Communicated by David GoldgarThe tumor suppressor gene TP53 is frequently mutated in human cancers. More than 75% of all mutations are missense substitutions that have been extensively analyzed in various yeast and human cell assays. The International Agency for Research on Cancer (IARC) TP53 database (www-p53.iarc.fr) compiles all genetic variations that have been reported in TP53. Here, we present recent database developments that include new annotations on the functional properties of mutant proteins, and we perform a systematic analysis of the database to determine the functional properties that contribute to the occurrence of mutational ''hotspots'' in different cancer types and to the phenotype of tumors. This analysis showed that loss of transactivation capacity is a key factor for the selection of missense mutations, and that difference in mutation frequencies is closely related to nucleotide substitution rates along TP53 coding sequence. An interesting new finding is that in patients with an inherited missense mutation, the age at onset of tumors was related to the functional severity of the mutation, mutations with total loss of transactivation activity being associated with earlier cancer onset compared to mutations that retain partial transactivation capacity. Furthermore, 80% of the most common mutants show a capacity to exert dominant-negative effect (DNE) over wild-type p53, compared to only 45% of the less frequent mutants studied, suggesting that DNE may play a role in shaping mutation patterns. These results provide new insights into the factors that shape mutation patterns and influence mutation phenotype, which may have clinical interest. Hum Mutat 28(6), 622-629, 2007. Published 2007

Inactivation of the tumor suppressor p53 by missense mutations is the most frequent genetic alteration in human cancers. The common missense mutations in the TP53 gene disrupt the ability of p53 to bind to DNA and consequently to transactivate downstream genes. However, it is still not fully understood how a large number of the remaining mutations affect p53 structure and function. Here, we used a comprehensive site-directed mutagenesis technique and a yeastbased functional assay to construct, express, and evaluate 2,314 p53 mutants representing all possible amino acid substitutions caused by a point mutation throughout the protein (5.9 substitutions per residue), and correlated p53 function with structure-and tumor-derived mutations. This high-resolution mutation analysis allows evaluation of previous predictions and hypotheses through interrelation of function, structure and mutation.

To investigate the clinical value of somatic TP53 mutations in breast cancer, we assembled clinical and molecular data on 1,794 women with primary breast cancer with long-term follow-up and whose tumor has been screened for mutation in exons 5 to 8 of TP53 by gene sequencing. TP53 mutations were more frequent in tumors of ductal and medullar types, aggressive phenotype (high grade, large size, node positive cases, and low hormone receptor content) and in women <60 years old. TP53 mutations within exons 5 to 8 conferred an elevated risk of breast cancer^specific death of 2.27 (relative risk >10 years; P < 0.0001) compared with patients with no such mutation. The prognostic value of TP53 mutation was independent of tumor size, node status, and hormone receptor content, confirming and reconciling previous findings in smaller series. Moreover, an interaction between TP53 mutation and progesterone receptor (PR) status was revealed, TP53 mutation combined with the absence of progesterone receptor being associated with the worst prognosis. Whereas previous studies have emphasized the fact that missense mutations in the DNA-binding motifs have a worse prognosis than missense mutations outside these motifs, we show that non^missense mutations have prognostic value similar to missense mutations in DNA-binding motifs. Nonetheless, specific missense mutants (codon 179 and R248W) seem to be associated with an even worse prognosis. These results, obtained on the largest series analyzed thus far, show that TP53 mutations identified by gene sequencing have an independent prognostic value in breast cancer and could have potential uses in clinical practice.The tumor suppressor gene TP53 plays a key role in many cellular pathways controlling cell proliferation, cell survival, and genomic integrity. It acts as a proliferation brake when cells experience stress conditions, such as DNA-damage, hypoxia, or oncogene activation. Disrupting TP53 function promotes checkpoint defects, genomic instability, and inappropriate survival, leading to the uncontrolled proliferation of damaged cells. The proliferative advantage given by its inactivation, and the fact that it is ubiquitously expressed, explains why it is frequently found to be mutated in almost every type of cancer (1). It has been shown in various experimental in vitro systems, as well as in mouse models, that cell cycle arrest or apoptosis induced by radiotherapy and various chemotherapeutic drugs depends on an intact TP53 pathway (2, 3). These results have raised the hypothesis that TP53 could be a key player in defining tumor sensitivity to a broad range of anticancer treatments in patients with cancer. Moreover, the presence of a TP53 mutation could be one of the underlying causes of drug resistance, the major cause of treatment failure and cancer death.Several studies have investigated the predictive value of TP53 mutation status for tumor response to treatment and patient outcome in various cancers. Different clinical and methodologic

Prediction of the biological effect of missense substitutions has become important because they are often observed in known or candidate disease susceptibility genes. In this paper, we carried out a 3-step analysis of 1514 missense substitutions in the DNA-binding domain (DBD) of TP53, the most frequently mutated gene in human cancers. First, we calculated two types of conservation scores based on a TP53 multiple sequence alignment (MSA) for each substitution: (i) Grantham Variation (GV), which measures the degree of biochemical variation among amino acids found at a given position in the MSA; (ii) Grantham Deviation (GD), which reflects the ‘biochemical distance’ of the mutant amino acid from the observed amino acid at a particular position (given by GV). Second, we used a method that combines GV and GD scores, Align-GVGD, to predict the transactivation activity of each missense substitution. We compared our predictions against experimentally measured transactivation activity (yeast assays) to evaluate their accuracy. Finally, the prediction results were compared with those obtained by the program Sorting Intolerant from Tolerant (SIFT) and Dayhoff's classification. Our predictions yielded high prediction accuracy for mutants showing a loss of transactivation (∼88% specificity) with lower prediction accuracy for mutants with transactivation similar to that of the wild-type (67.9 to 71.2% sensitivity). Align-GVGD results were comparable to SIFT (88.3 to 90.6% and 67.4 to 70.3% specificity and sensitivity, respectively) and outperformed Dayhoff's classification (80 and 40.9% specificity and sensitivity, respectively). These results further demonstrate the utility of the Align-GVGD method, which was previously applied to BRCA1. Align-GVGD is available online at .

The PI3K-AKT pathway is activated in a variety of human cancers, resulting in disturbance of cell growth, proliferation and survival. Among the factors affecting the pathway, the K-Ras mutation and PIK3CA mutation are the most common oncogenic alterations in colorectal cancer. We hypothesized that these two mutations are important in activation of the PI3K pathway and colorectal carcinogenesis. In this study, we aimed to examine the influence of PIK3CA mutation and K-Ras mutation on AKT activation, and to clarify whether PIK3CA mutation, K-Ras mutation and p-AKT expression may be used as parameters for predicting prognosis in colorectal cancer. Tissue samples from 158 colorectal cancer patients who underwent surgical resection were examined. The sequences of exon 1 of K-Ras and exons 9 and 20 of PIK3CA were determined by direct sequencing using genomic DNA extracted from paraffin-embedded blocks. Activation status of AKT was evaluated by immunohistochemical staining using phospho-specific AKT antibody (Ser473). Correlation between these factors and clinicopathologic findings/patient survival were examined. As a result, PIK3CA mutation was significantly associated with shorter relapse-free survival (RFS) in stage II/III patients (p 5 0.0216) and shorter disease-specific survival in all patients (p 5 0.0357). In the multivariate analysis, PIK3CA mutation was the only independent and significant prognostic factor for RFS in stage II/III patients (p 5 0.0433, HR 2.478). This study revealed the prognostic value of PIK3CA mutation in colorectal cancer patients. Patients with PIK3CA mutation should be followed up carefully. Moreover, our result suggests that inhibition of PIK3CA mutant may be a new molecular target therapy. ' 2007 Wiley-Liss, Inc.