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Clinical Cancer Research Vol. 11, 2502-2509, April 2005
© 2005 American Association for Cancer Research

Human Cancer Biology

The p53 Codon 72 Proline Allele Is Associated with p53 Gene Mutations in Non–Small Cell Lung Cancer

Yingchuan Hu1, Michael P. McDermott2 and Steven A. Ahrendt1

Authors' Affiliations: Departments of 1 Surgery and 2 Biostatistics and Computational Biology, University of Rochester Medical School, Rochester, New York

Requests for reprints: Steven A. Ahrendt, Department of Surgery, Box SURG, University of Rochester, 601 Elmwood Avenue, Rochester, NY 14642. Phone: 585-275-2147; Fax: 585-275-8513; E-mail: steven_ahrendt{at}urmc.rochester.edu.


   Abstract
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 Abstract
Materials and Methods
 Results
 Discussion
 References
 
The p53 gene plays a critical role in cell cycle control, the initiation of apoptosis, and in DNA repair. An Arg/Pro polymorphism at codon 72 of the p53 gene alters the ability of the p53 protein to induce apoptosis, influences the behavior of mutant p53, decreases DNA repair capacity, and may be linked with an increased risk of lung cancer. To further define the role of the p53 codon 72 polymorphism on DNA repair, lung cancer risk, and mutant p53 function, we examined the effect of this polymorphism on mutation of the p53 gene and patient survival in non–small cell lung cancer (NSCLC). Tumor and nonneoplastic (lung or lymphocyte) samples were collected from 182 patients with NSCLC. p53 mutations were detected by direct sequencing and/or the Gene Chip p53 assay in 93 of 182 (51%) tumors. p53 codon 72 polymorphisms were identified by PCR/RFLP analysis. p53 mutations were significantly (P = 0.01) associated with the number of codon 72 Pro alleles: Pro/Pro homozygotes, 17 of 26 (65%); Arg/Pro heterozygotes, 45 of 79 (57%); and Arg/Arg homozygotes, 31 of 77 (40%). The number of codon 72 Pro alleles was independently associated with p53 mutations (odds ratio, 1.97; 95% confidence interval, 1.14-3.40; P = 0.01) in a multiple logistic regression model. The codon 72 polymorphism did not influence patient survival in either the entire patient group or among patients with p53 mutant tumors. In summary, the p53 Pro allele is associated with an increased frequency of p53 mutations in NSCLC.

Key Words: lung cancer • p53 • alcohol • p53 codon 72 polymorphism

Lung cancer is the leading cause of death among smokers and is the leading cause of cancer deaths in the United States (1, 2). Despite the strong association between cigarette smoking and lung cancer, a small minority (11%) of lifelong smokers will develop lung cancer (3). Clearly, other factors play a role in determining outcome following exposure to tobacco carcinogens. Individual susceptibility to exogenous exposures such as tobacco smoke varies with the presence of single nucleotide polymorphisms in a variety of critical genes (4). These low-penetrance susceptibility genes have common variants and interact with environmental factors contributing a major effect to the population incidence of cancer (5–7). Polymorphisms in genes involved in the metabolic activation [cytochrome P4501A1 (CYP1A1)] and detoxification [glutathione S-transferase M1 (GSTM1)] of tobacco carcinogens as well as in the repair of DNA damage [8-oxoguanine DNA glycosylase 1 (OGG1), p53] have all been associated with an increased risk of lung cancer in individual case control studies (8–14). However, inconsistent results among published studies have yet to establish a definitive role for these polymorphisms in lung carcinogenesis (9, 15, 16).

The p53 tumor suppressor gene plays multiple integral roles in apoptosis, cell cycle control, and DNA repair. A polymorphism at codon 72 of the p53 gene results in an arginine-to-proline substitution and is common in African Americans and Caucasians. Several studies have examined the relationship between this polymorphism and lung cancer risk with conflicting results (12, 13, 17–21). However, large case control series have recently shown an increased lung cancer risk associated with the variant (Pro) allele (12, 13). Wu et al. identified an increased risk of lung cancer in carriers of the variant Pro allele at codon 72 as well as in carriers of two intronic (introns 3 and 6) polymorphisms (13). Similarly, Liu et al. also reported an increase in the risk of primary lung adenocarcinoma associated with the codon 72 Pro allele (12).

Tobacco carcinogens bind preferentially to mutational hotspots in several genes including p53 (22–24). However, it remains unclear whether the mutations produced by these carcinogens simply represent enhanced carcinogen binding or whether a failure to repair these lesions also plays a critical role in mutagenesis. Patients with lung cancer exhibit a diminished capacity to repair tobacco carcinogen-induced DNA damage. Cultured lymphocytes from patients with non–small cell lung cancer (NSCLC) have a diminished capacity to repair benzo(a)pyrene diol epoxide adducts in a transfected, benzo(a)pyrene diol epoxide–treated plasmid (host cell reactivation assay), when compared with lymphocytes from control subjects without cancer (25). The sheer magnitude of DNA damage produced on a daily basis (20,000 apurinic/apyrimidinic sites, 500 cytosine deaminations per cell per day) and the low mutation rate in nonneoplastic tissue (10 x 10–7) suggest that DNA repair pathways play a critical role in limiting mutagenesis (26–28). A decrease in DNA repair capacity increases the risk that mutations in critical genes such as p53 will be propagated during DNA replication.

The pattern of p53 mutations varies among cancer types and can provide clues to the pathogenesis of a tumor (29). GC -> TA transversions are the most common p53 mutation in lung cancer and their presence has been linked to tobacco use and polycyclic aromatic hydrocarbon exposure in lung cancer (24, 29–32). Defective DNA repair may also contribute to the pattern of p53 mutations in NSCLC (33–36). For example, inactivation of the DNA repair gene MGMT through promoter hypermethylation leads to a significant increase in G-to-A mutations in the p53 gene (33). We have also shown an increase in A-to-G transitions associated with the XRCC1 codon 399 Gln allele in cigarette smokers with NSCLC (36). Because the p53 codon 72 polymorphism has been associated with increased lung cancer risk, a decrease in DNA repair capacity, and the ability to modify mutant p53 behavior, we examined the relationship among p53 codon 72 genotype, the prevalence and spectrum of p53 mutations, and patient survival in NSCLC.


   Materials and Methods
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 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Sample collection. Primary tumor and normal lung and/or blood were collected from 182 patients undergoing surgical resection of NSCLC at the Johns Hopkins Hospital, the Johns Hopkins Bayview Medical Center, or the Medical College of Wisconsin. Subjects were selected from a larger group of samples collected prospectively to determine the prognostic significance of p53 gene mutations and included all available samples with sufficient DNA remaining to complete the p53 codon 72 genotyping (37). Any patient who received preoperative therapy (neoadjuvant chemotherapy and radiation therapy) was not eligible to participate. Pathologic stage was determined using the revised International System for Staging Lung Cancer (38). Histopathologic type was assigned using the WHO lung tumor classification in clinical use during patient accrual (39). Peripheral blood lymphocytes or nonneoplastic lung tissue was used as a source of genomic DNA. Tumor samples and normal lung tissue were promptly frozen at –80°C after initial gross pathologic examination. The Joint Committee on Clinical Investigation of the Johns Hopkins School of Medicine and the Institutional Review Board of the Medical College of Wisconsin approved this research protocol. Written informed consent was obtained from all subjects. Patient follow-up was obtained through the review of hospital and physician records, direct patient contact, the Johns Hopkins Hospital Tumor Registry, and the Social Security Death Index.

A history of cigarette smoking and alcohol consumption was obtained from patient interview, from clinical notes, and from the tumor registry as previously described (30). Patients were classified as alcohol drinkers if they consumed one or more drinks per day on average during the 20 years before being diagnosed with lung cancer and nondrinkers if they consumed less than one drink per day or abstained from alcohol (30). Nonsmokers were defined as patients who had smoked fewer than 100 cigarettes in their lifetime (30). All smokers had at least a 10 pack-year history of smoking.

DNA isolation. Portions of the primary tumor and nonneoplastic lung tissue were cut into 7-µm sections, stained with H&E, and examined by light microscopy. Additional 12-µm sections were cut and placed in a mixture of 1% SDS and proteinase K at 48°C overnight. Tumors with a low neoplastic cellularity (<70%) were further microdissected to remove contaminating normal cells. In these cases, nonneoplastic tissue was removed from individual 12-µm tumor sections using a 30-gauge needle under low power magnification on an inverted stage microscope. DNA was extracted with phenol/chloroform and precipitated with ethanol. Genomic DNA was also extracted from peripheral blood lymphocytes by proteinase K digestion and phenol/chloroform extraction.

p53 gene sequencing. Mutation analysis of the p53 gene was done on all 182 lung cancers by direct dideoxy nucleotide sequencing and/or the GeneChip p53 assay (Affymetrix, Inc., Santa Clara, CA) as reported previously (40). Results of the p53 sequence analysis for 178 of these 182 tumors have been published previously (30, 33, 36, 37, 40). Briefly, a 1.8-kb fragment of the p53 gene (exons 5-9) was amplified from primary tumor DNA by PCR. The PCR products were purified, sequenced directly using cycle sequencing (Amplicycle sequencing kit, Applied Biosystems, Foster City, CA), and the products of the sequencing reactions were then separated by electrophoresis on 6% polyacrylamide gels and exposed to film. Exons 2 to 11 of the p53 gene were also sequenced using the GeneChip p53 assay as described (40). All p53 mutations resulted in an amino acid substitution or truncated transcript (insertion/deletion), and the two known polymorphisms at codons 47 and 72 were not considered as mutations. All tumors without a detected p53 mutation were analyzed with both sequencing techniques to minimize the potential for undetected p53 gene mutations (40). Tumors with a mutation detected using one of the two techniques were not routinely examined with the alternate technique.

p53 codon 72 genotyping. Polymorphisms at codon 72 in the p53 gene were determined using a PCR-RFLP-based method. Prior studies have shown that the p53 GeneChip does not accurately detect the codon 72 polymorphism in the p53 gene (41). A 366-bp fragment of the p53 gene was amplified from nontumor lung DNA by PCR using forward primer 5'-GTCCTCTGACTGCTCTTTTCACCCATCTAC-3' and reverse primer 5'-GGGATACGGCCAGGCATTGAAGTCTC-3'. Each 25-µL PCR reaction contained 1x PCR buffer, 0.4 µmol/L of each primer, 2.0 mmol/L of MgCl2, 0.4 µmol/L of each deoxynucleotide triphosphate, 100 ng genomic DNA, and 2.5 units of Taq DNA polymerase (Invitrogen, Carlsbad, CA). The PCR reaction mixture was pretreated at 95°C for 10 minutes followed by 35 cycles of 95°C for 1 minute, 55°C for 1 minute, and 72°C for 1 minute. The final extension was at 72°C for 10 minutes. The PCR product was digested with 40 units of BstUI (New England Biolabs, Beverly, MA) at 60°C for 16 hours and electrophoresed on a 2% agarose gel and stained with ethidium bromide. The Arg/Arg homozygote is cleaved by BstUI and yields 215- and 151-bp bands (Fig. 1). The Pro/Pro homozygote is not cleaved by BstUI and yields a single 366-bp band. The Arg/Pro heterozygote contains all three bands (366, 215, and 151 bp) following restriction digestion.



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  		Fig. 1 PCR/RFLP analysis of the p53 codon 72 polymorphism. Arg/Arg homozygote is cleaved by BstUI and yields 215- and 151-bp bands. Pro/Pro homozygote is not cleaved by BstUI and yields a single 366-bp band. Arg/Pro heterozygote contains all three bands (366, 215, and 151 bp) following restriction digestion.

 
Statistical methods. Associations between codon 72 genotype and individual clinical and pathologic variables were assessed using {chi}2 tests and Fisher's exact tests, as appropriate. Genotype frequencies among Caucasians and African American subjects were compared with frequencies expected in Hardy-Weinberg equilibrium using the {chi}2 goodness of fit test. The associations between p53 mutation status and individual clinical and pathologic variables, including codon 72 genotype, were assessed using logistic regression. The primary analyses expressed the codon 72 genotype as the number of codon 72 Pro alleles; other analyses compared those with (Arg/Pro and Pro/Pro) and without (Arg/Arg) the codon 72 Pro allele. Interactions between codon 72 genotype and histologic cell type and pack-years smoking were also examined. A stepwise variable selection procedure was used to build a multiple logistic regression model for p53 mutation status; a significance level of 0.20 was used to determine whether a variable could be entered into, or removed from, the logistic regression model. We included gender, race, alcohol consumption, cigarette smoking, tumor cell type, tumor stage, pathologic grade, and codon 72 genotype as independent variables in the stepwise selection process. Patients were classified as light or heavy smokers if their lifetime cigarette exposure was below or above the median pack-years smoked for the entire group (50 pack-years), respectively. Associations were quantified using odds ratios (OR) and their 95% confidence intervals (95% CI).

Survival time was determined as the time from resection to death. Survival probabilities were estimated using the method of Kaplan and Meier. The associations between individual clinical and pathologic variables and survival were assessed using the Cox proportional hazard regression model. Of primary interest was the association between codon 72 genotype and survival, as well as the interaction between codon 72 genotype and p53 mutation status. We have previously shown that the decrease in survival in patients with p53 mutant NSCLC is limited to patients with stage I tumors (37). Therefore, the associations among p53 mutation status, codon 72 genotype, and survival were estimated separately for stage I patients using this model. We repeated all analyses after adjusting for age. Associations were quantified using hazard ratios (HR) and their 95% CIs. All statistical tests were two tailed.


   Results
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 Abstract
 Materials and Methods
 Results
Discussion
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p53 genotyping. The relationship among p53 codon 72 genotype and various clinical and pathologic variables previously shown to influence p53 mutational status is shown in Table 1 (30). The allelic frequencies of the Arg and Pro alleles were 0.64 and 0.36, respectively. Codon 72 genotype frequencies were significantly (P < 0.0001) different among Caucasians and African Americans. The frequency of the mutant Pro allele was greater among African Americans (0.62) than among Caucasians (0.30). The distribution of codon 72 genotypes was in agreement with Hardy-Weinberg equilibrium for both ethnic groups. No significant associations among age, gender, tumor histologic type, alcohol consumption, or tobacco smoking history, tumor stage, and p53 codon 72 genotype were observed.


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  		Table 1. Clinical and pathologic characteristics among p53 codon 72 genotypes in 182 subjects with NSCLC

 
p53 codon 72 genotype and p53 mutations. p53 mutations were present in 93 of the 182 tumors (51%). Two distinct p53 mutations were detected in each of three tumors, whereas the remaining 90 tumors contained a single mutation. Seventy-seven of the 96 identified mutations were only detected in a single tumor, whereas 19 mutations were detected in more than one tumor. The relationships among p53 mutations and other clinical and pathologic variables and p53 codon 72 genotype are shown in Table 2. Alcohol consumption, male gender, age, and tumor cell type were all associated with an increased risk of p53 mutations as reported previously (37). p53 mutations were significantly (P = 0.01) associated with the number of codon 72 Pro alleles: Pro/Pro homozygotes, 17 of 26 (65%); Arg/Pro heterozygotes, 45 of 79 (57%); and Arg/Arg homoygotes, 31 of 77 (40%). The OR for the number of codon 72 Pro alleles was 1.75 (95% CI, 1.13-2.70; P = 0.01).


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  		Table 2. Association of clinical, pathologic, and epidemiologic characteristics with p53 mutations in 182 patients with NSCLC

 
These clinical and pathologic variables were then examined in a multiple logistic regression model to define independent variables associated with mutation of the p53 gene in NSCLC (Table 3). The number of codon 72 Pro alleles was independently associated with p53 mutations (OR, 1.97; 95% CI, 1.14-3.40; P = 0.01). In addition, a history of alcohol intake (>1 drink/d) was independently associated with p53 mutations.


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  		Table 3. Logistic regression model defining independent variables associated with mutation of the p53 gene in patients with NSCLC

 
Next, we studied the interaction among p53 codon 72 genotype, histologic cell type, and the prevalence of p53 mutations. p53 mutations were significantly (P = 0.02) more common among Pro allele carriers with adenocarcinoma than among Arg/Arg homozygotes with this histology (Table 4). p53 mutations were more common among patients with squamous cell cancer (62%) than adenocarcinoma (43%) irrespective of codon 72 genotype. p53 mutation frequency was similar among Pro and Arg carriers with squamous cell cancer. However, the interaction between codon 72 Pro allele and cell type was not significant (P = 0.69); thus, we cannot conclude that the increased p53 mutation frequency seen in codon 72 Pro allele carriers is limited to just patients with adenocarcinoma. Similar results were obtained when considering the number of codon 72 Pro alleles instead of presence of the codon 72 Pro allele.


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  		Table 4. Associations among p53 codon 72 genotype, histologic cell type, and mutation of the p53 gene in NSCLC

 
The interaction among p53 codon 72 genotype, lifetime cigarette exposure, and the prevalence of p53 mutations was also examined in 146 of 169 cigarette smokers in whom a detailed smoking history was available. The increase in p53 mutations observed in carriers of the p53 codon 72 Pro allele was similar among patients with low and high lifetime smoking exposure. p53 mutations were more common (P = 0.04) among lighter lifetime smokers (<50 pack-years) who carried the Pro allele (25 of 41, 61%) than among light smokers with the Arg/Arg genotype (11 of 30, 37%). Similarly, among heavy smokers (≥50 pack-years), p53 mutations were also more common (P = 0.13) among Pro allele carriers (29 of 44, 66%) than among heavy smokers homozygous for the Arg allele (15 of 31, 48%).

No statistically significant differences in the p53 mutation spectrum were noted among patients with different p53 codon 72 genotypes. GC:TA and GC:AT mutations were the two most frequently identified mutations and were common both among patients with the wild-type Arg/Arg genotype (38% and 16%, respectively) and among carriers of the Pro allele (30% and 24%, respectively). The frequency of specific p53 mutations is shown in Fig. 2. The codon 72 Pro allele was associated with an increase in mutation frequency for each type of p53 mutation except AT:GC mutations. Age, ethnicity, gender, and histologic cell type did not statistically significantly influence the p53 mutation spectrum. GC:AT mutations were significantly (P = 0.017) more common among heavy smokers (≥50 pack-years; 13 of 76, 17%) than among light smokers (<50 pack-years; 3 of 69, 4%). Lifetime cigarette exposure did not influence the frequency of other p53 mutation types.



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  		Fig. 2 Frequency of specific p53 mutations among patients with NSCLC. p53 mutations were more common among patients with the Pro/Pro and Arg/Pro (62 of 105, 59%) genotypes than among patients with the wild-type Arg/Arg genotype (31 of 77, 40%). Codon 72 Pro allele was associated with an increase in mutation frequency for each type of p53 mutation except AT:GC mutations. Ins/del, insertion/deletion.

 
p53 codon 72 genotype, p53 mutations, and patient survival. Pathologic (tumor-node-metastasis) stage was the only variable significantly (P < 0.0001) associated with overall patient survival in the entire group of patients with NSCLC (stage II versus stage I: HR, 1.58; 95% CI, 0.92-2.74; and stage III or IV versus stage I: HR, 2.98; 95% CI, 1.82-4.89). A trend towards decreased patient survival was present among patients with p53 mutant NSCLC (HR, 1.43; 95% CI, 0.94-2.19; P = 0.10) and among older patients (>65 years old; HR, 1.47; 95% CI, 0.96-2.25; P = 0.08). Overall patient survival was not affected by the codon 72 Pro allele (HR, 1.20; 95% CI, 0.78-1.85; P = 0.41 versus Arg/Arg) or by the number of codon 72 Pro alleles (HR, 1.10; 95% CI, 0.82-1.48; P = 0.54). Gender, ethnicity, tumor cell type, tumor grade, cigarette or alcohol consumption, and p53 mutation type did not influence patient survival.

We have recently shown in a prospective study that p53 mutations predict decreased survival in stage I NSCLC (37). Among patients (n = 92) with stage I NSCLC in the present study, p53 mutations were significantly associated with a decrease in overall survival (HR, 2.77; 95% CI, 1.31-5.84; P = 0.008 versus p53 wild type). Patients ages >65 years also had a diminished overall survival (HR, 2.14; 95% CI, 0.99-4.60; P = 0.05) compared with younger patients. Among stage I patients, overall patient survival was not significantly affected by the codon 72 Pro allele (HR, 1.69; 95% CI, 0.83-3.44; P = 0.15 versus Arg/Arg, Fig. 3A). The effect of the codon 72 Pro allele on survival in these patients was not significantly different (P = 0.61) for those with p53 mutations (HR, 1.55; 95% CI, 0.61-3.95) and those without p53 mutations (HR, 1.03; 95% CI, 0.29-3.64). Several studies have reported different biological and biochemical activity between p53 mutant protein containing arginine at codon 72 compared with mutant p53 protein containing proline at this position (42, 43). Therefore, we also examined the effect of codon 72 genotype on overall patient survival among patients with stage I, p53 mutant NSCLC (n = 48). No statistically significant (P = 0.44) difference in overall survival was identified among Arg/Arg homozygotes with p53 mutant tumors and carriers of the codon 72 Pro allele (Fig. 3B). Similarly, no statistically significant influence on survival was noted among Arg/Pro heterozygotes with p53 mutant tumors (HR, 1.44; 95% CI, 0.54-3.85; P = 0.46 versus Arg/Arg) or among Pro/Pro homozygotes with p53 mutant tumors (risk ratio, 1.45; 95% CI, 0.44-4.77; P = 0.54 versus Arg/Arg).



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  		Fig. 3 Effect of p53 codon 72 genotype on overall survival in stage I NSCLC. A, no statistically significant difference (P = 0.15) in overall survival was noted among Arg/Arg homozygotes with stage I NSCLC and Pro allele carriers (Arg/Pro and Pro/Pro) with stage I NSCLC. B, overall survival among Arg/Arg homozygotes with p53 mutant stage I NSCLC was similar (P = 0.43) to that of Pro allele carriers (Arg/Pro and Pro/Pro) with p53 mutant stage I NSCLC.

 

   Discussion
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 Abstract
 Materials and Methods
 Results
 Discussion
References
 
Genetic factors are important in determining individual susceptibility to the damaging effects of cigarette smoke. An increased risk of lung cancer has been associated with the polymorphism at codon 72 of the p53 gene (12, 13). In the present study, p53 mutations were more common in NSCLC from carriers of the codon 72 Pro allele than in NSCLC from Arg/Arg homozygotes. This increase in p53 mutations observed in Pro allele carriers seemed more pronounced in adenocarcinomas and seemed independent of pack-years smoked. The codon 72 polymorphism did not influence overall survival either in the entire group of NSCLC patients, in patients with mutant p53 containing NSCLC, or in patients with stage I NSCLC.

The pattern of genetic damage in lung cancer differs from many solid tumors and has been linked to the carcinogens in tobacco smoke. Polycyclic aromatic hydrocarbons present in cigarette smoke bind to specific CpG sites, which strongly correlate with the pattern of mutational hotspots seen in the p53 gene in lung cancer (23). The overall prevalence of p53 mutations is also increased in lung cancer cases from cigarette smokers when compared with nonsmokers (30). Other epidemiologic factors associated with increased lung cancer risk such as a polymorphism in the CYP1A1 gene have also been shown to increase DNA damage including both p53 mutations and tobacco carcinogen adducts (17). The statistically significant increase in p53 mutation frequency associated with the number of Pro alleles in the logistic regression model supports an important role for this polymorphism in lung cancer. Our results are somewhat consistent with several case control series, which have suggested a stronger role for this polymorphism in the pathogenesis of primary lung adenocarcinoma than in squamous cell cancer (12, 18, 19). The association between the codon 72 Pro allele and p53 mutation seemed strongest in patients with adenocarcinoma; however, the dependence of this association on histologic cell type was not statistically significant. Fan et al. also reported that the increased cancer risk among Pro allele carriers was greater as the number of pack-years smoked increased (18). In the present study, the increase in p53 mutations was comparable among heavy and light smokers.

The p53 gene plays important roles in apoptosis, cell cycle control, and DNA repair, and these critical functions differ among p53 proteins encoded by the Arg and Pro alleles. Several of the functional differences among these two alleles could result in an increase in mutation of the p53 gene following tobacco carcinogen exposure. Lymphoblastoid cell lines carrying the variant codon 72 Pro allele exhibit a decrease in both apoptosis following radiation injury as well as DNA repair capacity following exposure to the tobacco carcinogen, benzopyrene, when compared with cells carrying the wild-type allele (13). Clearly, loss of p53 function through mutational inactivation or suppressed expression leads to a decrease in global genomic repair (44). Smaller decreases in DNA repair capacity associated with the codon 72 Pro allele may also lead to quantifiable increases in cellular DNA damage. The p53 Pro allele is also less effective at inducing apoptosis than the Arg allele (45). The Arg form of the p53 protein binds to MDM-2 with greater affinity, which leads to enhanced transport of p53 to the mitochondria (45). Finally, the Pro allele may also be less effective at inducing cell cycle arrest in response to DNA damage. The Arg form of p53 is more potent at inducing expression of the cell cycle inhibitor p21WAF1 and is more effective at suppressing the growth of transformed cells (46, 47). Although it is unclear from our results which of these functional differences among the codon 72 polymorphic variants is most important, each of these differences could account for the increase in p53 mutations observed in Pro allele carriers.

Several studies have examined the role of the codon 72 polymorphism in mutation of the p53 gene in cancer. Langerod et al. identified p53 mutations more commonly in breast cancer from Arg/Arg homozygotes (28.5%), than among Arg/Pro heterozygotes (21%) or Pro/Pro homozygotes (4%; ref. 48). No difference in p53 mutation frequency among codon 72 genotypes has been reported in colorectal or bladder cancer (48, 49). Several studies have also suggested that the codon 72 Arg allele is preferentially mutated and retained in Arg/Pro heterozygotes (48, 50). These authors have suggested that the codon 72 Arg containing mutants may have a selective growth advantage influencing the ratio of Arg and Pro containing mutants in tumors. Although we did not individually clone DNA from Arg/Pro heterozygotes to determine the mutated allele in this study, the significantly higher mutation rate of Pro/Pro versus Arg/Arg homozygotes does not support a selection pressure towards mutation of the Arg allele in NSCLC. Alternatively, we suggest that the significance of p53-dependent DNA repair in the maintenance of genomic stability may vary among different organ sites and in response to different carcinogen exposures. The p53 gene may play a much larger role in repairing DNA damage in the tobacco carcinogen-exposed respiratory tract than in the colon or breast.

The p53 codon 72 polymorphism has also been evaluated as a prognostic or predictive marker in human cancer. As outlined above, differences in p53 mutation rates among codon 72 genotypes could be due to growth differences among Arg and Pro containing p53 mutants. One small study did not show any survival differences among codon 72 genotypes in lung cancer patients (51). Wang et al. reported a decrease in survival among patients with the Pro/Pro genotype among 133 patients with lung cancer (21). In the present study, a trend towards decreased survival was noted among carriers of the Pro allele in stage I NSCLC. However, this trend disappeared when the higher frequency of p53 mutations among Pro allele carriers was included in the multivariate analysis. The codon 72 allele may play a more clinically meaningful role as a predictive rather than a prognostic marker. For example, 72Arg containing p53 mutants bind p73 more effectively than 72Pro mutants thereby limiting the efficacy of apoptosis-inducing cytotoxic chemotherapy (42). Head and neck cancers expressing Arg containing mutants had significantly lower response rates than those expressing Pro mutants (42). A predictive role for the codon 72 allele was not apparent in this study as the majority of stage I patients did not receive adjuvant therapy.

In summary, p53 mutations in NSCLC are increased in carriers of the codon 72 variant Pro allele. The codon 72 polymorphism did not seem to influence survival in this group of patients. These findings are consistent with impaired DNA repair associated with the 72Pro p53 protein.


   Footnotes
 
Grant support: National Cancer Institute Public Health Service grant K08 CA76452-01.

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 1734 solely to indicate this fact.

Received 9/17/04; revised 11/11/04; accepted 12/30/04.


   References
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 Results
 Discussion
 References
 

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