Nasopharyngeal Carcinoma: Recent Studies and Outlook for a Viral Etiology1

[CANCER RESEARCH 34, 1196-1206, May 19/4J Nasopharyngeal Carcinoma: Recent Studies and Outlook for a Viral Etiology1

Guy de-ThÃ©2and Anton Geser

International Agency for Research on Cancer, WHO, 150 Cours Alben Thomas, 69008, Lyon, France

Summary (8, 15, 31) make it a unique cancer in which one can investigate the respective roles of genetic and environmental Recent studies have brought new elements in assessing the factors (viruses and/or chemical carcinogens). role of the lymphotropic Epstein-Barr virus in the develop As the approach used in experimental viral oncology ment of nasopharyngeal carcinoma (NPC). In electron cannot be applied to humans, and as studies involving microscopy, we noted close contacts between lymphoid and humans are usually long and costly, the integration of epithelial cells in the normal nasopharyngeal mucosa, in epidemiological and laboratory studies is becoming a prom NPC biopsies, and in the in vitro growths derived from the inent path for the investigation of the relationship between NPC biopsies. Such close contacts with occasional loss of viruses and cancer in man. Table 1gives an example of such adjacent cell membrane continuity suggested that cell multiphasic integrated field and laboratory programs de cooperation might play an important role in NPC develop signed to study the role of the EBV in NPC and BL (10). ment if a lymphotropic virus is involved. The recent finding of a relatively high incidence of NPC In serology, we observed that NPC sera exhibited 10-fold in Tunisia (1, 62) makes it possible to conduct a compara higher antibody titers than Burkitt's lymphoma sera, tive study between 2 ethnic groups and geographical areas, against a soluble complement-fixing (CF) antigen. Further which might lead to the differentiation between important more, while CF antibody titers fluctuated in Burkitt's and unimportant factors in the development of NPC. lymphoma patients according to the clinical evolution, they We shall present recent laboratory studies and ongoing were very high and stable in NPC patients regardless of the seroepidemiological programs on NPC and discuss the stage of the disease and of the clinical evolution. CF hypotheses that can be formulated for the etiology of NPC, antibodies did not appear to be related to viral capsid or and then conclude with what we consider to be immediate early antigen activities. Preliminary results indicate the research priorities. presence of CF nuclear antigen in NPC biopsies. Our ongoing seroepidemiological program, although not Recent Laboratory Studies and Ongoing Seroepidemiolog completed, shows that adult Chinese males have lower viral ical Program capsid antigen antibody titers and higher CF antibody titers (against a soluble antigen) than Indian males. Etiological The debate on the possible role of a lymphotropic hypotheses and research priorities are presented which herpesvirus in the development of a carcinoma (NPC) include: completion and analysis of the ongoing field benefited from recent ultrastructural, serological, and mo studies, boosting of the laboratory investigations, i.e., the lecular virological studies. development of an experimental model, the assessment of Electron Microscopy. The epithelial nature of all histolÃ³g serological reactivities to disease development and continu ica! varieties of NPC has been established by Svoboda et al. ation of immunogenetic studies in Singapore. (57). We confirmed these studies (21) since, in the most undifferentiated tumors, the presence of cytoplasmic bun Introduction dles of tonofibrils and of desmosomes testified to the epithelial nature of the tumor cells. Our attention was The epidemiological characteristics of NPC3 (35, 54) (a focused on the relationship between epithelial cells and tumor mainly restricted to the Cantonese Chinese wherever lymphoid elements in the normal nasopharyngeal mucosa, they live in the world) and the serological association that in the NPC biopsies and, more recently, in the in vitro exists between this tumor and a DNA herpesvirus-type EBV growths leading eventually to lymphoblastoid transforma 1Presented at the International Symposium on Human Tumors Associ tion and establishment of permanent cultures (58). We ated with Herpcsviruses, March 26 to 28, 1973, Bethesda, Md. Supported found a close association between lymphoid cells and the by Contract NIH-NCI-E-70-2076, within The Special Virus Cancer epithelial cells of the basal layer of the normal nasopharyn Program, National Cancer Institute, NIH. geal mucosa. Similarly, in NPC, numerous lymphocytes 2Presented by. and a few lymphoblastoid cells were seen in close contact 'The abbreviations used are: NPC, nasopharyngeal carcinoma; EBV, Epstein-Barr virus; BL, Burkitt's lymphoma; VCA, viral capsid antigen; with the epithelial cells (Fig. 1) to such an extent that loss of GMT, geometric mean titer; EA, early antigen(s); CF, complement-fixing; adjacent membrane continuity was occasionally observed EA/R, EA detected by restricted staining; EA/D, EA detected by diffused (21). In NPC cultures, in which growth of epithelial cells stainine: EB. Epstein-Barr. was obtained (17), close association between epithelial and

1196 CANCER RESEARCH VOL. 34

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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1974 American Association for Cancer Research. Guy de-ThÃ©andAnton Geser lymphoid cells was also noted (Fig. 2). Later on, when tion by 5-iododeoxyuridine or 5-bromodeoxyuridine (19, fibroblastic elements dominated the cultures, a few lymph 22). According to G. Henle et al., 2 types of EA can be oid elements were present in close contact with or engulfed detected: an EA/R that disappears after methanol fixa in these fibroblasts. This special relationship between tion, and an EA/D that is resistant to this fixative and is lymphoid and fibroblastic cells was the rule at the time of best seen after acetone fixation. In NPC sera, antibodies lymphoblastoid transformation (Fig. 3), when the newly directed against anti-EA/D are usually present, although emerging lymphoblastoid cells were still dependent on the low levels of anti-EA/R are detectable while, in most BL presence of attached fibroblastoid cells acting probably as a patients, the dominant antibodies are directed against feeder layer. Close contact and apparent discontinuity of EA/R. In infectious mononucleosis patients, a transitory adjacent cell membranes between lymphoid cells and both anti-EA/D antibody is generally observed in two-thirds of epithelial and fibroblastic elements suggested that cell the patients (32). Recent results presented at this meeting by cooperation between these different cell types might be W. Henle et al. (33) show that the NPC sera have increasing highly relevant for tumor development, in view of the EA/D antibody titers from stages I to IV of the disease. association between a lymphotropic virus and NPC. When they studied long-term survivors, they found low Serology. NPC sera have immunological reactivities anti-EA/D reactivities with moderate titers of antibodies quite similar to those of BL sera (11, 40). Henle et al. (31), directed against an EA/R (W. Henle, presented at this using the EB3 cell line as antigen, showed that Chinese, meeting). African, and Caucasian NPC sera had titers against VCA Using a soluble CF antigen extracted from the virus-pro very similar to those of BL sera and much higher than those ducing QMIR-WIL cell line of Pope (51), we observed of other tumor-bearing patients presented at Ear, Nose and unexpected differences between NPC and BL sera (55). We Throat Clinics. In our laboratory, with the use of the Jijoye compared the evolution of CF titers in both diseases, before cell line as antigen, the VCA titers were somewhat higher and at different intervals after treatment. BL patients than those obtained by Henle et al. (31), but the ratio showed an increase in CF titers with clinical improvement between NPC and other tumors in Chinese was similar. As (56). In NPC patients, the CF titers were found to be an example, in a recent series, NPC sera had a GMT of 10-fold higher than in BL patients and showed an overall 1:1153, whereas the GMT's for other tumors varied from stability during the course of the disease (Ref. 18;Table 2). 1:124 for sera from cancers of the bronchus, to 1:140 for Before radiotherapy, CF titers were not related to the sera from cancers of the cervix, and to 1:110 for normal stage of NPC (CF:GMT = 167, 156, 309, and 181 in stages Chinese individuals (unpublished data). I, II, III, and IV, respectively). This contrasted with the NPC sera, like BL sera, possess antibodies against increasing value of VCA:antibody titers noted in NPC sera membrane-bound antigens detectable on both BL- and from stage I to stage IV (9, 18). After radiotherapy, NPC N PC-derived cell lines (8, 11) but, in contrast to the patients with a regressive tumor showed a moderate de situation in BL (45), these membrane-bound antigens are crease in CF titers, which might be an indication of not detectable in NPC tumor biopsies. NPC sera and BL localization of the soluble CF antigen in the tumor mass. sera also have in common immunoprecipitating antibodies Statistical analysis of the relationship between CF and VCA against an antigen extracted from the Jijoye line (47, 48), titers in NPC and BL sera showed that there was no but when 2 precipitating lines were observed with BL sera, 3 correlation between these 2 antibody reactivities (r = 0.064 were obtained with NPC sera (47). and r = 0.083, respectively, in the arrested and progressive Recent studies on antibodies directed against EBV- NPC tumor groups). The above results suggested that, while induced EA and CF antigens appear to be of significance in BL would behave like an acute viral infection (56), NPC differentiating between NPC and BL reactivities to EBV. would behave as though a long-standing infection was Both NPC and BL sera possess antibodies directed against taking place before the development of the clinical disease EA detectable in nonproducing lymphoblastoid lines, after (18). superinfection by semipurified virus (26-28) or after induc Recent studies using the anticomplement immunofluores-

Table 2 Comparison of the GMT of CF and VCA liters in BLÂ°andNPC" patients before and after treatmentNPCpatientsBL

treatment327 treatment161 treatment640710258treatment359806278

UncontrolledControlled2311736GMTAfter2441079VCABefore

patientsTumorsControlled UncontrolledCFBefore 490:GMTAfter 835 ' cf. RÃ©f.56(29 patients, 82 sera). "cf. RÃ©f.18(38 patients, 158 sera).

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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1974 American Association for Cancer Research. Recent Studies and Outlook on NPC cent test of Reedman and Klein (53) on touch smears of and his associates are investigating the human leukocyte fresh NPC biopsies showed that 15 to 50% of the lymphoid antigen pattern, as well as that of other immunogenetic cells and possibly some epithelial tumor cells were positive, markers in NPC patients versus the normal population. As i.e., contained the EBV nuclear antigen. the results to be presented by Dr. Simons at this meeting Tissue Culture. When NPC biopsies were put in culture, 4 will show, NPC patients have a strikingly different HL-A different types of growths were successively obtained (17). pattern from comparison groups. The pattern corresponds In the 1st phase, epithelial outgrowth took place simultane to a relative risk factor of approximately 5. ously with an early lymphocytic production. The latter A similar HL-A study on NPC patients and controls appeared to be related to the multiplication of the infiltrat from Tunisia is being carried out in collaboration with Dr. ing lymphoid cells from the tumor mass. Later, fibroblas- Betuel and Dr. Dausset. An international comparative study toid cultures developed and survived for as long as 400 days from various geographical areas with standardized sera and led eventually to lymphoblastoid transformation in should be conducted so that the results from these 2 approximately 30 to 40% of the NPC cultures (15, 17). The locations, as well as from others (United States, China), can presence of EBV infection in NPC-derived lymphoblastoid be compared. lines was shown by electron microscopy, immunofluores- Seroepidemiology. As presented in Table 1, the knowl cence, or nucleic acid hybridization tests. The NPC-derived edge of the epidemiological behavior of EBV in populations cell lines behaved in a manner quite similar to that of the at different risk of EBV-associated diseases (BL in African lines derived from BL biopsies and also from circulating children, NPC in adult Chinese males, and infectious lymphocytes in patients with infectious mononucleosis, mononucleosis in young Caucasian adults) is essential to leukemias, or from lymph nodes of tumorous or nontumor- design testable hypotheses for the role of EBV in human ous patients (20, 43, 45, 50). The establishment of perma tumors. As an example, the epidemiological behavior of nent lymphoblastoid lines therefore does not reflect a poliovirus in populations at different risk of poliomyelitis tumoral state of the starting material but rather the was essential for an understanding of the disease and the presence, in the originating material, of lymphoid cells development of the vaccination program. We are convinced infected by a herpesvirus having "transforming activities" that this applies to EBV-associated diseases. The sero- (16, 17, 45). Indeed, the herpesvirus purified from NPC- epidemiological program carried out in Hong Kong, Sin derived lymphoblastoid lines showed "transforming activi gapore, Uganda, and France (12), where representative ties in vitro" when cord blood lymphocytes or marmoset population samples are interviewed and bled, should give lymphocytes were used (G. de-ThÃ©,Hilgers, and C. Des- basic information on prevalence, incidence, and possibly granges, unpublished data) in a manner quite similar to that mode of transmission of the EBV infection in different observed with the EBV and BL (3, 23, 29, 42, 52, 59). geographical areas and among different ethnic groups. Our recent electron microscopic study on the various In such seroepidemiological surveys, it is hazardous to NPC growths in vitro leading to lymphoblastoid transfor give preliminary results, as they may not be confirmed when mation supported the hypothesis that lymphocytes closely the study is completed. Should we say, however, that associated with epithelial cells and later with fibroblastoid Chinese and Indo-Pakistanese in Singapore appear to cells survived up to the time of lymphoblastoid transforma behave differently with regard to VCA antibodies? From the tion. Such close association, with eventual loss of adjacent analysis of VCA titers in 709 sera from Singapore (332 cell membrane continuity, suggested that cell cooperation Chinese, 189 Indian, and 188 Malays), it appears that the was essential for establishment of permanent cultures in proportion of sera with titers >80 is significantly lower in vitro and possibly for tumor development in vivo (58). adult Chinese males than in Indian males (p < 0.001) or in Molecular Virology. The presence of EB viral DNA Malays (p < 0.01). These differences might point at genetic sequences has been demonstrated in biopsies from NPC and differences in susceptibility to virus infection or might be BL, as well as in derived lymphoblastoid cultures (46, 63, explained by differences in environmental or cultural fac 64). Whether the EB DNA is present in epithelial or in tors. Dr. Vonka in Prague is titrating these sera for CF lymphoid cells in NPC is still an open question. Wolf et al. antibodies against 5-antigen. Preliminary results indicate (61) recently claimed the presence of EBV genomes mostly differences between Chinese and Indians, the former having in epithelial cells from NPC biopsies, while we found EB significantly higher titers than Indians. It is essential to nuclear antigen mostly in lymphoid elements close to tumor await the completion of the seroepidemiological study epithelial cells (13). Further studies are obviously needed in before making any conclusions from these preliminary this area, as the steady association of EBV and epithelial findings. cells would sustain the hypotheses of direct involvement of The serum collection and serological testing should be EBV in the transformation of epithelial cells to induce completed next year, and the statistical analysis will help to NPC. determine the level of differences between Chinese, Indians, The recent findings of Kufe et al. (41) in NPC and BL and Malays living in Singapore. We also hope to evaluate biopsies of RNA sequences homologous to that of the precisely the possible difference in prevalence and incidence Rauscher leukemia virus RNA opened a new and exciting of infection between East Africa (Uganda) and South East field of investigation, and these results merit confirmation. Asia (Singapore and Hong Kong). Preliminary results from Immunogenetics. The epidemiolÃ³gica!characteristics of Hong Kong (H. C. Ho and Kwan, unpublished data) NPC suggest that genetic factors are important for the suggest that Chinese children of 1 year of age or less might development of the tumor (35, 36, 54). Dr. Malcolm Simons have lower anti-VCA tilers than African children in Uganda

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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1974 American Association for Cancer Research. Guy de-ThÃ©and Anton Geser (38). Great caution must prevail in the interpretation of such and controls in Hong Kong, Singapore, Tunis, and Los results, as the sera were tested in different laboratories, with Angeles. different cell lines. Since the virus is lymphotropic, it might be postulated In connection with the ongoing comparative EBV studies, that it lies dormant in the lymphoid cells of the nasopha- systematic efforts are made to standardize the laboratory ryngeal mucosa and that the virus-containing lymphoid cells methods for VGA antibody determination. VGA titers are stimulated to produce specific antibodies by the neigh obtained on the same sera in the Henle's laboratory in boring tumorous epithelial cells. If this were the case, it Philadelphia, our laboratory in Lyon, and the cooperating would be difficult to explain why NPC sera have high im- laboratories in Hong Kong and Singapore revealed consid munological reactivities against the virus, whereas sera erable variation of results. Subsequent duplicate testing has from patients with carcinoma of the tonsils and back of the enabled us to estimate the relative magnitude of the various tongue, where lymphoid tissue is also abundant, have sero sources of variation (inter- and intratechnician variation, logical reactivities within the normal range (45). This would variation in proportion of VGA-producing cells in the cell imply that a specific lymphoid cell population is present in lines over time and from batch to batch, and other the nasopharyngeal mucosa, which does not appear to be variations in the antigen). Geser et al. (submitted for the case (Yata et al., submitted for publication). publication) concluded that strict comparability of VGA Viral Carcinogenesis. Hypothesis 2 assumes that the antibody testing from different populations can be achieved herpesvirus associated with NPC is a necessary and suffi only if sera from all areas are tested in the same laboratory. cient factor for tumor development. The fact that all NPC patients of Chinese, African, and Caucasian origin have Etiological Hypotheses very strong and similar reactivities against this virus makes this hypothesis plausible. NPC biopsies do contain EBV If the above findings of a virological and serological viral genomes, and further investigations of epithelial cells association between EBV and NPC are taken into account, for presence of EBV-DNA sequences, separate from the 3 hypotheses can be postulated regarding the etiological tumor biopsies, should clarify this crucial point. The fact relationship between them, (a) The herpesvirus is a passen that lymphocyte precursors from bone marrow become ger parasite in the tumor which is induced by environmental T-lymphocytes, after their passage through the thymus factors (possibly associated with the Chinese way of life), on where they come in close contact with epithelial cells, sug the basis of a certain genetic susceptibility in Chinese to gests that cell cooperation leads to cell differentiation. A such chemical carcinogens, (b) The N PC-associated herpes- similar type of relationship between lymphoid cells infected virus is a necessary and sufficient factor for the development by the virus and epithelial cells in nasopharyngeal mucosa of the tumor, (c) The virus is 1 of the etiological factors might be critical for the development of NPC. The investi involved in a multistep transformation in which the virus gation of such cooperation cannot easily be made in humans, acts jointly with internal genetically controlled or external and experimental model systems must be developed to pro environmental factors. vide further understanding of NPC genesis. Marmoset Chemical Carcinogenesis. Clifford (4, 5) and Clifford and monkeys can be infected successfully with EBV and thus Beecher (6) have proposed that polynuclear hydrocarbons might provide such an experimental model. produced during cooking with exotic woods could be of EBV prevails all over the world, but NPC is mainly asso major importance in the etiology of NPC, together with ciated with Cantonese. Two alternatives can be proposed to hormonal factors such as estrogen-producing vasomotor solve this dilemma: (a) there is not 1 virus, but a group of rhinitis, and that genetic factors do not play any major role immunologically closely related viruses, each associated in NPC genesis. This viewpoint is hardly consistent with the etiologically with a specific disease (BL, NPC, infectious epidemiological data in South East Asia suggesting that mononucleosis); or (b) there is 1 virus, which induces differ important genetic factors are involved in the susceptibility ent diseases in genetically different ethnic groups. Further to NPC (10, 35, 36, 54). Dr. H. C. Ho stressed the fact that more, sociological habits may influence the mode and age of the members of the Hong Kong marine population, which infection which are known to be of critical importance in has the highest NPC incidence of all Chinese groups, spend certain experimental models in viral oncology. their whole lives on the sea, cooking in the open air, with The knowledge accumulated for decades on oncogenic very little exposure to hydrocarbon inhalation.- virus-host relationship suggests that both factors (viral in By contrast, nitrosamines might be of importance in the fection and genetic constitution of the host) are critical for development of NPC (1). This chemical carcinogen has a tumor development. This is certainly the case for the known tissue-specific oncogenic effect, and it seems possible Marek's disease herpesvirus (2, 7, 49, 60). The mechanism that the traditional Chinese habit of consuming considerable of the genetically determined susceptibility to oncogenic quantities of salted (cured, fermented, or "gamey") fish viruses is not fully understood, but the role of transplanta may lead to a high intake of nitrosamines or nitrosamine tion-type antigen (H2 for mice) appears to be of great im precursors (34). Dr. H. C. Ho, in Hong Kong, is now testing portance in the tumor induced by RNA tumor viruses in the hypothesis of an etiological association between salted mice. fish intake and NPC in a case control study there. Another Proving the involvement of EBV as the etiological agent chemical environmental factor could be present in tea, as for NPC will not be an easy task. Since the peak of NPC both Chinese and Arabs are heavy tea drinkers. This is incidence is about 45 years of age and since the incubation being investigated in a questionnaire study of NPC patients period of NPC is unknown, a prospective study of the type

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Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 1974 American Association for Cancer Research. Recent Studies and Outlook on N PC being carried out for BL is not feasible at present. However, mation by herpesvirus at the cellular levels, and (c) further CF antibodies being already at higher titers in Stage I of the assessment of the relevance of the various EBV antigens- disease, one can suggest that normal individuals with high antibodies reactivities to disease development. CF reactivities might be candidates for NPC development. Huebner and Todaro (37) postulated a universal role of References RNA viruses in mammals and human oncogenesis and, in this context, the recent Findings of Kufe et al. (41) are perti 1. Cammoun, M., Vogt-Hoerner, G., and Mourali, N. Les Tumeurs du nent. One can imagine EBV activating an endogenous RNA Nasopharynx en Tunisie. Etude Anatomo-clinique de 143 Observa virus. tions. Tunisie Med., 49: 131-141, 1971. Joint Viral and Chemical Carcinogenesis. The 3rd hy 2. Campbell, J. G. Discussion Summary. In: P. M. Biggs, G. de-ThÃ©,and L. N. Payne (eds.), Oncogenesis and Herpesviruses, pp. 54-55. Lyon, pothesis implies that NPC carcinogenesis is a multifactor France: International Agency for Research on Cancer, WHO, 1972. phenomenon in which chemical and biological carcinogens 3. Chang, R. S. Umbilical Cord Leucocytes Transformed by Lymphoid are involved in initiating the malignancy of epithelial cells Cell Filtrates from Healthy People. Nature New Biol., 233: 124, 1971. of the nasopharyngeal mucosa. There is as yet no adequate 4. Clifford, P. Carcinoma of the Nasopharynx in Kenya. E. African Med. experimental model system upon which NPC could be J., 42: 373 396, 1965. based. 5. Clifford, P. On the Epidemiology of Nasopharyngeal Carcinoma. Chinese migrants all over the world seem to keep the Intern. J. Cancer, 5: 287-309, 1970. Chinese way of life to a certain degree. The ongoing epi- 6. Clifford, P., and Beecher, J. L. Nasopharyngeal Carcinoma in Kenya. demiological studies may define the basic elements of "Chi- Clinical and Environmental Aspects. Brit. J. Cancer, 18: 25-43, 1964. neseness" maintained by various Chinese migrant popula 7. Cole, R. K. The Genetics of Resistance to Marek's Disease. In: P. M. Biggs, G. de-ThÃ©,andL. N. Payne (eds.), Oncogenesis and Herpes- tions and possibly shared by the Arabs in the Maghreb. The viruses, pp. 125 128. Lyon, France: International Agency for Research investigation of the presence of nitrosamines, nitrites, and on Cancer, WHO, 1972. nitrates in the food of Chinese and North Africans is an ob 8. de Schryver, A., Friberg, S. Jr., Klein, W., Klein, G., Henle, G., de- vious task in this context. ThÃ©,G.,Clifford, P., and Ho. H. C. Epstein-Barr Virus-associated Antibody Patterns in Carcinoma of the Post-Nasal Space. Clin. Exptl. Immunol., 5: 443-459, 1969. Outlook 9. de Schryver, A., Klein, G., and de-ThÃ©,G.Surface Antigens on Lym- phoblastoid Lines Derived from Nasopharyngeal Carcinoma. Clin. A year hence, the present seroepidemiological survey in Exptl. Immunol., 7: 161-171, 1970. Singapore, Hong Kong, and France will be completed. The 10. de-ThÃ©,G.The Etiology of Nasopharyngeal Carcinoma. In: H. L. testing of sera for the reactivities of various antibodies may loachim (ed.), Pathobiology Annual, Vol. 2, pp. 235-254. New York: take longer, and only after careful analysis will we be able to Appleton-Century-Crofts, 1972. conclude whether any testable hypothesis can be set forth 11. de-ThÃ©,G.Virology and Immunology of Nasopharyngeal Carcinoma: regarding the role of EBV in the development of NPC. For Present Situation and Outlook. In: P. M. Biggs, G. de-ThÃ©,andL. N. Payne (eds.), Oncogenesis and Herpesviruses, pp. 275-284. Inter instance, the findings of high CF antibodies in NPC patients at stage I of the disease suggest a long-standing and impor national Agency for Research on Cancer, WHO, 1972. 12. de-ThÃ©,G. Annual Report, 1972-1973, p. 51. Lyon, France: Inter tant infection prior to disease development. If the CF anti national Agency for Research on Cancer, WHO, 1973. body pattern in Chinese is different from that in Indo- 13. de-ThÃ©,G.,Ablashi, D. V., Liabeuf, A., and Mourali, N. Nasopha Pakistanese and if the number of normal individuals (within ryngeal Carcinoma. VI. Presence of an EBV Specific Nuclear Antigen the 35- to 40-year-old age group) with high CF titers is in Fresh Tumour Biopsies. Preliminary Results. Biomedicine, 19: small, one might suggest that such individuals should be 340-353, 1973. followed up, as they may be at highest risk for NPC. A 14. de-ThÃ©,G.,and Geser, A. A Prospective Seroepidemiological Study short-term prospective study on selected individuals could to Investigate the Role of EBV in Burkitt's Lymphoma. Unifying Con then be highly relevant and feasible. The prospective sero cepts of Leukemia. Bibliotheca Haematol., 39: 448-456, 1973. epidemiological study on Burkitt's lymphoma carried out 15. de-ThÃ©,G.,Geser, A., and Day, N. E. Problems Raised by the Asso ciation of a Herpesvirus with Two Different Human Tumors: Burkitt's in Uganda may then serve as a model (14, 24). In parallel Lymphoma and Nasopharyngeal Carcinoma. In: P. C. Vincent (ed.), with the serum collection, an epidemiological questionnaire The Nature of Leukemia, pp. 65-77. Sydney, Australia, 1972. is given to randomly selected families and NPC patients in 16. de-ThÃ©,G.,Ho, H. C., Greenland, T., Geser, A., and MuÃ±oz,N.Asso Singapore, Hong Kong, Tunis, and Los Angeles. Analysis ciation between an Herpes-type Virus and Nasopharyngeal Car of the answers will be carried out in 1974 and may help to cinoma. Present Status of Studies. In: W. Nakahara, K. Nishioka, T. focus on one or more environmental factors or cultural pat Hirayama, and Y. Ito (eds.). Recent Advances in Human Tumor Vi terns common to Cantonese Chinese and to Tunisians. rology and Immunology, pp. 297-308. Tokyo: Tokyo University Until careful analysis of the existing and presently col Press, 1971. lected seroepidemiological data is completed, no new major de-ThÃ©,G.,Ho, H. C., Kwan, H. C., Desgranges, C., and Favre, M. C. field project on NPC is envisaged in South East Asia. How Nasopharyngeal Carcinoma (NPC). I. Types of Cultures Derived ever, the population-based immunogenetic studies of Dr. from Tumor Biopsies and Nontumorous Tissues of Chinese Patients with Special Reference to Lymphoblastoid Transformation. Intern. Simons, in Singapore, should be continued. From the ex J. Cancer, 6: 189-206, 1970. perimental aspect, studies should be boosted in 3 directions, 18. de-ThÃ©,G.,Sohier, R., Ho, H. C. and Freund, R. J. Nasopharyngeal namely (a) development of an experimental model for NPC, Carcinoma. IV. Evolution of Complement-fixing Antibodies during (b) development of a better understanding of the transfor the Course of the Disease. Intern. J. Cancer, 12: 368-377, 1973.

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Fig. 1. NPC biopsy where lymphoid cells (L) are mixed with well-differentiated epithelial tumor cells. Note the cytoplasmic bundles of tonofibrils (thin arrows) and fully developed desmosomes (thick arrows), x 8,000. Fig. 2. NPC biopsy grown in vitro for 8 days. A lymphoid cell is inserted between 2 epithelial cells. Note the numerous close contacts between the 2 types of cells, x 17,400. Fig. 3. NPC-derived fibroblastoid culture after 4 months in vitro. A small lymphoid cell is completely engulfed within a giant fibroblastoid cell, with discontinuity of the adjacent plasma membranes.

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Guy de-Thé and Anton Geser

Cancer Res 1974;34:1196-1206.

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