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