1. health and fitness
2. disease
3. cancer

Expression of the Free Beta Subunit of
Human Chorionic Gonadotropin in Cancer of
the Urinary Bladder and Kidney
Kristina Hotakainen
Department of Clinical Chemistry
University of Helsinki
Helsinki, Finland
Academic Dissertation
To be publicly discussed with the permission of the Medical Faculty of
the University of Helsinki in Auditorium 2, Biomedicum Helsinki,
on May 24, 2002, at 12 o’clock noon.
Supervised by
Professor Ulf-Håkan Stenman, MD, PhD
University of Helsinki
Finland
Reviewed by
Docent Martti Nurmi, MD, PhD
University of Turku
Finland
and
Professor Kim Pettersson, PhD
University of Turku
Finland
Opponent
Professor Mirja Ruutu, MD, PhD
University of Helsinki
Finland
ISBN 952-91-4592-6 (Print)
ISBN 952-10-0514-9 (PDF)
Yliopistopaino
Helsinki 2002
Table of contents
List of original publications ..................................................................................... 5
Abbreviations .......................................................................................................... 6
Abstract .................................................................................................................. 7
Introduction ............................................................................................................ 8
Review of the literature ........................................................................................... 9
1. Chorionic gonadotropin and its subunits ............................................................. 9
1.1 Biochemistry and biology ..................................................................... 9
1.2 Biological function ............................................................................... 9
1.3 HCG in serum and urine of healthy subjects ......................................... 9
1.4 HCG in malignant disease .................................................................... 10
1.5 Clinical determinations ......................................................................... 10
1.5.1 Normal pregnancy ..................................................................... 10
1.5.2 Ectopic pregnancy ..................................................................... 10
1.5.3 Maternal screening for Down’s syndrome ................................... 11
1.5.4 Trophoblastic disease ................................................................. 11
1.5.5 Testicular cancer ........................................................................ 11
1.6 The beta subunit of chorionic gonadotropin .......................................... 11
1.6.1 Genes, mRNAs and proteins ..................................................... 11
1.6.2 HCGß expression in tissues and body fluids .............................. 12
1.6.3 HCGß expression in peripheral blood cells ................................ 12
1.6.4 HCGß in malignant disease ....................................................... 12
1.6.5 HCGß expression in bladder cancer ........................................... 13
1.6.6 HCGß expression in renal cell carcinoma (RCC) ........................ 14
2. Bladder cancer ..................................................................................................... 14
2.1 Epidemiology ....................................................................................... 14
2.2 Risk factors ........................................................................................... 14
2.3 Histology ............................................................................................. 14
2.4 Symptoms and signs ............................................................................. 14
2.5 Diagnostic procedures ........................................................................... 14
2.6 Staging and grading ............................................................................. 15
2.7 Treatment and prognosis ....................................................................... 15
2.8 Markers of bladder cancer ..................................................................... 16
2.8.1 Markers for screening and diagnosis ........................................... 16
2.8.2 Prognostic markers .................................................................... 17
3. Renal cell carcinoma ........................................................................................... 19
3.1 Epidemiology ....................................................................................... 19
3.2 Risk factors ........................................................................................... 19
3.3 Histology ............................................................................................. 19
Kristina Hotakainen
3.4 Symptoms and signs ............................................................................. 20
3.5 Diagnostic procedures ........................................................................... 20
3.6 Staging and grading ............................................................................. 20
3.7 Treatment and prognosis ....................................................................... 20
3.8 Markers of RCC .................................................................................... 21
3.8.1 Tissue markers ........................................................................... 21
3.8.2 Serum markers........................................................................... 22
Aims of the study .................................................................................................... 23
Material and methods .............................................................................................. 24
1. Subjects and samples (I-IV) ................................................................................ 24
2. Preparations and cultures of cells (I-III) .............................................................. 24
3. Leukocyte extracts (I) ......................................................................................... 25
4. Separation of mononuclear cells, granulocytes, monocytes, B- and T-cells (I) ...... 25
5. Isolation of RNA (I-III) ..................................................................................... 26
6. Removal of genomic DNA (I-III) ....................................................................... 26
7. Oligonucleotide primers (I-III) .......................................................................... 27
8. RT-PCR and gel electrophoresis (I-III) ............................................................... 27
9. Restriction enzyme analysis and sequencing of the PCR products (I-III) ............. 27
10.Determination of hCG, hCGß, hCGßcf, LH and LHß (I-IV) .............................. 28
11.Immunohistochemistry (III) ............................................................................... 28
12.Statistical methods (I-IV) ................................................................................... 29
Results and discussion ............................................................................................. 30
1. Expression of hCGß and LHß in peripheral blood cells (I) .................................. 30
1.1 Expression of hCGß- and LHß mRNA in peripheral blood
cells and cell lines ....................................................................................... 30
1.2 LH- and LHß expression in cultured lymphocytes................................. 30
1.3 Expression of hCG protein and hCGß mRNA and protein
in cultured lymphocytes ............................................................................. 30
1.4 Conclusions .......................................................................................... 32
2. Expression of hCGß in bladder cancer (II, III) .................................................... 33
2.1 Immunohistochemical expression of hCGß (III) .................................... 33
2.2 HCGß and hCG in serum of bladder cancer patients (II, III) ................. 34
2.3 HCGß, hCG and hCGßcf in urine of bladder cancer patients (II, III) .... 35
2.4 HCGß mRNA in urinary cells (II, III) .................................................. 36
3. HCGß expression in serum of RCC patients (IV) ............................................... 37
Summary
........................................................................................................... 39
Conclusions ........................................................................................................... 41
Acknowledgements ................................................................................................. 42
References
.......................................................................................................... 44
...........................................................................................................
Original publications .............................................................................................. 55
4
List of original publications
I. Hotakainen K, Serlachius M, Lintula S, Alfthan H, Schröder J, Stenman U-H. Expression of luteinising hormone and chorionic gonadotropin beta-subunit messenger-RNA
and protein in human peripheral blood leukocytes. Mol Cell Endocrinol 2000; 162:7985.
II. Hotakainen K, Lintula S, Stenman J, Rintala E, Lindell O, Stenman U-H. Detection
of messenger RNA for the ß-subunit of chorionic gonadotropin in urinary cells from
patients with transitional cell carcinoma of the bladder by reverse transcription-polymerase chain reaction. Int J Cancer 1999; 84: 304-308.
III. Hotakainen K, Haglund C, Paju A, Nordling S, Alfthan H, Rintala E and Stenman
U-H. Chorionic gonadotropin beta-subunit and core fragment in bladder cancer: mRNA
and protein expression in urine, serum and tissue (Submitted).
IV. Hotakainen K, Ljungberg B, Rasmuson T, Alfthan H, Stenman U-H. The free ßsubunit of human chorionic gonadotropin as a prognostic factor in renal cell carcinoma.
Br J Cancer, 2002; 86: 185-189.
5
Kristina Hotakainen
Abbreviations
ATCC
cDNA
ConA
CRCC
CRP
EGF
FCM
FDP
FGF
FISH
FRC
FSH
hCG
hCGα
hCGß
hCGßcf
IA
IFMA
IL
IRP
IS
LH
LHß
LSCM
MAb
MLC
mRNA
NMP
NSE
PCNA
PDGF
PHA
PRL
PWM
RCC
RT-PCR
TATI
TCC
TGF
Tis
TSH
US
UTI
VEGF
6
American Type Culture Collection
complementary DNA
Concanavalin A
Conventional RCC
C-reactive protein
Epidermal growth factor
Flow cytometry
Fibrinogen degradation products
Fibroblast growth factor
Fluorescence in situ hybridization
Finnish Red Cross
Follicle stimulating hormone
Human chorionic gonadotropin
α subunit of hCG
ß subunit of hCG
Core fragment of hCGß
Image analysis
Immunofluorometric assay
Interleukin
International Reference Preparation
International Standard
Luteinizing hormone
Beta subunit of LH
Laser scanning cytometry
Monoclonal antibody
Mixed lymphocyte culture
messenger RNA
Nuclear matrix protein
Neuron specific enolase
Proliferating cell nuclear antigen
Platelet derived growth factor
Phytohemagglutinin
Prolactin
Pokeweed mitogen
Renal cell carcinoma
Reverse transcription-polymerase chain reaction
Tumor-associated trypsin inhibitor
Transitional cell carcinoma
Transforming growth factor
Tumor in situ
Thyroid stimulating hormone
Ultrasonography
Urinary tract infection
Vascular endothelial growth factor
Abstract
Human chorionic gonadotropin (hCG) is
a glycoprotein hormone consisting of two
dissimilar subunits, α and ß. HCG is produced by the placental trophoblasts and
its function is to maintain pregnancy. The
free subunits have no known biological
function. The ß subunit (hCGß) is commonly produced at low concentrations by
many cultured cells and malignant tumors
of various origin, in which it frequently is
a sign of aggressive disease. Clinically
hCGß is a valuable marker for monitoring of trophoblastic tumors and testicular
carcinoma.
Reverse transcription-polymerase chain
reaction (RT-PCR) of hCGß mRNA was
used in this study to detect cells expressing
hCGß in blood and urine. HCGß expression was induced by culture of peripheral
blood cells, but no expression was observed
in unstimulated blood cells. HCGß mRNA
expression was detected in the urinary cells
of approximately 50% of patients with bladder cancer but not in healthy controls. The
expression was strongly associated with histologically proven carcinoma but not with
stage and grade of the tumor. Immunohistochemical expression of hCGß protein was
found in tumor tissue from one third of the
cancer patients but equally often in benign
epithelium. Current or previous instillation
therapies did not affect the detection of
hCGß mRNA in urinary cells or tissue expression detected by immunohistochemistry. The urine concentrations of the hCGß
core fragment, a degradation product of
hCGß, were higher in patients with hCGß
mRNA in urine and the urine to serum ratio of hCGß was strongly associated with
both stage and grade of the disease, and also
with immunohistochemical detection of
hCGß.
Our findings show that hCGß expression
is not cancer specific, but it may occur in
benign conditions. The ratio of urine to serum hCGß demonstrates that local production of hCGß by the tumor correlates with
stage and grade of the disease, supporting
previous data on the association of hCGß
with advanced disease.
To study whether hCGß has prognostic
significance in renal cell carcinoma (RCC)
preoperative serum samples from patients
with RCC were analyzed. The serum concentrations were elevated in 23% of the patients and hCGß was a prognostic marker
independent of stage and grade of the tumor. Our results suggest that hCGß expression characterizes a potentially aggressive
subgroup of tumors.
7
Kristina Hotakainen
Introduction
Bladder and kidney cancer are two fairly
common malignancies, for which few reliable tumor markers are available. Markers
that could be used for diagnosis, monitoring and evaluation of prognosis would be
of clinical value. The free ß subunit of human chorionic gonadotropin (hCG) in serum has proved to be useful as a prognostic
marker especially for ovarian and various
gastrointestinal cancers.
This study was undertaken to investigate whether hCGß expression could be
used as a marker for bladder and kidney
8
cancer. We studied whether hCGß mRNA
in urinary cells can be used to detect bladder cancer and if this finding correlates
with serum and urine concentrations of
hCGß and immunohistochemical staining
of hCGß in tumor tissue, as well as with
stage and grade of the tumor. The clinical
course of renal cell carcinoma (RCC) is
unpredictable, and no specific serum
markers are available. We therefore studied whether hCGß in serum of RCC patients is of diagnostic and prognostic value
for this disease.
Review of the literature
1. CHORIONIC GONADOTROPIN
AND ITS SUBUNITS
1.1 Biochemistry and biology
Human chorionic gonadotropin (hCG) is a
member of the glycoprotein hormone family, which also comprises luteinizing hormone (LH), follicle stimulating hormone
(FSH) and thyroid stimulating hormone
(TSH). These hormones are heterodimers
consisting of an α and a ß subunit. The free
subunits are devoid of hormonal activity
(Catt et al., 1973; Rayford et al., 1972). The
α subunit common to hCG, LH, FSH and
TSH contains 92 amino acids. The ß chains
are hormone specific and determine the biological activity. The ß subunit of hCG
(hCGß) contains 145 amino acids including a 24 amino acid C-terminal extension
lacking in LHß. The homology between the
ß chains of hCG and LH is about 80% and
hCG and LH exert their action through the
same receptor (McFarland et al., 1989). The
molecular weights of hCG, hCGß and
hCGα are 36700, 22200 and 14500 Da, respectively (Birken, 1984). HCGα is encoded
by a single gene on chromosome 12q21.123 and hCGß by a cluster of six nonallelic
genes on chromosome 19q13.3 (Boothby et
al., 1981; Talmadge et al., 1984b).
1.2 Biological function
HCG is produced at high concentrations by
placental syncytiotrophoblasts. HCG maintains pregnancy by stimulating progesterone production in the corpus luteum during the first trimester (Yoshimi et al., 1969).
It also stimulates testosterone production
by fetal testes (Huhtaniemi et al., 1977).
Receptors for hCG/LH have been identified
in the nonpregnant uterus (Reshef et al.,
1990), prostate (Dirnhofer et al., 1998a) and
several extragonadal sites such as leukocytes
(Lin et al., 1995), thyroid (Frazier et al.,
1990) and epidermal structures (Venencie
et al., 1999).
1.3 HCG in serum and urine of healthy
subjects
Low concentrations of hCG are produced
by the pituitary giving rise to measurable
plasma levels. The serum concentrations are
correlated with those of LH and thus they
increase with age both in men and in women
(Stenman et al., 1987). Age- and gender
specific reference values in serum and urine
are given in table 1. In healthy nonpregnant subjects the α subunit originates
mostly from the pituitary, but some pituitary adenomas may secrete hCGß (Gil-delAlamo et al., 1995). HCGα can be detected
at levels up to 3 ng/l in the serum of most
healthy individuals (87%), patients with
benign diseases (93%), and various nontrophoblastic cancers (96%) (Marcillac et al.,
1992).
HCG and hCGß are excreted into urine
and the concentrations are comparable to
those in plasma. The concentrations of
hCGα in urine are 3-5-fold those in serum
(Landy et al., 1990; Iles & Chard, 1991).
Much of hCG and hCGß is degraded during excretion and a variable part of the hCG
immunoreactivity in urine consists of a fragment of hCGß called the core fragment
9
Kristina Hotakainen
Table 1. Reference values for hCG, hCGβ, hCGβcf
and hCGα in serum and urine of women and men
(Alfthan et al., 1992a).
Upper reference limit (pmol/l)
Serum
Age (years)
hCG
hCGβ
hCGβcf
hCGα
Women
Men
<50
≥50
<50
≥50
8.6
1.6
1.1
301
15.5
2.0
1.1
602
2.1
1.9
1.1
6.1
2.1
1.1
8.8
1.7
8.1
11.5
4.3
9.5
2.9
1.3
6.7
8.4
3.6
8.5
1.5 Clinical determinations
Urine
hCG
hCGβ
hCGβcf
1
In subjects with a serum concentration of FSH < 20;
FSH ≥ 20 (Alfthan et al., unpublished data).
2
(hCGßcf) (Papapetrou & Nicopoulou,
1986).
1.4 HCG in malignant disease
The expression of hCG in serum of cancer
patients is a classic example of ectopic hormone production (Braunstein et al., 1973;
Weintraub & Rosen, 1973; Hattori et al.,
1978). Most cultured human fetal and cancer cells express hCGß, hCGα as well as intact hCG (Acevedo et al., 1992). However,
detectable serum concentrations of hCGα
and hCG occur in normal individuals also,
whereas elevated levels of hCGß are frequently associated with malignant tumors
(Marcillac et al., 1992; Alfthan et al.,
1992b). Most nontrophoblastic tumors produce hCGß exclusively, but isolated production of hCGα has recently been described
in an immunohistochemical study of neuroendocrine lung carcinoma (Dirnhofer et
al., 2000). Carcinoid tumors of the pancreas
frequently express hCGα (70%) in the absence of hCGß (Öberg & Wide, 1981), and
immunocytochemical staining of hCGα can
be observed equally often in endocrine pancreatic tumors (Heitz et al., 1983). Overexpression of hCGα in relation to hCGß at
the mRNA level has been shown in breast
cancer tissue, and these transcripts are also
10
translated into hCGß protein (Giovangrandi
et al., 2001). In addition, some gastrointestinal and gynecological cancers may produce
hCGα exclusively (Huang et al., 1989).
Various minor molecular forms of hCG may
occur in the urine of cancer patients, e.g.,
nicked and hyperglycosylated forms, and
variants lacking the C-terminal extension
of hCGß (Cole et al., 2001).
1.5.1 Normal pregnancy
During pregnancy the concentrations of
hCG become detectable 5-7 days after conception and increase exponentially with a
doubling time of about 1.5 days. Peak values are reached at 7-10 weeks of pregnancy.
After this the levels decrease slowly until
the 15th week, after which there is a small
gradual increase towards term. The concentrations of hCGß are about 0.5-1% of the
total hCG concentration, whereas those of
hCGα are less than 10% during the first
trimester increasing to 30-60% at term.
Low concentrations of hCGßcf occur in
pregnancy serum (<1% of the total hCG
concentration) (Alfthan & Stenman, 1990;
Kardana & Cole, 1990). In urine hCGßcf is
the major form of hCG immunoreactivity
and the concentration is about 4000-fold
that in serum (Wehmann et al., 1990). Pregnancy can be diagnosed by detecting elevated hCG concentrations in urine or serum by the time of the first missed menstrual period.
1.5.2 Ectopic pregnancy
Ectopic pregnancy can be diagnosed when
no intrauterine gestational sac is seen by
ultrasonography and the hCG level is greater
than 1000 IU/l (Cacciatore et al., 1995). The
hCG concentration at day 44 after the last
menstrual period can also be used to predict spontaneous resolution of an ectopic
pregnancy (Korhonen et al., 1994).
Review of the Literature
1.5.3 Maternal screening for Down’s syndrome
HCG and hCGß in serum or urine can be
used in the diagnosis of various pregnancyrelated disorders and chromosomal abnormalities of the fetus (Cuckle et al., 1999).
Serum concentrations of hCG or hCGß in
combination with other markers are used
in screening of fetal trisomy 21. The risk
increases with increasing concentration of
hCG and hCGß and decreasing levels of alpha fetoprotein (AFP) (Phillips et al., 1992).
mRNA expression has been demonstrated
by reverse transcription-polymerase chain
reaction (RT-PCR) in peripheral blood of
patients with germ cell tumors, and more
frequently in those with elevated serum concentrations of hCGß. However, the clinical
significance of this phenomenon is unclear
(Hautkappe et al., 2000).
1.6 The beta subunit of chorionic
gonadotropin
1.6.1 Genes, mRNAs and proteins
1.5.4 Trophoblastic disease
Elevated levels of both hCG and hCGß are
encountered in virtually all cases of molar
disease and choriocarcinoma. The proportion of hCGß to hCG may be used to differentiate between these. In benign disease
the proportion is below 5% (on a molar
basis) and in choriocarcinoma the proportion exceeds 6% (Ozturk et al., 1988;
Stenman et al., 1995; Vartiainen et al.,
1998).
1.5.5 Testicular cancer
Germ cell tumors of the testis represent
more than 95% of all testicular cancers.
These are classified as seminomas (30-40%)
and nonseminomatous germ cell tumors of
the testis. HCG and hCGß are important
markers for both tumor types. HCG is used
both as a prognostic factor, for monitoring
of the response to therapy and during follow up of the disease. Seminoma patients
have elevated serum levels of hCG in 1030% of the cases. Of all hCG-producing
seminomas 30- 40% have elevated serum
levels of both hCG and hCGß, 25% of hCG
only and 35% of only hCGß (Koshida et al.,
1996; Weissbach et al., 1997). Elevated serum concentrations of hCG are associated
with adverse prognosis (Bosl et al., 1981;
Droz et al., 1988; von Eyben et al., 2001),
and the risk increases with increasing concentrations (Vogelzang, 1987). HCGß
HCGß is encoded by a cluster of genes numbered ß1 to ß9 on chromosome 19q13.3.
The homology between the hCGß and LHß
genes is 94% (Talmadge et al., 1983;
Talmadge et al., 1984b), and the hCGß gene
has probably arisen through duplication of
an ancestral LHß gene (Boorstein et al.,
1982). The gene cluster comprises six nonallelic hCGß genes and the gene encoding
LHß. ß1 and ß2 are considered pseudogenes
that are not expressed, while ß7 and ß9 are
alleles to ß6 and ß3, respectively (Policastro
et al., 1983; Policastro et al., 1986). However, it is possible that ß1 and ß2 can produce alternatively spliced transcripts giving rise to a hypothetical protein 132 amino
acids in length (Dirnhofer et al., 1996).
Genes ß6 and ß7 (type I genes) encode a
protein with alanine at position 117 while
genes ß3/9, ß5 and ß8 (type II genes) encode a protein containing aspartic acid at
this position. The transcriptional activity
varies among the various hCGß genes, and
also among individuals. In the placenta,
gene 5 is the one with the strongest expression, followed by genes 3 and 8 with gene
7 accounting for less than 2% of the total
expression (Bo & Boime, 1992; MillerLindholm et al., 1997). A similar pattern is
common also in nontrophoblastic tissues;
Giovangrandi and coworkers recently
showed that the increased hCGß mRNA
expression observed in breast cancer tissue
is mainly due to overexpression of genes 5,
11
Kristina Hotakainen
8 and 3 (Giovangrandi et al., 2001). Transcripts of these genes have been demonstrated in testicular tumors also
(Madersbacher et al., 1994). The differential expression results from differences in
the promoter regions of the genes (Otani et
al., 1988). The mRNA of hCGß contains
879bp (Talmadge et al., 1984a).
HCGß is 145 amino acids in length. It
contains six carbohydrate chains and the
molecular weight is 22200 Da. The threedimensional structure of hCGß resembles
that of the so-called cysteine knot growth
factors, e.g. nerve growth factor, platelet
derived growth factor (PDGFß), transforming growth factor (TGFß) and vascular
endothelial growth factor (VEGF) (Lapthorn
et al., 1994; Muller et al., 1997). These
growth factors bind to their receptors both
as homo- and heterodimers. HCGß has been
shown to exist as a homodimer (Butler et
al., 1999), and thus it may also bind to a
hypothetical receptor as a homodimer.
However, no receptor for hCGß or hCGßß
has been identified.
1.6.2 HCGß expression in tissues and body
fluids
HCGß occurs in serum of men and nonpregnant females at levels 5 to 10-fold lower
than those of hCG. The serum levels are not
related to those of hCG or LH and they are
not age-dependent (Alfthan et al., 1992a).
Expression of hCGß mRNA has been demonstrated in several tissues, i.e., bladder,
adrenal, colon, testis, breast, thyroid and
uterus, in which the expression is about
10,000-fold lower than in the placenta
(Bellet et al., 1997). Expression of hCGß in
normal nontrophoblastic tissues is mostly
associated with type I gene expression but
testicular tissue also expresses type II genes.
The placenta, trophoblastic and other malignant tumors express predominantly type
II genes (Bellet et al., 1997).
Moderately elevated serum concentrations of hCGß protein occur in 30-70% of
12
most nontrophoblastic cancers (Alfthan et
al., 1992b; Marcillac et al., 1992) and it has
been suggested that activation of the hCGß/
LHß gene cluster is characteristic of malignant transformation (Acevedo et al., 1995;
Bellet et al., 1997; Krichevsky et al., 1995).
Membrane-bound hCGß has been found to
be characteristic of a metastatic phenotype
of cancer cells (Acevedo & Hartsock, 1996).
HCGß stimulates the growth of certain cancer cell lines in culture, and it is proposed
to act as an autocrine or paracrine growth
factor (Gillott et al., 1996). The growth
promoting effect of hCGß may partly result from inhibition of apoptosis (Butler et
al., 2000).
1.6.3 HCGß expression in peripheral blood
cells
Low level expression of several genes
thought to be specific for other organs can
be observed in peripheral blood cells (Azad
et al., 1993; Lintula & Stenman, 1997). Leukocytes have the potential of producing several peptide hormones (Blalock et al., 1985;
Harbour-McMenamin et al., 1986; Smith
et al., 1985), and receptors for LH/hCG have
been identified on leukocytes (Lin et al.,
1995). Cultured cells from leukemias and
lymphomas express membrane-bound hCG
and its subunits (Acevedo et al., 1992).
HCG immunoreactivity has been induced
in lymphocytes by mixed lymphocyte cultures, but not by other mitogenic stimuli
(Harbour-McMenamin et al., 1986). During pregnancy, peripheral blood mononuclear cells are capable of secreting hCG
(Alexander et al., 1998).
1.6.4 HCGß in malignant disease
HCGß expression can be detected by immunohistochemistry in 20-52% of colorectal carcinomas and the expression is associated with poorly differentiated and advanced tumors (Kido et al., 1996;
Yamaguchi et al., 1989; Campo et al., 1987).
Review of the Literature
In one study, only serum concentrations
were found significantly correlated with the
clinical outcome (Webb et al., 1995). The
prognostic significance has later been confirmed both for serum concentrations and
immunohistochemical detection of hCGß
(Lundin et al., 2001; Carpelan-Holmström
et al., 1996).
HCG immunoreactivity has been described in squamous cell carcinoma cell lines
(Cole et al., 1981; Cowley et al., 1985). In
immunohistological studies of the esophagus (Burg-Kurland et al., 1986; Trias et al.,
1991), oral cavity (Bhalang et al., 1999) and
uterine cervix, hCG expression has been
associated with poorly differentiated tumors. Preoperatively elevated serum levels
of hCGß predict shorter recurrence free survival of patients with squamous cell carcinomas of the oral cavity and oropharynx
(Hedström et al., 1999).
Serum levels of hCGß are rarely elevated
in vulvar carcinomas, but progressive disease is associated with rising levels (de
Bruijn et al., 1997). The concentrations of
hCGß in serum have a prognostic significance in ovarian carcinoma (Ind et al., 1997;
Vartiainen et al., 2001). Urinary hCGßcf has
been used as a marker of gynecological cancers (Cole et al., 1988; Wang et al., 1988).
HCGß mRNA is expressed in several
pancreatic cancer cell lines, and also in tissue samples from metastatic pancreatic cancer (Bilchik et al., 2000). Elevated serum
concentrations of hCGß have been described
in 40-70% of patients with pancreatic and
biliary cancer. In most of these, urinay concentrations of hCGßcf are also elevated
(Alfthan et al., 1992b; Motoo et al., 1996).
Elevated hCGß in serum has been reported
to correlate with adverse outcome in the
cancer patients (Syrigos et al., 1998).
RT-PCR of hCGß mRNA has been used
to detect circulating melanoma cells (Hoon
et al., 1995) and melanoma metastases in
lymph nodes (Doi et al., 1996). Recently,
Taback and coworkers showed that detection of hCGß mRNA expression in circu-
lating cells from patients with breast cancer is significantly associated with tumor
size (Taback et al., 2001). Hu and coworkers found an association between stage and
detection of hCGß mRNA in blood of breast
cancer patients (Hu & Chow, 2000). In both
studies the combination of hCGß with another marker significantly improved the
correlation (Hu & Chow, 2001). However,
hCGß mRNA expression also occurs in benign breast tumors (Reimer et al., 2000).
1.6.5 HCGß expression in bladder cancer
Approximately 70% of both benign and
malignant urothelial cells in culture express
hCG-like material, almost entirely consisting of hCGß (Iles et al., 1987; Iles & Chard,
1989; Iles et al., 1990b). Elevated levels of
hCGß, and rarely intact hCG, can be detected in serum and urine from patients
with transitional cell carcinomas (TCC) of
the bladder. Both serum and urine concentrations of hCGß are elevated in up to 70%
of the patients with metastatic disease (Iles
et al., 1989; Iles et al., 1996) but in less
than 10% of patients with local tumors (Iles
et al., 1989; McLoughlin et al., 1991; Smith
et al., 1994). The degradation of hCGß may
lead to increased concentrations of hCGßcf
in urine, but it is possible that some tumors synthesize and secrete hCGßcf into the
urine (Iles et al., 1990a; Dirnhofer et al.,
1998b).
Approximately 30% of TCCs express
hCGß and this expression seems to characterize an aggressive subgroup of tumors.
Elevated serum and urine concentrations of
hCGß in advanced disease have been shown
to predict the development of metastasis and
relapses as well as increased mortality (Iles
et al., 1996; Marcillac et al., 1993). HCGß
expression detected by immunohistochemistry is associated with a poor response to
radiotherapy (Martin et al., 1989; Jenkins
et al., 1990; Moutzouris et al., 1993) and
the serum concentrations of hCGß correlate with the response to chemotherapy
13
Kristina Hotakainen
(Dexeus et al., 1986; Mora, J. et al., 1996;
Cook et al., 2000).
1.6.6 HCGß expression in renal cell
carcinoma (RCC)
Many earlier studies have failed to show
any hCG immunoreactivity in serum or
tumor tissue of patients with RCC (Sufrin
et al., 1977; Kuida et al., 1988;
Dunzendorfer et al., 1981). However, hCG
immunoreactivity has been detected by radioimmunoassay in urinary concentrates
from patients with advanced or poorly differentiated tumors (Fukutani et al., 1983),
but the number of patients in the study was
very small. Elevated serum concentrations
of hCGß have been reported to occur in 10%
of the patients, and the levels correlate with
the clinical course (Dexeus et al., 1991).
2. BLADDER CANCER
2.1 Epidemiology
Bladder cancer is one of the most common
malignancies among men in the Western
world (Parkin et al., 1999). Bladder cancer
accounts for approximately 4% of all new
cancer cases in Finland, being the 3rd most
common malignancy in men and 16th in
women. The age-adjusted incidence rate has
increased from 10 to 19 cases per 100 000
inhabitants during the past 30 years (Finnish Cancer Registry, 2000), and it is still
expected to grow. Bladder cancer is a disease of the aged; mean age at presentation
in 1985-1994 was 69 years for males and
72 for females (Dickman et al., 1999).
2.2 Risk factors
Smoking increases the risk of bladder cancer (Sorahan et al., 1994), and more so in
women than in men (Castelao et al., 2001).
Occupational and other exposure to aromatic amines is an established risk factor
14
(Boffetta et al., 1997; Steineck et al., 1990)
and certain alkylating agents such as cyclophosphamide have also been reported to
induce bladder cancer (Durkee & Benson,
1980). Chronic inflammation and regeneration processes caused by schistosomal infection of the bladder induce squamous
metaplasia which may lead to development
of cancer (Johansson & Cohen, 1997).
2.3 Histology
TCC is by far the most common histological type of bladder cancer, accounting for
90-95% of the cases in industrialized countries (Dickman et al., 1999). The remainder are squamous cell carcinomas (3-6%),
adenocarcinomas (1-3%), or undifferentiated carcinomas (1%) (Mostofi FK, 1973).
Squamous cell carcinomas are frequently
associated with schistosomal infection, and
in endemic areas for schistosomiasis 75%
of bladder cancers are squamous cell carcinomas, 6% adenocarcinomas and the remainder transitional (Johansson & Cohen,
1997).
2.4 Symptoms and signs
The most frequently encountered sign of
bladder cancer is painless and intermittent
hematuria. This can be microscopic, but in
75% of the cases an episode of macrosopic
hematuria can be verified (Varkarakis et al.,
1974). Lower abdominal pain, frequency,
urgency and dysuria occur in approximately
one third of the patients with invasive disease (Utz et al., 1980).
2.5 Diagnostic procedures
Urinary cytology is the primary tool for
detection and follow up of bladder cancer
(Lewis et al., 1976; Papanicolaou &
Marshall, 1945). It is sensitive and specific
in high-grade or invasive tumors, but less
so in superficial and low-grade cancers
(Esposti et al., 1978; Rubben et al., 1979;
Review of the Literature
Badalament et al., 1987b; Hudson & Herr,
1995; Koss et al., 1985).
Cystoscopy is the gold standard for detecting bladder cancer. By diagnostic cystoscopy the extent and nature of the tumor
can be evaluated in addition to the state of
the urethral and bladder mucosae. Accurate
staging requires biopsies including the
muscular layer of the bladder wall (See &
Fuller, 1992). Concomitant upper urinary
tract disorders are excluded by intravenous
urography (Hatch & Barry, 1986). Larger
tumor masses in the bladder can be detected
by transabdominal sonography. Possible
muscle invasion can be evaluated by intravesical sonography. Computerized tomography and magnetic resonance imaging are
used as supplements to clinical staging and
evaluating metastatic spread of muscle-invasive tumors (Barentsz et al., 1996; See &
Fuller, 1992).
easier (Table 2). The tumors are graded into
three grades according to the World Health
Organization classification (Mostofi, 1973).
More recently, new grading systems for papillary carcinomas have been suggested, in
which the previous grades two and three
are classified low and high grade, respectively. The previous grade 1 carcinoma is
classified as a papillary tumor of low malignant potential (Table 3) (Epstein et al.,
1998). Carcinoma in situ (Tis) is a distinct
category of flat intraepithelial malignant
proliferation (UICC, 1978). This carcinoma
is an aggressive and potentially invasive
high grade tumor (Wolf & Hojgaard, 1983).
2.7 Treatment and prognosis
Superficial tumors (Ta and Tis) are treated
by transurethral resection alone or in combination with intravesical chemo- or immunotherapy (Soloway, 1983; Soloway &
Perito, 1992). Invasive and highly recurrent
tumors are managed by cystectomy together
with adjuvant treatments such as systemic
chemotherapy and radiotherapy (Soloway,
1990). After initial therapy, all patients enter a follow up scheme consisting of urinary cytology and cystoscopy every 3-4
months during the first two years, and later
2.6 Staging and grading
Stage classification of bladder cancer is performed according to the TNM classification
of bladder cancer (Sobin & Wittekind,
1997). However, a previous classification of
1978 (UICC, 1978) is used in many studies to make comparison with earlier reports
Table 2. TNM stage classification of bladder cancer.
3rd edition (UICC, 1978)
TX
T0
Tis
Ta
T1
T2
T3a
T3b
Primary tumor can not be assessed
No evidence of primary tumor
Carcinoma in situ (flat tumor)
Papillary non-invasive carcinoma
Tumor infiltrating the lamina propria
Tumor infiltrating the superficial muscle
Tumor infiltrating the deep muscle
Tumor infiltrating through the bladder wall
into the perivesical fat
T4a Carcinoma involving the prostate, uterus
or vagina
T4b Carcinoma invading the abdominal or
pelvic wall
N
Lymph node involvement
(N0=without, N1-4=with)
M
Distant metastasis (M0=without, M1=with)
5th edition (Sobin & Wittekind, 1997)
Tis
Ta
T1
T2
T2a
T2b
T3
T3a
T3b
T4
T4a
Carcinoma in situ (flat tumor)
Papillary non-invasive carcinoma
Tumor infiltrating the lamina propria
Tumor infiltrating the muscle
Tumor infiltrating the superficial muscle
Tumor infiltrating the deep muscle
Tumor infiltrating perivesical tissues
Microscopically
Macroscopically
Tumor infiltrating perivesical organs
Carcinoma involving the prostate, uterus
or vagina
T4b Carcinoma invading the abdominal or pelvic wall
N
Lymph node involvement
(N0=without, N1-3=with)
M
Distant metastasis (M0=without, M1=with)
15
Kristina Hotakainen
Table 3. Histologic grading of papillary urothelial
carcinoma.
WHO
19731
WHO/ISUP
1998 2
Papilloma
Grade 1
Grade 2
Grade 3
Papilloma
LMP
Low grade
High grade
Current
proposal3
Papilloma
Grade 1
Grade 2
Grade 3
1
Mostofi FK, 1973; 2Epstein et al., 1998; 3Cheng & Bostwick, 2000.
every 6-12 months (Nurmi & Rintala,
2002).
Most bladder cancers are superficial at
diagnosis, but approximately 75% recur,
and progression to more advanced stage
occurs in up to 40% by ten years (Heney et
al., 1983; Herr, 1997; Herr et al., 1995;
Pagano et al., 1987). In Tis the long-term
risk of progression to invasiveness or metastases is even higher, 60-70% (Cookson
et al., 1997; Herr, 2000). High grade and
tumor multiplicity are associated with a
higher recurrence rate (Heney et al., 1983).
However, 27% of patients with papillary
tumors of “low malignant potential” will
experience tumor recurrence and progression of grade occurs in 75% (Cheng et al.,
1999). Stage is the most accurate prognostic indicator; the 5-year survival rate declines with increasing stage, being only 021% in stage T4 and as high as 80-94% in
stage Ta (Malmström et al., 1987; Torti &
Lum, 1984). Age is also consistently associated with survival, with a more favourable
outlook in the younger age groups
(Dickman et al., 1999).
2.8 Markers of bladder cancer
The high recurrence rate and the risk of
progression necessitates a close surveillance
of bladder cancer. Furthermore, a significant number of recurrences occur more than
five years after primary diagnosis, which
justifies a lifelong follow up (Cheng et al.,
1999; Cookson et al., 1997; Leblanc et al.,
1999). An optimal marker would facilitate
noninvasive diagnosis and follow up of blad16
der carcinoma. The test should be economic,
well standardized and reproducible, easy to
perform and most importantly, sensitive and
specific. Furthermore, the ability to estimate stage and grade and to characterize
the malignant potential and prognosis
would be valuable.
Urinary cytology is the reference standard to which potential new tests are compared. The sensitivity of cytology ranges
from 70-100% in Tis and other high-grade
carcinomas, with a specificity of more than
95% (Badalament et al., 1987a; Bastacky et
al., 1999). However, in low-grade superficial tumors the sensitivity is at best no more
than 40%. Urinary calculi, infection, instillation therapies and other treatments may
cause cellular atypia leading to falsely positive cytology, but specificity is still better
than 80% regardless of stage and grade
(Murphy et al., 1984).
A multitude of biomarkers for bladder
cancer is being studied. These can roughly
be classified into two categories; markers
for screening and diagnosis of bladder cancer (Table 4) and potentially prognostic
markers (Table 5). Some tests are being
clinically used as complements to urinary
cytology and cystoscopy and others are under clinical evaluation. The best new markers give higher sensitivity than urinary cytology, but specificity is generally lower
(Brown, 2000; Burchardt et al., 2000).
However, the cytologic methods, to which
new tests are being compared, are not well
standardized. This makes evaluation of their
true performance difficult, and up to date
no noninvasive test is sensitive and specific
enough to replace cytology and cystoscopy.
However, the intervals between follow up
cystoscopies can be increased and the detection of relapse can be improved by using
the new markers.
2.8.1 Markers for screening and
diagnosis
Assays for nuclear matrix protein 22
Review of the Literature
(NMP22) and human complement factor
H-related protein (BTA stat and TRAK
tests) are the most widely used new markers for diagnosis and follow up of bladder
cancer. NMP22 is a urothelial cancer associated nuclear matrix protein (Soloway et al.,
1996). High NMP22 concentrations in
urine are associated with active disease
(Carpinito et al., 1996). The sensitivity is
at least twice that of cytology (Sozen et al.,
1999), but specifity (61-85%) (Serretta et
al., 1998; Zippe et al., 1999) is compromized by benign urologic disorders
(Miyanaga et al., 1997) and therapies (Öge
et al., 2000). This limitation also applies to
the various BTA-tests (Pode et al., 1999;
Sanchez-Carbayo et al., 1999). Furthermore,
elevated NMP22 concentrations in urine
have also been observed in patients with
renal cell carcinoma (Huang et al., 2000).
Previous (Bacillus Calmette-Guérin, BCG)
and current (any type) instillation treatments lower the specificity of the BTA stat
significantly limiting its use in the follow
up of bladder cancer (Raitanen et al., 2001).
Part of the telomeres in somatic cells are
degraded with every replication round
(Harley et al., 1990). Telomerase is a ribonucleoprotein enzyme with reverse transcriptase activity, which maintains the telomeres in replicating germ cells thus making repeated rounds of replication possible
without loss of parent DNA sequences
(Blackburn, 1991). Telomerase activity is
low or absent in normal cells but it is often
detected in voided urine of bladder cancer
patients regardless of grade (Kavaler et al.,
1998). Telomerase tests have been reported
to outperform both NMP22 and BTA assays as well as conventional cytology both
in sensitivity and specificity (Ramakumar
et al., 1999). However, the method is costly
and laborious, making it less feasible for
routine clinical use (Ross & Cohen, 2000).
Increased urinary levels of fibrinogen
degradation products (FDP) occur in patients with bladder cancer, and these can
be detected by an immunoassay. Initially,
Table 4. Markers for screening and diagnosis
of bladder cancer.
BTA (BTA TRAK; BTAstat)
NMP22
FDP
Telomerase
Cell surface antigens (M344, 19A211, LDQ10)
Hyaluronic acid/ hyaluronidase
Cytokeratins
the method was assigned a sensitivity of
of 68-100% and a specificity of 75-96%
(Johnston et al., 1997; Schmetter et al.,
1997) in detecting bladder neoplasia, but
later a sensitivity of merely 48% was reported (Ramakumar et al., 1999).
2.8.2 Prognostic markers
An increasing list of protein and genetic
alterations is under research as prognostic
markers of bladder cancer (Table 5). Of
these, assays for microsatellite instability,
VEGF and some tumor-associated antigens
can be performed on urinary cytology or
bladder washing specimens. This also applies to flow cytometry (FCM), by which
DNA-ploidy and S-phase fraction (SPF, fraction of cells in the population being in the
DNA-synthesis phase) are measured. Aneuploid cell populations and those with a high
SPF are typical of high grade and stage tumors (Klein et al., 1982; Koss et al., 1989;
Schapers et al., 1993). The reported sensitivity of DNA-ploidy analyzed by FCM for
detection of urothelial neoplasia ranges from
34% to 88% (Badalament et al., 1987a;
Gregoire et al., 1997; Murphy et al., 1986)
with a specificity exceeding 80% (Bakhos
et al., 2000; Gregoire et al., 1997). However, the number of cells needed can rarely
be obtained from voided urine, but a bladder washing sample is required. The test is
compromised by large amounts of non-malignant cells in the sample. In addition, only
gross genetic alterations leading to changes
in DNA-ploidy can be detected by FCM.
Thus, diploid tumors and those with bal17
Kristina Hotakainen
anced translocations pass undetected, that
is most of grade 1, stage 1 tumors and up
to half of grade 2 lesions (Bucci et al., 1995;
Kawamura et al., 2000; Liedl, 1995;
Tribukait & Esposti, 1978). Furthermore,
despite a higher overall sensitivity than for
cytology (Badalament et al., 1987c; Giella
et al., 1992; Song et al., 1995) FCM fails to
detect most in situ carcinomas which are
detected by urinary cytology. In summary,
analyses for DNA-ploidy can be useful in
differentiating benign atypia associated
with instillation therapies from recurrent
neoplasia (Bakhos et al., 2000), but the
prognostic utility is limited (Bittard et al.,
1996; Tetu et al., 1996) and the method is
too insensitive for screening (Gourlay et al.,
1995).
Combined with conventional cytology,
DNA-ploidy measured by static image
analysis (IA) can detect up to 85% of recurrent tumors (Cajulis et al., 1995; de la Roza
et al., 1996; Mora, L.B. et al., 1996;
Richman et al., 1998). Laser scanning
cytometry (LSCM) combines the advantages
of FCM and image analysis and measures
the DNA-content in individual cells. Small
cell populations with aberrant DNA-content can be detected by these newer methods, and thus they may also be used for
voided urine (Parry & Hemstreet, 1988;
Wojcik et al., 1998). Aberrant cellular
DNA-content can also be visualized by fluorescence in situ hybridization (FISH). This
method has been reported to be capable of
differentiating between Ta and T1 tumors
and also of predicting response to immunotherapy (Pycha et al., 1997; Sauter et al.,
1997). However, the sensitivity is highly
dependent on the number of centromeric
probes used, and due to the heterogenous
nature of bladder cancer multiple probes are
needed (Sauter et al., 1997). The possibility to also detect superficial tumors and to
obtain prognostic information makes the
method promising for the follow up of bladder cancer patients. Microsatellite analyses
can detect tumor-associated alterations in
18
Review of the literature
repetitive DNA sequences of the human
genome (microsatellites). In bladder cancer,
microsatellite instability and loss of heterozygosity is frequently observed in characteristic chromosomes. The analysis can be
performed on voided urine with a high sensitivity (74-95%) in detecting recurrent
tumors (Mao et al., 1996; Steiner et al.,
1997; van Rhijn et al., 2001) even months
before these are detected by cystoscopy. In
addition, the test can also be used on frozen
urine samples (Linn et al., 1997). However,
microsatellite instability and loss of heterozygosity in urine are frequently found
in benign urological disorders such as benign prostatic hyperplasia and cystitis also
(Christensen et al., 2000). Thus the high
specificity initially reported (Mao et al.,
1996; Mourah et al., 1998; Steiner et al.,
1997; van Rhijn et al., 2001) may be overestimated.
The expression of many growth factors
is altered in bladder cancers (Table 5) and
the concentrations of these can be determined in urine. Determination of VEGF has
been used to differentiate superficial (Ta)
and low grade (G1) tumors from invasive
(T1 or more) and high grade tumors (G23). High urinary concentrations of VEGF
predict recurrence (Crew et al., 1997).
Several monoclonal antibodies are being
studied as tools for detection of bladder cancer. These identify antigens that are mostly
absent in normal bladder epithelium. Some
differentiation between various grades and
stages as well as predicition of tumor recurrence can be achieved (Allard et al., 1995;
Huland et al., 1987). Combinations of antibodies can be used as immunocytochemical tests on voided urine (Mian et al., 1999),
and improve sensitivity and specificity when
used together with cytology.
P53 overexpression has been detected in
bladder cancer, and this is associated with
poor prognosis (Cordon-Cardo & Reuter,
1997; Sarkis et al., 1993; Tsuji et al., 1997).
However, distinct mutations of p53 occur
in bladder epithelium in smokers
Review of the Literature
Review of the literature
Table 5. Potentially prognostic markers of bladder
cancer.
Microsatellite instability assays*
DNA-ploidy/SPF *(FCM, IA, LSCM, FISH)
Blood group-related antigens (ABO, Lewis-ag)
Growth factors (EGF*, TGFβ, FGF, VEGF*)
Cellular adhesion molecules (cadherins, integrins)
Proliferation antigens (Ki-67, PCNA)
Tumor-associated antigens* (486 P3/12, M344,
19A211, T138, DD23),
Cell cycle regulatory proteins (p53, pRb, cyclins,
p15, p16, p21)
Oncogens (c-erb-B2, ras, c-myc, c-jun, mdm2)
Components of fibrinolysis system (urokinase
type I plasmin activator)
* detection on urinary sediment or bladder wash specimens
(Husgafvel-Pursiainen & Kannio, 1996),
and p53 overexpression is related to the
number of cigarettes smoked per day
(Zhang et al., 1994).
3. RENAL CELL CARCINOMA
3.1 Epidemiology
RCC or hypernephroma is the most common malignant tumor of the kidney accounting for more than 3% of the annual
new cancer cases in Finland. It is the 7th
most common cancer among men in Finland, and the 13th among women. The ageadjusted incidence rate has nearly doubled
in the past 30 years (Dickman et al., 1999).
3.2 Risk factors
Smoking doubles the risk for RCC in both
men and women (McCredie & Stewart,
1992; McLaughlin et al., 1990). Obesity is
another reported risk, and hypertension or
medication for it have also been suspected
(Chow et al., 1996; Messerli & Grossman,
1999). Certain dietary factors may play a
role, but alcohol and coffee consumption do
not (McLaughlin & Lipworth, 2000;
Mellemgaard et al., 1996; Wolk et al.,
1996). Occupational and other exposure to
several carcinogens are suspected risk factors (Boffetta et al., 1997). Genetic predisposition to RCC has been suggested to be
dependent on variations in metabolic pathways (Longuemaux et al., 1999). Nacetyltransferase 2 participates in the metabolism of drugs and carcinogens, of which
arylamines are found in tobacco smoke
(Hein et al., 1993). The so called slowacetylator genotype increases the risk associated with smoking. Toxification of
nephrocarcinogenic chlorinated hydrocarbons proceeds by conjugation with glutathione. This is mediated by glutathione
transferase, the activity of which may be
altered in RCCs to promote carcinogenesis
(Bruning & Bolt, 2000; Delbanco et al.,
2001; Grignon et al., 1994). The cytochrome P450 system may similarly be involved in carcinogenesis (Murray et al.,
1999).
Familial RCC accounts for less than 5%
of the cases, and most of these are associated with the von Hippel-Lindau syndrome.
This autosomal dominant disorder is caused
by errors in a tumor suppressor gene on
chromosome 3p (Latif et al., 1993). In addition to several other tumors, approximately 70% of the patients develop RCC
(Friedrich, 1999).
3.3 Histology
RCCs are adenocarcinomas arising in the
parenchymal epithelium. They are subclassified into five distinct types. Conventional
(CRCC) and papillary renal cell carcinomas account for approximately 85-90% of
RCCs, while the chromophobe and unclassified tumors account for up to 5% each.
The remainder are collecting duct tumors.
Transitional cell tumors may arise in the
transitional epithelium of the renal pelvis; these account for approximately 5%
of all kidney cancers. Oncocytoma is a benign renal tumor (Kovacs et al., 1997).
19
Kristina Hotakainen
3.4 Symptoms and signs
The symptoms caused by RCC can be either systemic or local. The classic triad of
local symptoms is hematuria, flank pain and
a palpable tumor mass. A rapidly developing varicocele may be caused by a tumor
invading the renal vein (left) or the inferior
vena cava. The hematuria can cause obstruction of the ureter leading to painful attacks.
Fatigue, fever and loss of weigth may occur
(Nurmi & Rintala, 2002; Nurmi et al.,
1985).
3.5 Diagnostic procedures
Renal tumors are investigated by ultrasonography (US), urography, computerized
tomography or magnetic resonance imaging, angiography and needle biopsies. The
differentiation between renal cysts and tumors is mostly possible by US. Very small
tumors can be detected by US and guided
needle biopsies can be obtained. Computerized tomography is an aid in evaluating
the extent of invasion into perirenal tissues
and veins. Angiography is useful in
localising and quantitating arteries in the
planning of surgery. Bone scintigraphy and
chest radiography are used to detect bone
metastases (Hilton, 2000).
3.6 Staging and grading
Staging of RCC is performed according to
the TNM classification (Table 6) (Sobin &
Wittekind, 1997) and nuclear grading according to Skinner and coworkers (Skinner
et al., 1971).
3.7 Treatment and prognosis
Curative treatment of RCC is usually possible only by surgical removal of the whole
tumor. This is usually achieved by radical
nephrectomy, but in selected cases nephronsparing surgery is an option. Response to
hormonal and cytotoxic therapies is ob20
Table 6. TNM classification of renal cell carcinoma
(Sobin & Wittekind, 1997).
Tx
T0
T1
Primary tumor can not be assessed
No evidence of primary tumor
Tumor 7 cm or less in greatest dimension,
limited to the kidney
T2 Tumor more than 7 cm in greatest dimension,
limited to the kidney
T3 Tumor extends into major veins, perinephric
tissues or the adrenal gland, but not beyond
Gerota fascia
T3a Tumor infiltrates adrenal gland or perinephric
tissues, but not beyond Gerota fascia
T3b Tumor grossly invades the renal vein(s) or
vena cava below the diaphragm
T3c Tumor grossly extends into the vena cava
above the diaphragm
T4 Tumor invades beyond Gerota fascia
N Lymph node involvement
(N0=without, N1-2=with)
M Distant metastasis (M0=without, M1=with)
tained in no more than 10% of the patients
(Motzer et al., 1997). Five to 20% of the
patients respond to immunotherapy based
on interferon-alpha or interleukin-2
(Malaguarnera et al., 2001; Minasian et al.,
1993; Motzer et al., 2000; Nanus, 2000;
Vogelzang et al., 1993). In approximately
one third of the patients distant metastases
are present at diagnosis (Dekernion et al.,
1978). A solitary metastasis can be removed
together with the primary tumor, and in
cases with multiple metastases removal of
the primary tumor may promote regression
of the metastases (Golimbu et al., 1986a;
Ljungberg et al., 2000; Wyczolkowski et al.,
2001).
Stage and nuclear grade are the most
important prognostic factors (Fuhrman et
al., 1982; Medeiros et al., 1988). The 5year survival rate in all stages is 50-60%,
but patients with metastatic disease at first
diagnosis survive only for approximately 12
months. Survival is consistently associated
with age being lowest in the highest age
groups (Dickman et al., 1999; Minasian et
al., 1993; Nurmi, 1984). A third of initially nonmetastatic tumors recur after surgery, mostly incurably (Ljungberg et al.,
Review of the Literature
1999; Levy et al., 1998). Relapses can occur even decades after primary diagnosis and
the 10-year relative survival rate is approximately 45% (Dickman et al., 1999). With
the increasing use of imaging techniques
such as US and computerized tomography
the proportion of incidentally detected renal tumors has been growing (Konnak &
Grossman, 1985; Smith et al., 1989). These
tumors are smaller and of lower grade and
stage. Thus, the 5-year survival rates for
patients with such tumors are significantly
higher (62-97%) than for those with symptomatic ones (42-83%)(Herr, 1994; Licht
et al., 1994; Tsui et al., 2000).
3.8 Markers of RCC
RCCs are a heterogenous group of tumors
with a distinct genetic background and biology associated with the histological type
(Takahashi et al., 2001; Young et al., 2001).
The clinical outcome in the individual RCC
patient is highly variable even within a
given stage (Golimbu et al., 1986b; Kovacs
et al., 1997). Several potential RCC markers have been described, but most of these
can be studied only in surgically removed
tissues (Table 7). No specific serum markers are available. Markers that could be used
before surgery would be of value in planning of treatment and follow up.
3.8.1 Tissue markers
Several genetic aberrations have been de-
tected in RCC by restriction fragment
length polymorphism analysis, comparative
genomic hybridization, cDNA microarray
screening, in situ hybridization and
microsatellite analysis. A frequently involved region is located on chromosome 3p
(Cohen et al., 1979), which also comprises
the von Hippel Lindau tumor suppressor
gene (Latif et al., 1993). Genetic losses of
3p occur commonly together with other
alterations (Velickovic et al., 2001;
Yamakawa et al., 1991). Some of these are
associated with tumor histology (Velickovic
et al., 2001), grade, stage (Morita et al.,
1991; Wada et al., 1998) or recurrence
(Moch et al., 1996; Thrash-Bingham et al.,
1995). Multiple genes are up- or down regulated in RCC, and a distinction between
histologic subtypes and clinical outcome is
possible based on the expression profiles
(Takahashi et al., 2001; Young et al., 2001).
P53 mutations are fairly infrequent occurring in 2-33% of RCCs (Gelb et al., 1997;
Ljungberg et al., 2001; Reiter et al., 1993;
Vasavada et al., 1998). However, p53 expression has been found to have a prognostic significance (Girgin et al., 2001) in some
papillary and chromophobe tumors, but not
in CRCCs (Gelb et al., 1997; Kamel et al.,
1994; Ljungberg et al., 2001). DNA-ploidy
has been studied as a prognostic indicator
in RCC, but its value is limited (Ljungberg
et al., 1996; Shalev et al., 2001; Tannapfel
et al., 1996). Vimentin expression can be
detected in some RCCs (Beham et al., 1992;
Dierick et al., 1991), and it may give addi-
Table 7. Potential markers of RCC.
Detected in
Serum
Tissue
VEGF
IL-10
CA-125
TATI
NSE
TNFβ
Ferritin
CRP
Basic FGF
Erythropoietin
Ki-67
PCNA
AgNOR
P53
Growth factors (basic FGF)
Adhesion molecules (CD44, cadherins, catenins)
Cyclins (A)
Vimentin
Matrix metalloproteinases (2 and 9)
Tissue inhibitors of MMPs (1 and 2)
21
Kristina Hotakainen
tional prognostic information especially
when analyzed together with tumor grade
(Nativ et al., 1997; Sabo et al., 1997). Markers of cell proliferation such as nucleolar
organizer regions analysis (AgNOR), Ki-67
index and proliferating cell nuclear antigen
(PCNA) expression are promising as prognostic markers (de Riese et al., 1993;
Delahunt et al., 1995; Hofmockel et al.,
1995; Tannapfel et al., 1996).
3.8.2 Serum markers
Of the many serum markers studied some
are of potential prognostic value; neuron
specific enolase (NSE), VEGF, interleukin10, CA-125, and tumor-associated trypsin
inhibitor (TATI) (Yaman et al., 1996;
Wittke et al., 1999; Grankvist et al., 1997;
Jacobsen et al., 2000; Paju et al., 2001).
Some of the general glycoprotein cancer
markers are detected in serum of RCC patients. CA 125 in serum is an independent
prognostic marker and CA 15-3 may also
be of some value; both markers are associ-
22
ated with tumor stage and grade (Grankvist
et al., 1997).
Anemia or polycythaemia may be encountered in RCC, suggesting involvement
of the erythropoietin processes. Elevated
serum concentrations of erythropoietin have
been described but although this is
prognostically significant the sensitivity of
this marker is too low for clinical use
(Ljungberg et al., 1992). Ferritin appears to
be produced by certain RCCs and can in
some cases be used for monitoring of the
disease (Essen et al., 1991; Kirkali et al.,
1995; Özen et al., 1995). Acute phase reactants such as C-reactive protein (CRP) and
inflammatory markers such as erythrocyte
sedimentation rate may also be useful
(Ljungberg et al., 2000; Nurmi et al., 1985).
Several other general serum analytes such
as calcium, lactate dehydrogenase and hemoglobin in combination with clinical features and other markers have been used in
prognostic models (Motzer et al., 1999).
However, none of these are specific for cancer in general and even less for renal cancer.
Aims of the study
The aims of the study were:
1. To study whether peripheral blood cells express hCGß and how the potential expression is regulated. Expression of hCGß mRNA in blood cells would limit the utility of
hCGß mRNA as an indicator of tumor cells in circulation (I).
2. To study whether hCGß mRNA expressing cells can be detected in the cells of voided
urine from bladder cancer patients (II).
3. To compare detection of hCGß mRNA in urinary cells with serum and urinary concentrations of hCGß and stage and grade of the disease, and with immunohistochemical
detection of hCGß in tumor tissue (III).
4. To study whether the preoperative serum concentrations of hCGß are of prognostic
significance in patients with RCC (IV).
23
Kristina Hotakainen
Material and methods
1. SUBJECTS AND SAMPLES (I-IV)
All studies were approved by the ethics
committees of Helsinki University Central
Hospital (HUCS) and the Finnish Red Cross
(FRC), Helsinki, Finland, and the University Hospital in Umeå, Sweden. Patients as
well as controls were recruited after informed consent.
Whole blood buffy coats from healthy
blood donors were obtained from the Blood
Service of the FRC (I). Samples of venous
peripheral blood (I) (Tables 8 and 9) control sera (III, IV) as well as urine (II, III)
(Table 10) were collected from healthy laboratory personnel from HUCS. Placental tissue (I, II, III) was obtained after normal
delivery from the Department of Obstetrics and Gynecology, HUCS. Urine and serum specimens of bladder cancer patients
and control patients (II, III) were obtained
from the Departments of Urology and Surgery, HUCS. All cases studied for this thesis were TCCs. TCC tissue specimens as well
as samples of benign bladder tissue were
obtained from the Department of Pathology, HUCS (Table 10). Staging of the tumors was performed according to the TNM
classification of bladder cancer (UICC,
1978) and grading according to the WHO
classification of bladder tumors (Mostofi
FK, 1973) (II). In study III the more recent
consensus classification of urothelial neoplasms was also used (WHO/ISUP 1998).
The histological diagnosis was based on
samples obtained within two weeks of urine
sampling. Cytology of voided urine was
classified according to Papanicolaou (Papanicolaou & Marshall, 1945). Sera of patients
24
with renal cell carcinoma (IV) were obtained
from the serum bank of the Department of
Urology and Andrology, University Hospital of Umeå (Table 10). Staging of RCC
was performed according to the 1997 TNM
classification (Sobin & Wittekind, 1997),
nuclear grading according to Skinner and
coworkers (Skinner et al., 1971), and DNAploidy according to Ljungberg and coworkers (Ljungberg et al., 1996).
2. PREPARATIONS AND CULTURES OF CELLS (I-III)
Blood samples from five apparently healthy
nonpregnant females and six males (Table
8) were drawn into 3 ml Vacutainer EDTA
tubes (Becton Dickinson, Rutherford, NJ)
(I). Erythrocytes in peripheral blood were
hemolysed by incubating blood with 1.5 vol
of diethylpyrocarbonate-treated water for 5
min. Mononuclear cells were isolated by
Ficoll-Paque centrifugation according to the
manufacturer’s instructions (Pharmacia,
Uppsala, Sweden) and total RNA was isolated (Chomczynski & Sacchi, 1987).
Urine samples (II, III) were stored at 4°
C for a maximum of six h before processTable 8. Peripheral blood cell preparations (I).
Leukocyte extracts
Mononuclear cells
B-cells
T-cells
Granulocytes
Monocytes
1
Mononuclear cells 1
Lymphocytes 2
Peripheral blood (11) 3
Buffy coats
Buffy coats
Buffy coats
Buffy coats
from peripheral venous blood of a healthy male; 2 from buffy coats;
five females and six males of healthy laboratory personnel.
All buffy coats were obtained from the Blood Service of the FRC.
3
Table 9. Cultured peripheral blood lymphocytes and
cell lines.All cell lines were obtained from the ATCC
and buffy coats from the FRC.
Cell type/ line Description
Lymphocytes
HL-60
Jurkat
K-562
U-937
JAR
UM-UC-3
TCC-SUP
Used in
Buffy coats
Acute promyelocytic leukemia
Acute T-cell leukemia
Chronic myelogenous leukemia
Histiocytic lymphoma
Choriocarcinoma
TCC cell lines
TCC cell lines
I
I
I
I
I
I
II
II
ing. The urinary cells were collected by centrifugation and washed once with phosphate
buffered saline (PBS, 50 mM sodium phosphate, 150 mM NaCl, pH 7.4; HyClone
Europe Ltd, Cramlington, UK). Total RNA
was then isolated from the cell pellet.
Peripheral blood lymphocytes from
whole blood buffy coats were purified on a
Ficoll-Paque gradient (I). 1 x 106 cells/ml
were cultured in 15 ml Dulbecco’s Modified Eagle’s Medium (DMEM) (HyClone)
supplemented with 5% fetal calf serum
(FCS, Biological Industries, Kibbutz Beit
Haemek, Israel), 100 IU/l penicillin, 100
mg/l streptomycin and 2 mM L-glutamine
(GibcoBRL, Paisley, Scotland). The cells
were stimulated with phytohemagglutinin
(PHA) 10 mg/ml, concanavalin A (Con A)
2,5 mg/ml, pokeweed mitogen (PWM) at
a final dilution of 1:100, and prolactin at
different concentrations (14, 28, 70, 100,
and 140 ng/ml) (all from Sigma Chemical
Co., St. Louis, MO). Control cultures were
performed without mitogens.
Two-way mixed lymphocyte cultures
(MLC, I) were performed in 25 cm2 tissue
culture flasks with 5 x 105 lymphocytes/ml.
The cultures were incubated at 37° C in a
humified atmosphere of 8% CO2 in air. The
cell number and the proliferative response
to each stimulus was measured by counting the proportion of lymphoblasts in the
culture by microscopy.
All cell lines (Table 9) were obtained from
Material and Methods
the American Type Culture Collection
(ATCC) and cultured according to instructions in RPMI 1640 medium (HyClone
Europe Ltd, Cramlington, UK) supplemented with 10% FCS (Flow Laboratories,
UK), 2 mM glutamine (GibcoBRL), 100
IU/l penicillin, 100 mg/l streptomycin
(HyClone) and 2.5 mg/l amphotericin B
(GibcoBRL).
3. LEUKOCYTE EXTRACTS (I)
Eight ml of peripheral blood was taken in a
Vacutainer Cell Preparation Tube (Becton
Dickinson, Franklin Lakes, NJ) and mononuclear cells were isolated according to the
manufacturer’s instructions (Table 8). Lymphocytes from whole blood buffy coats were
purified by Ficoll-Paque centrifugation. The
cells were washed twice with PBS and 108
cells were lysed in 100 µl of RIPA buffer
containing 10 mM Tris-HCl, 150 mM
NaCl, 1% sodium deoxycholate, 1% Triton X-100, 0.1% sodium dodecyl sulpahte,
1 mmol/l phenylmethanesulfonyl fluoride,
1 mM sodium vanadate, 10 mg/ml aprotinin (Sigma). The cell lysate was centrifuged
for 15 min at 10 000 x g and the supernatant was stored at -20° C until analyzed.
4. SEPARATION OF MONONUCLEAR CELLS, GRANULOCYTES,
MONOCYTES, B- AND T-CELLS (I)
Mononuclear cells and granulocytes were
isolated from buffy coats by density gradient centrifugation. Four ml of blood was
mixed with 6 ml of PBS, layered over 10
ml Histopaque 1077 (Sigma) and spun at
600 x g for 30 min. The mononuclear cell
layer was isolated and washed twice in PBS.
Granulocytes were separated by a triple discontinuous gradient (Bhat et al., 1993) by
layering 2 ml each of Histopaque 1119 at
the bottom, Histopaque 1107 (two parts
Histopaque 1119 and one part Histopaque
1083) in the middle and Histopaque 1077
on top. Two ml blood was diluted with 4
25
Kristina Hotakainen
ml PBS and layered on top of the gradient.
The tubes were centrifuged for 30 min at
600 x g. The granulocyte cell layer was then
aspirated and washed twice with PBS. Remaining erythrocytes were hemolysed by
incubating the cell pellet with 4 ml distilled water for 10 s, after which 1 ml 5 x
PBS was added. The granulocytes were collected by centrifugation, lysed in 4 M
guanidinium-thiocyanate buffer containing
0.1 M ß-mercaptoethanol and stored at
-20° C.
Magnetic cell sorting (MACS) was used
to separate monocytes, B- and T-cells. Cells
from the mononuclear cell layer (7,6 x 107)
were resuspended at a concentration of 107
cells per 80 µl buffer (PBS, 2 mM EDTA,
0,5% BSA), and incubated with mouse
monoclonal antibodies conjugated to magnetic microbeads (Miltenyi Biotec, Sunnyvale, CA) to human CD3, CD14 or CD19
(20 µl per 107 cells). CD3 antibodies were
used for separation of T-lymphocytes, CD14
for monocytes and CD19 for B-lymphocytes. The labelled cells were washed twice
in the buffer and immediately processed
with a VarioMACS using an RS+ column
(Miltenyi Biotec). The column was prewashed with the same buffer. Positive selection was done according to the manufacturer’s instruction. Cells from the positive fraction were eluted and washed in the
buffer. The purity of the monocyte and Bcell fractions was determined on the basis
of morphology and expression of MHC II
antigens (Schröder et al., 1982).
Table 10. Samples of patients and controls. The
number of patients is given in parentheses when
different from the number of samples used.
Serum samples
TCC patients (68)
TCC patients (63)
RCC patients
Control patients with benign diseases
Healthy controls
Healthy controls
n Used in
45
66
177
14
84
26
II
III
IV
III
IV
III
Tissue samples; source
Placental tissue; normal delivery
TCC tissue; TCC patients (66)
Benign bladder
Patients with hematuria
dysuria
pollacisuria
recurrent lower UTI
1 I, II, III
96
III
6
4
5
6
III
III
III
III
56
72
26
14
II
III
III
III
68
84
14
15
8
3
1
1
1
1
1
1
1
1
1
1
3
II
III
II
III
II, III
II
III
II, III
II
II
II, III
II, III
II, III
II, III
II
II, III
II, III
Urine samples
Urine
TCC patients (68)
TCC patients (63)
Healthy controls
Control patients with benign diseases
Urinary cells
TCC patients (68)
TCC patients (63)
Healthy controls
Healthy controls
Patients with acute appendicitis
urinary calculi
Patient with urinary calculi
BPH
impotence
epididymitis
inflammatory pseudotumor
chronic pyelonephritis
cystitis
hematuria
cervical carcinoma
renal carcinoma
Patients with prostate carcinoma
5. ISOLATION OF RNA (I-III)
6. REMOVAL OF GENOMIC DNA (IIII)
RNA was isolated from cells pelleted by
centrifugation of urine (II, III) as well as
from mononuclear blood cells and leukocyte subsets (monocytes, B- and T-lymphocytes, granulocytes) and cultured cells (I)
according to Chomczynski and Sacchi
(Chomczynski & Sacchi, 1987) with Heavy
Phase Lock Gel tubes (5 Prime->3 Prime,
Inc., Boulder, CO, USA).
To diminish the content of genomic DNA
in the RNA preparations each sample was
subjected to DNase-treatment before RTPCR. Ten µg of total RNA was incubated
at 37° C for 30 min in a 50-µl reaction
mixture consisting of 1 x DNase buffer (40
mM Tris-HCl, pH 7.9, 10 mM NaCl, 6 mM
MgCl2, 10 mM CaCl2), 33 U of RNasin
(Promega, Madison, WI) and 2 U of RQ1
26
Material and Methods
A
1
3
E2
hCGβ mRNA
E3
B
LHβ mRNA
E3
E2
2
4
1+2
3+4
Outer sense
Outer antisense
Nested sense
Nested antisense
404 bp
292 bp
3
1
E1
Primer
1
2
3
4
2
4
1+2
3+4
Sequence
CTG TTG CTG CTG CTG AG
GCC TTT GAG GAA GAG GAG
ACC CTG GCT GTG GAG AAG G
TCA TCA CAG GTC AAG GGG TG
401 bp
292 bp
Nucleotides
50-66
436-453
128-146
401-420
Figure 1. Schematic representation of the locations of the primers used for hCGβ (a). The
primers are specific to the sequence of hCGβ mRNA, but they are also able to amplify LHβ
mRNA (b). Primer 1 is homologous to both hCGβ- and LHβ mRNA, and the dashed primers (2,
3, and 4) contain mismatches with respect to LHβ mRNA. E; exon.
RNase-free DNase (Promega, Madison,
WI). The DNase was denatured for 10 min
at 55° C. RNA was precipitated with 140
µl ethanol and 5µl 2 M sodium acetate and
dissolved in diethylpyrocarbonate-treated
water.
7. OLIGONUCLEOTIDE PRIMERS
Oligonucleotide primers used for RT-PCR
of hCGß mRNA were chosen from exon 2
of the hCGß5 gene and the antisense primers from exon 3 (Figure 1).
8. RT-PCR AND GEL ELECTROPHORESIS (I-III)
For RT, 1 µg of total RNA and 1 pmol of
the outer antisense primer were denatured
for 10 min at 70° C in a 12-µl volume of
diethylpyrocarbonate-treated water. The
denatured RNA was incubated in a 20-µl
reaction mixture (Lintula & Stenman, 1997)
with 200 U SuperscriptTM reverse transcriptase (Gibco BRL) at 42° C for 50 min.
The RT enzyme was denaturated for 15 min
at 70° C. Contamination of RNA samples
was excluded by subjecting each sample to
the RT reaction without RT before PCR.
In the PCR, 5 µl of the cDNA mixture
was amplified in a 50-µl reaction volume
in 1x PCR buffer as previously described
(Lintula & Stenman, 1997) initially at 95°
C for 5 min, 35 cycles at 95° C for 1 min,
50° C for 1 min. One µl of the first PCR
product was further amplified with the
nested primers in a 50-µl reaction volume
for 35 cycles with an annealing temperature of 56° C but otherwise the same conditions as in the first PCR. Fifteen µl of
the product was electrophoresed on a 1.5%
agarose gel and visualized by ethidium bromide staining. RNA isolated from placental tissue was used as a positive control for
hCGß and water as a negative control in all
experiments. The detection limit of the RTPCR was estimated by mixing the cultured
cancer cells with PBS or urine from a healthy
male to dilutions of 102-106 cells per liter
PBS or urine and with blood to give dilutions up to 1 tumor cell per 107 peripheral
blood leukocytes.
9. RESTRICTION ENZYME ANALYSIS AND SEQUENCING OF THE
PCR PRODUCTS (I-III)
The homology between the genes for LHß
and hCGß is 94% (Talmadge et al., 1984b).
There are three mismatches between the
nucleotide sequences of the outer antisense
primer used in reverse transcription and the
27
Kristina Hotakainen
mRNA sequence of LHß. However, all mismatches are located in the 5'-end of the
primer, and amplification of LHß cDNA
occurs with these primers (Figure 1). The
identity of the PCR products produced by
the nested primers was therefore confirmed
by Hae III restriction enzyme digestion and
sequencing of the product. The 292 bp PCR
product of hCGß, but not that of LHß
mRNA, has a restriction site for Hae III
producing fragments of 108 and 184 bp,
respectively. Sequencing of the PCR products was performed using the nested sense
primer with the ABI PrismTM Dye Terminator Cycle Sequencing Core Kit With
AmpliTaq DNA Polymerase (Perkin Elmer,
Foster City, CA).
10. DETERMINATION OF hCG,
hCGß, hCGßcf, LH AND LHß (I-IV)
Serum (II-IV), urine (II, III) and cell culture medium (I, II) were stored at -20° C
before assay of the proteins. HCG (II, III)
and hCGß (II-IV) in serum and urine, and
hCGßcf in urine (II, III) were determined
by ultra sensitive time-resolved immunofluorometric assays (IFMA) (Alfthan et al.,
1992a). Cell culture media were collected
at days 3, 5 and 7 of culture and concentrated 14-60-fold with Centricon-3 concentrators (Amicon, Beverly, MA). HCG and
hCGß as well as LH and LHß in culture
media and leukocyte extracts (I) were determined by IFMA. The detection limit of the
hCG assay was 0.5 IU/l (WHO 3rd IS, 75/
537), 0.5 pmol/l for hCGß, and 0.44 pmol/
l for hCGßcf (WHO 1st IRP, 75/551). To
correct the results in urine for varying urine
concentrations the results were divided by
the concentration of urinary creatinine (II,
III). The LHß assay (LHspec, Delfia, Wallac)
detects LH, LHß, and a fragment of LHß
whereas the LH assay detects only intact LH.
The detection limit of the LH assay was 0.12
IU/l and that of the LHß assay was 0.05 IU/
l. The assays were calibrated against the 2nd
WHO IS coded 80/552.
28
11. IMMUNOHISTOCHEMISTRY(III)
HCGß immunoreactivity was analyzed using two monoclonal antibodies (MAbs) to
hCGß; clones 9C11 (Alfthan et al., 1988)
and 6G5 (Louhimo et al., 2001). MAb 9C11
is specific for hCGß, whereas MAb 6G5 also
reacts with intact hCG (Louhimo et al.,
2001). Immunoperoxidase staining was
performed with the Vectastain Elite ABC
Kit (Vector Laboratories Inc., Burlingame,
CA, USA). Four-µm thick, freshly cut sections of formalin-fixed, paraffin-embedded
tissue specimens were deparaffinized in xylene and rehydrated by sequential incubation in graded ethanol/water solutions. The
deparaffinized tissue sections were pretreated with 0.5% trypsin (DIFCO laboratories, Detroit, MI, USA) for 30 min at 37
°C, and washed with PBS. To quench endogenous peroxidase activity, the sections
were treated with 0.3% hydrogen peroxide
in methanol for 30 min at room temperature and then blocked with normal rabbit
serum (dilution 1:67) for 15 min to reduce
non-specific binding. The primary antibodies diluted 1:10 000 in PBS containing
0.5% BSA (Sigma) and 0.1% sodium azide
(Merck, Darmstadt, Germany) were added
to the sections and incubated overnight at
room temperature. After rinsing, biotinylated anti-mouse immunoglobulin was
added (1:200) for 30 min to the sections
followed by peroxidase labelled ABC reagent for 30 min. The peroxidase reaction
was developed for 15 min with 3-amino-9ethyl-carbazole in acetate buffer, pH 5, containing 0.03% hydrogen peroxide. The sections were rinsed with water, counterstained
with haematoxylin for 35 s and rinsed with
water for 10 min. Paraffin sections of placental tissue were used as positive controls.
As a negative control a placental section was
stained by replacing the primary antibody
with normal rabbit or mouse serum. Positive staining was graded 0-3 according to
the estimated percentage of positive cells
(negative <5%; +5-25%; ++ 25-50%;
Material and Methods
+++50-100%). Two persons (S. N. and K.
H.) evaluated the staining independently.
12. STATISTICAL METHODS (I-IV)
Student’s paired t-test, the chi-squared test
for trend, Fisher’s exact test for correlation,
Kruskal-Wallis test and the MannWhitney U test as well as receiver operat-
ing characteristic (ROC) curve analysis
were used in statistical analyses. Survival
curves were plotted using the KaplanMeier method, and comparison of survival
rates was performed with the log-rank test.
The independence of hCGß as a predictor
of survival was analysed by multivariate
analysis using the Cox proportional hazard model.
29
Kristina Hotakainen
Results and discussion
1. EXPRESSION OF hCGß- AND LHß
mRNA IN PERIPHERAL BLOOD
CELLS (I)
1.3 Expression of hCG protein and
hCGß mRNA and protein in cultured
lymphocytes
1.1 Expression of hCGß- and LHß
mRNA in peripheral blood cells and
cell lines
Culture of lymphocytes alone or with all
mitogens except PRL induced hCGß
mRNA expression (Table 12). The PCR
products were identified with Hae III,
which digests the cDNA of hCGß but not
that of LHß. However, a weak expression
of hCGß may be obscured by an excess of
LHß mRNA, potentially explaining why
no hCGß mRNA was detected in cultured
lymphocytes stimulated with PRL. No hCG
protein was detected by IFMA in the control cultures without mitogens. PHA- and
ConA stimulated cells secreted hCGß (Figure 3), but intact hCG protein was not detected in any of the cultures.
Our results show that normal blood cells
express LH and that the expression is stimulated by PRL. HCGß expression is not detectable in unstimulated cells but it is induced by culture and certain mitogens. LHß
mRNA is expressed in all the nucleated
blood cell fractions studied, and, when
stimulated with PRL, leukocytes also produced intact LH protein at readily detectable levels. The protein production occurred
LHß mRNA was detected by RT-PCR in
the nucleated cells isolated from blood from
nonpregnant females and healthy males and
also in the isolated B- and T-cell fractions,
monocytes and granulocytes. HCGß mRNA
was not detected in any of these cell fractions (Table 11). LH, LHß, hCG or hCGß
protein were not detected by IFMA in extracts from PBL or whole blood buffy coats.
The Jurkat and K562 cell lines expressed
mRNA for both LHß and hCGß (Table 12).
1.2 LH- and LHß expression in cultured lymphocytes
The effect of mitogens on the expression of
mRNA and protein was studied in lymphocyte cultures. LHß mRNA expression was
detected by RT-PCR in lymphocytes cultured with and without mitogens (Table
12). PRL induced maximal mitogenic
stimulation and LH-production at a concentration of 100 ng/ml (Figure 2). The LH
concentrations were similar when measured
by the assays detecting only LH or both LH
and LHß indicating that the immunoreactivity consisted of intact LH. The other
mitogens did not induce significant production of LH protein, and in control cultures
without mitogen no LH immunoreactivity
was detected.
30
Table 11. LHβ- and hCGβ mRNA detected by RTPCR in separated peripheral blood leukocytes without
stimulation.
Cell type
mRNA expression
LHβ
B-cell
T-cell
Granulocyte
Monocyte
+
+
+
+
hCGβ
-
Results and Discussion
Table 12. LHβ- and hCGβ mRNA detected by RT-PCR in cultured lymphocytes and cell lines
Days of culture
Mitogen
PHA
ConA
PWM
PRL
MLC
No mitogen
5
0
3
LHβ hCGβ
+
+
+
+
+
+
-
LHβ hCGβ
+
+
+
+
+
+
+
+
+
+
(+)
LHβ hCGβ
+
+
+
+
+
+
+
+
+
+
+
nd
nd
nd
nd
nd
nd
nd
nd
nd
nd
7
LHβ hCGβ
+
+
+
+
+
+
+
+
+
+
-
Cell line
nd
nd
nd
nd
nd
HL-60
Jurkat
JAR
K562
U937
+
(+)
-
(+)
+
+
+
(+)= weak positive, nd= not determined
early (after 6 h, data not shown), i.e. before
a mitogenic response was observed. This
suggests that LH expression was directly
induced by PRL and that it did not require
a mitogenic effect. In contrast, production
of hCGß protein was induced by the mitogens and the concentrations in the culture
Concentration (mIU/l per 107 cells)
LH , LHβ
LH
120
*
***
****
***
100
***
**
80
60
40
20
0
14
28
70
100
Prolactin (ng/ml)
140
Figure 2. The effect of PRL on the production
of LH protein by cultured lymphocytes. LH, as
well as LHβ and a fragment of LHβ in the culture
medium were determined by IFMA.
**** p<0.0001; *** p<0.001; ** p<0.01; * p>0.02
medium increased with time of stimulation.
PRL has been regarded predominantly a Tcell mitogen (Clevenger et al., 1990;
Pellegrini et al., 1992), but other T-cell specific mitogens such as Con A and PHA did
not induce translation of the genes for the
LH-subunits. The secretion of LH-protein
was highest at a PRL-concentration of 100
ng/ml, which also induced the strongest
mitogenic response. This concentration is
similar to that in pregnant and lactating
women and 5- to 10-fold that in males and
nonpregnant females. However, the concentrations of LH in the culture medium are
low in comparison with those of hCG occurring in blood during pregnancy. Thus
the LH produced can be expected to play a
physiological role only at a paracrine or
autocrine level.
PRL is produced by lymphocytes and it
is thought to act as a growth factor for
lymphoproliferation in a paracrine or
autocrine way by inducing expression of
genes associated with lymphocyte activation
(Sabharwal et al., 1992a). PRL-receptors
have been demonstrated in both B- and Tcells (Pellegrini et al., 1992; Dardenne et
al., 1994; Russell et al., 1985) and both cell
types respond to PRL-stimulation (Lahat et
31
Kristina Hotakainen
0.3
hCGβ (pmol/l per 107 cells)
p=0.05
0.2
0.1
0
3 5 7
control
3 5 7
PHA
3 5 7
MLC
3 5 7
ConA
3 5 7
PWM
Days
Figure 3. The effect of mitogens on hCGβ production
in cultured lymphocytes. The proteins were
determined in the culture medium by IFMA at days
3, 5, and 7 of stimulation. In control cultures no
mitogen was added. The mean concentrations and
S.D. of parallel cultures are shown.
al., 1993). Luteinizing hormone-releasing
hormone (LHRH) is also thought to participate in lymphocyte activation (Batticane
et al., 1991; Azad et al., 1993), and expression of its gene is regulated by PRL in lymphocytes (Wilson et al., 1995). Expression
of the hCG/LH receptor gene has been demonstrated in T-lymphocytes from pregnant
women (Lin et al., 1995). In line with these
results a mitogenic LH-like protein has been
observed in thymic extracts, suggesting that
this glycoprotein would be secreted by thymocytes and act as an autocrine stimulator
of lymphoproliferation (Sabharwal et al.,
1992b).
1.4 Conclusions
Together with the earlier described expression of the LH-receptor gene in T-lymphocytes (Lin et al., 1995) the expression of LH
in leukocytes shown here suggests that LH
may exert an autocrine function in blood
cells, possibly as a result of an autocrine or
paracrine effect of PRL. The expression of
LHß mRNA appears to occur in all peripheral blood leukocytes. The concentrations
of intact LH and LHß were similar when
determined separately indicating that the
32
immunoreactivity detected consisted of intact LH. LH protein production occurred
consistently and over a wide range of physiological PRL-concentrations, which supports
the notion of a physiological role of this
hormone in lymphocytes.
The expression of hCGß was induced
by mechanisms other than those stimulating LH expression, and only the free ß subunit, but not intact hCG protein, could
be reliably detected. Expression of hCGß
mRNA was detected only after culture of
PBL. This is in accordance with results
from other studies showing that culturing and certain other stimuli lead to production of hCG protein by blood cells
(Goldstein et al., 1990; HarbourMcMenamin et al., 1986). It has been proposed that mRNA for genes transcribed
in ectopic tissues at very low levels can be
detected by RT-PCR if an excessive number of amplification rounds are used (Hu
& Chow, 2000; de Graaf et al., 1997). We
tested our RT-PCR with various numbers
of cycles (20-45) and found the optimal
result for hCGß mRNA at 35 cycles,
whereas LHß mRNA could be detected
also by 30 cycles. In addition, we showed
translation of these transcripts in leukocytes, demonstrating that the mRNA expression is not only a non-functional background expression. During pregnancy, cultured monocytes have been reported to secrete hCG, and this has been suggested to
be related to the immunotolerance associated with early pregnancy (Alexander et al.,
1998). Although LH and hCGß have no
known function in lymphocytes, the expression of LH appears to be a further example of the involvement of pituitary hormones in immune modulation. Because
hCGß mRNA expression was induced by
culture it is conceivable that disorders leading to lymphocyte activation may also induce hCGß expression. This may lead to
positive results for hCGß mRNA in blood
from patients without cancer. This is a
potential limitation for the use of hCGß
Results and Discussion
expression for detection of cancer cells especially in blood and possibly also in urine.
2. EXPRESSION OF hCGß IN BLADDER CANCER (II, III)
2.1 Immunohistochemical expression
of hCGß (III)
We detected positive staining for hCGß
equally often in sections from noninvasive
(28%) and invasive tumors (35%), and in
histologically benign samples (25%) from
TCC patients (Table 13). There was no statistically significant association between
tumor stage (p=0.6) or grade (p=0.75) and
tissue staining for hCG antigen. Positive
staining was observed in five of 26 (19%)
cases with previous instillation therapy and
in one of the 12 patients currently receiving instillations. The results were identical
with both antibodies. Furthermore, 10 of
21 (48%) samples from control tissues from
patients with benign conditions stained
positive. Two of these samples were from
control patients with hematuria and in both
cases the epithelium was oedematous and
inflamed; squamous metaplasia was observed in one case with dysuria, and in a
patient with recurrent lower urinary tract
infections (UTI). One of the positively staining samples was an inflammatory pseudotumor from a patient with recurrent UTI
(Table 14). Two positive samples from patients with dysuria and pollacisuria were
judged to be histologically normal.
To our knowledge, immunohistochemical expression of hCGß in benign bladder
epithelium has not been reported before, but
mRNA expression has been observed in
normal and malignant bladder epithelium
(Lazar et al., 1995) and benign transitional
cells in culture secrete hCGß into the culture medium (Iles et al., 1990b). Five of the
ten positively staining control tissues
showed signs of inflammation or squamous
metaplasia of the epithelium. Thus, it is
Table 13. Immunohistochemical expression of
hCGβ-antigen in various stages and grades as
well as nonmalignant samples from TCC-patients.
Stage
Tis
Ta
T1
T2
T3
T4
Not available
Histologically benign
tissue*
n
hCGβ-positive %
8
46
12
4
3
1
2
20
1
14
5
0
1
1
1
5
12
30
42
0
33
100
50
25
22
31
23
76
5
11
7
23
23
35
30
Grade
1
2
3
All
30
n= number of patients
* Samples of histologically benign bladder epithelium from patients
with cystoscopically verified TCC.
possible that inflammation and hyperplasia may induce hCGß expression in benign
transitional epithelium. Grade and stage of
bladder cancer have been found to correlate
with immunohistochemical staining of
hCGß in most (Bacchi et al., 1993; Campo
et al., 1989; Dirnhofer et al., 1998b; Jenkins
et al., 1990; Martin et al., 1989; Moutzouris
et al., 1993; Oliver et al., 1988; Özkardes et
al., 1991), but not all earlier studies (Smith
et al., 1994). We detected immunohistochemical expression of hCGß antigen in
30% of the TCC specimens but equally often in benign samples from TCC patients
(25%) and in samples from patients with
benign bladder diseases. The results were
identical with both antibodies, one of which
is specific for hCGß (Alfthan et al., 1988)
whereas the other one also reacts with intact hCG. Therefore the staining most likely
originates from hCGß expression.
Expression of hCGß has been associated
with malignant transformation, lack of differentiation of nontrophoblastic cells (Bellet
et al., 1997) and aggressive disease (Bacchi
et al., 1993; Marcillac et al., 1993; Martin
et al., 1989; Moutzouris et al., 1993). Furthermore, the growth of bladder cancer cell
33
Kristina Hotakainen
Table 14. Immunohistochemical expression of hCGβantigen in bladder epithelium of patients with benign
conditions.
Diagnosis
Dysuria
Hematuria
Pollacisuria
Recurrent lower UTI
All
n
hCGβ-positive
%
4
6
5
6
21
3
2
2
3
10
75
33
40
50
48
lines in vitro is stimulated by hCGß, and it
has been proposed that hCGß acts as a
paracrine or autocrine growth factor in epithelial bladder tumors (Dirnhofer et al.,
1998b; Gillott et al., 1996). The stimulating action of hCGß in bladder cancer cell
lines has been associated with an antiapoptotic effect interfering with normal cell
turn over (Butler et al., 2000). While these
findings suggest a role of hCGß in tumor
progression, the frequently observed expression of hCGß in benign bladder tissue appears to need another explanation. Interestingly, tissue expression of hCGß has also
been observed in some other non-malignant
epithelial tissues (Iles et al., 1990b; Louhimo
et al., 2001). Thus this phenomenon is less
cancer specific than earlier thought.
2.2 HCGß and hCG in serum of bladder cancer patients (II, III)
Serum hCGß was moderately elevated in 18
of 45 samples (40%) in study II, and in 19
of 66 samples (29%) in study III. The concentrations were not significantly associated
with tumor stage or grade in either study,
or with tissue expression of hCGß. Slightly
elevated values in relation to the age and
gender-specific reference values of hCG were
observed in 4 of 43 patients (9%) in study
II, and in 12 of 66 cases (18%) in study III.
There was no difference in the serum levels
between patients with and without instillation therapies. Values exceeding the upper reference limit of postmenopausal
women (15.5 pmol/l) occurred in 8 patients.
The concentrations of hCG, hCGß and
34
hCGßcf in serum and urine of bladder cancer patients are summarized in table 3,
original publication III.
Earlier studies have shown that elevated
serum levels of hCGß occur in up to 50%
of patients with metastasized bladder cancer (Iles et al., 1989; Dexeus et al., 1986),
but only rarely in early stage disease. We
determined hCGß by a highly sensitive assay and found elevated serum concentrations in one third of the cases including
30% (15 of 49) of those with superficial
disease. This is probably explained by the
fact that we specifically measured hCGß
rather than “total hCGß”, i.e. hCG and
hCGß together as has been done in most
earlier studies (reviewed by Iles & Butler,
1998). The upper reference limit for hCGß
is 2.1 pmol/l while that for hCG is 2.115.5 pmol/l depending on age and gender. A much higher cutoff was used in the
studies of Iles et al., i. e. 25 IU/l roughly
corresponding to 75 pmol/l. The sum of
hCGß and hCG did not exceed this level
in any of the sera in this study. Marcillac
and coworkers determined hCGß in serum
with a specific and sensitive technique.
With a cutoff of 100 ng/l (4.5 pmol/l) they
found elevated values in 18 of 38 patients
(47%) with TCC (Marcillac et al., 1992)
most of which had advanced disease
(Marcillac et al., 1993). These results suggest that specific determination of hCGß
provides better diagnostic accuracy than
measurement of “total hCGß”. However,
we observed elevated serum levels also of
the intact hCG heterodimer in 18% of the
cases. Although the levels were only moderately elevated, they were higher than in
any case in our control material. This suggests that some tumors produce intact
hCG, i.e. both α and ß subunits. This is
supported by the finding of mRNA for
both subunits in some TCC tumors (Lazar
et al., 1995). Furthermore, Iles and coworkers showed that the urinary concentrations of hCGα in patients with hCGßpositive TCCs are higher than in controls
Results and Discussion
and patients with hCGß-negative TCCs
(Iles et al., 1990)
A
p < 0.00001*
Elevated levels of hCGß in urine were observed in only 5 of 55 cases (9%) in study
II, and in 6 of 72 (8%) cases in study III. In
10 urine samples the creatinine concentration was below 4 mmol/l and in seven of
these hCGß concentration was below detection limit. The median concentrations
were higher in patients with invasive than
with noninvasive tumors (p=0.03) and in
cases with suspicious or malignant urinary
cytology (Papa IV-V) (p=0.027). A slightly
elevated value (4.6 pmol/l) was observed in
one of the control patients, who had a benign inflammatory pseudotumor, which
also stained positive in immunohistochemistry (Table 14).
Urinary concentrations of hCGßcf were
elevated in 4 TCC samples (II, III); two of
which were from invasive grade 3 tumors.
The median urinary hCGßcf concentrations
were significantly higher in patients with
invasive than noninvasive tumors
(p=0.037), in cases with suspicious or malignant cytology (p=0.002), and in samples
positive for hCGß mRNA (p=0.03). Urinary hCG was elevated in only one of 56
(2%) samples in study II and in two of 72
(3%) cases in study III. There was no difference in the urinary levels of hCGß and
hCGßcf between patients with and without a history of instillation therapies, but
urinary hCG was significantly higher in
patients with previous or current instillation therapies (1.6 pmol/l) than in those
without instillations (0.4 pmol/l, p=0.04).
Theoretically, hCGß in urine might be
expected to best reflect local secretion of
hCGß from the tumor into urine. However,
the urine concentrations of hCGß were less
often elevated than those in serum. This
could be explained by large variations in
urinary flow rate leading to a wider refer-
1
0.1
0.01
Benign
Noninvasive
Invasive
B
p < 0.00001*
U/S- hCGβ / mmol creatinine
2.3 HCGß, hCG and hCGßcf in urine
of bladder cancer patients (II, III)
U/S- hCGβ ratio
10
1
0.1
0.01
Benign
Noninvasive
Invasive
Figure 4. Urine to serum-hCGβ ratio in TCC patients
with benign histology, superficial and invasive tumors
at the time of sampling (a). Panel b shows the ratio
corrected for urinary creatinine concentration and
expressed per mmol creatinine in urine. Samples
with a urinary creatinine < 4 mmol/l were omitted.
The horizontal lines indicate the medians in each
category.*) Kruskal-Wallis test.
ence range for hCGß in urine than in serum (Alfthan, 1994) This effect is often
corrected for by dividing the value of urinary proteins with creatinine. We calculated
this ratio for urinary hCGß but it did not
improve the diagnostic accuracy. Similarly,
correction for creatinine did not either improve the correlation between the serum and
urine concentrations of luteinizing hormone
in a study on prepubertal children (Demir
et al., 1994). Although urinary flow rate
affects its protein concentration, division by
creatinine does not appear to be an optimal
method of correction. We used morning
urine, which usually is fairly concentrated,
35
Kristina Hotakainen
but in spite of this ten samples had a low
creatinine value (<4 mmol/l) and in seven
of these hCGß was not measurable.
Circulating hCG and hCGß are excreted
into urine, and this is normally the source
of hCGß urine concentrations. Therefore we
studied whether the ratio of urine to serum
hCGß would provide information on local
secretion of hCGß in the urinary bladder.
This ratio was significantly associated with
histology (Kruskal-Wallis test for benign,
noninvasive and invasive disease p<0.0001,
Figure 4) and differentiated between high
and low grade tumors (Kruskal-Wallis test
for grades 1 to 3 p<0.00001; Mann –
Whitney test for grade 1 versus grade 3
p=0.002 and grades 1 and 2 versus 3
p=0.009). In addition, the ratio correlated
with tissue expression of hCGß detected by
immunohistochemistry (p=0.019). This
suggests that hCGß is directly released from
the tumor into urine, whereas secretion from
the tumor into the large plasma volume has
a smaller impact on the concentrations in
circulation, which are affected by secretion
from various normal tissues.
In accordance with earlier results (Baltaci
et al., 1995; McLoughlin et al., 1991), the
correlation between serum levels of hCGß
and tissue expression was poor. Tissue expression did not either correlate with urinary hCGß. The ß-core fragment has been
demonstrated by immunohistochemistry in
TCC of the bladder (Dirnhofer et al., 1998b)
and thus hCGßcf might be released directly
from the tumor into urine. Urinary hCGßcf
correlated with the invasiveness of the tumor and positivity for hCGß mRNA in
urinary cells. However, elevated urine levels were rare and hCGßcf in urine was not
useful as a marker.
2.4 HCGß mRNA in urinary cells (II,
III)
In study II, hCGß mRNA was detected in
29 of the 68 (43%) samples of urinary cells
form TCC patients and in none of the
36
healthy controls (n=14). There was a highly
significant association between histologically verified TCC and hCGß mRNA in
urinary cells (p=0.0014). In study III 42 of
84 (50%) urinary cell samples were positive for hCGß mRNA. The positive cases
tended to have a higher urine to serum ratio of hCGß (p=0.088) and hCGßcf concentrations in urine than the negative ones
(p=0.03), but there was no correlation with
tumor stage or grade (Table 15), urinary
cytology, immunohistochemical hCGß expression and serum marker levels. Previous
(more than 2 months earlier) or current instillation therapies did not either affect the
result; 12 of 23 (52%) cases were positive.
All healthy controls were negative, but in
four of the 23 control patients (II, III) hCGß
mRNA was present in the urinary cells.
Three of these had a UTI and one had hematuria of unknown origin.
HCGß mRNA in urinary cells is strongly
associated with TCC. However, this was also
detected in some patients with benign diseases, i. e. infection or hematuria. We found
that hCGß mRNA expression may be
incuced in leukocytes. Therefore hematuria
and inflammation may be expected to cause
a positive result in RT-PCR of hCGß
mRNA in urinary cells. However, although
hematuria occurred in several patients with
TCC it was not consistently associated with
Table 15. HCGβ mRNA in urinary cells from TCC
patients according to tumor stage and grade.
Stage
n
hCGβ mRNA
%
Tis
Ta
T1
T2
T3
T4
Not available
8
41
13
4
2
1
15
5
18
8
2
0
1
8
62
44
62
50
0
100
53
Grade
1
2
3
All
21
26
22
84
7
16
10
42
33
62
45
50
Results and Discussion
3. HCGß EXPRESSION IN SERUM
OF RCC PATIENTS (IV)
The concentration of hCGß in serum was
elevated (>2 pmol/l) in 23% of the patients
with RCC and 20 of these (11%) had values > 4pmol/l. The median concentration
of hCGß in serum was 1.2 pmol/l (range
0.2 - 18 pmol/l), which was significantly
higher (p<0.0001) than in controls (median
0.4 pmol/l, range 0.2 - 1.3 pmol/l). The
serum concentrations of hCGß were not related to serum creatinine. There was no difference in hCGß levels between males and
females, different age groups, different RCC
types, aneuploid and diploid tumors, or
tumors with and without venous invasion.
Serum hCGß concentrations were not either significantly correlated with tumor
stage or grade (Figure 5).
Clinical stage and grade were highly predictive of disease specific survival
(p<0.0001 each) in univariate analysis. Patients with serum hCGß concentrations
above the median value (1.2 pmol/l) had
significantly shorter survival than those
with lower levels (p=0.0029) (Figure 6A).
A difference in survival time was observed
also among patients with metastasized tu-
Table 16. Factors independently associated with
decreased cumulative survival of patients with RCC
(RR = Relative risk).
Coefficient (β)
Grade
Stage
hCGβ
1.15
2.34
0.48
RR
p
=0.032
<0.0001
=0.022
3.2 (1.1-9.0)
10.3 (4.8-22.3)
1.6 (1.1-2.5)
mors (stage IV, Figure 6B) (p=0.06). When
serum hCGß concentration was compared
as quartiles there was no difference in disease specific survival between the two lowest quartiles or between quartiles 3 and 4
(p>0.6). In multivariate analysis using the
Cox regression model with age, gender, serum hCGß, nuclear grade and stage as input variables, stage, grade, and serum hCGß
concentration were independently associated with the disease specific survival (Table
16).
RCC is known for its unpredictable clinical behavior and within stages and grades
the clinical course is highly variable. Recurrence can occur many years after apparently
successful surgery and metastases may spontaneously regress after removal of the primary tumor (Golimbu et al., 1986b). Several biomolecular markers have been studied as potential markers for RCC, but to date
there is no specific clinically useful serum
marker. Of the many serum markers studied, only a few, i. e. VEGF, interleukin-10,
12.8
6.4
(Serum hCGβ (pmol/l)
a positive RT-PCR. It is possible that infections, instillation therapies, instrumentation and other conditions causing cellular atypia and inflammation also induce
hCGß mRNA expression in transitional
epithelium. The more frequent expression
in patients with TCC could be explained
by increased shedding of TCC cells from
cancerous epithelium. However, half of the
patients with a histologically verified tumor had a negative RT-PCR. This could be
associated with a variable recovery of tumor cells in urine, or to selective expression of hCGß. Different hCGß genes are
expressed in benign and malignant bladder
epithelium. Our primers were designed according to the sequence of the hCGß5 gene
which is expressed in TCC (Lazar et al.,
1995).
3.2
1.6
0.8
0.4
0.2
0.1
I
II
III
IV
Stage
Figure 5. Boxplot demonstrating preoperative
serum hCGβ concentrations in RCC patients with
various stages. The horizontal line indicates the
upper reference limit for serum hCGβ (2.1 pmol/l).
37
Kristina Hotakainen
A
B
1
1
hCGβ ≥1.2 pmol/l
0.8
0.6
0.4
0.2
Cumulative survival
Cumulative survival
hCGβ <1.2 pmol/l
hCGβ <1.2 pmol/l
0.8
hCGβ ≥1.2 pmol/l
0.6
0.4
Log-rank p-value =0.06
0.2
Log-rank p-value=0.0029
0
0
0
Cumulative survival
A
25
50
75 100 125
Time (months)
0
150 175 200
24
48
72
96
120
Time (months)
144
168
1
0.8
hCGβ
<1.2
pmol/l
hCGβ
≥1.2
pmol/l
0.6
0.4
0.2
Log-rank
p-value=0.0029
0
CA-125, and TATI are of potential prognostic value (Wittke et al., 1999; Grankvist
et al., 1997; Jacobsen et al., 2000; Paju et
al., 2001). There is little data on hCGß expression in renal tumours. Dexeus and coworkers observed increased hCGß concentrations in serum of 10% of the patients,
and changes in the levels correlated with the
clinical course (Dexeus et al., 1991). We
found elevated concentrations in 23% of the
patients, and the median concentrations
were significantly higher than in controls
(p<0.0001). Patients with serum hCGß levels above the median value (1.2 pmol/l) had
significantly shorter survival than those with
lower levels (p<0.0029), and in multivariate analysis serum hCGß, tumour stage and
grade were independent prognostic variables. The fairly low frequency (23%) of elevated serum hCGß suggest that this marker
is expressed by only part of the RCCs. However, because normal levels between 1.2 and
2.1 pmol/l were associated with adverse
prognosis, it is possible that part of the
hCGß in these patients is derived from the
0
38
25
50
tumor. The absence of a correlation with
stage indicates that elevated levels are not
only a result of tumor burden but that hCGß
expression characterizes a subgroup of tumors. The lack of significant correlation
with established prognostic variables such
as grade and stage demonstrates the independent character of this marker.
The clinical utility of hCGß in the management of RCC is limited by the infrequent expression. About one fourth of the
patients had elevated levels and in half of
these the elevation was substantial. However, a prognostic value was observed even
with normal levels exceeding the median
value. Furthermore, the prognostic significance of hCGß in serum was seen not only
in patients with metastatic disease, but also
in earlier stages (stages I-III). Thus it appears worthwhile to study whether hCGß
could be used in identifying a subgroup of
patients with increased risk of aggressive
disease and as an aid in the selection of
therapy, as well as in monitoring of the disease after primary therapy.
75
Time
100
125
(months)
150
175
200
Summary
Methods for detection of cancer cells in
blood and body fluids have been extensively
studied in attempts to evaluate tumor
spread. The presence of cancer cells in circulation reflects the metastatic potential of
the tumor, although it is not necessarily a
sign of metastatic disease. RT-PCR is a
highly sensitive method of detecting cancer cells expressing a tumor- or tissue specific mRNA. With the best methods a
single tumor cell may be detected among
107 leukocytes. However, a prerequisite is
that peripheral blood cells do not express
the mRNA used as a marker of cancer cells.
Recently expression of several genes thought
to be specific for other organs has been detected in blood cells, and leukocytes have
the potential of producing many peptide
hormones and receptors for these. HCG is a
glycoprotein hormone consisting of two
subunits, α and ß. It is produced at high
concentrations by placental trophoblasts to
maintain pregnancy. Isolated low level expression of hCGß commonly occurs in malignant tumors and this is associated with
agressive disease.
We studied whether hCGß mRNA could
be used as a marker of cancer cells in blood
and urine. To confirm the specificity of the
method we first studied whether hCGß
mRNA is expressed by peripheral blood
cells. HCGß mRNA expression was detected by RT-PCR in cultured blood cells,
but not in isolated peripheral blood leukocytes. HCGß mRNA expression occurred
in the urinary cells from half of the patients
with bladder cancer, and this was strongly
associated with histological evidence of cancer. However, there was no association with
stage or grade of the disease. Urine concentrations of hCGß were elevated in less than
10% of the cases, while the serum concentrations of hCGß and of intact hCG were
elevated in 30-40% and 10-20%, respectively. However, the ratio of urine to serum
concentration of hCGß protein was significantly associated with both stage and grade
of the tumor, and also with immunohistochemical staining of hCGß in tumor tissue.
Immunohistochemical expression of
hCGß was observed in approximately one
third of the tumors, and this was not associated with stage and grade. We also detected hCGß protein in benign transitional
epithelium in 30% of the cases. In most of
these the expression was associated with
inflammation or squamous metaplasia.
In previous studies, the serum concentrations of hCGß have been found to have
prognostic value in bladder cancer, ovarian
cancer, cancer of the oral cavity, oropharynx and colon. The prognostic significance
of hCGß expression in bladder cancer was
not studied, but the strong correlation between urine to serum ratio of hCGß and
stage and grade of the disease suggest an
association between hCGß expression and
advanced and high grade tumors.
Because hCGß appears to be a universal
serum marker for aggressive cancers we studied the prognostic value of the preoperative
serum concentrations of hCGß in patients
with renal cell carcinoma (RCC). Serum
hCGß was elevated in 23% of the patients.
In addition to stage and grade, serum hCGß
was an independent prognostic marker, and
normal levels exceeding the median were
39
Kristina Hotakainen
also of prognostic value. In preliminary studies we have found that hCGß can be detected
by immunohistochemistry in approximately
15% of conventional RCCs and this is associated with survival.
Our results add further support to pre-
40
vious studies linking hCGß to a potentially
aggressive subgroup of tumors. A possible
explanation for this association is the recently described anti-apoptotic effect of
hCGß, but the detailed mechanism remains to be investigated.
Conclusions
1. HCGß expression was detected in cultured peripheral blood lymphocytes but not in
unstimulated cells. Because hCGß mRNA expression was induced by culture, disorders
leading to lymphocyte activation may also induce hCGß expression giving positive results for hCGß mRNA in blood from patients without cancer. This is a potential limitation for the use of hCGß expression for detection of cancer cells in blood and possibly also
in urine.
2. HCGß mRNA expression was detected in the urinary cells of 50% of the patients with
bladder cancer but not in healthy controls. There was no association with stage or grade of
the disease. The positive results in some benign diseases suggest that infections, instrumentation and other conditions causing cellular atypia and inflammation also induce hCGß
mRNA expression in transitional epithelium.
3. Immunohistochemical expression of hCGß in bladder cancer was not associated with
stage and grade of the tumor. The positivity in benign bladder tissues shows that the
phenomenon is not restricted to malignant tumors but inflammation and metaplasia may
also induce expression. While previous studies suggest a role of hCGß in tumor progression, these findings suggest that hCGß expression also is involved in growth stimulation.
4. Urinary hCGß and hCGßcf correlated with urinary cytology and the ratio of urine to
serum concentration of hCGß protein was significantly associated with both stage, grade,
and immunohistochemical staining. This suggests that hCGß is secreted into urine by
many bladder tumors and supports the previously reported association between hCGß
expression and advanced and high grade tumors.
5. Serum hCGß was elevated in 23% of the RCC patients and levels above the median
concentration were independently associated with shorter survival. The absence of a correlation with stage indicates that elevated levels are not only a result of tumor burden. The
fairly small proportion of RCC patients with elevated hCGß suggests that hCGß is not
expressed by all tumors, but the expression identifies a subgroup of RCCs with unfavourable
prognosis, supporting previous studies linking hCGß to aggressive tumors.
41
Kristina Hotakainen
Acknowledgements
This study was carried out at the Department of Clinical Chemistry in the University of Helsinki. I wish to express my sincere thanks to Professor Ulf-Håkan
Stenman, Head of the Department, (who is
fondly known as Uffen) for placing excellent working facilities at my disposal. I am
also deeply grateful to Uffen for supervising this work. His enthusiasm for research
and his profound knowledge of science are
admirable. His never-ending encouragement and interest in my work have been
invaluable. Despite numerous other ongoing projects and a tight schedule, Uffen has
always been ready to comment on my latest findings and to give clues to help solve
any problems. His kind personality, skill
and positive attitude towards his students
and life in general has made collaboration
with him a pleasure.
Docent Martti Nurmi and Professor Kim
Pettersson were the official reviewers of this
thesis. I wish to thank them for their thorough review and constructive criticism and
comments.
I am grateful for having had the opportunity to co-operate with Caj Haglund,
MD, PhD, Professor Börje Ljungberg, Professor Jim Schröder, Stig Nordling, MD,
PhD, Erkki Rintala, MD, PhD, and Ossi
Lindell, MD, PhD. I wish to thank my coauthors Susanna Lintula, MSc, Henrik
Alfthan, PhD, Annukka Paju, PhD, Jakob
Stenman, MD, Martina Serlachius, MSc and
Torgny Rasmuson, MD, PhD. Their participation has been most valuable.
I also wish to thank Mrs Sanna Kihlberg,
Mrs Anne Ahmanheimo, Mrs Taina
Grönholm, Ms Maarit Leinimaa, Mrs
42
Kristiina Nokelainen, Mrs Elina Laitinen
and Mrs Sari Nieminen for their friendship
and technical assistance throughout the
years. I also thank Mr Oso Rissanen for solving almost all the technical problems and
Leena Vaara for her help in preparing slides.
I am grateful to Jakob Stenman who took
me to Uffen’s office during my years of
medical studies to learn what research is all
about; the consequences are now evident. I
thank Susanna Lintula who introduced me
to scientific work; her support and friendship have been most valuable, and Annukka
Paju who has been an encouraging friend
both inside and outside the laboratory. I am
grateful to Doctors Jari Leinonen, Patrik
Finne, and Henrik Alfthan for their patience
with my questions about chemistry, statistics and everything else. I am indebted to
Heini Lassus for her thorough guidance in
the layout of this thesis. I also warmly thank
all my friends and colleagues in the laboratory who have contributed to the good
working atmosphere. Without you the work
would have been lacking many of the most
pleasant moments.
I am grateful for the financial support
from Finska Läkaresällskapet, the Foundation of K. Albin Johansson, the Helsinki
University Central Hospital Research Funds
and the Foundation of Else and Wilhelm
Stockmann.
Thanks go to all my friends and relatives
outside the laboratory who have provided
me with many joyful moments. I thank
Ruut and Olli for their company and enthusiasm in sports activities, and their
charming kids for being good examples and
friends to our son. My parents-in-law Aila
Acknowledgements
and Mauri are thanked for always being
available for baby-sitting and offering a
helping hand when needed. My heartfelt
thanks are directed to my mother Vuokko
and my sister Johanna for their constant love
and encouragement throughout the years.
Without their support and company in daily
matters from child-care to shopping, this
work would not have been possible.
I thank Toivo for his patience with our
son, a frequent visitor in mother’s and
Toivo’s home, and his interest in my work.
I devote my warmest thoughts to my
husband Markus. Your patience and encouragement towards my work and your love
has carried me through these years. Together
with our sunshine, Josia, you have brought
much happiness to my life.
Helsinki, April 2002
43
Kristina Hotakainen
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