The Bristol Experience of Molecular Genetic Analysis of Gliomas (LOH and MGMT)

for Optimisation of Treatment

Hilary SawyerBristol Genetics Laboratory

CMGS Spring Meeting 27th March 2009

The care of all patients with brain and other central nervoussystem (CNS) tumours should be coordinated through a specificmodel of multidisciplinary assessment and care

Outline

• Background to Gliomas• Clinical Trials suggesting prognostic indicators• Clinical Utility of 1p19q LOH and MGMT testing• Bristol team data• Case studies• How results are used clinically in Bristol• The future and laboratory issues

Brain tumours

• Common symptoms: headache with cognitive or behavioural symptoms, epilepsy, progressive focal neurological deficits, raised intracranial pressure

• Treatment - surgery, radiotherapy and more recently chemotherapy

• Tumours within the brain, such as gliomas, can rarely be completely removed because of their relation to critical structures and the infiltrating nature of the tumour

• Many do not demonstrate metastasis but invasion may preclude surgical resection generating ongoing management issues

• Slow growing tumours may transform into more aggressive tumours

• Classified by cell type, grade and location• 2007 WHO classification:

Grade I: low grade, well circumscribed, slowly progressing, can often be cured by resection Grade II: low grade, typically infiltrative, low proliferation but higher likelihood of

recurrence Grade III and IV: high grade, malignant, grow rapidly, aggressiveThere is considerable inter-observer variation in diagnosis and classification however.

• Molecular tests help understand pathogenesis and improve classification.• Evidence for two predictive molecular markers emerging for GLIOMAS:

1p/19q LOH and MGMT

1.6% of cancers in England and WalesHigh morbidity and mortality

Gliomas• Tumours arising from glial cells• Non-neuronal cells

• provide physical support and nutrition

• maintain homeostasis

• form myelin

• participate in signal transmission

• Glial cell types• Astrocytes – anchor neurons to blood supply, regulation and signalling ASTROCYTOMAS

• Oligodendrocytes - produce myelin sheath OLIGODENDROGLIOMAS

• Mixed Gliomas – OLIGOASTROCYTOMAS

• Ependymocytes - lining and secret CSF EPENDYMOMAS

• Glioblastomas are the most common form of astrocytic tumour

Average survival times:Grade II astrocytoma: 7 yearsAnaplastic astrocytoma: 3.5 yearsGlioblastoma: 9-11 months

High-grade glioma (WHO III and IV) includes: glioblastoma, anaplastic astrocytomas, anaplastic oligodendrogliomas and anaplastic ependymomas

well documented to progress to higher grade malignancy

Genetic pathways in gliomas

Adapted fromOhgaki H Neuropathology 2005;25 1-7

MGMT methylation

Oligodendrocyte

OligodendrogliomaGrade III

Chemosensitive

OligodendrogliomaGrade III

Chemoresistant

LOH 1p19q50-70%

p16 30%LOH 10q 50%EGFR 20%

No longer enough to defineoligodendrogliomas histologically

Astrocytes or precursor cells

Anaplastic AstrocytomaTP53 mutation (53%)

Low-grade astrocytomaTP53 mutation (59%)

WHO grade II

1° Glioblastomade novoLOH 10q (70%)EGFR amp (36%)p16INK4a deletion (31%)TP53 mutation (28%)PTEN mutation (25%)

2° Glioblastoma

LOH 10q (63%)EGFR amp (8%)p16INK4a deletion (19%)TP53 mutation (65%)PTEN mutation (4%)

WHO grade III

WHO grade IV

Oligodendroglioma

• Median age of onset between 40 & 50 years• Treatment by surgical resection, followed by radiotherapy and PCV

chemotherapy on recurrence. • Main aims of treatment and follow-up are to increase survival while

maximising a patient’s functional capability and quality of life

• Relatively good prognosis, survival of 3-10 years from diagnosis.

• Recurrence is common, often leading to disease progression and death.

• Oligodendroglia - specialised CNS myelin-forming cells.

• Rare primary brain tumours, frequency ~0.53 cases/100,000

• Around 25% primary brain tumours in adults

• ~20% are anaplasticAnaplastic oligodendroglioma (WHO grade III)

Typical ‘fried egg’ appearance (fixation artifact):

Molecular Markers in Oligodendrogliomas (ODs)Various trials support the clinical utility of analysis for 1p19 LOH:

LOH 1p/19q

No LOH

There had been general disillusionment with chemotherapy

Various trials have showed improved outcomesin patients with 1p19q LOH:

Initial trials: Cairncross G et al (1994 and 1998)

1p and 19q deletions were observed in ~66% ODs Reifenberger et al Neuropath Exp Neurol 2003

Oligodendrogliomas with 1p loss alone have an intermediate prognosis. Ino Y et al. Clin Cancer Res (2001).

I

The above were small trials or retrospective series suggesting predictive value. Further trials (Phase III) were needed:

From: McDonald et al (2005) Cancer. 1;104(7):1468-77

1) RTOG Trial 9402: Pure and Mixed Anaplastic Oligodendroglioma: PCV + RT vs RT aloneProgression-free survival time favoured PCV + RT but 65% of patients experienced toxicityTumours with LOH 1p19q have longer median survival times (>7 v 2.8 yrs) and longer PFSTumours with LOH 1p19q have longer median survival times (>7 v 2.8 yrs) and longer PFSBetter prognosis, time to relapse but no difference whether early v late chemotherapyno difference whether early v late chemotherapy

2) EORTC: In newly diagnosed AODs and OAs:Adjuvant chemotherapy improves PFS but does not affect OS, therefore timing is less relevant.

Multicentre randomised trial in Germany of sequential radiochemotherapyof oligoastrocytic WHO grade III tumours with PCV or temozolomideWolfgang WW et al (2008) J Clin Oncol 26

Patients with any grade III tumour: anaplastic oligodendroglioma, oligoastrocytoma or astrocytoma with or without deletions

Either 6 week course RT or 4x6 week cycles of either PCV or temozolomide

•LOH 1p/19q and hypermethylation of MGMT large risk reduction for time to treatment failure (TTF) regardless of histology

•Oligodendroglial histology better than astrocytic (NB 1p/19q LOH is present in higher % of cases of AOD than AOA, also low in mixed OAs with predominant OD)

•No difference in TTF between patients on RT v chemotherapy or which is used first

•MGMT methylation status may have been more important

3) NOA-4 study Phase III trial

These trials confirmed that 1p/19q loss improved outcome following RT with or without chemotherapyi.e. appears to be a prognostic factor for survival regardless of treatmenttype or timing

ODs with 1p19q LOH also respond more favourably to temozolomide(an alkylating agent)

Loss of Heterozygosity Analysis LOH (Bristol)

• Marker profile of tumour DNA compared to blood DNA to determine LOH. Panel of 8 markers in 3 multiplexes and one simplex

• Fresh/frozen tumours used in Bristol v PPFE

• As oligodendrogliomas may be diffuse tumours, problems with mixed tissue can occur (require 60-90% tumour tissue)

D1S468D1S214

D1S2736

D1S199

19q 1p

D19S408D19S412

D19S926D19S418

Ino Y et al. (2001) Clin Cancer Res 7, 839-845.

Smith J et al (2000). J Clin Oncol: 18 636-645

Cairncross et al (1998) J Natl Cancer Inst 90, 1473-1479

DNA from peripheral

blood:

DNA from tumour

tissue:

Allelic loss

Bristol LOH 1p19q Data 2005 to early March 2009 300 patients. Approx 50 per year but increasing

The majority of pure AODs carry 1p/19q deletions: predictive,prognostic

Patient with rare histologywith 1p/19q LOH and good response to PCV

There is a good response of glioblastomas with 19q deletion

Literature: Where co-deletion of 1p and 19q is present this is usually found throughout the tumour

Some OAs have loss andrespond well

Tumour

Type

No LOH

%

LOH

1p/19q

%

LOH 1p

%

LOH 19q

%

Partial

LOH 1p

Partial

LOH 19q

%

Partial LOH

1p+19q

%

Un-

Reportable

%

Total

Number

Oligodendroglioma 7.5 82.5 0 5 5 0 0 0 40

Oligoastrocytoma 34.6 46 0 7.7 3.8 3.8 3.8 0 26

Astrocytoma 56.8 9.8 5.9 7.8 7.8 5.9 5.9 0 51

Malignant astrocytic

glioma

50 0 0 50 0 0 0 0 2

Infiltrating astrocytic

tumour

85.7 7.1 0 0 7.1 0 0 0 14

Glioblastoma 57.6 5 1.2 16 3.8 10.1 5.1 0.6 158

Gliosarcoma 50 25 0 0 0 1 0 0 4

Neuroectodermal 50 0 0 25 0 0 25 0 4

Liponeurocytoma 0 100 0 0 0 0 0 0 1

Total 300

Grey = low no cases

Clinical Case 1

• Aug 02 Male aged 68 at presentation Confusion and left sided weaknessRight parieto-occipital massPartial resection

Histology : ganglioglioma with foci of mitotically active primitive neuroectodermal tumour

• Oct/Nov 02 Radical radiotherapy

• Dec 02 Massive recurrenceFurther surgery but prognosis poor

Clinical Case 1 (con)

• Dec 02 1p19q deletion detected and offered palliative PCV • Feb/Oct 03 PCV with complete radiological response

• Jun 06 relapse treated with stereotactic radiosurgery

• Dec 06Rapid decline

• Feb 07 Died

1p19q analysis correctly identified a chemosensitive tumour, even when the morphology was confusing and the clinical course appeared aggressive.

Patient had a 3.5 year remission with a good quality of life due to this intervention

Clinical Case 2• Dec 06 40 yr old female

Presented with headaches and drowsinessExtensive tumour in right hemisphere and thalamus

• Jan 07 Partial resection

Histology: Central liponeurocytomaRare tumour- c. 25 reported cases no clear guidance on treatment1p19q deletions detected

• Feb/Apr 07 Radical radiotherapy

• Oct 07 Recurrence –further surgeryHistology unchanged

• Nov/Jul 08 PCV chemotherapy

• Jul 08 MRI clear

1p19q encouraged the use of PCV where no data was available It has already proved of more durable adjuvant benefit than radiotherapy

Glioblastomas• Commonest primary brain

tumour: ~5/100,000 annum

• Either develop from lower malignancy grade tumour or de novo (different genes/same cell pathway)

• 50% respond to alkylating agent temozolamide

Glioblastoma (WHO grade IV)

• Responsive tumours show promoter methylation (inactivation) of the MGMT (O6-methylguanine-DNA methyltransferase) gene (10q26)

• MGMT is a DNA repair protein (suicide enzyme) that removes alkyl groups from guanine, reversing the effect of temozolamide

• MGMT methylation may predict responsiveness to temozolamide treatment.

The Stupp Trial

N=573 MS (months)

2 year survival (%)

3 year survival (%)

RT alone 12.1 10.4 3

RT and Temo

14.6 26.5 17

Recruited patients with GBM post surgery randomised to RT alone or RT with concomitant temozolomide

•Addition of chemotherapy to radiotherapy significantly prolongs survival among patients with newly diagnosed glioblastoma

•Increase in survival rate at 2 years

MGMT gene silencing and benefit from temozolomide in glioblastoma.

Hegi ME, et al New Eng J Med 2005;352:997-1003.

MGMT methylated tumours: Median Survival 18.2 monthsNo methylation: Median Survival 12.2 months

Randomized trial comparing RT alone with RT combined with concomitant and adjuvant temozolamide

206 tumours44.7% methylated55.3% unmethylated

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

From Hegi:

Hegi et al 2005 N=206 Methylated 45% RTOG study Methylated 50%

To late Jan 09388 cases

Methylated Unmethylated Equivocal Total % total samples Meth%Glioblastomas 165 116 291 75.00 56Astrocytomas 48 18 66 17.00 72

Oligoastrocytoma 9 2 11 2.83 81Oligodendrogliomas 14 0 14 3.60 100

Other/Unknown 5 1 6 1.50 83TOTAL 241 137 10 388 100

Bristol MGMT analysis data to Late Jan 09

Bristol MGMT methylation analysisMain assay is a diplex of unmeth and meth product

Unmethresult

Methresult

Muchof literatureas separatesimplexes

2005 MGMT Survival data n=21 • Methylated (n=12)• Median Survival 15.5 mths• Range 0-31 mths 1 patient alive

• Unmethylated (n=9)• Median survival 10 mths• Range 1-36 months 1 patient alive

The above date from 2005, before concomitant temozolomide was routinely available on the NHS

The above did not receive concomitant temozolomide

Now NICE approval obtained for concomitant temozolamide It is expected that survival gap will widen.

How are the clinicians using these MGMT data now?

• Subgroups in Hegi study were too small to exclude a possible un-seen benefit for unmethylated patients so NICE recommendation is that all patients with GBM receive concomitant treatment.

• However – the outlook is clearly poor for patients in the unmethylated group, therefore the challenge is either to enhance the effectiveness of temozolomide or to find a better option.

• RTOG ‘dose dense’ temozolomide study – can MGMT be saturated with more prolonged treatment schedule?– Bristol highest recruiters to trial in Western

Europe

Clinical Management in Bristol All patients with grade II-IV tumours have 1p19q analysis and MGMT analysed Management is not affected by morphology but determined by grade and genetic results Genetic results may be particularly useful when the histological diagnosis is unclear

• Grade II: Surgery and follow up 1p19q deletion informed of better prognosis At progression offer BR13 trial (randomised for radiotherapy or temozolamide

as initial treatment to look at OS,PFS and QOL ) Trial declined? PCV if 1p deletions, RT if no deletions

• Grade III: Surgery and immediate oncology treatment If have 1p19q deletions recommend PCV as first line treatment as patients with

good prognosis may suffer late effects if cranial radiotherapy No deletions recommend initial radiotherapy Patient factors and choice important

• Grade IV: Surgery and immediate radiotherapy with concomitant temozolamide Standard regimen whilst awaiting results of dose dense study.

Patients uncertain about chemotherapy are informed of methylation status and Hegi data to inform choice

Where MGMT promoter is unmethylated, patients advised that this tumour is particularly ‘aggressive’

May consider using chemotherapy in patients with rare tumours and 1p19q LOH

The Future

LOH and MGMT testing now indicated as valuable predictive markers in RCPath Dataset for tumours of the CNS (2nd edition) April 2008

Require robust funding streams and staffing – previous from charitable funds/training (NICE suggest 2500 annual tests and up to 30technical staff across England and Wales)

•Local clinicians are proud of the accurate information patients are given.•Bristol is one of the biggest testing centres in UK•It is hoped to have better therapies for all patients•Bristol patients in other trials –therapy for low grade gliomas (BR13 etc)•1p and 1p19q LOH appears to be a marker for improved PFS in grade II gliomas

• Routinely collecting fresh tissue for these assays has allowed Bristol to be prominent in national and international trials to inform future treatment (BR13 and CATNON trials etc). The local team will be able to implement any new guidelines speedily.

• Need to underpin this work through discussion of laboratory testing standards and guidelines to improve analysis, including tissue type (fresh v PPFE), choice of assay method(s), EQA and through availability of control reference materials

Laboratory-histopathology-genetics

Neurosurgeon

Neuropathologist

Neuro-radiologist

Specialist oncologist

Palliative care

Clinical nurse specialist

Epilepsy nurse

AHPs for speech therapy, OT, physio

Brain cancer networkMDT

Neuro-psychologist

We are part of thewider Bristol MDT networkas outlined by NICE

Thanks to the Bristol Team• BGL team Mark Greenslade

Elena MavrakiSarah Burton-JonesSuzanne O’SheaLaura YarramThais SimmonsPaula WaitsKayleigh McDonaghMaggie Williams

(Emma Ryan/Karen Meaney/Meera Parmar)

• Neuropathology Seth Love, Neuropathology, Frenchay• Neurooncology Kirsten Hopkins, Bristol Oncology Centre

Hugh Newman, UH Bristol