Epidermal Growth Factor Receptor Mutation Test V2 Research Articles

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.

L858R-K860I Doublet Mutation of EGFR gene in Non-Small Cell Lung Carcinoma: Cause of a False Negative Call for L858R on Initial Companion Diagnostic Test

With the advent of targeted therapy in non-small cell lung carcinoma (NSCLC), biomarker detection has become standard practice. US Food and Drug Administration (FDA) has approved a few companion diagnostics as single gene assays to find these driver mutations. We report a case of NSCLC where initial molecular tests to detect epidermal growth factor receptor ( EGFR)- sensitizing mutations by an FDA-approved assay (Cobas® EGFR Mutation Test v2) failed to identify the common EGFR mutation L858R. However, the latter was picked up on subsequent next-generation sequencing (NGS). Interestingly an additional single nucleotide variant K860I was also observed in the cis configuration. The false-negative result for L858R was assumed to be caused by the abrogation of complementarity for the probe binding site on the amplicon generated. The concurrent occurrence of L858R and K860I will not be detectable by Cobas® EGFR Mutation Test v2 (FDA-approved companion diagnostic) despite its real-world intra and inter-laboratory studies demonstrating high accuracy.

Possible False Results With cobas® EGFR Mutation Test v2 and Oncomine Dx Target Test for EGFR Mutation.

Disparities in the results of next-generation sequencing-based multiplex gene panel tests and those of single-gene tests when detecting epidermal growth factor receptor (EGFR) mutations in non-small-cell lung cancer (NSCLC) have been reported. However, the possible underlying causes have not been investigated. The aim of this study was to explore the possibilities and causes of false results obtained using cobas® EGFR Mutation Test v2 (cobas® EGFR) and Oncomine Dx Target Test (ODxTT). The data of patients with NSCLC who underwent gene assessment using both cobas® EGFR and ODxTT between April 2021 and May 2022 were retrospectively reviewed. Disparate results of EGFR mutation analyses were then reviewed. One hundred and sixteen patients were included in the analysis. The results of six samples were inconsistent. In four samples, exon 20 insertion mutations were detected using cobas® EGFR, but not identified using ODxTT. A fragment analysis was performed on three of the four samples, and all showed negative results for exon 20 insertion. Furthermore, one false negative result was obtained in the ODxTT for both exon 19 deletion and L858R mutations. For exon 19 deletion mutation, a single nucleotide variant from adenine to thymine was identified close to the mutation site. False positives for exon 20 insertion may occur when using cobas® EGFR, and false negatives for exon 19 deletion and L858R mutations may occur when using ODxTT.

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%).

Correlation between ALK, ROS1 Biomarkers and EGFR Oncogene Mutations in Lung Tumours: Our Observations in an Apex Oncopathology Laboratory

Introduction: Analysis of Anaplastic lymphoma receptor tyrosine kinase gene (ALK), Repressor of Silencing 1 (ROS1) gene are determined by immunohistochemistry (IHC) and it is an easily applicable, cost-effective assay for potential treatment with crizotinib. Mutations of Epidermal growth factor receptor (EGFR) genes are evaluated by IHC/Multiplex RT-PCR. The purpose of this study is to assess the frequencies of ALK, ROS1 and their association with EGFR fusion gene mutations in a spectrum of lung tumours. Materials and methods: A total of 202 cases of lung tumours reported at our Center for Oncopathology from September 1st 2020 to 31st August 2021, were retrospectively analyzed for ALK, ROS1 and EGFR fusion genes based on Immunohistochemistry (IHC) and Multiplex PCR findings. ALK was tested using D5F3 clone, and ROS1 was analyzed using Cell Signalling’s D4D6 clone on the Ventana immunohistochemistry platform. EGFR status was analyzed using EGFR mutation test V2 real-time multiplex PCR assay on Roche Cobas Z480. Results: 202 biopsy samples and cellblocks of fluid aspirates were analyzed. 175/202 were histologically and immunologically proved as Non-small cell lung carcinoma (Primary pulmonary adenocarcinoma) and its metastases. 09/199 (5.23%) were Positive for ALK IHC and 03/199 (1.74%) cases had equivocal results. 06/179 (3.85%) cases were Positive for ROS1 IHC and 03/179 (1.92%) cases had equivocal results. Other histo-morphological diagnoses i.e., adenosquamous, squamous, small cell, mucinous carcinoma etc (27 cases) were all ALK and ROS1 Negative. 188/202 tumours were analyzed for EGFR mutation status, which showed 70/188 (37.23%) had specific EGFR mutations. 118/188 (62.76%) cases were EGFR wildtype. Conclusion: We observed that age related incidence of EGFR mutations was highest in elderly females, of 61 to 70 years. ALK gene mutations occurred in 6.03% and ROS1 gene mutations occurred in 5.02% of lung tumours and their metastases. EGFR-mutations were associated with ROS1 mutated lung adenocarcinomas. There are no coexistent ALK-EGFR or ALK-ROS1 mutations. All ALK IHC positive pulmonary adenocarcinomas are ROS1 negative and are mutually exclusive.

Liquid Biopsy: A Multi-Parametric Analysis of Mutation Status, Circulating Tumor Cells and Inflammatory Markers in EGFR-Mutated NSCLC.

The liquid biopsy has the potential to improve patient care in the diagnostic and therapeutic setting in non-small cell lung cancer (NSCLC). Consented patients with epidermal growth factor receptor (EGFR) positive disease (n = 21) were stratified into two cohorts: those currently receiving EGFR tyrosine kinase inhibitor (TKI) therapy (n = 9) and newly diagnosed EGFR TKI treatment-naïve patients (n = 12). Plasma genotyping of cell-free DNA was carried out using the FDA-approved cobas® EGFR mutation test v2 and compared to next generation sequencing (NGS) cfDNA panels. Circulating tumor cell (CTC) numbers were correlated with treatment response and EGFR exon 20 p.T790M. The prognostic significance of the neutrophil to lymphocyte ratio (NLR) and lactate dehydrogenase (LDH) was also investigated. Patients in cohort 1 with an EGFR exon 20 p.T790M mutation progressed more rapidly than those with an EGFR sensitizing mutation, while patients in cohort 2 had a significantly longer progression-free survival (p = 0.04). EGFR exon 20 p.T790M was detected by liquid biopsy prior to disease progression indicated by computed tomography (CT) imaging. The cobas® EGFR mutation test detected a significantly greater number of exon 20 p.T790M mutations (p = 0.05). High NLR and derived neutrophil to lymphocyte ratio (dNLR) were associated with shorter time to progression and worse survival outcomes (p < 0.05). High LDH levels were significantly associated with shorter time to disease progression (p = 0.03). These data support the use of liquid biopsy for monitoring EGFR mutations and inflammatory markers as prognostic indicators in NSCLC.

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]

Analytical and clinical validation of an amplicon-based next generation sequencing assay for ultrasensitive detection of circulating tumor DNA.

Next-generation sequencing of circulating tumor DNA presents a promising approach to cancer diagnostics, complementing conventional tissue-based diagnostic testing by enabling minimally invasive serial testing and broad genomic coverage through a simple blood draw to maximize therapeutic benefit to patients. LiquidHALLMARK® is an amplicon-based next-generation sequencing assay developed for the genomic profiling of plasma-derived cell-free DNA (cfDNA). The comprehensive 80-gene panel profiles point mutations, insertions/deletions, copy number alterations, and gene fusions, and further detects oncogenic viruses (Epstein-Barr virus (EBV) and hepatitis B virus (HBV)) and microsatellite instability (MSI). Here, the analytical and clinical validation of the assay is reported. Analytical validation using reference genetic materials demonstrated a sensitivity of 99.38% for point mutations and 95.83% for insertions/deletions at 0.1% variant allele frequency (VAF), and a sensitivity of 91.67% for gene fusions at 0.5% VAF. In non-cancer samples, a high specificity (≥99.9999% per-base) was observed. The limit of detection for copy number alterations, EBV, HBV, and MSI were also empirically determined. Orthogonal comparison of epidermal growth factor receptor (EGFR) variant calls made by LiquidHALLMARK and a reference allele-specific polymerase chain reaction (AS-PCR) method for 355 lung cancer specimens revealed an overall concordance of 93.80%, while external validation with cobas® EGFR Mutation Test v2 for 50 lung cancer specimens demonstrated an overall concordance of 84.00%, with a 100% concordance rate for EGFR variants above 0.4% VAF. Clinical application of LiquidHALLMARK in 1,592 consecutive patients demonstrated a high detection rate (74.8% circulating tumor DNA (ctDNA)-positive in cancer samples) and broad actionability (50.0% of cancer samples harboring alterations with biological evidence for actionability). Among ctDNA-positive lung cancers, 72.5% harbored at least one biomarker with a guideline-approved drug indication. These results establish the high sensitivity, specificity, accuracy, and precision of the LiquidHALLMARK assay and supports its clinical application for blood-based genomic testing.

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.

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.

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...

Investigation of EGFR mutations in non-small cell lung cancer usually undetectable by PCR methods.

Epidermal growth factor receptor (EGFR) mutations are the most significant genomic drivers of non-small cell lung cancer (NSCLC) and determine the efficacy of EGFR tyrosine kinase inhibitor (EGFR-TKI) therapy. PCR methods are used clinically for the detection of EGFR mutations. The Scorpion Amplification Refractory Mutation System (Scorpion-ARMS) and the cobas® EGFR Mutation Test v2 (cobas v2) are widely used PCR methods. However, those PCR methods only selectively detect the common EGFR mutations. The aim of the present study was to reveal the true frequency of EGFR mutations in NSCLC by investigating EGFR mutations usually undetectable by PCR methods by using direct sequencing. A total of 70 Japanese patients who underwent lung resection for NSCLC between September 2016 and March 2019 were included in the present study. Subsequently, PCR methods and direct sequencing were performed. In total, 29 mutations were detected by cobas v2. In total, 41 patients were identified as EGFR wild-type by cobas v2, among whom direct sequencing detected mutations in 3 patients. Subsequent Scorpion-ARMS was performed in the 3 patients in whom direct sequencing detected mutations. In total, one exon 21 L858R + G863D compound mutation was identified as a L858R single mutation, and two other mutations were undetectable. Moreover, 1 patient who was ‘wild-type’ on cobas v2 but ‘EGFR mutation’ on direct sequencing developed recurrence after surgery and responded to EGFR-TKI treatment. In present study, the percentage of undetectable EGFR mutations by cobas v2 was 9.4% in 32 mutations. It was inferred that the cause of the discrepancy in the mutation type (L858R + G863D in exon 21, and L858R in exon 21) between cobas v2 and Scorpion ARMS was due to the different limit of detection between these two PCR methods. In conclusion, the findings of the present study suggested that a selective mutation detection method may decrease the opportunity of patients with NSCLC to receive EGFR-TKI therapy. Thus, the development of a screening test to determine the EGFR status as wild-type or mutant is required for EGFR-TKI therapy.

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.

A Real-World, Observational, Prospective Study to Assess the Molecular Epidemiology of Epidermal Growth Factor Receptor (EGFR) Mutations upon Progression on or after First-Line Therapy with a First- or Second-Generation EGFR Tyrosine Kinase Inhibitor in EGFR Mutation-Positive Locally Advanced or Metastatic Non-Small Cell Lung Cancer: The 'LUNGFUL' Study.

Simple SummaryNon-small cell lung cancer (NSCLC) accounts for approximately 85% of lung cancer cases, with few patients carrying driver mutations in the gene encoding for epidermal growth factor receptor (EGFR). Advances in translational research have established EGFR tyrosine kinase inhibitors (TKIs) as the standard first-line therapy for NSCLC patients with activating EGFR mutations. The aim of our observational study was to assess the frequency of T790M acquired resistance and predictors of its presence, in patients with EGFR-mutated locally advanced or metastatic NSCLC who have progressed in the first-line EGFR-TKI treatment setting with first- or second-generation TKIs and have undergone molecular testing in tissue and/or plasma biopsy. The study highlights the challenges of performing tissue re-biopsy in routine care settings, which can lead to patients considered non-eligible for certain therapies from which they can benefit, and merits further actions from the healthcare community, in order to establish re-biopsy as a standard procedure.Background: Real-world data on the molecular epidemiology of EGFR resistance mutations at or after progression with first- or second-generation EGFR-TKIs in patients with advanced NSCLC are lacking. Methods: This ongoing observational study was carried out by 23 hospital-based physicians in Greece. The decision to perform cobas® EGFR Mutation Test v2 in tissue and/or plasma at disease progression was made before enrollment. For patients with negative/inconclusive T790M plasma-based results, tissue re-biopsy could be performed. Results: Ninety-six (96) eligible patients were consecutively enrolled (median age: 67.8 years) between July-2017 and September-2019. Of the patients, 98% were tested upon progression using plasma and 2% using tissue/cytology biopsy. The T790M mutation was detected in 16.0% of liquid biopsies. Tissue re-biopsy was performed in 22.8% of patients with a T790M-negative plasma result. In total, the T790M positivity rate was 21.9%, not differing between patients on first- or second-generation EGFR-TKI. Higher (≥2) ECOG performance status and longer (≥10 months) time to disease progression following EGFR-TKI treatment initiation were associated with T790M positivity. Conclusions: Results from plasma/tissue-cytology samples in a real-world setting, yielded a T790M positivity rate lower than previous reports. Fewer than one in four patients with negative plasma-based testing underwent tissue re-biopsy, indicating the challenges in routine care settings.

Exosome-based detection of EGFR T790M in plasma and pleural fluid of prospectively enrolled non-small cell lung cancer patients after first-line tyrosine kinase inhibitor therapy

BackgroundThe exosomal nucleic acid (exoNA) from the plasma and pleural fluid can potentially provide means to identify genomic changes in non-small cell lung cancer (NSCLC) patients who develop resistance to targeted epidermal growth factor receptor (EGFR) inhibitor therapy.MethodsWe compared the performance of the following tools to detect EGFR mutations in 54 plasma samples and 13 pleural fluid using cfDNA, combined TNA (exoTNA + cfTNA), or total cellular DNA: droplet digital PCR (ddPCR), the Cobas® EGFR Mutation Test v2 (Cobas) and NGS with Oncomine Pan-Cancer Cell-Free Assay.ResultsAll three of these platforms demonstrated 100% specificity in the detection of EGFR mutations in the plasma. In the detection of an activating mutation (exon 19 deletion and L858R), Cobas using cfDNA, ddPCR using combined TNA, and NGS using combined TNA showed a sensitivity of 93, 95.3, and 93.8%, respectively. For T790M mutation detection, the Cobas, ddPCR, and NGS showed a sensitivity of 64.7, 88.2, and 93.3%, respectively. Pleural fluid analysis revealed enrichment of the T790M mutant copies in the exosomes. ddPCR using exoTNA showed higher sensitivity than did total cellular DNA from the pleural fluid.ConclusionThese results demonstrated that combined TNA in the plasma and exoTNA in the pleural fluid can be used to evaluate low-abundant EGFR mutant copies in NSCLC.

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]

Performance Characteristics of a Real-Time Polymerase Chain Reaction Assay for the Detection of Epidermal Growth Factor Receptor (EGFR) Mutations in Plasma Samples of Non-Small Cell Lung Cancer (NSCLC) Patients.

Circulating free DNA in plasma is an alternative source of tumor-derived DNA that can be a surrogate for tissue epidermal growth factor receptor (EGFR) testing. We evaluated the analytical performance of the cobas® EGFR Mutation Test v2 (cobas test), a real-time polymerase chain reaction assay designed to detect defined EGFR gene mutations in plasma from patients with advanced non-small cell lung cancer (NSCLC). We used K2-ethylenediaminetetraacetic acid plasma samples from NSCLC patients and healthy donors (HDs), along with cell line DNA. Results from a complete technical performance evaluation are described, including a comparison between NSCLC and HD plasma to support the use of surrogate samples and an independent confirmation of the limit of detection (LoD). The cobas test reported an overall percent agreement of approximately 88% for plasma samples when compared with a next-generation sequencing method. The LoD for all EGFR mutations was ≤ 100copies/mL for plasma samples. An external study confirmed the LoD for exon19 deletion, L858R, and T790M at ≤ 100copies/mL using samples derived from NSCLC patient specimens. The cobas test showed linearity between at least 50 and 10,000copies/mL for plasma samples. An internal repeatability study reported a correct call accuracy of 99.2% for plasma samples. The performance of the cobas test is equivalent when using sheared or intact cell line DNA diluted into either HD plasma or NSCLC patient plasma. The cobas test is a sensitive, robust, and accurate assay that delivers reproducible results.

Clearing of circulating tumour DNA predicts clinical response to osimertinib in EGFR mutated lung cancer patients

ObjectivesTyrosine kinase inhibitors (TKIs) are first line treatment choices for patients with epidermal growth factor receptor (EGFR) mutated non-small cell lung cancer (NSCLC). However, responses vary among patients, therefore good biomarkers predicting better responses are required. EGFR mutations are detected in the blood from patients as circulating tumour DNA (ctDNA). Studies have shown that clearing ctDNA during first line TKI treatment predicts outcomes for first and second generation TKI treatments. We aimed to investigate the effects on outcome measures of ctDNA clearing in subsequent treatment lines to treatment with the third generation TKI osimertinib. MethodsIn total, 225 patients were included in a prospective, multicentre study, where consecutive blood samples were monitored for EGFR mutations during systemic treatment lines, using the Cobas® EGFR mutation test v2. This study focused on EGFR mutations in ctDNA of 82 systemically pre-treated patients receiving osimertinib. ResultsClearing all EGFR mutations from the blood after osimertinib treatment, significantly predicted progression-free survival, objective response rates and disease control rates. Primary sensitising EGFR mutations were found in ctDNA in 70 % of patients, and were accompanied by the T790 M mutation in nearly two thirds of cases. The T790 M mutation was cleared in all cases, while the accompanying sensitising mutations did not necessarily clear. However, T790 M clearing without simultaneously clearing of the primary sensitising mutation did not predict clinical responses. Neither the detection of T790 M before osimertinib treatment, nor the presence of EGFR mutations at the time of osimertinib initiation predicted clinical outcomes. ConclusionThe clearing of EGFR mutations in ctDNA after osimertinib treatment initiation in patients with advanced NSCLC is useful as a positive predictor of clinical outcome.

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.