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Betapapillomavirus (betaPV) infections are often associated with squamous-cell carcinoma (SCC) and the prevalence of betaPV infections in (immunosuppressed) SCC patients is known to be high. The distribution and possible associated factors of betaPV infections in the general population, however, are largely unknown. To address this issue, betaPV infection was studied in 1405 SCC-free immunocompetent (n=845) and immunosuppressed (n=560) individuals from six countries of different latitudes. A standard study protocol was used to obtain information about age, sex, UV-irradiation and skin type, and from all participants eyebrow hairs were collected for detection and genotyping of 25 established betaPV types using the PM-PCR reverse hybridization assay (RHA) method. The frequency of betaPV-positive participants ranged from 84 to 91 % in the immunocompetent population with HPV23 as the most prevalent type, and from 81 to 98 % in the immunosuppressed population with HPV23 as the most or the second most prevalent type. The median number of infecting betaPV types ranged from four to six in the immunocompetent and from three to six in the immunosuppressed population. Increasing age in the immunocompetent participants and (duration of) immunosuppression in the immunosuppressed patients were associated with betaPV infection. In both groups, sex, skin phototype, sunburns and sun-exposure were not consistently associated with betaPV infection. This study demonstrates that betaPV infections are also highly prevalent in SCC-free individuals, with similar HPV types prevailing in both immunocompetent and immunosuppressed persons. Age and (duration of) immunosuppression were identified as betaPV infection-associated factors, whereas characteristics related to sun exposure and skin type were not.

Epidemiological studies have shown an association between infections by specific betapapillomaviruses, such as human papillomavirus (HPV) types 5 and 8, and cutaneous squamous cell carcinoma (SCC). The role of betapapillomaviruses in the development of cutaneous SCC is, however, still enigmatic. The ability to inhibit UVB-induced apoptosis, as demonstrated for HPV5 in vitro, may be important in this respect, as survival of DNA-damaged and mutated cells increases the risk of transformation. The aim of this study was to assess whether inhibition of UVB-induced apoptosis is a general property of betapapillomaviruses and to identify apoptotic factors that are potentially involved in this process. Primary human keratinocytes transduced with E6 and E7 of selected betapapillomaviruses (HPV5, HPV8, HPV15, HPV20, HPV24 and HPV38) were characterized and subjected to UVB irradiation. HPV8- and HPV20-expressing keratinocytes in particular showed fewer signs of apoptosis, as demonstrated by lower levels of active caspase 3, less enzymic caspase activity and less DNA fragmentation. The observed inhibition of UVB-induced apoptosis was mediated by E6 and coincided with reduced steady-state expression of the pro-apoptotic protein Bax. In conclusion, E6 of HPV8 and HPV20 reduces the apoptotic responses upon UVB irradiation when expressed in primary human keratinocytes. Infections with HPV8 and HPV20 may therefore augment the carcinogenic effect of UV radiation and potentially contribute to oncogenic transformation of the skin.

Separately, actinic keratosis (AK) and cutaneous squamous cell carcinoma (SCC) have been associated with cutaneous human papillomavirus (HPV) infections. To further explore the association between HPV infection and SCC development, we determined markers of cutaneous HPV infection within a single population in persons with precursor lesions (AK), cancerous lesions (SCC), and without. Serum and plucked eyebrow hairs were collected from 57 tumor-free controls, 126 AK, and 64 SCC cases. Presence of HPV L1 and E6 seroreactivity and viral DNA were determined for HPV types 5, 8, 15, 16, 20, 24, and 38. Significant positive associations with increasing severity of the lesions (controls, AK, and SCC, respectively) were observed for overall HPV L1 seropositivity (13%, 26%, and 37%) and for HPV8 (4%, 17%, and 30%). In parallel, the proportion of L1 seropositive individuals against multiple HPV types increased from 14% to 39% and 45%. The overall E6 seroreactivity, however, tended to decline with AK and SCC, especially for HPV8 (21%, 11%, and 2%). HPV DNA positivity was most prevalent in the AK cases (54%) compared with the SCC cases (44%) and the tumor-free controls (40%). Among all participants, there was a positive trend between overall HPV DNA positivity and L1 seropositivity, but not E6 seropositivity. Taken together, our data suggest that cutaneous HPV infections accompanied by detectable HPV DNA in eyebrow hairs and HPV L1 seropositivity, but not E6 seropositivity, are associated with an increased risk of AK and SCC. (Cancer Epidemiol Biomarkers Prev 2006;15(3):529–35)

A role for cutaneous human papillomaviruses (HPV) has been proposed in the development of skin cancer. Well-designed epidemiologic studies to demonstrate an association between HPV infection and skin cancer are extremely rare. To identify HPV infection as a potential risk factor, we investigated the association between the presence of HPV DNA in eyebrow hairs and a history of cutaneous squamous cell carcinoma. A case-control study was designed consisting of 155 immunocompetent individuals with a history of squamous cell carcinoma and 371 controls without skin cancer. DNA extracted from plucked eyebrow hairs collected from the study population was analyzed with a cutaneous HPV subgroup polymerase chain reaction and newly designed HPV type specific polymerase chain reactions for HPV 2, 5, 8, 15, 16, 20, 24, and 38. HPV DNA was detected in 63.1% of the total study population. The presence of HPV DNA was associated with age (p=0.0002) and male sex (p=0.02), but not with sun exposure, skin type, and smoking. After adjustment for age and sex, the presence of HPV DNA in eyebrow hairs was associated with a history of squamous cell carcinoma (odds ratio 1.7, 95% confidence interval 1.1; 2.7). HPV type specific analysis revealed that no HPV type stood out. The high-risk mucosal type HPV 16 and the skin wart type HPV 2 were rarely found in this study (0.2%). The positive association found between the presence of HPV DNA in eyebrow hairs and a history of squamous cell carcinoma warrants further research into the role that HPV infection plays in the development of cutaneous squamous cell carcinoma.

Warts and squamous cell carcinomas are important cutaneous complications in organ transplant recipients. The role of infection with human papillomaviruses (HPV) in the development of cutaneous squamous cell carcinoma is still unclear. An extremely diverse group of HPV types, mainly consisting of epidermodysplasia-verruciformis (EV)-associated HPV types, can be detected in benign, premalignant, and malignant skin lesions of organ transplant recipients. Frequently, there are multiple HPV types present in single skin biopsies. Typically, the prevalence of viral warts rises steadily after transplantation and a strong association exists between the number of HPV-induced warts and the development of skin cancer. The interval between the transplantation to the development of warts is clearly shorter than the interval from transplantation to the diagnosis of the first skin cancer. A comparison of transplant recipients with and without skin cancer, however, showed an equally high prevalence of EV-HPV DNA in keratotic skin lesions in both groups of patients and the detection rate and spectrum of HPV infection in hyperkeratotic papillomas, actinic keratoses, and squamous cell carcinomas was also similar. HPV DNA can frequently be detected in patients with hyperproliferative disorders like psoriasis and antibodies against HPV in patients with regenerating skin (e.g., after extensive second degree burns). Latent infection with EV-HPV seems to be widespread. The hair follicle region might be the reservoir of EV-HPV. The E6 protein from a range of cutaneous HPV types effectively inhibits apoptosis in response to UV-light induced damage. It is therefore conceivable that individuals who are infected by EV-HPV are at an increased risk of developing actinic keratoses and squamous cell carcinomas, possibly by chronically preventing UV-light induced apoptosis.

Epidermodysplasia-verruciformis-associated human papillomavirus DNA has been demonstrated in squamous cell carcinomas and plucked hair from immunocompetent patients and renal transplant recipients. This study investigated the association between infection with epidermodysplasia-verruciformis-associated human papillomavirus, identified by the detection of viral DNA in plucked eyebrow hairs, and solar keratoses. These lesions are strongly predictive of squamous cell carcinoma. In a cross-sectional study 518 individuals were enrolled from a randomly selected sample of a subtropical Australian community. Epidermodysplasia-verruciformis-associated human papillomavirus DNA in eyebrow hair was detected using a nested polymerase chain reaction specific for epidermodysplasia-verruciformis-associated human papillomavirus types. Epidermo dysplasia-verruciformis-associated human papillomavirus DNA was present in 121 (49%) of 245 men and 116 (44%) of 262 women. There was a strongly significant increase in epidermodysplasia-verruciformis-associated human papillomavirus infection with age (p < 0.00001), with prevalences of 29% in the 25–39 y age group, 42% at 40–59 y and 65% in the 60–79 y age group. Among men there was a strong association between epidermodysplasia-verruciformis-associated human papillomavirus and solar keratoses with an odds ratio, adjusted for age, skin color, and occupational sun exposure, of 3.40 (95% confidence interval, 1.77–6.53). No such association was found among women [odds ratio 1.03 (95% confidence interval 0.59–1.77, after adjustment for the same factors)]. Differences in occupational sun exposure and smoking histories could not explain these apparently different associations between epidermodysplasia-verruciformis-associated human papillomavirus infection and solar keratoses in men and women. In conclusion, epidermodysplasia-verruciformis-associated human papillomavirus infection is associated with solar keratoses in men suggesting that epidermodysplasia-verruciformis-associated human papillomavirus infection, in conjunction with sex specific factors (like androgens), may be involved in neoplastic changes of keratinocytes.

Human papillomavirus (HPV) infections play an important role in the development of cervical neoplasia. To get to a better understanding of the role of cytokines in the development of these neoplasias, we analysed the presence of various cytokines in cervicovaginal washings of healthy volunteers (n=22), cervical intraepithelial neoplasia (CIN) patients (n=63) and cervical cancer patients (n=33). IL-12p40, IL-10, TGF-beta1, TNF-alpha and IL-1beta levels were significantly higher in patients with cervical cancer than in controls and CIN patients. The levels of IFN-gamma were not different. Our data demonstrate alterations in the local cervical immune environment in cervical cancer patients. This could have important consequences for the further development of immune modulating therapies and vaccination strategies.

The association between human papillomavirus (HPV)–associated cervical cancer and cutaneous squamous cell carcinoma and codon 72 polymorphism in the p53 gene is not unequivocal. Especially, it is not known whether carriers of the arginine form have an increased risk of cancer that necessitates screening. The alternative is that the polymorphism is a tumor marker instead of a risk factor. We set out a case-control study to determine the risk of squamous cell carcinoma of the skin in individuals with the p53 codon 72 arginine genotype in order to establish the possible need for screening. The distribution of the different p53 codon 72 genotypes was examined in 86 subjects with a history of cutaneous squamous cell carcinoma and in 168 controls. Additionally, 121 subjects who had had histologically proven basal cell carcinoma and 108 subjects who had had non-familial malignant melanoma were tested. p53 polymorphism was evaluated by polymerase chain reaction (PCR) using DNA samples from peripheral blood lymphocytes. In a subgroup of patients with squamous cell carcinoma and controls, the presence of epidermodyplasia verruciformis human papillomavirus (EV-HPV) DNA was determined in plucked eyebrow hair. Differences in the distributions of the genotypes among cases and controls were calculated, and univariate and multivariate analyses were performed to assess the risk to develop cutaneous squamous cell carcinoma in the presence of the p53 codon 72 arginine genotype. Frequency distributions of the three different genotypes (homozygous for the arginine allele, heterozygous for the two alleles, and homozygous for the proline allele) were similar among the squamous cell carcinoma group and the control group: 47.1%, 46.0% and 6.9% versus 47.8%, 45.8% and 6.4%, respectively. Statistical analysis showed no significant differences between these groups. In patients with squamous cell carcinoma and controls who harbored EV-HPV DNA in their plucked eyebrow hair, similar results were obtained. The distributions of the p53 codon 72 genotypes in the basal cell carcinoma and malignant melanoma group were also not significantly different from the control group. p53 codon 72 arginine homozygosity does not appear to represent a significant risk factor for cutaneous squamous cell carcinoma and screening seems not to be indicated. Mol. Carcinog. 30:56–61, 2001. © 2001 Wiley-Liss, Inc.

By computer search, we identified one potential NF-kappaB binding site in the HPV16 long control region (LCR) at position 7554-7563 having two mismatches in comparison to the consensus NF-kappaB binding site of the Igkappa L promoter. Bandshift experiments with nuclear extracts from HeLa cells or purified glutathione S-transferase-p65 fusion protein clearly demonstrated that NF-kappaB is able to bind to this region of the LCR. However, in comparison to NF-kappaB binding on a consensus probe, the affinity of NF-kappaB for this site is about 250-fold reduced. When mutations were introduced into this NF-kappaB binding site, the activity of the LCR was increased, strongly suggesting that NF-kappaB was acting as a transcriptional repressor in the context of the HPV16 LCR. In addition, overexpression of NF-kappaB p65 repressed the activity of the HPV16 LCR, strengthening this conclusion., Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published

During genital human papillomavirus (HPV) infection several cytokines are released, such as interleukin-1 (IL-1), tumor necrosis factoralpha (TNFalpha), IL-6, and IL-8. These cytokines may play a role in the immune surveillance against viral infection. Two of these cytokines, IL-1 and TNFalpha, suppress the transcription of the HPV16 early genes. CAATT/ enhancer binding protein, (C/EBPbeta), which is activated by IL-1 and TNFalpha, has been suggested to act as a mediator of this transcriptional downregulation. C/EBPbeta contains three different translation initiation sites that can lead probably by leaky ribosome scanning to the generation of three isoforms of C/EBPbeta, namely full-length C/EBPbeta, liver enriched transcriptional activator protein (LAP), and liver enriched inhibitory protein (LIP). When transiently expressed in C33A and HeLa cells, the first two C/EBPbeta isoforms activate the HPV16 long control region (LCR). LIP, which acts as an antagonist of C/EBPbeta, represses the HPV16 LCR activity. Our observation that treatment of HeLa cells with IL-1 leads to induction of LIP supports the hypothesis that the LCR downregulation by IL-1 is mediated by LIP.

An extremely diverse group of human papillomavirus (HPV) types consisting of epidermodysplasia verruciformis (EV)-associated HPV types and other cutaneous HPV types (e.g., HPV types 2 and 3) is associated with nonmelanoma cancers and benign lesions of the skin. The frequent presence of multiple HPV types in single skin biopsy specimens of renal transplant recipients prompted us to develop PCR techniques for the detection of distinct (sub)groups of genotypically related cutaneous HPV types, i.e., three subgroups of EV-associated HPV types and two groups (A2 and A4) of other cutaneous HPV types. This approach generally allowed a reliable identification of HPV genotypes by direct sequencing of the PCR products, despite the frequent occurrence of multiple infections. The targeted spectrum of HPV types comprises 66 cutaneous HPV types including 21 putative novel HPV types. We also detected 17 putative novel HPV subtypes. We demonstrated that the skin of nearly all renal transplant recipients who developed various benign and (pre)malignant skin lesions was persistently infected with one or more EV-associated HPV types and/or HPV types belonging to groups A2 and A4. The frequency and distribution of EV-associated HPV and HPV types belonging to groups A2 and A4 were similar in biopsy specimens from hyperkeratotic papillomas (77.5%), squamous cell carcinomas (77.8%), and actinic keratoses (67.9%) but appeared to be lower in specimens of basal cell carcinomas (35.7%), benign lesions (38.5%), and clinically normal skin (32.3%). These findings suggest that renal transplant recipients are prone to persistent cutaneous HPV infection. Our data do not support the existence of high-risk cutaneous HPV types.

Keratinocyte cultures established from HPV containing skin cancers were described earlier to lose their HPV DNA after passaging in vitro. A different approach was therefore used in this study. Explant cultures were generated by depositing small pieces of various benign and (pre)malignant skin specimens of renal transplant recipients and non-immunosuppressed patients on fibroblast-populated collagen lattices or on de-epidermized dermis. Subsequently, the cultures were maintained at the air-liquid interface. At various time points, samples were collected for both HPV analysis, using a nested PCR approach, and morphology. The outgrowing keratinocytes developed into multilayered epithelial structures showing terminal differentiation. No histological differences were observed between cultures established from HPV positive and negative lesions. Eighteen biopsy specimens were tested for their HPV content before and after culture. Before culture 11 out of these skin specimens contained DNA of the Epidermodysplasia Verruciformis-related HPV types (EV-HPV). Comparison of the HPV types detected in two different parts of the same skin specimen before culture was strongly suggestive for a non-homogeneous distribution of EV-HPV in the lesions. From the explant cultures derived from the 11 HPV-positive biopsies, 31 samples from the originally explanted pieces of tissue and 38 samples from the outgrowing multilayered epithelial sections were collected. HPV DNA was detected in 10 of the 31 and in 3 of the 38 samples (Chi-square test, P = 0.01), respectively. These results indicate that EV-HPV positive keratinocytes do not efficiently proliferate or lose their HPV DNA in this culture system or EV-HPV DNA is present in only a few basal cells, making it improbable that these cells are located at the outgrowing margins. (C) 2000 Wiley-Liss, Inc.

A novel set of polymerase chain reaction (PCR) primers, designated SPF1 and SPF2 and located in the L1 region, was developed for universal detection of human papillomavirus (HPV). A short PCR fragment (SPF) of only 65 pb was synthesized. SPF amplimers were detected in a microtiter-based hybridization system, using a mixture of oligonucleotide probes. The SPF system allowed detection of at least 43 different HPV genotypes. The clinical performance of the novel SPF system was assessed in three different patient groups. 1) Analysis of 534 cervical scrapes, obtained from treated patients, showed that the detection rate in 447 (83.7%) scrapes with normal cytology was significantly higher using the SPF system as compared with the universal primer set GP5+/6+ (P < 0.001). 2) The SPF assay detected HPV DNA in 299 (98.4%) of 304 scrapes with cytological dyskaryosis. 3) The SPF system detected HPV DNA in 100% of 184 formalin-fixed, paraffin-embedded cervical carcinoma specimens. In conclusion, the novel SPF system permitted universal and highly sensitive detection of HPV DNA in diverse clinical materials and may improve the molecular diagnosis and epidemiology of this important virus.

OBJECTIVES: The aim of this study was to investigate the prevalence of human papillomavirus (HPV) after treatment for cervical dysplasia. STUDY DESIGN: The presence of HPV was investigated in cervical scrapes of 91 patients, before and after treatment, using consensus primers in the polymerase chain reaction. RESULTS: Before treatment for cervical dysplasia, 89 of 91 patients (98%) were HPV-positive compared with 28 of 91 patients (31%) after treatment. The HPV type present before treatment was found in the scrapes of only 9 of 89 patients (10%). Detection of HPV after treatment was associated with human immunodeficiency virus infection and incomplete conization. CONCLUSION: The HPV type present before treatment was frequently cleared by treatment for cervical dysplasia

We have previously detected a group of human papillomaviruses originally found in skin lesions of epidermodysplasia verruciformis (EV) patients in skin cancers from renal transplant recipients and from non-immunosuppressed patients. The reservoir of EV-HPVs is still unknown. In the current study we investigated whether EV-HPV DNA can be detected in plucked hairs from renal transplant recipients and healthy volunteers. Hairs were plucked from eyebrows, scalp, arms, and/or legs and DNA was subsequently isolated. To detect EV-HPV, we used nested PCR with degenerate primers located in the HPV L1 open reading frame. HPV DNA was detected in hairs from one or more sites in all 26 renal transplant recipients tested. Forty-five of 49 samples (92%) from these 26 patients were positive. The HPV type was successfully determined by sequencing in 38 samples, and all types belonged to the EV-HPVs. In ten of 22 healthy volunteers (45%), EV-HPV DNA was also detected in hairs from one or more sites. Twenty of 38 samples (53%) were positive, of which 17 samples were typed as EV-HPV types. These findings indicate that EV-HPV is subclinically present in the skin of the general population. Immunosuppression may lead to activation of the virus, explaining the finding that the apparent prevalence of EV-HPV in plucked hairs from renal transplant patients is higher than in those from the volunteers. If a dose-response situation exists for the carcinogenic potential of HPV infection, this finding may be relevant to the increased risk of skin cancer in this group of patients.

Previously, we have shown that immunization with human papillomavirus (HPV) type 16-derived cytotoxic T lymphocyte (CTL) epitope E7 49-57 (RAHYNIVTF) renders C57BL/6 mice insensitive to tumors formed by HPV16-transformed cells. In this study, we provide evidence that E7 49-57 is expressed as a subdominant CTL epitope on HPV16-transformed C57BL/6 cells. Using acid peptide elution, it is shown that HPV16-transformed cells express another CTL epitope, besides E7 49-57, which appears to be dominant. We demonstrate that a CTL line raised against the subdominant CTL epitope, offered as synthetic peptide E7 49-57, eradicates established HPV16-induced tumors in mice. Our data show that synthetic peptide-induced CTL can be applied successfully in vivo against (virus-induced) tumor, and emphasize that subdominant CTL epitopes are useful targets for immunotherapy. Furthermore, it is illustrated for the first time that HPV16-specific CTL interfere directly with HPV16-induced tumors.

Item does not contain fulltext BACKGROUND: Epidemiological data on cutaneous wart-associated HPV types are rare. OBJECTIVES: To examine the prevalence of cutaneous wart-associated HPV types and their relation with patient characteristics. STUDY DESIGN: Swabs were taken from all 744 warts of 246 consecutive immunocompetent participants and analysed by a broad spectrum HSL-PCR/MPG assay. Patient details including location, duration, and number of warts were recorded. RESULTS: No HPV DNA was detected in 49 (7%) swabs, a single HPV type in 577 (78%) swabs, and multiple HPV types in 118 (16%) swabs. HPV 2, 27 and 57 (alpha genus), HPV 4 (gamma genus) and HPV 1 (mu genus) were the most frequently detected HPV types, and HPV 63 (mu genus) was only frequently detected together with other HPV types. Less frequently detected HPV types were HPV 3, 7, 10 and 28 (alpha genus), 65, 88 and 95 (gamma genus) and 41 (nu genus). Warts containing HPV 1 showed the most distinct clinical profile, being related to children aged

Two sets of consensus PCR primers consisting of a common 3' primer CP-I and two 5'-primers, CP-IIG (primer set A) and CP-IIS (primer set B), in the E1 open reading frame of the human papillomavirus (HPV) genome are presented. These two primer sets enabled the detection of a 188 base pair (bp) fragment of HPV 1, 2, 3, 4, 5, 6b, 7, 8, 9, 10a, 11, 12, 14a, 16, 17, 18, 19, 20, 21, 22, 24, 25, 31, 33, 36, 37, 38, 39 and 46. HPV types 15, 23, 49 and 50 were poorly amplified and HPV type 41 was not amplified. The method is suitable for the detection of HPV DNA sequences in clinical samples of both cervical and cutaneous lesions.

A large number of human papillomavirus (HPV) types, distributed over five papillomavirus genera, are detectable in the skin. HPV types belonging to the alpha, gamma, and mu genera have been detected in cutaneous warts. A state-of-the-art HPV genotyping assay for these cutaneous wart-associated HPV types does not exist although warts constitute a highly prevalent skin condition, especially in children (33%) and organ transplant recipients (45%). Cutaneous warts are again the focus of attention as their clinical relevance rises with the increasing number of chronically immunosuppressed patients. The objective of this study was to develop and evaluate a DNA-based genotyping system for all known cutaneous wart-related HPV types using PCR and Luminex xMAP technology. The broad-spectrum PCR amplified DNA of all known wart-associated HPV types from the genera alpha (HPVs 2, 3, 7, 10, 27, 28, 29, 40, 43, 57, 77, 91, and 94), gamma (HPVs 4, 65, 95, 48, 50, 60, and 88), mu (HPVs 1 and 63), and nu (HPV41). The probes were evaluated using plasmid HPV DNA and a panel of 45 previously characterized cutaneous wart biopsy specimens showing high specificity. HPV was also identified in 96% of 100 swabs from nongenital cutaneous warts. HPV types 1, 2, 27, and 57 were the most prevalent HPV types detected in 89% of the swabs. In conclusion, this Luminex-based genotyping system identifies all known cutaneous wart HPV types including phylogenetically related types, is highly HPV type specific, and is suitable for large-scale epidemiological studies.

The human papillomavirus (HPV) type 16 enhancer-promoter has been shown to be active in human fibroblasts with a deletion on the short arm of one chromosome 11 (karyotype 46,del(11)(p11.11 p15.1)) but is virtually inactive in diploid human fibroblasts ( Smits, Smits, Jebbink, and ter Schegget, 1990b , Virology, 176, 158–165). In diploid human embryonic fibroblasts, activation of the HPV16 enhancer-promoter could be achieved by expression of the SV40 small t. By cotransfecting SV40 small t cDNA together with HPV16 DNA into diploid cells, it was possible to increase the transforming activity of HPV16 by 10- 15-fold. Furthermore, SV40 small t was essential for the SV40 large T-induced morphological transformation of human diploid fibroblasts, whereas SV40 small t was dispensable for transformation of del-11 cells. We propose that, as a result of the deletion of loci on the short arm of chromosome 11 in del-11 cells, functions are expressed that mimic those of SV40 small t in transformation and trans-activation.

Human papillomaviruses from the genus beta (betaPV) are a possible cause of cutaneous squamous cell carcinoma (SCC). We compared the betaPV infections in SCC and in sets of cutaneous tissues collected from a series of individual SCC patients to determine concordance and to assess the adequacy of eyebrow hairs as noninvasive markers of betaPV infection. Biopsies of SCC tumors, perilesional tissue, normal skin from the mirror image of nonfacial SCC and plucked eyebrow hairs were collected from 21 patients with incident SCC living in Queensland, Australia. These were tested for the presence of DNA from 25 different betaPV types. Overall prevalence of betaPV was high in every sample type, ranging from 81% to 95%. The median number of types was significantly higher in the SCC tumour (6), perilesional skin (5) and eyebrow hairs (5) than in normal skin (2). Comparing SCC tissue with other sample types within patients showed 63 overlapping infections with eyebrow hairs (71%; 95% CI: 60-80); 56 with perilesional skin samples (63%; 95% CI: 52-73) and 23 with normal skin samples (26%; 95% CI: 17-36). The sensitivity of eyebrow hair testing for detection of betaPV in the tumor was 82% (95% CI: 57-96) with concordance defined as 50% of betaPV types in common and 29% (95% CI: 10-56) for 100% concordance. These findings support the concept that perilesional skin represents an area of field change involving betaPV preceding SCC development and indicate that eyebrow hairs can serve to some degree as an easily collected marker of tumor betaPV status in epidemiological studies.

To determine the discriminative capacity of human papillomavirus (HPV) DNA testing for recurrent and residual cervical dysplasia, 43 patients with abnormal cytology after treatment for cervical dysplasia were tested for the presence of HPV DNA by PCR. An endocervical curettage was performed in all patients for histological examination. Sixteen of the 43 patients showed moderate or severe dysplasia. The HPV test was positive in all 16 patients with recurrent or residual dysplasia and negative in 12 of the 27 patients without dysplasia. The sensitivity and specificity of the HPV test were 100 and 44%, respectively. The likelihood ratio of a positive HPV test was 1.8, whereas a negative HPV test had a likelihood ratio of 0.12. Testing for the presence of HPV has the potential to select patients without recurrent or residual cervical dysplasia who have an abnormal cytological smear. This may have clinical implications, since unnecessary diagnostic conizations may be avoided in patients with abnormal cytology after treatment for cervical dysplasia and a negative HPV test.

Human papillomaviruses (HPV) are found in almost all squamous epithelia where they can cause hyperproliferative disease of mucosa and skin. Mucosal HPV types, such as HPV6 and HPV16, are known to cause anogenital warts and dysplasia or neoplasia, respectively. These HPV types have been studied extensively, and for some of them recently preventive vaccines have become available. Although HPV that populate the skin were the first identified HPV types, knowledge of the pathogenicity of HPV in the cornified epithelia stayed behind. What the majority of cutaneous HPV types do, for instance those belonging to the beta genus (betaPV), is largely unknown. As the number of reports that describe epidemiological associations between markers of betaPV infection and skin cancer gradually increases, the need for basic knowledge about these viruses grows as well. This review aims to picture what is currently known about betaPV with respect to infection, transmission and transformation, in order to envisage their potential role in cutaneous carcinogenesis.

Loci on the short arm of chromosome 11 between 11 p11 and 11 pl5 likely are involved directly or indirectly in the regulation of the HPV-16 enhancer-promoter strength and this may contribute to the control of the level of viral early gene expression. Transient expression assays have shown that the HPV-16 enhancer-promoter functions much stronger in fibroblasts in which this region of chromosome 11 (del-11 cells) is deleted than in normal diploid human embryonic fibroblasts. The differential regulation of the HPV-16 long control region may be due either to the presence or activity of a cellular transcription factor which up-regulates HPV-16 early gene expression in del-11 cells or to a factor which down-regulates expression in diploid cells. Since the HPV-16 enhancer containing fragment (nt 7003 to nt 57) in combination with the SV40 promoter functions equally well in del-11 cells as in diploid cells a target of this factor likely is located in the putative HPV-16 early promoter region. The relative resistance of diploid human embryonic fibroblasts to HPV-16 induced transformation could be explained by the inactivity of the HPV-16 early promoter as these cells could be transformed by HPV-16 DNA constructs in which the early gene expression was driven from a heterologous enhancer-promoter. These results may indicate that loci on the short arm could suppress HPV-16-induced transformation by down-regulating the activity of the viral early promoter.

Infections with human papillomaviruses (HPVs) belonging to the genus Betapapillomavirus have been linked to the development of non-melanoma skin cancer. Although persistence is expected, systematic investigation of this aspect of betapapillomavirus (β-PV) infection has not been conducted. This study investigated the prevalence and persistence of 25 known β-PV types in the skin of immunocompetent individuals. Over a 2 year period, eight consecutive plucked eyebrow hair samples taken from 23 healthy individuals were analysed for the presence of β-PV DNA. Using a recently published general β-PV PCR and genotyping method, 61 % of the individuals were β-PV DNA positive for one or more types at intake, whereas during follow-up this percentage rose to 96 %. HPV23 was the most frequently detected β-PV type. Type-specific β-PV DNA was detected over 6 months or longer in 74 % of the individuals. In 57 % of the individuals, DNA from multiple β-PV types was detected simultaneously for 6 months or longer. When the detection intervals of all β-PV type-specific infections in the study population were considered, a substantial proportion, 48 %, lasted at least half a year. The consistent β-PV patterns found over time in most individuals strongly suggested that β-PV DNA detection in plucked eyebrow hairs reveals true β-PV infection. If the minimum interval of detection was set at 6 months, persistent β-PV infections were found in the majority of the study population (74 %).

Human papillomavirus can be detected by amplification of viral DNA. A novel one-step PCR (PM-PCR) was evaluated for amplification of a 117-bp fragment from the E1 region. It permitted ultrasensitive detection of all 25 known human papillomavirus genotypes from the beta-papillomavirus genus. The intra- and intertypic sequence variations of the 77-bp interprimer region were studied. Genotype-specific probes as well as general probes were selected for the 25 established beta-papillomavirus types, and a reverse hybridization assay (RHA) was developed (PM-PCR RHA method). The analytical sensitivity of the PM-PCR RHA method was 10 to 100 viral genomes. The one-step PM-PCR turned out to be more sensitive than the previously described nested MaHa-PCR for beta-papillomavirus detection. The PM-PCR RHA method was able to detect and identify beta-papillomavirus types in frozen patient material as well as in poorly amplifiable material such as formalin-fixed, paraffin-embedded skin biopsy specimens. Inter- and intralaboratory variability experiments showed that the reproducibility of the assay was very high. In conclusion, the one-step PM-PCR together with the RHA allows extremely sensitive, specific, and reproducible detection of beta-papillomavirus DNA as well as reliable identification of beta-papillomavirus genotypes in both fresh and paraffin-embedded patient material.

Epidemiological studies, which address the role of human papillomavirus (HPV) in the pathogenesis of (pre)malignant cutaneous lesions, focus on the HPV B1 subgroup comprising the so-called epidermodysplasia verruciformis (EV)-associated HPV types. To detect and type HPV DNA in human materials, Polymerase Chain Reaction (PCR)-based assays are used. In this chapter, a nested, broad-spectrum PCR method using a mixture of primers and a type-specific PCR using specific primers are described. The broad-spectrum PCR detects the B1 subgroup of HPV types. HPV typing is performed by sequence analysis of the PCR product. The type-specific PCR detects and types HPV 5a, 8, 15, 17, 20, 24, 36, and 38. These HPV types are representative of the B1 subgroup, because they are evenly distributed over the phylogenetic tree of the B1 subgroup.

Epidemiological studies, which address the role of human papillomavirus (HPV) in the pathogenesis of (pre)malignant cutaneous lesions, focus on the HPV B1 subgroup comprising the so-called epidermodysplasia verruciformis (EV)-associated HPV types. To detect and type HPV DNA in human materials, Polymerase Chain Reaction (PCR)-based assays are used. In this chapter, a nested, broad-spectrum PCR method using a mixture of primers and a type-specific PCR using specific primers are described. The broad-spectrum PCR detects the B1 subgroup of HPV types. HPV typing is performed by sequence analysis of the PCR product. The type-specific PCR detects and types HPV 5a, 8, 15, 17, 20, 24, 36, and 38. These HPV types are representative of the B1 subgroup, because they are evenly distributed over the phylogenetic tree of the B1 subgroup.

Strong evidence has implicated human papillomaviruses (HPV) in the pathogenesis of anogenital cancers and a number of other mucosal and cutaneous lesions. Data concerning the involvement of HPV in esophageal cancers are controversial. Different investigators have detected HPV types (mainly types 16 and 18) in biopsy specimens of esophageal cancers. A study was undertaken to determine whether responses to chemotherapy of advanced squamous cell carcinomas could be correlated with the HPV status. Polymerase chain reaction (PCR) amplification was used for the detection of HPV DNA in biopsies of esophageal squamous cell carcinomas treated with either surgical resection alone (n = 42) or chemotherapy followed by surgical resection (n = 21). Different general and consensus PCR primer sets, which allow the detection of most of the known as well as a number of not yet characterized HPV types, were used. HPV DNA was not detected in any of the 61 esophageal squamous cell carcinomas, suggesting that HPV infections are not likely to play a major role in the etiology of this neoplasm. © 1995 Wiley-Liss, Inc.

DNA from epidermodysplasia verruciformis-related human papillomavirus (EV-HPV) types is frequently found in nonmelanoma skin cancer (squamous and basal cell carcinoma). Epidemiological studies that investigate the relation between EV-HPV infection and nonmelanoma skin cancer are scarce. We designed a case-control study in which we looked for HPV infection in 540 cases with a history of skin cancer and 333 controls. By measuring seroreactivity to L1 virus-like particles of EV-HPV types 5, 8, 15, 20, 24, and 38 and the genital type HPV16 and by estimating the skin cancer relative risk among HPV seropositives, we analyzed whether EV-HPV serorecognition is associated with nonmelanoma skin cancer. Seroreactivity to five of the six EV-HPV types tested (HPV5, 8, 15, 20, and 24) was significantly increased in the squamous cell carcinoma cases. After adjusting for age and sex, the estimated squamous cell carcinoma relative risk was significantly increased in HPV8 and HPV38 seropositives [odds ratio (OR) = 14.7 (95% confidence interval (CI), 1.6-135) and OR = 3.0 (95% CI, 1.1-8.4), respectively]. The estimated relative risk for nodular and superficial multifocal basal cell carcinoma was also significantly increased in the HPV8 seropositives [OR = 9.2 (95% CI, 1.1-78.2) and OR = 17.3 (95% CI, 2.1-143), respectively] and in the HPV20 seropositives [OR = 3.2 (95% CI 1.3-7.9) and OR = 3.4 (95% CI 1.2-9.5), respectively]. The relative risk of developing malignant melanoma was not increased among HPV seropositives, and no associations were found for HPV16. Restricted analyses among the HPV seropositives only, to exclude distortion by interindividual differences in seroresponsiveness, underscored the significance of our findings. Restricted analyses among patients with skin cancer only, however, revealed that EV-HPV seropositivity was not significantly more present in patients with nonmelanoma skin cancer than in those with melanoma skin cancer. Taken together, our results indicate that EV-HPV serorecognition is nonspecifically associated with nonmelanoma skin cancer and suggest that EV-HPV-directed seroresponses are induced upon skin cancer formation, rather than upon infection.

The MICA gene encodes for major histocompatibility complex class I chain-related proteins (MIC), which belong to a recently identified new family of nonclassical major histocompatibility complex molecules. The general structure of the MICA molecule resembles that of major histocompatibility complex class I molecules. MIC molecules are considered to be stress-induced antigens that are recognized by cytotoxic T cells and natural killer cells, which play an important role in the surveillance of transformed infected and damaged cells. Associations of major histocompatibility complex class I molecules with skin cancer have been described before. To evaluate the possible association of MICA gene polymorphism with the risk for nonmelanoma skin cancer we evaluated 153 cases with squamous cell carcinoma, 261 cases with basal cell carcinoma, 111 controls with malignant melanoma, and 247 controls without a history of skin cancer. Five distinct MICA alleles A4, A5, A6, A9, and A5.1 were studied. As the MICA 5.1 variant gene contains a four-nucleotide insertion that causes a stop codon in the trans membrane region, the resulting truncated MICA molecule does not reside on the cellular membrane. In the case of individuals who are homozygous for MICA 5.1 this results in cells that are naked for the MICA molecule. We therefore specifically addressed the possible association between MICA 5.1 homozygosity and skin cancer, as these individuals are expected to be at the highest risk for skin cancer if the MICA gene plays a role in skin carcinogenesis. Viral proteins may serve as antigens for recognition of skin cancer by the immune system. Human papillomavirus is the most likely candidate virus to be involved in the carcinogenesis of cutaneous squamous cell carcinoma. Hence, we also assessed the association between MICA polymorphism and squamous cell carcinoma in human-papillomavirus-positive and human-papillomavirus-negative individuals as identified by the presence of human papillomavirus DNA in hairs plucked from their eyebrows. Our analyses did not reveal any significant differences regarding the MICA allele frequencies between cases and controls. Also homozygotes and heterozygotes for the MICA 5.1 variant gene were not at an increased risk for skin cancer compared to individuals without this variant gene and infection with human papillomavirus did not materially influence these findings. The same group of cases and controls was large enough to show an association between melanocortin 1 receptor gene polymorphism and skin cancer and to reasonably exclude an association between p53 codon 72 polymorphism and skin cancer. Therefore, we conclude that an association between MICA gene polymorphism and nonmelanoma skin cancer is not likely.

Epidermodysplasia-verruciformis-associated human papilloma virus DNA has been detected in skin cancers, in premalignant and benign skin lesions, and in plucked hairs from immunocompetent and immunosuppressed patients. The role of epidermodysplasia-verruciformis-associated human papilloma virus in the pathogenesis of nonmelanoma skin cancer is still enigmatic. In organotypic cultures we investigated the effects of retroviral transduction of the E6 and E7 genes of epidermodysplasia-verruciformis-associated human papilloma virus types 5, 12, 15, 17, 20, and 38 on the growth and differentiation of human keratinocytes. Differentiation was disturbed to different degrees as revealed by histology and by the expression patterns of differentiation markers keratin 10 and small proline rich protein 2. Conversely, proliferating cell nuclear antigen was induced in some of the suprabasal, differentiated cells to varying extent. No unscheduled DNA synthesis was detected in these cells, however, as probed by 5′-bromo-2′-deoxyuridine incorporation. Most intriguingly, when the E6 and E7 genes of epidermodysplasia-verruciformis-associated human papilloma virus types 15 and 17 were transduced, a broadening layer of basal cells and an accelerated differentiation were observed. In addition, "papilla-like structures" comprising basal-like keratinocytes arose from the basal layer into the differentiated layers. These cells did not express the differentiation markers keratin 10 and small proline rich protein 2, but did actively replicate DNA. These observations warrant further research by using this system to elucidate the replication strategy of epidermodysplasia-verruciformis-associated human papilloma virus types in keratinocytes and to shed light on the role of these human papilloma virus types in the pathogenesis of skin cancer.

Genital human papillomavirus (HPV) types 6 and 11 are of clinical importance due to their role in the development of anogenital warts. A pilot study was performed to investigate whether DNAs from HPV types 6 and 11 are present in hairs plucked from the pubic and perianal regions and eyebrows of patients with genital warts at present and patients with a recent history of genital warts. Genital HPV DNA was detected in 9 of 25 (36%) pubic hair samples and in 11 of 22 (50%) perianal hair samples by the CPI/CPIIg PCR. After sequencing of 17 of 20 samples, HPV type 6 or 11 was detected in 6 of 25 (24%) hair samples from the pubis and 8 of 22 (36%) hair samples from the perianal region. These types were not detected in plucked eyebrow hairs. In contrast, the HPV types associated with epidermodysplasia verruciformis were detected in similar proportions (62%) in both samples of pubic and eyebrow hairs. Moreover, HPV type 6 and 11 DNAs were detected in pubic hairs plucked from two patients who had been successfully treated and who did not show any lesion at the time of hair collection; this finding is an argument that HPV DNA may persist in this region. The presence of genital HPV types in plucked pubic and perianal hair suggests that there is an endogenous reservoir for HPV which may play a role in the recurrences of genital warts.

Objective. Conflicting data exist on IL-6 production by human papillomavirus (HPV) immortalized cell lines and several cervical carcinoma cell lines. However, no information has been reported on the levels of cytokines in cervicovaginal washings in relation to cervical neoplasia. The aim of this study was to investigate whether local production of IL-6 could be found and whether the level of this cytokine was related to the severity of cervical neoplasia. IL-8 was measured to obtain additional information on an inflammatory cytokine with possible epithelial origin. Methods. Cervicovaginal washings and sera were obtained from 35 patients with invasive cervical cancer, 62 patients with cervical intraepithelial neoplasia (CIN), and 25 control subjects. IL-6 and IL-8 levels were determined by ELISA. HPV DNA in cervical smears was detected by a HPV-16-specific PCR method and additionally by CPI/IIG PCR. Histological analysis of the inflammatory infiltrate was performed on hematoxylin-eosin-stained tissue sections. Results. In the patients with cervical cancer, those with CIN, and the controls, the median IL-6 concentration in cervicovaginal washings was 171 pg/ml (interquartile range: 54-780), 22 pg/ml (

Mutations in p53 were detected in 11/23 (48%) of non melanoma skin cancers in renal allograft recipients and in 5/8 (63%) of sporadic tumours from immune competent patients. 9/12 (75%) of mutations in transplant patients and all 5 mutations in non transplant tumours were consistent with damage caused by ultraviolet (u.v.) irradiation. DNA sequences, predominantly of the epidermodysplasia verruciformis (EV) subgroup, were detected in 9/23 (39%) of transplant tumours and in 2/8 (25%) of eight non-transplant tumours. There was no relationship between HPV status and p53 mutation, HPV DNA being present in 5/16 (31%) of tumours with p53 mutation and 6/15 (40%) of tumours lacking p53 mutation. These data are consistent with an important role for sunlight in the development of post-transplant skin cancer, and with limited functional data suggesting that E6 proteins of the cutaneous and EV-related papillomaviruses do not target p53 for ubiquitin-mediated degradation.

BACKGROUND. The presence of human papillomavirus (HPV) DNA in relation to cervical cytology was evaluated after treatment of cervical dysplasia. METHODS. Forty patients, 22 with normal and 18 with abnormal cytology (mild or moderate dyskaryosis), with a history of cervical dysplasia were selected. Only patients with HPV DNA positive biopsies obtained before treatment were included. The presence of HPV was assessed in cervical smears at least 1 year after treatment of cervical dysplasia by using a polymerase chain reaction (PCR) with consensus primers (CPI/IIG). HPV typing was done by direct sequence analysis of the CPI/IIG PCR generated amplimers. RESULTS. Smears from 3 of the 22 patients with normal cytology after treatment were positive for HPV DNA (14%). HPV DNA positive smears were found in 13 of the 18 patients with abnormal cytology after treatment (72%) (relative risk : 5.3 ; 95% confidence interval : 1.78-15.75). In 11 of the 16 HPV DNA positive smears (69%), the HPV type was different from that before treatment. In 35 of 40 patients, the HPV type before treatment could not be detected after treatment (88%). CONCLUSIONS. A minority of the patients with normal cytology after treatment of cervical dysplasia had detectable HPV DNA. In contrast, a high prevalence of HPV DNA was found in cervical smears of patients with abnormal cytology after treatment of cervical dysplasia. After treatment, none of the patients with abnormal cytology but HPV DNA negative smears had recurrence of cervical intraepithelial neoplasia. This suggests the value of supplementary HPV DNA testing during follow-up of patients treated for cervical dysplasia.

Based on immunologic and epidemiologic data, it is plausible that skin cancer in renal transplant recipients is associated with human papillomaviruses (HPV). At present, conflicting evidence exists concerning the presence of HPV DNA in these cancers. We recently described a nested polymerase chain reaction method that enables the detection of all previously isolated epidermodysplasia verruciforasis (EV)-associated HPVs. We now describe the detection of EV-associated HPV DNA in 49 (80%) of 61 biopsies from squamous cell carcinomas, in four (50%) of eight basal cell carcinomas, in 14 (93%) of 15 actinic keratoses, in two (40%) of five cases of Bowen's disease, and in four (57%) of seven keratoacanthomas. HPV DNA typing revealed that all detected HPV types belonged to the EV-associated HPV types. A wide spectrum of EV-associated HPVs was found, including six putative new HPV types. In a high percentage of the lesions more than one HPV type was detected. We often found the same HPV types in different skin biopsies from both malignant and premalignant lesions from the same patient. The high frequency of detection of EV-associated HPV types in biopsies from malignant and premalignant lesions is in agreement with the hypothesis that EV-associated HPVs are involved in the pathogenesis of skin cancer in renal transplant recipients.

The naturally occurring sequence variation of human papillomavirus type 16 (HPV-16) was analysed by direct sequence analysis of the PCR products of the long control region (LCR), the E5 and E7 open reading frames (ORFs), a segment of the L2 ORF overlapping the early viral poly(A) signal and a small segment of the L1 ORF or clinical isolates from Barbados and The Netherlands. Despite the widely different geographical and ethnic origin of the two groups of specimens, sequence analysis revealed relatively few mutational differences. Analysis of the LCR and the E5 ORF appeared to be the minimum requirement for the correct positioning of these variants in the HPV-16 phylogenetic tree. Most of the Barbadian variants appeared to be located at a unique position in the HPV-16 phylogenetic tree, at the internal branch close to the point where the European and Asian branches diverge. In contrast, most of the Dutch samples were located on the European branch.

Human papillomavirus (HPV) DNA, predominantly of the HPV16 genotype, can he detected in more than 90% of the human cervical. carcinomas1. The “high risk” HPV types, including HPV16, are believed to play an important role in the pathogenesis of human cervical. cancer. The ability of HPV16 to in vitro immortalize human keratinocytes2and the dependence on HPV 16 expression for preservation of the transformed phenotype of cervical. cancer-derived cell lines3 suggests direct involvement of HPV 16 in the multi-step process of cervical. carcinogenesis. Cervical. cancer and other HPV-related cancers are more commonly seen in immunosuppressed individuals4,5. This suggests that proper immunosurveillance interferes with HPV-associated tumor development and that T cell immunity, in particular mediated by cytotoxic T lymphocytes (CTL), is important in the defense against virus induced tumors.

We have mapped using the RNA PCR the viral-cellular junctions of HPV16 viral-cellular cotranscripts expressed in CaSki cells and a cervical carcinoma to nt 3728 and 881, respectively. Both junctions were located within the E1–E2 region. Examination of the cellular sequences of the cotranscripts showed the presence of a polyadenylation signal in each of the transcripts. In CaSki cells and in the cervical carcinoma transcripts derived from the full-length early region including the E2 transcript were also detected. Our results suggest that the utilization of a cellular polyadenylation site could be important in the development of cancer by HPV.

We developed a polymerase chain reaction DNA amplification system using two distinct consensus oligonucleotide primer sets for the improved detection and typing of a broad spectrum of human genital papillomavirus (HPV) sequences, including those of novel viruses. The system incorporates one primer set designed to amplify a highly conserved L1 domain and a second primer set designed to amplify a domain within the E6 gene. We used this system to analyze 48 fixed, paraffin-embedded tissue sections (41 specimens from 33 cervical carcinomas, four normal cervical tissues, and several control tissues) for the presence of HPV DNA. HPV sequences were detected in all carcinoma samples and none of the control samples. Hybridization analyses showed that the results obtained with the two amplification schemes concurred completely. This approach allowed rapid confirmation of typing results and may improve the likelihood of detecting a wide variety of HPV sequences, including those of novel HPVs.

Background. The incidence of vulvar intraepithelial neoplasia Grade III (VIN III) is increasing and is diagnosed at a younger age than previously. VIN III is often multifocal and frequently coexists with multicentric dysplastic lesions in the cervix and vagina. Warty-type VIN III more often has been found to contain human papillomavirus (HPV) DNA than basaloid-type VIN III. The authors performed HPV DNA polymerase chain reaction (PCR) analysis in 48 VIN III biopsies and reverse transcriptase (RT)-PCR in 8 HPV-16 DNA-positive multifocal VIN III biopsies to detect E6/E7 transcripts. Methods. Human papillomavirus DNA detection and histologic analysis were performed on alternating slides of paraffin embedded biopsies. Polymerase chain reaction was performed with consensus primers, and HPV typing was performed by direct sequencing. Total RNA was isolated from frozen biopsies by centrifuging a guanidinium thiocyanate (GTC) lysate through a cesium chloride (CsCl) cushion. The RT reaction was performed using a 3′ primer, located just downstream of the E7 stop codon, and the PCR reaction was performed using the same 3′ primer and a 5′ primer located just downstream of the E6 start codon. Results. The mean age of the 48 patients was 37.7 years. Eighty-one percent had multifocal VIN III. Sixty-six percent had multicentric neoplasia. Forty-six percent of the biopsies were warty-type, 17% basaloid-type, 35% mixed-type and 2% differentiated-type. Ninety-two percent were HPV-positive and 83% contained HPV-16 DNA. Human papillomavirus DNA was more often present in multifocal VIN III lesions than in unifocal VIN III lesions and also more often in VIN III lesions coexisting with other dysplastic multicentric lesions than in unicentric VIN III lesions. Warty-type VIN III more often contained koilocytes than basaloid-type VIN III. A correlation between different morphologic forms of VIN III and the presence of HPV DNA was not found. Both types of VIN III often coexist in one lesion. In all the RT-PCRs, a 593-base-pair fragment was detected, corresponding to the expected length of the major E6-E7 mRNA. Conclusions. The observed high prevalence of transcriptionally active HPV DNA associated with multifocal and multicentric dysplasia suggests a role of HPV in the pathogenesis of these lesions. A positive correlation between different morphologic forms of VIN III and the presence of HPV DNA was not found. Cancer 1995;75:2879–84.

DNA of the epidermodysplasia-verruciformis associated subgroup of HPV (EV-HPV) is frequently detected in biopsies of premalignant lesions and nonmelanoma skin cancers of renal transplant recipients. The prevalence of EV-HPVs, however, has never been systematically studied in benign keratotic skin lesions of patients with or without a history of skin cancer. This study included 42 renal transplant recipients with and 36 without a history of skin cancer. A total of 176 skin biopsies were tested for the presence of EV-HPV DNA, using a nested polymerase chain reaction (PCR). Method. EV-HPV typing was done by comparison of the sequence of the amplified PCR products with the sequence of all known EV-HPVs. The natural history of the development of keratotic skin lesions was studied. The number of keratotic skin lesions rapidly increased after transplantation. This increase was most pronounced in patients who developed skin cancer. The prevalence of EV-HPV DNA in benign keratotic skin lesions was equally high in patients with and without a history of skin cancer, i.e., 55 and 53% in the two groups, respectively. A large variety of EV-HPV types was found, but of these none were predominantly present in either patient groups. A higher prevalence of EV-HPV DNA was found in benign skin lesions from sun-exposed sites, but only in patients with a history of skin cancer. The association between the number of keratotic skin lesions and the development of skin cancer strongly supports the hypothesis that EV-HPVs play a role in cutaneous oncogenesis. The equally high prevalence of EV-HPV infection in patients with and without a history of skin cancer, however, may indicate that besides EV-HPV infection, other factors, such as sun exposure may also be important. Renal transplant recipients are at an increased risk for warts and nonmelanoma skin cancer, of which squamous cell carcinomas are the most prevalent (1‐ 6). The prevalence and number of warts increase steadily after transplantation (4, 7‐10), and these lesions precede the development of skin cancer by some years (11). The number of keratotic skin lesions is strongly associated with the development of skin cancer (3, 11). DNA of human papillomaviruses (HPV*), which are mainly belonging to the epidermodysplasia-verruciformis- (EV) related subgroup, have been frequently detected in skin cancers and premalignant skin lesions of renal transplant recipients (12‐23). The prevalence of EV-HPVs in benign keratotic skin lesions of renal transplant recipients with and without a history of skin cancer has not been studied before. To address this question, we took biopsies of 176 clinically apparently benign keratotic skin lesions and normal skin in 42 patients with and 36 without a history of skin cancer. Keratotic skin lesions were counted in a group of 66 patients of whom keratotic skin lesions had been counted 7 years previously (11). The relationship between clinical and histological diagnoses was examined in a large number of skin lesions. In addition, the association of age, gender, length of time after transplantation, sun exposure, and skin type with the number of keratotic skin lesions, the presence of HPV DNA in these lesions, and skin cancer was studied.

The sensitivities of dot blot hybridisation and in situ filter hybridisation for the detection of HPV DNA were compared. Dot blot hybridisation was 10-50 times more sensitive than in situ filter hybridisation in detecting HPV 16 DNA in the cervical cancer cell lines SiHa and CaSki. Cervical smears collected from 51 women with a history of one or more abnormal cervical smears were tested by both hybridisation techniques for the presence of HPV 16 DNA; 11 were positive in the in situ filter hybridisation, 35 in the dot blot hybridisation. Thirty-five cervical biopsies available from this group of 51 women were processed for dot blot hybridisation. In 30 of the 35 cases the results of this hybridisation corresponded with the results of the dot blot hybridisation on the smears.

In this study we performed in situ hybridization using biotin-labelled total genomic DNA of Haemophilus influenzae type b as a probe on: (1) smears containing bacteria cultured in vitro: all haemophilus species that can be found in the human respiratory tract appeared to be positive and a large number of other bacterial species appeared to be negative in this in situ hybridization test; (2) sputum smears from 287 patients with bronchitis: the hybridization test was positive on all but 2 of the 44 smears derived from patients whose culture yielded haemophilus and additionally on 12 smears derived from patients, whose culture was negative; and (3) sputum smears from 7 patients suffering from cystic fibrosis (CF): the hybridization test was positive in all these 7 sputum smears, while the culture only yielded haemophilus in 3 cases. The higher sensitivity of the hybridization test compared to culturing could mainly be explained by the failure to detect haemophilus in culture caused by masking due to overgrowth by other bacteria. In conclusion the in situ hybridization test, which can be performed in only 4 h, is a sensitive and specific method for the detection of haemophilus in sputum and is particularly useful in CF patients, where overgrowth by pseudomonas often interferes with diagnosis by culturing.

Two nonculture methods, in situ hybridization and immunoperoxidase staining with monoclonal antibodies, were compared for the detection of Hemophilus influenzae in 184 sputa. For in situ hybridization, a biotin-labeled probe of total genomic DNA of H influenzae type b was prepared that hybridizes specifically with H influenzae, H parainfluenzae, H hemolyticus, and H parahemolyticus DNA. Immunoperoxidase staining was done with monoclonal antibody 8BD9 directed against outer membrane protein P6 of H influenzae. Both techniques detected Hemophilus in sputum equally well and were superior to culture: all 30 sputum samples culture-positive for H influenzae were positive on both nonculture tests, and 13 additional positive sputum samples were detected from which Hemophilus was not cultured. The higher sensitivity of the nonculture tests was mainly attributed to culture failure because of overgrowth of H influenzae by other bacteria, especially in patients with cystic fibrosis. The immunoperoxidase staining technique appeared slightly easier and quicker to perform than the in situ hybridization test. For the in situ DNA hybridization probe, DNA can be prepared from any strain of H influenzae. The immunoperoxidase test requires monoclonal antibody 8BD9 but has a higher specificity than the hybridization technique. Both techniques can be reliably applied, especially for the detection of Hemophilus in sputum of patients with cystic fibrosis.

Sodium-iodide density gradient centrifugation of lysates of BSC cells infected with herpes simplex virus type 2 was carried out in the presence of ethidium bromide. Two DNA bands could be visualized. The lower DNA band was further identified as herpes simplex virus type 2 DNA by its plaque-forming capacity and sedimentation behavior.