Multiplex assays, involving the simultaneous use of multiple circulating tumor DNA (ctDNA) markers, can improve the performance of liquid biopsies so that they are highly predictive of cancer recurrence. We have developed a single-tube methylation-specific quantitative PCR assay (mqMSP) that uses 10 different methylation markers and is capable of quantitative analysis of plasma samples with as little as 0.05% tumor DNA. In a cohort of 179 plasma samples from colorectal cancer (CRC) patients, adenoma patients, and healthy controls, the sensitivity and specificity of the mqMSP assay were 84.9% and 83.3%, respectively. In a head-to-head comparative study, the mqMSP assay also performed better for detecting early-stage (stage I and II) and premalignant polyps than a published SEPT9 assay. In an independent longitudinal cohort of 182 plasma samples (preoperative, postoperative, and follow-up) from 82 CRC patients, the mqMSP assay detected ctDNA in 73 (89.0%) of the preoperative plasma samples. Postoperative detection of ctDNA (within 2 wk of surgery) identified 11 of the 20 recurrence patients and was associated with poorer recurrence-free survival (hazard ratio, 4.20; P = 0.0005). With subsequent longitudinal monitoring, 14 patients (70%) had detectable ctDNA before recurrence, with a median lead time of 8.0 mo earlier than seen with radiologic imaging. The mqMSP assay is cost-effective and easily implementable for routine clinical monitoring of CRC recurrence, which can lead to better patient management after surgery.

Background Extrachromosomal circular DNAs (eccDNAs) increase the number of proto‐oncogenes by enhancing oncogene expression to promote tumorigenesis. However, there are limited reports on differential eccDNA expression and analysis in lung cancer, especially in lung adenocarcinoma (LAD). Methods Three LAD and three corresponding NT tissues samples were used for eccDNA next‐generation sequencing analysis, and an additional 20 were used for quantitative PCR (qPCR) evaluations. We further performed qPCR amplification using serum samples from LAD patients and healthy medical examiners. Results eccDNAs from LAD samples were mainly 200–1000 bp in length. Gene annotation analysis revealed that most eccDNAs were derived from chromosomes 1 and 2. The top‐ten increased and top‐ten decreased eccDNAs in LAD tissues were CircD‐ARPC1B, CircD‐ARPC1A, CircD‐FAM49B, CircD‐SDK1, CircD‐KCNG1, CircD‐POLR2F, CircD‐SS18L1, CircD‐SLC16A3, CircD‐CSNK1D, CircD‐KCTD1, and CircD‐TMIGD2, CircD‐PDIA5, CircD‐VAV2, CircD‐GATAD2A, CircD‐CAB39L, CircD‐KHDC1, CircD‐FOXN3, CircD‐SULT2B1, CircD‐DPP9, and CircD‐CSNK1D. qPCR demonstrated that the expression of CircD‐DZRN3 was higher in LAD tissues than in normal lung tissues, whereas CircD‐LGR6 and CircD‐UMODL1 expression levels were lower in LAD than in normal lung tissues. Furthermore, the serum CircD‐PDZRN3 level increased, while CircD‐LGR6 decreased in LAD. Receiver operating characteristic (ROC) analysis showed that area under curve (AUC) of serum CircD‐PDZRN3 (0.991), CircD‐LGR6 (0.916) was higher than that of serum carcinoembryonic antigen (CEA) (0.825), CY211 (cytokeratin 19 fragment) (0.842), SCCA(squamous cell carcinoma antigen) (0.857) for the diagnosis of LAD. Conclusions Our study first showed that several eccDNAs were aberrantly expressed in LAD, among which CircD‐PDZRN3 and CircD‐LGR6 clearly distinguished LAD patients from healthy controls, indicating their potential as biomarkers.

Lung adenocarcinoma (LAD) is a common malignancy; however, its underlying molecular mechanism is unclear. Circular RNAs (circRNAs) serve as significant cancer regulators. The overexpression of circRAPGEF5 in LAD tissues and cells indicated that it may be involved in promoting LAD progression. Analysis of 61 LAD tissues revealed that circRAPGEF5 was related to lymph node metastasis. Functionally, circRAPGEF5 promoted the proliferation, migration, invasion, and epithelial-mesenchymal transition of LAD cells in vitro and promoted LAD cells growth in vivo. Mechanistically, dual-luciferase reporter assays confirmed direct interaction of circRAPGEF5, miR-1236-3p, and ZEB1. miR-1236-3p was upregulated and ZEB1 expression reduced after circRAPGEF5 knockdown, and the proliferation, migration, and invasion of LAD cells was inhibited. circRAPGEF5 was significantly overexpressed in LAD cell exosomes, and co-culture experiments showed that exosomal circRAPGEF5 enhanced the metastatic ability of LAD cells. Further experiments found that serum exosomal circRAPGEF5 was overexpressed in LAD; moreover, the area under the receiver operator characteristic curve of exosomal circRAPGEF5 was superior to that of serum carcinoembryonic antigen (CEA). Jointly detected serum exosomal circRAPGEF5 and serum CEA had better diagnostic performance than when detected individually. Thus, exosomal circRAPGEF5 could promote the proliferation and metastasis of LAD via the miR-1236-3p/ZEB1 axis and serum exosomal circRAPGEF5 may serve as a promising biomarker for LAD.

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Background Lung adenocarcinoma (LUAD) is a highly malignant tumor with a very low five‐year survival rate. In this study, we aimed to identify differentially expressed long‐chain non‐coding RNA (lncRNAs) and mRNAs from benign and malignant pleural effusion exosomes. Methods We used gene microassay and quantitative real‐time reverse transcription polymerase chain reaction (RT‐qPCR) to detect and verify differentially expressed mRNAs and lncRNAs in benign and malignant pleural effusion exosomes. Gene Ontology (GO) functional significance and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway significance enrichment analyses were performed to identify the difference in biological processes and functions between different mRNAs. We selected the lncRNA ZBED5‐AS1 with an upregulated differential fold of 3.003 and conducted a preliminary study on its cellular function. Results Gene microassay results revealed that 177 differentially expressed lncRNAs were upregulated, and 215 were downregulated. The top 10 upregulated were FMN1, AL118505.1, LINC00452, AL109811.2, CATG00000040683.1, AC137932.1, AC008619.1, AL450344.1, AC092718.6, and ZBED5‐AS1. The top 10 downregulated were TEX41, G067726, JAZF1‐AS1, AC027328.1, AL445645.1, AL022345.4, AC008572.1, AC123777.1, AC093714.1, and PHKG1. For the mRNAs, 79 were upregulated, and 123 were notably downregulated. GO analysis revealed that the upregulated differential mRNAs were mainly involved in “cellular response to acidic pH” (biological processes), “endoplasmic reticulum part” (cellular components), and “at DNA binding, cyclase activity” (molecular functions). KEGG pathways were found to be related to V. cholerae infection, Parkinson's disease, and cell adhesion molecules. RT‐qPCR showed that ZBED5‐AS1 was highly expressed in LUAD tissues, cells, and benign and malignant pleural fluid exosomes. Overexpression of ZBED5‐AS1 could significantly promote the proliferation, migration, invasion, and colony formation of LUAD cells, and knockdown had the opposite consequence. Conclusion The pleural effusion exosomes from patients with LUAD include several improperly expressed genes, and lncRNA‐ZBED5‐AS1 is a new biomarker that aids in our understanding of the occurrence and progression of LUAD.