The studies that established the relationship between mutations in the EGFR gene and response to the small molecule EGFR TKIs gefitinib and erlotinib were done using analysis of DNA extracted from the tumor
. The recent availability of antibodies that are specific for the mutations most clearly associated with response to EGFR TKIs, L858R and E746_A750del, create the opportunity to exploit an alternative method to evaluate NSCLC for EGFR mutations to aid decisions with regard to EGFR TKI therapy
IHC has the advantage of being a method that is routinely applied in solid tumor diagnosis in pathology. Also, it can be used on specimens that are not optimal for DNA analysis such as small tissue samples or individual cells obtained from body fluids, bronchial washings, and fine needle aspirates. Although some studies have shown that EGFR gene sequencing could be successfully applied to cytological specimens
, it is still a problem to get enough DNA for sequencing from such samples in routine practice. Thus, the development of antibodies that specifically detect mutant EGFR protein by IHC could be a valuable addition to the current methods used to diagnose and predict response to treatment of lung cancer.
In 2009, Yu et al.
 reported generating two mAbs from New Zealand rabbits, one against the E746_A750del and the other against the L858R point mutation, and evaluated them by Western blotting, immunofluorescence and IHC. They tested these antibodies in a series of cell lines and in tumor tissues from patients with primary NSCLC, with known and unknown EGFR mutations, comparing the IHC results with DNA sequencing. They found that IHC with these mutation-specific antibodies showed a sensitivity of 92% and a specificity of 99%. Recently, several studies examined the presence of EGFR mutations in NSCLC by IHC using the same two antibodies and the reported sensitivity ranged from 24% to 100% and specificity ranged from 77% to 100%
[8–15]. IHC is known to sometimes suffer from high inter-laboratory variability in assay performance, and high inter-observer variability in assay interpretation. These drawbacks may explain the variability in results of the studies described above. There is still much work to be done before IHC can be considered an adequate substitute for direct analysis of mutations in the EGFR gene in NSCLC.
In our study we found that slides treated by EDTA (pH 8.0) showed the best histological pictures with strongly specific staining and minimal background. As a result when pathologists reviewed these slides the intra- and inter-observer agreement was better than those treated by sodium citrate (pH 6.0) and EDTA (pH 9.0). The difference was statistically significant (P < 0.05). In conclusion, EDTA (pH 8.0) is the preferred buffer for antigen retrieval for IHC using EGFR mutation specific antibodies.
Scoring is the final step involved in the IHC protocol, but is not the least one, because the scoring system plays a critical role in obtaining a reliable result. In our study, we compared three scoring systems that have been used by other investigators, using DNA sequencing as the gold standard
[10, 13, 15]. For L858R-specific IHC the agreement with DNA sequencing using Score A was superior to Score B and C. The difference was statistically significant. For E746_A750del-specific IHC the agreement with DNA sequencing was good for Score A, which was superior to Score B and C, but the difference was not statistically significant. In conclusion our study showed that Score A is the most appropriate way to interpret the EGFR mutation-specific IHC.
Based on Score A the specificity of EGFR mutation-specific IHC was very high, 100% for exon 19 deletions and 97% for L858R, while sensitivity was lower, 81% for L858R and 59% for exon 19 deletions. In another words, mutation-specific IHC demonstrated extremely high specificities, but much lower sensitivity. The low sensitivity of the exon 19 del IHC is mostly due to the presence of several exon 19 deletions other than the most common E746_A750del, which is the target of the exon 19 del antibodies. We conclude, based on our work, that NSLC cases positive by IHC could be selected as candidates for EGFR-TKI, while negative cases should be referred for further testing by DNA analysis.
In our study the majority of cases were either negative or positive in 100% of the tumor cells. The staining pattern showed characteristics of homogeneity more than heterogeneity. Consequently, we expect that evaluation of the mutation status by IHC should be reliable using small biopsy specimens or tissue microarray.
Our study also showed that all of the normal alveolar epithelial cells were completely negative and the intensity of mutation-specific immunostaining was much fainter in tumor cells with a lepidic pattern comparing to other patterns. This demonstrates that the EGFR mutations are tumor-specific, and likely an initiating event in lung cancer tumorigenesis.