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

Glioblastoma WHO Grade IV

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.

MGMT/LCA

MGMT(brown) MGMT(red)

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

Group 1 Group 2 Group 3

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.

H09 ICC assessment

GBM H&Ex20

H09 x20

H09 x20 Infiltrating tumour cells

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