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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
Brain and CNS tumour incidence
• 4300 new cases diagnosed per year in UK• 2% of all ‘cancers’ diagnosed
• 3rd 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
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
Brain tumours – the clinical problem
• Site • Raised intracranial pressure• Infiltrative pattern – surgical resection impossible• High grade transformation• Treatment resistance
MGMT
• Protein involved in DNA repair• Methylation of promoter region ‘silences’
gene and protein product reduced• Improved outcome with concomitant
chemoradiotherapy
MGMT gene silencing and benefit from Temozolomide in glioblastoma
Hegi, M.E et al (for EORTC), NEJM, 2005
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.
• 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
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
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.
ICC for MGMT/LCA
• Dako EnvisionTM 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.
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.
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.
MGMT promoter methylation status and outcome
0 20 40 600.00
0.25
0.50
0.75
1.00
Survivor
Times
U
M
MEDIAN SURVIVAL:Methylated (n=30) = 22.51Unmethylated (n=42) = 14.32
p = 0.002
Months
H09 (mutated IDH1) ICC and outcome
0 20 40 600.00
0.25
0.50
0.75
1.00
Survivor
Times
1
0
MEDIAN SURVIVALH09 positive (n=7) = 34.37H09 negative (n= 83) = 14.59
P = 0.012
Months
MGMT ICC and outcome
0 20 40 600.00
0.25
0.50
0.75
1.00
Survivor
Times
1
2
3
MEDIAN SURVIVALGroup 1 (n=25) = 19.91Group 2 (n=33) = 15.44Group 3 (n=14) = 11.83
P = 0.0007
Months
MGMT ICC, methylation status and outcome
0 10 20 30 400.00
0.25
0.50
0.75
1.00
Survivor
Times
1M
1U
MEDIAN SURVIVALMethylated (n=15) = 21.78Unmethylated (n=8) = 19.06
P = 0.674
Group 1
Months
1 6 11 16 21 260.00
0.25
0.50
0.75
1.00
Survivor
Times
3U
3M
MEDIAN SURVIVALMethylated (n=2) = 8.05Unmethylated (n=12) = 11.83
P = 0.371
Group 3
Months
0 10 20 30 400.00
0.25
0.50
0.75
1.00
Survivor
Times
2U
2M
Group 2
MEDIAN SURVIVALMethylated (n=22) = 22.51Unmethylated (n=8) = 12.45
P = 0.026
Months
Combination of ICC for MGMT and H09 with MS-PCR.
0 20 40 600.00
0.25
0.50
0.75
1.00
Survivor
Times
MEDIAN SURVIVAL:Group 1 (n= 23) = 21.59Group 2U (n=22) = 12.45Group 2M (n=8) = 22.51Group 3 (n=14) = 12.45H09 (n=7) = 34.37
P= <0.0001
Months
Summary: Glasgow GBM Protocol
GBM
Final report
H09 ICC
positivenegative
MS-PCR
methylated unmethylated
MGMT ICC
Group 1
Group 2
Group 3
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