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Molecular Biology and Genetics |
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
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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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To identify metastasis-associated genes, the first crucial step is to delineate the clonal relationship among the multiple tumor nodules, and to determine which tumor clones were derived from the primary. However, reliable parameters to define the clonality for multiple nodular HCC have not been established. Conventional discriminations are mostly based on clinical and pathological findings (5, 6, 7) , which cannot always provide a definitive answer. Commonly used approaches include comparison for the HBV integration site to differentiate between intrahepatic metastasis and independent occurrence (8, 9, 10) . However, this is only applicable to HCC with HBV integration, whereas tumors related to hepatitis C virus (HCV) or alcohol, which are the common associations in North America (11) , cannot be validated. Clonal analysis can be examined based on X chromosome inactivation (12 , 13) , which can be investigated only in female patients. In East Asia, including Hong Kong, HCC patients are predominantly male; thus, the approach based on X chromosome inactivation does not have a wide implication. DNA fingerprinting (14) and fractional allelic loss (15) analysis have also been used for clonality studies. However, the demanding technique involved combined with 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 examine the genome-wide expression profile of over 200 samples consisting of majority HCC and the tumor adjacent liver tissues (16) . A consistent expression difference was observed between the tumors and their adjacent nontumor livers. To investigate whether the expression profile can help to elucidate the clonal relationship of the multinodular liver cancers, samples from individual tumor foci were further examined with the conventional molecular genetic approach for tumor properties and genetic aberrations. The expression data of 22 individual tumor foci in six patients with multinodular HCC were compared with the conventional molecular genetic data including p53 protein overexpression and p53 gene mutation, HBV integration, and genetic aberrations as investigated by CGH. The data up until now had suggested that gene expression profiles were unique for each tumor and could provide sufficient information to delineate clonal relationships. The identity of primary and intrahepatic metastasis tumor nodules was further confirmed by conventional molecular genetic approaches, and the expression profiles of the original and metastatic tumor clones were compared with those of the genes associated with intrahepatic metastases.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. A total of 22 samples were obtained from individual tumor foci of the surgical specimens. Samples were snap-frozen in liquid nitrogen and stored at -70°C until use. Paralleled tumor specimens were formalin fixed and paraffin embedded for histological examination and immunohistochemical study. Total RNA was extracted with RNeasy kit (Qiagen, Hilden, Germany) and mRNA was isolated from total RNA using FastTrack (Invitrogen, Carlsbad, CA) or Poly(A)Pure (Ambion, Inc., Austin, TX) mRNA purification kit. DNA was extracted with DNAeasy kit (Qiagen) according to the manufacturer’s instructions.
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p53 Protein Staining and Gene Sequencing.
Immunohistochemistry was performed as described previously (21)
with modification. Briefly, antigen retrieval was performed by microwave with sections immersed in citrate buffer. Followed by endogenous peroxidase blocking and biotin blocking reagents (DAKO, Copenhagen, Denmark), antibody DO-7 (DAKO) in 1:100 dilution was applied. Signal was detected by horseradish peroxidase-conjugated secondary antibody and color development with diaminobenzidine (DAB) as the chromogen. Tissue sections were counterstained with hematoxylin.
Direct DNA sequencing was performed for exon 4 to exon 9, which resided with mutational hot-spot and accounted for over 80% of all of the mutations observed (22, 23, 24) . Primer sets and reaction conditions were adopted from Lehman et al. (25) . DNA was amplified by PCR, and direct DNA sequencing was performed with a BigDye sequencing kit (Applied Biosystems, Foster City, CA). Electrophoresis and sequence analysis were performed with ABI PRISM 310 (Applied Biosystems).
HBV Integration by Southern Blot Analysis.
DNA was digested overnight with restriction endonuclease (Ref. 10
; HindIII or EcoRI, Invitrogen) at 37°C with 10 units per µg of DNA. Fragments were electrophoresed on 1% agarose gel and transferred overnight to Hybond membrane (Amersham Pharmacia Biotech, Piscataway, NJ). The probe was full-length HBV fluorescein-labeled by random prime system (Amersham Pharmacia Biotech). After hybridization, stringency washes, and incubation with anti-fluorescein-horseradish peroxidase conjugate, ECL detection reagent (Amersham Pharmacia Biotech) was used, and a signal was developed by X-Omat AR film (Kodak, Rochester, NY).
CGH.
Protocols have been established and details described previously (26
, 27)
. In brief, DNA from tumor and normal reference were labeled with different fluorescent dUTPs by nick translation. Labeled probes were mixed and hybridized onto metaphase cells prepared from the lymphocytes of healthy donors. The slide was counterstained with 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) for digital image analysis using the Quips CGH program (Vysis, Downers Grove, IL). The thresholds used for the interpretation of gains and losses of a DNA sequence copy number were defined as the tumor:reference ratio and were >1.25 and <0.75, respectively. The thresholds applied for both the standard and the reverse hybridization methods.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The characteristic expression relationship of the 22 tumor foci from the six patients (Table 2)
with multinodular liver tumors is highlighted in Fig. 1B
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As a control, we investigated whether sampling the same nodule from the same tumor multiple times would also show variation similar to that seen from the multiple nodular tumors which were the discrete tumor nodules. Replicated samples, a total of 10, were taken from four patients from different regions of a single tumor nodule for comparison. All of the replicate samples from an individual were clustered into one terminal branch of the dendrogram with high correlation. (Fig. 1B)
. This demonstrates that each tumor nodule has its own distinct gene expression patterns, and the differences that we observed in the expression profiles in multiple nodular tumors are not attributable to a sampling artifact.
p53 Protein Expression and Mutation.
To investigate the molecular genetics of these multiple nodular HCC samples, we studied the expression of p53 in these samples by immunohistochemistry. Tissue sections from tumor foci of patients M1, M2, and M4 all showed a negative staining result for p53 protein. And all of the tumor foci from patient M3 showed a positive signal of p53. In patient M5, a heterogeneous staining pattern was observed (Fig. 2A)
. For M5T1, some of the tumor areas had less than 10% cells positive, whereas some of the tumor patches had more than 50% of cells positive. For M5T2 and T3, a homogeneous staining pattern was observed, and the majority of tumor cells showed overexpression of p53 protein. In patient M6, a similar heterogeneity pattern was observed. The majority of tumor cells in M6T2 were negative, although a rare positive signal could be observed in some of them (Fig. 2B)
. In M6T1, the majority of tumor cells showed overexpression of the p53 protein.
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. For patient M6, all three of the tumors exhibited a mutation at exon 5, amino acids 160 and 161 (Fig. 2B)
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HBV Integration Pattern.
To further confirm the clonality of those multiple tumor nodules with distinct gene expression profiles and heterogeneous p53 overexpression patterns, we applied Southern blot analysis for the HBV integration site. As the HBV integration is generally an early event during tumorigenesis, tumors arising from the same clone generally have the same integration pattern. The two tumor nodules, T1 and T2, of patient M1 had different HBV integration patterns (Fig. 3)
, which indicated that T1 and T2 were formed from two distinct clones. Tumor nodules T1 and T2 from patient M3 showed identical integration patterns. Patient M6 also demonstrated the same HBV integration pattern in both T1 and T2. This suggests that tumors from M3 and M6, respectively, were clonally related.
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; Table 3
). The CGH profiles of the two tumor foci T1 and T2 in patient M1 were basically distinct. T1 to T6 from patient M2 shared similar genetic aberrations that were different from T7. Patient M3 showed similar structural changes in both of the tumor foci, with T1 showing additional genetic aberrations compared with the common ones observed in T2. T1 to T5 in patient M4 showed a common structural change. T2 and T3 from patient M5 showed additional genetic aberrations when compared with T1. In patient M6, common genetic aberrations were observed in both T1 and T2, whereas T1 exhibited additional chromosomal changes.
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For patient M1, the two nodules T1 and T2 showed distinct gene expression patterns (Fig. 1B)
. The HBV integration site was different (Fig. 3)
and the genetic composition of the two nodules was discrete by CGH (Fig. 4)
. In addition, the two nodules were separated by a physical distance of 7 cm, which clinically would be classified as a multicentric occurrence (7)
or multicentric HCC (6)
. All of the data obtained supported the observation from the gene expression profile, which suggested that the two tumor nodules were independent clones.
For patient M2, the gene expression profiles of T1 to T6 were very similar (Fig. 1B)
. T7, however, segregated to a different major branch, and was likely to be different in clonality. Clinically, T1 to T6 fit the definition for unicentric HCC (6)
. They appeared as a solitary tumor mass surrounded by contiguously smaller nodules located within 1 cm of each other. However, T7 was an isolated nodule away from the main tumor mass. The histological parallel sections of T1 to T2 and T4 to T6 were all confirmed to be HCC. Interestingly, T3 was reported to have a mixture of tumor cell types, HCC and adenocarcinoma, in the same section. The expression profile by microarray and genetic changes by CGH of T3 were indistinguishable from the other closely surrounded nodules, which suggested that the HCC trait overwhelmed and masked the other tumor cell components. However, T7 was histologically confirmed to be adenocarcinoma without an intermix of HCC components. T7 was clearly demonstrated to be distinct with its gene expression by microarray and chromosomal aberrations by CGH. The data obtained suggested that T1 to T2, and T4 to T6, were clonally linked. T1 revealed the fundamental genetic aberrations common to all of the other tumor nodules and, therefore, was regarded as the primary tumor. T7 was an independent tumor clone.
For patient M3, T1 and T2 were clustered into one terminal branch with a high correlation coefficient of gene expression, suggestive of clonal linkage. Tumor cells from both nodules overexpressed p53 protein with identical mutation point. The HBV integration patterns were also identical. Common genetic aberrations were observed by CGH analysis with T2 showing the basic chromosomal changes. T1 exhibited additional alterations and, thus, should be the derived metastatic tumor nodule as defined by the genetic approach on cancer progression. The gross size of T1 (14 cm) was much bigger than that of T2 (5.5 cm). The difference in tumor size could be explained if the additional genetic changes had conferred it with growth advantage. Nonetheless, the two nodules were situated immediately adjacent and could be clinically classified as unicentric HCC. All of the data agreed well with the gene expression profiles: the two nodules were derived from the same clone and T1 was the metastatic nodule.
All of the tumor nodules from patient M4 showed similar expression profiles. They clustered in one terminal branch of the dendrogram with high values of correlation coefficient, which indicated intrahepatic spread from the original tumor clone. Chromosomal changes as examined by CGH were also similar and supported the clinical observation of clonal relationship. All of the tumor nodules revealed the fundamental genetic alterations specific to this patient, and subtle differences were observed among different nodules. Thus, for the designation of primary tumor, the conventional approach was used, which referred the major tumor mass T1 as the primary.
In patient M5, expression profiles of T2 and T3 were tightly linked and showed some distinct differences from T1. As shown in the dendrogram and by the correlation coefficient, T2 and T3 should be clonal with closer linkage to each other than to T1. The p53 expression pattern corresponded well with the overall gene expression 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 supported other genetic data and clinical observation that all three nodules arose from the same clone, and T2 and T3 were metastatic clones derived from the T1 tumor nodule.
Patient M6 presented a similar situation. The expression profiles of T2 and T4 were tightly linked suggesting definite clonal linkage, whereas T1 was not as tightly clustered but still resided on the same subbranch in the dendrogram. The p53 protein expression pattern coincided with the global gene expression profile. T2 and T4 were similar in the way that only rare tumor cells showed a positive signal, but T1 clearly demonstrated a positive expression for p53 (Fig. 2B)
. Identical gene mutations were observed in all three of the tumor foci. Coherent with the p53 protein expression pattern, the mutant sequence in T2 and T4 had just emerged, whereas a predominant mutant sequence was observed in T1. Similar with the situation in patient M5, the p53 aberration in patient M6 accumulated during tumor progression from T2 to T1. The genetic alterations as revealed by CGH indicated that both T1 and T4 had extra changes in addition to those observed in T2. The similar global gene expression patterns together with p53 aberration, HBV integration site, and common chromosomal aberrations, all implicated that the nodules had clonal relationship and that T1 and T4 were the metastatic tumor and that T2 was the primary.
Differential Genes between Primary and Metastatic Tumor.
Having established the clonal relationship of the multiple nodules and the delineation for the primary and metastatic tumors by the above investigations, the next step was to identify genes differentially expressed between the original and their derived clones. The expression profiles of the primary (T1, unless otherwise indicated that it was not the primary, as described in the following) and their metastatic nodules with the longest physical distance demonstrated with more genetic aberrations were compared. For patient M1, the two tumor foci were independent clones and thus were not included in this part of the study. For patient M2, T1 (primary) was compared with T6 (metastasis) because they indicated clonal linkage and the latter was the furthest away from T1. Similarly, M4T1 was compared with M4T5; and M5T1 was compared with M5T3. For patient M3, T2 was revealed as primary, and T1 would be the metastasis foci. In patient M6, T2 was the primary whereas T1 was the metastasis tumor foci.
The expression profiles of M2T1, M3T2, M4T1, M5T1, and M6T2 were considered as one group representing the primaries. M2T6, M3T1, M4T5, M5T3, and M6T1 were considered as another group representing the metastatic tumors. Student’s t test was used to select for genes that could differentiate between these two groups with a cutoff value at 0.05; 90 representative clones passed the criterion (Fig. 5)
. A total of 63 clones were down-regulated in the metastatic tumors compared with the primary tumors, where 39 clones represented 35 known genes; and 24 clones were ESTs with limited information for their biological function (Table 4)
. A total of 27 clones were up-regulated in the metastatic tumor, in which 14 clones were known genes and 13 clones were ESTs.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Comparison of the expression profiles from multiple specimens taken from an individual patient could indicate the unique characteristics of the tumor that represented the patient’s distinctive feature. The specific similarity and differences in gene expression pattern have provided fundamental information for the clonality relationship of individual tumor nodules. Tumor characteristics including the p53 tumor suppressor gene, HBV integration, and chromosomal alterations by CGH analysis all helped to elucidate the lineage of individual nodules. The present study indicated that the genome wide expression data contributed substantial information to the molecular characterization and clonality of HCC. Poor prognosis is frequently associated with a high recurrence rate and metastasis. Discrimination for independent multicentric occurrence or intrahepatic metastasis would, therefore, be essential for multiple nodular tumors and in patients with recurrence after curative surgery. Verification in clonality will affect prognosis and disease management. Conventional discriminations are mostly based on clinical and pathological findings, which cannot always provide a definitive answer. Using the present method, we confirmed that four patients demonstrated unicentric HCC with intrahepatic metastatic lesions (M3, T1–2; M4, T1–5; M5, T1–3; M6, T1, 2, and 4). One patient (M2) had intrahepatic metastasis (T1, 2, 4–6) as well as an independent tumor clone (M2, T7). Another patient (M1) had multicentric liver tumor (T1 and T2 were independent clones). The present approach can provide reliable parameters to define the clonality of multiple nodular liver cancer, which has not been established before.
The lineage of the multiple nodules had been elucidated through expression and genetic data. A number of metastasis-associated genes have been identified through gene expression profiling. The next crucial step is to identify the key gene/pathway responsible for metastasis. Although a large number of genes are associated with metastasis, some of the changes are believed to be the secondary events; the expression changes as a result of metastasis rather than as an initiator of the metastasis event. Furthermore, as the present data are based on the study on intrahepatic metastasis, further investigation and validation on extrahepatic metastasis are necessary to consolidate the clinical significance.
Primarily, special attention has been paid to the genes that were down-regulated in the metastatic tumors because they could be metastasis-suppressor genes (1
, 2)
, responsible for regulating the growth of disseminated cancer cells. This may lead to the identification of novel therapeutic targets for the treatment of metastatic disease. The present study has identified 63 clones, with 35 known genes that exhibited a lower expression level in metastatic tumors compared with the primaries. One of these genes, the breast-cancer metastasis suppressor 1 (BRMS1), has been shown to possess the functional capability of decreasing the metastatic potential of breast cancer cells that have been transfected with the gene (30)
. This would be very important in the investigation of its functional role in liver cancer cell lines and other cancer types, and to explore whether this gene, BRMS1, is the common key gene as "metastasis-suppressor." At least two other genes may have a key participation: the CD53 and the EMP3 (Fig. 5A)
. CD53 is a member of the transmembrane 4 superfamily (TM4SF), which is expressed in a number of different cell types and may be involved in transmembrane signal transduction, regulation of cell proliferation, and differentiation (31)
. EMP3 encodes for transmembrane protein and could be responsible for cell growth, differentiation, and apoptosis (32)
. Both of these genes, CD53 and EMP3, are membrane proteins that consist of four transmembrane domains, and possess an important feature of KAI1, a recently discovered metastasis-related gene the down-regulation of which had been associated with lymph node or distant metastases in prostate cancer (33)
, pancreatic cancer (34)
, and colon cancer (35)
. Reduced expression of KAI1 has also been observed in liver cancer (36)
. As the protein structure of both CD53 and EMP3 shows a high resemblance to that of KAI1, it is crucial to further investigate their functional role in liver cancer progression to consolidate their role in metastasis.
In summary, the present study demonstrated that expression profiles could provide a reliable method to delineate the clonal relationship of multiple nodules of liver cancer. Clonality verification is essential because prognosis and disease management are different for independent multicentric occurrence and intrahepatic metastasis. The same approach can be applied to recurrent disease and distant metastasis in various cancer types to elucidate the clonal lineage. Most importantly, metastasis-associated genes can be identified through the present approach. Identification of candidate genes can help to improve the ability to distinguish between unambiguously malignant lesions and indolent lesions, and to assist in the differentiation of tumors that are more likely to metastasize (1) . Through elucidation of the molecular pathways of cancer progression, these intrahepatic metastasis-associated genes will ultimately be targeted not only for the improvement of patient management but, more importantly, for the treatment of metastasis diseases.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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 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 Foundation, Stanford University (to X. C.). 
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: stcheung{at}hkucc.hku.hk
3 The abbreviations used are: HCC, hepatocellular carcinoma; HBV, hepatitis B virus; CGH, comparative genomic hybridization; EMP3, epithelial membrane protein 3.
Received 1/28/02. Accepted 6/13/02.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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1, 3-fucosyltransferase VII, and metastatic potential in a human hepatocarcinoma cell line. Eur. J. Biochem., 268: 3501-3512, 2001.[Medline]
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