Mutation and abnormal expression of the p53 gene in the viral skin carcinogenesis of epidermodysplasia verruciformis.

Patients suffering from epidermodysplasia verruciformis are prone to nonmelanoma skin cancers, due to an inherited abnormal susceptibility to the oncogenic human papillomavirus type 5. Genotoxic sunlight ultraviolet B radiations are likely to be a cofactor. Lesions of two human-papillomavirus-type-5-infected epidermodysplasia verruciformis patients collected during an 8 y period were retrospectively studied for p53 mutations in exons 5 through 8 by a polymerase chain reaction single-strand conformation polymorphism technique and/or by DNA sequencing of amplified exons. Mutations were detected in 11 of 26 (42.3%) specimens, including five (62.5%) squamous cell carcinomas, three (33.3%) Bowen's carcinomas in situ, two (40%) actinic keratoses, and one (33%) benign lesion. The nine mutations characterized by sequencing were shown to be missense and to affect mutational hotspots in human cancers. Five were C-->T transitions at dicytidine sites considered as ultraviolet signature mutations. Two were transversions (C-->G and C-->A) at dicytidine sites and two were C-->T transitions at nondipyrimidine sites. A marked p53 immunoreactivity was disclosed in 72.7% of 11 invasive carcinomas, 55.6% of nine carcinomas in situ, 37.5% of eight actinic keratoses, and one of three benign lesions. This includes 81.8% of 11 specimens with a p53 mutation but also 50% of 14 specimens with no mutation detected. A dysfunction of the p53 gene is thus likely to play a part in epidermodysplasia verruciformis carcinogenesis, either due to ultraviolet-B-induced p53 mutations, as in nonmelanoma skin cancers in the general population, or involving other mutagens or mechanisms. The part played by human papillomavirus type 5 proteins expressed in epidermodysplasia verruciformis keratinocytes remains to be determined.

Variation in the nucleotide sequence of cottontail rabbit papillomavirus a and b subtypes affects wart regression and malignant transformation and level of viral replication in domestic rabbits.

We previously reported the partial characterization of two cottontail rabbit papillomavirus (CRPV) subtypes with strikingly divergent E6 and E7 oncoproteins. We report now the complete nucleotide sequences of these subtypes, referred to as CRPVa4 (7,868 nucleotides) and CRPVb (7,867 nucleotides). The CRPVa4 and CRPVb genomes differed at 238 (3%) nucleotide positions, whereas CRPVa4 and the prototype CRPV differed by only 5 nucleotides. The most variable region (7% nucleotide divergence) included the long regulatory region (LRR) and the E6 and E7 genes. A mutation in the stop codon resulted in an 8-amino-acid-longer CRPVb E4 protein, and a nucleotide deletion reduced the coding capacity of the E5 gene from 101 to 25 amino acids. In domestic rabbits homozygous for a specific haplotype of the DRA and DQA genes of the major histocompatibility complex, warts induced by CRPVb DNA or a chimeric genome containing the CRPVb LRR/E6/E7 region showed an early regression, whereas warts induced by CRPVa4 or a chimeric genome containing the CRPVa4 LRR/E6/E7 region persisted and evolved into carcinomas. In contrast, most CRPVa, CRPVb, and chimeric CRPV DNA-induced warts showed no early regression in rabbits homozygous for another DRA-DQA haplotype. Little, if any, viral replication is usually observed in domestic rabbit warts. When warts induced by CRPVa and CRPVb virions and DNA were compared, the number of cells positive for viral DNA or capsid antigens was found to be greater by 1 order of magnitude for specimens induced by CRPVb. Thus, both sequence variation in the LRR/E6/E7 region and the genetic constitution of the host influence the expression of the oncogenic potential of CRPV. Furthermore, intratype variation may overcome to some extent the host restriction of CRPV replication in domestic rabbits.

[Human papillomaviruses and carcinogenesis of the uterine cervix: future prospects in the domain of detection and prevention]. / Papillomavirus humains et carcinogenèse du col utérin: perspectives dans les domaines du dépistage et de la prévention.

It is now admitted that certain genotypes of human papillomavirus (HPV), mainly HPV types 16 and 18, play an etiological role in the origin of the great majority of invasive carcinomas of the uterine cervix and their intraepithelial precursors. Such an evidence has modified our understanding of the natural history of cervical cancer and should result in new approaches for the early diagnosis and prevention of precursor lesions. Sensitive, specific and reliable HPV detection tests have been progressively designed but their use as routine tests requires multicentric studies, involving large series of women, to evaluate their usefulness in the clinical management or the screening of patients and to establish their limits and cost-effectiveness. It is already most likely that the association of HPV detection tests to cervicovaginal cytology would increase the detection rate of high-grade intraepithelial neoplasia and constitute a means for quality control in cytology. The viral origin of most cancers of the uterine cervix paves the way for their prevention by vaccination against the main oncogenic HPV genotypes and provides hope for specific immunotherapy of associated neoplasia.

A mouse model for studying epidermodysplasia-verruciformis-associated carcinogenesis.

Epidermodysplasia verruciformis (EV) is considered as a model of genetic cancer due to unusual susceptibility to EV-specific human papillomaviruses (HPVs). We established an in vivo experimental system for long-term propagation of EV tissue which should facilitate the study of tumor progression in EV. Skin fragments from benign and early pre-malignant lesions of 6 EV patients were grafted under the kidney capsule of athymic mice. After several months the grafted tissue formed epidermal cysts. These cysts showed typical EV cytopathic effect, in situ hybridization revealed the presence of HPV 5, 8, 9, 12 and 36 DNA, and immunoperoxidase staining detected papillomavirus-group-specific antigens in the permissive cells of the cysts. In some cysts, there were numerous mitoses and downward proliferation of the epidermis with slight dyskeratotic changes. The experimental system described constitutes a model for studies on the role of HPV and other co-factors in human cutaneous carcinogenesis.

Human papillomaviruses associated with cervical intraepithelial neoplasia. Great diversity and distinct distribution in low- and high-grade lesions.

All together, 30 genital human papillomavirus (HPV) types have been characterized so far. To evaluate the importance of HPV diversity in associated cervical diseases, we analyzed 188 biopsy specimens obtained from patients with a recent diagnosis of cervical HPV infection or intraepithelial neoplasia (CIN). Of these 188 specimens, 116 were classified as low-grade CIN (48 cases), high-grade CIN (53 cases), condylomata acuminata (10 cases), flat condylomas (five cases). Seventy-two specimens were considered nondiagnostic. Using probes specific for 18 genital HPV types, HPV DNA sequences were detected by Southern blot hybridization in 100 lesions and 21 nondiagnostic specimens. When further analyzed by the polymerase chain reaction, eight HPV-negative biopsy specimens, four CIN, and four nondiagnostic specimens were positive. Of the 129 positive biopsy specimens, 92 contained at least one of 18 known HPV types and 37 HPV that have not yet been identified. Nine specimens had more than one type. Thirteen HPV types were identified in CIN. The detection rate of HPV 16 increased from 21% in low-grade CIN to 57% in high-grade CIN. HPV 18 was detected in only 3% of CIN; HPV 31, 33, and 35 were found in 8%. HPV 30, 39, 45, 51, 52, 56, 58, and 61 were detected in 44% of low-grade CIN but in only 8% of high-grade CIN. Unidentified HPV were detected in about 25% of low-grade and high-grade CIN. Fifty-seven CIN positive for at least one HPV type were further analyzed by in situ hybridization. Thirty-five (65%) biopsy specimens were positive, including 21 of 24 low-grade CIN and 14 of 33 high-grade CIN. Ten of the 13 previously identified HPV types were detected. Thus, CIN represents an heterogeneous disease from a virologic viewpoint. This fact could explain their variable clinical evolution.

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-Löwenstein 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.

Human papillomavirus type 29 (HPV-29), an HPV type cross-hybridizing with HPV-2 and with HPV-3-related types.

The cloning and partial characterization of human papillomavirus (HPV) type 29 is presented. By hybridization analyses, this virus appears to be related to HPV types associated with common warts and HPV types associated with flat warts.

Rearrangement of a common cellular DNA domain on chromosome 4 in human primary liver tumors.

Hepatitis B virus (HBV) DNA integration has been shown to occur frequently in human hepatocellular carcinomas. We have investigated whether common cellular DNA domains might be rearranged, possibly by HBV integration, in human primary liver tumors. Unique cellular DNA sequences adjacent to an HBV integration site were isolated from a patient with hepatitis B surface antigen-positive hepatocellular carcinoma. These probes detected rearrangement of this cellular region of chromosomal DNA in 3 of 50 additional primary liver tumors studied. Of these three tumor samples, two contained HBV DNA, without an apparent link between the viral DNA and the rearranged allele; HBV DNA sequences were not detected in the third tumor sample. By use of a panel of somatic cell hybrids, these unique cellular DNA sequences were shown to be located on chromosome 4. Therefore, this region of chromosomal DNA might be implicated in the formation of different tumors at one step of liver cell transformation, possibly related to HBV integration.

Plurality of genital human papillomaviruses: characterization of two new types with distinct biological properties.

The genomes of two new genital human papillomavirus (HPV) types, tentatively named HPVs 39 and 42, have been cloned from biopsy specimens of penile Bowenoid papules and vulvar papillomas, respectively. Blot hybridization experiments, performed under stringent conditions (Tm -10 degrees), have revealed no cross-hybridization between the DNAs of HPVs 39 and 42, and between these DNAs and those of other genital and cutaneous HPVs. A significant cross-hybridization has been observed between the DNA of HPV42 and that of HPV32, the latter being associated with oral focal epithelial hyperplasia. The fraction of HPV32 and HPV42 hybrid molecules resistant to nuclease S1 treatment after hybridization in liquid phase at saturation has been evaluated to 20%, supporting the view that these HPVs constitute distinct types. In addition to HPV42 DNA, a 6.8-kb BamHI fragment, cross-hybridizing with HPV39 DNA, has been cloned from the vulvar papilloma DNA preparation. The cross-hybridization has been evaluated to 16%, pointing to the existence of an additional HPV39-related type. Electron microscope analysis of heteroduplex molecules formed between HPV32 and HPV42 DNAs showed paired regions over about 60 and 87% of their genome lenghts under stringent (Tm -18 degrees) and nonstringent (Tm -42 degrees) conditions, respectively. The 6.8-kb HPV DNA and HPV39 DNA formed paired regions over about 63 and 95% of the 6.8-kb fragment length at Tm -18 degrees and Tm -26 degrees, respectively. These data point to greater DNA sequence homologies than anticipated from the percentages of nuclease S1 resistance. Heteroduplex mapping has allowed the alignment of the physical maps of HPV39 and 42 DNAs and of the 6.8-kb HPV DNA with the map of the open reading frames of the HPV16 genome. So far, HPV42 has been detected only in benign genital lesions showing usually no cell atypia. HPV39 has been detected in a few cases of intraepithelial neoplasias and invasive carcinomas of the uterine cervix. The viral DNA sequences have been found integrated into the cell genome in all four HPV39-associated cervical cancers of our series. It seems most likely that HPV42 belongs to the low-risk group of genital HPVs, while HPV39 represents a potentially oncogenic genital HPV type.

Integration of human papillomavirus type 16 DNA sequences: a possible early event in the progression of genital tumors.

The keratinocyte line SK-v harbors only integrated human papillomavirus type 16 (HPV 16) DNA sequences, although it originated from vulvar Bowenoid papules predominantly containing multiple copies of free HPV 16 genomes. We have cloned a fragment of cell DNA that contains the integrated HPV 16 DNA sequences and have shown that integration interrupts the HPV 16 genome in open reading frames E2 and L2 and creates a deletion of 813 base pairs. This allows the expression of open reading frames E6 and E7, as actually substantiated by Northern (RNA) blot analysis of SK-v RNAs with subgenomic HPV 16 RNA probes. Using a unique flanking cellular DNA sequence as the probe, we have shown that the integration of HPV 16 sequences had already occurred in the premalignant lesions from which the SK-v cell line was derived.

High prevalence of papillomavirus-associated penile intraepithelial neoplasia in sexual partners of women with cervical intraepithelial neoplasia.

To determine whether neoplastic cervical lesions in women are associated with papillomavirus infections in their sexual partners, we used a colposcope to examine male sexual partners of women with cervical flat condyloma (294 cases) or cervical intraepithelial neoplasia (186 cases), before and after 5 percent acetic acid was applied to the penis and the anogenital area. Condylomata acuminata, papules, and macules were observed in 309 of the 480 men (64.4 percent). In 204 of them (42.5 percent), macules or slightly elevated papules were detected only after application of acetic acid. Condylomata acuminata or lesions showing histologic features of condyloma were found in 121 partners (41.2 percent) of women with condyloma, but in only 10 partners (5.4 percent) of women with cervical intraepithelial neoplasia. Penile lesions showing histologic features of intraepithelial neoplasia were found in 61 partners (32.8 percent) of women with cervical intraepithelial neoplasia, but in only 4 partners (1.4 percent) of women with flat condyloma. Thirty-six (60 percent) of the 60 macules or papules tested contained papillomavirus DNA sequences. Human papillomavirus types 16 and 33 were almost exclusively found in penile intraepithelial neoplasia. Type 6, type 11, and the recently recognized type 42 were found in lesions showing features of condyloma or minimal histologic changes. As yet uncharacterized papillomaviruses were found in 15 percent of the specimens. These data support the concept that cervical carcinomas and precancerous lesions in women may be associated with genital papillomavirus infection in their male sexual partners.

The morphology of butchers' warts as related to papillomavirus types.

Hand warts were studied in 160 butchers. Clinical and histological studies were performed in 190 warts and virological studies in 165 warts from 104 butchers. Since we found almost perfect correlation between the histological pattern and the type of infecting virus, it was possible to evaluate the virus types in a further 39 of 56 butchers without virological studies, on the basis of the histology of the warts. The most common infection was with HPV-2 (human papilloma virus) and HPV-7. Thirty-three butchers were infected with two types of viruses and three butchers with three HPVs. The morphology of warts varied considerably. The majority were similar to verrucae vulgares or verrucae planae. Some deep warts resembled myrmecia-type verrucae plantares. Often, several types of warts coexisted. Some clinical patterns were shown to be preferentially associated with distinct types of papillomaviruses: common warts with HPV-2, HPV-4, or HPV-7, plane and intermediate warts with HPV-3, HPV-10, HPV-28. HPV-7, previously identified for the first time in these butchers, was found to be associated with common warts or common wart-like, papillomatous lesions.

The L2 open reading frame of human papillomavirus type 1a encodes a minor structural protein carrying type-specific antigens.

The proteins encoded by the open reading frames of papillomavirus genomes and the minor polypeptides detected in purified virions are still poorly defined. We show here by its expression in Escherichia coli that the open reading frame L2 of human papillomavirus type 1a codes for a minor structural protein of Mr 76,000. Antisera raised against a truncated L2-beta-galactosidase fusion protein in which the conserved N-terminal region of L2 is missing are type specific for human papillomavirus type 1 virions and are reactive at high dilutions. Expression of the L2-encoded type-specific antigens thus provides a powerful new tool for the identification of papillomaviruses.

A novel type of human papillomavirus associated with genital neoplasias.

The role of human papillomaviruses (HPVs) in the development of genital neoplasias has been well documented. The genomes of two HPV types, HPV16 and HPV18, have been found to be associated with about 70% of invasive carcinomas of the uterine cervix. As, under non-stringent hybridization conditions, HPV DNA sequences have been detected in about 90% of cervical carcinomas, it seems likely that additional HPV types are associated with these tumours. Here we report the molecular cloning and characterization of a novel HPV type, tentatively named HPV33, whose sequences have been detected in 4-8% of biopsies of genital intra-epithelial neoplasias and cervical invasive carcinomas, usually as free monomeric or oligomeric molecules. However, in one specimen of vulvar Bowen's disease, HPV33 DNA sequences were integrated in the host-cell genome. Thus, HPV33 probably represents an additional type of potentially oncogenic genital HPV.

Characterization of a new type of human papillomavirus found in a lesion of Bowen's disease of the skin.

The genome of a human papillomavirus (HPV) found in a patient with Bowen's disease of the skin was molecularly cloned. Blot hybridization experiments, performed under stringent conditions, revealed no cross-hybridization between this HPV DNA and the other known HPV DNAs, showing that this HPV represents a new type, tentatively named HPV34. In relaxed hybridization conditions, the highest cross-hybridization was observed with HPV16 DNA. The physical map of HPV34 DNA was aligned with the genetic map of HPV16 DNA by heteroduplex mapping. HPV34 was not detected in 12 additional patients with Bowen's disease of the skin, but a closely related HPV DNA was found in one patient with penile Bowenoid papulosis.

[Human papillomaviruses in the epithelial cells of the cervix uteri: frequency of types 16 and 18. Preliminary results of a clinical, cytologic and viral study]. / Recherche de papillomavirus humains dans des cellules épithéliales du col utérin: fréquence des types 16 et 18. Résultats préliminaires d'une étude clinique, cytologique et virologique.

Between April 1 and June 30 1984, cervical scrapes were taken from 381 women attending the Gynecology Department of the Anticancer Center René-Huguenin. The scrapes were examined for the presence of human papillomavirus (HPV) DNA, by a molecular hybridization method, at the Pasteur Institute. The four HPV types involved in genital pathology, HPV 6, HPV 11, HPV 16 and HPV 18, were studied. Twenty four specimens (6.3%) were found positive: 19 for HPV 16, 3 for HPV 18, 2 for HPV 6 or HPV 11. Results of molecular hybridization were compared with cytological findings. HPV 6 or HPV 11 were detected in cases of mild dysplasia. HPV 16 or HPV 18 were mainly detected in cases diagnosed as severe dysplasia or carcinoma in situ (9 out of 14, i.e. 64.3%), and invasive carcinoma (3 out of 5 cases). The results were further confirmed when virological data obtained with cervical scrapes were compared with the histological diagnosis on biopsies: 14 out of 15 cases of severe dysplasia or carcinoma in situ (93%) and 3 out of 6 cases of invasive squamous carcinoma had been found positive for HPV 16 or HPV 18. Interestingly, four "normal" women (1.3%) with a negative cytology were found positive for HPV 16 or HPV 18. The data obtained by this sensitive and reliable method are useful to the clinician to identify women presenting a high risk of subsequent cervical intraepithelial neoplasia or invasive carcinoma, and, thus, to adapt the treatment and the follow-up of these patients.