variation of p53 mutational spectra between carcinoma of ... · vol. 1, 763-768, jo/v 1995 clinical...

Vol. 1, 763-768, Jo/v 1995 Clinical Cancer Research 763

3 The abbreviations used are: SCCHN, squamous cell carcinoma of the

head and neck; URT, upper respiratory tract; LRT, lower respiratory tract.

Variation of p53 Mutational Spectra between Carcinoma of the

Upper and Lower Respiratory Tract’

John C. Law,2 Theresa L. Whiteside,

Susanne M. Gollin, Joel Weissfeld,

Lobna El-Ashmawy, S. Srivastava,

Rodney J. Landreneau, Jonas T. Johnson,

and Robert E. Ferrell

Departments of Human Genetics [J. C. L., S. M. G., R. E. F.J and

Epidemiology [J. W.J, Graduate School of Public Health, University

of Pittsburgh and Pittsburgh Cancer Institute, Pittsburgh 15261;

Departments of Pathology [T. L. W., L. E-A.], Surgery [R. J. L.], and

Otolaryngology [J. T. J.], University of Pittsburgh Medical Center

and Pittsburgh Cancer Institute, Pittsburgh, Pennsylvania 15261 ; and

National Cancer Institute, Division of Cancer Prevention and Control,

Early Detection Branch, NIH, Bethesda, Maryland 20892 [S. S.]

ABSTRACT

Mutations of the p5.3 tumor suppressor gene are the most

common genetic alterations associated with human cancer.

Tumor-associated p5.3 mutations often show characteristic tis-

sue-specific profiles which may infer environmentally induced

mutational mechanisms. The p5.3 mutational frequency and

spectrum were determined for 95 carcinomas of the upper and

lower respiratory tract (32 lung and 63 upper respiratory

tract). Mutations were identified at a frequency of 30% in

upper respiratory tract (URT) tumors and 31 % in lung tu-mors. All 29 identified mutations were single-base substitu-

tions. Comparison of the frequency of specific base substitu-

tions between lung and URT showed a striking difference.

Transitions occurred at a frequency of 68% in URT, but only

30% in lung. Mutations involving G:C-�A:T transitions,which are commonly reported in gastric and esophageal hi-

mors, were the most frequently identified alteration in URT

(11119). Mutations involving G:C-*T:A transversions, which

were relatively common in lung tumors (3/10) and are repre-

sentative of tobacco smoke-induced mutations were rare in

URT tumors (1/19). Interestingly, G:C-�A:T mutations at

CpG sites, which are characteristic of endogenous processes,

were observed frequently in URT tumors (9/19) but only rarely

in lung tumors (1/10), suggesting that both endogenous and

exogenous factors are responsible for the observed differences

in mutational spectra between the upper and lower respiratory

systems.

Received 12/19/94; accepted 3/23/95.

1 This work was supported by NCI(MAO)CN-15393-02 and in part by

NCI-CN-24428-33, ACS Grant EDT-44, the Mary Hillman Jennings

Foundation, and the John R. McCune Charitable Trust Foundation.

2 To whom requests for reprints should be addressed, at Department ofHuman Genetics, Graduate School of Public Health, 130 DeSoto Street,

University of Pittsburgh, Pittsburgh, PA 15261.

INTRODUCTION

Two common cancers with poor prognosis, high mortality,

and clearly defined and overlapping environmental exposure

risk factors are SCCHN3 and lung cancer. SCCHN is the sixth

most frequent cancer worldwide (1). In the United States, ap-

proximately 11,000 individuals die annually from SCCHN (2).

There has been little improvement in the prognosis for SCCHN

oven the past 30 years, and it remains relatively poor, with an

overall 5-year survival rate of 54% (2, 3). Lung cancer is the

leading form of cancer diagnosed in the United States with an

overall incidence rate of 55.2/100,000 population (4). Cigarette

smoking is widely accepted as the major risk factor for the

development of lung cancer, with 80% of lung cancer incidence

attributed to exposure to tobacco smoke (5, 6). SCCHN shares

tobacco smoking as a common major risk factor with lung

cancer, although exposure to smokeless tobacco and alcohol are

additional significant risk factors for SCCHN (7, 8).

Mutations of the p53 tumor suppressor gene are the most

common genetic alteration associated with human cancer. Cur-

rent evidence suggests that the wild-type p53 protein is essential

for normal cell growth regulation and that its alteration or

inactivation is associated with the development of cancer (9). A

major function of the p53 gene is believed to be as a cell cycle

check point gene (10). Thep53 gene is induced by DNA damage

with a resultant transient cell cycle arrest at the G1-S interface

(1 1-13). Cells lacking wild-type p53 do not display this DNA

damage-induced cell cycle arrest (14, 15). The p53 gene has

recently been shown to act as a transactivator of a cell cycle-

associated protein that directly interacts with cyclin-dependent

kinases involved in G1 arrest (16, 17).

Many different point, deletion, and insertion mutations

have been described which can inactivate p53-mediated tumor

suppression (18, 19). The analysis of tumor DNA has revealed

that p53 mutations are usually missense mutations which lead to

amino acid substitutions in the protein, and are primarily found

in one of four evolutionanily conserved regions of nucleotide

sequence located in exons 5-8 (20, 21). Mutations of the p53

tumor suppressor gene are common in both lung cancer (22-26)

and in SCCHN (27-30).

The multistep process of carcinogenesis requires an accu-

mulation of multiple genetic alterations in order for normal cells

to progress to cancer. The role of mutations in the p53 tumor

suppressor gene in human cancer has been well established.

DNA damage may be induced by a number of factors including

endogenous metabolites and exogenous chemical and physical

carcinogenic compounds. It is known that various carcinogens

can induce specific DNA base changes (31, 32). Tissue-specific

mutational spectra may be indicative of the mutation-inducing

Research. on May 19, 2020. © 1995 American Association for Cancerclincancerres.aacrjournals.org Downloaded from

http://clincancerres.aacrjournals.org/

764 p53 Mutational Spectrum and Respiratory Tract Cancer

carcinogenic agents and may reflect underlying mutational

mechanisms. Therefore, the mutational spectra of the p53 gene

for lung cancer and for head and neck cancer may provide

information on the comparability of the underlying causes for

these cancers. In the present study, the p53 mutational spectra

(exons 5-8) were determined and compared for tumors of the

URT and LRT.

MATERIALS AND METHODS

The specimens were surgically resected, histologically con-

firmed tumors obtained from patients with non-small cell lung

cancer or head and neck cancer who were treated at the Uni-

versity of Pittsburgh Medical Center in conjunction with the

Pittsburgh Cancer Institute. Specimens for this study were se-

lected without regard to clinical stage, patient prognosis, therapy

regimen, metastasis, or primary origin of tumor site in the head

and neck region, except that tumors of the thyroid, esophagus,

and skin were not included. Cases were collected consecutively

and selection criteria were based on availability of tissue. Pa-

tients with prior treatment with radiation on chemotherapy were

not excluded from the study but comprised only a small portion

of the study population (15/95). Demographic information was

obtained by questionnaire at the time of tissue biopsy and was

entered into a computer data base by the personnel of the

Pittsburgh Cancer Institute Tissue and Serum Bank.

DNA was isolated from primary fresh-frozen tumor tissue

by guanidine thiocyanate extraction (33) using the commercially

available IsoQuick kit (MicroProbe, Garden Grove, CA). PCR

amplification and sequencing ofp53 exons 5 through 8 (includ-

ing exon-intron boundaries) were performed using previously

published PCR primers (34). All PCR reactions were carried out

in 100 pA total volume on an automatic thermocycler (Perkin

Elmer/Cetus 480 or 9600). The reaction conditions were: 10 msi

Tnis-HC1 (pH 8.3), 50 mM KC1, 1.5 mrvt MgCl2, 0.1% w/v

gelatin, 50 p.M each deoxynucleoside tniphosphate, 0.3 �.LM each

primer, and 1.25 units Taq polymerase (Perkin Elmer/Cetus,

Norwalk, CT). The PCR thermocycler parameters for all ampli-

fications consisted of an initial denaturation at 95#{176}Cfor S mm

followed by 28 cycles at 95#{176}Cfor 1 mm, 60#{176}Cfor 1 mm, and

72#{176}Cfor 1 mm. To confirm correct amplification, the products

were subjected to electrophoresis on 1.6% agarose minigels,

visualized by staining with ethidium bromide, and photographed

under UV light. Removal of primers and deoxynueleoside

tniphosphates as well as concentrations of the PCR products was

done by the use of Micron 100 microconcentrators (Amicon,

Beverly, MA). Direct sequencing of the double-stranded PCR

product was performed by a modification of the standard

dideoxynucleotide chain terminating method (35). PCR prod-

ucts were directly sequenced with fluorescent dye-labeled

dideoxynucleotides using Taq polymerase and cycle sequenc-

ing. The sequencing reactions were performed using the Taq

DyeDeoxy Terminator Cycle Sequencing kit (ABI, Foster City,

CA). Sequencing products were purified of unincorporated dye-

labeled dideoxynucleotides by processing through Centni-Sep

spin columns (Princeton Separations, Princeton, NJ). Sequenc-

ing products were electrophoretically fractionated through 6%

denaturing polyacrylamide gels (0.4-mm thick). Electrophore-

sis, band visualization, and sequence analysis were automati-

cally performed on the Applied Biosystems 373A automatic

sequencer. All mutations were confirmed by sequencing both

DNA strands.

RESULTS

Ninety-five tumor specimens were directly sequenced for

p53 mutations in exons 5-8. Mutations were identified in 29 of

these samples, for an overall frequency of approximately 31%.

All identified mutations were single-base substitutions. Four

were nonsense mutations leading to premature stop codons, 1

was a splice site mutation, 4 were same-sense mutations with no

change in amino acid, and 20 were missense mutations leading

to amino acid substitutions in the protein (Table 1). There was

a total of 16 base transitions (7 C-*T, 7 G->A, and 2 A-’G)

and 13 base transversions (8 G-�C, 3 G-+T, 1 C-�A, 1 C-+G).

The overall distribution of these mutations was relatively ran-

dom across p53 exons 5-8, with 8 mutations identified in exon

8, 3 in exon 7, 10 in exon 6, 7 in exon 5, and 1 splice site

mutation at the 3’ end of intron 4. The most frequently mutated

codons were codons 221, 273, and 299. The mutation at codon

273 was identified in three different URT tumor samples and

was always a G-*A transition at the second nucleotide position

of the codon. This missense mutation should lead to an amino

acid substitution of histidine for arginine in the protein. The

mutation at codon 221 was identified in four different tumor

samples (three URT and one lung) and was always the same

G-�C base substitution at the third nucleotide position of the

codon in the three lung tumors. This mutation leads to an amino

acid substitution of asparagine for glutamic acid in the protein.

The remaining codon 221 mutation was a G-�A base substitu-

tion at the third base position which leads to no amino acid

substitution. The codon 299 base substitution does not lead to an

amino acid substitution and is not located in one of the evolu-

tionanily conserved domains of p53. This silent mutation was

found in three different URT tumor specimens, was always a

G-#{247}Cbase substitution at the third nucleotide position of codon

299, and could not be identified in DNA extracted from the

nontumon tissue of patients. Most of the identified mutations

appeared to be hetenozygous, with both mutant and wild-type

peaks present on sequencing chrornatognaphs (Fig. 1). Tumor

specimen sectioning was performed with careful pathological

review but specimens were not microdissected. The true allelic

status of these mutations (homozygous or heterozygous) is

difficult to assess since normal tissue contamination of the gross

tumor specimen is unavoidable.

The mutational data was stratified for comparison between

the URT and LRT (Table 1). The frequency of identified mu-

tations was nearly identical at 30% (19/63) for URT tumors and

31% (10/32) for lung cancer. The functional nature of the

various types of mutations did not differ dramatically between

URT and LRT tumors (Fig. 2). Missense mutations made up a

significant majority of all identified mutations. Mutations lead-

ing to protein truncation (splice site and premature stop codons)

comprised approximately 16 and 20% of mutations identified in

URT and LRT tumors, respectively.

Differences in p53 mutational profiles were observed be-

tween URT and LRT tumors. Mutations identified in lung

cancer were primarily transversions (70%), whereas the muta-



URT Lung

Sample Exon Codon Mutation” Amino acid Sample Exon Codon Mutation’� Amino acid

5CC 006 8 282 IGG -� IGG Ang -� Trp EDRN 4877 5 167 �AG -� TAG Gln -� stop

5CC 007 5 175 C�jC -� CAC Arg -� His EDRN 5106 6 224 GA�I -p GAl Glu -� Asp5CC 015 8 273 C�1T -� CAT Arg -p His EDRN 5357 6 192 �AG - lAG Gln -� stop

5CC 027 7 248 1�GG -p TGG Arg -� Trp EDRN 5749 8 282 IGG - IGG Arg -� Trp

5CC 037 8 273 C�1T -� CAT Arg -� His EDRN 5791 5 158 C�1C -� CIC Arg -� Leu

5CC 041 8 299 Cr�1 -� Cit Leu -� Leu EDRN 6218 6 189 .�1CC -� �CC Ala -p Pro5CC 042 7 249 AGG -� �1GG Mg -� Gly EDRN 6334 6 221 GA� -* GA� Glu -p Asp5CC 066 6 189 11CC -� �CC Ala -k Pro EDRN 6377 6 221 GA� -� GA� Glu -� AspSCC 071 5 132 MG -� A�G Lys -� Arg EDRN 6425 7 249 AG�1 -� AGI Arg -� 5cr5CC 072 5 179 LAT -� �AT His -� Asn EDRN 6886 6 221 GA�1 -� GA� Glu -� Asp

5CC 074 6 221 GA�1 -� GA� Glu -A Glu5CC 078 5 158 .CGC -� �iGC Arg -� Gly

EDRN 4141 Intron - GT -� AT Splice siteEDRN 4135 6 196 IGA -� IGA Arg -� stop

EDRN 4149 6 196 IGA -� IGA Arg -* stop

EDRN 4882 8 299 CT�j -� C1� Leu -� Leu

EDRN 5176 8 299 CF�j -� CT� Leu -� Leu

EDRN 5700 5 175 C�jC -� CAC Arg -� His

EDRN5811 8 273 C�T-ACAT Arg-*His

Total analyzed (n = 63) Total analyzed (n = 32)

Mutations (19/63) Mutations (10/32)Transitions (13/19) 7 G-*A: 4 C-�T: 2 A-aG Transitions (3/10) 3 C-*T

Transversions (6/19) 4 G-sC: 1 C-+A: 1 C-Ki Transversions (7/10) 3 G-T : 4 G-sC

U LRTU UR

at

U

a

I

Fig. 2 Comparison of the frequency of types ofp53 mutation observed

in URT and lung tumors.

type of mutation

Clinical Cancer Research 765

a Coding strand sequence.

Table 1 p53 Mutational Spectra for URT and LRT

C � I A I C N M A C. 1 C C. A A

Al’� IA

1\! \ � / \f‘�‘J� � t “ \ 1’_g�’�j� ‘

Fig. I p53 DNA sequence eleetrochromatogram of an URT tumor(EDRN 4135). Sequence around p53 codon 196 demonstrating both

wild-type (C) and mutated (7) alleles.

tions observed in URT tumors were primarily transitions (68%).

Comparison of the frequency of specific base substitutions

between URT tumors and lung cancer clearly demonstrates a

difference in observed mutational spectra (Fig. 3). Lung cancer-

associated mutations included 3 C-�T, 3 G-*T, and 4 G-�C

base substitutions (coding strand). URT tumor mutations in-

eluded 7 G-�A, 4 C-�T, 2 A-�G, 4 G-�C, 1 C-�A, and 1

C-�G base substitutions (coding strand). Mutations involving

G:C-’A:T transitions were the most frequently identified alter-ation in URT tumors (1 1/19) and G:C-�A:T mutations at CpG

sites, which are characteristic of endogenous processes, were

observed frequently in URT tumors (9/19) but only rarely in

lung tumors (1/10). Additionally, G:C-�T:A tnansversions,

which were relatively common in lung tumors (3/10), were rare

in URT tumors (1/19). No significant associations were ob-

served when the mutational data were further stratified to look

for possible associations between specific nucleotide substitu-

tions by tumor site and occupation (data not shown). Two-

variable analyses failed to show any statistical relationship

between the type of mutation and gender, smoking history, or

history of alcohol consumption (Table 2).

DISCUSSION

The combined mutational data on URT and LRT tumors

suggest that p53 mutations are relatively frequent in respiratory

tract cancers, and that the nature of the mutations and their

distribution are not unusual when compared with p53 mutations

identified in other human cancers. However, comparison of the

frequency of specific base substitutions between URT and LRT

tumors showed a striking difference in p53 mutational profiles.

Among tissues with mutations, transversion mutations occurred

twice as often in lung tumors and transition mutations twice as

often in URT tumors. This difference achieved a borderline

level of statistical significance (P = 0.064). This difference

could not be explained by differences in environmental expo-



a UR

. LR

c->T - G->A � A->G � C->A C->G G->T G->Ctype of mutatIon


C0a

E0

UCa0�

Fig. 3 Specific p53 nucleotide substitutions observed in URT and lung

tumors (coding strand sequences).

sure as it relates to tobacco smoke, alcohol consumption, or

occupation.

Previous reports have found similar mutational profiles in

SCCHNs and squamous cell carcinoma of the lung (27, 29). The

difference observed in the p53 mutational spectra of URT and

LRT may be due, in pant, to the difference in the pathology of

the tumors. The majority of the URT tumors in which p.53 muta-

tions have been identified were squamous cell carcinomas (86%).

The two URT samples that were not squamous cell carcinomas

were 5CC 006 (adenoid cystic carcinoma) and 5CC 015 (muco-

epidermoid carcinoma), both of which displayed transition muta-

tions, whereas the majority of the lung cancer specimens (21/32)

and lung tumors identified with mutations (7/10) were primarily

adenocarcinornas (Table 3). The three lung tumors identified with

p53 mutations that were not adenocarcinomas were EDRN 4877,

EDRN 5791 (squamous cell carcinomas), and EDRN 6334 (large

cell carcinoma). However, the difference in pathology between

URT and lung tumors may not totally account for the observed

difference in mutational spectra, since comparison of base substi-

tutions identified in only the squamous cell carcinoma samples still

suggests a difference in the mutational profile between URT and

LRT tumors. One of the two lung squamous cell carcinoma sam-

ples identified with p53 mutations was a G:C-�T:A base substi-

tution, whereas this type of mutation was identified in only 1 of 17

URT squamous cell carcinoma mutations. Additionally, a recent

comparison of p53 mutations reported for adenocarcinoma and

squamous cell carcinoma of the lung show similar profiles with

approximately identical transversion frequencies (36).

An even more pronounced difference in the p53 mutational

spectra is observed between the URT and LRT when only

functionally relevant mutations are compared. Elimination of

the codon 299 (G-��C) same-sense mutation identified in three

URT tumors results in a URT mutational profile which consists

of 81% (13/16) base transitions, of which 85% (11/13) were

G:C-�A:T. A difference in mutational spectrum between head

and neck squamous cell carcinoma samples and lung cancer has

been previously reported (37). The p53 mutational profile of

URT tumors derived from our data more closely fits that of

previously reported p53 mutational spectra observed in gastro-

intestinal tract cancers (36, 38, 39). A recent, extensive compi-

lation and comparison of 2567 reported p53 mutations in human

Table 2 Com panison of m utation type t

drinking

o gender, smok ing, and

No mutation

(%)Transition

(%)Transversion

(%)x2

test

Gender

Men (54)

Women (41)

Smoking

Yes (80)

No (15)

Drinking

Yes (58)

No (33)

66.6

73.2

71.2

60.0

68.9

69.7

20.4

12.2

17.5

13.3

19.0

12.1

13.0

14.6

11.3

26.7

12.1

18.2

P = 0.57

P = 0.28

P = 0.75

cancers showed a similar, albeit not as pronounced, difference

between the profiles of 897 lung cancer mutations and 524 head

and neck tumor mutations (36). Approximately 1.5 times as

many transversions were observed in lung tumors as in head and

neck tumors with a preponderance of G:C-+T:A lung mutations

(40%) and a preponderance of G:C-�A:T (31%) head and neck

mutations (36). The reasons for the observed difference in

mutational profiles between the URT and LRT is uncertain, but

may reflect differences in both exogenously induced tissue-

specific mutational mechanisms and underlying endogenously

induced mutation rate variation between the tissues of the upper

and lower respiratory systems. The G:C-�A:T mutations at

CpG dinucleotides are due to frequent rnethylation of cytosine

to 5-methylcytosine and subsequent spontaneous deamination to

thyrnine, resulting in G:T mismatches which may not be me-

paired accurately (40). This mutational mechanism is an endo-

genous process for which no exogenous factors have yet been

identified that alter the frequency of methylation, deamination,

or the efficiency of repair leading to these CpG-related muta-

tions (41). In our study, G:C-�A:T mutations at CpG sites were

observed in 47% of URT tumors but only in 10% of lung

tumors. This suggests that endogenous biological factors may

play a role in determining some of the observed differences in

the p53 mutational spectra between the upper and lower respi-

ratory systems. The reasons for differences in endogenous tis-

sue-specific mutation rates for the p53 gene, as ascertained by

CpG mutations, is unknown but has been well documented

(reviewed in Ref. 41).

The higher frequency of G:C-#{247}T:A mutations in lung

tumors is representative of the type of mutation known to be

caused by polycyclic aromatic hydrocarbons, most notably ben-

zo(a)pyrene, that are present in tobacco smoke. The upper

aerodigestive and LRTs share tobacco smoke as a significant

common exogenous mutagenic risk factor. However, our data

suggest that either different tobacco-related mutagens or differ-

ent mutational mechanisms are involved in p53 mutation in

URT and LRT cancers. Tobacco smoke consists of many po-

tentially mutagenic substances, some of which have been well

characterized, such as N-nitrosamines and polycyclic aromatic

hydrocarbons, but tobacco smoke also consists of many other

compounds which have not been well defined and whose mu-

tagenic potentials are unknown. The concentration and duration

of exposure to the various compounds in tobacco smoke would

be expected to vary considerably between the URT and LRT.



Clinical Cancer Research 767

Table 3 Sample pathology, mutational status, and smoking history

Code’� Path” p53C Smoke Code Path p53 Smoke Code Path p53 Smoke

003 5CC wt + 048 5CC wt + L4789 ADC wt +

004 5CC wt + 055 5CC wt + L4841 ADC wt +

006 ACC mu - 058 5CC wt + L4877 5CC mu +

007 SCC mu + 059 MVC wt + L4899 5CC wt +

009 5CC wt + 060 5CC wt + L4926 5CC wt +

010 5CC wt + 061 5CC wt + L5011 5CC wt +

01 1 5CC wt + 066 5CC mu + L5037 ADC wt +

012 5CC wt + 067 5CC wt + L5106 ADC mu +

013 5CC wt + 069 5CC wt + L5224 ADC wt -

015 MEC mu + 070 5CC wt + L5229 SCC wt +

017 5CC wt + 071 5CC mu + L5317 ADC wt -

019 5CC wt + 072 SCC mu + L5357 ADC mu +

020 5CC wt - 073 5CC wt + L5506 ADC wt -

024 5CC wt + 074 5CC mu - L5517 5CC wt +

026 5CC wt - 075 5CC wt + L5749 ADC mu +

027 5CC mu + 078 5CC mu - L5791 SCC mu +

030 5CC wt + 079 5CC wt + L6020 5CC wt +

031 SCC wt - 080 5CC wt + L6041 ADC wt +

033 5CC wt + 4135 5CC mu + L6087 5CC wt +

034 5CC wt - 4141 5CC mu + L6120 ADC wt -

035 MED wt + 4149 5CC mu + L6218 ADC mu +

036 5CC wt + 4174 5CC wt + L6311 ADC wt +

037 SCC mu + 4684 5CC wt + L6334 LCC mu +

038 5CC wt + 4882 5CC mu - L6377 ASC mu -

039 5CC wt + 5176 5CC mu + L6425 ADC mu +

040 5CC wt - 5328 5CC wt + L6272 ADC wt +

041 5CC mu + 5416 5CC wt + L6879 ADC wt +

042 5CC mu + 5624 5CC wt + L6886 ADC mu -

043 5CC wt + 5700 5CC mu - L691 1 ASC wt +

044 5CC wt + 5710 5CC wt + L6906 ADC wt +

045 5CC wt + 5811 5CC mu + L6957 ADC wt +

046 5CC wt + L4763 5CC wt +

a Lung sample codes are preceded by L.b 5CC, squamous cell carcinoma; ACC, adenoid cystic cancer; MEC, mucoepidermoid cancer; MED, mild epithelial dysplasia; MVC, mixed

verrueous cancer; ADC, adenocarcinoma; ASC, adenosquamous carcinoma; LCC, large cell carcinoma.

C mu, mutation; wt, wild type.

Additionally, the upper aerodigestive tract is exposed to differ- ACKNOWLEDGMENTS

ent known environmental risk factors not associated with the We thank Diana Kerestan and Anee Deka for their assistance in

LRT. Some of these risk factors include alcohol, sodium nitrites, obtaining DNA sequencing data, Christa Lese and Jaya Reddy for their

and smokeless tobacco products. The fact that the p53 muta- assistance in the acquisition of specimens and associated patient infor-

tional profile observed for URT tumors was more similar to that mation, and the staff of the Pittsburgh Cancer Institute Tissue and Serum

previously reported in gastric cancer than to LRT tumors would Bank for coordination of specimen accrual and processing.

suggest that these additional factors may play a role in deter-

mining the underlying mutational events of these tumors. There REFERENCESis evidence to suggest that tumors of the head and neck are � � Parkin, D. M., Laara, E., and Muir, C. S. Estimates of the worldwide

associated with both alcohol and tobacco smoke (42), and that frequency ofsixteen major cancers in 1980. Int. J. Cancer, 41: 184-197.

there may be a synergistic effect between the two. . . . .2. Boring, C. C., Squires, T. S., and Tong, T. Cancer statistics. CA

Environmental agents that induce specific mutational Cancer J. Clin., 42: 19-38, 1992.

events may exert a general field cancenization effect on the �. Benninger, M. S., Roberts, J. K., Levine, H. L., Wood, B. G., and

exposed tissues (43, 44). Detection of critical mutational events Tucker, H. M. Squamous cell carcinoma of the head and neck in patients

in these exposed tissues, at an early stage (jreneoplastic le- 40 years of age and younger. Laryngoscope, 98: 531-534, 1988.

sions), may lead to earlier diagnosis and therapeutic intervention � National Cancer Institute Division of Cancer Prevention and Control,. Annual Cancer Statistics Review. Bethesda MD: National Institutes of

and to a better overall prognosis. A recent report has demon- Health 1988.

strated that elevated levels of p53 are present in premalignant . .

. . 5. Doll, R., and Hill, A. B. A study of the aetiology of carcinoma of thelesions of the head and neck (45). We are pursuing further lung. Br. Med J., 2: 1271-1286, 1952.

investigation to determine whether mutations of the p53 gene 6. Hammond, E. C., and Seidman, H. Smoking and cancer in the United

are associated with early lesions of the respiratory tract. States. Prey. Med., 9: 169-173, 1980.




7. Jacobs, C. D., Goffinet, D. R., and Fee, W. E., Jr. Head and neck

squamous cancers. Curr. Probl. Cancer, 14: 1-72, 1990.

8. Creath, C. J., Cutter, G., Bradley, D. H., and Wright, J. T. Oral

leukoplakia and adolescent smokeless tobacco use. Oral Surg. Oral

Med. Oral Pathol., 72: 35-41, 1991.

9. Donehower, L. A., Harvey, M., Slagle, B. L., McArthur, M. J.,

Montgomery, C. A., Jr., Butel, J. S., and Bradley, A. Mice deficient for

p53 are developmentally normal but susceptible to spontaneous tu-

mours. Nature (Land.), 356: 215-221, 1992.

10. Lane, D. P. p53, guardian of the genome. Nature (Land.), 358:

15-16, 1992.

1 1 . Maltzman, W., and Czyzyk, L. UV irradiation stimulates levels ofp53 cellular tumor antigen in nontransformed mouse cells. Mol. Cell.

Biol., 4: 1689-1694, 1984.

12. Kastan, M. B., Onyekwere, 0., Sidransky, D., Vogelstein, B., and

Criag, R. W. Participation of p53 protein in the cellular response to

DNA damage. Cancer Res., 51: 6304-6311, 1991.

13. Hall, P. A., McKee, P. H., Menage, H. D., Dover, R., and Lane,D. P. High levels of p53 protein in UV irradiated human skin. Onco-gene, 8: 203-207, 1993.

14. Livingstone, L. R., White, A., Sprouse, J., Livanos, E., Jacks, T.,

and Tlsty, T. D. Altered cell cycle arrest and gene amplification poten-

tial accompany loss of wild-type p53. Cell, 70: 923-935, 1992.

15. Yin, Y., Tainsky, M. A., Bischoff, F. Z., Strong, L. C., and Wahl,G. M. Wild-type p53 restores cell cycle control and inhibits geneamplification in cells with mutant p53 alleles. Cell, 70: 937-948, 1992.

16. El-Deiry, W. S., Tokino, T., Velculescu, V. E., Levy, D. B., Par-sons, R., Trent, J. M., Lin, D., Mercer, W. E., Kinzler, K. W., and

Vogelstein, B. WAF1, a potential mediator of p53 tumor suppression.

Cell, 75: 817-825, 1993.

17. Harper, J. W., Adami, G. R., Wei, N., Keyomarsi, K., and Elledge,S. J. The p21 Cdk-interacting protein Cipi is a potent inhibitor of G3cyclin-dependent kinases. Cell, 75: 805-816, 1993.

18. Harris, N., Brill, E., Shohat, 0., Prokocimer, M., Wolf, D., Arai, N.,

and Rotter, V. Molecular basis for heterogeneity of the human p53

protein. Mol. Cell. Biol., 6: 4650-4656, 1986.

19. Hinds, P., Finlay, C., and Levine, A. J. Mutation is required to

activate the p53 gene for cooperation with the ras oncogene and trans-

formation. J. Virol., 63: 736-746, 1989.

20. Nigro, J. M., Baker, S. J., Preisinger, A. C., Jessup, J. M.,

Hostetter, R., Cleary, K., Bigner, S. H., Davidson, N., Baylin, S.,

Devilee, P., Glover, T., Collins, F. S., Weston, A., Modali, R., Harris,

C. C., and Vogelstein, B. Mutations in the p53 gene occur in diverse

human tumour types. Nature (Land.), 342: 705-708, 1989.

21. Levine, A. J., Momand, J., and Finlay, C. A. The p53 tumour

suppressor gene. Nature (Land.), 351: 453-456, 1991.

22. Takahashi, M. M., Nau, I., Chiba, M. J., Birrer J., Rosenberg, R. K.,

Vinocour, M., Levitt, M., Pass, H., Gazdar, A. F., and Minna, J. D. p53:

A frequent target for genetic abnormalities in lung cancer. Science

(Washington DC), 246: 491-494, 1989.

23. Chiba, I., Takahashi, T., Nau, M. M., D’Damico, D., Curiel, D. T.,

Mitsudomi, T., Buchhagen, D. L., Carbone, D., Piantadosi, S., Koga, H.,

Reissman, P. T., Slamon, D. J., Holmes, E. C., and Minna, J. D.

Mutations in the p53 gene are frequent in primary, resected non-small

cell lung cancer. Oncogene, 5: 1603-1610, 1990.

24. Hollstein, M., Sidransky, D., Vogelstein, B., and Harris, C. C. p.53

Mutations in human cancers. Science (Washington DC), 253: 49-53,

1991.

25. Kishimoto, Y., Murakami, Y., Shiraishi, M., Hayashi, K., and

Sekiya, T. Aberrations of the p53 tumor suppressor gene in humannon-small cell carcinomas of the lung. Cancer Res., 52: 4799-4804,

1992.

26. Suzuki, H., Takahashi, T., Kurioshi, T., Suyama, M., Aniyoshi, Y.,Takahashi, T., and Ueda, R. p53 Mutations in non-small cell lung cancer

in Japan: association between mutations and smoking. Cancer Res., 52:

734-736, 1992.

27. Somers, K. D., Merrick, M. A., Lopez, M. E., Incognito, L. S.,

Schechter, G. L., and Casey, G. Frequent p53 mutations in head and

neck cancer. Cancer Res., 52: 5997-6000, 1992.

28. Yin, X-Y., Smith, M. L., Whiteside, T. L., Johnson, J. T., Herber-man, R. B., and Locker, J. Abnormalities in the p53 gene in tumors and

cell lines of human squamous-cell carcinomas of the head and neck. Int.

J. Cancer, 54: 322-327, 1993.

29. Boyle, J. 0., Hakim, J., Koch, W., van der Riet, P., Hruban, R. H.,

Roa, R. A., Correo, R., Yolanda, J., Eby, J., Ruppert, M., and

Sidransky, D. The incidence ofp53 mutations increase with progression

of head and neck cancer. Cancer Res., 53: 4477-4480, 1993.

30. Nees, M., Homann, N., Diseher, H., Andl, T., Enders, C., Herold-Mende, C., Schuhmann, A., and Bosch, F. X. Expression of mutated p53

occurs in tumor-distant epithelia of head and neck cancer patients: a

possible molecular basis for the development of multiple tumors. Cancer

Res. 53: 4189-4196, 1993.

31. Zarbl, H., Sukumar, S., Arthur, A. V., Martin-Zanca, D., and

Barbacid, M. Direct mutagenesis of Ha-ras-1 oncogenes by N-nitroso-

N-methylurea during initiation of mammary careinogenesis in rats.

Nature (Land.), 315: 382-385, 1985.

32. Quintanilla, M., Brown, K., Ramsden, M., and Balmain, A. Car-

cinogen-specific mutation and amplification of Ha-ras during mouse

skin carcinogenesis. Nature (Land.), 322: 78-80, 1986.

33. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J., and Rutter,

W. J. Isolation of biologically active ribonueleic acid from sourcesenriched in ribonuclease. Biochemistry, 18: 5294-5296, 1979.

34. Law, J. C., Strong, L. C., Chidambaram, A., and Ferrell, R. E. A

germ line mutation in exon 5 of the p53 gene in an extended cancer

family. Cancer Res., 51: 6385-6387, 1991.

35. Sanger, F., Nickley, S., and Coulson, A. R. DNA sequencing with

chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA, 74: 5463-

5476, 1977.

36. Greenblatt, M. S., Bennett, W. P., Hollstein, M., and Harris, C. C.

Mutations in the p53 tumor suppressor gene: clues to cancer etiology

and molecular pathogenesis. Cancer Res., 54: 4855-4878, 1994.

37. Brachman, D. G., Graves, D., Vokes, E., Beckett, M. Haraf, D.,

Montag, A., Dunphy, E., Mick, R., Yandell, D., and Weichselbaum,R. R. Occurrence of p.53 gene deletions and human papilloma virus

infection in human head and neck cancer. Cancer Res., 52: 4832-4836,

1992.

38. Yokozaki, H., Kuniyasu, H., Kitadai, Y., Nishimura, K., Todo, H.,

Ayhan, A., Yasui, W., Ito, H., and Tahara, E. p53 Point mutations in

primary human gastric carcinomas. Cancer Res. Clin. Oncol., 119:

67-70, 1992.

39. Renault, B., van den Broek, M., Fodde, R., Wijnen, J., Pellegata,N. S., Amadori, D., Khan, P. M., and Ranzani, G. N. Base transitions arethe most frequent genetic changes at p53 in gastric cancer. Cancer Res.,

53: 2614-2617, 1993.

40. Coulondre, C., Miller, J. M., Farabaugh, P. J., and Gilbert, W.

Molecular basis of base substitution hotspots in Escherichia coli. Nature

(Land.), 274: 775-780, 1978.

41. Jones, P. A., Buckley, J. D., Henderson, B. E., Ross, R. K., and

Pike, M. C. From gene to carcinogen: a rapidly evolving field inmolecular epidemiology. Cancer Res., 51: 3617-3620, 1991.

42. Blot, W. J., McLaughin, J. K., Winn, D. M., Austin, D. F., Green-

berg, R. S., Preston-Martin, S., Bernstein, L., Schoenberg, J. B., Stem-hagen, A., and Fraumeni, J. F. Smoking and drinking in relation to oral

and pharyngeal cancer. Cancer Res., 48: 3282-3287, 1988.

43. Strong, M. S., Ineze, J., and Vaughan, C. W. Field eancerization inthe aerodigestive tract-its etiology, manifestation, and significance. J.

Otolaryngol., 13: 1-6, 1984.

44. Slaughter, T. P., Southwiek, H. W., and Smejkel, W. Field cancer-

ization in oral stratefied epithelium. Cancer (Phila.), 6: 963-968, 1953.

45. Shin, D. M., Kim J., Ro, J. Y., Hittelman, J., Roth, J. A., Hong,W. K., and Hittelman, N. Activation of p53 gene expression in prema-

lignant lesions during head and neck tumorigenesis. Cancer Res., 54:

321-326, 1994.



1995;1:763-768. Clin Cancer Res J C Law, T L Whiteside, S M Gollin, et al. upper and lower respiratory tract.Variation of p53 mutational spectra between carcinoma of the

Updated version

http://clincancerres.aacrjournals.org/content/1/7/763

Access the most recent version of this article at:

E-mail alerts related to this article or journal.Sign up to receive free email-alerts

Subscriptions

Reprints and

[email protected] at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

Permissions

Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://clincancerres.aacrjournals.org/content/1/7/763To request permission to re-use all or part of this article, use this link



http://clincancerres.aacrjournals.org/cgi/alerts

mailto:[email protected]



variation of p53 mutational spectra between carcinoma of ... · vol. 1, 763-768, jo/v 1995 clinical...

Documents