Association between Cigarette Smoking and Mutation of the p53 Gene in Squamous-Cell Carcinoma of the Head and Neck
Joseph A. Brennan, M.D., Jay O. Boyle, M.D., Wayne M. Koch, M.D., Steven N. Goodman, M.D., Ph.D., Ralph H. Hruban, M.D., Yolanda J. Eby, M.S., Marion J. Couch, M.D., Ph.D., Arlene A. Forastiere, M.D., and David Sidransky, M.D.
Background Although epidemiologic studies have long associatedtobacco and alcohol use with the development of squamous-cellcarcinoma of the head and neck, the molecular targets of thesecarcinogens have yet to be identified. We performed a molecularanalysis to determine the pattern of mutations in the p53 genein neoplasms from patients with squamous-cell carcinoma of thehead and neck and a history of tobacco or alcohol use.
Methods Sequence analysis of the conserved regions of the p53gene was performed in tumor samples from 129 patients with primarysquamous-cell carcinoma of the head and neck. We then used statisticalanalysis to identify any patient characteristics associatedwith mutation of the p53 gene.
Results We found p53 mutations in 42 percent of the patients(54 of 129). Fifty-eight percent of the patients who smokedcigarettes and used alcohol (37 of 64; 95 percent confidenceinterval, 45 to 70 percent), 33 percent of the patients whosmoked but abstained from alcohol (13 of 39; 95 percent confidenceinterval, 19 to 50 percent), and 17 percent of the patientswho neither smoked nor drank alcohol (4 of 24, 95 percent confidenceinterval, 5 to 37 percent) had p53 mutations (P = 0.001). (Twopatients used alcohol but did not smoke, and neither had a p53mutation.) Furthermore, 100 percent of the mutations in thepatients who neither drank nor smoked occurred at sites containingcytidine phosphate guanosine dinucleotides (potentially representingendogenous mutations) within the p53 gene (5 of 5 mutations;95 percent confidence interval, 48 to 100 percent), whereasonly 23 percent of those in cigarette smokers consisted of suchchanges (12 of 53 mutations; 95 percent confidence interval,12 to 36 percent; P = 0.001).
Conclusions In our study, a history of tobacco and alcohol usewas associated with a high frequency of p53 mutations in patientswith squamous-cell carcinoma of the head and neck. Preliminaryevidence linked cigarette smoking to p53 mutations at nonendogenousmutation sites. Our findings suggest a role for tobacco in themolecular progression of squamous-cell carcinoma of the headand neck and support the epidemiologic evidence that abstinencefrom smoking is important to prevent head and neck cancer.
Epidemiologic data have strongly linked cigarette smoking andalcohol consumption to the development of certain cancers.1,2Smoking is the most common cause of cancer-related death inthe United States, and tobacco and alcohol use accounts forone third of all cancer-related deaths.1,2 Tobacco and alcoholare important etiologic agents in squamous-cell carcinoma ofthe head and neck.3,4,5,6 A large-scale prospective study determinedthat the relative risk of death due to cancer among smokersolder than 35 years of age, as compared with nonsmokers, was27.5 for oral and pharyngeal cancer and 10.5 for laryngeal cancer.7Repeated exposure to specific carcinogens in cigarette smokemay cause multiple neoplastic lesions in the mucosa of the aerodigestivetract (field carcinogenesis).8,9 The upper aerodigestive tract,the only area in the body in which the alimentary tract andthe airways form a common conduit, is an ideal site for evaluatingthe independent and synergistic effects of tobacco and alcohol.
The molecular targets of cigarette smoke and alcohol have notbeen firmly identified. Carcinogens may leave unique "fingerprints"in the form of specific mutations that cause the initiationor progression of cancer.10,11 Mutation of the p53 gene, themost common genetic alteration in human cancer, has been linkedto tobacco smoking in squamous-cell carcinoma of the head andneck, as well as esophageal, lung, and bladder cancer.11,12However, this conclusion rests on studies involving small numbersof patients and often immunohistochemical evaluation,13,14,15,16,17,18,19,20,21,22,23a method that, because of its high false positive and falsenegative rates, does not always identify mutations of the p53gene.24,25,26,27 Although technically difficult and time consuming,molecular sequencing is the gold standard for detecting p53mutations. It is the only means of identifying the pattern ofp53 mutations that may result from exposure to carcinogens.11We collected samples of invasive squamous-cell carcinomas ofthe head and neck, sequenced the p53 gene, and attempted todetermine whether any clinical characteristics correlated withmutation of the gene.
Methods
Patients
One hundred forty-four consecutive patients with squamous-cellcarcinoma of the head and neck who were undergoing biopsy orsurgical resection at Johns Hopkins Medical institutions wereprospectively entered into the study, which was approved bythe appropriate institutional review board. Sixty-nine of thesepatients had been part of a previous study investigating thevalue of p53 as a molecular marker of occult tumor cells inpathological samples.28 Demographic data were collected fromthe hospital charts, the cancer registry, and interviews withthe patient and treating physician as necessary. Demographicdata on each patient were collected by staff members who hadno knowledge of the status or the type of p53 mutation presentin the patient's tumor.
The history of use of tobacco and alcohol was carefully documented.Nonsmokers and nondrinkers were defined as patients who neverused, rarely used, or had stopped using tobacco and alcohol,respectively, more than 20 years before being treated for headand neck cancer. Smokers and drinkers were defined as patientswith moderate or heavy use of cigarettes (at least 20 pack-years)and alcohol (one or more drinks per day — one drink beingdefined as containing approximately 10 g of alcohol, which isequal to 1 oz [30 ml] of 86-proof hard liquor, one 3.6-oz [108-ml]glass of wine containing 12 percent alcohol, or one 12-oz [360-ml]can of beer), respectively, during the 20 years preceding theirtreatment for head and neck cancer.4 We intended to stratifythese patients according to whether they had quit using tobaccoor alcohol more than 15, 10, or 5 years before treatment orwere still using them at the time of our study.
The perioperative data included the tumor–node–metastasisstage of the head and neck cancer (stage I, II, III, or IV accordingto the staging system of the American Joint Committee on Cancer29),the site of the primary tumor, and the pathological grade ofthe neoplasm on light-microscopical examination. All patientswere assigned to subgroups according to whether the cancer wasnewly diagnosed or recurrent at the time of evaluation of thep53 gene.
Molecular Analysis
With the patient's consent, portions of the invasive tumorswere collected in the operating room and immediately frozenin liquid nitrogen. The frozen specimens were microdissectedto remove normal tissue (only specimens containing more than50 percent neoplastic cells were included in the analysis),and DNA was isolated.30 A 1.8-kb fragment of the p53 gene encompassingexons 5 through 9 was amplified from the frozen primary-tumorDNA by the polymerase chain reaction,31 cloned, and then sequenced.32The results were confirmed with repeated amplification, cloning,and sequencing of the tumor DNA (a complete list of the specificp53 mutations in these patients is available on request).
Statistical Analysis
The clinical and pathological findings were analyzed with respectto p53 mutations with use of the chi-square and Fisher's exacttests. The relation of multiple patient characteristics to mutationsof the p53 gene was also examined by logistic regression. Weused JMP 3.0 statistical software (SAS Institute, Cary, N.C.).
Results
Characteristics of the Patients
One hundred forty-four consecutive patients with invasive squamous-cellcarcinoma of the head and neck were enrolled in the study. Threepatients were excluded because of a lack of demographic data(their hospital charts could not be located), and 12 other patientswere excluded because cigarette-smoking and alcohol-consumptionhistories were not available. The demographic data were analyzedseparately for the 102 patients with newly diagnosed cancerand the 27 patients with recurrent cancer (Table 1).
Table 1. Characteristics of the Patients with Newly Diagnosed or Recurrent Squamous-Cell Carcinoma of the Head and Neck.
Of the patients with newly diagnosed squamous-cell carcinomaof the head and neck, 88 percent (90 of 102) presented withadvanced stage III or IV cancer, as is typical in most tertiarycancer centers. The most common primary sites were the larynx,the oral cavity, and the oropharynx. Light-microscopical examinationof the operative specimens, available for 86 patients, revealedthat approximately half the neoplasms were moderately differentiated;the others were evenly divided between well-differentiated andpoorly differentiated cancers. Most patients with newly diagnosedcancer currently smoked cigarettes (81 of 102, or 79 percent)or had smoked within the past 20 years (a history of at least20 pack-years). Fifty-seven percent of these patients (58 of102) also reported moderate to heavy intake of alcohol withinthe past 20 years. Very few patients had stopped using eithertobacco or alcohol within the past 20 years; therefore, we didnot subdivide the groups according to whether they had stoppedsmoking or drinking alcohol 5, 10, or 15 years before the studybegan.
Twenty-one percent of the patients (27 of 129) presented withpreviously treated recurrent squamous-cell carcinoma of thehead and neck (Table 1). Eighty-one percent of these patients(21 of 26) had been classified as having stage I or II lesionswhen the original diagnosis was made (in 1 patient there wasno documentation of the original stage). The stage of the recurrentneoplasms was not revised to reflect the occurrence of moreadvanced tumors, even though the patients typically presentedwith extensive locoregional disease. The primary sites of therecurrent and newly diagnosed neoplasms were similar, and thedegrees of histologic differentiation (available for 127 patients)were also similar. Among the patients with recurrent cancer,82 percent (22 of 27) smoked and 30 percent (8 of 27) were moderate-to-heavyusers of alcohol.
Molecular Analysis
The p53 gene was sequenced in tumor specimens from 129 patientswith squamous-cell carcinoma of the head and neck, and 42 percentof the neoplasms had at least one mutation of the p53 gene (Figure 1Aand Figure 1B). Four of these tumors had tandem mutationsof the p53 gene (2 apparently unrelated mutations), yieldinga total of 58 mutations in 54 head and neck cancers (Table 2).The most common p53 mutations were GCAT, GCTA, and AT GC. Twenty-eightpercent of the p53 mutations (16 of 58) included splice sites,frame shifts, deletions, or stops. These changes would be predictedto encode truncated p53 proteins that immunohistochemical analysisusually fails to detect.
Figure 1. Autoradiographs of mutations of the p53 Gene in Patients with Squamous-Cell Carcinoma of the Head and Neck.
Sequencing analysis of DNA from tumor samples is shown with the lanes grouped together to facilitate the identification of abnormal mutant bands (three tumors are shown in Panel A, and four tumors in Panel B). In Panel A, a tandem mutation consisting of base-pair changes (TAAT) at codons 253 and 254 (changing threonine to serine and isoleucine to phenylalanine) is shown in lane 2 (arrows). In Panel B, a change in a single base pair (GCAT) at codon 278 (changing proline to a stop codon) is evident in lane 3 (arrow).
Table 2. p53 Mutations Identified in 54 Patients with Squamous-Cell Carcinoma of the Head and Neck.
Statistical Analysis
Logistic-regression analysis did not reveal significant correlationsbetween the presence or absence of p53 mutations (P>0.50),the tumor–node–metastasis stage (P>0.50), thepathological tumor grade (P>0.50), or the primary site ofthe neoplasm in patients with either newly diagnosed cancer(P = 0.70) or recurrent cancer (P = 0.10).
By contrast, 47 percent of the tumors obtained from smokersin the group with newly diagnosed cancer (38 of 81) had p53mutations, whereas only 14 percent of the tumors from nonsmokers(3 of 21) had mutations of the p53 gene (P = 0.006). A significantassociation between alcohol use and mutation of the p53 genewas also found in patients with newly diagnosed cancer. Mutationsof the p53 gene were found in 55 percent of the carcinomas fromthe patients who drank alcohol (32 of 58), but in only 20 percentof the tumors from patients who did not drink (9 of 44, P<0.001).The association of cigarette smoking (P = 0.34) and alcoholuse (P = 0.42) with mutation of the p53 gene was not significantin the population of patients with recurrent squamous-cell carcinomaof the head and neck, possibly because of the small number ofpatients in that group (n = 27). Twelve of the 22 smokers withrecurrent cancer had mutations of the p53 gene, whereas thiswas true for only 1 of the 5 nonsmokers with recurrent cancer.Tumors from 5 of the 8 alcohol drinkers with recurrent cancerhad p53 mutations, whereas tumors from 8 of the 19 nondrinkerswith recurrent cancer had such mutations.
The characteristics of the patients with newly diagnosed cancerwere similar to those of the patients with recurrent cancer(Table 1). Almost all the patients with recurrent cancer (25of 27) had received radiation therapy before undergoing a secondtumor resection. In these patients, we did not see the deletionsof the p53 gene that exposure to radiation can cause.33,34,35,36Moreover, the proportions of p53 mutations in the patients withprimary (42 percent) and recurrent (50 percent) tumors and thepattern of these mutations were almost identical. Consequently,the two groups were combined for a more detailed analysis ofsmoking and drinking habits. In the total population of 129patients, the association of smoking and drinking with mutationsof the p53 gene was stronger than in the subgroups (Figure 2).Tumors from patients with head and neck cancer who smoked cigarettesand drank alcohol had a 58 percent incidence of p53 mutations(95 percent confidence interval, 45 to 70 percent), those frompatients who only smoked had a 33 percent incidence of p53 mutations(95 percent confidence interval, 19 to 50 percent), and thosefrom patients who neither smoked nor drank had a 17 percentincidence of p53 mutations (95 percent confidence interval,5 to 37 percent; P = 0.001). Only two nonsmoking patients usedalcohol, and neither had a p53 mutation.
Figure 2. Association of p53 Gene Mutations with Cigarette Smoking and Alcohol Consumption in 129 Patients with Squamous-Cell Carcinoma of the Head and Neck.
The frequency of p53 gene mutations in patients with invasive squamous-cell carcinoma of the head and neck was related to the patients' exposure to cigarette tobacco and alcohol (P = 0.001). Cigarette smokers who drank alcohol were 3.5 times more likely than nonsmokers who abstained from alcohol to have mutations of the p53 gene. The T bars represent the upper 95 percent confidence limit. Two nonsmokers who drank alcohol were excluded from the analysis (neither hada p53 mutation).
Sites containing cytidine phosphate guanosine (CpG) dinucleotidesare susceptible to endogenous mechanisms of mutation. Methylationat these sites can lead to spontaneous deamination and the misincorporationof nucleotides on the complementary DNA strand. All of the mutationsin tumors from the patients with head and neck cancer and p53mutations who neither smoked nor drank occurred at CpG sites(5 of 5 mutations; 95 percent confidence interval, 48 to 100percent), but such mutations were found in only 23 percent ofthe tumors from patients with cancer and p53 mutations who smokedcigarettes (12 of 53 mutations; 95 percent confidence interval,12 to 36 percent; P = 0.001).
Discussion
Patterns of mutations have been associated with certain environmentalcarcinogens.10,11,37,38,39 We sequenced the p53 gene in tumorspecimens from 129 patients with squamous-cell carcinoma ofthe head and neck and found that mutations of the gene correlatedstrongly with cigarette smoking, either alone or in combinationwith alcohol consumption. These mutations were 3.5 times morecommon among patients who both smoked cigarettes and drank alcoholthan among patients who neither smoked nor drank.
A significant minority of the patients (19 percent) neithersmoked nor drank, and 30 percent smoked but abstained from alcohol.We could thus analyze tobacco and alcohol use as independentrisk factors for mutation of the p53 gene. Since only two patientsdrank alcohol but did not smoke, we could not evaluate the effectof alcohol in the absence of smoking.
Preliminary data link mutation of the p53 gene with cigarettesmoking in patients with lung carcinoma.17,18,19,20 Most ofthe studies have used immunohistochemical analyses to evaluatethe p53 protein; this method has substantial false positiveand false negative rates as compared with those for molecularsequencing.24,25,26,27,40 In our patients, 28 percent of thep53 mutations could have resulted in a truncated p53 protein,which would not stain with labeled anti-p53 antibodies. Anotherstudy linking exposure to carcinogens with p53 mutations found14 mutations, most of which were GCTA.17 The authors suggestedthat benzo[a]pyrene in tobacco smoke specifically causes GCTAmutations in the p53 gene.17 In esophageal cancer, another neoplasmrelated to smoking and alcohol consumption, a wide range ofp53 mutations has been found, most commonly GC AT and GC TA.15,16Mutations of the p53 gene in patients with bladder cancer whosmoked typically consisted of GC CG and AT GC.22,41 These mutationsmay result from the aromatic amines and N-[4-(5-nitro-2-furyl)-2-thiaxolyl]formamide,both of which are present at increased levels in urothelialcells in cigarette smokers. all the patients in these earlierstudies were cigarette smokers, and their cancers had a widespectrum of base-pair changes in the p53 gene, similar to thosein our patients with squamous-cell carcinoma of the head andneck who used tobacco and alcohol (Figure 3).
Figure 3. The Pattern of p53 Base-Pair Mutations Resulting from Exposure to the Carcinogens in Cigarette Tobaccoand Alcohol.
Cigarette smokers who abstained from alcohol had five types of base-pair changes: ATTA, ATGC, GCAT, GCTA, and GCCG. Cigarette smokers who drank alcohol had the widest spectrum of alterations, involving all types of potential base-pair changes, including frame shifts. Frame shifts involve insertions or deletions of one or more base pairs.
Because both endogenous and exogenous mutagens generate specifickinds of base substitutions at preferred sites, the spectrumof p53 mutations in tumors may provide information about theircause.38,39,42 The pattern of p53 mutations in our patientswas notable in two respects. First, the highest incidence ofmutations was associated with exposure to tobacco and alcohol.In patients in Papua New Guinea who had squamous-cell carcinomaof the head and neck predominantly associated with betel-nutchewing,43 the incidence of p53 mutations was much lower (10percent), suggesting that tobacco (and perhaps alcohol) mayproduce carcinogens that increase the frequency of such mutations.Second, the location of the changes within the p53 gene withrespect to CpG sites was also associated with exposure to cigarettetobacco and alcohol. These changes at CpG sites have been implicatedas endogenous mutational "hot spots" resulting from methylationand deamination of cytosine by cellular enzymatic processes.42,44Consequently, a higher percentage of changes at CpG sites wouldbe expected in patients whose mutations occurred without substantialexposure to environmental carcinogens. In colon cancer, a neoplasmnot associated with smoking, the frequency of p53 mutationsis also high, but most mutations occur at endogenous CpG sites.42In the group of patients who neither smoked nor drank, we alsodetected a predominance of mutations at CpG sites. However,the small number of subjects in this group means that thesefindings must be regarded as preliminary.
Critical studies have shown that the loss of the protectivep53 cellular mechanism allows the evolution of a clonal populationof cells with a selective growth advantage that may eventuallyresult in the progression of cancer.45,46 Moreover, inactivationof the p53 gene may be an important step in the progressionof preinvasive lesions of the head and neck.23 The differenttypes of base-pair changes in squamous-cell carcinoma of thehead and neck suggest the involvement of many of the tobaccotoxins thus far identified, although experiments in animalshave suggested that tobacco-specific nitrosamine derived fromnicotine may be the main culprit.1 Our results also suggestthat alcohol may augment the effects of tobacco by further increasingthe frequency of p53 mutations. Researchers have suggested thatalcohol may cause mucosal injury and increase the absorptionof the mutagenic toxins present in cigarette smoke.3 Alcoholconsumption may also directly cause carcinogenesis by inducingmicrosomal enzymes involved in the metabolism of carcinogensby contributing to nutritional deficiencies, and by introducingcarcinogenic impurities that may have contaminated alcoholicbeverages.3
We have demonstrated that cigarette smoking and alcohol consumptionincrease the frequency of p53 mutations. Although such mutationsalso occur in cancers that are not related to smoking, our findingsprovide further evidence that such mutations are generally restrictedto endogenous hot spots in nonsmokers and that cigarette smokemay have a propensity to inactivate the p53 gene. Because inactivationof the gene appears critical for the progression of many headand neck cancers, our molecular data strongly support the epidemiologicevidence that abstinence from smoking is important for the preventionof such cancers. Moreover, the link between exposure to tobaccoand p53 mutations in squamous-cell carcinoma of the head andneck raises the possibility that a specific carcinogenic exposurecan serve as the etiologic agent in a particular patient's cancer.
Supported in part by grants from the Lung Cancer Spore (CA-58184-01and CA-54672) and by a collaborative research agreement withOncor, Inc., Gaithersburg, Md.
Source Information
From the Department of Otolaryngology–Head and Neck Surgery, Division of Head and Neck Cancer Research (J.A.B., J.O.B., W.M.K., Y.J.E., M.J.C., D.S.), the Oncology Center (S.N.G., A.A.F., D.S.), and the Division of Biostatistics (S.N.G.), Johns Hopkins University School of Medicine; and the Department of pathology, Johns Hopkins Hospital (R.H.H.) — both in Baltimore.
Address reprint requests to Dr. Sidransky at the Department of Otolaryngology–Head and Neck Surgery, 818 Ross Research Bldg., 720 Rutland Ave., Baltimore, MD 21205-2196.
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AT, GC
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