TP53 mutation, allelism and survival in non-small cell lung cancer
Heather H. Nelson *,
Magnus Wilkojmen,
Carmen J. Marsit 1 and
Karl T. Kelsey 1
Department of Environmental Health and 1 Department of Genetics and Complex Diseases, Harvard School of Public Health, Boston, MA 02115, USA
* To whom correspondence should be addressed at: Department of Environmental Health, Harvard School of Public Health, 665 Huntington Avenue, Building I, Room 603, Boston, MA 02115, USA. Tel: +1 617 432 0037; Fax: +1 617 432 0107; Email: hnelson{at}hsph.harvard.edu
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Abstract
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TP53 is well-recognized as a mutational target in cancers and common variation in the TP53 gene has been investigated as potentially contributing to cancer susceptibility. The codon 72 polymorphism has been proposed to alter the phenotype of TP53 mutations, and TP53 mutations have been reported to occur preferentially on the arginine allele. Using a consecutive case series of non-small cell lung cancer we have investigated whether TP53 mutations occur preferentially on the arginine or proline allele, and whether the combination of mutation and allelism confers differences in the clinical phenotype. The overall prevalence of TP53 mutation was 26% (76/293). The majority of mutations occurred on the arginine allele (51/60, 85%), and there was corresponding strong selection for loss of the proline allele [87% of loss of heterozygosity (LOH) events were loss of proline]. However, there was no statistically significant difference in the prevalence of mutation by constitutional genotype and among heterozygotes with LOH, TP53 mutation prevalence did not differ by the codon 72 polymorphism (48% on arginine versus 40% on proline). Importantly, patient survival did significantly differ: those patients having a TP53 mutation on the proline allele had the worst survival outcomes (hazards ratio = 2.6, P < 0.03). Further, this phenotype was limited to those patients with advanced disease, where mutation on the proline allele was associated with a significantly worse outcome compared with those without mutation or with mutation on the arginine allele (P < 0.001). Our data suggest that there are selective pressures for loss of the TP53 proline allele in non-small cell lung cancer. Further, the combination of mutation with the codon 72 proline variant predicts poorer patient survival, particularly in a disease that has progressed outside the lung, a finding that warrants further investigation.
Abbreviations: LOH, loss of heterozygosity; RFLP, restriction fragment length polymorphism; SSCP, single-strand conformational polymorphism
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Introduction
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The somatic alteration of TP53 in solid tumors has been well-documented. The majority of mutations are non-synonomous base substitutions occurring in the DNA-binding domain of the protein. Recent in vitro work suggests that the allelic background of TP53 mutations might have significant implications on the mutant protein phenotype (16). Specifically, defining alleles by polymorphic status at codon 72 (arginine versus proline) these studies demonstrate that when mutations occur on the arginine allele the resultant TP53 protein may have a dominant negative influence on TP73L/TP73 pathway that translates to a reduction in apoptosis. However, when mutations occur on the less common proline allele this dominant negative effect is absent. Congruent with these experiments, epithelial tumors (head and neck squamous cell carcinoma, skin, vulva and urinary transitional cell carcinoma) have been reported to show mutation bias for the arginine allele (1,710). Further, the combination of mutation on proline has been shown to have a better response to chemotherapy in head and neck cancers (6,9). We have investigated the relationship of TP53 allelism and mutation in non-small cell lung cancer, and examined whether there is a clinical phenotype associated with tumors defined by the joint classification of mutation and the codon 72 polymorphism.
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Subjects and methods
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Study design and patient population
Eligible cases consisted of all the newly diagnosed patients with resectable lung cancer who received treatment at the Massachusetts General Hospital Thoracic Surgery Service from November 1992 through December 1996 (11). All the patients involved provided written informed consent in accordance with the appropriate institutional review boards. Patients with recurrent disease or non-operable tumors were excluded. 293 patients with available tissue were analyzed for tp53 mutation and polymorphism. For these, tumor-derived DNA was isolated from archived pathology specimens, as described previously (12), and matched constitutive DNA was derived from circulating blood lymphocytes with the QIAmp DNA Blood Mini Kit (Qiagen, Valencia, CA). Demographic and epidemiologic data, including all the data on tobacco use and occupational asbestos exposure, were obtained from the interviewer review of a self-administered questionnaire completed by patients during the hospitalization for thoracic surgery and reviewed by a single reviewer.
Laboratory analyses
TP53 mutation status was determined by PCR-single-strand conformational polymorphism (SSCP) analysis of exons 510 in these tumors, as previously described (13). Genotyping of the TP53 Arg72Pro polymorphism was performed using a PCRrestriction fragment length polymorphism (RFLP) method as previously described (14). To determine the allelism of the mutation, informative patients (those heterozygous for the codon 72 polymorphism) were screened for LOH at this locus by performing the same genotyping PCRRFLP analysis, using a fluorescently labeled forward primer in matched blood DNA and tumor DNA-derived samples. Following restriction digest, the products were separated by capillary electrophoresis for detection using the ABI PRISM 310 Genetic Analyzer (Applied Biosystems, Culver City, CA). The allelic ratio was calculated by taking the ratio of the peak heights in the blood-derived sample divided by the ratio of the peak heights in the tumor-derived sample. Ratio values of <0.5 or >1.5 were taken as indicative of LOH. Visual analysis of the lost peak in the tumor sample provided the identity of the polymorphic allele that was lost. Assuming mutation as the second hit the allele mutated was considered the allele retained, in these heterozygous samples.
Statistical methods
Statistical analysis of the TP53 polymorphism and the allelism of the TP53 mutation with patient demographics, exposure information, tumor traits and outcome were performed with the SAS software (SAS Institute, Cary, NC). The
2 and Fisher's exact test were used to examine categorical data, whereas nonparametric tests (Wilcoxon ranks sums) were used to examine continuous data. For survival analysis, KaplanMeier survival curves were constructed for groups based on univariate predictors, and difference between the groups were tested by the log-rank method. To examine the simultaneous effects of several variables on patient outcome, the Cox proportional hazards model was employed and all the variables examined were consistent with the assumptions of Cox proportional modeling. All tests of significance are two-sided and considered significant at a P < 0.05 level.
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Results
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A total of 293 cases were included in the present analysis and are described in Table I. There were 118 cases heterozygous for the codon 72 polymorphism (40.3%). Of these informative cases, LOH was quantitated in 100 tumors at a prevalence of 38% (38/100). In 33/38 tumors with LOH the arginine allele was retained (87%).
TP53 mutation occurred in 76 of the 293 studied tumors (26%) (Table II). There were no differences in the prevalence of TP53 mutation by constitutional TP53 polymorphism status. Two strategies were used to assess the question of mutation bias; in the first we assigned known allelic status in the tumors, dropping the heterozygotes without LOH from the analysis. In this approach it was possible to clearly delineate on which allele the mutation had occurred for 60 mutation positive tumors. 85% of these tumors (51/60) had mutation on the arginine allele. The second method employed was to examine mutation prevalence among heterozygotes with LOH. In this case, the prevalence of mutation was not statistically different comparing those tumors that retained arginine (16/33, 49%) with those that retained proline (2/5, 40%). Tumors were classified into three groups: those without TP53 mutation, those with mutation on the proline allele and those with mutation on the arginine allele (there were 16 mutations that could not be mapped to a specific allele and were not included in the subsequent analysis). There were no statistically significant differences in demographic, exposure or pathologic descriptors (Table III). However, tumors with mutation on proline tended to be in males and to have smoked more packyears than those with mutation on arginine (Table III). Tumors with mutation on arginine were more likely to be squamous cell carcinomas and to occur in current smokers, although these were not statistically significant differences.
We next investigated whether the combination of mutation and polymorphism was associated with disease outcomes such as clinical stage and survival. The highest proportion of late stage disease occurred among cases with TP53 mutation on the proline allele (Table III). Consistent with this, those patients with TP53 mutation on the proline allele had the poorest overall survival in KaplanMeier analysis (P < 0.02, Figure 1). Examining survival by stage, we observed that the predictive value of the TP53-proline mutation was evident only among those cases with disease greater than stage 1 (Figure 2A and B). Finally, the results of the Cox proportional Hazards model, adjusting for histology (adenocarcinoma versus squamous cell carcinoma), stage (1 versus 2+), age and sex, demonstrated that the combination of TP53 mutation and codon 72 proline was associated with a hazards ratio of 2.6 (P < 0.03).

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Fig. 1. KaplanMeier survival probability curves of patients with NSCLC comparing patients with wild-type TP53 (n = 214), TP53 mutation on the proline allele (n = 9), and TP53 mutation on the arginine allele (n = 51). We used the log-rank method to test differences across the groups, and patients with TP53 mutation on the proline allele had significantly poorer overall survival (P 0.02). Tick marks represent censored data.
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Fig. 2. KaplanMeier survival probability curves of patients with NSCLC comparing patients with wild-type TP53, TP53 mutation on the proline allele, and TP53 mutation on the arginine allele. (A) Stage I disease (WT = 133, mutation on proline = 4, mutation on arginine = 28), log rank P 0.7 and (B) stage II, III, IV disease (WT = 78, mutation on proline = 5, mutation on arginine = 23), log-rank P 0.001. Tick marks represent censored data.
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Discussion
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Previous reports have indicated (i) an ability for some TP53 mutants on the arginine allele to alter p63/p73 mediated apoptosis, (ii) a bias for loss of the proline allele and for TP53 mutations to occur on the arginine allele and (iii) a significant clinical phenotype associated with mutation/allelism in head and neck cancers undergoing chemotherapy. Our results, from a surgical case series of non-small cell lung cancer differ from these prior findings. As with others, we see a strong bias for loss of the proline allele. However, although the majority of mutations occurred on the arginine allele we do not interpret this finding as necessarily indicative of mutation bias. When considering the constitutional heterozygotes, there is an equal likelihood of mutation occurring on the arginine or proline allele assuming no mutation bias. This is consistent with our data, where the prevalence of TP53 mutation was similar among those who retained arginine and those who retained proline. However, we cannot exclude the possibility that bias exists. For example, the literature suggests that when mutation occurs on arginine there may no longer be selective pressure for loss of the second allele. If true, then our analysis restricted to those heterozygotes with LOH would be biased against finding a relationship between mutation and the arginine allele. Although we are limited in our power to detect these differences, owing to our sample size, given that the prevalence of mutation was similar across constitutional genotype groups we feel this bias is unlikely and that in lung cancer there is equal likelihood of mutation occurring on the arginine or proline allele.
TP53 was assessed using SSCP and direct sequencing of exons 510, and the analysis of LOH using the codon 72 polymorphism as a marker. This limits our interpretation of the data as we do not have comprehensive data on mutation or LOH. Tada et al. (4), demonstrated that the association between mutation and arginine was restricted to the subset of mutations that were recessive, as measured by a yeast functional assay. While it was not possible to classify our data this way, we did sub-analyses restricted to substitution mutation events, excluding those that resulted in chain-terminations, however this classification did not alter our results (data not shown).
Prior studies investigating the clinical predictive value of TP53 mutation in lung cancer have been mixed (1519). And, in our present analysis there are no significant differences in patient outcome when the mutation data are considered without referring to the codon 72 polymorphism (data not shown). However, there was a strong association of mutation on the proline allele with the worse patient outcome, and this was not a function of later stage at diagnosis. This is in contrast to the findings of chemotherapy-treated head and neck squamous cell carcinomas where mutation on proline was associated with a better prognostic outcome (6,9). This discrepancy may be attributable to the differences in treatment between these two diseases.
In summary, although the majority of TP53 mutations occurred on the arginine allele we did not find strong evidence for mutation bias. However, consistent with prior reports we do see strong selection for loss of the proline allele. These data suggest that in addition to a potential gain-of-function attributable to the mutation on arginine, there may be a necessary loss of function linked to the proline allele. Further, in the small subset of cases that have TP53 mutation on the proline allele there is a strong correlation with adverse outcome, particularly in advanced stage cancers. Our surgical case series has an under-representation of advanced stage lung cancer and these findings warrant further testing in a larger population-based series that would capture all the stages of lung malignancy.
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Acknowledgments
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This work is supported by NIH grant ES00002.
Conflict of Interest Statement: None declared.
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References
|
---|
- Marin,M., Jost,C., Brooks,L. et al. (2000) A common polymorphism acts as an intragenic modifier of mutant p53 behaviour. Nat. Genet., 25, 4754.[CrossRef][ISI][Medline]
- Gaiddon,C., Lakshin,M., Ahn,J., Zhang,T. and Prives,C. (2001) A subset of tumor-derived mutant forms of p53 down-regulate p63 and p73 through a direct interaction with the p53 core domain. Mol. Cell. Biol., 21, 18741887.[Abstract/Free Full Text]
- Strano,S., Fontemaggi,G., Costanzo,A. et al. (2002) Physical interaction with human tumor-derived p53 mutants inhibits p63 activities. J. Biol. Chem., 277, 1881718826.[Abstract/Free Full Text]
- Tada,M., Furuuchi,K., Kaneda,M., Matsumoto,J., Takahashi,M., Hirai,A., Mitsumoto,Y., Iggo,R.D. and Moriuchi,T. (2001) Inactivate the remaining p53 allele or the alternate p73? Preferential selection of the Arg72 polymorphism in cancers with recessive p53 mutants but not transdominant mutants. Carcinogenesis, 22, 515517.[Abstract/Free Full Text]
- Thomas,M., Kalita,A., Labrecque,S., Pim,D., Banks,L. and Matlashewski,G. (1999) Two polymorphic variants of wild-type p53 differ biochemically and biologically. Mol. Cell. Biol., 19, 10921100.[Abstract/Free Full Text]
- Bergamaschi,D., Gasco,M., Hiller,L. et al. (2003) p53 polymorphism influences response in cancer chemotherapy via modulation of p73-dependent apoptosis. Cancer Cell, 3, 387402.[CrossRef][ISI][Medline]
- Brooks,L., Tidy,J.A., Gusterson,B., Hiller,L., O'Nions,J., Gasco,M., Marin,M.C., Farrell,P.J., Kaelin,W.G. and Crook,T. (2000) Preferential retention of codon 72 arginine p53 in squamous cell carcinomas of the vulva occurs in cancers positive and negative for human papillomavirus. Cancer Res., 60, 68756877.[Abstract/Free Full Text]
- Furihata,M., Takeuchi,T., Matsumoto,M., Kurabayashi,A., Ohtsuki,Y., Terao,N., Kuwahara,M. and Shuin,T. (2002) p53 mutation arising in Arg72 allele in tumorigenesis and development of carcinoma of the urinary tract. Clin. Cancer Res., 8, 11921195.[Abstract/Free Full Text]
- Schneider-Stock,R., Mawrin,C., Motsch,C. et al. (2004) Retention of the arginine allele in codon 72 of the p53 gene correlates with poor apoptosis in head and neck cancer. Am. J. Pathol., 164, 12331241.[Abstract/Free Full Text]
- McGregor,J., Harwood,C.A., Brooks,L. et al. (2002) Relationship between p53 codon 72 polymorphism and susceptibility to sunburn and skin cancer. J. Invest. Dermtatol., 119, 8490.[CrossRef]
- Nelson,H.H., Christiani,D.C., Mark,E.J., Wiencke,J.K., Wain,J.C. and Kelsey,K.T. (1999) Implications and prognostic value of K-ras mutation for early-stage lung cancer in women. J. Natl Cancer Inst., 91, 20322038.[Abstract/Free Full Text]
- Nelson,H.H., Wiencke,J.K., Gunn,L., Wain,J.C., Christiani,D.C. and Kelsey,K.T. (1998) Chromosome 3p14 alterations in lung cancer: evidence that FHIT exon deletion is a target of tobacco carcinogens and asbestos. Cancer Res., 58, 18041807.[Abstract]
- Toguchida,J., Yamaguchi,T., Ritchie,B. et al. (1992) Mutation spectrum of the p53 gene in bone and soft tissue sarcomas. Cancer Res., 52, 61946199.[Abstract]
- Ara,S., Lee,P.S., Hansen,M.F. and Saya,H. (1990) Codon 72 polymorphism of the TP53 gene. Nucleic Acids Res., 18, 4961.[CrossRef][ISI][Medline]
- Mehdi,S.A., Tatum,A.H., Newman,N.B., Gamble,G.P., Etzell,J.E., Weidner,N., Kern,J.A., Sorscher,S.M., Kohman,L.J. and Graziano,S.L. (1999) Prognostic markers in resected stage I and II non small-cell lung cancer: an analysis of 260 patients with 5 year follow-up. Clin. Lung Cancer, 1, 5967; discussion 6869.[Medline]
- Ahrendt,S.A., Hu,Y., Buta,M., McDermott,M.P., Benoit,N., Yang,S.C., Wu,L. and Sidransky,D. (2003) p53 mutations and survival in stage I non-small-cell lung cancer: results of a prospective study. J. Natl Cancer Inst., 95, 961970.[Abstract/Free Full Text]
- Tan,D.F., Li,Q., Rammath,N., Beck,A., Wiseman,S., Anderson,T., al-Salameh,A., Brooks,J. and Bepler,G. (2003) Prognostic significance of expression of p53 oncoprotein in primary (stage IIIIa) non-small cell lung cancer. Anticancer Res., 23, 16651672.[ISI][Medline]
- Grossi,F., Loprevite,M., Chiaramondia,M., Ceppa,P., Pera,C., Ratto,G.B., Serrano,J., Ferrara,G.B., Costa,R., Boni,L. and Ardizzoni,A. (2003) Prognostic significance of K-ras, p53, bcl-2, PCNA, CD34 in radically resected non-small cell lung cancers. Eur. J. Cancer, 39, 12421250.[CrossRef][ISI][Medline]
- Schiller,J.H., Adak,S., Feins,R.H. et al. (2001) Lack of prognostic significance of p53 and K-ras mutations in primary resected non-small-cell lung cancer on E4592: a Laboratory Ancillary Study on an Eastern Cooperative Oncology Group Prospective Randomized Trial of Postoperative Adjuvant Therapy. J. Clin. Oncol., 19, 448457.[Abstract/Free Full Text]
Received February 10, 2005;
revised April 14, 2005;
accepted May 10, 2005.