Human papillomaviruses are non-enveloped double-stranded DNA viruses which consist of ~8000 base pairs in length. Yet many species of HPVs have been identified. Infection by most papillomaviruses is either asymptomatic or causes warts. However, a small portion of HPVs is associated with malignant transformation (high risk types).
The viral genome expresses two late genes (L1 and L2) and six early genes (E1 to E7). The capsid consists of 72 star-shaped L1 viral proteins. The genome is packaged into the viral core along with cellular histones which are used to wrap and condense DNA.
HPVs replicate exclusively in keratinocytes and mucosal surfaces. Less-differentiated keratinocytes are the primary target of productive HPV infection and subsequent steps in the viral life-cycle are dependent on the process of keratinocyte differentiation. Viral entry is mediated through interaction of the viral L1 protein with sulfated sugars on the cell surface of the keratinocyte [1]. The virus enters the cell via interaction with a specific receptor (ï¡6-ï¢4 integrins) and is transported to the endosomes.The L2 capsid protein disrupts the endosomal membrane allowing the viral genome to be released from the endosome followed by translocation to the cell nucleus where the E1 and E2 genes are expressed first. High E2 levels repress E6 and E7 expression. After the HPV genome has integrated into the host genome E6 and E7 genes are derepressed. E6 and E7 are viral oncogenes which inactivate the tumor suppressor genes p53 and pRb causing the host keratinocyte to retain in a certain differentiation state that favours the replication of the viral genome and the expression of the late genes L1 and L2 in order to complete the viral life cycle.
HPV infection and cervical cancer development
HPV is proven to be the most important etiologic agent in cervical carcinoma. Cervical cancer is the second most common cause of cancer death in woman worldwide. Almost all cervical cancers are associated with HPV infection. HPV-16 and HPV-18 are the most common high risk types with a prevalence of 70-80% in all cervical cancers. HPV infection is very common in the female population, but only a very small fraction of HPV-infected woman will develop cervical cancer. Therefore, cervical carcinogenesis is most likely a multi-step transformation process. This process from infection to malignancy can take up to as long as 15 years [2]. The molecular mechanisms that lead to viral transformation are partly known, but the molecular mechanisms which lead to the progression of cervical cancer or regression and viral clearance are not yet very well understood.
The entire HPV genome or the E6/E7 proteins alone can immortalize primary human genital keratinocytes. Normally, in the presence of serum and calcium human, genital keratinocytes undergo terminal differentiation by activation of p53 and Rb pathways. The viral E6 inactivates host p53 by binding to the E6 associated protein which ubiquitinates p53, leading to its degradation. The viral E7 protein competes for pRb binding and thereby release of the transcription factor E2F which, in turn, transactivates its targets. Therefore, HPV-immortalized keratinocytes are unable to differentiate, but still do not exhibit a malignant phenotype. The progression to malignancy can be mimicked by transfection of the SV40 small-T antigen and therefore provides a model to study carcinogenesis [3]. HPV/SV40 small-T transformed keratinocytes show high cytoplasmic levels of ï¢-catenin levels, indicating that the Wnt-pathway is activated. SV40-small T antigen activates this pathway through interaction with protein phosphatase-2A. High cytoplasmic ï¢-catenin levels are also observed in malignant cervical lesions, but not in pre-malignant lesions. Even in the absence of SV40 small-T antigen, activation of the Wnt-pathway could initiate malignant transformation in HPV-immortalized cells, while activation of the Wnt-pathway alone is not sufficient to induce malignant transformation underlining that malignant transformation is a multi-step process of sequentially acquired genetic alterations in the cells. Also in cytology samples show a gradual staging of malignant transformation (see figure on cover) and premalignant lesions are usually indicated as CIN1 to 3. The mechanisms underlying either malignant transformation or reversal to normal tissue stills needs to be elucidated.
To obtain insight into the host cell alterations that play a decisive role in cervical carcinogenesis chromosomal and transcriptional profiles of normal tissue and cervical carcinonomas have been made [4, 5]. These studies show differential gene expression between normal epithelial controls, cervical adenocarcinomas (AdCa), cervical squamous cell carcinomas (SCC) and a panel of HPV transformed keratinocytes. Differentially expressed genes are predominantly down-regulated in cervical carcinomas. One mode for down-regulation is DNA methylation in the promoter regions of tumorsuppressor genes. These epigenetic alterations are of great value as novel biomarkers which enable to distinguish hrHPV positive women with high-grade disease from those with clinically irrelevant infections [6].
Repression of MAL tumor suppressor activity by promoter methylation during cervical carcinogenesis
Human DNA has about 80-90% of its CpG sites methylated, however certain areas, known as CpG islands, have non-methylated DNA. These are associated with the promoters of genes, including all ubiquitously expressed genes. Methylation within these CpG islands is a reversible process and controls gene-expression. Hypermethylation in promoterregions of tumorsuppressor genes results in epigenetic alterations that lead to oncogenic transformation.
Amongst the genes showing differential expression between invasive cancers and normal epithelial controls, the MAL gene is found to be the most significantly down-regulated [5]. MAL (T-lymphocyte maturation associated protein) is a 17 kD membrane protein which is located at chromosome 2q13, a region which is commonly retained in cervical carcinomas. MAL down-regulation is not correlated to gene mutations or genetic loss but is silenced by promoter methylation.
Promoter methylation can be monitored with a technique called quantitative methylation specific PCR (qMSP). In brief, genomic DNA is treated with bisulfite (changes unmethylated cytosines into uracils, while methylated cytosines are protected from this conversion and remain intact) and a quantitative PCR is performed with primer sets which target methylated areas within the promoter region. Housekeeping gene PCR, normal tissue DNA and tumor DNA are included as controls.
Two methylation sensitive regions in the MAL promoter (M1 and M2) are of great interest as a prognostic tool. Analysis of a large series of cervical biopsies has shown that the frequency as well as the level of methylation increase with the severity of the disease [7]. Moreover, MAL promoter methylation is histotype-indepent (AdCa versus SCC) in contrast to other genes of which promoter regions were found to be hypermethylated predominantly in one histotype during cervical carcinogenesis (e.g. CADM1, CCNA1, DAPK, APC, TFPI2, SPARC)[8]. The diagnostic value of MAL gene silencing by promoter methylation by qMSP as diagnostic marker for high-grade lesions is even further sustained by the fact that this test is highly specific and sensitive on cervical scrapings. Taking a cervical scraping is less invasive than taking a cervical biopsy. A biopsy enhances the chance on pre-term delivery in women in reproductive age. Furthermore, qMSP on a well-chosen panel of gene promoters can distinguish the patients with lesions who are in urgent need for therapeutic intervention and those who are not.
Future perspectives
To exploit the value of methylation markers like MAL-M1 and -M2 in population-based screening based on primary hrHPV testing, studies are presently ongoing on scrapings collected during prospective population-based studies combining classical histology and hrHPV testing (the dutch POBASCAM trial [9]). Representative sets of samples were analysed with qMSP for a panel of methylationmarkers in subsets of normal cervix tissue, CIN1 lesions, CIN3 lesions, SCC en AdCa retrospectively [7]. The frequency as well as the level of promoter methylation increases with the severity of the disease. Additional to population based screening and hrHPV testing in case of a CIN lesion these novel biomarkers can serve as a new triage tool to avoid under- and overtreatment of woman with CIN lesions. Women testing positive for hrHPV and methylation markers should be referred for immediate intervention and those who are methylation marker negative can have a follow-up test without a substantial risk. However, since genetic alterations are rather individual a very small group of patients, who eventually will develop malignant cervical carcinoma, are at risk to be missed. Therefore an optimized cut-off definition for the individual methylation markers should be determined by cross-validation in a training set with subsequent validation in an independent test set in order to implement this novel technique into the pathology laboratory.
Literature
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2. Snijders, P.J., et al., HPV-mediated cervical carcinogenesis: concepts and clinical implications. J Pathol, 2006. 208(2): p. 152-64.
3. Uren, A., et al., Activation of the canonical Wnt pathway during genital keratinocyte transformation: a model for cervical cancer progression. Cancer Res, 2005. 65(14): p. 6199-206.
4. Wilting, S.M., et al., Increased gene copy numbers at chromosome 20q are frequent in both squamous cell carcinomas and adenocarcinomas of the cervix. J Pathol, 2006. 209(2): p. 220-30.
5. Wilting, S.M., et al., Integrated genomic and transcriptional profiling identifies chromosomal loci with altered gene expression in cervical cancer. Genes Chromosomes Cancer, 2008. 47(10): p. 890-905.
6. Henken, F.E., et al., Sequential gene promoter methylation during HPV-induced cervical carcinogenesis. Br J Cancer, 2007. 97(10): p. 1457-64.
7. Overmeer, R.M., et al., Repression of MAL tumour suppressor activity by promoter methylation during cervical carcinogenesis. J Pathol, 2009. 219(3): p. 327-36.
8. Yang, N., et al., Gene promoter methylation patterns throughout the process of cervical carcinogenesis. Cell Oncol, 2010. 32(1-2): p. 131-43.
9. Bulkmans, N.W., et al., POBASCAM, a population-based randomized controlled trial for implementation of high-risk HPV testing in cervical screening: design, methods and baseline data of 44,102 women. Int J Cancer, 2004. 110(1): p. 94-101.
