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1997;3:3-9.Clin Cancer ResW Xia, Y K Lau, H Z Zhang, et al.survival of patients with oral squamous cell carcinoma.Strong correlation between c-erbB-2 overexpression and overal

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Vol. 3 , 3 -9 , Janu ary 1 997 C lin ic al C an cer R ese arch 3

Advan ces in Brie f

S trong C orrela tion be tw een c -erbB -2 O verexp ression and O vera llSu rv iva l o f Pa tien ts w ith O ra l Squam ous C ell C arcin om aW eiya X ia , Y iu -K eung L au , H ua-Z hong Z hang ,A i-R u L iu , L e i L i, N obu taka K iyokaw a,Ga ry L . C la ym an , Ru th L . K atz , andM ien -C h ie H ung D ep artm en t of Tum o r B io lo gy an d B reas t C ancer B as ic R esearchP rog ram 1W . X ., Y -K . L ., N . K ., M -C . H .}, Section s of C yto lo gy an dIm age Analys is, D epar tm ent o f P ath o lo gy [H -Z . Z ., R . L . K .], an dD epartm en t o f H ead an d N eck Surgery [G . L . C .], T he U niversity o fTexas M . D . A nderson C ancer C enter, H ouston , T ex as 7 7030 , an dD epartm en t o f O ra l P a th o logy , S hangha i S tom ato lo gy C olleg e ,Shangha i S eco nd M edical U nivers ity , 2 00016 People s R epub lic o fCh ina [A -R . L ., L . L .]

Abstrac tO verexp ress ion o f c-erbB -2 (a lso know n as HER -2ln eu )

has been found in many hum an cancers, in c lud ing head andneck squam ou s cell carc in om a (SCC ). W e th ere fo re ex am -m ed ex pres sion of the onco pro tein in o ral SC C pr imarytumor samples and com pared its rela tio nsh ip w ith clinicals tages and su rv iva l ra te. O u t o f 8 0 ca ses o f ora l SCC , h ighexpre ssion leve l (+ + or + + +) o f c -e rb B -2 w as fo und in 41cases . O f th e 80 cases w ith fo llow -up in form ation , 39 w erefu rth er in vestiga ted fo r the co rre la tion of ex press ion leve l o fc -erbB -2 and su rv iva l ra te. O v erexp ression of th e on copro -tein w as s ig nif ic an tl y co rre la ted w ith shor ter overa ll su r -v iva l, a nd th e patien ts w ith low and no expression o f c -erbB -2 had m uch h igh er su rv iv a l rates. Overexpression ofc -erbB -2 w as a ls o s ig ni fi ca ntl y corre la ted w ith noda l stag eand m eta stasis . W e found tha t h igh exp ression leve l o f c -erbB-2 w as frequ ently d etected in ora l can ce r ce ll lin es butnot in th e o th er h ead and neck SCC ce ll lin es. T hu s, w econ c lud e tha t o verexp ression of c - e rbB-2 is a frequen t even tin ora l SCC and is correla ted w ith poor su rv iv a l and m ay beu sed a s a poor p rognostic facto r.

Rece ived 6 /24 /96 ; rev ised 10 /419 6 ; accep ted 10 /9 /96 .T he cos ts of p ublica tion of th is art ic le w ere def ray ed in part by thepaym ent o f p age ch arg es. This a rtic le m ust the re fo re be hereby m arkedadver t i semen t in accordance w ith 18 U .S .C . S ec tion 1734 so le ly toind ica te th is fac t.I The resea rch w as supported in part by United S ta tes D epartm en t o fH ealth and Hum an Serv ices C an cer C en ter C ore S upport G ran t CA -16672 from the N ation al C ancer In stitu te, N IH G ran ts CA 58880 an dCA60856 ( to M -C . H .), and G ran t n o . D AM D 17-94- J4315 (to M -C . H .)from the D epartm en t o f D efense.2 To w hom requ ests fo r rep r in ts shou ld be ad dressed , a t B ox 079 ,D epartm en t o f T um or B io log y , T he U niversity o f T exas M . D . A nd er-son C ancer C en ter , 1515 Ho lcom be B lvd ., H ous to n , TX 77030 . P hone :(7 13) 792-3668 ; F ax : (7 13) 794-4 784 .

IntroductionT he W HO has desc ribed ora l cance r a s one of the 10 m ost

comm on cancers in the wor ld (1 ). O ra l SCC 3 is th e m ostcomm on h isto pa tho lo g ical type o f o ral can ce r , accoun ting fo rap prox im ate ly 91% of a ll o ra l m alignanc ies (1 , 2 ). Th e pro gno-s is of pat ien ts w ith oral SCC rem ains p oor ; th e 5-yea r surv iv alra te ave rages abo u t 50% and h as no t ch an ged m uch ove r th epast 1 0 years (3 ). T here fo re , the re is in te rest in de te rm in ingwhether spec if ic g ene a lte ra tions occur in ora l cancer andwheth er they are correla ted to d isease beh av io r. S uch stud iesm ay prov id e m o re accura te an d u sefu l p rognos tic m arkers andev en tua lly m ore e ffec tiv e trea tm en t.

T he c -e rbB -2 gene (a lso kn ow n as HER -2 /neu ) en co des atransm em bran e p ro tein o f M r 185 ,0 00 (8S ), w ith e xte ns iv esequence hom ology to EG FR (4-6 ). L ike EG FR , c-e rbB -2 hasin trins ic ty rosine k inase ac tiv ity (7 -10 ) and can in te ract w ithm any d iffe ren t ce llu la r p ro te in s, such as s rc -hom o lo gous co lla -gen , ph osp ho lip ase C -rny , an d G TPase -ac tiv atin g pro te in , w h ichm ed ia te a signa l tran sduc tion p athw ay (1 1 , 12). O verexp ress io nof c -e rbB -2 has b een show n to increase m etasta tic po ten tia l byprom o tin g th e m ultip le adh esion an d invas io n steps of them etas ta tic cascade (13). T h is sug gests tha t the c-e rbB -2 gen em ay p lay an im por tan t ro le in carc in ogenes is . c -e rbB -2 overex-p ress ion has been found to corre la te w ith poor su rv iva l o fsevera l hum an cancers , inc lud in g breas t, ovarian , lu ng , andstom ach can cers (14-20 ). A lthou gh c-e rbB -2 overexpressionhas been de tec ted in h ead and neck SCC (21-23 ), a co rre la tio nbe tw een c-erbB -2 overex press ion and surv iva l has no t beenrep orted in o ral SCC .

T he purposes o f th is study w ere to exam in e th e exp re ssio nof the c-e rbB -2 in ora l tum or spec im ens as w ell as in head an dneck SCC cell lin e s and to d ete rm ine th e rela tion sh ip be tw eenth e p ro te in expression and surv iva l in o ral SC C . For the latte r,we investiga ted re tro spec tive ly a g roup o f p atien ts w ho hadbeen trea ted b y su rg ery w ith o r w ithou t po stop era tive chem o -the rap y , rad io the rap y , o r b o th . O u r re su lts in d icate th a t ove rex -pression of c -e rbB -2 is sign ifican tly corre la ted w ith shorte rov era ll su rv iva l in o ral SCC .

M ater ia ls and M ethod sP atient S pec im en s. E ig h ty spec im ens of p rim ary ora l

SCC w ere ob ta ined from th e D epartm en t of O ral Pa tho lo gy ,N in th P eop le s H osp ita l, S hangha i S econd M edica l U n iv ersity ,Peop le s R epub lic o f C h ina . These pa tien ts (60 m a le and 20fem ale ) underw en t su rg ica l trea tm en t in th e hosp ita l be tw een1989 and 1990 . Th eir ag es rang ed from 10 to 76 years (m ed ian ,54 y ears ). T h e prim ary tum o rs w ere graded by the W HO clas -

3 The abbrev ia tions used are : 5CC , squam ou s ce ll ca rc inom a; EG FR ,ep iderm al g row th fac to r recep to r; MOD , m ean optica l densi ty ; T hM ,tumor-node-metas tas is .

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4 c-e rbB -2 Expression in O ra l SCC

Ta b le 1 R ela tio nsh ip betw een histo lo g ic al g ra d e an d c-e rbB -2 exp res sio n in o ral SC C s

H isto log ica l g rade N o. o f casesL ev el of exp res sio nb

% positive+ + + + +1 -IIIIII

Tota l

63 (78 .75 )14 (17 .5 )3 ( 3. 75 )

80(100)

8(10)7 (8 .75 )0 (0)

15 (18 .75 )

19(23 .75)4 (5.00)3 (3 .7 5)

26 (3 2.5 )

36(45)3 (3 .75 )0 (0)

39 (48 .75 )

85 .7092 .90

100 .0086 .40

a p < 0 .01 by th e x2 test .b No . of cases (%) .

sification of h isto log ica l d iffe ren tia tio n (24) . S ix ty -th ree casesw ere d ef in ed as grade I , 14 as grade II, and 3 as grade Ill. T h etis sue s inv o lv ed in tum or in clu ded to ng ue (40 ca ses ), gum (11ca ses) , flo or o f the m outh (8 cases), pala te (5 cases), ch eek (10cases) , m ucosa o f lip (3 cases ), m and ib le (1 case ), and m uco saof m and ib le m olar a reas (2 cases ). T he spec im ens w ere rou-tine ly fixed in fo rm alin , em bedded in para ffin , sec tioned seria llyevery 5 p .m , and sta ined w ith H& E . A ccura te and d e ta iledin fo rm atio n o n 39 of 80 pa tien ts w as ava ilab le . T h ese 39 pa-tien ts w ere the re fo re u sed to ana lyze the corre la tio n be tw eenovera ll su rv iva l and c-e rbB -2 expression . T hey rece iv ed e ithe rch em o th erapy or rad io the rapy or bo th afte r the ir su rg ica l trea t-m en t. S ince th e su rg ica l trea tm en t, 10 pa tien ts have d ied o f theprim ary tum o r and one h as d ied of m etastasis o f the p rim arytum o r to the lu ng .

Imm unoh istoch em ica l S ta in ing . T he imm unopero x i-dase s tain ing m ethod used w as m od if ied from the av id in -b io tincom plex techn iqu e desc ribed by H su and R ain e (25) and H su etal . (26 ). B r iefly , tissue sec tion s w ere deparaffin iz ed an d d eh y-dra ted in a graded series o f a lcoho l. T hen , they w ere d igested in0 .0 5% tryps in fo r 15 mm , b locked in 0 .3% H202 in m ethano l fo r1 5 mm , an d treated w ith 1% (vlv) norm al horse se rum for 30mm . Then , the s lides w ere incuba ted fo r 3 h at room tem pera tu rew ith 0 .003 p g /m l c -neu (A b-3) m onoc lo na l an tibody d ilu ted1 :3 200 . A fte r ex tensive w ash ing w ith PBS , the slides w ereincub ated fo r 30 mm at room tem pera tu re w ith b io tiny la ted goa tan tim ou se IgG an tibody d ilu ted 1 :200 in PB S. T he slides w eresubsequen tly incuba ted fo r 60 mm at room tem pera tu re w ithav id in -b io tin -perox id ase com plex d ilu ted 1 :10 0 in PB S. T hepero x id ase-ca ta lyzed pro duc t w as v isua lized w ith 0 .12 5% am i-no-e thy l ca rb azo le chrom ogen stock so lu tion (S igm a Chem ica lC o .). B e tw een s tep s, the s lid es w ere r in sed fo r 2 m inu tes in PB S(pH 7.6 ) th ree tim es. A fter lig h t coun te rs ta in ing w ith M ayersm odified hem ato xy lin (S igm a Chem ica l C o .), the s lides w eredehydra ted and m oun ted . N ega tive con tro ls , in w h ich PBS w asused ins tead of the prim ary an tibo dy , w ere ru n w ith each b a tchof s ta in in g . A prev io usly iden tified stron g ly sta in ing tum o rtissue sec tion w as u sed as a p ositive con tro l. In te r-assay andin tra -assay con sistency w as m ain ta ined by inc lud in g these pos-itive and nega tiv e con tro ls w ith each ba tch of slides sta ined . T heprepared s lid es w ere exam ined by lig h t m ic roscopy . T hosetum or ce lls tha t w ere imm unos ta in ed w ith red granu les w ereco nside red p ositive , and tho se ce lls w ith ou t any imm uno-s tain ing w ere cons ide red nega tive . A ll the cases w ere rev iew edind ep en den tly by tw o pa th o log is ts . Th e imm unoreac tiv ity o fc-e rbB -2 w as ranked in to four g ro ups accord ing to p ercen tageand sta in ing in tens ity o f pos itive ly s ta in ed tum or ce lls. B o th

cy to p lasm ic an d m em brane s ta in ing w ere ob served . T he casesw ith s tron g stain ing (> 50% cells sta in ed) w ere scored as h igh ,th ose w ith m od erate s ta in ing (20-50% cells sta ined) as in te r-m ed ia te , those w ith w eak s ta in in g (up to -20% cells s ta ined) aslow , and those w ith n o sta in ing as n eg a tive .

Quan tita tive C om pu ter ized Im age A nalysis . c-erbB -2imm unosta in ing w as ob jec tive ly qu an tified by com pute rizedim age an alys is w ith a SAMBA 4000 C ell Im ag e A nalysis S ys-tem (Im ag in g P rod uc ts In te rna tiona l, Inc ., C han tilly , VA ), and ax 20 obje ctiv e . T he im ag e analyze r w as an in te gra ted sy stem ofW indow s (M icroso ft, R edm ond , W A )-based softw are fo r den-s itom etric , m orphom etr ic , an d red-g reen -b lue to h ue sa tu ra tionin tens ity co lo rim etric an alys is o f ce lls and tissues an d cons istedo f N ikon m icrosco pe w ith X 10 , X 20 , an d X 40 ob jec tiv es and aJV C 3-C CD KY -17U v id eo cam era . T he SAMBA 4000 im ag eana lyzer consis ted of an IBM 80-386 /33-MH z clon e w ith aVGA graph ics bo ard , 80-M B hard d isc , 8 M B of RAM , Syques t88 -M B rem ovab le cartridg e sys tem , N ikon L abopho t-2 m icro -sco pe , H ew lett P ackard P a in tJe t co lo r p rin te r, M itsub ish i co lo rv ideo copy processo r (p rin te r), and Po la ro id f reeze-fram e v ideoreco rde r w ith 35-mm film processing and instan t p rin ts capa-bility.

T he softw are enab led the o pera to r, a fte r eva lu ating sev era lfie lds o n con tro l s lides , to se t a dens ity th resh o ld v alu e fo r theb es t d isc rim ina tio n be tw een ce ll nuc le i and cy top lasm , strom a,o r b ackg round . S im ila rly , a th resho ld fo r pos itive v alues w aso b tained b y averag ing th e resu lts from 10 fie lds o f nega tivecon tro l tis sues in w h ich the prim ary an tib ody w as rep laced w ithan iso type-m atched irre lev an t an tibody . T he th resh o ld va lu e w asthe m in im um abso rbance a t w hich the im age ana lyzer w ouldd isc rim in a te nega tiv ely from positive ly s ta ined cy to p lasm . A fte rtha t v a lu e w as read , b ackg round sub trac tion w as au tom atica llyp er fo rm ed on every tis sue afte r m easuring an em pty fie ld on thes lide . For each tissue , w e m easured the cy to p lasm ic area , MOD ,labe lin g index (lab eled nuc lear area /to ta l n uc lea r a rea m eas-u red ), an d qu ick score (labe ling index X MOD ). T o p erm its im u ltaneou s m easurem en t o f c -e rbB -2 imm uno sta in ing , thesam ples w ere imm unosta ined by av id in -b io tin com plex tech-n iqu e (25 , 2 6), w ith am ino-e thy l ca rb azo le chrom ogen as anind ica to r and M ay er s hem atoxy lin so lu tion as a coun ters ta in .

Stat is tica l A nalyses . Th e x2 te s t w as used fo r ana lys is o fcon tingen cy tab les, w ith P < 0 .0 5 as th e crite rion of sta tisticalsig n if ican ce . Su rv ival c urv es w ere c alc u la ted b y the m eth od o fKaplan an d M eie r, and d if fe rences b etw een cu rves w ere ana-lyzed b y the W ilcoxon test.

Cell L in es and Cu ltu re. S ev en head and neck SCC celllines w ere o b tain ed from th e D ep artm en t o f H ead and N eck

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.# { 248} .F ig. 1 Imm unoh istochem ica l s ta in in g o f o ra l SC C for c -e rbB -2 . A, h igh ; B, in te rmed ia te ; C, low ; D, no exp ress io n . X 400 .

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Su rgery o f th e U nivers ity o f T ex as M . D . A nd erson C ancerC en te r. 6861LN -l w as derived from lym ph nod e m etas tasis o fthe tongu e. T he o th er ce ll lin es w ere desc ribed prev io usly (27).T he hum an b reast cancer ce ll line M DA -M B -453 , w hich hasov erexpress ion o f c -e rbB -2 , w as used as a p ositive con tro l in theimm unob lo tting experim en t. A ll cells w ere grown in DM EM /F l2 (L ife T echno log ies , Inc ., G rand Island , N Y ) supp lem en ted

w ith 10% fe ta l bo v ine se rum and an tib io tics . C e lls w ere grow nin a h um id ified incub a to r a t 37#{176}Cnd er 5% CO 2 in a ir.

C ell L ysis and W estern B lot Ana lysis . T he cells w eregrow n to abou t 90% confluen ce befo re h arv esting . T hey w erew ashed tw ice w ith co ld PB S and then lysed on ice w ith 0 .5 m lof R IPA -B lys is bu ffe r (20 m N a2PO 4, pH 7.4 , 150 m N aC I,1% T riton X -lOO , 1% apro tin in , 1 mM pheny lm ethy su lfon y l

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6 c-e rbB -2 Exp ress io n in O ra l SCC

fluor id e, 100 m r N aF , an d 2 nm i N a3VO4 ). T he cells w ere thenscrap ed from the tissue cu ltu re d ishes , and the residua l ce lld eb ris w as rem oved by cen tr ifu ga tio n . F or imm unob lon ing , thesam p les w ere separa ted by 6% SD S-p o lyacry lam id e ge l e lec -to rph oresis and transfe rred to a n itrocellu lose m em brane . N on-spec ific m em b ran e b in d ing w as m in im ized w ith a b lock ingbuffe r (5 % nonfat d ry m ilk in Tw een 20-PBS ). T h e m em branew as then incub ated w ith prim ary an tibod ies (c -neu (A b-3)m onoc lon al an tibo dy , O ncogen e S c ien ce, Inc ., U n io nda le, N Y )fo llow ed by incuba ting w ith h orse rad ish perox id ase -goa t an ti-m ou se an tib ody (B oehr in ger M annhe im Corp ., Ind ianapo lis,IN ) .

ResultsImm unoh is tochem ical S ta in ing of O ral 5CC for

c -erbB-2 . The resu lts o f imm unos ta in in g of 8 0 prim ary ora lSC C s for c -e rbB -2 expression are sh ow n in T ab le 1 . There w asw ide varia tio n in the leve l o f c -e rbB -2 ex press ion in th e prim aryora l SCC sp ecim ens . F ifteen ( 1 8 .8% ) of them show ed h ighlev els o f cy top lasm ic an d m em b rane s ta in in g , 26 (3 2 .5% )show ed m edium leve ls , and 39 (4 8 .8% ) show ed low /no leve ls o fimm unoreac tiv ity . A represen ta tive exam ple is sh ow n in F ig . 1 .Fo r c-erbB -2 expression , 54 of 6 3 cases o f g rade I d isease(85 .7% ), 1 3 of 14 cases o f g rade II d isease (92 .9% ) and a ll 3cases o f g rade III d isease (100% ) dem ons tra ted pos itiv e s ta in -ing . S ta tis tica l ana lys is b y th e x2 tes t show ed tha t h isto log ica lg rade w as s ig n ifican tly assoc ia ted w ith expression leve l o fc -e rbB -2 exp ress ion (P < 0 .01 ; T ab le 1 ).

Corre la tion b etw een c-erbB -2 E xpression and P oorSurv iva l. T he s ign ifican ce o f the express ion of the oncopro -tein in hum an oral SC C was stud ied re trospec tiv e ly in 39 casestha t had been fo llow ed up s in ce the ir su rg ica l treatm en t. T ab le2 pro v ides c lin ica l da ta fo r the 39 ora l SCC patien ts . T heSAM BA 4000 ce ll im ag e analys is system w as u sed to ob jec-tiv ely qu an tita te cy to p lasm and m em brane sta in ing of the tum orspec im ens to d ete rm ine the exac t am oun t o f c -e rbB -2 pro te inexpression in ora l SCC . T he re liab ility o f the m etho d has beend iscussed prev io usly (28) . T he tum or spec im ens w ere observ edunder a ligh t m icroscop e conn ected w ith a com pute rized im ageana ly sis sy stem . T en m icroscop ic fie ld s from each of the spec-im en s w ere ana ly zed . T h e o ncopro te in leve l w as m easured asimm unos tain ing absorbency . N ex t, the ex press ion lev els w ered iv ided in to th ree g roups : h ig h ex press ion , 1 1 cases (28 .2 1% )w ith a M OD of 26 -70 ; in te rm ed ia te ex press ion , 8 cases(20 .51% ) w ith a MOD of 12-2 6 ; and low expression , 20 cases(51 .28% ) w ith a MOD of 0-10 . T h ere w as a s ign ifican t co rre -lation be tw een the M OD read ing an d v isua l q uan tifica tion o fp ro te in ex press ion leve ls in the tum or spec im en s (P < 0 .001) .

T he re lation sh ip b etw een TNM stag ing and c-e rbB -2 ex-p ress ion is shown in T ab le 3 . T he exp ress io n of c -e rbB -2corre la ted w ith nod al s tag e , as w ell as m etas tasis, w ith sta tis tica ls ign i f icance (P < 0 .05 b y the x2 te s t). T he resu lts su ggest tha tc -e rbB -2 exp ress ion m ay be assoc ia ted w ith ora l cancer m etas-tas is, con sisten t w ith an im al experim en ts tha t ind ica ted tha tenhanced c-erbB -2 expression increased m etas ta tic p o ten tia l(13 ). A lso , h igh expression leve l o f the oncop ro te in w as sign if-ican tly assoc iated w ith a shortened overa ll su rv iva l ra te, and th epa tien ts w ith low and no expression had s ign ifican tly h igher

Table 2 Charac te ristic s o f 3 9 o ra l SCC sP atien t charac te ristics N o . o f pa tien ts

A ge , y rs50 22< 50 17

Se xM ale 29Fem ale 10

Prim ary tum or siteTongue 19G um 7Floo r o f m outh 5O th er 8

H isto lo gica l g radesI 33II 6

Th MTum or size

T1 20I2 15T3 4

N Od al s tag ecN 0 29N 1 2N 2 2N 3

Metas tas is6

M 0 28M 1 11

c -e rb B- 2 e xp re ss io nLow or no ne 20In te rmed ia te 8H igh 11

Surv iva l, m on ths af te r d iagnos is60 29

a A ll p atien ts rece ived su rg ical trea tm en t.b T1, tum or 2 cm in grea tes t d im en sion ; T 2 , tum or >2 cm bu t 4

cm ; T 3 , tum or >4 cm .C N 0, n o reg iona l lym ph node m etastas is ; N 1 , m etas tas is to a s ing le

lym ph node (2 cm ); N 2 , m etas tas is to one or m ore lym ph nodes (>2cm but 5 cm ); N 3 , m etas tasis to a lym ph node (>5 cm ).

d d is tan t m etastasis ; M 1, d is tan t m etas tasis.

su rv iv a l ra tes, as shown in F ig . 2 (P < 0 .00 1 by th e W ilcoxontest).

Wes te rn B lot A nalysis o f H ead an d N eck SCC CellL in es . W este rn b lo t ana lys is revea led tha t a h igh leve l o fc -e rbB -2 w as fou nd in a ll th ree ora l cancer ce ll lines exam ined(Tu 138 , 68 6 /LN -l, and Tu 167 ) and th at th e o thers four lineshad on ly low or no ex press ion re la tive to th e b reas t cancer ce llline , M DA -M B -453 , w hich is know n to o verexpress c -e rbB -2(F ig . 3 ). T h is resu lt confirm s the h igh frequen cy of c -e rbB -2overexp ress io n in ora l SCC and a lso sug gests tha t c -e rbB -2expression m ay no t be comm on in o ther head and n eck SCC s.

DiscussionW e prev iou sly d em onstra ted tha t the re w as a s ign ifican t

asso c ia tion b e tw een c - e rbB-2 overexpression and ora l cancerp rogression (23). In th e s tudy rep orted here , w e used a d iffe ren tcohort to fu rthe r inves tiga te the re la tionsh ip b e tw een c-e rbB -2expression and pa tien t su rv iva l, h isto log ica l g rad e , and TNMstage . T he c-e rbB -2 g en e prod uc t w as ex pressed in 86 .4% of the

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Low /n one (n= 20)

I-ci)

80

60

40

20

L.In te rm ed ia te (n=8)

High (n=ll)

C lin ical C ancer R esearch 7

Table 3 Rela tio nsh ip b e t w een ThM stag ing a nd c-e rbB -2 expression in 39 ora l SCC s

TNM No . o f cases (% )L ev el o f e xp re ssio n

H igh In te rm ed ia te L ow /no neTum o r s ize

T1T2T3

21 (53 .85 )1 3 ( 33 .3 3)5 (12 .82)

4 ( 10 .2 6)4 ( 10 .2 6)3 (7 .69)

3 (7 .69 ) 14 (35 .90 )3 (7 .69 ) 6 (15 .38 )2 (5 .13 ) 0 (0 )

P

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cy ,L( )c

I

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C lin ical C ancer R ese arch 9

c -erbB -2 gen e in hum an head and n eck ca rcinom a. A n ticance r R es., 12 :2273 -22 76 , 19 92 .23 . H ou , L ., S h i, D ., Tu, S -M ., Z hang , H -Z ., H ung , M -C ., an d L ing , D .O ra l cancer p rogression and c-e rbB -2 /neu pro to -o ncogene exp ress io n .C an cer L ett ., 65 : 215 -22 0 , 1 99 2 .24 . W ashi , P . N ., C ohen , B ., Lutrh ra , U . K ., and T or lon i, H . H isto lo g ic altyp ing of ora l and o ro pha ryn gea l tum ors . In : In te rn at io na l H is to lo gic alC lass ific ation o f Tum o rs, N o. 4 , p p . 17-18 . G enev a: W HO , 1971 .25 . H su , S . M ., an d R aine, L . D iscov ery o f a h igh a ffin ity o f ph aseo lu svu lga ris a gg lu tin in (PH A ) w ith gas tro sec reting c ells . Am J . C lin .Patho l . , 77 : 396-4 00 , 198 2 .26 . H su , S . M ., R ain e, L ., a nd Fange r, H . U se o f av id in -b io tin -p erox -idase com plex (A BC ) in im m unop ero x id ase te chn iqu e: a com pa risonbe tween A BC and un labe lled antib ody (PA P) p rocedu res. J . H istoch em .Cy to ch em . , 2 9: 5 77 -5 80 , 1981.27 . Beckhart, R . N ., K iyok aw a, N ., X i, L . , L iu , T -J ., H ung , M -C .,E l-N agg ar, A . K ., Z hang , Z -H ., an d C laym an, G . L . HER -2/n eu on co -gene cha rac terization in head an d neck sq uam ou s c ell ca rcinom a. A rch .O to lary ngo l. H ead & Neck Su rg . , 121: 1265-1 270 , 1 99 5 .28 . E steban , J . M ., A hn , C ., Meh ta , P ., and B attifo ra , H . B io log icsign ificance o f qu an tita t ive es tro gen recep to r im m unohis tochem ica l as-say by im ag e analysis in bre ast c anc er. A m . J . C lin . Pa tho l., 102:158-162 , 199 3 .29 . E isbru ch , A ., B lick , M ., L ee, J . S ., S ack s, P . G ., an d Gutterm an , J.A nalysis o f th e ep id erm al grow th fa ctor re cep tor gen e in f resh h um anh ead an d n eck tum o rs. C ancer R e s., 47 : 3603-3605 , 1987 .30 . Ish itoy a, J ., T oriy am a, M ., O gu ch i, N ., K itam u ra, K ., O h sim a, M .,A san o , K ., and Y am am oto , T . G ene am plific ation an d ove rex pre ssio n ofE G F r ec ep to r in squam ous c ell ca rcin om as of the h ead and neck . B r. J.Cancer , 59 : 559-562, 1988.31 . San tin i, J ., F orm en to , J. L ., F ranco ual , M ., M ilano , G ., S chn eid er, M .,Dassonvil le , 0. , a nd D em ard , F . C ha rac te riz atio n, quantif icat ion , and po-ten t ia l c lin ical va lue o f th e ep iderm a l grow th fa cto r re cep tor in head andneck squ am ous ce ll carcin om as. H ead & N eck , 13 : 1 32 -1 39 , 1 99 1.

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