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Journal of Clinical Oncology, Vol 25, No 16 (June 1), 2007: pp. 2243-2247 © 2007 American Society of Clinical Oncology. DOI: 10.1200/JCO.2006.08.8914 MDM2 Polymorphism, Survival, and Histology in Early-Stage Non–Small-Cell Lung Cancer
From the Massachusetts General Hospital; Harvard School of Public Health; and the Brigham and Women's Hospital, Boston, MA Address reprint requests to David C. Christiani, MD, MPH, Department of Environmental Health, Harvard School of Public Health, 665 Huntington Ave, Boston, MA 02115; e-mail: dchris{at}hsph.harvard.edu
Purpose MDM2 is a negative regulator of p53. The MDM2 309T/G polymorphism has been associated with differential MDM2 expression levels and inhibition of the p53 pathway. We hypothesized that the MDM2 G/G genotype may be associated with worse survival outcomes in lung cancer, especially in squamous cell cancers where p53 abnormalities are more common. Patients and Methods We evaluated the relationship between MDM2 polymorphism status and overall survival (OS) among patients with early-stage non–small-cell lung cancer (NSCLC) treated with surgical resection at Massachusetts General Hospital from 1992 to 2000. Kaplan-Meier methods and the log-rank test were used to compare survival by polymorphism status. Cox proportional hazards models were used to adjust for possible confounding variables. Results There were 383 patients in the analysis. In the early-stage population as a whole, the G/G genotype seemed to be associated with worse OS on adjusted analysis (adjusted hazard ratio = 1.57; 95% CI, 1.03 to 2.40; P = .04). Among patients with squamous histology, OS was significantly worse among those with the G/G genotype (P = .0001 by log-rank test), with 5-year survival rates among the genotypes of 59% for T/T, 53% for T/G, and 7% for G/G. Conclusion Our findings suggest that the G/G genotype of the MDM2 polymorphism is associated with worse OS among early-stage NSCLC patients, particularly those with squamous cell histology.
Lung cancer is the leading cause of cancer-related death in the United States.1 Patients with early-stage non–small-cell lung cancer (NSCLC) have potentially curable disease. However, a significant proportion of patients with early-stage NSCLC will experience recurrence, and many ultimately die from their disease. Identifying prognostic factors in early-stage NSCLC can be useful to guide treatment decisions. The p53 tumor suppressor pathway regulates multiple downstream genes involved in DNA repair, cell cycle arrest, and apoptosis.2 There is evidence to suggest that p53 mutations are more associated with squamous cell carcinomas than adenocarcinomas, as well as with higher tumor, nodal, and overall stages.3 Smoking is also thought to play a role in p53 mutations.4-7 Murine double minute 2 (MDM2) is a negative regulator of p53 that normally acts to keep p53 levels under tight control; when p53 levels are elevated, MDM2 transcription increases. MDM2 binds p53, blocking its transcriptional activity and targeting it for degradation.8,9 This autoregulatory loop closely regulates p53 activity in normal cells. Under conditions of stress, such as DNA damage, p53 activity is enhanced via a variety of mechanisms, including increase in p53 levels, suppression of MDM2 levels, and blocking of MDM2-p53 interactions,9 thus enabling appropriate tumor suppressor responses. Overexpression of MDM2 is thought to interfere with p53-mediated apoptosis and growth inhibition, leading to cancer progression.10-12 MDM2 may also induce tumorigenesis independent of p53 pathways.13 However, the role of MDM2 in clinical outcomes in cancer is not clear.14 In NSCLC, studies have been contradictory as to whether MDM2 is associated with worse or better prognosis.15-18
Recently, a polymorphism in the promoter region of MDM2 has been described, which encodes a T We hypothesized that the MDM2 G/G genotype is associated with worse survival in patients with NSCLC. Because p53 aberrations vary by histology, we investigated whether the association of MDM2 and survival differed by histologic subtype. We investigated these hypotheses in our cohort of early-stage NSCLC patients.
Study Population Since 1992, patients with histologically confirmed NSCLC have been recruited prospectively into a molecular epidemiology study at Massachusetts General Hospital (MGH). These patients were part of a large ongoing case-control study being conducted to evaluate genetic polymorphisms and lung cancer risk. Blood samples for genotyping and patient and demographic information (including age, sex, and smoking status) were collected at the time of recruitment. A modified standardized American Thoracic Society respiratory questionnaire was used to collect detailed smoking information,21 with never smokers being defined as those who smoked 100 cigarettes or fewer over a lifetime, former smokers being defined as those who quit at least 1 year before diagnosis, and current smokers being defined as those who were smoking within a year of diagnosis. Informed consent was obtained to collect follow-up data. More than 85% of eligible patients were recruited in this cohort. We limited our analysis to all patients with stage I or II NSCLC, enrolled between December 1992 and September 2000, who had their surgical resection performed at MGH and had follow-up data. There were 456 patients who met these criteria. Of these patients, 73 did not have adequate blood for genotyping and were excluded from the analysis. There were no significant differences in demographic information between patients who had blood available for genotyping and patients who did not. The study was approved by the Institutional Review Boards of MGH and the Harvard School of Public Health.
Genotyping
Outcomes Collection
Statistical Analysis
Patient Characteristics There were 383 patients included in the analysis. Demographic and treatment characteristics are listed in Table 1. Median follow-up time was 5.7 years (range, 0.1 to 11.6 years). There were 191 deaths. The genotype frequencies for the MDM2 polymorphism were as follows: T/T, 161 patients (42%); T/G, 154 patients (40%); and G/G, 68 patients (18%). These frequencies were consistent with previously described genotype frequencies for this polymorphism.19 There were no significant differences by MDM2 genotype status in age, sex, stage, histology, or smoking status.
OS On univariate analysis, age, sex, stage, radiation, squamous cell histology, and pack-years of smoking were all significantly associated with survival in the population as a whole. The use of adjuvant radiation was significantly associated with stage but was not significant on multivariate models and was, therefore, excluded from the final models. There were too few patients who received adjuvant chemotherapy for meaningful analysis. After adjusting for covariates of age, sex, histology, stage, and smoking, the MDM2 G/G genotype was associated with worse OS (crude hazard ratio = 1.29; 95% CI, 0.87 to 1.91; P = .20; adjusted hazard ratio = 1.57; 95% CI, 1.03 to 2.40; P = .04; Table 2). For all histologies, five-year OS rates were 61% (95% CI, 53% to 69%) for patients with the T/T genotype, 58% (95% CI, 50% to 66%) for patients with the T/G genotype, and 44% (95% CI, 31% to 57%) for patients with the G/G genotype. Among patients with squamous cell cancer, the G/G genotype was associated with worse survival (P = .0001 by log-rank test; Fig 1). Five-year OS rates in patients with squamous cell carcinoma according to genotype were as follows: 59% for T/T genotype (95% CI, 42% to 72%), 52% for the T/G genotype (95% CI, 34% to 66%); and 7% for the G/G genotype (95% CI, 1% to 27%; Table 3).
OS in Subsets Because of the association of squamous histology with higher stage and smoking, we also investigated the effect of MDM2 genotype on survival by subsets of stage and smoking status. In our data set, squamous histology was significantly associated with having stage IB or II disease compared with stage IA disease (P = .0009 by  2 test) and with current smoking status (P = .01 by 2 test). The MDM2 G/G variant genotype was associated with a significantly worse OS among patients with stage IB or II disease and current smokers according to both log-rank tests and Cox regression models (Table 3). No difference was seen among patients with stage IA disease or noncurrent smokers. In addition, no difference was seen in subsets of age or sex. To test the hypothesis that having squamous histology was the most important factor for the survival differences seen with the G/G genotype, we looked at subsets of patients with squamous histology who were noncurrent smokers or had stage I disease. Interestingly, although no difference by genotype had been seen among noncurrent smokers in general, among noncurrent smokers with squamous histology, the G/G genotype was associated with significantly worse OS (P = .004 by log-rank test). Similarly, among patients with stage I disease who had squamous histology, the G/G genotype was associated with worse OS (P = .002 by log-rank test). Because of the small number of patients with stage IA disease and squamous cell subtype (n = 43), we could not draw meaningful conclusions from this subset, although there seemed to be a trend toward worse OS for the G/G genotype.
The G/G genotype of the MDM2 T/G polymorphism has been shown to attenuate p53 tumor suppressor response to DNA damage.19 Because p53 tumor suppression is important in cancer development, we hypothesized that a variant form of the MDM2 309T/G polymorphism would be associated with worse survival outcomes in lung cancer. We found that the homozygous variant G/G genotype is associated with worse OS in squamous cell carcinoma of the lung. In our analysis, it seems that squamous cell histology is an important determinant of the detrimental effect of the MDM2 309T/G polymorphism on survival. Subsets of stage and smoking status that correlate with squamous histology showed the same effect of MDM2 genotype on survival. Also, among noncurrent smokers and stage I patients with squamous cell histology, the MDM2 G/G genotype was associated with worse survival, whereas there was no such relationship among noncurrent smokers and stage I patients in general. This finding that the MDM2 polymorphism affects survival outcomes in squamous cell cancers is biologically plausible. MDM2 is a part of the p53 pathway of tumor suppression. There is evidence to suggest that p53 mutations are more associated with squamous cell carcinomas than adenocarcinomas.3 It is possible that when the p53 pathway is already dysregulated, any upregulation in MDM2, with its subsequent blocking of p53 tumor suppressor activity, may remove yet another critical brake on tumor progression. Studies of MDM2 in risk and prognosis in lung cancer have reported disparate results. Although some studies report an increased risk of lung cancer with the G/G genotype,24,25 others have reported a decreased risk.26 Studies in prognosis of lung cancer are similarly conflicting. Although there are no published reports of MDM2 polymorphism and lung cancer survival outcome, several groups have investigated gene amplification or mRNA or protein expression. Dworakwoska et al18 measured MDM2 gene amplification among 116 patients with resected NSCLC and found that gene amplification, which was found in 21% of patients, was associated with significantly worse survival. However, Higashiyama et al15 found a trend towards better prognosis among patients expressing MDM2 protein; among the 110 patients who were negative for p53 by immunohistochemistry (IHC), MDM2 expression was associated with significantly better prognosis. Ko et al16 reported that MDM2 mRNA expression, but not protein expression, was associated with better survival among 81 patients with resected NSCLC. Wang et al17 reported that, among 94 patients with early-stage lung cancer, the combination of p53 overexpression and low MDM2 expression was associated with squamous cell histology, higher stage, and worse prognosis. Of note, IHC studies use a variety of standards for measuring expression, and results across different studies can be difficult to interpret. In addition, the significance of an elevated level by IHC is unclear for both p53 and MDM2. For instance, IHC may not distinguish between various isoforms of MDM2, including the full-length protein, inactive breakdown products, or dominant-negative forms.14 To our knowledge, this is the first study investigating the association between the MDM2 309T/G polymorphism and survival outcomes in patients with early-stage NSCLC. The G/G genotype has been found to be a poor prognostic marker in other cancers, such as gastric carcinoma.27 Our finding that the G/G genotype is associated with worse survival in early-stage lung cancer patients is consistent with both this result and the expected function of the MDM2 309T/G polymorphism, because the variant genotype would be expected to lead to higher MDM2 levels and interference with p53-mediated apoptosis and growth inhibition. One of the strengths of this study is its large sample size; this is one of the largest case series of early-stage NSCLC described. The large sample size of our study allowed us to analyze the subset of squamous cell cancers separately. However, there are several limitations to this study. First, we were able to collect data on OS but not cancer-specific survival. The sources of survival data that we used did not reliably denote cause of death, although this would be helpful to know, particularly in the early-stage setting, where a significant proportion of patients ultimately die of non–cancer-related causes. Another limitation of this study is the fact that clinical outcomes were collected retrospectively. Our collection of survival outcomes is reasonably complete because we were able to contact local physicians or patients' families if there was any uncertainty regarding date of death. Finally, the tumorigenic effects of MDM2 in both p53-dependent and p53-independent pathways are complex, and analysis of one polymorphic variant may not address all of the intricacies of MDM2 function. Our data suggest that the G/G genotype is associated with poor prognosis among patients with squamous cell cancer of the lung. Further studies with even larger numbers of patients would be helpful to validate these findings. In addition, our results suggest that there may be different prognostic markers relevant to the different histologies of lung cancer, and further investigation along these lines is warranted.
Although all authors completed the disclosure declaration, the following author or immediate family members indicated a financial interest. No conflict exists for drugs or devices used in a study if they are not being evaluated as part of the investigation. For a detailed description of the disclosure categories, or for more information about ASCO's conflict of interest policy, please refer to the Author Disclosure Declaration and the Disclosures of Potential Conflicts of Interest section in Information for Contributors. Employment: N/A Leadership: N/A Consultant: David C. Christiani, Gentra Stock: N/A Honoraria: N/A Research Funds: N/A Testimony: N/A Other: N/A
Conception and design: Rebecca Suk Heist, Wei Zhou, Geoffrey Liu, David C. Christiani Financial support: Thomas J. Lynch, David C. Christiani Provision of study materials or patients: Li Su, Thomas J. Lynch, David C. Christiani Collection and assembly of data: Rebecca Suk Heist, Li Su, Thomas J. Lynch, John C. Wain, David C. Christiani Data analysis and interpretation: Rebecca Suk Heist, Wei Zhou, Lucian R. Chirieac, Thea Cogan-Drew, Geoffrey Liu, Donna Neuberg, David C. Christiani Manuscript writing: Rebecca Suk Heist, Wei Zhou, Lucian R. Chirieac, Thea Cogan-Drew, Geoffrey Liu, Li Su, Donna Neuberg, Thomas J. Lynch, John C. Wain, David C. Christiani Final approval of manuscript: Rebecca Suk Heist, Wei Zhou, Lucian R. Chirieac, Thea Cogan-Drew, Geoffrey Liu, Li Su, Donna Neuberg, Thomas J. Lynch, John C. Wain, David C. Christiani
Supported by National Institutes of Health Grants No. CA074386, CA092824, and CA090578; Flight Attendants Medical Research Institute Young Clinical Scientist Award; American Cancer Society Postdoctoral Fellowship; and the Doris Duke Charitable Foundation. Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.
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Copyright © 2007 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
G change at the 309th nucleotide in the first intron (rs2279744). The homozygous variant G/G genotype has been associated with increased binding affinity of the transcriptional activator Sp1, causing higher MDM2 mRNA and protein levels and ultimately leading to inhibition of the p53 pathway.
