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Thymic epithelial tumors are rare malignancies, and no optimal therapeutic regimen has been defined for patients with advanced disease. Patients with advanced thymic epithelial tumors, which were resistant or intolerable to prior therapies, were eligible for this study. Patients received 9 mer-WT1-derived peptide emulsified with Montanide ISA51 adjuvant via intradermal administration once a week as a monotherapy. After the 3-month-protocol treatment, the treatment was continued mostly at intervals of 2-4 weeks until disease progression or intolerable adverse events occurred. Of the 15 patients enrolled, 11 had thymic carcinoma (TC) and 4 had invasive thymoma (IT). Median period from diagnosis to the start of treatment was 13.3 and 65.5 months for TC and IT, respectively. No patients achieved a complete or partial response. Of the 8 evaluable TC patients, 6 (75.0%) had stable disease (SD) and 2 had progressive disease (PD). Of the 4 evaluable IT patients, 3 (75.0%) had SD and 1 (25.0%) had PD. Median period of monotherapy treatment was 133 and 683 days in TC and IT patients, respectively. No severe adverse events occurred during the 3-month-protocol treatment. As adverse events in long responders, thymoma-related autoimmune complications, pure red cell aplasia and myasthenia gravis occurred in two IT patients. Cerebellar hemorrhage developed in a TC patient complicated with Von Willebrand disease. Induction of WT1-specific immune responses was observed in the majority of the patients. WT1 peptide vaccine immunotherapy may have antitumor potential against thymic malignancies.

We previously evaluated Wilms’ tumor gene 1 (WT1) peptide vaccination in a large number of patients with leukemia or solid tumors and have reported that HLA‐A*24:02 restricted, 9‐mer WT1‐235 peptide (CYTWNQMNL) vaccine induces cellular immune responses and elicits WT1‐235‐specific cytotoxic T lymphocytes (CTLs). However, whether this vaccine induces humoral immune responses to produce WT1 antibody remains unknown. Thus, we measured IgG antibody levels against the WT1‐235 peptide (WT1‐235 IgG antibody) in patients with glioblastoma multiforme (GBM) receiving the WT1 peptide vaccine. The WT1‐235 IgG antibody, which was undetectable before vaccination, became detectable in 30 (50.8%) of a total of 59 patients during 3 months of WT1 peptide vaccination. The dominant WT1‐235 IgG antibody subclass was Th1‐type, IgG1 and IgG3. WT1‐235 IgG antibody production was significantly and positively correlated with both progression‐free survival (PFS) and overall survival (OS). Importantly, the combination of WT1‐235 IgG antibody production and positive delayed type‐hypersensitivity (DTH) to the WT1‐235 peptide was a better prognostic marker for long‐term OS than either parameter alone. These results suggested that WT1‐235 peptide vaccination induces not only WT1‐235‐specific CTLs as previously described but also WT1‐235‐specific humoral immune responses associated with antitumor cellular immune response. Our results indicate that the WT1 IgG antibody against the WT1 peptide may be a useful predictive marker, with better predictive performance in combination with DTH to WT1 peptide, and provide a new insight into the antitumor immune response induction in WT1 peptide vaccine‐treated patients., What's new? The Wilms' tumor gene 1 (WT1) antigen is a promising target for immunotherapeutic strategies against glioblastoma multiforme (GBM), a brain tumor with poor survival rates. The present study shows that vaccination with WT1‐235 peptide can induce WT1‐235‐specific humoral immune responses in GBM patients. WT1‐235 IgG antibody production was significantly associated with prolonged progression‐free survival and overall survival. Survival times were significantly longer in GBM patients with positive delayed‐type hypersensitivity (DTH) responses to WT1 peptide. Thus, in WT1 vaccine‐treated GBM patients, especially those exhibiting positive DTH responses, WT1‐235 IgG antibody production can predict long‐term survival.

Severe peripheral neuropathy and myelopathy are rare complications after stem cell transplantation (SCT). In our institution, seven patients of precursor T lymphoblastic leukemia/lymphoma without the central nervous involvement who had been treated by nelarabine to control their diseases received SCT from HLA-haploidentical familial donor (HLA-haploidentical SCT) with the conditioning regimen including high-dose cytarabine (HDAC). Three of evaluable six patients developed irreversible paresthesia and muscle weakness in both lower extremities after neutrophil engraftment. The results of nerve conduction studies and short latency somatosensory evoked potentials suggested axonal neuropathy of both lower extremities in all three patients and myelopathy in two patients. Negative findings of PET-CT, and analyses of repeated cerebrospinal fluid samples and the bone marrow also indicated that tumor involvement was improbable. In all three patients, the symptoms worsened or persisted despite administration of corticosteroid and intravenous immunoglobulin. The high frequency of the neurological symptoms in our patients previously treated by nelarabine strongly suggested the association of the nelarabine use. Furthermore, the HLA-haploidentical SCT setting and the use of a potentially neurotoxic agent, HDAC might augment the neurotoxicity of nelarabine. It may be desirable that HLA-haploidentical SCT candidates avoid receiving nelarabine.

Summary Monoclonal antibody (mAb) drugs are desirable for the improvement of multiple myeloma (MM) treatment. In this study, we found for the first time that CD48 was highly expressed on MM plasma cells. In 22 out of 24 MM patients, CD48 was expressed on more than 90% of MM plasma cells at significantly higher levels than it was on normal lymphocytes and monocytes. CD48 was only weakly expressed on some CD34+ haematopoietic stem/progenitor cells, and not expressed on erythrocytes or platelets. We next examined whether CD48 could serve as a target antigen for mAb therapy against MM. A newly generated in-house anti-CD48 mAb induced mild antibody-dependent cell-mediated cytotoxicity and marked complement-dependent cytotoxicity against not only MM cell lines but also primary MM plasma cells in vitro. Administration of the anti-CD48 mAb significantly inhibited tumour growth in severe combined immunodeficient mice inoculated subcutaneously with MM cells. Furthermore, anti-CD48 mAb treatment inhibited growth of MM cells transplanted directly into murine bone marrow. Finally and importantly, we demonstrated that the anti-CD48 mAb did not damage normal CD34+ haematopoietic stem/progenitor cells. These results suggest that the anti-CD48 mAb has the potential to become an effective therapeutic mAb against MM.

Tumor-specific immunotherapy with a Wilms' tumor 1 (WT1) peptide has been on clinical trial for leukemia, myelodysplastic syndrome, breast and lung cancers and is producing promising results. In this study, we treated three patients with renal cell carcinoma with an anchor modified, HLA-A*2402 binding WT1 peptide which was emulsified in Freund's incomplete adjuvant. In two patients tumor growth was suppressed and clinical response was evaluated as stable disease by the RECIST criteria after 3 months of weekly immunizations. Notably, development of new metastases has stopped in these patients for a prolonged period. No deleterious side effects were observed. Peptide-specific T cells were expanded in PBMCs of the patients and a substantial fraction of them bore the surface phenotype consistent with a CD8+ cytotoxic effector population. Although established tumors did not regress further, considering the component of the vaccine, i.e. peptide alone, the stabilization effect suggested the potential of WT1 peptide to develop into a more effective vaccine. To our knowledge, this is the first report of WT1 immunotherapy for renal cell carcinoma. Hopefully, the results will stimulate more extensive clinical studies.

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.

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.

Expression of the Wilms' tumor gene WT1 in de novo lung cancer was examined using quantitative real-time RT-PCR and immunohistochemistry. Overexpression of the WT1 gene was detected by RT-PCR in 54/56 (96%) de novo non-small cell lung cancers examined and confirmed by detection of WT1 protein with an anti-WT1 antibody. Overexpression of the WT1 gene was also demonstrated in 5/6 (83%) de novo small cell lung cancers by immunohistochemistry. Furthermore, when the WT1 gene was examined for mutations by direct sequencing of genomic DNA in 7 lung cancers, no mutations were found. These results suggest that the nonmutated, wild-type WT1 gene plays an important role in tumorigenesis of de novo lung cancers and may provide us with the rationale for new therapeutic strategies for lung cancer targeting the WT1 gene and its products.

The Wilms' tumour gene, WT1, is expressed at high levels in leukaemia cells and plays an important role in leukaemogenesis. WT1 is also expressed in human normal CD34+ bone marrow (BM) cells at about 100 times lower levels than in leukaemia cells. To identify and characterize WT1-expressing cells in CD34+ BM cells, they were sorted into single cells and analysed for WT1 expression using two kinds of single-cell reverse transcriptase polymerase chain reaction (RT-PCR) methods. Using the semiquantitative single-cell polyA-PCR + sequence-specific (SS)-PCR method, WT1 expression was detected in four (1.3%) out of 319 CD34+ BM single cells. To confirm the above results, a single-cell nested sequence-specific (NSS)-RT-PCR method that was less quantitative but more sensitive than the polyA-PCR + SS-PCR method was also performed, and WT1 expression was detected in 15 (1.1%) out of 1315 CD34+ BM single cells. In total, WT1 expression was found in 19 (1.2%) out of 1634 CD34+ BM single cells. No significant differences in the frequencies of WT1-expressing cells were found between CD34+CD38- and CD34+CD38+ BM single cells. Furthermore, WT1-expressing CD34+ BM single cells expressed WT1 at levels similar to those in K562 leukaemia single cells. Analysis of lineage-specific and cell cycle gene expression in WT1-expressing CD34+ BM single cells showed that the WT1 gene could be expressed in both uncommitted, dormant CD34+CD38- and lineage-committed, proliferating CD34+CD38+ BM cells. Our results could indicate that these WT1-expressing CD34+ BM cells were normal counterparts of leukaemia cells.

How to treat CML patients who are resistant to inhibitors of BCR-ABL tyrosine kinase such as Imatinib is a very important and urgent issue in clinical hematology. Here, we report a case of Imatinib-treated CML in which intradermally administered WT1 peptide vaccine elicited WT1-specific immune responses and the resultant reduction in the persistent residual disease in co-administration of Imatinib. BCR-ABL mRNA levels were being maintained under the detection limit for 8 months since week 77 of vaccination. No adverse effects except local erythema at the injection sites were observed. The tetramer assay revealed that the decrease in BCR-ABL mRNA levels was associated with the increase in frequency of WT1-specific cytotoxic T lymphocytes, notably effector-memory type of that, in the patient's peripheral blood. The case presented here indicates that WT1 peptide vaccine may become a safe and cure-oriented therapy for CML patients who have residual disease regardless of the treatment with Imatinib.

Summary There are few reports of unmanipulated HLA-haploidentical nonmyeloablative stem cell transplantation (NST) using only pharmacological acute graft-vs.-host disease (GVHD) prophylaxis. We present here a successful case of unmanipulated HLA-haploidentical NST for mediastinal large B cell lymphoma that was resistant to autologous peripheral blood stem cell transplantation (PBSCT). The conditioning regimen consisted of fludarabine, busulfan and rabbit anti-T-lymphocyte globulin (ATG) in addition to rituximab. GVHD prophylaxis was performed using tacrolimus and methylprednisolone 1 mg/kg. The patient had rapid engraftment, with 100% donor chimaerism in the lineages of both T cells and granulocytes on day +12, but developed no GVHD clinically. The patient is still in complete remission past day +1020, with no sign of chronic GVHD without receiving immunosuppressive agents. HLA-haploidentical NST may be performed without utilizing mixed chimaerism.