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Peptide vaccination constitutes a novel immunotherapeutical approach for the treatment of patients with solid tumors, lymphoma and leukemia. Moreover it might be of use in hematooncological patients for the prevention and therapy of infections like cytomegalovirus (CMV) reactivation due to immunosuppression. To meet good manufacturing practice (GMP) criteria, we introduce here a bio-assay to validate peptide vaccines for peptide content and bio-activity. As a paradigm for peptide vaccine preparation the immunogenic CMV peptide 495-503 NLVPMVATV lyophilisate was resolubilized in dimethyl sulfoxide, phosphate buffered saline and admixed with Montanide. Addition of different amounts of peptide (10-80 microg) to a mixed lymphocyte peptide culture (MLPC) resulted in the generation of interferon (IFN) gamma and granzyme B releasing CD8(+) CMV tetramer(+) T cells in a dose dependent manner. The combination of FACS and ELISPOT results allowed the definition of the peptide amount in a vaccine preparation. Storage at +/-4 degrees C over 24 h did not result in a significant change of the immunogenicity of the vaccine. In contrast, cryopreservation of the vaccine at -20 degrees C resulted in a loss of immunogenicity. Quantitation of tumor/viral antigen peptides admixed with adjuvants, such as incomplete Freund's adjuvant (IFA), is feasible through bio-assays as the modified ELISPOT/FACS assay described here, meeting GMP criteria for multi-center trials.

Unlabelled: Intensification of the conditioning regimen with a radioactively labeled anti-CD66 antibody is feasible before allogeneic stem cell transplantation. The use of radioimmunotherapy may deliver a significant dose of radiation to the kidneys. We therefore studied the incidence and clinical picture of bone marrow transplantation (BMT) nephropathy in our patients receiving radioimmunotherapy before allogeneic stem cell transplantation., Methods: This study was a clinical trial of 114 consecutive patients who received conditioning with a radiolabeled anti-CD66 antibody-188Re (n = 93) or 90Y (n = 21)-between 1998 and 2003., Results: Although BMT nephropathy has developed in none of the patients in the [90Y]anti-CD66 group, 6 of 93 patients receiving [188Re]anti-CD66 presented with signs of BMT nephropathy at a median of 11.5 mo after stem cell transplantation. The absorbed renal dose was significantly lower in the 90Y group (4 vs. 7 Gy, P < 0.0001). Of the patients receiving [188Re]anti-CD66 who are alive, BMT nephropathy developed in 19% (6/32). Five of 6 patients with BMT nephropathy received total-body irradiation. The patients presented with elevated serum creatinine, proteinuria, anemia, hypertension, and signs of microangiopathy. All 6 patients in whom BMT nephropathy has developed are alive at a median follow-up of 58 mo after stem cell transplantation, and 1 patient has entered a dialysis program., Conclusion: BMT nephropathy appears to be a significant problem after allogeneic stem cell transplantation with intensified conditioning using the 188Re-labeled anti-CD66 applied in this study, particularly when combined with total-body irradiation.

Regulation of sensitivity or resistance for apoptosis by death receptor ligand systems is a key control mechanism in the hematopoietic system. Dysfunctional or deregulated apoptosis can potentially contribute to the development of immune deficiencies, autoimmune diseases, and leukemia. Control of homeostasis starts at the level of hematopoietic stem cells (HSC). To this end, we found that CD34+ hematopoietic progenitor cells are constitutively resistant to CD95-mediated apoptosis and cannot be sensitized during short-term culture to death receptor-mediated apoptosis by cytokines. Detailed analysis of the death machinery revealed that CD34+ cells do not express caspase-8a/b, a crucial constituent of the death-inducing signaling complex (DISC) of death receptors. Instead, we found a smaller splice variant termed caspase-8L to be present in HSC. Forced expression of caspase-8L using a recombinant lentiviral vector was able to protect hematopoietic cells from death receptor-induced apoptosis even in the presence of caspase-8a/b. Furthermore, we found that caspase-8L is recruited to the DISC after CD95 triggering, thereby preventing CD95 from connecting to the caspase cascade. These results demonstrate an antiapoptotic function of caspase-8L and suggest a critical role as apoptosis regulator in HSC. Similar to CD34+ HSC, stem cell-derived leukemic blasts from AML(M0) patients only expressed caspase-8L. Additionally we found, caspase-8L expression in several AML and ALL samples. Thus, caspase-8L expression might explain constitutive resistance to CD95-mediated apoptosis in CD34+ progenitor cells and might participate in the development of stem cell-derived and other leukemias by providing protection from regulatory apoptosis.

In the present study the in vitro growth of CFU-GM from PBPC of patients with AML (n = 11), purged with mafosfamide alone or a combination of mafosfamide and amifostine, was compared to historical controls of mafosfamide-purged bone marrow (AML CR1, n = 16). Two patients were transplanted with mafosfamide and mafosfamide/amifostine pretreated PBPC autografts. The in vitro experiments demonstrated a significantly higher resistance of peripheral blood derived CFU-GM to mafosfamide (median ID95 190 microg mafosfamide/ml) compared with bone marrow derived CFU-GM (median ID95130 microg/ml). Preincubation with amifostine significantly further increased the median ID95 to 245 microg/ml. The clinical results showed short recovery times for neutrophils >500/microl (9 and 13 days) and platelets >20 000/microl (12 and 21 days) and stable long-term engraftment with one relapse at day +118 and one patient in CR at day 760 after transplantation. The in vitro results show a significant advantage of PBPC over bone marrow-derived progenitors for purging with mafosfamide. Furthermore, a protective effect from mafosfamide of amifostine on normal progenitors could be demonstrated. The clinical results demonstrate the clinical feasibility of using mafosfamide-purged autologous PBPCT without impairing the short-term and long-term repopulating capacities of the autografts.

The efficiency and toxicity of treatment regimens for nonintensive cytoreduction in 57 outpatients with refractory acute leukemia (mean age 56 years, 51 AML, six ALL/AUL) were retrospectively studied. Seventeen patients received one treatment regimen, 19 patients two treatment regimens, and 21 patients three or more treatment regimens. The treatment regimens analyzed were 6-thioguanine p.o. (daily) (T), 6-thioguanine p.o. (4-7 days/week) + cytarabine s.c./i.v. (once a week) (T+C), 6-mercaptopurine p.o. (daily) (MP), 6-mercaptopurine p.o. (daily) + methotrexate p.o./i.v. (once a week) (MP+MTX), etoposide p.o. (daily) (E), and mitoxantrone i.v. (M). The median leukocyte count was higher for M (73 x 10(9)/l) than for the other treatment regimens (T: 27 x 10(9)/l, T+ C: 37 x 10(9)/l, MP: 24 x 10(9)/l, MP + MTX: 30 x 10(9)/l, E: 31 x 10(9)/l). A cytoreduction >50% in the peripheral blood was achieved by T in 11/19, by T+C in 7/11, by MP in 5/8, by MP+MTX in 3/6, by E in 3/4, and by M in 16/22 patients. The period of cytoreduction was regarded as the duration of response - T: median 53 days, range 5-98; T+C: median 61 days, range 14-226; MP: median 37 days, range 4-192; MP + MTX: median 58 days, range 36-59; E: median 121 days, range 26-159; M: median 39 days, range 8-78. T and T + C were well tolerated by all but three patients (stomatitis, diarrhea, WHO grade 2). MP was accompanied by a rise of transaminases (WHO 1-3) in 5/6 patients. E led to stomatitis (WHO 1,2) in 4/5 and M to nausea/vomiting (WHO 1,2) in 5/22 and to stomatitis (WHO 2) in 4/22 cases. The mean survival time after start of palliative cytoreduction was 16 weeks (2-65). In summary, 6-thioguanine +/- cytarabine was best tolerated with effective but in oral monotherapy - often protracted cytoreduction in 60% of patients. Mitoxantrone showed tolerable side effects and potent cytoreduction in 73% of patients even after ineffective palliative pretreatment. Palliative cytoreductive therapy does not reduce the quality of life and can prevent complications of significant leukocytosis in refractory acute leukemia.

Chimerism analysis by DNA-based methods is a valuable diagnostic tool for monitoring engraftment and leukemic relapse after allogeneic BMT or PBPC transplantation (PBPCT). We investigated the chimerism after T-cell-depleted BMT (n = 32) in comparison with T-cell-depleted PBPCT (n = 39). BM grafts were T-cell depleted using the Campath-IgM antibody plus complement. For T-cell depletion of the PBPC grafts, a selection of CD34+ cells with or without a subsequent CD2/3 depletion was performed. In all patients, the T-cell dose of the transplant was < 10(6)/kg body weight. Between day 13 and day 120 after transplantation, chimerism analysis was done by RFLP or amplified fragment length polymorphism (PCR-AFLP), with a detection limit of 1%-5% recipient cells. In the BMT group, 8 of 32 (25%) patients showed a mixed chimerism, but only one graft rejection and no leukemic relapse occurred after a median follow-up of 41 (3-84) months. All patients with PBPCT revealed a complete chimerism of their granulocytes, and 38 of 39 patients showed complete chimerism of their lymphocytes. Follow-up time in these patients is 7 (2-21) months, with no graft rejection and two leukemic relapses. G-CSF-mobilized PBPC are superior to BM cells for full engraftment even after T-cell-depleted transplantation. The more relevant factor for developing complete chimerism seems to be the quantity and possibly the quality of the stem cells rather than the residual T-cell load of the graft. However, a mixed chimerism of the lymphocytes early after transplantation does not predict a higher rate of graft rejection or leukemic relapse.

Seventy-eight transfusions of autologous platelets were given to eight alloimmunized patients receiving curative chemotherapy for acute leukemia. Platelets were collected at regeneration of hematopoiesis after a chemotherapy cycle, cryopreserved with 5% dimethylsulfoxide in liquid nitrogen, and retransfused during bone marrow aplasia following the next treatment cycle. The in vitro platelet recovery after freezing, thawing, and washing was 85 +/- 4%. The in vivo corrected count increment 1 h after autologous platelet transfusions was 11 +/- 5 x 10(9)/l. With the exception of moderate urticaria and slight nausea each after one transfusion, no immediate or chronic side effects occurred. The bleeding time was shortened and hemorrhage during bone marrow aplasia was prevented in all alloimmunized patients by autologous platelet transfusions.

We have performed a non-randomised GVHD prophylaxis trial comparing cyclosporin/methotrexate with in vivo/ex vivo T cell depletion with the monoclonal antibodies Campath 1G/1M in patients with acute leukaemias in first complete remission. We observed significantly less acute and chronic GVHD, neutropenic fever and severe mucositis in the T cell depletion group. The incidence of graft rejection and relapses was no higher than in the cyclosporin/methotrexate group. There is a trend in favour of improved disease-free survival in the in vivo/ex vivo T cell depletion group (80% vs. 62%).

Worldwide, platelet transfusions have increased substantially, especially in hematological patients. The prophylactic and therapeutic indications for platelet substitution as well as the choice of platelet preparation are discussed for patients with acute leukemia or aplastic anemia who need the highest platelet support. Here we present our current strategy, i.e., prophylactic transfusions with random platelets in acute leukemia and therapeutic transfusions with filtered random platelets in aplastic anemia patients only.

40 patients with acute leukaemia and severe granulocytopenia were treated with granulocyte transfusions for bacteriologically documented or clinically suspected septicaemia. The patients received an average of 5 transfusions each with 2.1 X 10(10) granulocytes per m2 body surface area per day. 26 patients showed clinical benefit from transfusion therapy as documented by the course of blood cultures, local lesions and fever. Only patients with regeneration of granulopoiesis had definite benefit from granulocyte transfusions. All other patients died ultimately from septicaemia.

Forty-nine allogeneic and 14 autologous bone marrow grafts were processed with the Haemonetics V50 cell separator (Haemonetics Corp., Braintree, USA) for in vitro treatment with antibodies and cryopreservation respectively. The concentration of hemopoietic progenitor cells was performed without any sedimentation or density gradient agents. The recovery is given in percent (mean +/- sd) of the original marrow values: mononuclear cells (MNC) 74 +/- 10%, polymorphonuclear cells (PMC) 48 +/- 17%, red blood cells (RBC) 12 +/- 5%, granulocyte/monocyte progenitors (CFU-GM) 83 +/- 36%, and erythroid progenitors (BFU-E) 78 +/- 38%. The recovery of nucleated cells (NC) was 90 +/- 13% and the viability 82 +/- 11% after cryopreservation. The technique described provides a simple, rapid and efficient preparation of large bone marrow volumes for in vitro treatment and AB0-incompatible transplantation.

Graft-versus-host disease (GvHD) prophylaxis using methotrexate (23 patients) and T-cell depletion of the graft (40 patients) was compared in 63 allogeneic bone marrow transplantations (BMT) for leukaemia. T-cell depletion significantly reduced (p = 0.001) the incidence of GvHD from 68% to 11% and the GvHD-associated mortality from 79% to 5%. Actuarial disease-free survival for low-risk patients (57% with T-cell depletion and 47% with MTX) was not significantly improved, due to graft failure and possibly due to a higher leukaemic relapse rate after T-cell depletion. Prevention of graft failure after T cell-depleted BMT is essential and could also reduce the risk of leukaemic relapse by improved engraftment.