Liquid biopsies, based on cell free DNA (cfDNA) and proteins, have shown the potential to detect early stage cancers of diverse tissue types. However, most of these studies were retrospective, using individuals previously diagnosed with cancer as cases and healthy individuals as controls. Here, we developed a liquid biopsy assay, named the hepatocellular carcinoma screen (HCCscreen), to identify HCC from the surface antigen of hepatitis B virus (HBsAg) positive asymptomatic individuals in the community population. The training cohort consisted of individuals who had liver nodules and/or elevated serum α-fetoprotein (AFP) levels, and the assay robustly separated those with HCC from those who were non-HCC with a sensitivity of 85% and a specificity of 93%. We further applied this assay to 331 individuals with normal liver ultrasonography and serum AFP levels. A total of 24 positive cases were identified, and a clinical follow-up for 6–8 mo confirmed four had developed HCC. No HCC cases were diagnosed from the 307 test-negative individuals in the follow-up during the same timescale. Thus, the assay showed 100% sensitivity, 94% specificity, and 17% positive predictive value in the validation cohort. Notably, each of the four HCC cases was at the early stage (<3 cm) when diagnosed. Our study provides evidence that the use of combined detection of cfDNA alterations and protein markers is a feasible approach to identify early stage HCC from asymptomatic community populations with unknown HCC status.

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Cell-free DNA (cfDNA)–based liquid biopsy is a promising approach for the early detection of cancer. A major hurdle is the limited yield of cfDNA from one blood draw, limiting the use of most samples to one test of either mutation or methylation. Here, we develop a technology, Mutation Capsule Plus (MCP), which enables multiplex profiling of one cfDNA sample, including simultaneous detection of genetic and epigenetic alterations and genome-wide discovery of methylation markers. With this technology, we performed de novo screening of methylation markers on cfDNA samples from 30 hepatocellular carcinoma (HCC) cases and 30 non-HCC controls. The methylation markers enriched in HCC cfDNA were further profiled in parallel with a panel of mutations on a training cohort of 60 HCC and 60 non-HCC cases, resulting in an HCC detection model. We validated the model in an independent retrospective cohort with 58 HCC and 198 non-HCC cases and got 90% sensitivity with 94% specificity. Furthermore, we applied the model to a prospective cohort of 311 asymptomatic hepatitis B virus carriers with normal liver ultrasonography and serum AFP concentration. The model detected four of the five HCC cases in the cohort, showing 80% sensitivity and 94% specificity. These findings demonstrate that the MCP technology has potential for the discovery and validation of multiomics biomarkers for the noninvasive detection of cancer. This study also provides a comprehensive database of genetic and epigenetic alterations in the cfDNA of a large cohort of HCC cases and high-risk non-HCC individuals.

Background and aim: Acute severe ulcerative colitis (ASUC) is a life-threatening condition that requires timely referral for therapy. Sarcopenia has been associated with clinical outcomes of inflammatory bowel disease (IBD). This study investigated the role of sarcopenia in predicting the clinical course of ASUC. Methods: This retrospective cohort study included ASUC patients with abdominal CT scans. Univariate and multivariable regression analyses were performed to identify a practical predictive index for the clinical course of ASUC. Results: Of 233 included patients, 151 had intravenous corticosteroid (IVS) failure, among whom 32 received surgery without medical rescue therapy. Fifty patients underwent colectomy after medical rescue therapy failure. Of these 82 surgical patients, 42 suffered postoperative complications. Multivariable regression analysis showed that sarcopenia remained an independent risk factor for IVS failure (OR = 2.969; 95% CI, 1.547-5.701; p = 0.001), colectomy after medical rescue therapy failure (OR = 3.411; 95% CI, 1.147-10.141; p = 0.027), and postoperative complications after colectomy (OR = 4.157; 95% CI, 1.364-12.667; p = 0.012). During follow-up, patients with colectomy after first-line treatment had a lower comprehensive complication index and better health-related quality of life. Conclusion: Sarcopenia is useful in predicting the clinical course and postoperative outcomes of ASUC.

Background Averrhoa carambola L. is a traditional medicinal herb that has long been used to treat diabetes. Our previous studies found that 2-dodecyl-6-methoxycyclohexa-2,5-diene-1,4-dione (DMDD) isolated from A. carambola L . roots could ameliorate diabetic nephropathy (DN), but its exact mechanism remains unclear. Methods A DN model was established by streptozotocin (STZ, 100 mg/kg body weight) in TLR4 knockout (TLR4-/-, KO) mice and wild-type (WT) mice. Body weight and blood glucose were evaluated after oral administration of DMDD (12.5, 25, 50 mg/kg body weight/d) in diabetic mice. The levels of serum lipids, including TC, TG, HDL, and LDL and kidney function indexes Scr and BUN, were detected by biochemical equipment. The levels of inflammatory cytokines including IL-6 and TNF-α, were determined by ELISA kits. Furthermore, changes in renal ultrastructure were observed by electron microscopy. Western blot analysis and RT-PCR were used to assess the protein expression and mRNA levels of TLR4, MyD88 and NF-κB. Results DMDD treatment attenuated diabetic nephropathy, as a result of a decline in blood glucose, serum creatinine, and blood urine nitrogen levels and an increase in the quantity and density of podocytes, combined with improved dyslipidaemia. DMDD treatment inhibited the inflammatory response and downregulated the expression of the TLR4/MyD88/NF-κB pathway in diabetic mice, and these changes were significantly different in TLR4-/- mice. Conclusion DMDD alleviates diabetic nephropathy by mitigating kidney damage and inflammation via the inhibition of the TLR4/MyD88/NF-κB signalling pathway.

Pancreatic intraepithelial neoplasia (PanIN) is a precursor to pancreatic cancer and represents a critical opportunity for cancer interception. However, the number, size, shape, and connectivity of PanINs in human pancreatic tissue samples are largely unknown. In this study, we quantitatively assessed human PanINs using CODA, a novel machine-learning pipeline for 3D image analysis that generates quantifiable models of large pieces of human pancreas with single-cell resolution. Using a cohort of 38 large slabs of grossly normal human pancreas from surgical resection specimens, we identified striking multifocality of PanINs, with a mean burden of 13 spatially separate PanINs per cm3 of sampled tissue. Extrapolating this burden to the entire pancreas suggested a median of approximately 1000 PanINs in an entire pancreas. In order to better understand the clonal relationships within and between PanINs, we developed a pipeline for CODA-guided multi-region genomic analysis of PanINs, including targeted and whole exome sequencing. Multi-region assessment of 37 PanINs from eight additional human pancreatic tissue slabs revealed that almost all PanINs contained hotspot mutations in the oncogene KRAS, but no gene other than KRAS was altered in more than 20% of the analyzed PanINs. PanINs contained a mean of 13 somatic mutations per region when analyzed by whole exome sequencing. The majority of analyzed PanINs originated from independent clonal events, with distinct somatic mutation profiles between PanINs in the same tissue slab. A subset of the analyzed PanINs contained multiple KRAS mutations, suggesting a polyclonal origin even in PanINs that are contiguous by rigorous 3D assessment. This study leverages a novel 3D genomic mapping approach to describe, for the first time, the spatial and genetic multifocality of human PanINs, providing important insights into the initiation and progression of pancreatic neoplasia.

Background: 2-Dodecyl-6-methoxycyclohexa-2,5-diene-1,4-dione (DMDD) has been reported to inhibit a variety of cancer cell lines. The purpose of this study was to investigate the effects of DMDD on 4T1 breast cancer cells and the effects of DMDD on 4T1 breast cancer in mice and its molecular mechanisms. Methods: 4T1 breast cancer cells were treated with different concentrations of DMDD, and their proliferation, apoptosis, cell-cycle distribution, migration, and invasion were detected by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT, Acridine orange and ethidium bromide dual staining analysis (AO/EB) dual staining, flow cytometry, scratch test, and the Transwell assay. Relative quantitative real-time qPCR analysis and Western blot were applied to examine the expression levels of related genes and proteins. In animal experiments, we established a xenograft model to assess the anti-breast cancer effects of DMDD by evaluating the inhibition rate. The apoptotic activity of DMDD was evaluated by hematoxylin-eosin (HE) staining, transmission electron microscope (TEM) analysis and TdT-mediated dUTP nick end labeling (TUNEL) assays. The mRNA expression levels of MAPK pathway components were detected by relative quantitative real-time qPCR. In addition, the protein expression levels of MAPK pathway components were assessed through immunohistochemical assays and Western blotting. Results: Experiments showed that DMDD could inhibit the proliferation, migration, invasion of 4T1 cells and induce cellular apoptosis and G1 cell cycle arrest. Moreover, DMDD down-regulated the mRNA expressions of raf1, mek1, mek2, erk1, erk2, bcl2, and upregulated the mRNA expression of bax. DMDD reduced the protein expressions of p-raf1, p-mek, perk , p-p38, Bcl2, MMP2, MMP9 and increased the protein expressions of Bax and p-JNK. The results showed that DMDD can effectively reduce the tumor volume and weight of breast cancer in vivo, up-regulate the expression of IL-2, down-regulate the expression of IL-4 and IL-10, induce the apoptosis of breast cancer cells in mice, and regulate the expression of genes and proteins of the MAPK pathway. Conclusion: Our study indicates that DMDD can inhibit proliferation, migration, and invasion and induces apoptosis and cell-cycle arrest of 4T1 breast cancer cells. Also, our findings indicate that DMDD induces the apoptosis of breast cancer cells and inhibits the growth in mice. Its mechanism may be related to the MAPK pathway.

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