**2. Etiopathogenesis of HPV**

About 189 HPV genotypes have been sequence and classified according to their biological niche, oncogenic potential and phylogenetic position [25]. From them, about 40 can infect the genital tract [26]. HPV types are classified based on their association with cervical cancer and precursor lesion into low-risk types (**LR-HPV),** which are found mainly in genital warts, highrisk types (**HR-HPV)**, which are frequently associated with invasive cervical cancer and

4 Human Papillomavirus and Related Diseases – From Bench to Bedside A Diagnostic and Preventive Perspective

Worldwide, HPV-16 is the most common HPV type across the spectrum of HPV related cervical lesions. In women with ICC (invasive cervical cancer), the most common HPV types are HPV-16,18,33,45,31 and 58 [30, 31], but among these genotypes, certain variants have linked to different clinical outcomes. It is now generally accepted that HPV has co-existed with its human host over a very long period of time and has evolved into multiple evolutionary lineages [25, 32]. Intratypic variants of HPV16 have been identified from different geographic locations and are classified according to their host ethnic groups as European (including prototypes and Asian types), Asian American, African and North American [33]. Through epidemiological and in-vitro experimental studies, natural variants of HPV16 have shown substantial differences in pathogenicity, immunogenicity and tumorigenicity. IARC Study [34] and IARC Meta-analysis [31] are very robust in identifying that HPV-16 and 18 contibute approximately 70% of all ICC. HPV-16,18 and 45 are the three most relevant types in cervical adenocarcinoma [30]. The geographical variation in type distribution is of minor significance

Among men and women, cancers of the ano-genital tract and their precursor lesions have been strongly linked to infection with sexually transmited human papillomavirus. In men, HPV infection has been strongly associated with anal cancer and is associated with approximately 85% of the anal squamous cell cancers that accur annually worldwide. Likewise, approxi‐ mately 50% of cancers of penis have been associtated to HPV infection [35]. Genital warts are a common sexually transmitted condition with an estimated prevalence of 1-2% of young adults [36]. Although having genital warts is not associated with mortality, represent a significant public health problem (clinical symptoms and psychosocial problems) and healthcare costs for society [37-39]. More than 90% of genital warts are related to HPV-6 and 11 (low risk genotypes) in general these types are not associated with malignant lesions, however 20-50% of these also contained coinfection with oncogenic HPV types [39-41].

On the other hand, between 33-72% of oropharyngeal cancers, and 10% of cancer of the larynx

**Risk category HPV types** High-risk 16,18,31,33,35,39,45,51,52,56,58,59,68,73,82 Low-risk 6,11,40,42,43,44,54, 61,70,72, 81, 83, 89

Undetermined risk 26,53,66

**Table 1.** HPV types classification according their oncogenic potential

may be attributed to HPV infection [42-44].

variation.

undetermined risk types (table 1) [27, 28, 29].

The HPV virion has a double-stranded, circular DNA genome of approximately 7900bp, with eight overlapping open reading frames, comprising early (E), and late (L) genes and an untranslated long control region, within an icosahedral capsid. The L1 and L2 genes encode the mayor and minor capsid proteins. The capsid contains 72 pentamers of L1, and a pproxi‐ mately 12 molecules of L2. The early genes regulate viral replication and some have transfor‐ mation potential. Late genes L6 and L7 code for structural capsid proteins which encapsidate the viral genome. (Figure 1).

**Figure 1.** Organization of the HPV genome. Adapted from Doorbar J. [45]

Infection by papillomaviruses requires that virus particles gain access to the epithelial basal layer and enter the dividing basal cells. Having entered the epithelial tissues, the HPV virus enters the nucleus of a basal epithelial cell, where early genes E1 and E2 are expressed, replicating the viral genome and transcribing messenger RNA needed for viral replication; in addition to its role in replication and genome segregation, E2 can also act as a transcription factor and can regulate the viral early promoter and control expression of the viral oncogenes (E6 and E7). At low levels, E2 acts as a transcriptional activator, whereas at high levels E2 represses oncogene expression [45]. As the host cells differentiate, genes E4 and E5 assist in the production of the viral genome by controlling epidermal growth factor. E6 and E7 are viral oncogenes which now become important. E6 causes degradation of the tumour suppressor gene p53, while E7 completes for retinoblastoma protein (pRb), allowing the transcription factor E2F to drive cell proliferation processes. The p16 protein, encoded by the suppressor gene CDKN2A (MTS1, INK4A) at chromosome 9p21, is an inhibitor of cyclin dependent kinases (cdk)which slows cell cycle by inactivating the function of the complex-cdk4 and cdk6 cyclin D. These complexes regulate the control point of the G1 phase of the cell cycle with subsequent phosphorylation and inactivation of retinoblastoma (pRb), which E2F released and which allows cells to enter S phase. It has been demonstrated existence of a correlation between pRb and p16 reciprocal, which is why there a strong overexpression of p16 both in carcinomas as in lesions premalignant cervix. In cervical cancer, pRb is functionally inactivated from the initial stages of cervical carcinogenesis as a consequence of expression of HPV E7 gene. Genes E6 and E7 therefore act to remove two principle mechanisms of cell defence, and drive the cell replication machinery towards production of new virus particles. E6 and E7 are also known to promote oncogenesis. [45]

and histology, but two limitations of the Pap smear exist: low specificity leading to the need for repeat screening at relatively short intervals and cervical cancer screening, based on Pap smear, remains beyond the economic resources of nation in developing world. This econom‐ ic disparity has meant that cervical cancer incidence and mortality rates in the developing world have remained high, with large reductions in these rates being limited primarily to the industrialized world. Thus, the reduction of cervical cancer in developing nations remains an unmet need of high priority. Since the link between HPV and cervical cancer is known and numerous large scale studies have been done, molecular methods to detect HPV DNA in clinical specimens (vaginal, urethral, paraurethral, anal or pharyngeal exudates, biop‐ sies, and, especially, endocervical exudates) have been introduced into screening algorithms.

Molecular Diagnosis of Human Papillomavirus Infections

http://dx.doi.org/10.5772/55706

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Increased sensitivity has important clinical outcomes because reduce mortality and an elongation of screening, and implies better compliance with screening and lower cost [47]. An Italian study showed that HPV-based screening is more effective than cytology in preventing invasive cervical cancer, by detecting persistent high-grade lesions earlier and providing

HPV serves as paradigm for the use of NAATs for its diagnosis and typification due to how difficult it is to obtain the virus via cell cultures or to develop indirect diagnosis techniques [49].

The first protocols for detect HPV were described about 20 years ago, using L1 consensus primers PCR systems, particularly MY09/11 and GP5+/6+ [50-52]. These primer systems have been widely used to study the natural history of HPV and their rule in the development of genital cancer [53-55]. Nowadays, several kits are commercially available which allow for the detection of the virus or the detection and typification of the most relevant HPVs: Amplicor HPV test and Linear array HPV Genotyping test (Roche Diagnostics, Switzerland), Innolipa HPV Genotyping Extra (Innogenetics, Belgium), Biopat kit (Biotools, Spain) or Clart Papillo‐ mavirus 2 (Genómica, Spain). The latter uses microarray technology to increase the number of hybridizations in a reduced space. Besides genome amplification, direct hybridization protocols on the sample (hybrid capture) approved by the FDA for diagnosing HPV in women (Hybrid Capture II, Digene, USA) is also used. These protocols identify high and low-risk

The sensitivity of such methods has left out cytological methods (Papanicolau), which are less sensitive and specific. This high degree of sensitivity allows to extending the period between

The Hybrid Capture II system (HCII, Digene, USA) is a non radioactive signal amplifica‐ tion method based on the hybridization of the target HPV-DNA to labeled RNA probes in solution. The resulting RNA-DNA hybrids are captured onto microtiter wells and are detected by specific monoclonal antibody and chemiluminiscence substrate, providing a semiquantitative measurement of HPV-DNA. Two different probe cocktails are used, one containing probes for five low-risk gentypes: HPV 6, 11, 42,43 and 44 and the other contain‐ ing probes for 13 high-risk genotypes: HPV 16,18,31,33,35,39,45,51,52,56,58,59 and 68.

longer low-risk period [48].

genotypes without specifying the infecting genotype.

control visits of women to 5 or 6 years [56, 57].

**3.1. Signal amplification systems**

On the other hand, integration of HPV-DNA into the host DNA is a well known topic in cervical cancer. Integration of HPV 16 DNA correlates with dysfunction of HPV E1 or E2 ORF, which are active during HPV replication. E2 loss of function allows up-regulation of E6 and E7 oncoproteins, because E2 is a repressor of E6 and E7. (Figure 2).

**Figure 2.** The location in squamous epithelium of the main stages of the papillomavirus life cycle. [46]
