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0 Human Papillomavirus Involvement in Esophageal Precancerous Lesions and Squamous Cell Carcinomas as Evidenced by Microscopy and Different DNA Techniques F. CHANG, S. SYRJANEN, 0. SHEN, L. WANG, D. WANG & K. SYRJANEN Dept. of Pathology, Kuopio Cancer Research Centre, University of Kuopio, Kuopio, Finland, and Dept. of Precancerous Studies, Henan Medical University, Zhengzhou, Henan, China Chang F, Syrjanen S, Shen 0, Wang L, Wang D, Syrjanen K. Human papillomavirus involvement in esophageal precancerous lesions and squamous cell carcinomas as evidenced by microscopy and different DNA techniques. Scand J Gastroenterol 1992;27:553-563. A series of 71 surgically resected esophageal squamous cell carcinomas, including 51 cases of formalin- fixed samples and 20 cases of fresh biopsy specimens derived from the high-incidence area of esophageal cancer in China, were systematically analyzed for the presence of human papillomavirus (HPV) infections by light microscopy, electron microscopy (TEM), in situ DNA hybridization, Southern blot hybridization, and polymerase chain reaction (PCR) techniques. On light microscopy, HPV-suggestive lesions were found in a total of 49.0% (25 of 51) of the specimens, including the flat type (22 of 51) and, less frequently, an inverted one (2 of 51). Of the 51 formalin-fixed, paraffin-embedded specimens, 43.1% (22 of 51) contained HPV DNA sequences by in situ hybridization. Of the positive cases, HPV 6 was present in three (5.9%), HPV 11 in three (5.9%), HPV 16 in eight (15.7%r~), HPV 18 in six (11.8%), double infections with HPV 11/18 in one (2.0%.), and HPV 16/18 in one. In most cases the HPV- positive signals were localized in the hyperplastic and/or dysplastic epithelium adjacent to invasive carcinomas. In two specimens, however, HPV DNA sequences were found in the frankly invasive lesions, one being HPV 6 and the other HPV 18. On TEM, HPV-like particles located in the nuclei of koilocytotic cells were demonstrated in two of the five specimens previously shown to be IIPV-positive by in situ hybridization. By means of the PCR technique, all specimens positive for HPV by in situ hybridization also contained amplified HPV sequences. Moreover, three additional samples negative by in situ hybridization were found to contain HPV 1 l DNA sequences. Of the 20 DNA samples extracted from the fresh carcinoma samples (containing some surrounding tissues as well), 9 were shown to contain HPV DNA sequences by Southern blot hybridization under low-stringency conditions. Of these, eight samples remained positive when hybridized with the probe cocktail of HPV 11, 16, 18, and 30 DNA under high-stringency conditions. HPV DNA sequences in these carcinoma specimens appeared to be present mainly in an integrated form. The present results confirm the HPV involvement in esophageal squamous cell lesions and suggest that HPV infection might be an important etiologic factor in the pathogenesis of esophageal cancer, most probably acting synergistically with other carcinogenic factors. Key words: Carcinogenesis; electron microscopy; esophageal cancer; histopathology; human papil- lomavirus; in situ, hybridization; polymerase chain reaction; Southern blot hybridization Fuju Chang, M.D., Ph.D., Dept. of Pathology, University of Kuopio, P.O.B. 1627, SF-70211 Kuopio, Finland Worldwide, the incidence of esophageal cancer shows a great geographic variation, being remarkably high in certain regions including the northern parts of China (1-6), the Caspian littoral of Iran (4-6), the Turkmen area of the former Soviet Union (7-9), and the Transkei area of South Africa (10-12). In China the nationwide deaths due to esophageal cancer account for 27% of all cancer deaths among males and 20% in females, ranking among the two leading causes of cancer deaths, second only to that of stomach cancer (2-5). In Linxian (a county with a population of 800,000 in the Henan Province of North China), the age- adjusted average incidence rates were 161.33/105in males and 102.88/105 in females during 1971-74 (2-5). The deaths due to esophageal cancer in this area account for 16% of all deaths and 65% of deaths from cancers (4). Although the incidence and mortality fluctuate slightly from year to year, they do not appear to decline to any significant extent (2-4). The cause of these major regional variations in the inci- dence of esophageal cancer is not fully understood. No single etiologic factor(s) could account for the dramatic variation of esophageal cancer in all high-incidence areas. Cigarette smoking and excess alcohol intake may be risk factors in some areas (for example, Western countries and South ~

554 F. Chang et al. Africa), especially when the two factors are combined (12, 13). However, these agents do not appear to be a major problem in China and Iran (1-3, 6-9). In China attention has been focused on specific nutritional deficiencies, including those of vitamins A, B, and C, and on certain minerals and nitrosamines formed in moldy foodstuffs (1-6). In Iran the same nutritional deficiencies were also noted, and, in ad- dition, opium tar was blamed (7-9). Thus, it is still neces- sary to search for additional etiologic agents of this malig- nancy. Evidence for a possible viral etiology of esophageal cancer in cattle was first found in the Scottish Ilighlands, showing a remarkably high incidence of squamous cell carcinoma of the upper alimentary tract, especially the esophagus, among the animals (14-17). Two synergistic factors-that is, bovine papillomavirus type 4 (BPV 4) and bracken fern-have been regarded as the most important etiologic agents for the development of this malignancy (15, 17). The infection of the alimentary canal mucosa by BPV 4 causes squamous cell papillomas, which can become the focus for malignant transformation in animals grazing on bracken (14, 15, 17). Human papillomavirus (HPV) infections in the upper aerodigestive tract have been also established beyond doubt by morphologic studies showing HPV-suggestive lesions, by immunoperoxidase-peroxidase antiperoxidase (IP-PAP) staining for HPV antigens, and by DNA hybridization studies disclosing HPV DNA sequences (18-25). Esophageal HPV infection was first suggested in 1982 by Syrjanen et al. (26, 27), who found that 40% (24 of 60) of esophageal squamous cell carcinomas presented with histologic changes identical to those of condylomatous lesions. They sub- sequently demonstrated HPV structural proteins in one case of esophageal squamous cell papilloma (27, 28). These results have been confirmed by others demonstrating HPV- suggestive lesions (27, 29-35), HPV antigens (29-32, 34-36), and HPV DNA sequences (37-42) in esophageal squamous cell lesions, although some other reports failed to detect evidence of HPV involvement in esophageal lesions (43-47). Our previous studies have shown the presence of HPV DNA sequences in a high percentage of esophageal pre- cancerous lesions and cancer derived from the high-risk area of this malignancy in China (39, 40). Similar observations were also reported recently by Williamson et al. (41) and Miller et al. (42) from the high-incidence areas of South Africa and Alaska natives, respectively. These findings, in agreement with the currently available evidence indicating an etiologic role of HPV in the pathogenesis of squamous cell carcinomas at other mucosal sites (47-52), have led to the consideration of HPV as a potential etiologic agent in esophageal carcinogenesis as well (39-41, 53). To elucidate further the association of HPV and the dev- elopment of esophageal cancer, a series of esophageal pre- cancerous and cancerous samples collected from the high- risk area in Linxian, China, was systematically analyzed by means of light microscopy, transmission electron microscopy Table I. Human papillomavirus (HPV)-suggestive lesions found in the esophageal specimens Presence in the esophageal specimens Type of HPV lesions No./total % Papillary 0/51 0 Flat 25/51 49.0 Inverted* 2/51 3.9 * Inverted and flat types coexisted in these two cases. (TEM), in situ hybridization, Southern blot hybridization, and polymerase chain reaction (PCR) techniques in the present study. MATERIALS AND METHODS Specimen collection and preparation The material in the present study consisted of 71 surgical specimens (51 formalin-fixed biopsy specimens and 20 fresh specimens) derived from the same number of surgically operated patients with an invasive squamous cell carcinoma of the esophagus. All specimens were collected from the high-incidence area of Linxian County in Henan Province, China. The age-adjusted death rates of esophageal cancer in this area were 161.33/10` for males and 102.88/10g for females (4). The 51 formalin-fixed biopsy specimens were subsequently embedded in paraffin. Both the carcinoma foci and their surrounding epithelia from each patient were analyzed for the presence of HPV infections by histopathologic assess- ment, in situ DNA hybridization, and PCR amplification. To disclose HPV particles in these lesions, five specimens shown to be HPV-positive by in situ hybridization were further examined by TEM. Of the 20 fresh carcinoma samples, the high-molecular weight DNAs from the car- cinoma foci and their surrounding tissues were extracted and analyzed for the presence of HPV DNA by Southern blot hybridization. Morphologic evaluation Formalin-fixed, paraffin-embedded esophageal biopsy specimens were cut into 4-µm sections and routinely stained with hematoxylin and eosin (HE) for light microscopy. Epi- thelial lesions were graded in accordance with generally accepted criteria into normal, mild dysplasia, moderate dys- plasia, severe dysplasia, carcinoma in situ, and invasive carcinoma. The sections were in all cases evaluated with special emphasis on morphologic changes suggestive of HPV involvement, being classified into flat, inverted (or endo- phytic), and papillomatous (or exophytic) condylomas in accordance with the criteria described previously (26, 28, 29,39). . t m

Human Papillomavirus in Esophageal Cancer 555 HPV-DNA in situ hybridization In situ DNA hybridization on formalin-fixed, paraffin- embedded tissues, using j5S-labeled HPV DNA probes, was done as described by Syrjanen & Syrjanen (54). In brief, 4- µm-thick sections were cut from each specimen and mounted on microscopic slides pretreated with 1% aminopropyl- triethoxysilane (Sigma, St. Louis, Mo., USA). Sections were deparaffinized in xylene, rehydrated through graded ethanol, and digested with proteinase K. The specimens were hybridized in a mixture of 50% formamide, 10% dex- tran sulfate, 0.6 M NaCI, 0.01 M Tris-HCI (pH 7.4), 0.5 mM ethylenediaminetetraacetic acid (EDTA), 1 mg/ml bovine serum albumin (BSA), 0.02% Ficoll, 0.02% polyvinyl- pyrrolidone (PVP), 0.2 mg/mi salmon sperm DNA, 0.5 mM dithiothreitol, and 0.2 ng/l,tl of 35S-labeled HPV 6, 11, 16, or 18 DNA probes for 48 h at high-stringency conditions (Tm-17). Electron microscopy TEM on paraffin-embedded materials was performed as described by Widehn & Kindblom (55) with some modi- fications. In brief, the areas shown to be HPV-positive in the in situ hybridization were marked, and the corresponding regions in the paraffin block were cut out with a thin razor blade. The portions of interest were deparaffinized in xylene, rehydrated through graded ethanol, and postfixed in 1% Os04 in phosphate buffer for 1 h. After dehydration the specimens were embedded in epoxy resin and polymerized at 60°C overnight. Sections 1 µm thick were cut and stained with 0.1% toluidine blue and checked under the light micro- scope to confirm the correct regions. Ultrathin sections were subsequently cut on an LKB ultratome, double-stained with uranyl acetate and lead citrate, and examined under a Hitachi-700 electron microscope. Polymerase chain reaction The 51 formalin-fixed, paraffin-embedded biopsy speci- mens were further analyzed with PCR. One to several 7-µm sections with an average surface area > 1 cmZ were cut from each block. The sections were put directly into Eppendorf tubes and deparaffinized by adding 400 µl of xylene twice, followed by two washes with absolute ethanol and one with acetone. After air drying, 50 µl of sterilized double-distilled water was added to each tube. Sample DNAs were denatured at 100°C for 10 min before the PCR amplification. The primers chosen from the E5 or Ll regions of HPV 6, 11, 16, and 18 were synthesized and used as described previously (21, 46, 56, 57). Forty cycles of amplification of HPV target sequences were carried out in 50 µl of the reaction mixture with the Gene Ampli Taq kit and the Perkin Elmer-Cetus automatic thermal cycler (Perkin Elmer-Cetus, Emeryville, Calif., USA). Each cycle involved heating at 95°C for 30 sec (DNA denaturation), followed by cooling at 55°C for 30 sec (primer-annealing), and finally heating at 72°C for 1 min (chain elongation). Ten microliters of the amplified product were electrophoresed in a 3% NuSieve agarose gel, and the DNAs were visualized in the gel by ethidium bromide staining. The specific HPV DNA sequences amplified by Fig. 1. hligh-power view of esophageal dysplastic epithelium adjacent to a squamous cell carcinoma presenting as flat condylomatous lesion. The cells in the intermediate and superficial layers show extensive koilocytotic changes (hematoxylin and cosin; magnification, x 100).

d 556 F. Chang et al. Fig. 2. Human papillomavirus DNA in situ hybridization of esophageal specimens under high-stringency conditions. Black silver grains are superimposed on nuclei of numerous koilocytotic cells in the intermediate and superficial layers of hyperplastic epithelium adjacent to the carcinoma (hematoxylin counterstain; magnification, x 100). the PCR were subsequently confirmed by Southern blot hybridization and dot blot hybridization with 32P-labeled specific HPV probes as described earlier (21, 46, 57). Southern blot hybridization Fresh esophageal biopsy specimens derived from both of the carcinoma foci and their surrounding tissues were homogenized in mortars and lyzed in 10 mM Tris-HCI (pH 7.5), 0.1 M NaCl, 10 mM EDTA, 0.5% sodium dodecyl sulfate (SDS), and 0.5 mg/ml proteinase K. The total cellular DNAs were extracted by phenol-chloroform-isoamyl alcohol extraction and precipitated with ethanol (58). Five to ten Table II. Frequency of human papillomavirus (HPV) DNA in the esophageal specimens detected by in situ hybridization and polymerase chain reaction (PCR) In situ hybridization PCR HPV types No./total % No./total % 6 3/51 5.9 3/51 5.9 11 3/51 5.9 7/51 13.7 16 8/51 15.7 8/51 15.7 18 6/51 11.8 6/51 11.8 11/18 1/51 2.0 0/51 0 16/18 1/51 2.0 1/51 2.0 Total 22/51 43.3 25/51 49.0 micrograms of cellular DNAs were cleaved with Pstl, electro- phoresed in 0.8% agarose gels, and transferred onto nylon membranes. The filters were prehybridized in a solution containing 30% formamide, 5x sodium chloride, sodium citrate buffer (SSC), 50 mM NaPP, 1% SDS, 5x Denhardt's solution, and 500 µg/ml t-RNA at 42°C for 6 h at low- stringency conditions (Tm-35). Under the same conditions the filters were subsequently hybridized with a mixture of 106 cpm/ml each of HPV 11-, 16-, 18-, and 30-labeled DNA probes for 36 h. These filters were washed thrice at 56°C for 30 min each in 2x SSC/0.1% SDS and exposed to X-ray films for 3 days at -70°C with two intensifying screens. After autoradiography the radioactive-labeled probes were washed off with 0.4 N NaOH at 42°C for 30 min, followed by 0.1 x SSC, 0.1% SDS, and 0.2 M Tris-HCI (pH 7.5) once at 42°C for 30 min. The filters were then prehybridized and hybridized under high-stringency con- ditions (Tm-20) by increasing the concentration of form- amide from 30% to 50%. The filters were washed three times at 68°C for 30 min in 2 x SSC/0.1% SDS and exposed to X-ray films again for 3 days at -70°C with two intensifying screens. l RESULTS ~- - Condylornatous lesions in the esophagus The patients with esophageal cancers in the present series included 39 (54.9%) men and 32 (45.1%) women. The age W 4t:- ~

Fig. 3. Electron micrograph documenting intranuclear papil- lomavirus-like particles (*) in selected esophageal squamous epi- thelial cells, which have been demonstrated to contain human papillomavirus DNA sequences by in situ hybridization beforehand. N = nucleus; NM = nuclear membrane (transmission electron microscopy; magnification x 25,000). Fig. 4. Polymerase chain reaction amplification on paraffin-embed- ded tissue sections from esophageal carcinoma specimens with HPV 11-specific primers. The products wcrc electrophoresed on a 3% agarose gel stained with ethidium bromide. Lane A = DNA marker; lanes B and C = negative and positive controls; lanes D-R = esophageal specimens. Human Papillomavirus in Esophageal Cancer 557 of the patients ranged from 26 to 78 years, with a mean age of 54.6 years. Of the 51 cases examined on light microscopy, 28 cases were graded as well-differentiated, 17 cases as moderately, and 6 cases as poorly differentiated squamous cell carcinomas. Twenty-five of the 51 cases (49.0% of the total) were diagnosed as having HPV-suggestive lesions. Morphologic changes consistent with flat condyloma were seen in 25 of the 51 cases (49.0%), and 2 cases showed both the flat and inverted changes. No true squamous cell papilloma was identified among these cases (Table 1). The HPV-suggestive lesions were frequently found in the epithelia adjacent to the carcinomas, morphologically showing hyperplasias or dysplasias (Fig. 1). In general, the frequency of the mor- phologic changes suggesting HPV infection decreased with increasing epithelial atypia. HPV DNA demonstrated by in situ hybridization Twenty-two of the 51 specimens (43.1%) contained at least one type of HPV 6, 1 b, 16, and 18 DNA sequences. M N 0 Fig. 5. Autoradiogram of Southern blot of the polymerase chain reaction products corresponding to Fig. 3 after hybridization with ''P-labcled HPV 11 DNA probe. Signals indicating the presence of HPV I l DNA sequences in the esophagcal lesions are detected over lanes E, G, and Q. tV ~ C7 ' C1'+ 00

558 F. Chang et al. Among the 25 diagnosed as HPV-suggestive lesions by his- topathology, 16 showed HPV DNA-positive hybridization when further analyzed by in situ hybridization. HPV 16 was the most frequent type, being found in 8 of the 51 specimens (15.7%), followed by HPV 18 (11.8%), HPV 6(5.9%), and HPV 11 (5.9%). Double infections were noted in two cases, one with HPV 11/18 and the other with HPV 16/18. Alto- gether, the high-risk HPV types 16 and 18 DNA sequences were found in 16 (72.7%) of the 22 HPV DNA-positive cases. In most cases the HPV-positive signals were localized in the hyperplastic and/or dysplastic epithelia adjacent to invasive. carcinomas (Fig. 2). Two cases harbored HPV 6 or 18 DNA in malignant cells. In the latter case, HPV 18 DNA was simultaneously located both in the dysplastic area and in the cancer cells. The results of HPV DNA detection in the specimens are summarized in Table II. When the localization of HPV signals was correlated with the epithelial lesions, HPV DNA was detected in a total of 36.1% (13 of 36) of hyperplasia, 22.2% (8 of 36) of dysplasia, and 3.9% (2 of 51) of carcinoma lesions. Papillomaoirus-like particles on TEM On TEM, some structural details of the cells were lost because the specimens had been fixed in formalin. However, papillomavirus-like particles with round shape and about 50 nm in diameter were recognizable in the nuclei of squa- mous cells in two of five of the selected specimens (Fig. 3). These virus particles appeared as scattered or small aggregates throughout the nucleus. They were seen exclus- ively in the epithelial cells adjacent to the carcinoma foci with morphologic changes suggestive of HPV infection, par- ticularly in the nuclei of koilocytotic cells. These cells had a A B C 0 E F large perinuclear zone almost completely lacking cyto- plasmic components. At the periphery of the cells there was a narrow band of cytoplasmic debris condensed into a network of clumps and strands. HPV DNA detected by PCR Altogether, 51 specimens were analyzed for the presence of HPV DNA by the PCR, using the type-specific primers of HPV 6, 11, 16, and 18. When compared with the HPV- positive lesions of the genital tract, the amplified products of the esophageal lesions were more difficult to discern directly from the gels as visualized by ethidium bromide, because of the presence of some non-specific DNA bands. However, the positive signals could easily be recognized after the amplified products were detected by Southern blot and dot-blot hybridization using 3'-P-labeled specific HPV DNA probes. Figs. 4 and 5 show the results of 15 samples amplified with HPV 11 primers, in which the specific positive signals (for example, lanes E, G, and Q in Fig. 4) were confirmed by Southern blot hybridization (Fig. 5). Fig. 6 shows the result of PCR amplification among the 51 samples with HPV 6/11 primers and hybridization with the same types of HPV DNA probes. Of the 51 samples, a total of 25 (49.0%) were finally shown to contain at least one type of HPV 6, 11, 16, and 18 DNA sequences, including 22 samples previously positive by in situ hybridization and 3 samples negative by in situ hybridization. In one specimen a double infection with HPV types 11 and 18 was first demonstrated by in situ hybridization, whereas only HPV 11 DNA sequences were amplified by the PCR. The comparison of the results from in situ hybridization and the PCR is summarized in Table II. ~ 7 8 9 10 11 12 Fig. 6. Dot blot of specimens amplified with HPV 6 and I 1 primers, probed with `=P-labeled HPV 6/11 probes. A 1-A4, I-IPV DNA standards; B 1, positive control; B2, negative control; and B3-F5, esophageal samples. Samples B6, C5, C10, C12, D2, El, E3, E5, and E12 show distinct positive hybridization for I-IPV 6/11.

Human Papilloinavirus in Esophageal Cancer 559 HPV DNA detected by Southern blot hybridization Of the 20 DNA samples derived from esophageal car- cinomas and their surrounding tissues, 9 were shown to contain HPV DNA sequences when analyzed by Southern blot hybridization under low-stringency conditions (Tm-35), and 8 specimens remained positive with a probe cocktail of HPV 11, 16, 18, and 30 under high-stringency conditions (Tm-20) as well. The hybridization profiles of 10 specimens under low- and high-stringency conditions are shown in Figs. 7 and 8. Positive bands ranged from 3 to 21 kb and were mostly located at 3-5 kb. The digestion with Pstl did not yield any single band in these samples, and no Pstl pattern could be completely assigned to those of HPV 11, 16, 18, or 30, although certain samples (for example, lanes 3, 4, and 12 in Fig. 7) showed the Pstl enzyme pattern resembling that of the episomal form of HPV 30. DISCUSSION Although substantial evidence has been provided during the past few years implicating an etiologic role of HPV infection in anogenital and upper aerodigestive lesions (18-25, 47- 52), less data are so far available on the role of HPV infection in esophageal lesions. Our results indicated that HPV infec- tion in the human esophagus does occur and, indeed, seems 2.3-. 2.U-. 0.5 to be remarkably prevalent in the high-risk area for esopha- geal cancer in Henan Province of China. The histopathologic features and viral patterns of esophageal HPV infection are comparable with those described at other mucosal sites (18- 25, 47-52). HPV infection was involved in 49.0% (25 of 51) of the specimens derived from esophageal squamous cell carcinomas when analyzed histopathologically. All changes regarded as suggestive of HPV infection were found in the esophageal epithelium. As in the genital tract, such lesions were mostly of the flat type. Exophytic lesion-that is, squamous cell papilloma-seems to be a rare lesion, with 0.1%o-0.04% morbidity as estimated on the basis of autopsy data (59). In the present series no true papillomas were found. These data suggest that most esophageal HPV lesions must have been overlooked by endoscopists and pathologists in the past. The presence of HPV infection in the esophagus was confirmed in the present study by in situ hybridization. A total of 22 of 51 (43.1%) of the specimens derived from esophageal carcinoma were shown to contain at least one type of HPV 6, 11, 16, or 18 DNA sequences. Noteworthy is the finding of the high-risk HPV types 16 and 18 in 16 (72.7%) of the 22 HPV DNA-positive cases. The higher prevalence of these high-risk HPV types in the present series of esophageal precancerous and cancerous samples derived Fig. 7. Autoradiograph of Southern blot of the DNA samples extracted from fresh esophageal carcinoma specimens and cleaved with Pstl. The filter was hybridized with a mixture of "P-labelcd HPV 11, 16, 18, and 30 DNA probes under low-stringency conditions (Tm-35). Lane 1, HPV 16 DNA; lane 2, IIPV 11 DNA; lane 3-12, esophageal samples. O ~

560 F. Chang et al. Fig. 8. Autoradiograph of the same filter as in Fig. 7 after hybridization with a mixture of'zP-labeled HPV 11, 16, 18, and 30 DNA probes under high-stringency conditions (Tm-20). from the high-risk population might be correlated with the high morbidity and mortality of this malignancy in this area. The demonstration of papillomavirus-like particles in the HPV-suggestive and in situ HPV-positive lesions of the esophagus confirms HPV involvement in these cases. The presence of viral particles in benign esophageal lesions sug- gests the existence of productive infection, which leads to the synthesis of episomal viral DNA, the expression of late viral genes that encode capsid proteins, and the assembly and release of viral particles. The permissive infection of HPV in benign esophageal lesions was also suggested by the previous demonstration of HPV structural proteins in squamous cell papillomas (28-30), focal epithelial hyper- plasia (29), and epithelial lesions associated with esophageal precancerous and cancer lesions (31, 32, 34, 36). In full align- ment with the present results are the recent findings of genus- specific HPV antigens in 23% (7/23) of esophageal lesions adjacent to invasive carcinomas derived from the same high- incidence area of Henan Province of China, substantiated by the additional demonstration of 13% IIPV-antigen pos- itivity in a Japanese series (36). PCR has recently been applied to HPV DNA detection in formalin-fixed, paraffin-embedded biopsy specimens from genital lesions, and it has been shown to be more sensitive than other methods (56, 57, 60). With this technique 49.0% (25 of 51) of the present series of esophageal carcinomas were demonstrated to contain at least one type of HPV 6, 11, 16, and 18 DNA sequences. In addition to specific HPV bands, non-specific bands were found in several cases. The reason for this occurrence of a higher frequency of non- specific bands in'esophageal samples as compared with the genital lesions is unclear. It might well be due to the presence of new HPV types or subtypes which are not exactly identical with the prototypes of HPV 6, 11, 16, and 18 DNA sequences in these samples. The higher frequency of HPV 11 DNA detected by PCR suggested that this viral type was present at low copy number in esophageal precancerous lesions and carcinoma. The present results with Southern blot hybridization are fully consistent with the recent reports by Ni et al. (61) and Li et al. (62), who found HPV DNA in 57.1% (12 of 21) and 50% (12 of 24) of esophageal carcinomas derived from the population at high risk for esophageal cancer in China. Of interest was our frequent finding that the restriction enzyme patterns of HPV DNA were different with the episomal prototypes of HPV 6, 11, 16, 18, and 30. This could indicate the integration of viral DNA into cellular genomes. Similar observations have been demonstrated in the genital tract, where malignant tumors regularly contain HPV DNA in an integrated state, whereas the viral DNA is always present in episomal form and in multiple copies in benign lesions (63, 64). Therefore, the integration of viral DNA f O

into cellular DNA has been regarded as an important event in HPV oncogenesis. However, our results cannot rule out the possible presence of unidentified HPV types or subtypes in esophageal lesions (63, 64). There is some evidence to suggest that an HPV 16-related virus, which differs from the HPV 16 prototype in its enzyme pattern, is more closely related to oral precancerous lesions and carcinomas (65-67). This virus has recently been found in more than 80% of oral leukoplakias and in 46% of the carcinomas from the tongue and the floor of the mouth (66, 67). Therefore, further studies should be focusECd on identifying additional HPV types or subtypes in esophageal lesions. HPV infections of the anogenital tract are known to be sexually transmitted (48-52). However, the route by which HPV reaches the esophageal mucosa is not known as yet. Several conceivable ways have been proposed for the occur- rence of laryngeal and oral HPV infections. These include intrapartum infection during the passage through the infected birth canal, transplacental infection in utero before birth, and postnatal infection by contact with infected indi- viduals (25, 68--70). The recent findings of HPV DNA in the foreskin of normal newborns (58), in the oral and pharyngeal cavity of a large percentage of neonates vaginally delivered from HPV-infected mothers, and in the amniotic fluid (69, 70) favor the mechanisms for HPV transmission at birth. Recently, HPV DNA sequences were also found in some fetal esophagi in the high-risk areas for esophageal cancer in China (62, 71), suggesting that similar mechanisms may exist. The present results disclosing the same viral types- that is, HPV 6, 11, 16, and 18-as frequently associated with the genital lesions, in the esophagus further support the hypothesis that esophageal HPV infection may be acquired from infected mothers, either by intrapartum mechanisms or by the transplacental route. In parallel to that of human beings, cancer of the upper alimentary tract in cattle also shows a striking geographic distribution in the world. Studies in western Scdtland have shown that BPV 4 is the causative agent of papillomas of the alimentary canal, which frequently undergo malignant transformation after exposure to carcinogens and immuno- suppressants present in ingested bracken fern (14-17). Recently, Gaukroger et al. (72) observed one case of bovine papilloma induced by BPV 4 in the nude mouse renal capsule, which had progressed into an invasive squamous cell carcinoma with spleen metastasis. BPV 4 DNA in high copy number could be readily identified in the papillomas either naturally occurring or experimentally induced, but no viral DNA or viral antigens were detected in the carcinomas (14-17, 72), indicating that the viral genomes are not always required for maintenance of the malignant state. These data suggest that a) BPV 4 may perform one of the early events in cell transformation, and its genetic information may not be required for malignant progression; b) immunosuppression allows the spread and the persistence of BPV 4-induced papillomas; and c) bracken fern would supply carcinogens Hnman Papillomauirus in Esophageal Cancer 561 and/or cocarcinogens, leading to full transformation and progression (14-17, 72). Many similarities apparently exist between the patho- genesis of bovine alimentary papilloma-carcinoma sequence and human esophageal cancers. Both of these lesions show remarkable geographic distributions. In many instances, a high incidence of particular malignancy in well-defined geo- graphic areas appears to be associated with infectious etio- logic factors. This has been shown with hepatitis B virus, Epstein-Barr virus, and human T-lymphotropic virus (73- 78). The same is true of upper alimentary tract cancer in cattle, in which the higher incidence is ascribed to the synergistic actions between BPV 4 and the carcinogenic factors in bracken (14-17). Similarly, a high prevalence of papillomavirus infection has been recognized in the high- risk areas, such as in South Africa (31, 32, 35, 41), Alaska natives (42), and China (39, 40, 61, 62, 71). Our results show that the HPV DNA detection rate in esophageal carcinomas was much lower (3.9%) than that in hyperplastic (36.1%) and dysplastic (22.2%) lesions adjacent to carcinomas. This is fully consistent with the presence of BPV 4 DNA in papillomas and its absence in the frank malignancies. Cur- rent data suggest that HPV infections are necessary but insufficient in the development of human cancers, synergistic actions with other factors evidently being also required (47- 52). In addition to HPV infection, as discussed above, other high-risk factors including chemical carcinogens, physical trauma, and nutritional deficiencies have been associated with esophageal cancer in high-incidence areas (1-13). Therefore, the established mechanisms for the pathogenesis of bovine alimentary carcinomas may also be applicable to human beings, with regard to the pathogenesis of esophageal squamous cell carcinomas. In conclusion, by using light microscopy, electron mi- croscopy, in situ DNA hybridization, Southern blot hybrid- ization, and polymerase chain reaction techniques, we have demonstrated the presence of IIPV-suggestive lesions, pap- illomavirus-like particles, and HPV DNA sequences in a high percentage of esophageal precancerous and cancerous lesions derived from the high-incidence area of esophageal cancer in China. Our results confirm the involvement of IIPV in esophageal squamous cell lesions and suggest that HPV infections might be importantly involved in the patho- genesis of human esophageal squamous cell carcinomas, most probably acting synergistically with the other car- cinogens that have previously been associated with this malignancy. ACKNOWLEDGEMENTS This study has been supported by an award from the Inter- national Union Against Cancer (UICC) (for F. Chang) and in part by a research grant from the Finnish Cancer Society, a research contract (no. 1041051) from the Medical Research

562 F. Chang et al. Council of the Academy of Finland, and a joint research grant from Fabriques de Tabac Reunies S.A., and British- American Tobacco Company Ltd. The skillful technical assistance of Ms. Pirkko Karttunen, Mrs. Kaarina Hoffren, Ms. Helena Kemilainen, Mrs. Eija Sedergren-Varis, and Mrs. Ritva Sormunen is gratefully acknowledged. The authors extend their special thanks to Prof. Dr. Lutz Gissmann, Prof. Dr. Harald zur Hausen, DKFZ, Heidel- berg, Germany, and Prof. Gerald Orth, Pasteur Institute, Paris, France, for placing the HPV DNA probes at our disposal. REFERENCES 1. Coordinating Group for Research on Etiqlogy of Esophageal Cancer in North China. The epidemiology and etiology of esophageal cancer in China. Chin Med J 1975;1:167-83. 2. Li M, Li P, Li B. Recent progress in research on esophageal cancer in China. Adv Cancer Res 1980;33:173-249. 3. Yang CS, Research on esophageal cancer in China: a review. Cancer Res 1989;40:2633-44. 4. 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