The usefulness of mutation-specific antibodies in detecting epidermal growth factor receptor mutations and in predicting response to tyrosine kinase inhibitor therapy in lung adenocarcinoma
Introduction
Many human receptor tyrosine kinases mediate signals that promote the proliferation and survival of cancer cells. Activation of tyrosine kinases appears to be the causal event in many human malignancies [1]. The importance of this finding is reflected in the development of new anticancer drugs that specifically target these activated proteins. The clinical success of tyrosine kinase inhibitors (TKIs), such as imatinib for the treatment of chronic myeloid leukemia and gastrointestinal stromal tumors, has prompted intensive efforts to identify and target additional oncogene kinases as a broad therapeutic strategy for selected patient populations [2], [3].
A subset of non-small cell lung cancer (NSCLC), particularly adenocarcinomas, has activating mutations in the epidermal growth factor receptor (EGFR) gene [4], [5]. The most prevalent EGFR mutations are deletions in exon 19 (DEL) and a point mutation at codon 858 in exon 21 (L858R); together, these account for more than 90% of all EGFR mutations. These 2 types of EGFR mutations cause sustained activation of EGFR, followed by the selective activation of Akt and signal transduction, and the activation of transcription signaling pathways: altogether, these promote cell survival [4], [6].
EGFR-TKIs are competitive inhibitors of the adenosine triphosphate-binding clefts within the tyrosine kinase domain of EGFR [7]; they effectively inhibit the critical antiapoptotic signals transduced by the mutant receptors [6]. The clinicopathologic parameters of female gender, East Asian ethnicity, adenocarcinoma histology, and nonsmoking status are strong predictors of the response to EGFR-TKIs [4], [5], [8], [9]. Moreover, the DEL and L858R mutations were also revealed to be strong predictors [10], [11], [12], [13], [14]. Therefore, the detection of such mutations provides both patients and physicians with important information regarding the optimal choice for therapy.
Direct sequencing is the gold standard method to detect EGFR mutations. However, to obtain precise data, high-quality DNA extracted from an adequate amount of pure tumor cells is required, and this is expensive and time-consuming. Recently, other indirect methods were developed to detect EGFR mutations, including Scorpion ARMS, the peptide nucleic acid-locked nucleic acid PCR clamp, mutant-enriched PCR, the smart amplification process, and high-resolution melting analysis (HRMA) [15], [16]. These methods have high sensitivities, and can be applied to specimens in which cancer cell content is low. However, they invariably require technical labor and sophisticated instruments, and are therefore, not applied in most pathology laboratories.
Compared to molecular techniques, immunohistochemistry (IHC) is a fast and cost-effective method that can be performed in most pathology laboratories on not only fresh, but also archival, formalin-fixed tissue samples. Recently, some authors revealed the correlation between EGFR mutations and EGFR phosphorylation detected by IHC [17], [18]. Additionally, EGFR phosphorylation antibodies exhibited a correlation with response to EGFR-TKIs [18]. However, these antibodies recognize EGFR phosphorylation regardless of mutational status. More recently, highly sensitive and specific rabbit monoclonal antibodies against the 2 most common mutations were developed for detecting EGFR mutations [19], [20], [21], [22], [23], [24].
The main purpose of the present study was to explore the use of the 2 mutation-specific antibodies for DEL and L858R for detecting EGFR mutations. Additionally, we compared the molecular-based and the IHC-based EGFR mutational status to the response to EGFR-TKI.
Section snippets
Case selection
After obtaining institutional review board approval, the specimens used in the present study were obtained from 577 Japanese patients who underwent a surgical resection for primary lung adenocarcinoma at the National Cancer Center Hospital, Tokyo, Japan, between 1993 and 2009. Histological diagnosis was based on the latest World Health Organization classification of lung tumors [25].
Analysis of EGFR mutational status by molecular technique
The materials analyzed for the molecular-based mutational status were as follows: fresh frozen (in liquid
Clinicopathologic parameters
There were 319 males and 258 females with median age at surgery being 60 years (range, 30–82). A total of 343 patients had never/light smoking status with Brinkman index of <400, and 234 patients had smoking status with Brinkman index of ≥400. The pathological tumor stage (p-stage) was I in 331, II in 74, III in 164, and IV in 8 cases.
Molecular-based EGFR mutational status
After analyzing the EGFR mutational status by HRMA, DEL (m-DEL) was detected in 135 cases (23%), and L858R (m-L858R) was detected in 172 cases (30%). The
Discussion
In the present study, we investigated the clinical usefulness of IHC using 2 rabbit monoclonal antibodies against specific mutant EGFRs in lung adenocarcinomas. We found that the IHC-based EGFR mutational status detected by these antibodies was significantly correlated with the molecular-based EGFR mutational status. Furthermore, the IHC-based mutational status showed a significant correlation with the clinical response of tumors in conjunction with EGFR-TKI therapy.
The overall specificity of
Conflict of interest statement
All authors have no financial or personal relationship with other people or organization that could inappropriately influence our work.
Acknowledgements
We would like to thank Ms. Sachiko Miura, Ms. Chizu Kina, and Ms. Karin Yokozawa for their skillful technical assistance.
This work was supported in part by a Grant-in-aid for the Third-Term Comprehensive 10-Year Strategy for Cancer Control from the Ministry of Health, Labor and Welfare of Japan (J.Y., H.T), and a Grant-in-Aid for Cancer Research from the Ministry of Health, Labor and Welfare of Japan (J.Y., H.T.).
References (36)
- et al.
Immunohistochemical analysis of phosphorylated epidermal growth factor receptor might provide a surrogate marker of EGFR mutation
Lung Cancer
(2009) - et al.
Phosphorylation status of epidermal growth factor receptor is closely associated with responsiveness to gefitinib in pulmonary adenocarcinoma
Hum Pathol
(2008) - et al.
Assessment of EGFR mutation status in lung adenocarcinoma by immunohistochemistry using antibodies specific to the two major forms of mutant EGFR
J Mol Diagn
(2010) - et al.
Novel epidermal growth factor receptor mutation-specific antibodies for non-small cell lung cancer: immunohistochemistry as a possible screening method for epidermal growth factor receptor mutations
J Thorac Oncol
(2010) - et al.
The 2004 World Health Organization classification of lung tumors
Semin Roentgenol
(2005) - et al.
Effect of fixatives and tissue processing on the content and integrity of nucleic acids
Am J Pathol
(2002) - et al.
Tissue heterogeneity of EGFR mutation in lung adenocarcinoma
J Thorac Oncol
(2008) - et al.
Switching on kinases: oncogenic activation of BRAF and the PDGFR family
Nat Rev Cancer
(2004) - et al.
Efficacy and safety of imatinib mesylate in advanced gastrointestinal stromal tumors
N Engl J Med
(2002) - et al.
Efficacy of imatinib dose escalation in patients with chronic myeloid leukemia in chronic phase
Cancer
(2009)
Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib
N Engl J Med
EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy
Science
Gefitinib-sensitizing EGFR mutations in lung cancer activate anti-apoptotic pathways
Science
ZD1839 (Iressa): an orally active inhibitor of epidermal growth factor signaling with potential for cancer therapy
Cancer Res
Biological and clinical implications of EGFR mutations in lung cancer
Int J Clin Oncol
Multi-institutional randomized phase II trial of gefitinib for previously treated patients with advanced non-small-cell lung cancer (The IDEAL 1 Trial) [corrected]
J Clin Oncol
Predictive and prognostic impact of epidermal growth factor receptor mutation in non-small-cell lung cancer patients treated with gefitinib
J Clin Oncol
Predictors of the response to gefitinib in refractory non-small cell lung cancer
Clin Cancer Res
Cited by (67)
Molecular testing in lung cancer
2023, Diagnostic Molecular Pathology: A Guide to Applied Molecular Testing, Second EditionMolecular Testing in Lung Cancer
2018, IASLC Thoracic OncologyImmunohistochemistry for EGFR Mutation Detection in Non–Small-Cell Lung Cancer
2017, Clinical Lung CancerEGFR mutations in lung cancer: A morphological, immunohistochemical and molecular study of lung adenocarcinoma
2016, Revista Espanola de PatologiaMolecular Testing in Lung Cancer
2016, Diagnostic Molecular Pathology: A Guide to Applied Molecular Testing