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Molecular Oncology, Markers, Clinical Correlates |
Departments of Clinical Oncology [N. W., E. P., L. F. F., Z. S., V. W., P. J. J.] and Surgery [P. L., J. W-Y. L.], Sir Y. K. Pao Centre for Cancer, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong Special Administrative Region, China; Department of Pathology One, Kobe University School of Medicine, Kusunoki-cho, Kobe 650, Japan [W. W., Y. H.]; Department of Pathology, Johns Hopkins Hospital, Baltimore, Maryland 21211 [E. P.]; and Department of Pathology, Zhong Shan Hospital, Shanghai 200032, China [S. Y.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Initial data suggesting that there may, indeed, be specific genetic
consequences of particular etiological agents came from China and
Southern Africa. Circumstantial evidence was provided for a specific
AGGArg
AGTSer mutation in
codon 249 of the p53 tumor suppressor gene in HCC associated
with aflatoxin exposure (7, 8, 9)
. Others, however, have
emphasized that it is premature to regard any p53 mutations
as an established marker for aflatoxin exposure, particularly because
the association was only inferred on the basis of the reported high
levels of aflatoxin in the areas where the study was carried out,
rather than any direct assessment of aflatoxin exposure. Studies from
Japan and the United States (10
, 11)
, where aflatoxin
exposure is negligible, have, however, consistent with the
above-mentioned hypothesis, shown a very low percentage of codon 249
mutation in the liver.
We have attempted to expand this field of research by applying CGH (12) , a technique that permits a genome-wide chromosomal survey without either requiring cell culture (which is difficult with HCC tissue) or any prior knowledge of the aberrations involved to HCC associated with different etiological factors. In designing this study, we collected HCC specimens from carriers of the HBV (from two areas of China with different levels of aflatoxin exposure), carriers of the HCV (from Japan, where HCV infection is common and aflatoxin exposure low), and the United States. Because of the previously cited evidence that p53 mutations are a marker for aflatoxin exposure and that aflatoxin exposure may be one reason for any genetic aberrations detected by CGH, we have also examined, and sequenced, the entire p53 gene in all samples studied.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The diagnosis of HCC, with a tumor
cell content of >70%, was confirmed by histological examination. More
than 75% of the cases in each geographic location had underlying
chronic liver disease.
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CGH Analysis.
CGH experiments were carried out according to the method of Kallioniemi
et al. (14)
with modifications as described by
Chan et al. (15)
. Briefly, tumor and normal
reference DNA labeled with biotin-16-dUTP (Boehringer Mannheim,
Mannheim, Germany) and dig-11-dUTP (Boehringer Mannheim), respectively,
by nick translation were competitively cohybridized onto metaphase
preparations. After 2 days of hybridization at 37°C, biotin-labeled
signals were detected through avidin-conjugated FITC antibodies (Sigma
Chemical Co., St. Louis, MO), and dig-labeled probes were detected by
antibodies conjugated with TRITC (Sigma). The preparations were
counterstained with DAPI. Hybridized metaphases were captured with a
cooled CCD camera mounted on a Leitz DM RB (Leica, Wetzlar, Germany)
fluorescence microscope. Three band pass filter (DAPI, FITC, and TRITC)
sets arranged in an automated filter-wheel were used for image
acquisition. CGH software version 3.1 on Cytovision (Applied Imaging
Ltd., Sunderland, United Kingdom) was used for digital image analysis
of fluorescence intensity. Chromosome identification was performed on
the reverse DAPI banding images. The average ratio profiles were
calculated based on the analysis of 8–12 selected metaphases.
Thresholds for gains and losses were defined as the theoretical value
of 1.25 and 0.75, respectively. High-level gain of regional or whole
chromosome was considered to be present when ratios exceeded 1.5.
p53 Mutational Analysis by PCR-SSCP and Direct Sequencing.
One hundred ng of DNA were subjected to PCR in a mixture containing
0.125 pmol each of 5'-[
-32P]-end labeled
primer, 60 µM deoxynucleotide triphosphates, 2
mM MgCl2, 10 mM Tris-HCl
(pH 8.3), 50 mM KCl, and 0.125 unit AmpliTaq Gold (PE
Applied Biosystems, Foster City, CA). Initial denaturation was
performed at 94°C for 5 min, followed by 40 cycles of 94°C for
30 s, 55°C for 30 s, and 72°C for 45 s and final
extension at 72°C for 10 min. Primers, exons 1–11, of the entire
p53 gene were used (10)
.
SSCP analysis was performed on each PCR product. The PCR product in formamide dye mixture (95% formamide, 10 mM EDTA, 0.05% xylene cyanole, and 0.05% bromphenol blue) was subjected to electrophoresis in 6% nondenaturing polyacrylamide gel in Tris-borate buffer with and without 10% glycerol. Electrophoresis was performed at 300–500 V for 8–16 h at room temperature. Dried gels were then exposed onto Kodak X-Omat K film without intensifying screen at room temperature.
DNA fragments showing mobility shift by PCR-SSCP analysis were eluted from polyacrylamide gel by boiling in double distilled H2O. After ethanol precipitation, DNA fragments were subjected to 40 cycles of reamplification using the same set of primers and purified by High Pure PCR Product Purification kit (Boehringer Mannheim, Indianapolis, IN). Nucleotide sequences of these DNA fragments were determined using ABI PRISM 377 DNA sequencer using the dRhodamine Terminator Cycle Sequencing Ready Reaction kit (PE Applied Biosystems). DNA fragments were sequenced in both directions with sense and antisense primers.
Statistical Analysis.
Total copy number aberrations, including gains and losses, were
compared among the four groups of patients by the two-tailed unpaired
Student’s t test. Individual chromosome copy number changes
were compared by the nonparametric 
2
test. A
difference was considered significant in both tests when
P < 0.05.
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RESULTS |
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HBV-positive Cases (Hong Kong).
The mean (±SD) total copy changes per tumor was 6.1 ± 5.2. On
subdivision of the total aberrations into gains (including
amplifications) and losses, a mean number of 2.6 ± 2.8 gains and
3.4 ± 3.0 losses per tumor were determined (Fig. 1
A). There were frequent copy
number gains on chromosomes 1q (50%), 7q (33.3%), 8q (40%), and 20q
(30%) and common losses on 4q (27%), 8p (37%), and 13q (30%). Among
the 15 cases that showed 1q copy number gain, high level amplification
was identified in 5 (33%), and a common overlapping amplicon was
mapped to 1q21-q23 (Fig. 2
B).
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). An exon 7
mutation was detected in 5 patients, 4 of whom had the
"aflatoxin-associated" codon 249
AGGArgAGTSer mutation
(Fig. 2
D). Mutations on exon 5 codon 157 and exon 6 codon
192 were also identified. A p53 polymorphism
CGCArgCCCPro on exon 4
codon 72 mutation was also found frequently (7 of 30 cases; Fig. 2
C).
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A). Average copy number
gains was 6.1 ± 4.8. Common CGH aberrations detected included
those seen in the Hong Kong patients, but in addition there were also
frequent gains of 2q (25%), 3q (25%), 5p (40%), 6p (35%), 7p
(35%), 17q (25%), 19q (30%), and Xq (40%) and deletions of 1p
(30%), 7q (25%), 9 (30%), 17p (30%), and 18q (30%; Fig. 2
A). Percentage losses of 4q (75%), 8p (70%), and 16q
(65%) were found to be the highest of the four groups
(P < 0.035; Fig. 1
B).
p53 analysis indicated a mutation in 8 cases (40%). Four
cases had an exon 7 mutation, all of which, on subsequent sequencing,
were found to be codon 249 GT transversion. An exon 5 mutation was
detected in 4 cases, two being on codon 158 with one each on codons 174
and 181. Three cases showed the exon 4 codon 72
CGCArgCCCPro
polymorphism.
HCV-related HCC (Japan).
The mean total aberrations per sample was 4.8 ± 4.0, which
comprised 2.6 ± 2.5 copy number gains and 2.2 ± 2.5 losses
(Fig. 1
A). Frequent chromosomal gains were detected on 1q
(38%), 7q (25%), 11q (31%), 17q (31%) and 19q (25%), whereas
common losses were found on 4q (25%), 9q (25%), 13q (25%), and 17p
(25%). There was a significantly higher incidence of 11q copy number
gain compared with the other three groups (P < 0.036;
Fig. 1
B). Discrete hybridization between 11q12-11q14
was often observed, and an overlapping region was mapped to 11q13 (Fig. 2
B).
One or more p53 mutations were detected in 5 cases (31%).
Although 4 cases showed an exon 7 mutation, unlike cases from Hong Kong
and Shanghai, none were on codon 249. Instead, two mutations were found
on codon 245, and one each on codon 232 and 242. Mutations at exon 5
codons 146 and 159 were also identified (Table 2)
. Two cases displayed
the CGCArgCCCPro
polymorphism on codon 72 exon 4.
United States.
CGH analysis on 17 cases indicated common copy number gains on 1q
(53%), 6p (35%), 7p (41%), 8q (35%), and 20q (41%) and losses on
8p (35%). Compared with cases from Hong Kong and Japan, a higher
incidence of 6p (P < 0.041) and 7p (P < 0.019) gain was detected (Fig. 1
B). An amplicon on
chromosome 6, mapped to 6p21, was also noted frequently (Fig. 2
B). The average total aberration per sample detected was
5.5 ± 5.4, comprising 3.4 ± 2.9 copy number gains and
2.1 ± 2.8 copy losses/sample (Fig. 1
A). A single
instance of p53 mutation, at exon 5 codon 175, was
identified (Table 2)
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The present study serves as the first direct assessment and comparison of genetic aberrations, including the p53 mutational pattern, in HCC of differing etiologies, each typical of cases from a particular geographic region. CGH analysis revealed copy number aberrations in 84% of the cases. Despite a tumor cell content of >70%, the lack of detectable CGH abnormalities in the remaining 16% might reflect balanced translocations playing a role in the tumorigenesis. Although CGH analysis does not provide information on chromosomal translocations or clonal heterogeneity, our current study did reveal complex, but characteristic, patterns of genomic imbalances in the four groups.
The most striking feature from the analysis was the high number of
aberrations per sample in the HBV-related cases from Shanghai
(11.8 ± 7.4), which was significantly more than the other groups
(Fig. 1
A). On subsequent analysis, the differences were
accounted for mainly by a high percentage of copy number losses.
Deletions on chromosomes 4q (75%), 8p (70%), and 16q (65%) and a
gain of 5p (40%) were particularly frequent (Fig. 1
B).
Cases from Shanghai also had the highest frequency of p53
mutations (8 of 20 cases; 40%; Table 2
), and consistent with previous
reports in which the "aflatoxin-associated" codon 249 GT
transversion was identified in 33 and 60% of cases from Shanghai
(19
, 20)
, this figure was found to be 50% (4 of 8 cases)
in our series. Shanghai is known to be an area of higher aflatoxin
exposure than any of the other groups studied in this report (21
, 22)
. If, as initially suggested by Ozturk (7)
and
implied by the above-mentioned studies, the codon 249 GT
transversion is indeed related to aflatoxin exposure, then our data may
be interpreted to suggest that the genomic damage associated with
aflatoxin exposure may be more broadly based.
The HBV-related Hong Kong cases did not have as many genetic
alterations as the Shanghai samples and expressed a similar pattern of
sequence gains and losses to those from Japan and the United States
(Fig. 1
A). The cytogenetic dissimilarities between HBV
tumors from Shanghai and Hong Kong may thus be related to a lower
aflatoxin exposure in Hong Kong (22)
. In tumors from
Shanghai and the United States, despite the difference in aflatoxin
exposure (moderate versus negligible) and HBV status
(positive versus nil), the incidences of 6p (35%) and 7p
(35–41%) gain were similar. Both aberrations were also significantly
higher than the Hong Kong and Japanese tumors. This might be suggestive
of genes residing on 6p and 7p having a role in the non-HBV and
non-aflatoxin-related liver carcinogenesis.
The HCV-related cases from Japan displayed the highest incidence of 11q
copy number gain, with a minimal overlapping region mapped to 11q13
(Figs. 1
B and 2B). Significant 11q13 gains
detected may be suggestive of oncogenes, such as cyclin D-1
and Int-2, located in this region playing a role in the
hepatitis C-induced hepatocarcinogenesis. Although in a recent study
from Japan a higher incidence of 11q13 gain was suggested in
HBV-related HCC (36%) than HCV (23)
tumors, our finding
of a lower incidence in the HBV-infected tumors from China (7% in Hong
Kong and 5% in Shanghai) coincided with other studies in Chinese
subjects [4% from Taiwan (24)
; 0% from Shanghai
(25)
] and that reported by Marchio et al.
(Ref. 26
; 5% in 50 HBV-infected HCC cases from different
geographic origins). Such discrepancies may suggest that Japanese HCC
on the whole, whether type B and C viral infected, has a higher
incidence of 11q gain, which in turn will imply the presence of other
environmental factors in Japan that induce liver carcinogenesis via a
preferential target site on 11q13.
In the present study and previous studies involving Oriental subjects, we have shown that the most consistent aberration in HCC was the high incidence of 1q gain (23, 24, 25 , 27) . Other common imbalances in all four groups studied included gains of 8q, 17q, 20q, and Xq and losses on chromosomes 4q, 8p, 9, 13q, 16q, and 17p. Regions of DNA sequence losses found were in agreement with those reported by loss of heterozygosity studies (28, 29, 30, 31, 32) .
p53 mutational hotspots identified in the Chinese and
Japanese cases were largely confined to exon 7 (Table 2)
. A
CGCArgCCCPro change at
codon 72 exon 4 was also identified frequently. Although this
alteration is considered a polymorphism of the p53 gene, it
has been suggested to increase the susceptibility of Chinese females to
developing lung cancer (33)
. Although the use of codon 249
AGGArgAGTSer mutation as
a molecular marker for previous aflatoxin exposure remains
controversial, our finding of this mutation in only aflatoxin-exposed
regions (Hong Kong and Shanghai) strongly supports an association of
this mutation and aflatoxin.
A relation between specific cytogenetic aberrations and particular etiological agents in the development of HCC has been suggested in our present study. Although we cannot entirely rule out the possibility that some aspect of our sampling in the various groups could account for the differences, because overall surgical practice is similar between the regions (patients with metastatic disease or positive lymph nodes are not operated upon), and that we have reported previously no, or minimal, cytogenetic differences in tumors of different sizes (27) , it is therefore likely that the observed cytogenetic differences are associated with the etiological risk(s) of the region.
Our p53 mutational study is consistent with the hypothesis
that there is an association between dietary aflatoxin exposure and
codon 249 GT transversion. Furthermore, a more aberrant pattern of
genetic changes detected in the Shanghai series suggests that the
genotoxic effects of aflatoxin to be more broadly based than affecting
p53 alone. It also supports synergism between HBV infection
and high aflatoxin exposure in promoting HCC development. Such
synergism may have invoked oxidative damage to genomic DNA, resulting
in carcinogenic mutations in the liver cells. Other recurring genetic
alterations identified in all four groups, in particular 1q gain, may
reflect part of a common pathway in the liver carcinogenesis.
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FOOTNOTES |
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1 This work was carried out within the Hong Kong
Cancer Genetics Research Group that was supported by The Kadoorie
Charitable Foundations, Hong Kong, and the Research Grants Council of
the Hong Kong Special Administrative Region (RGC Ref. No. CUHK 4264/98
M). We are also thankful to the Providence Foundation Limited, Hong
Kong, for their continuing support. 
2 To whom requests for reprints should be
addressed, at Department of Clinical Oncology at the Sir Y. K. Pao
Centre for Cancer, The Chinese University of Hong Kong, Prince of Wales
Hospital, Shatin, N. T., Hong Kong SAR, China. Phone: 852-2632-2119;
Fax: 852-2649-7426; E-mail: pjjohnson{at}cuhk.edu.hk
3 The abbreviations used are: HCC, hepatocellular
carcinoma; CGH, comparative genomic hybridization; HBV, hepatitis B
virus; HCV, hepatitis C virus; dig, digoxigenin; TRITC,
tetramethylrhodamine isothiocyanate; DAPI,
4',6-diamidino-2-phenylindole; SSCP, single-strand conformational
polymorphism.
Received 3/ 2/00; revised 6/28/00; accepted 7/ 5/00.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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T transversions at codon 249: the fingerprint of aflatoxin exposure?. Environ. Health Perspect., 105: 392-397, 1997.[Medline]
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25% of cases in any one of the four groups studied.