8/8/2019 Identifying Markers for Pancreatic Cancer by Gene Expression Analysis

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Vol. 7, 109-112, February /998 Can cer E p id em io logy , B iom a rke rs & P reven tio n 10 9

3 The ab breviation s u sed are : C EA . carcin oem bryon ic antigen; SAGE . seria lan alys is of g ene ex pression; T IM P-l. tissue inh ib itor of m eta llopro teina se typ e I.

Id en tify ing M arke rs fo r P an crea tic C ancer by G eneExpress ion 1

W ei Zh ou , L or i J . S ok o ll, D ebra J . B ruzek , L in Z h an g ,V ic tor E . V e lcu le scu , S teven B . G o ld in , R a lph H . H ruban ,Sco t t E . K ern , S tan ley R . H am ilto n , D an ie l W . C han ,B ert V oge ls te in , an d K enn eth W . Kinz le r2H ow ard H ugh es M edical Ins titu te 1W . Z ., L . Z ., B . V .] and Jo hns HopkinsOncolo gy C ente r [V . E . V ., K . W . K .], Joh ns Hopkins Univ ers ity School ofM edic ine, B altim ore, M arylan d 21231 ; an d Departm ent of Pa tholog y, Jo hnsH opk ins U niv ers ity School of M ed icine , B a ltim ore , M aryland 21287 EL . J. S .,D . J . B ., S . B . G ., R . H . H ., S . E . K ., S . R . H ., D . W . C .]

Abstrac tT o beg in to id en tify n ew tum o r m arkers , w e recen tlyp erfo rm ed a system atic stud y o f gene exp ression incancers o f the co lo n an d p ancreas . O f th e 45 ,000 genesiden tif ied , 183 w ere fou n d to b e expressed a t sign ifican tlye leva ted leve ls in p an crea t ic cancer . O ne o f the gen es w astissue in h ib ito r o f m e ta iopro te inase type I (T IM P -1 ), wh i c hencod es a secre ted p ro te in . A n a ly s is o f T IM P -1 serumlev els revea led sign if ican t increases in p an crea t ic ca ncerp atien ts, bu t T IM P-1 by itse lf was inad equ a te as a serumm ark er for can cer. H ow ever , a com b ina tion o find iv idu a lly su bop tim a l m a rkers (T IM P -1 , C A 19-9 , an dcarcino em b ryon ic an tigen) de tected 60% of 85 pa tien tsw ith p an crea t ic ca ncers in a h igh ly spec ific m ann er .T h ese resu lts su gges t tha t a sy stem a tic ana ly sis o f gen eexpression can rev ea l n ove l serum m ark ers an d th a tind iv idu a lly su bop tim a l m a rkers can be com b ined toy ie ld h ig her sen sitiv ity an d spec ific ity .

In troduc t ionO ne key to effec tiv e can cer treatm en t is ea rly de tec tion . Inm any cancers , inc lu d ing those of th e pancreas, cu rren t m ethod sfo r ear ly de tec tio n are inadequ a te . P an crea tic cancer a ffec tsabo u t 27 ,000 in d iv idua ls in the U nited S ta tes each y ear ( 1 ) an dis usua lly asym p tom atic un til it has reached an advanced stage(2 , 3 ). T here fo re , s im p le and cos t-e ffec tiv e tests fo r ea rly de-tec tion of p an creatic can cer m igh t p rove v aluab le in decreasingm o rtality from th is d isease . H ere , w e presen t an approach fo rde tec ting pancrea tic cancers th rough serum assay s.

Serum tum o r m ark ers a re su bstances th at a re p roduced bytum or ce lls an d are sec re ted or re leased in to the b lood stream

Rece ived 7 /7 /97 ; rev ised 1 1 /1 1 /97; accepted 1 1 /2 0/97 .T he co sts o f p ublic ation of th is artic le w e re d efrayed in p art b y the paym ent ofp age ch arge s. T his a rticle m ust th erefore be hereby m arked ads er t i semen t inaccordance w ith 18 U .S .C . S ect ion 1734 sole ly to ind ica te th is fac t.1 Th is w ork w as su pported in part by th e C layto n Fund and by N IH G rantsGM 07 309 , CA 573 45 , and C A62924 . B . V . is an Inv es tiga tor of th e HowardH ughes M ed ica l Ins titu te . G en zym e M olecula r O nco logy provid ed re search su p-p ort to K . W . K .2 To w hom requ es ts fo r reprin ts sho uld be addressed , at T h e Oncolog y C ente r,T he Jo hns H opk ins Un ivers ity Schoo l of M ed icine , 424 N orth B ond S treet,B altim ore, M D 21231 .

(4 ). T h e m easurem en t o f such m arkers is non invas ive and isre la tive ly sim ple and inexpens ive to perfo rm , com pared tothose m ethod s tha t a re in vasive or requ ire spec ial ins tru rnen-ta tion and personn el. T he idea l tum or m arker fo r any cancertype has y e t to be found , a lthough severa l a re be ing usedc lin ically o r ac tiv ely inves tig ated . P rosta te -sp ec ific an tigen iscu rren tly in w idesp read u se fo r de tec ting pro sta te can cers ,a lth ough it is no t cancer spec ific (5 , 6 ). S erum a-fe topro te in isuse fu l in the d iagnos is o f h epa to ce llu la r carcinom a, bu t it a lsoinc reases in ben ign live r d isease (7 ). T here fo re . it is comm on lyused w ith abdom ina l u ltrasono graphy fo r sc reen ing p urposes(8 ). CEA 3 is used as a p rogno stic m arker fo r a varie ty of tum o rtypes , bu t it lacks the sens itiv ity an d sp ecific ity th at are re -qu ired fo r a p resym p tom atic m arker (9 , 1 0) . O f the se rum tum o rm arkers th at a re av a ilab le fo r p an crea tic cancers, CA 1 9-9 iscu rren tly the best, bu t lim ited spec ific ity and sens itiv ity hasrestric ted its app lica tio n ( 1 1 , 1 2). A dd itiona lly . 5% of thegen eral pop u lation are geno typ ica lly L ew is a-b - and canno tsyn thesize the carb ohyd ra te an tigen recogn ized by the CA 19-9an tibody (13).

M o st tum or m arkers have b een found serend ip itous ly orth rou gh m onoc lona l an tibod ies genera ted b y imm uniz in g m icew ith hum an tum or ce lls . F or exam ple , CA 19-9 w as o rig ina llyd iscovered th rough m onoc lona l an tib ody s tu d ies o f co lo rec ta lcancer cell lin es (14) and w as la te r fou nd to b e m o re spec ific fo rpancrea tic cancer ( 1 1 ). To deve lop new m arkers w ith po ten -tia lly h igher sens itiv ity and sp ec ific ity , w e h av e u sed a m olec-u la r b io logy -based approach . W e have used SA G E (15 -17 ) toinves tiga te express ion pa tte rn s in spec ific cancer typ es . as co rn -pared to norm al tis sues o r o ther cancers. O f th e d iffe ren tia llyexp ressed genes , those tha t a re abundan tly expressed in can cersand en co de secre ted or ce ll su rface pro te ins can be fu r th ereva lua ted as po ten tia l se rum tum or m ark ers. H ere . w e report thefirs t app lica tion of th is approach , us in g p an creatic cancer as apro to type .

M ater ia ls an d M ethodsT um o r and N orm al T is sue S am p le C o llectio n . Pr im ary pan-c rea tic adenocarc inom as w ere co llec ted from surg ica lly re -sec ted p an creas and frozen . T en spec im ens, judg ed to con ta in45 -65% tum o r ce lls b y h istop atho lo g ical exam ina tion , w ereused fo r m o lecu la r ana ly ses . N orm al k idney , pancreas , ske le ta lm usc le , b reast, b lood , p ros ta te , and th ym us tissues w ere co l-lected from biops ies o f th e corresp ond ing tis su es . N o rm al neo-p lastic co lon tissue w ere co llec ted as descr ib ed prev ious ly (1 7).T he resec tab ility o f pancrea tic cancers cou ld be de te rm inedth rou gh m edica l record s in 69 o f th e 85 p atien ts eva lu ated .Nor the rn B lo t A na ly sis. T o ta l R NA was iso la ted from bloodand tissues and used in N orthe rn ana lyses as d esc ribed p rev i-

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I 10 P ancr ea tic T um or M arker s

ous ly ( 1 8). E xpressed sequence tag c lone 265330 con ta in s theen tire co d ing sequence of T IM P -l an d w as ob ta ined fromG enom e Sys tem s, Inc . T he cod ing reg ion of T IM P-l w asPCR -arnp lified from th is p lasm id w ith th e a id o f M l3 fo rw ardand rev erse prim ers . an d the PCR p roduc t w as rad ioac tive lylab eled (19) and used as pro be .ELISA . P reopera tiv e se rum sam p les w ere co llec ted from pa-tien ts w ith p an crea tic cancer (,i = 85), b reast cancer (n 64) ,and norm al con tro ls (ii = 98) and sto red a t -8 0#{176}C . S erumm easurem en ts w ere perfo rm ed using the T IM P -l EL ISA sys-te rn (R PN 261 1 ; A m ersh am L ife S cience) o r the C EA and CA19-9 assays (C h iron D iag nos tics) . fo llow in g m anufac tu re rsp ro toco ls . B rie fly , a ll th ree assays a re sandw ich imm uno assay susing tw o pur ified an tib od ies aga ins t the sam e an tigen . Th e firs tan tib ody is used to cap tu re th e co rrespo nd ing an tigen , and th esecond an tibod y is co up led to a chem ica l reagen t to g ive as igna l. A d irec t rela tionsh ip ex is ts be tw een the co ncen tra tion o fthe an tig en in the serum and the re la tive s igna l de tec ted by th eassays . V a lues from 5 o f se rum w ere m easured and corn -p ared to standard cu rves cons truc ted w ith purified an tigen .

Resu l t sT IM P -1 E xp ression Is E leva ted in P ancrea tic T um ors . A srep orted prev io usly , SA G E tag lib ra ries w ere genera ted fromtw o prim ary p an crea tic can cers, tw o pancrea tic cell lines, tw oco lo rec ta l cancers, tw o co lo rec ta l cell lin es, an d tw o sam p les o fn orm al co lo rec ta l ep ithe liu rn ( 1 7). A to ta l o f 303 ,00 0 tran sc rip ttags , approx im ate ly 60 ,000 from each tissue type , w ere ana-lyzed an d fou nd to correspond to over 45 ,000 d iffe ren t genes .F urthe r ana lysis revea led I 83 transcr ip ts tha t w ere eleva ted inbo th prim ary pancrea tic tum ors and p ancreatic cancer ce ll lines(ava ilab le over the in te rne t at h ttp ://w elch link .w elch .jhu .edu /-rno lgen-g lhom e.h tm ). O ne of the m ost p rom in en t o f thesed ifferen tia lly expressed transc rip ts (H 560056 , tag G A GA G T -G TCT ). w as fo und to b e derived from the T !MP -1 gene . T h istag w as id en tified 32 tim es in th e 6 0 ,0 00 tags derived frompan crea tic cancers b u t o n ly 0 an d S tim es in an equ iva len tnum ber of tag s from no rm al co lo recta l m ucosa an d co lo rec ta lcancer tis su e, respec tive ly .

T o fu rthe r conf irm th is d if feren tia l expression , N orthe rnb lo t ana lys is w as perfo rm ed . T IMP -1 w as fou nd to be ex-p ressed a t h igh leve ls in each of 1 0 pancrea tic can cers (F ig . 1A ) .In c on tra st, T IMP - I expression w as no t de tec tab le o r w asex pressed a t re la tive ly low leve ls in norm al tissues (F ig . 1B ) .S erum T IM P -1 Is E leva ted in Pancrea tic C an cer P a tien ts .T IMP - J en codes a M r 30 ,00 0 serum pro tein th a t b inds to m atrixm eta llo p ro te inases and inh ib its the ir ac tiv itie s (20 , 21). A s aresu lt, T IM P - 1 c ircu la tes in b lood e ith er as a free fo rm or as anen zym e- in h ib ito r com plex . T h e u se of free T IM P -l as a se rumm arker has been p roposed fo r a v ar ie ty of d iseases bu t has hadlim ited su ccess. F or exam ple . T IM P-l is s ig n ifican tly e leva tedin synov ia l flu id f rom rheum ato id ar th ritis pa tien ts com pared tonorm al co n tro ls, b u t its free se rum lev el in th ese pa tien ts is on lysligh tly eleva ted ( 1 85 s ersus 1 57 .5 ng /m l; R ef . 22 ) . L ikew ise ,free se rum T IM P -l is o n ly m odera te ly e lev ated in pa tien ts w ithb ladd er and pro sta te cancer (23 , 24). R ecen tly , an EL ISAsystem w as deve lop ed th at can de tec t b o th the free and co rn -p lexed fo r rns o fTIM P -l (25) . Th is EL ISA w as used to eva lu atethe to tal T IM P- I concen tra tio n in preop era tive sera f rom pan-c rea tic cancer p a tien ts (,z = 85). b reas t cancer pa tien ts (,z = 64 )and hea lthy con tro ls (n = 98). T o ta l T IM P -l p ro te in concen-tra tion w as fou nd to be sign ifican tly h igher in the sera o fpan crea tic cancer pa tien ts (1736 864 ng /m l) than in tho se ofb reas t cancer pa tien ts (907 415 ng /rn l, P < 0 .00001 ) o r

A . 1 2 3 4 5 6 7 8 9 10

B .

*v Mt

U . . Ii i 0 t e a 0 0U Z

F ig . 1 . T IM P -/ exp re ss ion in tum o r an d no rm al tissue s. B ottom . T IM P-/ levelsw e re d ete rm ined by N orthe rn b lo t an alys is of to ta l RNA (5 g) iso la ted from 10prim a ry pancreatic cance rs (A ) or norm a l tissu es (B ). T o p . to ta l RNA w asvisualized by e th id ium brom ide sta in ing . T he n orm al tissues o f orig in are a sindica ted . C. , colorectal; P. , pr ima ry; N. , no rm al. T ota l R NA isolated fromprim a ry tum o rs 4 and 5 (A . La nes 4 an d 5) were a lso u sed a s po sitiv e co ntro ls inB (le ftm o st tw o lane s).

norm al co n tro ls (81 8 347 ng /m l, P < 0 .00 001 ; F ig . 2A ).Lev els g reate r than 1550 ng /m l w ere observed in 43 p an creaticpa tien ts (5 1% ) bu t o n ly in four (6% ) b reas t cancer pa tien ts andtw o (2% ) norm al con tro ls . F urthe rm ore, the re seem ed to be n ocorre la tion be tw een serum T IM P - 1 leve ls and tum o r resec tab il-ity ; leve ls in the resec tab le and un resec tab le g ro ups w ere1 687 772 ng /m l (range , 50 3-3809 ng /m l; n = 54) and1751 931 ng /m l (range , 60 1-3447 ng /m l; n = 1 2), res pe c-tive ly . H ence, se rum T IM P-l leve ls ap peared to be eleva tedre la tive ly early during p ancreatic cancer p ro gress ion . F in ally ,th ere w as n o corre la tion be tw een to ta l T IM P- 1 concen tra tio nan d the ag e or gend er o f no rm al co n tro ls (da te no t show n), afin d ing cons is ten t w ith prev io us o bserv ations (22 . 26 ).T IM P -1 a s a D iag nos tic T um o r M arker for P an crea t icC an cer . T h e e leva tion of T IM P-l expression in pancrea ticcancer pa tien ts p rom pted us to ex p lo re the possib ility o f us in gth is p ro te in as a tum or m arker. A t p resen t, C EA is the m o stw ide ly used d iagn ostic m ark er fo r cancer in genera l, andCA 1 9-9 is the best ava ilab le m arker fo r p ancreatic cancer(9 -I I). A s show n in F ig . 2 , B an d C , th e se rum concen tra tio nof each o f these m ark ers w as, on average , h igh er in pancrea ticcancer pa tien ts than it w as in hea lth y in d iv idua ls. H ow ev er ,th ere w as a large v ar ia tion am ong the cancer pa tien ts, com pro -m is in g bo th sens itiv ity and spec ific ity . B y com bin ing the m ark-ers an d using h igh cu to ffs , w e fo und tha t go od sp ec ific ity co u ldb e ob ta ined w h ile re ta in in g reasonab le sen sitiv ity . Fo r exam ple ,the sera f rom 81% of pancrea tic cancer p a tien ts bu t o n ly from

8/8/2019 Identifying Markers for Pancreatic Cancer by Gene Expression Analysis

3/4

A .6000

5000

4000

E 3000

2000

1000

A

B .30

25

201,15

10

5

A

5

!!-::; . . L

I

. tSt

N orm al Pancrea ticC on tro l C ancer

a

C ance r E p id em io logy , B iom a rke rs & P reven tion II!

4N orm al P ancrea ticC on tro l C ancer

C .100000

10000

1000

10 0

10

N orm al P ancrea ticC on tro l C ancer

F ig . 2 . Tumo r m arker serum levels ofp anc reat ic cance r pat ien ts an d norm al contro ls . lIM P-I (A ) . CE A (B ) , an d C AI9 -9 (C ) lev els in se rum from patien ts w ith panc reaticcance r (n = 85 ) or norm al contro ls (n = 98) w e re d ete rm ined as de sc ribed in M ate rials and M e thod s.

Tab le I C omb inati ons of tum or m arkers can im p rovea nd s pe ci fi ci ty

sensitivity

CAI9-9(uni ts /mI )

CE A(ng/ml)

T IM P-l Spec ificity(ng /m l) (% )

Sensi t iv i ty(% )

>136. 7

>136. 7

>3.3

>3.3

A . Sensitiv ity o ptim ized 9997

>1837 99> 1837 95

54414081

>400

>400>400

>400

>5

>5

>5>5

B . S pec if ic ity o ptim ize d10 010 0

> 2300 10010 0

> 2300 100>2300 10 0> 2300 100

34262248494160

, Cutoff va lues w ere set a t m ean + 3 SD s of the n orm al co ntro ls .h C utoff va lues w ere set abov e the highe s t norm a l con tro l va lue s.

5% of hea lth y con tro ls sco red positive fo r at leas t on e o f them arkers w h en the cu to ff va lues fo r each w as kep t a t a re la tive lyh igh va lue (T ab le 1A ). A ltho ugh these crite ria cou ld proveusefu l fo r sc reen ing sym ptom atic pop u lation s, they are ob v i-ous ly no t adequa te fo r sc reen in g a presym ptom atic popu la tionin w h ich n early 10 0% sp ec ific ity is requ ired . H ow ever, byusing h igh er cu to ff va lues, w e w ere ab le to d etec t 60% of thepa tien ts w ith pancrea tic can cer w hile m ain ta in ing 1 00% spec-ific ity (Tab le 1B ). H ow ever, as d iscussed be low , the c lin ica lv a lu e of th is test fo r sc reen ing presym ptom atic pop u la tion at itscu rren t s ta te is no t ce rta in .

Discuss ionW hat is the po ten tial use of m ark ers fo r pancrea tic cancer? T h iscancer typ e car ries a d ism al p rog nosis , w ith over 95% ofp a tien ts dy ing w ith in S years. H ow ever, som e pa tien ts w ithearly d isease su rv ive after su rgery . U nfortun a te ly , m ost p atien tsrem ain asym ptom atic u n til la te in the cou rse of the ir d isease .P resym ptom atic tests fo r th e presence of pancrea tic cancers

cou ld , the re fo re , be of su bstan tia l u se . W e env ision tha t acos t-ef fective and sim ple tes t to de tec t p resym p tom atic p an cre-a tic can cers cou ld fac ilita te ea rlier d iagn osis and surg ica l cu rein a subse t o f pa tien ts. S uch tests, h ow ev er, m us t m ee t stringen tc rite ria if they are to be prac tica l. O n ly 27 ,000 new cases a reexpec ted in the U n ited S ta tes th is year , and to avo id a h ighfrac tio n of fa lse pos itives in any genera lly adm in iste red test, thespec ific ity o f such a test m us t be ex trem ely h igh . E ach of them arkers stud ied here exh ib ited such sp ec ific ity if h igh cu to ffva lu es w ere cho sen , bu t the sensitiv ity o f an y sing le test w asre lative ly low at th ese cu to ff va lu es (34 , 26 , an d 22% fo rCA 19-9 , CEA , and T IM P -l, respec tive ly ). T he u se of a pane lin clud in g a ll th ree tests in creased the sens itiv ity to 6 0% w hilem ain ta in ing 100% spec ific ity . T here fo re, a lth ough add itiona lstud ies a re obv ious ly req u ired , our resu lts su ggest tw o u sefu lap proaches fo r cancer de tec tion in the fu tu re . F irs t, they showth at a p an e l o f ind iv idua lly su bop tim al m arkers m ay pro vem ore he lp fu l than any s in g le m arker. Second , th ey dem ons tra teth at the eva lua tion of g lob al gene exp ress io n pa tte rns in cancersw ith SA G E can rev ea l unexp ec ted transc rip ts tha t p rov ide newoppor tu n itie s fo r m arker d ev elopm ent. T h e in teg ra tion o f thesetw o s tra teg ies is a ttractive in tha t add itiona l m arkers can sim plybe added to the pane l as they are d iscovered , re tain ing sp ec i-fic ity th roug h h igh cu to ffs w h ile ex pand ing sensitiv ity . W eenv ision tha t o the r genes iden tified th roug h SA G E or o thertechn iqu es m igh t b e rec ru ited in th is regard . Fou r o ther genesth at a re exp ressed a t h igh leve ls in pancrea tic cancers com paredto o th er tissues o r cancer types have a lready been iden tified bySA GE and confirm ed by N o rth ern b lo t ana ly sis, and effo r ts tog en erate m onoc lona l an tibo d ies to th eir en co ded pro te ins a reunderway .

Ackn ow l e d gmen t sG en zym e M olecular On colo gy licensed the SAGE technolo gy from The Joh nsHopkin s U nive rs ity for com m erc ial p urposes ; the te ch nolog y is free ly availab leto acad em ia for re search purpose s. T h e Un ivers ity and re search ers ( K . W . K . andB . V .) h av e a fin an c ia l in te res t in G enzym e M ole cu la r O nco lo gy , th e ar ra nge-ments fo r which are m an aged by th e Un ive rs ity in acco rdan ce w ith its co nflict ofin teres t po licie s. C EA and CA I9-9 reag ents w e re provid ed by C h iron D iag nos-tic s . W e wou ld lik e to thank D r. K eith Y . L a i for provid ing the norm a l tissuesample s .

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