Search

Introduction: Data on the influence of different ATLG doses on immune reconstitution (IR) and GvHD in MUD allo-SCT is limited. In this study, we compared the impact of ATLG doses (30mg/kg vs 60 mg/kg) on IR and transplant outcomes. Methods: In this retrospective study we included 289 patients who received MUD allografts (HLA 10/10) between 2005-2019 in the University Cance Center University of Hamburg. All patients received PBSC-allo-SCT with MAC for various hematological malignancies. Seventy-three patients received 30mg/kg ATLG, and 216 patients received 60mg/kg (on days -3.-2 and -1) prior to allo-SCT. Periphereal blood samples were collected on days +30, +100 and +180 and analyzed by flow cytometry for following lymphocyte populations: T-cells (total and activated), T-helper cells (total, naïve and memory), cytotoxic T-cells (total, naïve and memory), B-Lymphocytes (total, naïve and memory), NK-cells, NKT-cells, γδT-cells and regulatory T-cells. Results: Neutrophil and platelet engraftments were significantly delayed after the 60mg/kg compared to 30mg/kg group with medians of 11 days (range, 8-23) vs 12 days (8-27) (p=0.009) for neutrophil and 14 days (range, 9-53) vs 16 days (range, 8-237) for platelets, respectively (p=0.002). We observed a higher incidence of EBV reactivation within the first 100 days in the 60mg/kg group (41% vs 21% in the 30mg/kg group, p=0.049). Higher cumulative incidence of Infections Day +100 was observed in the 60 mg/Kg group with an incidence of 75% vs that of 67% in the 30mg/Kg group respectively (p=0.002). At day +30 we observed a faster reconstitution of naïve-B cells (p The incidence of aGVHD grade II-IV was comparable between the groups: 63% and 59% in the 30mg/Kg and 60mg/Kg groups, respectively. We observed a higher incidence of grade IV aGvHD in the 30mg/kg group (8%) comparing with the rate of 0.5% in the 60mg/kg group (p=0.0002), this was confirmed in multivariate analysis: RR 0.65 (95%CI 0.005-0.363) p= 0.004. After a median follow up of 21 months (range, 1-161) there were no significant differences in OS, PFS, NRM, RI and cGVHD between the groups. Conclusion: The choice of ATLG dose has significant impact on IR after MUD-allo-SCT. Higher doses are associated with reduced severe aGVHD, however at the cost of delaying engraftment and increasing infections. Disclosures Ayuk: Celgene/BMS: Honoraria; Gilead: Honoraria; Janssen: Honoraria; Mallinckrodt/Therakos: Honoraria, Research Funding; Miltenyi Biomedicine: Honoraria; Novartis: Honoraria; Takeda: Honoraria.

Acute graft-versus-host disease (GVHD) is a life-threatening complication after allogeneic hematopoietic cell transplantation (allo-HCT). Although currently used GVHD treatment regimens target the donor immune system, we explored here an approach that aims at protecting and regenerating Paneth cells (PCs) and intestinal stem cells (ISCs). Glucagon-like-peptide-2 (GLP-2) is an enteroendocrine tissue hormone produced by intestinal L cells. We observed that acute GVHD reduced intestinal GLP-2 levels in mice and patients developing GVHD. Treatment with the GLP-2 agonist, teduglutide, reduced de novo acute GVHD and steroid-refractory GVHD, without compromising graft-versus-leukemia (GVL) effects in multiple mouse models. Mechanistically GLP-2 substitution promoted regeneration of PCs and ISCs, which enhanced production of antimicrobial peptides and caused microbiome changes. GLP-2 expanded intestinal organoids and reduced expression of apoptosis-related genes. Low numbers of L cells in intestinal biopsies and high serum levels of GLP-2 were associated with a higher incidence of nonrelapse mortality in patients undergoing allo-HCT. Our findings indicate that L cells are a target of GVHD and that GLP-2–based treatment of acute GVHD restores intestinal homeostasis via an increase of ISCs and PCs without impairing GVL effects. Teduglutide could become a novel combination partner for immunosuppressive GVHD therapy to be tested in clinical trials.

INTRODUCTION Allogeneic stem cell transplantation (alloSCT) is the only curative procedure for primary and secondary myelofibrosis (PMF, SMF). Elderly people are mainly affected, limiting the feasibility of intensive myeloablative chemotherapy regimens. The introduction of reduced-intensity conditioning (RIC) made alloSCT feasible and effective for old patients. Nevertheless, the incidence of PMF and SMF is not negligible in young patients, theoretically able to tolerate also high-intensity therapy. Very few data are available about the efficacy of RIC-alloSCT in the particular setting of young-aged MF patients. PATIENTS AND METHODS This study includes 56consecutive Myelofibrosis young patients (age RESULTS Engraftment rate was 98%, with a median neutrophil engraftment time of 15 days. Platelet engraftment was reached by 51 patients (91%) in a median time of 19 days. Four patients (7%) developed poor graft function, successfully treated with CD34+ selected PBSC boost. After a median follow up of 8.6 y estimated 5y PFS and OS was 68% and 82% respectively. DIPSS risk and donor HLA-matching resulted the only significant impacting factors on OS. Neither cytogenetic nor molecular abnormalities (included ASXL1) were significantly related to OS. Twenty-five patients (44%) experienced acute GVHD grade >1. Chronic GVHD was observed in 34 patients (65%), mostly (82%) beginning in the first 300 days after transplantation. Cumulative incidence of TRM was 7% at 1 year, with a plateau after the first year (5y TRM = 12%). TRM was observed only in patients with maximal grade (3) of marrow fibrosis (p=0.00). Furthermore, TRM never occurred in previously splenectomized patients (p=0.00), but no significant impact from splenectomy on OS was observed (p=0.32). After transplant, 11 patients (20%) relapsed: 1 died without any treatment because of infection, 9 received DLI with durable recovery of complete remission in 3 cases, 7 patients (6 after DLI) underwent a second alloSCT, with long-term survival in 5 cases. CONCLUSION Reduced intensity conditioning followed by allogeneic SCT is a curative treatment approach for younger patients with myelofibrosis with a low NRM. The most important outcome-determining factor is donor HLA-matching. Interestingly, marrow high grade fibrosis showed to significantly impact TRM. Biological markers such as ASXL1 mutation and cytogenetics, largely known as highly predictive for poor prognosis in the disease natural course, did not show any impact on survival, suggesting that patients harboring these abnormalities could get the greatest benefit from alloSCT. According to these data, RIC-alloSCT ensures optimal engraftment and low relapse rate, being able to lead also younger MF patients to cure. Further data collection, and a prospective randomized trial are needed to confirm our conclusion. Disclosures No relevant conflicts of interest to declare.

Introduction Myelofibrosis (MF) is predominantly a disease of the elderly with a median age of 65 years at diagnosis. Allogeneic stem cell transplantation (ASCT), which is associated with substantial treatment-related morbidity and mortality, is the only potentially curative option so far. The development of reduced intensity conditioning (RIC) regimens has enabled transplant to be performed successfully in older patients. To evaluate the outcome of transplantation among elderly patients, we conducted a retrospective analysis of results in 46 patients, aged 65 years or older, who were transplanted between 2002 and 2018 at the University Medical Center Hamburg-Eppendorf (UKE), Hamburg, Germany. Patients and methods Retrospective data from 46 patients with primary or secondary MF, who underwent ASCT, were collected. Median age at ASCT was 67 years (r: 65-74). 76% of patients were classified as MF-3 according to the WHO criteria. DIPSS risk status was intermediate-1 in 4% of patients, intermediate-2 in 61% and high in 33%. 70% of patients had mutation in JAK2, 17% in CALR and 9% in MPL. All patients received peripheral blood stem cell (PBSC) as graft source. Stem cell donor was related in 9% of patients, unrelated in 91%; 74% of patients had a completely HLA-matched donor, whereas 26% had at least 1 allele or antigen mismatch. 85% of patients received busulfan 10 mg/kg orally (or 8 mg/kg intravenously) plus fludarabine (150 mg/m2) reduced intensity conditioning regimen, whereas 15% received myeloablative conditioning with busulfan 16 mg/kg orally (or 12.8 mg/kg intravenously) plus fludarabine (150 mg/m2). 96% of the patients received anti-T-lymphocyte globulin (Grafalon®, Neovii, Germany) at a cumulative dose of 30 mg/kg for matched related donor (MRD) transplants and 60 mg/kg for matched/mismatched unrelated donor (MUD/MMUD) transplants. Further GVHD prophylaxis consisted of cyclosporine A and mycophenolate from day +1 to +28. Results Engraftment rate was 94%, with a median time to neutrophil engraftment of 13 days (r: 10-19). Two patients (4%) experienced primary graft failure (PGF), one received a second ASCT with successful engraftment, while the other had further PGF after second ASCT and died of infection after third ASCT. Platelet engraftment rate was 87% and was reached in a median time of 21 days (r: 10-293). Five patients (11%) developed secondary poor graft function and four received CD34+ selected PBSC boost, two achieving complete remission (CR) and one obtaining CR with incomplete platelet recovery (CRp). 52% of patients experienced acute GVHD grade I to IV, while the overall rate of chronic GVHD was 56%, which was moderate disease in 24% and severe disease in 13%. After a median follow-up of 4 years, 6-year estimated progression-free survival (PFS) and overall survival (OS) were 60% (95% CI: 42-78) and 64% (95% CI: 48-80), respectively. In the univariate analysis, male donor sex was the only significant factor for improved OS and PFS at 6 years (P=.001 and P=.003, respectively). A positive impact on OS was observed for mutation in CALR (P=.05), as previously reported. Interestingly, survival was not significantly different in patients aged 65-70 years compared with those aged 71-74 years. Cumulative incidence of non-relapse mortality (NRM) was 29% at 4 years (95% CI: 13-45). Major causes of death were infections (n=4) and GVHD (n=3). The only significant factor for lower NRM in the univariate analysis was male donor sex vs female (NRM 17% vs 58%, P=.004). No NRM occurred in CALR-mutated patients (p=0.00). Cumulative incidence of relapse at 6 years was 10% (95% CI: 0-22): one patient durably restored molecular response after early tapering of cyclosporine, one died because of GVHD without any further treatment, one achieved a long-lasting CR after donor lymphocytes infusions (DLI), while two patients underwent a second ASCT after DLI alone in one case and DLI plus azacitidine in the other, with long-term CR in the former. Conclusion Our results show that RIC regimen followed by allogeneic stem cell transplantation in older patients with myelofibrosis is a curative treatment option. These results are encouraging for older MF patients with minimal comorbidities. In addition to Hematopoietic cell transplantation-specific comorbidity index (HCT-CI), a comprehensive geriatric assessment could be a useful tool for a better selection of patients with the aim to further reduce NRM. Disclosures No relevant conflicts of interest to declare.

We investigated in a retrospective multicenter study the impact of chromosome arm 13q deletion (13q-) as detected by fluorescence in situ hybridization (FISH) on outcome after dose-reduced allografting in patients with multiple myeloma. In 68 of 140 patients, data on chromosome 13q status were available. Most patients included had advanced myeloma. At 2 years, patients with 13q deletion (n = 31) had a shorter event-free (18% vs 42%; P =.05) and overall survival (18% vs 67%; P =.03) than patients without 13q- (n = 37). Patients with 13q- experienced a higher relapse rate (77% vs 44%; P

We evaluated toxicity, engraftment, chimerism, graft-versus-host disease (GVHD), and response to a dose-reduced allograft after cytoreductive autografting in 17 patients with advanced stage II/III multiple myeloma (MM). After autografting with melphalan (200 mg/m2) the patients received after a median interval of 119 days (range 60-210) a dose-reduced regimen consisting of fludarabine (180 mg/m2), melphalan (100 mg/m2), and antithymocyte globulin (3 × 10 mg/kg) followed by allografting from related (n = 7), mismatched related (n = 2), or unrelated (n = 8) donors to induce a graft-versus-myeloma effect. After dose-reduced allografting all patients became neutropenic ( 1 × 109/L) and platelet (> 20 × 109/L) counts of 16 (range, 11-24) and 23 days (range, 12-43), respectively. Complete donor chimerism was detected after a median of 30 days (range, 19-38). Acute GVHD stage II occurred in 4 patients (25%) and grade III GVHD in 2 patients (13%). Chronic GVHD developed in 40% of the patients, but only 1 patient experienced extensive chronic GVHD requiring further immunosuppressive therapy. Two patients died of alveolar hemorrhage and pneumonia, resulting in a day 100 mortality rate of 11%. The rate of complete remission with negative immunofixation increased from 18% after autografting to 73% after allografting. After a median follow-up of 17 months after autologous and 13 months after allogeneic transplantation 13 patients are alive and 12 of them free of relapse or progression. The tandem auto-allotransplant protocol is highly active and provides rapid engraftment with complete donor chimerism and tolerable toxicity.

Allogeneic stem cell transplantation (ASCT) after reduced-intensity conditioning has become a reasonable treatment option for patients with advanced myelofibrosis. The role of characteristic molecular genetic abnormalities, such as JAK2V617F on outcome of ASCT, is not yet elucidated. In 139 of 162 myelofibrosis patients with known JAK2V617F mutation status who received ASCT after reduced-intensity conditioning, the impact of JAK2 genotype, JAK2V617F allele burden, and clearance of mutation after ASCT was evaluated. Overall survival was significantly reduced in multivariate analysis in patients harboring JAK2 wild-type (hazard ratio = 2.14, P = .01) compared with JAK2 mutated patients. No significant influence on outcome was noted for the mutated allele burden analyzed either as continuous variable or after dividing into quartiles. Achievement of JAK2V617F negativity after ASCT was significantly associated with a decreased incidence of relapse (hazard ratio = 0.22, P = .04). In a landmark analysis, patients who cleared JAK2 mutation level in peripheral blood 6 months after ASCT had a significant lower risk of relapse (5% vs 35%, P = .03). We conclude that JAK2V617F-mutated status, but not allele frequency, resulted in an improved survival and rapid clearance after allografting reduces the risk of relapse.

Introduction: Residual minimal disease (MRD) in hematological malignancies has become a valid tool to predict clinical relapse after allogeneic stem cell transplantation (ASCT). Methods and Patients: We screened 154 patients with myelofibrosis who underwent ASCT for molecular residual disease by qPCR or next-generation sequencing (NGS) for JAK2V617F, MPLW515L and MPLW515K or Calreticulin (L367fs*46, and K385fs*47) mutations in peripheral blood (PB) on days +100 and +180 after transplantation. Out of 154 pts, 103 were JAK2V617F (n=101), 31 Calreticulin (CALR), and 4 MPL mutated, while 13 pts were triple negative. 136 pts could be followed after ASCT with one molecular marker. The median age of the pts was 58 years (range, 32-75 y). Patients had either primary myelofibrosis (n= 90), post ET/PV myelofibrosis (n=40), myelofibrosis in acceleration or were transformed to AML (n=6). Conditioning mainly relied on a busulfan-based reduced-intensity regimen. Donor were HLA-identical sibling (n=26), matched unrelated (n=71) or mismatched unrelated (n=39). JAK2V617F, MPL, and CALR mutations were measured by usage of Taqman PCR or in case of CALR Type 2 mutation by digital PCR on day +100 and day +180 from PB as described elsewhere. Results: After a median follow up of 78 months (range, 49-101 months) the 5-year estimated overall survival was 60% (95% CI: 50-70%) and the cumulative incidence of relapse at 5 years was 26% (95% CI: 18-34%) for the entire study population. On days +100 and +180 after transplantation in 27% and 12% of the patients the underlying mutation was still detectable in peripheral blood. The percentage of complete clearance was higher in CALR-mutated patients (96%) in comparison to MPL (75%) and JAKV2617F (70%) mutated pts. Whereas there was a trend for better survival for CALR-mutated patients in comparison to JAK2-mutated patients (71% vs 57%; p=0.48), once a patient achieved molecular remission post-transplant the risk of relapse remained low independently of the underlying mutation. Patients who were alive and without relapse at days +100 or +180 but with still detectable mutation in PB had a significantly higher risk of relapse than those who were molecular negative (62% vs 10%, p Conclusions: JAK2V617F, CALR and MPL genetic lesions allow to monitor MRD in around 90% of the myelofibrosis patients after ASCT by qPCR or digital PCR in the PB. Persistence of MRD on days +100 or +180 in peripheral blood can be used to taper immunosuppressive drugs or to apply donor lymphocyte infusion to prevent clinical relapse. Disclosures No relevant conflicts of interest to declare.

Introduction Primary or post-ET/post-PV myelofibrosis is one of the Philadelphia chromosome-negative chronic myeloproliferative neoplasia characterized by significantly reduced overall survival. More recently several mutated genes have been detected, which allow, in addition to clinical factors, to identify patients with a significantly shorter overall survival and a higher risk of transformation into acute leukemia. Allogeneic stem cell transplantation is still the only curative treatment option for patients with myelofibrosis, and due to the inherent risk of the treatment procedure careful selection of the patients is required. Molecular genetics may help to select patients for allogeneic stem cell transplantation. Patients and methods To determine the impact of mutated genes in patients with myelofibrosis who underwent allogeneic stem cell transplantation we analyzed samples from 169 patients with a median age of 58 years (r: 18 - 75) who received allogeneic stem cell transplantation either from related (n = 36) or unrelated (n = 133) donor. Stem cell source were more often peripheral blood stem cells (n = 165) than bone marrow (n = 4). The intensity of conditioning was mainly reduced intensity (n = 166), rather than myeloablative conditioning (n = 3). Patients suffered from primary myelofibrosis (n = 110), post-ET/PV myelofibrosis (n = 46), while 13 patients were in acceleration or had transformed into acute myeloid leukemia. According to dynamic IPSS (DIPSS) (n = 165) the patients were either low (n = 7), intermediate-1 (n = 35), intermediat-2 (n = 91), or high risk (n = 32). Regarding molecular genetics we found JAK2V617F mutations in 62%, calreticulin (CALR) mutations in 20%, MPL mutations in 4%, U2AF1 in 7%, SRSF2 in 10%, SF3B1 in 4%, ASXL1 in 29%, IDH1 in 2%, IDH2 in 3%, CBL in 1%, DNMT3A in 4%, TET2 in 10%, EZH2 in 4%, while none of the patients showed mutations in ETV6 and PTPN11. Overall, only in 11 patients no mutation could be detected. One mutation could be detected in 41%, 2 mutations in 30%, 3 mutations in 11%, 4 mutations in 5%. Results During follow-up 39 patients experienced relapse and 46 patients experienced non-relapse mortality. From the non-molecular factors regarding disease-free survival in univariate analysis age < 58 (p < 0.01), intermediate-1 and low risk according to DIPPS (p = 0.002), HLA-matched vs. mismatched (p = 0.04) were significant factors for improved disease-free survival. Regarding molecular markers improved disease-free survival was seen for patients with mutations in CALR (p = 0.005), while negative impact on disease-free survival was seen for mutations in U2AF1 (p = 0.035), ASXL1 (p = 0.05), IDH2 (p = 0.006), DNMT3A (p = 0.029). No significant difference could be seen for patients with EZH2, IDH1, SRSF2, and SF3B1 mutations. There was no difference in disease-free survival for patients without any mutation vs. 1, and more than 1 mutation (p = 0.12). Regarding the previously described unfavorable mutations ASXL1, SRSF2, EZH2, IDH1, and IDH2, we found 40 patients who had at least 1 of these unfavorable mutations, 11 had 2 of these mutation, and 1 had 3 of these unfavorable mutations. However, the estimated 5-year disease-free survival did not differ significantly between patients without any of these unfavorable mutations, with 1 or with 2 of them (47 vs. 40 vs. 41%, p = 0.5). Conclusions These results suggest that some molecular marker such as ASXL1, U2AF1, IDH2 and DNMT3A negatively influence DFS in myelofibrosis after allogeneic stem cell transplantation in a univariate analysis. In contrast, the poor prognosis of the recently described unfavorable mutated genes SRSF2, EZH2, and IDH1 was not observed and may therefore be overcome by allogeneic stem cell transplantation. Disclosures No relevant conflicts of interest to declare.

The JAK2-V617F mutation occurs in about 50% of patients with myelofibrosis and might be a reliable marker to monitor residual disease after allogeneic stem cell transplantation. We describe a new, highly sensitive (≥ 0.01%) real-time polymerase chain reaction (PCR) to monitor and quantify V617F-JAK2–positive cells after dose-reduced allogeneic stem cell transplantation. After 22 allogeneic stem cell transplantation procedures in 21 JAK2-positive patients with myelofibrosis, 78% became PCR negative. In 15 of 17 patients (88%), JAK2 remained negative after a median follow-up of 20 months. JAK2 negativity was achieved after a median of 89 days after allograft (range, 19-750 days). A significant inverse correlation was seen for JAK2 positivity and donor-cell chimerism (r: −0.91, P < .001). Four of 5 patients who never achieved JAK2 negativity fulfilled during the entire follow-up all criteria for complete remission recently proposed by the International Working Group, suggesting a major role for JAK2 measurement to determine depths of remission. In one case, residual JAK2-positive cells were successfully eliminated by donor lymphocyte infusion. In conclusion, allogeneic stem cell transplantation after dose-reduced conditioning induces high rates of molecular remission in JAK2-positive patients with myelofibrosis, and quantification of V617F-JAK2 mutation by real-time PCR allows the detection of minimal residual disease to guide adoptive immunotherapy.

Suicide gene transfer into donor T lymphocytes is a promising strategy to avoid severe graft-versus-host disease (GvHD) after allogeneic stem cell transplantation (SCT).[1][1],[2][2] We conducted a phase 1/2 study[3][3] combining infusion of CD34-enriched peripheral blood stem cells (PBSCs) with

To improve the antimyeloma effect of donor lymphocyte infusion (DLI) after allogeneic stem cell transplantation in multiple myeloma, we investigated in a phase 1/2 study the effect of low-dose thalidomide (100 mg) followed by DLI in 18 patients with progressive disease or residual disease and prior ineffective DLI after allografting. The overall response rate was 67%, including 22% complete remission. Major toxicity of thalidomide was weakness grade I/II (68%) and peripheral neuropathy grade I/II (28%). Only 2 patients experienced mild grade I acute graft versus host disease (aGvHD) of the skin, while no grades II to IV aGvHD was seen. De novo limited chronic GvHD (cGvHD) was seen in 2 patients (11%). The 2-year estimated overall and progression-free survival were 100% and 84%, respectively. Adoptive immunotherapy with low-dose thalidomide and DLI induces a strong antimyeloma effect with low incidence of graft versus host disease. (Blood. 2004;104:3361-3363)

We investigated the feasibility of unrelated stem cell transplantation in 21 patients with advanced stage II/III multiple myeloma after a reduced-intensity conditioning regimen consisting of fludarabine (150 mg/m2), melphalan (100-140 mg/m2), and antithymocyte globulin (ATG; 10 mg/kg on 3 days). The median patient age was 50 years (range, 32-61 years). All patients had received at least one prior autologous transplantation, in 9 cases as part of an autologous-allogeneic tandem protocol. No graft failure was observed. At day 40 complete donor chimerism was detected in all patients. Grade II to IV acute graft-versus-host disease (GVHD) was seen in 8 patients (38%), and severe grade III/IV GVHD was observed in 4 patients (19%). Six patients (37%) developed chronic GVHD, but only 2 patients (12%) experienced extensive chronic GVHD. The estimated probability of nonrelapse mortality at day 100 was 10% and at 1 year was 26%. After allografting, 40% of the patients achieved a complete remission, and 50% achieved a partial remission, resulting in an overall response rate of 90%. After a median follow-up of 13 months, the 2-year estimated overall and progression-free survival rates are 74% (95% CI, 54%-94%) and 53% (95% CI, 29%-87%), respectively. A shorter progression-free survival was seen in patients who already experienced relapse to prior autograft (26% versus 86%, P = .04). Dose-reduced conditioning with pretransplantation ATG followed by unrelated stem cell transplantation provides durable engraftment and donor chimerism, reduces substantially the risk of transplant-related organ toxicity, and induces high remission rates.

To investigate impact of conditioning regimen, donor source, and Dynamic International Prognostic Scoring System (DIPSS) on outcome of allogeneic hematopoietic stem cell transplantation for myelofibrosis, we analyzed results in 388 patients transplanted between 1990 and 2011 in three major transplant centers (Essen, Germany: n = 63; Hamburg, Germany; n = 156; Seattle, USA: n = 169), including 216 male and 171 female patients, 18 – 79 (median 54) years old, with either primary (n = 283) or post ET/PV (n = 150) myelofibrosis who received myeloablative (n = 190), reduced intensity (n = 182), or non-myeloablative (n = 16) conditioning and were given stem cells from related (n = 156, including 152 HLA-identical siblings, 1 haplo-identical relative, and 3 identical twins ) or unrelated donors (n = 232; 175 were HLA-matched and 57 were HLA-mismatched). Stem cell source was bone marrow (n = 51) or peripheral blood (n = 337). According to DIPSS at time of transplantation patients were classified as low (n = 35), intermediate-1 (n = 106), intermediate-2 (n = 161), or high (n = 84) risk. The conditioning regimen consisted of busulfan 16 mg/kg and cyclophosphamide (n = 136), busulfan 10 mg//kg and fludarabine (n = 154), treosulfan-based (n = 26), TBI 12 Gy-based (n = 52), or low dose TB 2 Gy and fludarabine (n = 16), and melphalan 140 mg/m2 and fludarabine (n = 4). After a follow-up of 3.5 – 7.7 (median 5) years the 1-year non-relapse mortality (NRM) of the entire study cohort was 24% (95% CI: 20 – 28). In multivariate analysis significant factors for NRM were age as continuous variable (HR: 1.029; 95% CI: 1.011 – 1.048, p = 0.002), HLA mismatch (HR: 2.026; 95% CI: 1.318 – 3.113, p = 0.001), and TBI containing conditioning (HR 1.872; 95% CI: 1.198 – 2.926, p = 0.006). The cumulative incidence of relapse was 17% (95% CI: 13 – 21) at 5 years, negatively impacted by age as continuous variable (HR: 1.030; 95% CI: 1.003 – 1.057, p = 0.028), unrelated donors (HR: 0.560; 95% CI: 0.339 – 0.926, p = 0.024), and the use of ATG (HR: 2.425; 95% CI: 1.342 – 4.381, p = 0.003) or campath (HR: 3.257; 95% CI: 2.129 – 6.985, p < 0.0001 The estimated 3 and 5 year overall survival for the study group was 62% (95% CI: 56 – 68%) and 59% (95% CI: 53 – 65%), respectively. Significant factors for 5-year survival in the univariate analysis were HLA-matched vs. mismatched donor (61% vs. 47%, p = 0.05), related vs. unrelated donors (65% vs. 54%, p = 0.03), high dose conditioning vs. RIC vs. NMA (58% vs. 61% vs. 32%, p = 0.04), non TBI vs. TBI containing conditioning regimens (62% vs. 42%; p = 0.001), and DIPSS score at transplantation (low 83% vs. intermediate-1 64% vs. intermediate-2 58% vs. high risk 45%; p < 0.001). In the multivariate analysis for survival significant factors were age as continuous variable (HR: 1.027; 95% CI: 1.009 – 1.045; p = 0.004), DIPSS score: intermediate-1 (HR: 1.662, p = 0.3), intermediate-2 (HR: 2.069, p = 0.09), high (HR: 2.924, p = 0.02), and TBI-containing conditioning regimen (HR: 1.973; p < 0.001). Conclusion This analysis of results in a large cohort of patients confirms a high success rate of allogeneic hematopoietic stem cell transplantation for myelofibrosis significantly impacted by DIPSS classification at the time of transplantation. In addition the analysis confirms the risk factors of patient age and donor HLA-mismatch. Furthermore, the data show similar results between RIC and MAC, but less favorable outcome with TBI containing conditioning, regardless of the dose of TBI. Novel approaches need to be developed, particularly for patients with more advanced disease. Contribution The authors NK and MD contributed equally. Disclosures: No relevant conflicts of interest to declare.

Introduction Bone marrow fibrosis is a hallmark of primary or post ET/PV myelofibrosis. Recent studies have shown that allogeneic stem cell transplantation induces rapid bone marrow fibrosis regression suggesting that fibrosis is more a dynamic rather than a static process. So far, no data are reported if fibrosis regression influences outcome after transplantation. Methods and patients We studied 94 patients with myelofibrosis who received allogeneic stem cell transplantation at the University Medical Center Hamburg between 2002 and 2010. In 57 patients the complete bone marrow histology prior transplantation and/or on day+30 and +100 after transplantation were available. The median age was 57 years (r: 33–73) and patients were classified as IPSS low-risk (n = 1), intermediate-1 (n = 5), intermediate-2 (n = 18), and high-risk (n = 33). Donor source was unrelated (n = 46) or related (n = 11) and of these 38 were HLA-matched while 19 were HLA-mismatched. 41 had primary and 16 post ET or post PV myelofibrosis. The median number of blasts was 1% (r: 0–17%) and gender was male (n = 32) and female (n = 25). All patients received busulfan-based dose-reduced conditioning regimen. Bone marrow fibrosis was graded according to the European consensus and WHO (MF-0 – 3), respectively, and evaluated by experienced hematopathologists. Results Before transplantation 41 patients (72%) had MF grade 3 and 16 (28%) MF grade 2. After engraftment on day+30 (± 10 days) (n = 48), 3 (6%) had complete regression (MF-0) and 7 (15%) near complete regression of bone marrow fibrosis while 17 (35%) had MF-2 and 21 (44%) had MF-3. On day+100 (± 20 days) complete regression (MF-0) was noted in 11 (25%) and near complete regression (MF-1) in 13 (29%) while 12 (27%) and 8 (18%) had still MF-2 or MF-3, respectively. Patients with complete and near complete regression (MF-0 and MF-1) on day +30 had a 5-year estimated overall survival of 100% and those with MF-2 and MF-3 of 75% (p = 0.09). Patients with complete or near complete regression on day+100 had a 5-year estimated survival of 95% in contrast to 60% for those with MF-2 or MF-3 (p = 0.04). If analyzed by regression by grade at day+100 7 (16%) had a reduction of 3 grades (e.g. MF-3 to MF-0), 12 (27%) of 2 grades, 16 (36%) of 1 grade, and 9 (21%) had no grade reduction at day+100. Reduction of 2 or 3 grades at day+100 resulted in 95% survival at 5 years while reduction of 1 or no grade had a survival of 70% however this difference did not reach statistical significance (p = 0.1). There was no difference of fibrosis regression at day+100 between high risk IPSS and low/intermediate risk patients. Furthermore, in those patients with JAK2V617F mutation and complete or near complete regression 42% still had detectable JAK2V617F mutation level in peripheral blood. In contrast, 81% had complete donor cell chimerism if bone marrow fibrosis was MF-0/MF-1 while only 31% had complete chimerism at day+100 if fibrosis was classified as MF-2 or MF-3. Conclusion This data on fibrosis regression after allogeneic stem cell transplantation suggests that a more rapid regression - independently of IPSS risk score - resulted in a favorable survival and may be used as an early predictive factor for excellent survival. Disclosures: No relevant conflicts of interest to declare.

Introduction We investigate early outcome after allogeneic SCT in 22 patients – male (n = 13) and female (n = 9) – with myelofibrosis who received ruxolitinib prior to transplantation in order to reduce spleen size and constitutional symptoms. Patients and methods The median age of the patients was 59 years (r: 42 – 74 y) and ruxolitinib was given at doses between 2 x 5 mg (n = 5), 2 x 15 mg (n = 5), and 2 x 20 mg (n = 12) before first (n = 19) or second (n = 3) fludarabine-based reduced intensity conditioning from related (n = 2), and matched (n = 14), or mismatched (n = 6) unrelated donor. Thirteen patients had primary myelofibrosis and 9 post ET/PV myelofibrosis. Before ruxolitinib the patients were classified according to DIPSS as intermediate-1 (n = 3), intermediate-2 (n = 14), or high risk (n = 5). Stem cell source was PBSC (n = 21) or bone marrow (n = 1) with a median CD34+ cell count of 7.1 x 106/kg. Before ruxolitinib 21 patients (96%) had constitutional symptoms and all patients had splenomegaly. The median time from start of ruxolitinib to allogeneic SCT was 133 days (r: 27 – 324) and the median treatment duration was 97 days (r: 20 – 316). Most patients (n = 82%) received ruxolitinib until start of conditioning therapy. Four patients (18%) discontinued ruxolitinib between 28 and 167 days before transplantation due to progressive disease or no response (n = 3) or cytopenia (n = 1). Results At time of transplantation 86% had improvement of constitutional symptoms and 45% had major response (>50% palpable) of spleen size, 28% had response of spleen size which was less than 50%, and 27% had no response or progressive spleen size after ruxolitinib treatment. After discontinuation of ruxolitinib at first day of conditioning regimen no “rebound” phenomenon was seen. One patient transformed to sAML before transplantation despite response of spleen size and constitutional symptoms. After busulfan (n = 16), treosulfan (n = 3), or melphalan (n = 3) dose reduced conditioning no graft failure was observed and the median time for leukocyte and platelet engraftment was 15 days (r: 10 – 66) and 17 days (r: 8 – 122) respectively. Acute GvHD I-IV was seen in 50% of the patients which was severe (III/IV) in 18%. During follow-up 4 patients died, 1 patient with sAML at time of transplant due to relapse on day 102 and 3 patients due to therapy-related mortality. One female patient who received a second unrelated HLA-matched transplantation after treosulfan-based regimen died of CMV pneumonitis on day 75. She did not response to ruxolitinib regarding spleen size and constitutional symptoms. A second patient with iron overload and liver fibrosis died of liver toxicity on day 47. This patient initially responded to ruxolitinib but progressed regarding spleen size prior to transplantation. One patient who responded to ruxolitinib regarding constitutional symptoms and spleen size (< 50%) died of GvHD on day 77. The estimated 1-year OS and PFS was 76% (95% CI: 54 – 98%). Conclusion Ruxolitinib reduces spleen size and constitutional symptoms in the majority of patients before allogeneic stem cell transplantation. Discontinuation of ruxolitinib at start of conditioning did not induce rebound phenomenon and did not negatively impact engraftment after transplantation. Longer follow-up is needed to determine late outcome. Disclosures: Wolf: Novartis: Research Funding.

Introduction Myelofibrosis (MF) is a clonal hematological disorder classified as a myeloproliferative neoplasm (MPN). Bone marrow fibrosis, splenomegaly and extramedullary hematopoiesis are hallmarks of the disease. Symptomatic burden is present in most of the affected patients and can compromise quality of life (QoL) by its severity. Most patients suffer from constitutional symptoms such as fatigue, pruritus, night sweats, bone pain, fever, early satiety and weight loss. Currently allogeneic stem cell transplantation (HSCT) is the only curative therapy but it is associated with a high morbidity and mortality. Infectious complications, acute and chronic GvHD are major complications which influence quality of life after transplantation. Until now, no systematic evaluation of QoL after allogeneic stem cell transplantation for myelofibrosis patients exists. Methods In this cross-sectional study we examined 77 patients (male=37, female=40) with a median age of 61 years (range, 40 to 79 years) who received an allogeneic HSCT with a busulfan-based reduced-intensity conditioning from related (n=20) or unrelated donor (n=54) in the period from 1999 to 2011. Diagnosis was primary myelofibrosis in 47 patients and secondary myelofibrosis in 30 patients. At time of transplantation all patients had advanced disease and were classified by the Lille score as high-risk (n=18), intermediate-risk (n=34) and low-risk (n=12) patients. At time of evaluation, 72 patients were in complete remission, 5 patients were not in complete remission, 25 patients suffered from GvHD (limited=14, extensive=11). The patient population was divided into four groups according to the time between transplantation and evaluation of QoL. First group = 6-36 months (n=24); second group= 36-60 months (n=16); third group = 60-85 months (n=22); fourth group 85-146 months after transplantation (n=15). QoL was measured by using the two validated questionnaires FACT-BMT and MPN-SAF. By means of statistical analysis (SPSS; ANCOVA) it was examined whether there are significant differences between the groups. Results According to the MPN-SAF Total Symptom score (the lower the score the better is the Result: possible range= 0-100, the mean scores of the groups one to four are as followed: group1: 18.9 (SD =11.3), group2: 21.1 (SD=17.9), group 3:19.3 (SD=15.5) and group 4: 18.9 (SD=12.4). There were no significant differences between the groups (p=0,962). Unfortunately no MPN-SAF Total Symptoms score was available before transplantation, but according to the mean value of MPN described in the literature (mean=25.3; SD=17.2) the mean score after transplantation (mean=19.5; SD=14.0) of our study population was lower. The mean scores of the FACT-BMT from group one to four were 115.3 (SD=16.6); 120.9 (SD=17.5); 112.4 (SD=15.5) and 115.3 (SD=20.9) There were also no significant differences between the 4 groups (p=0.541). We investigated whether there are significant differences of the results in both questionnaires regarding gender (male/female), donor (unrelated/related), GvHD (yes/no), HLA (matched/mismatched), primary or secondary MF (post-PV/post-ET/PMF), remission (complete/not complete), but none of the variables did influence QoL significantly. The subscales BMTS, FACT-G, and its subscales Physical Well-Being (PWB), Functional Well-Being (FWB), Emotional Well-Being (EWB), Social Well-Being (SWB), of the FACT-BMT were calculated and analyzed by ANCOVA. No significant differences were found among the groups. Comparing the mean value of the FACT-G (mean=85.7; SD=13,1; range= 60-108)and its subscales PWB (mean=22.7; SD= 4.5), SWB (mean=22.4; SD=4.7), EWB (mean 19.9; SD=3.1), FWB (mean=20.6; SD=4.7) of our entire patient population with the mean value of a normal population FACT-G (mean=80.1; SD=18.1; range= 15.4-108), PWB (mean=22.7; SD= 5.4), SWB (mean= 19.1; SD=6.8), EWB (mean=19.9; SD=4.8) and FWB (mean=18.5; SD=6.8) our population has even the same or higher scores. Conclusion This cross-sectional study shows that after dose reduced allogeneic stem cell transplantation in MF-patients Physical and Emotional Well Being is almost as good as that of the normal healthy population whereas Social and Functional Well Being seem to be classified as even better than that of a normal population. Disclosures: No relevant conflicts of interest to declare.

Abstract 1740 Mutations in the additional sex combs like 1 gene (ASXL1) have been described recently in patients with myelodysplastic and myeloproliferative syndromes. While in MDS ASXL1 is associated with worse prognosis, the role in MPN is not well defined. The aim of this study was to investigate the prognostic impact of ASXL1 mutations in 136 patients with primary myelofibrosis or post ET/PV myelofibrosis who underwent allogeneic stem cell transplantation after reduced intensity conditioning. Out of 136 patients sufficient genomic DNA prior transplantation was available for 103 patients. The median age of 48 female and 55 male patients was 59 years (range, 33–75y). After busulfan (n= 97) or treosulfan (n=6) based dose-reduced conditioning, patients received a stem cell graft from a related (n= 17) or unrelated donor (n=48). ASXL1 was sequenced by Sanger sequencing, and positive patients were confirmed by an independent sequence analysis. Mutations in ASXL1 were found in 27 patients (26%), of whom 24 had frameshift mutations (23%). ASXL1 mutated and non mutated patients were equally distributed between patients with unrelated (19% pos/81% neg) and related (21% pos/79% neg) donors. ASXL1 mutations were found at the same frequency in Lille low risk (15%), intermediate risk (24%) and high risk (20%) patients. After a median follow up of 41 months (range 26–62) the cumulative incidence of non- relapse mortality at 1 year and relapse at 5 years were 26% (95% CI 16–36) and 26% (95% CI 16–36), respectively, and the 5-year OS was 56% (95% CI 44–68). Patients with ASXL1 mutation did not differ significantly to those without mutation in non relapse mortality (35% vs. 22% p=0.4), relapse incidence (24% vs. 27%, p=0.9) and regarding overall survival. Overall survival for ASXL1 mutated and wildtype patients was not significantly different (47% vs. 59%, p= 0.5) in patients with any ASXL1 mutation and in the subgroup of ASXL1 frameshift mutated patients (48% vs. 59%, p= 0.6). The rates of acute grade II-IV GvHD and of chronic GvHD were similar in ASXL1 mutated and wildtype patients (44% vs. 36%, p=0.5 and 52% vs. 46%, p=0.7, respectively). In summary, ASXL1 is one of the most frequently mutated genes in PMF patients. Patients with ASXL1 mutations have a similar outcome after dose reduced allogeneic HSCT as patients with wildtype ASXL1. Disclosures: No relevant conflicts of interest to declare.

Abstract 4212 Allogeneic stem cell transplantation remains the only curative treatment approach for chronic myelomonocytic leukemia (CMML), but relapse is the major cause of treatment failure. Here, we evaluated the impact of molecular mutations on outcome and the value of molecular monitoring post transplantation in patients with CMML. We screened 45 patients with a median age of 57 years (range: 24–73 years) who underwent allogeneic SCT pre transplant for molecular markers including a sensitive next-generation sequencing (NGS) technique. Of those, 7 were transformed to secondary acute myeloid leukemia, 5 were classified as MDS/MPN overlap and one patient as atypical CML. In 36 patients sufficient DNA was available for molecular analyses for ASXL1, CBL, NRAS and TET2, mutations. In particular, TET2 and CBL mutations were screened applying amplicon deep-sequencing (454 Life Sciences, Branford, CT). In 32 patients (89%) at least one mutation could be detected: ASXL1: n=18: 50%; CBL: n=7: 19%; TET2: n=15:42%; NRAS: n= 11: 32%. Allogeneic SCT was performed after reduced intensity (66%) or standard myeloablative (33%) conditioning from related (22%) or unrelated donors (78%). The overall survival at 5 years was 46% (95% CI 28–64%) and the cumulative incidence of relapse at 2 year was 35%. Neither TET2, ASXL1, CBL nor NRAS mutations influenced overall survival significantly. In 21 surviving patients with at least one positive marker pre transplantation, the corresponding marker was subsequently tested in peripheral blood after a median of 6 months (range 3–9 months) after transplantation. In 6 (33%) of the patients one of the molecular markers was still detectable: ASXL1: n=3; CBL: n= 3; TET2: n=2, NRAS: n=3) and in 66% the markers were undetectable. In patients who became negative for any molecular marker the cumulative incidence of relapse at 1 year was significantly lower than in the positive tested patients. (15% versus 50%, p = 0.04). In conclusion, this study shows that molecular monitoring post transplant with molecular markers detected also by NGS pre-transplant can help to predict the risk of relapse and may be used to guide post transplant immune-modulating therapies to prevent clinical relapse. In larger studies sequential monitoring of molecular markers after stem cell transplantation are mandatory to determine optimal timing for predicting relapse and for therapeutic intervention. Disclosures: Bacher: MLL Munich Leukemia Laboratory: Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Kohlmann:MLL Munich Leukemia Laboratory: Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.

Abstract 1406 Aiming to improve post-transplant monitoring strategies for patients with acute myeloid leukemia (AML) or myelodysplastic syndromes (MDS), approaches to monitor the minimal residual disease load are at present limited to distinct genetic subgroups, e.g. NPM1mut AML. In the conservative treatment setting, increased levels of physiologic B-cell precursors (>0.01% CD10+/CD19+ cells; “hematogones”) as determined by multiparameter flow cytometry (MFC) in the bone marrow (BM) were suggested to be favorable in AML pts (Chantepie et al., Blood, 2011). We investigated whether the prognostic impact of physiologic B-cell precursors can be confirmed in pts with myeloid malignancies in the transplant context. We quantified CD10+/CD19+ B-cell precursors by MFC in the post-transplant period in 76 pts with AML/MDS who received allogeneic hematopoietic stem cell transplantation (HSCT) at Hamburg University, and performed correlation with survival (41 m/35 f; 18–71 yrs; de novo AML: n=51; s-/t-AML: n=11; MDS: n=13). The majority (n=84, 73%) received reduced intensity conditioning (RIC), mainly based on the FLAMSA regimen. Hematogones were evaluated by MFC on days +30 (d30) and +100 (d100) post-HSCT including the following antibody panel: CD19+, CD10+, CD34+, TdT+. Median levels of CD10+/CD19+ cells were 0,06% on day 30 (mean, 0.25%; range, 0–2.4%) and 1.55% on day 100 (mean, 2.13%; range, 0–12.4%). OS from HSCT did not differ on the basis of the CD10+/CD19+ cells measured on d30 (mean±SD=61±6 weeks, for CD10+/CD19+ cells below the mean of 0.25% vs 74±13 weeks for CD10+/CD19+ cells above the mean, RR=0.73, p=0.685). Estimated 1-year-OS±SE was 63±10% for CD10+/CD19+ cells below the mean of 0.25% versus 71±17% for CD10+/CD19+ cells above the mean, respectively. EFS did not show a significant difference, either. Mean CD10+/CD19+ cells on day 100 associated positively with 1-year-OS. In the group below the mean CD10+/CD19+ cells of 2.13%, 1-year-OS±SE was estimated to be 69±9% (median±SEM=89±10 weeks) which was worse when compared to the group above the mean with a 1-year-OS±SE of 83±15% (median±SEM=96±8 weeks; RR=0.124, p=0.046). The effect of low versus high CD10+/CD19+ cells measured at day 100 on EFS was even more pronounced. 1-year-EFS was significantly worse with 60±10% in the group with lower CD10+CD19+ counts on day 100 versus 95±4% in the group with higher counts (RR=0.095, 95%CI=0.012–0.728, p=0.024). In univariate analysis, disease stage, remission status pre-HSCT, cytogenetic risk group and cGVHD had significant influence on OS and/or EFS. By multivariate analysis, CD10+/CD19+ cells on day 100 remained significantly associated with improved EFS (RR=0.998, p=0.001) but significance of the association with OS was lost (RR=0.145, p=0.07). Other factors significantly associated with superior OS and EFS were occurrence of cGVHD (OS: RR=0.997, p=0.049, EFS: RR=0.996, p=0.001), and low/intermediate cytogenetic risk group (OS: RR=0.995, p=0.005, EFS: RR=0.997, p=0.03). To the best of our knowledge, an association of CD10+/CD19+ cells measured by MFC with survival has not been performed after HSCT in pts with AML and MDS. Our study is the first one showing a favorable prognostic impact of increased CD10+/CD19+ cells 100 days after HSCT. It seems that the level of physiological precursors contributes to the prognostic parameters in patients with myeloid malignancies undergoing HSCT. In more detail, a proportion of more than 2.13% of hematogones at day 100 was associated with improved OS and EFS. The association remained positive in multivariate Cox regression analysis, when other parameters such as pre-transplant permission status, disease stage and cGvHD were included. In conclusion, the hematogones level at day 100 seems to be an independent prognostic parameter which should be further evaluated in pts with myeloid malignancies undergoing HSCT. Considering the experience needed for the determination of leukemia associated immunophenotypes by MFC, or the lack of molecular markers suitable for MRD monitoring in many AML and MDS patients, the introduction of hematogones measurement at defined time points may contribute to the identification of high-risk patients in the transplant context. Disclosures: Bacher: MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership.

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 This retrospective study shows that ATG can prevent severe chronic GvHD without obvious risk of relapse and similar PFS and OS in HLA identical sibling transplantation. A randomized trial including QoL measurement comparing ATG vs. no ATG in HLA identical sibling transplantation is ongoing (registered NCT 00678275). Disclosures: Kröger: Fresenius Biotech: Honoraria, Research Funding. Off Label Use: ATG in HLA identical sibling transplantation.

Abstract 4467 Background: Relapse incidence (RI) and non-relapse mortality (NRM) are competing risks limiting overall survival (OS) after allogeneic stem cell transplantation (SCT) in acute myeloid leukemia (AML). Disease and transplant specific factors predicting relapse like measurement of minimal residual disease (MRD) and chimerism analysis are widely used to aid prophylactic and preemptive treatment decisions. Prediction of NRM mostly relies on pretransplant features. Although most transplant centers routinely perform bone marrow (BM) cytomorphology after SCT for AML, the impact of factors beyond blast count is not well studied. Study Design: We analyzed frequencies and prognostic impact of dysplasia and cellularity upon BM cytomorphology of 112 patients (60 m/52 f, median age 53 [range 17–72] years) with AML at 1st manifestation/ relapse at day 30 (d30) and day 100 (d100) after SCT. Using peripheral blood as main graft source (n=106), donors were unrelated in 87 cases, related in 25. Conditioning was reduced (RIC, n=72) or myeloablative (MAC, n=40). All patients received G-CSF from day 5 until stable engraftment was achieved. Dysplasia was assessed following WHO criteria with different thresholds (10%, 20%, 50%) to define a hematopoietic lineage as dysplastic. We performed a correlation of dysplasia and age-adapted cellularity with outcome measures, calculating RI and NRM as competing risks. Only patients who achieved blast clearance on d30 after SCT were included in the study. Patients who developed hematological relapse between d30 and d100 were only evaluated for d30. At d30 (d100), BM aspirates from 75 (65) patients were available for morphologic evaluation. Result: Dysplasia was a frequent event both at d30 and d100, with ≥10% dysplastic features in granulopoiesis in 25.0% of cases at d30 (31% d100), in erythropoiesis in 34.6% of cases at d30 (43.6% d100) and in megakaryopoiesis in 47.7% of cases at d30 (63.5% d100). Overall, cellularity at d30 was increased in 17.3% (d 100: 6.5%), reduced in 37.3% (d100: 38.7), and normal in 45.3% (d100: 54.8%). No significant correlation with CMV reactivation or with the type of immunosuppression (cyclosporine/ methotrexate versus cyclosporine/ mycophenolic acid) was noted. Cumulative incidences of 2-year-RI and 2-year-NRM were 34% (95% CI, 24%-44%) and 17% (95% CI, 9%-25%). Dysplasia both at d30 and d100 did not correlate with OS or RI. Yet, a statistically significant correlation of normal overall cellularity at d30 with less relapses (RI 20.6%) when compared with reduced overall cellularity (RI 32.1%) or increased overall cellularity (RI 76.9%; p=0.001) was observed. Estimated 2-year-OS was 59% in pts with normal overall cellularity versus 31.4% (reduced) and 44.0% (increased), respectively (p=0.009). The same results, favoring normal cellularity, were observed for each lineage (granulopoiesis, erythropoiesis, megakaryopoiesis). Conversely, increased overall cellularity at d30 correlated with lower NRM (8.3%) when compared to normal (NRM 23.7%) and reduced overall cellularity (NRM 39.6%, p=0.031). Thus, whereas reduced overall cellularity at d30 correlated both with higher RI and higher NRM, the impact of increased cellularity on survival was less clear. The analysis of subdistributive hazards in the competing risk factor model revealed a cumulative RI of 62% (95%CI 35%-89%, HR 6.68, p=0.00014) for increased cellularity, making it the most potent hazard in this analysis. Presence of an informative sample was of prognostic value, too (2-year-OS/ NRM 54.7%/ 80.4% for “evaluable” versus 20%/ 36.9% for “not evaluable” due to low cellularity, p Conclusion: These data suggest that cellularity of BM cytomorphology at d30 after allogeneic SCT aids to assess risk of relapse and NRM in transplant recipients with AML. At this time, it can only be speculated whether underlying persistent leukemia below the microscopic level might be associated with disturbed BM cellularity. Disclosures: Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Abstract 1018 Introduction: There is conflicting data regarding the role of serum ferritin (SF) as an independent prognostic factor for outcome after allogeneic stem cell transplantation (allo-SCT). SF is a surrogate parameter for iron overload and - as an acute phase-protein - can be confounded by many factors. An independent, non-invasive measurement of iron overload is needed. Determining iron content through SQUID correlates well with results of liver biopsies (Nielsen et al. 1998). This method measures the interference of an exteriorly applied small but highly constant magnetic field by the paramagnetic liver storage iron of the patient. Methods: We conducted a single-center retrospective analysis from October 1994 to December 2012, comparing the effect of SF and liver iron content measured by SQUID shortly before transplantation on overall survival (OS), event-free survival (EFS) and transplant-related mortality (TRM) in 143 patients (median age 40 years) undergoing allo-SCT (73% reduced intensity regimen). The diagnoses were subdivided into 4 groups: myelodysplastic syndrome, secondary acute myeloid leukemia (AML), primary myelofibrosis, primary AML and other. Statistical calculations employed Pearson's correlation, ordinal logistic regression, Cox regression analysis and multiple Cox regression analyses with backward elimination. Results: Correlation between SF and SQUID showed a highly significant result of r=0.5 (p < 0.001). The chance of infection was increased 2.4-fold (CI 1.22,4.71) when SQUID values ranged > 1000 μg Fe/g liver. We found similar results for SF > 1000 ng/ml (p 0.003), where the risk of infection was increased 2.87–fold (CI 1.43;5.78). A significant association between SQUID und fungal infection was also seen (p 0.004). For patients with SQUID > 1000 the chance of proven fungal infection versus fungal infection of all other categories was increased 3.08-fold (CI 1.43,6.63). Similarly an association between SF > 1000 and fungal infection could be demonstrated (p 0.001), with a 4.04-fold increased chance of proven fungal infection versus lower fungus categories for patients with SF values > 1000. There was no association between continuous SQUID-, respectively SF values and VOD (veno-occlusive disease) or acute GvHD. The following variables were significantly related with OS: chronic GvHD (HR 0.326, CI 0.13–0.83, p 0.019,), sepsis (p 0.002, HR 2.94 CI 1.48–5.83), new onset cardiac abnormalities (p 0.001, HR 3.21, CI 1.64–6.27), SF > 1000 (p 0.033, HR 2.09, CI 1.06–4.11). For EFS we found statistically significant results for chronic GvHD (p 0.040, HR 0.45, CI 0.21–0.97), sepsis (p 0.012, HR 2.35, CI 1.21–4.58), cardiac abnormalities (p 0.001, HR 2.81, CI 1.50–5.29), SF > 1000 (p 0.016, HR 2.15, CI 1.15–4.10) and time to engraftment (p 0.038, HR 0.934, CI 0.89–1.00). For TRM, statistically significant results were found for sepsis (p 0.005, HR 3.23, CI 1.43–7.29), cardiac abnormalities (p < 0.001, HR 5.21, CI 2.41–11.27), and age (p 0.034, HR 1.04, CI 1.00–1.08). Fungal infection of all categories was not statistically significant (p=1,101), but proven fungal infection in comparison with no fungal infection was found significant (p 0.018, HR 3.12, CI 1.22–7.99. In the multivariate analysis SF and SQUID (categorical and continuous) were not significant factors for OS, EFS or TRM. Conclusion: Our data confirmed previous publications that SF > 1000 increases the risk of infection, moreover fungal infection. In the univariate analysis it is significantly associated with OS and EFS during allo-SCT. As SQUID values correlate well with SF, we could show that SF is indeed a good surrogate parameter for iron overload when measured shortly before allo-SCT. We are now in need of prospective trials investigating the effect of iron chelation before or during allo-SCT on transplant outcome. Disclosures: No relevant conflicts of interest to declare.

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.

Abstract 4147 Introduction: AML is one of the most common indications for allogeneic hematopoietic stem cell transplantation (allo-HSCT). A matched sibling donor (matched related donor, MRD) is generally considered preferable to an unrelated donor (UD). It has been reported before that donor age may influence outcome of allo-HSCT. Sibling donors of older patients will generally be old as well. How a young unrelated donor compares with an old sibling donor is unclear. In this retrospective study we evaluate the influence of donor age on outcome of MRD and UD allo-HSCT and compare the impact of young unrelated donors vs. old related donors on overall survival (OS). Patients and Methods: We performed a retrospective analysis of 298 consecutive patients transplanted for AML at our center between January 2000 and December 2009. Median follow-up was 56 months. The median age of donors and patients were 39 and 50 yrs. respectively. These two ages were used as cut-off points for young and old donors and patients respectively. Accordingly, four donor aged groups were assigned: UD ' 39 years, UD > 39 yrs, MRD ≤ 39 yrs and MRD > 39 yrs. Analyses were performed in the more homogeneous population of patients in CR at time of transplant (n = 170) as well for patients older than 50 years and in CR at time of transplant (n = 77). Patient characteristics for the 170 Patients in CR are summarized in table 1. Results: In the cox-regression multivariate analysis of all 298 patients including factors significant in the univariate analysis (conditioning intensity, cytogenetic risk group, remission status at time of transplant and donor age) remission status (RR: 1.88, p < 0.001) and cytogenetic risk (RR: 5.50, p = 0.004) significantly impacted overall survival (OS). Donor age (p = 0.08) and patient age (p = 0.063) tended to influence OS in this group. In the more homogenous group of 170 patients who were in CR at time of transplantation the Kaplan Meier estimated 5 yr OS was 66% (95% CI: 54%-78%) for patients transplanted from UD ≤ 39 yrs, 41% (95% CI: 25–57%) for UD >39 yrs, 61% (41–81%) for MRD ≤ 39 yrs and 33% (19–47%) for MRD > 39 yrs (p =0.002 comparing all 4 groups), fig. 1. OS of patients with UD ≤ 39yrs was significantly better than for those with MRD > 39 yrs (66% vs. 33%, p = 0.001). In the multivariate cox-regression analysis (including donor age, patient age and cytogenetic risk) only donor age (p = 0.001) and cytogenetic risk (p = 0.011) significantly impacted OS whereby MRD > 39 yrs. was associated with poorer OS compared to UD ≤ 39 yrs (RR: 3.07, p< 0.001). Further subgroup analyses of patients > 50 years old and in CR at time of transplant (n = 77) revealed similar findings with 5 yr OS of 62% for UD ≤ 39 yrs and 25% for MRD >39 yrs (p = 0.016). Conclusions: In patients undergoing allo-HSCT for AML our findings suggest that young unrelated donors may improve survival outcome as compared to old related donors. Further studies are necessary to confirm these findings and better define possible age limits for choosing young unrelated donors vs. older sibling donors. Disclosures: No relevant conflicts of interest to declare.

Abstract 912 Allogeneic stem cell transplantation from related and unrelated donors is now a reasonable treatment option for elderly patients with advanced MDS. Retrospective studies have shown that donor age is an independent risk factor for outcome of allogeneic stem cell transplantation. By nature there is a strong correlation between patient's age and age of an HLA-identical sibling. Therefore we investigated whether a young matched unrelated donor should be preferred as donor to an elderly HLA-identical sibling in elderly MDS patients who underwent allogeneic stem cell transplantation. We extracted from the MDS-registry of the EBMT 871 patients who received allogeneic stem cell transplantation between 1986 and 2009 and fulfilled the following criterias: 1. patient's age > 50 years, 2. advanced MDS: RAEB, RAEB-t, CMML or sAML, 3. HLA-identical sibling or fully matched unrelated donor transplantation. From those 871 patients with a median age of 57 years (r., 50–73) 706 received stem cells from an HLA-identical sibling and 168 from a matched unrelated donor, either after standard conditioning (n = 387) or after reduced intensity conditioning (n = 481). After a median follow-up of 30 months, the estimated 3 years overall survival did not differ between patients who received stem cells from HLA-identical sibling or from matched unrelated donor (36 % vs. 34 %, p = 0.8). However, the median donor age was significantly higher for HLA-identical sibling than for MUD transplantation: 56 years (r: 35 – 81 y) vs. 34 years (r: 19 – 64) (p < 0.001). While age as continuous variable did not influence survival in HLA-identical sibling transplantation (HR: 1.002, p = 0.8), but did in MUD transplantation (HR: 1.03, p = 0.009), we performed 4 groups according to donor age: HLA-identical sibling transplantation with donor age > 50 years (n = 535) and donor age 30 – 50 years (n = 169) and matched unrelated transplantation with donor age < 30y (n = 60) and donor age > 30y (n = 107). These groups were well balanced according to disease-status, abnormal cytogenetics, and intensity of the conditioning regimen. The estimated 5 years OS for MUD (donorage < 30y) was 41 % and better than for HLA-identical (donor > 50y) with 31 % (p = 0.03) and HLA-identical (donor 30 – 50 y) with 33 % (p = 0.06), but worse than MUD (donor > 30 y) 24 % (p = 0.003). For overall survival, younger donors (< 30y) remained an independent factor for improved survival in a multivariate Cox model: HR: 0.66 (p = 0.03). Other factors influencing survival were disease status (sAML: HR: 1.31, p = 0.03), standard myeloablative conditioning (HR: 1.40, p = 0.002), and abnormal cytogenetic (HR: 1.26, p = 0.07). In comparison to HLA-identical sibling transplantation the hazard ratio for survival remained less than 1 for matched unrelated donors aged 20–35 years (HR: 0.66–0.99), but the hazard ration increased >1 if matched unrelated donor age becomes greater than 35 years. We conclude that for elderly patients (> 50 years) with advanced MDS who need allogeneic stem cell transplantation a younger unrelated donor should be preferred to an HLA-identical sibling donor. Disclosures: No relevant conflicts of interest to declare.

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.

Abstract 3393 Poster Board III-281 Background: HLA-mismatched unrelated donors are increasingly becoming a graft source for both standard myeloablative (MAC) and reduced intensity conditioning (RIC) hematopoetic stem cell transplantation (HSCT). Patients and methods: We retrospectively analysed the effects of HLA-mismatching in 553 patients undergoing MAC (n = 342) or RIC (n = 211) unrelated donor HLA-matched (n = 289) or -mismatched (n = 264) HSCT with antithymocyte globulin as part of conditioning. Patient characteristics well matched between HLA-matched and –mismatched groups of MAC and RIC patients. Results: Median follow-up was 1946 days for MAC patients and 765 days for RIC patients. In MAC patients, there was no difference in the incidence of aGvHD, treatment related mortality (TRM) and overall survival (OS) between recipients of HLA-matched vs. –mismatched allografts. In RIC patients on the contrary, the incidence of aGvHD II-IV (46%, vs. 32.0% p = 0.05), III-IV (18%, % vs. 9.0 p = 0.07) and TRM (35% vs. 20%, p = 0.04) were higher and OS lower (31% vs. 50%, p = 0.001) for recipients of HLA-mismatched vs. –matched transplants. In the RIC patients, female donor gender (RR: 1.8; p = 0.02) and HLA-mismatch (RR: 1.8; p = 0.02) negatively influenced TRM while female donor gender (RR: 1.58, p = 0.03), HLA-mismatch (RR: 1.82, p = 0.003) and bad risk disease (RR: 2.14, p < 0.001) negatively impacted OS. Conclusion: Our analyses surprisingly reveal that the positive effect of ATG in HLA-mismatched transplants is only limited to patients undergoing standard myeloablative conditioning. HLA-mismatching and female donor gender negatively impact outcome of RIC allogeneic unrelated donor HSCT despite the use of ATG, highlighting the need for improved strategies. Acknowledgments We thank the staff of the BMT unit for providing outstanding care to our patients and the medical technicians for their excellent work in the BMT laboratory. Disclosures: Off Label Use: Antithymocyte globulin for prevention of severe GVHD.

Abstract 3387 Poster Board III-275 <>Introduction: Allogeneic stem cell transplantation after a dose-reduced conditioning has become a reasonable treatment option for elderly patients with MDS/sAML. For patients with high number of blasts prior transplantation, the risk of relapse is considerably. To reduce the risk of relapse after dose-reduced allograft we performed a study using an anthracycline based induction chemotherapy (amsacrine, cytosine-arabinoside, fludarabine) followed immediately by a reduced intensity conditioning therapy consisting of busulfan (8mg/kg). Patients and Methods: Between November 2005 and November 2008, 49 patients with MDS (n = 24), CMML (n = 8) and sAML (n = 17) and a median age of 61 years (r: 26 – 73) and a median number of 13% blasts were included. Stem cell source were unrelated (n = 43) or related donor (n = 6). Results: No graft failure was observed and the median time to leukocyte engraftment (> 1.0 × 109 /l) was only 10 days (r: 7 – 32). The incidence of acute graft-versus-host disease grade II to IV was 39 % and of grade III / IV was 14 %. Chronic GvHD was noted in 57 % of the patients, which was limited in 35 % and extensive in 32 % of the patients. After a median follow-up of 15 months (r: 3 – 35) the two-years estimated disease-free and overall survival was 49 % (95 % CI 33 – 65 %) and 54 % (95 % CI 39 – 69 %), respectively. The 1 year cumulative incidence of treatment-related mortality was 29 % (95 % CI 15 – 43 %). The 2 year cumulative incidence of relapse was 18 % (95 % CI 6 – 30 %). Patients with fully matched related or unrelated donor had a better survival than patients transplanted from mismatched donor (69 % vs. 37 %; p=0.06). Conclusions: A sequential approach using anthracycline based induction chemotherapy followed immediately by a busulfan based reduced conditioning regimen and allogeneic stem cell transplantation from related and unrelated donors resulted in a fast engraftment and a relative low risk of relapse in elderly patients with advanced MDS or sAML. To lower the therapy related mortality a careful donor selection is mandatory. Disclosures: No relevant conflicts of interest to declare.

For patients lacking an HLA-identical sibling, more than ten million unrelated donor (UD) are available for donation of hematopoietic stem cells. Despite this increasing number of UD, up to 30 % of the patients will not find a completely-HLA-matched donor. HLAmismatched unrelated stem cell transplantation (SCT) is associated with an increased risk of acute and chronic graft-versus-host disease and a higher transplant-related mortality resulting in a lower survival after allogeneic SCT. We analyzed on outcome of allele- and antigen-mismatch SCT from UD using pre-transplantation anti-thymocyte globulin (ATGFresenius ®) at a median dose of 60 mg/kg as part of the conditioning regimen. Between 08/1996 and 12/2004, 369 patients received SCT from UD in our institution. In 268, complete molecular typing (4-digit) of HLA-A, -B, -C, and DRB1, and DQB1 was available for donor as well as for recipient. According to high-resolution HLA-typing, 110 of the patients were completely matched for 10 alleles, whereas 91 patients had at least one allele-mismatch (9/10), 67 patients were mismatched for 2–4 alleles (6–8/10). The median age of patients was 41 years (range, 1–68). Diagnoses were acute leukemia or MDS in 135, chronic myeloproliferative disease (CML or PMF) (n=53), lymphoid malignancy (CLL, NHL, Hodgkin’s disease, MM) (n=60), or others (n=20). Stem cell source was either bone marrow (n=103) or peripheral blood stem cells (n=165). The matched and the mismatched group did not differ significantly between age, sex, risk category, graft source, CMV-status, conditioning, or CD34+ transplanted cells. Two patients (1 %) experienced primary graft-failure and were transplanted from 10/10-matched donors while in none of the mismatch-transplanted patients primary graftfailure occurred. The incidence of grade II–IV acute GvHD was 33 % in the 10/10-matched group, 41 % in the 9/10-mismatch-group, and 40 % in the 6–8/10-mismatch-group (p=0.1). The overall rate of chronic GvHD was 42 % in the 10/10-matched group, 46 % in the 9/10-mismatch group, and 46 % in the 6–8/10-mismatch group (p=0.8). Non-relapse mortality (NRM) in the 10/10-matched group was 27 %, in the 9/10-mismatch group 31 %, and in the 6–8/10-mismatch group 32 % (p=0.2). In a multivariate analysis, only age as continuous variable (HR 1.023) (p

Increasing numbers of patients with Non Hodgkin Lymphoma receive allogeneic stem cell transplantation (SCT) due to progressive disease or relapse after standard therapy protocols or after autologous stem cell transplantation. However, depending on the entity, variable proportions of lymphoma patients develop relapse after SCT. For the earliest detection of relapse with respect to immunotherapeutic approaches, monitoring of chimerism provides an option, but most studies were performed in patients with acute leukemias. This retrospective single-center analysis evaluated the performance of chimerism as assessed either by restriction length fragment polymorphisms (RFLP) or by quantitiative real-time PCR for nine different genetic polymorphisms (Alizadeh et al., 2001) and for Y-specific sequences (Fehse et al., 2001) in thirteen patients with mantle cell lymphoma after SCT. Allo-SCT was performed from related and unrelated donors (Jan 2000–Dec 2006) at the Interdisciplinary Clinic for Stem Cell Transplantation of the University of Hamburg, Germany. Thirteen patients received fourteen allo-SCTs (in one case due to relapse after allogeneic SCT being followed by a second SCT from a second donor). One patient received a stem cell boost after graft failure following SCT. There were eleven males and two females (median age: 52 years; range: 36–62 years). Four patients had Ann-Arbor Stage IIIA, eight had stage IVA–B (in one case the previous stage was unknown). Before allo-SCT, three cases were in complete remission, six in partial remission, two had progressive disease, and one case showed stable disease (data missing in one case). Four allo-SCTs were from related donors, nine from unrelated donors. Donors and recipients showed HLA-match in eleven transplantations and mismatch in two SCTs. At the time of this report, six patients were alive and in complete remission (6/13; 46%), four died due to transplant related mortality (TRM) (31%), and three due to relapse/progression of the lymphoma (23%). Median overall survival (OS) was 460 days (range 53–1380 days); median disease free survival (DFS) was 210 days (range: 49–1380 days). After SCT, all thirteen stem cell recipients achieved complete donor chimerism (being defined by >99.9% of donor cells). In eleven cases chimerism was stable, in seven cases in association to stable long-time remissions with a median of 145 days (range 36–1380 days). Five patients developed relapse with a median interval of 132 days after SCT (range 49–924 days), and one further patient achieved partial remission only. Four relapse cases maintained complete donor chimerism; only one of the five relapse patients had a significant decrease of donor chimerism on day +38 from 100% to 70%, while relapse was diagnosed on day +49. The patient with partial remission had complete loss of donor chimerism on day +171. This study points to the conclusion that measurement of chimerism as single diagnostic method in patients with NHL who undergo allogeneic SCT might not be as effective for early detection of relapse when compared to other hematological malignancies such as acute leukemias. Although the limited sample size of this study has to be considered, it should be investigated whether the combination of chimerism analyses with minimal residual disease detection as the specific reciprocal rearrangements e.g. BCL1-IgH in MCL might improve the early detection of relapse after SCT.

Introduction: In acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS), relapse after allogeneic stem cell transplantation (SCT) occurs with frequencies of 30–80% in dependence on risk profiles. Selected patients are offered a 2. allo-SCT. As the number of studies is limited, we here performed retrospective analysis of 20 consecutive MDS and AML pts who received at least 2 allografts from related/unrelated donors from 2000–2007. Patients: There were 4 children and 16 adults who received ≥2 allo-SCTs (10 males, 10 females; 2–63 years, median 38 yy). 14 pts had de novo AML, 3 pts s-AML, 2 MDS RAEB-1; one child had juvenile myelomonocytic leukemia (JMML). 19 pts received two allo-SCTs-18 due to another relapse after the 1. allo-SCT, one due to secondary graft failure after 1. SCT. One pt with MDS had 3 allo-SCTs due to relapses. 13/20 pts received treatment before the 2. allo-SCT, but only 6/20 achieved complete remission (CR). 10/20 pts had unfavorable cytogenetics. The median interval between the 1. and 2. allo-SCTs was 20 months (range 2–47 mo). Median disease free survival (DFS) after the 1. SCT was 16 months (range 3–45 mo). Characterization of 2. allo-SCTs: 4/20 second allo-SCTs were performed from related donors (3 HLA-identical, one haploidentical). 16 SCTs were performed from unrelated donors (HLA matched: n= 8, -mismatched: n=8). In 10 cases, the 2. allo-SCTs was performed from different, in 10 cases from the same donor. Reduced intensity conditioning was applied to 16 pts, myeloablative to 4 pts. Antithymocyte globuline was administered to 12/20 pts. One pt received bone marrow, 19 pts peripheral blood stem cells. Immunosuppression was performed in most pts by cyclosporine A plus short-course MTX or mycophenolate mofetil. Outcomes: Leukocyte engraftment was achieved in 16/20 cases with a median interval of 13 days (r. 10–24 d). 3 pts died prior to engraftment. One pt had secondary graft failure being followed by a 2. allo-SCT. CR was achieved by 16/20 pts. At the time of this report, 5/20 pts are still alive after a median follow-up of 15 months (r. 2–36 mo), three of those in CR with follow-ups of 7, 22, and 36 months. 9/20 pts developed relapse (45%), of whom 2 received donor lymphocytes. Median DFS after the 2. SCT was 7 months (range

Lenalidomide is an immunmodulatory drug, orally administered once daily, which is effective in relapsed multiple myeloma (MM). Here, we evaluated the efficacy and toxicity of lenalidomide in 24 MM patients with relapsed disease after allogeneic stem cell transplantation (allo-SCT). Median age was 59 (range, 37–70) years. The series included 8 females and 16 males. Prior to allo-SCT, all patients were heavily pretreated with a median of 6 chemotherapy cycles including autologous and allo-SCT in all patients. Also, prior to lenalidomide, salvage treatment included donor lymphocyte infusions (DLI) in 18 pts,, thalidomide in 11 pts and bortezomib in 13 pts. Lenalidomide was given at 15mg (n=4) or 25 mg (n=20) orally once daily on day 1–21 every 28 days and in 20 patients in combination with dexamethason.. No prophylactic anticoagulation was used. The median number of completed cycles was 5 (range 2–17). Myelotoxicity according NCI criteria was the primarily and major encountered side effect (leukopenia: 4% grade 4, 21% grade 3, 17% grade 2, thrombopenia: 17% grade 3, 29% grade 2) and led to dose reduction in 54% of the patients. Infectious complications were observed in 50%. Non-hematological toxicity consisted of cramps (n=9), fatique (n=5) and constipation (n=2). Thrombembolic complications (cerebral infarction) were observed in one patient, who received concomitant corticosteroid treatment for acute graft-vs.-host disease (GvHD), but neurological symptoms resolved completely. GvHD of the skin under lenalidomide treatment was seen in 3 patients (one grade 2, and 2 grade 1), with one case occurring shortly after an additional DLI.. Objective remission was achieved in 66% of the patients (CR: 8%, VGPR: 8%, PR: 50%) and stable disease (SD) in 13% of the patients, while in 21% progressive disease was noted. Prior treatment with thalidomide or with bortezomib did not influenced the rate of CR/PR. Surprisingly, patients with del 13q14 achieved a higher CR/PR rate than those without del 13q14 (p=0.02). The median time to progression was 9.7 months (95% CI: 7.5–11.9) and the median overall survival was 19.9 months (95% CI: 17.3–22.5). Lenalidomide is effective in relapsed patients with MM after allo-SCT. Major toxicity is myelotoxicity, which required dose reduction in the majority of patients. The optimal dose of lenalidomide after allo-SCT has to be investigated.

Dose-reduced conditioning followed by allogeneic stem cell transplantation (HSCT) is a potentially curative therapy in B non Hodgkin lymphomas (NHL). However, the number of patients who experienced an early relapse is significant, and the achievement of a prolonged second remission is usually a rare event. Posttransplant immunotherapy with DLI to induce graft versus lymphoma (GVL) effect is a reasonable option to prevent or treat relapse, however the risk of graft versus host disease (GvHD) is high. Since B-cells might serve as antigen presenting cells to induce GvHD, additional B-cell depleting therapy with monoclonal antibody (anti CD20) might reduce the risk of GvHD while inducing additional cytotoxicity to B-cells originating from B-cell lymphoma. In the current pilot study we investigated the feasibility of a combined rituximab (Rtx) plus DLI therapy in B-cell malignancies after allogeneic stem cell transplantation to prevent (n=10) or treat relapse (n=2). Twelve consecutive patients, 8 male and 4 female, affected by B cell malignancies and transplanted between July 2002 and February 2007, were included in this study. The median age at alloHSCT was 58 ys (range 27–64). The conditioning regimen consisted of melphalan (140 mg/m2), fludarabine (150 mg/m2) and ATG–Fresenius (30–60 mg/kg) (n=9) or busulfan (8–10 mg/kg), fludarabine (150 mg/kg) (n=3), donors were HLA identical sibling in the half of the group, with one HLA mismatched-unrelated donor. The source of stem cell was peripheral blood in all patients. Seven patients were not in disease remission at the time of HSCT. The indication of Rtx-DLI administration was prophylaxis of relapse in all but two patients. The schedule of this approach was: Rtx (375 mg/mq, in 4 consecutive weekly infusions), started at a median time of 194 dy from HSCT (range 77–895). DLI, in a single dose, was administered from a minimum of 1×105/Kg up to 5×106/Kg, after discontinuation of immuno-suppression, without signs of GvHD. Three patients developed acute GvHD after DLI, two with gastro-intestinal and one with cutaneous involvement, whereas 6/12 patients experienced chronic GvHD, limited in all but two. Two cases of bacterial and one case of Pneumocystis jirovecij pneumonia were diagnosed after the treatment, in 3 different patients. Two patients died in complete remission due to infectious complications. The median number of B-circulating lymphocytes before and after Rtx was 42/μL (range 0–477) and 4 (range 0–226) respectively, and the difference is not statistically significant in terms of incidence of infection in our group (χ2 test). After a median follow up of 21 mo (range 4–37) from the 1st Rtx infusion, 8/12 patients are alive at the last follow-up, six of them without disease, whereas 2 patients died due to progressive disease(table1). Rituximab and DLI post allogeneic HSCT seems to be a safe and effective approach to prevent early relapse in B malignancies in complete remission after allogeneic stem cell transplantation. Table 1 Pt no Indication of R-DLI Infection aGVHD cGVHD Relapse Last follow-up 1 prophylaxis pneumonia II (GI tract) n n alive in CR 2 prophylaxis n n n n alive in CR 3 prophylaxis n n n n alive in CR 4 prophylaxis n n n y alive with disease 5 prophylaxis n n lim n alive in CR 6 prophylaxis n n n n alive in CR 7 relapse n n lim y alive with disease 8 relapse n n n y dead with disease 9 prophylaxis PJP n ext n dead in CR 10 prophylaxis pneumonia n lim n dead in CR 11 prophylaxis n II (skin) lim y dead with disease 12 Prophylaxis N I (skin) ext n Alive in CR

Preclinical experiments have shown a strong anti-myeloma effect of polyclonal anti-thymo-cyte globulin (ATG). We evaluated ATG effect in a melphalan/fludarabine-based conditioning regimen in 138 multiple myeloma (MM) patients who underwent allogeneic stem cell transplantation with (n=79) or without (n=59) ATG. More patients in the group without ATG had experienced relapse to prior auto-transplant (63% vs. 47%, p = 0.08). Patient characteristics were otherwise well matched in both groups. A median ATG dose of 30 mg/kg BW (range, 10-90 mg/kg BW) was administered on days −3 to −1. GvHD prophylaxis was performed with cyclosporine A and short-course methotrexate. Overall response (93% vs. 78%, p=0.03) and complete response (59% vs. 39%, p=0.04) at day 100 were higher in the patients treated with ATG than in those without ATG. ATG led to a lower incidence of severe grade III/IV acute GvHD (11% vs. 22%, p=0.10), and chronic GvHD (23% vs. 65%, p 30 mg/kg BW. The rate of OR increased from 78% in the no-ATG group to 91% in patients who received ATG ≤ 30 mg/kg BW, and to 96% in patients who received ATG > 30 mg/kg BW (p=0.02). Separate analysis of patients transplanted from a sibling donor also showed a trend to better OR (86% vs. 78%) and CR (55% vs. 39%) for patients who received ATG (all patients received ATG at a dose ≤ 30 mg/kg) compared to the non-ATG group, however due to low numbers this difference did not reach statistical significance. Though the non-ATG group contained more patients who had relapsed to prior autografting, this as has been reported before, would not influence response rates. In the multivariate analysis of prognostic factors for achievement of a CR, ATG was the only significant prognostic factor (RR: 2.57; 95% CI: 1.17-5.64; P=0.02). The estimated overall survival (OS) at three years was 53% (95% CI: 40-66%) for the ATG group (p=0.46), and 43% (95% CI: 29-57%) for the non-ATG group (p=0.46), while progression-free survival (PFS) at three years was 39% (95% CI: 27-51%) for the ATG group, and 27% (95% CI: 14-40%), for the non-ATG group (p=0.55). The current results suggest a dose dependent beneficial effect of ATG in terms of myeloma cytotoxicity as well reduction of the incidence of chronic GvHD.

The major limitation of allogeneic stem cell transplantatiopn (SCT) in patients (pts) with myelofibrosis is the high treatment related mortality. We performed a prospective multicenter trial of a dose-reduced conditioning regimen, consisting of busulfan (10 mg/kg orally or 8mg/kg i.v), fludarabine (180 mg/m2) and anti-thymocyte globulin (ATG Fresenius: 30–60 mg/kg) followed by allogeneic SCT in pts with myelofibrosis. From 2002 to 2006, 104 pts with a median age of 55 years (range: 32–68) andlow risk with constitutional symptoms (18%) or intermediate risk (n= 58%) and high risk (n=19%) were included. Cytogenetic abnormalities and JAK2 mutation were noted in 22% and 48%, respectively. Bone marrow histology showed advanced fibrosis (MF 2 and 3) in all pts. All but 3 pts received peripheral blood stem cells as graft source either from related (n=33) or unrelated donor (n=71). All but one (1%) pts showed leukocyte and platelet engraftment after a median of 18 and 21 days, respectively. The median duration of leukocyte aplasia was 9 days (range: 3–21). Acute GvHD grade II to IV occurred in 19% and severe aGvHD III/IV in 7%, while chronic GvHD was seen in 32% of the pts. Non-relapse mortality at 1 year was 19% (95% CI: 11–27%) and significantly lower for pts younger than 50 years of age (0% vs 27%, p=0.004) and for pts with low risk vs intermediate/high risk disease (0% vs 27%, p= 0.02). The cumulative incidence of relapse at 3 years was 29% (95%CI: 15–43%). The 3 year overall (OS) and event-free survival (EFS) was 70% (95% CI 60–80%) and 55% (95% CI 42–68%). Significant factors for improved 3 year OS and EFS were age less than 50 years (92% vs 62%, p=0.003 and 79% vs 46%, p= 0,004) and low vs intermediate/high risk (100% vs 62%, p=0.01 and 72% vs 48%, p= 0.02), while no impact on survival was seen for cytogenetic abnormalities, JAK2 mutation status and donor (related vs unrelated). These prospective multicenter study show excellent outcome of an busulafan/fludarabine based reduced conditioning regimen followed by allogeneic SCT in pts with myelofibrosis.

Background: Lenalidomide is an immunmodulatory drug, orally administered once daily, which is effective in relapsed multiple myeloma (MM). Here, we evaluated the efficacy and toxicity of lenalidomide in 16 MM patients with relapsed disease after allogeneic stem cell transplantation (allo-SCT). Methods: Median age was 57 (range 37–68) years. The series included 5 females and 11 males. Prior to allo-SCT, all patients were heavily pretreated with multiple lines of chemotherapy, including high dose therapy with autologous stem cell support. Also, prior to lenalidomide treatment, other salvage therapies included donor lymphocyte infusions (DLI) in 93% of cases, thalidomide in 63%, and bortezomib in 88%. NCI-criteria were used to define toxicity. Lenalidomide was given 25 mg orally once daily on day 1–21 every 28 days. No prophylactic anticoagulation was used. Results: The overall median number of completed cycles was 5 (range 1–10). Very good partial response (VGPR) was achieved in 13%, partial remission (PR) in 53%, and stable disease (SD) in 27% of the patients. During the follow up period, disease progression was observed in 50% of cases, and death in one patient. The median progression-free survival was 8 months, while the median overall survival was not yet reached. Hematotoxicity was the primarily and major encountered side effect (leukopenia: 6% grade 4, 19% grade 3, 19% grade 2, 38% grade 1; thrombopenia: 19% grade 3, 19% grade 2, 31% grade 1). This myelotoxicity led to dose reduction (usually 10 mg) in 44% of the patients. Infectious complications were observed in 25%. Non-hematological toxicity was also seen in 75% of cases (56% grade 1, 19% grade 2), consisting of cramps in the leg, constipation, symptomatic fatigue, nausea and progressing polyneuropathy (n=1). Thrombembolic complications (cerebral infarction) were observed only in one patient, who was receiving concomitant corticosteroid treatment for acute graft-versus-host disease (GvHD), but neurological symptoms resolved completely. GvHD of the skin under lenalidomide treatment was seen in 25% of cases (one grade 2, and 3 grades 1), with one case occurring shortly after an additional DLI. Conclusions: Lenalidomide is effective in relapsed patients with MM after allo-SCT. Major toxicity is myelotoxicity, which required dose reduction in a majority of patients. With this background, a dose-finding study to determine the optimal lenalidomide dose as maintenance therapy after allo-SCT has already started.

Background: Aspergillosis is a lethal infection of immunocompromised patients that respond poorly to antifungal therapy. Caspofungin (CAS) or Posaconazole (POS) as salvage monotherapy for IA have response rates of 45% and 42% respectively. Preliminary results of in vitro studies, animal models and clinical studies suggest that combination therapy with azoles and echinocandins may have additive activity against Aspergillus species. Methods: Seven adult patients after unrelated haematopoietic stem cell transplantation, with proven, probable or possible IA (per EORTC/MSG criteria) and refractory to standard antifungal therapy received Caspofungin plus Posaconazole (standard monotherapy dosage). All patients had > 3 days of combination antifungal therapy. Underlying diseases were AML (n=5), Biphenotypic leukaemia (n=1), Mantle cell Lymphoma (n=1). Three patients were neutropenic (ANC Results: Success at end of combination therapy (EOCT) was seen in six patients (86%). One critically ill patient died (due to bacterial sepsis and severe acute GvHD) four days after first administration of combination therapy. One patient with complete radiographic response died 20 days after EOCT due to bacterial sepsis in relapse of AML. Combination therapy at 4 to 59 (mean 35.7) days was well tolerated. No patient discontinued combination therapy due to toxicity. Follow up from start of combination therapy was 4 to 204 (mean 79.8) days. Conclusion: Randomized trials are warranted to determine whether this combination should be used as primary therapy for aspergillosis.

The V617F-JAK2-mutation occurs in about 50 % of patients with myelofibrosis and is a reliable marker to monitor residual disease after allogeneic stem cell transplantation. The establishment of valid complete remission criteria for myelofibrosis especially after allogeneic stem cell transplantation remains a major issue. We developed a new, highly sensitive real-time PCR to monitor and quantify V617F-JAK2-positive cells after dose-reduced allogeneic stem cell transplantation. The mutated differs from the wild-type JAK2 allele by just one nucleotide exchange (G à T) leading to the valine to phenylalanine (V à F) transition. Using PrimerExpress® we designed a TaqMan® PCR where the reverse primer (RP) terminates at the (3′) nucleotide corresponding to this point mutation. Thus, this reverse primer should bind with higher affinity to the mutated than to the wild-type allele. To increase the specificity while conserving optimal sensitivity of the MRD-specific PCR we generated a set of primers shortened each time by one nucleotide at their 5’ end. In parallel, all those shortened primers were designed to contain an additional mutation at the third to last 3’ position. This allowed us to identify the reverse primer combining high specificity with so far not reported sensitivity(0.01 %). After 22 allogeneic stem cell transplantation procedures in 21 JAK2-positive patients with myelofibrosis, 78 % became PCR-negative. In 15 out of 17 patients (88 %), JAK2 remained negative after a median follow-up of 20 months. JAK2-negativity was achieved after a median of 89 days post allograft (range, 19 – 750 days). A significant inverse correlation was seen for JAK2 positivity and donor cell chimerism (r: −0.91, p

Purpose: The aim of our study was to evaluate the impact of genomic polymorphisms of methylene-tetrahydrofolate-reductase (MTHFR) and glutathione S-transferases (GST) on liver toxicities seen in patients receiving busulfan and cyclophoshamide conditioning followed by allogeneic HSCT. Background: Polymorphisms of methylene-tetrahydrofolate-reductase (MTHFR) have been shown to potentially influence toxicity (e.g. mucositis) in HSCT. Additionally, many chemotherapeutics that have been linked to veno-occlusive disease (VOD) and liver toxicity (e.g. Busulfan) are metabolised by glutathione S-transferases (GST). Little is known about the impact of gene polymorphisms of MTHFR and various GSTs on VOD and liver toxicity in allogeneic HSCT. Methods: 84 adult patients were enrolled in this retrospective study. The majority of the patients had chronic myelogenous leukemia (CML) in 1st chronic phase (94%). 2 patients suffered from acute myelogenous leukemia (AML-M4), 2 from myelodysplastic syndrome (MDS) and 1 patients from Farber’s disease. DNA was isolated from leucocytes of stem cell recipients collected prior to HSCT. Common gene polymorphisms of glutathione S-transferases (GSTP1-Ile105Val, GSTA1*A/B, GSTM1, GSTT1) and methylene-tetrahydofolate-reductase (MTHFR-C677T, MTHFR-A1298C) were detected by PCR-RFLP. All patients were treated with busulfan/cyclophosphamide as conditioning regimen and received cyclosporine and short-course MTX for GvHD prophylaxis. 67 patients (80%) received additionally anti-thymocyte globulin (ATG). Assessment of liver toxicities was based on appearance of VOD according to the Baltimore criteria, maximum bilirubin levels within 20 days after HSCT and duration of hyperbilirubinaemia > 2 mg/dl. Results: Analyses revealed statistical significant association between the MTHFR-A1298C polymorphism and liver toxicities. There was significant higher incidence of VOD in patients that are homozygous for the C allele (p = 0,04). Independently of VOD, patients carrying the MTHFR-1298CC genotype demonstrated higher median maximum levels of bilirubin with 6.8 mg/dl than MTHFR-1298AC and MTHFR-1298AA patients (3,0 mg/dl and 3,4 mg/dl) (p = 0,004). Furthermore, elevation of bilirubin levels > 2 mg/dl lasted longer in MTHFR-1298CC patients with a median of 14 days compared to 3 and 6 days in patients harbouring the MTHFR-1298AC and MTHFR-1298AA genotype (p = 0,004). Interestingly, the common MTHFR-C677T polymorphism did not reach statistical significance in accordance to liver toxicity in our study population. No significant associations between GSTP1-Ile105Val, GSTA1*A/B, GSTM1 and GSTT1 polymorphisms and liver toxicity were found in this study. Conclusion: Our data suggest that the MTHFR-A1298C polymorphism is associated with VOD and hyperbilirubinaemia in patients treated with busulfan/cyclophosphamide as conditioning regimen followed by allogeneic HSCT. To the best of our knowledge this the first report linking the MTHFR-A1298C polymorphism to liver toxicity in patients receiving allogeneic HSCT.

To investigate lineage-specific chimerism of plasma cells after allogeneic transplantation by real-time PCR based on bi-allelic single-nucleotide polymorphism (SNP) or, in case of female-to-male transplantation, on the detection of the DFFRY-gene, 48 samples from bone marrow samples and peripheral blood from 34 non-myeloma patients were analyzed at different times after transplantation. Plasma cells and T-cells were obtained with CD138- or CD3-microbeads and MiniMACS columns. The median chimerism for T-cells at day +100 was > 99.9 % and remained stable on day +180 and one year after transplantation. In contrast, the median donor-plasma-cell chimerism at day +100 was 95.5 %, at day +180 98,6 %, at day +360 99.8 %, and two or more years after transplantation > 99.9 %. There was a trend for more patients with full donor-plasma-cell chimerism at day 180 who received standard conditioning in comparison to reduced-intensity conditioning (56 % vs. 28 %, p=0.3) and who experienced acute graft-versus-host disease (43 % vs. 18 %, p=0.2). To evaluate whether the quantitative measurement of donor-plasma-cell chimerism after allogeneic stem cell transplantation for patients with multiple myeloma might be a useful tool to determine minimal residual disease, we investigated 48 samples from 21 myeloma patients at different times after allogeneic stem cell transplantation, and we compared it with immunofixation and, in some cases, with molecular methods by using patient-specific primers. All patients with molecular remission measured by patient-specific primers had a plasma-cell chimerism of more than 99.5 %. Furthermore, in all but one patient plasma-cell chimerism of more than 99.3 % was associated with a negative immunofixation. In six patients with positive immunofixation after allogeneic stem cell transplantation treatment with bortezomib or donor-lymphocyte infusion was started and monitored by plasma-cell chimerism and immunofixation. All patients experienced an increase of donor-plasma-cell chimerism with negative immunofixation if chimerism exceeded > 99.3 %. Dilution experiments with the TIB-196 cell line showed sensitivity of 104 using real-time PCR and SNPs, 105 in case of Y-PCR and 105 for nested PCR with patient-specific primers. We conclude that plasma-cell chimerism after allogeneic stem cell transplantation is delayed in comparison to T-cell chimerism, suggesting a reduced graft versus hematopoiesis effect on plasma cells. Quantitative measurement of plasma cells after allogeneic stem cell transplantation with highly sensitive real-time PCR allows monitoring of residual host-tumor cells in patients with multiple myeloma and allows guiding adoptive immunotherapy strategies to enhance remission status and to prevent relapse.

Long time of aplasie after allogeneic Stem cell- or Bone marrow-transplantation increase the risk of infections complications. Studies showed the positive effect of daily filgastrim to reduce the duration of aplastic time. The use of the Pegfilgastrim promise a simple handle with a single doses after transplantation. Between December 2004 and July 2005 we observed 19 patients with different diseases after SCT or BMT. All patients received a single doses subcutaneous injection of 6 mg pegfilgastrim on day 5 after transplantation. The median leucocytes engraftment was on day 16 after transplantation. The incidence of neutropenic infections was 84 percent with fever unknown origin, 21 percent with catheter induced infections and 10 percent with fungal pneumonia. Side effects of the cytokines had not been observed. The median hospitalisation was 33 days. In compared with the daily doses of Filgastrim 5 mg/kg/day was the duration to leucocytes engraftment and the infections profile similar. The use of the single injection doses of 6 mg pegfilgastrim shows a safe and comfortable profile for patients after allogenic transplantation. | Age | 17 – 61 years, median 47 years | || | Sex | 11 male, 8 female | | Diseases | 8 acute myeloid leukaemia, 1 acute lymphatic leukaemia, 1 chronic myeloid leukaemia, 1 Thalassaemia, 1 non hodgkin lymphoma, 1 osteomyelofibrosis, 5 multiple myeloma, 1 myelodysplastic syndrom | | Conditioning regime | 7 normal conditioning, 12 reduce intensity conditioning | | Type of transplantation | 12 MUD-PBSCT, 5 MMUD-PBSCT, 2 MUD-BMT | | CD34+ count | median 7,9 x 10 6 /kg KG | | Engraftment of leucocytes | median 16 days (10–25 days) | | Infection disease | 16 FUO (84%), 2 fungal-pneumonia (10%), 4 sepsis of catheter (21%) | | Duration of hospitalisation | median 33 days (17 – 47 days)

We investigated the toxicity and efficacy of early administration of bortezomib in 14 patients with residual disease (n=9) or complete remission (immunofixation-negative) (n=5) after a median of 383 days (range, 47–1317) after melphalan/fludarabine-based dose-reduced allogeneic SCT for multiple myeloma. The median age was 49 years (range, 32–62), and stem cell graft was from HLA-identical siblings (n=2) or unrelated donors (n=12). Three patients had received prior Thalidomide and donor lymphocyte infusion, and 2 of them still received low dose thalidomide (50 mg) during bortezomib treatment. The study consisted of two cycles bortezomib (1.3 mg/m2) given intravenously on day 1, 4, 8 and 14 (q21d). In case of response and low toxicity, two further cycles were applied. Eleven patients completed two cycles, and 4 patients completed four cycles. Two patients stopped after three cycles due to side-effects; 3 patients could not complete the proposed two cycles and stopped either after the first injection (n=1) of the first cycle, or after the second (n=1) or third (n=1) injection of the second cycle due to neurotoxicity. Major toxicity was thrombocytopenia grade IV (> 75 % reduction) which was seen in 6 patients (43 %), but none required platelet support. Neutropenia grade IV was seen in 2 patients (14 %), neurotoxicity grade III and IV was observed in 3 patients (21 %) while mild grade I-II neuropathy was seen in 10 patients (71 %). Other toxicity was fatigue grade I (85 %), nausea grade I (50 %), diarrhea grade I-II (93 %). Two patients with acute or chronic GvHD of the skin experienced deterioration of the manifestation, which could be treated succesfully with corticosteroids. A significant decrease during bortezomib-therapy was observed for CD3+ cells (p=0.02), while no effect was seen for B-cells (CD19+) (p=0.4). Two patients who received bortezomib on day 125 and 201 after allogeneic SCT experienced Herpes zoster infection during and 2 weeks after bortezomib treatment, respectively. All 9 patients with persistent disease showed further regression of the paraprotein including negative immunofixation in 5 patients. We conclude that early bortezomib after allogeneic SCT for patients with multiple myeloma is active and can induce further regression of the paraprotein. The toxicity is considerable especially if given early after allo SCT.

We analyzed the outcome of 28 patients who were treated within prospective treatment protocols to investigate the feasibility of unrelated stem cell transplantation for patients with haematological malignancies within the seventh decade of life. Twenty-eight patients with a median age of 62 years (range 60–70) were enrolled. Twenty-six received a dose-reduced conditioning regimen while two patients were transplanted after standard conditioning regimen, and eight of the patients had received at least one prior high-dose chemotherapy followed by autologous or allogeneic stem cell transplantation. Diagnoses were leukaemia (AML: n=10; ALL: n=1; CML: n=2), MDS (n=5), myelofibrosis (n=3), multiple myeloma (n=6) or non-Hodgkin’s lymphoma (n=1). No primary graft-failure was observed, and the median number of days to leucocyte and platelet engraftment was 18 days and 23 days, respectively. Acute GvHD grade II–IV was seen in 35% of the patients, chronic GvHD was seen in 56% of the patients. The one-year cumulative incidence of treatment-related mortality was 25%. The four-year estimated overall- and disease-free survival was 49% and 40%, respectively. Unrelated stem cell transplantation in patients within the seventh decade of life is a feasible treatment option and may induce long-term disease-free survival.

Introduction of a “suicide gene” into donor T-lymphocytes is a promising strategy to prevent severe graft-versus-host disease (GvHD) in the setting of allogeneic blood stem cell transplantation (B-SCT). We initiated a clinical phase I/II-study combining CD34-enriched peripheral B-SCT and Herpes simplex virus thymidine kinase- (HSV-tk) transduced donor T-cell infusions at days 0 and (optional) 60. Before our study was put on hold (after the leukemia cases in the French X-SCID trial) three patients were treated within the protocol each receiving >4x106/kg highly enriched CD34+ and appr. 5x106/kg suicide gene-modified (SGM) donor T-cells. All patients showed fast donor cell engraftment. Patient 2 (without signs of GvHD) received two SGM T-cell infusions and is well >2.5 years thereafter. Patient 1, showed an early (days 11–13) strong increase of SGM cell-counts peaking at 7.2x107/l, but did not develop GvHD. Unexpectedly, his blood was completely cleared from SGM T-lymphocytes a few days later. This patient received a second dose of SGM T-cells (day 65), which vanished within 2 days strongly indicative of their immune rejection. Indeed, MLR data confirmed anti-SGM reactivity of this patient’s PBL possibly related to an HSV reactivation during transplantation. Patient 3 showed a similar early sharp rise in SGM T-cells (days 14–17), in his case associated with acute skin GvHD grade II. Treatment with ganciclovir led to complete resolution of GvHD and rapid disappearance of SGM donor T-cells. Importantly, both patients 1 and 3 developed secondary graft failure at days 156 and 119. In summary, we confirmed previous reports that the use of SGM T-lymphocytes may allow control of acute GVHD. At the same time, we made the worrying observation that early total in vivo depletion of SGM donor T-cells may be associated with an increased risk of transplant rejection. This suggests that minimum numbers of donor CD3+ cells are required post-transplant not only to facilitate engraftment, but also to prevent late rejection.

Allogeneic stem-cell transplantation (SCT) is a potentially curative approach for patients (pts) with hematologic malignancies. However, it is associated with a high risk of treatment-related complications. Risks are significantly increased with advanced age, concurrent medical problems and with unrelated donors, therefore most centers limit unrelated-donor SCT to pts younger than 50–55 years. Moreover, hematological malignancies are more common and have a worse prognosis in the elderly, thus many pts who could benefit from SCT were often deferred from this potentially curative approach. Reduced-intensity conditioning allowed extension of allogeneic SCT to a wider pt population including the elderly, however there is only limited data on the feasibility and outcome of unrelated-donor SCT in elderly pts over age 55 years. In this study we report our experience with 40 pts over age 55 having unrelated-donor SCT following reduced-intensity conditioning. The median age was 58 years (range, 55–66). Eleven pts were over age 60 years. Diagnoses included AML (n=19; 7 CR1, 9 CR2, 3 refractory; 12 secondary to MDS or prior chemotherapy), MDS (n=4), myelofibrosis (n=2), CML (n=3), multiple myloma (n=8), non-Hodgkin’s lymphoma (n=4). The preparative regimen consisted of fludarabine combined with oral busulfan (8 mg/kg, n=8), intravenous busulfan (busulfex, 6.4 mg/kg, n=15), treosulfan (30 g/m2, n=5) or melphalan (100–150 mg/m2, n=12) and serotherapy, either ATG (n=36) or alemtuzumab (n=4). Thirty-six pts engrafted with a median of 14 days. Four pts died prior to engraftment. With a median follow-up of 14 months (range, 1– 54), 21 pts are alive and 19 have died, 13 of treatment-related causes and 6 of relapse. The probabilities of overall and disease-free survival at 1-year after SCT were 46% (95 CI, 28–63%) and 40% (95 CI, 23–57%), respectively. The cumulative incidence of non-relapse mortality (NRM) and relapse at 1 year were 35% and 25%, respectively. Acute GVHD grade II–IV and chronic GVHD occurred in 36% and 45%, respectively. The status of disease at SCT and the conditioning regimen used were the most significant predictors of outcome. Pts with chemosensitive or untreated malignancy had an OS of 52% whereas pts with refractory malignancy had an OS of 16% (p=0.05). Pts conditioned with fludarabine and busulfex or treosulfan had an OS of 57% compared with 35% in pts conditioned with fludarabine and melphalan or oral busulfan due to increased NRM with the later regimens (p=0.04). Multivariable analysis confirmed the independent impact of these factors with hazard ratios for decreases OS of 3.1 and 3.2, respectively. When a more homogenous subgroup of 23 pts with AML or MDS was analyzed, the 1-yaer OS and DFS were 49% (95 C.I. 24–75%) and 43% (95 C.I. 18–68%), respectively. NRM was 24% (95 C.I. 11–54%). In conclusion, unrelated-donor SCT is feasible in elderly pts, with outcomes that are similar to younger pts. Favorable outcome was observed in pts with myeloid malignancies, and those transplanted in remission and early in the course of disease. The newer regimens containing intravenous busulfan or treosulfan were less toxic translating into better outcome. Age alone should no longer be considered a contraindication to unrelated-donor SCT.

To improve anti-myeloma effect of donor lymphocyte infusion (DLI) after allogeneic stem cell transplantation in multiple myeloma, we investigated in a phase I/II study the effect of low dose thalidomide (100 mg) followed by DLI in 18 patients with progressive disease or residual disease and prior ineffective DLI after allografting. The median age was 53 years (range 31–64). Twelve patients received prior DLI and either relapsed again (n=1), or showed no response (PD: n=3; SD: n=8). The overall response rate was 67% including 22% complete remission. Major toxicity of thalidomide was weakness grade I/II (68%) and peripheral neuropathy grade I/II (28%). Only 2 patients experienced mild grade I acute graft versus host disease (aGvHD) of the skin, while no grade II-IV aGvHD was seen. De novo limited chronic GvHD (cGvHD) was seen in two patients (11%). The median time to response was 108 days (range, 36–266 days). In 5 patients not responding to 100 mg, the dose of thalidomide was increased (200 mg n=4; 300 mg n=1) and subsequently two of them responded with partial remission. In three patients dose escalation of DLI was performed, resulting in one minor and one partial remission. No difference regarding response was observed between unrelated and related donors (66% each). Five out of 6 patients with deletion 13 responded, while 5 out of 9 patients without deletion 13 responded to thalidomide plus DLI. In patients who failed to respond to prior DLI, the disease status could be converted in 6 patients with SD after first DLI into CR (n=2), PR (n=3), MR (n=1) while two with SD remainded SD after thalidomide plus DLI. In three patients with progressive disease after DLI the combination of thalidomide and DLI resulted in one CR, one PR and one minor remission. The two-years estimated overall and progression-free survival was 100 % and 84%, respectively. Adoptive immunotherapy with low dose Thalidomide and DLI induces strong anti-myeloma effect with low incidence of graft versus host disease.

We compared anti-thymocyte globulin (ATG-Fresenius median dose 60 mg/kg: n= 48) with alemtuzumab (Campath-1H 100mg: n=25) in 73 patients with multiple myeloma, who underwent dose-reduced conditioning with melphalan and fludarabine, followed by allogeneic stem cell transplantation from matched (n=63) or mismatched (n=10) unrelated donors. Patients of the ATG group had higher age (median 50 vs 47 years, p=0.05), more prior high-dose chemotherapies (p

Allogeneic stem cell transplantation (SCT) is a potentially curative treatment of otherwise incurable malignant hematological disorders and severe immunodeficiency diseases. The post-SCT immunoincompetence can last for up to several years and might cause significant morbidity and mortality. Immune reconstitution after SCT has been studied extensively in adults, but only a few reports have been published on a paediatric population or have compared paediatric and adult population. This study was carried out in order to determine the influence of age on quantitative reconstitution of peripheral blood lymphoid subsets. A cohort of 69 adult and 109 paediatric patients received allogeneic SCT from related or unrelated fully HLA matched or 1 locus -mismatched donors for different malignant and non-malignant diseases. Analysis of lymphocyte subsets was performed at various time points: until day 30, between days 31–60, 61–90, 91–180, 181–365, between 1–2 years, between 2–5 years and more than 5 years post transplant (at the last time point only paediatric patients were analysed). Comparing the paediatric with adult patients, we observed statistically significant faster recovery of CD19+, CD19+CD5+, CD4+CD45RA+ cells during the whole period of observation, and of total lymphocyte count (TLC) (< 60d and 1-2y), CD3+ (< 60d and 90d-2y), CD3+HLA-DR+ (31-60d), CD3+CD4+ (< 60d and > 90d), CD4+CD45R0+ (< 60d), CD3+CD8+ (< 60d and 90d - 2 y) and CD56+CD3−cells (< 31d) in the paediatric group. Interestingly, the recovery of CD56+CD3− cells was faster from day 61-365 in the adult group (significant faster on day 91–180). The paediatric patients were divided in two subgroups: 1) aged < 10 years; 2) aged > 10 years. The following lymphocyte subsets recovered significant faster in the younger group: TLC (31-60d, 91-365d and > 2y), CD3+ (91-180d and > 1y), CD3+HLA-DR+ (31-60d), CD3+CD4+ (31-60d and > day 90), CD4+CD45RA+ (61-90d and > day 180), CD4+CD45R0+ (91-180d and 2-5y), CD3+CD8+ (91-180d and > 2y), CD19+ (31-60d, 91-180d and 2-5y), CD19+CD5+ (31-60d, 181-365d and 2-5y) and CD56+CD3− cells (61-180d). The thymopoietic pathway of T helper cells is important throughout childhood; however, the thymus has a diminished capacity for the regeneration of these cells in adulthood. This can be the explanation of the faster recovery of T helper cells in the younger patients in this study. Besides, we found, that paediatric patients have a faster reconstitution of B cells and cytotoxic T cells. This might be because of the higher number of stem cells in the paediatric transplantation.

We evaluated in a prospective pilot- study the effect of reduced intensity conditioning with busulfan (10 mg/kg), fludarabine (150 mg/m²) and anti-thymocyte globulin followed by allogeneic stem cell transplantation from related and unrelated donors in 14 patients with myelofibrosis. Study objectives were engraftment, chimerism, treatment- related mortality (TRM) and response. The median age of the patients was 51 (range, 32 – 63) years. According the Lille score there were low risk (n=4), intermediare risk (n= 7) and high risk (n=3) The median time until leukocyte (> 1.0 x 109/l) and platelet (> 20 x 109/l) engraftment was 16 (range, 11 – 26) days and 26 (range, 9 – 139) days, respectively. No graft failure occurred. Complete donor chimerism on day 100 was seen in 13 patients (93%). Acute graft-versus host disease (GvHD) grade II-IV occurred in 50% and grade III/IV 29 % of the patients. The incidence of chronic GvHD was 54 %. Two patients died due to treatment complications, resulting in a TRM at one year of 15% (95% CI: 0–35%). Hematological response after allogeneic transplantation was seen in all patients and complete histopathological remission was observed in 90%of patients. After a median follow-up of 13 (range, 3 – 48) months, the 3-years estimated overall and disease-free survival is 85 % (95 % CI: 65–100 %).