Sara R. Selitsky

Identification of Driver Genes in Hepatocellular Carcinoma by Exome Sequencing

Genetic alterations in specific driver genes lead to disruption of cellular pathways and are critical events in the instigation and progression of hepatocellular carcinoma (HCC). As a prerequisite for individualized cancer treatment, we sought to characterize the landscape of recurrent somatic mutations in HCC. We performed whole‐exome sequencing on 87 HCCs and matched normal adjacent tissues to an average coverage of 59×. The overall mutation rate was roughly two mutations per Mb, with a median of 45 nonsynonymous mutations that altered the amino acid sequence (range, 2‐381). We found recurrent mutations in several genes with high transcript levels: TP53 (18%); CTNNB1 (10%); KEAP1 (8%); C16orf62 (8%); MLL4 (7%); and RAC2 (5%). Significantly affected gene families include the nucleotide‐binding domain and leucine‐rich repeat‐containing family, calcium channel subunits, and histone methyltransferases. In particular, the MLL family of methyltransferases for histone H3 lysine 4 were mutated in 20% of tumors. Conclusion: The NFE2L2‐KEAP1 and MLL pathways are recurrently mutated in multiple cohorts of HCC. (Hepatology 2013;58:1693–1702)

Small tRNA-derived RNAs are increased and more abundant than microRNAs in chronic hepatitis B and C

Persistent infections with hepatitis B virus (HBV) or hepatitis C virus (HCV) account for the majority of cases of hepatic cirrhosis and hepatocellular carcinoma (HCC) worldwide. Small, non-coding RNAs play important roles in virus-host interactions. We used high throughput sequencing to conduct an unbiased profiling of small (14-40 nts) RNAs in liver from Japanese subjects with advanced hepatitis B or C and hepatocellular carcinoma (HCC). Small RNAs derived from tRNAs, specifically 30–35 nucleotide-long 5′ tRNA-halves (5′ tRHs), were abundant in non-malignant liver and significantly increased in humans and chimpanzees with chronic viral hepatitis. 5′ tRH abundance exceeded microRNA abundance in most infected non-cancerous tissues. In contrast, in matched cancer tissue, 5′ tRH abundance was reduced, and relative abundance of individual 5′ tRHs was altered. In hepatitis B-associated HCC, 5′ tRH abundance correlated with expression of the tRNA-cleaving ribonuclease, angiogenin. These results demonstrate that tRHs are the most abundant small RNAs in chronically infected liver and that their abundance is altered in liver cancer.

Claudin-low bladder tumors are immune infiltrated and actively immune suppressed

We report the discovery of a claudin-low molecular subtype of high-grade bladder cancer that shares characteristics with the homonymous subtype of breast cancer. Claudin-low bladder tumors were enriched for multiple genetic features including increased rates of RB1, EP300, and NCOR1 mutations; increased frequency of EGFR amplification; decreased rates of FGFR3, ELF3, and KDM6A mutations; and decreased frequency of PPARG amplification. While claudin-low tumors showed the highest expression of immune gene signatures, they also demonstrated gene expression patterns consistent with those observed in active immunosuppression. This did not appear to be due to differences in predicted neoantigen burden, but rather was associated with broad upregulation of cytokine and chemokine levels from low PPARG activity, allowing unopposed NFKB activity. Taken together, these results define a molecular subtype of bladder cancer with distinct molecular features and an immunologic profile that would, in theory, be primed for immunotherapeutic response.

microRNA-122 Abundance in Hepatocellular Carcinoma and Non-Tumor Liver Tissue from Japanese Patients with Persistent HCV versus HBV Infection

Mechanisms of hepatic carcinogenesis in chronic hepatitis B and hepatitis C are incompletely defined but often assumed to be similar and related to immune-mediated inflammation. Despite this, several studies hint at differences in expression of miR-122, a liver-specific microRNA with tumor suppressor properties, in hepatocellular carcinoma (HCC) associated with hepatitis B virus (HBV) versus hepatitis C virus (HCV) infection. Differences in the expression of miR-122 in these cancers would be of interest, as miR-122 is an essential host factor for HCV but not HBV replication. To determine whether the abundance of miR-122 in cancer tissue is influenced by the nature of the underlying virus infection, we measured miR-122 by qRT-PCR in paired tumor and non-tumor tissues from cohorts of HBV- and HCV-infected Japanese patients. miR-122 abundance was significantly reduced from normal in HBV-associated HCC, but not in liver cancer associated with HCV infection. This difference was independent of the degree of differentiation of the liver cancer. Surprisingly, we also found significant differences in miR-122 expression in non-tumor tissue, with miR-122 abundance reduced from normal in HCV- but not HBV-infected liver. Similar differences were observed in HCV- vs. HBV-infected chimpanzees. Among HCV-infected Japanese subjects, reductions in miR-122 abundance in non-tumor tissue were associated with a single nucleotide polymorphism near the IL28B gene that predicts poor response to interferon-based therapy (TG vs. TT genotype at rs8099917), and correlated negatively with the abundance of multiple interferon-stimulated gene transcripts. Reduced levels of miR-122 in chronic hepatitis C thus appear to be associated with endogenous interferon responses to the virus, while differences in miR-122 expression in HCV- versus HBV-associated HCC likely reflect virus-specific mechanisms contributing to carcinogenesis. The continued expression of miR-122 in HCV-associated HCC may signify an important role for HCV replication late in the progression to malignancy.

Prioritization of genetic variants in the microrna regulome as functional candidates in genome-wide association studies

Comprehensive analyses of results from genome‐wide association studies (GWAS) have demonstrated that complex disease/trait‐associated loci are enriched in gene regulatory regions of the genome. The search for causal regulatory variation has focused primarily on transcriptional elements, such as promoters and enhancers. microRNAs (miRNAs) are now widely appreciated as critical posttranscriptional regulators of gene expression and are thought to impart stability to biological systems. Naturally occurring genetic variation in the miRNA regulome is likely an important contributor to phenotypic variation in the human population. However, the extent to which polymorphic miRNA‐mediated gene regulation underlies GWAS signals remains unclear. In this study, we have developed the most comprehensive bioinformatic analysis pipeline to date for cataloging and prioritizing variants in the miRNA regulome as functional candidates in GWAS. We highlight specific findings, including a variant in the promoter of the miRNA let‐7 that may contribute to human height variation. We also provide a discussion of how our approach can be expanded in the future. Overall, we believe that the results of this study will be valuable for researchers interested in determining whether GWAS signals implicate the miRNA regulome in their disease/trait of interest.

Model of fibrolamellar hepatocellular carcinomas reveals striking enrichment in cancer stem cells

The aetiology of human fibrolamellar hepatocellular carcinomas (hFL-HCCs), cancers occurring increasingly in children to young adults, is poorly understood. We present a transplantable tumour line, maintained in immune-compromised mice, and validate it as a bona fide model of hFL-HCCs by multiple methods. RNA-seq analysis confirms the presence of a fusion transcript (DNAJB1-PRKACA) characteristic of hFL-HCC tumours. The hFL-HCC tumour line is highly enriched for cancer stem cells as indicated by limited dilution tumourigenicity assays, spheroid formation and flow cytometry. Immunohistochemistry on the hFL-HCC model, with parallel studies on 27 primary hFL-HCC tumours, provides robust evidence for expression of endodermal stem cell traits. Transcriptomic analyses of the tumour line and of multiple, normal hepatic lineage stages reveal a gene signature for hFL-HCCs closely resembling that of biliary tree stem cells—newly discovered precursors for liver and pancreas. This model offers unprecedented opportunities to investigate mechanisms underlying hFL-HCCs pathogenesis and potential therapies.

Transcriptomic Analysis of Chronic Hepatitis B and C and Liver Cancer Reveals MicroRNA-Mediated Control of Cholesterol Synthesis Programs

ABSTRACT Chronic hepatitis B (CHB), chronic hepatitis C (CHC), and associated hepatocellular carcinoma (HCC) are characterized by cholesterol imbalance and dyslipidemia; however, the key regulatory drivers of these phenotypes are incompletely understood. Using gene expression microarrays and high-throughput sequencing of small RNAs, we performed integrative analysis of microRNA (miRNA) and gene expression in nonmalignant and matched cancer tissue samples from human subjects with CHB or CHC and HCC. We also carried out follow-up functional studies of specific miRNAs in a cell-based system. These studies led to four major findings. First, pathways affecting cholesterol homeostasis were among the most significantly overrepresented among genes dysregulated in chronic viral hepatitis and especially in tumor tissue. Second, for each disease state, specific miRNA signatures that included miRNAs not previously associated with chronic viral hepatitis, such as miR-1307 in CHC, were identified. Notably, a few miRNAs, including miR-27 and miR-224, were components of the miRNA signatures of all four disease states: CHB, CHC, CHB-associated HCC, and CHC-associated HCC. Third, using a statistical simulation method (miRHub) applied to the gene expression data, we identified candidate master miRNA regulators of pathways controlling cholesterol homeostasis in chronic viral hepatitis and HCC, including miR-21, miR-27, and miR-33. Last, we validated in human hepatoma cells that both miR-21 and miR-27 significantly repress cholesterol synthesis and that miR-27 does so in part through regulation of the gene that codes for the rate-limiting enzyme 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase (HMGCR). IMPORTANCE Hepatitis B virus (HBV) and hepatitis C virus (HCV) are phylogenetically unrelated hepatotropic viruses that persistently infect hundreds of millions of people world-wide, often leading to chronic liver disease and hepatocellular carcinoma (HCC). Chronic hepatitis B (CHB), chronic hepatitis C (CHC), and associated HCC often lead to cholesterol imbalance and dyslipidemia. However, the regulatory mechanisms underlying the dysregulation of lipid pathways in these disease states are incompletely understood. MicroRNAs (miRNAs) have emerged as critical modulators of lipid homeostasis. Here we use a blend of genomic, molecular, and biochemical strategies to identify key miRNAs that drive the lipid phenotypes of chronic viral hepatitis and HCC. These findings provide a panoramic view of the miRNA landscape in chronic viral hepatitis, which could contribute to the development of novel and more-effective miRNA-based therapeutic strategies. Hepatitis B virus (HBV) and hepatitis C virus (HCV) are phylogenetically unrelated hepatotropic viruses that persistently infect hundreds of millions of people world-wide, often leading to chronic liver disease and hepatocellular carcinoma (HCC). Chronic hepatitis B (CHB), chronic hepatitis C (CHC), and associated HCC often lead to cholesterol imbalance and dyslipidemia. However, the regulatory mechanisms underlying the dysregulation of lipid pathways in these disease states are incompletely understood. MicroRNAs (miRNAs) have emerged as critical modulators of lipid homeostasis. Here we use a blend of genomic, molecular, and biochemical strategies to identify key miRNAs that drive the lipid phenotypes of chronic viral hepatitis and HCC. These findings provide a panoramic view of the miRNA landscape in chronic viral hepatitis, which could contribute to the development of novel and more-effective miRNA-based therapeutic strategies.

tDRmapper: challenges and solutions to mapping, naming, and quantifying tRNA-derived RNAs from human small RNA-sequencing data.

BackgroundSmall RNA-sequencing has revealed the diversity and high abundance of small RNAs derived from tRNAs, referred to as tRNA-derived RNAs. However, at present, there is no standardized nomenclature and there are no methods for accurate annotation and quantification of these small RNAs. tRNA-derived RNAs have unique features that limit the utility of conventional alignment tools and quantification methods.ResultsWe describe here the challenges of mapping, naming, and quantifying tRNA-derived RNAs and present a novel method that addresses them, called tDRmapper. We then use tDRmapper to perform a comparative analysis of tRNA-derived RNA profiles across different human cell types and diseases. We found that (1) tRNA-derived RNA profiles can differ dramatically across different cell types and disease states, (2) that positions and types of chemical modifications of tRNA-derived RNAs vary by cell type and disease, and (3) that entirely different tRNA-derived RNA species can be produced from the same parental tRNA depending on the cell type.ConclusiontDRmappernot only provides a standardized nomenclature and quantification scheme, but also includes graphical visualization that facilitates the discovery of novel tRNA and tRNA-derived RNA biology.

Assembly-based inference of B-cell receptor repertoires from short read RNA sequencing data with V'DJer.

Motivation: B-cell receptor (BCR) repertoire profiling is an important tool for understanding the biology of diverse immunologic processes. Current methods for analyzing adaptive immune receptor repertoires depend upon PCR amplification of VDJ rearrangements followed by long read amplicon sequencing spanning the VDJ junctions. While this approach has proven to be effective, it is frequently not feasible due to cost or limited sample material. Additionally, there are many existing datasets where short-read RNA sequencing data are available but PCR amplified BCR data are not. Results: We present here V’DJer, an assembly-based method that reconstructs adaptive immune receptor repertoires from short-read RNA sequencing data. This method captures expressed BCR loci from a standard RNA-seq assay. We applied this method to 473 Melanoma samples from The Cancer Genome Atlas and demonstrate V’DJer’s ability to accurately reconstruct BCR repertoires from short read mRNA-seq data. Availability and Implementation: V’DJer is implemented in C/C ++, freely available for academic use and can be downloaded from Github: https://github.com/mozack/vdjer Contact: benjamin_vincent@med.unc.edu or parkerjs@email.unc.edu Supplementary information: Supplementary data are available at Bioinformatics online.

A Gene Expression Panel is Accurate for Diagnosis and Monitoring Treatment of Eosinophilic Esophagitis in Adults

OBJECTIVE: Eosinophilic esophagitis (EoE) can be difficult to diagnose. We aimed to evaluate whether a gene expression score could differentiate adult EoE cases from non‐EoE controls and to determine whether scores normalized after treatment for EoE. METHODS: We analyzed prospectively collected esophageal biopsies from EoE patients (diagnosed as per consensus guidelines and after a proton pump inhibitor trial) and non‐EoE controls. Gene expression for a previously constructed 94 gene panel was quantified for a single RNA‐later preserved biopsy. For diagnosis, a summary expression score and the area under the receiver operating characteristic curve (AUC) were calculated. For treatment response (defined as <15 eosinophils per high‐power field), pretreatment and posttreatment EoE samples were compared. RESULTS: For 91 EoE cases and 174 controls, gene scores for EoE cases were lower than non‐EoE controls (mean 198 vs. 420; P<0.001), with an AUC of 0.927. A score ≤263 yielded a positive predictive value=91%; a score ≥349 yielded a negative predictive value=90%; only 12% of subjects had an indeterminate score (264–348) by this classification scheme. For the 89 EoE cases with paired pretreatment and posttreatment samples, overall gene scores improved after treatment from 199 to 343 (P<0.001). This normalization was seen only in cases with histological response (202 vs. 425; P<0.001); scores were unchanged in non‐responders (189 vs. 226; P=0.25). CONCLUSIONS: A gene expression score has high diagnostic utility for distinguishing EoE patients from non‐EoE controls in adults and can be used in clinical algorithms. Because it is highly responsive to treatment, the test could be used to monitor disease status.