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. The WT1 gene was isolated as a gene responsible for Wilms' tumor, a pediatric renal cancer, and encodes a zinc finger transcription factor, which is involved in cell proliferation and differentiation, apoptosis, and organ development (3-6). Although the WT1 gene was first categorized as a tumor suppressor gene, we have proposed that the wild-type WT1 gene functions as an oncogene rather than a tumor-suppressor gene on the basis of the following findings. The first is high expression of the wild-type WT1 gene in both leukemias and solid tumors (7-18), the second is growth inhibition of leukemic and solid tumor cells by treatment with WT1 antisense oligomers (14,19), and the third is block of differentiation, but induction of proliferation, of wild-type WT1 gene-transfected myeloid progenitor cells in response to granulocyte colony-stimulating factor (20, 21). The last two are block of thymocyte differentiation but induction of thymocyte proliferation in the transgenic mice with the lck promoter-driven WT1 gene (22), and WT1 gene expression in the majority of dimethylbenzanthracene-induced erythroblastic leukemia and a stronger tendency of the cells with high levels of WT1 to develop into leukemias (23).Expression of the wild-type WT1 gene has been found in most cases of acute myelocytic leukemia (AML), acute lymphocytic leukemia, chronic myelocytic leukemia, and myelodysplastic syndrome (MDS) at higher levels than those in normal bone marrow (BM) or peripheral blood (7-13). Furthermore, various types of solid tumors, including lung, breast, thyroid, and colorectal cancers, expressed the wild-type WT1 gene at higher levels compared to those in corresponding normal tissues (15-18). These results indicated that the wild-type WT1 gene product may be a promising target for cancer immunotherapy (24,25).We tested the potential of the WT1 gene product to serve as a target antigen for tumor-specific immunotherapy. Human WT1-specific CTLs have been found to induce lysis of endogenously WT1-expressing tumor cells in vitro, but not to cause damage to physiologically WT1-expressing normal cells (24,(26)(27)(28). We used a mouse in vivo system to demonstrate that immunization of mice with either MHC class I-restricted WT1 peptide or WT1 cDNA induced WT1-specific CTLs. We also showed that the immunized mice rejected challenges of WT1-expressing tumor cells, whereas the induced CTLs did not affect normal healthy tissues that physiologically expressed WT1 nor damaged the normal tissues (25, 29). These results indicated that the WT1 protein could be a novel tumor rejection antigen for cancer immunotherapy (24)(25)(26)(27)(28)(29)(30)(31)(32).In...

A role of donor-specific HLA antibodies (DSA) in graft failure after SCT has been suggested, but the relevance of DSA in unmanipulated haploidentical SCT (haplo-SCT) remains unknown. We prospectively examined HLA antibodies using the Luminex-based single Ag assay for 79 adult patients undergoing unmanipulated haplo-SCT. Among them, 16 (20.2%) were HLA Ab-positive, including five patients with antibodies not corresponding to donor HLA Ags and 11 DSA-positive patients. Of the 11 DSA-positive patients, five received treatments to decrease DSA levels, including two, who received plasma exchange and rituximab, two who received platelet transfusions from healthy-related donors having DSAcorresponding HLA Ags and one who received bortezomib. Platelet transfusion was the most simple and effective treatment option for class I DSA. The cumulative incidence of neutrophil recovery was significantly lower in pretransplant (post-treatment) DSA-positive patients than in DSA-negative patients (61.9 vs 94.4%, P ¼ 0.026). Notably, three of five patients with high levels of DSA had graft failure. Donors should be selected on the basis of an evaluation of HLA antibodies. If haplo-SCT from donors with HLA Ags that correspond to high levels of DSA must be performed, then recipients should be treated for DSA to improve the chances of successful donor engraftment.

Expression of theThe Wilms' tumor gene WT1 was originally isolated as a tumorsuppressor gene responsible for Wilms' tumor, a kidney neoplasm of childhood. 1 However, we proposed that WT1 played an oncogenic role in leukemogenesis based on the following findings: 2 (i) the wild-type WT1 gene was expressed at high levels in leukemic blast cells, 3,4 (ii) there was a clear and inverse correlation between WT1 expression levels and prognosis in acute leukemia, 3 (iii) WT1 expression increased at relapse of acute leukemia, 5 (iv) growth of leukemic blast cells was inhibited by the treatment of WT1 antisense oligomers 6 and (v) constitutive expression of WT1 blocked differentiation and instead induced proliferation in response to granulocyte colony-stimulating factor in 32D cl3 myeloid progenitor cells 7 and normal myeloid progenitor cells. 8 Furthermore, we demonstrated that the wild-type WT1 as expressed in various types of cell line derived from lung cancer, gastric cancer, colon cancer and breast cancer and that growth of these WT1-expressing tumor cells was inhibited by the treatment of WT1 antisense oligomers. 9 These data suggested an oncogenic role of the WT1 gene in tumorigenesis. However, the involvement of the WT1 gene in de novo solid tumors remained unclear. In the present study, we examined WT1 expression in de novo lung cancer using quantitative real-time RT-PCR and immunohistochemistry and demonstrated that the wild-type WT1 was overexpressed in 54/56 (96%) de novo non-small cell lung cancers (NSCLCs) and 5/6 (83%) de novo small cell lung cancers (SCLCs) examined.

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 ؋ 10 ؊2 -5.0 ؋ 10 ؊2 , 44.4% for 4.0 ؋ 10 ؊3 -1.0 ؋ 10 ؊2 , 10.2% for 4.0 ؋ 10 ؊4 -4.0 ؋ 10 ؊3 , and 0.8% for < 4.0 ؋ 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

The Wilms tumor gene, WT1, is overexpressed not only in leukemias and myelodysplastic syndrome (MDS) but also in various types of solid tumors, including lung and breast cancer, and the WT1 protein is a tumor antigen for these malignancies. In clinical trials of WT1 peptide-based cancer immunotherapy, patients with overt leukemia from MDS or MDS with myelofibrosis were injected intradermally with 0.3 mg of an HLA-A*2402-restricted, 9-mer WT1 peptide emulsified with Montanide ISA51 adjuvant. Only a single dose of WT1 vaccination resulted in an increase in WT1-specific cytotoxic T-lymphocytes, which was followed by a rapid reduction in leukemic blast cells. Severe leukopenia and local erythema at the injection sites of WT1 peptide were observed as adverse effects. These results have provided us with the first clinical evidence suggesting that WT1 peptide-based immunotherapy is an attractive treatment for patients with leukemias or MDS.

Recipients of allogeneic HSCT had a significantly higher ∼2-fold risk of developing secondary solid cancers than the general population. Lifelong screening for high-risk organ sites, especially oral or esophageal cancers, is important for recipients with active, or a history of, chronic GVHD.

The major problems in human leukocyte antigen (HLA)-mismatched stem cell transplantation (SCT) are graft failure and graft-versus-host disease (GVHD). Less-intensive regimens should be associated with a lower release of inflammatory cytokines and possibly less GVHD. The objective of this study was to investigate whether HLA-haploidentical SCT can be performed using nonmyeloablative conditioning and pharmacologic GVHD prophylaxis, including glucocorticoids. Using conditioning consisting of fludarabine, busulfan, and anti-T-lymphocyte globulin and GVHD prophylaxis consisting of tacrolimus and methylprednisolone (1 mg/kg/day), 26 patients who had hematologic malignancies in an advanced stage or with a poor prognosis underwent transplantation using peripheral blood stem cells from an HLA-haploidentical donor (2-3 antigen mismatches in the graft-versus-host [GVH] direction) without T-cell depletion. All patients except for 1 achieved donor-type engraftment. Rapid hematologic engraftment was achieved (neutrophils > 0.5 x 10(9)/L on day 12 and platelets > 20 x 10(9)/L on day 12), with full donor chimerism achieved by day 14. Fifteen patients did not develop acute GVHD clinically, and only 5 patients developed grade II GVHD. The recovery of CD4+ T cells was delayed compared with that of CD8+ T cells. Sixteen of the 26 patients are alive in complete remission. Four died of transplantation-related causes, and 6 died of progressive disease. These data suggest that nonmyeloablative conditioning, GVHD prophylaxis consisting of tacrolimus and methylprednisolone, and early therapeutic intervention for the GVH reaction allow stable engraftment and effectively suppress GVHD in HLA 2-3 antigen-mismatched SCT.

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