ALBERT

Description: Interleukin-2 (IL-2) has a central role in immune tolerance thorough maintaining the homeostasis of CD4+CD25+Foxp3+ regulatory T cell (Treg). We recently reported that administration of low-dose IL-2 could preferentially enhance Treg in vivo and suppress clinical manifestations of chronic GVHD after allogeneic hematopoietic stem cell transplantation (NEJM2011). On the other hand, IL-2 is also necessary for the development of cytotoxic T cell function and has been used for the systemic immune therapy to amplify anti-tumor immunity. Thus, IL-2 administration after transplantation can induce the bipolar effects, namely, enhancement of Graft-versus-Leukemia effect (GVL effect) or prevention of Graft-versus-Host Disease (GVHD). For the appropriate therapeutic use of IL-2 for different purpose, we need to elucidate the factors to predict the effect of IL-2, however, the determinants except for the IL-2 dosage has not been characterized. To address this issue, we explored the impact of the immunological reconstitution after transplant on the effect of IL-2 therapy. First, we examined the in vivo reactivity to exogenous IL-2 in different lymphocyte subsets; CD8+ T cells, CD4+ conventional T cells (Tcon) and CD4+ regulatory T cells (Treg). We purified CD62L+ naïve lymphocytes and CD62L- lymphocytes from B6 spleen by cell-sorting and labeled with CFSE, adaptively transferred cells into irradiated B6 host and subcutaneously administrated 5000 IU of IL-2 or control vehicle from day1 to 5. At day6, spleen cells were harvested and the in vivo proliferation of each lymphocyte were evaluated by CFSE dilution assay. Proliferation of Tcons was just limited but both CD8 T cells and Tregs showed vigorous proliferations in response to IL-2. As expected, IL-2 induced the proliferation of CD62L- activated/memory CD8 T cells more than CD62L+ naïve CD8 T cells. In contrast, oppositely to CD8+ subsets, IL-2 induced the proliferation of CD62L+ naïve Tregs more than CD62L- Tregs, suggesting naïve Treg might be “primed” naturally and be ready to respond to IL-2 without antigenic TCR stimulation. Next, we examined the balance between naïve and memory phenotype in each lymphocyte subsets after allogeneic HSCT using murine BMT model. Lethally irradiated B6D2F1 mice were transplanted with 5x106 spleen cells from the CD45.2 B6 mice together with 5x106 TCD-BM from CD45.1 B6 donors. The balance between graft-derived cells (CD45.2) and BM-derived cells (CD45.1) in CD8+ T cell, CD4+ Tcon and Treg were monitored separately at day7, 14, 21,28 and 35. In the early phase of transplant, graft-derived cells showing CD62- were predominant, peaked at day21 and thereafter decreased in each subset. After day28, BM-derived cells generally emerged in each subset. Using this model, we compared the effects of IL-2 administration in the early phase (Day5-12) and in the late phase (Day35-42). Interestingly, IL-2 administration of daily 5000 IU of IL-2 in the early phase resulted in the dominant expansion of circulating CD62-CD44+ effector/memory CD8 T cell without Treg increase (CD8+T; 162 cells vs 75 cells/uL, P

Description: The TEL gene on 12p12-13 is a target for a number of translocations associated with various hematological malignancies. The fusion of the TEL gene to the Sykgene in a patient with myelodysplastic syndrome (MDS) with t(9;12)(q22;p12) is reported. Southern blot analysis of patient bone marrow cells with TEL and Syk gene probes detected rearranged fragments. Anchored polymerase chain reaction identified the Syk gene, a nonreceptor tyrosine kinase, on 9q22 fused downstream of TEL exon 5. The TELgene was fused in-frame to Syk and produced a fusion protein that was constitutively phosphorylated in tyrosine with dimerization that was mediated by the helix-loop-helix domain of TEL. A TEL-Syk fusion product transformed the murine hematopoietic cell line BaF3 to interleukin-3 growth factor independence. TEL-Syk is a novel transforming protein and leads to the transformation of hematopoietic cells. These data implicate that the rearranged Syk gene is involved in the pathogenesis of hematopoietic malignancies.

Description: Interaction of donor T cells and host antigen-presenting cells (APCs) in secondary lymphoid organs is critical for inducing graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation (BMT). Activation-induced cell death (AICD) is a chief mechanism of clonal deletion that is responsible for the rapid contraction of alloreactive donor T cells following an initial massive expansion. We hypothesized that sequestration of donor T cells in lymph nodes (LNs) would lead to prolonged interaction of donor T cells and host APCs in LNs and cause AICD of alloreactive donor T cells after allogeneic BMT. To test this hypothesis, recipient mice were given FTY720, which inhibits migration of T cells from LNs, after BMT. Lethally irradiated B6 (H-2b) mice were injected with 10 x 106 purified T cells and 5 x 106 bone marrow cells harvested from congeneic B6.Ly-5a (H-2b, CD45.1+) donors. FTY720 was administered by daily oral gavage at a dose of 0.3mg/kg from day 0. Six days after congeneic BMT, numbers of donor T cells in mesenteric LNs were greater in FTY720-treated mice as expected. By contrast, when B6D2F1 (H-2b/d) recipients were transplanted with 4 x 106 purified T cells and 5 x 106 bone marrow cells from allogeneic B6. Ly-5a donors, numbers of donor T cells in mesenteric LNs were greater in FTY720-treated mice than controls 4 days after allogeneic BMT but were subsequently less in FTY720-treated mice than controls (Figure). Five days after allogeneic BMT, frequencies of apoptotic donor T cells assessed by AnnexinV positivity were significantly greater in FTY720-treated mice (33±3 vs 24±5%, P

Description: Background: Invasive fungal infections (IFIs) are of serious concern in the management of immunocompromised patients (pts) with hematological disorders. Empiric antifungal therapy is recommended for neutropenic pts with persistent fever, because treatment after confirmation of fungal infection often produces poor outcomes. Micafungin (MCFG), one of the echinocandin families, was launched first in Japan in 2002, and has now been approved in more than 11 countries and areas including the USA and the EU. Although the efficacy and safety of MCFG against both Candida and Aspergillus infections has been shown in many clinical trials, there are few clinical study reports on the empiric therapy of a suspected fungal infection. Here, we report the multi-center study results of MCFG for the empiric antifungal therapy, which were conducted from April 2005 to September 2006 in Japan. Objective: This prospective study was performed to clarify the efficacy and safety of MCFG for the empirical antifungal therapy on suspected fungal infection in pts with hematological disorders and neutropenia. Methods: Study design: A multiple-center, open, uncontrolled study. The investigator registered pts with neutropenia (〈 1,000/μl) who met the following criteria to the Subject Registration Center. Suspected fungal infections were divided into two categories: possible fungal infection defined by positive clinical symptoms/findings and serological testing (beta-D-glucan or galactomannan) or diagnostic imaging (chest X-ray or CT scan), refractory fever defined by unexplained persistent fever (an axillary temperature higher than 37.5 °C) after the antibacterial treatment over 2 days and by positive clinical symptoms/findings. IFIs categorized as proven or probable were not included in this study. Efficacy evaluation was performed using an algorithm based on each of the evaluation of clinical symptoms/findings, imaging study findings, and serological tests. Results: 388 pts (M:234, F:154, mean age:57.8 years old) were registered. The mean dosage and duration of treatment with MCFG were 154.6±55.3 mg/day and 14.0±6.9 days, respectively. The main underlying hematological disorders were acute leukemia (61.3%), non-Hodgkin’s lymphoma (18.3%) and myelodysplastic syndrome (10.8%). The number of pts with hematopoietic stem cell transplantation (HSCT) was 76 (19.6%). The clinical response rate (CRR), excluding 4 non-evaluable pts was 63.3% (243/384): 60.1% (89/148) for pts with possible fungal infection and 65.3% (154/236) for pts with refractory fever, respectively. Even in persistent neutropenic pts whose neutrophil counts were 〈 500/μL throughout the treatment with MCFG, the CRR was high enough: 46.9% (61/130). No difference was observed in the CRR among the main underlying hematological disorders. The CRR in pts with HSCT and other conditions were 63.2% (48/76) and 63.3% (195/308), respectively. Drug-related adverse events (DAEs) were observed in 16.8% (65/388). Serious DAEs such as elevation of serum bilirubin and renal dysfunction was observed in 0.52% (2/388). Conclusion: MCFG was confirmed to have high clinical efficacy and be safe for the treatment of suspected fungal infection in pts with hematological disorders and neutropenia.

Description: Graft-versus-host disease (GVHD), the major complication of allogeneic hematopoietic stem cell transplantation, is mediated by donor T cells recognizing host alloantigens expressed on antigen-presenting cells (APCs). Total body irradiation (TBI) activates and damages host tissue, produces inflammatory cytokines and damage-associated molecular patterns, and allows translocation of microbial products into the systemic circulation. It has been assumed that these products bind to toll-like receptors (TLRs) on APCs and monocytes/macrophages, activate inflammatory pathways, and further amplify GVHD. On the other hand, TLRs are also expressed on T cells but their roles in the adoptive T cell response remain to be investigated in vivo. TLR signals are mediated by both MyD88-dependent and independent TRIF protein pathways. We evaluated the role of T cell TLR signaling in T cell alloreactivity in a mouse model of allogeneic bone marrow transplantation using mice with defective TLR signaling induced by deletions of the TLR adaptor proteins TRIF and MyD88 (TRIF/MyD88 DKO mice); in these mice, the TLR-dependent signaling pathway was eliminated. Lethally irradiated B6D2F1 mice were injected with 2 × 106 T cells from wild-type (wt) or TRIF/MyD88 DKO mice together with 5 × 106 T cell-depleted bone marrow (TCD-BM) from wt or DKO mice. Morbidity and mortality of GVHD was significantly less in recipients of TRIF/MyD88 DKO T cells than in recipients of wt T cells, irrespective of donor bone marrow type (Table). Reduction of GVHD was not seen in recipients of MyD88−/− T cells or TRIF−/− T cells, suggesting that inhibition of signaling through TRIF or MyD88 alone in donor cells is not sufficient to reduce GVHD. Histopathologic examination of the small intestine, liver, and skin showed significantly reduced GVHD pathology in recipients of DKO T cells (Table). Thus, effects of TLR signaling in donor cells on GVHD primarily reside in the T cell compartment of the donor graft, not in donor accessory cells. In recipients of TRIF/MyD88 DKO T cells, reduction of GVHD was associated with impairment of donor T cell expansion and production of TNF-□ and IFN-□. In marked contrast though, TRIF/MyD88 DKO T cells were activated equally to wt T cells in unirradiated B6D2F1 mice after adoptive transfer, thus activation of DKO T cells was impaired only in recipient mice heavily pretreated with TBI. These results suggest that T cell TLR signaling provides critical co-stimulation for T cells in an inflamed environment. T cell TLR signaling was also required for optimal graft-versus-leukemia activity. These results challenge the current paradigm that TLR functions are primarily attributable to their role in APCs and monocytes/macrophages in GVHD, and reveal a previously unrecognized critical role for T cell TLRs in inducing GVHD and GVL after hematopoietic stem cell transplantation. Pathological scores T BM Survivals(%) Clinical scores Serum IFN □ (ng/ml) Liver Intestine Skin *P

Description: Background: Unrelated cord blood transplantation (CBT) has emerged as an effective therapy for treating patients with advanced or high-risk hematologic diseases who have no suitable related or unrelated donor. In older patients or those with extensive prior therapy or other clinical features, nonmyeloablative regimens have been used to reduce the risk of regimen-related toxicity and transplantation-related mortality (TRM). Several studies have been reported on the use of unrelated CBT using various reduced-intensity conditioning regimens. Objectives: This study aimed to evaluate the safety and efficacy of unrelated CBT after nonmyeloablative conditioning using fludarabine, cyclophosphamide and single fraction of total body irradiation of 200 cGy, compared with bone marrow transplantation (BMT) or peripheral blood stem-cell transplantation (PBSCT) for adult Japanese patients with hematologic diseases and relate those to biological and procedural factors. Patients and Methods: Clinical data were collected retrospectively on 70 adults with hematologic disease who received unrelated CBT (n=19) or BMT from unrelated donors (n=27), or PBSCT from related donors (n=24) between August 2000 and June 2008 in our institution. All patients received nonmyeloablative conditioning regimens including fludarabine. The median period of follow-up for survivors was 253 days after CBT, 364 days after BMT and 692 days after PBSCT, respectively. We analyzed the hematopoietic recovery, incidence of graft-versus-host disease (GVHD), risks of TRM and relapse, and disease-free survival (DFS) using Cox proportional hazards models. Results: Comparisons of characteristics in 3 groups showed similar distributions for age, gender ratio, diagnosis, risk of disease at the transplant. Compared with BMT and PBSCT (BMT/PBSCT) recipients, CBT recipients had more previous history of hematopoietic stem cell transplantation (HSCT), less human leukocyte antigen-matched donor and less methotrexate (MTX)-containing GVHD prophylaxis. Multivariate analysis demonstrated slow neutrophil (P

Description: Exposure of offspring to noninherited maternal antigens (NIMAs) during pregnancy may have an impact on transplantations performed later in life. Using a mouse model, we recently showed that bone marrow transplantation (BMT) from NIMA-exposed offspring to the mother led to a reduction of graft-versus-host disease (GVHD). Since offspring can also be exposed to NIMAs by breastfeeding after birth, we tested whether breast milk could mediate the tolerogenic NIMA effect. We found that oral exposure to NIMAs by breastfeeding alone was sufficient to reduce GVHD, and that in utero exposure to NIMAs is required for maximum reduction of GVHD. The tolerogenic milk effects disappeared when donor mice were injected with CD25 monoclonal antibodies during the lactation period, suggesting a CD4+CD25+ regulatory T cell–dependent mechanism. Our results suggest a previously unknown impact of breastfeeding on the outcome of transplantation.

Description: Abstract 4688 Umbilical cord blood has been increasingly used as a source of hematopoietic stem cells. However, fetal blood recipients had slower hematopoietic engraftment and impaired immune reconstitution. To accelerate myeloid and lymphoid recovery, we used an animal model of newborn blood modeled for cord blood (CB), along with transiently reconstituting progenitor cells from congenic bone marrow with recipients, as sources of stem cells. According to the previous reports that murine transiently reconstituting progenitor cells express the c-kit molecule, but not Sca-1 and lymphohematopoietic lineage markers, Lin-sca-1-c-kit+(c-kit+) were isolated by MACS method. c-kit+ cells population consisted of exclusively of medium- or large-sized blast-like cells, which displayed relatively low proliferative potential in vitro than Lin-sca-1+ (sca-1+) population. After transplantation of CB from DBA/2 mice (H-2d/d, CD45.2), with or without graded numbers of either c-kit+ or sca-1+ cells isolated from BDF1 mice (H-2d/b, CD45.1) bone marrow into lethally irradiated CD45.2 congenic BDF1 mice, hematopoietic engraftment were dynamic investigated. The intermingled transplantation of CB and c-kit+ cells or sca-1+ cells at the dosages of 1×104 or 2.5×104 or 5×104 to recipient mice leads to the quantity of white blood cells and platelets increased to 1×109/L and 1×1012/L at day12, whereas the injection of CB alone resulted in day17. By 2 weeks post-transplantation, congenic BM-derived cells were dominantly found in granulocytes and B lymphocytes, while host cells were dominantly found in T lymphocytes in CB transplantation combined either with c-kit+cells or sca-1+ cells. In cotransplantation with CB and c-kit+cells – engrafted surviving mice, the degree of donor CB cells in the peripheral blood increased progressively over time, while congenic donor BM-derived cells decreased gradually. After 60 weeks cotransplantation with CB and c-kit+ cells, a complete chimerism frequency of CB–derived cells continued to maintain in granulocytes and B lymphocytes, while T lymphocytes were dominantly derived from CB. On the other hand, congenic bone marrow or host-derived cells were the dominant population and CB-derived cells in the peripheral blood were less than 10% after 60 weeks cotransplantation with CB and sca-1+cells. In conclusion, the cotransplantation of CB and congenic c-kit+ cells was able to accelerate early hematopoietic recovery due to congenic marrow cells. But complete or main chimerism of cord blood was formed without or with fewer residual cells of host origin and congenic BM origin in long-term multilineage reconstitution. Thus, this cotransplant model in vivo may be to bring useful information for improving hematopoietic and immune reconstitution in fetal blood recipients. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3101 Background: Natural killer (NK/) T-cell lymphoma, nasal type, is an aggressive form of extranodal lymphoma that is common in Asia, but rare in Europe and North America. Although NK/T-cell lymphoma involves upper aerodigestive sites such as the nasal cavity and nasopharynx, it frequently involves other extranodal sites, including the gastrointestinal tract, bone marrow, adrenal gland, and skin. Because of the frequency of extranodal site involvement, pretreatment evaluation of disease extension is important for staging and treatment. Recently, 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography/computed tomography (PET/CT) was evaluated for its usefulness in the prognosis and treatment response of aggressive B-cell lymphoma and Hodgkin's lymphoma, and relevant results were obtained. Many studies have evaluated the value of PET/CT for various types of B-cell lymphomas, but other similar studies involving T-cell and NK-cell lymphomas are rare. In the present study, we compared the utility of PET/CT and conventional modalities, particularly CT, in extranodal NK/T-cell lymphoma. Patients and Method: From January 2006 to April 2010, 19 untreated patients with extranodal NK/T-cell lymphoma (11 males and 8 females; median age 61 years; range 13–90 years) were included in the study. PET/CT and conventional procedures (e.g., CTs and biopsies) were compared and evaluated for their abilities to detect tumor lesions and their influence in staging and treatment strategies. PET/CT was performed as an initial staging procedure. In addition to PET/CT, all patients underwent initial staging workups, including whole-body CT with contrast media, biopsies from the bone marrow and other sites, and panendoscopies of the upper aerodigestive tract. Patients were evaluated for clinical stage by both evaluation modalities (PET/CT and conventional modalities) according to the Ann Arbor Staging System, and treatment strategies were first planned based on staging results. Following PET/CT, the clinical stage was reevaluated in each patient, and treatment strategies were decided based on the re-staging results. Result: Seven patients (37%) had bone marrow involvement, eight (42%) were in Ann Arbor stage I–II, and 11 (58%) had a systemic dissemination. Most patients with systemic dissemination (9/11, 82%) had cutaneous lesions. The median number of disease sites was 4 (range, 1–15). The median number of positive lesions was 3 (range, 1–15) by PET/CT compared to 2 (range, 0–14) by CT (p = 0.12). Using PET/CT, 108 lesions (93%) were detected, and at least one FDG-avid lesion was observed at initial staging workups in all patients. In contrast, 70 lesions (60%) were detected by CT, and three patients (16%) did not show any positive lesion. Two lesions (2%) at the nasal cavity that were detected by biopsy were undetectable by either PET/CT or CT. The nodal and extranodal regions were separately evaluated by PET/CT and conventional modalities. In total, 28 nodal lesions were detected: all (100%) were positive by PET/CT and 26 (93%) by CT. Conversely, 89 extranodal lesions were detected, and 83 (93%) and 44 (49%) were positive by PET/CT and CT (p = 0.003), respectively. For the detection of upper aerodigestive lesions, PET/CT and CT demonstrated similar results: 23 lesions (92%) vs. 17 (68%), respectively. Notably, PET/CT was superior to CT in detecting cutaneous lesions [31 lesions (100%) vs. 19 lesions (61%), respectively; p = 0.026]. Bone marrow involvement was confirmed pathologically in only seven patients; four cases (57%) were positive by PET/CT and none by CT. Using conventional modalities, 11 patients (58%) were in the localized stage and eight (42%) were in the advanced stage. Using PET/CT, eight (42%) were in stage I–II and 11 (58%) were in stage III–IV. Most patients received chemotherapy plus local irradiation in stage I–II, and intensive chemotherapy with or without hematopoietic stem cell transplantation in stage III–IV. One patient did not receive treatment because of unwillingness for treatment and older age. PET/CT findings altered the stage and treatment strategy in four (21%) and two cases (11%). Conclusion: In extranodal NK/T-cell lymphoma, PET/CT demonstrated a high detection rate for nodal and extranodal lesions, except in the bone marrow. PET/CT may have an impact on treatment strategy and is essential for risk-adapted treatment. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4484 Background: Dasatinib is a highly potent BCR-ABL kinase inhibitor. The previous report from the global DASISION trial showed dasatinib 100 mg once daily resulted in significantly higher and faster rates of complete cytogenetic response (CCyR) and major molecular response (MMR) compared with imatinib; both treatment arms were well-tolerated (N Engl J Med. 2010;362:2260-70). The objective of this subset analysis was to assess the efficacy and safety of dasatinib compared with imatinib in the Japanese population. Methods: Forty-nine Japanese patients (total 519 pts) with newly diagnosed CML-CP were randomly assigned to receive dasatinib 100 mg QD or imatinib 400 mg QD. Confirmed CCyR (cCCyR; CCyR on 2 consecutive assessments at least 28 days apart) was the primary efficacy endpoint with MMR as an important secondary endpoint. The safety profiles were also evaluated. Results: Minimum follow-up time and median treatment duration were 12 months and 15 months, respectively. Twenty-six patients with median age 56 (range, 21–70) years were treated with dasatinib and 23 patients with median age 52 (range, 22–77) years were treated with imatinib. Overall 89% of patients receiving dasatinib and 83% of patients receiving imatinib continue to receive treatment. The cCCyR rate by 12 months (primary endpoint), CCyR rate by 12 months and MMR rate at any time in dasatinib arm were higher than those in imatinib for Japanese patients (96% vs 70%, 96% vs 78%, and 73% vs 48%, respectively). Grade 3/4 cytopenias in dasatinib arm and imatinib arm were as follows: anemia (8% vs 4%), neutropenia (27% vs 39%), and thrombocytopenia (8% vs 9%). Non-hematologic and drug-related adverse events occurring in ≥10% of patients are shown as Table. No deaths were reported in either group. Drug-related serious adverse events were rarely reported and all events were not severe (Grade 1–2, including vomiting, hypoxia and cardiomyopathy in dasatinib arm). Conclusion: Dasatinib showed higher rates of cCCyR and MMR compared with imatinib. Both treatments were well tolerated. Given the predictive value of 12 months cCCyR, dasatinib may improve long-term outcomes in Japanese patients with newly diagnosed CML-CP. Disclosures: Ueda: Bristol-Myers K.K.: Employment. Seriu:Bristol-Myers K.K.: Employment. Bradley-Garelik:Bristol-Myers Squibb: Employment. Zhu:Bristol-Myers Squibb: Employment.