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Vo l. 4 , 117 7-1 18 2 , M cxv 19 98 C lin ic al C ance r R ese arch 1 177

The abbrev iations used is: N CF , na tive cellu la r fluo rescen ce .

n V ivo N ative C e llu lar F luo re scen ce and H isto log ica lC harac ter istic s o f H ead and N eck C an cer

S tim son P . S chan tz,2 V enka ta sw ara K o lli,H ow ard E . Savage , G uope i Y u , Jatin P . Shah ,D an iel E . H arris , A lv in K atz , R obert R . A lfano ,and A ndrew G . H uvosDep artm ents of S urgery [S . P . 5 ., V . K ., H . E . S ., G . Y ., J . P . 5 .,D . E . H .] and P a tho logy [A . G . H .J, M em oria l S loan-K ette ring C ancerC en te r, N ew Y ork , N ew Y ork 10021 ; and M edipho to nic L abora to ry .Institu te fo r U ltra fas t L ase rs and Spec troscop y , D epartm en t o fPhy sic s, C ity Co lle ge o f the C ity U niv ers ity o f N ew Yo rk , N ewYork , N ew Y ork 10031 [A . K ., R . R . A. l

ABSTRACTN ative ce llu la r fluo re scence N C F rep re sen ts th e in -nate cap ac ity o f tissu es to ab sorb and em it ligh t o f a spec i-

fled w ave length . T he ab ility to d efin e th e re la tion sh ip o f invivo N C F w ith b io log ica l ch aracte r is tics o f neop las tic d is ea sem ay allow for an im prov ed unde rs tan d ing of the c lin ica lcourse o f d isea se . H ead and neck ca ncers from 35 p atien tsw er e e va lu ate d in v ivo for NCF chara cterist ic s u sing a x enonlam p -ba sed sp ectrom eter coupled to a handhe ld fib erop ticp robe . Spec tra l a ssessm en t w as lim ited to A 450-nm em issionchara cter is tic s , in w h ich tis su es w ere exc ited a t v ar iou sw a ve len g th s, ran g ing from A 29 0 nm to A 415 nm , and th ein tensity o f A 450 nm em ission w as reco rded . E ach cancerw as subsequen tly b iop sied and assessed fo r h isto log ica l d if-fe ren tiation by a pa tho log is t w ho w as b linded to N CF an a l-ys is . C on sid era b le v ar ia tion in spec tra l ch arac ter is tic s be -tween head and neck cancers w as id en tified , w h ich wa sde te rm ined , in part, by NC F ch arac te ristic s o f the n o rma lm u cosa from th e sam e p atien t. P oor ly d ifferen tia ted tum o rsw ere m ore lik e ly th an w e ll- o r m od era te ly d ifferen tia tedtum ors to have low er exc ita tion max ima P < 0.0 5 byANOVA ). M ost s ign ifican tly , th e tum o r d ifferen tia tion sta -tu s , a s w e ll a s the p ro bab ility o f dem on stra ting recu rren td is ea se , cou ld a lso be re la ted to the N C F ch aracte r is tics o fth e p a tien ts no rm a l m uco sa from the sam e site w ith in theupper a erodig estiv e tra c t. NC F analys is m ay represen t ane ffec tive too l to iden tify b io log ica l ch arac ter is tic s o f headand neck tumors in v ivo w ith ou t the need fo r inva sive b iop -

R ec ei ve d 1 0/ 16 /9 7; r ev is ed 2 /5 /9 8; a cc ep te d 2 /9 /9 8.The costs o f pu b lica tion of th is a rtic le w ere defrayed in part by thepaym ent o f p ag e charges. T h is a rticle m us t the re fo re be hereb y m ark edadvert isemen t in a cco rdance w ith 1 8 U .S .C . S ection 1734 so lely toi nd ic at e t hi s f ac t . Suppo rted in p art by N ASA G rant 47 39 -A , O ffic e of N av al R ese archG ran t 49 547-C , an d a g if t from the M ed isc ien ce Techno logy C orpora -tion. To whom req uests fo r rep rin ts shou ld be ad dressed , a t M em or ialSloan -K ette ring C ancer C en te r. I 275 Y ork A venue . N ew Yo rk , N ewYork 10021 . Phon e: 212 639-6033; Fax: 212 717-3302; E- mail:schantzs@mskcc.org.

s ies . R esu lts sugg est th e n eed to exp lore th e d e term inan ts o fN C F cha rac ter is tic s exp re ssed b y c lin ically norm a l m ucosa .

INTRODUCT IONNC F3 represen ts the inna te capac ity o f tis sues to absorb

and em it lig h t o f spec if ied w ave len g ths. T he de te rm inan ts o fNCF are com plex bu t p rinc ipa lly re late to the qu alita tive an dquan tita tive s ta tes o f sp ec ific flu o rop hores, co nsis ting of v ario usp ro te in s, coenzym es, n uc leic ac id s, an d o ther ce llu la r com po-n en ts 1 . A lth ough the science of tissue fluo rescence has b eeninv es tiga ted fo r m any years, o n ly recen t adv an ces in op tica len g ineering an d com pute r techno logy have allow ed fo r c lin ica linve stig ations . Th ese latter s tud ies h ave con sisten t ly dem on -s tra ted tha t tis sues in vary ing stages o f cancer p rogress ion w illd iffer in the ir N CF ch aracte ristic s b o th in v itro an d in v ivo an dth a t su ch d if fe rences m ay be usefu l in cancer sc reen in g 2 -I 1 .

T h e m easurem en t o f N CF expressed b y upper ae ro d iges-tive m ucosa m ay represen t an im portan t ap p lica tion o f th istechno log y 12 . T he ora l cav ity can b e easily exam ined w ith inth e office p rac tice se ttin g , a fac to r tha t facilitates N CF inves ti-ga tio ns. Th e e tio log ica l agen t fo r o ra l cancer, i .e . tobacco , hasbeen w ell re cog niz ed , an d its use id en tifie s ind iv idua ls a t in -cre ased risk . T he n eed for im pro ved sc reen ing techn ique s in th isla tter pop u la tion is ev id en t d ue to the lim ited su ccess o f curren tsc reen ing stra teg ies. F ina lly , resea rch regard ing chem oprev en-tive agen ts ag ains t o ra l cancer sugges ts th e n eed fo r soph is ti-ca ted m onito ring techn iqu es th at record the subc lin ica l trea t-m en t e ffec t w ith ou t th e need fo r invas iv e b iops ies . O ur prev io usef fo rts have focu sed on th ree areas o f resea rch : a in v itrom odel system s tha t ex p lo re the im plica tion of ce llu la r d iffe ren -tia tion and pro life ra tio n on N CF ch arac te ristic s o f up per ae ro -d igesive m uco sa 13 -IS ; b th e use o f an im al ca rc inog en es ism ode ls tha t de fine the ch aracte ristic s o f N CF alterations in -duced by pro long ed n itrosam in e exposure 16 ; an d c initialobserva tion a l s tu d ies tha t ex p lo re d iffe rences be tw een head andneck cancers and norm al ae rod iges tive m ucosa w ith in an m di-v idua l p atien t I 0, 1 7 , 1 8 ). Each of these stud ies has dem on-stra ted tha t m uco sa th a t ex is ts in various b io lo g ical s ta tes w illd iffe r in its N CF charac te ris tic s . Fu rthe rm ore , a cons is ten t find -ing in th ese in itia l s tud ies rela tes to flu orescen ce abn orm alitie sinv o lv ing excita tion w aveleng ths rang ing from X 300 nm to s.340 nm and and em iss ion w av e leng th s a t X 450 nm . T heprinc ipa l fluo ro phore th at ab sorbs an d em its a t th is w ave leng this NA D H I .

In th is study , w e p erfo rm ed an in itia l exp lo ra to ry an alys iso f h is to pa th o log ica l charac ter is tic s o f head and neck can cer an dits re la tion sh ip to N CF. G iv en tha t our p rev ious e ffo rts sugges ta lte ra tions in N CF w ith pertu rba tions in ce llu lar d if feren tia tion13 , 15 , w e spec ifica lly ad dressed th is re lationsh ip in v iv o .

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2 6 28 0 3 00 3 20 34 0 36 0 38 0 4 0 0 4 20 43 0

1 1 7 8 N ative Ce llular Fluoresc enc e and He ad and N eck Cancer

PA T IEN T S , M A T ER IA L S , A ND M ET HO D SPat ien t P op ula t io n . T he p a t ien t p op u la t ion analy z ed

here co ns is ted o f 3 5 patients w ith squam o us ce ll carc ino m a o fthe head and neck. The patients co ns is ted o f 1 6 w o m en and 1 9m en. A s w ill be described be lo w , N CF analy se s in each instancew ere perfo rm ed in v ivo o n the patients cancer and o n theclinically no rm al tis sue fro m the co ntralateral site o f the cancer.The m edian ag e o f the po pulatio n w as 6 4 y ears rang e , 2 7 -8 7y ears ). B io ps ie s o f neo plas tic and co ntralateral no rm al m uco saw ere perfo rm ed after fluo rescence analy sis fo r ro utine his to pa-tho lo g y . A ll sam ple s w ere ex am ined by a s ing le patho lo g is t w how as blinded as to the fluo rescence pro file . Tum o rs w ere char-ac terized ro utine ly by m em bers o f the patho lo g y staff as w e ll-differentiated, m o derate ly differentiated, o r po o rly differenti-ated, depending o n the deg ree o f keratinizatio n. A ll patho lo g icalasse ssm ents w ere pe rforme d blinded as to fluore sce nce re sults.S ites of canc er w ithin the upper ae rodige stiv e trac t include d 18to ng ue cancers, 8 f lo o r-o f-m o uth cancers , S g ing iv al cancers , 1hard palate cancer, I buccal m uco sa cancer, and 2 phary ng ealcancers . These 3 5 patients w ere cho sen at rando m fro m the headand neck cancer po pulatio n w ho presented fo r treatm ent atM em o rial S lo an-Kettering Cancer Center. These patients w erecho sen w itho ut reg ard to prev io us treatm ent. Tw enty -nine pa-tients had prev io usly untreated disease , w hereas 6 patients haddisease recurrence o r a seco nd prim ary m alig nancy at the tim eo f N CF analy sis . This study w as appro v ed by the ins titutio nalrev iew bo ard o f M em o rial S lo an-K ettering C ancer Center, andeach patient s w ritten co nsent w as o btained prio r to study entry .

Fluor escen ce In st r um en ta t ion and in V ivo Ana l y s e s .N CF analy ses w ere perfo rm ed in v ivo in all circumstanc es usinga handhe ld fibero ptic pro be attached to a fluo rescent spec tro m -e ter CD S canner; M edisc ience Techno lo g y Co rpo ratio n,Cherry H ill, N J), as described prev io us ly in de tail 1 8 ). Thepro be w as 1 8 0 cm lo ng and had an inner diam eter o f 3 m m andan o uter diam eter o f 6 m m . The f ibers w ere rando m ly bifur-cated, w ith half to the em iss io n side o f the instrum ent. Theo ptical fibers w ere rece ssed 3 m m in a m etal sle ev e at the tip o fthe pro be , w hich prev ented the fibers fro m co m ing into co ntac tw ith the mucosal surface and also pre vented e xtrinsic w hitelig ht fro m interfering w ith the fluo rescence analy s is 1 8 ). Thecharac teris tic s o f the o ptical ins trum entatio n hav e , likew ise ,been described in de tail and co nsisted o f a spec ial x eno n flashtube ex c itatio n so urce that pro duced an intense , repe titiv e 5 0 -6 0 Hz), sho rt-duratio n 8 p.s ) discharg e o f radiatio n o v er aspectral range 18) . B ecause this m ethodolo gy m easured N CFcharacte ristics of tissue, no ex trinsic dy es o r suprav ital s tainsw ere used.

Em iss ion sc ans w ere perform ed by e xciting the tissues ato ne particular w av e leng th w hile the em iss io n w as m easuredo v er a v ariable w av e leng th. B y custo m , these scans w ere iden-tified firs t by the cho sen ex c itatio n w av e leng th, fo llo w ed by thespecif ic em issio n interv al. Conversely , the ex citatio n scans w ereperfo rm ed by ex c iting tis sues w ith a v ariable w av e leng th w hilethe em iss ion w as m easured at a fix ed w ave length. These scansw ere also identified f irst by the rang e o f tis sue ex c itatio n w av e-leng th used, fo llo w ed by the spec ific em issio n w av eleng th.A ltho ug h m ultiple em iss io n and ex citatio n analy se s hav e beenperfo rm ed, w e co nfined o ur analy ses to o ne ex c itatio n scan

W A V ELEN G TH nm )F ig . 1 Th e in v iv o spec tral pro file o f head and neck cancers . Resultsw ere g enerated by m easuring the intens ity o f is 4 5 0 -nm em iss io n afterthe tissue w as ex cited w ith mo noc hro matic light ranging from 2 60-43 0nm . The ex c itatio n pro file is presented fo r three tum o rs in w hich the Xex citatio n m ax imum, i e that w av e leng th that g enerate s m ax im um4 5 0 -nm em iss io n, v arie s fro m X 3 1 0 -3 3 8 nm . p oo rl y d if fe re nti -a te d c an ce r; - - - -, mo derate ly-differentiated cance r w ell-dif-f ere nti ate d c anc er.

e x c itatio n, X 2 9 0 -4 3 0 nm ; em issio n, X 4 5 0 nm ) fo r the pur-po ses of this study. Our pre vio us s tudies indicated that this scanm ay dis ting uish neo plas tic fro m no nneo plastic m uco sae w ithinspe cific o ral cavity s ites 10 , 1 7 , 18 ). It is of note that re sultsfro m this scan can be re lated to the sam e fluo ro pho re , N A D H,w hich ex c ite s at s. 3 4 0 nm and em its at 4 5 0 nm 1 , 1 9 , 2 0 ).Previous in v itro s tudie s hav e dem o nstrated abno rm alitie s in-v o lv ing this co enzy m e w ith transfo rm atio n o f tis sue 2 1 , 2 2 ).

RESULTSFo r the purpo ses o f scan analy s is, w e calculated the ex c i-

tatio n m ax im um , i .e . the w av e leng th that induces m ax im al X4 5 0 -nm em iss io n, the X 3 3 5 nm :X 3 7 5 nm ratio , and the areaunder the curv e , as w e ll as Fo urier analy sis , as w e repo rtedprev io us ly 1 0 , 1 7 , 1 8 ). The o nly fac to r that w e co uld identifythat re flec ted the tum o r bio lo g ical charac teristic s under co ns id-eratio n w as the ex c itatio n m ax im um Fig . 1 ) . Only the results o fthis m easure w ill be presented.

W e no ted that the ex c itatio n m ax im um o f the 3 5 tum o rsam ple s analy zed v aried fro m X 2 9 1 nm to 36 3 nm . In 3 3patients , the co rrespo nding no rm al m uco sa fro m the o ppo s itesite o f the cancer w as asse ssed. The rang e o f ex c itatio n in theselatter sam ple s w as X 3 2 0 -3 5 3 nm . O v erall, the ex c itatio n m ax -im a w ere sig nificantly hig her in the no rm al m uco sae than theyw ere in the cancers fro m the sam e patients I 3 3 4 8 nmv ersus X 3 27 nm 1 4 nm , respective ly; P < 0 .0 1 by pairedte s t). This pattern o f decreased ex c itatio n m ax im um w as seen in2 5 of the 3 3 patients. In eig ht indiv iduals, the e xcitation m axi-m um w as hig her than in the co rrespo nding cancer.

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NL TU NL TU NL TUA B C

a . W D x )M D ---x---P D x

I -310 320 33 0 34 0- E xc ita tio n M ax im umb. D .

W D x )M D - - - x - - -PD x ) ____

T - -____3 3 0 3 3 5 340 3 45

A E xc it atio n M ax im umF ig . 3 Th e It exc itation m axim um head and neck cancers and norm alm ucosa analy zed in v ivo . a , exc itation m ax im um exp ressed by thecancers o f 35 p atients in w hich the patients are categ oriz ed by the degreeof d ifferentiatio n of the ir tum ors. D ifferences be tw een the three groupsare s ignificant by A N O V A (P < 0 .05) . b, exc itation m axim um cx-p ressed by the norm al m ucosa of 33 p atients in w hich the patients arecatego riz ed by the degree of d ifferentiation of the ir tum ors . X m ean foreac h c ateg orical group ing ; SD . 5 , disease status; W D , w e l ldifferentiated; M D , m o d er at el y d if fe re nt ia te d; PD , p o or ly d if fe re nt ia te d.

0 .53; P < 0 .01). It should be em phasiz ed , how ev er, that con-s iderab le v ariance around the regress ion line still ex is ted andthat the f luorescence charac teristic s o f the norm al m ucosa d idno t exp lain all v ariance in tum or N C F v alues .

C atego riz ing the p atients b y histo log ical d ifferentiation ofthe ir respec tiv e cancer show ed that the the exc itation m ax im a ofthe po orly d ifferentiated tum ors tended to be low er than those ofthe w ell-d ifferentiated or m od erate ly d ifferentiated les io ns Fig .3 a). It should be em p hasiz ed that this relationship w as no tuniv ersal and that ov erlap in the exc itatio n m axim a co uld beid entified w hen all three g roups w ere co m pared . A n A N O V Afor the exc itation m ax im a of the three groups show ed them to bes ignificantly d ifferent (P < 0 .05) .

G iv en that d if ferentiation status could be asso c iated w ith atum ors exc itation m axim um and that the sam e tum ors exc ita-tio n m axim um corre lated w ith the m axim um of the no rm alm ucosa from w hich it w as deriv ed , w e assessed w hether theexc itation m ax im um of the norm al m ucosa from these patientscould be re lated to the his topatho log ical d ifferentiatio n of thesam e p atients tum ors Fig . 3b) . A s in the case of d irec t assess-m ent o f the tum or, the o v erall exc itation m axim a of the norm alm ucosa tended to d iffer, depend ing on the d ifferentiation s tatusof the cancer (P = 0 .17). In paired analy ses , these d ifferencesreached s tatistical d ifference w hen p atients w ith m oderatelyd ifferentiated d isease w ere com pared w ith those w ith w ell-d ifferentiated d isease (P < 0 .05). T his sug gests the possib ilitythat the b io log ical charac teristic s o f the norm al m ucosa w ithinan ind iv id ual patient could g ov ern the d ifferentiation status ofthe sam e p atients tum o r and that this capac ity m ay be reflectedin N C F charac teristic s o f the norm al aero d igestiv e ep ithe lium .

M ultip le consideratio ns ex is t w hen b io log ical traits o f headand neck cancer are defined . W e considered his topatho log ically

C linical C ancer R esearch 11 79

F ig . 2 A bo x-and-w hiskers p lo t o f the is m axim a of the no rm al m ucosaand cancerous tissue of 3 3 head and neck cancer patients . T he to talpo pulatio n w as d iv ided into tertile s based o n the X m axim a of theno rm al m uco sa, i .e . the lo we st (A) , midd le (B) , and upper (C ) tertiles I I patients in each gro up). W e then calculated the m ean X m axim umand S D for the co rrespo nd ing tum ors fro m the 1 1 p atients in each group .D ifferences be tw een the three groups are s ignificant by A NO V A (P