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High pre-treatment serum soluble interleukin-2 receptor (sIL-2R) levels are associated with poor overall survival (OS) of patients with newly diagnosed follicular lymphoma (FL). We evaluated the usefulness of pre-treatment sIL-2R levels in selecting a treatment regimen for advanced-stage FL with low tumor burden (FL-LTB). This retrospective, multicenter observational study enrolled consecutive patients who received a rituximab-containing regimen for newly diagnosed advanced stage FL-LTB (grade 1-3a) between 2008 and 2018. We applied a previously reported cut-off value of 1800 IU/mL for sIL-2R. A total of 211 patients were eligible for the analysis. Among patients with high sIL-2R (47 patients, 22.3%), the OS rates for patients treated by rituximab monotherapy (R-mono) (11 patients) were significantly lower than those treated by rituximab-combination chemotherapy (R-chemo) (36 patients): 5-year OS rates were 66.7% and 94.4%, respectively (P = 0.007). Among patients with low sIL-2R (164 patients, 77.7%), OS rates were comparably good between the R-mono group (34 patients) and the R-chemo group (130 patients): 5-year OS rates were 100% and 98.3%, respectively (P = 0.38). Our results suggest that R-chemo may yield better OS than R-mono for patients with newly diagnosed advanced-stage FL-LTB and high pre-treatment serum sIL-2R levels.

Background It is known that an immunoglobulin abnormality affects various clinical laboratory measurements and leads to abnormal values. We experienced a case of monoclonal gammopathy of undetermined significance (MGUS) showing a falsely low plasma glycated albumin (GA) level. Case report The patient was a 75-y-old male who visited our hospital for thrombocytosis identified during a medical checkup. Based on further examinations, he was diagnosed with MGUS (IgM-κ type). Laboratory examinations revealed that the plasma GA level was significantly low at −1.3% but the serum GA level was reasonable at 15.5%. We investigated the cause of the falsely low plasma GA level. Results The patient's plasma became turbid after mixing with the first reagent for GA measurement. The plasma GA level was increased by dilution of the plasma. The plasma GA level was falsely decreased only at the time of measurement on a sample collected using a blood-collecting tube with heparin sodium. The GA level was decreased by adding heparin sodium to the patient's serum, whereas the GA level was increased by neutralization of the patient's plasma with protamine sulfate. The GA level was increased after adding polyethylene glycol to the patient's plasma. Serum GA levels in healthy controls were decreased by adding purified M protein from the patient's serum. Conclusions We report a patient with MGUS whose plasma GA concentration was falsely decreased by M protein when blood was drawn in a heparin sodium-containing tube.

This retrospective, multicenter observational study investigated the prognostic value of pretreatment serum soluble interleukin-2 receptor (sIL-2R) level for outcomes of newly diagnosed follicular lymphoma (FL) grade 1-3a who required treatment at diagnosis. A total of 628 patients were recorded, and 502 of these were eligible for analysis. Patients were divided into four quartiles, based on their serum sIL-2R levels as follows: Q1 (sIL-2R520 IU/mL), Q2 (520 ≤ sIL-2R1030 IU/mL), Q3 (1030 ≤ sIL-2R2530 IU/mL) and Q4 (sIL-2R ≥ 2530 IU/mL). Using a multivariable Cox proportional-hazards model, we showed the adjusted probability of overall survival (OS) decreased with increasing serum sIL-2R levels (

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.

Introduction: Serum soluble interleukin-2 receptor (sIL-2R) is recognized as a tumor-related biomarker of malignant lymphomas, including follicular lymphoma (FL). The objective of this study is to assess the prognostic significance of pretreatment serum sIL-2R level for disease-specific overall survival (DSS) in patients with FL. Methods: We retrospectively analyzed medical records of our 10 individual institutions to identify all patients (pts) ≥ 18 years old with newly diagnosed FL between 01/01/2008 and 12/31/2018. Only pts with FL grade 1-3b, confirmed at pathological review, were included. Pts with histological transformation at diagnosis were excluded. Serum sIL-2R was determined by sandwich enzyme-linked immunosorbent assay. Clinical characteristics, therapies, response, relapse patterns and follow up status were collected and analyzed in the cohort. DSS was defined from the time of initial diagnosis to death due to lymphoma or last follow up. DSS was analyzed according to Kaplan Meier method and differences between subgroups were compared with log-rank test. Multivariate cox regression analysis was used to investigate associations of sIL-2R, FL International Prognostic Index (FLIPI) and progression or relapse within 24 months after diagnosis (POD24) with DSS. Serum levels of sIL-2R was measured before initiation of treatment and the data on the nearest date of the diagnostic biopsy within six months was adopted. Results: We recorded 565 pts and 535 pts of them with pretreatment serum sIL-2R levels available were included in the analysis with median age 64 years (range: 30-90) and 305 females (57%). Clinical characteristics are summarized in Table 1. The median of the serum sIL-2R level was 896 IU/mL (range: 145 - 23800). We decided an optimal cutoff value of 1800 IU/mL by using receiver operating characteristic (ROC) analysis, which showed it was 1830 IU/mL (AUC : 0.76, 95%CI : 0.67 - 0.84). Various poor prognostic indicators, such as bulky mass, increased LDH, ≥5 nodal lesions, poor performance status (PS), bone marrow invasion, FLIPI and FLIPI-2 high-risk, decreased Hb, advanced disease, high tumor burden, increased β2MG and existence of B symptoms, were strongly associated with high serum sIL-2R level (≥1800 IU/mL), and high sIL-2R level correlated with POD24 (P Conclusions: High sIL-2R level ( ≥1800 IU/mL) is a significant and independent adverse prognostic factor for DSS in pts with newly diagnosed FL. This study provides us useful information to predict population with worse DSS before initiation of treatment. Disclosures Nozaki: Chugai: Honoraria; Celgene: Honoraria. Kida:Eisai Co., Ltd: Honoraria. Kosugi:Novartis Pharma Co.: Honoraria; Bristol-Myers Squibb Co.: Honoraria; Eisai Co., Ltd: Honoraria; Takeda Pharmaceutical Company Limited: Honoraria; Kyowa Kirin Co., Ltd: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria; ONO PHARMACEUTICAL CO., LTD.: Honoraria; NIPPON SHINYAKU CO.,LTD.: Honoraria; Janssen Pharmaceutical K.K.: Honoraria; Celgene K.K.: Honoraria; Pfizer Inc.: Honoraria. Shibayama:Astellas, Teijin, MSD, Shionogi, Eisai, Sumitomo Dainippon, Taiho, Nippon Shinyaku: Research Funding; Celgene, Chugai, Eisai, AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Takeda, Novartis, Janssen, Chugai, Eisai, Mundi Pharma, Ono, Otsuka, Kyowa Kirin, Sumitomo Dainippon, AstraZeneca, Avvie, DaiichiSankyo, Fujimoto, Nippon Shinyaku, Sanofi, Bristol-Myers Squibb, Pfizer: Honoraria.

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.

Long-term WT1 peptide vaccination for patients with acute myeloid leukemia with minimal residual disease

Methotrexate (MTX) has been documented to accumulate in "third spaces'' such as pleural effusions or ascitic fluids, resulting in delayed clearance and severe toxicity. We present a case of Burkitt lymphoma possessing large liver cysts, up to the size of 7 x 7 cm, wherein clearance of high-dose MTX was severely delayed, despite normal renal and liver functions. The serum MTX concentration was higher than 0.1 microM on day 12 and remained at toxic levels, higher than 0.01 microM, even on day 25, resulting in severe neutropenia, anorexia, and diarrhea. It was presumed that MTX accumulated in the liver cysts over time and was slowly released back into the serum, resulting into prolonged high serum MTX concentrations. High dose of MTX in patients with large liver cysts induces severe toxicity by virtue of MTX accumulation in the cysts and its subsequent delayed clearance.

Object The object of this study was to investigate the safety and clinical responses of immunotherapy targeting the WT1 (Wilms tumor 1) gene product in patients with recurrent glioblastoma multiforme (GBM). Methods Twenty-one patients with WT1/HLA-A*2402–positive recurrent GBM were included in a Phase II clinical study of WT1 vaccine therapy. In all patients, the tumors were resistant to standard therapy. Patients received intra-dermal injections of an HLA-A*2402–restricted, modified 9-mer WT1 peptide every week for 12 weeks. Tumor size, which was obtained by measuring the contrast-enhanced area on magnetic resonance images, was determined every 4 weeks. The responses were analyzed according to Response Evaluation Criteria in Solid Tumors (RECIST) 12 weeks after the initial vaccination. Patients who achieved an effective response continued to be vaccinated until tumor progression occurred. Progression-free survival and overall survival after initial WT1 treatment were estimated. Results The protocol was well tolerated; only local erythema occurred at the WT1 vaccine injection site. The clinical responses were as follows: partial response in 2 patients, stable disease in 10 patients, and progressive disease in 9 patients. No patient had a complete response. The overall response rate (cases with complete or partial response) was 9.5%, and the disease control rate (cases with complete or partial response as well as those in which disease was stable) was 57.1%. The median progression-free survival (PFS) period was 20.0 weeks, and the 6-month (26-week) PFS rate was 33.3%. Conclusions Although a small uncontrolled nonrandomized trial, this study showed that WT1 vaccine therapy for patients with WT1/HLA-A*2402–positive recurrent GBM was safe and produced a clinical response. Based on these results, further clinical studies of WT1 vaccine therapy in patients with malignant glioma are warranted.

Objective The incidence of severe graft-vs-host disease (GVHD) in unmanipulated human leukocyte antigen (HLA) 2-3 antigen–mismatched bone marrow transplantation (BMT) using cyclosporine and methotrexate as GVHD prophylaxis is 80% to 90%. We investigated whether pharmacological GVHD prophylaxis consisting of four drugs, including a steroid, effectively suppressed GVHD in this transplantation setting. Materials and Methods Thirty patients who had hematologic malignancies at an advanced stage or with poor prognosis underwent allogeneic BMT using a myeloablative preconditioning regimen consisting of cyclophosphamide (60 mg/kg × 2), total body irradiation (8–10 Gy), and fludarabine (30 mg/m 2 × 4) with or without cytosine arabinoside (2 g/m 2 × 4), and GVHD prophylaxis consisting of a combination of tacrolimus, methotrexate, mycophenolate mofetil, and methylprednisone (2 mg/kg). Early therapeutic intervention for GVH reaction or grade I GVHD was performed, and steroid was slowly tapered. Results All patients achieved donor-type engraftment. Neutrophil (>0.5 × 10 9 /L) and platelet (>20 × 10 9 /L) engraftment was achieved on day 13 and on day 30, respectively. Seventeen patients (56.7%) had no GVHD. Eleven patients (36.7%) developed grade II–III acute GVHD. Seven patients (23.3%) died of transplant-related toxicity, including fungal or viral infections and thrombotic microangiopathy, and four patients died of disease progression. Estimated relapse rate at 3 years was only 20.9%. The probability of survival at 3 years was 49.9%. Conclusions These data suggest that, in unmanipulated HLA-haploidentical allogeneic BMT, this GVHD prophylactic regimen, which includes methylprednisolone 2 mg/kg, and early therapeutic intervention for GVH reaction suppress the incidence of severe GVHD to an acceptable level, while preserving the graft-vs-leukemia effect.

The Wilms' tumor gene WT1 is overexpressed in most of human leukemias regardless of disease subtypes. To characterize the expression pattern of WT1 during normal and neoplastic hematopoiesis, we generated a knock-in reporter green fluorescent protein (GFP) mouse (WT1(GFP/+)) and assayed for WT1 expression in normal and leukemic hematopoietic cells. In normal hematopoietic cells, WT1 was expressed in none of the long-term (LT) hematopoietic stem cells (HSC) and very few (1%) of the multipotent progenitor cells. In contrast, in murine leukemias induced by acute myeloid leukemia 1 (AML1)/ETO+TEL/PDGFbetaR or BCR/ABL, WT1 was expressed in 40.5 or 38.9% of immature c-kit(+)lin(-)Sca-1(+) (KLS) cells, which contained a subset, but not all, of transplantable leukemic stem cells (LSCs). WT1 expression was minimal in normal fetal liver HSCs and mobilized HSCs, both of which are stimulated for proliferation. In addition, overexpression of WT1 in HSCs did not result in proliferation or expansion of HSCs and their progeny in vivo. Thus, the mechanism by which expansion of WT1-expressing cells occurs in leukemia remains unclear. Nevertheless, our results demonstrate that the WT1(GFP/+) mouse is a powerful tool for analyzing WT1-expressing cells, and they highlight the potential of WT1, as a specific therapeutic target that is expressed in LSCs but not in normal HSCs.

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.

Wild-type Wilms' tumor geneWT1is expressed at a high level in hematopoietic malignancies including acute leukemia, chronic myelogenous leukemia, and myelodysplastic syndromes, as well as in various kinds of solid cancers. Human cytotoxic T lymphocytes (CTLs), which could specifically lyseWT1-expressing tumor cells with HLA class I restriction, were generatedin vitro. It was also demonstrated that mice immunized with the WT1 peptide rejected challenges byWT1-expressing cancer cells and survived with no signs of autoaggression to normal organs that physiologically expressedWT1. Furthermore, we and others detected IgM and IgG WT1 antibodies in patients with hematopoietic malignancies, indicating that the WT1 protein was highly immunogenic, and that immunoglobulin class-switch-inducing, WT1-specific, cellular immune responses were elicited in these patients. CD8+WT1-specific CTLs were also detected in peripheral blood or tumor-draining lymph nodes of cancer patients. These results provided us with the rationale for elicitation of CTL responses targeting the WT1 product for cancer immunotherapy. On the basis of these findings, we performed a phase I clinical trial of a WT1 peptide cancer vaccine for the patients with malignant neoplasms. These results strongly suggested that the WT1 peptide cancer vaccine had efficacy in the clinical setting because clinical responses, including reduction of leukemic blast cells or regression of tumor masses, were observed after the WT1 vaccination in patients with hematopoietic malignancies or solid cancers. The power of a tumor-associated-antigen (TAA)-derived cancer vaccine may be enhanced in combination with stronger adjuvants, helper peptide, molecular-target-based drugs, or some chemotherapy drugs, such as gemcitabine, which has been revealed to suppress regulartory T-cell function. In contrast, reduction of WT1 peptide dose may be needed for the treatment of patients with hematological stem cell diseases, because rapid and strong destruction of malignant cell-sustained hematopoiesis before recovery of normal hematopoiesis may lead to pancytopenia in these patients.

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 × 109/L on day 12 and platelets > 20 × 109/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.

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.

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.

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.

A 46-year-old woman with Hodgkin's disease who underwent nonmyeloablative allogeneic stem cell transplantation developed cortical blindness, seizures, and left hemiparesis on day 100 while receiving tacrolimus (FK506) and prednisone for the treatment of graft-versus-host disease (GVHD). Magnetic resonance imaging revealed multiple changes, mainly in the bilateral occipital lobes, suggesting FK506-related leukoencephalopathy. These abnormalities improved after discontinuation of FK506. However, 3 days after the episode, cerebral hemorrhage in the left occipital lobe with perforation to the left subdural space occurred. Although FK506-induced leukoencephalopathy with cerebral hemorrhage is considered the more severe form of such leukoencephalopathy, the patient's neurological symptoms almost completely resolved and radiographic findings improved after discontinuation of FK506, tapering of methylprednisolone, and initiation of mycophenolate mofetil. FK506-related leukoencephalopathy is a rare complication after allogeneic stem cell transplantation. Although the symptoms usually subside after discontinuation of FK506, therapeutic intervention in many cases may result in severe complications, including GVHD and vascular disease. We consider it important to use immunosuppressive agents without vascular endothelial toxicity for preventing the development of fatal GVHD after discontinuation of FK506.

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 WT1 gene is highly expressed in various types of leukemia, particularly in acute type leukemia. The extent of minimal residual disease (MRD) of leukemia can be evaluated by measuring the WT1 gene expression level using a quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) method. The WT1 transcript assay can be applied to almost all patients with leukemia regardless of the presence or absence of chimeric DNA markers. Furthermore, because of the fact that WT1 expression levels increase significantly at relapse compared with those at the time of diagnosis, and because of the decrease in background levels of WT1 expression in bone marrow following allogeneic SCT, the WT1 transcript assay possesses a high degree of sensitivity following allogeneic SCT. Frequent monitoring of the WT1 gene expression level predicts the risk of relapse following allogeneic SCT, and furthermore, the kinetics of WT1 transcripts predict the efficacy of immunomodulation therapy such as donor leukocyte infusion.

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.

A Wilms' tumor gene WT1 is expressed at high levels not only in most types of leukemia but also in various types of solid tumors, including lung and breast cancer. WT1 protein has been reported to serve as a target antigen for tumor-specific immunotherapy both in vitro in human systems and in vivo in murine models. We have shown that mice immunized with WT1 peptide or WT1 cDNA could reject a challenge from WT1-expressing tumor cells (a "prophylactic" model). However, it was not examined whether WT1 peptide vaccination had the potency to reject tumor cells in a "therapeutic" setting. In the present study, we demonstrated for the first time that WT1 peptide vaccination combined with Mycobacterium bovis bacillus Calmette-Guérin cell wall skeleton (BCG-CWS) was more effective for eradication of WT1-expressing tumor cells that had been implanted into mice before vaccination (a "therapeutic" model) compared with WT1 peptide vaccination alone. An intradermal injection of BCG-CWS into mice, followed by that of WT1 peptide at the same site on the next day, generated WT1-specific cytotoxic T lymphocytes (CTLs) and led to rejection of WT1-expressing leukemia or lung cancer cells. These results showed that BCG-CWS, which was well known to enhance innate immunity, could enhance WT1-specific immune responses (acquired immunity) in combination with WT1 peptide vaccination. Therefore, WT1 peptide vaccination combined with BCG-CWS may be applied to cancer immunotherapy in clinical settings.

On the basis of transplantation data from the Japan Society for Hematopoietic Cell Transplantation, we retrospectively analyzed the impact of cytogenetics at diagnosis on the outcome of transplantation in 628 patients with acute myeloid leukemia who underwent autologous (n = 200), allogeneic related (n = 363), or allogenic unrelated (n = 65) stem cell transplantation (SCT) at first complete remission. For autologous SCT, patients at good cytogenetic risk had a significantly lower relapse rate (P = .017) and a significantly higher event-free survival (EFS) (P = .013) compared with those at intermediate risk. For allogeneic SCT, patients at good cytogenetic risk had a significantly lower relapse rate (P = .019) and insignificantly higher EFS (P = .093) than those at poor risk. For unrelated SCT, there was no significant difference in relapse rate or EFS between patients at good risk and those at intermediate risk. Comparison of the 3 transplantation modalities revealed that autologous SCT patients had a significantly higher incidence of relapse compared with related or unrelated SCT patients in the intermediate-risk group but not in the good-risk group. However, there were no significant differences in EFS among the 3 transplant modalities in either of these 2 risk groups. In multivariate analysis, cytogenetics was found to be an independent predictor of relapse as well as of treatment failure.

Background: It was recently demonstrated that the WT1 gene was overexpressed in primary breast cancer and that the high expression levels of WT1 mRNA significantly correlated with poor prognosis. However, it remained undetermined whether or not the WT1 gene expressed in breast cancer had mutations. Methods: Breast cancer tissues were obtained from 36 patients with breast cancer. WT1 genomic DNA was PCR-amplified and examined for mutations by direct sequencing. Results: The sequencing analysis showed the absence of mutations through the whole 10 exons of the WT1 gene in the 36 cases of primary breast cancer. Two different single nucleotide polymorphisms (SNP) without an amino acid change (Pro42, C to T in exon 1, and/or Arg300, A to G in exon 7) were detected in the WT1 gene in 31 (86%) of the 36 cases examined. Conclusion: The results indicate that the wild-type WT1 gene plays an important role in the tumorigenesis of primary breast cancer.

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) γδ 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+ TCR78 T-cells were CD8as, 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.IntJHematol. 2003;77:463-470.

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 theWT1 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.

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.

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.

Background. Graft-versus-host disease (GVHD) is still a major problem in allogeneic bone marrow transplantation (BMT). Prophylactic regimens used against GVHD in unrelated BMT, including cyclosporine (CsA)-plus-methotrexate (MTX), CsA-plus-MTX-plus-prednisone, and tacrolimus (FK506)-plus-MTX, are still unsatisfactory (34–70% occurrence of grades II–IV GVHD). To address this problem, we examined the efficacy of FK506-plus-MTX-plus-methylprednisolone (mPSL) in 20 patients who underwent BMT from unrelated donors. Methods. All patients received FK506 beginning the day before transplantation at a dose of 0.03 mg/kg per day by continuous intravenous (IV) infusion. MTX was administered at a dose of 10 mg/m2 IV on day 1, and 7 mg/m2 on days 3, 6, and 11. Intravenous administration of mPSL was started at a dose of 2 mg/kg per day on day 1. In the absence of acute GVHD, mPSL was gradually tapered from day 29. Results. Development of acute GVHD was almost completely suppressed (one patient with grade I, none with grades II–IV). However, the incidence and severity of chronic GVHD did not decrease. Eight of 12 patients with extensive chronic GVHD died of thrombotic microangiopathy or infection. A vigorous fluctuation (>100 U/mL per 10 days) of the soluble interleukin 2 receptor level in the serum after engraftment was highly related to the occurrence of chronic GVHD. Conclusions. An FK506-plus(+)-MTX-plus(+)-mPSL prophylactic regimen could almost completely suppress acute GVHD but not chronic GVHD in unrelated BMT. In this GVHD prophylactic system, the extent of the change of soluble interleukin 2 receptor level may be a good predictor of development of chronic GVHD.

Wilms tumor gene WT1 is expressed at high levels in hematopoietic malignancies, such as leukemias and myelodysplastic syndromes (MDS), and in various kinds of solid tumors, including lung cancer, and it exerts an oncogenic function in these malignancies. IgM and IgG WT1 antibodies were measured by means of dot blot assay in 73 patients with hematopoietic malignancies (16 acute myeloid leukemia [AML], 11 acute lymphoid leukemia [ALL], 13 chronic myeloid leukemia [CML], and 33 MDS) and 43 healthy volunteers. Immunoglobulin IgM, IgG, and IgM+IgG WT1 antibodies were detected in 40 (54.8%), 40 (54.8%), and 24 (32.8%), respectively, of the 73 patients with hematopoietic malignancies, whereas 7 (16.2%), 2 (4.7%), and none of the 43 healthy volunteers had IgM, IgG, or IgM+IgG WT1 antibodies, respectively. Furthermore, immunoglobulin isotype class switching of WT1 antibodies from IgM to IgG occurred in conjunction with disease progression from refractory anemia (RA) to RA with excess of blasts (RAEB), and further to RAEB in transformation (RAEB-t) in MDS patients. These results showed that humoral immune responses against the WT1 protein could be elicited in patients with WT1-expressing hematopoietic malignancies, and they suggested that the helper T-cell responses needed to induce humoral immune responses and immunoglobulin isotype class switching from IgM to IgG were also generated in these patients. Our findings may provide new insight into the rationale for elicitation of cytotoxic T-cell responses against the WT1 protein in cancer immunotherapy using the WT1 vaccine.

We present a case of adenovirus (ADV) infection in a patient who had undergone nonmyeloablative stem cell transplantation (NST). A 50-year-old man with chronic myelogenous leukemia in the second chronic phase underwent NST from an HLA 2-loci-mismatched sibling. ADV hemorrhagic cystitis developed and progressed to lethal pneumonia. ADV was isolated from urine, bronchoalveolar lavage fluid, and postmortem specimens of kidney and liver. Because there are few reports of lethal pneumonia associated with ADV in Japan, we present the case and discuss the cause of and therapy for the infection.

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.

WT1 (Wilms' tumor gene 1) protein is a potent pan-tumor-associated antigen (TAA) and WT1-specific cytotoxic T lymphocytes (WT1 tetramer(+) CD8(+) T cells) are spontaneously induced in patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). We conducted a single-cell level comparative analysis of T-cell receptor beta-chain variable region (TCR-BV) gene families of a total of 1242 spontaneously induced WT1 tetramer(+) CD8(+) T cells in HLA-A*2402(+) patients with AML or MDS and those in healthy donors (HDs). This is the first report of direct usage analysis of TCR-BV gene families of individual TAA-specific CD8(+) T cells at single-cell level. Usage analysis using single-cell RT-PCR of TCR-BV gene families of individual FACS-sorted WT1 tetramer(+) CD8(+) T cells showed for the first time (i) that BVs 5, 6, 20, and 27 were commonly biased in both HDs and patients; (ii) that BV4 was commonly biased in HDs and MDS patients; (iii) that BV19 was commonly biased in the patients; and (iv) that BVs 7 and 28, BVs 9 and 15, and BVs 12 and 29 were specifically biased in HDs, AML, and MDS patients, respectively. However, statistical analysis of similarity among HD, AML, and MDS of individual usage frequencies of 24 kinds of TCR-BV gene families indicated that the usage frequencies of TCR-BV gene families in AML and MDS patients reflect those in HDs. These findings represent a novel insight for a better understanding of WT1-specific immune response.

Although tyrosine kinase inhibitors is effective for dramatically reducing CML cells, it might be difficult to eradicate completely the CML stem cells. We aimed to clarify the safety and effects of WT1 peptide vaccination in combination with imatinib therapy for a CML patient. A 51 year-old male with CML in CP, who showed a resistance against imatinib therapy for 2.5 years, began to be treated with 9 mer modified-type WT1 peptides in combination with standard dose of imatinib. Although every 2-week-administration of WT1 peptides for 22 weeks did not show definite effects on the quantification of bcr-abl transcripts, by changing the administration from every 2 weeks to 4 weeks bcr-abl transcripts decreased remarkably. After 11 months of every 4-week-administration of the peptides and 12 months post cessation of the peptides bcr-abl transcripts achieved to the level below detection by RQ/RT-PCR (complete molecular response). WT1/MHC tetramer(+)CD8(+) CTLs, which appeared after the second administration of WT1 peptides and remained more than 15 in number among 10(6) CD8(+) T cells throughout the administration of WT1 peptides, are still present in the blood on 14th month post cessation of the peptides. An in vitro study as to the cytotoxicity of lymphocytes induced by mixed lymphocyte peptide culture demonstrated that cultured lymphocytes possessed cytotoxicity against WT1 expressing leukemia cells and the cytotoxicity was WT1-specific and MHC class I restricted. The present study showed that WT1 peptide vaccination in combination with TKI is feasible and effective in the therapy for imatinib-resistant CML.

The product of the Wilms tumor gene, WT1, is a universal tumor antigen. We performed WT1 peptide-based immunotherapy for a patient with multiple myeloma (MM). This patient was a 57-year-old woman with chemotherapy-resistant MM (Bence Jones kappa type). The patient received weekly intradermal injections of an HLA-A*2402-restricted 9-mer WT1 peptide emulsified with Montanide ISA 51 adjuvant for 12 weeks and achieved a minimal response according to European Group for Blood and Marrow Transplantation criteria without experiencing systemic adverse effects. The proportion of myeloma cells in the bone marrow (BM) decreased from 85% to 25%, and the amount of M protein in the urine decreased from 3.6 to 0.6 g/day after WT1 vaccination. Furthermore, a bone scintigram showed an improvement after the vaccination. As for immunologic parameters, the frequency of WT1 tetramer-positive cells among CD8+ T-cells, which was higher than in healthy donors, temporarily decreased at weeks 4 and 8 but increased at week 12, whereas the frequency of WT1 peptide-responding CD107a/b+ cells among WT1 tetramer-positive T-cells increased from 27.0% to 38.6% after the vaccination. After WT1 vaccination, the frequency of CXCR4+ cells among WT1 tetramer-positive T-cells increased in the BM, where stromal cells expressed the ligand for CXCR4, stromal-derived factor 1 (SDF-1), but decreased in the peripheral blood (PB), implying that WT1-specific cytotoxic T-lymphocytes had migrated from the PB to the BM, a tumor site.

The wild-type Wilms tumor gene, WT1, is overexpressed in myelodysplastic syndrome (MDS) as well as acute myeloid leukemia. In a phase I clinical trial of biweekly vaccination with HLA-A*2402-restricted WT1 peptide for these malignancies, 2 patients with MDS developed severe leukocytopenia in association with a reduction in leukemic blast cells and levels of WT1 messenger RNA (mRNA) after only a single vaccination with 0.3 mg of WT1 peptide. These results indicated that the WT1-specific cytotoxic T-lymphocytes (CTLs) elicited by WT1 vaccination eradicated the WT1-expressing transformed stem or progenitor cells and that MDS patients with little normal hematopoiesis required a new strategy of WT1 vaccination to avoid severe leukocytopenia. We describe the first trial for a 57-year-old male patient with chronic myelomonocytic leukemia who was vaccinated biweekly with a small quantity (5 microg/body) of WT1 peptide. After the start of vaccination, the leukocyte and monocyte counts (13,780/microL and 1930/microL, respectively) gradually decreased to within the normal range in association with a reduction in the WT1 mRNA level. Simultaneously, the percentage of WT1-specific CTLs as measured by the HLA-WT1 tetramer assay increased. This case demonstrates for the first time that vaccination with as little as 5 microg of WT1 peptide can induce WT1-specific immune responses and resultant clinical responses.

Wild-type Wilms tumor gene WT1 is highly expressed not only in hematopoietic malignancies, including leukemia and myelodysplastic syndromes (MDS), but also in various kinds of solid tumors. Human cytotoxic T lymphocytes (CTLs) which could specifically lyse WT1-expressing tumor cells with HLA class I restriction were generated in vitro. We have also demonstrated that mice immunized with the WT1 peptide or WT1 cDNA rejected challenges by WT1-expressing tumor cells and survived with no signs of auto-aggression to normal organs which physiologically expressed WT1 in prophylactic and therapeutic models. Furthermore, we and others detected IgM and IgG WT1 antibodies in the patients with hematopoietic malignancies, indicating that WT1 protein was highly immunogenic, and that immunoglobulin class-switch-inducing WT1- specific cellular immune responses were elicited in the patients. CD8+ WT1-specific CTLs were also detected in peripheral blood or tumor-draining lymph nodes of cancer patients. These results provided us with the rationale for elicitation of CTL responses targeting the WT1 product for cancer immunotherapy. On the basis of the findings mentioned above, we performed a phase I clinical trial of WT1 peptide cancer vaccine for the patients with malignant neoplasms. These results strongly suggested that WT1 peptide cancer vaccine had efficacy in the clinical setting, because clinical responses, including reduction of leukemic blast cells or regression of tumor masses, were observed after the WT1 vaccination in patients with hematopoietic malignancies or solid cancers. The power of TAA-derived cancer vaccine may be enhanced by combination with stronger adjuvants, helper peptide, or conventional treatments such as molecular-target-based drugs.

Of late years, newly developed agents, such as bortezomib, lenalidomide, thalidomide, are widely used for treatment against multiple myeloma. After induction therapy, candidates for autologous stem cell transplantation are supposed to be followed by stem cell harvesting. There are several reports showing lenalidomide has a negative impact on stem cell mobilization. This opinion tends to let us refrain from using lenalidomide on the myeloma patients who are eligible for transplantation, even lenalidomide is expected promising. We experienced a series of three clinical cases presenting that stem cells were poorly mobilized with cyclophosphamide (CY) plus G-CSF after lenalidomide treatment, but sequential stem cell mobilization was incredibly improved with high-dose cytarabine plus G-CSF. One additional case who was treated with lenalidomide also presented successful stem cell mobilization with high-dose cytarabine plus G-CSF. Here we show all four cases in detail. Case 1: 66 years old male, symptomatic myeloma after smoldering period. After three course of bortezomib induction, the response was insufficient. Sequentially he was treated with lenalidomide (25mg/day, every day for three weeks with one week rest period) and dexamethasone (Dex) (40mg/day, weekly) for two courses, and finally achieved Partial response (PR). First peripheral blood stem cell harvesting was attempted with high-dose CY (2.0 g/m2, day1-2) + lenograstim (5mg/kg daily, on days 7 until leukapheresis), but mobilization was unsuccessful so harvesting was not performed. For subsequent mobilization, high-dose cytarabine was administered at a dose of 2.0 g/m2 twice daily (day1-2) + lenograstim. Second mobilization was markedly improved, and finally 33.0 x 106/kg CD34+ cells were obtained. Case 2: 63 years old male, symptomatic myeloma, IgG type. This patient was treated with bortezomib, CY and Dex but resulted in disease progression. As an alternative therapy, lenalidomide (10mg/day, daily for three weeks with one week rest) and Dex (40mg, weekly) were used for three cycles. The dose of lenalidomide was reduced due to renal dysfunction. PR was obtained, then first harvesting was attempted with high-dose CY + lenograstim, as case 1, and 0.088 x 106/kg of CD34+ cells were collected, which was not sufficient for transplantation. Second mobilization was performed with high-dose cytarabine as case 1, and consequently we could obtain 60.1 x 106/kg of CD34+ cells; the yield was dramatically improved. Case 3: 41 years old female, symptomatic myeloma after one year course of smoldering myeloma. As an induction therapy, bortezomib, CY and Dex were selected, but finally she could not achieved PR after three cycles. We gave up bortezomib-based induction, and then lenalidomide (15-25 mg on day1-21 with 1 week rest) and Dex (40 mg, weekly) were administrated for five courses, followed by PR. As previous two cases, the first peripheral stem cell collection was initiated with high-dose CY + lenograstim, and it was not sufficient (0.059 x 106/kg of CD34+ cells). And the second mobilization with high-dose cytarabine with lenograstim recovered the yield of stem cell up to 6.90 x 106/kg. Case 4: 63 years old male, symptomatic myeloma. He was treated with bortezomib, Dex with/without CY, but this regimen was not very effectual, and CY caused elevation of aminotransferase (CTCAE grade 3). Then lenalidomide (10-15 mg on day 1-21) and Dex (40 mg, weekly) were administrated for four courses, and the patient achieved PR. Due to the adverse effect of liver dysfunction, we could not use high-dose CY for mobilization. For this case, high-dose cytarabine was selected for first mobilization, and it was very successful, 50.2 x 106/kg of CD34+ cells were harvested. The yields of PBSC from all four cases are summarized on Table 1. These four cases suggest mobilization with high-dose cytarabine could be an alternative option for poor mobilizer of myeloma patient treated with lenalidomide-based induction. This fact may enable us to choose lenalidomide, not only bortezomib, for induction even for transplant-eligible cases. Figure 1 Figure 1. Disclosures Ishiko: Celgene: Honoraria.

The Wilms' tumor gene WT1 is highly expressed in leukemias and myelodysplastic syndrome (MDS), and WT1 expression levels increase along with the disease progression in chronic myeloid leukemia and MDS. We previously reported that IgM and IgG WT1 antibodies were detected with significantly higher detection rate and antibody titers in leukemias and MDS compared to those in healthy volunteers. In this study, whether IgG humoral immune responses against WT1 protein were Th1- or Th2-type were determined by measurement of four subclasses of IgG WT1 antibody, IgG1, IgG2, IgG3, and IgG4. In leukemias and MDS, Th1-type WT1 antibodies such as IgG1, IgG2, and IgG3 were significantly increased in both detection rate and antibody titers compared to those in healthy volunteers, whereas Th2-type WT1 antibody such as IgG4 did not increase. These results showed that Th1-biased humoral immune responses against WT1 protein were generated in leukemias and MDS. These results should allow us to consider that Th1-biased cellular immune responses against WT1 protein, which was essentially needed for cancer immunotherapy targeting WT1, should be elicited in patients with hematopoietic malignancies.

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.

Background Encephalitis as the result of human herpesvirus (HHV)-6 is usually fatal when it is resistant to antiviral drugs. Methods We describe a patient who developed HHV-6 encephalitis after human leukocyte antigen-haploidentical transplantation using a reduced intensity regimen. Results The patient developed severe disorientation, amnesia, and tremors on day 28. Magnetic resonance imaging of the brain revealed limbic encephalitis, and the cerebrospinal fluid sample was positive for only HHV-6 in polymerase chain reaction analysis. Neither ganciclovir nor foscarnet was effective. The patient recovered from the critical condition of HHV-6 encephalitis after donor lymphocyte infusion (DLI). Almost all of his symptoms resolved, polymerase chain reaction tests for HHV-6 in the cerebrospinal fluid were negative, and magnetic resonance imaging findings were normal. Conclusions This is the first report of DLI as a treatment for HHV-6 encephalitis and the first report of DLI from an human leukocyte antigen-haploidentical donor as a treatment for life-threatening viral infection.

A 57-year-old man with a history of renal cell carcinoma presented with presyncope. He underwent nephrectomy years earlier followed by HLA-matched allogeneic peripheral-blood stem-cell transplantation. Echocardiographic investigation revealed a solitary right ventricle mass without contiguous vena caval or right atrial involvement. The mass was pathologically confirmed to be metastatic carcinoma in the right ventricular cavity. This case highlights the need to consider an underlying neoplastic syndrome in patients presenting isolated right ventricle mass by echocardiography.

The Wilms' tumor gene WT1 is overexpressed in various types of solid tumors, including lung and breast cancer and WT1 protein is a tumor antigen for these malignancies. In phase I clinical trials of WT1 peptide-based cancer immunotherapy, two patients with advanced lung cancer were intradermally injected with 0.3 mg of an HLA-A*2402-restricted, 9-mer WT1 peptide emulsified with Montanide ISA51 adjuvant. Consecutive WT1 vaccination at 2-week intervals resulted in a reduction in tumor markers such as chorio-embryonic antigen (CEA) and sialyl Lewis (x) (SLX) and by a transient decrease in tumor size. No adverse effects except for local erythema at the injection sites of WT1 vaccine were observed. These results provided us with the first clinical evidence demonstrating that WT1 peptide-based immunotherapy should be a promising treatment for patients with lung cancer.

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.

Expression of the Wilms' tumor gene WT1 was examined in 59 cases of colorectal adenocarcinoma to examine the involvement of WT1 in tumorigenesis. Quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) showed that WT1 mRNA was expressed in the range from 7.2 x 10(-5) to 4.9 x 10(-1) levels (WT1 expression level in K562 leukemic cells was defined as 1.0) in all (100%) of the 28 cases of colorectal adenocarcinoma examined, and that the WT1 mRNA expression levels were higher in 20 (71%) of the 28 cases compared to those of normal-appearing mucosal tissues examined. Immunohistochemical analysis using an anti-WT1 antibody was performed on 46 cases of colorectal adenocarcinoma (15 of the 28 cases with WT1 mRNA expression and 31 newly collected cases), and the expression of WT1 protein was detected in 41 (89%) of the 46 cases. The direct sequencing analysis of the WT1 genomic DNA showed no mutations in any of the 10 exons of the WT1 gene in any of 5 different colorectal adenocarcinomas. These results may indicate an important role of the wild-type WT1 gene in tumorigenesis of colorectal adenocarcinoma.

The expression levels of the Wilms' tumor gene WT1 were examined in 34 primary thyroid cancers (24 papillary, 5 follicular, 1 anaplastic, and 4 medullary carcinomas), 17 thyroid follicular adenomas, and 6 normal-appearing thyroid tissues using quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR). In 33 of 34 thyroid cancers, the WT1 mRNA was expressed at levels ranging from 5.0 x 10 (-5) to 8.3 x 10 (-2) levels (WT1 expression level in K562 leukemic cells was defined as 1.0). The WT1 mRNA expression levels were significantly higher than those in either thyroid follicular adenomas (P < 0.001) or normal-appearing thyroid tissues (P < 0.01). Immunohistochemical analysis confirmed the expression of WT1 protein in 20 of 21 thyroid cancers with WT1 mRNA expression. WT1 protein was also detected in 6 of 7 follicular adenomas with WT1 mRNA expression. However, the intensity of staining of WT1 protein in adenoma cells was weaker than that in cancer cells and its expression was restricted to approximately 30-80% of adenoma cells in the tumors examined. The direct sequencing analysis of the WT1 genomic DNA showed no mutations in any of the 10 exons of the WT1 gene in all of the 9 different thyroid cancers. These findings indicate an important role of the wild-type WT1 gene in the tumorigenesis of primary thyroid cancer.

We describe a patient with bcr/abl-positive acute mixed lineage leukemia who successfully underwent transplantation in primary induction failure, using unmanipulated bone marrow from a human leukocyte antigen (HLA)-haploidentical cousin. The tumor burden was successfully reduced by the administration of imatinib mesylate (STI571) before transplantation. As graft-versus-host disease (GVHD) prophylaxis, a combination of tacrolimus and a short course of methotrexate, methylprednisolone, and mycophenolate mofetil was used. Hematopoietic reconstitution was rapid, and acute GVHD was limited to the skin (grade I). The patient is still in complete remission past day +400. This successful case suggests that HLA-haploidentical transplantation using unmanipulated marrow from a distantly related relative can be considered for patients in urgent situations who do not have HLA-identical donors.

We describe a patient with refractory T-cell acute lymphoblastic leukemia who successfully underwent unmanipulated stem cell transplantation from an HLA 3-loci mismatched (haploidentical) sibling. In order to avoid severe graft-versus-host disease (GVHD), we used intensified GVHD prophylaxis consisting of tacrolimus, a short course of methotrexate, methylprednisolone, and mycophenolate mofetil. Hematopoietic reconstitution was rapid, with neutrophil count >5 x 10(8)/l on day +16, and platelet count >2 x 10(10)/l on day +25. There was no evidence of clinical acute GVHD. Bacterial, fungal, and viral infections were well controlled with antibiotics. The patient is still in complete remission past day +400. We suggest that unmanipulated HLA-mismatched transplantation with intensified GVHD prophylaxis is an alternative option for patients who do not have an HLA-identical donor.