Original StudyImmunohistochemistry for EGFR Mutation Detection in Non–Small-Cell Lung Cancer
Introduction
For decades, platinum-based chemotherapy was the only systemic therapy for advanced non–small-cell lung cancer (NSCLC) resulting in a poor median overall survival (OS) of only 8 to 10 months.1 The discovery of activating epidermal growth factor receptor (EGFR) mutations in 2004 posed the first milestone for treatment improvement in patients with advanced NSCLC. EGFR mutations were the first discovered oncogene driver in NSCLC against which very effective targeted therapy with EGFR tyrosine kinase inhibitors (EGFR TKIs) has been developed.2, 3 Treatment with EGFR TKIs has doubled the response rates, significantly prolonged progression-free survival (PFS), and impressively extended OS up to nearly 3 years as compared with platinum-based chemotherapy in patients with advanced EGFR mutation–positive NSCLC.4 Based on this, EGFR mutation testing and first-line therapy with EGFR TKIs for patients who are EGFR mutation–positive are now becoming a standard for all patients with advanced lung cancer with non–squamous-cell carcinoma (NSCC) histology.5
Activating EGFR mutations are most frequently yet not exclusively detected in adenocarcinomas, nonsmokers, female patients, and in individuals of Asian ethnicity.3 They are mutually exclusive with some other gene alterations, such as ALK gene rearrangements and KRAS mutations. In White populations, activating EGFR mutations are found in approximately 15% of patients with NSCC histology, whereas in the Asian population their frequency is much higher.6 The most common activating EGFR mutations are deletions in exon 19 and point mutation in exon 21 (L858R), which represent approximately 90% of all activating EGFR mutations, thus being often referred to as common mutations.7 Most pivotal clinical trials confirming efficacy of EGRF TKIs in the first-line setting included only patients with common EGFR mutations.4 So-called activating mutations were found also in exon 18 and exon 20; however, those mutations are less common and predict only for a minor clinical response to currently used EGFR TKIs.8 Although there is only one detectable sensitizing mutation in exon 21 (point mutation L858R), there are multiple known deletions in exon 19, with specific deletion of 15 base pairs (ie, E746-A750 deletion [15bp-del19]), representing approximately 70% of all exon 19 deletions.9, 10
Several polymerase chain reaction (PCR)-based methods are available and are accepted as standard methods for EGFR mutation detection, with a wide range of sensitivity between them.6, 7, 11 Although direct sequencing allows for detection of a wide spectrum of EGFR mutations, it is a time-consuming procedure that requires large tumor samples.12 Recently developed, novel next-generation sequencing (NGS) enables highly sensitive and comprehensive molecular assessment of numerous predefined genetic alterations, including EGFR mutations (common and others) in a small tumor tissue sample. However, this technology is technically complex, labor demanding, and costly, thus making it often inaccessible in a real-world setting. To overcome these shortages, commercially available EGFR mutation detection kits were developed and are most widely used to detect a predefined set of common EGFR mutations, including exon 21 L858R mutation and various exon 19 deletions. Of note, most of those commercially available kits, such as Therascreen EGFR PCR Kit (Qiagen, Manchester, UK) used in the present work, cannot discriminate between different exon 19 deletions.
Immunohistochemistry (IHC) has been previously researched in EGFR mutation testing. It is an easy-to-access method routinely used in solid tumor diagnostics in all pathology laboratories that is relatively inexpensive and does not require a large amount of tumor tissue for biomarker determination. However, a major shortage of the IHC method for EGFR mutation detection in NSCLC comes from the fact that we are searching for multiple activating EGFR mutations, thus requiring multiple antibodies and more tissue. In 2009, the mutation-specific rabbit monoclonal antibodies against L858R mutation and against the most frequent deletion in exon 19 (15bp-del19) (clones 6B6 and 43B2 from Cell Signaling Technology, Inc, Danvers, MA, and SP125 and SP111 from Ventana Medical Systems, Tucson, AZ), have been developed. Shortly after that, Yu et al13 demonstrated a high sensitivity (92%) and specificity (99%) of these antibodies in recognizing the common EGFR mutations. However, several consequent studies that aimed to validate the sensitivity and specificity of those 2 mutant-specific antibodies in various tumor specimens of patients with NSCLC, with various cutoff scoring systems, failed to demonstrate such a high level of accuracy of those antibodies, with observed sensitivity ranging from 30% to 100% and specificity of approximately 90%.9, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38 Based on these data, IHC has not replaced conventional DNA sequencing PCR methods in EGFR mutation testing in NSCLC. However, according to the observations provided by Houang et al,22 Hasanovic et al,19 and Ragazzi et al,32 IHC can represent an adjunct to DNA sequencing in cases of small tumor samples with poor material and in patients requiring rapid therapy based on EGFR mutation status determination. In their series, EGFR mutation–specific antibodies performed equally well on small biopsies or on cytology samples.
The aim of the present study was to evaluate the sensitivity and specificity of IHC with commercially available monoclonal antibodies against common EGFR mutations compared with the routinely used PCR method. In addition, the predictive value of IHC EGFR mutation–positive status for EGFR TKI treatment outcome was studied, and a cost-effectiveness decision model for the upfront IHC testing was developed.
Section snippets
Patient Selection
The study included 79 consecutive PCR EGFR mutation–positive, histology-confirmed NSCC cases, harboring deletions in exon 19 and L858R mutations, as well as 29 EGFR mutation–negative NSCC cases, diagnosed at the University Clinic Golnik from October 2009 to December 2011. Reflex PCR-based EGFR mutation testing with a commercially available kit (Therascreen EGFR PCR Kit; Qiagen, Manchester, UK) was performed on all NSCC histology samples as a diagnostic procedure valid at our clinic in the
Sensitivity and Specificity of IHC-based Method
Results on sensitivity, specificity, NPV, and PPV of IHC-determined EGFR mutation status are summarized in Table 2.
As for exon 19 deletions, a positive IHC staining reaction was observed in 25 of 37 cases harboring exon 19 deletions detected by PCR method and in none of the EGFR mutation–negative or L858R-positive cases detected by PCR method. Sensitivity, specificity, PPV, and NPV of IHC testing for exon 19 deletions were 67.7% (95% confidence interval [CI] 50.1-81.4), 100% (95% CI 93.6-100),
Discussion
In our study, a good overall correlation between the IHC and PCR determined exon 19 deletions and L858R EGFR mutation status was found, with an overall IHC sensitivity of 84.8% and IHC specificity of 100% in a cohort of White patients with NSCLC. For L858R, excellent 100% sensitivity and 100% specificity were observed, whereas for exon 19 deletions, sensitivity of 67.6% and specificity of 100% were recorded. A lower specificity of IHC testing for exon 19 deletions was not surprising, because
Disclosure
The authors have stated that they have no conflicts of interest.
Acknowledgments
The authors thank the colleagues from the Department of Pulmonary Diseases at University Medical Center Maribor for their valued collaboration and sharing of clinical data. We also thank Mitja Rot for his extensive laboratory support. This work was financially supported by the Slovenian Research Agency (ARRS J3-4076).
References (45)
- et al.
2nd ESMO Consensus Conference on Lung Cancer: non-small-cell lung cancer first-line/second and further lines of treatment in advanced disease
Ann Oncol
(2014) - et al.
Second ESMO consensus conference on lung cancer: pathology and molecular biomarkers for non-small-cell lung cancer
Ann Oncol
(2014) - et al.
Molecular testing guideline for selection of lung cancer patients for EGFR and ALK tyrosine kinase inhibitors: guideline from the College of American Pathologists, International Association for the Study of Lung Cancer, and Association for Molecular Pathology
J Thorac Oncol
(2013) - 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.
High specificity but low sensitivity of mutation-specific antibodies against EGFR mutations in non-small-cell lung cancer
Mod Pathol
(2014) - et al.
Immunostaining with EGFR mutation-specific antibodies: a reliable screening method for lung adenocarcinomas harboring EGFR mutation in biopsy and resection samples
Hum Pathol
(2013) - et al.
Use of mutation specific antibodies to detect EGFR status in small biopsy and cytology specimens of lung adenocarcinoma
Lung Cancer
(2012) - et al.
Immunohistochemistry to identify EGFR mutations or ALK rearrangements in patients with lung adenocarcinoma
Ann Oncol
(2012) - et al.
EGFR mutation specific immunohistochemistry is a useful adjunct which helps to identify false negative mutation testing in lung cancer
Pathology
(2014) - 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)
Identification of non-small-cell lung cancer with activating EGFR mutations in malignant effusion and cerebrospinal fluid: rapid and sensitive detection of exon 19 deletion E746-A750 and exon 21 L858R mutation by immunocytochemistry
Lung Cancer
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
Lung Cancer
Novel EGFR mutation-specific antibodies for lung adenocarcinoma: highly specific but not sensitive detection of an E746_A750 deletion in exon 19 and an L858R mutation in exon 21 by immunohistochemistry
Lung Cancer
Effusion immunocytochemistry as an alternative approach for the selection of first-line targeted therapy in advanced lung adenocarcinoma
J Thorac Oncol
New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1)
Eur J Cancer
Association of the expression of mutant epidermal growth factor receptor protein as determined with mutation-specific antibodies in non-small cell lung cancer with progression-free survival after gefitinib treatment
J Thorac Oncol
Analysis of the effect of various decalcification agents on the quantity and quality of nucleic acid (DNA and RNA) recovered from bone biopsies
Ann Diagn Pathol
Comparison of four chemotherapy regimens for advanced non-small-cell lung cancer
N Engl J Med
Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib
N Engl J Med
EGF receptor gene mutations are common in lung cancers from “never smokers” and are associated with sensitivity of tumors to gefitinib and erlotinib
Proc Natl Acad Sci U S A
Epidermal growth factor receptor tyrosine kinase inhibitors vs conventional chemotherapy in non-small cell lung cancer harboring wild-type epidermal growth factor receptor: a meta-analysis
JAMA
Effectiveness of tyrosine kinase inhibitors on “uncommon” epidermal growth factor receptor mutations of unknown clinical significance in non-small cell lung cancer
Clin Cancer Res
Cited by (11)
Advanced non-small cell lung cancer: Rapid evaluation of EGFR status on fine-needle cytology samples using Idylla
2021, Pathology Research and PracticeCitation Excerpt :Therefore, a fast diagnosis and subsequent assessment of EGFR status, when requested, are crucial [11]. Since the introduction of tyrosine kinase inhibitors (TKIs) for the treatment of different tumors, including lung carcinoma, immunohistochemistry (IHC) was used to assess the EGFR status of corresponding tumors [12]. IHC is a simple, cheap and sensitive procedure but is not sufficiently specific for EGFR assessment; in fact IHC demonstrated a good specificity for common mutations only (L858R and exon 19 deletion) and not for all the remaining ones [13] In this study, diagnosis, and rapid evaluation of EGFR in patients with advanced NSCLC was performed by FNAC, ROSE and using the fully automated RT-PCR Idylla.
Recent advances in lung cancer genomics: Application in targeted therapy
2021, Advances in GeneticsCitation Excerpt :EGFR overexpression by IHC doesn't correlate with response to anti-EGFR treatment and should not be used as a predictive biomarker. Mutation specific antibodies against the most common EGFR mutations namely E746-A750 del (clone SP111) and L858R (clone SP125) have been shown to have good concordance with standard PCR based methods and acceptable sensitivity and specificity (84.8% and 100%) (Hitij et al., 2017; Jain et al., 2016). Use of mutation specific IHC to detect EGFR mutations is mostly restricted to screening in certain clinical situations.
Texture Analysis of Enhanced MRI and Pathological Slides Predicts EGFR Mutation Status in Breast Cancer
2022, BioMed Research InternationalEpidermal Growth Factor Receptor Gene Mutation Detection in Histology and Cytology Specimens of Primary Lung Adenocarcinoma: Immunohistochemistry Versus the Molecular Method
2021, Asian Pacific Journal of Cancer PreventionPrecision treatment for metastatic non-small cell lung cancer: A conceptual overview
2021, Cleveland Clinic Journal of Medicine