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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.

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.

The WT1 gene is a tumor-suppressor gene that was isolated as a gene responsible for Wilms' tumor, a childhood kidney neoplasm. We have previously reported that the WT1 gene is strongly expressed in leukemia cells with an increase in its expression levels at relapse and an inverse correlation between its expression levels and prognosis, thus making it a novel tumor marker for leukemic blast cells. Furthermore, WT1 antisense oligomers have been found to inhibit the growth of leukemic cells. These results strongly suggested the involvement of the WT1 gene in human leukemogenesis. The present study was performed to prove our hypothesis that the WT1 gene plays a key role in leukemogenesis and performs an oncogenic function in hematopoietic progenitor cells, rather than a tumor-suppressor gene function. 32D cl3, an interleukin-3-dependent myeloid progenitor cell line, differentiates into mature neutrophils in response to granulocyte colony-stimulating factor (G-CSF). However, when transfected wild-type WT1 gene was constitutively expressed in 32D cl3, the cells stopped differentiating and continued to proliferate in response to G-CSF. As for signal transduction mediated by G-CSF receptor (G-CSFR), Stat3alpha was constitutively activated in wild-type WT1-infected 32D cl3 in response to G-CSF, whereas, in WT1-uninfected 32D cl3, activation of Stat3alpha was only transient. However, most interesting was the fact that G-CSF stimulation resulted in constitutive activation of Stat3beta only in wild-type WT1-infected 32D cl3, but not in WT1-uninfected 32D cl3. Thus, WT1 expression constitutively activated both Stat3alpha and Stat3beta. A transient activation of Stat1 was detected in both wild-type WT1-infected and uninfected 32D cl3 after G-CSF stimulation, but no difference in its activation was found. No activation of MAP kinase was detected in both wild-type WT1-infected and uninfected 32D cl3 after G-CSF stimulation. These results demonstrated that WT1 expression competed with the differentiation-inducing signal mediated by G-CSFR and constitutively activated Stat3, resulting in the blocking of differentiation and subsequent proliferation. Therefore, the data presented here support our hypothesis that the WT1 gene plays an essential role in leukemogenesis and performs an oncogenic function in hematopoietic progenitor cells and represent the first demonstration of an important role of the WT1 gene in signal transduction in hematopoietic progenitor cells.

To clarify whether the expression of the WT1 gene in leukemic cells is aberrant or merely reflects that in normal counterparts, the expression levels of the WT1 gene were quantitated for normal hematopoietic progenitor cells. Bone marrow (BM) and umbilical cord blood (CB) cells were fluorescence-activated cell sorting (FACS)-sorted into CD34+ and CD34− cell populations, and the CD34+ cells into nine subsets (CD34+CD33−, CD34+CD33+, CD34+CD38−, CD34+CD38+, CD34+HLA-DR−, CD34+HLA-DR+, CD34+c-kithigh, CD34+c-kitlow, and CD34+c-kit−) according to the expression levels of CD34, CD33, CD38, HLA-DR, and c-kit. Moreover, acute myeloid leukemic cells were also FACS-sorted into four populations (CD34+CD33−, CD34+CD33+, CD34− CD33+, and CD34− CD33−). FACS-sorted normal hematopoietic progenitor and leukemic cells and FACS-unsorted leukemic cells were examined for the WT1 expression by quantitative reverse transcriptase-polymerase chain reaction. The WT1 expression in the CD34+ and CD34− cell populations and in the nine CD34+ subsets of BM and CB was at either very low (1.0 to 2.4 × 10−2) or undetectable (

Transforming growth factor-beta (TGF-beta) and macrophage inflammatory protein-l alpha (MIP-1 alpha) are both well-described inhibitors of committed and multipotential hematopoietic progenitors. The effect of these cytokines; on true stem cell activity in ex vivo culture systems as assayed by murine long-term repopulating activity (LTRA) has not been examined. We studied the stem cell effects of the addition of these cytokines to ex vivo cultures containing interleukin-3 (IL-3), IL- 6, and stem cell factor (SCF), using the murine competitive repopulation assay. We also tested the impact of adding an anti-TGF- beta neutralizing antibody, to ask whether abrogation of autocrine/paracrine TGF-beta may protect or enhance the survival of LTRA during ex vivo culture. TGF-beta 1 had significant suppressive effects on both short- and long-term repopulating activities, and anti- TGF-beta antibody had enhancing effects compared with control cultures containing IL-3, IL-6, and SCF alone. MIP-1 alpha had no significant effects on either short- or long-term repopulating ability. These data suggest that abrogation of TGF-beta during suspension culture may allow enhanced survival or even expansion of primitive cells ex vivo, with implications for many applications, including gene therapy.

Abstract 4203 The major drawback of HLA-haploidentical stem cell transplantation is graft-versus-host disease (GVHD): however, the GVHD can be overcome using substantial T cell depletion method (Perugia), the use of a high dose ATG (Beijing), or the posttransplantation cyclophophamide method (Johns Hopkins). These transplant procedures are found to be effective for the achievement of donor engraftment and immunological tolerance: however, graft-versus-leukemia (GVL) effects are not enough strong to prevent disease relapse. To harness GVL effect of HLA-haploidentical grafts, our regimen for unmanipulated haploidentical myeloablative transplantation is designed for GVH reaction to remain to some extent in the short time after transplantation using a small dose of ATG 2 mg/kg, coupled with GVHD prophylaxis containing a low dose of steroid (methylprednisolone 1 mg/kg). We hypothesize that allogeneic immunological response by donor T cells under low cytokine milieu exerts potent GVL effect without GVHD. This regimen was applied to patients with hematologic disease in an extremely high risk. From August 2008 to June 2012, 23 patients with high-risk or refractory hematologic diseases underwent unmanipulated peripheral blood stem cells using a graft from HLA-haploidentical related donor (11 cases were HLA 2 antigen-mismatched and 12 HLA 3 antigen-mismatched in the GVH direction). Patients' characteristics are sex: male 11, female 12, age: 17–46 years old (median 34), disease: AML/MDS 7, ALL 10, ML 5, others 1. All patients except for 5 underwent transplantation in non-complete remission (non-CR) status, including 5 patients in induction failure, and 10 in resistant relapse. Eleven patients with leukemia who underwent in non-CR state had a median of 66% blasts in the bone marrow (range, 10.6 – 97.0 %). Patients received a conditioning treatment consisting of fuludarabine, cytarabine, cyclophsphamide, TBI 8Gy and ATG (thymoglobuline) 2 mg/kg. GVHD prophylaxis consisted of tacrolimus and methylprednisolone (1 mg/kg). One patient dies of pneumonia on day 3. Among 22 patients that could be evaluated, the engraftment rate was 95.5% (one patient had graft rejection because of donor-specific HLA antibody). Neutrophil (>0.5×109/l) and platelet (>20×109/l) engraftment was achieved on day 11 and on day 32, respectively. All patients who underwent transplantation in non-CR achieved CR after transplantation. Eleven patients (52.4%) had no GVHD. Nine patients (42.9%) developed grade II-III acute GVHD. One patient died of cardiac failure on day 46, 1 VOD on day 74, and 1 GVHD on day 235. Two patients died of relapse on days 70 and 548. Four of the 8 patients with leukemia who had more than 50% blasts in the bone marrow at the time of transplantation survive in CR. The overall survival is 60% at 3 years (Figure 1). Our regimen for HLA-haploidentical transplantation using a small dose of ATG coupled with low dose of steroid exerts a strong GVL effect even for patients who had chemorefractory disease, and transplantation-related toxicities, including GVHD, are acceptable. As a result, patients, of whom the majority had a high tumor burden, achieved a high rate of long-term survival. Thus, our strategy for haploidentical stem cell transplantation is promising, although our results will have to be confirmed in a large-scale study. Disclosures: No relevant conflicts of interest to declare.

Abstract 3648 Background: Radioimmunotherapy (RIT) with 90Y-ibritumomab tiuxetan has been used for the treatment of relapsed or refractory indolent B-cell lymphoma. Early prediction of response to the therapy may offer the potential to identify patients who will benefit this therapy. We evaluated the efficacy of fluorine 18 fluorodeoxyglucose (FDG) combined positron emission tomographic-computed tomographic (FDG-PET/CT) imaging for assessment of therapy and predicting outcome in 90Y-ibritumomab tiuxetan radioimmunotherapy. Methods: Radioimmunotherapy with 90Y-ibritumomab tiuxetan was preformed in 52 patients with relapsed or refractory indolent B cell lymphoma (follicular lymphoma, 45; mucosa-associated lymphoid tissue lymphoma, 5; lymphoplasmacytic lymphoma, 2) at Hyogo College of Medicine Hospital from June 2009 through August 2012. FDG-PET/CT scanning was performed at two weeks and the later after 90Y-ibritumomab tiuxetan therapy. Results: In FDG-PET at 2 weeks after 90Y-ibritumomab therapy, 26 (50%) showed complete response (defined as early CR), 20 (38%) showed partial response (PR-2W) and 6 (12%) showed non response (NR-2W). Further follow-up revealed 10 later CR (8 CR out of 20 PR-2W and 2 CR out of 6 NR-2W), showing 69 %(36) of overall CR rate. Relapse was occurred in 4(17%) of 26 early CR and in 6 (60%) of 10 later CR, indicating significantly low relapse late in early CR (P= 0.0074, by Chi-square test). Patients with early CR (CR in 2 weeks after the RIT) showed significantly better progression free survival than those with later CR (P=0.0046, by Logrank test). In contrast, analysis at six weeks after the RIT showed 36 CR patients (10 of which eventually relapse), but failed to discriminate patients who had high risk of relapse. Conclusion: Our results suggest that CR assessed by FDG-PET/CT at 2 weeks after 90Y-ibritumomab tiuxetan therapy discriminates good responder to the RIT and predicts better progression free survival in relapsed or refractory indolent B cell lymphoma. Disclosures: No relevant conflicts of interest to declare.

Abstract 4117 Related haploidentical donors, as cord blood, can be alternative donor sources in stem cell transplantation (SCT). Severe GVHD, however, has interfered the progress of haploidentical SCT (haploSCT). To deal with this strong GVHD, T cell depletion has usually been used in US and European countries. In order to pursue the controllable GVL effect by T cells, we have performed unmanipulated haploSCT using myeloablative or reduced intensity preconditioning regimen accompanied with intensified GVHD prophylaxis, including steroids. In this meeting, we will summarize our experience of haploSCT for more than ten years. From August 1998 to September 2010, we have performed 351 cases of haploSCT (all cases were HLA 2–3 antigen mismatched in GVH direction). Patients' characteristics are sex: male 186, female 168, age: 16–65 years old (median 39), disease: AML/MDS 149, ALL 81, ML 67, others 54. Eighty-three percent of cases underwent SCT in non-complete remission (non-CR) status. Patients under 45 years old underwent myeloablative preconditioning regimen consisting of FLU/CA/CY/TBI 8Gy (haplo-full, n=100), and patients over 45 years old or with comorbidities or repetitive SCT (including second to fifth SCT) underwent reduced intensity preconditioning regimen consisting of FLU/(CA)/BU/ATG or FLU/(CA)/MEL/ATG (haplo-mini, n=251). High dose Ara-C (CA) was optional to reduce tumor burden. As ATG, ATG (Fresenius) 8mg/kg, or thymoglubulin (genzyme) 2–4mg/kg were integrated into conditioning treatments mainly for reduced-intensity transplantation. GVHD prophylaxis consisted of taclolimus (TAC), methylprednisolone (mPSL) 2mg/kg/day, short term MTX, and mycophenolate mofetil (MMF) 15mg/kg/day in haplo-full, and TAC, and mPSL 1mg/kg/day in haplo-mini, respectively. For elderly patients over 50 years old in haplo-mini, MMF was added. Hematopoietic engraftment in haploSCT was as rapid as that in HLA-identical SCT, except 10 cases of graft rejection. The median time to reach a neutrophil account of >0.5 × 109/l was 10 days for haplo-mini and 13 days for haplo-full. Platelet recovery was achieved in 66 % and 60% of patients undergoing haplo-mini and haplo-full, respectively. The median time to reach a nontransfused platelet count of 3 20 × 109/l was 22 days for haplo-mini and 33 days for haplo-full. Sixty percent of haplo-mini patients and 54 % of haplo-full patients did not develop acute GVHD. Acute GVHD (grade II-IV) was observed in 20% for haplo-mini and 36 % for haplo-full. Overall survival at five years was 30% for haplo-full and 40% for haplo-mini, respectively. If limited to CR cases, overall survival reached over 60% in haplo-mini. There is no difference in survival rate among patients' diseases. In multivariate analysis on survival using variables, including disease status before transplantation, haplo-full vs haplo-mini, mismatches in GVH direction, mismatches in HVG direction, patients' age, and the number of transplantation times, the disease status (CR) was found to be only a significantly favorable factor (P= 0.0026). Unmanipulated haploSCT is feasible and effective for refractory diseases. ATG dose used in haplo-mini is critical, and rather low compared with that of European cases reported so far. Although it should be too early to refer long term outcome, unmanipulated haploSCT could be considered as an option to control refractory diseases. Disclosures: No relevant conflicts of interest to declare.