For many decades, the microscopic observation of biopsied sampleshas been the mainstay of screening/diagnostic processes of cervical cancer, eventhough this technique suffers from intra-observational subjectivity.Despite numerous technical innovations that have beendeveloped to detect cancer in their earliest stages of formation, unfortunatelythe detection of many cancers at the microscopic level isoften too late for successful intervention(Fong, Fortner et al. 1999). Unlike many genitourinaryinfections, cervical cancer is not usually associated with immediatesymptoms such as itching, burning or vaginal discharge(Mao, Hughes et al. 2003).The initial changes that may occur in some cervical cells are notcancerous. However, these precancerous cells form dysplasia orSIL within the epithelial or outerlayer of cells.The majority of all mild dysplasias regress spontaneouslywithin less than a year (Figure 2.14). A proportion of the HR HPVinfections will however become persistent and, if left untreated,proceed to high-grade lesions and invasive cervical cancer.The long transit time from early cervical atypiato invasive cancer provides an opportunity to identify pre-cancerousat a stage where safe and affordable treatment is available making it appropriate disease for the application ofscreening principles.Taken together, diagnosis and prognosis of asymptomatic, invasive diseaseis very poor but treatment of pre-invasive lesions is highly effective(Dewar, Hall et al. 1992). It is well established that no single screening method exists that is highly sensitive, highly specific, affordable and practical (Gheit, Vaccarella et al. 2009). The field of detection has been plagued byproblems of over diagnosis, inadequate specificity of individualmarkers (Cancer antigen-125, Carcino embryonic antigen), lowcompliance (colonoscopy) and a lack of analytical tools for discoveringnew detection methods. Hence there is an obvious interestin identifying markers that could complement standard cyto/histopathologicevaluation to determine the presence of cancer cells in tissues.
Thus, in cervical cancer screening, biomarkers are needed that allow to identify persons at risk to develop cancer at a time point that still allows for a successful curative intervention before invasive cancer develops.In addition to screening markers,biomarkers could be used for a risk assessment of detectedlesions, to stratify intermediate lesions, to predictprogression and to monitor recurrences after treatment.A very interesting field for biomarkers couldbe the assessment of CIN1 and CIN2 lesions.
2.18.1 Biomarkers derived from the analysis of molecular key events of cervical carcinogenesis
The initial event in cervical transformation is an infection with HR-HPV.The major hallmark of progressionfrom HPV infected tissue to dysplastic lesions is the alteredexpression pattern of the HPV oncogenes (Duensing, Lee et al. 2000, Duensing and Münger 2001, Duensing and Münger 2002). Biomarkers are subdivided on the basis of the most important cellular and viralchanges from HPV infection to invasivecancer.
2.18.1.1 Surrogate biomarkers of deregulated HPV oncogene expression
p16INK4a: The p16 is a cell cycle regulatory protein which is the main target of HPV. p16 is an inhibitor of CDKs 4 and 6 and functions in the progression from G1 to S phaseof the cycle cell. In HPV infection with HR types, p16 is over expressed (Klaes, Friedrich et al. 2001). The p16 protein can be detected in both histology specimens as well as in LBc(Sahebali, Depuydt et al. 2004). Over-expression of p16 is correlated with HPV type 16 and 18 and can be detected in both squamous cell carcinoma and adenocarcinoma (Schorge, Lea et al. 2004, Tringler, Gup et al. 2004). In biopsies of suspicious cervical lesions p16 is used to discriminate the low grade lesions with potential to progress to high-grade lesions (Klaes, Benner et al. 2002, Passamonti, Gustinucci et al. 2010, Sung, Kim et al. 2010).
2.18.1.2 Markers of chromosomal instability
DNA aneuploidy: Earlier reports (Duensing and Münger 2001, Duensing and Münger 2002) have shown that disturbances of the mitotic spindle apparatus are induced early byderegulated expression of HPV oncogenes resulting in non-diploid nuclei (aneuploidy). Consequently, aneuploidy is characteristic for HPV transformed lesions even at precancerous stages (Bollmann, Bollmann et al. 2001, Bollmann, Méhes et al. 2003, Melsheimer, Vinokurova et al. 2004). The studies have shown an association between aneuploidy and increasing dysplasia.
HPV integration: It is believed that HPV DNA is integrated by chanceduring the cellular repair processes of double strandbreaks. As such, HPV integration is an indicator of severeongoing chromosomal instability and an advancedstage of the transformation process (Wentzensen, Vinokurova et al. 2004). The detectionof HPV DNA test alone is employed as a primary screening method toexhibit it as more sensitive than cytology among abundant clinical studies.Since HPV testing is more sensitive than cervical cytology in detectingCIN 2 and CIN 3, women with concurrent negative test results (Papand HPV test) can be reassured that they have no risk of unidentifiedCIN 2, CIN 3 or cervical cancer (Castle, Solomon et al. 2005, Walker, Wang et al. 2006). Widschwendter et al.suggest that the serum HPV DNA might be a useful additional markerfor early detection of recurrence in cervical cancer(Widschwendter, Blassnig et al. 2003).
Severalmethods exist to detect the integration of HPV DNA intothe host genome. The main HPV testing methods that have well defined clinical applications at present are polymerase chain reaction (PCR) and Hybrid Capture II (HC2). Real-time PCR and mRNA testing are techniques of growing interest (Koliopoulos, Valasoulis et al. 2009, Schiffman, Wentzensen et al. 2011). A simple approach is the real timePCR-based quantification of the E2 and E6/E7 gene ratio (Peitsaro, Johansson et al. 2002). E2 is frequently lost upon HPV integrationand the theoretical ratio of 1:1 between the two genesis expected to be shifted towards E6/E7.
Dysplastic cells show increased cell cycling. Therefore, markers of cell proliferation might be a logical choice as biomarkers for CIN.
ki67:The increased proliferation of cervical epithelial cells induced by deregulated HPV oncogene expressionis reflected by the activation of proliferation markerssuch as ki67 (MIB-1). This protein is strongly expressedin CIN lesions, but can also be found expressedin normal basal cells that retain proliferation capacity(Pirog, Baergen et al. 2002, Goel, Mehrotra et al. 2005).
MYC:The cellular oncogeneMYC is frequently found amplifiedand over-expressed in cervical cancer. Studies have shown MYC activation at premalignantstages, indicating that MYC detection might be usedto assess dysplastic lesions. (Golijow, Abba et al. 2001).
Cyclins:They are a large family of regulatory proteins withcentral functions in the coordination of the cell cycle.The expression of several cyclins has been analyzed in cervical cancer and pre-cancer. Cyclin D1 was foundto be over-expressed in low grade lesions induced byLR-HPV, while it was absent in HR-HPV induced lesions(Southern and Herrington 1998). Other cyclins such as A, B, and E werefound to be over-expressed in premalignant cervical lesions(Keating, Cviko et al. 2001, El-Ghobashy, Shaaban et al. 2004).
Telomerase:Telomerase expression is important to counteract theloss of sequences located at the outermost chromosomeendings that naturally occurs during every celldivision. Thegene encoding the RNA subunit of telomerase, TERC, is located onchromosome 3q, a region that is frequently amplifiedin cervical cancer and pre-cancer. Since telomerase is necessary to maintain telomere length in proliferatingcells, it is found over-expressed in many human cancers.Several groups have used a functional telomerase assayto evaluate telomerase activity on cervical smears(Jarboe, Thompson et al. 2004, Ault, Allen et al. 2005).
Replication complex proteins: MCM5andCDC6belong to theDNApre-replicationcomplex that is usually expressed in replicating, butnot in quiescent cells.
2.18.1.4 Markers of cellular stress and invasion
HSPs: These are chaperones protecting cellular functions inresponse to various cellular stresses that were foundto be overexpressed in a number of cancers. Incervical pre-cancer, over-expression of HSP40, HSP60and HSP70 were associated with increasing lesion grade(Castle, Ashfaq et al. 2005).
Carbonic anhydrase 9: It is a transmembraneprotein induced by lowered oxygen tension. TheCA9/MN antigen has been identified as a marker for allgrades of CIN (Resnick, Lester et al. 1996). Liao et al. have analysed CA9 expressionon cervical smears and found expression in allgrades of dysplasia as well as some slides exhibiting onlyatypical cells(Liao and Stanbridge 2000).
Laminin 5:It is part of cell adhesion complexes and isan important constituent of the extracellular matrix. Itwas found to be an invasion marker in various epithelialtumours, including cervical cancer (Skyldberg, Salo et al. 1999).
Hypoxia-inducible factors (HIFs): Inadequate oxygen supply, or hypoxia, is a characteristicof most tumors. (Wilson and Hay 2011). Hypoxia thus acts as aselective pressure that favours malignant pro-survival cellphenotypes such as migration, angiogenesis, and suppressionof apoptosis, and also promotes genetic instability due toincreased mitochondrial release of reactive oxygen species (ROS) (Guzy, Hoyos et al. 2005, Wilson and Hay 2011). HIFs are up-regulated in response to hypoxia and induce multiple neoplasticattributes upon cancer cell. Ishikawa et alidentified HIF1?as a biomarker of poor prognosis after radiation therapy incervical cancer patients; the 10-year recurrence-free survival(RFS) was only 22% in HIF1? positive tumours comparedto 68.7% in HIF1? negative tumours(Ishikawa, Sakurai et al. 2004).Similar findings were reported by Kim et al. where HIF2?was significantly associated with a higher recurrence risk(Kim, Kim et al. 2011).
Methylation of CpG islands is an epigenetic modifierof gene expression. In normal cells, DNA methylation plays a role in maintaining genome stability and in regulating gene expression. DNA methylation changes are an early event in carcinogenesis and are often present in the precursor lesions of various cancers. Such changes might therefore be used as markers of cervical neoplasia, either alone or in conjunction with cytology and/or HPV testing. The published data on these genes is highly heterogeneous. Three markersshowed elevated methylation in cervical cancers consistently across studies namely,
? Death-associated protein kinase 1(DAPK1),
? Cell adhesion molecule 1 (CADM1) and
? Retinoic acid receptor beta(RARB).
There is currently no methylation marker that can be readily translated for use in cervical cancer (Wentzensen, Sherman et al. 2009, Yang, Nijhuis et al. 2010).

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