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Objective: To determine the frequency and predictive value of p53 mutations in localized prostate cancer, comparing the accuracy of detection using immunohistochemistry (IHC) with a modified yeast assay, on archival tissue samples., Materials and Methods: Prostate cancer tissue was obtained from 98 patients who had >/= 2 years of clinical follow-up after radical prostatectomy. DNA sequencing was used to verify the presence of p53 mutations in samples that were immunopositive or that gave evidence for p53 alterations using the yeast assay. The IHC and yeast findings were compared with patient outcome to determine the predictive value of these two test types., Results: Fifty-five tumours (57%) were immunopositive, and 58 (59%) were positive using the yeast assay. Sequence-confirmed p53 mutations occurred in 44 (45%) cases. The IHC protocol generated 49% (27/55) false-positive and 36% (15/42) false-negative results, and was 65% sensitive and 50% specific, with an overall accuracy of 57%. The yeast assay resulted in 24% (14/58) false-positive results with a specificity of 74% and an accuracy of 86%. When the p53 status of these patients was correlated with their clinical outcome, patients who had sequence-confirmed p53 mutations had a 2.6-fold greater failure rate (P = 0.026) and a 2.5-fold greater risk of dying from prostate cancer (P = 0.05). Notably, mutations in exon 6 predicted a six-fold increase in treatment failure (P = 0.043) and a 5.3-fold increase in the chance of dying from prostate cancer (P = 0.009). Abnormal yeast-assay findings gave similar predictive results to those obtained for DNA sequencing, while immunopositivity did not correspond to patient outcome., Conclusions: Mutations of p53 occurred in 45% of localized prostate cancers. These alterations have important prognostic implications. The yeast assay was more accurate for detecting p53 mutations than the IHC protocol used and, unlike IHC, the results of the yeast assay were predictive of patient outcome.

Detection and functional evaluation of mutant p53 alleles using a yeast assay could yield significant information for predicting the prognosis of patients with prostate cancer (CaP). Since the current version of this yeast assay is not applicable to archival tissues, we developed a modified assay for use on formalin-fixed, paraffin-embedded tissue and have applied it to the study of patient samples. Using this modified assay, we examined archival CaP samples from 10 patients for mutations in exons 5-8 of p53 gene. Mutations were detected in four samples: three resulted in the formation of red yeast colonies indicating complete loss of function, while one gave pink yeast colonies, indicating that this mutant retained partial function. In parallel, we analyzed these samples for p53 abnormalities using a single-strand conformational polymorphism (SSCP) approach. Only three of the four yeast-positive samples gave abnormal SSCP bands. In each case where abnormal p53 was found by both methods, DNA sequencing revealed the identical base change. These results suggest that the modified yeast assay may be more sensitive than SSCP for detection of p53 mutations, and demonstrate that the modified method can be used to detect and evaluate the function of p53 mutants present in archival tissue.

Background: Few studies have used multiple assays to examine the functionality of mutant p53 in prostate cancer (CaP). We employed seven functional assays to study 16 representative mutant p53 alleles, six from localized and ten from metastatic CaP., Methods: Yeast assays were employed to determine loss of function (LOF), partial function (PF), and dominant-negative status. Assays using p53-null Saos2 cells were used to determine whether mammalian cells transfected with mutant p53 could up-regulate the MDR-1 or PCNA promoters, alter IL-6 expression or confer the ability to grow in soft agar. As a further test of gain of function (GOF), p53-null PC3 cells stably transfected with these mutant p53 alleles were examined for cell cycle distributions., Results: All 16 mutant p53 alleles demonstrated either total or partial LOF. All but one allele also had at least one gain of function; however, the pattern of GOF was different for each mutant allele. Alleles derived from both localized and metastatic CaP had similar GOF characteristics; however, only alleles from metastatic disease had significantly increased S-phase fractions., Conclusions: Different mutant p53 alleles from CaP had different, complex functional profiles. The lack of predictable patterns for these alleles suggest that each mutation may uniquely affect p53 function., (Copyright 2002 Wiley-Liss, Inc.)

Background: While many studies have suggested that p53 mutations are common in human cancers, the functional activity of these mutant alleles has not yet been fully addressed. We believe that information about the functional status of individual p53 mutants will prove to be important for a better understanding of the role of p53 in tumor development and progression. Ultimately, this information could also influence treatment decisions for individual cancer patients., Methods: A recently developed yeast functional assay can be used to assess the transactivational activity of p53 mutants. Furthermore, this assay is more sensitive than single strand conformational polymorphism (SSCP) for detection of p53 mutations. In this review, we summarize the mechanism of this new technique and describe its applications in cancer research, with an emphasis on prostate cancer., Results: The use of the yeast functional assay provides a simple, sensitive, and reproducible method for detecting p53 mutations and for determining the transactivational activity and dominant-negative role of individual p53 mutants., Conclusions: This method may be adapted to analyze other transcriptional factors, including the human androgen receptor., (Copyright 1999 Wiley-Liss, Inc.)

Background: The frequency of mutant p53 in bone marrow metastases of patients with carcinoma of the prostate (CaP) and in matched sets of metastatic and primary lesions from the same patients was investigated. The data were examined in relation to prior treatment with androgen ablation (AA) therapy and were compared with the frequency of mutant p53 reported for primary CaP., Methods: Seventeen patients with M1b (bone metastasis: TNM Stage IV) CaP had either unilateral or bilateral bone marrow biopsies taken for these studies. Specimens were divided and the outer one-third examined histologically to confirm the presence of CaP cells. Immunohistochemical (IHC) staining for accumulated p53 protein was performed by an antibody cocktail technique. RNA was extracted from the remaining portion of the biopsy, and p53 transcripts were amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) and screened for base sequence changes in the exon 4-11 region using nonisotopic single-strand conformation polymorphism (SSCP) analysis and direct DNA sequencing., Results: Ten of 17 metastases (59%) demonstrated accumulation of p53. Six of 7 (86%) of the p53 IHC positive bone marrow samples contained RT-PCR-SSCP abnormalities, as did 2 of 3 IHC negative samples. Overall, 12 of 17 metastases (71%) contained mutant p53. Four of 7 biopsies (57%) retrieved prior to AA contained mutant p53, whereas 8 of 10 post-AA biopsies (80%) contained mutant p53. One patient showed identical SSCP abnormalities in right and left iliac crest metastases after therapy, and in this patient DNA sequencing demonstrated a missense mutation at codon 126 (TAC --> GGC, Tyr --> Gly). Archival primary cancers from seven patients were retrieved. All seven were IHC positive for p53 accumulation., Conclusions: p53 mutations are associated with increased metastatic potential of CaP. Abnormalities are found at approximately twice the frequency in metastases than in unselected samples of primary CaP, whereas in matched specimens there is a high rate of consonance. Mutant p53 may contribute to systemic therapy resistance, due to increased association with post-AA CaP specimens.

Purpose: Tumor grade, deoxyribonucleic acid (DNA) ploidy, proliferation, p53 and bcl-2 expression were examined in clinically localized prostate cancers of black and white American men to learn whether these features showed racial differences., Materials and Methods: A total of 117 prostate cancers (43 black and 74 white patients) obtained at radical prostatectomy for clinically localized disease were assigned Gleason scores by a single pathologist. Enzymatically dissociated nuclei from archival prostate cancers were examined by DNA flow cytometry using propidium iodide staining and the multicycle program to remove debris and sliced nuclei and to perform cell cycle analysis. For immunostaining after microwave antigen retrieval we used a DO-1/DO-7 monoclonal antibody cocktail for p53 and the clone 124 antibody for bcl-2., Results: Significantly more black than white men had Gleason score 7 tumors. The DNA ploidy distribution of Gleason 6 or less tumors was similar for both races. As anticipated, the ploidy distribution of higher grade prostate cancer in white men was more abnormal but, unexpectedly, this was not found for higher grade prostate cancer in black men. No significant racial differences were found in S phase fractions, p53 or bcl-2 immunopositivity. However, for prostate cancer in black men there was a significant association between bcl-2 immunopositivity and higher S-phase fractions., Conclusions: The aggressive prostate cancers of black men may be characterized by the 2 features of high proliferation and a block to programmed cell death.

The prognostic value of the p53 gene (TP53), the most commonly mutated gene in human cancers, has been well established for several cancer types. However, because varying frequencies of TP53 mutations have been identified in prostatic adenocarcinoma (CaP) by genetic and immunohistochemical (IHC) studies, the role of TP53 in CaP tumorigenesis is currently unresolved. These experimental discrepancies could be caused by tissue heterogeneity within prostatic neoplasms, variations in experimental protocols, or other factors. Thus, the goal of this study was to develop a reliable IHC approach for the detection of p53 in archival prostate tissue. The authors evaluated four p53 antibodies, CM-1, 1801, DO-1, and DO-7, for their ability to reveal p53. They chose two reference CaP cell lines, 26 patient specimens (including eight benign prostatic hyperplasias (BPHs), 16 CaPs, and two lymph node metastases), one prostate and nine kidney cell lines for p53 analysis. The TP53 status of these samples was characterized using single-strand conformational polymorphism (SSCP) analysis of RNA/PCR products and sequencing. IHC detection of p53 was markedly enhanced by using the combination of microwave heat-induced antigen unmasking and a cocktail of the DO-1 and DO-7 antibodies. This approach identified 14 of 15 (93%) cell lines and patient samples having TP53 missense mutations in the exons 5 to 8 region. Of the 21 patient samples and cell lines that were either normal by SSCP or expressed p53 mutations that are not expected to stain, 18 (86%) were immunonegative. Because of this good correlation between molecular and IHC analysis, this approach may help to resolve the uncertainty about TP53 in CaP tumorigenesis.