This document discusses next generation epigenetic profiling and biomarkers. It provides an overview of epigenetics and methylation in oncology. MDxHealth is developing next-generation epigenetic biomarkers including methylation-based companion diagnostics. Deep sequencing techniques like MBD-Seq are being used to discover biomarkers from genome-wide methylation profiling and provide high resolution of methylation heterogeneity. Validation studies of biomarkers like MGMT promoter methylation are also discussed.
6. 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
7. 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
8. 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
9. 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
10. 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
25
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
2005
352(10):1036-‐8.
Study
with
207
pa*ents
11. 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
?
12. MBD_Seq
DNA
Sheared
Immobilized
Methyl
Binding
Domain
Condensed
Chroma*n
DNA
Sheared
13. Immobilized
Methyl
binding
domain
MgCl2
Next
Gen
Sequencing
GA
Illumina:
100
million
reads
MBD_Seq
19. GCATCGTGACTAGCGACTGATCGATGGATGCTAGCAT
25%
50%
25%
GCATCGTGACTAGCGACTGATCGATGGATGCTAGCAT
GCATCGTGACTAGCGACTGATCGATGGATGCTAGCAT
Dense
methylated
needed
for
transcrip*onal
silencing
Are
there
alleles
with
all
three
posi4ons
methylated
?
22. #
samples
#
markers
MBD_Seq
BT_Seq
Genome-‐wide
methyla,on
….
by
next
genera,on
sequencing
Discovery
Verifica*on
Valida*on
23. Assay
Technology:
Methyla,on-‐
Specific
PCR
(MSP)
DNA
Modification
1
2
Methyl
Cytosine 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
24. 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
change
per
cycle
Assay
Technology:
Methyla,on-‐
Specific
PCR
(MSP)