Received date January 09, 2016; Accepted date February 11, 2016; Published date February 15, 2016
Citation: Mäki-Nevala S, Knuuttila A, Knuutila S, Sarhadi VK (2016) Concordant Results of Epidermal Growth Factor Receptor Mutation Detection by Real-Time Polymerase Chain Reaction and Ion Torrent Technology in Non-Small Cell Lung Cancer. J Clin Respir Dis Care 2:107. doi: 10.4172/2472-1247.1000107
Copyright: © 2016 Mäki-Nevala S, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Nowaday screening of non-small cell lung cancer (NSCLC) patients for epidermal growth factor receptor (EGFR) activating mutations is carried out in routine diagnostics to select patients who could benefit from EGFR inhibitor therapies. We aimed to compare EGFR mutation testing by Ion Torrent PGM technology, using AmpliSeq Colon and Lung Cancer panel, with real-time PCR in order to evaluate the accuracy of next generation sequencing (NGS) in detecting clinically relevant EGFR mutations in NSCLC. In total, 368 NSCLC patient samples were tested for EGFR by PCR and were also sequenced by Ion Torrent PGM by using AmpliSeq Colon and Lung panel. Samples were formalin-fixed, paraffin-embedded tumor specimens of Finnish NSCLC patients. The mutations studied for comparison were G719X, S768I, T790M, L858R, L861Q, deletions in exon 19 and insertions in exon 20. Comparison of EGFR mutations detectable by both PCR kit and NGS panel, showed a high degree of concordance between the two methods. Out of 368 samples, 31 out of 32 positive by PCR were also positive by NGS, and 336 out of 336 negative by PCR for these mutations were also negative by NGS giving a concordance of 99.7%. Two negative samples by PCR showed insertions in exon 20, which were not detectable by PCR. In one sample NGS failed to detect G719X mutation that had a very weak signal in PCR. Our study shows that the Ion Torrent PGM technology gives highly comparable results with the golden standard PCR. Thus, this NGS methodology is sensitive and reliable while testing clinically and diagnostically significant EGFR mutations in FFPE samples.
Non-small cell lung cancer; Epidermal growth factor receptor; Mutation; Next generation sequencing; Ion Torrent; PCR
The differences in molecular features of tumors and their predictive role in the response to targeting treatment have become very evident in the last decade. The alterations can be divided into groups based on a type of study they are approached with: protein, RNA and DNA-based methods. The last is used to assess mutations in the genome. In 2004, activating EGFR mutations and their role in the response to EGFR tyrosine kinase inhibitors (TKI) were described [1-3]. Since then many driver mutations in multiple genes, such as in ALK, and RAS and HER family, have been described, but EGFR mutations are still among the most common biomarkers in non-small-cell lung cancer (NSCLC). Currently the US Food and Drug Administration (FDA) has approved three different targeted therapies, afatinib, erlotinib and gefitinib, for advanced NSCLC harboring activating mutations (most importantly EGFR exon 19 deletions or exon 21 substitution L858R) .
There are also some other EGFR mutations with a clinical interest, such as G719X, L861Q and S768I, which have been associated with a good or partial response to EGFR-TKIs [5,6]. However, the development of resistance to EGFR-TKIs is common in the course of treatment. For instance, the acquired EGFR mutation T790M or MET amplification can cause the insensitivity to EGFR-TKIs after the preliminary good response [7-9]. Also insertions in exon 20 involving the residues A767, S768, D770, P772 and H773 show lack of response when treated with gefitinib or erlotinib [5,10]. Thus, it would be important to test patients’ genetic alterations with the multiplex and sensitive methods.
PCR-based mutation testing kits and Sanger sequencing have become as golden standard methods for diagnostic purposes. Next generation sequencing (NGS) methods are fast, economical, sensitive and multiplexable, and they are slowly replacing the traditional methods. However before implementation of NGS in diagnostic settings, it is important to thoroughly test and compare results to those with standard routine diagnostic methods. Some previous studies have shown the Ion Torrent PGM system to be accurate in mutation analysis by using Ion AmpliSeq Colon and Lung cancer Panel and/or the Ion AmpliSeq Colon and Lung Cancer Research Panel V2 compared with Sanger sequencing [11,12], the Ion AmpliSeq™ Cancer Panel compared with direct sequencing of peptide nucleic acid-locked nucleic acid polymerase chain reaction (PNA-LNA PCR) product  and custom panel of cancer genes compared with Sanger sequencing , although these have been performed on smaller sample sizes. In this study, we compared the EGFR mutation statuses of 368 Finnish NSCLC tumor specimens detected by real-time PCR method and targeted ampliconbased Ion Torrent NGS by using Colon and Lung Cancer Panel performed on DNA extracted from formalin-fixed, paraffin-embedded (FFPE) tumor material in order to evaluate the accuracy of NGS in detecting clinically important EGFR mutations.
In total, we collected retrospectively 566 formalin-fixed, paraffinembedded (FFPE) tumor specimens obtained via either diagnosis or surgical procedures, of NSCLC patients treated at the Hospital District of Helsinki and Uusimaa (HUS), Finland, during 2006–2014. Of those, tumor DNA from 368 NSCLC specimens were studied for EGFR mutations by both real-time PCR and amplicon–based NGS using the Colon and Lung Cancer Panel with the Ion Torrent PGM sequencing technology. Tumor content and histological type of NSCLC were confirmed by pathologist. Tumor content ranged from 5 to 90%, in 90% of the samples, the content was at least 20%. Patient characteristics are presented in the Table 1.
DNA Extraction and mutation detection
DNA was isolated from FFPE sections using the QIAamp DNA Mini kit (Qiagen GmbH, Hilden, Germany), following the manufacturer’s instructions, with small modifications . DNA concentration was assessed using Qubit® 2.0 Fluorometer (Thermo Fisher Scientific).
In our previous study, the tumor DNA samples of over 500 NSCLC patients were tested for EGFR mutations by using the Therascreen EGFR PCR Kit (Qiagen®, Manchester, UK) according to the manufacturer’s protocol, on the ABI7500 platform or the cobas® EGFR Mutation Test (Roche Molecular Systems, South Branchburg, USA) according to the manufacturer’s protocol, on the cobas® z480 platform . Of those, 368 samples were studied also by Ion Torrent and in this study we compare the results.
|Other NSCLC||6 (2)|
|Median age, years||66|
|Light ex-smoker||29 (8)|
|Medium ex-smoker||133 (36)|
|Current smoker||170 (46)|
|Abbreviations: ADC:Adenocarcinoma; LCC:Large Cell Carcinoma; SCC:Squamous Cell Carcinoma; ADSQ:Adenosquamous Carcinoma; NSCLC:Non-Small Cell Lung Cancer|
alight ex-smoker: smoking <20 years, cessation >10 years ago; medium ex-smoker: smoking >20 years, ceased; and current smoker: current smokers, smoking >20 years
Table 1: The characteristics of the patients included into this study.
Ion torrent AmpliSeq sequencing
Ten ng of DNA were used to prepare the barcoded libraries with the Ion AmpliSeq™ Library kit 2.0 (Thermo Fisher Scientific). The Colon and Lung Cancer panel covered 504 mutational hotspots and targeted regions (totaling 14.6 kb) in 22 genes, including EGFR. Template preparation and enrichment was performed with the Ion OneTouch™ 2 System (Thermo Fisher Scientific). Finally, sequencing was carried out using Ion 316™ chips on the Ion Personal Genome Machine System (PGM™, Thermo Fisher Scientific) and with the Ion PGM™ Sequencing 200 kit v2.
Alignment to the hg19 human reference genome and variant calling was performed by the Torrent Suite Software v.4.0.2 (Thermo Fisher Scientific) by the default threshold settings. For quality scores, coverage and strand biases those were a quality score of minimum of 6, relative read quality of minimum of 6.5, coverage of minimum of 6 for SNP/ COSMIC variant and 15 for indel, and strand bias maximum of 95% for SNP/COSMIC variant and 90% for indel. Alignments were visually checked with the Integrative Genomics Viewer (IGV) (v.2.3.34, Broad Institute) .
We compared the EGFR mutation detection in 368 NSCLC FFPE tumor samples between the real-time PCR method and the ampliconbased Ion Torrent PGM sequencing technology (Tables 2 and 3). The mutations were detected in 9% (34/368) of the patients. The observed mutation status pattern was highly comparable with the results from other studies as illustrated in our previous study by standard PCR . We detected a very high concordance between the results by the two methods used. Comparing only those mutations detectable by PCR in 368 samples, 31 out of 32 positive by PCR were also positive by NGS, and 336 out of 336 negative by PCR for these mutations were also negative by NGS giving a concordance of 99.7%. When taking PCR results as true positive, the number of EGFR positive by NGS, were 31 (true positive=31; false positive=0) and negative 337 (true negative=336; false negative=1). The sensitivity and specificity of detecting clinically common mutations L858R, deletions in exon 19, insertions in exon 20, G719X, S768I by Ion AmpliSeq Colon and Lung cancer Panel as following: 96.9% and 100%, respectively.
Other studies carried on relatively smaller sample size have shown a good concordance between PCR and NGS mutation testing [15,18,19]. Our results from a larger cohort clearly indicate that clinically important EGFR mutations can be detected with high accuracy by amplicon based NGS and is suitable in diagnostics. Similarly, recent comparisons between Sanger sequencing and NGS have shown similar results, giving greater sensitivity to NGS [11,12,15,18-20]. Studies comparing Ion Torrent technology are also in concordance with this study, suggesting it to be eligible method in diagnostics [11,12,19].
|Mutation||Detected by PCR||Detected by NGS|
|Deletion in exon 19||12||12|
|G719X & S768I||1||1|
|Insertion in exon 20||0||2a|
|Other COSMIC mutationa||0||12|
aNot detectable by Therascreen EGFR mutation testing.
Table 2: The comparison of EGFR mutation result between PCR and AmpliSeq colon and lung panel.
|S.No||Patient||Mutation by PCR||Mutation by NGS||Mutant read frequency, % (NGS)||Tumor content,%|
|16||233||Del Ex19||Del Ex19||60||35|
|17||199||Del Ex19||Del Ex19||34||50|
|18||508||Del Ex19||Del Ex19||31||65|
|19||215||Del Ex19||Del Ex19 a||10||30|
|20||52||Del Ex19||Del Ex19||10||35|
|21||211||Del Ex19||Del Ex19||55||70|
|22||70||Del Ex19||Del Ex19||33||45|
|23||631||Del Ex19||Del Ex19b||28||55|
|24||653||Del Ex19||Del Ex19||12||15|
|25||587||Del Ex19||Del Ex19||26||60|
|26||646||Del Ex19||Del Ex19||54||70|
|27||632||Del Ex19||Del Ex19||18||10|
|32||672||G719X; S768I||G719A; S768I||12; 11||45|
|35||513||No mutation||S720F; A767V||3; 6||60|
|44||244||No mutation||A871T; G874S||4; 4||50|
aMissed by Ion Torrent Software, but seen clearly by visualization in 9.9% of the reads.
bNovel 2bp deletion followed by 10bp deletion.
cDetected by Therascreen, but is uncertain with weak signal.
dNot detectable by Therascreen EGFR mutation testing kit.
Table 3: The patients with EGFR mutations detected by either method.
The comparison of the two methods showed discrepant results in two samples. One patient with EGFR deletion by PCR, was missed by Ion Torrent software (probably filtered out by the variant calling setting), although it was clearly seen in 10% of reads with IGV. Another patient with G719X mutation in PCR was clearly negative for this mutation by NGS even though the read depth at this location was 2204x. Since, this sample had a very low concentration in PCR (Ct 38.97 and dCt 11.70) and was flagged as doubtful, it is not certain whether it is false positive in PCR or false negative in NGS.
One important parameter of considerable significance in detecting mutations in tumor samples by NGS is deciding appropriate cut-off for mutation allele frequency, so as it maximize sensitivity and minimize false positive rate. Since the proportion of tumor cells in tumor samples can vary greatly among different samples, it can greatly affect the mutant allele proportion. We therefore also analyzed the tumor content of sample, as estimated histologically, in relation to percent of mutant allele among total reads. Overall, the percentage of mutant reads ranged between 3 and 75% (average 28%) for all EGFR mutation. In this study, we could successfully detect EGFR mutations in samples with a tumor content of 10% that had mutant reads percentage ranging between 3 and 18. At a cut-off of 3% for reads with mutant allele, we could detect all clinically relevant mutations without any false positives. This suggests NGS to be a sensitive method and can detect mutant alleles as low as 3% of the reads without false positives. Previous study has reported the limit of detection 1.3% or less for Ion Torrent AmpliSeq panel . Percentage of mutant reads was positively co-related to tumor percentage, though not very strongly (Pearson’s correlation R=0.5). At least five samples had mutant reads more than 50% that could indicate a probable EGFR amplification.
In addition to EGFR mutations that could be detected by both PCR and NGS, 12 patients had other COSMIC mutations detected by NGS, but not seen by PCR as they are not included in the PCR panel. This is a great advantage of NGS to detect all kind of mutations and distinguish them. By NGS, for instance, exon 19 deletions and exon 20 insertions are detectable in more detail, as PCR cannot distinguish them. By NGS we were able to identify two samples with exon 20 insertions (p.D770_ N771insSVD) which were not detected by PCR, because it is not included in the PCR panel (Figure 1). Mutant reads were around 20% in both these cases by NGS. Similarly, we detected novel 2bp deletion in patient 631, which is not described previously. Although, this deletion was detected by PCR it did not identify it (since it only detects deletions without characterizing them). Moreover, we could clearly identify different variations of exon 19 deletion with NGS (Figure 2).
Another strength of amplicon based Ion Torrent NGS is the sufficiency of small amount of starting material (ten ng of DNA), and its applicability to FFPE specimens. Moreover, Ion Torrent NGS workflow is user-friendly and fast.
As conclusion, this study shows that the Ion Torrent technology gives highly comparable results with the golden standard PCR. Thus, this NGS methodology is sensitive and reliable while testing clinically and diagnostically important EGFR mutations from FFPE samples.
We are thankful to Milja Tikkanen and Tiina Wirtanen for the technical support. Grants: The study was funded by the Sigrid Jusélius Foundation, the Finnish Cancer Society, the Finnish Work Environment Fund and the Finnish Cultural Foundation.