The integration of HPV-18 DNA in cervical carcinoma.

AIMS: Little information is available on the patterns of integration into the host chromosomal DNA of cervical carcinomas of human papillomavirus type 18 (HPV-18) DNA, which is associated with up to 20% of these carcinomas. Because integration of the viral genome may be extremely important in the pathogenesis of cervical carcinoma, the aim of this study was to investigate which regions of HPV-18 DNA are integrated into the cellular DNA of cervical carcinomas. METHODS: Southern analysis using four subgenomic probes covering the entire HPV-18 genome was used to map viral DNA integrated within cellular DNA. The polymerase chain reaction (PCR) was used to confirm the presence of specific regions of the viral genome. RESULTS: In all 11 carcinomas there was a single major HPV-18 DNA integrant, retaining approximately 4000 bp of HPV-18 DNA, indicating that approximately half of the virus genome had been lost upon integration. Southern analysis suggested strongly that the viral breakpoint was within the E1/E2 gene boundary, with concomitant loss of part or all of the E2 ORF (open reading frame), all of the E4, E5, and L2 ORFs and part of the L1 ORF. These data were supported by the PCR results, which confirmed that the region of integrated HPV-18 DNA from nucleotides 6558 to 162 was present in all the carcinoma samples studied. Assuming that no genomic rearrangements, deletions, or insertions had occurred, 4131 bp of integrated HPV-18 DNA could be accounted for in eight cervical carcinoma samples. The results of Southern analysis also suggested that integration of HPV-18 DNA may have occurred at a specific host chromosomal site. CONCLUSIONS: Broadly, the viral sequences retained upon HPV-18 integration resemble those found when HPV-16 is integrated. However, it appears that the HPV-18 E2 region is more consistently deleted.     

Integration of human papillomavirus type 16 into cellular DNA of cervical carcinoma: preferential deletion of the E2 gene and invariable retention of the long control region and the E6/E7 open reading frames

The integration patterns of human papillomavirus (HPV) type 16 in the cellular DNA of six cervical carcinoma samples were analyzed by the Southern blot procedure. None of the HPV integrants retained the entire viral genome. Double HPV integration was found in one case while all other cases were single integrants. In some samples, internal deletion and selective amplification of the viral sequences were observed. On integration, the E2 open reading frame (ORF) was invariably lost but the E6/E7 ORFs and the long control region of the HPV-16 genome were retained in all seven integrations analyzed and may play a role in cellular transformation and/or maintenance of the transformed phenotype.     

DNA sequence of the HPV-16 E5 ORF and the structural conservation of its encoded protein

Infection of cervical epithelium by human papillomavirus type 16 (HPV-16) appears to be closely associated with the development of cervical dysplasia and carcinoma. By inference from genetic and biochemical studies of the bovine papillomavirus, the E5 ORF of the human papillomaviruses is anticipated to encode a "transforming" protein. In an effort to compare the E5 ORF of HPV-16 with other human papillomaviruses and bovine papillomavirus, we sequenced this region from a new isolate of HPV-16 which was derived from extrachromosomal viral DNA within a premalignant cervical lesion (cervical intraepithelial neoplasia, grade III, or CIN III). In addition, we also sequenced the original isolate of HPV-16 (derived from integrated viral DNA by Durst et al. [Proc. Natl. Acad. Sci. USA 80, 3812-3815 (1983)] and sequenced by Seedorf et al. [Virology 145, 181-185 (1985)]. Both HPV-16 isolates contained an additional nucleotide (T) at bp 3906. This nucleotide addition caused a frameshift in the E5 ORF such that it now contains an initiation codon at bp 3849; the frameshift also alters the predicted E5 NH2 terminus but retains the original COOH half of the protein. E5 proteins encoded by several HPVs which infect the genital region (e.g., types 6, 11, 16, 18, 33) exhibit a conserved trimodal hydrophobic structure, but not a conserved amino acid sequence.     

Recurrent integration of papillomavirus DNA within the human 12q14–15 uterine breakpoint region in genital carcinomas

Genital carcinomas are associated with human papillomaviruses, and the viral DNA is frequently integrated in the host cell genome. Recurrent chromosomal alterations are genetic markers for specific tumor phenotypes. To demonstrate that papillomavirus DNA integration is indeed a recurrent chromosomal aberration, we mapped two independent papillomavirus integration sites in the human 12q14–15 region, one containing HPV16 DNA and the other HPV18 DNA. The two HPV integration sites map approximately 10 kbp from each other within the cosmid LLNL12NCO1–196E1 clone. The integration site corresponding to HPV16 DNA in SK-v cells is proximal to the 5′ end of a DNA segment known to be rearranged by integration of HPV18 DNA in another cervical carcinoma cell line, SW756. Both integrations are located in the PAL2 locus within the uterine leiomyoma cluster region of translocation. Genes Chromosomes Cancer 23:55–60, 1998. © 1998 Wiley-Liss, Inc.     

Physical status of HPV-16 in esophageal squamous cell carcinoma.

BACKGROUND: Infection with high-risk human papillomavirus (HPV) has been implicated as one of the risk factors of esophageal squamous cell carcinoma (ESCC). Integration of viral DNA into host genome is essential for carcinogenesis since it promotes disruption of the HPV E2 gene, leading to abnormal expression of E6 and E7 oncoproteins. OBJECTIVES AND STUDY DESIGN: To investigate the viral integration status of HPV-16 infection in ESCC, 35 HPV-positive ESCC specimens collected from Chinese patients were subject to real-time quantitative PCR for determination of physical status of HPV-16 by analyzing the ratios of E2 to E6 genes. RESULTS: Our results showed that only 8.6% (3/35) of the HPV-16 positive specimens harbored exclusively the episomal form (i.e. E2/E6 ratio > or = 1), whereas the remaining 91.4% contained either only the integrated form (5.7%, with E2/E6 ratio = 0) or a mixture of episomal and integrated forms of viral molecules (85.7%, with E2/E6 ratios > 0 but < 1). Amongst the 30 cancer specimens carrying mixed integrated and episomal forms, 28 had E2/integrated E6 ratios of less than 1, indicating a predominance of integrated form of viral genes in these lesions. CONCLUSION: Our finding of frequent integration of viral DNA in the host genome suggests that integration HPV-16 is common in ESCC from Chinese patients and implies that HPV infection may play a role in the pathogenesis of ESCC.     

Correlation between laminin-5 immunohistochemistry and human papillomavirus status in squamous cervical carcinoma

BACKGROUND: Human papillomavirus (HPV) plays a critical role in the carcinogenesis of squamous cervical carcinoma. Integration of viral DNA into the host genome is a major contributing factor to malignant transformation. Viral load may influence integration. AIMS: To compare HPV status (type, viral load, integration status) between normal samples, carcinoma in situ and invasive carcinoma in order to elucidate the role of HPV in progression to invasive lesions. METHODS: The study population comprised 10 biopsy samples from each diagnostic group. Laminin-5 immunohistochemistry was performed to distinguish invasive carcinoma from non-invasive high-grade lesions. Real-time PCR was used to detect specific HPV types, viral load and integrated HPV, with quantification of viral E2 and E6 genes. RESULTS: Invasive carcinomas contained a higher number of laminin-5 immunoreactive cells as compared to non-invasive lesions. Almost all samples contained HPV, with a higher viral load and copy number of HPV16 integrated in E2 in cases of laminin-5 immunoreactivity and cases of invasive carcinoma. High HPV16 viral load was associated with more integrated copies in E2. CONCLUSIONS: HPV is important in progression from carcinoma in situ to invasive carcinoma. Viral load and HPV integration influence the development of cervical cancer towards invasiveness. Overall HPV status may be more predictive of patient outcome and may influence patient management.     

Analysis by Multiplex PCR of the Physical Status of Human Papillomavirus Type 16 DNA in Cervical Cancers

Integration of human papillomavirus (HPV) DNA occurs early in cancer development and is an important event in malignant transformation of cervical cancer. Integration of HPVs preferentially disrupts or deletes the E2 open reading frame, which results in the loss of its expression. The preferential disruption of the E2 gene causes the absence of the E2 gene sequences in the PCR product following integration. Twenty-two carcinomas positive for HPV type 16 (HPV-16) DNA were first tested for the disruption of the E2 gene by PCR. A specific fragment of the E2 gene was not amplified in 10 cases, suggesting integration of HPV DNA into the host genome. Next, multiplex PCR for the HPV E2 and E6 genes was carried out in the remaining 12 cases. Copy numbers of both genes should be equivalent in episomal forms, while the E2 gene copy number will be smaller than that for E6 following the preferential disruption of the E2 gene in concomitant forms. Although relative ratios of HPV E2 to E6 PCR products (E2/E6 ratios) ranged from 1.40 to 2.34 in 10 of 12 cases, multiplex PCR products from 2 cases displayed extremely low ratios of 0.69 and 0.61. Southern blot hybridization with an HPV-16 probe revealed that only in these two cases was both episomal and integrated HPV DNA being carried simultaneously. Thus, multiplex PCR for the E2 and E6 genes of HPV-16 DNA following PCR for the E2 gene can distinguish the pure episomal form from a mixed form of episomal and integrated HPV DNA. Clinical application of this technique will help researchers to understand the implication of the integration of HPV DNA for cervical carcinogenesis and cervical cancer progression.     

Human papillomavirus type 16 integration in cervical carcinoma in situ and in invasive cervical cancer

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.     

Frequent genomic structural alterations at HPV insertion sites in cervical carcinoma

To investigate whether integration of HPV DNA in cervical carcinoma is responsible for structural alterations of the host genome at the insertion site, a series of 34 primary cervical carcinomas and eight cervical cancer‐derived cell lines were analysed. DNA copy number profiles were assessed using the Affymetrix GeneChip Human Mapping 250K Sty array. HPV 16, 18 or 45 integration sites were determined using the DIPS‐PCR technique. The genome status at integration sites was classified as follows: no change, amplification, transition normal/gain, normal/loss or gain/LOH. A single HPV integration site was found in 34 cases; two sites were found in seven cases; and three sites in one case (51 sites). Comparison between integration sites and DNA copy number profiles showed that the genome status was altered at 17/51 (33%) integration sites, corresponding to 16/42 cases (38%). Alterations detected were amplification in nine cases, transition normal/loss in four cases, normal/gain in three cases, and gain/LOH in one case. A highly significant association was found between genomic rearrangement and integration of HPV DNA (p < 10^?10). Activation of the replication origin located in viral integrated sequences in a cell line derived from one of the primary cervical carcinomas induced an increase of the amplification level of both viral and cellular DNA sequences flanking the integration locus. This mechanism may be implicated in the triggering of genome amplification at the HPV integration site in cervical carcinoma. Structural alterations of the host genome are frequently observed at the integration site of HPV DNA in cervical cancer and may act in oncogenesis. Copyright © 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Study of viral integration of HPV-16 in young patients with LSIL

AIMS: To investigate the physical status of human papillomavirus 16 (HPV-16) in low grade squamous intraepithelial lesions (LSILs) as a means of determining the percentage of viral integration. METHODS: Ninety two LSIL/HPV positive Thin Prep(TM) samples were initially tested for the E6 gene by the polymerase chain reaction (PCR) to identify the HPV-16 virus. To avoid false positive results, the specificity of the bands obtained from PCR was confirmed by Southern blot hybridisation with internal oligonucleotide probes. Next, a PCR screen for the E2 gene was performed to identify those samples in which the virus was integrated. Viral integration was detected in just over half of them. RESULTS: Twenty of the 92 samples were HPV-16 positive, as shown by PCR for the E6 gene. Southern blot analysis confirmed that 13 of these samples were positive for the viral E6 gene. Thus, viral integration was detected in just over a half of the samples positive for HPV-16. CONCLUSIONS: These data show that HPV-16 integration occurs in a subset of LSILs. The measurement of HPV-16 integration would be a helpful complementary tool for cytological evaluation in primary cervical screening to identify those patients at risk of developing high grade squamous intraepithelial lesions and cervical cancer.     

The prevalence of HPV-18 and variants of E6 gene isolated from cervical cancer patients in Taiwan.

BACKGROUND: Cervical cancer is the second most common cancer in women worldwide. It has been considered that human papillomavirus (HPV) is associated with cervical cancer. Currently, more than 80 different serotypes of HPV have been characterized and they are divided into low- and high-risk groups. The most common types that lead to cervical cancer are HPV-16 and -18. The viral oncogenes E6 and E7 are associated with the development of cervical cancer. In previous study, the variants of HPV-16 E6 gene have been reported. It suggests that variants may influence the morbidity of carcinogenesis, but the variant study on HPV-18 remains unknown. OBJECTIVES: To identify the variants of integrated HPV-18 E6 gene in the prevalent infection of HPV-18 of cervical cancer patients. STUDY DESIGN: 25 cervical cancer patients were clinically identified and the biopsies were obtained. The infectious HPV types were identified by PCR and Southern blotting analysis. The DNA fragments of the integrated HPV-18 E6 were amplified by PCR and cloned. The nucleotide sequences were obtained by sequencing. RESULTS: The prevalence of HPV infection in our 25 cases was HPV-18 (100%) and 7 out of these 25 cases (28%) were co-infected with HPV-16. The most dominant mutation among 25 tested patients was a silence mutation C183G of the E6 coding region. CONCLUSIONS: The prevalent HPV infectious serotype is HPV-18, which differs from the worldwide prevalent type. The identified HPV-18 E6 variants had a unique silence mutation located on C183G in E6 coding region.     

Integrated human papillomavirus type 16 is frequently found in cervical cancer precursors as demonstrated by a novel quantitative real-time PCR technique

In contrast to cervical cancer, integration of human papillomavirus (HPV) DNA into the host genome has been considered a rare event in cancer precursor lesions (cervical intraepithelial neoplasia [CIN]). With our new real-time PCR method, we demonstrated that integrated HPV type 16 (HPV16) is already present in CIN lesions. The physical state of HPV16 and the viral load were simultaneously detected. A unique region of the E2 open reading frame (ORF) that is most often deleted during HPV16 integration is targeted by one set of PCR primers and a probe, and another set targets the E6 ORF. In episomal form, both targets should be equivalent, while in integrated form, the copy numbers of E2 would be less than those of E6. The method was tested with DNAs from 31 cervical lesions (non-CIN to CINIII) from 24 women prospectively followed up for 10 years. This report presents viral load and integration results from the largest series of CIN lesions described to date. Only one sample contained exclusively episomal HPV16 DNA, and this lesion regressed spontaneously. Samples from another patient, with only integrated HPV16, rapidly progressed from CINI to CINIII in 2 years. In all other patients, episomal and integrated forms of HPV16 DNA were found to coexist. Rapid progression of the CIN lesions was closely associated with a heavy load of integrated HPV16. Thus, the method described here is a very sensitive tool with which to assess the physical state of HPV, which is useful in predicting disease progression.     

A subtype of human papillomavirus 5 (HPV-5b) and its subgenomic segment amplified in a carcinoma: nucleotide sequences and genomic organizations.

A subtype of human papillomavirus 5 (HPV-5b) is closely associated with carcinomas in the disease epidermodysplasia verruciformis (EV). The complete genome was cloned from virus particles in benign lesions of a patient with EV and sequenced: it was 7779 nucleotides long and consisted of six open reading frames (ORFs) (E6, E7, E1, E2, E4, and E5) in the early region, three ORFs (L2, L3, and L1) in the late region, and a noncoding region, all existing on one DNA strand. The 40% segment of the HPV-5b genome specifically amplified in carcinomas was cloned from a primary carcinoma of the same EV patient and sequenced: it was 3143 nucleotides long and corresponded to a segment of the original HPV-5b genome containing the entire sequences of E6, E7, and the noncoding region and portions of E1 and L1. Compared to the whole genomic DNA, no mutations were detected in this probable malignancy-associated viral subgenomic segment cloned from carcinoma. These results suggest that amplification of the viral segment containing E6, E7, and the noncoding region may play a role in the malignant conversion of HPV-5b-infected benign lesions and that mutations in these genes or regions are not necessarily required.