van den berg 1995 clin canc res

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1995;1:11-18. Published online January 1, 1995.Clin Cancer ResC Van Den Berg, X Y Guan, D Von Hoff, et al.prognostic implications.identified by chromosome microdissection: potentialDNA sequence amplification in human prostate cancer

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Vo l. 1 , 11-18 , Jan ua ry /9 95 C lin ic al C ancer R esearch 11

DNA Sequ en ce Am p lifica tion in H um an Prosta te C an cer Iden tifiedb y C hrom osom e M icrod isse ction : P o ten tia l P rogno stic Im p lica tion s

C arla Van Den B erg ,2 X in -Y uan G uan ,2D an ie l V on H off, R obert J enk in s,M ichae l B ittn er, C on stance G riff in ,O lio K a llion iem i, T apio V isakorp i, J ohn M cG ill,John H erath , Jona than Epstein , M ichae l Sarosdy ,P au l M eltzer , and Jeffrey T ren t3U niv ers ity o f Tex as H ealth S cience C ente r, S an A n to n io , T ex as7 8284 [C . V . D . B ., D . V . H ., M . S .] ; L abo rato ry o f C ancer G en etic s,N CH G R , N IH , B ethesda , M ary land 20892 [X -Y . G ., M . B ., P . M .,J. T .]; L abora to ry of G en etic s, M ayo C lin ic , R o che ster , M in neso ta55905 [R . J ., J . H .]; Johns H opk ins O nco log y C en te r, B a ltim ore,M aryland 21287 [C . G ., J. E .]; D ep artm en t of Labo rato ry M ed icine,T am pe re U n ive rsity H o sp ita l, T am pere , F in la nd [0 . K ., T . V .]; an dC an cer T herapy and Re sea rch C ente r, San A n ton io , T exa s 78229[D . V . H ., J. M .]

ABSTRACTTh e p r im ary a im of th isrepo rt w as to exam in e th e s ig n if-

icance of inc reased DNA seq uen ce co py numbe r (g en e am p li-fica tion ) in hum an pros tate cancers. T h reeethodolog ies(chrom osom e m icrod is sec tion , com pa ra tive genom ic h yb r id -iza tion , and fluorescence init u hybr id iza t ion ) w ere com b in edto (a ) id en t ify a comm on reg io n o f gen e am p lifica tion in h um anpros ta te ce lls an d (b ) ev alu ate in pa tien t sam ples the preva lenceof th is gene tic chang e in bo th prim ary an d recurren t prosta tesamples . Th e resu lts o fch rom osom e m icro d issection revea led ac ommon ampl i f ied band reg ion (8q2 4 .1 -24 .2 ) in tw o prosta tecases with cy to log ica l ev id en ce of g en e am plifica tion (doub leminu tes ) . F lu ore scence in situ hybrid ization u sing th e 8 q m l-crod issec t ion probe w as p erfo rm ed on fresh tum o r tou ch p rep -ara t ions from 44 rand om ly selected prosta tectomy spec imens .Am plifica tion o f DNA seq uen ces from 8q 24as observ ed in 4( 9%) of44 cases. Fou r o f the 44 pa t ien ts inh is se ries presen tedw ith a po sitive lym ph node a t in itia l d iagn osis and 3 o f th ese 4pa tien ts sh ow ed 8qam p lifica tio n . B ecause of th is fin din g, c om -pa ra tive g enom ic h yb r id iza tion and fluorescence in situ hy -br id iza t ion wer e perfo rmed on tum or ce lls from n in e p rosta tecan cer pa t ien ts w ith recurren t d isea se . In e igh t o f n in e cases aga in o f D NA seq uen ces en com passing 8q 24w as observed .Taken toge ther with o th er ev id ence im p lica ting S q ga in inprosta te cance r p ro gre ssio n , theseesu l ts sugg est tha t th e ana l-ys is of th is g en e tic change m ay have d iagno stic u tility as am ark er o f pro sta te can cer p rogress ion .

Received 1 0/2 1 /9 4 ; ac cep ted 11 /17 /94 .1 Th is research w as supported , in p art , by G ran t P2 0CA58225 (R . J .) andG ran t U 01-CA -48405 (D . V . H .) from the N ationa l C ancer Ins titu te ,N IH and by the N ation al F ounda tion for C an cer R e sea rch (D . V . H .).2 C . V . D . B . and X -Y . G . con tribu ted equa lly to th is s tudy .3 To w hom correspond en ce sh ou ld b e ad dressed .

INTRODUCT IONA lthou gh pros tate cancer is the m ost comm only d iagn osed

cance r in m en in the U nite d S tates (-20 0 ,0 00 new ly diagn osedca ses /yea r; R ef. 1 ), the m olecu lar chang es und erly in g its g ene sisand progression rem ain poorly u nders to od . W ith the ex trem eva riab ili ty in the n atu ral h is to ry of th is d ise ase , co up led tofre qu en t inc iden ta l d ia gno sis of su bclin ic al d isea se [fo llow in gPSA testing o r tran sureth ral resec tion fo r u rina ry obs truc tion(2 ) ] , th e iden tifica tion of gene tic m arkers w hich cou ld iden tifyp ros ta te cancers like ly to p rogress to le tha l m etasta tic d isease isan im por tan t goa l.

R ecen t stud ies hav e iden tified severa l recurring g en e ticch an ges in pro sta te c anc er inc lud ing : (a ) a lle lic loss, p ar ticu la rlylo ss of chrom o som e 8p and 16q (3-5);b) g en eralized DN Ahyp erm ethy la tion (6 ) ; (c) po in t m utations or de le tions of th ere tin ob lastom a and p5 3 genes (7 -9) ; a nd (6 ) alte rat ion s in th eleve l o f sp ec ific ce ll to ce ll ad hesion m olecu les (i.e., E-cadher in /a -ca ten in ; R efs . 5 , 1 0 , an d 11 ) and aneup lo idy and aneusom y ofchrom osom es de tec ted by FISH 4, particu la rly chrom o som es 7and 8 (4 , 12 -15). It seem s cer ta in tha t a com bina tion of th esechang es is c ritica l to the acqu isition of m etas ta tic po ten tia l, andI ssacs e t a l. (6 ) have recen tly p roposed a m ode l p lac ing thesegene tic changes in th e con tex t o f p ros tate cancer d isease pro -gression.

A t the curren t tim e , a m ethod to accura te ly pred ic t them etasta tic sp read of an in d iv idua l tum o r is no t ava ilab le . H is -to n ica lly , c lin ica l an d p atho lo g ical stage and h isto log ica l g rad ingsy s t ems (e .g . , G leasons ) have been used to ind ica te the prog -nosis fo r group s of pa tie n ts b ased o n the d egree of tum o rd iffe ren tia tion or the type of g landu la r pa tte rn (16 , 1 7). T he u seo f a com pute r sys tem im ag e ana lys is o f h isto log ica l sections ofp rim ary le sion s has also b een su gge ste d a s an aid e in them anag em en t o f ind iv idua l pa tien ts (17). A dd itiona lly , D N Aconten t/p lo idy b y flow cy tom etry (18) and , v ery recen tly , F ISHu sin g c en trom ere-spec ific probe s hav e also b een dem onstra te dto hav e u tility as a m arker o f p ros ta te cancer aggressiveness(4 , 1 2-1 5 , i8 ). F in ally , the use of CGH has v ery rec en tly beenperfo rm ed on pros tate cancer spec im ens (19 , 20 ), enab linginve stigato rs to survey th e en tire g enom e for gains and losses ofD N A sequences, th us p inp o in tin g ch rom osom al reg ions lik elyto con tain genes im plica ted in th e deve lopm ent o r p rogress ionof p rosta te cancer.

F ina lly , fo r m any cancers the p resence of gene am plifica -tio n ha s been do cum en ted to rep resen t an im portan t pro gno sticind ica to r of d is ease pro gre ssion . Cyto log ica l ev idence of geneam plifica tion (recogn ized as ex trachrom osom al dm in) has beenprev ious ly repor ted in pro sta te tum o r spe cim en s (2 1-2 3), a l-

4 T he abb rev iations used are: F ISH , f luo rescen ce in situ hybr id izat ion ;CG H , com parative g enom ic hybrid iza tion ; dm in , d oub le m in u tes ; PCR ,po lym erase ch ain re act ion ; PSA , prosta te -sp ecif ic an tig en .

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12 DN A A m plif ic atio n in Prostate C ancer

thou gh no ev idence o f gene am p lif ication o f k now n o ncogeneshas y et b een repo rted . T he iden tif ication o f a com m on ly am p li-f ied chrom o som e reg ion in pro state cancer w o u ld b e o f cons id -erable im portan ce. T he p resence o f dm in in m etaphase sp readsf rom sh ort-te rm pro state cu ltu re s p rov ides a targ e t f o r the tech -niq ue o f chrom osom e m icrodisse ctio n . T his p rocedu re u ses aglass m icron eed le to dis sec t (u nde r the m ic roscop e) dm in andam plif y th is m inute am oun t o f DN A v ia PCR (24). T he produ ctis then hy brid iz ed to norm al-banded lym pho cy te m etaphasechrom o som es to de te rm ine the chrom osom al locatio n o f theam plif ied genes (25 , 26). U s ing th is app roach, w e hav e identi-f ied in the cu rrent stu dy a com m on chrom osom e band reg ionam plif ied in pro state cancer (8q24.1-2 4 .2 ).

T h is f ind ing led to th e design o f our cu rren t s tudy w ithth ree spec if ic goals: (a ) to generate a ch rom osom e m icrod issec -tion p rob e su itab le f or FIS H encom passin g th e 8q24 am p lif ie dreg ion in dm in-b ean ing prostate tum o r m etaphase cells ;b) toutil iz e th is probe in FISH analy sis o f in terp hase nuclei to d e-term in e the f requ en cy o f D N A copy num ber inc reases in anun se lec ted se rie s o f p rim ary pro s tate le s ion s ; andc) to u tiliz eCGH to asse s s th e D N A copy num ber chang es f o r 8q in pro statecan cer patien ts w ith re cu rren t d isease . T he unde rly ing presum p-tion of th is w ork is th at the ov e rex pre ssio n by gene am p lif ica-tion o f a gene (s ) on ch rom osom e 8 m ay pro v e to be a b io log i-cally and prog nos tically im portan t f ac to r in asse s sing p rostatecancer p rog ressio n .

MATER IALS AND M ETHODSSurg ica l S pec im en s. T is sue f rom prim ary d isease spec-

im en s for FISH o r m icrodis sec tio n w as obtained f rom patien tsun dergo ing rad ical p ro statec tom y at the M ay o C lin ic , T ex asH ealth S c ien ce C en te r o f S an A n to n io , o r Johns H opk in s U n i-v ers ity . A ll sam ple s w ere ob tained prio r to adm in is tration o f anyhorm onal the rapy , and all as part o f th e ir regu lar clin ical care .A ll recu rren t spec im ens cam e f rom T am pere U n iv e rs ity H osp i-tal f o llow ing tran su reth ral re sec tion s o f patien ts w ho had re -sponded f av orab ly to endocrine therapy (o rch iec tom y , s ix case s ;lu tein iz ing ho rm one -re leasing ho rm one ago n ist, tw oase s; es -trogen , one case ), bu t later show ed sy m p tom s o f lo cal recu r-rence.

Prep ara tion o f M etap hase C hrom osom es for M icro d is -sec tion . In ord er to ob tain m e taphase chrom osom es f o r m i-crodisse ctio n , c ells w ere analy z ed f rom a sho rt- term cu ltu re ofa p rim ary tum or spec im en (PR O -39) and f rom an es tab lishedpro state cance r cell line (M PC -3) .

Prim ary tum o r tissue w as obtain ed at the tim e of rad icalpros tatec tom y f rom case PR O -3 9 and a portion o f th e tum or w asused f o r d ete rm ination o f h isto log y and standard p atho lo gy . T h esp ec im en w as ob tain ed f rom a 63 -y ear-o ld m ale . T he patho log -ical f ind ing s conc lud ed 45% of the pro state g land w as inv o lv edby m oderately to poorly d if f erentiated ad eno carcin om a, G lea-so ns grade 8/1 0 (5+3/1 0) (T ab le 1 ) as: (a ) p oo rl y d if fe re nt iat edadenocarc inom a (prim ary p atte rn 5 in G leason s g rad ing sy s-tem ) an d (b) m oderate ly d if f e ren tiated adenocarc inom a (second -ary pattern 3 in G leasons gradin g sy stem ). O n ly 1 of 1 3 lym phnodes ex am ined dem ons trated m etas tatic adenocarc inom a.Pros tate tum o r ch ip s w ere transported in M cC oy s m ed ium w ith10% f e tal bov ine se rum , 2% H EPES bu f f e r, 500 u n its /m l pen -

ic illin /strep tom y cin , an d 2 m si sod ium py ruv ate . T is sue f o rcy togen etic an aly s is w as m inced in to 1 -2 -m m 3 f ragm en ts, th enf o rced th rough a 10 0 m esh siev e . A sing le -ce ll su spens ion w am ade by passing ce lls th rough a 22#{ 189 } -gaug e n eed le . C e lls w erecen trif uged and w ash ed , then re su sp en ded in M cC o y s S A m ed ium as describ ed ab ov e . C e lls w ere treated w ith co lc im id (0 .1 2p.g /m l) f or 60 m m . Ce lls w e re rem ov ed f rom the f lask using0 .25% try psin /m l mM ED T A , re su spen ded in 0 .075 M K C 1 atroom tem peratu re f o r 20 m m , cen trif ug ed and subseq uen tlyf ix ed in 3 :1 (m e thano l:g lac ial acetic ac id ). A f te r o v ern igh t f ixatio n at -20#{ 176} C , the ce lls w ere ref ix ed in f re sh f ix ativ e anm e tap hase sp reads w ere s tained w ith W righ ts s tain as d esc ribedprev iou s ly (27) .

T he M PC -3 pro state tum or ce ll lin e , p rev iou s ly repo rted tcon tain dm in an d p osse ss c-myc am p lif ication (28 , 29 ), w asgrow n in R PM ! 164 0 m ed ium w ith 10% f e tal bo v ine se rum ang lu tam ine . M CP-3 ce lls w ere ex posed to co lc im ed , h arv ested ,f ix ed , and prepared on slid e s as desc ribed abov e .

M icrod issec tion a nd Am p lifica tion o f dm in D NA . T hem icrod issec tio n and PCR am p lif ication o f d issec ted dm in DN Aw ere p erf o rm ed essen tially as desc ribed prev iou sly (30B rie f ly , m ic rod issec tion w as p erf o rm ed usin g g lass m ic ro -need les con tro lled by a m icrom an ipu lato r attached to anv ented m icro scope . T he targ et reg ion of d is sec tio n in bothprim ary spec im en an d the p ro state ce ll lin e w ere 3 -5 o f dm inT he d issec ted dm in f ragm en ts (w h ich adhere to the m icro -need le) w ere trans f e rred to a 5 - l co llec tion dro p and am p lif iedusing p rev iou sly p ublish ed m e thods fo r degen erate oligonuc le-o tide-pnim ed PCR . T h e am p lif ied m icrodissected D N A w aslab eled w ith b io tin -11 -dU T P in a secon dary PC R reac tion anth e p roduc ts w ere p urif ied and used f o r FIS H . H y brid iz ationth e FIS H prob es f o llow ed prev io u sly describ ed procedures (2 4and prov ided unequ iv o cal ev idence o f hy brid iz ation to th e dm inD N A . F ISH A nalysis o f T um or T is su e Sp ec im ens . D ua l-co lo r h y brid iz atio n s w ith a d irec tly labe led pro be f o r the centrom ere o f ch rom osom e 8 (S p ec trum G reen C EP-8 ; V Y S IS ,Fram ingham , M A ) and th e d irec tly lab e led S pec trum O range8q24 probe w ere p erf orm ed on prostate tum o r to uch p repara-tion s as p rev iou sly describ ed ( i8 ). Posthy bn id iz ation con d ition sand m e th ods o f m icro scop ic an aly s is hav e also been prev iou slyrepo rted (18 ).

Fo r each batch o f hy brid iz atio n s a neg ativ e co n tro l (ben ignp rostate tissue at M ay o , periph eral b lood ly m ph ocy te s at H o pk in s ) w as hy b rid iz ed an d scan ned to d em ons trate successf u l anappropriate no rm al hy b rid iz ation sign als . In the norm al con tro land in the app aren tly 8q 24 nonam p lif ied tum or cases, one sm all,distin ct orange -red sig nal ( f rom th e 8q24 p rob e) w as obse rv edto be assoc iated w ith each o f the larger g reen signals (th e CE8 p rob e) in 9 2% o f ce lls. For each batch of hy brid iz atio ns apo sitiv e con tro l (th e M PC -3 cancer ce ll lin e ) w as also hy brid -iz ed an d scanned to dem ons trate success f u l and app ropriateam plif ic atio n signals. A m p lif ic ation of 8 q24 w as de f in ed bym ultip le (o f ten d if f u se ) o rang e -red s ig nals assoc iated w ith onor m ore o f th e g reen sign als o r as large orange -red s igndom ain s w ith in a n uc leu s . T he o bserv ation o f f ou r 8q24 s ignalsand tw o C EP8 signals (e.g., f ou r cop ie s o f the 8q arm or 8q2 4and tw o cop ies o f the ch rom osom e 8 centrom ere) w as notde f ined as am p lif icatio n . W henev er possib le , b o th 8 chrom o -



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Pre -O p# { 176 } Pos t-O P D if f e ren tiationC lin ical PS A patho log ical G leason s S core (po or, m ed ium , w e ll) Plo idy 8q Probe

s tag ing prim ary second ary (M ay o 3 , 2 , 1 ) FCM resu lts (% )T um or s iz e

( c m)atien t In s titu tion state v alue24 M ay o C 13.4 0 3a-0 -0 2 3 2 D ip lo id N eg 4.2 .232 M ay o B 1 .0 0 2a-0 -0 2 3 2 D ip lo id N eg 2 1 x 120 M ay o C 6.20 3a-0 -0 3 4 3 D ip lo id N eg 2 X 1 x 1ii M ay o C 4 .0 0 3a-0 -0 3 3 2 D ip lo id N eg 2 .5 X 1 .7 X 3.513 M ay o B 5 .60 2c -0 -0 3 4 3 D ip lo id N eg 2 .1X 1.5 X 0. 55 M ay o B 7.80 2 c-0 -0 3 2 2 D ip lo id N eg 3 X 3 X I

16 M ay o C 4 .10 3a-0 -0 3 3 2 D ip lo id N eg 2 .4 X 1. 8 X 11 M ay o B 19.7 0 2 c-0 -0 3 3 3 D ip lo id N eg 2 X 2 X 1

17 M ay o C 4 .20 3 c-0 -0 3 2 2 D ip lo id N eg 2 .5 X 2 .5 X 1. 351 M ay o B 11 .70 2c -0 -0 2 3 2 D ip lo id N eg 4 .2 X 4 X 3.910 M ay o C N on e 3a-0 -0 3 3 3 D ip lo id N eg 1.5 X 1.5 X 128 M ay o C 22 .60 3b-0 -0 3 3 3 D ip lo id N eg 3 .5 X 3 X I .36 M ay o B 6 .20 2c -0 -0 2 2 2 D ip lo id N eg 0 .82 M ay o C 8.50 3a-0 -0 3 3 3 T etraplo id N eg 2. 5 X 2 X 1

34 M ay o C 6.10 3 -0-0 3 4 3 D ip lo id N eg 2 .3 X 1.8 X 0.823 M ay o C 3.90 3b -0-0 3 3 3 D ip lo id N eg 4 X 2. 8 X I .139 M ay o B 4 .60 2c -0 -0 3 3 2 T e trap lo id N eg 2 2 X 131 M ay o C 5 .70 3a-0 -0 2 3 2 T e trap lo id N eg 2 .2 X 1. 7 X 0. 99 M ay o B 5 .70 2b -0 -0 3 5 3 T e trap lo id N eg 2 X 2 X 18 M ayo C 3 .10 3a-0 -0 3 3 3 D ip lo id N eg 1 .5 X 1 X 0.8

18 M ay o C 35 .60 2b -0 -0 3 2 3 T e trap lo id N eg 2. 5 X 2 X 1.519 M ay o B 4 .90 2b -0 -0 3 4 3 T etraplo id N eg 2 X 2 X 121 M ay o C 19.2 0 3a-0-0 3 4 3 T e trap lo id N eg 2 X 1 .5 X 1 .525 M ay o C 26 .60 3 c -0 -0 3 4 3 T e trap lo id N eg 4 3 .5 X 2. 533 M ay o C 4 .50 3 c-1 -0 3 3 3 T etraplo id 10.50% 4 .5 X 3 X 235 M ayo C 1 6 .7 0 3 c-0 -0 2 4 2 D ip lo id N eg 3 .5X 2 X 1. 237 M ay o C 5 .70 3 c-0 -0 3 4 3 A neuplo id 18.50% 4X 3.5 X 343 M ay o C 9.30 3c -0-0 3 4 3 T etraplo id N eg 2 .5 X 2 X I .346 M ay o C 9 .30 3 -0 -0 3 3 3 T e trap lo id N eg 5 X 3 X 1 .548 M ay o C 6 .70 3a-0 -0 3 3 3 T etraplo id N eg 2 .5 X 2 X 249 M ay o C 2 8 .3 0 4a-0 -0 3 3 3 A neup lo id C EP8 /8q 24< 1 4 X 4 3 .353 M ay o C 8 .8 0 3c -0 -0 3 3 3 D ip lo id N eg 2 .5X 2.2 X 1.5

94 -0 84 H o pk in s B 22 .50 3 c-1 -0 4 4 3 A neup lo id 30% 1 .5X 0. 5 X 0. 594 -007 H opk in s B 7.60 3a-0-0 3 3 2 N D N eg 1 X 1 X 194 -0 02 H o pk in s B 3 .70 2b -0 -0 3 3 2 N D N eg I X I X 0.7 594 -008 H opk in s B 8 .90 2b-0 -0 3 4 2 A neup lo id N eg 1 I X I94-027 H opk in s B 10.1 0 2a-0 -0 3 3 2 N D N eg 2 X I X 194 -089 H opk in s B 1 .0 0 2a-0 -0 3 4 2 D ip lo id N eg I I X I94 -044 H opk in s B 6 .40 2a-0 -0 4 3 3 D ip lo id N eg I I .5 X 0.794 -0 24 H o pk in s B 4 .30 2c -0 -0 4 4 3 D ip lo id N eg 2 3 X I .594 -020 Hopk ins B 21 .80 2a-0 -0 3 3 2 A neuplo id N eg 2 X 2 X 194 -049 H opk in s B 12 .80 2b -0 -0 3 3 N D D ip lo id N eg 1 I x IPR O -39 U T S A B 31 .00 3b -1 -0 5 3 N D N D A m plif ied N D

a Pre -O p , p reo perativ e; Pos t-O p , po stoperativ e ; N eg , negativ e, N D, no t de term in ed .b Percen tage o f nu cle i dem ons trating am plif icatio n .

C lin ical C ancer R esearch 13

Table I P at ie nt i nf orm a ti on

som e h y brid iz atio n an d th e 8q m icro -FIS H pro be w ere s im u l- p ro teinase K (0 .1 m g /m l in 20 m si T n is -HC 1 , 2 m si C aC I2 ,tan eous ly assay ed . Fo r each o f the p ro state tum o rs s tu d ied , th e 7 .5 ) treatm en t at room tem peratu re and a second rounden tire hy brid iz ation s ite (22X 22 m m ) w as analy z ed . A ll nuc le i dehy d ration as desc ribed abo v e . D N A iso lated f rom tum o rs w asencount ered w hile scann in g w ere care f u lly ev aluated f o r the lab eled w ith FIT C -dU T P (D uPon t, B os ton , M A ) an d normpresen ce o f am p lif ication . If am p lif ication s w ere o bserv ed f o l- m ale D N A w ith T ex as R ed -dU T P (D uPon t) u s in g n ick translow ing th is com p le te scan , 200 -2 50 random nuc le i w ere sco red tion . H y brid iz atio n m ix tu re con tain ing 400 ng labe led tum orand the percen tage o f n uc lei dem ons trating am p lif ication w as n orm al D N A and 10 m g un labe led C o t-i D N A (G IB C O -B R L ,reco rded (T ab le 1 ). G aithe rsburg , M D ) in iO m l o f 50% form am ide -iO % d ex tran

C om para tiv e G enom ic H ybr id iza tion . CGHas per- su lf ate -2X S S C w as denatu rated an d app lied on norm al ly m -f o rm ed on th ree p ro state ce ll lin e s (du14 5 , L N C ap , and PC -3 ) ph ocy te m e taphase p reparation s. T h e hy brid iz ation w asand n ine recurren t tum ors u sing d irec tly f luo ro chrom e-con ju - u nder a cov ens lip in a h um id cham ber at 37 #{ 17 6} Co r 48 h . A f te rgated D N A S as describ ed p rev iou s ly (31 , 3 2 ). B rie f ly , n o rm al hy brid iz ation , th e slid e s w ere w ashed th ree tim es in 5 0%ly m pho cy te m e taphase p reparation s w ere den atu rated at 7 2 - m am ide -2X S S C (jH 7) , tw ice in 2X S S C , and on ce in74# { 176 } Cor 3 m m in 7 0% f orm am ide -2X S S C (pH 7) , and de - S S C at 45# { 176 } Cor 1 0 m m each f o llow ed by 2X S S C , 0 .hy d rated in a serie s o f 7 0 , 85 , and 100% ethano l f o llow ed by N aH 2PO 4-0 .i N a2H PO 4-0 .1% N P4O (pH 8), and d istilled



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14 DN A A m plif icatio n in Pros tate C an cer

w ater at ro om tem peratu re f o r 10 m m each . T h e s lid e s w ere thencou n te rstained w ith 4 ,6 -d iam id ino -2 -p heny lindo le (0 . 1 m g /m l)in an an tif ade solu tion . H y b rid iz ation s of FIT C -labe led norm alf em ale DN A w ith T ex as R ed - labe led norm al m ale DN A w ereu sed as contro ls.

D ig it a l Im a ge A n a lys is. D et ec t ion ofe lativ e DN A se -quen ce copy num ber chang es in chrom osom e 8 w as accom -p lish ed b y analy z ing hy brid iz atio n in tensit ies o f tum o r andno rm al DN A s alo ng chrom osom e 8 in m e taphase sp read s usinga dig ital im ag e analy sis sy stem as d esc rib ed prev io usly (32 ).T hree s in g le -co lo r im ages (m atch ing 4 ,6 -d iam id io -2 -pheny lin -do le , FIT C , an d T ex as R ed f luo re scence) w ere co llec ted f romf our to f iv e m e taphases f rom each hy brid iz ation us in g a N ik onS A ep if luo re scence m icro sco pe (N ik o n C o rporation , T ok y o ,Japan ) and a X illix CCD cam era (X illix T echno log ie s C orp o -ratio n , V ancouv er, B ritish C o lum b ia, C anada) in terf aced to aS u n L X w o rk station (S un M icro sy s tem s C om pu te r C o rporation ,M oun tain V iew , CA ). A f te r back g round sub trac tio n , th e g reenand red f lu ore scence in ten sitie s f rom pter to qter and the ratio o fth e tw o w ere calcu lated . T he abso lu te f luo re scen ce in ten s itie sw ere norm aliz ed f or each m etaphase sp read so th at the av erageg reen :red ratio o f all ch rom osom e obje cts in th e m e taphase w as1 .0 . T he f in al resu lt w as p lo tted as a m ean green :red ratio p ro f ileand its 1 S D f rom p ter to q te r alo ng chrom o som e 8 ob tainedf rom 4-8 chrom osom e hom o lo gues.

R E S U L T SI d en t i fica t ion of G en e A m p lif ica t io n in P r os t a t e C ells

b y C h r o m osom e M icr o d issec t ion . W e h a ve r ecen t ly deve l -op ed a strategy to iden tif y and charac te riz e g en e am p lif icationb y targeting dm in o r hsrs f o r m ic ro d issection (25 , 26) . In th isstud y w e report the resu lts o f m icro dis sec tin g dm in f rom tw oprostate can cers, id en tif y ing a com m on orig in o f am p lif iedDN A in bo th cases, an d us ing the m icro d issection prob e to te stin terp hase tum o r cells f rom patien t sam ples.

Dm in f rom the prim ary tum or case PR O -3 9 and f rom theM PC -3 prostate can cer ce ll lin e w ere m icrod issec ted (Fig . 1 ,A-C) and d irectly am plif ied in vitro by PCR . T h e PCR -am plif iedDN A sequen ces w ere th en secondarily lab eled w ith a f luo ro-chrom e and u sed f o r FIS H ag ain st m e taphase tum or ce lls f romw h ich the d issec tio n s w ere d eriv ed (Fig . 1 ,an d E) . T he resu ltso f FIS H to bo th dm in -bean ing tum o r ce ll m e tap hases c learlydo cum en ted h y brid iz atio n to the dm in , con f irm ing the presen ceo f D N A sequence am p lif ication (Fig . iF) . S ub sequen tly , th edm in m icrodissect ion p rob e w as used to hy b rid iz e agains t m e ta-phase sp reads f rom norm al p erip heral b lo od ly m ph ocy tes ino rde r to identi f y the ch rom osom al loci o f th e am plif ied DN Asequence s encoded w ith in the dm in (Fig . 2 ,an d B ). T he orig ino f the am p lif ied D N A w ith in PR O -39 dm in w as show n toencom p ass band reg io n 8 q24 (Fig . 2 ). T h e d issec ted dm in D N Af rom M PC -3 w as sh ow n to hy brid iz e to tw o d if f eren t bandreg ion s, 8 q24 and lO cen (Fig . 2 , and D ).

T he PR O -39 m icrod is sec tio n probe w as then hy brid iz ed toMPC - 3 m etaphases and hy b rid iz ation w as v isab le o n 100% o fthe recogn iz able dm in . A bio tin-labeled c-myc cosm id w as thenhy b rid iz ed to m ito se s f rom PR O -39 , PC -3 , and M PC -3 . In allthree cases, c-mvc seq uences w ere sh ow n to hy brid iz e to alldm in (Fig . 1E, inset).

F I S H A n a lysis o f P r os t a t e T u m o r S p ec im en s. Ba s e don th e id entif ic atio n o f a com m on ly am pli f ied band reg ion(8q24) , dual-co lor FIS H (us ing a direc tly labeled probe fo rcen trom ere o f 8 , labe led green , an d the d is sec ted band reg ion8q24, lab eled orang e-red) w as p erf orm ed on to uch p reparatio nsf rom the tum o rs o f 44 patien ts w ith p ro state can cer. T ab lesum m ariz es the clin ical in f orm ation and am p lif ication status8 q24 am p lif ication o f all 44 patien ts. W ith in th is un sele ctedp atien t popu lation 4 (9% ) o f 44 w ere sco red as am p lif ied [def ined as m ultip le d if f use (o range-red ) signals assoc iated w ione or m ore g reen s ig nals w ith in a nuc leu s , Fig . 3 ,an d B ]. O fin te res t, 4 o f 44 patien ts w ere also node pos itiv e at th e tim ein itial d iagn osis and in 3 o f 4 nod e-pos itiv e patien ts , 8q 24 amp lif ication w as observ ed .

Im po rtan tly , th e clin ical h is to ry o f all p atien ts w as unk now n to the labo rato ry p erf o rm ing th e FIS H ex perim en ts.A lso , each case am p lif ied f o r 8q2 4 w as reev aluated to con f irmth e p resence o f am p lif ied ce lls.

C G H A n a lysis of R ecu r r en t P r o st a t e T u m or T issu e .CGH w as used to delineate th e f req uen cy of 8q copy num bergain f rom three cell lines (du145, L N C ap, and PC -3) and nicase s of recurrent p ros tate cance r. N o cop y num ber changes8q w ere f ound in either the du145 or L N Cap cell lin es.co n trast, th e PC -3 ce ll lin e show ed a gain o f 8q13 -q te r an d lo so f 8p te r-q i2 (Fig . 3C ).

T he d e tailed C GH analy s is o f all ch rom o som al ch angesf rom th e n ine recurren t p ro state cancers is p re sen ted e lsew here(20 ). T his report f ocu ses ex clu siv e ly o n th e id entif ic atio n o fin creased copy num ber o n 8q . In th is s tu dy the in creased co pyn um ber o f 8q w as f o und in 8 (89% ) o f 9 recu rren t p ro statecan cers (Fig . 3C ). S ev en o f the se case s dem ons trated a gain o fth e en tire q arm . H ow ev er, in one case, th e gain w as lim ited8q24-q ter. S ev en tum ors also show ed lo ss o f S p . S ix tum o rsshow ed bo th 8p lo ss an d 8 q gain , w ith tw o dem ons tratingg ain and on ly one 8p lo ss .

D I S C U S S I O NT he w ork in th is rep ort f u rthe r ex tends o ur k now ledge o

g ene tic changes o f p ro state cancer by iden tif y ing DN A sq uence copy num ber ch an ges o f ch rom osom e 8q as a m ark erlik ely s ign if icance for the dev e lopm en t o f agg ressiv e p ros tatecancers.

U tiliz ation o f ch rom o som e m icrod is sec tio n prov id esrapid app roach to d ete ct and clone am plif ied DN A sequence sf rom cy to lo g ically reco gn iz ab le m ark ers su ch as dm in or hsr(26 ). O the r app roach es to the analy sis o f am p lif ied DN Aq uences hav e re lied on techn iqu es based on DN A e lec trop hore -si s (e .g . , in ge l ren atu ration or res triction landm ark genom icscannin g) . W hile these technique s su cce ssf u lly iden tif y am p li-f ied sequences, th ey are ex trem e ly lab oriou s an d can be conf o unded by am p lif ied sequences un re lated to the pheno ty pein terest . B y com bin ing m icrodis sec tion and FISH , th is app roachm ay be o f p rac tical im portance sin ce the m e th odo log y is av ail-able to p erf orm rap id FIS H pre treatm ent analy s is on p ros tateb io psy co re sp ecim en s that su bsequ ently can b e used fo r routineh is topatho log ical ex am inatio n .

In th is report 4 o f 4 4 prim ary pro s tate cancers and 8 o frecu rren t p ro state tum ors docum en ted DN A copy num b er gain s



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C linical C ancer R esearch 15

F ig. I U til iz ation of chrom o som e m icrodis-sec tio n to elucidate the chrom o som al com posi-tio n of dm in from a p rim ary pro state cancerm etaphase sp read . InA-C , m icro dis sec tion o f asingle dm in (arrows) from case PRO -39 . InDand E, afte r m ic rod issect ion of dm in (A-C),PC R am plif ication, and bio tin labeling of thedis sec ted D NA (see M aterials and M ethods),the PCR product w as purified and hy brid iz ed b yFISH back to the dm in to confirm that thedis sec tion product hy b rid iz es to the hsrs . Theidentical m etaphase tum or cell from casePRO-39 is show n follow ing G iem sa staining inD and fo llow ing FISH inE . N ote the FISHprobe c learly identifies m ultip le dm in not nec-ogniz ed by conv entio nal G iem sa s taining . Inset,FIS H o f a b io tin- lab ele d c-m yc cosm id to m eta-phase ce ll from PRO -39 , ind icating hy bnid iz a-tio n to dm in (see tex t).

F ig. 2 C hrom o som al localiz ation of am p lified dm in DNA from the p rim ary pro state cancer PRO -39A and B ) and the pros tate cancer ce ll line PC -3(C and D ) by FISH. A, G -banded no rm al partial m etaphase w ithr row to reg ion of hy brid iz ation on chrom osom e 8;, identical partial m etap haseas in A fo llow ing FISH id entify ing the norm al chrom o som al locus of the d issec ted dm in from PRO -39 as 8q24 .larrow); C, G -banded norm al partialm etaphase w ith black arrows to the reg ion o f hy brid iz atio n on chrom osom e 8, and thehite arrow s to the reg ion of hy b rid iz at ion on chrom osom e10 ; D , id entical partial m e taphase as in fo llow ing FISH identify ing the norm al chrom osom al locus of the d issec ted dm in from PC -3 as 8 q24 andlOcen .



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16 D NA A mplification in Pros tate Cancer

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F ig. 3 FISH (A and B) and CGH (C ) analys is indicating D NA sequence copy number increase for 8q in primaryB) a nd re c urre nt (C ) pro state c anc er.A, F I S H w ith a centromere probe for chromosome 8 (green) and a m icrodisse ction probe for 8q24 ( iso lated from PRO-39 dmin), on a touch prepfrom normal pro state cells demonstrating tw o signals for each probe in interphase nuclei;, U pper panels: F I S H w ith a centromere probe fo rchromosome 8 (green) and the m icrodissection probe for 8q24 (red) to interphase nuc lei from the amplif ied pro state case 33 (Table 1) . N o tepro state cancer cells show gains of 8q24 signals relative to the 8 centromere cons istent w ith amplification o f D NA sequences in this regionowerpanel: FISH using the m icrodissection probe for 8q24 (red) hybridiz ed to interphase nuc lei from the amplif ied pros tate case 94 -084 (Tabledemonstrating multiple copies of 8q24.C, C G H identify ing DN A sequence copy number increase along the long arm of chromosome 8 (8q)a case o f recurrent pros tate cancer.



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C lin ical C ancer R esearch 1 7

o f 8 q . For the p rim ary tum ors, a g ain o f ch rom osom al m ate rialat 8 q24 w as spec if ically id en tif ied by FIS H , w h ile in recurren ttum ors CG H recogn iz ed 8 q gain . B y CG H w ho le -arm g ain w asob serv ed in sev en o f n ine tum ors , w ith on e case dem on stratin gam p lif ication res tricted to 8 q24 -q ter. T h e com m on ly am p lif iedreg ion be tw een all can cers w as 8 q24 , and w h ile in som e tum orsthe degree o f m u ltip lication and siz e o f the reg ion inv o lv ed m ayind icate a gro ss ch rom o som al chang e such as an isochn om o-som e o f 8q , c lear do cum en tation o f g en e am p lif ication w asob serv ed in num erous cases . T he d ata f rom bo th chrom osom em icrod issec tion and C GH p rov ides ev idence f o r a com m onreg ion o f am p lif ication spann ing sev eral m egabases o f DN A .T he s iz e o f th is segm en t is su f f ic ien tly larg e to co n tain sev eraldo z en genes and on e o r m ore o f the se m ay con trib u te to th egrow th adv an tage during dev e lopm en t and reprog ress io n o fp ro s tate tum ors .

O f in te re s t, th e gain o f 8 q recog n iz ed by s tandard cy to ge -ne tic an aly s is has im p licated its in v o lv em en t in m an y o th ercancers inc lud ing acu te non ly m p hocy tic leuk em ia, hepatocellu -lar carcinom a, renal ce ll can cer, gastric cancer, uv eal m e lanom a,m y e lody sp lastic sy nd rom e , and co lon cancer and m ay ex p lain abroad en ro le f o r 8 q alte ration in carc in ogenes is . In th is regardthere is a stro ng cand id ate gene located w ith in 8q 24 w h ich m aybe the targe t o f th e 8q am p lif icatio n -c -m y c . A lth ough am p li-f ication o f c-myc h as no t p rev iou sly b een rep orted in p rim arypro s tate m alignanc ie s, ev idence f o r the ov erex press ion o f-ny cas a poss ib le m ak er o f a p oor pro gnos is in p ro s tate cancer hasappeared prev iou s ly (3 3 ). W e d irectly dem ons trated the pres -ence o f c-myc sequ ences w ith in dm in f rom the PR O -39 tum orm e tap hases and the PC -3 and M PC -3 cell lin e s u sing FIS H (Fig .1 E, inse t) . A lth ough th e c-myc gene is encoded w ith in th e dm in ,p rev iou s e s tim ation o f th e siz e o f the-myc am plicon (90 -3 00k ilobases ; R ef . 34 ) w ou ld acco un t f o r< 10 % o f the to tal dm inDN A . A lthough it is lik ely that-myc is th e targ et o f theam p lif ication o f 8q sequences, th e re m ay b e o ther gen e(s ) in -v o lv ed in the app aren t p hy s io lo g ical e f f e c ts o f am p lif ication o fth is ch rom osom al reg ion .

U tiliz ation o f f resh tum or preparation s f o r FIS H has ro u -tin e ly allow ed >50 0 non ov erlapp ing in tac t nuc le i to be ex am -m ed by FIS H f rom the sam e b iopsy spec im en u sed f o r h is to -log ical ev alu ation . T he u se o f a m icn od issec tion prob e f o r 8q 24prov id ed FIS H resu lts equ iv alen t to tho se f o r the m o re f re -quen tly stud ied cen trom en ic p robes. T h is s tudy f o cu sing on 8q isalso in goo d general agreem en t w ith o ther earlie r reports u tiliz -in g b o th FIS H and CGH w hich c lean ly d ocum en t ch rom osom e8 gain as a f req uen t chang e in pro s tate cancer p rogres sion(1 9 , 20) .

O ur s tudy illu strate s the u se f u lne s s o f ch rom osom e m icro -d issec tion in the analy sis o f d o sage abno rm ality o f ch rom osom echanges in p ro state can cers. W e hav e id en tif ied a com m onreg ion o f DN A seq uence am p lif icatio n (8 q24 ) an d docum en tedthat it o ccurs in a subse t o f patien ts w h ich m ay h av e a prop en -sity f o r d isease p ro gress ion . W e h av e f u rth e r ex am ined recu rren tpatien t sam p le s an d show n an ex trem e ly h igh in ciden ce o f 8 qgain , ind icating th is gene tic alteration m ay resu lt in a le ssf av o rab le p rog nosis . W h ile the se data h av e to b e tak en in th econ tex t o f the com p lex p rocess o f pros ta te carc ino genes is w h ichin v o lv e s no t on ly gain bu t lo s s o f ch rom osom al reg ions , if th e seprelim in ary re sults can b e ex ten ded and conf irm ed th e pre treat-

m en t d etec tion o f th is cy tog ene tic m ark er o f p oor prognos is m aybe use f u l in se lec ting p atien ts w ho w ou ld bene f it f rom aggres -s iv e th erap eu tic in te rv en tion .

R E F E R E N C E S1 . B oring , C . C ., S q u ire s, T . S ., and T ong , T . C ancer statis tic s 1 993 . C AC an cer J. C lin ., 43 : 7-29, 1 993 .2 . R uck le, H . C ., K lee , G . G ., and O este rlin g , J. E . Pro state-spec if icantig en: con cep ts f o r stagin g p ros tate can ce r and m onito rin g respon se toth e rapy . M ay o C lin . Proc ., 69 : 69 -79 , 1 994.3 . B ov a, G . S ., Carter, B . S . , B ussem ak ers, M . J. G .,t a l. Homo z y g o u sd elet ion and f requent allelic lo ss o f chrom o som e 8q22 lo ci in hum anprostate can cer. C an cer R es .,53 : 3869-3873, 1993.4 . M acosk a, J. A ., T ry bus , T . M ., S ak r, W . A ., W o lf , M . C ., B enson ,P. D ., Pow ell, I . J., and Pon te s, J. E . Fluorescence in si tu hybrid iza tionanaly sis o f 8p allel ic loss and ch rom osom e 8 in stab ility in hum anprostate cancer. C an cer R es .,54 : 3824-3830, 1994 .5. Carter, B . S ., Ew ing , C . M ., W ard, W . S t., T reiger, B . F., A ald er,T . W ., S chalk en , J. A ., Ep ste in , J. I ., and Isaac s, W . B . A lle lic lo ss o fchrom osom es 16q and IO q in hum an pro state can cer. Pro c . N atI. A cad .Sc i . USA, 87 : 8751 -8755 , 1 990 .6 . Isaacs , W . B ., B o v a, G . S ., M o rto n , R . A ., B ussem ak ers, M . J. G .,B rook s , J. D ., an d Ew ing , C . M . M o lecu lar b io logy o f p ro s tate cancer.S em in . O nco l., 21 : 1 - 1 8 , 1 9 9 4 .7 . B oo k stein , R ., S h ew , J., C hen, P.,t a l . S upp ression of tum onig enic-ity o f h um an p rostate carcinom a ce lls b y replacin g a m utabedb gene.S c ien ce (W ash ing ton D C ), 247: 712-715, 1990 .8 . B oo k stein , R ., R io , P., M adreperla, S .,t a l . Prom o ter de letion andlo s s o f re tinob las tom a g ene ex pres sion in h um an prostate carc in om a.Proc. N aIl. A cad . S ci . U S A , 87 : 7762 -7767, 1990 .9 . Isaac s, W . B ., C arter, B . S ., and Ew ing, C . M . W ild-ty pe53su pp resses g row th of hum an p ro state can cer cells contain ing m utant53alleles. C an cer R es ., 51 : 4716 -4720 , 1 991 .1 0 . M orton , R . A ., Ew ing , C . M ., N agaf uch i, A ., T suk ita, S ., an d Isaacs ,W . B . R eduction o f E -cad hen in lev e ls and d e le tion o f the a-caten in g en ein hum an prostate cance r cells. Cance r R es.,3: 3585 -3590 , 1 993.1 1 . U m bas , R ., S chalk en , J. A ., A alders, T . W ., Carter, B . S ., K arth aus ,H . F. M ., S chaaf sm a, H . E ., D eb ruy ne, F. M . J., and Isaacs, W . B .E x p ress io n o f the cellu lar ad hesion m o lecu le E -cadherin is reduced orabsent in high -grade p ros tate cance r. C an cer R e s.,2: 5104-5109,1 992 .1 2 . V isak o rpi, T ., H y y tinen , E ., K allion iem i, A ., Iso la, J., and K allio n-iem i, 0-P. S en sitiv e d ete ction o f ch rom o som e copy num ber ab erratio nsin pro state can cer b y f lu ore scence in s ity h y brid iz ation . A m . J. Path ol. ,145: 1-7 , 1994.1 3. T ak ah as hi, S ., Q ian , J., B row n, J. A ., A lcaraz , A ., B ostw ic k , D . G .,L ieber, M . M ., and Jenk in s , R . B . Po ten tial m ark ers o f p ro state canceraggress iv en ess d ete cted by f luo rescen ce in s itu h y b rid iz ation in needleb iop sies . Can ce r R es., 54 : 3574-3579, 1994.14 . A lcaraz , A ., T ak ahash i, S ., B row n , J. A ., H erath , J. F., B erg stralh ,E . J., L arson -K e lle r, J. J., L ieber, M . M ., and Jenk in s , R . B . A neup lo idyand aneusom y o f ch rom osom e 7 de tec ted by f lu o re scence in situ hy -b rid iz ation arc m ark ers of poo r p rog ress in pro state can cer. C ancer R e s.,55 : 3998-4002, 1 994.15 . B row n , J. A ., A lcaraz , A ., T ak ah ash i, S ., Persons , D . L ., L ieber,M . M ., and Jen k in s, R . B . C h rom osom al aneu som ies d etec ted b y f luo -re scen t in s itu hy brid iz atio n (FIS H ) analy s is in c lin ically localiz edprostate carcinom a. J. U ro l., /52 : 1 1 57 -1162 , 1 994.16 . Carter, H . B ., and C of f e y , D . S . Predictio n of tum or b ehav io r inp ro state c an ce r. In: J. P. K arn an d H . Y am anak (ed s.), Pro state C ancer:T h e S econd T o k y o S y m pos ium , pp . 1 9 -27 . N ew Y o rk : E lsev ie r, 1989 .17. D iam ond, D . A ., B erry , S . J., Jew ett , H . J., Eggle sto n , J. C ., andCo f f ey , D . S . A n ew m e thod to asse s s m e tastatic po ten tial o f hum anprostate cancer : re lativ e nu clear roun dness . J. U ro l.128: 729-734 , 1982 .18 . Pearsons , D . L ., G ibney , D . J., K atzm ann , J. A ., L ieber, M . M .,Farrow , G . M ., and Jenk ins , R . B . U se o f f lu ore scentin situ hybnid iza-



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lion for d eo xyr ibo nuc leic ac id p lo id y ana lys is of prosta tic ad eno carci-n o ma . J . Ur o l . , /50 : 120-1 25 , 1 993 .19 . C her, M . L ., M acG rogan , D ., B ookste in , R ., B row n , J . A ., Jenk ins ,R . B ., and Jensen , R . H . C om parative genom ic hyb rid izat ion , a lle licim b alance , an d f luo rescen ce in s itu hybrid iz atio n on ch rom osom e 8 inpro sta te c anc er. G en es, C hrom osom es, an d C ancer,1: 153-1 62 , 19 94 .20 . V isakorp i, T . K all ion iem i, A . H ., S yv anen , A -C ., H yytinen , E . R .,K arhu . R ., T amm ela, T ., Iso la , J. J., an d K allio n iem i, 0 -P . G ene ticchange s in prim ary an d recu rren t pro sta te can ce r b y com parat ivegen om ic hyb rid izat ion . C an cer R es.,5: in pres s, 1 99 5 .2 1 . B ro thm an, A . R ., Pe eh l , D . M ., P atel , A . M ., a nd M cN eal ,. E.F req uen cy and p atte rn of ka ryo typ ic abn orm ali ties in h um an p ros ta tecan cer . C ance r R es. , 50 : 3795 -38 03 , 1 990 .2 2 . L undgren , R ., M andahl, A ., Sakr, W ., Pow ell,. J., and W olm an , S .K . C ytogene tics o f p rim ary pros tatic ad en ocarc in om a. C lo na lity andchrom osom e instab i lity . C ancer G ene t. C yto gen et. , 61 : 165-173 , 1 99 2 .23 . M ilas in , J ., and M icic , S . D ouble m in ute chrom o som es in an in va-siv e adenocarc inom a of th e pro sta te . C ance r G enet . C ytogene t.,2:1 5 7 - 15 9 , 1 9 9 4 .24 . M eltze r, P . S . , G u an , X . Y ., B u rge ss, A ., and T ren t , J. M . M ic ro-F ISH : a nove l s tra teg y to iden tify cy tp tic ch rom osom al rea rrangem ents .N ature G en et. , I: 24-2 8 , 199 2 .25 . Z h an g , J ., T ren t, J . M ., and M eltze r, P . 5 . R ap id iso la tion an dcha rac terizat ion o f am plif ied D NA by chrom osom e m icro d issect ion :iden tificat ion of IG F IR am p lificat ion in m alig nan t m elanom a . O n co -gene, 8: 2827 -2 831 , 19 93 .2 6 . G uan , S . Y ., M eltzer, P . S ., D a lton , W . S ., and T ren t, J. M .Id en tific ation of c ryp tic si tes of DNA sequence am plific atio n in hum anbre ast can cer by chrom o som e m icro d is sec tion . N a ture G ene t.,: 1 55 -1 6 1 , 1 9 9 4 .

27 . T ren t, J. M ., a nd T hom p son , F . H . M e tho ds for chrom o som e band-ing of hum an and ex per im enta l tum o rsi n vi tr o. M ethod s E nzy mol., 151:267-2 79 , 198 7 .28 . W are, J. L ., Pau lso n , D . F ., P ark s, . F ., and W ebb , K . S . P ro duc tio nof m onoc lon al an t ibo dy alpha P ro3 recogn izing a hum an p ro sta t icca rcinom a antigen . C ance r R e s.,42 : 1215-1 222 , 1 982 .29 . F ukum oto , M ., S hev rin , D . H ., an d R on inson , I . B . A naly sis of gen eam plific atio n in hum an tum or c ell lin es. G enetics,85 : 6846-6850 ,1988 .30 . G u an , X . Y ., T ran t, J. M ., a nd M eltz er, P . 5 . G eneratio n o f bandsp ecific pa tin ing p rob es f rom a s ing le m ic rod issected chrom o som e.Hum. Mo l . Ge n e t . , 2: 1 1 1 7 - 11 2 1 , 1 9 9 3 .31 . K allion iem i, A ., K all ion iem i, 0 -P ., Sud ar, D ., R uto v itz , D ., G rayJ . W ., W aldm ann , F ., and P inke l, D . C om para tiv e genom ic hybn id iza-tio n for m olecu lar cy to gen etic analysis o f so lid tum o rs. Scienc e (W ash -ing ton D C ) , 258: 818-821 , 19 92 .3 2. K alli on ie mi, 0-P ., K a llion iem i, A ., P eper, J ., Iso la , J., W aldm ann ,F . M ., G ray , J . W ., and P inke l, D . O ptim iz in g com para tive gen om ichybr id iza tion fo r an alys is o f D N A sequence copy num ber chang es iso lid tum o rs. G en es C hrom osom es & C an cer,10: 231-2 43 , 19 94 .33 . F lem ing , W . H ., H am el, A ., M acDonald , R ., R am sey , E ., Pe ttig rew ,N . M ., Joh nston , B ., D odd , J . G ., and M atus ik , R . J . E xpression of thec-myc pro too ncogene in hum an p rosta tic carcinom a and gen ign pros ta tichyperp las ia . C an cer R es ., 46: 1535-1537, 1986 .34 . K in zler , K . W ., Z eh nbauer , B . A ., B rod eur , G . M ., S eeger , R .T ren t, J. M ., M e ltze r, P . S ., an d V oge lste in , B . A m plifica tio n unicon ta in ing h um an N-myc an d c-myc gen es. P ro c. N atl. A cad . Sc i., 3:1 0 3 1 - 10 3 5 , 1 9 8 6 .

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