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Antibodies t~o Epstein-Barr Virus in Naso- pharyngeal Carcinoma, Other Head and Neck Neoplasms, "and Control Groups 2,= * l~cccivc~ September 29, 1969: accepted Octobc~ 6, 1969. ~ Inv~6ga~o~ ~pportcd by Public H~th S~icc ~anU PH~3~77 ~d PH~2~5 ~ the Br~h Emp~c ~ Campai~ f~ R~ ~c Swc~h from ~= ~a~on~ Imt~ of H~. Ph~ade]phi~ ~d ~h~l o~ ~edicin~ Univ~;W of Pennsyh.ani~ Ph~addphla, P~n~ ~ Roy~ Hong gong 2~key Oub Im~t~tc of Que~ Eli~ Hmpi~, K~l~n, Hong gong. ~ Insti~t de Recherches $ci~d£qu~ ~dl~ju~ Franc~ ~ ]n~mt O~tavc Ro~ ~'~cj~, F~c~ I Dep~tm~t of H~d ~d ~e~ S~g~, ~yatta Radoa~ Hospi~, ~akobi, K~y~ t Dcp~t of Tumor 3;do~, ~{cdic~ ~oo~, St~hd~ Swcd~ IAgC, Lyon~ Fr~c~ ~ Pzs~ address: ~umhe~e~ K~o~nska Ins~tute, S~hd~ Swede. ~ We ~l~uHy ac~owledge the s~t~ce of ~c Adam, ~ AngOla Pactzd, and H. C. Kwh." .NOTICE Ir;;.; :Z - "" . B'- PROTECfED. : ~,~CO.P..Y.RIGIII" tA,,' UITLE IZ U, S. ~ _I).:~'_ T104132447

~n pat;enN w;l~ neoplasms o~h~r lhan NPC. ~e si~nffi~nce and lions o~these/indin~s are discu.ed.~J Nat Cancer In~ 44: ~5-~31,1970. THE EPSTEIN-BAILR virus (EBV) was shown to be etiologically related to infectious mononucleosis (I-3). This i:indin~ does not preclude the pos- sibility that this virus or a varlant of it is also involved under as yet undefined conditions in the gcnesls of two malignancies~ i.¢., Burkitt~s lymphoma (BL) and nasopharyngcal carcinoma (NPC). Such a link is suggested among others by the fact that African patients wlth either of these tumors ahd Chinese NPO paticnts,'~cncr.alIy possess anti- bodies to EBV-related. antige'.ns, usually at re- mark.ably higher titers than appropriate controls. These antibodies arc detectable by: a) indirect immunofluorescencc tests on acetone-fixed smears of cells from EBV-carrying ceil lines (d~ 5)..There is evidence that .this test mcasux'es~ essentlaIIy, antibodies to RB viral antigens (6); b) indirect immunofluorescencc or~ more dependable now, blocking of dlrcct immunofluorescencc reactions with llve cells from 1"~.BV.-positive blastoid cell lines (7~ ~). This technique detects antibodies to ceil membrane ant.igcns which are largdy distinct from viral antigens but apparently EBV induced (9~ 10);. and ~) doublc-di~uslon precipitation tests ~v'ith concentrated extracts from EBV-carrying cell lines (11). Thls technique most likdy measures yet other components of the EBV-related antigen- antibody spectrum (1~. In a previous stu.dy (13) and work to be reported elsewhere~ results obmlned by these three tests with sera from a limited number of African an.d immunofluoresccncc tests on fixed cell smears which measure antibodies mainly to Epsteln-Barr virus (EBV) antigens and is most readily suitable for large-scale testing. Numerous sera each from a) NPO patients, b) patients with other naso- pha.ryngeal neoplasms, or carcinomas or other neoplasms arising elsewhere in the head and neclq and ¢) various control groups were tltrated for antibodies to EBV. Results reaffirmed the remark- able predominance of high tlters in the N.P(3 group and showed also that the incidence of high titers increased as the disease advanced. MATERIALS AND METHODS ~ra.~Thesc were ~btalncd from patients with carcinomas and other neoplasms of the nasopharynx and other parts of the head and neck. ~ • "'.Th~ ~ were kindly fur- nighed by Dr. F. ]3. Bang and Dr. S. R. S. Rangan~ "Johns Hopkins University, School of Hygiene, Baltimorc~ /d'aryland. The patients wcrc classified as to the primar~ sltc and h|stopathology of the tumor. As a rul% these dam became available for correlation only after the. serological tests had been performed. "Control scra wcrc obtained from blood relatives and neighbors of East African NPC patients who Chinese i~aticnts with IN.PC~ other n.coplasm~ or resided near Fort Hall and IGambu in the high- on- eovlas c wen. h thy ia d oi r:e r . Chinesc.c~:~atrOl scra were secured donors, were corrdatcd with each other and with the histdogical diagnoses. Although the result~ of the scrolo~6cal tests were generMly "doncordant~ discrcpandes were noted~ as one might expect in tests measuring essentially different antigcrl-ant{- body systems. High antibody reaetivh7 by.any one of the tests was frequent in NPC but was occasional also in other head and neck neoplasms and con- trois. The present report is restricted to indlreet • from patients with .non-neoplastlc discases~ family members of patients, and pregnant women. Indian control sera were obtained from blood donors and patients with minor'ill.nesses. The sera werd .sent by air to Philadc.Iphla undch d~:y-icc rcfrigcration and used without inactivation. Titration ~f.antibodies to EBY.~Technlqucs have • been fully described (l~ JOURNAL OF "/"HE NATIONAL CANCER. T!04132448

NA$OPHARYI~GEAL CARCL~OMA 227 RESULTS The scrolo~cal results obtained with various groups of sera are prescnted in table l as to the distribution of anti-EBV titers~ the geometric mean ]evel~ and the percentages of negative scra (~1:10) and sera with high titers (~_ 1:160). All NPC patients from East Africa and Hong Kong had antibodies to EBV, 85% at fliers of _>-1 :160, and the geometric mean lords were'l:394 and 1:340, respectively. Among the French patients, only 6. were possible NPC cases---obviously too few for a meaningful analysis. In contrast, patients with carcinomas in other sites of the head and neck, or with other" types of tumor~ in the naso- pharynx or elsewhere in the head and neck, whether" from East Africa, Hong Kong, India, or France, had generally lower titers oi" even no antibodies to EBV. Only from 9-27% (mean 13%) had fiters >_ 1 : 160, and the geometric mean alters ranged from 1:28-1:80, with a mean of 1:36, which was nearly ten times lower than the mean alter of all NPC patients (1:348). Sera from control individuals in Africa (family members and neighbors of NPC. patients), Hong Kong (family members of patients, pregnant women, and l~atients with non-neoplastic diseases), and India (blood donors and patients with minor ilia.eases) gave results like those from scra of patients with neoplasms of the head and neck other than NPC. The percentages with anti-EBV titers _~1:160 ranged from 10-26 (mean I lA%) and the geo- metric mean titers from .1:35-I :46 (mean 1:57). The results obtained with the control group were comparable to'those in the controls for Burkitt's lymphoma patients (5"). The histology of NPC was described most fre- quendy as anaplastlc, undifferentiated, or poorly differentiated, and only occasionally as moderately differentiated. It was usually specified as squamous or epider.moid. Among patients grouped according to the histologlcal appearance of the carcinomas, no significant differences became apparent in the distribution of anti-EBV alters. Information on the NPC patients from Hong Kong included the stage of their disease (14, ]$). Serological data were correlated with this "disease- stage" classification (table 2), and the incidence of high anti-EBV thers showed successive increases. from 44.5% in stage I to 100% in stage V. The geometric mean anti-E, BV alters ros.e stepwise from 1 : 103-1 : 788 as the disease advanced. The results were not influenced by the sex or the age. of the Chinese NPC patients. In this series, male exceeded female patients by a factor of slightly greater than 2. Only 7% were under the. Tx~,~ 1.--Anti-EBV titers in patlent~ ~Sth nasophar3mgeal carcinoma (NPC), neoplasms at ot.her sites of head and neck (Olq), and controls (CO) 2~um- Number with mat~-EBV titers of Sourc~ Group bet Geomegrlo Pereen$ ~10 10 20 40 80 160 320 640 1280 2560 giter ~10 ~160 East A~rlca NPC 70 0 1 1 2 (; 9 • 14 18 18 1 1 :.394 0 85. 7 ON* 69 5 5 9 25 16 - 4 4 1 0 0 1:44 7.2 13.1 COt 105 7 18 24 29 21 8 3 .0 0 " " 0 1:38 6.7 10.5 Hong Kong NPC 165 0 '1 2 9 13 35 29 41 30 5 1:340 • 84. 8 ON 28 0 3 8 9 5 1 2 0 0 0 1:40 0 10. 6 CO 113 9 14 "29 34 16 8 1 1 1 0 1:35 8 9. 9 India ON 22 2 1 2 4 ? 3 2 0 1 0 1:'80 9 " 27. ~ CO 19 0 2 4 7 1 8 1 0 I 0 1:46 0 26. 2 France . NPC 6 0" 0 1 0 2 0 2 1 b 0 1: 61 0 50. 0 • ON. 66 10 19 6 13 2 3 2 1 0 0 1:28 15. 1 9, 1 All NPC 241 0 2 4 11 21 44 45 60 48 6 1:348 0 84.2 ON 185 • 17 28 25 51 30 11 .10 2 1 0 1:36 9.2 13.0 ' CO 23? 16 29 57 70 38 19 5 1 2 0 1:87 6.? 11.4 • Carelnom~ at slt~ other tbsn n~ophtt~r~ and neop~s t Relatlv~ of NPC p~tlents (Emt ~ and Hong and pregna~t~omen ~on¢ ~on~. VOL. 44~ NO. 1, JANUARY 1970 Tl04132449

age of 30 years and 94, the4th. 5th, 6th, and 7th de.de oflif~ Among neop]as~ o~er ~an no~ d~wh~e in ~e h~d and v~ons t~ of ~rco~ adenom~ m~anom~ fibrom~, etc. ~s group did not include lymph~ ~rcom~, redc~um ce~ sarcom~ or H~g~n's dis~e w~ had been s~died p~o~y for compa~son wi~ BL and rev~led an~-EBV r~c- tiom gene~ly like ~ose in con~ol ~oups (5). ~en ~e ~rcinomas other ~an NPO in ~e present seri~ were separated ~om neopl~ oth~ than c~cinom~ (table 8), ~e dis~b~fion ofand- EBV tite~ the incidence of high tit~ and the gcomcwic m~n Icvck were d~cly si~l~ ~o ~oups so obmlncd. ~rcinomas o~ ~an NPC wcrc subdiMd~ into ~osc a~slng in ~c p~an~ sinus~, 1ons~ and soft palate, ten.c, oral ~ (not further specified), and h~opha~ and la~. Tcxt- fibre 1 shows no difference ~ong ~c ~oups ~ m ~e dls~ibu~on of anfi-EBV fitch. ~rre- spending dam for the ~PC pa~cn~, included for comparison, revealed again ~c s~ingly high~ anfib~y levels in t~s mali~ancy. DISCUSSION The data confirm carllcr reports on the unusual frequency of high and-EBV titcrs among patients DIS'I'RIBUI"ION OF ANTI-EBV TITERS IN CARCINOMAS OF HEAD AND NECK ANTI- EBV TITER TZ.XT-rXO~RZ 1.wDLstribudon of anti-EBV tltcrs among patients with nasopharyngcal carcinomas and carcinomas elsewhere in head and neck. with NPC (8, 10, 13, 16). Most of these patients had carcJnomas described "as anaplasfic, undiffercnfi- " ated, or poorly differentiated. When further spedfi- cations were given, the squamous or epldermoid type predorrdnated. Similar types of carcinomas elsewhere in ~hc' head and neck were much less frequently accompanied by high and-EBV titers. ..~In fact~ the percentage of scra from th~s group of patients with fitcrs ~_1:160 was only slightly higher than that in the control series, i.e., relatives TA~ 2.--Rdation of stage of NPC to anti-EBV levels (Heat Kong patients) Number of Anti-gBV Geometric Stage patients tlters mean anti- ~l:-lO0, (~) EBV titer Confined ~o NP ~nucosa ~) 44. 5 1:103 Tumor extended to adjacent parts bdow base of skull without bone involvement and/or mobile nodes in upper cervical region ~ above skin crease extending from below laxyngeal prominence backward ~ound neck 16 8~. ~ 1:235 Bone aed/or cranial nerve involvement ~nd/or mobile nodes in reg|~ns between upper cervical and supraelavioular fossae ; and/or ~xed nodes above supraelavieular lessee 86 8L 5 1:288 ~nvolvem~nt of nodes in supraclavicular fossae irrespective • of local extent of primary tumor ~ 97. O 1:595 Hematogenous metastases or involvement of nodes or skin below the clavicles 10 100 1:788 165 8~. 8 1: 310 l.II. All paticnt~ i ~OURNAL OF THE NATIONAL CANCER INST1TO'I1~ T104132450

~ASO~n-Z~RYtCOZ~L C~CmO~.A 229 " TAZ~ 3.---Comparison of p~t~en~ with c~cinom~ of the n~oph~nx ~P~, p~ wi~h carcinom~ arising ' at oth~ si~ of bead and neck (OC), and patien~ with tumom oth~ than earc~nom~ (O~ Number with snti-EBV ti~ers of Geometric Neopl~m Number mea~ Percent ~10 10 20 40 80 160320 640 1280 2560 ~10 ~160 NPC 241 0 2 4 11 21 44 45 60 48 6 1:348 0 OC 129 15 22 14 84 26 ? 8 2 1 0 1:41 1L? 13.9 OT" 52 3 6 9 15 12 4 3 0 0 0 1:43 and nclghhors of NPC patients, patients with non- neoplastic d~e~ blo~ donom, and pre~ant women, M~t ~ were t~ted ~out ~ow]cdgc of &e ~ini~l or hlstolo#cM diagnose. F~ermor% ~c dlagnos~ of mmc pafien~ considered inifi~y to be NP~ mbsequenfly were .~anged when ~e ~stolo~ w~ r~iewed ~out prior ~owlcdge of the ~olo#~ data. ~en ~ ~fimatc dini~ and Mstol~ diagnos~ w=e co,dated wi~ ~c sero]o~, most pafienm who no longer qud~ for ~c ~ ~tcgo~ had low anfi-EBV titers (~:m). Scrim ~ ~om several ~i~n NPC pafienu were co,cacti for several mont~ and t~ted; as in patlcnts ~ BL (5~ ~$)~ the titers remained . constant or, at most~ showed minor, possibly tech- nic~ fluctmfions. B~ore any form of g~en~ the ~ouping of ~e Hong Kong NPC pa~enu into stag~ I-V (I4) revealed, however, successive creas~ in ~e incidence ~f ~gh anfi-EBV and in ~e geome~c m~n lev~ as ~e d~e advanced~ ~or~ ~e app~ent constancy of titem i~ ~e ~ri~n NPG pafien~ ~ght conceiv- ably be rdated to a reduction in ~e canc~ c~ populafio~ wi~ a eog~ponding de~e in EBV-relat~ an~gens for anfib~y and to i~osuppr~sion~ as a reset of chem~ • ~a~y or mdio~erapy. F~ hor~onml smdi~ of pafien~ ~e in pro~ to ass~ ~ po~ib~i~. In an~ ev~ ~e persistence of ~gh anfi-EBV leve~ ~ ~.~o m~ignanci~ con~asU wi~ ebse~afiom ~ ~ecfious mononucleosis. In ~ dls~s% w~ ~ ~ecog~zed mos@ ~ong adol~ cen~ and ad~ of ~gh socioeconomic antib~im to EBV are formed d~ no~ and, ~t~ passing p~ fit~ tend to ~t genera~y at low~ .~ough r~dfly detectable leveh fo~ y~, ~ not for lge (~). C~dho~ pri~ ~V ~ec~o~ VOL. 44~ NO. I~ ~ANUARY 1970 common under low socioeconomic conditions and generally unrecognized, seem to account for most of the persisting low antibody levcls in populations at large (S). The data presented suggest some association of • EBV with NPC. This suggestion is further based on the results of two other serological tests measur- ing chiefly antibodies to other EBV-rdatcd anti- gem: g) the precipitation of antigens extracted from EBV-earrylng Burkitt tumor cell lines and b) the demonstration of antibodies to mem- brane antigens of cultured blastoid cells of EBV- carrying lines derived from Burkitt's lymphomas (7-10), leukocytes of infectious mononucleosis patients (17), and now also _NPC (18). EBV replication presently appears restricted to cells of the lymphoreticular systcm and thus~ if oncoger~c or cocarclnogenic, could well be in- volved, in the etiology of BL. There is presendy no explanation why NPC should show the ap- parent association with EBV, whereas carcinomas elsewhere in the head and neck do not. A sug- gestion has been made that NPC might arise from thyrnlc remnants of the Waldeyer ring (19). NPC is known to be intcrsperscd~ as a rule, with variable amounts o.f lymphoid clcments~ but car- cin'bmas arising in other parts of the Waldeyer's ring~ such as tonsils and back of tongue~ in which the carcinoma cells are similarly interspersed with lymphoid tlssue~ are much less frequently associated with a high titer of antibodies to EBV-rdated antigens. Furthermore., according to-Teoh (~0), the presence of both lymphoid and epithelial structures was not observed in NPC metastases othcr than those in lymph glands. Posslbly~ EBV plays merely a passenger role in NPC. The increase in anti-EBV titers with ad- vancement of the disease does not differentiate between a passenger role and a causal relationship. o o • T!04132451

In ~th~ case~ |ncreas~ngly more viral antigens would pr~umably becom~ available for sfimulao ~on of antibody formation as the tumor rrm~ enlarges. The sarr/~ question has ~risnn with regard to BL. Pa~ent~ with ~is malignancy also reveal hlgh antibody ac6vhies to EBV-related antigens. • If the virus were merely a passenger in these lymphomas, other tumors, such as lcukcm|~s, rc~iculum cell sarcomas~ Hodgkln's d~scasc, and muhiplc myclomas should offer similarly favorable habitats for EBV, which, indeed, they do not (5). There is cvldcncc for a possibly genetically dctcr~ncd predisposition to develop NPC among Chinese (]5). Such a prcd|sposi6on could b¢ assoc|atcd with factors which pcrmh EBV to have either a direct function in" the transformation of target.cells or a priming role for subscqucnt action of carc.~nogcnlc or cocarclnogcnlc agcnts.~pr~- fcction~ and patterns of EBV-rclatfid scrologlcal rcactlons in a population at high risk, such as that of Hong Kong or "Singapore, mi~mh~p c''=~ ......... "-- ~ _.:.¥ LC ..... REFERENCES (I) I'~ O.~ Hr~z.~ W.~ and Dm~x~ V.: Rela~ou of B~kiR's ~umor-~iated h~-~ v~ to ~fio~ monvnude~..P~ Nat Acad ~ USA 59: ~I01, l~. ' (2) N~, J. C., Mc~, R. W., Hxsm, G., ~d ~ W.: Infccdo~ mononuclem~. Cl~ic~ m~mdom ~ rdadon m EB ~ andb~. "(3) G~ P., H~ D., Mov~ R. A, and ~ E.: I~6o~ ~ononudeo~: complem~t- ~g ~u~i~ to h~-Jike ~ ~iated B~ ]ymphom~ ~cnce 161 : 17~175, 1968. (~) ~ G, ~d H~ W.: I~unofluvr~cncc ~ d~ivcd ~m B~kiR'z lymphoma. J B~t 91: (5) H~ G., ~, W., ~roRv, P., D~u~ V., ~ ~" B~kiu'a lymphoma and con~ol ~ou~ J Nat ~nc~ I~t 43: 1147-I157, V~ ~d H~ O.: ~mp~a6~ studF of t~ B~kht minor c~h by I~ofluo~c~ • u~o~aphy, ~d ~on ~o~y. J V~ !: 8~7, 1~7. ET. AL. (7) Kr.zm, G., Kt.~m; E., and ~,~, P.: S~h for h~t d~ ~ BurHU lymphom~: mcanbrane immunofluo~c¢ t~ on blopsi~ and ~ue cul~ lin~ Can~ R~ 27: 251~20, 1~7. (~) K~, O., P~sos, G., H~, O., H~s~, ~., Go~s~m, G., ~nd ~rro~v, P.: Relation ~twc~ Epst~-B~ vir~ and c~l memb~e immunw fluo~ence in B~kht tumor c~l~ III. Comp~n ~f H~Hng of d~t membrane ~mmunofluor~ cencc and ~6-EBV rcacfivi6~ of di~c~nt J ~ ~cd 129: 697-705, 1~9. (9) K~m, G., P~, G., N~o~m, J. S., 3. 3-, K~m, E., H~, G., H~, W., and CL:rro~, P.: Rda~on be~e~ Epst~n-Ba~ viral and c~l memb~n¢ immunofluo~ence of Burkitt tumor celk. L Depend~ce of c~ membrane immunofluor~ence on pr~ce of EB ~p Meal 128:1011-1020, !~. (10) P~A~sos, G., ~, O., H~ G., H~, W., and CLarion, P.: R~a6on between Epst~-Ba~ ~r~ and c~ ~embrane i~munofiuor~cc ~ Burkltt minor c~is. IV, D~a6on be~e~ anu~o~ r~ponsible for membr~e and ~ i~un~ fluo~ce. J ~p Med 129: 707-718, E., G~o, O., W~L~sSO~, B.~ and P.: P~cipha~g ~fibody in human ~m an~g~ pr~t in "cuh~ed B~klu'z lymphoma c~ls. F~ Nat Acad Scl USA 55: 169~!7~, 19~. - (12) K~m, G., ~r~o, P., Hz~, G., H~, W., GEz~mo~ G.~ and O~, L. J.: EBV-~iated s~ologi~ pat~ns ~ a Bur~tt lymphoma patient d~g r~ion ~d recurrence. Int J Canc~ 4: 41~21, 1959. (13) ~ Sc~ ~., Fgme~¢, S., K~, G., W., H~, O., ~ ~, O., ~r~o~, P., ~d Ho, H. C.: Epst~n-B~ v~ ~6body pat1~ ~ carcinoma of ~¢ spa~. ~ Exp ~uno]. In (14) Ho, H. C.:. CHnlc~ $~mg of n~opha~'ngc~ no~ ~ use at ~e Medlc~ and H~ Depa~m~t' lns~mte of Ra~o]o~, Hong Kong. In pr~p~fion. (15) ~: Pa,~ II: C~c~ of N~opb~n~ UICC Mono~ S~I~, Vo[ 10.;'Nin~hIn~at Canc~ ~ngr ~a~, R. J. ~, ed.). B~]~, Heid~, New York, Spzing~ V~lag, 1~7, pp (16) H~, W., ~d ~, G.: ~t ~ of lhe" h~-group v~ ~a~ed wi~ c~m~ of ~e he~topole6c $yste~ In P~6v~ ~ ~. New Yor~ Academe Pr~ In~, 1~, pp l 0~124. E~t~-B~ v~ ~d c~l mem~r~c immun~ fiu~ence ~ B~Hu ~mor e~ls. II. ~mpa~on JOURNAL OF TIi~ NATIONAL CANER INSTITUTE i Ti04132452

NASOPHAP~3~'N~ EAL ~ARCINO}~A 231 of ~ and sera from pat~r_n~ w~th Burldtt', ]ymphoma and. infcct~ous rnononur-Icm~. J ~xp ]vfcd 128: 1021-1030, 19~8. (18) o~. T~ G.~ .AJ~R.OS~OHT, J. C., Ho, H. C.," and Kw.~, I'L C.: Lymphoblasto|d lransformat~on and prese.nc~ of herpes ~ viral par~de~ in a Chlnesc nasopharyngcal turnout cuhur~! h ~t~. Nature (London) RRl: 770-77}, 1g59. (1~) W~ss-r~m, 1¢. B.: The s|tc of o~q~n oF anaplas~ic tumors o1" ~hc pos~cr]or nasal space../'n Cancer in Africa (Clifford, P-I Linsdll C. A., and Timms, G. L., cds.). Nalrobl, East African Pub! Home., ! 968., p. 393. (20) Tzo~, T. B.: Epidcrrfioid cardnoma of the naso- pharynx a.mong Chinese. J Path Bact 73: 451-465~ 1957. VOL. 44~ NO. I~ ~]ANUARY 1970 T!0.4132453