Detection of EGFR Mutations in Lung Adenocarcinoma by Immunohistochemistry Using Mutant Specific Antibodies: Are We There Yet?
Komal Arora, Wei Zhang, Junya Fukuoka, Haruhisa Kitano, Jaishree Jagirdar. University of Texas Health Science Centre, San Antonio, TX; University of Toyama, Toyama, Japan; National Institutes of Health, Bethesda, MD
Background: Somatic mutations of the EGFR tyrosine kinase are the most prevalent and well studied in lung carcinoma. E746-A750 deletion in exon 19 and L858 point mutation in exon 21 account for ∼90% of these mutations. Immunohistochemistry (IHC) for EGFR mutation using mutant specific antibodies was studied recently in lung carcinoma and found to be ∼90% sensitive, thus allowing use as a screening tool.
Design: We evaluated the IHC expression of the two mutant specific EGFR antibodies in 38 cases of lung adenocarcinoma on a tissue microarray obtained from Japan. Ten cases had exon 19 mutation, 8 cases had exon 21 mutation, 1 case had both exon 19 and exon 21 mutation and 1 case had exon 18 mutation by PCR. In 18 cases no mutation was detected. Antibodies used included L858R specific for the exon 21 and E746-750 specific deletion for exon 19 EGFR mutation (Cell Signaling). The method followed for performing IHC was exactly same as the one previously published (antigen retrieval using EDTA pH9(Dako) for 30 minutes, dilution 1:100 for primary antibodies, and the EnVision+kit (Dako) for detection). In addition, various dilutions of primary antibody at 1:50 and 1:25 were used. Depending upon the intensity of cytoplasmic staining, cases were scored as 0, 1+, 2+ and 3+. Staining intensity of 0 to 1+ was counted negative and 2+ to 3+ was counted positive.
Results: Results are shown in Table 1. Using 1:100 dilution, the detection rate was 0% with both the antibodies. Using 1:50 dilutions the detection rate was 9% for exon 19 and 0% for exon 21 mutation. Using 1:25 dilution the detection rate was 73% for exon 19 with 22% false positivity and 11% for exon 21 with no false positivity.
|Primary antibody||Dilution||No Staining||Positive staining (2+ and 3+)|
|(0 -1+ )||True Positive||False Positive|
|Exon 19||1:100||11/11||0/11(0%)||0/27 (0%)|
|1:50||10/11||1/11 (9%)||0/27 (0%)|