A Highly Sensitive Next-Generation Sequencing-Based Genotyping Platform for EGFR Mutations in Plasma from Non-Small Cell Lung Cancer Patients
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Sel-CapTM, a digital enrichment next-generation sequencing (NGS)-based cancer panel, was assessed for detection of epidermal growth factor receptor (EGFR) gene mutations in plasma for non-small cell lung cancer (NSCLC), and for application in monitoring EGFR resistance mutation T790M in plasma following first-line EGFR-tyrosine kinase inhibitor (EGFR-TKI) treatment. Using Sel-Cap, we genotyped plasma samples collected from 185 patients for mutations Ex19del, L858R, and T790M, and compared results to those of PNAclampTM tumor biopsy (reference method, a peptide nucleic acid-mediated polymerase chain reaction clamping) and two other NGS liquid biopsies. Over two-thirds of activating mutations (Ex19del and L858R), previously confirmed by PNAclamp, were detected by Sel-Cap, which is 4–5 times more sensitive than NGS liquid biopsy. Sel-Cap showed particularly high sensitivity for T790M (88%) and for early-stage plasma samples. The relationship between initial T790M detection in plasma and progression-free survival (PFS) following first-line EGFR-TKIs was evaluated in 34 patients. Patients with T790M detected at treatment initiation (±3 months) had significantly shorter PFS than patients where T790M was first detected >3 months post treatment initiation (median PFS: 5.9 vs. 26.5 months; p < 0.0001). However, time from T790M detection to disease progression was not significantly different between the two groups (median around 5 months). In conclusion, Sel-Cap is a highly sensitive platform for EGFR mutations in plasma, and the timing of the first appearance of T790M in plasma, determined via highly sensitive liquid biopsies, may be useful for prediction of disease progression of NSCLC, around 5 months in advance.Keywords:
T790M
Liquid biopsy
The present pilot study assessed the usefulness of nanofluidic digital polymerase chain reaction (PCR) arrays in epidermal growth factor receptor (EGFR)-mutant lung adenocarcinoma after tyrosine kinase inhibitor (TKI) resistance.We enrolled 12 patients with primary lung adenocarcinoma with sensitive EGFR mutation-confirmed T790M status by re-biopsy after TKI resistance. Nanofluidic digital PCR arrays were used to quantify T790M in genomic DNA from the pre-treatment primary site and in serum cell-free DNA (cfDNA).On digital PCR, quantified T790M at the pre-treatment primary site was higher in re-biopsy-positive T790M patients (n=4) than in re-biopsy-negative patients (n=8) (0.78%±0.36% vs. 0.07%±0.09%, p<0.01). T790M at the pre-treatment primary site correlated with progression-free survival (PFS) after gefitinib therapy (r=0.67, p=0.016).Use of digital PCR to quantify T790M at the primary site of EGFR-mutant lung adenocarcinoma predicted T790M emergence in re-biopsies after TKI resistance and PFS after gefitinib therapy.
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// Eiji Iwama 1,2 , Koichi Takayama 2 , Taishi Harada 2 , Isamu Okamoto 3 , Fumihiko Ookubo 4 , Junji Kishimoto 5 , Eishi Baba 1 , Yoshinao Oda 6 and Yoichi Nakanishi 2,3 1 Faculty of Medical Sciences, Department of Comprehensive Clinical Oncology, Kyushu University, Fukuoka, Japan 2 Research Institute for Diseases of the Chest, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan 3 Center for Clinical and Translational Research, Kyushu University Hospital, Fukuoka, Japan 4 Division of Diagnostic Pathology, Kyushu University Hospital, Fukuoka, Japan 5 Department of Research and Development of Next Generation Medicine, Kyushu University, Fukuoka, Japan 6 Department of Anatomic Pathology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan Correspondence to: Eiji Iwama, email: // Keywords : EGFR, T790M, digital PCR, highly sensitive detection, quantification Received : January 09, 2015 Accepted : April 20, 2015 Published : May 10, 2015 Abstract The mutation of T790M in EGFR is a major mechanism of resistance to treatment with EGFR-TKIs. Only qualitative detection (presence or absence) of T790M has been described to date, however. Digital PCR (dPCR) analysis has recently been applied to the quantitative detection of target molecules in cancer with high sensitivity. In the present study, 25 tumor samples (13 obtained before and 12 after EGFR-TKI treatment) from 18 NSCLC patients with activating EGFR mutations were evaluated for T790M with dPCR. The ratio of the number of T790M alleles to that of activating mutation alleles (T/A) was determined. dPCR detected T790M in all 25 samples. Although T790M was present in all pre-TKI samples from 13 patients, 10 of these patients had a low T/A ratio and manifested substantial tumor shrinkage during treatment with EGFR-TKIs. In six of seven patients for whom both pre- and post-TKI samples were available, the T/A ratio increased markedly during EGFR-TKI treatment. Highly sensitive dPCR thus detected T790M in all NSCLC patients harboring activating EGFR mutations whether or not they had received EGFR-TKI treatment. Not only highly sensitive but also quantitative detection of T790M is important for evaluation of the contribution of T790M to EGFR-TKI resistance.
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Abstract A sensitive and convenient method for detecting epidermal growth factor receptor ( EGFR ) T790M mutations from circulating tumor DNA (ct DNA ) in advanced non–small cell lung cancer ( NSCLC ) patients with acquired EGFR ‐ TKI resistance would be desirable to direct patient sequential treatment strategy. A comparison of two platforms for detecting EGFR mutations in plasma ct DNA was undertaken. Plasma samples and tumor samples were collected from patients with acquired EGFR ‐ TKI resistance in Zhejiang Cancer Hospital from December 2014 to December 2015. Extracted ct DNA was analyzed using two platforms (Droplet Digital PCR and ARMS [ dPCR ]). A total of 108 patients were enrolled in this study. One hundred and eight patient plasma samples were detected by dd PCR and 75 were detected by ARMS . And 16 patients obtained tissue re‐biopsy, using ARMS assay for detecting EGFR T790M mutation. In all, 43.7% (47/108) had acquired T790M mutation by dd PCR . In 75 patient plasma samples, comparing dd PCR with ARMS , the rates of T790M mutation were 46.7% (35/75) and 25.3% (19/75) by dd PCR and ARMS , respectively. Of all, 16 patients both had tumor and plasma samples, the T790M mutation rates were 56.3% (9/16) by ARMS in tissue and 50.5% (8/16) by dd PCR in plasma ct DNA . The progression mode tended to gradual progression in T790M mutation patients (40.4%), but the T790M negative was inclined to the mode of dramatic progression (39.3%). The patients with T790M‐positive tumors had a longer time to disease progression after treatment with EGFR ‐ TKI s (median, 13.1 months vs. 10.8 months; P = 0.010) and overall survival (median, 35.3 months vs. 30.3 months; P = 0.214) compared with those with T790M‐negative patients. Our study demonstrates ddPCR assay may provide a highly sensitive method to detect EGFR T790M gene in plasma. And T790M‐positive patients have better clinical outcomes to EGFR ‐ TKI s than T790M‐negative patients.
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Abstract Purpose: The resistance to the EGFR tyrosine kinase inhibitors (TKI) is a major concern in non–small cell lung cancer (NSCLC) treatment. T790M mutation in EGFR accounts for nearly 50% of the acquired resistance to EGFR-TKIs. Earlier studies suggested that T790M mutation was also detected in TKI-naïve NSCLCs in a small cohort. Here, we use an ultra-sensitive droplet digital PCR (ddPCR) technique to address the incidence and clinical significance of pretreatment T790M in a larger cohort. Experimental Design: ddPCR was established as follows: wild-type or T790M mutation-containing DNA fragments were cloned into plasmids. Candidate threshold was identified using wild-type plasmid, normal human genomic DNA, and human A549 cell line DNA, which expresses wild type. Surgically resected tumor tissues from 373 NSCLC patients with EGFR-activating mutations were then examined for the presence of T790M using ddPCR. Results: Our data revealed a linear performance for this ddPCR method (R2 = 0.998) with an analytical sensitivity of approximately 0.001%. The overall incidence of the pretreatment T790M mutation was 79.9% (298/373), and the frequency ranged from 0.009% to 26.9%. The T790M mutation was detected more frequently in patients with a larger tumor size (P = 0.019) and those with common EGFR-activating mutations (P = 0.022), as compared with the others. Conclusions: The ultra-sensitive ddPCR assay revealed that pretreatment T790M was found in the majority of NSCLC patients with EGFR-activating mutations. ddPCR should be utilized for detailed assessment of the impact of the low frequency pretreatment T790M mutation on treatment with EGFR-TKIs. Clin Cancer Res; 21(15); 3552–60. ©2015 AACR.
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11054 Background: Patients with EGFR-mutant lung adenocarcinoma develop acquired resistance to EGFR-TKI (TKI) in 10-16 months. About 50% of patients with acquired resistance to TKI have T790M mutation (T790M). However, repeat biopsy to monitor mutation status is not practical. Digital PCR arrays limiting dilution of DNA and can detect single molecules, thus enabling extremely sensitive detection and quantification. We evaluated the usefulness of nanofluidic digital PCR arrays in EGFR-mutant lung adenocarcinoma. Methods: We enrolled 12 patients with primary lung adenocarcinoma and EGFR mutation (exon 19 deletion in 8; L858R in 4) at the pretreatment primary site who acquired resistance to gefitinib. Patients were divided into 2 groups according to T790M status after TKI resistance, as confirmed by rebiopsy. Nanofluidic digital PCR arrays (BioMark HD System, Fluidigm Japan K.K. Tokyo, Japan) were used to quantify T790M in genomic DNA from the pretreatment primary site and serum cell-free DNA (cfDNA) after TKI resistance. Numbers of mutant molecules were estimated by the number of positive chambers in the digital PCR chip and were corrected using the Poisson equation. We assessed the ratio of the number of positive T790M molecules to the number of positive exon 2 molecules. Results: The digital PCR array detected and quantified T790M (0.00-1.21%) in samples from the pretreatment primary site and serum cfDNA after TKI resistance. On digital PCR, the rebiopsy-positive T790M group (n=4) had a significantly higher quantified T790M at the pretreatment primary site than did the T790M-negative group (n=8) (0.59±0.58% vs 0.07±0.09%, p < 0.001). However, the results of analysis of serum cfDNA after TKI resistance did not significantly differ (0.09±0.14% vs 0.05±0.15%, respectively, p = 0.41). T790M at the pretreatment primary site as quantified by digital PCR significantly positively correlated with progression-free survival (PFS) after gefitinib therapy (r = 0.71, p < 0.001). Conclusions: Use of digital PCR to quantify T790M at the primary site of EGFR-mutant lung adenocarcinoma was useful for predicting T790M positivity in rebiopsies after TKI resistance and PFS after gefitinib therapy.
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Liquid biopsy allows the identification of targetable cancer mutations in a minimally invasive manner. In patients with advanced non-small cell lung cancer (NSCLC), droplet digital PCR (ddPCR) is increasingly used to genotype the epidermal growth factor receptor (EGFR) gene in circulating cell-free DNA (cfDNA). However, the sensitivity of this method is still under debate. The aim of this study was to implement and assess the performance of a ddPCR assay for detecting the EGFR T790M mutation in liquid biopsies.A ddPCR assay was optimized to detect the EGFR T790M mutation in plasma samples from 77 patients with NSCLC in progression.Our ddPCR assay enabled the detection and quantification of the EGFR T790M mutation at cfDNA allele frequency as low as 0.5%. The mutation was detected in 40 plasma samples, corresponding to a positivity rate of 52%. The number of mutant molecules per mL of plasma ranged from 1 to 6,000. A re-biopsy was analyzed for 12 patients that had a negative plasma test and the mutation was detected in 2 cases. A second liquid biopsy was performed for 6 patients and the mutation was detected in 3 cases.This study highlights the value of ddPCR to detect and quantify the EGFR T790M mutation in liquid biopsies in a real-world clinical setting. Our results suggest that repeated ddPCR tests in cfDNA may obviate tissue re-biopsy in patients unable to provide a tumor tissue sample suitable for molecular analysis.
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Background: The detection of the EGFR T790M (T790M) mutation in non-small cell lung cancer (NSCLC) patients who progressed under treatment with first- or second-generation EGFR-tyrosine kinase inhibitors (TKIs) is important to offer a subsequent therapy with a third-generation EGFR-TKI. Liquid biopsy is a powerful tool to determine the T790M mutation status. Several liquid biopsy platforms with varying degrees of accuracy are available to test for T790M mutations and sensitivities may differ among these methods. Methods: As no standard exists for the testing of T790M mutation in liquid biopsy, we performed a collaborative study to describe and compare the sensitivity of different in-house liquid biopsy platforms for the detection of the T790M mutation, EGFR exon 19 deletion (del19) and EGFR L858R mutation (L858R) across multiple participating laboratories in seven Central and Eastern European countries. Results: Of the 25 invited laboratories across Central and Eastern Europe, 21 centers participated and received 10 plasma samples spiked with cell-line DNA containing the T790M, del19, or L858R mutation in different concentrations. In-house PCR-based and NGS-based methods were used accordingly, and results reported as in routine clinical practice. Two laboratories, which used the AmoyDx® EGFR 29 Mutations Detection Kit (AmoyDx) with Cobas® cfDNA Sample Preparation Kit and QX200 Droplet Digital PCR (ddPCR) with the QIAamp Circulating Nucleic Acid Kit identified all ten samples correctly. Cobas® EGFR Mutation Test v2 (Cobas), the NGS methods, and the IdyllaTM detection method used in this study performed within the known sensitivity range of each detection method. Conclusions: If a negative result was obtained from methods with lower sensitivity (e.g. Cobas), repeated liquid biopsy testing and/or tissue biopsy analysis should be performed whenever possible, with the aim of identifying T790M-positive patients to allow them to receive the optimal second-line treatment with a third-generation EGFR TKI.
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Objective: To compare droplet digital PCR (ddPCR) and Super-amplification refractory mutation system (ARMS) in the detection of T790M mutation of epidermal growth factor receptor (EGFR) in the plasma of non-small cell lung cancer patients who had developed resistance to EGFR tyrosine kinase inhibitor (TKI) , and to investigate the clinical application of ddPCR. Methods: Plasma samples were collected from non-small cell lung cancer patients who had acquired EGFR-TKI resistance at Shanghai Pulmonary Hospital, Tongji University, from May 2017 to November 2017. Extracted ctDNA was analyzed by ddPCR and Super-ARMS to evaluate the T790M mutation status of EGFR gene. Results: A total of 37 patients with activating EGFR mutation that acquired resistance to EGFR-TKI were selected in the study, including 17 male and 20 female with a median age of 64 years (range 40-83 years). Before TKI treatment, all the patients harbored EGFR inhibitor sensitive mutations but without T790M mutation. After acquiring resistance to EGFR-TKI treatment, the T790M mutation rate detectable by ddPCR was 45.9% (17/37). In contrast, the mutation rate of T790M detectable by Super-ARMS was 35.1% (13/37, P<0.05). For the 13 positive cases detected by Super-ARMS (ΔCt<8), they were all positive by ddPCR assay; Among the 10 negative cases detected by Super-ARMS (ΔCt≥8), there were 3 cases positive by ddPCR assay. For patients without ΔCt by Super-ARMS assay, there was one weak positive case detectable by ddPCR assay. Among 17 EGFR T790M positive patients, 9 received EGFR inhibitor Osimertinib treatment, and 7 of them had good therapeutic response after the treatment. Conclusions: While a significant correlation between the two methods is shown. ddPCR is more sensitive than Super-ARMS in the detection of EGFR T790M mutation, indicating that it is a better method in guiding target drug therapy of non-small cell lung cancer patients after acquiring the resistance to EGFR-TKI.目的： 比较微滴数字PCR(ddPCR)与Super-扩增阻滞突变系统(ARMS)法检测表皮生长因子受体(EGFR)酪氨酸激酶抑制剂(TKI)治疗后耐药非小细胞肺癌患者血浆中EGFR基因耐药位点T790M突变的差异，并探讨ddPCR的临床应用价值。 方法： 收集同济大学附属上海市肺科医院2017年5至11月间37例经EGFR-TKI治疗后耐药的非小细胞肺癌患者血浆标本；分别采用ddPCR和Super-ARMS法检测血浆标本中EGFR基因T790M突变情况。 结果： EGFR-TKI治疗耐药后非小细胞肺癌患者，男性17例，女性20例，年龄范围40～83岁，中位年龄64岁。治疗前，37例患者肿瘤组织均检测出EGFR基因敏感突变，无T790M突变。靶向治疗耐药后，ddPCR检测患者血浆游离DNA EGFR基因T790M突变，阳性率为45.9%(17/37)，Super-ARMS检测阳性率为35.1%(13/37)，统计学分析发现，两种方法检测EGFR T790M突变阳性率差异具有统计学意义(P<0.05)。13例Super-ARMS法T790M突变病例(ΔCt值<8)，经ddPCR法检测均为阳性；Super-ARMS法10例翘尾病例(ΔCt值≥8，T790M突变阴性)，ddPCR法检测显示有3例T790M突变；Super-ARMS法14例完全阴性病例(无ΔCt值，T790M突变阴性)，ddPCR法检测显示有1例T790M突变弱阳性。ddPCR法检测T790M突变结果与Super-ARMS法检测判读的ΔCt值之间有显著相关性。在ddPCR检测出的17例T790M阳性的患者中，其中9例患者接受了奥西替尼治疗，7例患者都有良好的获益。 结论： ddPCR检测EGFR-TKI治疗耐药后非小细胞肺癌患者血浆EGFR基因T790M突变，比Super-ARMS法具有更高的灵敏度，这可能会在指导临床靶向药物治疗上具有广泛的应用价值。.
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