Single-cell multiomics reveals the interplay of clonal evolution and cellular plasticity in hepatoblastoma.

Hepatoblastomas (HB) display heterogeneous cellular phenotypes that influence the clinical outcome, but the underlying mechanisms are poorly understood. Here, we use a single-cell multiomic strategy to unravel the molecular determinants of this plasticity. We identify a continuum of HB cell states between hepatocytic (scH), liver progenitor (scLP) and mesenchymal (scM) differentiation poles, with an intermediate scH/LP population bordering scLP and scH areas in spatial transcriptomics. Chromatin accessibility landscapes reveal the gene regulatory networks of each differentiation pole, and the sequence of transcription factor activations underlying cell state transitions. Single-cell mapping of somatic alterations reveals the clonal architecture of each tumor, showing that each genetic subclone displays its own range of cellular plasticity across differentiation states. The most scLP subclones, overexpressing stem cell and DNA repair genes, proliferate faster after neo-adjuvant chemotherapy. These results highlight how the interplay of clonal evolution and epigenetic plasticity shapes the potential of HB subclones to respond to chemotherapy.

Region-specific DNA hydroxymethylation along the malignant progression of IDH-mutant gliomas.

The majority of low-grade isocitrate dehydrogenase-mutant (IDHmt) gliomas undergo malignant progression (MP), but their underlying mechanism remains unclear. IDHmt gliomas exhibit global DNA methylation, and our previous report suggested that MP could be partly attributed to passive demethylation caused by accelerated cell cycles. However, during MP, there is also active demethylation mediated by ten-eleven translocation, such as DNA hydroxymethylation. Hydroxymethylation is reported to potentially contribute to gene expression regulation, but its role in MP remains under investigation. Therefore, we conducted a comprehensive analysis of hydroxymethylation during MP of IDHmt astrocytoma. Five primary/malignantly progressed IDHmt astrocytoma pairs were analyzed with oxidative bisulfite and the Infinium EPIC methylation array, detecting 5-hydroxymethyl cytosine at over 850,000 locations for region-specific hydroxymethylation assessment. Notably, we observed significant sharing of hydroxymethylated genomic regions during MP across the samples. Hydroxymethylated CpGs were enriched in open sea and intergenic regions (p < 0.001), and genes undergoing hydroxymethylation were significantly associated with cancer-related signaling pathways. RNA sequencing data integration identified 91 genes with significant positive/negative hydroxymethylation-expression correlations. Functional analysis suggested that positively correlated genes are involved in cell-cycle promotion, while negatively correlated ones are associated with antineoplastic functions. Analyses of The Cancer Genome Atlas clinical data on glioma were in line with these findings. Motif-enrichment analysis suggested the potential involvement of the transcription factor KLF4 in hydroxymethylation-based gene regulation. Our findings shed light on the significance of region-specific DNA hydroxymethylation in glioma MP and suggest its potential role in cancer-related gene expression and IDHmt glioma malignancy.

Mutational signature, cancer driver genes mutations and transcriptomic subgroups predict hepatoblastoma survival.

BACKGROUND: Hepatoblastoma is the most frequent pediatric liver cancer. The current treatments lead to 80% of survival rate at 5 years. In this study, we evaluated the clinical relevance of molecular features to identify patients at risk of chemoresistance, relapse and death of disease. METHODS: All the clinical data of 86 children with hepatoblastoma were retrospectively collected. Pathological slides were reviewed, tumor DNA sequencing (by whole exome, whole genome or target) and transcriptomic profiling with RNAseq or 300-genes panel were performed. Associations between the clinical, pathological, mutational and transcriptomic data were investigated. RESULTS: High-risk patients represented 44% of our series and the median age at diagnosis was 21.9 months (range: 0-208). Alterations of the WNT/ß-catenin pathway and of the 11p15.5 imprinted locus were identified in 98% and 74% of the tumors, respectively. Other cancer driver genes mutations were only found in less than 11% of tumors. After neoadjuvant chemotherapy, disease-specific survival and poor response to neoadjuvant chemotherapy were associated with 'Liver Progenitor' (p = 0.00049, p < 0.0001) and 'Immune Cold' (p = 0.0011, p < 0.0001) transcriptomic tumor subtypes, SBS35 cisplatin mutational signature (p = 0.018, p = 0.001), mutations in rare cancer driver genes (p = 0.0039, p = 0.0017) and embryonal predominant histological type (p = 0.0013, p = 0.0077), respectively. Integration of the clinical and molecular features revealed a cluster of molecular markers associated with resistance to chemotherapy and survival, enlightening transcriptomic 'Immune Cold' and Liver Progenitor' as a predictor of survival independent of the clinical features. CONCLUSIONS: Response to neoadjuvant chemotherapy and survival in children treated for hepatoblastoma are associated with genomic and pathological features independently of the clinical features.

RNA Oncological Therapeutics: Intracellular Hairpin RNA Assembly Enables MicroRNA-Triggered Anticancer Functionality.

RNA therapeutics are of global interest because of their versatility in targeting a variety of intracellular and extracellular biomolecules. In that context, long double-stranded RNA (dsRNA) has been studied as an antitumor agent that activates the immune response. However, its performance is constrained by poor cancer selectivity and cell-penetration ability. Here, we designed and synthesized an oncolytic RNA hairpin pair (oHP) that was selectively cytotoxic toward cancer cells expressing abundant oncogenic microRNA-21 (miR-21). Although the structure of each hairpin RNA was thermodynamically metastable, catalytic miR-21 input triggered it to open to generate a long nicked dsRNA. We demonstrated that oHP functioned as a cytotoxic amplifier of information in the presence of miR-21 in various cancer cells and tumor-bearing mice. This work represents the first example of the use of short RNA molecules as build-up-type anticancer agents that are triggered by an oncogenic miRNA.

Transcriptomic and epigenetic dissection of spinal ependymoma (SP-EPN) identifies clinically relevant subtypes enriched for tumors with and without NF2 mutation.

Ependymomas encompass multiple clinically relevant tumor types based on localization and molecular profiles. Tumors of the methylation class "spinal ependymoma" (SP-EPN) represent the most common intramedullary neoplasms in children and adults. However, their developmental origin is ill-defined, molecular data are scarce, and the potential heterogeneity within SP-EPN remains unexplored. The only known recurrent genetic events in SP-EPN are loss of chromosome 22q and NF2 mutations, but neither types and frequency of these alterations nor their clinical relevance have been described in a large, epigenetically defined series. Transcriptomic (n = 72), epigenetic (n = 225), genetic (n = 134), and clinical data (n = 112) were integrated for a detailed molecular overview on SP-EPN. Additionally, we mapped SP-EPN transcriptomes to developmental atlases of the developing and adult spinal cord to uncover potential developmental origins of these tumors. The integration of transcriptomic ependymoma data with single-cell atlases of the spinal cord revealed that SP-EPN display the highest similarities to mature adult ependymal cells. Unsupervised hierarchical clustering of transcriptomic data together with integrated analysis of methylation profiles identified two molecular SP-EPN subtypes. Subtype A tumors primarily carried previously known germline or sporadic NF2 mutations together with 22q loss (bi-allelic NF2 loss), resulting in decreased NF2 expression. Furthermore, they more often presented as multilocular disease and demonstrated a significantly reduced progression-free survival as compared to SP-EP subtype B. In contrast, subtype B predominantly contained samples without NF2 mutation detected in sequencing together with 22q loss (monoallelic NF2 loss). These tumors showed regular NF2 expression but more extensive global copy number alterations. Based on integrated molecular profiling of a large multi-center cohort, we identified two distinct SP-EPN subtypes with important implications for genetic counseling, patient surveillance, and drug development priorities.

Multiancestry genomic and transcriptomic analysis of gastric cancer.

Gastric cancer is among the most common malignancies worldwide, characterized by geographical, epidemiological and histological heterogeneity. Here, we report an extensive, multiancestral landscape of driver events in gastric cancer, involving 1,335 cases. Seventy-seven significantly mutated genes (SMGs) were identified, including ARHGAP5 and TRIM49C. We also identified subtype-specific drivers, including PIGR and SOX9, which were enriched in the diffuse subtype of the disease. SMGs also varied according to Epstein-Barr virus infection status and ancestry. Non-protein-truncating CDH1 mutations, which are characterized by in-frame splicing alterations, targeted localized extracellular domains and uniquely occurred in sporadic diffuse-type cases. In patients with gastric cancer with East Asian ancestry, our data suggested a link between alcohol consumption or metabolism and the development of RHOA mutations. Moreover, mutations with potential roles in immune evasion were identified. Overall, these data provide comprehensive insights into the molecular landscape of gastric cancer across various subtypes and ancestries.

Loss of viral genome with altered immune microenvironment during tumour progression of Epstein-Barr virus-associated gastric carcinoma.

Epstein-Barr virus (EBV) is one of the major drivers of gastric carcinogenesis. EBV infection is established before tumour initiation and is generally maintained throughout tumour development; however, the significance of EBV in tumour maintenance and progression remains to be elucidated. Here, we report eight cases of EBV-associated gastric carcinoma (EBVaGC) with intratumoural heterogenous expression of EBV-encoded small RNA (EBER), a highly expressed latent gene of EBV, and demonstrate clinicopathological characteristics of these rare cases. By performing detailed histological assessment of EBER-positive and -negative components of each case, detection of EBV genome in tumour cells by fluorescence in situ hybridisation, TP73 methylation analysis, whole exome sequencing, and targeted gene panel sequencing, we identified tumours in two patients to be collision tumours of different origins. In the other six patients, some genetic/epigenetic alterations were shared between EBER-positive and -negative components, suggesting that EBV was eliminated from tumour cells during progression. Interestingly, in both tumour types, programmed death ligand 1 and intratumoural infiltration of CD8+ T lymphocytes were lower in EBER-negative than in EBER-positive components, suggesting an immunogenic role of EBV. To the best of our knowledge, this study is the first to demonstrate the detailed histological features and genetic/epigenetic alterations in EBVaGC with heterogenous EBER expression; the loss of EBV may benefit tumour progression and immune evasion and might be clinically important for selecting treatment strategies for such cancers. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Identification of the ultrahigh-risk subgroup in neuroblastoma cases through DNA methylation analysis and its treatment exploiting cancer metabolism.

Neuroblastomas require novel therapies that are based on the exploitation of their biological mechanism. To address this need, we analyzed the DNA methylation and expression datasets of neuroblastomas, extracted a candidate gene characterizing the aggressive features, and conducted functional studies. Based on the DNA methylation data, we identified a subgroup of neuroblastoma cases with 11q loss of heterozygosity with extremely poor prognosis. PHGDH, a serine metabolism-related gene, was extracted as a candidate with strong expression and characteristic methylation in this subgroup as well as in cases with MYCN amplification. PHGDH inhibition suppressed neuroblastoma cell proliferation in vitro and in vivo, indicating that the inhibition of serine metabolism by PHGDH inhibitors is a therapeutic alternative for neuroblastoma. Inhibiting the arginine metabolism, which is closely related to serine metabolism using arginine deiminase, had a combination effect both in vitro and in vivo, especially on extracellular arginine-dependent neuroblastoma cells with ASS1 deficiency. Expression and metabolome analyses of post-dose cells confirmed the synergistic effects of treatments targeting serine and arginine indicated that xCT inhibitors that inhibit cystine uptake could be candidates for further combinatorial treatment. Our results highlight the rational therapeutic strategy of targeting serine/arginine metabolism for intractable neuroblastoma.

Multi-omics analysis defines highly refractory RAS burdened immature subgroup of infant acute lymphoblastic leukemia.

KMT2A-rearranged infant acute lymphoblastic leukemia (ALL) represents the most refractory type of childhood leukemia. To uncover the molecular heterogeneity of this disease, we perform RNA sequencing, methylation array analysis, whole exome and targeted deep sequencing on 84 infants with KMT2A-rearranged leukemia. Our multi-omics clustering followed by single-sample and single-cell inference of hematopoietic differentiation establishes five robust integrative clusters (ICs) with different master transcription factors, fusion partners and corresponding stages of B-lymphopoietic and early hemato-endothelial development: IRX-type differentiated (IC1), IRX-type undifferentiated (IC2), HOXA-type MLLT1 (IC3), HOXA-type MLLT3 (IC4), and HOXA-type AFF1 (IC5). Importantly, our deep mutational analysis reveals that the number of RAS pathway mutations predicts prognosis and that the most refractory subgroup of IC2 possesses 100% frequency and the heaviest burden of RAS pathway mutations. Our findings highlight the previously under-appreciated intra- and inter-patient heterogeneity of KMT2A-rearranged infant ALL and provide a rationale for the future development of genomics-guided risk stratification and individualized therapy.

Genome-wide analysis of DNA methylation identifies the apoptosis-related gene UQCRH as a tumor suppressor in renal cancer.

DNA hypermethylation is frequently observed in clear cell renal cell carcinoma (ccRCC) and correlates with poor clinical outcomes. However, the detailed function of DNA hypermethylation in ccRCC has not been fully uncovered. Here, we show the role of DNA methylation in ccRCC progression through the identification of a target(s) of DNA methyltransferases (DNMT). Our preclinical model of ccRCC using the serial orthotopic inoculation model showed the upregulation of DNMT3B in advanced ccRCC. Pretreatment of advanced ccRCC cells with 5-aza-deoxycytidine, a DNMT inhibitor, attenuated the formation of primary tumors through the induction of apoptosis. DNA methylated sites were analyzed genome-wide using methylation array in reference to RNA-sequencing data. The gene encoding ubiquinol cytochrome c reductase hinge protein (UQCRH), one of the components of mitochondrial complex III, was extracted as a methylation target in advanced ccRCC. Immunohistochemical analysis revealed that the expression of UQCRH in human ccRCC tissues was lower than normal adjacent tissues. Silencing of UQCRH attenuated the cytochrome c release in response to apoptotic stimuli and resulted in enhancement of primary tumor formation in vivo, implying the tumor-suppressive role of UQCRH. Moreover, 5-aza-deoxycytidine enhanced the therapeutic efficiency of mammalian target of rapamycin inhibitor everolimus in vivo. These findings suggested that the DNMT3B-induced methylation of UQCRH may contribute to renal cancer progression and implicated clinical significance of DNMT inhibitor as a therapeutic option for ccRCC.

Genetic and epigenetic basis of hepatoblastoma diversity.

Hepatoblastoma (HB) is the most common pediatric liver malignancy; however, hereditary predisposition and acquired molecular aberrations related to HB clinicopathological diversity are not well understood. Here, we perform an integrative genomic profiling of 163 pediatric liver tumors (154 HBs and nine hepatocellular carcinomas) based on the data acquired from a cohort study (JPLT-2). The total number of somatic mutations is precious low (0.52/Mb on exonic regions) but correlated with age at diagnosis. Telomerase reverse transcriptase (TERT) promoter mutations are prevalent in the tween HBs, selective in the transitional liver cell tumor (TLCT, > 8 years old). DNA methylation profiling reveals that classical HBs are characterized by the specific hypomethylated enhancers, which are enriched with binding sites for ASCL2, a regulatory transcription factor for definitive endoderm in Wnt-pathway. Prolonged upregulation of ASCL2, as well as fetal-liver-like methylation patterns of IGF2 promoters, suggests their "cell of origin" derived from the premature hepatoblast, similar to intestinal epithelial cells, which are highly proliferative. Systematic molecular profiling of HB is a promising approach for understanding the epigenetic drivers of hepatoblast carcinogenesis and deriving clues for risk stratification.

Base-resolution analysis of 5-hydroxymethylcytidine by selective oxidation and reverse transcription arrest.

While 5-hydroxymethylcytidine in RNA (hm5C) is associated with cellular development and differentiation, its distribution and biological function remain largely unexplored because suitable detection methods are lacking. Here, we report a base-resolution sequencing method for hm5C in RNA by applying peroxotungstate-mediated chemical conversion of hm5C to trihydroxylated thymine (thT). Reverse transcription by SuperScript III terminated at the thT site, probably because of its unnatural nucleobase structure producing truncated cDNA. Consequently, base-resolution analysis of the hm5C sites in RNA was achieved with both Sanger sequencing and Illumina sequencing analysis by comparing sequencing data before and after peroxotungstate treatment.

Molecular classification and diagnostics of upper urinary tract urothelial carcinoma.

Upper urinary tract urothelial carcinoma (UTUC) is one of the common urothelial cancers. Its molecular pathogenesis, however, is poorly understood, with no useful biomarkers available for accurate diagnosis and molecular classification. Through an integrated genetic study involving 199 UTUC samples, we delineate the landscape of genetic alterations in UTUC enabling genetic/molecular classification. According to the mutational status of TP53, MDM2, RAS, and FGFR3, UTUC is classified into five subtypes having discrete profiles of gene expression, tumor location/histology, and clinical outcome, which is largely recapitulated in an independent UTUC cohort. Sequencing of urine sediment-derived DNA has a high diagnostic value for UTUC with 82.2% sensitivity and 100% specificity. These results provide a solid basis for better diagnosis and management of UTUC.

TET1 upregulation drives cancer cell growth through aberrant enhancer hydroxymethylation of HMGA2 in hepatocellular carcinoma.

Ten-eleven translocation 1 (TET1) is an essential methylcytosine dioxygenase of the DNA demethylation pathway. Despite its dysregulation being known to occur in human cancer, the role of TET1 remains poorly understood. In this study, we report that TET1 promotes cell growth in human liver cancer. The transcriptome analysis of 68 clinical liver samples revealed a subgroup of TET1-upregulated hepatocellular carcinoma (HCC), demonstrating hepatoblast-like gene expression signatures. We performed comprehensive cytosine methylation and hydroxymethylation (5-hmC) profiling and found that 5-hmC was aberrantly deposited preferentially in active enhancers. TET1 knockdown in hepatoma cell lines decreased hmC deposition with cell growth suppression. HMGA2 was highly expressed in a TET1high subgroup of HCC, associated with the hyperhydroxymethylation of its intronic region, marked as histone H3K4-monomethylated, where the H3K27-acetylated active enhancer chromatin state induced interactions with its promoter. Collectively, our findings point to a novel type of epigenetic dysregulation, methylcytosine dioxygenase TET1, which promotes cell proliferation via the ectopic enhancer of its oncogenic targets, HMGA2, in hepatoblast-like HCC.

Accumulation of Molecular Aberrations Distinctive to Hepatocellular Carcinoma Progression.

Cancer develops through the accumulation of genetic and epigenetic aberrations. To identify sequential molecular alterations that occur during the development of hepatocellular carcinoma (HCC), we compared 52 early and 108 overt HCC samples by genome sequencing. Gene mutations in the p53/RB1 pathway, WNT pathway, MLL protein family, SWI/SNF complexes, and AKT/PI3K pathway were common in HCC. In the early phase of all entities, TERT was the most frequently upregulated gene owing to diverse mechanisms. Despite frequent somatic mutations in driver genes, including CTNNB1 and TP53, early HCC was a separate molecular entity from overt HCC, as each had a distinct expression profile. Notably, WNT target genes were not activated in early HCC regardless of CTNNB1 mutation status because ß-catenin did not translocate into the nucleus due to the E-cadherin/ß-catenin complex at the membrane. Conversely, WNT targets were definitively upregulated in overt HCC, with CTNNB1 mutation associated with downregulation of CDH1 and hypomethylation of CpG islands in target genes. Similarly, cell-cycle genes downstream of the p53/RB pathway were upregulated only in overt HCC, with TP53 or RB1 gene mutations associated with chromosomal deletion of 4q or 16q. HCC was epigenetically distinguished into four subclasses: normal-like methylation, global-hypomethylation (favorable prognosis), stem-like methylation (poor prognosis), and CpG island methylation. These methylation statuses were globally maintained through HCC progression. Collectively, these data show that as HCC progresses, additional molecular events exclusive of driver gene mutations cooperatively contribute to transcriptional activation of downstream targets according to methylation status. SIGNIFICANCE: In addition to driver gene mutations in the WNT and p53 pathways, further molecular events are required for aberrant transcriptional activation of these pathways as HCC progresses.

A novel sensitive detection method for DNA methylation in circulating free DNA of pancreatic cancer.

Despite recent advances in clinical treatment, pancreatic cancer remains a highly lethal malignancy. In order to improve the survival rate of patients with pancreatic cancer, the development of non-invasive diagnostic methods using effective biomarkers is urgently needed. Here, we developed a highly sensitive method to detect DNA methylation in cell-free (cf)DNA samples based on the enrichment of methyl-CpG binding (MBD) protein coupled with a digital PCR method (MBD-ddPCR). Five DNA methylation markers for the diagnosis of pancreatic cancer were identified through DNA methylation microarray analysis in 37 pancreatic cancers. The sensitivity and specificity of the five markers were validated in another independent cohort of pancreatic cancers (100% and 100%, respectively; n = 46) as well as in The Cancer Genome Atlas data set (96% and 90%, respectively; n = 137). MBD-ddPCR analysis revealed that DNA methylation in at least one of the five markers was detected in 23 (49%) samples of cfDNA from 47 patients with pancreatic cancer. Further, a combination of DNA methylation markers and the KRAS mutation status improved the diagnostic capability of this method (sensitivity and specificity, 68% and 86%, respectively). Genome-wide MBD-sequencing analysis in cancer tissues and corresponding cfDNA revealed that more than 80% of methylated regions were overlapping; DNA methylation profiles of cancerous tissues and cfDNA significantly correlated with each other (R = 0.97). Our data indicate that newly developed MBD-ddPCR is a sensitive method to detect cfDNA methylation and that using five marker genes plus KRAS mutations may be useful for the detection of pancreatic cancers.

Defined lifestyle and germline factors predispose Asian populations to gastric cancer.

Germline and environmental effects on the development of gastric cancers (GC) and their ethnic differences have been poorly understood. Here, we performed genomic-scale trans-ethnic analysis of 531 GCs (319 Asian and 212 non-Asians). There was one distinct GC subclass with clear alcohol-associated mutation signature and strong Asian specificity, almost all of which were attributable to alcohol intake behavior, smoking habit, and Asian-specific defective ALDH2 allele. Alcohol-related GCs have low mutation burden and characteristic immunological profiles. In addition, we found frequent (7.4%) germline CDH1 variants among Japanese GCs, most of which were attributed to a few recurrent single-nucleotide variants shared by Japanese and Koreans, suggesting the existence of common ancestral events among East Asians. Specifically, approximately one-fifth of diffuse-type GCs were attributable to the combination of alcohol intake and defective ALDH2 allele or to CDH1 variants. These results revealed uncharacterized impacts of germline variants and lifestyles in the high incidence areas.

Two distinct modes of DNMT1 recruitment ensure stable maintenance DNA methylation.

Stable inheritance of DNA methylation is critical for maintaining differentiated phenotypes in multicellular organisms. We have recently identified dual mono-ubiquitylation of histone H3 (H3Ub2) by UHRF1 as an essential mechanism to recruit DNMT1 to chromatin. Here, we show that PCNA-associated factor 15 (PAF15) undergoes UHRF1-dependent dual mono-ubiquitylation (PAF15Ub2) on chromatin in a DNA replication-coupled manner. This event will, in turn, recruit DNMT1. During early S-phase, UHRF1 preferentially ubiquitylates PAF15, whereas H3Ub2 predominates during late S-phase. H3Ub2 is enhanced under PAF15 compromised conditions, suggesting that H3Ub2 serves as a backup for PAF15Ub2. In mouse ES cells, loss of PAF15Ub2 results in DNA hypomethylation at early replicating domains. Together, our results suggest that there are two distinct mechanisms underlying replication timing-dependent recruitment of DNMT1 through PAF15Ub2 and H3Ub2, both of which are prerequisite for high fidelity DNA methylation inheritance.

Spatial and temporal expansion of intrahepatic metastasis by molecularly-defined clonality in multiple liver cancers.

Multiple hepatocellular carcinoma (HCC) is divided into two categories: intrahepatic metastasis (IM), which is a true relapse of HCC, and multicentric origin (MO), which is a second primary tumor. Clinical diagnosis of multiple HCC is usually made based on tumor location and/or time to recurrence; however, it is often difficult to distinguish the two types of multiple HCC. Using 41 matched pairs of multiple HCC specimens, we confirmed the accuracy of clinical diagnoses using exome sequence data and investigated the importance of discriminating the type of multiple HCC. Genomic analysis revealed that 18 (43.9%) patients diagnosed as having genomic IM had common mutations in a pair of HCC tumors with the main tumor of these patients being more progressive compared to those with genomic MO. The accuracy of clinical diagnosis based on lobe (Definition 1) and segment (Definition 2) were 68.3% and 78.0%, respectively. Intriguingly, recurrence ≥2 years after initial surgery for 3 patients was IM. The survival of patients with clinical IM was significantly shorter than for those with clinical MO based on both Definition 1 (P = 0.045) and Definition 2 (P = 0.043). However, mean survival was not different between the patients with genomic IM and those with MO (P = 0.364). Taken together, genomic analysis elucidated that liver cancer may spread more extensively and more slowly than previously thought. In addition, distinguishing multiple HCC as IM or MC may have provided biological information but was not of clinical importance with respect to patient prognosis.

Comprehensive epigenome characterization reveals diverse transcriptional regulation across human vascular endothelial cells.

BACKGROUND: Endothelial cells (ECs) make up the innermost layer throughout the entire vasculature. Their phenotypes and physiological functions are initially regulated by developmental signals and extracellular stimuli. The underlying molecular mechanisms responsible for the diverse phenotypes of ECs from different organs are not well understood. RESULTS: To characterize the transcriptomic and epigenomic landscape in the vascular system, we cataloged gene expression and active histone marks in nine types of human ECs (generating 148 genome-wide datasets) and carried out a comprehensive analysis with chromatin interaction data. We developed a robust procedure for comparative epigenome analysis that circumvents variations at the level of the individual and technical noise derived from sample preparation under various conditions. Through this approach, we identified 3765 EC-specific enhancers, some of which were associated with disease-associated genetic variations. We also identified various candidate marker genes for each EC type. We found that the nine EC types can be divided into two subgroups, corresponding to those with upper-body origins and lower-body origins, based on their epigenomic landscape. Epigenomic variations were highly correlated with gene expression patterns, but also provided unique information. Most of the deferentially expressed genes and enhancers were cooperatively enriched in more than one EC type, suggesting that the distinct combinations of multiple genes play key roles in the diverse phenotypes across EC types. Notably, many homeobox genes were differentially expressed across EC types, and their expression was correlated with the relative position of each organ in the body. This reflects the developmental origins of ECs and their roles in angiogenesis, vasculogenesis and wound healing. CONCLUSIONS: This comprehensive analysis of epigenome characterization of EC types reveals diverse transcriptional regulation across human vascular systems. These datasets provide a valuable resource for understanding the vascular system and associated diseases.