Tumor mapping of regional immunostaining for p21, p53, and mdm2 in locally advanced bladder carcinoma

BACKGROUND: The aim of this study was to elucidate the associations among immunostaining for p53, p21, and mdm2; their respective expression within each tumor; and the value of these variables for predicting treatment outcome after cystectomy for patients with locally advanced bladder carcinoma. METHODS: The hospital records from all 173 patients treated with cystectomy for locally advanced urothelial bladder carcinoma between 1967 and 1992 were retrospectively reviewed. Three consecutive sections from biopsies taken before any treatment were stained using the standard immunohistochemical technique for p53, p21, and mdm2, respectively. The cutoff limit was 20% or more for positive p53 expression and 10% or more for positive p21 and mdm2 expression. RESULTS: Positive immunostaining was observed for p53 in 98 tumors (57%), for p21 in 89 tumors (51%), and for mdm2 in only 16 tumors (9%). The only association found between immunostaining for the three antibodies was that most mdm2-positive tumors had positive p21 expression. Tumor mapping of regional immunostaining showed no association between immunostaining for p53 and p21. In a proportional hazards analysis, no association was found between the results of immunostaining for the three antibodies and treatment outcome. CONCLUSIONS: Positive or negative expression of p53, p21, or mdm2, or combinations of these, was not associated with cancer specific mortality after cystectomy for bladder carcinoma. There was no association between immunostaining for p21 and p53, whereas positive immunostaining for mdm2 was observed in a minority of the tumors. These results indicate that, in addition to p21, p53, and mdm2, there are other oncoproteins and tumor suppressor proteins along the p53 pathway that are involved in tumor development and progression.     

EB病毒相关胃癌与mdm2，p53及p21基因异常的研究

 目的 探讨p53基因突变以及mdm2，p53和p21蛋白表达异常在EBV相关胃癌（EBVaGC）发生发展中的作用。方法 应用免疫组化技术检测13例EBVaGC、45例临床病理资料与之匹配的EBV阴性胃癌（EBVnGC）组织中 mdm2，p53和p21蛋白的表达；PCR-SSCP银染技术结合DNA序列分析检测p53基因exon 5~8突变；RT-PCR检测EBV相关基因的表达。结果 E-BVaGC组与EBVnGC组相比，两组间mdm2，p53和p21蛋白的阳性检出率差异无统计学意义（P = 0.830 0；P = 0.791 2；P = 0.353 1），但EBVaGC组p53蛋白过表达率（15.38 %）明显低于EBVnGC组（57.78 %），两组间差异有统计学意义（P = 0.008 5）。mdm2蛋白阳性表达与p53蛋白过表达呈显著正相关（P = 0.000 8，r = 0.439 1）；p21与p53蛋白共同表达率较高，但经计数资料相关性统计学分析表明两者无显著相关性（P = 0.2501，r = 0.202 5）。2例EBVnGC检测到p53基因突变，突变均位于exon 5，13例E-BVaGC和58例相应癌旁组织均未检测到p53基因突变。13例EBVaGC核抗原基因EBNA1均为阳性，潜伏膜蛋白基因LMP1均为阴性，即刻早期基因BZLF1，早期基因BARF1和BHRF1阳性率分别为46.15 %（6／13），46.15 %（6／13）和15.38 %（2／13），三者与EBVaGC组织中mdm2，p21和p53蛋白的表达均无显著相关性（P＞0.05）。 结论 EBV感染以及mdm2，p53和p21蛋白表达异常与胃癌发生有关； p53基因突变可能并非胃癌组织中p53蛋白异常累积的主要原因；胃癌组织中EBV感染与p53蛋白的异常表达有关，而与mdm2和p21蛋白的异常表达以及p53基因突变无显著相关性。     

Clinical application of oligonucleotide probe array for full-length gene sequencing of TP53 in colon cancer

OBJECTIVE: TP53 mutations are the most frequent genetic alterations in colon cancer. We studied whether the recently developed oligonucleotide microarray technique, GeneChip p53 assay, can be applied to sensitive detection of TP53 gene mutations in surgical specimens from colon cancer patients. METHODS: TP53 gene mutations in exons 2-11 in 20 colon cancers and the corresponding histopathologically normal mucosa at the surgical margins were assessed by GeneChip p53 assay, and the results were further evaluated by direct sequencing of the involved exon or by mutant-allele-specific amplification (MASA). The expression of TP53 protein was also evaluated immunohistochemically and the result was compared with the gene alteration. RESULTS: The GeneChip p53 assay detected TP53 mutations in 65% of primary cancers; 61% of the mutations were within the evolutionarily conserved regions, and 46% of the mutations were within the zinc-binding domains (regions of loop 2 and loop 3). Direct sequencing confirmed these mutations. Immunohistochemical examination detected TP53 protein overexpression in 47% of primary cancers, but this protein did not accumulate with all types of TP53 mutations. In addition, the GeneChip assay detected a mutation identical to that in the primary tumor in 2 samples from the surgical margins, and MASA confirmed both mutations, implying the presence of occult cancer cells. CONCLUSION: The GeneChip p53 assay is sufficiently sensitive to detect TP53 mutations in surgical specimens from colon cancers and may be applicable to screening examination in clinical laboratories as a routine procedure.     

mdm2 expression as a prognostic indicator in clear cell renal cell carcinoma: comparison with p53 overexpression and clinicopathological parameters.

The present study was designed to analyze the expression of p53 and mdm2 in clear cell renal cell carcinoma with special emphasis on their association with tumor grade and clinical outcome. In particular, the value of individual protein overexpression as well as combined p53/mdm2 positivity was evaluated because both proteins are functionally connected, and their expression is controlled by an autoregulatory feedback loop. A cohort of 97 clear cell renal cell carcinomas was analyzed. The overexpression of mdm2 and p53 proteins was investigated on paraffin-embedded material by using monoclonal antibodies. Eighteen tumors showed mdm2 positivity, whereas 35 of the tumors overexpressed p53. Whereas p53 and mdm2 positivity correlated significantly (P = 0.00004), no correlation could be found between mdm2 protein overexpression and tumor stage, lymph node involvement, and presence of distant metastases. mdm2 positivity was found significantly more frequently in tumors of higher grade. In univariate analysis, there was a statistically significant correlation between p53 and mdm2 overexpression in the same tumor and poor survival (P = 0.00179). Multivariate analysis revealed that coincident mdm2/p53 overexpression, the presence of distant metastases, and tumor grade were independent predictors for tumor progression. Our results indicate that mdm2/p53 co-overexpression, nuclear grade, and preoperative presence of distant metastasis are independent predictors for poor survival.     

骨巨细胞瘤mdm2、p185、p21及p53免疫组化研究

[目的]探讨mdm2、p185、p21及p53在骨巨细胞瘤（GCT）的表达及与GCT病理分级和复发的关系。[方法]应用SP免疫组织化学方法检测mdm2、p185、p21及p53在52例GCT中的表达（GCT按Jaffe分级：Ⅰ级15例、Ⅱ级25例、Ⅲ级12例）。[结果]52例GCT中mdm2、p185、p21及p53的阳性表达率分别为34.6%（18/52）,21.2%(11/52),13.5%(7/52)及26.f9%(14/52)。不同病理分级阳性表达均无显著性差异。mdm2、p185、p21、p53在复发和无复发的病例中,阳性表达率分别为61.5%(8/13)、25.6%(10/39);38.5(5/13)、15.4%（6/39）;23.1%(3/13)、10.3%(4/29);46.2%(6/13)、20.5%(8/39)。GCT复发与否之间的mdm2表达有显著性差异（P=0.018),而p185、p21及p53的表达无显著性差异。[结论]mdm2、p185、p21及p53在GCT中的表达与病理分级差异无关,而mdm2的表达与其复发与否有关。     

p53 alterations in recurrent squamous cell cancer of the head and neck refractory to radiotherapy

The aim of the study was to determine the incidence of p53 alterations by mutation, deletion or inactivation by mdm2 or human papillomavirus (HPV) infection in recurrent squamous cell cancer of the head and neck (SCCHN) refractory to radiotherapy. Twenty-two tumours were studied. The p53 status of each tumour was analysed by sequencing of exons 4-9 and by immunohistochemistry. Mdm2 expression was assessed by immunohistochemistry and HPV infection was assessed by polymerase chain reaction of tumour DNA for HPV 16, 18 and 33. Fifteen (68%) of the 22 tumours studied had p53 mutations, while seven had wild-type p53 sequence. p53 immunohistochemistry correlated with the type of mutation. HPV DNA was detected in 8 (36%) tumours and all were of serotype HPV 16. Of these, five were in tumours with mutant p53 and three were in tumours with wild-type p53. Mdm2 overexpression was detected in 11 (50%) tumours. Of these, seven were in tumours with mutant p53 and four were in tumours with wild-type p53. Overall, 21 of the 22 tumours had p53 alterations either by mutation, deletion or inactivation by mdm2 or HPV. In this study, the overall incidence of p53 inactivation in recurrent head and neck cancer was very high at 95%. The main mechanism of inactivation was gene mutation or deletion which occurred in 15 of the 22 tumours studied. In addition, six of the seven tumours with wild-type p53 sequence had either HPV 16 DNA, overexpression of mdm2 or both which suggested that these tumours had p53 inactivation by these mechanisms. This high incidence of p53 dysfunction is one factor which could account for the poor response of these tumours to radiotherapy and chemotherapy. Therefore, new therapies for recurrent SCCHN which either act in a p53 independent pathway, or which restore p53 function may be beneficial in this disease.     

In squamous cell carcinoma of the vulva, overexpression of p53 is a late event and neither p53 nor mdm2 expression is a useful marker to predict lymph node metastases.

To offer more tailored treatment to individual patients with squamous cell carcinoma of the vulva, more accurate prediction of lymph node metastases is required. As p53 and mdm2 are genes known to be involved in the development of other tumours, we studied expression of p53 and mdm2 in carcinogenesis of squamous cell carcinoma of the vulva and their clinical relevance. Archival material of 141 T1 and T2 vulvar tumours were used. Of the 141 primary tumours, the corresponding 39 lymph node metastases (LNM) were studied, and in 90 cases the pre-existent epithelia adjacent to the tumour (EAT) and in 14 cases vulvar intraepithelial neoplasia adjacent to the tumour (VIN) was also investigated. Detection of p53 and mdm2 protein was immunohistochemically performed. Scoring categories were: negative (1); weakly positive (2); moderately to markedly positive (3); and markedly positive (4). Overexpression of p53 was seen in 56% of the LNM, 39% of the primary tumours, 21% of the VIN lesions and 0% in the group of EAT. No relation was found between overexpression of p53 in the primary tumour and LNM. Expression of mdm2 was seen in 14% of the primary tumours, of which four cases were marked positive. In the group of LNM no mdm2-positive staining was observed. In the group of EAT, 25% was mdm2-positive, of which six cases were marked positive. In the group of VIN, 36% showed moderate (score 3) mdm2 expression. No relation was found between expression of mdm2 and LNM. In squamous cell carcinoma, overexpression of p53 is a late event in carcinogenesis. Marked expression of mdm2 is rarely seen in vulvar carcinomas, indicating that aberrant p53 cannot induce mdm2 expression. LNM cannot be predicted by detection of these proteins.     

Expression of p21/waf1 in oral squamous cell carcinomas--correlation with p53 and mdm2 and cellular proliferation index

The cyclin-dependent kinase inhibitor p21/waf1 is regulated by p53-dependent and p53-independent pathways. In addition, mdm2 is an oncogene which forms an auto-regulatory loop with the normal p53 protein and its role has been implicated in oncogenesis. To determine whether a correlation exists between the expression of these gene products, tumor differentiation, tumor staging and radiation therapy, we investigated the expression of p21, p53 and mdm2, and cellular proliferation by Ki-67 (MIB1) labeling index using immunohistochemistry in 88 human oral squamous cell carcinoma (SCC) samples from 56 patients. Tumor expression of all nuclear proteins was scored according to the percentage of positive cancer nuclei, both with the cancer tissue as a whole as well as in different epithelial compartments of differentiation. Positive p21, p53, mdm2 and MIB1 staining was present in 82.4, 67.8, 25.9 and 98.8% of the SCC samples. The staining in different epithelial compartments of differentiation varied: those of p21 and mdm2 present predominantly in suprabasal and upper regions of the tumors: those of p53 and MIB1 in basal and suprabasal regions. Higher levels of p21 expression were seen in actively proliferating tumors (P = 0.025). p21 expression positively correlated with mdm2 expression but not with p53 expression. Moreover, the level of p21 expression was higher in older patients (P = 0.024) and female patients (P = 0.008). There was no significant association among p53, mdm2 and MIB1. Expression of p53 was higher in tumors with poorer cellular differentiation and in younger patients (P = 0.038 and 0.028). There was no association between tumor stage by TNM classification and the expression of any of these gene products or proliferation index. Radiation therapy did not alter the expression of any of these. To conclude, p21 protein was overexpressed in oral SCCs, and this overexpression was related to cell proliferation index and mdm2 expression but independent of p53 protein alteration. Overexpression of p21 alone appeared to be insufficient to suppress tumor progression.     

Overexpression of c-met as a prognostic indicator for transitional cell carcinoma of the urinary bladder: a comparison with p53 nuclear accumulation.

PURPOSE: The c-met proto-oncogene encodes a receptor tyrosine kinase (Met) and has been shown to play a role in oncogenesis. Given that high titers of hepatocyte growth factor, the specific ligand for Met, are excreted in the urine and tend to reflect disease activity of bladder cancer, we performed this study to examine the clinical significance of Met in human bladder cancer. MATERIALS AND METHODS: We studied the mRNA expression and genomic alteration of c-met in five bladder cancer cell lines. Significance of Met overexpression was then compared with p53 nuclear accumulation (TP53) in primary bladder cancer (n = 142 patients). RESULTS: Expression of c-met mRNA tended to positively correlate with differentiation of cancer cell lines in the absence of point mutation. High expression of Met was found in seven cases (4.9%), low expression in 32 cases (22.5%), and negative expression in 103 cases (72.5%). Expression of Met was positively associated with histologic grade, stage classification, tumor size, and nodular tumor growth (P <.05, respectively); however, it was not related to TP53 status. Factors that predicted disease progression were tumor stage, Met status, and TP53 accumulation (P <.05, respectively). Indicators for poor long-term survival were invasive cancer, multiple tumors, and Met overexpression (P =.0006,.01, and.04, respectively). CONCLUSION: The c-met proto-oncogene plays a more important role in the progression of bladder carcinogenesis than p53. Evaluation of Met expression could identify a subset of bladder cancer patients who may require a more intensive treatment strategy.     

Analysis of p53 mutational events and MDM2 amplification in canine soft-tissue sarcomas.

Canine cancer is of major significance in terms of animal health and welfare and soft tissue sarcomas are an important group of tumours accounting for approximately 15% of all canine tumours presented. Abnormal p53 protein expression and gene mutations have been identified in a number of different canine tumour types. However, mdm2 gene amplification has only been investigated in a limited number of canine osteosarcomas. In this present study a series of canine soft-tissue sarcomas (STS) were examined for p53 mutations and/or mdm2 amplification. For p53 mutational studies polymerase chain reaction and direct DNA sequencing was used. Gene mutations were identified in 6 of 30 (20%) primary tumour cases including MPNST (n=3) leiomysarcoma (n=1), heamangiosarcoma (n=1) and sarcoma (n=1). mdm2 gene amplification was assessed by Southern Blot. Although there was no evidence for major gene rearrangements, gene amplification was detected in 4 of 35 (11.4 %) primary tumours including MPNST (n=2), rhabdomyosarcoma (n=2). A total of 33 cases were examined for both p53 mutations and mdm2 amplification. Seven of the tumours were positive for p53 mutations, while five were positive for mdm2 amplification. With the exception of one case, a reciprocal relationship between the presence of a p53 mutation and mdm2 gene amplification was demonstrated.     

TP53 alterations and patterns of carcinogen exposure in a U.S. population-based study of bladder cancer

The molecular pathology of bladder cancer has been the subject of considerable interest, and current efforts are targeted toward elucidating the interrelationships between individual somatic gene loss and both etiologic and prognostic factors. Mutation of the TP53 gene has been associated with more invasive bladder cancer, and evidence suggests that TP53 mutation, independent of stage, may be predictive of outcome in this disease. However, there is no consensus in the literature that bladder carcinogen exposure is associated with inactivation of the TP53 gene. Work to date has been primarily hospital based and, as such, subject to possible bias associated with selection of more advanced cases for study. We examined exposure relationships with both TP53 gene mutation and TP53 protein alterations in a population-based study of 330 bladder cancer cases in New Hampshire. Tobacco smoking was not associated with TP53 alterations. We found a higher prevalence of TP53 inactivation (i.e., mutation and nuclear accumulation) among hair dye users (odd ratio [OR] = 4.1; 95% confidence interval [CI] 1.2-14.7), and the majority of these mutations were transversions. Men who had "at risk" occupations were more likely to have mutated TP53 tumors (OR = 2.9; 95% CI 1.1-7.6). There also was a relative absence of TP53 mutation (OR = 0.4; 95% CI 0.0-2.9) and TP53 protein alterations (OR = 0.6; 95% CI 0.3-1.4) in bladder cancers from individuals with higher arsenic exposure. Our data suggest that there is exposure-specific heterogeneity in inactivation of the TP53 pathway in bladder cancers and that integration of the spectrum of pathway alterations in population-based approaches (capturing the full range of exposures to bladder carcinogens) may provide important insights into bladder tumorigenesis.     

Detection of TP53 mutation, loss of heterozygosity and DNA content in fine-needle aspirates of breast carcinoma.

Recent preclinical and clinical data suggest that TP53 status and TP53 mutations may be important in determining tumour aggressiveness and therapy response. In this study we investigate the feasibility of a structural and quantitative analysis of TP53 on fine-needle aspiration (FNA) material obtained from 31 consecutive female patients with breast carcinoma, enrolled in a primary chemotherapy protocol. Tumours were screened for p53 protein overexpression and TP53 mutations (exons 5-8) using immunocytochemistry, polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) and DNA sequencing analyses, and finally using fluorescence in situ hybridization (FISH) analysis. Positive nuclear staining was identified in six cases whereas mutations were detected in nine. Although the immunoreactive pattern fitted fully with the characterized TP53 mutation type, the considerable number of null p53 mutations (i.e. four) coupled with the lack of information regarding the localization of TP53 mutations make immunocytochemistry an inadequate indicator of TP53 function deregulation. Combining molecular and FISH analyses, we detected three cases with TP53 deletion and one case with deletion and mutation. Finally, DNA static-image analysis performed on 29 cases showed aneuploidy in 26 cases, which included all TP53-mutated cases. The present results show that FNA may assist clinical decisions by allowing the evaluation of a variety of biological parameters relevant for prognosis and treatment planning.     

p53 inactivation in chewing tobacco-induced oral cancers and leukoplakias from India

The inactivation of p53 tumour suppressor gene vis-á-vis point mutation, overexpression and degradation due to Human Papilloma virus (HPV) 16/18 infection, was examined in chewing tobacco-associated oral cancers and oral leukoplakias from India. The analysis of mutations was assessed by polymerase chain reaction (PCR) with single strand conformation polymorphism (PCR-SSCP) of exons 5-9 on DNA from 83 oral cancer cases, and the mutations confirmed by direct nucleotide sequencing of the PCR products. p53 protein expression was evaluated by immunohistochemical analysis on paraffin-embedded sections of 62 representative oral cancer biopsies and 22 leukoplakias, using p53-specific monoclonal antibody DO-7. The presence of HPV16/18 was detected in the 83 oral cancer cases by PCR analysis using HPV L1 consensus sequences, followed by Southern hybridization with type-specific oligonucleotide probes. Forty-six per cent (38/83) of oral cancer tumours showed p53 alterations, with 17% (14/83) showing point mutations, 37% (23/62) with overexpression and 25% (21/83) with presence of HPV16 wherein the E6 HPV16 protein degrades p53. HPV18 was not detected in any of the samples. Ninety-two per cent concordance was observed between missense point mutations and overexpression of p53 protein. A significant correlation was not observed between p53 alterations in oral cancer and clinico-pathological profile of the patients. Twenty-seven per cent (6/22) of oral leukoplakias showed p53 overexpression. The overall p53 alterations in oral cancer tissues and oral lesions are comparable to data from the oral cancers reported in the Western countries with smoking and alcohol-associated oral cancers, and suggest a critical role for p53 gene in a significant proportion of oral cancers from India. The overexpression of p53 protein in leukoplakias may serve as a valuable biomarker for identifying individuals at high risk of transformation to malignant phenotype.     

Comparison of TP53 mutations identified by oligonucleotide microarray and conventional DNA sequence analysis

As the rate of gene discovery accelerates, more efficient methods are needed to analyze genes in human tissues. To assess the efficiency, sensitivity, and specificity of different methods, alterations of TP53 were independently evaluated in 108 ovarian tumors by conventional DNA sequence analysis and oligonucleotide microarray (p53 GeneChip). All mutations identified by oligonucleotide microarray and all disagreements with conventional gel-based DNA sequence analysis were confirmed by re-analysis with manual and automated dideoxy DNA sequencing. A total of 77 ovarian cancers were identified as having TP53 mutations by one of the two approaches, 71 by microarray and 63 by gel-based DNA sequence analysis. The same mutation was identified in 57 ovarian cancers, and the same wild type TP53 sequence was observed in 31 ovarian cancers by both methods, for a concordance rate of 81%. Among the mutation analyses discordant by these methods for TP53 sequence were 14 cases identified as mutated by microarray but not by conventional DNA sequence analysis and 6 cases identified as mutated by conventional DNA sequence analysis but not by microarray. Overall, the oligonucleotide microarray demonstrated a 94% accuracy rate, a 92% sensitivity, and an 100% specificity. Conventional DNA sequence analysis demonstrated an 87% accuracy rate, 82% sensitivity, and a 100% specificity. Patients with TP53 mutations had significantly shorter overall survival than those with no mutation (P = 0.02). Women with mutations in loop2, loop3, or the loop-sheet-helix domain had shorter survival than women with other mutations or women with no mutations (P = 0.01). Although further refinement would be helpful to improve the detection of certain types of TPS3 alterations, oligonucleotide microarrays were shown to be a powerful and effective tool for TP53 mutation detection.     

P53 mutation analysis of colorectal liver metastases: relation to actual survival, angiogenic status, and p53 overexpression.

PURPOSE: To correlate TP53 mutations with angiogenic status of the tumor and prognosis after liver surgery in patients with colorectal liver metastases and to correlate immunohistochemical staining of p53 protein with TP53 gene mutations. EXPERIMENTAL DESIGN: Tumors of 44 patients with surgically treated colorectal liver metastases were analyzed for (a) TP53 mutations using denaturing gradient gel electrophoresis followed by sequencing, (b) microvessel density using the hot spot overlap technique, (c) apoptotic rate in tumor cells and endothelial cells of tumor microvessels using double immunostaining for anti-cleaved caspase 3 and anti-CD34, and (d) expression of p53 protein using immunohistochemistry. RESULTS: TP53 mutations were detected in 36% of the metastases and occurred more frequently in liver metastases from left-sided colon tumors than from right-sided colon tumors (P = 0.04). In metastases with TP53 mutations, microvessel density was higher compared with tumors with wild-type p53. Endothelial cell apoptosis was not different in tumor microvessels from TP53-mutated versus nonmutated tumors. The 5-year actual survival was not influenced by TP53 mutational status, microvessel density, or endothelial cell apoptotic rate of the tumors. Based on immunohistochemical p53 overexpression, the positive and negative predictive values of TP53 mutations were 61% and 82%. CONCLUSIONS: In patients with surgically treated colorectal liver metastases, TP53 mutations and angiogenic status did not influence prognosis. Immunohistochemistry is not a reliable technique for detecting TP53 mutations.     

P53和mdm2在骨巨细胞瘤中的表达

目的：探讨p53和mdm2在骨巨细胞瘤（GCT）的表达及与GCT病理分级和复发的关系。方法：应用SP免疫组织化学方法检测p53和mdm2在52例GCT（GCT按Jaffe分级：Ⅰ级15例、Ⅱ级25例、Ⅲ级12例）中的表达。结果：14例p53表达阳性,阳性率26．9％,18例mdm2表达呈阳性,阳性率达34．6％。其阳性表达与病理分级均无显著性差异（P值分别为0．699;0．871）。p53和mdm2在复发和无复发的病例中,阳性表达率分别为46．2％、20．5％和61．5％、25．6％。两者同时阳性表达有7例,p53和mdm2同时过表达与GCT复发有高度显著性差异（P＝0．008）。结论：p53和mdm2在GCT中的表达与病理分级无关而与其复发有关。