Project description:BackgroundRecent advances in circulating cell-free DNA (cfDNA) analysis from biofluids have opened new avenues for liquid biopsy (LB). However, current cfDNA LB assays are limited by the availability of existing information on established genotypes associated with tumor tissues. Certain cancers present with a limited list of established mutated cfDNA biomarkers, and thus, nonmutated cfDNA characteristics along with alternative biofluids are needed to broaden the available cfDNA targets for cancer detection. Saliva is an intriguing and accessible biofluid that has yet to be fully explored for its clinical utility for cancer detection.MethodsIn this report, we employed a low-coverage single stranded (ss) library NGS pipeline "Broad-Range cell-free DNA-Seq" (BRcfDNA-Seq) using saliva to comprehensively investigate the characteristics of salivary cfDNA (ScfDNA). The identification of cfDNA features has been made possible by applying novel cfDNA processing techniques that permit the incorporation of ultrashort, ss, and jagged DNA fragments. As a proof of concept using 10 gastric cancer (GC) and 10 noncancer samples, we examined whether ScfDNA characteristics, including fragmentomics, end motif profiles, microbial contribution, and human chromosomal mapping, could differentiate between these two groups.ResultsIndividual and integrative analysis of these ScfDNA features demonstrated significant differences between the two cohorts, suggesting that disease state may affect the ScfDNA population by altering nuclear cleavage or the profile of contributory organism cfDNA to total ScfDNA. We report that principal component analysis integration of several aspects of salivary cell-free DNA fragmentomic profiles, genomic element profiles, end-motif sequence patterns, and distinct oral microbiome populations can differentiate the two populations with a p value of < 0.0001 (PC1).ConclusionThese novel features of ScfDNA characteristics could be clinically useful for improving saliva-based LB detection and the eventual monitoring of local or systemic diseases.

Project description:PurposeTo seek novel diagnostic approaches, we improved the workflow of cell-free DNA (cfDNA) sequencing and evaluated its feasibility in vitreoretinal lymphoma (VRL) specimens; the profile of mutations was preliminarily analyzed for potential diagnostic value.MethodsThe study was a diagnostic trial. 23 eyes of 23 patients with VRL and 25 eyes of 25 patients with inflammatory eye diseases were enrolled. Approximate 500μl undiluted vitreous humor and 10ml diluted vitreous fluid was obtained through diagnostic vitrectomy and sent for cytopathological examinations. 500μl of the diluted vitreous fluid was spared for cfDNA sequencing. For cfDNA sequencing, DNA fragmentation procedure was added to the workflow to improve the extraction efficiency; mutations detected were analyzed for potential diagnostic model. The sensitivity and specificity of the cytopathology and cfDNA sequencing were compared. The clinical manifestations were preliminarily analyzed for potential correlations with the genotypes.ResultsCfDNA sequencing was accomplished in 23 eyes with VRL and 20 eyes with inflammatory eye diseases. VRL-related mutated genes included MYD88 (18 eyes, 78%), ETV6 (11 eyes, 48%), PIM1 (11 eyes,48%), BTG2 (7 eyes, 30%), IRF4 (7 eyes, 30%), CD79B (6 eyes, 26%), LRP1B (6 eyes, 26%), etc. Logistic regression based on the mutations of MYD88 and ETV6 was of the potential for the diagnosis of VRL (P<0.001, adjusted R2 = 0.789, sensitivity 0.913, specificity 0.950); by comparison, the sensitivity and specificity of the vitreous cytopathology were 0.826 and 1.000, respectively. Further analysis of the mutation profile showed that patients carrying CD79B mutation tended to have higher intraocular interleukin-10 level (P=0.030), that CARD11 mutation was correlated with younger age at ocular onset (P=0.039), and that patients with intracranial involvement carried more multiple-site mutations in the BTG2 gene (P=0.013).ConclusionsThe improved workflow of CfDNA sequencing is of sound feasibility in a limited amount of vitreous humor. The logistic model based on the mutations could help to provide reliable clues for the diagnosis of VRL.

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Precision cell-free DNA extraction for liquid biopsy by integrated microfluidics.

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Precision cell-free DNA extraction for liquid biopsy by integrated microfluidics.

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