1. Predicting glioblastoma outcome: the Glasgow experience Dr Sarah Bell Specialty Trainee Registrar Neuropathology Department of Neuropathology Southern General Hospital Ms Naomi Muir Biomedical Scientist Department of Pathology Southern General Hospital

2. Brain and CNS tumour incidence 4300 new cases diagnosed per year in UK 2% of all ‘cancers’ diagnosed 3 rd leading cause of death in men aged 15-50 and women aged 15-35 Around 10% 5 year survival Commonest tumour in children and the leading cause of cancer death

3. Classification 134 recognised types of primary brain tumour Glioma accounts for 65% -Astrocytomas -Oligodendrogliomas -Mixed -Subtyping and grading based on histological features and determines treatment

4. Brain tumours – the clinical problem Site Raised intracranial pressure Infiltrative pattern – surgical resection impossible High grade transformation Treatment resistance

5. Glioblastoma WHO Grade IV

6. MGMT Protein involved in DNA repair Methylation of promoter region ‘silences’ gene and protein product reduced Improved outcome with concomitant chemoradiotherapy

7. MGMT gene silencing and benefit from Temozolomide in glioblastoma Hegi, M.E et al (for EORTC), NEJM, 2005

8. IDH mutations Key enzymes in cell metabolism Mutations in IDH1 mainly found in ‘secondary’ glioblastomas R132H is commonest mutation Independent marker of improved OS OS 3.8yrs vs 1.1yr Parsons et al (2008) Science 321:1807- 1812.

9. Combination of immunocytochemistry for MGMT and H09 (mutated IDH1 protein) with assessment of MGMT promoter methylation in predicting glioblastoma outcome: the Glasgow Protocol SL Bell, N Muir, Z Hanzely, J Stewart, M MacKinnon, B Clark, R Rampling, W Stewart West of Scotland Neuro-Oncology Group

10. Background MGMT promoter methylation and IDH1 mutation are prognostic markers in glioblastoma (GBM) protein product of both can be detected with ICC AIM: combine molecular and ICC techniques and assess association with outcome in GBM. patients 100 consecutive GBM treated with concomitant chemoradiotherapy June 2005 – May 2009

11. Aims To develop and evaluate an ICC staining protocol for assessment of MGMT activity in GBM. To more accurately assess MGMT activity use sequential (double) staining using MGMT and LCA. Assess patterns of staining for MGMT and patient outcome.

12. ICC for MGMT/LCA Dako Envision TM G/2 Doublestain System (DAB/ Permanent Red) MGMT 1:20 and LCA 1:500 dilution. First step determine if MGMT better visualised using DAB or Permanent Red. Optimise timing of the protocol to give optimal staining.

13. MGMT/LCA MGMT(brown)MGMT(red)

14. ICC for MGMT/LCA Areas of solid tumour were selected and 300 cells counted. Total number of LCA positive cells subtracted from total number of MGMT positive cells. Cases were divided into 3 groups based on proportion of MGMT positive tumour cells. ICC Group 1 – Less than 10% tumour cells positive. ICC Group 2 – 10%-50% tumour cells positive. ICC Group 3 – More than 50% tumour cells positive.

15. ICC Group 1Group 2Group 3

16. ICC for IDH-1 Antibody from Prof. Von Deimling (Neuropathology Dept., Heidelberg, Germany). Vectastain Universal Elite ABC kit. 1:10 dilution Areas of solid tumour – positive or negative.

17. H09 ICC assessment GBM H&Ex20 H09 x20 H09 x20 Infiltrating tumour cells

18. MGMT promoter methylation status and outcome MEDIAN SURVIVAL: Methylated (n=30) = 22.51 Unmethylated (n=42) = 14.32 p = 0.002 Months

19. H09 (mutated IDH1) ICC and outcome MEDIAN SURVIVAL H09 positive (n=7) = 34.37 H09 negative (n= 83) = 14.59 P = 0.012 Months

20. MGMT ICC and outcome MEDIAN SURVIVAL Group 1 (n=25) = 19.91 Group 2 (n=33) = 15.44 Group 3 (n=14) = 11.83 P = 0.0007 Months

21. MGMT ICC, methylation status and outcome 010203040 0.00 0.25 0.50 0.75 1.00 Survivor Times 1M 1U MEDIAN SURVIVAL Methylated (n=15) = 21.78 Unmethylated (n=8) = 19.06 P = 0.674 Group 1 Months 1611162126 0.00 0.25 0.50 0.75 1.00 Survivor Times 3U 3M MEDIAN SURVIVAL Methylated (n=2) = 8.05 Unmethylated (n=12) = 11.83 P = 0.371 Group 3 Months

22. 010203040 0.00 0.25 0.50 0.75 1.00 Survivor Times 2U 2M Group 2 MEDIAN SURVIVAL Methylated (n=22) = 22.51 Unmethylated (n=8) = 12.45 P = 0.026 Months

23. Combination of ICC for MGMT and H09 with MS-PCR. MEDIAN SURVIVAL: Group 1 (n= 23) = 21.59 Group 2U (n=22) = 12.45 Group 2M (n=8) = 22.51 Group 3 (n=14) = 12.45 H09 (n=7) = 34.37 P= <0.0001 Months

24. Summary: Glasgow GBM Protocol GBM Final report H09 ICC positivenegative MS-PCR methylatedunmethylated MGMT ICC Group 1 Group 2 Group 3

25. Conclusions Molecular subtyping of gliomas predicts OS and treatment responses Molecular neuro-oncology services and EQA in Glasgow - 1p19q LOH, MGMT and IDH1 (H9) Combining ICC for MGMT and H09 with molecular assessment of MGMT promoter methylation allows stratification of patients –H09+ve > Gp1 = Gp2Me > Gp2Um = Gp3