ObjectiveThe lack of highly sensitive and specific diagnostic biomarkers is a major contributor to the poor outcomes of patients with hepatocellular carcinoma (HCC). We sought to develop a non-invasive diagnostic approach using circulating cell-free DNA (cfDNA) for the early detection of HCC.DesignApplying the 5hmC-Seal technique, we obtained genome-wide 5-hydroxymethylcytosines (5hmC) in cfDNA samples from 2554 Chinese subjects: 1204 patients with HCC, 392 patients with chronic hepatitis B virus infection (CHB) or liver cirrhosis (LC) and 958 healthy individuals and patients with benign liver lesions. A diagnostic model for early HCC was developed through case-control analyses using the elastic net regularisation for feature selection.ResultsThe 5hmC-Seal data from patients with HCC showed a genome-wide distribution enriched with liver-derived enhancer marks. We developed a 32-gene diagnostic model that accurately distinguished early HCC (stage 0/A) based on the Barcelona Clinic Liver Cancer staging system from non-HCC (validation set: area under curve (AUC)=88.4%; (95% CI 85.8% to 91.1%)), showing superior performance over α-fetoprotein (AFP). Besides detecting patients with early stage or small tumours (eg, ≤2.0 cm) from non-HCC, the 5hmC model showed high capacity for distinguishing early HCC from high risk subjects with CHB or LC history (validation set: AUC=84.6%; (95% CI 80.6% to 88.7%)), also significantly outperforming AFP. Furthermore, the 5hmC diagnostic model appeared to be independent from potential confounders (eg, smoking/alcohol intake history).ConclusionWe have developed and validated a non-invasive approach with clinical application potential for the early detection of HCC that are still surgically resectable in high risk individuals.

Robust and clinically convenient biomarkers for cancer diagnosis, early detection, and prognosis have great potential to improve patient survival and are the key to precision medicine. The advent of next-generation sequencing technologies enables a more sensitive and comprehensive profiling of genetic and epigenetic information in tumor-derived materials. Researchers are now able to monitor the dynamics of tumorigenesis in new dimensions, such as using circulating cell-free DNA (cfDNA) and tumor DNA (ctDNA). Mutation-based assays in liquid biopsy cannot always provide consistent results across studies due partly to intra- and inter-tumoral heterogeneity as well as technical limitations. In contrast, epigenetic analysis of patient-derived cfDNA is a promising alternative, especially for early detection and disease surveillance, because epigenetic modifications are tissue-specific and reflect the dynamic process of cancer progression. Therefore, cfDNA-based epigenetic assays are emerging to be a highly sensitive, minimally invasive tool for cancer diagnosis and prognosis with great potential in future precise care of cancer patients. The major obstacle for applying epigenetic analysis of cfDNA, however, has been the lack of enabling techniques with high sensitivity and technical robustness. In this review, we summarized the advances in epigenome-wide profiling of 5-hydroxymethylcytosine (5hmC) in cfDNA, focusing on the detection approaches and potential role as biomarkers in different cancer types.

Key Points Question Does data-driven phenotyping based on the trajectories of organ dysfunction in the acute phase of critical illness among children with multiple organ dysfunction syndrome uncover phenotypes with prognostic and therapeutic relevance? Findings In this 2-center cohort study of 20 827 pediatric intensive care encounters, a data-driven approach to phenotyping patients with multiple organ dysfunction syndrome using the trajectories of 6 organ dysfunctions uncovered 4 reproducible and distinct phenotypes with prognostic and potential therapeutic relevance. Meaning In this study, data-driven phenotyping based on the type, severity, and trajectory of 6 organ dysfunctions showed promising results in critically ill children with multiple organ dysfunction syndrome.

Many childhood Wilms tumors are driven by mutations in the microRNA biogenesis machinery, but the mechanism by which these mutations drive tumorigenesis is unknown. Here we show that the transcription factor ) is a microRNA target gene that is overexpressed in Wilms tumors with mutations in microRNA processing genes. Wilms tumors can also overexpress through copy number alterations, and expression correlates with prognosis in Wilms tumors. overexpression accelerates growth of Wilms tumor cells in vitro and induces neoplastic growth in the developing mouse kidney in vivo. In both settings, transactivates (), a key Wilms tumor oncogene, and drives mammalian target of rapamycin complex 1 (mTORC1) signaling. These data link microRNA impairment to the PLAG1-IGF2 pathway, providing new insight into the manner in which common Wilms tumor mutations drive disease pathogenesis.

We describe a low-input RNase footprinting approach for the rapid quantification of ribosome-protected fragments with as few as 1000 cultured cells. The assay uses a simplified procedure to selectively capture ribosome footprints based on optimized RNase digestion. It simultaneously maps cytosolic and mitochondrial translation with single-nucleotide resolution. We applied it to reveal selective functions of the elongation factor TUFM in mitochondrial translation, as well as synchronized repression of cytosolic translation after TUFM perturbation. We show the assay is applicable to small amounts of primary tissue samples with low protein synthesis rates, including snap-frozen tissues and immune cells from an individual's blood draw. We showed its feasibility to characterize the personalized immuno-translatome. Our analyses revealed that thousands of genes show lower translation efficiency in monocytes compared with lymphocytes, and identified thousands of translated noncanonical open reading frames (ORFs). Altogether, our RNase footprinting approach opens an avenue to assay transcriptome-wide translation using low-input samples from a wide range of physiological conditions.

Background Untangling the heterogeneity of sepsis in children and identifying clinically relevant phenotypes could lead to the development of enrichment strategies and targeted therapies. In this study, our aim was to analyze the organ dysfunction-based trajectories of children with sepsis-associated multiple organ dysfunction syndrome (MODS) to identify and characterize reproducible and clinically relevant sepsis phenotypes using a data-driven approach. Methods We collected data from patients admitted with suspected infections to 13 pediatric intensive care units (PICUs) in the U.S. between 2012-2018. We used subgraph-augmented nonnegative matrix factorization to identify candidate trajectory-driven phenotypes based on the type, severity, and progression of organ dysfunction in the first 72 hours of PICU admission. We analyzed the candidate phenotypes to determine reproducibility as well as prognostic, therapeutic, and biological relevance. Results Overall, 38,732 children had suspected infection, of which 15,246 (39.4%) had sepsis-associated MODS. Amongst patients with sepsis-associated MODS, 1,537 (10.1%) died in the hospital. We identified an organ dysfunction trajectory-based phenotype (which we termed persistent hypoxemia and shock) that was highly reproducible, had features of systemic inflammation and coagulopathy, and was independently associated with higher mortality. In a propensity score matched analysis, patients with the persistent hypoxemia and shock phenotype appeared to have a higher likelihood to benefit from adjuvant therapy with hydrocortisone and albumin than other patients. When compared to other high-risk clinical syndromes, the persistent hypoxemia and shock phenotype only overlapped with 50 to 60% of patients with septic shock, those with moderate-to-severe pediatric acute respiratory distress syndrome, or those in the top tertile of organ dysfunction burden, suggesting that it represents a distinct clinical phenotype of sepsis-associated MODS with a disproportionately high risk of mortality. Conclusions We derived and validated the persistent hypoxemia and shock phenotype, a trajectory-based organ dysfunction phenotype which is highly reproducible, clinically relevant, and associated with heterogeneity of treatment effect to common adjuvant therapies. Further validation is warranted. Future studies are needed to validate this phenotype, assess whether it can be predicted earlier in the course, study possible biological mechanisms underlying it, and investigate candidate therapeutic targets.

Germ cell tumors (GCT) are malignant tumors that arise from pluripotent embryonic germ cells and occur in children and young adults. GCTs are treated with cisplatin-based regimens which, while overall effective, fail to cure all patients and cause significant adverse late effects. The seminoma and nonseminoma forms of GCT exhibit distinct differentiation states, clinical behavior, and response to treatment; however, the molecular mechanisms of GCT differentiation are not fully understood. We tested whether the activity of the mTORC1 and MAPK pathways were differentially active in the two classes of GCT. Here we show that nonseminomatous germ cell tumors (NSGCT, including embryonal carcinoma, yolk sac tumor, and choriocarcinoma) from both children and adults display activation of the mTORC1 pathway, while seminomas do not. In seminomas, high levels of REDD1 may negatively regulate mTORC1 activity. In NSGCTs, on the other hand, EGF and FGF2 ligands can stimulate mTORC1 and MAPK signaling, and members of the EGF and FGF receptor families are more highly expressed. Finally, proliferation of NSGCT cells and is significantly inhibited by combined treatment with the clinically available agents erlotinib and rapamycin, which target EGFR and mTORC1 signaling, respectively. These results provide an understanding of the signaling network that drives GCT growth and a rationale for therapeutic targeting of GCTs with agents that antagonize the EGFR and mTORC1 pathways. .

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