Van criekinge next_generation_epigenetic_profling_vlille

Next Generation Epigenetic Profiling

Wim Van Criekinge10th octobre 2013, Lille (FR)

OverviewEpigenetics

– Introduction– Methylation & Oncology– Biomarkers

MDxHealth–NEXT-GENeration Epigenetic Biomarkers–PharmacoDX: Methylation Based Biomarkers

for Predictive and Prognostic Use

ActionableEpigenome

OutsideOncology ?

Historically, Cancer Was Considered to be Driven Mostly by Genetic Changes

Example: Replication errors

GENETIC

Altered DNA/mRNA/proteins

Altered DNA sequence

X X

Oncogenesis

Tumor

Mutations in p53

Activating mutations in RAS

Mutations or amplifications of the

HER-2 gene

Chromosomal translocations in

myeloid cells and the generation of

the BCR-ABL fusion protein

Epigenetic Changes are Important in Causing Cancer

Example: Replication errors

GENETIC EPIGENETIC

Example: Chromatin modification errors

Altered DNA/mRNA/proteins

Altered DNA sequence

Altered levels ofmRNA/proteins

Alteredchromatin structure

X X

Oncogenesis

Tumor

Source: Schuebel et al 2007

SYNE1

APC2

GPNMB

MM

P2EVL

STARD8

PTPRDCD109

LGR6

RETCHD%

RNF182

ICAM5

0

20

40

60

80

100

120

Methylated Mutated

76-100 51-75 21-50 1-20

Dx

CDx

Example of Methylation vs Mutation: Colon & Breast Cancer

MGMT BiologyO6 Methyl-Guanine Methyl Transferase

Essential DNA Repair Enzyme

Removes alkyl groups from damaged guanine bases

Healthy individual: - MGMT is an essential DNA repair enzymeLoss 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

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

Median 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 2005352(10):1036-8.Study with 207 patients

OverviewEpigenetics

– Introduction– Methylation & Oncology– Biomarkers

MDxHealth–NEXT-GENeration Epigenetic Biomarkers

Can we rediscover MGMT ? What does the epigenome look like ?….

MBD_Seq

DNA Sheared

Immobilized Methyl Binding Domain

Condensed Chromatin

DNA Sheared

Immobilized Methyl binding domain

MgCl2

Next Gen SequencingGA Illumina: 100 million reads

MBD_Seq

Confidential Information | ©2013 MDxHealth Inc. All rights reserved.

Quality evaluation of Methyl Binding Domain based kits for enrichment DNA-methylation sequencing

De Meyer et al (2013) Plos One


Quality evaluation of Methyl Binding Domain based kits for enrichment DNA-methylation sequencing

MBD_SeqMGMT = dual core

# samples

# markers

MBD_Seq

Genome-wide methylation …. by next generation sequencing

Discovery

1-2 millionmethylation

cores

BRCA1,a bidirectional promoter

18

19

Splice variants

Zooming into Exon 1

20

Zooming into Exon 1

21

Zooming into Exon 1

22

GCATCGTGACTAGCGACTGATCGATGGATGCTAGCAT

Where is the mC ?



25% 50% 25%



25% 50% 25%



Dense methylated needed for transcriptional silencingAre there alleles with all three positions methylated ?

GCATCGTGACTTACGACTGATCGATGGATGCTAGCAT

unmethylated alleles

less methylationmethylated alleles

more methylation

Deep Sequencing

Deep Sequencing MGMT Heterogenic complexity

Data integration with

DEEP Sequencing, Infinium, Reactivation, (directional) Expression …

Data integrationCorrelation tracks

methylation methylation

expression expression

Corr =-1 Corr = 1

Correlation trackin GBM @ MGMT

+1

-1

BRCA1

32

1 T47D2 BT5493 MCF74 MDAMB2315 HS578T6 NORMAL7 NORMAL8 NORMAL

9 BI T21 (BRCA1 MUT)10 BI T22 (BRCA1 MUT)11 BLC12 BLC13 BLC14 BLC15 BLC16 BLC17 BLC

13 BLC14 BLC15 BLC16 BLC17 BLC18 BLC

19 IVM20 DKO

# samples

# markers

MBD_Seq

BT_Seq

MSP

Genome-wide methylation …. by next generation sequencing

Discovery

Verification

Validation


Genetics

107 106 105 104 103 102 101 1108109

Full genome bp

Whole-genomeBisulphite seq

MSPProbes

(450-27K)Enrichment

Targeted Panels


Genetics

107 106 105 104 103 102 101 1108109

Full genome bp

GENETIC

Whole-genomesequencing

Enrichment seq(Exome)

PCREnrichment

Targeted Panels

Instrument and Assay providers

CLIA Lab service providers

Sequencing

DeepSeq

Molecular Unification

107 106 105 104 103 102 101 1108109

Full genome bp


RUO Clinical

EPI

GENETIC


Enrichment seq(MBD, RRBS)


Probes(450-27K)

Enrichment Targeted Panels


UltraDeep


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.


PubMeth.org Reviewed methylation database in cancer

Epigenetic Alterations Associated with Cancer Therapy Response

PCFT – folate transport (MTX)

WRN


440 cancer-related genes genes are known to be epigenetically altered in human solid cancers based on recent scientific and clinical literature.

Sequencing

DeepSeq

Molecular Unification

107 106 105 104 103 102 101 1108109

Full genome bp


RUO Clinical

EPI

GENETIC


Enrichment seq(MBD, RRBS)


Probes(450-27K)



UltraDeep

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Acknowledgments

Geert TrooskensSimon DenilKlaas MensaertJean-Pierre RenardSarah De KeulenaerEllen De MeesterTim De Meyer

Olivier Thas

Wim van den Berghe

Manon Van Engeland Jurgen Veeck

Monika HegiPierre BadySebastian Kurscheid

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biobixwvcrieki

biobix.bebioinformatics.be

http://www.bioinformatics.be/



Education

Van criekinge next_generation_epigenetic_profling_vlille