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Human papillomavirus-16 and -18 in penile carcinomas: DNA methylation, chromosomal recombination and genomic variation.

ABSTRACT: Penile carcinomas are frequently associated with high risk human papillomavirus (HPV) types. Because little is known about the molecular biology of this association, we investigated three properties of HPV genomes in penile carcinomas from Brazilian patients: (i) HPV DNA methylation, (ii) junctions between HPV and cellular DNA and (iii) genomic variation. In cervical carcinogenesis, recombination between HPV and chromosomal DNA is frequent and likely necessary for progression, and DNA hypermethylation-specifically of the L1 gene-is a biomarker for cancerous progression. The same mechanisms apparently occur during penile carcinogenesis, because 95 HPV-16 molecules derived from 19 penile lesions had 58% of the CpGs in L1 and 22% in the 5' part of the long control region methylated, more than the percentages found in cervical carcinomas. In addition, 2 out of 3 HPV-18 infections, all present in double infections with HPV-16, showed L1 specific methylation typical of malignant cervical lesions. In 11 out of 15 HPV-16 lesions, we confirmed chromosomal integration by reverse ligation inverted PCR, while 4 samples had concatemeric integrations or episomes. Nine of 17 penile carcinomas contained HPV-16 AA variants, and 8 E variants. As AA variants are relatively rare in Brazilian cohorts of asymptomatic women, the high prevalence in penile carcinomas may indicate a higher risk of progression of AA lesions, as suspected for cervical infections. Our observations of frequent viral DNA methylation, chromosomal integration and the prevalence of high risk variants suggest that HPV-dependent carcinogenesis of the penis and cervix follows similar etiological and epidemiological parameters.

SUBMITTER: Kalantari M

PROVIDER: S-EPMC2750853 | biostudies-literature | 2008 Oct

REPOSITORIES: biostudies-literature

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Human papillomavirus-16 and -18 in penile carcinomas: DNA methylation, chromosomal recombination and genomic variation.

Kalantari Mina M Villa Luisa L LL Calleja-Macias Itzel E IE Bernard Hans-Ulrich HU

International journal of cancer 20081001 8

Penile carcinomas are frequently associated with high risk human papillomavirus (HPV) types. Because little is known about the molecular biology of this association, we investigated three properties of HPV genomes in penile carcinomas from Brazilian patients: (i) HPV DNA methylation, (ii) junctions between HPV and cellular DNA and (iii) genomic variation. In cervical carcinogenesis, recombination between HPV and chromosomal DNA is frequent and likely necessary for progression, and DNA hypermethy ...[more]

PMID: 18688866

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Project description:BackgroundSquamous cell carcinoma is the most common genital, ocular and gastric tumour in horses. Equus caballus papillomavirus type 2 (EcPV2) DNA has been detected in several studies in equine penile squamous cell carcinomas (SCCs) and precursor lesions providing evidence of a causal role of EcPV2 in equine genital SCCs. Recently, EcPV2 E6/E7 nucleic acids were also detected in equine gastric SCCs, but further studies are required to determine the role of EcPV2 infection in the pathogenesis of gastric SCC. EcPV2 nucleic acids have been rarely described in ocular SCCs and precursor lesions.ObjectivesTo investigate the presence of EcPV2 nucleic acids with polymerase chain reaction (PCR) and in situ hybridisation (ISH) in penile hyperplasias, papillomas and SCCs in horses and to determine whether EcPV2 nucleic acids can be detected in SCCs affecting other locations, including the stomach, ocular tissues and larynx.MethodsTwenty-one archival formalin-fixed paraffin embedded (FFPE) tissue samples, including 12 genital lesions comprising penile hyperplasias, papillomas and SCCs, 6 ocular SCCs, 2 gastric SCCs and 1 laryngeal SCC, were screened by PCR and ISH for EcPV2 E6/E7 DNA and mRNA. Archival FFPE tissue samples (eyelid and penile mucosa and preputium) from six horses without a diagnosis or history of neoplastic or papillomavirus-associated disease were included as controls.ResultsEcPV2 nucleic acids were detected by PCR and ISH in all genital lesions (12/12) and gastric SCCs (2/2), in two ocular SCCs (2/6) and in one laryngeal SCC (1/1). In control horses, one eyelid sample was positive in PCR but not in ISH. The remaining control samples were negative for EcPV2 E6/E7 nucleic acids in PCR and ISH.ConclusionsThese results further support the role of EcPV2 infection in the development of equine genital SCCs and suggest that EcPV2 infection may also act as a predisposing factor for other SCCs in horses, including gastric, ocular and laryngeal SCCs.

Project description:It has been reported that >90% of women with cervical cancer are human papillomavirus (HPV)-positive, with HPV16 and 18 being the most 'highest-risk' HPV genotypes. However, in numerous women, HPV infection will not progress to cervical cancer. Accordingly, more appropriate screening markers need to be explored. In the present study, genome-wide DNA methylomic differences between cervical cancer tissues with HPV-16 or HPV-18 infection and normal cervical tissues were detected by using an Illumina Human Methylation 850 K BeadChip. The Gene Ontology functional enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses were conducted in order to define the nearest neighbouring genes of differentiated methylation sites. Moreover, differentiated methylation sites were verified using pyrosequencing. KEGG analyses suggested that the focal adhesion pathway and pathways in cancer were highly enriched. Bioinformatics and statistical analysis indicated that the nine CpG loci had the most significant differences amongst the genes involved in these pathways. Among these, six CpG sites in the CHRM2, LAMA4, COL11A1, FGF10, IGF1 and TEK genes were highly associated with HPV-16-positive cervical cancer, as validated using pyrophosphate sequencing. Additionally, 10 significantly different CpG sites of the HPV-18-positive group were selected and verified in The Cancer Genome Atlas, indicating their possible diagnostic roles in cervical cancer development and determination. In addition, eight hypermethylated CpG island sites that were associated with HPV-16-positive cervical cancer tissues and 10 hypermethylated CpG island sites that were associated with HPV-18-positive cervical cancer tissues were identified, highlighting their potential roles in screening and evaluating targeted therapy efficacy and prognosis. The main focus of the present study was to identify the genetic variability in HPV-16- and HPV-18-positive samples and to elucidate possible methylation biomarkers in HPV-positive women with a risk of developing cervical cancer.

Project description:BackgroundPersistent infection with oncogenic Human Papillomavirus (HPV) is associated with the development of cervical cancer with each genotype differing in their relative contribution to the prevalence of cervical disease. HPV DNA testing offers improved sensitivity over cytology testing alone but is accompanied by a generally low specificity. Potential molecular markers of cervical disease include type-specific viral load (VL), integration of HPV DNA into the host genome and methylation of the HPV genome. The aim of this study was to evaluate the relationship between HPV type-specific viral load, integration and methylation status and cervical disease stage in samples harboring HPV16, HPV18, HPV31 or HPV45.MethodsSamples singly infected with HPV16 (n=226), HPV18 (n=32), HPV31 (n=75) or HPV45 (n=29) were selected from a cohort of 4,719 women attending cervical screening in England. Viral load and integration status were determined by real-time PCR while 3'L1-URR methylation status was determined by pyrosequencing or sequencing of multiple clones derived from each sample.ResultsViral load could differentiate between normal and abnormal cytology with a sensitivity of 75% and a specificity of 80% (odds ratio [OR] 12.4, 95% CI 6.2-26.1; p<0.001) with some variation between genotypes. Viral integration was poorly associated with cervical disease. Few samples had fully integrated genomes and these could be found throughout the course of disease. Overall, integration status could distinguish between normal and abnormal cytology with a sensitivity of 72% and a specificity of 50% (OR 2.6, 95% CI 1.0-6.8; p=0.054). Methylation levels were able to differentiate normal and low grade cytology from high grade cytology with a sensitivity of 64% and a specificity of 82% (OR 8.2, 95% CI 3.8-18.0; p<0.001). However, methylation varied widely between genotypes with HPV18 and HPV45 exhibiting a broader degree and higher magnitude of methylated CpG sites than HPV16 and HPV31.ConclusionsThis study lends support for HPV viral load and CpG methylation status, but not integration status, to be considered as potential biomarkers of cervical disease.

Project description:Cervical cancer is caused by high-risk human papillomaviruses (HPV), in more than half of the worldwide cases by HPV16. Viral DNA integration into the host genome is a frequent mutation in cervical carcinogenesis. Because integration occurs into different genomic locations, it creates unique viral-cellular DNA junctions in every single case. This singularity complicates the precise identification of HPV integration sites enormously. We report here the development of a novel multiplex strategy for sequence determination of HPV16 DNA integration sites. It includes DNA fragmentation and adapter tagging, PCR enrichment of the HPV16 early region, Illumina next-generation sequencing, data processing, and validation of candidate integration sites by junction-PCR. This strategy was performed with 51 cervical cancer samples (47 primary tumors and 4 cell lines). Altogether 75 HPV16 integration sites (3'-junctions) were identified and assigned to the individual samples. By comparing the DNA junctions with the presence of viral oncogene fusion transcripts, 44 tumors could be classified into four groups: Tumors with one transcriptionally active HPV16 integrate (n = 12), tumors with transcribed and silent DNA junctions (n = 8), tumors carrying episomal HPV16 DNA (n = 10), and tumors with one to six DNA junctions, but without fusion transcripts (n = 14). The 3'-breakpoints of integrated HPV16 DNA show a statistically significant (p<0.05) preferential distribution within the early region segment upstream of the major splice acceptor underscoring the importance of deregulated viral oncogene expression for carcinogenesis. Half of the mapped HPV16 integration sites target cellular genes pointing to a direct influence of HPV integration on host genes (insertional mutagenesis). In summary, the multiplex strategy for HPV16 integration site determination worked very efficiently. It will open new avenues for comprehensive mapping of HPV integration sites and for the possible use of HPV integration sites as individualized biomarkers after cancer treatment of patients for the early diagnosis of residual and recurrent disease.

Dataset Information

Human papillomavirus-16 and -18 in penile carcinomas: DNA methylation, chromosomal recombination and genomic variation.

Publications

Human papillomavirus-16 and -18 in penile carcinomas: DNA methylation, chromosomal recombination and genomic variation.

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