Objective: The 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. Design: Applying 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. Results: The 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). Conclusion: We 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.

Misregulated alternative RNA splicing (AS) contributes to the tumorigenesis and progression of human cancers, including glioblastoma (GBM). Here, we showed that a major splicing factor, serine and arginine rich splicing factor 3 (SRSF3), was frequently upregulated in clinical glioma specimens and that elevated SRSF3 was associated with tumor progression and a poor prognosis for patients with glioma. In patient-derived glioma stem-like cells (GSC), SRSF3 expression promoted cell proliferation, self-renewal, and tumorigenesis. Transcriptomic profiling identified more than 1,000 SRSF3-affected AS events, with a preference for exon skipping in genes involved with cell mitosis. Motif analysis identified the sequence of CA(G/C/A)CC(C/A) as a potential exonic splicing enhancer for these SRSF3-regulated exons. To evaluate the biological impact of SRSF3-affected AS events, four candidates were selected whose AS correlated with SRSF3 expression in glioma tissues, and their splicing pattern was modified using a CRISPR/Cas9 approach. Two functionally validated AS candidates were further investigated for the mechanisms underlying their isoform-specific functions. Specifically, following knockout of SRSF3, transcription factor ETS variant 1 (ETV1) gene showed exon skipping at exon 7, while nudE neurodevelopment protein 1 (NDE1) gene showed replacement of terminal exon 9 with a mutually exclusive exon 9′. SRSF3-regulated AS of these two genes markedly increased their oncogenic activity in GSCs. Taken together, our data demonstrate that SRSF3 is a key regulator of AS in GBM and that understanding mechanisms of misregulated AS could provide critical insights for developing effective therapeutic strategies against GBMs. Significance: SRSF3 is a significant regulator of glioma-associated alternative splicing, implicating SRSF3 as an oncogenic factor that contributes to the tumor biology of GBM.

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.

An elevated level of circulating cell-free DNA (cfDNA) has been associated with tumor bulk and poor prognosis in diffuse large B-cell lymphoma (DLBCL), but the tumor-specific molecular alterations in cfDNA with prognostic significance remain unclear. We investigated the association between 5-hydroxymethylcytosines (5hmC), a mark of active demethylation and gene activation, in cfDNA from blood plasma and prognosis in newly diagnosed DLBCL patients. We used 5hmC-Seal, a highly sensitive chemical labeling technique, to profile genome-wide 5hmC in plasma cfDNA from 48 DLBCL patients at the University of Chicago Medical Center between 2010 and 2013. Patients were followed through 31 December 2017. We found a distinct genomic distribution of 5hmC in cfDNA marking tissue-specific enhancers, consistent with their putative roles in gene regulation. The 5hmC profiles in cfDNA differed by cell of origin and were associated with clinical prognostic factors, including stage and the International Prognostic Index. We developed a 29 gene–based weighted prognostic score (wp-score) for predicting event-free survival (EFS) and overall survival (OS) by applying the elastic net regularization on the Cox proportional-hazards model. The wp-scores outperformed (eg, prognostic accuracy, sensitivity, specificity) established prognostic factors in predicting EFS and OS. In multivariate Cox models, patients with high wp-scores had worse EFS (hazard ratio, 9.17; 95% confidence interval, 2.01-41.89; P = .004) compared with those in the low-risk group. Our findings suggest that the 5hmC signatures in cfDNA at the time of diagnosis are associated with clinical outcomes and may provide a novel minimally invasive prognostic approach for DLBCL.

BACKGROUND: Long-term complications of type 2 diabetes (T2D), such as macrovascular and microvascular events, are the major causes for T2D-related disability and mortality. A clinically convenient, noninvasive approach for monitoring the development of these complications would improve the overall life quality of patients with T2D and help reduce healthcare burden through preventive interventions.METHODS: A selective chemical labeling strategy for 5-hydroxymethylcytosines (5hmC-Seal) was used to profile genome-wide 5hmCs, an emerging class of epigenetic markers implicated in complex diseases including diabetes, in circulating cell-free DNA (cfDNA) from a collection of Chinese patients (n = 62). Differentially modified 5hmC markers between patients with T2D with and without macrovascular/microvascular complications were analyzed under a case–control design.RESULTS: Statistically significant changes in 5hmC markers were associated with T2D-related macrovascular/microvascular complications, involving genes and pathways relevant to vascular biology and diabetes, including insulin resistance and inflammation. A 16-gene 5hmC marker panel accurately distinguished patients with vascular complications from those without [testing set: area under the curve (AUC) = 0.85; 95% CI, 0.73–0.96], outperforming conventional clinical variables such as urinary albumin. In addition, a separate 13-gene 5hmC marker panel could distinguish patients with single complications from those with multiple complications (testing set: AUC = 0.84; 95% CI, 0.68–0.99), showing superiority over conventional clinical variables.CONCLUSIONS: The 5hmC markers in cfDNA reflected the epigenetic changes in patients with T2D who developed macrovascular/microvascular complications. The 5hmC-Seal assay has the potential to be a clinically convenient, noninvasive approach that can be applied in the clinic to monitor the presence and severity of diabetic vascular complications.