UV-B irradiation stimulates the promoter activity of the high-risk,cutaneous human papillomavirus 5 and 8 in primary keratinocytes

Summary. Human papillomaviruses (HPV) have been implicated in the development of non-melanoma skin cancer (NMSC). HPV types 5 and 8 are strongly associated with NMSC in patients with the inherited disease Epidermodysplasia verruciformis (Ev). In these patients tumours arise predominantly on sun-exposed skin and consistently harbour HPV DNAs. To determine whether UV-B irradiation modulates the noncoding region (NCR) promoter activity of the Ev-HPV types 5, 8, 9, 14, 23, 24, and 25 we performed transient transfection assays with NCR luciferase reporter gene constructs in primary human epithelial keratinocytes (PHEKs) and in p53-null RTS3b cells. Each of the HPVs showed different basal NCR activity in both cell types and reacted differently upon UVB treatment and p53 cotransfection in RTS3b cells. The NCR of HPV5 and 8 were the only ones to be activated by UV-B in PHEKs. The stimulation of the NCR activity of the high-risk cutaneous HPV types 5 and 8 by UV-B irradiation may point to a role of this interaction in the development of NMSC.     

Bead-based multiplex genotyping of 58 cutaneous human papillomavirus types

Cutaneous human papillomaviruses (HPVs) are a heterogeneous, nonmonophyletic assembly, comprising about 50 characterized types and at least 133 isolates putatively representing new types. Their natural history of infection and potential association with nonmelanoma skin cancer are not well understood. Several PCR systems have been developed that amplify a broad spectrum of cutaneous HPVs. However, amplicon genotyping by sequencing or reverse line blot assays are complex and not well suited for high-throughput analyses. We developed a novel multiplex cutaneous papillomavirus genotyping (McPG) assay for 38 defined and 20 putative cutaneous HPVs of the beta, gamma, mu, and nu genera. Viral DNA was amplified by the use of a modified single-tube nested "hanging-droplet" FAP PCR. The amplifiable papillomavirus (PV) spectrum was enlarged by the use of 9 outer and 13 inner primers. Biotinylated PCR products were hybridized to type-specific oligonucleotide probes coupled to fluorescence-labeled polystyrene beads and analyzed using Luminex technology. Analytical sensitivity was analyzed for 38 defined HPVs and was ≤100 genome copies for all types. Integrated β-globin primers allow for simultaneous DNA quality control. McPG is characterized by high reproducibility (κ= 0.84, 95% confidence interval = 0.79 to 0.88), good concordance with the original nested FAP PCR, followed by sequencing (70.2% complete or partial agreement) when 322 skin biopsy DNA samples were analyzed, and improved ability to detect multiple infections (on average 2.5 HPV types per HPV-positive sample compared to 1.7 HPV types with nested FAP-PCR). In conclusion, McPG is a powerful tool for genotyping multiple cutaneous HPVs in a high-throughput format and is thus suitable for large-scale epidemiological studies.     

Orientation-dependent toxic effect of human papillomavirus type 33 long control region DNA in Escherichia coli cells

Virus Genes - The functional analysis of human papillomavirus (HPV) sequence variation requires the molecular cloning of different genomic regions of virus variants. In this study, we report an...     

Diversity of cutaneous human papillomavirus types in individuals with and without skin lesion.

BACKGROUND: Human papillomavirus (HPV) is ubiquitous on the skin of normal and immunosuppressed hosts. OBJECTIVE: We describe the diversity of HPV types in skin specimens using PCR-sequencing directly and after cloning with FAP59/64 or HVP2/B5 primers. STUDY DESIGN: Cross-sectional analysis of skin swabs. RESULTS: Seventy-five (92.6%) of 81 subjects provided samples that could be analysed with PCR (34 healthy controls <50 years old, 13 healthy controls > or =50 years old, 12 with actinic keratosis (AK), 8 with squamous cell carcinoma (SCC) and 8 renal transplant recipients). HPV DNA was detected more frequently with FAP59/64 (68/75, 91%) than with HVP2/B5 (9/75, 12%) (p<0.001). Agreement of typing results using FAP59/64 primers with both sequencing strategies was fair (mean kappa 0.56+/-0.19, 95% CI: 0.46-0.65). HPV species 1 and 2 of the beta-papillomavirus genus were associated with the presence of AK (OR=24.8, 95% CI: 2.3-262.6). A greater number of HPV types per sample was found in individuals with AK or SCC (p=0.046) or AK alone (p=0.02), than in healthy participants. CONCLUSION: HPV infection on the skin is best evaluated with a combination of primers and sequencing strategies. Novel putative types were frequently detected in SCC. Skin lesions have a greater number of HPV types than normal skin.     

Purification and characterisation of the E7 oncoproteins of the high-risk human papillomavirus types 16 and 18

E7 proteins are major oncoproteins of human papillomaviruses (HPVs) which play a key role in virus-associated cervical carcinogenesis. The E7 oncoprotein of HPV-16 has been shown to interact with a variety of cellular target proteins and these interactions are considered essential for the transforming properties of this oncoprotein. Several additional HPV types associated etiologically to cervical cancer have been described, the second most common being HPV-18. Less is known about the biochemical functions and interactions of HPV-18 E7. As a first step to determine biochemical properties common to the E7 proteins of the high-risk HPV types 16 and 18 these E7 proteins were expressed in bacteria and purified to homogeneity. Purified E7 proteins were used to investigate the in vitro interaction with the pocket protein p107 and insulin-like growth factor-binding protein-3 (IGFBP-3) that are known to interact with the amino-terminal and the carboxyl-terminal part of IGFBP-3, respectively. Both purified E7 proteins interacted strongly with p107 and, as demonstrated here for the first time, HPV-18 E7 was capable of binding to IGFBP-3, albeit to a lesser extent than HPV-16 E7. These findings suggest that the purified recombinant E7 proteins retain, at least in part, their biochemical activities.     

HPV高危型别检测联合细胞学检查对宫颈病变筛查的研究

目的 研究人乳头瘤病毒HPV高危型别检测联合液基薄层细胞学检查（TCT）及阴道镜检查对宫颈癌及癌前病变筛查的诊断价值。方法 对1375例宫颈组织细胞样本进行HPV高危型别检测,对其中阳性样本进行TCT检查,有宫颈上皮内瘤变者（CIN）行阴道镜下活检病理组织学确诊。HPV高危型别检测采用双色荧光定量PCR方法进行8种高危型HPV DNA（主要高危型：HPV16,18,45,31）和次要高危型（HPV33,52,58,67）分型及病毒载量检测。结果 1375例样本高危型HPV DNA检测结果为阳性256例,阳性率为18.62％;TCT结果为WNL的样本高危型HPV的感染率为16.41％ （42/256）;TCT结果为ASCUS以上的样本高危型HPV的感染率为83.59％（ 214/256）。HPV各型别的病毒载量在TCT结果为WNL、ASCUS及LSIL/HSIL/SCC之间差异无统计学意义（P＞0.5）。TCT与阴道镜的阳性符合率分别为WNL-正常或炎症92.86％（ 39/42）,LISL-CIN I 81.36％（48/59）,HSIL-CIN Ⅱ＆Ⅲ 85.19％ （23/27）,SCC-宫颈癌9/10。结论 HPV高危型别检测联合TCT技术及阴道镜检查能显著提高宫颈病变的阳性检出率,可作为宫颈癌及宫颈上皮内瘤变（ CIN）筛查的可靠早期诊断方法,具有重要临床应用价值。     

Moderate variation of the oncogenic potential among high-risk human papillomavirus types in gynecologic patients with cervical abnormalities

The oncogenic potential of human papillomavirus (HPV) infection was assessed by following the disease course in 455 patients who had had a routine diagnostic Hybrid Capture HPV test due to squamous cell abnormalities of the uterine cervix as detected by cytology and/or colposcopy. At entry, 308 patients had cytologic atypia classified as P3 by the Papanicolau classification, 168 had a positive high-risk HPV test, and 23 were infected only with low-risk HPV. The patients were followed-up using the patient registry until the endpoint of histologically diagnosed cervical intraepithelial neoplasia (CIN). High-grade CIN was diagnosed in 75 surgical biopsies. High-risk HPV infection (relative risk: 76.8 CI(95): 23.7-249.5), cytologic atypia (RR: 16.2 CI(95): 3.9-66.6), and age above 35 (RR: 1.99 CI(95): 1.26-3.16) were independent risk factors for high-grade CIN, while the viral load did not predict oncogenic progression (P = 0.47). After PCR-RFLP typing, the high-risk types were classified into groups as follows: (1) types 16 and 18, (2) types 45, 52, and 56, (3) types 31, 33, 35, 51, and 58. The relative risks of high-grade CIN were 119.1 (CI(95): 36.2-390.9) for group 1, 44.4 (CI(95): 9.8-201) for group 2, and 39.7 (CI(95): 10.9-144.8) for group 3, respectively. The risk ratios between the groups of high-risk types were found to differ at most by a factor of 2.98 (corrected P value: 0.007) indicating that the oncogenic potential varies moderately within the high-risk group of HPVs.     

A comparison of the detection of biomarkers in infections due to low risk versus high-risk human papillomavirus types

Adjunctive immunohistochemistry tests for human papillomavirus (HPV) infection include p16 and Ki67 as well as the more recently discovered biomarkers importin-β, exportin-5, Mcl1, and PDL1. The purpose of this study was to compare the expression of these biomarkers in HPV infection due to the high-risk types such as HPVs 16, 18, 31, 33, 35, and 51 versus lesions that contain the low risk types HPV 2, 6 or 11. We studied 35 lesions with low risk HPV types (verruca vulgaris = 10 cases, condyloma acuminatum = 15 cases, CIN 1 with HPV 6/11 = 10 cases) and 25 CIN 1 or 2 lesions with a high-risk HPV type. The 25 high-risk positive CIN 1–2 cases had strong expression of the panel p16, Ki67, importin-β, exportin-5, Mcl1, and PDL1 where each protein localized to the cells in the parabasal aspect of the lesion. In comparison, neither p16, importin-β, exportin-5, Mcl1, nor PDL1 were increased in the epithelia of the lesions with the low risk HPV types; Ki67 showed variable expression. HPV viral capsid L1 protein and viral DNA were excellent markers of infection in the lesions with low risk types. Thus, p16, importin-β, exportin-5, Mcl1, and PDL1 are not only biomarkers of high-risk HPV infection but can also differentiate such lesions from those that contain low risk HPV types. Low risk HPV infections can be best differentiated from their mimics by viral L1 capsid detection and/or HPV DNA by in situ hybridization.     

Evaluation of high-risk human papillomavirus types PCR detection in paired urine and cervical samples of women with abnormal cytology.

BACKGROUND: During the last decade, increasing efforts have focused on HPV detection in self-obtained samples, to increase the overall proportion of patients participating in cervical cancer screening procedures. OBJECTIVES: A clinical evaluation study of an optimized protocol for PCR detection of high-risk human papillomavirus (HPV) types in urine compared with cervical samples in consecutive women referred to the colposcopy clinic with abnormal cervical cytology. STUDY DESIGN: Paired urine and cervical specimens were collected from 100 consecutive women referred to the colposcopy clinic with abnormal cervical cytology and normal urine parameters. In-house and a commercial PCR method for the detection of HPV types 16 and 18, and a commercial multiplex PCR for HPV types 6, 11, 16, 18, and 33 were performed. All HPV cervix-positive/urine-negative paired urine samples were spiked with serial dilutions of cell lines infected with HPV 16 or 18 to test the sensitivity of HPV detection in these urine samples. RESULTS: In all but two cases HPV type 16 was detected. In cancer cases, the urine/cervix HPV detection sensitivity was 88.8%; in cases with high-grade lesions it was 76.5%; and in cases with low-grade lesions it was 45.5%. In all concordant cases the same HPV type was detected in both samples. The urine/cervix HPV detection sensitivity was higher when urine samples contained two or more epithelial cells per field in urine microscopy. HPV detection in 9 cervix-positive but urine-negative urine samples spiked with serial dilutions of HPV-positive cell lines showed that in these cases urine PCR inhibitors did not affect PCR amplification. CONCLUSIONS: A higher urine/cervix HPV detection sensitivity in cancer and high-grade lesions suggests that urine testing could be used to detect HPV mainly when these lesions are present.     

Filaggrin expression in cutaneous and mucosal human papillomavirus induced lesions

A series of 32 human papillomavirus induced lesions derived from epidermis and mucosa was studied for the modulation of filaggrin-profilaggrin (F-PF) expression according to the degree of virus infection as compared to normal skin and mucosa biopsies. This investigation was carried out on frozen sections using indirect immunofluorescence for filaggrin detection and group specific viral antigen and by in situ hybridization with biotinylated probes for viral DNA detection and typing. The 9 cutaneous warts showed an increase of F-PF expression in upper layer cells as compared to normal epidermis, which could be related to the high production of virus (viral antigen and HPV types 1 or 2). The 5 condyloma acuminata displayed also an enhanced expression of these components which was located in several upper layers but virus infection was confirmed in 2 of them with HPV types 6, 11 or 16. The 6 laryngeal papillomas exhibited a granular reactivity pattern for F-PF in suprabasal cell layers with an increase in the upper layers; viral antigen was found in 4 cases and HPV DNA types 6, 11 or 16 were detected in 4 specimens. Conversely among 12 cervical intraepithelial neoplasia, F-PF was expressed only in very superficial layers in few cases, without any correlation with the DNA detection (6, 11 or 16, 18). Taken together these data are suggestive of an intense expression of F-PF in benign lesions which can replicate the virus and a discrete or an absent expression of these components in premalignant or malignant lesions.     

Numerical aberrations of chromosome 1 in cervical intraepithelial neoplasia are strongly associated with infection with high-risk human papillomavirus types

The aims of this study were to assess the relationships between numerical aberrations of chromosome 1 and the presence of high-risk human papillomavirus (HPV). Five normal samples, 11 CIN1, 13 CIN2, 18 CIN3, and nine carcinomas were studied by in situ hybridization (ISH), using a DNA probe for the centromere of chromosome 1 (cen#1) and a DNA probe cocktail for HPV types 16 and 18. A short fragment polymerase chain reaction hybridization line probe assay (SPF-PCR-LiPA) technique was used to detect 25 HPV types. The mean number of cen#1 per nucleus (chromosome index, CI) was measured, and the fractional areas of dysplastic epithelium with HPV16/18 infection and with cen#1 aneusomy were estimated. Disomy was found in all normal epithelium and in 36% of CIN1. Tetrasomy was observed in 64% of CIN1, 15% of CIN2, and 17% of CIN3. Hyper-tetrasomy was observed in 77% of CIN2, 83% of CIN3, and 100% of invasive carcinomas. High-risk HPVs were present in 20%, 75%, and 94% of disomic, tetrasomic, and hyper-tetrasomic lesions, respectively. The mean CI value was significantly higher in the lesions infected with high-risk HPV than in the lesions not infected by high-risk HPV (p < 0.001), due to the significantly higher prevalence of hyper-tetrasomy. The ISH study disclosed that HPV16/18 was exclusively found within dysplastically altered epithelium. The area with aneusomy is mostly enclosed within the area infected with HPV. In 83% of the HPV16/18-positive CIN lesions, the fractional area of HPV-infected epithelium was equal to, or larger than, the fractional area with aneusomy. In conclusion, aneusomy for chromosome 1 is strongly associated with high-grade CIN lesions and infection with high-risk HPV; it is likely that the occurrence of numerical aberrations of chromosome 1 is preceded by infection with high-risk HPV.