ALBERT

Description: A total of 68 adult patients with B-cell acute lymphoblastic leukemia (B-ALL) were treated in three consecutive adult multicenter ALL studies. The diagnosis of B-ALL was confirmed by L3 morphology and/or by surface immunoglobulin (Slg) expression with 〉 25% blast cell infiltration in the bone marrow (BM). They were characterized by male predominance (78%) and a median age of 34 years (15 to 65 y) with only 9% adolescents (15 to 20 y), but 28% elderly patients (50 to 65 y). The patients received either a conventional (N = 9) ALL treatment regimen (ALL study 01/81) or protocols adapted from childhood B-ALL with six short, intensive 5-day cycles, alternately A and B. In study B-NHL83 (N = 24) cycle A consisted of fractionated doses of cyclophosphamide 200 mg/m2 for 5 days, intermediate-dose methotrexate (IdM) 500 mg/m2 (24 hours), in addition to cytarabine (AraC), teniposide (VM26) and prednisone. Cycle B was similar except that AraC and VM26 were replaced by doxorubicin. Major changes in study B-NHL86 (N = 35) were replacement of cyclophosphamide by ifosphamide 800 mg/m2 for 5 days, an increase of IdM to high-dose, 1,500 mg/m2 (HdM) and the addition of vincristine. A cytoreductive pretreatment with cyclophosphamide 200 mg/m2, and prednisone 60 mg/m2, each for 5 days was recommended in study B-NHL83 for patients with high white blood cell (WBC) count (e 2,500/m2) or large tumor burden and was obligatory for all patients in study B-NHL86. Central nervous system (CNS) prophylaxis/treatment consisted of intrathecal methotrexate (MTX) therapy, later extended to the triple combination of MTX, AraC, and dexamethasone, and a CNS irradiation (24 Gy) after the second cycle. Compared with the ALL 01/81 study where all the patients died, results obtained with the pediatric protocols B-NHL83 and B-NHL86 were greatly improved. The complete remission (CR) rates increased from 44% to 63% and 74%, the probability of leukemia free survival (LFS) from 0% to 50% and 71% (P = .04), and the overall survival rates from 0% to 49% and 51% (P = .001). Toxicity, mostly hematotoxicity and mucositis, was severe but manageable. In both studies B-NHL83 and B-NHL86, almost all relapses occurred within 1 year. The time to relapse was different for BM, 92 days, and for isolated CNS and combined BM and CNS relapses, 190 days (P = .08). The overall CNS relapses changed from 50% to 57% and 17%, most probably attributable to the high-dose MTX and the triple intrathecal therapy. LFS in studies B-NHL83 and B-NHL86 was significantly influenced by the initial WBC count 〈 or 〉 50,000/microL, LFS 71% versus 29% (P = .003) and hemoglobin value 〉 or 〈 8 g/dL, LFS 67% versus 27% (P = .02). Initial CNS involvement had no adverse impact on the outcome. Elderly B- ALL patients (〉 50 years) also benefited from this treatment with a CR rate of 56% and a LFS of 56%. It is concluded that this short intensive therapy with six cycles is effective in adult B-ALL. HdM and fractionated higher doses of cyclophosphamide or ifosphamide seem the two major components of treatment.

Description: Skin is the most frequently affected organ in acute graft versus host disease (GVHD). Data from murine studies support the hypothesis that the interaction of residing host Langerhans cells of the epidermis (LC) and donor T cells is crucial for the initiation of acute GVHD. Donor T cells are also necessary to induce the switch of LC from host to donor origin after allogeneic stem cell transplantation (SCT). In an ongoing clinical protocol applying alemtuzumab-based T cell depleted (TCD) allogeneic SCT (Meyer, Blood2007; 109:374), we observed acute skin GVHD occurring early after transplantation. We therefore intended to analyse the LC chimerism in patients undergoing this protocol. So far, LC-chimerism analysis in humans has been performed by the detection of the Y-chromosome restricting it to sex-mismatched donor/recipient pairs. Here we introduce a new method to isolate LC from small skin samples at high purity for a sensitive STR-based chimerism analysis of general applicability. Epidermal skin layers were obtained from 6 mm punch biopsies by dispase I digestion. A small slice of epidermis was used for immunofluorescent staining. The remaining sample was digested by trypsin, and CD1a/MHC-class II-positive LC were sorted by flow cytometry. This approach resulted in a mean purity of 〉 96% with skin of healthy individuals. However, the density of LC early after SCT following non-TCD myeloablative regimens had previously been shown to be much lower compared to healthy individuals. By CD1a-staining, we were able to show that this is also the case after TCD reduced intensity SCT. Nevertheless, LC could be purified in all of 8 analyzed patients. The isolated LC numbers ranged from 10 to more than 1000. In 4 patients we performed a re-analysis of the isolated cells by flow cytometry and confirmed a purity exceeding 97%. We obtained reliable results for LC chimerism in 6 of 8 patients. After the RNA-isolation protocol was further improved, we were able to detect signals even with 35 isolated LC in patient MZ-47. In two patients, the majority of isolated LC were of donor origin whereas the other 4 patients had predominantly host LC (patients’ characteristics and chimerism results are summarized in Table 1). None of the patients developed spontaneous acute GVHD so far. For patients MZ-37 and MZ-43 LC-chimerism was also performed after day +50 post HSCT and showed a switch to 〉97% donor chimerism at that time. In summary, we have established a sensitive method that enables the chimerism analysis on highly purified LC independent of sex-mismatched donor/recipient pairs. Our results on a few patients’ samples can not yet be related to clinical events. This assay, however, allows the comprehensive investigation of the chimerism of LC and potentially of other tissue-resident antigen presenting cells to study their impact on GVHD in humans. Table 1 patient donortype sex (patient/donor) LC (n) purity (%) donor chimerism (%) MSD, matched sibling donor; MMUD, mismatched unrelated donor; n.a., not applicable; M, male; F, female 37 MSD F/M 1127 97,7 97 40 MMUD M/M 1800 98,3 13 43 MMUD M/M 10 n.a. n.a. 44 MSD M/M 157 n.a. n.a. 45 MSD M/F 325 〉 98 36 46 MMUD M/M 355 n.a. 48 47 MSD M/M 35 n.a. 〉90

Description: Allogeneic hematopoietic stem cell transplantation (allo-SCT) from an HLA-identical sibling donor (SIB) is considered the preferred postremission therapy for younger patients with HiRi AML in CR1. The role of allo-SCT from volunteer unrelated donors (VUDs) is less clear and randomized controlled trials addressing this issue do not exist. We performed an observational landmark analysis on parallel cohorts of patients aged

Description: To improve the results of allogeneic SCT for high-risk AML and MDS, the FLAMSA-RIC conditioning regimen for allogeneic SCT combines cytoreductive chemotherapy (fludarabine, HD AraC, amsacrine), followed three days later by reduced intensity conditioning (4Gy TBI/EDX). Since in particular patients with an unfavorable karyotype seemed to benefit from this approach (Schmid et al., JCO, 2005), we analysed the outcome of 172 patients with poor risk cytogenetics according to NCCN criteria, who had been allografted following FLAMSA-RIC conditioning in 11 European centres between 1999 and 2008. Median time from diagnosis to transplantation was 5 months. Donors were matched siblings, matched unrelated, or mismatched unrelated donors in 34%, 47%, and 19%. Patients suffered from progressive MDS (10%), de novo AML (47.5%), or secondary AML (43.5%). SCT was performed upfront, after primary induction failure, in CR1 and in relapsed disease in 17%, 33%, 22% and 28% of patients, respectively. Median patient age was 53 (18–71) years. 95 patients (56%) had a complex aberrant karyotype, 55 and 65 had abnormalities of chromosome 5 (−5/5q-) and 7 (−7/7q-), respectively. After a median follow up of 20 months, overall survival (OS) at 2 and 4 years was 46.4% and 40.5%, the respective leukemia-free survival was 37.7% and 32.0%. Causes of death were leukemia in 30%, and non-relapse mortality in 21%. Encouraging results were observed in patients with chromosome 7 aberrations or with a complex karyotype leukemia (4y OS=49.3% and 40.3%). In contrast, results were inferior in patients with chromosome 5 aberrations (4y OS=30%), mainly due to an increased rate of leukemia-associated death (p=.008). Patiens with MDS, who received allogeneic SCT as first line treatment, achieved a 4y OS of 80% despite unfavorable cytogenetics. Unlike, patients with secondary AML after MDS had an inferior outcome (4y OS=28%, p=.018). In a Cox regression model, a stage of remission at transplantation, a 8/8 or 10/10 matched family or unrelated donor, and lack of monosomy 5 or deletion 5q were associated with superior OS (p=.025, .05, and .05). In conclusion, allogeneic SCT following the FLAMSA-RIC regimen is a highly effective treatment for MDS and AML with unfavorable karyotype, comparing favourably with published data. In MDS, SCT should be performed before transformation into sAML. Long term remission is achieved in a substantial percentage of patients with complex karyotype disease and aberrations of chromosome 7. Aberrations of chromosome 5 may require alternative or additive strategies.

Description: Donor lymphocyte infusions (DLI) are increasingly used to treat minimal residual disease or mixed hematopoietic donor-recipient chimerism in T-cell depleted allogeneic stem cell transplantation (SCT). In addition, several clinical trials currently investigate the prophylactic application of DLI to promote donor T-cell reconstitution after transplantation. However, DLI carry a substantial risk of inducing graft-versus-host disease (GVHD). We investigate DLI heavily depleted of CD8 T cells using a clinical grade immunomagnetic in vitro procedure in an ongoing clinical study [Meyer et al., Blood2007, 109:374]. These DLI are administered in a prophylactic setting to patients with hematological malignancies who are off immunosuppressive treatment and free of GVHD early after allogeneic SCT. The reduced-intensity conditioning regimen consists of fludarabine and melphalan and in vivo T-cell depletion (TCD) by the anti-CD52 antibody Alemtuzumab. Up to now, 24 patients have been treated with 1 to 4 increasing doses of CD8-depleted DLI starting with 1x10^6 CD4+ T cells/kg bodyweight. The median time between SCT and first DLI was 119 days (range, 60–194). Seven of 24 patients (29%) developed acute GVHD of grade 2 to 4 or extensive chronic GVHD following CD8-depleted DLI. We did a longitudinal analysis of lineage-specific T-cell chimerism in 20 patients who received CD8-depleted DLI in comparison to 17 patients who did not qualify for DLI due to spontaneously occurring acute GVHD (n=14) or unavailable donors (n=3). The patients’ characteristics in both groups were comparable with a median age of 55 (range, 35–64) years in the DLI group and of 57 (range, 29–67) years in the non-DLI group. The donor types were matched sibling (DLI: n=6; non-DLI: n=2), matched unrelated (DLI: n=8; non-DLI: n=8), and unrelated with 1 HLA-mismatch (DLI: n=6; non-DLI: n=7), respectively. Twelve of the 20 patients in the DLI group and 6 of 17 patients in the non-DLI group showed a secondary decrease of donor T-cell chimerism to a median of 52% (range, 10–90%) between 7 and 35 weeks after transplantation (median, 12 weeks). Only one of the latter spontaneously reconverted to a full donor T-cell chimera. Of the remaining 5 non-DLI patients, 4 patients subsequently relapsed with their underlying disease and one patient still had a mixed T-cell chimerism of 50% two years after transplantation. In contrast, in patients receiving CD8-depleted DLI the proportion of donor T cells significantly increased and 11 of 12 patients converted to durable full donor chimerism. Three patients of the DLI group subsequently developed disease relapse. By monitoring CD52-expression on reconstituting T cells by flow cytometry, we were able to demonstrate the impact of CD8-depleted DLI on post-transplant T-cell reconstitution: In the non-DLI group, the majority of CD4 T cells remained CD52-negative 9 months after transplantation. Simultaneously, the proportion of CD52-expressing CD4 T cells was significantly higher in the DLI group (mean: 42% versus 86%; t-test, p

Description: Abstract 3351 Poster Board III-239 Objectives: The majority of patients with chronic lymphocytic leukemia (CLL) who receive allogeneic hematopoietic cell transplantation (HCT) have fludarabine-refractory disease. The most active single agent in this disease stage is alemtuzumab. Alemtuzumab has a long half-life and induces profound T-cell depletion (TCD). Since TCD may mitigate graft-versus leukemia effects we evaluated „pre-conditioning“ with alemtuzumab followed by a washout period in order to minimize in vivo T-cell depletion of the graft in a phase II study (NCT 00337519). Methods: Patients received cytoreductive treatment with 3 × 30 mg alemtuzumab weekly prior to HCT. The scheduled interval between last dose of alemtuzumab and HCT was increased from two weeks to one month during the study. The antibody level at the day of HCT was measured with an ELISA with a lower limit of detection of 31.25 ng/mL (BioAnaLab lim., Oxford, UK). The conditioning regimen contained fludarabine (150 mg/m2) and busulfan (8 mg/kg). Cyclosporine (CSA) and methotrexate (MTX) were applied as GVHD-prophylaxis. Medically fit patients with relapsed CLL were elible. Results: 62 patients with a median age of 57 years were included between April, 2004 and October, 2008. A median of 3 prior regimens had been given. 55% of the patients had fludarabine-resistant disease. Two patients failed to reach HCT due to progressive disease during alemtuzumab therapy. Donors were matched siblings for 26 and matched unrelated donors for 34 patients. The median level of alemtuzumab in peripheral blood after a washout period of two weeks was 62 ng/mL (interquartile range, 45 to 196 ng/mL; minimum below the limit of detection; maximum 490 ng/mL) compared to a median level below the limit of detection after a delay of four weeks (interquartile range, between the limit of detection and 77 ng/mL, maximum 256 ng/mL) (p=0.005). Despite one month time between the last dose of alemtuzumab and HCT 4 out of 30 patients (13%) had alemtuzumab levels greater than 200 ng/mL. No primary or secondary graft failure occurred. A linear relationship between the alemtuzumab level at HCT and the time to complete CD4-T-cell chimerism (TCC) was observed (p=0.003). At day +100 a CD4 positive T-cell-chimerism (TCC) 〉95% had been achieved by 84% of patients with alemtuzumab levels 200 ng/mL (p=0.006). All patients had a complete neutrophil-chimerism at day +100. After early taper of immunosuppression (N=2) or the application of donor lymphocyte infusions in incremental doses (N=5) mixed TCC has been converted to complete TCC in all patients. The median follow-up is 17 months (1 to 61 months). Day +100 non-relapse mortality was 2%. At two years non-relapse mortality and relapse incidence were 21% and 29%, respectively. Two-year overall survival and progression-free survival were 67% (95% CI, 51% to 83%) and 50% (95% CI, 31% to 69%). Conclusions: In patients who received alemtuzumab prior to HCT, residual drug levels may interfere with T-cell engraftment. Lineage specific T-cell chimerism should therefore be assessed prospectively in this group of patients. Persistent mixed T-cell chimerism can be converted by an early taper of immunosuppression and incremental doses of donor lymphocyte infusions. Disclosures: Schetelig: Bayer Schering: Research Funding. Platzbecker:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Description: 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.

Description: Inhibition of Janus-kinase 1/2 (JAK1/2) is a mainstay to treat myeloproliferative neoplasms (MPN). Sporadic observations reported the co-incidence of B-cell non-Hodgkin lymphomas during treatment of MPN with JAK1/2 inhibitors. We assessed 626 patients with MPN, including 69 with myelofibrosis receiving JAK1/2 inhibitors for lymphoma development. B-cell lymphomas evolved in 4 (5.8%) of 69 patients receiving JAK1/2 inhibition compared with 2 (0.36%) of 557 with conventional treatment (16-fold increased risk). A similar 15-fold increase was observed in an independent cohort of 929 patients with MPN. Considering primary myelofibrosis only (N = 216), 3 lymphomas were observed in 31 inhibitor-treated patients (9.7%) vs 1 (0.54%) of 185 control patients. Lymphomas were of aggressive B-cell type, extranodal, or leukemic with high MYC expression in the absence of JAK2 V617F or other MPN-associated mutations. Median time from initiation of inhibitor therapy to lymphoma diagnosis was 25 months. Clonal immunoglobulin gene rearrangements were already detected in the bone marrow during myelofibrosis in 16.3% of patients. Lymphomas occurring during JAK1/2 inhibitor treatment were preceded by a preexisting B-cell clone in all 3 patients tested. Sequencing verified clonal identity in 2 patients. The effects of JAK1/2 inhibition were mirrored in Stat1−/− mice: 16 of 24 mice developed a spontaneous myeloid hyperplasia with the concomitant presence of aberrant B cells. Transplantations of bone marrow from diseased mice unmasked the outgrowth of a malignant B-cell clone evolving into aggressive B-cell leukemia-lymphoma. We conclude that JAK/STAT1 pathway inhibition in myelofibrosis is associated with an elevated frequency of aggressive B-cell lymphomas. Detection of a preexisting B-cell clone may identify individuals at risk.

Description: Abstract 4109 We have previously demonstrated that the application of CD8-depleted donor-lymphocyte infusions (DLI) is feasible after reduced-intensity conditioning and in vivo T-cell depletion by alemtuzumab. DLI overcome slow lymphocyte recovery associated with alemtuzumab-administration and improve anti-infectious immunity and reliably convert a decreasing T-cell chimerism (Meyer et al. Blood 2007 & BMT 2010). Here we provide clinical follow up data of 117 patients with different hematological diseases and a median observation time of 1 year (range, 1–86 months) post hematopoietic stem cell transplantation (HSCT). The majority of patients either suffered from an acute leukemia / MDS (n=54), lymphoma (n=32), myeloma (n=17), or myeloproliferative neoplasms (n=12). Two patients suffered from non-malignant diseases. The median age of the patients was 56 years (range, 19–71) and none of them qualified for a conventional conditioning regimen. 50 patients had undergone previous transplantations (autologous: n=47, allogeneic: n=3). Donors were matched siblings (n=20), matched unrelated donors (n=55), or unrelated donors with a single HLA mismatch (n=42). Between days 60 and 120 after HSCT, without calcineurin-inhibitors and in the absence of graft-versus-host disease (GVHD), 1×106 CD8-depleted DLI per kg bodyweight were administered. Up to three further DLI were given in escalating doses in 60 to 90 day intervals. Following this procedure, 45 patients received at least one dose of DLI. Among those patients who did not qualify for DLI, 50 patients had primary GVHD. In 22 patients DLI were not administered for other reasons (donor unavailable, infections, relapse). In 64% of DLI induced acute GVHD, which was the major reason for withholding the next DLI-dose step. The rate of acute GVHD 〉 grade 2 was 30%. 10% suffered from extensive chronic GVHD. The 1 and 3 year overall survival was 63% and 43%, respectively. Survival significantly differed between the DLI and the non DLI group after 3 years (63% vs. 27%, p=0.002). Since this trial was not randomized, we also compared the DLI group to only those patients who did not receive DLI for other reasons than primary GVHD and found similar results (62% vs. 28%, p=0.01). As expected, the presence of GVHD at any time was associated with a reduced relapse rate in all patients (55.8% vs 30.8%, p=0.013). Although DLI was associated with a survival benefit, the relapse rate did not differ from that of the no-DLI cohort. AML/MDS patients represented the largest group of patients included in our study (n= 48). Among these, 41 patients achieved the time point for DLI administration, 16 of them received at least one dose of prophylactic DLI. 9 patients developed GVHD after DLI application. 20 patients had primary GVHD as major cause for not receiving DLI. The survival curves differed significantly between the DLI and non DLI group after 1 and 3 years (91.7% vs. 54%, and 82.5% vs 24% p=0.004). The estimated 5 year overall survival for all AML patients was 50.4%. There was no significant difference analyzing the relapse rate (20% vs 18.8%). In summary, the prophylactic application of CD8-depleted DLI in the absence of GVHD was associated with a survival benefit. However, we were not able to relate this benefit to a decreased relapse rate, and we assume a better control of infections. Our data strongly support a randomized trial, comparing prophylactic vs. preemptive / therapeutic DLI application in the context of T-cell depleted HSCT. Disclosures: Meyer: BMS: Membership on an entity's Board of Directors or advisory committees.

Description: We generated a transgenic mouse line that expresses the Cre recombinase under the control of the Ncr1 (p46) promoter. Cre-mediated recombination was tightly restricted to natural killer (NK) cells, as revealed by crossing Ncr1-iCreTg mice to the eGFP-LSLTg reporter strain. Ncr1-iCreTg mice were further used to study NK cell–specific functions of Stat5 (signal transducers and activators of transcription 5) by generating Stat5f/fNcr1-iCreTg animals. Stat5f/fNcr1-iCreTg mice were largely devoid of NK cells in peripheral lymphoid organs. In the bone marrow, NK-cell maturation was abrogated at the NK cell–precursor stage. Moreover, we found that in vitro deletion of Stat5 in interleukin 2–expanded NK cells was incompatible with NK-cell viability. In vivo assays confirmed the complete abrogation of NK cell–mediated tumor control against B16F10-melanoma cells. In contrast, T cell–mediated tumor surveillance against MC38-adenocarcinoma cells was undisturbed. In summary, the results of our study show that STAT5 has a cell-intrinsic role in NK-cell development and that Ncr1-iCreTg mice are a powerful novel tool with which to study NK-cell development, biology, and function.