A Meta-Analysis of Array-CGH Studies Implicates
Antiviral Immunity Pathways in the Development of
Xu Guo1., Yanna Ba2., Xi Ma1, Jiaze An3, Yukui Shang1, Qichao Huang1, Hushan Yang4, Zhinan Chen1*,
1State Key Laboratory of Cancer Biology, Department of Cell Biology, Cell Engineering Research Center, The Fourth Military Medical University, Xi’an, People’s Republic of
China, 2Department of Clinical Immunology, Xijing Hospital, The Fourth Military Medical University, Xi’an, People’s Republic of China, 3Department of Hepatobiliary
Surgery, Xijing Hospital, The Fourth Military Medical University, Xi’an, People’s Republic of China, 4Division of Population Science, Department of Medical Oncology,
Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania, United States of America
Background: The development and progression of hepatocellular carcinoma (HCC) is significantly correlated to the
accumulation of genomic alterations. Array-based comparative genomic hybridization (array CGH) has been applied to a
wide range of tumors including HCCs for the genome-wide high resolution screening of DNA copy number changes.
However, the relevant chromosomal variations that play a central role in the development of HCC still are not fully
Methods: In present study, in order to further characterize the copy number alterations (CNAs) important to HCC
development, we conducted a meta-analysis of four published independent array-CGH datasets including total 159
Results: Eighty five significant gains (frequency $25%) were mostly mapped to five broad chromosomal regions including
1q, 6p, 8q, 17q and 20p, as well as two narrow regions 5p15.33 and 9q34.2-34.3. Eighty eight significant losses (frequency
$25%) were most frequently present in 4q, 6q, 8p, 9p, 13q, 14q, 16q, and 17p. Significant correlations existed between
chromosomal aberrations either located on the same chromosome or the different chromosomes. HCCs with different
etiologies largely exhibited surprisingly similar profiles of chromosomal aberrations with only a few exceptions.
Furthermore, the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that the genes affected by
these chromosomal aberrations were significantly enriched in 31 canonical pathways with the highest enrichment observed
for antiviral immunity pathways.
Conclusions: Taken together, our findings provide novel and important clues for the implications of antiviral immunity-
related gene pathways in the pathogenesis and progression of HCC.
Citation: Guo X, Ba Y, Ma X, An J, Shang Y, et al. (2011) A Meta-Analysis of Array-CGH Studies Implicates Antiviral Immunity Pathways in the Development of
Hepatocellular Carcinoma. PLoS ONE 6(12): e28404. doi:10.1371/journal.pone.0028404
Editor: Shree Ram Singh, National Cancer Institute, United States of America
Received October 19, 2011; Accepted November 7, 2011; Published December, 2011
Copyright: ? 2011 Guo et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grant 30872927 from the National Natural Science Foundation of China and grant 2009CB521704 from the National Basic
Research Program. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org (JX); email@example.com (ZC)
. These authors contributed equally to this work.
The development and progression of hepatocellular carcinoma
(HCC) is significantly correlated to the accumulation of genomic
alterations . Therefore, it is important to have a clear landscape
of the genomic aberrations that occur during the multistep process
of hepatocarcinogenesis. Previous studies have used high-resolution
molecular karyotyping analyses to provide a comprehensive catalog
of structural aberrations of the whole chromosomes in HCC .
However, this method is highly specialized and time-consuming. As
a consequence, only a very limited number of HCC cases have been
evaluated in these studies. Moreover, the modest resolution of the
karyotyping analysis made it difficult to fully define the overall
genomic profiles of HCC in a more accurate manner. Comparative
genomic hybridization (CGH) has been developedin recent years to
monitor the DNA copy number changes at a global level .
However, traditional CGH techniques still have the limitation of
modest resolution (approximately 2 Mb for amplifications and 10–
20 Mb for deletions) and thus could not detect changes in smaller
chromosomal regions . In comparison, array-based CGH (array
CGH) is a newly developed technology that allows for high-
throughput and high-resolution (at 1 Mb) screening of genome-
wide DNA copy number changes (either amplifications or deletions)
at the gene level . Array CGH combines fluorescence techniques
PLoS ONE | www.plosone.org1 December 2011 | Volume 6 | Issue 12 | e28404
with the microarray platform that allows for the comparison of
DNA content in two differentially labeled genomes: a test genome
(patient)and a referencegenome(control).Themicroarray platform
also allows for the simultaneous scanning of thousands of individual
DNA sequences from the whole genome, and provides high-
resolution data on the locations of identified aberrations in a single
experiment. To date, array-CGH has been applied to a wide range
of solid tumors, including liver, breast, gastric, kidney and bladder
cancers [6,7,8,9,10]. Recently, another technology platform based
on single nucleotide polymorphism (SNP) array has been developed
to determine the copy number abnormalities of genomic DNA at
sub-kilobase resolution [11,12]. Except for an advantage of high
resolution, this platform also has a limitation of high signal-to-noise
ratio which is hard to improve.
Many investigators have made varying attempts to search for
genes implicated in hepatocarcinogenesis. Screening for chromo-
somal regions with frequent gains and losses is one of the first steps
toward the identification of genes. Using the traditional and array-
CGH, frequent DNA copy number gains at chromosomes 1q, 8q
and 20q, and frequent DNA copy number losses at 1p, 4q, 8p,
13q, 16q and 17p have been identified in HCC samples
[6,14,15,16,17,18,19]. Some of these regions contain known
candidate oncogenes or tumor suppressor genes, such as
ZNF217 (20q13) , TP53 (17p13), RB1 (13q14) and cyclin
D1 (11q13) . However, it is believed that the currently
identified genes represented only a small percentage of causal
elements in hepatocarcinogenesis and the vast majority of genes
with chromosomal aberrations that may play a central role in
HCC development are still unknown.
Meta-analysis is a systematic and quantitative synthesis of prior
evidence . It offers the opportunity to critically evaluate and
statistically combine the results of comparable studies or trials in
order to achieve more robust and reliable results as well as identify
novel findings that are not apparent in individual studies. In
previous reports, a meta-analysis of CGH data comprising of 785
HCCs has been carried out and identified significant correlations of
chromosomal deletions on 4q, 13q, and 16q with hepatitis B virus
(HBV) etiology . Recently, using the array-CGH technology,
several different studies have generated a wealth of data on more
than 100 analyzed HCC samples that await a more comprehensive
interpretation [16,25,26,27]. The aim of this study was to identify
potential genes and pathways important to HCC by utilizing the
available data from published array CGH studies of human HCC.
Materials and Methods
Data collection of array CGH studies in HCC
Datasets for HCC array CGH studies were identified from
public resources including the supplementary files of published
papers, NCBI Gene Expression Omnibus (GEO, http://www.
ncbi.nlm.nih.gov/geo), ArrayExpress (http://www.ebi.ac.uk) da-
tabase using hepatocellular carcinoma and array-based compar-
ative genomic hybridization as keywords. Datasets from studies
using HCC cell lines were excluded. We identified four datasets
with complete original data that are publicly available, including
two from the supplementary files of published HCC array CGH
studies and two from the GEO database (GSE8351 and
GSE22635) [16,25,26,27]. The detailed information was listed in
Table 1. Three of the four studies used BAC clone as the
hybridization probe while one used synthetic oligonucleotides as
the hybridization probe. A total of 159 HCC tissue samples in
these four datasets were collected, including 54 samples with HBV
infection, 57 with hepatitis C virus (HCV) infection, 6 with the
infections of both HBV and HCV, 3 with positive hepatitis B virus
X protein (HBx), and 39 samples without viral infection.
Data pre-processing for the integration across different
Because the four array CGH datasets in this study were
generated using different types of technical platforms that contain
different numbers of probes at varied spacing and resolution, they
cannot be directly compared and combined. To transform the
datasets from different platforms into a common format for the
purpose of meta-analysis, we pre-processed the original dataset of
each study based on a procedure previously described with minor
modification [24,28]. The detailed procedure used in this study is
First step: reconciliation of genome mapping data
In the original datasets, chromosomal positions of
BAC and oligo probes were assigned based on the different
versions of human genome assembly, such as hg15, hg17 and
hg18. Therefore, we used the annotation database of UCSC
human genome (hg19/GRCh37) to re-assign the start and end
chromosomal positions for all BAC and oligo probes from the four
datasets. The unmappable probes were excluded from further
Second step: assignment of chromosomal positioning
Because the copy number alterations (CNAs) of
chromosomal segments were detected on different scales by
different probes in the four datasets, it was difficult to directly
compare and integrate these datasets. To resolve this issue, we
assigned a set of chromosomal positioning anchors that were
composed of the start and end chromosomal positions of all the
probes used in the four datasets. The log2-transformed DNA copy
number ratio for each anchor was then determined for all the
samples from different datasets based on the following principles.
Table 1. Information of 4 collected public datasets (n=159).
1Mohini A.Patil et al.(2005)BAC44343——7
2Yasuyo Chochi et al.(2009)BAC426*34*2*——
3Christof Schlaeger et al.(2008)BAC6311144331
4Kazuya Taniguchi et al.(2010)Oligo1036——1
*Information of virus infection for individual samples is unavailable. HBV, hepatitis B virus; HCV, hepatitis C virus; HBx, hepatitis B virus X protein.
Array CGH Meta-Analysis in HCC
PLoS ONE | www.plosone.org2 December 2011 | Volume 6 | Issue 12 | e28404
and Jab1 as a potential target for 8q gain in hepatocellular carcinoma.
Carcinogenesis 26: 2050–2057.
26. Kakar S, Chen X, Ho C, Burgart LJ, Adeyi O, et al. (2009) Chromosomal
abnormalities determined by comparative genomic hybridization are helpful in
the diagnosis of atypical hepatocellular neoplasms. Histopathology 55: 197–205.
27. Schlaeger C, Longerich T, Schiller C, Bewerunge P, Mehrabi A, et al. (2008)
Etiology-dependent molecular mechanisms in human hepatocarcinogenesis.
Hepatology 47: 511–520.
28. Zhang NR, Senbabaoglu Y, Li JZ (2010) Joint estimation of DNA copy number
from multiple platforms. Bioinformatics 26: 153–160.
29. Chari R, Lockwood WW, Lam WL (2006) Computational methods for the
analysis of array comparative genomic hybridization. Cancer Inform 2: 48–58.
30. Kanehisa M, Goto S (2000) KEGG: kyoto encyclopedia of genes and genomes.
Nucleic Acids Res 28: 27–30.
31. Kaposi-Novak P, Libbrecht L, Woo HG, Lee YH, Sears NC, et al. (2009)
Central role of c-Myc during malignant conversion in human hepatocarcino-
genesis. Cancer Res 69: 2775–2782.
32. Yamamoto Y, Chochi Y, Matsuyama H, Eguchi S, Kawauchi S, et al. (2007)
Gain of 5p15.33 is associated with progression of bladder cancer. Oncology 72:
33. Wang T, Chen YH, Hong H, Zeng Y, Zhang J, et al. (2009) Increased
nucleotide polymorphic changes in the 59-untranslated region of delta-catenin
(CTNND2) gene in prostate cancer. Oncogene 28: 555–564.
34. Kang JU, Koo SH, Kwon KC, Park JW (2010) Frequent silence of chromosome
9p, homozygous DOCK8, DMRT1 and DMRT3 deletion at 9p24.3 in
squamous cell carcinoma of the lung. Int J Oncol 37: 327–335.
35. Yeh SH, Wu DC, Tsai CY, Kuo TJ, Yu WC, et al. (2006) Genetic
characterization of fas-associated phosphatase-1 as a putative tumor suppressor
gene on chromosome 4q21.3 in hepatocellular carcinoma. Clin Cancer Res 12:
36. Pehlivan D, Gunduz E, Gunduz M, Nagatsuka H, Beder LB, et al. (2008) Loss of
heterozygosity at chromosome 14q is associated with poor prognosis in head and
neck squamous cell carcinomas. J Cancer Res Clin Oncol 134: 1267–1276.
37. Zondervan PE, Wink J, Alers JC, JN IJ, Schalm SW, et al. (2000) Molecular
cytogenetic evaluation of virus-associated and non-viral hepatocellular carcino-
ma: analysis of 26 carcinomas and 12 concurrent dysplasias. J Pathol 192:
38. Villanueva A, Chiang DY, Newell P, Peix J, Thung S, et al. (2008) Pivotal role of
mTOR signaling in hepatocellular carcinoma. Gastroenterology 135:
1972–1983, 1983 e1971-1911.
39. Zaret KS, Grompe M (2008) Generation and regeneration of cells of the liver
and pancreas. Science 322: 1490–1494.
40. Thompson MD, Monga SP (2007) WNT/beta-catenin signaling in liver health
and disease. Hepatology 45: 1298–1305.
41. Sicklick JK, Li YX, Jayaraman A, Kannangai R, Qi Y, et al. (2006)
Dysregulation of the Hedgehog pathway in human hepatocarcinogenesis.
Carcinogenesis 27: 748–757.
42. Tada M, Kanai F, Tanaka Y, Tateishi K, Ohta M, et al. (2008) Down-
regulation of hedgehog-interacting protein through genetic and epigenetic
alterations in human hepatocellular carcinoma. Clin Cancer Res 14:
43. Guidotti LG, Chisari FV (2001) Noncytolytic control of viral infections by the
innate and adaptive immune response. Annu Rev Immunol 19: 65–91.
44. Kubler K, Arndt PF, Wardelmann E, Landwehr C, Krebs D, et al. (2008)
Genetic alterations of HLA-class II in ovarian cancer. Int J Cancer 123:
45. Chung MY, Wu JC, Chau GY, Lui WY, Tsay SH, et al. (2000) Preferentially
deleted chromosome region 9p21 in large hepatocellular carcinomas. Int J Mol
Med 5: 521–524.
46. Cai T, Nesi G, Dal Canto M, Tinacci G, Mondaini N, et al. (2010) Loss of
heterozygosis on IFN-alpha locus is a prognostic indicator of bacillus Calmette-
Guerin response for nonmuscle invasive bladder cancer. J Urol 183: 1738–1743.
47. Roman E, Meza-Zepeda LA, Kresse SH, Myklebost O, Vasstrand EN, et al.
(2008) Chromosomal aberrations in head and neck squamous cell carcinomas in
Norwegian and Sudanese populations by array comparative genomic hybrid-
ization. Oncol Rep 20: 825–843.
Array CGH Meta-Analysis in HCC
PLoS ONE | www.plosone.org9 December 2011 | Volume 6 | Issue 12 | e28404