Eur Arch Otorhinolaryngol (1990) 247: 296-299
European Archives of
Oto-RhinoLaryngology © Spnnger-Verlag1990
Presence of human papillomavirus type-6-related sequences in inverted nasal papillomas T. Ishibashi 1' 2, y . Tsunokawa 1, S. Matsushima 1, Y. Nomura 2, T. Sugimura 1, and M. Terada 1 1Genetics Division, National Cancer Center Research Institute, Tokyo 104, Japan 2Department of Otolaryngology, The University of Tokyo, Tokyo 113, Japan Received September I8, 1989 / Accepted October 10, 1989
Summary. Twenty D N A samples obtained from seven cases of inverted papillomas, eight cases of nasal polyps and five cases of chronic sinusitis were investigated by Southern blot hybridization for the possible presence of sequences homologous to h u m a n papillomavirus ( H P V ) types 6, 11, 16 and 18. H P V type-6-related D N A was identified in one of the seven inverted papillomas. The restriction endonuclease cleavage patterns showed that this latter D N A is a new subtype of H P V type 6 D N A . In the other six papillomas and in all cases of nasal polyps and chronic sinusitis, no H P V sequence could be demonstrated, even under low stringent conditions (T~-40°C). These results indicate that H P V infection might be one of the possible causative factors in the pathogenesis of inverted papillomas but is not essential for the induction of the tumor. Key words: Inverted nasal papilloma - H u m a n papillomavirus - Southern blot hybridization - Viral D N A
Introduction
ical studies [20]. It has been recently reported that human papillomavirus ( H P V ) type 11 D N A was detected by DNA-hybridization techniques in three isolated cases of inverted papilloma [2, 16]. In the present study, we used the Southern blot hybridization technique to examine the presence of H P V D N A sequences in lesions of the nose and paranasal sinuses, including seven inverted papillomas. We report here, for the first time to our knowledge, the presence of H P V type-6-related D N A in one out of seven inverted papillomas.
Materials and methods Collection of specimens and preparation of DNA. The present series consisted of 20 patients biopsied or surgically treated for inverted papillomas (7 cases), nasal polyps (8 cases), and chronic sinusitis (5 cases). All patients were managed at the Department of Otolaryngology at the University of Tokyo. Table 1 summarizes the major clinical findings of the seven patients with inverted papillomas. Parts of the fresh specimens were used for pathological diagnosis and the remaining tissues were stored at -70°C until used for further study.
The inverted papilloma is a benign neoplasm that arises in the nose and paranasal sinuses. It is a relatively unc o m m o n lesion, and is characterized by its tendency toward recurrence following removal and by its association with malignancy [7, 8, 11, 17, 21]. Viruses have long been suspected to be a causative agent in inverted papillomas. Kusiak and H u d s o n [10] found 40-50 nm intranuclear bodies consistent with an ultrastructural characteristic of papillomavirus particles. Other investigators, however, have failed to find evidence for virus by using light microscopic studies [7], electron microscopic studies [4, 17] or immunohistochem-
Table 1. Clinical findings of the patients with the inverted nasal
Offprint requests to: T. Ishibashi, Genetics Division, National Cancer Center Research Institute, 5-1-1, Tsukiji, Chuo-ku, Tokyo, 104, Japan
papillomas tested for HPV DNA Patient Age Sex Duration Site of lesion no. of illness tested (months)
Koilo- HPV cytosis DNA
1. 2. 3. 4. 5. 6.
77 69 44 80 61 52
F M F F M M
3 82 87 5 24 36
+ + -
+a -
7.
46
M
101
-
-
Middle turbinate Middle turbinate Maxillary sinus Maxillary sinus Nasal septum Lateral wall of nasal cavity Maxillary sinus
a Positive for HPV type-6-related DNA
T. Ishibashi et al.: HPV in inverted papillomas
297
High-molecular-weight DNA was extracted by sodium dodecyl sulfate-proteinase K treatment, followed by repeated phenol and chloroform extractions [15].
Southern blot hybridizations. About 5-10 gg of tissue DNA was cleaved with the restriction enzyme BamHI, electrophoresed in 0.8% agarose gels and transferred to nitrocellulose filters according to the method of Southern [19]. For "low" stringent conditions [40°C below the melting temperature (Tin)], the filters were hybridized with a "cocktail" of 32p-labeled denatured HPV types 6b, 11, 16 and 18 DNA probes in a solution of 20% formamide, 5 x SSPE (sodium chloride, sodium phosphate, EDTA) (1 × SSPE: 180mM NaC1/10mM NaH2PO4/lmM EDTA, pH 7.4), 0.1% sodium dodecyl sulfate, 5 x Denhardt's solution [12], and 100 gg/ ml denatured salmon testis DNA at 32°C. For "high" stringent conditions (18°C below Tin), the concentration of formamide was increased to 50% and the temperature for hybridization was increased to 42°C. Details of the hybridization procedure have been described previously [22]. After hybridization for 2 days, the filters were washed in 2 × SSC (sodium chloride, sodium citrate) (1 x SSC: 150mM NaC1/15 mM sodium citrate, pH 7.0) at room temperature, and in 2 × SSC, 0.1% SDS, at 52°C, and exposed to Kodak XAR films at -70°C using an intensifying screen. The filters were then rewashed in 0.1 × SSC, 0.1% sodium dodecyl sulfate at 65°C, and exposed to the film again. The filters showing positive hybridization signals with the mixtures of probes were washed out by incubation in a solution of 50% formamide and 10 mM phosphate buffer at 65°C for i h to remove the probes. The filter was hybridized with each HPV DNA probe respectively and was also hybridized with a pBR 322 DNA probe to rule out any false-positive signals. As a positive control, 10 pg of HPV type 6b DNA was used. DNAs used as probes were total sequences of HPV DNA types 6b, 11, 16 and 18, cleaved from the plasmid vectors, purified by electrophoresis, and electroeluted [12]. Each viral DNA was labeled with 32p by nick-translation [12]. Histological examination. Formalin-fixed materials were embedded in paraffin wax and routinely processed into hematoxylineosin-stained sections for light microscopic examination at magnifications ranging from x 40 to x 200. Results H P V D N A was d e t e c t e d in one case of an i n v e r t e d p a p illoma using the m i x e d p r o b e s of H P V types 6b, 11, 16 and 18 D N A s u n d e r low stringent conditions (Tin - 4 0 ° C ) . In the o t h e r six cases of inverted papillomas, and in all cases of nasal polyps and chronic sinusitis, no H P V D N A s e q u e n c e hybridizable to the D N A p r o b e s could be d e m onstrated, even u n d e r low stringent conditions (Tin - 4 0 ° C ) . T h e type of H P V D N A in the H P V D N A - p o s i tive sample of the inverted p a p i l l o m a (patient 1) was analyzed using individual D N A p r o b e s of H P V type 6b, H P V type 11, H P V type 16 and H P V type 18 u n d e r high stringent conditions (Tin - 1 8 ° C ) . This sample showed strong hybridization signals with H P V type 6b D N A (Fig. 1) and also showed w e a k hybridization signals with H P V type 11 D N A (data not shown), indicating crosshybridization with H P V type 6b D N A . This sample failed to hybridize with either H P V type 16 D N A or H P V type 18 D N A at low stringent conditions. T h e H P V type-6-positive D N A showed a m a i n b a n d of 7.9 kb and two additional bands after B a m H I cleavage (Fig. 1, lane a). T o characterize the nature of this sample further, we cleaved the sample D N A with two restriction
Fig. 1. Southern blot hybridization of the sample from the inverted papilloma of patient 1. Cleavage has been obtained with BamHI (lane a), HindIII (lane c), EcoRI (lane d) and 10pg of HPV type 6b DNA (lane b) with HPV type 6b DNA probe under high stringent conditions
e n z y m e s , HindIII and E c o R I . T h e resulting digestion patterns showed a main b a n d of 7 . 9 k b after H i n d I I I cleavage (Fig. 1, lane c), and two m a i n bands of 5 . 7 k b and 2.2 kb after E c o R I (Fig. 1, lane d). T h e additional bands after B a m H I digestion r e v e a l e d a shift after digestion with H i n d I I I and E c o R I (Fig. 1). T h e hybridization signals of these additional bands (Fig. 1, lanes c and d) were of almost the s a m e intensity as that of the 10 pg m a r k e r of H P V type 6 b D N A (Fig. 1, lane b), corresponding to one copy per cell. T h e seven cases of inverted p a p i l l o m a s were evaluated histopathologically for the p r e s e n c e of koilocytosis, which is characteristic for H P V infection. Koilocytosis was identified in two of these specimens (Fig. 2).
Discussion It is well k n o w n that h u m a n papillomaviruses can induce epithelial proliferation in various parts of the body. In this regard, H P V type 6 and H P V type 11 have b e e n found to have an etiological role in laryngeal p a p i l l o m a s [6, 13, 14]. T h e histological r e s e m b l a n c e s of inverted p a p i l l o m a s to laryngeal p a p i l l o m a s led us to suspect that
298
T. Ishibashi et al.: HPV in inverted papillomas
Fig. 2. Histopathological findings of HPV type 6 DNA positive in the inverted papilloma of patient 1. The koilocytic cells are shown by the arrows. Hematoxylin and eosin, x 200
inverted papillomas may result from a papillomavirus infection. Recently, H P V type 11 D N A has been found in three isolated cases of inverted papillomas when studied with Southern blot hybridization [2, 6]. The presence of H P V type 11 D N A in an inverted papilloma was also confirmed by an in situ DNA-hybridization technique [23]. However, there has been no previous report on the presence of H P V type 6 D N A in inverted papillomas. To our knowledge, our report is the first to describe the presence of H P V type-6-related D N A in an inverted papilloma, using Southern blot hybridization technique. H P V type 6 is known to have several subtypes. De Villiers et al. [3] cloned H P V type 6 D N A from human genital warts, and reported the existence of at least two D N A subtypes (HPV 6a and 6b). Mounts and Kashima [13] reported four subtypes of H P V 6 in laryngeal papillomas by their restriction endonuclease digestion pattern. These previously reported subtypes are not cleaved or cut once by the restriction enzyme EcoRI. However, H P V type-6-positive D N A in our present study showed two main bands of 5.7 kb and 2.2 kb after E c o R I cleavage, indicating that E c o R I cut twice the episomal H P V D N A of 7.9 kb in size. This D N A is probably a new subtype of H P V 6 DNA. Unfortunately, further characterization of the D N A was precluded by the lack of sufficient materials for further analysis. Some migration of additional bands was detectable by cutting the sample D N A with B a m H I or E c o R I or HindIII (Fig. 1). It is possible that some H P V D N A was integrated into the cellular genome while a majority of the H P V D N A remained in an episomal state having a 7.9 kb size. Additional fragments might represent junctional fragments between the tumor cell D N A and the viral DNA. There have been no reports indicating integration of H P V type 6 D N A into a cellular genome. Malignant tumors regularly contain H P V D N A in an in-
tegrated state, in contrast to viral D N A in an episomal state in benign lesions in the female genital tract [1, 18]. It is possible that the presence of some viral D N A in an integrated state in our inverted papilloma specimen might indicate the premalignant nature of this neoplasm. Of the seven inverted papillomas studied, only one demonstrated H P V type-6-related DNA. In contrast, H P V types 6 or 11 DNAs were detected in almost all of the laryngeal papillomas investigated [13, 14]. The other six papillomas in our study did not hybridize with H P V types 6b, 11, 16 and 18 D N A under low stringent conditions (Tin -40°C). Almost all types of H P V are detectable by cross-hybridization at Tm - 4 0 ° C [9]. Moreover, our specimens did not contain adjacent normal tissue or a large proportion of connective tissue. Instead, we selected only the tumorous areas for study. The possibility is low that other types of H P V are present in the six papillomas that do not hybridize with H P V types 6b, 11, 16 and 18 DNAs at Tm -40°C. Weber et al. [23] reported a high incidence of the HPV type 11 sequence in inverted nasal papillomas using the in situ hybridization technique, suggesting an etiological role of H P V in this tumor. The technique of Southern blot hybridization used in our present study is a more sensitive and reliable method for detecting H P V sequences than is the in situ hybridization technique. The results of our study suggest that H P V infection is not essential for the induction and maintenance of inverted papillomas, but H P V infection may be one of the causative factors in the tumor's pathogenesis. Acknowledgements. We thank Dr. Harald zur Hausen for his kind
gift of cloned HPV type 6, 11, 16 and 18 DNA. We also thank the following surgeons for obtaining clinical specimens for this study: Drs. Takashi Futaki, Kenbun Inoue, Mineko Chikuni and Masao Asai. This work was supported in part by a Grant-in-Aid for a comprehensive 10-Year Strategy for Cancer Control from the Ministry of Health and Welfare of Japan.
T. Ishibashi et al. : H P V in inverted papillomas References
1. Boshart M, Gissmann L, Ikenberg H, Kleinkeinz A, Scheurlen W, zur Hausen H (1984) A new type of papillomavirus DNA, its presence in genital cancer biopsies and in cell lines derived from cervical cancer. Eur Mol Biol Org J 3 : 1151-1157 2. Brandsma JL, Steinberg BM, Abramson AL, Winkler B (1986) Presence of human papillomavirus type 16 related sequences in verrucous carcinoma of the larynx. Cancer Res 46: 2185-2188 3. De Villiers EM, Gissmann L, zur Hausen H (1981) Molecular cloning of viral DNA from human genital warts. J Virol 40: 932-935 4. Gaito RA, Gaylord WH, Hilding DA (1965) Ultrastructure of a human nasal papilloma. Laryngoscope 75 : 144-152 5. Gissmann L, Diehl V, Schults-Coulon H, zur Hausen H (1982) Molecular cloning and characterization of human papillomavirus DNA derived from a laryngeal papilloma. J Virol 44: 393-400 6. Gissmann L, Walnik L, Ikenberg H, Koldovsky U, Schnurch HG, zur Hausen H (1983) Human papillomavirus type 6 and 11 DNA sequences in genital and laryngeal papillomas and in some cervical cancers. Proc Natl Acad Sci USA 80 : 560-563 7. Hyams VJ (1971) Papillomas of the nasal cavity and paranasal sinuses. A clinicopathological study of 315 cases. Ann Otol Rhinol Laryngol 80 : 192-206 8. Ishibashi T, Nomura Y, Inoue K (1987) Malignancy in nasal and paranasal 'transitional' papilloma. J Otolaryngol Jpn 90: 1927-1932 9. Kahn T. Schwarz E, zur Hausen H (1986) Molecular cloning and characterization of the DNA of a new human papillomavirus (HPV30) from a laryngeal carcinoma. Int J Cancer 37: 61-65 10. Kusiak RJ, Hudson WR (1970) Nasal papillomatosis. South Med J 63 : 1277-1280 11. Majumdar B, Beck S (1984) Inverted papilloma of the nose. Some aspects of etiology. J Laryngol Otol 98 : 467-470 12. Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
299 13. Mounts P, Kashima H (1984) Association of human papillomavirus subtype and clinical course in respiratory papillomatosis. Laryngoscope 94: 28-33 14. Mounts P, Shah KV, Kashima H (1982) Viral etiology of juvenile- and adult-onset squamous papilloma of the larynx. Proc Natl Acad Sci USA 79 : 5425-5429 15. Perucho M, Goldfarb M, Shimizu K, Lama C, Fogh J, Wigler M (1981) Human-tumor-derived cell lines contain common and different transforming genes. Cell 27: 467-476 16. Respler DS, Jahn A, Pater A, Pater MM (1987) Isolation and characterization of papillomavirus DNA from nasal inverting (schneiderian) papillomas. Ann Otol Rhinol Laryngo196:170173 17. Ridolfi RL, Lieberman PH, Erlandson RA, Moore OS (1977) Schneiderian papillomas: a clinico-pathologic study of 30 cases. Am J Surg Pathol 1 : 43-53 18. Schwarz E, Freese UK, Gissmann L, Mayer W, Roggenbuck B, Stremlau A, zur Hausen H (1985) Structure and transcription of human papillomavirus sequences in cervical carcinoma cells. Nature 314:111-114 19. Southern EM (1975) Detection of specific sequences among DNA fragments separated by gel elctrophoresis. J Mol Biol 98 : 503-517 20. Strauss M, Jenson AB (1985) Human papillomavirus in various lesions of the head and neck. Otolaryngol Head Neck Surg 93 : 342-346 21. Suh KW, Facer GW, Devine KD, Weiland LH, Zujko RD (1977) Inverting papilloma of the nose and paranasal sinuses. Laryngoscope 87 : 35-46 22. Wahl GM, Stern M, Stark GM (1979) Efficient transfer of large DNA fragments from agarose gels to diazobenzyloxymethyl-paper and rapid hybridization by using dextran sulfate. Proc Natl Acad Sci USA 76 : 3683-3687 23. Weber RS, Shillitoe E J, Robbins KT, Luna MA, Batsakis JG, Donovan DT, Adler-Storthz K (1988) Prevalence of human papillomavirus in inverted nasal papillomas. Arch Otolaryngol Head Neck Surg 114 : 23-26
European Archives of
Oto-RhinoLaryngology © Spnnger-Verlag1990
Presence of human papillomavirus type-6-related sequences in inverted nasal papillomas T. Ishibashi 1' 2, y . Tsunokawa 1, S. Matsushima 1, Y. Nomura 2, T. Sugimura 1, and M. Terada 1 1Genetics Division, National Cancer Center Research Institute, Tokyo 104, Japan 2Department of Otolaryngology, The University of Tokyo, Tokyo 113, Japan Received September I8, 1989 / Accepted October 10, 1989
Summary. Twenty D N A samples obtained from seven cases of inverted papillomas, eight cases of nasal polyps and five cases of chronic sinusitis were investigated by Southern blot hybridization for the possible presence of sequences homologous to h u m a n papillomavirus ( H P V ) types 6, 11, 16 and 18. H P V type-6-related D N A was identified in one of the seven inverted papillomas. The restriction endonuclease cleavage patterns showed that this latter D N A is a new subtype of H P V type 6 D N A . In the other six papillomas and in all cases of nasal polyps and chronic sinusitis, no H P V sequence could be demonstrated, even under low stringent conditions (T~-40°C). These results indicate that H P V infection might be one of the possible causative factors in the pathogenesis of inverted papillomas but is not essential for the induction of the tumor. Key words: Inverted nasal papilloma - H u m a n papillomavirus - Southern blot hybridization - Viral D N A
Introduction
ical studies [20]. It has been recently reported that human papillomavirus ( H P V ) type 11 D N A was detected by DNA-hybridization techniques in three isolated cases of inverted papilloma [2, 16]. In the present study, we used the Southern blot hybridization technique to examine the presence of H P V D N A sequences in lesions of the nose and paranasal sinuses, including seven inverted papillomas. We report here, for the first time to our knowledge, the presence of H P V type-6-related D N A in one out of seven inverted papillomas.
Materials and methods Collection of specimens and preparation of DNA. The present series consisted of 20 patients biopsied or surgically treated for inverted papillomas (7 cases), nasal polyps (8 cases), and chronic sinusitis (5 cases). All patients were managed at the Department of Otolaryngology at the University of Tokyo. Table 1 summarizes the major clinical findings of the seven patients with inverted papillomas. Parts of the fresh specimens were used for pathological diagnosis and the remaining tissues were stored at -70°C until used for further study.
The inverted papilloma is a benign neoplasm that arises in the nose and paranasal sinuses. It is a relatively unc o m m o n lesion, and is characterized by its tendency toward recurrence following removal and by its association with malignancy [7, 8, 11, 17, 21]. Viruses have long been suspected to be a causative agent in inverted papillomas. Kusiak and H u d s o n [10] found 40-50 nm intranuclear bodies consistent with an ultrastructural characteristic of papillomavirus particles. Other investigators, however, have failed to find evidence for virus by using light microscopic studies [7], electron microscopic studies [4, 17] or immunohistochem-
Table 1. Clinical findings of the patients with the inverted nasal
Offprint requests to: T. Ishibashi, Genetics Division, National Cancer Center Research Institute, 5-1-1, Tsukiji, Chuo-ku, Tokyo, 104, Japan
papillomas tested for HPV DNA Patient Age Sex Duration Site of lesion no. of illness tested (months)
Koilo- HPV cytosis DNA
1. 2. 3. 4. 5. 6.
77 69 44 80 61 52
F M F F M M
3 82 87 5 24 36
+ + -
+a -
7.
46
M
101
-
-
Middle turbinate Middle turbinate Maxillary sinus Maxillary sinus Nasal septum Lateral wall of nasal cavity Maxillary sinus
a Positive for HPV type-6-related DNA
T. Ishibashi et al.: HPV in inverted papillomas
297
High-molecular-weight DNA was extracted by sodium dodecyl sulfate-proteinase K treatment, followed by repeated phenol and chloroform extractions [15].
Southern blot hybridizations. About 5-10 gg of tissue DNA was cleaved with the restriction enzyme BamHI, electrophoresed in 0.8% agarose gels and transferred to nitrocellulose filters according to the method of Southern [19]. For "low" stringent conditions [40°C below the melting temperature (Tin)], the filters were hybridized with a "cocktail" of 32p-labeled denatured HPV types 6b, 11, 16 and 18 DNA probes in a solution of 20% formamide, 5 x SSPE (sodium chloride, sodium phosphate, EDTA) (1 × SSPE: 180mM NaC1/10mM NaH2PO4/lmM EDTA, pH 7.4), 0.1% sodium dodecyl sulfate, 5 x Denhardt's solution [12], and 100 gg/ ml denatured salmon testis DNA at 32°C. For "high" stringent conditions (18°C below Tin), the concentration of formamide was increased to 50% and the temperature for hybridization was increased to 42°C. Details of the hybridization procedure have been described previously [22]. After hybridization for 2 days, the filters were washed in 2 × SSC (sodium chloride, sodium citrate) (1 x SSC: 150mM NaC1/15 mM sodium citrate, pH 7.0) at room temperature, and in 2 × SSC, 0.1% SDS, at 52°C, and exposed to Kodak XAR films at -70°C using an intensifying screen. The filters were then rewashed in 0.1 × SSC, 0.1% sodium dodecyl sulfate at 65°C, and exposed to the film again. The filters showing positive hybridization signals with the mixtures of probes were washed out by incubation in a solution of 50% formamide and 10 mM phosphate buffer at 65°C for i h to remove the probes. The filter was hybridized with each HPV DNA probe respectively and was also hybridized with a pBR 322 DNA probe to rule out any false-positive signals. As a positive control, 10 pg of HPV type 6b DNA was used. DNAs used as probes were total sequences of HPV DNA types 6b, 11, 16 and 18, cleaved from the plasmid vectors, purified by electrophoresis, and electroeluted [12]. Each viral DNA was labeled with 32p by nick-translation [12]. Histological examination. Formalin-fixed materials were embedded in paraffin wax and routinely processed into hematoxylineosin-stained sections for light microscopic examination at magnifications ranging from x 40 to x 200. Results H P V D N A was d e t e c t e d in one case of an i n v e r t e d p a p illoma using the m i x e d p r o b e s of H P V types 6b, 11, 16 and 18 D N A s u n d e r low stringent conditions (Tin - 4 0 ° C ) . In the o t h e r six cases of inverted papillomas, and in all cases of nasal polyps and chronic sinusitis, no H P V D N A s e q u e n c e hybridizable to the D N A p r o b e s could be d e m onstrated, even u n d e r low stringent conditions (Tin - 4 0 ° C ) . T h e type of H P V D N A in the H P V D N A - p o s i tive sample of the inverted p a p i l l o m a (patient 1) was analyzed using individual D N A p r o b e s of H P V type 6b, H P V type 11, H P V type 16 and H P V type 18 u n d e r high stringent conditions (Tin - 1 8 ° C ) . This sample showed strong hybridization signals with H P V type 6b D N A (Fig. 1) and also showed w e a k hybridization signals with H P V type 11 D N A (data not shown), indicating crosshybridization with H P V type 6b D N A . This sample failed to hybridize with either H P V type 16 D N A or H P V type 18 D N A at low stringent conditions. T h e H P V type-6-positive D N A showed a m a i n b a n d of 7.9 kb and two additional bands after B a m H I cleavage (Fig. 1, lane a). T o characterize the nature of this sample further, we cleaved the sample D N A with two restriction
Fig. 1. Southern blot hybridization of the sample from the inverted papilloma of patient 1. Cleavage has been obtained with BamHI (lane a), HindIII (lane c), EcoRI (lane d) and 10pg of HPV type 6b DNA (lane b) with HPV type 6b DNA probe under high stringent conditions
e n z y m e s , HindIII and E c o R I . T h e resulting digestion patterns showed a main b a n d of 7 . 9 k b after H i n d I I I cleavage (Fig. 1, lane c), and two m a i n bands of 5 . 7 k b and 2.2 kb after E c o R I (Fig. 1, lane d). T h e additional bands after B a m H I digestion r e v e a l e d a shift after digestion with H i n d I I I and E c o R I (Fig. 1). T h e hybridization signals of these additional bands (Fig. 1, lanes c and d) were of almost the s a m e intensity as that of the 10 pg m a r k e r of H P V type 6 b D N A (Fig. 1, lane b), corresponding to one copy per cell. T h e seven cases of inverted p a p i l l o m a s were evaluated histopathologically for the p r e s e n c e of koilocytosis, which is characteristic for H P V infection. Koilocytosis was identified in two of these specimens (Fig. 2).
Discussion It is well k n o w n that h u m a n papillomaviruses can induce epithelial proliferation in various parts of the body. In this regard, H P V type 6 and H P V type 11 have b e e n found to have an etiological role in laryngeal p a p i l l o m a s [6, 13, 14]. T h e histological r e s e m b l a n c e s of inverted p a p i l l o m a s to laryngeal p a p i l l o m a s led us to suspect that
298
T. Ishibashi et al.: HPV in inverted papillomas
Fig. 2. Histopathological findings of HPV type 6 DNA positive in the inverted papilloma of patient 1. The koilocytic cells are shown by the arrows. Hematoxylin and eosin, x 200
inverted papillomas may result from a papillomavirus infection. Recently, H P V type 11 D N A has been found in three isolated cases of inverted papillomas when studied with Southern blot hybridization [2, 6]. The presence of H P V type 11 D N A in an inverted papilloma was also confirmed by an in situ DNA-hybridization technique [23]. However, there has been no previous report on the presence of H P V type 6 D N A in inverted papillomas. To our knowledge, our report is the first to describe the presence of H P V type-6-related D N A in an inverted papilloma, using Southern blot hybridization technique. H P V type 6 is known to have several subtypes. De Villiers et al. [3] cloned H P V type 6 D N A from human genital warts, and reported the existence of at least two D N A subtypes (HPV 6a and 6b). Mounts and Kashima [13] reported four subtypes of H P V 6 in laryngeal papillomas by their restriction endonuclease digestion pattern. These previously reported subtypes are not cleaved or cut once by the restriction enzyme EcoRI. However, H P V type-6-positive D N A in our present study showed two main bands of 5.7 kb and 2.2 kb after E c o R I cleavage, indicating that E c o R I cut twice the episomal H P V D N A of 7.9 kb in size. This D N A is probably a new subtype of H P V 6 DNA. Unfortunately, further characterization of the D N A was precluded by the lack of sufficient materials for further analysis. Some migration of additional bands was detectable by cutting the sample D N A with B a m H I or E c o R I or HindIII (Fig. 1). It is possible that some H P V D N A was integrated into the cellular genome while a majority of the H P V D N A remained in an episomal state having a 7.9 kb size. Additional fragments might represent junctional fragments between the tumor cell D N A and the viral DNA. There have been no reports indicating integration of H P V type 6 D N A into a cellular genome. Malignant tumors regularly contain H P V D N A in an in-
tegrated state, in contrast to viral D N A in an episomal state in benign lesions in the female genital tract [1, 18]. It is possible that the presence of some viral D N A in an integrated state in our inverted papilloma specimen might indicate the premalignant nature of this neoplasm. Of the seven inverted papillomas studied, only one demonstrated H P V type-6-related DNA. In contrast, H P V types 6 or 11 DNAs were detected in almost all of the laryngeal papillomas investigated [13, 14]. The other six papillomas in our study did not hybridize with H P V types 6b, 11, 16 and 18 D N A under low stringent conditions (Tin -40°C). Almost all types of H P V are detectable by cross-hybridization at Tm - 4 0 ° C [9]. Moreover, our specimens did not contain adjacent normal tissue or a large proportion of connective tissue. Instead, we selected only the tumorous areas for study. The possibility is low that other types of H P V are present in the six papillomas that do not hybridize with H P V types 6b, 11, 16 and 18 DNAs at Tm -40°C. Weber et al. [23] reported a high incidence of the HPV type 11 sequence in inverted nasal papillomas using the in situ hybridization technique, suggesting an etiological role of H P V in this tumor. The technique of Southern blot hybridization used in our present study is a more sensitive and reliable method for detecting H P V sequences than is the in situ hybridization technique. The results of our study suggest that H P V infection is not essential for the induction and maintenance of inverted papillomas, but H P V infection may be one of the causative factors in the tumor's pathogenesis. Acknowledgements. We thank Dr. Harald zur Hausen for his kind
gift of cloned HPV type 6, 11, 16 and 18 DNA. We also thank the following surgeons for obtaining clinical specimens for this study: Drs. Takashi Futaki, Kenbun Inoue, Mineko Chikuni and Masao Asai. This work was supported in part by a Grant-in-Aid for a comprehensive 10-Year Strategy for Cancer Control from the Ministry of Health and Welfare of Japan.
T. Ishibashi et al. : H P V in inverted papillomas References
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