Elsevier

Clinical Lung Cancer

Volume 18, Issue 3, May 2017, Pages e187-e196
Clinical Lung Cancer

Original Study
Immunohistochemistry for EGFR Mutation Detection in Non–Small-Cell Lung Cancer

https://doi.org/10.1016/j.cllc.2016.11.021Get rights and content

Abstract

Introduction

The sensitivity and specificity of immunohistochemistry (IHC) was compared with the standard polymerase chain reaction (PCR)-based method for detecting common activating epidermal growth factor receptor (EGFR) mutations in non–small-cell lung cancer (NSCLC). Additionally, we evaluated predictive value of IHC EGFR mutation–positive status for EGFR tyrosine kinase inhibitor (TKI) treatment outcome and estimated cost-effectiveness for the upfront IHC testing.

Methods

The trial included 79 consecutive EGFR mutation–positive and 29 EGFR mutation–negative NSCLC cases diagnosed with reflex PCR-based testing. Two mutation-specific antibodies against the most common exon 19 deletion, namely E746-A750del (clone SP111) and L858R mutation (clone SP125) were tested by using automated immunostainer. Sixty of 79 EGFR mutation–positive cases were treated with EGFR TKIs for advanced disease and included in treatment outcome analysis. A decision tree was used for the cost-effectiveness analysis.

Results

The overall sensitivity and specificity of the IHC-based method compared with the PCR-based method were 84.8% (95% confidence interval [CI] 74.6–91.6) and 100% (95% CI 85.4–100), respectively. The median progression-free survival (PFS) and overall survival (OS) of patients with IHC-positive EGFR mutation status were highly comparable to the total cohort (PFS: 14.3 vs. 14.0 months; OS: 34.4 vs. 34.4 months). The PCR and IHC cost ratio needs to be approximately 8-to-1 and 4-to-1 in White and Asian populations, respectively, to economically justify upfront use of IHC.

Conclusion

The trial confirmed an excellent specificity with fairly good sensitivity of IHC with mutation-specific antibodies for common EGFR mutations and the accuracy of IHC testing for predicting response to EGFR TKIs. The use of upfront IHC depends mainly on the population EGFR mutation positivity probability.

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).

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