Epigenetic - PhD Programme Uppsala

1. PhD programme
Slides available
www.bioinformatics.be

2. Lab for Bioinformatics and
computational genomics
10 “genome hackers”
mostly engineers (statistics)
42 scientists
technicians, geneticists, clinicians
>100 people
hardware engineers,
mathematicians, molecular biologists

3. Next Generation
Epigenetic Profiling
Wim Van Criekinge
12th december, Uppsala (SE)

4. Overview
Epigenetics
– Introduction
– Methylation & Oncology
– Biomarkers
MDxHealth
– NEXT-GENeration Epigenetic Biomarkers
– PharmacoDX: Methylation Based Biomarkers
for Predictive and Prognostic Use

5. The genome is just the start …

6. Defining Epigenetics
Genome
DNA


Chromatin
Epigenome
Gene Expression
Phenotype


Reversible changes in gene
expression/function
Without changes in DNA
sequence
Can be inherited from
precursor cells
Allows to integrate intrinsic
with environmental signals
(including diet)

7. DNA Methylation Differentiates Totipotent Embryonic
Stem Cells from Unipotent Adult Stem Cells
Alex Meissner, Henry Stewart Talks

8. Reprogramming the DNA methylome
Paula Vertino, Henry Stewart Talks

10. Transgenerational inheritence

11. Actionable
Epigenome

12. 250 different cell types
Epigenetic (meta)information = stem cells

13. Cellular programming
Epigenetic (meta)information = stem cells

14. Outside
Oncology ?

15. Historically,
Cancer Was Considered
to be Driven Mostly by Genetic Changes
GENETIC
 Mutations in p53
Example:
Replication errors
 Activating mutations in RAS
 Mutations or amplifications of the
X X
HER-2 gene
Altered
DNA sequence
 Chromosomal translocations in
Altered
DNA/mRNA/proteins
Oncogenesis
myeloid cells and the generation of
the BCR-ABL fusion protein
Tumor

16. Epigenetic Changes are
Important in Causing Cancer
GENETIC
EPIGENETIC
Example:
Chromatin modification errors
Example:
Replication errors
X X
Altered
chromatin structure
Altered
DNA sequence
Altered
DNA/mRNA/proteins
Oncogenesis
Altered levels of
mRNA/proteins
Tumor

17. Example of Methylation
vs Mutation: Colon & Breast Cancer
120
100
80
Dx
60
40
CDx
20
0
Methylated
Mutated
Source: Schuebel et al 2007
76-100 51-75
21-50
1-20

18. MGMT Biology
O6 Methyl-Guanine
Methyl Transferase
Essential DNA Repair Enzyme
Removes alkyl groups from damaged
guanine bases
Healthy individual:
- MGMT is an essential DNA repair enzyme
Loss of MGMT activity makes individuals susceptible
to DNA damage and prone to tumor development
Glioblastoma patient on alkylator chemotherapy:
- Patients with MGMT promoter methylation show
have longer PFS and OS with the use of alkylating
agents as chemotherapy

19. MGMT Promoter
Methylation Predicts
Benefit form DNA-Alkylating Chemotherapy
Post-hoc subgroup analysis of Temozolomide Clinical trial with primary
glioblastoma patients show benefit for patients with MGMT promoter methylation
25
Median Overall Survival
21.7 months
20
15
plus
temozolomide
12.7 months
radiotherapy
10
radiotherapy
5
0
Non-Methylated
MGMT Gene
Methylated
MGMT Gene
Adapted from Hegi et al.
NEJM 2005
352(10):1036-8.
Study with 207 patients

20. Overview
Epigenetics
– Introduction
– Methylation & Oncology
– Biomarkers
MDxHealth
– NEXT-GENeration Epigenetic Biomarkers
Can we rediscover MGMT ?
What does the epigenome look like ?
….

21. MBD_Seq
Condensed Chromatin
DNA Sheared
DNA Sheared
Immobilized
Methyl Binding Domain

22. MBD_Seq
Immobilized
Methyl binding domain
MgCl2
Next Gen Sequencing
GA Illumina: 100 million reads

23. Quality evaluation of Methyl Binding Domain
based kits for enrichment DNA-methylation
sequencing
De Meyer et al (2013)
Plos One
Confidential Information | ©2013 MDxHealth Inc. All rights reserved.

24. Quality evaluation of Methyl Binding Domain based
kits for enrichment DNA-methylation sequencing
Confidential Information | ©2013 MDxHealth Inc. All rights reserved.

25. MBD_Seq
MGMT = dual core

26. Genome-wide methylation
…. by next generation sequencing
# markers
1-2 million
methylation
cores
MBD_Seq
Discovery
# samples

27. BRCA1,
a bidirectional promoter
27

28. Splice variants
28

29. Zooming into
Exon 1
29

30. Zooming into
Exon 1
30

31. Zooming into
Exon 1
31

32. Where is the mC ?
GCATCGTGACTAGCGACTGATCGATGGATGCTAGCAT

33. GCATCGTGACTAGCGACTGATCGATGGATGCTAGCAT

34. GCATCGTGACTAGCGACTGATCGATGGATGCTAGCAT
25%
50%
25%
GCATCGTGACTAGCGACTGATCGATGGATGCTAGCAT

35. GCATCGTGACTAGCGACTGATCGATGGATGCTAGCAT
25%
50%
25%
GCATCGTGACTAGCGACTGATCGATGGATGCTAGCAT
GCATCGTGACTAGCGACTGATCGATGGATGCTAGCAT
Dense methylated needed for transcriptional silencing
Are there alleles with all three positions methylated ?

36. Deep Sequencing
unmethylated alleles
methylated alleles
less methylation
more methylation
GCATCGTGACTTACGACTGATCGATGGATGCTA

37. Deep Sequencing MGMT Heterogenic complexity

38. Data integration with
DEEP Sequencing, Infinium, Reactivation, (directional) Expression …

39. Data integration
Correlation tracks
expression
expression
Corr =-1
Corr = 1
methylation
methylation

40. Correlation track
in GBM @ MGMT
+1
-1

41. BRCA1
1
2
3
4
5
6
7
8
T47D
BT549
MCF7
MDAMB231
HS578T
NORMAL
NORMAL
NORMAL
41

42. 9
10
11
12
13
14
15
16
17
BI T21 (BRCA1 MUT)
BI T22 (BRCA1 MUT)
BLC
BLC
BLC
BLC
BLC
BLC
BLC

43. 13 BLC
14 BLC
15 BLC
16 BLC
17 BLC
18 BLC
19 IVM
20 DKO

44. Genome-wide methylation
…. by next generation sequencing
# markers
MBD_Seq
Discovery
BT_Seq
Verification
MSP
Validation
# samples

45. Genetics
Whole-genome
Bisulphite seq
Probes
(450-27K)
Enrichment
Targeted Panels
MSP
bp
Full genome
109
108
107
106
Confidential Information | ©2013 MDxHealth Inc. All rights reserved.
105
104
103
102
101
1

46. Genetics
Instrument and Assay providers
G
E
N
E
T
I
C
Whole-genome
sequencing
Enrichment seq
(Exome)
Enrichment
Targeted Panels
PCR
bp
Full genome
109
108
107
106
105
104
103
CLIA Lab service providers
Confidential Information | ©2013 MDxHealth Inc. All rights reserved.
102
101
1

47. Molecular Unification
E
P
I
Whole-genome
Bisulphite seq
G
E
N
E
T
I
C
Whole-genome
sequencing
Enrichment seq
(MBD, RRBS)
Probes
(450-27K)
Ultra
Deep
Enrichment
Targeted Panels
Enrichment seq
(Exome)
Enrichment
Targeted Panels
Deep
Seq
bp
Full genome
109
108
RUO
107
106
105
104
103
Sequencing
102
101
1
Clinical

48. 236 cancer-related genes
(3,769 exons) plus 47 introns
from 19 genes often
rearranged or altered in
cancer. These genes are
known to be somatically
altered in human solid
cancers based on recent
scientific and clinical
literature.
Confidential Information | ©2013 MDxHealth Inc. All rights reserved.

49. PubMeth.org
Reviewed methylation database in cancer
Confidential Information | ©2013 MDxHealth Inc. All rights reserved.

50. Epigenetic Alterations
Associated with Cancer Therapy Response
PCFT – folate
transport (MTX)
WRN

51. 440 cancer-related genes genes are known to
be epigenetically altered in human solid
cancers based on recent scientific and clinical
literature.
Confidential Information | ©2013 MDxHealth Inc. All rights reserved.

52. Molecular Unification
E
P
I
Whole-genome
Bisulphite seq
G
E
N
E
T
I
C
Whole-genome
sequencing
Enrichment seq
(MBD, RRBS)
Probes
(450-27K)
Ultra
Deep
Enrichment
Targeted Panels
Enrichment seq
(Exome)
Enrichment
Targeted Panels
Deep
Seq
bp
Full genome
109
108
RUO
107
106
105
104
103
Sequencing
102
101
1
Clinical

53. Acknowledgments
Geert Trooskens
Simon Denil
Klaas Mensaert
Jean-Pierre Renard
Sarah De Keulenaer
Ellen De Meester
Tim De Meyer
Wim van den Berghe
Manon Van Engeland
Jurgen Veeck
Monika Hegi
Pierre Bady
Sebastian Kurscheid
Olivier Thas
53

54. biobix
wvcrieki
biobix.be
bioinformatics.be
54