HPV infection and TERT gene expression levels. Association of the telomerase gene expression with cervical cancer and its precursor lesions and Human papillomavirus infection

Table of Contents

ABSTRACT

List of acronyms and abbreviations

1. InTRODUCTION

2. BACKGROUND

2.1. Cervix uteri and the squamocolumnar junction
2.2. CERVICAL DISEASE AND NEOPLASIA
2.3. CERVICAL PRECURSOR LESIONS
2.3.2. SCREENING
2.3.3. HPV testing in primary screening
2.3.4. Management of cervical precursor lesions - treatment
2.3.5 Follow-up after treatment
2.4. Human papillomaviruses (HPVs)
2.4.2. HPV genome organisation
2.4.3. Early genes E1, E2, E4-E7
2.4.4. Late proteins L1/L2
2.4.5. HPV life cycle
2.4.6. Epidemiology of the HPV infection and its role in malignant transformation
2.4.7. Host immune response and HPV infection
2.5. Evasion mechanism of HPV
2.5.1. HPV vaccines
2.6. Telomere structure
2.6.1. Telomerase activity and malignant transformation

3. MOTIVATION FOR THE RESEARCH

4. RESEARCH HYPOTHESIS

5. AIMS OF THE STUDY
5.1. The specific aims of the research

6. MATERIALS AND METHODS
6.1. Ethics
6.1.1. Inclusion and exclusion criteria
6.1.2. Preconditions for the screening
6.1.3. Population
6.1.4. Control group characteristics
6.1.5. Data collection
6.2. MOLECULAR analyses
6.2.1. DNA and RNA isolation
6.2.2. HPV detection and genotyping
6.2.3. Quantitative Real-Time PCR for telomerase gene expression
6.3. Statistical analysis

7. RESULTS

8. DISCUSSION

9. CONCLUSIONS

10. REFERENCES

ABSTRACT

Background: Cervical cancer is a preventable disease, however despite its avoidable nature it remains one of the leading causes of cancer mortality globally, with the highest incidence and mortality rates in low-resource countries. Human papillomavirus infection is certainly an underlying cause of cervical cancer and its precursor lesions with different prevalence of the genotypes depending on the patient's age, ethnicity, race, severity of the precursor cervical lesions and geographical region. Early events associated with disease progression and cervical cancer include hTERT up-regulation at the transcriptional level mediated by high-risk HPV E6 oncoproteins via indefinite mechanisms with concomitant immortal phenotype of the cell in vitro and increased replicative potential of cells in precancerous cervical lesions and cervical cancer.

Aim: The aim of this study is to evaluate the frequency of HPV infection and genotypes among women with normal and abnormal cytological results and its correlation according to the severity of the lesions as well as to investigate the correlation between the TERT gene expression levels and the cytological sub-group and to evaluate the correlation between the HPV infection and TERT gene expression levels as diagnostic and prognostic marker in cervical precursor lesions and cervical cancer.

Materials and methods: This study is a prospective observational case-control study conducted in Obstetrics and Gynaecology Clinic in Pristina and Molecular and Biology­Genetics Department in Skopje.Cervical samples from 214 women (median age 45.28 years; range 20-65) from the Outpatient Clinic in University Clinical Center in Kosovo were tested for HPV-DNAand quantitative TERT gene expression after performing conventional Pap smear. Among them 100 women had normal cytology ( NC) 45 cases were ASC-US, 37 LSIL,7 cases with ASC-H, 15 cases with HSIL and 10 cases with squamous cervical cancer. HPV detection and genotyping was performed by PCR using MY 09/MY11primers with subsequent genotyping by restriction -fragment length polymorphism (RFLP) analyses using combination of 3 endonucleases. Rna isolates were used for complementary DNA (cDNA) synthesis by reverse transcription. Amplification and relative quantitative real-time polymerase chain reaction (qRT-PCR) was performed using TERT-specific primer pair and gene specific fluorescent TaqMan probes. Quantitative TERT gene expression was calculated relative to the reference G3PDH gene and normalized to the normal cervical cell sample using AACt method. Two-tailed Mann-Whitney U-test was used for statistical analysis.

Results: HPV infection was detected in 109 cases (50.93%). A hundred (46.73%) out of 214 samples were found to have normal cytology (NC) and 114 cases showed some type of cytological abnormalities: 45(21.03%) ASC-US; 37(17.29%) LSIL; 7(3.27%) ASC-H; 15(7.01%) HSIL and 10(4.67%) with cervical cancer. The frequency of HPV positivity was dependent on the severity of the cervical lesions (62.22% for ASC-US; 85.71% for ASC-H; 86.49% for LSIL; 93.33% for HSIL and 100 % for cervical cancer. Multiple HPV infection did not correlate directly with the severity of the cervical lesions because in 44%, 72%, 71% and 80 % of samples with ASC-US, LSIL, ASC-H, and HSIL/CC respectively, only single HPV type was found. Multiple HPV infection with more than two genotypes was found in 18/109 patient samples or 8.41%.The most prevalent genotype found was HPV 53 (26.77%). HPV 16 was second most prevalent type ( 22.83%) followed by third most common type 31( 14.96%). The hTERT mRNA expression significantly correlated with the increased grade of the cervical lesion. Statistically high difference was found between the hTERT normal expression levels and hTERT over-expression in all cytological groups with abnormalities compared with normal control group ( p<0.01) hTERT gene overexpression was associated with 6.31 and 9.20 fold higher risk for developing ASC-US and LSIL compared to the patients with normal expression and normal cytology, respectively. In addition, there was a strong correlation between the hTERT gene expression levels and HPV infection notably, in the high-grade lesions and cervical cancer. hTERT gene relative expression values showed 98% specificity and 100 % sensitivity as indicator of cervical lesions particularly for the ACS-H, HSIL and cervical cancer.

Conclusions: This study highlights high HPV prevalence corresponding to high incidence rate of cervical cancer in Kosovo with the most common type HPV 53 and HPV 16 followed by type 31. Based on epidemiological profile of HPV genotypes in this region, those results might prove auxiliary to establish a platform for cervical cancer screening and prevention strategies in the future. Data from this study also suggest that hTERT expression occurs early in progressive cervical disease and correlates with disease severity and HPV infection. Therefore, it is useful as diagnostic and prognostic biomarker of the disease progression in HPV- infected subjects.

Keywords: Human papillomavirus, TERT gene, cervical cancer, telomerase activity, polymerase chain reaction.

LIST OF ACRONYMS AND ABBREVIATIONS

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1. INTRODUCTION

Cervical cancer is preventable disease but despite its avoidable nature it remains one of the leading causes of cancer mortality globally, with the highest incidence and mortality rates in low- resource countries (figure 1,2) (1). Cervical cancer is the fourth most common reason for cancer death among women worldwide with more than 527,000 new cases diagnosed in 2012(Ferlay et al., 2012). This disease is a foremost example of health inequality, where more than 80 % of women diagnosed with cervical cancer live in the developing countries and furthermore approximately 95% of them had no screening for cervical cancer (2). Countries with the highest incidence and mortality rate include low-resource countries particularly in sub-Saharan Africa, Central America, South Central Asia and Melanesia (3, 4). Significant disparities in the incidence and mortality rate of the disease are mainly due to the different prevalence of risk factors, limited access to health facilities in developing countries with poor organized screening programmes, with low coverage of high-risk groups, lack or insufficient referral system and non-effective interventions including screening, treatment and follow-up of the patients. Incidence and mortality rates are low in developed countries, due to effective and well-planned organised and opportunistic screening programmes.

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Figure 1: Estimated Cervical Cancer Incidence Worldwide in 2012

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Figure 2: Estimated Cervical Cancer Mortality Worldwide in 2012(1)

According to 2014 annual report of the National Institute of Public Health of Kosovo, prevalence of malignant disease was 186.5 per100.000 residents and incidence was 167.9 per 100.000 residents. More specifically malignant tumours of genital tract were presented with 251 cases or7.6 %. Importantly there were 67 new cases of cervical cancer diagnosed in 2014 or 2.0%. The cervical screening program was introduced in 1983/84s in Kosovo in order to detect and treat cervical precursor lesions which can progress to the cervical cancer. On international level, cervical cancer should be considered the major preventable disease since with early detection and treatment it is highly curable. In Kosovo the biggest challenge is to cover the highest risk group from 35 up to 45 years within opportunistic screening and reach the non-attending woman particularly in rural areas.

2. BACKGROUND

2.1. CERVIX UTERI AND THE SQUAMOCOLUMNAR JUNCTION

The cervix is the lower one-third of the uterus. The vaginal portion of the cervix is covered with stratified non-keratinized squamous epithelium (multi-layered) and resembles the vaginal epithelium. Endocervix or the upper two-thirds lies above the vagina and is covered by columnar epithelium, asingle layer of cells set on the basement membrane (2). Whilethe ectocervix is visible with a speculum, the lower portion of the endocervical canal is visualised by an endocervical speculum. The columnar epithelium which lines the cervical canal extends to a variable part of the ectocervix and this transition is called original squamocolumnar junction (SCJ) and its position depends on hormonal stimulation. The columnar epithelium when exposed to vaginal acidic milieu will be replaced through metaplasia into squamous epithelium thus giving rise to a new SCJ. The zone between the original and new SCJ is termed transformation zone, which for uncertain reasons is prone to develop precancerous and cancer lesions and more vulnerable to infections. In postmenopausal women squamocolumnar junction (SCJ) is rebound into the cervical canal while puberty, pregnancy and use of hormonal contraception are characterized by the enlarged transformation zone in the ectocervix (2).

2.2. CERVICAL DISEASE AND NEOPLASIA

2.2.1. Risk factors for cervical precursor lesions and cancer

In1976 Professor Harald zur Hausen after his several failed attempts to identify HSV-2 DNA in cervical cancer, and after he suggested a leading roleof a Human Papilloma Virus (HPV) in genital cancer, stated that “The condyloma (genital wart) agent has been entirely neglected thus far in all epidemiological and serological studies relating not only to cervical and penile, but also to vulvar and perianal carcinoma. This is particularly unusual in view of the localization of genital warts, their mode of venereal transmission, the number of reports on malignant transition, and the presence of an agent belonging to a well characterized group of oncogenic DNA viruses” (5). A Nobel Prize for Medicine was awarded to him in 2008 for his discovery of different HPV genotypes including types 16 and 18, most often observed in cervical cancer, and for indicating the integration of HPV DNA into the host genome (6, 7, 8).

Walboomers et al. after the report that 93 % of cervical cancer globally, consist of HPV, reanalysed HPV-negative cases from a prior study and confirmed that the worldwide HPV prevalence in cervical carcinomas is 99.7% (9). According to the International Agency for Research on Cancer, HPV types 16,18,31,33,35,39,45,51,52,56,58,59 are human carcinogens. HPV types 6 and 11 “are not classifiable as to their carcinogenicity to humans “ (10). Although the causal relationship between the high -risk HPV infection and cervical cancer is today well established, not all HPV infected patients are diagnosed with cervical cancer, thus indicating that HPV infection is obligate but not sufficient factorin cervical carcinogenesis.

HPV infection is the most common sexually transmitted disease in the world with different prevalence dependent on geographical areas and studied population. According to the de Sanjose and colleagues, inwoman with normal cytological results, worldwide estimated HPV prevalence was found to be 10.0% while regarding the age-specific groups, the highest HPV prevalence was found in women younger than 34 years with decreased prevalence in the age-group from 35-44.

An increased prevalence was found for the group-age 45-54(11). However other risk factors for cervical cancer should be considered in the complex process of cervical carcinogenesis since, not all HPV- infected women will be diagnosed with cervical cancer. Number of life-time sexual partners and a new sexual partner is a risk factor for HPV infection acquisition since the most ordinary route of transmission is sexual contact with infected person, although the majority of prior studies found a convincing association between non-penetrative sexual contact and HPV infection in virgins (12-13). Several studies consistently demonstrated no protective effect with condom use,possibly, since HPV is spread through skin-to skin contact (13-14).Other co-factors proved to be crucial for HPV infection and cervical cancer include early sexual debut, younger age at first full-time pregnancy and five or more full-term pregnancy, oral hormonal contraception for more than five years and previous exposure to other sexually transmitted disease predominantly, Chlamydia trachomatis, Herpes Simplex virus-2 and HIV. Association between cervical cancer and co-infection with Herpes Simplex Virus-2 and Chlamydia trachomatis in several epidemiological studies revealed from moderate to no association between HSV -2 serological response(antibodies) and cervical cancer. Finan and colleagues found that co-infection with HPV and Chlamydia trachomatis and co-infection with HPV and HSV -1 but not HSV-2, poses a higher risk for cervical cancer (15).

On the contrary, there is a strong evidence for the role of Chlamydia trachomatis in the cervical carcinogenesis as reported in several epidemiological studies(16-17). Seropositivity of prior Chlamydia trachomatis infection was associated with increased risk for invasive cervical cancer in HPV infected women, after adjustment for age, oral contraceptive use, herpessimplex virus-2 antibodies and number of full-term pregnancy(18).Recently, increased risk for CIN3 is reported in women with repeated chlamydial infections and prevalent or persistent HPV infection (19). Molecular mechanisms by which persistent chlamydial infections instigate or support cervical carcinogenesis are predominantly unclear. Antichlamydial activity in local mucosal tissues is modulated by interferon gamma-induced nitric oxide production (20) whereas through specific anti apoptotic factors this intracellular pathogen inhibits apoptotic activity in the host infected cells (21).Chronic cervical inflammation may precede to early steps in cervical carcinogenesis by several molecular mechanisms including malfunctioning inflammatory host response and increased level of pro-inflammatory cytokines (22).

Association between HIV infection and HPV prevalence, incidence, persistent infection with multiple HPV types and dysplastic cervical lesions was reported constantly in a large number of studies. The prevalence of CIN is high in HIV-infected women and increased risk for CIN positively associates with bacterial vaginosis but interestingly not with parity (23-24). Dataalso suggest that HIV-infected women have a higher risk for multiple HPV infection compared to HIV-negative women (25). Low CD4+ count as well as no treatment with antiretroviral drugs in HIV-positive women encompass an increased risk for squamous intraepithelial lesions (SIL) and HSIL albeit, no association was found between the high HIV viral load and risk for SIL (26). In a study from Stuardo and co-workers, 78.4% of HIV-1 positive women had multiple HPV infection hence, a strong association was found between the abnormal cytological findings, high­risk HPV infection and low CD4+ count in the studied subjects (27). Women infected with either HIV-1 or HIV-2 type are at increased risk for severe intraepithelial lesions (CIN 2/3) as well as invasive cervical cancer (28). Contrary to the moderate association of HIV infection and HPV persistence, a consistent association was reported between HPV incidence and CD4+ count and plasma HIV RNA level (29) Mechanism by which HIV infection modulates host response in HPV infected subjects and the usual course of the development of HPV related disease, is mainly vague but HIV related-immunosuppression particularly, CD4+count <200x10(6)/L) increases risk for the cervical dysplasia and HPV persistence (30).Those two infectious agents may also interact directly via their viral proteins which will result in compromised cellular and humoral immunity and cell cycle disruption (31).HIV-infected women treated with highly active antiretroviral therapy (HAART) showed a reduced progression and incidence rate of cervical precursor lesions (32).

Tobacco Smoking-Evidence that tobacco smoke causes lung cancer date as far back as early 1950.Today is generally confirmed that is one of the major causes of cancer-related death in the world and play a decisive role in cervical cancer and its precursor lesions. Relationship between cervical precursor lesions and cancer with smoking has been one of the most examined factor for decades in epidemiological studies. This way, evidence that smoking causes cervical cancer was regarded as “sufficient “by the International Agency for Research on Cancer (33). Compared to non-smokers cervical mucus of smokers was shown to be more mutagenic (34). Consistent findings were also reported in previous case-control studies, regarding the significant difference in the frequency of micro nucleated cervical epithelial cells among smokers and non-smokers (34)and a higher significant frequency of micronuclei (MN) found in CIN2 and CIN3 compared to CIN1 (35-36).

In a nested case-control study within Nordic Biobanks authors confirmed tobacco smoking as an independent risk factor for squamosus cervical cancer (SCC). Furthermore, they found 2-fold increased risk for cervical cancer in women with antibodies to HPV 16 and 18 (37). While the European Prospective Investigation into Cancer and Nutrition (EPIC) found 2-fold increased risk for cervical cancer in current smokers compared to non-smokers, after adjustment for HPV exposure (38) even a passive smoking was suggested to be an independent and significant factor for cervical cancer (39).

Combined hormonal contraception was accessible for use since late 1950s but after 1960s when usability was more sizable, their association with cervical cancer has been questioned and later reported in numerous epidemiological studies. Long-term use of oral hormonal contraception for 10 years or longer, was associated with 4-fold increased risk of cervical cancer in HPV-positive women (40). This association was consistently reported in large pooled case-control studies. Long-term use of oral contraceptives for 10 or more than 10 years was associated with increased relative risk for cervical cancer (RR=2.5, 95% CI; 1.6-3.9) in HPV infected women (41). Pooled analysis of 24 epidemiological studies globally, revealed that relative risk of invasive cervical cancer increases with long-term use of oral contraception and estimated that cumulative incidence of invasive cervical cancer increases from 7.3 to 8.3 per 1000 in developing countries in 10 year users of oral contraception (42). However, there are some conflicting results in this regard since several studies showed that oral contraceptives are not independent factor for precursor cervical lesions nor found consistent evidence for increased risk of cervical cancer or carcinoma in situ with oral contraceptive use. Shapiro et al. found no association between progestin-only contraceptive use or combined hormonal contraception use and increased risk for cervical cancer (43). Syrjanen et al. emphasize that since sexual behaviour differs among OC users, non-OC users and in non-users of contraception thus this factors determine the outcome of high-risk HPV infection and cervical disease (44). Whenthe effect of estrogen and progesterone on expression levels of E6 and E7 oncogenes in CaSki and SiHa HPV-16 positive cell lines, was evaluated, no significant effect was found in transcription levels ofthese oncogenes. Thus the ensuing conclusion was that these hormones can promote cell proliferation and make cells more vulnerable to mutations, but not through E6 and E7 mediated pathway. Furthermore, growth of HPV infected cells was a result of anti-apoptotic effect of estrogen (45). Upstream regulatory region (URR) of the HPV 16 viral genome contains enhancers, which are activated by steroid hormones, with consequent increased expression of HPV 16 oncogenes. HPV gene products lead to a degradation of p53 protein and cell cycle disruption (46).

Conflicting results were reported for high parity as an independent cofactor for cervical cancer. In several case-control studies a strong association was found between the high parity and cervical cancer whereas other studies observed no association between parity and CIN3. The IARC multicentre case control-study showed that high parity with seven or more full-term pregnancies is associated with an increased risk for cervical cancer in HPV-positive women compared to nulliparous women (OR=3.8, 95% CI;2.7-5.5) (47). Hildesheim et al, reported significant correlation between the oral contraception and high-grade lesions/cervical cancer in women with more than three pregnancies (48). Even though, risk for cervical adenocarcinoma is higher in HPV-infected women with eight or more pregnancies, evidence suggest that parity is significant co-factor for squamous cervical cancer and to a lesser extent for cervical adenocarcinoma (49). Multiparity and current smokingin HPV-positive women are major risk co-factors for CIN3 compared to CIN2 as previously reported (50). In this regard, there is also an increased risk for CIN2+ in women with high parity, infected with oncogenic HPV types (51).

Findings from Jensen and colleagues showed an increased risk for CIN3 especially for women with persistent high-risk HPV- infection (52). Potential factors that could explain the relation between parity and increased risk for cervical disease include not just increased steroid hormone levels which provide their effect through increased HPV oncogene expression, but also HPV- DNA integration into the host chromatin through cell oxidative stress. Impaired immune response and enlarged transformation zone in the extocervix of multiparous women which facilitates HPV exposure are also reported factors (53-54). Contrary to, other studies Castle and co-workers observed no association between the parity and increased risk for CIN2and CIN3 (55).

Socioeconomic status and level of education - Level of education and household income was found to be significant predictor of cervical cancer screening rate. Mostly, women with higher level of education and household income have a better screening rate for cervical cancer compared to those with low income and low level of education (56-57).

Antioxidants and cervical cancer - Several studies provided convincing data regarding the anti- carcinogenic activity of the polyphenols as epigallocatechin-3-gallate (EGCG) from the green tea, curcumin from turmeric and resveratrol from grapes. In this regard, EGCG which is the major compound of green tea, is mostly studied antioxidant. Ahn and colleagues studied the effect of the EGCG on the HPV-16 cervical cancer cell lines CaSki and reported that this compound demonstrates anti-proliferative effect throughout induced apoptosis, cell cycle arrest and through alteration of the gene expression in vitro (58). Study from Siddiqui et al, confirmed apoptotic effect of EGCG in the cervical carcinoma cells (59) while data provided by Noguchi and co­workers demonstrated that beyond apoptotic effect and cell cycle disruption, this compound also inhibits telomerase activity (60). Resveratrol has important role in apoptosis, inhibition of MMPs in CaSki cervical cancer cell line and can also suppress angiogenic activity in vitro (61). Although, their anti-carcinogenic effect was evidently demonstrated, further studies are needed before their routine therapeutic application. Myung et al, in meta-analyses of 22 case-control studies found that vitamin B12, vitamin C, vitamin E, beta-carotene, folate and lycopene have precautionary effect in cervical cancer while no such effect was found for selenium and vitamin A (62). Srivastava and co-workers in a prospective case-control study reported low levels of vitamin A and E and higher levels of lipid peroxide (LPO) in cervical cancer compared to the control group (63).

Male factor -Epidemiological studies showed evidence that male circumcision is associated with reduced risk for HPV infection and reduced risk for cervical cancer among women whose partners have had multiple sexual partners, decreased prevalence of HPV infection and implications in HPV transmission. Thus, a role of male circumcision as a cofactor in HPV infection and its natural history is strongly supported (64-66).

Genetic alterations and cervical cancer - Despite the well-established oncogenic role of the HPV infection in cervical cancer, interference with host genetic abnormalities is mainly uncertain. There is a marked latency period from HPV infection to clinical cervical disease and not all HPV-infected subjects will be diagnosed with cervical cancer, hence a partial explanation could be their genetic susceptibility. Magnusson et al. when comparing incidence of cervical disease in relatives of diseased women and controls found a 2-fold risk for the disease in relatives compared to non-relatives (67). Several studies focused their attention to the major histocompatibility complex (MHC) region, notably on the human leukocyte antigen (HLA) class II, and as reported by Kohaar and colleagues, HLA DR-DQ polymorphisms are connected with genetic susceptibility to HPV infection (68) whereas possibility that HLA polymorphism can modulate immune response to the virus is also reported (69). DNA repair genes were previously proposed as important factor in HPV persistence and disease progression to CIN 3 and cervical cancer (70).

The p53 polymorphism is largely studied, particularly polymorphic site at codon 72 of exon 4 concerning the increased risk for cervical cancer. Although the results on the subject of the p53 allelic polymorphism are generally indefinite, Gudleviciene and colleagues found an 2-fold increased risk for cervical cancer in p53 arg/arg allele (OR = 2.10, 95% CI 1.10-4.19) in Lithuanian women, while in the same line were results reported from Andersson and al. that there is an increased risk for adenocarcinoma in homozygotic women for arg/arg in codon 72 of this tumorsuppressor gene (71-72).

Progression from severe intraepithelial lesions to cervical cancer is certainly associated with chromosomal numerical abnormalities and genomic instability. This way, most frequent chromosomal abnormality which characterize progression from severe intraepithelial lesion to cervical cancer is gain of 3q chromosome (73) whereas in CIN frequently observed alterations as previously reported were loss-of-heterozygosity (LOH) at arms 3p,6p and 11q as well as LOH at arms 6q,17p and 18q in invasive cervical cancer (74).

2.3. CERVICAL PRECURSOR LESIONS

2.3.1. Definition and the pathophysiology of CIN

The terminology “Cervical Intraepithelial Neoplasia “(CIN) was introduced in 1973 by Richart This concept point out that dysplasia and carcinoma in situ are the different phases of disease progression but certainly present the same biological process. According to this system premalignant abnormalities are ranged from mild dysplasia (CIN1) to moderate dysplasia (CIN2) to severe dysplasia /carcinoma in situ (CIN3/CIS) (75). Later on, the terminology known as the Bethesda System reflected the need for unique reporting of cytological findings. The Bethesda System for reporting cervical cytological diagnoses was introduced in 1988, revised and modified in 1991 and moreover modified in 2001 in order to redefine the reporting system of the cervical smears and thus introduce uniformity in diagnostic approaches. The results are interpreted as (1) ASC (atypical squamous cells) which is further divided into ASC of “undetermined significance “ (ASC-US) and “cannot exclude high-grade squamous intraepithelial lesion (HSIL)” or (ASC­H), (2) LSIL (low-grade squamous intraepithelial lesions) (3) HSIL (high -grade squamous intraepithelial lesions), and (4) squamous cell carcinoma. The category of AGUS is replaced with AGC and absence or presence of endometrial cells should be reported in women 40 years or older. Glandular abnormalities are classified as atypical glandular cells (AGC), endocervical adenocarcinoma in situ (AIS) and adenocarcinoma (76-78). This classification is also recommended by WHO . CIN is characterized by the proliferation of atypical squamous cells with altered size, shape, polarity, higher mitotic index and hyperchromatic nuclei. While the lower third of the epithelium is involved in CIN1, 2/3 of the epithelium are involved in CIN2 and the entire thickness is involved in CIN3/CIS (79). Conclusions from review by Ostor, extracted by the published data from 1950 and 1990 regarding the natural history of CIN are presented in the table below (table 1) (80).

Table 1. Natural history of CIN (Ostor, 1993) (80)

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2.3.2. SCREENING

2.3.2.1 Conventional Papsmear and liquid-based cytology (LBC)

Utility of the exfoliative cytology for early detection of invasive cervical cancer was introduced as early as 1920s by Babes and Papanicolaou. Later was demonstrated that this exfoliative technique can be used to detect cervical precursor lesions (81). Pap smears can be performed as opportunistic and organised systematic screening. Organised screening programs which accomplish high coverage in predefined target population can decrease incidence of the disease and improve survival through early detection of cervical cancer because diagnosis at early stages improves survival rate. (82). Opportunistic screening which is carried out on demand from gynaecologist or from the women is less effective because of low sensitivity of the single Pap smear. Although, a marked reduction in the incidence of cervical cancer was achieved, mainly in developed countries, there is a wide range of performance of this test regarding the sensitivity and specificity with remarkably high variability reported regarding the sensitivity of the test.

Conventional PAP smear has a low sensitivity estimated as approximately 53 % (48-57 %) from a meta-analysis (83) whilein a systematic review carried out by Nanda et al, sensitivity of the PAP smear was estimated51 % (range 30-87%) (84) thusmaking use of ongoing Pap smear screening mandatory in order to avoid errors from false negative results in clinical management. Depending on the histological cut-off (CIN 1+ or CIN 2+) specificity of conventional PAP smear is reported up to 98% (85-86). Several reviews regarding the sensitivity and specificity of conventional cytology for the detection of CIN2 and CIN3 reported results that ranged from 47­62 % and 60-95% respectively (87).

Liquid-based cytology is another option method, introduced in the mid-1990s, where cervical cells are suspended in preservative solution with subsequent slide preparation. ThinPrep® and SurePath™, have been approved by the U.S. Food and Drug Administration (FDA) in 1996.

In Kosovo conventional PAP smear is a method of choice. Concerning the sensitivity and specificity of the two techniques, published results are noticeably controversial.

In a quantitative survey conducted form Abulafia and colleagues data from 10 studies showed that LBC is more sensitive and specific for detection of the LSIL and HSIL (88) Zhuet al, compared performance of the two methods and concordance with histological diagnosis and reported higher sensitivity of LBC in detecting the CIN 2 and 3 compared to the conventional PAP smear (66% vs 47%) and furthermore less ASCUS results were reported with LBC compared to conventional PAP smear ( 4.3% vs 8%) (89).

In a prospective study conducted by Sykes et al,the sensitivity of the two methods for detection of any epithelial abnormality was found to be 81 % while for detection of the high-grade lesions overall sensitivity was 92%(90).

Higher sensitivity of the liquid-based cytology vs conventional cytology for the detection of the cytological abnormalities confirmed consequently with histological diagnosis was reported also by Beerman and al, (96.2% vs. 92.0 %) (91).

Contrarily, a prospective randomized trial conducted by Obwegeser and colleagues showed no significant difference in performance of the two techniques regarding the sensitivity and the specificity (92). Recently, a study conducted by Zarchi et al, comparing the three methods (conventional PAP smear, liquid-based cytology and colposcopy) with cervical biopsy found no significant difference in sensitivity and specificity of the LBC vs conventional PAP smear (55,3 % vs 51 % and 77,7% vs 66,6%)(93).

United States Preventive Task Force (USPSTF) and the American Cancer Society (ACS) recommend against routine yearly screening. These guidelines recommend testing every three years for women ages 21-65 and no longer is recommended routine cervical cancer screening for women under 21 and over 65. The American Cancer Society(ACS), the American Society for Colposcopy and Cervical Pathology (ASCCP), the American Society for Clinical Pathology (ASCP) and the United States Preventive Task Force (USPSTF) recommend co-testing for screening women aged 30-65 every 5 years or testing with PAP test only, every 3 years (94).

2.3.3. HPV TESTING IN PRIMARY SCREENING

These methods are based on HPV-DNA detection and genotyping in vaginal or cervical smears.

In Europe, several randomized controlled trials showed that HPV-based screening is more sensitive in detecting CIN 2 and CIN3 in the first screening round thus making this approach a useful tool in primary screening. Rijkaart et al, in a population-based cohort study of 25,871 women age 29-61 years, (VUSA-Screen study) reported higher sensitivity of the hr-HPV testing vs cytology (91.9% vs 64.6%) for the detection of CIN3+. Thus, hr-HPV testing showed 3.1 % lower specificity for the detection of the CIN 3 compared to conventional cytology and 27.3 % higher sensitivity. However, false-positive results due to lower specificity of the HPV-testing can be managed with cytological triage and hence referring for colposcopy only HPV-positive women with abnormal PAP smear. In this study the CIN 3+ risk in negative hr-HPV women was very low (0.06%; 95% CI: 0.02-0.46 %) compared to women with normal cytology (0.26 %, 95% CI: 0.20-0.65 %) Also, a risk for CIN3+ was very low ( 5,22 %) for hr-HPV positive women with normal Pap smear compared to hr-HPV positive women with abnormal Pap smear ( 42.2%) thus supporting their conclusion that hr-HPV testing combined with cytology triage for HPV-positive women 30 or above 30 years of age, is efficient in identifying women with CIN3+ risk(95) .

In a large population-based randomized controlled trial (POBASCAM) with 5 years follow-up of the patients, final results showed that in the second screening round were all women were screened with co-testing, less CIN3 lesions were detected in the experimental arm (co-testing at baseline) compared to the control group (cytology at baseline) (RR 0.73, 95% CI 0.55-0.96; p=0.023) and also less cervical cancer in experimental arm compared to the control arm (RR 0.29, 0.10-0.87; p=0.031). This study also highlights that HPV-DNA testing can be started at 30 years since, in the two screening rounds no significant difference was observed between the control and experimental arm for the CIN 2 and CIN 3 detection rate for women aged 29-33 years and 34-56years (for CIN3 0.97 vs 0.95 and for CIN2 1.01 vs 1.11) (96)

Naucler and co-workers, performed a nested cross-sectional study within a population-based randomized controlled trial (Swedescreen) and included 6,257 women from the experimental arm age 32-38 years. The sensitivity of the HPV-based testing for the CIN3+ detection was 96.0 % (95% CI = 86.3% to 99.5%) and only 74.0 % for the cytology alone (95% CI = 59.7% to 85.4%).

In addition, co-testing with cytology and HPV-DNA testing compared with cytology alone showed increased sensitivity for the detection ofCIN2+ up to 40 % and up to 35 % increased sensitivity for the CIN3+ detection, while as reported, PPV was not significantly decreased (relative PPV = 0.76, 95% CI = 0.52 to 1.10) (97)

In a follow-up study of the four randomised controlled trials (NTCC, ARTISTIC, Swedescreen and POBASCAM) conducted by Ronco et al, although in the first 2.5 years no significant difference in cervical cancer detection rate was observed between the experimental arm (HPV- based) and control arm (cytology-based) after longer periodof follow-up (median 6.5 years) incidence of cervical cancer was significantly lower in experimental arm (RR, 0.45; 95% CI, 0.25-0.81) (98).

In 2011, the American Cancer Society, the American Society for Colposcopy and Cervical Pathology, and the American Society for Clinical Pathology recommended cytology and co­testing as primary screening alternatives, but hr-HPV testing as a primary screening test alone, was not recommended at that time.

In 2014, US FDA has approved for the first time the HPV testing as first line screening for cervical cancer for women 25 years and older, which was followed by debate and criticism among experts whether there is enough evidence to sustain changes in screening strategies.

A 2015 interim clinical guidance provided by Huh et co-workers regarding the hr-HPV inprimary screening after data review from several large trials recommends that hr-HPV testing mightbe considered as an option to cytology-based screening and co-testing, since negative hr-HPV women are at lower risk for CIN 3 (a lower detection rate of CIN 3) compared to those with normal Pap test (99). These recommendations were mainly based on the results from several RCTs conducted in Europe and also from the ATHENA trial. According to ATHENA -a large US prospective study which enrolled 47.208 subjects and evaluated performance of the primary HPV testing for CIN and cervical cancer, HPV primary testing in women >25 years is more sensitive for the CIN3+ detection compared with Pap testing and co-testing (100).

Meanwhile, appropriate age when to initiate hr-HPV screening is a very controversial issue although there is agreement between experts that HPV-based screening is not beneficial to young women, since HPV infections are highly prevalent and usually of a transient nature in this age group. In any case, severe lesions were detected more in the women 25-29 years than in women > 40 years (35.8% of CIN 2+ and 34.3% of CIN 3+ were found inthis age-group 25-29) (100).

Despite increased number of disease detection for this age group, at the same time increased number of colposcopy, patient anxiety and overtreatment of regressive lesions, make this approach quite unreasonable. Current guidelines recommend co-testing at 30 years of age while hr-HPV negative women should be re-screened every 3 years. HPV- negative women with ASCUS results should be followed with co-testing up to 3 years before returning to routine screening and as recently updated, with those results women cannot exit from the screening at age 65 years. HPV positive women with ASC-US result should be referred for colposcopy (94).

Regarding the clinical management of HPV-positive women there are several ongoing studies but the current recommendation based on the data from the ATHENA trial, support the algorithm according to which colposcopy is performed for HPV 16/18 positive women and reflex cytology for women tested positive for 12 other hr-HPV types with subsequent colposcopy if PAP test result is > ASC-US (100)

Recently, in a large retrospective study conducted by Zhou H and al, sensitivity of the cytology for the detection of the high grade lesions was reported to be 90.9 % (95% CI, 86.7%-94.1%) and only 91.3 % (95% CI, 87.1%-94.5%) for hr-HPV testing thus co-testing was proved as a better approach for detection of the severe cervical lesions (101).

Moreover, in a retrospective cross-sectional study performed by Blatt and al, 256.648 results (age-group 30 to 65) were reviewed and the conclusion was that co-testing is more sensitive for CIN 3 and cervical cancer detection (98.8 %) compared to positive cytology alone (91.3%) and positive HPV testing alone (94%) (102).

Based on these most recent data, co-testing is a more reliable alternative for CIN3+ and cervical cancer detection than either cytology or primary hr-HPV testing solely.

Some other visual inspection methods for cervical neoplasia screening include: visual inspection with acetic acid (VIA), visual inspection with Lugol's iodine (VILI), unaided visual inspection (VI) and colposcopy.

However, a policy of colposcopy examination after a single ASCUS result, in women of low socioeconomic status is supported in our Clinic.

2.3.4. MANAGEMENT OF CERVICAL PRECURSOR LESIONS - TREATMENT

Main purpose in the management of CIN lesions is certainly is to prevent progression of the disease while avoiding treatment of the low-grade lesions that can regress. Treatments options include ablative treatment (cryotherapy, laser vaporisation) and excisional treatment (cold knife conisation, LEEP and laser conisation). Both procedures have low morbidity rate but regarding the diagnostic sample, excisional methods perform better since they are not associated with thermal artefacts thus providing more appropriate specimen for histological examination. For women with CIN 1 and previous PAP results as LSIL or ASC-US and positive for HPV 16/18, co-testing at one yearis recommended. If previous Papsmear result is HSIL or ASC-H, then excisional procedure or observation with co-testing at 12 months and 24 months may be applied. Women with two years persistent CIN1 should undergo treatment with Loop Electrosurgical Excision Procedure (LEEP) (103). Suggested treatments for women with CIN 2/3 are LEEP or cold knife conisation, if the colposcopy is satisfactory and in cases with CIN 2/3 and non- satisfactory colposcopy, recurrent CIN2/3 or endocervical sampling with CIN2/3 than the recommended treatment is excision and not ablative methods (104). Ablative methods offer no information regarding the presence or absence of the disease in the margins of the treated zone. In meta-analyses of 29 randomized controlled trials (RCTs) which compared different surgical alternative treatments regarding the morbidity and treatmentfailure, authors concluded that none of the surgical technique was superior to other techniques regarding those criteria (105). However, regarding the future reproductive risks, excisional methods are followed by a higher rate of cervical stenosis, increased risk of second trimester pregnancy loss, increased risk of PPROM, and increased risk of preterm delivery and perinatal mortality (106-108).

For women with CIN2/3 who have completed childbearing and have residual disease after conisation the preferred route of treatment is hysterectomy (104).

Other treatment modalities like photodynamic therapy, topical agents and cyclooxygenase-2- inhibitors are under research (109-111).

2.3.5 FOLLOW-UP AFTER TREATMENT

Recommendations based on 2012 Updated Consensus Guidelines for the Management of Abnormal cervical cancer screening tests and Cancer Precursors, include co-testing at 12 months and 24 months for women treated for CIN2, CIN 3 and CIN2/3. After negative co-testing, then re-testing in three years is further recommended. Moreover, colposcopy with endocervical sampling are recommended if any of this tests are abnormal. Otherwise, for women with negative tests, routine screening is extended up to 20 years. Hysterectomy or repeated treatments are unacceptable based only on HPV positive testing as emphasised in this guidelines. Nevertheless, for women with recurrent or persistent CIN 2, CIN 3 or CIN2/3, repeated diagnostic excision or hysterectomy are acceptable (104).

2.4. HUMAN PAPILLOMAVIRUSES (HPVS)

2.4.1 Taxonomy, classification and genome organisation

Papillomaviruses (PVs) that belong to family Papillomaviridae, as proposed in 2004 by de Villiers and co-workers based on their nucleotide sequence identicalness, phylogenetic and pathologic properties, are small non-enveloped viruses with closed circular DNA genome (112). PVs are a large and dissimilar group of viruses that infect and have been detected in birds, mammals and reptiles. Phylogenetically, those viruses have evolved with humans, have tropism for species and genomic stability which is partly, because of absent intra and inter-type recombination (113). The taxonomy of the family is based on the L1 gene which is highly conserved and thus, newly identified HPV type is determined, if L1 gene sequence is a least 10 % diverse from any type which is already known (112). According to Bernard et al, Papillomaviridae family contains 29 genera which are created by 189 Papillomaviruses with 120 human types, 64 non-human mammalian types, 3 types from birds and 2 from reptiles (114).

Currently, there are 198 different Human papillomaviruses recognized, after withdrawn of four HPV types (HPV types 46, 55, 64 and 79) (115).About40 types infect anal and genital areain the both sexes (116).Human papillomaviruses belong to five genera-Alpha, Beta, Gamma, Mu and Nu. However, the most important species are considered alpha 9 and alpha 7 based on carcinogenic potential of types included in those species (a-9 with HPVs 16,35,31,52,67,33,58 and a-7 with HPVs 18,68,39,70,85,59,45,97)(117).HPVs can be further divided in mucosal and cutaneous types based on their tissue tropism. Thus, alpha papilomaviruses are divided in mucosal and cutaneous types. Low-risk cutaneous types infect basal epithelial cells and cause verruca vulgaris or common warts (types 1, 2, 4, 27, 57) andverruca plana or flat warts (types 3, 10) while high and low-risk mucosal types infect mouth, throat and anogenital epithelium. Regarding the oncogenic potential,they are classified in 14 high-risk types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66 and 68) based on their epidemiological relation with cervical cancer, assumed hr-types 26,53,67,70,73,82 and low-risk types 6,11,40 ,42,43,44,71 (118). Low-risk types (alpha 10 species) are associated with genital condylomata and laryngeal papillomatosis with type 6 being more frequent in genital condyloma and type 11 frequently foundin laryngeal papillomas (114). HPV 16 and 18 cause worldwide more than 70 % of cervical cancer, approximately 25 % up to 35 % of low-grade cervical lesions and more than 50 % of high-grade cervical lesions (119).Types from alpha 7 clade, especially type 18 are commonly found in adenocarcinomas (120).

2.4.2.HPV GENOME ORGANISATION

The small viral genome contains around 8,000 base-pairs (bp) and encodes eight or nine open reading frames (ORF). There are three regions identified, namely, the long control region (LCR) or upstream regulatory region (URR),early transcription region that comprise of the E1, E2, E4, E5, E6 and E7 open reading frames (ORF) and late transcription region which encodes for L1 and L2 capsid proteins . Even though, URR has no open reading frames, it contains binding sites for proteins E1 and E2 as well as enhancer and silencer sequences. While the promoter (p97) for the early genes is located in long control region, the late promoter (p670) is placed in the E7 region (121).

2.4.3. EARLY GENES E1, E2, E4-E7

E1 and E2 have a crucial role for the HPV genome replication hence, they are critical in the first genome amplification stage. In all papillomaviruses, replication genes (E1/E2) and wrapping genes (L1/L2) are highly conserved.

E1 is a specific-viral DNA helicase and in contrast to cellular helicase, it has ability to melt and unwind the DNA helix and it interacts with other replication factors (122).This viral helicase is efficiently recruited by E2 to its particular binding site or motif in the viral origin of replication (123)Therefore, replication begins after E2 binds to the LCR, recruits the E1-replication factor which further binds to its motif in the viral origin of replication (123).

E2 has various functions during the life cycle of the virus. In basal cells, E2 initiate viral replication and it is responsible for viral genome preservation (121). E2 is capable of modulating the viral gene expression through its numerous binding sites in the LCR so that, increased levels of E2 efficiently suppress E6/E7 expression (121). It also participates in viral transcription and ensures genome partitioning by binding the episomes or extrachromosomal viral genome with mitotic cell chromosomes, albeit binding target for a-PVs is not fully clarified. Full-length E2 gene product is polypeptide called E2 transactivator or E2-TA. It is important since by binding to specific sequences in E2 enhancers into the LCR, controls viral promoter activity (122). Integration of the viral genome randomly or more often at common fragile sites within the host chromosome willlead to loss or disruption of the E1, E2 and E4 gene function and subsequently unrestrained expression of E6 and E7 will occur (123). E2 has also pro-apoptotic activity and can induce apoptosis via p53-independent and through p53-dependent pathway in normal and in HPV infected cells(124-125).Thereis no E3 ORF identified in HPVs.

E4 and E5 facilitate viral genome amplification and L1/L2 expression. E4 is mainly important during the productive phase of the viral life cycle. E4 gene product E1A E4 is expressed in differentiating keratinocytes in upper layers and ensure infectivity by binding to cytoskeleton components and hence, disrupting keratin structure during the virion assembly and release(121). E5is ashort transmembrane protein (126) involved in maintaining a replication competent milieu in upper epithelial cells by enhancing EGF- mediated receptor signalling (127) and it is implicated inkoilocyte formation (127-128). During the early stages of viral infection, it supports replication and proliferation in basal cells through increasing mitogenic activity of the growth factor endothelin-1(129). It acts as a modulator of late viral functions, by promoting proliferation in mature cells and therefore is highly expressed in upper superficial layers (130).

It has a vital role in immunoevasion since interferes with antigen presentation and down-regulate expression of MHC class I genes (131). Furthermore, it can weaken the cell cycle control through inhibition of tumorsuppressor gene p21 (132). E5 anti-apoptotic activity is achieved by stimulating ubiquitin and proteasome-mediated degradation of the pro-apoptotic protein Bax (133).

E6 and E7 early HPV-oncoproteins have a crucial role in the progression of HPV-related disease through several molecular pathways.

E6-oncoprotein inactivates p53 gene product through E6/E6 associated protein(E6AP) complex, which catalysesp53 ubiquitination and hence targets it to the proteasome degradation. The p53 tumor suppressor gene product, which is important transcriptional regulator activated by DNA damage or stress, initiate DNA repair pathways, induce growth arrest and apoptosis. Importantly, more than 50% of human cancers bear mutation in this gene, therefore without p53 proper function; thecell is strongly determined to uncontrolled proliferation and cancer. All high-risk- HPVs contain a C-terminal PDZ binding domain which is mainly important since it is implicated in degradation of cellular targets that contain PDZ motifs and therefore actively regulate cell growth, attachment and cell polarity (134). Albeit, mechanism by which E6 activates catalytic subunit of telomerase (hTERT) is unknown, E6 contribute to an immortalized phenotype of the cells through telomerase up-regulation (135).

High-risk E6/E7 oncoproteins down-regulate the transcription of the toll-like receptor 9 gene (TLR9) and consequently inhibit TLR9 signalling pathways, thus efficiently evade innate immune response of the host (136). Immune response of the host is also modulated through E6- mediated decreased expression of interferon responsive genes, IFN-a and P, as well as nuclear STAT-1 protein down-regulation as reported by Nees et al, (137).

E6 interferes with extrinsic (ligand binding to corresponding death receptor and caspase activation) and intrinsic (via mitochondrial pro-apoptotic proteins) apoptotic pathways of p53- independent apoptosis through several apoptotic-related proteins like Bak, Bax, Myc and caspase 8. It can also up-regulate survivin promoter (138-139) There are also some indications that E6*, which is splice isoform might have a pro-apoptotic function (139).

E7 oncoprotein has numerous functions but primarily binds and inactivates retinoblastoma protein (Rb) (140) together with other associated proteins like p107, p130 and p105.These proteins, which bind to E2F, regulate transition from the G1 to S phase and therefore, disrupted association between the pRb and E2F transcription factors leads unequivocally to unrestrained S-phase progression and cellular proliferation. Inactivation of the Rb protein and activation of the E2F transcription factor transactivates cellular proteins like cyclin A and E necessary for viral replication. E7-mediated upregulation of cyclin and cyclin-dependent kinases (CDKs)is followed by increased cyclin-dependent kinase inhibitors (CDCIs) particularly, p16Ink4a which plays a role during the cell cycle progression by inhibiting the S-phase. p16Ink4aprevents E2F release and phosphorylation of Rbproteins (141). Another mechanism by which E7 protein causes cell cycle disruption, is through inhibition of the activity of cyclin-kinase inhibitors (CKIs) p21 and p27 (142-143).

E6 and E7 induce chromosomal instability due to centrosome amplification in proliferating cells, anaphase bridges, chromosome lagging, polyploidy and aneuploidy (144-145).

Regarding the effect of E7 in apoptosis, it is demonstrated that this protein acts as pro-apoptotic and anti-apoptotic factor depending on the cell type and virus type (139).

E7 inhibits TGF-P and tumour necrosis factor a (TNF- a) and furthermore interferes with IFN signalling via inhibition of IFN- a (146).

Finally, it plays a role in epigenetic programming via association with hystone deacetylases class I (HDACs)(146).

2.4.4. LATE PROTEINS L1/L2

L1, which is major capsid protein (55kD), is expressed in upper epithelial layers in mature differentiating keratinocytes during the late phase of the viral life cycle. L1 is capable of self­assembly into the virus-like particles(VLPs). VLPs are highly immunogenic thus; based in those properties VLP-based vaccines were developed and shown highly protective(147). Remarkably, L1 is responsible for viral entry mediated through its interactions with heparin sulphate proteoglycan on the basal membrane, which is a precondition for in vivo infection (148). L2 cleavage by furin protease is followed by virion binding to the secondary receptor on the keratinocyte with subsequent endocytosis and transfer to the nucleus(149-150).

L2 is a minor protein part of viral capsid. It is not markedly expressed in infected basal cells but has several roles like in a genome encapsidation during the virion assembly contributing to the virus infectivity and efficient packaging. After furin cleavage, L2 mediates viral genome transport into the nucleus and early gene expression for the establishment of infection(151).

2.4.5. HPV LIFE CYCLE

HPVs infect the basal epithelial cells through micro-abrasions of the epithelium. Two kinds of basal cells are present in the cervical basal layer, namely stem cells and transit amplifying cells (TA) Likely, HPV infects TA cells which pass through prophase of mitosis in order to initiate transcription of HPV genes (152). Despite the controversies regarding the second receptor, virions interact with heparin sulfat proteoglycan at the basal membrane and although not completely defined alpha 6 beta 4 integrin (153) and annexin A2 are possibly second receptors essential for efficient infection (154). TZ is fragile site for the cervical cancer formation most likely, because at these sites, hr- HPV have distorted gene expression during a productive infection (121).Thus, squamous cells of the transformation zone and even columnar or reserve cells have been defined as HPV target for long time, but recently Herfs et al, reported a cuboidal cells localized at squamocolumnar junction which present with unique morphologic features and gene expression profile, cells that do not regenerate after excision and are more prone to cancer progression after infection (155).Viral life cycle is exclusively dependent on the differentiation of the host epithelial cells. HPV life cycle goes through establishment, maintenance and amplification or productive phase. In establishment phase, after infection, E1/E2 gene expression is mandatory to maintain low copy number of episome, usually up to 200 copies per cell in this initial genome amplification stage in basal cells (123). Unlike, indecisive role of E6/E7 oncoproteins in basal cells, high-risk E6/E67 have a main role in cell proliferation and virion productivity in basal and suprabasal layers (121,123). E6/E7 are necessary for the re-entry of the infected cells in S-phase, although even E4/E5 contribute to genome amplification. E5 can modulate late viral functions by promoting proliferation in mature cells in upper epithelial layers (123). Productive phase is thus, characterised by genome amplification to a high copy number in differentiating keratinocytes and finally, expression of the major and minor capsid protein is mandatory for cell cycle exit, viral genome packaging and virus release in upper epithelial layers and thus propagation of the infection to the new host (128).

2.4.6. EPIDEMIOLOGY OF THE HPV INFECTION AND ITS ROLE IN MALIGNANT

TRANSFORMATION

HPV infection is one of the most common sexually transmitted diseases in the world with different incidence and prevalence dependent on geographical area and population. Most HPV infections are transient and occur after beginning of the sexual life with remarkably highest prevalence for the age group 25-35 years (156). Dunne et al. found that while the overall prevalence of HPV infection, including both low-risk and high-risk types, in US women between the ages of 14 and 59 years is 26.8% in a population of 1921 patients, the prevalence of high-risk (HR) HPVs is only 15.2% (157). The majority of HPV infections tend to clear or regress spontaneously after a few months or two years (158-160). IfHPV infection cannot be detected anymore-using sensitive tests methods than usually is assumed that infection is cleared although, latent state of this infection is poorly understood. Women infected with high-risk genotypes and persistent infection have increased risk of developing severe cervical lesions and cervical cancer (161). There are some data that HPV-16 has a longer persistence compared to other types (162).

Generally, cervical carcinogenesis includes several steps beginning with the persistent chronic infection with one or more oncogenic HPV types, clonal progression of the infected cells and invasion. Viral type, high virus load and infection with several oncogenic types, plays a crucial role in the viral persistence rather than clearance and hence progression of the disease. Risk of progression from precursor lesions to cervical cancer is decidedly related to HPV genotype (163­164). E6/E7oncoproteins through cell cycle disruption, genomic instability and consequent “mutator'' phenotype of the cell, have ability to strongly drive the cell to malignant transformation (165). After infection through methylation, viral expression might be suppressed with episome maintenance in basal layers. In CIN 1 lesions, HPV life cycle is completed. (123). In CIN2 and CIN3 lesions viral expression deregulation is present, which leads further to viral genome integration into the host chromosome with consequent E6/E7 overexpression and development of cervical cancer. Interestingly, 70 % of cervical cancer caused by HPV16 has integrated sequences and 30 % contains only episomes (123,166). Since the main aim of the screening programmes is identifying women at higher risk for cervical disease, several molecular markers can be analysed including E6/E7 mRNA, HPV sequencing, viral genome integration into the host chromosome and viral load.

2.4.7. HOST IMMUNE RESPONSE AND HPV INFECTION

2.4.7.1 The innate immune response

HPV clearance and eradication of HPV-infected cells and malignant transformed cells depends on the innate and adaptive effectors of the host immune system. Immune response comprises of innate and adaptive phases. The innate immune system which is a first line of defence comprises complement, lysozyme, natural killer cells (NK), macrophages and dendritic cells (DCs) as cellular and non-cellular effectors while the adaptive immune system executes its function through antibodies produced from B cells, cytokines and cytolytic molecules producedby T cells (167). DCs which are positioned in mucosa and epithelium after maturation in antigen-presenting cell (APCs) and expressing highly antigen/MHC complexes, activate naive T cells and through stimulatory signals promote T cell-mediated immune response and also activate B lymphocytes. This is the reason way innate and adaptive immune response are bridged by the APCs actions. Human a-defensins are innate peptides with antibacterial and antiviral action found in cervicovaginal secretions. A study conducted by Buck et al, revealed blocking effect of human a-defensins 1-3 and human a-defensin 5 on HPVs while human P-defensins1 and 2 showed no activity against HPVs (168).

Innate immune system detects the pathogens locally through transmembrane Toll-like receptors (TLRs) and therefore increased expression of TLR 2,3,7,8 and 9 was associated with HPV 16 viral clearance unlike viral persistence (169). In this line, Imiquimod or Aldara, which is TLR7 agonist, was shown effective for the treatment of genital warts, vulvar and vaginal intraepithelial neoplasia treatment (170).DCs, macrophages and T cells produce small proteins, which are called cytokines. They include interleukins, interferons and growth factors and are classified as pro-inflammatory or anti-inflammatory, which depends on their role during the immune response (167). High levels of pro-inflammatory cytokines such as IL-1 a, IL-1 P and IL-8 and low levels of anti-inflammatory mediators were found in women with CIN1 and CIN3 lesions thus showing that altered mucosal immunity in hr-HPV infected subjects can support viral persistence(171).

2.4.7.2 The adaptive immune response

Adaptive immunity which is second line of defence against pathogens is triggered by exposure to the specific antigen, acts through antigen-specific effectors and is characterised by immunological memory (167). CD4+ T cells or helper T cells (Th) are initially activated during immune adaptive response and act via cytokine production while helping other immune cells during the immunological response. Furthermore, CD4+ T cells are facilitators and CD8+ T cells are adaptive effectors during adaptive immune response. CD8+ T cells are capable to inhibit viral replication and limit pathogen spreading via their cytotoxic molecules. Numerous sub-sets of T helper cells are recognized as described below (167).

Th1 cells produce interferon -gamma(IFNY) which is important in pathogen clearance since plays a role in pathogen spreading and survival. TNFa are also produced by Th1 (167).

Th2cells produce interleukins IL-4, IL-5, IL-13 while IL-21which is involved in antibody production from the B cells, is produced by follicular T helper cells (Tfh) (167).

In general, hr-HPV evades host immune response through modulation of the T cell activity. A study from Peghini et al, found higher levels of Th1 cytokines for low-grade lesions and higher levels of Th2 in severe lesions while invasive cancer was associated with expression of Treg cells (172).

Regulatory T cells (T reg cells) which belong to CD4+ T-cell subcategory have central role in the suppression of the immune response and immunological tolerance (167). PD-1 and PD-L1 interactions inhibit T cell-mediated immunity hence; regulate peripheral immunological tolerance (173). Programmed cell death receptor (PD-1) or CD 279 and PD-L1 (programmed death ligand-1) or CD 274 belong to B7 family of co-stimulatory proteins and as already reported up-regulation of PD1/PD-L1pathway might result in viral persistence and can contribute to progression of hr-HPV-related lesions (173). This way, Yang et al, found increased expression of PD-1 on T cells and increased expression of PD-L1 on DCs in hr-HPV positive women with different CIN lesions (174).

B cell activation and differentiation is dependent on CD4+ cells, which differentiate in Tfh cells in order to help B cells to perform their task for antibody secretion. Although there is active secretion of IgA isotype in cervical mucus, (175) several studies found higher level of IgG compared to IgA in cervical secretions. Accurate source of cervical immunoglobulins is not clearly defined but local production, passive transfer and transudation from blood in HPV- vaccinated subjects are likely mechanisms. Safaeian and co-workers found no correlation when comparing total serum and cervical IgG and IgAs and no effect of hormones or menstrual cycle in serum levels of IgG and IgAs. (176).

Concerning humoral immunity executed by neutralising antibodies after HPV infection, Mbulawa et al, found a correlation between HPV-16 infection and serum HPV-16 neutralising antibodies while no significant correlation between HPV-16 infection and cervical antibodies was found and moreover, no correlation between cervical neutralising antibodies and disease severity was found. (177). A study from Passmore et al, which evaluated correlation between oral, cervical and serum HPV-16 antibody response, found significant increase in magnitude of HPV16 specific IgAs in women with CIN 2/3 compared to women with CIN1. Importantly, study pointed out that no immune linkage was found between mucosal compartments as well as between mucosal (cervical and oral) and serum or systemic compartments (178).

2.5. EVASION MECHANISM OF HPV

HPVs have evolved several mechanisms to avoid immune surveillance of sophisticated innate and adaptive effectors of the host immune machinery to ensure their survival, replication, dissemination and persistence. Infection and low viral expression is limited into the basal keratinocytes. T-cell mediated immune response with is crucial for HPV clearance is efficiently inhibited by E7 and E5 since they downregulate MHC class I/II complexes, thereby inhibit presentation of the viral peptides to the immune system (179-180). HPVs are non-lytic viruses with intraepithelial life cycle. They do not initiate inflammatory response and that is the reason why dendritic cell (DC) active migration to assure pro-inflammatory local microenvironment is not achieved in the early phases after infection (181). Due to E6 activity in E-cadherin mediated adhesion at the infected site number of the antigen-presenting cells or Langerhans cells is markedly reduced which further disable T-cell cytotoxic activity (182). There is no blood-borne phase and no viraemia, which further limits detection of the viral antigens bythe host immune system and impairs serological response (183). High-risk HPVs interfere with IFN pathway via down-regulation of IFN-a and IFN-P expression and in this manner disrupt antiviral, antiproliferative and antiangiogenic functions of the interferons (137). Other evasionmechanisms include cytokine and chemo-attractants inhibition, modulation of the intracellular signalling and

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