Laboratory of Human Carcinogenesis, NCI Center for Cancer Research, 37 Convent Drive, Bethesda, MD 20892-4255, USA, 1 Laboratory of Carcinogenesis and Biomarkers, CBCP-IRC, USUHS, Bethesda, MD, USA, 2 Lombardi Comprehensive Cancer Center, Georgetown University Medical Center, Washington, DC, USA and 3 Advance Technology Center, NCI, Gaithersburg, MD 20892, USA
* To whom correspondence should be addressed. Tel: +1 301 496 2048; Fax: +1 301 496 0497; Email: curtis_harris{at}nih.gov
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Abstract |
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Abbreviations: ORs, odds ratios; RFLP, restriction fragment length polymorphism; SSCP, single-stranded conformation polymorphism
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Introduction |
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Inactivation of the TP53 tumor suppressor gene is a key and thought to be an early event in lung carcinogenesis (3). The p53 protein functions to induce growth arrest, DNA repair or apoptosis in response to cellular stress, including DNA damage (46). Mutations in TP53 are present in >90% of small cell lung cancers and >50% of non-small cell lung cancers (79), suggesting that inactivation of p53 effector pathways often occurs as a consequence of mutation in p53. The majority of these mutations are missense mutations in exons 58, the DNA-binding domain of p53. Point mutations in TP53 more frequently occur at functionally and evolutionarily conserved residues of p53. These regions are considered hot-spots for p53 mutation (10).
The pattern (type or position) of somatic mutations in TP53 is distinct for particular cancers and carcinogenic exposures, providing clues to disease etiology (11). For example, G:CT:A mutations in TP53 are more frequently observed in tumors from smoking-associated cancers and mutations at codon 157 are relatively specific for smoking-associated lung cancer (11,12). Consistent with the observed mutation spectrum of TP53 in tumors, normal bronchial epithelial cells exposed to the active metabolite of benzo[a]pyrene, benzo[a]pyrene diol epoxide, developed mutations in TP53 at hot-spot positions which were predominantly G:C
T:A transversions (13).
Common functional variants in apoptosis and DNA repair genes, such as TP53 and XPD, may influence lung cancer susceptibility. The Arg72Pro polymorphism (rs1042522) in TP53 may modulate lung cancer risk (1417). This polymorphism in TP53 is located in a polyproline domain, which was recently shown to have an important role for transcription-independent function of p53 in apoptosis in the cytoplasm, as reviewed (18). The Arg72 form of p53 is more efficient at p53-dependent apoptosis compared with the Pro72 form, through more effective mitochondrial localization (19). Moreover, the Asn312 and Gln751 variants of XPD, a protein required for nucleotide excision repair with a role in p53-dependent apoptosis, are in linkage disequilibrium and are associated with reduced DNA repair capacity and increased lung cancer risk in some studies (2023).
In order to both investigate possible causes and characterize mechanisms of susceptibility differences, TP53 was sequenced in tumors from patients in a lung cancer case-only study. Patients with lung cancer (206 men and 103 women) were recruited at the time of diagnosis from hospitals in the greater Baltimore region. We investigated whether the TP53 mutation frequencies and distributions in these tumors were influenced by common functional polymorphisms in XPD and TP53.
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Materials and methods |
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Between 1974 and 1999, 1253 lung cancer cases were available as part of a large multi-organ study. The refusal rate of these cases for interviews was 6%. A case-only study was created using these historical cases. All women with confirmed primary lung cancer who were African-American or Caucasian and had completed questionnaires were recruited. For each woman, one man was pair-wise matched based on age (±5 years), race (African-American or Caucasian) and surgery date (±5 years) and a second man was pair-wise matched based on age (±5 years), race (African-American or Caucasian), surgery date (±5 years) and pack-years of smoking (±20 pack-years). From these patients, 371 were initially selected to be a part of the gender study. Because 19 patients refused to be in the study (16 personal and 3 physician refusals) and 43 patients did not have paraffin-embedded tissue available for study, only 309 patients (103 women and 206 men) were included into our case-only study. Matching for gender was performed in order to be able to examine gender-specific factors by increasing the proportion of women lung cancer cases in the study population. Pair-wise matching was incomplete for 6 subjects, 1 woman and 5 men, included in the study. Removing these individuals did not alter the results. Histology and staging were determined in a blinded fashion, according to WHO II guidelines, by three independent individuals (A.J.Marrogi, R.T.Jones and A.Borkowski).
DNA extraction
In order to isolate tumor DNA from the formalin-fixed tissue for the determination of TP53 mutation status, tumor foci were identified and tumor tissue was dissected away from normal tissue. Paraffin was removed from slides with 100% xylene and two washes with 100% ethanol. After paraffin removal, slides were dissected to enrich samples for majority tumor tissue. Frozen lung tissue was used for genotyping. For XPD 312 genotyping 89% of the tissue was uninvolved tissue and 11% was from tumor tissue. For XPD 751 and TP53 72 genotyping 88% of the tissue used was uninvolved and 12% was tumor tissue. Tissue samples obtained from slides or frozen samples were incubated in a SDS/proteinase K solution until tissue solution was homogeneous using methods outlined (24). DNA was then isolated using phenol/chloroform extraction followed by an overnight ethanol precipitation (20°C) using standard methods.
Mutational analysis of TP53
All mutation analyses were performed on coded samples lacking identifiers. Sequencing for TP53 mutations was performed on exons 58 of TP53. Screening for TP53 mutations was performed using the p53 GeneChip (Affymetrix), single-stranded conformation polymorphism (SSCP) and direct manual sequencing. One sample failed to amplify in both the GeneChip assay and the nested PCR experiments for SSCP and manual sequencing. Out of 1232 possible exons evaluated (308 participants and exons 58), 60% were successfully evaluated by GeneChip, 23% by SSCP, 18% by manual sequencing and 16% by a combination of the three methods.
The p53 GeneChip was used according to the instructions from the manufacturer (Affymetrix) and as described previously (25,26). DNA obtained from cell lines with known TP53 mutations was used as positive controls for each GeneChip experiment (SW480, codon 273 CGTCAT and Calu6, codon 196 CGA
TGA). GeneChip performed poorly on the archival paraffin-embedded tissue DNA samples. DNA obtained from 14% of participants in this study failed to amplify using the GeneChip and amplification using the GeneChip was not observed in at least one exon (exons 58) of TP53 in 41% of participants. In a pilot study a score of
13 was confirmed as mutant by manual sequencing in 60% of cases, a score of
13 and only a single mutation per GeneChip was confirmed by manual sequencing in 75% cases and a score of
15 plus one mutation per GeneChip was confirmed by manual sequencing in 86% of cases (J.Welsh and M.Khan, unpublished observations). Therefore, only mutations with a score of
13 were defined as mutations after confirmation by a second method. All GeneChip amplifications with a single TP53 mutation and a score <15 or multiple TP53 mutations >13 were verified by a second method (SSCP and/or manual sequencing) and GeneChip amplifications with a score of
15 were considered as mutant. Ninety three percent of GeneChip amplifications with a score of
15 and a single mutation were also verified using a second procedure. Fifty-four samples were scored with only one TP53 mutation per GeneChip with a score
15. Forty-eight of the 54 samples (89%) were coded as mutant, the other 6 samples being determined to be wild-type by manual sequencing. No TP53 mutations with a score of >13 were detected in 730 exons by GeneChip. All of these samples, except one, were defined as wild-type. One sample was determined to have a mutation by manual sequencing. Two percent of the GeneChip samples scored as wild-type for TP53 were evaluated by manual DNA sequencing with 93% concordance.
The mutational analysis of TP53 for exons that failed to amplify in GeneChip experiments was performed by SSCP and direct manual sequencing of exons 58 of TP53. Coding sequences and splice junctions were amplified using nested PCR reactions with an external and internal set of primers as described (27). SSCP was performed on amplified PCR products using procedures outlined (28). SSCP samples negative for a TP53 mutation were defined as wild-type. Samples scored as positive in SSCP experiments were evaluated by direct dideoxy sequencing of amplified products. Manual sequencing gels were evaluated by two independent readers (J.Welsh and M.Khan). For quality control, 10% of samples determined to be negative by SSCP were directly sequenced, with a 74% concordance rate. Nine of the negative SSCP samples were determined to contain TP53 mutations by manual sequencing.
XPD 312 and 751 genotyping
XPD 312 (rs1799793) and 751 (rs1052559) genotyping was performed by PCRrestriction fragment length polymorphism (RFLP). To genotype XPD at codon 312, DNA was amplified using the XPD312F (5'-CAGCTCATCTCTCCGCAGGATCAA-3') and XPD312R (5'-GTCGGGGCTCACCCTGCAGCACTTCCT-3') primers. Reactions contained 1x PCR buffer II, 500 nM each primer, 1.5 mM MgCl2, 5.2% dimethylsulfoxide, 0.2 mM dNTPs, 50 ng genomic DNA and 1 U AmpliTaq DNA polymerase (Perkin Elmer) in 25 µl. Amplifications were performed under the following conditions: 94°C for 4 min; 40 cycles of 94°C for 3045 s (denaturation), 61°C for 3045 s (annealing) and 72°C for 11.5 min (extension); followed by 72°C for 4 min. After PCR amplification, 10 µl of PCR products (165 bp) were digested overnight at 37°C using StyI restriction enzyme according to the manufacturer's (NEB) instructions. Digestion products were evaluated on 2% agarose gels stained with ethidium bromide. Genotypes were determined to be Asp/Asp (165 bp fragment), Asp/Asn (165, 139 and 26 bp fragments) or Asn/Asn (139 and 26 bp fragments) by the presence of appropriately sized DNA fragments.
To genotype XPD at codon 751, DNA was amplified using the XPD751F (5'-TCTGCAGGAGGATCAGCTG-3') and XPD751R (5'-GCAAGACTCAGGAGTCAC-3') primers. Reactions contained 1x PCR buffer II, 500 nM each primer, 1.5 mM MgCl2, 0.2 mM dNTPs, 50 ng of genomic DNA and 1 U AmpliTaq DNA polymerase (Perkin Elmer) in 25 µl. Amplifications were performed under the following conditions: 94°C for 4 min; 40 cycles of 94°C for 30 s (denaturation), 59°C for 3045 s (annealing) and 72°C for 11.5 min (extension); followed by 72°C for 4 min to generate 149 bp amplified DNA products. Aliquots of 10 µl of PCR products were digested overnight at 37°C using PstI restriction enzyme according to the manufacturer's (NEB) instructions. Digested products were evaluated on 2% agarose gels stained with ethidium bromide. The appearance of 143 and 6 bp bands indicated the Lys/Lys genotype, 143 plus 80, 63 and 6 bp bands the Lys/Gln genotype and 80 plus 63 and 6 bp the Gln/Gln genotype.
Samples that failed to amplify were repeated. Those samples that failed to amplify on the second run were scored as missing. Genotyping was performed on a subset of samples for XPD312 and XPD751, due to lack of available frozen tissue for genotyping. DNA was unavailable from 39 participants (13%) to genotype XPD 312 and from 36 (12%) participants for XPD 751 genotyping due to the lack of available frozen tissue. For both XPD genotypes genotyping of 20% of samples was repeated and the concordance between duplicate genotypes was 100%.
TP53 codon 72 genotyping
Genotyping of Arg72Pro of TP53 (rs1042522) was determined using a Taqman assay according to the instructions on the website (NCI Core Genotyping Facility, http://snp500cancer.nci.nih.gov). Twenty-five samples that failed to amplify using Taqman procedures were genotyped manually by PCRRFLP (n = 16). For quality control, 10% of the samples genotyped (n = 26) using the Taqman assay were repeated. The concordance of genotypes between duplicate samples was 100%. Eight percent of samples (n = 20) were also confirmed by PCRRFLP. Genotypes determined using both methods were identical. Genotyping was performed on a subset of samples for TP53 codon 72 due to a lack of frozen tissue for DNA genotyping and due to exhaustion of tissue samples. DNA was unavailable from 62 (20%) of participants for TP53 codon 72 genotyping.
Statistical methods
Differences in the characteristics of lung cancer patients were compared by the 2 or Fisher's exact test (when 20% of expected counts were <5) for categorical values or by Student's t-test for continuous measures as indicated. Never smokers were participants who had never smoked regularly for
6 month duration. Former smokers reported quitting smoking
1 year prior to the date of diagnosis. Current smokers had continued to smoke or quit smoking <1 year prior to the date of lung cancer diagnosis. Individuals were considered positive for cancer family history if they had parents or siblings with a previous diagnosis of lung cancer. Departures from HardyWeinberg equilibrium for the XPD or TP53 genotypes were evaluated by calculating expected genotype frequencies based on observed allele frequencies and comparing expected frequencies with observed genotype frequencies using the
2 test.
This study was originally designed to examine gender differences in the mutation spectrum of TP53 and so female and male lung cancer cases were pair-wise matched. For the purposes of examining associations of genetic polymorphisms and exposures other than gender with the TP53 mutation frequency, the data were treated as unmatched. Gender was not associated with mutation and was not a confounder or an effect modifier for any associations reported. Unconditional logistic regression models were used to calculate adjusted odds ratios (ORs) for XPD and TP53 genotypes in order to estimate the effect of these variables on the presence of a TP53 mutation or a G:CT:A TP53 mutation in lung tumors. PROC LOGISTIC in SAS version 8.1 (SAS Institute, Cary, NC) was used to perform unconditional logistic regression and to adjust for potential confounding variables, including race (African-American, Caucasian), age (quintiles) and smoking duration (
50 or >50 years), as indicated. Confounding factors were evaluated by determining whether variables resulted in changes in the OR of at least 5%. ORs estimated using conditional analyses were consistent with reported results. Participants with missing values for any of the variables in a regression model were omitted from the analysis.
Interaction between TP53 genotype and smoking or TP53 genotype and XPD genotype was examined by stratified analysis to investigate heterogeneity between strata. To further evaluate multiplicative interaction, the P value for the ß coefficient of an interaction term (TP53 x XPD) used in unconditional logistic regression models was calculated (Pint). Tests for trend were conducted by calculating P values for the ß coefficient in unconditional logistic regression models with XPD genotype or TP53 genotype coded as the ordinal variable (Ptrend).
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Results |
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Individuals with either the TP53 Arg/Pro72 or Pro/Pro72 genotype had increased odds of any TP53 mutation or a G:CT:A mutation in TP53. There was a suggestion of a doseresponse relationship of increased odds for TP53 mutations (Ptrend = 0.018) or G:C
T:A mutations (Ptrend = 0.047) in participants that correlated with increased copies of the Pro allele. The association of the Pro allele at codon 72 of TP53 with the presence of any TP53 mutations or G:C
T:A TP53 mutations in lung tumors was slightly stronger in individuals who smoked for shorter durations (5165 versus 050 years). Participants with the Pro/Pro or Arg/Pro genotype who had smoked for 050 years had an increased odds of TP53 mutations (OR 2.65, 95% CI 1.275.51) in comparison with participants with the Arg/Arg genotype. Meanwhile, an increased odds of TP53 mutation was less apparent in individuals who smoked for 5165 years with the Pro/Pro or Arg/Pro genotype when compared with Arg/Arg participants (any TP53 mutation OR 1.39, 95% CI 0.385.04; G:C
T:A mutation OR 1.26, 95% CI 0.305.28). Interaction on the multiplicative scale between the Arg72Pro TP53 genotype and smoking duration was not observed for TP53 mutations (Pint = 0.135).
In examining the association of variant XPD and TP53 genotypes with the presence of G:CT:A mutations in TP53, tumors without G:C
T:A mutations were used as the denominator. Some of these tumors showed other types of somatic TP53 mutations. When these tumors were excluded from analysis, variant XPD genotypes (any Asn312 or any Gln751 OR 2.98, 95% CI 1.058.42) or variant TP53 genotypes (OR 2.63, 95% CI 1.046.62) were associated with increased odds of somatic G:C
T:A mutations in TP53.
Since the TP53 Arg72Pro polymorphism was associated with the presence of a mutation in TP53 and there was a suggestion of an increased frequency of TP53 mutations in individuals with variant XPD genotypes (Asn312 or Gln751) in lung tumors in the preliminary analysis, these polymorphisms were examined in combination (Table III). Individuals with either the TP53 Arg/Pro72 or TP53 Pro/Pro72 genotype had increased odds of any TP53 mutation or a G:CT:A mutation attributable to the presence of any XPD Asn312 or XPD Gln751 genotype in comparison with the effect of the variant XPD alleles in participants with the TP53 Arg/Arg72 genotype. Evidence for an interaction between TP53 and XPD genotypes was detected on the multiplicative scale (any TP53 mutation Pint = 0.027; G:C
T:A TP53 mutation Pint = 0.041). These P values are consistent with a possible interaction between XPD and TP53 genotypes.
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Discussion |
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Cigarette smoking results in the formation of bulky adducts on DNA (38). The predominant pathway for repair of this form of DNA damage is nucleotide excision repair (39). XPD is an essential protein required for nucleotide excision repair (global genome and transcription coupled repair) and transcription (40). Some studies reported that XPD variant alleles (Asn312 or Gln751) were associated with reduced DNA repair capacity as measured by increased DNA adducts (22,31,41) or host cell reactivation assay (20). In our study a tendency towards an increased frequency of TP53 or G:CT:A mutations in TP53 was observed in participants with variant alleles in XPD (Asn312 or Gln751), although this increase was not statistically significant.
Several studies have investigated the possible association of XPD Asp312Asn and Lys751Gln polymorphisms with lung cancer, with inconsistent results. These polymorphisms were shown to be in linkage disequilibrium and were linked in our cases (20,21). Some studies failed to observe an association of the XPD Lys751Gln polymorphism with lung cancer (21,34,42). Meanwhile, other studies observed that participants with genotypes containing Asp312 or Lys751 had greater odds of lung cancer (21,43,44). In contrast, other reports noted an increased risk of lung cancer with variant alleles in XPD (20,23,45). The inconsistent associations in previous studies observed for the XPD polymorphisms could be due to differences in study populations, the small sample sizes of earlier studies and possible environmental interactions.
Two previous reports investigated the potential association of variant alleles in XPD with increased TP53 mutation frequency in lung cancer (46,47). Consistent with our observation of an association of Asn312 and Gln751 with an increased odds of somatic G:CT:A mutations in TP53, transversion mutations (including all types of transversions) were modestly increased in patients with XPD variant alleles (47). In contrast, in another report an increased TP53 mutation frequency was observed in participants with genotypes containing the Asp312 allele of XPD and no association was observed with the Lys751Gln polymorphism of XPD. This group also reported no difference in the distribution of TP53 mutation type related to XPD polymorphisms (46). Both of these previous studies focused on transversion versus transition mutations in TP53, yet our data indicate that the association of XPD polymorphisms with TP53 mutations seems stronger specifically for G:C
T:A transversion mutations. G:C
T:A mutations are the type of transversion most strongly related to smoking exposure (12). Therefore, Gao et al. (46) may not have observed a higher frequency of somatic TP53 mutations in participants with variant alleles in XPD due to the low frequency of G:C
T:A transversion mutations detected in their study in comparison with our study.
Furthermore, in our study, the Pro72 allele of TP53 was associated with an increased frequency of somatic mutations in TP53 (any TP53 mutation and G:CT:A TP53 mutations). Several studies have examined the association of the Arg72Pro polymorphism with lung cancer (16,17,32,33,4851). Some studies observed higher odds of lung cancer in individuals with the Arg/Pro or Pro/Pro genotype (17,33,52) or a greater risk of adenocarcinoma in particular (1517). Meanwhile, other studies reported no association of the Arg72Pro TP53 polymorphism with lung cancer (32,48,51) or higher odds of lung cancer in participants with Arg alleles at codon 72 (50). A recent meta-analysis concluded that previous studies of the association of lung cancer with the Arg72Pro polymorphism provide insufficient evidence for an effect of these variants on lung cancer susceptibility (37). Of note, many previous studies showed that there was a detectable violation of the HardyWeinberg equilibrium for the Arg72Pro TP53 polymorphism in at least one population subgroup (17,32,33,50). Violations of the HardyWeinberg equilibrium may be indicative of genotyping error, population stratification or selection bias (53). A recent review of the TP53 Arg72Pro polymorphism in cervical cancer suggested that some of the heterogeneity in previous studies could be attributed to violations of the HardyWeinberg equilibrium (36). We also observed a suggestion of a possible interaction between the Arg72Pro polymorphism with smoking duration. The Pro72 allele of TP53 was more strongly associated with somatic TP53 mutations in individuals who smoked for shorter durations, consistent with the observed stronger association of Pro72 with lung cancer in individuals who smoked for shorter pack-years (33).
While the potential contribution of the Arg72Pro TP53 polymorphism to lung cancer risk is unresolved in observational studies, Arg or Pro at codon 72 of TP53 alters the function of the p53 protein in apoptosis. Wild-type p53 with Arg72 was demonstrated to be more efficient at apoptosis (19,54,55). The higher rate of apoptosis by Arg72 p53 compared with Pro72 p53 was suggested to be through more effective localization to the mitochondria (19). Consistent with this model, codon 72 of p53 is in a polyproline domain of p53 which was shown to be required for transcription-independent apoptosis (18). In addition to transcription-independent mechanisms, a higher induction by Arg72 p53 versus Pro72 p53 of the apoptotic genes PIGPC1, PUMA and NOXA was observed, suggesting that the increased apoptosis by Arg72 p53 may be through both transcription-dependent and transcription-independent pathways (19,55). As well as the p53-dependent apoptotic pathways, the Arg72Pro polymorphism may modulate other apoptotic pathways. Several studies have indicated that mutated p53 with Arg at codon 72 is more effective at inhibiting p73-dependent apoptosis (5658). Taken together, these studies suggest that the effect of the Arg72 or Pro72 amino acid on p53 function is dependent on whether TP53 is wild-type or mutated in tissue. Consistent with this notion, when comparing individuals with somatic TP53 mutations, a poorer prognosis was observed for participants with mutated Arg72 alleles versus Pro72. Meanwhile, when comparing individuals with wild-type TP53, a poorer prognosis was associated with Pro72 alleles (55,56).
Previously, a few studies examined the association of the Arg72Pro polymorphism in TP53 with the presence of somatic TP53 mutations. Consistent with our observations, a recent study reported that the Pro72 allele of TP53 was associated with an increased frequency of somatic TP53 mutations in ovarian cancer (59). In other studies, somatic mutations in TP53 were associated with the Arg72 allele of TP53 (56,57,60,61). One study observed an association of Arg72 with TP53 mutations in breast cancer and another study failed to observe the same association (56,60). In tumor tissues from individuals with the Arg/Pro genotype, it was reported that the Arg72 allele of TP53 was preferentially mutated and the Pro72 allele of TP53 was more often the target of loss (56,57,60,61). However, in a few of these studies it was not possible to evaluate the association of the Pro/Pro genotype with the presence of somatic TP53 mutations, because only a small number of tumors were from individuals with the Pro/Pro genotype (<2 in each study) (50,56,61). The difference between the observed association in these studies and our results may also be due to differences in tissue type. Associations between the Arg72 polymorphism and somatic TP53 mutation appear to be tissue type-specific and the specificity may be related to expression levels of p73 (56,60). Another factor that may influence the association of the Arg72Pro polymorphism with TP53 mutations in tumors is the type of mutation. Primarily recessive conformational TP53 mutations, as evaluated using a yeast transdominance assay, were associated with Arg72 alleles (57).
An interaction between TP53 and XPD polymorphisms was detected on the multiplicative scale. Participants with the Arg/Pro72 or Pro/Pro72 TP53 genotype and XPD variant alleles had greater odds of TP53 mutation than expected if these polymorphisms functioned independently. The statistical interaction observed in our study is consistent with the observed physical interaction for XPD and p53 and functional interaction in nucleotide excision repair and apoptosis (6264). It is possible that the statistical interaction between the XPD and TP53 polymorphisms for the appearance of somatic p53 mutations is due to some combination of global genome nucleotide excision repair, transcription coupled repair or apoptosis pathways. It is important to note that in a case-only study examining interactions between polymorphisms, it is assumed that the frequencies of polymorphisms are independent in the source population. The previously reported frequencies of the XPD and TP53 polymorphisms were different (17,20,21,23,3036) and these genes are on separate chromosomes, suggesting that these polymorphisms are likely independent in controls.
A limitation of our study is the small number of African-American and Caucasian participants. There was no evidence of an interaction with race for any polymorphism examined. Therefore, all participants were combined to examine the influence of each polymorphism on TP53 mutation frequency and the models were adjusted for race. Combining Caucasian and African-American participants probably did not affect the results, because when limiting the analysis only to Caucasian participants similar ORs and interactions as reported were observed. Since there were only a small number of African-Americans in this study, limiting analysis only to African-Americans resulted in ORs close to the null value for any mutation in TP53 and could not be calculated for G:CT:A mutations in TP53. As a result, further study is needed to clarify the role of these polymorphisms in African-Americans. Moreover, due to the relatively small number of participants and mutations detected in this study, it is possible that the results observed are due to chance. These observed associations and interactions need to be confirmed in additional studies.
The frequency of TP53 mutations (25%) detected in our lung cancer case series was lower than the previously reported mutation frequency for TP53 in non-small cell lung cancer (42%, 95% CI 4045%) and in small cell lung cancer (59%, 95% CI 4870%) (65). The lower than expected frequency of TP53 mutations observed in our study may be due to assay sensitivity. The DNA for TP53 mutation determination in our study was obtained from archival paraffin-embedded tissues. These samples, in particular older samples, did not amplify well, reflecting difficulties with the DNA extraction and the 10 exon multiplex PCR required for successful GeneChip analysis. When limiting data analysis to cases from 1987 to 1999, the association of XPD and TP53 polymorphisms with increased somatic TP53 mutation frequency were the same as in the entire data set. Considering the low observed TP53 mutation frequency in our study and the suggested limitations of the assays used to evaluate the TP53 mutation frequency, it is possible that a portion of the cases defined as wild-type for somatic TP53 mutations contained TP53 mutations. A sensitivity analysis was performed assuming a 10% increase in actual mutation frequency. Using these numbers, the estimated ORs for the association of XPD and TP53 polymorphisms increased. A second sensitivity analysis was performed defining discordant (samples scored as mutant by GeneChip and wild-type by manual sequencing) samples as mutated (if no mutations were detected at other exons from the same person). In this analysis the same associations as reported were observed. Moreover, it is important to note that the overall trends in the mutation frequency of TP53 in our study are consistent with previous reports. The distribution of type of somatic TP53 mutations observed was similar to the type distribution in the IARC database and we observed an increased frequency of TP53 mutations in squamous cell carcinomas compared with adenocarcinomas, as reported previously (65,66).
A limitation of our study is the lack of genotyping data for some samples in our case series due to a lack of available frozen tissue or blood for DNA isolation. In addition, some tumor specimens were exhausted for the determination of TP53 mutation status or XPD genotyping. Since the genotyping data were not missing at random, this is a potential source of bias in this study population, even when performing complete case analyses as was done in this report. Missing genotyping data were related to surgery date; more recent samples had a significantly lower frequency of missing genotypes. Importantly, when limiting analysis to cases from 1987 to 1999, genotyping data were missing at random and overall associations of XPD and TP53 genotypes with somatic TP53 mutations were the same as in the entire data set. Therefore, it is unlikely that the missing genotyping data dramatically altered the reported results.
In conclusion, we have examined the association of smoking and polymorphisms in XPD and TP53 with somatic TP53 mutations in a lung cancer case series. The association between these factors and the TP53 mutation spectrum provides a valuable framework for developing new hypotheses about the mechanisms of lung carcinogenesis.
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Acknowledgments |
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References |
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