Identification of XAGE-1 isoforms: predominant expression of XAGE-1b in testis and tumors.

XAGE-1 is a cancer/testis (CT) antigen and has been shown to be immunogenic in lung cancer patients. Among 4 alternative splicing variants, XAGE-1a, b, c and d, XAGE-1b mRNA was dominantly expressed in cancer. In this study, we generated a XAGE-1b mAb, USO9-13. The B cell epitope recognized by the USO9-13 mAb was in the C-terminal region of the XAGE-1b protein and is also recognized by sera from patients with lung adenocarcinoma. Using USO9-13 and an anti-Flag mAb, we examined the translation products of the 4 transcripts. The XAGE-1a and b transcripts translated to the XAGE-1b protein. The XAGE-1c transcript possibly translated to 9- and 17-aa polypeptides. The XAGE-1d transcript translated to a protein consisting of 69 amino acids. Immunofluorescence analysis using USO9-13 mAb showed that the XAGE-1b protein is located in the nuclei of cells. Immunohistochemically, nuclear staining was heterogeneously observed in 25/47 lung adenocarcinomas, 1/12 hepatocellular carcinomas and 1/11 gastric cancers, but not in adjacent normal tissues. These findings suggested that XAGE-1b is a promising target molecule for a cancer vaccine against lung cancer.

In vitro stimulation of CD8 and CD4 T cells by dendritic cells loaded with a complex of cholesterol-bearing hydrophobized pullulan and NY-ESO-1 protein: Identification of a new HLA-DR15-binding CD4 T-cell epitope.

PURPOSE: NY-ESO-1 belongs to a class of cancer/testis antigens and has been shown to be immunogenic in cancer patients. We synthesized a complex of cholesterol-bearing hydrophobized pullulan and NY-ESO-1 protein (CHP/ESO) and investigated the in vitro stimulation of CD8 and CD4 T cells from peripheral blood mononuclear cells in healthy donors with autologous CHP/ESO-loaded dendritic cells as antigen-presenting cells. EXPERIMENTAL DESIGN: In vitro stimulation of CD8 or CD4 T cells was determined by IFNgamma ELISPOT assays against autologous EBV-B cells infected with vaccinia/NY-ESO-1 recombinant virus or wild-type vaccinia virus as targets and by ELISA measuring secreted IFNgamma. RESULTS: NY-ESO-1-specific CD8 and CD4 T cells were induced. In a donor expressing HLA-A2, CD8 T cells stimulated with CHP/ESO-loaded dendritic cells recognized naturally processed NY-ESO-1(157-165), an HLA-A2-binding CD8 T cell epitope. NY-ESO-1 CD4 T cells were Th1-type. We identified a new HLA-DR15-binding CD4 T cell epitope, NY-ESO-1(37-50). CONCLUSIONS: These findings indicate that CHP/ESO is a promising polyvalent cancer vaccine targeting NY-ESO-1.

2R regenerator based on a 2-m-long highly nonlinear bismuth oxide fiber.

We report the use of a 2-m-long Bismuth Oxide fiber with an ultra-high nonlinearity of ~1100 W(-1)km(-1) in a simple 2R regeneration experiment based on self phase modulation and offset filtering. Numerical simulations and experimental results confirm the suitability of this kind of fiber for 2R regeneration. An improvement in receiver sensitivity of more than 5 dB at 10 Gb/s and 2 dB at 40 Gb/s is achieved.

Quantitative real-time RT-PCR analysis of NY-ESO-1 and LAGE-1a mRNA expression in normal tissues and tumors, and correlation of the protein expression with the mRNA copy number.

We investigated NY-ESO-1 and LAGE-1a mRNA expression in normal tissues and various types of cancer by quantitative real-time RT-PCR. In addition to their high expression in the testis, we observed a low expression of NY-ESO-1 mRNA in the placenta, pancreas and liver, and no expression in 12 other normal tissues. We also observed a low expression of LAGE-1a mRNA in the placenta and ovary, and marginal expression in 13 other normal tissues. In contrast to the previous finding that NY-ESO-1 and LAGE-1a mRNAs were mostly co-expressed in solid tumors, we found an independent expression of NY-ESO-1 and LAGE-1a mRNAs. NY-ESO-1 mRNA expression was mostly associated with LAGE-1a mRNA expression in esophageal and liver cancers, but not in prostate cancer. Immunohistochemistry (IHC) using NY-ESO-1-specific ES121 mAb showed that NY-ESO-1 protein was detected in 6 of 9 and 3 of 10 NY-ESO-1 mRNA-positive specimens from esophageal and liver cancers, respectively. NY-ESO-1 protein expression was correlated with the copy numbers of NY-ESO-1 mRNA. IHC was also performed using ES121 mAb and B9.8 mAb recognizing both NY-ESO-1 and LAGE-1a in 4 esophageal and 6 liver cancer specimens preferentially expressing LAGE-1a mRNA. B9.8-specific staining was observed weakly and focally in one liver cancer specimen expressing >10(5) copies of LAGE-1a mRNA.

Long-term clinical outcomes of 420 consecutive prostate cancer patients in a single institute.

This study was undertaken to reveal the trends of prostate cancer and the outcome of treatment modalities for each disease stage in patients in a single institute over a 10-year period. From January 1994 through December 2003, 420 consecutive patients with previously untreated and histologically confirmed prostate cancer were analyzed for annual distributions of disease stages and treatment modalities and for long-term clinical progression-free survival, prostate cancer-specific survival, and prostate-specific antigen (PSA) failure-free survival rates for each stage and treatment modality. Annual trends showed that the number of patients, especially those with clinically localized cancer, increased dramatically. The 5-year disease-specific survival rates for patients with clinically localized disease were 100 percent for all treatment modalities, including hormonal therapy alone. Patients with PSA levels less than 10 ng/ml showed an 81 percent 5-year PSA failure-free survival rate with radical prostatectomy. Stage C patients treated by surgery or radiation-based therapy with concomitant hormonal therapy obtained 93 percent and 100 percent cause-specific survival rates, respectively, and those treated by hormonal therapy alone showed a 79 percent rate. The number of patients with localized prostate cancer was increasing in this decade. While long-term hormonal therapy alone was highly efficient in controlling localized prostate cancer, radical therapies in conjunction with neo-adjuvant hormonal therapy produced better survival rates in cases of locally advanced disease.

SSX expression in gynecological cancers and antibody response in patients.

The SSX genes are members of the cancer-testis (CT) antigen family. SSX2, the prototype SSX gene, was found by serological analysis of antigens by recombinant expression cloning (SEREX). Since little is known about SSX expression in gynecological malignancies, we investigated SSX mRNA expression in 115 gynecological cancer specimens and 25 normal control samples by RT-PCR. We also tested the humoral immune response to SSX2 and SSX4 using recombinant proteins. We found relatively high SSX4 mRNA expression in endometrial cancer (24%), ovarian cancer (13%), and cervical cancer (20%). In contrast, SSX1 and SSX2 mRNA expression was detected in not more than 4% of the gynecological cancer specimens analysed. No SSX mRNA expression was found in 25 normal specimens. Two gynecological cancer patients had antibodies against SSX4, whereas no antibody reactivity to SSX4 was found in the sera of 40 normal individuals. This suggests that SSX4 is a potential target for cancer vaccines in gynecological cancer patients.

XAGE-1 mRNA expression in prostate cancer and antibody response in patients.

To evaluate the feasibility of cancer vaccine targeting XAGE-1, we investigated the expression of 4 XAGE-1 transcript variants and the humoral immune response to XAGE-1 in prostate cancer patients. XAGE-1a, b, c, d mRNA expression was analyzed in 54 prostate cancer specimens and 8 specimens of benign prostate hypertrophy (BPH) by reverse transcription-polymerase chain reaction (RT-PCR). The humoral response to XAGE-1 was investigated in sera obtained from 278 patients with prostate cancer and 40 healthy volunteers by enzyme-linked immunosorbent assay (ELISA) using recombinant protein. XAGE-1b mRNA expression was observed in 14 of 54 (26%) prostate cancer specimens, while XAGE-1a, c, and d mRNA expressions were observed in 1, 1, and 3, respectively. None of the 4 XAGE-1 transcript variants was observed in the 8 BPH specimens. Antibody against XAGE-1 was detected in sera from 2 of 129 stage D2 patients, whereas none of sera from 149 patients with localized prostate cancer or lymph node metastasis had detectable XAGE-1 antibody. No reactivity to XAGE-1 was found in sera from the 40 healthy individuals.

Identification of glioma-specific RFX4-E and -F isoforms and humoral immune response in patients.

For regulatory factor X4 (RFX4), two alternatively spliced variants, RFX4-A and -B, were reported in the testis. In this study, we identified transcript variants RFX4-C, -D, -E, and -F, and demonstrated by reverse transcription-polymerase chain reaction (RT-PCR) that RFX4-A, -B and -C mRNAs were expressed only in the testis, and RFX4-D mRNA was expressed only in normal brain tissues. In tumors, RFX4-E and -F in addition to RFX4-D mRNA were expressed in gliomas by rapid amplification of cDNA ends and RT-PCR analyses. Expression of RFX4 mRNA was not observed in other tumors, such as lung, esophageal, stomach, colon or liver cancers. Quantitative real-time RT-PCR using common primer pairs detecting all of the variant transcripts showed high expression in normal testis, low expression in the brain (1% compared to the expression in testis), and overexpression in 17 of 61 gliomas (28%). Western blot analysis using DC28 monoclonal antibody (mAb) produced against recombinant RFX4-D C-terminus protein showed expression of RFX4-A and -C proteins, but not RFX4-B protein, in the testis, and expression of RFX4-D protein in the brain. Moreover, expression of RFX4-E and -F proteins, but not RFX4-D protein, was observed in gliomas. Immunohistochemistry analysis using DC28 mAb showed positive staining in the nuclei of spermatocytes in the testis and glioma cells. Antibody against RFX4 was detected in the sera of 3 of 58 (5%) glioma patients by enzyme-linked immunosorbent assay, suggesting the immunogenicity of RFX4-E and -F proteins in glioma patients.

Over-expression of the testis-specific gene TSGA10 in cancers and its immunogenicity.

The TSGA10 gene was originally isolated in normal testis by differential mRNA display. TSGA10 is located on chromosome 2q11.2 and consists of 19 exons extending over 3 kb. TSGA10 mRNA expression was investigated in normal and malignant tissues using quantitative real-time RT-PCR. It was predominantly expressed in the testis in adult normal tissues. In malignant tissues, TSGA10 was over-expressed in 4 of 20 hepatocellular carcinomas (HCC), 1 of 20 colon cancers, 7 of 20 ovarian cancers, 3 of 20 prostate cancers, 1 of 21 malignant melanomas, and 8 of 21 bladder cancers. Serological analysis revealed that 3 out of 346 patients with various types of cancer possessed antibody against recombinant TSGA10 protein. They included 2 patients with hepatocellular carcinoma and a patient with malignant melanoma.