Primary Motility  Disorders of the  Esophagus
 The Esophageal
 Esophagogastric  Junction

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Volume: Barrett's Esophagus
Chapter: Markers

What is the value of immunohistochemistry in addition to molecular analysis to explain the discordance that can exist between gene mutation and protein expression?

H.S. Garewal (Tucson)

Concerning immunohistochemistry (IHC) versus molecular biology methods, such as detection of mutations, I would like to address this from a standpoint that would be beneficial to clinicians. Traditionally, it has been common to criticize IHC with the more vociferous critics often simply negating the results obtained via IHC. The literature is replete with such comments, which are frequently made at meetings. Nevertheless, a closer look at the state of affairs will show that molecular biologic techniques have significant deficiencies in themselves and are by no means perfect. It is well known that methods such as gel electrophoresis depend on detection techniques that can give variable results depending on issues such as length of development of the gel, load of protein or nucleic acid, etc. Even a cursory look at a journal publishing the results of molecular biologic methods will show that the majority of figures, if they are shown at all, illustrate one or two bands cut out of the entire gel. It is rare to find a paper that mentions the number of times an experiment was conducted in order to obtain the "perfect" picture that appears in the publication. In summary, the methodology is complex and its reproducibility is variable from method to method and laboratory to laboratory.

Thus far, the favorite question of molecular biology aficionados has always been directed at IHC results questioning whether they have been confirmed by "demonstrating a mutation." Theoretically, these arguments are very legitimate, but the shortcomings of molecular biology are frequently overlooked. The question is generally posed as an issue of validating IHC results rather than genuinely comparing the two methods. In fact, I could only find a single paper in the literature that truly compared the two techniques for p53 alterations. This paper addressed head and neck cancer [1]. The molecular biology method used was single strand confirmational polymorphism (SSCP). A total of 85 specimens were studied using both methods for all specimens. The discrepancies could be IHC negative, SSCP positive for which the arguments are well known, e.g. IHC depends on production of a protein with a longer half-life, and therefore stained, in the absence of which it would be negative. On the contrary, IHC positive and SSCP negative cases are also possible for a variety of reasons including the fact that mutations could be outside the screened exons, the mutant confirmations could migrate like the wild-type leading to a band in the same position, non-mutational stabilization of the p53 would result in IHC positivity and, finally, the molecular methods may not be as good as "advertised" as mentioned above. Of the 85 cases, 23 (27%) were positive by both methods and 37 (44%) were negative by both methods. However, 12 (14%) of the cases were IHC positive and SSCP negative, while 13 (15%) were IHC negative and SSCP positive. In other words, an equal number of cases would have been missed by either technique.

In summary, molecular techniques and immunohistochemistry must be considered as complementary, rather than "antagonistic." The one clear advantage of IHC is the feasibility of using it with a quick turnaround time in the pathology laboratory. The methodology is also far better developed, provided a suitable antibody is available. Lastly, it is possible to visualize the tissue and see what is staining, e.g. the cytoplasm, nucleus or elsewhere.


1. Calzolaro, et al. Am J Clin Pathol 1997;107:7-11.

Publication date: August 2003 OESO©2015