Human Papillomavirus Type 16 Integration in Cervical Carcinoma In Situ and in Invasive Cervical Cancer

Department of Molecular Genetics and Microbiology, School of Medicine, Health Sciences Center, University of New Mexico, Albuquerque, NM 87111, USA.
Journal of Clinical Microbiology (Impact Factor: 3.99). 06/2006; 44(5):1755-62. DOI: 10.1128/JCM.44.5.1755-1762.2006
Source: PubMed


Integration of human papillomavirus type 16 (HPV-16) into the host DNA has been proposed as a potential marker of cervical
neoplastic progression. In this study, a quantitative real-time PCR (qRT-PCR) was used to examine the physical status of HPV-16
in 126 cervical carcinoma in situ and 92 invasive cervical cancers. Based on criteria applied to results from this qRT-PCR
assay, HPV-16 was characterized in carcinoma in situ cases as episomal (61.9%), mixed (i.e., episomal and integrated; 29.4%),
and integrated (8.7%) forms. In invasive cervical cancer samples, HPV-16 was similarly characterized as episomal (39.1%),
mixed (45.7%), and integrated (15.2%) forms. The difference in the frequency of integrated or episomal status estimated for
carcinoma in situ and invasive cervical cancer cases was statistically significant (P = 0.003). Extensive mapping analysis of HPV-16 E1 and E2 genes in 37 selected tumors demonstrated deletions in both E1 and
E2 genes with the maximum number of losses (78.4%) observed within the HPV-16 E2 hinge region. Specifically, deletions within
the E2 hinge region were detected most often between nucleotides (nt) 3243 and 3539. The capacity to detect low-frequency
HPV-16 integration events was highly limited due to the common presence and abundance of HPV episomal forms. HPV-16 E2 expressed
from intact episomes may act in trans to regulate integrated genome expression of E6 and E7.

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Available from: Hugo Arias-Pulido
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    • "In genital HPVs, the E6-E7 promoter (such as P97 in HPV16) is suppressed by E2, because binding of the E2 protein can displace important cellular transcriptional activators (SP-1 and TFIID) from their adjacent binding sites [12, 13]. HPV16 with intact E2 in the episomal form are often found in cervical carcinomas [14, 15], reflecting the lack of suppression of the E6 and E7 genes caused by the E2 protein. Therefore, a factor that could contribute to differences in biological behavior of hr-HPV is DNA sequence variation. "
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    ABSTRACT: HPV16 E2 variants have different effects on the transcriptional activity of the LCR. In this study, we examined the nucleotide and amino acid sequence variation within the HPV16 E2 gene and to correlate with disease progression. E2 gene disruption was detected by PCR amplification of the entire E2 gene using a single set of primers. Nucleotide variations were analyzed by bidirectional sequencing. mRNA expression patterns of E6 and E7 gene transcripts were evaluated by a reverse transcriptase-PCR method (RT-PCR). The detection of intact E2 genes was significantly higher among controls than cases (81.8% versus 37.5%, resp., P < 0.05). Among the E subgroup, variation at position 3684 C>A results in the amino acid substitution T310K and was more common among the E2 undisrupted cases (7/9; 77.7%), compared to controls (2/9; 22.2%). In addition, specific sequence variations identified in the E2 ORF at positions 3684 C>A were associated with increased viral oncogenes E6-E7 production. Besides HPV16 E2 disruption, the 3684 C>A variation within undisrupted E2 genes could be involved in an alternative mechanism for deregulating the expression of the HPV16 E6 and E7 oncogenes and appears to be a major factor contributing to the development of cervical cancer in Tunisian women.
    Full-text · Article · Jun 2014 · BioMed Research International
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    • "Of the fully integrated genomes (n = 12), samples lacking only the Ct E2 fragment or lacking both the Nt and Ct E2 fragments were more common (n = 4 [33%] and n = 7 [58%], respectively) than was disruption of the Nt E2 fragment alone (n = 1; 8%). Where this has been examined, studies report that disruption is commonly found in the Nt fragment of the E2 gene and entire E2 deletion is rare [13,15,17,35]. "
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    ABSTRACT: Background Persistent 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. Methods Samples 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. Results Viral 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. Conclusions This study lends support for HPV viral load and CpG methylation status, but not integration status, to be considered as potential biomarkers of cervical disease.
    Full-text · Article · May 2014 · BMC Cancer
    • "Unlike HPV18 which shows high frequency of integrated viral genomes in cervical carcinomas, only a proportion of cases ranging from 28 to 67 per cent, depending on the techniques used, demonstrate presence of integrated HPV16 in invasive cervical carcinoma1112. Some studies have demonstrated that integration of the HPV genome has also been found in low-grade lesions and even in normal cervices131415 whereas others have shown that not all invasive cancers carry the integrated HPV genome1416. However, besides a few sporadic reports417, data related to viral load and its physical status particularly with reference to HPV16- are lacking. "
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    ABSTRACT: Background & objectives: High-risk human papilloma virus (HR-HPV) infection and its integration in host genome is a key event in malignant transformation of cervical cells. HPV16 being a dominant HR-HPV type, we undertook this study to analyze if viral load and physical state of the virus correlated with each other in the absence of other confounding variables and examined their potential as predictors of progressive cervical lesions. Methods: Both, viral load and integration status of HPV16 were determined by real time URR PCR and estimation of E2:E6 ratio in a total of 130 PGMY-RLB -confirmed, monotypic HPV16-infected cervical DNA samples from biopsies of cytology-confirmed low grade (LSIL, 30) and high grade (HSIL, 30), and invasive carcinoma, (squamous cell carcinoma SCC, 70) cases. Results: Investigation of DNA samples revealed a gradual increase in HPV16 viral load over several magnitudes and increased frequency of integration from LSIL to HSIL and HSIL to invasive cancer in relation to the severity of lesions in monotypic HPV16-infected cervical tissues. In a substantial number of precancer (11/60) and cancer cases (29/70), HPV16 was detected in concomitant mixed form. The concomitant form of HPV16 genome carried significantly higher viral load. Interpretation & conclusions: Overall, viral load and integration increased with disease severity and could be useful biomarkers in disease progression, at least, in HPV16-infected cervical pre-cancer and cancer lesions.
    No preview · Article · Apr 2014 · The Indian Journal of Medical Research
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