Immunohistochemical detection of mutations in the epidermal growth factor receptor gene in lung adenocarcinomas using mutation-specific antibodies

Abstract

Background: The recent development of antibodies specific for the major hotspot mutations in the epidermal growth factor receptor (EGFR), L858R and E746_A750del, may provide an opportunity to use immunohistochemistry (IHC) as a screening test for EGFR gene mutations. This study was designed to optimize the IHC protocol and the criteria for interpretation of the results using DNA sequencing as the gold-standard.

Methods: Tumor sections from fifty lung adenocarcinoma specimens from Chinese patients were immunostained using L858R and E746_A750del-specific antibodies using three different antigen retrieval solutions, and the results were evaluated using three different sets of criteria. The same specimens were used for DNA purification and analysis of EGFR gene mutations.

Results: In this study the optimal buffer for antigen retrieval was EDTA (pH 8.0), and the optimal scoring method was to call positive results when there was moderate to strong staining of membrane and/or cytoplasm in >10% of the tumor cells. Using the optimized protocol, L858R-specific IHC showed a sensitivity of 81% and a specificity of 97%, and E746_A750del-specific IHC showed a sensitivity of 59% and a specificity of 100%, both compared with direct DNA analysis. Additionally, the mutant proteins as assessed by IHC showed a more homogeneous than heterogeneous pattern of expression.

Conclusions: Our data demonstrate that mutation-specific IHC, using optimized procedures, is a reliable prescreening test for detecting EGFR mutations in lung adenocarcinoma.

Virtual slides: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/2059012601872392.

Figure 1

Sample case of predominant solid adenocarcinoma immunostained with E746_A750del-specific antibody using different antigen retrieval buffers (original magnification x400). A Sodium citrate (pH 6.0). B EDTA (pH 8.0). C EDTA (pH 9.0).

Figure 2

Predominant acinar adenocarcinoma with adjacent normal alveoli. A H & E (original magnification x100). B The cytoplasm and membrane of the tumor cells were stained strongly with L858-specific antibody. In contrast, the adjacent normal alveolar epithelial cells were completely negative (original magnification x100).

Figure 3

Adenocarcinoma with acinar and lepidic patterns. A H & E (original magnification x100). B The cytoplasm and membrane of acinar component stained strongly, but lepidic tumor cells stained weakly with L858-specfic antibody (original magnification x100).

Figure 4

A case of predominant solid adenocarcinoma with the L858R mutation. A DNA sequencing of EGFR showing normal (upper panel) and L858R mutant (lower panel). The position of the mutation is boxed. The mutant sequence appears to be homozygous with complete absence of normal sequence. This is because of the use of a “clamp” strategy to suppress amplification of the normal sequence, as described in the methods section. B Immunohistochemical staining with L858R-specific antibody showing strong positivity (original magnification x200). C Immunohistochemical staining with E746-A750 del-specific antibody showing complete negativity (original magnification x200).

Figure 5

A case of predominant acinar adenocarcinoma with normal EGFR. A and B DNA sequencing shown normal EGFR exon 21 and 19, respectively, in the region that is frequently subject to mutation. C and D Tumor cells were not stained with either L858R-specfic or E746_A750del-specific antibodies, respectively (original magnification x200).

Figure 6

A case of predominant acinar adenocarcinoma with the exon 19 deletion mutation (E746-A750 del). A DNA sequencing of EGFR showing normal (upper panel) and the E746-A750 del mutant (lower panel). The position of the mutation is indicated. The mutant sequence appears to be homozygous with complete absence of normal sequence. This is because of the use of a “clamp” strategy to suppress amplification of the normal sequence, as described in the methods section. B Immunohistochemical staining with E746-A750 del-specific antibody showing strong positivity (original magnification x200). C Immunohistochemical staining with L858R-specific antibody showing complete negativity (original magnification x200).