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BackgroundHuman carboxylesterases (hCES) are key serine hydrolases responsible for the hydrolysis of a wide range of endogenous and xenobiotic esters. Although it has been reported that some ginsenosides can modulate the activities of various enzymes, the inhibitory effects of ginsenosides on hCES have not been well-investigated.MethodsIn this study, more than 20 ginsenosides were collected and their inhibitory effects on hCES1A and hCES2A were assayed using the highly specific fluorescent probe substrates for each isoenzyme. Molecular docking simulations were also performed to investigate the interactions between ginsenosides and hCES.ResultsAmong all tested ginsenosides, Dammarenediol II (DM) and 20S-O-β-(d-glucosyl)-dammarenediol II (DMG) displayed potent inhibition against both hCES1A and hCES2A, while protopanaxadiol (PPD) and protopanaxatriol (PPT) exhibited strong inhibition on hCES2A and high selectivity over hCES1A. Introduction of O-glycosyl groups at the core skeleton decreased hCES inhibition activity, while the hydroxyl groups at different sites might also effect hCES inhibition. Inhibition kinetic analyses demonstrated that DM and DMG functioned as competitive inhibitors against hCES1A-mediated d-luciferin methyl ester (DME) hydrolysis. In contrast, DM, DMG, PPD and PPT inhibit hCES2A-mediated fluorescein diacetate (FD) hydrolysis via a mixed manner.ConclusionThe structure–inhibition relationships of ginsenosides as hCES inhibitors was investigated for the first time. Our results revealed that DM and DMG were potent inhibitors against both hCES1A and hCES2A, while PPD and PPT were selective and strong inhibitors against hCES2A.

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e23062 Background: The analysis of EGFR, KRAS, and BRAF mutations and Alk fusion is critical for guiding targeted therapy selection, detecting drug resistance, and monitoring residual disease in patients with NSCLC. Designing next-generation sequencing (NGS) assays for detecting low-frequency variants, however, is an ongoing challenge. The limited availability of cfDNA combined with the breadth of coverage necessary to create meaningful, clinically-actionable results requires a solution with multiplex capacity which, in turn, requires greater technological sensitivity and specificity. Here we aim to develop such a solution: an ultra-accurate NGS technology using concatmer-based error correction with amplicon workflow for fast detection of rare mutations including SNV and fusion. Methods: We developed an amplicon-based panel covering variants of EGFR, BRAF, and KRAS, as well as a panel to detect Alk fusion. CfDNA simulate and cfDNA from healthy individuals were used to test assay sensitivity and specificity. Further validation was performed via a comparative analysis of 64 late-stage lung cancer patients using both Firefly -Comet and ddPCR. Results: Analytical sensitivity of the EGFR-TKI 3-gene panel was 100% detection at an allele frequency of 0.1% for 20ng of cfDNA input. Similarly, analytical sensitivity of the Alk fusion panel was 75% detection at an allele frequency of 0.1% and 100% at an allele frequency of 0.25% for the same input. Among our patient cohort, 5 EGFR variants (19del, T790M, L858R, G719X, L861X) and 2 KRAS variant (G12X) were detected. Firefly-Comet demonstrated strong per-variant detection-rate concordance ( > 99%) compared to ddPCR results. The PPV is 100% and the NPV is 98.7%. Statistical analysis of reported allele frequency concordance between Firefly-Comet and ddPCR reveals R-Sq > 0.9. Conclusions: In summary, we have developed Firefly-Comet, an easy-to-use amplicon-based NGS assay capable of detecting single-digit copies of somatic mutants and gene fusions in cfDNA. The multiplex capacity of Firefly-Comet makes it well-suited for supporting targeted therapy selection, drug resistance detection, and treatment monitoring.

e23067 Background: Circulating tumor DNA (ctDNA) is a promising biomarker for the detection of minimal residual disease and monitoring treatment in patients with CRC. The performance demands of any technology used for this purpose, however, are tremendous. Here we aim to develop a high-performance multiplex NGS platform suitable for cancer MRD using ctDNA. Methods: We have developed Firefly, a NGS method capable of detecting low-frequency variants with high precision in plasma cfDNA. In our protocol, denatured double-stranded cfDNA is circularized and converted into long tandem repeats using rolling-circle amplification enabling consensus-based concatemer error correction. We demonstrated Firefly’s performance sensitivity and specificity by testing our technology on cfDNA samples with known variant frequencies and cfDNA collected from healthy individuals (n = 82). Further analysis of Firefly as a tool for MRD and treatment monitoring was performed by tracking ctDNA mutation profile concordance between 81 CRC tumor samples and their corresponding plasma samples collected from patients before and after treatment. Results: Performance sensitivity of Firefly NGS was 0.1% with an error-rate was 1 in 1 Million for 20ng of input ctDNA. Concordance analysis was performed on CRC tumor/plasma pairings derived from patients with CRC using, Accu-Act, a 61-gene assay. The number of tumor-matching mutations detected in plasma varied greatly on a per-patient basis (range, 0-28). Pre and post-treatment ctDNA profiling was performed on all 81 patients included in our study (surgery, n = 56; chemotherapy/radiotherapy, n = 30). Among patients who underwent surgery, 46% had detectable tumor-matching mutations in their plasma. Among patients who received neoadjuvant therapy, 70% ctDNA fluctuations consistent with tumor reduction based on surgical tumor regression grades evaluation(TRG1-3). Conclusions: We report a novel ultra-accurate NGS-based ctDNA assay suitable for MRD and monitoring in CRC patients. Firefly should ultimately make a significant contribution in the development of personalized cancer treatment.