MONIKA E. HEGI

Laboratory of Tumor Biology and GeneticsCentre Romand de Neurochirurgie,

Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland

&« NCCR Molecular Oncology », ISREC

ENASCO Meeting, November 15-17, 2007

Biomarkers of Brain Tumors to Temozolomide Treatment

Biomarkers of Brain Tumors to Temozolomide Treatment

Predictive Factor(s) for Temozolomide (TMZ) Derived Benefit for Glioblastoma Patients

Predictive Factor(s) for Temozolomide (TMZ) Derived Benefit for Glioblastoma Patients

months0 6 12 18 24 30 36 42

0102030405060708090

100

TMZ/RT

RT

%

RT TMZ/RTMedian OS, mo: 12.1 14.62-yr survival: 10% 26%HR [95% CI]: 0.63 [0.52-0.75]

Logrank, p <0.0001

Who are the patients who benefit from TMZ ?Who are the patients who benefit from TMZ ?

573 patients enrolled

Weeks

Concomitant

TMZ/RT Adjuvant TMZ

6 10 14 18 22 26 30

RT Alone

R0

Stupp et al. N Engl J Med, 352: 987-996, 2005

NNNN

NN

NN NHNH22

OO

dRdR

O6-methylguanine

CHCH33

06-Methylguanine-DNA Methyltransferase (MGMT)06-Methylguanine-DNA Methyltransferase (MGMT)

•TMZ, alkylating agent

Guanine

N

N

N NH2

O

dR

NH

•irreversible inactivationinactivation•degradation

NH2

NHCH CH

CO

COOH

S 2CHCH33

MGMT• CHR 10q26

• linked with resistance to alkylating agent therapy

• inducible :•RT•genotoxic agents•glucocorticosteroids

MGMT• CHR 10q26

• linked with resistance to alkylating agent therapy

• inducible :•RT•genotoxic agents•glucocorticosteroids

MGMT

NH2

NHCH2 CH

CO

COOH

HS

repair

MOUSE MODELS & Alkylating Agents

•MGMT-/- hypersensitive

•MGMT-Tg resistant to tumor formation

MOUSE MODELS & Alkylating Agents

•MGMT-/- hypersensitive

•MGMT-Tg resistant to tumor formation

MGMT GENE

expressionexpression

Silencing of the MGMT Repair Gene by Methylation of the Gene Promoter

Silencing of the MGMT Repair Gene by Methylation of the Gene Promoter

Promoter region expressionexpression

Normal

Tumor

06-Methylguanine-DNA Methyltransferase (MGMT)

Tumor

•No repair protein•No repair of TMZ treatment induced DNA damage

•Response to tumor treatment•Improved survival of glioblastoma patient

methylatednonono« turned off »

Hegi_et al_06

months

Ove

rall

Surv

ival

0 5 10 15 20 25 30 35 400

10

20

30

40

50

60

70

80

90

100

UnmethylatedN = 114 (55%)

Methylated, N = 92 (45%)

MGMT Promoter Methylation Predicts Better Outcome in Glioblastoma Patients of this TrialMGMT Promoter Methylation Predicts Better

Outcome in Glioblastoma Patients of this Trial

Total N=206

MGMT Unmeth MethMedian OS, mo: 12.2 18.22-yr survival: 7.8% 34.1%

HR [95% CI]: 0.45 [0.32-0.61]Logrank test: p <0.0001

Risk of death reduced by 55%

Hegi et al. N Engl J Med, 352: 997-1003, 2005

[bp]

200 100

300

93 81

[bp]

H2O 569 555 549 LU M U M U M U M U M U M U M U M

529 527 PBL MethPBL

Hegi_et al_06

months

0 4 8 12 16 20 24 28 32 36 400

10

20

30

40

50

60

70

80

90

100

Ove

rall

Surv

ival

TMZTMZ/ RT

RT alone

Unmethylated MGMT

MGMT Promoter Methylation Predicts Benefit from TMZ Treatment

MGMT Promoter Methylation Predicts Benefit from TMZ Treatment

Randomization: RT TMZ/RTMedian OS mo: 11.8 12.72-yr survival: 1.9% 13.8%

Logrank : p = 0.062

Hegi et al. N Engl J Med, 352: 997-1003, 2005

Methylated MGMT

0 5 10 15 20 25 30 35 400

10

20

30

40

50

60

70

80

90

100

TMZTMZ/ RT

RT alone

months

Randomization: RT TMZ/RTMedian OS mo: 15.3 21.72-yr survival 22.7% 46.0%

Logrank : p = 0.0074

0 3 6 9 12 15 18 21 24 27 30 330

10

20

30

40

50

60

70

80

90

100

months

Overall Wald test : p < 0.0001 (df=3)

Predictive Value of MGMT Methylationfor Overall Survival

Predictive Value of MGMT Methylationfor Overall Survival

Hegi et al. N Engl J Med, 352: 997-1003, 2005

36 39

SurvivalRandomization median mo 2-yr

Unmeth, RT alone 11.8 <2.0 %Unmeth, RT/TMZ 12.7 13.8 %

Unmeth, TMZ/RTUnmeth, RT

Logrank : p = 0.062

Meth,Meth, RTRT

Meth, TMZ/RT

Meth,Meth, RTRT alone 15.3 15.3 22.7 %22.7 %Meth, RT/TMZ 21.7 46.0 %

Logrank : p = 0.0074

Stupp et al NEJM 2005

Palliative Care only

Repeat RTSurgery

Temozolomide

Any AdditionalChemotherapy

22

523

25

58

TMZ/RT [%]n=287

17

423

60

72

RT [%]n=286

Treatment at progressionat discretion of treating physician

Randomization

months0 4 8 12 16 20 24 28 32 36 40

0

10

20

30

40

50

60

70

80

90

100

O N Number of patients at risk :54 54 28 9 0 0 0 0 0 0 053 60 44 18 8 8 8 7 5 3 145 46 33 15 7 3 2 1 0 0 040 46 35 28 18 14 10 6 3 1 0

Unmeth, RT aloneUnmeth, TMZ/ RTMeth, RT aloneMeth, TMZ/ RT

Prog

ress

ion

Free

Sur

viva

l Overall Wald test: p <0.0001 (df=3)

Meth, TMZTMZ/ RT

Meth, RTUnmeth, TMZTMZ/ RTUnmeth

RT

Progression Free Survival SupportsMGMT Methylation Status as

Predictive Factor for Benefit from TMZ

Progression Free Survival SupportsMGMT Methylation Status as

Predictive Factor for Benefit from TMZ

Hegi_et al_06

0

10

20

30

40

50

60

70

80

90

Review of the literature, August 2007data from 1376 patients from 15 publications

WHO grade 1 2 3 4 4 4 2 3 2 3Brain Pilocytic LA AA GB PrGB ScGB O AO OA AOA

% M

GM

Tm

eth

Astrocytoma Oligodendro-glioma

Mixed Oligoastrocytoma

30 145

672

169 18155 45 73 51 13

n=5

MGMT Promoter Methylation Ranges from 20 to >80% Depending on Glioma Subtype

Validation of MGMT as Predictive FactorDepletion of MGMT in Tumor Cells by a Dose Dense Schedule

Validation of MGMT as Predictive FactorDepletion of MGMT in Tumor Cells by a Dose Dense Schedule

TMZ daily x 6 wksR

RTOG/EORTC/NCICPhase III Study

Radiotherapy (30 x 2 Gy)

Concomitant Phase Adjuvant Phase (6-12 mo)

Dose dense TMZ (100 mg/m2 daily x 21d)

Stratify by:MGMT methylation

TMZ daily x 6 wksR

RTOG/EORTC/NCICPhase III Study

Radiotherapy (30 x 2 Gy)

Concomitant Phase Adjuvant Phase (6-12 mo)

Dose dense TMZ (100 mg/m2 daily x 21d)

Stratify by:MGMT methylation

TMZ daily x 6 wksR

Validation: RTOG/EORTC/NCIC Phase III Study

Radiotherapy (30 x 2 Gy)

Concomitant Phase Adjuvant Phase (6 ( -12) mo)

Dose dense TMZ(100 mg/m 2 daily x 21d)

Stratify by:MGMT methylationTi

ssue

open since January 06, will enroll 1153 patients

Integrated TranslationalResearch Program:Identification of other resistance factors and new targets

Study Chairs:Mark R. Gilbert, M.D. (Medical Oncology)Minesh Mehta, M.D. (Radiation Oncology)Ken Aldape, M.D. (Neuropathology and Correlative Biology)Arnab Chakravarti, M.D. (Neuropathology and Correlative Biology)

EORTCRoger Stupp, M.D. (Medical Oncology)Monika Hegi, Ph.D. (Neuropathology and Correlative Biology)

Trials in GBM based on the RT/TMZ→TMZ scheme

Trials in GBM based on the RT/TMZ→TMZ scheme

Stupp et al JCO, 2007

Frequency of MGMT Methylation in Glioblastoma

Frequency of MGMT Methylation in Glioblastoma

Tumor type # samples MGMT-meth % Reference

GBM 29 10 34 {Watanabe, 2005 #3670}GBM 21 8 38 {Balana, 2003 #3672}GBM 29 12 41 {Esteller, 2000 #1455}GBM 12 5 42 {Yu, 2004 #2165}GBM 74 33 45 {Kamiryo, 2004 #3677}GBM 206 92 45 {Hegi, 2005 #2000}GBM 44 30 68 {Blan, 2004 #3674}GBM 38 26 68 {Hegi, 2004 #1721}GBM 219 126 58 {Criniere, 2007 #6708}TOTAL 672 342 51

Hegi 9-07

Range published for GBM 34 to 68%(gel based, mostly on frozen tissue)

Comparison of qMSP and Classic Gel Based Nested MSP for determination of the MGMT status

Comparison of qMSP and Classic Gel Based Nested MSP for determination of the MGMT status

Quantitative MSP

OncoMethylomeSciences

Ilse VlassenbroeckStéphane CalificeJosef StraubIvano Di StefanoFabrice MoreauIsabelle Renard, Bruno FlamionJames DiGuiseppiKatja Bierau

Tissues from Trials : Lausanne, R. Stupp; Rotterdam, M. van den BentRegensburg, P. Hau

Vlassenbroeck et al submitted

Gel Based MSP

Lab of Tumor Biology and Genetics, Neurosurgery, CHUV

Annie-Claire Diserens Marie-France HamouMonika E. Hegi

Statistics

NCCR Molecular Oncology & Swiss Institute of Bioinformatics

Eugenia MigliavaccaMauro Delorenzi

GEL BASED ASSAY CHUV• Bisulfite treated DNA• Nested MSP : 1st PCR (298bp)• 2nd PCR (discriminating)

– PCR for meth MGMT (81bp)

– PCR s for unmeth MGMT (93bp)

• Results visualized on gel

GEL BASED ASSAY CHUV• Bisulfite treated DNA• Nested MSP : 1st PCR (298bp)• 2nd PCR (discriminating)

– PCR for meth MGMT (81bp)

– PCR s for unmeth MGMT (93bp)

• Results visualized on gel

qMSP OncoMethylome Sciences

• Bisulfite treated DNA• quantitative MSP :

– methMGMT (136bp)– beta Actin (125bp)

• Ratio of mMGMT/Actin *1000

qMSP OncoMethylome Sciences

• Bisulfite treated DNA• quantitative MSP :

– methMGMT (136bp)– beta Actin (125bp)

• Ratio of mMGMT/Actin *1000

Experimental Workflow of the Assays

•Evaluation of tissue (H&E slide, tumor content, amount)

•4 sections / sample for each center

•Evaluation of tissue (H&E slide, tumor content, amount)

•4 sections / sample for each center

Comparison of resultsComparison of resultsHegi 9-07

Vlassenbroeck et al submitted

Non-disclosed Unpublished Data

MGMT MGMT immunohistochemistryimmunohistochemistry in GBM:in GBM:InterobserverInterobserver agreementagreement

in EORTC/NCIC trial26981/22981in EORTC/NCIC trial26981/22981

PreusserPreusser M, M, JanzerJanzer RC, RC, Felsberg J, Felsberg J, ReifenbergerReifenberger G, G, HamouHamou MM--F, F, DiserensDiserens AA--C, C, MarosiMarosi C, C, HeinzlHeinzl H,H, Stupp R, Stupp R, HainfellnerHainfellner JA, Hegi MEJA, Hegi ME

Methods Methods : : 2 anti2 anti--MGMT MGMT antibodiesantibodies, , DakoDako MT3.1, MT3.1, ZymedZymed MT23.2MT23.2

•• TissueTissue micromicro arrayarray (TMA): 163 (TMA): 163 tissuetissue samplessamples from from GBM GBM trialtrial 26981/2298126981/22981

-- 4 4 neuropathologists neuropathologists -- 3 3 laboratorieslaboratories (RJ, GR, JAH, MP)(RJ, GR, JAH, MP)

-- StatisticalStatistical analysisanalysis

0.6 mm0.6 mm

TMA

Preusser et al submitted

Non-disclosed Unpublished Data

Hegi et al. N Engl J Med, 352: 997-1003, 2005

MGMT methylationStatusMGMT methylationStatus

Epigenetic Inactivation of the MGMT Repair Gene Predicts Benefit from TMZ

0 3 6 9 12 15 18 21 24 27 30 33 360

Meth

Unmeth

RT/TMZ

SurvivalMGMT median 2-yr

Meth 21.7mo 46.0 %Unmeth 12.7mo 13.8 %

monthsFrozen TissueFrozen Tissue

#3 #4#6

Tissue Array

Array-CGHArray-CGH

Genomewide View- %frequency of CNAs in 46 GBMs

-100

-75

-50

-25

0

25

50

75

100

0 500000 1000000 1500000 2000000 2500000 3000000

Genomewide View- %frequency of CNAs in 46 GBMs

-100

-75

-50

-25

0

25

50

75

100

0 500000 1000000 1500000 2000000 2500000 3000000

CHR 7

New Molecular Targets

Gene Expression Profiles

Gene Expression Profiles

18S

28S

Fluo

resc

ence

Time (seconds)

0

1

2

3

4

5

6

7

19 24 29 34 39 44 49 54 59 64 6918S

28S

Fluo

resc

ence

Time (seconds)

0

1

2

3

4

5

6

7

19 24 29 34 39 44 49 54 59 64 6918S

28S

Fluo

resc

ence

Time (seconds)

0

1

2

3

4

5

6

7

19 24 29 34 39 44 49 54 59 64 69

18S

28S

Fluo

resc

ence

Time (seconds)

0

1

2

3

4

5

6

7

8

19 24 29 34 39 44 49 54 59 64 69

Murat et al submitted

Non-disclosed Unpublished Data

Conclusions• The MGMT methylation status predicts benefit from the

alkylating agent TMZ

• Standardized MGMT-testing required – Quantitative MSP is reproducible, prospective testing ongoing– IHC is not useful for diagnostic MGMT-testing

• New trials will select patients based on MGMT status

The Team in the LabAnastasia MuratWanyu Louis LambivIsabelle DesbailletsAnnie-Claire DiserensMarie-France HamouYan LachatSophie Shnaper

Monika HegiNicolas de TriboletMarc Levivier

85 CENTERS85 CENTERSMichael WellerMichael WellerTübingenTübingen, D, D

Max Max KrosKrosRotterdam, NLRotterdam, NL

Johannes Johannes HainfellnerHainfellnerViennaVienna, A, A

Warren Warren MasonMason,,Toronto, CAToronto, CA

Luigi Luigi MarianiMarianiBerne, CHBerne, CH

JacolineJacoline BrombergBrombergUtrecht, NLUtrecht, NL

Peter Peter HauHauRegensburgRegensburg, D, D

Gregory CairncrossGregory CairncrossLondon, CALondon, CA

René René MirimanoffMirimanoffLausanne, CHLausanne, CH

Roger StuppRoger Stupp

Oncology, Oncology, CePOCePO, CHUV, CHUV

Brain Brain TumorTumor GroupGroupRadiotherapy GroupRadiotherapy Group

EORTCEORTCThierry Thierry GorliaGorlia

NationalCancer Instituteof Canada

Patients and their FamiliesPatients Patients and their Familiesand their Families

WeizmannWeizmann Institute Institute of Scienceof ScienceTalTal ShayShayEytan DomanyEytan Domany

NCCRNCCRMolecular OncologyMolecular OncologyISRECISRECEugenia Eugenia MigliavaccaMigliavaccaMauro Mauro DelorenziDelorenzi

NCCR NCCR Frontiers in GeneticsFrontiers in GeneticsGenèveGenèvePatrick Patrick DescombesDescombesDidier Didier CholletChollet

UCSFUCSFAnjan MisraAnjan MisraBurt FeuersteinBurt Feuerstein

85 CENTERS85 CENTERS