Early detection of hepatocellular carcinoma (HCC) can greatly improve the survival rate of patients. We aimed to develop a novel marker panel based on cell-free DNA (cfDNA) methylation for the detection of HCC. The differentially methylated CpG sites (DMCs) specific for HCC blood diagnosis were selected from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases, then validated by the whole genome bisulphite sequencing (WGBS) of 12 paired HCC and paracancerous tissues. The clinical performance of the panel was evaluated using tissue samples [32 HCC, chronic liver disease (CLD), and healthy individuals] and plasma cohorts (173 HCC, 199 CLD, and 98 healthy individuals). The combination of G protein subunit beta 4 (GNB4) and Riplet had the optimal area under the curve (AUC) in seven candidates through TCGA, GEO, and WGBS analyses. In tissue validation, the GNB4 and Riplet showed an AUC of 100% with a sensitivity and specificity of 100% for detecting any-stage HCC. In plasma, it demonstrated a high sensitivity of 84.39% at 91.92% specificity, with an AUC of 92.51% for detecting any-stage HCC. The dual-marker panel had a higher sensitivity of 78.26% for stage I HCC than alpha-fetoprotein (AFP) of 47.83%, and a high sensitivity of 70.27% for detecting a single tumour (size ≤3 cm). In conclusion, we developed a novel dual-marker panel that demonstrates high accuracy in detecting HCC, surpassing the performance of AFP testing.

Background: Aberrant DNA methylation patterns play a critical role in the development of hepatocellular carcinoma (HCC). However, the molecular mechanisms associated with these aberrantly methylated genes remain unclear. This study aimed to comprehensively investigate the methylation-driven gene expression alterations in HCC using a multi-omics dataset. Methods: Whole genome bisulfite sequencing (WGBS) and RNA sequencing (RNA-seq) techniques were used to assess the methylation and gene expression profiles of HCC tissues (HCCs) and normal adjacent tissues (NATs). The candidate genes’ potential function was further investigated using single-cell RNA sequencing (scRNA seq) data. Results: We observed widespread hypomethylation in HCCs compared to NATs. Methylation levels in distinct genomic regions exhibited significant differences between HCCs and NATs. We identified 247,632 differentially methylated regions (DMRs) and 4,926 differentially expressed genes (DEGs) between HCCs and NATs. Integrated analysis of DNA methylation and RNA-seq data identified 987 methylation-driven candidate genes, with 970 showing upregulation and 17 showing downregulation. Four genes involved in the retinol metabolic pathway, namely ADH1A, CYP2A6, CYP2C8, and CYP2C19, were identified as hyper-downregulated genes. Their expression levels could stratify HCCs into three subgroups with distinct survival outcomes, immune cell infiltration, and tumor microenvironments. Validation of these findings in an independent dataset yielded similar outcomes, confirming the high concordance and potential prognostic value of these genes. ScRNA seq data revealed the low expression of these genes in immune cells, emphasizing their role in promoting malignant cell proliferation and migration. In conclusion, this study provides insights into the molecular characteristics of HCC, revealing the involvement of retinol metabolism-related genes in the development and progression of HCC. These findings have implications for HCC diagnosis, prognosis prediction, and the development of therapeutic targets.

Background and objective: The outbreak of COVID-19 has become a global health concern. In this study, we evaluate the effectiveness and safety of convalescent plasma therapy in patients with severe and critically ill COVID-19. Methods: Sixteen COVID-19 patients received transfusion of anti-COVID-19 antibody-positive convalescent plasma. The main outcome was time for viral nucleic acid amplification (NAA) test turning negative. Clinical laboratory parameters were measured at the baseline (d0) before plasma transfusion, and day 1 (d1), day 3 (d3) after transfusion as well. Results: Among the 16 patients, 10 of them had a consistently positive result of viral NAA test before convalescent plasma transfusion. Eight patients (8/10) became negative from day 2 to day 8 after transfusion. Severe patients showed a shorter time for NAA test turning negative after transfusion (mean rank 2.17 vs 5·90, P = 0.036). Two critically ill patients transfused plasma with lower antibody level remained a positive result of NAA test. CRP level demonstrated a decline 1 day after convalescent plasma treatment, compared with the baseline ( P = 0.017). No adverse events were observed during convalescent plasma transfusion. Conclusions: Viral NAA test of most patients with COVID-19 who received convalescent plasma transfusion turned negative on the 2nd to 8th days after transfusion, and the negative time of severe patients was shorter than that of critically ill patients. Trial Registration: Chinese Clinical Trial Registry; No.: ChiCTR2000030627 URL: http://www.chictr.org

Screening for colorectal cancer (CRC) using plasma methylation is challenging due to the low abundance of cell-free DNA (cfDNA). Therefore, the development of signal amplification assays based on appropriate markers is essential to increase sensitivity. In this study, we employed an epigenome-wide approach for de novo identification differentially methylated CpGs (DMCs) common to CRC and adenoma using 17 public datasets. A sense-antisense and dual MGB probe (SADMP) assay was then developed. Subsequently, the biomarkers were validated in 712 plasma samples based on SADMP. A total of 2237 DMCs showed overlap between CRC and adenoma. Of these, 75 were hypomethylated in 30 other non-CRC cancers. Following LASSO regression, WBC validation and primer/probe design evaluation, NTMT1 and MAP3K14-AS1 were identified as the most informative candidate biomarkers. At preset template concentrations, the SADMP assay for NTMT1 or MAP3K14-AS1 could reduce the cycle threshold by 1. The NTMT1 and MAP3K14-AS1 dual-target SADMP assay demonstrated a sensitivity of 84.8% for CRC (stage I: 75.0%), a sensitivity of 32.0% for advanced adenomas (AA), and a specificity of 91.5% in controls. The dual-target assay showed high performance for CRC early detection in plasma, suggesting that it may serve as a promising noninvasive tool for CRC screening.