Including Total EGFR Staining in Scoring Improves EGFR Mutations Detection by Mutation-Specific Antibodies and EGFR TKIs Response Prediction

Shang-Gin Wu,Chong-Jen Yu,Jin-Yuan Shih,Yung-Chie Lee,Meng-Feng Tsai,Yih-Leong Chang,Hsuan-Yu Chen,Jou-Wei Lin,Chen-Tu Wu,Alfons Navarro

doi:10.1371/journal.pone.0023303

Abstract

Epidermal growth factor receptor (EGFR) is a novel target for therapy in subsets of non-small cell lung cancer, especially adenocarcinoma. Tumors with EGFR mutations showed good response to EGFR tyrosine kinase inhibitors (TKIs). We aimed to identify the discriminating capacity of immunohistochemical (IHC) scoring to detect L858R and E746-A750 deletion mutation in lung adenocarcinoma patients and predict EGFR TKIs response. Patients with surgically resected lung adenocarcinoma were enrolled. EGFR mutation status was genotyped by PCR and direct sequencing. Mutation-specific antibodies for L858R and E746-A750 deletion were used for IHC staining. Receiver operating characteristic (ROC) curves were used to determine the capacity of IHC, including intensity and/or quickscore (Q score), in differentiating L858R and E746-A750 deletion. We enrolled 143 patients during September 2000 to May 2009. Logistic-regression-model-based scoring containing both L858R Q score and total EGFR expression Q score was able to obtain a maximal area under the curve (AUC: 0.891) to differentiate the patients with L858R. Predictive model based on IHC Q score of E746-A750 deletion and IHC intensity of total EGFR expression reached an AUC of 0.969. The predictive model of L858R had a significantly higher AUC than L858R intensity only (p = 0.036). Of the six patients harboring complex EGFR mutations with classical mutation patterns, five had positive IHC staining. For EGFR TKI treated cancer recurrence patients, those with positive mutation-specific antibody IHC staining had better EGFR TKI response (p = 0.008) and longer progression-free survival (p = 0.012) than those without. In conclusion, total EGFR expression should be included in the IHC interpretation of L858R. After adjusting for total EGFR expression, the scoring method decreased the false positive rate and increased diagnostic power. According to the scoring method, the IHC method is useful to predict the clinical outcome and refine personalized therapy.

Highlights

Epidermal growth factor receptor (EGFR), a member of the ErbB family, is a transmembrane glycoprotein [1]
Informed consent about the use of these specimens for future molecular studies was obtained before surgery after approval of the Institutional Review Board (IRB). The paraffin-embedded tissues were collected for EGFR sequencing and IHC staining of EGFR mutation-specific antibodies
This is the first study to demonstrate the influence of total EGFR expression on the IHC interpretation of mutation-specific antibodies, especially L858R, and compare different IHC scoring methods of mutation-specific antibodies by statistical analysis

Summary

Introduction

Epidermal growth factor receptor (EGFR), a member of the ErbB family, is a transmembrane glycoprotein [1]. Non-small cell lung cancer (NSCLC) patients with EGFR mutations have had a dramatic response to EGFR tyrosine kinase inhibitors (EGFR TKIs) [2,3]. The patients who have shown a good response to EGFR TKIs have been mainly from particular groups, including female, adenocarcinoma histology, non-smokers and Asian ethnicity [3,4,5]. 90% of EGFR mutation types have been found to be a point mutation of L858R in exon 21 and an in-frame deletion in exon 19 (Del-19), especially the E746-A750 deletion [6]. They are the most well-known EGFR TKI sensitive mutations and are known as ‘‘classical mutations’’. For EGFR mutation analysis, different molecular techniques such as direct DNA sequencing and scorpion amplified refractory mutation systems (ARMS) have been used [7], but they are time-consuming, expensive and complicated, and not routinely used in general hospitals or clinical laboratories

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