P53 overexpression in epiderm oid carcinom a
of the head and neck
L u d w ig ln s titu te fo r C a n c e r R e s e a r c h
D e p a r t1 1 1 e n t o f H e a d a n d N e c k S u r g e r y , A .C . C a m a r g o H o s p ita l, F u n d a ç ã o A n to n io P r u d e n te - S ã o P a u lo , B r a z il
T h e th e o ry o f fie ld ca n ce riza tio n in tu m o rs o f th e h e a d a n d n e ck re fle cts th e co m p le x o n co g e n e sis th a t o ccu rs in th is re g io n . T h e m e ch a n ism s th a t co n tro l ce ll p ro life ra tio n a t th e m o le cu la r le ve i in e p id e rm o id ca rcin o m a s (E C s) o f th e u p p e r a e ro d ig e stive tra ct a re still u n cle a r. M u ta tio n s in p 5 3 a re th e g e n e tic a lte ra tio n s m o st o fte n d e te cte d in E C s o f th e h e a d a n d n e ck a n d se e m to co n trib u te a ctive ly to th e ca rcin o g e n ic p ro ce ss trig g e re d b y p 5 3 a s a tu m o r-su p p re sso r g e n e a n d to its a sso cia tio n w ith to b a cco . T h e o b je ctive o f th e p re se n t stu d y w a s to in ve stig a te th e e xp re ssio n o f p 5 3 p ro te in in e p id e rm o id h e a d a n d n e ck ca rcin o m a s b y im m u n o h isto ch e m istry a n d its im m u n o h isto ch e m ica l co rre la tio n w ith o th e r p ro g n o stic fa cto rs. T h e stu d y w a s co n d u cte d o n 6 3 co n se cu tive E C s ca se s n o t su b m itte d to p re vio u s tre a tm e n t. S p e cim e n s o f th e tu m o r a n d o f th e n o rm a l a d ja ce n t m u co sa w e re co lle cte d d u rin g su rg e ry a n d su b m itte d to im m u n o h isto ch e m ica l re a ctio n fo r th e d e te rm in a tio n o f th e e xp re ssio n o f a n ti-p ro te in p 5 3 a n tib o d y (M 7 0 0 1 D A K OA IS , D e n m a rk). A n a to m o -c1 in ica l a n d d e m o g ra p h ic d a ta w e re n o t sig n ifica n tly co rre la te d w ith th e p re se n ce o f Iym p h n o d e m e ta sta se s o r p 5 3 e xp re ssio n in th e tu m o r o r in th e a d ja ce n t n o rm a l m u co sa . T u m o r lo ca liza tio n in th e la ryn x w a s sig n ifica n tly co rre la te d w ith p 5 3 e xp re ssio n . H isto lo g ica l g ra d in g a s g ra d e s I, 1 1 ,1 1 1a n d IV w a s co rre la te d w ith sig n ifica n t p 5 3 e xp re ssio n (p
=
0 .0 2 5 ). C o n clu sio n s: 1 ) in th e stu d ie d m a te ria l o b ta in e d fro m 6 3 ca se s o f h e a d a n d n e ck E C s, w e d e te cte d a 4 8 p e rce n t ra te o f im m u n o h isto ch e m ica lly d e te cta b le p 5 3 o ve re xp re ssio n ; 2 ) w e d id n o t d e te ct a re la tio n sh ip b e tw e e n d e m o g ra p h ic p a tie n t d a ta a n d p 5 3 e xp re ssio n in th e tu m o r o r in th e n o rm a l a d ja ce n t m u co sa ; 3 ) p 5 3 o ve re xp re ssio n w a s sig n ifica n tly m o re fre q u e n t in E C s m a te ria l fro m th e la ryn x; a n d 4 ) T h e p re se n ce o f 1 2 ca se s w ith p 5 3 o ve re xp re ssio n in th e n o rm a l a d ja ce n t m u co sa a n d w ith a p 5 3 -n e g a tive tu m o r is in a g re e m e n t w ith th e th e o ry o f fie ld ca n ce riza tio n . F o llo w -u p o f th is p a tie n t se rie s fo r a lo n g e r p e rio d o f tim e w ill p e rm it a b e tte r a n a lysis o f th e se va lu e s.U N IT E R M S : P 5 3 , sq u a m o u s ce ll ca rcin o m a , h e a d a n d n e ck ca n ce r, im m u n o h isto ch e m istry
IN T R O D U C T IO N
E
90P iderm oid carcinom aspercent of the neoplasias(E C s) representof them ore thanupperaerodigestive trace) and their incidence increases
am ong subjects w ho sm oke cigarettes and consum e
alcoholic beverages.12
E C s correspond to the sixth m ost
frequent form of cancer in the w orldY
A d d r e s s fo r c o r r e s p o n d e n c e : A n g e la F la v ia L o g u llo
R u a P r o t. A n to n io P r u d e n te , 2 1 1 S ã o P a u lo /S P - B r a s il- C E P 0 1 5 0 9 - 0 1 0
P atients w ith head and neck E C s in the sanle stage
IT Iay respond differently to different form s of treatlnent.2
C urrently recognized prognostic factors are not sufficiently
objective or standardized, and do not correspond to the
biological behavior of the tum or at the cellular leveI.
T he theory of field cancerization in head and neck
neoplasias reflects the com plex oncogenesis that occurs
in this region.33 T he frequent occurrence of IT Iultiple
prim ary tum ors in the upper aerodigestive tract Inay be
explained by continuous exposure of the IT IU C O S ato variolls
carcinogens. H ow ever, the sequential stages of this
com plex carcinogenesis have not been flllly elucidated and
the m olecular m echanism s that control cell proliferation
and the aberrant behaviorofE C s in the upper aerodigestive
tract are still unknow n.
L O G U L L O , A .F .; K O W A L S K I, L .P .; N O N O G A K I, S . e a I. - P 5 3 o ve re xp re ssio n in e p id e rm o id ca rcin o m a o f th e h e a d a n d n e ck
1350
lmmunohistochemistry
M A -T E R IA L A N D
M E T H O D S
The study w as conducted
on 63 consecutive
EC s
cases not subm itted
to previous
treatm ent
registered
at
the D epartm ent
of H ead and N eck Surgery
of the A .C .
C am argo H ospital in 1994 and 1995. Tum or and nonn~l
adjacent m ucosa specim ens w ere collected during surgery
and fixed in C arnoy solution. A fter fixation, the m aterial
w as em bedded
in paraffin
and 4 ~m thick sections
w ere
obtained
and stained
w ith H E. The m aterial
w as then
subm itted
to im m unohistochem ical
reaction
for the
determ ination
of the expression
of anti-protein
p53
antibody
(anti-hum an
p53 protein
clone
D 07,
M 700
ID A K O ,
AIS,
D enm ark).
p = 0 .4 2 4 p = 0 .4 5
p = 0 .4 8 5 p = 0 .8 9 p = o .5 1
p = 1 .0 0
8 1 9 5 1 2 1 5
23
925
The m aterial w as deparaffinized
and im m ersed
in a
bath containing
decreasing
concentrations
of xylol and
ethyl alcohol. Tne antigen w as recovered
by treatm ent
in
a m icrow ave
oven under
im m ersion
in a nitric
citrate
solution, w ith 4 baths of 5 m in each at lnaxilnum
potency.
Endogenous
peroxidase
(H 202) was blocked
and the
m aterial
w as incubated
1: 100 w ith prim ary
antibody
overnight
at 4°C , follow ed
by incubation
w ith
I:200
biotinylated
secondary
antibody for 30 lnin at 37°C . A fter
incubation
w ith 1 :800 StrepA B com plex:H R P
for 30 lnin
at 37°C (D A K O
StrepA B
cO lnplex/G R P
D uet,
m ouse/rabbit
kit),
the
m aterial
w as ilnm ersed
in
60 m g percent
D A B
(2,3
diam inobenzidine
tetra-hydrochloride,
Siglna
C hem ical
C o., D 5637) plus
1 percent
dilnethyl
sulfo-xide PA (Labsynth)
for 5
m in
at 37°C
and
coun-terstained
w ith
helna-toxylin.
W hen
m ore
than
20
percent
of the tum or
cell
nuclei
stained
dark brow n,
the sam ple
w as considered
positive. The sam e criterion
w as used for the evaluation
of norm al adjacent m ucosa. _
Positive
and
negative
controls
w ere
systelna-tically
perform ed
for each
reaction.
4 1 3 1 3 24 6 2 6 2 6 22p 5 3 e x p re s s io n
(+ ) (-)
26
25
4 7
2 8 3 0
1 O
1 1
p re s e n t
a b s e n t
p re s e n t
a b s e n t
p re s e n t
a b s e n t C a te g o rie s
m a le
fe m a le
W h ite
A s ia n
B la c k
< 4 5
4 6 -6 0
> 6 0
T a b le 1
C o rre la tio n b e tw e e n p 5 3 e x p re s s io n a n d d e m o g ra p h ic d a ta
A g e
F a m ily
H is to ry
S m o k in g
h a b it
a lc o h o lis m R a c e V a ria b le s
S e x
G enes involved
in carcinogenesis
m ay be classified
as proto-oncogenes
and tum or-suppressor
genes according
to their action
m echanism .23,35 Tum or-suppressor
genes
co de for proteins
w ith functions
related to the control and
suppression
of
cell
grow th
and
proliferation
by
transactivation
or transrepression
of specific genes. These
genes
actively
affect
the cell cycle,
regulating
it in a
repressi ve m anner.
IIThe role of gene p53 has been the subject of several
studies
on solid
hum an
tum ors.5,6,15,16,IX ,19,25,26,30
P53
m utations
are the genetic
alterations
m ost frequently
detected
in head
and neck EC S,2X J4 specifically
in the
larynx, and seem to actively contribute to the carcinogenic
process
of the gene31 as a tum or
suppressor
and to its
association
w ith tobacco.4,IO ,17
The accum ulation
of lnutation of gene p53 associated
w ith head and neck EC s has been reported in the literature,
although
it has not been correlated
w ith other prognostic
factors.1.2,7.13 A better
understanding
of the role
and
m echanism
of action of gene p53 in the various stages of
carcinogenesis
m ay
be of help
in term s
of specific
therapeutic
approaches,
and m ay identify
new tum or
m arkers for early diagnosis.
The objective
of the present study w as to investigate
the expression
of p53 protein
in head anC l neck EC s by
ilnm unohistochem istry
and to determ ine
its relation
to
other prognostic
factors.
F ig u re 1 - p 5 3 e x p re s s io n in g ra d e II e p id e rm o id c a rc in o m a o f th e to n g u e .
Statistica/ ana/ysis
D o u b le - e n tr y c o n v e r g e n c e ta b le s w e r e c o n s tr u c te d f o r th e a n a ly s is o f c o r r e la tio n s b e tw e e n th e c lin ic a l-p a th o lo g ic v a r ia b le s s tu d ie d a n d th e im m u n o h is to c h e m ic a l e x p r e s s io n o f m u ta te d p 5 3 p r o te in . S ta tis tic a l s ig n if ic a n c e w a s c a lc u la te d b y th e c h i - s q u a r e te s t o r b y th e e x a c t F is h e r te s t w h e n a p p lic a b le .
R E S U L T S
E x p r e s s io n o f m u ta te d p 5 3 p r o te in w a s c o n s id e r e d to b e p o s i ti v e in 3 0 o f th e 6 3 s p e c im e n s o f p r im a r y tu m o r s ( 4 8 p e r c e n t) . T h e s tu d y d id n o t s h o w a s ig n if ic a n t c o r r e la tio n b e tw e e n p r o te in p 5 3 o v e r e x p r e s s io n a n d p a tie n t d e m o g r a p h ic v a r ia b le s ( f a m ily h is to r y o f c a n c e r , c ig a r e tte s m o k in g o r a lc o h o l c o n s u m p tio n ) ( T a b le 1 ) . C a s e s f o r
w h o m d e m o g r a p h ic d a ta w e r e n o t a v a ila b le w e r e e x c lu d e d f r o m th e ta b le .
F ig u r e 1 s h o w s a g r a d e 1 1E C o f th e to n g u e w ith p 5 3 o v e r e x p r e s s io n .
T o f a c ilita te d a ta a n a ly s is , th e tu m o r s w e r e s c h e m a tic a lly a s s ig n e d to th e th r e e f o llo w in g to p o g r a p h ie s : p h a r y n x , in c lu d in g th e c e r v ic a l e s o p h a g u s ; o r a l c a v ity , in c lu d in g p a la te , m o u th f I o o r , to n g u e a n d g in g iv a ; a n d la r y n x . T a b le 2 s h o w s th e c o r r e la tio n b e tw e e n p 5 3 e x p r e s s io n a n d a n a to m ic a l lo c a tio n , h is to lo g ic g r a d e a n d ly m p h n o d e s ta tu s .
E p id e r m o id c a r c in o m a o f th e la r y n x w a s s ig n if ic a n tly c o r r e la te d w ith p 5 3 e x p r e s s io n w h ic h d id n o t o c c u r in o th e r lo c a tio n s .
H is to lo g ic a l g r a d in g w a s s c h e m a tic a lly d iv id e d in to g r a d e s 1 1 . 1 1 1a n d IV . U s u a lly , g r a d e s 1 1 1a n d IV h a v e th e b e s t p r o g n o s is . C o r r e la tio n w ith p 5 3 e x p r e s s io n w a s s ig n if ic a n t ( p = 0 .0 2 5 ) . N o c o r r e la tio n w a s o b s e r v e d b e tw e e n p 5 3 e x p r e s s io n a n d p r e s e n c e o f ly m p h n o d e m e ta s ta s e s .
L O G U L L O , A .F .; K O W A L S K I, L .P .; N O N O G A K I, S . e t a I. - P 5 3 o v e re x p re s s io n in e p id e rm o id c a rc in o m a o f th e h e a d a n d n e c k
1352
p=0.20 p=0.025
28
4
14
19 27
3
17
13 negative
positive
D IS C U S S IO N
The hypothesis
of the participation
of gene p53 in
the process
of carcinogenesis
of head and neck tum ors
has been discussed
in severaI
studies.7-lJ.'3.'4.21.24.27.2lJ,32.34.37
A fter the discovery
of anti-protein
p53 antibodies,
severaI
retrospective
studies
have been published,
w ith variable
results,
i.e., rates of p53 positivity
of 30 to 67 percent.
Thirty to 65 percent of the carcinom as
have characteristic
l11utations in gene p53, w ith an allele usually having a point
Inutation
and producing
a m is-sense
p53 protein
that
accum ulates
at high leveIs in cancer cells. The second allele
w as Iost by conversion
and/or genetic deletion.22
C elllines
from EC s of the upper aerodigestive
tract
have
revealed
increased
p53
expression
in vitro.1.3ó
D ata
concerning
p53
expression
in the norm al
adjacent
l11ucosa and in the tum or of the sam e patient are
presented
in Table 3. The results show that p53 expression
at these tw o sites w as not coincident in 29 cases. The tum or
w as p53+ and the lnucosa p53- in 17 cases, and 12 cases
presented
opposite
results
(a p53-
tum or
and a p53+
adjacent
lnucosa).
Figure 2 show s an area ofthe nO rInal adjacent m ucosa
w ith
lnoderate
epithelial
dysplasia
and strongly
p53
positive. Figure 3 show s an area of non11al adjacent l11ucosa
that is negative
for p53 overexpression.
A lthough
the follow -up
til11e of the present series is
still short, three patients
w ho presented
recurrences
had
p53+
tU l11ors, w hereas
only one case had a recurrence
am ong patients w ithout p53 expression. This sl11allnU lnber
of recurrences
prevents
statisticaI
analysis.
Tum or topography
O ral cavity
P harynx
Larynx
H istological grade
I + II
111 + IV
Lym ph node
m etastases
M utations,
deletions
anel
Table 2A ssociation betw een p53 expression and tum or topography, histological grade
al11plification ofthe undeleteel
and Iym ph node involvem ent
allele
of
gene
p53
are
---
C O l11111on
Iy fou n eI in these
p53 expression p=0.0225
lines.:
n(+ ) (-)
C o n s i d e r in g t h e 111
u
1-15
18
ticentric etiology of tun10rs of
6
12
the
aerodigestive
tract,
10
2
several
investigators
have
a Is o e x a 111
i n e d t h e n o r n1a
Iprelesional
adjacent
ll1ucosa
of
som e
cases
and
have
detected
an irregular
pattern
of p53 expression,
although
they
elid
not
detect
a
correlation
betw een
p53
expression
in the non11al m ucosa
adjacent
to the turnor
and
the
appearance
of
a
second
neoplasia
or
recurrence.14.20.27.2lJ,32
B rachlnan
et aI.2 investigated
31 specir11ens of head
and neck EC s and observed
rnutations
of gene p53 in 53
percent of cases, dem onstrated
by polY lnorphisll1 of single
strand confon11ation. The salne investigators
later detecteel
m utations
in gene p53 by the polY lnerase
chain re£1ction
in 63 percent of the cell lines studied.
O f the 3 I cases of
head and neck EC s studied by C hung et £11.,72 I presenteei
at least one m utation
of gene p53, analyzed
by the single
chain polym orphisl11 confon11ation
l11ethod. Field et aI.,1O
in a study of73 EC s cases, observed the presence of protein
p 5 3
o ver e x p r e s s i o n
i n
67
p e r c e n t
o f
t h e In
b y
im m unohistochem istry.
In another il11l11unohistochelnical
study, Frank
et aI.I3 detected
p53 overexpression
in 37
percent of cases of EC s of the hypopharynx.
Shin et £11.32
obtained
a 43 percent
index in an il11111unohistocheI11ic£11
study of head and neck EC s. The presence
of p53+ in 48
percent
of the 63 head and neck EC s cases positive
for
p53 in our study is com patible
w ith literature
data.
In vitro studies carried out by Y in et aI.,37 S0l11erS et
aI. 34 and W einberg
et aI. 3ó show ed
abnorI11alities of gene
p53 in celllines
froln head and neck carcinolnas,
such as
overexpression
of
protein
p53
del110nstrated
by
il11111unohistochel11istryand m utations
and deletions
of the
gene detected
by sequential
analysis
by the polY lnerase
chain reaction.
M aestro et aI.24investigated
58 cases of larynx tunlors
and found
p53 overexpression
in 60 percent
using
£1n
im l11unohistochem icallnethod
and l110lecular analysis
by
the polym erase
chain reaction
and in situ hybridization.
The authors concluded
that this is the genetic
£1lteration
m ost frequently
occurring
in these tU I11ors. O f 89 cases of
EC s of the larynx
studied
by N adaI et aI.,27 64 percent
presented
il11m unohistochelnically-d~tecteel
p53
Figure 2 -
N o r m a l a d ja c e n t m u c o s a w ith m o d e r a te e p ith e lia l d y s p la s ia a n d s tr o n g ly p 5 3 p o s itiv e .overexpression. T he specific localization for these case.s
of cancer of the larynx w as statisticalIy significant in term s
of p53 expression, a fact not previously reported.
T he p53 overexpression observed in 10 of the 12
cases of cancer of the larynx studied here w as m ore
frequent than in the study discussed above. N ees et aI.
(1993) found p53 overexpression in only 44 percent of
their cases of E C s of the larynx and in 80 percent of the
cases of cancer of the oropharynx. W e are currently
expanding our study to determ ine p53 expression in the
various topographies of the head and neck.
P rospective studies w ith appropriate m aterial for
analysis of p53 have only perm itted a sim ple statistical
correlation w ith som e isolated factors such as sm oking4 .IO and hU lT Ian papilIom avirus.3 T he absence of a correlation
betw een sm oking and p53 gene expression in the present
study disagrees w ith the data reported by others.2,4
W ith a longer foIlow -up tim e, few studies w ere
consistent in show ing the absence of a correlation betw een
the prognostic factors norm alIy used and p53 expression
in head and neck tum ors. F rank et aI.I3 did not detect a
correlation betw een p53 expression in E C s of the
hypopharynx and other data such as incidence of secondary
tum ors, survival, ploidy or histological grade. N adaI et
aI.27 did not find a prognostic significance of p53
expression in neoplastic and dysplastic lesions of the Iarynx
in a study of 89 cases, and concluded that the presence of
lT Iutations of gene p53 detected by the poIY lT IeraS e chain
reaction is not correlated w ith radiosensitivity in these
tum ors.
T aking into account the theory of field cancerization,
N ees et aI.29 identified m utations of gene p53 in tU lT IO rS
and in the norm al adjacent m ucosa and suggested the
participation of p53 in the carcinogenesis of lT IultipIe
tL llT IO rs. S hin et aI.32 and G usterson et aI.I4 studied
prem alignant lesions of the squam ous epitheIiulT I,
including dysplasia, hyperplasia, B ow en disease, and the
norm al adjacent m ucosa and found 21 to 45 percent and
15 to 16.6 percent p53 positivity by im m unochem istry in
their respective series. T he scarcity of data concerning
L O G U L L O , A .F .; K O W A L S K I, L .P .; N O N O G A K I, S . e t a I. - P 5 3 o v e r e x p r e s s io n in e p id e r m o id c a r c in o m a o f th e h e a d a n d n e c k
1354
su rv iv al
p rev en ted
th ese
au th o rs
fro m
co rrelatin g
th eir
resu lts
w ith
p ro g n o stic
facto rs.
H o w ev er,
th ere
is n o
o b jecti v e ev id en ce o f a co rrelatio n b etw een p 5 3 ex p ressio n
an d p ro g n o stic
facto rs fo r h ead an d n eck carcin o m as.
2 7T h e ab sen ce o f a co rrelatio n
b etw een th e ex p ressio n
o f p 5 3 in th e n o rm al ad jacen t lT IU C O S a
an d in th e tu m o r area
o f th e sam e p atien ts d etected h ere ag rees w ith literatu re d ata.
N ees
et
£11.2 9fo u n d
p 5 3
o v erex p ressio n
in th e n o rm al
ad jacen t m u co sa an d ab sen ce o f p 5 3 p ro tein in p atien ts w ith
h ead an d n eck E C s T h is ap p aren t d iscrep an cy w as o b serv ed
in
1 2o f o u r cases. T h e p resen ce
o f p 5 3 ex p ressio n
in th e
ad jacen t m u co sa m ay in d icate th e p o ssib le o rig in o f m u ltip le
tu m o rs o r th e lo cal recu rren ce in th is area th ro u g h d ifferen t
carcin o g en ic
p ro cesses,
in v o lv in g
o r n o t m u tatio n
o f th e
p 5 3 g en e. T h e sp ecific lo calizatio n
in can cer o f th e lary n x
w as statistically
sig n ifican t
in term s o f p 5 3 ex p ressio n ,
a
fact th at is n o t u n co m m o n
in literatu re rep o rts. O u r series
m u st b e ex p an d ed
fo r a b etter an aly sis o f th e to p o g rap h ic
d istrib u tio n
o f tu m o rs acco rd in g
to p 5 3 ex p ressio n .
Table 3
Expression of p53 in the primary tumor and in the
adjacent mucosa.
CONCLUSIONS
1 .
In m aterial o b tain ed
fro m 6 3 cases o f h ead an d n eck
E C s, w e o b serv ed 4 8 p ercen t im ln u n o h isto ch elT Iically
-d etectab le
p 5 3 o v erex p ressio n ,
a rate co m p atib le
w ith
li teratu re d ata.
2 .
W e d id
n o t
o b serv e
a relatio n
b etw een
p atien t
d em o g rap h ic
d ata an d p 5 3 ex p ressio n
in th e tU ln o r o r
in th e n o rm al ad jacen t
m u co sa.
3 .
p 5 3 o v erex p ressio n
w as sig n ifican tly
m o re freq u en t
in m aterial fro m E C s o f th e lary n x .
4 .
T h e p resen ce
o f
1 2cases w ith p 5 3 o v erex p ressio n
in
th e n o rm al ad jacen t m u co sa an d a p 5 3 -n eg ativ e
tU ln o r
ag rees
w ith th e th eo ry
o f field
can cerizatio n .
T h is
fin d in g
m ay b e a facto r
fo r th e early
d etectio n
o f
p atien ts
at h ig h risk to d ev elo p
o th er
p 5 3 -p o sitiv e
tu m o rs.
A lo n g er fo llo w -u p
o f th is series \v ill p en n it a b etter
an aly sis
o f th ese
v alu es
in term s
o f recu rren ce
an el th e
p o ssib le
ap p earan ce
o f o th er
tu m o rs,
w h ich
is ab o L It 6
p ercen t (u n p u b lish eel
d ata).
p 5 3 ( + ) e x p re s s io n p 5 3 ( -) e x p re s s io n
in th e tu m o r
p 5 3 (+ )
e x p re s s io n
p 5 3 (-)
e x p re s s io n
1 3
1 7
in m u c o s a
1 2
2 0
ACKNOWLEDGMENTS
W e w ish to th an k M y iu k i F u k u ela d a S ilv a an el C arlo s
F erreira N ascim en to
fo r tech n ical
assistan ce.
A .F .I. is th e
recip ien t o f a D o cto ral
fello w sh ip
fro m F A P E S P .
REFERENCES
1. B urns lE , B aird M C , C lark L I, et aI. G ene m utations and increased leveIs of p53 protein in hum an squam ous-cell carcinom as and their celllines. B r 1 C ancer 1993;67: 1274-84.
2. B rachm an D G , B eckett M , G raves D , et aI. p53 m utation does not correlate w ith radiosensitivity in 24 head and neck cancer cell lines. C ancer R es 1993;53:3667-9.
3. B rachm an D G , G raves D , V okes E , et aI. O ccurrence of p53 gene deletions and hum an papillom a virus infection in hum an head and neck cancer. C ancer R es 1992;52:4832-6.
4. B rennan IA , B oyle 10, K och W M , et aI. A ssociation betw een cigarette sm oking and m utation of the p53 gene in squam ous-cell carcinom a of the head and neck. N E ngl 1 M ed
1995;332(11 ):712-17.
5. C hang F, Syrjanen A , T ervahauta A , et aI. T um origenesis associated w ith the p53 tum or-suppressor gene. B r 1 C ancer
1993;68:653-61.
6. C hen PL ,C hen Y , B ookstein R , L ee W H . G enetic m echanism s of tum or suppression by the hum an p53 gene. Science 1990;250:576-9.
7. C hung K Y , M ukhopadhyay T , K im 1, et aI. D iscordant p53 m utations in prim ary head and neck cancers and corresponding second prim ary cancers of upper aerodigestive tract. C ancer R es 1993;53: 1676-83.
8. D onehow er L A , B radley A . T he tum or suppressor p53. B iochem ica et B iophysica A cta 1993;155:181-205.
9. Field lK . O ncogenes and tum or-suppressor genes in squam ous cell carcinom a of the head and neck. O ral oncol E ur 1 C ancer 1992;28B ( 1)67-76.
10. Field lK , Spandidos D A , M alliri A . E levated p53 expression correlates w ith a history of heavy sm oking in squam ous-cell carcinom a of the head and neck. B r 1 C ancer 1991 ;64:573-77. 11. Finlay C A , H inds PW , L evine A I. T he p53 proto-oncogene
can act as a suppressor oftransform ation. C ellI989;57: 1
083-93.
12. Franco L E , K ow alski L P, K anda lL . R isk factors for second cancers of the upper respiratory and digestive system s: A case-control study. 1 C lin E pidem ic 1991 ;4(7):615-25. 13. Frank, lL B ur M E , G arb JL , et aI. p53 tum or suppressor
oncogene expression in squam ous-cell carcinom a of the hypopharinx. C ancer 1994;73( 1): 181-6.
14. G uterson B A , A nbazhagan R , W arren W , et aI. E xpression of p53 prem alignant ad m alignant squam ous epitheIium . O ncogene 1991 ;6:85-9.
15. H arris C C , H ollstein M . C linicaI im plications of the p53 tum or suppressor gene. N E ngl 1 M ed 1993; O ct 28: 1318-25.
16. H ollstein M , Sidransky D , V ogeIstein B , H arris C C . p53 m utations in hum an cancers. Science 1991 ;253 :49-53. 17. K nudson A , G enetics of tum ors of the head and neck. A rch
O tolaryngol H ead N eck Surg 1993; 119:735-7.
18. L am b P, C raw ford M L . C haracterization of the hum an p53 gene. M oI C el B iol 1986;6: 1379-85.
19. L ake D . W orrying about p53. C urrent B ioI 1992;2(11):581-3.
20. L ee N K , Y e Y W , C hen 1. p53, retinoblastom a, and hum an papillom a virus in squam ous-cell carcinom a and adjacent norm al m ucos of the upper aerodigestive tract. A rch O tolaringol H ead N eck Surg 1993; 119: 1125-31. 1992; 21. L eonard H L , K earsley IH , C henovix- T rench G , et aI. A nalysis
of gene am plification in head and neck squam ous-cell carcinom as. Int 1 C ancer 1991 :48:511-15.
22. L evine A I. T he road to the discovery of the p53 protein. lnt 1 C ancer 1994;56:775-6.
23. L evine A I, M om and 1, Finlay C A , T he p53 tum or suppressor gene. N ature 1991 ;351 :453-6.
24. M aestro R , D olcetti R , G asparotto D , et aI. H igh frequency of p53 gene alterations associated w ith protein overexpression in hum an squam ous-cell carcinonla o the larynx. O ncogene 1992, 7: 1159-66.
25. M alkin D , 1011y K W , B arbier N , et aI. G erm line m utations of the p53 tum or-suppressor gene in children and young adults w ith second m alignant neoplasm s. N E ngl J M ed 1992;326: 1309-25.
26. M cB ride O W , M erry D , G ivol D . T he gene for the p53 cellular tum or antigen is localized on chrom osom e 17 short ann (17pI3). Proc N atl A cad Sci U SA 1986;83: 130-4.
27. N adaI A , C am po E , Pinto 1, et aI. p53 expression in norm al, dysplastic, and neoplastic laryngeal epithelium . A bsence of a correlation w ith prognostic factors. 1 Path 1995; 175: 181-8. 28. N agai M A , M iracca E C , Y am am oto L , K ow alski L P, B rentani
R R . T P53 m utations in upper aerodigestive squam ous-cell carcinom as from a group of B razilian patients. A 1 Surg
1996; 170:92-4.
29. N ees M , H om ann N , D ischer H , et aI. E xpression of m utated p53 in tum or-distant epithelia of head and neck cancer patients: A possible m olecular basis for the developm ent of m ulti pIe tum ors. C ancer R es 1993;53:4189-96.
30. N igro 1M , B aker SI, Preisinger A C , et aI. M utations in the p53 gene occur in diverse hum an tum or types. N ature
1989;342:705-9.
31. R eiss M , V ellucci V F, Z hou Z L . M utant p53 suppressor gene causes resistance to transform ing grow th factor in m urine keratinocytes. C ancer R es 1993;53:899-04.
32. Shin D M , K im 1, R o lY , et aI. A ctivation of p53 gene expression in prem alignant lesions during head and neck tum origenesis. C ancer R es 1994;54:321-6.
33. Slaughter D P, Southw ick H W , Sm ejkal, W . "Field C ancerization" in oral stratified squam ous epithelium . C ancer
1953;6:963-8.
34. Som ers K D , M errick A , L opez M E , et aI. Frequent p53 m utations in head and neck cancer. C ancer R es 1992;52:5997-6000.' .
35. W einberg R A . O ncogenes, antioncogenes and m olecular basis of m ultistep carcinogenesis. C ancer R es 1989;49:3713-8.
36. W einberg W C , A zzoli C G , C hapm an K , et aI. p53-m ediated transcriptional activity increases in ditferentiating epidernlal keratinocytes in association w ith decreased p53 protein. O ncogene 1995;10:2271-9.
37. Y in X Y , Sm ith M L , W itesade T L , et aI. A bnorm alities in ,the p53 gene in tum ors and celllines of hum an squam
ous-cell carcinom as of the head and neck. lnt J C ancer 1993;54:322-7.
L O G U L L O , A .F .; K O W A L S K I, L .P .; N O N O G A K I, s .e t a I. - P 5 3 o v e r e x p r e s s io n in e p id e r m o id c a r c in o m a o f th e h e a d a n d n e c k
