This document describes a study that used circulating cell-free DNA (cfDNA) isolated from cerebrospinal fluid (CSF) to diagnose and monitor leptomeningeal disease. The researchers used a targeted sequencing panel to analyze 341 cancer genes in cfDNA from 53 patients. Results from CSF cfDNA analysis were compared to standard CSF cytopathology and MRI findings. CSF cfDNA analysis detected tumor-derived genetic alterations and was more sensitive than cytopathology. The study demonstrates that genomic analysis of CSF cfDNA can help facilitate early diagnosis of central nervous system tumors and guide treatment.

1. Diagnosis
and
monitoring
of
Leptomeningeal
Disease
using
Circula5ng
free
DNA
in
the
cerebrospinal
fluid
(CSF
cfDNA)
R.
H.
Shah1,
E.
I.
Pentsova2,
J.
Tang5,
A.
Boire2,
D.
You5,
S.
Briggs2,
A.
Omuro2,
X.
Lin2,
M.
Fleisher3,
C.
Grommes2,
F.
Meng5,
S.
D.
Selcuklu5,
S.
Ogilvie4,
N.
Distefano4,
L.Shagabayeva2,
M.Rosenblum2,
L.
M.
DeAngelis2,
A.
Viale5,
I.
K.
Mellinghoff2,,
M.
F.
Berger1,5,
1Department
of
Pathology,
Memorial
Sloan
KeTering
Cancer
Center,
New
York,
NY
10065,
USA.2Department
of
Neurology,
Memorial
Sloan
KeTering
Cancer
Center,
New
York,
NY
10065,
USA.
3Department
of
Laboratory
Medicine,
Memorial
Sloan
KeTering
Cancer
Center,
New
York,
NY
10065,
USA.
4Department
of
Neurosurgery;
Memorial
Sloan
KeTering
Cancer
Center,
New
York,
NY
10065,
USA.
5Center
for
Molecular
Oncology,
Memorial
Sloan
KeTering
Cancer
Center,
New
York,
NY
10065,
USA
Background
Conclusion
Leptomeningeal
metastases
(LM)
in
solid
tumors
(ST)
represent
a
devasta]ng
complica]on
of
cancer
with
a
median
survival
of
only
12-­‐14
weeks
a_er
diagnosis
[1];
however,
establishing
the
diagnosis
of
LM
can
be
difficult,
par]cularly
at
early
stages
before
the
pa]ent
is
disabled.
The
diagnosis
is
based
on
CSF
cytologic
analysis
and/or
MRI
findings.[2-­‐4]
Brain
and
spine
MRIs
have
been
increasingly
preferred
for
the
ini]al
evalua]on
of
LM
because
of
their
non-­‐invasive
nature
and
convenience
to
pa]ents.
However,
MRI
findings
are
nega]ve
in
25%-­‐50%
of
pa]ents
[3,
4],
and
unequivocal
findings
may
only
appear
in
late-­‐stage
disease
when
the
pa]ent
is
already
debilitated.
CSF
cytologic
analysis
provides
diagnos]c
confirma]on
of
LM
but
is
associated
with
a
rela]vely
low
sensi]vity
(approximately
50%
on
the
first
lumbar
puncture)
and
is
highly
examiner-­‐dependent.
Improved
diagnos]c
tools
are
required
to
facilitate
early
diagnosis.
To
this
end,
we
explored
whether
sufficient
quan]ty
and
quality
of
DNA
can
be
isolated
from
CSF
for
genomic
study
and
whether
the
CSF
pellet
or
CSF
supernatant,
would
be
more
suitable
for
detec]ng
cfDNA.
We
used
an
in-­‐house
sequencing
assay,
MSK-­‐IMPACT
[5],
to
interrogate
341
clinically
relevant
cancer
genes
in
tumor-­‐
derived
cfDNA
from
53
pa]ents.
Results
of
CSF
cfDNA
were
compared
to
standard
CSF
cytopathologic
analysis
from
that
same
CSF
sample
and
with
MRI
findings
performed
at
the
same
]me.
When
possible,
we
compared
CSF
cfDNA
with
DNA
from
tumor
]ssue
(primary
tumor
and
non-­‐CNS
sites)
to
determine
similari]es
and
differences
in
gene]c
altera]ons
between
these
different
compartments.
Acknowledgements
Introduc5on
Methods
References
Our
study
demonstrates
that
genomic
analysis
of
CSF,
using
a
sufficiently
sensi]ve
and
comprehensive
plaiorm,
may
be
useful
to
facilitate
diagnosis
of
tumor
in
the
CNS,
monitor
the
evolu]on
of
the
cancer
genome
during
treatment
of
CNS
cancers,
guide
the
choice
of
second-­‐line
agents,
and
perhaps
iden]fy
pathways
that
are
uniquely
associated
with
cancer
spread
to
the
central
nervous
system.
Center
of
Molecular
Oncology,
Department
of
Pathology
&
Department
of
Neurology
Targeted
Capture,
Sequencing
&
Genomic
Analysis
Captures
all
protein-­‐coding
exons
of
341
cancer-­‐associated
genes
Sequence
pair-­‐end
reads
(2x100)
on
HiSeq
2500
Analyse
genomic
data
using
methods
described
previously
[5].
Extrac]on
of
cfDNA.
Centrifuged
at
10,000
g
for
30
min
at
4℃
to
remove
residual
precipitated
cellular
components
QIAamp
Circula]ng
Nucleic
Acid
Kit
Cerebrospinal
Fluid
Collec]on
and
Prepara]on.
Lumbar
Puncture
Centrifuged
at
1,000
x
g,
4°C
for
5
min
to
separate
supernatent
&
pellet
Image
1:
Comparison
of
tumor-­‐derived
DNA
from
CSF
cell
pellet
and
supernatant.
(A)
Schema]c
of
separa]on
of
CSF
pellet
and
supernatant.
Cellular
DNA
is
isolated
from
the
pellet,
and
cfDNA
is
isolated
from
the
supernatant.
(B)
Variant
allele
frequencies
for
known
muta]ons
in
CSF
cfDNA
and
pellet
DNA.
(C)
Log2
ra]os
of
normalized
sequence
coverage
for
target
exons
in
CSF-­‐cfDNA
and
pellet
DNA
for
pa]ent
8
.
Greater
than
10-­‐fold
amplifica]on
of
HER2
was
observed
in
CSF-­‐cfDNA,
whereas
HER2
amplifica]on
was
barely
detectable
in
pellet
DNA.
(D)
Evidence
of
EML4-­‐ALK
gene
fusion
in
CSF
cfDNA
and
pellet
DNA
for
pa]ent
6.
Read-­‐pairs
suppor]ng
the
fusion
(red)
are
visualized
using
the
Integra]ve
Genomics
Viewer.
Results
Muta5ons
detected
in
most
pa5ents
with
LM
disease
Image
2:
(A)
Schema]c
showing
paTerns
of
CNS
involvement
in
pa]ents
with
solid
tumors.
(B)
Percentage
of
pa]ents
for
which
high-­‐confidence
soma]c
altera]ons
were
detected
by
MSK-­‐IMPACT.
Pa]ents
are
grouped
according
to
the
presence
or
absence
of
intraparenchymal
brain
metastases
and
leptomeningeal
metastases
(LM)
Drug-­‐resistance
mechanisms
in
CSF
in
pa5ents
with
CNS
relapse
Image
3:
Summary
of
genomic
profiling
results
from
CSF
and
other
tumor
sites
in
pa]ents
who
developed
progressive
CNS
disease
during
treatment
with
the
indicated
kinase
inhibitors.
Tumor
evolu5on
in
pa5ents
with
primary
brain
tumors.
Image
4:
Tumor
evolu5on
in
pa5ents
with
primary
brain
tumors.
(A)
Spa]al
and
temporal
heterogeneity
between
samples
obtained
at
diagnosis,
at
recurrence
and
from
CSF
in
pa]ent
42
with
recurrent
glioblastoma.
CSF
cfDNA
harbors
a
PTEN
R130*
muta]on
(VAF=0.25),
while
resec]on
#2
harbors
a
PIK3CA
H1047R
muta]on
(VAF=0.441).
(B)
CSF
molecular
profile
for
a
pa]ent
45
with
anaplas]c
oligodendroglioma
contains
the
IDH1
R132H
muta]on
and
1p/19q
dele]on
found
in
]ssue
resec]on
#2,
as
well
as
454
non-­‐silent
soma]c
muta]ons.
448
SNVs
represent
C>T/G>A
muta]ons
demonstra]ng
temozolomide-­‐induced
mutagenesis.
A
B
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51-­‐7.
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Cheng,
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et
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Sloan
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of
Ac4onable
Cancer
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A
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Pilot:
CSF
cell
pellet
vs.
CSF
cfDNA