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Int. J. Cancer.• 55, 171-173 (1993) ® 1993 Wiley-Liss, Inc. LETTER TO THE EDITOR t I Dear Sir, Publication of the International Union Against Cancer Publicatfon de I'Union Internationale Contre le Cancer Demonstration of human papillomavirus (HPV) type 30 in esophageal squamous-cell carcinomas by in situ hybridization Papillomaviruses are a group of DNA tumor viruses which are epitheliotrophic and primarily affect the skin and mucous membranes of the genitourinary and upper aerodigestive tracts, inducing hyperplastic, papillomatous and verrucous lesions of the squamous epithelium (Syrjanen et a1.,1987a; zur Hausen, 1991). With the rapidly developing molecular biological tech- niques, 68 distinct types of human papillomavirus (HPV) have been recognized from human epithelial lesions in less than 10 years (de irlliers, 1989). Some of them show remarkably specific host- and target-tissue tropism (de Villiers, 1989). Kahn et ai. (1986) isolated and cloned a novel HPV type, designated HPV 30, from a laryngeal squamous-cell carci- noma. This new HPV type was found to be most closely related to HPV types 16 and 53 (de Villiers, 1989; Gallahan et al., 1989). Using HPV 30 DNA as a probe, Kahn et al. (1986) detected this virus in 2 genital condylomas, but failed to demonstrate it in any of the 41 laryngeal carcinomas, 10 bronchial carcinomas and 31 other head-and-neck tumors examined. This is consistent with our previous negative results on a series of 116 laryngeal carcinomas (Syrjanen et al., 1987b), and 73 oral pre-cancerous lesions and squamous-cell carcinomas examined by in situ hybridization (ISH) with HPV 30 DNA as one of the probes (Syrjanen et al., 1988b). Similarly, we were unable to find in the literature any other reports on the discovery of HPV 30 in human squamous-cell lesions. We have recently begun a large-scale survey to detect HPV infections in esophageal carcinomas by ISH (both screening and specific typing) (Chang et al., 1993), and have been able to show the presence of HPV 30 DNA sequences in esophageal carcinomas. A total of 776 esophageal biopsies dertved from 363 patients with invasive squamous-cell carcinoma of the esophagus were included in this study. Biopsies were taken from the invasive carcinomatous tissue, from epitheliufn adjacent to the carcino- mas and from surgically resected margins. All specimens were collected from the high-incidence area for esophageal cancer in Linxian, Henan province of north China. Specimens were fixed in neutral formalin and embedded in paraffin. They were first screened for the presence of HPV DNA by screening ISH with a commercial kit (Biohit HPV Screening Kit, Biohit, Helsinki, Finland), according to the manufacturer's protocol. The hybridization mixture for ISH HPV screening test con- tained biotin-labelled HPV cocktail pr•obes (a mixture of differ- ent HPV DNAs in 30% fonnaj*nide). By using this test, many HPV types, including both mucosal and skin types, can be detected (Biohit HPV Screening Kit, Biohit, Helsinki, Fin- land). The HPV DNA positive samples were further analyzed by ISH HPV typing using biotin-labelled HPV DNA probes under high-stringency conditions (Tm-17). HPV typing ISH was performed as described earlier with minor modifications (Syrjanen et al., 1988a). Briefly, 4-pin-thick sections were cut from each biopsy and mounted on microscope slides pre- treated with 1% aminopropyltriethoxy-silane (Sigma, St. Louis, MO). Sections were deparaffinized inxylene, rehydrated through graded ethanol, and digested with proteinase K The specimens were hybridized in a mixture of 50% formamide, 2 X SSC, 400 µg/ml herring sperm DNA, 10% dextran sulfate and 1.0 p,g/ml biotinylated HPV 30 DNA probes. Hybridization was carried out in a 55°C incubation oven overnight. Post- hybridization washes consisted of 2 x SSC, twice for 5 min at room temperature; 0.2 x SSC/0.1 % SDS once at 55°C for 5 min and finally one 5-rnin wash in 2 x SSC at room tempera- ture. The slides were incubated with streptavidin alkaline phosphatase complex, and successively developed with nitro- blue tetrazolium and bromo-ehloro-indoxyl phosphate. Of the 363 esophageal carcinomas examined so far, 85 (23.4%) were shown to contain HPV DNA sequences. Positive signals were found on the nuclei of cancer cells in 71 cases (19.6%), in the surrounding hyperplastic or dysplastic epithe- lial cells exclusively in 13 cases (3.6%), in both the cancer cells and surrounding epithelial cells in 10 cases (2.8%), and in the resection margins in 1 case (0.3%). Of these 81 cases with HPV DNA in the cancer cells, 27 were well-, 39 moderately- and 15 poorly-differentiated squamous-cell carcinomas. Thirty- four (40%) of the 85 HPV-positive cases contained at least one type of HPV 6/11, 16, 18 and 30 DNA sequences. A single HPV type was detected in 29/85 (34.1 %) carcinomas; HPV 6/11 (a mixed probe) in 6, HPV 16 in 12, HPV 18 in 6, and HPV 30 in 5 cases. Double or multiple infections were noted in 5 cases. The results of the HPV screening and typing ISH on these biopsies have been reported in detail elsewhere (Chang et al., 1993). Here we draw attention to the presence of HPV 30 DNA sequences in 8 (9.4%) of the 85 HPV-positive esophageal carcinomas. Among the HPV-30 positive cases, 5 patients were infected with the HPV 30 as the single type, and 3 patients were co-infected with otherHPV types, 1 with HPV 6/11, and 2 with HPV 16. The characteristics of the HPV-30 positive esophageal carcinomas are shown in Table I. The hybridization signals were exclusively confined to the nuclei of cancer cells (Fig. 1). Patterns of the positive signals were variable and, in most sections, the highest signal intensity was present in areas with the highest squamous-cell differentia- tion (Fig. 1). No cross-hybridization was found when the sections were hybridized with HPV type 53, which shows the highest DNA homology (25%) to HPV 30 (de Villiers, 1989; Gallahan et al., 1989). Tltis indicates that the ISH typing technique employed in the present study has an extremely high specificity for the HPV types detected.

172 CHANG ET AL. TABLE I - CHARACTERIZATION OF THE HPV-30-POSITIVE ESOPHAGEAL CARCINOMAS Case Tumor Screening Sex Age Yearof number biopsy differentiation ISH '~yp ing iSH 1 F 39 1988 Good + HPV 30 2 F 53 1988 Moderate + HPV 30 3 M 60 1988 Moderate + HPV 30 4 M 41 1988 Moderate + HPV 30 5 M 50 1990 Moderate + HPV 30 6 F 62 1988 Good + HPV 16 + 30 7 F 58 1988 Moderate + HPV 16 + 30 8 F 44 1988 Poor + HPV 6/11t + 30 tA mixture of HPV 6 and 11 probes was used for ISH typing. Infections with the high-risk HPV types have been closely linked with specific human cancers, especially those of the anogenital tract. HPV involvement in both benign and malig- nant squamous-cell lesions has been demonstrated by morpho- logical studies showing condylomatous changes, by immuno- histochemical detection of HPV structural proteins, and by DNA hybridization disclosing HPV DNA sequences in these lesions (Chang et a1.,1990a, b, 1992; Williamson et a1.,1991; Benamouzig et al., 1992; Toh et al., 1992). Increasing num- bers of reports suggest that esophageal HPV infection could be a risk factor for the development of esophageal squamous-cell carcinoma (Chang et al., 1990a, b, 1992; Williamson et al., 1991; Benamouzig et al., 1992; Toh et al., 1992). Of the known HPV types, HPV 6, 11, 16, 18, 31133135 have been detected in esophageal lesions so far. HPV 16 and 18 seem to be the 2 types most frequently associated with esophageal carcinoma. Our results confirm the previously reported HPV involvement in esophageal lesions, and furthersupport a causal association of HPV infection with esophageal carcinoma. The identification of HPV 30 DNA sequences in esophageal lesions is of special interest. Since the original isolation of HPV 30 from a laryngeal carcinoma and identification of the viral DNA in 2 genital condylomas (Kahn et al., 1986), no other authors have detected this HPV type either in head-and-neck tumors or in anogenital lesions, suggesting that the primary sites for HPV 30 infection have not yet been identified. In view of the close anatomical proximity of the esophagus to the laty~n.x, the possibility is raised that esophageal mucosa may be one of pritnary mucosal sites for HPV 30 infection. The original isolation of HPV 30 from a laryngeal squamous- cell carcinoma indicates that this virus can exert an oncogenic potential in epithelial cells. The identifzcation of HPV 30 DNA FIGURE 1- Identiflcation of HPV 30 DNA sequences in a moderately differentiated esophapal squamous-cell carcinoma by DNA in situ hybridization. Positive stgnais are localized in a number of cancer-cell nuclei (in situ hybridization). in 2.2% (8/363) of esophageal squamous-cell carcinomas points to a causal role for this HPV type in the pathogenesis of esophageal carcinoma. Further studies on the association between HPV 30 and esophageal carcinomas are clearly war- ranted. Yours sincerely, Fuju CHANG, Stina SS7ZlANEN and Kari SYR.tANEN Department of Pathology and Kuopio Cancer Research Centre, University of Kuopio, POB 1627, SF-70211 Kuopio, Finland. March 18,1993. ACKNOWLEDGEMENTS The authors thank Drs. L. Gissmann and H. zur Hausen, Heidelberg, Germany, for providing the HPV 30 DNA probes. This study was supported in part by a research grant from the Savo Cancer Fund (F. C.) and in part by a research grant from the Finnish Cancer Society and a joint research grant from Fabriques de Tabac Reunies and the British-American To- bacco Company (BAT). REFERENCES BENAMOUZIG, R., PIGOT, F., QUIROGA, G., DALIDIRE, P., CHAUSSADE, S., CATALAN, F. and COUTURIER, D., Human papillomavirus infection in esophageal squamous-cell carcinoma in Western countries. Int. J. Cancer, 50, 549-552 (1992). CHANG, F., SHEN, Q., ZHOU, J., WANG, C., WANG, D., SYRJANEN, S. and SYRJANEN, K., Detection of human papillomavirus DNA in cytologic specimens derived from esophageal precancer lesions and cancer. Scand. J. Gastroenterol., 25, 383-388 (1990a). CHANG, F., SYRJANEN, S., SHEN, Q., Ji, 11. and SYRJANEN, K., Human papillomavirus (HPV) DNA in esophageal precancer lesions and squamous cell carcinomas from China. lnt. J. Cancer, 45, 21-25 (1990b). CHANG, F., SYRJANEN, S., SHEN, Q., WANG, L. and SYRJANEN, K., Screening for human papillomavirus (IIPV) infections in esophageal squamous cell carcinomas by in situ hybridization. Cancer (1993) (In press). CHANG, F., SYRJANEN, S., SHEN, Q., WANG, L., WANG, D. and SYRJANEN, K., Human papillomavirus (HPV) involvement in esopha- geal precancer lesions and squamous cell carcinomas as evidenced by microscopy and different DNA-techniques. Scand. J. GastroenteroL, 27, 553-563 (1992). DE VILLIERS, E.-M., Heterogeneity of the human papillomavirus group..I. Virot., 63, 4898-4903 (1989). GALLAHAN, D., MULLER, M., SCHNEIDER, A., DELIUS, H., KAHN, T., DE VILLIERS, E.-M. and GISSMANN, L., Human papillomavirus type 53.J. Virol., 63, 4911-4912 (1989). KAHN, T., SCHwARZ, F. and zuR HAUSEN, H., Molecular cloning and C (

HPV TYPE 30 IN ESOPHAGEAL CARCINOMA characterization of the DNA of a new human papillomavirus (HPV 30) from a laryngeal carcinoma. Int. J. Cancer, 37, 61-65 (1986). SYRJANEN, K.J., GISSMANN, L. and Koss, L.G., Papillomavinuses and human disease. Springer, Heidelberg (1987a). SYRJANEN, S., PARTANEN, P., MANTYJARVI, R. and SYRJANEN, K., Sensitivity of in situ hybridization techniques using biotin and 35S- labeled human apillomavirus (HPV) DNA probes. J. virol. Meth., 19, 225-238 (1988a~ SYRJANEN, S., SYRJANEN, K., MANTYJARVI, R., COLLAN, Y. and KARJA, J., Human papillomavirus DNA in squamous cell carcinomas of the larynx demonstrated by in situ DNA hybridization. ORL J. Otorhinolar- yngo[. Relat. Spec., 49, 175-186 (1987b). 173 SYRJANEN, S.M., SYRJANEN, K.J. and HAPPONEN, R.-P., Human papillomavirus (HPV) DNA sequences in oral precancerous lesions and squamous cell carcinoma demonstrated by in situ hybridization. J. oral Pathol., 17, 273-278 (1988b). TOH, Y., KUWANO, H., TANAKA, S., BABA, K., MATSUDA, H., SUGIMA- cHl, K. and MORI, R., Detection of human papillomavirus DNA in esophageal carcinoma in Japan by polymerase chain reaction. Cancer, 70, 2234-2238 (1992). WILLIAMSON, A.I., JASKIESICZ, K. and GUNNING, A., The detection of human papillomavirus in oesophageal lesions. Anticancer Res., 11, 263-266 (1991). ZUR HAUSEN, H., Human papillomaviruses in the pathogenesis of anogenital cancer. Virology, 184, 9-13 (1991). ~ ~