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PubMed2004

Transition of high-grade cervical intraepithelial neoplasia to micro-invasive carcinoma is characterized by integration of HPV 16/18 and numerical chromosome abnormalities.

Cervical intraepithelial neoplasia (CIN I, II, and III) and cases of CIN III associated with micro-invasive cervical carcinoma (CIN III & mCA) were analysed for evidence of episomal or integrated human papillomavirus (HPV) 16/18 DNA by fluorescence in situ hybridization (FISH). In parallel, numerical aberrations of chromosomes 1, 17, and X were determined in these lesions as indicators of genomic instability. HPV 16/18 DNA was present in 2 of 12 CIN I, 19 of 23 CIN II/III, and 10 of 12 CIN III & mCA. None of the CIN I and only two of the 19 HPV 16/18-positive solitary CIN II/III showed an integrated HPV pattern. However, all ten cases of HPV-positive CIN III & mCA showed this pattern. Transition of CIN II/III to CIN III & mCA therefore correlates strongly with viral integration (p<0.001). Chromosomal aberrations were detected in 23 of 31 HPV 16/18-positive lesions (14 solitary CIN I-III and nine CIN III & mCA) and 5 of 16 HPV-negative lesions. Nine of 21 HPV 16/18-positive solitary CIN I-III showed tetrasomy for all chromosomes tested, while trisomies for a single chromosome were seen in a further five of these HPV-positive lesions. In eight of ten HPV-positive CIN III & mCA, predominantly aneusomies and/or polysomies were detected. A significant correlation (p<0.02) was found between the chromosome copy number and the physical status of HPV, indicating that in its episomal form HPV induces genomic changes such as tetrasomies and single trisomies, while HPV integration correlates with aneusomies and polysomies, predominantly detected in CIN III & mCA. These data indicate that integration of HPV 16/18 DNA is a pivotal step in the transition of CIN to micro-invasive carcinoma.

Adult↗

PubMed1989

[Biological significance of HPV sequences integrated into cancer cells].

In cervical cancer with integrated human papillomavirus (HPV) type 16 or 18 DNA, E6 and E7 regions of HPV 16 or 18 were always present, suggesting that these regions play an important role in development of cervical cancer. Structure and biological activity of cDNAs representing two types of HPV 18 transcripts in HeLa cells were analyzed. Three open reading frames (E6, E6* and E7) were identified. These open reading frames were introduced into the expression vectors. Among them, E7 alone provided NIH3T3 cells with the ability of anchorage-independent growth. Furthermore, introduction of E7 into mouse primary keratinocytes lead to immortalization of the cells. We also isolated cDNA clones of HPV 16 transcripts from the NIH3T3 transformant which had been obtained by transfection of genomic DNA from a cervical cancer tissue containing HPV 16 DNA. The structure of some of these cDNA clones were analyzed. The clones consisted of joined viral and human sequences and had entire E7 open reading frame together with spliced or truncated E6 open reading frame. These clones were subcloned into the expression vectors. NIH3T3 cells transfected separately by these constructs grew in soft agar but were not tumorigenic in nude mice.

Animals↗

PubMed2006

Distribution and viral load of eight oncogenic types of human papillomavirus (HPV) and HPV 16 integration status in cervical intraepithelial neoplasia and carcinoma.

Current human papillomavirus (HPV) DNA testing using pooled probes, although sensitive, lacks specificity in predicting the risk of high-grade cervical intraepithelial neoplasia (CIN 2/3) progression. To evaluate selected HPV genotyping, viral load, and viral integration status as potential predictive markers for CIN progression, we performed HPV genotyping in formalin-fixed, paraffin-embedded cervical tissue with cervical carcinoma (29 cases) and CINs (CIN 1, 27 cases; CIN 2, 28 cases; CIN 3, 33 cases). General HPVs were screened using consensus primers GP5+/GP6+ and PGMY09/11. HPV genotyping and viral load measurement were performed using quantitative real-time PCR for eight oncogenic HPV types (16, 18, 31, 33, 35, 45, 52, and 58). HPV 16 viral integration status was evaluated by measuring HPV 16 E2/E6 ratio. We observed that HPV DNA positivity increased in parallel with the severity of CINs and carcinoma, with 59% positivity in CIN 1, 68% in CIN 2, 76% in CIN 3, and 97% in carcinoma (P trend=0.004). The eight oncogenic HPV types were significantly associated with CIN 2/3 (81%) and carcinoma (93%) (odds ratio (OR), 15.0; 95% confidence interval (CI), 5.67-39.76; P<0.0001) compared with the unknown HPV types (OR, 2.87; 95% CI, 0.89-9.22; P=0.08). HPV 16 was the predominant oncogenic HPV type in CIN 2/3 (51%) and carcinoma (71%) and integrated significantly more frequently in carcinoma than in CIN 2/3 (P=0.004). No significant differences in viral load were observed across the disease categories. Our findings suggest that selected genotyping for the eight oncogenic HPV types might be useful in separating women with a higher risk of CIN progression from those with a minimal risk. We also conclude that the HPV 16 integration status has potential to be a marker for risk assessment of CIN progression.

Adenocarcinoma↗

PubMed1992

Analysis by polymerase chain reaction of the physical state of human papillomavirus type 16 DNA in cervical preneoplastic and neoplastic lesions.

Integration of human papillomavirus (HPV) DNA into the host cell genome is believed to be essential for malignant progression. However unambiguous detection of the physical state of HPV is a difficult and time-consuming procedure. To resolve this issue a simple, rapid and highly sensitive technique of polymerase chain reaction (PCR) has been utilized for detecting the physical state of HPV-16 DNA. Investigations were carried out in 122 cervical specimens comprising the whole spectrum of cervical lesions starting from cervical dysplasia to invasive carcinoma including HPV-16-positive normal controls. A pair of oligonucleotide primers specific to the E2 open reading frame, which is often deleted or disrupted following HPV integration, was used for the study. Distinction between episomal and integrated forms of viral DNA was accomplished by detecting amplification of the E2-specific fragment (1139 bp) in the PCR product. The PCR results were compared with those obtained by the conventional methods of Southern blotting, two-dimensional gel electrophoresis and chromosomal in situ hybridization; a high degree of agreement was observed between the methods. The findings indicate that although integrated forms of HPV-16 DNA were detected in more than 70% of cervical cancer specimens, integration was less frequent (23%) in severe dysplasia and carcinoma in situ. Only 2.5% of cases showed both episomal and integrated forms of HPV-16 DNA. The difference between episomal and integrated forms was statistically significant (P less than 0.01). The absence of integration in about 30% of cancer cases suggests that integration of HPV may not be necessary for malignant progression and alternative mechanism(s) of malignant transformation may occur without HPV integration. The PCR test thus provides an effective complement to Southern blotting and two-dimensional gel electrophoresis for accurate detection of the integration of HPV DNA.

Base Sequence↗

PubMed1987

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.

Base Sequence↗

PubMed1999

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.

Adult↗

PubMed1995

Jun-B oncogene aberrations in cervical cancer cell lines.

We describe here structural and expression analysis of the jun-B oncogene in two cervical cancer cell lines. In the CC7T-a cell line, results from both Southern analysis and cDNA cloning studies revealed the existence of two structurally altered jun-B alleles besides the normal gene. One of the altered alleles was due to a type 16 human papillomavirus (HPV-16) integration event, whereas the other allele was a consequence of a chromosomal translocation involving chromosome 19 (jun-B) and an EST182 locus residing in chromosome 15. In the HeLa cell line, which contains integrated HPV-18, an apparent structural aberration, a 3-fold amplification and a 3-fold overexpression of the jun-B gene were observed. Our observations suggest that deregulation of the jun-B gene expression may have contributed to the transformation process in these two cervical cancer cell lines.

Alleles↗

PubMed1989

A novel deletion within the upstream regulatory region of episomal human papillomavirus type 16.

The pattern of human papillomavirus type 16 (HPV-16) integration was studied in 23 invasive carcinomas of the cervix using subgenomic probes. Seventeen tumours contained integrated HPV-16 and in 13 of these there was evidence of disruption within the E1-E2 open reading frames (ORFs). In all cases the upstream regulatory region (URR)-E6-E7 ORFs was maintained intact. Two independently derived tumours were infected with episomal wild-type HPV-16 and an episomal variant of HPV-16 containing a 325 bp deletion within the URR (positions 7598 to 17) and a point mutation at position 20 (A to C). This is the first report of a variant HPV-16 which is likely to be both defective and transmissible. Loss of E2 expression and deletion of a large portion of the URR may be two of the mechanisms leading to altered HPV-16 early gene expression in cervical tumours.

Adenocarcinoma↗

PubMed2004

Positioning of cervical carcinoma and Burkitt lymphoma translocation breakpoints with respect to the human papillomavirus integration cluster in FRA8C at 8q24.13.

Molecular cytogenetic analysis frequently shows human papillomavirus (HPV) integration near translocation breakpoints in cervical cancer cells. We have recently described a cluster of HPV18 integrations in the distal end of the common fragile site FRA8C at 8q24 in primary cervical carcinoma samples. Chromosome band 8q24 contains the MYC gene (alias c-MYC), FRA8C, and FRA8D. The MYC gene is frequently deregulated--usually by translocation or amplification--in various tumor types. In the present study, we performed a molecular cytogenetic analysis of HPV18 integration patterns and the 8q24 translocation in a primary cervical carcinoma and in HeLa cells using combined binary ratio-fluorescence in situ hybridization. Our aim was to determine how the chromosomal breaks involved in these events relate physically to the MYC gene; whether they map to the FRA8C site, the FRA8D site, or both; and how they correlate with the occurrence of DNA flexibility domains. The 8q24 translocation breakpoints mapped between stretches of integrated HPV18 sequences in the distal end of FRA8C. This region contained DNA helix flexibility clusters, several of which mapped in the vicinity of HPV integration sites and translocation breakpoints in cervical carcinomas. DNA helix flexibility clusters were also found near known MYC translocation breakpoints in Burkitt lymphomas (BL), but most BL breakpoints mapped clearly outside FRA8C. Our data revealed that FRA8C is involved in HPV integration and chromosomal translocations in cervical carcinoma; however, this fragile site is not involved in classical MYC translocations in most BLs. In the context of the familial nature of cervical cancer, FRA8C may be considered a candidate susceptibility region for cervical carcinoma.

Burkitt Lymphoma↗

PubMed2006

Use of multiple displacement amplification in the investigation of human papillomavirus physical status.

BACKGROUND AND AIMS: The investigation of human papillomavirus (HPV) physical status in pre-invasive cervical lesions has been restricted by the small amounts of tissue available for study. Multiple displacement amplification (MDA), a phi29 DNA polymerase based whole genome amplification technique, has the potential to help resolve this problem by yielding large amounts of high molecular weight DNA from tiny starting quantities. METHODS: Firstly, a comparison was made of restriction endonuclease fragment patterns of DNA from seven different HPV types and corresponding MDA products. Secondly, E6/E7 and LCR sequencing data from HPV16 recombinant plasmid and MDA copy DNA were correlated. Thirdly, DNA and MDA products from cervical cell lines (CaSki, HeLa, and SiHa that contain integrated HPV) and an invasive cervical carcinoma were analysed by Southern blot hybridisation. Fourthly, MDA product from CaSki cell DNA mixed with HPV18-plasmid DNA was tested for the demonstration of both episomal and integrated HPV. Finally, MDA products from HPV16 positive abnormal cervical cytological samples were assayed for integration by Southern blot hybridisation. RESULTS: DNA templates and MDA products yielded analogous data. Episomal and integrated HPV DNA were successfully detected by Southern blot assay of the cell line/HPV-plasmid model, and in MDA products of clinical samples. CONCLUSIONS: These data show that MDA has considerable potential to assist in the investigation of HPV physical status; abundant (>40 microg) DNA can be generated with high fidelity from minuscule (50 ng) starting quantities, and both episomal and integrated HPV DNA are distinguishable in MDA products from solid tumours and cytological materials.

Base Sequence↗

PubMed1995

Human papillomavirus and disease-free survival in FIGO stage Ib cervical cancer.

To determine if human papillomavirus (HPV) in the primary tumor was associated with disease-free survival of stage Ib cervical cancer patients, archival tissues from 47 patients were analyzed for HPV DNA by in situ hybridization (ISH) and polymerase chain reaction. HPV integration was determined by ISH signal pattern. Laboratory data were correlated with clinical parameters and disease-free survival. Kaplan-Meier estimates of 4-year disease-free survival were 56% in women with HPV detected in the primary tumor by ISH and 100% for women in whom HPV was not detected (P = .02). Four-year disease-free survival was 39% for patients with integrated HPV in the primary tumor (P = .005 vs. HPV-negative tumors and .05 vs. HPV episomal or episomal/integrated). HPV detection and integration state was not associated with any other clinical variable. Detection of integrated HPV DNA in the primary tumor was strongly associated with treatment failure.

Adult↗

PubMed2003

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.

Adult↗

PubMed1995

Human papillomavirus DNA in oesophageal carcinomas in South Africa.

Using morphological criteria, the presence of human papillomavirus (HPV) in oesophageal carcinomas has been inferred in patients from Finland and South Africa. However, studies to demonstrate the viral antigen in tissue sections of these tumours have proved disappointing. This study investigates 48 archival oesophageal carcinoma biopsies from South Africa for the presence of HPV DNA using non-isotopic in situ hybridization (NISH) with HPV DNA probes to HPV 6, 11, 16, 18, 31, and 33. HPV DNA sequences were detected in 25/48 (52 per cent) oesophageal cancers. HPV 16 was present in 84 per cent of the HPV-positive cancers. A NISH type 2 signal pattern (punctate/dot) was present in all HPV-positive tumours. This signal pattern was previously shown to represent integrated HPV DNA within host chromosome. Integrated HPV DNA in oesophageal cancers has also been demonstrated in patients from China and Japan. In addition, the prevalence of HPV DNA in oesophageal cancers from high-risk countries like South Africa (52 per cent) and China (49 per cent) would appear to be consistent.

Base Sequence↗

PubMed2002

Human papilloma viruses and cervical tumours: mapping of integration sites and analysis of adjacent cellular sequences.

BACKGROUND: In cervical tumours the integration of human papilloma viruses (HPV) transcripts often results in the generation of transcripts that consist of hybrids of viral and cellular sequences. Mapping data using a variety of techniques has demonstrated that HPV integration occurred without obvious specificity into human genome. However, these techniques could not demonstrate whether integration resulted in the generation of transcripts encoding viral or viral-cellular sequences. The aim of this work was to map the integration sites of HPV DNA and to analyse the adjacent cellular sequences. METHODS: Amplification of the INTs was done by the APOT technique. The APOT products were sequenced according to standard protocols. The analysis of the sequences was performed using BLASTN program and public databases. To localise the INTs PCR-based screening of GeneBridge4-RH-panel was used. RESULTS: Twelve cellular sequences adjacent to integrated HPV16 (INT markers) expressed in squamous cell cervical carcinomas were isolated. For 11 INT markers homologous human genomic sequences were readily identified and 9 of these showed significant homologies to known genes/ESTs. Using the known locations of homologous cDNAs and the RH-mapping techniques, mapping studies showed that the INTs are distributed among different human chromosomes for each tumour sample and are located in regions with the high levels of expression. CONCLUSIONS: Integration of HPV genomes occurs into the different human chromosomes but into regions that contain highly transcribed genes. One interpretation of these studies is that integration of HPV occurs into decondensed regions, which are more accessible for integration of foreign DNA.

Binding Sites↗

PubMed2003

Leptomycin B enhances CDDP-sensitivity via nuclear accumulation of p53 protein in HPV-positive cells.

Cervical cancer, which commonly contains a wild-type p53 gene, is highly correlated with human papilloma virus (HPV) infection. Because the oncoprotein E6, derived from HPV, inhibits the function of p53 protein, the inhibition of apoptosis via the p53 pathway by HPV may be related to cisplatin (CDDP)-sensitivity in cervical cancer. We conducted the present study to determine whether and how HPV is related to CDDP-sensitivity in HPV-positive cervical cancer cells. We used cervical carcinoma cell lines HeLa with integrated HPV 18 and SiHa with integrated HPV 16. An HPV-negative cell line, Yumoto, with wild-type p53 gene was used as a control. Leptomycin B (LMB) enhanced sensitivity to CDDP and CDDP-induced apoptosis in HeLa and SiHa cells, but not in Yumoto cells. After exposure to LMB or CDDP alone, we observed weak p53 staining in HeLa, SiHa and Yumoto cells. Nuclear p53 staining was significantly increased by combined treatment with CDDP and LMB in HeLa and SiHa cells, but not in Yumoto cells. The expression of p53 and Bax protein increased with exposure to CDDP and was enhanced by LMB in HeLa and SiHa cells. The present study demonstrated that LMB enhanced CDDP-sensitivity via nuclear accumulation of p53 protein in HPV-positive cells.

Antineoplastic Combined Chemotherapy Protocols↗

PubMed2005

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.

Adult↗

PubMed2026

Charting host structural variations in cervical cancer by long-read sequencing pinpoints a functional deletion in PIAS1.

Host structural variations (SVs) are critical in cancer development but their landscape and interaction with HPV integration in cervical carcinogenesis remain unclear. In this study, we performed Nanopore long-read sequencing on five HPV-positive cervical cancer tissues and two cell lines to profile host SVs. We identified thousands of SVs and statistically demonstrated their significant enrichment in genomic windows ±25 to ±50 kb from HPV integration sites. Cross-sample analysis revealed 60 shared SVs, including a recurrent deletion within the PIAS1 gene. Multi-omics integration (Hi-C, H3K27ac ChIP-seq, and TCGA data) showed that this deletion is associated with reduced PIAS1 expression, disruption of local topologically associating domains, advanced pathological tumor stage, and poorer overall survival. Functional assays confirmed that PIAS1 deficiency inhibits cervical cancer cell proliferation and migration. Our findings identify a PIAS1 deletion as a candidate driver event, and underscore the pivotal role of host genomic instability in HPV-associated oncogenesis.

Cervical cancer↗

PubMed2003

Common fragile sites are preferential targets for HPV16 integrations in cervical tumors.

The development of cervical cancer is highly associated with human papillomavirus (HPV) infection. HPV integration into the genome of infected cervical cells is temporally associated with the acquisition of the malignant phenotype. A relationship between the sites of HPV integration in cervical cancer and the position of the common fragile sites (CFSs) has been observed at both the cytogenetic and molecular levels. To further explore this relationship at the molecular level, we used RS-PCR to rapidly isolate cellular sequences flanking the sites of HPV16 integration in 26 primary cervical tumors. Human bacterial artificial chromosome clones were isolated based on these flanking sequences and used as probes for fluorescence in situ hybridization on aphidicolin-stimulated metaphases. Our data demonstrate that 11/23 HPV16 integrations in cervical tumors occurred within CFSs (P&<0.001). In addition, we show that deletions and complex rearrangements frequently occur in the cellular sequences targeted by the integrations and that integrations cluster in FRA13C (13q22), FRA3B (3p14.2), and FRA17B (17q23). Finally, our data suggest that cellular genes, such as Notch 1, are disrupted by the HPV16 integrations, which may contribute to the malignant phenotype.

Carcinoma, Squamous Cell↗