Zhao L, Mou DC, Leng XS, Peng JR, Wang WX, Huang L, Li S, Zhu JY. Expression of cancer-testis antigens in hepatocellular carcinoma. World J Gastroenterol 2004; 10(14): 2034-2038 [PMID: 15237429 DOI: 10.3748/wjg.v10.i14.2034]
Corresponding Author of This Article
Dr. Xi-Sheng Leng, Center of Hepatobiliary Surgery, People’s Hospital, Peking University, 11 XiZhimen Nandajie, West District, Beijing 100044, China. lengxs2003@yahoo.com.cn
Article-Type of This Article
Liver Cancer
Open-Access Policy of This Article
This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/
Li Zhao, Xi-Sheng Leng, Ji-Run Peng, Wan-Xiang Wang, Lei Huang, Shu Li, Ji-Ye Zhu, Center of Hepatobiliary Surgery, People’s Hospital, Peking University, Beijing 100044, China
Dong-Cheng Mou, Department of General Surgery, First Affiliated Hospital of TsingHua University, Beijing 100016, China
ORCID number: $[AuthorORCIDs]
Author contributions: All authors contributed equally to the work.
Supported by the National Natural Science Foundation of China, No. 30200271, and the National Key Technologies Research and Development Program of China during the 9th Five-Year Plan Period, No. 2001BA703B04, and the Clinical Program of Ministry of Health of China (2001) and Center for Human Disease Genomics of Peking University (2001-6)
Correspondence to: Dr. Xi-Sheng Leng, Center of Hepatobiliary Surgery, People’s Hospital, Peking University, 11 XiZhimen Nandajie, West District, Beijing 100044, China. lengxs2003@yahoo.com.cn
Telephone: +86-10-68314422-3500
Received: February 2, 2004 Revised: February 15, 2004 Accepted: February 21, 2004 Published online: July 15, 2004
Abstract
AIM: To investigate the expression of cancer-testis (CT) antigens MAGE-1, SSX-1 ,CTp11 and HCA587 genes in hepatocellular carcinoma (HCC) and the possibility of applying these antigens as targets for specific immunotherapy for HCC.
METHODS: Expression levels of MAGE-1, SSX-1, CTp11 and HCA587 mRNA were detected with reverse transcription polymerase chain reaction (RT-PCR) in HCC tissues and corresponding adjacent non-cancerous tissues from 105 HCC patients, 40 samples of cirrhosis and normal liver tissues. Genes of five samples with positive PCR results were sequenced.
RESULTS: Of 105 HCC tissues, MAGE1, SSX-1 ,CTp11 and HCA587 mRNA expressions were detectable in 75.2% (79/105), 72.4% (76/105), 62.9% (66/105) and 56.2% (59/105) of HCC samples, respectively. About 93.3% (98/105), 72.4% (76/105), 48.6% (51/105) and 37.1% (39/105) of HCC tissues positively expressed at least one, two, three, and four members of CT antigens, respectively. Conversely, only SSX-1 could be detectable in 2.9% (3/105) of the corresponding adjacent non-HCC tissues in which no metastatic lesion was found. Of the latter 3 patients, biopsy samples far from tumor were obtained in 2 patients and RT-PCR indicated no expression of SSX-1 mRNA in these two samples. In addition, none of 40 samples of cirrhotic and normal liver tissues expressed CT antigen gene mRNA. DNA sequences confirmed that the RT-PCR products were true target cDNA. No relationship was found between expression of CT antigens and clinico pathological indicators such as age, gender, tumor size, degree of tumor differentiation, serum α-fetoprotein level and infection of hepatitis B virus or hepatitis C virus (P > 0.05).
CONCLUSION: CT antigens genes (MAGE-1, SSX-1, CTp11 and HCA587) are expressed with high percentage and specificity in HCC and their products are promising targets for antigen-specific immunotherapy of HCC. High frequent co-expression of multiple members of CT antigens in HCC provides possibility of polyvalent vaccinations for HCC.
Key Words: $[Keywords]
Citation: Zhao L, Mou DC, Leng XS, Peng JR, Wang WX, Huang L, Li S, Zhu JY. Expression of cancer-testis antigens in hepatocellular carcinoma. World J Gastroenterol 2004; 10(14): 2034-2038
Hepatocellular carcinoma (HCC) is a common tumor with a poor prognosis, irrespective of a variety of treatment options[1,2]. So, there is a need to develop additional therapeutic approaches for the management of this disease. A proposed strategy is immunotherapy, which has the potency to eradicate systemic tumor cells in multiple sites in the body and the specificity to discriminate between neoplastic and non-neoplastic cells[3,4]. Genes or antigens, which are either exclusively or preferentially expressed in malignant tissues, are a prerequisite for antigen-specific cancer immunotherapy[4,5].
Recently, the integration of molecular and immunological techniques has led to the identification of a new category of tumor-specific antigens called cancer-testis (CT) antigens, such as MAGE superfamily, SSX family, GAGE, BAGE, SCP-1, NY-ESO-1, CTp11 and HCA587[6,7]. The CT antigens share several distinct features including: (1) CT antigen mRNA is expressed predominantly in tumors of different origins with various frequencies but not in normal tissues except for testis; and (2) the genes encoding CT antigens locate at the X chromosome[8,9]. The combination of CT antigen peptides and human leukocyte antigen (HLA), when efficiently presented by antigen presenting cells to cytotoxic T lymphocytes (CTL), is capable of eliciting cellular immune responses in cancer patients[10,11]. Also, some members of CT antigens are capable of inducing humoral immunity[12,13]. Both cellular and humoral immune response can kill tumor cells specifically. The testis is an immune privileged organ because spermatogenic cells do not express HLA class I antigen at their surface. Concomitantly, the testis has a so-called blood-testis barrier, which limits contact between testicular and immune cells, in the seminiferous tubuli generated by the Sertoli cells[7,14]. Because of these features, CT antigens have the potential to provide specific antitumor immunity directly to malignant cells without harm to normal cells. At present, the application of tumor antigenic peptide vaccine based on CT antigens for cancer immunotherapy has become a hot spot[15,16].
The screening of tumor antigens recognized by autologous cytotoxic T cells led to the isolation of MAGE-1, which has been identified as a member of MAGE-A genes family[17]. Clinical trials targeting MAGE-1 antigen are in progress for malignant melanoma and have shown favorable curative effect[18,19]. SSX genes and HCA genes were originally identified using serological analysis of recombinant cDNA expression libraries (SERAX). SEREX was to define B cell epitopes in antigens that were specifically recognized by antibodies in cancer sera, suggesting that SSX and HCA antigens were immunogenic and capable of inducing an antibody response[6,20]. In a recent report, Li et al[25] have proved HCC patients were able to develop humoral immune response to HCA587 antigen. Based on representational difference analysis of cDNA, Zendman et al[21] first identified the expression of CTp11 in melanoma cell lines. In another study (unpublished data), our group predicted HLA-A2-restricted CTL epitopes of CTp11 by peptide supermotif prediction combined with quantitative motif [22,23] and found the potential HLA-A2-restricted CTL epitopes of CTp11 antigen (HLA-A2 is the most common HLA-A allele in Asian population, especially in Chinese, with an estimated frequency of more than 50%[24,25]), suggesting the possibility of CTP11 antigen to trigger cellular immunity and a new potential vaccine candidate for HCC immunotherapy.
Several studies reported high frequent expression of CT antigens in the HCC tissues, such as MAGE-1[26,27] and SSX-1[4,28]. However, the expression of CT antigens is heterogenous in a variety of human tumors, which may be a way for malignant cells to evade from immunosurveillance[7,29-31]. Screening highly and specifically expressed multiple CT antigens in tumors and using them as targets for immunotherapy are promising means to overcome the heterogenous expression of tumor cells, thus expanding patients’ opportunities for cancer specific immunotherapy and establishing the basis of polyvalent vaccinations[29].
In an effort to improve the efficiency of tumour vaccines and prevent immune escape, we investigated the expression of CT antigens MAGE1, SSX-1, CTp11 and HCA587 in HCC tissues.
MATERIALS AND METHODS
Data of patients
From October 2000 to September 2003, 105 patients receiving operation for HCC, including hepatectomy or orthotopic liver transplantation, at the 2nd Hospital of Peking University Health Science Center, were enrolled in this study. There were 90 men and 15 women with a mean age of 47.2 ± 8.4 years (range: 18-75). The hepatitis B surface antigen was positive in 98 cases. Among 105 cases, anti-HCV was positive in 5 cases. The serum α-fetoprotein (α-FP) level was normal (< 20 ng/mL) or elevated slightly (< 40 ng/mL) in 35 patients. Histopathological examination indicated that 18 HCC samples were well differentiated, 61 moderately and 26 samples poorly differentiated, respectively. According to TNM classification of the International Union Against Cancer[32], there were 13 cases at stage I, 30 at stage II, 12 at stage III and 50 at stage IV, respectively. Seventy-nine patients had large-sized tumors (> 5 cm), while the tumor size of the other 26 patients was equal to or less than 5 cm (≤ 5 cm). The control samples included 20 tissues from cirrhosis patients and 20 normal liver tissues from patients without liver disease by surgical biopsy. Testis tissues (kindly provided by Urological Department of the 2nd Hospital of Peking University Health Science Centre) were used as the positive control. Each sample was immediately frozen in liquid nitrogen after a surgical resection and stored at -80 °C until the extraction of total RNA. Informed consent was obtained from each patient before the study was conducted. The Ethic Committee of Peking University approved the study protocol.
Total RNA extraction and synthesis of cDNA
Total RNA was extracted from frozen tissue specimens (50-100 mg) using TRIzol reagent (GIBCOL BRL) according to the protocol provided by the manufacturer. Total RNA (2.5 μg) was primed with an Oligo (dT) 15 oligonucleotide (Promega) and reverse-transcribed according to manufacturer’s instructions.
PCR amplification of CT antigens
The amplification reaction contained 5 μL of a 1:5 dilution of reverse-transcribed products, 1 μL each of 10 μmol/L specific primers, 1 μL 10 mmol/L dNTP mixture (dATP, dGTP, dCTP, dTTP), 2.5 U Taq DNA polymerase (Gibco BRL) and PCR-buffer solution. The total volume was brought to 50 μL using water. The PCR amplifications were performed in a UNO II thermocycler (Perkin-Elmer, USA) under the following conditions: After an initial denaturation for 5 min at 94 °C, samples were subjected to 35 cycles of amplification, followed by a final extension of 8 min at 72 °C. The length of PCR products was 421 base pair (bp) (MAGE-1), 422 bp (SSX-1), 297 bp (CTp11) and 238 bp (HCA587), respectively. To verify that the RNA had not degraded, a PCR assay with specific primers for the gene β2-microglobin (Β2-MG) was performed on each cDNA sample under the following conditions: After an initial heating for 2 min at 94 °C, samples were subjected to 30 cycles of amplification, followed by a final extension of 8 min at 72 °C. The product size was 335 bp. The PCR conditions and specific primer sets for MAGE-1[33], SSX-1[4], CTp11[21], HCA587[34] and Β2-MG[26] used in this study are shown in Table 1.
Temperature, duration of denaturation, annealing, extension
Primer sequence
Product size (bp)
MAGE-1
94 °C, 45 s
f: 5’-CGGCCGAAGGAACCTGACCCAG-3’
421
65 °C, 45 s
r: 5’-GCTGGAACCCTCACTGGGTTGCC-3’
72 °C, 45 s
SSX-1
94 °C, 60 s
f: 5’-CTAAAGCATCAGAGAAGAGAAGC-3’
422
57 °C, 60 s
r: 5’-AGATCTCTTATTAATCTTCTCAGAAA- 3’
72 °C, 60 s
CTp11
94 °C, 45 s
f: 5’-CTGCCCCAGACATTGAAGAA-3’
297
57 °C, 60 s
r: 5’-TCCATGAATTCCTCCTCCTC-3’
72 °C, 90 s
HCA587
94 °C, 30 s
f: 5’-AGGCGCGAATCA AGTTAG-3’
238
60 °C, 30 s
r: 5’-CTCCTCTGCTGTGCTGAC-3’
72 °C, 30 s
Β2-MG
94 °C, 45 s
f: 5’-CTCGCGCTACTCTCTCTTTCTGG -3’
335
55 °C, 45 s
r: 5’-GCTTACATGTCTCGATCCCACTTA-3’
72 °C, 45 s
At last, 10 μL of reaction product was analyzed by electrophoresis on a 20 g/L agarose gel (Promega, USA), followed by ethidium bromide staining and digital camera photographing (Korda D3.5, USA).
Sequence analysis of PCR products
To confirm the specificity of the RT-PCR products of the CT antigen genes, we performed the sequence analysis. In brief, purified PCR products were cloned into pGEM-T Easy Vector (Promega) by T4 DNA ligase and amplified in E. coli, JM109. Five cases of positive colonies were selected and assessed using EcoRI digestion of mini-prepared DNA. The putative MAGE-1, SSX-1, CTp11 and HCA587 cDNA samples were sequenced with T7 sequencing primers in Sangon Co, Shanghai, China.
Statistical analysis
The statistical analysis was assessed using the chi-square test and the Fisher’s exact test, and the significant level was set at P < 0.05.
RESULTS
Expression of CT antigen genes
Among the 105 HCC tissues investigated, MAGE1, SSX-1, CTp11 and HCA587 mRNA was expressed in 75.2% (79/105), 72.4% (76/105), 62.9% (66/105) and 56.2% (59/105) of tumor samples from HCC patients, respectively. At least one CT antigen mRNA was positive in 93.3% (98/105) of HCC tissues. Conversely, only SSX-1 could be detected in 2.9% (3/105) of the corresponding adjacent non-HCC tissues in which no metastatic lesions were found. Of the latter 3 patients, biopsy samples far from tumor were obtained in 2 patients and RT-PCR indicated no expression of SSX-1 mRNA was detectable in these 2 samples. No expression of MAGE-1, CTP11 and HCA587 was detected in the samples of corresponding adjacent non-cancerous tissues. In addition, none of 40 samples of cirrhosis and normal liver tissue expressed each CT antigen mRNA. In consistent with Chen et al[4]’s findings, the electrophoretogram analysis revealed the intensities of PCR products varied considerably among different samples, indicating HCC cells expressing tumor antigens at mRNA level are heterogeneous. The typical electrophoresis of RT-PCR products amplified from cDNA of tissue samples of some HCC patients is shown in Figure 1.
Figure 1 Electrophoresis of RT-PCR products amplified from cDNA of HCC tissues.
M: Molecular marker, 100-bp DNA lad-der (Gibco); P: Positive control, testis tissues; Ne: Negative control, PCR amplification in the absence of template; Β2-MG: 335-bp cDNA quality control; T: HCC tissues, arrows indicate the RT-PCR products of CT antigens; N: Corresponding adja-cent non-tumor tissues. All the samples shown here except for the negative control are positive.
Highly synchronous expression of multiple CT antigens in HCC samples
Highly frequent co-expression of multiple members of CT antigens was observed in HCC samples, with synchronous expression of at least two, three, and four of these gene in 72.4% (76/105), 48.6% (51/105) and 37.1% (39/105) of HCC tissues, respectively. We also analysed the cases with expression of neither MAGE-1 nor SSX-1 genes to determine targets for cancer immunotherapy other than MAGE-1 and/or SSX-1. Among 18 HCC patients with expression of neither MAGE-1 nor SSX-1, 61.1% (11/18) of HCC samples expressed CTp11 and/or HCA587.
Sequence analysis of PCR products
Sequence analysis of PCR products verified the nucleotide sequences of MAGE-1, SSX-1, CTp11 and HCA587 cDNA fragments were identical to that in the database of the GenBank. It confirmed that the RT-PCR products were true MAGE-1, SSX-1, CTp11 and HCA587 cDNA (data not shown).
Expression of CT antigens and clinical indicators
No relationship was found between the expression of CT antigens and clinico pathological indicators such as age, gender, differentiation degree of tumour, serum α-FP level, TNM classification, tumor size, infection of hepatitis B virus or hepatitis C virus (P > 0.05). However, among 35 patients with the normal (< 20 ng/mL) or slightly elevated (< 40 ng/mL) serum α-FP level, 32 patients had MAGE-1, SSX1, CTp11 or HCA587 gene transcripts detected in their HCC tissues. The results are demonstrated in Table 2.
Table 2 Expression of CT antigens and clinico pathological indicators.
Group
n1
MAGE-1
SSX-1
CTp11
HCA587
CT2(+)
+
-
+
-
+
-
+
-
+
-
Total number
105
79
26
76
29
66
39
59
46
98
7
Gender
Male
90
70
20
66
24
57
33
48
42
85
5
Female
15
9
6
10
5
9
6
11
4
13
2
Tumor diameter
≤ 5 cm
26
18
8
16
10
14
12
15
11
22
4
> 5 cm
79
61
18
60
19
52
27
44
35
76
3
TNM stage
I + II
43
29
14
31
12
27
16
22
21
39
4
III + IV
62
50
12
45
17
39
23
37
25
59
3
Hepatitis B virus
HBV (+)
98
74
24
71
27
62
36
55
43
91
7
HBV (-)
7
5
2
5
2
4
3
4
3
7
0
Hepatitis C virus
HCV (+)
5
5
0
4
1
4
1
4
1
5
0
HCV (-)
100
74
26
72
28
62
38
55
45
93
7
AFP (ng/mL)
< 40
35
28
7
22
13
20
15
18
17
32
3
≥ 40
70
51
19
54
16
46
24
41
29
66
4
Differentiation
Well
18
13
5
12
6
12
6
9
9
16
2
Moderately
61
44
17
44
17
39
22
34
27
58
3
Poorly
26
22
4
20
6
15
11
16
10
24
2
DISCUSSION
Tumor antigens should demonstrate high frequency expression in the tumor tissues and restricted expression in normal tissues, which is the prerequisite for being an ideal candidate antigen for antigen-specific cancer immunotherapy[35]. The growing class of CT antigens is of particular interest for antitumor immunotherapy because of their specific expression on tumor cells. It has demonstrated high frequent expression of MAGE-1 and SSX-1 in HCC tissues, but little in nontumor tissues [4,26-28]. However, the heterogeneous expression of MAGE-1 and SSX-1 antigens may cause problems for immunotherapy targeting these antigens, as tumor cases with a low percentage or negative expression of MAGE-1 and/or SSX-1 antigens may be less suitable for immunotherapy targeting these antigens. Furthermore, to our knowledge, so far little information is available on the CTp11 and HCA587 expression in HCC tissues. Therefore, we investigated the expression of CT antigens MAGE1, SSX-1, CTp11 and HCA587 in HCC tissues.
In the present study, we found highly positive rate of MAGE-1, SSX-1, CTp11 and HCA587 transcripts in HCC tissues (56.2%-75.2%). No expression of MAGE-1, CTP11 and HCA587 was detected in the samples of corresponding adjacent non-cancerous tissues, while SSX-1 mRNA could be detected in 2.9% (3/105) of the corresponding adjacent non-HCC tissues where no metastatic lesions were found, similar to Chen et al[4]’s findings. Of these 3 patients, biopsy samples far from tumor were obtained in 2 patients and RT-PCR indicated expression of SSX-1 mRNA was not detectable in these two samples. The sensitivity of RT-PCR assay was much higher than that of conventional methods[36], such as histochemistry and cytology, so we speculate that there might be micrometastasis in the adjacent non-tumor liver tissue. In addition, none of 40 samples of cirrhosis and normal liver tissues expressed each of these 4 CT antigen mRNAs. As MAGE-1, SSX-1, CTp11 and HCA587 were expressed with a high percentage and specificity in HCC, their products might be ideal antigen targets for HCC immunotherapy. A high proportion (93.3%, 98/105) of the HCC tissue samples was positive for at least one of these 4 CT antigen genes, indicating using multiple CT antigens as targets will greatly increase the number of candidates for CT antigen-based HCC immunotherapy.
In the present study, we found relatively high frequency of expression of CTp11 and HCA587 mRNA, being 62.9% (66/105) and 56.2% (59/105) of tumor samples from HCC patients, respectively, while no expression of CTP11 and HCA587 was detected in the samples of corresponding adjacent non-cancerous tissues and control liver tissues, indicating their products may also be ideal antigen targets for HCC immunotherapy. Though frequency of expression of CTp11 and HCA587 mRNA was relative lower than that of MAGE-1 and SSX-1, being 75.2% (79/105) and 72.4% (76/105), respectively, we found that samples with negative expression of MAGE-1 and SSX-1 showed expression of CTp11 and HCA587. With respect to these HCC patients, vaccines based on CTp11 and HCA587 should be a desirable choice.
We also found a high frequency of synchronous expression of multiple CT antigens in HCC samples, with synchronous expression of at least two, three, and four of these genes in 72.4% (76/105), 48.6% (51/105) and 37.1% (39/105) of HCC tissues, respectively. High frequent co-expression of multiple members of CT antigens in HCC provides possibility of polyvalent vaccinations for HCC. The polyvalent vaccinations with multiple antigens, which avoid the heterogenous expression of tumor cells, might obtain better clinical results than a single antigen-based vaccination in cancer immunotherapy. Patients’ opportunities for cancer specific immunotherapy can thus be expanded[37]. However, our results showed no expression of CT antigens in 6.7% (7/105) of HCC tissues. For these patients, it is necessary to screen other CT antigens or tumor specific antigens serving as immune targets for HCC immunotherapy.
Concerning the issues of heterogenous expression of CT antigens and possibility of polyvalent vaccinations for HCC, Tahara et al[27] investigated the 10 CT antigens expression in HCC with RT-PCR and found that 86% (19/22) of HCC tissue samples expressed at least one CT antigen gene. Comparably, Luo et al[28] showed that 81% (17/21) of HCC tissue samples expressed at least one of the 10 CT genes. While in our present study, selecting just 4 CT antigens, we proved that 93.3% (98/105) of HCC patients were detectable of at least one positive CT antigen expression. Notably, the sample number of our study is 105, which is much more than the case number in the study of Tahara et al[27] (22 cases) or Luo et al[28] (22 cases), therefore our results should be more convincing.
Our previous report showed the prognostic value of MAGE-1 and MAGE-3 as tumor specific markers to detect blood dissemination of HCC cells after hepatectomy[33]. In this study, our results indicated SSX-1, CTp11 and HCA587 were also expressed in HCC tissues with high percentage and sensitivity, and some cases with negative MAGE expression were positive for SSX-1, CTp11 and/or HCA587 transcripts. Therefore, we can combine SSX-1, CTp11 and HCA587 genes with MAGE genes and apply them as multiple-markers to detect hepatoma cells in circulation, which may improve the sensitivity of the assay.
In this study, the serum α-FP was normal (< 20 ng/mL) or slightly elevated (< 40 ng/mL) in 30.0% (35/105) of HCC patients. In these 35 patients, 32 had MAGE-1, SSX1, CTp11 or HCA587 transcripts in their HCC tissues, suggesting applying their mRNAs as tumour-specific markers to detect HCC cells in circulation might be an adjuvant diagnostic tool. This assay combined with the examination of serum α-FP level may improve the diagnosis of HCC patients.
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