**4. Screening and progression prognostic biomarkers technologies**

Because molecular testing for HR-HPV DNA may detect infection too early in the process, with only a small subset of women developing disease that progresses to cancer, there is interest in defining secondary markers that have potential application in identification of women who need to be followed more closely because they are at higher risk of developing high-grade lesions [77]; especially, when the positive predictive value of current screening strategies will be diminished in a vaccinated population [78]. Then, the impetus for new screenig or progre‐ sion technologies in the developed world is thus predominately driven by the need to increase positive predictive value and reduce over-manegement of low-grade and often transient abnormalities.

developed: NucliSENS-EasyQ® HPV E6/E7 mRNA assay (Biomerieux, USA) and Aptima HPV test (Gen-Probe, USA) both are a type-specific E6/E7 mRNA test for HR-HPV types performed in one NASBA reaction NucliSENS-EasyQ® HPV E6/E7 mRNA assay detected HPV 16,18,31,33 and 45 with detection and genotyping and Aptima HPV test detects E6/E7 mRNA

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Most HR-HPV infections are either latent or permissive. Latent infections are not very well defined, but it is assumed that the viral genome is maintained as an episome in the basal and parabasal cells of the epithelium without inducing obvious phenotypic alterations in the

The transformation process is characterized by the deregulation of viral oncogenes E6 and E7 in cycling cells which ultimately results in chromosomal instability and the accumulation of mutations. The underlying mechanisms for deregulation are manifold. Integration of the HPV genome is a characteristic step in cervical carcinogenesis and its appearance correlates with

However, integration is not mandatory in this process and was shown to be HPV-type dependent. Vinokurova and colleagues observed that HPV16, 18 and 45 were substantially

The loss of the viral E2 gene is a common consequence of HPV integration. This event may lead to an elevated expression of the oncogenes E6 and E7 due to the fact that E2 is no longer able to repress the expression of the viral oncogenes in trans [102, 103 ]. However, in a recent analysis of biopsy material no correlation between the expression levels of viral oncogene

Several investigators have also focussed on the impact integration may have on the host genome. Methods for detection of integrated HPV have been described [87, 105. However, they are affected by similar limitations described for HPV viral load. On the other hand, cervical epithelial cells for women with CIN may simultaneously countain episomal and integrated HPV DNA. Recent data suggest that integration frequency in CIN3 and ICC is variable by HPV

A variety of HPV types have been characterized on the basis of differences greater than 10% in L1 gene sequence [25]. Isolates of the same type are referred to as "variants" when the nucleotide sequences of their coding genes differ by less than 2%, or when the non-coding region (LCR) differs by as much as 5% [106]. HPV 16 is one of the most important HPV genotypes wich cause serius cervical disorders, but amoung these genotypes, certains variants have been linked to different clinical outcomes. HPV 16 variants have been grouped into six distinct phylogenetic branches: E (European), AA (Asian-American), Af1(African 1), Af2 (African 2), NA (North American), As (Asian) with different geographic distributions. Most

the progression of precancerous lesions (CIN2/3) to invasive carcinoma [98-100].

more often present in an integrated state compared with HPV types 31 and 33 [101].

transcripts and the physical state of the viral genome was found [104.

genotype, further reducing the desired gains in specificity [101].

**4.4. E6-T350G HPV 16 variant**

of 14 oncogenic types HPV16,18,31,33,35,39,45,51,52,56,58,59,66, and 68.

**4.3. HPV integration (E2/E6-7 ratio)**

host cell.

In these situations, several surrogate markers are in research.

#### **4.1. HPV viral load**

Several studies have suggested that a high HPV-DNA viral load may be a candidate marker that could help identify women at greater risk of CIN progression [64, 65, 79, 80]. It has been reproted that average HPV DNA copy number increases significantly with the grade of CIN mainly for HPV 16, but not for other HR-HPV types [81-83]. Some studies have pointed out that high viral load in cytological normal epithelium could also be a risk factor for neoplasic progression but other studies suggested an important limitation to the utility in screening algorithms for the sustancial overlap oh HPV load values between women without and with CIN and the common presence of more than one carcinogenic HPV type [64, 84].

Real- time PCR techniques have been developed to quantify HPV in clinical samples. More‐ over, the HCII provides semiquantitative measurement of HPV–DNA, and some studies have demonstrated that the estimated HCII load correlated well with the precise load generated by RT-PCR [85-86]. However, real-time PCR assays more accurately measure HPV 16 viral load by adjusting the signal obtained for HPV 16 DNA with the amount of cellular DNA calculated for amplification of a human gene, therefore providing a more accurate viral load [64, 65, 87, 88]. However, due to low multiplicity for different HR-HPV types, real-time PCR methods are not suitable as a high-throughput screening tool.

#### **4.2. HPV mRNA**

Although HR-HPV genotypes are associated with any grade of dysplasia, these types can be detected in a significant proportion of women with normal cytology. It is konwn that HPV E6 and E7 genes are overexpressed throughout the thickness of epithelial cells in high-grade lesions and cancer. Then, mRNA could be more efficient than cytology for the triage of HPV DNA-positive women, and provides high speficity for high grade cervical intraepithelial neoplasia identification [69, 89-93].

Some authors have developed a real time reverse transcriptase amplificatios (RT–PCR) for HPV detection strategies and suggested that it may be more specific for the detection of symtomatic infections and quantitative increased coordinately with severity of the lesion [94, 95].

These assays incorporates NASBA amplification of E6/7 mRNA transcripts prior to type specific detection via molecular beacons for HPVs 16,18,31,33,and 45. Initial data, on the pronsotic value and specificity for underlying disease, is promising, but the value of this method compared with DNA based assays remains to be determined in large-scale prospective studies [96,97].

Detection of human papillomavirus (HPV) E6/E7 oncogene expression may be more predictive of cervical cancer risk than test HPV-DNA.Commercial test targeting HPV mRNA has been developed: NucliSENS-EasyQ® HPV E6/E7 mRNA assay (Biomerieux, USA) and Aptima HPV test (Gen-Probe, USA) both are a type-specific E6/E7 mRNA test for HR-HPV types performed in one NASBA reaction NucliSENS-EasyQ® HPV E6/E7 mRNA assay detected HPV 16,18,31,33 and 45 with detection and genotyping and Aptima HPV test detects E6/E7 mRNA of 14 oncogenic types HPV16,18,31,33,35,39,45,51,52,56,58,59,66, and 68.

### **4.3. HPV integration (E2/E6-7 ratio)**

positive predictive value and reduce over-manegement of low-grade and often transient

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

Several studies have suggested that a high HPV-DNA viral load may be a candidate marker that could help identify women at greater risk of CIN progression [64, 65, 79, 80]. It has been reproted that average HPV DNA copy number increases significantly with the grade of CIN mainly for HPV 16, but not for other HR-HPV types [81-83]. Some studies have pointed out that high viral load in cytological normal epithelium could also be a risk factor for neoplasic progression but other studies suggested an important limitation to the utility in screening algorithms for the sustancial overlap oh HPV load values between women without and with

Real- time PCR techniques have been developed to quantify HPV in clinical samples. More‐ over, the HCII provides semiquantitative measurement of HPV–DNA, and some studies have demonstrated that the estimated HCII load correlated well with the precise load generated by RT-PCR [85-86]. However, real-time PCR assays more accurately measure HPV 16 viral load by adjusting the signal obtained for HPV 16 DNA with the amount of cellular DNA calculated for amplification of a human gene, therefore providing a more accurate viral load [64, 65, 87, 88]. However, due to low multiplicity for different HR-HPV types, real-time PCR methods are

Although HR-HPV genotypes are associated with any grade of dysplasia, these types can be detected in a significant proportion of women with normal cytology. It is konwn that HPV E6 and E7 genes are overexpressed throughout the thickness of epithelial cells in high-grade lesions and cancer. Then, mRNA could be more efficient than cytology for the triage of HPV DNA-positive women, and provides high speficity for high grade cervical intraepithelial

Some authors have developed a real time reverse transcriptase amplificatios (RT–PCR) for HPV detection strategies and suggested that it may be more specific for the detection of symtomatic infections and quantitative increased coordinately with severity of the lesion

These assays incorporates NASBA amplification of E6/7 mRNA transcripts prior to type specific detection via molecular beacons for HPVs 16,18,31,33,and 45. Initial data, on the pronsotic value and specificity for underlying disease, is promising, but the value of this method compared with DNA based assays remains to be determined in large-scale prospective

Detection of human papillomavirus (HPV) E6/E7 oncogene expression may be more predictive of cervical cancer risk than test HPV-DNA.Commercial test targeting HPV mRNA has been

CIN and the common presence of more than one carcinogenic HPV type [64, 84].

In these situations, several surrogate markers are in research.

not suitable as a high-throughput screening tool.

neoplasia identification [69, 89-93].

abnormalities.

**4.1. HPV viral load**

**4.2. HPV mRNA**

[94, 95].

studies [96,97].

Most HR-HPV infections are either latent or permissive. Latent infections are not very well defined, but it is assumed that the viral genome is maintained as an episome in the basal and parabasal cells of the epithelium without inducing obvious phenotypic alterations in the host cell.

The transformation process is characterized by the deregulation of viral oncogenes E6 and E7 in cycling cells which ultimately results in chromosomal instability and the accumulation of mutations. The underlying mechanisms for deregulation are manifold. Integration of the HPV genome is a characteristic step in cervical carcinogenesis and its appearance correlates with the progression of precancerous lesions (CIN2/3) to invasive carcinoma [98-100].

However, integration is not mandatory in this process and was shown to be HPV-type dependent. Vinokurova and colleagues observed that HPV16, 18 and 45 were substantially more often present in an integrated state compared with HPV types 31 and 33 [101].

The loss of the viral E2 gene is a common consequence of HPV integration. This event may lead to an elevated expression of the oncogenes E6 and E7 due to the fact that E2 is no longer able to repress the expression of the viral oncogenes in trans [102, 103 ]. However, in a recent analysis of biopsy material no correlation between the expression levels of viral oncogene transcripts and the physical state of the viral genome was found [104.

Several investigators have also focussed on the impact integration may have on the host genome. Methods for detection of integrated HPV have been described [87, 105. However, they are affected by similar limitations described for HPV viral load. On the other hand, cervical epithelial cells for women with CIN may simultaneously countain episomal and integrated HPV DNA. Recent data suggest that integration frequency in CIN3 and ICC is variable by HPV genotype, further reducing the desired gains in specificity [101].

#### **4.4. E6-T350G HPV 16 variant**

A variety of HPV types have been characterized on the basis of differences greater than 10% in L1 gene sequence [25]. Isolates of the same type are referred to as "variants" when the nucleotide sequences of their coding genes differ by less than 2%, or when the non-coding region (LCR) differs by as much as 5% [106]. HPV 16 is one of the most important HPV genotypes wich cause serius cervical disorders, but amoung these genotypes, certains variants have been linked to different clinical outcomes. HPV 16 variants have been grouped into six distinct phylogenetic branches: E (European), AA (Asian-American), Af1(African 1), Af2 (African 2), NA (North American), As (Asian) with different geographic distributions. Most HPV16 variants from European and North American samples were classified as European prototype (EP) [107]. Several studies have shown that the infection by the European L83V HPV16 variant, harbouring a nucleotide substitution at position 350 in the E6 gene (E6-T350G), is a risk factor for advanced cervical disease although some discrepant results have also been found [21, 104, 108, 109 ].

**4.7. Human telomerase RNA component (hTERC)-gain**

[119], including cervix of the uterus [120, 121].

**4.8. Other proliferation/cell cycle markers**

have a role as "biomarkers" of dysplastic cells.

between their presence or absence and the grade of dysplasia [132].

quencing or allelic discrimination techniques [24, 109, 135].

development.

**5. Summary**

It has been generally accepted that carcinogenesis involves the progressive accumulation of genetic abnormalities. Gain at 3q is a common feature of squamous-cell carcinoma (SCC), with an overlapping area of gain at 3q26 having been reported in SCC at different anatomic sites

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The human telomerase RNA component (hTERC) gene, localized on chromosome 3q26, encodes the RNA component of human telomerase, and acts as a template for the addition of the repeat sequence [122]. Genetic studies have shown that amplification of *hTERC* gene might be an early event commonly involved in the progression of CIN to cervical cancer [123-127].

Amplification of hTERC gene has been identified in many tumor samples and immortalized cell lines using techniques such as fluorescence in situ hybridization (FISH) and Southern blot analysis, suggesting that transcription is upregulated during tumorigenesis [128]. Lan YL et al. confirm that measuring hTERC gene gain could be a useful biomarker to predict the progression of CIN-I or –II to CIN-III and cervical cancer [129]. The present limitation to this assay is the technical complexity and requeriment of highly trained individuals to interpret the FISH staining, however automated methods for reading TERCH FISH slides are under

HPV contributes to neoplastic progression predominantly through the action of two viral oncoproteins (E6 and E7) and is manifested by changes in the expression of host cell cycle regulatory proteins [130]. Such differentially expressed host proteins and nucleic acids may

To date, a wide array of molecular markers has been evaluated. Three markers that have shown the greatest potential are the cyclin dependant kinase inhibitor p16INK4 [131, 132] and the DNA replication licensing proteins CDC6 (cell division cycle protein 6) and MCM5 (mini chromo‐ some maintenance 5) [133]. Some authors found that three markers showed a linear correlation

In summary, the relevance of HPV infections requires a close monitoring, especially in certain groups o individuals (e. i. Women older than 30 years old). The accuracy of methos using NAATs has emerged as election in the control of HPV infection. But the search is ongoing for safer: more precise markers which may allow for a better control of the infection [134]. These markers include genome quantification via real-time PCR, viral integration into the human genome via E2-E1/E6-E7 genes ratio or the search of viral variants by sequencing, pyrose‐

Detection of HPV variant has been performed mainly by Sanger sequencing, pyrosequencing or high resolution melting analysis [110, 111]. A new one-step allelic discrimination real time PCR assay to detect the E6-T350G HPV 16 variant was evaluated in clinical samples, this novel allelic discrimination assay is a fast sensitive and specific method [24].

#### **4.5. p16 enzyme linked inmunosorbent assay**

Protein p16 is a cell cycle regulation protein which accumulates in abnormal epithelial cells infected with HR-HPVs as a result of a loss of negative regulation by the retinoblastoma protein induced by E7 expresion [112]. In immunostaining studies, p16 (INK4a) has shown potential as a marker of high grade cervical intraepithelial neoplasia (CIN) and invasive cervical cancer [113, 114]. A recent literature report demonstrates different p16 accuracy according to different anatomical sub-sites. In this complex scenario the p16-IHC test alone or in association to CDKN2a promoter methylation could be used only as screening methods but need to be associated with molecular tests in order to detect HPV-DNA and to assess its integration status. Furthermore, non-dysplastic cells, particulary methaplastic, atrophic and endocervical cells, may display p16 immunoreactivity, thereby reducing specificity [115].

#### **4.6. Methylation profile**

Methylation of CpG islands within gene promoter regions can lead to silencing of gene expression. Methylation of tumor-relevant genes has been identified in many cancers: p16 methylation is the paradigm for epigenetic inactivation of a tumor suppressor gene, leading to abrogation of cell cycle control, escape from senescence, and induction of proliferation.

Methylation has been detected already at precancerous stages, suggesting that methylation markers may have value in cervical cancer screening [116]. Furthermore, methylated DNA is a stable target and allows for flexibility of assay development.The detection of methylated genes from cervical specimens is technically feasible and represents a source for detecting potential biomarkers of relevance to cervical carcinogenesis. In particular, there is the ultimate hope of finding methylation markers that, among HPV-infected women, would indicate the presence of CIN2+ and risk of cancer.

A clear role of methylation in carcinogenesis has been demonstrated only for 6 genes (DAPK1, RASSF1, CDKN2A, RARB, MLH1, and GSTP1 [117].

During the last years, several new platforms have been developed that allow for accurate highthroughput genome-wide DNA methylation profiling [118]. Markers or marker panels identified in these approaches could be translated to smaller scaled assays such as Methylight to be used in cervical cancer screening, but their use is in research.
