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Unlabelled: OBJECTDIVES: Mouse double minute 2 is a key negative regulator of the p53 protein, a central node in the mediation of tumor suppression. The MDM2 gene contains 2 differently regulated promoters, MDM2-P1 and MDM2-P2, which differ strongly in their biological and clinical importance., Methods: We assess the clinical significance of the expression of messenger RNA (mRNA) transcripts originating from both MDM2 promoters, measured with quantitative reverse transcription polymerase chain reaction in microdissected tissues from 57 patients with pancreatic ductal adenocarcinoma (PDAC). Furthermore, we determine the clinical relevance of p53 mRNA transcript expression and incorporate the somatic p53 mutational status into our analyses., Results: Interestingly, elevated transcript levels from the P1 promoter, but not the P2 promoter, associate significantly with up to 6.3-fold increased relative risk for tumor-related death (Cox multivariate analysis: P = 0.013). Furthermore, transcripts originating from both MDM2 promoters are found to correlate significantly with p53 mRNA levels (up to r = 0.315; P = 0.017). In addition, low p53 mRNA expression associates with worse PDAC prognosis (relative risk = 2.28; P = 0.021)., Conclusions: This study presents the first differentiated analysis of the MDM2-P1, MDM2-P2, and p53 transcript expression in human PDAC and demonstrates the significant clinical implications of those transcripts. Furthermore, it suggests an additional facet in the regulation of MDM2 via its P1 promoter in this malignancy.

Accumulation of the p53 gene product can lead to its immunogenic appearance and the generation of p53 serum antibodies (p53ab). In different cancer types the occurrence of detectable p53ab has an independent prognostic impact. In spite of the known p53 protein overexpression rate in soft tissue sarcomas (STS), up to 70%, there have been no investigations done on p53ab in serum in STS patients. In this prospective study of 50 STS patients, we investigated the presence of serum p53ab using an enzyme-linked immunosorbent assay system and the presence of p53 overexpression in the appropriate tissue specimen immunohistochemically. Using Kruskal-Wallis chi(2) and Kaplan-Meier tests the results were then correlated to histopathological and clinical data. Six of the 50 patients (12%) showed p53ab detectable in the serum, and 56% (28/50) of the tumors were p53 immunohistochemically positive. Four of the six p53ab positives (66%) had immunohistochemically p53 positive and two (33%) had negative tumors. Altogether four of the 50 patients (8%) were positive for p53ab in serum as well as for p53 immunohistochemistry in tumor tissue specimens. Twenty patients (40%) were negative for both. All of the p53ab positive patients had stage I or II tumors. Excluding tumor stage there was no p53ab correlation to histopathological, clinical or prognostic parameters. We conclude that in STS patients, p53ab also occurs but in contrast to other tumor types at a relatively low frequency. According to our results, the clinical value of p53ab seems to be limited in STS patients.

Soft tissue sarcomas (STS) are mostly resistant after radiation treatment and are characterized by a rather low rate of apoptosis. The aim of this study was to test, in the p53 mutant STS cell line US8-93, the effect of a combined treatment with DNA transfection--either with mdm2 antisense oligodesoxynucleotides (mdm2-AS) or with a wild-type p53-plasmid (wtp53)--and the effects of irradiation on radiosensitivity. Mdm2-sense oligodesoxynucleotides (mdm2-SE) and a GFP-plasmid (GFP) were applied as controls. In order to evaluate the treatment radiation sensitization (clonogenic survival), apoptotic cell death and P53/MDM2-protein expression were determined. A moderately increased radiation sensitization was observed when comparing clonogenic survival after 2 Gy irradiation between cells transfected either with the control mdm2-SE (48%) or with mdm-2 AS (30%). At the same irradiation dose, clonogenic survival of wtp53-plasmid transfected cells (32%) was about 2-fold less than in the cells transfected with the control GFP-plasmid (61%). This enhancement factor of radiation sensitization was increased by about 3-fold at 4 Gy irradiation. Furthermore, an increase in apoptotic cells was already detectable by up to 7.7% (mdm2-AS) in comparison to 3.1% (mdm2-SE control) 72 hours after transfection. In parallel, the percentage of apoptotic cells could be further elevated after subsequent irradiation with 12 Gy by up to 15% (mdm2-AS) compared to 5.7% (mdm2-SE control). A striking result was obtained with the combined treatment of a wtp53 and 12 Gy irradiation which produced in 25% and 38.9% of apoptotic cells 48 hours and 72 hours after transfection, respectively. We can therefore conclude that the sensitivity of radiation therapy is enhanced by DNA transfection with wtp53 or mdm-2 AS ODNs for the correction of the p53-mdm2 balance in STS in vitro.

Soft tissue sarcomas (STS) are malignant mesenchymal lesions with a high degree of prognostic variability. Different prognostic markers such as grading, staging, tumour type and localisation are known. The establishment of these markers was based on the evaluation at results of extensive cohorts of patients. Therefore, only the established markers provide us with information about probabilities in relation to other qualities. Considering as many different markers as possible in one prognostic statement should increase the value of the resultant information. Therefore, we developed a model involving known prognostic markers to formulate an individual prognostic index. In a retrospective analysis, different prognostic factors of 198 adult STS patients with histological tumour free resection margins were evaluated using a multifactorial analysis. On the basis of a Cox-Regression-Model with proportional hazards, the prognostic factors (tumour type, staging, localisation and type of surgical resection) were selected using previous knowledge and a statistical step backward selection procedure adjusting the immunohistochemical status of p53/Mdm2 expression. On the basis of the baseline survival function of our cohort (S0 (t)), the cumulative probability of survival for two S (2) and five S (5) years was estimated. As a result of our analysis the equations S (2) = (e-00393)P and S (5) = (e-00869)P can be used to estimate the individual two and five-year probability of survival in our cohort. Here p is the result of the amount of the estimated regression-coefficients of the exact variables of the respective individual patient. This model makes it possible to include all the evaluated prognostic factors which, in turn, increases the accuracy of the prognostic information for individual patients underlining the proportional hazards assumption.

The goal of this study was to examine the relationship between immunohistochemical pRb detectability and p53 overexpression in 198 soft tissue sarcomas (STS) with regard to its clinical relevance. Distinct pRb detectability multivariately shows a correlation to survival rate (relative risk (RR)=1.59, P=0.037). p53 positivity was also multivariately correlated to poor prognosis (RR=2.17, P=0.0014). Stratification of pRb staining to p53 results shows a prognostical graduation. Patients with negativity for both proteins have the most favorable prognosis (projected 5-year survival rate (psr)=54.5%). In contrast to this, positivity for both antibodies has the highest risk (RR=2.48, P=0.02) and the poorest prognosis (psr=17.4%). To conclude, these results explain that the clinical relevance of immunohistochemical pRb positivity in STS is connected with p53 in the form of having an increasing effect on the known prognostic relevance of p53 overexpression.

To determine the role of p53 in G2/M arrest, G2/M transition and apoptosis, we investigated five human sarcoma cell lines with different p53 gene status in their response to X-rays. The p53 status of the cell lines was mutant (US 8-93, LMS 6-93 and RD), null (SAOS-2) and wildtype (A-204). Clonogenic survival of the cell lines varied as the survival fraction at 2 Gy (SF2) ranged from 0.28 to 0.79. Compared with the mutated p53 cell lines (SF2 with a range from 0.46 to 0.79) the clonogenic survival of the wildtype p53 (wt-p53) cell line A-204 (SF2 = 0.34) was lower. The p53 null cell line (SAOS-2) was also sensitive to X-rays (SF2 = 0.28). We detected, in all cell lines a similar behavior in their response to irradiation with G2/M arrest and apoptosis. However, the maximal rate of apoptosis with a range from 7.0 to 18.0% was rather small. The decrease of G2/M cells was coupled with an increased percentage of apoptotic cells. However, a different delay in G2/M did not result in a change of radiation sensitivity. Western analyses showed an increased P53 level only for the cell line A-204 (wt-p53) after irradiation. Our results point out that there is not always a simple relationship between p53 gene status and radiation sensitivity. We suggest, that wt-p53 plays an active role in G2/M arrest and in decreasing the number of G2/M cells as a response to apoptosis. Therefore, p53-dependent regulation in G2/M may be as important as p53-independent mechanisms.

Background: Following therapeutic irradiation after a latency period of many years radiation-induced tumors, often sarcomas, can arise. Results of radiation-induced DNA damage can be 1. p53 over-expression, inducing growth arrest or apoptosis, and 2. occurrence of mutations, frequently including the p53 gene, as one molecular promotor for carcinogenesis. We were interested whether radiation-induced sarcomas are associated with alterations of the p53 status., Material and Methods: Samples from 11 radiation-induced soft-tissue sarcomas (STS) were studied by a non-radioactive PCR-SSCP sequencing analysis and by immunohistochemistry with five antibodies for their p53 status., Results: A tumor of one patient possessed a G->A transition in codon 280 (exon 8). Of 11 tumors, 9 showed nuclear p53 positivity, detected by monoclonal antibody DO-1. Of these 9 patients, 7 died during the observation period, whereas the 2 patients with DO-1 negative tumor samples are still alive., Conclusions: p53 over-expression and p53 mutation occur in radiation-induced STS. p53 status is expected to have prognostic impact for radiation-induced STS.

Soft tissue sarcomas are a heterogeneous group of neoplasms with various histological subtypes. Up to now, no individual causal molecular markers for prognosis and therapeutic success have been identified. A tumorigenic connection between the oncogene product Mdm2 and tumor suppressor p53 is generally accepted, but their possible clinical relevance has not yet been investigated sufficiently in soft tissue sarcoma. In 86 primary soft tissue sarcoma of the extremities (RO-resected, T1/2 N0 M0), Mdm2 and p53 overexpression were investigated by immunohistochemistry. The results were adjusted to clinico-pathological characteristics and evaluated for their prognostic relevance by multivariate analysis. In Cox's multivariate analysis with stratification of Mdm2 to p53 results, we determined four groups which had different prognostic values for relapse-free and overall survival (Mdm2-/p53- < Mdm2-/p53+ < Mdm2+/p53- < Mdm2+/p53+). The most striking finding was a relative risk (rr) for overall survival of 18.77 (P=0.006) for patients with Mdm2/p53 co-overexpression (n=40). It is noticeably higher than the additive risk from both factors. Coincident Mdm2/p53 overexpression is an independent molecular marker with the highest prognostic relevance described for soft tissue sarcoma. Thus, a high risk sarcoma group has been defined which we believe requires alternative therapeutic approaches.

Most changes in p53 result in a protein with prolonged half life. This permits immunohistochemical detection. P53-overexpression seems to have prognostical relevance in soft tissue sarcomas (STS). The goal of this study was to compare the prognostic relevance of five different p53 antibodies in immunohistochemistry of primary STS using a Cox regression model with adjustment to staging, localization, tumor type, and surgical therapy. We investigated 198 primary STS of six different tumor types for p53-overexpression using the antibodies DO-1, DO-7, Pab1801, Pab240, and CM-1. The rate of positivity (cut of point 10% positive tumor cells) was between 36.2% and 62.6% dependent on the applied antibody. Prognostic significance could be determined only for the N-terminal binding monoclonal antibodies (DO-1, p = 0.0014; DO-7, p = 0.005; Pab1801, p = 0.02) with the highest level for combination of DO-7 and Pab1801 positivity (RR = 2.57, p = 0.0098). Pab240 (epitope amino acids 213-217) and CM-1 (polyclonal) showed no prognostic relevance in the multivariate analysis. We therefore suggest that for immunohistochemical evaluation of p53-overexpression in STS a pannel of two N-terminal antibodies should be used at least.

Background: Changes in the p53 gene product can be immunogenic and enable the formation of p53 serum antibodies (p53ab), detectable in patients with different cancer types. So far, there have been no reports describing the detectability of p53ab in gastric cancer patients., Methods: We investigated the presence of p53ab and their clinical relevance in a cohort of 74 gastric cancer patients, using an enzyme-linked immunosorbent assay system., Results: In our investigation 20.3% of all patients (15 of 74) and 46.9% of the patients with immunohistochemically (IHC) p53-positive tumors (15 of 32) showed detectable p53ab in serum. All p53ab-positive patients had IHC p53-positive tumors. We have found a significant correlation of p53ab with a higher tumor stage (P = 0.002) and also with a poor prognosis of survival (P = 0.04)., Conclusion: We have shown that in gastric cancer patients p53ab are also detectable and that p53ab positivity is a predictor of an unfavorable prognosis.

We examined the clinical value of immunohistochemical (IHC) Mdm2 detection by an N-terminal (IF2) and a C-terminal (19E3) binding monoclonal antibody (Ab) in soft tissue sarcomas (STSs) with regard to the p53 status. Therefore, we investigated a cohort of 198 patients with STSs of six entities with known p53 IHC by using a multivariate Cox regression model to determine the prognostic value of Mdm2 staining. Only positivity with the 19E3 Ab correlated multivariately significantly with survival (RR = 2.32, p = 0.0035). We stratified the C-terminal Mdm2 staining (19E3) according to p53 IHC (DO-1) and found patients could be divided into three groups with an increasing risk: (a) patients with Mdm2 (19E3)-negative as well as p53 (DO-1)-negative tumors, (b) patients with tumors that were either Mdm2 (19E3) or p53 (DO-1) positive, and (c) patients with tumors that were Mdm2 (19E3) as well as p53 (DO-1) positive. Positive staining for both Mdm2 and p53 meant a very poor prognosis with a relative risk of 4.63 (p = 0.00001). This points to the possibility that--in addition to the p53-dependent pathway--Mdm2 could have an effect through a p53-independent pathway. Thus, our results indicate that C-terminal Mdm2 staining (19E3) constitutes an independent prognostic marker in STS.

Most changes of tumor suppressor p53 and its pathway involve a protein with prolonged half-life that permits immunohistochemical detection. The goal of this study was to compare the prognostic relevance of five different p53 antibodies in primary soft-tissue sarcomas (STS) with known p53 mutation status, using a multivariate Cox regression model (adjusted to tumor grading, staging, localization, tumor type, and therapy). A group of 198 primary STS of six types were investigated for p53 overexpression, using p53 antibodies DO-1, DO-7, Pab1801, Pab240, and CM-1. A positive marker frequency between 36.2% and 62.6% was detected. Out of 65 patients whose primary tumor reacted positively to all five antibodies, 52 (80%) died within the study period. Only the N-terminal-binding monoclonal antibodies DO-1, DO-7 and Pab1801 showed a multivariate correlation with survival (P = 0.0014, 0.0048 and 0.02). CM-1 and Pab240 had a univariate, but not a multivariate correlation, with a confounding effect of grading. The prognostic relevance for the five p53 antibodies was: DO-1 > Pab1801 > DO-7 > CM-1 > Pab240. This is the first study that investigates multivariately the prognostic relevance of p53 immunostaining in STS. If monoclonal antibodies with an epitope in the N-terminal region of the p53 protein (DO-1, Pab1801, DO-7) are applied, p53 immunohistochemistry provides an independent prognostic marker in STS.

Background: p53 mutations are the most frequently observed tumor-related genetic changes. Mutational analysis concerns mostly carcinomas and is not comprehensive for soft tissue sarcomas. Among soft tissue sarcomas, malignant fibrous histiocytoma (MFH) and liposarcoma represent the most frequent tumor types. Most of the few identified mutations for soft tissue sarcomas are localized in the core domain of p53. A correlation between p53 positive immunoreactivity, missense mutations, and a poor prognosis is generally assumed. However, the character of p53 mutations and their functional importance for the clinical process is still unknown., Methods: Sixty-two soft tissue sarcoma samples were investigated for the presence of p53 mutations and for p53 immunoreactivity. Exons 4-9 of the p53 gene were amplified from genomic DNA with the polymerase chain reaction. A prescreen for mutations was performed by nonradioactive single strand conformation polymorphism analysis; striking cases were sequenced directly. For an evaluation of the immunohistochemical status, five p53 antibodies were used., Results: In 10 tumor samples 7 new p53 mutations and one polymorphism were identified. Mutations were detected for five liposarcomas (four patients) and four MFHs (three patients). Of the seven mutations, three were missense point mutations, three were deletions, and one was a complex conversion. All mutations but one were localized in the core domain of p53. Of 62 tumor samples, 56% (14 of 32 liposarcomas and 21 of 30 MFHs) were positive for p53 immunostaining., Conclusions: The mutations identified in the core domain affect codons that are structurally or functionally involved in DNA binding. A relation between p53 positive immunoreactivity and a poor prognosis, but not with an exclusively high tumor grade, is evident. p53 mutations in soft tissue sarcomas have a similar spectrum to those in carcinomas.