At the cancer steering wheel: Defining key genomic drivers of liver cancer with next generation sequencing
Authors: Anuradha Budhu, Xin Wei Wang
Journal: Journal of Hepatology (2012)

COMMENTARY ON: Inactivating mutations of the chromatin remodeling gene ARID2 in hepatocellular carcinoma. Li M, Zhao H, Zhang X, Wood LD, Anders RA, Choti MA, Pawlik TM, Daniel HD, Kannangai R, Offerhaus GJ, Velculescu VE, Wang L, Zhou S, Vogelstein B, Hruban RH, Papadopoulos N, Cai J, Torbenson MS, Kinzler KW. Nat Genet. August 7 2011;43(9):828–9. doi:10.1038/ng.903. Copyright (2011). Abstract reprinted with permission from Macmillan Publishers Ltd. http://www.ncbi.nlm.nih.gov/pubmed/21822264 Abstract: Through exomic sequencing of 10 hepatitis C virus (HCV)-associated hepatocellular carcinomas (HCC) and subsequent evaluation of additional affected individuals, we discovered novel inactivating mutations of ARID2 in four major subtypes of HCC (HCV-associated HCC, hepatitis B virus (HBV)-associated HCC, alcohol-associated HCC and HCC with no known etiology). Notably, 18.2% of individuals with HCV-associated HCC in the United States and Europe harbored ARID2 inactivation mutations, suggesting that ARID2 is a tumor suppressor gene that is relatively commonly mutated in this tumor subtype. Cancer is thought to develop from certain genetic and epigenetic alterations that constitute 10 established and emerging hallmarks that enable tumor initiation and progression through tumor suppressor inactivation and oncogene activation [[1]Hanahan D. Weinberg R.A. Hallmarks of cancer: the next generation.Cell. 2011; 144: 646-674Abstract Full Text Full Text PDF PubMed Scopus (44228) Google Scholar]. Recently, advances in DNA sequencing technology, particularly next generation sequencing (NGS), have provided an outlet for a detailed and global perspective on the driving alterations in cancer through high resolution analysis of tumor genomes [2Su Z. Ning B. Fang H. Hong H. Perkins R. Tong W. et al.Next-generation sequencing and its applications in molecular diagnostics.Expert Rev Mol Diagn. 2011; 11: 333-343PubMed Google Scholar, 3Schweiger M.R. Kerick M. Timmermann B. Isau M. The power of NGS technologies to delineate the genome organization in cancer: from mutations to structural variations and epigenetic alterations.Cancer Metastasis Rev. 2011; 30: 199-210Crossref PubMed Scopus (59) Google Scholar]. Over the last few years, NGS has allowed for detailed analysis of DNA structural changes, chromosomal translocations, duplications and deletions in cancer driver or susceptibility genes in over 16 tumor types [[4]Sastre L. New D.N.A. Sequencing technologies open a promising era for cancer research and treatment.Clin Transl Oncol. 2011; 13: 301-306Crossref PubMed Scopus (14) Google Scholar]. Over one-third of these studies rely on sequencing select regions of the genome, such as the exome, to reduce sequencing time as well as cost and to focus on relevant protein-coding regions. While the benefits of NGS technologies include increased throughput and ever-reducing cost, several challenges remain in translating sequencing information to clinical practice. NGS studies utilize various methods to define true mutations, since a systematic general strategy to adequately mine this massive volume of data is not in place. For example, among the three published NGS studies for hepatocellular carcinoma (HCC), a lethal form of liver cancer that impacts global health, the authors use different methods to capture exons, various bioinformatic tools or algorithms to align sequences to reference genomes and diverse criteria for selecting mutations based on coverage, reads and frequency [5Tao Y. Ruan J. Yeh S.H. Lu X. Wang Y. Zhai W. et al.Rapid growth of a hepatocellular carcinoma and the driving mutations revealed by cell-population genetic analysis of whole-genome data.Proc Natl Acad Sci USA. 2011; 108: 12042-12047Crossref PubMed Scopus (112) Google Scholar, 6Totoki Y. Tatsuno K. Yamamoto S. Arai Y. Hosoda F. Ishikawa S. et al.High-resolution characterization of a hepatocellular carcinoma genome.Nat Genet. 2011; 43: 464-469Crossref PubMed Scopus (241) Google Scholar, 7Li M. Zhao H. Zhang X. Wood L.D. Anders R.A. Choti M.A. et al.Inactivating mutations of the chromatin remodeling gene ARID2 in hepatocellular carcinoma.Nat Genet. 2011; 43: 828-829Crossref PubMed Scopus (332) Google Scholar]. Thus, the mutations that pass these selection criteria differ between studies. Moreover, mutation calling by NGS is simply not perfect, with false positives generated from machine errors or through the difficulty in differentiating somatic mutations from more frequently occurring germline inherited single nucleotide polymorphisms. Since the somatic mutation rate per gene per cell is approximately 10−7, the probability of single or multiple mutations occurring within one cell is highly unlikely within its lifespan [[8]Nowak M.A. Komarova N.L. Sengupta A. Jallepalli P.V. Shih I. Vogelstein B. et al.The role of chromosomal instability in tumor initiation.Proc Natl Acad Sci USA. 2002; 99: 16226-16231Crossref PubMed Scopus (391) Google Scholar]. Thus, identification of true mutations requires improved understanding of cancer biology coupled with enhanced bioinformatics and validation using traditional Sanger DNA sequencing methods. Tumor heterogeneity also influences the value of NGS data. Distinguishing whether multiple mutation events occur within each tumor cell (clonal evolution) vs. compilations of mutation events contributed by different cells within a tumor remains a challenge. NGS may play a significant role in defining such intra-heterogeneous and inter-heterogeneous tumor types. In order to begin distinguishing these differences, one must more closely define the samples to be analyzed. Of the three published HCC-related NGS studies, two have only examined a single individual for mutation calling and failed to confirm these putative mutants in additional samples [5Tao Y. Ruan J. Yeh S.H. Lu X. Wang Y. Zhai W. et al.Rapid growth of a hepatocellular carcinoma and the driving mutations revealed by cell-population genetic analysis of whole-genome data.Proc Natl Acad Sci USA. 2011; 108: 12042-12047Crossref PubMed Scopus (112) Google Scholar, 6Totoki Y. Tatsuno K. Yamamoto S. Arai Y. Hosoda F. Ishikawa S. et al.High-resolution characterization of a hepatocellular carcinoma genome.Nat Genet. 2011; 43: 464-469Crossref PubMed Scopus (241) Google Scholar]. Hence, the importance of these mutations is not clear in the context of the general HCC population due to the lack of multiple discovery and validation samples. In their recent work, Li et al., have conducted an elegant study to address this issue through an exomic sequencing screen of ten hepatitis C virus (HCV) positive HCC patients, followed by validation in 129 patients [[7]Li M. Zhao H. Zhang X. Wood L.D. Anders R.A. Choti M.A. et al.Inactivating mutations of the chromatin remodeling gene ARID2 in hepatocellular carcinoma.Nat Genet. 2011; 43: 828-829Crossref PubMed Scopus (332) Google Scholar]. The authors found novel inactivating ARID2 mutations in HCC and infer that ARID2 is a HCC tumor suppressor, since 18.2% of discovery samples harbored inactivating mutations of this gene. Chromatograms of ARID2 mutations in additional cases however, show a heterozygous nature which may result from intra-tumor heterogeneity or normal cell contamination, whose origination and interpretation require further exploration. An examination of the contribution of CAMSAP1L and EPS8L1 mutants would be germane since these genes also met the authors’ stringent criteria for calling tumor mutations. Furthermore, the DMXL1 and NLRP1 mutations found in the discovery cases did not validate in the 23 additional HCV–HCC cases and thus, may not be global driving mutations for this etiological subset of HCC, which reinforces the necessity of data validation and cohort design in NGS studies. By constructing a study to consider mutations consistently occurring in several individuals from multiple centers, the authors provide noteworthy data on common mutations that impact a disease on a macro level, rather than mutants that exist in a single individual, which may have more nominal effects. We congratulate the authors on effectively managing this complex and timely study addressing the mutations which drive this aggressive disease. Although the findings from Li’s study are important to the liver cancer field and to the cancer NGS efforts as a whole, an additional factor that must be considered in such studies is patient diversity. For HCC in particular, one must consider that HCC is a more aggressive disease in males vs. females, has diverse racial/ethnic incidence rates with significant bearing in Asians and has several underlying risk factors including viral hepatitis B and C, alcoholic disease and obesity. While the authors have performed some initial analyses of these potential confounding factors, well-defined studies constructed with homogeneous populations must be developed to address their impact on tumor mutations (Fig. 1). Follow-up studies are also necessary to determine the functional consequence of these mutations. Our ability to identify the mutations which drive tumorigenesis and progression has recently been expanded by the growing field of high-throughput sequencing. While recent endeavors, such as those described in Li et al., have shown the power of NGS technology to accelerate our understanding of cancer on the genomic scale, we are only at the tip of the iceberg in terms of our capacity to interpret the extensive resulting data. As ARID2 is a member of the chromatin remodeling complex, it is important to note that NGS technology, via whole-genome bisulfate sequencing methods, is extremely useful in defining the epigenetic alterations, such as DNA methylation and histone modifications, that occur in cancer [[9]Meaburn E, Schulz R. Next generation sequencing in epigenetics: insights and challenges. Semin Cell Dev Biol 2011, in press.Google Scholar]. Ever-improving NGS-based protocols are being employed to catalog the dynamics of the epigenome and its impact on cancer initiation and progression. In the coming years, NGS and large-scale cancer genome studies such as those launched by the International Cancer Genome Consortium [[10]Hudson T.J. Anderson W. Artez A. Barker A.D. Bell C. Bernabe R.R. et al.International network of cancer genome projects.Nature. 2010; 464: 993-998Crossref PubMed Scopus (1726) Google Scholar], along with novel technologies and informatics will undoubtedly allow us to better translate research findings as clinical tools to benefit cancer patients. The authors declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.