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: The role of allogeneic stem cell transplantation in chronic myeloid leukemia is being reevaluated. Whereas drug treatment has been shown to be superior in first-line treatment, data on allogeneic hematopoietic stem cell transplantation (allo SCT) as second-line therapy after imatinib failure are scarce. Using an interim safety analysis of the randomized German CML Study IV designed to optimize imatinib therapy by combination, dose escalation, and transplantation, we here report on 84 patients who underwent consecutive transplantation according to predefined criteria (low European Group for Blood and Marrow Transplantation [EBMT] score, imatinib failure, and advanced disease). Three-year survival after transplantation of 56 patients in chronic phase was 91% (median follow-up: 30 months). Transplantation-related mortality was 8%. In a matched pair comparison of patients who received a transplant and those who did not, survival was not different. Three-year survival after transplantation of 28 patients in advanced phase was 59%. Eighty-eight percent of patients who received a transplant achieved complete molecular remissions. We conclude that allo SCT could become the preferred second-line option after imatinib failure for suitable patients with a donor. The study is registered at the National Institutes of Health, http://clinicaltrials.gov: NCT00055874.

Beschreibung: Abstract 1900 Cancer-testis (CT) antigens show an expression restricted to malignancies and the human germline among healthy tissues and, therefore, represent attractive targets for cancer vaccines. CT antigen SSX-2 is expressed in about 20% of multiple myeloma patients (MM), however, little is known about the occurrence of spontaneous humoral immune responses against SSX-2 in these patients. Moreover, no information is available regarding the functionality of anti-SSX2 antibody responses and a possible impact on the course of the disease. Screening 1098 peripheral blood samples and 200 bone marrow samples of 194 MM patients, as well as 100 healthy donors serving as controls, we found six patients (3%) to present with SSX-2 specific antibodies. When we mapped the target epitopes using overlapping peptides for the whole SSX-2 sequence, we found the antibodies to be exclusively directed against amino acids 81–90. Remarkably, all antibodies were of the IgG3 subtype. In addition, SSX-2 antibodies were strictly antigen-specific and represented potent activators of the complement system. Correlating the antibody responses with clinical events, we found that the majority (5 out of 6) of seropositive patients had been antibody-negative before allogeneic stem cell transplantation (alloSCT) and had only developed anti-SSX2 antibodies after transplantation. Donor-derived antibody responses increased with depth of remission and, in case of recurrence of the disease, dropped to low or undetectable levels. Our data suggest that alloSCT is able to induce immune responses against myeloma specific SSX-2 antigen which is of low immunogenicity under normal conditions. Such antibody responses are of an effective phenotype and seem to correlate with protection from disease recurrence. To further characterize the biological role of transplantation-induced anti-SSX2 immune responses, we will analyze our patients for the presence of SSX-2-specific T cells which might be induced together with serological responses in the framework of an integrated immune response. Active immunotherapy might contribute to amplifying and shaping anti-SSX2 immune responses in myeloma patients, particularly after alloSCT. Disclosures: No relevant conflicts of interest to declare.

Beschreibung: Abstract 1327 Total body irradiation (TBI) is an essential part of the conditioning regimen for acute lymphablastic leukaemia (ALL) patients who undergo allogeneic stem cell transplantation. Due to high toxicity and mortality induced by TBI, we investigate in a multicenter prospective phase II study the toxicity and efficacy of a non TBI-based regimen consisting of treosulfan (3 × 12 g/m2), etoposide (30 mg/kg), and cyclophosphamide (120 mg/kg) in patients with ALL who underwent allogeneic stem cell transplantation. Major inclusion criteria were complete remission, indication for allogeneic stem cell transplantation according GMALL, non eligibility for TBI or patient's wish to avoid TBI. Graft-versus host prophylaxis consisted of cyclosporine A and short course methotrexat and in case of unrelated donors also of anti-lymphocyte globulin (ATG). This preliminary analysis is based on 36 patients (female: n=17; male: n=19) with a median age of 42 years (range: 18–60y). Remission-status at transplantation was either 1. CR (n=27), 2.CR (n=7) or 〉2.CR (n=2). Donors were HLA-identical sibling (n=6), matched unrelated (n=21) or mismatched unrelated (n=9). 39% of the patients were Philadelphia chromosome positive. Primary graft failure was observed in 1 pts. The median time to achieve leukocyte (〉 1×109/l)) and platelet (〉20 × 109/l) engraftment was 22 (range, 11–47) and 24 days (range, 11–95), respectively. The toxicity was moderate including VOD (6%) and liver toxicity grade III (8%) and IV (3%). Acute graft versus host disease grade II-IV and grade III/IV was noted in 22% and 11%, respectively. Chronic graft versus host disease at 1 year was seen in 27%, which was extensive in 7% of the patients. After a median follow up of 12 months, the cumulative incidence of non-relapse mortality (NRM) at 1 year was only 9% (90% CI: 1–17%) and for relapse 37% (90% CI: 21–52%). The estimated 1-year disease free survival was 56% (90% CI:40-71%) and significantly better for patients transplanted in 1.CR vs 2. or higher CR (68% vs 15%, p=0.05). The estimated 1-year overall survival was 81% (90% CI: 69–92%). This preliminary results of a treosulfan, non TBI based conditioning regimen followed by allogeneic stem cell transplantation shows favourable toxicity profile with low non-relapse mortality. Longer follow up is needed to determine long-term freedom from disease. Disclosures: Kröger: MEDAC: Research Funding. Off Label Use: Treosulfan is not approved as conditioning regimen for stem cell transplantation.

Beschreibung: Abstract 2373 NK-cell alloreactivity determined by donor killer-cell immunoglobulin-like receptors (KIRs) has been shown to be predictive for relapse-free survival in myeloid leukemia after allogeneic stem cell transplantation. The role role of NK-cell allo reactivity in patients with multiple myeloma is unclear. Although the ligands for activating KIRs remain elusive, inhibitory KIRs on donor derived NK-cells and HLA class I molecules on the recipient cell determine NK cell alloreactivity. KIR genes on chromosome 19 segregate independently from HLA genes KIR haplotypes can be distinguished in two groups: group A has a fixed number of inhibitory receptors and only one activating receptor (KIR2DS4), while group B haplotype includes more activating receptor genes. Therefore individuals can be categorised as having A-KIR haplotype (A/A), or B-KIR haplotype which contains either A/B heterocygotes or B/B homocygotes KIRs. To analyse the effect of donor KIR-haplotype on outcome in multiple myeloma, we analysed 118 patients with a median age of 51 years (r: 29 – 68) who underwent allogeneic stem cell transplantation. Gender of the study population was predominantly male (n = 75, female: n = 33). Stem cell source was mainly peripheral blood stem cells (n = 105) and derived from unrelated donors (n = 81) or HLA-identical sibling (n = 37). 46 patients received graft from HLA-mismatched donor. The majority of the patients received a melphalan (100 – 140 mg/m2)/fludarabine-based reduced conditioning regimen (N = 106), either as part of an auto-allo-tandem protocol or as an allogeneic salvage protocol after relapse to an autograft. 46 patients had experienced relapse to an autograft prior to allogeneic stem cell transplantation. 79 patients were transplanted from KIR haplotype Bx and 39 from KIR haplotype A. Both groups were well balanced regarding age, HLA match, stem cell source, CMV seropositivity, remission status prior to transplantation, del 13q14, and relapse to prior autograft. After a median follow-up of 5 years the 5-year estimated disease-free and overall survival was better for KIR haplotype B donors in comparison to KIR haplotype A donors (30 % vs. 14 %, p = 0.008, and 49 % vs. 36 %, p = 0.07). The disease-free survival benefit for KIR haplotype B was mainly seen in HLA-matched patients (4y DFS: 39 % vs. 18 %, p = 0.005), while in HLA mismatch transplantation no difference according to KIR haplotype could be detected (26 % vs. 18 %, p = 0.5).The difference in survival in the HLA-matched group was due to higher risk of relapse for KIR haplotype A in the HLA-matched group (cumulative incidence at 1 year 46 % vs. 17 %, p = 0.005). In a multivariate Cox model KIR haplotype A remained a significant factor for worse disease-free (HR: 1.82, p = 0.008) and overall survival (HR: 1.69, p = 0.042). Other significant factors in the multivariate analysis were female donor sex (HR: 1.67, p = 0.04) and chemo-refractory disease at transplantation (HR: 1.76, p = 0.03) for OS and factors for DFS were unrelated donor (HR: 1.83, p = 0.02) and patient's age as continous variable (HR: 1.03, p =0.008) and donor female sex (HR: 1.62, p = 0.03). We conclude that female donor sex and KIR haplotype A are important risk factors for outcome of allogeneic SCT in multiple myeloma and should be considered for donor selection. Disclosures: No relevant conflicts of interest to declare.