[CANCER RESEARCH 53, 4477-4480, October 1, 1993]
Advances in Brief
The Incidence of p53 Mutations Increases with Progression of Head and Neck
Cancer
Jay O. Boyle, John Hakim, Wayne Koch, Peter van der Riet, Ralph H. Hruban, R. Arturo Roa, Russell Correo,
Yolanda J. Eby, J. Michael Ruppert, and David Sidransky2
Otolaryngology-Head and Neck Surgery [J. 0. B., J. H., W. K., P. v. d. R., R. A. R., Y J. E., J. M. R., 1). S.] and Department of Oral Surgery JR. C.] the Johns Hopkins
University, and Department of Pathology [R. H. H.], the Johns Hopkins Hospital, Baltimore, Maryland 21205
Abstract
To establish a genetic model of the progression of head and neck squamous carcinoma we have defined the incidence and timing of p53 mutations in this type of cancer. We sequenced the conserved regions of the p53
gene in 102 head and neck squamous carcinoma lesions. These included 65
primary invasive carcinomas and 37 noninvasive archival specimens consisting of 13 severe dysplasias and 24 carcinoma in situ lesions. The incidence of p53 mutations in noninvasive lesions was 19% (7/37) and increased to 43 % (28/65) in invasive carcinomas. These data suggest that p53
mutations can precede invasion in primary head and neck cancer. Furthermore, the spectrum of codon hotspots is similar to that seen in squamous carcinoma of the lung and 64% of mutations are at G nucleotides,
implicating carcinogens from tobacco smoke in the etiology of head and
neck squamous carcinoma.
Introduction
Cancers arise through a series of well defined genetic changes that
accumulate during histopathological progression (1-3). This process
has been best delineated through an analysis of colorectal cancer in
which the progression from adenoma to carcinoma was correlated
with specific genetic changes (4). Alterations of p53 are the most
common mutation in human cancers (5) and often occur in the transition from early to invasive lesions in colorectal cancer (6) and other
epithelial tumors (7). However, recent reports have suggested that
alterations of p53 may occur even earlier in squamous epithelium
during progression to cancer (8, 9).
HNSC 3 kills more than 11,000 Americans each year (10), yet little
is known of the genetic events involved in its progression (11). We
examined the genetic progression for HNSC by determining the timing and incidence of p53 mutations in noninvasive lesions as well as
in primary invasive carcinomas. We found that p53 mutations occur in
noninvasive lesions and continue to increase in frequency in invasive
carcinomas. We have also catalogued the spectrum of p53 mutations
in HNSC for comparison with p53 mutations in other types of carcinomas. As expected, HNSC shares a p53 mutation spectrum similar to
that found in squamous carcinoma of the lung. Moreover, the types of
p53 mutations in HNSC also implicate tobacco-derived carcinogens in
the etiology of this disfiguring and often fatal disease.
Materials a n d M e t h o d s
Surgically resected specimens of invasive HNSCs were collected with consent from patients at Johns Hopkins Hospital. Tumors were fresh frozen and
later carefully cryostat-microdissected to enrich for neoplastic cells. Cases with
less than 50% neoplastic cells were not included in the analysis. More than 50
Received 7/21/93; accepted8/19/93.
The costs of publication of this article were defrayedin part by the paymentof page
charges. This article must therefore be hereby marked advertisement in accordancewith
18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by NCI CORE Grant CA58184-01.
2 To whom requests for reprints should be addressed.
3 The abbreviations used are: HNSC, head and neck squamous carcinoma; PCR,
polymerase chain reaction; CIS, carcinomain situ.
12-~m sections were cut and placed in sodium dodecyl sulfate/Proteinase K
followed by extraction with phenol/chloroform and ethanol precipitation as
described (6). Archival lesions consisting of dysplasia or CIS were identified
retrospectively (1991-1993) through a systematic search in the files of the
surgical pathology division of the Department of Pathology. These formalinfixed, paraffin-embedded lesions were microdissected to enrich for neoplastic
cells and then deparaffinized in xylene. DNA from tissue was then digested,
extracted, and precipitated with ethanol as described above.
p53 Gene Mutations. From primary fresh frozen tumor DNA a 1.8-kilobase segment of the p53 gene encompassing exons 5-8 was amplified by the
PCR as described (12). A UDP cloning site was added to the 5' end of the
primers (a = 5'-CAU CAU CAU CAU TIC ACT TGT GCC CTG ACT T-3'
and d -- 5'-CUA CUA CUA CUA CTG GAA ACT TTC CAC TIG AT-3') to
allow cloning into a Cloneamp (BRL) plasmid vector (pSPORT) (13). From
archival samples the p53 gene was amplified in two segments. One segment
included exons 5 and 6 utilizing primers a and b (b = 5'-CUA CUA CUA CUA
CCA CTG ACAACC ACC CTF-3') and the other segment included exons 7
and 8 utilizing primers c (c = 5'-CAU CAU CAU CAU CCAAGG CGCACT
GGC CTC-3') and d. Following amplification with uracil-containing primers,
the PCR products were extracted with phenol/chloroform and run on a 1%
agarose gel. The product was extracted from the gel and treated with 1 unit of
uracil DNA glycosolase, and one-half of the total product was annealed to the
plasmid vector according to the manufacturer's instructions (13). Competent
DH5-ot cells were transfected with plasmid by heat shock, plated on ampicillin
plates, and incubated overnight at 37~ More than I00 colonies were pooled,
and plasmid DNA was isolated by alkaline lysis.
Sequencing. Double-stranded DNA obtained from plasmid was sequenced
by the dydeoxy method utilizing Sequenase (USB) and [33p]dATP or [35S]dATP (12). Prior to termination, a 30-min incubation with 0.5 units of Klenow
fragment (USB) was added to eliminate "stop" bands. Sequencing reaction
products were then separated on a 6% urea/polyacrylamide gel and exposed to
film. All mutations were confirmed by a second PCR reaction followed by
recloning and resequencing.
Clinical Data. All information was obtained from patient records and
assessed as follows. Tobacco exposure was classified as: heavy, >1 pack/day;
moderate, <1 pack/day or quit 5-20 years ago; and mild, nonsmoker or quit
>20 years ago. Alcohol exposure was classified as: light, nondrinker, quit >20
years ago, or special occasions only; moderate, <12 ounces/week or quit 5-20
years ago; and heavy, >12 ounces/week.
Results
To determine the relative timing of p53 gene mutations in HNSC,
we sequenced 65 fresh primary invasive tumor samples and 37 archival preinvasive lesions. Nineteen % (7 of 37) of the early preinvasive
lesions contained p53 mutations, compared to 43% (28 of 65) of the
primary invasive HNSCs (Table 1). Only 7 p53 mutations were found
in preinvasive lesions: 5 of 24 in CIS lesions (21%); and 2 of 13 in
dysplastic lesions (15%). Both of the latter mutations were in lesions
of severe dysplasia; no lesions of mild or moderate dysplasia contained p53 mutations. The difference in incidence of p53 mutations
between noninvasive and invasive lesions was found to be statistically
significant (P < 0.02 by X2 analysis).
Closer analysis of the specific p53 mutations found in all lesions
reveals that 72% (26 of 36) were missense mutations and 28% (10 of
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p53
MUTATIONS
AND
HEAD
AND
NECK
CANCER
Table 1 Timingof p53 mutations in HNSC"
Tab/ETOH
Site
Age
Sex
Codon
Mut
Amino acid
Primary invasive HNSC tumors with p53 mutations
H/M
H1
T2No
H/L
H2
T3No
M/M
H3
T3No
H/H
H4
T3N25
H/H
H5
T2N2
H/H
H6
T2No
H/H
H7
T4NzC
H/L
H8
T3No
H9
N/A
N/A
H/L
HI0
TaNo
Stage
L
L
L
L
OP
OP
OP
L
L
HP
41
67
80
65
36
55
55
58
N/A
59
M
F
M
M
M
F
M
M
N/A
M
OC
OC
L
OC
OC
L
L
OC
OP
OP
L
OC
NE
L
L
OC
OC
OC
68
84
64
65
66
66
46
64
70
46
47
85
61
58
66
59
58
58
M
M
M
M
F
F
M
F
F
M
M
F
M
M
M
F
F
F
275
296
163
175
294
234
278
228
298
175
216
b
220
198
205
245
257
220
258451
278
220
279
248
237
251
236
273
278
281
TGT-~TAT
CAC-~CCC
TAC~ CAC
CGC~ CAC
GAG---,TAG
TAC~ TGC
T insertion
GAC--~GAG
GAG--~TAG
CGC--~CAC
GTG---,ATG
Acceptor G-*T
TAT-->TCT
GAA~AAA
TAT-*GAT
GGC~ GTF
CTG---~CCG
TAT~ TGT
9-base pair deletion
CCT-*TCT
TAT-->TGT
G insertion
CGG--~CAG
ATG~ ATI"
ATC---~AAC
TAC---~TAA
CGT~ GGT
CCT---~ACT
GAC~ CAC
Cys~ Tyr
His--->Pro
Tyr~ His
Arg~ His
Glu-* Term
Tyr-* Cys
FS
Asp~ Glu
G|u~Term
Arg---~His
Val-Met
Splice site
Tyr---~Set
Glu~Lys
Tyr-*Asp
Gly-* Val
Leu---~Pro
Tyr---~Cys
FS
Pro-*Ser
Tyr-* Cys
FS
Arg---~Gin
Met-* lie
Ile-*Asn
Tyr--~Term
Arg~ Gly
Pro--->Thr
Asp-~His
Hll
H12
H13
H14
H15
H16
H17
H18
H19
H20
H21
H22
H23
H24
H25
H26
H27A
27B
TaN1
T3N1
T3N1
TINo
T1N1
T2No
T2N2A
T2No
T3No
T2No
T4N2C
T4No
TxN2
T3N2C
TzN2C
TzNo
T3No
TaN2C
Pathology
H/H
M/H
H/L
M/H
M/H
H/L
H/H
H/H
H/H
H/M
H/M
L/L
H/M
H/H
H/NA
H/L
H/H
H/H
Noninvasive tumors
C1
CIS
N/A
OC
42
M
200
A deletion
FS
C2
CIS
N/A
OC
81
M
146
TGG--~TGA
Trp~Term
C3
dys
N/A
OC
68
M
249
A insertion
FS
C4
dys
N/A
OC
34
M
169
ATG---,ACG
Met-->Thr
C5
CIS
N/A
OC
54
M
278
CCT---~ACT
Pro-*Thr
C6
CIS
N/A
OC
53
M
158
CGC~TGC
Arg-*Cys
C7
CIS
N/A
OC
64
M
176
TGC--~TTC
Cys--,Phe
a Tab, tobacco; ETOH, ethanol; L, light; M, moderate; H, heavy; N/A, not available; FS, frame shift; L, larynx; OP, oropharynx; OR, oral cavity; NE, neck; dys, dysplasia.
b Intron 5 (-3 base pairs from exon 6). For assessment of tobacco and ETOH exposure see "Materials and Methods."
36) w o u l d produce truncated proteins. This represents a high percentage o f mutations resulting in truncations, similar to that seen in
esophageal cancer (5). O f these, four were nonsense mutations, four
were frame shift mutations, and one was an altered splice site mutation. Interestingly, one o f these mutations (H18) was a deletion that
occurred near small repeating sequences, perhaps secondary to replication errors as described by others (14). Additionally, one tumor
(H15) contained an unusual pyrimidine dimer 2-base pair mutation at
codon 245. Although c o m m o n in UV-induced skin tumors (15, 16),
similar mutations have been previously seen in bladder cancer and
m a y also be induced by reactive o x y g e n free radicals or severe exposure to carcinogens (17). Eighty-two % o f the invasive tumors (23
o f 28) had lost the wild type allele, suggesting loss o f the remaining
p53 allele during tumor progression. One tumor (H10) contained two
point mutations, and analysis of individual clones revealed that each
occurred on a different allele. In contrast, 5 of 7 p53 mutations in early
lesions appeared to have retained the wild type allele. The presence o f
this allele m a y represent s o m e residual contamination from surrounding nonneoplastic tissue, or it m a y m e a n that the second allele had not
yet been lost in these early lesions. Mutations were spread over a wide
range o f codons yet occurred in the most highly conserved regions of
p53 (Fig. 1) as previously reported (5). The mutations also occupied
a spectrum similar to that seen in squamous cell c a r c i n o m a o f the lung
(18), but they differed f r o m mutations seen in other epithelial tumors
(5).
Clinical data w e r e available for 26 o f the 27 patients with primary
invasive carcinomas containing mutations in the p53 gene (Table 1).
O f these, 16 o f 25 point mutations (64%) w e r e at guanine nucleotides,
including 8 G---,A, 5 G--~T, or 3 G - * C ; these are changes often
A~
B~
. . . . . ~,~,~,,,~
.._~ . . . . . 12.. ??
A
C
G
-~
;
,dL
T
ACGT
Fig. 1. p53 mutations in HNSC. In A, sequencing gel audioradiographfrom 6 invasive
tumor DNA samples are shown with lanes grouped together for rapid identification of
novel bands upon visual comparison.Arrow,T-~C mutation (codon 220) in Lane 1 from
the tumor of patient H17. In B, arrowpoints to C--,T mutation (codon 146) in the tumor
DNA of patient C2 with CIS. Antisense sequencing primers have been previously described (12).
associated with benzopyrenes, nitrosamines, and possibly o x y g e n
radicals f r o m cigarette smoke (18-21). Thirteen o f these 16 patients
w e r e heavy smokers, and five o f them also had histories o f heavy
drinking. Only one patient with a G----~A transition was a n o n s m o k e r
and nondrinker. Overall, 25 of 26 patients with mutations had histories
o f moderate to heavy smoking. In contrast, 11 o f 38 patients without
p53 mutations had had only minimal exposure to tobacco and alcohol.
A l t h o u g h the exact amount was quite variable and difficult to quantify
accurately, carcinogen exposure was significant in all but one o f the
patients with missense mutations. Exposure was particularly heavy in
the group with specific mutations previously found to be associated
with k n o w n carcinogens in cigarette smoke. The presence or absence
o f p53 mutations did not correlate with age or sex of the patient nor
4478
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p53 MUTATIONS AND HEAD AND NECK CANCER
Fig. 2. Immunohistochemical staining of p53 demonstrating intense nuclear staining in
the carcinomatous portion of a carcinoma arising in an inverted papilloma from patient C7.
The inverted papilloma portion did not stain. The staining was performed using the CM-1
polyclonal anti-p53 antibody and the ABC VectaStain kit (Vector Laboratories) with
methods previously described (33).
with the site, size, or stage of the tumor. Clinical data were not
available for patients with noninvasive (dysplastic or CIS) lesions.
One patient (H27) had two separate tumors on opposite sides of the
oral cavity (Table 1). Each tumor had a distinct p53 mutation as
previously seen in patients with separate primaries (22). One lesion
from another patient (C7) was identified as an inverting papilloma
with carcinoma arising in the papilloma (Table 1). As shown in Fig. 2,
only the carcinomatous portion of the neoplasm stained with an antip53 antibody (usually indicative of p53 mutation) (23). Microdissection of the distinct histological regions followed by DNA extraction
and p53 sequencing revealed a codon 176 mutation in the carcinoma,
while no mutation was detected in the papilloma. This case was
stained simply to illustrate the histopathological progression to cancer
associated with a new p53 mutation.
centage of mutations occur outside the conserved regions, we did not
sequence all the exons within p53 (5). Therefore, the total number of
p53 mutations in these tumors is probably slightly higher. The validity
of our approach is supported by recent articles suggesting similar p53
mutations in a smaller number of HNSC tumors (29, 30). Our findings
in turn support recent case reports demonstrating p53 staining and/or
mutations in selected lesions with severe dysplasia in squamous epithelium (8, 9). The significant frequency of p53 mutations in early
lesions suggests that direct damage of DNA may be important in
HNSC progression because of the role of wild type p53 in inducing a
G1-S arrest following DNA damage (31). It appears that ongoing DNA
damage exerts selective pressure on p53 to mutate early, allowing
clonal outgrowth and progression. The pattern of p53 mutations with
regard to affected codons and specific nucleotide changes implicates
cigarette smoke and is reminiscent of changes seen in lung cancer (5,
18). Additionally, codons 220, 245-248, and 278-281 appear to be
particular "hot spots" for p53 mutations in HNSC, based on previous
reports (22, 29, 30) and our own data. Our work further supports
growing evidence that cigarette smoke is a prime mutagenic agent in
cancers of the aerodigestive tract.
We have recently described an assay capable of detecting a small
percentage of mutant-containing cancer cells among an excess percentage of normal cells (12, 32). Establishing the genetic steps in the
progression of HNSC may allow us to target early genetic events for
novel screening techniques in saliva from these HNSC patients. Our
results suggest that p53 mutations can be detected in a significant
minority of early lesions including severe dysplasia and CIS. However, preliminary observations suggest that second primary tumors
may have different p53 mutations as reported by Roth et al. (22) and
as seen in patient H27. Furthermore, our information suggests thatp53
mutations peak somewhat later in the progression to invasive lesions,
which would seem to negate the use of this assay for initial screening.
Rather, testing for reemergence of the initial p53 mutation might be
indicative of recurrence with need for immediate intervention. The
availability of additional molecular markers for screening will depend
on the development of a reasonable model that identifies early steps in
the genetic progression of HNSC.
Discussion
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since other screening modalities, such as RNase protection (26),
single-strand conformation analysis (27), or denaturing gel electrophoresis (28), can miss certain mutations. Because only a small per-
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Research.
The Incidence of p53 Mutations Increases with Progression of
Head and Neck Cancer
Jay O. Boyle, John Hakim, Wayne Koch, et al.
Cancer Res 1993;53:4477-4480.
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