Association of a p73 exon 2 G4C14-to-A4T14 polymorphism with risk of squamous cell carcinoma of the head and neck

Guojun Li1, Erich M. Sturgis1,2, Li-E. Wang1, Robert M. Chamberlain1, Christopher I. Amos1, Margaret R. Spitz1, Adel K. El-Naggar3, Waun K. Hong4 and Qingyi Wei1,5

1 Department of Epidemiology, 2 Department of Head and Neck Surgery, 3 Department of Pathology and 4 Department of Thoracic and Head and Neck Medical Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, TX 77030, USA

5 To whom correspondence should be addressed Email: qwei{at}mdanderson.org


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
p73, a novel p53 homolog, has some p53-like activity and plays an important role in modulating cell-cycle control, apoptosis and cell growth. p73 regulates differentiation of head and neck squamous epithelium, and changes in p73 may lead to the development of squamous cell carcinoma of the head and neck (SCCHN). Two linked non-coding exon 2 polymorphisms (designated as G4C14-to-A4T14) were identified recently but their functional relevance is unknown. We hypothesized that this p73 polymorphism plays a role in the etiology of SCCHN. Therefore, in this hospital-based case-control study of 708 patients newly diagnosed with SCCHN and 1229 cancer-free controls, we evaluated the association between the p73 AT variant allele and risk of SCCHN. The controls were frequency-matched to the cases by age (±5 years), sex and smoking status, and all subjects were non-Hispanic whites. Our results showed that the frequencies of variant AT allele and genotypes were more common in the cases than in the controls (P = 0.029 and P = 0.009, respectively). Compared with the GC/GC genotype, the variant genotypes (GC/AT + AT/AT) were associated with a statistically significantly increased risk for SCCHN [odds ratio (OR) = 1.33, 95% confidence interval (CI) = 1.10–1.60]. Further stratification analyses by age, sex, smoking and alcohol status and by cancer sites within the head and neck region indicated that this significantly increased risk was more pronounced in younger (≤50 years) individuals (adjusted OR = 1.70; 95% CI, 1.19–2.43), women (1.61; 1.09–2.37), current smokers (1.77; 1.25–2.51) and patients with oral cancer (1.54; 1.15–2.07). Our results suggest that this p73 polymorphism may be a risk marker for genetic susceptibility to SCCHN.

Abbreviations: CI, confidence interval; MDACC, M. D. Anderson Cancer Center; MPP, multi-specialty physician practice; OR, odds ratio; PCR, polymerase chain reaction; SCCHN, squamous cell carcinoma of the head and neck


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Squamous cell carcinoma of the head and neck (SCCHN), including cancers of the oral cavity, pharynx and larynx, is one of the five most common cancers worldwide (1). The incidence of the disease is increasing, and it is estimated that >500 000 new cases will occur annually worldwide (2). In the USA, it is estimated that 38 538 new SCCHN cases and 11 060 deaths will occur in 2003 (3). Many factors contribute to the development of SCCHN, including smoking (4,5), alcohol drinking (5,6), viral infection (7,8) and genetic factors (8). Although smoking and alcohol use play a major role in the etiology of SCCHN, only a fraction of smokers and drinkers develop SCCHN, suggesting that there is inter-individual variation in genetic susceptibility to SCCHN in the general population. For instance, genetically determined DNA repair capacity may contribute to the variation in susceptibility to SCCHN (911). Therefore, identification of factors that modulate the risk of SCCHN could help identify at-risk subgroups that will benefit from primary prevention programs.

Although no major susceptibility genes for SCCHN have been identified, gains and loss in several loci and altered expression of p53 and DNA repair genes suggest involvement of altered oncogenes and tumor suppressor genes in SCCHN tumorigenesis (12,13). p73, a novel member of the p53 family, was identified and mapped to chromosome 1p36 (14). p73 activates transcription of p21- and p53-responsive genes and inhibits cell growth in a p53-like manner by inducing apoptosis or G1 cell-cycle arrest both in cell lines (15,16) and mice (17). In response to DNA damage by modulating p53 response to DNA damage, p73 may play an important role in cell-cycle control and DNA repair activities (18,19).

Extensive molecular analyses of p73 in various tumors suggested that mutations in this gene are rare, because they occur in <2% of all cancers (2022). However, loss of heterozygosity at the p73 locus has been reported at various frequencies in different tumors (2225). Furthermore, increased p73 expression was found in human malignancies associated with p53 mutations (21,2629). These suggest that p73 may act as a tumor suppressor with some of the same functions as p53 or may compensate loss of p53 function (21,2628,30). p73 over-expression is believed to be an early event in head and neck tumorigenesis, and p73 may function as an oncogene in the development of SCCHN, playing a role in SCCHN progression (30).

It is unknown whether the alteration of p73 expression has any genetic basis such as sequence variations or polymorphisms. At least 17 single-nucleotide polymorphisms have been identified in p73 (some in exons and others in introns), but none cause an amino acid change (14,31). However, two linked single-nucleotide polymorphisms at p73 positions 4 (G -> A) and 14 (C -> T) are thought to affect p73 function by altering gene expression, perhaps by altering the efficiency of translational initiation (14). Given the roles of p73 and p53 in early development of SCCHN (30), this p73 polymorphism may contribute to the risk of SCCHN.

Few studies have investigated the role of the p73 G4C14-to-A4T14 polymorphism in risk of cancers (32,33) and none examined SCCHN. To test the hypothesis that the p73 G4C14-to-A4T14 polymorphism is associated with risk of SCCHN, we evaluated this association in a large hospital-based case-control study of 708 patients with incident SCCHN and 1229 cancer-free controls frequency-matched on age, sex and smoking status.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Study subjects
Between May 1995 and September 2003, 1008 patients with histologically confirmed SCCHN were consecutively recruited at our Head and Neck Surgery Clinic. The participation rate of eligible incident cases was ~95% of those who were initially contacted for participation. Patients with second SCCHN primary tumors, primary tumors of the nasopharynx or sinonasal tract, primary tumors outside the upper aerodigestive tract, cervical metastases of unknown origin, or histopathologic diagnoses other than squamous cell carcinoma were excluded. Because genotype frequencies can vary between ethnic groups, the few minority patients enrolled were also excluded from this analysis. As a result, this study included 708 non-Hispanic white subjects with primary tumors of the oral cavity (n = 216; 30.5%), oropharynx (n = 321; 45.3%), hypopharynx (n = 36; 5.1%) and larynx (n = 135; 19.1%).

We also included 1229 cancer-free control subjects who were recruited from two populations during a similar period of time. One group (n = 607) were enrolled from a multi-specialty physician practice (MPP), the Kelsey Seybold Foundation, with multiple clinics throughout the Houston metropolitan area; and we designated this as ‘MPP controls’, which are part of an ongoing lung cancer study. MPP controls provided older male former and current smokers for our frequency-matching purpose. Another group (n = 622); was genetically unrelated and were M. D. Anderson Cancer Center visitors accompanying patients to our cancer center outpatient clinics and we designated these as ‘MDACC controls’. For both MPP and MDACC controls, we first surveyed the potential control subjects by using a short questionnaire to determine their willingness to participate in research studies and to obtain information about their smoking behavior and demographic factors. Among the willing respondents we contacted for recruitment, the response rate was >80%. We frequency matched the controls to the cases by age (±5 years), sex and smoking status. Those subjects who had smoked >100 cigarettes in their lifetimes were defined as ever smokers. Ever smokers who had quit smoking >1 year previously were defined as former smokers and the other smokers as current smokers. The purpose of frequency matching was to control confounding in order to evaluate the main effect of the p73 polymorphism. We interviewed each eligible subject to obtain data on age, sex, ethnicity, smoking status and alcohol consumption (before the onset of disease for the cases and at the time of interview for the controls). Subjects who had drunk alcoholic beverages at least once a week for >1 year previously were defined as ever drinkers. Ever drinkers who had quit drinking >1 year previously were defined as former drinkers and the others as current drinkers. After we obtained informed consent, each subject provided 30 ml of blood that was collected in heparinized tubes. The research protocol was approved by the M. D. Anderson and Kelsey Seybold institutional review boards.

Genotyping
We extracted genomic DNA from the buffy-coat fraction of the blood samples by using a QIAGEN DNA Blood Mini Kit (QIAGEN, Valencia, CA) according to the manufacturer's instructions. We typed for the p73 G4C14-to-A4T14 (confirmed by the antisense sequence of GenBank entry no. AL136528: nucleotide 86885 being the mutated G and nucleotide 86895 the mutated C) genotype by polymerase chain reaction (PCR) with confronting two-pair primers, which makes genotyping possible by electrophoresis without restriction digestion (34). We performed the PCRs with a PTC-200 DNA Engine (Peltier Thermal Cycler, MJ Research, Watertown, MA) in 10 µl of PCR mixture. This PCR mixture included ~20 ng of genomic DNA, 0.1 mM each dNTP, 1x PCR buffer (50 mM KCl, 10 mM Tris HCl and 0.1% Triton X-100), 1.5 mM MgCl2, 0.5 U of Taq polymerase (Sigma-Aldrich Biotechnology, Saint Louis, MO), and 2 pmol of each of four primers. The A4T14 allele was amplified with primers F1 [5'CCACGGATGGGTCTGATCC3'] and R1 [5'GGCCTCCAAGGGCAGCTT3'], which produced a 270-bp fragment, and the G4C14 allele was amplified with primers F2 [5'CCTTCCTTCC-TGCAGAGCG3'] and R2 [5'TTAGCCCAGCGAAGGTGG3'], which amplified a 193-bp fragment. F1 and R2 also produced a common 428-bp fragment in each PCR (33,34). The amplification conditions were 10 min of initial denaturation at 95°C followed by 35 cycles of 1 min at 95°C, 45 s at 62°C, and 1 min at 72°C and a final 5-min step at 72°C for final extension. All PCR products were visualized on a 2% agarose gel containing a 0.25 µg/ml of ethidium bromide. Figure 1 shows a representative example of the genotyping. A water control having all assay agents but the DNA template was included in the PCRs. Because these two variant alleles are in complete linkage equilibrium (14), only three genotypes are available: the homozygote GC/GC, the heterozygote GC/AT and the homozygote AT/AT. PCRs were conducted and the results evaluated without knowledge of the subjects' case-control status. At least 10% of the samples were randomly re-tested, and the results were 100% concordant.



View larger version (33K):
[in this window]
[in a new window]
 
Fig. 1. PCR-based CTPP genotyping for the p73 G4C14-to-A4T14 exon 2 polymorphism. Lane M, 100-bp DNA ladder. Lanes 1, 3 and 5, GC/AT genotype, with 193-, 270-, and 428-bp bands; lanes 2 and 11, AT/AT genotype with 270- and 428-bp bands; lanes 4, 6, 7, 8, 9, 10 and 12, the GC/GC genotype, with 193- and 428-bp bands; lane 13, a water control that had everything but DNA template.

 
Statistical analysis
We first evaluated the differences in the distributions of selected demographic variables (such as age and sex), smoking, alcohol consumption and p73 allele and genotype frequencies between cases and controls by using the {chi}2 test. We estimated the association between p73 genotypes and risk of SCCHN by computing the odds ratios (ORs) and their 95% confidence intervals (CIs) by both univariate and multivariate logistic regression analyses. We further stratified the genotype data by subgroups of age, sex, smoking, alcohol drinking and site of SCCHN. For logistic regression analysis, p73 genotype was also recoded as a dummy variable. All of the statistical analyses were performed with Statistical Analysis System software (Version 8e; SAS Institute, Cary, NC).


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Selected characteristics of the 708 cases and 1229 cancer-free controls are summarized in Table I. Because the cases and controls were frequency-matched for age and sex, there were no significant differences in the distributions of age and sex between the cases and controls (P = 0.446 for age and 0.553 for sex). The median age was similar for the cases (57 years, mean 56.9, range 18–90) and for the controls (58 years, mean 57.2, range 20–88). The data confirmed that the frequency matching on age and sex was adequate. However, the frequency matching by smoking status was imperfect. There were more current smokers and drinkers among the cases than among the controls, and the differences were statistically significant (P < 0.001) (Table I). However, there was no difference in sex distribution among ever smokers between the cases and controls (males: 77.5 and 78.9%, respectively; P = 0.526). These frequency-matching variables were further adjusted for in the multivariate regression analyses.


View this table:
[in this window]
[in a new window]
 
Table I. Frequency distributions of selected variables in SCCHN patients and cancer-free controls

 
We compared p73 genotype distributions between the two controls groups, i.e. MPP and MDACC controls, because they were selected from two separate control populations. The frequency of p73 AT allele was 0.201 for MPP controls and 0.226 for MDACC controls, and the difference was not statistically significant ({chi}2 = 2.14, P = 0.144). The frequencies of the p73 GC/GC, GC/AT and AT/AT genotypes were 64.6, 30.6 and 4.8%, respectively, for the MPP controls and 61.3, 32.3 and 6.4%, respectively, for the MDACC controls (Table II), and this difference was also not statistically significant ({chi}2 = 2.31, P = 0.315). p73 genotype distribution of the two control groups were in agreement with Hardy–Weinberg equilibrium ({chi}2 = 1.28, P = 0.258 for MPP controls and {chi}2 = 3.59, P = 0.058 for MDACC controls). Therefore, to increase study power these two groups were combined and used as one control group in the final analysis. However, p73 genotype distribution of the combined controls was not in agreement with Hardy–Weinberg equilibrium ({chi}2 = 4.82, P = 0.028) (Table II). Therefore, we presented the ORs and 95% CIs using each set of these controls as well as the combined control group as the comparison group (Table II).


View this table:
[in this window]
[in a new window]
 
Table II. p73 genotype and allele frequencies of the cases and controls and their association with risk of SCCHN

 
As shown in Table II, the frequency of p73 variant AT allele in the combined controls was 0.214, which was lower than in the cases (0.245) ({chi}2 = 4.78, P = 0.029), suggesting the variant AT allele may be a risk allele. The difference in p73 genotype distributions between the cases and controls was statistically significant ({chi}2 = 9.32, P = 0.009) (Table II). Compared with the p73GC/GC homozygotes, the adjusted OR associated with risk of SCCHN was 1.36 (95% CI = 1.12–1.66) for the p73GC/AT genotype and 1.11 (95% CI = 0.73–1.69) for the p73 AT/AT genotype. However, these adjusted ORs were greater when the MPP controls were used as the comparison group (1.42, 1.12–1.80 the p73GC/AT genotype and 1.22, 0.73–2.04 for the p73 AT/AT genotype) than when the MDACC controls were used as the comparison group (1.27, 0.99–1.61 the p73GC/AT genotype and 0.99, 0.61–1.62 for the p73AT/AT genotype). Because the AT/AT genotype was relatively uncommon, it was combined with the GC/AT genotype. A significantly increased risk associated with this combined variant genotype (GC/AT + AT/AT) compared with the GC/GC genotype was observed when using either MPP or combined controls as the reference (1.40, 1.11–1.75 and 1.33, 1.10–1.60, respectively) but this risk was only borderline significant (1.22, 0.97–1.54) when using the MDACC controls as the reference (Table II).

To evaluate the risk for subgroups, we further stratified the associations between the p73 genotypes (GC/AT + AT/AT versus GC/GC) and risk of SCCHN by age, sex, smoking status, drinking status and cancer sites. As shown in Table III, the associations between the p73 genotype and risk of SCCHN were also evident for several subgroups. After adjustment for age, sex, smoking and alcohol use, whenever appropriate within each stratum, the risk associated with the combined variant genotypes (GC/AT + AT/AT) was more pronounced in subjects who were younger (≤50 years) at diagnosis (OR = 1.70, 95% CI = 1.19–2.43), women (1.61, 1.09–2.37) and current smokers (1.77, 1.25–2.51) and had oral cavity cancer (1.54, 1.15–2.07) (Table III). Among the smokers, the risk was higher in women (1.67, 1.01–2.78) than in men (1.29, 1.00–1.67), but this sex difference was not statistically significant (Pint = 0.501) (Table III). We also observed non-significantly elevated risk for both hypopharynx (1.52, 0.78–2.95) and larynx (1.39, 0.96–2.00) but a relatively lower risk for oropharynx (1.15, 0.89–1.49), and never drinkers tended to have a higher risk as well (1.46, 0.99–2.14), but it was only borderline significant (Table III). However, we did not find any evidence for multiplicative interactions between the p73 variant genotypes and age (P = 0.166), sex (P = 0.322), smoking status (P = 0.194) or alcohol use (P = 0.488) in the risk of SCCHN as evaluated in the multivariate logistic regression models.


View this table:
[in this window]
[in a new window]
 
Table III. Stratification analysis of p73 genotype frequencies, ORs and 95% CIs for their associations with risk of SCCHN by selected variables

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In this hospital-based case-control study of a non-Hispanic white population, we found that the p73 AT variant allele is a risk allele and that there is an association between the p73 variant genotype and risk of SCCHN. The variant genotypes (GC/AT + AT/AT) were associated with a >30% increased risk of SCCHN. Although how this p73 polymorphism influences development of SCCHN is unknown, one possible explanation is that the GC to AT change may lead to formation of a stem–loop structure and so may influence the translation efficiency of p73 (14). Another possibility is that the p73 polymorphism is in linkage disequilibrium with other functional polymorphisms that affect either the expression or activity levels of enzymes involved in tumorigenesis (35).

p73 shares similar sequences with p53 in their central DNA binding and C-terminal oligomerization domains (17) and partially compensates for the early loss of p53 function in some pre-malignant lesions (36). p73 is considered a putative tumor suppressor gene because it is imprinted in some human tissues (14,20,21,24,37). Difference in p73 function or splice variant expression, perhaps related to the GC/AT genotype, could play a critical role in the development or progression of early cancerous lesions (32). More importantly, p73 has also been associated with homeostasis and control of differentiation of head and neck squamous epithelium, probably together with p53 and p63, and therefore down-regulation of p73 expression could contribute to carcinogenesis of head and neck epithelium (38).

Several studies investigated the association between the p73 exon 2 polymorphism and cancer risk. In an early Irish study of 84 cases of esophageal cancer and 152 healthy, age- and sex-matched normal population controls, the AT/AT homozygous genotype appeared to protect against the development of esophageal cancer (32). However, there was only one AT/AT homozygote found among the cases, but GC/AT heterozygotes were more frequent among the cases than the controls (32), similar to what we found in our subjects. Later, a Japanese study did not find any cancer risk associated with the AT/GC genotype in 102 esophageal cancer patients, 144 stomach cancer patients, 147 colorectal cancer patients and 241 outpatients without cancer (33). More recently, the same Japanese research team did not find any cancer risk associated with the p73 polymorphism in one study of 192 non-small cell lung cancer and 241 non-cancer controls (39) and another study of 200 breast cancer and 282 non-cancer controls (40).

The genotype frequencies were 47.3% for GC/GC, 42.8% for GC/AT and 9.9% for AT/AT among 152 Irish non-cancer controls (32) and 55.3% for GC/GC, 40.4% for GC/AT and 4.3% for AT/AT among 241 Japanese non-cancer controls (33), whereas our 1229 controls had a higher proportion of the GC/GC genotype (62.9%) but a lower proportion of the GC/AT genotype (31.5%). However, the 31.5% of the GC/AT genotype in our controls is more comparable with that (27.7%) from the National Cancer Institute, Cancer Genome Anatomy Project SNP500 Cancer Database (http://snp500cancer.nci.nih.gov/snplist.cfm). The overall departure from the Hardy–Weinberg equilibrium of the p73 genotype distribution in our combined controls in the present study could be due to possible selection bias from both MPP and MDACC controls that were not random samples from the general population. The discrepant results of the Irish study may be due to the small number of subjects included, but the results of three Japanese studies of various kinds of cancers suggest a different role for this p73 polymorphism in different ethnic groups. However, this hypothesis needs to be tested further in studies of different cancer sites with larger sample sizes in which the controls and cases should be from the same population.

The greater risk of SCCHN associated with the p73 genotype in younger subjects (≤50 years) compared with other age groups is consistent with the notion of genetic susceptibility, which is often associated with early age of onset. We have observed similar findings with polymorphisms in the DNA repair gene XRCC1 in SCCHN (10). We found that the risk appeared to be higher in women in this study, particularly among woman smokers. A possible explanation for this finding is that women with the p73 variant genotypes may be more sensitive to tobacco carcinogens, which is also consistent with previous findings that women tend to have a higher risk of lung cancer than men with the same level of tobacco exposure and that women tend to have lower DNA repair capacity than men do (41). We also found that the risk associated with the p73 variant genotypes was higher in current smokers than in former and never smokers. These results may indicate that gene–environment interaction may be involved in SCCHN carcinogenesis, particularly in individuals with the p73 variant genotypes.

In addition, we found that the risk associated with the p73 variant genotypes was higher for patients with cancers of the oral cavity, hypopharynx and larynx but not for oropharynx, suggesting that oropharyngeal carcinoma may have a different etiology. Other studies have demonstrated that among SCCHN sites, oropharyngeal cancers are strongly associated with human papillomavirus-type 16 and laryngeal cancers have variable associations, while oral cavity cancers do not (7). It is possible that the p73 variant genotype is a risk factor for tobacco-induced tumors but not for human papillomavirus-initiated tumors. While we did not find any statistically significant gene–environment interactions in this study, these hypotheses will need to be tested further in future studies, including data on human papillomavirus exposure.

In this hospital-based case-control study, our SCCHN cases were enrolled from the M. D. Anderson Cancer Center, and the controls were recruited either from a multi-specialty physician practice or from among hospital visitors genetically unrelated to the cases. Therefore, the controls were not selected from the same population as the cases. In addition, as in all case-control studies, we could not rule out the possibility of selection and recall bias. Also, we restricted our analysis to non-Hispanic white subjects, so it is uncertain whether these results are generalizable to the general population. However, by matching age, sex and ethnicity, potential demographic confounding factors might have been minimized. The inadequacy in matching was controlled by further adjustments during data analysis. Nevertheless, to confirm the role of this p73 AT/GC polymorphism in cancer risk requires further studies in different populations and with other types of tumors. Because the p73 polymorphism is in a non-coding region, it will be difficult, if not impossible, to unravel the precise mechanisms by which it modulates p73 function. Future studies on potential mechanisms should focus on regulation of p73 expression at both the transcriptional and translational levels.

In conclusion, our study provides evidence that variant genotypes of a newly identified p73 exon 2 polymorphism were significantly associated with a >30% increased risk of SCCHN in a non-Hispanic white population. This association was especially noteworthy in younger individuals, women, current smokers and those with oral cancer, but the findings from subgroup analysis may be by chance due to small numbers of observations in each subgroup. Although the reason for the higher risk associated with the genotypes remain unknown, it is possible that the AT/GC genotype is functionally relevant itself or in linkage disequilibrium with alleles at other susceptibility loci. To further explore the role of the p73 gene in the etiology of SCCHN, we are currently testing other functional polymorphisms of the p53, p63 and MDM2 genes, which are members of the p53 family, to determine whether the variants of these genes interact in the etiology of SCCHN.


    Acknowledgments
 
We thank Margaret Lung and Peggy Schuber for their assistance in recruiting the subjects, Dr Zhensheng Liu, Dr Luo Wang, Jianzhong He, John I.Calderon and Kejin Xu for their laboratory assistance, Dr Maureen Goode for her scientific editing and Joanne Sider for manuscript preparation. This study was supported by National Institutes of Health grants ES 11740 (to Q.W.) and in part by CA 86390 and CA 97007 (to M.R.S. and W.K.H.), CA 16672 (to M. D. Anderson Cancer Center), CA 57730 (to R.M.C.) and ES11047 and ES07784 (center grants from the National Institute of Environmental Health Sciences).


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Pisani,P., Parkin,D.M., Bray,F. and Ferlay,J. (1999) Estimates of the worldwide mortality from 25 cancers in 1990. Int. J. Cancer, 83, 18–29.[ISI][Medline]
  2. Johnson,N.W., Ranasinghe,A. and Warnakulasuriya,K.A.A.S. (1993) Potentially malignant lesions and conditions of mouth and oropharynx: natural history-cellular and molecular markers of risk. Eur. J. Cancer Prev., 2, 31–51.[Medline]
  3. American Cancer Society, Inc. (2004) Cancer Facts & Figures. American Cancer Society, Atlanta, p. 4,
  4. Kabat,G.C., Chang,C.J. and Wynder,E.L. (1994) The role of tobacco, alcohol use, and body mass index in oral and pharyngeal cancer. Int. J. Epidemiol., 23, 1137–1144.[Abstract]
  5. Blot,W.J., McLaughlin,J.K., Winn,D.M., Austin,D.F., Greenberg,R.S. and Preston-Martin,S. (1998) Smoking and drinking in relation to oral and pharyngeal cancer. Cancer Res., 48, 3282–3287.
  6. Sankaranarayanan,R., Nair,M.K., Mathew,B., Balaram,P., Sebastian,P. and Dutt,S.C. (1992) Recent results of oral cancer research in Kerala, India. Head Neck, 14, 107–112.[ISI][Medline]
  7. Dahlstrom,K.R., Adler-Storthz,K., Etzel,C.J., Liu,Z., Dillon,L., El-Naggar,A.K., Spitz,M.R., Schiller,J.T., Wei,Q. and Sturgis,E.M. (2003) Human papillomavirus type 16 infection and squamous cell carcinoma of the head and neck in never-smokers: a matched pair analysis. Clin. Cancer Res., 9, 2620–2626.[Abstract/Free Full Text]
  8. Sturgis,E.M. and Wei,Q. (2002) Genetic susceptibility—molecular epidemiology of head and neck cancer. Curr. Opin. Oncol., 14, 310–317.[CrossRef][ISI][Medline]
  9. Cheng,L., Eicher,S.A., Guo,Z., Hong,W.K., Spitz,M.R. and Wei,Q. (1998) Reduced DNA repair capacity in head and neck cancer patients. Cancer Epidemiol. Biomarkers Prev., 7, 465–468.[Abstract]
  10. Sturgis,E.M., Castillo,E.J., Li,L., Zheng,R., Eicher,S.A., Clayman,G.L., Strom,S.S., Spitz,M.R. and Wei,Q. (1999) Polymorphisms of DNA repair gene XRCC1 in squamous cell carcinoma of the head and neck. Carcinogenesis, 20, 2125–2129.[Abstract/Free Full Text]
  11. Sturgis,E.M., Zheng,R., Li,L., Castillo,E.J., Eicher,S.A., Chen,M., Strom,S.S., Spitz,M.R. and Wei,Q. (2000) XPD/ERCC2 polymorphisms and risk for the head and neck cancer: a case-control analysis. Carcinogenesis, 21, 2219–2223.[Abstract/Free Full Text]
  12. Gollin,S.M. (2001) Chromosomal alterations in squamous cell carcinomas of the head and neck: window to the biology of disease. Head Neck, 23, 238–253.[CrossRef][ISI][Medline]
  13. Friedlander,P.L. (2001) Genomic instability in head and neck cancer patients. Head Neck, 23, 683–691.[CrossRef][ISI][Medline]
  14. Kaghad,M., Bonnet,H., Yang,A. et al. (1997) Monoallelically expressed gene related to p53 at 1p36, a region frequently deleted in neuroblastoma and other human cancers. Cell, 90, 809–819.[ISI][Medline]
  15. Jost,C.A., Marin,M.C. and Kaelin,W.G.,Jr (1997) p73 is a simian [correction of human] p53-related protein that can induce apoptosis. Nature, 389, 191–194.[CrossRef][ISI][Medline]
  16. Zhu,J., Jiang,J., Zhou,W. and Chen,X. (1998) The potential tumor suppressor p73 differentially regulates cellular p53 target genes. Cancer Res., 58, 5061–5065.[Abstract]
  17. Yang,A., Walker,N., Bronson,R. et al. (2000) p73-deficient mice have neurological, pheromonal and inflammatory defects but lack spontaneous tumours. Nature, 404, 99–103.[CrossRef][ISI][Medline]
  18. Wang,X.Q., Ongkeko,W.M., Lau,A.W., Leung,K.M. and Poon,R.Y. (2001) A possible role of p73 on the modulation of p53 level through MDM2. Cancer Res., 61, 1598–1603.[Abstract/Free Full Text]
  19. Husgafvel-Pursiainen,K., Boffetta,P., Kannio,A., Nyberg,F., Pershagen,G., Mukeria,A., Constantinescu,V., Fortes,C. and Benhamou,S. (2000) p53 mutations and exposure to environmental tobacco smoke in a multicenter study on lung cancer. Cancer Res., 60, 2906–2911.[Abstract/Free Full Text]
  20. Mai,M., Yokomizo,A., Qian,C., Yang,P., Tindall,D.J., Smith,D.I. and Liu,W. (1998b) Activation of p73 silent allele in lung cancer. Cancer Res., 58, 2347–2349.[Abstract]
  21. Yokomizo,A., Mai,M., Tindall,D.J., Cheng,L., Bostwick,D.G., Naito,S., Smith,D.I. and Liu,W. (1999) Overexpression of the wild type p73 gene in human bladder cancer. Oncogene, 18, 1629–1633.[CrossRef][ISI][Medline]
  22. Takahashi,H., Ichimiya,S., Nimura,Y., Watanabe,M., Furusato,M., Wakui,S., Yatani,R., Aizawa,S. and Nakagawara,A. (1998) Mutation, allelotyping, and transcription analyses of the p73 gene in prostatic carcinoma. Cancer Res., 58, 2076–2077.[Abstract]
  23. Ichimiya,S., Nimura,Y., Kageyama,H. et al. (1999) p73 at chromosome 1p36.3 is lost in advanced stage neuroblastoma but its mutation is infrequent. Oncogene, 18, 1061–1066.[CrossRef][ISI][Medline]
  24. Nomoto,S., Haruki,N., Kondo,M., Konishi,H., Takahashi,T., Takahashi,T. and Takahashi,T. (1998) Search for mutations and examination of allelic expression imbalance of the p73 gene at 1p36.33 in human lung cancers. Cancer Res., 58, 1380–1383.[Abstract]
  25. Cai,Y.C., Yang,G.Y., Nie,Y., Wang,L.D., Zhao,X., Song,Y.L., Seril,D.N., Liao,J., Xing,E.P. and Yang,C.S. (2000) Molecular alterations of p73 in human esophageal squamous cell carcinomas: loss of heterozygosity occurs frequently; loss of imprinting and elevation of p73 expression may be related to defective p53. Carcinogenesis, 21, 683–689.[Abstract/Free Full Text]
  26. Nimura,Y., Mihara,M., Ichimiya,S. et al. (1998) p73, a gene related to p53, is not mutated in esophageal carcinomas. Int. J. Cancer, 78, 437–440.[CrossRef][ISI][Medline]
  27. Zaika,A.I., Kovalev,S., Marchenko,N.D. and Moll,U.M. (1999) Overexpression of the wild type p73 gene in breast cancer tissues and cell lines. Cancer Res., 59, 3257–3263.[Abstract/Free Full Text]
  28. Kang,M.J., Park,B.J., Byun,D.S., Park,J.I., Kim,H.J., Park,J.H. and Chi,S.G. (2000) Loss of imprinting and elevated expression of wild-type p73 in human gastric adenocarcinoma. Clin. Cancer Res., 6, 1767–1771.[Abstract/Free Full Text]
  29. Han,S., Semba,S., Abe,T. et al. (1999) Infrequent somatic mutations of the p73 gene in various human cancers. Eur. J. Sur. Oncol., 25, 194–198.[CrossRef][ISI][Medline]
  30. Choi,H.R., Batsakis,J.G., Zhan,F., Sturgis,E., Luna,M.A. and El-Naggar,A.K. (2002) Differential expression of p53 gene family members p63 and p73 in head and neck squamous tumorigenesis. Hum. Pathol., 33, 158–164.[CrossRef][ISI][Medline]
  31. Peters,M.A., Janer,M., Kolb,S., Jarvik,G.P., Ostrander,E.A. and Stanford,J.L. (2001) Germline mutations in the p73 gene do not predispose to familial prostate-brain cancer. Prostate, 48, 292–296.[CrossRef][ISI][Medline]
  32. Ryan,B.M., McManus,R., Daly,J.S., Carton,E., Keeling,P.W., Reynolds,J.V. and Kelleher,D. (2001) A common p73 polymorphism is associated with a reduced incidence of oesophageal carcinoma. Br. J. Cancer, 85, 1499–1503.[ISI][Medline]
  33. Hamajima,N., Matsuo,K., Suzuki,T. et al. (2002) No associations of p73 G4C14-to-A4T14 at exon 2 and p53 Arg72Pro polymorphisms with the risk of digestive tract cancers in Japanese. Cancer Lett., 181, 81–85.[CrossRef][ISI][Medline]
  34. Hamajima,N., Saito,T., Matsuo,K., Kozaki,K., Takahashi,T. and Tajima,K. (2000) Polymerase chain reaction with confronting two-pair primers for polymorphism genotyping. Jpn. J. Cancer Res., 91, 865–868.[ISI][Medline]
  35. Haber,D.A. and Fearon,E.R. (1998) The promise of cancer genetics. Lancet, 351, 1–8.[CrossRef][ISI]
  36. Montesano,R., Holestein,M. and Hainaui,P. (1996) Genetic alterations in esophageal cancer and their relevance to etiology and pathogenesis: a review. Int. J. Cancer, 69, 225–235.[CrossRef][ISI][Medline]
  37. Mai,M., Qian,C., Yokomizo,A., Tindall,D.J., Bostwick,D., Polychronakos,C., Smith,D.I. and Liu,W. (1998) Loss of imprinting and allele switching of p73 in renal cell carcinoma. Oncogene, 17, 1739–1741.[CrossRef][ISI][Medline]
  38. Faridoni-Laurens,L., Bosq,J., Janot,F., Vayssade,M., Le Bihan,M.L., Kaghad,M., Caput,D., Benard,J. and Ahomadegbe,J.C. (2001) p73 expression in basal layers of head and neck squamous epithelium: a role in differentiation and carcinogenesis in concert with p53 and p63? Oncogene, 20, 5302–5312.[CrossRef][ISI][Medline]
  39. Hiraki,A., Matsuo,K., Hamajima,N., Ito,H., Hatooka,S., Suyama,M., Mitsudomi,T. and Tajima,K. (2003) Different risk relations with smoking for non-small-cell lung cancer: comparison of TP53 and TP73 genotypes. Asian Pac. J. Cancer Prev., 4, 107–112.[Medline]
  40. Huang,X.E., Hamajima,N., Katsuda,N. et al. (2003) Association of p53 codon Arg72Pro and p73 G4C14-to-A4T14 at exon 2 genetic polymorphisms with the risk of Japanese breast cancer. Breast Cancer, 10, 307–311.[Medline]
  41. Wei,Q., Cheng,L., Amos,C.I., Wang,L.E., Guo,Z., Hong,W.K. and Spitz,M.R. (2000) Repair of tobacco carcinogen-induced DNA adducts and lung cancer risk: a molecular epidemiologic study. J. Natl Cancer Inst., 92, 1764–1772.[Abstract/Free Full Text]
Received January 13, 2004; revised May 18, 2004; accepted May 25, 2004.