Deconvolution of immune cell composition and biological age of hepatocellular carcinoma using DNA methylation.

Hepatocellular carcinoma (HCC) has been an approved indication for the administration of immunotherapy since 2017, but biomarkers that predict therapeutic response have remained limited. Understanding and characterizing the tumor immune microenvironment enables better classification of these tumors and may reveal biomarkers that predict immunotherapeutic efficacy. In this paper, we applied a cell-type deconvolution algorithm using DNA methylation array data to investigate the composition of the tumor microenvironment in HCC. Using publicly available and in-house datasets with a total cohort size of 57 patients, each with tumor and matched normal tissue samples, we identified key differences in immune cell composition. We found that NK cell abundance was significantly decreased in HCC tumors compared to adjacent normal tissue. We also applied DNA methylation "clocks" which estimate phenotypic aging and compared these findings to expression-based determinations of cellular senescence. Senescence and epigenetic aging were significantly increased in HCC tumors, and the degree of age acceleration and senescence was strongly associated with decreased NK cell abundance. In summary, we found that NK cell infiltration in the tumor microenvironment is significantly diminished, and that this loss of NK abundance is strongly associated with increased senescence and age-related phenotype. These findings point to key interactions between NK cells and the senescent tumor microenvironment and offer insights into the pathogenesis of HCC as well as potential biomarkers of therapeutic efficacy.

Metabolomic Analysis of Liver Tissues for Characterization of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) causes more than half a million annual deaths worldwide. Understanding the mechanisms contributing to HCC development is highly desirable for improved surveillance, diagnosis, and treatment. Liver tissue metabolomics has the potential to reflect the physiological changes behind HCC development. Also, it allows identification of biomarker candidates for future evaluation in biofluids and investigation of racial disparities in HCC. Tumor and nontumor tissues from 40 patients were analyzed by both gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS) platforms to increase the metabolome coverage. The levels of the metabolites extracted from solid liver tissue of the HCC area and adjacent non-HCC area were compared. Among the analytes detected by GC-MS and LC-MS with significant alterations, 18 were selected based on biological relevance and confirmed metabolite identification. These metabolites belong to TCA cycle, glycolysis, purines, and lipid metabolism and have been previously reported in liver metabolomic studies where high correlation with HCC progression is implied. We demonstrated that metabolites related to HCC pathogenesis can be identified through liver tissue metabolomic analysis. Additionally, this study has enabled us to identify race-specific metabolites associated with HCC.

Protein network construction using reverse phase protein array data.

In this paper, we introduce a novel computational method for constructing protein networks based on reverse phase protein array (RPPA) data to identify complex patterns in protein signaling. The method is applied to phosphoproteomic profiles of basal expression and activation/phosphorylation of 76 key signaling proteins in three breast cancer cell lines (MCF7, LCC1, and LCC9). Temporal RPPA data are acquired at 48h, 96h, and 144h after knocking down four genes in separate experiments. These genes are selected from a previous study as important determinants for breast cancer survival. Interaction networks are constructed by analyzing the expression levels of protein pairs using a multivariate analysis of variance model. A new scoring criterion is introduced to determine relevant protein pairs. Through a network topology based analysis, we search for wiring patterns to identify key proteins that are associated with significant changes in expression levels across various experimental conditions.

INDEED: Integrated differential expression and differential network analysis of omic data for biomarker discovery.

Differential expression (DE) analysis is commonly used to identify biomarker candidates that have significant changes in their expression levels between distinct biological groups. One drawback of DE analysis is that it only considers the changes on single biomolecule level. Recently, differential network (DN) analysis has become popular due to its capability to measure the changes on biomolecular pair level. In DN analysis, network is typically built based on correlation and biomarker candidates are selected by investigating the network topology. However, correlation tends to generate over-complicated networks and the selection of biomarker candidates purely based on network topology ignores the changes on single biomolecule level. In this paper, we propose a novel approach, INDEED, that builds sparse differential network based on partial correlation and integrates DE and DN analyses for biomarker discovery. We applied this approach on real proteomic and glycomic data generated by liquid chromatography coupled with mass spectrometry for hepatocellular carcinoma (HCC) biomarker discovery study. For each omic data, we used one dataset to select biomarker candidates, built a disease classifier and evaluated the performance of the classifier on an independent dataset. The biomarker candidates, selected by INDEED, were more reproducible across independent datasets, and led to a higher classification accuracy in predicting HCC cases and cirrhotic controls compared with those selected by separate DE and DN analyses. INDEED also identified some candidates previously reported to be relevant to HCC, such as intercellular adhesion molecule 2 (ICAM2) and c4b-binding protein alpha chain (C4BPA), which were missed by both DE and DN analyses. In addition, we applied INDEED for survival time prediction based on transcriptomic data acquired by analysis of samples from breast cancer patients. We selected biomarker candidates and built a regression model for survival time prediction based on a gene expression dataset and patients' survival records. We evaluated the performance of the regression model on an independent dataset. Compared with the biomarker candidates selected by DE and DN analyses, those selected through INDEED led to more accurate survival time prediction.

LC-MS/MS-based serum proteomics for identification of candidate biomarkers for hepatocellular carcinoma.

Associating changes in protein levels with the onset of cancer has been widely investigated to identify clinically relevant diagnostic biomarkers. In the present study, we analyzed sera from 205 patients recruited in the United States and Egypt for biomarker discovery using label-free proteomic analysis by LC-MS/MS. We performed untargeted proteomic analysis of sera to identify candidate proteins with statistically significant differences between hepatocellular carcinoma (HCC) and patients with liver cirrhosis. We further evaluated the significance of 101 proteins in sera from the same 205 patients through targeted quantitation by MRM on a triple quadrupole mass spectrometer. This led to the identification of 21 candidate protein biomarkers that were significantly altered in both the United States and Egyptian cohorts. Among the 21 candidates, ten were previously reported as HCC-associated proteins (eight exhibiting consistent trends with our observation), whereas 11 are new candidates discovered by this study. Pathway analysis based on the significant proteins reveals upregulation of the complement and coagulation cascades pathway and downregulation of the antigen processing and presentation pathway in HCC cases versus patients with liver cirrhosis. The results of this study demonstrate the power of combining untargeted and targeted quantitation methods for a comprehensive serum proteomic analysis, to evaluate changes in protein levels and discover novel diagnostic biomarkers. All MS data have been deposited in the ProteomeXchange with identifier PXD001171 (http://proteomecentral.proteomexchange.org/dataset/PXD001171).

LC-MS profiling of N-Glycans derived from human serum samples for biomarker discovery in hepatocellular carcinoma.

Defining clinically relevant biomarkers for early stage hepatocellular carcinoma (HCC) in a high-risk population of cirrhotic patients has potentially far-reaching implications for disease management and patient health. Changes in glycan levels have been associated with the onset of numerous diseases including cancer. In the present study, we used liquid chromatography coupled with electrospray ionization mass spectrometry (LC-ESI-MS) to analyze N-glycans in sera from 183 participants recruited in Egypt and the U.S. and identified candidate biomarkers that distinguish HCC cases from cirrhotic controls. N-Glycans were released from serum proteins and permethylated prior to the LC-ESI-MS analysis. Through two complementary LC-ESI-MS quantitation approaches, global profiling and targeted quantitation, we identified 11 N-glycans with statistically significant differences between HCC cases and cirrhotic controls. These glycans can further be categorized into four structurally related clusters, matching closely with the implications of important glycosyltransferases in cancer progression and metastasis. The results of this study illustrate the power of the integrative approach combining complementary LC-ESI-MS based quantitation approaches to investigate changes in N-glycan levels between HCC cases and patients with liver cirrhosis.

RNA-Seq Data Analysis.

RNA-Seq data analysis stands as a vital part of genomics research, turning vast and complex datasets into meaningful biological insights. It is a field marked by rapid evolution and ongoing innovation, necessitating a thorough understanding for anyone seeking to unlock the potential of RNA-Seq data. In this chapter, we describe the intricate landscape of RNA-seq data analysis, elucidating a comprehensive pipeline that navigates through the entirety of this complex process. Beginning with quality control, the chapter underscores the paramount importance of ensuring the integrity of RNA-seq data, as it lays the groundwork for subsequent analyses. Preprocessing is then addressed, where the raw sequence data undergoes necessary modifications and enhancements, setting the stage for the alignment phase. This phase involves mapping the processed sequences to a reference genome, a step pivotal for decoding the origins and functions of these sequences.Venturing into the heart of RNA-seq analysis, the chapter then explores differential expression analysis-the process of identifying genes that exhibit varying expression levels across different conditions or sample groups. Recognizing the biological context of these differentially expressed genes is pivotal; hence, the chapter transitions into functional analysis. Here, methods and tools like Gene Ontology and pathway analyses help contextualize the roles and interactions of the identified genes within broader biological frameworks. However, the chapter does not stop at conventional analysis methods. Embracing the evolving paradigms of data science, it delves into machine learning applications for RNA-seq data, introducing advanced techniques in dimension reduction and both unsupervised and supervised learning. These approaches allow for patterns and relationships to be discerned in the data that might be imperceptible through traditional methods.

Short-Read RNA-Seq.

RNA sequencing (RNA-Seq) has emerged as a powerful and versatile tool for the comprehensive analysis of transcriptomes and has been widely used to investigate gene expression, copy number variation, alternative splicing, and novel transcript discovery. This chapter outlines the methodology for conducting short-read RNA-Seq, starting from RNA enrichment to library preparation and sequencing. Throughout the chapter, practical tips and best practices are provided to guide researchers in order to optimize each step of the RNA-Seq workflow. Multiple quality control steps throughout the workflow that are critical to obtain high-quality RNA-Seq data are also discussed.

Biomarker Discovery for Hepatocellular Carcinoma in Patients with Liver Cirrhosis Using Untargeted Metabolomics and Lipidomics Studies.

Hepatocellular carcinoma (HCC), the most prevalent form of liver cancer, is the third leading cause of mortality globally. Patients with HCC have a poor prognosis due to the fact that the emergence of symptoms typically occurs at a late stage of the disease. In addition, conventional biomarkers perform suboptimally when identifying HCC in its early stages, heightening the need for the identification of new and more effective biomarkers. Using metabolomics and lipidomics approaches, this study aims to identify serum biomarkers for identification of HCC in patients with liver cirrhosis (LC). Serum samples from 20 HCC cases and 20 patients with LC were analyzed using ultra-high-performance liquid chromatography-Q Exactive mass spectrometry (UHPLC-Q-Exactive-MS). Metabolites and lipids that are significantly altered between HCC cases and patients with LC were identified. These include organic acids, amino acids, TCA cycle intermediates, fatty acids, bile acids, glycerophospholipids, sphingolipids, and glycerolipids. The most significant variability was observed in the concentrations of bile acids, fatty acids, and glycerophospholipids. In the context of HCC cases, there was a notable increase in the levels of phosphatidylethanolamine and triglycerides, but the levels of fatty acids and phosphatidylcholine exhibited a substantial decrease. In addition, it was observed that all of the identified metabolites exhibited a superior area under the receiver operating characteristic (ROC) curve in comparison to alpha-fetoprotein (AFP). The pathway analysis of these metabolites revealed fatty acid, lipid, and energy metabolism as the most impacted pathways. Putative biomarkers identified in this study will be validated in future studies via targeted quantification.

Deep Learning Based Metabolite Annotation.

Metabolite annotation is a major bottleneck in untargeted metabolomics studies by liquid chromatography coupled with mass spectrometry (LC-MS). This is in part due to the limited publicly available spectral libraries, which consist of tandem mass spectrometry (MS/MS) data acquired from just a fraction of known compounds. Machine learning and deep learning methods provide the opportunity to predict molecular fingerprints based on MS/MS data. The predicted molecular fingerprints can then be used to help rank candidate metabolite IDs obtained based on predicted formula or measured precursor m/z of the unknown metabolite. This approach is particularly useful to help annotate metabolites whose corresponding MS/MS spectra cannot be matched with those in spectral libraries. We previously reported application of a convolutional neural network (CNN) for molecular fingerprint prediction using MS/MS spectra obtained from the MoNA repository and NIST 20. In this paper, we investigate high-dimensional representation of the spectral data and molecular fingerprints to improve accuracy in molecular fingerprint prediction.

Formalin Fixation, Delay to Fixation, and Time in Fixative Adversely Impact Copy Number Variation Analysis by aCGH.

Although molecular profiling of DNA isolated from formalin-fixed, paraffin-embedded (FFPE) tumor specimens has become more common in recent years, it remains unclear how discrete FFPE processing variables may affect detection of copy number variation (CNV). To better understand such effects, array comparative genomic hybridization (aCGH) profiles of FFPE renal cell carcinoma specimens that experienced different delays to fixation (DTFs; 1, 2, 3, and 12 hours) and times in fixative (TIFs; 6, 12, 23, and 72 hours) were compared to snap-frozen tumor and blood specimens from the same patients. A greater number of regions containing CNVs relative to commercial reference DNA were detected in DNA from FFPE tumor specimens than snap-frozen tumor specimens even though they originated from the same tumor blocks. Extended DTF and TIF affected the number of DNA segments with a copy number status that differed between FFPE and frozen tumor specimens; a DTF ≥3 hours led to more segments, while a TIF of 72 hours led to fewer segments. Importantly, effects were not random as a higher guanine-cytosine (GC) content and/or a higher percentage of repeats were observed among stable regions. While limiting aCGH analysis to FFPE specimens with a DTF <3 hours and a TIF <72 hours may circumvent some effects, results from FFPE specimens should be validated against fresh or frozen specimens whenever possible.

MicroRNA-483-5p Inhibits Hepatocellular Carcinoma Cell Proliferation, Cell Steatosis, and Fibrosis by Targeting PPARα and TIMP2.

MicroRNAs (miRNAs) are small non-coding RNA molecules that bind with the 3' untranslated regions (UTRs) of genes to regulate expression. Downregulation of miR-483-5p (miR-483) is associated with the progression of hepatocellular carcinoma (HCC). However, the significant roles of miR-483 in nonalcoholic fatty liver disease (NAFLD), alcoholic fatty liver diseases (AFLD), and HCC remain elusive. In the current study, we investigated the biological significance of miR-483 in NAFLD, AFLD, and HCC in vitro and in vivo. The downregulation of miR-483 expression in HCC patients' tumor samples was associated with Notch 3 upregulation. Overexpression of miR-483 in a human bipotent progenitor liver cell line HepaRG and HCC cells dysregulated Notch signaling, inhibited cell proliferation/migration, induced apoptosis, and increased sensitivity towards antineoplastic agents sorafenib/regorafenib. Interestingly, the inactivation of miR-483 upregulated cell steatosis and fibrosis signaling by modulation of lipogenic and fibrosis gene expression. Mechanistically, miR-483 targets PPARα and TIMP2 gene expression, which leads to the suppression of cell steatosis and fibrosis. The downregulation of miR-483 was observed in mice liver fed with a high-fat diet (HFD) or a standard Lieber-Decarli liquid diet containing 5% alcohol, leading to increased hepatic steatosis/fibrosis. Our data suggest that miR-483 inhibits cell steatosis and fibrogenic signaling and functions as a tumor suppressor in HCC. Therefore, miR-483 may be a novel therapeutic target for NAFLD/AFLD/HCC management in patients with fatty liver diseases and HCC.

LC-MS based serum metabolomics for identification of hepatocellular carcinoma biomarkers in Egyptian cohort.

Although hepatocellular carcinoma (HCC) has been subjected to continuous investigation and its symptoms are well-known, early stage diagnosis of this disease remains difficult and the survival rate after diagnosis is typically very low (3-5%). Early and accurate detection of metabolic changes in the sera of patients with liver cirrhosis can help improve the prognosis of HCC and lead to a better understanding of its mechanism at the molecular level, thus providing patients with in-time treatment of the disease. In this study, we compared metabolite levels in sera of 40 HCC patients and 49 cirrhosis patients from Egypt by using ultraperformance liquid chromatography coupled with quadrupole time-of-flight mass spectrometer (UPLC-QTOF MS). Following data preprocessing, the most relevant ions in distinguishing HCC cases from cirrhotic controls are selected by statistical methods. Putative metabolite identifications for these ions are obtained through mass-based database search. The identities of some of the putative identifications are verified by comparing their MS/MS fragmentation patterns and retention times with those from authentic compounds. Finally, the serum samples are reanalyzed for quantitation of selected metabolites as candidate biomarkers of HCC. This quantitation was performed using isotope dilution by selected reaction monitoring (SRM) on a triple quadrupole linear ion trap (QqQLIT) coupled to UPLC. Statistical analysis of the UPLC-QTOF data identified 274 monoisotopic ion masses with statistically significant differences in ion intensities between HCC cases and cirrhotic controls. Putative identifications were obtained for 158 ions by mass based search against databases. We verified the identities of selected putative identifications including glycholic acid (GCA), glycodeoxycholic acid (GDCA), 3ß, 6ß-dihydroxy-5ß-cholan-24-oic acid, oleoyl carnitine, and Phe-Phe. SRM-based quantitation confirmed significant differences between HCC and cirrhotic controls in metabolite levels of bile acid metabolites, long chain carnitines and small peptide. Our study provides useful insight into appropriate experimental design and computational methods for serum biomarker discovery using LC-MS/MS based metabolomics. This study has led to the identification of candidate biomarkers with significant changes in metabolite levels between HCC cases and cirrhotic controls. This is the first MS-based metabolic biomarker discovery study on Egyptian subjects that led to the identification of candidate metabolites that discriminate early stage HCC from patients with liver cirrhosis.

Using a spike-in experiment to evaluate analysis of LC-MS data.

BACKGROUND: Recent advances in liquid chromatography-mass spectrometry (LC-MS) technology have led to more effective approaches for measuring changes in peptide/protein abundances in biological samples. Label-free LC-MS methods have been used for extraction of quantitative information and for detection of differentially abundant peptides/proteins. However, difference detection by analysis of data derived from label-free LC-MS methods requires various preprocessing steps including filtering, baseline correction, peak detection, alignment, and normalization. Although several specialized tools have been developed to analyze LC-MS data, determining the most appropriate computational pipeline remains challenging partly due to lack of established gold standards. RESULTS: The work in this paper is an initial study to develop a simple model with "presence" or "absence" condition using spike-in experiments and to be able to identify these "true differences" using available software tools. In addition to the preprocessing pipelines, choosing appropriate statistical tests and determining critical values are important. We observe that individual statistical tests could lead to different results due to different assumptions and employed metrics. It is therefore preferable to incorporate several statistical tests for either exploration or confirmation purpose. CONCLUSIONS: The LC-MS data from our spike-in experiment can be used for developing and optimizing LC-MS data preprocessing algorithms and to evaluate workflows implemented in existing software tools. Our current work is a stepping stone towards optimizing LC-MS data acquisition and testing the accuracy and validity of computational tools for difference detection in future studies that will be focused on spiking peptides of diverse physicochemical properties in different concentrations to better represent biomarker discovery of differentially abundant peptides/proteins.

Ion annotation-assisted analysis of LC-MS based metabolomic experiment.

BACKGROUND: Analysis of multiple LC-MS based metabolomic studies is carried out to determine overlaps and differences among various experiments. For example, in large metabolic biomarker discovery studies involving hundreds of samples, it may be necessary to conduct multiple experiments, each involving a subset of the samples due to technical limitations. The ions selected from each experiment are analyzed to determine overlapping ions. One of the challenges in comparing the ion lists is the presence of a large number of derivative ions such as isotopes, adducts, and fragments. These derivative ions and the retention time drifts need to be taken into account during comparison. RESULTS: We implemented an ion annotation-assisted method to determine overlapping ions in the presence of derivative ions. Following this, each ion is represented by the monoisotopic mass of its cluster. This mass is then used to determine overlaps among the ions selected across multiple experiments. CONCLUSION: The resulting ion list provides better coverage and more accurate identification of metabolites compared to the traditional method in which overlapping ions are selected on the basis of individual ion mass.

Convolutional Neural Network-Based Compound Fingerprint Prediction for Metabolite Annotation.

Metabolite annotation has been a challenging issue especially in untargeted metabolomics studies by liquid chromatography coupled with mass spectrometry (LC-MS). This is in part due to the limitations of publicly available spectral libraries, which consist of tandem mass spectrometry (MS/MS) data acquired from just a fraction of known metabolites. Machine learning provides the opportunity to predict molecular fingerprints based on MS/MS data. The predicted molecular fingerprints can then be used to help rank putative metabolite IDs obtained by using either the precursor mass or the formula of the unknown metabolite. This method is particularly useful to help annotate metabolites whose corresponding MS/MS spectra are missing or cannot be matched with those in accessible spectral libraries. We investigated a convolutional neural network (CNN) for molecular fingerprint prediction based on data acquired by MS/MS. We used more than 680,000 MS/MS spectra obtained from the MoNA repository and NIST 20, representing about 36,000 compounds for training and testing our CNN model. The trained CNN model is implemented as a python package, MetFID. The package is available on GitHub for users to enter their MS/MS spectra and corresponding putative metabolite IDs to obtain ranked lists of metabolites. Better performance is achieved by MetFID in ranking putative metabolite IDs using the CASMI 2016 benchmark dataset compared to two other machine learning-based tools (CSI:FingerID and ChemDistiller).

Cell-type Deconvolution and Age Estimation Using DNA Methylation Reveals NK Cell Deficiency in the Hepatocellular Carcinoma Microenvironment.

Hepatocellular carcinoma (HCC) has been an approved indication for the administration of immunotherapy since 2017, but biomarkers that predict therapeutic response have remained limited. Understanding and characterizing the tumor immune microenvironment enables better classification of these tumors and may reveal biomarkers that predict immunotherapeutic efficacy. In this paper, we applied a cell-type deconvolution algorithm using DNA methylation array data to investigate the composition of the tumor microenvironment in HCC. Using two publicly available datasets with a total cohort size of 57 patients, each with tumor and matched normal tissue samples, we identified key differences in immune cell composition. We found that NK cell abundance was significantly decreased in HCC tumors compared to adjacent normal tissue. We also applied DNA methylation "clocks" which estimate phenotypic aging and compared these findings to expression-based determinations of cellular senescence. Senescence and epigenetic aging was significantly increased in HCC tumors, and the degree of age acceleration and senescence was strongly associated with decreased NK cell abundance. In summary, we found that NK cell infiltration in the tumor microenvironment is significantly diminished, and that this loss of NK abundance is strongly associated with increased senescence and age-related phenotype. These findings point to key interactions between NK cells and the senescent tumor microenvironment and offer insights into the pathogenesis of HCC as well as potential biomarkers of therapeutic efficacy.

A Bayesian two-step integrative procedure incorporating prior knowledge for the identification of miRNA-mRNAs involved in hepatocellular carcinoma.

Recent studies have confirmed the role of miRNA regulation of gene expression in oncogenesis for various cancers. In parallel, prior knowledge about relationships between miRNA and mRNA have been accumulated from biological experiments or statistical analyses. Improved identification of disease-associated miRNA-mRNA pairs may be achieved by incorporating prior knowledge into integrative genomic analyses. In this study we focus on 39 patients with hepatocellular carcinoma (HCC) and 25 patients with liver cirrhosis and use a flexible Bayesian two-step integrative method. We found 66 significant miRNA-mRNA pairs, several of which contain molecules that have previously been identified as potential biomarkers. These results demonstrate the utility of the proposed approach in providing a better understanding of relationships between different biological levels, thereby giving insights into the biological mechanisms underlying the diseases, while providing a better selection of biomarkers that may serve as diagnostic, prognostic, or therapeutic biomarker candidates.

Metabolomic Analysis of Plasma from Breast Cancer Patients Using Ultra-High-Performance Liquid Chromatography Coupled with Mass Spectrometry: An Untargeted Study.

Breast cancer (BC) is one of the leading causes of cancer mortality in women worldwide, and therefore, novel biomarkers for early disease detection are critically needed. We performed herein an untargeted plasma metabolomic profiling of 55 BC patients and 55 healthy controls (HC) using ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC/Q-TOF-MS). Pre-processed data revealed 2494 ions in total. Data matrices' paired t-tests revealed 792 ions (both positive and negative) which presented statistically significant changes (FDR < 0.05) in intensity levels between cases versus controls. Metabolites identified with putative names via MetaboQuest using MS/MS and mass-based approaches included amino acid esters (i.e., N-stearoyl tryptophan, L-arginine ethyl ester), dipeptides (ile-ser, met-his), nitrogenous bases (i.e., uracil derivatives), lipid metabolism-derived molecules (caproleic acid), and exogenous compounds from plants, drugs, or dietary supplements. LASSO regression selected 16 metabolites after several variables (TNM Stage, Grade, smoking status, menopausal status, and race) were adjusted. A predictive conditional logistic regression model on the 16 LASSO selected ions provided a high diagnostic performance with an area-under-the-curve (AUC) value of 0.9729 (95% CI 0.96−0.98) on all 55 samples. This study proves that BC possesses a specific metabolic signature that could be exploited as a novel metabolomics-based approach for BC detection and characterization. Future studies of large-scale cohorts are needed to validate these findings.

Integrative Analysis of DNA Methylation and microRNA Expression Reveals Mechanisms of Racial Heterogeneity in Hepatocellular Carcinoma.

Pathologic alterations in epigenetic regulation have long been considered a hallmark of many cancers, including hepatocellular carcinoma (HCC). In a healthy individual, the relationship between DNA methylation and microRNA (miRNA) expression maintains a fine balance; however, disruptions in this harmony can aid in the genesis of cancer or the propagation of existing cancers. The balance between DNA methylation and microRNA expression and its potential disturbance in HCC can vary by race. There is emerging evidence linking epigenetic events including DNA methylation and miRNA expression to cancer disparities. In this paper, we evaluate the epigenetic mechanisms of racial heterogenity in HCC through an integrated analysis of DNA methylation, miRNA, and combined regulation of gene expression. Specifically, we generated DNA methylation, mRNA-seq, and miRNA-seq data through the analysis of tumor and adjacent non-tumor liver tissues from African Americans (AA) and European Americans (EA) with HCC. Using mixed ANOVA, we identified cytosine-phosphate-guanine (CpG) sites, mRNAs, and miRNAs that are significantly altered in HCC vs. adjacent non-tumor tissue in a race-specific manner. We observed that the methylome was drastically changed in EA with a significantly larger number of differentially methylated and differentially expressed genes than in AA. On the other hand, the miRNA expression was altered to a larger extent in AA than in EA. Pathway analysis functionally linked epigenetic regulation in EA to processes involved in immune cell maturation, inflammation, and vascular remodeling. In contrast, cellular proliferation, metabolism, and growth pathways are found to predominate in AA as a result of this epigenetic analysis. Furthermore, through integrative analysis, we identified significantly differentially expressed genes in HCC with disparate epigenetic regulation, associated with changes in miRNA expression for AA and DNA methylation for EA.