Mutagenesis, Vol. 15, No. 6, 457-458,
November 2000
© 2000 UK Environmental Mutagen Society/Oxford University Press
Discussion Forum |
Analysis of different versions of the IARC p53 database with respect to G
T transversion mutation frequencies and mutation hotspots in lung cancer of smokers and non-smokers
Analytisch-Biologisches Forschungslabor, Goethestrasse 20, D-80336 Munich, Germany
Abstract
Analysis of the IARC p53 database revealed a large number of discrepancies in the classification of smoking status for identical lung cancer entries in different versions of the database. In addition, no statistically significant differences in G
T transversion mutation frequencies or in mutational hotspots at codons 157, 248 and 273 were found in the R3 version of the database between p53 sequences from smoking and non-smoking lung cancer patients. The possible influence of confounding factors on p53 mutation spectra was demonstrated as illustrated by the impact of ethnicity on GT transversion mutation frequencies.
The p53 gene encodes a tumour suppressor protein with negative regulatory functions in cell proliferation processes. Activation of wild-type p53 as a result of DNA damage leads either to cell cycle arrest or to apoptosis (Kastan et al., 1995
). Missense mutations in the coding region of the gene, which lead to inactivation of p53, are often found in various human cancers. The comparison of in vitro mutagenesis data with the mutational pattern of p53 in cancer has led to the conviction that evaluation of mutational spectra in human tumours may provide information about the molecular basis of carcinogenic mechanisms (Hollstein et al., 1991; Greenblatt et al., 1994).
One often-cited example of the `fingerprint' of a mutagenic substance in the p53 tumour suppressor gene is the distribution and frequency of GT transversion mutations detected in lung cancers of smokers. These mutations are presumed to result from DNA adducts of polycyclic aromatic hydrocarbons (PAHs) such as benzo[a]pyrene (BaP) present in tobacco smoke (Greenblatt et al., 1994; Hussain and Harris, 1998; Pfeifer and Denissenko, 1998) based on in vitro studies showing that BaP and its mutagenic metabolite, benzo[a]pyrene-diolepoxide (BPDE), predominantly induce GT transversions (Chen et al., 1990; Ruggeri et al., 1993; Mazur and Glickman, 1998).
Using the January 1998 version of the International Agency for Research on Cancer (IARC) p53 database (R1), which contains 8131 entries for p53 mutations in human tumours and cell lines (Hainaut et al., 1998; http://www.iarc.fr/p53/homepage.htm#p53), Hernandez-Boussard and Hainaut (1998) found significantly more G T transversions in lung cancers of smokers than in those of non-smokers (
2 = 7.83; P < 0.002). The authors concluded that their analysis was a clear indication for the carcinogenic action of BaP present in tobacco smoke, yet failed to define the exclusion criteria used in the analysis, which was based on only 236 of a total of 284 entries for smokers (83%) and 36 of 95 entries for non-smokers (38%). Taking into consideration all lung cancer entries, except patients who were listed twice in the p53 database because the same study population had been reported in two separate publications, reanalysis of the R1 version of the database showed a statistically significant difference in GT transversion frequencies in smokers and non-smokers (2 = 3.98; P < 0.046), although the difference was less clear than that reported by Hernandez-Boussard and Hainaut (1998).
The R1 version of the IARC p53 database was updated in July 1998. The updated version, R2, contained information from 1046 lung cancer cases, whereas the earlier version had 900 entries. Surprisingly, only 221 of the entries in the R2 version were classified for smoking status (131 smokers, 90 non-smokers), whereas in the earlier R1 version 379 lung cancer cases with information on smoking status had been included (284 smokers, 95 non-smokers).
Careful analysis of the R2 version of the database revealed that 179 entries listed as smokers and 34 as non-smokers in the R1 version of the database were no longer classified for smoking status. Analysis of the original publications showed that most of these entries with missing information on smoking status in the R2 version of the p53 database had been correctly classified as smokers and non-smokers and included in the R1 version. In addition, a total of 26 new entries for smokers and 29 new entries for non-smokers were included in the R2 version of the database. The current, R3, version of the p53 database, which was initially released in January 1999 and modified in April 1999, again contains most of these entries with the correct assignment of smoking status as reported in the original publications. The changes in classification of identical entries in updated versions of the database are difficult to understand and complicate the comparison of studies based on different versions of the p53 database. The basic properties of all three versions of the IARC p53 mutation database are summarized in Table I.
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In response to these discrepancies in the presentation of data in the R1, R2 and R3 versions of the IARC p53 mutation database, an analysis is presented to determine the validity of the conclusions previously reported by Hernandez-Boussard and Hainaut (1998). Analysis of the R2 version and the modified R3 version of the database using all entries for lung cancer revealed non-significant differences in the G
T transversion frequencies between smokers and non-smokers (Figure 1; R2 version: 2 = 0.44, P = 0.51; R3 version: 2 = 0.69, P = 0.41). Exclusion of entries listing exposure to radon (which may have been performed by Hernandez-Boussard and Hainaut (1998) in their analysis of the R1 version of the database) did not substantially change the results of the analysis (R2 version: no change; R3 version: 2 = 0.68, P = 0.41).
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Hernandez-Boussard and Hainaut (1998) further reported the presence of mutation hotspots at codons 157, 248 and 273 in lung cancers from smokers which were not evident in nonsmokers. Corresponding hotspots of adduct formation have previously been reported to occur in HeLa cells and bronchial epithelial cells treated in vitro with BPDE (Denissenko et al., 1996
). Thus, the causal link between exposure to BaP in tobacco smoke and lung cancer was further stressed by Hernandez-Boussard and Hainaut (1998). However, the current (R3) version of the database does not support this hypothesis since the differences in these hotspot mutations found in both smokers and non-smokers are not statistically significant [Fisher's exact test, smokers compared with non-smokers; codon 157: 3.2% and 1.5%, respectively (P = 0.37); codon 248: 4.1% and 1.5%, respectively (P = 0.26); codon 273: 5.8% and 3.7%, respectively (P = 0.49); see Figure 2]. Although the mutation frequencies at codons 157, 248 and 273 of the p53 gene are slightly lower in non-smokers, these hotspots are not exclusively found in lung cancer of smokers as reported in earlier publications (Hernandez-Boussard and Hainaut, 1998; Pfeifer and Denissenko, 1998).
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It is possible that differences in lung p53 mutation spectra between smokers and non-smokers are influenced by other factors such as histological tumour type and gender, age and ethnic origin of the patients. Unfortunately, complete data are not available for these factors for all of the entries in the IARC p53 database since they were not always reported in the original publications. However, comparison of the available data for ethnic status in the current (R3) version shows, for example, a significantly (
2 = 83.23, P < 0.000) lower prevalence of entries of Asian origin (Chinese, Japanese and Taiwanese) among smokers (42.3%; n = 343) than among non-smokers (88.2%; n = 136). To validate the consequences of this unequal distribution on GT transversion frequencies in smoking and non-smoking patients, separate analyses of Asian and non-Asian populations were performed. Interestingly, no significant differences in frequencies of GT transversions between smokers and non-smokers were found in either ethnic group [Figure 1
; 21.4% and 22.5% for Asian smokers and non-smokers, respectively (2 = 0.05, P = 0.83); 33.8% and 37.5% for non-Asian smokers and non-smokers, respectively (2 = 0.09, P = 0.77)]. Thus, the slight but non-significant increase in GT mutation frequencies in smokers found in the analysis of all cases in the R3 version (Figure 1) can be completely explained by confounding due to the high prevalence of entries of Asian origin in the listed non-smokers, since the frequencies of GT transversion mutations in Asian smokers and Asian non-smokers (21.4% and 22.5%, respectively) are substantially lower than in the corresponding non-Asian groups (33.8% and 37.5%, respectively).
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Received on June 1, 2000; accepted on August 15, 2000.
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