Desmetilació genòmica com a biomarcador de predisposició a càncers múltiples
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Colorectal
cancer
(CRC)
is
one
of
the
most
frequent
cancers
in
human
populations,
particularaly
in
the
western
countries.
Both
genetic
and
epigenetic
alterations
are
germain
to
CRC
develpoment
and
progression.
In
a
previous
collaborative
work
conducted
in
Japanese
population,
it
was
reported
that
global
genomic
methylation
of
the
normal
colonic
mucosa
(NCM)
of
CRC
patients
was
predictive
of
of
the
risk
to
develop
multiple
colonic
cancers,
both
synchrounous
and
metachronous.
In
that
work,
global
methylation
was
measured
on
the
LINE--‐1
repetitive
elements,
which
constitute
about
17%--‐20%
of
the
human
genome
and
are
frequently
employed
as
surrogate
marker
of
global
methylation
levels.
This
sequences
are
mayoritarily
methylated
in
virtually
all
somatic
adult
healthy
tissues,
including
the
colonic
epithelial
cells.
However,
cancer
cells
have
been
known
for
a
long
time
to
exhibit
global
genomic
hypomethylaton
which
affects
also
LINE--‐1
elements.
LINE--‐1
methylation
was
analyzed
by
MethyLight,
a
QPCR--‐
based
method
capable
of
quantify
the
relative
proportion
of
hypomethylated
molecules.
While
the
hypomethylation
in
cancer
cells
was
known,
the
strong
association
of
genomic
demethylation
in
NCM
with
multiple
cancer
risk
was
novel,
and
suggested
the
existence
of
an
epigenetic
field
for
cancerization,
i.e.
a
region
of
normal
colonic
mucosa
that
harbours
epigenetic
alterations
preceding
and
promoting
tumour
development.
That
analysis,
however,
could
not
test
the
extension
of
that
epigentic
field
because
only
one
normal
sample
was
analyzed
in
every
patient
and,
in
most
cases,
the
distance
between
the
normal
biopsy
to
the
tumor
location
was
unreported.
Moreover,
both
the
normal
and
the
tumor
samples
consist
of
different
cell
types
(cancer
cells,
epithelial
cells,
muscle
layer)
with
different
epigenomic
profiles.
This
work
was
aimed
to
explore
the
extent
of
DNA
hypomethylation
using
FFPE
samples
from
multiple--‐CRC
patients
from
the
Hospital
Germans
Trias
i
Pujol
in
Catalonia,
and
frozen
samples
from
single--‐CRC
from
the
CHTN
in
USA.
FFPE
samples
were
microdissected
to
facilitate
ba
more
precise
determination
of
their
cellular
content.
In
addition,
in
this
work
two
normal
samples
taken
from
differnet
locations
of
the
colon,
plus
non--‐cancer
cells
directly
adjacent
to
the
tumor
samples
have
been
analyzed.
We
employed
a
real--‐time
methylation--‐specific
PCR
(MS--‐QPCR)
technique
based
on
that
used
in
the
previously
published
work.
The
technique
has
been
slighty
modified
to
reduce
the
cost
of
the
analysis,
by
substituting
the
specific
fluorescent
probes
by
SYBR--‐Green
for
the
quantification
of
the
amplified
products,
and
including
a
melting--‐curve
analysis
to
determine
the
differences
between
amplicons.
Our
results
demonstrate
the
suitability
of
the
expermental
approach
to
analyze
global
genomic
hypomethylation
in
FFPE
samples.
Tumor
cells
exhibited
significant
lower
levels
of
methylation
than
their
non--‐tumoral
matching
samples,
in
agreement
with
previous
observations.
We
also
observed
a
high
degree
of
coordination
of
the
demethylation
level
in
normal
samples
from
the
same
individual,
suggesting
that
the
demethylation
is
homogeneous
along
the
colon.
This
coordination
might
facilitate
the
estimation
of
the
global
genomic
demethylation
level
in
the
whole
colonic
tract
from
a
single
biopsy