clin cancer res 1997 bednarek 11 6

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1997;3:11-16.Clin Cancer ResA K Bednarek, A Sahin, A J Brenner, et al.detection at the in situ breast carcinoma stage.Analysis of telomerase activity levels in breast cancer: positive

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Vo l. 3 , 11 -16 , Janu ary 1 997 C lin ica l C ancer R esearch 11

A dvan ces in B rie f

Analy sis o f T elom erase A ctiv ity L evels in B reas t C an cer : P ositiv eD e tection a t th e in S itu B rea st C arc in om a S tage A nd rzej K . B edn arek , A ysegu l Sah in ,A nd rew J . B renner , D en n is A . John ston ,and C . M arcelo A ldaz2Depa r tment of C arc ino genesis , U n ivers ity o f T ex as M . D . A nd ersonC ancer C en ter , S c ien ce Park -R esearch D iv is ion , Sm ithv ille , T ex as78957 [A . K . B ., A . J. B ., C . M . A .], and D epartm en ts o f P a tho logy[A . S .] an d B iom athem atics [D . A . J.], U n ivers ity o f T exas M . D .A nderso n C an cer C en te r, H ouston , Tex as 7 7030

Abs trac tTelom erase activ ity h as been im p lica ted to b e assoc i -

a ted w ith m ost hum an m alignant tum ors, in c lud in g breastcan cer. T o eva lu ate possib le asso c ia tio n s w ith w e ll-know nprognost ic fac to rs in b reast cancer, w e perfo rm ed a s em i -quantita t iv e ana lys is o f te lom erase activ ity leve ls us ing th every sen sitiv e PCR -m ed ia ted te lom er ic rep eat am plifica tionp rotoco l. T elom erase a c tiv ity w as d e tec ted in 99 o f 10 4breas t can cer sam p les ana lyzed (95 .2% ), w herea s no ac tiv ityw as d e tec ted in 10 of 10 ad ja cen t nonm alignan t b reast tis -su es. A na lysis o f f ive b reast fib road enom a sam p les revea ledtelom erase ac tiv ity in on e (20% ). In con trast to p rev iou sob serva tio ns , w e ob serv ed th at 100% of stag e I breast tu -mo r s were posit ive fo r telom erase a ctiv ity . M ore in terest-in g ly , w e d etected te lom era se ac tiv ity in six o f six du c ta lcarc inoma in si tu samp les (i.e., sta ge 0) . In ou r sem iquan ti-ta t ive ana lysis o f lev els o f en zym a tic ac tiv ity , w e found nosta tistica lly s ig n ifican t correla tio n a t th e P < 0 .0 5 leve lbe tw een te lom erase levels and lym ph node meta s ta s is sta tus,e stro gen and progesteron e receptor sta tu s, tum or size, S -phase fra ction , and p lo id y . T he on ly sta tist ica lly sig n ifican tcorre la tion w as found with patien t age (rho -0 .3 ; P =0.03) . W e ob served no sta tis tica lly sign if ican t d ifferen ce inth e te lom era se a ctiv ity lev els o f ear ly tumor s (sta ges 0 and I)versus m ore advan ced le sion s (sta ges H to IV ). N everth e les s ,stag e IV tum ors d isp layed a tend en cy for h igher t e l omera sea ctiv ity leve ls . In summ ary , n o clear a ssoc ia t ion w as ob -se rved be tw een te lom erase leve ls and know n brea st can cerp rogno stic ind ica tors. H ow ever , telom erase d etect ion by th etelom er ic rep eat am p lifica tion p ro to co l m ethod , du e to itsh igh sen sit iv ity , m ay be o f v a lu e in ear ly b reast can cerd ia gnosis and d etec tion , becau se our data ind ica te that

Rece ived 7 /10 /96 ; rev ised 10 /9 /96 ; accep ted 10 /2 3 /96 .T he costs o f pu b lica tion of th is a rtic le w ere defrayed in p ar t by thep aym en t o f page charges. T h is a rticle m ust the re fo re b e hereby m ark edadv e r t i s emen t in accord an ce w ith 18 U .S .C . S ec tio n 1734 so le ly toi nd i ca te t hi s f a ct .I Supported in p ar t by D ep ar tm en t o f D efense B reast C ancer U . S . A rm yG ran t DA M D 17-94 -J-4 078 ( to C . M . A .).2 To w hom reques ts fo r rep rin ts sho u ld be ad dressed . P hone : (5 12)2 37-24 03 ; F ax : ( 512) 237-2475 .

te lom erase reac tiv a tion appears to con s titu te a re la t ive lyearly ev en t in b reast ca rc in og en esis .

Introduc t ionThe spec ia lized syn thesis o f telom eric repea ts , TTAG G G

in verteb ra tes, w as dem ons tra ted to b e perfo rm ed by the ribo-nuc leopro te in te lom erase us ing its R NA com pon en t as a tem -p la te (1 -3 ). I t has b een po stu la ted tha t te lom erase ac tiv ity isassoc iated w ith acqu is itio n o f an im m o rtal pheno ty pe in vitro ,and it w as show n tha t m o st im m orta l cell lines express th isenzym e (rev iew ed in R ef. 4 ). U sing a very sen sitive PCR -b asedTR AP assay ,3 it h as been d em on strated tha t m ost hum an adu ltsom atic tissues do no t show ev idence of active te lom erase (5) .Activ ity o f th is enzym e, how ever, has b een ob served in germ -line cells, b one m arrow , ac tiva ted periphera l b lood lym pho-cy tes, and possib ly s tem ce lls (rev iew ed in R ef. 4 ). In te res ting ly ,telom erase ac tiv ity w as de tec ted in num erous hum an cancertypes, su ggesting tha t tum o r ce lls m ay need reac tiva tion of th isenzym e to rem ain v iab le (4 , 6 -14).

B reas t can cer is on e of th e tum or types in w hich telom eraseactiv ity h as been dem ons tra ted (12) . Fu rth e rm ore , it has beensu ggested recen tly tha t te lom erase de tection cou ld have pro g-nos tic im plica tions in breast can cer (5 , 12 ). E xac tly w h en in th eprocess o f b reast ca rc inogenes is reac tiva tion of telom erase oc-curs rem ain s to be de term in ed .

A lthough num erou s som atic m uta tions a ffec ting variousgenes have been desc ribed in sporad ic b reas t cancer, it stillrem ain s to be de te rm ined which anom alies cou ld b e cons id eredcau sa tive of b reas t carcino genesis . C erta in spec ific aberra tio ns,such as ERBB 2 o r EG FR am plifica tion and overexp ress io n andP53 m uta tion , h ave been po stu la ted an d exp lo red as of possib lea id in de term in in g breast cancer p rog nos is (1 5-18) .

In th is repo rt , w e ana lyze p oss ib le assoc ia tion s of te lom -erase w ith w ell-kn own prog nostic fac to rs in b reast cancer tofu rthe r ev alua te w he ther te lom erase de tection and quan tifica tioncou ld have po ten tia l im pac t on breas t can cer p ro gnos is.

M ateria ls and M ethod sU nse lec ted breas t cancer sam ples w ere ob ta ined from the

Coopera tive Hum an T issue N etw ork (abo u t o ne-ha lf o f thesam ples ) an d from the D ep ar tm en t o f P atho lo gy , U n iversity o fT exas M . D . A nderso n C an cer C en te r. B reas t cancer sam plesand ad jacen t nonm align an t b reast tissues w ere ob ta ined from104 p a tien ts , frozen , and s to red a t - 80#{176 }Cn til use . W e alsoana lyzed a group of five breast fib roadenom as ob ta ined fromCoopera tive H um an T issue N etw o rk . Fo r m ost o f th e breastcan cer sam ples, in fo rm atio n ab ou t estrogen and pro geste rone

3 The abbrev ia tions u sed are : TRA P , te lom eric repea t am p lifica tio nprotocol ; ITA S, in te rna l telom erase assay s tan dard ; D C IS , duc ta l ca rc i-n oma in situ .

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12 T elom er ase A ct iv it y in B reast C ancer

receptor status , lymph node metastas is status, S -phase , plo idy ,tumor stage , and age of patients w ere available . Tumor stag ingw as perfo rmed according to the guidelines o f the AmericanJo int Committee on Cancer (19 ).

The TRA P assay allow s the detec tion of in vitro telomeraseproducts (5, 20 ). W e used a modified vers ion of this assay . Inbrie f, te lomerase adds 1TA GGG repeats to the 3 end of TSprimer (5 -A A TCCGTCGAGCA GAGTF-3 ; R efs. 5 and 21 ).The number and amount of the repeats added is dependent uponte lomerase activ ity . In a second step, te lomerase products areamplified using the C X p ri me r ( 5 -C C CT FA C CC TF A CC C T-T A C C C T A A - 3 ) and Taq DN A polymerase . A s pos itiv e con-tro l, a ce ll ex tract from a sample w ith know n te lomerase activ ity(a rat mammary tumor line) w as used. A s negative contro l, ly s isbuffer w as substituted for ce ll ex tract (20). To compare the leve lo f te lomerase activ ity in dif ferent tumor samples , w e used asem iquantitative analys is based on the use of an internal stand-ard ( I T A S ), w h ich am p lif ie s f r om th e sam e p r im er s (22). Thisinternal standard, w hich cons is ts o f a l50-bp D N A product,allow s identification of false -neg ative tumor samples that couldcontain Taq polymerase inhibito rs.

Ce ll ex tracts w ere obtained from 10-5 0-mg samples o f thetumors, and te lomerase assay w as perfo rmed according to amethod described prev iously (5 , 20), w ith m inor modifications(2 2 , 23 ). In a standard procedure , w e used 2 pA of tissue extract(prote in concentration, 0 .5 pg /pJ) per assay . The CX primer,I T A S , and Taq D NA polymerase (7 units/assay) w ere added toeach sample at a hot start after S mm incubation at 90#{ 176} C .B ecause te lomerase has an RN A component, S l o f the tumorce ll ex tract from the same samples and the pos itive ce ll ex tractw ere incubated w ith 1 .l o f RN ase A (1 mg/m l) as an additionalex perim ental contro l.

A liquots (10 p.1 ) o f the PCR mixture w ere analyzed on 8%nondenaturing , 0 .4 -mm acry lam ide ge ls (20 X 40 cm) run in0 .5 x TBE buffer until the xy lene cyano l had migrated 17 cmfrom the orig in. The ge ls w ere then dried and exposed for 20 hto hyperfilm MP films (A mersham Corp., A rlington Heights,I L ). F o llow in g au t or ad iogr ap h y , each ge l w a s an a ly z ed a f t e rovernight expo sure us ing a Molecular D ynamics Phosphorlm-ager (Sunnyvale , CA ). This scan w as used to perform themeasurements o f the te lomerase ladder amplificatio n intens ity .Thus, area integration of all peaks (ex cept the firs t band from thebottom) w ere normalized to the s ignal from the internal standardand then, after background subtraction, expressed as re lative tothe positive contro l signal that w as run w ith each experiment.The first band from the bottom w as not inc luded, because itusually incorpo rates background from primer-dimer formation(8 , 1 3). The method described is only sem iquantitativ e , but it issuffic ient for the comparative analys is o f the tumors re lative tothe same pos itive -contro l ce ll ex tract.

A naly sis o f the leve ls o f telomerase activ ity and otherc linico -patho log ical characteris tics w as perfo rmed us ing non-parametric Spearman rank corre lation and t test.

Results an d D iscu ssionT o con f ir m t h e lin ear it y o f t h e T R A P assa y , w e p er f o r m ed

a dilution experiment of the te lomerase-pos itive breast cancerce ll line MD A -MB -157 (Fig . 1 ). The activ ity of the te lomerase

F ig. I Quantif ication of te lomerase activ ity from breast cancer cell lineMD A -MB -157 . The activ ity leve l w as calculated by summation ofinteg rated areas of the te lomerase ladder and normalized to internalstandard (ITA S). A fter background subtractio n, leve ls w ere expressed asre lativ e to a 100 -ce ll equivalent sample . The solid line w as c om pute rfitted according to a calculated alg orithm .

ladder w as no rmalized to that o f the internal s tandard (ITA S;Ref. 22), express ing the results relative to the activ ity found inthe 1 00-ce ll equiv alent sample . Linearity of activ ity w ith cellnumber w as observed (Fig . 1 ), in ag reement w ith observationspublished prev ious ly (22) .

Te lomerase activ ity w as then analyzed and detected in 99of the 104 breast cancer samples (95%). It w as undetectable in10 o f 10 adjacent nonmalignant breast samples (Fig . 2 ). A sexpected, the enzymatic activ ity detec ted is abo lished if samplesare pretreated w ith RN ase A (Fig . 2 ).

Interesting ly , w e detec ted te lomerase activ ity in six o f sixDC I S sam p les an a ly z ed (F ig . 3A ; stage 0 in Fig . 4A ). Four ofthese D CIS samples w ere c lass ified as high nuclear grade andshow ed high te lomerase activ ity , w hereas the tw o samples clas -sified as low nuclear grade show ed very w eak te lomerase ac-tiv ity (Fig . 3A ). These finding s sugg est that the reactivation oftelomerase activ ity o ccurs re lative early in breast carc inogene-sis . W e also analyzed a small group of breast fibro adenomas fo rtelomerase activ ity and observed one pos itive tumor of fiv etes ted (20%).

S everal c linical, his to log ical, and bio log ical indicators o fprognos is are commonly used to determ ine the therapeutic man-agement of breast cancer patients. Marker combinatio ns areusually more accurate than sing le markers (1 8 , 24). A majorgoal o f our study w as to evaluate the ro le o f te lomerase detec -tio n as a possible additional prognostic indicator. Our overallinc idence of telomerase-pos itive breast cancer samples (9 5 .2%)is s im ilar to that reported prev ious ly by Hiyama et al. (Ref. 12 ;93%). How ever, w hereas H iyama et al. (12 ) found 68% of stageI b r eas t ca r c in om as t o b e p o sit iv e f o r t e lom er ase ac t iv it y , w efound 100% of the 17 stage I breast carc inomas to be pos itive .The few tumors negative for te lomerase activ ity w ere found tobe advanced-stage (II and III) rather than early -s tag e tumors , ascan be observed in Fig . 4A , in w hich breast cancer samples aregrouped by tumor stag e. Furthermore , w e did not find anycorre lation betw een the very few tumors (five samples) found to



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be negative for te lomerase activ ity and any o f the know n prog -no sti c i ndi cato rs .

In ou r sem iq uan t it a t iv e ana ly sis o f lev e ls o f en z ym at icactiv ity , w e found no statistically signif icant corre lation atthe P < 0 .05 leve l betw een telomerase leve ls and tumor s ize,lymph node metastas is, es trogen and progesterone receptorstatus, S -phase fractio n, and plo idy , (Fig . 4 and Table 1).A x illary lymph node status is generally accepted as one of the

best prognostic indicators fo r breast cancer recurrence (2 4).W e observ ed as much te lomerase activ ity in lymph node-negative tumors as in tumors positive for lymph node metas-tas is (Fig . 4E). Interes ting ly , w e only found statis ticallys ignificant corre lation w ith patient age (Fig . 4C ; Table 1). Itappears that leve ls o f te lomerase activ ity are higher in tumorsfrom younger patients . How ever, although P = 0 .0 3 , theSpearman rank correlation analysis indicates that s ince the



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14 Telom erase A ctiv ity in B reas t C ancer

A _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _5000 x I n = 1 0 4 1 xS .,- x0) x *.2 x . 5 0 0 x x D Eti * x Ln 1 1 n=104nn ____ -< x x x x x xa ) x x x x 50 x x x x x x x x x x xx xa) 5

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F ig . 4 Com parison of re lative te lom erase ac tiv ity leve ls to p ro gnos tic ind ica to rs in b reast cancer.

rh o va lue is -0 .3 (Tab le I); approx im ate ly o n ly 9% o f th e s ig n ifican t d iffe rence in te lom erase ac tiv ity leve ls o f ea rlyvariab ility in te lom erase leve ls is exp la in ed by age . A s m en - tum ors (stages 0 and I) versus m ore advanced lesions (F ig .tioned earlie r, w e observ ed th at a ll stage 0 and stage I tum o rs 4A and T ab le 1). H ow ever, as can be observed in F ig . 4A andexp ress telom erase ac tiv ity . W e did n o t observe a sta tistically T ab le 1 , s tage IV tum ors appear to show a tendency fo r



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C lin ical C ancer R esearch 1 5

Ta b le 1 S tatistical analy s is o f te lom erase activ ity 1ev e lsA g e Plo idy S iz e S -phase

rho (S pearm an rank co rre lation ) -0 .3P 0 .03

0 .020 .88

0 .060 .56

0. 10 .39

C om parison o f breast tum or statu s P(t-test)ER + (318 67 ) versus ER - (333 91 )PR + (27 6 60 ) ve rsu s PR - (405 101)ER +/PR + (2 77 68 ) versus ER -/PR - (34 5 1 1 1 )L N + (3 19 49 ) versus L N - (37 1 113)S tages 0 and I (249 52) versu s II-IV (3 67 70 )S tages 0 -IIIB (2 90 45 ) versus IV (10 83 623)

0. 90. 20. 60. 70. 40 .001

a ER + and ER -, e strog en recep tor p ositiv e an d negativ e , respec tiv e ly ; PR + and PR -, p roges tero ne po sitiv e and n egativ e , re spectiv e ly ; L N ,ly m ph node . V alues in paren theses represen t m ean S E . T he analy s is w as perf o rm ed us in g S tatV iew 4 .0 (A bacus C on cep ts ).

h ig her te lom erase lev els . T he sm all num ber o f s tage IVtum o rs prec ludes f u rth er specu lation on th is observ atio n .

A ltho ugh patien t f o llow -u p w as no t analy z ed in our s tudy ,the lack o f a c lear assoc iation be tw een te lom erase activ ity lev e lsan d p rov en p rogno stic ind icato rs in breas t cancer ind icate s thatan aly s is o f th e lev els o f th is en z y m e by m eans o f th e PC R -m ed iated T R A P assay in hum an breast cancer m ay hav e lim itedv alu e as a progn ostic too l. T h is appears in con tras t to pre lim i-n ary observ ation s by ano ther laboratory (25 ) . It is u nc lear at th isp o in t w he ther the hy p o thesis that a w orse prog nos is sho u ldco rre late w ith h igh er te lom erase lev e ls is incorrec t, o r alterna-tiv e ly that the lack o f correlation o bserv ed is the resu lt o f them e tho do log ical app roach . It is th en poss ib le that f u tu re d ev e l-o pm en t o f non -PC R -m ed iated m e thodo lo g ie s f o r m easurem en to f te lom erase ac tiv ity m ay be b ette r su ited f o r s tud ie s o f p rog -n osis . O n the o ther hand , because w e de tec ted te lom erase ac -tiv ity at p reinv as iv e s tages o f b reas t can cer, the h igh ly sen sitiv eT RA P assay m ay be of valu e in ear l y b r east cancer detect i onan d d iagn osis , as recen tly su ggested b y H iy am a e t a l. (26 ). O urf ind ings are in agreem en t w ith o bserv ations o f o ther neop las iasin w h ich te lom erase ac tiv ity w as de tec ted at pre inv asiv e s tageso f tum or d ev e lo pm en t (13 , 1 4 ). Fo r in stance , te lom erase ac tiv ityh as b een dem on strated in prem alignan t pros tatic hy perp lasias(13 ), and recen tly te lom erase activ ity has been also o bserv ed inco lo n ad enom as (14 ). W e hav e also reported a s ign if ican t in -c rease in te lom erase ac tiv ity at p rem align an t s tages using am ouse m u ltis tag e carc in ogenesis m ode l (2 3 ).

A s m en tioned prev ious ly , w e also o bserv ed that o ne o f f iv eb reas t f ib roadenom as analy z ed show ed te lom erase ac tiv ity . T h isinc idence appears low er th an that rep orted prev ious ly (45% ;R e f . 12 ) . D esp ite th e d if f e rence in inc idence , bo th stud ie s m d i-cate that som e breast f ib roadenom as show reac tiv atio n o f te -lom erase . It is un clear at th is p o in t the p u tativ e ro le f o r te lom -erase reac tiv ation in th is ben ign n eo p las ia.

In sum m ary , te lom erase ac tiv ity w as observ ed in alm os t allb reast can cer sam p les, regard le ss o f tum o r s tage . N o te lom eraseactiv ity w as de tec ted in the n orm al breas t sam p les te sted , an dte lom erase ac tiv ity w as de tected in 20% of f ib roadenom as. N ocorre lation w as f oun d be tw een te lom erase de tec tio n or lev el o factiv ity and k now n b reast cancer progn ostic ind icato rs, w h ichappears to lim it th e po ten tial v alu e o f ev aluating the lev e l o f th ispu tativ e b iom ark er in m anag ing p atien ts w ith inv as iv e breastcancer. N ev erth ele ss, the f inal co nc lu sion on th is is su e w ill o n ly

be reso lv ed af te r cons idering the corre lation be tw een patien tou tcom e and te lom erase lev e ls. Probab ly the m os t im po rtan tco nc lu sion f rom our s tudy is that telom erase reac tiv atio n ap -pears to be an early ev en t in breas t carcino genesis . T h is f ac to rh igh ligh ts the po ten tial f o r us in g te lom erase de tec tio n as aposs ib le aid in early tum or d etec tio n .

A c k n ow l e d gmen t sW e th ank D r. Jerry S hay f or the IT A S s tan dard sam ples.

Note A dded in Pr oofW hile th is m anuscrip t w as be ing rev iew ed , S ug ino e t a l. reported

s im ilar con clus ions on the po ten tial v alu e o f te lom erase de tec tio n as anearly d iagnos tic m ark er in b reas t cancer rather than as a progn osticind icato r (S ug ino , T ., Y osh ida, K ., B o lodeok u , J., T ahara, H ., B uley , I.,M anek , S ., W ells , C ., G ood ison , S ., Ide , T ., S u z u k i, T ., T ahara, E ., andT arin , D . T e lom erase ac tiv ity in hum an breast cancer an d ben ig n b reas tle s io ns: d iagno stic app lication s in c lin ical spec im ens, in clud in g f in eneed le asp irate s. In t. J. C ancer, 69 : 3 01-30 6 , 19 96).

References1 . G re id er, C . W ., and B lack bu rn , E . H . T he te lom ere te rm inal trans -f erase o f te trah y m ena is a ribon uc leo pro te in enz y m e w ith tw o k ind s o fprim er spec if ic ity . C ell, 51 : 8 87-8 98 , 19 87 .2 . M orn , G . B . T h e hum an te lom ere te rm inal tran sf e rase enz y m e is arib onuc leopro te in that sy n thesiz e s T T A GGG repeats. C e ll, 59 : 521-5 29 , 1 98 9.3 . Fen g , J., Fun k , W . D ., W ang , S -S ., W einrich , S . L ., A v ilion , A . A .,C h iu , C -P., A dam s , R . R ., C hang , E ., A llso pp , R . C ., Y u , J., L e , S .,W es t, M . D ., H arley , C . B ., A nd rew s , W . H ., G reid er, C . W ., andV illep on teau , B . T h e R N A com ponen t o f h um an te lom erase . S c ience(W ash ing to n D C), 269 : 1 2 3 6 - 1 2 4 1 , 1 9 9 5 .4 . H o lt, S . E ., S hay , J. W ., an d W rig h t, W . E . R ef in ing the te lom e re-te lom erase hy po th es is o f ag ing and cancer. N at. B io techno l., 14 : 836-8 39 , 1 99 6.5. K im , N . W ., Piaty sz ek , M . A ., Prow se, K . R ., H arley , C . B ., W es t,M . D ., H o , P. L . C ., C ov ie llo , G . M ., W righ t, W . E ., W einrich , S . L ., andS hay , J. W . S pec if ic assoc iation o f hum an te lom erase ac tiv ity w ithim mo rtal ce lls and cancer. S c ien ce (W ash ing ton D C ), 266: 2011-2015 ,1994 .6 . C ou n ter, C . M ., H irte , H . W ., B acche tti, S ., and H arley , C . B .T e lom erase activ ity in hum an ov arian carc inom a. Proc . N atI . A cad . S c i.U S A , 91 : 2 9 0 0 - 2 9 0 4 , 1 9 9 4 .7 . H iy am a, E ., Y ok oy am a, T ., T atsum o to , N ., H iy am a, K ., Im am ura,Y ., M u rak am i, Y ., K odam a, T ., Piaty sz ek , M . A ., S h ay , J. W ., and



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17 . N akopoulou , L . L ., an d A lex iado u , A . P rog nos tic sig n ificance ofthe co-exp ress io n of p53 and c-e rb b-2 p ro te in s in breast cancer.J . P at ho l. , 179: 3 1 - 3 8 , 1 9 9 6 .18 . S cho ll, S ., B ieche , I ., Pa llud , C ., C ham pem e, M . H ., B eu von , F .,and H acene , K . R e levance of m ultip le b io log ica l pa ram ete rs in b reastcanc er p ro gno sis . B reas t, 5: 21 -30 , 1 996 .19 . B eahrs, 0 . H ., H enson , D . E ., H u tte r, R . V . P ., and K ennedy , B . J .(ed s.) . A m eric an Jo in t Com m itte e on Cancer. M anu al for S tag in g ofCancer, E d . 4 . P h ilade lph ia : J . B . L ipp inco tt C o ., 199 2 .20 . P ia tysz ek , M . A ., K im , N . W ., W ein ric h , S . L ., H iy am a , K .,H iy am a, E ., W righ t, W . E ., and Shay , J. W . D etec tion of te lom eraseac tiv i ty in hum an cells and tum ors by a te lom eric repea t am plifica tio npro to co l (T RA P). M e tho ds C ell S ci. , 17: 1 -15 , 1995 .21 . M orn , G . B . R eco gn itio n of a chrom osom e tru nca tion site asso ci-a ted w ith a -tha lassaem ia by hum an te lom erase . N a tu re (L ond .) 3 5 3 :4 5 4 - 4 5 6 , 1 9 9 1 .22 . W righ t, W . E ., S hay , J . W ., and P iatyszek . M . A . M odifica tion s of ate lom er ic repea t am plifica tion pro toco l (TR AP ) resu lts in inc reased reli-a bi li ty , l in ea ri ty and sen sitiv ity . N u cle ic A c ids R es ., 23 : 3794-3795 , 1995 .23 . B ednarek , A ., B udunov a, I., S la ga, T . J., an d A lda z, C . M . Inc rea sedtelom era se act iv i ty in m ouse sk in p rem alignan t pro gression . C ance rRes . , 55 : 4 5 6 6 - 4 5 6 9 , 1 9 9 5 .24 . D on eg an , W . L . P rog no stic fa cto rs: sta ge and re cep tor sta tus inb re ast c an ce r. C an ce r (P hila .)(S up pl.), 70 : 1755-1764 ., 1992 .25 . K im , N . W ., L ev itt, D ., H uang , G ., W u, F ., O sbo rne , K ., an d C lark ,G . C or re la t ion o f t elo m er ase w ith pro gnost ic in d ic ato rs of b rea st cance r.Pro c. A m . A ssoc . C ancer R es., 37 : 562 , 199 6 .26 . H iy am a, E ., G ollaho n , L ., K atao ka , T ., K u ro i , K ., Y okoyam a , T .,G azdar, A . F ., H iyam a, K ., P ia tyszek , M . A ., and Shay , J. W . T e lom -era se a ctiv ity in hum an breas t tum ors . J . N a il. C ance r Ins t., 88 : 839-8 4 0 , 1 9 9 6 .

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