The document summarizes a study analyzing a single nucleotide polymorphism (SNP) in the actin filament associated protein (AFAP-110) gene in patients with head and neck cancer. The SNP leads to an amino acid change that enhances activation of c-Src kinase, which promotes tumor growth. The study found that all 50 patients had the polymorphic AFAP-110403C genotype, with 13 patients being heterozygous and 37 being homozygous. Further analysis is underway to determine if the AFAP-110 SNP correlates with tumor measurements, biomarkers of response, and clinical outcomes in patients treated with dasatinib, a Src inhibitor, and erlotinib, an EGFR inhibitor.

1. Background
Future
Direc1ons
`
Acknowledgements
§ The
author
would
like
to
thank
the
following
contributors
for
their
invaluable
help
and
guidance:
§ The
members
of
the
Ferris
lab
§ The
Sobol
Lab
for
allowing
use
of
the
Applied
Biosystems
StepOnePlus
Real-­‐Time
PCR
System
§ Dr.
Michael
Lotze
and
the
UPCI
Summer
Academy
Conclusions
Abstract
u EGFR
§ Epidermal
Growth
Factor
Receptor:
AcKvated
by
the
binding
of
its
specific
ligands
§ Majority
of
SCCHN
overexpress
EGFR,
and
is
associated
with
poorer
clinical
outcomes
§ UpregulaKon
can
lead
to
constant
acKvaKon
and
uncontrolled
cell
division
§ Recent
findings
show
overexpression
in
EGFR,
as
well
as
c-­‐Src
in
SCCHN
§ Results
in
synergisKc
increase
in
EGF-­‐induced
DNA
synthesis/
tumorigenesis
u c-­‐Src
§ non-­‐receptor
protein
tyrosine
kinase
protein
§ Src-­‐family
kinases
(SFKs):
can
regulate
gene
expression
and
affect
cell
adhesion
through
integraKons
with
integrins,
acKns,
and
focal
adhesion
kinases
§ High
expressions
of
the
c-­‐Src
non-­‐receptor
tyrosine
kinase
is
correlated
with
malignancy
progression
of
cancer
§ overexpression
of
c-­‐Src
increases
the
response
of
EGFR-­‐mediated
processes
§ TransacKvaKon
of
EGFR
via
c-­‐Src
promotes
tumorigenesis
and
survival
u AFAP-­‐110
§ AcKn
Filament
Associated
Protein
§ Src
binding
partner:
links
SFKs
to
acKn
filaments,
which
relays
signals
from
PKCα
that
acKvate
c-­‐Src
leading
to
alteraKons
in
acKn
filaments
(Figure
1)
§ SNP
in
exon
9
(C1210G)
leads
to
change
in
amino
acid
403
from
Serine
to
Cysteine
(S403C)
§ In
study
of
ovarian
cancer,
AFAP-­‐110403C
acKvated
c-­‐Src
and
increased
invasiveness
§ Sequence:
§ CAGCCGGAACGTCAGAGGATGTTTA[C/G]AATCCAAACCCGGGATCACCTCGCA
Results
Materials
and
Methods
Demographic
Data
Introduc1on:
Squamous
Cell
Carcinoma
of
the
Head
and
Neck
(SCCHN)
is
the
sixth
most
common
cancer
worldwide,
with
a
median
survival
of
6-­‐9
months
for
paKents
with
recurrent
or
metastaKc
disease.
The
majority
of
SCCHN
overexpress
epidermal
growth
factor
receptor
(EGFR),
and
EGFR-­‐targeted
therapies
have
shown
effecKveness
in
a
subset
of
paKents.
To
improve
clinical
responses,
studies
are
underway
that
combine
anK-­‐EGFR
therapies
with
therapeuKc
agents
targeKng
other
molecules
in
the
EGFR
signaling
pathway.
One
potenKal
target
is
c-­‐Src,
a
nonreceptor
protein
tyrosine
kinase
downstream
of
EGFR.
AcKvaKon
of
c-­‐Src
promotes
tumor
survival,
proliferaKon,
invasion,
and
angiogenesis.
Recently,
a
mulKcenter
clinical
trial
combining
erloKnib,
an
EGFR
inhibitor,
with
dasaKnib,
a
Src
inhibitor,
completed
accrual.
We
analyzed
genomic
DNA
from
this
cohort
for
a
single
nucleoKde
polymorphism
(SNP)
within
the
acKn
filament
associated
protein
(AFAP-­‐110),
an
adaptor
protein
that
relays
signals
through
c-­‐Src,
promoKng
alteraKons
in
the
acKn
cytoskeleton.
Previous
studies
idenKfied
a
polymorphic
variant,
AFAP-­‐110403C,
that
intrinsically
enhances
c-­‐Src
acKvaKon.
Results:
The
AFAP-­‐110403C
genotype
was
present
in
all
paKents,
where
13/50
were
heterozygous
SC,
and
37/50
were
homozygous
CC.
Further
study
is
underway,
including
correlaKon
of
AFAP-­‐110
SNP
genotype
with
tumor
measurements,
tumor
biomarkers
of
response
such
as
Src
acKvaKon,
proliferaKon,
and
apoptosis,
as
well
as
clinical
outcome.
Clinical
Trial
Is
there
a
Correla1on
between
a
Single
Nucleo1de
Polymorphism
in
AFAP-­‐110
and
Dasa1nib
Response
in
Head
and
Neck
Cancer
Pa1ents?
Rebekah
L.
Madrid1,
Sandra
P.
Gibson3,4,
David
A.
Clump5,
Robert
L.
Ferris2,3,4
,
Jennifer
R.
Grandis2,3,4
University
of
Pifsburgh
Cancer
InsKtute
Summer
Academy1;
Departments
of
Immunology2,
Otolaryngology3,
University
of
Pifsburgh;
University
of
Pifsburgh
Cancer
InsKtute4;
Department
of
RadiaKon
Oncology,
University
of
Pifsburgh
School
of
Medicine5,
Pifsburgh,
PA
Arms
No.
of
Pa<ents
Tumor
Site
Mean
Age
(yrs.)
Males
Females
Smoking
Alcohol
Blue
9
OC
4
60.1
9
0
8
6
OP
2
L
2
HP
1
O
0
UNK
PRIM
0
Red
3
OC
2
62.6
1
2
3
2
OP
0
L
1
HP
0
O
0
UNK
PRIM
0
Yellow
7
OC
6
64.6
5
2
5
6
OP
1
L
0
HP
0
O
0
UNK
PRIM
0
Green
8
OC
6
45.25
6
2
7
6
OP
2
L
0
HP
0
O
0
UNK
PRIM
0
PKCα
DAG
Ca++
PMA
AFAP-­‐11
0
Src
AcKn
PKCα
DAG
Ca++
AFAP-­‐11
0
AFAP-­‐11
0
AFAP-­‐11
0
Src
Altera1ons
in
ac1n
filament
integrity
1
2
3
4
5
Genotype
SCCHN
Pa1ents
Healthy
Donors
SS
0/50
(0%)
0/9
(0%)
SC
13/50
(26%)
2/9
(22%)
CC
37/50
(74%)
7/9
(78%)
Figure
1
SchemaKc
showing
the
interacKng
of
AFAP-­‐110
with
c-­‐Src
and
acKn.
Figure
2
UPCI
07-­‐124,
a
“Comparison
of
Biomarker
ModulaKon
by
InhibiKon
of
EGFR
and/or
Src
Family
Kinases
using
ErloKnib
and
DasaKnib
in
Head
and
Neck
and
Lung
Cancers,”
was
a
4-­‐arm
randomized
trial
looking
at
the
effects
of
short-­‐term
preoperaKve
therapy
with
EGFR
(erloKnib)
and
Src
(dasaKnib)
inhibitors.
Prior
to
surgery,
paKents
either
received
placebo,
erloKnib,
erloKnib+dasaKnib,
or
placebo+
dasaKnib
for
14
–
21
days
prior
to
surgery.
Figure
3
The
TaqMan
SNP
Genotyping
assay
is
a
variaKon
of
the
polymerase
chain
reacKon
(PCR).
PCR
is
an
innovaKve
way
of
amplifying
a
single
piece
of
DNA
and
has
3
disKnct
steps:
i) Denatura1on-­‐
separates
double
stranded
DNA
into
2
strands
ii) Annealing-­‐
targeKng
the
sequence
by
using
primers,
which
mark
the
ends
of
the
target
sequence
iii) Extension-­‐
begins
the
synthesis
process
at
the
region
previously
marked
by
the
primers;
synthesizes
new
double
stranded
DNA
molecules
(both
idenKcal
to
the
original
double
stranded
target
DNA
region)
The
SNP
genotyping
assay
requires
several
components:
" Primers
w Forward
and
reverse
primers
for
amplifying
region
of
AFAP-­‐110
containing
the
SNP
" Probes
w VIC
dye
is
linked
to
the
5’
end
of
the
Allele
1
probe
w FAM
dye
is
linked
to
the
5’
end
of
the
Allele
2
probe
w Nonfluorescent
Quencher
(NFQ)
quenches
dye
signals
unKl
DNA
polymerase
cleaves
the
probe
to
generate
dye
signal
" Genotyping
Master
Mix
w Contains
essenKal
reagents,
including
AmpliTaq
Gold
DNA
polymerase
to
catalyze
PCR
reacKon
u DNA
extrac1on
" DNA
was
extracted
from
50
clinical
trial
peripheral
blood
mononuclear
cells
(PBMC)
samples
using
QIAGEN
DNeasy
Blood
and
Tissue
kit
" DNA
previously
extracted
from
9
healthy
donor
PBMC
samples
(Central
Blood
Bank)
was
also
tested
" BioTek
Epoch
Plate
Reader
was
used
to
read
DNA
concentraKon
of
each
sample
u SNP
Genotyping
" TaqMan
SNP
Genotyping
assay
for
AFAP-­‐110
S403C,
Assay
ID:
C_25623820_10
" Water
was
used
as
a
no
template
control
(NTC)
to
rule
out
cross
contaminaKon
" Aser
loading
96-­‐well
plate,
samples
were
run
on
an
MJ
Research
PTC-­‐100
thermal
cycler
u Analysis
" Applied
Biosystems
StepOnePlus
Real-­‐Time
PCR
System
was
used
for
reading
the
plate
" StepOne
Sosware
v2.1
and
TaqMan
Genotyper
sosware
was
used
to
generate
allelic
discriminaKon
plots
Figure
4
Allelic
discriminaKon
plot
of
AFAP-­‐110
S403C
in
SCCHN
and
healthy
donors
DNA
samples.
Dark
blue
dots
represent
homozygous
allele
2
(CC)
and
green
dots
represent
heterozygous
allele
1/2
(SC).
No
red
dots
(homozygous
allele
1
(SS)
were
observed.
Figure
5
Summary
of
AFAP-­‐110
S403C
genotyping
results
in
SCCHN
paKents
and
healthy
donors.
Similar
frequencies
were
observed
in
each
group.
Figure
6
Demographic
informaKon
on
27
of
the
50
SCCHN
paKents.
AbbreviaKons
for
tumor
sites:
OC=Oral
Cavity;
OP=Oropharynx;
L=Larynx;
HP=Hypopharynx;
O=Other;
UNK
PRIM=
Unknown
Primary.
u The
AFAP-­‐110403C
polymorphism
is
present
in
50/50
paKents
(homozygous
CC
or
heterozygous
SC)
u Similar
results
were
obtained
with
healthy
donors
u More
samples
needed
to
determine
SNP
frequency
in
SCCHN
as
well
as
healthy
populaKon
u Validate
results
by
repeaKng
AFAP-­‐110
S403C
genotyping
in
these
samples
u Genotype
more
healthy
PBMC
Samples
and
SCCHN
paKents
to
determine
frequency
of
SNP
in
these
populaKons
u Correlate
SNP
genotype
with
results
of
the
UPCI
07-­‐124
trial
(in
progress):
§ Tumor
measurements
pre-­‐
and
post-­‐treatment
§ Tumor
biomarkers
of
response
(phospho-­‐Src),
proliferaKon
(Ki67),
and
apoptosis
(TUNEL
assay)
§ Clinical
Outcome
`
References
§ Clump,
D.
A.,
Yu,
J.
J.,
&
Cho,
Y.
(2010).
A
polymorphic
variant
of
AFAP-­‐110
enhances
c-­‐Src
AcKvity.
TranslaKonal
Oncology,
3.4,
276-­‐285.
§ Egloff,
A.
M.,
Grandis,
J.
R.,
&
,
(2009).
Improving
Response
Rates
to
EGFR-­‐Targeted
Therapies
for
Head
and
Neck
Squamous
Cell
Carcinomas.
Journal
of
Oncology,
1-­‐11.
§ Kim,
L.
C.,
Song
,
L.,
&
Haura,
E.
B.
(2009).
Src
Kinases
as
TherapeuKc
Targets
for
Cancer.
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Myers,
J.
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&
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M.
(2008).
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in
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and
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AcKvaKon
With
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Cadherin
Down-­‐regulaKon,
VimenKn
Expression,
and
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112.9,
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CC
SC
SS