Next Generation Epigenetic Profiling
Wim Van Criekinge 28th March 2014, UGent (BE)
Overview Epigene,cs
– Introduc*on – Methyla*on & Oncology – Biomarkers
MDxHealth – NEXT-‐GENera*on Epigene*c Biomarkers – Methyla*on Based CDx
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Actionable Epigenome
Outside Oncology ?
Historically, Cancer Was Considered to be Driven Mostly by Gene,c Changes
Example: Replica,on errors
GENETIC
Altered DNA/mRNA/proteins
Altered DNA sequence
X X
Oncogenesis
Tumor
§ Muta*ons in p53
§ Ac*va*ng muta*ons in RAS
§ Muta*ons or amplifica*ons of the
HER-‐2 gene
§ Chromosomal transloca*ons in
myeloid cells and the genera*on of
the BCR-‐ABL fusion protein
Epigene,c Changes are Important in Causing Cancer
Example: Replica,on errors
GENETIC EPIGENETIC
Example: Chroma,n modifica,on errors
Altered DNA/mRNA/proteins
Altered DNA sequence
Altered levels of mRNA/proteins
Altered chroma,n structure
X X
Oncogenesis
Tumor
Source: Schuebel et al 2007
0
20
40
60
80
100
120
Methylated Mutated
76-100 51-75 21-50 1-20
Dx
CDx
Example of Methyla,on vs Muta,on: Colon & Breast Cancer
MGMT Biology O6 Methyl-Guanine Methyl Transferase
Essential DNA Repair Enzyme Removes alkyl groups from damaged guanine bases Healthy individual:
-‐ MGMT is an essen*al DNA repair enzyme Loss of MGMT ac*vity makes individuals suscep*ble to DNA damage and prone to tumor development
Glioblastoma pa,ent on alkylator chemotherapy: -‐ Pa*ents with MGMT promoter methyla*on show have longer PFS and OS with the use of alkyla*ng agents as chemotherapy
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
0
5
10
15
20
25Median Overall Survival
21.7 months
12.7 months
radiotherapy
plus temozolomide
Methylated MGMT Gene
Non-Methylated MGMT Gene
radiotherapy
Adapted from Hegi et al. NEJM 2005 352(10):1036-‐8. Study with 207 pa*ents
Overview Epigene,cs
– Introduc*on – Methyla*on & Oncology – Biomarkers
MDxHealth – NEXT-‐GENera*on Epigene*c Biomarkers
Can we rediscover MGMT ? What does the epigenome look like ?
MBD_Seq
DNA Sheared
Immobilized Methyl Binding Domain
Condensed Chroma*n
DNA Sheared
Immobilized Methyl binding domain
MgCl2
Next Gen Sequencing GA Illumina: 100 million reads
MBD_Seq
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Quality evalua*on of Methyl Binding Domain based kits for enrichment DNA-‐methyla*on sequencing
De Meyer et al (2013) Plos One
MBD_Seq MGMT = dual core
GCATCGTGACTAGCGACTGATCGATGGATGCTAGCAT
Where is the mC ?
GCATCGTGACTAGCGACTGATCGATGGATGCTAGCAT
GCATCGTGACTAGCGACTGATCGATGGATGCTAGCAT
25% 50% 25%
GCATCGTGACTAGCGACTGATCGATGGATGCTAGCAT
GCATCGTGACTAGCGACTGATCGATGGATGCTAGCAT
25% 50% 25%
GCATCGTGACTAGCGACTGATCGATGGATGCTAGCAT
GCATCGTGACTAGCGACTGATCGATGGATGCTAGCAT
Dense methylated needed for transcrip*onal silencing Are there alleles with all three posi4ons methylated ?
GCATCGTGACTTACGACTGATCGATGGATGCTAGCAT!
unmethylated alleles
less methyla*on methylated alleles
more methyla*on
Deep Sequencing
Deep Sequencing MGMT Heterogenic complexity
# samples
# markers
MBD_Seq
BT_Seq
Genome-‐wide methyla,on …. by next genera,on sequencing
Discovery
Verifica*on
Valida*on
Assay Technology: Methyla,on-‐Specific PCR (MSP)
DNA Modification
1
2
MethylCytosine Methyl Cytosine
Uracil
Primer Choice
DNA modification
chemical Cytosine
To design PCR primers to differen*ate methylated from unmethylated we take advantage of the base pair binding proper*es of Uracil
3 amplification occurs when methylation specific primers bind
to the promoter DNA
Amplification and Detection by quantitative
PCR
MSPrimer MSPrimer
Methylated CPG Islands
Ct value
Signal cha
nge pe
r cycle
Assay Technology: Methyla,on-‐Specific PCR (MSP)
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Gene,c tes,ng
107 106 105 104 103 102 101 1 108 109
Full genome bp
Whole-genome Bisulphite seq MSP Probes
(450-27K) Enrichment
Targeted Panels
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Gene,c tes,ng
107 106 105 104 103 102 101 1 108 109
Full genome bp
G E N E T I C
Whole-genome sequencing
Enrichment seq (Exome)
PCR Enrichment Targeted Panels
Instrument and Assay providers
CLIA Lab service providers
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§ An epigene*c assay to help dis*nguish pa*ents who have a true-‐nega*ve biopsy from those who may have occult cancer.
§ Provides urologists with ac*onable informa*on to help:
− RULE OUT prostate-‐cancer-‐free men from undergoing unnecessary repeat biopsies
− RULE IN those who require repeat biopsies and poten4al treatment
Addressing False-‐Nega,ve Biopsy Concerns