AOGD Bulletin - Volume 11, December 2011 : A Note from the Editors’ Team

Introduction
Persistent infections with carcinogenic human papillomavirus (HPV) genotypes have long been established as the necessary, but not sufficient cause of invasive cervical cancer. Organized prevention programs in industrialized settings have relied on early detection of HPV-associated dysplastic changes in exfoliated cervical
cells (‘Pap smear’) that reflect underlying precancerous lesions. Cervical cancer screening has been a success owing to the long natural history, typically extending
over many years and the availability of relatively safe and effective methods of treatment of cervical precancer. Yet concerns about the substantial cost burden associated with screening, limited accuracy of cytology and complications of unnecessary treatment have prompted research and development of more efficient approaches for cervical cancer prevention. Over the past two decades, substantial improvements in understanding the natural history of HPV-associated cervical carcinogenesis as well as advancements in molecular technologies have led to the availability of novel screening tests that provide alternatives or adjunctive methods to cytology. Prominent among these are the HPV-DNA based screening assays, already widely used as adjunctive methods for primary screening and for triage of equivocal cytology.

New biomarkers may have potential use in primary screening, as triage tests for primary cytology screening, and as triage tests for primary HPV screening. For any biomarker to be useful, the test result has to influence clinical management. Management options include direct referral for treatment, referral to colposcopy to confirm precancer histologically, increased surveillance through more intensive screening or release to routine screening. The management options should be chosen based on an individual’s risk of precancer and cancer, indicated by screening test results and other risk indicators such as age.

HPV Carcinogenesis & the Basis for Biomarker Selection
The HPV genome consists of a circular double-stranded, 8000 bp long DNA with three regions:

The upper regulatory region which functions as a transcription and replication control region; An ‘early’ region encoding proteins (E1, E2, E4, E5, E6, E7) for replication, regulation and modification of the host cytoplasm and nucleus and a ‘late’ region encoding the viral capsid proteins (L1, L2). The prominent areas of research focused on biomarker discovery and validation are conceptually based on events in the HPV life cycle and natural history of HPV-dependent cervical carcinogenesis. While the phylogenetic taxonomy and classification of papillomaviruses continues to be refined, 13 HPV genotypes (HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68) are considered carcinogenic while some others (HPV types 26, 53, 66, 67, 70, 73, 82) are considered possibly carcinogenic in humans. The molecular mechanisms of how HPV causes cancer have been extensively studied. Two viral oncoproteins, E6 and E7, interfere with key cellular pathways that control cell proliferation and apoptosis. Specifically, E7 disrupts pRb from its binding to E2F and triggers uncontrolled cell cycling. E6 interferes with p53 and abrogates apoptosis, which would normally occur in cells with uncontrolled cell proliferation. E6 and E7 induce substantial chromosomal instability in transformed cells, even at precancerous stages. While biomarker discovery continues in multiple directions, current biomarker candidates can be broadly categorized into two groups, viral or cellular markers. The biomarker research pipeline extends from discovery (in vitro/preclinical studies) to early stage validation, and then to validation in randomized clinical trials.

HPV DNA Testing in Cervical Cancer Screening
The recognition of the strong causal relationship between persistent infection of the genital tract with high-risk HPV types and occurrence of cervical cancer has resulted in the development of a number of HPV DNA or RNA detection systems for screening. But, the main drawback of HPV-DNA testing as a screening method is that it identifies infection but not disease. In the first phase of the infection, viral gene expression and replication are restricted to terminally differentiated cells in the intermediate or superficial epithelial cell layers. These infections usually are transient and resolve spontaneously. Therefore, the mere detection of a high risk HPV (HR-HPV) infection has only limited specificity. In less than 10% of cases, the infection persists. In these cases, usually after persistence of the infection for several months or years, expression of the viral gene products E6 and E7 occurs in basal or parabasal cells of the epithelium, leading to chromosomal instability
in replicating host cells and inducing transformation of the infected epithelium. Thus, screening for women who have this transforming type of HR-HPV infection may be an appropriate way to identify patients who require medical intervention.

Since the FDA approval of Digene HC2 as a test for triage of ASC-US cytology in 2000, its use has increased steadily in the USA. In the ALTS trial, it was found that while HPV testing was deemed to have utility in distinguishing women with ASC-US who were at risk for precancer, it was limited in its discriminating capacity for mildly abnormal (LSIL) cytology given the high background prevalence of carcinogenic HPV in this population. The availability of genotype-specific information for HPV could potentially provide additional risk stratification in HPV-positive women. This may be of particular relevance in the detection of HPV types 16 and 18, since HPV 16-associated lesions are more likely to be persistent and have higher carcinogenicity than other HPV types, and since HPV 18 is more associated with lesions within the endocervical canal that are frequently missed by cytology. Indeed, some newer HPV DNA detection assays are able to provide type-specific information for HPV 16/18. A typical application is HPV16/18 genotyping in HPV-positive, cytology-negative women. Positivity for HPV16/18 may warrant earlier referral to colposcopy because of the higher risk associated with these types. However, it remains to be determined in clinical studies and cost-effectiveness analyses whether HPV genotyping provides sufficient risk stratification in a screening population.

New Biomarkers in Cervical Cancer Prevention
Given limitations in use of both cytology and HPV DNA based approaches as standalone tests for screening, the focus of cervical cancer prevention research has been on development and validation of new disease-specific biomarkers of HPV-associated transformation.

E6/E7 mRNA Detection
The progression from a transient to a transforming HPV infection is characterized by a strong increase of HPV E6/ E7 mRNA and protein expression. Multiple studies have evaluated the role of detection of mRNA transcripts in cervical scrapings to identify cervical precancers. At least two commercial platforms are currently available: PreTect® Proofer (Norchip [marketed as NucliSENS EasyQ® by BioMerieux in some European markets]) and APTIMA® (GenProbe). In a recent meta-analysis by Burger and colleagues, 11 studies that evaluated HPV E6/E7-based mRNA detection against HPV DNA testing for detection of CIN2+ reference standard were summarized. Given the considerable heterogeneity, pooling of data was not possible. A ‘best evidence synthesis’ for E6/E7 mRNA HPV testing accuracy was provided, that reflected a sensitivity ranging between 0.41 to 0.86 for the PreTect Proofer/NucliSENS Easy Q assays while a higher range – from 0.90 to 0.95 – for the APTIMA assay. The specificity ranged from 0.63 to 0.97 and from 0.42 to 0.61 for the PreTect Proofer/NucliSENS EasyQ and APTIMA assays, respectively. The considerable difference in sensitivity (and specificity) between PreTect Proofer/EasyQ and APTIMA may in part be explained by the difference in type coverage: The former tests detect only five types (HPV16, 18, 31, 33, 45), while the latter covers 14 types (HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68).

p16ink4a
The biomarker most widely evaluated is p16ink4a, a cyclin-dependent kinase inhibitor that is markedly overexpressed in cancerous and precancerous cervical tissue. p16ink4a is a cellular correlate of the increased expression of the viral oncoprotein E7 that disrupts a key cell cycle regulator, pRb, in transforming HPV infections. The disturbance of the Rb pathway leads to a compensatory overexpression of p16ink4a through a negative feedback loop. The resultant overexpression and cellular accumulation of p16ink4a is a specific marker of cervical precancerous lesions and can be measured through immunocytochemical staining of histology and cytology slides and using ELISA assays.

A commercially available CE-marked assay (CINtec®, mtm Laboratories) has been widely validated. Liquidbased cytology systems such as ThinPrep®, SurePath™, CYTO-screen system® and others have been used in these studies. p16ink4a has been evaluated as a standalone test and as an adjunct to cytology19 or HPV testing. The role of p16ink4a based detection in screening and triage has been reviewed in previous articles. These reviews noted substantial heterogeneity in methods used for defining p16ink4a positivity in the cytology application, including quantitative and morphologic approaches. The sensitivity has ranged between 0.59 and 0.96 and the specificity has ranged between 0.41 and 0.96 for the detection of CIN2+ lesions in clinical studies, reflecting the heterogeneity in test interpretation and analyzed populations. Recently, a dual immunostain of p16ink4a with Ki-67 (CINtec® PLUS) has been introduced that is supposed to substantially simplify and standardize the evaluation of stained slides.

Markers of Aberrant S-phase Induction

The cell cycle activation mediated by HPV oncogenes in transforming infections is characterized by aberrant S-phase induction. An assay detecting two proteins indicating aberrant S-phase induction, topoisomerase IIA (TOP2A) and minichromosome maintenance protein 2 (MCM2) is commercially available (ProEx™ C by Becton Dickinson). Few clinical studies with limited sample size have shown that it has a sensitivity ranging between 0.67 and 0.99 and specificity ranging between 0.61 and 0.85.

Other Biomarkers Undergoing Clinical Validation
Other cellular makers such as CK13 and CK14, MCM5 and CDC6, Survivin and CEA have also been evaluated in various stages of development. Most are marked by nonuniformity in determination of end points and limited sample sizes. Other viral markers such as HPV L1 capsid protein and E6 oncoprotein detection have been
evaluated in a limited number of small studies, but more evidence is needed to determine their utility.

Biomarkers for Low-resource Settings
In the context of resource-constrained settings, the failure to establish and sustain cytology-based screening has necessitated research on operationally simple and
less resource-intensive approaches for cancer prevention and control. Visual methods such as visual inspection with acetic acid and visual inspection with Lugol’s Iodine provide immediate in vivo detection of visually apparent precancerous cervical lesions and the potential to link screening results and same-visit treatment by cryotherapy (or appropriate referral for cryotherapy-ineligible lesions). While visual inspection with acetic acid/visual inspection with Lugol’s Iodine have been extensively evaluated and have high operational feasibility in the hands of nonphysician health providers, they miss anywhere between 20 and 50% of true disease due to variations in definitions of disease positivity, inherent subjectivity in test results, and challenges in quality assurance and control. There is a huge need for utilizing novel biologically-based approaches in resource-constrained settings of the developing world for improving access and accuracy of screening. careHPV™ is a new assay developed by Qiagen that is a low-cost adaptation of the Digene HC2 assay and can be performed rapidly (<2 h) without access to running water or electricity, an ideal solution for operation in field settings. This assay has been shown to have performance characteristics approaching those of HC2, and in conjunction with simpler alternatives like visual inspection it may permit effective single visit strategies (‘screen-and-treat’) by same-day results and linkage to cryotherapy. Yet, further research on adaptation of these strategies is needed to avoid overtreatment, given the different age distributions of HPV prevalence worldwide. Novel biomarkers that reflect measurement of an advanced disease process end point, such as overexpression of p16ink4a or HPV E6 protein detection, are also being evaluated in these settings, with the goal of achieving an optimal balance of sensitivity and specificity for very infrequent testing. Additional efforts are also being undertaken to evaluate biomarker assays using noninvasive and user-operated screening methods (e.g., self-sampling or urine-based sampling) that can address challenges in improving access to cervical cancer prevention services in these settings.

Biomarkers in Discovery & Early Validation Phases
Epigenetic Markers: DNA Methylation
Methylation of CpG sites within the genome occurs at varying levels during carcinogenesis. While tumors are often hypomethylated in repetitive regions of DNA such as LINE elements, promoter regions of tumor suppressor genes may become hypermethylated, frequently leading to decreased expression of important regulatory proteins. Since DNA methylation is a stable analyte that can be detected in many biospecimens, and changes in methylation patterns that occur early in carcinogenesis are often retained in invasive tumors, they represent potentially clinically useful biomarkers.

Most work in the cervical cancer field has focused on candidate genes that were identified by gene expression profiling in cervical cancer cell lines and tissue or have been suggested to play a role in tumor development in sites other than the cervix. Very few studies have taken advantage of microarray technologies or other profiling approaches to identify differentially methylated genes. Broadening the scope of research to include previously unknown genes offers an opportunity to identify novel markers that could be useful clinically. In addition, most methylation markers that have been studied extensively come from studying the host genome. However, there is growing evidence that methylation of HPV DNA may also be important in cervical carcinogenesis and could provide additional biomarkers for screening and prognosis.

Host Methylation: To date, methylation of many genes has been studied in cervical cancer. There is a great diversity in the roles these genes play in normal cellular processes, ranging from apoptosis to cell–cell interactions. In general, these genes are negative regulators of cell growth and motility, therefore it is conceivable that they are more frequently methylated, and presumably silenced, in cervical cancer and its precursor lesions.

A few differentially methylated genes have been formally studied as diagnostic tools for detection of cervical precancer, including single markers and marker panels. Although some candidates have shown promising results, further studies are needed to confirm that host methylation markers can be useful for cervical cancer prevention.

Viral Methylation: Preliminary work has suggested that understanding methylation of the HPV genome could lead to additional biomarkers for the detection of cervical cancer and its progression. The promoter regions of E6 and E7 are more frequently methylated in the later stages of tumor progression and the methylation level has been correlated to E6 mRNA expression. In addition, methylation of CpGs within L1 have been shown to be elevated in high grade lesions. The functional relevance of this phenomenon is currently not known.

The data for methylation markers, both host and viral, in cervical cancer screening has come from small, heterogeneous studies, limiting the evidence of their clinical utility. Although some small panels such as CADM1 and MAL have promise as triage tests for HPVpositive women, the best panel or marker combination has yet to be identified and validated. The addition of other genes such as DAPK, RARb, TWIST or other viral markers to CADM1 and MAL may be necessary to increase the diagnostic performance of the panel.

Chromosomal Abnormalities
Cervical carcinomas are characterized by a high degree of genomic instability with many recurrent chromosomal amplifications and deletions. Based on studies in clinical cervical cancer samples, several regions are typically lost in cervical carcinogenesis (2q, 3p, 4p, 5q, 6q, 11q, 13q and 18q) while other regions are amplified (1q, 3q, 5p and 8q). Some of these alterations can be detected in precancerous lesions (CIN3).

Gain of 3q is the most consistently reported chromosomal abnormality in cervical cancer. One gene within this region that is of particular interest in carcinogenesis is TERC. A large multicenter study in China recently confirmed previous small studies and showed that TERC amplification could serve as an effective triage test for HPV-positive women who have ASC-US or LSIL cytological diagnoses. In addition, in a small study of women who had repeat pap smears available, TERC amplification was only seen in patients who progressed to a diagnosis of CIN3+ in follow-up tests from an initial diagnosis of CIN1/2. A separate study showed that amplification of 3q had a high negative predictive value for the development of CIN2+ in women with LSIL cytology results. Together this suggests that amplification of this region could be useful in determining which women have clinically relevant HPV infections and need to be referred to coloposcopy and which women can be monitored by continued screening.

On the Horizon
miRNAs: miRNAs, short noncoding RNAs, are responsible for negatively regulating the expression of genes by binding to the mRNA and preventing its translation. Abnormalities in miRNA expression patterns have been seen in a number of tumors and these changes have been suggested to have prognostic value for other cancers. Profiles of cervical tumors and cancer cell lines have identified miRNAs that have increased (miR-21, miR-127 and miR-199a) and decreased (miR-143, miR214, miR-218 and miR-34a) expression in cancer compared with normal tissue, suggesting a role for miRNAs in cervical carcinogenesis. Since these changes in expression are seen in early, precancerous lesions, they hold promise as biomarkers for cervical cancer screening; however, they have not been formally studied in this way.

Proteomics: The field of proteomics and the identification of differentially expressed proteins in biospecimens is a growing area of research. Most proteomic work in cervical cancer has focused on comparing cancer specimens to normal samples to identify potential markers for tumors. A serum-based study with 165 patients identified three peaks by MALDI-TOF that were different between cancer patients and healthy volunteers. In a validation data set, these biomarkers showed a sensitivity of 87.5% and specificity of 90% in the detection of cervical cancer.

Some studies have successfully used alternative biospecimens for the identification of protein markers, including cervical–vaginal fluid from colposcopy exams and cervical mucus. suggesting that proteomic research does not need to be restricted to serum- or tissue-based assays. However, most proteomic studies have been small, few have studied precancerous lesions, and any differentially expressed proteins need to be validated in larger studies.