A diagnostic algorithm using EGFR mutation-specific antibodies for rapid response EGFR-TKI treatment in patients with...

We previously reported that EGFR mutation-specific antibodies performed well in immunohisto/cytochemical analysis. Assessment of EGFR mutation status for EGFR-TKIs (tyrosin kinase inhibitors) treatment of non-small cell lung cancer (NSCLC) patients can be performed by DNA-based assay. To establish a testing algorithm for EGFR mutation status in NSCLC patients, we utilized the peptide nucleic acid-locked nucleic acid (PNA-LNA) PCR clamp assay to determine the EGFR mutation, and immunostaining to detect the delE746-A750 and L858R mutation protein.

One hundred thirty-three patients with NSCLC were examined by immunostaining with EGFR mutation-specific antibodies, and by the PNA-LNA PCR clamp assay, using histological or cytological samples. The expression levels of immunostaining were classified as positive (score 2+), negative (score 0) or equivocal (score 1+), indicating questionable, negative or weak expression. Of these patients, 42 NSCLC patients received EGFR-TKIs treatment and we analyzed whether expression of mutant EGFR proteins was correlated with progression-free survival in patients with NSCLC treated with EGFR-TKIs.

In the 133 samples, positive, negative and equivocal results for EGFR mutation-specific antibodies were observed in 37 patients (27.8%), 85 patients (63.9%) and 11 patients (8.3%), respectively. Thirty-seven patients showed positive EGFR expression by immunostaining, and 35 (94.6%) of these also tested positive in the DNA-based assay. Of 85 EGFR-negative patients by immunostaining, 77 (90.6%) also tested negative in the DNA-based assay. Of the 11 patients with equivocal immunostaining results, DNA-based assay detected EGFR mutation in 8 (72.7%) patients. Immunostaining for the EGFR mutation-specific antibodies showed a sensitivity of 81.4%, specificity of 97.5%, positive predictive value of 94.6% and negative predictive value of 90.6%, excluding the 11 patients with equivocal results. In NSCLC patients treated with EGFR-TKIs, the progression-free survival after the start of EGFR-TKIs treatment was significantly longer in patients with EGFR positive expression than in those with equivocal and negative expression (P=0.002).

Our results suggest that patients testing positive for EGFR mutations by immunostaining are good candidates for rapid response EGFR-TKI therapy. Immunostaining for EGFR mutation-specific antibodies is a new test for EGFR-TKI inhibition and could be a useful indicator with regard to patient management.