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ORIGINAL ARTICLE
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Correlation of epidermal growth factor receptor mutation status in plasma and tissue samples of patients with non-small cell lung cancer


1 Department of Pathology, College of Medicine, Dong-A University, Busan, South Korea
2 Department of Internal Medicine, Division of Pulmonology, Dongnam Institute of Radiological and Medical Sciences, Busan, South Korea
3 Department of Internal Medicine, Division of Pulmonology, Bong Seng Memorial Hospital, Busan, South Korea
4 Department of Internal Medicine, Division of Pulmonology, College of Medicine, Dong-A University, Busan, South Korea

Correspondence Address:
Choonhee Son,
Department of Internal Medicine, Division of Pulmonology, College of Medicine, Dong-A University, Daesingongwon-ro 26, Seo-gu, Busan 49201
South Korea
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jcrt.JCRT_515_18

 > Abstract 


Background: Somatic mutations of the gene encoding epidermal growth factor receptor (EGFR) are detected in approximately 30%–50% of patients with non-small cell lung cancers (NSCLC), so detection of EGFR mutation is the pivotal step of treatment in patients with advanced NSCLC. However, difficulty in obtaining sufficient tissue and bias from the heterogeneity of the tumor samples are the major obstacles. Although analyzing EGFR with circulating tumor DNA (ctDNA) in plasma is a breakthrough, accuracy is the problem in variable methods. Peptide nucleic acid (PNA) clamping-assisted fluorescence melting curve analysis (PANAMutyper®) is a novel and highly sensitive method of detecting EGFR mutation in tumor tissues.
Aims and Objectives: This study was designed to evaluate PANAMutyper® for detecting EGFR mutation with ctDNA of patients with lung cancer.
Materials and Methods: EGFR mutation status detected by PNA clamp with tissue samples and by PANAMutyper® with ctDNA was compared. Tissue biopsy was done in 158 patients with lung tumor, in which 23 cases were excluded and 135 cases were enrolled. EGFR mutation rate was 23.0% (31/135) in overall patients. All the plasma samples of the cases with mutant EGFR in tissue samples were verified by an already known highly sensitive method of droplet digital polymerase chain reaction (ddPCR).
Results: The concordance rate of tissue and plasma samples was 91.9% (124/135). The sensitivity, specificity, negative predictive value, and positive predictive value were 64.5%, 100%, 90.4%, and 100%, respectively, according to the tissue samples as a standard. PANAMutyper® method was not inferior to ddPCR for the detection of EGFR mutation including T790M with ctDNA. These results suggest that the detection of EGFR mutation status using ctDNA in plasma by PANAMutyper® is a feasible test prior to tissue biopsy.

Keywords: Epidermal growth factor receptor, liquid biopsy, non-small cell lung cancer, PANAMutyper®, plasma



How to cite this URL:
Roh MS, Yoon NB, Lee S, Kang BH, Um SJ, Lee DH, Son C. Correlation of epidermal growth factor receptor mutation status in plasma and tissue samples of patients with non-small cell lung cancer. J Can Res Ther [Epub ahead of print] [cited 2019 Nov 13]. Available from: http://www.cancerjournal.net/preprintarticle.asp?id=264700




 > Introduction Top


Somatic mutations in the gene encoding epidermal growth factor receptor (EGFR) are detected in approximately 30%–50% of non-small cell lung cancers (NSCLCs) worldwide, especially high in Asian patients,[1],[2],[3] and EGFR-tyrosine kinase inhibitors (EGFR-TKIs) are proved superior treatment over conventional cytotoxic chemotherapy for these patients.[4],[5] Detecting EGFR mutation is the first step of systemic therapy in patients with advanced NSCLC. The major obstacle of EGFR mutation detection is the difficulty of obtaining sufficient tissue samples, and analyzing it in circulating tumor DNA (ctDNA) from plasma sample is an important breakthrough. However, the concordance with tissue sample is the problem to be solved.

Peptide nucleic acid (PNA) is an artificially synthetic polymer, which has the characteristics of both nucleic acid and protein. There is no electrostatic repulsion in PNA; the binding capacity is more powerful than DNA. PNA probe can selectively amplify mutant-type DNA because it is designed to suppress the amplification of wild-type DNA.[6] PNA clamping-assisted fluorescence melting curve analysis (PANAMutyper ®) is a novel technology that integrates PNA clamp and PNA S-melting, known to be more sensitive compared with PNA clamp alone.[7]

This study was designed to evaluate the accuracy and feasibility of EGFR mutation detection by PANAMutyper ® using ctDNA by means of comparing with EGFR mutation status in tissue samples.


 > Materials and Methods Top


Study design

This prospective study was conducted from January 2016 to March 2017. Within 2 days after obtaining tissue samples, plasma samples were collected. Patients with insufficient samples, metastatic cancers from other organs, and small cell carcinoma were excluded from the study. EGFR mutation status was analyzed using PNA clamping with tissue samples and by PANAMutyper ® with ctDNA of plasma samples. Cases with mutant EGFR in tissue samples were verified by droplet digital polymerase chain reaction (ddPCR) with ctDNA of plasma samples [Figure 1]. Tissue samples were formalin-fixed, paraffin-embedded (FFPE) materials. Concordance rate and negative and positive predictive values were analyzed. This study was approved by the Institutional Review Board of Dong-A University Hospital (study ID number: DAUHIRB-16-151) and was performed in accordance with the Declaration of Helsinki.
Figure 1: Flow of the study. *EGFR = Epidermal growth factor receptor, PNA = Peptide nucleic acid, PANAMutyper®=Peptide nucleic acid clamping-assisted fluorescence melting curve analysis, §ddPCR = Droplet digital polymerase chain reaction

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Sample preparation

Formalin-fixed, paraffin-embedded preparation

DNA extraction was carried out using the automated Maxwell 16 Instrument with the Maxwell ® 16 FFPE Plus LEV DNA Purification Kit (Promega, Korea), following the manufacturer's instructions. DNA concentration was determined by spectrophotometer (Nanodrop, DE, USA).

ctDNA preparation from plasma

Blood samples from patients were collected in 10-mL BCT ® tubes (Streck, NE, USA) and within 4 h, plasma was separated from the blood by centrifugation at 4°C It was stored at −80°C as soon as the plasma was separated. The ctDNA was extracted using QIAamp ® Circulating Nucleic Acid kit (Qiagen, CA, USA) from 2 mL of the plasma. The eluted volume was 100 μL per sample. The extracted ctDNA was stored at −70°C until use.

Detection of epidermal growth factor receptormutation

Peptide nucleic acid clamp

We used PNA Clamp ® EGFR mutation detection kit (PANAGENE, Korea) to detect EGFR mutations in real-time PCR. All reactions were done in 20-μL volumes using 7-μL template DNA, 3-μL primer and PNA probe set, and 10-μL SYBR ® Green PCR master mix (Life Technologies, CA, USA) per sample. All reagents were included in the kit. Real-time PCR reactions were performed using a CFX 96® (Bio-Rad, CA, USA). PCR cycling commenced with a 5-min hold at 94°C, followed by 40 cycles of 94°C for 30 s, 70°C for 20 s, 63°C for 30 s, and 72°C for 30 s. Three EGFR mutation types were detected using PNA-mediated real-time PCR. The efficiency of PCR clamping was determined by measuring the threshold cycle (Ct) value.

PANAMutyper ®

PANAMutyper ® (PANAGENE, Korea) contains a PNA clamp and PNA detection probes in each reaction tube. All reactions were done in 25-μL volumes using 5-μL template DNA, 19-μL primer and PNA probe master mix set, and 1-μL taq polymerase per sample. All reagents used were included in the kit. Real-time PCR reactions were performed using a CFX 96® (Bio-Rad, CA, USA). PCR was performed under the following conditions: 50°C for 2 min and 95°C for 15 min as two holding periods; 15 cycles of 95°C for 30 s, 70°C for 20 s, 63°C for 60 s; 35 cycles of 95°C for 10 s, 53°C for 20 s, 73°C for 20 s; and a melting curve step (from 35°C to 75°C with gradual increment for 0.5°C for 3 s). Fluorescence was measured on all the four channels (FAM, ROX, Cy5, and HEX). Melting peaks were derived from the melting curve data. Mutations were detected by the melting temperature of each tube for each fluorescent dye.

Droplet digital polymerase chain reaction

For the confirmation of discrepancy result, the samples were tested using ddPCR. The ddPCR assays were commercial products designed for the detection of EGFR gene mutations, namely Exon 19 deletion, T790M, and L858R. There were totally four types, namely C.2240_2257del18/p. L747_P753>S (dHsaCP2500546, Bio-Rad, CA, USA), c.2235_2249del15/p. E746_A750del (dHsaCP2000039, Bio-Rad, CA, USA), c.2369C>T/p. T790M (dHsaCP2000019, Bio-Rad, CA, USA), and c.2573T>G/p. L858R (dHsaCP2000021, Bio-Rad, CA, USA). According to the manufacturer's method, all assays were performed on the QX200® (Bio-Rad, CA, USA).

Statistical analysis

Statistical analysis of DNA measured semiquantitatively in tissue samples by using PANAMutyper ® in concordant and discordant cases was performed using the Student's t-test. Statistical significance was determined by a two-tailed P < 0.05.


 > Results Top


Patient characteristics

Tissue biopsy was done in 158 patients with lung tumor: 23 cases were excluded because their tumors were small cell carcinoma, 6 cases were tuberculoma, and 4 cases were metastatic cancers from other organs (two from breast and two from colon). The clinical characteristics of 135 patients are shown in [Table 1]. The mean age was 68.4 years (range: 38–94). Male and female patients constituted 110 (69.6%) and 48 (30.4%), respectively. Ninety-nine patients were current or recent smokers, of which 91 were male. Most of the patients were in advanced stage. Thirty-one out of the 135 patients had EGFR mutations (23.0%). In females and males, EGFR mutation rates were 47.7% (21 out of 44) and 11.0% (10 out of 91), respectively.
Table 1: Patient characteristics of the study population for epidermal growth factor receptor testing

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Concordance of the epidermal growth factor receptorstatus in tissue and plasma samples

There were 31 cases (19.6%) with EGFR mutations in the tissue samples (TmEGFR). Of these 31 cases, 20 cases had the same mutation, but 11 cases had wild-type mutations, which was found by PANAMutyper ® method using plasma samples (PMwEGFR) [Table 2]. The concordance rate of tissue and plasma samples was 91.9% (124/135). The sensitivity and specificity were 64.5% and 100.0%, negative predictive value (NPV) and positive predictive value (PPV) were 90.4% and 100.0% according to the tissue samples as a standard.
Table 2: Correlation of the epidermal growth factor receptor mutation status in tissue samples by peptide nucleic acid clamp and in plasma samples by peptide nucleic acid clamping-assisted fluorescence melting curve analysis method

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Verification of discrepancy

For the verification of discordant EGFR mutation test, highly sensitive ddPCR was used with ctDNA extracted from plasma samples. In 9 out of 11 discordant plasma samples, the EGFR mutations were similarly wild by ddPCR (PDwEGFR) and two were mutant by ddPCR (PDmEGFR) [Table 3]. Whereas in four concordant cases, PMmEGFR and TmEGFR were discordant in ddPCR (PDwEGFR) [Table 4]. The response to EGFR TKIs was in accordance with the tissue samples. PANAMutyper® with tissue was the most sensitive method for detecting T790M mutation [Table 5].
Table 3: Comparison of the results of epidermal growth factor receptor mutation status by peptide nucleic acid clamping-assisted fluorescence melting curve analysis and droplet digital polymerase chain reaction in discordant cases

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Table 4: Comparison of the results of epidermal growth factor receptor mutation status by peptide nucleic acid clamping-assisted fluorescence melting curve analysis and droplet digital polymerase chain reaction in discordant cases

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Table 5: The results of epidermal growth factor receptor mutation status by variable methods in cases with T790M

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In addition, to find why the mutation was not detected in plasma, a retest was performed with PANAMutyper ® using tissue samples [Figure 2]. Semiquantitatively measured levels of tumor DNA were significantly lower in discordant cases than that in concordant cases (34 ± 50 vs. 296 ± 241, P = 0.005) [Figure 3].
Figure 2: Comparison of PANAMutyper®* results with tissue and plasma samples. *PANAMutyper®=Peptide nucleic acid clamping-assisted fluorescence melting curve analysis. A concordant case showed identical EGFR exon 21 L858R mutations in both plasma (a) and tissue (b) samples. A discordant case showed no EGFR mutation in plasma sample (c), but EGFR exon 21 L858R mutation in tissue (d) sample. EGFR = Epidermal growth factor receptor

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Figure 3: Levels of mutant tumor DNA in concordant and discordant cases. Levels of mutant tumor DNA measured semiquantitatively in tissue samples by using peptide nucleic acid clamping-assisted fluorescence melting curve analysis (PANAMutyper®) in concordant and discordant cases. The amount of mutant tumor DNA was significantly lower in discordant cases than that in concordant cases (34 ± 50 vs. 296 ± 241, P = 0.005)

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 > Discussion Top


The identification and characterization of molecular biomarkers has helped to revolutionize NSCLC management, and the determination of EGFR mutation status represents a critical step in the diagnostic process. Although tissue biopsy techniques remain the gold standard of detecting initial EGFR mutation status and acquired resistance to EGFR TKIs through multiple mechanisms, including T790M and HER2 or MET amplifications, the emergence of the so-called liquid biopsies, using ctDNA and advances in analytical techniques, may eventually allow real-time monitoring of tumor evolution.[8]

Detection of ctDNA was used for the early diagnosis of cancer,[9] and technical advances allowed to use it for the selection of the best treatment.[10] At present, improving the accuracy of novel molecular diagnostics is the issue. In PNA clamping method, an optimized PNA clamp can tightly bind to only wild-type DNA sequences and then suppress amplification during the PCR reaction [Figure 4].[6] This method was proved as a sensitive and simple procedure, compared with direct DNA sequencing, and a useful tool for the detection of EGFR mutations in tissue samples.[11]
Figure 4: The PNA clamping system. The PNA oligomer was designed to bind to the bottom strand of the wild-type sequence, spanning mutational hotspots in exons 18–21 of the epidermal growth factor receptor gene. The forward PCR primer partially overlapped the PNA-binding site. (a) A PNA/DNA hybrid with a perfect match prevents annealing of the PCR primer and amplification of wild-type DNA. (b) A PNA/DNA hybrid with a single base pair mismatch does not suppress annealing of the PCR primer or amplification of mutant alleles (J Thorac Oncol 2012;7:355-64). PNA = Peptide nucleic acid, PCR = Polymerase chain reaction

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In addition, by incorporating melting curve analysis to PNA-mediated PCR clamping method [Figure 5], the sensitivity of this platform (PANAMutyper ®) increased up to nearly 70%.[12] This result was comparable to those of nondigital amplification refractory mutation system assay,[13] allele-specific PCR assay,[14] next-generation sequencing,[15] and droplet digital PCR (ddPCR) assay.[16] Furthermore, it can simultaneously detect multiple mutations while maintaining high sensitivity and specificity.[17]
Figure 5: Diagram of PANAMutyper® technology peptide nucleic acid clamping-assisted fluorescence melting curve analysis (PANAMutyper®) technology combines the advantages of PNA clamp technology (highly sensitive detection) and fluorescence melting curve analysis technology (advanced multiplex detection). PNA = Peptide nucleic acid

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In comparison with other methods, PANAMutyper ® can be performed easily with real-time PCR and can be finished in a brief time.[7]

PANAMutype ®, a novel method using PNA detection probe conjugated with a fluorescent dye and a quencher, can detect a specific target mutant-type DNA and each mutation can be genotyped by melting peak analysis. This method can detect 47 different mutations in exons 18, 19, 20, and 21 of EGFR gene with detection limits as low as 0.01% [Table 6]. In this study, the concordance rate of tissue and plasma samples was feasible (91.9%) for clinical use. The sensitivity was too low (64.5%) for the exclusion of EGFR mutation, consistent with other studies using plasma samples.[18] However, the specificity was high (100.0%) enough to the selection of EGFR TKIs as a treatment choice. It means ctDNA is a useful alternative for screening, which decreases the need for more dangerous and difficult tissue biopsy.
Table 6: Comparison of methods for epidermal growth factor receptor mutant detection

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Plasma samples of patients with TmEGFR or PMmEGFR by ddPCR were verified. Fifteen results were discrepant between in tumor tissue and in plasma samples either PANAMutyper ® or ddPCR. Two out of 15 cases only the results of ddPCR were consistent with tumor tissue, whereas 4 out of 15 cases only the results of PANAMutyper ® were consistent with tumor tissue. Altogether, PANAMutyper ® method seems not an inferior technology compared with ddPCR.

Tissue samples were also verified by PANAMutyper ®. The levels of semiquantitatively measured tumor DNA were lower in discordant cases when compared with concordant cases [Figure 2]. Although this analysis was done only in a limited number of cases, this result suggested very small amount of ctDNA as the cause of low sensitivity and, to overcome this problem, highly sensitive novel methods are necessary in the future for replacing all the tissue biopsies. The promising results of next-generation sequencing in tissue [19],[20] are not yet consistently proved in plasma samples and moreover is not an easily accessible technology in most hospitals.

T790M is well known as the most common resistant mechanism after use of the 1st- and 2nd- generation EGFR TKIs. Recently, many studies reported that this mutation is not only detected after EGFR TKIs as a result of acquired resistance, but also exists at the time of the diagnosis. The meaning and frequency of mixed mutation with T790M and other sensitive EGFR mutations is not clearly known.[21] PANAMutyper ® with tissue sample detected both T790M and L858R in 2 cases. PANAMutyper ® and ddPCR with plasma detected both mutations in one of these two cases, whereas PNA clamp with tissue detected only L858R in 2 cases. In the other four cases of mixed T790M mutations by PANAMutyper ® with tissue samples, two mixed mutations were missed by PNA clamp [Table 5]. Although in this small study we could not conclude, liquid biopsy may be more reliable method of detecting multiple mutations. Larger study is necessary to prove whether PANAMutyper ® can overcome tumor heterogeneity.

This study has several limitations. One is small sample size. In this study, cases with mutant EGFR were only 31 in tissue samples. The prevalence of EGFR mutation is very low (23.0%) compared with that of other studies,[2],[3] which may be due to the high rate of smoking males. In addition, because of insufficient tissue samples, all the cases could not be verified by PANAMutyper ®. Another limitation is that ddPCR analysis with ctDNA was done only in cases with either TmEGFR or PMmEGFR. However, the possibility of detecting mutant EGFR is very low in concordant cases with TwEGFR and PMwEGFR, and that newly detected mutation may be false positive. Furthermore, the purpose of proving the noninferiority of PANAMutyper ® to ddPCR can be achieved in this limited number of tests.

Although detecting EGFR mutation status is a pivotal step for the selection of the treatment modality in patients with advanced NSCLC, there are two major obstacles for clinicians. One is the difficulty of getting sufficient tumor tissue and the other is sampling bias due to the heterogeneity of tumor tissue. Using ctDNA in plasma, known as liquid biopsy, is a breakthrough. However, the sensitivity and specificity should be improved in various methods of liquid biopsy. In this study, we verified highly sensitive PANAMutyper ® for the detection of EGFR mutation in ctDNA. The concordance rate of EGFR mutation status in tissue and plasma samples was 91.9%. The sensitivity, specificity, NPV, and PPV were 64.5%, 100.0%, 90.4%, and 100.0%, respectively, according to the tissue samples as a standard. These results suggest that the detection of EGFR mutation status using ctDNA in plasma by PANAMutyper ® is a feasible test prior to tissue biopsy.

Acknowledgement

  1. The biospecimens for this study were provided by the Bio-Resource Bank at Dong-A University Medical Center, which were obtained with informed consent, as per the Institutional Review Board-approved protocol
  2. This work was supported by Dong-A University research fund.


Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

[22]



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]



 

 
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