigations for prostate cancer: 1.7.1 Transrectal ultrasound guided prostate biopsy (TRUS biopsy): TRUS biopsy of the prostate is performed when there is an abnormal DRE and/or an elevated PSA, in addition to men with previous biopsies showing isolated prostatic intraepithelial neoplasia (PIN) or atypical small acinar proliferation (ASAP). It is also indicated in men with previous normal biopsies, but rising PSA or abnormal DRE. In less common conditions, it is used to confirm viable prostate cancer following treatment if further treatment is being considered. In general, adverse findings on needle biopsy accurately predict adverse findings in the radical prostatectomy specimen, whereas favourable findings on needle biopsy do not necessarily predict favourable findings in the radical prostatectomy specimen. For example, in terms of grade, a Gleason score of 5 to 6 on biopsy corresponds to the same grade in the radical prostatectomy specimen in only 64% of cases. When the Gleason score is greater than 7 on biopsy, the radical prostatectomy grade is the same in 88% of cases. Similarly, with extent of tumour on biopsy, when there is only one positive core, as many as 40% of the tumours may show extraprostatic extension (Badalament et al., 1996). Even when less than 3 mm of cancer is present on one core and the cancer is not high grade (Gleason score of less than 7), 21% of the radical prostatectomy specimens still have moderate or advanced tumour (Epstein et al., 1994). 37 1.7.2 CT scan and MRI scan: Both CT and MRI are now of a high technical standard, but neither modality is sufficiently reliable to make it mandatory to use them to assess local tumour invasion. MRI of the prostate appears to be the most accurate non-invasive method of identifying locally advanced disease (EAU Guidelines: www.uroweb.org). 1.7.3 Isotope bone scan: Bone scintigraphy remains the most sensitive method of assessing bone metastases, being superior to clinical evaluation, bone radiographs, serum alkaline phosphatase measurement and PAP determination (EAU Guidelines: www.uroweb.org). 1.8 Management of prostate cancer: 1.8.1 Localized prostate cancer: 1.8.1.1 Active Surveillance: Active Surveillance is reserved for people at any given age with Gleason score 24 disease, in addition to men with Gleason score 5 and 6 disease who are more than 75 years old. It is also applied to men with significant co morbidity and those in whom life expectancy considered to be less than 10 years and occasionally to those with stage T1a disease with normal PSA (Nam, 1998). 38 Most men with localized prostate cancer on active surveillance are seen every 6 months for clinical history, examination (including a DRE), and a serum PSA test. 1.8.1.2 Radical prostatectomy (RP): RP (open and laparoscopic) is indicated for the treatment of fit men with localized prostate cancer whose life expectancy exceeds 10 years, with curative intent. The patient should consider all available treatment options and the complications of RP prior to proceeding. Multidisciplinary team discussion of each case is the normal practice in the UK. 1.8.1.3 Radical external beam radiotherapy and brachytherapy: This is indicated in men with life expectancy of more than 5 years. A 6-week course of daily treatments amounting to a dose of 60-72Gy for the radiotherapy is the recommended treatment. A higher dose of approximately 150Gy is delivered in one session using brachytherapy. Results of both treatments are comparable to radical prostatectomy in terms of progression-free survival (EAU guidelines: www.uroweb.org). 1.8.1.4 Cryotherapy and high frequency focused ultracound (HIFU): Cryotherapy and HIFU have emerged as alternative therapeutic options in patients with clinically localized prostate cancer. Whereas HIFU is still considered 39 to be an experimental treatment, cryotherapy has been recognized as a true therapeutic alternative. Both techniques have been developed as minimally invasive procedures potentially resulting in the same therapeutic efficacy as the established surgical and non-surgical options associated with reduced therapyassociated morbidity (EAU guidelines: www.uroweb.org). 1.8.2 Management of locally advanced prostate cancer: 1.8.2.1 External beam radiotherapy: External beam radiotherapy has been used alone or in combination with hormone therapy. Better outcomes are obtained using combined treatment with a 15-30% 10-year survival (EAU guidelines: www.uroweb.org). 1.8.2.2 Hormone therapy: Hormone therapy alone is another option in elderly patients or those unwilling to consider radiotherapy. The treatment would be in the form of a non-steroidal antiandrogen (e.g. bicalutamide 150mg), in addition to androgen deprivation by orchidectomy or luteinizing hormone-releasing hormone (LHRH) analogue. It should be made clearly that hormone therapy is not a treatment offered with curative intent (EAU guidelines: www.uroweb.org). 40 1.8.2.3 Active surveillance: This is also an option for non-metastatic T3 disease in an elderly asymptomatic man who may wish to avoid side effects of treatment. 1.8.3 Management of advanced prostate cancer: Metastatic disease is the cause of nearly all prostate cancer-related deaths. Advanced prostate cancer is currently incurable. The overall 5-year survival is 25%. The extremes are 10% with <6 months survival, while <10% survive more than 10 years. The mainstay of treatment is hormone therapy. Cytotoxic chemotherapy is reserved in selected cases. Novel treatments such as growth factor inhibitors, angiogenesis inhibitors, immunotherapy, and gene therapy are in the process of development (EAU guidelines: www.uroweb.org). 1.9 Gonadotropins as tumour markers: Tumour markers in general can play a crucial role in detecting disease and assessing response to therapy in selected groups of patients. In monitoring patients for disease recurrence, tumor marker levels should be determined only when there is a potential for meaningful treatment. In addition, normalization of tumor marker values may indicate cure despite radiographic evidence of persistent disease. In this circumstance, the residual tumor is frequently nonviable. Conversely, tumor marker levels may rise after effective treatment (possibly related to cell lysis), but the increase may not portend treatment failure. However, a consistent increase in tumor marker levels, 41 coupled with lack of clinical improvement, may indicate treatment failure. Residual elevation after definitive treatment usually indicates persistent disease. Following tumor marker response is particularly useful when other evidence of disease is not readily accessible (Perkins, 2003). Gonadotropins are a group of glycoprotein hormones secreted by the anterior pituitary gland. Their end organ where they exert their action is the gonad, hence their name. These are luteinizing hormone (LH) and follicle-stimulating hormone (FSH). Gonadotropins are not necessary for life, but are essential for reproduction. Most anterior pituitary cells secrete only LH or FSH, but some appear to secrete both hormones. Another glycoprotein hormone, with a close similarity to LH, called human chorionic gonadotropin (hCG) also belongs to the glycoprotein family but is produced by the placenta. The glycoprotein hormones consist of a common -subunit but all differ in their individual ß-subunits. Both subunits are connected via disulphide bonds. The -subunit consists of 92 amino acid residues and is encoded by a single gene that is localized on chromosome 6q12.21. The ß-subunits differ slightly in their molecular structure among the different hormones, yet they have some similarity in their amino acid constituents. There is 83% similarity in the molecular structure of the ß subunits in LH and hCG. The genes encoding for the ß-subunit genes are located on different chromosomes, as will be described later (Themmen et al., 2000). There is increasing evidence of para/autocrine functions of the gonadotropic glycoprotein-hormones (GPH), as well as the genetic encoding of their receptors in non-gonadal tissues. This led researchers to investigate intraprostatic GPH 42 and GPH receptor gene expression in prostatic tissue. In this study we aim at highlighting the association of those gonadotropins in the development and prognosis of prostate cancer. 1.9.1 Gonatotropin receptors: The gonadotropin hormone receptors belong to the large family of G proteincoupled receptors, whose members all have a transmembrane domain that is connected by three extracellular and three intracellular loops. The glycoprotein hormone receptors share a common character of having a large extracellular hormone-binding domain at the N terminus. Whilst FSH binds to its own FSH receptor, LH and hCG both bind to the same LH receptor. Genes for the latter receptor are located on chromosome 2p21. Several parts (exons) of the genes encode specifically for the extracellular, transmembrane and intracellular parts of the gonadotropin hormone receptors and confer their specific binding sites for their corresponding hormones. The extracellular domain of the glycoprotein hormone receptors may have a similar structure. This might explain the possible interaction of hCG with the LH receptor. The extracellular domain of the gonadotropin receptors is connected to its transmembrane domain by a hinge region. The transmembrane domain is in turn connected with an intracellular G protein, which upon stimulation activates the various adenylyl cyclase isoenzymes, resulting in elevation of intracellular cAMP levels. This will lead to increased phosphatidylinositide turnover, elevated 43 intracellular Ca2+, and activation of mitogen-activated protein kinases (see diagram below). In case there is persistent stimulation of the LH receptors, they undergo down regulation via several mechanisms. These are uncoupling of the receptor from the intracellular transducing G proteins, in addition to internalization extracellular domain of the receptor, resulting in decreased density of extracellularly exposed hormone binding sites. This may explain the principle of treatment of prostate cancer by LHRH analogues (Themmen et al., 2000). LH/hCG Stimulation LH receptor Extracellular domain Transmembrane portion Cell membrane G protein Intracellular domain phosphatidylinositide turnover intracellular Ca2+ mitogen-activated protein kinases Diagram 1.1 demonstrating gonadotropins and their receptors. 44 1.9.2 Gonadotropins and prostate cancer: 1.9.3 Human Chorionic Gonadotropin (hCG): It is a heterodimeric glycoprotein hormone, produced by the Syncytiotrophoblast cells of placenta during pregnancy. Its essential function is maintaining the production of progesterone during pregnancy by maintaining the corpus luteum after fertilization. This is achieved by delaying apoptosis (programmed cell death). It is composed of two dissimilar subunits, and ß, joined noncovalently. The -subunit of hCG has a similar molecular structure to that of the other glycoprotein hormones. It is composed of 92 amino acids linked by five disulfide bridges. The ß-subunit is unique, and distinguishes hCG from the other glycoprotein hormones. It is composed of 145 amino acids linked by six disulfide bridges. The ß-subunit contains two oligosaccharide side chains (linked to each other via N-linkage). It also has four O-linked oligosaccharide units, located in the C-terminal extension. This confers its three dimensional structure (Laurence, 1997). Although the intact hormone hCG is produced by the placenta and by germ-cell tumors, the free ß-subunit (hCGß) is independently produced by epithelial tumors (Stephen et al., 2003). 45 1.9.3.1 Metabolism and excretion of hCG: hCG undergoes a series of complex and yet partially understood metabolic pathways leading to the dissociation of the α and ß subunits. The α-subunit is predominantly excreted unaltered in urine and seminal fluid, while the ß subunit is further degraded in the circulation to “nicked” hCGß and ultimately undergoes renal degradation to a urinary breakdown product called hCGß core fragment (hCGß-cf). The intact hCGß and the intact hCG are also found in urine and seminal fluid to a lesser extent. (Stephen et al., 2003; Berger et al., 2007). 1.9.3.2 Genetic encoding of hCGß: The genetic mapping for the hCG was originally described in 1983. hCGß is encoded by a cluster of six genes. They were found later to be located in Chromosome 19q13.3, in close proximity with the single gene encoding for the ß fragment of LH. These genes are divided into two groups. Whilst the expression of group II genes (hCGß 3/9, 5, and 8) was shown to be associated with aggressive epithelial tumours (Hotakainen et al., 2007), group I genes (hCGß 6/7) over expression was also shown as independent prognostic markers in other epithelial tumours (Talmadge et al., 1983; Hotakainen et al., 2006). 1.9.3.3 hCGß and cancers: The subunit of the hCG is produced by a wide range of epithelial tumours and has been suggested as a potential tumour marker. Thus, hCG has been identified by immunohistochemical methods in prostate, bladder, testicular, 46 colorectal, ovarian, and other cancers (Rao, 2004; Weissbach, 1999; Dirnhofer, 1998; Dirnhofer, 2000, Noeman, 1994; Crawford et al., 1998). 1.9.3.4 hCGß and Prostate cancer: In prostate cancer, hCG detection by immunohistochemical methods has been variable. In a previous study by Sheaff and associates, hCG expression was correlated with poor prognosis. In that series, hCG was detected in 12 of 80 adenocarcinoma of prostates, with 11 of these 12 patients not surviving at 18 months’ follow up (Sheaff et al., 1996). These results were supported by Zhang et al. They investigated the presence of a number of potential target antigens and their expression for the possibility of using them in immunotherapy on primary and metastatic prostate cancers. Among the different antigens they studied, they found that hCG is present in 8 out of 9 prostate cancer specimens and in only 3 out of 10 normal prostate specimens (Zhang, 1998). On the other hand, genetic analysis was conducted to study the expression of genes hCG and its relation to prostate cancer. Span et al compared the levels of the different hCG transcripts in concurrent normal and cancerous prostate tissues obtained from 17 patients. Surprisingly, median expression levels of hCG mRNA were lower in prostate cancer when compared with normal tissue from the same patient. In contrast, hCG 3, 5 and 8 transcripts were found in normal tissue as much as in prostate cancer, arguing against a specific role of these gene transcripts in the development of prostate cancer (Span et al., 2002). 47 In addition, researchers indirectly investigated the association of the hCG in prostate tissue by assessing its presence in seminal fluid. It was found that hCG was well expressed in prostate cancer patients. Interestingly, the protein was associated with low-grade disease. Hence, contradicting previous results of Sheaff et al and Daja. (Sheaff et al., 1996; Daja, 2000). hCG has been explored as a potential target for treatment. In vitro studies have shown that it would be possible to inhibit the growth of refractory prostate cancer cells using hCG receptors in these cells as a target as mentioned above (Devi, 2002). However, the association of the hCG and the prognosis of the prostate cancer in relation to different ethnic groups has not yet been studied. 1.9.4 Luteinizing Hormone (LH): LH is one of the heterodimeric glycoprotein hormones (FSH, hCG and TSH). It has a molecular weight of ~29 400 Daltons. Similar to the hCG, it consists of noncovalently associated α and β subunits. The alpha subunit of LH is composed of 92 amino acids and is similar in structure to, and immunologically indistinguishable from the α subunits of the other glycoprotein hormones mentioned above. It contains two N-linked oligosaccharides. The β subunit confers physiological specificity and is unique to LH. It comprises 121 amino acids, has a molecular weight of ~14,800 Daltons and contains one N-linked oligosaccharide attachment. The glycosylation of LH is characterised by a lower 48 sialic acid content than the other glycoprotein hormones. It has been postulated that this leads to more rapid clearance of LH from the circulation. Hence the halflife of LH is only 20 minutes (Veldhuis et al. 1987). 1.9.4.1 Physiology of action: Normal LH secretion from the gonadotrophs of the anterior pituitary is dependent on pulsatile Gonadotropin releasing hormone (GnRH) discharge. In contrast, non-pulsatile administration of GnRH causes pituitary desensitisation after an initial “surge”, hence the principle of hormonal ablation in prostate cancer treatment. Release of LH from the pituitary is controlled by a complex balance of positive and negative feedback mechanisms (Counis, 2005). In females LH stimulates rising oestrogen levels in the first part of menstrual cycle, which in turn amplifies the LH discharge. In the luteal phase, LH stimulates the release of progesterone from the corpus luteum and the latter provides negative feedback. The biological activity of LH in the ovary is mediated via rapid and reversible binding to specific membrane-bound receptors on the granulosa and theca cells. In men, LH acts on the leydig cells of the testis to produce testosterone. The latter will have a negative feedback to the hypothalamic gonadotropin-releasing hormones. As mentioned earlier, binding of LH stimulates the enzyme adenylate 49 cyclase, resulting in an increased synthesis of cyclic AMP (cAMP) and providing the stimulus for androgen biosynthesis. 1.9.4.2 Genetic encoding for LH and its variant: The gene for the α subunit of LH is located on chromosome 6p21.1-23. It is expressed in different cells type. The gene for the LH β subunit is located on chromosome 19q13.3 and expressed in gonadotrophs of the pituitary cells and controlled by GnRH. A genetic variant of the LH-beta protein (vLH) exists in up to 40% of Caucasians and is more bioactive than the wild-type protein (Elkins, 2003). It results from two point mutations in the genes encoding for the β chain; in locus (8), resulting in replacement of the amino acid tryptophan by arginine (Trp8 Arg), and in locus (15) in which isoleucine is replaced by threonine (Ile15 Thr). While the Trp8 Arg mutation has been proposed as responsible for the altered immunoreactivity of the vLH, the Ile15 Thr mutation was suggested to alter the biological activity by enhancing its in vitro bioactivity/binding affinity but conversely decreasing its half-life in vivo (Haavisto et al., 1995; Nilsson et al., 2001). Structurally, vLH was shown to contain more complex carbohydrate chains when compared to the wild-type LH (wtLH) (Papandreou et al., 1993). The same 50 polymorphism was found in Japanese patients presenting with reproductive disorders, therefore suggesting a worldwide distribution of the vLH (Furui et al., 1994; Okuda et al.,1994). Interestingly, these same changes are present as part of the evolution of hCGß subunit from the ancestral LHß gene (Talmadge et al., 1984). 1.9.4.3 vLH bioactivity: vLH bioactivity was studied extensively with conflicting results. It was found that in breast cancer patients, vLH appears to have higher in vitro bioactivity but a shortened circulatory half-life (Akhmedkhanov, 2000). In addition, gonadotropinreleasing hormone stimulation of the pituitary in healthy females resulted in a relatively enhanced response from vLH when compared with wild-type LH (Takashi et al., 2000a). Moreover, vLH appeared to have a higher androgenic action than wtLH in healthy postmenopausal women (Hill et al., 2001). Although immunologically distinct, the vLH was still bioactive (Pettersson et al., 1992). Also, in vitro experiments have shown that the procession of vLH gene does not affect receptor binding and bioactivity of the hormone, compared to the wild type LH (Lamminen and Huhtaniemi, 2001). However, in a cohort study it was found that vLH is associated with delayed testicular growth and slower growth rate in pre-pubertal boys, suggesting a lower bioactivity of the variant allele (Raivio, 1996). 51 1.9.4.4 Luteinizing Hormone and Prostate cancer Prostate cancer has been always thought of as being androgen-dependent. Hence, therapy involves removing androgens and/or opposing their effects using antiandrogens (Bevan, 2005). Circulating testosterone plays an important role in maintenance and growth of prostate cells. More importantly, its active metabolite; dihydrotestosterone is the principal hormone acting upon this tissue. Therefore, enzymes or hormones involved in the synthesis and metabolism of androgens have been investigated. Four proteins; CYP17, CYP19, CYP11A1, and LH are found to be involved in the synthesis and conversion of testosterone to dihydrotestosterone and estradiol (Douglas, 2005). In addition, LH has been investigated as a target in treating prostate cancer cells. Lytic peptides, conjugated with LH beta subunit, targeted at androgen sensitive and insensitive prostate cancer cells selectively destroy both androgen sensitive and insensitive human prostate cancer cells transplanted in athymic mice (Hansel, 2001). These interesting findings were also noticed by Bodek et al, who found that lytic peptides combined with hCGß subunit selectively destroyed cells possessing LH receptors. In their study, they described functional characteristics of the conjugate and the molecular mechanism of the cell death pathway in prostate 52 cancer cells. Based on their in vitro studies, they concluded that the conjugate kills cells possessing luteinizing hormone receptors (LHR) faster than the lytic peptides alone (Bodek, 2005). Epidemiological studies have been made to investigate whether the vLH function might affect susceptibility to prostate cancer via altered testosterone secretion. Elkins et al studied the frequency of the vLH-ß gene in familial prostate cancer patients (n = 446), in sporadic prostate cancer patients (n = 388), and in population-based controls without prostate cancer (n = 510) in order to assess the role of this polymorphism in susceptibility to prostate cancer. They observed a higher frequency of vLH-ß gene polymorphism in familial prostate cancer patients (18.6%). than in controls (13.7%). After taking into account the correlation of the familial cases and adjusting for age and body mass index (BMI), there was a weak positive association between the variant LH-ß genotype, and risk of familial prostate cancer. The sporadic case group was also slightly more likely to have a variant genotype (15.2%) compared to the controls (13.7%), and after adjustment for age and BMI, a similar association with this variant was found. Their data are consistent with the hypothesis that the vLH- is a weak risk factor for prostate cancer (Elkins, 2003). 53 1.10 HYPOTHESIS: Previous studies have shown that black men have the highest incidence and mortality from prostate cancer in the world. Even after adjusting for stage at diagnosis, black men have higher mortality rates than white men (Freedland 2005). One of the postulated theories is the elevated androgen levels found in African – Caribbean men (Mohler, 2004; Abdelrahaman, 2005). In fact, a recent study showed that African-Caribbean men had a three times greater risk than European men, and South Asian men had the lowest risk of prostate cancer (Chinegwundoh, 2006). hCG has been identified by immunohistochemical methods to be expressed by prostate, bladder, testicular, colorectal, ovarian, and other cancers (Weissbach, 1999; Dirnhofer 2000; Rao, 2004). It was suggested that hCG have some form of biological role, acting on the cells from which it is secreted. Furthermore, given that the LH/hCG receptor is not expressed by these tissues, any activity observed must be occurring via an as yet unidentified pathway (Davies, 2001). Several theories have been postulated with regards the hCG involvement in cancer biology. There is an increasing evidence of para/autocrine functions of the hCG and LH/hCG-receptor (R) gene expression in non-gonadal tissues (Rao, 2004). It may act as autocrine growth factor by inhibiting apoptosis. Structural homology and in vitro studies suggest that it may achieve this by inhibition of the transforming growth factor (TGF) receptor complex (Iles, 2007). Finally the 54 close similarity of the molecular structure of the hCG to LH could explain its action on the LH receptor in the target organs, probably leading to increased androgenic activity and possibly contributing to the development of prostate cancer (Iles et al., 2003). LH is one of the hormones responsible for the conversion of testosterone into its active metabolite in the prostatic tissue (Douglas, 2005). Thus, in addition to ectopic expression of homologous hCG genes, previous studies conducted on the vLH suggest that it has a possible higher bioactivity and therefore could result in higher basal androgens and hence an increased susceptibility to the development of prostate cancer (Akhmedkhanov, 2000). Therefore, for the reasons explained above it may be hypothesized that ectopic hCG expression and the incidence of vLH genotype are more prevalent in prostate cancer patients and in particular the African-Caribbean, in whom baseline androgen levels are known to be higher than other men of different ethnic origin (Mohler, 2004). We attempt to test this hypothesis. 55 1.11 AIM OF THESIS Primary aims: 1. Compare the hCGβ protein expression in BPH and prostate cancer tissue. 2. Investigate the clinical significance of hCGβ expression by prostate cancer on disease progression and patient survival. 3. Investigate the expression of hCGβ in prostate tissue of patients from different ethnic groups. 4. Study the relation between the degree of immunohistochemical expression of the hCGß in prostate cancer tissue, with the presenting PSA in the three ethnic groups. 5. Study the relation between the degree of immunohistochemical expression of the hCGß in prostate cancer tissue, with the disease progression and patient survival in the three ethnic groups. 6. Compare the incidence of vLH genotype in people with BPH and those with prostate cancer. 7. Compare the incidence of vLH in the three ethnic groups in the study. Secondary aims: 1. Determine the age at presentation in the three distinct ethnic groups. 2. Investigate if there is any difference in the Gleason Scoring of the prostate cancer and its relationship with survival in the three groups involved in the study. 56 CHAPTER II PATIENTS’ DEMOGRAPHICS AND EQUIPMENT 57 2.1 Introduction: In this pilot study, patients with various stages of prostate cancer were selected. These were compared with age-matched patients with benign conditions. Archival prostate tissues obtained from TURP were used as a control. 2.2 Case selection: Specimens from the Middle East population (Group I) were obtained from Sheikh Khalifa Medical City and Tawam Hospitals in the UAE. These were histology specimens obtained from TURP, TRUS biopsy and radical prostatectomy specimens. Both hospitals treat UAE nationals only. Ethical approval from the Local Research Ethic Committees of both hospitals was obtained. Prostate archival tissues were sent to the Barts and the London University Hospital in the UK, where the other two groups of Caucasians (Group II) and African-Caribbean men (Group III) from the East London Region were selected from the prostate cancer database available at Barts and the Royal London Hospitals. Those with sufficient relevant clinical information were included in the study. Ethics approval was obtained from the Multi-center Research Ethics Committee (MREC) Northern and Yorkshire Committee and then the East London and City Local Research Ethics Committee (Reference: 05/MRE03/52). The archival tissues from the three groups were then anonymised thus avoiding any bias in the data collection. The following diagram summarizes the steps involved in our study. 58 THE RELATIONSHIP OF hCGβ EXPRESSION AND vLH WITH PROSTATE CANCER IN THREE POPULATION GROUPS OF MULTI-ETHNIC ORIGIN Patients assessed for eligibility n = 550 Excluded n = 192 Lack of enough clinical data n = 181 Different ethnic origins n = 11 Group I Middle East population (United Arab Emirates) n = 148 Cancers n = 49 Controls n =99 Group II Caucasians (UK) n = 118 Cancers n = 41 Group III African-Caribbean men (UK) n = 92 Controls n =77 Cancers n = 28 Controls n = 64 PCR for detection of vLH in the three ethnic Immunohistochemistry for hCGß expression groups in the three ethnic groups Statistical analysis Results Diagram 2.1 Consort diagram showing the steps involved in our study 59 2.3 The study (prostate cancer) population: Prostate tissue samples from the UK population (n=69)-subdivided into groups of Caucasians (n=41) and African-Caribbean men (n=28) were studied along with the Middle East population (n=49). The first two groups from the UK population were randomly selected from the prostate cancer database over a five-year period (between1997-2002). However, because of the low prevalence of prostate cancer in the UAE population, the time frame was expanded to ten years (between19902000). All patients were selected using pathology reports, theatre reports, hospital episode statistics and cancer registry data. Ethnicity of all the cases was determined from the clinical notes, death certificates and patient questionnaires and questionnaires to relatives of deceased patients. Standardized data were extracted from hospital records on stage and grade of disease at presentation (table 2.1), investigation and treatments. Follow up status were obtained from the hospital records. Inclusion criteria: Patients with prostate cancer of Caucasians, African-Caribbean men and from the Middle East region were included in the study, from whom archival tissues were available for research. 60 Exclusion criteria were: Patients with insufficient clinical information were excluded from the study. Also patients from other ethnic groups were also excluded from the study. Tumour staging and Gleason scores of patients in the three groups is shown in the following table (Table 2.1). Stage (TNM) No. of Gleason score Organ- Locally Metastatic patients and confined advanced T2-4Nx/+M1 Groups disease disease T1-2N0M0 T3- N0/xM0 6 7 8-10 M.East 47% 37% 16% 35% 30% 35% n=49 ( 23) (18) (8) (17) (15) (17) Caucasians 73% 12% 15% 63% 20% 17% (30) (5) (6) (26) (8) 64% 25% 11% 32% 50% 18% (18) (7) (3) (9) (14) n=41 Af.Caribbeans n=28 Table 2.1 Clinical tumour stage and Gleason score of patients with prostate cancer in the three groups: X2 of Stage p=0.08, X2 of Gleason scores p=0.008 61 (7) (5) 2.4 The control population: Similarly, Prostate tissue samples from all three population groups were obtained from the same resources; Middle East (control n=99), Caucasian (control n=77) and African-Caribbean (control n=91). Controls were selected upon histology specimens obtained from TURP. Those with relatively high PSA were followed, at least, to the time of this study and cancer of the prostate was excluded, by TRUS biopsy of the prostate and/or series of PSA follow up, relevant to each case. Inclusion criteria were: Patients with BPH-proven histology from TURP specimens of Caucasians, AfricanCaribbean men and from the Middle East region were included in the study, from whom archival tissues were available for research. Exclusion criteria were: Patients from other ethnic groups and those with insufficient clinical information were excluded. 62 Age distribution: The mean age of patients in the Middle East group (I) was 69.1 years (range 51 – 98). In the Caucasian group (II) the mean age was 72.3 years (range 48 – 82). The mean age in African-Caribbean group (III) was 71.1 years (range 49 – 90) as indicated in table 2.2. Age M. East M. East groups (Cancer) (Control) Caucasians Caucasians Af.Caribbeans Af.Caribbeans (Cancer) (Control) (Cancer) (Control) 0 3% 0 2% (years) 40-49 0 0 (2) 50-59 60-69 70-79 80-89 90-99 (1) 12% 14% 3% 9% 4% 10% (6) (14) (1) (7) (1) (6) 47% 40% 19% 26% 46% 35% (23) (39) (8) (20) (13) (23) 31% 27% 59% 48% 39% 35% (15) (27) (24) (37) (11) (23) 10% 16% 19% 14% 11% 16% (5) (16) (8) (11) (3) (10) 0 3% 0 0 0 2% (3) Table 2.2 (1) Age distribution of Study (prostate cancer) and Control population in the three groups. 63 2.5 Specimen collection and storage: The archival tissue samples were obtained from the pathology departments of the involved hospitals. In the UAE: 1. Sheikh Khalifa Medical City, PO Box 51900, Abu Dhabi, United Arab Emirates. 2. Tawam Hospital, PO Box 15258, Al−Ain, United Arab Emirates. In the UK: Barts and the Royal London NHS Trusts. Archival paraffin-fixed tissue blocks from previous interventions (transurethral resection of prostate, transrectal ultrasound-guided prostate biopsy and radical prostatectomy specimens were used. Samples from all the hospitals were then anonymised by allocating each with a randomly distributed research reference number (identifier). This particular number was then used for the relevant clinical data for each tissue. Samples were stored in specially allocated storage areas, at the Molecular Biology laboratory, Middlesex University. 64 2.6 Materials and equipment: The different reagents and solutions used are described in the methodology sections of chapters III, IV and V as well as in the appendices. Below is a list of major and minor equipment used. 2.6.1 EQUIPMENT CENTRIFUGES Heraeus Megafuge 1.0R Heraeus Instruments, Brentwood, Essex WATER PURIFICATION SYSTEM Aquatron A4S Sigma Chemical Company, Poole, Dorset. INCUBATOR Heraeus B5060 EK Heraeus Instruments, Btrentwood, Essex. FREEZERS/FRIDGES Lab Cold 1250 65 Boro Labs Ltd, Derby Freezers, Denmark VORTEX MIXERS Rotamixer Hook & Tucker instrument Ltd, Croydon PIPETTES Exelpette Elkay Laboratory Products Ltd, Basingstoke, Hampshire. Eppendorf pipettes Amersham Pharmacia Biotech, Little Chalfont, Bucks. PLASTICS Pipette tips Starlab (UK), Milton Keynes, Bedfordshire. Gloves NHS supplies, London GLASSWARE Jencons PLS, 66 Leighton Buzzard, Beds. STATIONERY Guilbert Niceday, Erith, Kent 2.7 STATISTICAL ANALYSIS Data was collected in a Microsoft Excel file. Statistical analysis was performed using StatsDirectTM computer software (StatsDirect Ltd, Altrincham, UK). Nonparametric tests such as Box and Wisker plots and Kruskal Wallis tests were the main tests used for general comparison of data. P < 0.05 was taken as the threshold of statistical significance. Simple linear regression and Chi2 tests were also used where necessary. Survival/outcome was analyzed using Kaplan Meier plots and estimates of survival curves were compared with Logrank and Wilcoxon tests. 67 2.8 RESULTS 2.8.1 Age at presentation in the three ethnic groups: 2.8.1.1 Age at presentation in cancer cases in the three groups: There is a significant difference between the three groups in terms of age at presentation for the Caucasian population who presented at a median greater age of 76 as apposed to 70 for African-Caribbeans and 68 in the Middle East population. Kruskal Wallis test (Conover-Inman): Middle East vs. Caucasians p= 0.0.001, Middle East vs. African-Caribbean men p= 0.60, Caucasians vs. AfricanCaribbean men p= 0.02. Fig 2.1 Age distribution of Cancer patients in study groups Middle East Caucasians Af.Caribbean 50 60 70 80 90 min -[ lower quartile - median - upper quartile ]- max Fig 2.1 Age distribution at presentation in cancer patients the three groups 68 2.8.1.2 Age at presentation in controls in the three groups: Similarly using Kruskal Wallis (Conover-Inman) tests we found that the median age of the BPH control patient samples was very similar for each of the three ethnic grouping and there was no significant difference. See fig 2.2 Of greater study significance is that there was no significant difference in the age distribution of BPH control patients with their respective ethnic study population of patients (presenting with prostate cancer). Age distribution of BPH in the three ethnic groups Middle East Caucasian Afro-Carribian 40 60 80 100 120 min -[ lower quartile - median - upper quartile ]- max Fig 2.2 Age distribution in the control populations in the three groups 69 2.8.2 PSA values in the cancer and control groups: Serum PSA was measured in the patients with prostate cancer at presentation. For the control group, PSA was routinely measured as part of the investigations for LUTS. The Bayer Immulite systemTM was the system used for the PSA measurement. As mentioned earlier in the text, men presenting with PSA values higher than the normal for their age were followed, at least, to the time of this study and cancer of the prostate was excluded, by TRUS biopsy of the prostate and/or series of PSA follow up accordingly. PSA values in all the groups are indicated in the table 2.3. Median PSA (μg/L) Patients M.East Caucasians Af.Caribbeans Cancer 6.6 9.5 26.2 Control 3.88 6.95 5.7 Table 2.3: PSA distribution in the three groups 70 The PSA values in the Control groups: There was a marginally significant difference in the presenting PSA between the Caucasians and the Middle East population, being higher in the former (Caucasians vs. Middle East P = 0.02), while there was no significant difference between the other groups (Middle East vs. African-Caribbean men P =0.07, Caucasians vs. African-Caribbean men P = 0.7). Fig 2.3. The PSA values in the Cancer groups: There is significant difference between the African-Caribbean group, being highest (median: 26.2) and the Caucasians (median: 9.5) (p= 0.002). whilst there was no significant difference between the African-Caribbean group and Middle East group (p=0.4). There was also a significant difference between Caucasian group, being higher, and Middle East group (p= 0.5). Fig 2.3 71 PSA in the three groups 30 26.2 25 20 PSA values 15 10 9.5 PSA 6.95 6.6 5.7 3.88 5 0 Group I ca Group I BPH Group II Group II ca BPH Ethnic groups Group III Ca Group III BPH Group I = Middle East population Group II = Caucasians Group III= African-Caribbean men Fig 2.3 PSA values (median) in the three ethnic groups 2.8.3 Progression-free survival and Gleason scoring in the three groups: Progression-free survival is defined as the time recorded until a clinical evidence (Pains, weight loss, generalized weakness, etc), biochemical evidence (rising PSA) or radiological evidence (newly positive bone scan, new osteolytic bone lesions, CT findings of metastases, etc.) of progression of the disease in the three groups was noticed (EAU guidelines: www.uroweb.org). 72 The Gleason scoring of the archival prostate cancer tissues for the three ethnic groups was independently re-scored by a single uro-pathologist (D.O.), thus omitting the potential bias for different Gleason scoring systems in the three different places. Gleason score of six was found in 25.5%,25.8%,28.5% (in Middle East, Caucasian and African-Caribbean groups respectively). Gleason score of seven was found in 58.1%,58.1%,36.7% in the abovementioned groups respectively. Gleason score eight-ten was found in 34.6%, 16.1%, 16.2% respectively. There was a higher percentage of patients with Gleason Score 7 in the AfricanCaribbean men (50%). Also there was a higher percentage of men with lower Gleason scores in the Caucasian group (63%). Whilst in the Middle East group Gleason score was evenly distributed amongst the cancer patients. Fig 2.4 n=49 n=41 17% n=28 Gleason 8-10 Gleason 7 Gleason 6 18% patients (%) 35% 20% 50% 30% 63% 35% M.East 32% Caucasians Ethnic groups Fig 2.4 Gleason scoring in the three ethnic groups. 73 Af.Caribbeans Using Kaplan-Meier survival estimates, there was a significant difference in survival between the three groups, which was inversely proportional with the Gleason scoring (P=0.0004). In addition, with regards to its relation with the ethnicity, we found the following: Gleason 6: the median survival time for the Middle East group was 79 months (95% CI 62-95). For Caucasians was 42 months (95% CI 35-49). For AfricanCaribbean men was 37 months (95% CI 31-42). There is a statistically significant difference in survival between Middle East men and the other two groups (p 0.0124, 0.0029 respectively), whereas there is no significant difference between Caucasians and African-Caribbean men (p 0.3842). Fig 2.5 Gleason 7: the median survival time in Middle East men was 58 months (95% CI 55-70). For Caucasian men was 35 months (95% CI 33-36). For AfricanCaribbean men was 35 months (95% CI 31-38). There is a statistically significant difference in survival between Middle East men and the other two groups (p0.0006, 0.0002 respectively), whereas there is no significant difference between Caucasians and African-Caribbeans (p0.1846). Fig 2.6 Gleason 8-10: the median survival time in Middle East men was 46 months (95% CI 39-52). For Caucasian men was 15 months (95% CI 10-19). For AfricanCaribbean men was 15 months (95% CI 12-18). There is a statistically significant difference in survival between Middle East men and the other two groups (p 74 0.0139, 0.0003 respectively), whereas there is no significant difference between Caucasian mend and African-Caribbean men (P 0.5057). Fig 2.7 Proportion of progressionfree survivors Survival Plot (PL estimates) 1.00 0.75 Af.Caribbeans Caucasians M.East 0.5 0.25 0.00 0 20 40 60 80 100 Time(months) Fig 2.5 Progression-free survival, Gleason score 6 in the three groups 75 Survival Plot (PL estimates) Proportion of progression-free survivors 1.00 Caucasians 0.75 M.East Af.Caribbeans 0.50 0.25 0.00 0 30 60 90 Time(months) Fig 2.6 Progression-free survival, Gleason score 7 in the three groups. Survival Plot (PL estimates) Proportion of progression-free survivors 1.00 1(8-10) 2(8-10) 3( 810 Caucasians M.East 0. 75 Af.Caribbeans 0.50 0.25 0.00 0 20 40 60 80 Time(months) Fig 2.7 Progression-free survival, Gleason score (8-10) in the three groups 76 2.9 Discussion: This study shows an earlier age at presentation in the Middle East population, followed by the Caucasian population and lastly the African-Caribbean group. These results differ from Shavers et al, who have shown that there is no significant difference in age at presentation with prostate cancer (Shavers et al., 2004). Also, our results differ from Karami et al, who found in their Surveillance, Epidemiology, and End Results (SEER) Program that African American men tend to have an earlier age in presentation in many cancers, even though their finding with regards to prostate cancer was not statistically significant (Karami et al., 2007). The fact that Caucasians and African-Caribbean men tend to have closely similar ages in our results may reflect the similarity in accessing health care by these two groups, who come from the same geographic area in the UK and hence may share similar access facilities to health care. However, our results concur with Ghafoor et al by showing a relatively early age at presentation in men from the Middle East, which may reflect the relatively affluent socio-economic status, and hence the easier access to health centres in the gulf region (Ghafoor et al., 2003). This study shows that presenting PSA in the cancer patients tends to be highest in the African-Caribbean group. These results coincide with Pan et al., who have shown that men of African origin tend to present with higher PSA. The differences are now less pronounced, possibly owing to the improvement in 77 social care (access to medical care, socioeconomic status, and educational level) (Pan et al., 2003). PSA was lowest in the Middle East men. Kamal et al. have also demonstrated that PSA tends to be lower in men from the Middle East (Kamal et al., 2003). This study shows that men diagnosed with prostate cancer from the Middle East have a significantly better prognosis, as opposed to Caucasian and AfricanCaribbean men, sharing the same histological characters of the disease. These findings coincide with other similar data in the literature showing that black men have the highest incidence and mortality from prostate cancer in the world. Even after adjusting for stage at diagnosis, black men have higher mortality rates than white men (Freedland, 2005). In fact earlier studies in the UK population showed that prostate cancer is approximately treble in men of African-Caribbean origin than in white men; it is lowest in Asian and oriental men, suggesting a strong ethnic influence (Chinegwundoh, 2006). Multiple reasons have been postulated to explain these findings including access to care, attitudes about care, socio-economic and education differences, differences in type and aggressiveness of treatment, dietary, and genetic differences. While each reason may contribute to the higher incidence or higher mortality, likely combinations of reasons will best explain all the findings. Also, with recent advances in the understanding of genetic variation in the human 78 genome, in general, and in the genes involved in pathways relevant to prostate cancer biology, in particular, a number of genes with alleles which differ in frequency between black and white men have been proposed as a genetic cause or contributor to the increased prostate cancer risk in black men. However, the clinical significance of these genetic differences is not fully known yet (Shavers, 2004; Oakley-Girvan, 2003; Meyer, 2007). 79 CHAPTER III IMMUNOHISTOCHEMICAL DETECTION OF THE hCG EXPRESSION IN PROSTATE CANCER AND CONTROLS IN THE THREE GROUPS 80 3.1 INTRODUCTION Immunohistochemistry is a process aimed at identification of antigens in tissues via using specific reagents in the form of labelled antibodies . Through antigenantibody interactions, the resultant complexes are visualized by a marker such as fluorescent dye, enzyme, radioactive element or colloidal gold. Immunohistochemistry has become a widely used technique in many medical research laboratories as well as clinical diagnostics. As it involves specific antigen-antibody reaction, it became superior to traditionally used enzyme staining techniques that identify only a limited number of proteins and enzymes. There are various methods in immunohistochemistry that are used to localize antigens. The selection of a suitable method is based on several factors such as tissue type being tested in addition to the degree of sensitivity required. However, the standard goal remains to be unmasking of antigens hidden by formalin crosslinks or other fixation (Cregger et al., 2006). In this chapter the methodology used for the immunohistochemical detection of the hCG in the archival prostate is described. The relation between detection of 81 hCG with the presenting PSA, Gleason score and the prognosis of prostate cancer in the three ethnic groups is investigated. We aim to: 1 Compare the hCGβ protein expression in BPH and prostate cancer tissue. 2 Investigate the clinical significance of hCGβ expression by prostate cancer on disease progression and patient survival. 3 Investigate the expression of hCGβ in prostate tissue of patients from different ethnic groups. 4 Study the relation between the degree of immunohistochemical expression of the hCGß in prostate cancer tissue, with the presenting PSA in the three ethnic groups. 5 Study the relation between the degree of immunohistochemical expression of the hCGß in prostate cancer tissue, with the disease progression and patient survival in the three ethnic groups. PATIENTS Three hundred and ninety patients, divided into three groups according to their ethnicity were included. Each group in turn is subdivided, as previously outlined in chapter II. 82 MATERIALS 3.2.1 First step (Primary Antibody): Rabbit Anti-Human Chorionic Gonadotropin (hCG) (DakoCytomation, Cat# A0231). Optimal dilution 1:6,000. 3.2.2 Second step (Secondary Antibody): Goat Anti-Rabbit IgG (H+L), biotinylated (Vector Laboratories, Cat# BA-1000). Optimal dilution 1:500. 3.2.3 Third step (enzyme labelling) : Avidin and biotinylated horseradish peroxidase macromolecular complex solution. 3.2.4 Detection Reagent: HRP-Streptavidin (Vector Laboratories, Cat# SA-5004). Optimal dilution 1:500 METHODS The method of immunohistochemistry staining for the hCG is described below, but the step by step protocol is described in details in appendix II. 83 We aimed at visualizing the peroxidase complex (as a red color) using 3,3'diaminobenzidine and hydrogen peroxidase. Mayer's solution was used as a counter stain (blue color). The presence of brown dots in the membrane and cytoplasm of prostate epithelial cells against a blue background (Haematoxylin) is diagnostic of being an hCG positive sample. 3.4.1 Deparaffinization: Using the microtome, five (4 µm slices) slides are made from each of the archival prostate tissues. One is stained with Haematoxylin-Eosin (H&E) and the remaining four slides are used for the hCG staining. The four slides were deparaffinized in 2 changes of xylene, 5 minutes each, then they were rehydrated in 2 changes of absolute alcohol, 3 minutes each. This is continued in 90% alcohol for 3 minutes and 70% alcohol for 3 minutes. 3.4.2 Peroxidase blocking: 3% hydrogen peroxide (H2O2) (Sigma-Aldrich Co. Ltd, Poole, UK) in methanol is put for 10 minutes to block endogenous peroxidase. The section is then washed briefly in running tap water, and then rinsed in phosphate-buffered saline (PBS) for 2 minutes. 3.4.3 Epitope retrieval: 0.1% TRITON-100 in PBS is added for 10 minutes and then washed in 2 changes of PBS, 5 minutes each. 84 3.4.4 Serum blocking: Section is incubated with normal horse serum (50% in PBS) (Harlan, Sera-Lab, Bolney, UK) for 10 minutes to block non-specific binding of immunoglobulin. 3.4.5 Primary antibody: Sections are incubated with rabbit anti-human chorionic gonadotropin (hCG) polyclonal antibody diluted in PBS 1:6000 overnight at 4 oC and then rinsed in PBS twice for 5 minutes. 3.4.6 Secondary antibody: Section is incubated with biotinylated goat anti-rabbit IgG from the universal streptavidin biotin kit (Sigma-Aldrich Co. Ltd), diluted in PBS with addition of horse serum for 30 minutes at room temperature and then rinsed in PBS twice for 5 minutes. 3.4.7 Enzyme labelling: Section is incubated with avidin and biotinylated horseradish peroxidase macromolecular complex solution for 20 minutes at room temperature followed by rinsing in PBS twice for 5 minutes. 85 3.4.8 Homogen/Substrate: Section is incubated in DAB peroxidase substrate solution for 5 minutes and then rinsed briefly in distilled water. It is then counterstained with Gill’s haematoxylin solution (Sigma-Aldrich Co. Ltd), followed by differentiation in 1% acid alcohol for few seconds. It is then washed in running tap water for 5 minutes. The specimen is then dehydrated through 70% alcohol for 2 minutes, 90% alcohol 2 minutes, 2 changes of absolute alcohol 3 minutes each. This is followed by clearing in 2 changes of xylene, 3 minutes each and then mounting with xylene based mounting medium. 3.4.9 Controls: Positive and negative controls were included with each batch tested. Positive control was placental tissue (figure 3.1, 3.2), while negative control was a slide in which the first step of primary antibody treatment was omitted (figure 3.3, 3.4). 3.4.10 Scoring and evaluation of results: The slides from each specimen were examined to look for cytoplasmic/membrane staining. The immunohistochemical results were scored guided by the published criteria (Kawaguchi K et al., 2004; Li et al., 2008). The immunoreactivity score for hCGß immunohistochemistry was graded using a modified semi-quantitative scoring system, which scored the average number of positive cells seen at four medium power fields (at X200 magnification). However, due to the limited number of hCGß positive cells in the tissues examined, it was 86 not possible to score the tissues according to the intensity of immuno-positivity. A numerical score was given as follows: score 1, and average of 1-10 stained cells per medium power field are found (figures 3.5, 3.6); score 2, an average of 11-20 stained cells per medium power field (figures 3.7, 3.8) and score 3, an average of more than 20 stained cells are found per medium power field (figures 3.9, 3.10). All slides were scored independently by three individuals (D.L., L.G. and A.T.). 87 Figure 3.1 immunostaining of placenta tissue for hCG beta as positive control (x 100) Figure 3.2 immunostaining of placenta tissue for hCG beta as positive control (x 200) 88 Figure 3.3: negative control of prostate using immunostaining. Note the primary antibody was excluded as (x200) Figure 3.4: negative control of prostate using immunostaining. Note the primary antibody was excluded as (x400) 89 Figure 3.5 H&E staining of prostate cancer showing hCG beta positive (score 1) (x400) Figure 3.6 hCG beta staining of prostate cancer showing hCG beta positive (score 1) (left: x200; right: x400) 90 Figure 3.7 H&E staining of prostate cancer showing hCG beta positive (score 2) (left: x200; right: x400) Figure 3.8 immunostaining of prostate cancer showing hCG beta positive (score 2) (x400) 91 Figure 3.9 H&E staining of prostate tissue showing hCG beta positive (score 3) (left: x200; right: x400) Figure 3.10 hCG beta staining of prostate tissue showing hCG beta positive (score 3) (x400) 3.5 RESULTS 92 3.5.1 hCG EXPRESSION IN THE THREE GROUPS (Cancers and Controls): We used Mann Whitney U and Kruskal Wallis tests as non-parametric tests for our statistical analysis. We found that there is significant difference between the African-Caribbean group and the other two groups (African-Caribbean group vs. Middle East Group: P = 0.0009, African-Caribbean vs. Caucasians: P = 0.0082, while there is no significant different between the other two groups (Middle East Group vs. Caucasians: P = 0.653). Table 3.1 illustrates the distribution of the hCGβ cases and controls in the three groups. Figure 3.11 demonstrates the above as well. M.East Caucasians Group III Controls (+) 37% 40% 58% (n) (37) (31) (37) Controls (-) 63% 60% 42% (n) (62) (46) (27) Cancers (+) 22% 27% 74% (n) (11) (12) (21) Cancers (-) 78% 73% 26% (n) (38) (29) (7) Table 3.1: The distribution of the hCGβ in cancers and controls among the three ethnic groups. 93 Figure 3.11 hCG expression in the three groups Immunohistochemical- grading of cancer cases and controls in the three groups: The majority of the hCG positive slides were Grade I in the three groups and very few cases were graded under Grades II and III. Grade I was found in 71%, 74%, 70% in Groups I, II and III respectively; Grade II was found in 15%, 12%, 17% in Groups I, II and III respectively and Grade III was found in 15%, 14%, 12% in Groups I, II and III respectively. There is no statistically significant difference in the presentation of different grades between the three groups (p=0.17893, p=0.157299 and p= 0.334644 in relation to the three grades respectively). Figure 3.12 demonstrates the different grades in cancer patients and controls in the three groups. 94 Scoring of hCGB + cells in the three ethnic groups 100% 15% 14% 12% 80% 15% 12% 17% 70% 74% 60% Score 3 % 40% 71% Score 2 Score 1 20% 0% M.East Caucasians Af.Caribbeans Ethnic groups Score 1= 1-10 stained cells per medium power field Score 2= 11-20 stained cells per medium power field Score 3= 20< stained cells per medium power field. Figure 3.12 the distribution of the hCG positive cells according to their immunochemical scoring in (cancers and controls) in the three ethnic groups. 3.5.2 hCG EXPRESSION IN THE CONTROLS OF THE THREE GROUPS: There is significant difference between the African-Caribbean group and the other two groups (African-Caribbean group vs. Middle East Group: p = 0.0002, African-Caribbean vs. Caucasians: p = 0.0034, while there is no significant 95 different between the other two groups (Middle East Group vs. Caucasians: p = 0.3885). Figure 3.13 In addition, 58% of the controls were hCGβ positive in group III, which is higher than group II (40%) and group I (37%) (table 3.1). M.East Caucasians Af.Caribbeans Figure 3.13 hCG expression in the controls (BPH) of the three groups Immunohistochemical scoring of controls in the three groups: Score 1 was the main finding in the hCG positive slides in the three groups. Small number of cases were graded under scores 2 and 3; score1 68%, 71%, 88% in Groups I, II and III respectively; score 2 18%,15%, 6% in Groups I, II and III respectively; score 3 14%, 14%, 6% in Groups I, II and III respectively . There is no statistically significant difference in the presentation of different 96 scores between the three groups (p=0.553997, p=0.071898 and p= 0.171797 in relation to the three scores respectively). Figure 3.14 demonstrates the different scores in the three control groups. Scoring of hCGB + cells in controls 100% 80% % 14% 14% 18% 15% 68% 71% 6% 6% 60% 40% 88% Score 3 20% Score 2 0% Score 1 Middle East Caucasians AfricanCarribean Ethnic groups Score 1 = 1-10 stained cells per medium power field Score 2= 11-20 stained cells per medium power field Score 3= 20< stained cells per medium power field Figure 3.14 the distribution of the hCGß positive cells according to their immunochemical scoring in controls in the three ethnic groups 3.5.3 hCG EXPRESSION IN THE PROSTATE CANCER CASES OF THE THREE GROUPS: hCGβ expression is significantly higher in African-Caribbean group than the other two groups (African-Caribbean group vs. Middle East Group: P = 0.0004, African-Caribbean vs. Caucasians: P = 0.0025, while there is no significant 97 difference between the other two groups (Middle East Group vs. Caucasians: P = 0.6001). Figure 3.15. Also in the African-Caribbean group,74% were hCGβ positive, while 22.4% and 27.9% were positive in groups I and II respectively, as shown in table 3.1 and figure 3.15. hCG beta, Cancer and Ethnicity 60 50 40 %30 78% 71% 25% 20 75% 10 22% 29% M.East Group I Caucasians Group II Ethnic Groups 0 Af.Caribbeans Group III negative positive Figure 3.15 hCG expression in the study cases (prostate cancer) of the three groups 98 Immunohistochemical scoring of cancer cases in the three groups: The majority of the hCG positive slides were score 1 in the three groups and very few cases were graded under Grades II and III; score 1 82%, 84%, 87% in Groups I, II and III respectively; score 2 0%, 8%, 4% in Groups I, II and III respectively; score 3 18%, 8% and 9% in Groups I, II and III respectively. There is no statistically significant difference in the presentation of different scores between the three groups (p=0.17893, p=0.157299 and p= 0.334644 in relation to the three scores respectively). Figure 3.16 demonstrates the different grades in cancer patients in the three cancer groups. Scoring of hCGB+ cells in cancer cases 100% 80% 18% 0% 8% 8% 9% 4% 84% 87% 60% % 40% Score 3 82% Score 2 Score 1 20% 0% Middle East Caucasians AfricanCarribean Score 1 = 1-10 stained cells per medium power field Score 2= 11-20 stained cells per medium power field Score 3= 20< stained cells per medium power field Figure 3.16 the distribution of the hCGß positive cells according to their immunochemical scoring in cancers in the three ethnic groups. 99 Relationship between Immunohistochemical grading system and Gleason scoring in the three ethnic groups: Score 1 was the most common finding in prostate cancer archival tissues with Gleason 6, 7, and 8-10 (79%, 82% and 90% respectively). Score 2 was present in 5%, 9% and 0% in Gleason 6, 7 and 8-10 respectively. Score 3 was present in 16%, 9% and 10% of samples with Gleason 6,7, and 8-10 respectively. There was no statistically significant difference in the presence of scores 1,2,3 between the three Gleason categories (6,7, and 8-10 respectively) (p=0.38574, p=0.105141 and p=0.105804 respectively). Figure 3.17 illustrates the above results. Scoring of hCGB positivity and Gleason scoring 100% 80% 16% 5% 9% 9% 10% 0% 60% % 40% 71% 90% 82% Scroe 3 Score 2 Score 1 20% 0% Gleason 6 Gleason 7 Gleason 8-10 Gleason Score 1 = 1-10 stained cells per medium power field Score 2= 11-20 stained cells per medium power field Score 3= 20< stained cells per medium power field Figure 3.17 hCG positivity scoring characteristics in relation to Gleason system. 100 3.5.4 The PSA DISTRIBUTION IN THE THREE GROUPS IN RELATION TO THE hCG STATUS: PSA distribution in the hCG (positive versus negative) cases in the three groups: There is no statistically significant difference in the presenting PSA median among the hCGβ positive cases in the three groups (p=0.213750). Figure 3.18 demonstrates this relationship. Relation of PSA and hCG beta status in the three groups 20 15 7.35 PSA value 10 6.4 hCG + hCG - 5 0 1 hCG status in the three groups Figure 3.18: PSA distribution in hCG positive and negative cases in the three ethnic groups. PSA in the hCG positive and negative cancers in Middle East group: There is no statistical significant difference in the presenting PSA in the hCG positive cancers (p=0.082388). Similarly there is no significant difference in the PSA among the hCG positive, compared with the hCG negative controls in Middle East group (p=0.721921). Figures 3.19 and 3.20 demonstrate this relationship. 101 Median PSA in hCGB + & - cancers in M.East group 100 90 80 70 60 PSA value 50 40 30 20 10 45 41 hCGB + hCGB - 1 hCGB status Figure 3.19: PSA in hCGβ positive and negative cancers in the Middle East group. Median PSA in the hCGB +&- controls in M.East group 20 15 PSA value 10 5.14 hCGB + 3.21 hCGB - 5 0 1 hCGB status Figure 3.20: PSA in hCGβ positive and negative controls in Middle East group. 102 PSA in the hCG positive cancers and controls in Caucasians: There is no statistical significant difference in the presenting PSA in the hCG positive and negative cancers in Caucasians (p=0.326977). Again, we found no significant difference in the PSA between the hCGB positive and negative controls in the same group (p=0.245522 respectively). Figures 3.21 and 3.22 demonstrate this relationship. Median PSA values in hCGB +&- cancers in Caucasians 15 11.2 8.8 10 hCGB + PSA values hCGB - 5 0 1 hCGB status Figure 3.21: PSA in hCGβ positive and negative cancers in Caucasians 103 Median PSA in the hCG +&- controls in Caucasians 10 8 PSA values 7 5.85 6 hCGB + 4 hCGB - 2 0 1 hCG status Figure 3.22: PSA in hCGβ positive and negative controls in Caucasians PSA in the hCG positive cancers and controls in African-Caribbean men: Between hCG beta positive and negative cases in group III, there is no statistically significant difference in the presenting PSA in cancers (p=0.995559), nor controls (p=0.128927). Figure 3.23 and 3.24 demonstrate this relationship. 104 Median PSA in hCGB +&- cancers in Af.Caribbean group 25 20.4 16.75 20 15 PSA values 10 hCGB+ hCGB- 5 0 1 hCGB status Figure3.23: PSA in hCGβ positive cancer cases in the African-Caribbean group Median PSA in hCGB +&- controls in Af.Caribbeans 10 8 PSA values 6.4 5.6 6 hCGB+ 4 hCGB- 2 0 1 hCGB status Figure 3.24: PSA in hCGβ positive controls in African-Caribbean group 105 3.5.5 THE RELATION BETWEEN THE PROGRESSION-FREE SURVIVAL AND hCG IN ALL THE ETHNIC GROUPS: When all cancer patients of the three groups were pooled together, there was no statistically significant difference in progression-free survival between the hCG positive and negative cases (p = 0.8881). Figure 3.25. Survival Plot (PL estimates) Proportion of progression-free survivors 1.00 * (missing) neg 0.75 0.50 hCG –ve 0.25 hCG+ve 0.00 0 20 40 60 80 Time(months) 100 Figure 3.25: Progression-free survival between the hCG positive vs. hCG negative 106 The relation between the progression-free survival and the scoring system in all groups: When all cancer patients were divided according to the immunostaining grades used in this study, no significant difference was found regarding the progressionfree survival of patients (p=0.09), as shown in figure 3.26. Survival in relation to immunochemical scoring system 1.00 Score1 1I Score2 2Score30.75 0.50 0.25 0.00 0 30 60 90 Times Score 1 = 1-10 stained cells per medium power field Score 2= 11-20 stained cells per medium power field Score 3= 20< stained cells per medium power field Figure 3.26 Progression-free survival in relation to the immunochemical scoring system used in this study. 107 3.6 DISCUSSION Being a hormone dependant cancer, one of the postulated theories to explain increased incidence of prostate cancer in African-Caribbean men was the elevated androgen level. While reports of racial differences in gonadal steroid hormone levels in middle-aged men have produced conflicting results, there is evidence that high SHBG and androstenedione levels are more common among young adult African American men than white men (Mohler, 2004). Abdelrahaman and colleagues conducted a cross-sectional study of 47 healthy pre-pubertal African American and white boys aged 5 to 9 years of age. Mean SHBG levels were found to be 25% higher in African American males. This difference and its effect on the ratio between bound and free steroid hormones may contribute to racial differences in disease risk in adult men (Abdelrahaman et al., 2005). Similarly, the relation between prostate cancer and androgen levels may well explain the low incidence of prostate cancer among the people from the Middle East. It has been found that the mean total testosterone (TT) and sex hormone binding globulin (SHBG) was significantly lower in Arab men compared to Caucasian men especially in early adulthood. Caucasians have significantly higher serum levels of the precursor androgens dehydroepiandrosterone sulphate (DHEAS) and androstenedione (ADT) especially in early adulthood compared to Arab men. These observations of low circulating androgens and their adrenal precursors in Arab men may partially account for the decreased risk for prostate cancer among Arab men (Kehinde, 2006). 108 Another fact is that hCG plays a paracrine role in the intratesticular regulation of testosterone secretion (Caroppo, 2003), since the beta subunit of hCG possesses 85% sequence homology with the first 114 amino acids of the beta subunit of pituitary human LH (Gadkari, 2005). The association of the hCG and prostate cancer has two possibilities; the first one is its relation with the production of male hormones and hence leading to an increase in the incidence and progression of prostate cancer. This is also supported by Daehlin who found that hypophysectomized rats which were treated for 8-9 days with human chorionic gonadotropin, maintained testicular blood flow within the physiological range (Daehlin, 1985). The other theory is the association of the hCG with the poor prognosis, proved by its para/autocrine effect on the tumourogenesis, possibly via the inhibition of apoptosis (Li et al., 2008). Our results demonstrate clearly the statistically significant expression of the hCGβ among the African-Caribbean men (both in prostate cancer and BPH), as opposed to the Caucasian, and men from the Middle East population. However, it should be born in mind that many of the archival tissues obtained from the different ethnic groups were biopsy specimens (47.8% in African-Caribbean, 44.8% in Caucasians, 16.9% in men from Middle East), representing only a very small percentage of the tissues studied. Bearing in mind the heterogenicity of prostate cancer, higher number of potentially hCG beta positive tissues might have been found (BPH and Cancer, in the different groups). Although our 109 findings showed a higher incidence of hCGβ positivity in African-Caribbean population, no significant association between hCGβ and survival was found. These findings concur with those of Otite et al. (2006) 110 CHAPTER IV LH-BETA GENE ISOLATION 111 4.1 DNA extraction from paraffin-embedded prostate tissues: 4.1.1 Introduction: Archival, formalin-fixed, paraffin-embedded tissue is an invaluable resource for molecular genetic studies. Extraction of nucleic acid from archival tissue allows retrospective analysis and correlation of clinical end points or histological appearance structures with molecular biological markers (www.protocolonline.org). Three steps are involved: 1. Deparaffinization (getting rid of the paraffin wax): this is achieved via dissolution of the wax in xylene and ethanol. 2. Digestion of the cellular proteins; this is performed by the use of certain proteinases. 3. Purification (dehydration of tissue processing). 4.1.1.1 REAGENTS: Chloroform EDTA, 0.5 M Ethanol, absolute Isoamyl alcohol Phenol Proteinase K Sodium acetate, pH 5.2 TE Buffer (Tris-EDTA), pH 7.4 Xylene 112 4.1.1.2 PREPARATION: Chloroform/Isoamyl alcohol 24:1 Chloroform 24 ml Isoamyl alcohol 1 ml DNA extraction buffer 1.5 ml 5M NaCL 5.0 ml 0.5M EDTA 0.5 ml Tween 20 Fill up to 100 ml with sterile water. Proteinase K (10 mg/ml) Dissolve 100 mg Proteinase K in 10 ml TE for 30 min at RT. Aliquot and store at –20°. 4.1.2 Methods: Day 1: Slices (20 µm) of formalin-fixed and paraffin-embedded tumor samples are cut using microtome. Samples are then put in 2 ml Eppendorf collection tubes. Tissues are incubated in xylene at 45°C for 15 minutes allowing the wax to dissolve. Samples are then centrifuged 10 min at 14,000 rpm. Resultant supernatant is pipetted off, leaving the wax in the bottom of the tube. The latter 113 steps are repeated once more. Then 1 ml 100% ethanol added to tissue pellet, vortexed and then centrifuged for 10 min at 14,000 rpm, and supernatant pipetted off. 1 ml 90% ethanol added to tissue pellet, vortexed, centrifuged for 10 min at 14,000 rpm, and supernatant pipetted off. The same step is repeated, with 70% ethanol this time. The resultant pellet is then dried in speed vac. Pellet is resuspended in 400 µl of DNA extraction buffer. 40 µl of Proteinase K (10 mg/ml) added, vortexed briefly, and incubated at 55°C overnight in order to get rid of the protein. Day 2: 440 µl of phenol is added, shook vigorously by hand for 5 min, and centrifuged for 5 min at 8000 rpm. Supernatant pipetted into a new tube, a solution of 220 µl phenol plus 220 µl chloroform/isoamyl alcohol (24:1 )is then added, shook vigorously by hand for 5 min, and centrifuged for 5 min at 8000 rpm. The aim is to get rid of the Phenol. Again, the supernatant is pipetted into a new tube, 440 µl chloroform/isoamyl alcohol (24:1) is added, shook vigorously by hand for 5 min, and centrifuged for 5 min at 8000 rpm, to get rid of chloroform/isoamyl alcohol. Then, the resultant supernatant pipetted into a new tube (2 ml Eppendorf tube), 1/10 volume of sodium acetate (pH 5.2)added. In addition, 3 volumes of ice-cold 100 % ethanol add, and tube kept overnight at –20°C. 114 Day 3: The tubes are centrifuged for 30 min at 4°C and the supernatant removed. The pellet is dried in speed vac. 20-50 µl sterile H20 is then added. Tubes are then shook gently in thermo mixer at 37°C for 2 hours. Following DNA extraction, DNA concentration was confirmed via the use of spectrophotometer (Camspec M550, Cambridge, UK). 1µl of the resultant DNA was diluted in 100μl of water (1:100). The DNA concentration measurement relies upon its Ultraviolet light absorbance at 260 nm wavelength (DNA A 260). The DNA concentration is measured acceding to the following formula: DNAconcentration= Optical Density (measured at 260nm)X 50X100(dilution factor) 4.2 4.2.1 PCR: Introduction: A method for amplifying a DNA base sequence using a heat- stable polymerase and two 20-base primers, one complementary to the (+ve)-strand at one end of the sequence to be amplified and the other complementary to the (-ve)-strand at the other end. Because the newly synthesized DNA strands can subsequently serve as additional templates for the same primer sequences, successive rounds of primer annealing, strand elongation, and dissociation produce rapid and highly specific amplification of the desired sequence (www.dnalc.org). 115 PCR, as currently practiced, requires several basic components. These components are: DNA template, which contains the region of the DNA fragment to be amplified Two primers, which determine the beginning and end of the region to be amplified (see following section on primers) Taq polymerase (or another durable polymerase), a DNA polymerase, which copies the region to be amplified Deoxynucleotides-triphosphate, from which the DNA Polymerase builds the new DNA Buffer, which provides a suitable chemical environment for the DNA Polymerase The PCR process is carried out in a thermal cycler. This is a machine that heats and cools the reaction tubes within it to the precise temperature required for each step of the reaction. To prevent evaporation of the reaction mixture (typically volumes between 15-100µl per tube), a heated lid is placed on top of the reaction tubes or a layer of oil is put on the surface of the reaction mixture. The DNA fragment to be amplified is determined by selecting primers. Primers are short, artificial DNA strands — often not more than 50 and usually only 18 to 25 base pairs long — that are complementary to the beginning or the end of the DNA fragment to be amplified. They anneal by adhering to the DNA template at 116 these starting and ending points, where the DNA polymerase binds and begins the synthesis of the new DNA strand. The choice of the length of the primers and their melting temperature (Tm) depends on a number of considerations. The melting temperature of a primer -not to be confused with the melting temperature of the template DNA -- is defined as the temperature at which half of the primer binding sites are occupied. Primers that are too short would anneal at several positions on a long DNA template, which would result in non-specific copies. On the other hand, the length of a primer is limited by the maximum temperature allowed to be applied in order to melt it, as melting temperature increases with the length of the primer. Melting temperatures that are too high, i.e., above 80°C, can cause problems since the DNA polymerase is less active at such temperatures. The optimum length of a primer is generally from 15 to 40 nucleotides with a melting temperature between 55°C and 65°C. 4.2.1.1 Nested PCR: Using the conventional PCR, over 1 billion copies of the PCR product can be produced very quickly from a single template molecule. However, if the primers used in the PCR have the affinity to combine with more than one locus on the DNA template, especially if these loci are in close proximity in the DNA template. This results in amplification of “undesired” DNA fragments, rendering non-specific results. To overcome this, nested primers are used to ensure specificity (www.bio.davidson.edu). 117 Nested PCR means that two pairs of PCR primers were used for a single locus. The first pair amplified the locus as seen in any PCR experiment. The sequence amplified in the first PCR contains the targeted sequence and specifically excludes the unwanted sequences containing similar primers of the targeted sequence. The first PCR product is composed mainly of over one billion sequences containing (not exclusively) the targeted sequence. The second pair of primers (nested of “short” primers) binds within the first PCR product and produces the final desired PCR product that will be shorter than the first one. The logic behind this strategy is that if the wrong locus were amplified by mistake, the probability is very low that it would also be amplified a second time by a second pair of primers. 118 The following diagram (4.1) demonstrates the nested PCR: Unwanted segment targeted sequence Example. Segment of DNA with dots represents a DNA sequence of unspecified length. The segment between the double arrows represent the targeted sequence to be amplified. The rest are unwanted sequences. Unwanted segment targeted sequence long primers Step1. The first pair of PCR “long” primers (red squares) binds to the outer pair of primer binding sites and amplify the entire DNA in between these two sites. 119 Step2. PCR product after the first round of amplification. Notice that the bases outside the PCR primer pair are not present in the product. short primers Step3. Second pair of nested “short” primers (green squares) binds to the first PCR product. The binding sites for the second pair of primers are a few bases "internal" to the first primer binding sites. Step4. Final PCR product after second round of PCR. The length of the product is defined by the location of the internal primer binding sites. Diagram 4.1 Nested PCR 120 We used nested PCR as a definitive test for mutation. In an earlier experiment performed on blood samples in females with polycystic ovarian syndrome (unpublished series) it was clearly demonstrated that the nested PCR is superior to other methods of obtaining the vLH gene mutation. Nested PCR was compared with restriction fragment length polymorphisim; a method adopted widely in the literature for obtaining the vLH gene. We have demonstrated that the latter method could overestimate the prevalence of the vLH gene variant. This is simply due to the close proximity of the location of the LHβ and hCGβ genes on chromosome 19q13.3. In the nested PCR we used two sets of primers with specific mismatches for the hCGβ genes (Furui et al., 1994; Elter et al., 1999). The amplified region with the first set of primers was 786 bp in length. 4.2.2 PCR using LH long primers (LH-LP): Materials: MgCl2 (25mM) Buffer (75mM Tris-HCl (pH 8.8, 20mM (NH4)2SO4 dNTPs (300μM of each of the dNTPs: dATP, dGTP, dCTP, dTTP). Primers (LH-LP: forward and reverse: 2.5 pmol each) Taq polymerase (5 units/μl Abgene) H2O 121 4.2.3 Methods: PCR was performed in a 25μl reaction buffer containing 20 ng DNA. Amplification was varied using Omn-E thermal cycler (Hybaid) with the following temperature profiles: 950C for five minutes: 1 cycle. 950 for 30 seconds, 580C 30 seconds and 720C 1 minute: 30 cycles. 720C for 5 minutes: 1 cycle. We used the following PCR mix: 2.0μl of DNA 2.5μl of MgCl2 (25mM) 2.5μl of Buffer (75mM Tris-HCl (pH 8.8, 20mM (NH4)2SO4 0.3μl of dNTPs (300μM of each of the dNTPs: dATP, dGTP, dCTP, dTTP). 2.0μl of Primers (LH-LP: forward and reverse: 2.5 pmol each) 0.2μl of Taq polymerase (5 units/μl Abgene) 15.5μl of H2O The PCR products were electrophoresed on a 1.5% Agarose gel made in 0.5 x TBE buffer (45 mM Tris, 45 mM boric acid and 1mM EDTA) and 2μl of 10mM ethiduim bromide per 100ml gel were then added (Bjornsti and Megonigal, 1999). Samples to be loaded on the gel were mixed with 6 x loading dye (0.09% 122 bromophenol blue, 0.09% xylene cyanol FF, 60% glycerol and 60mM EDTA: Promega). The gel was run at 100V for 40 minutes and subsequently visualized under UV light. Fig 4.1. 768 bp LH-LP DNA bands controls DNA Ladder Fig 4.1 Gel electrophoresis of the first PCR (LH-LP): 123 4.3 DNA EXTRACTION AND PURIFICATION FROM AGAROSE GEL: The resulting DNA band which is about 786 bp was then excised from the gel and was purified using the QIAquick gel extraction kit (Qiagen) according to the following method: 1. The gel slice containing the DNA band with a clean is excised with a sharp scalpel. 2. 3 volumes of Buffer QG are added to 1 volume of supernatant and mix. 8. QIAquick Spin Column is placed in a provided 2 ml collection tube. 9. To bind DNA, the QIAquick Spin Column containing the sample is centrifuged for one minute. 10. The flow-through is discard and QIAquick Spin Column is placed back into the same collection tube. 11. To wash, we add 0.75 ml Buffer PE to column and then centrifuge for 30–60 seconds. 12. The flow-through is discarded and QIAquick Spin Column is placed back into the same collection tube. We then centrifuge column for an additional 1 min at maximum speed. (IMPORTANT: Residual ethanol from Buffer PE will not be completely removed unless the flow-through is discarded before this additional centrifugation.) 13. QIAquick Spin Column is placed into a clean 1.5 ml micro centrifuge tube. 124 14. To elute DNA, we add 20 µl Buffer EB (10 mM Tris·Cl, pH 8.5) to the center of the QIAquick Spin Column. We let it stand for 1 min and then centrifuge for 1 min., 4.4 PCR using LH Short primers (LH-SP): PCR is then performed using the purified 786 bp DNA band, using the second set of primers known to produce a 194 bp DNA product (Mengual et al., 2003). 4.4.1 MATERIALS: MgCl2 (25mM) Buffer (75mM Tris-HCl (pH 8.8, 20mM (NH4)2SO4 DNTPs (300μM of each of the dNTPs: dATP, dGTP, dCTP, dTTP). Primers (LH-SP: forward and reverse: 2.5 pmol each) Taq polymerase (5 units/μl Abgene) H2O 125 We used the following PCR mix 4.0μl of DNA 2.5μl of MgCl2 (25mM) 2.5μl of Buffer (75mM Tris-HCl (pH 8.8, 20mM (NH4)2SO4 0.3μl of dNTPs (300μM of each of the dNTPs: dATP, dGTP, dCTP, dTTP). 2.0μl of Primers (LH-LP: forward and reverse: 2.5 pmol each) 0.2μl of Taq polymerase (5 units/μl Abgene) 13.5μl of H2O 4.4.2 METHODS: PCR was performed in a 25 μl reaction buffer containing 20 ng DNA. Amplification was varied using Omn-E thermal cycler (Hybaid) with the following temperature profiles: 950C for five minutes: 1 cycle. 950 for 30 seconds, 600C 30 seconds and 720C 1 minute: 35 cycles. 720C for 5 minutes: 1 cycle. The PCR products were electrophoresed on a 2% Agarose gel made in 0.5 x TBE buffer (45 mM Tris, 45 mM boric acid and 1mM EDTA) and 2μl of 10mM ethiduim bromide per 100ml gel were then added (Bjornsti and Megonigal, 1999). Samples to be loaded on the gel were mixed with 6 x loading dye (0.09% 126 bromophenol blue, 0.09% xylene cyanol FF, 60% glycerol and 60mM EDTA: Promega). The gel was run at 100V for 40 minutes and subsequently visualized under UV light. Fig 4.2. 194 bp LH-SP DNA bands DNA ladder control Fig 4.2 Gel electrophoresis of the second PCR (LH-SP): The resultant 194 bp DNA band was then excised from the Agarose gel and then purified using the above technique. The samples were then ready for sequencing. 127 4.5 DNA SEQUENCING: 4.5.1 INTRODUCTION: DNA sequencing, the process of determining the exact order of the 3 billion chemical building blocks (A, T, C, and G bases) that make up the DNA of the 24 different human chromosomes, was the greatest technical challenge in the Human Genome Project. Achieving this goal has helped reveal the estimated 20,000-25,000 human genes within our DNA as well as the regions controlling them. In essence, the DNA is used as a template to generate a set of fragments that differ in length from each other by a single base. The fragments are then separated by size, and the bases at the end are identified, recreating the original sequence of the DNA. The most commonly used method of sequencing DNA - the dideoxy or chain termination method - was developed by Fred Sanger in 1977. The key to the method is the use of modified bases called dideoxy bases; when a piece of DNA is being replicated and a dideoxy base is incorporated into the new chain, it stops the replication reaction. (http://nobelprize.org/nobel_prizes/chemistry/laureates/1980/sanger-lecture.pdf). Gel-based sequencers use multiple tiny (capillary) tubes to run standard electrophoretic separations. These separations are much faster because the 128 tubes dissipate heat well and allow the use of much higher electric fields to complete sequencing in shorter times (figure 4.3). Fig 4.3 a diagram showing DNA sequencing using Capillary Array Electrophoresis (CAE): (www.ornl.gov/sci/techresources/Human_Genome). 4.5.2 METHODS: DNA samples are introduced into the 96-capillary array; as the separated fragments pass through the capillaries, they are irradiated all at once with laser light. Fluorescence is measured by a charged coupled device that acts as a simultaneous multichannel detector. Because every fragment length exists in the sample, bases are identified in order according to the time required for them to reach the laser-detector region. BigDye v2 (applied Biosystems) was used for 129 labeling DNA and samples were subsequently sequenced by Prism 3700 sequencer* (applied Biosystems). Both forward and reverse strand sequencing using the primer set 2 (LH-SP) was performed and matched in order to confirm sequence polymorphisms detected. The ABI PRISM™ 3700 DNA analyser is a fully automated, multi-capillary electrophoresis instrument designed for use in production-scale DNA analysis. Fig 4.4 (A) (B) Fig 4.4 DNA sequencing of LHβ gene (using second set-forward PCR primer). LHβ specific amino acids Arg2 and His10 are underlined. (A) shows a patient DNA with wild type LHβ sequence, the boxes triplet are showing Trp8 and Ile15 of the wild type LHβ sequence. (B) shows a patient DNA with Variant LHβ (vLH) allele, the boxed triplet showing the point mutation in codon 8 and 15 TGG (Trp) to CGG (Arg) and ATC (Ile) to ACC (Thr) respectively 130 4.6 RESULTS Interestingly, only four cases were found positive for the vLH among all the ethnic groups (Fig 4.3). These were two controls (Middle East: n=1, Caucasians n=1) and two study cases (prostate cancer) (Middle East n=1, Caucasians n=1). The first patient presented with a PSA of 8.4 and was diagnosed with Gleason 3+3=6. He received radical radiotherapy and his disease is still under control. The second patient had a presenting PSA of 17, Gleason 4+4=8 and is currently under hormonal treatment. Fig 4.5, 4.6 There was no correlation with the prognosis of those with prostate cancer, who were positive for the vLH. vLH -ve vLH +ve Fig 4.5: The prevalence of vLH in the three study groups 131 No. of patients Ethnic groups Fig 4.6: The prevalence of vLH in the three study group 4.7 DISCUSSION LH is a member of glycoprotein hormone family. Like other members of the family, it consists of two subunits: an alpha- subunit (common among Glycoprotein hormones) associated with a unique B-subunit. LH signals testicular Leydig cells to secrete testosterone, which maintains growth of prostate cells. Several epidemiological studies have shown its prevalence in normal population groups (Pettersson and Soderholm, 1991; Pettersson, 1992). Other studies, however, indicated that small increases of LH may be associated with increase of 132 testosterone and therefore may be associated with an increased risk of prostate cancer, though this association has not been universal (Shaneyfelt, 2000). The prevalence of this variant (vLH) allele has been estimated worldwide at a carrier frequency reaching up to 53%. Screening for vLH has primarily relied on relative immunoreactivity in variant-sensitive and insensitive assays. Restriction Fragment Length Polymorphism (RFLP) studies have largely been used to confirm immunoassay results. However, both methods present various limitations and potential bias, due to the close similarity in the molecular structure, immunological characteristics and its gene location in close proximity with the six hCG genes on chromosome 19q13.3. While the Trp8 ---Arg mutation has been proposed as responsible for the altered immunoreactivity of the vLH, the Ile15 ---Tr mutation was suggested to alter the biological activity by enhancing its in vitro bioactivity/binding affinity but conversely decreasing its half-life in vivo (Haavisto et al., 1995; Nilsson et al., 2001). Gonadotropin-releasing hormone stimulation of the pituitary in healthy females resulted in a relatively enhanced response from vLH when compared with wtLH (Takashi et al., 2000a). Moreover, vLH appeared to have a higher androgenic action than wtLH in healthy postmenopausal women (Hill et al., 2001). 133 Generally, there is some controversy about the involvement of vLH in reproductive disorders among different ethnic populations (Jameson, 1996; Huhtaniemi et al., 1999; Huhtaniemi and Petterson, 1999; Begendah and Veldhius, 2001; Lamminen and Huhtameini, 2001). A confusing fact is the presence of the vLH in both healthy individuals as well as those presenting with certain pathophysiologies of the reproductive system. Kaleva M, et al studied the association between the vLH and improper testicular position in late pregnancy. They found an increased prevalence of vLH among cryptorchid boys with GA >40, suggests that the lower hormonal efficacy of vLH predisposes for improper testicular descent in late pregnancy. (Kaleva, 2005). Similarly, the association of the vLH with infertility was studied by many researchers. Liao and associates investigated the Functional characterization of a natural variant of luteinizing hormone with regards to infertility. Their studies found a significant association in between the two, suggesting that the vLH may be a contributing factor to the pathogenesis of infertility in the carriers of this variant. (Liao, 2002) We used nested PCR and DNA sequencing of the LH-Beta gene region as a definitive test for mutations in our population. We have shown that the occurrence of the vLH is extremely low in our three population groups (0.0107%), contradicting the previous studies in this field. We relied in our work on the 134 nested PCR method, by which we excluded the hCG genes (which are six in number and are very similar to the LH gene). Our initial hypothesis was based on prior theories of a higher bioactivity and a shorter half-life of the vLH (Akhmedkhanov, 2000), hence the possibility of its association with increases androgen levels, which in turn may be associated with higher incidence of prostate cancer. However, the association of the vLH with undescended testis (which –in part- is dependant on testosterone) and infertility (in which, the endogenous testosterone is an integral part of the spermatogenesis) suggesting that the vLH is associated with less bioactivity, thus disregarding our hypothesis of its role in the development of prostate cancer (Liao, 2000; Kaleva, 2005). 135 CHAPTER V GENERAL DISCUSSION 136 Prostate cancer is the most common cancer in men. It affects 1 in 12 men in the UK. The highest prostate cancer incidence rates are in the developed world and the lowest rates in Africa and Asia (Parkin, 2002). The extremely high rate in the USA (125 per 100,000), more than twice the reported rate in the UK (52 per 100,000), is likely to be due to the particularly high rates of PSA testing in the USA (Gann, 2002). Men of African-Caribbean origin have the highest incidence and mortality from prostate cancer in the world. Men of African origin in the UK have three times the chance of developing the disease, as compared with Asian and Caucasians. This fact suggests a strong ethnic influence. (Chinegwundoh, 2003; Chinegwundoh, 2008) Multiple genetic, environmental and socio-economic reasons have been postulated to explain these findings (Shavers,2004; Oakley-Girvan, 2004; Meyer, 2007). However, socio-economic factors seem to have a limited role in cancer outcome in men of African origin, re-enforcing the genetic factors’ role in the disease process (Dash et al., 2008). Previous studies have earlier shown that the prevalence of prostate carcinoma in the Middle East, is very low compared to Western Countries. However, genetic and environmental factors believed to be involved in the complex aetiology of 137 prostate cancer in UAE are not clear yet, and awaiting investigation (Segev, 2006). In our study we have shown that there was no significant difference in age at presentation in the three ethnic groups. Our results concur with Shavers et al, who have shown that there is no significant difference in age at presentation with prostate cancer (Shavers et al., 2004). However, our results differ from SEER study in their findings that African American men tend to have an earlier age in presentation in many cancers, even though their finding with regards to prostate cancer was not statistically significant. Our results may reflect the similarity in accessing health care by the three population groups, and indeed between the UK population groups (Caucasians and men from African-Caribbean origin) (Karami et al., 2007). It has been shown that men of African origin tend to present with higher PSA, compared to Caucasian men. This was thought to be partly related to the socioeconomic status and partly to biological factors. We have demonstrated in our pilot study that presenting PSA in the cancer patients tends to be higher in the Caucasian group, then African-Caribbean men and lastly the Middle East men. Our results vary from prior studies in this field. This may be explained by the fact that the difference in socio-economic status in the UK (particularly the fact that all UK men in our study were recruited from the same geographic area) is less pronounced in men with different ethnic background. However this does not 138 explain the biological factors affecting this outcome (Pan et al., 2003; Wolf et al., 2007; Moul et al., 1995; Henderson et al., 1997). Our findings coincide with Kamal et al. in their study which have demonstrated that PSA tends to be lower in men from the Middle East (Kamal et al., 2003). We have shown that men with prostate cancer from the Middle East have a significantly better prognosis, compared to Caucasian and African-Caribbean men, sharing the same histological characters of the disease. Our findings concur with other studies in the literature showing that black men have the highest incidence and mortality from prostate cancer in the world (Freedland, 2005). In fact we have shown earlier in the UK population that prostate cancer is proximately treble in men of African-American origin than in white men, and lowest in Asian and oriental men, suggesting a strong ethnic influence (Chinegwundoh, 2006). Being a hormone dependant cancer, one of the postulated theories was the elevated androgen level in African-Caribbean men. While reports of racial differences in gonadal steroid hormone levels in middle-aged men have produced conflicting results, there is evidence that high SHBG and androstenedione levels are more common among young adult African American men than white men (Mohler, 2004). 139 Similarly, the relation between prostate cancer and androgen levels may well explain the low incidence of prostate cancer among the people from the Middle East; an ethnic group with lower TT and SHBG. In contrast, Caucasians have significantly higher serum levels of the precursor androgens, especially in early adulthood compared to Arab men. These may partially account for the decreased risk for prostate cancer among Arab men (Kehinde, 2006). hCG plays a paracrine role in the intratesticular regulation of testosterone secretion (Caroppo, 2003), since the beta subunit of hCG possesses 85% sequence homology with the first 114 amino acids of the beta subunit of pituitary human LH (Gadkari, 2005). Therefore, we hypothesized that hCG may be associated with prostate cancer through two possible mechanisms; firstly its relation with the production of male hormones and hence leading to an increase in the incidence and progression of prostate cancer. Secondly its para/autocrine effect on the tumourogenesis and its inhibitory effect on the apoptosis (Daehlin, 1985; Iles, 2007). We were able to demonstrate that there is statistically significant expression of the hCG among the African-Caribbean men (both in prostate cancer and BPH), as opposed to Caucasian men and then men from the Middle East population. However, we found no statistically significant association between the positive hCG status and the prognosis of prostate cancer. Our findings concur with an 140 earlier study in our department by Otite et al. who showed, by Immunoassay measurement of serum or urinary hCGβ that it has no significant association with the prognosis of prostate cancer (Otite et al., 2006). With regards to the vLH, we used nested PCR and DNA sequencing of the LHβ gene region as a definitive test for mutations in our population. We have shown that the occurrence of the vLH is extremely low in our three population groups (0.0107%), contradicting the previous studies in this field. We relied in our work on the nested PCR method, by which we excluded the hCG genes (which are six in number and are very similar to the LH gene). Our initial hypothesis was based on prior theories of a higher bioactivity and a shorter half-life of the vLH (Akhmedkhanov, 2000), hence the possibility of its association with increases androgen levels, which in turn may be associated with higher incidence of prostate cancer. However, the association of the vLH with undescended testis, a process relying on testosterone, at least in the first half of gestational life, and infertility, in which lack of endogenous testosterone might be a predisposing factor affecting spermatogenesis, may suggest that the vLH is associated with less bioactivity, thus disregarding our hypothesis of its role in the development of prostate cancer (Liao, 2002; Kaleva, 2005). 141 LIMITATIONS OF THE STUDY: The following factors were encountered at different stages of our work. These are summarized below: 1. Unequal number of the population groups in the study: this is attributed to the facts that there is a relatively low number of African-Caribbean men in the East London region presenting with LUTS and /or prostate cancer. 2. Discrepancy in the timescale for collection of samples between the three population groups: this is due to the fact that the prevalence of prostate cancer is extremely low in the Middle East population. This has lead the researchers to include participants, known with LUTS and/or prostate cancer over a longer time scale. 3. As a result of the above, there was a difference in the selection methods used in including the patients in the Middle East group (consecutive) as opposed to the Caucasian and the African-Caribbean groups. 4. Lack of standardized diagnostic and therapeutic measures in the three population groups: this is due to the fact that the study was carried out on participants from two different countries, with different standards of health care. This includes access to the health care and the screening methods used for prostate cancer in the two countries. This includes the Gleason scoring. However, the latter has been overcome by having the scoring repeated by a single independent uro-pathologist in the UK. 142 SUMMARY AND CONCLUSIONS: The association of the hCG beta, prostate cancer and ethnicity was studied in this thesis. The prevalence of the hCGβ in the African-Caribbean men is significantly higher (44%) than the Caucasians (36%) and the Middle East population (27%). Similarly, the same association exists with regards to its presence in the prostate cancer group. (72% vs. 28% vs. 20% respectively). However, there is no relation of the hCG beta with prognosis in the prostate cancer in the three groups. The prevalence vLH is extremely low in the population groups. There is no significant relation of the mutant gene with the neither incidence nor prognosis of prostate cancer. Similarly, there is no relation of the vLH with ethnicity in the groups included in the study. 143 RECOMMENDATIONS FOR FUTURE STUDIES: We believe that future studies can be performed on the association of the hCGβ and prostate cancer, in relation to the ethnicity, highlighting its action on the prostate cancer cells in terms of post translational modifications that in turn lead to its effects on the androgen secretion and its effects on apoptosis in prostate cancer cells. We have shown earlier in our department the effect of the hCGβ on apoptosis in bladder cancer (Butler et al., 2000). We aim to study its association with apoptosis in prostate cancer. In addition, our findings may suggest an important application of the use of the hCGβ as a potential target as a “vaccine” against tumours. Earlier studies at our labs showed reduction in tumour growth in a vaccinated rat serum (Butler et al., 2003). 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Expression of potential target antigens for immunotherapy on primary and metastatic prostate cancers. Clin Cancer Res. 4(2):295-302. 163 APPENDICES 164 APPENDIX I DATA EXTRACTION SHEET (information to be extracted from patients’ notes) PATIENT DETAILS Patient name: DOB: Place of birth: Ethnic Origin: White African-Caribbean Middle East Region Hospital number: Name of consultant: Name of Hospital: CLINICAL HISTORY Previous prostate problems? Yes/No If yes: 165 delete as appropriate Mode of presentation: 1. GP referal 2. Inpatient referal 3. Emergency 4. Incidental 5. others (please specify) 6. Abscent information Clinical presentations: 1. lower urinary tract symptoms acute urinary retention 3. sexually transmitted diseases nocturia 4. weight loss frequency 5. non of the above dysuria hesitancy Family history of prostatic cancer? urgency Yes/No poor stream haematuria other(please specify) delete as appropriate Past medical history: 1. diabetes 2. urinary tract infection 166 2. hypertension 6 CT SCAN YES NO 3. cardiac 7 MRI YES NO 4. others(please specify) 8 OTHERS YES NO Prostate cancer? Yes/No delete as appropriate If yes: Rectal examination findings: 1. normal prostate 2. enlarged benign prostate 3. clinically suspicious prostate Clinical stage: Investigations (please circle appropriate response) 1 TRUS YES NO 2 CXR YES NO 3 PSA YES NO 4 BONE SCAN YES NO 5 US KUB YES NO 167 APPENDIX II IMMUNOHISTOCHEMICAL DETECTION OF THE hCG EXPRESSION IN PARAFFIN EMBEDED ARCHIVAL PROSTATE TISSUES MATERIALS Primary Antibody: Rabbit Anti-Human Chorionic Gonadotropin (hCG) (DakoCytomation, Cat# A0231). Optimal dilution 1:20,000. Species Reactivity: Human (refer to antibody datasheet for more information). Secondary Antibody: Goat Anti-Rabbit IgG (H+L), biotinylated (Vector Laboratories, Cat# BA-1000). Optimal dilution 1:500. Tertiary antibody: Avidin and biotinylated horseradish peroxidase macromolecular complex solution Detection Reagent: HRP-Streptavidin (Vector Laboratories, Cat# SA-5004). Optimal dilution 1:500 METHODS 1. Deparaffinize section in 2 changes of xylene, 5 minutes each. 2. Re-hydrate in 2 changes of absolute alcohol, 3 minutes each. 3. Continue re-hydrating in 90% alcohol for 3 minutes and 70% alcohol for 3 minutes. 4. Peroxidase blocking: put in 3%H2O2 in methanol for 10 minutes to block endogenous peroxidase. 5. Wash briefly in running tap water. 6. Rins in PBS for 2 minutes. 7. Epitope retrieval: put into 0.1% TRITON-100 in PBS for 10 minutes. 8. Wash in 2 changes of PBS, 5 minutes each. 9. Serum blocking: incubate section with normal horse serum (50% in PBS) for 10 minutes to block non-specific binding of immunoglobulin. 10. Primary antibody: Incubate sections with rabbit anti-human chorionic gonadotrophin (hCG) polyclonal antibody diluted 1:6000 overnight at 4oC. 11. Rinse in PBS for 2x5minutes 12. Secondary antibody: incubate section with biotinylated anti-rabbit IgG diluted in PBS with addition of horse serum for 30 minutes at room temperature. 13. Rinse in PBS 2x5 minutes. 169 14. tertiary antibody: incubate section with avidin and biotinylated horseradish peroxidase macromolecular complex solution for 20 minutes at room temperature. 15. Rins in PBS 2x5 minutes. 16. Homogen/Substrate: incubate sections in DAB peroxidase substrate solution for 5 minutes. 17. Rinse briefly in distilled water. 18. Counterstain with Gill’s hematoxylin solution. 19. Differentiate in 1% acid alcohol for few seconds. 20. Wash in running tap water for 5 minutes. 21. Dehydrate through 70% alcohol for 2 minutes, 90% alcohol 2 minutes, 2 changes of absolute alcohol 3 minutes each. 22. Clear in 2 changes of xylene, 3 minutes each. 23. Mount with xylene based mounting medium. 170 APPENDIX III DNA EXTRACTION FROM PARAFFIN-EMBEDDED PROSTATE TISSUES PROTOCOL: 1. Slices (20 µm) of formalin-fixed and paraffin-embedded tumor samples were cut using microtome. Samples are then put in 2 ml Eppindorf collection tubes. (4 µm slices before and after each 20 µm slice were cut for HaematoxylinEosin staining to insure that the tissue is still representative.) 2. Tissues are incubated in xylene at 45°C for 15 min. 3. samples centrifuged 10 min at 14,000 rpm. 4. Resultant supernatant is pipetted off. 5. steps 2-4 repeated. 6. 1 ml 100% ethanol added to tissue pellet, vortexed and then centrifuged for 10 min at 14,000 rpm, and supernatant pipetted off. 7. 1 ml 90% ethanol added to tissue pellet, vortexed, centrifuged for 10 min at 14,000 rpm, and supernatant pipetted off. 8. Add 1 ml 70% ethanol to tissue pellet, vortexed, centrifuged for 10 min at 14,000 rpm, and supernatant pipetted off. Pellet then dried in speed vac. 171 9. Pellet resuspended in 400 µl of DNA extraction buffer. 10. 40 µl of Proteinase K (10 mg/ml) added, vortexed briefly, and incubated at 55°C overnight. 11. 440 µl of phenol added, shook vigorously by hand for 5 min, and centrifuged for 5 min at 8000 rpm. 15. Supernatant pipetted into a new tube, a solution of 220 µl phenol plus 220 µl chloroform/isoamyl alcohol (24:1 )is then added, shook vigorously by hand for 5 min, and centrifuged for 5 min at 8000 rpm. 16. Supernatant pipetted into a new tube, 440 µl chloroform/isoamyl alcohol (24:1) added, shook vigorously by hand for 5 min, and centrifuged for 5 min at 8000 rpm. 17. Supernatant pipetted into a new tube (2 ml Eppindorf tube), 1/10 volume of sodium acetate (pH 5.2)added. In addition, 3 volumes of ice cold 100 % ethanol add, and tube kept overnight at –20°C. 18. tubes centrifuged for 30 min at 4°C. 19. Supernatant removed. 20. Pellet dried in speed vac. 21. 20-50 µl sterile H20 added. 22. Shake gently in thermomixer at 37°C for 2 hours (DNA should be dissolved, 172 but if you have doubts put on a rotating shaker in the cold room overnight). 23. Measure DNA concentration with a spectrophotometer and run around 200 ng on a 1% agarose gel. 173 APPENDIX IV PCR using LH long primers (LH-LP) PCR conditions: 950C for five minutes: 1 cycle. 950 for 30 seconds, 580C 30 seconds and 720C 1 minute: 30 cycles. 720C for 5 minutes: 1 cycle. PCR mix: 2.0μl of DNA 2.5μl of MgCl2 (25mM) 2.5μl of Buffer (75mM Tris-HCl (pH 8.8, 20mM (NH4)2SO4 0.3μl of dNTPs (300μM of each of the dNTPs: dATP, dGTP, dCTP, dTTP). 2.0μl of Primers (LH-LP: forward and reverse: 2.5 pmol each) 0.2μl of Taq polymerase (5 units/μl Abgene) 15.5μl of H2O 174 APPENDIX V DNA EXTRACTION AND PURIFICATION FROM AGAROSE GEL PROTOCOL: 1. The gel slice containing the DNA band with a clean is excised with a sharp scalpel. 2. 3 volumes of Buffer QG are added to 1 volume of supernatant and mix. 8. QIAquick Spin Column is placed in a provided 2 ml collection tube. 9. To bind DNA, the QIAquick Spin Column containing the sample is centrifuged for one minute. 10. The flow-through is discard and QIAquick Spin Column is placed back into the same collection tube. 11. To wash, we add 0.75 ml Buffer PE to column and then centrifuge for 30–60 s. 12. The flow-through is discard and QIAquick Spin Column is placed back into the same collection tube. We then centrifuge column for an additional 1 min at maximum speed. (IMPORTANT: Residual ethanol from Buffer PE will not be completely removed unless the flow-through is discarded before this additional centrifugation.) 13. QIAquick Spin Column is placed into a clean 1.5 ml microcentrifuge tube. 14. To elute DNA, we add 20 µl Buffer EB (10 mM Tris·Cl, pH 8.5) to the center of the QIAquick Spin Column. We let it stand for 1 min and then centrifuge for 1 min., 175 APPENDIX VI PCR using LH short primers (LH-SP): PCR conditions: 950C for five minutes: 1 cycle. 950 for 30 seconds, 580C 30 seconds and 720C 1 minute: 35 cycles. 720C for 5 minutes: 1 cycle. PCR mix: 4.0μl of DNA 2.5μl of MgCl2 (25mM) 2.5μl of Buffer (75mM Tris-HCl (pH 8.8, 20mM (NH4)2SO4 0.3μl of dNTPs (300μM of each of the dNTPs: dATP, dGTP, dCTP, dTTP). 2.0μl of Primers (LH-LP: forward and reverse: 2.5 pmol each) 0.2μl of Taq polymerase (5 units/μl Abgene) 13.5μl of H2O 176 RESEARCH COMMITTEE 25th July 2005 Northern and Yorkshire MREC Ms. Sandy Brunton-Shiels Sunderland Teaching Primary Care Administration Corridor Ryhope Hospital Ryhope, Sunderland, England SR2 8AE RE: THE ASSOCIATION BETWEEN THE SUB-UNIT OF HUMAN CHORIONIC GONADAPTROPHIN AND THE GENETIC VARIANT WITH THE PROGNOSIS OF PROSTATE CANCER IN MULTI-ETHNIC POPULATION GROUPS Dear Ms. Brunton-Shiels, I am pleased to inform you that the above protocol has been reviewed by the SKMC Research Committee and approved on 7th June 2005. Yours sincerely, PATRICK KILLORN, MD, FRCPC Chairman, Research Committee cc: Dr. Ali Thwaini, England` Dr. Donald Morgan, SKMC Research Committee Members 177 178 Sunderland Teaching Primary Care Trust (South Office) Administration Corridor Ryhope Hospital Ryhope Sunderland SR2 0LY Tel: 0191 569 9559 Fax: 0191 569 9545 15 July 2005 Mr Ali Thwaini Research Fellow - Urology Barts and the London TRUST West Smithfield London E1 1BB Dear Mr Thwaini Full title of study: REC reference number: The relationship between Beta subunits of Human Chorionic Gonadotropin(hCG beta)and the genetic variant of Luteinizing Hormone, with the prognosis of prostate cancer in different population groups of multi-ethnic origin. 05/MRE03/52 The Research Ethics Committee reviewed the above application at the meeting held on 08 July 2005. Documents reviewed Provisional opinion The Committee would be content to give a favourable ethical opinion of the research. The formal approval letter documentation will arrive by post shortly. Good luck with the research! Peter Carey Consultant Haematologist E floor, Sunderland Royal Hospital SR4 7TP tel 0191 5656256 ext 47479 fax 0191 5699007 email: [email protected] Miss Sandy Brunton-Shiels MREC Administrator. 179 180