Tissue tumor marker expression in normal cervical tissue and

Digital Comprehensive Summaries of Uppsala Dissertations
from the Faculty of Medicine 1137
Tissue tumor marker expression
in normal cervical tissue and in
cervical intraepithelial neoplasia,
for women who are at high risk of
human papilloma virus infection,
are smokers, contraceptive users
or in fertile age
RAGHAD SAMIR
ACTA
UNIVERSITATIS
UPSALIENSIS
UPPSALA
2015
ISSN 1651-6206
ISBN 978-91-554-9348-6
urn:nbn:se:uu:diva-262889
Dissertation presented at Uppsala University to be publicly examined in Rosénsalen, ingång
95/96, Akademiska sjukhuset, Uppsala, Monday, 16 November 2015 at 13:00 for the
degree of Doctor of Philosophy (Faculty of Medicine). The examination will be conducted
in English. Faculty examiner: Prof. Pär Sparen (Department of Medical Epidemiology and
Biostatistics (MEB), C8).
Abstract
Samir, R. 2015. Tissue tumor marker expression in normal cervical tissue and in cervical
intraepithelial neoplasia, for women who are at high risk of human papilloma virus infection,
are smokers, contraceptive users or in fertile age. Digital Comprehensive Summaries of
Uppsala Dissertations from the Faculty of Medicine 1137. 74 pp. Uppsala: Acta Universitatis
Upsaliensis. ISBN 978-91-554-9348-6.
The aim of this research was to study the correlation between tissue tumor marker expression
and HR-HPV infection, smoking, hormonal contraceptive use and sex steroids in women with
cervical intraepithelial neoplasia or normal epithelium. The study investigated the expression
of 11 tumor markers in cervical biopsies obtained from 228 women with different diagnoses
ranging from normal cervical epithelium to various stages of CIN. 188 women were recruited
at our colposcopy clinic (out-patient surgery, Department of Obstetrics and Gynecology, Falun
Hospital) for laser cervical conization or a directed punch biopsy, either because of a vaginal
smear (Pap smear) that showed cytological findings suggesting CIN, or because of repeated
findings showing atypical squamous cells of undetermined significance (ASCUS). For 40
volunteers, punch biopsies were taken from the normal cervical epithelium. The time period for
this study was 2005-2007.
Study I : 228 women, of whom 116 were tested, 64 were positive to HR-HPV. The results
showed that Ki67 tumor cell proliferation index was the only marker that independently
correlated to both the presence of HR-HPV and the severity of cervical lesions.
Study II: 228 women, of whom 83 were smokers (36, 9%). Smokers showed lower expression
of p53, FHIT (tumor suppressor markers) and interleukin-10 .Higher expression of Cox-2 and
Ki-67 (tumor proliferation markers).
Study III: 195 women who were premenopausal. There was increased p53 expression (tumor
suppressor) in the progestin-IUD users compared to non-users. Decreased IL-10 expression
(immunological marker) was observed in both COC users and any progestin-only users.
Study IV: Serum from 80 premenopausal women was available. The main finding was that
the increased levels of serum progesterone and estradiol were associated with increased Cox-2
expression (proliferation marker). Serum progesterone and estradiol levels influence cellular
and extracellular proteins which have been associated with neoplastic development in normal
epithelium and CIN.
Conclusion: The results of these studies support previous epidemiological findings on the
role of smoking, contraceptive use and sex steroids as co-factors in development of CIN and
that tumor marker expression varies in different grades of CIN.
Keywords: tumor markers, cervical intraepithelial neoplasia, smoking, contraceptive use, sex
steroid hormones, HPV infection
Raghad Samir, Department of Women's and Children's Health, Obstetrics and Gynaecology,
Akademiska sjukhuset, Uppsala University, SE-75185 Uppsala, Sweden.
© Raghad Samir 2015
ISSN 1651-6206
ISBN 978-91-554-9348-6
urn:nbn:se:uu:diva-262889 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-262889)
To my family
List of Papers
This thesis is based on the following papers, which are referred to in the text
by their Roman numerals.
I
Samir R, Asplund A, Tot. T, Pekar G, Hellberg D. High-risk
HPV infection and CIN grade correlates to the expression of cmyc, CD4+, FHIT, E-cadherin, Ki-67, and p16INK4a. J Low
Genit Tract Dis. 2011 Oct; 15(4):280-6.
II
Samir R, MD; Anna Asplund, PhD; Tibor Tot, MD, PhD;
Gyula Pekar, MD; Dan Hellberg, MD, PhD. Tissue tumor
marker expression in smokers, including serum cotinine concentrations, in women with cervical intraepithelial neoplasia or
normal squamous cervical epithelium. Obstet Gynecol
2010;202:579.e1-7.
III
Samir R, Anna Asplund, Tibor Tot., Gyula Pekar, Dan Hellberg. Oral contraceptive and progestin-only use correlates to
tissue tumor Marker expression in women with cervical intraepithelial neoplasia. Contraception. 2012 Mar; 85(3):288-93.
IV
Samir R, Tibor Tot., Anna Asplund, Gyula Pekar, Dan Hellberg. Increased Serum Progesterone and Estradiol Correlate to
Increased COX-2 Tissue Expression in Cervical Intraepithelial
Neoplasia. Anticancer Res. 2010 Apr; 30(4):1217-22.
Reprinted with permission from the respective publishers.
Contents
Introduction ................................................................................................... 11
Screening programs: ................................................................................. 12
Cytology tests/screenings: ................................................................... 14
HPV tests/screening ............................................................................. 15
Cell biology .............................................................................................. 17
Etiology, co-factors and Risk factors ....................................................... 18
Human papillomavirus infection. ........................................................ 19
Smoking ............................................................................................... 25
Oral contraceptives .............................................................................. 26
Genetic predilection ............................................................................. 26
Ageing ................................................................................................. 27
Tumor markers ......................................................................................... 27
Present investigation ..................................................................................... 32
Aims of this thesis .................................................................................... 32
Material and methods .................................................................................... 33
Paper 1 ...................................................................................................... 36
Paper 2 ...................................................................................................... 37
Paper 3 ...................................................................................................... 38
Paper 4:..................................................................................................... 38
Results ........................................................................................................... 40
Paper 1 ...................................................................................................... 40
Paper 2 ...................................................................................................... 42
Paper 3 ...................................................................................................... 44
Paper 4 ...................................................................................................... 45
Discussion ..................................................................................................... 47
Tumor markers ......................................................................................... 47
Co-factors ................................................................................................. 52
Smoking ............................................................................................... 52
Hormonal Contraceptives .................................................................... 53
Progesterone and Estrogen .................................................................. 54
Summary ....................................................................................................... 56
Acknowledgments......................................................................................... 57
Sammanfattning på Svenska ......................................................................... 60
References ..................................................................................................... 62
Abbreviations
ASCUS
CIN
CPS
CI
COC
DAB
HPV
HR.-HPV
LR-HPV
HSIL
LSIL
IHC
IL 10
LBC
PCR
PPV
MID
SCJ
Syst-p
TZ
TMA
Atypical squamous cell of underdetermined origin
Cervical intraepithelial neoplasia.
Cytology pap smear
Confidence interval
Combined oral contraceptives
3, 3-diaminobenidine
Human papilloma virus
High risk HPV
Low risk HPV
High grade squamous intra-epithelial lesion
low grade squamous intra-epithelial lesion
Immunohistochemistry
Cytokine, immunosuppressive
Liquid based cytology
Polymerase chain reaction
Positive predictive value
Medicated intrauterine device
Squamouscolumnar junction.
Systemic progesterone users.
Transformation zone
Tissue microarray
Introduction
Cervical cancer is the second most common female cancer worldwide and
according to the most recent data, an estimated 528,000 new cases of cervical cancer occur among women each year, the vast majority of them in developing countries (1) . In 2008 an estimate of 275,000 deaths from cervical
cancer was registered, placing cervical cancer as the fourth most common
cause of cancer death among females worldwide(2, 3) .The main cause of
cervical cancer is the persistent human papillomavirus infection(4), but invasive cancer is a rare complication of HPV infection, itself a rather common
type of viral infection (5) . HPV virus infection is the main cause of cervical
cancer but it needs co-factors such as smoking, sex steroids and contraceptive use in order to invade the immune system of the host.
Since the beginning of cervical screening programs in the early sixties,
mortality and morbidity from cervical cancer has been reduced. The aim of
screening programs is to recognize cervical intraepithelial lesions and establish the right diagnosis in order to treat those lesions which are liable to lead
to cancer. The need for more than diagnostic cytology appears reasonable, in
order to decrease the unnecessary over-treatment of non-specific cervical
lesion. The main purpose of many studies done in this field is to find the
tumor markers which can be used as diagnostic tools or surrogate markers
for HPV infection. These surrogate markers might also aid the differential
diagnosis among different cervical intraepithelial lesions and help to distinguish those lesions liable to lead to cervical cancer from non-specific changes, such as atypical squamous cells of underdetermined origin ASCUS.
Two things must be taken in consideration: firstly, the risk of subjecting
women with low grade or non-specific lesions to repeated testing, biopsies
and colposcopies, and secondly, the cost and time required in order to establish the right diagnosis.
Anatomy:
The cervix is the most inferior part of the uterus protruding into the vagina.
The cervix is divided anatomically into a vaginal portion and a supra-vaginal
portion. This cervical layer consists of the inner mucosa, smooth muscles.
Normally in non-pregnant females the cervix length is about 3cm. The end
cervix is a mucosal lining of the cervical canal (covered with a single layered
columnar epithelium) while the ectocervix is the vaginal part of the cervix
(covered with stratified squamous epithelium).
11
The term SCJ refers to the squamocolumnar junction where the metaplastic changes occur. The majority of carcinomas begin at the transformation
zone of the cervix uteri where the metaplastic changes convert the columnar
mature cells to a different mature cell type. The process usually involves
conversion from a columnar cell to a stratified squamous cell.
The area between the congenital junction (the original squamocolumnar
junction) and functional junction (post puberty) is called the transitional
zone TZ.
Screening programs:
Organized screening programs using cervical cytology (pap smears or liquid
based cytology) have been shown to be effective in decreasing mortality and
incidence of invasive cervix cancer. Mass screening has also reduced the
present incidence by more than half since the 1960s and 1970s in many highincome countries (6-8).
Cervical cancer screening was implemented in Sweden in the mid-60s and
since then a significant decrease in cervical cancer has been observed. Annually around 700,000 pap smears are taken in Sweden, with a total population of 9,000,000. Approximately 600,000 are taken within the massscreening program, the remainder being taken as part of other gynecological
examinations. The overall incidence of cervical cancer declined by 67% over
a 40-year period, from 20 cases per 100,000 women (world standard rate) in
1965 to 6.6 cases per 100,000 women in 2005. During the last decade, however, the incidence has stabilized (Cancer incidence in Sweden. National
Board of Health and Welfare 2008).
One explanation for the stability in the cervical cancer incidence is the
nonparticipation in the regular screening programs; another explanation is
the poor management of abnormal smears found during the primary screening(9) . According to the Swedish nationwide audit of the cervical screening
program, 32% of women with invasive cervical cancer had a pre-diagnosis
of abnormal smear finding under their participation in the normal screening
interval (10).
Dysplasia
Dysplasia is used to describe the premalignant squamous cervical cellular
changes which range from mild to moderate and severe, but dysplasia has
been replaced by the term CIN which follows the Richart’s description system (7). The European guidelines for quality assurance in cervical cytology
terminology recommend CIN classification for histopathological diagnosis
(11).
CIN is used to describe the histological changes which are detected with
biopsy and is divided into three degrees of severity. CIN I is a low grade
12
lesion (formerly called mild dysplasia) manifesting mildly atypical cellular
changes in the lower third of the epithelium. HPV viral cytopathic effect
(koilocytotic atypia) is often present. CIN II is a high grade lesion (moderate
dysplasia) referring to moderately atypical cellular changes confined to the
basal two-thirds of the epithelium with preservation of epithelial maturation.
CIN III is a high grade lesion (formerly called severe dysplasia or carcinoma
in situ). It refers to severely atypical cellular changes encompassing more
than two-thirds of the epithelial thickness, and includes full-thickness lesions.
Another terminology used to describe these changes at the cytological and
histopathological level is LSIL (Low-grade squamous intraepithelial lesion)
and HSIL (High –grade squamous intraepithelial lesion). LSIL corresponds
to CIN I and HSIL comprises CIN II and CIN III (12, 13).
Background to cervical cytology screening
The Pap smear (vaginal cytology) was developed by George Papanicolaou in
the 1920s. Later, in 1941, G. Papanicolaou and the gynecologist Herbert
Traut published the first major article on the use of vaginal smears for the
diagnosis of cancer of the uterus (14) . Soon thereafter, the pap smear
(named after Papanicolou), was born and is still considered a sensitive, specific, simple and effective cancer screening test. Simultaneously, Hans Hinselman and Leitz technicians devised the first working binocular colposcopy
(hinselmann H Einfurhrung in die kolposopi.Hamburg 1933). In 1925, Hinselman published the first paper on colposcopy, and in 1933 the book ‘Einfuehrung in die Colposcopy’ was published .Colposcopy was developed
further in1925 by Hinselman and Eduardwirth, but routine colposcopy examinations were confined to Germany until the 1960s. In the United States,
as early as 1929, Levy described the importance of studying the genital tract
with some degree of magnification and subsequently Emmert published an
article introducing colposcopy to North American physicians (Emmert F.The
recognition of cancer of the uterus in its earlier stages .J AMA 1031;
97:1684). By 1932 the colposcopy technique was used in a few centers. The
present form of colposcopy started in 1953 when Bolten introduced modern
colposcopy in United States. Initially, it served as a tool to identify women
with asymptomatic early invasive disease. Subsequently, it has also helped
physicians identify pre-invasive squamous neoplasia of the cervix. At a
meeting of the American College of Obstetricians and Gynecologists in Miami in 1964, a group of enthusiastic colposcopists identified the need for a
Colposcopy Society. Thereafter, through the dedicated efforts of many
members of the Society, various colposcopy courses were initiated. In 1981,
Hamou introduced the microlpohystrescope for the examination of the cervix
and endocervical canal. This provided a panoramic and contact microscopical observation of stained cells in vivo at high magnification.
13
Cytology tests/screenings:
The conventional cytology Pap smear (CPS) has been successfully used
since the sixties. Cells are taken from the cervical canal and the uterine cervix, these cells smeared on a glass slide and fixed in 95% ethyl alcohol to
prevent air drying. The slide is then sent to the lab for analysis. The sensitivity of this test depend on the number of the tests done within a period of
time, a single test has a sensitivity for CIN II+ (70-80%) (13, 15-17).
The specificity is high, 90-96%, and the positive value PPV is about 42%.
Repeating the test within an interval of 3-5 years increases its sensitivity.
In order to evaluate the sensitivity and specificity of the test we need to
understand the differences between test threshold (the point when the test is
positive) and the reference standard threshold (the point when the reference
standard is positive). In CPS the most accurate testing is to consider LSIL as
the test threshold and CIN2-3 as a reference standard (this will give as a
sensitivity of 70-80% and specificity 95%)(18) .
According to 2 different studies done by Soost. H, et al 1991 and Benoit
.AG, et al 1984 (19, 20) comparing single and repeating Pap testing, the
sensitivity of any abnormality on a single test for detecting high-grade lesions was 55% to 80%. Because of the slow-growing nature of cervical cancer, the sensitivity of the repeating Pap testing is likely to be higher.
Liquid –based cytology (LBC) is another type of test that has been used
recently based on the idea that cells will be sampled with a cerveix–brush or
with a plastic spatula and endo-cervical brush. The smear will directly immersed into a special liquid solution (20 ml preservCytSolution) and send to
the lab for analysis .This method provides a thin monolayer of the cells on
the slides which are easier and quicker to read than the CPS and the material
can be used to do more testing such as human papillomavirus (HPV) DNA
testing.
LBC has been compared with CPS in a wide range of studies, most of
these studies showing an increase in the sensitivity for detection of pathological changes (LBC eliminates blood and extra derbies from the specimen)
(21-24).
A meta-analysis done by E.Davey et al (24)showed that in high–quality
studies CPS classified more slides than HSIL and fewer slides than ASCUS
as compared to LBC. The benefits and disadvantages of these tests can be
discussed as a matter of economy and accuracy. Many RCT have been done
to find which test is the more reliable; Fröberg et al 2013 found no significant differences in screening performance between LBC +HPV triage and
CPS(25, 26).
Sigurdsson K et al published a comparative study done in Reykjavik, Iceland which included 98477 women aged between 20-69 years old. The result
of the study showed that LBC tests are no more sensitive than Pap smear in
detection of high grade cytology or CIN2+histology, irrespective of age.
14
LBC test increased the frequency of low grade lesion below the age of 40,
but decreased the total number of abnormal cytology tests over the age of
40(27).
The usefulness of cytology smear only use (CPS,LBC) in establishing the
right diagnosis in cases of HILS (CIN 11-CIN111) is well known, while this
usefulness is questioned in cases of atypical squamous cells of undetermined origin (ASCUS) or in LSIL. In order to establish the correct diagnosis
or to reach subsequent diagnostic procedures, many different health guidelines have been introduced such as repeating the cytology, testing for HRHPV infection, colposcopy and cervical punch biopsies or a combination of
2 or more of these.
It is well known that the progression risk of these intraepithelial lesions
from CIN1 to CIN111 within 5 years is 2,6% (28), but also to be considered
are the hazards of over-treatment and subjecting these women to invasive
procedure (over-treatment) such as bleeding, pain, sexual dysfunction (29)
and pregnancy–related morbidity. The rate of invasive cervical cancer in
women with high –grade squamous abnormal cytology within 24 months is
1,44% (30). The rate in histologically verified CIN III without treatment was
31% after 30 years follow up (31). In recent years surrogate markers have
been used to estimate the high risk precursor or invasive cervical cancer
lesion such as P16/Ki67.
HPV tests/screening
Since the progression of cervical intraepithelial lesion to cervical cancer is
considered to be quite slow, and the period between the establishment of
HPV infection and invasive cervical cancer development is many years, it
will be logical to find an effective screening test. It is important to understand that CIN lesions have a 30% risk to progress to invasive cancer if untreated within many years (31) while CIN 2 is considered as the threshold
between treatment or not. This fact shows the need of finding an effective
test to be used primarily or as a part of triage system. In this way the screening for HR-HPV will make it easier to decide which lesions we should treat
and will help to identify women at risk (32) (33, 34).(35).
The low risk HPV testing does not identify women at risk for low risk
CIN II, III (36, 37) . HPV testing has been considered as co-test for screening cytology in many countries. Primary cervical screening for HR-HPV has
been found to be more sensitive in detecting CIN II+ compared to cytology
but unfortunately with less specificity (38). HPV testing has been used as a
part of triage screening including both cytology and HPV testing and has
showed a higher sensitivity in detecting CIN III+ than cytology by itself (
(39). It is important to shed light on the benefit of triage screening since each
test alone (as a single test) has its own problems regarding specificity. HPV
testing shows less specificity among younger women (over-diagnosis regard15
ing CIN II (40), whilst cervical cytology alone is more dependable among
women over the age of 50. (41).
A Swedish study done in 2011by Gyllensten. U and et al (42) found that
repeating the self-sampling HPV test can increase the specificity of cervical
cancer screening of women aged 30-65. The prevalence of CIN II+ lesions in
women with two positive HPV tests varied from 49% in women aged 30-39
years to 24% in women aged 50-65 years. Short-time repeat HPV testing
increased the specificity for detection of CIN II+ lesions from about 94.2%
to 97.8%.
Different HR-HPV testing types
It must be taken into consideration that HPVs cannot be cultured and the use
of serology assays has its own limitations concerning the accuracy of the
test. There are three types of HPV test: those tests that can detect HPV nucleic acid DNA, RNA, or the cellular markers of HPV-associated malignant
transformation in clinical samples. (43, 44).
There are many types of assays available nowadays:
HPV tests are divided into two categories:
DNA detectors:
1 -HC2 test (Hybrid capture 2 assay).
2-PCR. (Polymerase chain reaction).
3-LA. (Linear array HPV genotyping test).
RNA detectors:
1-HPV mRNA
Home screening test (self-collection of cervical (and vaginal) fluid
for HPV testing).
This is a new approach regarding HR-HPV testing by using an FTA card
which provides a solid support that stabilizes the DNA. The sampling kits
can be used accurately and the risk of damaging the sample will be very low.
HR-HPV testing there with the PCR method has been found to be more sensitive for CIN II ,CIN II+ compared to conventional cytology (45)
Histopathology:
The most common types of cervical cancer are squamous cell carcinomas
and these represent about 75%% of all cervical cancer - about 15% are adenocarcinomas. Neuroendocrine and other histological types constitute about
10% (46, 47).
Dysplasia is the premalignant squamous cell abnormalities that range
from mild, moderate to severe dysplasia, and eventually to carcinoma in situ,
but this classification has been replaced by cervical intraepithelial neoplasia
(CIN). CIN is also used for histological abnormalities that are histopathologically diagnosed in cervical biopsies.
16
Cell biology
The cell cycle (eukaryotic cells) is programmed so that the cell will replicate
its own DNA, correctly leading to the formation of two identical cells. In
order to ensure that this will happen, there are many check points at which
the control and repair of the DNA can be permitted. Alteration of this mechanism is the key factor in the initiation of cancer changes in all organs in the
human body (Charlotte E, Gullberg U .Cell biology .Chapter 15.Denmark
2003.ISBN91-44-20047-3).
Three main phases in the cell cycle are:
G0 (the resting phase): in this stage the cell is considered to be inactive but
is still metabolically and functionally active.
G1: The first check point (the cell growing phase): in this stage the cell
starts to grow as a response to external factors (growth factors, cytokines
etc.). The cell cycle will progress to S phase according to two mechanisms:
1- E2F transcription factor activation.
E2F (a family of transcription factors (TF)). Three of them are activators:
E2F1, 2 and E2F3a. Six others act as suppressors: E2F3b, E2F4-8. All of
them are involved in cell cycle regulation and DNA synthesis.
The E2F1 is normally controlled or inhibited by the Rb protein. The Rb
tumor suppressor protein (Rb) binds to the E2F1 transcription factor, preventing it from interacting with the transcription machinery of the cell. In the
absence of Rb, E2F1 facilitates the trans-activation of E2F1 target genes that
enable the G1/S transition and S-phase.
2 -Activation of the CDK4/6-cyclin D complex:
In human cells the presence of cyclins is important since their major role as
eukaryotic proteins is the activation of the CDK4/6 –cyclin D complex.
This complex phosphorylates the Rb (tumor suppressor) leading to inactivation of Rb protein and enhancing the transcription factor E2F which allows the cell to enter G1-S phase.
The alteration of these two mechanisms is the key factor in the development of cancerous changes at cell level. Rb is one of the targets of the oncogenic human papilloma virus protein E7. By binding to Rb, they stop the
regulation of E2F transcription factors and drive the cell cycle to enable virus genome replication. The other mechanism that plays a major role in cell
cycle is the activation of P53.
P53 is considered to be the guardian of genome in the human body. Normally p53 is controlled by the negative regulator, mdm2, its main function
being to inactivate P53.
17
P53 activates as a response to cell injury (viral infection or hypoxia) allowing the cell to repair itself and enter what is called cell arrest. Apoptosis
is another form of response to injury where p53 prompts cell death instead.
The mechanism behind the decision between apoptosis or repair is still considered to be complicated and difficult to explain. P53 acts also in another
way by stimulating the p21 and p16 which act as inhibitors to the CDK4/6
complex, leading to the inactivation of cyclin D and preventing it from affecting Rb protein.
S (DNA synthesis).
In this phase DNA synthesis begins, where the Cyclin/cdk2 complex controls the phosphorylation of DNA polymerase, at the same time stopping
another replication of DNA.
G2: the second check point is G2, in which the cell will be controlled in
order to enter the mitosis phase. This process is controlled by the CDKs
which are activated by phosphorylation by Mitosis Promoting Factor (MPF).
M (mitosis).
Etiology, co-factors and Risk factors
HPV infection.
Co- factors:
Smoking.
Contraceptive Use.
Sex steroids.
Genetic predilection
Risk factors:
Age.
HIV/AIDS, Immunodeficiency.
Low socioeconomic status
Poor access to health care
Multiple sex partners/ first intercourse at an early age.
High parity
18
Cofactors/risk factors
Exogenous/Enviromental
Endogenous /Host
•Smoking
•contracepve use
•Mulple partners
•Co-infecon with HIV
•Low soicaleccnomic status.
•Sex steroids
•Genecs
•High Party
Figure 1. Cofactors and risk factors for development of cervical intraepithelial neoplasia.
Human papillomavirus infection.
Human papillomavirus belongs to the family Papillomaviridae, which includes viruses infecting many different vertebrates. They are strictly speciesand organ specific. The HPV life cycle in the cervix is confined to the epithelium (48).At the border between squamous cell epithelium covering the
vagina and glandular epithelium covering the uterus, the transformation zone
is the target for HPV invasion (26). HPV is transmitted in desquamated genital epithelial cells which after their own degeneration leave the HPV capsids
free to bind themselves to the target tissue such as TZ of the cervix, anal
verge or oropharynx (the areas most exposed to micro trauma) (49, 50). HPV
infection is one of the most common sexually transmitted genital infections.
It is mostly clinically silent and self-limiting. Some women may remain persistent carriers of the viral infection and develop a high risk of progression to
CIN and invasive cervical cancer (26). Most HPV-related lesions resolve
spontaneously, and progression to cervical neoplasia is relatively rare (5153). A key factor in permitting the disease to progress is the ability of HPV
to evade the immune system and establish a persistent infection. Even when
immune response is achieved there is no full protection from future HPV
infections. The lifetime risk of genital HPV infection is approximately 80%.
For many HPV- infected women (90%), their immune defense will generally
eliminate the infection within 12 to 36 months (54).In remaining cases, progression to cytological abnormalities and CIN is observed in 5% to 10% of
persistent HPV infections(26). Within a period of 7-15 years less than 1% of
these infections lead to carcinoma (55, 56). According to a study by Kleter et
19
al, other factors such as genetic predisposition, frequency of reinfection,
genetic variation within HPV type, co-infection with more than one HPV
type and hormone levels may influence the clarity of the primary HPV infection.(57).
The HPV virus
The HPV has a relatively small genome (double –stranded DNA) of 8000
base pairs which are organized in 3 regions: The long control region (upper
regulatory region URR) and 2 coding regions (the late L and the early region
E)(58).
The virion is encased in a non-enveloped 72-sided icosahedral protein
capsid. The URR is a non-coding region that is responsible for regulation of
viral replication and transcription in the early region. The functional HPV
genes are found only in the early region and the late region, they referred as
ORF (open reading frames). Each ORF is read by the RNA polymerase. The
early region is responsible for viral replication in the early HPV life cycle
while the late region is responsible for capsid production late in the life cycle.
The early coding
The early coding region is divided into E1-E7.
The life cycle of HPV in the cervix mucosa and its main target in the
transformation zone started with the integration of the HPV DNA with the
host cell genome. Because it has no DNA polymerase, it needs to bind itself
with host polymerase via E1 and E 2. The cell transformation starts at this
point with the help of E6 and E7 (8) . E7 binds to and degrades Rb and initiates the cell division via E2F. E 6 binds to and degrades p53. E6 and E7 will
disable the anti-oncogene effect of p53 and Rb and will result in cell proliferations and immortalization. Thus, impairment of the tumor suppressor
genes p53 and Rb is the key step in cell immortalization (8).
The late region
The two late regions L1 and L2 are responsible for the construction of the
capsid of the infected HPV genome. The capsid is crucial for HPV because
without the surrounding capsid the HPV DNA is considered as noninfective. The production of capsids appears to happen in the superficial
layer of cervix to allow delivery of HPV infection from host till host (59).
High- and low-risk HPV infections
More than 174 HPV types are known and new types are continually being
discovered (59). Of these, 13-18 HPV types are considered high-risk (HRHPV) and 5 HPV types as moderate-risk for cervical neoplasia. Globally,
HPV 16 and HPV 18 dominate in CIN and cervical cancer. According to a
retrospective cross-section worldwide study, published in The Lancet by de
20
Sanjose S et al, approximately 70% of cervical cancer was caused by HrHPV-16 and-18 while HPV -31,-33,-35,-45,51,-52 and -58 contribute to the
remaining 20% of cases(60).
Co-infection with both HR-HPV and LR-HPV
Infection with low risk HPV (HPV6,HPV11) causes condyloma (genital
warts) .It is well-known that LR-HPVs have low carcinogenic ability but a
Swedish study by Sundström et al .2015 found that co- infection with both
high-risk HPV and low –risk HPV can decrease the risk for future invasive
squamous cancer development (61). This finding can be explained by the
ability of low-risk HPV 6 and HPV11 to act as antagonists to the infection
with high-risk HPV 16 resulting in the hampering of the risk for invasive
cancer to be lower than expected (62).
The life cycle of human papillomavirus in the cervix:
As mentioned above, HPVs are intracellular parasites which attack the basal
layer of the cervix through minor abrasions in order to start their own life
cycle. The initial attachment of HPV to the cells starts when L1 protein interacts with HSPG (heparin sulfate proteoglycans) receptors, up-regulators
on the basement membrane during wound repair. This interaction will promote the internalization of the virion into the cell by causing viral structure
changes that allow HPV L2 to be exposed to the local enzymes, furin or PC
5/6. These changes allow previously hidden epitopes of L1 molecules to
become exposed to receptors on the newly-formed basement membrane (63).
This gives the HPV its unique privilege in only infecting the basement layers
of the cervical epithelium. It has been shown that by using antibodies targeting the capsid proteins L1, L2, the virion will lose its ability to attach to the
basal membrane and enter the cells. The virus will be transported into the
cell nucleus where an episomal reaction will start (HPV DNA is selfreplicating plasmid which is not integrated with human chromosomes) and
this will result in the genome replicating itself once per cell cycle during Sphase (64). The infected basal cells, which show signs of cell disruption as a
result of the viral infection, continue their differentiation and migration to
the epithelial surface(65). During the early stages of infection, the copy
number of viral genomes is between 50 to100, and the viral genome exists as
extrachromosomal plasmid or an episomal from the replicates, as the host
cell chromosomes replicate (66) .This viral infection prompts the host cell to
proliferate abnormally inducing various kinds of lesion (Flat warts, papillary
warts, CIN, cervical cancer). As it is well-known that not all individuals who
have been exposed to HPV infection exhibit these changes, a question arises
about other cofactors that play a major role in the outcome of the HPV infection.
These cofactors can be internal, such as genetic effects, or external, such
as environmental. HPV integration into the host genome will initiate a chain
21
of events involving the impairment of the tumor suppressor genes P53 and
Rb. E7 and E6 start to express by affecting their target proteins. E7 binds to
Rb, a tumor suppressor that, as mentioned above, inactivates E2F (Transcription factor). The activation of E2F causes the transcription of genes responsible for DNA replications and cell division. E6 virus protein binds to P53.
Normally P53 is responsible for triggering cell repair and even apoptosis.
The main principle in the HPV infection is the need to induce DNA synthesis in the host cells in order to replicate its own viral DNA. This process is
usually started with the interference of E7 with Rb, which is normally responsible for inducing apoptosis of the infected cell. The difference in the
mode of action between the HR-HPV and LR-HPV is the mode of E6 and
E7capacity (67). LR-HPV is incapable of degradation of p53 and of inactivating Rb and thus cannot induce cell immortalization and oncogenesis.
Single amino acid differences of the E7 protein in LR-HPV and HR-HPV
lead to higher affinity of HR-HPV for RB protein and p53. This induces an
alteration in the normal cell cycle, in that the cell will be totally controlled
by the virus and the process of immortalization of the epithelial cells will be
initiated. These changes will lead to the start of cancerous and precancerous
lesions in the cervix (68, 69). Genome amplification starts in the mid-layers
of the cervical epithelium. The cells will then express viral E4 and enter the
cell cycle phase S to G2. When the cell cycle is finished in the upper epithelial layers, the viral L1 and L2 proteins start forming the capsid of the virus
and packaging of the viral genome begins finalizing.
HPV infection
Microabression in the
cervix
Viral particles
targeting the host
cells in the basement
membrane
Viral genome
replicated in
conjunction with host
DNA
Formation of
episomes /low level
(escape the immune
system)
Cell divison
Daughter cells starting
differentiation
increasing expression
of viral genome
Generation of new
viral particles
shedding of
differentiated cells+
spreading of the virus
Figure 2. The pathway for HPV from the cervical basal cells to the shedding of new
virus particles.
22
Figure 3. This illustration shows the effect of HPV virus on the basal cervical cells
and the integration into the host genome.
•mdm2.
•P53-mdm2 complex(inactive form av P53).
•Activiation of P53 by infection/Hypoxia.
P53
P16/P18
Cdk4/Cdk6
•Inactiviation of P53by cancer(mutation).
•degredation of P53 by HPV oncogen E6 (inactive form)
•Activiatation of P16/P 18 pathway by P53.
•Cell repair/Apoptosis.
•P16/p18 inactivt the Cdk pathway.
•Cyclin D.
•Cdk4/Cdk-CyclinD.
•Rb inactiviation by phosphorylation.
•The cell will be activited and enter G1/S phase.
• Rb inactivitation by protolysis (HPV oncogen E7).
Figure 4. Cell biology and the pathway of tissue markers in the cell cycle during
HPV infection.
23
Host immunity
The important factor in determining the risk factor of malignancy is the
clearness of the viral infection by the host immune system. When there is a
defect in the host immune system, HPV does not clear within a short period.
HPV is mostly a transient infection with the median time to clearance of
incident cervical HPV being just over 9 months, and nearly 91% of infections becoming undetectable within 2 years (70). The likelihood of persistent
infection depends on the time required for elimination, so the longer the
infection lasts the higher the risk of persistence (71, 72) . HPV can escape
the immunity of the host by different mechanisms (73):
-As the virus is not causing viremia (it is not cytolytic), there is no
inflammation and activation of the host immune system.
-The area of replication is avascular area, so the accessibility of immune cells to the affected area will be limited.
-HPV E6, E7 onco-proteins interfere with type 1 interferon antiviral
response (Interferon 1 stimulate both macrophages and natural killers’ cells to start an antiviral response).
-The lack of inflammation cytokine released from infected keratinocytes limits the activation of adaptive immunity.
-HPV E5 proteins promote entrapment of peptide –loaded HLA class
1 receptors in the Golgi body, and the lack of HLA surface expression allows the non-detection of the virus from CD8+cytotoxic Tcells.
Prevention:
The identification of the HPV oncogenes E6 and E7 led to the development
of effective vaccines with immunological activation of HPV antibodies, but
these vaccinations are at present still only directed against HPV 16 and HPV
18. However, some cross-reactions have been observed against other high
risk HPV types (74). As 13-18 HPV types are considered as high-risk, conclusive results will not be available for the next 10-20 years. The HPV vaccine is made of Virus-like particles (VLPs) which are empty non-infectious
viral shells consisting of recombinant L1 capsid proteins without containing
any viral DNA. Immune response will be demonstrated by the formation of
antibodies against L1.
The two main vaccines that are used nowadays are quadrivalent HPV
vaccine Gardasil (Merck Sharp & Dohme Corp) against new infections with
HPV 6, 11, 16, and 18, and bivalent HPV vaccine Cervarix (Glaxo Smith
Kline Biologicals) which protects against new infections with HPV 16 and
18. Both HPV vaccines may offer some cross-protection against other HPV
strains. The vaccines do not protect against strains which already have been
infected.
24
In Sweden, qHPV vaccine is considered part of the organized school vaccination programs since May 2007 (75) and is given to girls aged 9-13 years.
Catch up vaccination to girls aged 18-26 has been offered in some counties
in Sweden (Stockholm). According to a Swedish study in 2012 by Leval A
et al, where the effectiveness of qHPV vaccine was tested against Genital
warts (short incubation period), the qHPV vaccine offered high protection
against genital warts (caused by HPV 6, 11) among girls and women younger than 20 years. The effectiveness of the vaccine was 93% in the group of
girls who received vaccination before the age of 14. The study could prove
that the effectiveness declined with higher age at the first vaccination (76) .
Smoking
Since the early 80s, smoking has been considered a co-factor in cervical
neoplasms, involved in cervical carcinogenesis and the initiation of precancerous changes in the cervix (77, 78), although the biological mechanism is
still unclear. Earlier it was seen as a surrogate for lifestyle factors, in particular promiscuity. In the early 80s some studies appeared that were able to
adjust for sexual risk behavior, and the correlation between smoking and
CIN remained. As in all epidemiological studies, residual confounding could
not be ruled out. In the mid-80s cervical mucus in smokers with CIN was
found to have very high concentrations of nicotine and its metabolite, cotinine. Thus, the nicotine levels were 40 times increased in cervical mucus
compared to serum levels (79). It was a turning point and further studies
have been published, essentially confirming the first report. There are now
few objections to smoking being a biological co-factor in cervical carcinogenesis. Possible biological mechanisms however, by evaluating expression
of tumor markers in CIN such as in the present study, have been poorly studied and in general included only single tumor markers.
Recent data showed that the carcinogenic effect of smoking combined
with the alteration of the immunological response at the cell level can explain the poor prognosis of cervical cancer among smokers. A study in 2008
by Simen-Kapeu et al (80) found that smoking has the ability to alter the
immunological response to HPV16/18, impairing the antibody response in
HPV16/18-infected young women. Although the exact mechanism behind
the effect of cotinine on immunological activity is not well understood at cell
level, some studies had shown that the reduction in NK cells activity and
Langerhans cell was noted in smokers compared to non-smokers (81-83).
One study by Vaccarella et al (84) showed that the prevalence of HR-HPV
infection is higher among smokers and is dose-dependent. Another study
done in Finland followed 150 women with ASCUS or LSIL baseline cytology and normal colposcopy for up to 2 years with an examination interval of
6 months (these women were examined with cytology test, HPV testing).
The result showed the increasing risk for medium-term development of high
25
grade CIN in women who smoked with baseline ASCUS/LSIL cytology
compared to non-smokers.(85) The smoking effect persists over several
years after smoking cessation, and smokers or former smokers have a 3-fold
higher risk of developing CINII+ than non-smokers (HR=3.6;95%CI 1,58,6) (86).
Oral contraceptives
A well-known epidemiological risk factor in the development of cervical
cancer is the long-term use of combined hormonal contraceptives and there
is some evidence that they serve as a co-factor to human papillomavirus
(HPV) in cervical carcinogenesis (87). There is little evidence that the use of
oral contraceptives (OC) facilitates HPV infection as such, but rather modulates the HPV-induced progression of cervical neoplasia (6, 7) .In general,
large epidemiological studies suffer from inability to control the effect of
confounding factors. Even when the most important confounder, measures of
sexual risk behavior, is included, there is the possibility of residual confounding. Biological evidence on the effects on cervical epithelium in OC
users, compared to non-users, of any hormonal contraceptive in similar age
groups, is required (79, 87-89). Few studies, if any, have investigated the
effects of OC use on tumor marker expression in human cervical epithelium,
normal or neoplastic, and then only for single biological markers.
The effects of gestagenic contraceptives, if any, have been poorly studied.
The WHO Collaborative Study of Neoplasia and Steroid Contraceptives
found a relative risk of 2.4 with at least five years’ use of injectable depomedroxyprogesterone acetate, but results have been contradictory (90). Medicated intrauterine devices (MID), such as the levonorgestrel-releasing intrauterine system, are increasingly used. These differ from systemic use of progestin as the steroid is released close to the cervix. No studies on the expression of tumor markers in cervical tissue by systemic or intrauterine progestogen-only contraceptives have, to our knowledge, been reported. In
addition few, if any, studies have evaluated possible effects of serum levels
and sex steroid levels on normal cervical epithelium and CIN.
Genetic predilection
Variation in the rate of cervical cancer development between many racial
groups can be explained by genetic differences. According to a Swedish
study by Patrick K, et al. in 1999, there is a genetic predilection to cervical
cancer and its precursor forms, and the family risk for developing a cervical
tumor has the same degree as those for other cancers such as prostate cancer(91).
26
A recent study by Wang SS et al. in 2010 showed that there are different
genetic profiles that might be associated with persistent CINIII or the rapid
progression to cervical cancer (92).
Ageing
Ageing is considered as one of the risk factors in the development of cervical
cancer. The organised Swedish cervical screening program is divided into
two categories according to European and national guidelines. The first
group is aged between 23-50 years old and offered screening every 3 years;
the second group is aged between 51-60 years and offered screening every 5
years. The benefit is high but the limitation of the screening test must be
considered, especially for women in post-menopausal status.
The cause of this limitation is the low sensitivity of the test in detecting
CIN III in these women. The older the women the higher the risk for insufficient test due to the high location of CIN changes in the endo- cervical canal.
In order to improve the sensitivity of the screening, it has been suggested to
include HPV testing as a useful test with high sensitivity in post-menopausal
women - HPV infection is more common in young females and decreases
with age (95) .
Other risk factors have an indirect effect and can be explained by their
link to HPV infection - such as multiple sexual partners, frequency of intercourse, anal sex and early sexual debut (53,93, 94).These risk factors are
beyond the aim of this thesis.
Cervical and vaginal cancer risk in previously treated women
The changing in the treatment modalities since the 1990s may play a major
role in the increasing risk for the development of cervical or vaginal cancer
in women who were previously treated for cervical intraepithelial grade 3. It
was proven in a study by Strander B, et al that women previously treated for
cervical intraepithelial neoplasia grade 3 are at risk of developing invasive
cervical or vaginal cancer. According to this study both cancers are caused
by HR-HPV infection, and by the incomplete treatment of the intraepithelial
changes grade 3 in the cervix, regardless of whether hysterectomy was done
or not (96).
Tumor markers
Most tumor markers are proteins that are produced either by tumor cells or
by the host cells as a response to the presence of cancer cells. As such, they
indicate the presence of cancer and may also reflect the severity of these
cancerous changes. Tumor markers can be used to identify the risk of developing cancer in asymptomatic subjects. Tumor markers assist the
27
pathologists in making the correct diagnosis, typing and grading the tumors,
and in tracing the origin of metastasis of unknown primary tumor. In addition, some of them are used as prognostic markers while the others as predictive markers indicating the usefulness of target therapy.
A number of biomarkers (tumor markers) have been studied in CIN. The
objective has generally been to find tumor marker expression that could discriminate between normal cervical epithelium and CIN, aiding the histological diagnosis of CIN grade, or as a surrogate for HPV infection. Other studies have focused on determining the aggressiveness of CIN, but with inconclusive results. During recent years much attention and extensive research
has been directed towards p16, retinoblastoma protein (Rb), p53 and Ki67/MIB1 (97). The present study evaluated expression of eleven tumor
markers and their correlation, if any, to HPV infection, smoking, oral contraceptive use, and levels of serum progesterone and serum estradiol. As this
was practically virgin soil we analyzed some tumor markers based on our
previous results on invasive cervical cancer (98, 99) and tumor markers of
possible clinical importance that were previously studied in cervical neoplasia. Tumor markers were chosen to represent different mechanisms in
carcinogenesis. In our study we investigated the correlation between tumor
marker expression in cervical intraepithelial neoplasia with HR-HPV infection and suspected co-factors such as smoking, serum levels of sex steroid
hormones, and hormonal contraceptives. Possible response of tumor marker
expression after exposure to co-factors would provide biological support for
epidemiological associations. Tumor markers were divided into different
groups according to their mode of action, such as proliferation markers, tumor suppressors, apoptosis, cell-to-cell interactions, angiogenesis and oncoproteins.
Proliferation markers
EGFR (epidermal growth factor receptor)
A cell surface receptor for a member of the epidermal growth factor family Stanley Cohn received the Nobel Prize in medicine 1986 for his discovery of
EGFR.
The mechanism of activation of EGFR is by binding to EGF or TGF
(TGFα), resulting in auto-phosphorylation of Tyrosines inside the cell (the
ability to interact with specific cell-surface receptors and transduce intracellular signals). This activation normally results in DNA synthesis and cell
division.
Mutation of the EGFR gen leads to overexpression of EGFR which results in constant activation.
This constant activation produces uncontrolled cell proliferation. This is
why EGFR has been associated with poor prognosis in different kinds of
28
cancers and is considered to be a prognostic marker especially in cervical
cancers (100-103).
Ki-67/MIB-1 (non-histone protein) is a cellular marker for proliferation.
It is present during all active cell cycle phases (G1, S, and G2) but not G0
(the resting phase). It has also been shown that Ki-67 expression increased
with advanced stages of cervical neoplasia. (104) .
Ki -67 has been found to be more intensely stained in HPV-positive than
in HPV-negative epithelium (105, 106). Although Ki-67 is mostly expressed
in proliferative cells, a recent study by Bullwinkle J, et al showed that Ki-67
can be detected at sites linked to ribosomal RNA (r RNA) synthesis in the
non-proliferative stage of the cell cycle. This finding may call into question
the use of Ki-67 as a proliferative marker (107)
Apoptosis inhibitors
COX- 2
Its main action is to inhibit apoptosis by enhancing proliferation, inflammation response to tumor, angiogenesis and tumor invasion, the mode of action
depending on a variety of events. The mechanism of action is through the
cyclooxygenase pathway which regulates the production of prostaglandins
from arachidonic acid in inflammatory conditions. Cox2 acts as a mediator
in this process. A high expression might indicate poor prognosis in cervical
cancer.(108-110)
Tumor suppressors
P53: P53 is considered to be the guardian of genome in the human body. P53
activates as a response to cell injury causing G0/G1 cell cycle arrest allowing cell repair or apoptosis (programmed cell death)(111). P53 can be inactivated or deactivated by different kinds of cancer in which these changes led
to the disability of the p53 to bind to target DNA sequences and the beginning of the repairing process. Inactive p53 mutants with a subsequent loss of
tumor suppression are often found in cancer cells allowing for increased
proliferation (112).
HPV E6 oncogene binds itself and inactivates P53 as the first step of viral
DNA replication. Mutations of p53 that rarely occur in the cervical cancer
have been found in other types of cancer, leading to the loss of the tumor
suppressor function of p53 and the beginning of oncogenic activity(113).
Rb (Retinoblastoma susceptibility gene)
Rb is encoded as a nucleoprotein that plays a major role in the cell cycle at
the G1-S phase (114, 115). The Rb tumor suppressor protein (Rb) binds to
the E2F1 transcription factor, preventing it from interacting with the transcription machinery of the cell. In the absence of Rb, E2F1 facilitates the
29
trans-activation of E2F1 target genes that enable the G1/S transition and Sphase (116) . Rb is one of the targets of the oncogenic human papilloma
virus protein E7. By binding to Rb, they stop the regulation of E2F transcription factors and drive the cell cycle to enable virus genome replication.
FHIT
Fragile histidine triad (FHIT) acts mainly as tumor suppression and control
of apoptosis. The gene involves the common fragile site on chromosome p3,
where carcinogen-induced damage can lead to translocations and uncharacteristic transcripts of the gene.
These abnormal transcripts from the gene have been found in about half
of esophageal, stomach, cervical and colon carcinoma.(117) .
P16
HPV 16 has been found to integrate at FRA3B fragile site and is frequently
demonstrated in cervical carcinoma.(118, 119)
P16 (cyclin-dependent kinase inhibitor 2A, multiple tumor suppressor 1):
p16 (p16INK4a) is a tumor suppressor protein, that in humans is encoded by
the CDKN2A gene. The CDKN2A gene is frequently mutated or deleted in a
wide variety of tumors. p16 plays an important role in cell cycle regulation
by decelerating cell progression from G1 phase to S phase, and therefore acts
as a tumor suppressor implicated in the prevention of cancers such as melanoma, oropharyngeal squamous cell carcinoma, and esophageal cancer. P16
is an inhibitor of cyclin dependent kinases such as CDK4 and CDK6. These
latter kinases phosphorylate retinoblastoma protein (Rb) which eventually
results in progression from G1 phase to S phase.
Several studies have investigated the usefulness of p16 INK4a expression
in HR-HPV infections (112, 119, 120). In an early study, p16INK4a expression showed a high sensitivity for HR-HPV infection (83%), but a low specificity (57%).
Cell-cell Adhesion
E-cadherin
The calcium-dependent interactions among E-cadherin molecules are critical
for the formation and maintenance of adherent junctions in areas of epithelial
cell-cell contact. Loss of E-cadherin-mediated adhesion characterizes the
transition from benign lesions to invasive, metastatic cancer (121)
Immunological marker
Interleukin 10
L-10 or human cytokine synthesis inhibitory factor (CSIF) is a cytokine considered to be a potent immuno-suppressor that depresses local virus-specific
immunological responses.
30
Normally the human body needs both innate and adaptive immunity to
identify viral infections (122). The key mechanism in HPV infection is that
the HPV goes through different steps to avoid recognition by the innate and
adaptive immunity. The life cycle of human papillomavirus inside the cells
is in itself a good way to avoid recognition by the innate immunity APC
(Antigen presenting cell).
The adaptive immunity system depends on the T lymphocytes (CD4+,
CD8+) action in identifying the viral infection as a threat.
CD4+ T cells have two subsets, the th1 and th2 cytokines.
The main action for th1 (e.g. IFN-γ) is to start the cell-mediated immunity
reaction (CMI), while th2 cytokines (IL –interleukins) IL-4, IL-5, IL-6, IL10 and IL-13 activate the humoral immune responses. IL-10 is a potent immuno-suppressive cytokine and the overexpression of IL10 has been found
in a varied range of cancers.
IL10 inhibits the activity of APCs and decreases the production of th1
(thus to say that IL 10 has an inhibitory effect on T-cell proliferation and
inflammation) (123). In the present study IL-10 expression is neither correlated to HR-HPV status, nor to increasing histopathological grade.
Oncoprotein
C-myc oncoprotein induces the transcription. The HPV E6 gene
increases telomerase expression through induction of c-myc and
subsequent TERT gene activation (124).
•Inflammaon
•TNF,IL 1
•ki67+cMYC
•Acviaon of COX 2.
•EGFR acviates the
phosphorylaon of
tyrosin in the
nucleous
•Repair
Phosphryaon
of tyrosin DNA
P53+mdm
P53 Acviaon
P16
COX 2
CDK
Rb
E2F
•Cell proliferaon
•Apoptosis
inhibiaon.
•Cell repair + cell
arrest.
•Apostosis
acviaon.
Figure 5. Main function of biomarkers and their effect on the cell cycle.
31
Present investigation
Aims of this thesis
To study whether epidemiological findings of a correlation between smoking, sex steroids and contraceptive, and CIN are reflected in the expression
of tissue tumor markers relevant for cervical neoplasia, thus supporting a
biological role of these risk factors. The presence of HPV in tissue from the
study population in women with normal cervical epithelium will also be
studied for tissue tumor marker expression.
Correlations to tumor marker expression will be studied:
- Between a panel of biomarkers/tumor markers as above and HRHPV-positive versus HR-HPV-negative cervical lesions.
- Between smoking and serum cotinine levels respectively, and tumor marker expression in cervical intraepithelial neoplasia (CIN) and
normal epithelium.
- Between hormonal contraceptives - combined oral contraceptives
(COC), any progestin-only contraceptives, medicated IUD (MID) or
systemic progestin-only contraceptive use (Syst-P), compared to
non-users, and tumor marker expression in cervical intraepithelial
neoplasia (CIN).
- Between serum progesterone and serum estradiol levels and expression of tissue tumor markers in CIN and normal epithelium.
32
Material and methods
The study population comprised 228 women of whom 188 were recruited at
our colposcopy clinic for laser cervical conization or a directed punch biopsy, because of a vaginal smear (Pap smear) that showed CIN, cytological
findings suggesting CIN, or repeated findings showing atypical squamous
cells of undetermined significance (ASCUS). They were consecutively recruited from the out-patient surgery, Department of Obstetrics and Gynecology, Falun Hospital, Falun.
In addition 40 healthy volunteers in fertile ages and with normal Pap
smears were recruited to ensure that the study included a sufficient number
of women with normal epithelium. The mean and median ages of the entire
study population were 36.6 years and 34.0 years, respectively. These samples were collected between 2005 -2007.
A structured questionnaire included birthday, age, last menstruation, cycle
day, menopausal status, history of abnormal Pap smear, contraceptive use, if
any, present or past smoking, cigarettes per day and duration, and climacteric status.
Paentes in
the cohort
188
155in ferle
age
60 noncontracepve
users
33 post
menopausal
76 smokers
volunteers in
the cohort
40
40in the ferle
age
20 non contracepve
users
0
7 smokers
Figure 6. The study population.
33
The paraffin embedded punch biopsies or cones were stored at the Department of Pathology at Falun hospital.
The original histology slides were microscopically reviewed by a
pathologist of our team and the most representative area(s) was marked for
tissue microarray (TMA). Two-millimeter punch biopsies were taken from
the blocks corresponding to the marked area and joined into TMA paraffin
blocks, containing 24 to 30 punch biopsies. Each TMA block also included
one control and one empty square to avoid diagnostic mistakes.
Immunohistochemistry was performed. In brief, glass slides were deparaffinized in xylene (2x15 min), dehydrated through graded alcohols and
endogenous peroxidase was blocked (using H2O2 in 70% ethanol). Antigen
retrieval was performed using Target Retrieval Solution (TRS pH 6.0 or
pH9.0, Lab vision) in a de-cloaking chamber (Bio care Medical, Walnut
Creek, CA) for 4 minutes at 125 °C. Thereafter the slides were immunostained in the automated staining instrument, where primary antibodies and
secondary reagent were each incubated for 30 minutes at room temperature
(RT). Finally, the slides were incubated with diaminobenzidine (DAB) as
chromogen for 10 minutes and counter-stained with Mayer’s hematoxylin
(Sigma-Aldrich, St Louis, MO) for 15 min. Slides were washed in distilled
water for 10 min, dehydrated through graded alcohols to xylene, and mounted in Pertex organic mounting medium (Histolab, Gothenburg, Sweden).
Figure 7. The making of punch biopsies for TMA examination
Two-millimeter punch biopsies were taken from the blocks corresponding to
the marked area and joined into TMA paraffin blocks, containing 24 to 30
punch biopsies.
Each TMA block also include one control and one empty square to avoid
diagnostic mistakes.
34
Figure 8. The making of the slide for TMA examination.
The making of the slides using water fall microtome. The typical section is
4um. Sections were de-paraffinized in xylene (2x15 min), dehydrated
through graded alcohols and endogenous peroxidase was blocked (using
H2O2 in 70% ethanol).
Antigen retrieval was performed using Target Retrieval Solution (TRS pH
6.0 or pH9.0, Labvision) in a de-cloaking chamber (Biocare Medical, Walnut Creek, CA) for 4 minutes.
The slides were immuno-stained in the automated staining instrument,
where primary antibodies and secondary reagent were each incubated for 30
minutes at room temperature (RT).
The slides were incubated with diaminobenzidine (DAB) as chromogen
for 10 minutes and counter-stained with Mayers hematoxylin (SigmaAldrich, St Louis, MO) for 15 min. Slides were washed in distilled water for
10 min, dehydrated through graded alcohols to xylene, and mounted in Pertex organic mounting medium (Histolab, Gothenburg, Sweden).
25 °C.
DAB staining
Counter staining
Figure 9. The final stage the staining with DAB.
35
Occasionally, it was not possible to evaluate expression of a specific tumor
marker in a specific subject. As it was not systematic, the whole study population was included and the number of evaluations thus differed with a few
diagnoses between the tumor markers.
One external senior pathologist, who was blinded for clinical details,
evaluated all biopsies. Frequency of stained cells and intensity of staining
were diagnosed. A four-grade semi-quantitative score was used for 0 in absence of biomarker expression, 1 if 1-19 % of cancer cells, 2 if 20-49% and
3 if 50% or more cells expressed of the tumor marker. Intensity of staining
was graded in four steps: absent, mild, moderate and strong. In the analyses,
there was in general a good correlation between proportion and intensity and
the best discriminatory evaluation was used for presentation in all tables.
Biological marker
Fragile histidine triad
(FHIT)
p53
Functions
Clone
Species
Tumor
RB-9232
Rabbit
suppressor
Tumor suppresM7001
Mouse
sor, apoptosis
p16
Tumor
NCL-p16-432 Mouse
suppressor
Retinoblastoma
Tumor
554136
Mouse
protein (Rb)
suppressor
CD4+
Immunologi- NCL-CD4-1F6 Mouse
cal marker
Interleukin 10 (IL-10)
Immunologi- RHCIL1000 Rat
cal marker
Epidermal growth factor Proliferation
28-0005
Mouse
Ki-67 (MIB-1)
Proliferation
M7240
Mouse
Cytokeratin10
Cytoskeleton
MS-611
Mouse
E-Cadherin
Cell-cell
13-1700
Mouse
adhesion
Cyclooxygenase-2
Inflammation
18-7379
Mouse
(COX-2)
Antigen
retrieval
HIER pH6
HIER pH6
HIER pH9
HIER pH9
HIER pH9
HIER pH6
Proteinase K
HIER pH9
HIER pH9
HIER pH9
HIER pH9
Figure10. Tumor markers included in the study and their major functions.
Paper 1
High-risk HPV infection and CIN grade correlates to expression of c-myc,
CD4+, FHIT, E-cadherin, Ki-67 and p16INK4a
PCR (polymerase chain reaction) for HPV detection from eight 5-µm sections from the original cervical cone was performed. In brief, standard
36
curves ranging from 102 to 105 copies were established for each HPV type
using plasmids containing the full genome of different HPV types. A highly
significant linear relationship was seen between HPV copy number and
threshold cycle for all HPV types. The threshold for a positive HPV type
was set to 10 copies per PCR. Sufficient and representative material from
cone biopsies, in contrast to punch biopsies, was available from 116 women
for analyses of 12 HR-HPV types, i.e. HPV 16, 18, 31, 33, 35, 39, 45, 51,
52, 56, 58, and 59.
Ninety-four (82.5%) of the women were premenopausal while 20 women
(17.5%) women were postmenopausal. The menopausal status of two women was uncertain. HPV-positivity was more common in the former group
(59.6% vs. 35.0%). When postmenopausal women were excluded there were
no significant differences in oral contraceptive (total 34.8%) and gestagenic
contraceptive (total 24.2%) use between HPV-positive and HPV-negative
women. In the entire study population 36.0% were smokers with no difference by HPV status.
The distribution of HR-HPV positivity in different stages of CIN and in
normal epithelium shows a sharp increase from LCIN (mean 39.2% HPV
pos) to HCIN (mean 89.2% HPV pos)
Paper 2
Tissue tumor marker expression in smokers, including serum
Cotinine concentrations, in women with cervical intraepithelial
Neoplasia or normal squamous cervical epithelium
Cotinine was analyzed at the Advanced Bioanalytical Services Laboratories,
London.
Smokers averaged 34.9 years and non-smokers 37.7 years (p=0.06). Eightythree (36.9%) of the women were smokers, defined as daily smoking and
serum cotinine levels above 12 ng/mL, and 142 (63.1%) women were nonsmokers. There were no findings of measurable serum cotinine in the latter
group.
The histopathological diagnoses were distributed on 87 (38.2%) normal
squamous epithelium, 31 (13.6%) ASCUS, 49 (21.5%) CIN I, and 61
(26.8%) CIN II-III. In women of fertile ages (n=195), 74 had normal epithelium, 23 ASCUS and 94 were diagnosed as CIN
37
Paper 3
Oral contraceptive and progestin-only use correlates to tissue tumor marker
expression in women with cervical intraepithelial neoplasia
The study population (n=195) distribution was 57 OC users, 15 users of
MID, 24 users of systemic progestin-only contraceptives, and 99 non-users
(Fig 15). Mean age of OC users, and any progestin-only contraceptive users
(Syst-P or MID) was 28.4 years and 35.5 years, respectively, compared to
35.2 years among non-users (p=0.01 and p=0.89). Distribution of histopathological diagnoses among oral contraceptive users, any progestogen-only use
and the comparison groups.
CIN including borderline cases was diagnosed in 121 (62.1%) women, while
in 74 (37.9%) women the cervical epithelium was considered normal. There
were small and non-significant differences regarding histopathological diagnoses between OC users and any progestogen-only use, compared to nonusers
Paper 4:
Increased levels of serum progesterone and estradiol correlate to increased
Cox-2 tissue expression in CIN – and some pitfalls in evaluation of expression of other tumor markers.
Serum hormone levels were analyzed by standard methods at the biochemical laboratories of Falun Hospital (progesterone) and Uppsala (estradiol).
80 women - 60 patients and 20 volunteers.
Mean serum progesterone was 20.1 nmol/L, and the corresponding serum
estradiol 449.2 pmol/L.
Serum hormone levels were analyzed by standard methods at the biochemical laboratories of Falun Hospital (progesterone) and Uppsala (estradiol).
Statistical analysis:
For the analyses the most explanatory cut-off level was used when the results were dichotomized.
When there was no evidence of any correlation to serum progesterone
and/or estradiol, HPV status, smoking habits, contraceptive use data were
dichotomized so that an equal number of patients, if possible, were included
in the two groups.
38
For adjustments according to CIN grade, the material was divided into
high-grade (CIN II and CIN III) and low-grade (borderline and CIN I)
squamous intraepithelial lesions
(HSIL and LSIL, respectively) and logistic regression was used. For statistical analyses, the JMP statistical package (SAS Institute) was used. Student’s t-test was used for significance Testing. The study was approved by
the Research Ethical Committee, Uppsala University.
39
Results
Paper 1
High-risk HPV infection and CIN grade correlates to expression of cmyc, CD4+, FHIT, E-cadherin, Ki-67 and p16INK4a.
The aim of study I was to look for a correlation between a panel of biomarkers and high risk human papilloma virus (HPV) - positive versus HRHPV negative in cervical lesions.
From the original cohort of 116 tissue samples which were tested for
HPV, 64/116(55.2%) were positive for one more of the 12 HR-HPV subtypes. Most common were HPV-16 (N=21), HPV-18(N=14) AND hpv33/52/58(n=15).None was positive for HPV-39, whereas occasional biopsies
were positive for HPV-31,-35,-56 AND -59.
The prevalence of premenopausal status in the cohort was 94/114 (82.5%)
while the prevalence of postmenopausal status was 20 /114 (17.5%). The
menopausal status of two women was uncertain. HPV-positivity was more
common in the former group (59.6% vs. 35.0%). When postmenopausal
women were excluded there were no significant differences in oral contraceptive (total 34.8%) and gestagenic contraceptive (total 24.2%) use between
HPV-positive and HPV-negative women. In the entire study population
36.0% were smokers with no difference by HPV status.
When the distribution of HR-HPV in different stages of CIN and in normal epithelium was analyzed , there was a sharp increase of HR-HPV positivity from LCIN ( ASCUS+ CIN I) to HCIN (CIN11-CIN111) by the difference in the mean from 39,2% to 89,2% HPV-positive.
40
Table I. Expression of biological markers in HR-HPV- positive and –negative cervical epithelium of women with low grade or high grade CIN (frequency of stained
cells.0=no cells, 1=1%-19% cells, 2=20%-49% cells, 3=50% to 100% cells. Staining
of c-myc could only be evaluated in 56 cases. P*adjusted for squamous intraepithelial lesion status.
Frequency
of stained
cells
HPVpositive
HPVnegative
OR
95%
CI
P
P*
c-myc
11(33.3)
1(4,6)
10,50
1,80200,4
,006
,13
8(14,8)
1(2,7)
62,60
1,08118,8
0,04
,19
36(67,9)
5(14,3)
12,71
4,4942,64
0,0001
,004
33(61,1)
7(18,9)
6,73
2,6219,26
0,0001
0,34
1-3 vs 0
Cd4+
3 vs 0-2
Ki-67
2-3 vs 0-1
P16
3 vs 0-2
The analysis for biomarkers showed, there were a high expression of C-myc,
CD4+, Ki-67 and p16 which correlated with HR-HPV positivity but after
adjustment was made for CIN grade only Ki-67 remained significant.
When the subcategorizing was done according to the histopathological assessed diagnosis and because of the small proportion of HCIN in HPVnegative tissue (n=4), only normal epithelium and LCIN were analyzed in
this group. In HR-HPV negative tissue, a high expression of E-cadherin
(P=0, 03) and Epidermal growth factor receptor (P=0, 03) correlated significantly to LCIN compared to normal epithelium. When the HPV-positive
group was adjusted for normal/LCIN/HCIN, in the univariate analyses of
HR-HPV positive tissue, showed that Ki-67(p=0.001), p16 (0.001), FHIT
(p=0.002), E-cadherin (p=0.0004) and Rb (p=0.04) expression increased
significantly with severity of the lesion.
P16 expression did not correlate to HPV status (p=0, 34) but correlated
significantly to histological grade (p=0,001). Ki-67 expression remained
significantly correlated to HPV status also after adjustment (p=, 01) but also
to histopathological diagnosis (P=0, 02).
41
Ki-67 was the only marker that independently predicted both the
presence of HR-HPV and the severity of cervical lesions.
Paper 2
Tissue tumor marker expression in smokers, including serum Cotinine
concentrations, in women with cervical intraepithelial Neoplasia or
normal squamous cervical epithelium.
The aim of study II was to investigate the correlation between biomarkers
expression and smoking in cervical intraepithelial lesions. In this cohort the
Smokers averaged 34.9 years and non-smokers 37.7 years (p=0.06).
Smoking was defined as daily smoking and serum cotinine level above 12
ng/ ml. Eighty-three (36.9%) of the women were smokers, and 142 (63.1%)
women were non-smokers. There were no findings of measurable serum
cotinine in the latter group.
The histopathological diagnoses were distributed on 87 (38.2%) normal
squamous epithelium, 31 (13.6%) ASCUS, 49 (21.5%) CIN I, and 61
(26.8%) CIN II-III. None of the 40 volunteers’ hade CIN.
Smoking in the whole cohort:
The result in general showed that smoking associated with lower P53 in
CIN, but not in those women with normal epithelium, compared to nonsmokers. Lower Interleukin -10 expression was also noted in smokers with
CIN than in non-smokers
42
Table II. Smoking habits and correlation to tumor marker expression in cervical
intraepithelial neoplasia and normal epithelium. (Frequency of stained cells was
semi quantitatively 0=0%, 1=1-19% cells, 2=20-49 cells, 3=50-100% cells).
variable
Smokers
Nonsmokers
0R
95%
CI
Normal
Normal
epithelium epithelium
P53
2-3 vs 0-1
N (%)
N (%)
3(10)
5(11.6)
0,84
(0,163,74)
11(25,6)
1,01
(0,342,90)
Interleukin 8(25)
-10
1-3 vs 0-2
P
Smoker Nonvalue CIN
smokers
CIN
OR
P
95%CI value
N (%)
N (%)
,83
7(17)
28(35) 0,38
0,140,93)
,98
15(34,9) 47(58) 0,39
(0,180,82)
0,03
0,01
There was no significant correlation for p53 between smokers and nonsmokers in normal epithelium in contrast to those with CIN where a significantly lower p53 expression was found (p=0.03). There was also a lower
interleukin 10 expression in smokers with CIN (p=0.01), than in nonsmokers.
There was a negative but insignificant dose-response relationship between
EGFR expression and serum cotinine levels in normal epithelium (p=0.09),
while there was a positive correlation between EGFR expression and serum
cotinine levels in CIN (p=0.049) in women who were non-smokers.
Smoking in fertile ages
All cervical tissue of women in fertile ages was analyzed separately. No
significant correlations between tumor marker expression and serum cotinine
levels was found.
The tumor suppressors P53 (p=0, 06) showed a significant under expression in smokers compared to nonsmokers in normal epithelium, the same is
true regarding Fragile histidine triad (p=0.05) and immunologic marker interleukin-10 (p=0.11) they were under-expressed in smokers compared to
non-smokers. The tumor markers COX 2 (p=0.04) and Ki 67 (p=0.005) were
over-expressed in smokers compared till non-smokers in women in fertile
ages. Women with CIN had similar patterns, but the statistical significance
decreased.
43
Paper 3
Oral contraceptive and progestin-only use correlates to tissue tumor
marker expression in women with cervical intraepithelial neoplasia.
The purpose of this study was to look for the correlation between contraceptive use as combined oral contraceptive (COC), any progestin-only contraceptive, medicated intrauterine device (MID)or systemic progestin –
only(Syst-P) and tumor marker expression in cervical intraepithelial compared to non-user.
The study population (n=195) distribution was 57 OC users, 15 users of
MID, 24 users of systemic progestin-only contraceptives, and 99 non-users.
Mean age of OC users, and any progestin-only contraceptive users (Syst-P or
MID) was 28.4 years and 35.5 years respectively, compared to 35.2 years for
non-users (p=0.01 and p=0.89).
CIN including borderline cases was diagnosed in 121 (62.1%) women,
while in 74 (37.9%) women the cervical epithelium was considered normal.
There were small and non-significant differences regarding histopathological
diagnoses between OC users and any progestogen-only use, compared to
non-users.
COC users:
The results showed that a significantly increased expression of cyclooxygenase-2 (Cox-2) in cervical tissue in COC users with normal epithelium (OR
7.8, 95% CI 2.4-28.0) and in those with CIN (OR 5.7, 95% CI 1.8-20.1),
compared to non-users of hormonal contraception.
A lower expression of IL 10 was found in OC users with CIN than in
non-users among (OR 0.32, 95% CI, 0.10-0.90).
Progestin –only users
A lower expression of IL10 was found in any progestogen –only contraceptive users with CIN than non-users (OR 0, 37, 95% 0, 12-1, 03). The same is
true regarding CK 10(0, 17, 95% CI0, 03-0, and 71).
Comparison between MID users and COC users
When a comparison was made between MID users and COC users, COX2
and Rb (Retinoblastoma protein) expression were increased in COC users,
while P53 expression was increased in MID users.
44
Table III. OC vs MID use and correlation to tumor marker expression in the total
study population (frequency of stained cells was semi quantitatively 0=0%, 1=119% cells, 2=20-49 cells, 3=50-100% cells).
MID users
N (%)
OC users
N (%)
OR
95% CI
Cox2
2-3 vs 0-2
5(35,7)
40(74,1)
0,19
0,05-0,66
P53
3 vs 0-2
4(28,6)
3(6,5)
5,73
1,10-33,2
Rb
2-3 vs 0-1
2(14,3)
20(40,8)
0,24
0,04-1,01
There was no tendency for correlation between P53 or Rb expressions when
COC and SYST-P users where compared, but only a similar correlation to
COX2-expression.
Paper 4
Increased levels of serum progesterone and estradiol correlate to
increased Cox-2 tissue expression in CIN.
The aim of this study was to investigate the correlation between serum progesterone and serum estradiol levels and expression of tissue tumor markers
in cervical intraepithelial neoplasia (CIN) and normal epithelium.
The mean and medium age was 35. 7 years and 34.5 years, respectively,
with a range of 22-52 years. Twenty –four (30%) women were smokers in
the whole cohort. The histopathological diagnoses were distributed as 25(31,
3%) cases of normal epithelium, 10 (12, 5%) borderline, 18(22, 5%) CIN I
and 27(33, 8%) CIN II-III.
The mean and medium menstrual cycle day at serum sampling was day
18, 3 and 19, 0, respectively.
The mean serum progesterone was 20, 1 nmol/l, and the corresponding
serum estradiol was 449, 2 pmol/l.
Progesterone level and tumor markers expression:
-In normal epithelium
EGFR (15, 4 vs 36,5nmol/l; p=0,002) and CD4+ expression (22.2 vs 39, 8;
p=0,048) correlated to low serum progesterone levels.
-In CIN
Positive or negative correlations between serum progesterone levels and
intensity of expression were evident in CIN for Rb, p16, and Cox-2 in crude
45
analysis. After adjustment for CIN grade, only increased Cox-2 expression
remained significantly correlated to serum progesterone levels.
Estrogen level and tumor markers expression:
When estradiol levels were analyzed there were no differences in tumor
marker expression by serum levels in the normal epithelium, while in the
CIN group high serum estradiol levels correlated weakly, but significantly,
to increased Cox-2 expression.
46
Discussion
This thesis shows novel results on the correlations between tumor marker
expression and HR-HPV. It is particularly designed to evaluate tumor marker expression in smokers, hormonal contraceptive users and with serum sex
steroid hormone levels in cervical intraepithelial neoplasia and in normal
epithelium. HPV infection is regarded as a necessary but not sufficient factor
in transforming CIN to invasive cancer, and the presence of co-factors might
also be one reason when there is a rapid progress of early lesions to HSIL
and cervical cancer (4). Several previous studies focused on the correlation
between expression of Ki-67, p16 and HPV infection in cervical intraepithelial neoplasia, while others focused on direct exposure to smoking and its
unfavorable effect on tissues like lung or oral cavity. It is important to note
that smoking, contraceptive use and sex steroids have been subjects of interest in many studies regarding their effect on tissue tumor markers in different types of cancers. In order to simplify our findings on a possible association between HPV infection, smoking, contraceptive and sex steroids, respectively, and tumor marker expression we will discuss findings in some
tumor markers separately and thus include all studies. The possible correlations have rarely, if ever, been studied.
Tumor markers
Cox 2
Cox 2 has been used as a tumor marker in several cancer types and is involved in different inflammatory and mitogenic processes. The finding of
high expression of Cox 2 might indicate poor prognosis in cervical cancer.
The link between the initiation of inflammation process and cancer development has been recently explained by the discovery of the infiltration of
tumor micro-environment with different types of immune cells such as macrophages, lymphocytes and dendritic cells. These cells are responsible for
the release of cytokines (125) and the role of cytokines in the development
of cancer in different organs is well-known. The chronic use of NSIDs (nonsteroidal anti-inflammatory drugs) which are COX- 2 inhibitors has been
found to decrease the incidence of cancer such as colorectal cancer (126)
and lung cancer (127). In this study, high COX 2 expression was close to
47
significant (p=0.08) in HPV-positive tissue compared to HPV-negative tissue in high-grade or low-grade CIN. This finding may indicate the early
oncogenic effect of HR-HPV infection on cervical tissue. Smoking correlated significantly with high expression of Cox 2 (p=0. 04) in the subgroup of
fertile women in the entire study. Similar results have been found for the
effect of exposure to smoke extract in other tissues such as the lung, where
the exposure led to increased Cox-2 expression in fibroblast (128).
The finding of a high COX 2 expression (OR 7.8 and 5.7) in OC users is
interesting and can be explained as a response to an inflammatory process or
as susceptibility of cervical cells to inflammation. In many studies it has
been found that the OC users are at more risk of developing cervical atopies
than other contraceptive users (129).
Cox 2 expression also showed a strong staining intensity in HSIL with elevated serum progesterone levels. Progesterone has immuno-suppressive
effects and there might be an increased susceptibility for HPV infection
(130). Our findings indicate an adverse effect of progesterone on CIN that
could confirm findings in invasive cancer. A study by Lindström et al. (131)
showed increased cell proliferation measured as the number of cells in the S
phase, with high serum progesterone levels in invasive cervical cancer.
Many researchers have reported similar findings with high estradiol levels in
HSIL. In all, a possible clinical role for Cox-2 expression could be to indicate an association to poor prognosis in cervical cancer.
Ki 67
Ki-67 is a non-histone protein which is specific for cells with active DNA
replication. Increased proliferation has been found during pregnancy, in inflammatory processes and cancer. Ki- 67 is expressed during all phases in
the cell cycle except in resting stage (G0). It is unclear whether Ki-67 expression carries prognostic information in invasive cervical cancer (132). In
the first paper we were able to demonstrate that Ki-67 correlated to HPV
status and to the histopathological diagnosis. These results can be explained
by the effect of HPV oncogene on the cell cycle, as it promotes dysregulation of the infected cells leading to proliferation and growth acceleration.
This will lead to over-expression of Ki-67 as a proliferative antigen which is
specific for active DNA replication. There is some uncertainty whether Ki67 has the ability to distinguish between normal proliferating cells and infected cells (dysplastic cells). (32) (133). The finding of overexpression of
Ki-67 in women smokers in fertile age indicates that there is a tendency to
increased cell proliferation in smokers in general. This relation between
smoking and increased cell proliferations has been noticed even in other
studies done on the effect of smoking in other organs such as lungs or oral
cavity. (134). Ki67 was close to significant in elevated serum progesterone
in normal epithelium (P=0, 06). This, however, might be explained by the
increased proliferation of cells in the luteal phase of menstruation.
48
EGFR (epidermal growth factor receptor)
EGFR has the ability to interact with specific cell-surface receptors and
transduce intracellular signals to stimulate DNA synthesis and cell division.
EGFR is considered as one of the prognostic markers in different types of
cancer (135, 136). The finding of high EGFR expression in CIN and low
expression in normal epithelium in smokers compared to non-smokers might
be explained by the increased cell proliferation in smokers in general. The
high expression of EGFR in LCIN compared to normal epithelium in HPVnegative tissue indicates the early dysregulation of EGFR in the cervical
squamous metaplasia and epithelial hyperplasia. Intense EGFR staining correlated with low progesterone level in normal epithelium but not in CIN. In a
previous study by Lindström .A.K et al (137) the finding of a strong correlation between low EGFR staining and high serum estradiol was interesting, as
was the question whether the effect of these sex steroids on the cervix epithelium reverse their effect on the endometrium. It is well-known that estradiol is associated with cell proliferation and thatProgesterone has a suppressor effect on the endometrial growth, but its effect on the cervical epithelium
has been poorly studied.
p53 (cellular tumor antigen p53, phospho-protein p53)
P53 is a tumor suppressor which is activated as a response to DNA damage
leading to cell arrest at the G0/G1 phase. This arrest leads to DNA repair and
the prevention of mutations. HPV E6 oncogenes bind to p53, leading to the
degradation of P53 and resulting in the loss of the ability of apoptosis and
tumor suppression function. In cervical cancer the wild type 53 (active p53)
dominates but is degraded by HPV DNA, explaining why the expression of
mutant p53 is low in cervical cancer. The finding of a correlation between
p53 and HPV positivity was not conclusive in our study and it disappeared
when adjusted to CIN grade. Our other results regarding smoking showed
lower expression of P53 in smokers compared to non-smokers in CIN analysis. This finding of lower expression of p53 in smokers is consistent with the
overall unfavorable molecular expression pattern of smoking in this study.
The suppressor effect of smoking on p53 also started early in the precancerous process according to our results.
P53 and progesterone
The malignant proliferation of squamous cervical epithelium induced by
HPV infection may be associated with increased progesterone receptor expression (138). Previous studies suggest that p53 function is inactivated in
cervical carcinoma either by Complex Formation with HPV-E6 viral oncoprotein product in HPV-positive cervical carcinoma or, rarely, by gene mutation in HPV-negative cervical carcinoma.(139, 140)
49
A most interesting finding in this study was the increased expression of
p53 in MIUD users compared to non-users. This correlation has never been
studied previously. The results were surprising as it would indicate a protecting effect of MIUD use. HPV has a tendency to transfect cells with progesterone receptors. Progesterone has previously been suggested as the major
candidate hormone in cervical neoplasia due to its immunosuppressive effect
and to a possible connection with HPV infection. Both HPV 16 and HPV 18
contain progesterone and glucocorticoid response elements that increase the
expression of HPV E6 and E7 oncogenes, considered crucial in cell transformation, with gestagenic stimuli (141) .
A study by Orbo et al 2009 (142) showed that there is up-regulation of
P53 pathway in patients treated with Mifepristone. This may be of interest as
potential new therapeutic targets in endometrial hyperplasia and endometrial
cancer, as candidate markers for tumor progression. Local progestin-only
contraceptives are known to have a potent effect on the endometrium, and a
reduced thickness of the endometrium from 6.1 to 4 mm after 4 months of
use has been reported (143). The endometrial concentration of LNG with an
LNG-MID, one common type of progestin used in MIDs, is 200 to 800 times
higher than that of SystP (144, 145). Little is known about the effects on
cervical epithelium, but it is evident by the scanty and viscous cervical mucus that there are such effects (145). There are only two small studies on
cervical cytology. In one study, 162 users of LNG-MID were followed up
for 5 years and were compared with users of subdermal progestin-releasing
implants. Although CIN was more than twice as common in the former
group, it was not a significant difference (146). Another small study did not
find significantly increased rates of abnormal smears compared to expected
rates, but there was no control group (147) . Substantially larger studies are
needed.
p 16 cyclin-dependent kinase inhibitor 2A, multiple tumor suppressor 1
P16 is a gene product which acts as a tumor suppressor protein, its main
action being to inhibit the inactivation of Rb by cyclin dependent kinase.
Many studies showed that P16 immuno-reactivity is lost or decreased as a
part of the immunological reaction towards the external threat (carcinogenic
effect) (148-150) . In cervical dysplasia another mechanism is established
where P16 expression is increased as a response to the effect of HPV infection. HPV oncogenes E6, E7 alter the cell cycle mechanism by binding to the
2 main proteins which regulate the cell cycle. E7 binds itself to Rb protein,
leading to degradation of E2F transcription factor (activation of E2F leads to
cell progression). E7 causes the Rb dysfunction (151-153). The cells will
react by increasing the expression of P16 in the cell and, since E2F is released by E7 and not by the effect of CDK4/6, p16 loses its effect on cell
cycle progression (p16 has no more effect on the cell progression). P16
would accumulate in the nucleus and cytoplasm of the affected cell and can
50
be detected by immuno-staining. P16 has been used as tumor marker in cervical carcinoma. Our results of a concomitant increase of p16INK4a and Rb
expression in HR-HPV positive specimens may indicate that p16INK4 in
these early neoplasms still influences Rb expression. This is instead of the
feedback loop with increasing p16INK4 expression as a result of decreased
Rb expression caused by HPV induced inactivation, while increased Rb expression leads to decreased p16 activity. HPV-E7 onco-protein inactivates
Rb protein leading to over-expression of p16. The results of p16 expression
were surprising. The poor correlation between p16INK4a over-expression
and positive HPV status in LCIN, and the lack of over-expression in nearly
20% of HCIN, might reflect non-integration of HPV DNA in the host genome. As we also found almost 10% over-expression in HR-HPV-negative
LCIN, p16INK4a our results do not support the idea that p16INK4a is a useful surrogate for HR-HPV infection.
In this study there was an excellent correlation to severity of CIN in those
who were HR-HPV positive, but not in HR-HPV negative lesions. No expression was found in HR-HPV positive normal epithelium, while a high
level of expression was evident in HCIN. A substantial part (9%) of LCIN
also had p16INK4a expression. Thus, the role in differential diagnosis was
limited. Our findings of the use of p16INK4 expression as a histopathological adjunct are similar as those in a meta-analysis (119).
In the present study the significant correlation to HR-HPV infection disappeared in the multivariate analyses. In analyses of tumor marker expression HR-HPV positive and negative tissue have rarely been separated (154),
but it would be easier to interpret the results if specific tumor marker expression is significantly correlated to HR-HPV infection
C-myc
c –myc is an onco-protein that is predominantly located in the cell nucleus
and involved in carcinogenesis through the activation of transcription of m
RNA (effect proliferation, prevent differentiation, sensitize the cells for
apoptosis). It has been used as prognostic marker in HPV positive
cancer(155). C-myc overexpression has been detected in many cases of colorectal, hepatocellular, endometrium, mammary and prostate cancer .ref
C-myc activation occurs in advanced cases of cervical carcinoma (indicating the beginning of cancer progression, relapse and metastasis) (156). In a
study published by C.D. Golijow et al (157) in 2001, there is cmyc amplification found in cellular atopies, low grade intraepithelial and high grad intraepithelial lesions (157). In this study, C-myc was highly expressed in
HPV positive in the univariate analyses, which can be explained by the previous finding in a study by Bernard et al. (158) where a correlation between
c-myc and HPV status was observed .
51
IL10
Interleukin-10 (IL-10), or human cytokine synthesis inhibitory factor (CSIF).
IL-10 is a cytokine and is considered to be a potent immuno-suppressor
that depresses local virus-specific immunological responses. In the present
study IL-10 expression correlated neither to HR-HPV status, nor to increasing histopathological grade.
IL-10 pattern in women with HR-HPV-associated CIN has been investigated in blood (159), cervical secretions (160) and in abnormal vaginal
smears (161). These previous studies indicate a correlation to the presence of
HR-HPV infections but independency of CIN grade has not been investigated. It has been reported that IL-10 expression was independently positively
correlated to HCIN compared to less severe lesions, but was not useful as a
surrogate marker for HPV (161).
Decreased IL-10 expression was observed both in OC-users and any progesterone-only users, possibly reflecting an effect on the immunological
defense mechanism. Two studies evaluated women with normal cervical
epithelium. Increased levels of IL-10 in cervical cytology of OC users were
found in both studies OC (124, 162). It is therefore of particular interest that
we observed an increase of IL-10 expression in users of progestogen-only
contraceptives in women with normal epithelium, in contrast to those with
CIN. This could indicate a switch in immuno-suppression when CIN is established. From the present results it can be concluded that exogenous steroid sex hormones influence IL10 expression, but whether the immunological
balance is influenced positively or negatively remains unknown.
Co-factors
Smoking
The present study included evaluation of smoking habits and serum cotinine
level in women with cervical intraepithelial lesion or normal cervical epithelium. Smoking seems to be associated with a negative molecular pattern.
Lower expression of p53 and FHIT and higher expression of COX2 and Ki
67 have been found in smokers compared to non-smokers and this is true for
women at a fertile age. Significant difference between smokers and nonsmokers was found also in IL 10 expression in CIN only.
Low expression correlated to cotinine levels, which suggests that cotinine
(and thus nicotine) are generally bystanders and that other substances affect
cervical cells. It must be stressed, however, that accumulation of cotinine has
been found in cervical mucus and that we had no possibility to perform such
analyses. The findings of lower EGFR expression in normal epithelium and
higher expression in CIN in smokers, compared to non-smokers, are interesting and deserve further studies. Our results showed positive correlations
52
between smoking and tumor marker expression, and we could determine
objectively that smoking status was true through serum cotinine.
In a previous study on invasive cervical cancer, we found significantly
lower p53 expression in smokers compared with non-smokers, as in the present study (137). A novel tumor marker, LRIG1, which is probably a tumor
suppressor in cervical cancer, was found to exhibit lower expression in
smokers than in non-smokers (163). The present study showed that the expression of Ki-67 is increased in smokers with CIN, which was confirmed in
the previous study.
The p53 activation after several signals of cellular stress or DNA damage
needs to be compromised during tumorigenesis. In cervical cancer, the human papillomavirus oncogene E6 is able to promote wild type p53 degradation, and in contrast to other cancer types, the expression of mutant p53 is
low in cervical cancer (69). Our results indicate that suppression of p53 occurs very early in the presence of smoking.
Ki-67 (or MIB1) is, like EGFR, a well-known and thoroughly studied
proliferation factor in cancer. It is unclear whether Ki-67 expression carries
prognostic information in invasive cervical cancer (164). Our results showed
a significant correlation between Ki-67 expression and smoking in fertile
women which indicates an increased cell proliferation in smokers. Smoking
correlated to increased Cox-2 expression in the subgroup of fertile women in
this study. In tissues directly exposed to smoke, like the lung and the oral
cavity, smoke extract on human lung fibroblasts has induced Cox-2 (187).
Smokers have increased Cox-2 messenger RNA levels in oral mucosa, compared with non-smokers (165) .
Interleukin-10 showed a significant difference in expression between
smokers and non-smokers in women with CIN. Interleukin-10 is an immunosuppressor that is involved in immune response escape in cancer (166). Interleukin-10 concentrations in cervical secretions of smokers, compared with
non-smokers, have been measured, but the differences were non-significant
(99).
Hormonal Contraceptives
This study provides evidence that hormonal contraceptives influence expression of molecular markers that are used in investigations of cervical cancer.
We cannot speculate whether these changes indicate an overall favorable or
unfavorable pattern. Carcinogenesis is complex, and the present study only
evaluates expression of 11 (although carefully selected) tumor markers. One
of the main indications that there is a switch in the immuno-suppression
mechanism when CIN is established is the finding of increased IL10 expression in any progesterone-only users in women with normal epithelium in
contrast to those with CIN. The finding of increased IL10 expression in cervical HPV infections is well known (160, 167) . From the present results, it
53
can be concluded that exogenous steroid sex hormones influence IL10 expression, but whether the immunological balance is influenced positively or
negatively remains unknown. Cytokeratin 10 is a major marker of keratinocytes and is involved in cell differentiation. Promotion of keratinocytes and
enhancement of differentiation in cervical cell lines have been associated
with growth suppression (168). Cytokeratin 10 expression was also reported
to be increased in thick cervical epithelium associated with cervical cytology
showing mild dysplasia or ASCUS. Other studies have found decreased expression with progressive CIN and invasive cancer (169, 170), but the clinical importance of CK10 expression in cervical carcinogenesis is poorly studied(171). As CK10 expression is associated with differentiation, our findings
of decreased expression in progestin-only users in CIN compared to nonusers might be unfavorable. When users of MID and Syst-P were analyzed
separately, no significant correlations to expression of tumor markers were
observed for systemic users. In the present study, there was increased p53
expression in the progestin-IUD users compared to non-users. The role of
p53 in response to DNA damage and the correlation to MID use are intriguing, but could indicate favorable effects.
Progesterone and Estrogen
Progesterone hormone is considered to be an important risk factor in the
development of cervical neoplasia, although the present study is the first to
investigate molecular biological mechanisms of these hormones.
It has been shown that HPV has a tendency to target cells with progesterone receptors. Both HPV 16 and HPV 18 contain progesterone and glucocorticoid response elements that increase expression of the HPV E6 and E7
oncogenes, which are considered crucial in cell transformation(89). Such a
transformation has been reported to take place when progesterone or OC
gestagens were added to cell cultures (130).
Progesterone and glucocorticoid hormones increase HPV mRNA and
significantly stimulate viral replication(172,32).An increased cell proliferation with high serum progesterone levels in .Invasive squamous cell cervical
cancer has also been reported in a study which included more than 100
women (173). Estrogen has been reported to reduce susceptibility to primary
HPV infection, but might be of no importance once an HPV infection has
been established (174).Estrogens might also have a role in increasing the
levels of HPV-induced apoptosis (175). Sex steroid hormones might also be
involved in single steps in the neoplastic progression, similar to that of many
tumor markers. Once invasive cancer has been established, high serum estrogen levels might have a positive effect on outcome, while high serum
progesterone levels have been found to have a deleterious effect (173).
Some previous studies have studied sex steroid hormone interactions with
HPV, in vitro experiments on cervical cancer cell lines, and animal experi54
ments. This clinical study on women with normal or neoplastic epithelium
indicates that serum progesterone and estradiol levels influence cellular and
extracellular proteins which have been associated with neoplastic development, in normal epithelium and CIN.
55
Summary
The main findings of this study were the observations that exposure to cofactors, as well as HR-HPV infection, was associated with molecular changes in cervical tissues. There was a tendency towards a “negative” pattern of
tumor marker expression with exposure to co-factors. This supported the
main goal of the study, i.e. lending biological support to previous epidemiological studies.
Thus, our findings included the following:
I
Smoking correlated to decreased suppressor expression, i.e.
p53, FHIT and IL-10, while the proliferation markers Ki-67 and
Cox2 were over-expressed. Serum cotinine levels, the main metabolite of nicotine, correlated with increased EGFR expression.
II
Hormonal contraceptives. In combined oral contraceptive users
both Cox-2 and Ki-67 expression was increased, and IL-10 expression decreased. In MIB-users, p53 expression was increased.
III
Serum sex steroid levels. Increased levels of serum progesterone and estradiol were associated with increased Cox-2 expression.
IV
HR-HPV infection. Several correlations to tumor marker expression were found. Independent correlations could not be
conclusively established when CIN grade was adjusted for. Ki67 was over-expressed independently of CIN grade, but other
candidates need to be further studied.
56
Acknowledgments
I would like to express my sincere gratitude to all the people who have
helped and supported me throughout my whole journey.
My special thanks to
Dan Hellberg, my primary supervisor, for supporting, encouraging, and
guiding me throughout the whole research. I want to thank him for his patience, kindness and being there for me even when I had problems outside
the research fields.
Anna Asplund, my co- supervisor for helping me and introducing me to the
Protein Atlas Project and TMA.
Tibor Tot, my co-supervisor for his expertise in histopathology.
Gyula Pekar, for his expertise and assisting with diagnostics of tumor marker
expression.
Staffan Nilsson, for convincing me to enter the scientific research field, for
annoying me by challenging me all the time, being a father-like friend. I
enjoy every minute being with you and appreciate the time we have together.
Annika Lindström, for her support and advice. I want to thank her for being
there when I had struggled through my research.
Gunnel Janeslätt for her support and advice. I want to thank you for your
help and encouragement .When I Felt that everything was falling down, you
corrected it with a smile on your face and one click on the keyboard.
All participating women in the study for their contributions.
Maria Pilawa, for her administrative support and kindness.
Associate professor Ulrika Schytt and all friends and colleagues at the center
for Clinical Research Falun for their help and support.
57
Maria Hårdstedt, for her encouragement and support .
Agneta Romin, my first mentor, for helping, supporting and introducing me
to the Swedish health system. I want to thank her for being a good friend,
being there from the beginning, and most importantly for being her.
Thomas Riman, PhD, for support and encouragement, for helping me to
achieve my goals in the IVF field and clinical researches.
Katarina Ekman, for her participation in collecting some materials for the
research.
My colleagues in the Department of Obstetrics and Gynecology at Falun
hospital, for their kindness and caring, for being so welcoming from the first
time I started working with them (the best clinic in the world).
The staff at Falun hospital, mid-wives, nurses, assistant nurses, secretaries,
for encouraging me in my work
The staff of IVF Falun, my colleagues. Special thanks to all midwives, nurses, laboratory staff and secretaries.
The staff of Falun hospital medical library, for providing me all the articles I
needed and helping me gain access to my sources.
My friends Helena Stenport, Hanna Sahlgren, Maria Enberg for their support
and encouragement.
Bente Simonssen and Klaus Grimstrup for their love and support. For being
there from the beginning to the end.
Eva-Britta Råsjö and Bengt Korpås for the wonderful friendship and support.
For their love and kindness and being there when I needed them.
My best friend Nadja Alhashemi for all the help and support, for being you
and because you mean a lot to me.
Sarab Nadim, my cousin - thank you for reminding me that we are fighters
and pushing me up when I felt down and remember cousin as far as anyone
know we are a normal family.
I want to thank my husband Ahmed Salim for being so supportive all the
way through our life together. I want to thank him for his understanding and
58
patience and his kindness and love. I want to thank him for the wonderful
journey that we started together from 1998 till now and on ahead.
To my wonderful girls Noor (light) and Farah (joy) the greatest achievement
in my life. I want to thank you both for the light and the warmth that you
bring to my life and I also want to thank you for each minute that I spend
with you, for each smile and laugh together that we share. Without you my
life would have no meaning.
59
Sammanfattning på Svenska
Infektion med hög risk-human papillomavirus (HR-HPV) anses vara en viktig orsak i utvecklingen av cellförändringar i livmoderhalsen (cervical
intraepithelial neoplasia, CIN) och invasiv livmoderhalscancer. Trotts att
HPV infektionen är den viktigaste orsaken, kan förändringar i immunsvar i
livmoderhalsens celler förklara de olika graderna av cell förändringar och
deras utveckling till invasiv cancer. Dessa förändringar i immunsvaren kan
förklaras av närvaron av ytterligare faktorer som rökning, preventivmedel
och könshormoner.
Syftet med denna forskning var att undersöka sambandet mellan vävnadstumörmarkörers uttryck och HR-HPV-infektion, rökning, hormonella preventivmedel och könshormoner hos kvinnor med CIN eller normalt epitel.
Studien undersökte uttrycket av 11 tumörmarkörer i cervikala biopsier som
erhållits från 228 kvinnor med olika diagnoser, allt från normalt cervikal
epitel till olika stadier av CIN. 188 kvinnor rekryterades på vår kolposkopi
klinik (poliklinisk kirurgi, Institutionen för obstetrik och gynekologi, Falu
lasarett) för laser behandling (konisering) eller en riktad stansbiopsi. Proverna togs på grund av ett vaginalt cellprov som visade cytologiska fynd som
tyder på CIN, eller på grund av upprepade fynd som visar atypiska skivepitelceller (squamous cells) av obestämd betydelse (ASCUS). För 40 frivilliga,
var stansbiopsier tagna från normalt cervixepitel. Material för studierna samlades in under perioden 2005-2007.
Studie I: 228 kvinnor, varav 116 var testad, bara 64 var positiva till HRHPV. Resultaten visade att Ki67 tumörcellproliferationsindex var den enda
markör som på ett oberoende sätt var korrelerad med både HR-HPV och
svårighetsgraden av cellförändringar i cervix. Detta innebär att HR-HPVinfektion var associerad med ett negativt mönster av uttryck för tumörmarkörer i cervix och att Ki-67 kan användas som en surrogatmarkör för HPVinfektion.
Studie II: 228 kvinnor, varav 83 var rökare (36,9 %). Rökare uppvisade
lägre uttryck av p53, FHIT (tumör suppressor markörer) och interleukin-10
och högre expression av Cox-2 och Ki-67 (tumörproliferationsmarkörer).
Det sammanlagda resultatet tyder på en negativ inverkan av rökning på cervikal epitel.
Studie III: 195 premenopausala kvinnor. 57 var COC (kombinerat ppiller) användare, 15 MID (medicinsk/hormonell spiral) användare, 24 var
progestin enbart användare och 99 icke-användare. Det fanns en ökad p5360
expression (tumörsuppressor) i progestin-spiralanvändare, jämfört med ickeanvändare. Minskat IL-10-uttryck (immunologisk markör) observerades hos
både kombinerade p-piller och eventuella progestinbaserade endast användare. Detta resultat visar att hormonella preventivmedel påverkar uttrycket
av de mest undersökta molekylära markörer för livmoderhalscancer.
Studie IV: Serum från 80 premenopausala kvinnor var tillgängliga, varav
25 (31,3 %) med normalt epitel, 10 (12,5 %) med epitel på gränsen till normalt, 18 (22,5 %) med CIN I och 27 (33,8 %) med CIN II-III. Den viktigaste
slutsatsen var att de ökade nivåerna av serum progesteron och estradiol var
associerade med ökad Cox-2-uttryck (spridningsmarkör). Upptäckten av en
omvänd korrelation mellan höga Rb-uttryck och låg P16-uttryck med höga
progesteron nivåer, när justering för CIN grad gjordes, kunde bekräfta sambandet mellan dessa tumörmarkörer i HPV-infektioner.
Serum progesteron och östrogennivåer påverkar cellulära och extracellulära proteiner som har satts i samband med tumörutveckling i normalt epitel
och CIN.
Slutsats: Resultaten av dessa studier stöder tidigare epidemiologiska rön
angående rollen som rökning, preventivmedel och könshormoner har som
bidragande faktorer i utvecklingen av CIN och att tumörmarkörers uttryck
varierar oavsett graden av CIN.
Nyckelord: tumörmarkörer, cervikal intraepitelial neoplasi, rökning, preventivmedel, könssteroidhormoner, HPV-infektion.
61
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Acta Universitatis Upsaliensis
Digital Comprehensive Summaries of Uppsala Dissertations
from the Faculty of Medicine 1137
Editor: The Dean of the Faculty of Medicine
A doctoral dissertation from the Faculty of Medicine, Uppsala
University, is usually a summary of a number of papers. A few
copies of the complete dissertation are kept at major Swedish
research libraries, while the summary alone is distributed
internationally through the series Digital Comprehensive
Summaries of Uppsala Dissertations from the Faculty of
Medicine. (Prior to January, 2005, the series was published
under the title “Comprehensive Summaries of Uppsala
Dissertations from the Faculty of Medicine”.)
Distribution: publications.uu.se
urn:nbn:se:uu:diva-262889
ACTA
UNIVERSITATIS
UPSALIENSIS
UPPSALA
2015