diffuse gliomas of human adults part 1 · dysembryoplastic neuroepith. tumor 0 ganglioglioma 1...

Diffuse Gliomas of Human Adultspart 1

7. April 2009

Luigi Mariani Neurochirurgie

WHO 2007: 132 Entities, Metastases excluded!

Tumoren des Hirns„neuroepithelial“

Tumoren der Nerven

Tumoren der Hirnhäute„Meningen“

Tumore der Hypophysenregion„Hirnanhangdrüse“

Keimzelltumore

Blutzelltumore

Symptome

Fokal:

“minus” an Funktion: Ausfall

“plus”: zB Epilepsie, Hormone

Hirndruck

“Zufall”: asymptomatisch

Schmerzhafte Strukturen im/am KopfSchmerzhafte Strukturen im/am Kopf

Hirnhäute

AugenZahnwurzelnInnenohrRachenäussere Haut nach P. Duus, 1995

SchmerzprojektionSchmerzprojektion

Nebenhöhlen(Sinus)

Gefässe

??Kopfschmerzen: harmlos?

World Cancer Report 2003

(Metastases excluded)

Grad I

�No Growth or over Years

�Well delimited

�Life expectancy usually not

reducedGrad III und IV

�Growth (weeks/months)

�Infiltration, Invasion

�Life expectancy reducedGrad II

�Growth over Months/Years

�Infiltration, Malignant transformation

�Life expectancy in danger

Localization

Transformation / Proliferation

Infiltration / Invasion

WHO I

WHO II-IV

Prognosis

1. Kinder

2. Erwachsene

NeuroepithelialTumors

HypophysenadenomKraniopharyngeom

Neurinome(Schwannome) Meningeome

Neuroepitheliale Tumoren� Astrocytic tumors� Diffuse astrocytoma 3*

fibrillary, protoplasmic, gemistocytic� Anaplastic astrocytoma 3� Glioblastoma 3

Giant cell glioblastoma 3Gliosarcoma 3

� Pilocytic astrocytoma 1� Pleomorphic xanthoastrocytoma 3� Subependymal giant cell astrocytoma 1�

� Oligodendroglial tumors� Oligodendroglioma 3� Anaplastic oligodendroglioma 3�

� Mixed gliomas� Oligoastrocytomas 3� Anaplastic oligoastrocytoma 3

� Ependymal tumors� Ependymoma 3� cellular, papillary, clear cell,

tanycytic� Anaplastic ependymoma 3� Myxopapillary ependymoma 1� Subependymoma 1�

� Choroid plexus tumors� Choroid plexus papilloma 0� Choroid plexus carcinoma 3

Glial tumors of uncertain originAstroblastoma 3Gliomatosis cerebri 3Chordoid tumor of the 3rd ventricle 1

Neuronal and mixed neuronal-glialtumorsGangliocytoma 0Dysplastic gangliocytoma of cerebellum

(Lhermitte-Duclos) 0Desmoplastic infantile astrocytoma/ganglioglioma

1Dysembryoplastic neuroepith. tumor 0Ganglioglioma 1Anaplastic ganglioglioma 3Central neurocytoma 1Cerebellar liponeurocytoma 1Paraganglioma of the filum terminale 1

Neuroblastic tumorsOlfactory neuroblastoma 3Olfactory neuroepithelioma 3Neuroblastoma of the adrenal glandand sympathetic nervous system 3

Pineal parenchymal tumorsPineocytoma 1Pineoblastoma 3Pineal parenchymal tumor of

intermediate differentiation 3

Embryonal tumorsMedulloepithelioma 3Ependymoblastoma 3Medulloblastoma 3

desmoplasticlarge cellMedullomyoblastomamelanotic

Supratentorial primitiveneuroectodermal tumor(PNET) 3

NeuroblastomaGanglioneuroblastoma

Atypical teratoid/rhabdoidtumor 3

*Behaviour:0 = benign;1 = low or uncertain malignancy3 = malignant

Medulloblastoma

�Age peak <10years�Males>Females�Cerebellar vermis�Positive Prognostic Factors:

�Higher age�No dissemination�Total removal achieved�Expression of Neurotrophin Receptor TrkC*

�Negative Prognostic factor: myc Ampl.-Expr.�Survival at 5 years 50-75%, Side effects of ttt.�Cell of Origin:

Granular cell neurons cerebellum�Role of transcription factors in tumorigenesis**:

Olig2, Tlx3

*Segal und Pomeroy PNAS 1994, mRNA Northern; Grotzer JCO 2000, mRNA ISH)

** Schüller et al, 2007, transgenic mice

Diffuse Gliomas (WHO 2007)

•Astrocytic tumors WHO grade•Diffuse astrocytoma (A) II

fibrillary, protoplasmic, gemistocytic•Anaplastic astrocytoma (AA) III•Glioblastoma (GBM) IV

Giant cell glioblastoma IVGliosarcoma IV

•Pilocytic astrocytoma I•Pleomorphic xanthoastrocytoma II•Subependymal giant cell astrocytoma I

•Oligodendroglial tumors WHO grade•Oligodendroglioma (O) II•Anaplastic oligodendroglioma (AO) III

•Mixed gliomas WHO grade•Oligoastrocytomas (OA) II•Anaplastic oligoastrocytoma (AOA) III

Diffuse Gliome

Grad II Grad III Grad IV = Glioblastom

Diffuse Gliome des Erwachsenen

WHO Grad2 Jahres-

Überleben (~)

Alter

(~)Relative

Häufigkeit (~)

II 85% 40 J 20%

III 70% 50 J 15%

IV

Glioblastom25% 60 J 65%

2JInselspital, Bern, 2007

Ursachen

�Assoziert mit genetischen Krankheiten (selten)

�Ionisierende Strahlen (z.B. nach Radiotherapie)

�Häufigkeitsgipfel bei Kinder , dann wieder zunehmende Häufigkeit im Alter

�Geschlecht

�Rasse (kaukasisch weiss)

�Keine „moderne“ Erkrankung = „konstante“ Epidemiologie

�Keine „Noxen“ bekannt:

?

Familiäre TumorsyndromeSyndromSyndromSyndromSyndrom GenGenGenGen ChromosomChromosomChromosomChromosom NervensystemNervensystemNervensystemNervensystem HautHautHautHaut Sonst. GewebeSonst. GewebeSonst. GewebeSonst. Gewebe

Neurofibromatose 1Neurofibromatose 1Neurofibromatose 1Neurofibromatose 1 NF1 17q11 Neurofibrom, MPNST, optisches Gliom, Astrozytom

Café-au-lait Flecken, axilläre Epheliden

Iris-Hamartome, ossäre Läsionen, Pheokromozytom, Leukämie

Neurofibromatose 2Neurofibromatose 2Neurofibromatose 2Neurofibromatose 2 NF2 22q12 Bilaterales Akustikusneurinom, peripheres Schwannom, Meningeom, Meningionangiomatose, spinales Ependymom, Astrozytom, gliales Hamartom, zerebrale Verkalkungen

- Linsentrübung, retinale Hamartome

Von HippelVon HippelVon HippelVon Hippel----LindauLindauLindauLindau----KrankheitKrankheitKrankheitKrankheit

VHL 3p25 Hämangioblastom - Retinale Hämangioblastome, Nierenzellkarzinom, Pheokromozytom, viszerale Zysten

TuberTuberTuberTuberööööse Hirnsklerosese Hirnsklerosese Hirnsklerosese Hirnsklerose(M. Bourneville)(M. Bourneville)(M. Bourneville)(M. Bourneville)

TSC1TSC2

9q3416p13

Subependymales Riesenzellastrozytom, „cortical tubers“

Kutane Angiofibrome (adenoma sebaceum), peau chagrin, subungueale Fibrome

Kardiales Rhabdomyom, Dünndarm-Polypen, Lungen-und Nierenzysten, Lymphangioleiomyomatose, Angiomyolipom der Niere

LiLiLiLi----FraumeniFraumeniFraumeniFraumeni----SyndromSyndromSyndromSyndrom TP53 17p13 Astrozytom, PNET - Mamma-Karzinom, Leukämie, Sarkom, adrenokortikales Karzinom

CowdenCowdenCowdenCowden----KrankheitKrankheitKrankheitKrankheit PTEN (MMAC1)

10q23 Dysplastisches Gangliocytom des Cerebellums (Lhermitte-Duclos), Megalenzephalie

Multiple Trichilemmome, Fibrome

Kolon-Polypen,, Schilddrüsentumore, Mamma-Karzinom

TurcotTurcotTurcotTurcot----SyndromSyndromSyndromSyndrom APChMLH1hPSM2

5q213p217p22

MedulloblastomGlioblastom Café-au-lait Flecken

Kolorektale-PolypenKolorektale-Polypen

Naevoid Basal Cell Naevoid Basal Cell Naevoid Basal Cell Naevoid Basal Cell Carcinoma syndrome Carcinoma syndrome Carcinoma syndrome Carcinoma syndrome (Gorlin)(Gorlin)(Gorlin)(Gorlin)

PTCH 9q31 Medulloblastom Multiple Basaliome, palmar and plantar pits

Unterkiefer-Zysten, Fibrome des Ovars, Skelettanomalien

Neurofibromatose 1

Diagnostische Kriterien der NF1Diagnostische Kriterien der NF1Diagnostische Kriterien der NF1Diagnostische Kriterien der NF1Zwei oder mehr der folgenden Merkmale:Zwei oder mehr der folgenden Merkmale:Zwei oder mehr der folgenden Merkmale:Zwei oder mehr der folgenden Merkmale:

≥≥≥≥6 caf6 caf6 caf6 caféééé----auauauau----lait Flecken lait Flecken lait Flecken lait Flecken von >5 mm (prepubertär), resp. >15 mm Durchmesser (postpubertär)

≥≥≥≥2 Neurofibrome2 Neurofibrome2 Neurofibrome2 Neurofibrome, oder≥≥≥≥1 plexiformes Neurofibrom1 plexiformes Neurofibrom1 plexiformes Neurofibrom1 plexiformes Neurofibrom

AxillAxillAxillAxillääääre und/oder inguinale Ephelidenre und/oder inguinale Ephelidenre und/oder inguinale Ephelidenre und/oder inguinale Epheliden

Gliom des N. opticusGliom des N. opticusGliom des N. opticusGliom des N. opticus

OssOssOssOssääääre Lre Lre Lre Lääääsion sion sion sion (sphenoidale Dysplasie, Ausdünnung der kortikalis eines langen Knochen)

Verwandte/r ersten Grades mit NF1Verwandte/r ersten Grades mit NF1Verwandte/r ersten Grades mit NF1Verwandte/r ersten Grades mit NF1

Hauptmanifestationen der NF1Hauptmanifestationen der NF1Hauptmanifestationen der NF1Hauptmanifestationen der NF1

TumoralTumoralTumoralTumoral AndereAndereAndereAndere

Neurofibrome (dermal, nodulär, plexiform) Ossäre Läsion (Skoliose, Minderwuchs, Makrocephalie, Pseudarthrose, Dysplasie des Sphenoids)

Gliome (N. opticus, Astrozytom, Glioblastom)

Nervensystem (Minderintelligenz, Epilepsie, Neurophatie, Hydrocephalus (Aqueduktstenose)

Sarkome (Neurofibrosarkom, Rhabdomyosarkom, Triton-Tumor)

Vaskuläre Läsionen (Fibromuskuläre Hyperplasie (Nierenarterie)

Neuroendokrine Tumore (Pheochromozytom, Carcinoid-Tumor)

Hämatologische Tumore (juvenile CML)

Treatment

1. Surgery (Resection, local Therapies)

2. Radiation

3. Antitumoral Drugs

Glioblastoma multiforme

HE

GFAP

Mikroneurochirurgie Navigation

13j MädchenKomplex partielleAnfälle

Optimierung der Chirurgischen Extirpation von Glioblastomen

„5-ALA directed“ surgery„white light“ surgery

GBM resection

Speech activation

The 4. Dimension: Function

Motor activation

Fibre connectionsDiffusor Tensor Imaging (Diffusion Weighted MRI)

Cortical areasFunctional MRI

MRT im OP

Stummer et al 2008

Role of Surgery for GBM

„komplette“ Extirpation

inkomplette Extirpation

Stummer 2008

Role of Surgery in GBMKlinik für Neurochirurgie

Infiltration von glialen Tumorzellen bei GBM

Entfernung des proliferativen Tumorknotens ...

… es bleiben unzählige infiltrierende Tumorzellen in der Oedemzone

Radiation

Glioblastoma – Radiation therapyClass 1 evidence

From: Radiotherapy for newly diagnosed malignant glioma in adults: a systematic review. Laperriere et al, Radiotherapy and Oncology, 2002

R. Stupp et al, NEJM 352(10):987-996, 2005

Progression-free Survival Overall Survival

Glioblastoma – ChemotherapyClass 1 evidence

Alter und Genetische Tumor-Marker

Ohgaki al, NEJM 2007

57 yo, GBMearly postop. MRI

5 months later despite radio-chemotherapy

Recurrence

80-90% of GBM recur within 2 cm from resection cavity!

Invasion - Necroscopic evidence

Preoconditions for more effective therapies

Local strategiesLocal control of proliferative bulkSurgery - Radiation – local drugsLow systemic and neurologic toxicity

Systemic strategiesControl of invasive cellsApoptosis inducing drugs through blood-brain-barrierLow systemic and neurologic toxicity

Biology of Glioblastoma

�Tumorigenesis

�Invasion

�Models

Zhu and Parada (2002), nature reviews 2; 616

Neurons

Oligodendrocytes

Astrocytes

Neuronal-restrictedprogenitor cells

Glial-restrictedprogenitor cells

Neural stem cells

Medulloblastoma

Oligodendroglioma

Oligoastrocytoma

Astrocytoma

Gliomas and Meduloblastomas – Cell of Origin?

NeuralProgenitorcells

Glioblastoma - Cell of origin

Primary GBM

Primary GBM – Gain

Amplification

Double minute chrom.

Overexpressed in 70-90% of amplified cases. EGFR expression profile distinguishes different for more than 90 genes

EGFRvIII constitutively active in 20-50% of amplified cases

Proliferation/invasion/survival

PI3-kinase converting PIP2 to PIP3, activating AKT and mTOR (proliferation/survival)

RAS pathway

MAP-kinase pathways

Gain of 7

EGFR (7)

40% of GBM (not young patients)

GeneChrom

Genomics Transcriptomics / Proteomics / Signalling / Pathways

Catalytic subunit of PI3-kinase, activating AKTPIK3CA (Chrom 3q), Ampli. in 0-68% of GBM

Overexpression distinguishing 1. GBM from 2. GBM

Accumulation of IGFBP2 in GBM cells, Invasion

Dozens of genes (e.g. IGFBP2, IGFBP5)

Strong Overexpression leading to transcriptional activation of more than 100 genes

Induces transcription of VEGF, VEGFR, angiopoietin … (angiogenesis, vascular permeability, homing of lymphocytes)

carbonic anhydrase, LDH (cellular metabolism), BNIP (survival),c-met, CXCR4 (migration) …

Hypoxia master regulator HIF1α

Dozens of genes

MDM2 overexpressed in >50% of 1. GBM by IHC

MDM2 binds to mutant and wt p53 in autoregulatory loop and disrupts P53/MDM2/p14ARF pathway. Cell cycle, response to cell damage, cell death, cell differentiation, angiogenesis

Loss of activation of p53, thus loss of p21Waf1/Cip1

MDM2 (12q14.3-q15)

50% of 1. GBM

Primary GBM – LossGeneChrom

Genomics

Mutations Loss of inhibition of PIP3 , thus activation of AKT and mTOR (proliferation/survival)

Loss of dephosphorylation of FAK (migration)

PTEN (Chr. 10q)

15-40% of GBM

Catalytic subunit of PI3-kinasePIK3CA (3q), Mutation <10-27% of GBM

Complete loss of 1 copy

LOH 10p14-p15 in 1.GBM

Unknown tumor suppressor genes

Loss of inhibition of PIP3 , thus activation of AKT and mTOR (proliferation/survival)


Chromosome 10

60-80% of GBM

(Loss containing the PTEN gene in 75-95%)

Homozygous deletions, promoter methylation

Underexpression p14ARF

p53/mdm2/p14ARF pathway cell cycle/response to cell damage/ celldeath / cell differentiation / angiogenesis

p14ARF

76% of GBM

Transcriptomics / Proteomics / Signalling / Pathways

LOH 1p 12-15% of 1. GBM

LOH 19q (19q13.3) 6% of 1.GBM

Mutations/Deletions p16INK4a

CDK4 amplification

Loss of 13q including RB1 locus in 38% of 2. GBM

Promoter methylation of RB1 in 43% in 2. GBM

p16INK4a/CDK4/RB1 pathway

Control of progression through G1 into S phase.

RB1 107 kDa, phosphorylated by CDK4/cyclinD1 complex to induce release of E2F, TF for G1 into S.

P16INK4a binds and inhibits CDK4/cyclinD1 complex

P16INK4a (9p.21)

CDK4 (12q13-14)

RB1 (13q14)

In 40-50% of GBM

Genetic heterogeneity

CGH and genetic groups of primary GBM

Gain 7Loss 10qEGFR ampPTEN mutp16INK4a del

No gain 7No loss 10No EGFR ampNo p53 mutNo PTEN mut

No gain 7Loss 10No EGFR ampNo p53 mutNo PTEN mut

Misra et al, Clin Can Res 2005

EGFR

EGFR amplification and overexpression

EGFR

PTEN loss

p53 / mdm2 / p14ARF pathway


Targeting of TK Receptors

Anti-VEGF therapy for GBM recurrence:Bevacizumab (Avastine) + Camptothecin (Irinotecan)

61% respondersPFS 6 mos= 38 months!

Vredenburgh 2007

EGFR~40% amplification and ~ 60% overexpression(chr. 7p12, double minute chromosomes)

~ 30% PTENmutation (10q24)

MDM2 <10% amplification and ~50% overexpression

30-40% p16deletionINK4A loss (9q26)

Signal transduction activation(migration, invasiveness, …)

Signal transduction activation(apoptosis resistance, …)

Disrupt p14ARF and p16INK4apathways (G1 to S transition)

Tumorigenesis: primary GBMM

ON

TH

SCell of origin

WHO Grade IV

Diffuse Gliomas of Human Adultspart 2

7. April 2009

Luigi Mariani Neurochirurgie

Gliom Grad II - Management?

�„... we do not know: which patients we should treat; when and how we should treat them.“

Edward Laws, Lausanne, 1987

Klinik für Neurochirurgie

Diffuse Gliome des Erwachsenen

WHO Grad2 Jahres-

Überleben (~)

Alter

(~)Relative

Häufigkeit (~)

II 85% 40 J 20%

III 70% 50 J 15%

IV

Glioblastom25% 60 J 65%

2JInselspital, Bern, 2007

Gliom WHO Grad II

Gesamtüberleben, in Monaten

% Ü

berle

bend

e

SEER Review, Cancer, 2006


Role of Surgery?

Surgery

Biopsy


1. Age <40 J

2. Diameter <6 cm

3. Unilateral

4. Oligodendroglial

5. No neurological deficit

Pignatti et al (EORTC), JCO 2002

Better Prognosis(1% significance level, multivariate analysis)

Score 0-5

Low-risk0-2

High-risk3-5

Surgery/Biopsy

watch treat

Prognostic Factors

Oligodendroglial?

Claus, Cancer 2006

Oligodendroglioma

0

0,2

0,4

0,6

0,8

1

1,2

0 50 100 150 200

Time after diagnosis (months)

Sur

viva

l

astrocytomas and oligoastrocytomas n=73

oligodendrogliomas n=6

p=0.02

E

Oligoastrocytoma?

0

0,2

0,4

0,6

0,8

1

1,2

0 50 100 150 200


Sur

viva

l . others n=42

fibrillary astrocytomas n=37

p=0.03

F

Copyright © American Society of Clinical Oncology

Mariani, L. et al. J Clin Oncol; 24:4758-4763 2006

Markers of microsatellite polymorphism used to dete ct loss of heterozygosity for 1p and 19q

Prevalence of LOH 1p in LGG

0

5

10

15

20

25

30

35

40

Oligodendrogliom Oligoastrozytom Astrozytom

no LOH 1p

LOH 1p

100% 54% 16%


Prevalenz von LOH 1p in LGG

0

5

10

15

20

25

30

35

40

LOH 1p no LOH 1p

Astrozytom

Oligoastrozytom

Oligodendrogliom

32%

36%

32%


0

0,25

0,5

0,75

1

0 50 100 150 200


S

urvi

val

.

Astrocytomas with LOH 1p19q, n=6

Astrocytomas without LOH 1p19q, n=39

p=0.09

F

„Pure“ Astrocytoma and LOH 1pKlinik für Neurochirurgie

0

0,25

0,5

0,75

1

0 50 100 150 200


Sur

viva

l .

LOH 1p 19q (+) n=17

LOH 1p 19q (-) n=49

p=0.003

A

0

0,25

0,5

0,75

1

0 50 100 150 200


Sur

viva

l .

1p36 (+) n=21

p=0.01

1p36 (-) n=46

B

0

0,25

0,5

0,75

1

0 50 100 150 200


Sur

viva

l .

19q13 (+) n=22

19q13 (-) n=42

p=0.002

C

LOH 1p/19q LOH 1p LOH 19q

LOH 1p und 19q – Overall survival

JCO 2006 Keine Chemotherapie!


LOH 1p/19q – Overall Survival

LOH 1p und 19q

∅∅∅∅LOH 1p/19q

p < 0.01

Update 2007

0

0,25

0,5

0,75

1

0 50 100 150 200


Sur

viva

l .

LOH 1p 19q (+) n=17

LOH 1p 19q (-) n=49

p=0.003

A Klinik für Neurochirurgie

LOH 1p/19q = Predictive Factor

November 2004 Februar 2006

Chemotherapie


Neoadjuvant Chemotherapy

November 2003 Juni 2004 Dezember 2005

Chemotherapie Operation


Epigenetic gene silencing by DNA methylation

DNA methylation is an important epigenetic mechanism of tumors to silence tumor suppressor genes

Role of MGMT during chemotherapy with alkylating agents (unmethylated MGMT promoter)

XXGXXG

Alkyl

Alkyl

MGMT promoter unmethylated: chemotherapy

MGMT

promoter coding regiongenomic DNA

Role of MGMT during chemotherapy with alkylating agents (unmethylated MGMT promoter)

XXGXXG

MGMT promoter unmethylated: chemotherapy

Alkyl

Alkyl

MGMT

promoter coding region

tumor

genomic DNA

Role of MGMT during chemotherapy with alkylating agents (methylated MGMT promoter)

XXGXXG

MGMT promoter methylated: chemotherapy

Alkyl

Alkyl

MGMT

promoter coding region

tumor

genomic DNA

CH3CH3CH3

Quantitative MSP assay

MGMT Promoter

methylation-positive

methylation-negative

PCR

HEX

FAM

2:1

Primer extension

2:1

Linearity of the Assay

U87:PBMC

U87 = methylated PBMC = unmethylated

Signal ratio

1:5

1:1

5:1

360

1000

1600

1400

1600

400

1:4

1:1.6

4:1

methylated unmethylated

M-Primer

U-Primer

U-Primer

Case 2: hypermethylated DNA (from FFPE tissue)

Qualitative analysis:

Quantitative analysis:M-Primer

Case 2 (PCR)

Case 2 (Extension)

U87:PBMC=1.1(Extension)

M-Primer U-Primer

methylated

methylated

unmethylated

unmethylated

Results

Glioma type n %methylated %hypomethyl. % PCR failed %methyl. Lit

O+OA II 29 86% 21% (24%) 0% 60%

O+OA III 18 55% 6% (10%) 0% 80%

A II 19 37% 5% (14%) 0% 46%

A III 25 40% 12% (30%) 0% 50%

G IV 135 44% 17% (39%) 0% 45%

Glioma type methylated LOH 1p no loss

O+OA II+III yes 74% (26/35) 26% (9/35)no 8% ( 1/12) 92% (11/12)

A II+III yes 15% ( 2/12) 85% (11/13)no 0% ( 0/28) 100% (28/28)

G IV yes 10% ( 4/39) 90% (35/39)no 4% ( 2/55) 96% 53/55)

Gliomas with unmethylated MGMT promoter rarely have a LOH 1p/19q

MGMT promoter methylation: a predictive factor for chemosensitivity in glioblastoma patients?

Glioblastoma tumours analysed: 135(patients with chemotherapy)PCR failed: 0/135 (0%)No MGMT methylation: 76/135 (56%)Methylation-positive (qualitative): 59/135 (44%)Hypermethylated (quantitative): 36/135 (27%)Hypomethylated (quantitative): 23/135 (17%)Survival data available (dead patients): 36/135 (27%)

Median survival MGMT-positive (qualitative): 24.4 monthsMedian survival MGMT-negative (qualitative): 14.3 months

Hegi M, Diserens AC, Gorlia T, Hamou MF, de Tribolet N, Weller M, Kros JM, Hainfellner JA, Mason W, Mariani L, Bromberg JEC, Hau P, Mirimanoff RO, Cairncross JG, Janzer RC, Stupp R. MGMT Gene

Silencing and Benefit from Temozolomide in Glioblastoma NEJM 352(10):997-1003, 2005

Gliom WHO IVTMZ und MGMT status

Progression free survival

0 12 24 36 48 600

20

40

60

80

100

month

prop

ortio

n pr

ogre

ssio

n fr

ee (%

)

22 Patients with WHO Grade II Glioma

water fall plot response at 6 month (LOH)

res

pon

se (

% c

hang

e in

tum

or v

olum

e)

LOH 1p19qLOH 1p

LOH 19qneg.not done

-60

-40

-20

0

20

40

60

water fall plot best response (LOH)

-100

-50

0

50

max

imal

res

pon

se (%

cha

nge

in tu

mor

vol

ume)

LOH 1p19qLOH 1p

LOH 19qneg.not done

res

pon

se (

% c

han

ge

in t

umo

r vo

lum

e)

MGMT promotor methylation (%)

p=0.012

Correlation response 6 month vs. MGMT

20 40 60 80 100

-60

-40

-20

0

20

40

60

res

pon

se (

% c

han

ge

in t

umor

vo

lum

e)

MGMT promotor methylation (%)

p=0.045

Correlation best response vs. MGMT

10 20 30 40 50 60 70 80 90 100

-80

-60

-40

-20

0

20

40

Grades 1-2 Grades 3-4Event No of

patients% No of

patients%

Blood/bone marrowAnemia 13 59Leucopenia 9 41 2 9Thrombocytopenia 3 14 5 23

LaboratoryALT/AST 1 5Bilirubin 2 9Glucose 6 27Hyponatremia 1 5Hypernatremia 5 23

GastrointestinalNausea / vomitting 14 64Constipation 9 41

OthersMucositis 2 9Rash / Desquamation 2 9

Table 2 Toxicity

Number %No of patients 22GenderMale 13 59Female 9 41Age, yearsMedian 53Range 27-72HistologyOligodendroglioma 10 45Oligoastrozytoma 7 32Astrozytoma 5 23Prior SurgeryBiopsy only 15 68Subtotal resection 7 32Prior Radiotherapy 6 27Tumorvolume (cm3)Median 69,7Range 21-332SideLeft 14Right 7Right and left 1Tumor locationFrontal lobe 9 41Insula 1 5Temporal lobe 4 18Parietal lobe 8 36Clinical symptomsseizure 18 82aphasia 7 32cognitive deficiency 9 41paralysis 1 5vertigo 1 5KPS at start of treatmentMedian 0Range 0-2Cycles of TMZMedian 12Range 4-22TMZ regimen (pts)Day 1-5 22 100Dose dense 7 32

Table 1 Patient characteristics

Malignant Progression of WHO Grade II gliomas

Grade II Grade III Grade IV = GBM

1994 1995 1998 2002

Secondary GBM

Secondary GBM – Gain

Amplification

Double minute chrom.

Overexpressed in 70-90% of amplified cases

EGFRvIII constitutively active in 20-50% of amplified cases

Proliferation/invasion

PI3-kinase converting PIP2 to PIP3, activating AKT and mTOR (proliferation/survival)

RAS pathway

MAP-kinase pathways

EGFR (Chr. 7)

8% of GBM

GeneChrom

Genomics

Strong Overexpression leading to transcriptional activation of more than 100 genes

Induces transcription of VEGF, VEGFR, angiopoietin … (angiogenesis, vascular permeability, homing of lymphocytes)

carbonic anhydrase, LDH (cellular metabolism)

BNIP (survival)

c-met, CXCR4 (migration)

…

Hypoxia master regulator HIF1α


Secondary GBM – LossGeneChrom

Genomics

Mutations Loss of inhibition of PIP3 , thus activation of AKT and mTOR (proliferation/survival)


PTEN (Chr. 10q)

4% of 2. GBM

Mutations, hot spots codons 248 and 273

P53/MDM2/p14ARF pathway. Accumulation of p53 protein (ICH)

MDM2 binds to mutant and wt p53 in autoregulatory loop. Cell cycle, response to cell damage, cell death, cell differentiation, angiogenesis

Loss of activation of p53, thus loss of p21Waf1/Cip1

p53 (17p13.1)

65% of 2. GBM

LOH10q23-24

LOH 10q25-qter in 2. GBM, e.g. DMBT1, homozygous deletion in 13-38%)

Unknown tumor suppressor genes

Loss of inhibition of PIP3 , thus activation of AKT and mTOR (proliferation/survival)


Chromosome 10q

63% of 2. GBM

Mutations

Deletions p16INK4a

CDK4 amplification

Loss of 13q including RB1 locus in 38% of 2. GBM

Promoter methylation of RB1 in 43% in 2. GBM


Control of progression through G1 into S phase.

RB1 107 kDa, phosphorylated by CDK4/cyclinD1 complex to induce release of E2F, TF for G1 into S.

P16INK4a binds and inhibits CDK4/cyclinD1 complex

P16INK4a (9p.21)

CDK4 (12q13-14)

RB1 (13q14)

In 40-50% of GBM


Tumor Progression

Grade IIPrecursor cell Grade IIIGrade IVSecondary GBM

PDGF-A/PDGFR-α ↗ (~ 60%) FGF2 ↗…

p53p53p53p53 mutation (17p13) (~ 60%)mutation (17p13) (~ 60%)mutation (17p13) (~ 60%)mutation (17p13) (~ 60%)17p loss (~ 60%) 22q13.3 loss (~ 30%) 1p/19q LOH (~ 50%)…

~60% 10q LOHCDK4 amplification (12q13)VEGF Overexpression~ 25% Rb alteration (13q13) (disrupts p16INK4A)pathway)~50% DCC loss of expression~5% PTEN mutat. (10q24)~8% EGFR amplification…

Time5 years in mean

9p21 LOH9p21 LOH9p21 LOH9p21 LOH

Integrated genomic analysis of GBM

Parsons et al, Science 2008 321:1807-1812

20‘661 protein coding genes in 22 GBMs:-12 % with mutations of IDH1 gene-5 out of the 6 patients with secondary GBM had a mutated IDH 1:

position 395codon 132, G to AArg to His

Original Article

IDH1 and IDH2 Mutations in Gliomas

Hai Yan, M.D., Ph.D., D. Williams Parsons, M.D., Ph.D., Genglin Jin, Ph.D., Roger McLendon, M.D., B. Ahmed Rasheed, Ph.D., Weishi Yuan, Ph.D., Ivan Kos, Ph.D.,

Ines Batinic-Haberle, Ph.D., Siân Jones, Ph.D., Gregory J. Riggins, M.D., Ph.D., Henry Friedman, M.D., Allan Friedman, M.D., David Reardon, M.D., James Herndon, Ph.D., Kenneth W. Kinzler, Ph.D., Victor E. Velculescu, M.D., Ph.D., Bert Vogelstein,

M.D., and Darell D. Bigner, M.D., Ph.D.

N Engl J MedVolume 360(8):765-773

February 19, 2009

IDH1 and IDH2 Mutations in Human Gliomas

Yan H et al. N Engl J Med 2009;360:765-773

Enzymatic Activity of Wild-Type and Mutant IDH1 and IDH2 Proteins


Study Overview

• Isocitrate dehydrogenases, encoded by the IDH1 and IDH2 genes, catalyze the reduction of NADP+ to NADPH in the brain

• One or the other of these two genes was found to be mutated in 70% of 445 gliomas of World Health Organization grade II or III

• The mutations abolished the enzymatic activity of the IDH1 and IDH2 proteins

• The evidence suggests that mutation of an IDH gene is an early event in the development of gliomas

Survival of Adult Patients with Malignant Gliomas w ith or without IDH Gene Mutations


IDH1 and IDH2 somatic Mutations in Astrocytomas

Yan H et al, NEJM 2009Confirmed by Watanabe et al, AJP 2009

Grade IIPrecursor cell Grade IIIGrade IVSecondary GBM

Time5 years in mean

IDH1 mutation codon R132 or IDH2 mutation codon 172: 90%Among IDH1/2 mutated tumors:TP53 mutation 84%PTEN 0%EGFR 0%CDKN2A and CDKN2B 0%1p loss 5%

IDH1 mutation codon R132 orIDH2 mutation codon R172: 75%Among IDH1/2 mutated tumors:TP53 mutation 82%PTEN 0%EGFR 0%CDKN2A and CDKN2B 0%1p loss 5%

IDH1 mutation codon R132IDH2 mutation codon 172: 73%Among IDH1/2 mutated tumors:TP53 mutation 73%PTEN 0%EGFR 0%CDKN2A and CDKN2B 12%

IDH1 and IDH2 Mutations in Oligodendroglial Tumors

Yan H et al, NEJM 2009Confirmed by Watanabe et al, AJP 2009

Grade IIPrecursor cell Grade III

IDH1 mutation codon R132 or IDH2 mutation codon 172: 85%Among IDH1/2 mutated tumors:TP53 mutation 21%PTEN 0%EGFR 0%CDKN2A and CDKN2B 5%1p/19q loss 78%

IDH1 mutation codon R132 orIDH2 mutation codon R172: 95%Among IDH1/2 mutated tumors:TP53 mutation 10%PTEN 0%EGFR 0%CDKN2A and CDKN2B 9%1p loss 90%

Watanabe et al, AJP 2009

IDH1 and IDH2 Mutations = early event in gliomagenesis

Yan H et al, NEJM 2009

Grade IIAstrocytomaOligodendrogliomaPrecursor cell

Grade IIIAstrocytomaOligodendroglioma

IDH 1 or 2 Mutation p53 Mutation (astrocytoma) orLOH 1p/19q (oligodendroglioma)

IDH1 and IDH2 = isocitrate dehydrogenase

�Chromosome 2q33

�„Krebs` cycle“: Metabolic pathway converting carbohydrates, fats and proteins into carbon dioxide and water to generate energy

�Cytosolic enzyme

�Biology of mutated IDH in Grade II gliomas needs to be elucidated

>80% IDH1 and IDH2 Mutations, function?~ 60% PDGF-Aor PDGFR-α overexpr., FGF2 overexpression (autocrineactivation of signal transd. pathways)

~ 60% p53mutation (17p13) (disrupts p14ARF pathways, inhibits apoptosis, promotesgenomic instability), ~ 60% 17ploss, ~ 30% 22q13.3loss, ~ 50% 19qLOH

10qLOH, CDK4 amplification (12q13), ~ 25% Rbalteration (13q13) (disrupts p16INK4Apathway), ~50% DCC loss of expression

~5% PTENmutat. (10q24) (enhanced AKT activity, Forkhead transcription, promoted survival)

WHO Grade II

WHO Grade III

WHO Grade IV

2-10 YE

AR

STumorigenesis: secondary GBM Cell of origin

HW Zheng et al. Nature 455, 1129-1133 (2008) doi:10.1038/nature07443

p53 and Pten inactivation cooperate to induce high-grade malignant gliomas

Thank you!

Glioma cell motility - in vitro evidence

ab

ab

ab

_ase

RTK growthfactors

ECM/myelin

LOSS OF:

NCAMN-Cadherin ?

DCCgap junctions

Cx-43

GAIN OF:GF / GFR

ECM / IntegrinsBEHAB

CD44SPARC

proteases

invasion

_ase

Tumorbiopsat1 Patient

PixCell II LCMor Clones

Astrozytom Zellline

Differential Display / cDNA MicroArray

Tumorzentrum Invasive Zellen

UnbehandeltMigrations-Stimuliert

Genetik der Astrozytom-Invasion

Tumorbiopsate> 1 Patient

“nicht”invasive Tumore

Invasive Tumore

Kandidat-Gene/Gen-Sequenzen verantwortlich für den invasiven Phenotyp

Identifizierung von invasiven Astrozytomzellen in gefrorenen Preparate

Unter 20x Vergrösserung.“Laser-capture”von Astrozytomzellen

Entfernung von dissezierten Astrozytomzellen

Mariani et al, Cancer Res

RNA Differential Display

AP1-AP7 different arbitraryprimer used in combination with H-T(11)A oligo-dT primer

C- Tumor CoreR- Invasive Rim


P311Ein neues Invasionsgen, das ausgeschaltet werden kann

Antisense [µM]

0 1 2 3 4 5

Cha

nne

l Pea

k Fl

uore

scen

ce

400

800

1200

1600

0 1 2 3 4 5 6

Nor

mal

ized

Cop

y N

umbe

r

0

100

200

300

400

500

600

0 1 2 3 4 5

Mig

ratio

n R

ate

(µm

/day

)

0

30

60

90

120

150


Death Associated Protein 3 (Dap-3)

Ein Invasions- und Apoptosegen, das ausgeschaltet werden kann

No coating

Coating with ECM

Control CAM 1uM 24 hrs

Mariani, Clin Cancer Res

Astrozytom Zellline G112

cDNA MicroArray

UnbehandeltMigrations-Stimuliert



Mariani et al, J Neurooncol

Matrix-aktivierte Gliom-Migration


CD44

EGF, PDGF HGF/SF

ECM proteins

LPA

ankyrin

FAK

Rac

EGFR

CTGF

N-WASP IQGAP

Arp2/3

α-cateninβ-catenin

E-cadherin

tenascinC

integrins

GAP43

Nucleation of actin atmembrane

Stabilizationof adherensjunctions

PTPL1associatedRho GAP1

ROK

Adducin 3γγγγ

Moesin profilin

Corticalactin

network

Actinmembranelinkage

Actinpolymerization

stress fibers

Neuropilin 2

VEGF

Plexin 5FN-14

HA Osteopontin

Elastin

MSE 55

PAI-1

Cdc42

CIP 4

αγ

β

G proteins Caveolin 2

Syntaxin 1

paxillinAnnexin 1

Actin

Osteopontin

Caveolin 2

GPCRs

Ras

Rho

PLCγγγγ

EzrinRadixin

Up regulated

Down regulated


Cdc2

Cyclin B

Cdc2 Cyclin B

Cdc2

Cyclin ACDK2

P107E2F

G1 Phase

Mitosis

G2 PhaseS Phase

Rb

Rb

E2F

P

P

P

CDK 4/6

Cyclin D

P107E2F

CDK2Cyclin E

p16

Ras

TGF ββββ DNA damage,other insults

Bax

P53-independentapoptosis

P53-dependentapoptosis

Bcl-2

Free E2F*

ARF

MDM2

PCNA

p18

Myc

E2CUbiquitinCarrierProtein

p15

p21

p53

p27

Up regulated

Down regulated

Cyclin A,B

Free E2F*


Fas

Fas Ligand

FADD

Caspase-8

FAF

Sentrin

UBC9

Annexin

Activation of Downstream-Caspases

Caspase-3 Caspase-6 Caspase-7

TNF

TNFR-1

FADD

Caspase-8

RAIDD

Caspase-2

Cleavage of Death Substrate

Fragmentation of DNA

DNA repair

Cell cycleKinases

Structural proteins

PARPpRbMdm-2

MuMaPITSLREDNA-PkFAK

PAK2MEKK-1

lamin A,B

Nucleus

Caspase-1

RICKCARDIAK

RIP3

JNK

FLIP

PKCδδδδG-actin

presenilins

gelsolinfodrin

TRAF1/2

RIP

Topoisomerase

Bcl-x

Bcl-2

Baxbadbid

Caspase-9PKR

PKR inhibitor

ICAD / CAD

Up regulated

Down regulated

TNFRsuperfamily

NF κκκκB

Cyt-C

Apaf-1



RNA-Amplifikation

cDNA Microarray (das Ganze Transkriptom!)



ParentalMigrations-Stimuliert




Invasive Tumore


0.01

0.1

1

10

100EH

D3*

KIA

A05

13**

RY

R2*

PTPR

N2*

OPC

ML*

*EF

NB3

**BE

X1*

*A

TX

*BC

LW**

CA

SP7

KLK

6**

CH

N1*

*EM

AP2

**D

AP3

**D

TR

MEF

2CPY

K2*

*V

IMIG

FPB5

IGFP

B2M

T1L

microarray profile QRT-PCR profile white matter profile

Hoelzinger, Mariani et al, submitted

Invasion gradient

HE Sampleholes

Serialsamples

Tumour core

Tumour close

Close rim

Far rim

Midway

Gli o

ma

WH

O II

I(n

=2)

GliomaWHO IV

(n=5)

Glioblastoma(n=21)

Glio

ma

WH

O IV Oligo-

astro-cytoma (n=3)

Epileptogenicbrain(n=5)

Pilocytic Astrocytoma

(n=5)

GliomaWHO III

(n=5)

Anaplastic astrocytoma(n=11)

Glioma (WHO II )(n=10)

Low grade astrocytoma(n=10)

Foetaltissue(n=3),

Medullo-blastoma

(n=5)

Gliotic controls(n=7)

Autotaxin (ATX)in autokrines, Motilitätstimulierendes Ligand, überexprimiert in invasiven Astrozytomzellen

Differential Display / cDNA MicroArray


PixCell II LCMor Clones



ParentalMigrations-Stimuliert




Invasive Tumore


Genetik der Gliom-Invasion

Gewebe von“nicht” invasiven Tumoren

WHO Grad II


cDNA Microarray

Gewebe voninvasiven TumorenWHO Grad II

ab

ab

ab

_ase

RTK growthfactors

ECM/myelin

LOSS OF:

NCAMN-Cadherin ?

DCCgap junctions

Cx-43

GAIN OF:GF / GFR

ECM / IntegrinsBEHAB

CD44SPARC

proteases

invasion

_ase Autotaxin

Dap-3p311

Inherited mutations in families at increased risk of glioma

Syndrome Gene name Function

Neurofibromatosis 1NF1 Cytoskeletal protein with GTPase-

activating protein (GAP) domain,

regulates RASNeurofibromatosis 2NF2 Member of ERM family with growth- inhibitory functionTuberous sclerosis TSC1, TSC2 mTOR pathway

Retinoblastoma RB1 cell cycle

Li-Fraumeni syndrome TP53 cell cycle, apoptosis

Common chromosomal alterations in gliomas*

*array-based comparative genomic hybridizationSchwartzbaum et al. (2006), Nature clicical practice neurology 2; 494

Predictive markers for chemotherapy?

Astrocytoma II

Astrocytoma III

Secondary glioblastoma

Primary glioblastoma

Oligodendroglioma II

Oligodendroglioma IIIT

umour progression

Tum

our progression

PDGFR overexp.

EGFR amplif.p16/p14ARF del

CDK4 ampl

TP53 mutation, 60%

MDM2 amplif.

PTEN mutation

LOH 1p and 19q

LOH 10q

LOH 10qMGMT methyl 40%

MGMT methyl 46%

MGMT methyl 50%

MGMT methyl 70%

MGMT methyl 60%

MGMT methyl 80%telomerase 60%

telomerase 20%

telomerase 40%

telomerase 100%

telomerase 100%

In vivo Model of diffuse Glioma

Figure 1

Zheng et al, Nature, 2008, 455:1129-1133

Implication of the MGMT promoter methylation status for chemotherapy (qualitative MSP)

• Glioblastoma patients with unmethylated MGMT do not significantly benefit from the addition of temozolomide to the standard treatment with radiation therapy.

• Patients with methylated MGMT promoter have a significantly longer progression-free and overall survival with the combination of radiation therapy and temozolomide.

• Irrespective of treatment, MGMT promoter methylation was an independent favorable prognostic factor

• Success rate of the methylation-specific PCR was 67% (n=307)

Esteller et al. (2003) N Engl J med 343, 1350Hegi et al. (2004) Clin Cancer Res 10, 1871Hegi et al. (2005) N Engl J med 352, 997

A large number of tumors are heterogeneous forMGMT expression.

Hypomethylation of the MGMT promoter?

Biological relevance?

Establishment of a quantitative MGMT promoter methylation assay by primer extension (modification of the commonly used gel-based assay)

tumorchemotherapy

MGMT-Promoter Methylation: a quantitative assay

Bisulfite treatment of DNA and PCR

XXXCXXCXXCGXXCGXXCGCGXXXX

Bisulfite treatment

XXXUXXUXXCGXXCGXXCGUGXXXX

CH3 CH3 CH3

CH3 CH3 CH3

PCR

XXXTXXTXXCGXXCGXXCGTGXXXX

Commonly used gel-based MSP assay

MGMT Promoter

Potential methylation sites (CpG islands)

290 bp

PCR 1

PCR 2

A Primer specific formethylated DNA

B Primer specific forunmethylated DNA

Commonly used gel-based MSP assay

methylatedunmethylated

Hegi et al. (2005) N Engl J med 352, 997

M-Primer

U-Primer

U-Primer

Case 1: unmethylated DNA (from FFPE tissue)


Quantitative analysis:

M-Primer

Case 1 (PCR)

Case 1 (Extension)

U87:PBMC=1:1(Extension)

M-Primer U-Primer

methylated

methylated

unmethylated

unmethylated

M-Primer

U-Primer

U-Primer

Case 12: hypomethylated DNA (from FFPE tissue)



M-Primer

Case 12 (PCR)

Case 12 (Extension)

U87:PBMC=1.1(Extension)

M-Primer U-Primer

methylated

methylated

unmethylated

unmethylated

Case 125: hypermethylated DNA (from FFPE tissue)

Qualitative PCR:

Nested PCR:

Case 125 (PCR)

Case 125 (Ext.)

M-Primer U-Primer



U-PrimerM-Primer

M-Primer U-Primer


methylated

Case 125 (nested PCR)

unmethylatedmethylated

p53 mutation (17p13) (~60%) 17p loss (~ 60%) 22q13.3 loss (~ 30%) 1p/19q LOH (~ 50%)…

Grad II

Tumor Progression

Vorläufer-Zelle Grad III Grad IVSekundäres Glioblastom

IDH1 and IDH2 Mutation >70%P53 MutationPDGF-A/PDGFR-α ↗ (~ 60%) FGF2 ↗…

10q LOHCDK4 amplification (12q13)~ 25% Rb alteration (13q13) (disrupts p16INK4A)pathway)~50%DCC loss of expression~5% PTEN mutat. (10q24)…

Ohgaki et al, AJP 2009

Grade II

Tumor Progression

Precursor cell Grade III Grade IVSecondary Glioblastoma

PDGF-A/PDGFR-α ↗ (~ 60%) FGF2 ↗…

p53p53p53p53 mutation (17p13) (~ 60%)mutation (17p13) (~ 60%)mutation (17p13) (~ 60%)mutation (17p13) (~ 60%)17p loss (~ 60%) 22q13.3 loss (~ 30%) 1p/19q LOH (~ 50%)…

~60% 10q LOHCDK4 amplification (12q13)VEGF Overexpression~ 25% Rb alteration (13q13) (disrupts p16INK4A)pathway)~50% DCC loss of expression~5% PTEN mutat. (10q24)~8% EGFR amplification…

Time4-5 years in mean


Attenuated Myc expression restores hGFAP-Cre+;p53lox/lox;Ptenlox/+ TNS differentiation potential and reducestumorigenic potential.


p53 and Pten coordinately regulate Myc protein level as well asNSC self-renewal and differentiation.


hGFAP-Cre+;p53lox/lox;Ptenlox/+ gliomas mirror key features ofhuman malignant gliomas.

Summary of Genetic and Clinical Characteristics of Brain Tumors in the Study


Frequency of Common Genetic

Alterations in Gliomas with

Mutated or Wild-Type IDH1 and

IDH2 Genes


Conclusion

• Mutations of NADP+-dependent isocitrate dehydrogenases encoded by IDH1 and IDH2 occur in a majority of several types of malignant gliomas

diffuse gliomas of human adults part 1 · dysembryoplastic neuroepith. tumor 0 ganglioglioma 1...

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