ALBERT

Beschreibung: Abstract 5178 Background: Natural Killer cells (NK cells) are part of the innate immune system. These cells have the ability to recognise and kill malignant cells, like myeloma cells. NK cell activation is tightly regulated by different activating or inhibiting receptors. Killer immunoglobulin like receptors (KIR, CD158) are a family of receptors which have activating as well as inhibitory function. KIR molecules are thought to recognize the HLA-C molecules, which then lead to NK cell signal transduction. Our knowledge about KIR expression and impact on tumor cell control has developed over the last years, but still our understanding of how the receptors are activated in multiple myeloma is limited. We therefore, investigated three different model systems for NK cell alloreactivity: 1) HLA-C/ HLA-C interaction model (KIR-Ligand model), 2) HLA-C/ KIR receptor interaction model, and 3) impact of donor KIR haplotype. Material and Methods: Three different myeloma cell lines (KMS12BM [C1/C1], MOLP8 [C1/C2] and RPMI8266 [C2/C1]) and a NK cell line (NKL) were cultured under standard conditions. NKL cells were transfected with human KIR2DL1 and KIR2DL3 alleles, respectively. For RNAi experiments two siRNA and two control siRNA were used. NK cells from healthy donors were isolated by magnetic end labeling. Enriched NK cells were HLA-typed for expression of HLA-C molecules, KIR receptor expression or KIR haplotype. Thereafter, NK cells were transiently transfected with the siRNA or control siRNA against the KIR2DL1 or KIR2DL3 receptors for up to 48 h. Functional analysis of the NK cell cytotoxicity was measured using a LDH release assay, based on their killing ability against the three fore mentioned myeloma cell lines. Results: Using NK cells that have been HLA-C genotyped as HLA-C1/C1, we observed a rescue of C1/C1 positive meyeloma cell lines in contrast to the C2/C1 myeloma cell line (12% cytotoxicity vs. 38% and 45% cytotoxicity, respectively, p

Beschreibung: Abstract 3496 Introduction: Allogeneic hematopoietic stem cell transplantation (allogeneic HSCT) has found entrance into treatment of patients (patients) with poor-risk T-cell malignancies, but post-transplant relapse rates of ~30% were reported (Marks et al. Blood 2009). Improvement of the pre-transplant remission status in relapsed/refractory disease might lead to better outcomes. Nelarabine - a novel purine antimetabolite - has been approved by the FDA in 2005 as salvage approach for patients with T-cell malignancies, but data on pre-transplant use are limited (Goekbuget et al. ASH Annual Meeting 2005). Patients/Methods: Aiming to estimate its clinical applicability and efficacy in the pre-transplant period, we evaluated its use in 8 patients (5 males, 3 females; 22–56 years) with precursor/mature T-cell neoplasms who had failed to ≥2 previous conventional/intensified chemotherapy regimens (including a history of autologous/allogeneic HSCT in 2 patients) and were consequently proceeded to allogeneic HSCT (in 6 cases the 1st, in 2 cases the 2nd allogeneic HSCT). Five had T-ALL (1 pre-T-, 3 cortical, 1 mature), 3 had mature T-cell lymphomas (angioimmunoblastic lymphoma, n=1; peripheral T-cell lymphoma not otherwise specified, n=2), all with bone marrow involvement. Patients entered conventional chemotherapy in 6 cases at the 1st manifestation of disease, in 2 cases at the 1st relapse. Following conventional chemotherapy, 5 patients developed an early relapse, while 2 patients were refractory to treatment; one patient was minimal residual disease (MRD) positive as assessed by multiparameter flow cytometry. Therefore, all 8 patients were offered an allogeneic HSCT (1st allogeneic HSCT: n=6; 2nd allogeneic HSCT: n=2) with pre-treatment with nelarabine. The median interval from the last chemotherapy to 1st nelarabine administration was 8 weeks (range, 1– 252 weeks). Nelarabine (1,500 mg/m2 i.v.) was administrated on days 1, 3, and 5 at a median of 11 weeks (range, 2–53 weeks) before allogeneic HSCT. Three patients received a 2nd cycle of nelarabine after a median of 4 weeks (range, 2–9 weeks) from the 1st. Six patients had unrelated (HLA-match, n=4; mismatch, n=2) and 2 patients matched related donors. Most patients received myeloablative conditioning (TBI, 12 Gy; cyclophosphamide 120 mg/kg; n=3; treosulfan 36 g/m2; etoposide 30 mg/kg; cyclophosphamide 120 mg/kg; n=4). In the 8th patient who had already received 2 courses of nelarabine, a 3rd nelarabine application was incorporated as pre-phase into reduced-intensity conditioning (nelarabine 2 × 1,500 mg/m2; fludarabine 90 mg/m2; TBI 8 Gy). Results: i) Pre-transplant application of nelarabine: Immediate neurotoxicity was observed in 1 patient (grand mal seizure day +3 after the last nelarabine administration). Grade II hematotoxicity was observed in 3/8 patients. There were no treatment-related deaths. Seven of 8 patients showed response to nelarabine (86%; CR, n=5; PR, n=2) after a median of 4 weeks (1 – 35 days) from the 1st cycle. One patient had stable disease (SD). ii) Peri-/post-transplant period: there were no uncommon adverse events. After HSCT, prolonged leukocytopenia (WBC 20 days) and thrombocytopenia (thrombocytes 30 days) were observed in 3 and 5 patients, respectively. At the 1st post-transplant bone marrow control, 3 patients with residual disease (PR or SD) prior to allogeneic HSCT achieved CR, and all other patients maintained CR. Acute (grade II) and chronic GvHD developed in 2/8 (25%) and in 2/6 (33%) patients, respectively; there was no case of grade III-IV acute GvHD. At a median follow up of 9 months (range, 1–24), 3 patients had died (38%; relapse, n=1, t-AML, n=1; transplant-related toxicity, n=1). All 5 patients being alive were in CR; 4 patients required no further treatment, and one patient received donor lymphocyte infusions (DLIs) due to mixed chimerism. Conclusions: Nelarabine improves pre-transplant remission in patients with relapsed/refractory T-cell neoplasms and seems to be well tolerated immediately before allogeneic HSCT even in heavily pre-treated patients. The compound might be used as pre-phase or be incorporated into conditioning for allogeneic HSCT which should be further evaluated. Disclosures: No relevant conflicts of interest to declare.

Beschreibung: Abstract 4056 Background: Multiple myeloma (MM) is a malignancy characterized by the expansion of a plasma cell (PC) clone that localizes to the bone marrow (BM). Myeloma cells and BM stromal cells both produce soluble factors promoting the survival and progression of MM. Interleukin-(IL)-16 is involved in regulating migration and proliferation of normal leukocytes, however, it has been unclear whether IL-16 also plays a role in the pathophysiology of human cancers. Methods: Using an antibody array we screened supernatants of myeloma cell lines for the presence of a variety of cytokines/chemokines. We confirmed IL-16 expression in myeloma cell lines as well as in malignant PC and BM plasma from MM patients applying real-time PCR, western blots, ELISA, and flow cytometry. We applied inhibitory RNA to analyze IL-16 function and we used anti-IL-16 antibodies to evaluate possible therapeutic options for MM. Results: We found IL-16 to be strongly overexpressed in the BM of myeloma patients. Myeloma cell lines as well as primary tumor cells from MM patients constitutively expressed IL-16 RNA and protein and spontaneously secreted soluble IL-16. Functional analyses revealed that IL-16 supports the proliferation of myeloma cells. Accordingly, silencing of IL-16 expression had an anti-proliferative effect on the tumor cells. Most importantly, the application of a monoclonal antibody directed against IL-16had a strong growth-inhibiting influence on myeloma cells. Conclusions: These findings suggest that cytokine IL-16 is an important growth-promoting factor in MM and might represent a novel diagnostic and therapeutic target for this incurable human malignancy. Disclosures: No relevant conflicts of interest to declare.

Beschreibung: Abstract 212 Background: Chronic GvHD (cGvHD) is the leading late complication after allogeneic HSCT. Most previous randomized studies in GvHD prophylaxis failed to demonstrate reduced incidence and severity of cGvHD, and shorter time to discontinuation of immunosuppressive therapy (IST). Aims: We previously reported that addition of ATG-F to standard cyclosporine, methotrexate GvHD prophylaxis (control group) significantly reduced severe acute and chronic GvHD (Finke et al., Lancet Oncology, 2009). Here we present final data and unpublished results on cGvHD with extended follow-up [median of 3 (25%-quartile 2.5, 75%-quartile 3.9) years] on 201 patients with median age of 40 (range 18–60) years, transplanted between 2003 and 2007, with AML (n=101), MDS (n=10), ALL (n=70), CML (n=17), OMF (n=3) in early (1st CR or MDS-RA, n=107), or advanced status of disease (all other, n=94). Results: With extended follow-up the cumulative incidence (CI) of extensive cGvHD after three years was 12.2% in the ATG-F group versus 45.0% in the control group (p

Beschreibung: Abstract 4689 Introduction: Allogenic stem cell transplantation (allo SCT) offers a potential curative approach for many malignant and non malignant haematological diseases. Despite its therapeutic benefit, long term immunodeficiency, poor immune reconstitution and Graft vs. Host Disease (GvHD) can often be limiting drawbacks. Since the nineties, regulatory T cell subsets (Treg) have been described and several lines of evidence indicated their implication on GvHD occurrence and progression. We analysed the immune reconstitution of 184 patients who underwent allo SCT at our Transplant Center from 2007 till 2009. Patients, Materials and Methods: Differential lymphocyte subsets were analysed by flow cytometry. Antigens were stained by usage of the following mAb: CD3, CD4, CD8, CD19, HLA-DR, CD56/CD16, CD45RA, CD45RO, CD45, γδ TCR, CD25, and CD127. Tregs were evaluated on simultaneous expression of CD4/CD25hi/CD127low. Data were obtained in monthly intervals for the first six months and thereafter every six months for the next 3 years. Data were analysed for three different subgroups: Multiple Myeloma (MM: n=83), Myelofibrosis (PMF: n=22) and AML/MDS (n=51). Smaller number subgroups of patients with CML (n=11), NHL (n=10) and ALL (n=7) were included into the overall analysis but not evaluated separately. Results: The mean value of Treg cell number before allo SCT was 2,5% of the total leukocyte number in all patients. There was no significant difference in the Treg level in any of the three major groups (MM: 2,2%; PMF: 2,1% and AML/MDS: 2,03%). All patients exhibited a significant reduced number of Treg cells during the first 30 days after allo SCT (MM: 0,79%; p= 0,009; PMF: 0,41%; p= 0,01; MDS/AML: 0,6%; p=0,01). Between day 30 and 60 after allo SCT patients with MM had a transient Treg recovery to baseline level (2,4%) while Tregs of patients with PMF or MDS/AML remained significantly lower in comparison to baseline value (PMF: 0,72%, p=0,002 and MDS/AML 0,81%, p=0,01 respectively). One year after allo SCT a faster Treg recovery (1,3% and 1,8% respectively) was observed in MM and MDS/AML patients while patients with PMF still maintained a significant reduction (0,65%; p=0,01). Interestingly, in the second year after allo SCT, Treg cell levels were decreased in all investigated subgroups (MM: 1,1%, p=0,008; PMF: 0,7%, p=0,02 and MDS/AML: 0,7%, p

Beschreibung: Abstract 3090 Background: Previously, in 201 adult patients with allogeneic hematopoietic cell transplantation from matched unrelated donors, we demonstrated that addition of ATG-F to standard cyclosporine, methotrexate GvHD prophylaxis (control) significantly reduces acute and chronic GvHD without negatively affecting non-relapse mortality (NRM), relapse, disease-free survival (DFS) and overall survival (OS) [1,2]. Methods: Now, we present updated results of extended follow-up (median 5.5, Q1 5.0, Q3 6.5 years). Furthermore, we investigated whether the European Group for Blood and Marrow Transplantation (EBMT) risk score (0 to 7 points) based on the sum of points for patient age (20–40y: 1 point, 〉40y: 2 points), disease stage (intermediate: 1 point, late: 2 points), time interval from diagnosis to transplant (〉12m: 1 point), donor type (unrelated: 1 point), and donor-patient sex combination (donor female, patient male: 1 point) [3] was able to predict outcome with respect to OS in our trial. The explained variation R2 of the risk score was calculated by setting the prediction error (Integrated Brier Score) of the score in relation to the prediction error of an overall estimate of OS without dividing the patients into different risk groups. Results: The incidence of extensive chronic GvHD after 5 years was 13.6% in the ATG-F group vs 48.9% in the control group (p

Beschreibung: Patients with advanced chronic myeloid leukemia (CML) in accelerate (AC) or blastic phase (BC) have a dismal prognosis despite the use of tyrosine kinase inhibitors (TKI). Here we report our experience for allogeneic stem cell transplantation (ASCT) in advanced and phase of CML. Between July 1990 and May 2012 88 patients with a median age of 36 years (range 7-76) received allogeneic stem cell transplantation from related (n=34) or unrelated (n=54) donors , including 19 HLA-mismatched donors after myeloablative (n=54) or reduced intensity (n= 34) conditioning. Stem cell source was bone marrow (n=37) or peripheral blood stem cells (n=51). GvHD prophylaxis consisted of calcineurin inhibitor plus short course MTX or MMF. Most of the patient received additional ATG as GvHD prophylaxis (84%).The majority of patient (75%) received TKIs before ASCT. 50% received one TKI, 8 received 2 TKIs and 2 received 3 TKI before ASCT. At time of transplantation 34 patient achieved a second or subsequent chronic phase, 28 were in accelerate and 25 in blastic phase. Overall the median number of blasts at time of ASCT was 18.75% (range 5-58%) and the time from diagnosis to transplantation was 27 months (range 3-296). No primary graft failure was observed. The incidence of acute graft versus host disease (GvHD) grade II to IV was 43% and of severe grade III/IV GvHD was 28%. Forty-two percent of the patient experienced chronic GvHD. The non-relapse mortality (NRM) at 1 and 5 years was 22% (95% CI; 14-30%) and 23% (95%CI: 13-33%), respectively. In a multivariate analysis (MVA) only higher number of transplanted CD34+ cells were associated with a lower risk of NRM (HR 0.850, 95%CI: 0.729-0.992, p=0.04). The cumulative incidence of relapse at 5 years was 43% (95% CI: 31-55%). In a MVA age 〉 40 years (HR 2.272, 95%CI: 1.112-4.645, p=0.024) and Reduced intensity conditioning (HR 2.034, 95%CI: 0.998-4.144, p=0.051) were significant factors for higher risk of relapse. After a median follow-up of 91 months (r., 52-133) the estimated 5 and 10 year overall survival was 44% (95% CI: 32-56) and 40% (95% CI: 28-52%), respectively. In an univariate analysis overall survival was significantly influenced by stem cell source, gender, CD34 transplanted cell number and blastic phase at time of transplantation. However, the only significant factor for improved survival in a MVA was a higher number of transplanted CD34+ cells (HR 0.916, 95%CI: 0.844-0.916, p=0.038). Disclosures No relevant conflicts of interest to declare.

Beschreibung: Previous randomized graft-versus-host disease (GVHD)-prophylaxis trials have failed to demonstrate reduced incidence and severity of chronic GVHD (cGVHD). Here we reanalyzed and updated a randomized phase 3 trial comparing standard GVHD prophylaxis with or without pretransplantation ATG-Fresenius (ATG-F) in 201 adult patients receiving myeloablative conditioning before transplantation from unrelated donors. The cumulative incidence of extensive cGVHD after 3 years was 12.2% in the ATG-F group versus 45.0% in the control group (P 〈 .0001). The 3-year cumulative incidence of relapse and of nonrelapse mortality was 32.6% and 19.4% in the ATG-F group and 28.2% and 33.5% in the control group (hazard ratio [HR] = 1.21, P = .47, and HR = 0.68, P = .18), respectively. This nonsignificant reduction in nonrelapse mortality without increased relapse risk led to an overall survival rate after 3 years of 55.2% in the ATG-F group and 43.3% in the control group (HR = 0.84, P = .39, nonsignificant). The HR for receiving immunosuppressive therapy (IST) was 0.31 after ATG-F (P 〈 .0001), and the 3-year probability of survival free of IST was 52.9% and 16.9% in the ATG-F versus control, respectively. The addition of ATG-F to standard cyclosporine, methotrexate GVHD prophylaxis lowers the incidence and severity of cGVHD, and the risk of receiving IST without raising the relapse rate. ATG-F prophylaxis reduces cGVHD morbidity.

Beschreibung: Abstract 3055 Acute and chronic graft versus host disease (GvHD) are severe complications after allogeneic stem cell transplantation but occurrence of GvHD is associated with reduced risk of relapse. Therefore, strategies to prevent GvHD without increasing the risk of relapse are urgently needed. We investigate retrospectively whether anti-lymphocyte globulin (ATG Fresenius®, Fresenius Biotech, Graefelfing, Germany) as part of the conditioning regimen may reduce the risk of GvHD without increasing the risk of relapse after peripheral blood stem cell transplantation from HLA-identical siblings or HLA-compatible relatives. Out of 462 patients who received HLA-identical stem cell transplantation between 1990 and 2011 in our institution, we selected 238 consecutive patients who received allogeneic peripheral blood stem cell grafts after the year 2000. The median age of the patients was 48 years (r.,18–73y) and diagnosis were: AML (n=93), ALL (n=24), CML (n=25), MDS (n=23) or lympho-proliferative disorders (n=73). Patients were classified as good risk (n=95) or bad risk (n=143). 79 patients did receive ATG within the conditioning regimen with a median dose of 30mg/kg (r., 20–90mg/kg) and 159 patients did not receive ATG. In the ATG group there were more HLA mismatch donors (6% vs. 1%, p=0.02), more bad risk patients (70% vs. 50%, p=0.04), more reduced intensity conditioning regimens (65% vs. 34%, p〈 0.001) and older patients (median age 50 vs. 46 years, p=0.03). Acute GvHD grade I to IV was less observed in the ATG group (27% vs. 40%, p= 0.04), but the difference in severe GvHD grade III and IV did not show statistical significance (10% vs. 18%, p=0.1). Chronic GvHD was less observed in the ATG group (30% vs. 52%, p=0.002), which was most obvious for extensive chronic GvHD (14% vs. 40%, p

Beschreibung: Abstract 3024FN2 Reduced-intensity conditioning (RIC) regimen followed by allogeneic stem cell transplantation (SCT) has become a reasonable treatment option for patients with multiple myeloma who are refractory or have relapse to an autograft. However, in comparison to standard myeloablative conditioning RIC resulted in higher risk of relapse. Maintenance therapy after autologous transplantation has shown to improve survival while after allogeneic SCT data are lacking so far. We here report the results of myeloablative toxicity-reduced allograft consisting of intravenous busulfan (12.2 mg/kg) and cyclophosphamide (120 mg/kg) followed by lenalidomide maintenance in 33 patients with multiple myeloma who relapsed to an autograft. A total of 32 patients had received one (n=16), two (n=15), or even three (n=1) autografts, and 1 patient was refractory to 2 induction therapies and failed to collect autologous stem cells. The median duration of remission after the autograft was 12 months. The primary endpoint of the study was non-relapse mortality at 1 year and secondary objectives were evaluation of response, incidence of acute and chronic graft-versus-host disease as well as progression and overall survival. To prevent graft-versus-host disease antithymocyte globulin (ATG Fresenius®) was given at a median dose of 20 mg/kg on day -3, -2, and -1. Lenalidomide was started earliest at day 120 after transplantation if there were no signs of infection or graft-versus-host disease. The median time between last autograft and allogeneic transplantation was 20 months. 19 patients were treated with fully HLA-matched unrelated donor, 8 patients had a mismatch donor, and 6 patients were transplanted from an HLA-identical sibling. 2 patients died of treatment-related complications resulting in a cumulative incidence of non-relapse mortality at 1 year of 6% (95% CI: 0–14%). After transplantation 27% developed grade II graft-versus-host disease, and severe grade III graft-versus-host disease was seen in 6% of the patients. Complete remission was noted in 46% of the patients, partial remission was seen in 48% and stable disease in 3%. The median interval between allogeneic transplant and start of lenalidomide was 168 days. The median starting dose was 5 mg (range 5–15 mg) without dexamethasone for 21 day followed by 1 week rest. 9 patients did not receive lenalidomide maintenance due to ongoing graft-versus-host disease, cytopenia or patient's wish. The median number of lenalidomide cycles was 6 (range 1–30). During follow-up 13 patients discontinued lenalidomide treatment due to progressive disease (n=6), GvHD (n=3), thrombocytopenia (n=2), or fatigue (n=2). In 10 patients lenalidomide dose could be increased to 10 or 15 mg, respectively. The major toxicities of lenalidomide were acute graft-versus-host disease grade I – III (21%), viral reactivation (12%), thrombocytopenia grade III-IV (12%), neutropenia grade III-IV (6%), peripheral neuropathy grade I-II (12%), or other infectious complications (6%). During follow-up 9 patients experienced relapse resulting in a cumulative incidence of relapse at 3 years of 42% (95% CI: 18–66%). The 3 year estimated probability of progression-free and overall survival was 52 % (95% CI: 28–76%) and 79 % (95% CI: 63–95%), respectively. In the current trial neither the deletion 13q14 nor the use of mismatch donor nor the chemosensitivity prior allogeneic SCT could be identified as risk factor for survival. This study showed that toxicity-reduced myeloablative conditioning regimen is feasible and highly effective in relapsed patients with multiple myeloma resulting in an acceptable treatment-related mortality. Lenalidomide as maintenance therapy is feasible early after transplantation but toxicity especially the induction of graft-versus-host disease should be considered. Disclosures: Kröger: Celgene: Research Funding. Kropff:Janssen-Cilag: Consultancy, Honoraria; Celgene: Consultancy, Honoraria.