Low grade amplification of MDM2 gene in a subset of human breast cancers without p53 alterations

MDM2 protein is thought to bind to p53 tumor suppressor protein leading to inhibition of p53-mediated transactivation. Amplification of the MDM2 gene has been frequently observed in human sarcoma, and relevant overexpression of the MDM2 protein is assumed to contribute to tumorigenesis through inactivation of the p53 function. In order to determine whether MDM2 amplification plays a role in the development of human breast cancer without genetic alteration of p53, we analyzed, MDM2 gene amplification by quantitative hybridization and genetic alteration of p53, in 32 primary tumors and 26 metastatic lymph nodes. Low grade amplification of the MDM2 gene (2-6 fold) was observed in four cases, none of which showed even subtle genetic alterations of p53 or loss of alleles on 17p. Moreover, in three of the four cases with MDM2 gene amplification, the level of gene amplification in the metastatic lymph nodes was slightly higher than that in the primary tumors. These results, taken together with previous findings, suggest that a subset of breast cancers without genetic alteration of p53 may also arise by inactivation of the p53 function through interaction with the overexpressed MDM2 protein induced by gene amplification.     

Wild-type p53 overexpression and its correlation with MDM2 and p14ARF alterations: an alternative pathway to non-small-cell lung cancer.

PURPOSE: We found a relatively reduced frequency of p53 mutation with a much greater frequency of p53 protein overexpression, which reflected stabilization of p53 protein in the absence of p53 gene mutation. Therefore, we investigated the possibility of alternative mechanisms leading to p53 protein stabilization. PATIENTS AND METHODS: We performed gene and protein alteration studies on p53 and its upstream effectors, MDM2 and p14ARF, in tumors from 94 non-small-cell lung cancer (NSCLC) patients. RESULTS: Immunohistochemical and sequencing analyses indicated that 37 tumors showed overexpression of wild-type p53. An absence of nuclear staining of MDM2 protein was found in 95% of these tumors (35 of 37; P < .001). The tumors with negative MDM2 staining showed a significantly high concordance of loss of Akt activity and low MDM2 mRNA expression (P < .001). Sequencing analysis revealed five distinct MDM2 splicing variants disrupting the conserved p53 binding domain. Corresponding variant proteins were detected in three lung cancer cell lines using the Western blot analysis. Our results also indicated that among the tumors with overexpression of the wild-type p53, 92% (34 of 37) showed immunoreactivity to p14ARF (P = .001). In addition, the deregulation of p53 and MDM2 genes was significantly associated with squamous lung cancer (P < .05) and was correlated with advanced stages (P < .05) and poor prognosis (P < .05). CONCLUSION: Our data suggest that immunopositivity of p14ARF together with a low expression of MDM2 contributes to accumulation of the wild-type p53, and that deregulation of the p53-MDM2-p14ARF pathway is important in the pathogenesis and outcome of a subset of NSCLC.     

Growth reduction of a xenotransplanted human soft tissue sarcoma by MDM2 antisense therapy via implanted osmotic minipumps

The MDM2 oncogene plays an important role in tumorigenesis and especially in soft tissue sarcomas (STS). Overexpression of the MDM2 protein is associated with a poorer prognosis for STS patients. An MDM2 antisense approach to reduce MDM2 protein levels has been successfully applied on several carcinomas in vitro and used on a few in vivo cases. However, antisense treatment not only resulted in an MDM2 protein reduction but also in a wild-type P53 gene (wt-P53) mediated tumor growth suppression due to its genetic wt-P53 status. In this study, we used a clinically relevant xenotransplanted STS model with a mutated P53 gene (mt-P53) in order to exclude the influence of wt-P53. The human STSs were surgically implanted and one week later osmotic pumps were implanted intraperitoneally into nude rats releasing MDM2-antisense ODNs (AS ODNs) continuously for one week. As controls MDM2-sense ODN (SE ODN) or a 0.9% NaCl solution (saline solution) were administered. After one week animals treated with MDM2-AS ODN (100 or 200 microg) showed a reduction in tumor mass in comparison to animals treated with MDM2-SE ODN. The reduction in tumor mass was significant in animals treated with MDM2-AS ODNs in comparison to the saline solution treated ones (p=0.018 or p=0.007). Furthermore, a significant reduction in macroscopically visible tumor number with MDM2-AS ODN treatments (100 or 200 microg) in comparison to MDM2-SE ODN or saline solution treatment (both p=0.009) was observed for the first time. As expected a reduction in MDM2 protein expression in the MDM2-AS ODN treated tumors when compared to the MDM2-SE ODN or saline solution treated tumors was detected in Western blot analyses and immunohistochemically. In addition, an unexpected reduction in mt-P53 protein expression after AS ODN therapy was also observed. In short, we have demonstrated in vivo for the first time that MDM2-AS ODN treatment of xenotransplanted STS (mt-P53) reduces tumor mass, tumor number, MDM2 protein and mt-P53 protein expression. Our findings support the hypothesis that MDM2-AS ODN treatment may exert a tumor inhibiting effect on all MDM2 expressing tumors regardless of the P53-status, this in turn may be of general importance in gene therapy of cancer.     

Molecular features of adult glioma associated with patient race/ethnicity, age, and a polymorphism in O6-methylguanine-DNA-methyltransferase.

BACKGROUND: Risk factors for adult glioma in the San Francisco Bay Area include well-known demographic features such as age and race/ethnicity, and our previous studies indicated that these characteristics are associated with the TP53 mutation status of patients' tumors. We enlarged our study to assess the relationships of risk factors with TP53 as well as epidermal growth factor receptor (EGFR) and murine double minute-2 (MDM2) gene amplification and expression and the germ line Leu84Phe polymorphism in the DNA repair protein O6-methylguanine-DNA-methyltransferase (MGMT). MGMT expression may depend on the TP53 status of cells. METHODS: Molecular analyses were carried out on 556 incident astrocytic tumors. MGMT genotype data were collected on germ line DNA from 260 of these cases. RESULTS: The tumor data confirm the inverse relationships between TP53 mutation and MDM2 (P = 0.04) or EGFR (P = 0.004) amplification and that patients whose tumors contain TP53 mutations are younger than those without (P < 0.001). Although there was little difference in age of patient by EGFR amplification or expression among glioblastoma multiforme cases, EGFR gene amplification was associated with much older age of onset of anaplastic astrocytoma; for example, EGFR-amplified anaplastic astrocytoma cases were on average 63 years old compared with 48 years for nonamplified cases (P = 0.005). An increased prevalence of TP53 mutation positive glioblastoma multiforme was noted among nonwhites (African American and Asian) compared with whites (Latino and non-Latino; P = 0.004). Carriers of the MGMT variant 84Phe allele were significantly less likely to have tumors with TP53 overexpression (odds ratio, 0.30; 95% confidence interval, 0.13-0.71) and somewhat less likely to have tumors with any TP53 mutation (odds ratio, 0.47; 95% confidence interval, 0.13-1.69) after adjusting for age, gender, and ethnicity. Interestingly, EGFR gene amplification and EGFR protein overexpression were also inversely associated with the MGMT 84Phe allele. CONCLUSIONS: Our results are consistent with ethnic variation in glioma pathogenesis. The data on MGMT show that an inherited factor involving the repair of methylation and other alkylation damage, specifically to the O6 position of guanine, may be associated with the development of tumors that proceed in their development without TP53 mutations or accumulation of TP53 protein and possibly also those that do not involve amplification of the EGFR locus.     

Overexpression of MDM2 oncoprotein correlates with possession of estrogen receptor alpha and lack of MDM2 mRNA splice variants in human breast cancer

To evaluate the significance of murine double minute 2 (MDM2) oncoprotein in human breast cancer as a nuclear-cytoplasmic shuttling protein, an estrogen receptor (ER) alpha regulator, and a prognostic marker and to study how MDM2 is overexpressed, we investigated its status in tissue samples and examined the correlation between overexpression and MDM2 gene abnormalities, status, and clinicopathological parameters. We detected MDM2 oncoprotein in both nucleus and cytoplasm by frozen-section immunohistochemistry. There was a significant correlation between MDM2 overexpression and low-grade nuclear atypia, absence of lymph node involvement, and increased levels of ER alpha protein. Our molecular assays found no point mutations in Ser¹⁷, but there was a correlation between MDM2 overexpression and the lack of splice variant mRNAs. These results suggest that the distribution of MDM2 reflects its nuclear-cytoplasmic shuttling ability; that interaction between p53 and MDM2 for tumor progression is not enhanced by point mutations at codon 17; and that the expression of MDM2 splice variants is a reason for the lack of its overexpression. MDM2 overexpression correlates with favorable prognostic parameters. A decreased level of MDM2 will lead to a deviation from the ER alpha signaling pathway.     

MDM2 gene amplification and expression in non-small-cell lung cancer: immunohistochemical expression of its protein is a favourable prognostic marker in patients without p53 protein accumulation.

MDM2 is an oncoprotein that inhibits p53 tumour-suppressor protein. Amplification of the MDM2 gene and overexpression of its protein have been observed in some human malignancies, and these abnormalities have a role in tumorigenesis through inactivation of p53 function. To determine the clinicopathological and prognostic value of MDM2 abnormalities in non-small-cell lung cancer (NSCLC), MDM2 gene amplification and its protein expression status were analysed in surgically resected materials. MDM2 gene amplification was detected in only 2 (7%) of the 30 tested patients. MDM2 protein was found immunohistochemically in a total of 48 (24%) of the 201 patients. MDM2 protein was slightly frequently observed in patients with adenocarcinoma, but its presence or absence was not associated with clinicopathological factors such as T-factor, N-factor, stage, tumour size, differentiation or p53 protein status. Overall, MDM2-positive patients tended to have a better prognosis (P = 0.062). In particular, among immunohistochemically p53-negative patients (n = 110), those with positive MDM2 protein expression showed significantly better prognosis (P = 0.039) and, in a multivariate analysis, MDM2 protein status was a favourable prognostic factor (P = 0.037). In contrast, among p53-positive patients (n = 91), there was no difference in prognosis depending on MDM2 protein status. Thus, in the NSCLC patients studied, MDM2 gene amplification was a minor event, but expression of its protein, which was often observed immunohistochemically, was a favourable prognostic marker, especially among patients without p53 protein accumulation. Further study is needed to determine how MDM2 protein expression results in a better prognosis.     

MDM2 overexpression is associated with short survival in adults with medulloblastoma

In adult medulloblastoma, postoperative radiotherapy is significantly effective in prolonging time to recurrence and survival time; however, the response of individual cases to radiotherapy, that is the total survival, is different. Apoptosis is an important cellular response to radiation. It can be hypothesized that the individual radiosensitivity of medulloblastomas depends on the individual capability to undergo apoptosis. p53 protein is involved in the apoptotic response to ionizing radiation; loss of function of p53 can be the consequence not only of TP53 mutations, but also of amplification and/or overexpression of the MDM2 gene. We have analyzed cerebellar medulloblastomas from 51 adults (>16 years of age) for MDM2 gene amplification (by differential polymerase chain reaction assay), TP53 gene mutation (by polymerase chain reaction single-strand conformation polymorphism analysis of exons 5-8), and immunohistochemical expression of p53 (clone DO1) and MDM2 (clone IF2). The results have been evaluated in relation to age, tumor location, classic or desmoplastic type, MIB-1 labeling index, and total survival. No tumor had MDM2 amplification. Ten tumors had MDM2 positive tumor cells. One case had a mutated TP53 gene; 16/51 cases had intense p53 immunostaining. Only 2 MDM2 protein-positive tumors were also p53-positive. Both subgroups of MDM2 - and p53-positive tumors had a significantly shorter postoperative survival. In conclusion, the overexpression of MDM2 protein and the accumulation of wild-type p53 are unrelated in adult medulloblastoma; they may result in a reduced apoptotic response after radiotherapy and contribute to a shortened survival. Also, MDM2 amplification and TP53 gene mutation are rare events in medulloblastomas of adults.     

A comparative study of P53/MDM2 genes alterations and P53/MDM2 proteins immunoreactivity in soft-tissue sarcomas

In the present study, the expression of P53 and MDM2 proteins were examined in 94 soft-tissue sarcomas (35 malignant fibrohistiocytomas, 15 neurosarcomas, 14 liposarcomas, 13 leiomyosarcomas, 11 fibrosarcomas and 6 dermatofibrosarcomas) by immunohistochemistry. The immunohistochemical findings were correlated with P53 mutation analysis using PCR-SSCP, PCR-HDF and direct sequencing, and MDM2 amplification studies by differential PCR. P53 immunopositivity was found in 25 out of 94 (26.6%) cases. Alterations of the P53 gene were detected in 12 (12.8%) tumors; eight of these tumors revealed P53 immunoreactivity. A high number of P53 positive and P53 mutated tumors were histologically defined as poorly differentiated G3 (64.0% and 75.0%, respectively). MDM2 immunopositivity was revealed in 36 out of 94 (38.3%) cases. MDM2 amplification occurred in 17 tumors (18.1%); only nine of these tumors exhibited MDM2 immunoreactivity. Overall, MDM2 positivity was not associated with MDM2 amplification in 27 out of 94 tumors (28.7%). There was no significant correlation between MDM2 overexpression and histological grade. However, when the samples were stratified by immunophenotype, the majority of tumors (52.5%) with isolated MDM2 overexpression (dissociated from P53 positivity) were defined histologically as low grade (G1 + G2). These results support the notion that besides P53 alterations, MDM2 gene deregulation seems to be an important event in sarcomas evolution. Additionally, the mechanism of MDM2-mediated degradation of P53 protein, without involving stabilization and inactivation of P53 gene, should be considered for better understanding of all features of tumor progression processes.     

Novel mdm2 splice variants identified in pediatric rhabdomyosarcoma tumors and cell lines

Mdm2 is an oncogene that binds to and inactivates the tumor suppressor p53. However, the presence of oncogenic splice variants of mdm2 in human tumors that lack the p53 binding site has suggested a p53-independent transforming function for this protein. This report describes expression of 11 different mdm2 splice variants in pediatric rhabdomyosarcoma (RMS) cell lines and tumors at a frequency of 75% and 82%, respectively. Five of these isoforms have previously been described in other tumor histiotypes but six are novel and may be unique to RMS. There was no association between expression of splice variants and mdm2 gene amplification or p53 status. In addition, the frequency of splice variants was much higher than the incidence of mdm2 amplification or p53 mutations. These variants may be important to consider with respect to RMS tumor progression and therapeutic response.     

Association of p53 codon 72 polymorphism and MDM2 SNP309 with clinical outcome of advanced nonsmall cell lung cancer

BACKGROUND: The purpose of the study was to investigate whether polymorphisms of p53 codon 72 (Arg72Pro) and MDM2 SNP309 (309T>G) affect p53 expression and the clinical outcome of patients with advanced nonsmall cell lung cancer (NSCLC). METHODS: A total of 148 NSCLC patients, previously enrolled in 2 different prospective clinical trials, were genotyped for the p53 Arg72Pro and MDM2 309T>G polymorphisms. Immunohistochemical staining of p53 protein was performed on 61 tumor samples. Genotypes were correlated with p53 expression, clinicopathologic factors, tumor response, and survival. Multivariate logistic or Cox regression analyses were used to adjust for possible confounding variables. RESULTS: The distribution of sex, age, performance status, stage, tumor histology, and smoking habit was not significantly different among polymorphism variants. However, a significant association was observed between p53 Arg72Pro polymorphism and primary resistance to chemotherapy. Patients with the Pro/Pro variant were more likely to be resistant to first-line chemotherapy, especially the irinotecan plus cisplatin regimen, than those with Arg/Arg or Arg/Pro variants (60% vs 27%, P = .014). In multivariate analysis, the Pro/Pro genotype was strongly predictive for shorter progression-free survival (PFS) (hazard ratio [HR] = 1.952, P = .01). The p53 overexpression was associated with MDM2 SNP309. The TT genotype showed more p53 overexpression than TG or GG genotypes (P = .036). In multivariate analysis, the MDM2 TT genotype was independently predictive for longer survival (HR = 1.742, P = .032). CONCLUSIONS: The p53 72Pro/Pro variant was predictive for primary resistance to chemotherapy and shorter progression-free survival. The MDM2 SNP309 was associated with less p53 overexpression and prognostic for worse survival. Genotyping these polymorphisms may be useful for predicting the clinical outcome of advanced NSCLC.     

Alterations of G1-S checkpoint in chordoma: the prognostic impact of p53 overexpression

BACKGROUND: To the authors' knowledge, little is known regarding the alterations of G(1)-S checkpoint and their significance in chordoma, a rare bone tumor. The authors investigated the clinicopathologic relevance of cell cycle abnormalities in chordoma. METHODS: The expression levels of p53, murine double minute 2 (MDM2), retinoblastoma protein (pRb), cyclin D1, p16(INK4a), and p27(Kip1) were investigated using immunohistochemical techniques; p53 mutations were studied by polymerase chain reaction (PCR)-single-strand conformation polymorphism, and mdm2 amplification was analyzed using real-time quantitative PCR. The results were compared with clinicopathologic parameters in 101 lesions. RESULTS: Approximately 10-45% of primary tumors presented alterations of p53, MDM2, cyclin D1, and pRb proteins; most tumors lacked expression of p16(INK4a) and p27(Kip1). Alterations of p53, MDM2, cyclin D1, and pRb proteins were found to have cooperative effects on both higher proliferative ability (MIB-1 labeling index [LI]) and increased nuclear pleomorphism, a previously described prognostic indicator for patients with chordoma. Multivariate analyses revealed that, among these alterations, p53 overexpression was the only independent factor for higher MIB-1 LI. At the genetic level, mdm2 gene amplification was detected in 15.4% of the lesions but did not correlate with MDM2 overexpression or other clinicopathologic parameters. No p53 mutations were detected in the current series. Survival analysis revealed that p53 overexpression, but no other cell cycle alterations, was associated with a reduced overall survival. CONCLUSIONS: Accumulation of cell cycle alterations led to an increased MIB-1 LI and nuclear pleomorphism, a previously described prognostic indicator in chordoma. The authors believe that p53 overexpression in particular is associated with an unfavorable prognosis in patients with chordoma.