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

1. The Bristol Experience of Molecular Genetic Analysis of Gliomas (LOH and MGMT) for Optimisation of Treatment CMGS Spring Meeting 27th March 2009 The care of all patients with brain and other central nervous system (CNS) tumours should be coordinated through a specific model of multidisciplinary assessment and care Hilary Sawyer Bristol Genetics Laboratory

2. 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

3. Brain tumours 1.6% of cancers in England and Wales High morbidity and mortality • 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, aggressive There 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

4. 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 years • Anaplastic astrocytoma: 3.5 years • Glioblastoma: 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

5. Genetic pathways in gliomas Astrocytes or precursor cells Oligodendrocyte Low-grade astrocytoma TP53 mutation (59%) WHO grade II LOH 1p19q 50-70% p16 30% LOH 10q 50% EGFR 20% Anaplastic Astrocytoma TP53 mutation (53%) WHO grade III Oligodendroglioma Grade III Chemosensitive Oligodendroglioma Grade III Chemoresistant MGMT methylation 1° Glioblastoma de novo LOH 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 IV No longer enough to define oligodendrogliomas histologically Adapted from Ohgaki H Neuropathology 2005;25 1-7

6. Typical ‘fried egg’ appearance (fixation artifact): Oligodendroglioma • 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 anaplastic • 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. Anaplastic oligodendroglioma (WHO grade III)

7. Molecular Markers in Oligodendrogliomas (ODs) LOH 1p/19q No LOH Various trials support the clinical utility of analysis for 1p19 LOH: There had been general disillusionment with chemotherapy Various trials have showed improved outcomes in 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 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 alone • Progression-free survival time favoured PCV + RT but 65% of patients experienced toxicity • Tumours with LOH 1p19q have longer median survival times (>7 v 2.8 yrs) and longer PFS • Better prognosis, time to relapse but no 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. Oligodendrogliomas with 1p loss alone have an intermediate prognosis. Ino Y et al. Clin Cancer Res (2001). I

8. 3) NOA-4 study Phase III trial • Multicentre randomised trial in Germany of sequential radiochemotherapy • of oligoastrocytic WHO grade III tumours with PCV or temozolomide • Wolfgang 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 These trials confirmed that 1p/19q loss improved outcome following RT with or without chemotherapy i.e. appears to be a prognostic factor for survival regardless of treatment type or timing ODs with 1p19q LOH also respond more favourably to temozolomide (an alkylating agent)

9. 1p 19q D1S468 D1S214 D1S2736 D1S199 DNA from peripheral blood: D19S408 D19S412 DNA from tumour tissue: D19S926 D19S418 Allelicloss Loss of Heterozygosity Analysis LOH (Bristol) 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 • 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)

10. 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 Some OAs have loss and respond well Grey = low no cases There is a good response of glioblastomas with 19q deletion Patient with rare histology with 1p/19q LOH and good response to PCV Literature: Where co-deletion of 1p and 19q is present this is usually found throughout the tumour

11. Clinical Case 1 • Aug 02 Male aged 68 at presentation Confusion and left sided weakness Right parieto-occipital mass Partial resection Histology : ganglioglioma with foci of mitotically active primitive neuroectodermal tumour • Oct/Nov 02 Radical radiotherapy • Dec 02 Massive recurrence Further surgery but prognosis poor

12. 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 06 Rapid 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

13. Clinical Case 2 • Dec 0640 yr old female Presented with headaches and drowsiness Extensive tumour in right hemisphere and thalamus • Jan 07 Partial resection Histology: Central liponeurocytoma Rare tumour- c. 25 reported cases no clear guidance on treatment 1p19q deletions detected • Feb/Apr 07 Radical radiotherapy • Oct 07 Recurrence –further surgery Histology 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

14. 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.

15. The Stupp Trial 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

16. MGMT gene silencing and benefit from temozolomide in glioblastoma.Hegi ME, et alNew Eng J Med 2005;352:997-1003. Randomized trial comparing RT alone with RT combined with concomitant and adjuvant temozolamide From Hegi: 206 tumours 44.7% methylated 55.3% unmethylated MGMT methylated tumours: Median Survival 18.2 months No methylation: Median Survival 12.2 months Irrespective of treatment, MGMT promoter methylation was an independent favorable prognostic factor

17. Bristol MGMT methylation analysis Main assay is a diplex of unmeth and meth product Much of literature as separate simplexes Meth result Unmeth result Bristol MGMT analysis data to Late Jan 09 To late Jan 09 388 cases Hegi et al 2005 N=206 Methylated 45% RTOG study Methylated 50%

18. 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.

19. 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

20. 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 1p19qLOH

21. 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 30 • technical 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

22. Neurosurgeon Clinical nurse specialist Neuro-psychologist Neuropathologist Specialist oncologist Brain cancer network MDT Epilepsy nurse Neuro-radiologist Palliative care Laboratory -histopathology -genetics AHPs for speech therapy, OT, physio We are part of the wider Bristol MDT network as outlined by NICE

23. Thanks to the Bristol Team • BGL team Mark Greenslade • Elena Mavraki • Sarah Burton-Jones • Suzanne O’Shea • Laura Yarram • Thais Simmons • Paula Waits • Kayleigh McDonagh • Maggie Williams • (Emma Ryan/Karen Meaney/Meera Parmar) • Neuropathology Seth Love, Neuropathology, Frenchay • Neurooncology Kirsten Hopkins, Bristol Oncology Centre • Hugh Newman, UH Bristol