Dataset Information

A circulating tumor cell-based digital assay for the detection of EGFR T790M mutation in advanced non-small cell lung cancer.

ABSTRACT: Determining the status of epidermal growth factor receptor (EGFR) T790M mutation is crucial for guiding further treatment intervention in advanced non-small cell lung cancer (NSCLC) patients who develop acquired resistance to initial EGFR tyrosine kinase inhibitor (TKI) treatment. Circulating tumor cells (CTCs) which contain plentiful copies of well-preserved RNA offer an ideal source for noninvasive detection of T790M mutation in NSCLC. We developed a CTC-based digital assay which synergistically integrates NanoVelcro Chips for enriching NSCLC CTCs and reverse-transcription droplet digital PCR (RT-ddPCR) for quantifying T790M transcripts in the enriched CTCs. We collected 46 peripheral arterial and venous blood samples from 27 advanced NSCLC patients for testing this CTC-based digital assay. The results showed that the T790M mutational status observed by the CTC-based digital assay matched with those observed by tissue-based diagnostic methods. Furthermore, higher copy numbers of T790M transcripts were observed in peripheral arterial blood than those detected in the matched peripheral venous blood. In short, our results demonstrated the potential of the NanoVelcro CTC-digital assay for noninvasive detection of the T790M mutation in NSCLC, and suggested that peripheral arterial blood sampling may offer a more abundant CTC source than peripheral venous blood in advanced NSCLC patients.

SUBMITTER: Wang J

PROVIDER: S-EPMC8136811 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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A circulating tumor cell-based digital assay for the detection of EGFR T790M mutation in advanced non-small cell lung cancer.

Wang Jing J Sun Na N Lee Yi-Te YT Ni Yiqian Y Koochekpour Rose R Zhu Yazhen Y Tseng Hsian-Rong HR Wang Shuyang S Jiang Liyan L Zhu Hongguang H

Journal of materials chemistry. B 20200701 26

Determining the status of epidermal growth factor receptor (EGFR) T790M mutation is crucial for guiding further treatment intervention in advanced non-small cell lung cancer (NSCLC) patients who develop acquired resistance to initial EGFR tyrosine kinase inhibitor (TKI) treatment. Circulating tumor cells (CTCs) which contain plentiful copies of well-preserved RNA offer an ideal source for noninvasive detection of T790M mutation in NSCLC. We developed a CTC-based digital assay which synergistical ...[more]

PMID: 32525199

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Project description:The present study aimed to explore the influence of T790M neighboring single nucleotide polymorphism (SNP) on the sensitivity of amplification refractory mutation system (ARMS)-based T790M mutation assay. Three ARMS-quantitative polymerase chain reaction (qPCR) systems (system 1 had a forward ARMS primer without rs1050171, system 2 included a forward ARMS primer with rs1050171 and system 3 contained the above two forward ARMS primers) were used to detect the T790M mutation in two series plasmid samples and genomic DNA (gDNA) of the cell line H1975. A total of 670 formalin-fixed paraffin-embedded (FFPE) tumor samples from non-small cell lung cancer patients were used to detect the epidermal growth factor receptor (EGFR) gene T790M mutation by direct sequencing and ARMS-qPCR. The ARMS-qPCR system 1 effectively detected samples with as low as 1% T790M mutant plasmid 1 (without rs1050171) and with 50% T790M mutant plasmid 2 (with rs1050171), while the ARMS-qPCR system 2 detected samples with 20 and 50% T790M mutant plasmid 1, in addition to samples with 1% T790M mutant plasmid 2. For the ARMS-qPCR system 3, samples with as low as 1% T790M mutant plasmids 1 or 2 were effectively detected. For gDNA analysis of the cell line H1975, the T790M mutation was effectively detected by the ARMS-qPCR systems 2 and 3 (~50% mutation rate), but was detected with a low mutation abundance by the ARMS-qPCR system 1 (~1% mutation rate). Of the 670 FFPE samples, 5 cases were identified to have the T790M mutation by sequencing and by the ARMS-qPCR system 1. One sample (named N067), which was considered as T790M-negative by sequencing, was demonstrated to have the T790M mutation using the ARMS-qPCR system 1. Sample N094, which was variant homozygous for rs1050171 and was indicated to be T790M-negative by sequencing and by the ARMS-qPCR system 1, was identified to have the T790M mutation with the ARMS-qPCR system 3. The A-variant allele frequency of rs1050171 was observed to be 28.2% in the 670 FFPE tumor samples, while the presence of rs148188503 (c. C2355T, p. T785T) was observed in sample N558, and a novel SNP with a base substitution (c. T2375C) at position 792 (p. L792P) in exon 20 of the EGFR gene was observed in sample N310. rs1050171 is a high-frequency SNP located near T790M, and the mutation statuses of rs1050171 appear to influence the sensitivity of the ARMS-based T790M detection system, thus generating a 14.3% false-negative rate (1/7). The present study proposes the risk that target neighboring SNPs (as far as 8 bp away in the present study) may exert on the sensitivity of ARMS-based detection methods.

Dataset Information

A circulating tumor cell-based digital assay for the detection of EGFR T790M mutation in advanced non-small cell lung cancer.

Publications

A circulating tumor cell-based digital assay for the detection of EGFR T790M mutation in advanced non-small cell lung cancer.

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