Dataset Information

Comparative assessment of NOIR-SS and ddPCR for ctDNA detection of EGFR L858R mutations in advanced L858R-positive lung adenocarcinomas.

ABSTRACT: Genotyping epidermal growth factor receptor (EGFR) is an essential process to indicate lung adenocarcinoma patients for the most appropriate treatment. Liquid biopsy using circulating tumor DNA (ctDNA) potentially complements the use of tumor tissue biopsy for identifying genotype-specific mutations in cancer cells. We assessed the performance of a high-fidelity sequencing method that uses molecular barcodes called the nonoverlapping integrated read sequencing system (NOIR-SS) for detecting EGFR L858R-mutated alleles in 33 advanced or recurrent patients with L858R mutation-positive lung adenocarcinoma revealed by matched tissue biopsy. We compared NOIR-SS with site-specific droplet digital PCR (ddPCR), which was taken as the reference, in terms of sensitivity and ability to quantify L858R variant allele fractions (VAFs). NOIR-SS and ddPCR had sensitivities of 87.9% (29/33) and 78.8% (26/33) for detecting L858R alleles, respectively. The VAFs measured by each assay were strongly correlated. Notably, one specimen was positive with a VAF of 30.12% for NOIR-SS but marginally positive with that of 0.05% for ddPCR because of a previously poorly recognized mechanism: two-base substitution-induced L858R (c.2573_2574delinsGA). These results indicate that NOIR-SS is a useful method for detecting ctDNA, potentially overcoming a limitation of ddPCR which highly depends on the binding ability of primers to specific targeting sequences.

SUBMITTER: Akahori D

PROVIDER: S-EPMC8298558 | biostudies-literature | 2021 Jul

REPOSITORIES: biostudies-literature

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Comparative assessment of NOIR-SS and ddPCR for ctDNA detection of EGFR L858R mutations in advanced L858R-positive lung adenocarcinomas.

Akahori Daisuke D Inoue Yusuke Y Inui Naoki N Karayama Masato M Yasui Hideki H Hozumi Hironao H Suzuki Yuzo Y Furuhashi Kazuki K Fujisawa Tomoyuki T Enomoto Noriyuki N Nakamura Yutaro Y Suda Takafumi T

Scientific reports 20210722 1

Genotyping epidermal growth factor receptor (EGFR) is an essential process to indicate lung adenocarcinoma patients for the most appropriate treatment. Liquid biopsy using circulating tumor DNA (ctDNA) potentially complements the use of tumor tissue biopsy for identifying genotype-specific mutations in cancer cells. We assessed the performance of a high-fidelity sequencing method that uses molecular barcodes called the nonoverlapping integrated read sequencing system (NOIR-SS) for detecting EGFR ...[more]

PMID: 34294857

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Project description:BackgroundEarly data has indicated that EGFR 19Del mutation and EGFR L585R mutation are two different types of non-small cell lung cancer (NSCLC). However, how the different molecular mechanisms participate in the process of mediastinal lymph node metastasis (MLNM) in lung adenocarcinoma (LA) harboring EGFR 19Del and EGFR L858R mutation remains unknown. We thus explored the genes responsible for MLNM in LA with EGFR 19Del or L858R mutation.MethodsWe performed transcriptome sequencing and bioinformatics analysis from 10 patients with LA resection specimens of primary tumors. Quantitative reverse transcription-polymerase chain reaction was used to validate gene expressions.ResultsThere were 69 mRNAs upregulated and 100 mRNAs downregulated in five samples with MLNM compared with samples without MLN metastasis. EEF1A2 and ERN2 were observed exhibiting different expression patterns in EGFR 19Del and EGFR L858R samples with MLNM. In samples harboring EGFR 19Del mutation, the expression of EEF1A2 gene in samples with MLNM was significantly lower compared with samples without MLN metastasis, and in samples with EGFR L858R, it was significantly higher in samples with MLNM. The expression pattern of ERN2 was opposite to EEF1A2. In addition, several other genes including SLC6A11, IGHV3-48, IGHV3-43, DUSP9, and HOXA9 were also shown to be associated with invasion and metastasis and exhibited an expression pattern similar to EEF1A2 and ERN2 in EGRF 19Del and L858R mutation tumors.ConclusionsEEF1A2 and ERN2 were for the first time observed exhibiting distinct expression patterns in MLNM in lung adenocarcinomas harboring EGFR 19Del and EGFR L858R mutation by interindividual DEGs analysis.Key pointsSignificant findings of the study In our study, we focused on the mechanisms of metastasis and invasion that different EGFR mutations conferred and identified two critical genes separately involved in this process in EGFR 19Del and L858R mutation tumors. What this study adds Our findings not only reinforced theoretical foundations that the EGFR 19Del and L858R mutation tumors should be considered as two kinds of diseases, but also laid the fundamentals for precise determination of the mediastinal lymph node radiation field and improvement of clinical outcome.

Project description:Activating double mutations L858R/T790M in the epidermal growth factor receptor (EGFR) region are often observed as the cause of resistance to tyrosine kinase inhibitors (TKIs). Third-generation EGFR-TKIs, such as osimertinib and rociletinib (CO-1686), was developed to target such resistance mutations. The detection of activating L858R/T790M mutations is necessary to select sensitive patients for therapy. Hence, we aimed to develop novel radiobromine-labeled CO-1686 as a positron emission tomography (PET) imaging probe for detecting EGFR L858R/T790M mutations. Nonradioactive brominated-CO1686 (BrCO1686) was synthesized by the condensation of N-(3-[{2-chloro-5-(trifluoromethyl)pyrimidin-4-yl}amino]-5-bromophenyl) acrylamide with the corresponding substituted 1-(4-[4-amino-3-methoxyphenyl]piperazine-1-yl)ethan-1-one. The radiobrominated [77Br]BrCO1686 was prepared through bromodestannylation of the corresponding tributylstannylated precursor with [77Br]bromide and N-chlorosuccinimide. Although we aimed to provide a novel PET imaging probe, 77Br was used as an alternative radionuclide for 76Br. We fundamentally evaluated the potency of [77Br]BrCO1686 as a molecular probe for detecting EGFR L858R/T790M using human non-small-cell lung cancer (NSCLC) cell lines: H1975 (EGFR L858R/T790M), H3255 (EGFR L858R), and H441 (wild-type EGFR). The BrCO1686 showed high cytotoxicity toward H1975 (IC50 0.18 ± 0.06 µM) comparable to that of CO-1686 (IC50 0.14 ± 0.05 µM). In cell uptake experiments, the level of accumulation of [77Br]BrCO1686 in H1975 was significantly higher than those in H3255 and H441 upon 4 h of incubation. The radioactivity of [77Br]BrCO1686 (136.3% dose/mg protein) was significantly reduced to 56.9% dose/mg protein by the pretreatment with an excess CO-1686. These results indicate that the binding site of the radiotracers should be identical to that of CO-1686. The in vivo accumulation of radioactivity of [77Br]BrCO1686 in H1975 tumor (4.51 ± 0.17) was higher than that in H441 tumor (3.71 ± 0.13) 1 h postinjection. Our results suggested that [77Br]BrCO1686 has specificity toward NSCLC cells with double mutations EGFR L858R/T790M compared to those in EGFR L858R and wild-type EGFR. However, the in vivo accumulation of radioactivity in the targeted tumor needs to be optimized by structural modification.

Project description:Epidermal growth factor receptor EGFR major driver mutations may affect downstream molecular networks and pathways, which would influence treatment outcomes of non-small cell lung cancer (NSCLC). This study aimed to unveil profiles of mutant proteins expressed in lung adenocarcinomas of 36 patients harboring representative driver EGFR mutations (Ex19del, nine; L858R, nine; no Ex19del/L858R, 18). Surprisingly, the orthogonal partial least squares discriminant analysis performed for identified mutant proteins demonstrated the profound differences in distance among the different EGFR mutation groups, suggesting that cancer cells harboring L858R or Ex19del emerge from cellular origins different from L858R/Ex19del-negative cells. Weighted gene coexpression network analysis, together with over-representative analysis, identified 18 coexpressed modules and their eigen proteins. Pathways enriched differentially for both the L858R and Ex19del mutations included carboxylic acid metabolic process, cell cycle, developmental biology, cellular responses to stress, mitotic prophase, cell proliferation, growth, epithelial to mesenchymal transition (EMT), and immune system. The IPA causal network analysis identified the highly activated networks of PARPBP, HOXA1, and APH1 under the L858R mutation, whereas those of ASGR1, APEX1, BUB1, and MAPK10 were highly activated under the Ex19del mutation. Interestingly, the downregulated causal network of osimertinib intervention showed the highest significance in overlap p-value among most causal networks predicted under the L858R mutation. We also identified the causal network of MAPK interacting serine/threonine kinase 1/2 (MNK1/2) highly activated differentially under the L858R mutation. Tumor-suppressor AMOT, a component of the Hippo pathways, was highly inhibited commonly under both L858R and Ex19del mutations. Our results could identify disease-related protein molecular networks from the landscape of single amino acid variants. Our findings may help identify potential therapeutic targets and develop therapeutic strategies to improve patient outcomes.

Dataset Information

Comparative assessment of NOIR-SS and ddPCR for ctDNA detection of EGFR L858R mutations in advanced L858R-positive lung adenocarcinomas.

Publications

Comparative assessment of NOIR-SS and ddPCR for ctDNA detection of EGFR L858R mutations in advanced L858R-positive lung adenocarcinomas.

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