FULLTEXT01

UMEÅ UNIVERSITY MEDICAL DISSERTATIONS
New series No. 991

ISBN 91-7305-962-5 ISSN 0346-6612
Department of Radiation Sciences, Oncology, Umeå University
Prostate Cancer and Inflammatory

Genes
Fredrik Lindmark
Umeå 2005
Copyright © 2005 by Fredrik Lindmark
ISSN 0346-6612
ISBN 91-7305-962-5
Printed by Print & Media, Umeå University, Umeå, 2005

To my Family
Prostate Cancer and Inflammatory Genes 1
Table of contents
Abstract, 3
Populärvetenskaplig sammanfattning, 5
Original studies, 7
List of Abbreviations, 8
Introduction, 9
Risk factors for prostate cancer, 10
Dietary factors, 10
Hormonal and other physiological factors, 11
Familial and genetic factors, 11
Inflammation and cancer, 15
The role of inflammation in the pathogenesis of prostate cancer, 17
Molecular pathology, 17
Epidemiology, 19
Genetics, 22
Cytokines in prostatic inflammation and prostate cancer, 24
Aims, 29
Material and methods, 30
Hereditary prostate cancer families used for mutation screening, 30
Patients with prostate cancer and controls, 30
The Northern Sweden Health and Disease Cohort, 30
CAPS, 30
Genetic analysis, 33
Genomic PCR, 33
Mutation screening, 33
Common sequence variants of the MSR-1 gene, 34
Association of MIC-1 sequence variants and prostate cancer, 35
Association of IL1-RN haplotype with prostate cancer risk, 36
Determination of MIC-1 serum levels, 38
Statistical analysis, 38
Tests for Hardy-Weinberg Equilibrium, 38
Association analysis, 39
Haplotype analysis, 39
Assessment of serum levels, 39
Results and comments, 40
Genetic analysis of MSR1 (Study I), 40
Genetic analysis of IL-1RN (Study II), 43
Genetic analysis of MIC-1 (Study III), 46
Analysis of MIC-1 serum levels (Study IV), 48
Discussion, 55
2 Prostate Cancer and Inflammatory Genes
Association studies, 55
Cytokine polymorphisms in prostate cancer, 58
Future perspectives, 61
Conclusions, 62
Acknowledgements, 63
References, 64
Original studies
I
II
III
IV
Abstract
Prostate cancer remains a significant health cancer and identified 18 sequence variants.
concern for men throughout the world. One previously reported truncation
Accumulating epidemiologic and molecular mutation was found more frequently in men
evidence suggests that inflammation is an with prostate cancer than in unaffected men,
important component in the aetiology of in accordance with previously published
prostate cancer. Supporting this hypothesis, results. However, the difference in
population studies have found an increased frequencies we found between these groups
risk of prostate cancer in men with a prior was not statistically significant. In addition,
history of certain sexually transmitted we genotyped five common polymorphisms
infections or prostatitis. More general in MSR1 in 215 men with unselected
evidence of a relationship between prostate cancer and 425 controls. No
inflammation and prostate cancer has been association between any of the five common
provided by reports indicating that daily use variants and prostate cancer were found.
of non steroidal anti-inflammatory drugs We then performed a comprehensive
(NSAIDs) may be associated with a lower genetic study using extensive population-
incidence of prostate cancer. The exact based case-control material to evaluate
mechanism whereby inflammation might act possible associations between sequence
in tumour development and progression variants in inflammation-related genes and
remains to be elucidated, but is likely to be prostate cancer. The first gene to be
complex. The genetic contribution to examined was interleukin-1 receptor antagonist
inflammatory responses involved in the (IL-1-RN), encoding a cytokine that plays an
development of prostate cancer has not yet important role in regulation of the
been extensively or systematically studied. inflammatory response by binding to the IL-
However, this thesis evaluates the role of 1 receptor and thus inhibiting the binding of
various inflammation-related genes in the the pro-inflammatory cytokines IL-1α and
pathogenesis of prostate cancer. IL-1β. Collectively, these three cytokines
The macrophage scavenger receptor 1 exert a central role in the protection against
(MSR1) is a transmembrane protein that is diverse lesions, ranging from microbial
mainly expressed by macrophages. This colonisation to infection and malignant
receptor mediates the binding, transformation. The genetic analysis of IL-
internalization and processing of a wide 1RN revealed that the most common
range of macromolecules, and is suggested haplotype was significantly associated with
to play a major role in the recognition and prostate cancer risk for patients with
clearance of pathogenic and damaged cells. prostate cancer, and further this association
Recent reports have suggested MSR1 to be a appears to be stronger in cases with
candidate gene for hereditary prostate advanced disease.
cancer. Therefore, we screened the MSR1 The macrophage inhibitory cytokine-1 (MIC-
gene among men with hereditary prostate 1), a member of the transforming growth-
factor-β superfamily has been shown to associated with an increased risk for prostate
exert diverse biological functions, including cancer. Further, the clear relation between
regulation of macrophage activity in the clinical stage and MIC-1 level also suggest
inflammatory response and both growth that MIC-1 may be useful as a prognostic
inhibition and induction of apoptosis in factor, where high serum concentration is
epithelial and other tumour cell lines. The associated with a poor prognosis.
genetic analysis of MIC-1 revealed that a In summary, our results provide further
seuqence variant (H6D) appears to be support for the assumption that
associated with a decreased prostate cancer polymorphisms in inflammatory genes play
risk. We also performed measurements of critical roles in prostate cancer susceptibility.
MIC-1 serum levels among patients with Additional studies are needed to elucidate
prostate cancer and healthy controls. These the mechanisms whereby the demonstrated
data indicate that serum MIC-1 levels are variations contribute to prostate cancer
development.
Populärvetenskaplig sammanfattning
Antalet män som insjuknar i prostatacancer försvar mot invaderande patogener har blivit
har ökat markant under de två senaste utförligt studerade.
decennierna, vilket lett till att den blivit den I det första arbetet undersöktes om
vanligast diagnostiserade cancerformen sekvensvariationer i en gen kallad Macrophage
bland män i de flesta industrialiserade Scavenger Receptor 1 (MSR1), kan påverka
länderna. År 2002 diagnostiserades 679,000 risken att insjukna i prostatacancer. MSR1 är
nya fall av prostatacancer världen över, ett protein lokaliserat i cellens membran och
medan 221,000 män avled av samma är i huvudsak uttryckt av makrofager.
sjukdom. Orsaken till incidensökningen Proteinet fungerar som en receptor med
beror delvis på förbättrade diagnosmetoder, möjlighet att binda till sig en mängd olika
men detta förklarar inte hela stegringen utan substanser, som lipopolysackarider
andra okända faktorer är också involverade. (fragment från bakterier), och oxiderade
De senaste decennierna har ett flertal studier lipoproteiner. Efter inbindning tas dessa upp
visat att inflammationsprocesser i av den aktuella cellen. Funktionen hos
prostatakörteln är en betydande faktor i MSR1 är inte helt klarlagd men den är bland
utvecklingen av prostatacancer. Den exakta annat involverad i igenkänning och
mekanismen för hur inflammation eliminering av patogener och skadade celler.
medverkar till utveckling och progression av Tidigare studier i en amerikansk population
tumörer i prostatakörteln är fortfarande har visat att mutationer i denna gen skulle
höljd i dunkel men är antagligen en komplex kunna ha betydelse för utvecklingen av
process. prostatacancer. Vi screenade MSR1 genen
Det mänskliga genomet består av ungefär hos män med ärftlig prostatacancer och
30000 olika gener. Alla dessa gener uppvisar identifierade ett antal sekvensvariationer. Ett
olika grad av strukturell variation mellan urval av dessa sekvensvariationer
olika individer. Denna variation kan i sin tur undersöktes bland 215 män med oselekterad
leda till en förändrad funktion hos det prostatacancer och 425 kontroller, men
protein som en gen kodar för, eller att ingen av dem medförde en förändrad risk att
mängden protein som produceras varierar. insjukna i prostatacancer. Våra resultat
Även gener involverade i inflammations- stödjer ej tidigare publicerade resultat där
processer uppvisar en sådan genetisk sekvensvariationer i MSR1-genen skulle ha
variation. Om denna variation har någon betydelse för uppkomsten av prostatacancer.
betydelse för risken att utveckla I nästföljande studier använde vi oss av
prostatacancer har inte tidigare blivit ett stort populationsbaserat
utförligt studerat. I denna avhandling har vi fall/kontrollmaterial med 780
studerat hur genetisk variation i olika prostatacancerfall och 1383 friska kontroller
inflammationsrelaterade gener påverkar för att studera om genetisk variation hos
risken att utveckla prostatacancer. Tre gener, inflammationsrelaterade gener medför en
vilka alla är involverade i reglering av ökad eller minskad risk att insjukna i
inflammationsresponsen eller kroppens prostatacancer.
Den första genen kallad Interleukin-1 Den genetiska analysen av MIC-1 visade att
receptor antagonist (IL1-RN), är ett lösligt en genetisk variant i genen (H6D), medför
protein, en cytokin, som reglerar kroppens en minskad risk att insjukna i prostatacancer.
inflammationsrespons genom att binda till Vidare mättes nivåerna av MIC-1-proteinet i
en Il-1 receptor och därigenom förhindra serum hos patienter med prostatacancer och
inbindning av två andra cytokiner, IL-1α och hos friska män för att se om MIC-1 nivåerna
Il-1β, som normalt driver på var förändrade hos patienter med
inflammationsresponsen. Tillsammans spelar prostatacancer. Analyserna visade att
dessa tre cytokiner en central roll i kroppens serumnivåerna var tydligt förhöjda hos
skydd mot många olika skador, från invasion patienter med prostatacancer. Nivåerna var
av mikrober till malign transformation. också positivt korrelerade med ökande ålder
Balansen mellan pådrivande och inhiberande och tumörstadie. Resultaten indikerar att
cytokiner bestämmer utvecklingen av en MIC-1 skulle kunna vara en användbar
inflammationsrespons. Den genetiska prognostisk faktor, där höga serumnivåer är
analysen av IL-1RN visade att bärare av den associerade med en dålig prognos.
vanligast förekommande sekvens- Således visar våra resultat att genetisk
kombinationen (haplotypen) av IL1-RN- variation i inflammationsrelaterade gener har
genen medför en ökad risk att insjukna i betydelse för uppkomst av prostatacancer.
prostatacancer. För att förstå med vilka mekanismer denna
Macrophage inhibitory cytokine-1 (MIC-1) är variation kan påverka utvecklingen av cancer
en medlem i en stor familj av proteiner är vidare studier nödvändiga. Vi fann också
kallade transforming growth factor β att mätning av MIC-1-nivåer i serum skulle
superfamily, som har visat sig medverka i en kunna vara av kliniskt värde som
mängd olika viktiga biologiska funktioner, prognostisk markör vid prostatacancer.
inkluderat reglering av inflammations-
responsen, tillväxthämning, men också
induktion av apoptosmekanismer i olika
tumörcellinjer.
Original studies
This thesis is based on the following studies.
I. Lindmark F, Jonsson BA, Bergh A, III. Lindmark F, Zheng SL, Wiklund F,

Stattin P, Zheng SL, Meyers DA, Bensen J, Augustsson Bälter K,
Xu J, Grönberg H. Analysis of the Chang KA, Hedelin M, Clark J,
macrophage scavenger receptor 1 Stattin P, Meyers DA, Adami HO,
gene in swedish hereditary and Isaacs W, Grönberg H, Xu J. H6D
sporadic prostate cancer. Prostate. polymorphism in macrophage-
2004 May 1;59(2):132-40. inhibitory cytokine gene-1
associated with prostate cancer.
II. Lindmark F, Zheng SL, Wiklund F, JNCI 2004 Aug 18;96(16):1248-54.
Augustsson Bälter F, Sun J, Chang
B, Hedelin M, Clark J, Johansson J- IV. Lindmark F, Wiklund F, Hunter M,
E, Meyers DA, Adami HO, Isaacs Xu J, Bälter K, Adami HO, Breit S,
W, Grönberg H, Xu J. Interleukin-1 Grönberg H. Serum levels of
receptor antagonist haplotype macrophage inhibitory cytokine-1 in
associated with prostate cancer risk. prostate cancer cases and controls.
Br J Cancer. 2005 Aug 22;93(4):493- Manuscript
7.
Reprints are reproduced with the permission of the

respective publishers
List of Abbreviations
AR Androgen receptor NSHDC Northern Sweden health and

BPH Benign prostatic hyperplasia disease cohort
BSA Bovine serum albumin OR Odds ratio
CAPS Cancer prostate in Sweden PCR Polymerase chain reaction
CHEK2 Checkpoint kinase 2 PIA Proliferative inflammatory
CEPH Centre d’Etude du atrophy
Polymorphisme Humain PIN Prostatic intraepithelial
CI Confidence interval neoplasia
DHT Dihydrotestosterone PSA Prostate specific antigen
DNA Deoxyribonucleic acid ROC receiver operator
DZ Dizygotic characteristic
ELISA Enzyme-linked ROS Reactive oxygen species
immunosorbent assay RNASEL Ribonuclease L gene
fPSA free PSA RNS Reactive nitrogen
GSTP1 Glutathione-S-transferase P1 species
htSNP Haplotype tagging SNP RR Risk ratio
HPC Hereditary prostate cancer SNP Single nucleotide
HPLC High performance liquid polymorphism
chromatography SPC Sporadic prostate cancer
HPV Human papilloma virus SR-A Class A scavenger receptor
HWE Hardy Weinberg Equilibrium SRD5A2 5α-Reductase type 2
IL-1α Interleukin-1 alpha STI Sexually transmitted
IL-1β Interleukin-1 beta infection
IL1-RN Interleukin-1 receptor TEAA Trietylammonium acetate
antagonist TGF-β Transforming growth factor
LD Linkage disequilibrium β
LPS Lipopolysacharide TMHA Temperature modulated
MIC-1 Macrophage inhibitory heteroduplex analysis
cytokine-1 TNM Tumour-node-metastasis
MSR1 Macrophage scavenger TNF-α Tumour necrosis factor α
receptor 1 tPSA total PSA
MZ Monozygotic VNTR Variable number of tandem
repeats
Introduction
In most developed countries, prostate lesions are found in the transition zone,
cancer is not only the most frequently which is located in the periurethral region
diagnosed malignancy in men, but also the (McNeal, 1968, 1988). Prostate cancer is
second leading cause of cancer-related commonly multifocal, i.e. there is generally
deaths in males. In 2002, an estimated more than one tumour with different origins
679,000 prostate cancer diagnoses were in the prostate at the time of diagnosis
made worldwide, accompanied by an (McNeal, 1988). Each of these tumours can
estimated 221,000 prostate cancer deaths show remarkable differences in gene
(Parkin et al., 2005). In Sweden, over 9000 expression and behaviour, which are
new diagnoses of prostate cancer were associated with varying prognoses. In its
recorded in the year 2003, accounting for clinical form, prostate cancers are diagnosed
35.3 % of cancer diagnoses among men that upon histological evaluation of needle
year (fig.1). Further, 2350 men died from biopsy samples of prostate tissue, and
this disease 2003. Although the incidence of classified as either local or advanced. In
prostate cancer is increasing steadily in localized disease, the treatments range from
almost all countries, the aetiology and radical prostatectomy, radiotherapy and
molecular mechanisms underlying the application of anti-androgens to “watchful
development and progression of the disease waiting” (Afrin et al., 2000; Drachenberg,
is still large unknown. 2000). If the cancer has spread outside the
Prostate cancer is often regarded as a prostate, with lymph nodes and bone being
disease of older men, but nearly a third of the most common sites for metastasis, the
men aged between 30 and 40 years have most common treatment is surgical or
been found to harbour small foci of prostate chemical castration, to lower androgen
cancer (Sakr et al., 1994). The majority of levels, causing reductions in tumour growth
these lesions are diagnosed at a much later and symptomatic relief, but not a cure.
age or do not progress to clinically However, after one to two years most
detectable tumours within the lifetime of the tumours will relapse to an androgen-
affected men. Cancer lesions can develop in independent, but still androgen receptor-
two different regions of the prostate gland, dependent, state that normally kills the
most commonly (in ~80% of cases) in the patient (Afrin et al., 2000; Drachenberg,
periphery zone, while most of the remaining 2000; Feldman et al., 2001).
Figure 1. Cancer incidence in Sweden 2003. The ten most frequently specified
cancer sites among men, according to the National Board of Health and Welfare.
Risk factors for prostate cancer
The etiological factors associated with variations in prostate cancer rates. A large
prostate cancer are varied and particularly number of epidemiological studies have
poorly understood compared to other investigated the association between dietary
common cancers. factors and prostate cancer, but the findings
The observation that prostate cancer risk have been mixed.
increases for Japanese immigrants to Hawaii Dietary fat. There is considerable
and Japanese immigrants to Los Angeles, consistency across studies indicating that a
suggests that diet and environmental high intake of fat, especially total fat and
differences play important roles (Shimizu et saturated fat, is a risk factor for prostate
al., 1991; Minami et al., 1993). However, the cancer (Kolonel, 2001). The mechanisms
increase is only to about 50% of the rate for mediating the effects of fat on prostate
Caucasian people and to 25% of that for carcinogenesis are not known, but it is
African-American people in the USA, hypothesized that the effect of dietary
suggesting that differences between ethnic factors, such as fat, may be mediated
populations are substantial and that through endogenous hormones (Bosland,
differences in incidence rate cannot be 2000).
explained solely by differences in the Phytoestrogens. Soy products contain
environment and lifestyle (Grönberg, 2003). isoflavonoids that have been shown to have
a prophylactic effect on prostate cancer
Dietary factors (Holzbeierlein et al., 2005). People in Asian
countries eat more soy products than people
Dietary habits are probably an important in Western countries and higher levels of
factor that contributes to the geographic phytoestrogen metabolites have been
observed in Asian men compared to Hormonal and other physiological

European men. This may provide a partial factors
explanation for the low incidence of prostate
cancer in Asia (Adlercreutz et al., 1993). Mice The growth and development of the
studies with human androgen-sensitive prostate is dependent on androgens.
prostate cancer cells have shown that a diet Ablation of androgens by surgical or medical
based on rye bread and soy protein reduces castration is an effective strategy in the
tumour size and secreted PSA levels, while treatment of advanced prostate cancer.
increasing apoptosis, and may thus inhibit Moreover, men with congenital
tumour growth (Landström et al., 1998; abnormalities in androgen metabolism and
Bylund et al., 2000). A subsequent pilot study those who undergo castration before
of men with prostate cancer indicated that a puberty do not develop prostate cancer
high intake of rye bread increases apoptosis (Haas et al., 1997).
in prostate tumour cells (Bylund et al., 2003). Several epidemiologic studies have
Vegetables and fruits. While chronic evaluated the possible association between
consumption of animal fat and red meats plasma testosterone concentrations and
may promote prostate cancer, intake of prostate cancer risk, but the results have
vegetables and fruit may protect against been inconsistent (Hsing, 2001). A recent
prostate cancer development (Chan et al., well designed study published in 2004
2001). A large prospective study of 130,544 reported that high levels of circulating
men found no association between the total testosterone are not associated with
consumption of fruits and vegetables and increased prostate cancer risk (Stattin et al.,
risks for prostate cancer among 1104 2004). However, if testosterone is important
prostate cancer cases. However, it still for the onset of prostate cancer, then it may
remains possible that there may be an need to be measured in early adulthood,
association with specific types of fruits and years before the prostate cancer is actually
vegetables (Key et al., 2004). detected (Crawford, 2003).
Vitamins and trace elements. A variety of Other factors such as smoking, weight,
vitamins, trace elements have been alcohol consumption, physical activity,
elucidated for their role in prostate cancer socioeconomic status, and vasectomy have
pathogenesis, but the results of the also been evaluated for their role in prostate
epidemiology studies are inconsistent (Chan pathogenesis, but the results have generally
et al., 2001). A promising substance is indicated that they are not significant
selenium; an essential, non-metallic trace (Grönberg, 2003).
element that has been demonstrated to
promote apoptosis in prostate cancer cells Familial and genetic factors
and, possibly, impairs their proliferation
through antiangiogenic activity. Selenium
Over the past two decades multiple pieces of
intake has been significantly associated with
evidence have been accumulated supporting
a decreased risk for prostate cancer, for a
the view that genetics plays a role in prostate
review see Combs GF, 2004 (Combs, 2004).
cancer susceptibility and pathogenesis.
However, the evidence also indicates that
the genetic basis of prostate cancer is much Twin studies

more complex than was initially anticipated. The most straightforward way to estimate
Large families have been observed in which the impact of genetic factors on the
prostate cancer cluster together. Early development of prostate cancer is to analyze
observations were made in large families its incidence in mono- and di-zygotic twins.
collected and studied in Utah, but the first This is because monozygotic twins (MZ)
description of familial aggregation of derive from the fission of a single fertilized
prostate cancer was reported as far back as egg, thus they inherit identical genetic
1956, (MORGANTIG et al., 1956). This material and the only genetic differences
initiated a series of case-control studies, between them originate from somatic
cohort studies, and twin studies that changes. By contrast, dizygotic (DZ) twins
generated evidence of a familial component. derive from two distinct fertilized eggs and
thus have the same genetic relationship as
full siblings, although they may be more
Case-control and cohort studies “biologically” related because they share the
Several published reports have demonstrated same prenatal intra-uterine experience. Since
an increased risk for prostate cancer among the environmental factors affecting pairs of
men with a positive family history for MZ and DZ twins are generally very similar,
prostate cancer. In 2003, Bruner et al. since they are usually raised in the same
published a meta-analysis of 16 case-control environment, it is assumed that any
studies and eight cohort studies supporting differences in concordance rates between
this working theory, in which family history them solely reflect genetic factors. Two large
was defined as cases where a father, brother, twin studies from America and Scandinavia
any first- or second-degree relative or other have been published and the results they
relative had been affected by prostate cancer present show surprisingly consistency (Page
(Bruner et al., 2003). Pooled Risk ratios et al., 1997; Lichtenstein et al., 2000). Both
(RRs) for men with any affected family studies found that an MZ twin whose
member, one affected first-degree relative brother has been reported as having had
and one affected second-degree relative were prostate cancer is nearly four times more
1.9 (95% CI, 1.6-2.2), 2.2 (95% CI, 2.1-2.4) likely to have prostate cancer than a DZ
and 1.9 (95% CI, 1.6-2.3), respectively. twin whose brother has reportedly had
Further increases in RR have consistently prostate cancer. Furthermore, Page et al. and
been observed (i) when more than one Lichtenstein et al. estimated that the
relative is affected, (ii) as the closeness of the proportion of the risk of prostate cancer
affected relative to the unaffected individual that can be explained by heritable factors
increases, and (iii) as the age of onset of the was estimated to 57% and 42%, respectively.
affected cases decreases. Taken together,
this is all strong evidence for the
involvement of a genetic component in Family-based segregation analyses
familial disease. Further support for a genetic component in
prostate cancer comes from family-based
segregation analyses, which have been used
to derive models for the prevalence (rare,
common), mode of inheritance (dominant, Carriers of mutations of another gene,

recessive, autosomal, or sex chromosome BRCA2, are known to be at high risk of
linked), and penetrance (the likelihood of breast and ovarian cancer, but BCRA2 has
carriers developing the disease by a certain also been implicated in prostate cancer
age) of genes that may be involved in predisposition. A large collaborative study
familial prostate cancer. In 2004, Daniel from the Breast Cancer Linkage Consortium
Schaid reviewed the eight segregation estimated, based on a survey of 173 families
analyses that had published thus far (Schaid, harbouring BRCA2 mutations, a relative risk
2004). In summary, the segregation analysis of 4.65 (95% CI, 3.48-6.22) for prostate
of prostate cancer give strongest support for cancer in male BRCA2 gene carriers (The
autosomal dominant inheritance of Breast Cancer Linkage Consortium,
susceptibility genes, but there are also 1999). However, since BRCA2 mutations
evidence for autosomal recessive or X- are rare in families with hereditary prostate
linked mode of inheritance, a model with 2 cancer, they only account for a very small
or 3 genes contributing to the disease and a fraction of hereditary prostate cancers.
multifactorial mode of inheritance. The discovery of highly penetrant
Nevertheless, it has become obvious that prostate cancer genes has coupled problems
prostate cancer is most likely caused by a for at least two major reasons. First, due to
number of genes – each with different the late onset of prostate cancer, it is
modes of inheritance, population difficult to identify representatives of more
frequencies, and penetrance. than two generations of a family that could
be included in molecular studies. Increases
in the number of generations that can be
Prostate cancer susceptibility genes examined increases the possibility of finding
Although more than 40% of prostate cancer relevant and true susceptibility loci that
cases are estimated to be an effect of genetic affect prostate cancer risks. Another
variation (Lichtenstein et al., 2000), rare, significant problem is the lack of clear
highly penetrant genes probably account a distinguishing features between hereditary
minority of prostate cancer cases. Mutations and sporadic forms of the disease, making it
in high-penetrance susceptibility genes difficult to distinguish, within high-risk
increase the risk of cancer several fold, and pedigrees, men who have developed cancers
tumours with such mutations are often that are sporadic rather than due to an
called hereditary cancers. So far three high- inherited germline mutation (Rubin et al.,
risk candidate genes have been identified; 2004).
HPC/ELAC2 on 17p, RNASEL on 1q25,
and MSR1 on 8p22-23 (see descriptions
below). Attempts to confirm these findings Polymorphisms associated with prostate cancer
in different populations have been The remainder of the genetic influence is
problematic, and the importance of these most likely mediated by more common
genes in prostate cancer pathogenesis is still genetic variants or polymorphisms. Low-
obscure (Schaid, 2004). Nevertheless, these penetrance polymorphisms increase the risk
three genes clearly do not account for the of cancer only modestly, but the prevalence
majority of hereditary prostate cancer cases. of such polymorphisms may be higher than
the prevalence of mutations in high- conversion of testosterone into the more

penetrance susceptibility genes, and thus active form androgen dihydrotestosterone
their overall impact or attributable risk can and maps to 2p22-23. SRD5A2 is primarily
be substantial. The list of these variants is expressed in androgen-sensitive cells of
long, but the major pathways currently genital tissues and the prostate gland.
under investigation include those involved in Certain SRD5A2 polymorphisms may
androgen action, DNA repair, carcinogen contribute to different enzyme activities of
metabolism, and inflammation pathways. 5α-reductase variants. A dinucleotide (TA)
Numerous polymorphisms have already repeat polymorphism in the untranslated
been suggested to be associated with the risk region of SRD5A2 has been identified.
of prostate cancer. Although no clinical significance for these
Androgen receptor (AR). The most widely polymorphisms has yet been determined,
studied polymorphic gene may be the some TA repeat alleles may promote an
androgen receptor (AR) gene. The AR gene, elevation of enzyme activity, which may in
located on chromosome Xp11-12, encodes a turn increase the level of DHT. Thus, these
ligand-activated transcription factor that alleles may increase the risk of prostate
mediates the androgenic response, and has cancer. (Coughlin et al., 2002)
been shown to play an important role in In addition, two nonsynonymous
prostate development and function. Exon 1 polymorphisms, A49T and V89L, have been
of this gene contains two polymorphic identified. These two polymorphisms may
microsatellites: a highly polymorphic CAG also alter the DHT levels and consequently
repeat and a less polymorphic GGC repeat. influence prostate cancer risks. However,
Since the length of the polymorphic CAG the evidence for prostate cancer risk
trinucleotide repeat has been shown to be associated with these three SRD5A2 variants
inversely correlated with the transcriptional has been conflicting (Ntais et al., 2003).
activity of the AR gene, it has been Other genes with sequence variants that
hypothesized that variation in the length of have suggested association with the risk of
the repeat are also associated with variations prostate cancer include CHEK2 and the
in prostate cancer risks. Several genetic vitamin D receptor (VDR) and 17α-
epidemiological studies have shown a hydroxylase (CYP17) (Hughes et al., 2005).
correlation between an increased risk of However, a typical feature of many of these
prostate cancer and the presence of short genes is that highly conflicting findings
androgen-receptor CAG repeats, but other regarding their putative involvement in
studies have reported inconsistent results prostate cancer have been published
(Simard et al., 2003). At present, no study (personal communication, Sara Lindström).
has detected any correlation between GGC Thus, at the moment, none of the sequence
repeats and functional activity of the AR variants can be considered to be definitely
gene. Furthermore analyses of the possible associated with prostate cancer.
association between GGC repeats and In conclusion, a large number of studies
prostate cancer have been inconclusive have evaluated the risk of dietary,
(Gsur et al., 2004). environmental and genetic risk factors, with
SRD5A2. The 5α-reductase type II gene mixed results. Since all these risk factors are
(SRD5A2) is a membrane protein of the likely to cluster within families,
endoplasmatic reticulum that catalyzes the discriminating genetic from non-genetic
risks is difficult, especially since the genetic and non-genetic risk factors (Schaid,
magnitude of the risks is similar for both 2004).
Inflammation and cancer
Various carcinomas (including cancers of cell type, orchestrating and directing the
the liver, bladder, colon, stomach, and healing process. Normally, inflammation is a
oesophagus) have been shown to arise from self-limiting process due to the production
areas of infection and inflammation. Over of anti-inflammatory cytokines that buffer
15% of all malignancies worldwide are the effect of pro-inflammatory cytokines.
attributable to infectious agents, and The cytokine/chemokine pattern persisting
inflammation is a major component of these at the inflammatory site is important in the
chronic infections (Kuper et al., 2000). development of chronic disease.
Colon cancers arising in individuals with Dysregulation of any of the cooperating
inflammatory bowel disease (e.g. chronic factors can lead to prolonged inflammation
ulcerative colitis or Crohn’s disease) and with chronic exposure to cytotoxic
stomach cancers caused by chronic mediators. (Coussens et al., 2002). Chronic
Helicobacter pylori infection are among the inflammation can be caused by a variety of
most intensively studied and well established factors, including bacterial, viral, and
types of cancer associated with inflammation parasitic infections, chemical irritants, and
of different origins (Coussens et al., 2002). non-digestible particles, but often the
Inflammation involves the induction of underlying cause is unknown. The longer the
complex, coordinated chemical signals and inflammation persists, the higher the risk of
associated physiological processes following associated carcinogenesis (Shacter et al.,
injury that promote “healing” of damaged 2002).
tissues. Early responses include increases in At the site of inflammation, caused by
vascular permeability and activation, either wounding or infection, phagocytic
together with the directed migration of cells (e.g. neutrophils and macrophages)
leukocytes (neutrophils, monocytes and generate reactive oxygen and nitrogen
eosinophils) towards the site of injury, where substances, but these cells also synthesize
the ground-work is being laid for the and secrete large quantities of growth factors
formation of a new extracellular matrix. The and a number of potent angiogenic factors,
directional migration is mediated by secreted cytokines, and proteases, all of which are
chemokines that form a concentration important mediators in the tissue
gradient towards the site of inflammation. regeneration, but can also potentiate
The extracellular matrix provides the neoplastic tumorigenesis. The focus of
structure upon which cells (fibroblasts and interest has been the individual function of
endothelial cells) can migrate and proliferate, the different mediators, but in the
regenerating new tissue and a vascular inflammation response there is significant
network. In later stage of the inflammatory interaction and synergy among them – for
response, the macrophages are the dominant example, prostaglandins induce the
expression of certain inflammatory cytokines 2002). Two different cyclooxygenase (COX)

which can in turn, induce the production of enzymes catalyse the rate-limiting first step
reactive oxygen and nitrogen species. The in prostaglandin synthesis. COX-1 is
following description summarises the role of constitutively expressed in most cell types,
three types of inflammatory mediators in while COX-2 is primarily expressed by
inflammation and tumour development. monocytes and macrophages during
Reactive oxygen and nitrogen species. inflammation. Long-term intake of non-
At the site of inflammation activated steriodal anti-inflammatory drugs (NSAIDs;
phagocytes produce large amounts of drugs that inhibit the activity of both COX-
reactive oxygen (ROS) and nitrogen species 1 and COX-2) have been shown to reduce
(RNS). These substances are normally the incidence and progression of colon
produced in response to infection, killing caner (Baron et al., 2000), and similar effects
invading agents. Nevertheless, they can also has been indicated for prostate cancer
cause tissue damage and thereby act as (Basler et al., 2004). However, believing that
initiators of tumorigenesis. ROS and RNS the tumour-repressing effect is mediated
have been shown to alter protein structure solely through the inhibition of
and function, cause lipid peroxidation, and cyclooxygenase activity is an over
damage DNA, thereby contributing to the simplification (Zha et al., 2004). Finally,
carcinogenic process. ROS/RNS-induced other metabolites derived from arachidonic
damage to DNA may result in mutagenesis acid, such as leukotrienes and tromboxanes,
or altered expression of transcriptional may also be important mediators in the
factors involved in carcinogenesis. Damage development of cancer, but the knowlegde
may also occur to vital DNA repair of these aspects is still very limited.
enzymes, altering their activity, which may Cytokines. Cytokines are generally small
increase mutagenesis (Wiseman et al., 1996). molecules that alter the behaviour of the
These processes can cumulatively lead to an cells that secrete them and/or other cells,
imbalance between the rate of mutagenesis generally within the heamatopoietic system.
and the ability to repair mutations, resulting They act in a in a complex coordinated
in the accumulation of multiple mutations network in which they initiate or suppress
and, thus, increases the risk for their own synthesis as well as that of other
carcinogenesis. cytokines and cytokine receptors. The
Prostaglandins. In addition to ROS and complexity arises from the fact that there is
RNS, prostaglandins are synthesized in large often substantial overlap and redundancy
amounts by inflammatory cells in response between the functions of individual
to both acute and chronic inflammatory cytokines (Howell et al., 2002). In response
stimuli. Prostaglandins are lipid mediators of to both antigen-specific and non-specific
the inflammatory immune response that stimuli, inflammatory cells secret a wide
have a wide range of physiological functions, array of cytokines, which play critical roles in
such as vasodilatation/constriction and the generation of both pro- and anti-
inhibition/enhancement of inflammatory inflammatory immune responses. For
cell functions. Studies on humans and example, cytokines such as TNFα and IL-1
animals have generated evidence that are usually classed as key pro-inflammatory
suggesting that prostaglandins contribute to cytokines, while cytokines such as IL-10 are
the development of cancer (Shacter et al., anti-inflammatory. The balance between the
effects of pro-inflammatory and anti- inflammation with chronic exposure to

inflammatory cytokines determines the cytotoxic mediators.
outcome of the inflammatory process. In conclusion, multiple pieces of
Cytokines can contribute to cancer evidence have demonstrated that
development in several ways. TNFα has inflammatory cells have powerful effects on
been shown to enhance the formation of tumour development, but recruitment of
ROS by inflammatory cells, and thereby inflammatory cells may also be inhibitory to
increase the risk for DNA damage and tumour development, and may represent an
inhibition of DNA repair in tumour cells. attempt by the host to suppress tumour
Other mechanisms include direct growth. This process requires further
stimulation of cell growth, induction of evaluation. One approach is to elucidate
angiogenesis, and recruitment of whether functional polymorphisms in genes
inflammatory neutrophils (Shacter et al., that regulate inflammatory processes, such
2002). In addition, an imbalance of pro- and as cytokines, confer altered risks for
anti-inflammatory cytokines, caused by point developing cancer or are potential
mutations or polymorphisms, may prevent prognostic factors. See (Shacter et al., 2002)
the normal self-limiting nature of the and (Coussens et al., 2002) for a
immune response, leading to prolonged comprehensive review.
The role of inflammation in the pathogenesis of prostate cancer
Although it has been established that processes and the development of prostate
chronic inflammation plays a causative role cancer.
in the development of many human cancers,
the contribution of inflammatory processes
to the development of prostate cancer has Molecular pathology
not been extensively studied. However, data
from epidemiological, genetic Focal areas of epithelial atrophy in the
epidemiological and molecular pathology prostate were first observed by pathologists
studies have been accumulated recently more than fifty years ago (FRANKSLM,
suggesting that inflammatory processes are 1954; Rich, 1979). Atrophy of the prostate is
also involved in the development of prostate identified as a reduction in the volume of
cancer. The chronic inflammatory the glands and can be divided into two
microenvironment, characterized by the major patterns: diffuse and focal. Diffuse
accumulation of macrophages and atrophy is a reduction that involves the
lymphocytes, is extremely common in the entire prostate and is caused by a decreased
tissue stroma and epithelium of the prostate level of circulating androgens. Focal atrophy,
and may support both the initiation and on the other hand, occurs primarily, but not
progression of prostate cancer (Lucia et al., exclusively, in the outer part of the gland,
2004). The following description highlights where prostate cancer normally arises
connections between inflammatory
(McNeal, 1988; Feneley et al., 1996; Ruska et prostate cancer tissue are approximately
al., 1998; De Marzo et al., 1999). Focal 70% and 90%, respectively (Jerónimo et al.,
atrophy is not related to reduced levels of 2001; Lin et al., 2001).
circulating androgens, compared with In addition, chromosome 8
normal prostatic epithelium, and the abnormalities, which are common in
condition is highly proliferative without any prostate cancer, have been found in areas of
increase in apoptosis. Focal atrophy is often PIA lesions. Acquired somatic chromosome
associated with acute or chronic 8 abnormalities have been noted in about
inflammation, reflected by infiltration of 1% of normal prostate tissue, 4% of PIA
inflammatory cells (lymphocytes, lesions, and about 6% of adenocarcinomas
macrophages and polymorphonuclear cells) (Shah et al., 2001). Other studies have
(Ruska et al., 1998; De Marzo et al., 1999). reported p53 mutations in 5% of PIA
Due to the combined findings of high lesions, a rate similar to that seen in high-
proliferation indices and inflammatory grade PIN, compared to 20% of prostate
infiltrations seen in these lesions, the term cancer cases (Tsujimoto et al., 2002). Thus,
proliferative inflammatory atrophy, or PIA, these are genetic changes that are associated
was introduced (De Marzo et al., 1999). The with PIN and prostate cancer.
underlying causes of PIA lesions are still not De Marzo et al. examined expression
clearly understood, but they may arise either patterns of three molecular markers
as a consequence of epithelial damage (from implicated in prostatic carcinogenesis: p27,
infection, ischemia, or toxin exposure) or as Bcl-1 and GSTP1 (De Marzo et al., 1999).
a direct consequence of inflammatory p27 is a cyclin-dependent kinase inhibitor
oxidant damage to the epithelium (Platz et whose expression suppresses the cell cycle
al., 2004). and is usually reduced in prostatic
Accumulating evidence from adenocarcinomas and high-grade PIN. The
morphological, immunohistochemical and cited authors reported a down-regulation of
genetic studies have provided support for p27 in PIA. They also observed an increase
the concept that PIA lesions may be in Bcl-2 expression, as previously reported
precursors to prostate cancer, partly because by Ruska et al. (Ruska et al., 1998), which is
the vast majority of such lesions are found consistent with the very low levels of
adjacent to or near early adenocarcinomas or apoptosis. Moreover, they found increased
high grade prostatic intraepithelial levels of GSTP1 in many lesions suggesting,
neoplasms, or both (Feneley et al., 1996; De together with the elevated levels of GSTA1
Marzo et al., 1999; Putzi et al., 2000). Further and COX-2 reported in other studies
support for the hypothesis comes from the (Parsons et al., 2001; Zha et al., 2004), that a
fact that PIA shares several molecular stress-induced response has occurred in
alterations found in both Prostate these cells. Accumulating evidence also
Intraepithelial Neoplasia (PIN) and prostate indicates that many of the atrophic luminal
cancer. Nakayama et al. reported that cells in PIA represent a form of intermediate
approximately 6% of PIA lesions show epithelial cell with high proliferative activity.
evidence of somatic hypermethylation of the These cells share features of both basal and
GSTP1 gene promoter (Nakayama et al., luminal secretory cells, and hence are
2003). Correspnding levels of GSTP1 postulated to be targets of neoplastic
hypermethylation in PIN lesions and
transformation in the prostate (van Leenders 2-10% of men experience it during their
et al., 2003). lifetime (Habermacher et al., 2005). The
Even though PIA lesions may be an early clinical presentation is characterised by
histological precursor to prostate cancer, it is uncomfortable symptoms, such as dysuria,
important to note that some studies have and rectal and suprapubic pain (Roberts et
not found an association between PIA and al., 1998) Prostatitis is classified into four
prostate cancer (De Marzo et al., 2003) and, categories: I, acute bacterial; II, chronic
moreover, not all cancers occur within or in bacterial; III, chronic nonbacterial/chronic
the vicinity of PIA. In fact, PIA may simply pelvic pain syndrome; and IV, asymptomatic
indicate an intraprostatic environment that prostatitis. Both acute bacterial prostatitis
favours to prostate cancer development. (which is usually caused by Escherichia coli)
Nevertheless, further studies are needed to and chronic bacterial prostatitis (caused by
elucidate the relationship between focal E. coli and several other infectious agents)
atrophy and cancer in the prostate. are normally treated with antibiotics.
Chronic inflammation, with a Chronic non-bacterial prostatitis/chronic
predomination of lymphocytes and pelvic pain syndrome, is the most common
macrophages, is extremely common in the form, affecting 90-95% of patients with
prostate. However, examination of prostatitis. Men with this type of prostatitis
prostatectomy specimens seldom shows may experience symptoms for weeks to
inflammatory infiltrates in the malignant years, and current therapy has limited
tissue, whereas directly adjacent benign success in alleviating the pain that the
tissue can be heavily infiltrated (Lucia et al., patients experience. Asymptomatic
2004). To date, the relationship between prostatitis is a histological diagnosis of
lymphocytic infiltration in the tumour and prostate inflammation that is found
progression of tumour development in the subsequent to pathological examination of
prostate is uncertain, but the results from the prostate tissue. This category is found in
two studies evaluating the connection a significant number of patients;
between inflammatory infiltration and epidemiologic studies have estimated the
survival indicate that lymphoid aggregates prevalence to be as high as 32.2% in a
within the tumour are associated with a poor population of men with elevated PSA (van
outcome in patients with prostate cancer Leenders et al., 2003).
(Irani et al., 1999; McArdle et al., 2004). Several case-control studies have
Furthermore, increased macrophage activity investigated the association between prostate
in the tumour has also been found to be cancer and prostatitis, with variable results.
related to poor prognosis in prostate cancer In 2002, Dennis et al. performed a meta-
(Lissbrant et al., 2000). Why the presence of analysis of 11 case-control studies that
increased in lymphocytic aggregates would evaluate the possible relationship between
be associated with poor cancer specific prostatitis and prostate cancer. The meta-
survival rates remains unexplained. analysis found an increased risk of prostate
cancer among men with a history of clinical,
Epidemiology or symptomatic prostatitis (OR=1.6; 95%
CI, 1.0-2.4), particularly in population-based
Prostatitis and prostate cancer. Prostatitis case-control studies (OR=1.8; 95% CI, 1.1-
is a common urological disorder worldwide; 3.0) (Dennis et al., 2002b). However, the
complexities involved in diagnosing was provided by reports of an association

prostatitis and the risks for potential recall between the use of anti-inflammatory drugs
or detection bias when performing (NSAIDs) and prostate cancer risk.
association studies between prostatitis and However, the results have been inconsistent
prostate cancer make the analyses of the (Basler et al., 2004). The most interesting
results problematic. The results could reflect data showing an inverse association with
an effect of detection bias, since patients NSAID use come from a large (n=1362)
with prostatitis may be more likely to be population-based longitudinal study in
monitored by a urologist and thus more Olmsted County, MN, USA, the results of
likely to be screened for prostate cancer, or which suggest that daily use of NSAIDs may
the results could reflect a recall bias, since be associated with a lower incidence of
patients with prostate cancer may try to prostate cancer among ³60-year-old men
remember more about potential exposure (RR 0.45; 95% CI, 0.28-0.73) (Roberts et al.,
than controls do (Dennis et al., 2002b). 2002). Furthermore, the inverse association
Epidemiological studies investigating the with NSAID use increased with increasing
possible association between prostatitis and age.
prostate cancer have relied on patient Sexually transmitted infections and
reports for information on prostatitis prostate cancer. Sexually transmitted
history. Therefore, there are serious infections (STIs) have long been
problems in using studies of this type to hypothesized to increase the risk for
identify valid relationships between category prostate cancer. STIs are theorized to
IV prostatitis and prostate cancer. The increase the risk of prostate cancer by
reason why inflammation leads to symptoms causing inflammation of the prostate, which
in some men but not others are not known. may in turn lead to the initiation of
It is possible that a distinguishing feature carcinogenesis (Nelson et al., 2003). Several
between symptomatic and asymptomatic studies have investigated the potential
prostatitis may be the anatomical localisation association between prostate cancer and
of the prostatic inflammation. Inflammation STIs, using various approaches, including
in the peri-urethtral region, or transition self-reports, serology, and detection of
zone, may be more likely to manifest in infectious agents in the prostate.
urinary symptoms and pain than Self-report: In a meta-analysis (Dennis et al.,
inflammation in the outer areas of the gland, 2002a) reviewed 36 independent analytical
or the peripheral zone, where prostate studies, published between 1971 and 2000,
cancer more commonly develops (Palapattu that measured some aspect of sexual activity
et al., 2004). in relation to prostate cancer. The data
In addition, according to two suggest there was an elevated relative risk of
epidemiological studies, the prevalence of prostate cancer among men who reported a
prostatitis correlates with the prevalence of history of any STI (RR=1.44; 95% CI, 1.24-
prostate cancer worldwide, with rates of 1.66). The risk associated with syphilis alone
prostatitis and prostate cancer being much was higher (RR=2.30; 95% CI, 1.34-3.94),
higher in North America than Asia (Roberts while the risk associated with gonorrhoea
et al., 1998; Tan et al., 2002). was not as strong as that for the presence of
More general evidence of a relationship all STIs (RR=1.36; 95% CI, 1.15 -1.61). The
between inflammation and prostate cancer association between prostate cancer and
STIs was further supported by RR have found variable results, ranging from no
increasing with increasing frequency of association to statistically significantly higher
sexual activity (RR=1.20; 95% CI, 1.11- risks for HPV-seropositive men (reviewed in
1.30), for men reporting more than 30 sexual (Palapattu et al., 2004)).
partners (RR=1.27; 95% CI, 1.08-1.49), and The meta-analysis by Taylor et al. also
for men who visited prostitutes (RR=1.19; examined the associations between HPV
95% CI, 1.01-1.41). infection and prostate cancer risk detected in
Recently, Taylor et al. performed a new ten studies using either serological or PCR-
meta-analysis, including 29 case-control based evidence of HPV infections. The
studies, which corroborated the previous results showed an OR of 1.39 (95% CI,
findings (Taylor et al., 2005). The study 1.13-1.71). Notably, while HPV is known to
demonstrated a signficantly elevated OR for be an oncogenic virus, its influence on
prostate cancer for all STIs (OR=1.48, 95% prostate carcinogenesis may be independent
CI 1.26-1.73), and for gonorrhea (OR=1.35; of inflammation.
95% CI, 1.05-1.83). However, the results Detection of infectious agents in prostate tissue:
should be interpreted with caution, since Several studies have investigated the
most of these studies were retrospective presence of infectious agents in the prostate
case-control studies, and thus may have tissue. Various viruses and bacteria have
been affected by bias in recall between cases been found, but the results have been
and controls. Also, a history of STIs may inconsistent, possibly due to methodological
not be causally associated with prostate differences (such as differences in the tissue
cancer, but may be simply be a surrogate for handling and detection methods), or
other lifestyle risk factors for prostate cancer possible contamination from agents in areas
(Platz et al., 2004) Furthermore, since these close to the prostate, e.g., the urethra. It is
two meta-analyses, a number of studies have also worth noting that STIs detected in the
been published reporting inconsistent prostate cancer tissues could have been
association between STIs and prostate acquired after the initiation of
cancer risk (Giles et al., 2003; Fernández et carcinogenesis. Furthermore, the failure to
al., 2005; Patel et al., 2005). detect infectious agents in the prostate tissue
Serological markers: Several epidemiological does not necessarily mean that such agents
studies have also reported associations play no role in prostate carcinogenesis, as
between prostate cancer and circulating some may be cleared after causing damage
serum IgG antibodies against agents (Platz et al., 2004). Human papilloma viruses,
infecting the genito-urinary tract. One study human herpes virus-8, Herpes simplex virus-
reported an increased risk for prostate 2, cytomegalovirus and Epstein-Barr virus
cancer among men who showed serological are some of the agents that have been
evidence of syphilis (OR=1.8; 95% CI, 1.0- found, see the review by Palapattu et al. for
3.5) (Hayes et al., 2000), while studies further information (Palapattu et al., 2004).
investigating possible serological links In 2005, Cohen and co-workers
between Chlamydia trachomatis and performed an exploratory study to detect the
prostate cancer reported null results (Dillner presence of bacterial agents in prostate
et al., 1998). Five studies investigating tissue from patients with prostate cancer
possible associations between human (Cohen et al., 2005). They reported a
papilloma virus (HPV) and prostate cancer predominant presence of the gram-positive
bacterium Propionibacterium acnes, which been identified, some of which indicate that
moreover showed a positive association with inflammation may be an important mediator
prostatic inflammation. P. acnes is a slow- in the development of prostate cancer. The
growing microbe that is known to be a following sections summarise some of the
potent stimulant of the immune system, and germline and somatic genomic changes
it is highly resistant to killing and suggesting that infection or inflammation of
degradation by human neutrophils and the prostate contributes to the development
monocytes, a characteristic which allows it of prostate cancer.
to establish long-term low-grade infections
that may persist for years to decades. A GSTP1
independent study using PCR-based GSTPs represent a superfamily of enzymes
technique, found P.acnes twice as common that play an important role in detoxification
in prostate tissue from patients with benign by catalyzing the conjugation of many
prostatic hyperplasia that later developed hydrophobic and electrophilic compounds
cancer compared to those that did not with reduced glutathione. GSTP1 appears to
(personal communication, Oleg Alexyev). act as a “caretaker” gene, defending prostate
cells against genome damage mediated by
Genetics carcinogens. In the normal prostate
epithelium, GSTP1 is expressed
As described above, multiple pieces of predominantly in basal cells, but not in the
evidence have been accumulated supporting columnar secretory cells, although the
the view that genetic factors play a role in enzyme may be induced in columnar
prostate cancer susceptibility and epithelial cells that are subjected to genome-
pathogenesis. Unlike some carcinomas such damaging stresses. An elevated level of
as those of the colon (Klump et al., 2004) GSTP1 is a histological characteristic of
and pancreas cancer (Jaffee et al., 2002), proliferative inflammatory atrophy, strongly
where relatively common somatic genetic indicating that a stress-induced response has
alterations are observed (e.g. mutations in been induced in these cells (De Marzo et al.,
p53 and K-ras), gene mutations in prostate 1999). GSTP1 can undergo somatic
cancer display a great deal of heterogeneity, alterations, especially hypermethylation of
not only from case to case, but also from CpG islands of regulatory sequences at the
lesion to lesion in a single cases. The GSTP1 locus, which prevents the
heterogeneity of genomic defects in prostate transcription of GSTP1. Abnormal
cancer suggests that prostate cancers may hypermethylation of CpG islands of
arise as a consequence of either chronic or regulatory sequences at the GSTP1 locus is
prolonged exposure to genome-damaging associated with decreased transcriptional
stresses, defective maintenance of genome activity. Such hypermethylation occurs in
integrity, or a combination of both, and thus many types of human cancers (Jones et al.,
no single dominant molecular pathway is 2002), it has been reported to be the most
responsible for prostate carcinogenesis (De common somatic genome alteration in
Marzo et al., 2004). prostate cancer and it represents an early
To date, a substantial number of event in neoplastic transformation. The
germline prostate cancer susceptibility genes hypermethylation occurs in approximately
as well as somatic genome alterations have 70% of PIN lesions and in over 90% of
prostate carcinomas, but not in normal receptor (MSR1, also known as SR-A, an
prostate tissue or benign prostatic important component of the innate immune
hyperplasia (Jerónimo et al., 2001; Lin et al., system), which occurs in three different
2001). isoforms (I, II, and III) generated by
Recent published data have suggested alternative splicing of a single 11-exon
that heterocyclic amine carcinogens present mRNA. Isoforms I and II are functional
in well-done meat as well as oxidants may be while isoform III is a non-functional protein
detoxified by GSTP1 (Nelson et al., 2001). acting as a dominant negative isoform by
Experimental studies in which LNCaP cells blocking modified low-density lipoprotein
(that do not normally express GSTP1) have uptake (Gough et al., 1998). The functional
been modified to express GSTP1, show that MSR1 protein is a trimeric transmembrane
cells expressing GSTP1 more readily molecule composed of three identical
withstand DNA damage when exposed to protein chains. It has six distinct structural
promutagenic substances and oxidative domains: the amino-terminal cytoplasmic
stress, compared to unmodified LNCaP cells domain, a transmembrane domain, a spacer
(Nelson et al., 2001, 2002). However, in domain, an α-helical coiled coil domain, a
response to oxidant stress, unmodified collagenous domain, and the scavenger
LNCaP cells survive better than LNCaP receptor, cysteine-rich, carboxy-terminal
cancer cells that have been modified to domain (Kodama et al., 1990). MSR1 shows
express high levels of GSTP1. Thus, for unusually broad ligand-binding properties. It
LNCaP prostate cancer cells, loss of the binds a diverse array of macromolecules
GSTP1 function appears to increase genome including bacterial lipopolysaccharide and
damage and decrease cell mortality rates lipoteiochic acid, and both oxidized high-
following exposure to oxidants. This finding density lipoprotein and low-density
of tolerance to oxidative genomic damage in lipoprotein in the serum (Platt et al., 2001).
the absence of functional GSTP1 may be a The function of the receptor in vivo is still
clue regarding the function of somatic not entirely clear, but studies have suggested
GSTP1 inactivation in prostate cancer that these receptors may play a role in
progression (Palapattu et al., 2004). macrophage–host cell interactions,
macrophage adhesion to substrata,
MSR1 endocytosis of ligands, and the recognition
In 2001, Xu et al. published a study with and clearance of foreign pathogenic
suggestive linkage to chromosome 8p22-23 substances and damaged or apoptotic cells
among 159 families (Xu et al., 2001). Further (Platt et al., 2001).
mutation screening studies of hereditary The MSR1 receptor is mainly expressed
prostate cancer families identified a number in macrophages and related cells, and its
of mutations in the macrophage scavenger expression is regulated by a number of
receptor 1 gene (MSR1) that co-segregate factors including cytokines such as M-CSF,
with prostate cancer (Xu et al., 2002). IFNg, and TNFα (Shirai et al., 1999). Mice
MSR1 is a large 11-exon gene located on that are deficient in MSR1 are highly
chromosome 8p22, an area that is frequently susceptible to infection by Listeria
lost in prostate cancer (Matsumoto et al., monocytogenes, Staphylococcus aureus, and herpes
1990). It encodes a Class A scavenger simplex type 1 (Thomas et al., 2000; Ishiguro
et al., 2001), which may be relevant in
prostate cancer susceptibility, since an apoptosis (Silverman, 2003). Defects in the

infectious aetiology of prostate cancer has RNASEL gene have been shown to result
been proposed. Moreover, areas within the in reduced immunity to viral infections and
prostate that show evidence of inflammation cancer (Hassel et al., 1993).
are often populated by macrophages that A genome-wide scan for linkage in
express MSR1 (Nelson et al., 2003). prostate cancer families found evidence for a
Several germ-line mutations in MSR1 prostate cancer susceptibility locus on
have been linked to prostate cancer in some chromosome 1q24-25 (Smith et al., 1996). In
families with hereditary prostate cancer. In 2002, Cartpen et al. identified RNASEL as a
the mutation screening study by Xu et al., six candidate gene for the HPC1 locus through
rare missense and one nonsense mutation a positional cloning and candidate approach.
(R293X) were detected in MSR1 (Xu et al., A truncating mutation (E265X) and an
2002). A family-based linkage and initiation codon mutation (M1I) were
association test indicated that these reported to co-segregate within two specific
mutations cosegregate with prostate cancer prostate cancer families (Carpten et al.,
(P=0.0007). Of these mutations, the 2002). In addition to the rare mutations, a
nonsense mutation R293X was detected in number of relatively common RNASEL
approximately 2.5% of men with non-HPC variants have been associated with prostate
compared to a 0.4% of unaffected men cancer risk. There is some confirmatory
(P=0.05). In a follow-up study of 301 non- evidence for the association between
HPC cases and 250 controls, by the same variants of RNASEL and prostate cancer
group, five common variants within MSR1 risk (Rennert et al., 2002), but other studies
were found, with statistically significant have showed no association (Wang et al.,
differences in allele and haplotype 2002; Kotar et al., 2003; Wiklund et al., 2004).
frequencies (Xu et al., 2003). In summary, the role of RNASEL in the
A number of other independent studies pathogenesis is still very unclear, due to
have evaluated the association between inconsistencies in the association studies.
prostate cancer and the MSR1 gene, and the However, as Palapattu et el. mention in a
generated data have created doubt review from 2004, it is tempting to speculate
concerning the role of MSR1 sequence that genetic variants of the RNASEL gene
variants in prostate cancer susceptibility increase the risk for prostate cancer only in
(Miller et al., 2003; Seppälä et al., 2003; Wang the presence of some environmental factor
et al., 2003). (e.g. viral infection) that may vary between
different study populations (Palapattu et al.,
2004).
RNASEL
RNASEL is a constitutively expressed latent Cytokines in prostatic inflammation
endoribonuclease that mediates the antiviral and prostate cancer
and proapoptotic activities of the interferon-
inducible 2-5A system. Once activated by As described above, cytokines are a diverse
interferon, cells containing a functional group of intracellular signalling peptides and
RNASEL gene produce an enzyme that glycoproteins that regulate local and
degrades single-stranded RNA, leading to systemic inflammatory and immune
responses. Several pro- and anti- TNFα-308, and VEGF-1154) were analyzed
inflammatory cytokines have been identified among 247 prostate cancer cases and 263
in patients with prostatic inflammation and controls. The results showed that IL-8-251
prostate cancer, and their roles have been TT and VEGF-1154 AA genotypes were
studied. In 1998, Alexander et al. measured lower among patients compared with
the levels of the pro-inflammatory cytokines controls (OR=0.66; 95%, CI 0.44-0.99 and
TNFα and IL-1β in the semen of men with OR=0.45; 95% CI, 0.24-0.86, respectively),
chronic prostatitis and compared them with whereas the IL-10-1082 AA genotype was
levels in normal men (Alexander et al., 1998). significantly increased in patients compared
They found that men with prostatitis had with controls (OR=1.78; 95% CI, 1.14-2.77).
higher mean levels of TNFα and IL-1β than Although the influence of cytokine
normal men, as would be expected in an polymorphism on prostatic inflammation
ongoing inflammatory response. These and prostate cancer is likely to be complex,
results were confirmed by Nadler and co- the few relevant studies published to date
workers, who showed that IL-1β was indicate that SNPs in cytokine genes may
measurable in ~90% of patients with type have a significant impact on disease
III and type IV prostatitis, but rarely development. These results also highlight
detectable in controls (Nadler et al., 2000). the critical need to further elucidate the
The level of TNFα could be measured in impact of cytokines and their sequence
both cases and controls, but levels were variants on prostate cancer susceptibility.
significantly elevated in patients with type
III and type IV prostatitis.
Genetic polymorphisms that alter MIC-1
cytokine gene expression or protein function The type β transforming growth factor
could have an important impact on prostatic (TGF-β) superfamily has more than 40
inflammation and the further development members, which are involved in regulation
of prostate cancer. A polymorphism in the of many cellular functions and biological
promoter of the anti-inflammatory cytokine processes, including proliferation, apoptosis,
IL-10 has been identified; and the IL-10 - extracellular matrix secretion and adhesion,
1082 AA genotype is associated with low terminal differentiation, and development
IL-10 production. In a study by Shoskes et (Derynck et al., 1997).
al., patients with chronic prostatitis were To identify novel molecules that
found to have higher proportions of the participate in the local inflammatory
allele associated with low IL-10 production response, Bootcov et al. subtracted cDNA
compared to controls, indicating a pro- library enriched for genes associated with
inflammatory state in those patients macrophage activation. The following
(reviewed in Pontari M and Ruggieri M screening resulted in the identification of a
(Pontari et al., 2004)). novel transforming growth factor β
Only a few studies have examined the superfamily cytokine, designated
association between prostate cancer risk and macrophage inhibitory cytokine-1 (MIC-1).
sequence variants in cytokine genes such as The MIC-1 gene, located on 19p13, is about
TNFα, IL-8 and IL-10 (McCarron et al., 6 kb long and is composed of two exons.
2002). In the cited study, cytokine sequence The protein is synthesized as a 308 amino
variants (IL-1β-511, IL-8-251, IL-10-1082,
acid long precursor molecule. The at position 7 that has been indicated to be
propeptide is separated from the mature important for the stability of this protein
protein by a furin-like protease acting on a (Fairlie et al., 2001). Due to the differences in
conserved cleavage site at amino acids 193- the properties of these two amino acids, this
196. Following processing, the mature change may alter the stability of the protein
protein of 112-aa is secreted as a disulfide- and thus the function of MIC-1. Some
linked homodimer. The mature protein studies have examined the possible
subunits contain a conserved pattern of relationship between MIC-1 genotypes and
seven cysteine residues, which is a hallmark different diseases, including cancer, with
of the TGF-β superfamily (Bootcov et al., variable results. A study by Brown et al.
1997). Since MIC-1 is a secretory protein it indicated that the presence of the HH
can function in cells secreting it as well as in genotype of MIC-1 was associated with a
their neighbouring cells (i.e. in both decreased risk of metastasis of colorectal
autocrine and paracrine fashions). It has caner at presentation of the disease, but it
been reported that MIC-1 activity (like that should be noted that the numbers of
of TGF-β), requires an intact signalling patients in the investigated groups were
pathway mediated by type I and type II relatively small (Brown et al., 2003).
TGF-β receptors, as well as receptor It has been shown that MIC-1 has
activated Smad4 (Tan et al., 2000). diverse biological functions in distinct
The MIC-1 gene is also known by various cellular contexts. A number of direct and
names, including growth/differentiation indirect lines of evidence suggest there is a
factor-15 (GDF-15) (Böttner et al., 1999), link between MIC-1 and cancer. The
placental bone morphogenetic protein promoter region of this gene contains two
(PLAB) (Hromas et al., 1997; Thomas et al., p53 target sites; and a number of studies
2001), prostate-derived factor (PDF) have demonstrated that MIC-1 is strongly
(Paralkar et al., 1998), and the non-steroidal induced by transactivation of p53 (Kannan et
anti-inflammatory drug-activated gene al., 2000; Li et al., 2000; Tan et al., 2000). A
(NAG-1). Like many TGF-β superfamily well-studied function of p53 is its activity as
cytokines, MIC-1 is expressed very widely, a transcription factor, regulating genes
but under resting conditions the placenta is whose products are involved in a variety of
the only tissue expressing large amounts of cellular processes including growth arrest,
MIC-1 (Fairlie et al., 1999). Small amounts of apoptosis, senescence and genome stability
MIC-1 mRNA have been detected in (Ko et al., 1996). Loss-of-function of DNA
epithelial cells in the kidney, pancreas, colon, target recognition by mutated p53 is an
and prostate as well as macrophages. important step in cell transformation, and
However, in cases of inflammation, injury or over 50% of human cancers contain various
malignancy, MIC-1 expression is inactivated p53 mutants (Hollstein et al.,
dramatically increased. 1994). This indicates that some of the genes
Shortly after cloning of the MIC-1 gene, that are transactivated by p53, including
a polymorphism was identified, resulting MIC-1, may play an important role in the
from alteration of the basic amino acid development of cancers. Furthermore, two
histidine (H), to the acidic amino acid studies by Tan et al. (Tan et al., 2000) and Li
aspartic acid (D), located at position 6 of the et al. (Li et al., 2000) both show that MIC-1
mature MIC-1 protein, next to the cysteine inhibits tumour cell growth through the
TGF-β signalling pathway following demonstrated that overexpression of MIC-1

activation by p53. in prostate cancer cell lines reduces cell
More direct evidence for links between adhesion and induces apoptosis (Liu et al.,
MIC-1 and cancer, have been obtained from 2003). These findings are supported by
serial analysis of gene expression, which has observations of increased rates of apoptotic
indicated that MIC-1 is highly expressed in death in mammary carcinoma cells where
both prostate cancer and benign prostatic MIC-1 is overexpressed (Li et al., 2000;
hyperplasia tissue compared to normal Graichen et al., 2002). Moreover, Baek et al.
prostate tissue, with a higher expression in showed that NSAIDs that inhibit tumour
cancer tissue than benign prostatic development also induce colon cancer cells
hyperplasia tissue (Welsh et al., 2001, 2003; to undergo apoptosis, mediated by
Nakamura et al., 2003). A recently published autocrine/paracrine induction of MIC-1
exploratory proteomic analysis of prostate (Baek et al., 2001).
cancer tissue and benign prostatic Apart from playing an important role in
hyperplasia tissue originating from the same inhibiting tumour progression, MIC-1 has
prostate tissue block supports this been shown to exert anti-inflammatory
expression pattern (Hood et al., 2005). effects by inhibiting the late phase of
Elevated MIC-1 expression is also coupled macrophage activation (Bootcov et al., 1997).
with a number of other cancers, including Since MIC-1 is a potent p53 target gene and
colon, breast, and pancreas cancers has anti-inflammatory activity, it has been
(Buckhaults et al., 2001; Koopmann et al., suggested that increased p53 expression in
2004; Wollmann et al., 2005). inflammatory tissues may reflect a tissue
Analyses of serum samples of patients defence mechanism that triggers a signalling
have indicated that there is a relationship pathway, leading to activation of MIC-1 and
between elevated serum MIC-1 and the ultimately inhibition of inflammation (Tan et
metastatic progression of colorectal, prostate al., 2000). Understanding of the biological
and breast (Welsh et al., 2003) (Brown et al., function of MIC-1 in the prostate may help
2003). MIC-1 levels are also elevated in the delineate its role in prostatic physiology and
serum of patients with pancreatic ductal pathobiology.
cancer (Koopmann et al., 2004). These
findings have led to the suggestion that
measurement of MIC-1 levels may be useful IL-1RN
for the diagnosis and surveillance of cancer. The interleukin-1 (IL-1) family consists of
Serum MIC-1 levels can be markedly three different cytokines; the two
elevated in metastatic cancer and seem to proinflammatory cytkines IL-1α and IL-1β,
parallel the stage and extent of disease, and the IL-1 inhibitor, IL- 1 receptor
particularly in colorectal cancer (Brown et al., antagonist (IL-1RN) (Dinarello, 1994). The
2003). Furthermore, despite the relationship IL-1RN gene is located on the long arm of
between elevated serum MIC-1 levels and human chromosome 2 at band 2q14.2 in
metastatic progression, a number of studies close proximity to the genes coding for IL-
have shown MIC-1 to have an 1α and IL-1β, distributed over 430 kb
antitumorigenic function, since it induces (Steinkasserer et al., 1992). IL-1α, IL-1β and
apoptosis and inhibits proliferation of IL-1RN are all produced in a wide variety of
several tumour cell lines. Liu et al.
cells - including monocytes, macrophages, two repeats) has been associated with a
neutrophils and epithelial cells of many variety of human diseases. Several studies
organs (Arend et al., 1998) – and they bind have shown that people with allele 2 of the
to the same IL-1 receptors. IL-1α and IL-1β VNTR have a more prolonged and severe
are pro-inflammatory cytokines, and their pro-inflammatory immune response than
binding to the receptors initiates a cascade usual, and that carriers of this genotype
of events leading to the recruitment and more efficiently combat microbial infection
activation of macrophages and neutrophils, or colonization (Hu et al., 2005). The
vascular dilation and fever, and a potent pro- majority of studies relating IL-1RN gene
inflammatory immune response (Dinarello, polymorphisms to disease have focused on
1988). When the anti-inflammatory cytokine patients with autoimmune diseases or
IL-1RN binds to the same receptors the disorders associated with chronic
activity of IL-1α/β is blocked. Consequently, inflammation, including inflammatory bowel
the biological function of IL-1α and IL-1β disease, psoriasis, lichen sclerosus, and
cytokines is neutralized in both physiological multiple sclerosis (Witkin et al., 2002). A
and pathophysiological immune and number of studies have evaluated the
inflammatory responses. The relative levels presence of the VNTR in malignant
of IL-1RN, IL-1α and IL-1β at an diseases, and the results have been
inflammatory site determine whether a pro- conflicting rather than conclusive. Studies
inflammatory response will be initiated and from Caucasian populations have shown
persist or will be terminated (McIntyre et al., that homozygous carriage of allele 2 is
1991). Typically, the level of IL-1RN associated with an increased risk of gastric
increases in the later stages of inflammatory cancer (El-Omar et al., 2000; Machado et al.,
events, thereby promoting their termination 2001; Glas et al., 2004), however this
and limiting the risks that inflammation will association was not replicated in Asian
become chronic and cause damage to populations (Wu et al., 2003; Zeng et al.,
healthy cells (Granowitz et al., 1991). The 2003; Chang et al., 2005). In addition to
central role of the IL-1 system is protection gastric cancer, a few studies have evaluated
against many different types of lesion, possible links between VNTR and risks of
ranging from microbial colonisation to other kinds of cancers. Recently, Sehouli et
infection and malignant transformation al. found in a case-control study of 162
(Witkin et al., 2002). women with ovarian cancer and 121
In the second intron of IL1-RN, there is controls that patients who were
a variable number of tandem repeat heterozygous for allele 2 had a significantly
(VNTR), consisting of two to six copies of a higher risk for ovarian cancer (Sehouli et al.,
86-bp sequence (Tarlow et al., 1993). The 2003).
frequency of the individual alleles varies Although the role of genetic variation in
among different ethnic and geographical IL-1RN has been investigated in many types
populations, but allele 1 (containing four of cancers, the role of IL-1RN
repeats) is always the most common allele, polymorphisms in prostate cancer
while the less common allele 2 (containing pathogenesis has not been examined.
Aims
The specific aims of the work underlying this thesis were:
ü To investigate whether mutations in ü To evaluate the possible role of

the Macrophage Scavenger Receptor 1 genetic sequence variants in the
gene influence the risk of prostate Macrophage Inhibitory Cytokine-1 gene
cancer in both Swedish families on prostate cancer risk in a Swedish
with hereditary prostate cancer and population.
in a cohort of unselected prostate
cancer.
ü To evaluate the possible role of ü To study the serum levels of MIC-1

genetic sequence variants in the in relation to prostate cancer and
Interleukin-1 Receptor Antagonist gene MIC-1 genotypes and whether MIC-
on prostate cancer risk in a Swedish 1 levels are associated with prostate
population. cancer risk.
Material and methods
Hereditary prostate cancer families used for mutation screening

(Study I)
Since 1995 our research group has been and the mean age of diagnosis was 67 years.
identifying (mainly on the basis of referrals Nearly two-thirds of the cases were
from urologists and oncologists) and diagnosed before PSA measurement was
collecting information on Swedish families introduced as a diagnostic tool in Sweden.
with a history of HPC. In the analysis of the The majority of the cases (79%) had clinical
Macrophage Scavenger Receptor I gene we symptoms at diagnosis and 60% of the cases
examined material from 83 of these families. were diagnosed with advanced or metastatic
The 83 families included in this study disease.
had an average of 4.5 affected men/family
Patients with prostate cancer and controls (Studies I, II, III, IV)
The Northern Sweden Health and months), and date (± 2 months) at

Disease Cohort recruitment. Controls were randomly
selected from each group of eligible subjects
In study I, we used a study population from if more than two subjects matched the
Northern Sweden Health and Disease identified case. If fewer suitable control
Cohort (NSHDC) to test for associations subjects were found by this procedure, less
between eight selected sequence variants and than two control subjects was accepted. We
prostate cancer risk. The design of the identified 425 eligible control subjects, with
NSHDC has been presented in detail before an average age of 66 years at the time of
(Stattin et al., 2000). For this study 215 recruitment in this study. All participants
incident cases of prostate cancer were were residents of the county of
identified by cross-reference to cancer and Västerbotten.
all-cause mortality registries. Data on patient
and tumour characteristics were obtained
through medical records. Two control CAPS
subjects were selected for each case subject
from all members who were alive and free For the association analyses in Studies II,
of cancer at the time of diagnosis of the case III, IV we utilized men included in the
in each subcohort, and matched the index population-based CAPS (CAncer of the
case subject in terms of sex, age (± 6 Prostate in Sweden) case-control study. The
case participants, diagnosed for prostate additional questionnaires and record linkage
cancer between January 2001 and September to the Swedish Cancer Registry or medical
2002, were recruited from four of the six records. The families were subsequently
regional cancer registries that cover the classified as hereditary prostate cancer
entire population of Sweden. Each of these families, where three or more relatives had
registries serves one health care region prostate cancer (52 cases), or familial
(Northern, Central, Stockholm, and South prostate cancer families: where two relatives
Eastern) and they collectively encompass had prostate cancer (130 cases). In total
approximately 6 million inhabitants (67% of 1961 prostate cancer cases were invited for
Sweden’s population). The source-person- participation in the CAPS study, of those
time was divided into two age–specific study 1444 (73.6%) approved to participate by
groups. The first group included men 35-65 donating a blood sample and answering the
years of age, living in all regions mentioned questionnaire. At the time of the studies in
above. The second study base included men this thesis DNA was available for 1383 of
66-79 years of age at the time of the study the cases who accepted to participate.
entry, living only in the central and northern Control subjects were randomly selected
region. Swedish law requires both the from the continuously updated Swedish
attending physician and pathologist to report Population Registry, frequency matched
newly diagnosed cancer cases to the cancer according to the expected age distribution
registries. Therefore, the registries include (within five years), geographic origin of the
records of almost 100% of all cancers cases and sex. Of the 1697 randomly
diagnosed in Sweden. The cases were linked selected controls that were invited for
to the National Prostate Cancer Registry and participation in the study, 866 (52.0%)
clinical information on aspects such as TNM accepted to participate with blood donation
(tumour-node-metastasis) stage, Gleason and questionnaire. At the time of the studies
sum, PSA level at the time of diagnosis, in this thesis DNA was available for 780 of
methods of diagnosis and primary treatment the controls that approved to participate.
were obtained for 95.3% of the cases. The Mean age (age at diagnosis for case patients
cases were then classified as either and age at inclusion for control subjects) for
Localised (T1-2, N0/NX, M0/MX, Grade the cases and controls were 66.6 and 67.9
I-II/Gleason sum 2-7, and PSA<100) or years, respectively. Blood (4 x 10 ml) was
Advanced (having or being prone to collected from all cases and controls and
progressive disease; T3/4 or N+ or M+ or separated into serum, plasma, and buffy
Grade III or Gleason sum 8-10 or coat. A detailed description of the study
PSA>100). For cases where at least one sample is presented in Study III. Clinical
reported family member with prostate characteristics of the prostate cancer cases in
cancer a more detailed family history of the study are summarized in Table 1.
prostate cancer was obtained through
Table 1. Clinical characteristics of the 1383 cases with prostate cancer included in
the CAPS study.
Age
45-65 (n=704) 66-80 (n=679)
Number (%) Number (%)
T-stage
T0 3 (0.4) 3 (0.4)
T1 259 (36.8) 200 (29.5)
T2 234 (33.2) 208 (30.6)
T3 144 (20.5) 207 (30.5)
T4 20 (2.8) 28 (4.1)
TX 44 (6.3) 33 (4.9)
N-stage
N0 157 (22.3) 66 (9.7)
N1 26 (3.7) 19 (2.8)
NX 521 (74.0) 594 (87.5)
M-stage
M0 328 (46.6) 250 (3.8)
M1 67 (9.5) 64 (9.4)
MX 309 (43.9) 365 (53.8)
Gleason score
<=4 29 (4.1) 24 (3.5)
5 79 (11.2) 63 (9.3)
6 236 (33.5) 185 (27.2)
7 177 (25.1) 188 (27.7)
8 53 (7.5) 70 (10.3)
9 40 (5.7) 37 (5.4)
10 7 (1.0) 3 (0.4)
Missing 83 (11.8) 109 (16.1)
Tumour grade
GI 35 (5.0) 34 (5.0)
GII 154 (21.9) 151 (22.2)
GIII 70 (9.9) 72 (10.6)
GX 445 (63.2) 422 (62.2)
PSA levels
<4 49 (7.0) 26 (3.8)
4-9.99 264 (37.5) 169 (24.9)
10-19.99 139 (19.7) 152 (22.4)
20-49.99 92 (13.1) 128 (18.9)
50-99.99 49 (7.0) 77 (11.3)
>=100 64 (9.1) 87 (12.8)
Missing 47 (6.7) 40 (5.9)
Genetic analysis
Genomic PCR (Studies I and II) column is proportional to the number of ion
pairs formed between the negatively charged
For the mutation screening in Study I and nucleic acids and the positively charged
genotyping of the VNTR in IL1-RN (Study TEAA ions absorbed to the stationary
II), intron complementary primer pairs that phase.
could amplify all exons, exon-intron The second step is elution of the nucleic
junctions, the promoter region, and 3´-UTR acids bound to the column using a linear
in the MSR1 and the VNTR in IL-1RN were gradient of Acetonitrile, which weakens the
used for amplification from genomic DNA. bonding between the column and TEAA by
competive hydrophobic interactions and
thus releases the nucleic acids. The helical
Mutation screening (Study I) nature of the duplex is disrupted in the
heteroduplexes due to the presence of
incorrect base pairing at the site of the
The analysis of the MSR-1 gene was started
mutation, which reduces the number of ion-
by screening the entire coding sequence -
pairing bonds with the column and thus
including exon-intron junctions, promoter
results in the heteroduplex eluting earlier (at
regions and 5´- and 3´-untranslated regions -
a lower acetonitrile concentration) than the
for mutations by Temperature Modulated
homoduplex (Figure 2). This approach to
Heteroduplex Analysis (TMHA) using High
detecting heteroduplexes is most suitable
Performance Liquid Chromatography
when screening for rare mutations and
(HPLC) and a DNA-binding column
mutations, for which homozygotes of the
(DNAsepTM). This technique is based on the
rare allele are unlikely to be present. Since
principle that heteroduplexes formed
this was our aim, we did not modify the
between mutated DNA and normal DNA
method to enable common polymorphisms
molecules can be distinguished from
(for which homozygotes of both alleles are
homoduplexes consisting entirely of normal
likely to present in the study population) to
DNA molecules, by a differences in their
be detected. A detailed description of the
elution profiles at a given optimized
method is presented elsewhere (Xiao et al.,
temperature. The experimental procedure
2001).
includes two main steps:
The sequence variants identified using
First, the nucleic acids of interest (for
the HPLC analyses were confirmed by direct
example PCR-products) are denatured at
sequencing. The amplicon of interest was
95°C for 5 minutes, then gradually
amplified from corresponding genomic
reannealed by reducing the temperature to
DNA. PCR products were purified and then
20°C over 50 minutes, a process that enables
sequenced with a dye terminator kit. To
formation of mismatched DNA
ensure high sequence quality, all fragments
(heteroduplexes). The samples are
were sequenced from both directions.
subsequently bound to the column using
Primers used for amplification of the PCR
trietylammonium acetate (TEAA). The
product were used as sequencing primers.
binding strength of nucleic acids to the
Wild
Mutant Heteroduplexes Homoduplexes
type
Wild type Homoduplexes
heat heat
cool cool
AT AT AT GC A C G T A T GC
10
3
9
Intensity (mV)
Intensity (mV)
8
7
2
6
5
4
3 1
2
1
0
0
0
Retention
2
time
4
(min)6 8 -0,2 0,8
Retention time (min)

1,8 2,8 3,8 4,8 5,8 6,8
Figure 2. Schematic illustration over homo/heteroduplex formation and the following

elution profiles.
Common sequence variants of the Thermo Sequenase (both from

MSR-1 gene (Study I) SEQUENOM, Inc.) and 600 nM of
extension primer. The cycling conditions
Five common sequence variants identified were 94°C for 2 minutes followed by 55
of the MSR-1 gene together with three rare cycles of 94°C for 5 seconds, 52°C for 5
mutations were further evaluated for seconds and 72°C for 5 seconds. After
possible association with prostate cancer cleaning up the hME reaction products with
risk. For the genotyping of the five common SpectroCLEAN the products were
SNPs (PRO3, INDEL1, IVS5-57, P275A, transferred to a SpectroCHIP using
and INDEL7) and the rare nonsense SpectroPOINT, and then scanned through
mutation (R293X), the MassARRAY system SepctroREADER. Genotyping was done
was used (SEQUENOM, Inc. Valencia, CA). using SpectroTYPER (all from
PCR reactions were performed in a total SEQUENOM, Inc.).
volume of 5 µl with 10 ng of genomic In addition to the MassARRAY
DNA, 2.5 mM of MgCl2, 0.1 U of genotyping system we utilized HPLC
HotStarTaq polymerase, (QIAGEN analyses (described above) to genotype the
Inc.Valencia, CA), 200 µM of each dNTP two splice site mutations located adjacent to
and 200 nM of each primer. The PCR exon 4. Since both were rare among the
conditions were as follows: 95°C for 15 HPC families and we did not expect any
minutes followed by 45 cycles of 95°C for homozygotes of the rare allele HPLC was a
20 seconds, 50°C for 30 seconds and 72°C suitable method.
for 1 minute with a final extension of 72°C
for 3 minutes. The hME reactions were
performed in a total volume of 9 µl with 50
µM of each d/ddNTP, 0.063 U/µl of
Association of MIC-1 sequence nuclease assay with TaqMan MGB probes.

variants and prostate cancer (Study The SNP genotyping assays were designed
III) using Applied Biosystems’ Assay-by-Design
service (Applied Biosystems Inc., Foster
To fully elucidate possible associations City, CA). All reactions were performed in
between prostate cancer risk and SNPs in 25 µl mixtures consisting of 10 ng genomic
the inflammatory gene MIC-1, the following DNA, 900 nM of each primer, 200 nM of
approach was used. First, the target region each probe and 12.5 µl of TaqMan universal
for selection of SNPs was defined as 2 kb of master-mix. PCR cycling conditions were as
the promoter plus all exons, introns, and the follows, 50°C for 2 min, 95°C for 10 min
3’UTR. The SNP information for this region followed by 40 cycles of 92°C for 15 sec and
was obtained from public databases (NCBI 60°C for 1 min. The samples were analyzed
dbSNP, and SNPer) or from data obtained using an ABI 7700 sequence detection
by re-sequencing samples from 24 control system. For quality control, two CEPH
subjects when detailed SNP information was DNA samples and additional water blanks
not publicly available. Two criteria were use were included on each 96-well plate.
to select SNPs for further analyses: Following the first genotyping, the SNP
1) the minor allele frequency had to be MIC1-1576 was excluded from further
at least 5%, at a resolution of 1 SNP analysis since it deviated significantly from
per kb of DNA across the genome HWE (p=0.003). Haplotype frequencies for
region of MIC-1. The density of the the remaining seven SNPs were estimated
selected SNPs was chosen to ensure using the statistical method proposed by
that they sufficiently reflected the Stephens et al. (2001), as implemented in the
haplotype block pattern throughout computer program PHASE
the genome (Wall et al., 2003). (http://www.stats.ox.ac.uk/mathgen/softwa
2) all SNPs that lead to an amino acid re.html). A subset of “Haplotype Tagging
substitution, since such changes are SNPs” that could uniquely represent at least
more likely to have an effect on 95% of the haplotype information observed
prostate cancer susceptibility and among the 94 control individuals was
pathogenesis. selected using the computer software
From the public database, six SNPs htSNP2 (http://www-
(Exon1+25, Exon1+142, IVS1+904, gene.cimr.cam.ac.uk/clayton/software/stata
IVS1+1809, Exon2+2423, 3’-UTR+2816) ). Four SNPs (Exon1 +25, Exon1 +142,
located in the exons, introns and 3’UTR IVS1 +1809, and Exon2 +2423 (H6D)) that
were selected. In addition, two SNPs captured 98.6% of the haplotype variation
(MIC1-1576, and MIC1-893) located in the among the 94 controls were selected as
promoter region that fulfilled the criteria haplotype tagging SNPs. Each of these four
were selected by the re-sequencing samples SNPs was in HWE among both the cases
from 24 randomly selected CAPS controls and controls. These four htSNPs were
(Figure 3). genotyped for all 1383 cases and 780
DNA from 94 control subjects selected controls from the CAPS study. The
at random from CAPS was genotyped with genotyping was performed using the
respect to the eight chosen SNPs using a 5’ MassARRAY system (SEQUENOM Inc.,
Valencia, CA), described in detail above.
The DNA samples were labelled blindly and assays were designed using the Assay-by-
shipped from Umeå University, Sweden, to Design service (Applied Biosystems Inc.,
the core genotyping laboratory in the Center Foster City, CA) and the samples were
for Human Genomics, Wake Forest analyzed using an ABI 7700 sequence
University, USA. In total, 37 controls from detection system. Since it was not possible
the CEPH foundation (1331-01, 1331-02), to design a 5’ nuclease assay for the 86-bp
and 29 blind repeats were spread among the VNTR in intron 2, it was genotyped by
DNA samples. In addition, every DNA plate temperature modulated heteroduplex
contained two water blanks. analysis using a High Performance Liquid
Chromatography system, performed as
described above (Study I). Haplotypes of
Association of Interleukin-1 receptor these SNPs were estimated using a Markov
antagonist haplotype with prostate Chain Monte Carlo approach as
cancer risk (Study II) implemented in the PHASE software
package
The procedure described above for selecting (http://www.stats.ox.ac.uk/mathgen/softwa
MIC-1 sequence variants for analysis was re.html). Haplotype-tagging SNPs, which
also used to select SNPs of the IL1-RN captured at least 95% of the haplotype
gene. In total, 16 SNPs and two repeats (the variation among the 94 controls, were
86-bp VNTR and an 8-bp repeat) were selected using the htSNP2 computer
identified by scrutinizing public databases program (www-
(NCBI dbSNP, and SNPper) (Figure 3). gene.cimr.cam.ac.uk/clayton/software/stat).
Three different methods were used for Four htSNPs were identified and they were
genotyping. genotyped in all 1383 cases and 779
First, the 16 identified SNPs and the 8- controls. The htSNPs (rs878972, rs315934,
bp repeat were genotyped in 94 control rs3087263, rs315951) were then genotyped
subjects using a 5’ nuclease assay with using the MassARRAY system
TaqMan MGB probes. The SNP genotyping (SEQUENOM, Inc. Valencia, CA).
1 kb
MIC-1
-893 T/C
1809 G/T
-1576 A/G
V9L 25 C/G
H6D 2423 C/G

S48T 142 A/T
904 A/G
2816 C/G
2423 C/G
1809 G/T
142 A/T
25 C/G
Haplotype-tagging
SNPs (htSNPs)
1 kb
IL1-RN
8599C/T
3701A/G
6429-/+
-1129C/T
1631G/A
9726A/G
10173G/A
8111G/A
15818A/G
12985C/T
11864A/C
13875T/G
14992G/C
2118C/A
-12C/G
4696C/G
5351T/C
618A/C
10909A/G
8111G/A
10173G/A
14992G/C
2118C/A
Haplotype-tagging
SNPs (htSNPs)
Figure 3. A) Single nucleotide polymorphisms (SNPs) of the Macrophage Inhibitory

Cyokine-1 gene and Interleukin-1 Receptor Antagonist gene. The diagrams of the genes
show exons (marked in red), the location of selected SNPs (relative to the transcriptional
start site) and SNPs that result in an amino acid substitution. Arrows represent
transcription start sites. B) Location of the haplotype-tagging SNPs that were selected to
represent at least 95% of the haplotype information.
Determination of MIC-1 serum levels (Study IV)
MIC-1 serum levels were determined by a substrate (1 mg/mL o-phenylenediamine

MIC-1 sandwich ELISA, using the mouse dihydrochloride from Sigma) in 0.05 mol/L
monoclonal antibody (MAb) 13C4H3 for phosphate-citrate buffer containing 0.014%
antigen capture and a sheep polyclonal H2O2, pH 5.0 (Sigma). Colour development
antibody (PAb), 233B3-P, for detection. The was allowed to proceed for 5-15 min and
optimum concentration of both antibodies was terminated by the addition of 100
was determined and then used for all mL/well of 4N H2SO4. The absorbance was
subsequent studies. Ninety-six-well measured at 490 nm in a microplate reader.
Maxisorp ELISA plates were coated with The concentration of hMIC-1 in the
MAb 13C4H3 supernatant diluted 1:5 (final samples was determined by comparison with
concentration, approximately 20 ng/mL) in an rhMIC-1 standard curve constructed
coating buffer at 4°C for 24 hours. The using standard curve-fitting software
plates were then washed three times with supplied with the microplate reader (Pasteur
300 mL/well 1% (wt/vol) BSA in PBS for 2 Diagnostics). The level of rhMIC-1 in the
h at 37°C. rhMIC-1 standards, tissue culture standard curve was determined on the basis
supernatant and patient serum were then of a comparison of this standard to a
added to the plates (100 mL/well) and master standard of highly purified
incubated for 1 h at 37°C. The plates were recombinant MIC-1. The master standard
washed three times, 100 mL/well of the protein concentration was determined by
sheep PAb 233B3-P diluted 1:5000 in averaging eight estimates of total amino acid
antibody diluent (Ab dil) was added and they composition. All samples were assayed in
were incubated for 1 h at 37°C. The plates triplicate.
were then washed three times, 100 mL/well The serum samples were labelled blindly
of biotinylated donkey antisheep IgG and shipped from Umeå University, Sweden,
diluted 1:5000 in Ab dil was added and they to the Centre for Immunology, St Vincent's
were incubated for 1 h at 37°C. The plates Hospital & University of New South Wales,
were washed four times, followed by the Australia, where the serum concentrations
were determined.
addition of 100 mL/well of peroxidase
Statistical analysis
Tests for Hardy-Weinberg replication method, as described by (Weir,

Equilibrium (Studies I, II, III) 1996). For each test, 10,000 permutations
were performed and the Fisher probability
Hardy-Weinberg Equilibrium (HWE) tests test statistic for each replicate was calculated
for each sequence variant and pair-wise from the new corresponding multilocus
linkage disequilibrium (LD) tests for all table. Empirical p-values for each test were
sequence variants were performed using a estimated as the proportion of replicate data
sets found to be as probable as, or less Haplotype analysis (Studies II, III)
probable than, the observed data set, as
implemented in the software package Tests of association between haplotypes and
Genetic Data Analysis (GDA). prostate cancer risk were performed using a
score test developed by Schaid et al. (Schaid
et al., 2002), as implemented in the software
Association analysis (Studies I, II, HAPLO.STAT for the R programming
III, IV) language. This method, based on the
generalized linear model framework, allows
Associations between genotypes/MIC-1 adjustment for possible confounding
serum and prostate cancer risk were assessed variables and provides both global and
by the score tests in conditional logistic haplotype-specific tests. In these analyses,
regression of a covariate equal to the age and geographic region were adjusted for
number of rare alleles (0, 1, 2) (Studies II using indicator variables representing each
and III) and by dichotomized MIC-1 serum combination of age category (5-year age
levels (with a cut-off value of 1000 pg/ml, groups) and geographical region was
based on the median level among unaffected adjusted as described earlier. Haplotypes
controls) (study IV). Genotype specific risks with estimated frequencies less than 0.005
were estimated as Odds Ratios (OR) with were pooled into a single group. Empirical
associated 95% confidence intervals by P-values, based on 10,000 simulations, were
conditional logistic regression. Both when computed for the global score test and each
testing for association and estimating ORs, of the haplotype-specific score tests.
the conditional logistic regression was
stratified by each combination of age (5-year
age groups) and geographical region (the Assessment of serum levels (Study
northern part of Sweden vs. the south IV)
eastern part of Sweden and the Stockholm
area) to adjust for the matching conducted Serum MIC-1 levels of prostate cancer cases
in collecting control subjects. Besides age and controls were presented as means +/-
and geographical region, no other factors standard deviation (SD). Formal comparison
were included in the regression analysis. In of MIC-1 levels between different subject
Study I unconditional logistic regression was groups were conducted using analysis of
used to test for association between variance (ANOVA) methods. To evaluate
genotypes and affection status. We adjusted the diagnostic value of MIC-1 serum levels,
for age and geographical region using nonparametric receiver operating
indicator variables representing each characteristic (ROC) analysis was performed.
combination of age of onset (5-year age
groups) and geographical region.
Results and comments
Genetic analysis of MSR1 (Study I)
In order to evaluate the role of MSR1 gene In agreement with our findings, R293X
mutations in prostate cancer in the Swedish mutation was observed in the same
population we carried out a genetic analysis frequency (3.2%) among Caucasian HPC
in a large number of subjects from two families in the study from the United States,
different study populations in Sweden. We (Xu et al., 2002). In addition, Seppälä et al.
initially screened a set of DNA samples reported the same frequency (2.5%) among
representing one affected individual from 120 families affected with HPC (Seppälä et
each of 83 Swedish families affected by al., 2003), as well as Wang et al. among 163
HPC. Among these individuals we identified families affected with familial prostate
18 sequence variants in total, including two cancer (3.1%) (Wang et al., 2003) (Table 2).
exonic variants, four intronic and nine
variants located in the 5’- or 3’-uncoding We further evaluated the association
regions. A nonsense mutation at codon 293 between this mutation and prostate cancer
(R293X) and five common sequence by screening a group of 215 unrelated men
variants (PRO3, INDEL1, IVS5-57, P275, with prostate cancer and 425 age-matched
INDEL7) identified in this study, were also controls. The R293X mutation was found
reported by Xu et al (Xu et al., 2002). more frequently in men with prostate cancer
The truncating mutation R293X results (10 individuals, 4.9%) than their matched
in a deletion of most of the collagen-like controls (10 individuals, 2.7%), however this
domain, including the ligand-binding region difference was not statistically significant
and the cysteine-rich domain. Experimental (P=0.16) (Table 2). In contrast, Xu et al.
studies have demonstrated that an MSR1 reported a significant difference in
mutant harbouring a similar truncating frequencies of the R293X mutation between
mutation to R293X has a dominant-negative men with non-HPC and unaffected men
phenotype when expressed in vitro (Dejager (2.5% versus 0.39%, P=0.047) (Xu et al.,
et al., 1993). R293X was found in two 2002). The choice of control population
probands from two different families among might explain some of the differences
the 83 HPC families (2.4%). One family had between our results and the previous report.
one affected (the proband) and three The men in the control population in the
unaffected R293X carriers, while the other present study were on average 66 years old
family had one affected (the proband) and at recruitment, an age at which only 15% of
two unaffected R293X carriers. However, all prostate cancers have been diagnosed.
due to the low numbers of affected men in Thus, some of the unaffected R293X
these two families, it was not possible to carriers are likely to develop prostate cancer
determine whether the mutation segregated eventually. Another difference is that Xu et
with the disease. al. only included men with PSA values <4
and a normal prostate examination.
Table 2. Reported studies of the association between the MSR1 nonsense
mutation R293X and prostate cancer risk
No. of carriers/total (frequency )
Study Families C ases with C ontrols P-value

with HPC unselected
prostate
cancer
Xu et al. 2002 6/190 8/317 1/256 0.047*
(3.2%) (2.52%) (0.39%)
Seppälä et al. 2003 3/120 6/537 5/480 0.91

(2.5%) (1.1%) (1.0%)
Wang et al. 2003 5/163 14/496 16/492 0.69

(3.1%) (2.8%) (3.3%)
Lindmark et al. 2004 2/83 (2.4%) 10/215 10/425 0.16*

(4.9%) (2.7%)
* Fisher exact test (two-sided) between cases and controls.
Two other studies that investigated the mutations located in similar positions to
possible association between the R293X IVS3-4 and IVS4+3 can lead to exon
mutation and prostate cancer did not find skipping, intron retention or insertions and
any statistically significant difference deletions due to utilization of cryptic splice
between unselected prostate cancer cases sites (Margaglione et al., 2000; Attanasio et
and controls (Seppälä et al., 2003; Wang et al., 2001, 2003). Further functional studies
al., 2003) (Table 2). If the R293X mutation are needed to assess the functional
increases the risk for prostate cancer by a significance, if any, of these mutations.
factor of two and the frequency is ~3% in Besides the three rare mutations
the general population, as our results described above, we decided to elucidate the
suggest, a much larger case-control study importance of five common sequence
would be needed to significantly verify this. variants found in the mutation screening of
In addition, we detected two novel MSR1: an SNP in the promoter region
potential pathogenic mutations, IVS3-4 (PRO3), a 15-bp insertion/deletion of
A>G and IVS4+3 A>G, respectively “GAATGCTTTATTGTA” in intron 1
located in the splice donor region and splice (INDEL1), an SNP in intron 5 (IVS5-57), a
site acceptor region of exon 4. Both of these SNP in Exon 6 (P275A), and a 3-bp
mutations were observed in single families, insertion/deletion of “TTA” in intron 7
and neither of them was detected when (INDEL7). The initial study evaluating the
screening the group of unselected men with role of the five sequence variants in the
prostate cancer or age-matched healthy MSR1 gene, reported a significant difference
controls. Point mutations which alter a in the allele frequencies of each of the five
conserved sequence in the splice donor variants between patients with non-HPC
region, the splice acceptor region or a region and unaffected control subjects, which led
nearby may cause aberrant splicing of a to the suggestion that these common MSR1
gene. Several studies have shown that variants are associated with prostate cancer
risk in the general population (Xu et al., patients with prostate cancer and 425 age–
2003). To investigate whether these variants matched controls. None of the variants were
are associated with prostate cancer risk we found to be associated with prostate cancer
decided to genotype them in 215 unrelated (Table 3).
Table 3. Allele frequencies of common MSR1 sequence variants in unselected

prostate cancer cases and unaffected control subjects
Allele frequencies (%) Allele frequencies (%)
215 cases / 425 controls; 301 cases / 250 controls;
Lindmark et al. 2004 Xu et al. 2003
Sequence Allele Controls Cases P- Controls Cases P-
variant values* values*
PRO3 G 6.1 6.0 1.00 7.6 12.3 0.01
INDEL1 + 5.9 5.9 0.94 7.9 11.8 0.04
IVS5-57 A 3.2 2.9 0.80 3.6 7.0 0.02
P275A C 96.9 96.3 0.55 91.2 95.0 0.01
INDEL7 - 97.2 96.7 0.62 91.3 94.4 0.04
* χ2-test for the allele
Two other studies have evaluated the macrophages by oxidative stress and may
common sequence variant in exon 6 (P275) modify the amounts of reactive oxygen
for its role in prostate cancer risk in the intermediates. The inflammation and
general population. Carrier frequencies of proliferative regeneration of prostate
the P275A variant were compared between epithelium in the presence of increased
unselected prostate cancer cases and oxidative stress that are associated with
controls. Their data are consistent with our MSR1 expression may have a role in the
results because they found no statistically development of prostate cancer (Xu et al.,
significant difference in the carrier 2002).
frequencies between the different sample In conclusion, we found no evidence to
groups (Seppälä et al., 2003; Wang et al., support the hypothesis that the sequence
2003). variants in the MSR1 gene play a role in the
It is not known how MSR1 could development of hereditary or sporadic
contribute to the development of prostate prostate cancer.
cancer. However, MSR1 is induced in
Genetic analysis of IL-1RN (Study II)
Several studies have shown that IL1-RN acts to be less sensitive to departure from HWE
as an important regulator of the than the asymptotically distributed score
inflammatory response by inhibiting the statistic.
action of IL-1α and IL-1β. Since chronic The SNP analysis showed that there were
inflammation in the prostate appears to be a no significant differences in proportions of
co-factor in the pathogenesis of prostate any of the four htSNPs between the controls
cancer, we hypothesized that functional and prostate cancer cases. Assuming that the
polymorphisms in inflammation-related SNPs had a dominant or recessive allelic
genes, such as IL-1RN may be associated effect on prostate cancer risk did not alter
with prostate cancer risk. To test this these findings. Furthermore, stratified
hypothesis, we performed genotype analyses analyses based on age (<65 or ³65), tumour
for four IL1-RN gene polymorphisms in a stage (localized or locally advanced) and
large population-based case-control study of family history (sporadic or
1383 prostate cancer case patients and 779 familial/hereditary) detected no significant
control subjects. differences in genotype frequencies between
Quality control of the genotype results cases and controls.
(based on included CEPH controls, repeated We also performed a haplotype analysis
study samples and water blanks) provided an of the four htSNPs, which identified the
estimated error rate of 0%. Except for the presence of eight major haplotypes. Global
SNP rs315951, which significantly deviated tests for association between haplotypes and
from HWE in cases (P=0.04), but not prostate cancer risk all yielded non-
controls (P=0.34), the genotype data for the significant results. However, individual
remaining three SNPs were consistent with haplotype analyses revealed that one
HWE (all P>0.05). The significant deviation haplotype was statistically significantly
of the genotype frequencies of SNP associated with prostate cancer risk (Table
rs315951 from expected proportions among 4). The frequency of the ATGC haplotype
the prostate cancer subjects may be a result of SNPs rs878972, rs315934, rs3087263, and
of genotyping errors, population rs315951 was significantly higher among
stratification, selection, or statistical cases (38.7%) compared to controls (33.5%)
fluctuations. However, given the high quality (haplotype-specific P=0.009). Furthermore,
of the genotyping results and the ethnic in the stratified analysis the frequency of the
homogeneity of the Swedish population, it “ATGC” haplotype was higher in sporadic
seems most plausible that chance alone was (39.3%) than in familial (34.8%) prostate
responsible for the observed departure from cancer cases. Likewise, cases with advanced
HWE. Since the departure from HWE was disease had a higher prevalence (40.0%) than
in the direction of excessive homozygosity, patients with localised disease (37.5%). The
the error in haplotype estimations (based on fact that the association was strengthened in
the EM algorithm) was not increased (Fallin cases with advanced disease is particularly
et al., 2000). Moreover, all statistical noteworthy. While it is important to identify
inferences regarding haplotypes were based disease susceptibility genes, it is equally or
on simulated P-values, which are expected
probably even more important to identify that only alter protein function significantly
genes that play roles in disease progression, when they occur together. Second, the
especially for prostate cancer since the ATGC-haplotype may be in strong linkage
disease has a late average age of onset and is disequilibrium (LD) with a risk-conferring
life threatening in a relatively small sequence variant in IL1-RN, or in close
proportion of cases (aggressive cases). vicinity to the gene, while the degree of LD
Polymorphisms in genes mediating between this risk-conferring sequence
progression of prostate cancer could variant and each of the four htSNPs is much
function as genetic markers predicting an weaker.
increased risk of progressive disease. In this study the overall haplotype test
The observation that the most common gave a non-significant result, while the
haplotype was associated with prostate haplotype-specific score was significant for
cancer risk but none of the individual the most common haplotype. However, the
htSNPs, highlights the advantages of estimated odds ratios show significantly
studying all variations in a gene with the use increased risk only for homozygous carriers
of a haplotype-tagging approach instead of (OR = 1.6; 95% CI, 1.2-2.2) of the “ATGC”
testing a limited number of single variants. haplotype and not for heterozygous carriers
Two different scenarios may explain this (OR = 1.0: 95% CI, 0.8-1.2). Since the
observation. First, the association may be a proportion of individuals that are
result of two or more sequence variants in homozygous for this haplotype is
the region that do not individually confer a considerably lower than the overall
detectably increased risk for prostate cancer, frequency of the haplotype, the power of the
but do confer a statistically significant risk in global test will be considerably lower than if
combination, which can be detected using heterozygous carriers were also at increased
the haplotype-association strategy. For risk.
example, there may be two SNPs in the gene
Table 4. Estimated haplotype frequencies in the IL1-RN gene in controls, patients with sporadic prostate cancer (SPC) and
45
patients with advanced prostate cancer.

Haplotype Controls Cases SPC Advanced PC
rs3087263
rs878972
rs315934
rs315951
% % Score* P† % Score* P† % Score* P†

A T G C 33.5 38.7 2.60 0.009 39.3 2.85 0.006 40.0 2.67 0.009
A T G G 17.6 15.5 -1.01 0.301 15.0 -1.24 0.215 15.0 -1.15 0.246
Prostate Cancer and Inflammatory Genes
C T G C 17.5 15.3 -1.16 0.234 15.2 -1.22 0.208 14.1 -1.78 0.080
A C G G 14.3 13.6 -0.58 0.560 14.0 -0.41 0.667 14.9 0.45 0.655
C T A C 8.4 7.7 -0.80 0.428 7.9 -0.64 0.513 8.4 -0.20 0.839
A C G C 5.8 5.4 -0.38 0.713 4.9 -0.89 0.367 4.8 -0.86 0.401
C T G G 1.8 2.7 0.97 0.337 2.9 1.06 0.274 2.4 0.17 0.872
C T A G 0.6 0.7 0.33 0.737 0 0
Overall 0.116 0.052 0.024
*
Score test statistics for association between haplotype and prostate cancer risk.
†
Empirical P values based on 10,000 replications.
Several studies have evaluated the role of characteristics, raises the possibility that an
IL-1RN in a number of malignancies by association between a haplotype in IL-1RN
investigating the frequency of the 86-bp and prostate cancer may in fact be due to an
VNTR in various study populations. association between another gene or genes
However, since the VNTR only covers a in the cluster and prostate cancer.
minor part of the variance in the gene, it is Knowledge of the degree of LD across this
not enough to solely analyze the repeat to region is vital to our understanding of the
evaluate the role of IL-1RN. Therefore, we combinations of genotypes that are
used another approach, in which we studied important in disease. Several analyses have
a set of tagging SNPs that captured almost been performed to determine the degree of
all the genetic information in the entire gene. LD across this region, all of which have
We genotyped four htSNPs in the study found moderate LD in this gene cluster that
population, which explained >98% of is not strictly correlated with distances
diversity in the gene. The VNTR was not between markers (Cox et al., 1998; Bensen et
included in the genotyping, but it was almost al., 2003). Thus, additional SNPs in this
equivalent to the htSNP rs878972 (Pearson region need to be identified and analysed to
correlation coefficient = 0.99). The htSNP determine whether apparent associations
rs878072 was not associated with prostate between IL-1RN and prostate cancer reflect
cancer risk, thus it is unlikely that the VNTR a causative relationship, or are due to the
in intron 2 by itself contributes significantly effects of another gene in the cluster.
to prostate cancer risk. Our results suggest that (a) prostate
The IL-1RN gene belongs to the IL-1 cancer risk variant(s) may be located
gene cluster, which spans a 360-kb region of somewhere in the region of IL1-RN. The
chromosome 2 (Steinkasserer et al., 1992). exact location and biological function of
This cluster of genes contains several pro- this/these sequence variant/variants remain
and anti-inflammatory cytokine genes that to be identified. Further studies are needed
are expressed in both physiological and to replicate our findings in other populations
pathological conditions and play a key role and to elucidate the biological function of
in the inflammatory immune response. The the IL-1RN haplotype in relation to prostate
high density of IL-1 genes in this region, cancer risk.
which share similar functional
Genetic analysis of MIC-1 (Study III)
Due to the potential importance of MIC-1 As a first step, we genotyped four

in tumour development and regulation of common SNPs (Exon1+25, Exon1+142,
inflammation responses, we decided to IVS1+1809, and Exon2+2423 [H6D]) in a
further elucidate the role of MIC-1 by large population-based case-control study of
investigating possible associations between 1383 prostate cancer patients and 780
sequence variants in the gene and prostate control subjects. With these four SNPs it
cancer risk. was possible to capture >98% of the
haplotype variation in the gene, and each of mechanisms involved remain unclear. The
them were in HWE among both cases and substituted amino acid at position six is
controls (all P>0.05). These SNPs were in located adjacent to the cysteine at position
strong LD, as most of the pair-wise D’ seven, which has been indicated to be
estimates were 1.0, with the lowest at 0.94. important for the stability of MIC-1 (Fairlie
Testing for genotype frequency et al., 2001). Because of the different
differences between cases and controls biochemical properties of aspartic acid and
revealed one SNP (H6D) for which there histidine, the H6D polymorphism may alter
was a significant difference between cases MIC-1’s stability and function. If it abolishes
and controls (P=0.006); proportions of or reduces MIC-1 activity, inflammation in
homozygotes for the common CC genotype the prostate may go unchecked in carriers
encoding the wild-type protein being higher with the risk genotype, leading to an
among prostate cancer cases than among increased risk for tumour development. On
controls (53.0% versus 48.5%). In an the other hand, expression of a MIC-1
assessment of genotype-specific risk we protein with increased activity or stability
found that the OR for carriers of the GC or may lead to a reduced pro-inflammatory
GG genotype, which encodes the H6D response, which in turn could reduce the
mutant, was lower than the OR for the CC capacity to eradicate certain pathogens
genotype carriers (OR=0.83; 95% CI, 0.69- effectively. This is in line with the infectious
0.99). The decreased risk for carriers of the aetiology of prostate cancer that has been
GC or GG genotype compared with that of proposed (Dennis et al., 2002a).
the CC genotype carriers was further Furthermore, altered functionality of MIC-1
accentuated both in patients with advanced due to the H6D polymorphism may also
disease (OR=0.79; 95% CI, 0.63-0.99) and in lead to decreased tumour cell growth
patients with a positive family history of inhibition mediated through the TGF-β
prostate cancer (OR=0.68; 95% CI, 0.48- signalling pathway. Other types of
0.96) (Table 5). The observation that the investigations, such as in vitro and in vivo
association was further strengthened in functional studies of the H6D variant, are
patients with a positive family history is needed to address these possibilities.
consistent with the hypothesis that risk In conclusion, this is the first published
genotypes may co-segregate with unknown study evaluating the possible association
gene alterations with low penetrance within between sequence variants in the MIC-1
families. In summary, the findings that an gene and prostate cancer risk. Our study
association between the H6D variant and provided evidence for such an association,
prostate cancer is present in sporadic cases but more studies are needed to confirm or
and further accentuated in familial cases are refute this finding in independent
consistent with current knowledge. populations and to elucidate the mechanism
Although this study provided strong whereby the H6D sequence variant affects
evidence for an association between the the expression and function of MIC-1 in the
H6D sequence variant in the MIC-1 gene signalling pathways that seem to control
and prostate cancer, the underlying macrophage regulation and tumour growth.
Table 5. Association between prostate cancer and the H6D sequence variant of the MIC-1
gene in a large population-based study (CAPS).
OR* (95% CI)
Study subset No. of GC vs. CC GG vs. CC GC or GG vs. CC
subjects
All PC 1340 0.77 (0.64, 0.93) 1.22 (0.84, 1.75) 0.83 (0.69, 0.99)
Advanced PC 0.75 (0.59, 0.95) 1.11 (0.70, 1.74) 0.79 (0.63, 0.99)
FPC and 159 0.61 (0.42, 0.89) 1.18 (0.61, 2.29) 0.68 (0.48, 0.96)
HPC†
Advanced PC 57 0.44 (0.24, 0.81) 0.62 (0.18, 2.05) 0.47 (0.26, 0.82)
*Conditional logistic regression stratified by age and geographical region.
†FPC= Familial Prostate Cancer, HPC= Hereditary Prostate Cancer.
Analysis of MIC-1 serum levels (Study IV)
The development of sensitive immunoassays available for analyses. 19 prostate cancer

for MIC-1 has made it possible to study cases were excluded from further statistical
MIC-1 protein levels in human serum and analyses since clinical data were missing for
other body fluids. Brown et al. found the these subjects.
MIC-1 serum concentration to be higher in Examination of serum MIC-1 levels
patients with colorectal cancer than in among control subjects revealed that MIC-1
controls, and production of the protein was concentrations increased with increasing age
also up-regulated in tumour tissues (P<0.001) (Figure 4). This is an interesting
compared to its normal counterparts (Brown and important finding since it influences the
et al., 2003). The same pattern was also interpretation of the serum analyses. A
observed in a small study in patients with number of investigations of potential serum
breast and prostate cancer (Buckhaults et al., markers for cancer (including MIC-1), have
2001). Based on these findings, together used a small number of control subjects,
with the results from study III, we decided drawn from volunteers or blood donors,
to determine the serum MIC-1 limiting the scope to identify possible
concentration in the CAPS population to correlations between serum levels and age.
elucidate whether the circulating MIC-1 Further, using a control group that differs
levels are related to prostate cancer and significantly in mean age from the case
MIC-1 genotype and whether MIC-1 levels subjects could result in significant difference
are associated with prostate cancer risk. At in serum concentration between cases and
the time of the study sera from 620 controls controls that are solely due to the
and 1116 cases from the CAPS study were differences in mean age between the two
groups. The reason for the relationship but may be related to an increased general
between age and serum MIC-1 levels found inflammation burden.
among the controls in this study is unclear,
3000
2500
2000
MIC-1 (pg/ml)
1500
1000
500
0
<60 60-64 65-69 70-74 75-79
Age at inclusion (years)

Figure 4. MIC-1 concentration in 620 controls without prostate cancer with respect to age
(P<0.001).
The mean serum MIC-1 level among all (watchful waiting, hormonal treatment,
prostate cancer patients analysed was 1357 radical prostatectomy and radiotherapy) they
pg/ml, SD=1534, which was significantly received. For 43 patients, treatment was not
higher (P= 0.004) than the MIC-1 level specified so these patients could not be
among the control subjects (1190 pg/ml, classified. Significant differences in serum
SD=942) (Table 6) In addition, MIC-1 levels MIC-1 level between the different treatment
were significantly higher (P<0.001) among options were observed (P<0.001). However,
patients with advanced disease than among multiple ANOVA analyses of MIC-1 levels
patients with localised prostate cancer, with age, disease stage and treatment as
(mean 1666 pg/ml, SD=2138 and 1121 independent factors revealed significant
pg/ml, SD=733, respectively (Table 6). The effects of age and disease stage, while no
prostate cancer group was further divided significant effect was observed for
into subgroups depending on the treatment treatment.
Table 6. Serum MIC-1 level of all prostate cancer patients and controls according to
stage and treatment
Status n Mean ± S.D P-value*
MIC-1 (pg/ml) Controls 620 1190±942

Prostate cancer cases (all) 1097 1357±1534 0.004
Stage
Localized 622 1121±733
Advanced 475 1666±2138 <0.001
Treatment
Watchful waiting 245 1278±773
Radical prostatectomy 219 1059±929
Hormonal treatment 506 1575±2057
Radiotherapy 84 1153±573 <0.001
* Analysis of variance (ANOVA)
To further refine the correlation between relationship between MIC-1 serum level and
serum MIC-1 levels and disease stage, the tumour burden. These data are consistent
prostate cancer group was stratified based with previous reports that patients with
on the TNM classification. The stratified metastatic cancer had significantly elevated
analysis revealed a significant increase in serum MIC-1 levels compared with other
serum MIC-1 levels with increasing TNM- cancer patients (Brown et al., 2003).
stage (P<0.001, age adjusted)(Figure 5). The However, if the elevation is a secondary
same trend was observed when only effect of the tumour growth or wether MIC-
untreated cases (n=245) were included in the 1 palys an active role in the disease
analysis (P=0.029) (data not shown), clearly progression, where an increase in serum
indicating that this phenomenon is not concentration enhances tumous growth, is
related to treatment effects. All together still needed to be investigated.
these results clearly demonstrate a
4000
3500
3000
MIC-1 (pg/ml)
2500
2000
1500
1000
500
0
I II III IV
TNM tumour stage

Figure 5. Correlation between MIC-1 serum concentrations and disease stage (P<0.001)
among 1097 men with prostate cancer.
A possible association between serum showed that there is no diagnostic

levels and prostate cancer risk were also usefulness MIC-1 (area under ROC curve:
assessed. Using logistic regression analysis, a 0.56) (Figure 6). On the other hand, the
serum MIC-1 level above the median of the clear relation between clinical stage and
controls is indicated to be a significant serum MIC-1 levels indicates that MIC-1
predictor for development of prostate may be useful as a prognostic factor, where
cancer. Thus, considering a cut-off serum high serum concentrations at diagnosis of
level of > 1000 pg/ml, the relative risk for prostate cancer are associated with a poor
development of prostate cancer was 1.5 prognosis. For this purpose, serial
(95% CI, 1.1-2.0). To test the ability of the measurements of MIC-1 among diagnosed
serum assay to differentiate prostate cancer cancer patients would probably be of greater
cases from control subjects, ROC analyses value than a single observation.
were performed. The ROC curve clearly
1.0
0.8
0.6
Sensitivity
0.4
0.2
0.0
0.0 0.2 0.4 0.6 0.8 1.0

1 - Specificity
Figure 6. Receiver operator characteristic (ROC) curve of MIC-1 for differentiation of

patients with untreated prostate cancer (n=245) from control subjects (n=620). Area
under ROC curve: 0.56.
Finally, to test for possible association the H allele and thereby alter the risk for
between MIC-1 genotype and serum levels, prostate cancer development.
we compared serum levels between control Another important question needed to
subjects with different genotypes according be answered, but that is beyond the result
to the H6D polymorphism. No significant from this study, involves the role that MIC-1
differences in MIC-1 serum levels between may play in the development of prostate
the genotypes was observed (P=0.41) cancer. The increase in serum MIC-1 level
(Figure 7). We therefore suggest that the associated with the presence of prostate
H6D polymorphism may alter the function cancer implies that an inflammatory process
or stability may of the protein, for example in the prostate may be involved in the
the D allele may be more biologically active development or progression of prostate
or function in a different way compared to cancer, since in vitro studies have shown that
MIC-1 may function as a suppressor of
macrophage-mediated proinflammatory together with the striking correlation

activity. However, studies of several tumour between elevated MIC-1 levels and
cell lines have also shown that MIC-1 has metastatic progression of prostate, breast
proapoptotic and antitumorigenic activities and colorectal cancer – is related to MIC-1’s
(Bauskin et al., 2005). Brown et al. proposed similarity to the TBF-β protein. It has been
the following explanation for this dual role reported that MIC-1, like TGF-β, requires
of MIC-1 in cancer progression. As the an intact signaling pathway mediated by type
prostate cancer tumour progresses, MIC-1 I and type II TGF-β receptors, as well as
secretion may rise, leading to an inhibitory receptor-activated Smad4 to exert its effects
effect on tumour growth. However, this (Tan et al., 2000). TGF-β is considered to act
inhibitory effect may be negated by the as a tumor supressor during the early stages
decrease in local tumour immunity, of cancer (by inhibiting cell proliferation),
mediated by the immune suppressor effect and as a promoter of growth and metastasis
of MIC-1, and thus providing a mechanism in later stages, thereby enhancing
for tumour escape (Brown et al., 2003). angiogenesis, immunosupression and
Another explanation for the incongruous synthesis of extracellular matrix (Dumont et
indications of both proapoptotic and al., 2003).
antitumorigenic functions for MIC-1 –
3000
2500
2000
MIC-1 (pg/ml)
1500
1000
500
0
CC CG GG
H6D genotype
Figure 7. Correlation between MIC-1 genotypes and serum level in 620 controls without
prostate cancer. No significant difference in MIC-1 serum levels among control subjects
with different genotypes was observed (P=0.41).
In conclusion, a clear association data revealed that the differences in serum

between disease stages and serum MIC-1 MIC-1 levels between cases and controls are
levels was found, indicating that MIC-1 may not large enough to provide a discriminatory
be involved in prostate cancer test for the presence of prostate cancer.
tumorigenesis. Moreover, our results However, the clear relation between clinical
indicate that serum MIC-1 levels are stage and serum MIC-1 levels indicates that
associated with an increased risk for prostate MIC-1 may be useful as a prognostic factor,
cancer. We also assessed the clinical value of high serum concentrations being associated
the MIC-1 protein as a serum marker. Our with a poor prognosis.
Discussion
Association studies
Association studies offer a potentially harbour tumours in the prostate by the time
powerful approach to identify genetic they are 60 years old, which means that a
variants that influence susceptibility to larger proportion of men die with prostate
complex diseases, but are afflicted by the cancer rather than from it (Sakr et al., 1994).
impression that they are not consistently This makes it difficult to find case-control
reproducible. Nevertheless, a number of populations where cases and controls differ
complex diseases have been consistently significantly in the phenotype of interest,
shown to be associated with specific genetic thus reducing the probability of detecting
variants, including thrombophilia, cancer-associated factors.
Alzheimer’s disease, and diabetes types 1 Another problem that arises when
and type 2 (Hattersley et al., 2005). The performing association studies of prostate
susceptibility variants identified to date cancer, as well as other complex diseases, is
either common, and carry a modest risk, or the heterogeneous nature of these diseases.
uncommon and carry a substantial relative Prostate cancer patients may be predisposed
risk. However, few of the many possible to the condition, due to the inheritance of
associations investigated in published different sequence variants of the same or
cancer-genetic association studies have been different genes from different founders.
subsequently established beyond reasonable This causes two major problems. First,
doubt (Pharoah et al., 2004). In other cases, inherited susceptibility could vary
the results of further investigations have significantly among ethnic groups. Second,
generally been conflicting, for several different distributions of ethnicity among
possible reasons, including population cases and controls could lead to population
stratification, small sample sizes, and stratification, so observed differences in
misclassification of study subjects. genotype frequencies of a variant may
One of the major factors hindering partially reflect differences in the genetic
association studies of prostate cancer are background of cases and controls. However,
that prostate cancer is often detected by if cases and controls are well matched,
PSA measurements in cases where there are differences in the frequency of genotypes
no clinically distinguishing features. A should only be seen at predisposition loci.
further complication is that the presence of A further substantial problem is that a
prostate cancer is also frequently undetected large number of genes and genetic variants
among controls, partly due to the fact that may affect the risk for prostate cancer
prostate cancer is commonly a late age of through different mechanisms, and the
onset disease, and there are often long effect of each given gene, or variant of a
periods in which it is present but there are gene, is likely to be minor in the general
no obvious symptoms. Evidence from population. Such polygenic effects could
autopsies indicate that over 50% of men apply even in a homogenous population.
This phenomenon is observed in other number of possible candidate genes and

complex diseases, as reported in a recent multiple sequence variants with in a gene,
meta-analysis of genetic association studies there is the substantial problem of multiple
in complex diseases (Lohmueller et al., 2003). tests. To overcome this problem, correction
After evaluating 301 published studies that methods have been suggested that
attempted to replicate reported disease compensate for multiple testing. However,
associations for 25 different genes, the study in analysis of variance, the multiple testing is
confirmed the associations for only eight of further complicated by the fact that the
the 25 genes. Strikingly, seven of these eight comparisons are not independent. Due to
genes were associated with modest estimated linkage disequilibrium among closely spaced
genetic effects (ORs between 1.07 and 1.76 sequence variants the different tests of the
in the pooled analyses). This highlights the variants within a gene or other defined
need to use studies with large and well region will not be independent. At present
characterised populations to identify such there is a lack of appropriate methods to
effects convincingly. For example, in order adjust for multiple, dependent tests (Cordell
to detect a sequence variant with a frequency et al., 2005).
of 20% in the population conferring a In addition, a candidate variant that
relative risk of 1.5 (50% increased risk) a segregates with a specific condition may not
study of approximately 1000 cases and 1000 be causally linked to it, but could be in
controls will be needed (Pharoah et al., linkage disequilibrium (LD) with the causal
2004). Historically, the majority of genetic variant or variants. A number of variants
association studies have analyzed a relatively that are in LD with a specific variant of
small number of study subjects, leading to interest are likely to be associated with a
both false positive and false negative disease even if there is only one underlying
reports. variant that causes the risk. Systematically
The genotyping quality is an important studying a set of htSNPs that may or may
factor for a successful and reliable not be functionally relevant to a disease, but
association study (Hattersley et al., 2005). capture all the sequence variation in the
Robust quality control needs to be entire genes, makes it possible to assess
implemented in the genotyping procedure, whether sequence variants in the genes are
including both positive and negative associated with a particular disease (Johnson
controls and at least 5% repeated study et al., 2001). If the association is confirmed
samples. To assure reliable results both cases in an independent population, the region
and control samples should be included on that is represented by the associated htSNP
the same plate and, furthermore, the case- or htSNPs needs to be sequenced. After
control status of each sample should be identifying all genetic variants within the
blinded during genotyping. Most reported region, in vivo and in vitro functional studies
deviations from Hardy-Weinberg of the identified sequence variants will be
equilibrium originate from problems with needed to finally establish the identity of the
genotyping methods. causal variant.
Another issue highlighting a problem in Finally, performing an association study
interpreting the findings of an association with one candidate SNP also has another
study is the risk of type I error due to major drawback when no association is
multiple testing. As a result of a large found, since the lack of association does not
exclude the possibility that there may be control study that recruited subjects from
another important variant in the same gene. 70% of the Swedish population. The genetic
By using the tagging SNP approach, in background of the Swedish population is
which the variance in the entire gene is homogenous, which significantly reduces the
evaluated, it is easier to obtain evidence that risk for population stratification. In addition,
could exclude the possibility that a candidate the population-based study was carefully
gene plays a role in prostate cancer designed, and almost all patients who met
pathogenesis (Pharoah et al., 2004). the inclusion criteria enrolled in the study.
Control subjects were frequently matched to
We have performed association studies to case subjects on the basis of residence area,
evaluate the role of sequence variants in sex and age. Further, the full clinical
three different genes in prostate cancer spectrum of prostate cancer was well
susceptibility, and have therefore taken into represented, with over 45% of cases having
account the issues described above. The advanced disease. The large number of study
criteria needed to perform a well-designed subjects increases the statistical power to
association study have been fulfilled with detect a modestly altered risk for prostate
varying degrees in the three studies. cancer. However, it should be noted that the
In study I, we used a homogenous statistical power to detect a risk genotype for
population with minimal risks for an allele present at low frequencies is still
population stratification drawn from the limited unless the study is very large. For
NSHDC. Each case had two controls example, to have 80% power to detect a risk
matched in terms of sex, age, date at genotype that confers an OR of 1.4 (at a 5%
recruitment and geographical residency. significance level according to two-sided
However, the NSHDC case-control study tests), the risk genotype need to be present
sample is too small to detect modest genetic in 5% of the population. If the risk genotype
risks (OR 1.5-2.0) such as those expected for only confers an OR of 1.2, it needs to be
the five common sequence variants present in 20% of the population to have
evaluated (PRO3, INDEL1, IVS5-57, 80% power to detect the genotype (Figure
P275A, INDEL7). For instance, to evaluate 8). Finally, a careful quality control was
convincingly the effects of the rare mutation implemented in the genotyping procedure
R293X, which is present at a frequency of by including both cases and controls
3% in the general population, according to (including CEPH samples, blinded repeats
our observations, and increases prostate and water blanks on the same DNA plates).
cancer risk by a factor of two (OR ~2.0), a No indication of genotyping error was
much larger case-control study would be observed in Studies II and III, the genotype
needed. consistency was 100% for the CEPH
In Studies II and III we used 780 control DNA samples, and results from
controls and 1383 cases drawn from the duplicated samples were 100% concordant,
CAPS study, a population-based case- giving an estimated error rate of 0%.
1.0
MAF = 5%
0.9 MAF = 10%
MAF = 20%
0.8
0.7
0.6
Power
0.5
0.4
0.3
0.2
0.1
0.0
1.0 1.1 1.2 1.3 1.4 1.5

Relative Risk
Figure 8. Graph illustrating the dependence of the power to detect a risk genotype on
minor allele frequency (MAF), at a significance level of 5% according to two-sided tests.
Cytokine polymorphisms in prostate cancer
In 1863, Virchow first suggested that cancer between the effects of pro-inflammatory and
originates at sites of chronic inflammation anti-inflammatory cytokines strongly
(Balkwill et al., 2001). Chronic inflammation influences the outcome of inflammatory
has also been proposed recently to be an events. Cytokine genes are highly
important factor in prostate cancer polymorphic and the stability and function
pathogenesis (Platz et al., 2004). of the cytokines that specific variants
Inflammatory processes involve the action encode, which in turn can affect the
of a number of mediators, including inflammation response, depend on the
metabolites of arachidonic acid, cytokines, location and nature of the polymorphisms.
chemokines, and free radicals. The balance
The role of sequence variants in two some situations and harmful in others. For
genes encoding cytokines (MIC-1, IL-1RN) example, decreased stability of the mRNA
was evaluated in the studies underlying this or protein encoded by the MIC-1 gene could
thesis. Both of these cytokines are important lead to a prolonged inflammatory response,
regulators of the inflammatory response, which could be advantageous for combating
although MIC-1 has also been documented infectious agents. However, chronic
to act as an inhibitor of tumour growth. The exposure of inflammatory mediators leads to
haplotype-tagging approaches used enabled increased cell proliferation, mutagenesis,
us to elucidate most of the genetic variation oncogene activation, and angiogenesis - the
with a limited number of SNP genotypes. ultimate results of which will be
The anti-inflammatory cytokine IL-1RN proliferation of cells that have lost normal
is well known for its role as a competitive growth control (Shacter et al., 2002).
inhibitor of the pro-inflammatory cytokines Another interesting observation
IL-1α/β, and thus as a “neutralizer” of the regarding the association between these two
physiological and pathophysiological genes and prostate cancer risk is the fact that
inflammatory responses. Our results both genes are expressed in macrophages.
revealed that the most common haplotype Macrophages, together with lymphocytes,
of IL-1RN was associated with an increased are the predominant cell types in areas of
risk of prostate cancer. The genetic analysis chronic inflammation, a condition that is
of the MIC-1 gene showed that a non- extremely common in the prostate, but are
synonymous polymorphism in exon two was rarely found in tumour tissues. Furthermore,
associated with prostate cancer. The increased macrophage activity and
polymorphism alters an amino acid in the infiltration of lymphocytes in the tumour
mature protein, but although this variant has has been found to be related to poor
been previously reported, no functional prognosis in prostate cancer (Irani et al.,
studies have evaluated its biological role. 1999; Lissbrant et al., 2000; McArdle et al.,
Further studies of other types are needed to 2004). Both MIC-1 and IL1-RN are anti-
elucidate the biological rationale for the inflammatory cytokines; MIC-1 has been
association between sequence variants in shown to down-regulate the overall activity
these two genes and prostate cancer, but it is of macrophages, whereas IL1-RN is known
tempting to speculate that these variations to inhibit the function of IL1-α/β, and
could be linked to an imbalance in the thereby decrease the pro-inflammatory
inflammatory process. activity of the macrophage. Since
Polymorphisms affecting susceptibility to macrophages produce a number of
complex disorders, like prostate cancer, are mediators that strongly influence cell
likely to be fairly common in the population, proliferation and differentiation under both
and therefore should not be deleterious for physiological and pathological
the carrier. They are unlikely therefore to circumstances, as well as being the key cells
disrupt gene activity or protein function regulating reactions leading to and driving
completely, but instead affect gene chronic inflammation, increases in their
expression levels, mRNA stability, protein activity could enhance tumour initiation and
folding or the affinity of the encoded progression. Sequence variants in these two
proteins to their receptors or substrates. genes could be the causes of such increases
These polymorphisms might be beneficial in in activity.
In collaboration with a US research statistical power to evaluate the combined

group at Wake Forest University we effect of several genetic polymorphisms.
systematically evaluted associations between However, there is a need to assess the
sequence variants of 20 inflammatory genes effects of multiple genes simultaneously, as
and prostate cancer. The genes were selected clearly demonstrated by considering how
based on their roles in inflammation and the cytokines function. Cytokines act in a highly
results of gene expression profiling studies complex coordinated network in which they
in prostate cancer tissue. Sequence variants induce or repress their own synthesis as well
of eight of these genes – MIC-1 IL1-RN, as that of other cytokines and cytokine
TLR-1, TLR-4, TLR-6, TLR-10, IL-10, and receptors (Howell et al., 2002). Furthermore,
COX-2 (Lindmark et al., 2004, 2005; Zheng there is often considerable overlap and
et al., 2004; Sun et al., 2005) – were found to redundancy between the functions of
be associated with prostate cancer risk. The individual cytokines. Due to their highly
high frequency of positive results obtained interrelated functions, it is very important to
for this group of genes clearly indicate that simultaneously evaluate polymorphisms in
genes related to inflammation processes may the genes encoding cytokines, their receptors
play an important role in the development and downstream effectors. In an attempt to
of prostate cancer. do so, Xu et al. explored the joint effects of
So far, most studies evaluating possible 20 genes (mentioned above) involved in the
associations between sequence variants and inflammation pathway on prostate cancer
prostate cancer risk have tended to focus on risk. Using a data-mining method (MDR),
one gene at a time, even though the they found that the interactions of four
aetiology of prostate cancer cannot be SNPs in these inflammatory genes (one SNP
explained by genetic variability in a single each from IL-10, IL-1RN, TIRAP, and
gene. One reason for the focus on single TLR5) are significant indicators of prostate
genes is probably that most studies lack the cancer risk (Xu et al., 2005).
Future perspectives
Many questions arise from the studies MIC-1. This effect could be reversed in a
appended to this thesis, but the overall dose-dependent manner by injecting the
findings are in concordance with the mice with a monoclonal antibody specific
growing body of evidence that chronic for human MIC-1 (personal communication,
inflammation plays an important role in the Samuel Breit). Collectively, these
development of prostate cancer. In the two observations raise the possibility that the
association studies reported in Papers II and large variations in MIC-1 values seen among
III we detected sequence variations in two stage IV patients could influence the
inflammatory genes that alter the risk for outcome for these patients. Performing a
developing prostate cancer. Since these follow-up study in which clinical parameters
findings are novel, there is a need for large- and disease-progression-data are collected
scale, independent confirmatory studies. regarding the cases included in the CAPS
However, if the results are corroborated, the study, could help assess this possibility.
next steps would be to identify the causal MIC-1 has been reported to be expressed
varints in these regions and to elucidate the in both epithelial cells and macrophages, two
underlying mechanism(s) responsible for types of cells that are abundant in tissues in,
these associations. Since the cytokine and adjacent to, prostate tumours. To
pathways are highly interrelated it is possible further elucidate the role of MIC-1 in cancer
that polymorphisms in several related pathogenesis, it would be valuable to
cytokines are needed to cause disease. determine how much MIC-1 (if any) these
Furthermore, the effect of variations in the cell types produce in normal prostate tissue
ability to influence an inflammatory and the various prostate cancer stages. This
response may not be manifest unless could be done by analyzing protein
exposure to certain infectious agents or expression in prostate tissue sections from a
oxidants occurs. These issues may have to selected number of cases and controls drawn
be addressed in order to identify the casual from the CAPS study. Another approach
variant or variants in the cytokine genes. would be to use sections of prostate tissue
The results of Study IV clearly showed a with various disease stages from TRAMP
positive correlation between serum MIC-1 mice to examine the localisation and
levels and disease stages. Among the group expression pattern of the MIC-1 protein.
of patients with TNM-stage IV there were In summary, although multiple pieces of
large variations in MIC-1 values. A recent evidence clearly indicate that prostatic
study found that mice transfected with inflammation plays an important role in the
prostate cancer cell line DU145 expressing development of prostate cancer, much work
human MIC-1 dramatically lost weight, and remains to be done to elucidate the
the degree of weight loss was proportional underlying mechanims.
to serum levels of tumour-derived human
Conclusions
Based on the findings in Paper I-IV, the following conclusions can be made:
ü Genetic analysis in a large number of subjects from two different study populations
in Sweden failed to support a role of sequence variants in the MSR1 gene in the
development of hereditary or sporadic prostate cancer.
ü A comprehensive evaluation of a possible association between sequence variants of

IL1-RN gene and prostate cancer susceptibility revealed that the most common
haplotype in the I L1-RN gene confers an increase risk for prostate cancer. These
findings highlight the advantages of studying all variation in a gene with the use of a
haplotype-tagging approach instead of testing a limited number of single sequence
variants.
ü A large population-based association study provided strong genetic data supporting

an association between the nonsynonymous polymorphism H6D in the MIC-1 gene
and prostate cancer risk. More studies are needed to understand the mechanism by
which sequence variation in this gene affect the function of the MIC-1 protein
which in turn confers an altered prostate cancer risk.
ü MIC-1 serum levels are elevated in prostate cancer patients compared to control
subjects. In addition, MIC-1 serum levels were significantly associated with age and
disease stage. The differences in serum concentration between prostate cancer
patients and healthy individuals are not large enough to provide a discriminatory
diagnostic test for the presence of this disease. However, serial measurements of
MIC-1 may be useful in the management of prostate cancer.
ü The findings of the studies underlying this thesis are in overall concordance with the
growing evidence from epidemiological, molecular, and histopathological studies
that chronic inflammation plays an important role in the development of prostate
cancer. The results indicate that further research is required on the possible roles of
genetic variation in genes involved in the inflammatory process and on how this
variation may affect susceptibility to, and development of, prostate cancer.
Acknowledgements
The work for this thesis was performed at populations, Kristina C, for god company
the department of Radiation Sciences, during the writing procedure, and Camilla
Oncology, Umeå University. I hereby wish T, Beatrice M, Sara L, Stina for valuable
to sincerely thank all of those involved in comments and enthusiasm in my research.
helping me to complete it. Mark Hunter, for introducing me into the
In particular I would like to thank: tricky business of MIC-1 ELISA.
Henrik Grönberg, my supervisor, for Anders Bergh, for all interesting
excellent scientific guidance and for helpful discussions and for valuable comments on
discussions throughout my research studies. the final draft of this thesis.
Also thanks for your great understanding in All the colleagues at the Department of
how I have organized the research studies Medical Biosciences, for providing a nice
together with my medical studies. working-atmosphere.
Jianfeng Xu and Lilly Zheng, for your Carina Ahlgren and Anna Wernblom for
excellent collaboration and assistance in excellent help no matter what kind of
experiments and for stimulating the problems I have brought to you.
scientific discussions. To all I have forgotten to mention
All co-authors, for contributing to my Gun-Brith and Royne, my parents in law,
work. for all your support both of my family and
All the people in the research group Björn- me.
Anders J for friendship and for all My parents, Monica and Jörgen for
interesting discussions in many different devoted support and for always believing in
areas, and for always being there with a me.
helping hand, Fredrik W for your invaluable Margareta and Freddie for being part of
and comprehensive statistical help and for given the family and for your support.
me self-confidence at the squash court, Benjamin and Tuva, our wonderful kids,
Ingela G for truly skilful laboratory for always helping me keep things in
assistance and Lena L for keeping track on perspective and for bringing so much joy
everything and helping me when I have run into our lives.
out of time, Monica E, Elisabeth S and Anna, my love, for sharing your life with
Karin A for all your work with the study me, and for your endless support.
References
Adlercreutz H, Markkanen H, Watanabe S (1993) Plasma concentrations of phyto-

oestrogens in Japanese men. Lancet 342:1209-1210
Afrin LB, Ergul SM (2000) Medical therapy of prostate cancer: 1999. J S C Med Assoc
96:77-84
Alexander RB, Ponniah S, Hasday J, Hebel JR (1998) Elevated levels of
proinflammatory cytokines in the semen of patients with chronic
prostatitis/chronic pelvic pain syndrome. Urology 52:744-749
Arend WP, Malyak M, Guthridge CJ, Gabay C (1998) Interleukin-1 receptor antagonist:
role in biology. Annu Rev Immunol 16:27-55
Attanasio C, David A, Neerman-Arbez M (2003) Outcome of donor splice site mutations
accounting for congenital afibrinogenemia reflects order of intron removal in
the fibrinogen alpha gene (FGA). Blood 101:1851-1856
Attanasio C, de Moerloose P, Antonarakis SE, Morris MA, Neerman-Arbez M (2001)
Activation of multiple cryptic donor splice sites by the common congenital
afibrinogenemia mutation, FGA IVS4 + 1 G-->T. Blood 97:1879-1881
Baek SJ, Kim KS, Nixon JB, Wilson LC, Eling TE (2001) Cyclooxygenase inhibitors
regulate the expression of a TGF-beta superfamily member that has
proapoptotic and antitumorigenic activities. Mol Pharmacol 59:901-908
Balkwill F, Mantovani A (2001) Inflammation and cancer: back to Virchow? Lancet
357:539-545
Baron JA, Sandler RS (2000) Nonsteroidal anti-inflammatory drugs and cancer prevention.
Annu Rev Med 51:511-523
Basler JW, Piazza GA (2004) Nonsteroidal anti-inflammatory drugs and cyclooxygenase-2
selective inhibitors for prostate cancer chemoprevention. J Urol 171:S59-62
discussion S6
Bauskin AR, Brown DA, Junankar S, Rasiah KK, Eggleton S, Hunter M, Liu T,
Smith D, Kuffner T, Pankhurst GJ, Johnen H, Russell PJ, Barret W,
Stricker PD, Grygiel JJ, Kench JG, Henshall SM, Sutherland RL, Breit
SN (2005) The propeptide mediates formation of stromal stores of
PROMIC-1: role in determining prostate cancer outcome. Cancer Res
65:2330-2336
Bensen JT, Langefeld CD, Hawkins GA, Green LE, Mychaleckyj JC, Brewer CS,
Kiger DS, Binford SM, Colicigno CJ, Allred DC, Freedman BI, Bowden
DW (2003) Nucleotide variation, haplotype structure, and association with
end-stage renal disease of the human interleukin-1 gene cluster. Genomics
82:194-217
Bootcov MR, Bauskin AR, Valenzuela SM, Moore AG, Bansal M, He XY, Zhang HP,
Donnellan M, Mahler S, Pryor K, Walsh BJ, Nicholson RC, Fairlie WD,
Por SB, Robbins JM, Breit SN (1997) MIC-1, a novel macrophage
inhibitory cytokine, is a divergent member of the TGF-beta superfamily. Proc
Natl Acad Sci U S A 94:11514-11519
Bosland MC (2000) The role of steroid hormones in prostate carcinogenesis. Journal of the
National Cancer Institute Monographs 27:39-66
Böttner M, Suter-Crazzolara C, Schober A, Unsicker K (1999) Expression of a novel
member of the TGF-beta superfamily, growth/differentiation factor-
15/macrophage-inhibiting cytokine-1 (GDF-15/MIC-1) in adult rat tissues.
Cell Tissue Res 297:103-110
The Breast Cancer Linkage Consortium (1999) Cancer risks in BRCA2 mutation carriers.
J Natl Cancer Inst 91:1310-1316
Brown DA, Ward RL, Buckhaults P, Liu T, Romans KE, Hawkins NJ, Bauskin AR,
Kinzler KW, Vogelstein B, Breit SN (2003) MIC-1 serum level and
genotype: associations with progress and prognosis of colorectal carcinoma.
Clinical Cancer Research 9:2642-2650
Bruner DW, Moore D, Parlanti A, Dorgan J, Engstrom P (2003) Relative risk of prostate
cancer for men with affected relatives: systematic review and meta-analysis.
Int J Cancer 107:797-803
Buckhaults P, Rago C, St Croix B, Romans KE, Saha S, Zhang L, Vogelstein B,
Kinzler KW (2001) Secreted and cell surface genes expressed in benign and
malignant colorectal tumors. Cancer Res 61:6996-7001
Bylund A, Lundin E, Zhang JX, Nordin A, Kaaks R, Stenman UH, Aman P,
Adlercreutz H, Nilsson TK, Hallmans G, Bergh A, Stattin P (2003)
Randomised controlled short-term intervention pilot study on rye bran bread
in prostate cancer. Eur J Cancer Prev 12:407-415
Bylund A, Zhang JX, Bergh A, Damber JE, Widmark A, Johansson A, Adlercreutz H,
Aman P, Shepherd MJ, Hallmans G (2000) Rye bran and soy protein delay
growth and increase apoptosis of human LNCaP prostate adenocarcinoma in
nude mice. Prostate 42:304-314
Carpten J, Nupponen N, Isaacs S, Sood R, Robbins C, Xu J, Faruque M, Moses T,
Ewing C, Gillanders E, Hu P, Bujnovszky P, Makalowska I, Baffoe-
Bonnie A, Faith D, Smith J, Stephan D, Wiley K, Brownstein M, Gildea
D, Kelly B, Jenkins R, Hostetter G, Matikainen M, Schleutker J,
Klinger K, Connors T, Xiang Y, Wang Z, De Marzo A, Papadopoulos
N, Kallioniemi OP, Burk R, Meyers D, Grönberg H, Meltzer P,
Silverman R, Bailey-Wilson J, Walsh P, Isaacs W, Trent J (2002)
Germline mutations in the ribonuclease L gene in families showing linkage
with HPC1. Nat Genet 30:181-184
Chan JM, Giovannucci EL (2001) Vegetables, fruits, associated micronutrients, and risk of
prostate cancer. Epidemiol Rev 23:82-86
Chang YW, Jang JY, Kim NH, Lee JW, Lee HJ, Jung WW, Dong SH, Kim HJ, Kim
BH, Lee JI, Chang R (2005) Interleukin-1B (IL-1B) polymorphisms and
gastric mucosal levels of IL-1beta cytokine in Korean patients with gastric
cancer. Int J Cancer 114:465-471
Cohen RJ, Shannon BA, McNeal JE, Shannon T, Garrett KL (2005) Propionibacterium
acnes associated with inflammation in radical prostatectomy specimens: a
possible link to cancer evolution? J Urol 173:1969-1974
Combs GF (2004) Status of selenium in prostate cancer prevention. Br J Cancer 91:195-199
Cordell HJ, Clayton DG (2005) Genetic association studies. Lancet 366:1121-1131
Coughlin SS, Hall IJ (2002) A review of genetic polymorphisms and prostate cancer risk.
Annals of Epidemiology 12:182-196
Coussens LM, Werb Z (2002) Inflammation and cancer. Nature 420:860-867
Cox A, Camp NJ, Nicklin MJ, di Giovine FS, Duff GW (1998) An analysis of linkage
disequilibrium in the interleukin-1 gene cluster, using a novel grouping
method for multiallelic markers. Am J Hum Genet 62:1180-1188
Crawford ED (2003) Epidemiology of prostate cancer. Urology 62:3-12
De Marzo AM, DeWeese TL, Platz EA, Meeker AK, Nakayama M, Epstein JI, Isaacs
WB, Nelson WG (2004) Pathological and molecular mechanisms of prostate
carcinogenesis: implications for diagnosis, detection, prevention, and
treatment. J Cell Biochem 91:459-477
De Marzo AM, Marchi VL, Epstein JI, Nelson WG (1999) Proliferative inflammatory
atrophy of the prostate: implications for prostatic carcinogenesis. Am J
Pathol 155:1985-1992
De Marzo AM, Meeker AK, Zha S, Luo J, Nakayama M, Platz EA, Isaacs WB,
Nelson WG (2003) Human prostate cancer precursors and pathobiology.
Urology 62:55-62
Dejager S, Mietus-Snyder M, Friera A, Pitas RE (1993) Dominant negative mutations of
the scavenger receptor. Native receptor inactivation by expression of
truncated variants. J Clin Invest 92:894-902
Dennis LK, Dawson DV (2002a) Meta-analysis of measures of sexual activity and prostate
cancer. Epidemiology 13:72-79
Dennis LK, Lynch CF, Torner JC (2002b) Epidemiologic association between prostatitis
and prostate cancer. Urology 60:78-83
Derynck R, Feng XH (1997) TGF-beta receptor signaling. Biochim Biophys Acta
1333:F105-F150
Dillner J, Knekt P, Boman J, Lehtinen M, Af Geijersstam V, Sapp M, Schiller J,
Maatela J, Aromaa A (1998) Sero-epidemiological association between
human-papillomavirus infection and risk of prostate cancer. Int J Cancer
75:564-567
Dinarello CA (1988) Biology of interleukin 1. FASEB J 2:108-115
Dinarello CA (1994) The interleukin-1 family: 10 years of discovery. FASEB J 8:1314-1325
Drachenberg DE (2000) Treatment of prostate cancer: watchful waiting, radical
prostatectomy, and cryoablation. Semin Surg Oncol 18:37-44
Dumont N, Arteaga CL (2003) Targeting the TGF beta signaling network in human
neoplasia. Cancer Cell 3:531-536
El-Omar EM, Carrington M, Chow WH, McColl KE, Bream JH, Young HA, Herrera
J, Lissowska J, Yuan CC, Rothman N, Lanyon G, Martin M, Fraumeni
JF, Rabkin CS (2000) Interleukin-1 polymorphisms associated with
increased risk of gastric cancer. Nature 404:398-402
Fairlie WD, Moore AG, Bauskin AR, Russell PK, Zhang HP, Breit SN (1999) MIC-1 is
a novel TGF-beta superfamily cytokine associated with macrophage
activation. J Leukoc Biol 65:2-5
Fairlie WD, Russell PK, Wu WM, Moore AG, Zhang HP, Brown PK, Bauskin AR,
Breit SN (2001) Epitope mapping of the transforming growth factor-beta
superfamily protein, macrophage inhibitory cytokine-1 (MIC-1): identification
of at least five distinct epitope specificities. Biochemistry 40:65-73
Fallin D, Schork NJ (2000) Accuracy of haplotype frequency estimation for biallelic loci,
via the expectation-maximization algorithm for unphased diploid genotype
data. Am J Hum Genet 67:947-959
Feldman BJ, Feldman D (2001) The development of androgen-independent prostate
cancer. Nat Rev Cancer 1:34-45
Feneley MR, Young MP, Chinyama C, Kirby RS, Parkinson MC (1996) Ki-67
expression in early prostate cancer and associated pathological lesions. J Clin
Pathol 49:741-748
Fernández L, Galán Y, Jiménez R, Gutiérrez A, Guerra M, Pereda C, Alonso C, Riboli
E, Agudo A, González C (2005) Sexual behaviour, history of sexually
transmitted diseases, and the risk of prostate cancer: a case-control study in
Cuba. Int J Epidemiol 34:193-197
FRANKSLM (1954) Atrophy and hyperplasia in the prostate proper. J Pathol Bacteriol
68:617-621
Giles GG, Severi G, English DR, McCredie MR, Borland R, Boyle P, Hopper JL
(2003) Sexual factors and prostate cancer. BJU International 92:211-216
Glas J, Török HP, Schneider A, Brünnler G, Kopp R, Albert ED, Stolte M, Folwaczny
C (2004) Allele 2 of the interleukin-1 receptor antagonist gene is associated
with early gastric cancer. J Clin Oncol 22:4746-4752
Gough PJ, Greaves DR, Gordon S (1998) A naturally occurring isoform of the human
macrophage scavenger receptor (SR-A) gene generated by alternative splicing
blocks modified LDL uptake. J Lipid Res 39:531-543
Graichen R, Liu D, Sun Y, Lee KO, Lobie PE (2002) Autocrine human growth hormone
inhibits placental transforming growth factor-beta gene transcription to
prevent apoptosis and allow cell cycle progression of human mammary
carcinoma cells. J Biol Chem 277:26662-26672
Granowitz EV, Santos AA, Poutsiaka DD, Cannon JG, Wilmore DW, Wolff SM,
Dinarello CA (1991) Production of interleukin-1-receptor antagonist during
experimental endotoxaemia. Lancet 338:1423-1424
Grönberg H (2003) Prostate cancer epidemiology. Lancet 361:859-864
Gsur A, Feik E, Madersbacher S (2004) Genetic polymorphisms and prostate cancer risk.
World J Urol 21:414-423
Haas GP, Sakr WA (1997) Epidemiology of prostate cancer. CA Cancer J Clin 47:273-287
Habermacher GM, Chason JT, Schaeffer AJ (2005) Prostatitis/Chronic Pelvic Pain
Syndrome. Annu Rev Med.
Hassel BA, Zhou A, Sotomayor C, Maran A, Silverman RH (1993) A dominant negative
mutant of 2-5A-dependent RNase suppresses antiproliferative and antiviral
effects of interferon. EMBO J 12:3297-3304
Hattersley AT, McCarthy MI (2005) What makes a good genetic association study? Lancet
366:1315-1323
Hayes RB, Pottern LM, Strickler H, Rabkin C, Pope V, Swanson GM, Greenberg RS,
Schoenberg JB, Liff J, Schwartz AG, Hoover RN, Fraumeni JF (2000)
Sexual behaviour, STDs and risks for prostate cancer. Br J Cancer 82:718-725
Hollstein M, Rice K, Greenblatt MS, Soussi T, Fuchs R, Sørlie T, Hovig E, Smith-
Sørensen B, Montesano R, Harris CC (1994) Database of p53 gene
somatic mutations in human tumors and cell lines. Nucleic Acids Res
22:3551-3555
Holzbeierlein JM, McIntosh J, Thrasher JB (2005) The role of soy phytoestrogens in
prostate cancer. Current Opinion in Urology 15:17-22
Hood BL, Darfler MM, Guiel TG, Furusato B, Lucas DA, Ringeisen BR, Sesterhenn
IA, Conrads TP, Veenstra TD, Krizman DB (2005) Proteomic analysis of
formalin fixed prostate cancer tissue. Mol Cell Proteomics.
Howell WM, Calder PC, Grimble RF (2002) Gene polymorphisms, inflammatory diseases
and cancer. Proc Nutr Soc 61:447-456
Hromas R, Hufford M, Sutton J, Xu D, Li Y, Lu L (1997) PLAB, a novel placental bone
morphogenetic protein. Biochim Biophys Acta 1354:40-44
Hsing AW (2001) Hormones and prostate cancer: what's next? Epidemiol Rev 23:42-58
Hu Z, Shao M, Chen Y, Zhou J, Qian J, Xu L, Ma H, Wang X, Xu Y, Lu D, Shen H
(2005) Allele 2 of the interleukin-1 receptor antagonist gene (IL1RN*2) is
associated with a decreased risk of primary lung cancer. Cancer Lett
</Pagination><Ab-1 receptor antag
Hughes C, Murphy A, Martin C, Sheils O, O'Leary J (2005) Molecular pathology of
prostate cancer. J Clin Pathol 58:673-684
Irani J, Goujon JM, Ragni E, Peyrat L, Hubert J, Saint F, Mottet N (1999) High-grade
inflammation in prostate cancer as a prognostic factor for biochemical
recurrence after radical prostatectomy. Pathologist Multi Center Study
Group. Urology 54:467-472
Ishiguro T, Naito M, Yamamoto T, Hasegawa G, Gejyo F, Mitsuyama M, Suzuki H,
Kodama T (2001) Role of macrophage scavenger receptors in response to
Listeria monocytogenes infection in mice. Am J Pathol 158:179-188
Jaffee EM, Hruban RH, Canto M, Kern SE (2002) Focus on pancreas cancer. Cancer Cell
2:25-28
Jerónimo C, Usadel H, Henrique R, Oliveira J, Lopes C, Nelson WG, Sidransky D
(2001) Quantitation of GSTP1 methylation in non-neoplastic prostatic tissue
and organ-confined prostate adenocarcinoma. J Natl Cancer Inst 93:1747-

1752
Johnson GC, Esposito L, Barratt BJ, Smith AN, Heward J, Di Genova G, Ueda H,
Cordell HJ, Eaves IA, Dudbridge F, Twells RC, Payne F, Hughes W,
Nutland S, Stevens H, Carr P, Tuomilehto-Wolf E, Tuomilehto J,
Gough SC, Clayton DG, Todd JA (2001) Haplotype tagging for the
identification of common disease genes. Nat Genet 29:233-237
Jones PA, Baylin SB (2002) The fundamental role of epigenetic events in cancer. Nat Rev
Genet 3:415-428
Kannan K, Amariglio N, Rechavi G, Givol D (2000) Profile of gene expression regulated
by induced p53: connection to the TGF-beta family. FEBS Lett 470:77-82
Key TJ, Allen N, Appleby P, Overvad K, Tjønneland A, Miller A, Boeing H, Karalis
D, Psaltopoulou T, Berrino F, Palli D, Panico S, Tumino R, Vineis P,
Bueno-De-Mesquita HB, Kiemeney L, Peeters PH, Martinez C,
Dorronsoro M, González CA, Chirlaque MD, Quiros JR, Ardanaz E,
Berglund G, Egevad L, Hallmans G, Stattin P, Bingham S, Day N,
Gann P, Kaaks R, Ferrari P, Riboli E (2004) Fruits and vegetables and
prostate cancer: no association among 1104 cases in a prospective study of
130544 men in the European Prospective Investigation into Cancer and
Nutrition (EPIC). Int J Cancer 109:119-124
Klump B, Nehls O, Okech T, Hsieh CJ, Gaco V, Gittinger FS, Sarbia M, Borchard F,
Greschniok A, Gruenagel HH, Porschen R, Gregor M (2004) Molecular
lesions in colorectal cancer: impact on prognosis? Original data and review of
the literature. Int J Colorectal Dis 19:23-42
Ko LJ, Prives C (1996) p53: puzzle and paradigm. Genes Dev 10:1054-1072
Kodama T, Freeman M, Rohrer L, Zabrecky J, Matsudaira P, Krieger M (1990) Type I
macrophage scavenger receptor contains alpha-helical and collagen-like coiled
coils. Nature 343:531-535
Kolonel LN (2001) Fat, meat, and prostate cancer. Epidemiol Rev 23:72-81
Koopmann J, Buckhaults P, Brown DA, Zahurak ML, Sato N, Fukushima N, Sokoll
LJ, Chan DW, Yeo CJ, Hruban RH, Breit SN, Kinzler KW, Vogelstein
B, Goggins M (2004) Serum macrophage inhibitory cytokine 1 as a marker
of pancreatic and other periampullary cancers. Clinical Cancer Research
10:2386-2392
Kotar K, Hamel N, Thiffault I, Foulkes WD (2003) The RNASEL 471delAAAG allele
and prostate cancer in Ashkenazi Jewish men. J Med Genet 40:e22.
Kuper H, Adami HO, Trichopoulos D (2000) Infections as a major preventable cause of
human cancer. J Intern Med 248:171-183
Landström M, Zhang JX, Hallmans G, Aman P, Bergh A, Damber JE, Mazur W,
Wähäla K, Adlercreutz H (1998) Inhibitory effects of soy and rye diets on
the development of Dunning R3327 prostate adenocarcinoma in rats.
Prostate 36:151-161
Li PX, Wong J, Ayed A, Ngo D, Brade AM, Arrowsmith C, Austin RC, Klamut HJ
(2000) Placental transforming growth factor-beta is a downstream mediator
of the growth arrest and apoptotic response of tumor cells to DNA damage
and p53 overexpression. J Biol Chem 275:20127-20135
Lichtenstein P, Holm NV, Verkasalo PK, Iliadou A, Kaprio J, Koskenvuo M, Pukkala
E, Skytthe A, Hemminki K (2000) Environmental and heritable factors in
the causation of cancer--analyses of cohorts of twins from Sweden, Denmark,
and Finland. N Engl J Med 343:78-85
Lin X, Tascilar M, Lee WH, Vles WJ, Lee BH, Veeraswamy R, Asgari K, Freije D,
van Rees B, Gage WR, Bova GS, Isaacs WB, Brooks JD, DeWeese TL,
De Marzo AM, Nelson WG (2001) GSTP1 CpG island hypermethylation is
responsible for the absence of GSTP1 expression in human prostate cancer
cells. Am J Pathol 159:1815-1826
Lindmark F, Zheng SL, Wiklund F, Bälter KA, Sun J, Chang B, Hedelin M, Clark J,
Johansson JE, Meyers DA, Adami HO, Isaacs W, Grönberg H, Xu J
(2005) Interleukin-1 receptor antagonist haplotype associated with prostate
cancer risk. Br J Cancer 93:493-497
Lindmark F, Zheng SL, Wiklund F, Bensen J, Bälter KA, Chang B, Hedelin M, Clark
J, Stattin P, Meyers DA, Adami HO, Isaacs W, Grönberg H, Xu J (2004)
H6D polymorphism in macrophage-inhibitory cytokine-1 gene associated
with prostate cancer. J Natl Cancer Inst 96:1248-1254
Lissbrant IF, Stattin P, Wikstrom P, Damber JE, Egevad L, Bergh A (2000) Tumor
associated macrophages in human prostate cancer: relation to
clinicopathological variables and survival. International Journal of Oncology
17:445-451
Liu T, Bauskin AR, Zaunders J, Brown DA, Pankhurst S, Russell PJ, Breit SN,
Panhurst S (2003) Macrophage inhibitory cytokine 1 reduces cell adhesion
and induces apoptosis in prostate cancer cells. Cancer Res 63:5034-5040
Lohmueller KE, Pearce CL, Pike M, Lander ES, Hirschhorn JN (2003) Meta-analysis
of genetic association studies supports a contribution of common variants to
susceptibility to common disease. Nat Genet 33:177-182
Lucia MS, Torkko KC (2004) Inflammation as a target for prostate cancer
chemoprevention: pathological and laboratory rationale. J Urol 171:S30-4
discussion S35
Machado JC, Pharoah P, Sousa S, Carvalho R, Oliveira C, Figueiredo C, Amorim A,
Seruca R, Caldas C, Carneiro F, Sobrinho-Simões M (2001) Interleukin
1B and interleukin 1RN polymorphisms are associated with increased risk of
gastric carcinoma. Gastroenterology 121:823-829
Margaglione M, Santacroce R, Colaizzo D, Seripa D, Vecchione G, Lupone MR, De
Lucia D, Fortina P, Grandone E, Perricone C, Di Minno G (2000) A G-
to-A mutation in IVS-3 of the human gamma fibrinogen gene causing
afibrinogenemia due to abnormal RNA splicing. Blood 96:2501-2505
Matsumoto A, Naito M, Itakura H, Ikemoto S, Asaoka H, Hayakawa I, Kanamori H,

Aburatani H, Takaku F, Suzuki H (1990) Human macrophage scavenger
receptors: primary structure, expression, and localization in atherosclerotic

lesions. Proc Natl Acad Sci U S A 87:9133-9137
McArdle PA, Canna K, McMillan DC, McNicol AM, Campbell R, Underwood MA
(2004) The relationship between T-lymphocyte subset infiltration and survival
in patients with prostate cancer. Br J Cancer 91:541-543
McCarron SL, Edwards S, Evans PR, Gibbs R, Dearnaley DP, Dowe A, Southgate C,
Easton DF, Eeles RA, Howell WM (2002) Influence of cytokine gene
polymorphisms on the development of prostate cancer. Cancer Res 62:3369-
3372
McIntyre KW, Stepan GJ, Kolinsky KD, Benjamin WR, Plocinski JM, Kaffka KL,
Campen CA, Chizzonite RA, Kilian PL (1991) Inhibition of interleukin 1
(IL-1) binding and bioactivity in vitro and modulation of acute inflammation
in vivo by IL-1 receptor antagonist and anti-IL-1 receptor monoclonal
antibody. J Exp Med 173:931-939
McNeal JE (1968) Regional morphology and pathology of the prostate. Am J Clin Pathol
49:347-357
McNeal JE (1988) Normal histology of the prostate. Am J Surg Pathol 12:619-633
Miller DC, Zheng SL, Dunn RL, Sarma AV, Montie JE, Lange EM, Meyers DA, Xu J,
Cooney KA (2003) Germ-line mutations of the macrophage scavenger
receptor 1 gene: association with prostate cancer risk in African-American
men. Cancer Res 63:3486-3489
Minami Y, Staples MP, Giles GG (1993) The incidence of colon, breast and prostate
cancer in Italian migrants to Victoria, Australia. Eur J Cancer 29A:1735-1740
MORGANTIG, GIANFERRARIL, CRESSERIA, ARRIGONIG, LOVATIG (1956)
[Clinico-statistical and genetic research on neoplasms of the prostate.]. Acta
Genetica et Statistica Medica 6:304-305
Nadler RB, Koch AE, Calhoun EA, Campbell PL, Pruden DL, Bennett CL, Yarnold
PR, Schaeffer AJ (2000) IL-1beta and TNF-alpha in prostatic secretions are
indicators in the evaluation of men with chronic prostatitis. J Urol 164:214-
218
Nakamura T, Scorilas A, Stephan C, Yousef GM, Kristiansen G, Jung K, Diamandis
EP (2003) Quantitative analysis of macrophage inhibitory cytokine-1 (MIC-1)
gene expression in human prostatic tissues. Br J Cancer 88:1101-1104
Nakayama M, Bennett CJ, Hicks JL, Epstein JI, Platz EA, Nelson WG, De Marzo
AM (2003) Hypermethylation of the human glutathione S-transferase-pi gene
(GSTP1) CpG island is present in a subset of proliferative inflammatory
atrophy lesions but not in normal or hyperplastic epithelium of the prostate: a
detailed study using laser-capture microdissection. Am J Pathol 163:923-933
Nelson CP, Kidd LC, Sauvageot J, Isaacs WB, De Marzo AM, Groopman JD, Nelson
WG, Kensler TW (2001) Protection against 2-hydroxyamino-1-methyl-6-
phenylimidazo[4,5-b]pyridine cytotoxicity and DNA adduct formation in
human prostate by glutathione S-transferase P1. Cancer Res 61:103-109
Nelson WG, De Marzo AM, Isaacs WB (2003) Prostate cancer. N Engl J Med 349:366-
381
Nelson WG, DeWeese TL, DeMarzo AM (2002) The diet, prostate inflammation, and the
development of prostate cancer. Cancer Metastasis Rev 21:3-16
Ntais C, Polycarpou A, Ioannidis JP (2003) SRD5A2 gene polymorphisms and the risk of
prostate cancer: a meta-analysis. Cancer Epidemiol Biomarkers Prev 12:618-
624
Page WF, Braun MM, Partin AW, Caporaso N, Walsh P (1997) Heredity and prostate
cancer: a study of World War II veteran twins. Prostate 33:240-245
Palapattu GS, Sutcliffe S, Bastian PJ, Platz EA, De Marzo AM, Isaacs WB, Nelson
WG (2004) Prostate carcinogenesis and inflammation: emerging insights.
Carcinogenesis </Pagination><Ab-disciplinary bod
Paralkar VM, Vail AL, Grasser WA, Brown TA, Xu H, Vukicevic S, Ke HZ, Qi H,
Owen TA, Thompson DD (1998) Cloning and characterization of a novel
member of the transforming growth factor-beta/bone morphogenetic
protein family. J Biol Chem 273:13760-13767
Parkin DM, Bray F, Ferlay J, Pisani P (2005) Global cancer statistics, 2002. CA Cancer J
Clin 55:74-108
Parsons JK, Nelson CP, Gage WR, Nelson WG, Kensler TW, De Marzo AM (2001)
GSTA1 expression in normal, preneoplastic, and neoplastic human prostate
tissue. Prostate 49:30-37
Patel DA, Bock CH, Schwartz K, Wenzlaff AS, Demers RY, Severson RK (2005)
Sexually transmitted diseases and other urogenital conditions as risk factors
for prostate cancer: a case-control study in Wayne County, Michigan. Cancer
Causes Control 16:263-273
Pharoah PD, Dunning AM, Ponder BA, Easton DF (2004) Association studies for
finding cancer-susceptibility genetic variants. Nat Rev Cancer 4:850-860
Platt N, Gordon S (2001) Is the class A macrophage scavenger receptor (SR-A)
multifunctional? - The mouse's tale. J Clin Invest 108:649-654
Platz EA, De Marzo AM (2004) Epidemiology of inflammation and prostate cancer. J Urol
171:S36-S40
Pontari MA, Ruggieri MR (2004) Mechanisms in prostatitis/chronic pelvic pain syndrome.
J Urol 172:839-845
Putzi MJ, De Marzo AM (2000) Morphologic transitions between proliferative
inflammatory atrophy and high-grade prostatic intraepithelial neoplasia.
Urology 56:828-832
Rennert H, Bercovich D, Hubert A, Abeliovich D, Rozovsky U, Bar-Shira A, Soloviov
S, Schreiber L, Matzkin H, Rennert G, Kadouri L, Peretz T, Yaron Y,
Orr-Urtreger A (2002) A novel founder mutation in the RNASEL gene,
471delAAAG, is associated with prostate cancer in Ashkenazi Jews. Am J
Hum Genet 71:981-984
Rich AR (1979) Classics in oncology. On the frequency of occurrence of occult carcinoma
of the prostate: Arnold Rice Rich, M.D., Journal of Urology 33:3, 1935. CA
Cancer J Clin 29:115-119
Roberts RO, Jacobson DJ, Girman CJ, Rhodes T, Lieber MM, Jacobsen SJ (2002) A
population-based study of daily nonsteroidal anti-inflammatory drug use and
prostate cancer. Mayo Clin Proc 77:219-225
Roberts RO, Lieber MM, Rhodes T, Girman CJ, Bostwick DG, Jacobsen SJ (1998)
Prevalence of a physician-assigned diagnosis of prostatitis: the Olmsted
County Study of Urinary Symptoms and Health Status Among Men. Urology
51:578-584
Rubin MA, De Marzo AM (2004) Molecular genetics of human prostate cancer. Mod
Pathol 17:380-388
Ruska KM, Sauvageot J, Epstein JI (1998) Histology and cellular kinetics of prostatic
atrophy. Am J Surg Pathol 22:1073-1077
Sakr WA, Grignon DJ, Crissman JD, Heilbrun LK, Cassin BJ, Pontes JJ, Haas GP
(1994) High grade prostatic intraepithelial neoplasia (HGPIN) and prostatic
adenocarcinoma between the ages of 20-69: an autopsy study of 249 cases. In
Vivo 8:439-443
Schaid DJ (2004) The complex genetic epidemiology of prostate cancer. Hum Mol Genet
13 Spec No 1:R103-R121
Schaid DJ, Rowland CM, Tines DE, Jacobson RM, Poland GA (2002) Score tests for
association between traits and haplotypes when linkage phase is ambiguous.
Am J Hum Genet 70:425-434
Sehouli J, Mustea A, Koensgen D, Lichtenegger W (2003) Interleukin-1 receptor
antagonist gene polymorphism in epithelial ovarian cancer. Cancer Epidemiol
Biomarkers Prev 12:1205-1208
Seppälä EH, Ikonen T, Autio V, Rökman A, Mononen N, Matikainen MP, Tammela
TL, Schleutker J (2003) Germ-line alterations in MSR1 gene and prostate
cancer risk. Clinical Cancer Research 9:5252-5256
Shacter E, Weitzman SA (2002) Chronic inflammation and cancer. Oncology (Huntingt)
16:217-26 229 discussio
Shah R, Mucci NR, Amin A, Macoska JA, Rubin MA (2001) Postatrophic hyperplasia of
the prostate gland: neoplastic precursor or innocent bystander? Am J Pathol
158:1767-1773
Shimizu H, Ross RK, Bernstein L, Yatani R, Henderson BE, Mack TM (1991)
Cancers of the prostate and breast among Japanese and white immigrants in
Los Angeles County. Br J Cancer 63:963-966
Shirai H, Murakami T, Yamada Y, Doi T, Hamakubo T, Kodama T (1999) Structure
and function of type I and II macrophage scavenger receptors. Mech Ageing
Dev 111:107-121
Silverman RH (2003) Implications for RNase L in prostate cancer biology. Biochemistry
42:1805-1812
Simard J, Dumont M, Labuda D, Sinnett D, Meloche C, El-Alfy M, Berger L, Lees E,
Labrie F, Tavtigian SV (2003) Prostate cancer susceptibility genes: lessons
learned and challenges posed. Endocrine-Related Cancer 10:225-259
Smith JR, Freije D, Carpten JD, Grönberg H, Xu J, Isaacs SD, Brownstein MJ, Bova
GS, Guo H, Bujnovszky P, Nusskern DR, Damber JE, Bergh A,
Emanuelsson M, Kallioniemi OP, Walker-Daniels J, Bailey-Wilson JE,
Beaty TH, Meyers DA, Walsh PC, Collins FS, Trent JM, Isaacs WB
(1996) Major susceptibility locus for prostate cancer on chromosome 1
suggested by a genome-wide search. Science 274:1371-1374
Stattin P, Bylund A, Rinaldi S, Biessy C, Déchaud H, Stenman UH, Egevad L, Riboli
E, Hallmans G, Kaaks R (2000) Plasma insulin-like growth factor-I, insulin-
like growth factor-binding proteins, and prostate cancer risk: a prospective
study. J Natl Cancer Inst 92:1910-1917
Stattin P, Lumme S, Tenkanen L, Alfthan H, Jellum E, Hallmans G, Thoresen S,
Hakulinen T, Luostarinen T, Lehtinen M, Dillner J, Stenman UH,
Hakama M (2004) High levels of circulating testosterone are not associated
with increased prostate cancer risk: a pooled prospective study. Int J Cancer
108:418-424
Steinkasserer A, Spurr NK, Cox S, Jeggo P, Sim RB (1992) The human IL-1 receptor
antagonist gene (IL1RN) maps to chromosome 2q14-q21, in the region of
the IL-1 alpha and IL-1 beta loci. Genomics 13:654-657
Sun J, Wiklund F, Zheng SL, Chang B, Bälter K, Li L, Johansson JE, Li G, Adami

HO, Liu W, Tolin A, Turner AR, Meyers DA, Isaacs WB, Xu J,
Grönberg H (2005) Sequence variants in Toll-like receptor gene cluster
(TLR6-TLR1-TLR10) and prostate cancer risk. J Natl Cancer Inst 97:525-532
Tan JK, Png DJ, Liew LC, Li MK, Wong ML (2002) Prevalence of prostatitis-like
symptoms in Singapore: a population-based study. Singapore Med J 43:189-
193
Tan M, Wang Y, Guan K, Sun Y (2000) PTGF-beta, a type beta transforming growth
factor (TGF-beta) superfamily member, is a p53 target gene that inhibits
tumor cell growth via TGF-beta signaling pathway. Proc Natl Acad Sci U S A
97:109-114
Tarlow JK, Blakemore AI, Lennard A, Solari R, Hughes HN, Steinkasserer A, Duff
GW (1993) Polymorphism in human IL-1 receptor antagonist gene intron 2 is
caused by variable numbers of an 86-bp tandem repeat. Hum Genet 91:403-
404
Taylor ML, Mainous AG, Wells BJ (2005) Prostate cancer and sexually transmitted
diseases: a meta-analysis. Fam Med 37:506-512
Thomas CA, Li Y, Kodama T, Suzuki H, Silverstein SC, El Khoury J (2000) Protection
from lethal gram-positive infection by macrophage scavenger receptor-
dependent phagocytosis. J Exp Med 191:147-156
Thomas R, True LD, Lange PH, Vessella RL (2001) Placental bone morphogenetic
protein (PLAB) gene expression in normal, pre-malignant and malignant
human prostate: relation to tumor development and progression. Int J Cancer
93:47-52
Tsujimoto Y, Takayama H, Nonomura N, Okuyama A, Aozasa K (2002) Postatrophic

hyperplasia of the prostate in Japan: histologic and immunohistochemical
features and p53 gene mutation analysis. Prostate 52:279-287
van Leenders GJ, Gage WR, Hicks JL, van Balken B, Aalders TW, Schalken JA, De
Marzo AM (2003) Intermediate cells in human prostate epithelium are
enriched in proliferative inflammatory atrophy. Am J Pathol 162:1529-1537
Wall JD, Pritchard JK (2003) Haplotype blocks and linkage disequilibrium in the human
genome. Nat Rev Genet 4:587-597
Wang L, McDonnell SK, Cunningham JM, Hebbring S, Jacobsen SJ, Cerhan JR,
Slager SL, Blute ML, Schaid DJ, Thibodeau SN (2003) No association of
germline alteration of MSR1 with prostate cancer risk. Nat Genet 35:128-129
Wang L, McDonnell SK, Elkins DA, Slager SL, Christensen E, Marks AF,
Cunningham JM, Peterson BJ, Jacobsen SJ, Cerhan JR, Blute ML,
Schaid DJ, Thibodeau SN (2002) Analysis of the RNASEL gene in familial
and sporadic prostate cancer. Am J Hum Genet 71:116-123
Weir BS (1996) Anonymous Genetic data analysis II. Sinauer, Sunderland (MA)
Welsh JB, Sapinoso LM, Kern SG, Brown DA, Liu T, Bauskin AR, Ward RL,
Hawkins NJ, Quinn DI, Russell PJ, Sutherland RL, Breit SN,
Moskaluk CA, Frierson HF, Hampton GM (2003) Large-scale delineation
of secreted protein biomarkers overexpressed in cancer tissue and serum.
Proc Natl Acad Sci U S A 100:3410-3415
Welsh JB, Sapinoso LM, Su AI, Kern SG, Wang-Rodriguez J, Moskaluk CA, Frierson
HF, Hampton GM (2001) Analysis of gene expression identifies candidate
markers and pharmacological targets in prostate cancer. Cancer Res 61:5974-
5978
Wiklund F, Jonsson BA, Brookes AJ, Strömqvist L, Adolfsson J, Emanuelsson M,
Adami HO, Augustsson-Bälter K, Grönberg H (2004) Genetic analysis of
the RNASEL gene in hereditary, familial, and sporadic prostate cancer.
Clinical Cancer Research 10:7150-7156
Wiseman H, Halliwell B (1996) Damage to DNA by reactive oxygen and nitrogen species:
role in inflammatory disease and progression to cancer. Biochem J 313:17-29
Witkin SS, Gerber S, Ledger WJ (2002) Influence of interleukin-1 receptor antagonist gene
polymorphism on disease. Clin Infect Dis 34:204-209
Wollmann W, Goodman ML, Bhat-Nakshatri P, Kishimoto H, Goulet RJ, Mehrotra
S, Morimiya A, Badve S, Nakshatri H (2005) The macrophage inhibitory
cytokine integrates AKT/PKB and MAP kinase signaling pathways in breast
cancer cells. Carcinogenesis 26:900-907
Wu MS, Wu CY, Chen CJ, Lin MT, Shun CT, Lin JT (2003) Interleukin-10 genotypes
associate with the risk of gastric carcinoma in Taiwanese Chinese. Int J
Cancer 104:617-623
Xiao W, Oefner PJ (2001) Denaturing high-performance liquid chromatography: A review.
Hum Mutat 17:439-474
Xu J, Lowey J, Wiklund F, Sun J, Lindmark F, Hsu FC, Dimitrov L, Chang B, Turner

AR, Liu W, Adami HO, Suh E, Moore JH, Zheng SL, Isaacs WB, Trent
JM, Grönberg H (2005) The interaction of four genes in the inflammation
pathway significantly predicts prostate cancer risk. Cancer Epidemiol
Biomarkers Prev 14:2563-2568
Xu J, Zheng SL, Hawkins GA, Faith DA, Kelly B, Isaacs SD, Wiley KE, Chang B,
Ewing CM, Bujnovszky P, Carpten JD, Bleecker ER, Walsh PC, Trent
JM, Meyers DA, Isaacs WB (2001) Linkage and association studies of
prostate cancer susceptibility: evidence for linkage at 8p22-23. Am J Hum
Genet 69:341-350
Xu J, Zheng SL, Komiya A, Mychaleckyj JC, Isaacs SD, Chang B, Turner AR, Ewing
CM, Wiley KE, Hawkins GA, Bleecker ER, Walsh PC, Meyers DA,
Isaacs WB (2003) Common sequence variants of the macrophage scavenger
receptor 1 gene are associated with prostate cancer risk. Am J Hum Genet
72:208-212
Xu J, Zheng SL, Komiya A, Mychaleckyj JC, Isaacs SD, Hu JJ, Sterling D, Lange
EM, Hawkins GA, Turner A, Ewing CM, Faith DA, Johnson JR,
Suzuki H, Bujnovszky P, Wiley KE, DeMarzo AM, Bova GS, Chang B,
Hall MC, McCullough DL, Partin AW, Kassabian VS, Carpten JD,
Bailey-Wilson JE, Trent JM, Ohar J, Bleecker ER, Walsh PC, Isaacs
WB, Meyers DA (2002) Germline mutations and sequence variants of the
macrophage scavenger receptor 1 gene are associated with prostate cancer
risk. Nat Genet 32:321-325
Zeng ZR, Hu PJ, Hu S, Pang RP, Chen MH, Ng M, Sung JJ (2003) Association of
interleukin 1B gene polymorphism and gastric cancers in high and low
prevalence regions in China. Gut 52:1684-1689
Zha S, Yegnasubramanian V, Nelson WG, Isaacs WB, De Marzo AM (2004)
Cyclooxygenases in cancer: progress and perspective. Cancer Lett 215:1-20
Zheng SL, Augustsson-Bälter K, Chang B, Hedelin M, Li L, Adami HO, Bensen J, Li
G, Johnasson JE, Turner AR, Adams TS, Meyers DA, Isaacs WB, Xu J,
Grönberg H (2004) Sequence variants of toll-like receptor 4 are associated
with prostate cancer risk: results from the CAncer Prostate in Sweden Study.
Cancer Res 64:2918-2922

FULLTEXT01

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

FULLTEXT01

Uploaded by

Copyright:

Available Formats

UMEÅ UNIVERSITY MEDICAL DISSERTATIONS

New series No. 991

Department of Radiation Sciences, Oncology, Umeå University

Prostate Cancer and Inflammatory

Printed by Print & Media, Umeå University, Umeå, 2005

This thesis is based on the following studies.

I. Lindmark F, Jonsson BA, Bergh A, III. Lindmark F, Zheng SL, Wiklund F,

Reprints are reproduced with the permission of the

AR Androgen receptor NSHDC Northern Sweden health and

Risk factors for prostate cancer

observed in Asian men compared to Hormonal and other physiological

the genetic basis of prostate cancer is much Twin studies

common), mode of inheritance (dominant, Carriers of mutations of another gene,

the prevalence of mutations in high- conversion of testosterone into the more

Inflammation and cancer

expression of certain inflammatory cytokines 2002). Two different cyclooxygenase (COX)

effects of pro-inflammatory and anti- inflammation with chronic exposure to

The role of inflammation in the pathogenesis of prostate cancer

complexities involved in diagnosing was provided by reports of an association

prostate cancer susceptibility, since an apoptosis (Silverman, 2003). Defects in the

TGF-β signalling pathway following demonstrated that overexpression of MIC-1

The specific aims of the work underlying this thesis were:

ü To investigate whether mutations in ü To evaluate the possible role of

ü To evaluate the possible role of ü To study the serum levels of MIC-1

Material and methods

Hereditary prostate cancer families used for mutation screening

The Northern Sweden Health and months), and date (± 2 months) at

Retention time (min)

Figure 2. Schematic illustration over homo/heteroduplex formation and the following

Common sequence variants of the Thermo Sequenase (both from

Association of MIC-1 sequence nuclease assay with TaqMan MGB probes.

H6D 2423 C/G

Figure 3. A) Single nucleotide polymorphisms (SNPs) of the Macrophage Inhibitory

Determination of MIC-1 serum levels (Study IV)

MIC-1 serum levels were determined by a substrate (1 mg/mL o-phenylenediamine

Tests for Hardy-Weinberg replication method, as described by (Weir,

Results and comments

Genetic analysis of MSR1 (Study I)

Study Families C ases with C ontrols P-value

Seppälä et al. 2003 3/120 6/537 5/480 0.91

Wang et al. 2003 5/163 14/496 16/492 0.69

Lindmark et al. 2004 2/83 (2.4%) 10/215 10/425 0.16*

Table 3. Allele frequencies of common MSR1 sequence variants in unselected

Genetic analysis of IL-1RN (Study II)

patients with advanced prostate cancer.

% % Score* P† % Score* P† % Score* P†

Genetic analysis of MIC-1 (Study III)

Due to the potential importance of MIC-1 As a first step, we genotyped four

Analysis of MIC-1 serum levels (Study IV)

The development of sensitive immunoassays available for analyses. 19 prostate cancer

Age at inclusion (years)

MIC-1 (pg/ml) Controls 620 1190±942

TNM tumour stage

A possible association between serum showed that there is no diagnostic

0.0 0.2 0.4 0.6 0.8 1.0

Figure 6. Receiver operator characteristic (ROC) curve of MIC-1 for differentiation of

macrophage-mediated proinflammatory together with the striking correlation

In conclusion, a clear association data revealed that the differences in serum

This phenomenon is observed in other number of possible candidate genes and

1.0 1.1 1.2 1.3 1.4 1.5

Cytokine polymorphisms in prostate cancer

In collaboration with a US research statistical power to evaluate the combined

ü A comprehensive evaluation of a possible association between sequence variants of