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r Nasopharyngeal'Carcinoma in Alaskan Eskimos, Indians, and Aleuts: A Review of Cases and Study of Epstein-Barr Virus, f-f! A, and Environmental Risk Factors ANNE LANIER, MD, MPH,' THOMAS BENDER, MD, MPH,' MARION TALBOT, MD, MPH,t SALLY WILMETH,' CHARLES TSCHOPP, MD,t WERNER HENLE, MD,$ GERTRUDE HENLE, M0,$ DONALD RITTER,§ AND PAUL TERASAKI, MD" ~ ~ ~ I The records of thirty-one patients with nasopharyngeal carcinoma (NPC) diagnosed from 1966 through 1976 among the Alaskan rfattive 19ofialation (Eskinffo, Aleut, Indianj were reviewed. There were 25 males and six females, which results in relatively high incidence rates per 100,000 of 13.5 for males and 3.7 for females. Clinical and pathologic features were similar to those found among southern Chinese NPC patients. Five-year survival rate was 48%. Antibodies to Epstein-Barr virus were higher in NPC patients than in tients witht other tumo or matched controls. On histocompatt tlity testing Sin-2 was not etected, nor was there significantly increased frequency of A2. Instead, B W40 and a second locus blank occurred more often among NPC patients than among other groups. In response to a questionnaire, NPC patients more often reported use of salt fish in the childhood diet, smoking of cigarettes, and / exposure to noxious inhalants than did controls, but the differences were not statistically stgnificant. V Cancer 46:2100-2106, 1980. N ~ 1 >r I\ IASOPHARYNGEAL CARCINOMA (NPC) occurs at high rates in certain provinces of southern China and at low rates in America, Europe, Japan, and India.'='-" It has also been reported to occur at high rates in Alaskan natives (Eskimos, Indians, Aleuts) and Canadian Eskimos.'~'~•" Genetic, environmental, artd viral factors have been postulated as important in the cause of this disease. Specifically, there has been recent interest in the association of certain histocom- patibility antigens and the Epstein-Barr virus (EBV) with NPC. ` This report describes the epidemiologic and clinical features of nasopharyngeal cancer in Alaskan natives and the results of viral serology, histocompatibility antigen typing, viral cultures, and a questionnaire From the •Alaska Investigations Division. Bureau of Epidemiol- ogy, Center for Disease Control, Anchorage, Alaska: tAlaska Native Medical Center, Anchorage, Alaska: +:he Joseph Stokes, Jr.. Research Institute,` The Ctildren's HospitaJ of Philadelphia. Philadelphia, Pennsylvania: §Virology-Rabies Unit, Alaska Divi- sion of Public Health, University of Alaska. Fairbanks, Alaska: 'Department of Surgery, University of California. Los Angeles, California. Address for reprints: Anne Lanier, MD. Center for Diseasa Control. Bureau of Epidemiology. Alaska Investigations Division, 225 Eagle Street. Anchorage. AK 99501. Accepted for publication November 21, 1979. the administered to patients with nasopharyngeal cancer, patients with benign and malignant tumors of he head and neck (grouped as other tumors), and selected controls. Methods According to the 1970 census, there were 50,900 natives (Eskimos, Indians, Aleuts) who constituted 17% of the total population of Alaska. Between 1966 and 1976 all Alaskan natives with the diagnosis of cancer of the nasopharynx and the majority of patients with benign and malignant tumors of the salivary glands or other cancers of the head and neck (excluding thyroid) were identified from the pathology files and tumor registry of the Alaska Native Medical Center, a regional tumor registry, and death certificates. The medical records of each patient, including surgical pathology and autopsy reports, were_ reviewed. In the spring of 1975 the residence- and'health status of each patient were determined, and during the following 18 months an effort was made to locate each living patient and enroll him/her in the study. Of 31 patients diagnosed with NPC during 11 years (1966- 1976), 21 were alive at the onset of the study and 17 of these patients were studied. Forty-seven patients 0008-543X/80/1101/?100 50.85 C American Cancer Society TITX 0004445 2100 r~ , ~. , , .... . I Wi4jsAi ~01

No. 9 NPC IN ALASKAN NATIVES • Lanier et al. 2101 I were identified with other tumors of the head and. neck, 35 of whom were alive at the onset of the study. Diagnoses of those studied included benign salivary gland tumors (16 patients), malignant salivary gland tumors (six patients), squamous call carcinoma of the oropharynz and larynx- (three patients), and transitional cell tumors of the sinuses (two patients). - For each tumor patient an apparently healthy indi- vidual was selected as a control. Each control was matched to a case by age (±2.5 years), sex, race (Eskimo, Indian, Aleut), and village of residence. The control was not a close relative (first cousin or closer) of the patient and did not have cancer. An attempt was made to obtain blood specimens, swabs for viral cultures, and a completed questionnaire for all patients and controls. In some instances it was not possible to complete all aspects 'of the project. Serum samples were tested for antibodies to Epstein- Bair antigens-IgG and IgA antibodies to viral capsid antigen (VCA), IgG antibodies to the diffuse (D) and restricted (R) components of the EBV-induced early antigen (EA) complex, and antibodies to the EBV- associated nuclear antigen (EBNA).ie•"•t9 Human leukocyte antigcn testing was done by the micro- cytotoxicity method for 35 different antigens, including Sin-2." Saliva and swabs of the nasopharytix, throat, and rectum were inoculated into newborn Swiss albino inice and into four cell culture lines (Vero, W138, RMK, and HEp2). , The questionnaire included questions concerning chronologic history of village of residence; type and size of housing, type of stove and lamp fuel used; occupational history, including exposures to chemicals, dust, fumes; foods consumed as an adult and as a child, specifically salted, smoked, fermented, dried, and raw frozen foods; use of tobacco, alcohol, 'incense, and mosquito powders; frequency of steam bathing; fre- quency of transportation in small unpressurized air- planes; medical history of tonsillectomy, cold sores, tuberculosis, and antituberculosis drugs; and family history of cancer. ' Results Clinical and Epidemiologic Thirty-one patients from the Alaskan ative popula- tion with NPC were diagnosed during t)F years 1966- 1976. There were 25 males and six females. Annual crude rates per 100,000 were 8.8 for males and 2.2 for females. When adjusted to the world standard popula- tion, rates were 13.5 and 3.7 per .100,000 for males and females, respectively. Age at diagnosis for males and females is shown in Figure 1. The age range was 32-80 years, and the 16 14 - 1o 0 4) 6 E ~ z 4 I ~ Females 25-34 35-44 / 45-54 1 55-6.4 I Males 65 * Age at Diagnosis. Fta. 1. Age at diagnosis of Alaskan native patients with naso- pharynQeal cancer. median age was 54 for males and 56 for females. No malignant tumors of the inasopharynx are known to have occurred in thig population in anyone under 30 years of age. Seventy-one percent of the patients diagnosed were between the ages of 45-64 years. Rates were highest for males in the age group 45-54 years and for females in the age group 55-64 years. Nine patients were diagnosed in the first 51/`z years of the study and 22 patients 'in the second 511,-; years: Of the 31 cases, there were 21 Eskimos, four Indians, and six Aleuts (Table 1). Ethnic status was based on patients' self-classification. No female Aleut wiih NPC was identified. All three ethnic groups appear to be at increased risk relative Yo U. S. Whites, although the magnitude of the risk is greater for Eskimos and male• Aleuts than Indians. Cases were diagnosed in TASLB 1. NPC in Alaskan Natives by Sex and Ethnic Group Eskimo Aleut Indian No. of cases Male 16 6 , 3 Female 5 0 1 Rate110'lyr Male 10.2 16.2 3.2 Female 3.3 0.0 1.2 Relative risk' Male 20 31 6 Female 30 0 11 • Based on rates for Connecticut. 1966- 1968 (Connecticut Health Bulletin. Cancer in Connecticut. 1966-1968. Connecticut State De• partment of Health, Hanford, 1977). r TITX 0004446 _ ^:

2102 CANCER November 1 1980 Vol. 46 TABLE 2. NPC in Alaska Natives. Pretreatment EBV Titers (Reciprocal) by Stage' of Disease Anti-VCA Anti-EA-D Pa- tient Stage IgA IgG IgA IgG Anti- EBNA 01 I <10 80 - <10 320 11 I <10 80 - <10 80 07 II <10 160 <10 10 80 16 II 10 640 - <10 80 19 1I 20 320 < 10 40 320 08 III 40 640 10 40 ~320 18 III 40 320 20 40 40 17 IV 40 640 - <10 z320 15 V 10 320 <10 40 160 ' Staging as defined in Reference 18. both Inupiat and Yupik-speaking Eskimos and in Indian tribes of both interior and southeastern Alaska. No definite geogfaphic clustering was noted: Medical records were available for all but one of the 31 patients. The symptom that brought the patient to medical attention in nearly every case was a lump in the neck: .The•lump•was•.nontender.and had been present. from one month to one year prior to diagnosis. Half of the patients noted an increase in the size of the lump between the time of discovery and diagnosis..A history of recent epistaxis was noted in four patients, nasal obstruction and discharge in three, and headache and occipital pain in one patient. In 29 of the 31 patients, diagnosis was confirmed by biopsy of the nasopharynx. Tissue from the.remain- ing two patients was obtained from cervical nodes.' All patients had poorly differentiated epidermoid carcinomas variously described as transitional cell; lymphoepithelial; undifferentiated, or poorly differ- entiated squamous cell carcinomas. At the time of diagnosis, tumor had spread to the following sites- nasopharynx only-2; the upper cervical lymph nodes-11; bone andlor cranial nerves and mid-cervical nodes-10; supraclavicular nodes-4; below the clavicle-3; and unknown-1: All but one patient were treated by irradiation. Eight patients underwent radical neck dissection in addition to irradiation. Five-year survival determined by life table method was 48% (95% confidence intervals 28-68To). Death in all instances was due to recurrent or metastatic nasopharyngeal carcinoma. Epstein-Barr Viral Antibodies Antibodies to the various EBV antigens were deter- mined in 17 patients with NPC, 27 with other tumors, and 33 controls. Among NPC patients tested at or within six months of diagnosis, antibody titers to VCA and EA appear to increase with advanced stage of disease except for the one patient in Stage V (Table 2). NPC patients, especially those with clinical evidence of cancer, had higher antibody titers than the other groups (Table 3). The differences between NPC patients and others are most striking for IgA anti-VCA (Fig. 2). All three.NP.C .patients.wish IgA anti.VCA aatibody titer i' 1:10 have developed clinical evidence of recur- rent or metastatic disease. since the blood was drawn. IgA anti-VCA titers of 1:10 and 1:40 were found in two patients who did not have NPC. In these two a mixed parotid tumor and a lymphoepithelial lesion of the. parotid gland had been diagnosed several years prior to antibody testing, but the patients were without evidenc.e of tumor when blood for EBV testing was obtained. ?Red Blood Cell Type and Histocornpatibility Antigens Among patients with nasophaFyngeal carcinoma, 33~7b were blood group A, 17% B, 6% AB, and 44%'0. Among ail other subjects tested the percentages by blood group were 36, 9, 6, and 49, respectively. All patients tested were Rh+. HLA testing was done for 51 patients _(NPC-16 patients, other tumors-14 patients, controls-21). A2 occurred in patients with NPC more often than in controls, but less often in patients with other tumors (Table 4). B W40 and a single antigen at the second locus occurred more often in NPC cases than in controls or in patients with other tumors. The differences were not statistically significant. TABLE 3. NPC in Alaska Natives: Antibody to Epstein-Barr Antigens Frequency elevated titer (%) GMT (reciprocal) Group No. IBG anti-V z 1:32 ._ ° ; . . IgAA CA anti-VCA 0 z 1:10 .2tnti-. , EA-D z1:40 Anti- EBNA Z1:80 IgG anti-VCA IgA anti-VCA Anti- EA-D Anti- EBNA NPC-active disease' 9 78 78 56 78 320 16 22 137 NPC-ail cases 17 59 59 44 77 283 ~ 12 18 120 Other tumors 27 33 7 7 44 168 6 6 43 Controls 33 45 3 9 33 170 5 7 39 ' NPC patients studied at the time of diagnosis or with known recurrent or metastatic disease. rF .~~ . _ . I TITX 0004447

t No. 9 NPC IN ALASKAN NATIVES • Lanier et al. FtG. 2. IgA anti-VCA Epstein-Barr viral antibodies in Alaskan native patients with nasopharyngeal carcinoma, other tumors of the head and neck, and controls. (Cases with tumor include patients tested pretreatment or with known recurrent or metastatic disease; those patients without tumor were clinically cured at the time of testing.) L Y V C A total of 30 patients were tested for SIN-2, and all tests were negative. Of these, five NPC patients and five patients with other tumors had a single second locus antigen. Viral Cultures Specimens for culture were obtained from 13 NPC .patients, 21 other tumor patients, and- 27 controls. Three viral isoiates were obtained. 'A herpesvirus hominis Type I(lyiayo 1814) and a low-titer viral agent (cytopathic effect resembled an enterovirus group) were isolated from two female controls and an adeno- virus type 9 (Hicks) was isolated from a female who had' undergone parotidectomy for mixed' tumor six years previously. Nq-viral agents were isolated frotn any patients with malignant_tumors of the head and neck, including a group of five patients with active nasopharyngeal caricer sampled on eight occasions. ' Questionnaire Risk factor questionnaires were obtained for 13 NPC patient-contro' pairs and on 17 other tumor patient- control pairs. ' '.c sponses from patients and their village- matched controls were generally similar, although some differences were noted. No differences were statistically significant by matched-pair analysis. Among NPC matched pairs, more patients than controls were found to live in a different village at diagnosis than at birth, or to have lived at some time in a village in Alaska other than the village of birth; to have eaten salted fish more frequently in childhood; to have smoked ci_arettes; 2103 Carcinoma of the Nasopharynx Cases Cases With Without Controls Tumor ' Tumar Other Tumors of the Head & Neck Cases Contr9ls 40 20 and to have been exposed to chemicals, fumes, or dust as an adult.* Compared with their controls, patients with other tumors also reported more mobility within Alaska, did not differ in 'the use of salt fish in the child- hood diet or exposure to inhalants, and used cigarettes and tobacco less often. Discussion ' Tliis study confirins previous reports of high;rates of = NPC in the Alaskan native population.`•=' The rates of 13.5 per 100,000 in males and 3.7 in females would be even higher if calculations were based on the second half off the: study in which more than twice as many pa[ients== were identified as in the first half.9 From 1960-1970, although the percentage of people in the age group 40 years and over increased from 18 to =07c, this was not enough to account for the increase in the number of cases identified in the latter half of the study. Whether there has been a true rate increase or simply improvement in case finding and diagnosis cannot be determined. Because some Asian groups are at very high risk for NPC (southern Chinese) and others are at low nsk (Japanese, Koreans) it is not enough to simply ascn.-e the high risk for Iti'PC in Alaskan Natives to their prehacie Asian origin. The increased risk among Alaskan Es}::.mos ' Patient-control pairs reporting nonhomoseneous responses.. e.; case yes/control no vs. control yesicase no were: dII-ferent '•.~.a2e at dia¢nosis as birth-3 vs. 1, residence at any time out of ecrnal village-3 vs. 0, more frequent use of salted fish in childhooe 4 vs. 1, use of cigarettes-3 vs. I, occupational exposure-4 ~s. 1. tll 0 ~3'1 _) : 103 TITX 0004448

Vol. 46 CANCER November 1 1980 2104 T.ats 4. HLA Frequencies' among Alaska Natives: NPC, Other Tumors and Controls NPC (N - 16) Other tumors (N = 14) All controls (N - 21) Num- ber Per- cent - Num- ber Per- cent Num- ber Per- cent First locus 2 7 44 7 30 8 38 11 24 11 69 12 86 13 62 28 4 25 3 21 8 38 Only one antigen 4 25 4 29 5 24 Second locus i25 l 6 3 1 21 7 5 24 15 3 19 2 14 5 24 27 4 25 5 36 6 29 35 2 12 5 36 4 19 40 12 75 6 43 11 52 Only one antigen 7.. 44 3 21 _ .. 5.. . 24 ' Only the more frequently occurring antigens are.reported. . and among Aleuts to whom the Eskimos are belieyed to be related is consistent with the report of high risk of NPC reported among Canadian-Bskimos.'= How- ever, an increased risk among Alaskan Indians was not anticipated because in two studies among South- western American Indians, to whom some groups of Alaskan Indians are related, an increased risk was noted for nasal and sinus cancers but not for NPC.°•'Z Age distribution, sex ratio, clinical presentation, and pathologic findings for patients with NPC in• this study appear similar to those of Chinese patients.~•' To date there has been no Alaskan native patient under 30 years of age. This is in contrast to reports from Tunisia and Uganda where nearly 25% of the patients with primary NPC are under 20 years of age.'-1e The five-year survival rate of 48% for patients in our study is comparable to other reports.i'st The serologic data presented confirm the association of the Epstein-Barr virus with NPC in patients of a different ethnic origin and in a different geographic location than previously reported.lo.~t.,s.~e~o =e.zsas The NPC patients in this study showed a greater prevalence of high titers and/or higher geometric mean titers of IgG and IgA anti-VCA specific antibodies and of anti-EA-D than patients with other tumors of the head and neck, or healthy controls. In addition, the antibody pattprns found in Alaskan natives with NPC appear to cerrelate with stage of disease as well as disease activity, again as previously. reported.lg•'s•=0 The ratio of the EBV geometric mean titers in NPC as I compared with controls was not as high in this study as in other studies.'e•'e•=° This was largely a result of high levels of EBV anti- bodies in controls; the reasons for this are unknown. Regarding the sensitivity and specificity of EBV serology, particularly IgA anti-VCA in this situation, it is important to note that patients with`early stage NPC and those in remission have antibody levels as low as those patients with other tumors and the controls. Furthermore, other patients in this study (two patients with benign salivary gland tumors and one control) had antibody levels as high as patients with NPC. It is essential, therefore, to also study the tumors for evidence of their association with EBV by, either the demonstration of viral DNA in biopsies or` of EBNA- positive cells in touch preparations of biopsies.=3's•" To date, EBY DNA or EBNA-positive carcinoma cells have been found almost uniformly in biopsies of poorly differentiated carcinomas of the nasopharynx and iri some lymphomas, but not in other tumors.'-1o Similar studies are now in progress op tumor specimens from Alaskan patients. Among Chinese NPC patients in Singapore, Simons reported an increased frequency of HLA A2, a second locus blank (Sin-2), and a lymphocyte-determined antigen (Sin-2a)."''Z Among Tunisian patients, an increased frequency of a second Idcus'blank'was found; although this was not statistically significant, and A2 appeared less 'often among NPC cases than controls.' Typing of four of six Bermuda NPC patients and a Canadian family with two cases of NPC and two cases of Burkitt lymphoma did not show the A2-Sin 2 patterns.s•'! Our study failed to demonstrate the A2-Sin 2 haplo- type among NPC patients. NPC patients had a higher frequency of BW40 (75%) and a second locus blank (44%) than did controls, though neither difference was statistically significattt. BW40 has been reported in 35% of Greenland Eskimos, Chinese, Japanese, and in•75%a of Canadian Eskimos.=51 The frequencies Qf HLA antigens in the various ethnic groups of Alaskan natives are not known. Further testing on NPC patients and controls"would be necessary to detect significant differences in the frequency of HLA antigens. No viral agents were isolated more frequently from NPC patients than from controls. Leukocyte trans- formation for detection of EBV was not done. Oral excretion of EBV has been demonstrated in patients with infectious mononucleosis, Burkitt's lymphoma, other cancers, and healthy seropositive subjects.g•" However, in studies of NPC F a'.ients, oral excretion of EBV was not detected by leu:.ocyte transformation even though the virus was seen under electron micros- copy.4== It is suggested that this is due to the pres- ence of EBV-specific IgA antibodies in the saliva. A case-control study in Taiwan suggested that increased risk of NPC was associated with smoking and TITX 0004449 1 ',-Y r' y"' ~, 1

t No. 9 NPC IN ALASKAN NATIVEs - Lanier et al. working in poorly ventilated environments; in another study of NPC patients of various ethnic origins in California, however, increased risk of NPC was asso- ciated with prior history of ear, nose, and throat disease, and occupational exposure to fumes, smoke or chem- icals, although not with snioking.ts=' Ho has proposed `` that early use of salt fish by the Chinese may increase the risk for NPC.21 In our study patients and controls were questioned regarding approximately 40 items. Responses were most remarkable for the similarity between patients and their village-matched controls, a finding not unexpected in view of the fact that most patients lived in villages that were culturally homogenous. However, there were some discrepant responses. NPC patients were more likely to have used cigarettes, to have been exposed to noxious inhalants,_and.uported eating salt fish.more_. frequently as children than the controls. These factors have been implicated in other studies. ' Despite the report of NPC in three Canadian bush pilots,2 our study did not detect a difference. in the use of small, unpressurized airplanes. In conclusion, rates calculate rnm his study con- firm previous reports of increased risk of NPC for Alaskan natives. The reported association of EBV and NPC also appears to exist in this culturally and geographically unique population. Further studies that document the presence of EBV in tissue and the correlation of EBV antibody titers with activity and stage of disease are warranted. The histocompatibility antigen pattern A2-Sin 2 was not found tobe a risk factor. in this population, although other HLA antigens may be important. No environmental factor unique to this population was identified to account for.the increased rates of NPC. . REFERENCES 1. Andersson-Anvret M, Forsby N, Klein G, Henle W. 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. 2106 CANCER November 1 1980 31. Loring MF. Malignant tumors of the nasopharynx. Am J Roentgenol Radium Ther Nuclear.Wed 1%S; 93:36-43. 32. Mallen. RW, Shandro WG. Nasopharyngeal carcinoma in Eskimos. Can J Orolaryngol 1974; 3:175-179. 33. McAlpine PJ, Chen SH, Cox DW et a!. Genetic markers in blood in a Canadian Eskimo population with a comparison of allele frequencies in circumpolar populations. Hum Hered 1974; 24: 114=143. 34. Muir CS. Nasopharyngeal carcinoma in non-Chinese popula- tions with special reference to southeast Asia and Africa. Br J Cancer 1971; 8:351-363. 35. Nonoyama M, Huang CH, Pagano JS, Klein G, Singh S. DNA of Epstein-Barr virus detected in tissue of Burkitt's lym- phoma nad nasopharyngeal carcinoma. Proc Nail Acad Sci 1973; 70:3265-3268. 36.. Sako K. Minowada J, Marchetta FC. Epstein-Barr virus antibodies in patients with carcinoma of the nasopharynx and car- cinoma of other sites in the head and neck. Am J Surg 1975; 130: 437-439. 37. Schaefer O, Hildes JA, Medd LM, Cameron DG. The changing pattern of neoplastic disease in Canadian Eskimos. Can Med Assoc J 1975: 112:1399-1404. Vol. 46 38. Schmauz R. Templeton AC. Nasopharyngeal carcinoma in Uganda. Cancer 1972; 29:610-621. 39. Simons MJ, Chan SH, Ho JHC, Cbau JCW, Day NE, de-The GB. A Singapore 2-associated LD antigen in Chinese patients with nasopharyngeal carcinoma. In: Histocompatibility Testing-1975. Copenhagen, Denmark: Munksgaard, 1976; 809-812. 40. Simons HJ, Chen SH, Wes GB, Shanmugaratnam K. Probable identification of an HLA second-locus antigen associated with a high risk of NPC. Lancet 1975; 1:142-143. 41. Simons MJ, Wee GB. Day NE. Morris. PJ, Shanmugaratnam K, de-The GB. Immunogenetic aspects of nasopharyngeal car- cinoma: I. Difference in HLA antigen profiles between patients and c ntrol groups. Int J Cancer 1974; 13:1?-234. 4Simons MJ, Wee GB, Goh EH et a!. Immunogenetic aspects PC IV. Increased risk in Chinese of NPC associated with a Chinese-related HLA profile (A2. Singapore 2). 1 Narl Cancer Insr. 1976; 57:977-980. 43. Terasaki PI, McClelland JD. Microdroplet assay of human. serum cytotoxins. Nature 1964; 204:998- 1000. 44. zur Hausen H. Schulte-Holthausen H. Klein G er a!. EBV- DNA in biopsies of Burkitt tumors and anaplastic carcinomas of e the nasopharynx. Nature 1970; 228:1056-1058. TITX 0004451 - I "~ ~ 10 'ij) < -sv