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