40 20 L in z. 1 7. O k tober 2 0 0 7

Neurochirurgische Abteilung T heoretische Neurochirurgie M ag a. Sabine Spiegl-Kreinecker Tel: + 43 (0)50 554/62-26092 E -mail: sabine.spiegl-kreinecker@ gespag.at 1 7. O k tober 2 0 0 7 A n d e n V e re in z u r F o rs c h u n g s fö rd e ru n g d e r Ö s te rre ic h is c h e n K re b s h ilfe OÖ z H U n iv.-d o z. D r. P e te r S c h re n k H arrac h s traß e 1 3 40 20 L in z Betreff A n trag u m P ro je k tfö rd e ru n g au s d e n M itte ln d e r Ob e rö s te rre ic h is c h e n K re b s h ilfe W e rte s K u rato riu m d e r Ö s te rre ic h is c h e n K re b s h ilfe Ob e rö s te rre ic h! S e h r g e e h rte r H e rr D o z. S c h re n k! In d e r A n lag e e rlau b e ic h m ir, Ih n e n d e n A n trag u m F ö rd e ru n g d e s P ro je k ts MGMT Status, Chemotherapy Response and Stem-Cell-Ness of Glioblastoma Multiforme (" Therapieversagen im Glioblastom: Rolle von Tumorstammzellen und MGMT Status" ) m it d e r B itte u m F ö rd e ru n g d u rc h d e n F o rs c h u n g s v e re in d e r Ob e rö s te rre ic h is c h e n K re b s h ilfe OÖ, v o rz u le g e n. D ie D u rc h fü h ru n g d e s P ro je k te s s o ll im R ah m e n e in e r D is s e rtatio n im Z e ll-u n d m o le k u larb io lo g is c h e n L ab o r d e r T h e o re tis c h e n N e u ro c h iru rg ie d e r N e u ro c h iru rg is c h e n A b te ilu n g an d e r L an d e s n e rv e n k lin ik W ag n e r J au re g g in L in z e rfo lg e n u n d 24 M o n ate d au e rn. Ic h b itte S ie s e h r u m U n te rs tü tz u n g u n s e re s P ro je k te s. E s h an d e lt s ic h b e i d e n b e an trag te n M itte ln in e rs te r L in ie u m d ie K o s te n fü r e in e P e rs o n alan s te llu n g (D an ie la L ö ts c h ) u n d u m M ate rialk o s te n. D ie e n ts p re c h e n d e In fras tru k tu r s o w ie d ie w e ite re n M ate rial- u n d P e rs o n alk o s te n w e rd e n v o n d e r N e u ro c h iru rg is c h e n A b te ilu n g /T h e o re tis c h e n N e u ro c h iru rg ie g e trag e n. D ie D u rc h fü h ru n g d e r V e rg le ic h e n d e n G e n o m is c h e n H y b rid is ie ru n g (C G H ) s o w ie d ie S e q u e n z ie ru n g v o n p 53 s o ll, m an g e ls g e rä te te c h n is c h e r A u s s tattu n g, im K re b s fo rs c h u n g s in s titu t W ie n d u rc h g e fü h rt w e rd e n. M it fre u n d lic h e n G rü ß e n, M ag a. S ab in e S p ie g l-k re in e c k e r Landes-Nervenklinik Wagner-Jauregg, Wagner-Jauregg-Weg 15, 4020 Linz, eine Gesundheitseinrichtung der Oö. Gesundheits- und Spitals-AG, Hafenstraße 47-51, 4020 Linz, DVR 2107870, ATU51928204, Firmenbuchgericht: Landesgericht Linz, FN 210146 p, Bankverbindung: Oberbank AG, Kto-Nr.: 711-0426/06, BLZ 15000, www.gespag.at

A n tra g u m P ro jek tfö rd eru n g d u rc h d en F o rs c h u n g s fö rd eru n g s v erein d er Ö s terreic h is c h en K reb s h ilfe O Ö MGMT STATUS, CHEMOTHERAPY RESPONSE AND STEM-CELL-NESS OF GLIOBLASTOMA MULTIFORME TH E RA P IE V E RSA GE N IM GL IO B L A STO M: RO L L E V O N TU MO RSTA MMZ E L L E N U ND MGMT STA TU S A N T R A G S T E L L E R I N M ag a.s ab in e S p ie g l-k re in e c k e r OÖ L N K W ag n e r J au re g g N e u ro c h iru rg is c h e A b te ilu n g ; T h e o re tis c h e N e u ro c h iru rg ie W ag n e r J au re g g W e g 1 5 40 20 L in z K O O P E R A T I O N S P A R T N E R A N D E R L N K W A G N E R J A U R E G G D e p artm e n t o f N e u ro s u rg e ry : P rim. U n iv -P ro f. D r. J o h an n e s F is c h e r D r in. J o h an n a B u c h ro ith n e r P riv.-d o z in. D r in.g ab rie le W u rm D e p artm e n t o f In te rn al M e d ic in e : D r. J o s e f P ic h le r In s titu te o f P ath o lo g y : P rim. D r. R e n é S ily e K O O P E R A T I O N S P A R T N E R I M I N S T I T U T F Ü R K R E BS F O R S C H U N G D E R M E D.U N I V E R S I T Ä T W I E N A o.p ro f.d r.w alte r B e rg e r 2

Z U S A M M E N F A S S U N G G lio b las to m e s in d, tro tz n e u e r E rk e n n tn is s e u n d V e rb e s s e ru n g e n, w e n ig e rfo lg re ic h th e rap ie rb ar u n d d ie m ittle re Ü b e rle b e n s z e it b e trä g t n ac h w ie v o r 1 2 M o n ate. D as th e rap e u tis c h e S tan d ard s c h e m a b e s te h t au s c h iru rg is c h e r R e s e k tio n m it n ac h fo lg e n d e r k o n k o m itan te r R ad io -C h e m o th e rap ie m it d e m A lk y lan tiu m T e m o z o lo m id. E in b e w ie s e n e r c h e m o th e rap ie lim itie re n d e r F ak to r is t d ie E x p re s s io n d e s D N A R e p aratu re n z y m s O 6 - M e th y lg u an in -D N A - M e th y ltran s fe ras e (M G M T ), d e s s e n A k tiv itä t d e r E ffe k tiv itä t alk y lie re n d e r S u b s tan z e n e n tg e g e n w irk t. E in e fe h le n d e E x p re s s io n au fg ru n d e p ig e n e tis c h e r H e m m u n g d u rc h M e th y lie ru n g am P ro m o te r d e s M G M T G e n s g e h t h in g e g e n m it e in e r E rh ö h u n g d e r S e n s itiv itä t g e g e n ü b e r d ie s e r G ru p p e v o n C h e m o th e rap e u tik a e in h e r. Z u d e m h at s ic h d ie M G M T P ro m o te rm e th y lie ru n g als u n ab h ä n g ig e r p rä d ik tiv e r F ak to r fü r d as T h e rap ie an s p re c h e n v o n T e m o z o lo m id, v e rb u n d e n m it e in e m d e u tlic h v e rlä n g e rte n Ü b e rle b e n, h e rau s g e s te llt. D e n n o c h k o m m t e s tro tz m ax im ale r T h e rap ie b e i n ah e z u alle n G lio b las to m e n z u r B ild u n g v o n R e z id iv tu m o re n, d av o n au s g e h e n d d as s e in e S u b g ru p p e v o n re s is te n te n T u m o rz e lle n au c h n ac h rad ik ale r Op e ratio n im G e h irn v e rb le ib t. T u m o rs tam m z e lle n, c h arak te ris ie rt d u rc h d e n M ark e r C D 1 33, z e ic h n e n s ic h d u rc h R e s is te n z g e g e n ü b e r e in e m b re ite n S p e k tru m v o n C h e m o th e rap e u tik a au s. In G lio b las to m e n k o n n te n A n te ile d ie s e r C D 1 33 + S tam m z e lle n in v ariie re n d e m A u s m aß e rs t k ü rz lic h g e z e ig t w e rd e n. Z ie l d ie s e r A rb e it is t e s 1 ) C D 1 33 + T u m o rs tam m z e lle n in G lio b las to m e n z u id e n tifiz ie re n b z w. z u s e le k tio n ie re n u n d 2) z u an aly s ie re n o b u n d in w ie w e it C D 1 33 + T u m o rs tam m z e lle n z u r T h e rap ie re s is te n z g e g e n ü b e r T e m o z o lo m id in G lio b las to m e n b e iträ g t. Z u d e m s o ll g e k lä rt w e rd e n o b d ie U n te rd rü c k u n g d e r M G M T A k tiv itä t u n te r V e rw e n d u n g v o n n e u e n tw ic k e lte n M G M T -In h ib ito re n d ie S e n s itiv itä t g e g e n ü b e r T e m o z o lo m id in d u z ie re n k an n. H au p tau g e n m e rk s o ll d ab e i au f d ie R o lle d e r T u m o rs tam m z e lle n g e le g t w e rd e n. 3

S U M M A R Y D e s p ite n e w re s e arc h fin d in g s re s u ltin g in im p ro v e m e n t o f th e rap y, in g lio b las to m as th e rap e u tic s u c c e s s re m ain s m o d e s t an d m e d ian s u rv iv al d o e s s till n o t e x c e e d 1 2 m o n th s. T h e s tan d ard tre atm e n t re g im e n c o n s is ts o f o p e ratio n fo llo w in g c o n c o m itan t rad io -c h e m o th e rap y w ith te m o z o lo m id e. L im itin g fac to r o f c h e m o th e rap e u tic s u c c e s s is th e e x p re s s io n /ac tiv ity o f th e D N A re p air e n z y m e O 6 -m e th y lg u an in e -D N A - m e th y ltran s fe ras e (M G M T ). M G M T ac ts as a c o u n te rp art o f alc y latin g ag e n ts re s u ltin g in re c o n s titu tio n o f an in tac t D N A, le av in g b e h in d v iab le tu m o u r c e lls. In c o n tras t, lac k o f M G M T e x p re s s io n d u e to e p ig e n e tic s ile n c in g o f th e M G M T g e n e p ro m o te r w as p ro v e n to b e as s o c iate d w ith in c re as e o f s e n s itiv ity ag ain s t th is g ro u p o f c h e m o th e rap e u tic s. M o re o v e r M G M T p ro m o te rm e th y latio n tu rn e d o u t to b e an in d e p e n d e n t p re d ic tiv e fac to r in g lio b las to m as lin k e d w ith e n h an c e d o v e rall s u rv iv al. N e v e rth e le s s, als o in c as e o f s u c c e s s fu l tre atm e n t w ith te m o z o lo m id e g lio b las to m as alm o s t g e n e rally re c u r in d ic atin g th at a s u b g ro u p o f tu m o u r c e lls is g e n e rally re s is tan t to c h e m o th e rap y. R e c e n tly, g lio b las to m as w e re s h o w n to c o n tain d iffe rin g am o u n ts o f s o -c alle d tu m o u r s te m c e lls c h arac te ris e d b y th e s te m c e ll m ark e r C D 1 33. T h e s e c e lls are re s is tan t ag ain s t a w id e s p e c tru m o f c h e m o th e rap e u tic ag e n ts. A im o f th e s tu d y is to 1 ) id e n tify an d s e le c t re s p e c tiv e ly C D 1 33 + tu m o u r c e lls in g lio b las to m as an d 2) to in v e s tig ate th e c o n trib u tio n o f tu m o u r s te m c e lls o r a s te m -c e ll lik e c e ll c o m p artm e n ts to th e lim ite d tre atm e n t s u c c e s s w ith te m o z o lo m id e in h u m an G B M. M o re o v e r, it w ill b e an aly s e d w h e th e r s u p p re s s io n o f M G M T b y n e w ly s y n th e s is e d M G M T in h ib ito rs w ill b e ab le to in d u c e c h e m o s e n s itiv ity in G B M w ith a s p e c ific fo c u s o n th e tu m o r s te m c e lls. 2

E X P O S E 1 ) I n tro d u c tio n, S ta tu s o f R es ea rc h H u m an g lio b las to m a (as tro c y to m a W H O g rad e IV, G B M ) re p re s e n t th e v e ry c o m m o n an d h ig h ly ag g re s s iv e p rim ary m alig n an t b rain tu m o r. G B M is ty p ic ally c o m p o s e d o f d iffe re n t c e ll ty p e s d is p lay in g a w id e s p e c tru m o f h e te ro g e n e ity (re fe rre d to b y th e te rm m u ltifo rm e ) (1 ). G lio b las to m as aris e e ith e r de novo w ith o u t e v id e n c e o f an an te c e d e n t le s io n (p rim ary g lio b las to m a) o r th ro u g h an an ap las tic p ro g re s s io n fro m a lo w e r-g rad e as tro c y to m a (s e c o n d ary g lio b las to m a). D e s p ite in te n s iv e re s e arc h re g ard in g th e g e n e tic s an d b io lo g y, th e p ro g n o s is o f g lio b las to m a p atie n ts is s till p o o r. E v e n at m ax im al th e rap y, m e d ian s u rv iv al o f g lio b las to m a p atie n ts re m ain s lo w an d ran g e s b e tw e e n 1 0 to 1 2 m o n th s, o n ly (2). T h e c u rre n t s tan d ard o f c are fo r p atie n ts w ith h ig h -g rad e m alig n an t g lio m a u s e d to b e re s e c tio n fo llo w e d b y rad io th e rap y (R T ). H o w e v e r, th e u s e o f ad ju v an t an d /o r c o n c o m itan t c h e m o th e rap y an d th e s tan d ard o f c are at firs t re lap s e are s till u n d e r d e b ate (3). C h e m o th e rap e u tic tre atm e n t s u c c e s s in b rain tu m o rs is m ain ly lim ite d b y th e b lo o d -b rain - b arrie r an d th e e x p re s s io n o f c h e m o re s is tan c e -re late d p ro te in s lik e P -g ly c o p ro te in (P -g p ), m u ltid ru g re s is tan c e p ro te in 1 (M R P 1 ), an d m ajo r v au lt p ro te in (M V P ) als o k n o w n as lu n g re s is tan c e p ro te in (L R P ) (4-6). B as e d o n th e ir ab ility to c irc u m v e n t th e b lo o d -b rain -b arrie r, alk y latin g ag e n ts re p re s e n t th e g o ld s tan d ard in th e tre atm e n t o f m alig n an t g lio m as. B e s id e s n itro s o u re as, te m o z o lo m id e is th e m o s t p ro m is in g d ru g in th e c u rre n t tre atm e n t o f p atie n ts w ith m alig n an t g lio m as. T e m o z o lo m id e h as d e m o n s trate d a g o o d tis s u e d is trib u tio n in c lu d in g p e n e tratio n o f th e b lo o d -b rain -b arrie r an d th e c e re b ro s p in al flu id (7). R e c e n tly, a s ig n ific an t s u rv iv al b e n e fit fo r g lio b las to m a p atie n ts tre ate d w ith te m o z o lo m id e (T M Z ) c o m b in e d w ith R T w as s h o w n (8). H o w e v e r, p ro te c tio n ag ain s t th e to x ic e ffe c t o f alk y latin g ag e n ts m ig h t re ly o n th e ac tiv ity o f O 6 -m e th y lg u an in e -D N A m e th y ltran s fe ras e (M G M T )(9), a k e y e n z y m e in th e D N A re p air n e tw o rk (1 0 ). T h u s, th e M G M T le v e ls in tu m o u r c e lls c o rre late n e g ativ e ly w ith th e e ffic ac y o f T M Z tre atm e n t, in p artic u lar w h e n alte ratio n s o f o th e r D N A re p air s y s te m s are ab s e n t. A d d itio n ally, d e fic ie n c ie s o f th e p o s t-re p lic atio n m is m atc h re p air s y s te m re n d e r tu m o u r c e lls re s is tan t to m e th y latin g ag e n ts in d e p e n d e n t o f M G M T le v e ls. O 6 -m e th y lg u an in e D N A 3

m e th y ltran s fe ras e e x p re s s io n is re tain e d in m o s t g lio m as (1 1-1 3). L o s s o f M G M T p ro te in as a c o n s e q u e n c e o f M G M T g e n e p ro m o te r m e th y latio n p re d ic ts a g o o d re s p o n s e to alk y latin g ag e n t-b as e d c h e m o th e rap y (1 4). A c c o rd in g ly, in h ib ito rs o f M G M T lik e O 6 -b e n z y lg u an in e (O 6 -B G ), re s to re s T M Z -in d u c e d c y to to x ic ity in g lio m a c e lls in v itro an d in v iv o (F rie d m an e t al. 20 0 2) an d th e th e rap e u tic ap p lic atio n o f O 6 -B G in c o m b in atio n w ith d iffe re n t alk y latin g ag e n ts h as b e e n te s te d in c lin ic al s tu d ie s (1 5, 1 6). R e c e n tly it h as b e e n s h o w n th at th e tu m o r s u p p re s s o r p 53 is in v o lv e d in th e re g u latio n o f M G M T ac tiv ity in d e p e n d e n t o f p ro m o te r m e th y latio n (1 7)an d lo s s o f M G M T e x p re s s io n c o m b in e d w ith w ild -ty p e p 53 is s tro n g ly p re d ic tiv e fo r e n h an c e d s u rv iv al in G B M p atie n ts (1 8). N e v e rth e le s s, als o in c as e o f s u c c e s s fu l tre atm e n t w ith te m o z o lo m id e G B M alm o s t g e n e rally re c u r in d ic atin g th at a s u b g ro u p o f tu m o r c e lls is g e n e rally re s is tan t to c h e m o th e rap y. R e c e n tly, G M B w e re s h o w n to c o n tain d iffe rin g am o u n ts o f s o -c alle d tu m o r s te m c e lls c h arac te ris e d b y th e s te m c e ll m ark e r C D 1 33. T h e s e c e lls are re s is tan t ag ain s t a w id e s p e c tru m o f c h e m o th e rap e u tic ag e n ts. V e ry re c e n tly it h as b e e n in d ic ate d th at th e s e c e lls are ab le to p re fe re n tially ac tiv ate th e D N A d am ag e re s p o n s e. H o w e v e r n o th in g is c u rre n tly k n o w n o n th e re latio n s h ip b e tw e e n M G M T p ro m o te r m e th y latio n, s te m -c e ll-n e s s an d c h e m o th e rap y re s p o n s e in c e ll s u b p o p u latio n s o f h u m an G B M. 2 ) A im s o f th e S tu d y A im o f th e s tu d y is to in v e s tig ate th e c o n trib u tio n o f tu m o u r s te m c e lls o r a s te m -c e ll lik e c e ll c o m p artm e n ts to th e lim ite d tre atm e n t s u c c e s s w ith te m o z o lo m id e in h u m an G B M. M o re o v e r, it w ill b e an aly s e d w h e th e r s u p p re s s io n o f M G M T b y n e w ly s y n th e s is e d M G M T in h ib ito rs w ill b e ab le to in d u c e c h e m o s e n s itiv ity in G B M w ith a s p e c ific fo c u s o n th e tu m o u r s te m c e lls. 3 ) P la n n ed E x p erim en ts U n d e r p o in t 5) th e p re v io u s w o rk o f o u r g ro u p w ith re g ard to G B M an d th e m u ltip le c e ll m o d e ls av ailab le fo r th is an aly s is is d e s c rib e d. 4

* A s a firs t c h arac te ris atio n s te p, aro u n d 40 G B M c e ll c u ltu re s at e arly p as s ag e n u m b e r w ill b e an aly s e d fo r th e p re s e n c e o f C D 1 33-p o s itiv e c e lls s u p p o s e d to b e c an c e r s te m c e lls b y m ag n e tic s o rtin g. A b o u t 5 ap p ro p riate c e ll m o d e ls w ill b e s e le c te d fo r fu rth e r an aly s is. * F ro m th e s e p rim ary G B M c e ll c u ltu re s s u b p o p u latio n s w ill b e is o late d b y th re e d iffe re n t p ro c e d u re s fo r fe atu re s all as s o c iate d w ith s te m c e ll-lik e p ro p e rtie s : 1 ) C e lls e x p re s s in g th e n e u ro n al s te m c e ll m ark e r C D 1 33 (m ag n e tic s o rtin g ) 2) C e lls ab le to e fflu x th e flu o re s c e n t A B C -tran s p o rte r s u b s trate s D A P I an d /o r d o x o ru b ic in 3) T h o s e c e ll lin e s s u rv iv in g s h o rt-te rm e x p o s u re to te m o z o lo m id e o r B C N U. T o d o s o, d ru g - s e n s itiv e c e ll lin e s w ill b e e x p o s e d to in c re as in g c o n c e n tratio n s o f te m o z o lo m id e /B C N U. A n id e al c u ltu re c o n d itio n allo w in g s u rv iv al o f 5-1 0 % o f c e lls w ill b e u p s c ale d to g e t an ap p ro p riate c e ll n u m b e r fo r fu rth e r an aly s e s. * T h e to tal c e ll c u ltu re s an d th e th re e d iffe re n t c e ll s u b p o p u latio n s w ill b e an aly s e d fo r e x p re s s io n o f s te m c e ll m ark e rs (C D 1 33, n e s tin ) an d p ro p e rtie s (s p h e ro id fo rm atio n ). A d d itio n ally, th e p re s e n c e o f ac tiv e d ru g e fflu x p u m p s (A B C B 1, A B C C 1, A B C C 2, an d A B C G 1 ) as s o c iate d w ith d iffe re n t s te m c e ll c o m p artm e n ts w ill b e an aly s e d b y d iffe re n t s u b s trate /in h ib ito r c o m b in atio n s all av ailab le fo r an aly s is in th e lab. M o re o v e r it w ill b e an aly s e d w h e th e r th e s e s u b p o p u latio n e x h ib it d iffe re n t s e n s itiv itie s to c h e m o th e rap e u tic ag e n ts lik e B C N U an d te m o z o lo m id e. F u rth e r, it w ill b e d e te rm in e d w h e th e r th e s e c e lls h av e a c h an g e d M G M T s tatu s in te rm s o f p ro m o te r m e th y latio n an d o r g e n e e x p re s s io n. * In o rd e r to in v e s tig ate w h e th e r th e id e n tic al s te m -c e ll-lik e c e lls h av e b e e n is o late d b y th e s e d iffe re n t m e th o d s th e s u b p o p u latio n s w ill b e c o m p are d w ith th e p are n tal c e ll c u ltu re w ith re s p e c t to c h ro m o s o m al ab e rratio n s b y C G H. A d d itio n ally, th e m u tatio n s tatu s o f p 53 w ill b e in v e s tig ate d b y d ire c t s e q u e n c in g. * L as tly, in h ib itio n o f M G M T e n z y m e ac tiv ity b y n e w ly d e v e lo p e d M G M T in h ib ito rs (O6- B G o r g lu c o s e -c o n ju g ate d d e riv ativ e s (8-az a-o(6)-b e n z y lg u an in e, O(6)-(4-b ro m o th e n y l)- g u an in e an d th e c o rre s p o n d in g s p ac e r-lin k e d b e ta-d -g lu c o s e c o n ju g ate s ) w ill b e te s te d as a s trate g y to s e n s itiz e M G M T p ro m o te r-u n m e th y late d tu m o r c e lls an d tu m o r s te m c e lls to alk y latin g ag e n ts. 5

4 ) M a teria l/m eth o d s : A ro u n d 40 g lio m a c ell lin es a n d p rim a ry c u ltu res e s tab lis h e d fro m tu m o r tis s u e s (s e e als o c h ap te r 4.) w ill b e in c lu d e d an d an aly s e d w ith re g ard to M G M T p ro m o te r an d p ro te in e x p re s s io n s tatu s. A s e le c tio n o f aro u n d 20 g lio b las to m a c e ll lin e s d e riv e d fro m p atie n ts re s p o n s iv e (N = 1 0 ) an d u n re s p o n s iv e (N = 1 0 ) to te m o z o lo m id e c h e m o th e rap y w ill b e s e le c te d fo r fu rth e r an aly s is. A b o u t th re e to fiv e c e ll c u ltu re s w ill b e s e le c te d fo r p re p aratio n o f c e ll s u b p o p u latio n s w ith re g ard to th e e x p re s s io n o f C D 1 33. T em o z o lo m id e a n d O 6 -Ben z y lg u a n in e (O 6 -BG ) as w e ll as d iv e rs e c h e m o th e rap e u tic ag e n ts are av ailab le in th e lab. T h e O6-B G d e riv ativ e s g lu c o s e -c o n ju g ate d d e riv ativ e s 8-az a-o(6)- b e n z y lg u an in e, O(6)-(4-b ro m o th e n y l)-g u an in e an d th e c o rre s p o n d in g s p ac e r-lin k e d b e ta-d - g lu c o s e c o n ju g ate s are s u p p lie d b y F au s tu s, V ie n n a. M ag n e tic c e ll s o rtin g w ill b e d o n e afte r C D 1 3 3 s ta in in g (an tib o d y fro m M ilte n y ) w ith th e m ag n e tic C D 1 33 c e ll is o latio n k it (M ilte n y ). F o r c h em o s en s itiv ity tes tin g M T T -as s ay s (E as y fo r y o u, B io rad ) an d ³H -th y m id in e in c o rp o ratio n as s ay s w ill b e u s e d as p u b lis h e d. C ell c y c le an aly s is w ill b e d o n e b y P I s tain in g. T h e s tatu s o f th e M G M T p ro m o ter m eth y la tio n w ill b e fo llo w e d b y m e th y latio n - s p e c ific P C R as e s tab lis h e d in o u r lab. W es tern b lo ts an d R T -P C R w ill b e p e rfo rm e d b y s tan d ard m e th o d s. D iv e rs e a p o p to s is as s ay s (D A P I/P I, J C 1, an n e x in V ) w ill b e u s e d as p u b lis h e d. C o m p a ra tiv e g en o m ic h y b rid iz a tio n (C G H ) an d array C G H is e s tab lis h e d at th e lab o f P ro f. B e rg e r (In s titu te o f C an c e r R e s e arc h, M e d ic al U n iv e rs ity V ie n n a). 5 ) S ta tu s o f O w n R es ea rc h W ith in th e o n g o in g g lio m a re s e arc h at o u r lab w e h av e e s tab lis h e d p rim ary c e ll c u ltu re s o f 259 G B M tu m o rs (in c lu d in g re c u rre n c e s ) w h ic h w e re o p e rate d at th e D e p artm e n t o f N e u ro s u rg e ry b e tw e e n 20 0 0 an d 20 0 6. 23 % d e v e lo p e d in to s tab le c e ll lin e s. A ro u n d 40 o f th e s e c e ll c u ltu re s an d th e re s p e c tiv e tu m o u rs h av e b e e n c h arac te ris e d fo r c h ro m o s o m al c h an g e s b y C G H. M o re o v e r s e v e ral o f th e s e an d ad d itio n ally in te rn atio n al G B M c e ll m o d e ls h av e b e e n c h arac te ris e d fo r th e ir s e n s itiv ity to d iv e rs e c h e m o th e rap e u tic ag e n ts an d th e im p ac t o f k n o w n d ru g re s is tan c e g e n e s in c lu d in g A B C -tran s p o rte rs an d lu n g re s is tan c e p ro te in L R P. 6

M o re o v e r, in ab o u t 50 c e ll lin e s an d th e re s p e c tiv e tu m o r tis s u e s m e th y latio n o f th e M G M T p ro m o te r h as b e e n e s tab lis h e d b y m e th y latio n -s p e c ific P C R. M G M T p ro m o te r m e th y latio n is als o in o u r s y s te m an d ac c o rd in g to th e lite ratu re in d ic ativ e fo r te m o z o lo m id e an d B C N U s e n s itiv ity in G B M c e lls (m an u s c rip t in p re p aratio n ). A ll th e s e c e ll m o d e ls an d d ata are th e b as is o f th e c u rre n t s tu d y. P u b lic a tio n s w ith res p ec t to b ra in tu m o rs S p ieg l-k rein ec k er, S., P irk e r C, P ic h le r J, B u c h ro ith n e r, J, S ily é R, F is c h e r, J., M ic k s c h e M., an d B e rg e r, W. M G M T is p re re q u is ite b u t n o t c o n d itio n fo r c h e m o s e n s itiv ity to te m o z o lo m id e in g lio b las to m as in v itro an d in v iv o : a s in g le c e n te r e x p e rie n c e (in p re p aratio n ) A lle rs to rfe r, S., S o n v illa, G., F is c h e r, H., S p ieg l-k rein ec k er, S., S e tin e k, U., C z e c h, T., M aro s i, C., B u c h ro ith n e r, J., P ic h le r, J., S ily e, R., M o h r, T., H o lz m an n, K., G ras l-k rau p p, B., M arian, B., G ru s c h, M., F is c h e r, J., M ic k s c h e, M, B e rg e r, W. F G F 5 as an On c o g e n ic F ac to r in H u m an G lio b las to m a M u ltifo rm e : A u to c rin e an d P arac rin e A c tiv itie s. O nc ogene, in re v is io n, 20 0 7 S p ieg l-k rein ec k er, S., P irk e r, C., M aro s i, C., B u c h ro ith n e r, J., P ic h le r, J., S ily e, R., F is c h e r, J., M ic k s c h e, M. & B e rg e r, W. (20 0 7). D y n am ic s o f c h e m o s e n s itiv ity an d c h ro m o s o m al in s tab ility in re c u rre n t g lio b las to m a. B r J Canc er, 96, 960-9 S p ieg l-k rein ec k er, S., B u c h ro ith n e r, J., E lb lin g, L., S te in e r, E., W u rm, G., B o d e n te ic h, A., F is c h e r, J., M ic k s c h e, M. & B e rg e r, W. (20 0 2). E x p re s s io n an d fu n c tio n al ac tiv ity o f th e A B C -tran s p o rte r p ro te in s P -g ly c o p ro te in an d m u ltid ru g -re s is tan c e p ro te in 1 in h u m an b rain tu m o r c e lls an d as tro c y te s. J Neuroonc ol, 5 7, 27-36. B e rg e r, W., S p ieg l-k rein ec k er, S., B u c h ro ith n e r, J., E lb lin g, L., P irk e r, C., F is c h e r, J. & M ic k s c h e, M. (20 0 1 ). Ov e re x p re s s io n o f th e h u m an m ajo r v au lt p ro te in in as tro c y tic b rain tu m o r c e lls. Int J Canc er, 94, 377-82. G ra n t reg a rd in g b ra in tu m o rs O b erö s terreic h is c h e K reb s h ilfe 0 1 2/20 0 3-0 5/20 0 6 C h arak te ris ie ru n g q u an titativ e r g e n o m is c h e r V e rä n d e ru n g e n in p rim ä re n H irn tu m o re n 7

6 ) T im e P la n TASK MONTHS 1-1 2 MONTHS 1 3-2 4 Tu m o r c e ll c u ltu r e CD1 3 3 s ta in in g Ma g n e tic c e ll s o r tin g Ch e m o s e n s itiv ity te s tin g MGMT e x p r e s s io n a n a ly s is Ap o p to s is a s s a y s Co m p a r a tiv e g e n o m ic h y b r id iz a tio n Re a l-tim e PCR, W e s te n b lo t Da ta a n a ly s is, m a n u s c r ip t p r e p a r a tio n 7 ) F in a n c ia l P la n P e rs o n n e l C o s t (S alary fo r D a n iela L ö ts c h fo r 24 M o n th ) 24 x 2570.- 61,720.- M a teria l C o s ts C D 1 33 C e ll Is o latio n k it (á 490,-/20 s am p le s ) 1,960.- M ate rial fo r M G M T e x p re s s io n an aly s is 50 0.- C e ll c u ltu re m ate rial 1,0 0 0.- R T -P C R, w e s te rn b lo t 60 0,- A n tib o d ie s 2,0 0 0.- S e q u e n c in g 1,0 0 0.- T o ta l 6 8,7 8 0.- 8

R eferen c es 1. M ao, X., an d R. A. H am o u d i. 20 0 0. M o le c u lar an d c y to g e n e tic an aly s is o f g lio b las to m a m u ltifo rm e. Canc er Genet Cytogenet 1 22:87-92. 2. S c o tt, C. B., C. S c aran tin o, R. U rtas u n, B. M o v s as, C. U. J o n e s, J. R. S im p s o n, A. J. F is c h b ac h, an d W. J. C u rran, J r. 1 998. V alid atio n an d p re d ic tiv e p o w e r o f R ad iatio n T h e rap y On c o lo g y G ro u p (R T OG ) re c u rs iv e p artitio n in g an aly s is c las s e s fo r m alig n an t g lio m a p atie n ts : a re p o rt u s in g R T OG 90-0 6. Int J Radiat O nc ol B iol P hys 40 :51-55. 3. v an d e n B e n t, M. J., M. E. H e g i, an d R. S tu p p. 20 0 6. R e c e n t d e v e lo p m e n ts in th e u s e o f c h e m o th e rap y in b rain tu m o u rs. E ur J Canc er. 4. S p ie g l-k re in e c k e r, S., J. B u c h ro ith n e r, L. E lb lin g, E. S te in e r, G. W u rm, A. B o d e n te ic h, J. F is c h e r, M. M ic k s c h e, an d W. B e rg e r. 20 0 2. E x p re s s io n an d fu n c tio n al ac tiv ity o f th e A B C -tran s p o rte r p ro te in s P -g ly c o p ro te in an d m u ltid ru g -re s is tan c e p ro te in 1 in h u m an b rain tu m o r c e lls an d as tro c y te s. J Neuroonc ol 57:27-36. 5. B e rg e r, W., S. S p ie g l-k re in e c k e r, J. B u c h ro ith n e r, L. E lb lin g, C. P irk e r, J. F is c h e r, an d M. M ic k s c h e. 20 0 1. Ov e re x p re s s io n o f th e h u m an m ajo r v au lt p ro te in in as tro c y tic b rain tu m o r c e lls. Int J Canc er 94:377-382. 6. B re d e l, M. 20 0 1. A n tic an c e r d ru g re s is tan c e in p rim ary h u m an b rain tu m o rs. B rain Res B rain Res Rev 35:1 61-20 4. 7. D e A n g e lis, L. M. 20 0 3. B e n e fits o f ad ju v an t c h e m o th e rap y in h ig h -g rad e g lio m as. Semin O nc ol 30 :1 5-1 8. 8. S tu p p, R., W. P. M as o n, M. J. v an d e n B e n t, M. W e lle r, B. F is h e r, M. J. T ap h o o rn, K. B e lan g e r, A. A. B ran d e s, C. M aro s i, U. B o g d ah n, J. C u rs c h m an n, R. C. J an z e r, S. K. L u d w in, T. G o rlia, A. A llg e ie r, D. L ac o m b e, J. G. C airn c ro s s, E. E is e n h au e r, an d R. O. M irim an o ff. 20 0 5. R ad io th e rap y p lu s c o n c o m itan t an d ad ju v an t te m o z o lo m id e fo r g lio b las to m a. N E ngl J Med 352:987-996. 9. H arris, L. C., an d G. P. M arg is o n. 1 993. E x p re s s io n in m am m alian c e lls o f th e E s c h e ric h ia c o li O6 alk y lg u an in e -D N A -alk y ltran s fe ras e g e n e o g t re d u c e s th e to x ic ity o f alk y ln itro s o u re as. B r J Canc er 67:1 1 96-1 20 2. 9

1 0. P e g g, A. E. 1 990. M am m alian O6-alk y lg u an in e -D N A alk y ltran s fe ras e : re g u latio n an d im p o rtan c e in re s p o n s e to alk y latin g c arc in o g e n ic an d th e rap e u tic ag e n ts. Canc er Res 50 :61 1 9-61 29. 1 1. P re u s s, I., I. E b e rh ag e n, S. H aas, R. H. E ib l, M. K au fm an n, G. v o n M in c k w itz, an d B. K ain a. 1 995. O6-m e th y lg u an in e -D N A m e th y ltran s fe ras e ac tiv ity in b re as t an d b rain tu m o rs. Int J Canc er 61 :321-326. 1 2. P re u s s, I., S. H aas, U. E ic h h o rn, I. E b e rh ag e n, M. K au fm an n, T. B e c k, R. H. E ib l, P. D all, T. B au k n e c h t, J. H e n g s tle r, B. M. W ittig, W. D ip p o ld, an d B. K ain a. 1 996. A c tiv ity o f th e D N A re p air p ro te in O6-m e th y lg u an in e -D N A m e th y ltran s fe ras e in h u m an tu m o r an d c o rre s p o n d in g n o rm al tis s u e. Canc er D etec t P rev 20 :1 30-1 36. 1 3. S ilb e r, J. R., M. S. B o b o la, S. G h atan, A. B lan k, D. D. K o ls to e, an d M. S. B e rg e r. 1 998. O6-m e th y lg u an in e -D N A m e th y ltran s fe ras e ac tiv ity in ad u lt g lio m as : re latio n to p atie n t an d tu m o r c h arac te ris tic s. Canc er Res 58:1 0 68-1 0 73. 1 4. E s te lle r, M., J. G arc ia-f o n c illas, E. A n d io n, S. N. G o o d m an, O. F. H id alg o, V. V an ac lo c h a, S. B. B ay lin, an d J. G. H e rm an. 20 0 0. In ac tiv atio n o f th e D N A -re p air g e n e M G M T an d th e c lin ic al re s p o n s e o f g lio m as to alk y latin g ag e n ts. N E ngl J Med 343:1 350-1 354. 1 5. F rie d m an, H. S., J. P lu d a, J. A. Q u in n, R. B. E w e s u e d o, L. L o n g, A. H. F rie d m an, I. C o k g o r, O. M. C o lv in, M. M. H ag lu n d, D. M. A s h le y, J. N. R ic h, J. S am p s o n, A. E. P e g g, R. C. M o s c h e l, R. E. M c L e n d o n, J. M. P ro v e n z ale, E. S. S te w art, S. T o u rt- U h lig, A. M. G arc ia-t u rn e r, J. E. H e rn d o n, 2n d, D. D. B ig n e r, an d M. E. D o lan. 20 0 0. P h as e I trial o f c arm u s tin e p lu s O6-b e n z y lg u an in e fo r p atie n ts w ith re c u rre n t o r p ro g re s s iv e m alig n an t g lio m a. J Clin O nc ol 1 8:3522-3528. 1 6. Q u in n, J. A., J. P lu d a, M. E. D o lan, S. D e lan e y, R. K ap lan, J. N. R ic h, A. H. F rie d m an, D. A. R e ard o n, J. H. S am p s o n, O. M. C o lv in, M. M. H ag lu n d, A. E. P e g g, R. C. M o s c h e l, R. E. M c L e n d o n, J. M. P ro v e n z ale, S. G u ru ran g an, S. T o u rt-u h lig, J. E. H e rn d o n, 2n d, D. D. B ig n e r, an d H. S. F rie d m an. 20 0 2. P h as e II trial o f c arm u s tin e p lu s O(6)-b e n z y lg u an in e fo r p atie n ts w ith n itro s o u re a-re s is tan t re c u rre n t o r p ro g re s s iv e m alig n an t g lio m a. J Clin O nc ol 20 :2277-2283. 1 0

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