Cobas Epidermal Growth Factor Receptor Research Articles

The Advantage of Targeted Next-Generation Sequencing over qPCR in Testing for Druggable EGFR Variants in Non-Small-Cell Lung Cancer.

The emergence of targeted therapies in non-small-cell lung cancer (NSCLC), including inhibitors of epidermal growth factor receptor (EGFR) tyrosine kinase, has increased the need for robust companion diagnostic tests. Nowadays, detection of actionable variants in exons 18-21 of the EGFR gene by qPCR and direct DNA sequencing is often replaced by next-generation sequencing (NGS). In this study, we evaluated the diagnostic usefulness of targeted NGS for druggable EGFR variants testing in clinical NSCLC material previously analyzed by the IVD-certified qPCR test with respect to DNA reference material. We tested 59 NSCLC tissue and cytology specimens for EGFR variants using the NGS 'TruSight Tumor 15' assay (Illumina) and the qPCR 'cobas EGFR mutation test v2' (Roche Diagnostics). The sensitivity and specificity of targeted NGS assay were evaluated using the biosynthetic and biological DNA reference material with known allelic frequencies (VAF) of EGFR variants. NGS demonstrated a sufficient lower detection limit for diagnostic applications (VAF < 5%) in DNA reference material; all EGFR variants were correctly identified. NGS showed high repeatability of VAF assessment between runs (CV% from 0.02 to 3.98). In clinical material, the overall concordance between NGS and qPCR was 76.14% (Cohen's Kappa = 0.5933). The majority of discordant results concerned false-positive detection of EGFR exon 20 insertions by qPCR. A total of 9 out of 59 (15%) clinical samples showed discordant results for one or more EGFR variants in both assays. Additionally, we observed TP53 to be a frequently co-mutated gene in EGFR-positive NSCLC patients. In conclusion, targeted NGS showed a number of superior features over qPCR in EGFR variant detection (exact identification of variants, calculation of allelic frequency, high analytical sensitivity), which might enhance the basic diagnostic report.

The association of EGFR amplification with aberrant exon 20 insertion report using the cobas EGFR Mutation Test v2.

Determining the exact type of epidermal growth factor receptor (EGFR) exon 20 insertion (ex20ins) mutation in lung cancer has become important. We found that not all ex20ins mutations reported by cobas EGFR test v2 could be validated by Sanger sequencing even using surgical specimens with high tumor contents. This study aimed to validate the ex20ins results reported by the cobas test and to determine whether there were clinicopathological factors associated with aberrant cobas ex20ins report. In total, 123 cobas-reported cases with ex20ins were retrospectively collected and validated by Sanger sequencing and Idylla assay. Clinicopathological features between ex20ins cobas+/Sanger+ group (n = 71) and cobas+/Sanger- group (n = 52) were compared. The Idylla assay detected ex20ins in 82.6% of cobas+/Sanger+ cases but only in 4.9% of cobas+/Sanger- cases. The cobas+/Sanger- group was significantly associated with higher tumor contents, poorly differentiated patterns, tumor necrosis, and a lower internal control cycle threshold value reported by the Idylla which suggesting the presence of increased EGFR gene copy numbers. EGFR fluorescence in situ hybridization (FISH) revealed the majority of cobas+/Sanger- group had EGFR high copy number gain (16%) or amplification (76%) according to the Colorado criteria. Among cases reported to have concomitant classic EGFR and ex20ins mutations by the cobas, the classic EGFR mutations were all detected by Sanger sequencing and Idylla, while the ex20ins mutations were undetected by Sanger sequencing (0%) or rarely reported by Idylla assay (3%). FISH revealed high EGFR copy number gain (17.9%) and amplification (79.5%) in cases reported having concomitant classic EGFR and ex20ins mutations by the cobas. This study demonstrated an unusually high frequency of EGFR amplification in cases with aberrant cobas ex20ins report which could not be validated by Sanger sequencing or Idylla assay. Ex20ins reported by the cobas test should be validated using other methods especially those reported having concomitant ex20ins and classic EGFR mutations.

Clinical Validation of the Unparalleled Sensitivity of the Novel Allele-Discriminating Priming System Technology-Based EGFR Mutation Assay in Patients with Operable Non-Small Cell Lung Cancer.

Recently, we developed allele-discriminating priming system (ADPS) technology. This method increases the sensitivity of conventional quantitative polymerase chain reaction up to 100 folds, with limit of detection, 0.01%, with reinforced specificity. This prospective study aimed to develop and validate the accuracy of ADPS epidermal growth factor receptor (EGFR) Mutation Test Kit using clinical specimens. In total 189 formalin-fixed paraffin-embedded tumor tissues resected from patients with non-small cell lung cancer were used to perform a comparative evaluation of the ADPS EGFR Mutation Test Kit versus the cobas EGFR Mutation Test v2, which is the current gold standard. When the two methods had inconsistent results, next-generation sequencing-based CancerSCAN was utilized as a referee. The overall agreement of the two methods was 97.4% (93.9%-99.1%); the positive percent agreement, 95.0% (88.7%-98.4%); and the negative percent agreement, 100.0% (95.9%-100.0%). EGFR mutations were detected at a frequency of 50.3% using the ADPS EGFR Mutation Test Kit and 52.9% using the cobas EGFR Mutation Test v2. There were 10 discrepant mutation calls between the two methods. CancerSCAN reproduced eight ADPS results. In two cases, mutant allele fraction was ultra-low at 0.02% and 0.06%, which are significantly below the limit of detection of the cobas assay and CancerSCAN. Based on the EGFR genotyping by ADPS, the treatment options could be switched in five patients. The highly sensitive and specific ADPS EGFR Mutation Test Kit would be useful in detecting the patients who have lung cancer with EGFR mutation, and can benefit from the EGFR targeted therapy.

Performance of Ultra-Rapid Idylla™ EGFR Mutation Test in Non-Small-Cell Lung Cancer and Its Potential at Clinical Molecular Screening.

The Idylla™ EGFR Mutation Test is an ultra-rapid single-gene test that detects epidermal growth factor receptor (EGFR) mutations using formalin-fixed paraffin-embedded specimens. Here, we compared the performance of the Idylla EGFR Mutation Test with the Cobas® EGFR Mutation Test v2. Surgically resected NSCLC specimens obtained at two Japanese institutions (N = 170) were examined. The Idylla EGFR Mutation Test and the Cobas EGFR Mutation Test v2 were performed independently and the results were compared. For discordant cases, the Ion AmpliSeq Colon and Lung Cancer Research Panel V2 was performed. After the exclusion of five inadequate/invalid samples, 165 cases were evaluated. EGFR mutation analysis revealed 52 were positive and 107 were negative for EGFR mutation in both assays (overall concordance rate: 96.4%). Analyses of the six discordant cases revealed that the Idylla EGFR Mutation Test was correct in four and the Cobas EGFR Mutation Test v2 was correct in two. In a trial calculation, the combination of the Idylla EGFR Mutation Test followed by a multi-gene panel test will reduce molecular screening expenses if applied to a cohort with EGFR mutation frequency >17.9%. We demonstrated the accuracy and potential clinical utility of the Idylla EGFR Mutation Test as a molecular screening platform in terms of turnaround time and molecular testing cost if applied to a cohort with a high EGFR mutation incidence (>17.9%).

Efficacy of different platforms in detecting EGFR mutations using cerebrospinal fluid cell-free DNA from non-small-cell lung cancer patients with leptomeningeal metastases.

Cell-free tumor DNA (ctDNA) obtained through liquid biopsy is useful for the molecular analysis of advanced non-small-cell lung cancer (NSCLC). Few studies have directly compared analysis platforms in terms of their diagnostic performance in analyzing ctDNA obtained from the cerebrospinal fluid (CSF) of patients with leptomeningeal metastasis (LM). We prospectively analyzed patients with epidermal growth factor receptor (EGFR)-mutant NSCLC who were subjected to CSF analysis for suspected LM. To detect EGFR mutations, CSF ctDNA was analyzed using the cobas EGFR Mutation Test and droplet digital polymerase chain reaction (ddPCR). CSF samples from osimertinib-refractory patients with LM were also subjected to next-generation sequencing (NGS). Significantly higher rates of valid results (95.1% vs. 78%, respectively, p = 0.04) and EGFR common mutation detection (94.3% vs. 77.1%, respectively, p = 0.047) were obtained through ddPCR than through the cobas EGFR Mutation Test. The sensitivities of ddPCR and cobas were 94.3% and 75.6%, respectively. The concordance rate for EGFR mutation detection through ddPCR and the cobas EGFR Mutation Test was 75.6% and that for EGFR mutation detection in CSF and plasma ctDNA was 28.1%. In osimertinib-resistant CSF samples, all original EGFR mutations were detected through NGS. MET amplification and CCDC6-RET fusion were demonstrated in one patient each (9.1%). The cobas EGFR Mutation Test, ddPCR, and NGS appear to be feasible methods for analyzing CSF ctDNA in patients with NSCLC and LM. In addition, NGS may provide comprehensive information regarding the mechanisms underlying osimertinib resistance.

Osimertinib treatment based on plasma T790M monitoring in patients with EGFR-mutant non-small-cell lung cancer (NSCLC): EORTC Lung Cancer Group 1613 APPLE phase II randomized clinical trial

The APPLE trial aimed to evaluate the feasibility of longitudinal plasma epidermal growth factor receptor (EGFR) T790M monitoring for the best sequencing strategy of gefitinib and osimertinib. APPLE is a randomized, non-comparative, phase II study in patients with common EGFR-mutant, treatment-naive non-small-cell lung cancer including three arms: arm A (osimertinib upfront until RECIST progression, PD), arm B [gefitinib until emergence of circulating tumor DNA (ctDNA) EGFR T790M mutation by cobas EGFR test v2 or RECIST PD], and arm C (gefitinib until RECIST PD), and then switch to osimertinib in both arms. The primary endpoint is the progression-free survival (PFS) rate 'on osimertinib' at 18 months (PFSR-OSI-18) after randomization in arm B (H0: PFSR-OSI-18 of ≤40%). Secondary endpoints include response rate, overall survival (OS), and brain PFS. We report the results of arms B and C. From November 2017 to February 2020, 52 and 51 patients were randomized into arms B and C, respectively. Most patients were females (70%) and had EGFR Del19 (65%); one-third had baseline brain metastases. In arm B, 17% of patients (8/47) switched to osimertinib based on the emergence of ctDNA T790M mutation before RECIST PD, with a median time to molecular PD of 266 days. The study met its primary endpoint of PFSR-OSI-18 of 67.2% (84% confidence interval 56.4% to 75.9%) in arm B versus 53.5% (84% confidence interval 42.3% to 63.5%) in arm C, with a median PFS of 22.0 months versus 20.2 months, respectively. The median OS was not reached in arm B versus 42.8 months in arm C. Median brain PFS in arms B and C was 24.4 and 21.4 months, respectively. The serial monitoring of ctDNA T790M status in advanced EGFR-mutant non-small-cell lung cancer during treatment with first-generation EGFR inhibitors was feasible, and a molecular progression before RECIST PD led to an earlier switch to osimertinib in 17% of patients with satisfactory PFS and OS outcomes.

Epidemiology and clinical impact of EGFR mutation in patients with lung cancer after radical surgical treatment.

8552 Background: The epidemiology of epidermal growth factor receptor (EGFR) mutation in lung adenocarcinoma and its clinical impact are well-known. However, most studies have focused on advanced-stage inoperable cancer. Data on the frequency of EGFR mutation in surgically resected lung cancer are limited. Recent studies have shown a promising effect of Osimertinib in adjuvant settings. Hence, the need to estimate the target population in the real-world data. This study aimed to assess the occurrence of EGFR mutation in patients after radical surgical treatment of lung adenocarcinoma and explore its prognostic impact compared to EGFR negative group. Methods: This single-center retrospective analysis included the group of 732 consecutive Caucasian patients with histopathologically confirmed lung adenocarcinoma, evaluated for EGFR mutations expression, who underwent anatomical resection between January 2016, and December 2020. EGFR status was assessed by cobas EGFR mutation test v2. The frequency of EGFR mutations, disease-free survival (DFS) and overall survival (OS) in EGFR positive and EGFR negative groups were analyzed. Results: EGFR mutations were found in 65 surgical patients (8.9%) and did not differ from patients with advanced stages of lung adenocarcinoma (7.9%). EGFR mutations occurred more frequently in females than males, 48 out of 344 (14%) and 17 out of 388 (4.4%), respectively. Deletions within exon 19 and the L858R mutation in exon 21 constituted 49.2% and 24.6% of all mutations, respectively, while others comprised 26.2%. One case of L858R mutation coincided with T790M in exon 20 mutation. Detailed results divided by stages are presented in the Table. The occurrence of EGFR mutation had no significant influence on DFS and OS in patients after radical resection. Conclusions: The frequency of EGFR mutation in postoperative lung cancer was comparable to the occurrence in the general lung cancer population. EGFR mutation did not affect DFS and OS in patients after radical resection. [Table: see text]

Efficacy of liquid biopsy for disease monitoring and early prediction of tumor progression in EGFR mutation-positive non-small cell lung cancer.

15–40% of non-small cell lung cancer (NSCLC) patients harbor epidermal growth factor receptor (EGFR)-sensitizing mutations. Tyrosine kinase inhibitors (TKIs) provide significant clinical benefit in this population, yet all patients will ultimately progress. Liquid biopsy can reliably identify somatic tumor-associated EGFR mutations in plasma. This study aimed to assess the feasibility and value of the quantitative assessment of EGFR driver mutations in plasma in EGFR-mutated NSCLC patients treated with EGFR-TKIs as a tool to evaluate therapeutic response to TKIs and monitor for disease progression. The study included 136 patients with tissue biopsy-confirmed EGFR-sensitizing, mutation-positive lung adenocarcinoma with plasma collected prior to TKI treatment and at least two post-initiation TKI treatment/follow-up blood samples. Plasma samples were tested with the cobas® EGFR Mutation Test v2 (cobas EGFR Test), and semi-quantitative index (SQI) values for each identified mutation were reported by the assay software. The most common baseline EGFR mutations detected in tissue were L858R (53.7%) and exon 19 deletion (39.7%). Plasma cell-free DNA analysis detected EGFR mutations in 74% of the baseline samples. Objective response rate by RECIST 1.1 was achieved in 72% of patients, while 93% had a molecular response (defined as disappearance of the EGFR mutation from plasma). 83% of patients had molecular progression (MP; 1.5X SQI increase or new T790M mutation), and 82% who had a clinical response had clinical progression. On average, MP occurred 42 days prior to clinical progression. Patients who progressed while on first-line TKI showed MP of the original EGFR-sensitizing mutations prior to the emergence of a T790M mutation, which was detected in 27% of the EGFR plasma-positive patients. Longitudinal monitoring of EGFR mutational load in plasma is feasible and can predict both response and clinical progression in EGFR-mutated NSCLC patients treated with EGFR-TKIs, as well as detect treatment-emergent EGFR mutations.

Observational study of rebiopsy in EGFR-TKI-resistant patients with EGFR mutation-positive advanced NSCLC

The identification of acquired resistance mutations has been essential in non-small-cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) active mutations. Rebiopsy plays a pivotal role in selecting the optimal treatment for patients who develop resistance to initial EGFR-tyrosine kinase inhibitors (EGFR-TKIs). This multicenter, observational study was conducted to investigate the details of rebiopsy in Japanese clinical practice. The primary endpoints were the implementation rate of rebiopsy and the concordance rate for T790M mutation detection between histological and cytological specimens using the cobas EGFR Mutation Test, version 2. One hundred ninety-four patients with EGFR-mutant NSCLC were enrolled, and 120 patients developed acquired resistance to EGFR-TKIs. The median age was 68 years (range 20–87), and 52.5% of the patients were women. Rebiopsy was performed in 109 patients, and the implementation rate of rebiopsy was 90.8%. The success rates of rebiopsy in the total, histology, cytology and liquid biopsy populations were 67.9%, 81.3%, 66.7% and 43.8%, respectively. The positive percent agreement and the negative percent agreement in the detection of the T790M mutation between the histological and cytological specimens were both 90.9%. Obtaining histological or cytological tissue samples at rebiopsy may contribute to improving the detection rate of the T790M mutation (trial registration number: UMIN000026019).

374 Therapeutic Class Labeling of EGFR Companion Diagnostics (CDxs)

OBJECTIVES/GOALS: Review all approved companion diagnostics (CDxs) labeling for therapeutic class language. Examine the regulatory pathways of CDx products whose labels contain therapeutic class labeling. Develop recommendations for pharmaceutical industry professionals on best practices in the co-development of CDxs and oncology therapeutic products. METHODS/STUDY POPULATION: Literature discussing companion diagnostics was reviewed from EBSCOhost, PubMed, and OVID. The Intended Use language within CDx labels on the Food and Drug Administrations “List of Cleared or Approved Companion Diagnostic Devices (In Vitro and Imaging Tools)” website were reviewed on November 1, 2021 for therapeutics class language. For CDx products with therapeutic class label language, the regulatory history was evaluated to determine the development approach taken to achieve the language. RESULTS/ANTICIPATED RESULTS: A total of 45 CDxs were identified, of which only 2 contained therapeutic class labeling, both of which were devices for the identification of epidermal growth factor receptor (EGFR) mutations for the treatment of non-small cell lung cancer (NSCLC). Three additional EGFR CDxs were approved; however, they did not contain therapeutic class labeling. The first CDx was the Cobas EGFR Mutation Test V2, which received therapeutic group label language as an update on October 27, 2020; however, prior to the therapeutic class labeling, three oncology products were named in the Intended Use: Tarceva (erlotinib), Tagrisso (osimertinib), and Iressa (gefitinib). The second CDx to incorporate therapeutic class labeling was the ONCO/Reveal Diagnostic Lung and Colon Cancer Assay upon initial approval on July 30, 2021. DISCUSSION/SIGNIFICANCE: EGFR CDxs are the first to shift towards therapeutic class labeling. Indication, molecular alterations, and mechanism of action of the approved therapeutic class products, number of products approved, as well as CDx analytical and clinical validation influence class label relevance. Discussions with the FDA are encouraged early in development.

Evaluation of molecular methods for plasma detection of EGFR mutations in non-small cell lung cancer.

Epidermal growth factor receptor (EGFR) mutations are detected in non-small cell lung cancer (NSCLC) and associated with responses to therapy with tyrosine kinase inhibitors (TKIs). We compared the analytical performances of two real-time PCRs and droplet digital PCR (ddPCR) to detect EGFR mutations using plasma. Plasma EGFR tests were performed using 86 plasma samples from 75 prospectively enrolled NSCLC patients with early and advanced stages. Analytical performances of plasma-using two real-time PCR, Cobas EGFR mutation v2 and PANAMutyper, EGFR kit, and ddPCR were evaluated based on the tissue EGFR test results. The frequencies of EGFR mutations and acquired T790M mutation after TKI therapy were also assessed. The incidence of all EGFR mutations was 52.3% (23/44) in tissue and was up to 43.2% (19/44) in plasma. The Cobas detection rates of three EGFR mutations (exon 19 deletions, L858R, and T790M) in plasma were similar to those in tissue. The Cobas showed a higher detection rate (76.7%) than that by the PANAMutyper (60.5%). Sensitivity for T790M mutation was lower than the sensitivity for the exon 19 deletions or L858R in both tests. Mutant allele frequency measured by ddPCR was significantly correlated with the semi-quantitative values of the Cobas. Plasma EGFR tests showed similar detection rates for common EGFR mutations compared to the tissue EGFR tests. Cobas showed higher sensitivity in detection of EGFR mutations in body fluids than the PANAMutyper. Real-time PCR using plasma or body fluids could be a suitable first test for the detection of EGFR mutations.

Application of liquid biopsy-based targeted capture sequencing analysis to improve the precision treatment of non-small cell lung cancer by tyrosine kinase inhibitors

BackgroundTargeted therapy of patients with non-small cell lung cancer (NSCLC) who harbour sensitising mutations by tyrosine kinase inhibitors (TKIs) has been found more effective than traditional chemotherapies. However, target genes...

FP12.05 The Intrinsic Limitation and Clinical Impact of Mutant Allele-Specific Real-Time PCR-Based EGFR Mutation Assay in NSCLC

The detection of driver gene mutations, such as epidermal growth factor receptor (EGFR) mutations, plays a decisive role in the treatment of non-small cell lung cancer (NSCLC). Cobas EGFR mutation test is a mutant allele-specific real-time PCR assay that is commonly used in daily practice. It has a notable intrinsic detection limitation caused by its primer designs. Next-generation sequencing (NGS) is a comprehensive mutation assay that does not have the constraint. It will have a higher mutation detection rate but the clinical impact remains uncertain.

Detection of EGFR mutations in liquid biopsy samples using allele-specific quantitative PCR: A comparative real-world evaluation of two popular diagnostic systems

PurposeThe detection of epidermal growth factor receptor (EGFR) mutations in plasma cell-free DNA (cfDNA) is an auxiliary tool for the molecular diagnosis of non-small cell lung cancer (NSCLC), especially when an adequate tumor tissue specimen cannot be obtained. We compared the diagnostic accuracy of two commonly used in vitro diagnostic-certified allele-specific quantitative PCR assays for detecting plasma cfDNA EGFR mutations. MethodsWe analyzed EGFR mutations in plasma cfDNA from 90 NSCLC patients (stages I–IV) before treatment (n ​= ​60) and after clinical progression on EGFR tyrosine kinase inhibitors (n ​= ​30) using the cobas EGFR mutation test v2 (Roche Molecular Systems, Inc.) and therascreen EGFR Plasma RGQ PCR kit (Qiagen GmbH). ResultsThere was higher concordance between plasma cfDNA and matched tumor tissue EGFR mutations with cobas (66.67%) compared with therascreen (55.93%). The concordance rate increased to 90.00% with cobas (Cohen's kappa coefficient, κ ​= ​0.80; p ​< ​0.0001) and 73.33% with therascreen (κ ​= ​0.49; p ​= ​0.0009) in advanced NSCLC patients. In treatment-naïve patients, cobas was superior to therascreen (sensitivity: 82.35% vs. 52.94%; specificity: 100% vs. 100%). In patients with clinical progression on EGFR tyrosine kinase inhibitors, EGFR exon 20 p.T790M was detected in 30% and 23% of cfDNA samples by cobas and therascreen, respectively. ConclusionsCobas was superior to therascreen for detection of plasma EGFR mutations in advanced NSCLC. Plasma cfDNA EGFR mutation analysis is complex; therefore, the diagnostic accuracy of commercially available assays should be validated.

Lungful: An observational prospective study to assess the molecular epidemiology of EGFR mutations upon progression on or after first line EGFR-TKI therapy, in real-life clinical settings in Greece.

e21641 Background: Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are the gold standard 1st line strategy for non-small-cell lung cancer (NSCLC) patients with activating EGFR mutations (EGFRm), associated with improved survival outcomes and quality of life compared to chemotherapy. Despite the high response rate with first- and second- generation TKIs, most patients develop resistance to treatment and progress. The acquisition of T790M mutation in exon 20 is considered the most common resistance mechanism. This study aims to investigate the molecular epidemiology of EGFR resistance mutations, focusing on T790M in EGFRm NSCLC patients treated with TKIs. Methods: The study included patients with locally advanced/metastatic EGFRm NSCLC who have progressed on or after 1st line treatment with first- or second- generation TKI. Samples either from plasma-based liquid biopsy and/or tissue re-biopsy were analysed using the Cobas EGFR Mutation Test v2. All patients signed informed consent and were enrolled between July 2017 and September 2019. Statistical analyses were performed using SAS software, Version 9.4. Results: Ninety-six eligible patients were enrolled. At the time of progression, T790M mutation was detected in 16.7%of the patients using plasma-based liquid biopsies. Among patients with negative T790M result, in plasma, tissue re-biopsy was performed in 22,7% with evaluable/valid results in 72.2% of them. T790M mutation was identified in 38.5% of re-biopsy samples. According to Cobas EGFR Mutation test results (combined plasma and tissue), T790M mutation was identified in 21.9% of the patients. Of T790M-positive patients 42.9% had previously received first and 57.1% second generation EGFR-TKI. Conclusions: Results from this study in real world clinical setting in Greece, show that EGFR-T790M acquired resistance positivity rate in plasma is lower compared to previous reports. Moreover, these data underline the challenges of implementing precision medicine using tissue re-biopsy in advanced/metastatic NSCLC. Clinical trial information: D133FR00126. [Table: see text]

Predictive impact of low-frequency pretreatment T790M mutation in patients with EGFR-mutated non-small cell lung cancer treated with EGFR tyrosine kinase inhibitors

ObjectivesLow-frequency epidermal growth factor receptor (EGFR) T790M mutation could be detected by ultrasensitive methods in EGFR tyrosine kinase inhibitor (TKI)-naïve non-small cell lung cancer (NSCLC). However, the impact of pretreatment T790M (preT790M) on the efficacy of EGFR-TKIs and on resistance remains unclear. Materials and methodsTwo independent cohorts consisting of advanced EGFR-mutated NSCLC patients treated with first-line EGFR-TKIs, a derivation cohort that started treatment between August 2013 and July 2016 (cohort A, n = 44) and a validation cohort between August 2016 and December 2017 (cohort B, n = 22), were examined in this study. Among these, 28 patients underwent re-biopsy at disease progression. DNAs from pretreatment tumor biopsy samples and re-biopsy samples were assessed to detect T790M by the Cobas EGFR Mutation Test v2 (Cobas) and for quantitating T790M by droplet digital polymerase chain reaction (ddPCR). ResultsDetection rates of preT790M were 40.9% (18/44) in cohort A and 45.5% (10/22) in cohort B by ddPCR, and none by Cobas. A cutoff value of 0.3% for dividing into high- vs. low-preT790M allele frequency was determined by receiver operating characteristic curve analysis in cohort A. Progression-free survival (PFS) was significantly shorter in the high- preT790M group (n = 12) than in the low-preT790M (n = 6) and negative (n = 26) groups (combined low-preT790M) (median: 6.9 vs. 13.8 months, P = 0.00073). These observations were validated in cohort B [median: 6.2 (n = 5) vs. 15.3 months (n = 17), P = 0.0029]. In 28 paired biopsies, Cobas detected post-progression T790M in 60% (3/5) of the high-preT790M, in 57% (4/7) of the low-preT790M, and in 56% (9/16) of the negative-preT790M groups. ConclusionEGFR-mutated NSCLC with high preT790M had significantly shorter PFS on EGFR-TKIs. However, preT790M abundance may not necessarily confer post-TKI T790M resistance.

1415P - Feasibility study of a ctEGFR prototype assay on the fully automated Idylla™ platform

BackgroundTo predict the response to EGFR tyrosine kinase inhibitor (TKI) therapy in non-small cell lung cancer (NSCLC) patients, formalin-fixed paraffin-embedded (FFPE) tumor tissue is routinely tested for the presence of somatic mutations in the epidermal growth factor receptor (EGFR) gene. Sufficient tumor tissue is not always available and ctEGFR testing from plasma is an alternative approach for the detection of EGFR mutations. Therefore, a fast and fully automated ctEGFR assay with minimal hands-on time should allow laboratories to quickly generate EGFR testing results. MethodsIdyllaTM (Biocartis) is a fully integrated molecular diagnostics platform that combines speed and ease of use with high sensitivity and high multiplexing capabilities. In terms of ctDNA testing, it overcomes the time-consuming step of ctDNA extraction from plasma. After insertion of 2ml of plasma into the cartridge, the complete process of ctDNA extraction, real-time PCR, data analysis and reporting is fully automated. The ctEGFR prototype assay allows the detection of 49 mutations including insertions and deletions in exons 18, 19, 20 and 21. The results obtained by the ctEGFR prototype assay were compared with NGS (sensitivity 2-5%). ResultsSixty-four NSCLC samples were tested with both assays. Overall, 34 mutations were detected by NGS and confirmed by the ctEGFR prototype assay. In 33 samples, NGS detected no mutation. The ctEGFR prototype assay detected 7 additional mutations in this cohort. Retesting with the cobas EGFR Mutation Test v2 confirmed the presence of these mutations. Analytical sensitivity was assessed for 20 mutations using plasma spiked with synthetic targets. Analytical sensitivities ranging from 1 to 4% were obtained for the tested mutations. Inclusivity was demonstrated for 49 mutations in total. The average turnaround time of a run was <2h 40min and the hands-on time for the assay was <2min. ConclusionsThis study shows that the Idylla™ platform enables the development of a prototype ctEGFR assay with high sensitivity and ease of use combined with a fast turnaround time for the testing of 49 relevant EGFR mutations in 2ml of plasma from NSCLC patients. Legal entity responsible for the studyBiocartis. FundingBiocartis. DisclosureM. Reijans: Full / Part-time employment: Biocartis. S.V. Gestel: Full / Part-time employment: Biocartis. E.D. Haes: Full / Part-time employment: Biocartis. C. Vandesteene: Full / Part-time employment: Biocartis. J.M. Castro Gomez: Full / Part-time employment: Biocartis. C. Gouedard: Full / Part-time employment: BioPath Innovations SA. S. Patera: Full / Part-time employment: BioPath Innovations SA. S. Murray: Full / Part-time employment: BioMarker Solutions Ltd. G.G. Maertens: Full / Part-time employment: Biocartis.

Five Technologies for Detecting the EGFR T790M Mutation in the Circulating Cell-Free DNA of Patients With Non-small Cell Lung Cancer: A Comparison

Third-generation tyrosine kinase inhibitors (TKIs) were developed to overcome T790M-mediated resistance to earlier generations of epidermal growth factor receptor (EGFR)-targeted TKIs. We compared four well-established and one in-house method for the analysis of the EGFR T790M mutation in plasma cell-free DNA (cfDNA), in hope to find a better way to select non-small cell lung cancer (NSCLC) patients appropriate for 3rd-generation TKI therapy. For sensitivity levels of each method, plasmid DNA with EGFR T790M mutations was serially diluted with cfDNA from healthy controls with wild type EGFR. The clinical performance was analyzed in a clinical cohort of EGFR mutation-positive NSCLC patients with acquired EGFR TKI resistance (n = 40). All methods except the therascreen kit (Qiagen) had a sensitivity level of 10 copies of T790M plasmid DNA in the spiked specimen. The detection rates of the EGFR T790M mutation in plasma cfDNA from the clinical cohort were 42.5, 35, 32.5, 22.5, and 17.5% for the in-house ARMS method, Bio-Rad droplet digital PCR, PANAMutyper, Therascreen EGFR Plasma RGQ PCR Kit and Cobas EGFR Mutation kit (with suboptimal template amounts), respectively. Osimertinib was given to 17 of 20 patients with EGFR T790M mutations. The best treatment responses, based on the RECIST criteria, included 6 partial responses (PR) and 7 stable diseases (SD). The PANAMutyper and the Bio-Rad droplet digital PCR were comparable, the Cobas EGFR Mutation kit required significantly more template for testing. The best combination would be the in-house ARMS method plus the PANAMutyper or Bio-Rad droplet digital PCR, which would have a detection rate of 50% (20/40) and a disease control rate of 76% (13/17).

Liquid biopsy for detection of EGFR T790M mutation in nonsmall cell lung cancer: An experience of proficiency testing in Taiwan.

The use of liquid biopsy to detect epidermal growth factor receptor (EGFR) T790M mutation in nonsmall cell lung cancer (NSCLC) is a promising method to screen patients eligible for third-generation EGFR inhibitors. Proficiency testing (PT) programs involving liquid biopsy are currently lacking. In this study, we conducted a PT program to assess the quality assurance of liquid biopsy tests for detecting EGFR T790M mutation in molecular pathology laboratories in Taiwan. Whole blood samples (2 mL) with various concentrations of the EGFR T790M mutation were prepared and analyzed in six participating laboratories using their clinically validated assays. For circulating cell-free DNA (cfDNA) isolation, three of the six participating laboratories used the cobas cfDNA Sample Preparation Kit, and three used the QIAamp Circulating Nucleic Acid Kit. For testing platforms, two of the six participating laboratories used mass spectrometry, three used the cobas EGFR mutation test, and one used a laboratory-developed test. There was 100% concordance in detection of all the given concentrations of EGFR T790M mutation between the participating laboratories and different testing platforms. The testing platforms used by all participating laboratories could successfully detect EGFR T790M mutation to an expected frequency of 1%. In this first PT program using liquid biopsy in Taiwan, local clinical laboratories were suitably equipped and proficient in the use of cfDNA to test for the EGFR T790M mutation. Establishing a routine PT system to ensure the reliability and accuracy of liquid biopsy in clinical practice in Taiwan would be helpful.

Determination of plasma EGFR mutations from non-small cell lung cancer patients at Bach Mai Hospital.

e13121 Background: Lung cancer patients with mutations in the epidermal growth factor receptor (EGFR) are primary candidates for EGFR-targeted therapy. Reliable analyses of such mutations have previously been possible only in tumor tissue. Here, we demonstrate that mutations can be detected in plasma samples of non-small cell lung cancer patients at Bach Mai Hospital and relation of different variables to the frequency of EGFR mutations. Methods: 193 patients with non-small cell lung cancer were identified with EGFR mutation by cobas EGFR Mutation Test v2 kit (Roche Diagnostics, USA), evaluating the EGFR mutation status with different variables. Results: The EGFR mutation rate was 43%, common in patients with adenocarcinoma, female, non-smoking, late stage disease. The rate of EGFR T790M mutation was 36.1%. Among 78 patients who was previous treated TKIs, EGFR mutation rate was 73.1% and the rate of EGFR T790M mutation was 50.8% at the time of progression. The concordance of EGFR mutation from plasma sample with tissue sample was 81%. Conclusions: EGFR mutation in plasma sample results was not different from those in tissue samples and similar with result of studies in Vietnam and worldwide.