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Identification of reliable biomarkers of human papillomavirus 16 methylation in cervical lesions based on integration status using high-resolution melting analysis.

ABSTRACT: Background:The dynamic methylation of human papillomavirus (HPV) 16 DNA is thought to be associated with the progression of cervical lesions. Previous studies that did not consider the physical status of HPV 16 may have incorrectly mapped HPV 16 methylomes. In order to identify reliable biomarkers for squamous cervical cancer (SCC), we comprehensively evaluated the methylation of HPV 16 depending on the integration incidence of each sample. Methods:Based on the integration status of 115 HPV 16-infected patients (50 SCC, 30 high-grade squamous intraepithelial lesion [HSIL], and 35 low-grade squamous intraepithelial lesion [LSIL]) and HPV 16-infected Caski cell lines by PCR detection of integrated papillomavirus sequences, we designed a series of primers that would not be influenced by breakpoints for a high-resolution melting (HRM) PCR method to detect the genome methylation. Results:A few regions with recurrent interruptions were identified in E1, E2/E4, L1, and L2 despite scattering of breakpoints throughout all eight genes of HPV 16. Frequent integration sites often occurred concomitantly with methylated CpG sites. The HRM PCR method showed 100% agreement with pyrosequencing when 3% was set as the cutoff value. A panel of CpG sites such as nt5606, nt5609, nt5615, and nt5378 can be combined in reweighing calculations to distinguish SCC from HSIL and LSIL patients which have high sensitivity and specificity (88% and 92.31%, respectively). Conclusions:Our research shows that combination of CpG sites nt5606, nt5609, nt5615, and nt5378 can be used as potential diagnosis biomarkers for SCC, and the HRM PCR method is suitable for clinical methylation analysis.

SUBMITTER: Liu L

PROVIDER: S-EPMC5781301 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Identification of reliable biomarkers of human papillomavirus 16 methylation in cervical lesions based on integration status using high-resolution melting analysis.

Liu Lu L Ying Chunmei C Zhao Zhen Z Sui Long L Zhang Xinyan X Qian Chunyan C Wang Qing Q Chen Limei L Guo Qisang Q Wu Jiangnan J

Clinical epigenetics 20180123

<h4>Background</h4>The dynamic methylation of human papillomavirus (HPV) 16 DNA is thought to be associated with the progression of cervical lesions. Previous studies that did not consider the physical status of HPV 16 may have incorrectly mapped HPV 16 methylomes. In order to identify reliable biomarkers for squamous cervical cancer (SCC), we comprehensively evaluated the methylation of HPV 16 depending on the integration incidence of each sample.<h4>Methods</h4>Based on the integration status ...[more]

PMID: 29410710

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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.

Project description:HPV16 accounts for 50-70% of cervical cancer cases worldwide. Characterization of HPV16 variants previously indicated that they differ in risks for viral persistence, progression to cervical precancer and malignant cancer. The aim of this study was to examine the association of severity of disease with HPV16 variants identified in specimens (n = 281) obtained from a Cervical Pathology and Colposcopy outpatient clinic in the University Hospital of Espírito Santo State, Southeastern Brazil, from April 2010 to November 2011. All cytologic and histologic diagnoses were determined prior to definitive treatment. The DNA was isolated using QIAamp DNA Mini Kit and HPV was detected by amplification with PGMY09/11 primers and positive samples were genotyped by RFLP analyses and reverse line blot. The genomes of the HPV16 positive samples were sequenced, from which variant lineages were determined. Chi2 statistics was performed to test the association of HPV16 variants between case and control groups. The prevalence of HR-HPV types in <CIN1, CIN2 and CIN3+ were 33.7%, 84.4% and 91.6%, respectively. Thirty-eight of 49 (78%) HPV16 positive samples yielded HPV16 sequence information; of which, 32 complete genomes were sequenced and an additional 6 samples were partially sequenced. Phylogenetic analysis and patterns of variations identified 65.8% (n = 25) as HPV16 European (E) and 34.2% (n = 13) as non-European (NE) variants. Classification of disease into CIN3+ vs. <CIN3 indicated that NE types were associated with high-grade disease with an OR = 4.6 (1.07-20.2, p = 0.05). The association of HPV16 NE variants with an increased risk of CIN3+ is consistent with an HPV16 genetically determined enhanced oncogenicity. The prevalence of genetic variants of HPV16 is distributed across different geographical areas and with recent population admixture, only empiric data will provide information on the highest risk HPV16 variants within a given population.

Dataset Information

Identification of reliable biomarkers of human papillomavirus 16 methylation in cervical lesions based on integration status using high-resolution melting analysis.

Publications

Identification of reliable biomarkers of human papillomavirus 16 methylation in cervical lesions based on integration status using high-resolution melting analysis.

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