Papillomavirus infections and human genital cancer

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ONCOLOGY

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Papillomavirus Infections and Human Genital Cancer HARALD ZUR HAUSEN, ETHEL-MICHELE Institut

DE VILLIERS, AND LUTZ GISSMANN

fir Virologie, Zentrum fir Hygiene, Universitiit Freiburg Hermann-Herder-Strasse II, 7800 Freiburg, West Germany Presented at the Conference on Early Cervical Neoplasia, March 23-25, 1981

Human papillomas are induced by at least eight distinct types of papillomaviruses. They are listed in Table 1. Condylomata acuminata, human genital warts, represent a group of fibropapillomatous benign tumors with exuberant exophytic growth which are induced by a papillomavirus (reviewed in [l]). They are mainly located at the foreskin of the penis, the glans, the vulva, the introitus vaginae, and intravaginally (see review [l]). They have also been reported on the cervix uteri, perianally, and even within the urethra [2-41. Epidemiological studies demonstrate a venereal mode of transmission [5]. They are prevalent in groups of high sexual promiscuity, such as homosexuals [3, 61 and prostitutes, and are by no means a rare disease. According to British statistics [7] they comprise 6.1% of all reported cases of venereally transmitted diseases (Table 2). Malignant conversion of condylomata acuminata has been reported repeatedly (reviewed in [l]). Anecdotal reports were published mainly from cases of long duration but exceptionally also from young individuals with a rather recent history of genital warts (reviewed in [l]). Malignant tumors developed within genital warts of the vulva, the penis, and also of the vagina. The role of the virus found electron microscopically in a few nuclei of the benign condylomata acuminata in the induction of such malignant tumors has not been established. Our own investigations on a possible role of human genital wart virus in genital cancer were initiated after a number of unsuccessful attempts to demonstrate herpes simplex type II DNA in biopsies of such tumors by nucleic acid hybridization [8]. Since epidemiological features of genital cancer point in a number of parameters (reviewed in [9]), such as dependence on the number of sexual partners, early onset of sexual relations, existence of marital clusters, and correlation in the incidence between cervical and penile cancer [9-151, to an infectious etiology, we started to analyze additional candidate viruses for their possible involvement in this disease. The presence of a virus belonging to a group of clearly oncogenic agents in genital warts and occasional reports on their malignant conversion stimulated the interest in these viruses. Initial experiments were designed to clarify the question of whether the papillomavirus found in genital warts is identical with papillomaviruses observed in other skin papillomas. This led to the identification of distinct types of papilS124 0090-8258/81/05!3124-05$01.00/O Copyright All rights

0 1981 by Academic Press, Inc. of reproduction in any form reserved.

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HUMAN PAPILLOMAS

Responsible virus type

Papilloma Verruca vulgaris common wart Venuca plana flat wart Epidermodysplasia verruciformis

Condyloma acuminatum genital wart Laryngeal papilloma Condyloma planum

HPV-1 HPV-2 HPV4 HPV-7 HPV3

(plantar wart virus) (hand wart virus) (virus of mosaic warts) (butcher wart virus) (juvenile flat wart virus)

HPV-3 (warts without reported malignant conversion) HPV-< (warts with malignant conversion) HPV-8 I HPV-6 (genital wart virus) Unidentified papillomavirus types

lomaviruses by our group as well as by Orth and his colleagues in Paris [ 16-221. Up to now, eight distinct types of human papillomaviruses have been identified. It is interesting to note that individual types are responsible for histologically typical papillomas (Table 1). Moreover, it is of considerable interest to state that the risk of malignant conversion is specifically correlated with specific types of papillomaviruses. In man, infection of patients with epidermodysplasia verruciformis with HPV types 5 and 8 represents a rather well-evaluated risk for the development of squamous cell carcinomas within those papillomas located in light-exposed regions of the skin [23]. Viral DNA has been demonstrated within malignant tissue and seems to persist in a nonintegrated form in multiple copies per tumor cell [20]. Observations on synergistic or potentiating interactions between a specific papillomavi.rus-type infection and environmental carcinogens are further emphasized by similar reports in animal systems, notably, in esophageal papillomatosis of cattle, where consumption of bracken fern containing a powerful carcinogen leads to frequent malignant conversion [24]. Synergistic effects between Shope papillomavirus infections of rabbits and chemical carcinogens were already recorded by Rous and his associates 40 to 45 years ago [25, 261. The purification of papillomavirus particles from human genital warts turned TABLE 2 CLINIC RETURNS M)R THE UNITED KINGDOM OF NEW CASESBY DIAGNOSIS’

Disease Gonorrhea Genital warts Herpes simplex Syphilis

Number of cases 63,080 (14.1) 27,133 ( 6.1) 8,957 ( 2.0) 4,802 ( 1.1)

’ Source: Sexually transmitted disease surveillance 1978 (grit. Med. J. 2, 1375-1376 (1979)).

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out to be virtually impossible in view of minute quantities of virus present in those papillomas. In the very few instances where larger quantities of virus have been recovered, they invariably turned out to represent HPV-1 infections, being most likely local variants of common warts (verrucae vulgares). Nucleic acid hybridizations performed with in vitro labeled DNA of characterized human papillomavirus types and total DNA derived from condylomata acuminata failed to reveal significant cross-hybridization, suggesting that a different virus should be responsible for the genital lesions [17, 27-291. Selective extraction of circular DNA from genital warts circumventing viral purification procedures proved to be more promising. From some condylomata sufficient DNA was obtained for contour length measurements [29] and gel analysis [28]. Our group [21] reported the first cleavage patterns of such DNA and demonstrated that this DNA annealed specifically with fractions of CsCl gradients used for purification of papillomavirus particles from genital warts at a buoyant density of about 1.34 g/ml-corresponding to the expected density of papillomavirus particles. According to these data the virus was tentatively designated as HPV-6. The availability of restriction endonuclease cleavage data indicating the existence of one EcoRI or one BumHI cleavage site in some HPV 6-DNA preparations, prompted experiments to clone such DNA in plasmid vectors. Cloning experiments were performed by using pBR 322 as vector and by cutting the molecules with EcoRI and BumHI. Clones were expected carrying a small HPV6-DNA sequence of about 1.6 x IO6 in molecular weight and additional ones harboring the other part of the viral DNA. In initial tests the small segment of viral DNA has been cloned successfully and only very recently clones containing the large segment were also obtained [30]. The viral DNA isolated from the clones has been mapped [30] and corresponds in its cleavage sites to viral DNA isolated directly from genital warts. In vitro labeled, cloned HPV-6 DNA was used to analyze 44 genital warts by Southern blots for cross-hybridizing sequences under stringent conditions of hybridization. Forty-one of these DNA preparations were positive, three, however, failed to anneal. These data indicate that the majority of typical genital warts harbor sequences homologous to HPV-6. HPV-6 DNA also hybridized with HPV-1, -2, -4, and -8 DNA under conditions of low stringency revealing some degree of relatedness to these viruses. Meisels and his colleagues [2, 311, Laverty el al. [32], and Della Torre et al. [33] described the presence of typical papillomavirus particles in cervical and vaginal lesions which they designated as condylomata plana, flat genital papillomas. Shah and his group [34] recently demonstrated antigens reacting with antibodies directed against papillomavirus group-specific determinants in about one-third of all mild dysplastic lesions of the cervix tested. This seems to underline the frequency of this papillomavirus infection. With the generous help of Dr. Meisels we have been able to test six of these typical flat condylomas, all provided by the Quebec group. They were annealed with HPV-6 DNA under stringent and relaxed conditions of hybridization. Three of these materials clearly hybridized with HPV-6 DNA under conditions of low stringency, one of them even under stringent conditions, indicating that this

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probe indeed contained HPV-6 DNA. The other two should contain DNA of a related but not identical papillomavirus. The three negative tumors are presently difficult to interpret. They may contain DNA of an unrelated virus or may be totally devoid of viral DNA. The availability of cloned HPV-6 DNA permitted also the analysis of biopsies from cervical, penile, and vulva1 cancer for the presence of this DNA. Although initial experiments performed at low sensitivity failed to demonstrate HPV-6 sequences in such materials, a recent experiment performed under conditions which still permit detection of one HPV-6 genome in 10 cells (Gissmann et al., in preparation) was more successful. Some tumors tested in this series hybridized in Southern blots with HPV-6 DNA. It should be noted that this hybridization test was performed under stringent conditions. These data are still preliminary and based on a single experiment only. However, they boost considerably our interest in a possible role of genital papillomaviruses in genital cancer. Although the presence of viral DNA within malignant tumors does not prove an etiological relationship, the available data justify the increasing interest in the possible role of papillomaviruses in human malignant tumors. Moreover, it is likely that more than one papillomavirus type infects the human genital tract and only preliminary data are available on the characterization of the viruses involved. The role of these agents in cancer induction also remains to be elucidated. ACKNOWLEDGMENT Original data cited in this paper were supported by the Deutsche Forschungsgemeinschaft (SFB 3 1: Medizinische Virologie-Tumorentstehung und -Entwicklung).

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