Assessment of the Transcriptional Activity of p53 Improves the Prediction of Recurrence in Superficial Transitional Cell Carcinoma of the Bladder

Abstract

Purpose:To investigate the value of p53 functional analysis of separated alleles in yeast (FASAY) as a witness of p53/p21pathway alteration and as a predictor of recurrence in superficial transitional cell carcinomas. Experimental Design: p53 transcriptional activity was prospectively analyzed in 52 newly diagnosed transitional cell carcinoma using FASAY competent for the transactivation of p21 and bax promoters.TP53 and p21gene expression was quantified by real-time PCR, and expression of corresponding proteins was assessed by immunohistochemistry. In addition to tumor stage and grade, the predictive value of FASAY, real-time PCR, and immunohistochemistry for tumor recurrence was assessed by Cox survival analysis. Results: A total (p21 and bax) or partial (bax only) loss of transcriptional activity was observed in 15 of 52 (29%) and 4 of 52 (7.7%) cases, respectively, a partial loss being consistently associated with R283H mutation. p53 nuclear overexpression grossly overestimated (f40%) or underestimated (f10%) the true incidence of p53 transcriptional abnormalities, especially inT a -T 1 grade 1 to 2 tumors. Loss of p21 transactivation significantly correlated with decreased p21 gene expression and lack of expression of p21 (P = 0.001). FASAY had a better predictive value for recurrence than p53 immunohistochemistry (Cox hazard ratio, 6.57 versus 3.95; P = 0.0002 versus 0.019, respectively), whereas neither p21 immunohistochemistry (hazard ratio, 1.9; P = 0.29) norTP53 or p21 gene expression were significant predictors of recurrence. The prognostic difference between FASAY and p53 immunohistochemistry was maintained in the subgroup of T a -T 1 grade 3 tumors. Conclusions: FASAY is a valuable surrogate marker for assessing p53/p21 pathway alteration and predicts transitional cell carcinoma recurrence better than p53 immunohistochemistry.Transitional cell carcinoma (TCC) of the bladder is the fourth most common malignancy in men and the eight most common cancer in women. Eighty percent of TCCs are initially superficial (TMN97: T a -T 1 ) and easily treated by transurethral resection of bladder tumor. Despite initial complete resection of the tumor, recurrence occurs in 30% to 90% of the cases (1, 2). Progression to muscle invasive and/or metastatic stages requires radical and/or systemic therapies in 15% to 20% of the cases. To decrease recurrence and progression rates, intravesical administration of chemotherapy and/or Bacillus CalmetteGuerin is considered as standard therapy (2, 3). Because these therapies are not devoid of local and systemic toxicity, their use should better be guided by individual risks for recurrence and progression (4).Over the years, many attempts have been made to identify molecular markers that would supersede clinical features, such as tumor stage, grade, and multifocality (5, 6). Expression of TP53 gene has been one of the most frequently assessed genetic alterations in bladder TCC. In an overwhelming majority of studies, the prognostic significance of TP53 alterations in...

Preservation of RNA for functional analysis of separated alleles in yeast: comparison of snap-frozen and RNALater® solid tissue storage methods

“…Although the kinetics of RNA degradation at room temperature is largely tissue-dependent, a sample inadequately stored often produces false positive FASAY results, giving a large number of red colonies that contain a spectrum of p53 genetic alterations (unpublished data). To avoid this, immediate snap freezing of the tissue biopsy remains a gold standard that allows reliable and specific detection of non-functional p53 protein in tumour cells (1). Unfortunately, snap freezing is not always feasible in routine clinical practice, a limitation that considerably hampers the use of FASAY on samples from remote origin or from clinical institutions without liquid nitrogen facilities.…”

“…The FASAY assay was used as previously described (1,2). Briefly, the transcriptional activity of human p53 in tumour cells is assessed in Saccharomyces cerevisae, where it activates a p53 target gene (Ade2).…”

“…Recently, this assay has emerged as a tool for predicting recurrence in bladder tumours (1). However, FASAY is an mRNA-based assay that can be greatly affected by RNA degradation (2).…”

Unambiguous Detection of Multiple TP53 Gene Mutations in AAN-Associated Urothelial Cancer in Belgium Using Laser Capture Microdissection

“…Abbreviations: RAL: Relative adduct labelling, Nx: Nephrectomy, Nux: Nephroureterectomy, Ux: Ureterectomy, Cx:Cystectomy, ND: not done, NA: not available.a Mean of at least three determinations in reported separate experiments [15], [16], [18].* Mutations previously reported [10] and confirmed by the FASAY method [25] in the Center for Applied Molecular Technology of the Université Catholique de Louvain.° No CiS found.…”

Quantitative Assessment of Human β-Globin Gene Expression In Vitro by TaqMan Real-Time Reverse Transcription-PCR: Comparison with Competitive Reverse Transcription-PCR and Application to Mutations or Deletions in Noncoding Regions

“…For each cDNA, a triplicate amplification was carried out using 2.5 L of cDNA, 12.5 L of Universal PCR Master Mix (2ϫ; Applied Biosystems), 300 nM each of the primers, and 100 nM probe in a total reaction volume of 25 L. TaqMan PCR was performed in a 25-L total volume containing 12.5 L of Universal PCR Master Mix (PE Applied Biosystems), 300 nM each of the primers, and 100 nM fluorescent probe. The human ␤-globin and mGAPDH genes were amplified in parallel, and the reaction was carried out as described previously (9 ).…”