Treosulphan has recently demonstrated antileukaemic activity and potent haematopoietic stem cell toxicity. Dose-escalated treosulphan (3 × 12 or 3 × 14 g/m2) combined with cyclophosphamide (Cy) was chosen for a new preparative regimen before allogeneic haematopoietic stem cell transplantation in 18 patients (median age 44, range 19–64 years) with haematological malignancies, considered ineligible for other myeloablative preparative regimens. Pharmacokinetic studies demonstrated rapid treosulphan plasma clearance and a dose-dependent increase of its maximum plasma concentrations and area under the concentration–time curves. Rapid and sustained white blood cell and platelet recovery and full donor chimerism was attained in all evaluable patients. Nonhaematological regimen-related CTC grades 3–4 adverse events were transient and predominantly consisted of cardiac (28%), gastrointestinal (39%), and hepatic (39%) toxicities. The 1-year nonrelapse mortality was 22%. Principal causes of transplant-related lethal events were infections in three of four affected patients. Only one patient died from regimen-related cardiac toxicity. The 1-year relapse estimate is 22%, overall and progression-free survival estimates are 67 and 56%, respectively. In conclusion, this new treosulphan and Cy combination is an effective, comparatively well-tolerated myeloablative preparative regimen even in patients with an increased risk for regimen-related toxic complications.
Treosulphan (L-treitol-1,4-bis-methanesulphonate) is a water-soluble bifunctional alkylating agent registered in several European countries for the treatment of ovarian cancer. In conventionally dosed chemotherapy, haematotoxicity is limiting at an intravenous (i.v.) dose of 10 g/m2.1 If combined with autologous haematological stem cell rescue, a substantial dose escalation in the range of five times of the conventional maximum tolerated dose is feasible. At this escalated dose, mucositis, diarrhoea, skin toxicity, and metabolic acidosis have been found to be dose-limiting at doses of 47 and 49 g/m2, respectively.2,3 Pharmacokinetic studies demonstrated linear pharmacokinetic characteristics of treosulphan up to single doses of 47 g/m2 with a comparably low intra- and interindividual variability.2
Recently, preclinical evaluation of the agent revealed further evidence for its feasibility as a component of preparative regimens for allogeneic haematopoietic stem cell transplantation (HSCT) in that treosulphan demonstrated pronounced haematopoietic stem cell toxicity in vitro.4 In a murine transplantation model, repeated dose-regimens of treosulphan proved to be at least as effective as busulphan or total body irradiation (TBI), and further allowed the development of stable donor chimerism in recipient animals.5,6,7,8,9 Beside its potent haematopoietic stem cell toxicity, treosulphan also demonstrated in vitro activity against a variety of haematological malignancies including acute leukaemias, chronic myelogenous leukaemia, and multiple myeloma.10,11,12 The antileukaemic efficacy of treosulphan in human acute lymphoblastic leukaemia (ALL) xenograft models was superior compared to equitoxic doses of cyclophosphamide (Cy) or busulphan.13 In addition, immunosuppressive characteristics of treosulphan were elucidated in a rat model of experimental autoimmune encephalomyelitis.14 These preclinical data thus provide a rationale for the clinical application of treosulphan in preparative regimens for allogeneic transplantation.
Most recently, in a series of 30 patients with various haematological malignancies ineligible for standard preparative regimens, a combination of treosulphan and fludarabine showed a comparatively favourable toxicity and efficacy profile.15
In the present prospective multicentre phase I trial, the combination of treosulphan and Cy prior to allogeneic HSCT from HLA-identical sibling donors has been investigated. The treosulphan/Cy combination was chosen to allow a more direct comparison of its properties to well-established preparative regimens like TBI and Cy or busulphan and Cy. Only those patients regarded ineligible for these standard preparative regimens by predefined criteria were entered in this study. It was the intention of this study to evaluate engraftment, donor chimerism, and safety of this regimen rather than to define a maximum tolerable dose (MTD) in the allogeneic setting. Based on previous experience in autologous and allogeneic transplantation studies, cumulative treosulphan doses of either 36 or 42 g/m2 in combination with standard-dose cyclophosphamide were applied.2,3,15 De-escalation was not planned to keep maximum antileukaemic activity. Pharmacokinetic parameters were assessed for the two different treosulphan doses. The present study report summarises safety and survival data obtained after a follow-up of 12 months, as well as pharmacokinetic results.
Patients and methods
Between July 2000 and April 2002, 18 patients with haematological neoplasms indicated for allogeneic SCT were enrolled in this study. All patients had been judged to carry an unacceptable high risk for regimen-related toxicities according to predefined risk criteria if treated by an established myeloablative preparative regimen. These criteria included severe liver or lung toxicities, organ or infectious complications during preceding radiochemotherapy, disease recurrence after autologous transplantation, or patient age greater than 55 years. Other eligibility criteria were standard. Patients and disease characteristics are summarised in Tables 1 and 2.
The protocol was conducted according to the principles of the Declaration of Helsinki, the Good Clinical Practise guidelines, and the German drug law. Ethical committees of the participating institutions had given their protocol approval. Written informed consent to all aspects of the study treatment had to be obtained from all patients and donors prior to study enrolment. Compliance to the study protocol, adverse events, and outcome of enrolled patients were monitored by an external clinical research associate.
The study was designed as a dose-escalation trial. Initially, six eligible patients at the dose level of 12 g/m2 treosulphan once daily i.v. on three consecutive days (total dose 36 g/m2) were planned. Owing to the additional recruitment of two further patients, a total of eight patients was available on this dose level. Since no treosulphan-dependent dose-limiting toxicity was observed within this dose level, the dose was escalated to 14 g/m2 treosulphan once daily i.v. on three consecutive days (total dose 42 g/m2). An amendment was filed to allow enrolment of a total of 10 patients within this dose level. Thereafter, dose was not further escalated as it was not intended to define a maximum tolerable dose, but to evaluate a dose which allows safe and reliable engraftment as well as high antileukaemic activity considering safety data of other treosulphan-based transplantation protocols.2,3,15
Donors and grafts
All patients received grafts from HLA-identical sibling donors. Two patients were transplanted with unmanipulated bone marrow and 16 patients with G-CSF-mobilised peripheral blood stem cell grafts. A median of 4.9 × 106 CD34+ cells/kg of recipient body weight (range 1.0–22.4) was transplanted on day 0.
Patients received 12 g/m2 (eight patients) or 14 g/m2 (10 patients) treosulphan (medac, Hamburg, Germany) as a daily 2-h infusion from days −6 to −4 (total dose 36 or 42 g/m2, respectively). Cy (Baxter Oncology, Frankfurt, Germany) at a dose of 60 mg/kg was given as a 1-h infusion from day −3 to −2 (total dose 120 mg/kg). The protocol was amended after inclusion of nine patients to start on day −7.
All patients were treated in laminar HEPA filtration rooms. Prophylaxis and treatment of infections, blood component substitution, and other supportive measures were performed according to the institutional standards, which were essentially uniform in the participating institutions. Prophylaxis for acute graft-versus-host disease (GvHD) consisted of methotrexate (10 mg/m2 i.v. on days 1, 3, and 6) in combination with cyclosporin (CSP) from day −1 to day 100. In the absence of GvHD, the dose of CSP was tapered thereafter.
Evaluation of GvHD
Diagnosis of acute and chronic GvHD was based on the characteristic clinical appearance of the symptoms of organ involvement. The staging of organ involvement and grading of the degree of severity followed the commonly accepted criteria.16 Patients who survived longer than 100 days after transplantation were regarded evaluable for chronic GvHD.
Adverse events evaluation
Adverse events and symptoms were documented from start of the preparative regimen to day 28 after transplantation according to the Common Toxicity Criteria (CTC) version 2.0 (National Cancer Institute, Bethesda, MD, USA). The BMT-specific appendix was not applied.
Definition of engraftment
Engraftment of white blood cells (WBCs) and neutrophils was defined as the first of three consecutive days with WBC counts exceeding 1 × 109/l and neutrophil counts exceeding 0.5 × 109/l. Platelets engraftment was defined by sustained transfusion-independent platelet counts exceeding 20 × 109/l. Graft failure would have been assumed if these end points of haematological reconstitution were not reached until day 28 after transplantation.
Analysis of chimerism in peripheral blood or marrow mononuclear cells was performed according to the established methods of the participating institutions. For the detection of chimerism in gender-mismatched transplants, interphase fluorescence in situ hybridisation using X and Y chromosome-specific probes (dual-colour FISH) was generally applied according to the manufacturer's instructions (Vysis, Downers Grove, IL, USA). In gender-matched transplants, chimerism was analysed by variable number of tandem repeats (VNTR) polymerase chain reaction (PCR) amplification or by single-nucleotide tandem repeats PCR amplification. Chimerism analyses were generally performed on day 28 and at 3 and 6 months after transplantation. Patients were classified as complete chimera, if the proportion of donor cells exceeded 90%.
Blood and urine samples were collected to measure treosulphan concentrations and to assess pharmacokinetic parameters for comparison of interday and interindividual variation of treosulphan exposure. Based on previous data, limited numbers of blood samples were collected at 0, 2, 4, 5, 6, and 24 h after treosulphan dosing on each of the three consecutive days.17 Urine was collected at 4 h intervals and corresponding aliquots were taken during the entire treosulphan treatment period. All samples were adjusted with citrate to a final pH of 5.5 in order to avoid pH-dependent ex vivo degradation of treosulphan. Plasma was separated by centrifugation at 4°C and 1000 × g for 10 min preceded by microfiltration (cutoffr10000) and then applied to the HPLC analysis system. Urine samples were centrifuged (4°C; 14 000 g for 15 min) and directly analysed. Treosulphan was separated by a validated RP-HPLC method and quantified by refractometrical detection as previously published.17 The limit of quantification for treosulphan was given as 1 μg/ml in plasma and 20 μg/ml in urine. Reproducibility was 99±3%, recovery was 96±4%, and linearity was given from 1 μg/ml to 50 mg/ml treosulphan (correlation coefficient: 0.99).
Data entry and data validation was performed with the clinical data management system Clintrial V. 4.3. All statistical analyses were performed using SAS software package V. 8.02 (SAS Institute, Cary, NC, USA).
Categorical data were analysed in contingency tables with frequencies and percentages. Probabilities of nonrelapse mortality (NRM), acute and chronic GvHD, and complete chimerism were calculated as cumulative incidences, while overall survival (OS) and progression-free survival (PFS) were estimated by the product-limit method. For cumulative incidence rates, 95%-confidence intervals (95% CI) were calculated.18 Tabulations of adverse events documented from the start of the preparative regimen to day 28 consist of the number and percentage of patients involved per CTC category, CTC term, and the maximum severity. In addition, analysis of adverse events judged to be at least possibly related to treosulphan was performed. Two-sided Mann–Whitney tests were used to compare distributions of grades 0–4 adverse events.
Individual pharmacokinetic parameters were evaluated by two compartment disposition modelling using optimised initial model parameters within the data analysis system TopFit 2.0.19 Single dose and multiple dose models were applied. All pharmacokinetic data were normalised to body surface area (1 m2). Intraindividual interday variations with regard to area under the concentration–time curve (AUC) and maximum concentration (Cmax) as well as interindividual variations of AUC and Cmax after the first treosulphan dose were calculated. For the analyses of dose-dependency and day to day intrapatient variance of the calculated parameters, descriptive statistics as well as paired T-test analysis were performed.
WBC counts dropped below 1.0 × 109/l at a median of 2.5 days after transplantation in all patients, and at 2 and 3 days in the two treosulphan dose level groups, respectively (NS). The median duration of leukopenia was 9 days in all patients, irrespective of the treosulphan dose applied (Figure 1). Neutrophils and WBCs engrafted in the 16 evaluable patients after a median of 16 (range 11–25) and 15 (range 11–19) days, respectively. Transfusion-independent platelet counts beyond 20 × 109/l were reached after a median of 16 days (range 10–57) with a single delayed platelet engraftment at day 57. Thus, the new regimen allowed rapid and sustained engraftment in all evaluable patients.
In all patients surviving more than 28 days after transplantation (n=16), complete donor chimerism was demonstrable at day 28. Donor chimerism remained stable at 6 months after transplantation in 11 out of 14 patients without haematological relapse. Three patients (Nos. 9, 10, 15) experienced a transient decrease of chimerism, which converted back to complete donor chimerism without any intervention (No. 9), after treatment with imatinib-mesylate for incipient cytogenetic relapse of CML (No. 10), or after withdrawal of immunosuppressive therapy (No. 15). No difference in the degree of chimerism was notable between the two dose groups.
Haematologic CTC grades 3 and 4 adverse events
As expected, all patients experienced CTC grade 4 leuko- and thrombocytopenia irrespective of the administered treosulphan dose and received a median of 10.5 (range 1–34) platelet and 10 (range 2–58) red cell transfusions. Nonfatal grade 3 haemorrhage due to thrombocytopenia developed in four patients (22%).
Nonhaematological CTC grades 3 and 4 adverse events
All severe nonhaematological adverse events documented between the start of the preparative regimen and day 28 after transplantation are summarised in Table 3. A detailed evaluation by the clinical investigators suggested that the majority of these events were not only attributable to acute toxic effects of the preparative regimen but were also caused by the toxicity of methotrexate applied as acute GvHD prophylaxis in the immediate post transplant course and by pre-existing organ functional impairments. Overall CTC grades 3 and 4 gastrointestinal events included diarrhoea, nausea, vomiting, mucositis, and esophagitis. Cardio-vascular events mainly presented as arrhythmias, hyper- or hypotension. Severe skin reactions included transient rash or desquamatous skin lesions. One case of erythema multiforme was most probably caused by accompanying amphothericin B treatment. CTC grades 3–4 fever and infections were frequently observed, and three patients succumbed to infectious complications (see below). Transient increases of transaminases, gamma-GT, or bilirubin (three patients) were recorded, accompanied by multiorgan failure due to bacterial septicemia in two patients and by a severe infection in one patient (Figure 2). No episode of hepatic veno-occlusive disease was observed in this study. One patient experienced grade 3 seizures due to reversible cerebro-vascular ischaemia. Severe pulmonary events were generally associated with pulmonary infectious complications (three patients) or cardiac toxicity (one patient). A single patient experienced haemorrhagic alveolitis, judged to be possibly regimen-related, although invasive fungal pneumonia was present at this time. Two cases of renal failure developed in association with multiorgan failure, but no isolated renal toxicity was recorded. Comparing the reported CTC grades 3 and 4 events, no significant differences were detectable between the two treosulphan dose levels (Table 3).
Four of the 18 patients (22%) died within the 12-months-observation period from causes not related to their underlying disease, of whom two patients died within the first month post transplantation. The 1-year NRM estimate is 22% (95% CI: 3–41%) for all patients, 13% (95% CI: 0–34%) for the lower treosulphan dose level, and 30% (95% CI: 3–57%) for the higher levels, respectively (NS) (Figure 3). Leading causes of nonrelapse-related deaths were infections alone (Nos. 8, 11, 17) or in combination with acute heart failure (No. 1) (Table 1).
Response, relapse, and survival
A total of 16 patients were evaluable for disease response and 15 of these patients were in complete haematological remission after transplantation. A single patient with refractory aggressive lymphoma (No. 7), who underwent transplantation in a second partial remission, had rapid disease progression leading to death on day 37 after transplantation. Another patient with advanced ALL (No. 16) later relapsed and died from his disease on day 269. The PFS and OS estimates for all 18 patients at 1 year after transplantation were 56% (95% CI: 33–79%) and 67% (95% CI: 55–88%), respectively (Figure 3). Rates were comparable between the 3 × 12 and 3 × 14 g/m2 treosulphan dose levels with PFS estimates of 50 and 60%, and OS estimates of 75 and 60%, respectively.
The cumulative incidence of grades 2–4 acute GvHD was 22%. No difference was observed between the two treosulphan dose levels. Chronic GvHD affected five patients at the time of last follow-up (two patients limited chronic GvHD, three patients extensive chronic GvHD), which results in a cumulative incidence of chronic GvHD of 33%.
Mean pharmacokinetic parameters for the two treosulphan dose levels are summarised in Table 4. AUCs and Cmax were significantly different (P=0.01) and were clearly dose-dependent. Day to day variation of pharmacokinetic parameters was evaluated within the daily dose levels of 12 and 14 g/m2 treosulphan (Figure 4). The interday variations of parameters did not reach statistical significance. Pharmacokinetic evaluation of repeated treosulphan dosing revealed no evidence for drug accumulation or increased elimination (Figures 4 and 5).
The present study for the first time combined dose-escalated treosulphan and cyclophosphamide as a myeloablative preparative regimen prior to allogeneic HSCT. Treosulphan was chosen for this combination because previous dose escalation studies of intravenous treosulphan as a single agent revealed potent myelotoxic properties together with comparatively few nonhaematologic toxicities, and a very favourable pharmacokinetic profile.2 These desirable characteristics for a myeloablative component together with the well-established immunosuppressive properties of high-dose Cy justified the expectation that this combination is especially suitable as a myeloablative regimen even in patients who carry a substantially increased risk for regimen-related toxicities in the setting of allogeneic HSCT. The results of the present study support this expectation, because comparatively low rates of severe organ toxicities attributable to this preparative regimen were documented in this study. Most remarkably, fatal regimen-related toxicities occurred only in a single patient with pre-existing congestive heart failure. NRM was attributable to infectious complications in three of four affected patients in this study, and the 1-year NRM estimate of 22% compares favourably to that reported for standard myeloablative regimens in a patient population, which was deemed to be ineligible for an established myeloablative protocol by the investigators.20
Nearly all CTC grades 3 and 4 nonhaematological toxicities recorded after this new regimen were transient and encompassed the typical patterns of functional organ impairments observed in the early time phase after allogeneic HSCT, which are not unequivocally attributable to the preparative regimen alone, but also to adverse effects of post transplant immunosuppressants, early infectious complications, and the treatment with anti-infectious agents.20 Although the rates of all adverse events appeared somewhat reduced after the lower treosulphan dose, a dose effect was not corroborated by the analysis on dose-dependent regimen-related adverse events. The most prevalent organ-specific adverse events included reversible gastrointestinal mucositis and transient elevations of liver functional parameters, but this did not exceed the extent, which has been previously described for the applied short-course methotrexate plus ciclosporin acute GvHD prophylaxis.21 In particular, no case of hepatic veno-occlusive disease (VOD) was observed in this study, which otherwise has to be anticipated in a significant proportion of patients with a risk profile similar to that of the present study population.22 This strongly suggests that high-dose treosulphan does not increase, but may even reduce the risk of hepatic VOD in the combination with Cy, whose toxic metabolites have now been identified as a major determinant of this life-threatening complication when applied in combination with other myeloablative components like TBI or busulphan.23,24,25 A sparing treosulphan effect on liver functional impairment is further supported by a phase I dose escalation trial in which single doses up to 47 g/m2 were not associated with significant hepatotoxicity.2 In accordance with the favourable treosulphan toxicity profile reported in the phase-I trial, which was performed with autologous stem cell support, significant other regimen-related toxicities were infrequent in the present study, and, with one exception, reversible. This particular patient who died from acute cardiac failure was found to have toxic cardiomyopathy at autopsy consistent with myocardial damage from cytotoxic agents applied as part of the induction chemotherapy or the preparative regimen before allogeneic HSCT. Therefore, this single fatal event was classified as possibly regimen-related, although the impact of pre-existing cardiac damage remains undetermined.
In contrast to the mandatory application of anticonvulsive prophylaxis during busulphan-based preparative regimens, this is dispensable in high-dose treosulphan regimens, because, as in the present study, no case of regimen-related seizures has so far been observed.1,2,15
Dose escalation was terminated at a cumulative dose of 42 g/m2 treosulphan in this study, because the primary objective was to define a safe and reliable dose level which allows rapid and stable donor cell engraftment. On both treosulphan dose levels, rapid WBC and platelet regeneration was documented. In addition, analysis of chimerism demonstrated early achievement of complete donor haematopoiesis in all evaluable patients, which remained stable in those without haematological disease recurrence. Thus, it appears justified to assume that this new regimen has myeloablative properties as demonstrated by the durable eradication of the physiologic recipient lympho-haematopoiesis.
The daily intravenous infusion of aqueous treosulphan solutions, which lack any organic solvent components, induced plasma levels as expected from previous studies.2,17 The repeated dose regimen was selected for this study on the basis of preclinical studies which assessed dose- and regimen-dependent haematopoietic stem cell toxicity of treosulphan in mice.4,6,7,9 The Cmax and AUC values after each treosulphan dose were predictable and showed a low interpatient and interday variability. We detected no evidence for accumulation of treosulphan plasma levels or an increased drug elimination. Accordingly, dose adjustments of daily treosulphan infusions are dispensable. This can be explained by the nonenzymatic activation and processing of treosulphan. In addition, renal elimination of about 35% of the unchanged parent compound within the first hours after administration is predictable. The reliable pharmacokinetic characteristics of treosulphan can be considered related to its pharmacodynamic activity resulting in rapid and complete engraftment, and may thereby also contribute to disease eradication. Most recently, potent antileukaemic effects against xenografted human leukaemias have been demonstrated after single as well as repeated treosulphan doses.13 In addition, one recent clinical trial in which a lower cumulative dose of treosulphan (30 g/m2) was combined with fludarabine (150 mg/m2) before allogeneic HSCT demonstrated strong antineoplastic activity even in patients with advanced haematological malignancies.15 This is corroborated by the results of the present study, in which only four of 16 evaluable patients had disease progression or relapsed in the observation period. Thus, the present available data on the antineoplastic efficacy of treosulphan-based preparative regimens for allogeneic HSCT warrant additional disease-specific trials. Further, the advantageous toxicity profile together with the 1-year overall survival estimate of nearly 70% in an otherwise unfavourable patient population provide a basis for a broader application of this new regimen in comparable patient subsets. This may be especially suitable in clinical situations, in which reduced-intensity conditioning regimens lead to an increased risk for early disease recurrence due to high proliferative activity of the underlying haematological malignancy or its resistance to conventional radiochemotherapy.26 In addition, this regimen should be further evaluated in standard risk patients who may likewise benefit from the comparatively low rates of severe regimen-related toxicities and fatal events as demonstrated in the present study.
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We acknowledge the significant contribution of C Buchsteiner and C Peiske for data management.
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Beelen, D., Trenschel, R., Casper, J. et al. Dose-escalated treosulphan in combination with cyclophosphamide as a new preparative regimen for allogeneic haematopoietic stem cell transplantation in patients with an increased risk for regimen-related complications. Bone Marrow Transplant 35, 233–241 (2005) doi:10.1038/sj.bmt.1704784
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