Abstract
Background/Aim: Disparities in the results of next-generation sequencing-based multiplex gene panel tests and those of single-gene tests when detecting epidermal growth factor receptor (EGFR) mutations in non-small-cell lung cancer (NSCLC) have been reported. However, the possible underlying causes have not been investigated. The aim of this study was to explore the possibilities and causes of false results obtained using cobas® EGFR Mutation Test v2 (cobas® EGFR) and Oncomine Dx Target Test (ODxTT). Patients and Methods: The data of patients with NSCLC who underwent gene assessment using both cobas® EGFR and ODxTT between April 2021 and May 2022 were retrospectively reviewed. Disparate results of EGFR mutation analyses were then reviewed. Results: One hundred and sixteen patients were included in the analysis. The results of six samples were inconsistent. In four samples, exon 20 insertion mutations were detected using cobas® EGFR, but not identified using ODxTT. A fragment analysis was performed on three of the four samples, and all showed negative results for exon 20 insertion. Furthermore, one false negative result was obtained in the ODxTT for both exon 19 deletion and L858R mutations. For exon 19 deletion mutation, a single nucleotide variant from adenine to thymine was identified close to the mutation site. Conclusion: False positives for exon 20 insertion may occur when using cobas® EGFR, and false negatives for exon 19 deletion and L858R mutations may occur when using ODxTT.
- Non-small-cell lung cancer
- epidermal growth factor receptor
- exon 20 insertion mutation
- Oncomine Dx Target Test
- cobas® EGFR Mutation Test v2
Molecular targeted therapies for non-small cell lung cancer (NSCLC) have advanced over the recent years. Epidermal growth factor receptor (EGFR) mutations occur in 47% of patients with adenocarcinoma in the Asia-Pacific area. EGFR-thyroxine kinase inhibitors (TKIs) are effective in improving prognosis in patients with EGFR mutations (1, 2). For a long time, the conventional single-gene test based on real-time polymerase chain reaction (PCR) was the only method available for EGFR mutation detection. The cobas® EGFR Mutation Test v2 (cobas® EGFR; Roche Molecular Systems, Pleasanton, CA, USA), which can detect 42 different EGFR mutations in exons 18, 19, 20, and 21, is a conventional single-gene test (3). Single-gene tests are being replaced by next-generation sequencing (NGS), which enables the detection of multiple driver oncogenes simultaneously (4). The Oncomine Dx Target Test (ODxTT; Thermo Fisher Scientific, Waltham, MA, USA) is the first NGS-based multiplex gene panel test approved by the US Food and Drug Administration. It was approved in Japan for detecting multiple driver mutations, including EGFR mutations in exons 18-21, in 2019 (5).
NGS-based multiplex gene panel tests can detect multiple driver oncogenes and are, therefore, superior to conventional single-gene tests in terms of cost performance (6). However, NGS-based multiplex gene panel tests generally require large tissue samples in terms of volume and tumour content and involve extended processing times compared to single-gene tests (7-9). Unfortunately, inconsistencies in the results of EGFR variant detection between single-gene and NGS-based multiplex gene panel tests have been reported (10-13). The difference in detectable variants between these tests can be attributed to these discrepancies. For example, in a previous study, two variants of exon 19 deletion mutations, p.E746_P753delinsVS and p.E746_p753delinsLS, were detected only by cobas® EGFR because they were not covered variants of ODxTT. In contrast, L861R and a variant of exon 20 insertion mutations, p.P772_H733insV were detected only by ODxTT because these were not covered by cobas® EGFR (13). Furthermore, it is hypothesized that the low sensitivity of ODxTT could be a reason (10). Therefore, it is uncertain whether these disparities are caused by false positives from single-gene tests or false negatives from NGS-based multiplex gene panel tests (11). The relative reliability of both tests must be determined, considering that these results often guide therapeutic decisions for critically ill patients. Therefore, the aim of this study was to explore the possibilities and causes of false results of cobas® EGFR and ODxTT.
Patients and Methods
Study design and ethics approval. This single-centre retrospective cohort study was approved by the ethics committee of National Hospital Organization Kinki-Chuo Chest Medical Center (No. 2022-042). The ethics committee waived the need for informed consent from patients as the data were collected retrospectively and anonymised. This study was conducted according to the tenets of Helsinki Declaration.
Patients. Patients who were diagnosed with NSCLC and had received results regarding EGFR mutation status from both cobas® EGFR and ODxTT between April 2021 and May 2022 were considered eligible. Patients who had invalid results or did not complete one of the tests because of a low quantity or quality of their specimens were excluded.
Sample processing and genetic tests. Tumour biopsy samples were immediately placed in 10% neutral buffered formalin, and then fixed for 12-24 h at 20°C-27°C. The formalin-fixed tissues were subjected to serial processing, and then embedded in paraffin to generate formalin-fixed, paraffin-embedded blocks. The number of nucleated cells (tumour and inflammatory cells) in haematoxylin and eosin-stained sections was evaluated to determine tumour content percentage. If the tumour cell percentage was below 20%, macro-dissection was performed. Furthermore, if the tumour content was below 20% after macro-dissection and number of nucleated cells was less than 5,000, the samples were not subjected to ODxTT. Next, 15-30 sections of small biopsy samples and 5 sections of surgical resection samples were assessed using ODxTT at SRL Diagnostics Laboratory (Tokyo, Japan). We then carried out cobas® EGFR testing on duplicates of tissue samples subjected to ODxTT. EGFR mutations were subsequently analysed following the manufacturer’s instructions.
Data collection and outcome. Data pertaining to demography, smoking history, specimen sampling methods, histological patterns, tumour content percentage of tissue samples, and cobas® EGFR and ODxTT results were collected. When disparities between cobas® EGFR and ODxTT results were identified, we isolated the related cases and reviewed the patients’ information, including the concentration of DNA and RNA, disease staging, and treatments received. Subsequently, the original sequencing results were reanalysed to identify potential error sources.
Fragment analysis. When disparities were identified between the cobas® EGFR and ODxTT results regarding the presence of exon 20 insertion mutations in EGFR in the isolated samples, these samples were subjected to a fragment analysis at the BML laboratory (Tokyo, Japan) to examine whether any exon 20 insertions existed. EGFR exon 20 was amplified via PCR using two primers: 5′-FAM-TGAAACTCAAGATCGCATTCAT-3′ and reverse, 5′-ATCTCCCTTCCCTGATTACCTT-3′. The length of the PCR products was then examined using the Applied Biosystems 3130 Genetic Analyser (Thermo Fisher Scientific).
Statistical analysis. Patient background information and histological characteristics were reviewed. Data are described as median and interquartile range for quantitative variables and as count and percentage for qualitative variables. The results regarding EGFR mutation status from cobas® EGFR and ODxTT were then compared. We performed Fisher’s exact test to compare the positivity rates between the tests. Statistical significance was set at p<0.05. All statistical analyses were conducted using EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan), a graphical user interface for R (The R Foundation for Statistical Computing, Vienna, Austria).
Results
Inclusion cohort. Two-hundred and fifty-seven patients were diagnosed with NSCLC during the inclusion period, whilst 117 specimens were subjected to both cobas® EGFR and ODxTT. One patient was excluded from this analysis because RNA defects prevented the use of ODxTT. Consequently, 116 patients were included in the analysis.
Patient characteristics. The patient characteristics are summarised in Table I. The median age of the patients was 72.8 years, and there were 82 male patients (71%); 90 patients (77%) had smoking history. The specimen sampling methods included transbronchial biopsy (n=80, 69%), endobronchial ultrasound-guided transbronchial needle aspiration (n=15, 13%), computed tomography-guided biopsy (n=5, 4.3%), surgical lung biopsy (n=7, 6.0%), and pleural effusion (n=4, 3.4%). The most common histological pattern was adenocarcinoma (n=59, 51%), followed by squamous cell carcinoma (n=43, 37%).
Patient characteristics.
Differences between the tests. A comparison of EGFR mutation results between cobas® EGFR and ODxTT is shown in Table II. cobas® EGFR detected 21 EGFR mutations, whereas ODxTT detected 15 mutations. cobas® EGFR results were positive for exon 20 insertion in four patients, whereas ODxTT showed negative results in all four patients. With regard to exon 19 deletion and L858R mutations, cobas® EGFR detected one more positive result, for both mutations, compared to ODxTT. However, the positivity rates were not significantly different between the tests for any mutation.
Results of cobas® EGFR and ODxTT.
Analysis of discordant cases. Data of patients with disparate EGFR test results of cobas® EGFR and ODxTT are shown in Table III. A fragment analysis was performed on three of the four samples, which tested positive for exon 20 insertion in cobas® EGFR but negative in ODxTT. One patient sample was excluded from this analysis owing to RNA defects. None of the samples tested positive for the mutation in the fragment analysis. Furthermore, all were male patients and current smokers, and had squamous cell carcinoma histology. When we referred to the original ODxTT sequencing results of cases for which the cobas® EGFR result was positive for an EGFR exon 19 deletion mutation but the ODxTT result was negative, a mutation (p. E746_A750del) was identified with an allele frequency of 28% in the original sequencing results of ODxTT (Figure 1). Furthermore, there was a single nucleotide variant (SNV) from adenine to thymine close to the deletions, which likely caused the negative ODxTT result. In the final disparate case, which showed a positive result for an EGFR-L858R mutation in cobas® EGFR but negative in ODxTT, the mutation could be identified with an allele frequency of 1.7% in the original sequencing results of ODxTT. The allele frequency of the mutation was below the limit of detection in ODxTT, and therefore, this was considered to be a negative result.
Clinical and pathological characteristics of discordant cases.
Original sequencing results from ODxTT for a case in which the cobas® EGFR result was positive for the EGFR exon19 deletion mutation, whereas the ODxTT result was negative. Arrowhead and arrow indicate exon 19 deletion and the single nucleotide variant from adenine to thymine, respectively.
Discussion
Here, we investigated the potential regularity of disparities between cobas® EGFR and ODxTT. We found that cobas® EGFR could generate false positive results for exon 20 insertion mutations in current smokers and patients with squamous cell carcinoma. ODxTT may generate false negative results for exon 19 deletion mutations even if they are detectable variants, and this might be because of an SNV from adenine to thymine, close to the deletion. These two test results were different for L858R mutations owing to differences in their respective lower limits of detection. Exon 20 insertion mutations are the third most common subtype of EGFR mutations, following exon 19 deletion and L858R, and account for 4%-12% of all EGFR mutations in lung cancer (14). Patients with exon 20 insertion mutations have been reported to be refractory to conventional EGFR-TKIs, and invasive platinum-based chemotherapy remains the standard treatment (15). However, patients with EGFR exon 20 insertion mutations administered amivantamab, which is an EGFR/mesenchymal–epithelial transition (MET) bispecific antibody with an immune cell-directing activity designed to engage these two distinct driver pathways, showed a high response rate and long progression-free survival (16). Therefore, it is clinically important to correctly identify exon 20 insertions.
To our knowledge, this is the first study to demonstrate that cobas® EGFR could show false positives for exon 20 insertion mutations. Disparity between a single gene test and ODxTT in exon 20 insertion mutation detection has been reported (10). However, this did not mean a false result of either test, as exon 20 insertions were not the target mutations of ODxTT at the time of the study. Here, the fragment analysis results indicated false positives of cobas® EGFR. All false positive results were in male patients who were current smokers and had squamous cell carcinoma. False positives of this nature have not been previously reported. This may be because of the limited number of studies comparing the results of single-gene tests and NGS-based multiplex gene panel tests in squamous cell lung carcinoma. We could not determine the mechanism underlying the observed false positives in samples from smokers and patients with squamous cell carcinoma when testing for exon 20 insertion mutations, warranting further research. It is established that clinicians must remain vigilant when encountering positive results for exon 20 insertion mutations in squamous cell carcinoma and to validate all results using secondary methods, such as fragment analysis.
Here, we also found a disparity between cobas® EGFR and ODxTT results for exon 19 deletion mutations in one case. A previous study also showed disparities between cobas® EGFR and ODxTT results for two variants of exon 19 deletion mutations, p.E746_P753delinsVS and p.E746_p753delinsLS (13). However, these were not the target variants of ODxTT originally; thus, these disparities were not false negatives of ODxTT. The mutation under consideration in our study, p. E746_A750del, is one of the variants that can be detected using ODxTT. Furthermore, the detection rate for this mutation was 28% in the review of original sequencing results; thus, we conclude that ODxTT presented false negatives. For ODxTT, the tumour content percentage should be ≥20% and DNA concentration should be ≥0.83 ng/μl, and in this case, both of them were sufficient for NGS-based multiplex tests (17). An SNV from adenine to thymine was detected close to the deletions in the review of the original sequencing results; no other abnormalities were found. Therefore, we thought that this SNV could be the cause of failure in the detection of exon 19 deletion mutations using the ODxTT software. To our knowledge, this is the first study to show the possibility and the underlying cause of false negatives of ODxTT in the detection of exon 19 deletion mutations.
Furthermore, we found that the cobas® EGFR and ODxTT results for L858R mutations contradicted in one case because of differences in their detection limits. The tumour content percentage of the specimen, in this case, was 21.0%, which was relatively low compared with that of other samples in this study. However, the minimum tumour content percentage required for ODxTT has been reported to be ≥20% (17). This case demonstrates that a tumour content percentage of less than 30% may be a risk factor for false negative results when using ODxTT. This finding is clinically relevant, as it is currently unknown whether EGFR-TKIs are effective for cases in which cobas® EGFR and ODxTT show discordant results for L858R mutations because of the difference in sensitivity. However, given the clinical course of this patient, who responded to osimertinib for several months, EGFR-TKIs could be considered as a first-line treatment for similar cases.
There are several limitations to this study. First, the retrospective nature of our analysis may have been a source of bias. Second, this study was conducted in a single centre. The methods of specimen sampling, sample processing, and prescription for ODxTT may differ among institutes. Additionally, the number of samples was small. While the discrepancies in testing were likely multifactorial, we could only present some of the possibilities based on our initial findings.
In conclusion, we detected the possibilities and determined the causes of false positives for EGFR exon 20 insertion mutations with cobas® EGFR and false negatives for exon 19 deletion and L858R mutations with ODxTT. In the future, it is important to consider the possibility of false results such as the results of this study.
Acknowledgements
The Authors thank Mr. Tomoaki Teramoto, Mr. Yasunori Tsuruta, Ms. Koutaka Masami, and Ms. Nagi Matsumura for carrying out cobas® EGFR testing. The Authors received no funding for this study.
Footnotes
Authors’ Contributions
K.K.: Conceptualisation, Data curation, Formal Analysis, and Writing–original draft. A.T.: Conceptualisation, Writing–review and editing. Y.I.: Writing–review and editing. Y.T.: Writing–review and editing. K.N.: Writing–review and editing. M.T.: Writing–review and editing. Y.M.: Writing–review and editing. K.O.: Writing–review and editing. S.S.: Conceptualisation, Data curation, Writing–review and editing, and Supervision.
Conflicts of Interest
Dr. Kanaoka reports receiving personal fees from AstraZeneca outside the submitted work. Dr. Tamiya reports receiving personal fees from Thermo Fisher Scientific within the submitted work and receiving grants from AstraZeneca, Beigene, and Daiichi-Sankyo, in addition to receiving personal fees from Eli Lilly, Ono Pharmaceutical, Chugai Pharmaceutical, Boehringer Ingelheim, AstraZeneca, Bristol-Myers Squibb, MSD, Taiho, Pfizer, Takeda Pharmaceutical, Nihon Kayaku, Novarits, and Merk Biopherma outside the submitted work. Dr. Inagaki reports receiving personal fees from AstraZeneca, Chugai, Chugaiigakusya, and Pfizer outside the submitted work. Dr. Taniguchi reports receiving personal fees from Chugai Pharmaceutical, Ono Pharmaceutical, and AstraZeneca outside the submitted work. The remaining Authors declare no conflicts of interest.
- Received April 6, 2023.
- Revision received April 17, 2023.
- Accepted April 18, 2023.
- Copyright © 2023 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
