Discriminating functional and non-functional p53 in human tumours by p53 and MDM2 immunohistochemistry

Abstract

Mutation and/or loss of the TP53 tumour suppressor gene is the single most common genetic abnormality in human cancer. The majority of TP53 mutations lead to stabilization of the protein, so that immunohistochemical staining for p53 can suggest mutation status in many cases. However, various false-positive and false-negative situations mean that simple immunostaining for p53 is not informative in a substantial number of tumours. In the present study, a series of 119 human cancers were immunostained using a highly sensitive technique that detects the low levels of wild-type protein expressed in normal cells, such that homozygous gene deletion or non-sense TP53 mutation can be identified by an absence of staining. TP53 gene status was also assessed using FASAY as a genetic/functional screen and in selected cases by direct sequencing. A quantitative scoring system was employed to assess p53 levels, and p53 post-translational modification was evaluated using antibodies that detect specific phosphorylation sites. Phosphorylated p53 correlated with total p53 levels and did not improve the prediction of TP53 mutation status. The transcriptional activity of TP53 was determined by staining for two downstream target genes, p21(WAF1) and MDM2, and statistical correlations between MDM2/p21(WAF1) and p53 were found in tumours with wild-type, but not mutant TP53. Measurement of staining for p53 and MDM2 accurately identifies the TP53 status of tumours. This simple and cost-effective method, applicable to automated staining and quantitation methods, improves the identification of TP53 status over standard methods for p53 immunostaining and provides information about tumour p53 phenotype that is complementary to genotyping data.

Survivin is an independent predictor of short-term survival in poor prognostic breast cancer patients

“…It is recognised that TP53 mutations can predict poor response to tamoxifen (Berns et al, 2000). Phosphorylation of Ser 15 has also been detected in breast cancers with mutant p53 (Nenutil et al, 2005) and would merit further analysis.…”

“…The biological role of ser 392 phosphorylation of wild-type p53 is unclear, but it may regulate the oncogenic function of mutant p53 (Yap et al, 2004). Immunohistochemical detection of phosphorylated Ser 392 is more frequent in breast cancers with mutant rather than wild-type p53 (Nenutil et al, 2005). In other tumours the presence of p53 phosphorylated at Ser 392 has been associated with poorer survival (Matsumoto et al, 2004).…”

Calculator for ovarian carcinoma subtype prediction

“…8,13,14 and added eight markers reported as differentially expressed in the literature: cyclin-dependent kinase inhibitor 2A (p16) (CDKN2A), mouse double minute 2 (MDM2), catenin (cadherin-associated protein), beta 1, 88 kDa (CTNNB1), phosphatase and tensin homolog (PTEN), mucin 5AC, oligomeric mucus/gel-forming (MUC5AC), paired box 2 (PAX2), secretoglobin, family 1A, member 1 (SCGB1A), CD44 molecule, splice variant 6 (CD44v6). [15][16][17][18][19][20][21] Three additional markers: growth differentiation factor 15 (GDF15), trefoil factor 3 (intestinal) (TFF3), dickkopf homolog 1 (DKK1) were derived from comprehensive gene expression profiling data generated using Human Exonic Evidence Based Oligonucleotide microarray (Stanford, CA, USA) or whole transcriptome sequencing, as recently described. 22,23 Immunohistochemistry Three tissue microarrays were constructed, as described previously 13 from representative tumor areas using a tissue arrayer (Beecher Instruments, Silver Spring, MD, USA and Pathology Devices, Westminster, MD, USA).…”

Updates on p53: modulation of p53 degradation as a therapeutic approach

“…This picture is now changing rapidly with the discovery of p53 isoforms whose mRNAs show more variable patterns of expression. Quantitative analysis of large numbers of human tumours using panels of antibodies has allowed a variety of expression phenotypes to be classified, including post-translational modifications and examination of the expression of p53-induced gene products (Nenutil et al, 2005). The relationship between these expression patterns and the genetic mutations in the p53 pathway is not straightforward.…”