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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). We aim to conduct in vivo studies the hCGβ-combined vaccine in prostate
cancer.
144
REFERENCES:
Abdelrahaman E, Raghavan S, Baker L, Weinrich M, Winters SJ (2005). Racial
difference in circulating sex hormone-binding globulin levels in prepubertal boys.
Metabolism.54(1):91-6.
Akhmedkhanov A, Toniolo P, Zeleniuch-Jacquotte A, Pettersson KS, Huhtaniemi
IT (2000). Genetic Variant of Luteinizing Hormone and Risk of Breast Cancer in
Older Women. Cancer Epidemiology Biomarkers & Prevention.9: 839-842.
Aumuller G, Adler G. Experimental studies of apocrine secretion in the dorsal
prostate epithelium of the rat (1979). Cell Tissue Res. 198(1):145-58.
Bendell JJ, Dorrington JH. Epidermal growth factor influences growth and
differentiation of rat granulosa cells (1990). Endocrinology. 127(2):533-40.
Ben-Shlomo Y, Evans S, Ibrahim F, Patel B, Anson K, Chinegwundoh F,
Corbishley C, Dorling D, Thomas B, Gillatt D, Kirby R, Muir G, Nargund V, Popert
R, Metcalfe C, Persad R; PROCESS study group (2008). The risk of prostate
145
cancer amongst black men in the United Kingdom: the PROCESS cohort study.
Eur Urol. 53(1):99-105.
Bergendah M, Veldhuis JD (2001). Is there a physiological role for gonadotropin
oligosaccharide
heterogeneity
in
humans?
III.
Luteinizing
hormone
heterogeneity: a medical physiologist's perspective. Hum Reprod.16(6):1058-64.
Berger P, Gruschwitz M, Spoettl G, Dirnhofer S, Madersbacher S, Gerth R, Merz
WE, Plas E, Sampson N (2007). Human chorionic gonadotropin (hCG) in the
male reproductive tract. Mol Cell Endocrinol. 2:260-262.
Bevan CL (2005). Androgen receptor in prostate cancer: cause or cure? Trends
Endocrinol Metab.16(9):395-7.
Bodek G, Kowalczyk A, Waclawik A, Huhtaniemi I, Ziecik AJ (2005). Targeted
ablation of prostate carcinoma cells through LH receptor using Hecate-CGbeta
conjugate: functional characteristic and molecular mechanism of cell death
pathway. Exp Biol Med.230(6):421-8.
Bratt O (2002). Hereditary prostate cancer: clinical aspects. J Urol.168(3):906-13.
146
Burchardt M, Burchardt T, Chen MW (2000). Vascular endothelial growth factor a
expression in the rat ventral prostate gland and the early effects of castration.
Prostate.43:184–194.
Butler, S.A., Ikram, M.S., Mathieu, S., Iles, R.K (2000). The increase in bladder
carcinoma cell population induced by the free beta subunit of human chorionic
gonadotropin is a result of an anti-apoptosis effect and not cell proliferation. Br. J.
Cancer. 82:1553–1556.
Butler, S.A., Staite, E.M., Iles, R.K (2003). Reduction of bladder cancer cell
growth in response to hCG_ CTP37 vaccinated mouse serum. Oncol. Res.14: 1–
8.
Chinegwundoh F I, Enver M K, Lee A, Ben-Shlomo Y, Kapoor S, Pati J (2003).
Increased risk of prostate cancer in UK African Caribbean immigrants. BJU Int.9:
supplement 2, abstract 79.
Chinegwundoh F, Enver M, Lee A, Nargund V, Oliver T, Ben-Shlomo Y (2006).
Risk and presenting features of prostate cancer amongst African-Caribbean,
South Asian and European men in North-east London. BJU Int. 98(6):1216-20.
Chan TY, Epstein JI (1999): Follow-up of atypical prostate needle biopsies
suspicious for cancer. Urology. 53:351–355.
147
Counis R, Laverriere JN, Garrel G, Bleux C, Cohen-Tannoudji J, Lerrant Y,
Kottler ML, Magre S (2005). Gonadotropin-releasing hormone and the control of
gonadotrope function. Reprod Nutr Dev. 45(3):243-54.
Cregger M, Berger AJ, Rimm DL (2006). Immunohistochemistry and quantitative
analysis of protein expression. Arch Pathol Lab Med.130(7):1026-30
Cunha CR, Chung LWK, Shannon JM (1983). Hormone-induced morphogenesis
and growth: Role of mesenchymal-epithelial interactions. Recent Prog Hormone
Res.39:559.
Daehlin L (1985). On the effects of oestrogen in the male. Some effects of
different oestrogenic substances in rats and men with prostatic carcinoma. Scand
J Urol Nephrol Suppl. 91:1-44.
Daja MM, Aghmesheh M, Ow KT, Rohde PR, Barrow KD, Russell PJ (2000).
Beta-human chorionic gonadotropin in semen: a marker for early detection of
prostate cancer? Mol Urol. 4(4):421-7.
Dash A, Lee P, Zhou Q, Jean-Gilles J, Taneja S, Satagopan J, Reuter V, Gerald
W, Eastham J, Osman I (2008). Impact of Socioeconomic Factors on Prostate
Cancer Outcomes in Black Patients Treated with Surgery. Urology.
148
Davies, S. Ectopic expression of glycoprotein hormones and their receptors by
urogenital cancers London, United Kingdom: PhD thesis. University of London,
2001.
Devi GR, Oldenkamp JR, London CA, Iversen PL (2002). Inhibition of human
chorionic gonadotropin beta-subunit modulates the mitogenic effect of c-myc in
human prostate cancer cells. Prostate. 53(3):200-10.
Dirnhofer S, Berger C, Hermann M, Steiner G, Madersbacher S, Berger P
(1998). Coexpression of gonadotropic hormones and their corresponding FSHand LH/CG-receptors in the human prostate. Prostate. 35(3):212-20.
Dirnhofer S, Freund M, Rogatsch H, Krabichler S, Berger P (2000). Selective
expression of trophoblastic hormones by lung carcinoma: neurendocrine tumors
exclusively produce human chorionic gonadotropin alpha-subunit (hCGalpha).
Hum Pathol. 31(8):966-72.
Douglas JA, Zuhlke KA, Beebe-Dimmer J, Levin AM, Gruber SB, Wood DP,
Cooney KA (2005). Identifying susceptibility genes for prostate cancer-a familybased association study of polymorphisms in CYP17, CYP19, CYP11A1, and
LH-beta. Cancer Epidemiol Biomarkers Prev.14(8):2035-9.
149
EAU guidelines
(http://www.uroweb.org/professional-resources/guidelines/)
Epstein JI, Walsh PC, CarMichael M, Brendler CB (1994). Pathological and
clinical findings to predict tumor extent of non-palpable (stage T1c) prostate
cancer. JAMA. 271:368–374.
Elkins DA, Yokomizo A, Thibodeau SN, J Schaid D, Cunningham JM, Marks A,
Christensen E, McDonnell SK, Slager S, J Peterson B, J Jacobsen S, R Cerhan
J, L Blute M, J Tindall D, Liu W (2003). Luteinizing hormone beta polymorphism
and risk of familial and sporadic prostate cancer. Prostate. 15;56(1):30-6.
Elter K, Erel CT, Cine N, Ozbek U, Hacihanefioglu B, Ertungealp E (1999) Role
of the mutations Trp8 => Arg and Ile15 => Thr of the human luteinizing hormone
beta-subunit in women with polycystic ovary syndrome. Fertil Steril. 71(3):42530.
Fowler JE Jr, Platoff GE, Kubrock CA, Stutzman RE (1982). Commercial
radioimmunoassay for beta subunit of human chorionic gonadotropin: falsely
positive determinations due to elevated serum luteinizing hormone. Cancer
49:136-9.
150
Forest MG (1979). Plasma androgens (testosterone and 4-androstenedione) and
17-hydroxyprogesterone in the prenatal, prepubertal and peripubertal periods in
the human and the rat: Differences between species. J Ster Biochem. 11:543–
548.
Freedland SJ, Isaacs WB (2005) Explaining racial differences in prostate cancer
in the United States: sociology or biology? Prostate.15;62(3):243-52.
Furui K, Suganuma N, Tsukahara S, Asada Y, Kikkawa F, Tanaka M, Ozawa T,
Tomoda Y (1994) Identification of two point mutations in the gene coding
luteinizing
hormone
(LH)
beta-subunit,
associated
with
immunologically
anomalous LH variants. J Clin Endocrinol Metab. 78(1):107-13.
Gadkari RA, Roy S, Rekha N, Srinivasan N, Dighe RR (2005). Identification of a
heterodimer-specific epitope present in human chorionic gonadotrophin (hCG)
using a monoclonal antibody that can distinguish between hCG and human LH. J
Mol Endocrinol. 34(3):879-87.
Gann PH (1997). Interpreting recent trends in prostate cancer incidence and
mortality. Epidemiology. 8(2):117-20.
Getzenberg RH, Pienta KJ, Coffey DS (1990). The tissue matrix: Cell dynamics
and hormone action. Endocr Rev.11:399–416.
151
Ghafoor M, Schuyten R, Bener A (2003). Epidemiology of prostate cancer in
United Arab Emirates. Med J Malaysia. 58(5):712-6.
Gleason DF, Mellinger GT, Veterans Administration Cooperative Urological
Research Group (1974). Prediction of prognosis for prostatic adenocarcinoma by
combined histologic grading and clinical staging. J Urol.111;58–64.
Haavisto AM, Pettersson K, Bergendahl M, Virkamaki A, Huhtaniemi I (1995).
Occurrence and biological properties of a common genetic variant of luteinizing
hormone. J Clin Endocrinol Metab. 80(4):1257-63.
Habib FK, Ross M, Tate R, Chisholm GD (1997). Differential effect of finasteride
on the tissue androgen concentrations in benign prostatic hyperplasia. Clin
Endocrinol. 46:137–144.
Habuchi T, Suzuki T, Sasaki R, Wang L, Sato K, Satoh S, Akao T, Tsuchiya N,
Shimoda N, Wada Y, Koizumi A, Chihara J, Ogawa O, Kato T (2000).
Association of vitamin D receptor gene polymorphism with prostate cancer and
benign prostatic hyperplasia in a Japanese population. Cancer Res. 60:305–308.
152
Hansel W, Leuschner C, Gawronska B, Enright F (2001). Targeted destruction of
prostate cancer cells and xenografts by lytic peptide-betaLH conjugates. Reprod
Biol. 1(1):20-32.
Hassan MO, Maksem J (1980). The prostatic perineural space and its relation to
tumor spread. Am J Surg Pathol. 4:143–148.
Henderson RJ, Eastham JA, Culkin DJ, Kattan MW, Whatley T, Mata J, Venable
D, Sartor O (1997). Prostate-specific antigen (PSA) and PSA density: racial
differences in men without prostate cancer. J Natl Cancer Inst. 15;89(2):134-8
Hill M, Huhtaniemi IT, Hampl R, Starka L (2001). Genetic variant of luteinizing
hormone: impact on gonadal steroid sex hormones in women. Physiol Res.
50(6):583-7.
Hotakainen K, Lintula S, Ljungberg B, Finne P, Paju A, Stenman UH, Stenman J
(2006). Expression of human chorionic gonadotropin beta-subunit type I genes
predicts adverse outcome in renal cell carcinoma. J Mol Diagn. 8(5):598-603.
Hotakainen K, Lintula S, Jarvinen R, Paju A, Stenman J, Rintala E, Stenman UH
(2007). Overexpression of human chorionic gonadotropin beta genes 3, 5 and 8
in tumor tissue and urinary cells of bladder cancer patients. Tumour Biol.
28(1):52-6.
153
http://biology.clc.uc.edu/courses/bio105/sexual.htm
http://nobelprize.org/nobel_prizes/chemistry/laureates/1980/sanger-lecture.pdf
Huhtaniemi I, Jiang M, Nilsson C, Pettersson K (1999). Mutations and
polymorphisms in gonadotropin genes. Mol Cell Endocrinol. 25;151(1-2):89-94.
Caroppo E, Niederberger C, Iacovazzi PA, Correale M, Palagiano A, D'Amato G.
Human chorionic gonadotropin free beta-subunit in the human seminal plasma
(2003): a new marker for spermatogenesis? Eur J Obstet Gynecol Reprod Biol.
2003 Feb 10;106(2):165-9.
Iehlé C, Délos S, Guirou O, Tate R, Raynaud JP, Martin PM (1995). Human
prostatic steroid 5-alpha-reductase isoforms—a comparative study of selective
inhibitors. J Steroid Biochem Mol Biol. 54:273–279.
Iles RK (2007). Ectopic hCGbeta expression by epithelial cancer: malignant
behaviour, metastasis and inhibition of tumor cell apoptosis. Mol Cell Endocrinol.
2;260-262:264-70
154
James E. Montie, M.D. Staging of prostate cancer (1994). Current TNM
classification and future prospects for prognostic factors. Cancer.75(S7):1814 1818
Jameson JL (1996). Inherited disorders of the gonadotropin hormones. Mol Cell
Endocrinol. 20;125(1-2):143.
Kaleva M, Virtanen H, Haavisto AM, Main K, Skakkebaek NE, Huhtaniemi I, Irjala
K, Toppari J (2005). Does variant luteinizing hormone (V-LH) predispose to
improper testicular position in late pregnancy? Pediatr Res. 58(3):447-50.
Kamal BA, Ali GA, Taha SA (2003). Prostate specific antigen reference ranges in
Saudi men. Saudi Med J. 24(6):665-8.
Karami S, Young HA, Henson DE (2007). Earlier age at diagnosis: another
dimension in cancer disparity? Cancer Detect Prev. 31(1):29-34.
Kawaguchi K, Oda Y, Saito T, Yamamoto H, Takahira T, Tamiya S, Iwamoto Y,
Tsuneyoshi M (2004). Death-associated Protein kinase (DAP kinase) alteration in
soft tissue leiomyosarco: promoter methylation or homozygous deletion is
associated with a loss of DAP kinase expression. Hum. Pathol. 35(10): 1266–
1271.
155
Kehinde EO, Akanji AO, Memon A, Bashir AA, Daar AS, Al-Awadi KA, Fatinikun
T (2006). Prostate cancer risk: the significance of differences in age related
changes in serum conjugated and unconjugated steroid hormone concentrations
between arab and caucasian men. Int Urol Nephrol. 38(1):33-44.
Lamminen T, Huhtaniemi I (2001). A common genetic variant of luteinizing
hormone; relation to normal and aberrant pituitary-gonadal function. Eur J
Pharmacol. 23;414(1):1-7.
Lamminen T, Jiang M, Manna PR, Pakarinen P, Simonsen H, Herrera RJ,
Huhtaniemi I (2002). Functional study of a recombinant form of human LHbetasubunit variant carrying the Gly(102)Ser mutation found in Asian populations. Mol
Hum Reprod. 8(10):887-92.
Laurence A. Cole (1997). Immunoassay of human chorionic gonadotropin, its
free subunits, and metabolites. Clinical Chemistry.43:2233-2243.
Li D, Wen X, Ghali L, Al-Shalabi FM, Docherty SM, Purkis P, Iles RK (2008).
hCG beta expression by cervical squamous carcinoma--in vivo histological
association with tumour invasion and apoptosis. Histopathology. 53(2):147-55
Liao WX, Goh HH, Roy AC (2002). Functional characterization of a natural
variant of luteinizing hormone. Hum Genet. 111(2):219-24. Epub 2002 Jul 16.
156
Mengual L, Oriola J, Ascaso C, Ballescà JL, Oliva R (2003). An increased CAG
repeat length in the androgen receptor gene in azoospermic ICSI candidates. J
Androl. 24(2):279-84.
Meyer TE, Coker AL, Sanderson M, Symanski E (2007). A case-control study of
farming and prostate cancer in African-American and Caucasian men.
Occup Environ Med. 64(3):155-60.
Mohler JL, Gaston KE, Moore DT, Schell MJ, Cohen BL, Weaver C, Petrusz P
(2004). Racial differences in prostate androgen levels in men with clinically
localized prostate cancer. J Urol. 171(6 Pt 1):2277-80.
Moul JW, Sesterhenn IA, Connelly RR, Douglas T, Srivastava S, Mostofi FK,
McLeod DG (1995). Prostate-specific antigen values at the time of prostate
cancer diagnosis in African-American men. JAMA. 25;274(16):1277-81
Nasseri K, Mills PK, Allan M (2007). Cancer Incidence in the Middle Eastern
Population of California, 1988–2004. Asian Pac J Cancer Prev.8(3): 405–411
Nilsson CH, Kaleva M, Virtanen H, Haavisto AM, Pettersson K, Huhtaniemi IT
(2001). Disparate response of wild-type and variant forms of LH to GnRH
stimulation in individuals heterozygous for the LHbeta variant allele. Hum
Reprod. 16(2):230-5.
157
Noeman SA, Sharada K, el Dardiry S, Rahim AA, Zaki Y (1994). Significance of
tumour markers in colorectal carcinoma associated with Schistosomiasis.
Bangladesh Med Res Counc Bull. 20(1):12-20.
Oakley-Girvan I, Feldman D, Eccleshall TR, Gallagher RP, Wu AH, Kolonel LN,
Halpern J, Balise RR, West DW, Paffenbarger RS Jr, Whittemore AS (2004).
Risk of early-onset prostate cancer in relation to germ line polymorphisms of the
vitamin D receptor. Cancer Epidemiol Biomarkers Prev. 13(8):1325-30.
Oakley-Girvan I, Kolonel LN, Gallagher RP, Wu AH, Felberg A, Whittemore AS
(2003). Stage at diagnosis and survival in a multiethnic cohort of prostate cancer
patients. Am J Public Health. 93(10):1753-9.
Otite U, Baithun S, Chinegwundoh F, Nargund VH, Iles RK (2006). Detection of
human chorionic gonadotrophin-beta in serum or urine of prostate cancer
patients is of no clinical significance. Tumour Biol. 27(4):181-6.
Pan CC, Lee JS, Chan JL, Sandler HM, Underwood W, McLaughlin PW (2003).
The association between presentation PSA and race in two sequential time
periods in prostate cancer patients seen at a university hospital and its
community affiliates. Int J Radiat Oncol Biol Phys. 1;57(5):1292-6.
158
Papandreou MJ, Asteria C, Pettersson K, Ronin C, Beck-Peccoz P (1993).
Concanavalin A affinity chromatography of human serum gonadotropins:
evidence for changes of carbohydrate structure in different clinical conditions. J
Clin Endocrinol Metab. 76(4):1008-13.
Parkin DM, Bray F, Ferlay J, Pisani P (2005). Global cancer statistics, 2002. CA
Cancer J Clin. 55(2):74-108
Perkins GL, Slater ED, Sanders GK, Prichard JG (2003). Serum tumor markers.
Am Fam Physician. 15;68(6):1075-82.
Pettersson K, Ding YQ, Huhtaniemi I (1992). An immunologically anomalous
luteinizing hormone variant in a healthy woman. J Clin Endocrinol Metab.
74(1):164-71.
Potischman N, Troisi R, Thadhani R, Hoover RN, Dodd K, Davis WW, Sluss PM,
Hsieh CC, Ballard-Barbash R (2005). Pregnancy hormone concentrations across
ethnic
groups:
implications
for
later
cancer
risk.
Cancer Epidemiol Biomarkers Prev. 14(6):1514-20.
Raivio T, Huhtaniemi I, Anttila R, Siimes MA, Hagenas L, Nilsson C, Pettersson
K, Dunkel L (1996). The role of luteinizing hormone-beta gene polymorphism in
159
the onset and progression of puberty in healthy boys. J Clin Endocrinol Metab.
81(9):3278-82.
Rao ChV, Li X, Manna SK, Lei ZM, Aggarwal BB (2004). Human chorionic
gonadotropin decreases proliferation and invasion of breast cancer MCF-7 cells
by inhibiting NF-kappaB and AP-1 activation. J Biol Chem. 11;279(24):25503-10.
Reed A, Ankerst DP, Pollock BH, Thompson IM, Parekh DJ (2007). Current age
and race adjusted prostate specific antigen threshold values delay diagnosis of
high grade prostate cancer. J Urol. 178(5):1929-32.
Crawford RA, Iles RK, Carter PG, Caldwell CJ, Shepherd JH, Chard T (1998).
The prognostic significance of ‚ human chorionic gonadotrophin and its
metabolites in women with cervical carcinoma. J Clin Pathol. 51:685–688.
Shaneyfelt T, Husein R, Bubley G, Mantzoros CS (2000). Hormonal predictors of
prostate cancer: a meta-analysis. J Clin Oncol. 18(4):847-53.
Shavers VL, Brown ML, Potosky AL, Klabunde CN, Davis WW, Moul JW, Fahey
A (2004). Race/ethnicity and the receipt of watchful waiting for the initial
management of prostate cancer. J Gen Intern Med. 19(2):146-55.
160
Sheaff MT, Martin JE, Badenoch DF, Baithun SI (1996). Beta hCG as a
prognostic marker in adenocarcinoma of the prostate. J Clin Pathol. 49(4):32932.
Span PN, Thomas CM, Heuvel JJ, Bosch RR, Schalken JA, vd Locht L, Mensink
EJ, Sweep CG (2002). Analysis of expression of chorionic gonadotrophin
transcripts in prostate cancer by quantitative Taqman and a modified molecular
beacon RT-PCR. J Endocrinol. 172(3):489-95.
Steinberg DM, Sauvageot J, Piantadosi S, Epstein JI (1997). Correlation of
prostate needle biopsy and radical prostatectomy Gleason grade in academic
and community settings. Am J Surg Pathol. 21:566–576.
Stephen A., Butler, Ray K. Iles (2003). Ectopic Human Chorionic Gonadotropin
Secretion by Epithelial Tumors and Human Chorionic Gonadotropin beta-Induced
Apoptosis in Kaposi’s Sarcoma: Is There a Connection?. Clinical Cancer
Research. 9: 4666–4673.
Talmadge K, Boorstein WR, Fiddes JC (1983). The human genome contains
seven genes for the beta-subunit of chorionic gonadotropin but only one gene for
the beta-subunit of luteinizing hormone. DNA. 2(4):281-9.
161
Talmadge K, Vamvakopoulos NC, Fiddes JC (1984). Evolution of the genes for
the beta subunits of human chorionic gonadotropin and luteinizing hormone.
Nature. 5-11;307(5946):37-40.
Themmen A, Huhtaniemi I (2000). Mutations of Gonadotropins and Gonadotropin
Receptors: Elucidating the Physiology and Pathophysiology of Pituitary-Gonadal
Function. Endocrine Reviews. 21 (5): 551-583.
Thompson TC, Chung LW (1986). Regulation of overgrowth and expression of
prostatic
binding
protein
in
rat
chimeric
prostate
gland.
Endocrinology. 118(6):2437-44.
Thompson AA, Cunha GR (1999). Prostatic growth and development are
regulated by FGF10. Development. 126(16):3693-701.
Veldhuis JD, Guardabasso V, Rogol AD, Evans WS, Oerter KE, Johnson ML,
Rodbard D (1987). Appraising the nature of luteinizing hormone secretory events
in men. Am J Physiol. 252(5):599-605.
www.bio.davidson.edu
www.cancerresearchuk.org
162
www.cancerscreening.nhs.uk
www.dnalc.org
www.protocol-online.org
Wagenlehner FM, Elkahwaji JE, Algaba F, Bjerklund-Johansen T, Naber KG,
Hartung R, Weidner W (2007). The role of inflammation and infection in the
pathogenesis of prostate carcinoma. BJU Int. 100(4):733-7.
Weissbach L, Bussar-Maatz R, Lohrs U, Schubert GE, Mann K, Hartmann M,
Dieckmann KP, Fassbinder J (1999). Prognostic factors in seminomas with
special respect to HCG: results of a prospective multicenter study. Seminoma
Study Group. Eur Urol. 36(6):601-8.
Zhang S, Zhang HS, Reuter VE, Slovin SF, Scher HI, Livingston PO (1998).
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
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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