© The Author 2006. Published by Oxford University Press.
ARTICLE |
Association of Breast Cancer Outcome With Status of p53 and MDM2 SNP309
Affiliations of authors: Laboratory of Human Carcinogenesis, National Cancer Institute, Bethesda, MD (BJB, TMH, SA); Gradient Corporation, Cambridge, MA (JEG); Pathology and Laboratory Medicine, Baltimore Veterans Affairs Medical Center, Baltimore, MD (HGY, DHL); Section on Genomic Variation, Pediatric Oncology Branch, National Cancer Institute, Bethesda, MD (SJC)
Correspondence to: Stefan Ambs, PhD, Laboratory of Human Carcinogenesis, National Cancer Institute, Bldg. 37/Rm. 3050B, Bethesda, MD 20892-4258 (e-mail: ambss{at}mail.nih.gov).
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ABSTRACT |
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Background: A common single-nucleotide polymorphism (SNP) in the promoter region of the MDM2 gene, known as T-309G and referred to as SNP309 for this study, leads to increased expression of Mdm2 protein and attenuated function of the p53 tumor suppressor protein. We investigated whether genetic variants in MDM2 were associated with breast cancer incidence and survival and whether the variant status could interact with the tumor p53 status to modify breast cancer survival. Methods: We used multivariable logistic and Cox regression analyses to study the relationship of SNP309 status and the status of a second MDM2 SNP in exon 12 at codon 354 (SNP354) with breast cancer incidence and with disease-specific survival among 293 case patients and 317 cancer-free control subjects. Survival analysis included 248 of the 293 case patients who had known tumor p53 status. All statistical tests were two-sided. Results: We did not observe an association between SNP309 status and breast cancer incidence in the unstratified analysis, but we did find a statistically significant association between SNP354 status and breast cancer incidence (odds ratio = 3.34, 95% confidence interval [CI] = 1.88 to 5.93). We also discovered a statistically significant interaction between SNP309 status and tumor p53 expression for breast cancer survival (Pinteraction = .002). Among homozygous carriers of the common MDM2 SNP309 allele (T/T), a mutant p53 status (risk ratio [RR] of death = 2.33, 95% CI = 1.08 to 5.03) and aberrant p53 protein expression (RR = 2.61, 95% CI = 1.22 to 5.57) in breast tumors were associated with poor survival. Tumor p53 status was not associated with breast cancer survival among carriers of the variant MDM2 SNP309 allele (G/T or G/G), which is consistent with a dominant effect of the variant allele. Conclusion: A strong interaction between SNP309 status and tumor p53 status appears to modify the association between p53 status and breast cancer survival.
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INTRODUCTION |
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Breast cancer has a hereditary component that is insufficiently explained by high-penetrance genetic risk factors, such as germline mutations in the BRCA1 and BRCA2 genes (1). Allele variants in oncogenes are candidate genetic risk factors that may alter breast cancer onset and outcome. MDM2 is a known human oncogene that is amplified and overexpressed in various human malignancies (2,3). Mdm2 protein is an E3 ubiquitin ligase that is a key member of both the p53 pathway and the pathway that connects the tumor suppressor functions of PTEN and p53 (4,5). The most important role of Mdm2 protein is the regulation of p53 (6–8). The functions of Mdm2 and p53 are linked through an autoregulatory negative-feedback loop that maintains low p53 protein levels in the absence of stress (9). However, this feedback loop is disrupted in many human tumors that contain somatic p53 mutations. A loss of p53 function, as indicated by a p53 mutation or by nuclear accumulation of functionally impaired p53 protein, is observed in 20%–40% of all breast cancers, depending on disease stage, and is associated with poor disease outcome (10–14).
It has recently been shown (15) that a common polymorphism in the MDM2 promoter region, a T 
G change at nucleotide 309 in the first intron (National Center for Biotechnology Information Single-Nucleotide Polymorphism [SNP] Identification Number = rs2279744) leads to increased expression of Mdm2 and accelerated tumor formation in hereditary and sporadic cancers. Cells that harbor the variant G allele of MDM2, which is also known as SNP309, have a compromised p53 response pathway and form transcriptionally inactive p53–Mdm2 complexes in response to stress (15,16). These observations are consistent with an oncogenic function for the variant SNP309.
We investigated whether SNP309 and a second MDM2 polymorphism, SNP354 (rs769412), are associated with breast cancer incidence and outcome. SNP354 leads to an A G base change at codon 354 and creates a Sp1 binding site, but it does not cause an amino acid substitution. There are no previous reports, to our knowledge, that link this polymorphism to MDM2 RNA stability or splicing, to a MDM2 haplotype, or to cancer. Because of the possibility that MDM2 polymorphisms change the function of p53 protein in breast tumors, we also studied whether SNP309 status and SNP354 status interact with the tumor p53 status to modify breast cancer survival.
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SUBJECTS AND METHODS |
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Study Population
Case patients (n = 293) with incident breast cancer and cancer-free control subjects (n = 317) were women who were recruited between February 15, 1993, and August 27, 2003, in the greater Baltimore area. Case patients were selected from a group of 361 breast cancer patients who had been recruited at the University of Maryland Medical Center, the Baltimore Veterans Affairs Medical Center, Union Memorial Hospital, Mercy Medical Center, and the Sinai Hospital in Baltimore during this time period. The 293 case patients had pathologically confirmed breast cancer, were of African American descent or Caucasian white descent (and not Hispanic white descent), had been diagnosed with breast cancer within the last 6 months before recruitment, and had, by self-report, no previous history of breast cancer. We collected tumor specimens and, if available, noncancerous breast tissue from 248 unselected surgical case patients. The control subjects were frequency matched to case patients by race and age and had, by self-report, no history of breast cancer. They were either hospital based (n = 230), recruited from a participating hospital during their stay for reasons other than breast cancer (pulmonary clinics, family/internal medicine, thoracic surgery, and breast reduction surgery), or population based (n = 87). Population-based control subjects were identified from Maryland Vehicle Administration records and contacted by mail and then by telephone. The participation rates among eligible hospital-based control subjects were 87% for patients with breast reduction surgery (296 participants among 340 eligible patients) and 88% for other hospital-based control subjects (293 participants among 334 eligible patients). Population-based control subjects had been recruited as participants of a lung cancer case–control study in the greater Baltimore area. The participation rate among eligible population-based control subjects in this study was 90% (17). We collected fresh-frozen normal breast and adipose tissue, if available, from the hospital-based control subjects. Blood was collected at the subjects' convenience, but generally at the conclusion of the interview. Additional information to determine the estrogen receptor-
status of the tumor, disease stage, treatment, and survival was obtained from medical records and pathology reports, the Social Security Death Index, and the National Death Index. The institutional review boards at the participating institutions approved the study.
Histology Evaluation, Disease Staging, and Immunohistochemistry
A pathologist reviewed each tumor specimen to confirm the presence of a tumor and the histology. Disease staging was performed according to the tumor–node–metastasis (TNM) system of the American Joint Committee on Cancer/the Union Internationale Contre le Cancer. Nuclear p53 expression in tumor sections was determined immunohistochemically with a p53 monoclonal antibody (DO-7, 1:100 diluted [DakoCytomation]; the antibody recognizes the p53 amino terminus); p53 expression was scored positive if more than 10% of the tumor cells expressed nuclear p53, as described (18). Nuclear cyclin E expression in tumor sections was determined immunohistochemically with a cyclin E monoclonal antibody (Ab-2; Lab Vision Corp., Fremont, CA). The antibody recognizes the carboxyl terminus of cyclin E and detects both wild-type cyclin E and the low molecular weight isoforms of cyclin E. After an overnight incubation with the primary antibody at 4 °C, slides were washed in phosphate-buffered saline (PBS) and incubated with a corresponding horseradish peroxidase–labeled secondary antibody and DakoCytomation Envision System reagents (Dako, Carpinteria, CA). Slides were washed in PBS after the 30-minute incubation at room temperature, stained with diaminobenzidine, and counterstained with methyl green. A combined score of intensity and distribution was used to categorize the immunohistochemical staining for protein expression as described (19,20). Intensity received a score of 0–3 if the staining was negative, weak, moderate, or strong, respectively. The distribution received a score of 0–4 if the staining distribution was less than 10% positive cells, 10%–30%, more than 30%–50%, more than 50%–80%, and more than 80%, respectively. A sum score was then divided into four groups as follows: 1) negative = 0–1; 2) weak = 2–3; 3) moderate = 4–5; and 4) strong = 6–7. Negative and weak nuclear cyclin E staining was scored as being negative. Moderate to strong nuclear cyclin E staining indicated cyclin E overexpression and was scored as being positive.
Genotyping
Genomic DNA was isolated from fresh-frozen normal tissue, if available (i.e., 299 noncancerous breast tissue specimens from case patients and control subjects), from 280 buffy coat specimens from blood collected at the time of recruitment from case patients and control subjects, and from 31 fresh-frozen breast tumor samples. The SNP309 genotype of these samples was determined by use of a custom MGB Eclipse By Design assay from Nanogen, Inc. (San Diego, CA). The following primers and probes were used: forward primer was 5'-CGGGAGTTCAGGGTAAAGGT-3' (product EV3260-E1), and reverse primer was 5'-CGACAGGCACCTGCGATCAT-3' (product EV3260-L1). For the assay, we also used a 20x mixture (product EV3260-MIX1), the G/C probe was 5'-CCGCGCCGCAGC-FAM (product EV3260-FAM1), and the A/T probe was 5'-CCGCGCCGAAGC-TET (product EV3260-TET1) (where FAM is 6-carboxyfluorescein and TET is tetrachlorofluorescein). Both probes had an MGB Eclipse Dark Quencher on the 5' end. In addition, we used a 20x probe mixture (product EV3260-FAMTET1). Primers and probes were added to 30 ng of genomic DNA in MGB Eclipse PreMix (Sigma M4443; Sigma, St. Louis, MO) containing 1.25 units of JumpStart Taq DNA polymerase (product D6558; Sigma) and molecular grade water to a total volume of 25 µL. As controls for the assay, we used the following DNA from Coriell Cell Repositories (Camden, NJ): TET (T) control, NA10860; FAM (G) control, NA13607; and hetero (G/T) control, NA17068. Samples were amplified on the PerkinElmer 9700 PCR apparatus or the ABI PRISM 7700 Sequence Detection System under the following conditions: 50 °C for 2 minutes, 95 °C for 2 minutes, followed by 50 cycles of 95 °C for 5 seconds, 58 °C for 20 seconds, and 76 °C for 30 seconds. A dissociation curve was created with the amplified product by use of the ABI PRISM 7700 Sequence Detection System. The conditions for this assay were 95 °C for 15 seconds, 35 °C for 15 seconds, and 80 °C for 15 seconds, with data collection occurring during a 5-minute increase in temperature from 35 to 80 °C. After completion of the assay, the data were imported into the MGB Eclipse Melt Macro (Version 2.330) and analyzed by following the manufacturer's directions. The assay was validated in-house by use of genotyped controls provided by Nanogen and direct sequencing with published primers (15) and with Coriell DNA from Mormon family members. Briefly, we genotyped three pedigree families of DNA from Coriell Cell Repositories and verified that the genotype followed Mendelian genetics in the Mormon family trees. SNP354 was genotyped at the National Cancer Institute's Genotyping Core Facility in Gaithersburg, MD. The assay is described at http://snp500cancer.nci.nih.gov (21) and uses 5'-AAGCCAAACTGGAAAACTCAACAC-3' as the forward primer, 5'-GACTCTCTGGAATCATTCACTATAGTTTTT-3' as the reverse primer, 5'-FAM-AGCTGAGGAGGGC-mgb as the G/C probe, and 5'-VIC-AGCTGAAGAGGGCT-MGB as the A/T probe. (MGB and VIC are proprietary moieties, e.g., fluorescent dye and quencher, manufactured by Applied Biosystems [Foster City, CA].) Our genotype assays contained negative and positive control DNAs and 10% blinded duplicates consisting of DNA samples from case patients and control subjects. We successfully genotyped 98%–100% of the case patients and control subjects for SNP309 and SNP354, and we found a 100% concordance among blinded duplicates for the two assays. The observed genotype frequencies for SNP309 and SNP354 were in Hardy–Weinberg equilibrium for the control subjects.
p53 Mutational Analysis
Tumor DNA was screened by single-stranded conformation polymorphism analysis for the presence of somatic p53 mutations, as previously described (22,23). p53 exons 5–8 were amplified by polymerase chain reaction (PCR) from microdissected, paraffin-embedded tumor tissue, as described (24). PCR products were denaturated and loaded on a Gene Gel Excel gel (Amersham Pharmacia Biotech, Piscataway, NJ). The single-stranded DNA was separated by electrophoresis by use of the GenePhor System (Amersham Pharmacia Biotech, Piscataway, NJ) and visualized by DNA silver staining. When an aberrant DNA band pattern was detected, the PCR product was sequenced to determine whether a mutation was present. Most tumors were also screened for mutations in p53 exons 2–11 with the GeneChip p53 assay (Affymetrix, Santa Clara, CA), as previously described (25,26). Predicted mutations were scored as described (27). We validated the assay in-house with DNA from tumor cell lines with known p53 mutational status. A total of 48 tumors harbored a p53 mutation. One tumor contained two mutations. Missense mutations accounted for 73% of the mutations, insertions or deletions for 19%, and nonsense mutations for 8%. The occurrence of a p53 mutation (n = 49) was associated with aberrant p53 protein expression (n = 70) in the tumors (P<.001, two-sided 
2 test).
Statistical Analysis
We used multivariable logistic and Cox regression analyses to study the relationship of either SNP309 status or SNP354 status with breast cancer incidence and disease-specific survival. Data analysis was performed with Stata Version 7.0 (Stata Corp, College Station, TX) and SAS version 8.0 (SAS Institute, Cary, NC) statistical software packages. All statistical tests were two-sided, and an association was considered statistically significant with P values <.05. The 2 and Fisher's exact tests and multivariable logistic regression were used to analyze dichotomized data and to calculate odds ratios (ORs), respectively. A t test was applied to analyze the relationship between MDM2 genotypes and mean age at diagnosis. A trend test for the SNP354 genotype data was performed in SAS. We calculated a Ptrend for the risk of developing breast cancer with the three ordinal variables—homozygous common allele (A/A), heterozygous alleles (G/A), and homozygous variant allele (G/G)—for SNP354. Survival was determined for the period from the date of hospital admission to the date of the last completed search for death entries in the Social Security Index (date of search: August 18, 2004) for the 248 case patients with available tumor specimens. The mean follow-up time for breast cancer survival was 55 months (range: 12–140 months). A total of 59 (24%) of these 248 case patients died during this period. We obtained death certificates for the deceased case patients and censored all patients whose causes of death, such as accidents, violent crimes, stroke, heart attack, and liver cirrhosis, were not related to breast cancer. The Kaplan–Meier method and the log-rank test were used for univariate survival analysis. Cox regression was used for multivariable survival analysis to calculate adjusted risk ratios (RRs). The following covariates were included into the analyses: age at diagnosis (as a continuous variable), race (categorized as African American or Caucasian), estrogen receptor status (categorized as positive versus negative), TNM stage (categorized as 
stage II versus > stage II), chemotherapy (categorized as yes versus no), and tumor p53 status, with p53 protein expression (categorized as positive versus negative) and p53 mutation (categorized as positive versus negative). Proportional hazards assumptions were verified by log–log plots and with the nonzero slope test of the scaled Schoenfeld residuals. A statistical test for interaction was performed in Stata to determine whether the association of the two MDM2 genotypes with breast cancer survival was modified by other factors. For this test, the result was coded as zero for the common MDM2 genotype (T/T for SNP309 and A/A for SNP354) and one for the variant genotype (G/T or G/G for SNP309 and G/A or G/G for SNP354). A power analysis to detect associations between the two MDM2 genotypes and survival was conducted with the Sample Size software, Version 2.1.31, developed by Dupont and Plummer at Vanderbilt University Medical Center (28,29). The software is available at http://biostat.mc.vanderbilt.edu/twiki/bin/view/Main/PowerSampleSize. We had 80% power to detect a relative risk of breast cancer of 1.6 and 1.8 with SNP309 and SNP354, respectively, at an of .05, if we assumed a dominant effect of the variant alleles.
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RESULTS |
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MDM2 Genotypes and Breast Cancer Incidence
We investigated the relationship between two MDM2 genotypes, represented by SNP309 and SNP354, and breast cancer incidence in a case–control study of 293 case patients and 317 control subjects. Demographic features of the case patients and control subjects are shown in Table 1, and clinicopathologic features of the case patients are shown in Table 2. The study population consisted of mostly postmenopausal women, of whom 57% were self-identified African American women and 43% were self-identified Caucasian women. The recruited African American women had, on average, a higher body mass index, a lower household income, and less education than the Caucasian women. We analyzed the genotype data with multivariable logistic regression, with adjustments for age at diagnosis, race, menopausal status, and family history of breast cancer (Table 3). A total of 211 (67%) of the 314 genotyped control subjects harbored the common SNP309 MDM2 genotype (T/T) and 103 (33%) were carriers of the variant allele (G/T or G/G). The variant SNP309 G allele was less common in African American women than in Caucasian women (P<.001; two-sided 2 test). SNP309 was not in linkage with SNP354, as indicated by a genetic linkage analysis with the Haploview software (D' = 0.46 for African American women; D' = 0.6 for Caucasian women). Only 31 (10%) of the 310 genotyped control subjects carried the variant SNP354 allele (G/A or G/G); in contrast with SNP309, the variant SNP354 allele was found to be more prevalent among African American women than among Caucasian women.
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In the unstratified analysis and the analysis stratified by race, SNP309 was not statistically significantly associated with the breast cancer incidence among African American or among Caucasian women. In the analysis stratified by menopausal status, SNP309 was also not associated with the risk of breast cancer among premenopausal or postmenopausal women (data not shown). Because of the previously described functional relationship between SNP309 and p53, we also examined the association between SNP309 status and breast cancer risk, after stratification of the case patients by tumor p53 status. In this analysis, the homozygous variant SNP309 MDM2 genotype (G/G), compared with the common SNP309 MDM2 genotype (T/T), was associated with a marginally increased risk of developing breast cancer when case patients had tumors that did not have a p53 mutation (OR = 2.05, 95% confidence interval [CI] = 0.91 to 4.60) or did not aberrantly express p53 protein (OR = 2.47, 95% CI = 1.04 to 5.88).
When MDM2 SNP354 status was analyzed by logistic regression, a statistically significant association was observed between the variant SNP354 G allele and an increased incidence of breast cancer (OR = 3.34, 95% CI = 1.88 to 5.93). The trend for an increased risk of breast cancer with increasing numbers of G alleles was statistically significant (Ptrend = .002 for G allele). The variant SNP354 G allele was statistically significantly associated with risk of breast cancer among African-American women (OR = 4.22, 95% CI = 1.99 to 8.98; Ptrend = .004 for G allele). We did not find the same strong association between the variant allele and breast cancer incidence among the Caucasian women. Furthermore, among both premenopausal and postmenopausal African American women, carrying the variant SNP354 G allele was associated with an increased incidence of breast cancer (data not shown). The association between SNP354 and an increased incidence of breast cancer was independent of the tumor p53 status in an analysis that was stratified by the tumor p53 status of the case patients but not by race/ethnicity (for a tumor with a p53 mutation, OR = 2.82, 95% CI = 1.02 to 7.84; for a tumor without a p53 mutation, OR = 3.49, 95% CI = 1.93 to 6.31; for a p53-positive tumor, OR = 3.64, 95% CI = 1.65 to 8.01; and for a p53-negative tumor, OR = 3.28, 95% CI = 1.72 to 6.27).
MDM2 Genotype and Age at Diagnosis
Because an association between MDM2 SNP309 status and age at colorectal cancer diagnosis according to the p53 mutation status has recently been reported (30), we hypothesized that MDM2 SNP309 may also modify the onset of breast cancer in a manner that is dependent on the tumor p53 status. We investigated the relationships of SNP309 status and SNP354 status with age at diagnosis in an unstratified analysis and in an analysis stratified by the p53 mutational status of the tumor. In this analysis, SNP309 status was not statistically significantly associated with the mean age at diagnosis (data not shown), but SNP354 status appeared to modify the age at onset of breast cancer in a manner that was dependent on the tumor p53 status (Table 4). Among case patients whose tumors did not have a p53 abnormality, the variant SNP354 G allele (G/A or G/G), compared with the common SNP354 genotype (A/A), was associated with developing breast cancer at a younger age. Patients with the variant SNP354 developed breast cancer 5–6 years earlier than carriers of the common SNP354 genotype.
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MDM2 Genotypes and Breast Cancer Survival
We investigated the association between the two MDM2 genotypes and breast cancer survival by use of Cox regression analyses with adjustments for age at diagnosis, race, estrogen receptor status, tumor p53 status, TNM stage, and chemotherapy (Table 5). The causes of death that were not related to breast cancer were censored in our analysis. We did not find an association between breast cancer survival and SNP309 or SNP354 status. In contrast, lymph node involvement, having an estrogen receptor–negative tumor, or having a high TNM stage were statistically significantly associated with poor survival, as expected. Race was not associated with outcome in our study, and the association between SNP309 status or SNP354 status and survival also did not differ by race (data not shown).
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Our hypothesis was that MDM2 genotypes modulate p53 function. Thus, we examined whether the status of MDM2 SNP309 or SNP354 modified the association between tumor p53 status and poor disease outcome. p53 mutations were associated with decreased survival in the study population (Table 6). The expression of p53 protein was statistically significantly associated with poor survival in the univariate analysis (P = .02; two-sided log-rank test) but not in the multivariable analysis, unless the analysis was stratified by MDM2 SNP309 status (Table 6). The SNP309 status modified the association between the tumor p53 status and survival, as shown by Kaplan–Meier analyses (Fig. 1) and by multivariable Cox regression analyses (Table 6). The combination of the common SNP309 genotype (T/T) and either a p53 mutation or aberrant p53 protein expression was associated with statistically significantly poorer survival than the combination of the common SNP309 genotype and the lack of a p53 mutation or a p53-negative status (for the comparison of case patients with a p53 mutation with case patients without p53 mutation as the reference group, RR of death = 2.33, 95% CI = 1.08 to 5.03; and for the comparison of case patients with a p53-positive tumor with case patients with a p53-negative tumors as the reference group, RR = 2.61, 95% CI = 1.22 to 5.57). These results are consistent with the functionally impaired p53 pathway having a dominant effect on breast cancer survival in the presence of the common SNP309 genotype. In contrast, the tumor p53 status was not statistically significantly associated with breast cancer survival in the presence of the variant SNP genotype (G/T or G/G). This result is consistent with the variant SNP309 allele having a dominant effect on the association between p53 and breast cancer survival. SNP354 status did not statistically significantly modify the association between tumor p53 status and breast cancer survival (data not shown).
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We next performed a statistical test to determine whether a statistical interaction between SNP309 status and p53 protein expression status or the status of other risk factors modified breast cancer survival (Table 7). Consistent with the survival analysis, we found a statistically significant interaction between SNP309 status and p53 protein status for breast cancer survival (Pinteraction = .002). The variant SNP309 was associated with increased survival among case patients whose tumors have aberrant nuclear p53 expression (RRinteraction of death = 0.11, 95% CI = 0.03 to 0.46), but it was associated with decreased survival among case patients whose tumors were p53-negative (RRinteraction of death = 8.92, 95% CI = 2.18 to 36.6). However, we did not find the same strong relationship between SNP309 status and the p53 mutational status. In addition, we found statistically significant interactions for breast cancer survival between SNP309 status and both nuclear cyclin E protein expression (Pinteraction = .005) and age of diagnosis (Pinteraction = .015) but not between SNP309 status and menopausal status at diagnosis, estrogen receptor status, or race. Because of the interaction between SNP309 status and age, the survival hazard associated with the variant SNP309 genotype (G/T or G/G) increased with the age at diagnosis. The interaction between the variant SNP309 genotype and cyclin E decreased the relative risk of death associated with cyclin E expression in breast tumors (RRinteraction of death = 0.05, 95% CI = 0.01 to 0.4), whereas the interaction between the common SNP309 genotype (T/T) and cyclin E increased the relative risk of death associated with cyclin E expression in breast tumors (RRinteraction of death = 21.4, 95% CI = 2.5 to 181). Among those tumors that expressed cyclin E, the presence of the variant SNP309 genotype was associated with a statistically significantly better survival than the presence of the common SNP309 genotype in the Cox regression analysis with adjustments for age at diagnosis, race, estrogen receptor status, tumor p53 status, TNM stage, and chemotherapy (RR of death = 0.07, 95% CI = 0.01 to 0.88). Among those tumors that did not express cyclin E, the presence of the variant SNP309 genotype was associated with a statistically significantly poorer survival than the presence of the common SNP309 genotype in the Cox regression analysis with adjustments for age at diagnosis, race, estrogen receptor status, tumor p53 status, TNM stage, and chemotherapy (RR of death = 2.27, 95% CI = 1.02 to 5.04).
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SNP309 and Expression of p53 and Cyclin E
Because the survival analysis suggested that SNP309 regulates p53 function, we investigated the association between SNP309 genotype and nuclear p53 protein expression in tumor tissue (Table 2). We found an association between SNP309 status and tumor p53 protein expression (P = .024; two-sided 2 test, with SNP309 genotype coded as T/T, G/T, and G/G). The association between tumor p53 protein expression and SNP309 status was stronger when we compared carriers of the homozygous variant genotype (G/G) with carriers of the common genotype (T/T) (P<.01; two-sided Fisher's exact test); case patients with the homozygous variant genotype had fewer tumors that aberrantly expressed p53 (one [5%] of 21 tumors) than case patients with the common genotype (49 [32%] of 153 tumors; OR = 0.12, 95% CI = 0.01 to 1.05 after adjustments for race and neoadjuvant therapy). We did not find an association between the heterozygous variant genotype (G/T) and tumor p53 protein expression. Thus, the Mdm2 protein encoded by the homozygous variant SNP309 genotype (G/G) may more efficiently target p53 protein for degradation than the Mdm2 protein encoded by the common SNP309 genotype (T/T). Alternatively, carriers of the homozygous variant genotype may acquire p53 mutations less frequently than carriers of the common genotype. Indeed, case patients with the homozygous variant genotype had fewer tumors with a p53 mutation (two [
10%] of 21 tumors) than case patients with the common genotype (31 [20%] of 153 tumors), but the association was not statistically significant (P = .24; two-sided 2; OR = 0.78, 95% CI = 0.14 to 4.3 after adjustments for race and neoadjuvant therapy).
Because cyclin E overexpression has been found to be associated with p53 expression in breast cancer (31), we also examined cyclin E expression in relation to both SNP309 status and p53 status. We found a statistically significant association between the variant SNP309 genotype (G/T or G/G) and cyclin E expression in only those tumors with aberrant p53 expression (P<.01; two-sided Fisher's exact test, with SNP309 coded as T/T, G/T, and G/G). In this subgroup, case patients with the variant SNP309 genotype had statistically significantly fewer tumors that expressed cyclin E (seven [27%] of 26 case patients) than case patients with the common genotype (30 [61%] of 49 case patients) (OR = 0.22, 95% CI = 0.08 to 0.64 after adjustments for race and neoadjuvant therapy). The SNP309 genotype was not associated with cyclin E expression in p53-negative tumors (OR = 1.0, 95% CI = 0.47 to 2.1 after adjustment for race and neoadjuvant therapy). These data are consistent with the hypothesis that the Mdm2 protein encoded by a variant SNP309 allele can suppress nuclear accumulation of cyclin E by targeting either aberrantly expressed p53 protein or a protein downstream of aberrantly expressed p53.
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DISCUSSION |
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TopNotesAbstractIntroductionSubjects and methodsResultsDiscussionReferences |
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Our investigations indicate that the two MDM2 genotypes, represented by SNP309 and SNP354, could be functionally significant in human breast cancer. We found that the SNP354 status was associated with breast cancer incidence and disease onset. The SNP309 status was not associated with breast cancer incidence or with disease onset in an unstratified analysis; however, this variant was statistically significantly associated with breast cancer outcome, because it appeared to modify the association between tumor p53 status and breast cancer survival. This finding is consistent with the previously reported function of SNP309 (15,16).
Our study has several limitations. We used a case–control design to investigate the association between two MDM2 genotypes and breast cancer. Although the case patients and control subjects were from the same geographic area and were frequency matched by age, we cannot exclude the possibility that the selection process for our control subjects introduced a bias that may confound the association between the genotypes and breast cancer. We believe that such a bias is unlikely, because when associations were examined by use of hospital-based and population-based control subjects either separately or combined into one group, the risk association of the MDM2 genotypes with breast cancer were similar. Furthermore, our study population consisted of African-American and Caucasian women. Although this design strengthened the study, it also required stratification by race. Stratifying on both race and tumor p53 status resulted in small numbers of participants in some categories and also generated concerns regarding multiple comparisons. Because these analyses addressed specific hypotheses and because the observed interactions between tumor p53 status and MDM2 genotypes are consistent with previous biological observations, it is unlikely that our results are due to chance observations. Nevertheless, the combined effects of the MDM2 genotypes and tumor p53 status on breast cancer will require further confirmation in investigations in other study populations.
Other recent studies have not found an association between the SNP309 MDM2 genotype and the incidence or onset of breast cancer (32–35), indicating that SNP309 may not contribute substantially to breast cancer development in the general population. However, there is evidence that SNP309 may accelerate tumor formation in populations with an inherited predisposition toward cancer (15). In contrast, we found that the less frequently observed synonymous SNP354 MDM2 genotype was associated with increased breast cancer incidence among carriers of the variant allele and modified disease onset in a p53-dependent manner. To the best of our knowledge, this is the first report describing SNP354. It is possible that this SNP may affect the function of Mdm2 protein by regulating Mdm2 expression or mRNA stability. It is also possible that the presence of the SNP354 variant of MDM2 is not causal for breast cancer but may be linked to another, yet unknown, functional polymorphism in the MDM2 gene.
We found a statistically significant interaction between SNP309 status and p53 status that modified breast cancer survival. We did not observe this interaction between SNP354 status and p53 status, perhaps because of the low frequency of the variant SNP354 genotype in the study population. Our data show that the association between breast cancer survival and p53 status was modified by SNP309 status. Tumor p53 status, as expressed by both p53 mutational status and the level of p53 protein expression, was associated with outcome only among case patients who had the common SNP309 MDM2 genotype (T/T) and not among carriers of the variant SNP309 MDM2 genotype (G/T or G/G). These results indicate that the variant allele acts as a dominant oncogene. The mechanism by which the variant SNP309 Mdm2 protein exhibits an oncogenic function has been described (15,16); i.e., it leads to increased Mdm2 protein expression, abrogation of p53 tumor suppressor function, and the formation of a transcriptionally inactive p53–Mdm2 complex. These functions are consistent with our survival data.
We also found evidence that the variant SNP309 MDM2 allele is associated with increased survival in the subgroup of case patients with a tumor p53 abnormality. Both the test for interaction and the Kaplan–Meier survival curves indicated that, among case patients with a p53-positive tumor, improved survival was associated with the variant SNP309 MDM2 genotype, compared with the common SNP309 genotype. A similar, but weaker, relationship was found for p53 mutations. These data suggest that variant SNP309–encoded Mdm2 protein may target wild-type p53, as well as some mutant forms of p53 protein that cause increased p53 protein stability and nuclear protein accumulation and have a gain of function. However, this interpretation is preliminary.
We examined the association between either SNP309 status or SNP354 status and breast cancer in the context of tumor p53 mutations and p53 protein expression. p53 mutations and aberrantly expressed p53 protein are surrogates for an impaired p53 pathway and have been associated with poor survival in breast cancer (10–12). In our study, 75 (30%) of the 248 tumors had p53 protein that accumulated in the nucleus, and only 48 (19%) of the 248 tumors carried a p53 mutation. It is possible that our analysis missed p53 mutations, although we used three methods (single-stranded conformation polymorphism, the GeneChip p53 assay, and direct sequencing) to detect p53 mutations in microdissected tumor samples. The p53 expression detected in a subset of the breast tumors could be the result of stress-induced p53 expression or of p53 sequestration in a complex with a chaperone (9,36). Nevertheless, there has been a report showing that human Mdm2 protein is capable of inhibiting the transcriptional activation of tumor-derived p53 mutants (37). We also found a statistically significant association between the variant SNP309 genotype and low cyclin E expression that was restricted to tumors with aberrant p53 expression. Nuclear accumulation of cyclin E protein in breast tumors is associated with aberrant p53 expression and poor prognosis (31). We made the same observation in our study. Thus, the observation that the variant SNP309 genotype was associated with low cyclin E protein expression in p53-positive tumors suggests that this genotype may abrogate the gain of function by aberrantly expressed p53 that leads to cyclin E overexpression. Alternatively, it may target the activity of a cell cycle–related protein that is downstream of aberrantly expressed p53.
Although p53 mutations are associated with poor outcome in breast cancer, as shown by a recent meta-analysis of 16 studies (13) and by this study, the prognostic significance of an aberrant p53 expression in breast tumors has been controversial, and some studies have not found a statistically significant association between aberrantly expressed p53 and poor survival (13,14). In addition, different studies have produced widely different risk estimates. Our study indicates that the risk estimates for both p53 mutations and p53 expression depend on the prevalence of the variant SNP309 genotype in a population. It should be noted that, in our study, the variant genotype was less common among African-American women than among Caucasian women. Thus, the risk estimate for a p53 mutant status may be less confounded by SNP309 status in an African-American population than in a Caucasian population. Finally, we found that the association between SNP309 status and breast cancer survival was dependent on the age at diagnosis. The survival hazard associated with the variant allele increased with increasing age at diagnosis.
In summary, our data show that survival associated with the tumor p53 status is associated with the MDM2 SNP309 genotype, which should be investigated further as a prognostic marker of breast cancer outcome. The data are consistent with the previously reported oncogenic function of the variant SNP309 G allele that leads to the overexpression of Mdm2 protein and the abrogation of p53 tumor suppressor activity.
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This research was supported by the Intramural Research Program of the National Institutes of Health, National Cancer Institute, Center for Cancer Research.
We thank Neil Caporaso and Christopher Loffredo for their help with the study design, Leah Mechanic for help with the data analysis, Curt Harris for critically reviewing the manuscript, and Dorothea Dudek-Creaven for editorial assistance. Sharon Pine assisted us with the prediction analysis for transcription factor binding sites. We also thank Raymond Jones, Audrey Salabes, Leoni Leondaridis, Glennwood Trivers, and the personnel at the University of Maryland and the Baltimore Veterans Administration and the Surgery and Pathology Departments at the University of Maryland Medical Center, Baltimore Veterans Affairs Medical Center, Union Memorial Hospital, Mercy Medical Center, and Sinai Hospital for their contributions.
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