Intra-tumoral epigenetic heterogeneity and aberrant molecular clocks in hepatocellular carcinoma

There is limited understanding of the epigenetic drivers of tumor evolution in hepatocellular carcinoma (HCC). We quantify epigenetic intra-tumoral heterogeneity (ITH) using regional enhanced reduced-representation bisulfite sequencing (eRRBS) DNA methylation data from 47 early stage, treatment-naive HCC biopsies across 9 patients. Integrating these data with matching RNAseq, targeted DNA sequencing, tumor-infiltrating lymphocyte (TIL) and hepatitis-B viral (HBV) expression, we computed regional differential methylation (DM) ITH signatures across 19,327 promoter regions, and 654,133 CpG islands, while overlapping with known methylation age marker genes (240/354). We found substantial ITH signatures in promoter and enhancer sites across 4/9 patients highlighting novel molecular pathways of tumor progression not otherwise detectable from RNA analysis alone. Additionally, we identify an epigenetic tumoral aging measure that reflects a complex tumor fitness phenotype as a potential proxy for tumor evolution. In order to compute clinical associations with epigenetic tumoral age, we use 450k array data from 377 HCC patients in the TCGA-LIHC single-biopsy cohort to calculate tumoral age and find evidence implying that epigenetically old tumors have lower fitness yet higher TIL burden. Our data reveal a novel, unique epigenetic ITH axis in HCC tumors that furthers our understanding of tumor evolution and may serve as a potential avenue for enhancing patient stratification and treatment.

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Identification of the PML-RARα Associated DNA Methylome Using Reduced Representation Bisulfite Sequencing in Primary Patient Samples

Blood ◽

10.1182/blood.v118.21.2436.2436 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2436-2436

Author(s):

Christian Rohde ◽

Till Schoofs ◽

Katja Hebestreit ◽

Hans-Ulrich Klein ◽

Anika Witten ◽

...

Keyword(s):

Binding Sites ◽

Bisulfite Sequencing ◽

Cpg Islands ◽

Primary Diagnosis ◽

Promoter Regions ◽

Reduced Representation ◽

Cpg Sites ◽

Reduced Representation Bisulfite Sequencing ◽

Canonical Pathways ◽

High Degree

Abstract Abstract 2436 The origin of altered DNA methylation in cancer and more specifically in leukemia remains incompletely understood. One favored hypothesis suggests that aberrant methylation is recruited to direct genomic target regions of transcriptional repressor oncogenes. PML-RARα, as a transcriptional repressor oncogene, mediates a repressed chromatin structure at its genomic targets and leads to a rather uniform leukemia subtype. In the current study we aimed to identify patterns and variability of DNA methylation changes in PML-RARα associated acute promyelocytic leukemia (APL). We used reduced representation bisulfite sequencing to investigate a large number of samples at single CpG-site resolution with a focus on CpG islands and related promoters. Overall, 20 APL patient samples were sequenced from patients at primary diagnosis. As controls we used 10 remission bone marrow samples and three sets of MACS-purified CD34+ hematopoetic progenitor cells from healthy donors as well as in vitro differentiated promyelocytes. Using Illumina next generation sequencing we obtained about 1 × 107 uniquely mapped bisulfite sequencing reads per patient in total. More than 10 sequencing reads were available for on average 1.2 million distinct CpG sites per sample. Unsupervised hierarchical clustering revealed that APL samples clustered separately from remission bone marrow, CD34+ progenitors and promyelocytes. Using Poisson-Regression (p<10−5) and at least a 2-fold change in methylation as threshold for analysis, primary diagnosis samples where markedly hypomethylated versus controls. 2160 promoter regions were more than 2-fold hypomethylated in primary diagnosis samples compared to controls. However, 716 promoter regions were also 2-fold hypermethylated. A microarray data analysis (www.leukemia-gene-atlas.org) was carried out. The top hypomethylated genes, as sorted by fold changes in methylation and p-values, showed differential expression. Using published PML-RARα binding sites, we also examined methylation differences within these. A total of 11026 CpG-sites comprising 368 promoters could be taken into account. Notably, there was a high degree of hypomethylation at PML-RARα binding sites when comparing primary diagnosis samples with controls, while only a few binding sites appeared hypermethylated. Differential hypomethylation at PML-RARα binding sites was accentuated in both CpG islands and single CpGs. We also carried out a pathway analysis using Ingenuity IPA Software. Several canonical pathways appeared differentially methylated. This included, for example, 32 genes of the Wnt-signaling pathway as well as CDK5-signaling genes. Cancer-related genes such as CTNNA2 were also differentially methylated. Taken together, our data provide evidence of a specific PML-RARα associated APL-Methylome. Unexpectedly, primary patient samples exhibited a high degree of relative hypomethylation within published PML-RARα binding sites and across promoter regions in general. Methylation differences were associated with deregulated canonical pathways and especially Wnt-signaling. Disclosures: No relevant conflicts of interest to declare.

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Prognosis of Early-Stage Hepatocellular Carcinoma: Comparison between Trans-Arterial Chemoembolization and Radiofrequency Ablation

Cancers ◽

10.3390/cancers12092527 ◽

2020 ◽

Vol 12 (9) ◽

pp. 2527

Author(s):

Byung-Yoon Yun ◽

Hye Won Lee ◽

In Kyung Min ◽

Seung Up Kim ◽

Jun Yong Park ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Radiofrequency Ablation ◽

Early Stage ◽

Complete Response ◽

Long Term Outcomes ◽

Structural Problems ◽

Treatment Naïve ◽

Tace Group ◽

Arterial Chemoembolization

Radiofrequency ablation (RFA) is a curative treatment for early-stage hepatocellular carcinoma (HCC) ineligible for surgery or liver transplantation. However, trans-arterial chemoembolization (TACE) might be an alternative when RFA is contraindicated due to structural problems. Here, we aimed to compare their long-term outcomes. Treatment-naive HCC patients fulfilling the Milan criteria who underwent RFA (n = 136) or TACE (n = 268) were enrolled. Complete response (CR) and 5-year recurrence-free survival (RFS) rates were higher in the RFA group than in the TACE group (94.1% vs. 71.6% and 35.8% vs. 17.0%, respectively; both p < 0.001), whereas 5-year overall survival (OS) rates were not significantly different (65.5% vs. 72.3%, respectively; p = 0.100). Multivariate analysis showed that RFA was associated with better RFS (adjusted hazard ratio [aHR] 0.628; p = 0.001) than TACE, but not with better OS (aHR 1.325; p = 0.151). The most common 1st-line treatment after recurrence were TACE (n = 53), followed by RFA (n = 21) among the RFA group and TACE (n = 150), followed by RFA (n = 44) among the TACE group. After propensity-score matching, similar results were reproduced. Hence, TACE could be an effective alternative to RFA in terms of OS rates. However, TACE should be confined only to RFA-difficult cases, given its lower CR and RFS rates and multi-disciplinary approaches are desirable in decision-making.

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Wisp1 as an early stage marker of human hepatocellular carcinoma (HCC)?

Zeitschrift für Gastroenterologie ◽

10.1055/s-0035-1568093 ◽

2015 ◽

Vol 53 (12) ◽

Author(s):

A Dropmann ◽

T Feng ◽

T Dediulia ◽

I Ilkavets ◽

B Hofmann ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Early Stage ◽

Human Hepatocellular Carcinoma

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miR-224 is an early-stage biomarker of hepatocellular carcinoma with miR-224 and miR-125b as prognostic biomarkers

Biomarkers in Medicine ◽

10.2217/bmm-2020-0099 ◽

2020 ◽

Vol 14 (15) ◽

pp. 1485-1500

Author(s):

Lichao Yang ◽

Chunmeng Wei ◽

Yasi Li ◽

Xiao He ◽

Min He

Keyword(s):

Hepatocellular Carcinoma ◽

Systematic Review ◽

Survival Analysis ◽

Sensitivity And Specificity ◽

Poor Prognosis ◽

Early Stage ◽

Meta Analysis ◽

Benign Lesion ◽

Diagnostic Efficiency ◽

Low Expression

Aim: The aim was to systematically investigate the miRNA biomarkers for early diagnosis of hepatocellular carcinoma (HCC). Materials & methods: A systematic review and meta-analysis of miRNA expression in HCC were performed. Results: A total of 4903 cases from 30 original studies were comprehensively analyzed. The sensitivity and specificity of miR-224 in discriminating early-stage HCC patients from benign lesion patients were 0.868 and 0.792, which were superior to α-fetoprotein. Combined miR-224 with α-fetoprotein, the sensitivity and specificity were increased to 0.882 and 0.808. Prognostic survival analysis showed low expression of miR-125b and high expression of miR-224 were associated with poor prognosis. Conclusion: miR-224 had a prominent diagnostic efficiency in early-stage HCC, with miR-224 and miR-125b being valuable in the prognostic diagnosis.

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The Treatment Effect of Liver Transplantation versus Liver Resection for HCC: A Review and Future Perspectives

Cancers ◽

10.3390/cancers13153730 ◽

2021 ◽

Vol 13 (15) ◽

pp. 3730

Author(s):

Berend R. Beumer ◽

Roeland F. de Wilde ◽

Herold J. Metselaar ◽

Robert A. de Man ◽

Wojciech G. Polak ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Liver Transplantation ◽

Liver Resection ◽

Early Stage ◽

Disease Free Survival ◽

Milan Criteria ◽

Term Survival ◽

Equal Distribution ◽

Intention To Treat ◽

Treat Analysis

For patients presenting with hepatocellular carcinoma within the Milan criteria, either liver resection or liver transplantation can be performed. However, to what extent either of these treatment options is superior in terms of long-term survival is unknown. Obviously, the comparison of these treatments is complicated by several selection processes. In this article, we comprehensively review the current literature with a focus on factors accounting for selection bias. Thus far, studies that did not perform an intention-to-treat analysis conclude that liver transplantation is superior to liver resection for early-stage hepatocellular carcinoma. In contrast, studies performing an intention-to-treat analysis state that survival is comparable between both modalities. Furthermore, all studies demonstrate that disease-free survival is longer after liver transplantation compared to liver resection. With respect to the latter, implications of recurrences for survival are rarely discussed. Heterogeneous treatment effects and logical inconsistencies indicate that studies with a higher level of evidence are needed to determine if liver transplantation offers a survival benefit over liver resection. However, randomised controlled trials, as the golden standard, are believed to be infeasible. Therefore, we suggest an alternative research design from the causal inference literature. The rationale for a regression discontinuity design that exploits the natural experiment created by the widely adopted Milan criteria will be discussed. In this type of study, the analysis is focused on liver transplantation patients just within the Milan criteria and liver resection patients just outside, hereby ensuring equal distribution of confounders.

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Epigenetic Effects of Benzene in Hematologic Neoplasms: The Altered Gene Expression

Cancers ◽

10.3390/cancers13102392 ◽

2021 ◽

Vol 13 (10) ◽

pp. 2392

Author(s):

Giovanna Spatari ◽

Alessandro Allegra ◽

Mariella Carrieri ◽

Giovanni Pioggia ◽

Sebastiano Gangemi

Keyword(s):

Tumor Suppressor ◽

Cpg Islands ◽

Hematologic Malignancies ◽

Genetic Alterations ◽

Dna Hypomethylation ◽

Negative Effects ◽

Promoter Regions ◽

Hematological Neoplasms ◽

Altered Gene ◽

Hematological Tumors

Benzene carcinogenic ability has been reported, and chronic exposure to benzene can be one of the risk elements for solid cancers and hematological neoplasms. Benzene is acknowledged as a myelotoxin, and it is able to augment the risk for the onset of acute myeloid leukemia, myelodysplastic syndromes, aplastic anemia, and lymphomas. Possible mechanisms of benzene initiation of hematological tumors have been identified, as a genotoxic effect, an action on oxidative stress and inflammation and the provocation of immunosuppression. However, it is becoming evident that genetic alterations and the other causes are insufficient to fully justify several phenomena that influence the onset of hematologic malignancies. Acquired epigenetic alterations may participate with benzene leukemogenesis, as benzene may affect nuclear receptors, and provoke post-translational alterations at the protein level, thereby touching the function of regulatory proteins, comprising oncoproteins and tumor suppressor proteins. DNA hypomethylation correlates with stimulation of oncogenes, while the hypermethylation of CpG islands in promoter regions of specific tumor suppressor genes inhibits their transcription and stimulates the onset of tumors. The discovery of the systems of epigenetic induction of benzene-caused hematological tumors has allowed the possibility to operate with pharmacological interventions able of stopping or overturning the negative effects of benzene.

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The APAC Score: A Novel and Highly Performant Serological Tool for Early Diagnosis of Hepatocellular Carcinoma in Patients with Liver Cirrhosis

Journal of Clinical Medicine ◽

10.3390/jcm10153392 ◽

2021 ◽

Vol 10 (15) ◽

pp. 3392

Author(s):

Joeri Lambrecht ◽

Mustafa Porsch-Özçürümez ◽

Jan Best ◽

Fabian Jost-Brinkmann ◽

Christoph Roderburg ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

At Risk ◽

Liver Cirrhosis ◽

Early Stage ◽

Treatment Options ◽

Growth Factor Receptor ◽

Serum Samples ◽

Disease Etiology ◽

Risk Patients ◽

Scoring Tool

(1) Background: Surveillance of at-risk patients for hepatocellular carcinoma (HCC) is highly necessary, as curative treatment options are only feasible in early disease stages. However, to date, screening of patients with liver cirrhosis for HCC mostly relies on suboptimal ultrasound-mediated evaluation and α-fetoprotein (AFP) measurement. Therefore, we sought to develop a novel and blood-based scoring tool for the identification of early-stage HCC. (2) Methods: Serum samples from 267 patients with liver cirrhosis, including 122 patients with HCC and 145 without, were collected. Expression levels of soluble platelet-derived growth factor receptor beta (sPDGFRβ) and routine clinical parameters were evaluated, and then utilized in logistic regression analysis. (3) Results: We developed a novel serological scoring tool, the APAC score, consisting of the parameters age, sPDGFRβ, AFP, and creatinine, which identified patients with HCC in a cirrhotic population with an AUC of 0.9503, which was significantly better than the GALAD score (AUC: 0.9000, p = 0.0031). Moreover, the diagnostic accuracy of the APAC score was independent of disease etiology, including alcohol (AUC: 0.9317), viral infection (AUC: 0.9561), and NAFLD (AUC: 0.9545). For the detection of patients with (very) early (BCLC 0/A) HCC stage or within Milan criteria, the APAC score achieved an AUC of 0.9317 (sensitivity: 85.2%, specificity: 89.2%) and 0.9488 (sensitivity: 91.1%, specificity 85.3%), respectively. (4) Conclusions: The APAC score is a novel and highly accurate serological tool for the identification of HCC, especially for early stages. It is superior to the currently proposed blood-based algorithms, and has the potential to improve surveillance of the at-risk population.

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Identification of BHLHE40 expression in peripheral blood mononuclear cells as a novel biomarker for diagnosis and prognosis of hepatocellular carcinoma

Scientific Reports ◽

10.1038/s41598-021-90515-w ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Pattapon Kunadirek ◽

Chaiyaboot Ariyachet ◽

Supachaya Sriphoosanaphan ◽

Nutcha Pinjaroen ◽

Pongserath Sirichindakul ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Peripheral Blood Mononuclear Cells ◽

Peripheral Blood ◽

Mononuclear Cells ◽

Early Stage ◽

High Sensitivity ◽

Peripheral Blood Mononuclear ◽

Diagnosis And Prognosis ◽

Transcription Profiles ◽

Blood Mononuclear Cells

AbstractNovel and sensitive biomarkers is highly required for early detection and predicting prognosis of hepatocellular carcinoma (HCC). Here, we investigated transcription profiles from peripheral blood mononuclear cells (PBMCs) of 8 patients with HCC and PBMCs from co-culture model with HCC using RNA-Sequencing. These transcription profiles were cross compared with published microarray datasets of PBMCs in HCC to identify differentially expressed genes (DEGs). A total of commonly identified of 24 DEGs among these data were proposed as cancer-induced genes in PBMCs, including 18 upregulated and 6 downregulated DEGs. The KEGG pathway showed that these enriched genes were mainly associated with immune responses. Five up-regulated candidate genes including BHLHE40, AREG, SOCS1, CCL5, and DDIT4 were selected and further validated in PBMCs of 100 patients with HBV-related HCC, 100 patients with chronic HBV infection and 100 healthy controls. Based on ROC analysis, BHLHE40 and DDIT4 displayed better diagnostic performance than alpha-fetoprotein (AFP) in discriminating HCC from controls. Additionally, BHLHE40 and DDIT4 had high sensitivity for detecting AFP-negative and early-stage HCC. BHLHE40 was also emerged as an independent prognostic factor of overall survival of HCC. Together, our study indicated that BHLHE40 in PBMCs could be a promising diagnostic and prognostic biomarker for HBV-related HCC.

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