Abstract
Background: Tyrosine kinase inhibitors (TKIs) targeting epidermal growth factor receptor (EGFR) represent novel, effective tools in the management of advanced-stage non-small cell lung cancer (NSCLC). We aimed to evaluate the incidence and predictive role of EGFR gene amplification in patients with advanced-stage NSCLC treated with EGFR-TKIs. Patients and Methods: The study included 290 patients with advanced-stage (IIIB or IV) NSCLC. Multiplex ligation-dependent probe amplification (MLPA) and polymerase chain reaction (PCR) were used for detection of EGFR gene amplification and EGFR mutations, respectively. Results: EGFR amplification was detected in 26 (9.0%) patients. EGFR amplification was found more frequently in patients harboring the EGFR mutation (p<0.001). No significant corelation between EGFR gene amplification and survival was observed. Conclusion: EGFR gene amplification is associated with EGFR gene mutation. EGFR gene amplification is not a feasible predictive biomarker for treatment with EGFR-TKIs in patients with advanced-stage NSCLC.
Lung cancer is the most common cause of cancer-related death worldwide, while non-small cell lung cancer (NSCLC) is the most frequent histological type (1, 2). Treatment with low-molecular weight tyrosine kinase inhibitor (TKI) targeting epidermal growth factor receptor (EGFR) signaling pathway represents a novel, effective tool in the management of locally advanced or metastatic NSCLC. Amplification of the human epidermal growth factor receptor 2 (HER2)/NEU gene has since long been used as a predictive biomarker for targeted therapy of breast and gastric cancer by effective blockage of HER2-signaling by anti-HER2 antibody (3, 4). Considering HER2/NEU and EGFR are members of the same human epidermal growth factor receptor (ERBB) superfamily, it is reasonable to expect that EGFR gene amplification indicates a better response to anti-EGFR therapy in NSCLC. The aim of the present study was, therefore, to evaluate the predictive role of EGFR gene amplification in patients with advanced-stage NSCLC treated with EGFR-TKIs.
Patients and Methods
Patients and treatment. The study included 290 patients with cytologically or histologically confirmed locally advanced (stage IIIB) or metastatic (stage IV) NSCLC tested for EGFR gene amplification and activating EGFR gene mutations (exon 19 deletion or exon 21 L858R mutation). A sub-group including 177 patients was treated with EGFR-TKIs (erlotinib or gefitinib). All patients were diagnosed and treated between 2004 and 2012. The baseline patient characteristics are summarized in Table I.
Both erlotinib and gefitinib were administered orally at the standard approved doses of 150 mg and 250 mg daily, respectively. The treatment was continued until disease progression or development of intolerable adverse effects. Dose interruption or reduction was permitted in the event of treatment-related toxicity.
Clinical monitoring and statistics. The treatment was prospectively monitored and the clinical course of patients was continuously assessed at specific time points. Clinical follow-up including physical examination, plain chest X-ray and routine laboratory tests was performed every 3-4 weeks; computed tomography (CT) or positron-emission tomography-CT was performed after two or three months of the treatment. Progression-free survival (PFS) was determined from the date of erlotinib or gefitinib initiation until the date of first documented progression or death. Overall survival (OS) was determined from the date of erlotinib or gefitinib initiation until the date of death.
Baseline patient characteristics.
Data source. The clinical registry TULUNG (http://tulung.registry.cz/), in which Faculty Hospital Pilsen has participated since its creation, is a non-interventional post-registration database of epidemiological and clinical data of patients with advanced-stage NSCLC treated with targeted therapies in the Czech Republic. The registry contains anonymized individual patient data including demographic parameters, initial staging and disease characteristics, baseline patient information at the start of targeted therapy, as well as data on survival and adverse events which is updated at least twice a year. The protocol was approved by the independent Ethics Committee (130515_ TULUNG_IBA_ FNBRNO) and complied with the International Ethical Guidelines for Biomedical Research Involving Human Subjects, Good Clinical Practice guidelines, the Declaration of Helsinki, and local laws.
Statistical analysis. Standard summary statistics were used to describe the sample data set. The significance of differences in the incidence of EGFR gene amplification according to patient clinical characteristics was assessed using Fisher's exact test. PFS and OS were estimated using Kaplan–Meier method and all point estimates were accompanied by 95% confidence intervals. Statistical significance of the differences in Kaplan–Meier estimates was assessed using the log-rank test. As a level of statistical significance α=0.05 was used.
EGFR mutation and amplification analysis. The tumor specimens acquired during initial bronchoscopy were evaluated by a senior cytologist using standard giemsa staining. In a few cases, a tumor biopsy was processed into formalin-fixed paraffin-embedded (FFPE) histological sections. The cytology slides or, eventually, the FFPE sections, were submitted for molecular genetic testing, which included detection of somatic mutations in EGFR exons 19 and 21 and detection of EGFR gene amplification. If necessary, tumor cells were carefully selected and removed from the samples by laser microdissection using a P.A.L.M. microlaser instrument (Carl Zeiss MicroImaging GmbH, Jena, Germany). The microdissected cells were collected directly into the polymerase chain reaction buffer and processed without a special DNA extraction step. In all other cases, the DNA was extracted from tissue cells by a standard spin-column procedure using JetQuick Tissue DNA Issolation Kit (Genomed GmbH, Loehne, Germany). Mutations in exons 19 and 21 of the EGFR gene were tested by Genoscan mutation detection kits (Genomac International, Prague, Czech Republic) utilizing a denaturing capillary electrophoresis technique on an ABI PRISM 3100 16-capillary genetic analyzer (Applied Biosystems, Foster City, CA, USA). Detected mutations were confirmed by Sanger DNA sequencing using a BigDye v 3.0 chemistry (Applied Biosystems). In rare cases, where the overall fraction of mutated DNA was below the 20% threshold for DNA sequencing, mutation was identified indirectly after forming only a homoduplex fragment with a given known mutation reference standard. EGFR amplification was evaluated by Multiplex ligation-dependent probe amplification (MLPA) technique using SALSA MLPA KIT P315-A1 (MRC Holland, Amsterdam, the Netherlands), which contains 28 probes for each of the EGFR exons (chromosome 7) in addition to nine reference probes on other chromosomes. A standard manufacturer protocol was followed to generate MLPA products, which were then again analyzed on the ABI PRISM 3100.
Results
Incidence of EGFR gene amplification. EGFR gene amplification was detected in 26 (9.0%) patients and EGFR gene mutation was detected in 17 (5.86%) patients out of all 290 tested. EGFR mutations included 10 (58.8%) cases of exon 19 deletion and 7 (41.2%) cases of exon 21 L858R substitution. EGFR gene amplification was found more frequently in patients harboring EGFR mutation compared to those harboring wild-type EGFR gene (41.2 vs. 7.0%); the difference was statistically significant (p<0.001) (Figure 1). We did not observe any significant correlation between the incidence of EGFR gene amplification and clinical characteristics including: sex (7.3% in women vs. 9.9% in men; p=0.529), smoking status (8.4% in current or former smokers vs. 12.5% in non-smokers; p=0.331) and histological type of NSCLC (11.2% in adenocarcinomas vs. 7.5% in squamous-cell carcinomas; p=0.135).
Relation between EGFR gene amplification and survival. The median PFS and OS for patients with EGFR gene amplification (n=22) was 3.9 and 13.6 compared to 2.1 and 9.8 months (p=0.078 and p=0.587) for those without EGFR gene amplification (n=155) (Figure 2). Among patients harboring wild-type EGFR gene, the median PFS and OS for patients with EGFR gene amplification (n=18) was 2.1 and 13.6 compared to 1.9 and 9.8 months (p=0.172 and p=0.737) for those without EGFR gene amplification (n=145) (Figure 3).
Comparison of incidence of epidermal growth factor receptor (EGFR) gene amplification according to EGFR mutation.
Kaplan–Meier plots showing progression-free (PFS) (A) and overall (OS) (B) survival according to epidermal growth factor receptor (EGFR) gene amplification.
Discussion
The efficacy and safety of EGFR-TKIs in the treatment of patients with advanced-stage NSCLC have been demonstrated in various phase III clinical trials (5-9). Several molecular biomarkers predicting the treatment efficacy of EGFR-TKIs have been established in recent years. EGFR gene mutations, occurring approximately in 5-20% of patients (predominantly exon 19 deletions or a point-mutation in exon 21 termed L858R), represent the strongest predictor of a good treatment response to EGFR-TKIs (10-13). Based on that finding, patients with advanced-stage NSCLC are selected for first-line treatment according to the presence of activating EGFR gene mutation. Because the response to other targeted agents, such as trastuzumab and tamoxifen, depends mainly on the level of expression of the target, several studies focused on the predictive role of EGFR protein expression and EGFR gene copy number have recently been conducted, although the results are conflicting.
Kaplan–Meier plots showing progression-free (PFS) (A) and overall (OS) (B) survival according to epidermal growth factor receptor (EGFR) gene amplification in patients harboring wild-type EGFR gene.
In the present study, we aimed to evaluate the incidence and predictive role of EGFR gene amplification in patients with advanced-stage NSCLC treated with EGFR-TKIs. We were able to detect EGFR gene amplification in 9% of patients, that is similar to the findings by Hirsch et al. (14) and Dacic et al. (15), who detected EGFR gene amplification in 9% and 10% of patients, respectively.
Fluorescence in situ hybridization (FISH) has since long been the golden standard for detection of gene amplification in various tumor types, including breast, stomach and brain tissues (16). More recently multiplex ligation-dependent probe amplification (MLPA) has been demonstrated as an alternative semi-automatic approach with low demand on sample consumption (17). MLPA analysis can also be seemingly integrated with other molecular-genetic testing, most importantly the predictive testing of activating EGFR mutations (18). Using MLPA in the present study, we obtained results equivalent to FISH in terms of the frequency of EGFR amplifications reported in the literature (14-21). We observed higher frequency of EGFR gene amplifications in patients harboring EGFR gene mutation compared to those harboring wild-type EGFR gene (41.2 vs. 7.0%, p<0.001), which is in agreement with the findings previously reported by Hirsch et al. (22). Such a finding is important for evaluation of the predictive role of EGFR gene amplification. We did not observe any significant correlation between the incidence of EGFR gene amplification and basic clinical characteristics such as sex (p=0.529), smoking status (p=0.331) and histological type of NSCLC (p=0.135). Our results did not confirm higher frequency of EGFR gene amplification in patients with squamous cell carcinoma, which has been reported by others (14, 18, 21).
We observed a trend for longer PFS and OS for patients with EGFR amplification (3.9 and 13.6 months) compared to those without EGFR amplification (2.1 and 9.8 months), although the differences did not reach statistical significance (p=0.078 and p=0.587). Given the previously mentioned finding, we suggest that the trend for longer survival may be caused by the frequent coincidence of EGFR gene amplification and EGFR gene mutation. The basis for such a hypothesis becomes more apparent when evaluating survival data for the sub-group of patients harboring wild-type EGFR gene, where we recorded almost equivalent PFS (2.1 vs. 1.9 months, p=0.172). Thus, we did not confirm the predictive role of EGFR gene amplification reported in several previous studies (19, 22, 23). Our results are similar to those reported by Fukuoka et al., who analyzed biomarkers in phase III randomized study comparing gefitinib with chemotherapy in patients with advanced NSCLC (IPASS), the study results suggested that the predictive value of EGFR gene copy number was driven by coexisting EGFR gene mutation (24). The principal limitations of our study are its retrospective design and limited number of patients included.
In conclusion, using MLPA, we confirmed the incidence of EGFR gene amplification reported in previous studies using FISH. We recorded frequent coincidence of EGFR gene amplification and EGFR gene mutation. The results of this study suggest that EGFR gene amplification is not a feasible biomarker for prediction of treatment efficacy EGFR-TKIs in patients with advanced-stage NSCLC.
Acknowledgements
The Authors would like to thank all patients voluntarily taking part in this study. This study was supported by the National Sustainability Program I (NPU I) No. LO1503 provided by the Ministry of Education Youth and Sports of the Czech Republic.
Footnotes
Conflicts of Interest
JF has received honoraria from Astra Zeneca, Roche and Novartis for consultations and lectures unrelated to this project. OF, MP, MM, LB, OS, MS, ZB, RK and OT declare that they have no actual or potential conflict of interest including any financial, personal or other relationships with other people or organizations that could inappropriately influence this work.
- Received October 27, 2015.
- Revision received December 1, 2015.
- Accepted December 4, 2015.
- Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
