Verification of Wild-Type EGFR Status in Non–Small Cell Lung Carcinomas Using a Mutant-Enriched PCR on Selected Cases

Chen, Yi Lin; Lu, Chuanyong; Yang, Shu Ching; Su, Wen; Lin, Ya Lan; Chen, Wan Li; Huang, Wenya; Su, Wu Chou; Chow, Nan Haw; Ho, Chung Liang

doi:10.1016/j.jmoldx.2014.05.007

Cited by 14 publications

(23 citation statements)

References 33 publications

(21 reference statements)

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“…The EGFR PCR Kit (EGFR RUO Kit) and the therascreen EGFR RGQ PCR Kit (EGFR IVD Kit, Qiagen) were used to identify mutated EGFR DNA. These kits utilized a combination of Scorpions real‐time PCR technology and amplification‐refractory mutation system (ARMS) technology for the detection of the mutations with real‐time quantitative PCR …”

Section: Methodsmentioning

confidence: 99%

Real‐world outcomes of NSCLC patients receiving tissue or circulating tumor DNA‐guided osimertinib treatment

Yang

Chen

et al. 2019

Cancer Medicine

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Background Osimertinib yields significant tumor responses and durations of progression‐free survival (PFS) in patients with acquired T790M mutations. However, the evidence supporting liquid biopsy‐guided treatment is still limited. This study examined the real‐world benefits of osimertinib in patients with tissue or plasma T790M mutations. Methods From January 2016 to June 2018, a total of 183 non‐small‐cell lung cancer patients were enrolled. The presence of the T790M mutation was assessed by either tissue or plasma. The PFS, overall survival, and tumor response rates of the patients were calculated and compared with those of previous clinical trials. Results T790M mutations were detected in 51.5% of the patients, including 64 of 140 (45.7%) who underwent liquid biopsies and 23 of 29 (79.3%) who underwent tumor biopsies. After excluding those in clinical trials, 46 patients received osimertinib, including 33 with positive plasma and 13 with positive tissue results for T790M mutations. The median PFS was 11.3 months (interquartile range: 5.2‐NR) in all the T790M‐positive patients and 10.1 months (interquartile range: 5.9‐NR) in the plasma T790M‐positive patients. The overall survival, meanwhile, was not reached, whereas the one‐year survival rate was 66.1% in all the patients and 61.4% in those who were plasma T790M‐positive. The objective response rate and disease control rate were 37.8% and 91.9% in all the patients and 34.6% and 92.3% in the plasma T790M‐positive group, respectively. Using a Cox proportional hazards regression, we determined that male gender was a poor prognostic factor for PFS. Conclusions In this retrospective real‐world analysis, it was determined that both tissue and plasma T790M mutations can be used to guide treatment with osimertinib. Similar disease control rates and survival durations were observed in comparison to those of phase 3 clinical trials.

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Section: Methodsmentioning

confidence: 99%

Real‐world outcomes of NSCLC patients receiving tissue or circulating tumor DNA‐guided osimertinib treatment

Yang

Chen

et al. 2019

Cancer Medicine

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“…Disease progression was determined based on the radiographic evidence according to Response Evaluation Criteria in Solid Tumors version 1.1. 12 …”

Section: Methodsmentioning

confidence: 99%

“…These kits combine Scorpion’s and the amplification-refractory mutation system (ARMS) technologies to detect the mutations using real-time quantitative PCR. 12 …”

Section: Methodsmentioning

confidence: 99%

Preventing and treating brain metastases with three first-line EGFR-tyrosine kinase inhibitors in patients with EGFR mutation-positive advanced non-small cell lung cancer

Chang

et al. 2018

Ther Adv Med Oncol

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Introduction:Brain metastases (BM) are common in advanced non-small cell lung cancer (NSCLC), and the prognosis is poor with few therapeutic options. This study evaluated the efficacy of three epidermal growth factor receptor–tyrosine kinase inhibitors (EGFR-TKIs) in preventing and treating BM in patients with EGFR mutation-positive advanced NSCLC.Methods:Patients with EGFR mutation-positive advanced NSCLC who visited a tertiary referral center from 1 December 2013 to 30 November 2017 were analyzed retrospectively. They received gefitinib, erlotinib, or afatinib until disease progression, death, or intolerable adverse events. The cumulative incidence of subsequent BM of initial non-BM patients, progression-free survival (PFS), and overall survival (OS) of the BM and non-BM patients were estimated and compared using the Kaplan–Meier and log-rank tests.Results:306 NSCLC patients were enrolled, with 116, 75, and 115 receiving first-line gefitinib, erlotinib, and afatinib, respectively. The afatinib group had a better PFS [12.7 versus 9.8 months; hazard ratio (HR) 0.59, p = 0.001] and OS (39.1 versus 22.0 months; HR 0.64, p = 0.035) than the gefitinib group. Afatinib tended to provide better BM prevention than gefitinib (BM cumulative incidence, HR 0.49; 95% confidence interval 0.34–0.71, p < 0.001) according to a Cox model adjusted for possible confounders. Patients with initial BM had a shorter PFS (p < 0.001) and OS (p = 0.015) than those without initial BM. Among the former, there were no differences in median PFS (p = 0.34) and median OS (p = 0.46) in the three EGFR-TKI groups.Conclusions:Our data suggested that, compared with gefitinib, afatinib provided better benefits significantly in terms of PFS and OS. Both had the same effectiveness in preventing subsequent BM.

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“…To increase sensitivity of mutant detection, especially in samples like liquid biopsies, various techniques are used, such as sophisticated sequencing methods [40][41][42] , droplet digital PCR 43 , allele-specific PCR 7 , SNPase-AMRS qPCR 44 , COLD-PCR (co-amplification at lower denaturation temperature-PCR) 45 , HRM assay 30,32,34,46,47 , competitive probe blocking 48 , peptide nucleic acid-mediated PCR clamping 49 , and DNA terminal structure-mediated enzymatic reaction 50 . However, none of these contain all the optimal qualities: high sensitivity and specificity, simplicity and cost-efficiency, feasibility in clinical settings.…”

Section: Discussionmentioning

confidence: 99%

SLAM-MS: Mutation scanning of stem-loop amplicons with TaqMan probes by quantitative DNA melting analysis

Kondratova

Botezatu

Shelepov

et al. 2020

Sci Rep

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DnA Melting Analysis (DMA) with a taqMan probe covering the mutation "hot spot" is a simple, sensitive, and "closed tube" method of mutation detection. However, DMA requires asymmetric pcR to produce single-stranded amplicons capable of interacting with taqMan probes. this makes quantitative analysis impossible owing to low amplification efficiency. Moreover, bi-strand mutation detection necessitates two independent pcRs. the SLAM-MS (Stem-Loop AMplicon Mutation Scanning) assay, in which symmetric PCR is performed using primers with 5'-universal primer sequence (UPS), has been developed to detect KRAS mutations. Some of the resulting amplicons, sense and antisense, adopt single-stranded stem-loop conformation and become unable to renature, but able to hybridize with taqMan probes. Hybrids of stem-loops and complementary taqMan probes are suitable for melting analysis and simultaneous bi-strand mutation scanning. in addition, the areas under the melting peaks are determined by the PeakFit software, a non-linear iterative curve fitting program, to evaluate the wild-type/mutant allele ratio. Thus, the SLAM-MS assay permits quantification of both the number of copies of the target sequence and the percentage of mutant alleles. for mutant enrichment, the SLAM-MS assay uses TaqMan probes as PCR blocking agents allowing an ~10 times higher mutation detection sensitivity than High Resolution Melting (HRM) assay. In basic cancer research, priority is given to large-scale (genome-and exome-wide) next generation sequencing (NGS) methods, which give a panoramic picture of defects in the cancer genome. However, for clinical diagnostics, a "targeted approach" is more often required, i.e. screening of previously known clinically important mutations, in particular in the KRAS, NRAS, BRAF, EGFR, and PIK3CA genes 1-7. In this case, the priorities are the feasibility of the method in a clinical laboratory, and its simplicity, performance, and sensitivity. These requirements are largely met by asymmetric PCR with a TaqMan probe covering the mutation "hot spot" and subsequent DMA (DNA Melting Analysis), which is one of the most simple, fast, and least costly methods to detect DNA mutations. Moreover, it is implemented in the "closed tube format" that avoids any additional manipulations and eliminates sample cross-contamination 8,9. The short length of the TaqMan probes (20-30 nucleotides) facilitates strong mismatch-induced changes in melting curves and clear discrimination of wild-type and mutant melt peaks. Computation of the areas under the peaks using specialized software permits quantification of the ratio of wild and mutant alleles 9. Furthermore, under special (suboptimal) PCR conditions, the TaqMan probes serve as blocking agents contributing to the enriched synthesis of mutant alleles 10. An inherent drawback of this method is the asymmetric mode of PCR necessary for accumulation of single-stranded amplicons ("targets" for TaqMan probes). This leads to a number of limitations: (i) a decrease in the amplification efficiency, ...

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Verification of Wild-Type EGFR Status in Non–Small Cell Lung Carcinomas Using a Mutant-Enriched PCR on Selected Cases

Cited by 14 publications

References 33 publications

Real‐world outcomes of NSCLC patients receiving tissue or circulating tumor DNA‐guided osimertinib treatment

Real‐world outcomes of NSCLC patients receiving tissue or circulating tumor DNA‐guided osimertinib treatment

Preventing and treating brain metastases with three first-line EGFR-tyrosine kinase inhibitors in patients with EGFR mutation-positive advanced non-small cell lung cancer

SLAM-MS: Mutation scanning of stem-loop amplicons with TaqMan probes by quantitative DNA melting analysis

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