Introduction

Infections during neutropaenia contribute still significantly to morbidity and mortality of high-dose therapy and autologous stem cell transplantation.¹ The use of growth-factor-mobilized peripheral blood stem cells instead of bone marrow has shortened period of cytopaenia significantly owing to the large number of infused primed progenitors. However, even when a large number of progenitor cells is available for transplantation, an engraftment before day +8 to day +10 can hardly be reached.²

Since the concept of high-dose therapy following autologous bone marrow transplantation has been developed years after the introduction of allogeneic bone marrow transplantation, some conditioning protocols have been easily transferred for use in the autologous setting. High-dose cyclophosphamide has been used within a variety of conditioning protocols. Although it has considerable activity against both Hodgkin's and non-Hodgkin's lymphoma, acute and chronic leukaemia and a variety of childhood's solid tumours, the main reason for use is the excellent immunosuppression to permit the engraftment of allogeneic haemopoiesis. However, this immunosuppressive activity is associated with rapid development of severe neutropaenia.³

The facts that immunosuppression is not desired in the autologous setting and that the antineoplastic effect is mainly based on concomitant cytostatics such as busulphan or etoposide or on total body irradiation raise the question whether cyclophosphamide can be omitted to reduce toxicity. Therefore we performed a retrospective analysis comparing classical busulphan/cyclophosphamide (Bu/Cy) conditioning with single-agent busulphan conditioning in 30 patients undergoing high-dose therapy and autologous stem cell reinfusion for haematological malignancies.

Patients and methods

Patients

Fifteen patients with follicular lymphoma, four patients with diffuse large-cell lymphoma, three patients each with acute myeloid leukaemia and chronic myeloid leukaemia (CML), two patients each with mantle-cell lymphoma and Hodgkin's disease, and one patient with MALT lymphoma were included in this study. Their median age was 53 years with a range from 17 to 69 years. Seventeen patients (56.7%) were male and 13 (43.3%) were female. The patients were treated with the following high-dose protocols: Bu/Cy (busulphan: 4 mg/kg per os (p.o.) in four divided doses daily, day 7 to 4, total dose 16 mg/kg; cyclophosphamide 60 mg/kg once daily intravenously, day 3 to 2, total dose 120 mg/kg) or Bu-mono (busulphan alone: 4 mg/kg p.o. in four divided doses daily, day 7 to 4, total dose 16 mg/kg) (Table 1). All patients were transplanted with granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood stem cells at day 0 and received rhG-CSF once daily subcutaneously from day 0 until the leucocyte count raised above >5 10⁹/l (<65 kg body weight: 300 g; >65 kg body weight: 480 g; Neupogen^®, Amgen, Munich, Germany). Stem cell aphaeresis and analysis were carried out following the International Society for Cellular Therapy (ISCT) criteria.

Table 1 - Characteristics of patients treated with single-agent busulphan and matched patients treated with busulphan/cyclophosphamide.

Full table

For any patient treated with single-agent busulphan, two corresponding patients treated with combined busulphanBu/Cy were selected as a control based on the following criteria: number of transplanted CD34⁺ cells, underlying disease and age (Table 1). The median age was 59 years (range 37–69) in the Bu-mono group and 50 years (range 17–66) in the Bu/Cy group (P<0.05; Mann–Whitney U-test). The mean CD34⁺ cell dose was slightly higher in the Bu/Cy group (5.54 10⁶/kg, range 2.50–24.00 10⁶/kg) than in the Bu-mono group (4.38 10⁶/kg, range 2.65–8.25 10⁶/kg). The leucocyte count per nl on the day before the beginning of high-dose therapy was 4.6 (median, range 3.7–5.9) in the Bu-mono group and 4.7 (median, range 2.3–11.9) in the Bu/Cy group. Both differences were statistically nonsignificant (P=0.71; Mann–Whitney U-test). In summary, pre-condition for the selection of the control patients was to have comparable or slightly better parameters predicting a favourable outcome after high-dose therapy. The significant difference of age was due to the fact that older patients had more often an indication to withdraw cyclophosphamide from the conditioning protocol.

Supportive care, toxicity and haematological recovery

Supportive care and antimicrobial prophylaxis was carried out with quinolones by following standard procedures as described.⁴ In the case of fever above 38.3°C, broad-spectrum antibiosis consisting of ceftazidime, vancomycin or carbapenems was initiated after immediate diagnostics following standard procedures. Parameters of haematology and clinical chemistry were analysed at least once daily. Erythrocytes were substituted when haemoglobin decreased below 8 mg/dl, and platelets were given when these were less than 20/nl. The haematological recovery was related to the day of stem cell reinfusion (day 0). Leucocyte engraftment was defined as the first of 3 consecutive days that the absolute leucocyte count exceeded 1.0 cells/nl of blood and steps of platelet count recovery were defined as the first day that the platelet count exceeded 20 and 50 thrombocytes/nl of blood, respectively, independent of platelet transfusions. Non-haematological toxicity was graded according to the WHO criteria and overall and relapse-free survival was calculated using the log-rank test and the Kaplan–Meier analysis.

Statistics

Data were collected using the computer software MS-Office (Microsoft, Munich, Germany) and analysed with GraphPad Prism (GraphPad Software, San Diego, CA, USA). The statistical tests used were the Mann–Whitney U-test, Fisher's exact P-test and the Kaplan–Meier survival analysis.

Results

Haematological parameters

Leucocytes

Leucocytes decreased significantly faster below 1/nl in patients conditioned with Bu/Cy (median: day +4; range 2–4) than in patients conditioned with busulphan alone (median: day +5.5; range 3–7) (P=0.0001, Mann–Whitney U-test). Differences in the time interval until neutropaenia was below 0.5 leucocytes/nl remained nonsignificant, however, this observation is due to the fact that deep leucopaenia was observed in only 4/10 patients after single-agent busulphan conditioning, but in all patients after high-dose busulphanBu/Cy (P=0.004; Fisher's exact P-test).

All patients recovered with 0.5 and 1 leucocyte/nl on day +9 (median, ranges 7–10 and 8–10, respectively) without significant differences between both groups (Table 2). The effect of slower leucocyte decrease led to significantly shorter leucopaenic intervals in patients after single-agent conditioning than in patients after combination therapy (L<1.0/nl: 4 days (median, range 1–6) vs 5.5 days (median, range 4–8), P<0.001; Mann–Whitney U-test; L<0.5/nl: 0 days (median, range 0–5) vs 5 days (median, range 3–7), P=0.0001; Mann–Whitney U-test).

Table 2 - Kinetics of leucocytes (WBC) and platelets (PLT) related to the day of stem cell reinfusion (day 0).

Full table

Platelets

The platelets decreased below 50/nl on day +5 after stem cell reinfusion and below 20/nl 6.5 and 6 days after single-agent and combined conditioning therapy, respectively. Data for platelet recovery were also comparable for both groups (Table 2). Subsequently, no differences were seen with respect to length of thrombopaenic intervals.

The choice of high-dose therapy had an influence neither on decrease and engraftment of platelets after high-dose therapy and stem cell reinfusion nor on the number of platelet or red-cell transfusions required. Figure 1 and Figure 2 show a graphical overview about kinetics of leucocytes and platelets after high-dose therapy with busulphanBu/Cy (top) or busulphan alone (bottom) related to the day of stem cell reinfusion (day 0).

Figure 1.

Kinetics of leucocytes (WBC) after high-dose therapy with busulphan alone (top) and busulphan/cyclophosphamide (bottom). The mean and s.e. are shown.

Full figure and legend (21K)

Figure 2.

Kinetics of platelets after high-dose therapy with busulphan alone (top) and busulphan/cyclophosphamide (bottom). The mean and s.e. are shown.

Full figure and legend (18K)

Transfusion of blood derivates

Transfusions of blood derivates were necessary in both groups without significant differences. After Bu-mono conditioning patients received 4 U of red cells (median, range 0–6) and 3 U of platelets (median, range 1–8) compared to 4 U of red cells (median, range 0–12) and 3 U of platelets (median, range 1–15) after combined BuCy conditioning.

Non-haematological toxicity and infections

One patient died from a traumatic spleen rupture during thrombocytopaenia, other therapy-related deaths were not seen. With regard to fever episodes, the use of antibiotics after transplantation and incidence of mucositis, there was no difference between the two groups. Patients after Bu-mono conditioning experienced 1 day of fever (median, range 0–3) compared to 1 day (median, range 0–15) after combined therapy with BuCy (P=0.68; NS, MannWhitney U-test). Administration of systemic antimicrobial therapy was necessary for median duration of 4.5 days (range 0–12) after Bu-mono high-dose therapy when compared to 7 days (range 0–16) after combined therapy (P=0.21; NS, Mann–Whitney U-test).

To evaluate further non-haematological toxicity, we determined bilirubin, alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT) and creatinine. An increase in these parameters was often detected during the first 20 days after transplantation, but there were no significant differences between the two groups. Generally, toxicity was mild to moderate. In addition, there was no significant difference in time to discharge (data not shown).

Survival

Analysis of event-free and overall survival was calculated according to the method from Kaplan–Meier and was compared using the log-rank test. Progression-free survival was 30 months (median, range 0–73) after single-agent busulphan as compared to 20 months (median, range 2–91) after combined Bu/Cy conditioning (P=0.31; log-rank test) (Figure 3, top). The median overall survival was 51 months (range 5–77) after single-agent busulphan conditioning and 32 months (range 5–104) after combined Bu/Cy conditioning (P=0.63; log-rank test) (Figure 3, bottom). Overall, survival analysis did not give any hint for inferiority of single-agent conditioning.

Figure 3.

Progression-free survival (PFS, top) (P=0.31; log-rank test) and overall survival (OS, bottom) (P=0.63; log-rank test) after single-agent busulphan (Bu) and busulphan/cyclophosphamide (BuCy)-conditioning therapy in months.

Full figure and legend (33K)

Discussion

The present matched-triplet analysis shows that the choice of conditioning therapy can significantly shorten or avoid severe neutropaenia after autologous stem cell reinfusion. This observation is of importance because infections during aplasia contribute still significantly to morbidity and mortality after autologous stem cell transplantation despite the introduction of modern antimicrobial substances, especially newer antifungal drugs, and growth factors. Major risk factors for acquisition of life-threatening infections are neutropaenia below 0.5 leucocytes/nl and prolonged neutropaenia over 10 days.^{5, 6, 7}

The switch from non-stimulated marrow grafts to G-CSF-mobilized peripheral blood stem cells led to a significant faster haematopoietic regeneration, however, an earlier engraftment than on day +8 to day +10 cannot be reached for biological reasons.² Although patients undergoing myeloablative high-dose therapy and autologous stem cell transplantation are not at highest risk for severe infections, the shortening of neutropaenia by choosing of appropriate high-dose protocols could contribute to further reduction of complications in these patients.

In addition, pharmaco-economic considerations have become extremely important during the last decade. Patients undergoing high-dose therapy and autologous stem cell transplantation are commonly nursed under special, personnel-intensive conditions on special wards. Furthermore, these patients require parenteral nutrition for several days and often expensive diagnostics and antimicrobial drugs in the case of suspected or proven infection. The daily use of growth factors is common practice and can be discontinued after haematological recovery.⁸ To shorten the neutropaenic interval could be a way to increase efficiency of autologous stem cell transplantation during current time of limited resources.

One could criticise that the take out of cyclophosphamide from the high-dose protocol might be associated with a considerable loss of antineoplastic activity. Here we would argue that the outcome of patients from both collectives is comparable in our investigation. In this context, we would like to mention that the patients conditioned with busulphan alone were significantly older than those from BuCy group and that higher age is a well-known negative prognostic factor for treatment-related morbidity and mortality.⁹ A significant activity of busulphan-containing high-dose protocols has been shown in several investigations.^{10, 11} In addition, successful single-agent busulphan conditioning before autologous blood stem cell transplantation has been reported by Anderson et al.¹² in patients with myeloid metaplasia, and Olavarria et al.¹³ and others in patients with CML. These investigators concluded that single-agent busulphan conditioning can provide sufficient antineoplastic activity in combination with mild to moderate toxicity in patients undergoing high-dose protocols.

We would like to conclude that single-agent busulphan conditioning therapy could reduce infectious complications after high-dose therapy by shortening or avoidance of leucopaenia and might offer a way to increase the efficiency of autologous stem cell transplantation during times of limited resources without loss of antineoplastic activity. The investigators want to encourage comparison of both protocols in a prospectively randomized trial.

References

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Acknowledgements

We thank Dr BP Jäger from the Department of Biostatistics for his advices and the nurses from the BMT unit for the excellent patient care.