Human Papillomavirus Type 18 in Conjunctival Intraepithelial Neoplasia Simeon A. Lauer, M.D., James S. Malter, M.D., and J. Ralph Meier, M.D. Human papillomaviruses are oncogenic viruses that have been found in a variety of epithelial neoplasias. We sought to confirm their presence in conjunctival intraepithelial neoplasia. Five tumors were studied with a polymerase chain-reaction assay designed to detect the E6 region of human papillomavirus types 16 and 18. Human papillomavirus type16 DNA was found in four of the five tumors, including two tumors that contained both type-16 and type-18 DNA. Viral DNA was not present in the fifth tumor.
T HE CLINICAL and histopathologic features of nonpigmented conjunctival and corneal epithelial neoplasias were classified by Pizzarello and [akobiec in 1978. 1 They adopted the nomenclature of anogenital epithelial neoplasias, which have similar histopathologic characteristics. In a conjunctival or corneal intraepithelial neoplasm, dysplastic cells are confined to the epithelium. When these cells have invaded the epithelial basement membrane, the tumor is considered to be a squamous cell carcinoma. Human papillomaviruses are oncogenic viruses that are present in up to 90% of anogenital epithelial neoplasms.! Their presence has recently been demonstrated in conjunctival epithelial neoplasms" and in a squamous cell carcinoma of the eyelid.' Papillomaviruses induce tumor formation in animals" and malignant transformation of cell cultures." The mechaAccepted for publication April 13, 1990. From the LSU Eye Center (Dr. Lauer) and the Department of Pathology (Dr. Meier), LSU Medical Center School of Medicine, and the Department of Pathology (Dr. Malter), Tulane Medical Center, New Orleans, Louisiana. This study was supported in part by U.S. Public Health Service grants EY08482 (Dr. Lauer) from the National Eye Institute and CA01427 (Dr. Malter) from the National Cancer Institute, National Institutes of Health, Bethesda, Maryland, and a grant from the ARVOI Alcon Fellowship Program (Dr. Lauer). Reprint requests to Simeon A. Lauer, M.D., LSU Eye Center, 2020 Gravier St., New Orleans, LA 70112.
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nism of oncogenesis is not completely understood.' Human papillomaviruses are divided into types based on homologous characteristics of the DNA sequences." In the anogenital region, types 169 and 18 10 have been found predominantly in carcinomas." Thus far, human papillomavirus type 16 is the only type demonstrated in conjunctival epithelial neoplasms." In this study, we confirmed the presence of human papillomavirus DNA in conjunctival intraepithelial neoplasia. Human papillomavirus type 18 was present in two of these tumors.
Material and Methods Specimens for study were collected from formalin-fixed, paraffin-embedded tissue. The pathologic characteristics were reviewed and the histopathologic diagnosis confirmed for each block by one of us (l.R.M.). Five sections, 10 J.Lm thick, were removed with a clean microtome blade and placed in a microcentrifuge tube under sterile conditions. Paraffin was removed with two xylene extractions, and xylene was removed with 95% ethanol. The tissue was pelleted, residual ethanol was removed, and the specimen was vacuum dried for five minutes before resuspension in 100 J.Ll of water. The specimen was boiled for 20 minutes and the DNA content determined by spectrophotometry. Approximately 2.0 J.Lg of total nucleic acid was used for the polymerase chain-reaction assay. The polymerase chain-reaction assay was performed in a total volume of 50 J.Ll. Buffer consisted of 17.0 mM of ammonium sulfate, 67.0 mM of Tris-HCl pH 8.5, 10.0 mM of 2mercaptoethanol, and 170 J.Lg/ml of bovine serum albumin. Magnesium chloride 7.0 mM, a biologic detergent composed of various polyoxyethylene ethers and other surface-active compounds (Triton-X 100, Sigma Chemical Co., St. Louis, Missouri) 0.1 %, deoxynucleotides (1.5 mM each), and three units of Taq DNA 1990
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polymerase (Promega, Madison, Wisconsin) were added to each reaction mixture. Each specimen was run with type-I6 and type-I8 oligonucleotide (20 base) primers synthesized according to the sequences reported by Shibata and associates." Aliquots (1 ""g) of DNA extracted from SiHa and HeLa cell culture lines were used as positive controls for human papillomavirus types 16 and 18, respectively. Solutions and primers without DNA served as negative controls. Amplification consisted of denaturation for one minute at 93 C, annealing for one minute at 47 C, and extension for 30 seconds at 72 C. The amplification process was continued for 35 cycles. Two separate 10-",,1 aliquots from each of the amplification products were loaded on an ethidium bromide-stained 2% agarose gel, separated by electrophoresis for 45 minutes at 100 V, and transferred to a nylon membrane. Each amplification product was hybridized independently with human papillomavirus type 16 or 18 oligonucleotide (40 base) probes as described by Shibata and assoctates." Prehybridization was performed for three hours at 42 C in 50% formamide, five times the standard concentration of sodium chloride, sodium phosphate, ethylene-diaminetetraacetic acid (EDTA) buffer, 0.1 % Denhardt's solution, 10 mgjml of salmon-sperm DNA, and 1% glycine. Hybridization was performed for 24 hours at 42 C in 50% formamide, five times the standard concentration of sodium chloride, sodium phosphate, EDTA buffer, 10 mgjml of salmon-sperm DNA, and 1% sodium dodecyl sulfate. Membranes were washed with two times the standard concentration of sodium chloride, sodium citrate buffer sequentially at room temperature, 37 C, and 42 C until background radioactivity was undetectable. Autoradiography with Kodak X-AR film and two intensifying screens was performed for three to 12 hours at -70 C.
Results We studied five conjunctival intraepithelial neoplasms (Table). Representative autoradiograms for three of the tumors showed the presence of type-I6 (Fig. 1) and type-I8 (Fig. 2) DNA. In Figure 1, the amplified products were hybridized with the human papillomavirus type-I6 probe. In Figure 2, the type-I8 probe was used for hybridization. Type-I6 primers
TABLE VIRAL DNA IN CONJUNCTIVAL INTRAEPITHELIAL NEOPLASMS HUMAN PAPILLOMAVIRUS DNA SPECIMEN NO.
TYPE 16
TYPE 18
1
Absent Present Present Present Present
Absent Present Absent Absent Present
2 3 4
5
were used for amplification in odd-numbered lanes, and type-I8 primers were used in evennumbered lanes. DNA extracted from the SiHa and HeLa cervical cell lines, which are known to contain human papillomavirus DNA, were used as positive controls. The SiHa DNA, which contains type 16, was used in lane 1. In lane 2, He La DNA, which contains type 18, was used. These positive controls yielded a strong single band of the expected size (109 base pairs) as visualized after agarose gel electrophoresis. These amplified fragments hybridized strongly with appropriate probes. A band was not always visible in the agarose gel for the specimens but the autoradiographic bands corresponded precisely with those of the positive control. No DNA was added to the negative controls in lanes 3 and 4. Autoradiography continued to yield negative results even after prolonged exposure. Amplified DNA from the first specimen was
•
1 2 3 4 5 6 7 8
•
9 10
Fig. 1 (Lauer, Malter, and Meier). Southern blot hybridization of polymerase chain reaction product with human papillomavirus type-I6 32P-radiolabeled oligonucleotide probe. Type-I6 primers were used in odd-numbered lanes, and type-I8 primers were used in even-numbered lanes. The primers were incubated with the following: SiHa DNA (lane 1), HeLa DNA (lane 2), without DNA (lanes 3 and 4), specimen 1 (lanes 5 and 6), specimen 2 (lanes 7 and 8), and specimen 3 (lanes 9 and 10). Lanes 1 (type 16, control), 7, and 9 contain type-16 DNA.
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Fig. 2 (Lauer, Malter, and Meier). Southern blot as in Figure 1 hybridized with human papillomavirus type-I8 probe. Lanes 2 (type 18, control) and 8 contain type-I8 DNA.
subjected to electrophoresis in lanes 5 and 6. No band was visualized with either papillomavirus probe even after prolonged exposure. The amplification product from the second specimen hybridized strongly with the type-18 probe (Fig. 2, lane 8), which showed the presence of human papillomavirus type-18 DNA. A weaker band was also seen with the probe specific for type-16 DNA (Fig. 1, lane 7). The amplification product from the third specimen hybridized strongly with the type-16 probe (Fig. 1, lane 9) but not with the probe for type 18 (Fig. 2, lane 10). The fourth specimen was strongly positive for human papillomavirus type 16 and did not hybridize with the type-18 probe. The fifth specimen hybridized strongly with both probes.
Discussion
Human papillomaviruses are epitheliotropic oncogenic viruses." They have been found in a variety of epithelial neoplasias, including more than 90% of uterine cervical carcinomas.! Human papillomavirus DNA induces malignant transformation of cultured cells in vitro." The ability to transform cultured cells has been traced to the E6 and E7 early open-reading frames of the human papillomavirus genome." The E6-encoded transforming proteins confer tumorigenicity. The E7-encoded proteins confer malignant growth properties. There is evidence that E6- and E7-transforming proteins interact with nuclear proteins." Alternatively, the E6-encoded protein may be a hormonereceptor protein," which is consistent with the hormone dependence of some human papillomavirus-transformed cells." Human papillomaviruses are divided into types based on homologous characteristics of
25
their DNA sequences." Low-risk types such as 6 and 11 are found in premalignant and benign tumors.! These types have been identified in conjunctival papillomas.":" High-risk types such as 16 and 18 are more common in carcinomas." The E6 region of types 16 and 18 differs from that of other types." The potential for malignant transformation may be linked to the E6* intron, which is unique to these types. Shibata and associates" have developed a polymerase chain-reaction assay that is specifically designed to identify the E6 region of human papillomavirus types 16 and 18. In a polymerase chain-reaction assay (Fig. 3), target DNA is amplified by the enzyme DNA polymerase.f This process starts with the denaturation of double-stranded DNA into two single strands. Two oligonucleotide primers hybridize to single-stranded complementary regions of the target DNA. DNA polymerase then extends the primer by using the double-stranded region of the target DNA as a template. The newly synthesized double-stranded DNA is denatured and the cycle repeated. With each cycle, the amount of target DNA is doubled. After 20 to 40 cycles, a small quantity of target DNA can be replicated a billion times." As little as 100 attograms of target DNA can be detected (Pietro Lampertico, M.D., Tulane University College of Medicine, New Orleans, Louisiana, unpublished data) when the assay is coupled with Southern blotting" as in this study. The target DNA is identified by hybridization with a probe. The probe is a radiolabeled oligonucleotide that is complementary to a unique internal region of the amplified target DNA. The presence of bound probes is demonstrated by autoradiography (Figs. 1 and 2). We used a polymerase chain reaction to demonstrate the presence of human papillomavirus DNA in four of five conjunctival intraepithelial neoplasms. McDonnell, Mayr, and Martin" reported finding human papillomavirus type-16 DNA in nine of 13 (69%) conjunctival intraepithelial neoplasms and in three conjunctival squamous cell carcinomas. The two studies combined found human papillomavirus DNA in 13 of 18 (72%) conjunctival intraepithelial neoplasms. In our study, human papillomavirus type 18 was found in two tumors. Type 16 was found in four tumors including two tumors that contained both types. The significance of finding both types in the same tumor is not known." We provide evidence for the presence of hu-
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Denaturation 93C
Whether human papillomavirus DNA plays an active role in the development of conjunctival intraepithelial tumors cannot be stated until the mechanism of papillomavirus oncogenesis is better understood.
References
Annealing 47C
Extension 72C
Fig. 3 (Lauer, Malter, and Meier). Schematic diagram of the polymerase chain-reaction sequence. Top, Step 1. Double-stranded target DNA is denatured to single-stranded DNA at 93 C for one minute. Middle, Step 2. Oligonucleotide primers complementary to flanking regions of the target DNA are allowed to anneal at 47 C for one minute. Bottom, Step 3. Primers are extended by the enzyme DNA polymerase by using the single-stranded target DNA as a template.
man papillomavirus type-18 DNA. The band produced by the tumor we studied was identical to that produced by DNA extracted from the type 18-containing HeLa cell line. The assay produced no amplification product when no DNA was added, which confirms that the target DNA was not a contaminant in the reagents. The first tumor did not produce an amplification product, which confirms that the contaminating DNA was not added during processing of the specimens. The third specimen was exclusively positive for human papillomavirus type 16, confirming that cross-hybridization did not occur. The fifth tumor studied had equally strong bands with both type-16 and -18 probes. This specimen, therefore, contained both types. The weak band that developed (Fig. 1, lane 7) probably represents low-level type-16 infection.
1. Pizzarello, L. D., and [akobiec, F. A.: Bowen's disease of the conjunctiva. A misnomer. In [akobiec, F. A. (ed.): Ocular and Adnexal Tumors. Birmingham, Aesculapius, 1978, pp. 553-571. 2. Howley, P. M., and Schlegel, R.: The human papillomaviruses. An overview. Am. J. Med. 85(supp!. 2A):155, 1988. 3. McDonnell, J. M., Mayr, A. J., and Martin, W. J.: DNA of human papillomavirus type 16 in dysplastic and malignant lesions of the conjunctiva and cornea. N. Eng!. J. Med. 320:1442, 1989. 4. McDonnell, J. M., McDonnell, P. J., Stout, W. c., and Martin, W. J.: Human papillomavirus DNA in a recurrent squamous carcinoma of the eyelid. Arch. Ophthalmo!. 107:1631, 1989. 5. Lancaster, W. D., and Olson, c.: Animal papillomaviruses. Microbio!' Rev. 46:191,1982. 6. Matlashewski, G., Schneider, J., Banks, L., Jones, N., Murray, A., and Crawford, L.: Human papillomavirus type 16 DNA cooperates with activated ras in transforming primary cells. EMBO J. 6:1741, 1987. 7. Yutsudo, M., Okamoto, Y., and Hakura, A.: Functional dissociation of transforming genes of human papillomavirus type 16. Virology 166:594, 1988. 8. Coggin, J. R., and zur Hausen, H.: Workshop on papillomaviruses and cancer. Cancer Res. 39:545, 1979. 9. Durst, M., Cissmann, L., Ikenberg, H., and zur Hausen, H.: A papillomavirus DNA from a cervical carcinoma and its prevalence in cancer biopsy samples from different geographic regions. Proc. Nat!. Acad. Sci. 80:3812, 1983. 10. Boshart, M., Gissmann, L., Ikenberg, H., Kleinheonz, A., Scheurlen, W., and zur Hausen, H.: A new type of papillomavirus DNA, its presence in genital cancer biopsies and in cell lines derived from cervical cancer. EMBO J. 3:1151, 1984. 11. Pfister, H.: Human papillomaviruses and genital cancer. Adv. Cancer Res. 48:113, 1987. 12. Shibata, D. K., Fu, Y. S., Gupta, J. W., Shah, K. V., Arnheim, N., and Martin, W. J.: Detection of human papilloma virus in paraffin-embedded tissue using the polymerase chain reaction. J. Exp. Med. 167:225, 1988. 13. Munger, K., Phelps, W. c.. Bubb, V., Howley, P. M., and Schlegel, R.: The E6 and E7 genes of the human papillomavirus type 16 together are necessary and sufficient for transformation of primary human keratinocytes. J. Virol. 63:4417, 1989.
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14. Campo, M. S.: Viral and cellular oncogenes in papillomavirus-associated cancers. Br. J. Cancer 9(suppl.):80, 1988. 15. Grossman, S. R., and Laimins, L. A.: E6 protein of human papillomavirus type 18 binds zinc. Oncogene 4:1089,1989. 16. Crook, T., Almond, N., Murray, A., Stanley, M., and Crawford, L.: Constitutive expression of c-myc oncogene confers hormone independence and enhanced growth-factor responsiveness on cells transformed by human papilloma virus type 16. Proc. Natl. Acad. Sci. USA 86:5713, 1989. 17. Nagashafar, Z., McDonnell, P. J., McDonnell, J. M., Green, W. R., and Shah, K. V.: Genital tract papillomavirus type 6 in recurrent conjunctival papilloma. Arch. Ophthalmol. 104:1814, 1986. 18. Lass, J. H., Foster, C. S., Grove, A. S., Rubenfeld, M., Lusk, R. P., Jenson, A. B., and Lancaster, W. D.: Interferon-alpha therapy of recurrent con-
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junctival papillomas. Am. J. Ophthalmol. 103:294, 1987. 19. McDonnell, P. J., McDonnell, J. M., Kessis, T., Green, W. R., and Shah, K. V.: Detection of human papillomavirus type 6/11 DNA in conjunctival papillomas by in situ hybridization with radioactive probes. Hum. Pathol. 18:1115, 1987. 20. Schochetman, G., Ou, c.. and Jones, W. K.: Polymerase chain reaction. J. Infect. Dis. 158:1154, 1988. 21. Southern, E. M.: Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. BioI. 98:503,1975. 22. Kiyabu, M. T., Shibata, D., Arnheim, N., Martin, W. J., and Fitzgibbons, P. L.: Detection of human papillomavirus in formalin-fixed, invasive squamous carcinomas using the polymerase chain reaction. Am. J. Surg. Pathol. 13:221, 1989.
OPHTHALMIC MINIATURE
It was an old woman's room, and above the smell of beeswax and the faint summer scent from a bowl of pot-pourri on the Pembroke table, his sensitive nose could detect a whiff of the sour smell of old age. Their eyes met and held. Hers were still remarkable, immense, well spaced and heavily lidded. They must once have been the focus of her beauty, and although they were sunken now, he could still see the glint of intelligence behind them. P. D. James, A Taste for Death New York, Alfred A. Knopf, 1986, p. 96
T HE CLINICAL and histopathologic features of nonpigmented conjunctival and corneal epithelial neoplasias were classified by Pizzarello and [akobiec in 1978. 1 They adopted the nomenclature of anogenital epithelial neoplasias, which have similar histopathologic characteristics. In a conjunctival or corneal intraepithelial neoplasm, dysplastic cells are confined to the epithelium. When these cells have invaded the epithelial basement membrane, the tumor is considered to be a squamous cell carcinoma. Human papillomaviruses are oncogenic viruses that are present in up to 90% of anogenital epithelial neoplasms.! Their presence has recently been demonstrated in conjunctival epithelial neoplasms" and in a squamous cell carcinoma of the eyelid.' Papillomaviruses induce tumor formation in animals" and malignant transformation of cell cultures." The mechaAccepted for publication April 13, 1990. From the LSU Eye Center (Dr. Lauer) and the Department of Pathology (Dr. Meier), LSU Medical Center School of Medicine, and the Department of Pathology (Dr. Malter), Tulane Medical Center, New Orleans, Louisiana. This study was supported in part by U.S. Public Health Service grants EY08482 (Dr. Lauer) from the National Eye Institute and CA01427 (Dr. Malter) from the National Cancer Institute, National Institutes of Health, Bethesda, Maryland, and a grant from the ARVOI Alcon Fellowship Program (Dr. Lauer). Reprint requests to Simeon A. Lauer, M.D., LSU Eye Center, 2020 Gravier St., New Orleans, LA 70112.
©AMERICAN
JOURNAL OF OPHTHALMOLOGY
110:23-27,
JULY,
nism of oncogenesis is not completely understood.' Human papillomaviruses are divided into types based on homologous characteristics of the DNA sequences." In the anogenital region, types 169 and 18 10 have been found predominantly in carcinomas." Thus far, human papillomavirus type 16 is the only type demonstrated in conjunctival epithelial neoplasms." In this study, we confirmed the presence of human papillomavirus DNA in conjunctival intraepithelial neoplasia. Human papillomavirus type 18 was present in two of these tumors.
Material and Methods Specimens for study were collected from formalin-fixed, paraffin-embedded tissue. The pathologic characteristics were reviewed and the histopathologic diagnosis confirmed for each block by one of us (l.R.M.). Five sections, 10 J.Lm thick, were removed with a clean microtome blade and placed in a microcentrifuge tube under sterile conditions. Paraffin was removed with two xylene extractions, and xylene was removed with 95% ethanol. The tissue was pelleted, residual ethanol was removed, and the specimen was vacuum dried for five minutes before resuspension in 100 J.Ll of water. The specimen was boiled for 20 minutes and the DNA content determined by spectrophotometry. Approximately 2.0 J.Lg of total nucleic acid was used for the polymerase chain-reaction assay. The polymerase chain-reaction assay was performed in a total volume of 50 J.Ll. Buffer consisted of 17.0 mM of ammonium sulfate, 67.0 mM of Tris-HCl pH 8.5, 10.0 mM of 2mercaptoethanol, and 170 J.Lg/ml of bovine serum albumin. Magnesium chloride 7.0 mM, a biologic detergent composed of various polyoxyethylene ethers and other surface-active compounds (Triton-X 100, Sigma Chemical Co., St. Louis, Missouri) 0.1 %, deoxynucleotides (1.5 mM each), and three units of Taq DNA 1990
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polymerase (Promega, Madison, Wisconsin) were added to each reaction mixture. Each specimen was run with type-I6 and type-I8 oligonucleotide (20 base) primers synthesized according to the sequences reported by Shibata and associates." Aliquots (1 ""g) of DNA extracted from SiHa and HeLa cell culture lines were used as positive controls for human papillomavirus types 16 and 18, respectively. Solutions and primers without DNA served as negative controls. Amplification consisted of denaturation for one minute at 93 C, annealing for one minute at 47 C, and extension for 30 seconds at 72 C. The amplification process was continued for 35 cycles. Two separate 10-",,1 aliquots from each of the amplification products were loaded on an ethidium bromide-stained 2% agarose gel, separated by electrophoresis for 45 minutes at 100 V, and transferred to a nylon membrane. Each amplification product was hybridized independently with human papillomavirus type 16 or 18 oligonucleotide (40 base) probes as described by Shibata and assoctates." Prehybridization was performed for three hours at 42 C in 50% formamide, five times the standard concentration of sodium chloride, sodium phosphate, ethylene-diaminetetraacetic acid (EDTA) buffer, 0.1 % Denhardt's solution, 10 mgjml of salmon-sperm DNA, and 1% glycine. Hybridization was performed for 24 hours at 42 C in 50% formamide, five times the standard concentration of sodium chloride, sodium phosphate, EDTA buffer, 10 mgjml of salmon-sperm DNA, and 1% sodium dodecyl sulfate. Membranes were washed with two times the standard concentration of sodium chloride, sodium citrate buffer sequentially at room temperature, 37 C, and 42 C until background radioactivity was undetectable. Autoradiography with Kodak X-AR film and two intensifying screens was performed for three to 12 hours at -70 C.
Results We studied five conjunctival intraepithelial neoplasms (Table). Representative autoradiograms for three of the tumors showed the presence of type-I6 (Fig. 1) and type-I8 (Fig. 2) DNA. In Figure 1, the amplified products were hybridized with the human papillomavirus type-I6 probe. In Figure 2, the type-I8 probe was used for hybridization. Type-I6 primers
TABLE VIRAL DNA IN CONJUNCTIVAL INTRAEPITHELIAL NEOPLASMS HUMAN PAPILLOMAVIRUS DNA SPECIMEN NO.
TYPE 16
TYPE 18
1
Absent Present Present Present Present
Absent Present Absent Absent Present
2 3 4
5
were used for amplification in odd-numbered lanes, and type-I8 primers were used in evennumbered lanes. DNA extracted from the SiHa and HeLa cervical cell lines, which are known to contain human papillomavirus DNA, were used as positive controls. The SiHa DNA, which contains type 16, was used in lane 1. In lane 2, He La DNA, which contains type 18, was used. These positive controls yielded a strong single band of the expected size (109 base pairs) as visualized after agarose gel electrophoresis. These amplified fragments hybridized strongly with appropriate probes. A band was not always visible in the agarose gel for the specimens but the autoradiographic bands corresponded precisely with those of the positive control. No DNA was added to the negative controls in lanes 3 and 4. Autoradiography continued to yield negative results even after prolonged exposure. Amplified DNA from the first specimen was
•
1 2 3 4 5 6 7 8
•
9 10
Fig. 1 (Lauer, Malter, and Meier). Southern blot hybridization of polymerase chain reaction product with human papillomavirus type-I6 32P-radiolabeled oligonucleotide probe. Type-I6 primers were used in odd-numbered lanes, and type-I8 primers were used in even-numbered lanes. The primers were incubated with the following: SiHa DNA (lane 1), HeLa DNA (lane 2), without DNA (lanes 3 and 4), specimen 1 (lanes 5 and 6), specimen 2 (lanes 7 and 8), and specimen 3 (lanes 9 and 10). Lanes 1 (type 16, control), 7, and 9 contain type-16 DNA.
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Fig. 2 (Lauer, Malter, and Meier). Southern blot as in Figure 1 hybridized with human papillomavirus type-I8 probe. Lanes 2 (type 18, control) and 8 contain type-I8 DNA.
subjected to electrophoresis in lanes 5 and 6. No band was visualized with either papillomavirus probe even after prolonged exposure. The amplification product from the second specimen hybridized strongly with the type-18 probe (Fig. 2, lane 8), which showed the presence of human papillomavirus type-18 DNA. A weaker band was also seen with the probe specific for type-16 DNA (Fig. 1, lane 7). The amplification product from the third specimen hybridized strongly with the type-16 probe (Fig. 1, lane 9) but not with the probe for type 18 (Fig. 2, lane 10). The fourth specimen was strongly positive for human papillomavirus type 16 and did not hybridize with the type-18 probe. The fifth specimen hybridized strongly with both probes.
Discussion
Human papillomaviruses are epitheliotropic oncogenic viruses." They have been found in a variety of epithelial neoplasias, including more than 90% of uterine cervical carcinomas.! Human papillomavirus DNA induces malignant transformation of cultured cells in vitro." The ability to transform cultured cells has been traced to the E6 and E7 early open-reading frames of the human papillomavirus genome." The E6-encoded transforming proteins confer tumorigenicity. The E7-encoded proteins confer malignant growth properties. There is evidence that E6- and E7-transforming proteins interact with nuclear proteins." Alternatively, the E6-encoded protein may be a hormonereceptor protein," which is consistent with the hormone dependence of some human papillomavirus-transformed cells." Human papillomaviruses are divided into types based on homologous characteristics of
25
their DNA sequences." Low-risk types such as 6 and 11 are found in premalignant and benign tumors.! These types have been identified in conjunctival papillomas.":" High-risk types such as 16 and 18 are more common in carcinomas." The E6 region of types 16 and 18 differs from that of other types." The potential for malignant transformation may be linked to the E6* intron, which is unique to these types. Shibata and associates" have developed a polymerase chain-reaction assay that is specifically designed to identify the E6 region of human papillomavirus types 16 and 18. In a polymerase chain-reaction assay (Fig. 3), target DNA is amplified by the enzyme DNA polymerase.f This process starts with the denaturation of double-stranded DNA into two single strands. Two oligonucleotide primers hybridize to single-stranded complementary regions of the target DNA. DNA polymerase then extends the primer by using the double-stranded region of the target DNA as a template. The newly synthesized double-stranded DNA is denatured and the cycle repeated. With each cycle, the amount of target DNA is doubled. After 20 to 40 cycles, a small quantity of target DNA can be replicated a billion times." As little as 100 attograms of target DNA can be detected (Pietro Lampertico, M.D., Tulane University College of Medicine, New Orleans, Louisiana, unpublished data) when the assay is coupled with Southern blotting" as in this study. The target DNA is identified by hybridization with a probe. The probe is a radiolabeled oligonucleotide that is complementary to a unique internal region of the amplified target DNA. The presence of bound probes is demonstrated by autoradiography (Figs. 1 and 2). We used a polymerase chain reaction to demonstrate the presence of human papillomavirus DNA in four of five conjunctival intraepithelial neoplasms. McDonnell, Mayr, and Martin" reported finding human papillomavirus type-16 DNA in nine of 13 (69%) conjunctival intraepithelial neoplasms and in three conjunctival squamous cell carcinomas. The two studies combined found human papillomavirus DNA in 13 of 18 (72%) conjunctival intraepithelial neoplasms. In our study, human papillomavirus type 18 was found in two tumors. Type 16 was found in four tumors including two tumors that contained both types. The significance of finding both types in the same tumor is not known." We provide evidence for the presence of hu-
26
July, 1990
AMERICAN JOURNAL OF OPHTHALMOLOGY
Denaturation 93C
Whether human papillomavirus DNA plays an active role in the development of conjunctival intraepithelial tumors cannot be stated until the mechanism of papillomavirus oncogenesis is better understood.
References
Annealing 47C
Extension 72C
Fig. 3 (Lauer, Malter, and Meier). Schematic diagram of the polymerase chain-reaction sequence. Top, Step 1. Double-stranded target DNA is denatured to single-stranded DNA at 93 C for one minute. Middle, Step 2. Oligonucleotide primers complementary to flanking regions of the target DNA are allowed to anneal at 47 C for one minute. Bottom, Step 3. Primers are extended by the enzyme DNA polymerase by using the single-stranded target DNA as a template.
man papillomavirus type-18 DNA. The band produced by the tumor we studied was identical to that produced by DNA extracted from the type 18-containing HeLa cell line. The assay produced no amplification product when no DNA was added, which confirms that the target DNA was not a contaminant in the reagents. The first tumor did not produce an amplification product, which confirms that the contaminating DNA was not added during processing of the specimens. The third specimen was exclusively positive for human papillomavirus type 16, confirming that cross-hybridization did not occur. The fifth tumor studied had equally strong bands with both type-16 and -18 probes. This specimen, therefore, contained both types. The weak band that developed (Fig. 1, lane 7) probably represents low-level type-16 infection.
1. Pizzarello, L. D., and [akobiec, F. A.: Bowen's disease of the conjunctiva. A misnomer. In [akobiec, F. A. (ed.): Ocular and Adnexal Tumors. Birmingham, Aesculapius, 1978, pp. 553-571. 2. Howley, P. M., and Schlegel, R.: The human papillomaviruses. An overview. Am. J. Med. 85(supp!. 2A):155, 1988. 3. McDonnell, J. M., Mayr, A. J., and Martin, W. J.: DNA of human papillomavirus type 16 in dysplastic and malignant lesions of the conjunctiva and cornea. N. Eng!. J. Med. 320:1442, 1989. 4. McDonnell, J. M., McDonnell, P. J., Stout, W. c., and Martin, W. J.: Human papillomavirus DNA in a recurrent squamous carcinoma of the eyelid. Arch. Ophthalmo!. 107:1631, 1989. 5. Lancaster, W. D., and Olson, c.: Animal papillomaviruses. Microbio!' Rev. 46:191,1982. 6. Matlashewski, G., Schneider, J., Banks, L., Jones, N., Murray, A., and Crawford, L.: Human papillomavirus type 16 DNA cooperates with activated ras in transforming primary cells. EMBO J. 6:1741, 1987. 7. Yutsudo, M., Okamoto, Y., and Hakura, A.: Functional dissociation of transforming genes of human papillomavirus type 16. Virology 166:594, 1988. 8. Coggin, J. R., and zur Hausen, H.: Workshop on papillomaviruses and cancer. Cancer Res. 39:545, 1979. 9. Durst, M., Cissmann, L., Ikenberg, H., and zur Hausen, H.: A papillomavirus DNA from a cervical carcinoma and its prevalence in cancer biopsy samples from different geographic regions. Proc. Nat!. Acad. Sci. 80:3812, 1983. 10. Boshart, M., Gissmann, L., Ikenberg, H., Kleinheonz, A., Scheurlen, W., and zur Hausen, H.: A new type of papillomavirus DNA, its presence in genital cancer biopsies and in cell lines derived from cervical cancer. EMBO J. 3:1151, 1984. 11. Pfister, H.: Human papillomaviruses and genital cancer. Adv. Cancer Res. 48:113, 1987. 12. Shibata, D. K., Fu, Y. S., Gupta, J. W., Shah, K. V., Arnheim, N., and Martin, W. J.: Detection of human papilloma virus in paraffin-embedded tissue using the polymerase chain reaction. J. Exp. Med. 167:225, 1988. 13. Munger, K., Phelps, W. c.. Bubb, V., Howley, P. M., and Schlegel, R.: The E6 and E7 genes of the human papillomavirus type 16 together are necessary and sufficient for transformation of primary human keratinocytes. J. Virol. 63:4417, 1989.
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14. Campo, M. S.: Viral and cellular oncogenes in papillomavirus-associated cancers. Br. J. Cancer 9(suppl.):80, 1988. 15. Grossman, S. R., and Laimins, L. A.: E6 protein of human papillomavirus type 18 binds zinc. Oncogene 4:1089,1989. 16. Crook, T., Almond, N., Murray, A., Stanley, M., and Crawford, L.: Constitutive expression of c-myc oncogene confers hormone independence and enhanced growth-factor responsiveness on cells transformed by human papilloma virus type 16. Proc. Natl. Acad. Sci. USA 86:5713, 1989. 17. Nagashafar, Z., McDonnell, P. J., McDonnell, J. M., Green, W. R., and Shah, K. V.: Genital tract papillomavirus type 6 in recurrent conjunctival papilloma. Arch. Ophthalmol. 104:1814, 1986. 18. Lass, J. H., Foster, C. S., Grove, A. S., Rubenfeld, M., Lusk, R. P., Jenson, A. B., and Lancaster, W. D.: Interferon-alpha therapy of recurrent con-
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junctival papillomas. Am. J. Ophthalmol. 103:294, 1987. 19. McDonnell, P. J., McDonnell, J. M., Kessis, T., Green, W. R., and Shah, K. V.: Detection of human papillomavirus type 6/11 DNA in conjunctival papillomas by in situ hybridization with radioactive probes. Hum. Pathol. 18:1115, 1987. 20. Schochetman, G., Ou, c.. and Jones, W. K.: Polymerase chain reaction. J. Infect. Dis. 158:1154, 1988. 21. Southern, E. M.: Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. BioI. 98:503,1975. 22. Kiyabu, M. T., Shibata, D., Arnheim, N., Martin, W. J., and Fitzgibbons, P. L.: Detection of human papillomavirus in formalin-fixed, invasive squamous carcinomas using the polymerase chain reaction. Am. J. Surg. Pathol. 13:221, 1989.
OPHTHALMIC MINIATURE
It was an old woman's room, and above the smell of beeswax and the faint summer scent from a bowl of pot-pourri on the Pembroke table, his sensitive nose could detect a whiff of the sour smell of old age. Their eyes met and held. Hers were still remarkable, immense, well spaced and heavily lidded. They must once have been the focus of her beauty, and although they were sunken now, he could still see the glint of intelligence behind them. P. D. James, A Taste for Death New York, Alfred A. Knopf, 1986, p. 96
