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Cytogenetic and Molecular Cytogenetic Studies of Uroepithelial Carcinomas

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CYTOGENETIC AND MOLECULAR CYTOGENETIC STUDIES OF

UROEPITHELIAL CARCINOMAS

IMAD MOHAMED FADL-ELMULA

DEPARTMENT OF CLINICAL GENETICS

LUND UNIVERSITY HOSPITAL
LUND 2001

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To the memory of my father

© 2001 Imad Mohamed Fadl-Elmula

ISBN 91-628-4739-2
Lund, Sweden 2001

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PREFACE
One in three persons develops cancer at some time during their lives, and one in four dies
from this disease. Today, it is widely accepted that cancer arises through a multistep
accumulation of inherited and/or acquired mutations of the genetic material leading to
clonal selection of neoplastically transformed cells showing unrestrained proliferation and
the ability to infiltrate locally and to set up distant metastases. Many of the genetic
alterations acquired by neoplastic cells are visible as chromosomal aberrations which are
clearly nonrandom in distribution and sometimes occur in highly specific patterns (Heim &
Mitelman, 1995). These chromosome aberrations are therefore highly informative with
regard to the pathogenetic events of carcinogenesis, and their identification and
classification have proved to be of great diagnostic and prognostic value for patients with
malignant disorders (Heim & Mitelman, 1995; Sandberg & Berger, 1994).

Uroepithelial cancer is a common disease, presently ranking as the eleventh most

frequent cancer worldwide. Although the urinary system is a relatively simple one, its
malignancies are highly heterogeneous both with regard to their natural history and their
clinical, histologic, and geographic characteristics. As for most cancers and other complex
diseases, the attempts to find answers as to their etiology and pathogenesis often lead but to
more questions. It seems a never-ending process in which a comprehensive understanding
of uroepithelial cancer probably depends on a better understanding of both normal
proliferation and differentiation control and the phenomenon of cancer in general.

The aim of the present study was to characterize the acquired genetic alterations in
different etiologic and histologic types of uroepithelial carcinomas, and to study the clonal
composition of synchronous and metachronous multifocal uroepithelial tumors. The thesis
is based on 7 articles and divided into three chapters. The first chapter provides an
introductory background to the cytogenetic and genetic basis of human neoplasia. The
second chapter gives an overview of clinical, pathologic, and genetic characteristics of
uroepithelial carcinomas. In the third chapter, finally, the findings of the present study are
summarized and an attempt is made to integrate them into a broader clinical context.

Lund, May 2001

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PREFACE...........................................................................................................................................3
LIST OF ARTICLES.........................................................................................................................5
1 INTRODUCTION...........................................................................................................................6
1.1 THE GENETIC BASIS OF HUMAN CANCER................................................................................ 6
1.1.1 The somatic mutation theory of cancer...............................................................................6
1.1.2 The origin of mutations .......................................................................................................6
1.1.3 Multistep carcinogenesis.....................................................................................................7
1.2 HUMAN CYTOGENETICS................................................................................................................. 7
1.2.1 Historical background ........................................................................................................7
1.2.2. Nomenclature .....................................................................................................................8
1.3 CANCER CYTOGENETICS ............................................................................................................. 12
1.3.1 Nonrandom chromosome aberrations in cancer ..............................................................12
1.4 MOLECULAR CYTOGENETICS.................................................................................................... 14
1.4.1 Fluorescence in situ hybridization (FISH)........................................................................14
1.4.2 Comparative genomic hybridization (CGH).....................................................................14
1.4.3 Multicolor FISH................................................................................................................14
1.5 MOLECULAR CONSEQUENCES OF CHROMOSOMAL ABERRATIONS............................ 15
1.5.1 Neoplasia-associated genes ..............................................................................................16
2 UROEPITHELIAL CARCINOMAS..........................................................................................18
2.1 CLINICAL ASPECTS ........................................................................................................................ 18
2.1.1 Epidemiology ....................................................................................................................18
2.1.2 Etiology and risk factors ...................................................................................................18
2.1.3. Histopathology .................................................................................................................20
2.1.4. Natural history .................................................................................................................21
2.1.5. Diagnosis .........................................................................................................................23
2.1.6 Treatment ..........................................................................................................................24
2.2 GENETIC ASPECTS OF UROEPITHELIAL CANCER............................................................... 25
2.2.1 Cytogenetics ......................................................................................................................25
2.2.2 Molecular genetics............................................................................................................29
3 THE PRESENT STUDY ..............................................................................................................31
3.1 AIMS..................................................................................................................................................... 31
3.2 MATERIALS AND METHODS......................................................................................................... 32
3.2.1 Materials ...........................................................................................................................32
3.2.2 Methods.............................................................................................................................32
3.3 RESULTS AND DISCUSSION .......................................................................................................... 35
3.3.1 Transitional cell carcinoma of the bladder.......................................................................35
3.3.2 Upper urinary tract carcinomas .......................................................................................39
3.3.3 Post-radiation uroepithelial carcinomas ..........................................................................40
3.3.4 Multifocal uroepithelial carcinomas.................................................................................40
3.3.5 Squamous cell carcinoma of the bladder..........................................................................41
3.3.6 Bilharzia-associated bladder lesions ................................................................................41
3.4 CONCLUSIONS.................................................................................................................................. 43
SAMMANFATTNING PÅ SVENSKA..........................................................................................44
ACKNOWLEDGMENTS ...............................................................................................................46
REFERENCES.................................................................................................................................47
APPENDIX .......................................................................................................................................55

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LIST OF ARTICLES
This thesis is based on the following articles, which will be referred to in the text by
their Roman numerals:

I. Fadl-Elmula I, Gorunova L, Lundgren R, Mandahl N, Forsby N, Mitelman F, and

Heim S (1998). Chromosomal abnormalities in two bladder carcinomas with
secondary squamous cell differentiation. Cancer Genet Cytogenet 102:125-130.

II. Fadl-Elmula I, Bonaldi L, Gorunova L, Mandahl N, Elfving P, and Heim S (1998).

Cytogenetic heterogeneity in a second primary radiation-induced bladder carcinoma:
Ten karyotypically unrelated clones. Cancer Genet Cytogenet 105:134-137.

III. Fadl-Elmula I, Gorunova L, Mandahl N, Elfving P, Lundgren R, Mitelman F, and

Heim S (1999). Cytogenetic monoclonality in multifocal uroepithelial carcinomas:
Evidence of intraluminal tumor seeding. Br J Cancer 81:6-12.

IV. Fadl-Elmula I, Gorunova L, Mandahl N, Elfving E, Radomak C, Mitelman F, and

Heim S (1999). Cytogenetic analysis of upper urinary tract transitional cell
carcinomas. Cancer Genet Cytogenet 115:123-127.

V. Fadl-Elmula I, Gorunova L, Mandahl N, Elfving P, Lundgren R, Mitelman F, and

Heim S (2000). Karyotypic characterization of urinary bladder transitional cell
carcinomas. Genes Chromosomes Cancer 29 (3):256-265.

VI. Fadl-Elmula I, Kytölä S, Pan Y, Weng-Onn L, DeRienzo G, Forsberg L, Mandahl

N,Gorunova L, Bergerheim U, Heim S, and Larsson C (2001). Characterization of
chromosomal abnormalities in uroepithelial carcinomas by G-banding, spectral
karyotyping, and FISH analysis. In J Cancer. In press.

VII. Fadl-Elmula I, Kytölä S, El Leithy M, Abdel-Hameed M, Mandahl N, El Agib A,

Ibrahim M, Larsson C, and Heim S (2001). Chromosomal aberrations in benign and
malignant bilharzia-associated bladder lesions analyzed by comparative genomic
hybridization. Submitted.

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1 INTRODUCTION
1.1 THE GENETIC BASIS OF HUMAN CANCER
The description of cancer goes back to many ancient civilizations, including those of Asia, South
America, and the Middle East. The earliest records of cancer, from 2500 BC, come from the
physicians of ancient Egypt, who in seven Hieroglyphic papyri gave detailed descriptions of
surgical, mechanical, and magical treatments of several forms of this disease. The oldest known
cases of cancer (osteosarcomas) have been verified in Egyptian mummies that died 4000 years
earlier still.
The first known statement on cancer etiology came from Hippocrates (~400 BC) who
claimed that the disease stemmed from an imbalance between the black bile (from the spleen) and
the other three bodily fluids; blood, phlegm, and yellow bile. This old humoral theory was largely
discarded when new optical instruments were invented paving the way for the cellular theories of
disease that have dominated much of pathogenetic thinking at least since the mid 1800s. The first
real scientific observation on cancer etiology was made in 1775 when Percival Pott, an English
physician, described a high incidence of cancer of the scrotum in men working as chimney sweeps.
1.1.1 THE SOMATIC MUTATION THEORY OF CANCER
In the late 19th century, genetic alterations were first observed as mitotic abnormalities in
histological preparations of tumors (Arnold, 1879). Theodor Boveri observed that multipolar
mitoses in sea urchin eggs resulted in abnormal chromosome numbers followed by abnormal
development of the larvae. As cancer cells, too, often display multipolar divisions, Boveri, in what
would become known as the somatic mutation theory of cancer, suggested that the chromosomal
abnormalities were the cause of this disease (Boveri, 1914; Burnet, 1974). According to this theory,
acquired genetic changes bring about the malignant transformation of target cells. The first real
evidence in favor of Boveri´s hypothesis had to wait until 1960, when the first consistent
chromosome abnormality in human neoplasia, the Philadelphia chromosome in chronic myeloid
leukemia, was identified (Nowell & Hungerford, 1960). Later support for the somatic mutation
theory of cancer has come from a very large number of studies showing that many cancers have at
least one distinctive, abnormal chromosome never present in normal cells; that carcinogens can
cause tumors in both humans and animals; that cancer can be inherited; and that many tumors are of
monoclonal origin.
The recent development of molecular techniques have led to the disclosure of the DNA-
level consequences of many cancer-specific chromosomal changes and has also revealed additional
submicroscopic genetic changes in human neoplasms (Bishop, 1991; Heim & Mitelman, 1989;
Rabbitts, 1994; Weinberg, 1989a). In all sporadic tumors investigated, the mutations are present in
the individual’s cancer cells only. Only in the 1% of human cancer which is part of a hereditary
cancer syndrome, can a predisposing constitutional mutation be found in neoplastic and non-
neoplastic cells alike (Fearon, 1997).
1.1.2 THE ORIGIN OF MUTATIONS
The hallmark of cancer is deranged growth control, continuous cell proliferation and disturbed
apoptotic weeding, eventually leading to expansion of one or more clones of neoplastically
transformed cells. The primary cause of such transformation is the accumulation of genetic
mutations by relevant genes and chromosomes in the target cells. A mutation may be defined as an
acquired, stable alteration in the normal DNA sequence or in the amount of DNA causing harmful,
beneficial or neutral effects on the individual’s health. Mutations may also be broadly divided into
length mutations with gain or loss of genetic material, and point mutations with alteration of the
genetic code without gain or loss of genetic material. Most carcinogens, be they chemical, physical
or biological, be they exogenous or endogenous, are mutagens capable of inducing the said changes
in somatic cells (Mrtini, 1989).

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1.1.3 MULTISTEP CARCINOGENESIS
More than half a century ago, Berenblum and Shublik (1949) appreciated that carcinogenesis is, at
least, a two-stage process. Further evidence in support of the view that carcinogenesis consists of
multiple steps was provided by Armitag and Doll (1954). Using the age/incidence curves of 17
common types of cancer, they concluded that carcinogenesis had to proceed via at least 6-7 steps
(Figure 1.1). These indirect studies could not identify the exact number and nature of pathogenetic
events behind the malignant transformation in individual human tumors, however.
Although it is evident that multiple changes are necessary for the development of most
tumors and that the genetic alterations of the neoplastic cells often occur in preferred sequences, it
is not completely clear whether the total sum of changes or their sequential order is critical in
determining the tumor’s biological properties. Most evidence suggests, however, that it is the
accumulation of events that is important in both tumor initiation and progression rather than the
sequential order (Weinberg, 1989b). The most famous example comes from colon cancer in which a
specific series of genetic alterations appears to occur during the progression from benign adenoma
to carcinoma (Fearon & Vogelstein, 1990). In this sequence, mutation of the familial adenomatous
polyposis gene on chromosome arm 5q is an early event, followed by mutation of the RASK
oncogene and loss of the tumor suppressor genes (TSGs) DCC and TP53 from chromosome arms
18q and 17p, respectively.
The sequential, multistep nature of the development of human spontaneous cancers has its
parallel in experimental cancers in animals, e.g., skin cancer in mice, in which carcinogenesis can
be divided into different phases corresponding to initiation, promotion, progression, and metastasis
(Goodfellow, 1994) (Figure 1.1). The initiation step is believed to be induced by a genotoxic agent
such as radiation or a chemical carcinogen. At this stage, the cell nevertheless retains its normal
phenotype in spite of the DNA alteration. Further mutations affect genes responsible for the control
of cell growth and lead to the appearance of clones possessing the capacity to progress. Additional
changes permit the outgrowth of clones with metastatic ability.
1.2 HUMAN CYTOGENETICS
1.2.1 HISTORICAL BACKGROUND
More than a century has elapsed since human chromosomes first evoked scientific interest
(Fleming, 1882). The term chromosome was introduced by Waldeyer (1888) to describe the visible
structures that separate during mitosis (Miller & Therman, 2001). Painter (1921) was able to
demonstrate that human females had an XX and human males an XY chromosome complement.
Later methodological improvement in tissue culturing, the use of colchicine as a mitotic inhibitor of
mammalian cells, and the discovery that hypotonic treatment greatly facilitated the spreading of
metaphase chromosomes had opened the door for a crucial breakthrough when Tjio and Levan
(1956) reported that the correct number of normal human chromosomes was 46 and not 48. Soon
afterwards, several numerical chromosome aberrations in humans corresponding to Down’s
syndrome (Lejeune et al., 1959), Turner’s syndrome (Ford et al., 1959), and Klinefelter’s syndrome
were described (Jacobs et al., 1959), and also many more subtle but rare constitutional chromosome
abnormalities were described in the following years. In 1960, Nowell and Hungerford described the
Philadelphia chromosome (Ph) in bone marrow and blood cells from patients with chronic myeloid
leukemia (CML), the first consistent karyotypic abnormality detected in a human neoplastic
disorder, thus ushering in the singularly fertile modern era of cancer cytogenetics that continues to
this day. All these early studies of human chromosomes were limited to a relatively crude
evaluation of chromosome size and number until chromosome banding techniques were introduced
around 1970 allowing the identification of each chromosome by its unique series of alternating dark
and light bands (Caspersson et al., 1970a, b). Shortly afterwards, Rowley (1973) showed that the
Philadelphia chromosome in chronic myeloid leukemia arose through a translocation between
chromosomes 9 and 22. Since then, cytogenetic information on some 37.000 human neoplasms with
abnormal karyotypes has been accumulated (Mitelman et al., 2000).

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 Looking back on the history of human cytogenetics, Hsu (1981) divided it into four eras: the
dark ages before 1952, the hypotonic era from 1952 to 1958, the trisomy period that lasted for a
decade from 1959 to 1969, and finally the banding era that began in 1970. The recent crossbreed
between conventional banding cytogenetics and molecular techniques has given birth to various
important molecular cytogenetic screening techniques such as comparative genomic hybridization
(CGH) (Kallioniemi et al., 1992), spectral karyotyping (SKY) (Schröck et al., 1996), and multiplex
FISH (M-FISH) (Speicher et al., 1996), thus opening the door to what could be called the color
chromosomes era.

1.2.2. NOMENCLATURE
Chromosomes are named so because of their ability to take up certain stains, as in Greek "chromos"
means color and "soma" means body. The genome of a human diploid cell contains 3x109
nucleotide pairs arranged in 46 chromosomes, 22 pairs of autosomes numbered 1-22 and the sex
chromosomes which are referred to as X and Y (Tjio & Levan, 1956). The centromere divides each
chromosome into a short (p) and a long (q) arm. Chromosomes are classified according to their size,
the location of the centromere, and the banding pattern along each arm, which is a unique pattern of
light and dark transverse bands that are numbered from the centromere outward (Figures 1.2 and 1.3).

Chromosomal aberrations are only detectable when alterations involve large parts of the
genome, more than approximately 4 million base pairs (0.13% of the genome). If we consider the
distance between London and New York equal to the length of the haploid human DNA, then 4
million base would be equivalent to 8 km. Chromosomal aberrations observed in neoplastic cells
are of two main types; numerical changes, i.e., gain or loss of whole chromosomes, and structural
aberrations, which may be balanced (no resulting loss or gain of genetic material), or unbalanced
(with loss or gain of genetic material) (Table 1.1).

Table 1.1 Abbreviations and descriptions of the most common chromosome abnormalities

Rearrangement Abbreviation Description

Addition add Addition of unknown material to a chromosome
Deletion del Interstitial or terminal loss of chromosomal material
Structurally rearranged chromosome resulting from more than one
Derivative der
change within a single, two, or even more chromosomes
Double minute dmin Multiple copies of acentric chromosomal material
Duplication dup Duplication of chromosomal segment
A chromosomal segment has moved into an interstitial position
Insertion ins
within the same or another chromosome
Inversion inv A chromosomal segment has rotated 180 degrees
Isochromosome i Mirror image chromosome with two identical arms
Marker mar Rearranged chromosome in which no part can be identifie
Monosomy - Loss of one chromosome copy
Translocation t Transfer of material between two or more chromosomes
Ring r Break and fusion of the two chromosome arms
Trisomy + Gain of one chromosome copy

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 Normal

Initiation

Normal phenotype

Promotion

Premalignant cell

Promotion

Expansion

Progression

Malignant cells

Expansion

Metastasis

Figure 1.1 Schematic model of multistep carcinogenesis view

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 10
Figure 1.2 Normal G-banded human male karyotype
 6
5 6
5
4 5
3 3 4 4
2 3
2 3 2 2
2
1 1
1
6
2 6
5 4
2 4 5
1 1 3 1 3 1 5 1 4
2 2 4
3
1 2 1 3 3
1 21 21
1 1
1 1 2
1 1 21 1
2 2 1
1 3 3 3 2
1 4 1 3
1 1
2 1 2 4
2 3 2
3 3 5
4 2 4 4
2 2 5 1
5 3 2 5 6
4 2 2
1 6 7 3
3 1 8
7 1
2 2 8
9 1
1 3 3 3 2
4 2
3
4
5
3 3 2
3
3
4
6 4 5
4 5
1
7
4 5
2
5
4
3 2
2 2 5
2 1 3 4 5
2 2 3 4
5 2 2 3 3
1 1 1 1 4
1 4 1 2 3 1 2
3 1 2 3 2
1 2 2 1 1 2 1 1
1 1 1
2
1 1
1
1 1 1 1 21
3 1 1 1 2 2
1 4 1 2 1 3
3 1
5 3 1 3

11
4
6 1 5
2 1 1 4
1 2 2
2 1 1
2 2 2
2 3
2 2 2
1 1 4 2 3 2 3
2 3 3
2
4 3 2 3 3 5 4
3 5 4
4 4
5 4 6
6 5
6 11 12
7
8 9 10
6 7
3 3 3
1 2 3 1 2 1 2
1 1 2 3 1 1
1 1 2 1
1 1 1
2 1 1 1
1 1 2 1
1 3 1 2 2 1 2 1
1 3 1 2
4 3 4 3
5 1
1
2 2 2 1
1 1 3
2 2 2 2 3
4 2
2 3 2 4 2
5
4 2 3 3
1 4
16 17
3 2
3
4
3
1
2
5
6 18
13 14 15
2
2
1
3
1 3 1 2 3 3 1 1
1 2
2
1 1 1 1 2
1
1 1 1 1 1 1
2 1 1 1 2
1 2 1 3
1 2
3
3
2 1 1
2 1
3 1
19 20 21
2
2
22
3
2 4
5
6 Y
7
8
Figure 1.3 Idiogram of human G-banded chromosomes X
International guidelines exist for the description of karyotypes. The 1985 version of the
International System for Human Cytogenetic Nomenclature (ISCN) (ISCN, 1985) was followed in
1991 by the Supplement Guidelines for Cancer Cytogenetics (ISCN, 1991), which deal mainly with
karyotypic description of neoplastic disorders. Later the two guidelines have been fused into one
version (ISCN, 1995).
A clone is defined as a population of cells derived from a single progenitor. A clonal origin
is inferred whenever two cells are found to have the same structural chromosome abnormality or
gain of the same chromosomal copy, or when three cells exhibit loss of the same chromosome. The
term cytogenetic noise, introduced by Heim and Mitelman (1995), is used to describe the extensive
but nonclonal abnormalities often seen in solid tumors. The biologically more interesting clonal
chromosomal changes can be classified as primary or secondary. Primary chromosome
abnormalities are believed to be essential in establishing the neoplasm. They are frequently found as
the sole anomaly and are usually specific for a certain type of tumors. However, submicroscopic
mutations, unseen at cytogenetic level, may precede them. Secondary chromosome aberrations arise
in cells already carrying a primary change. They, too, are usually nonrandom and reflect the clonal
evolution during the tumor progression process.
1.3 CANCER CYTOGENETICS
Over the past few decades, cytogenetic analyses have come to play an important role in both cancer
research and in the clinical management of cancer patients. In addition to their ability to identify
specific areas of the genome containing cancer-related genes, cytogenetic analysis also provides
crucial diagnostic and prognostic information in several cancers such as leukemias, lymphomas, and sarcomas.

1.3.1 NONRANDOM CHROMOSOME ABERRATIONS IN CANCER

Since the introduction of the banding techniques in the early 1970s, more than 37,000 cases of
human neoplasia with acquired clonal chromosome abnormalities have been reported (Mitelman et
al., 2000) (Figure 1.4). Excluding the numerical anomalies and aberrations involving unidentified
chromosomes, chromosome regions or bands, a total of 215 balanced and 1,588 unbalanced
recurrent neoplasia-associated aberrations have been recognized in the Cancer Chromosome Data
Bank (Mitelman et al., 2000).
In many malignancies, cytogenetic studies have revealed nonrandom, often disease-specific,
neoplasia-associated chromosome rearrangements. Although carcinomas such as those of the lung,
prostate, breast, and large bowel are the main malignancies contributing to human cancer mortality,
these diseases only contribute 26% of the database, and few tumor-specific aberrations have been
identified among them. In contrast, a large number of disease-specific abnormalities have been
identified in hematologic malignancies (Table 1.2) (Heim & Mitelman, 1995).
Table 1.2 Examples of characteristic cytogenetics changes in hematologic malignancies
Cytogenetic abnormality Disease
t(9;22)(q34;q11) Chronic myeloid leukemia
t(8;21)(q22;q22) Acute myeloid leukemia
t(11;19)(q23;p13) Acute lymphoid leukemia
t(8;14)(q24;q32) Burkitt's lymphoma

The discrepancy, in quantity and quality, between the collected data on hematologic malignancies
and solid tumors reflects the technical difficulties encountered in the cytogenetic analysis of the
latter, and probably also that much of the cytogenetic information on solid cancer is based on
studies of advanced carcinomas, in which massive secondary rearrangements overshadow the
important abnormalities or early-stage neoplastic growth. Karyotypically unrelated clones
encountered in some carcinomas such as those of the skin (Heim et al., 1989b), pancreas (Gorunova
et al., 1998), and breast (Teixeira et al., 1994) further complicate the interpretation of cytogenetic

12
 Figure 1.4 Overview of the cancer cytogenetics database (Mitelman, 2000)

findings in solid tumors. The recent addition to the cytogenetic arsenal of fluorescence in situ
hybridization (FISH), including methods such as comparative geneomic hybridization (CGH) and
the various modifications of multicolor FISH, have only partially remedied this situation.
Nevertheless, the picture has improved in later years and several characteristic chromosomal
aberrations in both epithelial and, especially, mesenchymal solid tumors have been identified (Table 1.3).

Table 1.3 Examples of characteristic cytogenetics changes in solid tumors

Mesenchymal tumors Epithelial tumors

Cytogenetic change Tumor type Cytogenetic change Tumor type
t(12;16)(q13;p11) Myxoid liposarcoma t(X;1)(p11;q21) Kidney cancer
t(11;22)(q24;q12) Ewing sarcoma -9 Bladder cancer
t(2;13)(q35;p14) Rhabdomyosarcoma der(1;16)(q10;p10) Breast cancer
t(X;18)(p11;q11) Synovial sarcoma inv(10)(q11q21) Thyroid cancer

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1.4 MOLECULAR CYTOGENETICS
1.4.1 FLUORESCENCE IN SITU HYBRIDIZATION (FISH)
In situ hybridization (ISH) is based on the principle that target nucleic acid sequences can be
hybridized with appropriately labeled DNA or RNA probes (Gall & Pardue, 1969). Initially,
radioactive isotopes were used as signal substances, but these were latter replaced by fluorochromes
that are safer and also require a shorter reaction time. In 1986, Pinkel and coworkers introduced
FISH as a new method to explore alterations in specific nucleic acid sequences in individual cells
and chromosomes.
FISH has many advantages, including the ability to assess sample cells for their integrity at
specific nucleic acid sequences even in non-dividing cells (interphase FISH) (Lawrence et al.,
1988). The increased resolution provided by FISH enables also the study of minute chromosomal
deletions, usually too small to be detected by conventional banding techniques. Although FISH thus
is an extremely useful and powerful technique, some prior knowledge of the nature of the
rearrangement to be investigated is usually needed to aid in the selection of FISH probes. If a FISH
screening technique is relied upon, on the other hand, the resolution is no better than that obtained
by banding cytogenetics. The probes for FISH can be chromosome painting probes, centromere-
specific repetitive DNA probes or locus-specific probes.
1.4.2 COMPARATIVE GENOMIC HYBRIDIZATION (CGH)
CGH is a relatively new, powerful molecular cytogenetic technique that allows the screening of the
whole tumor genome in one experiment (du Manoir et al., 1993; Speicher et al., 1993). The
technique used differentially labeled test (tumor) DNA and normal tissue DNA as competing probes
and normal metaphases as templates to detect and localize gains and/or losses of genetic material
across the entire tumor genome. Like in FISH, tumor and normal DNA can be labeled directly by
fluorochromes or with reporter molecules, e.g., biotin-dUTP and digoxigenin-dUTP, followed by
visualization with different fluorochromes. The labeled DNAs are mixed to allow competition
between the two when hybridized to normal human metaphase chromosomes. The hybridization
profile along each chromosome is then analyzed. To determine the losses and/or gains, the
fluorescence ratios of the two fluorochromes are calculated. The differences in fluorescence
intensities on the chromosomes reflect the copy number of the corresponding sequences in the
tumor DNA. Usually the normal DNA is labeled with a red fluorochrome and tumor DNA with a
green fluorochrome. In case of deletion (loss of genetic material), less green is seen in contrast to
the intense red signal, whereas in cases of gain or amplifications, a stronger green signal
corresponding to the gained segment is seen. The ability of CGH to detect gains requires that a
segment more than 2Mb is gained, whereas for deletions, the region lost has to be at least 10 Mb
(Bentz et al., 1998). Although CGH cannot detect balanced chromosomal changes and low
frequency aberrations, the technique is capable of identifying genomic imbalances in both fresh and
frozen tumor samples as well as in paraffin-embedded materials (Isola et al., 1994). This makes it a
uniquely well-suited technique in cancer cytogenetics especially when fresh samples are not
available. For optimal results, the specimen should contain a high proportion (>75%) of malignant
cells (tumor parenchyma) since CGH detects only genomic imbalances present in major tumor
clones. In cases with excessive stromal growth, microdissection of the tumor may be necessary.
1.4.3 MULTICOLOR FISH
Two novel multicolor FISH techniques, spectral karyotyping (SKY) and multiplex FISH (M-FISH),
are equally capable of examining the entire genome in one experiment, thus combining the
screening ability of banding approaches with FISH specificity. In both techniques, 24 chromosome-
specific probes, labeled with 3-5 fluorochromes, are used alone and in different combinations. The
main difference between SKY and M-FISH is the analysis system used. Complete emissions are
acquired by an interferometer-based spectral imaging system in SKY and by a set of filters in M-FISH.

14
 Multicolor FISH enables the rapid characterization and identification of most chromosomal
rearrangements in hematologic malignancies and solid tumors with a resolution limit of
approximately 1.5 Mb (Fleischman et al., 1999; Kytölä et al., 2000). Whereas conventional FISH
analyzes only specific chromosomes or regions of chromosomes and CGH visualizes only changes
resulting in gains or losses of genomic material, multicolor FISH permits the visualization of all
chromosomes by painting each pair with specific combinations of flurochromes. Multicolor FISH is
particularly suitable for the identification of subtle translocations of telomeric chromatin and of
small markers, both difficult to discern using conventional banding analyses (Haddad et al., 1998).
Recently, SKY was used with promising results in the cytogenetic analysis of hematologic
malignancies (Veldman et al., 1997), of cell lines with complex genomic rearrangements (Ghadimi
et al., 1999; Kytölä et al., 2000; Padilla-Nash et al., 1999; Pan et al., 1999), of primary solid tumors
(Adeyinka et al., 2000), and even of murine tumors (Liyanage et al., 1996). However, as every
other technique, also SKY has its limitations, as it cannot detect the centromeric regions, satellite
translocations, and intrachromosomal rearrangements such as small deletions, duplications, and
inversions. In addition, multicolor FISH is expensive, although future reductions in the amount of
DNA and fluorochromes necessary for single hybridizations may turn it into a more cost-effective technique.

1.5 MOLECULAR CONSEQUENCES OF CHROMOSOMAL ABERRATIONS

Recent advances in cancer cytogenetics have been crucial in delineating those genomic sites and
areas that harbor cancer-related genes. The activation of the MYC oncogene in Burkitt’s lymphoma
provided the first example of oncogene activation resulted from a chromosomal rearrangement in
neoplasia (Klein, 1989). Through one of the Burkitt-specific translocations, t(8;14), t(2;8) or
t(8;22), MYC (from 8q24) is brought into close physical proximity of elements whose normal
function is to ensure a high transcription rate on an immunoglobulin gene. Excessive production of
a principally normal MYC protein is the end result. The second typical example was from Ph-
positive CML, in which translocation of the ABL oncogene (in 9q34) to the BCR gene (in 22q11)
leads to the formation of a BCR/ABL fusion gene. In this situation, production of a qualitatively new
BCR/ABL protein with abnormal tyrosine kinase activity results with leukemogenic consequences.

In more general terms, the various genetic alterations in neoplastic cells can be divided into four
main categories:

1. Subtle sequence changes that involve base substitutions, deletions or insertions of a few
nucleotides. Unlike the other type of alterations described below, these changes cannot be
detected by conventional cytogenetic analyses.

2. Loss of chromosome material brought about by loss of an entire chromosome copy, deletions,
or unbalanced translocations. The putative pathogenetic consequences of such changes are loss
of TSG(s) or gene(s) involved in DNA-repair.

3. Gain of chromosome material brought about by gain of an entire chromosome copy or low- or
high-level amplification of smaller genomic regions through a variety of chromosomal
mechanisms. Special situations of this kind are the occurrence of homogeneously staining
regions (hsr) and double minutes (dmin), which often reflect high-grade amplification of
oncogenes.

4. Structural balanced chromosomal rearrangements, i.e., no gain or loss of genomic material

occurs. The molecular consequences in all or nearly all such instances in the neoplastic context
have been qualitative or quantitative activation of oncogenes as in the two examples discussed
above. Altogether more than 100 fusion genes have until now been described as a result of
translocation in hematologic disorders and solid tumors (Mitelman et al., 1997).

15
1.5.1 NEOPLASIA-ASSOCIATED GENES
Cancer-associated genes can be roughly categorized as belonging to 2 main groups: (1) genes
involved in the tumorigenic process, and (2) genes involved in progression and the metastatic
process (Tables 1.4 and 1.5).

Table 1.4 Genes involved in tumorigenesis

Gene group Example Chromosomal location
Oncogenes MYC, ABL 8q24
Tumor suppressor genes TP53, RB1 17p13, 13q14
DNA repair genes MSH2, PMS2 2p21, 3p21
Apoptosis regulating genes BCL2 18q21
Cell cycle regulator genes CDKN2A, MDM2 9p21, 12q14

Table 1.5 Genes involved in progression and metastasis

Gene group Example Chromosomal location
Anti-metastasis genes NM23 17q
Genes for proteases and protease inhibition NMP1, NMP2 11q22-23, 16q13
Genes for cell adhesion CDH5, CTNA1 16q22, 5q31
Multi-drug resistance genes MDR1 7q

Progress in molecular genetics is extremely fast and often much more is known about the genes of
tumorigenesis than about their essential biological effects. In addition, several reports have
suggested that some cancer-related genes cut across the functional classification listed above (Evan
& Littlewood, 1993). For example, TP53 is considered a TSG but in its mutant form, it may behave
as an oncogene (Harvey et al., 1995), and although both BRCA1 and BRCA2 are traditionally
considered as TSGs, they have also been associated with DNA repair (Scully et al., 1997).

1.5.1.1 Oncogenes
Oncogenes are qualitatively or quantitatively activated proto-oncogenes, i.e., cellular genes that
promote normal growth and differentiation. The operational definition of an oncogene rests on its
capacity to transform cells in vitro after transfection (Ponten, 1987).
Qualitative activation of oncogenes can be obtained by point mutations or chromosomal
translocations giving rise to an altered DNA primary structure and, ultimately, an altered
oncoprotein. Quantitive activation can be obtained by amplification, i.e., the presence of multiple
copies of a given oncogene allele, or by interference with normal transcriptional control
mechanisms. In both instances, increased production of an otherwise normal oncoprotein results.
Examples of qualitative oncogene activation are ABL and RAS activation via, respectively,
chromosomal and point mutation changes, whereas MYC activation in Burkitt’s lymphoma (see
above) is a typical example of quantitative activation.
Oncoproteins can be classified according to the biologic activity of their products into: (1)
secreted growth factors (e.g., SIS), (2) cell surface receptors (e.g., ERBB), (3) proteins involved in
signal transduction (e.g., ABL), and (4) DNA-binding nuclear regulatory proteins (e.g., MYC).

16
1.5.1.2 Tumor suppressor genes
TSGs or antioncogenes, are believed to be physiologically involved in the inhibition of cell
proliferation (Levine & Momand, 1990). The first evidence of their existence came from somatic
cell hybrid experiments, in which the fusion of normal and tumorigenic cells almost invariably
resulted in non-tumorigenic hybrids (Harris et al., 1969; Klein, 1987). Mutation or loss of both
alleles of a suppressor gene during tumorigenesis leads to loss, reduction or abnormalities of the
proteins they encode, contributing to oncogenesis. It has been hypothesized that the development of
any tumor requires, at least, two separate mutational events (Knudson, 1971). This "two hit
hypothesis" was confirmed for retinoblastoma development and has later been extended to apply
also to other tumors. In hereditary cases of retinoblastoma, the first hit is inherited as a germline
mutation in one RB1 allele, whereas the second hit occurs later as a somatic mutation of the other
allele in one of the retinal cells. In sporadic cases of retinoblastoma (60% of the cases), the two
mutational events occur only in a somatic cell (Knudson, 1971). In principle, any genomic region
consistently lost in cancer should be considered to contain one or more loci for TSG. This principle
opened the door for loss of heterozygosity (LOH) analyses of tumors (Strachan & Read, 1999).
However, few, if any, TSGs involved in sporadic tumors have actually been cloned as a direct result
of LOH mapping (Hilgers & Kern, 1999).
According to their location, TSG proteins can be grouped into: (1) cytoplasmic molecules
that regulate signal transduction, e.g., APC involved in colonic and gastric carcinoma, NF1 in
neurofibroma, and NF2 involved in meningioma, (2) molecules that regulate transcription, e.g.,
TP53 involved in a wide range of tumors, WT1 involved in Wilms tumor, and RB1 involved in
retinoblastoma, bladder cancer, and small-cell lung carcinoma (Harris & Hollstein, 1993; Leong &
Leong, 1998).

1.5.1.3 DNA repair genes

DNA is dynamically stable due to the large variety of DNA enzymes that continuously scan the
DNA and replace damaged nucleotides. Human DNA mismatch repair (MMR) genes are
responsible for maintaining the integrity of the genome. Mutations in mismatch repair (MMR)
genes, MSH2, MLH, PMS1 and PMS2, lead to an increase in the frequency of other mutations, and
have been detected in sporadic colon cancer and in individuals affected by hereditary non-polyposis
colorectal cancer (HNPCC) (Bronner et al., 1994; Moslein et al., 1996).

17
2 UROEPITHELIAL CARCINOMAS
2.1 CLINICAL ASPECTS
2.1.1 EPIDEMIOLOGY
2.1.1.1 Incidence
Bladder cancer (BC) is a common malignancy in Europe and the USA, especially among men,
where it holds the 4th place after cancer of the lung, prostate, and large bowel (Silverman et al.,
1992). It is the 12th leading cause of cancer death (Heney et al., 1983). In areas where infestation
with Schistosoma haematobium is endemic, it is even more common and accounts for 25% of all
cancers in men (El-Bolkainy et al., 1981).
Upper urinary tract carcinomas (UUTC), i.e., tumors of the renal pelvis and ureter, are
relatively uncommon, constituting 2-4% of all uroepithelial tumors. The low incidence of UUTC in
contrast to transitional cell carcinoma (TCC) of the bladder may reflect the rapid transit of chemical
carcinogens through the upper urinary tract and their stasis in the urinary bladder.
2.1.1.2 Gender and race
Uroepithelial carcinomas are 3 times more common in men than in women (Batata & Grabstald,
1976). The sex discrepancy was previously explained by the fact that men are usually more exposed
to industrial and environmental toxins than women. However, this may not explain the whole
difference since during the last 30 years women were increasingly exposed to the same carcinogenic
substances, but still the annual incidence in males remains 50% higher than that in females. The
higher incidence in men may therefore be due also to other factors than carcinogenic exposure, be
they genetic (Risch et al., 1993), hormonal (Horn et al., 1995), anatomical or others. Evidence of
racial incidence differences between, e.g., blacks and whites has been reported even within the same
country (Groeneveld et al., 1996).
2.1.1.3 Age
Whereas UUTC rarely occur before the age of 40 years, BC may occur at any age. However, it is
usually a disease of middle-aged and elderly patients, with the exception of post-bilharzial
squamous cell carcinoma (SCC), which often occurs in adolescents. Younger patients tend to have
well-differentiated tumors and a more favorable prognosis with less risk of recurrence and
progression (Benson et al., 1983). The risk of progression in younger and older patients
nevertheless remains the same in case of tumors of the same grade (Wan & Grossman, 1989).
2.1.1.4 Regional differences
The incidence of uroepithelial cancer varies considerably among countries. A higher incidence of
UUTC in the Balkans has been related to Balkan nephropathy. In Europe and the USA, TCC is the
dominant form (90%), whereas SCC is rare. This picture is reversed in the Middle East and Africa,
where SCC dominates. Histologic variations may be seen even within the same country;
Groeneveld et al. (1996) reported that bladder SCC was more frequent among blacks in South
Africa (53%) than among Caucasians (2%).

2.1.2 ETIOLOGY AND RISK FACTORS

Several etiologic factors have been associated with the development of uroepithelial carcinomas of
which occupational exposure to chemicals, cigarette smoking, physical agents, parasitic and
bacterial infections, high consumption of certain analgesics, bladder calculus, genotoxic
chemotherapeutic agents, Balkan nephropathy, and heredity are the most important.

18
2.1.2.1 Occupational exposure
In the late 19th century, three cases of BC in workers in the German dye industry were described,
establishing for the first time the relationship between BC and industrial exposure (Rehan, 1895).
The known chemical carcinogens involved in bladder carcinogenesis include 2-naphthylamine, 4-
nitrobiphenyl, benzidine, and 2-amino-1-naphthol (Morrison & Cole, 1976). Today, occupational
exposure is well documented and accounts for 20% of all BC in the USA (Cole et al., 1972),
typically with long latency periods (25-30 years) in most cases. Several occupations have been
reported to be associated with an increased risk of uroepithelial cancer (Table 2.1).
Table 2.1. Occupations associated with an increased risk of bladder cancer

High risk group Low risk group

Dye workers Taxi drivers
Painters Cement and concrete finishers
Leather workers Architects
Truck drivers Aluminum processing workers
Chemical workers Miners
Petroleum workers Butchers

2.1.2.2 Cigarette smoking

Cigarette smoking has been suggested to increase the incidence of BC four-fold in smokers
compared with non-smokers (Morrison & Cole, 1976). Studies have shown that the risk correlates
with the number of cigarettes smoked, the duration of smoking, and the level of smoke inhalation.
The increased risk has been equally noticeable in both sexes (Augustine et al., 1988). Others forms
of tobacco use are also associated with a slightly increased risk for BC (Burch et al., 1989).
Although some reports suggest that one-third of BC is smoke-related (Howe et al., 1980), the
putative specific chemical carcinogen for smoke-induced BC has not been identified. At the
moment, it is not clear to what extent the malignant transformation arises spontaneously or is
induces by a purported chemical carcinogen (Jones et al., 1991). Studies comparing TP53 mutations
in tumors from smokers with tumors from nonsmokers have been equivocal; although an increased
number of mutations is observed in smokers, no difference is seen with regard to their exact type or
site within the gene (Spruck et al., 1993).
For UUTC, cigarette smoking has been found to be strongly associated with an increased
risk of developing tumors. The risk varies depending on the tumor site, being higher for ureteral
than for renal pelvis tumors (Jensen et al., 1988; McLaughlin & Harrison-Stewart, 1992).
Moreover, a dose-response relationship was found between the total amount of tobacco smoked and
the risk of developing UUTC. In long-term (45 years) smokers, the risk was 7.5-fold compared with
non-smokers (McLaughlin & Harrison-Stewart, 1992). Many investigators have therefore suggested
that smoking causes 70% of all UUTC in men and 40% in women (Morrison & Cole, 1976). The
risk seems to decline after cessation of smoking but, unfortunately, as for other smoking-related
cancers, it declines slowly and only partially, and a 10 years cessation results in only 50% to 60%
reduction of the risk. Former smokers still show a two times higher risk of developing UUTC than
non-smokers of the same age (Ross et al., 1989).
2.1.2.3 Urinary bilharziasis
Urinary schistosomiasis is endemic in 74 tropical developing countries in Northern Africa, the
Middle East, Southeast Asia, and Latin America. The disease affects 200 million individuals
worldwide, causing 250,000 annual deaths.
In 1911, Ferguson suggested that infestation with Schistosoma haematobium is associated
with an increased risk of developing BC, especially of the SCC type. In areas where urinary
bilharziasis is endemic, BC is the most common cancer in men (25% of all cancers). Evidence of

19
urinary bilharziasis was found in 69% of bladder SCC of Sudanese patients (Sharfi et al., 1992) and
in 80% of Egyptian patients (El-Bolkainy et al., 1981). However, the mechanisms whereby urinary
bilharziasis induces BC are not fully understood. Elevated urinary N-nitroso compounds (Tricker et
al., 1991), elevated levels of B-glucuronidase (Norden & Gelfand, 1972), and chronic mechanical
irritation of the urothelium by calcified eggs deposited in the bladder wall have all been suggested
to be important (Matanoski & Elliott, 1981). Histologic examination has shown that the initial
chronic inflammation of the bladder develops into a destructive granulomatous reaction with an
increased rate of cell division, which gives way to the formation of a hyperplastic urothelium
containing immature cells. The disturbed differentiation pattern is thought to give rise to various
non-malignant morphologic changes, which then are prone to subsequent malignant transformation
(Cotran et al., 1999).
2.1.2.4 Physical agents (radiation)
Physical agents can be important etiologic factors in uroepithelial carcinogenesis. The carcinogenic
effect of ionizing radiation on the urinary tract is well documented (Kaplan & Loftus, 1985).
Radiation-induced uroepithelial carcinoma is the most frequent neoplasm following pelvic radiation
for gynecologic malignancies, occurring at a 50 times higher frequency than in the general
population (Kleinerman et al., 1982; Pettersson et al., 1985).
2.1.2.5 Balkan nephropathy
As the name of this familial degenerative interstitial nephropathy implies, it is confined to rural
areas of Balkan countries where the incidence of UUTC is reported to be 100-200 times higher than
elsewhere. These tumors differ from UUTC of other etiologies, however, and are often low-grade,
multiple and/or bilateral.
2.1.2.6 Analgesics
In 1983, McCredie reported an increased risk for UUTC in individuals with analgesic abuse
(McCredie et al., 1983). The risk was 3.6 times higher after use of phenacetin, and increased to 20-
fold in patients having papillary necrosis. The mechanism suggested is that analgesic abuse induces
capillary sclerosis (thickening of the basement membrane around subepithelial capillaries), which
has been reported to occur in 16% of UUTC (Piper et al., 1985).
2.1.2.7 Heredity
There are few reports of familial BC (Schulte, 1988), and hereditary cases account for only 1% of
all urological cancers (Kantor et al., 1985). UUTC has been reported in some familial cancer
syndromes (Lynch & Cohen, 1995) such as Lynch syndrome II in which synchronous and
metachronous colonic and extra-colonic cancer, including UUTC, may be seen.
2.1.3. HISTOPATHOLOGY
2.1.3.1 Histology
Uroepithelial tumors exhibit a wide range of histologic types. Non-epithelial urinary tract tumors
are exceedingly rare (Table 2.2). Most epithelial tumors can be classified into four main groups:
• Transitional cell carcinoma (TCC)
• Squamous cell carcinoma (SCC)
• Adenocarcinoma (AC)
• Undifferentiated carcinoma (UC)
Intermediary histologies are not uncommon, and as many as 20% of TCC contain foci of squamous
cell differentiation, whereas 7% contain glandular differentiation.
Table 2.2 Non-epithelial urinary tract tumors
Bladder Upper urinary tract
Sacromatoid carcinoma Leiomyosarcoma
Carcinomas with lymphoid stroma Plasmacytoma
Neurofibroma
20
2.1.3.2 Tumor grade
This is based mainly on the degree of anaplasia, nuclear crowding, degree of differentiation, mitotic
count, presence of giant cells, and irregularity in cell size.
Uroepithelial tumors are divided into three grades (Koss, 1975):
1. Well-differentiated carcinoma (G1)
2. Moderately differentiated carcinoma (G2)
3. Poorly differentiated carcinoma (G3)
Although the grading system for BC is known to be highly subjective, a strong correlation
nevertheless exists between tumor grade and stage, with most well- and moderately differentiated
tumors being superficial and most poorly differentiated tumors being muscle invasive.
2.1.3.3 Tumor stage
The first important task of staging is to determine whether the patient has superficial or muscle-
invasive disease. In case of a superficial cancer, more elaborated staging procedures, such as bone
scan and computed tomography, are not usually needed. They are reserved for patients with muscle-
invasive disease. The second task in staging is to identify patients with locally invasive disease who
may benefit from aggressive, radical therapy. The TNM system developed by the International
Union Against Cancer (UICC) remains the most widely used classification system in clinical
practice (Hermanek & Sobin, 1988) (Figure 2.1).

2.1.4. NATURAL HISTORY

2.1.4.1 High incidence of recurrence
Although 70% of newly diagnosed bladder urothelial lesions are superficial and papillary tumors,
i.e., the tumor is confined to the lamina propria, the disease represents a major therapeutic challenge
and a continuous threat to the patients since the tumors recur after transurethral resection of the
primary in more than 70% of the cases (Heney et al., 1983; Thompson et al., 1993). Of these
recurrent cases, 80% remain superficial throughout the life of the patient, whereas 16% to 25%
eventually recur as higher grade and/or stage tumor(s) (Kiemeney et al., 1993b). The possibility of
new tumor(s) arising as the result of field cancerization was rendered unlikely by molecular
investigations (Sidransky et al., 1992). Epidemiologic and clinical data have shown that patients
with ureteral or renal pelvis tumors have a higher (40%) risk of having tumor(s) also of the bladder,
and if carcinomas of both the renal pelvis and the ureter are present, the probability of having BC
increases to 75% (Kakizoe et al., 1980). In contrast, patients with BC have only a 3% risk of having
upper urinary tract tumors (Babaian & Johnson, 1980).

2.1.4.2 Heterogeneity of the clinical course

Uroepithelial TCC, even tumors of same pathologic stage, may follow very different clinical
courses. As prognostic markers, the ones currently used are tumor grade, multiplicity, tumor shape,
location, and presence of carcinoma in situ (Cis), are of limited value (Messing & Catalona, 1997),
it is difficult to predict the clinical course, especially for superficial bladder tumors. This may
suggest that TCC are of at least two main subgroups with distinct genetic make-ups and,
consequently, clinical behavior patterns. Good prognostic markers and a better understanding of the
pathogenic mechanisms behind local and distant disease spreading are therefore urgently needed.
Such information might help design new therapeutic strategies geared towards a more aggressive
approach in progression-prone cases.

21
 Outer muscle layer

Inner muscle layer

Lamina propria
T3a
T2

T3b
T1

T4a
Ta

Tis T4b

N: Lymph nodes
Mucosa

Prostate gland

T: Primary tumor N: Lymph node

T0 No tumor present
Tis Carcinoma in situ
Ta Papillary tumor limited to mucosa
T1 Tumor extends into lamina propria
T2 Tumor invades superficial muscle
T3a Tumor invades deep muscle
T3b Tumor invades perivesical fat
T4a Tumor invades adjacent organ(s)
T4b Tumor fixed to pelvis or abdominal wall
N0 No lymph node involvement
N1 Involvement of a single lymph node
N2 Involvement of multiple regional lymph nodes
N3 Involvement of regional lymph nodes creating
a fixed mass
N4 Involvement of juxtaregional lymph nodes
M: Distant metastasis
M0 No evidence of distant metastasis
M1 Evidence of distant metastasis

Figure 2.1 TNM staging system of bladder cancer (Hermanek P and Sobin LH, 1988)

22
2.1.4.3 Multifocality

Around 30% of urinary tract TCC are found as multiple tumors at the time of diagnosis (Kiemeney
et al., 1993b). The multifocal nature of uroepithelial cancer, together with a propensity for
recurrence (polychronotopicity) and the dysplastic changes that are usually found in the mucosa
surrounding bladder tumors (Wolf & Hojgaard, 1983), have been interpreted as strong
circumstantial evidence that TCC represents a field disease (Ross et al., 1989). According to this
field disease theory, the entire epithelium is tumor-prone in the sense that multiple polyclonal
primary lesions are likely to emerge from it, either synchronously or metachronously (Yao &
Rubin, 1994). The alternative, monoclonal view presupposes a common clonal origin of all
uroepithelial tumors in each patient, even the multifocal ones, implying that these macroscopically
distinct lesions develop as the result of intraluminal seeding of cancer cells shed from the original
tumor. Support for the monoclonal hypothesis has come from analyses of X-chromosome
inactivation patterns (Sidransky et al., 1992) and the precise identification of TP53 mutations as
clonal markers in multifocal uroepithelial tumors (Xu et al., 1996).
UUTC are less often multicentric than BC, with bilateral involvement (synchronous or
asynchronous) occurring in 2-5% of sporadic cases (Steffens & Nagel, 1988). Almost all UUTC
show transitional cell differentiation. Ureteral tumors are located more frequently in the lower
ureter (73%) than in the middle (24%) or upper parts of the ureter (3%) (Anderström et al., 1989).

2.1.5. DIAGNOSIS

2.1.5.1 Signs and symptoms

Painless macroscopic hematuria is the alarming sign in 85% of patients with BC, whereas
microscopic hematuria is present in almost all patients (Messing & Vaillancourt, 1990). Other
urinary symptoms such as frequency, urgency, and dysuria are usually associated with carcinoma in
situ and invasive bladder carcinomas.
2.1.5.2 Cystoscopy
A careful cystoscopic examination should be performed in all patients complaining of hematuria. In
addition to the visual information like the shape, number, and site of tumor(s), cystoscopy provides
material for histologic examination. However, cystoscopic examination has its limitations as a
diagnostic tool because small papillary tumors are easily overlooked, cases of Cis and dysplasia
cannot be detected, and when random biopsies are taken, only 24% of the cases can be diagnosed.

2.1.5.3 Excretory urography

Although excretory urography is not a sensitive diagnostic tool, especially for small bladder tumors,
its remains an important modality for detecting ureteral associated tumors. It therefore should be
performed in all patients complaining of hematuria. Large bladder tumors, when present, may
appear as filling defects in the cystogram phase. The appearance of a dilated, obstructed ureter
strongly indicates muscle invasion.

2.1.5.4 Urine cytology

Urine cytology cannot provide information about the depth of the tumor (T stage) and has low
sensitivity for well- and moderately differentiated cells. In the absence of a good molecular marker,
urine cytology and histologic examination remain the only clinical modalities to diagnose Cis.
Positive urine cytology is obtained in 10% of patients with G1, 50% with G2, and 90% with G3
tumors (Soloway, 1985).

23
2.1.5.5 Staging tests
These can be divided into the following three groups:

1. Tests to assess local extension (T stage)

• Examination under anesthesia
• Transurethral resection of the tumor
• Excretory urography
• Mucosal biopsy
• Cytology

2. Tests to assess regional extension (N stage)

• Lymphadenectomy
• CT or MRI

3. Tests to assess systemic extension (M stage)

• Chest X-ray
• Bone and liver MRI
• Chest, liver, and bone scan

2.1.6 TREATMENT

As for other malignancies, the treatment of BC patients first and foremost depends on the grade
and/or stage of the tumor. Several studies have suggested the risk of disease progression to be 4-
10% and 25-30% for Ta and T1, respectively (Chen et al., 1996; Prout et al., 1986). However, some
studies have questioned the accuracy of pathologic examination in superficial disease, claiming that
33% of the tumors are being understaged and 10% overstaged (Wijkström et al., 1984). The
discrepancies among pathologists are mainly due to confusion of the smooth muscle fibers of the
tunica muscularis mucosa in the lamina propia with detrusor muscle (Younes et al., 1990). Usually
patients are classified into two main groups:

1. Patients with superficial disease (Ta, T1) who generally are treated by:
• Transurethral resection of tumor (TUR-T)
• TUR-T + Adjuvant intra-vesical immune- or chemotherapy

2. Patients with invasive disease (T2) who generally are treated by:
• Radical surgery with pre- or post-operative radiation therapy
• Cytostatic and palliative treatment in cases with systemic metastasis

Most bladder carcinomas (70%), especially TCC, are superficial at diagnosis and can be adequately
treated by transurethral resection with 5-year survival ranging between 70% and 95% (Gilbert et al.,
1978; Heney et al., 1982). However, considerable treatment limitations and prognostic
heterogeneity exist. After standard transurethral resection, about 60% of superficial tumors recur as
tumors of the same grade and stage, whereas 25% relapse with more advanced and aggressive forms
of cancer. This requires prolonged and close postoperative monitoring of the disease.

24
2.2 GENETIC ASPECTS OF UROEPITHELIAL CANCER
2.2.1 CYTOGENETICS
2.2.1.1 Overview
Although uroepithelial carcinomas are among the most common malignancies, the existing
knowledge about their karyotypic characteristics is clearly insufficient. Only 144 urinary bladder
TCC and 3 TCC of the ureter with abnormal karyotypes had been reported prior to this study
(Mitelman et al., 2000). The reported data are not only quantitatively limited but are also lacking in
precision. Of the 144 bladder TCC, 35 (29%) had grossly incomplete karyotypes, 47 (38%)
displayed altogether 132 unidentified marker chromosomes as well as 10 unidentified ring
chromosomes and a total of 190 uncertain breakpoint, as is evident from how the karyotypes were
written (Mitelman et al., 2000).
Although no chromosomal aberration completely specific for BC has been identified, a
clearly nonrandom pattern of chromosomal changes has emerged, albeit with considerable
karyotypic heterogeneity among cases ranging from the presence of sole anomalies in early tumors
to very complex karyotypes in advanced ones. Translocations are rarely seen, at least in early
stages, and seem to play no important role in the initiation of uroepithelial carcinomas. Instead, the
cytogenetic profile is dominated by nonrandom chromosome gains and, especially, losses, the latter
indicating that loss of tumor suppressor gene(s) may be the most crucial event in the pathogenesis
of uroepithelial carcinomas (see below).
All chromosomes have been involved in numerical and/or structural changes in BC (Gibas
et al., 1984; Gibas et al., 1986; Granberg-Öhman et al., 1984; Smeets et al., 1987; Vanni et al.,
1988). Chromosomal imbalances (Figure 2.1) are dominated by losses of or from chromosome arms
1p, 2p, 5q, 6q, 8p, 11p, and 17p, as well as loss of an entire copy of chromosomes 4, 9, 10, 14, 15,
16, 18, 21, 22, X, and Y. The dominating gains are of chromosome arms 1q, 5p, 8q, and 17q as well
as of an entire copy of chromosomes 7 and 16. A total of 290 chromosomal breakpoints were
identified in the 100 cases reported with structural aberrations (Figure 2.2), with the highest number
of breakpoints (≥18) seen in chromosomes 1, 3, 5, 6, 9, 11 and 13. All chromosomes have been
involved in structural rearrangements except chromosomes 20 and Y. The chromosome bands most
frequently involved were 1p12, 1q21, 5q10, 8q11, and 11p15 (each involved at least 6 times).
Below is given a more extensive survey of the chromosomal aberrations most frequently seen
(Mitelman et al., 2000) in BC prior to this study.
Chromosome 1
Alterations involving chromosome 1 had been seen in 35% of the investigated BC cases (Mitelman
et al., 2000). The alterations are diverse and include deletions, translocations, duplications, and
isochromosome formations. However, regardless of the type of change, in most cases the net
resulting imbalance was gain of 1q material and loss from 1p. Changes involving chromosome 1 are
rarely if ever seen as the sole aberration but nevertheless may form part of relatively simple
karyotypes. Support for the view that changes of chromosome 1 are secondary in BC tumorigenesis
also comes from molecular cytogenetic investigations showing that gain of 1q material is more
frequent in pT1 than in pTa tumors (Sauter et al., 1997; Voorter et al., 1995). Regardless of when
they occur, the molecular consequences and biological significance of these changes in uroepithelial
carcinomas remain uncertain, although some investigators suggest that they may be implicated in
tumor progression and in in vitro immortalization of human cells (Paraskeva et al., 1989).
Chromosome 3
Rearrangement of chromosome 3 resulting in loss of material from the short arm had been seen in
44% of the reported BC with abnormal karyotypes (Mitelman et al., 2000), usually in advanced
tumors with complex karyotypes (Dalbagni et al., 1993a; Knowles et al., 1994). Deletion of 3p is
also frequent in other carcinomas such as renal cell cancer, breast cancer, and small cell lung cancer
(Andersen et al., 1992). Although many potential target genes are located on 3p, the crucial
molecular-level consequence of these chromosomal aberrations in BC carcinogenesis remains unknown.

25
 6
5 5
4 4 6
5
33 23 4
2 2 3
1 22
1
6 1
5
2 4 6
21 13 4 5
2 3 5
1 1 4 14
2
1 2 1 3 3
21
1 1 21
1 2 1
12 13 2 21 1
4 3 2
3
1 1 13
1
2 1 4
2 2 2 2
3 3
4 3 3 5
4
5 4 4 25 1
5 6 22
1 1
7 3
3 2 6 8
2 7 1
3 8 1
1 4 9 2
5 33
43
2 6 3 32
3 4
4 7 4 5
5
1 2 4 5
5
4
3 2
21 5
2 4 5
2 3 3 4
5 22 22 3 4
1 1 1
4 1 12 3
13 3 2
2
12 1
2
1
11 12 1 1
1
1
21 1 1 11 2
3 11 12
4
15 12
3 3 1 13
6 1 1 4
1
2 1
2 2 1
2 2 2
2 23
2 2
1 1 23
3 2 4
23 4
33

26
4 32
3
5
5 4 4 4
6
6 5
6 11
7
8 9 10
6 7
3 3
3 3
12 3 3 2 12
2
11 12
1 1
12
1 1 1
1
1 1
21 2 1 2
3
1 1
12
12 1 4 12
13 13 3 5 3 1
4 4 1
1 1 2 2
5 23 23
1 2 2
1 2 23 3 4 4
2
2
4 5
2
23 1
4
5 16
32
1
32
6 17
3
4
4 15
13 14
12
2
2
1
3
2 3 3
11 13 2
11 2
11 11 11
2 1
1
1 1 11 1 21
1 13
12 2 1 1
2 2
13 2 1
3 2 3 1 1
1 2
22
19 20 22
2
3
3 24
21 5 Y
18 1 2
6
7
8
X
Figure 2.1 Karyotypic imbalances caused by numerical and structural chromosomal
aberrations in 144 TCC of the bladder. Losses are shown to the left, and gains to the right
 5
6 4
5 23
4 2 6 6
5
33 1
4
5
2 6 5
3 14
5 22 4
1 4 3 3
13 1 21 21
2
2 21 1
1 3 2
2 4
1 1 13
3 1
2 1
1 2
13 2 2 4
1 4 1 3 5
1 1 4
1 2 25 1
1 2 6
2 3
22
1 7 3
3 8
2 4 1
23 2 1
4 1 3 1
2
5 4 33
2
5
32
1 3 4
3 4 5
3 4 6 5
2 5
6
7
8 4 5
1 9
7
2
43
2 3
4
1
5 3 5
4 2 4 5 3
3 4
21 22 3 3
22 1 22 12 12
5 2 1 14 1
11 1 3
1 4 3 2 1
13 1 2
1 21 11 1
2 2 13
11 1 12 1 1
2 4
21
3 12 1
11 3 5
3
1
13
14 1 2 4 1
5
6 1 2
2 2 23 1 2
2 2 2 23
1 2
3 4

27
1 23
2 2 5 4
1
23 4 33 4
6
4
2
33 4
11 12
5
4
5 8 10
6
7
6 9
6 7 3
12 11
3 3 1 1
3 3 12 12 11 12
2
11 12 1 2
1 1 1 1
1 1 2
13 1 1
2 12 4 2
5 12 23 22
13 3 3
4 1 1 4 3
1 5
2 2
2
1 2
23 23
23 18
2 4
4
5
4
17
1 1 6 16
32 32
3
4
15
14 2
13 2
1
11
11
1
2
3 3 13 1
13 2
2
3 2
11
2 1
11 1 1
1
1 11 1 2
2 1 2 2
2 1 1 3
13 2 3 24
3 2 5
6
Y
19 20 21 22 7
8
X
Figure 2.2 Distribution of the 290 breakpoints observed in structural chromosomal aberrations in 100 TCC of the bladder
Chromosome 5
Cytogenetic alterations involving chromosome 5 had been reported in 22% of BC with abnormal
karyotypes (Mitelman et al., 2000). In the majority of cases, the change was the formation of an
isochromosome for 5p, i(5)(p10), which makes this the single most common structural
chromosomal abnormality in BC. The net imbalance of i(5)(p10) is gain of the short arm and loss of
the long arm of this chromosome. Recent CGH studies have confirmed the cytogenetic findings
showing imbalances at 5p and/or 5q in 18-28% of BC cases (Simon et al., 1998). A strong
correlation between chromosome 5 involvement and tumor grade and stage was shown in several
studies (Simon et al., 1998).
Chromosome 7
Trisomy 7 has been described in near-diploid karyotypes and as the sole chromosomal change in
BC in several cytogenetic studies (Babu et al., 1987; Smeets et al., 1987; Vanni et al., 1988). The
importance of trisomy 7 as a tumor-associated aberration remains controversial, however, as it has
been found repeatedly also in some unquestionably nonneoplastic lesions (Johansson et al., 1993).
The important molecular consequence of trisomy 7 is unknown but an increased number of alleles
for the epidermal growth factor (EGF) receptor gene could play a role.
Chromosome 8
Aberrations involving chromosome 8 usually result in loss of material from the short arm, and have
been repeatedly detected in BC by both cytogenetic and molecular cytogenetic methods (Mitelman
et al., 2000; Zhao et al., 1999). Similar aberrations occur frequently also in many other tumor types
such as carcinoma of the liver and pancreas (Mitelman et al., 2000). The rearrangements of
chromosome 8 have been associated with advanced tumor grade/stage (Knowles, 1995). Studies of
patients with non-muscle-invasive bladder cancers have shown more frequent loss of 8p in
minimally invasive (pT1) tumors than in non-invasive (pTa) ones (Wagner et al., 1997). These
findings are consistent with the view that loss of a putative TSG on 8p plays a role in the
progression and tumor invasion in BC.
Chromosome 9
Changes involving chromosome 9 are the most common chromosomal aberrations in TCC of the
bladder. Prior to this study, loss of an entire chromosome copy was seen in around 45% of the
reported cases, loss from 9p only was seen in 5%, and loss from 9q was seen in 6% of cases with
abnormal karyotypes (Mitelman et al., 2000). Loss of chromosome 9 material had therefore been
widely accepted as a nearly ubiquitous, pathogenetically important and early event in urinary tract
transitional cell carcinogenesis (Gibas & Gibas, 1997). The losses are seen in both early and
superficial carcinomas and in muscle invasive ones. The fact that material is often lost from either
of chromosome 9’s two arms, as well as of the entire chromosome, argues that at least one
pathogenetically important TSG may be present in each arm (Simoneau et al., 1999; Williamson et
al., 1995). Recent molecular data suggest that 9q abnormalities are more common in Ta compared
with T1 tumors, in which a mixture of aberrant 9p and 9q genotypes are seen (Simoneau et al.,
2000). These observations indicate that loss of 9p material may be associated with the development
of tumors with more aggressive biological behavior or, alternatively, they may be related to early
disease progression (Orlow et al., 1994). Although several attractive candidates such as
p16/CDKN2 in 9p21 and TSC1 in 9q34 have been reported to be homozygously deleted in
superficial TCC of the bladder (Balazs et al., 1997; van Tilborg et al., 1999), the crucial gene-level
consequences of these chromosomal aberrations remain unknown.
Chromosome 11
Several cytogenetic banding studies have revealed nonrandom involvement of chromosome 11 in
BC, mostly leading to loss of genetic material from 11p (Mitelman et al., 2000). Additional
evidence to the same effect has come from CGH analyses (Voorter et al., 1995). The frequency of
11p loss may be higher in pT1 than in pTa tumors, and even higher in pT2-4b tumors (Sauter et al., 1997).
28
In contrast, some FISH studies using centromeric probes have shown an increased copy
number of chromosome 11 (Hopman et al., 1991). However, these studies did not assess the tumors'
ploidy level and the seemingly increased copy number may therefore have reflected general
polyploidization. Loss of 11p therefore appears to be an early but secondary change, associated with
tumor progression. The putative TSG on 11p lost through the chromosomal rearrangements remain unknown.

Chromosome 13
Aberrations involving this chromosome are known to occur in muscle invasive tumors and often
result in net loss of material from the long arm (Mitelman et al., 2000). LOH studies seem to be
more informative than cytogenetic studies in this prospect as LOH at the RB locus in band 13q14
was seen in 90% of muscle invasive tumors (Cairns et al., 1991).
Chromosome 17
Rearrangement of chromosome 17 resulting in loss of material from the short arm is rarely seen in
superficial BC, but is common in more advanced and aggressive tumors (Olumi et al., 1990;
Williamson et al., 1994). The cytogenetic data probably underestimate 17p involvement; whereas
karyotypic analyses indicate the involvement of this arm in less than 10% of the analyzed cases,
molecular genetic studies have shown LOH in up to 42% of the tumors (Dalbagni et al., 1993b).
Most deletions of 17p therefore seem to be submicroscopic. On the other hand, several FISH
analyses using centromeric probes have revealed frequent chromosome 17 copy-gain in bladder
carcinomas of high grade and stage. Such apparent gains of whole chromosome copies in FISH
analyses may reflect polyploidization in advanced tumors rather than a specific role of chromosome
17. The target of del(17p) could be the TP53 gene, which resides at 17p12 and is known as the most
generally important TSG in human neoplasia.
Chromosome Y
Loss of the Y chromosome is common in bladder tumors of male patients. It has been reported in all
stages and even as the sole change in several reports (Sandberg, 1992; Sandberg & Berger, 1994;
Smeets et al., 1987). In contrast, bladder tumors obtained from female patients do not reveal the
same incidence of X chromosome loss. FISH studies have shown that loss of the Y is infrequent in
normal urothelial cells obtained from healthy males (Bentz et al., 1998). This observation does not
exclude, however, the possibility that loss of chromosome Y in cultured cells could reflect changes
in stromal elements, in particular since –Y has been demonstrated in non-neoplastic disease lesions
of several tissues and organs such as kidney, bone marrow, and brain (Elfving et al., 1990; Heim et
al., 1989a). In conclusion, one cannot be certain that loss of the Y chromosome really signifies a
pathogenetically important event in neoplastic cells (Heim & Mitelman, 1995).

2.2.2 MOLECULAR GENETICS

2.2.2.1 Tumor suppressor genes
Consistent loss of chromosomal material is generally supposed to work pathogenetically via loss of
a TSG. As alluded to repeatedly above, the actual identity of the putative TSGs operative in BC
tumorigenesis remains unknown, but a lot of interest has focused on the following candidate genes.
P16 (CDKN2/MTS-1)
This was claimed to be the most frequently inactivated TSG in BC by Serrano et al. (1993). P16
maps to 9p21 and has been found to be mutated in familial melanoma and pancreatic cancer (Caldas
et al., 1994). Although many bladder tumors, perhaps 50%, have a hemizygous deletion of 9p that
includes P16, few cases have shown mutation of this gene (Baud et al., 1999).
TP53
Alteration of the TP53 gene, either by deletion or mutation, is the most common genetic
abnormality found in human cancer cells (Aprikian et al., 1993; Harris & Hollstein, 1993). The
gene maps to chromosome band 17p12. The function of its product is to act as a cell cycle regulator
29
and guardian of the genome, directing cells with genetic abnormalities toward apoptosis. TP53
mutations have been associated with genomic instability and progressive development of further
mutations at other loci. Several groups have closely correlated mutations of TP53 with
accumulation of the p53 protein in cell nuclei, which can be readily detected by
immunohistochemical techniques. Loss of the gene is a rare event in superficial BC unless the
tumors are high-grade papillary lesions or carcinoma in situ. On the other hand, as many as 70% of
advanced stage tumors have abnormal TP53 (Sidransky et al., 1992). Mutation of TP53 has been
shown to correlate with an increased risk of recurrence and a decreased overall survival in patients
with uroepithelial carcinomas (Sarkis et al., 1995). However, its independent prognostic value
remains controversial. Although some studies have shown an increased rate of TP53 mutation in
current smokers (56%) and previous smokers (38%) in comparison with non-smokers (29%), no
statistical significance was shown (Kannio et al., 1996).
RB1
The retinoblastoma (RB1) gene acts to control cell proliferation through regulation of the cell cycle
at the G1-S-phase transition (Cordon-Cardo, 1995). The gene maps to chromosome band 13q14 and
loss of its normal function occurs in a variety of human epithelial cancers (Fradet & Cordon-Cardo,
1993). Mutation or loss of RB1 is usually a late event in BC and is associated with high-grade
invasive disease (Xu et al., 1993). Similar to TP53, alterations of RB1 have been shown to be
associated with an increased risk of recurrence and progression of bladder cancer. Concomitant
alterations in both TSGs have a further negative effect on recurrence and survival compared to
alterations in either gene alone. Patients showing no alteration of TP53 or RB1 demonstrate very
low rates of recurrence and increased survival, indifferent to the stage of disease (Richter et al.,
1998). This indicates that alterations in TP53 and RB1 act in a synergistic fashion to promote tumor
progression.
2.2.2.2 Oncogenes
The actual pathogenetic role of oncogenes in TCC carcinogenesis is uncertain, but probably smaller
than that of TSG loss. Below are mentioned but two of the oncogenes that have commanded interest
in the BC setting.
C-erbB-2 (HER-2/neu)
This oncogene is located in chromosome band 17q21, shows structural homology with the
epidermal growth factor receptor gene (EGFR), and its protein product is presumed to function as a
growth factor receptor with ability to stimulate cell growth (Lee & Droller, 2000). Gene
amplification and increased protein expression of c-erbB-2 in BC are closely associated with a poor
prognosis, response to therapy, and poor overall survival in BC patients (Kohlberger et al., 1996).
There appears to be a discrepancy between gene amplification and protein expression in BC,
however (Coombs et al., 1991). Although the value of c-erbB-2 amplification as a prognostic
marker remains controversial (Lipponen, 1993), its use together with tumor grade and stage could
provide a more accurate basis for determining the prognosis of BC patients (Miyamoto et al., 2000).
RAS
The human ras gene family consists of three closely related genes: RASH, RASK, and RASN (Land
et al., 1983). Activation of the ras proteins usually occurs as a result of a single amino acid change
at positions 12, 13 or 61 following point mutations in the gene (Tabin et al., 1982). There is
evidence of specificity of activation of the RAS oncogenes in particular tumors types, with activated
RASH being found predominantly in urothelial carcinomas, RASK in lung and colon carcinomas,
and RASN in hematologic malignancies. A mutation frequency of 50% was reported for human
bladder tumor DNA samples using direct screening of PCR amplified tumor DNA with sequence-
specific oligonucleotide probes (Czerniak et al., 1999).

30
3 THE PRESENT STUDY

3.1 AIMS

The aims of this study were to explore and characterize the genetic alterations in different
etiologic, anatomic, and histopathologic types of uroepithelial cancer to determine the
clonal origin and composition of synchronous and metachronous multifocal uroepithelial
carcinomas, and to identify chromosomal abnormalities of possible pathogenetic and/or
clinical importance. These goals were approached from different angles, namely by:

1. Analyzing BC samples of different histologic types (TCC and SCC) to identify genetic
alterations involved in both tumor initiation and progression, and to see if the phenotypic
differences corresponding to these two main BC types are brought about by acquired
genetic differences.

2. Analyzing and characterizing the karyotypic profile of uroepithelial tumors from

different anatomic sites, i.e., the renal pelvis, ureter, and urinary bladder. Do these
tumors develop via the same or different pathogenetic mechanisms?

3. Characterizing the karyotypic profiles of uroepithelial carcinomas associated with

various etiologies such as parasite infestation, radiation, and chemical exposure. Do
differences in etiology between tumors arising in the same organ correlate with
pathogenetic differences in genetic alterations as well as with distinctive clinical
phenotypes?

4. Analyzing synchronous and metachronous uroepithelial carcinomas in order to determine

their clonal origin and composition, and to identify the mechanisms behind frequently
recurring tumors in this part of the urinary tract. Are multifocal and recurrent tumors
clonally related (indicative of intraluminal spreading) or unrelated (indicative of field
disease)?

31
3.2 MATERIALS AND METHODS

3.2.1 MATERIALS

3.2.1.1 Material for cytogenetic analysis

Sixty-eight uroepithelial carcinoma specimens (corresponding to 2 tumors of the renal pelvis, 8
ureteral, and 58 bladder tumors) were obtained from 55 patients, 44 males and 11 females (age 35-
89, mean 77 years) following surgery at the departments of urology at the Lund and Helsingborg
hospitals, between January 1996 and March 1999 (Tables 3.1-3.7). Of the 68 tumors, 47 were
solitary and 21 multifocal. Except 2 post-radiation and 7 recurrent tumors, all were primary
uroepithelial carcinomas and none of the patients had received prior radiation or cytotoxic
medication. Twelve tumors were obtained after radical cystectomy, 34 after TUR-T, and 12 were
cold cup biopsies. All ureteral and renal tumors were obtained after nephrouterectomy except one
ureteral tumor that was removed by segmental ureteral resection followed by ureter reimplantation.
When the tumor was obtained after T-TUR, the distal part of the tumor was cultured and the
proximal part was used for histologic examination. In tumors obtained following radical
cystectomies or cold cup biopsies, a piece of tumor tissue was taken close to that used for
histopathologic examination. In multifocal tumors, great care was taken to avoid contamination or
mixing when the macroscopically separate tumors were sampled or removed.

Five established cell lines from poorly differentiated, muscle invasive bladder carcinomas
(HTB-1, HTB-2, HTB-4, HTB-5, and HTB-9) were obtained from the American Type Culture
Collection (ATCC, Rockville, MD) (Table 3.2).
3.2.1.2 Material for SKY and FISH analyses
Metaphase spreads were obtained from 24 short-term cultured uroepithelial carcinomas and 5
established bladder cell lines for SKY and FISH analyses (Tables 3.2 and 3.3). Of the 24 short-term
cultures, 15 were from solitary tumors (13 primary and 2 recurrent), 4 were multiple tumors (1
primary and 3 recurrent), and one was from a post-radiation primary carcinoma.
3.2.1.3 Material for CGH and interphase FISH analyses
Fourteen formalin-fixed, paraffin-embedded blocks of post-bilharzial bladder carcinomas (8 SCC
and 6 TCC) and 6 non-neoplastic bilharzia-associated bladder lesions from 20 Sudanese patients
(12 males and 8 females) were obtained from the pathology archives of the Ibn Sina hospital and
the National Laboratory at Khartoum, Sudan (Table 3.8). All patients had a history of chronic
urinary bilharziasis. No radiation therapy had been given before tumor sampling.

3.2.2 METHODS

3.2.2.1 Cytogenetic analysis

All tumors were sampled in the operating room under aseptic conditions and transferred to 10 ml
sterile tubes containing 5ml of RPMI 1640 medium supplemented with 17% fetal bovine serum,
penicillin (100 IU/ml), and streptomycin (200 µg/ml). The specimens were brought directly to the
cytogenetics laboratory and processed for short-term culture within 30 minutes (tumors from Lund)
or 4 hours (tumors sent from Helsingborg). The specimens were washed in RPMI 1640, minced
with scalpels, and enzymatically disaggregated at 37° in 0.08 % collagenase II dissolved in Ham’s
F-10 medium for 30-60 minutes; for the cold cup biopsy specimens no collagenease treatment was
performed. The resulting cells and cell clumps were washed three times in RPMI 1640 medium and
plated out in 25 cm2 plastic flasks coated with vitrogen 100. The culture medium used was
Dulbecco’s MEM/F12 (1:1) supplemented with insulin (3 µg/mL), hydrocortisone (0.5 µg/mL),
cholera toxin (0.5 µg/mL), ascorbic acid (10 µg/mL), dibutyl cyclic AMP (10 nM), epidermal
growth factor (100 ng/mL), penicillin (100 IU/mL), streptomycin (200 µg/mL), fetuin (20µg/mL),
32
fibronectin (100 ng/mL), glutamine (2 mM), transferrin (25 µg/mL), phosphoethanolamine (0.1
mM), pituitary gland extract (80 µg/mL), and 5% fetal bovine serum.

All cultures were harvested within 10 days. Colcemid was added 3-4 hours before
harvesting and the cells were detached by trypsinization. After hypotonic shock in 0.05 M KCl, the
cells were fixed three times in methanol:acetic acid (3:1). G-banding was obtained with Wright’s
stain. Between 25 and 100 metaphase cells were analyzed for each tumor. The clonality criteria and
the karyotypic descriptions were according to the ISCN (1995) recommendations.
The 5 cell lines were cultured according to the protocols recommended by the ATCC. In
brief, cell lines were thawed rapidly by placing the ampule in a water bath at 37°C. The contents
were then transferred to a culture flask containing 5 ml of modified Dulbecco’s MEM/F12 (1:1)
medium. Culturing, harvesting, and metaphase spreads for FISH and SKY analyses were prepared
according to standard protocols.

3.2.2.2 Spectral karyotyping (SKY)

The probe cocktail containing 24 differently labeled chromosome specific painting probes and Cot-
1 blocking DNA (SKY kit, Applied Spectral Imaging, ASI, Migdal Haemek, Israel) was denatured
and hybridized to denatured tumor metaphase chromosomes according to the recommended
protocol. After hybridization and washing, the chromosomes were counterstained with DAPI.
Image acquisitions were performed using a SD200 Spectracube system (ASI) mounted on a Zeiss
Axioskop microscope with a custom-designed optical filter (SKY-1, Chroma Technology,
Brattleboro, VT, USA). The conversion of emission spectra to the display colors was achieved by
assigning blue, green, and red colors to specific sections of the emission spectrum.

3.2.2.3 Fluorescence in situ hybridization (FISH)

FISH analysis of metaphase spreads was performed using whole chromosome painting probes for
chromosomes 1, 3, 5, 6, 7, 9, 10, 11, 12, 14, 17, 18, and 22 labeled directly by nick translation using
FITC-12-dUTP and Texas Red-5-dUTP (DuPont, Boston, MA, USA) or Spectrum Orange-dUTP
(Vysis, Downers Grove, IL, USA). The results were analyzed using a Zeiss Axioplan 2 (Carl Zeiss
Jena GmbH, Jena, Germany) epifluorescence microscope and a Sensys (Photometrics Ltd, Tucson,
AZ, USA) charge-coupled-device camera interfaced to an IPLab Spectrum 10 workstation (Signal
Analytics Corporation, Vienna, VA, USA).
Interphase FISH was performed using pericentromeric probes for chromosomes X
(pBAMX7), 9 (pHuR98), and 17 (p17H8) labeled with FITC (DuPont) and digoxigenin using nick
translation. Prior to hybridization, xylene and absolute ethanol were used to remove the paraffin
from the slides. After air-drying, the samples were pretreated with 0.01 M sodium citrate (pH7.3) at
92ºC for 10 min, followed by proteinase K digestion (10 mg in SSC) at 45ºC for 30 min. The slides
were then rinsed in 2xSSC, dehydrated in an ethanol series, and air-dried. The probes with human
placental DNA were denatured together with the slides on a hot plate at 75ºC for 5 min, and
hybridized at 37ºC overnight. After washing and detection of digoxigenin with anti-digoxigenin
rhodamine, the slides were counterstained with 0.1 µM 4,6-diamino-2-phenylindole (DAPI) in an
antifade solution (Vectashield, Vector Laboratories, Burlingame, CA, USA). The analysis was
performed using an Olympus epifluorescence microscope equipped with a CCD camera
(Photometrics).

3.2.2.4 Comparative genomic hybridization (CGH)

From each case, 20-30 paraffin sections (thickness 3-4 µm) were prepared for DNA extraction
using the QIAamp Tissue Kit (QIAGEN GmbH, Germany). The yield of DNA was maximized with
prolonged proteinase-K digestion according to standard protocols. Tumor DNA samples were
labeled with FITC-dUTP (DuPont, Boston MA, USA) by nick translation, whereas normal
reference DNA was labeled with Texas Red (Vysis Inc., Downers Grove, IL, USA). In all cases, the
tumor and reference DNA samples were sex-matched. Tumor and reference DNA were mixed with
33
unlabeled Cot-1 DNA (Gibco, BRLTM), denatured, and applied onto slides with denatured
metaphases of normal lymphocytes (Vysis Inc.). After hybridization at 37°C for 48 h, the slides
were washed in 0.4x SSC/0.3% NP-40 at 74°C for 2 min and in 2x SSC/0.1% NP-40 at room
temperature for 1 min. After air-drying, the slides were counterstained with DAPI (Vysis Inc.). Two
control hybridizations were also performed. In the first, DNAs from a normal female and a normal
male were labeled and hybridized to normal male metaphases. For the second experiment, DNA
from a previously characterized breast cancer cell line (MPE 600, Vysis Inc.) and DNA from a
normal female were labeled and hybridized to normal male metaphases.
Six to 10 three-color digital images (DAPI, FITC, and Texas Red fluorescence) were
collected from each hybridization using a Zeiss Axioplan 2 (Carl Zeiss Jena GmbH, Jena, Germany)
epifluorescence microscope and a Sensys (Photometrics, Tuscon, AZ, USA) charge-coupled-device
camera interfaced to an IPLab Spectrum 10 workstation (Signal Analytics Corporation, Vienna,
VA, USA). Interpretation of CGH results was carried out as previously described (Weiss et al.,
1999). Green-to-red ratios >1.20 were considered to signify gains of genetic material and ratios <
0.80 losses. Heterochromatic regions, the short arms of the acrocentric chromosomes, and
chromosome Y were not included in the evaluation.

3.2.2.5 Histopathologic examination

The diagnosis, classification, and tumor grading were done without prior knowledge of cytogenetic
results. Grading was according to the WHO (1973) grading system and staging in accordance with
the UICC tumor-node-metastasis (TNM) classification system (Hermanek & Sobin, 1988).

3.2.2.6 Maps of breakpoints and imbalances

To help identify chromosomal changes in a more comprehensive manner, the data were also
presented as breakpoint and karyotypic imbalance maps (Figures 3.1 and 3.2). In order to report the
basic and most representative range of cytogenetic changes and to avoid cytogenetic noise brought
about by possible ploidy shifts, we included only 2n-4n clones for each tumor in the maps. In case
of duplicated aberrations found within a clone or in more than one related clone, the breakpoints
were plotted once only. If any aberrant chromosome was involved twice in the same clone or in a
related clone, only additional breakpoints were considered. When the same chromosome was
involved in both numerical and structural aberrations, we recorded only the total net imbalance, and
if the same imbalance was found in more than one related clone, it was recorded only once. If
additional gains or losses of the same chromosome or chromosomal segment were found in related
clones, only the largest imbalance was recorded.

34
3.3 RESULTS AND DISCUSSION

3.3.1 TRANSITIONAL CELL CARCINOMA OF THE BLADDER

3.3.1.1 General profile

The karyotypic profile of the 73 bladder TCCs (68 tumor specimens and 5 established cell lines)
examined in this study was dominated by clearly nonrandom chromosomal changes but
nevertheless demonstrated considerable heterogeneity, with the cytogenetic complexity ranging
from cases with only a single chromosomal anomaly (e.g., cases 13 and 25 in Table 3.1) to those
with massive rearrangements (e.g., cases 6 and 9 in Table 3.1). Deletions and losses of chromosome
copies were most frequently seen. Only 5 balanced translocations were seen, in contrast to 120
numerical changes and 86 unbalanced structural rearrangements.

In our first large series of bladder TCC (Table 3.1), all chromosomes were involved in
numerical and/or structural aberrations at least once. The most commonly involved were
chromosomes 9 (22 cases), Y (12 cases), 11, 15, and 18 (11 cases each), 1 (10 cases), 6 (9 cases),
and 5, 7, 8, 14, and 17 (8 cases each). A total of 15 unbalanced recurrent chromosomal
rearrangements were detected: del(1)(q21), i(1)(q10), i(5)(p10), del(6)(q21), i(11)(q10),
del(1)(p11), add(3)(q21), add(5)(q11), del(6)(q13), i(13)(q10), del(14)(q24), and i(17)(q10), of
which the latter 7 have not been described previously as recurrent aberrations in TCC of the bladder.

3.3.1.2 Chromosomal imbalances and breakpoints

A total of 443 chromosomal breakpoints were identified in the 50 uroepithelial TCC showing
structural chromosomal abnormalities (Figure 3.1), with the highest number of breakpoints (≥20)
being seen in chromosomes 1, 2, 3, 5, 6, 7, 8, 9, 10, 11, 13, and 17. The chromosome bands most
frequently involved were 1q21, 3q27, 5q10, 6q21, 8p11, 9q22, 10q22, 11p11, and 11q21 (each
involved at least 7 times).
The karyotypic imbalances resulting from the structural and numerical chromosomal
aberrations (Figure 3.2) were dominated by loss of an entire copy of chromosomes 9, 10, 16, 18, X,
and Y and gain of chromosomes 7, 19, and 20. Losses of the entire arm or parts of 1p, 6q, 8p, 9p,
11p, 13p, 15p and 17p and gain of 1q, 3q, 5p, 8q, and 13q were also common. The fact that
chromatin losses dominated the imbalance pattern indicates that loss of TSGs is the most important
pathogenetic consequence of the chromosomal aberrations.

3.3.1.3 Clonal origin

The absence of clonal heterogeneity within individual tumors in this study is in sharp contrast to the
massive cytogenetic intratumor variability observed in many other epithelial neoplasms, such as
tumors of the skin (Heim et al., 1989b), breast (Pandis et al., 1995), and pancreas (Gorunova et al.,
1998). It constitutes strong evidence that uroepithelial carcinomas are of monoclonal origin, a
conclusion also reached previously based on molecular genetic investigations (Sidransky et al., 1992).

3.3.1.4 Chromosome 9
Our findings present further support for the view that chromosome 9 is of primary importance in the
pathogenesis of bladder TCC. Changes involving chromosome 9 were the most frequent
cytogenetic alterations in our series, being seen in almost all tumors with abnormal karyotypes, be
they non-invasive, well-differentiated tumors or poorly differentiated tumors. Rearrangement of
chromosome 9 could be present as the sole change but also persisted in the massively complex
karyotypes of advanced tumors (Tables 3.1-3.7). Rearrangements of chromosome 9 were seen in
90% of the informative cases. Besides losses of material from either arm (cases 13 and 25; Table
3.1), which indicates the presence in both of different, pathogenetically important tumor TSGs, loss
35
of the entire chromosome 9 was the dominating change seen in 30 of the cases (60 %). By
comparison, loss from 9p only was seen in 10 cases (20%) and loss from 9q only in 5 cases (10%).
Molecular data are to some extent at odds with the cytogenetic picture in finding complete
monosomy for chromosome 9 only rarely in TCCs of the bladder, which has led some investigators
to conclude that loss of heterozygosity on chromosome 9 and loss of an entire chromosome copy
are separate events in TCC pathogenesis (Van Tilborg et al., 1998). The initial G-banding analyses
revealed monosomy 9 in 10 cases (9, 10, 12, 13, 15, 18, 19, 20, HTB-1, and HTB-2; Tables 3.1-
3.3). Subsequent SKY and FISH analyses of the same cases revealed true monosomy 9 in 7 cases,
and in cases 4, 9, 18, and 20, monosomy was the only aberration involving chromosome 9 (Table
3.3). Our findings thus further underscore the cytogenetic conclusion that loss of one copy of
chromosome 9 is a common and early change in uroepithelial carcinogenesis.
3.3.1.5 Karyotypic findings in superficial tumors
Apart from chromosome 9 (see above), chromosomes 1, 8, and 11 seemed to be preferentially
involved and were found to be rearranged in 18 cases (33%), 19 cases (35%), and 24 cases (44%),
respectively (Tables 3.1-3.7). The most common consequences of the changes in chromosome 1
were gain of 1q material and loss of 1p material. Losses from 8p and gain of 8q were the dominant
net results of the changes affecting that chromosome. Although often found in simple karyotypes in
low-grade tumors (Tables 3.1-3.7), the aberrations involving chromosomes 1, 8 and/or 11 were
never the sole change in our series but typically accompanied chromosome 9 loss (e.g., cases 10 and
29; Table 3.1). Thus, losses of material from chromosome arms 1p, 11p, and 8p and gains of 1q and
8q seem to be early, but nevertheless secondary, changes appearing in superficial and well
differentiated tumors. Our findings are in agreement with the molecular genetic results obtained by
Sauter (1997), who found 1q gain and 11p loss to be more frequent in pT1 than in pTa tumors. The
important molecular consequences of these chromosomal changes are unknown.

3.3.1.6 Chromosomes associated with aggressive biological tumor behavior

The formation of an isochromosome for 5p, i(5)(p10), leading to net gain of material from the short
arm of chromosome 5, was associated with more aggressive tumor phenotypes (Table 3.1). An
i(5)(p10) has previously been reported in 9 bladder TCCs (Babu et al., 1987; Gibas et al., 1984,
1986; Koo et al., 1999), making it the single most common recurrent structural chromosomal
abnormality in BC. In most of the reported cases as well as those of the present study, the tumors
were muscle-invasive and poorly differentiated. Considering the fact that this marker has not been
consistently linked to any other malignancy and that it is seldom seen in association with
rearrangements of chromosome 17, it appears to characterize a subgroup of advanced TCC of the
bladder arisen via a unique pathogenetic pathway.
Rearrangement of chromosome 17 resulting in loss of material from the short arm was seen
in 21 cases, of which only 13 were high-grade, muscle-invasive tumors, whereas the remaining 8
were papillary and superficial (Tables 3.1-3.7). Previous cytogenetic studies have indicated that 17p
losses are restricted to advanced TCC (Olumi et al., 1990). We believe that our findings, too, may
fit such a cytogenetic-pathologic correlation. Prognostic heterogeneity in superficial BC is massive
and it has been suggested that early loss of 17p, combined with mutational inactivation of the TP53
allele on the other chromosome 17, could be responsible for the aggressive behavior seen in 16-25%
of superficial BCs (Kiemeney et al., 1993b). It is an attractive possibility, therefore, that the 15
cases we observed with loss of 17p belong to a subgroup of superficial BCs harboring a tendency to
more aggressive clinical behavior in the future. Naturally, it will be of great clinical interest to
follow these patients closely and to see whether the natural history of their disease indeed displays
such features.

36
 5
6 4
5 23
4 2 6 6
5
33 1
4
5
2 6 5
3 14
5 22 4
1 4 3 3
13 1 21 21
2 1 1
2 2
1 3 2
2 4
1 1 13
3 1
2 1
12 13 2 2 4
1 4 1 3 5
1 1 4
25
2 1
12 1
6 22
2 3 7
1 3
3 8
2 4 1
23 2 1
4 1 3 1
5 4 32
2 32 3
5 3 4
1
3 4 5
3 4 6 5
2 5
6
7
8 4 5
9
1 7
42 3
3
4
2
1
5 3 5
4 2 4 5 3
3 4
21 22 3 3
22 12
22 1 12 14
5 2 1 1
11 1 3
1 4 3 2 1
13 1 2
1 2 11 1
2 2 1
11 1 12 1 1 13
2 4
3 12
21 11 3
1
5
3 13
14 1
5 1 2 4 1
6 1 2
2 2 23 1 2
2 2 2 23

37
1 2
3 1 4
23 4
2 1 2 5
4 33 4
23 6
4
32
3
4
11 12
5
4
5 8 10
6
7
6 9
6 7 3
12 11
3 3 1 1
3 3 12 12 1
12 12
2
11 12 1
1 1 1 1
1 1 2
3
14 1 1
2 12 2
5 12 23 2
13 3 3 2
4 1 1 4 3
1 5
2 2
2
1 2
23 23
23 18
2 4
4 4 17
1 1
5
6 16
32 32
3
4
15
13 14 2
2
1
11
11
2 1
3 3 13 1
13 2 3 2
11
2
1
12 1 1
1 1
1 11 1 2
2 1 2 2
2 1 1 3
13 2 24
3
3 2 5
6
Y
19 20 21 22 7
8
X
Figure 3.1 Distribution of the 443 breakpoints observed in structural chromosomal aberrations in 50 uroepithelial carcinomas
 6
5 5
4 4 6
5
33 23 4
2 2 3
1 22
1
6 1
5
2 4 6
2 13 4 5
1 2 5
3
1 1 4
14
2
12 1 3 3
1 1 21 21
1 2 1 1
12 13 2 2 1
4 3 2
3
1 1 1 13
2 2 1 4
23 2 2
4 3 3 3 5
4
5 4 4 25 1
5 6 22
1 1
7 3
3 2 6 8
2 7 1
3 8
9 1
4 2
1
5 33
4
2
3
6 3 32
3 4
4 7 4 5
5
1 2 4 5
5
4
3 2
21 5
22 3 4
3
5
4
22 22
1 5 1 1
3 14
4 12 3
13 2 3 2
2
11 2 11 1 1
21 1
1 1
1 1 1
2 11 2
3 11 12 12
14 3 3 1 13
5
6 1 1 1 4
1 2 2 2
2 1
2 2
2 23
2 2
1 3 1 23
23 2 4
33 4
4 2
33 5
4

38
5 4 4
6
6 5
6 11
7 8 9 10
6 7
3 3
3 3
12 3 3 12
2
11 12 12 12
1 1 1 1
1 1
1
21 2 1 2 1 1
12
12 13
4 12
13 13 3 5 3 1
4 4 1
1 1 2 2
5 23 23
1 2 2
2 23 4 4
1 23
2 4 5
2
23 1
4
5 16
32
1
32
6 17
4 3
4 15
13 14
12
2
2
1
3
2 3 3
11 13 2
11 2
11 11
2 1 11
1 11 1
1 1 1 1 2
12 2 1 13 1
2 2
13 2 1
3 2
3 1 1
1 2
22
19 20 21 22
2
3
3 24
5 Y
18 1 2
6
7
8
X
Figure 3.2 Karyotypic imbalances caused by numerical and structural chromosomal
aberrations in 54 uroepithelial carcinomas. Losses are shown to the left, gains to the right
3.3.1.7 Correlation between karyotype and tumor grade and stage
Although the present grading and staging systems for BC are often unable to predict the prognosis
in superficial disease (Wijkström et al., 1984), a strong correlation nevertheless exists between the
grade and stage of the tumors. Most well- and moderately differentiated tumors are superficial and
non-invasive, whereas poorly differentiated tumors tend to be muscle-invasive. An interesting
perspective on the present material was to examine whether the tumors’ stage and grade correlate
with their karyotypic complexity. In our series, all superficial and well-differentiated tumors (TaG1)
were pseudo- or near-diploid and exhibited simple karyotypes (5 or fewer chromosomal changes).
A progressive increase in the number of chromosome aberrations with tumor grade and/or stage was
evident (Tables 3.1-3.6), with TaG1 tumors showing less abnormal karyotypes than did those that
were T1G2, which, in their turn, were less abnormal than T2G3 tumors, all in agreement with the
view that the clinical progression of uroepithelial tumors is steered by the synergistic effect of
accumulated genetic alterations. If systematic, genome-wide cytogenetic screening of BC is
commenced to quantify the genetic differences between various disease grades and stages, the
information thus obtained may make possible a pathogenetic classification of the disease that may
have an impact on the treatment as well as follow-up of patients suffering from these malignancies.

3.3.1.8 Combined G-banding, SKY, and FISH analyses

Combined analysis was used in the present study to characterize a heterogeneous set of uroepithelial
carcinomas that included primary and post-radiation tumors, solitary and multiple lesions, and
primary cultures with simple karyotypes as well as cell lines with complex rearrangements, in order
to assess the efficiency of such a multipronged cytogenetic approach. The cytogenetic information
was greatly enhanced by using the combined analysis compared with what was obtained by G-
banding analysis alone, resulting in a more comprehensive description of all examined tumors by
identifying 32 additional numerical changes, by establishing the chromosomal origin of 27 markers
and 2 ring chromosomes, by redefining 53 aberrations, and by detecting 15 hidden chromosomal
rearrangements. The descriptions of the cell lines were also enhanced by redescribing 23 structural
changes, leading to the identification of 6 novel structural aberrations and 15 numerical aberrations.
However, no recurrent translocation was detected and of the 25 tumors analyzed, complete
cytogenetic description was possible only in 8 cases (cases 2-4, 5, 8, 9, 12, and 17; Tables 3.2 and
3.3), whereas in the remaining 17 (5 cell lines and 12 uroepithelial carcinomas), uncertainties with
regard to breakpoint positions remained.
We conclude that the combination of G-banding, SKY, and other FISH techniques
significantly enhances the information obtainable with any one technique used alone. Thus SKY
and similar methods should be used to complement, not to replace, banding cytogenetics in the
analysis of TCC as well as other malignant tumors.

3.3.2 UPPER URINARY TRACT CARCINOMAS

Because of the great similarities between upper urinary tract carcinomas (UUTC) and bladder TCC
with regard to etiology, histology, and natural disease history, one would assume that also the
tumorigenic mechanisms, including the karyotypic profile, are more or less identical. However, the
very limited information on UUTC cytogenetics published prior to our study (Berger et al., 1986;
Grammatico et al., 1996; Sandberg et al., 1986) indicated substantial karyotypic differences
between UUTC and bladder TCC. Ours was the first large series of UUTC successfully analyzed by
chromosome banding technique (Table 3.4), and we found UUTC to have karyotypic features
indistinguishable from those of bladder TCC.
Loss of material from chromosome 9 was seen in all informative UUTC in our series. The
ubiquity of chromosome 9 involvement thus seems to be no less pronounced in UUTC than BC, in
agreement with the view that this is an early and crucial event in the genesis of all or nearly all
urinary tract TCC, regardless of their site and stage. The high frequency (all informative cases) of
39
chromosome 9 involvement in upper urinary tract transitional cell carcinogenesis makes this a
logical target for a possible genetic marker to be used in the follow-up of patients with such tumors,
something that would be particularly useful considering how frequent downstream disease
spreading is in these patients.

3.3.3 POST-RADIATION UROEPITHELIAL CARCINOMAS

The two (bladder and ureteral) post-radiation tumors examined in the course of this study fulfill the
criteria for second primary radiation-induced cancers (Ravi, 1993). They developed in an irradiated
field after radiotherapy. The histological type was different from that of the previous primary
tumors; they were already poorly differentiated, and one had infiltrated the underlying muscle at the
time of diagnosis. To our knowledge, no previous report on the cytogenetics of post-radiation
uroepithelial carcinomas exists. The extensive cytogenetic analysis of the tumors revealed 10
mostly unrelated, near-diploid, karyotypically abnormal clones characterized by rather simple
balanced and/or unbalanced translocations in each tumor (Table 3.5). The intratumor karyotypic
heterogeneity seen in these cases indicates massive polyclonality in contrast to the cytogenetic
monoclonality otherwise consistently demonstrated in urinary tract TCC (Sidransky et al., 1992).
Although the tumors we describe were advanced as far as grade and clinical stage are
concerned, most of the clones were pseudo- or near-diploid with relatively few karyotypic
aberrations. Again, this is seemingly at odds with the strong association that has been shown
between tumor ploidy or karyotypic complexity and the tumor’s clinical stage and degree of
differentiation. In all likelihood, this reflects a carcinogenic field effect of the previous radiation,
illustrating that post-radiation urinary tract tumors are distinct from other urinary tract TCC not only
etiologically but also with regard to the pathogenetic mechanisms involved.

3.3.4 MULTIFOCAL UROEPITHELIAL CARCINOMAS

Around 70% of bladder carcinomas are superficial at diagnosis (Thompson et al., 1993). Of these,
30% are found as multiple tumors and as many as 60% of the patients develop recurrence(s) after
transurethral resection of the primary tumor(s) (Kiemeney et al., 1993a). Three mechanisms have
been suggested for the polychronotopicity of uroepithelial carcinomas: (1) Implantation of cancer
cells shed from the primary tumor, (2) growth of new carcinoma(s) at remote sites, and (3) regrowth
of an incompletely resected primary tumor.
All examined cases (n=6) showed consistent cytogenetic monoclonality among
macroscopically distinct lesions in each patient, including the recurrent tumor (Table 3.6). In two of
the patients (cases 3 and 4), bladder tumor(s) were associated with a ureteral tumor. Since all the
tumors in each case, irrespective of their site, were cytogenetically related demonstrating their
common clonal origin, and since downstream rather than upstream shedding of cancer cells is more
likely, the ureteral tumors should be considered the primary ones. The data speak strongly against
the regrowth of a new tumor at a site different from the primary and instead favor the shedding and
intraluminal seeding hypothesis. Epidemiological data are also consistent with this conclusion. The
presence of an upper urinary tract tumor is associated with a 75% probability of finding tumor(s)
also in the bladder, whereas the risk of developing a ureteral tumor following the diagnosis of a
bladder tumor is only 4% (Kenworthy et al., 1996).
The extensive cytogenetic similarity observed among all tumors from the same patient, each
tumor sharing 5-8 numerical and/or structural chromosome aberrations, suggests, in addition to the
common clonal origin, that seeding is a rather late event; such similarity is hardly compatible with
an early splitting up of the monoclonal, neoplastic cell population followed by the acquisition of
similar secondary genetic changes by all cell colonies. The limited cytogenetic information allows
no conclusion as to whether tumor cell shedding with subsequent implantation and growth is more
likely to occur from the first or later (second) primary tumors, but judging from the clinical and
epidemiological data, both situations are common (Herr, 1997).

40
 Rearrangement of chromosomal 9, leading to loss of material from the short and/or the long
arm, was seen in all multifocal cases, again indicating that this is an early, pathogenetically
important event in transitional cell carcinogenesis which precedes the shedding and seeding of
cancer cells. The findings in our study have considerable potential clinical relevance as they open
the door for a molecular marker test based on these changes for the follow-up of patients with
uroepithelial carcinomas. The fact that intraluminal shedding and implantation of tumor cells is the
mechanism responsible for both synchronous and metachronous multifocal BC, may also be of
considerable relevance for therapeutic strategies. Complete endoscopic removal of the primary
tumor is often not enough even in the treatment of superficial BC. Additional measures should be
directed toward stopping tumor cell seeding and the ability of already implanted tumor cells to
divide. This implies more emphasis on intravesical adjuvant therapy in superficial BC, possibly
including the use of anti-inflammatory drugs, antagonists of cell adhesion, and intravesical
chemotherapy or immunotherapy with BCG.

3.3.5 SQUAMOUS CELL CARCINOMA OF THE BLADDER

Two secondary SCC of the bladder (i.e., tumors with squamous cell differentiation but which began
as primary transitional cell carcinomas), were examined cytogenetically. Both tumors displayed
complex karyotypes with numerous numerical as well as structural chromosomal rearrangements
similar to what has been seen in advanced bladder TCC (–9, +7, loss of 8p, 11p, 17p and formation
of i(5)(p10); Table 3.7). The detected chromosomal aberrations therefore provide no clear-cut
explanation for the SCC differentiation. When TCC undergo squamous changes, this is usually
associated with increasingly aggressive tumor behavior. Due to the meager data available on genetic
characteristics of bladder SCC, and the highly complex karyotypes in these tumors, it is still unclear
what controls this aspect of tumor phenotypes. We cannot rule out the possibility that epigenetic
factors are important for the altered differentiation observed.

3.3.6 BILHARZIA-ASSOCIATED BLADDER LESIONS

Seven of the 20 examined lesions (6 carcinomas and one granuloma; Table 3.8) showed
chromosomal imbalances varying from 1 to 6 changes. The most common imbalances detected
were losses of 1p21-31, 8p21-pter, and 9p and gain of 19p material, seen in three cases each,
including the benign lesion. The FISH analysis of the same cases yielded findings consistent with
those obtained by CGH.
Bilharzia-associated bladder cancer (BAC) is a major health problem in countries where
urinary schistosomiasis is endemic. At the time of the first diagnosis, more than 80% of patients
with BAC present with muscle infiltrating disease. Unfortunately, at this late stage, even extensive
therapeutic measures, such as radical cystectomy with urinary diversion, give long-term survival
only in 27–39% of the cases. Thus, any improvement in disease outcome probably depends mainly
on earlier detection of the disease. The main objective of the present study was to determine the
genetic imbalances involved in post-bilharzial carcinogenesis, to see whether the unique etiology of
this cancer yields a distinct cytogenetic profile compared to chemically induced bladder tumors. We
also wanted to find out if preneoplastic bilharzia lesions carry chromosomal aberrations resembling
those seen in carcinomas. Such changes, if present, would presumably constitute the earliest steps
of a putative multistage cascade (Heim & Mitelman, 1989) of post-bilharzial carcinogenesis,
possibly eventually leading to a molecular genetic test based on such changes capable of detecting
early tumors in high-risk individuals.
With the exception of the gain of chromosome material from 19p observed in three cases
(2228-99, 476-98, and 479-98), almost all changes detected in post-bilharzial tumors (complete or
partial loss of 1p, 8p, 9p, and 13q, and gain of 5p, 17q, and 20q) have been previously reported as
recurrent changes in chemically induced TCC of the bladder (Heim & Mitelman, 1995). For the
latter aberrations, the available data therefore indicate that they form part of a pathogenetic pathway
followed by both BAC and chemical carcinogen-induced BC. The data also indicate loss of material
from 9p in both malignant and non-malignant bilharzial lesions; this may constitute an initiating
event in BAC. Accordingly, one can envisage the use of a molecular genetic test based on the
41
consistent loss of 9p in cells detached into urine for screening purposes. The data are of course too
meager to draw firm conclusions about differences in the karyotypic profile corresponding to two
etiologies, namely parasite infestation versus chemically induced bladder carcinomas, but viewed in
concert with what has already been reported by other investigators (Ghaleb et al., 1996; Gonzalez-
Zulueta et al., 1995; Tsutsumi et al., 1998), some features come across as potentially meaningful.
BAC appears to follow a narrower pathogenetic pathway (loss of 9p) than is the case for BC in the
industrialized world (the earliest genomic change in these tumors is loss of 9p, loss of 9q or loss of
the entire chromosome). More data are obviously needed to resolve this issue, and the same also
goes for the possibility that 19p gains may constitute an avenue for progressional, evolutionary
changes taken by BAC exclusively.

42
3.4 CONCLUSIONS
1. The karyotypic profile of bladder TCCs is characterized by nonrandom chromosomal
aberrations varying from one or few changes in low-grade and early stage tumors to
massively rearranged karyotypes in muscle invasive ones. In general, the karyotypic
profile is dominated by losses of chromosomal material. There is little intratumor
cytogenetic heterogeneity and cytogenetically unrelated clones are never observed,
strongly indicating that tumorigenesis is monoclonal.
2. Rearrangements of chromosome 9 resulting in loss of material from 9p, 9q, or of the
entire chromosome are the most frequent cytogenetic alterations. These observations are
seen as the sole change as well as in massively complex karyotypes, indicating that loss
of TSG(s) from this chromosome is of major importance in uroepithelial carcinogenesis.
3. Whereas loss of material from chromosome arms 1p, 11p, and 8p and gains of 1q and 8q
seem to be early, but secondary, changes appearing in superficial and well differentiated
tumors, the formation of an isochromosome for 5p and loss of material from 17p are
associated with more aggressive tumor phenotypes.
4. The karyotypic profile of upper urinary tract TCC is identical to that of bladder TCC,
indicating that the same pathogenetic mechanisms are at work in both locales.
5. Post-radiation uroepithelial carcinomas have a distinct karyotypic and clonal pattern that
is characterized by massive intratumor heterogeneity (cytogenetic polyclonality) with
near-diploid clones characterized by rather simple balanced and/or unbalanced
translocations.
6. Secondary SCCs of the bladder are karyotypically indistinguishable from advanced
TCC of the same organ. The putative genetic changes that steer the differentiation of the
neoplastic epithelium in the direction of squamous cells thus remain unknown.
7. Although the cytogenetic profiles of chemical- and bilharzia-induced carcinomas are
largely similar, bilharzia-associated bladder tumors seem to follow a more narrow
pathogenetic pathway characterized by loss of material from 9p as the initiating event
and gain of material from 19p as a later, progressional event.
8. Multifocal uroepithelial tumors have a monoclonal origin and arise via intraluminal
seeding of viable cancer cells shed from the original tumor. Later lesions may develop
also from cells shed from so-called second primary tumors. The relatively complex
karyotype seen in all lesions from most cases argues that the seeding of tumor cells is a
comparatively late event that succeeds the acquisition by them of multiple secondary
genetic abnormalities. Rearrangement of chromosome 9 precedes the intraluminal
dissemination of the disease.
9. Because intraluminal shedding and implantation of tumor cells is the mechanism
responsible for both synchronous and metachronous multifocal uroepithelial
carcinomas, complete endoscopic removal of the primary tumor is often not enough in
the treatment of even superficial BC. In addition to endoscopic removal of the
superficial tumors, measures against the intraluminal seeding of the cancer cells and
their ability to divide should be taken.
10. The high frequency of chromosome 9 involvement in urinary tract transitional cell may
form the basis for a genetic marker to be used in the follow-up of patients with BC,
something that would be particularly useful considering the high frequency of
recurrence in these patients.
43
SAMMANFATTNING PÅ SVENSKA
Cancer är på cellulär nivå en genetisk sjukdom, som uppkommer genom ansamling av icke -letala
mutationer. Dessa mutationer, som kan ske på både DNA- och kromosomnivå, ger cellen
tillväxtfördelar och leder till klonal expansion av en cellpopulation som står utanför normal
tillväxtkontroll. Utvecklingen frå n normal till malign fenotyp sker stegvis, där en ökad ansamling
av genetiska skador är associerad med allt mer aggressivt växtsätt. Cancerassocierade gener kan
grovt sett indelas i två huvudgrupper: (1) gener involverade i uppkomsten av tumörer och (2) gener
involverade i tumörprogression och sjukdomsförlopp. För närvarande har man mycket mer
detaljerad kunskap om de genetiska händelser som är av betydelse för uppkomsten av tumörer än
om de som styr den vidare tumörutvecklingen.
Cancer i urinvägarna som är en vanlig tumörsjukdom i Europa och USA, särskilt hos män,
intar en fjärdeplats efter cancer i lunga, prostata och tjocktarm och är den tolfte vanligaste orsaken
till dödsfall orsakad av cancer. I områden med endemisk kontakt med sugmaskar, Schistosoma
haematobium, är urinvägscancer ännu vanligare och svarar för 25% av all cancer hos män. I Europa
och USA är carcinom av uroepitelial typ (transitional cell carcinoma, TCC) den dominerande
formen (90%), medan skivepitelcancer (squamous cell carcinoma, SCC) är sällsynt. I Mellersta
Östern och Afrika är bilden den omvända, med en dominans av SCC. Cirka 30% av TCC i
urinvägarna uppträder som multipla tumörer vid diagnostillfället. Trots att 70% av
nydiagnosticerade TCC i blåsa är ytliga, d.v.s. begränsade till lamina propria, utgör sjukdomen en
betydande terapeutisk utmaning och ett ständigt hot mot patienterna eftersom tumörerna recidiverar
efter transuretral resektion av primärtumören i mer än 70% av fallen. Dessutom uppträder
progression till mer avancerade stadier och/eller malignitetsgrader i 30% av fallen.
I delarbete I i avhandlingen beskrivs karyotypen i två blåscarcinom med sekundär
skivepitelsdifferentiering, en typ av tumörer som inte tidigare undersökts cytogenetiskt. Mönstret av
kromosomavvikelser skiljde sig inte markant från det som förekommer i TCC. I delarbete II
analyserades ett strålinducerat blåscarcinom. I motsats till vad som kännetecknar spontana
blåscarcinom uppvisade denna tumör multipla, cytogenetiskt obesläktade kloner, vilket tyder på
polyklonal carcinogenes. I delarbete III beskrivs cytogenetiska analyser av multifokala
blåscarcinom. Förekomst av samma klon i multipla lesioner hos samma patient visar att tumörerna
är monoklonala och följaktligen måste ha uppstått genom metastasering från en första, ursprunglig
lesion. I delarbete IV kunde det visas att carcinom i de övre urinvägarna, i motsats till vad som
tidigare har rapporterats, har samma karyotypmönster som uroepiteliala blåscarcinom. I delarbete V
presenteras resultaten från en omfattande cytogenetisk analys av totalt 34 primär a uroepiteliala
blåscarcinom. Mönstret av de observerade kromosomavvikelserna pekar på karakteristiska
patogenetiska utvecklingsvägar hos dessa tumörer. Vidare visade det sig åter att varje enskild tumör
var monoklonal. I delarbete VI användes spektralkaryotypering och fluorescens in situ hybridisering
som supplement till G-bandsanalys för att kunna göra en så komplett cytogenetisk karakterisering
som möjligt av uroepiteliala carcinom. I delarbete VII beskrivs molekylärcytogenetiska analyser
och jämförelser med hjälp av komparativ genomisk hybridisering av bilharzia-inducerade (Från
Afrika) och sporadiska (från Sverige) blåslesioner. Även om många kromosomavvikelser var
gemensamma för de båda grupperna av tumörer, fanns också enstaka skillnader som möjligen kan
peka på att olika patogenetiska utvecklingsvägar följs beroende på etiologin.
Följande slutsatser har kunnat dras:
1. Profilen av kromosomavvikelser hos TCC i blåsa kännetecknas av icke slumpmässiga
förändringar, som kan variera från ett fåtal förändri ngar i låggradiga tumörer och tumörer i ett
tidigt stadium till massivt rearrangerade karyotyper hos invasivt växande tumörer. I allmänhet
domineras karyotypen av förlust av kromosomsegment. De enskilda tumörerna uppvisar
obetydlig cytogenetisk heterogenitet och cytogenetiskt obesläktade kloner påträffas aldrig (med
undantag av strålinducerade tumörer), vilket starkt antyder en monoklonal tumorigenes.

44
2. Rearrangemang av kromosom 9, som leder till förlust av material från 9p, 9q eller hela
kromosomen är de vanligaste cytogenetiska förändringarna. Dessa avvikelser kan uppträda såväl
som den enda förändringen som en av många avvikelser i massivt komplexa karyotyper, vilket
antyder att förlust av en eller flera tumörsuppressorgener belägna i denna kromosom är av
väsentlig betydelse för den uroepiteliala carcinogenesen.
3. Medan förlust av kromosomarmarna 1p, 11p och 8p samt tillskott av 1q och 8q tycks vara tidiga,
men sekundära, förändringar som uppträder i ytliga och väldifferentierade tumörer, så tycks
bildningen av en isokromosom för 5p och förlust av material från 17p vara associerat med mera
aggressiva tumörfenotyper.
4. Karyotypprofilerna hos TCC i de övre urinvägarna respektive blåsa är identiska, vilket antyder
att samma patogenetiska mekanismer är aktiva i de båda fallen.
5. Strålinducerade uroepiteliala carcinom tycks uppvisa speciella särdrag vad gäller karyotyp - och
klonmönster, nämligen en omfattande intratumör-heterogenitet (cytogenetisk polyklonalitet)
med närdiploida kloner kännetecknade av tämligen enkla balanserade och/eller obalanserade
translokationer.
6. Sekundära SCC i blåsa är ur karyotypsynpunkt ourskiljaktiga från avancerade TCC i samma
organ. De tänkbara genetiska förändringar som styr differentieringen av neoplastiskt epitel i
riktning mot skivepitelceller är ännu okända.
7. Även om de cytogenetiska profilerna hos kemiskt respektive bilharzia-inducerade carcinom i
grova drag är likartade, så tycks bilharzia -associerade blåstumörer följa en smalare, mera
enhetlig patogenetisk utvecklingsväg karakteriserad av förlust av material från 9p som en
initierande händelse och tillskott av material från 19p som en senare, progressionsrelaterad
händelse.
8. Multifokala uroepiteliala tumörer har ett monoklonalt ursprung och uppkommer genom
intraluminal spridning av viabla cancerceller som stöts ut från ursprungstumören. Senare
uppkomna lesioner kan även utvecklas från celler som stötts ut från s.k. sekundära
primärtumörer. De relativt komplexa karyotyper som i de flesta fall förekommer i samtliga
lesioner hos en patient ger vissa belägg för att utstötning av tumörceller är en förhållandevis sen
händelse som inträffar sedan de förvärvat multipla sekundära genetiska förändringar.
Rearrangemang av kromosom 9 föregår den intraluminala spridningen av sjukdomen.
9. Eftersom intraluminal spridning och implantation av tumörceller är den mekanism som ligger
bakom både synkrona och metakrona multifokala uroepiteliala carcinom, så är fullständigt
endoskopiskt avlägsnande av primärtumören ofta inte ett tillräckligt ingrepp vid behandlingen,
inte ens vid ytliga blåscarcinom. I tillskott till endoskopiskt avlägsnande av ytliga tumörer borde
även åtgärder riktade mot intraluminal spridning av cancerceller och deras förmåga att dela sig
tillgripas.
10. Den höga frekvensen av förändringar av kromosom 9 hos TCC i urinvägarna skulle möjligen
kunna användas som en genetisk markör och utgöra grunden i en förbättrad uppföljning av
patienter med blåscarcinom, något som skulle vara synnerligen användbart med tanke på den
höga frekvensen av recidiv hos dessa patienter.

45
ACKNOWLEDGMENTS
I would like to express my sincere gratitude to:
Professor Sverre Heim, my scientific supervisor who has been a source of knowledge, advice,
support, and inspiration. His immense knowledge in the field of cancer cytogenetics and his positive
criticism throughout this study have been extremely valuable.

Professor Felix Mitelman, for introducing me to the fascinating field of cancer cytogenetics. His
friendly and professional attitude has surrounded him with pleasant and qualified people and has
created an excellent working atmosphere.

Ludmila Gorunova, my co-supervisor for introducing me to cell culturing and sharing with me her
unlimited laboratory knowledge.

Professor Nils Mandahl for practical help with karyotyping and for allowing me to take up a lot of
his valuable time.

My fellows Ph. D. students and all the guest researchers for stimulating discussions and nice
company.

The personnel at the Department of Clinical Genetics, Lund University hospital for help and for the
scientific and friendly atmosphere.

My colleagues and co-authors at the Departments of Urology and Pathology at Helsingborg and
Lund hospitals. I am also very grateful to the operating-theatre staff at the same hospitals for
assistance in obtaining the tumor samples. Their support was crucial for the completion of this
study.

My co-authors and collaborators at the Departments of Molecular Medicine and Urology at the
Karolinska hospital, Stockholm, Sweden and at the Laboratory of Cancer Genetics, University of
Tampere and Tampere University hospital, Tampere, Finland;

My co-authors and collaborators at the Department of Molecular Biology, Institute of Endemic

Diseases, University of Khartoum, Sudan and at the Tropical Medicine Research Institute,
Khartoum, Sudan for their fruitful collaboration.

For financial support I wish to thank the Medical Faculty of Lund University, the Swedish Cancer
Society, the Norwegian Cancer Society, the Swedish Society for Medical Research, the Swedish
Match AB, and the John and Augusta Perssons Fund for Medical Research.

Finally, yet importantly, I thank my entire family for their love and support.

46
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APPENDIX
Table 3.1 Clinical and cytogenetic data on 30 transitional cell carcinomas of the bladder
Case Sex/age Stage/grade Karyotype
1 M-55 pT4aG3 62~71,XXY,+Y,del(1)(p21),+del(2)(p13),i(5)(p10),+6,del(6)
(q21)x2,-9,-9,del(10)(p12),del(14)(q24),-15,-15,del(16)(p12),
-17,+18,+del(20)(p11),+21,+22,+der(?)t(?;2)(?;p13)[cp86]
2 M-77 pTaG2 45,X,-Y[15]/46,XY[25]
3 M-73 pT4aG3 45~46,X,-Y,t(5;10)(q35;q22),+7,inc[cp24]/46,XY[18]
4 M-54 pT4aG3 57~90,XY,+der(2)t(2;4)(q33;q21),i(13)(q10),add(13)(p11),inc[cp6]
5 M-75 pT1G2 45,X,-Y[10]/46,XY[23]
6 M-67 pT1G2 44,X,-Y,del(2)(q11q21),del(7)(p13p15),inv(8)(p21q24),-9,
del(10)(q23),der(11)del(11)(p11)del(11)(q22q23),del(14)
(q24),-17,der(18)t(17;18)(q11;q12),+mar[30]/45,X,-Y,
+del(1)(p11),del(2)(q11q21),-9,del(10)(q23),der(11)del(11)
(p11)del(11)(q22q23),del(14)(q24),-17,der(18)t(17;18)
(q11;q12),+mar[25]/45,X,-Y,+del(1)(p11),del(2)(q11q21),
del(5)(q11q22),-9,del(10)(q23),der(11)del(11)(p11)del(11)
(q22q23),del(14)(q24),-17,der(18)t(17;18)(q11;q12),+mar[50]
7 M-78 pTaG1 45,X,-Y[7]/46,XY[25]
8 M-76 pT1G2 47,XY,t(5;9)(q31;q12),+6,-9,der(11)t(1;11)(q12;p11),+20[19]
9 M-77 pT2G3 72~76,XXY,-1,del(1)(q21),add(2)(q11)x2,+der(2)t(1;2)(q11;q13)
ins(2;?)(q13;?)x2,+der(2;3)(q10;q10),del(4)(p14),i(5)(p10),
+der(5)t(5;12)(q11;q11),+add(6)(q11),+del(6)(p12),+del(6)
(q13),add(7)(q11),+add(7)(q11),-9,-10,-11,add(11)(q13),-12,
add(12)(q11),-13,-14,-15,-17,-18,add(19)(p13),i(19)(q10),+20,
-21,-22,+der(?)t(?;1)(?;p13),+r,+8mar[cp73]
10 M-74 pTaG1 45,XY,-9,-11,+15,-18,+mar[53]
11 M-73 pT1G2 90,XXY,-Y,-6,-6,-9,-14,i(17)(q10),+3mar[3]
12 F-71 pT1G2 71~103,XX,-X,-X,add(1)(p13),-2,-4,der(5)t(5;6)(q31;p21),
der(5)t(5;15)(q22;q15),add(6)(q11)x2,dup(8)(q21q24)x2,-9,
-9,i(11)(q10),i(13)(q10),-15,-15,-18,-20,-20,+4mar,inc[cp4]
13 M-69 pTaG1 46,XY,del(9)(q12q22)[5]
14 M-80 pT2G3 51~53,XY,+7,+8,del(9)(q13q22),+15,+18,+20,+2mar[cp30]
15 M-35 pTaG1 46,XY[12]
16 M-65 pT1G2 87,XXYY,i(1)(q10),-2,del(6)(q23q24)x2,-9,-9,-10,del(10)
(q22q24),-11,-16,+r[9]/46,XY[23]
17 M-85 pT1G2 88~90,XX,-Y,-Y,-2,add(5)(q11),-6,der(6)t(2;6)(q21;p23),+7,+7,
add(8)(q24),der(9)t(1;9)(q12;p12)x2,i(10)(p10),-14,-16,+22,+22,+r[cp5]
18 M-79 pT1G2 43,X,-Y,add(1)(q11-12),del(2)(p21),add(6)(q13),i(7)(q10),
-9,-12,-12,inc[cp5]/86,idemx2[cp4]/46,XY[24]
19 M-60 pT1G2 46,XY[10]
20 F-83 pTaG1 46,XX[17]
21 M-81 pT1G2 49~52,XY,+Y,+6,+8,-9,del(11)(p11),-13,-14,+15,-18,+19,
-20,+der(?)t(?;5)(?;q13),+4mar[cp15]/46,XY[25]
22 F-89 pT1G2 46,XX,del(2)(q11q13),add(3)(p23),add(3)(q21),+7,-8,-9,
der(9)t(8;9)(q21;p13),+13,+14,der(15)t(9;15)(q13;p1?),der(17)t(17;19)
(p11;p11),-18,der(19)t(18;19)(q11;p11),der(22)t(9;22)(p13;p1?)[25]
23 M-71 pT1G2 43,Y,-X,der(8)t(X;8)(q13;p21),-9,add(11)(p11),der(13;13)(q10;q10),
+15,-20,add(22)(q13)[4]/46,XY[14]
24 M-44 pT1G2 44,X,-Y,-9,-17,der(19)t(8;19)(q13;q13)×2,+der(?)t(?;8)
(?;q13)[25]/88,idemx2[2]
25 M-47 pTaG1 46,XY,del(9)(p11)[20]
26 F-81 pT1G2 44,XX,+add(1)(p12),-9,i(11)(q10),-13,-14,-14,der(15)t(14;15)
(q13;q13),+17,add(17)(p11-12)x2,der(18)t(13;18)(q12;p11),
-21,+mar[32]/88,idemx2[9]
27 M-71 pT1G2 46,XY,del(2)(p11p21),-9,-15,+der(?)t(?;9)(?;p11),+mar[8]
28 M-64 pT1G2 59~71,XX,-Y,+6,der(7)t(1;7)(q12;q36),+der(7)t(1;7)(q12;q36),
-9,+10,-11,-12,der(15)t(12;15)(q13;q24)x2,-16,-18,+20,+20,
+der(?)t(?;11)(?;q13),+mar[cp13]
29 M-73 pTaG1 45,XY,-9,-11,+mar[20]
30 F-58 pT2G3 46~48,XX,der(3)add(3)(p11)add(3)(q27),der(9)del(9)(p13)del(9)
(?q13q22),add(12)(p13),ins(15;?)(15;?),-17,+18,+20,+2mar[cp40]

55
Table 3.2 Clinical data and karyotype descriptions based on G-banding,SKY and FISH
on five bladder carcinoma cell lines

Name Sex/Age Stage Grade Composed karyotype

HTB-1 M/58 T3 G3 66~74,XXY,der(2)t(2;9)(p11;q13),inv(2)(q33q37),+3,der(3;5)

(p10;p10),der(3;4)(q10;p10),-4,+5,-7,der(8)t(3;8)(q21;p11),
+der(8)t(3;8)(?;p11),der(10)t(4;10)(p14;q11),der(11)t(6;13)
(p21.1;?)t(11;13)(q21;?)x2,+del(13)(q12q14),+13,
-14,-18,der(19)t(14;19)(q11;p12),?dup(19)(q13.3q13.4),
der(20)dup(20)(q?)t(6;20)(p21.3;q13.3),-21,-22[cp19]

HTB-2 M/63 T3 G3 87~90,XXYY,der(1)t(1;19)(p35;p12)x2,-4,del(6)(q21),der(6)

t(6;15)(q25;q24)x2,t(8;17)(p21;p11),-9,-10,del(10) (p13),
+del(12)(q13q15),-13,+14,+15,-16,der(17)t(8;17)(p21;p11),
-21[cp20]

HTB-4 F/81 T3 G3 83~87,XXXX,der(1)t(1;19)(q11;?)t(9;19)(q?;?),-2,der(3)t(1;3)

(p13;p13),-4,-6,-7,der(8;13)(q10;q10),del(9)(p21)x2,-10,+11,
-13,+del(15)(q21),-17,-18,-18,der(18)t(15;18)(q22;p11.3),
-19,der(19)t(9;19)(q?;q11),+20,+20,+20,-21,i(22)(q10),
del(22)(q12)[cp19]

HTB-5 F/67 T4 G4 72~74,XXX,der(1;18)(p10;p10),+der(1;21)(q10;q10)x2,-2,

+der(3)t(3;10)(?;q11)x2,-4,i(5)(p10)x3,+i(5)(p10)x2,+i(5)
(q10)x2,der(6)t(6;12)(p23;q23)x2,del(6)(q11),+del(6)(q11),
+7,i(10)(p10),+del(13)(q12q14),+16,-18,+20,-21,-21[cp20]

HTB-9 M/68 ? ? 65~69,XXY,der(3)t(3;6)(p12;q21),der(3)t(3;21)(q25;q11),

-4,t(4;8)(q25;p21),+der(5;21)(p10;q10),-6,+7,der(8)t(4;8)
(q25;p21),+9,der(10)t(?5;10)(?;p14)x2,-10,+11,
del(13)(q12q14)x2,i(13)(q10),der(15;17)(q10;q10),
-16,i(17)(q10),+r(18)(?),+20,-21,-21[cp19]

56
Table 3.3 Karyotype descriptions of 20 uroepithelial carcinomas based on
G-banding, SKY and FISH
Case Composed karyotype
1 48,X,-Y,+7,der(9)t(1;9)(q12;q34)x2,del(11)(?p or ?q),+17,+19[29]
2 46,XX,der(11)t(11;16)(p15;q11),del(16)(q11),der(17)t(14;17)(q11.2;q25)[24]
3 52,XY,t(2;11)(q31;q21),+i(8)(q10),+15,+16,+19,+20,+20[9]
4 45,XY,-9[3]/46,XY[18]
5 46,XY,i(8)(q10)[23]
6 46,XY[9]
7 49~74,XY,add(4)(q31),+5,+5,+del(6)(?q23q27),del(9)(q22q34),del(11)(q21q23),-17,-17,add(19)(p13),
+marx2[cp2]*
8 49~52,XY,t(5;9)(p10;p10),+7,+8,+15,+18,+20[cp35]
9 44,XX,+der(1)ins(1;14)(p13;q11-13q32)del(1)(p22),-9,i(11)(q10),-13,-14,del(14)(q13),
der(15)t(14;15)(q13;p13),+17,der(17)t(17;21) (p11;q11)x2,der(18)t(13;18)(q12;p11),-21[42]
10 46,XY,del(2)(p11p21),-9,dup(15)(q?),+der(20)t(9;20)(p11;p11)[18]
11 93,XXXX,dup(1)(q21q44)x2,del(7)(p21),del(9)(?p or ?q)x2,del(12)(?p),-16,+20,+20[11]
12 70~82,XX,Y,+6,der(7)t(1;7)(q12;q36),+der(7)t(1;7)(q12;q36),der(9)t(9;17)(q13;q21)x2,+10,-11,
-12,ins(15;12)(q24;q13q24)x2,-16,-18,+20,+20[cp17]
13 45,XY,-9,-11,+mar[20]†, 46,XY,t(12;13)(q24;q33)[2]/46,XY[14]‡
14§ C1: 47,XX,t(3;4)(q29;q25),add(7)(p22),del(15)(q22),inv(16)(p11q24),+18[5]
C2: 46,XX,inv(1)(p11q32),t(7;12)(p22;q22),t(15;17)(p11;q21)[10]
C3: 46,XX,del(2)(p11),der(9)t(2;9)(p13;q13)ins(9;?)(q13;?),t(9;20)(q22;q11),der(11)inv(11)
(q13q25)ins(11;?)(q13),del(17)(p12)[10]
C4: 46,XX,t(8;11)(q11;q21),t(9;20;10)(q22;q11;q22)[12]
C5: 46,XX,der(4)t(4;11)(p11;q21)ins(4;?)(p11;?),t(9;12)(q22;q24),der(11)t(4;11)(p15;q21)[14]
C6: 47,XX,add(3)(q21),+mar[5]
C7: 46,XX,ins(10;7)(q22;q11q22)[5]
C8: 46,XX,t(1;8)(q25;q24)[6]
C9: 46,XX,t(1;22)(q25;p11)[3]
C10: 46,XX,t(8;22)(q11;q13)[6]
C11: 49,XX,der(4)t(4;13)(q3?1;q21-22),der(6)t(4;6)(?;p25),+7,del(8p)(p11),
der(13)t(8;13)(?;q22),+20[2]
C12: 49,X,der(1)t(1;11)(q32;?)t(1;14)(p3?;6;?),t(2;18)(q31-32;q21),t(6;11)(q14;21;q21-22),
+der(7)t(9;7)(p13-15;p13),+del(8p)(p11-12),del(9)(p13),ins(12;15)(q22-24;q1?5q22-25),
der(14)t(1;14)(?;q31-32),del(15)(q?),+20,del(X)(?)[2]
C13: 49,X,der(2)t(2;9)(q3?;?),del(3)(q21),der(4)t(4;6)(q?;?),t(5;8)(q13-15;q22),
+7,t(9;18)(p13;q21),der(9)t(4;9)(?;p11-12)t(4;10)(?;?),der(10)t(3;10)(?;q2?4),
der(11)t(X;11)(q23-24;q11-12),der(X)t(X;11)(q13-21;q21-22),+der(20)t(X;20)(?;q13)[2]
C14: 49,X,t(1;3)(q?31;q2?9),+der(5)t(5;8)(+;?),der(5)t(X;5)(?;q11-12),+t(7;20)(q22-13;q13),
der(X)t(X;8)(q22;?)[2]
15 65~67,XXY,add(1)(p36),+3,-5,-6,+7,-9,-10,-17,-18,+19,+20,-21,+mar[cp30]†,
46,XY,del(9)(p?)[2]/46,XY[10 ]‡
16 T1: 46,X,t(X;9)(p11;p11),der(1)t(1;13)(p10;q10),+del(1)(p10)x2,der(2)t(2;13)(q?;q?)x2,der(7)t(7;14)
(q10;?),i(8q)(q10),-9,der(11)t(9;11)(?;p?),-13,der(13)t(2;13)(?;q?),der(14)t(7;14)(q?;q?)[110]
T2: 46,X,t(X;9)(p11;p11),der(1)t(1;13)(p10;q10),+del(1)(p10)x2,der(2)t(2;13)(q?;q?)x2,der(7)t(7;14)

57
 (q10;?),i(8q)(q10),-9,der(11)t(9;11)(?;p?),-13,der(13)t(2;13)(?;q?),der(14)t(7;14)(q?;q?)[108]
T3: 46,X,t(X;9)(p11;p11),der(1)t(1;13)(p10;q10),+del(1)(p10)x2,der(2)t(2;13)(q?;q?)x2,der(7)t(7;14)
(q10;?),i(8q)(q10),-9,der(11)t(9;11)(?;p?),-13,der(13)t(2;13)(?;q?),der(14)t(7;14)(q?;q?)[105]
17 46~48,X,t(X;9)(p11;q22),der(1)t(1;3)(q25;q11)inv(1)(p31p34),der(3)t(1;3)(q21;q11),t(5;12)(q31;p13),
t(8;17)(q13;q21),t(14;15)(q22;q15),+18,+20[cp47]
18 50,XY,+Y,-3,der(5)t(5)(5;14)(p?;?),del(5)(?),+6,+8,-9,del(11)(p11),t(13;20)(?;?),-14,+15,der(18)
?del(18)(q11q21)t(5;18)(q13;q23),+19,+22,der(22)t(14;22)(q11-13;q11-12)t(7;14)(?;q2?4)[27]
19 67~76,XXY,+der(1;9)(p10;p10),del(1)(q21),der(1)t(1;8)(p?;q?)t(1;9)(q?;q?),der(2)t(2;13)
(q13;q12)t(10;13)(q11;q22)x2,+der(2)t(2;11)(q11;q13)t(1;11)(q11;q23)x2,der(2;3)(q10;q10),
+der(3)t(1;3)(p?;q?),del(3)(p?orq?),del(4)(p14),+i(5)(p10)x2,der(5;12)(p10;p10),del(6)(p12),
+del(6)(q13),+der(6)t(4;6)(p1?4;q1?3)x2,+7,der(7)t(5;7)(p?orq?;q11)x2,del(9)(p?),-11,
der(11)t(9;11)(p13;q12),-12,der(12)t(3;12)(p?orq?;p?orq?),-13,+14,-15,-16,-17,+18,
del(18)(q?)x2,der(19)t(12;19)(q13;q13)inv dup(12)(q13q24)t(1;12)(p22;q13),der(19)t(5;19)
(q13;p13),i(19)(q10),+20,-21,der(21)t(5;21)(p?;q?),der(22)t(19;22)(p?orq?;q?)[cp85]
20 T1: 44,XX,-9,del(10)(q22q24),der(11)del(11)(p?)del(q?),t(12;17)(q10;q10),-17[31]
T2: 44,XX,-9,del(10)(q22q24),der(11)del(11)(p?)del(q?),t(12;17)(q10;q10),-17[33]
T3: 44,XX,-9,del(10)(q22q24),der(11)del(11)(p?)del(q?),t(12;17)(q10;q10),-17[35]

*based only on G-banding.

†
detected by G-banding.
‡
detected by SKY.
§
C1-10 detected by G-banding; C11-14 by SKY.

58
Table 3.4 Clinical and cytogenetic data on the 10 upper urinary tract TCC
Tumor Sex/Age Site Stage/Grade Karyotype
1 F/76 Renal pelvis pT1GI 45,X,-X,+del(1)(p13),-2,-9,+r[15]/45,XX,del(1)
(q42),dup(1)(q21q44),-9[14]/44,X,-X,del(1)(q42),
dup(1)(q21q44),-2,+7,-9[12]
2 M/75 Ureter pT1GII 48,XY,+1,der(1;14)(q10;q10),del(3)(q27),
add(4)(q34),-9,+ mar,+r[25]
3 M/77 Ureter pT1GII 48~50,XY,del(3)(p13p23),+8,add(8)(p11)×2,
add(9)(p11),del(11) (q13q21),i(11)(q10),
add(14)(q21),add(16)(q22),-18,add(20)(q11),
+3-4mar[cp14]/97-100,idem×2[5]/46,XY[10]
4 M/69 Renal pelvis pT2GII 67,XXY,add(1)(p36),+3,-5,-6,+7,-9,-10,-17,-18,
+19,+20,-21,+mar[30]
5 M/85 Ureter pT1GI 45,X,-Y[39]/46,XY[25]
6 M/70 Ureter pT1GI 45,X,-Y[49]/46,XY[27]
7 M/62 Ureter pT1GI 46,XY[41]
8 M/60 Ureter pT1GI 46,XY[41]
9 M/58 Ureter pTaGI 43~44,XY,-9[cp8]
*10 F/69 Ureter pT1GII-III C1 43~44,XX,t(1;3)(q25;q27),add(5)(p13),
+add(5)(q11),add(7)(q22),add(17)(q25)[cp7]
C2 47~48,XX,t(1;3)(q25;q27),add(5)(p13),
+add(5)(q11),add(7)(q22),add(17)(q25),
+?der(?)t(7;?)(q22;?)[5]
C3 49,XX,add(6)(p23),+7,-13,+15,+20,
+?der(?)t(8;?)(q13;?)[8]
C4 46~47,XX,t(3;7)(q29;p11),ins(5;?)(q13;?),
+7,-9[cp12]
C5 49,XX,del(2)(p21),add(3)(q27),add(4)(q22),-5,
+inv(7)(p11q36),add(9)(p22),add(13)(q32),+20,
+del(20)(q12),+der(?)t(X;?)(q13;?),
+der(?)t(18;?)(q11;?)[14]
C6 48,XX,der(4)t(4;5)(p14;q14),-5,+7,+8,+20[6]
C7 47,XX,t(2;13)(q21;q12),-5,+7,
+der(8)t(5;8)(q14;p11),+20[5]
C8 92~96,XXXX,+1,t(1;3)(p36;q21)x2,+7,+7,+8,
der(11)t(5;11)(q15;p15),marx2,inc[cp2]
C9 49,XX,+7,+8,+20[6]
C10 46,XX[9]

Abbreviations; F, female; M, male; C, clone; *, post-radiation tumor

59
Table 3.5 Clinical and cytogenetic data on 2 post-radiation carcinomas

Case Sex/Age Stage Grade Karyotype

1 F-80 pT2 G3 C1 47,XX,t(3;4)(q29;q25),add(7)(p22),del(15)(q22),inv(16)

(p11q24),+18[5]
C2 46,XX,inv(1)(p11q32),t(7;12)(p22;q22),t(15;17)
(p11;q21)[10]
C3 46,XX,del(2)(p11),der(9)t(2;9)(p13;q13)ins(9;?)(q13;?),
t(9;20)(q22;q11),der(11)inv(11)(q13q25)ins(11;?)(q13),
del(17)(p12)[10]
C4 46,XX,t(8;11)(q11;q21),t(9;20;10)(q22;q11;q22)[12]
C5 46,XX,der(4)t(4;11)(p11;q21)ins(4;?)(p11;?),t(9;12)
(q22;q24),der(11)t(4;11)(p15;q21)[14]
C6 47,XX,add(3)(q21),+mar[5]
C7 46,XX,ins(10;7)(q22;q11q22)[5]
C8 46,XX,t(1;8)(q25;q24)[6]
C9 46,XX,t(1;22)(q25;p11)[3]
C10 46,XX,t(8;22)(q11;q13)[6]

2 F/69 pT1 G2-3 C1 43~44,XX,t(1;3)(q25;q27),add(5)(p13),+add(5)(q11),

add(7)(q22),add(17)(q25)[cp7]
C2 47~48,XX,t(1;3)(q25;q27),add(5)(p13),+add(5)(q11),
add(7)(q22),add(17)(q25),+?der(?)t(7;?)(q22;?)[cp5]
C3 49,XX,add(6)(p23),+7,13,+15,+20,+?der(?)t(8;?)(q13;?)[8]
C4 46~47,XX,t(3;7)(q29;p11),ins(5;?)(q13;?),+7,-9[cp12]
C5 49,XX,del(2)(p21),add(3)(q27),add(4)(q22),-5,
+inv(7)(p11q36),add(9)(p22),add(13)(q32),+20,+del(20)
(q12),+der(?)t(X;?)(q13;?),+der(?)t(18;?)(q11;?)[14]
C6 48,XX,der(4)t(4;5)(p14;q14),-5,+7,+8,+20[6]
C7 47,XX,t(2;13)(q21;q12),5,+7,+der(8)t(5;8)
(q14;p11),+20[5]
C8 92~96,XXXX,+1,t(1;3)(p36;q21)x2,+7,+7,+8,
der(11)t(5;11)(q15;p15),marx2,inc[cp2]
C9 49,XX,+7,+8,+20[6]
C10 46,XX[9]

60
Table 3.6 Clinical and cytogenetic data on 19 multifocal uroepithelial carcinomas

Case Sex/Age Tumour/Site Stage/Grade Karyotype

1 M/54 BT1/Bladder Ta/G2 47,X,-Y,del(2)(q21q31),t(3;5)(q27;q31),del(5)(q11q13),+7,
-9,+r+mar[50]
BT2/Bladder T1/G2 46,X,-Y,del(2)(q21q31),t(3;5)(q27;q31),del(5)(q11q13),+7,-9,
der(9;13)(q10;q10),+13,+r[45]

2 M/75 BT5/Bladder Ta/G1-2 46,X,t(X;9)(p11;p11),del(1)(p11),+del(1)(p11),del(7)(q11),

i(8)(q10),add(9)(q12),der(11)t(7;11)(q11;p15)del(11)
(q12),der(13)t(1;13)(p11;p11),del(13)(q22q13),-14[100]
BT6/Bladder Ta/G1-2 46,X,t(X;9)(p11;p11),del(1)(p11),+del(1)(p11),del(7)(q11),
i(8)(q10),add(9)(q12),der(11)t(7;11)(q11;p15)del(11)
(q12),der(13)t(1;13)(p11;p11),del(13)(q22q13),-14[100]
BT7/Bladder T1/G3 46,X,t(X;9)(p11;p11),del(1)(p11),+del(1)(p11),del(7)(q11),
add(9)(q12),der(11)t(7;11)(q11;p15)del(11)(q12),der(13)t(1;13)
(p11;p11),-14[30]/46,X,t(X;9)(p11;p11),del(1)(p11),del(1)(p11),
del(7)(q11),i(8)(q10),add(9)(q12),der(11)t(7;11)(q11;p15)del(11)
(q12),der(13)t(1;13)(p11;p11),del(13)(q22q13),-14[40]/46,
X,t(X;9)(p11;p11),del(1)(p11),del(1)(p11),del(7)(q11),i(8)(q10),
add(9)(q12),der(11)t(7;11)(q11;p15)del(11)(q12),
der(13)t(1;13)(p11;p11),del(13)(q22q13),-14,del(17)(p11)[26]

3 M/77 UT1/Ureter Ta/G1-2 46,XY,+1,der(1;14)(q10;q10),del(3)(q27),add(4)(q34),-9,+mar[30]

BT8/Bladder Ta/G1 45,XY,+1,der(1;14)(q10;q10),del(3)(q27),add(4)(q34),-9,+[25]

4 M/64 UT2/Ureter T1/G2 48~50,XY,del(3)(p13p23),+8,add(8)(p11)x2,add(9)(p11),

del(11)(q13q21),i(11)(q10),add(14)(q21),del(16)(q22),-18,
add(20)(q11),+3-4mar[cp27]/97-100,idemx2[10]/46,XY[35]
BT9/Bladder T1/G2 48~50,XY,del(3)(p13p23),+8,add(8)(p11)x2,add(9)(p11),
del(11)(q13q21),i(11)(q10),del(16)(q22),-18,+19,
+3-4mar[cp60]/97-100,idemx2[cp20]/46,XY[25]
BT10/Bladder T1/G2 48~50,XY,del(3)(p13p23),+8,add(8)(p11)x2,-9,del(11)
(q13q21),i(11)(q10),der(18)t(1;18)(q21;q21),
+3-4mar[cp23]/97-100,idemx2[cp9]/46,XY[15]
BT11/Bladder T1/G2 48~50,XY,del(3)(p13p23),+8,add(8)(p11)x2,add(9)(p11),
del(11)(q13q21),i(11)(q10),del(16)(q22),-18,
+3-4mar[cp37]/97-100,idemx2[cp6]/46,XY[14]
BT12/Bladder T1/G2 48~50,XY,del(3)(p13p23),+8,add(8)(p11)x2,add(9)(p11),
del(11)(q13q21),i(11)(q10),add(14)(q21),del(16)(q22),-18,
+3-4mar[cp22]/96-100,idemx2[cp11]/46,XY[24]
RT1/Bladder T1/G2 48-50,XY,del(3)(p13p23),+8,add(8)(p11)x2,add(9)(p11),
del(11)(q13q21),i(11)(q10),add(14)(q21),del(16)(q22),
-18,+3-4mar[cp44]/97-100,idemx2[cp10]/46,XY[20]

5 75/F RT2/Bladder T1/G2 43,XX,-9,del(10)(q22q24),-11,der(12;17)(q10;q10)[25]

RT3/Bladder T1/G2 43,XX,-9,del(10)(q22q24),-11,der(12;17)(q10;q10)[25]
RT4/Bladder T1/G2 43,XX,-9,del(10)(q22q24),-11,der(12;17)(q10;q10)[25]

6 74/M RT5/Bladder Ta/G2 88~93,XXYY,add(6)(p21),add(8)(p11),del(8)(p22),

-9,add(16)(p11),der(17)t(1;17)(q12;p11), +mar[cp28]
RT6/Bladder Ta/G2 89~94,XXYY,add(6)(p21),add(8)(p11),del(8)(p22),
-9,add(16)(p11),der(17)t(1;17)(q12;p11),+1-2mar[cp25]
RT7/Bladder Ta/G2 89~93,XXYY,add(6)(p21),add(8)(p11),del(8)(p22),
-9,add(16)(p11),der(17)t(1;17)(q12;p11),+mar[cp30]

61
Table 3.7 Clinical and cytogenetic data on 2 squamous cell carcinomas of the bladder

No Age/Sex Site Stage Grade Karyotype

1 F-77 Bladder pT3 G3 76~87,XX,X,+1,der(1)add(1)(p22)t(1;9)(q42;q22)×2,+2,

del(2)(q13)×2,+3,del(3)(q27)×2,+4,+5,+6,der(6)t(6;10)
(p21;q11),del(6)(q21;q23)×2,+7,+del(7)(q11)x2,add(8)
(p11)x2,der(8)t(x;8)(q13;q24),-9,-9,-9,+10,add(10)(q26)x2,
del(11)(p11),der(11)t(3;11)(q11;p11)del(3)(q27),der(11)
del(11)(p11)t(3;11)(q21;q23),+12,-13,der(13)t(1;13)
(p12;p32),der(15)t(15;17)(p11;q11),del(16)(q21),-17,-17,
-17,add(18)(q27)x2,add(18)(q22),+19,der(19)t(2;19)
(q13;p13)x2,+20,-21,+add(22)(p11),+der(?)t(?;9)(?;q13),
+8mar[cp240]/46,XX[67]

2 M-55 Bladder pT3 G3 94~109,XX,-X,-X,-1,-1,-1,-1,2,add(2)(q35)x2,der(2)add(2)

(q35)t(2;13)(p11;q13),add(3)(p11),der(3)t(3;5)(p12;q13)x2,
+4,der(4)t(1;4)(q25;q31)x2,add(5)(q11)x2,der(5)t(5;17)
(q11;q21)ins(5;?)(q11;?),+i(5)(p10)x2,+7,+add(7)(q11),
+der(7)t(1;7)(p22;q21),+8,del(8)(p12)x2,der(8)t(1;8)
(q21;p11)add(1)(q32)x2,+der(8)t(2;8)(q11;q11)x2,-9,
i(9)(q10),add(9)(p11),der(9;17)(q10;q10),+der(10)t(3;10)
(p21;q22)x2,-12,-12,-13,-13,-15,der(16)t(8;16)(q13;p12)x2,
+der(16)t(3;16)(q11;q11-12),der(17)t(13;17)(q14;p11)x2,
+add(17)(p11),+19,der(19)t(1;19)(q12;p12)x2,add(19)
(q13)x2,+add(19)(p11-12)x2,-20,-20,der(21)t(21;22)
(p13;q11)x2,der(21)t(21;22)(p13;q11)t(7;22)(q11;q13)t(1;7)
(p22;q21),der(21)t(21;22)(p13;q11)t(7;22)(q11;q13)t(7;12)
(q21;q13),+der(21)t(3;21)(q11;p13),-22,-22,der(22)t(1;22)
(p13;p11),+der(?)t(?;2)(?;p11),+der(?)t(?;3)(?;p14),
+2-4r,+6mar[cp210]/190220,idem(2[40]/46,XX[76]

62
Table 3.8 Clinical and molecular cytogenetic data in benign and malignant post-bilharzial bladder lesions

Histopathology1 FISH signals3 CGH imbalances

Lab No Sex/Age Typ Grade Stage Other2 9cen 17cen Xcen Losses Gains
Benign
747-98 M/65 Chr. inflammation

738-98 F53 Chr. inflammation

162-99 M/70 Chr. inflammation,

moderate dysplasia

548-98 M/51 Granuloma CBO

352-99 F/55 Granuloma CBO 3
286-98 M/45 Granuloma 1 2 9p, 16p, 22q
with
inflammatory
polyp
Malignant
453-99 F/49 SCC G1 ? CBO
2228-99 F/60 SCC G1 CBO 1 3 3 1cen-p31, 3p, 19p
9pter-q13
229-99 M/70 SCC G2 T3 KCs 2 2
210-99 M/70 SCC G3 T3 KCs
471-99 M/60 SCC G3 ? CBO+SM 3 2
217-98 F/51 SCC G3 T3 CBO 9p
476-98 M/53 SCC G3 T3 CBO 2 3 1p21-p22, 2q13- 19p
q21,8p,13q, 18q

479-98 M/47 SCC CBO 2q13-q21, 8p 19p

345-98 M/61 TCC G1 ? CBO+SM
240-98 F/55 TCC G2 ? 2 2 1
746-98 F/60 TCC G2 2-3 2-6∗ 2-3
4540-98 M/62 TCC G2 ?
5605-97 M/50 TCC G3 T3 SM 2 8p21-pter
5653-98 F/50 TCC G3 T3 2 2 1-3 1cen-p31 5p,
17q,
20q
1
Chr, chronic; SCC, squamous cell carcinoma ; TCC, transitional cell carcinoma ; G1, well differentiated;
G2, moderately differentiated; G3, poorly differentiated
2
CBO, calcified bilharzial ova; KCs, keratinized cells; SM, squamous metaplasia
3
the number indicates the number of signals

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