Analytical and Clinical Validation of Cell-Free Circulating Tumor DNA Assay for the Estimation of Tumor Mutational Burden.

BACKGROUND: Ultra-deep sequencing to detect low-frequency mutations in circulating tumor-derived DNA (ctDNA) increases the diagnostic value of liquid biopsy. The demand for large ctDNA panels for comprehensive genomic profiling and tumor mutational burden (TMB) estimation is increasing; however, few ctDNA panels for TMB have been validated. Here, we designed a ctDNA panel with 531 genes, named TMB500, along with a technical and clinical validation. METHODS: Synthetic reference cell-free DNA materials with predefined allele frequencies were sequenced in a total of 92 tests in 6 batches to evaluate the precision, linearity, and limit of detection of the assay. We used clinical samples from 50 patients with various cancers, 11 healthy individuals, and paired tissue samples. Molecular barcoding and data analysis were performed using customized pipelines. RESULTS: The assay showed high precision and linearity (coefficient of determination, r2 =0.87) for all single nucleotide variants, with a limit of detection of 0.24%. In clinical samples, the TMB500 ctDNA assay detected most variants present and absent in tissues, showing that ctDNA could assess tumor heterogeneity in different tissues and metastasis sites. The estimated TMBs correlated well between tissue and blood, except in 4 cases with extreme heterogeneity that showed very high blood TMBs compared to tissue TMBs. A pilot evaluation showed that the TMB500 assay could be used for disease monitoring. CONCLUSIONS: The TMB500 assay is an accurate and reliable ctDNA assay for many clinical purposes. It may be useful for guiding the treatment of cancers with diverse genomic profiles, estimating TMB in immune therapy, and disease monitoring.

Toxicogenomic study to identify potential signaling alterations related to nasal inflammatory damages induced by diesel exhaust particles in primary human nasal epithelial cells.

In this study, we aimed to identify signaling alteration caused by exposure to diesel exhaust particles (DEPs) using primary human nasal epithelial cells (PHNECs). Global gene expression profiles in PHNECs following 50 and 200 µg/ml of DEP exposure were identified using microarray analysis. To cover the limitation of array-based mRNA expression analysis, text-mining-based software was used to analyze the integrative biological networks and relevant disease-focused functions among identified DEP-responsive genes. The confidence was valued based on the connectivity between the analyzed pathway and marker candidates. Through a literature-based pathway analysis, the stimulation of inflammation- and immune response-related processes mediated by TNF were predicted as major signaling alterations in PHNECs caused by DEP exposure. CSF3, CXCL8, MMP1, and VEGFA were identified as key hub genes in the predicted pathway. Significant expression level changes in the five key genes following DEP exposure were validated in terms of protein and mRNA expression. Although further studies are required, our toxicogenomic investigation provides key clues to the exact mechanism underlying DEP-induced nasal inflammatory damage. It also suggests an efficient approach for other research on adverse effects occurring in the upper respiratory tract following DEP exposure.

A Putative Adverse Outcome Pathway Relevant to Carcinogenicity Induced by Sulfuric Acid in Strong Inorganic Acid Mists.

Based on epidemiological studies, an International Agency for Research on Cancer Working Group determined that strong inorganic acid mists containing sulfuric acid are carcinogenic to human even though, sulfuric acid, per se, is not. Accumulative studies indicate that there is a link between chronic occupational exposure to sulfuric acid mists and an increased risk of laryngeal cancer. Unintended, acute exposure to sulfuric acid mists can cause corrosive damage to target tissues depending on the route of exposure. This review compares the toxicity and carcinogenicity of sulfuric acid mists compared to other strong inorganic acid mists. It also examines the routes and duration of exposure (short-term, prolonged, and long-term). In vivo evidence does not support or refute the carcinogenicity of sulfuric inorganic mists even though its co-carcinogenic or promoting potential has been considered. On the basis of existing evidence on sulfuric acid mist toxicity, we suggested a putative adverse outcome pathway (AOP) relevant to carcinogenicity caused by mists containing sulfuric acid. A possible key factor involved in sulfuric acid mist carcinogenesis is the genotoxic effects of low pH since it can increase instability in chromosomes and DNA. A putative AOP for sulfuric acid mist carcinogenicity would help generate better risk assessments and more accurate predictions regarding the risk of developing cancer due to prolonged exposure. Establishing an AOP would also be useful for future studies examining the carcinogenicity of other strong inorganic mists.

Application of the Adverse Outcome Pathway Framework to Risk Assessment for Predicting Carcinogenicity of Chemicals.

As industry develops in modern society, many chemicals are being used. The safety of chemicals is an important issue because humans are constantly exposed to chemicals throughout their daily life. Through a risk assessment, the hazardous human effects of chemicals can be identified. Recently, the adverse outcome pathway (AOP) framework has been used to predict the adverse effects of chemicals. As a conceptual framework for organizing existing biological knowledge, the AOP consists of a molecular initiating event, key events, and an adverse outcome. These independent elements represent biological responses and are connected by key event relationships. This AOP framework provides intuitive hazard identification that can be helpful for carcinogenic risk assessment of chemicals. In this review, we introduce the application of the AOP framework to risk assessment for predicting carcinogenicity of chemicals and illustrate the utility of this approach for cancer prevention.