Ex vivo characterization of tumor-derived melanoma antigen encoding gene-specific CD8+cells in patients with hepatocellular carcinoma
Introduction
Hepatocellular carcinoma (HCC) is the most common primary liver cancer. It has a worldwide distribution with the highest prevalence in Africa and Southeast Asia and a rising incidence in the last two decades in Europe and United States due to a wide exposure to hepatitis B virus (HBV) and hepatitis C virus (HCV) in the 1960s and 1970s. Therefore, occurrence of HCC may still continue to rise for long time because of the large pool of subjects infected by HBV and HCV [1].
Surgery is often effective for treatment of early stage HCC, but patients with advanced HCC remain without therapeutic options since chemotherapy has shown only marginal benefit. Recently, a randomized trial showed that adoptive immunotherapy with autologous lymphocytes activated in vitro by recombinant interleukin-2 and antibodies to CD3 can lower recurrence and improve recurrence-free outcomes after surgery for HCC [2]. However, the antigen-specificities and the anti-tumor mechanisms involved in suppression of HCC growth by adoptive transfer of polyclonally activated autologous T lymphocytes remain still undefined.
There is now a consensus that cytotoxic T lymphocytes (CTL) recognize tumor rejection antigens in the context of major histocompatibility complex-I (MHC) molecules. In melanoma, evidence of a tumor specific T cell response is provided by the identification of several tumor-associated antigens (TAAs), which can stimulate T cell responses. Recently TAAs have been reported in several other solid tumors. Of particular interest are cancer testis antigens of the melanoma antigen encoding gene (MAGE) family. They are silent in normal cells, with the exception of male germline cells that do not express HLA class I and are therefore unable to present antigens to CTL. For these reasons, they are of particular interest in cancer immunotherapy. MAGE-1 and -3 genes are expressed in a high percentage (40–80%) of resected HCC tissues [3], [4], [5], [6]; other antigens of the MAGE family (-2, -4, -6, -8 -10, -11 and -12) are expressed at lower and variable degree in different studies [5], [7]. In particular, the immune response to MAGE-1 and -3 has been defined by identification of several HLA class I restricted epitopes in melanoma patients [8], but MAGE-specific T cell responses have never been demonstrated in patients with HCC.
The objective of our study was to analyze the expression of MAGE-1 and -3 antigens in HCC, the phenotype of HCC infiltrating lymphomononuclear cells and their capacity to recognize MAGE-1 and -3 antigens. In contrast to previous studies in melanoma that showed a MAGE-specific cytotoxic T cell response from tumor infiltrating lymphomononuclear cells only after in vitro antigen stimulation [9], [10], our study provides evidence ex vivo of MAGE-specific CD8 cells within the tumoral tissue in patients with HCC. These observations provide the theoretical basis to further investigate whether exogenous stimulation of the MAGE-specific immune response can prevent HCC recurrence after tumor resection or local ablative treatments.
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Patients
Twenty patients with HCC in liver cirrhosis undergoing surgical resection were studied. Etiology of liver cirrhosis, HCC differentiation grade and size are shown in Table 1. Liver cirrhosis was HCV related in 17 patients, HBV related in two patients and induced by alcohol in one patient. Patients gave written informed consent before entering the study and the study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki.
MAGE-1 and -3 mRNA and MAGE antigen expression in HCC
Total RNA was extracted from the tumor and
MAGE-1 and -3 mRNA and antigen expression in HCCs
Expression of MAGE-1 and -3 mRNA in liver tissues and HCCs was tested by RT-PCR. The specificity of the amplified PCR fragments was confirmed by hybridization with oligonucleotide probes (DEIA) and by sequencing of RT-PCR products (patients 3 and 10). Sequencing showed a nucleotide cDNA fragment identical to those of the GenBank database (not shown).
MAGE-1 transcripts were positive in 80% of HCC samples and MAGE-3 transcripts in 45% (Table 1). MAGE-1 and -3 mRNAs were not detected in the tissue
Discussion
Since the identification of the first gene encoding for a tumor-associated antigen in melanoma [12], there have been several reports of successful in vitro induction of HLA class I-restricted anti-tumor CTLs using MAGE peptides. These immunogenic peptides have also been used in clinical trials as peptide vaccines with positive results in some patients with melanoma [16]. While CD8+ cells specific for melanocyte differentiation antigens (Melan-A/MART-1, tyrosinase) have been detected ex vivo in
Acknowledgements
The study was supported in part by grant EC Biomed (grant n. BMH4-98-2239), grant from Fondazione Cassa di Risparmio di Parma, Italy, and from Fondazione Oretta Bartolomei Corsi, Italy.
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2017, Cytokine and Growth Factor ReviewsCitation Excerpt :On the contrary, another study did not confirm this finding but showed instead that SSX-1 and GAGE CT genes were found in 38%, HOM-TES-14/SCP-1 in 29%, MAGE-3 in 24%, HOM-TES-85 and MAGE-1 in 19% of HCC samples analyzed [38]. Spontaneous T cell responses against specific CT antigens have been detected in HCC patients [39–41]. Based on such observations, an early stage clinical trial has been performed in 5 HCC patients to evaluate the safety and immunogenicity of autologous DCs-pulsed with a peptide mix including MAGE-1, AFP and GPC3 TAAs.
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