Elsevier

Lung Cancer

Volume 77, Issue 2, August 2012, Pages 299-305
Lung Cancer

Use of mutation specific antibodies to detect EGFR status in small biopsy and cytology specimens of lung adenocarcinoma

https://doi.org/10.1016/j.lungcan.2012.04.004Get rights and content

Abstract

Background

EGFR mutation status is the best predictor of response to tyrosine kinase inhibitors (TKIS) in primary lung adenocarcinoma. Approximately 70% of lung cancers are diagnosed in advanced stages where small biopsies and cytological specimens are the only source of material for both diagnosis and mutation testing. Specific antibodies that can detect mutant EGFR protein were evaluated for the detection of EGFR mutation by immunohistochemistry (IHC) in cytology and small biopsy specimens.

Methods

Assessment of EGFR mutation status was performed by using antibodies specific to the two major forms of mutant EGFR, exon 21 L858R and exon 19 deletion (15 bp). The study was performed in 145 lung adenocarcinomas, including cytology material, core biopsy, and decalcified bone biopsy. Stains were scored as negative (0), 1+ (weak and focal), 2+ (moderate intensity and focal), and 3+ (strong and diffuse). The result of the IHC stains was correlated with mutations status determined by standard molecular methods.

Results

Validation using clinical material showed deletions in exon 19 were detected in 35% and L858R mutation in 17.6% of all cases by standard molecular methods. A cutoff value of 2+ was used as positive by IHC. No wild type cases were immunoreactive. The positive predictive value (PPV) and specificity for both antibodies was 100%. The antibodies performed well in cytology, core biopsies and decalcified bone biopsies.

Conclusion

Immunostaining to detect specific mutant EGFR shows a good correlation with mutation analysis and can be used as a screening method to identify patients for TKI therapy. IHC methodology is potentially useful when molecular analysis is not available and for use in small biopsies when material is too scant for molecular tests. Importantly mutation specific antibodies are useful in determining EGFR status in tissues obtained from bone biopsy as decalcification processes used in molecular based studies often result in DNA degradation hindering mutation detection.

Introduction

Lung cancer presents a major public health problem and is responsible for the largest number of cancer related deaths worldwide for both men and women, causing approximately 1.2 million deaths per year. In the United States alone, there are more than 200,000 new lung cancer cases diagnosed per year with more than 150,000 deaths [1], [2].

The discovery that activating mutations in the epidermal growth factor receptor (EGFR) are associated with response to treatment with the small molecule tyrosine kinase inhibitors (TKI) has revolutionized the field of thoracic oncology [3], [4], [5]. Gefitinib and erlotinib, two TKI drugs approved for the treatment of patients with lung adenocarcinoma have been shown to significantly extended progression free and overall survival time in patients that harbor activating EGFR mutations [6], [7], [8]. The presence of mutation is closely associated with the histological diagnosis of adenocarcinoma, but not other histological subtypes. The overall incidence of EGFR mutations varies by gender, ethnicity and smoking status [9], [10], [11].

The two most common EGFR mutations associated with adenocarcinoma are in-frame deletions in exon 19 (the most common E746-A750 15-bp deletion) and the point mutation replacing leucine with arginine at codon 858 in exon 21 (L858R). These two mutations are responsible for 90% of the EGFR mutations in lung adenocarcinoma patients [12]. EGFR mutational analysis in lung adenocarcinoma is used to guide treatment decisions, to aid in selecting therapy and to stratify patients for clinical trials [13]. Direct DNA sequencing and PCR based assays have been used as methods for detection of EGFR mutations in tumor tissue [14]. Mutation testing in the clinical setting has become standard of care [15]. Nonetheless, technical complexity, and relative high cost of the test have challenged the wide-spread use of molecular techniques in everyday clinical settings. As a result, many patients that could benefit from targeted therapy with EGFR-TKI inhibitors are not tested.

Most patients with pulmonary carcinoma are diagnosed with advanced disease, and are not candidates for surgical based therapy. In many cases, cytology, small biopsy material or samples from metastatic site may be the only tissue available for diagnostic, prognostic and predictive testing.

Recently we have shown that EGFR mutational status can be accurately determined in small biopsy material including cytology fine needle aspiration biopsies (FNA) [16]. Some of these limited samples, however, fail molecular testing mostly due to scant cellularity. In addition, molecular analysis of bone metastasis is difficult due to the decalcification process that negatively influences the quality of DNA [17], [18]. Recently two monoclonal antibodies, specific to the two most common forms of mutated EGFR protein have become commercially available [19]. Early studies have shown promising results regarding the use of these antibodies in screening patients for TKI therapy [20], [21], [22], [23], [24], [25], [26], [27], [28].

In our current study we report our experience with these new antibodies in identifying mutant EGFR protein in cytology and small biopsy specimens, including decalcified bone biopsies. The results of mutation detection using immunohistochemistry were correlated to results obtained by molecular testing.

Section snippets

Specimen selection

This study was done in two steps; first the antibodies were tested retrospectively in a research setting, and later, validated in a clinical setting. Initially, 94 cases were identified by searching for small biopsies and cytology cases that corresponded to resection material already analyzed in the Diagnostic Molecular Laboratory. Material used included needle biopsies, cytology cell blocks and Thin-prep cytology fluids. All cases were diagnosed as adenocarcinoma both in the surgical and in

Results

The samples in our study were enriched for EGFR mutant cases in order to provide adequate evaluation of the sensitivity of the mutant EGFR protein specific antibodies. EGFR mutations determine by standard molecular analysis were present in 87% (82/94) in the initial research evaluation and in 53% (27 of 51) in clinical/validation cases, compared with a frequency of approximately 20% in adenocarcinoma samples found in routine testing [30], [31].

Discussion

Approximately 20% of North American and European patients and 40% of Asian patients with adenocarcinoma of the lung harbor EGFR mutations [30]. Molecular testing of patients with adenocarcinoma for selection of specific therapy is standard of care in clinical practice.

Mutation testing has been shown to be cost-efficient for first line treatment option in patients with pulmonary adenocarcinoma [32]. Although molecular based mutational analysis of EGFR is the mainstay of current laboratory

Conflict of interest

None declared.

Acknowledgments

The author's thank Miriam Fayad and Marina Ascher for immunohistochemical technical support.

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