scholarly journals The use of COLD-PCR and pyrosequencing for sensitive detection of EGFR T790M mutation

2021 ◽  
Vol 271 ◽  
pp. 03026
Author(s):  
Huimin Chen ◽  
Yang Zhang ◽  
Dan Pu ◽  
Kunxian Shu
Keyword(s):  
Kinase Inhibitors ◽  
Acquired Resistance ◽  
Growth Factor Receptor ◽  
Cost Effective ◽  
Fold Increase ◽  
Conventional Pcr ◽  
T790m Mutation ◽  
Detection Rates ◽  
Nsclc Patients ◽  
Egfr T790m

A sensitive and convenient method for the detection of epidermal growth factor receptor (EGFR) T790M mutation in non-small cell lung cancer (NSCLC) patients with acquired resistance to tyrosine kinase inhibitors (TKIs) would be desirable to guide treatment strategy. Consequently, studies have focused on sensitive characterization of EGFR T790M mutation. Herein, two methods of co-amplification at lower denaturation temperature PCR (COLD-PCR) and pyrosequencing were combined (COLDPCR/ pyrosequencing) for detecting EGFR T790M mutation. Evaluation of mutation-containing dilutions revealed that the sensitivities of COLD-PCR/pyrosequencing and conventional PCR/pyrosequencing assays for the detection of the T790M mutation were 0.1 and 5%, respectively, indicating a 50-fold increase in sensitivity. When the T790M mutation in 20 clinical NSCLC samples who had relapsed under firstgeneration EGFR TKI were further determined using COLD-PCR/pyrosequencing and conventional PCR/pyrosequencing, the detection rates were 35% (7/20) and 25% (5/20), respectively. All patients who were positive for the T790M mutation with conventional PCR/pyrosequencing were also found to be positive with COLD-PCR/pyrosequencing. The discordant cases were 2 samples with no T790M mutation detected with conventional PCR/pyrosequencing, but which were positive with COLD-PCR/pyrosequencing. COLD-PCR/pyrosequencing is a sensitive and cost-effective tool for detecting the T790M mutation which will permit an improvement of therapeutic management.

Novel resistance mechanisms to first-generation EGFR tyrosine kinase inhibitors: A perspective study in NSCLC patients using targeted next generation sequencing.

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. e20576-e20576
Author(s):  
Ying Jin ◽  
Jianjun Zhang ◽  
Ming Chen ◽  
Yang Shao ◽  
Xun Shi ◽  
...  
Keyword(s):  
First Generation ◽  
Kinase Inhibitors ◽  
Acquired Resistance ◽  
Resistance Mechanisms ◽  
Egfr Mutations ◽  
T790m Mutation ◽  
Targeted Next Generation Sequencing ◽  
Egfr Tyrosine Kinase Inhibitors ◽  
Nsclc Patients ◽  
Egfr T790m

e20576 Background:Patients with non-small-cell lung cancer (NSCLC) harboring sensitive epithelial growth factor receptor (EGFR) mutations invariably develop acquired resistance to EGFR tyrosine kinase inhibitors (TKIs). Identification of actionable mutations conferring drug-resistance can be helpful for guiding the subsequent treatment decision. Currently, the known mechanisms of acquired resistance includes: the secondary gatekeeper EGFR-T790M mutation, activation of members of downstream signaling pathways such as PI3K/AKT/mTOR pathway, activation of bypass signaling such as MET, and changes in tumor histology. However, the mechanisms in the remaining patients are still unknown. Methods:In this prospective study, thirty-one advanced NSCLC patients initially carrying sensitive EGFR mutations and subsequently developing acquired resistance to the first-generation EGFR-TKIs were enrolled. Pre-treatment tumor samples as well as re-biopsies of tumor and plasma when the patients were diagnosed with EGFR-TKI resistance were acquired, followed by mutation profiling using targeted next generation sequencing (NGS) on 416 cancer-related genes. Results: In total, 55% of patients were identified to carry acquired secondary EGFR-T790M mutation. Three patients (~10%) harbor EGFR-T854A mutation, which has been reported as another TKI resistant mutation. 26% and 19% of cases accumulated TP53 and RB1 mutations, respectively. In T790M/T854A-negative cases, 30% of patients acquired MET amplification. Other potential acquired resistance mechanisms includes single nucleotide variants (SNVs) in genes such as SMAD4, DNMT3A, GNAS, ATM, KRAS, PIK3CA and TET2, and copy number variations (CNVs) in genes such as CDK4, MDM2, MYC, RICTOR and ERBB2. Conclusions:The study depicted the genetic landscapes comprehensively in matched pre- and post-EGFR-TKIs samples of NSCLC population resistant to first generation TKI treatments. Our analysis demonstrates new perspectives for further study of resistance and putting forward corresponding relevant tactics against the challenge of disease progression. Clinical trial information: NCT02804217.

Sensitivity of TargetSelector in clinical experience in ctDNA profiling of NSCLC 2000 cases.

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. e23033-e23033
Author(s):  
Veena M. Singh ◽  
Anthony J. Daher ◽  
Jeffery J. Chen ◽  
Lyle Arnold ◽  
Cecile Rose T. Vibat
Keyword(s):  
Kinase Inhibitors ◽  
Acquired Resistance ◽  
Growth Factor Receptor ◽  
Egfr Mutations ◽  
Wild Type ◽  
T790m Mutation ◽  
Blood Samples ◽  
Copy Numbers ◽  
Activating Mutation ◽  
Nsclc Patients

e23033 Background: Targeted cancer therapy relies on identifying specific DNA mutations from a patient’s tumor. Tyrosine kinase inhibitors (TKIs) tend to be effective for non-small cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) activating mutations, of which exon 19 deletions (Del19) and L858R are most common. Acquired resistance to TKI therapy is associated with a T790M mutation. Standard biomarker analyses may not reflect tumor heterogeneity; they entail tissue biopsies often with surgical complications. To address these limitations, Biocept developed a minimally invasive method to characterize cancer biomarkers in blood. Biocept's proprietary TargetSelector assays selectively amplify relevant mutations from circulating tumor DNA (ctDNA). Clinical validations demonstrated high concordances between molecular tests in blood vs tissue. As further validation, EGFR mutation detection frequencies were compared to US averages (mycancergenome.org). Here we analyze 2000 blood samples received at Biocept from 1Mar 2016 to 4Jan 2017 from late stage NSCLC patients. Methods: Blood was collected in Biocept OncoCEE BCT validated to preserve DNA ≤ 8 days. TargetSelector was used to detect ctDNA L858R, Del19 and T790M.EGFR allele copy numbers for wild type and each mutant were calculated. The prevalence of each mutation was compared to US averages. Results: Del19, L858R, and T790M mutations were detected in 12.9%, 8.5%, and 9.9% of the analyzed blood samples, respectively. This is concordant with US averages, which are 10% for each mutation. Median copy numbers/ml of blood were 30 for Del19 (range: 1 – 91974), 15 for L858R (range: 1 – 91200), and 10 for T790M (range: 1 – 137360). The median wild type EGFR copy number detected/ml blood was 2304 (range: 8 – 2498725). In ~80% of T790M cases, ≥ 1 concomitant activating mutation was detected. Conclusions: Biocept's TargetSelector detects EGFR mutations (Del19, L585R, and T790M) at a very high level of sensitivity down to 1 mutant copy/ml in advanced NSCLC patients at frequencies consistent with cited US rates. Moreover, the underlying activating mutation was detected in ~80% of T790M cases.

scholarly journals Development of epidermal growth factor receptor tyrosine kinase inhibitors against EGFR T790M. Mutation in non small-cell lung carcinoma

Open Medicine ◽  
2016 ◽  
Vol 11 (1) ◽  
pp. 68-77 ◽  
Author(s):  
Yuli Wang ◽  
Zhitao Guo ◽  
Yang Li ◽  
Qinghua Zhou
Keyword(s):  
Epidermal Growth Factor Receptor ◽  
Small Cell Lung Carcinoma ◽  
Acquired Resistance ◽  
Growth Factor Receptor ◽  
Small Cell ◽  
Small Cell Lung ◽  
T790m Mutation ◽  
Cell Lung Carcinoma ◽  
Epidermal Growth ◽  
Egfr T790m

AbstractIndividualized therapies targeting epidermal growth factor receptor (EGFR) mutations show promises for the treatment of non small-cell lung carcinoma (NSCLC). However, disease progression almost invariably occurs 1 year after tyrosine kinase inhibitor (TKI) treatment. The most prominent mechanism of acquired resistance involves the secondary EGFR mutation, namely EGFR T790M, which accounts for 50%–60% of resistant tumors. A large amount of studies have focused on the development of effective strategies to treat TKI-resistant EGFR T790M mutation in lung tumors. Novel generations of EGFR inhibitors are producing encouraging results in patients with acquired resistance against EGFR T790M mutation. This review will summarize the novel inhibitors, which might overcome resistance against EGFR T790M mutation.

scholarly journals Clinical Characteristics of Osimertinib Responder in Non-Small Cell Lung Cancer Patients with EGFR-T790M Mutation

Cancers ◽  
2019 ◽  
Vol 11 (3) ◽  
pp. 365 ◽  
Author(s):  
Akihiro Yoshimura ◽  
Tadaaki Yamada ◽  
Naoko Okura ◽  
Takayuki Takeda ◽  
Kazuki Hirose ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Small Cell Lung Cancer ◽  
Cell Lung Cancer ◽  
Kinase Inhibitors ◽  
Small Cell ◽  
Small Cell Lung ◽  
T790m Mutation ◽  
Nsclc Patients ◽  
Egfr T790m ◽  
Egfr Tkis

Osimertinib is a mutant-selective EGFR inhibitor that is effective against non-small cell lung cancer (NSCLC) in patients with the EGFR-T790M mutation, who are resistant to EGFR-tyrosine kinase inhibitors (EGFR-TKIs). However, the factors affecting response to osimertinib treatment are unknown. In this retrospective study, 27 NSCLC patients with the EGFR-T790M mutation were enrolled at five institutions in Japan. Among several parameters tested, the progression-free survival (PFS) associated with the initial EGFR-TKIs was positively correlated with the PFS after osimertinib treatment (p = 0.021). The median PFS following osimertinib treatment and the overall survival (OS) were longer in patients who responded to osimertinib than in those who did not (17.7 months versus 3.5 months, p = 0.009 and 24.2 months versus 13.5 months, p = 0.021, respectively). A multivariate analysis demonstrated that the PFS with initial EGFR-TKIs was significantly related to the PFS with osimertinib treatment (p = 0.035), whereas osimertinib response was significantly related to the PFS and OS with osimertinib treatment (p = 0.016 and p = 0.006, respectively). Our retrospective observations indicate that PFS following the initial EGFR-TKI treatment and the response rate to osimertinib might be promising predictors for effective osimertinib treatment in NSCLC patients with the EGFR-T790M mutation.

scholarly journals MET Gene Dysregulation as a Promising Therapeutic Target in Lung Cancer—A Review

2021 ◽  
Vol 11 (12) ◽  
pp. 1370
Author(s):  
Paulina Terlecka ◽  
Paweł Krawczyk ◽  
Anna Grenda ◽  
Janusz Milanowski
Keyword(s):  
Lung Cancer ◽  
Clinical Trials ◽  
Kinase Inhibitors ◽  
Acquired Resistance ◽  
Point Mutations ◽  
Growth Factor Receptor ◽  
Molecular Abnormalities ◽  
Promising Therapeutic Target ◽  
Tumorigenic Activity ◽  
Nsclc Patients

Several molecular abnormalities in the MET gene have been identified, including overexpression, amplification, point mutations, and “skipping mutation” in exon 14. Even though deregulated MET signaling occurs rarely in non-small cell lung cancer (NSCLC), it possesses tumorigenic activity. Since the discovery of the significant role played by MET dysregulations in resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKI), many clinical trials have been focused on mechanisms underlying this acquired resistance. Therefore, new therapeutic strategies are being considered in the personalized therapy of NSCLC patients carrying MET abnormalities. First, MET kinase inhibitors (tepotinib and capmatinib) have been shown to be effective in the first and subsequent lines of treatment in NSCLC patients with “skipping mutations” in exon 14 of MET gene. In this article, the authors show the role of MET signaling pathway alterations and describe the results of clinical trials with MET inhibitors in NSCLC patients.

Mutational analysis of the EGFR gene in lung cancer with acquired resistance to gefitinib

2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 7074-7074 ◽  
Author(s):  
T. Mitsudomi ◽  
T. Kosaka ◽  
H. Endoh ◽  
K. Yoshida ◽  
T. Hida ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Kinase Inhibitors ◽  
Mutational Analysis ◽  
Acquired Resistance ◽  
Growth Factor Receptor ◽  
Myelogenous Leukemia ◽  
Ras Gene ◽  
T790m Mutation ◽  
Egfr Gene ◽  
Activating Mutations

7074 Background: Non-small cell lung cancer (NSCLC) harboring activating mutations in the epidermal growth factor receptor (EGFR) gene is usually highly sensitive to EGFR tyrosine kinase inhibitors (TKI), gefitinib or erlotinib. However, it is common to develop acquired resistance to TKI after presenting an initial striking response. It has been reported that secondary mutation of threonine to methionine at codon 790 (T790M) of the EGFR gene is related to this acquired resistance. Methods: We sequenced exons 18–21 of the EGFR gene in 14 NSCLC patients exhibiting acquired resistance to gefitinib following the initial good response. This region of the EGFR gene corresponds with that of the ABL gene where various secondary mutations have been reported in patients with chronic myelogenous leukemia (CML) with acquired resistance to imatinib. To raise sensitivity of the assay, we also subcloned the PCR products into plasmids and sequenced, or we used CyCleave method (real-time PCR combined with fluorescence labeled mutant specific probe) in addition to usual sequencing. We also searched for secondary K-ras mutations. Results: All the 14 patients had activating mutations of the EGFR gene (9 with exon 19 deletions, 5 with L858R). In addition, we found that 7 of 14 patients had a T790M mutation, but there were not any other novel secondary mutations. In these seven patients, T790M mutant bands were smaller than wild-type bands. Patients with T790M tended to be never smoking female, but there was no difference in the period of gefitinib administration by the presence or absence of T790M. We could not detect any T790M in tumors before gefitinib treatment at the sensitivity of 1%. There were no patients with acquired mutation of the K-ras gene. Conclusions: Secondary T790M mutation of the EGFR gene accounted for half of the cases with acquired resistance to gefitinib. Unlike the cases with CML, various kinds of secondary mutations were not likely to exist in the EGFR gene as a mechanism of acquired resistance. No significant financial relationships to disclose.

Cost-effectiveness analysis of icotinib versus whole-brain irradiation with or without chemotherapy in EGFR-mutant NSCLC patients with brain metastases.

2019 ◽  
Vol 37 (15_suppl) ◽  
pp. e20556-e20556
Author(s):  
Wenqian Li ◽  
Rilan Bai ◽  
Lei Qian ◽  
Naifei Chen ◽  
Yuguang Zhao ◽  
...  
Keyword(s):  
Cost Effectiveness ◽  
Brain Metastases ◽  
Kinase Inhibitors ◽  
Growth Factor Receptor ◽  
Cost Effective ◽  
Sensitivity Analyses ◽  
Net Benefit ◽  
Whole Brain Irradiation ◽  
Nsclc Patients ◽  
The Impact

e20556 Background: Non-small-cell lung cancer (NSCLC) patients with brain metastases had a poor prognosis. Despite the traditional methods including radiotherapy and chemotherapy, epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) might benefit patients on survival and quality of life. We investigated the cost-effectiveness of icotinib compared with WBI with or without chemotherapy for NSCLC patients with brain metastases. Methods: A markov model was conducted based on the data of BRAIN trial. We compared the economic benefit between icotinib and the combination of WBI and WBI plus chemotherapy group. We considered disease progression as intracranial progression and overall progression separately. Sensitivity analyses were performed to observe the stability of the model. The willingness-to-pay (WTP) was set as 3× per capita gross domestic product ($25929/quality-adjusted life year [QALY]). Results: When considering progression as intracranial progression and overall progression respectively, the incremental cost-effectiveness ratio (ICER) was $930.17/QALY and $842.76/QALY between icotinib and WBI/WBI-chemotherapy. Besides, both of the average cost-effective ratio (average CE) and net benefit showed advantage of icotinib (average CE: $2157.59/QALY for intracranial progression, $2285.16/QALY for overall progression; net benefit: $372153.35 for intracranial progression, $349938.32 for overall progression). One-way sensitivity analyses demonstrated the impact of the utilities of icotinib group. The probabilistic sensitivity analyses showed even at a WTP under $6000/QALY, icotinib could be cost-effective. Conclusions: Icotinib was cost-effective compared with WBI with or without chemotherapy. [Table: see text]

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