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Beschreibung: Article Activating mutations of the tyrosine kinase Kit are commonly found in mast cell neoplasms and gastrointestinal stromal tumours. Here the authors show that mutant Kit, through the activation of PI3K and STAT3 pathways, elicits proliferative and survival signals from endolysosomes and from the endoplasmic reticulum. Nature Communications doi: 10.1038/ncomms6715 Authors: Yuuki Obata, Shota Toyoshima, Ei Wakamatsu, Shunichi Suzuki, Shuhei Ogawa, Hiroyasu Esumi, Ryo Abe

Beschreibung: 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), Stat3α was constitutively activated in wild-type WT1-infected 32D cl3 in response to G-CSF, whereas, in WT1-uninfected 32D cl3, activation of Stat3α was only transient. However, most interesting was the fact that G-CSF stimulation resulted in constitutive activation of Stat3β only in wild-type WT1-infected 32D cl3, but not in WT1-uninfected 32D cl3. Thus, WT1 expression constitutively activated both Stat3α and Stat3β. 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.

Beschreibung: 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.

Beschreibung: Abstract 4707 Reduced-intensity stem cell transplantation (RIST) has come to be generally accepted as a method of allogeneic stem cell transplantation (SCT) for patients considered ineligible for myeloablative preparative regimens because of advanced age or comorbidities. We have recently reported unmanipulated nonmyeloablative HLA-haploidentical SCT using a conditioning treatment consisting of fludarabine, busulfan and anti-T-lymphocyte globulin and graft-versus-host disease (GVHD) prophylaxis consisting of tacrolimus and methylprednisolone (1 mg/kg) (Biol Blood Marrow Transplant 2006; 12:1073). In that study, the incidence of severe GVHD was only 10%. One of the mechanisms for such a low incidence of GVHD may have been caused by a reduced intensity of conditioning. Less intensive regimens should be associated with lower toxicity, a lower release of inflammatory cytokines, and potentially less GVHD; however, the mechanisms remain to be determined. Thus, using a murine MHC-haploidentical BMT model, BDF1(H-2b/d)®B6C3F1 (H-2b/k), that we established, we examined the influence of an intensity of conditioning treatment on GVHD. Recipient mice received T-cell-depleted bone marrow (5×106) and spleen cells (2×107) after total body irradiation (TBI) 13 Gy (myeloablative group) or 5 Gy (RIST group). Both groups of mice rapidly achieved donor engraftment. Recipients in the RIST group showed significantly fewer GVHD signs than those in the myeloablatve group. Histopathological examination of the myeloablative group on day 14 revealed various pathological changes in intestine (in particular large intestine) compatible to GVHD. In contrast, intestine samples from the RIST group showed few pathological changes with much less infiltration of donor T cells. Consequently, all recipients in the myeloablative group had died of GVHD by day 60, while all recipients survived for more than 3 months. These results clearly showed that the intensity of conditioning treatment influenced on the severity of GVHD and survival of recipients. Next, we investigated the mechanisms by which reduced intensity of conditioning ameliorated GVHD. Transplantation was performed using spleen cells that were labeled with the fluorescent cytoplasmic dye, carboxyfluorescein diacetate succinimidyl ester (CFSE), and cells in secondary lymphoid organs were analyzed by flow cytometry. The number of donor T cells in mesenteric lymph nodes on day 7 of the RIST group was significantly lower than that of the myeloablative group. In addition, a significantly increased number of host CD4+ T cells were recruited to secondary lymphoid organs on day 4 in the RIST group compared with the myeloablative group. An increased number of donor or host regulatory (Foxp3+CD4+) T cells were also observed in the RIST group. The levels of IFN gamma or IL-4 in lymphoid organs of the RIST group were higher than those of the myeloablative group. These results strongly suggest that host immune cells that survived conditioning treatment or cytokine milieu in secondary lymphoid organs contributed to the suppression of donor T cells during the initiation of GVHD. In addition, the expression of Th1 chemokine receptor, CXCR3, on donor T cells in secondary lymphoid organs and the expression levels of CXCL9, CXCL10, and CXCL11, ligands for CXCR3, in the large intestines were relatively lower in the RIST group, suggesting that the migration ability of donor T cells into GVHD target organs was negatively influenced by the intensity of conditioning. In conclusion, we showed that reduced intensity of conditioning improved the severity of GVHD, and that recipient immune cells, including regulatory T cells, together with reduced expression of inflammatory cytokines or chemokines, contributed to the improvement of GVHD in RIST. Disclosures: No relevant conflicts of interest to declare.

Beschreibung: Lipopolysaccharide (LPS) has been implicated in the pathogenesis of acute graft-versus-host disease (GVHD). The toll-like receptor (TLR)-4 has been recently identified as a major receptor for LPS. It is possible that mutations of TLR-4 have been associated with LPS hyporesponsiveness, thereby reducing the risk of acute GVHD in allogeneic hematopoietic stem cell transplantation (HSCT). However, stimulation of TLRs may also initiates a signaling cascade for induction of various pro-inflammatory cytokines, leading to microbial eradication. Therefore, mutations of TLR-4 may increase risk of microbial infection, thereby accelerating acute GVHD in allogeneic HSCT recipients. Retrospective studies to determine the frequency of TLR-4 mutations and their possible association with acute GVHD contradict each other. In the present study, we examined the association of TLR-4 mutations in host and donor cells and the risk for acute GVHD after allogeneic HSCT. To examine the role of TLR-4 in host cells, we analyzed TLR-4 expression in the intestine by immunohistochemistry. Lethally irradiated C3H/HeN mice received allogeneic HSCT from C57BL/6 donors. TLR-4 expression in the intestinal epithelial cells was significantly increased after GVHD induction. Next, we used a mouse strain (C3H/HeJ) that has a single amino acid substitution in its TLR-4 as recipients in an allogeneic HSCT. Lethally irradiated either C3H/HeN or C3H/HeJ hosts received HSCT from C57BL/6 donors. C3H/HeN recipients receiving C57BL/6 donors developed significantly less GVHD as measured by mortality and intestinal histopathology compared with C3H/HeJ recipients receiving C57BL/6 donors. TNF-a mRNA expression in C3H/HeJ recipients was stronger in the intestine but weaker in the spleen and the liver comparing to that in C3H/HeN recipients. These results suggest that TLR-4 mutation in host cells reduces the expression of pro-inflammatory cytokines in the spleen and the liver, while it also reduces microbial eradication in the intestine, increasing the risk of LPS stimulation of macrophages in the intestine, thereby augmenting TNF-a expression and damages of the intestine. Next, we examined the role of TLR-4 in donor cells. Lethally irradiated C57BL/6 hosts received allogeneic HSCT from either C3H/HeN or C3H/HeJ donors. Mice receiving C3H/HeJ donors developed significantly less GVHD as measured by mortality and intestinal histopathology compared with mice receiving C3H/HeN donors. Taken together, these results suggest that TLR-4 in host cells is crucial in the microbial eradication in the intestine and the protection of intestinal GVHD, while responsiveness of donor cells to LPS may be an important risk factor for acute GVHD.

Beschreibung: Introduction: Acute myeloid leukemia (AML) with chromosomal translocation t(7;11)(p15;p15) [t(7;11)], which results in a fusion between NUP98 and HOXA9, is uncommon, and classified as intermediate risk (IR) in National Comprehensive Cancer Network (NCCN) Guidelines. Previous studies have reported that the patients with AML harboring t(7;11) exhibited a worse clinical outcome than the other AML groups. These reports included mostly patients who underwent chemotherapy rather than allogeneic hematopoietic stem cell transplantation (allo-HSCT), and the number of patients in each study was very small. Moreover, the transplant outcomes of patients with AML harboring t(7;11) compared to patients with other AML have not been reported. Therefore, we investigated the transplant outcomes among 91 patients with AML harboring t(7;11) compared to the patients with IR (n=7,308) and poor risk (PR) (n=2,406) AML, except for t(7;11). Moreover, we evaluated the risk factors for survival in patients with AML harboring t(7;11) who underwent allo-HSCT. Patients and Methods: During 1997 and 2014, 91 patients with AML harboring t(7;11) were identified from the nationwide registration data of the Japan Society for Hematopoietic Cell Transplantation. Cytogenetic risk group stratification was performed using the NCCN guidelines for AML in 2016. Univariate and multivariate analysis for survival were performed in all patients cohort and only among patients with AML harboring t(7;11). Univariate models for overall survival (OS) and disease-free survival (DFS) included age at allo-HSCT (age 55 ≥ years vs. 55 〈 years), sex, performance status (PS) (2-4 vs. 0-1), the hematopoietic cell transplantation-comorbidity index (0-2 vs. ≥ 3), additional chromosomal change, disease status at allo-HSCT (first complete remission [CR]: CR1 vs. second CR:CR2 or non-CR at allo-HSCT), conditioning regimen (total body irradiation [TBI] ≥ 8Gy vs. others), and stem cell source (related donor vs. cord blood or unrelated donor). Factors associated with at least borderline significance (p 〈 0.20) on univariate analyses were subjected to multivariate analysis. Results: Patient Characteristics We evaluated the clinical characteristics of patients with t(7;11), IR, and PR.The median follow-up period for survivors was 1,124 days (range, 1-8,758). Patients with t(7;11) were younger (median age: 45 vs. 48 vs. 50 years, p

Beschreibung: 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.

Beschreibung: 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.

Beschreibung: We analyzed 338 adult patients with acute myeloid leukemia (AML) with t(8;21) and inv(16) undergoing stem cell transplantation (SCT) who were registered in the Japan Society for Hematopoietic Cell Transplantation database. At 3 years, overall survival (OS) of patients with t(8;21) and inv(16) was 50% and 72%, respectively (P = .002). Although no difference was observed when restricted to allogeneic SCT in first complete remission (CR; 84% and 74%), OS of patients with t(8;21) and inv(16) undergoing allogeneic SCT in second or third CR (45% and 86% at 3 years; P = .008) was different. OS was not different between patients in first CR who received allogeneic SCT and those who received autologous SCT for both t(8;21) AML (84% vs 77%; P = .49) and inv(16) AML (74% vs 59%; P = .86). Patients with inv(16) not in CR did better after allogeneic SCT than those with t(8;21) (70% and 18%; P = .03). Patients with t(8;21) and inv(16) should be managed differently as to the application of SCT. SCT in first CR is not necessarily recommended for inv(16). For t(8;21) patients in first CR, a prospective trial is needed to clarify the significance of autologous SCT and allogeneic SCT over chemotherapy.