Biomedical subjects

Sumiyuki Nishida

Publications and source records attributed to Sumiyuki Nishida.

15 recordsLinked to original sources

PubMed2006

A phase I/II trial of a WT1 (Wilms' tumor gene) peptide vaccine in patients with solid malignancy: safety assessment based on the phase I data.

OBJECTIVE: We conducted a phase I study to investigate the safety of a weekly WT1 tumor vaccine therapy in patients with solid tumors that had been refractory to all other anti-cancer therapies. METHODS: Skin-test-negative patients were intradermally injected weekly for 12 weeks with 3.0 mg of an HLA-A*2402-restricted modified 9-mer WT1 peptide emulsified in Montanide ISA51 adjuvant. We estimated the Bayesian posterior probability of the occurrence of grade 3 or 4 toxicity when receiving the weekly WT1 vaccination. This analysis provided the basis for making a decision to terminate the phase I study and switch to phase II. Moreover, we performed an exploratory assessment of the anti-tumor effects of WT1 treatment. RESULTS: Ten patients received 114 vaccinations with WT1 on a weekly schedule. No grade 3 or 4 toxicities were observed. Based on the Bayesian approach, it was highly likely that the probability of grade 3 or 4 toxicity was below 20% (the posterior probability = 0.914). Fifteen grade 2 and two grade 1 toxicities were observed; all of these incidents, however, were determined by the Independent Data and Safety Monitoring Committee to be unrelated to the WT1 treatment. One patient exhibited a partial response; five additional patients had stable disease while receiving weekly WT1 treatment. CONCLUSION: This paper confirms that the potential toxicities of the treatment schedule of weekly WT1 vaccination are acceptable and suggested a potential anti-tumor effect. Consequently, we validated the decision to continue to the phase II trial.

Adolescent↗

PubMed2006

The extracellular domain of p185(c-neu) induces density-dependent inhibition of cell growth in malignant mesothelioma cells and reduces growth of mesothelioma in vivo.

EGFR is involved in the density-dependent inhibition of cell growth, while coexpression of EGFR with erbB2 can render normal cells transformed. In this study, we have examined the effect of a species of p185 that contains the transmembrane domain and the extracellular domain of p185(c-neu), on growth properties of a human malignant mesothelioma cell line that coexpresses EGFR and erbB2. The ectodomain form of p185(c-neu) enhanced density-dependent inhibition of cell growth and we found that p21 induction appeared to be responsible for this inhibitory effect. Previously, the extracellular domain species was shown to suppress the transforming abilities of EGFR and p185(c-neu/erbB2) in a dominant-negative manner. The ability of this subdomain to affect tumor growth is significant, as it reduced in vivo tumor growth. Unexpectedly, we found that the domain did not abrogate all of EGFR functions. We noted that EGFR-induced density-dependent inhibition of cell growth was retained. Tyrosine kinase inhibitors of EGFR did not cause density-dependent inhibition of cell growth of malignant mesothelioma cells. Therefore, simultaneously inhibiting the malignant phenotype and inducing density-dependent inhibition of cell growth in malignant mesothelioma cells by the extracellular domain of p185(c-neu) may represent an important therapeutic advance.

Animals↗

PubMed2006

Wilms' tumor gene WT1 17AA(-)/KTS(-) isoform induces morphological changes and promotes cell migration and invasion in vitro.

The wild-type Wilms' tumor gene WT1 is overexpressed in human primary leukemia and in a wide variety of solid cancers. All of the four WT1 isoforms are expressed in primary cancers and each is considered to have a different function. However, the functions of each of the WT1 isoforms in cancer cells remain unclear. The present study demonstrated that constitutive expression of the WT1 17AA(-)/KTS(-) isoform induces morphological changes characterized by a small-sized cell shape in TYK-nu.CP-r (TYK) ovarian cancer cells. In the WT1 17AA(-)/KTS(-) isoform-transduced TYK cells, cell-substratum adhesion was suppressed, and cell migration and in vitro invasion were enhanced compared to that in mock vector-transduced TYK cells. Constitutive expression of the WT1 17AA(-)/KTS(-) isoform also induced morphological changes in five (one gastric, one esophageal, two breast and one fibrosarcoma) of eight cancer cell lines examined. No WT1 isoforms other than the WT1 17AA(-)/KTS(-) isoform induced the phenotypic changes. A decrease in alpha-actinin 1 and cofilin expression and an increase in gelsolin expression were observed in WT1 17AA(-)/KTS(-) isoform-transduced TYK cells. In contrast, co-expression of alpha-actinin 1 and cofilin or knockdown of gelsolin expression by small interfering RNA restored WT1 17AA(-)/KTS(-) isoform-transduced TYK cells to a phenotype that was comparable to that of the parent TYK cells. These results indicated that the WT1 17AA(-)/KTS(-) isoform exerted its oncogenic functions through modulation of cytoskeletal dynamics. The present results may provide a novel insight into the signaling pathway of the WT1 gene for its oncogenic functions.

Actinin↗

PubMed2005

AML1-ETO rapidly induces acute myeloblastic leukemia in cooperation with the Wilms tumor gene, WT1.

AML1-ETO, a chimeric gene frequently detected in acute myelogenous leukemia (AML), inhibits the differentiation of myeloid progenitors by suppressing genes associated with myeloid differentiation and increases the replating ability of clonogenic myeloid progenitors. However, AML1-ETO alone cannot induce AML and thus additional genetic events are required for the onset of AML. The Wilms tumor gene (WT1), which has been identified as the gene responsible for Wilms tumor, is expressed at high levels in almost all human leukemias. In this study, we have generated transgenic mice (WT1-Tg) that overexpress WT1 in hematopoietic cells to investigate the effects of WT1 on AML1-ETO-associated leukemogenesis. AML1-ETO-transduced bone marrow (BM) cells from WT1-Tg mice exhibited inhibition of myeloid differentiation at more immature stages and higher in vitro colony-forming ability compared with AML1-ETO-transduced BM cells from wild-type mice. Most importantly, all of the mice that received a transplant of AML1-ETO-transduced BM cells from the WT1-Tg mice rapidly developed AML. These results demonstrate that AML1-ETO may exert its leukemogenic function in cooperation with the expression of WT1.

Animals↗

PubMed2005

The Wilms' tumor gene WT1 is a common marker of progenitor cells in fetal liver.

It is well known that the Wilms' tumor gene WT1 plays an important role in cell proliferation and differentiation, and in organ development. In this study, to examine the role of the WT1 gene in lineage determination, fetal liver cells from LacZ-transgenic mice, in which WT1 expression was marked by the expression of the LacZ gene driven by WT1 promoter, were FACS-sorted according to LacZ expression of high (LacZ(++)) or undetectable (LacZ(-)) levels, which paralleled endogenous WT1 expression levels. LacZ(++) fetal liver cells were enriched by hepatocyte and endothelial progenitor cells. These results indicated that WT1 expression is a common marker of both hepatocyte and endothelial progenitors. These results also implied a role of the WT1 gene in lineage determination.

Animals↗

PubMed2004

Induction of WT1 (Wilms' tumor gene)-specific cytotoxic T lymphocytes by WT1 peptide vaccine and the resultant cancer regression.

The Wilms' tumor gene WT1 is overexpressed in leukemias and various types of solid tumors, and the WT1 protein was demonstrated to be an attractive target antigen for immunotherapy against these malignancies. Here, we report the outcome of a phase I clinical study of WT1 peptide-based immunotherapy for patients with breast or lung cancer, myelodysplastic syndrome, or acute myeloid leukemia. Patients were intradermally injected with an HLA-A*2402-restricted, natural, or modified 9-mer WT1 peptide emulsified with Montanide ISA51 adjuvant at 0.3, 1.0, or 3.0 mg per body at 2-week intervals, with toxicity and clinical and immunological responses as the principal endpoints. Twenty-six patients received one or more WT1 vaccinations, and 18 of the 26 patients completed WT1 vaccination protocol with three or more injections of WT1 peptides. Toxicity consisted only of local erythema at the WT1 vaccine injection sites in patients with breast or lung cancer or acute myeloid leukemia with adequate normal hematopoiesis, whereas severe leukocytopenia occurred in patients with myelodysplastic syndrome with abnormal hematopoiesis derived from WT1-expressing, transformed hematopoietic stem cells. Twelve of the 20 patients for whom the efficacy of WT1 vaccination could be assessed showed clinical responses such as reduction in leukemic blast cells or tumor sizes and/or tumor markers. A clear correlation was observed between an increase in the frequencies of WT1-specific cytotoxic T lymphocytes after WT1 vaccination and clinical responses. It was therefore demonstrated that WT1 vaccination could induce WT1-specific cytotoxic T lymphocytes and result in cancer regression without damage to normal tissues.

Amino Acid Substitution↗

PubMed2004

Overexpression of the Wilms' tumor gene W T1 in primary astrocytic tumors.

Expression of the Wilms' tumor gene W T1 in primary astrocytic tumors was examined using a quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) or immunohistochemistry. Real-time RT-PCR showed that W T1 mRNA was expressed at various levels in all of the 25 astrocytic tumors examined. Immunohistochemical analysis showed that W T1 protein was expressed in 5 of 6 low-grade astrocytic tumors (grade I-II) and all of 18 high-grade ones (grade III-IV), and that expression levels of W T1 protein in high-grade tumors were significantly higher than those in low-grade ones. W T1 protein was not detected in the normal glial cells contained in the tumor specimens. Furthermore, treatment with W T1 antisense oligomers specifically inhibited growth of glioblastoma cell lines, U87-MG, A172, and T-98G. These results may indicate that the W T1 gene plays an important role in tumorigenesis of primary astrocytic tumors.

Adolescent↗

PubMed2004

Overexpression of the Wilms' tumor gene WT1 in pancreatic ductal adenocarcinoma.

The expression of the Wilms' tumor gene WT1 was examined by immunohistochemistry in 40 cases of pancreatic ductal adenocarcinoma. WT1 protein was expressed in 30 (75%) of the 40 pancreatic ductal adenocarcinomas, but not in the remaining 10 (25%). In normal pancreatic ductal cells, WT1 protein was undetectable. No correlations between WT1 expression and clinicopathological parameters such as age, sex, T or N stage, tumor location, and tumor differentiation were observed. Treatment with WT1 antisense oligomers significantly inhibited the growth of five human pancreatic cancer cell lines, PSN1, MiaPaCa2, ASPC1, BxPC3, and PCI6, expressing the WT1 gene. These results indicate an important role of the WT1 gene in the tumorigenesis of pancreatic ductal adenocarcinoma expressing WT1 and provide a rationale for new treatment strategies to treat pancreatic ductal adenocarcinoma by targeting the WT1 gene and its product.

Adenocarcinoma↗

PubMed2004

Unmanipulated reduced-intensity stem cell transplantation from a haploidentical donor mismatched at 3 HLA antigens to a patient with leukemic transformation of myelodysplastic syndrome: successful second transplantation after graft rejection.

We present the case of a patient with myelodysplastic syndrome who experienced leukemia transformation and subsequently underwent transplantation of unmanipulated peripheral blood stem cells from a haploidentical sibling mismatched at 3 HLA antigens, along with a reduced-intensity regimen (fludarabine, busulfan, and anti-T-lymphocyte globulin) and tacrolimus-containing graft-versus-host disease (GVHD) prophylaxis. The patient experienced graft rejection but successfully underwent a second transplantation from the same donor with a slightly intensified conditioning regimen. Although the patient developed life-threatening cytomegalovirus (CMV) pneumonia following the second transplantation, he recovered completely from the pneumonia with intensive supportive therapy. He is still in complete remission past day 1000 in the absence of GVHD. As far as we know, this report is the first to describe a successful second transplantation that was performed for graft rejection following HLA-haploidentical nonmyeloablative stem cell transplantation. Furthermore, we emphasize that patients should be carefully monitored for CMV infection when reduced-intensity conditioning is given repeatedly over a short period.

Adult↗

PubMed2003

Monitoring minimal residual disease in leukemia using real-time quantitative polymerase chain reaction for Wilms tumor gene (WT1).

We previously showed that Wilms tumor gene (WT1) expression level, measured by quantitative reverse transcriptase polymerase chain reaction (RT-PCR), was useful as an indicator of minimal residual disease (MRD) in leukemia and myelodysplastic syndrome. However, in conventional quantitative RT-PCR (CQ-PCR), RT-PCR must be performed for various numbers of cycles depending on WT1 expression level. In the present study, we developed a new real-time quantitative RT-PCR (RQ-PCR) method for quantitating WT1 transcripts. Results of intraassay and interassay variability tests demonstrated that the real-time WT1 assay had high reproducibility. WT1 expression levels measured by the RQ- and the CQ-PCR methods were strongly correlated (r = 0.998). Furthermore, a strong correlation was observed among WT1 transcript values normalized with 3 different control genes (beta-actin, ABL, and glyceraldehyde-3-phosphate dehydrogenase) and between relative WT1 transcript values with WT1 expression in K562 cells as the reference and absolute WT1 transcript copy numbers per microgram RNA. When WT1 expression and minor bcr-abl expression were concurrently monitored in 2 patients with bcr-abl-positive acute lymphoblastic leukemia, both MRDs changed mostly in parallel, indicating the reliability and validity of our RQ-PCR method. In conclusion, this RQ-PCR method is convenient and reliable for monitoring MRD and enables routine clinical use of a WT1 assay.

Gene Dosage↗

PubMed2003

The lck promoter-driven expression of the Wilms tumor gene WT1 blocks intrathymic differentiation of T-lineage cells.

In the thymi of WT1-transgenic (Tg) mice with the 17AA+/KTS- spliced form of the Wilms tumor gene WT1 driven by the lck promoter, the frequencies of CD4-CD8- double-negative (DN) thymocytes were significantly increased relative to those in normal littermates. Of the 4 subsets of CD4-CD8- DN thymocytes, the DN1 (CD44+CD25-) subset increased in both frequency and absolute cell number, whereas the DN2 (CD44+CD25+) and DN3 (CD44-CD25+) subsets decreased, indicating the blocking of thymocyte differentiation from the DN1 to the DN2 subsets. Furthermore, CD4-CD8+ T-cell receptor (TCR) -gammadelta T-cells increased in both frequency and absolute cell number in the spleen and peripheral blood of the WT1-Tg mice relative to those of normal littermates. The CD8 molecules of these CD4-CD8+ TCRgammadelta T-cells were CD8alphabeta, suggesting that they originated from the thymus. These results are the first direct evidence demonstrating that the WT1 gene is involved in the development and differentiation of T-lineage cells.

Animals↗

PubMed2002

The usefulness of monitoring WT1 gene transcripts for the prediction and management of relapse following allogeneic stem cell transplantation in acute type leukemia.

In acute-type leukemia, no method for the prediction of relapse following allogeneic stem cell transplantation based on minimal residual disease (MRD) levels is established yet. In the present study, MRD in 72 cases of allogeneic transplantation for acute myeloid leukemia, acute lymphoid leukemia, and chronic myeloid leukemia (accelerated phase or blast crisis) was monitored frequently by quantitating the transcript of WT1 gene, a "panleukemic MRD marker," using reverse transcriptase-polymerase chain reaction. Based on the negativity of expression of chimeric genes, the background level of WT1 transcripts in bone marrow following allogeneic transplantation was significantly decreased compared with the level in healthy volunteers. The probability of relapse occurring within 40 days significantly increased step-by-step according to the increase in WT1 expression level (100% for 1.0 x 10(-2)-5.0 x 10(-2), 44.4% for 4.0 x 10(-3)-1.0 x 10(-2), 10.2% for 4.0 x 10(-4)-4.0 x 10(-3), and 0.8% for < 4.0 x 10(-4)) when WT1 level in K562 was defined as 1.0). WT1 levels in patients having relapse increased exponentially with a constant doubling time. The doubling time of the WT1 level in patients for whom the discontinuation of immunosuppressive agents or donor leukocyte infusion was effective was significantly longer than that for patients in whom it was not (P <.05). No patients with a short doubling time of WT1 transcripts (< 13 days) responded to these immunomodulation therapies. These findings strongly suggest that the WT1 assay is very useful for the prediction and management of relapse following allogeneic stem cell transplantation regardless of the presence of chimeric gene markers.

Acute Disease↗

PubMed2002

Enhanced induction of human WT1-specific cytotoxic T lymphocytes with a 9-mer WT1 peptide modified at HLA-A*2402-binding residues.

The Wilms' tumor gene WT1 is overexpressed in most types of leukemias and various kinds of solid tumors, including lung and breast cancer, and participates in leukemogenesis and tumorigenesis. WT1 protein has been reported to be a promising tumor antigen in mouse and human. In the present study, a single amino-acid substitution, M-->Y, was introduced into the first anchor motif at position 2 of the natural immunogenic HLA-A*2402-restricted 9-mer WT1 peptide (CMTWNQMNL; a.a. 235-243). This substitution increased the binding affinity of the 9-mer WT1 peptide to HLA-A*2402 molecules from 1.82 x 10(-5) to 6.40 x 10(-7) M. As expected from the increased binding affinity, the modified 9-mer WT1 peptide (CYTWNQMNL) elicited WT1-specific cytotoxic T lymphocytes (CTL) more effectively than the natural 9-mer WT1 peptide from peripheral blood mononuclear cells (PBMC) of HLA-A*2402-positive healthy volunteers. CTL induced by the modified 9-mer WT1 peptide killed the natural 9-mer WT1 peptide-pulsed CIR-A*2402 cells, primary leukemia cells with endogenous WT1 expression and lung cancer cell lines in a WT1-specific HLA-A*2402-restricted manner. These results showed that this modified 9-mer WT1 peptide was more immunogenic for the induction of WT1-specific CTL than the natural 9-mer WT1 peptide, and that CTL induced by the modified 9-mer WT1 peptide could effectively recognize and kill tumor cells with endogenous WT1 expression. Therefore, cancer immunotherapy using this modified 9-mer WT1 peptide should provide efficacious treatment for HLA-A*2402-positive patients with leukemias and solid tumors.

HLA-A Antigens↗

PubMedDate not supplied

The effect on the proliferation and apoptosis of alloreactive T cells of cell dose in a murine MHC-mismatched hematopoietic cell transplantation model.

Activation-induced cell death (AICD) of lymphocytes is an apoptotic pathway involved in the control of T-cell homeostasis. The magnitude of graft-vs.-host disease (GVHD) following allogeneic hematopoietic cell transplantation may be attenuated by the enhancement of AICD. The aim of the present study was to clarify the effect of T cell dose upon the fate (proliferation or apoptosis) of individual activated T cells in a murine GVHD model. To this end, we investigated the kinetics of the proliferation and apoptosis of donor T cells in recipient spleens in the early stage of a fully major histocompatibility complex (MHC)-mismatched murine transplantation model from C57BL/6 (H-2(b)) to lethally-irradiated (8.5 Gy) BALB/c (H-2(d)) mice. To track the behavior of alloreactive lymphocytes in vivo, we used the fluorescent cytoplasmic dye carboxyfluorescein diacetate succinimidyl ester in combination with flow cytometry. Engraftment of donor T cells to recipient spleens was almost completed within 24 h after transfer. After that, at higher doses of transferred cells, the donor T cells actively divided for up to 72 h resulting in a 30-fold increase in cell number at the maximum cell dose (2.0 x 10(7)). As the transferred cell dose decreased, the proliferation of T cells tended to be suppressed. At cell doses of 0.5 x 10(7) or less, the proliferation of T cells was profoundly suppressed, ultimately resulting in little proliferation of donor T cells observed from 24 to 72 h at the minimum cell dose (0.1 x 10(7)). The frequency of Annexin-V-positive cells was found to increase gradually as the transferred cell dose decreased. Thus, an increase in apoptotic events appeared to play an important role in the suppression of the proliferation of T cells at lower splenocyte doses. Further analyses revealed that Fas ligand (FasL)-positive T cells were observed exclusively among T cells that divided at least 5 times, and that all of them were positive for Annexin-V, indicating that they were in the process of apoptosis. Together with our finding that the frequency of apoptosis increased with the progression of cell division, these findings strongly suggest that AICD occurred through the Fas/FasL system and that AICD increased as the dose of donor T cells participating in the allogeneic response decreased. When relatively small numbers of T cells are confronted with an excess of antigen, they disappear. This process is called clonal exhaustion-deletion. Our results support the idea that AICD is involved in the process of clonal exhaustion-deletion. Relevant to the clinical aspects of hematopoietic cell transplantation, our findings indicate that AICD may be associated with tolerance induction in T-cell-depleted transplantation from HLA-mismatched donors, in which T cells contaminating marrow grafts do not need to be completely removed for achieving tolerance between donors and recipients. Furthermore, our results indicate that a small change in the quantitative balance between antigens and T cells responding to them leads to a large difference in the fate of T cells activated in response to MHC-incompatible antigens. Thus, the size of the T cell dose is one of the important considerations in tolerance induction, GVHD and rejection.

Adoptive Transfer↗

PubMedDate not supplied

Overexpression of the Wilms' tumor gene WT1 in esophageal cancer.

BACKGROUND: The Wilms' tumor gene WT1 is overexpressed in various kinds of solid cancers. However, it remains unclear whether WT1 is expressed in esophageal squamous cell carcinoma. MATERIALS AND METHODS: Expression of the WT1 gene was examined by real-time RT-PCR in 12 esophageal squamous cell carcinoma (ESCC) and by immunohistochemistry in 9 of these 12 and another 29. RESULTS: Real-time RT-PCR showed that the WT1 mRNA was overexpressed in all of the 12 ESCC examined Immunohistochemical analysis showed that the WT1 protein was overexpressed in ESCC cells in 36 (95%) of the 38 examined Furthermore, expression of the WT1 protein was examined in 20 esophageal squamous dysplasia. The WT1 protein was overexpressed in 5 (45%) out of 11 mild dysplasia and in 8 (89%) out of 9 moderate to severe dysplasia. CONCLUSION: These results may indicate an important role of the WT1 gene in the tumorigenesis of ESCC.

Adult↗