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From the Department of Surgery, Medical Institute of Bioregulation, Kyushu University, 4546 Tsurumibaru, Beppu, 874–0838 Japan.
ABSTRACT
Background: Cancer-testis antigens (CTA), such as MAGE, are selectively expressed in various types of human neoplasms but not in normal tissues other than testis. This characteristic feature of CTA makes them promising antigens for cancer-specific immunotherapy.
Methods: We investigated the expression of five genes, including MAGE-1, MAGE-3, NY-ESO-1, SCP-1, and SSX-4, in 20 surgical samples of intrahepatic cholangiocarcinomas (IHCC) using reverse transcription-polymerase chain reaction. To visualize the localization of MAGE proteins, we performed immunohistochemical studies. Furthermore, the correlation between the CTA expression and DNA methylation status was studied in three bile duct cancer cell lines.
Results: Expression of MAGE-1, MAGE-3, NY-ESO-1, SCP-1, and SSX-4 was recognized in 4, 4, 2, 6, and 3 of all 20 cases, respectively. In contrast, the expressions of five genes were not recognized at all in the corresponding normal tissues. In 10 cases (50%), the tumors expressed at least one of the five CTA. An immunohistochemical analysis of MAGE proteins demonstrated homogenous or focal distributions in cytoplasm of the IHCC. Using a demethylating agent, MAGE-1, NY-ESO-1, SCP-1, and SSX-4 were induced in two of three cell lines, whereas MAGE-3 was not.
Conclusions: Half of the tumor tissues of IHCC expressed at least one of the CTA. Some of the patients with IHCC, therefore, should be candidates for potentially useful cancer-specific immunotherapy.
Key Words: MAGE • Cancer-testis antigen • Immunotherapy • Cancer vaccine • Cholangiocarcinoma
Intrahepatic cholangiocarcinoma (IHCC) is the second most common primary malignant tumor of the liver, comprising approximately 5% to 10% of all malignant tumors of the liver.1 It is rarely diagnosed at an early stage, because no characteristic symptoms or signs are seen in the early stage of IHCC. Most patients, thus, have advanced disease at the time of diagnosis, thereby resulting in a poor survival. The therapeutic options include surgery, radiation therapy, or chemotherapy; however, these modalities do not yield good results. For example, the most effective treatment may be surgery, however, the 1- and 5-year survival rates are approximately 50% and 20%, respectively.2 A great need, therefore, exists for the development of novel therapeutic approaches for patients with IHCC.
Recently, several genes coding tumor rejection antigens such as MAGE,3 BAGE,4 and GAGE,5 have been isolated from melanoma cell lines. These antigens are recognized by autologous cytotoxic T lymphocytes (CTL), which are restricted by human leukocyte antigen (HLA) class I molecules. Some of these antigens are expressed in various tumors, but not in normal tissues other than testis. They, therefore, have been designated as cancer-testis antigens (CTA),6 and their characteristics make them promising candidates for cancer-specific immunotherapy.
A novel method termed SEREX (serological analysis of antigens by recombinant expression cloning)7 permits the direct molecular determination of new tumor antigens that elicit an IgG antibody response in tumor patients. With this method, several novel genes with tumor specificity (e.g., NY-ESO-18, SCP-19, and SSX10) were identified. These antigens are also expected to become new candidates for cancer-specific immunotherapy (e.g., MAGE).
We previously reported that MAGE-1 and MAGE-3 genes were expressed in gastrointestinal carcinomas.11–15 To the best of our knowledge, however, no information is available on the expression of CTA in IHCC. If they are expressed, then we should be able to develop potentially useful cancer-specific immunotherapy for both gastrointestinal carcinomas and IHCC. Previous evidence has clearly defined the regulatory role of DNA methylation in the constitutive expression of CTA by cancer cells, thereby showing that in vitro treatment with demethylation agents induced or up-regulated their expression in cancer cells.13,16–19 If an up-regulation of the expression of CTA genes can be induced with a demethylation agent, 5-Aza-2'-deoxycytidine (DAC), in IHCC, the number of potential clinical cases appropriate for specific immunotherapy might increase. This study was conducted to determine whether CTA are expressed in IHCC. Furthermore, the effect of DAC on the regulation of CTA gene expressions in human bile duct cancer cell lines was also examined.
MATERIALS AND METHODS
Clinical Cases: Tissue Samples
We studied 20 patients with IHCC who had surgery at the Medical Institute of Bioregulation Hospital and the Department of Surgery and Science, Kyushu University. All 20 patients had hepatectomies for primary tumors. No patients demonstrated complications from either hepatolithiasis or primary sclerosing cholangitis. Following the hepatectomies, the tumor and corresponding nontumor tissue specimens were immediately frozen in liquid nitrogen, and kept at –90°C until use. Informed consent was obtained in writing from all patients. The study was performed according to the latest revision of the Helsinki’s Declaration (1989) for human research.
Extraction of RNA and RT-PCR Analysis
The acid guanidinium thiocyanate-phenol-chloroform extraction procedure was used to extract total RNA, and the complementary DNA (cDNA) was synthesized from 8.0 µg of total RNA as described previously.20 The presence of MAGE-1 and MAGE-3 cDNA in the reverse transcription products was detected by polymerase chain reaction (PCR) amplification in separate reactions, using oligonucleotide primers located in the different exons of the each gene.21 NY-ESO-1 was determined by PCR amplification using oligonucleotide primers under the same conditions as reported previously.22 SSX–4 and SCP-1 were also detected by PCR amplification reactions using the same conditions as previously reported.23 Briefly, a 1/100 aliquot of the reverse transcription products was amplified in a 30-µl reaction mixture containing 10 nmol each of dNTP (dATP, dTTP, dCTP, dGTP), 1 µl of each oligonucleotide primer at 10 µM, 3 µl of 10xPCR buffer (Perkin-Elmer, Branchburg, NJ), and 1U of AmpliTaq DNA polymerase (Perkin-Elmer). To detect SCP-1, 2 U of AmpliTaq Gold (Perkin-Elmer) were substituted for AmpliTaq DNA polymerase. The reaction mixtures were then subjected to the appropriate PCR programs as listed in Table 1. To confirm the specificity of the PCR products of the genes, we cloned the PCR product into pCRII vector (Invitrogen, San Diego, CA) and then sequenced the cDNA by using the chain-termination DNA sequencing method; determined the nucleotide sequence of representative samples of PCR products; and confirmed them to be identical to the expected fragments of cDNA in each CTA gene. Next, an 8-µl aliquot of each PCR product was separated on a 1.5% agarose gel and visualized with ethidium bromide staining. The pattern of expression and the transcription level of the CTA genes were established semiquantitatively using reverse transcription-PCR (RT-PCR) assays by evaluating the intensity of a band in agarose gel. The integrity of the RNA was confirmed by performing PCR amplification of each cDNA with primers for the gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH).24
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Immunohistochemistry
The primary antibodies used in this study were 57B and 77B mouse monoclonal antibody (mAb) (both kindly provided by Dr G. C. Spagnoli).25,26 The 57B mAb stains tumor tissues that express MAGE-325 and MAGE-4,26 and 77B mAb stains tumor tissues that express MAGE-1.27 The frozen tumor sections were thawed, washed in phosphate-buffered saline (PBS), fixed in formalin and incubated overnight at 4°C in the presence of hybridoma supernatant from 57B or 77B. For both preparations, bound antibodies were visualized using a Dako LSAB Peroxidase Kit, Peroxidase (Dako, CA), according to the manufacturer’s recommendations.
Clinicopathologic Data
All data, including sex, age, stage of disease, and pathologic factors, were available from the clinical and pathologic records. The tumors with or without an expression of these genes were then compared.
Statistical Analysis
For continuous variables, the data were expressed as the means ± standard deviation. To compare the clinicopathologic characteristics between the two patient groups, either the unpaired Student t test or the 
2 test was used. A two-sided P value < .05 was considered to be significant.
Experimental Studies: Tumor Cell Lines
The human bile duct cancer cell lines, HuCC-T1, TFK-1, and HuH28, were provided from the Cell Resource Center for Biomedical Research Institute of Development, Aging and Cancer, Tohoku University, Japan. All cell lines were maintained in an RPMI 1640 medium supplemented with 10% of fetal calf serum and antibiotics.
Treatment of Cells with 5-Aza-2'-Deoxycytidine
Tumor cell lines were grown in Roswell Park Memorial Institute (RPMI) 1640 in T-75 flasks (Costar Inc., Cambridge, MA) in a 5% co2 incubator at 37°C; flasks were wrapped in aluminum foil to protect them from light, and 1 mM DAC (Sigma Chemical Corp., St. Louis, MO) was added for 48 h as described previously.13
Preparation of RNA and cDNA
The cells were harvested before and after (48 h) the DAC treatment. Total RNA isolated by the acid guanidine phenol chloroform (AGPC) procedure was treated with deoxyribonuclease (DNase) (Promega, Madison, WI) at 37°C for 15 minute followed by phenol/chloroform treatment. The cDNA was synthesized as mentioned above.
Assay for CTA Gene Expression
The presence of MAGE-1, MAGE-3, NY-ESO-1, SCP-1, and SSX-4 cDNA was detected by PCR amplification in separate reactions, using oligonucleotide primers. The sequences of the primers for the amplification of these CTA genes were the same as those noted above. The amplification and analysis were the same as the assays in the tissue samples.
RESULTS
Clinical Cases
No expression of the five CTA was observed in any of the matched control samples of normal tissue. The expression of MAGE-1, MAGE-3, NY-ESO-1, SCP-1, and SSX-4 was recognized in 4 (20%), 4 (20%), 2 (10%), 6 (30%), and 3 (15%) of all 20 cases of IHCC, respectively. One case (case 20) expressed all five genes, one case (case 12) expressed three genes, three cases (cases 5, 13, and 17) expressed two genes, and five cases (cases 3, 4, 7, 8, and 9) expressed one gene (Fig. 1). Consequently, 10 of 20 (50%) expressed at least one of these five genes. In addition, the remaining 10 showed no expression of these genes. Several pathologic factors were compared between the cases with (n = 10) or without (n = 10) such gene expression. Although the number of cases examined in this study was relatively small, no significant differences between the two groups were observed regarding such factors as age, sex, tumor size, lymph node metastasis, lymphatic vessel permeation, perineural invasion, and histologic grade (Table 2).
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We investigated the distribution of carcinoma cells expressing MAGE proteins by an immunohistochemical analysis for representative cases. Homogeneous (Fig. 2A) and heterogeneous (Fig. 2B) immunoreactivity of 57B were detected in the IHCC cells of the resected tissue specimens. By analyzing in the consecutive sections, the MAGE-1 gene product (Fig. 2C) was detected in the IHCC cells not expressing either the MAGE-3 or MAGE-4 gene product (Fig. 2D).
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Experimental Studies
HuH-28 expressed three genes, including MAGE-1, MAGE-3, NY-ESO-1, whereas HuCC-T1 or TFK-1 expressed none of the five CTA genes. Following 48 hours of incubation with DAC, the gene expressions of MAGE-1, NY-ESO-1, SCP-1, and SSX-4 were induced in both HuCC-T1 and TFK-1. No induction, however, was recognized for SCP-1 and SSX-4 in HuH-28 (Table 3). Figure 3 shows the expression status of NY-ESO-1, SCP-1, and SSX-4 genes before and after the DAC treatment.
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DISCUSSION
We previously reported that MAGE-1 and MAGE-3 genes were expressed in 62% and 57% of esophageal carcinomas,11 41% and 38% of gastric carcinomas,12 30% and 20% of colorectal carcinomas,13 24% and 31% of breast carcinomas,14 and >65% of hepatocellular carcinoma.15 We also identified MAGE-1 and MAGE-3 gene-encoded peptides recognized by CTL.28,29 Based on these findings, clinical trials of cancer-specific immunotherapy have been ongoing for patients with gastrointestinal carcinomas, using antigen-presenting cells (dendritic cells) pulsed with MAGE-1 or MAGE-3 peptides. The prerequisite for therapy is MAGE gene expressions in tumor tissues and adaptation of patient HLA class I type to peptide-binding specificity. HLA-A24 is the most popular type and it is seen in 60% of all Japanese, so we originally identified28,29 and have used MAGE-1, or MAGE-3 peptides that have been adapted to HLA-A24. Until January 2004, 21 patients with recurrent disease or far-advanced disease had this therapy (the results of 12 patients have been published elsewhere30). Good results were observed in some clinical cases with no side effects. We, therefore, are convinced that this therapy can be applied safely both to patients with gastrointestinal tract carcinoma and to those with other carcinomas (e.g., IHCC), if the above-stated prerequisites are satisfied.
This study demonstrated that five patients showed IHCC with MAGE-1, MAGE-3, or both genes. Four of the five patients showed HLA-A24, so these patients were good candidates for cancer-specific immunotherapy. On the other hand, almost four-fifths of the patients were excluded from this therapy. To expand the number of candidate patients, CTA other than MAGE were evaluated. Consequently, 10 (50%) patients expressed at least one of the five CTA examined. This showed that if the peptides that are adapted to a patient’s HLA type are identified, about a half of the patients with IHCC can become candidates for cancer-specific immunotherapy. We recently identified NY-ESO-1 peptides31 that can be adapted to HLA-A24.
We found that SCP-1 was expressed more frequently than MAGE-1 or MAGE-3. Six tumors expressed the SCP-1 gene; four of these expressed SCP-1 only and the other two expressed both MAGE genes also. The result suggests that if SCP-1 is a new target antigen, in addition to MAGE-1 and MAGE-3, the number of candidates for CTA-based cancer immunotherapy for IHCC will increase. SCP-1 was identified as HOM-TES-14, during the screening of a cDNA library enriched for testis-specific clones with serum from a renal cell carcinoma patient. High expression levels of SCP-1 in tumor tissue and normal testis were recognized at the mRNA and protein levels by RT-PCR and a Western blot analysis, respectively.9 The antigenic peptides encoded by these CTA will be identified in the future studies. Patient’s opportunities for cancer-specific immunotherapy, thus, can be expanded.
Another problem of cancer-specific immunotherapy is the heterogeneous expression pattern of CTA. Others and we have reported the heterogeneous expression of MAGE genes in tumor tissue specimens using immunohistochemical analyses.32,33 This means that even antigen-expression positive tumors by RT-PCR showed more or less negative areas within the tumor as shown by immunohistochemical studies. This may be a strategy for the evasion of immunosurveillance by malignant cells. We previously reported the heterogeneous expression of MAGE-4 and NY-ESO-1 in breast carcinoma tissue.23 In such cases, a combined vaccination based on both MAGE-4 and NY-ESO-1 may cause immunologic reactions in a larger area of tumor tissues than a single peptide-based vaccination. This means that polyvalent vaccinations with multiple antigens might be necessary to obtain good clinical results when performing cancer immunotherapy.
In the present study, we noticed that the incidence of IHCC expressing MAGE genes (20%) was relatively lower than that of other digestive organ carcinomas, such as esophageal carcinoma (60%),11 gastric carcinoma (40%),12 and hepatocellular carcinoma (>65%).15 To increase the number of patients who are appropriate for tumor-specific immunotherapy, it is desirable to induce the expression of CTA before performing immunotherapy in patients whose tumor does not express such genes. In fact, the expression of CTA genes such as MAGE, NY-ESO-1, SSX, or CAGE has been reported to be induced by demethylation agents.13,16–19 For example, Weber et al.16 demonstrated that DAC can induce MAGE-1 gene expression in MAGE-1 negative melanoma cell lines but not in normal blood cells or diploid cell lines. Our study of three bile duct cancer cell lines disclosed that MAGE-1, NY-ESO-1, SCP-1, and SSX-4 gene expressions were induced in two cell lines which otherwise showed a negative expression of these CTA genes. Another way to increase the number of patients who may successfully respond to cancer-specific immunotherapy, therefore, is to give demethylation agents to patients whose tumors do not express the CTA gene. These findings raise the possibility that CTA gene-specific immunotherapy in combination with the prior use of a demethylation agent might be beneficial in patients with IHCC.
After preparation of this manuscript, we learned that Tsuneyama et al.34 reported the frequent expression (47%) of MAGE-3 in IHCC, which they suggested is a promising target molecule for the specific immunotherapy of IHCC.
Acknowledgments:
We thank to Dr. G. C. Spagnoli for kindly providing the CTA-specific antibodies. We are also grateful to Ms. J. Miyake, Ms. K. Ogata, and Ms. T. Shimooka for their excellent technical assistance.
FOOTNOTES
Received January 30, 2004; accepted July 5, 2004.
This work was supported in part by Grants-in-Aid for Scientific Research (B) (15390398 and 14370358) and for Scientific Research (C) (15591412 and 15591411), Japan Society for the Promotion of Science and Grants-in-Aid for Exploratory Research (14657286), the Ministry of Education, Culture, Sports, Science and Technology of Japan.
Address correspondence and reprint requests: Masaki Mori, MD, PhD, Department of Surgery, Medical Institute of Bioregulation, Kyushu University, 4546 Tsurumibaru, Beppu, 874–0838, Japan; Fax: 81-977-27-1651; E-mail: mmori{at}beppu.kyushu-u.ac.jp.
REFERENCES
7 expression as a new clinicopathological marker in patients with intrahepatic cholangiocarcinoma. Arch Surg 2002;137:181–5.
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