The search for a culture system for papillomavirus.

Papillomaviruses induce tumors of keratinocytes. Vegetative viral DNA replication and virion assembly are seen in those cells which are in the process of keratinizing or are keratinized. To date, no cell culture system has been developed that permits expression of the complete viral life cycle. Keratinocytes infected in culture may harbor the virus as a stable, replicating episome, but they do not support vegetative viral growth, nor do they become immortalized or transformed. The major obstacle in using keratinocyte cultures may be related to a dual need for transformation and full differentiation. Some animal papillomaviruses have been shown to be capable of transforming cultured murine fibroblasts. The fibroblast model is useful for identifying the viral-transforming gene(s) and their products.

Identification of papillomaviruses in butchers' warts.

We have studied the papillomaviruses found in the hand warts of 60 butchers, most of them from 2 distant slaughterhouses. Warts differing in morphology and location were studied separately. The viruses were identified by molecular hybridization, restriction enzyme analysis and immunofluorescence. Four known human papillomaviruses (HPV-1, HPV-2, HPV-3, HPV-4) were detected and one hitherto unknown papillomavirus was identified in 9 butchers. The DNA of the latter virus did not anneal with any of the RNAs complementary to either HPV-1 to HPV-5 or bovine papillomavirus type 1 (BPV-1) DNAs, and showed a Hind II + III restriction enzyme cleavage pattern distinct from those of known HPVs and BPVs. This virus showed distinct antigenic properties, as shown by immunofluorescence, using HPV-1, -2, -3, -5, and BPV-1 antisera. It may represent a new type of human papillomavirus (HPV-7) or a yet unidentified animal papillomavirus. In addition, 6 butchers were found to be infected with a papillomavirus, distinct from the known skin HPVs and from BPV-1, which could not be characterized by restriction enzyme analysis. Eleven butchers were found to be infected by 2 viruses. A characteristic histological pattern was found to be associated with the different papillomaviruses.

A potentially oncogenic human papillomavirus (HPV-5) found in two renal allograft recipients.

We have observed 2 immunosuppressed renal allograft recipients with skin lesions induced by human papillomavirus type 5 (HPV-5). One recipient had multiple pityriasis versicolor-like (PV-like) skin lesions on his arms and trunk, and multiple Bowenoid in-situ skin cancers. The other had 2 warty lesions on the back of her fingers. Structural antigens of human papillomavirus type 5 (HPV-5) were identified in benign lesions from both patients by immunofluorescence. The histologic and ultrastructural features observed in lesions from both patients were similar to those previously seen in HPV-5-induced lesions occurring in patients with the rare disease epidermodysplasia verruciformis (EV). The severe form of EV is characterized by HPV-5-induced PV-like lesions, multiple skin cancers, and usually depressed cell-mediated immunity. The picture seen in one of our renal allograft recipients recalls this severe form of EV. HPV-5, until now, has been found only in EV patients. The role of this potentially oncogenic virus in skin cancers which are known to occur with increased frequency in immunosuppressed renal allograft recipients must be determined.

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.

Epidermodysplasia verruciformis versus disseminated verrucae planae: is epidermodysplasia verruciformis a generalized infection with wart virus?

Recently it has been shown that epidermodysplasia verruciformis is induced by human papilloma/virus different from the papilloma/virus of warts, and that 2 distinct viruses-designated HPV-3 and HP-4--are responsible for it. Ten cases of epidermodysplasia verruciformis were found to have been caused by HPV-3. Clinically and histologically, as well as in the often depressed cell-mediated immunity they closely resembled long-standing verrucae planae, also caused by HPV-3. Contrariwise, in epidermodysplasia verruciformis caused by HPV-4 there are characteristic red, red-brown, and depigmented, pityriasis versicolor-like plaques, and malignant transformation seems almost inevitable. Cases due to HPV-3 may be abortive or even regressive, or stationary, and hard to distinguish from flat warts. No malignant conversion was seen in patients infected only with HPV-3, whereas it occurred in 2 patients infected with both viruses: HPV-3 and HPV-4. Pigmented plaques are the most important adverse prognostic sign in EV induced by HPV-3.

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.

Different papillomaviruses as the causes of oral warts.

We have observed four patients with oral papillomas. Two children had oral mucosal lesions characteristic of focal epithelial hyperplasia, a young man had common, wart-like lesions on his hard palate, and a male immunosuppressed renal allograft recipient had condyloma-like lesions on his gingivae. Papillomavirus-like particles were seen by electron microscopy in lesions from both patients with focal epithelial hyperplasia. No structural antigens for human papillomavirus (HPV) 1, 2, 3, or 5 were found by immunofluorescent microscopy, but further evidence of the presence of a papillomavirus was found by immunoperoxidase microscopy using a cross-reacting sodium lauryl sulfate-disrupted bovine papillomavirus 1 anti-rabbit serum sample. The distinct histologic pattern seen in focal epithelial hyperplasia suggests that a yet undescribed HPV type might be associated with this disease. Histologic, ultrastructural, and immunofluorescent microscopy and restriction endonuclease analysis all gave evidence of HPV 2 in the palatal lesions in patient 3. Evidence of papillomavirus antigen was found by immunoperoxidase microscopy in the oral condylomas from our immunosuppressed patient.

An unusual wart-like skin lesion found in a renal allograft recipient.

Immunosuppressed renal allograft recipients have an increased tendency to acquire warts. While studying such patients, we found a virus-induced, wart-like lesion that had an unusual histologic picture. Light microscopic studies showed bizarre keratinocytes with cytoplasmic, juxtanuclear, giant, crescentic bodies and round, nuclear inclusions, By electron microscopy, the giant cytoplasmic bodies were found to be composed of tonofilaments, and the nuclear inclusions were found to be composed of papillomavirus-like particles in a filamentous matrix. Typical papillomavirus particles were observed in a wart extract by the negative-staining method. Although virus was abundant in infected cells, no structural viral antigens of the human papillomavirus (HPV) types 1, 2, 3, or 5 could be detected by immunofluorescence microscopy, indicating infection by HPV 4 or some other, as yet undescribed, HPV type.

Two anatomoclinical types of warts with plantar localization: specific cytopathogenic effects of papillomavirus. Type I (HPV-1) and type 2 (HPV-2).

In this study, the clinical and histopathological aspects of 50 plantar warts are reported in relation to the type of papillomavirus present in the lesions, as detected by immunofluorescence tests, using specific guinea pig fluorescein-labelled IgG. Warts of plantar localization are not caused by the same human papillomavirus (HPV) since they are found to be associated with both HPV type 1 (HPV-1) and HPV type 2 (HPV-2). HPV-1 is always associated with deep and painful plantar warts (myrmecia), whereas HPV-2 is found to be associated with superficial, painless plantar warts (vulgaris or often mosaic type). Histologically, these two types of plantar warts are quite different. In myrmecia (HPV-1), characterized by an endophytic growth, large eosinophilic, keratohyaline-like granules are observed in the cytoplasm and nucleus of infected, often vacuolated cells. These granules appear early in stratum spinosum and are very numerous in stratum granulosum. In the mosaic type (HPV-2), the histopathological aspect is not different from that of common warts; these lesions have an exophytic growth and are characterized by foci of clear vacuolized cells which are found in stratum granulosum. Their cytoplasm contains round, basophilic keratohyalin granules which often have a heterogenous aspect. These differences are observed in other localizations of morphologically related warts associated with HPV-1 and HPV-2 and seem to be related to a specific cytopathogenic effect of HPV-1 an HPV-2 in human papillomas.

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.

[Condylomatous lesions of the uterine cervix: their course in 2466 patients]. / Lésions condylomateuses du col utérin: évolution chez 2 466 patientes.

2466 women with cervical condylomatous lesions out of the general consulting (0.7%) have been followed by the authors. The cytological and histological criteria of these lesions and the detection of the viral antigen by immunoperoxidase (positive in only about 50% of the cases), are recalled. The flat condylomas are often associated with dysplasia (CIN I, II, III). The condylomas appear in women before the age of 20. These cases increased in number between the ages of 25 to 30 and stayed high until 35. The number of condylomas associated with CIN II have their maximum between 36 and 38 years of age and decrease afterwards to age 48. The graphic is the same for CIN III. The evolution of these condylomatous lesions studied during 42 months, shows that in 1269 women with condyloma and nuclear atypia, regression occurred in 53 per cent, persistence in 37 per cent and aggravation in 10 per cent of the cases. In 762 women with CIN II, regression appeared in 39 per cent, persistence in 44 per cent and aggravation in 17 per cent of cases. In a group of 764 closely followed women, regression and aggravation in CIN I and II appeared between the 3rd and the 6th months of follow-up. Condyloma associated with CIN III were not observed after the 3rd month. Recurrence appeared however in 55 cases after insufficient ablation. Lastly, the histogenesis of these lesions and the relationship between the viral action and the host are discussed.

The human papillomaviruses.

Recent biochemical and serological studies have shown the existence of at least four distinct types of human papillomaviruses (HPVs) causing benign skin lesions. These viruses show hardly no antigenic relationships; their DNAs differ by their sensitivity to restriction endonucleases, and show little, if any, sequence homology, as detected by molecular hybridization using complementary RNAs transcribed in vitro. Data on the pathogenicity of HPVs are still incomplete but indicate that some types of benign skin lesions (plantar warts, common warts, flat warts) may be preferentially associated with some types of HPV. Most interesting is that epidermodysplasia verruciformis has been found associated with two types of virus, and that malignant conversion of some lesions has been observed in all the patients infected with one of them. This suggests that at least a HPV may have a higher oncogenic potential, as do rabbit (Shope) papillomavirus and bovine alimentary tract papillomavirus. Much remains to be known on human papilloma-viruses and further studies may lead to the characterization of additional types of HPVs, especially in genital condylomata acuminata and laryngeal papillomas whose malignant conversion, although rare, may be observed. Progress in this field has been and remains hampered by the lack of cell culture systems allowing replication of these highly host and tissue specific viruses, and by the widely variable virus content of the different human lesions known to be associated with a papillomavirus. Further studies are warranted by the possible role of these widespread and epitheliotropic viruses in the origin of some carcinomas in man.

Immunological studies in epidermodysplasia verruciformis.

Immunofluorescence and cell mediated immunity studies have been performed in 14 cases of epidermodysplasia verruciformis (EV), 3 of those abortive or regressing in members of the families of the patients with EV. Two different types of human papillomavirus (HPV)--HPV3 and HPV4--have been found in cases of EV. HPV3 was detected also in flat warts without features of EV. There was no cross-reactivity between these two viruses, neither with HPV1 responsible for plantar warts nor with HPV2 inducing common warts. There was a relationship between the type of HPV and the clinical picture of EV as well as the malignant transformation, namely HPV4 has been found to be more oncogenic. Cell mediated immunity (CMI) seems to be an important factor because it was depressed in a vast majority of active cases and preserved in regressing and abortive cases (in the members of the families of EV patients). However, low CMI has been found in EV cases infected with HPV3 and in persistent flat warts also due to HPV3, which did not undergo malignant transformation. In contrast, in a case of EV due to HPV4 a malignant transformation occured in spite of still preserved, although lowered CMI. Various human papillomaviruses seem to differ in their oncogenic potential. HPV1 responsible for plantar warts, and HPV2 for common warts have no evident oncogenic potential, HPV3 inducing both EV and flat warts has a low oncogenicity, whereas HPV4 inducing some cases of EV seems more oncogenic.

[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.

[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.