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, ~ EXHIBIT NO., /1 npCt[Or14s a'Mf( Kr[nr r•A l,l. ~,t•-•l O.l`. ..o MIDK.[ N4.:.:-••1 10-• Homotransplontdtion of Lorynxes of Cigarette Smoke-Exposed Syrian Hamsters' (38760) Boirorr, thut.achuxru Oa/A F. HOMBCRGER, .a. H. HA`DLER. A. B. RUSSFIELD? .vo P. BERNFELD Bio-Rntocrh CumrdrJnrt dnJ B:•+-R,•t:,r.c4 1•icrruy, C,t•.rA.,,hr, ll.rctorbn.ttrt O:IJI. J•rl r Porholor<)'. Boiron Cnnniuy &•!wd nr trrlrcnr. B,nlaI C'nnPrtu,r ScGoo/ o/ GraJuorr Drnns.••.. In =0" of BIO inbred Syrian hamsters. carcinoma of the laryngeal respiratory epi- thelium developed after 70-100 wk of daily exposure to smoke generated from IRI Ken- tucky Reference cigarettes Even larger numbers of smoke-e!eposed animals de~el- oped less severe epttheltal changes in the area of the larynx bordering the .ocal cords and including the ventricle of %1orgigrtt ! I). Ilnder similar experimental condtuons in smaller numbers of BIO 87:0 hamsters. laryngeal carcinoma occurred in j" of the animals. epithelial changes lesser than car- cinoma in 13'%. and metal+l.tsia in 16":. Similar rates for lar.ngeal epithelial changes and carcinoma had pre% iousl% been obser%ed b% German investigators using German Ref- erence cigarettes and noninbred hamsters i:) The earliest malignant Ieswn induced by cigarette smoke in the hamster lar.ni was obser%ed 7) wk after the start of inhalatton, when the aged animal Mas close to death from degenerati.e disease attendant upon old age: therefore. little time remained for growth of the neoplasttc lesion and metasta- tic spread in this animal ! I 1. Actuall,v, no metastases of laryngeal cancer were found in these experiments, although metastases of a smoke-induced nasophar.ngeal sarcoma did oceur. It was postulated that transplantation of a cigarette smoke-induced early laryngeal carcinoma might lead to further growth of the tumor in a new host. 810 13.16 inbred hamsters, which had proved to be the most receptive hosts for homo- and isotransplantatton (3), were chosen for the present study. Because of the microscopic nature of the induced laryngeal cancers, it was impossible to select by pre- c Thrs trorlt w•as supported in p.n by l: SPHS Gen- enl Resnrch Suppon Grant No S01-FRdSS23 (D+vt- sion of Research Facihues arxt Resources). c rreaent address St. vincent Hosptal, Worcater, Mauachtuetts 01610. .itws microscopic study the best ;+ossic:e le:ions for transplantatton: rather, the irreeit possible number of smoke-exposed' Irr.nce; was taken for tr•rnsplantatwn into is mam 810 15. 16 eheek pouches as poiiible J%t :h: end of our smoke tnhalrtton cxren-r:e^ts 14 lar%nxes of smoke-exposed !}I0 IS 16 a-i- mals and 34 lar.nxes of B10 S7 :0 crn.,ke• exposed animals were a%ailable .ts iurc;nz material. .Nerhods. .I almals orid rro, ; d:rr, c Ft},)- cure of orrrmoh to cntnke Thcse e%pen-n:r:ts are described in detail e?ie%knere I In brief: 102 male BIO 15 16 hum,t:rs und ln equal number of m.tle B10 S7 :lls •cr: exposed S eonsecuti~vd.,%s each Aeek to 19 3: ei:arette smoke generated in .t modi- fled Walton-%1ornsse% re%erse smvker start- mg at age 90 da.s. Smoke exposure sessiens took place twtce ada% t.Ir % n;in. danrz whieh time the animals r:c;i~eJ t I:•sec exposure each minute to sn;ol.a .:nerateJ b% :S ml pulTs of :-sec dur,lr.on !""E"aed 5% exposure to room air for 1: iec t^ei,re the next putT. All animais wed ;or transrl.t.^.ta- tion of lar)ngeal tissue recet%ed thii treit- ment for 76-90 wk: at the end of which time they were killed. Sham-smoked animals underwent the same manipulation .ts t~e smoke-exposed animals. except that there were no cigarettes in the machine. Traniplantatlon of lurtngeal tissue B10 15.16 males were chosen as hosts because o' their great tolerance to transplanted ussue These animals, all in good health. were 60-a, : days old at the time of transplantation The larynxes of the smoke•exposed an: mals were remo.ed and cut hurizonwlly v sections of approximately 0.5 mm thickness Such ring-shaped sections, often contatnin parts of the .'ocal cords and the ventrtcle L Siorgagni, were inserted through a sm., incision made in the everted chcek pouch : the lightly anesthetized BIO 15.16 hosts. 142 e.rr~t O 1971 w w satt r.r 4trensw ta.lra0 w MMor wuwor....a t..r 1' R i i N 0422600

a /1 HOMOTRANSPLA`TAT/ON OF HAMSTER LARYNXES 143 TaBLE I animals, the epithelium lining these cysts was I 1 i D ir..r a-..T.a s H•Kt antma!s train o reacment HistoloSy of transplants. ~ur.wtn8 s .wmous :•pithclium IS 16 15 Smoke exposed IS I 13 16 - Sham.,moked - 13 16 - Cige hcyJ - 87 20 ]4 Smoie e%vosed 67 1) 87 20 17 Sham-,moned 17 0 d7 :0 9 Cage neW 9 0 In the case of the 810 15.16 srnoke-ex- posed .tntmals, onl% 13 ie¢ments from 15 larNnxes of smoke-exposcd .,n,mals were ,ttatlable :'or tr.tnapl.tntattJn. All sham~ i-moked and untreated ca.e-held control 810 15 16 antm.tls h.td been ,,tcrtticed prior to our decision to :.,rr,r out transpl.tnt.ttton studies Among the 810 smoke-esiposed animals and their controli. :> >mohe-etposed ant- muls }telded 67 trinsDiint.tble l.trvn, itC- trons. and 11 sham-)mokeJ .tnmmals ga.e 17 larynx sections Al;o nine cage-held control antmals yte:dcd nine transplants At Meekly tnter%ais. the transplants were inspected by e.erting the cheek pouch of the lightly etherized host animal. Of the 108 transplants in as many host animals, :; had to be retransierred into new hosts because of signs of infection or rqectton. In only three cases was this done more than once. After the transplants had been left rn s+ru for at least 10 mo. the host animals were killed and the transplants and cheek pouch envelopes Mere remo%cd and fixed in Tellyesniczky's Autd for hutologscal examination of hems- toxyltn-eostn-statned secttons- Rrsulrs. Htsrological r.caminarlon. With the exception of ai-ery small number of transplants (or retransplants) lost by rejec- tion or infection, all transplants had become well vascularized and had grown larger than when originally inserted into the cheek pouch. Common to .tll transplants, whether from smoke-cxposed larynxes or from con- trols, was the formation of occasional multi- laculor cysts Mhtch were usually lined by i single layer of low cubo,d.+l cells In the case of all transplants from nonsmoke-exposed usually atrophtc, sometimes cubotdal, but ne%er multtstratt6ed squamous. In most of the sections from smoke-exposed animals. the c%sts contained desquamated epuhetiat cells, and squamous eptthelium was often observed (Figs. 1.nd 2). Six specimens (9'',*) from smoke-exposed 810 87.:0 ham- sters contained squamous epithelium with some degree of epithelial hyperplasia or nuclear at)pia tFigs. 3 and ;). One section showed a nest of squamous cells apparently in the middle of cartilage which might well be an, artifact. There Mas no clearcut e.i• dence of malignant transformation 'Fig. $). In the case of the 810 37.:0 hamsters. statts- ttcal e.aluauon by Fisher's exact test (4) showed that the incidence of squamous ept- thelium in the smoke-expoied antmals was st;ntficantly higher than in controls not exposed to smoke at a probability le~el of less than I" see Table I1. All spectmens !those from smoke-exposed animals as well as controls) contained well presev,ed cartilage, often with areas of cal- cification or osseous metaplasta In a few cases. the cartilage pattern was characteristic of the upper larynx, the region where hyper- plastic changes occurred in the intact. smoke- exposed hamsters. In some spectmens, the cartilage pattern was that of normal tracheal , t I rtngs. In most specimens it was impossible to determine the level of the upper respiratory tract whence the transplant originated. Two specimens included a well preser%ed, normal thyroid gland (Fig. 5). Thete was a limited amount of surrounding connective tissue, which was not remarkable. Any organ of the host which showed gross anomalies was studied histologically. The commonest abnormalities seen were renal amyloidosis and reactive hyperplasia of the lymph nodes. The remaining pathology ob- served was comparable to that generally seen in aging hamsters of the 810 15. 16 stratn. Orscussron. Hyperplasia of the laryngeal epithelium caused by cigarette smoke per- ststs for up to one year in a host animal without further exposure to smoke. No hyperplasttc epithelium was seen in tran.- planu of tracheas which had not becn c~- posed to smoke prior to impl,tnt.,ttoty It was also ubsered th.,t uth:r aJult normal tissues such ls thynaJy l.,r.ny~.tl ,,nJ , l.r T>j"~'. { 11 S "i 0`"i' 2" [J 0S.

a /1 144 CTR HN 042602

/k HO%tOTRANSPLaNT+T10I~ OF HAsrSTER L1RY%XES tracheai caralaee anJ norm.ti tracheo•lann- gcal r^tthcltum sursi., tutJ:r thc,r c.,nJt• uons. hence. mere ;urst%jl of a mctwpljsuc tlssue is no proQl of its m,lllenanc)'. HOM'• eser% it is of interest that esen in the absence od,i further >tumulus such .is smoke. a trans- Flanted alttred tissue mjy retJtn its anomal- ous characteristics 143 I Bernfeld. P. Homburger. F, and R ussAeld A. B, 1 Nat Cancer Inst SJ a'. 11:1 tv'a, 2 Oonmcn-dl. w. J •2: Cancer Inst 51:6~, 17,81 t97-41 ) HanJler. A. N, and Cosman. E 8, Life Sci 14. i1S7~1971i J OlJham. P t) ."Measurement in Medicine," p 94, Philadclphia. Ltppincolt Companr i 1968, Recerved September 6. 19'J P 5 E 8M 1975, vol. 149 Fic I Th siIlustrates a metaplastic tracheal tube from a smokt«posed 81017 20 nvk hamster fcantlage on eitrenx lo*er lefti in a transplant havtn{ resided I yr in the cheek pouch of a femak B(O 13 16 hamster H E. A _00 ) Fic 2 Section from tracheal tmplant from smokee.posed BIO 17 220 nyk hamster placed I yr earlie,r tnto check poucn of male B!O IS 16 host (HE, x:00). In the rtudss of a chron+c mAsmnutory taudast (which was contained in a large cysU, then is a well prcscrved tubular structure lined with a tall columnar eptthelwm which in places has changed into a metaplasuc squamous epuhelwm. Fic 3 Section from a eracheal transplant from a smokes.posed mak 81017 20 hsnsster tn cheek pouch of a fcmak 810 13 16 hamster after I yr,HE, x2001 The tracheal tube, bordered by carttlage shows porttotu with miss n6 or atroph+c epithelium itopl as well as a nliated, taU colurtwr eptthehum and a nodulat a{pcptt of meuplastic cells bulging into the submtxosa tat anow/. Fic a Same tield as Fig 5 tHE, x1701, ckarly showtn6 cilia and eptthelutl chan6a descrtbtd iu legend 3. above Fic S Section from implant of tracha or laryn: from sham•smoked BIO IS 16 male hamster havtnM re• mursed for to mo n cheek pouch of 810 15 16 male host cHE, X 2001, sho*m6 wrvtvin6 thyroid {lusd and an tage The tar>nacal epuhehum has atrophied CTR HN i..d426.-+ ti..d ...Jt