Specificity of TP53 mutation screening methods in cancerous tissues.

摘要

Yamanoshita et al. recently published a TP53 mutation pattern obtained by analysing oesophageal cancerous tissues from 207 Chinese patients.1 With great interest, we read their article, which was mainly dedicated to the comparison of 2 mutation screening approaches: single-strand conformation polymorphism (SSCP) and denaturing high-performance liquid chromatography (DHPLC).Reliable TP53 mutations screening and identification methods are needed in numerous fields of cancer research. In molecular epidemiology and environmental toxicology, the establishment of mutational patterns provides interesting clues in the involvement of cancer risk factors. Two of the most famous examples are tandem CC to TT substitutions related to UV radiations in skin cancers and G to T transversions at codon 249 in aflatoxin Bl-related liver cancers. Another application is the use of TP53 mutations as a tool to improve diagnosis, prognosis or prediction of response to therapy. In these areas, the precise determinations of mutation type and localisation inside the gene seem to bring more relevant information than a simple immunohistochemical determination of the p53 protein overexpression. Finally, the identification of TP53 mutations could be of great value if new therapies aiming to restore the protein p53 functionality confirm their promising preliminary results in the future.4 For all these reasons, many approaches are optimised to improve the detection and the following identification of TP53 mutations in human tissues. These last few years, the most popular of them have been SSCP, functional assay for the separation of alleles in yeast (FASAY), DNA microarrays dedicated to TP53 analysis, restriction methods and techniques based on double-stranded DNA denaturation such as DHPLC, denaturing gradient gel electrophoresis (DGGE) or temporal temperature gradient gel electrophoresis (TTGE). DHPLC has already been used to detect TP53 alterations in different cancerous tissues including oesophagus, stomach, ovary, blood cells, bladder or conjunctiva.10 As mentionned by Yamanoshita et al., this technique is sensitive, easy to use (in comparison to electrophoretic methods) and offers an interesting throughput. However, the authors reported a disappointing specificity with 50% of false-positives and considered this performance as "not yet understood." As this issue is essential to obtain reliable results, we would like to provide here some clues to explain at least one part of this lack of specificity and some practical solutions for clinicians and researchers beginning to screen TP53 mutations. Indeed, we will demonstrate that some false-positives attributed to a lack of DHPLC specificity could in fact constitute genuine mutated DNA fragments. The inclusion of these additional positive results in tumor series could improve the sensi-tivity of the whole procedure (screening .+ sequencing) and increase mutation rates found in studies similar to that of Yamanoshita et al. These comments are based on our own experience and on technical information collected in other laboratories and published studies.