Power play: Scoring our goals for liver cancer with better GWAS study design
Authors: Anuradha Budhu, Xin Wei Wang
Journal: Journal of Hepatology (2010)

COMMENTARY ON: Genome-wide association study identifies 1p36.22 as a new susceptibility locus for hepatocellular carcinoma in chronic hepatitis B virus carriers. Zhang H, Zhai Y, Hu Z, Wu C, Qian J, Jia W, Ma F, Huang W, Yu L, Yue W, Wang Z, Li P, Zhang Y, Liang R, Wei Z, Cui Y, Xie W, Cai M, Yu X, Yuan Y, Xia X, Zhang X, Yang H, Qiu W, Yang J, Gong F, Chen M, Shen H, Lin D, Zeng YX, He F, Zhou G. Nature Genetics Sep 2010;42(9):755–8. Abstract reprinted by permission from Macmillan Publishers Ltd. Copyright (2010). http://www.ncbi.nlm.nih.gov/pubmed/20676096 Abstract: To identify susceptibility variants for hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC), we conducted a genome-wide association study by genotyping 440,794 SNPs in 355 chronic HBV carriers with HCC and 360 chronic HBV carriers without HCC, all of Chinese ancestry. We identified one intronic SNP (rs17401966) in KIF1B on chromosome 1p36.22 that was highly associated with HBV-related HCC and confirmed this association in five additional independent samples, consisting of 1962 individuals with HCC, 1430 control subjects, and 159 family trios. Across the six studies, the association with rs17401966 was highly statistically significant (joint odds ratio=0.61, P=1.7×10(−18)). In addition to KIF1B, the association region tagged two other plausible causative genes, UBE4B and PGD. Our findings provide evidence that the 1p36.22 locus confers susceptibility to HBV-related HCC, and suggest that KIF1B-, UBE4B-, or PGD-related pathways might be involved in the pathogenesis of this malignancy. Hepatocellular carcinoma (HCC) is the third most deadly and fifth most common cancer worldwide. The majority of HCC cases occur in China. The etiology of liver cancer is known with risk factors including hepatitis B virus (HBV), hepatitis C virus (HCV), cirrhosis, gender, diabetes, obesity, alcohol, and smoking, all which contribute to the development of chronic liver disease (CLD). While over 80% of HCC occurs in the background of inflammation, mainly caused by viral hepatitis [[1]Roessler S. Budhu A. Wang X.W. Future of molecular profiling of human hepatocellular carcinoma.Future Oncol. 2007; 3: 429-439Crossref PubMed Scopus (22) Google Scholar] only a fraction of patients with CLD develop HCC, suggesting that the individual genetic background contributes to HCC risk. Patients with HCC currently have a very poor outcome, partly due to the advanced stages of disease at clinical presentation and to its heterogeneity. HCC prognosis is also complicated by both the tumor stage and the condition of the liver when compromised by hepatitis virus, making it difficult to provide clinical recommendations. At this time, only 10–20% of HCC patients are eligible for potentially curative therapies, such as resection and liver transplantation. However, many patients have a propensity to develop post-surgical recurrence [[2]Zhou X.D. Tang Z.Y. Yang B.H. Lin Z.Y. Ma Z.C. Ye S.L. et al.Experience of 1000 patients who underwent hepatectomy for small hepatocellular carcinoma.Cancer. 2001; 91: 1479-1486Crossref PubMed Scopus (261) Google Scholar]. Thus, the identification of genetic susceptibility loci and the development of new biomarkers and stratification programs based on these loci are needed to refine diagnosis and improve patient outcome. There are a few studies that have utilized a candidate approach to select promising genes carrying single nucleotide polymorphisms (SNP) and attempted to link these genetic components to HCC susceptibility and/or prognosis [3Kim Y.J. Lee H.S. Single nucleotide polymorphisms associated with hepatocellular carcinoma in patients with chronic hepatitis B virus infection.Intervirology. 2005; 48: 10-15Crossref PubMed Scopus (34) Google Scholar, 4Wu L. Xu X. Shen J. Xie H. Yu S. Liang T. et al.MDR1 gene polymorphisms and risk of recurrence in patients with hepatocellular carcinoma after liver transplantation.J Surg Oncol. 2007; 96: 62-68Crossref PubMed Scopus (56) Google Scholar]. These types of studies are limited due to their cohort-specific nature and to a design which produces high false positive data requiring additional independent validation. Recently, genome-wide association studies (GWAS) have been utilized to perform large-scale interrogation of genetic variants in cancer. While these studies have identified susceptibility loci for common cancers types, such as breast and prostate, a GWAS for liver cancer has been lacking. In their recent work, Zhang and colleagues have conducted the first liver GWAS for HCC in chronic HBV carriers of Chinese ancestry [[5]Zhang H. Genome-wide association study identifies 1p36.22 as a new susceptibility locus for hepatocellular carcinoma in chronic hepatitis B virus carriers.Nat Genet. 2010; 42: 755-758Crossref PubMed Scopus (295) Google Scholar]. Using the Affymetrix Genome-Wide Human SNP Array 5.0 to assess 355 HBV HCC cases and 360 HBV controls, they identified rs17401966, as a new susceptibility SNP, located on chromosome 1p36.22, for HBV-related HCC. As described in their manuscript, the association of this intronic SNP with HBV-related HCC was confirmed in five additional replication cohorts (joint odds ratio = 0.61, p= 1.7 × 10−18). This study is timely and important to the field of HCC research. It provides a good example of mobilizing resources and bringing clinicians, basic researchers, and statisticians together towards a collaborative effort. The authors are congratulated on effectively managing a complicated study and addressing this difficult disease with a global health impact. Although the findings from this study are of importance to the field of liver cancer and to the cancer GWAS efforts as a whole, the initial study by Zhang et al. is underpowered, and, given this fact, remarkably produced an SNP of interest. However, rs17401966 only had a statistical level of 10−6 in the initial GWAS scan which, as noted in the literature and by the authors, is below what is considered to be genome-wide significance (p<10−7), a cutoff level which takes multiple testing into account. Therefore, the choice to move forward with replication of this SNP in additional cohorts is debatable and worrisome. With this in mind, this SNP did not reach significance in unadjusted conditions in any of the five case-control studies or family trios used in the follow-up studies. It is not surprising therefore, that associations were not found between the SNPs identified in this HCC study and other SNPs of interest found in other GWAS. The seemingly 'cherry-picking' approach of selecting this SNP raises the question of whether this study should be considered a GWAS approach since the selection of this SNP was not based on a global unbiased rule. In addition, based on the results shown in Table 2 of the Zhang manuscript, rs17401966 was the third ranking SNP in the initial scan and it would be interesting to see how the top two SNPs fared in the replication and pooled studies. GWAS-based statistical significance of rs17401966 was met upon pooling the data of these cohorts; however, the pooled cohorts were recruited from different regions of China with differing eligibility, recruitment, and enrollment criteria and represent un-matched sample sets. It would therefore be of interest to determine whether ethnic and clinical parameter differences are represented within the pooled dataset. This also becomes an issue since pooled samples were used in stratification analyses based on age and gender. It is also unclear whether the multiple cohorts used in this study represent a selected population of cases since the majority of cases were hospital-based. The authors also genotyped rs17401966 in two control populations, one of which contained patients with no HBV and the other where HBV infection status was unknown and found a similar frequency of the rs17401966[G] allele between these control groups and chronic HBV carriers. However, it is unclear, whether this analysis was performed with the HBV carriers from the initial scan or from the pooled data. Regardless, an analysis of odds ratio between the population controls and the HBV carriers or HCC cases was not presented in this study and would shed some light on the relevance of HBV infection to their findings. While the findings of this HCC GWAS are of interest to the field, several factors noted above impede the interpretation of the results. Therefore, we recommend the following in future liver GWAS: (1) An appropriate sample size is needed to ensure sufficient power to identify significant GWAS SNPs. (2) Since allele frequency can differ among subgroups and populations, a concerted effort should be made to design studies in uniform ethnic groups. (3) Since each of the several etiologies that underlie HCC can individually contribute to disease onset and progression, GWAS should be constructed to specifically address each etiological type. Thus, we advocate that amply powered GWAS should be conducted in homogeneous populations that are well-matched and address the effect of a single variable on the association of a SNP with disease incidence and/or outcome. Our ability to assess liver cancer and, importantly, to identify new biomarkers for diagnosis, prognosis, and prediction has recently been expanded by the growing field of knowledge based on high-throughput genomics. While many cancer types have also been interrogated for genetic alterations by candidate-gene studies and GWAS, the liver cancer arena has lacked much of this information. While recent endeavors, such as those described in Zhang et al., have raised the bar, much effort is still needed to ensure that GWAS are well-designed and well-powered to achieve robust results which can be applied to improve the care of patients suffering from this disease. The authors who have contributed to this work declare that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.