Identify Metastasis-Associated Genes in Hepatocellular Carcinoma Through Clonality Delineation for Multinodular Tumor1

[CANCER RESEARCH 62, 4711–4721, August 15, 2002] Identify Metastasis-associated Genes in Hepatocellular Carcinoma through Clonality Delineation for Multinodular Tumor1

Siu Tim Cheung,2 Xin Chen, Xin Yuan Guan, San Yu Wong, Lai Shan Tai, Irene O. L. Ng, Samuel So, and Sheung Tat Fan Departments of Surgery [S. T. C., S. Y. W., S. T. F.], Clinical Oncology [X. Y. G., L. S. T], Pathology [I. O. L. N.], Centre for the Study of Liver Disease, The University of Hong Kong Medical Centre, Queen Mary Hospital, Hong Kong, China, and Departments of Biochemistry [X. C.] and Surgery [S. S.], Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, California 94305

ABSTRACT specimens in some cancers. To study clinical metastasis, HCC3 may be the best choice because tumor invasion into blood vessels and Disease recurrence and metastasis are frequently observed in many intrahepatic metastasis are frequently observed and presented as mul- successfully treated localized cancers, including hepatocellular carcinoma tiple nodules at the time of surgical resection (5). in which intrahepatic and extrahepatic recurrence (metastasis) are fre- To identify metastasis-associated genes, the first crucial step is to quently observed after curative resection. The present study aimed at delineate the clonal relationship among the multiple tumor nodules, identifying metastasis-associated genes through delineation of the clonality for multinodular liver cancer. The clonal relationship of 22 tumor foci and to determine which tumor clones were derived from the primary. from six patients was investigated by the genome-wide expression profile However, reliable parameters to define the clonality for multiple via cDNA microarray consisting of 23,000 genes. Tumor molecular prop- nodular HCC have not been established. Conventional discriminations erties including p53 protein overexpression and gene mutation, hepatitis B are mostly based on clinical and pathological findings (5–7), which virus integration pattern, and genetic alteration examined by comparative cannot always provide a definitive answer. Commonly used ap- genomic hybridization were compared. Results indicated that gene expres- proaches include comparison for the HBV integration site to differ- sion patterns could serve as the molecular fingerprint for clonality iden- entiate between intrahepatic metastasis and independent occurrence tification. Together with the molecular data from p53, hepatitis B virus (8–10). However, this is only applicable to HCC with HBV integra- integration and comparative genomic hybridization profiles, tumor nod- tion, whereas tumors related to hepatitis C virus (HCV) or alcohol, ules from five patients were confirmed with clonal relationship, and the which are the common associations in North America (11), cannot be expression profiles of the primary nodules were compared with their validated. Clonal analysis can be examined based on X chromosome corresponding intrahepatic metastatic nodules. A total of 90 clones were inactivation (12, 13), which can be investigated only in female pa- found to be correlated with intrahepatic metastasis by Student’s t test tients. In East Asia, including Hong Kong, HCC patients are predom- (P < 0.05). With reference to the primary tumor, 63 clones (39 known inantly male; thus, the approach based on X chromosome inactivation genes and 24 express sequence tags) were down-regulated whereas 27 clones (14 known genes and 13 express sequence tags) were up-regulated does not have a wide implication. DNA fingerprinting (14) and in the metastatic nodules. These metastasis-associated genes may provide fractional allelic loss (15) analysis have also been used for clonality clues to reveal patients with increased risk of developing metastasis, and studies. However, the demanding technique involved combined with to identify novel therapeutic targets for the treatment of metastasis. the problem of reproducibility and the subjective data analysis all hinder their wide application in clonality examination. In a recent study, we used the cDNA microarray approach to INTRODUCTION examine the genome-wide expression profile of over 200 samples consisting of majority HCC and the tumor adjacent liver tissues (16). Metastasis is the major cause of cancer morbidity and mortality. A consistent expression difference was observed between the tumors Comprehensive analysis of gene expression profiles between a pri- and their adjacent nontumor livers. To investigate whether the expres- mary tumor and its derived tumors can identify differential genes sion profile can help to elucidate the clonal relationship of the multi- associated with the metastatic phenotype, which help to elucidate the nodular liver cancers, samples from individual tumor foci were further molecular mechanism of cancer metastasis (1, 2). There are a number examined with the conventional molecular genetic approach for tumor of experimental models for the examination of genes responsible for properties and genetic aberrations. The expression data of 22 individ- the enhancement or suppression of cancer metastasis (3, 4). However, ual tumor foci in six patients with multinodular HCC were compared the clinical relevance of these genes is sometimes difficult to consol- with the conventional molecular genetic data including p53 protein idate. The most direct method to identify genes associated with overexpression and p53 gene mutation, HBV integration, and genetic clinical metastasis is to examine the human specimens, and to com- aberrations as investigated by CGH. The data up until now had pare the primaries with their corresponding metastatic tumors. Sample suggested that gene expression profiles were unique for each tumor availability can be the major hurdle, as reflected in the long time and could provide sufficient information to delineate clonal relation- interval between the primaries and the appearance of secondaries. The ships. The identity of primary and intrahepatic metastasis tumor tumor type or anatomical site may not allow for the collection of nodules was further confirmed by conventional molecular genetic approaches, and the expression profiles of the original and metastatic Received 1/28/02; accepted 6/13/02. tumor clones were compared with those of the genes associated with The costs of publication of this article were defrayed in part by the payment of page intrahepatic metastases. charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported in part by grants from the Sun Chieh-Yeh Research Foundation for Hepatobiliary and Pancreatic Surgery, and Seed Funding Programme, The University of MATERIALS AND METHODS Hong Kong (to S. T. C. and S. T. F.), and H. M. Liu Foundation, Stanford University (to X. C. and S. S.), and a fellowship from Howard Hughes of the Life Science Research Patients and Samples. Informed consent was obtained from the patients Foundation, Stanford University (to X. C.). for the collection of liver specimens, and the study protocol was approved by 2 To whom requests for reprints should be addressed, at Department of Surgery, Centre for the Study of Liver Disease, The University of Hong Kong Medical Centre, Queen Mary Hospital, Hong Kong, China. Phone: 852-2855-3995; Fax: 852-2818-4407; E-mail: 3 The abbreviations used are: HCC, hepatocellular carcinoma; HBV, hepatitis B virus; [email protected]. CGH, comparative genomic hybridization; EMP3, epithelial membrane protein 3. 4711

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2002 American Association for Cancer Research. METASTASIS-ASSOCIATED GENES IN LIVER CANCER the Ethics Committee of The University of Hong Kong. Between April and washes, and incubation with anti-fluorescein-horseradish peroxidase conjugate, July 2000, six patients with multiple nodular liver tumors were treated by ECL detection reagent (Amersham Pharmacia Biotech) was used, and a signal was surgical resection (Table 1). A total of 22 samples were obtained from developed by X-Omat AR film (Kodak, Rochester, NY). individual tumor foci of the surgical specimens. Samples were snap-frozen in CGH. Protocols have been established and details described previously liquid nitrogen and stored at Ϫ70°C until use. Paralleled tumor specimens (26, 27). In brief, DNA from tumor and normal reference were labeled with were formalin fixed and paraffin embedded for histological examination and different fluorescent dUTPs by nick translation. Labeled probes were mixed immunohistochemical study. Total RNA was extracted with RNeasy kit (Qia- and hybridized onto metaphase cells prepared from the lymphocytes of healthy gen, Hilden, Germany) and mRNA was isolated from total RNA using Fast- donors. The slide was counterstained with 4Ј,6-diamidino-2-phenylindole di- Track (Invitrogen, Carlsbad, CA) or Poly(A)Pure (Ambion, Inc., Austin, TX) hydrochloride (DAPI) for digital image analysis using the Quips CGH program mRNA purification kit. DNA was extracted with DNAeasy kit (Qiagen) (Vysis, Downers Grove, IL). The thresholds used for the interpretation of gains according to the manufacturer’s instructions. and losses of a DNA sequence copy number were defined as the tumor: Microarray Expression Study. The cDNA microarray slides were printed reference ratio and were Ͼ1.25 and Ͻ0.75, respectively. The thresholds with 23,075 cDNA clones. Protocols have been established and the details applied for both the standard and the reverse hybridization methods. described previously (17, 18). In brief, a fluorescent-labeled (Cy5) cDNA probe was synthesized by reverse transcription using the Superscript II reverse transcription kit (Invitrogen) from each experimental tumor RNA sample to RESULTS serve as the “test”’ case. The “reference” mRNA was prepared by a different Gene Expression Profile. We applied a cDNA microarray with fluorescent (Cy3) from a pool of mRNAs isolated from different cultured cell 23,075 cDNA clones to study the gene expression patterns in 103 liver lines. This common reference provided an internal standard against which the gene expression of each experimental sample was compared. The two fluo- cancer samples from 81 patients. The tumors and genes were grouped rescent-labeled probes were combined, purified, and put onto the arrays. After on the basis of their similarity in expression pattern by hierarchical hybridization, arrays were scanned with a microarray scanner for the fluores- clustering algorithm (19, 20). A total of 7,830 cDNA clones were cent images. The image files were analyzed with the program GenePix Pro 3.0 shown to have significant variation (genes with at least 3-fold of (Axon Instruments, Inc., Union City, CA) to quantitate the relative amount of expression difference in at least one array and 75% valid data points; mRNA between the two samples. Data were deposited into the Stanford Fig. 1A). The characteristic expression relationship of the 22 tumor Microarray Database at: http://genome-www4.stanford.edu/MicroArray/SMD/ foci from the six patients (Table 2) with multinodular liver tumors is index.html. Areas of the array with obvious blemishes were flagged. Each highlighted in Fig. 1B. fluorescent signal in the array element with intensity 1.5-fold greater than local The gene expression patterns of the two tumor foci from patient M1 background was considered as valid data, and those genes with less than 75% were very different, and they are separated into different major sub- valid data points in the sample set were excluded. Genes with expression levels that differed by at least 3-fold from the mean in at least one sample were branches in the dendrogram. The correlation coefficient of the overall selected. Hierarchical clustering algorithm (19, 20) was applied to both the gene expression patterns between T1 and T2 was 0.28. In patient M2, genes and the arrays using the Pearson correlation coefficient as the measure T1 (tumor nodule 1, as assigned according to gross size and proximity of similarity. The results were further analyzed with TreeView (Eisen; http:// to the main tumor mass) to T6 demonstrated similar gene expression rana.lbl.gov). profiles and they were clustered into one terminal branch. Correlation p53 Protein Staining and Gene Sequencing. Immunohistochemistry was coefficient ranged from 0.45 to 0.89. Tumor nodule T7 of patient M2 performed as described previously (21) with modification. Briefly, antigen showed a distinct pattern of gene expression and it segregated into retrieval was performed by microwave with sections immersed in citrate another major branch different from T1 to T6 of the same patient, with buffer. Followed by endogenous peroxidase blocking and biotin blocking an overall very low correlation, around 0.1 to 0.2 with other nodules reagents (DAKO, Copenhagen, Denmark), antibody DO-7 (DAKO) in 1:100 from patient M2. The two tumor foci from patient M3 showed similar dilution was applied. Signal was detected by horseradish peroxidase-conju- gated secondary antibody and color development with diaminobenzidine gene expression patterns, residing in one terminal branch with a (DAB) as the chromogen. Tissue sections were counterstained with hema- correlation of 0.52. Similarly, in patient M4, T1 to T5 clustered into toxylin. one terminal branch with an overall correlation coefficient ranging Direct DNA sequencing was performed for exon 4 to exon 9, which resided from 0.79 to 0.85. Expression patterns of T2 and T3 from patient M5 with mutational hot-spot and accounted for over 80% of all of the mutations were more similar. They were clustered into one terminal branch with observed (22–24). Primer sets and reaction conditions were adopted from a correlation of 0.80. Whereas T1 of the same patient M5 was less Lehman et al. (25). DNA was amplified by PCR, and direct DNA sequencing tightly linked to T2 and T3, they nonetheless resided closely together was performed with a BigDye sequencing kit (Applied Biosystems, Foster in the same major branch of the dendrogram with a correlation of City, CA). Electrophoresis and sequence analysis were performed with ABI 0.51. Correspondingly, in patient M6, T2 and T4 showed a more PRISM 310 (Applied Biosystems). similar expression pattern with a correlation of 0.51. Tumor nodule T1 HBV Integration by Southern Blot Analysis. DNA was digested overnight with restriction endonuclease (Ref. 10; HindIII or EcoRI, Invitrogen) at 37°C with of the same patient M6 resided in the same subbranch with the 10 units per ␮g of DNA. Fragments were electrophoresed on 1% agarose gel and correlation of 0.30 for T2 and 0.28 for T4. These data suggest a transferred overnight to Hybond membrane (Amersham Pharmacia Biotech, Pis- complex gene expression program between the multiple tumor nod- cataway, NJ). The probe was full-length HBV fluorescein-labeled by random ules from the same patients. prime system (Amersham Pharmacia Biotech). After hybridization, stringency As a control, we investigated whether sampling the same nodule

Table 1 Clinical patient details TNM Edmonson Venous Survival Patienta Sex/Age stage HBsAgb grade invasion (mo) Current status M1 F/66 II ϩ 2 absent 14.9 Alive, disease free M2 F/54 III ϩ 2–3 present 0.2 Deceased M3 F/43 IVA ϩ 3 present 5.1 Deceased M4 M/66 III ϩ 3 absent 13.1 Alive, disease free M5 M/13 IVA ϩ 3 present 6.2 Deceased M6 M/70 IVB ϩ 2–3 absent 8.5 Deceased a Patients M1 to M5 had primary liver tumors, and M6 had recurrent HCC after primary HCC resection and TOCE treatment for recurrence. b HBsAg, hepatitis B surface antigen; TOCE, transarterial oily chemoembolization.. 4712

Fig. 1. Hierarchical clustering of 7830 genes in 103 liver cancer tissues (from 81 patients, 6 of whom had multiple tumor foci and contributed 22 samples). In A, the figure is turned 90° for ease of presentation. Rows, results of individual samples; columns, individual genes. The color in each cell of the cluster reflects the mean-centered expression level of the gene. The scale bar extends from fluorescence ratios of 0.25 to 4 (Ϫ2toϩ2inlog base 2 units). Red, a high expression level as compared with the mean; green, a low expression level as compared with the mean; grey, missing or excluded data. In B, the dendrogram at the left of the figure in A is magnified. The samples were color- coded for individual patients; blue, replicate specimens that were taken from single tumor nodules.

from the same tumor multiple times would also show variation similar exon 6, amino acid 213. In patient M5, all three of the tumors to that seen from the multiple nodular tumors which were the discrete contained a single mutation at exon 7, amino acid 249 (Fig. 2A). For tumor nodules. Replicated samples, a total of 10, were taken from four patient M6, all three of the tumors exhibited a mutation at exon 5, patients from different regions of a single tumor nodule for compar- amino acids 160 and 161 (Fig. 2B). ison. All of the replicate samples from an individual were clustered HBV Integration Pattern. To further confirm the clonality of into one terminal branch of the dendrogram with high correlation. those multiple tumor nodules with distinct gene expression profiles (Fig. 1B). This demonstrates that each tumor nodule has its own and heterogeneous p53 overexpression patterns, we applied Southern distinct gene expression patterns, and the differences that we observed blot analysis for the HBV integration site. As the HBV integration is in the expression profiles in multiple nodular tumors are not attribut- generally an early event during tumorigenesis, tumors arising from the able to a sampling artifact. same clone generally have the same integration pattern. The two p53 Protein Expression and Mutation. To investigate the molec- tumor nodules, T1 and T2, of patient M1 had different HBV integra- ular genetics of these multiple nodular HCC samples, we studied the tion patterns (Fig. 3), which indicated that T1 and T2 were formed expression of p53 in these samples by immunohistochemistry. Tissue from two distinct clones. Tumor nodules T1 and T2 from patient M3 sections from tumor foci of patients M1, M2, and M4 all showed a showed identical integration patterns. Patient M6 also demonstrated negative staining result for p53 protein. And all of the tumor foci from the same HBV integration pattern in both T1 and T2. This suggests patient M3 showed a positive signal of p53. In patient M5, a hetero- that tumors from M3 and M6, respectively, were clonally related. geneous staining pattern was observed (Fig. 2A). For M5T1, some of CGH. Using CGH, we further explored the genetic alterations the tumor areas had less than 10% cells positive, whereas some of the among the multiple tumor nodules (Fig. 4; Table 3). The CGH profiles tumor patches had more than 50% of cells positive. For M5T2 and T3, of the two tumor foci T1 and T2 in patient M1 were basically distinct. a homogeneous staining pattern was observed, and the majority of T1 to T6 from patient M2 shared similar genetic aberrations that were tumor cells showed overexpression of p53 protein. In patient M6, a different from T7. Patient M3 showed similar structural changes in similar heterogeneity pattern was observed. The majority of tumor both of the tumor foci, with T1 showing additional genetic aberrations cells in M6T2 were negative, although a rare positive signal could be compared with the common ones observed in T2. T1 to T5 in patient observed in some of them (Fig. 2B). In M6T1, the majority of tumor M4 showed a common structural change. T2 and T3 from patient M5 cells showed overexpression of the p53 protein. showed additional genetic aberrations when compared with T1. In The p53 gene sequence was further analyzed in those patients who patient M6, common genetic aberrations were observed in both T1 had exhibited p53 protein overexpression to confirm the presence of and T2, whereas T1 exhibited additional chromosomal changes. gene mutation. Direct DNA sequencing was performed for exon 4 to Determination and Delineation for the Clonality of Multinod- exon 9, in which Ͼ80% of all mutations were observed (22, 24). Both ular Liver Cancer Samples. Through the above analysis, we were of the tumor foci T1 and T2 from patient M3 showed a mutation at able to determine the clonality of these multinodular liver cancer 4713

Table 2 Clonality details for the multinodular liver tumors Array p53

Tumor Gross Size Separation Correlation Protein Mutation, HBV Derived clonal Patient nodulea appearanceb (cm) distancec Clustering coefficient overexpression exon 4–9/a.a.d integration Histology relationship M1 T1 Nodular 2 Different branch 1 and 2 ϭ 0.28 Negative Different site HCC Separate clone T2 2 7 Negative Multicentric occurrence M2 T1 Massive 3.5 T1 to 6, same branch 1 and 2 ϭ 0.58 Negative HCC T1, 2, 4, 5 and 6 T2 3 0 2 and 3 ϭ 0.45 Negative HCC Same clone T3 2.5 0 T7: far away 3 and 4 ϭ 0.52 Negative Mixede Intrahepatic metastasis T4 3 0 4 and 5 ϭ 0.69 Negative HCC T5 2.5 0 5 and 6 ϭ 0.89 Negative HCC T7 T6 2 0 6 and 7 ϭ 0.18 Negative HCC Separate clone T7 1 5 1 and 7 ϭ 0.15 Negative adenoCA Multi-centric occurrence M3 T1 Massive 14 Same branch 1 and 2 ϭ 0.52 Positive E6/213 Same site HCC Same clone T2 5.5 0 Positive E6/213 Intrahepatic metastasis M4 T1 Massive 5 Same branch 1 and 2 ϭ 0.79 Negative HCC Same clone T2 3 0 2 and 3 ϭ 0.79 Negative HCC Intrahepatic metastasis T3 3 0 3 and 4 ϭ 0.84 Negative HCC T4 3 0 4 and 5 ϭ 0.80 Negative HCC T5 3 0 1 and 5 ϭ 0.85 Negative HCC M5 T1 Massive 8 T1 further away 1 and 2 ϭ 0.51 Positive (some) E7/249 HCC Same clone T2 1 4 1 and 3 ϭ 0.51 Positive E7/249 HCC Intrahepatic metastasis T3 3 6 T2 and 3 same branch 2 and 3 ϭ 0.80 Positive E7/249 HCC M6 T1 Nodular 2 0 T1 further away 1 and 2 ϭ 0.30 Positive E5/160 and Same site HCC Same clone 161 T2 2 0 1 and 4 ϭ 0.28 Rare positive E5/160 and HCC Intrahepatic metastasis 161 T4 2 0 T2 and 4 same branch 2 and 4 ϭ 0.51 Rare positive E5/160 and HCC 161 a For each patient, tumor nodule 1 (T1) was the largest tumor foci on gross morphology and T2 was the tumor next to it, and so forth. b Gross appearance: Eggel’s classification. c Separation distance: calculated with reference to the major tumor mass. 0 cm ϭ immediately adjacent with the main tumor mass but grossly separated foci. d a.a., amino acid; adenoCA, adenocarcinoma. e Mixed: presence of both HCC and adenocarcinoma cell types. samples. It also helped to determine the primary tumor nodules versus obtained supported the observation from the gene expression profile, metastatic nodules. which suggested that the two tumor nodules were independent clones. For patient M1, the two nodules T1 and T2 showed distinct gene For patient M2, the gene expression profiles of T1 to T6 were very expression patterns (Fig. 1B). The HBV integration site was different similar (Fig. 1B). T7, however, segregated to a different major branch, (Fig. 3) and the genetic composition of the two nodules was discrete and was likely to be different in clonality. Clinically, T1 to T6 fit the by CGH (Fig. 4). In addition, the two nodules were separated by a definition for unicentric HCC (6). They appeared as a solitary tumor physical distance of 7 cm, which clinically would be classified as a mass surrounded by contiguously smaller nodules located within 1 cm multicentric occurrence (7) or multicentric HCC (6). All of the data of each other. However, T7 was an isolated nodule away from the

Fig. 2. p53 protein overexpression and p53 gene mutation analysis. In patient M5, different patterns of positive signals were observed in the tumor nodules (A). T1 was heterogeneous with respect to the p53 protein expression. Some areas of the tumor cells had less than 10% of the population showing protein accumulation, whereas some had more than 50%. T2 (and T3) were similar in that the majority of tumor cells showed overexpression. Sequence analysis supported the observation that the mutant sequence had already emerged in T1. This population was further selected and expanded in T2, which had a larger proportion of cells demonstrating mutant sequence at exon 7, amino acid 249. Similar observation for the p53 aberration in patient M6 (B). T2 was heterogeneous in p53 protein overexpression with positive signal shown only rarely. Sequence analysis demonstrated that mutation had developed at exon 5, amino acid 160–161, in the background of wild-type sequence. The p53 aberration was further accumulated in T1, with the majority of tumor cells overexpressing the protein and exhibiting predominantly the mutant sequence. 4714

difference, in which the majority of tumor cells in T2 and T3 were positive, and only a proportion of tumor cells in T1 were positively stained for the p53 protein (Fig. 2A). However, all of the nodules showed an identical point mutation of the p53 gene at exon 7, amino acid 249. Consistent with the p53 protein overexpression pattern, the mutant sequence in T1 barely emerged, whereas a predominant mutant sequence was observed in T2. The p53 data here indicated that this gene mutation and protein accumulation had conferred growth and/or metastasis advantage to the tumor cells, and such property was selected for and expanded during tumor progression. In addition, common chromosomal aberrations were observed with T1 demonstrating the fundamental changes, whereas T2 and T3 showed additional alterations. Grossly, T1 was the major tumor mass, whereas T2 and T3 were smaller surrounding nodules. Expression profiles sup- Fig. 3. Southern blot analysis of integrated HBV DNA in liver tumors. The separate ported other genetic data and clinical observation that all three nod- tumor nodules from patient M1 showed different patterns of HBV integration. For patients ules arose from the same clone, and T2 and T3 were metastatic clones M3 and M6, identical HBV integration patterns were observed for the individual tumor derived from the T1 tumor nodule. foci from each patient. Patient M6 presented a similar situation. The expression profiles of T2 and T4 were tightly linked suggesting definite clonal linkage, main tumor mass. The histological parallel sections of T1 to T2 and whereas T1 was not as tightly clustered but still resided on the same T4 to T6 were all confirmed to be HCC. Interestingly, T3 was subbranch in the dendrogram. The p53 protein expression pattern reported to have a mixture of tumor cell types, HCC and adenocar- coincided with the global gene expression profile. T2 and T4 were cinoma, in the same section. The expression profile by microarray and similar in the way that only rare tumor cells showed a positive signal, genetic changes by CGH of T3 were indistinguishable from the other but T1 clearly demonstrated a positive expression for p53 (Fig. 2B). closely surrounded nodules, which suggested that the HCC trait over- Identical gene mutations were observed in all three of the tumor foci. whelmed and masked the other tumor cell components. However, T7 Coherent with the p53 protein expression pattern, the mutant sequence was histologically confirmed to be adenocarcinoma without an inter- in T2 and T4 had just emerged, whereas a predominant mutant mix of HCC components. T7 was clearly demonstrated to be distinct sequence was observed in T1. Similar with the situation in patient M5, with its gene expression by microarray and chromosomal aberrations the p53 aberration in patient M6 accumulated during tumor progres- by CGH. The data obtained suggested that T1 to T2, and T4 to T6, sion from T2 to T1. The genetic alterations as revealed by CGH were clonally linked. T1 revealed the fundamental genetic aberrations indicated that both T1 and T4 had extra changes in addition to those common to all of the other tumor nodules and, therefore, was regarded observed in T2. The similar global gene expression patterns together as the primary tumor. T7 was an independent tumor clone. with p53 aberration, HBV integration site, and common chromosomal For patient M3, T1 and T2 were clustered into one terminal branch aberrations, all implicated that the nodules had clonal relationship and with a high correlation coefficient of gene expression, suggestive of that T1 and T4 were the metastatic tumor and that T2 was the primary. clonal linkage. Tumor cells from both nodules overexpressed p53 Differential Genes between Primary and Metastatic Tumor. protein with identical mutation point. The HBV integration patterns Having established the clonal relationship of the multiple nodules and were also identical. Common genetic aberrations were observed by the delineation for the primary and metastatic tumors by the above CGH analysis with T2 showing the basic chromosomal changes. T1 investigations, the next step was to identify genes differentially ex- exhibited additional alterations and, thus, should be the derived met- pressed between the original and their derived clones. The expression astatic tumor nodule as defined by the genetic approach on cancer profiles of the primary (T1, unless otherwise indicated that it was not progression. The gross size of T1 (14 cm) was much bigger than that the primary, as described in the following) and their metastatic nod- of T2 (5.5 cm). The difference in tumor size could be explained if the ules with the longest physical distance demonstrated with more ge- additional genetic changes had conferred it with growth advantage. netic aberrations were compared. For patient M1, the two tumor foci Nonetheless, the two nodules were situated immediately adjacent and were independent clones and thus were not included in this part of the could be clinically classified as unicentric HCC. All of the data agreed study. For patient M2, T1 (primary) was compared with T6 (metas- well with the gene expression profiles: the two nodules were derived tasis) because they indicated clonal linkage and the latter was the from the same clone and T1 was the metastatic nodule. furthest away from T1. Similarly, M4T1 was compared with M4T5; All of the tumor nodules from patient M4 showed similar expres- and M5T1 was compared with M5T3. For patient M3, T2 was sion profiles. They clustered in one terminal branch of the dendrogram revealed as primary, and T1 would be the metastasis foci. In patient with high values of correlation coefficient, which indicated intrahe- M6, T2 was the primary whereas T1 was the metastasis tumor foci. patic spread from the original tumor clone. Chromosomal changes as The expression profiles of M2T1, M3T2, M4T1, M5T1, and M6T2 examined by CGH were also similar and supported the clinical ob- were considered as one group representing the primaries. M2T6, servation of clonal relationship. All of the tumor nodules revealed the M3T1, M4T5, M5T3, and M6T1 were considered as another group fundamental genetic alterations specific to this patient, and subtle representing the metastatic tumors. Student’s t test was used to select differences were observed among different nodules. Thus, for the for genes that could differentiate between these two groups with a designation of primary tumor, the conventional approach was used, cutoff value at 0.05; 90 representative clones passed the criterion (Fig. which referred the major tumor mass T1 as the primary. 5). A total of 63 clones were down-regulated in the metastatic tumors In patient M5, expression profiles of T2 and T3 were tightly linked compared with the primary tumors, where 39 clones represented 35 and showed some distinct differences from T1. As shown in the known genes; and 24 clones were ESTs with limited information for dendrogram and by the correlation coefficient, T2 and T3 should be their biological function (Table 4). A total of 27 clones were up- clonal with closer linkage to each other than to T1. The p53 expres- regulated in the metastatic tumor, in which 14 clones were known sion pattern corresponded well with the overall gene expression genes and 13 clones were ESTs. 4715

Fig. 4. CGH profiles. In A, in patient M1, the chromosomal alterations in T1 were distinctly different from T2. In patient M2, common genetic changes were observed from both tumor foci at chromosomes 1, 4, 8, 11, 13, and 17; and T6 revealed additional alterations at chromosomes 6, 12, and 22. In patient M3, similar genetic changes were observed from both tumor foci at chromosomes 2, 3, 4, 5p, 8, 10, 12, 17, and 22; and T1 exhibited additional aberrations at chromosomes 1, 5q, 11, 13, 14, 16, 17, 18, 20, and 21. In B, in patient M4, comparable alterations were observed at chromosomes 1, 5, 8, 16, 17, 19, 20, 21, and 22; and T5 exhibited extra aberrations at chromosome 4. In patient M5, similar genetic changes were observed at chromosomes 1, 5, 6pter, 15, 16, 17p, 19, and 21; and T3 exhibited extra alterations at chromosome 4, 6p, 8, 13, 17q, 18, 19, and 22. In patient M6, similar genetic alterations were observed from both tumor foci at chromosomes 1, 4, 6q, 7, 8p, 16, and 17; and additional genetic aberrations of T1 were observed at chromosomes 8q, 9, and 20; additional genetic aberrations of T4 were observed at chromosomes 6p, 7, 8q, 11, and 16.

DISCUSSION gration and chromosomal content involved complicated protocols and procedures. However, expression profiles examined by microarray Examination of the HBV integration site is the conventional involved standard labeling, hybridization, and a data analysis ap- method to confirm HCC clonality. Southern blotting was commonly proach that were researcher-independent. Microarray technique had used (6, 9) but was labor intensive, time consuming, and applicable the potential of standardization and automation (including array man- only to tumors with HBV infection. Cloning and PCR of the inte- ufacture, probe synthesis, hybridization, and signal detection), sug- grated HBV DNA could examine archive tissue in paraffin (28). gesting a wider application compared with conventional investigation However, it encountered a similar restriction in applicability. Molec- approaches to confirm the clonal relationship of separate tumor foci. ular cytogenetic studies by CGH had also been used to determine Comparison of the expression profiles from multiple specimens tumor clonality (29). It is noteworthy that assessment of HBV inte- taken from an individual patient could indicate the unique character- 4716

Fig. 4. Continued

4717

Table 3 Genetic aberrations by CGH Tumors from individual patients were compared for their differential similarities and differences. Tumor Patient nodule Differential chromosomal changes Common alterations in the same patient M1 T1 Ϫ1pter-p31.2, ϩ2q22-q32.3, ϩ4p, Ϫ4q21.1-q27, ϩ5p, ϩ7q11.23-qter, Ϫ16, Ϫ18q ϩ1q31-qter, Ϫ6q22.3-qter, Ϫ8pter-p12, Ϫ17p, Ϫ19, Ϫ22q T2 ϩ3p, ϩ5q13.3-qter, Ϫ6q13-qter, ϩ8q, ϩ10p, ϩ13q31-qter, ϩ17q22-qter M2a T1, 3, 4, 6, 7 Ϫ4q21.1-q26, Ϫ11p ϩ1q21.1-qter, Ϫ8p, Ϫ17p, ϩ17q21-qter T1, 2, 3, 4, 6 Ϫ13q T2, 3, 4, 6 Ϫ22q T3, 6 ϩ12q13.2-qter T3, 4, 6 ϩ6pter-p21.1 T1, 4 Ϫ6q22.1-q25.1 T3 Ϫ6q21-q25.3 T6 Ϫ6q16.1-q25.3 T4 Ϫ10q11.2-q23.3 T7 ϩ2q12-qter, ϩ3q25.1-qter, Ϫ10, Ϫ13q12.1-q21.2, ϩ14q11.2-q21, Ϫ14q24.1-qter, Ϫ18q, Ϫ20p, Ϫ21p, ϩ22q M3 T1 ϩ1p32.3-p13.1, ϩ1q21.1-q44, ϩ5q31.1-q35.3, ϩ6, Ϫ11p14-q25, ϩ12, Ϫ13q14.1-q22, Ϫ2q, ϩ3p, Ϫ4, ϩ5p, Ϫ8p, Ϫ10q22.1-qter, ϩ12, Ϫ17p, Ϫ22q Ϫ14q, ϩ16q12.2-qter, ϩ17q22-qter, ϩ18, ϩ20, ϩ21q T2 Ϫ1pter-p34.2, ϩ11pter-p15.1 M4 T1, 2, 3, 5 ϩ1q21.1-q41, ϩ5p ϩ5, Ϫ8p, ϩ8q, Ϫ16, ϩ17q, Ϫ19, Ϫ20 T1, 4, 5 Ϫ17p, Ϫ21, Ϫ22 T2, 3, 5 ϩ4p12-q13.1 T1 ϩ2q12-q32.3 T3 ϩ2q21.1-q32.3 T4 Ϫ1q21.1-qter, ϩ5p T2 Ϫ18pter-q12.1 M5 T1 ϩ4q28-q32, ϩ13q14.1-qter ϩ1q23-qter, ϩ5p, ϩ6pter-p21.1, Ϫ15q, Ϫ16, Ϫ17p, Ϫ19p, Ϫ21p T1, 2 Ϫ22 T1, 3 Ϫ1pter-p22.2 T2, 3 Ϫ4q13.2-q27, ϩ6p12-p11.1, Ϫ8p, ϩ8q12-qter, Ϫ13q, ϩ18p, ϩ18q21.3-qter, Ϫ18q11.2-21.2, ϩ19q T3 Ϫ13q12.3-qter, ϩ17q24-qter, ϩ22q13.1-qter M6 T1 ϩ6q22.3-qter, ϩ8q22.1-qter, Ϫ9p, ϩ20q ϩ1q21-qter, Ϫ4, Ϫ6q11-q22.1, ϩ7q31.1-qter, Ϫ8p, Ϫ16q, Ϫ17p T4 ϩ6p, ϩ6q22.1-qter, ϩ7, ϩ8q, Ϫ11q, Ϫ16p a M2, T1 to T4, and T6 to T7 were examined.

istics of the tumor that represented the patient’s distinctive feature. tastasis, some of the changes are believed to be the secondary events; The specific similarity and differences in gene expression pattern have the expression changes as a result of metastasis rather than as an provided fundamental information for the clonality relationship of initiator of the metastasis event. Furthermore, as the present data are individual tumor nodules. Tumor characteristics including the p53 based on the study on intrahepatic metastasis, further investigation tumor suppressor gene, HBV integration, and chromosomal alter- and validation on extrahepatic metastasis are necessary to consolidate ations by CGH analysis all helped to elucidate the lineage of individ- the clinical significance. ual nodules. The present study indicated that the genome wide ex- Primarily, special attention has been paid to the genes that were pression data contributed substantial information to the molecular down-regulated in the metastatic tumors because they could be characterization and clonality of HCC. Poor prognosis is frequently metastasis-suppressor genes (1, 2), responsible for regulating the associated with a high recurrence rate and metastasis. Discrimination growth of disseminated cancer cells. This may lead to the identifica- for independent multicentric occurrence or intrahepatic metastasis tion of novel therapeutic targets for the treatment of metastatic dis- would, therefore, be essential for multiple nodular tumors and in ease. The present study has identified 63 clones, with 35 known genes patients with recurrence after curative surgery. Verification in clonal- that exhibited a lower expression level in metastatic tumors compared ity will affect prognosis and disease management. Conventional dis- with the primaries. One of these genes, the breast-cancer metastasis criminations are mostly based on clinical and pathological findings, suppressor 1 (BRMS1), has been shown to possess the functional which cannot always provide a definitive answer. Using the present capability of decreasing the metastatic potential of breast cancer cells method, we confirmed that four patients demonstrated unicentric HCC that have been transfected with the gene (30). This would be very with intrahepatic metastatic lesions (M3, T1–2; M4, T1–5; M5, T1–3; important in the investigation of its functional role in liver cancer cell M6, T1, 2, and 4). One patient (M2) had intrahepatic metastasis (T1, lines and other cancer types, and to explore whether this gene, 2, 4–6) as well as an independent tumor clone (M2, T7). Another BRMS1, is the common key gene as “metastasis-suppressor.” At least patient (M1) had multicentric liver tumor (T1 and T2 were independ- two other genes may have a key participation: the CD53 and the ent clones). The present approach can provide reliable parameters to EMP3 (Fig. 5A). CD53 is a member of the transmembrane 4 super- define the clonality of multiple nodular liver cancer, which has not family (TM4SF), which is expressed in a number of different cell been established before. types and may be involved in transmembrane signal transduction, The lineage of the multiple nodules had been elucidated through regulation of cell proliferation, and differentiation (31). EMP3 en- expression and genetic data. A number of metastasis-associated genes codes for transmembrane protein and could be responsible for cell have been identified through gene expression profiling. The next growth, differentiation, and apoptosis (32). Both of these genes, CD53 crucial step is to identify the key gene/pathway responsible for me- and EMP3, are membrane proteins that consist of four transmembrane tastasis. Although a large number of genes are associated with me- domains, and possess an important feature of KAI1, a recently dis- 4718

Fig. 5. Hierarchical clustering of 90 metastasis-associated genes. The primary and metastasis tumors were analyzed by Student’s t test with a cutoff value at P Ͻ 0.05 to identify differentially expressed genes. A decreased level of gene expression was observed in 63 clones (A), and increased expression was shown in 27 clones (B) with reference to the primary tumors.

covered metastasis-related gene the down-regulation of which had essential because prognosis and disease management are different for been associated with lymph node or distant metastases in prostate independent multicentric occurrence and intrahepatic metastasis. The cancer (33), pancreatic cancer (34), and colon cancer (35). Reduced same approach can be applied to recurrent disease and distant metas- expression of KAI1 has also been observed in liver cancer (36). As the tasis in various cancer types to elucidate the clonal lineage. Most protein structure of both CD53 and EMP3 shows a high resemblance importantly, metastasis-associated genes can be identified through the to that of KAI1, it is crucial to further investigate their functional role present approach. Identification of candidate genes can help to im- in liver cancer progression to consolidate their role in metastasis. prove the ability to distinguish between unambiguously malignant In summary, the present study demonstrated that expression pro- lesions and indolent lesions, and to assist in the differentiation of files could provide a reliable method to delineate the clonal relation- tumors that are more likely to metastasize (1). Through elucidation of ship of multiple nodules of liver cancer. Clonality verification is the molecular pathways of cancer progression, these intrahepatic 4719

Table 4 Metastasis-associated genes Table 4 Continued Metastasis-suppressed genes (decreased expression in metastatic nodules), known hypothetical protein MGC4604 genes: 39 clones representing 35 genes KIAA0513 gene product Signal transduction likely ortholog of mouse testis expressed gene 27, Hs.6120 docking protein 1, Mr 62,000 (downstream of tyrosine kinase 1) ESTs: 13 clones 3-phosphoinositide-dependent protein kinase-1 110609 phospholipase D2 118053 AA229650 protein tyrosine phosphatase, nonreceptor type 7 DKFZP566C134 protein, Hs.20237 ribosomal protein S6 kinase, Mr 90,000, polypeptide 2 ESTs, Hs.290259 Others ESTs, Hs.36034 ankylosis, progressive (mouse) homolog ESTs, Hs.303520 ankylosis, progressive (mouse) homolog clone LNG02039, Hs.186180 breast cancer metastasis-suppressor 1 clone HEP02442, Hs.28465 crystallin, ␣ A clone RP11-16L21 on chromosome 9, Hs.297143 EMP3 hypothetical protein FLJ22174 FAST kinase hypothetical protein FLJ10036 HIV-1 inducer of short transcripts binding protein KIAA0090 protein fibroblast growth factor 12B KIAA1530 protein cell membrane glycoprotein, 110,000 Mr (surface antigen) anchor attachment protein 1 (Gaa1p, yeast) homolog interleukin 1 receptor, type II mitogen-activated protein kinase kinase 2 macrophage stimulating 1 (hepatocyte growth factor-like) metastasis-associated genes will ultimately be targeted not only for brain-specific protein p25 ␣ the improvement of patient management but, more importantly, for protein tyrosine phosphatase, receptor type, F tumor necrosis factor (ligand) superfamily, member 10 the treatment of metastasis diseases. Transcription factor nuclear receptor corepressor 1 nuclear factor I/X (CCAAT-binding transcription factor) ACKNOWLEDGMENTS transcription factor AP-2 ␣ Transporter/Kinase We thank the members of Hepatobiliary and Pancreatic Surgery at The cytochrome P450, subfamily IIA (phenobarbital-inducible), polypeptide 7 3-hydroxyanthranilate 3,4-dioxygenase University of Hong Kong and members of Stanford Microarray Database at ketohexokinase (fructokinase) Stanford University. peroxisome biogenesis factor 10 phosphomevalonate kinase solute carrier family 2, (facilitated glucose transporter) member 8 REFERENCES solute carrier family 4, anion exchanger, member 2 Leukocyte-related 1. Yoshida, B. A., Sokoloff, M. M., Welch, D. R., and Rinker-Schaeffer, C. W. CD3D antigen, ␦ polypeptide (TiT3 complex) Metastasis-suppressor genes: a review and perspective on an emerging field. J. Natl. CD4 antigen (p55) Cancer Inst. (Bethesda), 92: 1717–1730, 2000. CD53 antigen 2. Yokota, J. Tumor progression and metastasis. Carcinogenesis (Lond.), 21: 497–503, immunoglobulin heavy constant ␥ 3 (G3m marker) 2000. immunoglobulin ␭ locus 3. Liu, F., Qi, H. L., Zhang, Y., Zhang, X. Y., and Chen, H. L. Transfection of the ␣ immunoglobulin ␭ locus c-erbB2/neu gene upregulates the expression of sialyl Lewis X, 1, 3-fucosyltrans- immunoglobulin ␭ locus ferase VII, and metastatic potential in a human hepatocarcinoma cell line. Eur. immunoglobulin ␭ locus J. Biochem., 268: 3501–3512, 2001. Metastasis-enhanced genes (increased expression in metastatic nodules), Known genes: 4. Guo, H. B., Liu, F., Zhao, J. H., and Chen, H. L. Down-regulation of N-acetylglu- 14 clones cosaminyltransferase V by tumorigenesis- or metastasis-suppressor gene and its BUB3 (budding uninhibited by benzimidazoles 3, yeast) homolog relation to metastatic potential of human hepatocarcinoma cells. J. Cell Biochem., 79: centrin, EF-hand protein, 2 370–385, 2000. choline dehydrogenase 5. Hirohashi, S., Bulm, H. E., Ishak, K. G., Deugnier, Y., Kojiro, M., Laurent Pulg, P., eyes absent (Drosophila) homolog 3 Wanless, I. R., Fischer, H. P., Theise, N. D., Sakamoto, M., and Tsukuma, H. hepatic leukemia factor Hepatocellular carcinoma. In: S. R. Hamilton and L. A. Aaltonen (eds.), Classifica- 3-hydroxy-3-methylglutaryl-Coenzyme A reductase tion of Tumours. Pathology and Genetics of Tumours of the Digestive System, pp. kinesin family member 5B 158–172. Geneva, Switzerland: World Health Organization, 2000. mitochondrial ribosomal protein S28 6. Hsu, H. C., Chiou, T. J., Chen, J. Y., Lee, C. S., Lee, P. H., and Peng, S. Y. Clonality oculocerebrorenal syndrome of Lowe and clonal evolution of hepatocellular carcinoma with multiple nodules. Hepatology, profilin 2 13: 923–928, 1991. nuclear phosphoprotein similar to S. cerevisiae PWP1 7. Shimada, M., Hasegawa, H., Gion, T., Shirabe, K., Taguchi, K., Takenaka, K., STRIN protein Tanaka, S., and Sugimachi, K. Risk factors of the recurrence of hepatocellular tumor protein, translationally-controlled 1 carcinoma originating from residual cancer cells after hepatectomy. Hepatogastroen- tumor rejection antigen (gp96) 1 terology, 46: 2469–2475, 1999. ESTs: 24 clones 8. Blum, H. E., Offensperger, W. B., Walter, E., Offensperger, S., Wahl, A., Zeschnigk, 107377 T64192 C., and Gerok, W. Hepatocellular carcinoma and hepatitis B virus infection: molec- 114260 AA598950 ular evidence for monoclonal origin and expansion of malignantly transformed EPI64, Hs.17719 hepatocytes. J. Cancer Res. Clin. Oncol., 113: 466–472, 1987. LOC51295, Hs.22199 9. Chen, P. J., Chen, D. S., Lai, M. Y., Chang, M. H., Huang, G. T., Yang, P. M., Sheu, ESTs Hs.14425 J. C., Lee, S. C., Hsu, H. C., and Sung, J. L. Clonal origin of recurrent hepatocellular ESTs Hs.273741 carcinomas. Gastroenterology, 96: 527–529, 1989. ESTs Hs.128436 10. Sheu, J. C., Huang, G. T., Chou, H. C., Lee, P. H., Wang, J. T., Lee, H. S., and Chen, ESTs Hs.339725 D. S. Multiple hepatocellular carcinomas at the early stage have different clonality. ESTs Hs.260644 Gastroenterology, 105: 1471–1476, 1993. Homo sapiens gene from PAC 426I6, Hs.105911 11. El-Serag, H. B., and Mason, A. C. Risk factors for the rising rates of primary liver cDNA DKFZp586E1624, Hs.94030 cancer in the United States. Arch. Intern. Med., 160: 3227–3230, 2000. clone MGC:17259 IMAGE:4149333, Hs.348136 12. Ochiai, T., Urata, Y., Yamano, T., Yamagishi, H., and Ashihara, T. Clonal expan- clone MGC:20737 IMAGE:4563636, Hs.38163 sion in evolution of chronic hepatitis to hepatocellular carcinoma as seen at an clone MGC:15403 IMAGE:4126342, Hs.337772 X-chromosome locus. Hepatology, 31: 615–621, 2000. Human TB1 gene mRNA, 3Ј end, Hs.75639 13. Paradis, V., Laurendeau, I., Vidaud, M., and Bedossa, P. Clonal analysis of ma- hypothetical protein DKFZp547O146 cronodules in cirrhosis. Hepatology, 28: 953–958, 1998. hypothetical protein FLJ10479 14. Sirivatanauksorn, Y., Sirivatanauksorn, V., Bhattacharya, S., Davidson, B. R., hypothetical protein FLJ10761 Dhillon, A. P., Kakkar, A. K., Williamson, R. C., and Lemoine, N. R. Evolution of hypothetical protein FLJ20186 genetic abnormalities in hepatocellular carcinomas demonstrated by DNA fingerprint- hypothetical protein MGC3181 ing. J. Pathol., 189: 344–350, 1999. hypothetical protein MGC4172 15. Roncalli, M., Bianchi, P., Grimaldi, G. C., Ricci, D., Laghi, L., Maggioni, M., Opocher, E., Borzio, M., and Coggi, G. Fractional allelic loss in non-end-stage 4720

cirrhosis: correlations with hepatocellular carcinoma development during follow-up. detected by comparative genomic hybridization. Genes Chromosomes Cancer, 29: Hepatology, 31: 846–850, 2000. 110–116, 2000. 16. Chen, X., Cheung, S. T., So, S., Fan, S. T., Barry, C., Higgins, J., Lai, K. M., Ji, J., 27. Kallioniemi, A., Kallioniemi, O. P., Piper, J., Tanner, M., Stokke, T., Chen, L., Smith, Ng, I. O., van de Rijn, M., Botstein, D., and Brown, P. O. Gene expression profiles H. S., Pinkel, D., Gray, J. W., and Waldman, F. M. Detection and mapping of in human liver cancers. Mol. Biol. Cell, 2002. amplified DNA sequences in breast cancer by comparative genomic hybridization. 17. DeRisi, J., Penland, L., Brown, P. O., Bittner, M. L., Meltzer, P. S., Ray, M., Chen, Proc. Natl. Acad. Sci. USA, 91: 2156–2160, 1994. Y., Su, Y. A., and Trent, J. M. Use of a cDNA microarray to analyse gene expression 28. Yamamoto, T., Kajino, K., Kudo, M., Sasaki, Y., Arakawa, Y., and Hino, O. patterns in human cancer. Nat. Genet., 14: 457–460, 1996. Determination of the clonal origin of multiple human hepatocellular carcinomas by 18. Perou, C. M., Sorlie, T., Eisen, M. B., van de Rijn, M., Jeffrey, S. S., Rees, C. A., cloning and polymerase chain reaction of the integrated hepatitis B virus DNA. Pollack, J. R., Ross, D. T., Johnsen, H., Akslen, L. A., Fluge, O., Pergamenschikov, Hepatology, 29: 1446–1452, 1999. A., Williams, C., Zhu, S. X., Lonning, P. E., Borresen-Dale, A. L., Brown, P. O., and 29. Chen, Y. J., Yeh, S. H., Chen, J. T., Wu, C. C., Hsu, M. T., Tsai, S. F., Chen, P. J., Botstein, D. Molecular portraits of human breast tumours. Nature (Lond.), 406: and Lin, C. H. Chromosomal changes and clonality relationship between primary and 747–752, 2000. recurrent hepatocellular carcinoma. Gastroenterology, 119: 431–440, 2000. 19. Eisen, M. B., Spellman, P. T., Brown, P. O., and Botstein, D. Cluster analysis and 30. Seraj, M. J., Samant, R. S., Verderame, M. F., and Welch, D. R. Functional evidence display of genome-wide expression patterns. Proc. Natl. Acad. Sci. USA, 95: 14863– for a novel human breast carcinoma metastasis suppressor. BRMS1, encoded at 14868, 1998. chromosome 11q13. Cancer Res., 60: 2764–2769, 2000. 20. Eisen, M. B., and Brown, P. O. DNA arrays for analysis of gene expression. Methods 31. Carmo, A. M., and Wright, M. D. Association of the transmembrane 4 superfamily Enzymol., 303: 179–205, 1999. molecule CD53 with a tyrosine phosphatase activity. Eur. J. Immunol., 25: 2090– 21. Ng, I. O., Chung, L. P., Tsang, S. W., Lam, C. L., Lai, E. C., Fan, S. T., and Ng, M. 2095, 1995. p53 gene mutation spectrum in hepatocellular carcinomas in Hong Kong Chinese. 32. Jetten, A. M., and Suter, U. The peripheral myelin protein 22 and epithelial membrane Oncogene, 9: 985–990, 1994. protein family. Prog. Nucleic Acid Res. Mol. Biol., 64: 97–129, 2000. 22. Hollstein, M., Sidransky, D., Vogelstein, B., and Harris, C. C. p53 mutations in 33. Dong, J. T., Lamb, P. W., Rinker-Schaeffer, C. W., Vukanovic, J., Ichikawa, T., human cancers. Science (Wash. DC), 253: 49–53, 1991. Isaacs, J. T., and Barrett, J. C. KAI1, a metastasis suppressor gene for prostate cancer 23. Hsu, I. C., Metcalf, R. A., Sun, T., Welsh, J. A., Wang, N. J., and Harris, C. C. on human chromosome 11p11.2. Science (Wash. DC), 268: 884–886, 1995. Mutational hotspot in the p53 gene in human hepatocellular carcinomas. Nature 34. Guo, X., Friess, H., Graber, H. U., Kashiwagi, M., Zimmermann, A., Korc, M., and (Lond.), 350: 427–428, 1991. Buchler, M. W. KAI1 expression is up-regulated in early pancreatic cancer and 24. Hussain, S. P., and Harris, C. C. Molecular epidemiology and carcinogenesis: en- decreased in the presence of metastases. Cancer Res., 56: 4876–4880, 1996. dogenous and exogenous carcinogens. Mutat. Res., 462: 311–322, 2000. 35. Lombardi, D. P., Geradts, J., Foley, J. F., Chiao, C., Lamb, P. W., and Barrett, J. C. 25. Lehman, T. A., Bennett, W. P., Metcalf, R. A., Welsh, J. A., Ecker, J., Modali, R. V., Loss of KAI1 expression in the progression of colorectal cancer. Cancer Res., 59: Ullrich, S., Romano, J. W., Appella, E., and Testa, J. R. p53 mutations. ras mutations, 5724–5731, 1999. and p53-heat shock 70 protein complexes in human lung carcinoma cell lines. Cancer 36. Guo, X. Z., Friess, H., Di Mola, F. F., Heinicke, J. M., Abou-Shady, M., Graber, Res., 51: 4090–4096, 1991. H. U., Baer, H. U., Zimmermann, A., Korc, M., and Buchler, M. W. KAI1, a new 26. Guan, X. Y., Fang, Y., Sham, J. S., Kwong, D. L., Zhang, Y., Liang, Q., Li, H., Zhou, metastasis suppressor gene, is reduced in metastatic hepatocellular carcinoma. Hepa- H., and Trent, J. M. Recurrent chromosome alterations in hepatocellular carcinoma tology, 28: 1481–1488, 1998.

4721

Downloaded from cancerres.aacrjournals.org on September 28, 2021. © 2002 American Association for Cancer Research. Identify Metastasis-associated Genes in Hepatocellular Carcinoma through Clonality Delineation for Multinodular Tumor

Siu Tim Cheung, Xin Chen, Xin Yuan Guan, et al.

Cancer Res 2002;62:4711-4721.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/62/16/4711

Cited articles This article cites 34 articles, 8 of which you can access for free at: http://cancerres.aacrjournals.org/content/62/16/4711.full#ref-list-1

Citing articles This article has been cited by 5 HighWire-hosted articles. Access the articles at: http://cancerres.aacrjournals.org/content/62/16/4711.full#related-urls

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/62/16/4711. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.