High-dose sequential chemotherapy followed by autologous stem cell transplantation in relapsed and refractory aggressive non-Hodgkin's lymphoma: results of a multicenter phase II study.

BACKGROUND: Combination chemotherapy can cure patients with non-Hodgkin's lymphoma (NHL), but those who suffer treatment failure or relapse still have a poor prognosis. High-dose chemotherapy (HDCT) with autologous stem cell transplantation (ASCT) can improve the outcome of these patients. We evaluated an intensified high-dose sequential chemotherapy program with a final myeloablative course. PATIENTS AND METHODS: Inclusion criteria were age 18-65 years, histologically proven primary progressive or relapsed aggressive NHL and eligibility for HDCT. The therapy consists of two cycles DHAP: dexamethasone 40 mg (day 1-4), high-dose cytarabine 2 g/m2 12q (day 2), cisplatin 100 mg/m2 (day 51); patients with partial (PR) or complete remission (CR) received cyclophosphamide 4 g/m2 (day 37), followed by peripheral blood stem cell (PBSC) harvest; methotrexate 8 g/m2 (day 1) plus vincristine 1.4 mg/m2 (day 51); and etoposide 500 mg/m2 (day 58-62). The final myeloblative course was BEAM: cytarabine 200 mg/m2 12q (day 81-84), etoposide 150 mg/m2 12q (day 81-84), melphalan 140 mg/m2 (day 80), carmustin 300 mg/m2 (day 80) followed by PBSCT. RESULTS: Fifty-seven patients (median age 43 years, range 24-65) were enrolled: 23 (40%) patients were refractory to primary therapy and 34 (60%) patients had relapsed NHL. The response rate (RR) after 2 cycles of DHAP was 72% (9% CR, 63% PR) and at the final evaluation (100 days post transplantation) 43% (32% CR, 11% PR). Toxicity was tolerable. Median follow-up was 25 months (range 1-76 months). Freedom from second failure (FF2F) and overall survival (OS) at 2 years were 25% and 47% for all patients, respectively. FF2F at 2 years for patients with relapse and for patients refractory to primary therapy were 35% and 9% (P=0.0006), respectively. OS at 2 years for patients with relapse and for patients refractory to primary therapy were 58% and 24% (P=0.0044), respectively. CONCLUSIONS: We conclude that this regimen is feasible, tolerable and effective in patients with relapsed NHL. In contrast, the results in patients with progressive disease are unsatisfactory. This program is currently being modified by addition of rituximab for patients with relapsed aggressive NHL.

Cologne high-dose sequential chemotherapy in relapsed and refractory Hodgkin lymphoma: results of a large multicenter study of the German Hodgkin Lymphoma Study Group (GHSG).

BACKGROUND: We designed a dose- and time-intensified high-dose sequential chemotherapy regimen for patients with relapsed and refractory Hodgkin lymphoma (HD). PATIENTS AND METHODS: Eligibility criteria included age 18-65 years, histologically proven primary progressive (PD) or relapsed HD. Treatment consisted of two cycles DHAP (dexamethasone, high-dose cytarabine, cisplatinum); patients with chemosensitive disease received cyclophosphamide followed by peripheral blood stem cell harvest; methotrexate plus vincristine, etoposide and BEAM plus peripheral blood stem cell transplantation (PBSCT). RESULTS: A total of 102 patients (median age 34 years, range 18-64) were enrolled. The response rate was 80% (72% complete response, 8% partial response). With a median follow-up of 30 months (range 3-61 months), freedom from second failure (FF2F) and overall survival (OS) were 59% and 78% for all patients, respectively. FF2F and OS for patients with early relapse were 62% and 81%, for late relapse 65% and 81%; for PD 41% and 48%, and for multiple relapse 39% and 48%, respectively. In multivariate analysis response after DHAP (P <0.0001) and duration of first remission (PD and multiple relapse versus early and late relapse; P=0.0127) were prognostic factors for FF2F. Response after DHAP (P <0.0081), duration of first remission (P=0.0017) and anemia (P=0.019) were significant for OS. CONCLUSION: Based on the promising results of this study, a prospective randomized European intergroup study was started comparing this intensified regimen with two courses of DHAP followed by BEAM (HD-R2 protocol).

New insights into the clinical pharmacokinetics of trofosfamide.

OBJECTIVE: This study focuses on the pharmacokinetics of trofosfamide (TRO) and metabolites after oral administration of TRO. METHODS: Twelve patients with solid tumors and non-Hodgkin lymphomas were treated with 450 mg TRO orally for 7 days. TRO and the stable metabolites ifosfamide (IFO), cyclophosphamide (CYC), 2- and 3-dechloroethylifosfamide (2-DCE, 3-DCE) were determined by GC and the sum of the 4-OH-metabolites was measured by HPLC. RESULTS: A fast metabolism of TRO with a half-life of about 1 h was observed. IFO was the main stable metabolite, whereas CYC was only detected in minor quantities. The peak levels and the AUC of the 4-OH-metabolites were 9.5 and 4.3 times higher than observed after an equimolar IFO dose. Only 6% of the administered dose was recovered in urine within 24 hours as stable metabolites. TRO was under limit of detection. CONCLUSIONS: Our results confirm that dechloroethylation of TRO to IFO is a major metabolic pathway. Additionally, we found considerable 4-hydroxylation not shown previously. With respect to the low levels of IFO and CYC observed, the sum of 4-OH-metabolites cannot be explained by hydroxylation of these metabolites only. Hence, we assume a direct 4-hydroxylation of TRO occurring to a high extent. Bioavailability of TRO could not be calculated directly, because TRO is only available as an oral formulation. The bioavailability of oral IFO, however, is reported to be almost 100%. Therefore, after normalization of the dose, a bioavailability of 32% for IFO after oral TRO could be calculated. Thus, in contrast to previous reports, direct 4-hydroxylation of TRO seems to be the main metabolic pathway.

Collection efficiencies of CD34+ progenitor cells and mononuclear cells in leukapheresis products quantified by flow cytometry and calculated on the basis of a new formula.

BACKGROUND AND OBJECTIVES: Optimal mobilization and harvest of hematopoietic progenitors are essential for peripheral blood stem cell transplantation after myeloablative high-dose chemotherapy. Conflicting data have been published concerning the most useful, cost-effective collection strategy which is also convenient for patients. MATERIALS AND METHODS: A total of 66 leukaphereses in 20 patients were retrospectively evaluated. We assessed the predictive value of the number of white blood cells, mononuclear cells (MNCs) and CD34+ cells in peripheral blood for the yield of CD34+ cells in leukapheresis products. The concentrations of MNCs and CD34+ cells were quantified simultaneously by a flow cytometric procedure using fluorescent microparticles. Their collection efficiencies were calculated based on a newly developed formula. RESULTS: The collected hematopoietic progenitor concentration could be predicted only by the number of peripheral blood CD34+ cells prior to apheresis (r = 0.902; p<0.01). Furthermore, the mobilization of at least 30 CD34+ cells/microl peripheral blood was a good predictor that a single leukapheresis would yield a minimum of 2.0x10(6) CD34+ cells/kg body weight. The collection efficiencies calculated by the new formula were 55.2+/-10.7% and 57.7+/-11.2% for MNCs and CD34+ cells, respectively. CONCLUSION: The precise quantification of MNCs and CD34+ cells by a direct flow cytometric assay, as well as the new formula to determine the collection efficiencies, has an impact on optimizing high-quality stem cell products.

Metabolism of ifosfamide to chloroacetaldehyde contributes to antitumor activity in vivo.

Metabolic activation of ifosfamide (IFO) leads to the active 4-hydroxy-metabolite and to a substantial liberation of chloroacetaldehyde (CAA). CAA has been presumed responsible for side effects of IFO. We recently have shown cytotoxic effects of CAA against human tumor cells in vitro. The aim of this study was to demonstrate antitumor effects of CAA in vivo, and to compare its potency to 4-OH-IFO. Pharmacokinetics of IFO and metabolites were evaluated after infusion of 250 mg/kg IFO in mice. The area under the curve (AUC) for 4-hydroxyifosfamide (4-OH-IFO) and CAA were 138. 5 and 102.4 micromol. h/liter, respectively. To compare pharmacokinetics and antitumor effects, the mice received isolated infusion of 4-OH-IFO or CAA in equimolar doses to IFO. Administration of 4-OH-IFO yielded AUC values comparable with those obtained after administration of the parent drug. In contrast, infusion of isolated CAA via tail vein gave a low AUC value of 51.5 micromol. h/liter due to slow flow in the tail vein and rapid degradation. Administration of the parent drug gave highly cytotoxic intratumoral peak concentrations of 25 and 12 micromol/kg tumor weight for 4-OH-IFO and CAA in MX1 xenotransplanted nude mice. Both IFO and isolated 4-OH-IFO led to complete remissions. Administration of isolated CAA (75 mg/kg) delayed tumor growth significantly. The equitoxic dose of isolated 4-OH-IFO was 40 mg/kg. On a molar basis CAA was seven times less potent as 4-OH-IFO. However, on the basis of achieved AUC values, CAA seems to exhibit a similar antitumor activity to 4-OH-IFO.

The cytotoxicity of mafosfamide on G-CSF mobilized hematopoietic progenitors is reduced by SH groups of albumin--implications for further purging strategies.

The efficacy of mafosfamide purging depends on factors like incubation time, drug and erythrocyte concentration. To determine the influence of protein-bound SH groups in the incubation medium, the cytotoxicity of mafosfamide on G-CSF mobilized CD34+/- cells was evaluated by short-term culture assays and drug concentration measurements. 100 micromol/ml mafosfamide was incubated for 30 min in five buffers (PBS, PBS with 1%, 5% and 10% BSA and plasma). The mean calculated areas under the concentration-time curves (AUC) were 2489 +/- 198, 1561 +/- 286, 976 +/- 201, 585 +/- 62 and 605 +/- 196 micromol/l/min. The mean reductions of CFU-GM growth were 79.4%, 73.0%, 62.5%, 30.3%, 6.2% respectively. Similar results were obtained for BFU-E. Regression analysis showed a good correlation between cytotoxicity and AUCs (CFU-GM: r = 0.8195; BFU-E: r = 0.8207). This effect is well explained by the different concentrations of SH moieties in the incubation medium resulting in a higher drug binding capacity. The profound difference between AUCs and CFU-GMs in plasma and 10% BSA cannot be explained by the quantity of SH-groups. It is probably due to an additional enzymatic drug degeneration by the 3'-5'exonuclease subsite of plasma DNA polymerase. In conclusion, the concentration of albumin-associated SH groups strongly influences the cytotoxicity of mafosfamide. It has to be considered as a new and important aspect in ex vivo bone marrow purging.

Ifosfamide cytotoxicity on human tumor and renal cells: role of chloroacetaldehyde in comparison to 4-hydroxyifosfamide.

The initial metabolism of ifosfamide (IFO) consists of two different pathways, which lead to the alkylating metabolite 4-hydroxy-IFO and to chloroacetaldehyde (CAA). CAA has been reported to cause side effects, such as neuro- and nephrotoxicity, whereas no direct antitumor effect has been described thus far. Therefore, two human tumor cell lines (MXI and S117) and a renal tubular cell line (Landa Leiden) were exposed to 4-hydroxy-IFO, CAA, and a combination of both. The concentrations used were in the same range as measured in the blood of 10 patients treated with 5 g/m2 IFO. The cell survival was measured using the MTT assay. Similar dose-response curves were found for both metabolites. For the MX1 tumor, the IC50s of 4-hydroxy-IFO and CAA were 10.8 and 8.6 microM, respectively. For the reduction of S117 cell survival, higher concentrations of the metabolites were needed (25.0 microM 4-hydroxy-IFO and 15.3 microM CAA). Combination treatment of the cells resulted in an approximately additive effect. Both metabolites exhibited similar toxicity against Landa Leiden cells. Our results indicate that CAA has its own cytotoxic profile against tumor cells. Hence, we conclude that the molecular mechanism of action of IFO seems to be only in part an alkylating effect and that CAA may play an important role in the therapeutic efficacy of IFO.

Whole-body hyperthermia increases plasma-levels of transforming growth-factor-Beta.

The low bone marrow toxicity of high dose alkylating agents when given in combination with whole body hyperthermia (WBH) may be explained in part by the parallel induction of the granulocyte colony stimulating factor. Since transforming growth factor beta (TGF beta) is known to act synergistically with colony stimulating factors, we performed studies of TGF beta expression under WBH in 12 patients with histologically confirmed metastatic sarcoma. Each patient was given ifosfamide, carboplatin, and etoposide combined with WBH (41.8 degrees C, 1 h). Plasma specimens far determination of TGF beta levels were taken prior to WBH and at different time points after start of WBH. Immunoreactive TGF beta 1 and TGF beta 2 were measured by ELISA. Follow-up revealed a significant increase in TGF beta 1 and TGF beta 2 in 9 of 12 patients, starting 2 h after begin of WBH and peaking 10 h later. The mean value for TGF beta 1 prior to therapy was 3.3 ng/ml (range: 0.9-7.3 ng/ml), rising after 12 h to 5.3 ng/ml (range: 2.8-11,5 ng/ml) (p<0.05). The values for TGF beta 2 were 3.1 ng/ml (range: 1.9-4.1 ng/ml) and 3.9 ng/ml (range: 3.2-4.5 ng/ml) (p<0.05), respectively, Both TGF beta 1 and TGF beta 2 had to be activated in vitro by acidification, Initial TGF beta 1 and TGF beta 2 levels did not differ from those in healthy controls. Collectively, our data indicate enhanced TGF beta 1 and TGF beta 2 expression under WBH and support the thesis that TGF beta is a myeoloprotective factor because it stimulates granulopoiesis. The transient growth arrest of very early stem cells by TGF beta may be an additional factor contributing to the low myelotoxicity of alkylating agents when given under WBH.