Primary Motility  Disorders of the  Esophagus
 The Esophageal
 Esophagogastric  Junction

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Volume: Barrett's Esophagus
Chapter: Markers

How can the contradictory findings of p53 gene mutation be explained? Can differences in technique be sufficient reason?

S.C. Evans, A.G. Casson (Halifax)

Techniques widely employed to screen tumors for p53 mutations include single-strand conformation polymorphism analysis, denaturation gradient gel electrophoresis (DGGE), and temporal temperature gradient electrophoresis (TTGE). Single-strand conformation polymorphism, DGGE, and TTGE all entail the amplification of individual p53 exons using the polymerase chain reaction (PCR), with assessment of electrophoretic migration. DNA sequencing is still necessary to confirm the site and nature of a suspected p53 mutation. Although these assays are relatively time consuming and expensive, recent advances in automated technology should facilitate increased DNA sequencing. It should be remembered however, that although DNA sequencing is the most objective method available to characterize p53 mutations, false-negative results are to be expected if the proportion of mutated tumor cells in a specimen is low. This may be improved by selective tumor microdissection, particularly using laser-capture technology. Further limitations of sequencing alone include the potential for PCR mutation artefacts, and the finding of mutations that do not necessarily result in a functional change.

Immunohistochemistry (IHC) is a convenient and inexpensive technique to evaluate cell nuclear p53 protein accumulation in frozen or paraffin-embedded tissue sections. Several polyclonal and monoclonal antip53 antibodies are now widely available commercially, each with well-defined sensitivity and specificity for p53 epitopes. Normal levels of p53 protein, usually between 103 and 104 molecules with short half-life, are usually undetectable by IHC. As most missense mutations extend the p53 protein half-life, IHC is a useful surrogate marker. However, significant variation in results may arise from use of different antibodies, technical issues (i.e. antigen retrieval), and interpretation (related to intensity of immunoreactivity and tissue heterogeneity). Furthermore, IHC positivity may arise (see above) in the absence of a point mutation.

In summary, IHC to assess protein distribution, and DNA sequencing to detect point-mutations should be considered complimentary techniques to evaluate the p53 tumor suppressor gene. Their combined use should not only provide important biological information about p53 in esophageal tumorigenesis, but in addition, a comprehensive analysis of a clinically relevant molecular biomarker for this disease.

Publication date: August 2003 OESO©2015