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

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

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

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

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,

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

A clear role of methylation in carcinogenesis has been demonstrated only for 6 genes (DAPK1,

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

allelic discrimination assay is a fast sensitive and specific method [24].

may display p16 immunoreactivity, thereby reducing specificity [115].

**4.5. p16 enzyme linked inmunosorbent assay**

found [21, 104, 108, 109 ].

**4.6. Methylation profile**

presence of CIN2+ and risk of cancer.

RASSF1, CDKN2A, RARB, MLH1, and GSTP1 [117].

to be used in cervical cancer screening, but their use is in research.

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

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 development.

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

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 have a role as "biomarkers" of dysplastic cells.

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