[CANCER RESEARCH 52. 4832-4836, September I. 1992]

Advances in Brief

Occurrence of p53 Gene Deletions and Human Papilloma Virus Infection inHuman Head and Neck Cancer1

David G. Brachman,2 Deborah Graves, Everett Yokes, Michael Beckett, Daniel Haraf, Antony Montag,

Edward Dunphy, Rosemarie Mick, David Yandell, and Ralph R. Weichselbaum

Departments of Radiation and Cellular Oncology fD. G. B., D. G., E. V.. M. B., D. H., E. D., R. R. W.], Pathology [A. M.J. and Medicine [R. M.J, University ofChicago Hospitals. Chicago, Illinois 60637, and Department of Ophthalmology, Massachusetts Eye and Ear Infirmary and Harvard Medical School, Boston,Massachusetts 02114 [D. Y.J

Abstract

Little is known regarding the molecular genetic events in head andneck carcinoma. Epidemiológica! evidence suggests that both alcoholand tobacco use are related to the development of these neoplasms, andviral infections have also been postulated to play a role in some tumors.Loss of pSi tumor suppressor gene function has been found in manymalignancies and can occur through either gene mutation or by interaction with the E6 protein of oncogenic human papilloma viruses(HPV). Because the mucosa! surfaces of the head and neck are exposedto mutagens and HPVs, we studied DNA derived from 30 stage I-IVsquamous cell carcinomas of the head and neck (9 primary tumorsand 21 early passage cell lines) for p53 gene mutations as well as forthe presence of oncogenic HPV DNA. Exons 2 through 11 of the p53gene were examined using single strand conformation polymorphismanalysis followed by direct genomic sequencing of all variants. HPVdetection was done using polymerase chain reaction amplificationwith HPV E6 region type specific primers as well as LI region degenerate ("consensus") primers; HPV type was determined by restriction

fragment length polymorphism analysis of the amplified fragment aswell as by Southern blotting of genomic DNA. Sixteen of 30 tumors(53%) had p53 mutations and oncogenic HPV DNA was detected in3 of 30 (10%) tumors, none of which had p53 mutations. The pS3mutational spectrum observed was characterized by equal frequenciesof transversions (6 of 16), transitions (5 of 16), and deletions (5 of16). This distribution of mutations differs from the spectrum ofp53 mutation reported in esophageal (/' = 0.05) and lung (/' = 0.02)cancers, two other tobacco associated neoplasms. A previously unde-scribed clustering of 3 mutations at codon 205 was also observed. Atrend toward a shorter time to tumor recurrence after treatment wasnoted for those patients with tumors exhibiting p53 gene mutations,and no relationship between p53 mutations and tumor stage or nodestatus was noted. Alteration in p53 gene function appears commonin head and neck cancer, and the mutational spectrum observedmay reflect the role of different mutagens or mutagenic processesthan those responsible for the p53 mutations in lung and esophagealneoplasms.

Introduction

Human malignancies arising in the head and neck regionrepresent a significant cause of morbidity and mortality worldwide. Primarily of squamous cell histology, these neoplasmsaccount for ~5% of malignancies in the United States (1) to40-50% in parts of Southeast Asia and India (2). Tobacco andalcohol use, viral infections, nutritional deficiency, and localdietary customs have all been implicated in the etiology of head

Received 6/18/92; accepted 7/27/92.The costs of publication of this article were defrayed in part by the payment of

page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 17.14 solely to indicate this fact.

' This work was supported by NIH Grant CA 41068. The Chicago TumorInstitute, and the Center for Radiation Therapy.

2 To whom requests for reprints should be addressed.

and neck cancer (1). Whereas the epidemiology of HNSCC3has been well described, the molecular steps involved in induction of these common neoplasms are poorly understood.

Loss of function of tumor suppressor genes has been implicated in the etiology and/or progression of a variety of humantumors (3). In the case of the p53 tumor suppressor gene, lossof function may confer a proliferati ve advantage and subsequentclonal expansion of tumor cells with altered p53 gene function(4). Several mechanisms of p53 inactivation have been described, including gene mutation and viral interaction. In thecase of gene mutation, the resulting DNA sequence change maylead to either an absence of p53 protein production or an abnormal protein product (3). Gene mutation appears to be thepredominant mechanism ofp53 inactivation in lung (5-10) andesophageal cancer (11-14). Abrogation ofp53 function can alsooccur via complex formation with certain viral proteins, including the E6 protein produced by oncogenic HPVs (3). OncogenicHPV DNA is detectable in over 80% of cervical squamous cellneoplasms and it has been suggested this association with thep53 protein may play a role in the transforming ability of theseHPV types (15).

Since the mucosal surfaces of the head and neck region areexposed to both mutagens and HPVs, we examined 30 tumor-derived DNA specimens for both p53 gene mutations and thepresence of HPV DNA. Furthermore, inasmuch as the specifictype and location of mutations found within the p53 gene insome tumors may reflect specific mutagen exposure or mutagenic processes (16), we compared the mutational spectrumobserved in our study to that reported for lung and esophagealcancers, two neoplasms closely linked to smoking.

Materials and Methods

Tumor Specimens. Tumor specimens were collected at the time ofbiopsy or surgical excision. We chose to study 30 head and neck tumorsof squamous cell histology for which clinical follow-up was available.All patients reported regular use of tobacco and alcohol. Specimenswere obtained prior to any therapy in 20 patients and at the time ofdisease recurrence in 10 patients. In 21 cases, the available tumor tissuewas scant due to the limited extent of the surgical procedure performed;these tumors were placed into short-term cell culture and DNA wasobtained from early passage cell lines as previously described (17).Histological review of the remaining 9 cases disclosed 50-90% tumortissue and DNA was extracted from frozen specimens using publishedtechniques (12). Materials for study were selected without regard toclinical stage, site of primary origin within the head and neck region,therapy received, or patient outcome. All samples were numericallycoded and the clinical details were unknown to the principal investigators until completion of the laboratory analyses.

3 The abbreviations used are: HNSCC. head and neck squamous cell cancer;HPV. human papilloma virus; PCR. polymerase chain reaction; SSCP, singlestrand conformation polymorphism.

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pS3 AND HPV IN HEAD AND NECK CANCER

p53 SSCP and Direct Genomic Sequencing. Exons 2-11 of the p53gene were examined for alterations in DNA sequence using PCR amplification (18) from genomic DNA followed by SSCP analysis (19) anddirect genomic DNA sequencing (20) of all variants as described previously (21). This approach has been shown to be a rapid and sensitivemeans of identifying DNA sequence variations as small as a single basesubstitution (19). For SSCP, exon-specific PCR primers were chosen soas to include 10-20 base pairs of intron both 5' and 3' to the exon of

interest. Electrophoresis was carried out twice for each sample, once ona 6% nondenaturing polyacrylamide gel and once on a similar gel butwith the addition of 10% glycerol. Generation of amplified DNA fordirect genomic sequencing was accomplished using a second PCR reaction with at least one new primer external to that used for SSCPanalysis. Amplified DNA samples were combined with one of severaldifferent 32P-end labeled primers internal to the amplified fragment,

and both the coding and noncoding strands were examined. All mutations were confirmed by repeat sequencing utilizing a separate PCRreaction.

HPV Detection and Typing. All 30 tumor specimens were examinedfor the presence and type of HPV DNA with 2 independent PCR-basedmethods (22, 23). Using the first method, oligonucleotide primers corresponding to a region of the E6 gene of HPV-16 and a separate set ofprimers with homology to the E6 region of HPV-18 were used inindependent PCR reactions (22). In the presence of the E6 gene ofHPV-16or-18, this technique generates an amplified HPV DNA fragment of type-specific length and restriction-enzyme digest pattern (22).The second method used was that of HPV LI gene consensus primeramplification of Manos et ai. (23). Triple restriction enzyme digest ofthe resulting approximately 450 base-pair amplified fragment withHaein, Pstl, and Rsal restriction endonucleases allows detection andtyping of HPVs 16, 18, and 33 as well as 1, 2, 5,6, 8, 11,31, 39,41,47,51, and 57 (24). With both techniques results are visualized on ethidiumbromide stained agarose gels. Amplifications, digests, and gel electro-phoresis were repeated in triplicate. Known HPV positive and HPV

negative controls were utilized for all amplifications, digests and gels;"no template" reactions were included with each PCR amplification.

Southern blot analysis was performed on HPV positive samples using5 Mgof genomic DNA digested by Hindlll restriction endonuclease andelectrophoresed through a 0.8% agarose gel. The digested DNA wastransferred to a GeneScreen Plus membrane (NEN) and sequentiallyhybridized to •¿�t2P-endlabeled probes made from oligonucleotide primers homologous to the E6 regions of HPV-16 and HPV-18, respectively(22). The membranes were washed twice under stringent conditions andexposed to X-ray film with an intensifying screen at -80°C for 1-4

days. This procedure was repeated on the same specimens using Dde\restriction endonuclease; all other conditions remained the same asdescribed above.

Statistical Analysis. Analysis of time to disease recurrence frominitiation of therapy was estimated by the Kaplan-Meier method (25)and the p53 mutation groups compared by the log-rank test (26). Association of p53 mutation with disease recurrence and with site ofprimary tumor were assessed by the Fisher exact test (27). Comparisonof tumor site (lung, esophagus, head and neck) by distribution of mu-tational spectrum was also assessed by the Fisher exact test. Associationof p53 mutational status and tumor and node stage were performed bythe exact Wilcoxon rank sum test (28). All P values were two-sided.Exact testing was performed with StatXact software (Cytel Software,Cambridge, MA).

Results

Using a combination of SSCP and direct genomic sequencing, we examined exons 2 through 11 of the p53 gene in 30squamous cell head and neck neoplasms. Mutations of the genewere identified in 16 of 30 (53%) tumors. The presence ofoncogenic HPV DNA was detected in 3 additional tumors(10%), none of which had p53 mutations (Table 1). The 16

Table I Summary of primary site, TNM" stage, p53 status, HPV status, outcome, and initial treatment for 30 patients with head and neck squamous cell carcinoma

TumorHNSCC

42SQ20BSQ3IdHNSCC

294HNSCC313JSQ

3</HNSCC

151HNSCC152HNSCCUS'*HNSCC29JSCC25dHNSCC

80SQ9G''HNSCC

28dSCCIS"HNSCC166HNSCC257HNSCC

340HNSCC349JSQ13HNSCC296BHNSCC324HNSCC104HNSCC143HNSCC\i\dHNSCC

297HNSCC58SCC61HNSCC

30IBHNSCC308Primary

siteOral

tongueLarynxPyriform

sinusOraltongueParotidNasal

vestibuleOraltongueParotidHard

palateFloorofmouthFloorofmouthNasopharynxTonsilLarynxPyriform

sinusHardpalateHardpalateEthmoid

sinusPyriformsinusAlveolarridgeLarynxTonsilPyriform

sinusBaseoftongue/tonsilOral

tongueTonsilBase

oftongueOraltongueUnknownprimaryLarynxTNM

stage*T.NoMoT2N0M0T2N0M0T2N„M0T2N0MoT,N0M0T,N0M0T3N0M0T,N,MOT,N,MOT2N,M0T2N,M0TjN,M0TjN.MoT4N0MoT4NoM0T4N0M0T4N0M0T4NoM0T4N,M0T2N2M0T3N2cM0TjNjaMoT,N3bM0T4N2aMoT4N2aMoT4N2bM0T4N2bM0T,N2M,T4N3M0p53mutationYesYesNoYesNoYesYesYesYesYesYesYesYesNoNoNoNoNoNoYesYesNoNoNoYesNoNoNoYesYesHPVpresent/typeNoNoNoNoNoNoNoNoNoNoNoNoNoNoNoYes/

18NoNoNoNoNoYes/

16NoNoNoYes/

16NoNoNoNoOutcomes'LF5

moLF4moLF7moNED

15moDiedNED 3moLF4moLF3moLF

6moLF6moLF6moPersistent

diseaseNED37moLF8

moLF9moLF

25moDiedNED 43moPersistent

disease*NED

10moPersistentdiseaseDied

duringRT*NED12moNED13moDied

NED 26moLF12moLF7

moNED19moDied

NED 12moPersistentdiseaseLF

4moNED17 moInitial

treatmentSRTRT

—¿�'CxS—¿�RTSRTRTSS

—¿�RTS-RTRTCx+^RTRTS-RTRTS

—¿�RTPartialsurgicalremoval—

protractedRTCx—¿�S —¿�RTRTRTS

—¿�RTCx- S - RT +CxCx—¿�S —¿�RT +CxCx—¿�RtS-RTCx

—¿�RTCx—¿�S —¿�RT +CxRTRT

+CxCx—¿�S —¿�RT + Cx

" TNM, tumor-nodes-metastasis; LF, local failure: NED, no evidence of disease: S, surgery; RT. radiotherapy: Cx. chemotherapy.* Staging according to American Joint Committee on Cancer. 1988.1 Interval since treatment initiation: all patients with local failure ultimately died of their disease.J Denotes specimens collected at time of local failure.' Sequential treatments.•¿�^Concurrenttherapy.* Excluded from outcome analysis due to incomplete therapy.

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pS3 AND HPV IN HEAD AND NECK CANCER

mutations were found between codons 126 and 307, corresponding to exons 5 through 9 of the p53 gene. Five of 16(31%) mutations were small deletions, and 11 of 16 (69%) weresingle base pair substitutions (6 transversions and 5 transitions)(Table 2). Four of 5 deletions were homozygous, and all causeframe-shift errors that would result in premature stop codonformation. Three of 6 transversions were G:C to T:A changes,2 of 6 produced A:T to C:G mutations, and one produced anA:T to T:A mutation. The transitions were G:C to A:T in 3tumors and A:T to G:C in 2 tumors. All but 2 base pair substitutions were clear homozygous changes; in two primary tumors(HNSCC 301B and HNSCC 308) we could not fully differentiate between the possibility of a true heterozygous mutationand a homozygous mutation with stromal contamination simulating a heterozygous state. One tumor (SQ 20B) demonstrated an altered exon 5 splice-acceptor site caused by a G to Atransition; the remaining base substitutions alter the amino acidcoding sequence in 9 cases and result in a premature stop codonin 1 case (Table 2). Of the 11 base substitution mutations observed, 3 of 11 (27%) occurred at codon 205, all involvingseparate nucleotide substitutions (Table 2; Fig. 1). Nine of 16(56%) mutations in these head and neck neoplasms occurred intwo distinct regions of the gene, codons 203-209 (5 mutations)and 245-249 (4 mutations).

The same 30 DNA samples were analyzed for the presence ofoncogenic HPV with two independent, PCR-based methods.Using type-specific (HPV-16 or -18) oligonucleotide PCRprimers which anneal to the E6 region, HPV-16 was detected intwo tumors (HNSCC 297, HNSCC 324) and HPV-18 in onetumor (HNSCC 166) (Table 1). The second method used wasthe HPV LI region consensus primer technique combined with

RFLP analysis of the resulting amplified DNA fragment. Usingthis technique, no additional cases of HPV infection were detected. For samples HNSCC 297 and HNSCC 324, RFLP analysis demonstrated HPV-16, in agreement with the E6 regiontype-specific technique results. In the case of HNSCC 166 nodigestion of the amplified DNA was seen with any of the threeenzymes used, despite the generation of an appropriate lengthPCR fragment. Further evaluation of this tumor using Southern blot analysis of genomic DNA disclosed a restriction digestenzyme pattern that does not correspond to any known HPVtype (data not shown). One explanation for this finding may bethat rearrangement or loss of a portion of the viral genomelying outside the E6 region may have occurred during or afterintegration but we cannot completely rule out the possibility ofthe detection of a novel HPV type.

p53 gene mutations occurred most commonly in tumors ofthe oral cavity (8 of 11) and larynx (3 of 4); only 1 of 8 oro- orhypopharyngeal lesions had sequence alterations (Table 1). Mutations were equally distributed among all tumor stages and noassociation between mutational status and node stage wasnoted; only 1 patient had distant métastasesat the time ofdiagnosis. Follow-up was available on all 30 patients studied.Analysis of the time to tumor recurrence from the start oftreatment was performed on 28 patients; 2 patients (1 each withand without p53 mutation) did not undergo curative therapyand were excluded from outcome analysis (Table 1). All recurrences were at the primary site, and all patients in whom thetumor recurred ultimately died of their disease. Eleven of 15(73%) patients with p53 mutations had tumor recurrence compared to 6 of 13 (46%) patients without p53 mutations(P = 0.25). Statistical analysis of local failure rates by the

Table 2 p5S mutation summary in 16 head anil neck squamous cell carcinomas

SpecimenSQ20BHNSCC

135HNSCC308JSQ13HNSCC152HNSCC29HNSCC131HNSCC151SCC25HNSCC42HNSCC294HNSCC

301BHNSCC296BSQ9GJSQ

3HNSCC80Primary

siteLarynxHard

palateLarynxAlveolar

ridgeParotidFloor

ofmouthOraltongueOraltongueFloor

ofmouthOraltongueOraltongueUnknownprimaryArytenoidTonsilNasal

vestibuleNasopharynxTNM"T2N0M0T,N,MOT4N3M0T4N,M0TjNoMoT,N,MoT4N2aM0T,N0M0T2N,M0T.NoMoT2N0M0TXN2M,T2N2M0TjN.MoT,N0M0T2N,M0Exon55556666677778g9CodonJust

before 126132154176203205205205209245246248249271298307Sequence

changeAG—¿�AAAAG

—¿�AGG1-basepairdeletionTGC

—¿�TTC1-basepairdeletionTAT

—¿�GATTAT-TCTTAT-TGT2-base

pairdeletionGGC-GACATG—¿�TTGCGC-TGGAGG—¿�ACT1-basepairdeletionGAG

—¿�TAG16-basepair deletionResultAltered

spliceacceptorsiteLys

—¿�ArgFrame-shiftCys

—¿�PheFrame-shiftTyr

—¿�AspTyr—¿�SerTyr—¿�LysFrame-shiftGly

—¿�AspMet—¿�LeuArg—¿�TyrArg—¿�TyrFrame-shiftGlu

—¿�•¿�stopFrame-shift

' Tumors were staged according to American Joint Committee on Cancer, 1988. TNM, tumor-nodes-metastases.

Fig. I. Autoradiograph of p53 gene coding-strand sequence in the region of codon 205 forPCR-amplificd DNA from normal fibroblasts(a) and head and neck squamous cell carcinomas (b-d). a, control (normal fibroblast) DNAdemonstrating wild-type codon 205 sequence(TAT); b, sequence of oral tongue tumorHNSCC 131 showing A —¿�C transversion; c,sequence of oral tongue tumor HNSCC 151;both a mutated G band and normal A band atposition 2 of codon 205 are seen; d, sequenceof floor of mouth tumor HNSCC 29 which hada T —¿�G transversion at the first position ofcodon 205.

a) Normalfibroblast

GATC

codon 205TAT

b) HNSCC 131 c) HNSCC 151 d) HNSCC 109

GATC GATC GATC

TCT

T TA/G A

T •¿�. G

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pS3 AND HPV IN HEAD AND NECK CANCER

Table 3 Comparison of type (transition, transversion, or deletion) of mutationsin the p53 gene in tumors linked to the use of alcohol and/or tobacco

(Head and neck, esophagus) and tobacco alone (lung)P = 0.05, head and neck versus esophagus; P = 0.02, head and neck versus lung;

P —¿�0.85, esophagus versus lung.

Transition Transversion DeletionsTumor site (n) Rcf.

Head and neck(16)Esophagus(37)Lung

(55)3141343855613145Present

studv11-145-10

Kaplan-Meier method showed tumors with p53 gene mutationsrecurred at a median time of 6 months compared to a mediantime to recurrence of 17.4 months for those tumors withoutmutations (P = 0.10). All 3 patients with HPV infections hadstage IV lesions (2 oral cavity, 1 oropharynx); none had p53mutations and none experienced disease recurrence (Table 1).

Discussion

Our finding that p53 mutations occurred in 53% of head andneck tumor samples is similar to the frequency of p53 genemutation reported for both lung (5-10) and esophageal cancer(11-14). Smoking is a common risk factor for these three neoplasms, and therefore it is not surprising to find a similar percentage of p53 mutations; the patients in our study were allregular users of both tobacco and alcohol. What was unexpected was the spectrum and location of the mutations in headand neck carcinomas compared to these other tobacco-relatedcancers. Unlike the findings reported for lung (5-10) andesophageal (11-14) cancers, small p53 gene deletions appear tobe common in head and neck cancer specimens, occurring at thesame relative frequency as transitions or transversions (Tables 2and 3). This spectrum of mutations in head and neck cancerdiffers from that seen in esophageal cancer (P = 0.05) or lungcancer (P = 0.02), whereas a comparison of the distribution ofthe types of p53 mutations seen in lung versus esophageal neoplasms reveals no such differences (P = 0.85) (Table 3). Thereason(s) for the difference observed between the mutationalspectrum for head and neck and these other tobacco-relatedneoplasms is unclear. The mutational spectrum that we describe is similar to that reported in other genes for certain typesof free-radical mediated oxidative damage (29, 30). Ethanolmetabolism has been shown to increase free-radical production(31), and high levels of oxidants are present in both cigarettesmoke and tar (32, 33). Because epidemiological evidence suggests that both alcohol and tobacco use are related to the development of these tumors (34), it is tempting to speculate thatfree-radical induced p53 damage may play a role in head andneck cancer. In terms of location of mutations within the p53gene, we observed a clustering of 3 mutations at codon 205;furthermore, over one-half of the the mutations we report occurin 2 distinct regions of the gene. These two regions, corresponding to codons 203-209 and 245-249, make up only 6% of a200-codon stretch in which greater than 95% of all somaticmutations have been found to occur (16). This distribution maybe the result of chance or be related to specific etiologic factors;separate p53 "hotspots" have previously been proposed forhepatocellular (16), lung (5), and ovarian4 carcinomas.

A major determinate of survival in head and neck cancer isthe ability of treatment to prevent disease reoccurrence at theprimary site (1). Reappearance of disease at the primary site isusually due to incomplete tumor elimination, because of either

inadequate therapy or tumor resistance. In our series, thosepatients with tumors in which p53 DNA sequence changes weredetected had a shorter median time to recurrence at the primarysite than those patients whose tumors had no p53 mutations(6.0 months versus 17.4 months, P = 0.10). This trend towardearlier disease recurrence does not appear to be associated withtumor stage, nodal status, clinical stage, or type of treatmentreceived (Table 1). p53 gene mutations have been shown toconfer a proliferative advantage both in vitro (35) and in vivo(4). Taken together with these findings, our data suggest thatthe local growth advantage a p53 gene mutation confers mayresult in a shorter disease free interval following treatment inhead and neck cancer. Of interest is that none of the threepatients with oncogenic HPV infections detected had recurrence of their tumors despite being stage IV at presentation.Whereas the expected result of a p53 gene mutation is theproduction of either no protein or an abnormal protein, cellsinfected with oncogenic HPV appear to produce a normal p53protein (36) and the degree to which normal p53 function isinhibited may depend in part upon the relative amounts ofwild-type p53 and E6 oncoprotein present. Thus, although either p53 gene mutation or oncogenic HPV infection appearcapable of producing altered p53 "pathway" function in

HNSCC, the clinical consequences of these two mechanismsmay differ. A parallel situation may exist in cervical cancerwhere HPV-negative tumors appear to carry a worse prognosis(37, 38). Both HPV-16 infections in our study occurred inpatients with tonsillar carcinoma, and it has been suggested thatthis anatomic subsite may be more frequently infected byHPV-16 than other sites within the head and neck region (39,40). The possibility that patients with oncogenic HPV-medi-

ated p53 abrogation of function represent a distinct clinicalsubset of head and neck cancer is under investigation in ourlaboratory.

It is unlikely that the results we present are due to either priortreatment or cell culture artifact. We have shown previouslythat while p53 gene mutations can occur in human radiation-induced tumors, such neoplasms usually become clinically detectable only after a latent period of several years from irradiation (41); given the short time to tumor recurrence in thiscurrent study it is doubtful that treatment produced the DNAchanges observed. The 53% frequency of p53 mutations described herein is consistent with two recent studies demonstrating increased p53 protein expression by immunofluorescence in34% (42) and 67% (43) of head and neck squamous cell carcinomas. Furthermore, in colorectal cancer, Baker et al. (44)noted a complete concordance between the/?55 mutations identified in tumor cell lines and those identified in the primarytumors from which they were derived.

Our study suggests that alteration of p53 tumor suppressorgene function is common in squamous cell head and neck cancer and may occur through either gene mutation or oncogenicHPV infection. The location and spectrum of p53 gene mutations we report in head and neck cancer differ from those reported for other smoking-associated neoplasms and may be dueto exposure to both alcohol and tobacco. Further study is necessary to clarify the role of p53 gene mutation and oncogenicHPV infection on clinical outcome.

Acknowledgments

4 D. W. Yandell. unpublished results.We wish to thank Laimonis Laimins for his valuable technical advice

and Denise Adams for her editorial assistance.4835

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1992;52:4832-4836. Cancer Res   David G. Brachman, Deborah Graves, Everett Vokes, et al.   Infection in Human Head and Neck Cancer

Gene Deletions and Human Papilloma Virusp53Occurrence of

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