Characterization of a new human papillomavirus (HPV 41) from disseminated warts and detection of its DNA in some skin carcinomas

A new human papillomavirus type (HPV 41), distantly related to known HPV prototypes, was isolated from a patient with disseminated facial, peri-anal and foot warts (epidermodysplasia verruciformis was not diagnosed). The viral DNA was molecularly cloned in 2 BamH1 restriction fragments with sizes of 6.5 kb and 0.98 kb, respectively. The classification of this cloned DNA as a new type is based on the degree of cross-hybridization with 40 HPV types under conditions of varying stringency. A total of 106 benign and malignant skin lesions, as well as 71 malignant tumours of different origins, were screened for the presence of HPV-related sequences. In 2 out of 10 squamous-cell carcinomas and in 1 of 3 arsenic keratoses HPV 41 DNA sequences could be detected.     

Two new human papillomavirus types (HPV54 and 55) characterized from genital tumours illustrate the plurality of genital HPVs

The genomes of two new human papillomavirus (HPV) types, named HPV54 and HPV55, were cloned from penile lesions of 2 patients. HPV54 was isolated from a verrucous carcinoma (Buschke-L?wenstein tumour) together with full-length HPV6 genomes and HPV6 DNA molecules with a deletion of about 0.3 kb located in the non-coding region. HPV55 was isolated from a condyloma acuminatum. No cross-hybridization was observed between HPV54 DNA and the DNAs of the known cutaneous and genital HPVs by blot hybridization experiments performed under stringent conditions. In contrast, significant cross-hybridization was detected between HPV55 DNA and the DNA of HPV13, associated with benign oral lesions, and, to a lesser extent, with the DNAs of HPV6, 11, and 44, associated with benign genital proliferative lesions. The DNA sequence homology between HPV55 and HPV6, 11, and 13 was estimated at 12%, 12%, and 20%, respectively, by hybridization in liquid phase at saturation, followed by nuclease S1 analysis. The physical maps of HPV54 and 55 were aligned with the genetic maps of HPV16 and 11, respectively, by heteroduplex mapping and partial DNA sequencing. HPV54 is thus only weakly related to the known HPVs, while HPV55 represents an additional HPV6-related HPV type. HPV54 and HPV55 are uncommon genital HPV types since, in a survey of a large series of specimens of benign, pre-malignant or malignant anogenital and orolaryngeal tumours, HPV54 was not detected, and HPV55 was found in another case of condyloma acuminatum.     

Presence of HPV 16 sequences in laryngeal carcinomas

Human papillomavirus types HPV 16 and HPV 11 DNA sequences were analyzed in normal and neoplastic tissues of the larynx, using the technique of polymerase chain reaction (PCR). An amplified region of E6 ORF was hybridized with 3' end-labelled oligonucleotide probe. Twenty six out of 48 (54%) squamous-cell carcinomas, and 3 out of 3 verrucous-cell carcinomas hybridized with HPV 16 DNA sequences, whereas we did not detect HPV 11 sequences. HPV 16 DNA sequences were also found in normal, autologous mucosa and lymphnode metastases, although these were absent in other tissues analyzed. HPV-16-positive tumors were most frequently poorly differentiated squamous-cell carcinomas.     

Expression of p16INK4a and MIB-1 in relation to histopathology and HPV types in cervical adenocarcinoma

A total of 101 primary cervical adenocarcinomas were analyzed for the presence of p16INK4a and MIB-1 expression in correlation with the presence of 'high-risk' types of human papillomavirus (HR-HPV) and clinical outcome.We found that adenocarcinoma grading showed a significant negative correlation to p16INK4a levels (p=0.001): i.e. we found less intense p16 staining in poorly differentiated tumors than in more highly differentiated tumors as well as a highly significant correlation between HPV infection and higher levels of p16INK4a staining (p=0.00), which was similar for different HPV-types. Tumor suppressor protein p16INK4a levels were higher in HPV positive than in HPV negative tumors. Higher levels of the proliferation marker MIB-1 were associated with poorer outcome. Higher MIB-1 levels were seen in tumors with a lower grade and higher stage at diagnosis. Moreover, MIB-1 levels seem to be higher in tumors due to infection with HPV 16 and 18 compared with HPV 45. MIB-1 may be a helpful marker in grading adenocarcinoma: a high level of expression of MIB-1 indicates a low grade of tumor, whereas high expression of p16INK4a indicates a highly differentiated of the tumor. Thus, immunostaining for p16INK4a appears to be a useful diagnostic tool for cervical adenocarcinoma.     

Prevalence of human papillomavirus type 16-related DNA in cutaneous Bowen's disease and squamous cell cancer

Human papillomavirus (HPV) type 16 and related types are frequently found in genital located Bowen's disease but they are only rarely identified in non-genital cutaneous Bowen's disease and squamous cell cancer (SCC). We used the polymerase chain reaction to detect HPV 16 and related DNA sequences in 205 formalin-fixed non-genital cutaneous Bowen's disease and SCC specimens from 159 non-immunosuppressed patients. HPV 16 and related DNA sequences could be detected in 12 of 198 cutaneous specimens from extra-genital sites other than the fingers and in 2 of 7 specimens from digital lesions. Our study suggests a minor but still not negligible role of HPV 16 and related HPV-types in the etiology of non-genital non-melanoma skin cancer of the general population.     

Human papilloma virus (HPV) is rarely detected in malignant melanomas of sun sheltered mucosal membranes

Human papillomavirus (HPV) has been associated with some types of human cancer. The aim of this study was to investigate if HPV could be associated with human primary malignant melanoma in non sun-exposed body areas like mucous membranes. Through the Swedish National Cancer Registry, in compliance with the rules of the Human Ethical Committee, histopathological specimens were collected from different pathological laboratories throughout Sweden. The histopathological diagnosis was reviewed, and from 45 primary melanomas, tumour tissue was micro-dissected and analysed further. A protocol for detection of HPV DNA using general HPV primers GP5 + /GP6+ or CPI/IIG, which together identify 36 different HPV subtypes, was developed. This protocol could detect presence of HPV DNA in less than 10 ng of DNA of a control cell that contained 1-2 copies of HPV type 16/cell. Before HPV testing the melanoma samples were examined for amplifiable DNA by a β-microglobulin PCR and 39 were positive. Thirty-five of these could be evaluated for HPV DNA and no samples were positive according to all five defined criteria for HPV positivity although two were positive according to 4/5 criteria. In conclusion, HPV is rarely detected in primary malignant melanomas of non-sun exposed body areas.     

Search for HSV DNA in genital,cerebral and labial tumors

DNA sequences homologous to HSV-1 and HSV-2 DNA fragments were searched in 64 genital, 35 labial and 34 cerebral tumors. Southern blot transfers of tumor and control DNAs were hybridized in stringent conditions with ³²P labelled probes from HSV-1 and HSV-2 cloned DNA fragments. Specific hybridization to HSV-2 BgIII N fragment was observed in six (9.4%) genital tumors. Labial and cerebral tumors did not show hybridization to any of the probes used. The technique employed allowed the detection of 0.1 copies of viral fragments per diploid genome.     

Mapping common deleted regions on 5p15 in cervical carcinoma and their occurrence in precancerous lesions

Previous studies have shown that the short arm of chromosome 5 (5p) exhibit frequent genetic changes in invasive cervical carcinoma (CC), and that these changes arise early during the carcinogenesis, in precancerous lesions. These data therefore suggest that loss of candidate tumor suppressor genes located on 5p is associated with the development of CC. However, the precise location of 5p deletions is not known. We performed a detailed deletion mapping of 5p in 60 cases of invasive CC. We found that 60% of the tumors exhibit a 5p loss of heterozygosity (LOH). The patterns of LOH allowed us to identify two minimal regions of deletions, one at 5p15.3 spanning a 5.5 cM genetic distance and a second site of 7 cM at 5p15.2-15.3. In addition, we also identified 5p deletions in 16% lesions of high-grade cervical intraepithelial neoplasia (CIN). 5p LOH was found in 63% of HPV 16 positive tumors, while only 33% tumors with other HPV-types had 5p LOH. The differences in frequency of 5p LOH between tumors harboring HPV16 in combination with other HPV types and tumors harboring HPV16 DNA alone were significantly higher, suggesting a synergistic effect of high-risk types in causing genomic instability. These findings implicate the presence of tumor suppressor gene(s) on 5p relevant to CC tumorigenesis.     

Characterization of a human papillomavirus from epidermodysplasia verruciformis lesions of a patient from Upper-volta

A case of epidermodysplasia verruciformis in a patient from Upper-Volta is described. Slightly elevated, flat warts were observed on hands, feet, arms and legs, and pityriasis versicolor-like lesions were found mainly on the trunk. The patient showed no malignant tumors. Histological examination revealed hyperkeratosis, granulosis and moderate acanthosis with large, foamy, basophilic keratinocytes in stratum granulosum and stratum spinosum. Papillomavirus particles could be prepared from these lesions and were differentiated from known papillomavirus types by immune electron microscopy with monospecific antisera and by DNA-DNA hybridization. The viral DNA was characterized by cleavage with several restriction endonucleases and a physical map of the resulting fragments was established. The virus is designated as HPV 8. Preliminary seroepidemiologic studies with human sera indicate a rather wide distribution of HPV 8. Blot hybridization of DNA from human carcinomas with 32P-labelled virus DNA detected no HPV 8-specific sequences.     

Human papillomavirus infection and anal cancer

To study the association of human papillomavirus (HPV) infection with anal cancer, we examined tissue specimens from 126 patients with malignant lesions of the anal skin or mucosa. The patients were enrolled in a population-based, case-control study of ano-rectal cancer which is being conducted in the state of Washington and the Province of British Columbia. Histologic sections from formalin-fixed, paraffin-embedded tissues were tested for the presence of HPV DNA by in situ hybridization with biotin-labelled HPV 6, 11, 16, 18 and 31 DNA probes. HPV DNA sequences were found in tumor tissues from 24 of the 126 subjects (19.0%). When only squamous neoplasms are considered, 23 of 70 subjects (32.9%) had lesions which contained detectable HPV DNA. One HPV-positive patient had a cloacogenic carcinoma that contained regions of squamous differentiation and it was in these squamous cells that HPV DNA was localized. Of the 23 squamous lesions that harbored detectable HPV DNA, 8 contained HPV 6, 10 contained HPV 16, 1 contained HPV 18 and 4 contained an unclassified virus type(s). HPV DNA was found in tissues from 14 patients with carcinoma-in situ and 10 subjects with invasive carcinoma. These results demonstrate that some malignant tumors of the anus, in both men and women, are associated with HPV infection. We conclude that the anal squamous epithelium is another site where infection with the common genital tract HPVs may carry a risk of malignant transformation.     

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

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

Loss of heterozygosity on chromosomal segments 3p, 6q and 11p in human ovarian carcinomas

The genetic events involved in human ovarian carcinoma development are still largely unknown. We have used DNA recombinant technologies to examine the genome of normal and neoplastic tissues from 12 different patients with such tumors. We used cloned DNA sequences homologous to loci showing restriction fragment length polymorphisms on chromosomal segments 3p, 5p, 6q, 11p, or 21q to determine the frequencies of losses of constitutional heterozygosity at these loci in tumor DNAs. Losses affecting loci on 3p, 6q, and 11p were found in a high percentage of the cases examined. In contrast, losses of heterozygosity were not found when we used DNA probes for the other 2 above chromosomal segments. Thus, genetic losses involving chromosomal segments 3p, 6q and 11p occurred at non-random frequencies in ovarian carcinomas, suggesting that inactivation of genes located on these chromosomes played a role in their development.     

New human papillomavirus sequences in female genital tumors from Japanese patients

Tissues from 52 cervical carcinomas, 15 cervical intraepithelial neoplasias III (CINs III), and 3 vulvar carcinomas were examined for the presence of human papillomavirus (HPV) sequences by Southern blot hybridization with HPV 16 or 18 DNA as the probe. HPV 16 or 18 DNA was detected in only 17 cervical carcinomas (33%), 5 CINs III (33%), and 1 vulvar carcinoma (33%). These frequencies are lower than those reported by others. However, new, as yet unidentified, HPVs were also detected at rather high frequency under less stringent conditions of hybridization using HPV 16 and 18 DNAs as probes. These HPVs did not hybridize with HPV 6, 11, 16, or 18 under stringent conditions, and were different from two other known types of genital tumor-associated HPVs, HPV 31 and HPV 33, judging from the patterns of their restriction enzyme digests. The total frequencies of HPV sequences were 48% (25/52) for cervical carcinomas, 53% (8/15) for CINs III, and 67% (2/3) for vulvar carcinomas.     

Human papillomavirus DNA in genital tumours

Human papillomaviruses (HPV) types 16 and 18 have been identified in two different human cervical carcinomas. The viral DNAs were molecularly cloned and used as probes to screen a large number of genital tumours by Southern blot analysis. HPV 16 or HPV 18 sequences were found in a high percentage of cervical carcinomas but only in a small number of condylomata acuminata or flat condylomas. The majority of the latter lesions, however, contained HPV 6 or HPV 11 sequences, respectively, which, in contrast, were detected only rarely in carcinoma in situ or invasive carcinomas. A similar distribution of the different papillomaviruses was observed when cell scrapings taken from the cervix were tested by in situ hybridization.     

Alterations of the p53 gene in human primary cervical carcinoma with and without human papillomavirus infection.

A previous report using cervical carcinoma cell lines suggests that the inactivation of two tumor suppressor gene products, p53 and pRB, either by complex formation with the E6 and E7 proteins of oncogenic human papillomaviruses (HPVs) or by mutation, may be an important step in cervical carcinogenesis (M. Scheffner et al., Proc. Natl. Acad. Sci. USA, 88: 5523-5527, 1991). The present study was designed to clarify the association between p53 inactivation and infection with oncogenic HPVs in primary carcinomas of human uterine cervix. We examined 36 primary cervical carcinomas for the presence of HPV DNAs by Southern blot analysis with probes specific for HPV-16, -18, -31, -33, -52, -56, and -58. HPV DNA sequences were detected in 19 of 36 tumors: 10 cases with HPV-16; 3 cases with -18; 3 cases with -58; 2 cases with -56; and one case with -52. The presence of HPV-16 and -18 in cervical carcinomas was further reexamined using polymerase chain reaction. HPV DNA sequences were detected in an additional 10 cases: 9 cases with -16 and one case with -18. The inactivation of the p53 gene by allelic loss or by point mutation was also examined. No allelic loss at the polymorphic site in codon 72 of the p53 gene was detected in any of 10 informative cases. Missense point mutations in the highly conserved regions of the p53 gene were demonstrable as single-stranded conformational polymorphisms of polymerase chain reaction-amplified DNA fragments and subsequently identified by direct DNA sequencing. Point mutations were detected in only two cases: one with an ATG----CTG transversion in codon 133 of exon 5, resulting in a Met----Leu substitution, and another with a CGG----TGG transition in codon 248 of exon 7, resulting in an Arg----Trp substitution. Both tumors with point mutations in p53 genes were among 10 tumors which contained a small copy number of HPV-16 DNA sequences (1 copy of HPV/10(1) to 10(5) cells) detectable by polymerase chain reaction amplification but not by Southern blot analysis of genomic DNAs derived from the tumors. None of 19 tumors with a large copy number of HPV DNA sequences detectable by Southern blot analysis (more than 1 copy of HPV/2 to 10 cells) nor any of 7 tumors with undetectable HPV DNA sequences contained p53 gene mutations in the regions examined.(ABSTRACT TRUNCATED AT 400 WORDS)     

Papillomavirus DNA in human tongue carcinomas

Seven biopsies from different carcinomas of the tongue were analyzed for human papilloma virus (HPV) sequences by Southern blot analysis. After hybridization with various types of human papilloma viruses, 3 tumors were found to be positive. Whereas DNA from one tumor hybridized with HPV 2 DNA under conditions of high stringency, the 2 other positive biopsies contained HPV 16 DNA. All positive tumors revealed high copy numbers of the respective DNA. The cleavage pattern of the HPV-2-positive tumor differed from the established HPV 2 prototypes. Minor differences from the HPV 16 prototype were also noted in one of the HPV-16-positive tongue carcinomas.