Ronghua Chen

Genome-wide survey of recurrent HBV integration in hepatocellular carcinoma

To survey hepatitis B virus (HBV) integration in liver cancer genomes, we conducted massively parallel sequencing of 81 HBV-positive and 7 HBV-negative hepatocellular carcinomas (HCCs) and adjacent normal tissues. We found that HBV integration is observed more frequently in the tumors (86.4%) than in adjacent liver tissues (30.7%). Copy-number variations (CNVs) were significantly increased at HBV breakpoint locations where chromosomal instability was likely induced. Approximately 40% of HBV breakpoints within the HBV genome were located within a 1,800-bp region where the viral enhancer, X gene and core gene are located. We also identified recurrent HBV integration events (in ≥4 HCCs) that were validated by RNA sequencing (RNA-seq) and Sanger sequencing at the known and putative cancer-related TERT, MLL4 and CCNE1 genes, which showed upregulated gene expression in tumor versus normal tissue. We also report evidence that suggests that the number of HBV integrations is associated with patient survival.

Molecular analysis of gastric cancer identifies subtypes associated with distinct clinical outcomes

Gastric cancer, a leading cause of cancer-related deaths, is a heterogeneous disease. We aim to establish clinically relevant molecular subtypes that would encompass this heterogeneity and provide useful clinical information. We use gene expression data to describe four molecular subtypes linked to distinct patterns of molecular alterations, disease progression and prognosis. The mesenchymal-like type includes diffuse-subtype tumors with the worst prognosis, the tendency to occur at an earlier age and the highest recurrence frequency (63%) of the four subtypes. Microsatellite-unstable tumors are hyper-mutated intestinal-subtype tumors occurring in the antrum; these have the best overall prognosis and the lowest frequency of recurrence (22%) of the four subtypes. The tumor protein 53 (TP53)-active and TP53-inactive types include patients with intermediate prognosis and recurrence rates (with respect to the other two subtypes), with the TP53-active group showing better prognosis. We describe key molecular alterations in each of the four subtypes using targeted sequencing and genome-wide copy number microarrays. We validate these subtypes in independent cohorts in order to provide a consistent and unified framework for further clinical and preclinical translational research.

Whole genome sequencing identifies recurrent mutations in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most deadly cancers worldwide and has no effective treatment, yet the molecular basis of hepatocarcinogenesis remains largely unknown. Here we report findings from a whole-genome sequencing (WGS) study of 88 matched HCC tumor/normal pairs, 81 of which are Hepatitis B virus (HBV) positive, seeking to identify genetically altered genes and pathways implicated in HBV-associated HCC. We find beta-catenin to be the most frequently mutated oncogene (15.9%) and TP53 the most frequently mutated tumor suppressor (35.2%). The Wnt/beta-catenin and JAK/STAT pathways, altered in 62.5% and 45.5% of cases, respectively, are likely to act as two major oncogenic drivers in HCC. This study also identifies several prevalent and potentially actionable mutations, including activating mutations of Janus kinase 1 (JAK1), in 9.1% of patients and provides a path toward therapeutic intervention of the disease.

Genome sequencing and comparison of two nonhuman primate animal models, the cynomolgus and Chinese rhesus macaques

The nonhuman primates most commonly used in medical research are from the genus Macaca. To better understand the genetic differences between these animal models, we present high-quality draft genome sequences from two macaque species, the cynomolgus/crab-eating macaque and the Chinese rhesus macaque. Comparison with the previously sequenced Indian rhesus macaque reveals that all three macaques maintain abundant genetic heterogeneity, including millions of single-nucleotide substitutions and many insertions, deletions and gross chromosomal rearrangements. By assessing genetic regions with reduced variability, we identify genes in each macaque species that may have experienced positive selection. Genetic divergence patterns suggest that the cynomolgus macaque genome has been shaped by introgression after hybridization with the Chinese rhesus macaque. Macaque genes display a high degree of sequence similarity with human disease gene orthologs and drug targets. However, we identify several putatively dysfunctional genetic differences between the three macaque species, which may explain functional differences between them previously observed in clinical studies.

Comprehensive Characterization of Oncogenic Drivers in Asian Lung Adenocarcinoma

Introduction: The incidence rate of lung adenocarcinoma (LUAD), the predominant histological subtype of lung cancer, is elevated in Asians, particularly in female nonsmokers. The mutation patterns in LUAD in Asians might be distinct from those in LUAD in whites. Methods: We profiled 271 resected LUAD tumors (mainly stage I) to characterize the genomic landscape of LUAD in Asians with a focus on female nonsmokers. Results: Mutations in EGFR, KRAS, erb‐b2 receptor tyrosine kinase 2 gene (ERBB2), and BRAF; gene fusions involving anaplastic lymphoma receptor tyrosine kinase gene (ALK), ROS1, and ret proto‐oncogene (RET); and Met Proto‐Oncogene Tyrosine Kinase (MET) exon 14 skipping were the major drivers in LUAD in Asians, exhibiting mutually exclusive and differing prevalence from those reported in studies of LUAD in non‐Asians. In addition, we identified a novel mutational signature of XNX (the mutated base N in the middle flanked by two identical bases at the 5′ and 3′ positions) that was overrepresented in LUAD tumors in nonsmokers and negatively correlated with the overall mutational frequency. Conclusions: In this cohort, approximately 85% of individuals have known driver mutations (EGFR 59.4%, KRAS 7.4%, ALK 7.4%, ERBB2 2.6%, ROS1 2.2%, RET 2.2%, MET 1.8%, BRAF 1.1%, and NRAS 0.4%). Seventy percent of smokers and 90% of nonsmokers had defined oncogenic drivers matching the U.S. Food and Drug Administration–approved targeted therapies.

Genome-wide identification of RNA editing in hepatocellular carcinoma.

We did whole-transcriptome sequencing and whole-genome sequencing on nine pairs of Hepatocellular carcinoma (HCC) tumors and matched adjacent tissues to identify RNA editing events. We identified mean 26,982 editing sites with mean 89.5% canonical A→G edits in each sample using an improved bioinformatics pipeline. The editing rate was significantly higher in tumors than adjacent normal tissues. Comparing the difference between tumor and normal tissues of each patient, we found 7 non-synonymous tissue specific editing events including 4 tumor-specific edits and 3 normal-specific edits in the coding region, as well as 292 edits varying in editing degree. The significant expression changes of 150 genes associated with RNA editing were found in tumors, with 3 of the 4 most significant genes being cancer related. Our results show that editing might be related to higher gene expression. These findings indicate that RNA editing modification may play an important role in the development of HCC.

Identification of important long non-coding RNAs and highly recurrent aberrant alternative splicing events in hepatocellular carcinoma through integrative analysis of multiple RNA-Seq datasets

Hepatocellular carcinoma (HCC) is an aggressive and deadly cancer. The molecular pathogenesis of the disease remains poorly understood. To better understand HCC biology and explore potential biomarkers and therapeutic targets, we investigated the whole transcriptome of HCC. Considering the genetic heterogeneity of HCC, four datasets from four studies consisting of 15 pairs of HCC and adjacent normal samples were analyzed. We observed that the number of lncRNAs expressed in each HCC sample was consistently greater than the adjacent normal sample. Moreover, 15 lncRNAs were identified expressed in five to seven HCC tissues but were not detected in any adjacent normal tissue. Differential expression analysis detected 35 up- and 80 down-regulated lncRNAs in HCC samples compared with adjacent normal samples. In addition, five differentially expressed lncRNAs were predicted to play a role in oxidation and reduction process. With regard to splicing alterations, we identified nine highly recurrent differential splicing events belonging to eight genes USO1, RPS24, CCDC50, THNSL2, NUMB, FN1 (two events), SLC39A14 and NR1I3. Of them, splicing alterations of SLC39A14 and NR1I3 were reported for the association with HCC for the first time. The splicing dysregulation in HCC may be influenced by three splicing factors ESRP2, CELF2 and SRSF5 which were significantly down-regulated in HCC samples. This study revealed uncharacterized aspects of HCC transcriptome and identified important lncRNAs and splicing isoforms with the potential to serve as biomarkers and therapeutic targets for the disease.

Systematic identification of cancer-related long noncoding RNAs and aberrant alternative splicing of quintuple-negative lung adenocarcinoma through RNA-Seq

OBJECTIVESnLung adenocarcinoma (LUAD) is a common subtype of non-small cell lung cancer prevalent in Asia. There is a dearth of understanding regarding the transcriptome landscape of LUAD without primary known driver mutations. In this study, LUAD samples without well-known driver mutations occurring in EGFR, KRAS, ALK, ROS1 or RET (quintuple-negative) were used for transcriptome study with a focus on long noncoding RNAs (lncRNAs), alternative splicing and gene fusions.nnnMATERIALS AND METHODSn24 pairs of LUAD and adjacent normal samples and 13 tumor-only samples derived from 37 quintuple-negative patients were used. Differentially expressed lncRNA transcripts were detected by paired t-test and were validated by qPCR. Functions of lncRNAs were predicted by co-expressed mRNAs. Aberrant splicing events in LUAD were identified using MISO. In addition, gene fusions were screened by SOAPfuse.nnnRESULTS AND CONCLUSIONnIn total, 90 and 153 up- or down-regulated lncRNA transcripts were detected in LUAD samples in comparison with the adjacent normal samples. The most significantly differentially expressed lncRNA transcript was ENST00000598996.1 (FENDRR) down-regulated in LUAD. By lncRNA-mRNA co-expression analysis, functions of 14 lncRNAs were predicted. The predicted functions included vasculature development, immune response, cell cycle and respiratory gaseous exchange. Furthermore, six co-expressed pairs of lncRNAs and their nearby protein coding genes were identified as associated with lung development. This study also identified two highly recurrent (22 in 24) differential exon skipping events occurring in MYH14 and ESYT2 with exon including isoforms of both genes up-regulated in isoform percentage in LUAD samples. On the other hand, two out of 24 LUAD samples possessed the driver mutation exon 14 skipping of MET. The transcriptional alterations of LUAD samples without well-known driver mutations identified in the study can be used as references for future research. The translational values of these transcriptional changes are also worthy of further investigation.

Species specific exome probes reveal new insights in positively selected genes in nonhuman primates

Nonhuman primates (NHP) are important biomedical animal models for the study of human disease. Of these, the most widely used models in biomedical research currently are from the genus Macaca. However, evolutionary genetic divergence between human and NHP species makes human-based probes inefficient for the capture of genomic regions of NHP for sequencing and study. Here we introduce a new method to resequence the exome of NHP species by a designed capture approach specifically targeted to the NHP, and demonstrate its superior performance on four NHP species or subspecies. Detailed investigation on biomedically relevant genes demonstrated superior capture by the new approach. We identified 28 genes that appeared to be pseudogenized and inactivated in macaque. Finally, we identified 187 genes showing strong evidence for positive selection across all branches of the primate phylogeny including many novel findings.

Abstract LB-229: Whole genome sequencing reveals genetic landscape of hepatocellular carcinoma.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DCnnHepatocellular carcinoma (HCC) is one of the most deadly cancers worldwide and has no effective treatment, yet the molecular basis of hepatocarcinogenesis remains largely unknown. Here we report findings from a whole genome sequencing (WGS) study of 88 matched HCC tumour/normal pairs, 81 of which are HBV positive, seeking to identify genetically altered genes and pathways implicated in HBV-associated HCC. We find β-catenin to be the most frequently mutated oncogene (15.9%) and TP53 the most frequently mutated tumour suppressor (35.2%). The Wnt/β-catenin pathway, altered in 62.5% of cases, is likely to act as the major oncogenic driver in HCC. TP53 alterations appear to cause increased levels of genomic arrangement and chromosomal instability. We identified chromothripsis in 5 HCC genomes (5.7%) recurrently affecting chromosomal arms 1q and 8q. We also identified recurrent HBV integration events at the known and putative cancer-related genes such as TERT, MLL4 and CCNE1, which showed upregulated gene expression in tumour versus normal tissue. The frequently altered genes and pathways in HCC reflect classical cancer hallmarks. This study identified several prevalent and actionable mutations that provide a path towards therapeutic intervention of the disease.nnCitation Format: Mao Mao, Hancheng Zheng, Zhengyan Kan, Jiangchun Xu, Xiao Liu, Shuyu Li, Thomas Barber, Zhuolin Gong, Huan Gao, Ke Hao, Melinda Willard, Robert Hauptschein, Paul Rejto, Julio Fernandez, Guan Wang, Qinghui Zhang, Bo Wang, Ronghua Chen, Jian Wang, Nikki Lee, Wei Zhou, Zhao Lin, Zhiyu Peng, Kang Yi, Shengpei Chen, Lin Li, Xiaomei Fan, Jie Yang, Rui Ye, Jia Ju, Kai Wang, Heather Estrella, Shibing Deng, Ping Wei, Ming Qiu, Isabella Wulur, Jiangang Liu, Mariam Ehsani, Chunsheng Zhang, Andrey Loboda, Wing Kin Sung, Amit Aggarwal, Ronnie Poon, Sheung Tat Fan, Jun Wang, James Hardwick, Christoph Reinhard, Hongyue Dai, Yingrui Li, John Luk. Whole genome sequencing reveals genetic landscape of hepatocellular carcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-229. doi:10.1158/1538-7445.AM2013-LB-229