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PubMedDate not supplied

Integration of HPV-16 DNA in cervical carcinoma cell line CC3/CUHK3 and its xenografts.

A recently established cell line, designated CC3/CUHK3, derived from a squamous cell carcinoma of the uterine cervix of a Hong Kong Chinese patient was investigated for its association with human papillomavirus (HPV) by Southern blot hybridization studies. A "grafted tumour" or xenograft of CC3/CUHK3 has also been successfully established in athymic mice, which showed similar histology to that of the original biopsy. The epithelial nature of the xenografts, like the original biopsy. The epithelial nature of the xenografts, like the original tumour specimen, was also confirmed by transmission electron microscopy which demonstrated the presence of desmosomes and tonofilaments. Moreover transmission electron microscopic examination revealed no HPV particle in either the tumour biopsy or the xenograft tissues. Analysis of the DNA samples extracted from the cervical cancer cell line and the xenograft tissues derived from it showed the presence of HPV type 16 DNA. No DNA sequence related to HPV type 6, 11, or 18 was demonstrated. The viral DNA was found to be integrated into the cellular genome at multiple sites as single copy or head-to-tail tandem repeats. Deletion or rearrangement of the HPV DNA had probably occurred on or subsequent to integration. Viral sequence deletion has also been observed in the grafted tumours derived from CC3/CUHK3.

Animals↗
PubMed2005

Subvisual chromatin changes in cervical epithelium measured by texture image analysis and correlated with HPV.

OBJECTIVES: In this study, we are testing the hypothesis that human papillomavirus (HPV) positivity is correlated with chromatin texture in the cell. Interim analyses are important since this study involves 2000 patients and generates 6000 biopsy specimens that will be subjected to quantitative histopathological analysis and correlated to HPV positivity as measured by the Hybrid Capture II test (Digene; Gaithersberg, MD) and both HPV-DNA and mRNA by the polymerase chain reaction (PCR). The studies of optical technologies, from which we derive this sample, use the colposcopically directed and histopathologically classified cervical biopsy as the gold standard. In this report, we describe the results of an interim analysis of quantitative histopathology and chromatin texture as correlates of HPV infection using the cyto-savant system in cytologically and histopathologically negative specimens. METHODS: A group of 1544 patients entered the optical technology trials, generating 3275 biopsies and 1544 Papanicolaou readings. Two hundred forty-eight patients were cytologically and histopathologically negative. Study pathologists reviewed histologic samples 3 times in a blinded fashion. Non-overlapping, quantitatively stained nuclei were selected from the samples by the pathologists. HPV testing was done using the PCR method and the Hybrid Capture II test. Statistical analysis involved the creation of a classification matrix using a linear discriminant analysis. The matrix was trained on HPV-positive cells by PCR. The analysis included the random creation of both a training set and a validation set that were classified based on the discrimination score obtained by correlating nuclear texture with HPV positivity. RESULTS: The sensitivity of the classification was 52-54% and the specificity was 77-78%. Overall, a 68% predicted accuracy was achieved for both the training set and the test set. The agreement of a test and training set shows that the sets created randomly are indeed similar, and that the discrimination score worked equally well in both sets of cells. Once a cell-by-cell algorithm for HPV positivity was derived, HPV positivity was recalculated on the basis of cell-by-cell texture features. HPV positivity was then recalculated on both a per-biopsy basis and a per-patient basis. For HPV 16 and 18, the positivity rate was 70% on a per-biopsy basis and 73% on a per-patient basis. CONCLUSIONS: Although these results are preliminary, they suggest that texture features reflecting chromatin condensation may correlate with HPV positivity. The current sample is histologic, the analysis suggests that in a cytologic sample, HPV positivity could be detected or confirmed by texture features computed as part of an HPV-associated score. Additional biologic markers could be used as needed. While this study was performed on histologic samples, a study of cytologic samples would be more useful. Future studies will examine chromatin texture compared to HPV integration and mRNA HPV expression.

Adolescent↗
PubMed2000

Human papillomavirus type 16 integrations in cervical tumors frequently occur in common fragile sites.

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 the cytogenetic level. To explore this relationship at the molecular level, we used a PCR-based method to rapidly isolate cellular sequences flanking the sites of HPV16 integrations in 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 metaphases derived from cells cultured in the presence of aphidicolin. Our data demonstrate that HPV16 integrations in cervical tumors frequently occur within CFSs at the molecular level. In addition, we have determined the precise molecular locations of the CFSs FRA6C and FRA17B.

Base Sequence↗
PubMed2005

Integration of high-risk human papillomavirus DNA correlates with HLA genotype aberration and reduced HLA class I molecule expression in human cervical carcinoma.

In human cervical cancer (CC), local immunity against this human papilloma virus (HPV)-associated neoplasia has been signified. To stratify the possibility of HPV integration on HLA mutations, we measured the genotypic and phenotypic integrity of all available HLA class I loci in 30 cases of CC. Paired normal and cancer genomic DNA was analyzed with DNA typing trays, including 57 subtypes of HLA-A, 120 subtypes of HLA-B, and 60 subtypes of HLA-C. We demonstrated significant mutations of HLA genotype with reduced HLA molecule expression in CC. HPV coincide in > 70% cases of aberrant HLA genes. Southern blot analysis revealed the presence of HPV DNA within the mutated HLA foci. Our study reveals a plausible role of HPV integration in the contexts of aberrant HLA genotypes in CC cells. Disruptions of the HLA genes can be possible tactics of HPV to attain the potential carcinogenetic purposes, and thus the cancer immune escape.

Carcinoma↗
PubMed1992

Conservation of HPV-16 E6/E7 ORF sequences in a cervical carcinoma.

A cervical carcinoma that contained human papillomavirus (HPV)-16 homologous DNA was analyzed. Each tumor cell genome contained a single, incomplete copy of HPV-16 DNA. The E6 and E7 open reading frames (ORFs) were completely conserved relative to other published HPV-16 sequences. Much of the non-coding region (NCR) was free of base changes, including complete conservation of several regulatory elements. Multiple mutations were identified in the remaining integrated HPV-16 DNA, which was composed of parts of the L1 and E1 ORFs. The extraordinary conservation of the E6/E7 DNA sequence, as compared with other regions of the integrated HPV-16 DNA, supports the role of E6/E7 in tumorigenesis.

Adult↗
PubMedDate not supplied

[Status of the human DNA papillomavirus in cervical tumors].

Cervical carcinoma is etiologically associated with the human papilloma virus (HPV), HPV 16 and HPV 18 being the most common. Viral DNA is thought to persist mostly in the episomal form in early tumor development, and in the integrated form in carcinomas. This assumption was checked with a new method that discriminated between RNAs transcribed from episomal and integrated HPV DNAs. Both forms were detected in carcinomas of Russian patients regardless of the disease stage. The data were verified by two other methods. RNA with sequences of the HPV transforming gene E7 proved to be transcribed from either DNA form. The results suggest that HPV integration is not crucial for carcinoma progression.

DNA, Viral↗
PubMed2006

[Detection of physical status of human papillomavirus 16 in cervical cancer tissue and SiHa cell line by multiplex real-time polymerase chain reaction].

BACKGROUND & OBJECTIVE: The integration of high-risk human papillomavirus (HPV) into host cell genome is one of the major contributing factors to cervical malignant transformation. The detection of HPV integration is helpful for understanding its role in cervical carcinogenesis and tumor progression. However, there is no ideal detection method of HPV physical status for clinical use. The study was to explore an ideal method of detecting the physical status of HPV-16. METHODS: Setting HPV-16 plasmid as standard, multiplex real-time polymerase chain reaction (PCR) using 2 different fluorescent report radicals was used to quantify the copy numbers of E2 and E6 genes for analysis of the physical status of HPV-16 according to E2/E6. Multiplex real-time PCR test and Southern blot results of cervical cancer cell line SiHa and 27 specimens of HPV-16-positive cervical squamous cell carcinoma were compared. RESULTS: There was a linear relationship between the threshold cycle values and the copy numbers of E2 and E6 in both standard curves, with the correlation coefficients of 1.00 and the amplification efficiencies of above 95%. The 95% reference range of plasmid E2/E6 ratio, in which the amount of E2 DNA was equal to that of E6 DNA, was 0.81-1.29. The cut-off value of E2/E6, which was used to distinguish the pure episomal form from a mixed form of episomal and integrated HPV-16, was 0.81 in the multiplex real-time PCR test. HPV-16 was observed to be integrated into the host genome of SiHa cells by multiplex real-time PCR and Southern blot. The coincidence rate of multiplex real-time PCR and Southern blot was 81.5% (22/27) in the cervical squamous cell cancer tissues (kappa=0.844, P<0.001). CONCLUSION: Multiplex real-time PCR test is a rapid, sensitive and reliable method for detecting the physical status of HPV-16 DNA, and is convenient to be applied in paraffin-embedded tissue and small preneoplastic or early neoplastic cervical lesions, even in cervical scrapes which contain a small amount of DNA.

Adult↗
PubMed1993

p53 antigen in cervical condylomata, intraepithelial neoplasia, and carcinoma: relationship to HPV infection and integration.

It has been proposed that wild-type p53 cell-regulating functions are annulled in human cervical carcinomas, either by mutations in the human papillomavirus (HPV)-negative cases or as a consequence of their complexing with HPV E6. The aim of this study was to test this hypothesis on 39 fresh cervical biopsies by p53 immunocytochemistry (ICC) with antibody PAb 240 and with NISH (non-isotopic in situ hybridization) and PCR (polymerase chain reaction) for HPV detection. p53 protein was present in the basal layer of pure wart virus infection; the basal to middle third of CIN (cervical intraepithelial neoplasia); in 19/22 (86 per cent) HPV-positive cervical carcinomas, ten of which contained integrated HPV; and in 4/8 (50 per cent) HPV-negative cervical carcinomas. Dual detection of p53 antigen and HPV 16 DNA in the same sections demonstrated either p53 protein or integrated HPV 16 alone in the majority of cells. Co-localization of both signals was only evident in isolated cells. These data suggest that PAb 240 immunoreactivity is not mutant-specific. They are, however, consistent with the conformation hypothesis which proposes that wild-type p53 changes from a suppressor (PAb 240-negative) to a promoter (PAb 240-positive) form during cell growth response. Hence, according to this hypothesis, p53 protein expression may represent either the wild-type promoter form or mutant p53 protein, both of which share the same conformation. This may explain co-localization of p53 and HPV in some tumours. However, the absence of p53 protein in 50 per cent HPV-negative squamous cell carcinomas suggests that not all HPV-negative tumours accumulate p53 protein.

Condylomata Acuminata↗
PubMed2007

Whole genome tiling path array CGH analysis of segmental copy number alterations in cervical cancer cell lines.

Cervical cancer is the second most common malignancy in women worldwide, with high risk subtypes of human papillomavirus (HPV) constituting the major etiological agent. However, only a small percentage of women infected by the virus develop disease, suggesting that additional host genetic alterations are necessary for disease progression. In this study we examined the genomes of a panel of commonly used model cervical cancer cell lines using a recently developed whole genome tiling path array for CGH analysis. Detailed analysis of genomic profiles enabled the detection of many novel aberrations, which may have been missed by conventional cytogenetic methods. In total, 27 minimal regions of recurrent copy number alteration were identified that are potentially involved in tumorigenesis. Interestingly, fine mapping of the 3q gain, which is associated with the progression of precursor lesions to invasive cervical cancer, identified a minimal region of alteration harboring genes distinct from previous candidates. Novel regions of gene amplification, including the coamplification of both the Birc and MMP gene clusters on 11q22, were also evident. Lastly, characterization of genomic structure at sites of HPV integration identified the copy number gain of host cellular sequences between the viral-host genomic boundaries in both SiHa and SW756, suggesting a direct role for HPV integration in the development of genetic abnormalities that initiate cervical cancer. This work represents the highest resolution look at a cervical cancer genome to date and offers definitive characterization of the alteration status of these cancer cell lines.

Cell Line, Tumor↗
PubMed1997

Association of human papillomavirus type 16 integration in the E2 gene with poor disease-free survival from cervical cancer.

To determine the clinical relevance of human papillomavirus (HPV) integration and E2 function suggested by in vitro studies, we investigated 50 patients with HPV 16-positive primary cervical carcinoma (stage Ib-IV) diagnosed and treated at one institution. The physical state of HPV was determined by colorimetric in situ hybridization and was not found to vary by stage. Overall, 62% of tumors had integrated HPV, 16% had episomal and 22% had both integrated and episomal. The E1/E2 region was evaluated by 8 separate polymerase chain reactions, which resulted in overlapping products. There was no significant variation in ability to amplify the E1/E2 region with stage. E1/E2 amplification correlated with physical state. Nearly all tumors with episomal or mixed HPV 16 DNA amplified all 8 E1/E2 fragments. Half of the tumors with integrated HPV 16 DNA failed to amplify one or more E1/E2 fragments. Disruptions were most frequent in the E2 region. For all 46 patients receiving curative therapy, the Kaplan-Meier estimate of disease-free survival was determined for those whose primary tumors had amplifiable E2 compared with those lacking one or more E2 DNA fragments. Disruption of E2 was associated with significantly shortened disease-free survival.

Adenocarcinoma↗
PubMed2001

[Aspects of molecular pathogenesis of cervical cancer in establishing new tumor markers for early detection and diagnosis].

The mortality rates of cervical cancer could be drastically reduced by the implementation of population wide cytological screening test for women (Pap-Test). However, these screening tests are hampered by high rates of false positive and false negative results, pointing to the urgent demand for improved screening technologies. High risk human papillomaviruses were identified as the causal agents of cervical cancer. The detection of the viral infection allows to identify patients at risk, however, about 5-30% of the normal female population harbours these viruses and only very few of these develop clinically relevant lesions. The activity of two viral oncogenes E6 and E7 initiates in a long term process neoplastic transformation in few of the HPV harbouring cells. As consequence of the expression of E7 a cellular marker protein (p16) is increasingly expressed in dysplastic cells. Monoclonal antibodies directed against p16 allow therefore to specifically identify dysplastic cells and derived invasive cancers in histological slides but also cytological smears (CINtec Assay). In advanced preneoplastic lesions HPV genomes are often integrated into cellular chromosomes. This leads to enhanced expression of the viral oncogenes. The detection of specific viral mRNA transcripts derived from integrated HPV genomes allows to identify preneoplastic lesions with a particularly high risk for progression to invasive cancers (APOT-assay). These findings will allow to establish highly sensitive, but specific and cost efficient new cancer early detection assays.

Biomarkers, Tumor↗
PubMed2004

Role of human papillomavirus in the development of head and neck squamous cell carcinomas.

OBJECTIVE: To review the literature on the role of oncogenic human papillomaviruses (HPVs) in the carcinogenesis of the head and neck mucosa. MATERIAL AND METHODS: Molecular and epidemiological studies concerning the high-risk HPV types and their role in carcinogenesis in the head and neck region were screened. RESULTS: Different studies revealed that: (i) 15-25% of head and neck squamous cell carcinomas (HNSCCs) are clonally associated with high risk HPV types (type 16); (ii) the oropharynx and particularly the tonsils are the most susceptible sites; (iii) patients with HPV-positive tumours present with more advanced stages of disease, are relatively younger, do not have extravagant tobacco and alcohol intake and seem to have a better survival; (iv) HPV-positive tumours are characterized by poor differentiation grade and a basaloid appearance; and (v) HPV-positive tumours exhibit integrated HPV DNA, wild-type p53, pRb downregulation and overexpression of p16INK4A. CONCLUSION: Taken together, these data support the view that HPV-harbouring HNSCC can be considered a discrete tumour entity with, moreover, a favourable prognosis. Screening of patients, especially those with tonsillar cancers, for the presence of HPV may help to further optimize treatment protocols and to provide more accurate prognostic information.

Alcohol Drinking↗
PubMed1997

Demonstration of human papillomavirus (HPV) genomic amplification and viral-like particles from CaSki cell line in SCID mice.

We demonstrate that from the CaSki cervical cancer cell line, integrated HPV-16 genome was amplified and viral-like particles were generated in an in vivo SCID mouse model. The in vivo tumor growth of several HPV-containing cell lines and 2 HPV-negative cell lines was examined in SCID mice. Tumor growth was noted with the HeLa, CaSki, ME-180, and MS751 cell lines within 2 months after subcutaneous injection. Squamous differentiation was appreciated in focal areas of tumors derived from CaSki and ME-180. In the CaSki tumors, DNA in situ hybridization revealed homogeneous staining of nuclei in some cells in the differentiated areas, suggesting HPV genomic amplification. In contrast, punctate or speckled patterns of hybridization were identified in the less differentiated areas, suggesting continued integration of the HPV genome. Immunocytochemical staining for HPV-16 L1 capsid protein showed it to be concentrated in cells from the differentiated areas, correlating with the results of hybridization. Electron microscopic studies revealed 50 nm uniform particles, consistent with HPV viral-like particles, in the nuclei of some cells in well-differentiated areas. Furthermore, Southern transfer and hybridization of the Hirt's extract from the CaSki tumors was positive for HPV-16 DNA, indicating non-integrated, low molecular weight HPV-16 DNA. Our results show HPV genomic amplification of integrated viral DNA and generation of HPV viral-like particles in CaSki cancer cells in SCID mice and that viral DNA amplification and the formation of viral-like particles are coupled to cellular differentiation. This experimental model provides a potential system for studying the molecular pathogenesis of HPV infections.

Animals↗
PubMed2005

Superficial (early) endocervical adenocarcinoma in situ: a study of 12 cases and comparison to conventional AIS.

Although established histologic criteria for the diagnosis of endocervical adenocarcinoma in situ (AIS) have been published, some lesions are not readily classified or present with more subtle degrees of epithelial atypia. Lesions confined to the surface mucosa may be particularly challenging, possibly because they represent early disease. Twelve cases of superficial AIS (SAIS) confined to the surface mucosa or crypt openings culled from the in-house and consultation practices were examined histologically, immunostained for MIB-1 and p16, and analyzed (when possible) for HPV nucleic acids by DNA-DNA in situ hybridization (INFORM). The mean age was 26.7 years for SAIS versus 37.0 years for 42 consecutive cases of conventional AIS from the same practice (P < 0.001). Seven and five were biopsies and conization specimens, respectively. Five coexisted with CIN, four arose in endocervical papillae, and two arose in endocervical polyps. Nuclear hyperchromasia was conspicuous in 10 and mitoses were present in all; however, apoptosis was rare or absent in four, and six exhibited only mild nuclear atypia. Mib-1 staining exceeded 40% in 5 of 7 cases tested, and all (8 of 8) were strongly positive for p16(ink4). Five of five were positive for HPV by ISH with an "integrated" dot-like pattern. SAIS is an early variant of AIS that 1) occurs at a younger mean age, 2) exhibits variable atypia, and 3) arises adjacent to morphologically normal columnar epithelium. Diffuse p16 expression and integrated HPV pattern are identical to that seen in more extensive forms of the disease. Superficial AIS should be suspected in endocervical columnar epithelium with segmental nuclear hyperchromasia with mitotic activity, and confirmed by biomarker staining (p16 and Mib-1) if the pathologist is uncertain of the diagnosis.

Adenocarcinoma↗
PubMed2000

Human papilloma virus status and chromosomal imbalances in primary cervical carcinomas and tumour cell lines.

Human papilloma virus (HPV) infection is the crucial step in the initiation of cervical carcinomas. In addition, HPV18 has been implicated in tumour progression and adverse clinical outcome. We determined the HPV types in 12 primary cervical carcinomas and 12 cell lines and compared the findings with the comparative genetic hybridisation (CGH) pattern of chromosomal alterations. The most frequent alteration was the deletion at 3p14 followed by the loss of 2q34-q36 along with 3q gain. High risk HPV types were detected in all samples except one primary tumour. In contrast to the normal distribution, HPV18 was present in 75% of cases including all cell lines. The cell lines carried a higher number of genetic alterations and a different CGH pattern for several chromosomes than the primary tumours, despite microdissection. Purely HPV18 positive cases indicated a high incidence of imbalances at specific loci with peaks of the histogram coinciding with known HPV integration sites. The study suggests that HPV infection is associated with a recurrent pattern of chromosomal changes in cervical carcinomas and that the development and progression of these alterations is triggered by integration into the host genome.

Carcinoma, Squamous Cell↗
PubMed1997

Establishment and characterization of 12 uterine cervical-carcinoma cell lines: common sequence variation in the E7 gene of HPV-16-positive cell lines.

A total of 12 carcinoma cell lines of the human uterine cervix were established from 5 keratinizing and 5 nonkeratinizing squamous-cell carcinomas, and 2 small-cell carcinomas. Of these, 10 lines grew as adherent cells and 2 as floating aggregates. All lines showed (i) similarity in morphology to the primary tumor from which they were derived; (ii) high viability with relatively long doubling times (48-96 hr); (iii) absence of Mycoplasma and other bacteria, apart from one Mycoplasma-contaminated line; (iv) genetic heterogeneity by DNA-fingerprinting analysis; (v) absence of p53 mutation from exon 4 through 9; and (vi) the presence of HPV DNA sequence. Among the lines, 7 were infected by HPV-16, 3 by HPV-18, 1 by HPV-31, and 1 by HPV-33; the 2 cell lines derived from small-cell carcinomas contained HPV-18. Interestingly, 6 of the 7 cell lines containing HPV-16-type DNA harbored the same alteration of E7 at nucleotide position 647 (amino acid 29, AAT --> AGT, Asn --> Ser), whereas the 3 HPV-18-positive lines did not; 3 cell lines proved to have intact E1/E2 of HPV, suggesting the presence of episomally replicating HPV DNA as well as the integrated form, whereas the other 9 lines were shown to have integrated HPV. Taken together, these cell lines would be very useful for studying the biology of uterine cervical carcinoma.

Carcinoma↗
PubMed1994

Human papillomavirus 18-immortalized endocervical cells with in vitro cytokeratin expression characteristics of adenocarcinoma.

OBJECTIVE: To determine whether human papillomavirus (HPV) 18 has a role in the development of adenocarcinoma from human endo- or ectocervical cells. METHODS: Secondary cultures of human endo- and ectocervical cells were assayed for immortalization by HPV 18 DNA using lipofection. The effects of immortalization on the patterns of cytokeratin expression were determined by indirect immunofluorescence using monoclonal antibodies. The differentiation phenotype of the immortalized cells was investigated by a modified in vivo implantation system. RESULTS: Both endo- and ectocervical cells were immortalized by HPV 18. The immortalized cells contained integrated HPV 18 DNA and expressed E6-E7 RNA. The immortalized endocervical cells had a cytokeratin phenotype characteristic of adenocarcinoma, whereas the immortalized ectocervical cells retained a distinct cytokeratin expression pattern of normal parental cells. In an in vivo implantation system, endocervical cells formed a lesion resembling severe dysplasia or carcinoma in situ, whereas ectocervical cells developed into a lesion resembling mild dysplasia. Both cell lines were nontumorigenic in nude mice. CONCLUSION: Both endo- and ectocervical cells are targets for immortalization by HPV 18. Based on cytokeratin expression patterns, immortalized endocervical cells, but not ectocervical cells, may be useful as a model for premalignant lesions that progress into adenocarcinoma.

Adenocarcinoma↗