Detection Of EGFR T790M Mutation Research Articles

Abstract 3750: Dual isolation and profiling of circulating tumor cells and circulating tumor DNA from a single liquid sample using the Genesis Cell Isolation System and Droplet Digital PCR

Abstract Introduction: Recent advances in technology support the use of liquid biopsy as a noninvasive method for cancer monitoring and diagnosis. Circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) are both regarded as key liquid biopsy biomarkers, offering significant disease-related molecular insights. Despite their potential, dual isolation and profiling have been challenged in a clinical setting by the lack of established platforms and optimized protocols that support this approach. The Genesis System with Celselect SlidesTM captures CTCs based on size and deformability, then the enriched cells can be recovered for subsequent downstream analysis. Concurrently, Droplet Digital PCR (ddPCRTM) can be utilized to interrogate cell-free DNA (cfDNA) to determine the presence of a range of DNA aberrations present in ctDNA. This study, using a model lung CTC-ctDNA sample, illustrates the feasibility of this dual-biomarker approach to detect cancer-associated nucleic acid mutations simultaneously with CTC enumeration, using commercially available kits and instruments. Methods: To model lung cancer samples, blood samples were prepared by spiking in two model circulating markers, cultured cancer cells (model CTCs, human ㅣlung cancer cell line A549) and fragmented genomic DNA containing EGFR mutations (model ctDNA, multiplexed 23 ctDNA [~145 bp band > 95%]), into whole blood from a healthy donor prior to enrichment/extraction. Plasma isolation from the whole blood model samples was performed using either two-step centrifugation or Ficoll-Paque PLUS solution for plasma/buffy coat separation. The extracted plasma layer was used for ctDNA separation, while the remaining cell layer was used for CTC isolation on the Genesis System with a modified protocol. Results: The Genesis System isolated 78-79% of spiked cancer cells in whole blood at a concentration of 40 cells/ml. Despite additional plasma separation steps and liquid handling, the system isolated CTCs spiked in with minimal cell loss (<6%). With an optimized ctDNA extraction, more than 70% of spiked ctDNA in blood samples in the size range of 120-220 bp was extracted. Using the Bio-Rad QX200 ddPCR System and ddPCR EGFR T790M Mutation Detection Assay, blood samples with the model ctDNA spiked in showed positive droplets (17 copies) for EGFR T790M while control blood samples without model ctDNA spiked in showed no positive droplets. Discussion and Conclusions: This work illustrates an optimized protocol for the dual isolation and profiling of CTCs and ctDNA from a single blood sample by using an automated CTC isolation platform and high-sensitivity ddPCR based ctDNA mutation detection. This protocol enables comprehensive liquid biopsy studies using a single blood sample from cancer patients with readily available instruments and kits, which can be applied to many other cancers. Citation Format: Yoon-Tae Kang, Abiodun Bodunrin, Errile Pusod, Kris Simonyi, Adam Corner, David Coe, Michelle Racey, Elizabeth J. Dreskin. Dual isolation and profiling of circulating tumor cells and circulating tumor DNA from a single liquid sample using the Genesis Cell Isolation System and Droplet Digital PCR [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3750.

RLP system: A single-tube two-step approach with dual amplification cascades for rapid identification of EGFR T790M

BackgroundThe detection of cancer gene mutations in biofluids plays a pivotal role in revolutionizing disease diagnosis. The presence of a large background of wild-type sequences poses a challenge to liquid biopsy of tumor mutation genes. Suppressing the detection of wild-type sequences can reduce their interference, however, due to the minimal difference between mutant and wild-type sequences (such as single nucleotide variants differing by only one nucleotide), how to suppress the detection of wild-type sequences to the greatest extent without compromising the sensitivity of mutant sequence detection remains to be explored. SignificanceThe RLP system addresses the incompatibility between RPA and RT-PCR reactions through a physical separation strategy. Besides, due to the remarkable flexibility of locked nucleic acid probes, the RLP system emerges as a potent tool for detecting mutations across diverse genes. It excels in sensitivity and speed, tolerates plasma matrix, and is cost-effective. This bodes well for advancing the field of precision medicine. ResultsThe recombinase-assisted locked nucleic acid (LNA) probe-mediated dual amplification biosensing platform (namely RLP), which combines recombinase polymerase amplification (RPA) and LNA clamp PCR method in one tube, enabling highly sensitive and selective detection of EGFR T790M mutation under the help of well-designed LNA probes. This technique can quantify DNA targets with a limit of detection (LoD) at the single copy level and identify point mutation with mutant allelic fractions as low as 0.007 % in 45 min. Moreover, RLP has the potential for the direct detection of plasma samples without the need for nucleic acid extraction and the cost of a single test is less than 1USD. Furthermore, the RLP system is a cascading dual amplification reaction conducted in a single tube, which eliminates the risk of cross-contamination associated with opening multiple tubes and ensures the reliability of the results.

Abstract 780: Digital PCR-based EGFR T790M mutation detection from human blood samples collected in PAXgene Blood ccfDNA Tubes

Abstract Introduction: Circulating cell-free DNA (ccfDNA) is used as an analyte in liquid biopsy-based cancer research with promising applications in the fields of cancer screening, therapeutic decision making and monitoring and minimal residual disease control. However, despite the multitude of future applications, challenges regarding the sensitivity of analytical tests remain, thereby limiting the routine use of ccfDNA within the above mentioned fields. In this study, we investigated the compatibility of the PAXgene® Blood ccfDNA Tube and the QIAcuity® Digital PCR System as an optimal “sample to insight” workflow with regard to the detection of EGFR T790M mutations spiked into apparently healthy human donor blood specimens. Methods: Blood from 22 apparently healthy consented donors was collected into PAXgene Blood ccfDNA Tubes* (PreAnalytiX). Within 2 h of blood draw, blood samples were either spiked with an EGFR-Multiplex 5% AF cfDNA standard (SensID) containing an EGFR T790M mutation or left unspiked as a control. Tubes were incubated for 3 d at room temperature to simulate transport over a weekend and plasma was generated by double centrifugation. Plasma pools were generated from spiked and unspiked samples and ccfDNA was isolated with the QIAamp® Circulating Nucleic Acid Kit (QIAGEN). CcfDNA quantity was measured by Qubit® dsDNA HS Assay (Thermo Fisher Scientific) and fragment size distribution was determined by TapeStation® Cell-free DNA ScreenTape® (Agilent Technologies). EGFR T790M mutation detection was performed using the dPCR Mutation Assay EGFR 6240 Human (QIAGEN) with a 26K 24-well Nanoplate (QIAGEN) on the QIAcuity Digital PCR System (QIAGEN). Data analysis was conducted using the QIAcuity Software Suite (QIsAGEN). Results: CcfDNA isolated from PAXgene Blood ccfDNA Tubes had a fragment size distribution profile with distinct mononucleosomal peaks at ~180 bps without high-molecular weight DNA, indicating successful nucleated cell stabilization over the simulated transport duration and extraction of high-quality ccfDNA. Digital PCR analysis of extracted ccfDNA revealed positive detection of EGFR T790M mutations in spiked blood samples at a relative mutation fraction of approximately 2% in the investigated samples. Conclusion: PAXgene Blood ccfDNA Tubes enabled high-quality ccfDNA isolation by stabilizing nucleated cells during simulated transport condition. Spiked EGFR T790M mutations were successfully detected by dPCR following sample storage. This proof of principle study shows the combined usability of both systems for mutation detection in human blood specimens, highlighting the potential of both technologies for a broader application in the liquid biopsy field to detect oncogenic driver as well as tumor suppressor gene mutations in a multitude of tumor types. *The PAXgene Blood ccfDNA Tube is for Research Use Only in the US. Not for use in diagnostic procedures. Citation Format: Christian Neander, Andrea Huxhold, Maike Schoenborn, Thorsten Voss. Digital PCR-based EGFR T790M mutation detection from human blood samples collected in PAXgene Blood ccfDNA Tubes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 780.

EGFR T790M Mutation Detection in Patients With Non-Small Cell Lung Cancer After First Line EGFR TKI Therapy: Summary of Results in a Three-Year Period and a Comparison of Commercially Available Detection Kits.

EGFR mutation in non-small cell lung cancer (NSCLC) offers a potential therapeutic target for tyrosine kinase inhibitor (TKI) therapy. The majority of these cases, however eventually develop therapy resistance, mainly by acquiring EGFR T790M mutation. Recently, third-generation TKIs have been introduced to overcome T790M mutation-related resistance. Cell free circulating tumor DNA (liquid biopsy) has emerged as a valuable alternative method for T790M mutation detection during patient follow up, when a tissue biopsy cannot be obtained for analysis. In this study, we summarized our experience with Super-ARMS EGFR Mutation Detection Kit (AmoyDx) on 401 samples of 242 NSCLC patients in a 3-year period in Hungary, comprising 364 plasma and 37 non-plasma samples. We also compared the performance of two commercially available detection kits, the cobas EGFR Mutation test v2 (Roche) and the Super-ARMS EGFR Mutation Detection Kit (AmoyDx). The same activating EGFR mutation was detected with the AmoyDx kit as in the primary tumor in 45.6% of the samples. T790M mutation was identified in 48.1% of the samples containing activating EGFR mutation. The detection rate of T790M mutation was not dependent on the DNA concentration of the plasma sample and there was no considerable improvement in mutation detection rate after a second, subsequent plasma sample. The concordance of EGFR activating mutation detection was 89% between the two methods, while this was 93% for T790M mutation detection. The AmoyDx kit, however showed an overall higher detection rate of T790M mutation compared to the cobas kit (p = 0.014). T790M mutation was detected at 29.8% of the patients if only plasma samples were available for analysis, while the detection rate was 70.2% in non-plasma samples. If the activating EGFR was detected in the plasma samples, the detection rate of T790M mutation was 42.4%. Although non-plasma samples provided a superior T790M mutation detection rate, we found that liquid biopsy can offer a valuable tool for T790M mutation detection, when a tissue biopsy is not available. Alternatively, a liquid biopsy can be used as a screening test, when re-biopsy should be considered in case of wild-type results.

Direct digital polymerase chain reaction chip for the detection of EGFR T790M mutation in plasma

Nucleic acid extraction and purification before amplification is considered an essential step for nucleic acid amplification testing. However, this may cause losses or introduce errors that can lead to inaccurate results, especially when using samples with a small nucleic acid concentration. Here, we developed a direct digital chip that enabled us to detect nucleic acid without DNA extraction and purification. We have developed a self-priming liquid-dispensing digital PCR chip that does not require any external power. This is a robust anti-evaporation digital PCR chip with fast sampling and accurate quantification performance. Using this chip, we have established an on-chip direct nucleic acid amplification method that does not require nucleic acid extraction and purification for liquid biopsy samples. In order to verify the feasibility of this chip for clinical samples, we detected the EGFR T790M mutation from plasma. Results showed that EGFR T790M mutation could be detected with an accuracy of 100% and a sensitivity of 0.01%. Without nucleic acid extraction and purification, the assay avoids complex pre-processing, thus saving time and achieving precise quantification. We expect our direct digital PCR chip to have practical applications in diagnosis, screening, and research, especially in resource-deprived regions.

A Pan-Canadian Validation Study for the Detection of EGFR T790M Mutation Using Circulating Tumor DNA From Peripheral Blood

IntroductionGenotyping circulating tumor DNA (ctDNA) is a promising noninvasive clinical tool to identify the EGFR T790M resistance mutation in patients with advanced NSCLC with resistance to EGFR inhibitors. To facilitate standardization and clinical adoption of ctDNA testing across Canada, we developed a 2-phase multicenter study to standardize T790M mutation detection using plasma ctDNA testing. MethodsIn phase 1, commercial reference standards were distributed to participating clinical laboratories, to use their existing platforms for mutation detection. Baseline performance characteristics were established using known and blinded engineered plasma samples spiked with predetermined concentrations of T790M, L858R, and exon 19 deletion variants. In phase II, peripheral blood collected from local patients with known EGFR activating mutations and progressing on treatment were assayed for the presence of EGFR variants and concordance with a clinically validated test at the reference laboratory. ResultsAll laboratories in phase 1 detected the variants at 0.5 % and 5.0 % allele frequencies, with no false positives. In phase 2, the concordance with the reference laboratory for detection of both the primary and resistance mutation was high, with next-generation sequencing and droplet digital polymerase chain reaction exhibiting the best overall concordance. Data also suggested that the ability to detect mutations at clinically relevant limits of detection is generally not platform-specific, but rather impacted by laboratory-specific practices. ConclusionsDiscrepancies among sending laboratories using the same assay suggest that laboratory-specific practices may impact performance. In addition, a negative or inconclusive ctDNA test should be followed by tumor testing when possible.

Detection of EGFR T790M Mutation by Droplet Digital Polymerase Chain Reaction in Lung Carcinoma Cytology Samples.

Advanced-stage non-small cell lung carcinoma patients on EGFR-targeted tyrosine kinase inhibitors frequently present with an acquired EGFR T790M resistance mutation. Early detection using a high-sensitivity assay is critical to allow patients to switch to third-generation tyrosine kinase inhibitors. The detection of EGFR T790M mutation is often challenging because of low tumor fraction in posttreatment specimens. Because a large fraction of non-small cell lung carcinoma patients are given a diagnosis by cytology, evaluating a high-sensitivity technique for EGFR T790M detection in these specimens is essential. To evaluate a high-sensitivity droplet digital polymerase chain reaction (ddPCR) assay for EGFR T790M using different cytologic specimen preparations. A total of 42 cytology samples, including smears and cell block preparation, were evaluated for EGFR T790M using ddPCR. The results of the mutation assay were compared to the patient's known EGFR T790M mutation status. The ddPCR assay successfully determined the EGFR T790M mutation status in 36 of 42 samples (86%), including samples with low tumor fraction (≤20%). In 4 cases the results of the ddPCR assay could not be compared because the mutation status was unknown at the time of collection of the cytology sample. There was 1 false-positive result, with borderline positivity, and 1 false-negative result. Overall sensitivity and specificity of the ddPCR assay were 93% and 96%, respectively. Our results indicate that EGFR T790M ddPCR is a highly sensitive and specific mutational assay that can be used reliably in cytologic specimens, including samples with low tumor fraction.

P2.01-71 Comprehensive Analysis of EGFR T790M-Mutant Abundance of Different Technologies and Its Effect on Efficacy of Osimertinib in Advanced NSCLC

Osimertinib has been adopted as the standard of care for T790M-mediated acquired epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) resistance. And detecting EGFR T790M mutation can be challenging in routine care, low abundance of the mutation and difficulty for re-biopsy in patients with advanced disease. We aim to compare droplet digital PCR (ddPCR) and next generation sequencing (NGS) in T790M mutation testing, and analyze whether the abundance of T790M mutation is associated with the efficacy of osimertinib in advanced Non–small cell lung cancer (NSCLC). We retrospectively studied 132 T790M-positive advanced NSCLC patients who have received osimertinib after acquired TKI resistance from April 2017 to August 2018 at our institution. Treatment response was evaluated and survival data were collected and analyzed. Among the 132 patients, 18(13.6%) had adopted amplifcation refractory mutation system (ARMS), 58(43.9%) adopted ddPCR, 43(32.6%) used NGS in T790M testing, and the detection methods of 13(9.9%) patients were unknown. Data of T790M-mutatnt abundance were obtained in 64 patients, 57 of whom were tested by ddPCR in plasma and the other 7 by NGS. The median progression free survival (PFS) was 15 months, median OS was not reached, objective response rate (ORR) was 88%. We found that ARMS, ddPCR, NGS have no difference in T790M testing (p=0.114). Among the 57 ddPCR testing patients with the abundance of T790M, there was no difference in PFS of T790M abundance in the cutoff value of 0.5 (p=0.303). Our results suggest that ARMS, ddPCR and NGS are all useful and reliable methods in EGFR T790M mutation detection. And the association between the abundance of T790M mutation using ddPCR in plasma and the efficacy of osimertinib seemed not strong and remained to be explored.

Abstract 4899: The impact of the EGFR-T790M mutation detection by re-biopsy in EGFR mutant NSCLC patients in the retrospective analysis

Abstract Background: EGFR-TKIs show a good anticancer effect in to most of patients with advanced non-small cell lung cancer (NSCLC) harboring EGFR activating mutations. However, it ultimately acquires resistance to EGFR-TKIs. We currently attempt to detect EGFR-T790M mutation by re-biopsy after disease progression, because the third generation EGFR-TKI osimertinib was effective against the refractory tumors with EGFR-T790M mutation. However, the re-biopsy from tumors is relatively invasive and some cases are impossible to biopsy. Therefore, it is a beneficial if we predict the EGFR-T790M mutation before re-biopsy. In this study, we analyzed the patients with disease progression after initial EGFR-TKIs to address the issues. Methods: 78 advanced NSCLC patients with EGFR mutations were enrolled from five institutions in Japan. In all patients, re-biopsy samples were obtained successfully after the resistance to initial EGFR-TKI treatment We analyzed the relationship between the emergence of EGFR-T790M mutation and clinical parameters including clinical outcomes with EGFR-TKI treatment and EGFR activating mutation status. Results: Of 78 advanced NSCLC patients with EGFR mutations, 39 patients were EGFR-T790M positive and 39 were negative based on the re-biopsy samples. Of 39 EGFR-T790M positive patients, 2 patients achieved complete response (CR), 33 partial response (PR), and 4 stable disease (SD) by initial EGFR-TKI treatment. In contrast, 1 patient experienced a CR, 19 a PR, 18 SD, and 1 progressive disease (PD) in 39 T790M negative patients. The objective response rate was higher in patients with T790M positive mutations than those with T790M negative mutations (89.7% versus 51.2%, p< 0.01). There was no statistically significant difference in progression free survival and time to failure treated with initial EGFR-TKIs between both groups. Conclusion: The response to initial EGFR-TKIs treatment might be one of good predictors for emerging of refractory tumors with EGFR-T790M mutation. Citation Format: Akihiro Yoshimura, Tadaaki Yamada, Naoko Okura, Junji Uchino, Koichi Takayama. The impact of the EGFR-T790M mutation detection by re-biopsy in EGFR mutant NSCLC patients in the retrospective analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4899.

Tag-based next generation sequencing: a feasible and reliable assay for EGFR T790M mutation detection in circulating tumor DNA of non small cell lung cancer patients

BackgroundThe demonstration of EGFR T790M gene mutation in plasma is crucial to assess the eligibility of Non Small Cell Lung Cancer (NSCLC) patients, who have acquired resistance to first or second generation Tyrosine Kinase Inhibitors (TKIs), to receive a subsequent treatment with osimertinib. Since circulating tumor DNA (ctDNA) is present in very low amounts in plasma, high sensitive and specific methods are required for molecular analysis.Improving sensitivity of T790M mutation detection in plasma ctDNA enables a larger number of NSCLC patients to receive the appropriate therapy without any further invasive procedure.MethodsA tag-based next generation sequencing (NGS) platform capable of tagging rare circulating tumor DNA alleles was employed in this study for the identification of T790M mutation in 42 post-TKI NSCLC patients.ResultsCompared to Real Time PCR, tag-based NGS improved the T790M detection rate (42.85% versus 21.4%, respectively), especially in those cases with a low median mutation abundance (i.e. 0.24, range 0.07–0.78). Moreover, the tag-based NGS identified EGFR activating mutations more efficiently than Real Time PCR (85.7% versus 61.9% detection rate, respectively), particularly of the L858R variant type (0.06–0.75 mutation abundance range). Patients in whom the T790M mutation was detected in plasma, achieved an objective response to osimertinib (9/14, 64.28%).ConclusionsTag-based NGS represents an accurate and sensitive tool in a clinical setting for non-invasive assessment and monitoring of T790M variant in NSCLC patients.

The impact of the tumor shrinkage by initial EGFR inhibitors according to the detection of EGFR-T790M mutation in patients with non-small cell lung cancer harboring EGFR mutations

BackgroundThe EGFR-T790M mutation is clinically detected using re-biopsy in approximately 50% of patients with acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) in advanced non-small cell lung cancer (NSCLC) who harbor EGFR mutations. However, little is known about the population of NSCLC patients who develop acquired resistance due to the T790M mutation. In this study, we focused on the emergence of the T790M mutation and analyzed patients refractory to initial EGFR-TKIs with successful re-biopsy samples.MethodsSeventy-eight advanced NSCLC patients with EGFR mutations who had successful re-biopsy samples after resistance to initial EGFR-TKI treatment were enrolled at 5 institutions in Japan. We validated the association between the emergence of the T790M mutation and their clinical profiles.ResultsThirty-nine patients tested positive for T790M and 39 tested negative in the re-biopsy samples. The objective response rate to initial EGFR-TKIs was higher in patients with the T790M mutation than in those without the mutation (89.7% versus 51.2%, p < 0.001). Moreover, there was a significant difference in the maximal tumor shrinkage rate relative to baseline in T790M-positive tumors compared with T790M-negative tumors (42.7% versus 24.0%, p = 0.001). Multivariate analysis demonstrated that the maximum tumor shrinkage rate was a significant predictive factor for the detection of the T790M mutation (p = 0.023, odds ratio 1.03, 95% confidence interval 1.00–1.05).ConclusionsOur retrospective observations suggested that the maximum tumor shrinkage rate with initial EGFR-TKI treatment might be one of the promising predictive biomarkers for emerging refractory tumors with the EGFR-T790M mutation.

P1.13-16 The Diagnostic Accuracy of Circulating Tumor DNA for the Detection of EGFR-T790M Mutation in NSCLC: A Systematic Review and Meta-Analysis

P1.13-16 The Diagnostic Accuracy of Circulating Tumor DNA for the Detection of EGFR-T790M Mutation in NSCLC: A Systematic Review and Meta-Analysis

P3.01-80 Retrospective Analysis of the Impact of EGFR T790M Mutation Detection by Re-Biopsy in Patients with NSCLC Harboring EGFR Mutations.

EGFR-TKIs show a good response to most of patients with advanced non-small cell lung cancer (NSCLC) harboring EGFR activating mutations. However, it ultimately becomes the acquired resistance to EGFR-TKIs after various periods. We currently attempt to detect EGFR-T790M mutation by re-biopsy that induced a half of acquired resistances to them, because the third generation EGFR-TKI osimertinib had an effective response against the refractory tumors with EGFR-T790M mutations. However, the re-biopsy from tumors is relatively invasive and some cases are impossible to perform them. Therefore, it is a critical issue to select the population with EGFR-T790M mutations. In this study, we analyzed the refractory cases to initial EGFR-TKIs with successful re-biopsy samples to disclose these clinical questions. 78 advanced NSCLC patients with EGFR mutations who had successful re-biopsy samples after the resistance to initial EGFR-TKI treatment are enrolled at five institutions in Japan. We validated for the association between the emergence of EGFR-T790M mutation and their profiles, such as clinical outcomes with EGFR-TKI treatment and EGFR activating mutation status. s Of 78 advanced NSCLC patients with EGFR mutations, 39 cases were EGFR-T790M positive and 39 were negative in the re-biopsy samples. Of EGFR-T790M positive patients, 2 cases achieved a complete response (CR), 33 a partial response (PR), and 4 stable disease (SD). In contrast, 1 patient experienced a CR, 19 a PR, 18 SD, and 1 progressive disease (PD) in T790M negative patients. The objective response rate was higher in patients with T790M positive mutations than in those with T790M negative mutations (89.7% versus 51.2%, p< 0.01). There were no differences between each patients in progression free survival and time to failure treated with initial EGFR-TKIs. The response to initial EGFR TKI treatment might be one of good predictors for emerging of refractory tumors with EGFR-T790M mutations. Further experiments are needed to identify them.

The diagnostic accuracy of circulating tumor DNA for the detection of EGFR-T790M mutation in NSCLC: a systematic review and meta-analysis

This pooled analysis aims at evaluating the diagnostic accuracy of circulating tumor (ct) DNA for the detection of EGFR-T790M mutation in NSCLC patients who progressed after EGFR-TKIs. Data from all published studies, reporting both sensitivity and specificity of plasma-based EGFR-T790M mutation testing by ctDNA were collected by searching in PubMed, Cochrane Library, American Society of Clinical Oncology, European Society of Medical Oncology and World Conference of Lung Cancer meeting proceedings. A total of twenty-one studies, with 1639 patients, were eligible. The pooled sensitivity of ctDNA analysis was 0.67 (95% CI: 0.64–0.70) and the pooled specificity was 0.80 (95% CI: 0.77–0.83). The pooled positive predictive value (PPV) was 0.85 (95% CI: 0.82–0.87) and the pooled negative predictive value (NPV) was 0.60 (95% CI: 0.56–0.63). The positive likelihood ratio (PLR) and negative likelihood ratio (NLR) were 2.67 (95% CI: 1.86–3.82) and 0.46 (95% CI: 0.38–0.54), respectively. The pooled diagnostic odds ratio (DOR) was 7.27 (4.39–12.05) and the area under the curve (AUC) of the summary receiver operating characteristics (sROC) curve was 0.77. The ctDNA analysis represents a promising, non-invasive approach to detect and monitor the T790M mutation status in NSCLC patients. Development of standardized methodologies and clinical validation are recommended.

Abstract B32: RADIANCE: An open-label, nonrandomized, prospective biomarker study to assess analytic concordance between noninvasive testing and tissue testing for EGFR T790M mutation detection in patients with non-small cell lung cancer

Abstract Background: EGFR-TKIs are the recommended first-line therapy of patients (pts) with advanced non-small cell lung cancer (NSCLC) who have an EGFR-sensitizing mutation (EGFRm). However, most pts develop resistance to EGFR-TKIs, with the EGFR T790M resistance mutation observed in &amp;gt;50% of pts on first- and second-generation EGFR-TKIs. Osimertinib is a third-generation, CNS-active EGFR-TKI that potently and selectively inhibits both EGFRm and T790M resistance mutations, recommended for pts with T790M-positive advanced NSCLC who have progressed on first-line EGFR-TKI therapy. Tumor tissue is the preferred sample source for T790M testing; however, often tumor biopsy is not feasible following disease progression due to inaccessible sites and/or concerns about safety. Testing using plasma samples offers a less invasive, albeit less sensitive, alternative method for the detection of T790M-positive circulating tumor (ct) DNA. Additional noninvasive options, including urine ctDNA analyses, are also being investigated. The RADIANCE trial is designed to determine if urine and plasma tests can provide similar detection rates to tissue testing for T790M detection when combined. Pts identified as T790M-positive may be offered treatment with osimertinib. Trial Design: RADIANCE (NCT03137264) is an open-label, prospective biomarker study with a primary objective of assessing the analytic concordance between noninvasive testing (Guardant360 Plasma Assay and Trovera Liquid Biopsy Test using urine) and tissue testing (cobas® EGFR Mutation Test v2) for T790M in pts with NSCLC. Approximately 470 pts will be enrolled. The study consists of two parts, Diagnostic Analytic Validity (Part 1) and Clinical Outcomes (Part 2). In Part 1, eligible pts will provide a tumor biopsy (cobas tissue test), plasma (cobas plasma and Guardant360 tests) and urine sample (Trovera test) for T790M testing. Eligibility criteria include a primary diagnosis of EGFRm-positive NSCLC with evidence of disease progression during or following treatment with a first- or second-generation EGFR-TKI. Pts must not be enrolled on another clinical trial, or have previously received osimertinib or another T790M-directed therapy. In Part 2, pts with T790M-positive status by cobas tissue and/or cobas plasma test will be treated according to standard of care, and may receive osimertinib. Pts will be followed for clinical outcomes for up to 18 months, and evaluated for objective response, progression-free survival, and duration of response (investigator-assessed using RECIST v1.1) and safety. Pts who are T790M negative or do not receive osimertinib will have completed the study after Part 1. Citation Format: Hatim Husain, Martin Dietrich, Steven Dubinett, David E. Gerber, Jeffrey Gregg, Michelle Shiller, Howard West, Kim Thai, Alvin Milner, Nabil Chehab, Christina Baik. RADIANCE: An open-label, nonrandomized, prospective biomarker study to assess analytic concordance between noninvasive testing and tissue testing for EGFR T790M mutation detection in patients with non-small cell lung cancer [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr B32.

A CODAR-PCR assay using nucleic acids extracted from whole blood samples to provide superior sensitivity in EGFR T790M mutation detection and accurate prediction of osimertinib response in advanced NSCLC patients.

e24221Background: Plasma-derived circulating tumor DNA (ctDNA) has been the predominant source of nucleic acids for tumor mutation detection in today’s liquid biopsy technologies. However tumor-der...

Validation and comparison of two NGS assays for the detection of EGFR T790M resistance mutation in liquid biopsies of NSCLC patients.

Analysis of circulating cell-free DNA (cfDNA) derived from peripheral blood (“liquid biopsy”) is an attractive alternative to identify non-small cell lung cancer (NSCLC) patients with the EGFR T790M mutation eligible for 3rd generation tyrosine kinase inhibitor therapy.We evaluated two PCR-based next generation sequencing (NGS) approaches, one including unique molecular identifiers (UMI), with focus on highly sensitive EGFR T790M mutation detection. Therefore, we extracted and sequenced cfDNA from synthetic plasma samples spiked with mutated DNA at decreasing allele frequencies and from 21 diagnostic NSCLC patients. Data evaluation was performed to determine the limit of detection (LoD), accuracy, specificity and sensitivity of both assays.Considering all tested reference dilutions and mutations the UMI assay performed best in terms of LoD (1% vs. 5%), sensitivity (95.8% vs. 81.3%), specificity (100% vs. 93.8%) and accuracy (96.9% vs. 84.4%). Comparing mutation status of diagnostic samples with both assays showed 81.3% concordance with primary mutation verifiable in 52% of cases. EGFR T790M was detected concordantly in 6/7 patients with allele frequencies from 0.1% to 27%. In one patient, the T790M mutation was exclusively detectable with the UMI assay.Our data demonstrate that both assays are applicable as multi-biomarker NGS tools enabling the simultaneous detection of primary EGFR driver and resistance mutations. However, for mutations with low allelic frequencies the use of NGS panels with UMI facilitates a more sensitive and reliable detection.

P2.03-040 EGFR T790M Mutation Detection and Osimertinib Treatment Response Evaluation by Liquid Biopsy in Advanced NSCLC Patients

Lung cancer remains the leading cause of cancer-related death worldwide. Though most patients with EGFR activating mutations are sensitive to EGFR tyrosine kinase inhibitors (TKIs), tumors will inevitably acquire resistance to first generation EGFR TKIs. EGFR T790M mutation accounts for about 50% of these resistance cases. Droplet digital PCR (ddPCR) and cobas enables quantification of specific mutated ctDNA. In this study, these methods were used to detect and evaluate the ctDNA response after Osimertinib treatment. In the screening period of ASTRIS (D5160C00022) study in single center, tumor and blood samples from 69 stage IIIB-IV NSCLC patients defined as acquired resistance to first generation EGFR TKIs (Gefitinib, Elortinib or Ecotinib) were collected. The cobas® Mutation Test v2 kit was used to detect EGFR mutations in FFPE or plasma samples. Plasma T790M mutation of both Osimertinib naïve and treated patients were quantified by Droplet digital PCR (ddPCR). The T790M mutation rate of FFPE tissue cobas, plasma cobas and plasma ddPCR testing were 54.5%, 21.3% and 30.4% respectively. Taking the FFPE tissue cobas testing as gold standard, the sensitivity and specificity of plasma ddPCR to detect T790M mutation was 66.7% and 63.6%. Taking all testing methods into account, the T790M positive rate was 52.2%. In all of the plasma cobas positive cases, T790M mutation was detected by ddPCR. In 10 tumor biopsy cobas negative cases, 3 were positive defined by ddPCR. In 23 patients received Osimertinib treatment, the OOR was 60.9%, quantification of T790M after 6 weeks of treatment decreased to very low level, no association was observed between response status and T790M mutation decrease. ddPCR is more sensitive in plama ctDNA testing and should be performed even tumor tissue biopsy yielded negative results in certain cases. Osimertinib significantly decreased plasma T790M level, but not associated with clinical response.

MA 12.10 Clinical Utility of Plasma EGFR T790M Mutation Detection in Advanced Non-Small Cell Lung Cancer Patients According to RECIST Criteria

MA 12.10 Clinical Utility of Plasma EGFR T790M Mutation Detection in Advanced Non-Small Cell Lung Cancer Patients According to RECIST Criteria

85PD - Ultrasensitive detection of EGFR T790M mutation by droplet digital PCR (ddPCR) in TKI naïve non-small cell lung cancer (NSCLC) harboring EGFR mutation: Results of the nationwide program Biomarkers France of the French Cooperative Thoracic Intergroup (IFCT)

85PD - Ultrasensitive detection of EGFR T790M mutation by droplet digital PCR (ddPCR) in TKI naïve non-small cell lung cancer (NSCLC) harboring EGFR mutation: Results of the nationwide program Biomarkers France of the French Cooperative Thoracic Intergroup (IFCT)