Imaging, Diagnosis, Prognosis Effect of TP53 Arg72Pro and MDM2 SNP309 Polymorphisms on the Risk of High-Grade Osteosarcoma Development and Survival

Purpose: The germ-line polymorphisms TP53 Arg72Pro and MDM2 SNP309 T>G are risk factors for tumor development and affect response to chemotherapy and survival in several cancers, but their prognostic and predictive value in patients with high-grade osteosarcomas is not yet defined. The purpose of this study was to investigate the effect of the TP53 Arg72Pro and the MDM2 SNP309 on the risk of osteosarcoma development and survival. Experimental Design: The relative risk to develop osteosarcomas and the overall survival associated to TP53 Arg72Pro and MDM2 SNP309 polymorphisms were investigated in 201 patients. Correlations with event-free survival (EFS) were analyzed in a homogeneous subgroup of 130 patients with high-grade osteosarcomas of the limbs, nonmetastatic at diagnosis, which underwent neoadjuvant chemotherapy. Results: Multivariate analysis showed that the MDM2 polymorphism T309G was associated with an increased risk of developing osteosarcomas [GG versus TT; odds ratio, 2.09; 95% confidence interval (95% CI), 1.15-3.78]. A case/control gender approach evidenced a significant increased risk only for female osteosarcoma patients (GG versus TT; odds ratio, 4.26; 95% CI, 1.61-11.25). Subjects carrying the TP53 Arg72Pro polymorphism were found to have a significantly increased death risk (Pro/Pro versus Arg/Arg; hazard ratio, 2.90; 95% CI, 1.28-6.66). In the subgroup of 130 high-grade osteosarcomas, the TP53 Arg72Pro was an independent marker of EFS (Pro/Pro versus Arg/ Arg; hazard ratio, 2.67; 95% CI, 1.17-6.11). Conclusion: The study provides evidence supporting the association of MDM2 SNP309 with high-grade osteosarcoma risk in females and shows that TP53 Arg72Pro has a prognostic value for overall survival and EFS in osteosarcoma patients. The products of TP53 and MDM2 oncosuppressive genes are related with a feedback loop for regulating localization and activation of TP53 downstream signal. MDM2 gene codes for a protein that binds TP53 and inhibits its transcriptional activity (1). TP53 shows frequent somatic mutations in human cancers and is commonly associated with a more aggressive tumor phenotype, with worse clinical outcome (2–4) and with an increased risk for developing cancers (5–7). Positive correlations between TP53 status and response to chemotherapy or disease progression have been reported in osteosarcomas or soft tissue sarcomas (STS) but with contradictory evidence (8–11). A meta-analysis of 16 studies in human osteosarcomas evaluating the correlation between TP53 protein expression in tumor cells, histologic response to chemotherapy, and 2-year survival failed to find a relationship, whereas the TP53 gene mutations were found to be variably associated with decreased survival (12). A common polymorphism of TP53 is located at codon 72 of the exon 4 leading to an amino acid change (Arg72Pro, rs1042522). The presence of this genetic polymorphism brings to the loss of one of the five PXXP repeats (P represents Pro and X represents any amino acid) altering the protein structure and decreasing induction of apoptosis (13). Patients with the Pro allele seem to have a poor prognosis and survival in different tumors, such as breast (14, 15), ovarian (16), lung (15), head and neck cancer (15), and esophageal squamous cell carcinoma (17). However, these correlations are not unequivocal (7, 18). Authors' Affiliations: Experimental and Clinical Pharmacology Unit, Centro di Riferimento Oncologico-National Cancer Institute, Aviano, Italy; Unit of Epidemiology and Biostatistics, “San Carlo Borromeo” Hospital, Milan, Italy; and Laboratory of Oncologic Research and Department of Pathology, “Istituto Ortopedico Rizzoli,” Bologna, Italy Received 8/29/08; revised 1/23/09; accepted 2/13/09. Grant support: Associazione Italiana per la Ricerca sul Cancro; EuroBoNeT, an European Commission–granted Network of Excellence for studying the pathology and genetics of bone tumors (LSHC-CT-2006-018814); and Italian Ministry of Health “Progetto Oncologia.” The costs of publication of this article were defrayed in part by the payment of page charges. This articlemust therefore be herebymarked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Giuseppe Toffoli, Experimental and Clinical Pharmacology Unit, Centro di Riferimento Oncologico-National Cancer Institute, via Franco Gallini 2, 33081 Aviano, Italy. Phone: 39-0434-659612; Fax: 390434-659659; E-mail: [email protected]. F 2009 American Association for Cancer Research. doi:10.1158/1078-0432.CCR-08-2249 3550 Clin Cancer Res 2009;15(10) May 15, 2009 www.aacrjournals.org Research. on April 2, 2017. © 2009 American Association for Cancer clincancerres.aacrjournals.org Downloaded from In humans,MDM2was shown to be overexpressed in a subset of tumors. This overexpression is frequently associated with an accelerated cancer progression and lack of response to therapy (19, 20). Amplification and overexpression ofMDM2 in human sarcomas (21–23) can stimulate cell proliferation, enhance cell survival, and induce resistance to conventional chemotherapy. A common polymorphism of MDM2 gene occurs at the nucleotide 309 and determines the substitution of T with G (rs2279744). This polymorphism, which involves the second MDM2 promoter-enhancer region, extends the length of Sp1 binding site, increasing theMDM2 basal level. This effect attenuates the increase of TP53 protein levels, with a consequent decrease of the TP53 apoptotic response, leading to a faster and more frequent tumor formation (24–28). The effect of 309G allele on the age of tumor onset seems to be gender specific, decreasing the age of cancer diagnosis in women but not in men affected by STS, B-cell lymphoma (26), and breast cancer (29). These results could be explained by the location of SNP309 in a region that is regulated by estrogen signaling (30, 31). The aim of this study was to investigate the association of the TP53 Arg72Pro and the MDM2 T309G on the risk of high-grade osteosarcoma development and their effect on survival. Moreover, this work was done to evaluate the effect of these polymorphisms on development of STS. Materials and Methods Patients. The study included 201 Caucasian patients with high-grade osteosarcomas from the Istituto Ortopedico Rizzoli (Bologna, Italy) and from the Centro di Riferimento Oncologico-National Cancer Institute (Aviano, Italy) collected between March 1992 and December 2004. DNA was isolated from frozen normal muscular tissues of diagnostic biopsy or peripheral blood collected at the time of the first operation. Cases had conventional histopathologic parameter analysis, including stage (tumor-node-metastasis), tumor type, and grade of differentiation, defined following the WHO guidelines (32). All these patients were checked on a regular follow-up basis (median, 41 mo; range, 1-180) from the time of diagnosis or until death. Overall survival was calculated from the time of diagnosis until death or last follow-up. Among the 201 osteosarcomas, a homogeneous subgroup of 130 patients was considered to be eligible for event-free survival (EFS) analyses. These 130 cases included patients newly diagnosed for high-grade intramedullary osteosarcomas of the limbs without prior treatment and with no evidence of metastasis at diagnosis. All these cases were treated with neoadjuvant chemotherapy protocols (based on doxorubicin, high-dose methotrexate, cisplatin, and ifosfamide) and surgery (33). EFS was calculated from the first day of preoperative chemotherapy to the most recent follow-up examination, local or systemic recurrence, or death unrelated to the tumor (33). Twohundred and fifty healthyCaucasian control subjectswere randomly selected froma pool of healthy volunteers (age range, 18-40 y), who had signed the informed consent to use their biological material for genetic analysis. The study was approved by both the local ethics committees. Additional 129 STS patients from Centro di Riferimento Oncologico were also included in the study to investigate the effect of the TP53 Arg72Pro and theMDM2 SNP309 on STS risk of development. The characteristics of STS patients were as follows: histotype (leiomyosarcoma, 23.2%; malignant fibrous histiocytoma, 17.8%; liposarcoma, 15.5%; fibrosarcoma, 5.4%; synovial sarcoma, 15.5%; other miscellaneous, 22.6%), site (64.3% extremities and 35.7% other), median age (51 y; range, 10-82), and gender (44.2% males and 55.8% females). Genotyping assays. Genotyping analyses of TP53 and MDM2 genes were done after DNA extraction by High Pure PCR Template Preparation kit (Roche Diagnostic GmbH) using Pyrosequencing technology with specific 5′-biotinylated primers. For TP53 analysis, we used a 50 μL volume reaction containing 2.5 mmol/L MgCl2, 125 μmol/L each of deoxynucleotide triphosphates, 200 nmol/L each of primer (forward, 5′-AGACCCAGGTCCAGATGAAGC; reverse, 5′-biotin-GAAACCGTAGCTGCCCT), and 1 unit of Taq polymerase for 38 cycles of amplification (30 s at 95°C, 30 s at 59°C, and 30 s at 72°C) obtaining a 160-bp fragment. Differently, for a 103-bp fragment of MDM2, the reaction was done in a 50 μL volume with 2 mmol/L MgCl2, 125 μmol/L each of deoxynucleotide triphosphates, 200 nmol/L each of primer (forward, 5′-GGGG‐ TGGTTCGGAGGTCTC; reverse, 5′-biotin-AGGCACCTGCGATCATCC), and 1 unit of Taq polymerase for 35 cycles of amplification (30 s at 95°C, 30 s at 62°C, and 30 s at 72°C). Sequencing primers (TP53-CAGAGGCTGCTCCCC and MDM2-GGGCTGCGGGGCCGCT) were developed using SNP Primer Design software (version 1.0) from Biotage AB. Genotyping was done using a PSQ96 pyrosequencer according to the manufacturer's instructions. Samples that were difficult to interpret because of an unclear heterozygous status for Arg72Pro polymorphism were confirmed with a validated Taqman assay. Statistical analysis. The distribution of individual characteristics was assessed by simple descriptive statistics. Differences among distributions of selected variables were evaluated by using Fisher's exact test for categorical data. The Hardy-Weinberg equilibrium assumption was assessed by the standard method of matching the observed numbers of individuals in the different genotype categories with those expected under Hardy-Weinberg equilibrium for the estimated allele frequency and comparing the Pearson goodness-of-fit statistic with the χ distribution with 1 degree of freedom. Genotype distributions were compared with the use of contingency table analysis. Odds ratio (OR) and corresponding 95% confidence intervals (95% CI) were computed by multiple logistic regression models using as dependent variable case/control status. Overall survival was estimated using the Kaplan-Meier product limit method. Cumulative survival probability was calculated at 60 mo within each of the three genotypes. Differences were tested using the log-rank test. To assess the relative excess risk of relapse/death in patients with different genotypes and to control for confounding factors, proportional hazards models (including sex, age, primary sites, and neoadjuvant chemotherapy) were fitted computing hazard ratios (HR) and the corresponding 95% CIs. Translational Relevance The study analyzes the role of two common polymorphisms, the TP53 Arg72Pro and the MDM2 SNP309 T>G, on the risk of developing high-grade osteosarcomas and their effect on overall survival and event-free survival after neoadjuvant treatment. The prognostic and predictive value of TP53 Arg72Pro and the MDM2 SNP309 T>G in cancer patients is an emerging issue, but at present, inconsistent data have been reported for osteosarcomas. Our study provides evidence supporting the association of MDM2 SNP309 T>G with high-grade osteosarcoma risk in females [GG versus TT; odds ratio, 4.26; 95% confidence interval (95% CI), 1.61-11.25] and shows that TP53 Arg72Pro had a prognostic value for overall survival (Pro/Pro versus Arg/Arg; hazard ratio, 2.90; 95% CI, 1.28-6.66) and event-free survival (hazard ratio, 2.67; 95% CI, 1.17-6.11, compared with Arg/ Arg) after neoadjuvant treatment of osteosarcoma patients. This could lead to consideration of this genetic variant as a possible new candidate prognostic marker for high-grade osteosarcomas. 3551 Clin Cancer Res 2009;15(10) May 15, 2009 www.aacrjournals.org TP53 Arg72Pro and MDM2 SNP309 in Osteosarcomas Research. on April 2, 2017. © 2009 American Association for Cancer clincancerres.aacrjournals.org Downloaded from The proportional assumption was examined with log-log survival plots or by adding time-dependent interaction terms to the model.