PREDICCIÓN DE RESPUESTA AL TRATAMIENTO EN...
36
UNIVERSIDAD DE BARCELONA FACULTAD DE FARMACIA DEPARTAMENTO BIOQUIMICA Y BIOLOGIA MOLECULAR PREDICCIÓN DE RESPUESTA AL TRATAMIENTO EN PACIENTES CON CARCINOMA ESCAMOSO DE CABEZA Y CUELLO MIGUEL ANGEL PAVÓN RIBAS 2009
Transcript of PREDICCIÓN DE RESPUESTA AL TRATAMIENTO EN...
UNIVERSIDAD DE BARCELONA
FACULTAD DE FARMACIA
DEPARTAMENTO BIOQUIMICA Y BIOLOGIA MOLECULAR
PREDICCIÓN DE RESPUESTA AL TRATAMIENTO EN PACIENTES CON
CARCINOMA ESCAMOSO DE CABEZA Y CUELLO
MIGUEL ANGEL PAVÓN RIBAS 2009
IX. ANEXOS
IX ANEXOS ANEXO 1. Genes expresados diferencialmente al comparar los tumores del cluster 1 con los tumores del cluster 2 y 3
ANEXO 2. Genes expresados diferencialmente al comparar los tumores del cluster 3 con los tumores del cluster 1 y 2
ANEXO 3. Genes expresados diferencialmente al comparar los tumores del cluster 2 con los tumores del cluster 1 y 3
ANEXO 4. Genes expresados diferencialmente en función de la rediviva local a los 2 años ANEXO 5. Genes expresados diferencialmente en función de la supervivencia a 3 años
ANEXO 6. Genes expresados diferencialmente en función de la respuesta a la quimioterapia de inducción
ANEXO 7. Genes expresados diferencialmente tras comparar los niveles de expresión de los tumores respecto a las mucosas normales ANEXO 8. Publicación de los resultados obtenidos en el estudio de los niveles de expresión de los genes del sistema NHEJ en biopsias pre-tratamiento de pacientes con carcinoma escamosos de cabeza y cuello localmente avanzado tratados con quimioterapia de inducción (Int J Cancer. 2008 Sep 1;123(5):1068-79)
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AN
EX
O 1
: Gen
es e
xpre
sado
s di
fere
ncia
lmen
te a
l com
para
r lo
s tu
mor
es d
el c
lust
er 1
con
los
tum
ores
del
clu
ster
2 y
3 (
p a
just
ada<
0,05
)
Prob
eSy
mbo
lD
escr
ipti
onC
ytob
and
t-st
atis
tic
p-va
lue
adjp
2026
27_s
_at
SER
PIN
E1
serp
in p
eptid
ase
inhi
bito
r, cl
ade
E (n
exin
, pla
smin
ogen
act
ivat
or in
hibi
tor t
ype
1),
mem
ber 1
7q21
.3-q
226.
8453
41e
-04
9e-0
4
2011
09_s
_at
TH
BS1
thro
mbo
spon
din
115
q15
6.77
397
1e-0
49e
-04
2105
11_s
_at
INH
BA
inhi
bin,
bet
a A
7p15
-p13
6.76
322
1e-0
49e
-04
2124
73_s
_at
MIC
AL
2m
icro
tubu
le a
ssoc
iate
d m
onox
ygen
ase,
cal
poni
n an
d LI
M d
omai
n co
ntai
ning
211
p15.
36.
5396
61e
-04
0.00
1722
1471
_at
SER
INC
3se
rine
inco
rpor
ator
320
q13.
1-q1
3.3
6.48
186
1e-0
40.
0023
2026
20_s
_at
PLO
D2
proc
olla
gen-
lysi
ne, 2
-oxo
glut
arat
e 5-
diox
ygen
ase
23q
23-q
246.
4366
11e
-04
0.00
2620
1105
_at
LG
AL
S1le
ctin
, gal
acto
side
-bin
ding
, sol
uble
, 1 (g
alec
tin 1
)22
q13.
16.
3553
91e
-04
0.00
3520
2949
_s_a
tFH
L2
four
and
a h
alf L
IM d
omai
ns 2
2q12
-q14
6.20
728
1e-0
40.
0061
2119
45_s
_at
ITG
B1
inte
grin
, bet
a 1
(fib
rone
ctin
rece
ptor
, bet
a po
lype
ptid
e, a
ntig
en C
D29
incl
udes
MD
F2,
MSK
12)
10p1
1.2
6.16
237
1e-0
40.
0074
2011
08_s
_at
TH
BS1
thro
mbo
spon
din
115
q15
6.10
825
1e-0
40.
0087
2038
89_a
tSC
G5
secr
etog
rani
n V
(7B
2 pr
otei
n)15
q13-
q14
6.09
843
1e-0
40.
0089
2026
19_s
_at
PLO
D2
proc
olla
gen-
lysi
ne, 2
-oxo
glut
arat
e 5-
diox
ygen
ase
23q
23-q
246.
0384
21e
-04
0.01
02
2075
43_s
_at
P4H
A1
proc
olla
gen-
prol
ine,
2-o
xogl
utar
ate
4-di
oxyg
enas
e (p
rolin
e 4-
hydr
oxyl
ase)
, alp
hapo
lype
ptid
e I
10q2
1.3-
q23.
15.
9989
92e
-04
0.01
14
2196
55_a
tC
7orf
10ch
rom
osom
e 7
open
read
ing
fram
e 10
7p14
.15.
9905
51e
-04
0.01
1720
3735
_x_a
tP
PF
IBP
1PT
PRF
inte
ract
ing
prot
ein,
bin
ding
pro
tein
1 (l
iprin
bet
a 1)
12p1
1.23
-p11
.22
5.87
329
1e-0
40.
0175
2222
35_s
_at
GA
LN
AC
T-2
chon
droi
tin su
lfate
Gal
NA
cT-2
10q1
1.21
5.85
546
1e-0
40.
0182
2041
40_a
tT
PST
1ty
rosy
lpro
tein
sulfo
trans
fera
se 1
7q11
.21
5.82
066
1e-0
40.
0197
2183
68_s
_at
TN
FR
SF12
Atu
mor
nec
rosi
s fac
tor r
ecep
tor s
uper
fam
ily, m
embe
r 12A
16p1
3.3
5.79
667
1e-0
40.
0212
2007
55_s
_at
CA
LU
calu
men
in7q
325.
7953
91e
-04
0.02
1221
2472
_at
MIC
AL
2m
icro
tubu
le a
ssoc
iate
d m
onox
ygen
ase,
cal
poni
n an
d LI
M d
omai
n co
ntai
ning
211
p15.
35.
7233
71e
-04
0.02
5320
6026
_s_a
tT
NF
AIP
6tu
mor
nec
rosi
s fac
tor,
alph
a-in
duce
d pr
otei
n 6
2q23
.35.
7205
71e
-04
0.02
5620
3736
_s_a
tP
PF
IBP
1PT
PRF
inte
ract
ing
prot
ein,
bin
ding
pro
tein
1 (l
iprin
bet
a 1)
12p1
1.23
-p11
.22
5.64
557
1e-0
40.
0335
2120
97_a
tC
AV
1ca
veol
in 1
, cav
eola
e pr
otei
n, 2
2kD
a7q
31.1
5.62
559
1e-0
40.
036
2186
18_s
_at
FN
DC
3Bfib
rone
ctin
type
III d
omai
n co
ntai
ning
3B
3q26
.31
5.62
314
1e-0
40.
0361
mpavon
183
2030
65_s
_at
CA
V1
cave
olin
1, c
aveo
lae
prot
ein,
22k
Da
7q31
.15.
6083
91e
-04
0.03
7520
1110
_s_a
tT
HB
S1th
rom
bosp
ondi
n 1
15q1
55.
6055
41e
-04
0.03
7721
4845
_s_a
tC
AL
Uca
lum
enin
7q32
5.56
538
1e-0
40.
0425
2099
35_a
tA
TP2
C1
ATP
ase,
Ca+
+ tra
nspo
rting
, typ
e 2C
, mem
ber 1
3q22
.15.
5318
81e
-04
0.04
6920
2395
_at
NSF
N-e
thyl
mal
eim
ide-
sens
itive
fact
or17
q21
-5.5
2875
1e-0
40.
047
2039
14_x
_at
HP
GD
hydr
oxyp
rost
agla
ndin
deh
ydro
gena
se 1
5-(N
AD
)4q
34-q
35-5
.584
651e
-04
0.04
0521
3236
_at
SASH
1SA
M a
nd S
H3
dom
ain
cont
aini
ng 1
6q24
.3-5
.588
21e
-04
0.03
9837
201_
atIT
IH4
inte
r-al
pha
(glo
bulin
) inh
ibito
r H4
(pla
sma
Kal
likre
in-s
ensi
tive
glyc
opro
tein
)3p
21-p
14-5
.612
571e
-04
0.03
721
9727
_at
DU
OX
2du
al o
xida
se 2
15q1
5.3
-5.7
9467
1e-0
40.
0212
2093
89_x
_at
DB
Idi
azep
am b
indi
ng in
hibi
tor (
GA
BA
rece
ptor
mod
ulat
or, a
cyl-C
oenz
yme
A b
indi
ngpr
otei
n)2q
12-q
21-5
.829
481e
-04
0.01
91
2181
86_a
tR
AB
25R
AB
25, m
embe
r RA
S on
coge
ne fa
mily
1q22
-5.8
4865
1e-0
40.
0183
2200
17_x
_at
CY
P2C
9cy
toch
rom
e P4
50, f
amily
2, s
ubfa
mily
C, p
olyp
eptid
e 9
10q2
4-5
.891
951e
-04
0.01
6620
6004
_at
TG
M3
trans
glut
amin
ase
3 (E
pol
ypep
tide,
pro
tein
-glu
tam
ine-
gam
ma-
glut
amyl
trans
fera
se)
20q1
1.2
-5.9
2236
1e-0
40.
0154
4164
4_at
SASH
1SA
M a
nd S
H3
dom
ain
cont
aini
ng 1
6q24
.3-5
.933
581e
-04
0.01
4320
7935
_s_a
tK
RT
13ke
ratin
13
17q1
2-q2
1.2
-6.0
2933
1e-0
40.
0107
2139
29_a
t-6
.121
361e
-04
0.00
83
2110
70_x
_at
DB
Idi
azep
am b
indi
ng in
hibi
tor (
GA
BA
rece
ptor
mod
ulat
or, a
cyl-C
oenz
yme
A b
indi
ngpr
otei
n)2q
12-q
21-6
.462
661e
-04
0.00
25
2024
28_x
_at
DB
Idi
azep
am b
indi
ng in
hibi
tor (
GA
BA
rece
ptor
mod
ulat
or, a
cyl-C
oenz
yme
A b
indi
ngpr
otei
n)2q
12-q
21-6
.503
241e
-04
0.00
2
2053
79_a
tC
BR
3ca
rbon
yl re
duct
ase
321
q22.
2-6
.549
641e
-04
0.00
1720
8126
_s_a
tC
YP
2C18
cyto
chro
me
P450
, fam
ily 2
, sub
fam
ily C
, pol
ypep
tide
1810
q24
-6.7
3238
1e-0
40.
0011
2128
41_s
_at
PP
FIB
P2
PTPR
F in
tera
ctin
g pr
otei
n, b
indi
ng p
rote
in 2
(lip
rin b
eta
2)11
p15.
4-6
.804
131e
-04
9e-0
421
5103
_at
CY
P2C
18cy
toch
rom
e P4
50, f
amily
2, s
ubfa
mily
C, p
olyp
eptid
e 18
10q2
4-7
.606
741e
-04
1e-0
421
4070
_s_a
tA
TP
10B
ATP
ase,
Cla
ss V
, typ
e 10
B5q
34-7
.63
1e-0
41e
-04
mpavon
mpavon
184
AN
EX
O 2
: Gen
es e
xpre
sado
s di
fere
ncia
lmen
te a
l com
para
r lo
s tu
mor
es d
el c
lust
er 3
con
los
tum
ores
del
clu
ster
1 y
2 (
p aj
usta
da<0
,05
)
Prob
eSy
mbo
lD
escr
ipti
onC
ytob
and
t-st
atis
tic
p-va
lue
adjp
2115
97_s
_at
HO
Pho
meo
dom
ain-
only
pro
tein
4q11
-q12
8.57
654
1e-0
41e
-04
2049
52_a
tL
YPD
3LY
6/PL
AU
R d
omai
n co
ntai
ning
319
q13.
318.
4423
51e
-04
1e-0
421
4536
_at
SLU
RP
1se
cret
ed L
Y6/
PLA
UR
dom
ain
cont
aini
ng 1
8q24
.38.
3415
31e
-04
1e-0
420
5185
_at
SPIN
K5
serin
e pe
ptid
ase
inhi
bito
r, K
azal
type
55q
327.
8147
51e
-04
1e-0
422
0620
_at
CR
CT
1cy
stei
ne-r
ich
C-te
rmin
al 1
1q21
7.69
724
1e-0
41e
-04
2061
25_s
_at
KL
K8
kalli
krei
n-re
late
d pe
ptid
ase
819
q13.
3-q1
3.4
7.41
616
1e-0
41e
-04
2195
29_a
tC
LIC
3ch
lorid
e in
trace
llula
r cha
nnel
39q
34.3
7.32
736
1e-0
41e
-04
2204
13_a
tSL
C39
A2
solu
te c
arrie
r fam
ily 3
9 (z
inc
trans
porte
r), m
embe
r 214
q11.
27.
0241
81e
-04
2e-0
420
3747
_at
AQ
P3
aqua
porin
3 (G
ill b
lood
gro
up)
9p13
6.49
846
1e-0
46e
-04
2049
71_a
tC
STA
cyst
atin
A (s
tefi
n A
)3q
216.
4276
71e
-04
0.00
121
9554
_at
RH
CG
Rh
fam
ily, C
gly
copr
otei
n15
q25
6.42
418
1e-0
40.
0011
3924
8_at
AQ
P3
aqua
porin
3 (G
ill b
lood
gro
up)
9p13
6.24
541e
-04
0.00
23
2197
22_s
_at
GD
PD3
glyc
erop
hosp
hodi
este
r pho
spho
dies
tera
se d
omai
nco
ntai
ning
316
p11.
26.
2016
81e
-04
0.00
29
2126
57_s
_at
IL1R
Nin
terle
ukin
1 re
cept
or a
ntag
onis
t2q
14.2
6.19
893
1e-0
40.
0029
2057
78_a
tK
LK
7ka
llikr
ein-
rela
ted
pept
idas
e 7
19q1
3.33
6.17
859
1e-0
40.
0031
3924
9_at
AQ
P3
aqua
porin
3 (G
ill b
lood
gro
up)
9p13
6.12
927
1e-0
40.
004
2057
59_s
_at
SUL
T2B
1su
lfotra
nsfe
rase
fam
ily, c
ytos
olic
, 2B
, mem
ber 1
19q1
3.3
6.07
189
1e-0
40.
0048
2195
97_s
_at
DU
OX
1du
al o
xida
se 1
15q1
5.3
6.04
634
1e-0
40.
0051
2068
84_s
_at
SCE
Lsc
ielli
n13
q22
5.94
222
1e-0
40.
0082
2076
02_a
tT
MP
RSS
11D
trans
mem
bran
e pr
otea
se, s
erin
e 11
D4q
13.2
5.92
51e
-04
0.00
8722
2223
_s_a
tIL
1F5
inte
rleuk
in 1
fam
ily, m
embe
r 5 (d
elta
)2q
145.
8776
1e-0
40.
0120
3021
_at
SLP
Ise
cret
ory
leuk
ocyt
e pe
ptid
ase
inhi
bito
r20
q12
5.85
279
1e-0
40.
0112
2034
07_a
tP
PL
perip
laki
n16
p13.
35.
8515
91e
-04
0.01
1221
4599
_at
IVL
invo
lucr
in1q
215.
8476
11e
-04
0.01
12
2060
08_a
tT
GM
1tra
nsgl
utam
inas
e 1
(K p
olyp
eptid
e ep
ider
mal
type
I,pr
otei
n-gl
utam
ine-
gam
ma-
glut
amyl
trans
fera
se)
14q1
1.2
5.81
622
1e-0
40.
0122
2012
01_a
tC
STB
cyst
atin
B (s
tefin
B)
21q2
2.3
5.77
212
1e-0
40.
0132
2148
38_a
tSF
T2D
2SF
T2 d
omai
n co
ntai
ning
21q
24.2
5.74
449
1e-0
40.
0148
mpavon
185
2216
65_s
_at
EPS
8L1
EPS8
-like
119
q13.
425.
7257
21e
-04
0.01
5721
0397
_at
DE
FB
1de
fens
in, b
eta
18p
23.2
-p23
.15.
7131
81e
-04
0.01
6220
5470
_s_a
tK
LK
11ka
llikr
ein-
rela
ted
pept
idas
e 11
19q1
3.3-
q13.
45.
7040
61e
-04
0.01
726
6_s_
atC
D24
CD
24 m
olec
ule
6q21
5.69
613
1e-0
40.
0174
2216
67_s
_at
HSP
B8
heat
shoc
k 22
kDa
prot
ein
812
q24.
235.
6486
41e
-04
0.02
1420
8650
_s_a
tC
D24
CD
24 m
olec
ule
6q21
5.63
916
1e-0
40.
0222
2064
00_a
tL
GA
LS7
lect
in, g
alac
tosi
de-b
indi
ng, s
olub
le, 7
(gal
ectin
7)
19q1
3.2
5.62
061e
-04
0.02
3420
1939
_at
PLK
2po
lo-li
ke k
inas
e 2
(Dro
soph
ila)
5q12
.1-q
13.2
5.61
335
1e-0
40.
0243
2188
53_s
_at
MO
SPD
1m
otile
sper
m d
omai
n co
ntai
ning
1X
q26.
35.
5937
71e
-04
0.02
5520
5627
_at
CD
Acy
tidin
e de
amin
ase
1p36
.2-p
355.
5419
61e
-04
0.03
0120
9792
_s_a
tK
LK
10ka
llikr
ein-
rela
ted
pept
idas
e 10
19q1
3.3-
q13.
45.
5186
21e
-04
0.03
2321
0128
_s_a
tL
TB
4Rle
ukot
riene
B4
rece
ptor
14q1
1.2-
q12
5.51
518
1e-0
40.
0326
2047
33_a
tK
LK
6ka
llikr
ein-
rela
ted
pept
idas
e 6
19q1
3.3
5.51
391e
-04
0.03
2722
0782
_x_a
tK
LK
12ka
llikr
ein-
rela
ted
pept
idas
e 12
19q1
3.3-
q13.
45.
5120
91e
-04
0.03
2920
5863
_at
S100
A12
S100
cal
cium
bin
ding
pro
tein
A12
1q21
5.51
139
1e-0
40.
0329
2145
49_x
_at
SPR
R1A
smal
l pro
line-
rich
prot
ein
1A1q
21-q
225.
5101
61e
-04
0.03
2922
2242
_s_a
tK
LK
5ka
llikr
ein-
rela
ted
pept
idas
e 5
19q1
3.3-
q13.
45.
5089
1e-0
40.
0331
2098
00_a
tK
RT
16ke
ratin
16
(foc
al n
on-e
pide
rmol
ytic
pal
mop
lant
arke
rato
derm
a)17
q12-
q21
5.50
785
1e-0
40.
0332
2204
12_x
_at
KC
NK
7po
tass
ium
cha
nnel
, sub
fam
ily K
, mem
ber 7
11q1
35.
4610
91e
-04
0.03
7220
1454
_s_a
tN
PE
PP
Sam
inop
eptid
ase
puro
myc
in se
nsiti
ve17
q21
5.45
814
1e-0
40.
0373
2053
63_a
tB
BO
X1
buty
robe
tain
e (g
amm
a), 2
-oxo
glut
arat
e di
oxyg
enas
e(g
amm
a-bu
tyro
beta
ine
hydr
oxyl
ase)
111
p14.
25.
4556
51e
-04
0.03
73
2066
05_a
tP
1126
serin
e pr
otea
se12
q13.
15.
4324
61e
-04
0.04
0621
8779
_x_a
tE
PS8L
1EP
S8-li
ke 1
19q1
3.42
5.42
746
1e-0
40.
0414
2057
83_a
tK
LK
13ka
llikr
ein-
rela
ted
pept
idas
e 13
19q1
3.3-
q13.
45.
4188
71e
-04
0.04
2920
3535
_at
S100
A9
S100
cal
cium
bin
ding
pro
tein
A9
1q21
5.39
759
1e-0
40.
0455
2061
99_a
tC
EA
CA
M7
carc
inoe
mbr
yoni
c an
tigen
-rel
ated
cel
l adh
esio
nm
olec
ule
719
q13.
25.
3896
11e
-04
0.04
64
2136
80_a
tK
RT
6Cke
ratin
6C
12q1
3.13
5.38
696
1e-0
40.
0465
2181
86_a
tR
AB
25R
AB
25, m
embe
r RA
S on
coge
ne fa
mily
1q22
5.37
569
1e-0
40.
0485
9182
6_at
EPS
8L1
EPS8
-like
119
q13.
425.
3675
1e-0
40.
049
mpavon
186
AN
EX
O 3
: Gen
es e
xpre
sado
s di
fere
ncia
lmen
te a
l com
para
r lo
s tu
mor
es d
el c
lust
er 2
con
los
tum
ores
del
clu
ster
1 y
3 (
p a
just
ada<
0,05
)
Prob
eSy
mbo
lD
escr
ipti
onC
ytob
and
t-st
atis
tic
p-va
lue
adjp
2016
50_a
tK
RT
19ke
ratin
19
17q2
1.2
6.65
757
1e-0
42e
-04
2184
40_a
tM
CC
C1
met
hylc
roto
noyl
-Coe
nzym
e A
carb
oxyl
ase
1 (a
lpha
)3q
276.
0847
91e
-04
0.00
1520
9205
_s_a
tL
MO
4LI
M d
omai
n on
ly 4
1p22
.36.
0346
31e
-04
0.00
221
8546
_at
C1o
rf11
5ch
rom
osom
e 1
open
read
ing
fram
e 11
51q
415.
7837
41e
-04
0.00
6220
1908
_at
DV
L3
dish
evel
led,
dsh
hom
olog
3 (D
roso
phila
)3q
275.
3920
31e
-04
0.02
620
4734
_at
KR
T15
kera
tin 1
517
q21.
25.
1976
21e
-04
0.04
99
2086
36_a
tA
CT
N1
actin
in, a
lpha
114
q24.
1-q2
4.2
14q2
4 14
q22-
q24
-5.2
4627
1e-0
40.
043
2088
98_a
tA
TP
6V1D
ATP
ase,
H+
tran
spor
ting,
lyso
som
al 3
4kD
a, V
1 su
buni
t D14
q23-
q24.
2-5
.257
641e
-04
0.04
120
2693
_s_a
tST
K17
Ase
rine/
thre
onin
e ki
nase
17a
7p12
-p14
-5.4
0438
1e-0
40.
0251
2032
34_a
tU
PP
1ur
idin
e ph
osph
oryl
ase
17p
12.3
-5.4
3455
1e-0
40.
0233
2009
85_s
_at
CD
59C
D59
mol
ecul
e, c
ompl
emen
t reg
ulat
ory
prot
ein
11p1
3-5
.510
341e
-04
0.01
8620
8899
_x_a
tA
TP
6V1D
ATP
ase,
H+
tran
spor
ting,
lyso
som
al 3
4kD
a, V
1 su
buni
t D14
q23-
q24.
2-5
.543
911e
-04
0.01
6320
5627
_at
CD
Acy
tidin
e de
amin
ase
1p36
.2-p
35-5
.563
291e
-04
0.01
5121
9165
_at
PDL
IM2
PDZ
and
LIM
dom
ain
2 (m
ystiq
ue)
8p21
.2-5
.565
71e
-04
0.01
5
2158
13_s
_at
PT
GS1
pros
tagl
andi
n-en
dope
roxi
de sy
ntha
se 1
(pro
stag
land
in G
/Hsy
ntha
se a
nd c
yclo
oxyg
enas
e)9q
32-q
33.3
-5.6
5713
1e-0
40.
0104
2178
73_a
tC
AB
39ca
lciu
m b
indi
ng p
rote
in 3
92q
37.1
-5.6
6127
1e-0
40.
0103
2057
67_a
tE
RE
Gep
iregu
lin4q
13.3
-5.6
8622
1e-0
40.
0091
2099
46_a
tV
EG
FCva
scul
ar e
ndot
helia
l gro
wth
fact
or C
4q34
.1-q
34.3
-5.7
6699
1e-0
40.
007
2186
44_a
tPL
EK
2pl
ecks
trin
214
q23.
3-5
.805
51e
-04
0.00
5520
2949
_s_a
tFH
L2
four
and
a h
alf L
IM d
omai
ns 2
2q12
-q14
-6.0
2845
1e-0
40.
0021
2101
38_a
tR
GS2
0re
gula
tor o
f G-p
rote
in si
gnal
ing
208q
12.1
-6.3
7452
1e-0
44e
-04
2019
39_a
tPL
K2
polo
-like
kin
ase
2 (D
roso
phila
)5q
12.1
-q13
.2-6
.490
471e
-04
4e-0
4
mpavon
187
AN
EX
O 4
: G
enes
exp
resa
dos
dife
renc
ialm
ente
en
func
ión
de la
rec
idiv
a lo
cal a
los
2 añ
os (p
<0,0
01)
Prob
eSy
mbo
lD
escr
ipti
onC
ytob
and
t-st
atis
tic
p-va
lue
adjp
2179
93_s
_at
MA
T2B
met
hion
ine
aden
osyl
trans
fera
se II
, bet
a5q
34-q
35.1
4.57
716
2e-0
40.
4089
2007
48_s
_at
FTH
1fe
rriti
n, h
eavy
pol
ypep
tide
111
q13
4.36
626
3e-0
40.
5913
2178
65_a
tR
NF
130
ring
finge
r pro
tein
130
5q35
.34.
2445
63e
-04
0.70
0520
7277
_at
CD
209
CD
209
mol
ecul
e19
p13
4.22
255e
-04
0.71
7621
9318
_x_a
tM
ED
31m
edia
tor c
ompl
ex su
buni
t 31
17p1
3.2
4.13
949
2e-0
40.
7866
2061
29_s
_at
AR
SBar
ylsu
lfata
se B
5q11
-q13
4.13
633
7e-0
40.
7885
2092
87_s
_at
CD
C42
EP
3C
DC
42 e
ffec
tor p
rote
in (R
ho G
TPas
e bi
ndin
g) 3
2p21
4.06
079
4e-0
40.
8417
3798
6_at
EP
OR
eryt
hrop
oiet
in re
cept
or19
p13.
3-p1
3.2
4.04
038
4e-0
40.
8536
2089
64_s
_at
FA
DS1
fatty
aci
d de
satu
rase
111
q12.
2-q1
3.1
3.93
975
1e-0
40.
9101
2092
88_s
_at
CD
C42
EP
3C
DC
42 e
ffec
tor p
rote
in (R
ho G
TPas
e bi
ndin
g) 3
2p21
3.91
379e
-04
0.92
4221
4211
_at
FTH
1fe
rriti
n, h
eavy
pol
ypep
tide
111
q13
3.91
065
3e-0
40.
9249
2083
54_s
_at
SLC
12A
3so
lute
car
rier f
amily
12
(sodi
um/c
hlor
ide
trans
porte
rs),
mem
ber 3
16q1
33.
9089
43e
-04
0.92
56
2188
52_a
tP
PP
2R3C
prot
ein
phos
phat
ase
2 (f
orm
erly
2A
), re
gula
tory
subu
nit
B'',
gam
ma
14q1
3.2
3.89
281
4e-0
40.
9308
2026
23_a
tE
AP
PE2
F-as
soci
ated
pho
spho
prot
ein
14q1
3.1
3.89
032
6e-0
40.
9313
2041
85_x
_at
PPID
pept
idyl
prol
yl is
omer
ase
D (c
yclo
phili
n D
)4q
31.3
3.88
913
4e-0
40.
9318
2028
92_a
tC
DC
23ce
ll di
visi
on c
ycle
23
hom
olog
(S. c
erev
isia
e)5q
313.
8614
54e
-04
0.94
1820
4140
_at
TPS
T1
tyro
sylp
rote
in su
lfotra
nsfe
rase
17q
11.2
13.
8470
43e
-04
0.94
5720
0013
_at
RP
L24
ribos
omal
pro
tein
L24
3q12
3.83
201
9e-0
40.
9511
2013
30_a
tR
AR
Sar
giny
l-tR
NA
synt
heta
se5q
35.1
3.81
124
3e-0
40.
9584
2043
49_a
tM
ED
7m
edia
tor c
ompl
ex su
buni
t 75q
33.3
3.80
747e
-04
0.95
92
2081
98_x
_at
KIR
2DS1
kille
r cel
l im
mun
oglo
bulin
-like
rece
ptor
, tw
o do
mai
ns,
shor
t cyt
opla
smic
tail,
119
q13.
43.
7956
48e
-04
0.96
24
2090
46_s
_at
GA
BA
RA
PL
2G
AB
A(A
) rec
epto
r-as
soci
ated
pro
tein
-like
216
q22.
3-q2
4.1
3.79
255
5e-0
40.
9629
2007
30_s
_at
PTP4
A1
prot
ein
tyro
sine
pho
spha
tase
type
IVA
, mem
ber 1
6q12
3.77
523
8e-0
40.
9669
2015
03_a
tG
3BP
1G
TPas
e ac
tivat
ing
prot
ein
(SH
3 do
mai
n) b
indi
ng p
rote
in 1
5q33
.13.
7294
79e
-04
0.97
8320
6307
_s_a
tFO
XD
1fo
rkhe
ad b
ox D
15q
12-q
133.
7057
66e
-04
0.98
1422
1597
_s_a
tH
SPC
171
HSP
C17
1 pr
otei
n16
q22.
13.
7056
7e-0
40.
9814
2183
39_a
tM
RP
L22
mito
chon
dria
l rib
osom
al p
rote
in L
225q
33.1
-q33
.33.
6819
88e
-04
0.98
48
mpavon
188
2204
16_a
tA
TP
8B4
ATP
ase,
Cla
ss I,
type
8B
, mem
ber 4
15q2
1.2
3.67
825
4e-0
40.
985
2012
72_a
tA
KR
1B1
aldo
-ket
o re
duct
ase
fam
ily 1
, mem
ber B
1 (a
ldos
ere
duct
ase)
7q35
3.65
533
7e-0
40.
9874
2091
81_s
_at
RA
BG
GT
BR
ab g
eran
ylge
rany
ltran
sfer
ase,
bet
a su
buni
t1p
313.
6481
34e
-04
0.98
8321
0278
_s_a
tA
P4S
1ad
apto
r-re
late
d pr
otei
n co
mpl
ex 4
, sig
ma
1 su
buni
t14
q12
3.60
072
9e-0
40.
993
2214
23_s
_at
YIP
F5Y
ip1
dom
ain
fam
ily, m
embe
r 55q
323.
5815
19e
-04
0.99
46
2116
71_s
_at
NR
3C1
nucl
ear r
ecep
tor s
ubfa
mily
3, g
roup
C, m
embe
r 1(g
luco
corti
coid
rece
ptor
)5q
31.3
3.55
595
8e-0
40.
996
2203
32_a
tC
LD
N16
clau
din
163q
283.
5502
58e
-04
0.99
6520
4191
_at
IFN
AR
1in
terf
eron
(alp
ha, b
eta
and
omeg
a) re
cept
or 1
21q2
2.1
21q2
2.11
3.51
619
5e-0
40.
9976
2175
85_a
tN
EB
Lne
bule
tte10
p12
-3.0
8968
9e-0
41
2215
07_a
tT
NPO
2tra
nspo
rtin
2 (im
porti
n 3,
kar
yoph
erin
bet
a 2b
)19
p13.
13-3
.256
338e
-04
0.99
9921
5063
_x_a
tL
RR
C40
leuc
ine
rich
repe
at c
onta
inin
g 40
1p31
.1-3
.481
829e
-04
0.99
82
2154
04_x
_at
FGFR
1fib
robl
ast g
row
th fa
ctor
rece
ptor
1 (
fms-
rela
ted
tyro
sine
kina
se 2
, Pfe
iffer
synd
rom
e)8p
11.2
-p11
.1-3
.537
477e
-04
0.99
69
2089
65_s
_at
PYH
IN1
pyrin
and
HIN
dom
ain
fam
ily, m
embe
r 11q
23.1
-3.6
141
3e-0
40.
9922
2027
25_a
tP
OL
R2A
poly
mer
ase
(RN
A) I
I (D
NA
dire
cted
) pol
ypep
tide
A,
220k
Da
17p1
3.1
-3.6
203
8e-0
40.
9912
2145
94_x
_at
AT
P8B
1A
TPas
e, C
lass
I, ty
pe 8
B, m
embe
r 118
q21-
q22
18q2
1.31
-3.6
3359
9e-0
40.
9898
2035
46_a
tIP
O13
impo
rtin
131p
34.1
-3.6
4354
7e-0
40.
9886
2104
21_s
_at
SLC
24A
1so
lute
car
rier f
amily
24
(sodi
um/p
otas
sium
/cal
cium
exch
ange
r), m
embe
r 115
q22
-3.6
651
3e-0
40.
9862
2207
20_x
_at
FA
M12
8Bfa
mily
with
sequ
ence
sim
ilarit
y 12
8, m
embe
r B2q
21.1
-3.6
8598
9e-0
40.
9841
2215
06_s
_at
TN
PO2
trans
porti
n 2
(impo
rtin
3, k
aryo
pher
in b
eta
2b)
19p1
3.13
-3.7
0103
4e-0
40.
9818
2122
80_x
_at
AT
G4B
ATG
4 au
toph
agy
rela
ted
4 ho
mol
og B
(S. c
erev
isia
e)2q
37.3
-3.7
0414
9e-0
40.
9814
2206
59_s
_at
C7o
rf43
chro
mos
ome
7 op
en re
adin
g fr
ame
437q
22.1
-3.7
2321
9e-0
40.
9796
2210
71_a
t-3
.753
313e
-04
0.97
1921
5455
_at
TIM
EL
ESS
timel
ess h
omol
og (D
roso
phila
)12
q12-
q13
-3.9
2562
3e-0
40.
9181
2120
51_a
tW
IPF2
WA
S/W
ASL
inte
ract
ing
prot
ein
fam
ily, m
embe
r 217
q21.
2-3
.941
793e
-04
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buni
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7822
2159
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2pl
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85e
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0.85
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se E
3 co
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nent
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0.85
21
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189
2024
24_a
tM
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gen-
activ
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pro
tein
kin
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825
2042
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thre
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nase
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96
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759
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3875
93e
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2041
60_s
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quen
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40.
9011
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736
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40.
9432
2137
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3.75
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gam
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742
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41
2132
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cyst
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8745
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40.
9999
2042
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0.99
9920
2115
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tN
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98
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40.
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0.99
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40.
9994
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0.99
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9949
2138
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inas
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0.99
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2019
79_s
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phos
phat
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nit
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40.
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41_a
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221
1002
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5555
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989
2034
31_s
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6820
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0.97
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2121
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6e-0
40.
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0.94
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7821
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40.
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2190
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8145
2032
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40.
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2134
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0.77
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7701
2192
78_a
tM
AP3
K6
mito
gen-
activ
ated
pro
tein
kin
ase k
inas
e ki
nase
61p
36.1
1-4
.443
873e
-04
0.74
7221
6641
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tL
AD
1la
dini
n 1
1q25
.1-q
32.3
-4.4
851
4e-0
40.
7155
5899
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CC
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coile
d-co
il an
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mai
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-04
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_at
SMA
RC
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NF
rela
ted,
mat
rix a
ssoc
iate
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ctin
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ende
ntre
gula
tor o
f chr
omat
in, s
ubfa
mily
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embe
r 419
p13.
2-4
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543e
-04
0.68
06
2157
92_s
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(Hsp
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omol
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embe
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8501
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6402
2125
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SMA
RC
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SWI/S
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ted,
mat
rix a
ssoc
iate
d, a
ctin
dep
ende
ntre
gula
tor o
f chr
omat
in, s
ubfa
mily
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r 419
p13.
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194e
-04
0.62
56
2107
91_s
_at
RIC
SR
ho G
TPas
e-ac
tivat
ing
prot
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11q2
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332e
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0.61
35
2137
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_at
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A11
guan
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eotid
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ndin
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tein
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pha
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q cl
ass)
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531
2e-0
40.
5862
2034
11_s
_at
LM
NA
lam
in A
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-04
0.58
3122
1506
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tT
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rtin
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porti
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kar
yoph
erin
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a 2b
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p13.
13-4
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982e
-04
0.54
1522
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_at
TN
PO2
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porti
n 2
(impo
rtin
3, k
aryo
pher
in b
eta
2b)
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3.13
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6858
1e-0
40.
4975
2119
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tU
BR
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iqui
tin p
rote
in li
gase
E3
com
pone
nt n
-rec
ogni
n 4
1p36
.13
-4.7
9467
1e-0
40.
4773
2006
01_a
tA
CT
N4
actin
in, a
lpha
419
q13
-5.9
9771
1e-0
40.
0346
2179
03_a
tST
RN
4st
riatin
, cal
mod
ulin
bin
ding
pro
tein
419
q13.
2-6
.019
861e
-04
0.03
3
mpavon
194
AN
EX
O 6
: Gen
es e
xpre
sado
s di
fere
ncia
lmen
te e
n fu
nció
n de
la r
espu
esta
a la
qui
mio
tera
pia
de in
ducc
ión
( p<
0,00
1 )
Prob
eSy
mbo
lD
escr
ipti
onC
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and
t-st
atis
tic
p-va
lue
adjp
2020
65_s
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A1
prot
ein
tyro
sine
pho
spha
tase
, rec
epto
r typ
e, f
poly
pept
ide
(PTP
RF)
, int
erac
ting
prot
ein
(lip
rin),
alph
a 1
11q1
3.3
4.37
776
4e-0
40.
5335
2169
03_s
_at
CB
AR
A1
calc
ium
bin
ding
ato
py-r
elat
ed a
utoa
ntig
en 1
10q2
2.1
3.90
132
5e-0
40.
9114
2112
62_a
tPC
SK6
prop
rote
in c
onve
rtase
subt
ilisi
n/ke
xin
type
615
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33.
6870
63e
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0.98
2121
62_a
tK
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kina
se D
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ract
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243.
6632
19e
-04
0.98
3821
7921
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MA
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lpha
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ss 1
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embe
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133.
2617
39e
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1
2019
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CT
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ase
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all p
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2216
3e-0
40.
8979
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195
AN
EX
O 7
: Gen
es e
xpre
sado
s di
fere
ncia
lmen
te tr
as c
ompa
rar
los
nive
les
de e
xpre
sión
en
los
tum
ores
res
pect
o a
las
muc
osas
nor
mal
es( p
aju
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a <
0,05
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obe
Sym
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Des
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fect
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ster
chr
omat
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15.1
934
1e-0
48e
-04
2068
58_s
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HO
XC
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meo
box
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12q1
3.3
14.4
493
1e-0
40.
0017
2044
75_a
tM
MP
1m
atrix
met
allo
pept
idas
e 1
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erst
itial
col
lage
nase
)11
q22.
313
.701
91e
-04
0.00
48
2107
76_x
_at
TC
F3tra
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iptio
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2A im
mun
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r bin
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fact
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014
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40.
0068
2039
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Npo
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ease
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p13
12.1
953
1e-0
40.
0182
2140
39_s
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LA
PT
M4B
lyso
som
al a
ssoc
iate
d pr
otei
n tra
nsm
embr
ane
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ta8q
22.1
12.0
178
1e-0
40.
021
2194
93_a
tSH
CB
P1
SHC
SH
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mai
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ndin
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1.2
12.0
057
1e-0
40.
021
2105
11_s
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INH
BA
inhi
bin,
bet
a A
7p15
-p13
11.8
133
1e-0
40.
0254
2137
30_x
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TC
F3tra
nscr
iptio
n fa
ctor
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2A im
mun
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bulin
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fact
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19p1
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11.7
075
1e-0
40.
0277
2054
83_s
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ISG
15IS
G15
ubi
quiti
n-lik
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odifi
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311
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21e
-04
0.02
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AT
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tain
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_at
IFI6
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rfer
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lpha
-indu
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e pr
otei
n 6
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11.6
833
1e-0
40.
0282
2038
56_a
tV
RK
1va
ccin
ia re
late
d ki
nase
114
q32
11.5
896
1e-0
40.
0313
2085
46_x
_at
HIS
T1H
2BH
hist
one
clus
ter 1
, H2b
h6p
21.3
11.5
361e
-04
0.03
3120
4033
_at
TR
IP13
thyr
oid
horm
one
rece
ptor
inte
ract
or 1
35p
15.3
311
.404
81e
-04
0.03
7520
4197
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tR
UN
X3
runt
-rel
ated
tran
scrip
tion
fact
or 3
1p36
11.3
576
1e-0
40.
0387
2137
54_s
_at
PA
IP1
poly
(A) b
indi
ng p
rote
in in
tera
ctin
g pr
otei
n 1
5p12
11.2
728
1e-0
40.
0431
2035
14_a
tM
AP3
K3
mito
gen-
activ
ated
pro
tein
kin
ase k
inas
e ki
nase
317
q23.
3-1
1.11
731e
-04
0.04
9920
7935
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tK
RT
13ke
ratin
13
17q1
2-q2
1.2
-11.
194
1e-0
40.
0467
2114
65_x
_at
FUT
6fu
cosy
ltran
sfer
ase
6 (a
lpha
(1,3
) fuc
osyl
trans
fera
se)
19p1
3.3
-11.
2168
1e-0
40.
0455
2103
98_x
_at
FUT
6fu
cosy
ltran
sfer
ase
6 (a
lpha
(1,3
) fuc
osyl
trans
fera
se)
19p1
3.3
-11.
2419
1e-0
40.
0443
2023
13_a
tP
PP
2R2A
prot
ein
phos
phat
ase
2 (f
orm
erly
2A
), re
gula
tory
subu
nit B
, alp
hais
ofor
m8p
21.2
-11.
2567
1e-0
40.
0438
2044
84_a
tP
IK3C
2Bph
osph
oino
sitid
e-3-
kina
se, c
lass
2, b
eta
poly
pept
ide
1q32
-11.
5077
1e-0
40.
0339
2134
51_x
_at
TN
XB
tena
scin
XB
6p21
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1.55
141e
-04
0.03
2321
5288
_at
LO
C65
0465
sim
ilar t
o Sh
ort t
rans
ient
rece
ptor
pot
entia
l cha
nnel
2 (T
rpC
2)-1
1.70
32e
-04
0.02
78
mpavon
196
(mTr
p2)
2138
95_a
tE
MP1
epith
elia
l mem
bran
e pr
otei
n 1
12p1
2.3
-11.
8069
1e-0
40.
0255
2158
00_a
tD
UO
X1
dual
oxi
dase
115
q15.
3-1
2.00
361e
-04
0.02
121
0609
_s_a
tT
P53I
3tu
mor
pro
tein
p53
indu
cibl
e pr
otei
n 3
2p23
.3-1
2.02
11e
-04
0.02
0720
8126
_s_a
tC
YP
2C18
cyto
chro
me
P450
, fam
ily 2
, sub
fam
ily C
, pol
ypep
tide
1810
q24
-12.
1425
1e-0
40.
0191
2094
98_a
tC
EA
CA
M1
carc
inoe
mbr
yoni
c an
tigen
-rel
ated
cel
l adh
esio
n m
olec
ule
1(b
iliar
y gl
ycop
rote
in)
19q1
3.2
-12.
148
1e-0
40.
0189
2101
55_a
tM
YO
Cm
yoci
lin, t
rabe
cula
r mes
hwor
k in
duci
ble
gluc
ocor
ticoi
d re
spon
se1q
23-q
24-1
2.20
111e
-04
0.01
8220
8116
_s_a
tM
AN
1A1
man
nosi
dase
, alp
ha, c
lass
1A
, mem
ber 1
6q22
-12.
2206
1e-0
40.
018
2118
82_x
_at
FUT
6fu
cosy
ltran
sfer
ase
6 (a
lpha
(1,3
) fuc
osyl
trans
fera
se)
19p1
3.3
-12.
2423
1e-0
40.
0176
2144
21_x
_at
CY
P2C
9cy
toch
rom
e P4
50, f
amily
2, s
ubfa
mily
C, p
olyp
eptid
e 9
10q2
4-1
2.26
921e
-04
0.01
721
2230
_at
PP
AP
2Bph
osph
atid
ic a
cid
phos
phat
ase
type
2B
1pte
r-p2
2.1
-12.
2907
1e-0
40.
0165
2134
78_a
tK
IAA
1026
kazr
in1p
36.2
1-1
2.29
921e
-04
0.01
6421
8035
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tF
LJ2
0273
RN
A-b
indi
ng p
rote
in4p
13-p
12-1
2.65
951e
-04
0.01
1621
5103
_at
CY
P2C
18cy
toch
rom
e P4
50, f
amily
2, s
ubfa
mily
C, p
olyp
eptid
e 18
10q2
4-1
2.75
571e
-04
0.01
07
2036
58_a
tSL
C25
A20
solu
te c
arrie
r fam
ily 2
5 (c
arni
tine/
acyl
carn
itine
tran
sloc
ase)
,m
embe
r 20
3p21
.31
-12.
9394
1e-0
40.
0089
2163
33_x
_at
TN
XB
tena
scin
XB
6p21
.3-1
2.95
891e
-04
0.00
8722
0026
_at
CL
CA
4ch
lorid
e ch
anne
l, ca
lciu
m a
ctiv
ated
, fam
ily m
embe
r 41p
31-p
22-1
3.03
071e
-04
0.00
83
2142
35_a
tC
YP
3A5P
2cy
toch
rom
e P4
50, f
amily
3, s
ubfa
mily
A, p
olyp
eptid
e 5
pseu
doge
ne 2
7q21
.3-q
22.1
-13.
0555
1e-0
40.
0082
2131
72_a
tT
TC
9te
tratri
cope
ptid
e re
peat
dom
ain
914
q24.
2-1
3.18
911e
-04
0.00
7220
9763
_at
CH
RD
L1
chor
din-
like
1X
q22.
3-1
3.74
191e
-04
0.00
4320
4829
_s_a
tFO
LR
2fo
late
rece
ptor
2 (f
etal
)11
q13.
3-q1
3.5
-13.
7673
1e-0
40.
0042
2083
35_s
_at
DA
RC
Duf
fy b
lood
gro
up, c
hem
okin
e re
cept
or1q
21-q
22-1
3.95
061e
-04
0.00
33
2096
87_a
tC
XC
L12
chem
okin
e (C
-X-C
mot
if) li
gand
12
(stro
mal
cel
l-der
ived
fact
or1)
10q1
1.1
-14.
0868
1e-0
40.
0028
2014
12_a
tL
RP
10lo
w d
ensi
ty li
popr
otei
n re
cept
or-r
elat
ed p
rote
in 1
014
q11.
2-1
4.18
491e
-04
0.00
2621
7846
_at
QA
RS
glut
amin
yl-tR
NA
syn
thet
ase
3p21
.3-p
21.1
-15.
1385
1e-0
48e
-04
2094
87_a
tR
BPM
SR
NA
bin
ding
pro
tein
with
mul
tiple
splic
ing
8p12
-p11
-15.
5269
1e-0
46e
-04
2094
88_s
_at
RB
PMS
RN
A b
indi
ng p
rote
in w
ith m
ultip
le sp
licin
g8p
12-p
11-1
5.78
71e
-04
6e-0
4
mpavon
197
2052
00_a
tC
LE
C3B
C-ty
pe le
ctin
dom
ain
fam
ily 3
, mem
ber B
3p22
-p21
.3-2
3.08
841e
-04
1e-0
421
9747
_at
C4o
rf31
chro
mos
ome
4 op
en re
adin
g fr
ame
314q
27-2
4.12
491e
-04
1e-0
4
mpavon
198
ANEXO 8. Los resultados obtenidos en el estudio de los niveles de expresión de losgenes del sistema NHEJ en biopsias pre-tratamiento de pacientes con carcinomaescamoso de cabeza y cuello localmente avanzado tratados con quimioterapia deinducción han sido publicados en la revista “International Journal of Cancer”.
“Ku70 predicts response and primary tumor recurrence after therapyin locally advanced head and neck cancer”
Miguel Angel Pavón, Matilde Parreño, Xavier León, Francesc J. Sancho, MariaVirtudes Céspedes, Isolda Casanova, Antonio Lopez-Pousa, Maria Antonia Mangues,Miquel Quer, Agustí Barnadas and Ramón Mangues.
Int J Cancer. 2008 Sep 1;123(5):1068-79.
mpavon
199
Ku70 predicts response and primary tumor recurrence after therapy
in locally advanced head and neck cancer
Miguel Angel Pav�on1, Matilde Parre~no1*, Xavier Le�on2, Francesc J. Sancho3, Maria Virtudes C�espedes1, Isolda Casanova1,Antonio Lopez-Pousa4, Maria Antonia Mangues5, Miquel Quer2, Agust�ı Barnadas4 and Ram�on Mangues1*
1Grup d’Oncogenesi i Antitumorals (GOA), Networking Research Center on Bioengineering,Biomaterials and Nanomedicine (CIBER-BBN) and Institut de Recerca, Hospital de la Santa Creu i Sant Pau,Barcelona, Spain2Department of Otorhinolaryngology, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain3Department of Pathology, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain4Department of Medical Oncology, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain5Department of Pharmacy, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain
5-Fluorouracil and cisplatin-based induction chemotherapy (IC) iscommonly used to treat locally advanced head and neck squamouscell carcinoma (HNSCC). The role of nonhomologous end joining(NHEJ) genes (Ku70, Ku80 and DNA-PKcs) in double-strandbreak (DSB) repair, genomic instability and apoptosis suggest apossible impact on tumor response to radiotherapy, 5-fluorouracilor cisplatin, as these agents are direct or indirect inductors ofDSBs. We evaluated the relationship between Ku80, Ku70 or DNAPKcs mRNA expression in pretreatment tumor biopsies, andtumor response to IC or local recurrence, in 50 patients withHNSCC. Additionally, in an independent cohort of 75 patientswith HNSCC, we evaluated the relationship between tumor Ku70protein expression and the same clinical outcomes or patient sur-vival. Tumors in the responder group had significantly highermRNA levels for Ku70, Ku80 and DNA-PKcs than those in thenonresponder group. Ku70 mRNA was the marker most signifi-cantly associated with response to IC. Moreover, high tumorKu70 mRNA expression was associated with significantly longerlocal recurrence-free survival (LRFS). Ku70 protein expressionwas also significantly related to response, and patients with higherpercentage of tumor cells expressing Ku70 had longer LRFS. Inaddition, the percentage of Ku70 positive cells, tumor localizationand node involvement were significantly associated with overallsurvival of patient. Therefore, Ku70 expression is a candidate pre-dictive marker that could distinguish patients who are likely tobenefit from chemoradiotherapy or radiotherapy after the induc-tion chemotherapy treatment, suggesting a contribution of theNHEJ system in HNSCC clinical outcome.' 2008 Wiley-Liss, Inc.
Key words: Ku70; Ku80; DNA-PKcs; NHEJ; predictive marker;HNSCC; induction chemotherapy
Therapy for locally advanced head and neck squamous cell car-cinoma (HNSCC) aims at organ preservation, having displacedradical surgery. Concomitant platinum-based chemoradiotherapy(CRT) has become the standard treatment for this disease.1 5-Flu-orouracil (5-FU) and cisplatin-based induction chemotherapy (IC),followed by CRT or radiotherapy (RT), has also been used to treatlocally advanced HNSCCs, after demonstrating a benefit for organpreservation, loco-regional control and overall survival.2,3
Tumor response to IC predicts response to RT and patient sur-vival in locally advanced HNSCC.4–6 The IC response distin-guishes sensitive carcinomas, which will follow a conservativetreatment, from nonsensitive carcinomas, which will be treatedwith surgery. Conservative treatment consists of the administra-tion of CRT (formerly RT alone) aimed at preventing the mutilat-ing effect of surgery.
Despite conventional TNM information having a strong prog-nostic value in HNSCC,7 few predictive molecular markers ofresponse to therapy exist in this pathology. Consequently, uncov-ering predictors of response to IC may improve our ability toanticipate tumor response to subsequent CRT, identifying patientswho could benefit from a conservative treatment.
The cytotoxicity of the classic antineoplasic agents (5-FU, cis-platin, radiotherapy) depends of their ability to produce DNAdamage in tumor cells. Despite these agents producing differenttypes of DNA damage, all of them directly or indirectly produceDNA double strand breaks (DSB).8–10 CDDP produces interstrandcrosslinks, stops DNA replication and generates DSBs.11 5-FU isan analog of uracil that is converted intracellularly to fluorodeoxy-uridine monophosphate (FdUMP), a potent inhibitor of theenzyme thymidylate synthetase. This results in a deoxynucleotide(dNTP) pool imbalance, which disrupts DNA replication, generat-ing DSBs.10 DNA DSB is the most toxic and mutagenic of allDNA lesions. Two mechanisms exist for DSB repair: homologousrecombination and nonhomologous end joining (NHEJ). NHEJhas been described as the predominant DSB repair mechanism inmammalian cells.12
We hypothesized that the expression of NHEJ DNA repair(Ku70, Ku80 and DNA-PKcs) genes will influence tumorresponse to therapy on the basis of the following: (i) A highfrequency of chromosomal translocations and alteration ofdouble strand break (DSB) DNA repair in HNSCCs13–15; (ii)Direct induction of DSBs by radiation9 or indirectly by5-FU10,16 or cisplatin8,11; (iii) The major importance of NHEJin DSB repair, genomic stability maintenance and suppressionof translocations in mammalian cells17,18; (iv) An increasedin vitro sensitivity to radiation by NHEJ protein inactivation andrecovery of resistance by restoration of activity19–21 and (v) Involve-ment of Ku70, Ku80 and DNA-PKcs in apoptotic signaling afterradiation- or chemotherapy-induced DSBs.22–24
Grant sponsor: Spanish Ministerio de Educaci�on y Ciencia; Grant number:SAF03-07437. Grant sponsor: Fondo de Investigaciones Sanitarias; Grantnumbers: FIS PI052591, FIS PI051776, FIS 98/3197, FIS01/3085. Grantsponsor: Fundaci�on Mutua Madrile~na; Grant number: FMM 2006/169. Grantsponsor: Fundaci�o Marat�o de TV3; Grant number: MaratoTV3 04/050510.Grant sponsor: AGAUR; Grant number: SGR1050. Grant sponsor: ISCIIICIBER-BBN; Grant number: CB06/01/1031.*Correspondence to: GOA, Laboratori d’Investigaci�o Gastrointestinal,
Institut de Recerca, Hospital de la Santa Creu i Sant Pau, Av. SantAntoni M. Claret, 167, 08025 Barcelona, Spain. Phone: 134-93-2919106,Fax:134-93-4552331. E-mail: [email protected] or [email protected] 26 October 2007; Accepted after revision 14 March 2008DOI 10.1002/ijc.23635Published online 10 June 2008 in Wiley InterScience (www.interscience.
wiley.com).
Abbreviations: 5-FU, 5-fluorouracil; CDDP, cis-diamminedichloridopla-tinum; CRT, chemoradiotherapy; CT, computed tomography; dNTP, deox-ynucleotide; DSB, double strand break; ES, embryonic stem; FdUMP,fluorooxyuridinine monophosphate; HNSCC, head and neck squamous cellcarcinoma; HPV, human papillomavirus; HR, hazard ratio; HSCSP, Hospi-tal de la Santa Creu i Sant Pau; IC, induction chemotherapy; IHC, immu-nohistochemistry; LRFS, local recurrence-free survival; MEFs, mouseembryo fibroblasts; MR, magnetic resonance; NHEJ, nonhomologous endjoining; OS, overall survival; ROC, receiver-operating-characteristics; RT,radiotherapy.
Int. J. Cancer: 123, 1068–1079 (2008)' 2008 Wiley-Liss, Inc.
Publication of the International Union Against Cancer
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Patients with locally advanced (Stage III or IV) HNSCC, treatedat our institution with induction chemotherapy followed by eitherCRT/RT or surgery depending on tumor response, participated ina prospective mRNA study (n 5 50) or in an independent retro-spective immunohistochemistry (IHC) study (n 5 75). We eval-uated the relationship between Ku70, Ku80 or DNA-PKcs mRNA,or Ku70 protein expression in pretreatment tumor biopsies, andthe degree of tumor response to IC, local recurrence and patientsurvival. Our aim was to test the capacity of the expression ofthese genes in identifying patients likely to benefit from a conserv-ative treatment.
Patients and methods
Patient characteristics and treatment plan
Accrual for the mRNA prospective study (n 5 50) was initiatedin 2002. Patients in the IHC retrospective study (n 5 75) weretreated during the 1995–2003 period at the Hospital de la SantaCreu i Sant Pau (HSCSP). All patients had pathologically con-firmed, untreated, locally advanced (Stage III or IV) HNSCCs.The HSCSP Ethics Committee approved the study.
In both studies, patients were treated with IC consisting of theadministration of cisplatin at a dose of 100 mg/m2 on day 1, and5-FU at a dose of 1,000 mg/m2/day by continuous intravenousinfusion on days 2–6 every 3 weeks, per 3 courses. Patients whopresented a high tumor response after IC followed a conservativetreatment, which consisted of the administration of RT or CRT.
Patients treated from 1995 to 2002 who presented a high tumorresponse after IC followed treatment with radiotherapy (RT). CRTwas introduced in 2003 as an alternative to RT to treat patientswith a substantial IC response. Progressively, CRT displaced RTas the treatment of choice after IC.
RT was administered to the primary tumor and clinically posi-tive nodes in 35 fractions of 2 Gy, each over a 7-week period at atotal dose of 70 Gy. Nodal areas not clinically involved by tumorreceived a total dose of 50 Gy. CRT consisted of RT at the samedoses plus 3 cycles of cisplatin at a dose of 100 mg/m2 on day 1every 3 weeks.
Patients without significant response to IC were usually treatedwith surgery followed by RT. The characteristics of patientsincluded in both studies are summarized in Table I.
RNA extraction, cDNA synthesis and quantitative PCR
The prospective study was performed using fresh pretreatmentprimary tumor biopsies obtained from patients with HNSCC. Asample aliquot was used for pathological diagnosis of the malig-nancy. Samples with a low content of tumor tissue were excludedfrom the study. Another aliquot was frozen immediately after tak-ing the biopsy, in cold isopentane (histobath), and placed in liquidnitrogen until RNA processing.
Total RNA was extracted with Trizol1 (Invitrogen, Paisley,UK) and the phenol–chloroform method. Samples were then pre-cipitated twice with isopropanol and 70% ethanol, and cleaned-upusing RNeasy1 Spin columns (Qiagen, Valencia, CA). TotalRNA was quantified espectrophotometrically. Reverse transcrip-tion was performed using 1.5 lg of total RNA and the HighCapacity cDNA Archive Kit (Applied Biosystems, Foster City,CA), in a 50 lL final reaction volume, containing 5 lL of 103 RTbuffer, 2 lL of 253 dNTPs mixture, 5 lL of 103 Random Hex-amer Primers, 125 U of Multiscribe Reverse transcriptase and 40U of RNase inhibitor (Invitrogen, Paisley, UK). These mixtureswere incubated at 25�C for 20 min, and then at 37�C for 2 hr.Finally, heating at 95�C for 3 min inactivated the reverse tran-scriptase.
mRNA expression was measured on an ABIPrism 7000 SequenceDetection System (Applied Biosystems, Foster City, CA), using pre-designed Taqman1 Gene Expression Primer and probe Assays(Applied Biosystems, Foster City, CA), which were available forKu70, Ku80, and b-actin (http://www.appliedbiosystems.com). Allprobes were FAM-labeled, except for b-actin that was VIC-labeled.For DNA-PKcs detection, we used Primer Express software v2.0(Applied Biosystems, Foster City, CA) to design the forward (50-TGGGAGCATCACTTGCCTTTAATAA-30) and reverse (50-CAAACTGTTCCACCAGAGACTCTT-30) primers and a Taqman1
probe (50-CTTCCCTGAATTCCC-30) (Table II).mRNA quantitation was performed, in duplicates, in 20 lL total
volume PCR reactions, using 2 lL of each sample cDNA, 10 lLof 23 Universal Taqman Master Mix, 1 lL of primers and probemixture and 7 lL of H2O, following the manufacturer’s recom-mendations (Applied Biosystems, Foster City, CA). Thermal cy-cling conditions were 2 min at 50�C, 10 min at 95�C, and 40cycles at 95�C for 15 s and 60�C for 1 min.
TABLE I – CHARACTERISTICS OF PATIENTS INCLUDED IN THEPROSPECTIVE AND RETROSPECTIVE STUDIES
Patients characteristics Prospective mRNAstudy (n 5 50)
Retrospective IHCstudy (n 5 75)
SexMen 45 (90%) 71 (95%)Women 5 (10%) 4 (5%)
Tumor localizationOral cavity 11 (22%) 7 (9.3%)Oropharynx 7 (14%) 18 (24%)Hypopharynx 14 (28%) 12 (16%)Larynx 18 (36%) 38 (51%)
Lynph node involvementPresent 40 (80%) 41 (55%)Absent 10 (20%) 34 (45%)
Tumor size (T)2 8 (16%) 9 (12%)3 24 (48%) 50 (67%)4 18 (36%) 16 (21%)
Histological tumor gradeGood 5 (10%) 2 (3%)Moderate 39 (78%) 65 (87%)Poor 6 (12%) 8 (11%)
Tumor staging (TNM)III 12 (24%) 40 (53%)IV 38 (76%) 35 (47%)
Treatment subsequent to ICRadiotherapy 16 (32%) 38 (51%)Chemoradiotherapy 15 (30%) 4 (5%)Surgery 19 (38%) 33 (44%)
TABLE II – DESCRIPTION OF THE AMPLIFICATION AND HYBRIDIZATION REGIONS FOR mRNA GENEEXPRESSION ANALYSIS BY qPCR IN THE PROSPECTIVE STUDY
Gene Assay1 RefSeq2 Interexonic union Amlicon size (bp)
Ku70 Hs00750856_s1 NM_001469 Exon 1 99Ku80 Hs00221707_m1 NM_021141 Exon 2/Exon 3 72DNA-PKcs –3 NM_006904 Exon 27/Exon 28 72b-actin Hs99999903_m1 NM_00101 Exon 1 171
1Number assigned to predesigned Taqman Gene Expression Assay (Applied Biosystems; http://www.appliedbiosystems.com).–2mRNA Reference Sequence amplified using Taqman Gene ExpressionAssays. Available at the NCBI reference Sequence Database (Refseq).–3Custom-made sequence (seePatients and Methods section).
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Gene expression results were calculated applying the compara-tive CT method (22(DDCt)) as previously described.25,26 Using the22(DDCt) method, the data are presented as the fold change ingene expression normalized to an endogenous gene and relative toa calibration sample. b-actin was used as the endogenouscontrol to normalize the PCR results for the amount ofRNA added to the reverse transcription reaction. We extractedRNA from the HNSCC UM-SCC-22A cell line (a gift fromDr. R.H. Brakenhoff), which was used as the calibrationsample.27 First, we subtracted the endogenous gene expression fromtarget gene expression (DCT 5 CTtarget gene(Ku80, Ku70 or DNA-PKcs) 2CTendogenous gene(b-actin)) and, afterwards, calculated the expres-sion in the tumor relative to the calibration sample (DDCT 5DCTtumor sample 2 DCTcalibrator(UM-SCC-22A)). Each gene mea-sure was expressed in relation to the level of the same gene inthe UM-SCC-22A cell line. This cell line was maintained in10% FBS DMEM (Invitrogen, Paisley, UK), with 2 mmol/Lglutamine, 50U/mL penicillin and 50U/mL streptomycin, at37�C in a humidified atmosphere containing 5% CO2.
Immunohistochemistry
We used pretreatment formalin-fixed and paraffin-embeddedbiopsies of primary HNSCCs, with high tumor tissue content, toperform the retrospective study. Four-micron sections were depar-affinized in xylol and rehydrated using decreasing ethanol concen-trations (100, 96, 80, 70 and 50%) and distilled H2O. The sampleswere immersed in 13 Target Retrieval Solution pH 5 6 (DakoCytomation S.A., St Just Desvern, Spain) and autoclaved over10 min at 121�C for antigenic retrieval. Endogenous tissue peroxi-dase was inactivated by incubating samples in a 3% H2O2 solutionfor 10 min. Samples were then incubated with a mouse monoclo-nal antibody against Ku70 (Ab-4) (Lab Vision, Freemont, CA) ata 1:200 dilution. For primary antibody detection, we used theEnVison 1 Dual Link System-HRP Kit in an automatic Autos-tainer System (DakoCytomation S.A., St Just Desvern, Spain),according to manufacturer’s instructions. Counterstaining was per-formed with hematoxylin (DakoCytomation S.A., St Just Desvern,Spain). Samples were dehydrated in a growing ethanol and xylolgradient, and mounted with DPX media (Sigma Aldrich, TresCantos, Spain). Negative controls were processed substituting theprimary antibody by nonimmunized mouse serum. A normalmucousa and 2 HNSCC samples were used to control for IHCbatch staining variability.
Immunohistochemistry analysis
We took three 1003-magnified images per sample, using aDP50 camera and an Olympus DX51 microscope, under the samelighting and time exposure conditions. The ViewFinder Lite v1.0and Studio Lite v1.0 software (Olympus, USA) were used to cap-ture and store all images. Afterwards, we eliminated all areas con-taining no tumor cells, using AdobePhotoshop software v7.0(AdobePhotoshop systems, USA). We quantitated the IHC stain-ing of the areas containing tumor cells, using the Metamorph v 5.0(Universal Imaging, Downingtown, PA) software and applied theHSI (HUE-saturation-intensity) model,28,29 which selects a partic-ular area according to its color, as defined by its HUE (the domi-nant wavelength of transmitted light), saturation and intensity. Foreach pixel in an image, the values of HUE, saturation and intensityare independently transformed to one of 256 integral values withina 0–255 range. Setting a range between 0 and 255 for each of these3 parameters makes it possible the selection of the particular areasof interest.28,29
Evaluation of tumor response, local recurrence andpatient survival
In both, the prospective and retrospective studies, tumorresponse was evaluated by comparing tumor volume before ICand after the third cycle of IC. Response was defined as a reduc-tion in primary tumor volume, as measured by physical examina-
tion, fiber optic laryngoscopy, and CT scan/MR imaging followingRECIST criteria.30
The responder group was composed of tumors presenting acomplete response or a partial response higher than 50%. The non-responder group included all tumors presenting a stable disease(<25% progression or <50% reduction) or progressive disease(>25% of progression in tumor volume).
We recorded local recurrence-free survival (LRFS), which wasdefined as time from diagnosis to recurrence at the primary loca-tion of the tumor. In addition, we recorded overall survival,defined as the time from diagnosis to patient death. The medianfollow-up time in the prospective study was 2.1 years. The medianfollow-up time for the retrospective study was 4.0 years. Themolecular analysis was performed with no knowledge of clinicaloutcomes.
Statistical analysis
We compared tumor mRNA levels and the percentage of posi-tively stained tumor cells between the responder and nonrespondergroups by applying the Mann–Whitney U test. We performed anonparametric receiver-operating characteristics (ROC) analysisto evaluate the diagnostic usefulness of Ku80, Ku70 and DNA-PKcs mRNA levels and Ku70 protein levels to discriminatebetween responding and nonresponding tumors. We established acut-off level for mRNA and protein levels for each studied vari-able to distinguish tumors with high or low expression levels.These cut-off values were determined selecting the most accuratevalues obtained from the nonparametric ROC analysis, taking intoaccount the best balances between sensitivity and specificity.Logistic regression analysis was used to evaluate the associationsof Ku70, Ku80 and DNA-PKcs expression above or below thedefined cut-off values, tumor size (T), node involvement andtumor localization, with IC response. Adjusted LRFS curves wereestimated using the Kaplan–Meier method. We applied a 2-tailedlog-rank test to evaluate the differences in LRFS between patientswith tumor expression above or below the defined cut-off values.The association of Ku70, Ku80 and DNA-PKcs gene expression,node involvement, tumor size and localization with LRFS and OSwas assessed applying a univariate and a multivariate Cox regres-sion model analysis.
Differences were considered significant at p values <0.05 in allapplied statistical tests. All statistical analyses were performedusing the SPSS software v.14.01 (SPSS, Chicago, IL).
Results
Differences in Ku70, Ku80 and DNA-PKcs mRNA levels betweenresponder and nonresponder tumors (prospective study)
The characteristics of the patients included in the prospectivestudy are summarized in Table I. Twenty-eight out of 50 patientshad a tumor response to IC higher than 50% (responder group),whereas 22 tumors showed responses lower than 50% (nonres-ponder group). The median mRNA level was 4.4 (range0.5–309.8) for Ku70, 2.3 (0.1–36.8) for Ku80 and 3.3 (0.1–59.9)for DNA-PKcs when considering all samples (responders and non-responders) included in the prospective study. The median mRNAlevel in the responder group was 6.6 (range 1.2–236.4) for Ku70,3.2 (range 0.7–36.8) for Ku80 and 4.2 (range 0.2–59.9) for DNA-PKcs. The median mRNA level in the nonresponder group was3.3 (range 0.5–309.0) for Ku70, 1.8 (range 0.1–3.3) for Ku80 and2.4 (range 0.1–11.5) for DNA-PKcs.
We observed significantly higher mRNA tumor values for Ku70(p 5 0.005), Ku80 (p 5 0.002) or DNA-PKcs (p 5 0.017) in res-ponders than in nonresponders (Figs. 1a–1c). Afterwards, we per-formed a ROC analysis to test the sensitivity and specificity ofKu70, Ku80 or DNA-PKcs mRNA levels in evaluating responseto IC (Fig. 1d). The areas under the curve (AUC) were 0.73 [CI(95%) 5 0.59–0.88, p 5 0.005] for Ku70, 0.76 [CI (95%) 50.63–0.90, p 5 0.002] for Ku80 and 0.70 [CI (95%) 5 0.55–0.88,
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p 5 0.017] for DNA-PKcs. We next established a cut-off levelbetween high and low mRNA levels for each gene by selecting themost accurate value obtained in the ROC analysis. Thus, we dis-tributed all patients between 2 groups, one above and anotherbelow the following mRNA thresholds: 3.6 for Ku70, 2.6 forKu80 and 5.0 for DNA-PKcs. The sensitivity, specificity and accu-racy in predicting IC response with the established cut-offs wereas follows: Ku70 (sensitivity 86%, specificity 68%, accuracy78%), Ku80 (sensitivity 57%, specificity 77%, accuracy 66%) andDNA-PKcs (sensitivity 43%, specificity 86%, accuracy 62%).
Logistic regression analysis was conducted to evaluate the asso-ciations of mRNA levels, tumor localization, lymph node involve-ment and tumor size (T) with IC response. Only high (above 3.6)Ku70 mRNA levels were significantly associated with response toIC higher than 50% [p 5 0.03; odds ratio 5.9; CI (95%) 5 1.28–29.6]. Therefore, ROC and logistic regression analysis indicatedthat Ku70 mRNA gene expression was the best marker to discrim-inate between responding and nonresponding tumors.
Ku70 and Ku80 mRNA levels are associated with localrecurrence-free survival (prospective study)
We next analyzed whether the mRNA expression of these geneswas associated with primary tumor recurrence (Fig. 2a). Patientswhose tumors expressed Ku70 mRNA levels above the established3.6 threshold had a significantly higher probability of survivingwithout having primary tumor recurrence (increased LRFS) thanpatients bearing tumors with lower mRNA levels (p 5 0.043).
Similarly, patients whose tumors expressed Ku80 mRNA levelsabove the 2.6 threshold had a higher LRFS than patients bearingtumors with lower mRNA levels (p 5 0.04). DNA-PKcs mRNAlevels did not show an association with patient’s LRFS (p 50.457).
We also applied a Cox model analysis to study the associationof mRNA levels, tumor size (T), localization, lymph node involve-ment and IC response, with LRFS (Table III). On a Cox univariateanalysis, we found that Ku70 mRNA levels (p 5 0.05) and Ku80mRNA levels (p 5 0.05) were associated with LRFS. On a multi-variate Cox analysis, Ku70 mRNA levels (p 5 0.04) and tumorsize (p 5 0.03) were significant independent risk factors of LRFS.Therefore, in our study, Ku70 gene expression remains the onlymarker able to consistently predict local control of the disease.Thus, patients with tumors showing high Ku70 mRNA levels pre-sented a higher probability of surviving without primary tumor re-currence than patients with tumors showing low expression of thisgene. Ku80 mRNA, despite displaying the same trend as Ku70and being also a predictive marker of response, showed a weakerassociation with LRFS.
After applying a Cox model analysis to assess the association ofmRNA levels, tumor size (T), localization, lymph node involve-ment and IC response with overall survival, we observed that highKu70 levels displayed the same trend towards associating withlonger overall survival, as it happened with LRFS; however, theobserved differences did not reach statistical significance (p 50.14).
FIGURE 1 – Significant differences in Ku70 (a), Ku80 (b) and DNA-PKcs (c) mRNA levels in pretreatment biopsies between tumors with aresponse to induction chemotherapy higher than 50% (Resp > 50%) and tumors with response lower than 50% (Resp < 50%) in the prospectivestudy. Differences in NHEJ gene expression between the responder group and the nonresponder group were compared using the nonparametricMann–Whitney U test. (d) Curves obtained applying receiver-operating-characteristics (ROC) analysis for Ku70, Ku80 and DNA-PKcs.
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FIGURE 2 – Analysis of patient LRFS in the prospective study. Differences in adjusted local recurrence-free survival (LRFS) between patientsbearing tumors expressing pretreatment Ku70, Ku80 or DNA-PKcs mRNA levels above or below the depicted cut-off levels. (a) Analysis per-formed including all patients treated with induction chemotherapy, followed by either surgery or conservative treatment. (b) Analysis performedonly with the group of patients who followed a conservative treatment after IC.
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Ku70 mRNA levels are associated with local recurrence-freesurvival in patients who followed a conservative treatment(prospective study)
We next searched for the possible differences in adjusted LRFSwithin the subset of patients who received CRT or RT after IC (n531), excluding those treated with surgery after IC (n 5 19) (Fig.2b). Our objective was to study Ku70, Ku80 and DNA-PKcs as pos-sible markers of tumor response and primary tumor recurrence aftergenotoxic therapy. Surgery is applied to patients with poor responseto IC, and their inclusion could have altered the LRFS registered inpatients under genotoxic treatment. All patients (n 5 28) of the re-sponder group followed a treatment with CRT or RT after IC. Nine-teen patients of the nonresponder group underwent surgical excisionof their tumors after IC. Three patients of the nonresponder grouprefused mutilating surgery and, contrarily to medical advice, weretreated with RT or CRT. Out of the 31 IC patients who followed aconservative treatment (CRT or RT) after IC, those whose tumorsexpressed Ku70 mRNA levels above the 3.6 threshold had a signifi-cantly higher probability of surviving without having a primary tu-mor recurrence (increases LRFS) than patients bearing tumors withlower mRNA levels (p 5 0.001). Moreover, patients whose tumorsexpressed Ku80 mRNA levels above the 2.6 threshold showed atrend towards increased LRFS, but it did not reach statistical signifi-cance (p 5 0.113). DNA-PKcs mRNA levels did not show signifi-cant differences in patient’s LRFS (p5 0.442).
A univariate Cox model analysis confirmed the significant asso-ciation between Ku70 mRNA levels and LRFS (p < 0.01) (TableIII). Moreover, a multivariate Cox model analysis showed thatKu70 mRNA was the only independent risk factor for local recur-rence-free survival in patients who followed conservative treat-ment after IC (p 5 0.02) (Table III).
On a multivariate analysis, the difference in relative risk of pri-mary tumor recurrence between high and low Ku70 mRNA tumorpatients was 6 times higher when only conservatively treatedpatients were included in the analysis than when all patients wereincluded (HR: 28.2 vs. 4.7, Table III). A univariate (p 5 0.23) anda multivariate (p 5 0.64) Cox model analysis in patients treatedwith surgery after IC showed no association between Ku70mRNA levels and LRFS. The capacity of Ku70 mRNA in predict-ing local recurrence after conservative treatment was improvedwhen the data on LRFS after mutilating surgery were excludedfrom the analysis, indicating that Ku70 mRNA is a better markerof local recurrence after CRT/RT than of recurrence after surgery.
Differences in Ku70 protein expression between responder andnonresponder tumors (retrospective study)
Patient characteristics in the retrospective study are summarizedin Table I. Out of 75 analyzed patients, 39 had a tumor response to
IC greater than 50% (responder group), whereas 36 patientsshowed a tumor response lower than 50% (nonresponder group).Ku70 immunohistochemical analysis showed an exclusively nu-clear staining pattern. Using the ‘‘Set color threshold’’ tool ofMetamorph v5.0 software and the HSI color model (Fig. 3a), weestablished a 180–255 HUE range to select all brown primary anti-body-stained areas (positive nuclei) (Fig. 3c), which ensured theabsence of any selected area in negative control samples. More-over, we established a 160–255 HUE range to select all (brown 1blue) tumor cell nuclei present in the sample (Fig. 3d), leaving outcell cytoplasm, membrane or keratin deposits. Since all tumornuclei present in each sample image displayed a similar size, wecalculated the percentage of positive tumor cells dividing the areaoccupied by positive tumor nuclei by the area occupied by allnuclei. Two experienced pathologists validated this method.
Figure 4a shows Ku70 staining in 2 tumors from patients withdifferent responses to IC treatment. The median of the percentageof Ku70 positively stained cells was 63.31 (range 7.4–92.8) for allsamples (responders and nonresponders) included in the retrospec-tive study. The median of the percentage of Ku70 positively stainedcells in the responder group was 70.7% (range 19.0–92.8%) and inthe nonresponder group was 57.7% (range 7.4–88.3%). Weobserved a significantly higher percentage of Ku70 positive tumorcells in responders than in nonresponders (p 5 0.036) (Fig. 4b).Using ROC analysis, we obtained an AUC of 0.64 [CI (95%) 50.51–0.77, p 5 0.036] for Ku70 positive cells (Fig. 4c). The cut-offvalue between high and low percentage of positive Ku70 cells wasestablished at 74%, which was the most accurate value obtained inthe ROC analysis. Its sensitivity, specificity and accuracy in predict-ing IC response was 47, 69 and 59%, respectively. Thus, in pretreat-ment tumor samples, the percentage of Ku70 positive tumor cellswas significantly associated with response to IC.
Association between the percentage of Ku70 positive cells andlocal recurrence-free survival (retrospective study)
Patients whose tumors contained a percentage of Ku70 positivecells above the 74% threshold had a significantly increased proba-bility of surviving without having a primary tumor recurrence(longer LRFS), as compared to patients whose percentage of posi-tive tumor cells were below this threshold (p 5 0.013) (Fig. 4d).Cox univariate (p 5 0.03) and multivariate (p 5 0.02) analysesrevealed the percentage of Ku70 positive tumor cells as the mostsignificant factor associated with LRFS (Table IV).
Similar to the mRNA prospective study, in this study, we ana-lyzed the adjusted LRFS within the subset of patients whoreceived RT or CRT after IC, excluding patients who underwent asurgery procedure after IC. All patients (n 5 39) of the respondergroup followed a treatment with CRT or RT after IC. Thirty-three
TABLE III – HAZARD RATIOS FOR LOCAL RECURRENCE OBTAINED APPLYING COX MODEL ANALYSIS IN THEPROSPECTIVE STUDY
Local recurrence-free survival
Univariate Multivariate
HR (95% CI) p value HR (95% CI) p value
All patientsKU70 mRNA (<3.6 vs. >3.6) 2.8 (1.0–7.7) 0.05 4.7 (1.1–19.8) 0.04KU80 mRNA (<2.6 vs. >2.6) 3.5 (1.0–12.4) 0.05 2.1 (0.5–9.3) 0.34DNA-PKcs (<5.0 vs. >5.0) 1.5 (0.5–4.9) 0.46 1.7 (0.2–12.6) 0.59Tumor size (T) (T3–T4 vs. T1–T2) 2.3 (0.8–6.6) 0.14 7.4 (1.3–42.9) 0.03Node involvement (N1 vs. N0) 2.1 (0.5–9.3) 0.33 1.0 (0.2–6.0) 0.97Localization 1.4 (0.4–4.3) 0.60 2.1 (0.5–8.6) 0.32
Conservative treatmentKu70 mRNA (<3.6 vs. >3.6) 6.9 (1.9–26.5) <0.01 28.2 (1.7–47.0) 0.02Ku80 mRNA (<2.6 vs. >2.6) 2.9 (0.7–11.8) 0.13 0.9 (0.0–8.6) 0.95DNA-PKcs (<5.0 vs. >5.0) 1.7 (0.4–6.9) 0.45 0.4 (0.02–7.3) 0.53Tumor size(T) (T3–T4 vs. T1–T2) 1.8 (0.4–7.2) 0.42 4.9 (0.7–36.1) 0.12Node involvement (N1 vs. N0) 2.6 (0.3–20.6) 0.37 1.3 (0.1–16.5) 0.85Localization 1.1 (0.2–5.1) 0.94 0.5 (0.1–3.2) 0.45
The bold values indicate the existence of statistically significant differences.
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patients of the nonresponder group followed a surgery treatmentafter IC. Three patients of the nonresponder group, contrarily tomedical advice, rejected mutilating surgery and followed CRT orRT treatment. A total of forty-two patients followed a conserva-tive treatment after IC. Patients whose tumors contained a percent-age of Ku70 positive nuclei above the established 74% thresholdhad a significantly longer LRFS as compared to patients whosepercentage of positive tumor nuclei were below this threshold(p 5 0.008) (Fig. 4e). Cox univariate (p 5 0.03) and multivariate(p 5 0.02) analyses confirmed the association between the per-centage of Ku70 positive tumor cells and LRFS in patients whofollowed conservative treatment after IC (Table IV). A univariate(p 5 0.51) and a multivariate (p 5 0.48) Cox model analyses inpatients treated with surgery after IC showed no associationbetween the percentage of Ku70 positive tumor cells and LRFS.
In summary, high percentage of Ku70 positive cells was signifi-cantly associated with a longer local primary-recurrence-free sur-vival. In addition, the predictive capacity of Ku70 protein expres-sion became more significant when analyzing only patients underconservative treatment than when including all patients (conserva-tive plus surgical) in the analysis (see Figs. 4d and 4e).
Association between the percentage of Ku70 positive cells andoverall survival (retrospective study)
On a Cox univariate analysis, we found that the percentage oftumor Ku70 positive cells (p 5 0.03), node involvement (p 50.03) and tumor localization (p < 0.01) were significantly associ-ated with patient’s overall survival (Table IV). Moreover, on amultivariate Cox analysis, only the percentage of Ku70 positive
cells (p < 0.01) and tumor localization (p < 0.01) were significantindependent risk factors of overall survival (Table IV). Perform-ance of the same analysis only on patients who underwent con-servative treatment after IC yielded similar results (Table IV).
Discussion
The aim of this work was to obtain predictive markers ofresponse to therapy in locally advanced HNSCCs. In the prospec-tive study, we have described that tumors showing a responsehigher than 50% to induction chemotherapy (IC) had significantlyhigher pretreatment Ku70, Ku80 or DNA-PKcs mRNA levels thantumors with a response lower than 50%. In the retrospective study,which was carried out in a distinct cohort of patients, we analyzedKu70 tumor expression, because it was the best marker ofresponse and local recurrence in the prospective analysis. Thisstudy included a higher number of patients and longer follow-uptimes allowing to perform a more extensive local recurrence andoverall survival analysis. Here, our goal was to know if theobserved relationship between Ku70 expression and the clinicalvariables described in the prospective study was also found in theretrospective study and whether these findings went in the samedirection, despite using a different methodology (IHC instead ofqPCR).
In the retrospective study, Ku70 was the most significant inde-pendent factor in predicting LRFS, being also associated with OS.In contrast, node involvement and tumor localization were associ-ated with overall survival, but they did not predict LRFS. Theobserved differences between the LRFS and OS analyses suggest
FIGURE 3 – Ku70 Immunohistochemistry quantification used in the retrospective study. (a) We used the ‘‘Set Color Threshold’’ tool, imple-mented in Metamorph software v5.0, to establish a suitable HUE range that selects areas presenting specific immunostaining characteristics. (b)Immunohistochemistry quantification was performed in 1003 magnified images. Ku70 shows a nuclear staining pattern. (c) All positive immu-nostained nuclei, present in the image, were selected using a 180–255 HUE range (green areas). (d) All the nuclei (positive and negative), pres-ent in the image, were selected using a 165–255 HUE range (green areas).
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that clinical factors other than primary tumor recurrence areinvolved in determining overall survival. Indeed, node involve-ment and distant metastases, together with primary tumor recur-rence, are the factors showing the highest association with poorprognosis in patients with HNSCC.7,31
These results support the notion that Ku70 expression is a pre-dictive marker of response and recurrence after adjuvant therapy.In this sense, it differs from classical prognostic factors such as tu-mor localization or node involvement, which associate with clini-cal outcomes independently of the applied treatment. Thus, prog-nostic markers are related to tumor aggressiveness and to the na-ture of the mutations driving cell growth, motility anddissemination capacity, which may not necessarily relate to tumorresponse to therapy. In contrast, predictive markers are related tothe interaction between the tumor and the therapeutic agents andanticipate tumor response. In HNSCC, prognosis largely dependson conventional TNM information, which stratifies patients interms of tumor aggressiveness, and indicates the requirement ornot for systemic therapy. However, this information has so far pro-
ven inadequate in predicting response to nonsurgical therapy.7,32
Thus, despite lymph node involvement is the single most adverseindependent prognostic factor31; it is, however, not useful in pre-dicting response to adjuvant therapy.32
Our finding of Ku70 gene or protein expression as a marker oftumor response to IC, as well as a marker of local disease controland patient survival after genotoxic therapy, suggest that this pro-tein contributes to determine tumor response. These associationsare consistent with the previous demonstration that tumor responseto IC predicts response to subsequent RT4,33 and patient survival.5
To our knowledge, no previous report has addressed the predictionof response to therapy by NHEJ genes/proteins in patient biopsiesof locally advanced head and neck squamous cell carcinoma(HNSCC) treated with chemoradiotherapy. We are aware of only2 related studies, 1 performed in primary cultures from mostlyStage IV head and neck carcinoma biopsies, which found no cor-relation between DNA-PKcs protein levels and in vitro radiosensi-tivity.34 Another study evaluated DNA-PKcs, Ku70 and Ku80 ex-pression in head and neck cancer patients treated with radiotherapy
FIGURE 4 – Analysis of Ku70protein expression in the retro-spective study. (a) Differences inKu70 immunostaining in pre-treatment biopsies between a rep-resentative tumor in the re-sponder group and a representa-tive tumor in the nonrespondergroup. (b) Significant differencesin the percentage of Ku70 posi-tive tumor cells between tumorswith a response to inductionchemotherapy higher than 50%(Resp >50%) and tumors withresponse lower than 50% (Resp<50%). (c) Receiver-operating-characteristics (ROC) curvesapplied to the percentage ofKu70 positive cells. (d, e) Differ-ences in adjusted local recur-rence-free survival (LRFS)between patients bearing tumorswith a percentage of Ku70 pro-tein staining higher or lower than74%. (d) Analysis performedincluding all patients treated withinduction chemotherapy, fol-lowed by either surgery or con-servative treatment. (e) Analysisperformed only in the group ofpatients who followed a conserv-ative treatment after IC.
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and found no relationship with radiosensitivity.35 Nevertheless,there are some clinical reports regarding NHEJ protein predictionof response to therapy on related tumor types. Thus, in agreementwith our results, high expression of DNA-PKcs, detected byimmunohistochemistry, is associated with response to chemora-diation in esophageal carcinomas.36 Similarly, high expression ofKu80 or DNA-PKcs protein associates with increased survival intonsillar carcinoma patients treated with radiotherapy.37 In con-trast to these and to our results, high levels of Ku70 or DNA-PKcs, measured by IHC, associate with lower locoregional controlafter concurrent chemoradiotherapy in patients with nasopharyn-geal carcinoma38; nevertheless, this is considered a tumor entitydifferent from HNSCCs.39 In addition, results predicting responseby Ku70 and/or Ku80 proteins by IHC, in a direction opposed toour findings, are also found in other tumor types such as cervicalcarcinoma, since high levels of these proteins associate with lowerresponse and survival to radiotherapy in patients with cervicalcancer.40,41
Cell type-dependent response to therapy involvingNHEJ proteins
Despite our identification of Ku70 and, to a lesser degree, Ku80or DNA-PKcs as possible predictive markers of response to CRTin HNSCC, our findings went in a direction contrary to that antici-pated. We were expecting higher NHEJ protein levels, orincreased DNA-PK complex activity, being related to enhancedDNA damage repair, which in turn would lead to lower tumorresponses to therapy. This assumption is based on previous in vitroand clinical reports involving DNA repair proteins. For instance,in vitro inactivation of NHEJ proteins associates with higherradiosensitivity, whereas restoration of repair activity restores re-sistance.21 Moreover, high Ercc1 levels predict for poor responsein nonsmall cell lung42 or ovarian43 carcinoma patients.
Searching for a possible mechanistic explanation of the unanti-cipated direction of our findings, we have exhaustively reviewedthe in vitro and in vivo literature that evaluates the role of NHEJproteins in response to DNA damage. Despite recognizing incon-sistency of our results with some previous work, we also foundsolid work agreeing with our findings. Overall, the literaturereports that response to genotoxic therapy by DNA repair proteinsdepends on the studied cell type,44 on its differentiation state45 oreven on its degree of functional activation.46 In the following, weare describing findings, in specific cell types, in which inactiva-tion, or low level of activity, of the NHEJ system decreases theirsensitivity to genotoxic agents, and its possible mechanistic basis.Afterwards, we are describing results in other cell types, in whichinactivation of NHEJ proteins leads to increased cell sensitivity.
In agreement with the direction of our findings, inactivation ofKu70, in the chicken B lymphocyte cell line DT40, significantlyincreases their viability when exposed to high doses of doublestrand break (DSB) inducers, such as g-radiation or methyl metha-nesulfonate, as compared with wild type cells.47 Similarly,enhanced cell survival, through suppression of p53-dependent ap-optosis, has been reported in thymocytes of DNA-PKcs2/2 micetreated with whole body-ionizing radiation, as compared to wildtype mice.48 In addition, DNA damage-induced apoptosis by ion-izing g-radiation is abolished in DNA-PKcs2/2 mouse embryofibroblasts (MEFs) expressing E1A.49 Moreover, cisplatin inducesmarked cell death in Ku801/1 immortalized MEFs, Ku801/1Chinese hamster ovary-derived (CHO) cells or SCID cells com-plemented with human DNA-PK, as compared with their matcheddeficient counterparts. This cisplatin induced-death is mediated bythe kinase function of the DNA-PK complex and conveyed toneighboring cells through gap junctions, whereas cells deficient inKu80 or DNA-PKcs are markedly resistant to the drug.24 In agree-ment with a role for the DNA-PK complex proteins in apoptosis,the exposure of MEFs or glioma cell lines to g-radiation leads toDNA-PK and Chk2 phosphorylation of p53, which mediates sub-sequent induction of apoptosis.50,51 Similarly, the apoptosis
TABLE
IV–HAZARD
RATIO
SFOR
LOCAL
RECURRENCE
AND
CANCER
DEATH
OBTAIN
ED
APPLYIN
GCOX
MODEL
ANALYSIS
INTHE
RETROSPECTIV
ESTUDY
Localrecurrence-freesurvival
Overallsurvival
Univariate
Multivariate
Univariate
Multivariate
HR(95%
CI)
pvalue
HR(95%
CI)
pvalue
HR(95%
CI)
pvalue
HR(95%
CI)
pvalue
Allpatients
%Ku70positivecells(<
74%
vs.>74%)
5.2
(1.2–22.9)
0.03
5.6(1.3–24.3)
0.02
3.9
(1.2–13.3)
0.03
5.1(1.5–17.4)
<0.01
Tumorsize
(T)(T3–T4vs.T1–T2)
2.7
(0.4–20.1)
0.34
2.7(0.4–21.4)
0.34
1.4
(0.3–6.0)
0.65
2.2(0.5–9.6)
0.30
Nodeinvolvem
ent(N
1vs.N0)
1.1
(0.5–2.8)
0.78
1.0(0.4–3.0)
0.97
3.1
(1.1–8.3)
0.03
1.9(0.6–5.8)
0.26
Localization
1.9
(0.8–4.9)
0.17
2.3(0.8–6.7)
0.12
5.9
(2.0–17.4)
<0.01
5.6(1.7–18.4)
<0.01
Conservativetreatm
ent
%Ku70positivecells(<
74%
vs.>74%)
9.5
(1.3–73.1)
0.03
12.3
(1.6–96.6)
0.02
2.0
(0.7–13.9)
0.16
5.5(1.1–26.5)
0.03
Tumorsize
(T)(T3–T4vs.T1–T2)
3.3
(0.4–25.0)
0.26
4.6(0.6–38.2)
0.16
2.4
(0.3–18.4)
0.42
5.6(0.7–46.9)
0.11
Nodeinvolvem
ent(N
1vs.N0)
1.0
(0.4–2.9)
0.99
1.7(0.5–5.2)
0.37
2.2
(0.6–8.1)
0.26
2.5(0.6–10.2)
0.19
Localization
2.0
(0.7–6.0)
0.22
2.8(0.9–9.0)
0.08
12.3
(1.6–96.6)
0.02
16.4
(2.0–134.3)
<0.01
Thebold
values
indicatetheexistence
ofstatisticallysignificantdifferences.
1076 PAV�ON ET AL.
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induced by IGFBP-3 in glioblastoma or prostate cancer cell linesis blocked when DNA-PK is knocked out or chemically inhib-ited.52 In addition, in breast cancer cells exposed to ionizing radia-tion, a nuclear trimeric protein complex, including clusterin, Ku70and Ku80, is formed that constitutes a death signal for severelydamaged cells.53
In contrast to the results described above, the role of the NHEJproteins in DNA repair is consistent with the association betweenlow Ku70 expression or DNA-PK activity and sensitization to radi-ation in fourteen esophageal cancer cell lines.54 Similarly, downre-gulation of Ku70 or DNA-PKcs by siRNA induces sensitization tocisplatin, etoposide or topotecan in the human cervical carcinomaHeLa cell line.20 Also, in low-passage normal human fibroblasts,siRNA knockdown of DNA-PKcs resulted in increased radiosensi-tivity.19 Moreover, enhanced NHEJ DNA repair activity is associ-ated with lower sensitivity to genotoxic agents in primary culturesof human B-chronic lymphocytic leukemia cells, so that treatmentwith DNA-PK inhibitors increases their sensitivity to the DSBinducers g-radiation, etoposide or neocarzinostatin.55 Similarly,Ku702/2 embryonic stem cells have showed an increased sensi-tivity to g-radiation as compared to Ku70 heterozygous or wildtype embryonic stem cells.56 These findings suggest that NHEJ pro-teins may function in DNA repair.46,57 Moreover, DNA-PKcs mayalso participate in a NFkB-dependent antiapoptotic pathway thatprotects cells from death induced by toposisomerase inhibitors, inSV40-transformed human fibroblasts or CHO cells or p53 nullMEFs.58 Similarly, DNA-PKcs inhibits apoptosis induced by heatshock treatment in HeLa, CHO or mouse lung fibroblast cells, a dis-tinct function from its involvement in DNA repair.59
Therefore, the apparent contradiction between the results sup-porting and opposing our findings is solved when considering theexistence of a cell type-dependent response to genotoxic therapy.Consistent with this notion, response to DNA damage by doublestrand break (DSB) inducers, involving the NHEJ system, is celltype-dependent.46 Thus, a cell type-dependent response to DNAdamage, mechanistically involving the DNA-PK complex, hasbeen proposed on the basis of 2 main and alternate functions. Theactivation of the DNA-PK complex by radiation or other geno-toxic agents could trigger signaling pathways that result in apopto-sis or cell cycle arrest.44 Cells, whose physiological functioninvolves their rapid proliferation (e.g., lymphocytes), may requireactive apoptotic pathways to avoid tumorogenesis after genotoxicdamage; in contrast, cells playing a supportive role to other celltypes (e.g., stroma providing growth factors to epithelial cells)may instead enter senescence, to maintain their function whileavoiding transformation.44 In agreement with the described celltype dependency, cells with the same genetic background, but atdistinct differentiation states, respond differently to genotoxicagents, in the same mouse model. Thus, DNA-PKcs2/2 embry-onic stem (ES) cells show a similar level of sensitivity to IR thanwild type ES, whereas DNA PK2/2 fibroblasts (differentiated)cell line derived from the same mice are significantly more sensi-tive to IR than its wild type counterpart.45 On the other hand,some cell types change the NHEJ protein function, as they changetheir activation status. Thus, in nonactivated human multiple my-eloma (MM) cells, Ku80 confers sensitivity to DNA damage;however, CD40 activation of MM cells induce Ku80 and Ku70translocation to the plasma membrane changing their functiontowards antiapoptosis, which then leads to protection against apo-ptosis triggered by irradiation or doxorubicin.60
Further support for a cell type-dependent role of NHEJ inresponse to genotoxic therapy comes from findings in some celltypes, in which the DNA complex does not appear to play any rolein determining response to genotoxic agents. Thus, the sensitivityto IR in mouse ES cells knock out for Artemis, a member of theNHEJ pathway, does not vary as compared to ES wild type cells.61
Similarly, DNA-PK-deficient murine SCID embryogenic fibro-blast or the DNA-PKcs2/2 human glioma cell line MO59J showsa similar sensitivity to etoposide as wild type MEFs or the DNA-PKcs1/1 MO59K glioma cell line.62 Moreover, in human lym-
phoblastic cell lines, siRNA knockdown of DNA-PKcs results inno significant increase in radiosensitivity.19
We also reviewed whether other proteins involved in DNA repairfollowed a cell type-dependent response to genotoxic agents. Wechoose to focus on p53 role, because it is the most extensively stud-ied gene regarding response to chemotherapy and/or radiotherapy,and because of its involvement in processes similar to those of theNHEJ proteins (DNA repair, apoptosis, genomic stability) and ofits functional link with NHEJ signaling.48–50 We observed a patternof response to DNA damage similar to that described above for theNHEJ system. Thus, p53 mutation may be a predictive marker ofresponse to 5-FU and cisplatin-based neoadjuvant chemotherapy inHNSCC patients, since tumors with no response have a signifi-cantly higher prevalence of p53 mutations than responder tumors.63
Moreover, we also found a clear cell-type-dependent pattern ofresponse to genotoxic agents in tumors other than HNSCC, as afunction of their p53 status. Thus, inactivation of p53 shows eithersensitivity or resistance to DNA damage depending on the tumortype, cancer cell line or primary cell line being tested.57 Tumorsbearing a p53 wild type gene show higher response to radiotherapy/chemotherapy, which associates with longer patient survival, whileits mutational inactivation leads to lower response and survival innonsmall cell lung,64 breast65 or ovarian66 carcinoma. In contrast tothese results, an increased response in p53 mutant testicular,67 gli-oma68 or bladder69 tumors has been observed, as compared to p53wild type tumors.67 Moreover, and similar to NHEJ proteins, p53functions in DNA repair as well as in apoptotic regulation,57 whichcould lead to contrary outcomes regarding response to genotoxictherapy. Therefore, some of the p53 results that have been reportedcould be unexpected if only the participation of p53 in DNA repairwas considered rather than acknowledging a complex and cell-de-pendent role in DNA repair and apoptosis.
Overall, the literature reports that cell response to genotoxictherapy in human tumors, or in in vitro and in vivo models, afterthe inactivation of NHEJ proteins (and also of p53) depends onthe studied cell type, and or its differentiation state or functionalactivation. These studies demonstrate the increasing complexity ofthis area of research; thus, in addition to its function in DNArepair and stress response, the proteins of the DNA-PK complex(DNA-PKcs, Ku70 and Ku80) are implicated in multiple and/orseparated pathways that regulate distinct cell death pathways.
In summary, our results in HNSCCs could be explained consid-ering that, in addition to their expected DNA repair function, theNHEJ proteins participate in cell apoptotic regulation. Thus, in aparticular cell type (e.g., head and neck carcinoma), higher levelsof each of these proteins could enhance apoptosis (signalingthrough the pathways available for this cell type, considering thatthey remain active after transformation) and lead to a higher tumorresponse. In contrast, in other cell types, in which apoptotic path-ways are blocked, or in which NHEJ proteins participate only inrepair functions, higher levels of these proteins would lead toincreased repair and lower tumor responses. Therefore, the cross-talk between the available repair and apoptotic functions of thespecific repair system (e.g., NHEJ) in a specific cell type (e.g.,HNSCC) could determine if the treated cell is sensitive or resistantto the damaging agent (e.g., DSB inducer). Nevertheless, the com-plexity of this area requires the mechanistic dissection of the can-didate pathways, as they relate to our results, before we could es-tablish a definite role for some of the NHEJ proteins in HNSCCcells. We are now starting to address this issue.
Clinical implications of our findings
Independent of the exact role that Ku70 gene plays, our resultssupport its use as a predictor of response to therapy, primary tumorrecurrence and patient survival. Moreover, our results need to bevalidated in independent studies before their clinical introduction,as it should happen with other proposed markers (e.g., p53 orHPV infection), which are not still in use.63,70,71 Finally, in anattempt to extend their possible predictive capacity, we are now
1077Ku70 PREDICTS RESPONSE AND PRIMARY TUMOR RECURRENCE
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evaluating whether NHEJ genes may also predict HNSCCresponse to primary concomitant CRT.
In summary, our results suggest that Ku70 mRNA could be agood candidate to be validated as a predictive marker of responseto genotoxic therapy. Thus, in biopsies of locally advancedHNSCCs, the levels of tumor mRNA or the percentage of proteinpositive tumor cells for Ku70 can identify patients with high prob-ability of response to induction chemotherapy and longer local re-currence-free survival, leading to longer overall survival. Thesepatients would benefit from a conservative treatment based on
chemoradiotherapy or radiotherapy and would differ from thosewho would not respond and would require surgical resection orthe exploration of alternative therapies.
Acknowledgements
We would like to thank Mr. Luis Carlos Navas for his technicalsupport and Dr. Montserrat Lopez for her collaboration in biopsysampling. We also wish to thank the patients who gave permissionto use their tissue for research.
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