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Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Medicine 1080
Molecular Pathogenesis of
Cervical CarcinomaAnalysis of Clonality, HPV16 Sequence Variations
and Loss of Heterozygosity
BY
XINRONG HU
ACTA UNIVERSITATIS UPSALIENSISUPPSALA 2001
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Dissertation for the Degree of Doctor of Philosophy (Faculty of Medicine) inPathology presented at Uppsala University in 2001
Abstract
Hu, X. 2001. Molecular Pathogenesis of Cervical Carcinoma: Analysis ofClonality, HPV16 Sequence Variations and Loss of Heterozygosity. ActaUniversitatis Upsaliensis. Comprehensive Summaries of Uppsala Dissertations fromthe Faculty of Medicine 1080. 76pp. Uppsala. ISBN 91-554-5127-6
A previous model of morphological pathogenesis assumed that cervical carcinomais of monoclonal origin and progresses through multiple steps from normal epitheliumvia CINS into invasive carcinomas. The aim of this study was to investigate themolecular mechanism of pathogenesis of cervical neoplasia.
In the clonality study, we found that 75% (6/8) of informative cases of cervicalcarcinoma had identical patterns of loss of heterozygosity (LOH) in the multiplesynchronous lesions, while the remaining cases had different LOU patterns. In anextensively studied "golden case", the multiple carcinoma and cervical intraepithelialneoplasia (CIN) lesions could be divided into several different clonal groups by theX-chromosome inactivation patterns, HPV 16 mutations and LOH patterns. Thebiggest clonal family included one CIN II, one CIN III and four carcinoma samples,while four other monoclonal families of carcinoma did not include CIN lesions. Theseresults suggested that cervical carcinoma can be either monoclonal or polygonal andcontains clones developing either directly or via multiple steps. In the study of HPVtypes and HPV16 variations, the results confirmed that specific HPV types are thecause of cervical carcinoma but failed to support the previous opinion that HPV16 E6variants are more malignant than the prototype. We established a novel classificationcalled oncogene lineage of HPV16, and found that additional variations of HPV 16oncogenes might be a weak further risk factor for cervical carcinoma. In the study ofLOH, we found that interstitial deletion of two common regions of chromosome 3p,i.e., 3p2l.1-3p2l.3, and 3p22, was an early event in the development of cervicalcarcinoma. The results showed that the hMLH1 gene, located in 3p22 and showingLOH in 43% of the studied cases, was not involved in the development of cervicalcarcinoma because neither the expression level of protein nor the gene sequence wasaltered in these cases.
In summary, a suggested model of molecular pathogenesis of cervical carcinoma isas follows. Specific types of HPV infect one or more committed stem cells in thebasal layer of the epithelium. Fully efficient LOH events turn one (monoclonal origin)or more (polyclonal origin) HPV-infected stem cells into carcinoma cells without CINsteps. Less efficient LOH events would lead to CIN steps where some other unknownfactors require to be added to facilitate the formation of carcinoma. In the absence ofLOH events no carcinoma develops from the HPV-infected stem cells.
Keywords: cervical carcinoma, CIN, HPV, LOH, X-chromosome inactivation,clonality, molecular pathogenesisXinrong Hu, Department of Genetics and Pathology, Rudbeck Laboratory, UppsalaUniversity, SE-751 85, Uppsala, Sweden © Xinrong HuISSN 0282-7476ISBN 91-554-5127-6
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Contents
Abstract ………………………………………………………………………… 1
Contents ………………………………………………………………………… 2
Publications and Manuscript included …………………………………… 5
Abbreviations …………………………………………………………………… 6
1 Introduction ……………………………………………………… 7
1.1 Morphological Biology …………………………………… 71.1.1 Anatomy and histology of cervix ……………………………………… 7
1.1.2 Cell kinetics in the transitional zone …………………………………… 7
1.1.3 Benign tumors …………………………………………………………… 8
1.1.4 Cervical intraepithelial neoplasia (CIN) ………………………………… 8
1.1.5 Cervical invasive carcinoma …………………………………………… 9
1.1.6 Assumed morphological pathogenesis ………………………………… 10
1.2 Etiology ………………………………………………………………… 11
1.2.1 Human Papillomavirus (HPV) ………………………………………… 11
1.2.1.1 Epidemiology ………………………………………………………… 12
1.2.1.2 Organization of HPV ……………………………………………… 12
1.2.1.3 HPV detection and typing …………………………………………… 13
1.2.1.4 HPV types and infection of the human tissue ………………………… 13
1.2.1.5 Prevalence of the presence of HPV DNA in cervical neoplasia ……… 14
1.2.1.6 HPV is the main cause of cervical neoplasia ………………………… 15
1.2.2 Cigarette smoking ……………………………………………… 15
1.2.3 Hormonal contraception …………………………………………… 16
1.2.4 Inheritance ……………………………………………………………… 16
1.3 Molecular Biology ……………………………………… 17
1.3.1 Clonality of cervical neoplasms ……………………………………… 17
1.3.1.1 Strategies of clonality analysis ……………………………………… 17
1.3.1.2 Polymorphism of inactivation of X-chromosome linked androgen
receptor gene ……………………………………………………… 17
1.3.1.3 Clonality features of cervical neoplasms ………………………… 19
1.3.1.4 Clonality status of some other common human tumors ………… 21
1.3.2 HPV16 ……………………………………………………………… 22
1.3.2.1 The structure of the HPV16 genome and the function of HPV16 genes 22
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1.3.2.2 HPV16 variants in progression of cervical carcinoma ………………… 25
1.3.2.3 Physical stage of HPV16 ……………………………………………… 26
1.3.3 Loss of heterozygosity (LOH) ………………………………………… 28
1.3.3.1 LOH on chromosome 3p ……………………………………………… 28
1.3.3.2 LOH on chromosome 6p ……………………………………………… 29
1.3.3.3 LOH on chromosomes 4, 5p, 11, 17, 18 and 19 ………………………. 29
1.3.4 Point mutations and gene polymorphisms …………………………… 30
1.3.4.1 Point mutations in TP53 ……………………………………………… 30
1.3.4.2 Susceptibility of patients with TP53 codon 72 polymorphism to HPV
infection ……………………………………………………………… 31
1.3.4.3 Susceptibility of patients with different HLA class II genotypes
to HPV infection ……………………………………………………… 31
1.3.5 Assumed molecular pathogenesis …………………………………… 32
2 Specific aims of study ………………………………………………… 33
3 Experimental Design ………………………………………………… 33
3.1 Cross-sectional case control study system ……………………… 33
3.2 Synchronous lesion study system …………………………………… 34
4 Materials and Methods ……………………………………………… 34
4.1 Patients ……………………………………………………………… 34
4.1.1 Paper 1 ……………………………………………………………… 34
4.1.2 Paper 2 ………………………………………………………………… 35
4.1.3 Paper 3 …………………………………………………………… 35
4.1.4 Paper 4 ……………………………………………………………… 35
4.1.5 Paper 5 ……………………………………………………………… 36
4.1.6 Paper 6 …………………………………………………………… 36
4.1.7 Paper 1-6 …………………………………………………………… 36
4.2 Microdissection (Paper 1 –6) ……………………………………… 37
4.3 Immunohistochemistry (Paper 5) …………………………………… 37
4.4 DNA preparation (Paper 1-6) and purification (Paper 6) …………… 37
4.5 Methylation sensitive Hpa II restriction enzyme digestion (Paper 6) … 38
4.6 Polymerase chain reaction (PCR) (Paper 1-6) ………………………… 38
4.7 Polyacrylamide gel electrophoresis with single-strand conformation
polymorphism (SSCP) and silver staining for HPV typing (Paper 5) … 39
4.8 Fragment analysis (Paper 4 and 6) …………………………………… 39
4.9 DNA sequence analysis (Paper 1-6) ………………………………… 40
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5 Results and Discussions ………………………………………… 40
5.1 Paper 1 …………………………………………………………… 40
5.1.1 Results …………………………………………………………… 40
5.1.2 Discussion ………………………………………………………… 42
5.2 Paper 2 …………………………………………………………… 43
5.2.1 Results …………………………………………………………… 43
5.2.2 Discussion ………………………………………………………… 44
5.3 Paper 3 …………………………………………………………… 46
5.3.1 Results ……………………………………………………………… 46
5.3.2 Discussion ………………………………………………………… 47
5.4 Paper 4 …………………………………………………………… 47
5.4.1 Results …………………………………………………………… 47
5.4.2 Discussion ………………………………………………………… 48
5.5 Paper 5 ……………………………………………………………… 49
5.5.1 Results ……………………………………………………………… 49
5.5.2 Discussion ………………………………………………………… 49
5.6. Paper 6 ……………………………………………………………… 50
5.6.1 Results ……………………………………………………………… 50
5.6.2 Discussion ………………………………………………………… 52
6 General discussion and summary of conclusions ………………… 53
7 Acknowledgements ………………………………………… 57
8 References ………………………………………………………… 59
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Publications and Manuscript included
This dissertation is based on the following original publications and manuscript,
which are referred to in the text by their Arabic numerals:
1. Xinrong Hu, Zhongmin Guo, Pang Tianyun, Fredrik Pontén, Erik Wilander, Sonja
Anderson and Jan Pontén. HPV Typing and HPV16 E6-Sequence Variations in
Synchronous Lesions of Cervical Squamous-Cell Carcinoma from Swedish Patients.
Int. J. Cancer 1999; 83:34-37.
2. Xinrong Hu, Pang Tianyun, Zhongmin Guo, Natalia Mazurenko, Fjodor Kisseljov,
Jan Pontén and Monica Nistér. Human Papillomavirus Type 16 E6 Gene Variations in
Invasive Cervical Squamous Cell Carcinoma and Cancer in Situ from Russian
Patients. Br. J. Cancer 2001; 84(6): 791-795.
3. Xinrong Hu, Pang Tianyun, Zhongmin Guo, Jan Pontén, Monica Nistér and Gijs
Afink. Oncogene Lineage of Human Papillomavirus Type 16 E6, E7 and E5 in Pre-
invasive and Invasive Cervical Squamous Cell Carcinoma. J. Pathol. 2001, in press.
4. Zhongmin Guo, Xinrong Hu, Gijs Afink, Fredrik Pontén, Erik Wilander and Jan
Pontén. Comparison of Chromosome 3p Deletion between Cervical Pre-cancers
Synchronous with and without Invasive Cancer. Int. J. Cancer 2000; 86:518-523.
5. Xinrong Hu, Zhongmin Guo, Pang Tianyun, Qing Li, Gijs Afink and Jan Pontén.
Immunohistochemical and DNA Sequencing Analysis on Human Mismatch Repair
Gene MLH1 in Cervical Squamous Cell Carcinoma with LOH of this Gene.
Anticancer Research 2000; 20:171-176.
6. Xinrong Hu, Pang Tianyun, Anna Asplund, Jan Pontén and Monica Nistér.
Clonality Analysis of Synchronous lesions of Cervical Carcinoma based on X-
chromosome Inactivation Polymorphism, HPV16 Genome Mutations and Loss of
Heterozygosity. Submitted.
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Abbreviations
CIN: cervical intraepithelial neoplasia
MPU: minimum proliferative unit
FIGO: international Federation of Gynaecology and Obstetrics
HPV: human papillomavirus
Nt: nucleotide
LTR: long terminal region
LOH: loss of heterozygosity
HLA: human Leukocyte Antigen
PGK: phosphoglucokinase
G6PD: glucose-6-phosphate dehydrogenase
FHIT: fragile histidine triad
PCR: polymerase chain reaction
SSCP: single strand conformational polymorphism
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1 Introduction
Perhaps what ordinary people nowadays know about cervical cancer may be the
following. It is one of the most common malignancies in women; It can be sexually
transmitted by men (with HPV); Vaginal cytology can be used to detect it; Cervical
conization can be used to treat and prevent it; And unproperly treated, patients will be
killed by it. But cervical carcinoma is much more if you set your sight into it.
1.1 Morphological Biology
1.1.1 Anatomy and histology of cervix
Cervix is a hollow cone. On the hollow surface, the cervical os is lined with
squamous epithelium and endocervix with columnar epithelium. The former consists
of several layers of squamous cells with short cubic cells in the basal layer. The latter
contains only one layer of columnar cell. The border area between the squamous
epithelium and columnar epithelium is called transitional zone (1) or transformation
zone (2). On the average, the transitional zone is populated by a 3-4 cell deep layer of
epithelium of squamous cells presumed to be deriving from ectocervical squamous
epithelium. It is the meeting ground of infection, regeneration, squamous metaplasia
and neoplasia (1-8).
1.1.2 Cell kinetics in the transitional zone
In the basal layer, there are a limited number of committed stem cells in the
transition zone (9). They are reversibly sleeping in G0 and capable of self-renewal.
Normally, the committed stem cells take the way of asymmetrical division by giving
rise to two daughter cells. One of the daughters inherits all natures of the mother and
stays in the basal layer serving as the resource of stem cells, while another is able to
divide and differentiate into more mature epithelial cells and is called precursor cell
of the epithelium. It is postulated that the transitional zone is built up by a number of
minimum proliferative unit (MPU)s (9). Each MPU contains a precursor cell that
proceeds through several rounds of DNA replication and division and differentiates
into mature epithelial cells, moving outwards to form a cell lineage. Different MPUs
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stand together unidentifiable by morphology. Older MPUs will finally die out by
shedding off and is replaced by the new ones. When the epithelium is injured, the
committed stem cells are responsible for repairing it by regeneration of MPUs, in
which process the structures and functions of the epithelial cells and epithelium
recover completely (10).
If the MPUs proliferate faster than they die, a lesion of hyperplasia will arise (11).
The lesion protrudes above the cervical surface and the cells of the hyperplastic lesion
look normal. It is often the result of a responsive proliferation to inflammation. When
inflammation is eliminated, it will disappear. In some cases, the committed stem cells
in the glands may produce precursor cells that differentiate into squamous cells that
occupy the glands partly or fully. This is the course we all know as metaplasia (11,
12). The metaplasia has normal-looking cells and can be cured as long as the
stimulation is removed. Neither the hyperplasia nor the metaplasias represent any true
tumors.
Challenged by some factors such as HPV, the committed stem cells become tumor
stem cells. It is suggested that, in this way, tumors on the cervix are initiated.
Neoplasia of the cervical squamous epithelium is classified as papilloma/condyloma,
cervical intraepithelial neoplasia (CIN) and invasive carcinoma.
1.1.3 Benign tumors
Benign tumors of the cervix are of the type squamous cell papilloma, also called
condyloma (13). The cells of a condyloma do not show conspicuous atypia but often
show obvious koilocytosis as a sign of HPV synthesis (14-18). The condylomas are
divided into two types: exophytic and flat. Exophytic condylomas grow outward with
an appearance of nipples (15-17, 19). It is believed that an exophytic condyloma
never develops into dysplasia. Flat condylomas grow deep wards with a slightly
raised plaque on the cervical surface (20, 21). At a low frequency, flat condylomas
may develop into dysplasia (21-26).
1.1.4 Cervical Intraepithelial Neoplasia (CIN)
CIN is a malignant tumor growing within the epithelium. It has been considered a
pre-cancerous lesion. In CIN lesions, the number of layers of tumor cells increase and
the order of the cell stratum is confused. Similar to the cells of invasive carcinoma,
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the cells of CIN typically show atypia: bizarre cell body, enlarged bulk of cells,
enlarged nuclei with thick membrane and strongly stained pieces of chromatin, high
frequency of mitotic figures and abnormal mitotic figures. CIN lesions are often
demarcated from the adjacent normal epithelium. CIN I occupies one third of the
epithelial stratum, CIN II two thirds, and CIN III the whole epithelial layer. All CIN
lesions have not yet broken through the basal membrane and never kill the patient
because they do not metastasis (27, 28). When CIN cells break through the basal
membrane, they become invasive carcinoma and invade into stroma and neighboring
tissues.
According to the clinical and epidemiological surveys, the prevalence of CIN
attains its peak around the age at 35, with about 3,500/100,000 women in Sweden
(29). Although the reported percentages show a wide range, in the absence of
intervention, roughly one-third of CIN lesions disappear spontaneously, one-third
persist and one-third progress (30). Bos et al. screened a population in Denmark
(1966-82) and found that 24% of CINs would progress, 39% regress, and 38%
remain, if all pre-clinical lesions in women aged 25-50 were included (31). In a
Swedish series of 555 women with CIN I followed up for an average of 39 months,
progression to CIN II or III took place in 16% of the cases, whereas 62% regressed
and 22% persisted (32). In another study, 894 women with CIN II were followed for
78 months. Here, progression to CIN III was seen in 30%, regression in 54% and
persistence in 16% (33). The time from discovery of CIN I and II to the observation
of CIN III was around 3.5-4.5 years. The mechanisms that determine the fate of CIN
lesions have not been clarified at all.
The non-invasive tumor of cervix, i.e., intra epithelial neoplaia, was once
categorized into four types: mild dysplasia, moderate dysplasia, severe dysplasia and
carcinoma in situ (30). Nowadays these subdivisions have to a large extent been
replaced by the simplified scheme of CIN. CIN I corresponds to mild dysplasia, CIN
II to moderate dysplasia, and CIN III to severe dysplasia and carcinoma in situ.
1.1.5 Cervical invasive carcinoma
The incidences of cervical carcinoma show a highly global variation, ranging from
4.2 to 54.6/100, 000 (34). Weighted age-specific incidence of invasive carcinoma
from European countries shows that the peak of incidence is around 45 years of age
(30). Cervical invasive carcinoma includes adenocarcinoma and squamous cell
10
carcinoma. Since squamous cell carcinoma accounts for over 85% of cervical
invasive carcinoma and is thought to have relationship with CIN, in my dissertation
only squamous cell carcinoma is considered.
Unlike CIN, invasive carcinoma can metastasize and cause death of the patients
(35, 36). Based on the differentiation of the carcinoma cells, invasive carcinoma is
graded into three degrees: well, moderate and low differentiation. A lower
differentiated invasive carcinoma might easier give rise to metastases and have poorer
prognosis, but some contradictions have been encountered.
Based on invasion and metastasis, the FIGO (the standard of the International
Federation of Gynecology and Obstetrics subdivides the cervical carcinoma cases into
stage I to IV) stages (37) have been used to predict the prognosis of the patients.
Stage I: growth is strictly limited to the cervix. Stage II: carcinoma extends beyond
the cervix but does not reach the pelvic wall and/or involves the vagina but not the
lower third of vagina. Stage III: carcinoma reaches the pelvic wall and/or involves the
lower third of the vagina. Stage IV: carcinoma involves the bladder, rectum or far
organs.
1.1.6 Assumed morphological pathogenesis
Invasive carcinoma often coexists with one or more CIN lesions in the same
cervix. The scene of CIN III breaking through the basal membrane and invading into
stroma can be seen under the microscope some time. The risk of subsequent cervical
carcinoma increases in the patients with CINs. Introduction of cytological screening
and consequent removal of CIN lesions diminishes the incidence of cervical cancer.
CIN occurs with a peak incidence in women about 10 years younger than those with
invasive carcinoma. The CIN lesions commonly are demarcated from surrounding
normal epithelium and show uniform morphology within the lesion, which suggests
that the neoplastic expansion of cells is “locked” in a fixed scheme of differentiation
and expand sideways at the expense of normal cells. Due to these phenomena, a
model of morphological carcinogenesis of cervical carcinoma has been assumed. It is
said that cervical carcinoma originates form a single stem cell and develops via
different degrees of CIN, i.e. putative pre-cancerous lesions, into invasive carcinoma.
However, these arguments are not entirely convincing. Morphological coexistence as
well as subsequent development of carcinoma in patients with CINs could be
explained by a common etiologic process acting over the whole cervical mucosa
11
rather than by progression from one lesion to the other. Treatment of CINs by
conization or laser will not selectively remove CINs but also most of the target
cervical mucosa susceptible to future development of carcinoma in a putative “pre-
cancerous” but morphologically normal field.
The question if cervical carcinoma originates from a single stem cell and develops
via CINs into invasive carcinoma, or the invasive carcinoma and CINs in the same
cervix originate and develop independently, is crucial for understanding the cause of
cervical carcinoma, the mechanism of carcinogenesis, and the rational design of
treatment and prevention. Nevertheless, morphology limits itself when it comes to
resolving these problems. Molecular studies, especially the clonality analysis has
proven to be a powerful way to address these issues. For example, if invasive
carcinoma and CIN are monoclonal, it will favor the interpretation that cervical
carcinoma originates from a single stem cell and takes multiple steps of
carcinogenesis. Thus, genetic deficiency is likely the initial cause, or more efforts
should be made to search for cellular genetic markers, and it is rational to remove
CINs and expect to have prevented progression to more severe forms of the neoplasm.
Otherwise, if CIN and cervical carcinoma are polyclonal lesions, this will favor the
interpretation that invasive carcinoma and CINs originate and develop independently.
Based on this, the common field factors such as HPV should be considered the cause,
and elimination of the field factors is the best way to treat and prevent this cancer.
1.2 Etiology
Epidemiological investigations provide some proposed risk factors for cervical
carcinoma. Some of the proposed risk factors included are early age at first
intercourse, large number of sexual partners, a male partner himself with many sexual
partners, tobacco smoking, diet poor in fruit, vegetables and some micronutrients
(vitamin C, beta-carotene, folate), use of oral contraceptives, infection by HPV,
presence of HPV DNA in cervical specimens, infection by genital herpes virus, and
presence of certain host cellular genotypes (30). Among those proposed risk factors,
HPV has been proven to be an etiological agent by both the clinical and experimental
evidences. So HPV is one of the main subjects focused on in my review and study.
1.2.1 Human Papillomavirus (HPV)
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1.2.1.1 Epidemiology
Early epidemiological data have long suggested that cervical neoplasia is a
venereally transmitted infectious disease. Brinton found that the risk of cervical
neoplasia is increased among women who initiate sexual intercourse at early ages,
who have great numbers of sexual partners and who have non-related diseases (38).
Later on, epidemiologists have studied the prevalence of HPV infection in normal
control women (39-41) and women with neoplasms (42). They have also studied the
shared risk factors of HPV and cervical neoplasia (40, 42-44), and whether HPV
infection predicts the development, recurrence, or pathological progression of the
cervical neplasia (45-47). If HPV is the venereally transmissible cause, positivity of
HPV should have a higher prevalence in the cases with neoplasia than in the normal
control individuals, HPV should share many of the same risk factors as cervical
neoplasia, and HPV infection should be a predictor of the neoplasia. Most although
not all studies of these kinds support that HPV is the cause of cervical neoplasia. The
results from different studies varied a lot and proven to be strikingly dependent on
methods of cervical tissue sampling and DNA detection (48-51).
1.2.1.2 Organization of HPV
The virion consists of a small capsid and a one-coding DNA strand. The capsid is
about 55nm in diameter consisting only of protein. Analysis of electron micrographs
reveals 12 five-coordinated and 60 six-coordinated capsomers arranged on a T= 7
surface lattice (52). After negative staining the capsomers appear as hollow cylinders
of equal height and width, which are connected at their base by fibrous structures
(53). The HPV capsid consists of two structural proteins. One protein with a Mr in the
range of 53, 000-59, 000 (54-56) represents 80% of the total viral protein. A minor
component has an average Mr of 70, 000. The two proteins are coded for by two open
reading frames of the HPV DNA.
Mature HPV particles contain the viral genome in the form of a double-stranded
circular DNA molecule. For the majority of HPVs, the size of the genome is about 7.9
kb (55, 57-59). Some variations have been observed in the range of ±10% (60). The
GC content of the HPV genome is rather low. The average value is 42.6%, the
extreme cases being HPV16 (36.5%) (61). There is no clear GC distribution pattern
13
throughout the genome with the exception of the non-coding regions that show
slightly lower GC values as a rule.
HPV genomes reveal a well-conserved general organization. All putative open
reading frames (ORFs) are restricted to one DNA strand. The second, presumably
non-coding strand contains only short ORFs, which are conserved neither with regard
to localization nor composition. This is in line with all known data on viral
transcription, which uniformly point to a single sense strand. Most major ORFs
occupy similar positions relative to each other and there is no substantial difference in
the length of homologous ORFs, which can be used to align the genomes of different
HPVs. The individual frames are classified as “early” (E) or “late” (L) genes in
analogy with other DNA viruses, where genes are turned on according to a specific
time schedule in the course of a productive infection. The so-called early genes are
expressed shortly after infection and prior to the onset of DNA replication. Products
of these genes mediate specific functions controlling replication and expression of
viral DNA. In the case of tumor viruses, early gene products are also involved in
transformation of the host cell. The late genes code for structural proteins of viral
particles and are activated during the final stages of the viral cycle. Up to six early
genes and two late genes can be detected in HPV (62).
1.2.1.3 HPV detection and typing
Epidemiologists often use at least four different techniques of collecting
exfoliated cervical cells for HPV testing: swab, scrape, cytobrush and lavage.
Cervical lavage has been shown to be the most sensitive non-tissue method to harvest
cells for molecular identification of HPV (48). Pathologists would either use cervical
tissue. Once cervical samples are collected, multiple methods of light microscopy,
electron microscopy, immunohistochemistry and molecular biology can be used to
identify HPV. Molecular biology is now commonly used. Southern blot (48, 63, 64),
Northern blot (50), Dot (Spot) blot (63, 65), Filter In Situ (66) , and In Situ (63, 64,
67) hybridization are applied for HPV detection and typing. Recently PCR has been
developed to amplify DNA sequences shared by many of the 20 or so different types
of genital HPVs (51). However, no “gold standard” has yet been established (51, 68-
70).
1.2.1.4 HPV types and infection of the human tissue
14
A new type of HPV is defined by having less than 90% homology with other types
when comparing the DNA sequence of the complete L1 coding region (71). HPVs
reveal a remarkable plurality of different genotypes. Until now, 85 HPV types have
been identified and fully characterized. Thus far, all identified types appear to be
strictly epitheliotropic (72). They infect the skin (73, 74) or epithelium of external
genital and anal regions (75), oral cavity (76), larynx (77, 78), lung (79), and
esophagus (80-82). No evidence for HPV infections has yet been found in the gastric,
ileojejunal, or colon mucosa. They may cause warts, verruciform, benign tumors and
malignant tumors. Only a limited number of the approximately 40 types infect the
anogenital tract. This recalls the different biological activities of individual HPV
types (72).
1.2.1.5 Prevalence of HPV DNA in cervical neoplasia
Most early investigators have observed an elevated prevalence of HPV in women
with existing cervical neoplasia compared with control women. Because the
prevalence of HPV among cytologically normal women has ranged unbelievably from
5% (83) to 80% (84), the estimated magnitude of the increase varied substantially,
primarily. The impression is that misclassification of HPV status may have limited
the first generation of epidemicological studies. With new and better methods for the
HPV assay, recent case-control data have firmly established that a great majority of
women with existent cervical neoplasia concurrently has detectable HPV DNA. In
contrast, the control women have a much lower percentage of HPV DNA (85-89). In
India, 98% of cases with cervical neoplasia and 18% of normal control women are
HPV positive (89); in Taiwan, 92% of patients with cervical neoplasia and 9% of
normal control women are HPV positive (86).
The relation between specific HPV types and different degrees of cervical
neoplasia has been investigated. Although up to 40 congenital types of HPV have
been identified, the distribution of the types of HPV to the cervical neoplasia is
uneven. HPV6, 11, 42, 43, and 44 mainly distribute in condyloma and CIN I (90-94);
HPV 30, 31, 33, 35, 39, 40, 49, 52, 59 and 68 are often seen in CIN II and III (91, 92,
95, 96); and HPV16, 18, 45 and 56 are the major types of HPV seen in CIN III and
invasive carcinoma (90-94, 97), with about 50% of invasive squamous cell
carcinoma infected by HPV16 (98). The HPVs, such as HPV6 and 11, mainly present
15
in low grade cervical neoplasia are defined as low risk types of HPV. The HPVs, such
as HPV31, 33, 59, often seen in moderate grade of cervical lesions are classified into
intermediate risk types of HPV. The high-risk types of HPV, such as HPV16 and 18,
are the ones found to be present in the high-grade lesions of cervical neoplasia.
HPV infections in normal cervix are often transient. Many HPV infections
disappear in about two years (71, 99). Most cases of HPV infected women have been
found with only one type of HPV present, but a low percentage of the cases has
double or multiple types of HPV (47, 100). Fewer cases of cervical carcinoma or high
grade CIN lesions than cases with normal cervix or cervix with low grade CIN lesions
can be found with double or multiple types of HPV infection (47). This indicates that
the immunogenicity of the original infection of HPV may be efficient to prevent
against infection of other types of HPV in many cases.
1.2.1.6 HPV is the main cause of cervical neoplasia
So far no specific oncogenes or tumor suppressor genes have been strongly
associated with cervical neoplasia. HPV considered the major cause of cervical
neoplasia has drawn most attention from the scientists who engage in studies of this
issue. Due to the plenty of efforts, data accumulate to prove that HPV is the cause of
cervical neoplasia. The evidence include the following. Cervical neoplasia is a
venereally transmitted infectious disease. The prevalence of presence of HPV in
patients with cervical neoplasia is higher than in normal control women. HPV shares
many of the risk factors with cervical neoplasia. Specific types of HPV infection
predict the development of the cervical neoplasm (47, 71, 99). Experimental data
reinforce the conclusion. The proteins of the viral oncogenes E6 and E7 of some types
of HPV can degrade p53 and pRb and transform epithelial cells in vitro (101-104).
However, only a minority of women with HPV infection develop any cervical lesions
(71, 105). This leads to an adjustment of the conclusion suggesting that HPV is the
necessary but insufficient cause of cervical neoplasia. More than HPV infection is
needed to cause cervical neoplasia. In other words, there must be some further risk
factors or cofactors that take part in the process of cervical carcinogenesis.
1.2.2 Cigarette smoking
16
Although studies show no direct association between smoking and cervical
neoplasia (106), there are evidence that cigarette smoking increases the risk (87, 107-
111). Some observations have also suggested that there may be an interaction
between HPV and smoking in the causation of cervical neoplasia (112, 113).
However, Reeves reported that smoking is significantly associated with cervical
carcinoma (52% of cases vs. 27% controls) but is not associated with HPV infection
(110). The confusion about the possible relation between smoking and cervical
neoplasia and between smoking and HPV infection indicates that smoking might be a
weak factor to impact the development of cervical neoplasia and the infection of
HPV. It is difficult to control the investigative settings of epidemiological studies to
get consistent results. Seemingly, cigarette smoking at the most is a minor cofactor of
some specific types of HPV in certain populations.
1.2.3 Hormonal contraception
It has been shown that long-term use of hormonal contraceptives tends to increase
the risk of cervical neoplasia (111, 112, 114-116) or increase the HPV infection (117).
By contrast, women using barrier contraceptives and spermicidal foams or jellies
seem to be at lower risk for this neoplasm (118). However, Pike reported that
hormonal contraceptive reduces the risk of cervical neoplasm (119). So, similarly
with smoking, hormonal contraception might be another weak cofactor of HPV
infection in the development of cervical neoplasia.
1.2.4 Inheritance
Since only a small number of women infected by HPV (considered the main cause
of cervical carcinoma) develop any lesions, it prompts people to search for further
risk factors. Genetic factors are an attractive field although so far no specific
oncogenes or tumor suppressor genes have been found. Magnusson et al. found a
significant family clustering of Swedish cases among biological, but not adoptive,
relatives of patients with cervical tumors, which provided an epidemiological
evidence of a genetic predisposition to cervical carcinoma (120). Several other
articles with similar conclusion were also published (121-124). They explained that
genetic susceptibility to HPV infection appears to be important in determining the
individual risk to develop this virally induced cancer.
17
1.3 Molecular Biology
1.3.1 Clonality of cervical neoplasms
It is generally believed that a single cell accumulates a randomly occurring set of
somatic mutations and then alone is responsible for developing a tumor because it is
unlikely for a number of cells to catch the same set of genetic events simultaneously.
Human tumors are therefore thought to be monoclonal and the monoclonality has
been taken to serve as a hall-mark of neoplastic proliferation and a diagnostic criteria
for some tumors. Clonality analysis will be a key issue to understand the cause and
mechanism of carcinogenesis of cervical neoplasia.
1.3.1.1 Strategies of clonality analysis
Currently the strategies of clonality analysis of tumors include X-chromosome
inactivation based analysis, somatic mutation detection, genetic rearrangement based
assay of lymphocytes and viral integration analysis. They are applied in different
situations. The former two are used for clonality analysis of most types of tumors. In
X-chromosome inactivation analysis, the restriction fragment length polymorphism
(RFLPs) of the G6PD (glucose-6-phosphate dehydrogenase) gene, PGK
(phosphoglycerokinase) gene, HPRT (hypoxanthine phosphoribosyltransferase) gene
(125-128) and microsatellite repeat polymorphism of the AR (androgen receptor)
gene (129) have been used. For somatic mutation analysis, genetic deletions,
chromosomal translocations and specific point mutations are often detected. The
immunoglobulin assay is used for analysis of B- lymphocyte tumors and T-cell
receptor gene assay is used for T-cell tumors. Genetic EBV termini, HIV and HPV
integration sites have been used to analyze the clonality status of the tumors harboring
the corresponding virus.
1.3.1.2 Polymorphism of inactivation of X-chromosome linked androgen receptor
gene
Because of the fact that up to 90% of the cases turn out to be informative for the
androgen receptor gene, the polymorphism of this gene is used the broadest for the
18
clonality analysis of tumors. Thus, here is introduced the working principle of this
analysis.
One X-chromosome, the one from the mother or the one from the father in an
embryonic stem cell is randomly inactivated by methylation of CpG sites at a stage
with about 50 cells in the female embryo to avoid double dosage of the genes on X-
chromosomes. The inactivation pattern of the X-chromosome in a stem cell is stably
inherited by subsequent descendants forming a cell lineage (130-132). Therefore, a
female body including cervical epithelium develops into a mosaic of Xm- and Xp-
inactivated cells. Cervical carcinoma cells derived from a committed epithelial stem
cell with a certain Xm- or Xp-inactivation pattern are equipped with the same pattern
as their progenitor, and this pattern serves as the clonality sign of the carcinoma.
Located at the first exon of the human androgen receptor gene, a highly polymorphic
microsatellite consisting of a short tandem repeat, [CAG]n (n=11-13), is about 100bp
away from HpaII and HhaI (methylation sensitive) restriction sites. This
polymorphism of the microsatellite allows the use of enzyme cleavage of DNA and
PCR-fragments in an assay to identify the X-chromosome inactivation pattern of the
cells tested (133, 134). Figure 1 shows how the analysis works and how the
interpretation is done. However, the interpretation of the clonality information for a
XX Embryonic stem cell
XX XX Random inactivation
XX XX.XX XX Mosaic of cell lineage
Enzymatic digestion
Bands of PCR product
Monoclonal Polyclonal Monoclonal
19
Fig. 1. Technique of X-chromosome inactivation analysis. Before digestion with
methylation sensitive restriction enzyme HpaII, the informative sample gives two
bands. After digestion, the active allele of an X-chromosome linked gene is destroyed.
Only the inactivated allele is amplified and will give a band. One band indicates the
presence of one type of cell population in the analyzed sample (monoclonality). Two
bands indicate more than one type of cell populations in the analyzed sample
(polyclonality).
few samples with the same X-chromosome inactivation pattern in an individual
requires further markers.
To get reliable and reproducible results, one has to take notice of some facts (135).
In larger cell populations, the average numbers of paternally- and maternally-
inherited inactivated X-chromosomes is close to 50:50 due to the random inactivation
of the X-chromosomes, but in small cell populations, it would be possible to find
skewing towards one allele to an extent that meets the criteria for clonal derivation.
This kind of skewing is different from tissue to tissue in the same patient and
probably from case to case. So in every case, the tumor sample must be compared
with matched controls from the same patient to test the heterozygosity for the marker.
Fine microdissection to separate the tumor cells and normal cells as much as possible
is one of the requirements. The sample size is also a limiting factor. The lower the cell
number, the higher is the probability to detect a monoclonal pattern based on patch
size mosaic in normal cells. More than 100 cells each in multiple microdissected
samples from different tumor areas and from controls are required. The sample
number from the same patient is one among other impact factors. As many samples as
possible should be taken to represent the whole situation of the tumor. Good
purification of DNA samples and optimal enzymatic digestion of the samples are
always important factors.
1.3.1.3 Clonality features of cervical neoplasms
Evidence shows that cervical neoplasia can be monoclonal or polyclonal. More
cases with monoclonal origin than cases with polyclonal origin have been reported.
This actually depends on the type of cervical neoplasm analyzed. The lower grade of
the cervical neoplasia (toward CIN I) has higher frequency of polyclonality.
20
With inactivation polymorphism of the X-chromosome linked androgen receptor
gene as the clonality marker, some efforts on clonality analysis of cervical neoplasia
have been made. Park et al. demonstrated that 100% of CIN III (25/25) and 68% of
CIN II (54/79) are monoclonal, while 32% of CIN II (25/79) are polyclonal (136).
Enomoto et al. found that 100% CIN III (30/30) are monoclonal and found 1 case of
CIN II to be polyclonal. Six cases of cervical invasive carcinoma were found to be
monoclonal and their synchronous CIN lesions shared the clonality composition with
the corresponding invasive carcinoma (137). In another study, Enomoto et al. found
all 13 cases of cervical squamous cell carcinoma and 6 cases of adeneocarcinoma to
be monoclonal (138). Based on the finding of a 100% monoclonality in cervical
carcinoma (12/12) including two early invasive carcinomas, Guo et al. pointed out
that genetic events are critical in the transition of pre-malignant epithelia to invasive
cancer in cervical carcinogenesis and that monoclonality of human tumors is not a
late event during the process of clonal competition or selection (139). In a series of
studies, however, Guo et al. found that among 22 cases of cervical invasive carcinoma
and synchronous CIN lesions two thirds of these cases were monoclonal, while the
remaining CINs were polyclonal (140). Later on, they found that 25% of cervical
invasive carcinoma ((2/8) were polyclonal (141). In line with these results, Ko et al.
reported that 33.3% of cervical invasive carcinoma cases (6/18) are polyclonal (142).
These reports indicate that the pathogenesis of cervical carcinoma is probably even
more complicated than that of many other types of cancers involving selection of sub-
clones of different clonal composition. In a study with 64 cases of cervical neoplasms,
Chuaqui et al. showed that invasive carcinoma and CIN III had high percentages of
cases with monoclonality and identical LOH patterns on chromosome 6q, while CIN I
and II had low percentages of cases with monoclonality and identical LOH patterns
on chromosome 6q, suggesting that CINs develop into invasive carcinoma through
progression from polyclonal lesions to monoclonal lesions (Table 1) (143).
PGK is also an X-chromosome linked gene. Sawada et al. used the X-
chromosome inactivation polymorphism of PGK to analyze the clonality of 25 cases
of cervical invasive and metastatic carcinoma. They found that all analyzed cases
were monoclonal and the same allele of the PGK gene was inactivated in the primary
invasive carcinoma and the metastatic carcinoma in each case (144).
LOH is another commonly used clonality marker (145). However, LOH patterns
are not always identical in all cases in other studies. A distinct type of cervical
carcinoma, adeno-squamous cell carcinoma, contains an adenocarcinoma component
21
Table 1. Monoclonal patterns as seen in analysis of the AR inactivationand identical LOH patterns on chromosome 6q in cervical neoplasms(ref. 143). AR: X-chromosome linked androgen receptor geneinactivation.
ICC (%) CIN III (%) CIN II (%) CIN I (%)
AR 92 93 20 0
6q 55 40 37 10
and a squamous cell carcinoma component in the same case. Kersemaekers et al.
performed LOH analysis on nine chromosomes, i.e., chromosomes1, 2, 3, 6, 11, 15,
17, 18, and X, in two cases of adeno-squamous cell carcinoma. Many genetic
alterations were the same in each component in both cases, which indicates that the
adeno-squamous cell carcinoma most likely has one cell origin. But the presence of
some differences of genetic changes associated with the component in one cases favor
a diversion of different development (146). With the combination of LOH analysis
and X-chromosome inactivation analysis, Guo et al. found two of eight cases of
cervical invasive carcinoma to be of polyclonal origin (141).
Park et al. even associated the clonality status with HPV types in cervical
neoplasia (136). All cases of 24 high-grade CIN and 47 out of 71 cases of low-grade
CIN were found to be monoclonal and contain HPV types 16, 18, 33, 35, 45, 56, 58 or
65. In contrast, 22 of 71 cases of low-grade CIN were polyclonal and contained other
types of HPV. Their findings suggest that the histopathological entity termed low-
grade CIN consists of two different types of lesions that are biologically distinct.
1.3.1.4 Clonality status of some other common human tumors
Most cases of other kinds of human tumors show monoclonality (147-151). This
favors the theory that genetic defects cause most tumors in a model of multiple steps.
Simultaneously randomly occurring multiple genetic events in multiple cells are very
rare. One progenitor cell that undergoes a series of genetic changes required for the
initiation of lesions will give rise to a monoclonal neoplasia. So monoclonality is
believed to be a hallmark of neoplastic proliferation. In some tumors, clonality
22
analysis has been used as a tool to diagnose the tumor, to exclude hyperplasia or
responsive proliferation, and to monitor the response to treatment (148, 152, 153).
However, there are a significant number of reported cases of different kinds of tumors
with polyclonality. In 11 cases of multiple squamous cell carcinomas of the
aerodigestive tract the p53 mutations and LOH patterns were stable during tumor
progression, and different p53 mutations or LOH patterns in multiple tumor areas
were observed in each case (154). In a study of bilateral breast carcinoma, four of 12
cases were scored as being of polyclonal origin (155). Yamamoto et al. found that two
of eight cases of multiple human hepatocellular carcinomas were monoclonal (had
intrahepatic metastases) and six of eight were polyclonal (showed independent
multicentric occurrence) (156). Sporadic medullary thyroid carcinoma has often been
found to result from a mutational event occurring at the single cell level and therefore
should be monoclonal, however, 10 of 11 such cases showed polyclonal patterns
(157). Polyclonal origin in some cases can also be seen in other tumors such as
“Kaposi” sarcoma (158), benign breast tumors (159, 160), and multifocal urothelial
papillary tumors (161).
While the origin of tumors, whether from one cell or many, has been a source of
fascination for experimental oncologists for many years, in recent years there has
been an explosion of information. Although these results have apparently confirmed
the monoclonal origin of tumors, there are some studies in which this conclusion just
cannot be made. The potential impact of such considerations as the patch size of a
normal clonal cell population and clonal evolution on the determination of clonality
has largely been ignored, with the result that a number of these determinations are
confounded. It is clear, for many reasons, that more efforts should be put into the
techniques of analysis (162).
1.3.2 HPV16
HPV16 is the most commonly seen type of HPV in cervical squamous cell
neoplasia, so this chapter mainly describes the features of HPV16.
1.3.2.1 The structure of the HPV16 genome and the function of HPV16 genes
The DNA sequence of the HPV16 genome contains 7905bp (61). HPV16 has a
long terminal region (LTR) and eight genes including six early genes and two late
23
genes. The six early genes are E6 (495bp, nt 65-559), E7 (315bp, nt 544-858), E1
(1955bp, nt 859-2813), E2 (1128bp, nt 2725-3852), E4 (287bp, nt 3332-3619), and
E5 (296bp, nt 3804-4100). The two late genes are L2 (1523bp, nt 4134-5657) and L1
(1630bp, nt 5527-7155). The LTR is 815bp long located between E6 and L1. Fig.2
shows the structure of the HPV16 genome. E6, E7 and E5 are viral oncogenes
encoding proteins with growth-stimulatory and transforming properties.
The E6 protein is an approximately 150 amino acid protein that is localized to the
nuclear matrix, as well as to non-nuclear membrane (163). E6 is not normally capable
of inducing transformation by itself, but has been shown to induce immortalization of
primary human keratinocytes in conjunction with E7 (164), as well as to promote
anchorage-independent growth of rat cells (165). Binding and degrading cellular p53,
E6 intervenes with the cell cycle and functions as an anti-apoptosis factor (104). E6
can also destabilize chromosomes (166), enhance foreign DNA integration and
mutagenicity (167) and activate telomerase (168).
The E7 protein is a 98 –amino acid protein that is located in the cytoplasm of cells
(169). Expression of E7 alone in epithelial cells is sufficient for transformation, but
E7-mediated transformation is much more efficient when co-expressed with the HPV
E6 protein (170). E7 can bind and inactivate Rb protein and Rb-related pocket
E6
E7
E1
E2
E4L2
L1
LTR
Fig. 2. The genome of HPV16
24
proteins to deregulate the cell cycle (101, 171). To intervene with the cell cycle, E7
also inhibits cyclin-dependent kinase inhibitors (172) and activates cyclins E and A
(173). Like E6 protein, E7 is able to enhance foreign DNA integration and
mutagenicity (174).
The E5 protein is a small hydrophobic protein located in cytoplasmic and plasma
membranes (175, 176). It complexes with a variety of other trans-membrane proteins,
such as the epidermal growth factor receptor, platelet-derived growth factor β-
receptor, and colony-stimulating factor-1 receptor (175). The E5 protein also binds to
adenosine triphosphatase (ATPase) in membrane (177). The HPV16 E5 protein
possesses weak transforming activity and induces a protein kinase-mediated, PKC-
independent, activation of membrane-associated protein kinase (178, 179). Its
transient induction in mouse 3T3 cells or immortalized human keratinocytes results in
suppression of the cyclin-dependent kinase inhibitor p21 and in the induction of c-jun
expression (180), and enhances endothelin-1-induced keratinocyte growth (181). The
expression pattern of E5 is less well established, but like E6 and E7, it is also
correlated with abnormal cervical cytology and can be detected in all stages of
development of cervical carcinoma (182, 183).
The E2 gene codes for two proteins with a transcriptional regulatory function. The
E2 protein has a transcriptional activation domain in the N-terminus and a DNA
binding domain in its C-terminus. The E2 gene can be expressed in two forms, a
complete protein and a short protein with only the DNA binding domain (184). Both
E2 and E1 proteins are necessary and sufficient in vivo for efficient viral DNA
replication. The ability of E2 and E1 to complex with each other appears to be
essential for efficient viral DNA replication (185, 186). E1 may play a role to
maintain HPV in episomal form, while E2 may regulate expression of E6 and E7
(163). Mutation of E2 increases immortalization capacity (187). The E4 protein
localizes to cytoplasmic inclusion granules and results in the collapse of the epithelial
cell intermediate filament network (188, 189). E4 expression is abundant in CIN and
condyloma, but the significance of this is unknown. L1 protein is the major capsid
protein and L2 is the minor one. Both L1 and L2 constitute the viral coat protein that
surrounds the viral DNA genome to assemble a complete infectious HPV particle
(190). LTR is a region of the genome adjacent to the E6 open reading frame. It does
not encode any protein but rather contains promoters and enhancers that influence
viral gene transcription (191).
25
1.3.2.2 HPV16 variants in progression of cervical carcinoma
HPV variants are defined as those HPV DNA sequences differing from each other
by less than 2% of nucleotides (71). Variations refer to any change of nucleotides
compared to the “prototype” nucleotides at the same positions. Based on the
variations in E6, L1 and LTR, HPV16 has been classified into six classes: E-350T, E-
350G, As, AA, Af1, and Af2. The geographical distribution of HPV16 variant classes
by continents is shown in table 2 (192). This classification can not reflect the
Table 2. Distribution of HPV16 variant classes by continent (Ref. 192)
No. (%) of specimens from:
Variantclass
Europe NorthAmerica
CentralSouthAmerica
SoutheastAsia
Africa Total no.
E-350T 20(40.0) 16(53.3) 56(24.6) 19(54.3) 5(7.8) 116E-350G 22(44.0) 12(40.0) 119(52.2) 2(5.7) 1(1.6) 156As 1(2.0) 1(3.3) 0(0.0) 9(25.7) 0(0.0) 11AA 7(14.0) 0(0.0) 45(19.7) 2(5.7) 0(0.0) 54Af1 0(0.0) 0(0.0) 5(2.2) 0(0.0) 39(60.9) 44Af2 0(0.0) 1(3.3) 3(1.3) 3(8.6) 19(29.7) 26
Total 50 30 228 35 64 407
biological function of HPV16 variants. When studying the clinical significance of
HPV16 variants, some people classified the HPV16 sequence into “prototype” and
variants groups, or “prototype”-like and “non-prototype”-like groups. Some other
studies use more precise categories.
The HPV16 sequence has frequent variations in the whole genome, particularly in
E6 (47, 71, 100, 192-195). The variants are reported to have different biological and
biochemical properties. In prospective studies, women with HPV16 “non-prototype”-
like variants were 6.5 times more likely to develop CIN II-III than those with
“prototype”-like variants (71). Londesborough et al. found that only 1 of 16 women
infected with the HPV16 prototype developed CIN or invasive cancer; in contrast,
26
10/12 women infected with HPV16 E6 variants had persistent infection which was
associated with development of CIN and invasive cancer (47). Zehbe et al. concluded
that sequence variation in HPV16 E6 predicted risk of progression from CIN III,
because 15/16 cases of ICC contained variant E6 in contrast to CIN III where only
11/25 had variant E6 (196). Alvare-Salas et al. showed that variants of HPV16 E6
correlated positively with clinical aggressiveness (193). Stöppler et al. described that
variants of HPV16 E6 protein differed in the abilities to suppress keratinocyte
differentiation and to induce p53 degradation in vitro (197). HPV16 E6 variants
represent a significant risk factor is common in both Western and Japanese women
despite the different distribution of each variant in them (198). Variants of HPV16 E7
could affect the properties of E7, including the binding to pRb (199).
However, the results are not geographically uniform. In women living in the
South London area, the E6 variants were identified in only 3/95 (3%) individuals, two
of whom had normal histology and one had a CIN II lesion. Wild type E6 sequence
was identified in the remaining 92/95 (97%) individuals. These data suggest that the
E6 variant does not play a major role in the pathogenesis of HPV16 related cervical
disease (200). Fujinaga et al. found that E7 variants were distributed uniformly among
CIN lesions and invasive carcinoma and the variant proteins had transforming
potential similar to the “prototype” E7 (201). Terry et al. reported that there were no
co-relation between HPV16 E2 variants and the grades of cervical neoplasia (202).
There was no significant correlation between HPV16 variant incidence and disease
progression or viral persistence and no significant correlation with any HLA allele
(203).
1.3.2.3 Physical stage of HPV16
Generally, HPV16 DNA is a circular molecule and remains episomal, i.e., it
remains separate from the host chromosome. When the circular molecule breaks at a
point it may linearize to integrate into the chromosome by two end-points. HPV16
can be in an episomal or an integrated form inside the infected cells. Theoretically the
two different physical stages would represent different biological status because the
breaking point must destroy the integrity of the viral genome and the integrated viral
genome must be out of control by itself and be controlled by or interact with the
cellular DNA elements. The E1/E2 gene region is the common place to be broken
when HPV16 linearizes to integrate into the host chromosome. Since E2 protein can
27
regulate the expression of E6 and E7, loss of E2 function may release the control of
E6 and E7 expression. High expression of E6 and E7 increases the immortalizing
capacity of HPV16. It is believed that one of the important events contributing to the
development of cervical neoplasia is viral integration, with subsequent loss of the E2
expression, and enhanced E6 and E7 expression. The integration status of the viral
DNA may also be important because the viral genome may integrate into a site that
results in alteration of human gene expression or its regulation (163). Additional
evidence for contributing effects of E2 loss of function comes from the studies in
vivo. In cells transfected with HPV16, the viral DNA was integrated in the genome of
the host cell, and these cells acquired a growth advantage that could not be explained
by the known effects of other retained viral genes, E6 and E7 (204). Later on, it was
demonstrated that the reintroduction of a normal E2 protein in Hela cells, suppressed
the growth of these cells (184). For this effect to take place, both the transactivating
and DNA binding domains of this E2 protein have to be intact (184).
Although HPV16 integration into host DNA is found in all cases of cervical
carcinoma (98), their metastases and derived cell lines (205), many cases of cervical
carcinoma contain both episomal form and integrated form of HPV (206-209). The
deficient function of the genes in the integrated form of HPV16 can be compensated
for easily by the corresponding function of the genes in the episomal form of HPV. In
addition, some cases of cervical carcinoma retain only episomal form of HPV16
(209). Hall et al. established a cervical carcinoma cell line that harbors episomal
copies of HPV16 DNA of approximately10 kb. Restriction enzyme and two-
dimensional gel analysis confirmed that HPV16 DNA was extra-chromosomal with
both monomeric and multimeric forms present. HPV16 was maintained as episomes
during passage both in culture and after subcutaneous growth in nude mice. The10 kb
viral genome, consisting of a full-length copy of HPV16 and a partial duplication of
the long terminal region and the L1 open reading frame, exhibited transforming
activity comparable to prototype HPV16 (210). These studies suggest that HPV16 can
transform target cells by mechanisms that do not require viral integration. In contrast,
many CIN cases contain an integrated form of HPV16 (211, 212).
The consequences of HPV16 integration on the cellular genome seem not to be
important because there is a lack of homology between the locations of integration
sites. Several integration sites and their corresponding normal target sequences have
been cloned and studied (213-216). Sequence comparison does not detect any
significant homology among different cellular target regions. This lack of homology
28
suggests that the integration of HPV16 is a random event. Taken together, HPV16
integration seems not as important in the carcinogenesis of cervical neoplasia as
people ever thought it was.
1.3.3 Loss of heterozygosity (LOH)
The LOH means that a particular DNA region has been lost, and therefore, if this
loss is recurrent in a significant number of cases of a particular tumor type we can
conclude that this chromosome region plays a role in that tumor. LOH is generally
thought of as an intermediate step in the inactivation of tumor suppressor genes, such
as p53 or pRb, which requires the inactivation of both alleles in order to display their
phenotype. However, in most cases, the LOH reflects a hemizygous situation and its
effect might be a dose effect rather than total inactivation of a tumor suppressor gene.
Analysis of LOH has been broadly applied to cervical neoplasms. Like many other
tumors, cervical neoplasms have extensive deletions in multiple chromosomes.
1.3.3.1 LOH on chromosome 3p
Chromosome 3p is one of the most frequently deleted chromosome regions in
cervical neoplasms. A broad region, 3p12-24 was identified as a target for LOH. The
data show that the prevalence of LOH in 3p varies in different studies, ranging from
35% to 100% depending on the number of markers and the different 3p loci selected
(217-221). The LOH in 3p might be an indicator of tumor progression and involve
tumor suppressor genes located at the region. Larson et al. reported that the frequency
of LOH in 3p increases from 25% in FIGO stage I cases to 100% in stage IV cases
(220). Some studies of CIN cases found that LOH in 3p occurred in about 30% of the
patients (222-224). These results suggest that the LOH at many loci is just a neutral
sign of evolution of cervical neoplasia, only those that occur during progression from
the early to the late stage lesions are non-random genetic events. Several studies agree
that within the region 3p14-22 there are two sub-regions where the deletions
concentrate, and frequently both are altered in the same tumor. These two regions are
3p14.2 and 3p21 (218-221), where tumor suppressor genes might harbor.
Recently, the FHIT gene has been identified within the chromosome region
3p14.2. A high incidence of LOH was also described at this region and FHIT is
altered in several other types of human tumors, such as lung (225), breast (226), and
29
esophageal carcinoma (227). The FHIT gene comprises the FRA3B fragile site at
which HPV16 spontaneously integrated (228). Although aberrant FHIT transcripts
were detected in 11 (63%) cervical carcinoma cell lines and in 35 out of 74 (47%)
primary cervical carcinomas of different types and stages, similar aberrant transcripts
were detected in normal tissues in 12/31 (39%) of the cases (229). A similar
phenomenon that both tumor and normal tissues contained aberrant transcripts could
also be detected in cases with some other types of solid tumors, and thus the
functional importance of the FHIT gene as a tumor suppressor gene has been
challenged.
In the 3p21 region the affected gene is not known. This region contains the
hMLH1 gene. The hMLH1 gene is one of the DNA mismatch repair genes which are
indispensable for DNA replication fidelity including the stabilization of short
nucleotide repeats (230-232). When the functions of the DNA mismatch repair system
are deficient, carcinogenesis might occur with the phenotype of microsatellite
instability in some types of tumors such as colon cancer (233), breast cancer (234)
and so on. Two mechanisms can inactivate those DNA mismatch repair genes, i.e.,
hypermethylation and mutation. Although the LOH at an hMLH1 locus can be
detected in up to 22% of cervical carcinomas (235) and the microsatellite instability
frequency is about 15% in cervical carcinoma (236), it is not clear if alterations of
hMLH1 expression and mutation in this gene occur in cervical carcinoma.
1.3.3.2 LOH on chromosome 6p
In cervical carcinoma studies have shown a high incidence of LOH at
chromosome 6p21.3-25 where HLA is located (237-240). As seen in in vitro
functional studies, chromosome 6 transferred into breast and ovarian cancer cells can
successfully suppress the tumorigenesis and metastatic ability of the target cells,
indicating potential location of tumor suppressor genes and cell scenescene genes on
this chromosome (241, 242). However, it remains to be determined if allelic losses of
chromosome 6 represent an early genetic event and play a role in the progression of
cervical pre-cancer.
1.3.3.3 LOH on chromosomes 4, 5p, 11, 17, 18 and 19
30
LOH on chromosomes 4, 5p, 11, 17, 18 and 19 can be seen in cervical neoplasia
(237, 238, 240). The introduction of chromosome 4 into Hela cells conferred a
senescence phenotype, which suggests that this chromosome might be important in
cervical carcinoma (243). Tumor suppressor genes might harbor at two regions, 4p16
and 4q21-35, frequently showing LOH in cervical neoplasms (240, 244). Two
different regions with LOH have been identified on chromosome11. The
chromosome11p15 region showed LOH in 12 out of 43 cervical carcinomas (28%)
(238) and LOH at 11q23 has a strong correlation with the presence of an invasive
carcinoma in FIGO stage I and II (240). Chromosome 17 was the first to attract
attention in cervical carcinoma because it contains the TP53 gene in the 17p3.3 band,
which is frequently mutated in many types of tumors. The frequency of LOH it
17p13.3 is 24%, and often it dose not affect the TP53 locus, thus, suggesting that the
candidate tumor suppressor gene in this region must be different from TP53 (245).
Although LOH occurs frequently on many chromosomes in cervical carcinoma and
some regions with a high frequency of LOH have been identified, no tumor
suppressor genes causally related to cervical neoplasia have been found so far.
1.3.4 Point mutations and gene polymorphisms
In contrast to loss of genetic material, recurrent amplifications and gain at
chromosomes can be seen in cervical carcinoma. Microsatellite instability is another
type of genetic change seen in cervical neoplasms, but the incidences of those
changes are relatively low. This chapter is going to briefly review some point
mutations and polymorphisms, which might be involved in cervical carcinoma.
1.3.4.1 Point mutations in TP53
Some studies have attempted to detect mutations in genes well known to have
point mutations in other tumors, including TP53, RB, H-RAS, and other cell cycle
genes. TP53 has been shown with a rather low frequency (less than 10%) of mutation
(246, 247), while others have reproted even much lower frequency of mutations in
cervical carcinoma. TP53 appears to be more frequently mutated in HPV-negative
tumors, but this negativity has been questioned since nowadays all tumors are
considered HPV-positive (98). This has lent support to the interpretation that the
mechanism implicated in these tumors is likely to be a consequence of the interaction
31
of the HPV E6 viral protein with the p53 protein, and by which they could achieve a
biological situation resembling a partial p53 defect.
1.3.4.2 Susceptibility of patients with TP53 codon 72 polymorphism to HPV
infection
Storey et al. first reported the effect of p53 polymorphism on the susceptibility to
HPV infection (248). The E6 oncoprotein derived from tumor-associated HPVs binds
to and induces the degradation of the cellular tumor-suppressor protein p53. A
common polymorphism that occurs in the p53 amino-acid sequence results in the
presence of either a proline or an arginine at position 72. They found that the arginine
form of p53 is significantly more susceptible than the proline form to be degraded by
E6 mediation. Moreover, allelic analysis of patients with HPV-associated tumors
revealed a striking overrepresentation of homozygous arginine-72 p53 compared with
the normal population, which indicated that individuals homozygous for arginine 72
are about seven times more susceptible to HPV-associated tumorigenesis than
heterozygotes. The arginine-encoding allele therefore represents a significant risk
factor in the development of HPV-associated carcinoma. Although both forms of p53
variants are morphologically wild type and do not differ in their ability to bind to
DNA in a sequence-specific manner, in another study, they showed that there are a
number of differences between the p53 variants in their abilities to bind components
of the transcriptional machinery, to activate transcription, to induce apoptosis, and to
repress the transformation of primary cells. This may have implications for the
development of cancers which harbor wild-type p53 sequences and possibly for the
ability of such tumors to respond to therapy, depending on their p53 genotype (249).
There are some other supportive data (250-258). However, also more objecting results
have been reported (247, 248, 259-269). This far, this argument is still open.
1.3.4.3 Susceptibility of patients with different HLA class II genotypes to HPV
infection
An association between HLA type and cervical neoplasia or HPV infection has
been reported. In one study the likelihood of developing invasive cervical carcinoma
was increased about 7-fold among female Caucasian carriers of HLA DQw3
compared with women who lacked this antigen (270). HLA DQA1 *0311 increases
32
the risk for CIN III and invasive carcinoma whereas DQA1 *0501 protects the
women from development of cervical neoplasia (271). The DQB1 ’03 is associated
with CIN III probably through being permissive for HPV infection (272). A higher
proportion of women without HPV infection carry DQB1 profiles than women with
HPV infection (273). Cuzick et al. showed that HLA DQB1 *0501 is protective
against development of cervical carcinoma in HPV16 positive patients while DRB1
*1301 has no protective effect (274). Allen et al. associated HLA 5 DQ-DR
haplotypes with development of cervical carcinoma in individuals infected with
HPV18, suggestive of a deficiency in antigen presentation by the HLA molecules
encoded by these haplotypes (275). Probably, HLA class II polymorphism is involved
in genetic susceptibility to cervical carcinoma and HPV infection (122). It can be
explained that viral infection is immunologically influenced. However, such a risk
figure was not confirmed in some other reports (276-278).
1.3.5 Assumed molecular pathogenesis
There are some assumed models for the pathogenesis of cervical carcinoma.
Many of them are similar in principle. Recently, Walboomers et al. suggested a model
of molecular pathogenesis of cervical neoplasia (279). They think of cervical
carcinogenesis as a multi-step process. First, high-risk HPV infects normal squamous
epithelium. In most cases this will not lead to a lesion or at worst give rise to a
regressing low grade CIN (CIN I). Both phenomena involve viral clearance. Second,
only persistent infections with high-risk HPV will lead to a high-grade lesion (CIN
II), subset of which may undergo malignant transformation. Third, at the transition of
CIN II to CIN III deregulated expression of the viral oncogenes E6 and E7 takes
place, resulting in genetic instability. Subsequently, activation of the telomere-
lengthening enzyme, telomerase occurs, as a result of which cells obtains a replication
capacity. Fourth, ultimately, successive allelic losses occur at different chromosomal
locations which, followed by a clonal outgrowth results in an invasive carcinoma.
This model sounds reasonable, but the process is too arbitrary to claim a strict and
fixed hierarchy for CINI, CIN II and CIN III and to stress high-risk HPV as the only
actively functioning factor during the process of carcinogenesis in different individual
patients. Actually, most of the cases with persistent high-risk HPV infection can stay
long in the stages of morphologically normal epithelium or different grades of CINs.
Some moderate-risk HPV can also cause cervical carcinoma. Even high grades of
33
CINs do not have to progress, most of them can indeed regress. The model neglects
the fact that the neoplasia probably originates from one or more progenitor cells (stem
cells) but not from the common normal epithelial cells. This means that the model
ignores that the cellular genetic background can play a role during the carcinogenesis.
2 Specific Aims of the Study
(1) To investigate if all studied cases of cervical carcinoma are monoclonal.
(2) To investigate if all isolated CIN lesions and all cases of CIN are monoclonal.
(3) To investigate if cervical carcinoma and synchronous CIN lesions share the
clonality status.
(4) To determine if all lesion areas and areas of normal epithelium in the same cervix
have the same type of HPV or HPV16 variant.
(5) To determine the biological significance of HPV16 variants.
(6) To find out if all areas of lesions in the same case have LOH and have the same
pattern of LOH.
(7) To find out if there are specific chromosome loci with LOH in early step of
cervical neoplasia.
(8) To determine if the hMLH1 gene, which shows LOH in 43% of the cases is
involved in cervical carcinoma.
(9) Finally, to suggest potential models for the molecular pathogenesis of cervical
carcinoma.
3 Experimental Design
3.1 Cross-sectional case control study system
CIN shares some cytological features with cervical invasive carcinoma, but the two
lesions differ in histology and biological behavior. Invasive carcinoma can invade
into neighboring tissues or organs and metastasize to other parts of the body. CIN
grows on the basal membrane and does not invade or metastasize. A proportion of
CINs can break the basal membrane and become invasive carcinomas, but a higher
proportion of CINs would persist or regress. So CIN is different from but related to
invasive carcinoma. If one takes a suitable number of CIN cases as a group and
invasive carcinoma cases as another group to compare the variables from the two
34
groups with each other with a statistics power, an event occurring at a higher
frequency in invasive carcinoma than in CIN would be said to be a factor involved in
the evolution process from CIN to invasive carcinoma. Contrarily, if the frequency is
the same, the event might not be involved in. Thus, this study system consists of two
group of cases, CIN groups and invasive carcinoma groups. For this kind of study,
random collection of enough number of studied cases is essential to draw a safe
conclusion.
3.2 Synchronous lesion study system
Some cases of cervical squamous cell carcinoma contain one or multiple
independent CIN lesions in the same cervix, which are called synchronous lesions of
cervical carcinoma. Since invasive carcinoma and CIN lesions are present in the same
cervix, they share many basic backgrounds of genetic factors and environment.
Comparing the variables between CIN lesions and invasive carcinoma in such cases
would make it possible to obtain the best precise and correct results. With such kinds
of results, one can often draw reliable and sound conclusions. Therefore, the use of
synchronous lesions of cervical carcinoma to conduct the study is called synchronous
lesion study system. Sometimes the statistics power in such a stusy can be
unimportant.
4 Materials and Methods
4.1 Patients
4.1.1 Paper 1
Fifteen cases of cervical squamous cell carcinoma from Sweden were studied. The
extirpated uteri with one or more synchronous CIN lesions were serially selected from
1996 to 1998 in the Uppsala province without any other selection. Tissue from 10
cases (L1-L10) was fixed by neutral formalin. Specimens from 5 cases (H1-H5) were
fresh-frozen. The mean age for those cases was 45 years (32-62 years). Twelve
squamous cell carcinoma cases were moderate, two poor and one high in
differentiation. The FIGO stage for the cases belonged to Ia to IIb.
35
4.1.2 Paper 2
One hundred and four cases, including 55 cases of primary invasive squamous
cell carcinoma and 49 cases of CIN, from patients undergoing radical hysterectomy at
Blokhin Cancer Research Center, Moscow, Russian in the period from 1994 to 1998
were studied. The age range was 30 - 80 (mean 43.8) for the patients with ICC and 31
- 43 (mean 37.2) for those with CIN. The FIGO stage and degree of differentiation
(high, moderate or low) were recorded at the Blokhin Cancer Research Center. Tumor
samples were snap-frozen in liquid nitrogen. Part of each sample was transferred on
dry ice to the Department of Genetics and Pathology, Uppsala University, Sweden.
4.1.3 Paper 3
Sixty-one HPV16 positive cases, among which 30 were CIN III and 31 were
invasive squamous cell carcinomas, from 141 Swedish patients undergoing radical
hysterectomy or cervical conization at the Uppsala University Hospital in the period
from 1996 to 1999 were studied. The age range was 24-69 years (mean 40.9 years)
for those with CIN and 26-83 years (mean 48.5 years) for ICC patients. The FIGO
stages for ICC patients were from Ia to IIa. Of the 61 cases, nine were freshly-frozen
specimens and the remaining 52 were neutral formalin-fixed paraffin-embedded
specimens.
4.1.4 Paper 4
Thirty cases of CIN or cervical invasive carcinoma were collected from neutral
formalin-fixed and paraffin-embedded archival material at the Department of
Genetics and Pathology, Uppsala University Hospital, Sweden. Among the cases, 20
were CIN without invasive carcinoma, 10 were invasive carcinoma with one or more
synchronous CIN lesions in the same samples. The morphological diagnoses for the
morphology of the cases were confirmed by two experienced pathologists
independently.
36
4.1.5 Paper 5
Eleven women with invasive cervical squamous cell carcinoma came from
Sweden. Their age ranged from 26 to 63 (mean 43.7), and the FIGO stage was Ia or
Ib. Ten cases carried high-risk HPV and the remaining one case had no detectable
HPV DNA. The paired tumor and normal samples from the same patient were from
the Department of Genetics and Pathology, Uppsala University Hospital, Sweden.
The cases, including seven cases with and four cases without LOH at the hMLH1
locus were subjected to immunohistochemical staining. 8/11 cases, seven with and
one without LOH of the hMLH1 gene, were used for DNA sequence analysis of the
hMLH1 gene. The invasive carcinoma was synchronous with CIN lesions in all but
one case.
4.1.6 Paper 6
One case (H2) was used. The patient was a Swedish woman when at the age of 33
had her uterus with cervical carcinoma removed. Macroscopically, tumor grew within
the cervix and around the external os with no involvement of uterus body or vagina.
The histopathological diagnosis in this case was cervical invasive squamous cell
carcinoma (moderate differentiation) with invasion of local vessels and metastases to
local lymph nodes. One month before the surgical procedure, the patient had been
found with cervical malignancy by vaginal cytology and latter on the diagnoses had
been confirmed by biopsy. HPV routine test showed HPV16 positively. Before this
HPV test, the HPV infectious situation was not known. In two previous vaginal
cytological examinations 11 and 8 years ago, no abnormality was found. The entire
fresh cervix was cut from external os to endocervix into six parts designated A, B, C,
D, E and F, respectively. Parts A, C and D were used for histopathological routine
examination, while B, D and F were frozen at –80°C for research.
4.1.7 Papers 1-6
All projects had received official institutional and local ethical approval from
Uppsala, Sweden and Moscow, Russia.
37
4.2 Microdissection (Paper 1–6)
Serial cryosections (6 µm) were prepared from the freshly-frozen tissues and
stained shortly with Mayer´s hematoxylin. Ordinary sections were made from
paraffin-embedded tissues and deparaffinized in xylene then stained with Mayer´s
hematoxylin. According to the purpose and design of the experiments, only one
sample were picked up each case or multiple samples were picked up from different
areas and different lesions in the individual cervix.
In paper 6, the microdissection was used most extensively. Separate
microdissections of invasive cancer nest, CINs, normal epithelium, gland and stroma
in one section for each tissue block were performed. All lesions were sharply
demarcated from stroma or adjacent normal epithelium. Admixture of normal
epithelial or stroma cells was insignificant as judged by careful examination under the
microscope. The blade of the scalpel was changed after each microdissection. The
microdissected pieces were transferred to Eppendorf tubes containing 50µl 1 x PCR
buffer II (PE, Roche Molecular System, NJ). Each sample contained 500-1000 cells.
4.3 Immunohistochemistry (Paper 5)
Slides were deparaffinized in xylene, washed in gradients of ethanol and water,
then placed in a Tissue-Tek® Slide Staining Holder, the empty slots of which were
filled with blank slides. Slides were microwaved in citrate buffer pH 6.0 and treated
by 250 ml of working strength Antigen Retrieval solution according to instructions by
the manufacturers. The slides were allowed to cool for 20 minutes and then treated
with 0.3% hydrogen peroxide in methanol for 30 min. to suppress endogenous
peroxide. The anti-hMLH1 antiserum (Labora) diluted 1:100 was applied to the
sections and incubated overnight at 4oC. The slides were then incubated with
biotinylated IgG and with ABC each for 30 min, at room temperature. As a positive
control, normal colon mucosa known to display hMLH1 protein immunoreactivity
was used. Negative controls were obtained by replacing the primary antibody with a
non-immune rabbit serum.
4.4 DNA preparation (Papers 1-6) and purification (Paper 6)
38
Lysis of cells by proteinase K (500 µg/ml) at 56°C over night was interrupted by
incubation of the sample at 95°C for 10 min. The roughly prepared DNA from each
sample was ready to be used for PCR-sequencing of HPV16 and PCR-genescanning
detection of LOH with microsatellite markers. A portion of roughly prepared DNA
sample (20µl) in paper 6 was further purified for X-chromosome inactivation
analysis. 50µl of 95% EtOH was added to 20µl roughly prepared DNA sample and
the sample was incubated at –70°C for 5 hours. After centrifugation at 13,000 rpm for
30 min, the precipitate was washed with 250µl of 70% EtOH and spinned at 13,000
rpm for 20 min and then air-dried.
4.5 Methylation sensitive Hpa II restriction enzyme digestion (Paper 6)
The pellet gained at step 4.4 was dissolved in 20µl reaction buffer for
methylation-sensitive restriction enzyme HpaII analysis (Promege, Madison, USA)
and halved into two tubes. One portion was added 15 Units of HpaII and incubated at
37°C over night. The reaction was terminated by heat inactivation at 95°C for 5 min.
Another portion of DNA, as a control, was not exposed to HpaII digestion. The non-
HpaII digested and the HpaII digested DNA portions were then ready to be used for
PCR amplification of androgen receptor gene.
4.6 Polymerase chain reaction (PCR) (Papers 1-6)
Information on the sequences of PCR primers for the genes of HPV16 and HPV
typing, the androgen receptor gene, the hMLH1 gene, and the microsatellite DNA
sequences have been described in the different papers. The PCR reaction mixture and
the PCR program for each PCR primer pair have also been described in
corresponding papers. The PCR was performed on a RoboCycler Gradient 96
(STRATAGENE) or GeneAmp PCR Systems 9600 (PE, Norwalk, CT).
An example shows the PCR procedure. To amplify HPV16 E6, 30µl volume (1 x
PCR bufferII, 3.0mM MgCl2, 200µM of each deoxynucleotide, 0.5U Taq DNA
polymerase, 0.5µM of each sense and anti-sense primer, and 1µl DNA solution) was
used. 35 cycles (1min at 95°C, 2min at 55°C and 3min at 72°C) with 10min initial
denaturation at 95°C and 7min final elongation at 72°C were used.
39
To avoid contamination, we prepared a PCR master mix in an isolated room under
a hood, where UV-light was used to destroy any potential contaminating DNA or
PCR product at the working area before and after this manipulation and then added
template DNA under similar working conditions in another separate room.
4.7 Polyacrylamide gel electrophoresis with single-strand conformation
polymorphism (SSCP) and silver staining for HPV typing (Paper 5)
After PCR amplification, part of the PCR product from primer pair GP5+/GP6+
were electrophoresed in agarose gel and stained by ethidium bromide to roughly
check if the samples contained specific fragments. The samples with specific
fragments were subjected to SSCP on PhastSystem with semi-automated
electrophoresis in pre-formed 12.5% homogeneous polyacrylamide Phast gel and
native buffer strips (Pharmacia, Uppsala, Sweden). A pre-run at 400 V, 10.0 mA, 4.0
W, 10°C, was executed until it paused at 99 accumulated Volt hours (Avh). Then 1µl
of a denatured solution, consisting of 99% formamide, 0.05% bromphenol blue and
0.05% xylene cyanol, were blended in a 1.5-ml Eppendorf tube, centrifuged briefly,
de-natured for 5 min at 95°C in an Eppendorf thermomixer and cooled rapidly on ice.
A 0.8 -µl sample was pipetted onto a sample comb fitting the holders of the
PhastSystem intrument, and the comb was inserted into the holders. Electrophoresis
was carried out up to 230 Avh. The gel was stained with 0.5% silver nitrate in the
development chamber of the PhastSystem according to standard procedure. To
determine the HPV type of the samples, a reference picture made by Zehbe et al.
(280) was used. The 12 most common HPV types present in cervix could be clearly
distinguished.
4.8 Fragment analysis (Papers 4 and 6)
The inactivation pattern of the X-chromosome linked androgen receptor gene and
LOH of microsatellite loci were subjected to fragment analysis. An example presents
the procedure of fragment analysis. 1.5µl of the final PCR product of the androgen
receptor gene or a microsatellite locus was mixed with 0.5µl loading buffer, 0.5µl
internal size standard (GENESCAN-350 ROX, Perkin Elmer) and 2.5µl formamid,
and denatured at 95°C for 5min followed immediately by cooling on ice before
40
loading on a 4.5% acrylamid gel. Electrophoresis was performed on an ABI Prism
377 sequencer (Perkin Elmer). Size and quantification of allelic fragments and LOH
were analyzed and determined by GeneScan Analysis 2.0.2 (PE Applied Biosystems).
A 60% or higher allelic signal reduction between any two alleles after HpaII digestion
was considered a homogenous pattern representing a sample with paternally or
maternally inactivated X-chromosome. The same criterion of allelic reduction was
adopted for judgement of LOH.
4.9 DNA sequence analysis (Papers 1-6)
The whole genome of HPV16 and 19 exons of the hMLH1 gene were subjected to
DNA sequence analysis. An example represents the DNA sequencing procedure. The
PCR amplicons of HPV16 E6 were electrophoretically separated on 1.5 % agarose gel
and stained with ethidium bromide. Desired bands were cut out with subsequent
purification on GenElute Minus EtBr Spin Columns (SUPELCO, Bellefonte, PA).
The purified PCR products were quantified and then applied to enzymatic extension
reactions for DNA sequencing using the Cycle Sequencing Ready Reaction Kit (Big-
Dye terminator reagent [PE Applied Biosystems] containing dye-labeled terminators)
in GeneAmp PCR Systems 9600 (PE, Norwalk, CT). The same forward and reverse
primers as for the PCR amplification of HPV16 were used separately in cycle
sequencing. The extension products were purified by Ethanol/Sodium Acetate
precipitation, then electrophoresed on an ABI Prism 377 sequencer. The sequence and
variations were analyzed and determined by the FacturaTM and Sequence Navigator
version 2.0 (PE Applied Biosystems).
With respect to all analyses performed, the test for each sample was repeated at
least once starting from DNA-PCR amplification with the same result.
5 Results and Discussions
5.1 Paper 1
5.1.1 Results
41
One hundred and twenty-two samples from 15 cases of cervical squamous cell
carcinoma with synchronous CIN lesions were analyzed with HPV typing. By
comparison with the reference picture (Fig. 3) (280), the HPV types in the samples
were interpreted from the SSCP patterns (Fig. 4). 13/15 cases were HPV-positive.
Fig. 4. Representative SSCP patterns of HPV types. Lane 1 and 7: Molecular weight
marker. Lane 2 and 3: HPV16. Lane 4 and 5: HPV33. Lane 6: HPV18
HPV16 was the most common type (10/15). HPV31 was found in 2/15 and HPV18 in
1/15 cases. There was concordance between different synchronous lesions with regard
42
to type of HPV or absence of HPV. HPV found in normal squamous epithelium was
always of the same type as that of any positive lesions in the same patient.
All 10 of the cases with HPV16 positivity were analyzed for E6 variation. By
comparison with the “prototype” of the HPV16 E6 sequence (61), three variations
were found (Fig. 5). One variation present only in case L10, was A→G at nt 131,
Fig. 5: Electropherograms of HPV16 E6 sequence variations. Upper line: Prototype
sequence from case H2. Lower line: Variations. Left column: A → G variation at nt
131 in case L10. Middle column: A → G variation at nt 276 in case L5. Left column:
T → G at nt 350 in case L9.
which altered arg→gly in codon 10. The second, only present in case L5, was A→G
at nt 276 which changed asn→ser in codon 58. The third variant, T→G at nt 350
which changed leu→val in codon 83, was common, seen in 5/10 cases. The “
prototype”, as well as the variant E6, was identical in the different lesions within any
individual patient. Multiple samples from the lesions and also the normal epithelium
were consistently positive either for the “prototype” or for the variant E6.
5.1.2 Discussion
The studies of single lesions of different grades of CINs and invasive carcinoma
have for instance shown that benign neoplasms, CIN I and CIN II are predominantly
associated with low-risk HPV but only rarely with high-risk HPV. CIN III and
invasive carcinoma are predominantly associated with high-risk HPV (90, 91). It is
43
important to elucidate if the types of HPV would be different in normal epithelium,
different grades of CIN lesions and invasive carcinoma in the same case. In our cases
of cervical carcinoma with synchronous CIN lesions, one important result of the
present study is the regular persistence of one type only of HPV in simultaneously
present normal epithelium, CINs and invasive carcinoma. High-risk HPV, as
expected, dominated invasive carcinoma and CIN III. However, they were equally
prevalent in concomitantly present lower grades of CIN and normal epithelium. This
observation could be extended to the HPV16 E6 variants. Homogeneity in HPV type
was also seen for these lesions where multiple samples were compared. The results
strongly indicate that the original infection of HPV persists during progression from
normal via CIN lesions to invasive carcinoma, thus that HPV is a necessary and
inefficient cause of cervical neoplasia. The results also indicate that multiple types of
HPV infection may be very rare, or new types of HPV may for immunological or
other reasons have difficulty in establishing themselves.
Zehbe et al. concluded that the HPV16 E6 variants (most of the reported cases are
all E6 variant (nt 350 G) are particularly strongly associated with progression from
CIN to invasive carcinoma (195, 196). However, our results do not support the
previous conclusions since “prototype” HPV16 E6 was also common in invasive
carcinoma (50%) and, more importantly, the E6 variants were present in normal
epithelium, CIN lesions and invasive carcinoma in the same case. This indicates that
HPV16 E6 variants are not the strong risk factor to drive the CIN lesions into invasive
carcinoma either.
5.2 Paper 2
5.2.1 Results
Thirty-two cases of cervical invasive carcinoma and 18 cases of CIN were
analyzed for HPV16 E6 sequence variations. The E6 variants were classified as three
groups, A, B and M. A and B were identical except the polymorphic nt 350, which
was either guanine (A) or thymidine (B). Group M was a mixture of variants with
sequence departures from group A and/or B at other sites than nt 350. Group A
dominated both in CIN (10/18) and ICC (19/32). Group B, which corresponded to the
international “prototype,” occurred in 4/18 cases of CIN and 5/32 cases of ICC. The
third group (M) was represented in 4/18 cases of CIN and 8/32 cases of ICC. There
44
was no statistically significant difference in the distribution of groups A, B or M
between CIN and ICC.
However, group B was mainly associated with cancers detected in early clinical
stages. Group A, the most common one, was represented in early as well as advanced
stages. The M group was not seen in FIGO stage I. Differences between the groups
were statistically significant indicating that the order of clinical malignancy between
the groups would be M > A > B.
Fourteen out of 32 cases of invasive carcinoma with synchronous lesions or
multiple samples were available. Among these cases, 10 contained a single HPV16
E6 isolate. The E6 Variant in these cases was concordant from different parts of each
ICC case. When CIN was also present (N3, M 2, M 21), all samples from these
precursors had the same variant as the simultaneously found invasive cancer. Normal
squamous epithelium sampled in case M19 and N3 showed the same variants as in the
invasive cancers.
Four cases (M4, M12, M13, M23) bore double/multiple variants. M4 had A and B
variants in CIN II lesions and invasive carcinoma. M12 contained both A and B
variant in carcinoma. M13 presented the B variant in 3/3 CIN II samples, and in
invasive cancer, one sample showed the A variant and another sample had an A
variant with an additional missense variation from T to G at nt 310. In M23, the
normal epithelium showed the B variant, the two CIN II samples showed either A or
B variant, the invasive carcinoma had either A (two samples) or B (two samples), and
one of the invasive cancer areas contained a B variant with additionally changed C to
T at nt 374 which created a stop signal.
5.2.2 Discussion
HPV16 E6 variants are reported to have different biological and biochemical
properties and the “variants” are more malignant than the “protorype” (47, 193, 195-
197). However, the rate, the type and the biological and clinical significance of the
variants of HPV16 E6 have not been geographically uniform. Besides, the definition
of “variant” has been a source of confusion in previous studies, because it has been
based on all departures from the original “prototype” once isolated randomly from a
case of cervical invasive carcinoma.
45
We found that the variations at different nucleotide sites differ principally from
each other. Nt 350 behaves as a polymorphic site where roughly two thirds of the
isolates have a G and the remainder a T. The other sites register as classical variants
with one or occasionally two departures from a predominant configuration. This
classification permits a logical division into two homogenous groups (A and B)
supplemented by one heterogeneous group M. Failure to have noted this is the major
reason for a confused literature based on lumping together all “pure non -T” at nt 350
with all other departures from the sequence of the “prototype”. When our groups A, B
and M are applied, the distribution of the three groups in invasive cancer and CIN is
identical. This contrasts with claims that E6 variants at nt 350 have a higher
prevalence in invasive cancer than in CIN III (195, 196). Therefore our findings do
not support the conclusion that “variants” of E6 are more likely to cause progression
to invasive cancer than “prototype” E6. Even if the previous scheme (195, 196) which
classified E6 variants as two groups, “prototype” and “variants” group, is applied on
our cases, no difference in the distribution of the E6 groups can be seen between CIN
and invasive carcinoma. Since this investigation was done on samples from Russian
patients, our results might reflect the fact that the human genetic susceptibility
probably determined by HLA or TP53 polymorphisms of the Russian population to
different HPV16 variants differs from that of other ethnic populations (122, 248).
Since A and B variants of HPV16 E6 are so commonly distributed in cervical
neoplasms, they might be stable polymorphic variants having been mutated very long
ago and undergone natural selection. It is unlikely that the variation of A variant and
the variation of B variant would be substituted for each other. The four cases
containing both A and B may be the result of double infections because it should not
be difficult for the patients to pick up the E6 variants prevalent in the infection pool.
The two additional variants in cases M13 and M23 probably represent mutations of
the B variants within the host because the additional variants are very rare in nature.
The reason for such mutations of HPV16 E6 is unclear. Since cervical neoplasms
have pronounced genomic instability with allelic losses, mutated variants of E6 would
arise in vivo within cancer cells subject to genomic instability. The positive
association of the M group with clinical malignancy would then be an
epiphenomenon explained by an influence of the cancer cells on a residing viral
genome, rather than the reverse conventional hypothesis that different E6 variants
have a different potential to drive progression to invasive cervical carcinoma.
46
5.3. Paper 3
5.3.1 Results
Sixty-one cases of HPV16 positive CIN III (30 cases) and cervical invasive
carcinoma (squamous cell type, 31 cases) out of 141 Swedish cases of cervical
neoplasms were selected. The nucleotide (nt) sequences of the full open reading
frames of HPV16 E6 (477 base pairs), E7 (315 base pairs) and E5 (297 base pairs)
from these 61 cases were determined, and they were compared with the HPV16
reference DNA sequence (61). Nucleotides at any position different from the
reference nucleotide at the corresponding position were recorded as variations. The
variations covered nearly the whole length of the open reading frame of each gene: E6
showed 13 variations with 9 being missense and 4 silent, E7 had 3 variations with
only 1 being missense and 2 silent, and E5 had 9 variations with 5 being missense and
4 silent. There was no evidence of premature stop codons or deletions. The highest
variation rate for the whole sequence length of the combined E6, E7 and E5 in a
single case was 1.5%, indicating that the analyzed HPV types should be regarded as
variants.
The nucleotide variations were not randomly distributed. There were clearly three
prevalent variation sites: nt 350 in E6 (41/61 T, 20/61 G), and in E5 nt 3979 (16/61 A,
45/61 C) and nt 4042 (14/61 A, 47/61 G). All these variations were missense
variations. Clustering of the prevalent variations in the order E6 nt 350, E5 nt 3979,
E5 nt 4042, gave five lineages: Va (T-A-A), Vb (T-C-G), Vc (T-A-G), Vd (G-A-A),
and Ve (G-C-G), accounting for 21.3%, 41.0%, 3.3%, 1.6%, and 32.8% of the cases,
respectively. The 3 major lineages combined (Va, Vb, and Ve) accounted for 95.1%
of the cases.
The distribution of the lineages showed no statistically significant difference
between CIN and invasive carcinoma. However, additional missense variations
occurred in 3 cases of CIN and 11 cases of invasive carcinoma, and a simple
correlation of the proportion of additional missense variations shows a statistically
significant difference of 3.7 times more additional missense variations in invasive
carcinoma than in CIN (P <0.025, chi-square test). If we analyze the number of
additional missense variations for the different oncogenes lineage, we do not detect
any significant correlation for most of the lineages, with the exception of Vb. In the
47
Vb lineage we can see a strong prevalence of additional missense variations in ICC.
The Vc and Vd lineages were found in too few cases to allow us to assess any
difference in additional missense variations between CIN and ICC.
5.3.2 Discussion
Some of the previous studies on sequence variations of HPV16 often considered
only one of the oncogenes of the virus. This may be another source of confusion and
reason for inconsistent conclusions such as that E6 variants are more malignant than
the prototype (195, 196, 200, 203). Since genetic experiments have assigned
oncogenic activity to the viral genes E6, E7 and E5, and their co-operative action may
make a major contribution to the development of cervical neoplasia, we hypothesize
that HPV16 has different oncogene lineages at genetic level with different biological
functions. Clustering of the three prevalent variation sites in E5 and E6 theoretically
gives eight lineages, but only five lineages were identified with the three major ones
accounting for 95%. This indicated that the lineages have undergone natural selection
and become stable. The additional missense variations appear more frequently in
invasive carcinoma than in CIN. The Vb group, one of the oncogene lineages, has
more additional missense variation in invasive carcinoma than in CIN. This suggests
that the HPV16 oncogene lineages may represent different groups of HPV16 variants
with different biological functions and therefore present a further risk factor
contributing to the development of cervical carcinoma.
5.4 Paper 4
5.4.1 Results
Twenty cases of CIN and 10 cases of invasive carcinoma synchronous CIN lesions
were analyzed with 11 microsatellite markers covering chromosome 3p from 3p24 to
3p13. No LOH at the tested 3p loci was found in normal epithelium, squamous
metaplasia and CIN I lesions. Allelic losses at one or more 3p markers were recorded
in 33% (3/9) of CIN II and 36% (5/14) of CIN III lesions in the CIN cases. A
statistically significant increase of the percentage of LOH was observed in the
synchronous CIN lesions of invasive carcinoma, showing 71% (5/7) in CIN II and
76% (13/17) in CIN III.
48
Patterns of LOH were compared between multiple synchronous CIN lesions and
corresponding invasive carcinoma in 10 cases. 75% (6/8) of informative cases had
identical LOH patterns between multiple CIN lesions and invasive carcinoma. The
remaining one case showed LOH in the invasive carcinoma but not in the two co-
existing CIN III lesions. In another remaining case, LOH was detected in the invasive
carcinoma and the CIN II lesion but not in the CIN I lesion.
Two common interstitially deleted regions with incidence of LOH higher than 50%
of the cases of invasive carcinoma were identified. The six cases with identical LOH
patterns showed a common deletion in the region between D3S1076 and D3S663 that
maps to 3p21.1-3p21.3, and the region between D3S1260 and D3S1611 that maps to
3p22. In contrast to the complex and extensive pattern of allelic deletions in invasive
carcinoma, LOH in CIN cases was more restricted to small regions on 3p. The
frequently deleted loci among the CIN cases were at D3S1260 (25%) and the locus
between markers D3S1076 and D3S1289 (33% and 27%, respectively).
5.4.2 Discussion
LOH in tumors represents a genotype of recurrent genetic deletions, some
occurring in the early stage of carcinogenesis, others during the process of tumor
development or in the late stage of the tumor (281). Two regions 3p22-21-3 and
3p21.1 showing frequent LOH in both groups of the cases studied promise to contain
novel potential tumor suppressor loci, which may play a role in early transition of
CIN into invasive carcinoma. Most cases of cervical neoplasms were reported to be of
monoclonal origin (136-138). Identical LOH patterns of these microsatellite markers
between synchronous multiple CIN lesions and invasive carcinoma in 75% of the
informative cases suggest that different cervical CIN lesions and invasive carcinoma
are genetically linked and most likely originate from a single progenitor cell.
Monoclonal origin of cervical neoplasms favors the probability that the genetic
defects are the causes of this neoplasm. However, no oncogenes or tumor suppressor
genes strongly related to cervical neoplasia have been found. The two cases (25%)
showing different LOH patterns between the synchronous CIN lesions and invasive
carcinoma in this study indicate that some of the cervical carcinomas and the
synchronous CIN lesions can originate independently from multiple progenitor cells.
This is consistent with other reports (142) and supports that HPV as a field factor is
the main cause of cervical neoplasia.
49
5.5 Paper 5
5.5.1 Results
Seven cases with LOH within the hMLH1 gene and four without were stained
with the hMLH1 antibody. All cases were positive in CIN cells, carcinoma cells and
normal epithelial cells. The staining was strongest in the order: CIN > carcinoma >
normal cells.
The hMLH1 gene sequences of the carcinoma were compared with the ones of
their matching normal tissues and the wild type sequence of the hMLH1 gene from
the Gene bank database (282). No mutation was found in any exon in samples from
the normal epithelium, CIN or cancer regardless of the presence of LOH or not. Exon-
intron boundaries had no changes but in the exon 13 and 15 regions. For exon 15
fragments, 3 bp TTT in all sequenced samples of the cases substituted for the AAA of
the wild type sequence at a position 42 bp from the 3´end of the exon. For exon 13
fragments, change was seen at a nt 12bp away from 3´end of the exon in only one
case. No alteration was found in the remaining cases. Case 4 was represented by 6
samples, which were 1 stroma, 1 normal epithelium, 3 CIN III and 1 invasive
carcinoma. LOH of the hMLH1 gene was present in all CIN and invasive carcinoma
samples from this case, but not in stroma and normal epithelium (223). In this study
of the same case, stroma and epithelium had double signals, G (wild type) plus A,
while all CIN and invasive carcinoma had only A.
5.5.2 Discussion
The hMLH1 gene is one of the DNA mismatch repair genes. It has been mapped
to chromosome 3p21-23 (283). This gene covers about one million base pairs and
contains 19 exons (282, 284, 285). Inactivation of the gene by mutation and/or
hypermethylation causes microsatellite instability in sporadic and familial colorectal
tumors (282, 284, 285). Microsatellite instability is also found in some female-
specific tumors or corresponding cell lines such as breast cancer, ovarian cancer,
endometrial cancer and cervical cancer (236, 286, 287). Using a microsatellite marker
D3S1260 which is located within one of the introns of the hMLH1 gene, 22% - 43%
of cervical carcinoma cases were found with LOH in this gene. However, our results
50
from the analysis of immunohistochemistry and DNA sequencing showed no
alteration of the hMLH1 gene or of the gene expression in the cases of cervical
carcinoma with LOH in this gene, indicating that the hMLH1 gene is not involved in
the development of cervical neoplasia.
One mutation within an intron of the hMLH1 gene was found in one case. All CIN
and invasive carcinoma lesions lost the wild type allele, while the normal epithelium
and stroma retained two alleles, which matched the finding of LOH analysis and
indicates that the multiple CIN lesions and invasive carcinoma shared the clonal
origin in this case.
5.6 Paper 6
5.6.1 Results
Twenty-four samples named H2-1 to H2-24 from one “golden” case of cervical
carcinoma with multiple synchronous CIN lesions were analyzed with respect to
clonality with the X-chromosome inactivation marker, HPV genome sequencing and
microsatellite markers.
The X-chromosome inactivation patterns were informative in this case. After
HpaII digestion, three common X-chromosome inactivation patterns were seen. The
microdissected samples with X-chromosome inactivation patterns are presented in
Table 3.
H2-9, a CIN II sample, was special. It seemed to have three alleles of the
androgen receptor gene, one was a, the second was b and the extra allele was the
shortest named –a. After HpaII digestion, b was cut down by 63% and a by 25% of –
a.
DNA sequence variations or mutations of the HPV16 genome were analyzed as an
assistant clonality marker. The entire genome of HPV16 with 7905 bp was PCR-
sequenced for 20 out of 24 samples (the other four were gland and stroma and other
HPV16 negative samples). Any nucleotide found at any position to be different from
that of the reference HPV16 sequence (61) is here described as variation (probably
occurring before infecting a specific host) or mutation (probably occurring inside a
specific host). Three common variations in all HPV16 positive samples and, in
51
addition, two mutations in only some of the samples were found in this case (Table
3). If an HPV16 variant is defined as an HPV16 sequence with at least one differing
Table 3. X-chromosome inactivation pattern, HPV16 variations and mutations, and
LOH of the microdissected samples. Normal: normal squamous epithelium. Sup.
Inv.: superficially invasive carcinoma. Xc. pattern: X-chromosome inactivation
patterns (a, b, ab, -aa). The a: only short allele remains (inactivation), indicating
52
single type of X-chromosome inactivation pattern in the cell population. The b: only
long allele remains, indicating another single type of X-chromosome inactivation
pattern in the cell population. The ab: two alleles retained, indicating two types of X-
chromosome inactivation patterns in the cell population. The –aa: an additional
allele remains (-a is shorter than a) in the cell population. -: the same nucleotide as
the reference nucleotide at this position. ng: negative for HPV. q57q: left q is
reference amino acid, 57 is codon position and right q is amino acid found at this
codon with a nucleotide variation, thus, q57q is a silent variation, while the other
three are missense variations or mutations. s: short allele remains. l: long allele
remains. d: both alleles remain, thus normal.
nucleotide within the whole genome, three HPV16 variants were found. The V1 was
the variant with the three common variations and was distributed among most of the
samples. The V2 with all the three variations and in addition a mutation in E2 (nt
3800), was seen in three samples (H2-1, -4 and –5). The V3 also with all the three
variations and in addition a mutation in L1 (nt 6318), was found in another three
samples (H2-9, -15 and –16).
Loss of heterozygosity was analyzed as another assistant clonality marker.
Detection was successful for D3S659 (3p13), D3S1283 (3p24.2-22) and D6S311
(6q22-23) in all samples of this case. The LOH patterns for all samples are shown in
Table 3.
As shown in Table 3 where all data were compiled, five different monoclonal
families were found in the samples from CIN II (H2-13), CIN III and 11/12 invasive
carcinoma. The samples from all normal epithelium, three out of four CIN II samples
and a superficial invasive carcinoma were polyclonal.
5.6.2 Discussion
Inactivation of the X-chromosome occurs at the embryonic stage (130-132), and
the polymorphism of the X-chromosome linked androgen receptor gene is neutral to
the proliferation of the cells. This makes it an ideal clonality marker. However, only
three kinds of result (a, b or ab) can be obtained. The a and b represent monoclonality,
while ab shows polyclonal. If a skewed X-chromosome inactivation or some technical
53
problem occurs or samples are taken in a biased way, the same inactivation pattern
would probably be presented by cells with different clonal origin in the same cervix
(135). Therefore, assistant clonality markers such as HPV variation or mutation and
LOH are required to resolve this problem.
With the X-chromosome inactivation marker, we identified four types of clonality
in the samples of cervical neoplasia. The a and b represented different monoclonal
samples. The ab and –aa indicated polyclonality of the respective sample. The HPV16
mutation in E2 and the LOH patterns further divided the three b samples of carcinoma
into two groups. The LOH patterns further classified the ten a samples of CIN II, CIN
III and invasive carcinoma into three groups. The X-chromosome inactivation
patterns, the HPV16 mutation in L1 and LOH patterns together divided the four CIN
II samples into four groups: one with monoclonal a pattern and, three different
polyclonal groups. The X-chromosome inactivation and LOH patterns together pull
one CIN II, one CIN III and four invasive carcinoma samples into one monoclonal
group. These findings strongly indicate that this case is of polyclonal origin. Also, in
this case, the neoplasia seems to have developed in two ways: one from normal
epithelium via CIN II, CIN III to invasive carcinoma such as the a family with six
members, and another from normal epithelium possibly directly into invasive
carcinoma such as the two other a clones and the two b clones.
6 General discussion and summary of the conclusion
Cervical carcinoma is one of the most common malignancies in women.
Epidemical, clinical and experimental studies have suggested that HPV may be the
main cause of cervical carcinoma (90, 288). However, only a minority of HPV
infected patients develop any cervical lesions. Specific types of HPV are considered
the major risk factor because several studies associate HPV types with different
morphologies of cervical neoplasia (289). High-risk types of HPV such as HPV16
and18 associate with invasive carcinoma and CIN III. Moderate-risk types of HPV
such as HPV31 and 33 can be seen most frequently in CIN I and CIN II. Low-risk
types of HPV such as HPV6 and 11 present only in benign tumors, i.e., condyloma
and in CIN I. Thus, HPV6 and 11 have been used as a tool to diagnoses condyloma in
some pathology laboratories. Other risk factors associated with the virus may include
persistence of HPV infection, HPV copy number, expression level and patterns of
54
HPV E6 and E7 genes, HPV physical stage and HPV sequence variations (47, 72,
196, 212, 290).
Since HPV16 is the most commonly seen HPV type in cervical carcinoma and
HPV variations are one of the currently most active subjects in the studies on the
further risk factors, we analyzed HPV types and HPV16 sequence variations or
mutations in cervical neoplasms. In cross sectional case control studies of Swedish
and Russian women, HPV16 E6 variants were distributed nearly even in CIN cases
and invasive carcinoma cases, but when viral oncogene lineages were considered,
additional variations were more frequently seen in invasive carcinoma than in CIN
cases. In a synchronous lesion study, most cases contained an identical type of HPV
or of HPV16 E6 variant from normal epithelium, different grades of CIN lesions to
invasive carcinoma. The results strongly support the opinion that specific infection
with HPV is the cause of cervical carcinoma because identical types of HPV and
HPV16 variants persist from the normal epithelium via CIN lesions to invasive
carcinoma. The results also suggest that a specific HPV16 variant might not be the
further risk factor but that the additional variations of HPV16 oncogene lineages
might be a weak further risk factor.
Recurrent genetic aberrations, such as those observed as LOH, occur frequently in
cervical neoplasms (281), however, no specific oncogene or tumor suppressor gene
has been found to be involed so far. We identified that two common interstitially
deleted regions of chromosome 3p with incidence of LOH higher than 50% of the
informative cases of invasive carcinoma. The six cases with identical LOH patterns
among the synchronous CIN lesions and invasive carcinoma showed a common
deletion in the region between D3S1076 and D3S663 that maps to 3p21.1-3p21.3, and
the region between D3S1260 and D3S1611 that maps to 3p22. The deletions
involving these two regions might contain potential tumor suppressor genes and their
loss may be the early event in the development of the cervical neoplasm. The hMLH1
gene is located in 3p22 and analysis of a microsatellite marker in this gene showed
LOH in 43% of the studied cases, but neither the expression level of protein nor the
genetic sequence of this gene was altered in the cases with LOH in the hMLH1 locus,
suggesting that the hMLH1 gene is not involved in the development of cervical
carcinoma in spite of its frequent involvement in colon carcinoma and other female
tumors.
Cervical invasive carcinoma often co-exists histologically with CINs in the same
patient, and it is assumed that cervical carcinoma is of monoclonal origin and
55
progresses by multiple steps from normal epithelium via different grades of CINs into
invasive carcinoma. If this assumption is true, all the independent CIN cases and the
invasive carcinoma cases should be monoclonal and synchronous lesions of
carcinoma in a certain patient should share the clonality status. Several studies have
shown that CIN and invasive carcinoma are monoclonal (140) while some studies
supplied the possibility of polyclonality for a fraction of cases of cervical neoplasia
(136, 142). With microsatellite markers for chromosome 3p, 75% (6/8) of informative
cases of cervical carcinoma were found to have identical LOH patterns in these
multiple synchronous lesions, but 25% (2/8) cases had different LOH patterns among
the multiple synchronous lesions. In the “golden case” H2, the clonality status was
investigated extensively. The multiple carcinoma and CIN samples from the lesions
could be divided into several different clonal groups by the X-chromosome
inactivation patterns, HPV16 mutations and LOH patterns. The biggest clonal family
included both CIN II, CIN III and invasive carcinoma samples, while another four
families with a monoclonal pattern of carcinoma did not have associated CIN lesions.
These results indicated a complex clonality status and suggested that cervical
carcinoma can be either monoclonal or polyclonal and develop either directly or via
multiple steps.
Both HPV infection and LOH are known to be common events in cervical
carcinoma (281). Our results and the results of others show that HPV infection occurs
earlier than does LOH because HPV can be seen from normal epithelium and CIN
lesions through invasive carcinoma, while LOH can be found only in the two latter
(223, 291). A suggested molecular mechanism of the carcinogenesis of cervical
neoplasia is that one or more target committed stem cells in the basal layer of the
epithelium are infected by specific types of HPV. The HPV infected stem cells then
go along different paths. One path is that chromosomal deletion occurs in these stem
cells to further equip them with the capacities to give rise to neoplasms. If the event
of deletion has a strong enough biological effect, the stem cells would directly give
rise to carcinoma without CIN lesions, otherwise with CIN lesions. During the
process of carcinoma development, some other risk factors can be added, for instance,
additional variations of HPV16 oncogene lineage, some specific mutations or other
unknown factors, which can increase or reduce the tendency of tumor initiation and
progression. If the HPV infected stem cells do not acquire internal gene deletions,
they will continue to give rise to morphologically normal epithelium. If only one HPV
infected stem cell randomly acquires a strong cellular genetic event, it will
56
independently cause a cervical neoplasia with monoclonal status, otherwise with
polyclonal status.
Conclusions:
(1) Cervical invasive carcinoma and CINs in the same case may originate either from
a single committed stem cell or from multiple committed stem cells.
(2) Invasive carcinoma may develop directly from the progenitor cell or via different
grades of CINs.
(3) HPV (as a field factor) is the cause of cervical neoplasia.
(4) Additional variations in specific HPV16 oncogene lineages might be one among
other weak further risk factors of cervical carcinoma.
(5) Chromosomal deletions involving 3p22 and 3p21.1-21.3 may be early events in
the development of cervical carcinoma and these areas probably harbor potential
tumor suppressor genes.
(6) The chromosomal deletions, occurring after HPV infection, may be very
important to assist HPV to accomplish the carcinogenesis of cervical neoplasia.
(7) hMLH1, one of the DNA mismatch repair genes, is not involved in the
carcinogenesis of cervical carcinoma.
57
7 Acknowledgements
During the days and nights working on my Ph.D. project and this dissertation, I
have gotten a lot of help and encouragement from many persons. Here I would like to
express my most sincere thanks to:
Monica Nistér, current supervisor, for kindly helping, encouraging and supporting
me to finish the last half of my Ph.D. project. She scrutinizes the experimental work
patiently and discusses about the interpretation of results repeatedly with me. She
revises the manuscripts and dissertation so seriously that they can become perfect
even without a small punctuation error. From her correction work, I have learnt a lot
of written English which will benefit my future scientific career.
Jan Potén, primary supervisor, for scientific guiding and kindness to me and my
family. I do wish he could read this dissertation and give us comments. For this
dissertation, he already contributed many efforts to supervise me to work on it. Git,
for the hospitality and kindness to us in her family party.
Fredrik Pontén, for being a friendly colleague and a nice referee, for all the help
and encouragement.
Gijs Afink, for being a nice co-author and a nice referee. He speaks and writes
English much better than his native Dutch language. I wish I had that good English
skills like his. He likes to “argue” about scientific stuff in our project with me. I wish
we could have some opportunities to continue the “argument” in the future.
Zhongmin Guo and Feng Wu, for many years of friendship and encouragement.
We share many feeling about life, science, career and society. Guo is also a nice co-
author. Xiang Zou, for good friendship although we have a lot of different ideas about
politics. Xiaoqun Zhang, for the co-operation in the p53 transgenic mouse project.
The Chinese friends who worked or work with me in this department, for their
friendship, Siwei Peng, Zhiping Ren, Qin Li, Weiwei Zhang, Wenru Yu, Siqin Wu
and Guiling Li . The Chinese friends who shared or share a corridor with me, for the
friendship: Xuxia Wu–liang Liu, Ling Ling, Kiu Huang, Xiaoli Liu, Yi Wang, Li Li,
Yuman Zhang, Hao He, Sizhong Bu and Shao Hong.
Former group members: Anna Asplund, Ling Gao, Helena Bäckvall, Cecilia
Wassberg and Natalia Mazurenko, for wonderful co-operation, nice group meetings,
free talks and laughs, delicious food and stimulating alcohol at the group dinner
parties. Anna Asplund is also a nice co-author.
58
Current group members in Monica´s group for the nice scientific presentations at
the group meeting.
Other co-authors: F Kisseljov, Sonja Andersson and Erik Wilander, for the co-
operation.
All other colleagues who work in the department of Genetics and Pathology, for
the great help.
Finally and particularly, my lovely daughter Xian Hu, my lovely son Ruiwen Hu,
my beloved wife Tianyun Pang and my parents, for their forever love and strong
support. Pang is also a nice co-author.
59
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