ALBERT

Description: Purpose In patients with relapsed acute myeloid leukemia (AML) 〉 60 years of age we analyzed age at relapse, interval from first complete remission (CR1) to relapse, cytogenetic risk at initial diagnosis, prior allogeneic stem cell transplantation (alloSCT) and FLT3/NPM1 mutational status as possible prognostic factors for overall survival (OS). Introduction After achieving CR1 more than 50% of elderly AML patients eventually relapse. Prognostic factors for OS are poorly defined in this patient population. For younger patients with relapsed AML a risk score has been described including age at relapse, interval from CR1 to relapse, cytogenetic risk at initial diagnosis and prior stem cell transplantation (SCT) as prognostic factors. We sought to investigate whether these are also prognostic factors in elderly patients with relapsed AML. In addition, we assessed the prognostic impact of FLT3- and NPM1 mutational status (wild-type (wt) or mutated (mut)) at diagnosis. Patients and methods In the ongoing multicenter OSHO trial #69 for AML patients 〉 60 years we evaluated data of all relapsed patients. Overall survival was calculated from the day of first relapse until the day of death using the Kaplan Meier method. Univariate analysis was performed to test for the influence of age at relapse, interval from CR1 to relapse, cytogenetic risk at initial diagnosis, prior alloSCT and FLT3/NPM1 mutational status. Subsequently, independent prognostic factors were defined in a multivariate analysis with age at relapse, time from CR1 to relapse, cytogenetic risk at initial diagnosis and prior alloSCT as covariates. Results From April 2005 until April 2013 904 patients were registered. 733 of these received intensive induction chemotherapy which resulted in CR1 in 447 (61%) pts. In this patient group 260 relapses were observed after a median interval, calculated from the day of CR1, for living patients of 2.7 years (range 0.1 to 7.5). Median age at relapse was 69 years (range 60 – 85) with 129 (49.6%) pts. being 60 to 68 years old, 102 (39.2%) pts. being 69 to 74 years old and 29 (11.1%) pts. being 75 to 85 years old. Median interval from CR1 to relapse was 0.58 years (0.07 – 6.28). 114 (43.8%) relapses occurred up to 6 months after CR1, 119 (45.8%) between 7 and 18 months after CR1 and 27 (10.4%) later than 18 months after CR1. Only five (1.9%) relapsed pts. showed good risk cytogenetics at diagnosis, whereas it was of intermediate risk in 159 (61.1%) pts., of poor risk in 68 (26.2%) pts. and unknown in 28 (10.8%) pts. Forty-one (15.8%) pts. had received prior alloSCT in CR1. Information on FLT3- and NPM1 mutational status at diagnosis was available in 194 (74.6%) pts. 110 (42.3%) pts. had FLT3/NPM1 wt/wt, 48 (18.5%) pts. had FLT3/NPM1 wt/mut, 23 (8.8%) pts. had FLT3/NPM1 mut/wt and 13 (5.0%) pts. had FLT3/NPM1 mut/mut. OS rate at 2 years of all relapsed pts. was 13 ± 2%. For patients younger than 69 years and for those 69 years of age or older OS rate at 2 years was 17 ± 4% and 9 ± 3%, respectively (p=0.03). The interval between CR1 and first relapse also affected 2 year-OS with 7 ± 3%, 15 ± 4% and 36 ± 12% for pts. with relapse up to 6 months, 7 to 18 months and later than 18 months after CR1, respectively ( 18 months: p=0.009). OS rate at 2 years was also influenced by cytogenetic risk at initial diagnosis with 17 ± 3% for pts. having good or intermediate risk cytogenetics and 3 ± 2% for those with poor risk cytogenetics (p〈 0.0005). Prior alloSCT had a negative influence on OS. Two-year OS rate was 10 ± 5 and 13 ± 3% (p= .015) for patients with prior alloSCT vs. those without prior alloSCT, respectively. FLT3/NPM1 mutational status at diagnosis had no impact on OS. In univariate analysis age at relapse (p

Description: Mitochondria are complex cell compartments characterized by a nuclear genome independent own genome referred to as mitochondrial genome (mtDNA). MtDNA encodes 13 proteins which are part of the five enzyme complexes of the mitochondrial respiratory chain. The respiratory chain is responsible for ATP synthesis and is the main source of reactive oxygen species (ROS) in the cell. During cellular aging, mutations in mtDNA accumulate leading potentially to respiratory chain deficiency. Due to the lack of DNA repair mechanisms within the mitochondrion itself, the mtDNA is especially vulnerable towards ROS. Until now it is not fully understood whether intracellular ROS levels increase with aging in all cell types, and whether this process is due to a potential impaired function of the respiratory chain. Thus, the influence of mtDNA mutations on oxidative stress and aging of hematopoietic cells remains to be investigated. Herein, we compared two conplastic mouse strains differing in one point mutation in the mtDNA affecting the respiratory chain. The C5BL/6Ntac-mtFVB/NJ (mtFVB) strain carries a point mutation at nt7778G/T leading to a D〉Y replacement in ATP8 protein, while the control stain C5BL/6Ntac-mtAKR (mtAKR) does not. The targeted protein is part of the F0sub unit of complex V in the respiratory chain and we hypothesize that the mutation affects the transport of protons within the complex V. In our study, we analyzed bone marrow cells of both strains at four different aging stages (3 to 24 months) for ROS and ATP levels by DCFH-fluorescence and luminescence staining, respectively. Additionally, the subpopulations of bone marrow cells were analyzed by flow cytometric immunophenotyping. Further, blood counts of the mice treated with a single dose of 5 Fluorouracil (5-FU, 150mg/kg BW, i.p.) were performed every 3 days during a total time span of 21 days. The mtAKR strain showed increasing levels of ROS ranging from 7.1 x 103 to 12 x 103 relative fluorescence units (RFU) and decreasing levels of ATP (from 0.99 to 0.36 µM) during the measurement time points 3 to 24 months. In comparison, the mtFVB strain showed a significant decrease in ROS levels ranging from 5.5 x 103 to 2.4 x 103 RFU and a significant increase of ATP levels (from 0.23 to 0.64 µM) during the same period. Hematopoietic cells of aged mtFVB mice (24 months) contained significantly more ATP than cells of mtAKR mice. Analysis of bone marrow cell composition of both strains showed an increase of hematopoietic stem cells (LSK cells: lineage-, sca-1+, c-kit+) during experimental time span from 3 to 24 months (mtAKR: from 2.13 to 2.81 % of lineage- cells, mtFVB: from 2.13 to 3.52 %). However, only in the mtFVB strain this increase was significant. Interestingly, the amount of LSK cells was significant higher in mtFVB compared to the mtAKR strain (mtFVB: 2.36 %, mtAKR: 1.23 %) at 6 months. Furthermore, erythroide cells (Ter119+) in mtAKR increased with aging (from 25.22 % to 32.69 %), while the mtFVB strain showed a decreased rate (from 25.88 % to 18.9 %). Blood counts of 3 months old mtFVB mice treated with 5-FU were similar to those of the mtAKR strain in the myelosuppression phase after application, but demonstrated higher regeneration peaks at day 15 in white blood cells (mtAKR: 20.24 x 103/µl, mtFVB: 25.72 x 103/µl), platelets (mtAKR: 2016 x 103/µl, mtFVB: 2848 x 103/µl), neutrophilic (mtAKR: 0.8 x 103/µl, mtFVB: 2.36 x 103/µl) and eosinophilic granulocytes (mtAKR: 0.28 x 103/µl, mtFVB: 0.76 x 103/µl) and lymphocytes (mtAKR: 17.12 x 103/µl, mtFVB: 21.84 x 103/µl). The mtDNA polymorphism in complex V of the respiratory chain in mtFVB strain influences the development of ROS and ATP levels in hematopoietic cells during aging, and seems to have a protective impact concerning ROS and ATP levels in aged mice. Furthermore, the capability of LSK cells to expand with age seems to be enhanced in mtFVB strain. Moreover, the regeneration capacity after 5-FU myelosuppression seems also to be increased in 3 months old mice. Disclosures: No relevant conflicts of interest to declare.

Description: Background: Viral infections contribute significantly to morbidity and mortality following allogeneic HSCT. Whereas incidences and risk factors for CMV infections are well studied data for other viral infections are more limited. Here, we present data on 202 alloHSCT recipients that received predominantly reduced intensity conditioning (RIC). Patients and Methods: Data of 202 consecutive patients (pts) who received HSCT during 1/1999 to 12/2006 were retrospectively analysed with regards to viral infections caused by adenovirus (AdV), herpes simplex virus (HSV), varicella zoster virus (VZV), human herpesvirus 6 (HHV6), epstein-barr virus (EBV) and BK-virus (BKV). Other factors included were gender, age, underlying disease, disease status, year of diagnosis, year of HSCT, time from diagnosis to HSCT, donor type, gender-matching between donor and recipient, stem cell source, conditioning-regimen, T-cell-depletion, prior autologous HSCT, acute and chronic GvHD, prior fungal infection, and viral serostatus of the recipients. Results: Median follow-up was 9.2mths (mean 21.7mths, range 8days to 95.3mths). Median pt. age was 45yrs (range 15 to 69yrs). Underlying diseases were ALL (n=20), AML (n=63), CLL (n=7), CML (n=32), MDS (n=21), MM (n=17), NHL (n=28), OMF (n=5), others (n=9). 137 pts received RIC most commonly based on treosulfan/fludarabine (n=112). 65 pts received myeloablative conditioning mainly based on 12Gy TBI containing regimen (n=48). GvHD-prophylaxis consisted of CSA/MTX (n=106), other CSA based regimen (n=83) or other (n=13). The overall infection incidences of AdV were 4.0%, of HSV 7.4%, of VZV 5.9%, of HHV6 8.9%, of EBV 4.5% and of BKV 5.4%. Median time of onset of AdV infections were 64 days after HSCT, of HSV 34 days, of VZV 542 days, of HHV6 97 days, of EBV 83 days, of BKV 50 days. Pts with AdV or HHV6 infections had a inferior survival compared to pts w/o the according viral infection (p=0.024 and p=0.048, respectively). 3/8 pts (37%) with AdV and 5/18 pts (28%) with HHV6 infections died within 30 days after infection diagnosis. Pts with EBV or BKV infections tended to have an inferior survival compared to pts w/o the according infection (p=0.084, p=0.152, respectively). 4/9 pts (44%) with EBV and 2/11 pts (18%) with BKV infection died within 30 days after infection diagnosis. Whereas pts with HSV infections had a similar survival compared to pts w/o infections, mean survival for pts with VZV infections was significantly better compared to pts w/o VZV infections (p=0.003). In multivariate analyses VZV infections remained to be associated with better survival (p=0.046, HR 0.132, CI 0.018–0.962). The other viral infections did not remain significant risk factors when analysed adjusted for competing risk factors. In order to determine the influence of late viral infections we performed an additional multivariate analyses solely including pts surviving beyond day 90. EBV infections (p=0.024, HR 2.807, CI 1.147–6.873) and HHV6 infections (p=0.013, HR 2.594, CI 1.227- 5.484) were significant risk factors for inferior survival besides unrelated donor HSCT (p=0.013), acute GvHD III/IV (p=0.001) and fungal infection prior to HSCT (p=0.005). Conclusion: Non-CMV viral infections are commonly observed following myeloablative and RIC alloHSCT. Since they are associated with significant mortality early broad viral screening seems advisable if virus infections are clinically suspected.

Description: Clinical trials on different cytarabine doses for treatment of AML provide evidence of a dose response effect, but also for increase toxicity after high dose AraC (HDAC). Pharmacokinetic measurements of cytarabine-triphosphate (AraC-CTP), which is the most relevant cytotoxic metabolite of AraC, have revealed its formation in leukemic cells to be saturated with infusion rates above 250 mg/m2/h, this being significantly lower than used in HDAC schedules. Methods: Based on a pharmacological model and encouraging results of a phase II study we conducted a prospective randomized multicenter clinical trial comparing the effects of two different application modes of AraC in patients up to 60 years with untreated newly diagnosed AML. Patients were randomized to receive AraC at two different infusion rates (IR) during induction and consolidation treatment: arm A/experimental: 1 × 2 g/m2/d AraC over 8 hours (IR 250 mg/m2/h) arm B/standard: 2 × 1 g/m2/d AraC over 3 hours (IR 333 mg/m2/h). Induction and first consolidation consisted of AraC (days 1, 3, 5, 7) in combination with an anthracycline (Idarubicine 12 mg/m2 or Mitoxantrone 10 mg/m2, days 1–3). The final dosage points (AraC day 7 and anthracycline day 3) were excluded from the second consolidation. The third consolidation consisted of either allogeneic or autologous stem cell transplantation or of chemotherapy identical to second consolidation. Results: From 02/97 to 04/02 419 patients were enrolled in the study. The present analysis is based on 361 eligible and evaluable patients with a median follow up of 7 years. CR was reached in 249/361 (69%; 95%CI: 65%–74%) patients. No statistically significant differences were detected between arms A and B with regard to CR-rate (69% vs 69%) or early death rate (11% vs 8%). Hematological recovery of median white blood cell count (WBC) 〉 109/l and median platelets (plt) 〉 50 × 109/l revealed no difference between arms A and B after induction (WBC day 22 vs 22, p=0,68; plt day 25 vs 26, p=0,41) and consolidation (WBC day 28 vs 27, p=0,07; plt day 42 vs 40, p= 0,58). The event free survival (EFS) after 5 years is 0,25 ± 0,03 % for all patients with an overall survival of 0,31 ± 0,03 % after 5 years. For the purposes of analysis, the 83 transplant patients (23 allogeneic MRD, 14 allogeneic MUD and 46 autologous) were censored at time of transplant. No statistically significant difference between arms A and B in regard to EFS (0,25 ± 0,04 vs 0,25 ± 0,04, p=0,99), relapse incidence (0,63 ± 0,06 vs 0,60 ± 0,06, p=0,89), overall survival (0,32 ± 0,04 vs 0,30 ± 0,04, p=0,44) and therapy associated mortality (0,18 ± 0,04 vs 0,17 ± 0,03, p=0,95) were detectable after adjustment of prognostic factors. An analysis of risk factors by multivariate cox regression model confirmed cytogenetics at diagnosis to be the most important risk factor for CR rate (p

Description: Abstract 3711 Introduction: Successful engraftment following transplantation of hematopoietic stem cells (HSCT) depends mainly on pre- and posttransplant immunosuppression, graft type and composition as well as on the HSC numbers infused. Whereas some of the aforementioned parameters can be influenced in the clinical setting, the latter one is more difficult to address. HSCTs of grafts with limited HSC numbers are accompanied by increased graft failure rates, longer cytopenias and increased morbidity. Current concepts to overcome low HSC numbers include the combination of two unrelated grafts, expansion techniques, modification of the graft composition or the site of graft infusion. In preliminary rodent studies intra-bone marrow (IBM) compared to intravenous (IV) HSCT led to faster engraftment which might be explained by closer location of the HSC to the stem cell niches. Aims: To investigate the feasibility and efficiency of IBM-HSCT following a non-myeloablative conditioning regimen in a dog-leukocyte antigen (DLA) identical canine HSCT model. Method: DLA-identical siblings were used as donor/recipient pairs for HSCTs. Recipients received a single dose of 2 Gy total body irradiation before HSCT (day 0). Pre- and postgrafting immunosuppression consisted of CSA (d-1 to d+35) and MMF (d0 to d+27). Two IBM-HSCT cohorts were investigated and data compared to IV controls (CON). BM-grafts of the respective donors were infused unmodified IV (CON, n=9) or IBM after HSC enrichment using a buffy coat followed by ficoll density centrifugation (IBM-I, n=6; 5ml total volume) or IBM after HSC enrichment using buffy coat centrifugation only (IBM-II, n=6; 25 ml total volume). In the CON group the graft was infused in the cubital vein. In the IBM-groups the grafts were infused through a BM aspiration needle into the BM of the left humerus and femur over a period of 5 minutes. In 4 IBM animals graft migration analyses were performed using technecium99 marking. Chimerism and BM cellularity were determined at injection and opposite sides. Analyses of chimerism were performed via polymorphic nucleotide repeat analyses weekly. BM cellularity was determined biweekly. Complete blood count was performed daily. Result: Infusion of grafts directly into the BM was feasible: both volumes (5ml, 25ml) could be infused without any leakage at the injection sites. Tc99-marked BM cells stayed predominately at the injection site for the first 24 hours. All animals engrafted. Mean TNC numbers infused were 2.6 ×108/kg (range: 1.6–11.4; CON), 1.6 ×108/kg (range: 1–2.4; IBM-I), 3.7 ×108/kg (range: 2.1–5.8; IBM-II) (IBM-I vs CON: p=0.08, IBM-II vs CON: p=0.9, IBM-I vs II: p

Description: Introduction: Direct intra bonemarrow (IBM) infusion of hematopoietic stem cells (HSC) is assumed to improve the homing efficiency and to accelerate the early engraftment in comparison to the conventional intravenous application of HSC. Especially for transplantation of low cell numbers i.e. "weak grafts" that is generally associated with delayed engraftment. The direct infusion of HSC in close proximity to the HSC niche by intra bone marrow transplantation (IBMT) might be a promising way. Whether the HSC infusion rate might influence the homing process and therefore the outcome after IBMT is so far unknown. Aims: Herein, we analyzed in a canine DLA-identical littermate model the impact of different graft infusion rates on the hematopoietic recovery as well as on the engraftment kinetics after IBMT following reduced intensity conditioning. Methods: Recipient dogs received IBMT following a 4.5 Gy total body irradiation (TBI). From day (d) -1 until d+35 Cyclosporin A (15mg/kg) was administered orally twice a day as immunosuppression. For IBM transfusion the graft volume was reduced by buffy coat centrifugation and dogs obtained 2x25 ml simultaneously into the humerus and femur. The infusion rate of the graft was 25ml/10 min in group 1 (IBM10, n = 8) and 25 ml/60 min in group 2 (IBM60, n = 7). A 28 day follow-up is currently available for twelve dogs (IBM10 n = 7; IBM60 n = 5). The development of the peripheral blood mononuclear cell (PBMC) and granulocyte chimerism was tested weekly. Blood count, kidney and liver enzymes were monitored routinely. Results: All animals engrafted. One dog of the IBM10 group died at d+15 (infection) and was therefore not included into analysis. The median number of infused total nucleated cells were in IBM10 4.1*108/kg (range 2.3-6.0*108/kg) and in IBM60 3.2*108/kg (range 1.8-4.4*108/kg; p=0.4). The infused CD34+ numbers were median 3.2*106/kg (range: 1.2-10.0*106/kg; IBM10) and 3.6*106/kg (range: 1.5-6.8*106/kg; IBM60; p=0.7). Time of leukocyte recovery was median d+11 after IBMT in both groups (range: d+4 to d+11, IBM10; d+8 to d+14, IBM60; p= 0.5). Median leukocytes nadirs amounted to 0.2*109/l for IBM10 and 0.3*109/l for IBM60 (p= 0.08). The median duration of leukopenia (

Description: Background The German AML Intergroup conducted two randomized studies in younger (

Description: Purpose: To define prognostic factors for overall survival in adult patients (pts) with relapsed acute myeloid leukemia (AML). Introduction: Prognostic factors for overall survival after AML relapse are poorly defined. Here, we investigate patient and disease related factors in terms of their impact on prognosis after AML relapse in a cohort of 495 adult AML relapse patients treated in two prospective AML trials of the East German Society of Hematology and Oncology (OSHO). Patients and methods: We retrospectively evaluated all consecutive relapsed AML pts treated in two OSHO trials (OSHO #61 (pts 〈 60 years) and OSHO #69 (pts 〉 60 years)). Age, cytogenetic risk at initial diagnosis, FLT3/NPM1 mutational status, type of AML (de novo versus secondary to myelodysplastic syndrome or myeloproliferative neoplasia (MDS/MPN) versus therapy related), time interval from first complete remission (CR1) to relapse and allogeneic stem cell transplantation (alloSCT) as consolidation in CR1 were evaluated in univariate and multivariate analysis. Results: Between March 2002 and July 2014, a total of 862 and 968 patients (pts) were enrolled in the OSHO #61 and #69 trial, respectively. Five hundred and thirty two of 690 (77%) documented pts achieved first complete remission in the #61 and 501 of 813 (62%) pts in the #69 trial. Of these, 495 pts (252 male, 243 female) experienced AML relapse, 207(39%) pts in #61 and 288(57%) pts in #69. Median age at relapse was 63 years (range 18 to 86 years). Initial diagnoses were de novo AML, secondary AML to MDS/MPN and therapy related AML in 332(67%) pts, 129 (25.9%) pts and 30 (6%) pts, respectively. Time from CR1 to relapse was 〈 = 6 months in 198 (40%) pts, 7 to 18 months in 226 (45.7%) pts and 〉 18 months in 71 (14.3%) pts. Initial karyotpe was available for 449 pts (90.7 %). It was favorable, intermediate and poor in 20 (4.5%) pts, 301(67%) pts and 128(28.5%) pts, respectively. Sixty two (13.9%) relapsed pts had a monosomal karyotype at initial diagnosis. NPM1/FLT3 mutational status at initial diagnosis was available in 354 (78.8%) pts, 378 (71%) pts in #61 and 370 (74%) pts in #69. One hundred and three (20.8%) had allogeneic stem cell transplantation as consolidation in CR1 (56 pts) in #61 and (47 pts) in #69. Relapse therapy was documented in 450 (91%) pts. Six pts that had immunosuppression withdrawn as the only therapy and nine pts that had received a tyrosine kinase inhibitor as monotherapy were excluded from further analysis due to small numbers. All other treatments were as follows: intensive chemotherapy (INT) n=225, alloSCT with or without prior INT n=50, donor lymphocyte infusions (DLi) with or without prior chemotherapy n=22, palliative mild cytoreductive chemotherapy (mCT) n=66, azacitidine (Aza) n=52, best supportive care (BSC) n=20. With these, CR was achieved in 78 (36%), 34 (68%), 8 (36%), 6 (9%), 1 (2%), 0 (0%), respectively. Median overall survival probability (OS) for all 495 relapsed patients was 6 months. It was 11.3 months, 5.7 months, 4.5 months and 4.6 months for patients aged 18 to 50 years, 51 to 60 years, 61 to 70 years and 71 to 86 years, respectively (p

Description: Introduction: Successful engraftment following hematopoietic stem cell transplantation (HSCT) depends on factors like immunosuppression, graft composition and number of infused HSC. Whereas the immunosuppression as well as the type and composition of the graft are influenceable low numbers of available HSCs i.e. “weak grafts” remain a clinical challenge. Weak grafts are accompanied by increased graft failure rates and longer cytopenias associated with increased morbidity. Intra bone marrow (IBM) infusion of HSC might be an approach to overcome these problems. Studies in rodents demonstrated faster engraftment with an IBM HSCT approach compared to intravenous (IV) HSCT following myeloablative conditioning. Studies of IBM HSCT following non-myeloablative or reduced intensity conditioning (RIC) are missing. Aims: Exploring the feasibility and efficiency of IBM allogeneic HSCT in comparison to IV HSCT in dog leukocyte antigen (DLA) identical canine littermates using a RIC regimen. Methods: DLA-identical siblings were used as donor/recipient pairs for HSCT. Recipient dogs were conditioned with 4.5 Gy total body irradiation before HSCT (d0) and received 15 mg/kg Cyclosporin A BID as pre- and postgrafting immunosuppression (d-1 to d+35). BM grafts were harvested at d0. In the control group (CON, n=7) unmodified BM was transplanted IV. In the IBM group (n=7) BM harvests were centrifuged and buffy coat of the BM was then transfused simultaneously into the recipient humeri and femura (50 ml, 10 min). 10 dogs are currently evaluable. Chimerism of the peripheral blood mononuclear cells (PBMC) and granulocytes (G) were tested weekly until week 8 and afterwards in larger intervals. Blood cell counts and clinical toxicities such as weight loss were monitored. Results: Infusion of BM directly into the bone was feasible. All animals engrafted. Median number of infused total nucleated cells was 4.0*108/kg (range 2.3-6.0*108/kg, IBM) and 3.3*108/kg (range 1.9-5.0*108/kg, CON, IBM vs CON: p=0.4). Median CD34+ numbers infused were 3.1*106/kg (range:1.2-10.0*106/kg, IBM) and 3.9*106/kg (range: 1.0-7.2*106/kg, CON; IBM vs CON: p= 0.8). Hematopoietic recovery in the IBM and CON groups were similar. Leukocytes recovery (〉1.0*109/l) occurred at median d+11 (range: d+10 - d+16, IBM) and d+10 (range: d+9-d+12, CON; IBM vs CON: p=0.3). Median leukocytes nadirs amounted to 0.23*109/l (IBM) and 0.28*109/l (CON; IBM vs CON: p=0.3) and median duration of leukopenia (