Human Papillomavirus Genomic DNA Research Articles

Genome of Helicobacter pylori and Serotype of HPV Detected in Oropharyngeal and Laryngeal Cancer and Chronic Inflammation Patients.

Objective: Oropharyngeal/laryngeal carcinoma are common cancers of the upper aerodigestive system. Human papillomavirus (HPV) is described as the most frequent in the cancer of unknown primary. The presence of Helicobacter pylori (HP) in the oral cavity is discussed in some papers. The aim of study: To analyze the incidence of HPV and HP in oropharyngeal/laryngeal cancer persons versus persons with chronic tonsillar inflammation and healthy persons. Methods: The samples were taken in three groups: (1) tissue of oropharynx/larynx cancer (103 specimens); (2) tissue of palatine tonsils (85 specimens); and (3) healthy control group (50 specimens). We analyzed the presence of HP (PCR) and HPV genomic DNA (Sacace HPV High-Risk Screen Real-TM Quant) in the samples. Results: HP was detected in 86 samples (83.5%) and high-risk HPV in 62 samples (60.2%). We found a very high incidence of HP. In the cancer group, HP was detected in 82.5% cases and HPV positivity in 57.8%. In total, 7.2% of the cancer patients were negative for HP and HPV together. In turn, 53.6% of the cancer patients were positive for HP and HPV together. Four cases (4.2%) were positive for HPV only. VacA positivity was detected in 82 (79.6%) of the cancer cases and VacA negativity in 21 (20.4%) if the cancer cases. The incidence of HP in chronic inflammation (n = 85) was 65 cases (76.5%) and the incidence of HPV was 38 cases (44.7%). VacA positivity was detected in 59 (69.4%) of the chronic inflammation cases and VacA negativity was found in 26 (30.6%) of the chronic inflammation cases. Regarding the control group, we found HP positivity in 5 cases (11.1%) and HPV positivity in 19 cases (42.2%). There was VacA positivity in 6 cases (50.0%) of the control group. Statistically significantly lower prevalence of HP (p < 0.001) and HPV (p = 0.006) was found in the control group. Conclusions: We suggest that the palatine tonsils are colonized by HP. In our study, HP was present in oropharyngeal cancer in more cases in comparison with HPV infection. The presence of VacA from HP can have an influence on the human epithelial and immune cells’ regulation ways. Our results do not support idea that the CagA-positive HP is a primary carcinogen in oropharyngeal area.

Almost Complete Lack of Human Cytomegalovirus and Human papillomaviruses Genome in Benign and Malignant Breast Lesions in Shiraz, Southwest of Iran

Breast cancer ranks as the most common cancer among women worldwide. There have been controversial reports regarding contributions of human papillomaviruses (HPVs) and human cytomegalovirus (HCMV) to its development. The aim of this study was to determine the frequency of HPV and HCMV positivity in benign and malignant breast tumors.Materials and Methods:Formalin fixed paraffin-embedded tissue specimens of 150 breast cancers (invasive ductal and lobular carcinomas) and 150 non-malignant breast lesions (fibroadenomas, fibrocystic disease and adenosis) were examined. All samples were first deparafinized then subjected to commercial DNA extraction. The β-globin gene fragment was amplified using polymerase chain reaction (PCR) to confirm the quality of extracted DNA. The presence of HPV and HCMV genomic DNA was determined using PCR and Real time PCR techniques, respectively.Results:The mean ages of the test and control groups were 35.2 and 45 years, respectively. For HCMV, none of the malignant lesions were positive and only 2 of the 150 benign samples demonstrated presence of the virus. No HPV genomic DNA was found in either malignant or benign cases.Conclusion:The results of this study indicated no relationship between HCMV or HPV infection with breast cancer development. Whether investigations in larger populations with longer follow-up might demonstrate any role remains unclear.

Human papillomavirus in oral leukoplakia is no prognostic indicator of malignant transformation

Background: Oral leukoplakia is considered as a premalignant lesion for the development of oral squamous cell carcinoma (OSCC); several risks factors have been reported to contribute to this step-wise carcinogenesis; including human papillomavirus (HPV). Nevertheless, few reports have analyzed both the HPV status and the genotype in a single individual who develops OSCC from pre-existing oral leukoplakia. In this study, we surveyed the HPV status, genotype and clinicopathological risk factors in cases of malignant transformation from pre-existing oral leukoplakia. Methods: HPV genomic DNA was detected by PCR (MY09/MY11 in conjugation with nested primer-GP05+/GP06+) from paraffin sections, and the genotype was determined by direct DNA sequencing. Fisher's exact test and logistic regression were used to analyze risk factors for malignant transformation of oral cavity leukoplakia. Results: One hundred and sixty-seven patients with oral leukoplakia were enrolled; including 12 who had malignant transformation from the pre-existing oral leukoplakia. HPV prevalence was 22.8% in cases with oral leukoplakia. The risk factor associated with malignant transformation was recurrence of leukoplakia after treatment ( p = 0.03), nevertheless, HPV status was not statistically significant by logistic regression analysis. Among these 12 patients with malignant transformation from pre-existing oral leukoplakia, the status or genotype of HPV was chaotic; the oral habits of these patients might contribute to malignant transformation. Conclusions: Our data suggest that HPV in oral leukoplakia is no prognostic indicator of malignant transformation.

Rapid detection of human papilloma virus using a novel leaky surface acoustic wave peptide nucleic acid biosensor

A novel leaky surface acoustic wave (LSAW) bis-peptide nucleic acid (bis-PNA) biosensor with double two-port resonators has been constructed successfully for the quantitative detection of human papilloma virus (HPV). The bis-PNA probe can directly detect HPV genomic DNA without polymerase chain reaction (PCR) amplification, and it can bind to the target DNA sequences more effectively and specifically than a DNA probe. When the concentrations varied from 1 pg/L to 1000 μg/L, with 100 μg/L being the optimal, a typical linearity was found between the quantity of target and the phase shifts. The detection limit was 1.21 pg/L and the clinical specificity was 97.22% of that of real-time PCR. The bis-PNA probe was able to distinguish sequences that differ only in one base. Both the intraassay and interassay coefficients of variance (CVs) were <10%, and the biosensor can be regenerated for ten times without appreciable loss of activity. Therefore, this technical platform of LSAW biosensor can be applied to clinical samples for direct HPV detection.

HPV infection and oral carcinogenesis

To the Editor,High risk human papilloma viruses (HPV) have been found in head and neck squamous cell carcinomas (HNSCC), particularly in oropharyngeal carcinomas (1). Nevertheless, there are some controversial aspects regarding this issue (2) such as whether the HPV infec-tion is a temporary or a persistent oral infection in these patients.Recently, Chuang et al. (3) have associated the presence of HPV-16 DNA in surveillance salivary rinses with a significant risk for recurrence in HNSCC.We hypothesized that the improved prognosis of many patients with HPV-related oropharyngeal carcinoma is due to the temporary nature of the infection, and there-fore in the absence of lesions no HPV genomic DNA is detected in oral smears.We have analyzed a longitudinal cohort of 47 patients who had been treated for a previous oral squamous cell carcinoma (OSCC) without any precancerous or cancer-ous oral lesion when the oral smear was collected. The group was composed of 29 men and 18 women with a mean age of 62.02 years (45-87 years). Primary OSCC sites were tongue (44.6%), floor of the mouth (31.9%), oropharynx (17%), gingiva (12.7%) and buccal muco-sa (8.5%). Tumours were classified as T1 (53.3%), T2 (37.7%), T3 (2.2%) and T4 (6.6%).A cytological brush (CytobrushTM) was vigorously swabbed over the oral cavity. Cytological samples were centrifuged at 1500 rpm for 15 minutes and washed with phosphate-buffered saline (PBS) to obtain the cellular pellet. DNA was extracted following the clas-sical proteinase-K, phenol/chloroform method and pre-cipitated with ethanol. The detection of HPV genomic DNA was performed using the Papitype kit (Progenie-Molecular S.L, Valencia. Spain). In brief, a polymerase-chain reaction (PCR) targeting the L1 region of the viral genome, harbouring an internal control to avoid false negatives, was carried out. Subsequently, and in HPV+ samples, typing was performed using a restriction frag-ment length polymorphism (RFLP).To our surprise, no HPV genomic DNA was detected in any of the samples. These results agree with other published data (4, 5) which indicate that the HPV infec-tion, when present in oral cancer patients, is temporary and probably related with the presence of a malignant or premalignant lesion.Conscious of the limitations of our study, we think, as other authors (3), that the presence of HPV genomic DNA in oral smears may be used as a biomarker in

Routine human papillomavirus genotyping by DNA sequencing in community hospital laboratories

BackgroundHuman papillomavirus (HPV) genotyping is important for following up patients with persistent HPV infection and for evaluation of prevention strategy for the individual patients to be immunized with type-specific HPV vaccines. The aim of this study was to optimize a robust "low-temperature" (LoTemp™) PCR system to streamline the research protocols for HPV DNA nested PCR-amplification followed by genotyping with direct DNA sequencing. The protocol optimization facilitates transferring this molecular technology into clinical laboratory practice. In particular, lowering the temperature by 10°C at each step of thermocycling during in vitro DNA amplification yields more homogeneous PCR products. With this protocol, template purification before enzymatic cycle primer extensions is no longer necessary.ResultsThe HPV genomic DNA extracted from liquid-based alcohol-preserved cervicovaginal cells was first amplified by the consensus MY09/MY11 primer pair followed by nested PCR with GP5+/GP6+ primers. The 150 bp nested PCR products were subjected to direct DNA sequencing. The hypervariable 34–50 bp DNA sequence downstream of the GP5+ primer site was compared to the known HPV DNA sequences stored in the GenBank using on-line BLAST for genotyping. The LoTemp™ ready-to-use PCR polymerase reagents proved to be stable at room temperature for at least 6 weeks. Nested PCR detected 107 isolates of HPV in 513 cervicovaginal clinical samples, all validated by DNA sequencing. HPV-16 was the most prevalent genotype constituting 29 of 107 positive cases (27.2%), followed by HPV-56 (8.5%). For comparison, Digene HC2 test detected 62.6% of the 107 HPV isolates and returned 11 (37.9%) of the 29 HPV-16 positive cases as "positive for high-risk HPV".ConclusionThe LoTemp™ ready-to-use PCR polymerase system which allows thermocycling at 85°C for denaturing, 40°C for annealing and 65°C for primer extension can be adapted for target HPV DNA amplification by nested PCR and for preparation of clinical materials for genotyping by direct DNA sequencing. HPV genotyping is performed by on-line BLAST algorithm of a hypervariable L1 region. The DNA sequence is included in each report to the physician for comparison in following up patients with persistent HPV infection, a recognized tumor promoter in cancer induction.

Six‐month natural history of oral versus cervical human papillomavirus infection

Human papillomavirus (HPV) infection is etiologically associated with a subset of oral cancers, and yet, the natural history of oral HPV infection remains unexplored. The feasibility of studying oral HPV natural history was evaluated by collecting oral rinse samples on 2 occasions at a 6-month interval from 136 HIV-positive and 63 HIV-negative participants. Cervical vaginal lavage samples were concurrently collected for comparison. HPV genomic DNA was detected in oral and cervical samples by consensus primer PCR and type-specified for 37 HPV types. The six-month cumulative prevalence of oral HPV infection was significantly less than for cervical infection (p < 0.0001). HIV-positive women were more likely than HIV-negative women to have an oral (33 vs. 15%, p = 0.016) or cervical (78 vs. 51%, p < 0.001) infection detected. Oral HPV infections detected at baseline were as likely as cervical infections to persist to 6 months among HIV-negative (60% vs. 51%, p = 0.70) and HIV-positive (55% vs. 63%, p = 0.27) women. Factors that independently elevated odds for oral HPV persistence differed from cervical infection and included current smoking (OR = 8, 95% CI = 1.3-53), age above 44 years (OR = 20, 95% CI = 4.1-83), CD4 < 500 (OR = 6, 95% CI = 1.1-26), use of HAART therapy (OR = 12, 95% CI = 1.0-156), and time on HAART therapy (trend p = 0.04). The rate of oral HPV infections newly detected at follow-up was significantly lower than cervical infection among HIV-positive (p < 0.001) and HIV-negative women (p < 0.001). Our study not only demonstrates that it is feasible to study the natural history of oral HPV infection with oral rinse sampling, but also indicates that oral and cervical HPV natural history may differ.

Human papillomavirus type 45 propagation, infection, and neutralization

The organotypic (raft) culture system has allowed the study of the entire differentiation-dependent life cycle of human papillomaviruses (HPVs), including virion morphogenesis. We introduced linearized HPV45 genomic DNA into primary keratinocytes, where it recircularized and maintained episomally at a range of 10–50 copies of HPV genomic DNA. Following epithelial stratification and differentiation in organotypic culture, virion morphogenesis occurred. HPV45 virions were purified from raft cultures and were able to infect keratinocytes in vitro. By testing a panel of HPV VLP antisera, we were able to demonstrate that the infection was neutralized not only with human HPV45 VLP-specific antiserum, but also with human HPV18 VLP-specific antiserum, demonstrating serological cross-reactivity between HPV18 and HPV45.

Chapter 7: Human papillomavirus and cancer of the upper aerodigestive tract.

We discuss current evidence of the role of human papillomavirus (HPV) in some cancers of the upper aerodigestive tract, including the oral cavity, the pharynx, and the larynx. Cancers of the oral cavity and the pharynx are associated mainly with tobacco and alcohol exposure, but there is evidence from case series, from case-control studies, and from cohort studies that HPV plays a role in a fraction of these cancers, particularly cancer in the oropharynx and tonsil. The HPV type most commonly associated with cancers in these locations is HPV 16. Laboratory evidence indicates that the virus is integrated and that HPV oncogenes are transcriptionally active in these tumors. Many aspects of the association remain to be investigated, including the epidemiology and natural history of HPV infection in the mouth, the role of cofactors, and the potential use of HPV testing and vaccines in the prevention of these tumors. An analogous role for the virus at other anatomic sites in the upper aerodigestive tract such as the larynx is less clear. The relationship between HPV infection and laryngeal cancer is of particular interest, given that recurrent respiratory papillomatosis is clearly caused by benign proliferative growths induced by HPV 6 or 11 infection of the laryngeal epithelium. Although HPV genomic DNA has been detected in a proportion of laryngeal cancers and despite the many efforts made during the last 15 years, there is not yet compelling evidence that HPV plays a substantial role in laryngeal cancer.

Infectious virions produced from a human papillomavirus type 18/16 genomic DNA chimera.

The organotypic raft culture system has allowed the study of the differentiation-dependent aspects of the human papillomavirus (HPV) life cycle. However, genetic strategies to more completely understand the HPV life cycle are limited. The generation of chimeric viruses has been a useful tool in other virus systems to analyze infection and replication. To investigate the specificity of the interaction of nonstructural genes of one HPV type with the structural genes of another HPV type, we have replaced the L2 and L1 open reading frames (ORFs) of HPV type 18 (HPV18) with the L2 and L1 ORFs of HPV type 16 (HPV16). The resulting HPV18/16 chimeric construct was introduced into primary keratinocytes, where it was stably maintained episomally at a range of 50 to 100 copies of HPV genomic DNA, similar to that typically found in HPV-infected cells in vivo. The integrity of the HPV18/16 genomic DNA chimera was demonstrated. Upon differentiation in raft cultures, late viral functions, including viral DNA amplification, capsid gene expression, and virion morphogenesis, occurred. Chimeric HPV18/16 virions were purified from the raft cultures and were capable of infecting keratinocytes in vitro. Additionally, infection was specifically neutralized with human HPV16 virus-like particle (VLP)-specific antiserum and not with human HPV18 VLP-specific antiserum. Our data demonstrate that the nonstructural genes of HPV18 functionally interact with the structural genes of HPV16, allowing the complete HPV life cycle to occur. We believe that this is the first report of the propagation of chimeric HPV by normal life cycle pathways.

Nuclear import and DNA binding of human papillomavirus type 45 L1 capsid protein

During the life cycle of human papillomaviruses (HPVs), the L1 capsid proteins seem to enter the nucleus twice: once after the virions infect the cells, and later during the productive phase when they assemble the replicated HPV genomic DNA into infectious virions. We established for the high-risk HPV45 that when digitonin-permeabilized HeLa cells were incubated with L1 homopentameric capsomers, the HPV45 L1 protein was imported into the nucleus in a receptor-mediated manner. In contrast, intact capsids were not able to enter the nucleus. Immunoisolation assays showed that HPV45 L1 capsomers interact with cytosolic karyopherin alpha 2 beta 1 heterodimers. HPV45 L1 bound strongly to karyopherin alpha 2, and weakly to karyopherin beta 1, as did its nuclear localization signal (NLS). Nuclear import of HPV45 L1, or of a GST-NLS(HPV45L1) fusion protein was efficiently mediated by karyopherin alpha 2 beta 1 heterodimers, and only weakly by karyopherin beta 1. Nuclear import required RanGDP, but was independent of GTP hydrolysis by Ran. Together, these data suggest that the major nuclear import pathway for HPV45 L1 major capsid protein in infected host cells is mediated by karyopherin alpha 2 beta 1 heterodimers and that GTP hydrolysis by Ran is not required for import. Remarkably, HPV45 L1 capsomers can interact nonspecifically with different types of HPV-DNA, and the DNA binding region of HPV45 L1 overlaps with its NLS sequence.

Nuclear import of HPV11 L1 capsid protein is mediated by karyopherin α2β1 heterodimers

L1 major capsid proteins of human papillomaviruses (HPVs) enter the nuclei of host cells at two times during the viral life cycle: 1) after infection and 2) later during the productive phase, when they assemble the replicated HPV genomic DNA into infectious virions. L1 proteins are stable in two oligomeric configurations: as homopentameric capsomers, and as capsids composed of 72 capsomers. We found that intact L1 capsids of HPV type 11 cannot enter the nucleus, suggesting that capsid disassembly may be required for HPV11 L1 nuclear import. We established that HPV11 L1 is imported in a receptor-mediated manner into the nuclei of digitonin-permeabilized HeLa cells. HPV11 L1 docked at the nuclear pore complexes via karyopherin α2β1 heterodimers. Anti-karyopherin-β1 and anti-karyopherin α2 antibodies specifically inhibited nuclear import of HPV11 L1. Moreover, nuclear import of HPV11 L1 could be reconstituted using karyopherin α2, β1, RanGDP and p10. In agreement with the docking and import data, we found that HPV11 L1 binds to karyopherin α2 and that this interaction is inhibited by a peptide representing the classical nuclear localization signal of SV40 T antigen. These results strongly suggest that HPV11 L1 enters the nucleus of the infected host cell via the karyopherin α2β1 pathway. J. Cell. Biochem. 74:628–637, 1999. © 1999 Wiley-Liss, Inc.

Nuclear import of HPV11 L1 capsid protein is mediated by karyopherin ?2?1 heterodimers

L1 major capsid proteins of human papillomaviruses (HPVs) enter the nuclei of host cells at two times during the viral life cycle: 1) after infection and 2) later during the productive phase, when they assemble the replicated HPV genomic DNA into infectious virions. L1 proteins are stable in two oligomeric configurations: as homopentameric capsomers, and as capsids composed of 72 capsomers. We found that intact L1 capsids of HPV type 11 cannot enter the nucleus, suggesting that capsid disassembly may be required for HPV11 L1 nuclear import. We established that HPV11 L1 is imported in a receptor-mediated manner into the nuclei of digitonin-permeabilized HeLa cells. HPV11 L1 docked at the nuclear pore complexes via karyopherin alpha2beta1 heterodimers. Anti-karyopherin-beta1 and anti-karyopherin alpha2 antibodies specifically inhibited nuclear import of HPV11 L1. Moreover, nuclear import of HPV11 L1 could be reconstituted using karyopherin alpha2, beta1, RanGDP and p10. In agreement with the docking and import data, we found that HPV11 L1 binds to karyopherin alpha2 and that this interaction is inhibited by a peptide representing the classical nuclear localization signal of SV40 T antigen. These results strongly suggest that HPV11 L1 enters the nucleus of the infected host cell via the karyopherin alpha2beta1 pathway.

Adult rhabdomyoma: A light microscopic, ultrastructural, virologic, and immunologic analysis

Adult rhabdomyoma is a rare benign tumor of skeletal muscle origin that frequently arises from musculature of the head and neck region. In this study we report five cases of adult rhabdomyoma. Light and electron microscopic examination revealed the typical cellular features as described by others. The results of immunophenotyping and quantitation of nucleolar organizer regions were consistent for a benign skeletal muscle tumor. Epstein-Barr virus and human papillomavirus genomic DNA could not be detected by in situ hybridization.

Reduction of vector contamination in detection of human papillomavirus DNA using full-length genomic DNA probes

Nucleic acid hybridization techniques are currently the most specific and sensitive procedures available for diagnosing and typing human papillomavirus (HPV) infection. HPV genomic DNA cloned into vector pBR322 or related vectors are commonly used as probes for detection of HPV. When isolating HPV insert DNA however, it is difficult to remove all pBR322 DNA. This can result in false positive readings when clinical specimens harbouring sequences homologous to pBR322 are screened. It was found that up to 200 ng of vector-like sequences in a clinical sample could be blocked by the addition of non-labelled, digested pBR322 sequences to the hybridization reaction.