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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.

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predicting glioblastoma outcome the glasgow experience

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
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
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
Brain tumours – the clinical problem
  • Site
  • Raised intracranial pressure
  • Infiltrative pattern – surgical resection impossible
  • High grade transformation
  • Treatment resistance
slide6
MGMT
  • Protein involved in DNA repair
  • Methylation of promoter region ‘silences’ gene and protein product reduced
  • Improved outcome with concomitant chemoradiotherapy
slide7
MGMT gene silencing and benefit from Temozolomide in glioblastomaHegi, M.E et al (for EORTC), NEJM, 2005
idh mutations
IDHmutations
  • 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.

slide9

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
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
slide11

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

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/LCA

MGMT(brown) MGMT(red)

icc for mgmt lca1

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.

slide15

ICC

Group 1 Group 2 Group 3

icc for idh 1

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
H09 ICC assessment

H09 x20

H09 x20 Infiltrating tumour cells

GBM H&Ex20

mgmt promoter methylation status and outcome
MGMT promoter methylation status and outcome

MEDIAN SURVIVAL:

Methylated (n=30) = 22.51

Unmethylated (n=42) = 14.32

p = 0.002

Survivor

1.00

U

M

0.75

0.50

0.25

0.00

Months

0

20

40

60

Times

h09 mutated idh1 icc and outcome

Survivor

1.00

1

0

0.75

0.50

0.25

0.00

0

20

40

60

Times

H09 (mutated IDH1) ICC and outcome

MEDIAN SURVIVAL

H09 positive (n=7) = 34.37

H09 negative (n= 83) = 14.59

P = 0.012

Months

mgmt icc and outcome

Survivor

1.00

1

2

3

0.75

0.50

0.25

0.00

0

20

40

60

Times

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

mgmt icc methylation status and outcome

Survivor

Survivor

1.00

1.00

1M

3U

1U

3M

0.75

0.75

0.50

0.50

0.25

0.25

0.00

0.00

0

10

20

30

40

1

6

11

16

21

26

Times

Times

MGMT ICC, methylation status and outcome

Group 3

Group 1

MEDIAN SURVIVAL

Methylated (n=2) = 8.05

Unmethylated (n=12) = 11.83

P = 0.371

MEDIAN SURVIVAL

Methylated (n=15) = 21.78

Unmethylated (n=8) = 19.06

P = 0.674

Months

Months

slide22

Survivor

1.00

2U

2M

0.75

0.50

0.25

0.00

0

10

20

30

40

Times

Group 2

MEDIAN SURVIVAL

Methylated (n=22) = 22.51

Unmethylated (n=8) = 12.45

P = 0.026

Months

combination of icc for mgmt and h09 with ms pcr
Combination of ICC for MGMT and H09 with MS-PCR.

Survivor

1.00

0.75

0.50

0.25

0.00

0

20

40

60

Times

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

summary glasgow gbm protocol
Summary: Glasgow GBM Protocol

GBM

H09 ICC

MS-PCR

MGMT ICC

methylated

unmethylated

negative

positive

Group 1

Group 3

Group 2

Final report

conclusions
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