Mutated p53 gene encodes a nonmutated epitope recognized by HLA-B*4601-restricted and tumor cell-reactive CTLs at tumor site

Mutations of p53 gene occur in approximately 50% of human cancers, and accumulated p53 protein may be an appropriate target molecule to use for cancer immunotherapy. Indeed, mutated or nonmutated p53-derived peptides can induce HLA class I-restricted and tumor cell-reactive CTLs in vitro. However, to our knowledge, evidence that p53-derived peptides are truly recognized by CTLs at tumor sites has not yet been obtained. This study revealed that a mutated p53 gene encoded a nonmutated nonapeptide recognized by a HLA-B46-restricted and tumor cell-reactive CTL line that was established from T cells infiltrating a colon cancer lesion with the p53 mutation. This p53 peptide, at amino acid positions 99-107, had the ability to induce HLA-B46-restricted and peptide-specific CTLs reactive to tumor cells with the p53 mutation from the peripheral blood mononuclear cells of cancer patients, but not from those of healthy donors. These peptide-induced CTLs did not react to either HLA-B46(+) tumor cells without the p53 mutation or to HLA-B46(+) phytohemagglutinin-blastoid cells. These results provide a scientific basis for the development of p53-directed specific immunotherapy for HLA-B46(+) cancer patients.     

Assay of serum hyaluronic acid in clinical application

A sandwich-binding protein assay to determine the concentration of hyaluronic acid (HA) in body fluids has been developed. In this method, a hyaluronic acid binding protein (HABP) was adsorbed to the surface of a solid phase, and HA bound to HABP on the solid phase was detected by biotin-conjugated HABP. The method could assay HA levels within 6 hours using precoated microwells with HABP. HA could be determined in the range of 2-500 micrograms/l by this method using 50 microliters of serum. Within-run precision (CV) was 5.2-10.2%. The specificity of HABP to HA was confirmed by the elimination of the reaction with treatment by hyaluronidase digestion. Serum HA levels (median; range) of patients with rheumatoid arthritis (34; 2-187 micrograms/l) were shown to be higher than those with osteoarthritis (1; 1-21 micrograms/ml) and healthy controls (2; 1-8 micrograms/ml). No correlation between levels of HA and rheumatoid factor was found. HA was demonstrated to be a potential diagnostic marker for rheumatoid arthritis, and this HABP assay could be useful for determination of HA in clinical laboratory tests.     

Diagnostic value of ganglioside patterns in plasma of human diseases

A method that could be facilitated for the quantitation and qualitation of gangliosides in human plasma was developed and applied in the present study for characterization of ganglioside patterns in plasmas of patients with neoplastic diseases including gastric cancer, adult T-cell leukemia (ATL), and acute lymphocytic leukemia (ALL). As a retrovirus-infected disease with different clinical entity, human T-cell lymphotropic virus type I-associated myelopathy (HAM) was also subjected to this study. The results were compared with the patterns obtained from normal control plasmas. The analytical data revealed that GM3 increased in gastric cancer, GT1b decreased in HAM and ATL, and GD3, GM1 and GM3 decreased in ALL. There were close correlations between various human diseases and the presence of gangliosides with their specific patterns. Furthermore, gangliosides purified from plasma of patients with HAM significantly inhibited the expression of CD4 antigen on human T lymphocyte membrane. Therefore, analytical studies of plasma gangliosides could provide diagnostic and therapeutic values in retroviral infections.     

Ran, a small GTPase gene, encodes cytotoxic T lymphocyte (CTL) epitopes capable of inducing HLA-A33-restricted and tumor-reactive CTLs in cancer patients.

PURPOSE: The purpose is to identify a gene coding for tumor-associated antigen and peptide capable of inducing CTLs reactive to tumor cells with a HLA-A33-restricted fashion to provide scientific basis for specific immunotherapy to HLA-A33+ cancer patients. EXPERIMENTAL DESIGN: An expression gene-cloning method was used to identify the tumor-associated antigen gene. Northern blot analysis and immunohistochemistry were used to examine the mRNA and protein expression levels in various cells and tissues, respectively. Synthetic peptides were examined for their ability to induce HLA-A33+ tumor-reactive CTLs in peripheral blood mononuclear cells from cancer patients. RESULT: A gene of small GTPase, Ran, which controls the cell cycle through the regulation of nucleocytoplasmic transport, mitotic spindle organization, and nuclear envelope formation, was found to encode epitopes recognized by the HLA-A33-restricted CTLs established from T cells infiltrating into gastric adenocarcinoma. The expression of the Ran gene was increased in most cancer cell lines and cancer tissues at both the mRNA and protein levels. However, it was not enhanced in the surrounding normal cells or tissues. It was also undetectable in normal tissues as far as tested. Ran-derived peptides at positions 48-56 and 87-95 could induce CD8+ peptide-specific CTLs reactive to tumor cells from HLA-A33+ epithelial cancer patients in a HLA class I-restricted manner. CONCLUSIONS: Because of its increased expression in cancer cells and involvement in malignant transformation and/or the enhanced proliferation of cancer cells, the two Ran-directed peptides could be potent candidates in use for specific immunotherapy against HLA-A33+ epithelial cancers.     

A new tumor-rejection antigen recognized by cytotoxic T lymphocytes infiltrating into a lung adenocarcinoma

Lung cancer is the most commonly occurring malignancy worldwide and one of the few that continues to show an increasing incidence. To understand the molecular basis of host immunity against lung cancer, we investigated tumor antigens recognized by HLA-A24-restricted CTLs established from T cells infiltrating into lung adenocarcinoma and report a new gene coding for antigens recognized by the CTLs. The mRNA of this gene was expressed at different levels in all of the malignant cells tested (high in adenocarcinomas and gliomas and low in esophageal cancers and malignant hematological disease). It was also expressed at the different levels in each of a panel of normal tissues (high in the thymus, low in peripheral blood mononuclear cells, and lowest in the stomach, small intestine, and skeletal muscle). This gene encodes a Mr 60,000 nuclear protein with 414 deduced amino acids. The three peptides at positions 158-165, 170-179, and 188-196 were recognized by the CTLs. One peptide at position 188-196 had the ability to induce HLA-A24-restricted and tumor-specific CTLs in peripheral blood mononuclear cells of lung cancer patients. These CTLs, however, did not lyse HLA-A24+ PHA-activated T cells in which the mRNA of this gene was highly expressed, even in the presence of excess amounts of a corresponding peptide in culture. These results suggest that this gene product and peptide could be applicable to specific immunotherapy of lung cancer patients.     

Identification of a SART-1-derived peptide capable of inducing HLA-A24-restricted and tumor-specific cytotoxic T lymphocytes

We have described the SART-1 gene-encoding peptides recognized by HLA-A2601-restricted and tumor-specific cytotoxic T lymphocytes (CTLs). We now have investigated whether SART-1 encodes peptides capable of inducing the HLA-A24-restricted CTLs. Among the 18 different peptides with HLA-A24-binding motifs, the SART-1(690-698) peptide (EYRGFTQDF) was most strongly recognized by the HLA-A24-restricted and tumor-specific CTLs established from an esophageal cancer patient. After a third stimulation in vitro, this peptide induced HLA-A24-restricted CTLs recognizing the SART-1(259)+ tumor cells in PBMCs of all HLA-A24 homozygous and the majority of HLA-A24 heterozygous cancer patients and healthy donors tested. A similar activity, induction of CTLs from PBMCs, was observed in the Saccharomyces cerevisiae-derived nonapeptide (EYRGFTPMF) that shares 7 amino acids with the SART-1(690-698) peptide. The SART-1(690-698) peptide-induced CTL activity was significantly higher in PBMCs of HLA-A24 homozygotes than in HLA-A24 heterozygotes. The CTL precursor frequency in PBMCs after a third stimulation in vitro with the SART-1(690-698) peptide was high (>1/200) in both cancer patients and healthy donors. The SART-1(690-698) peptide could thus be useful for specific immunotherapy of HLA-A24+ cancer patients.     

Expression of the tumor-rejection antigen SART1 in brain tumors

We have reported a tumor-rejection antigen, SART1(259), possessing tumor epitopes capable of inducing cytotoxic T lymphocytes (CTLs) in epithelial-cancer patients. This study investigated the expression of SART1(259) antigen in brain tumors, to explore for a potential molecule for use in specific immunotherapy of patients with brain tumors. The SART1(259) antigen was detected in the cytosol fraction of 13 of 18 (72%) glioma cell lines and in 12 of 34 (35%) brain-tumor tissues, with a higher rate of expression among malignant gliomas (5/10, 50%) and schwannomas (3/4). HLA-A24-restricted and SART1-specific CTLs recognized the HLA-A24+ and SART1(259)+ glioma cells, and the levels of recognition correlated both with HLA-A24-antigen expression level and with the concentration of the SART1 peptide antigen. Therefore, the SART1(259) antigen could be a target molecule for specific immunotherapy of patients with brain tumors expressing HLA-class-1 antigens.     

Human tumor-rejection antigens and peptides from genes to clinical research

One of the most significant advances in the field of modern tumor immunology is the identification of genes encoding tumor-rejection antigens that are recognized by human leukocyte antigen (HLA) class I-restricted and tumor-specific cytotoxic T lymphocytes (CTLs). Several peptides encoded by these genes are now under clinical trial as cancer vaccines, and major tumor regression has been observed in some melanoma patients. These results indicate that identification of the peptides capable of inducing CTLs may provide a new modality of cancer therapy. We investigated tumor-rejection antigens from epithelial cancers, and reported 7 genes encoding tumor-rejection antigens and peptides available for specific immunotherapy of HLA-A26 or -A24 patients with epithelial cancers. Furthermore, we identified more than 10 genes encoding tumor-rejection antigens and peptides available for specific immunotherapy of HLA-A2 patients with epithelial cancers. Therefore these new antigens and peptides could be applicable to the treatment of numerous epithelial cancer patients worldwide. Phase I clinical trials of cancer vaccine with these peptides for epithelial cancer patients are in progress at our university. Basic and clinical research will provide new insights for a better understanding of the molecular basis of T cell-mediated recognition of cancer cells and be important for the development of cancer vaccines.     

Management of local recurrence after endoscopic resection of neoplastic colonic polyps

A proportion of neoplastic polyps are incompletely resected, resulting in local recurrence, especially after resection of large polyps or piecemeal resection. Local recurrences that develop after endoscopic resection of intramucosal neoplasms that lacked risk factors for lymph node metastasis or positive vertical margins are usually treated endoscopically. Endoscopic submucosal dissection(ESD) is indicated for local residual or recurrent early carcinomas after endoscopic resection. However, ESD for such recurrent lesions is technically difficult and is typically a lengthy procedure. Underwater endoscopic mucosal resection(UEMR), which was developed in 2012, is suitable for recurrent or residual lesions and reportedly achieves superior en bloc resection rates and endoscopic complete resection rates than conventional EMR. However, a large recurrent lesion is a negative independent predictor of successful en bloc resection and of complete endoscopic removal. We therefore perform UEMR for relatively small(≤ 10-15 mm) recurrent lesions and ESD for larger lesions.