keratinocytes (Demeret et al., 1997). In addition to modulating viral gene expression, HPV E2 proteins associate with the viral DNA helicase E1. This interaction ...
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4. Discussion The prevalence of Human Papillomavirus (HPV) has not been well studied in Iraq especially in Anbar Governorate. The present study is the pioneer study, which utilized Real-Time Polymerase Chain Reaction (PCR) technique as molecular tool for the detection of low and high -risk genotype; Human Papillomavirus (HPV). In addition, to detect of low and high -risk genotype Human Papillomavirus (HPV) in fixed formalinparaffin embedded (FFPE) tissues for patients diagnosed with cervical cancer previously.
4.1 Human Papilloma Virus infection according to age Regarding the increased rate of HPV infection in correlation to age group. (Carestiato et al., 2006) revealed in their study that the highest rate was among the age group of 21-30 years whereas, Paulo and associates, 2007 reported that the rate was raised in 18-39 years age range population. On the other hand, Eslami and co-workers, 2008 documented that the highest rate was among cases with the age range of 35 - 44 years. However, Rama and coworkers., 2008 reported that the highest prevalence of HPV infection was in women under 25 years, then the rate declines after the age of 25 and remains below low (5.0%) after the age of 55 years and they suggest that this decreasing rates are unrelated to sexual practices, but it seems to be more related to the development of a typespecific immune response to HPV infection. Chan and colleagues, 2009 also observed that the second minor peak of HPV prevalence in women aged 46-55. This dissimilarity represents the variation in incidence and prevalence of genital HPV infections in the different geographical area. In addition to several other factors such as the study design, the sensitivity of virus detection test, 74
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virus subtypes studied and sexual behaviors. Superior to these aforementioned factors, adopting and the effectiveness of cervical cancer screening programs for the diagnosis and treatment of women with HPVinduced cervical lesions (Speich et al., 2004). The highest rates occur in sexually active women among 18-28 year-olds (Koutsky, 1997). The estimated prevalence of HPV infection in the Hungarian female population is approximately 17.6%, with the highest rates occur in sexually active women among 17-22 year - olds (Kornya et al., 2002). The result obtained from the present study showed that high rate of HPV infection at the age group 29-38 and an older age group 49-58 years. This result is inconsistent with the reported high prevalence rates of cervical cancer among older ages as reported by (Chang et al., 1997), who clarified that the patients with cervical cancer are significantly older than patients with normal cervical histology or those with squamous intraepithelial lesion (SIL). Most of the studies suggested that above 35 years of age HPV infection might represent viral persistence, probably caused by oncogenic HPV and induced the risk of cervical cancer development. In other study by (Dunne et al., 2007), Human Papilloma Virus prevalence was 24.5 % among females ages 14-19 years, 44.8% among women ages 20-24 years, 27.4% among women ages 25-29 years, 27.5% among women ages 30-39 years, 25.2% among women ages 40- 49 years, and 19.6% among women ages between 50 to 59 years. There was a statistically significance trend for increasing HPV prevalence with each year of age from 14 to 24 years P value < 0.001 followed by a gradual decline in prevalence through 59 years (P = 0.06). Al-Shabanah and 75
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coworker., 2013, find that the patient’s mean age was 50 ± 11 years (range, 25-78 years). The patients’ age distribution was analyzed and the prevalence of HPV genotypes was detected among them. There was no significant difference (P > 0.5) observed in HPV infection among patients with age < 45.0 year compared with more than 45 years old, in which 15/35 (42.8 %) of the patients with age < 45 years old and 27/65 (41.5%) were positive for HPV. Regarding HPV infection versus histological grade, HPV was detected in 25 % (7/28) of cases with grade I, 50 % (21/42) of cases with grade II and 46.7% (14/30) of cases with grade III.
4.2 Cervical lesion findings according to age Warts (benign skin papilloma) are common, benign skin tumors. Malignant genital warts contained HPV-6 or -11 DNA five of 27 cervical cancer biopsies were examined, contained HPV-11. Similar findings were made by other groups. While, this evidence was suggested a role for HPV in cervical cancer, the low-risk HPV types (mainly 6 and 11) may cause clinically apparent, benign genital warts, the most recognizable signs of genital HPV infection in less than 10% of all infections (Marek, 2013). The mean age and standard deviation for the fixed formalinparaffin blokes (50.00 ±9.64) for Adenocarcinoma (ADCA), (57.17 ±7.41) for cervical carcinoma was type (44.36 ±5.35) a type of High grade squamous intraepithelial lesions (HSIL). These results were in agreement with the findings of other researcher, who stated that patients with cervical malignant lesions were in majority with the age ranging from 50-65 years (Maleknejad et al., 2006). Moreover, it has been suggested that the reason for this post-menopausal peak incidence of cervical cancer may be related to reactivation of latent HPV infection acquired at earlier life and progress with gradual loss of type-specific
76
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immunity with aging or to a sudden loss of hormonal influences during post -menopausal years (Dutra et al., 2008).
4.3
Risk co-factors
and
Human
Papillomavirus
infection HPV infection is a necessary cause of cervical cancer, but it is not sufficient cause. Other co-factors are necessary for progression from cervical HPV infection to cancer which are sexual behavior, particularly the number of sexual partners during lifetime has been identified as a significant risk factor for acquiring HPV infections and developing genital cancer as well as the early age at first intercourse. In addition, cigarette smoking, high parity and co-infection with other STDs (e.g. Chlamydia trachomatis and Herpes Simplex Virus type-2, HIV) have been also identified as cofactors (Munoz et al., 2006; Flores et al., 2008).
4.3.1 Contraceptives A non-significant statistical correlation was found between the types of contraceptive and the incidence of Human Papillomavirus DNA positive among the studied patients (Table 3.5). Green and associates., 2003 suggest that the prolonged use of oral contraceptives is associated with an increased incidence of cervical ectropion changes (the site where HPV infection preferentially induces neoplastic lesions) at the SCJ (squamocolumnar junction). This site is more prone and exposed to potential carcinogens effects of estrogen and progestogens and increasing cell proliferation and stimulating the transcription of HPVs. The study could conclude that no significant differences were observed between the types of contraceptive and the incidence of Human Papillomavirus DNA positive. This could
77
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attribute to many factors, some of these the limited period of usage among the majority of women, second the fluctuation in period of usage between pregnancies as method control posterity indifference with other community either aiming to have no offspring or limited numbers. In addition, there may be jumping and intermingling from one contraceptive-style to another. Shepherd and colleagues, (2000) considered 10 studies of different duration providing evidence for a positive effect on sexual risk reduction, typically with an increased use of condoms for vaginal intercourse, while a majority of the experimental group (68.1%) had learned that the use of a condom does not give them full protection from HPV infection. Since it is the generally accepted view of the scientific community, that although condoms may not give 100% protection, still, their use is highly recommended as they may reduce the risk of contracting HPV infection, also protect from other STDs as well as they may prevent undesired pregnancy. Thus, promoting the use of a condom during HPV education could, therefore, provide an opportunity to educate about a range of sexual health issues (Kollar and Kahn, 2008; Kwan et al., 2011). Cervical cancer risk is 45% lower in women who have ever used an intrauterine device (IUD) versus never-users, a meta-analysis showed; IUD use may reduce the risk of HPV progression to cervical cancer (Castellsagué et al., 2011). Longatto-Filho and colleagues (2011), also observed that there is no evidence referring for association between the use of hormonal contraception (HOC-oral, injections, patches, implants, the vaginal ring and progesterone intrauterine system) with an increased risk for High Risk - HPV infection or high - grade CIN. Further, Marks and associates, 78
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(2011) reported that the women diagnosed with cervical cancer had longer duration and more recent use of combined oral contraceptives (COCs). It is unclear whether COC use is associated with the upstream events of human papillomavirus (HPV) infection prior to the development of clinical disease.
4.3.2 Education level A non-significant association was concluded regarding the level of education and HPV infection in our study as reflected in Table 3.6. This finding is in agreement with other major studies that are not found any correlation between HPV cervical cancer and the educational level among the studied cases (Balakrishnan et al., 2006). In contrast to our result, Marais and coworkers (2008) founded that there was significant difference between of cervical HPV infection and higher education levels. Education level was inversely and consistently associated with cervical cancer risk, but not with HPV prevalence, in a broad range of world populations included in the IARC studies (Franceschi et al., 2009).
4.4 Molecular Analysis 4.4.1 Genomic DNA extraction Spectrophotometer measurements revealed that the differences in DNA concentration and purity according to the origin of tissue. The OD260/OD280 ratio values satisfied those suggested by (Sambrook et al., 2004), ≥ 1.8, the variability in DNA quality and purity can be explained by tissue -specific structural complexity (Biase et al., 2005). The DNA concentration was determined by optical density 79
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measurement in a spectrophotometer using 50µg/mL as 1 OD260. The 260/280 ratio should be in the range of 1.7-1.9 and should not exceed 2 as this indicates the contamination of the preparation of RNA or low molecular weight nucleic acids that lead to overestimation of DNA concentration (WHO, 2010). The result of this study in Fixed Formalin Paraffin Embedded tissue extraction of DNA may be due to the fact that formalin is Known to fix protein in tissue samples including all proteins on each cellular level which makes the reach for the DNA difficult and incomplete (Sambrook et al., 2004). In addition, it is unsuitable for molecular techniques a slow degradation of DNA occurring with time. Al-Khalidi, (2006) demonstrated in her thesis disagreement with our result regarding DNA extraction. Other study mentioned that formalin preservation does not break up DNA but in fact may preserve it more completely. Despite the use of traditional protocols, kits for genomic DNA extraction are becoming very popular, mostly because they are ready to use and contain non-toxic reagents, a genomic DNA isolation reagent which uses a guanidine-detergent lysing solution for selective precipitation of DNA from cell lysate. It is recommended for purification of DNA from solid and liquid samples from animal, yeast, bacteria and plant sources. An advantage of the solution is to allow samples to be stored for longer periods of time without compromising DNA integrity. It also produced good DNA suitable for HPV detection and identification. The method is simple, fast and generates DNA of high molecular weight. Another advantage is that no griding is necessary to obtain sufficient 80
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DNA (Wanderlei-Silva et al., 2005).
4.4.2 Real Time PCR for detection of Low-Risk human papilloma virus (6 and 11) analysis Genital warts are the most frequent benign tumors in the anogenital
region
of
both
males
and
females. Human
papillomaviruses are etiologically associated with the development of virtually all Genital warts. HPV-6 and HPV-11 are the most commonly detected Human papillomavirus genotypes but at least 20 other alphaHPV genotypes have occasionally been found in Genital warts tissue specimens (Kocjan et al., 2007). HPV types are non-oncogenic, or low-risk HPV types, such as HPV 6 or 11, can be caused that (1) benign or low-grade abnormalities of cervical cells, (2) anogenital warts, and (3) a disease of the respiratory tract called recurrent respiratory papillomatosis (RRP) (Lacey et al., 2006; Giuliano et al., 2007). Dunne and associates, 2007 reported the prevalence for Low-Risk HPV 6 1.3% while HPV11 was 0.1% Manhart and associates, (2006) documented that in a populationbased assessment of sexually active 18- to 25-year-old women using a urine sample found the prevalence of HPV-6 2.2% and HPV-11 to be 1.0%. These differences may be due to differences in the type of test used for the HPV detection, differences in the study population, or both. The variables associated with HPV DNA detection that was significant in the bivariate analysis were age, race, poverty index, education, marital status, and sexual behavior (Dunne et al., 2007).
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Chapter Four 4.4.3
Quantitative
Discussion detection
of
High-Risk
Human
Papillomavirus (16, 18, 31, 33, 35, 39, 45, 52, 58 and 59). Eluf-Neto and colleagues in (1994) reported that the most common high-risk types HPVs are 16, 18, 31, 33, 35, 51, and 52, which have been associated with CIN II, CIN III and invasive cervical cancer. While the other study in (2005) by Wanderlei-Silva and associates in preliminary data shows that the predominant types in the State of Alagoas are HPV 16 and HPV 33, both of them presenting high oncogenic potential. The investigation further supports the role of these high-risk types HPVs in the development of cervical neoplasia. Al-Shabanah and co-workers, (2013) report that in their study HPV-16 was yielded in 18/42 (42.9%), which was the most predominant genotype followed by HPV-18 in 11out of 42 (26.2%), followed by HPV45 in 7.1%. HPVs have classified a high and low risk, according to their relationship with benign or malignant proliferative lesions (Varsani et al., 2003). The oncogenic activity of high-risk HPV types occurred when they integrated into the host genome. In many studies, molecular assays were used to identify different types of HPV in cells and tissues (Kosel et al., 2003). Only one of the two strands of the circular papillomavirus DNA genome is passively transcribed. The genome can be divided into three major portions: a -4-kb early (E) region that encodes nonstructural proteins, a -3-kb late (L) region that encodes the two capsid proteins, and a -1-kb noncoding long control region (LCR) that contains a variety of cis elements, which regulate viral replication and gene expression. E and L
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genes are numbered according to size; the higher the number, the smaller the corresponding open reading frame (Mu¨nger et al., 2004). The papillomavirus life cycle is tightly linked to the differentiation program of the infected epithelium. Papillomaviruses initially infect basal epithelial cells, which constitute the only cell layer in an epithelium that is actively dividing. The nature of the HPV receptor(s) remains unclear, although integrin -4-6 has been implicated (Evander et al., 1997). Similarly, the processes that mediate virus uptake, decapsidation, and nuclear import of the viral genome remain largely unknown. The viral DNA is maintained at a low copy number in the nuclei of infected host cells as they undergo differentiation and move toward the surface of the epithelium. In terminally differentiated cells, the virus replicates to a high copy number, late genes are expressed, and progeny virus is produced. HPVs are non-lytic viruses, and progeny virus is shed into the environment as a cargo within epithelial squamous. The HPV E4 protein associates with keratin intermediate filaments, which affects the mechanical stability of the keratin network and may facilitate the release of viral particles. The papillomavirus E1 and E2 proteins each plays important roles in viral genome replication. E2 is a DNA binding transcription factor that interacts with ACCN6GGT motifs in the viral LCR. High-risk HPV E2 proteins have the capacity to act as transcriptional activators, but they function as transcriptional repressors of viral gene expression in keratinocytes (Demeret et al., 1997). In addition to modulating viral gene expression, HPV E2 proteins associate with the viral DNA helicase E1. This interaction is necessary for efficient
origin
recognition 83
and
viral
genome
replication.
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Discussion
Papillomavirus E2 proteins cell division by tethering viral genomes to mitotic chromosomes. The association of E2 with mitotic chromosomes is mediated by interaction with the human bromodomain protein Brd4. Since HPVs do not encode other enzymes that are rate limiting for DNA replication, production of viral genomes is critically dependent on the host cellular DNA synthesis machinery. Papillomaviruses are replicated in differentiated squamous epithelial cells that are growth arrested and thus intrinsically incompetent to support genome synthesis. Hence, HPVs encode functions that create and/or maintain a replication competent cellular milieu in infected differentiated keratinocytes (Mu¨nger et al., 2004). The relatively low prevalence of HPV 16 and 18 is also reported in several studies. The study has been done in Saudi Arabia, the most prevalent types were HPV68/73 followed by HPV 6 and 18 (Al-Obaid et al., 2014). In several studies, HPV 16 is the leading type but HPV 18 does not come right in the second place. In China, HPV 16 is followed by HPV 52 and 58 (Wang et al., 2015). HPV 16 is followed by HPV 51 and 53 in Australia but it is followed by HPV 58 and 18 in Tunisia (Tabrizi et al., 2014). All this data shows the low prevalence of HPV 16 and 18 in women with normal cytology in comparison to cervical cancer cases. In effect, around 70% of cervical cancer cases are associated with these two genotypes (Khair et al., 2009; IARC, 2012). The logistical regression models show clearly that HPV infections are responsible for cervical abnormalities. An increased number of pregnancies and menopause are both significantly related to HPV infections and cervical abnormalities. Most of the women (93.3%) in the present study have only a one-lifetime sexual partner but women who had two or more lifetime sexual partners have an increased risk of HPV infection. However, the lifetime number of sexual partners was not 84
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associated with cellular abnormalities. Therefore, this result shows that women have two or more sexual partners increasing the risk of HPV infection but it is not responsible for HPV persistence and cellular transformation. Cellular lesions result from HPV persistence after escaping from the immune system which is related to the hormonal balance. That pregnancy and menopause are responsible for many psychological, physiological and hormonal changes that can weaken the immune system. The reduced immunity dose not only allow HPV infection but also constitutes a favorable medium for HPV persistence and cellular lesions appearance (Souho et al., 2016). 4.4.4 Real Time PCR for quantitative detection of High-Risk Human Papillomavirus (16, 18, 31, 33, 35, 39, 45, 52, 58 and 59) in fixed formalin -paraffin embedded samples The extracted DNA from formalin-fixed and paraffin embedded (FFPE) tissue remains a real challenge, despite numerous attempts to develop more effective method. Polymerase chain reaction (PCR) success rates with DNA extracted using current methods remain low (Lin J et al., 2009). Matsukura and Sugase, (2001) documented the HPV 16 was the most frequently identified type among CIN II/III tissues. This result is in agreement with the results of the previous studies. The significantly uneven distribution of HPV 16 prevalence between CIN grades supports the contention that CIN II/III lesions can arise de novo and/or that HPV 16-infected CIN I may rapidly progress to high grade.
Ahmed, 2008 reported that HPV 16 and 18, HPV 16 was identified in 57% of the positives were comparable or in agreement with those reported before (Clifford et al., 2003) detect that HPV16 in 46-63% of 85
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study specimens, HPV 16 was yielded in 50% in the study laid down by Zur Housen, 1987, 6% by (Stanczuk et al., 2003) in of the cervix squamous cell carcinomas. While the result of HPV 18 was identified in 29% of the positives, which is more than that reported by (Clifford et al., 2003) (10-14%) and Zur Housen, (1987) (20%). However, HPV18 was not detected in a study in Iran (Farjadian et al., 2003). Accordingly, HPV16 is the most common HPV type associated with cervical carcinoma in Central Sudan (Ahmed, 2008). Practically, formalin fixation may cause extensive DNA damage, including cross-linking and fragmentation. Consequently, it has been reported that successful amplification of HPV sequences from archival FFPE specimens is inversely correlated to the length of the amplicon of the PCR method and that specimen age may contribute to degradation (Baay et al., 1996). Furthermore, differences in sample processing and DNA extraction of FFPE materials may explain discrepancies observed in the performance of specific genotyping methods for FFPE specimens (Dona et al., 2013). Several recent reports suggest that the use of robust extraction methods can improve the performance of nucleic acid tests when applied to fixed specimen types. Regarding to the result high risk genotype, the result of the two cervical lesion and FFPE was variables. Reflected that there is no significant difference. Statistically, this may be attributed to the fact that the mechanism X2 parameter will delete a square labeled zero.
86
