Patients and methods

This analysis was performed as part of a therapeutic trial of HPC transplantation for patients with grade III and IV malignant glioma achieving optimal cytoreduction by surgery. With the first design of the program, 2 days after the surgery, the patient received a course of 100 mg/m² carmustine (BCNU) intravenously plus 50 mg/m² cisplatin by the intracarotid route. It should be noted that the purpose of this CT was cytoreduction, not mobilization. At 1 month after this CT, patients were mobilized with filgrastim, 12 g/kg subcutaneously, and underwent PB HPC collection by apheresis. In a further review of the program, the course of standard-dose BCNU and cisplatin mentioned was eliminated, and patients commenced mobilization directly 2 weeks after the surgery, with the same dose of filgrastim (see Figure 1). The goal of this protocol amendment was two-fold: to reduce the delay for the beginning of the planned post transplant radiation therapy and to attempt to improve the HSC mobilization results, found to be surprisingly poor in an interim analysis. We compared the mobilization, collection and engraftment kinetics achieved in the patients entered into this program before and after this change.

Figure 1.

Therapeutic schedule in the two groups of patients compared. With the first design of program, the patients received a course of cytoreductive CT after surgery, and were mobilized with filgrastim after they had recovered from it. After protocol revision, the course of CT mentioned was eliminated and patients commenced mobilization directly after surgery. WK/WKS: week/weeks; BCNU: carmustine; CDDP: cisplatin; IV: intravenous route; IA: intra-arterial route.

Full figure and legend (68K)

HPC collection

Daily leukaphereses were performed from the fourth day of filgrastim administration. A dual-lumen central catheter was placed specifically for this purpose. The collections were performed with either a Cobe Spectra (software version 4.0, MNC collection program) or a Fenwall CS3000plus (5-special program, with reverse of flow during spillover and 50-ml chamber) cell processor. Between 2.5 and 5 blood volumes were processed per session. A minimum of two leukaphereses was scheduled. All products collected were cryopreserved, and then thawed and infused during transplantation, with no back-up product retained.

Conditioning regimen and further management

The conditioning CT consisted of 900 mg/m² BCNU intravenously, plus 50 mg/m² cisplatin by the intracarotid route. HPCs were re-infused 48–72 h after the end of the high-dose CT. Radiotherapy to both tumor cavity and the margins was administered from the seventh day post infusion. After reaching a dose of 55 Gy, the tumor was exposed to a further 20–25 Gy of stereotaxic radiosurgery.

Prophylactic single-donor platelet transfusions were administered when the PB count fell below 20 10⁹/l. Red blood cell transfusions were given if the hemoglobin level was lower than 8.5 g/l.

Statistical analysis

As the distribution of most variables was significantly different from normal (tested with Kolmogorov–Smirnoff's test with Liliefor's Correction), descriptive statistics were given as median with the interquartile range (IQR) in brackets, and Mann–Whitney's test was used for group comparisons. A SPSS 9.0 statistical package was used for data management and statistical calculations.

Results

A total of 46 consecutive patients entered this program from July 1997 to September 1999. Three of them were excluded from the analysis because of different violations of the mobilization program. Among the remaining 43 patients, the first 21 received the course of standard-dose BCNU plus cisplatin 1 month before mobilization, and the remaining 22 were mobilized directly after surgery. The main characteristics of the patients in both groups are outlined in Table 1.

Table 1 - Patient characteristics.

Full table

The median levels of CD34+ cells reached in PB were significantly lower in the group previously treated with one course of CT, both after 4 days of filgrastim administration (15 vs 36 10⁶/l cells; P=0.01) and after 5 days (25 vs 58 10⁶/l cells; P=0.003). The median peak PB CD34+ cell count was 27 10⁶/l cells (IQR: 33) in the treated group vs 57 10⁶/l cells (IQR: 42) among the untreated (P=0.003) (Figure 2).

Figure 2.

Median level of PB CD34+ cells ( 10⁶/l) reached along the mobilization treatment. CT: chemotherapy; PB: peripheral blood.

Full figure and legend (197K)

Cell-collection results were similarly different between the two groups. The median absolute number of CD34+ cells collected was 3.4 10⁶/kg (IQR: 4) in the group previously treated with CT vs 6.9 10⁶/kg (IQR: 8.2) in the untreated group (P<0.0005). The CD34+ cell yield per processed blood volume was 0.5 10⁶/kg (IQR: 1) among the treated vs 1.3 10⁶/kg (IQR: 1.7) in the untreated patients (P<0.0005). Table 2 summarizes the collection results achieved.

Table 2 - Overview of collection results.

Full table

Despite the small number of patients in each group, these differences remained significant or nearly significant after adjusting the analyses for the pathological stage of the tumor. The median peak PB CD34+ cell counts were 29 vs 50 10⁶/l cells (P=0.05) among stage IV (glioblastoma multiforme) patients and 24 vs 64 10⁶/l cells (P=0.017) among stage III (anaplastic astrocytoma or oligodendroglioma). The median absolute numbers of CD34+ cells collected were 3.7 vs 6.3 10⁶/kg (P=0.026) in the stage IV patients and 2 vs 7.8 10⁶/kg (P=0.007) among stage III patients. The CD34+ cell yields per processed blood volume were 0.5 vs 1.2 10⁶/kg (P=0.006) among stage IV, and 0.6 vs 1.6 10⁶/kg (P=0.014) in stage III patients.

Conversely, the engraftment kinetics were similar in the patients previously treated with CT as compared to those never treated before the transplant. The median numbers of days with less than 0.5 10⁹/l granulocytes were 5 vs 5 (P=0.89) and median numbers of days post infusion to the last platelet transfusion were 9 vs 9 (P=0.94). Median numbers of platelet transfusions needed were 1 vs 1 (P=0.37), and median number of red blood cell packs transfused were 2 vs 2 (P=0.35).

Discussion

The small likelihood of bone marrow tumor infiltration in untreated stage III–IV malignant glioma makes this population of patients especially suitable for a study of factors influencing mobilization kinetics. In this setting, the behavior of patients never treated with CT from our series should be superimposable upon that of healthy individuals. In fact, the mobilization and collection results achieved in this group are similar to those described after filgrastim mobilization of normal donors for allogeneic PB HPC transplantation.^6,7

Exposure to cytotoxic agents has been said to produce different degrees of residual marrow damage.² Neben et al,⁸ in a model of murine syngeneic transplantation, demonstrated a decrease in HSC self-renewal in mice previously exposed to drugs such as BCNU or cisplatin. In the clinical setting, Drake et al⁹ described a scoring system to grade the amount of previously received CT in patients eligible for HPC transplantation. Previous exposure to drugs classified as 'factor 4' in their score (the most toxic for stem cells, including BCNU) emerged as the only significant factor influencing CD34+ cell yield in a multivariate analysis. Likewise, Gandhi et al¹⁰ and Clark et al¹¹ found similar results in two independent series of patients using Drake's Score. Other studies analyzing the influence of different variables on mobilization and collection also found worse results among heavily pretreated patients,^3,4,5 with a remarkable effect among patients treated with regimens including BCNU in combination with melphalan such as dexa-BEAM.¹¹ Nevertheless, all these studies analyzed patients treated with several courses of combination CT and, therefore, drew conclusions mainly associated with this very fact, such as the significant negative effect of more than six previous courses of CT, more than 24 months of prior therapy or a score of previous CT greater than 60.^3,4,5 To the best of our knowledge, no data are available about the actual effect of a single course of CT administered to untreated patients with an otherwise healthy bone marrow. The median summation score of previous CT in the study by Drake et al⁹ was 38, while that of the patients treated with the single BCNU-cisplatin course in our series was only 6.

Based on experimental data and theoretical considerations, the nitrosoureas have been classically considered more toxic for the stem cell than cisplatin.^9,10,11,12 Nonetheless, the former drug is far from being harmless to the bone marrow.^8,13 Lee et al¹³ found prior treatment with cisplatin to be a significant independent factor predicting worse HPC collection. Cisplatin was administered by the direct intra-arterial route in our study, but this does not rule out the possibility of a systemic effect of this drug contributing to the hematopoietic toxicity of BCNU.

The hematopoietic depression induced by the nitrosoureas is characteristically delayed.^14,15 In the clinical setting, it is not unusual to need an interval of 5–6 weeks between BCNU-based CT courses. This characteristic could provide a possible explanation for the dramatic impairment of mobilization capacity observed in our study among the treated patients. The generally accepted interval of 1 month from the last CT course to the beginning of mobilization was not enough after nitrosourea-based CT.

Several studies have described the effect of both HPC dose and prior CT received on engraftment kinetics.^3,5,12,16,17 Early leukocyte and platelet recovery is reasonably guaranteed in most patients receiving more than 2 10⁶/kg CD34+ cells, providing they had not been extensively pretreated.^3,10 Even though the previously treated patients in our series received a significantly lower dose of CD34+ cells, the median dose in this group was 3.4 10⁶/kg and it was above 2 10⁶/kg in 16 cases of 21 (76%). This number of HPC seems to have been enough for early hematopoietic recovery, and, thus, no difference in engraftment kinetics was found with respect to the previously untreated group.

In conclusion, even a single course of standard-dose CT, containing stem cell toxic agents, had a significant negative effect on filgrastim-based HPC mobilization. As a direct consequence, there was a significantly greater difficulty in collecting stem cells, although it did not preclude the procurement of an appropriate amount in most cases. This effect seems to be the consequence of a certain degree of bone marrow damage induced by the small cumulative amount of CT. This toxicity was sufficient to reduce the mobilization response, but not to preclude early engraftment, provided that an appropriate cell dose was given at transplantation.

References

1. Del Cañizo MC, López N, Vázquez L et al . Hematopoietic damage prior to PBSCT and its influence on hematopoietic recovery. Haematologica 1999; 84: 511–516. | PubMed |
2. Trainor KJ, Morley AA. Screening of cytotoxic drugs for residual bone marrow damage. J Natl Cancer Inst 1976; 57: 1237–1239. | PubMed | ChemPort |
3. Tricot G, Jagannath S, Vesole D et al. Peripheral blood stem cell transplants for multiple myeloma: identification of favorable variables for rapid engraftment in 225 patients. Blood 1995; 85: 588–596. | PubMed | ISI | ChemPort |
4. Haas R, Möhle R, Frühauf S et al. Patient characteristics associated with successful mobilizing and autografting of peripheral bold progenitor cells in malignant lymphoma. Blood 1994; 83: 3787–3794. | PubMed | ISI | ChemPort |
5. Bensinger W, Appelbaum F, Rowley S et al. Factors that influence collection and engraftment of autologous peripheral blood stem cells. J Clin Oncol 1995; 13: 2547–2555. | PubMed | ISI | ChemPort |
6. De la Rubia J, Martínez C, Solano C et al. Administration of recombinant human granulocyte colony-stimulating factor to normal donors: results of the Spanish National Donor Registry. Bone Marrow Transplant 1999; 24: 723–728. | Article | PubMed | ISI | ChemPort |
7. Schmitz N, Dreger P, Suttorp M et al. Primary transplantation of allogeneic peripheral blood progenitor cells mobilized by filgrastim (granulocyte colony-stimulating factor). Blood 1995; 85: 1666–1672. | PubMed | ISI | ChemPort |
8. Neben S, Hemman S, Montgomery M et al. Hematopoietic stem cell deficit of transplanted bone marrow previously exposed to cytotoxic agents. Exp Hematol 1993; 21: 156–162. | PubMed | ISI | ChemPort |
9. Drake M, Ranaghan L, Morris TCM et al. Analysis of the effect of prior therapy on progenitor cell yield: use of a chemotherapy scoring system. Br J Haematol 1997; 98: 745–749. | Article | PubMed | ISI | ChemPort |
10. Gandhi MK, Jestice K, Scott MA et al. The minimum CD34 threshold depends on prior chemotherapy in autologous peripheral blood stem cell recipients. Bone Marrow Transplant 1999; 23: 9–13. | Article | PubMed | ISI | ChemPort |
11. Clark RE, Brammer CG. Previous treatment predicts the efficiency of blood progenitor cell mobilization: validation of a chemotherapy scoring system. Bone Marrow Transplant 1998; 22: 859–863. | Article | PubMed | ISI | ChemPort |
12. Dreger P, Klöss M, Petersen B et al. Autologous progenitor cell transplantation: previous exposure to stem cell-toxic drugs determines yield and engraftment of peripheral blood progenitor cell but not of bone marrow grafts. Blood 1995; 86: 3970–3978. | PubMed | ISI | ChemPort |
13. Lee JL, Kim SB, Lee GW et al. Collection of peripheral blood progenitor cells: analysis of factors predicting the yields. Transfusion Apher Sci 2003; 29: 29–31. | Article | ISI |
14. De Vita VT, Carbone PP, Owens Jr AH et al. Clinical trials with 1,3-bis(2-chloroethyl)-1-nitrosurea, NSC-409962. Cancer Res 1965; 25: 1876–1885. | PubMed | ChemPort |
15. Tew KD, Colvin M, Chabner BA. Alkylating agents. In: Chabner BA, Longo DL (eds). Cancer Chemotherapy and Biotherapy, 2nd edn. Lippincott-Raven Publishers: Philadelphia, 1996, pp 297–332.
16. Watts MJ, Sullivan AM, Jamieson E et al. Progenitor-cell mobilization after low-dose cyclophosphamide and granulocyte colony-stimulating factor: an analysis of progenitor-cell quantity and quality and factors predicting for these parameters in 101 pretreated patients with malignant lymphoma. J Clin Oncol 1997; 15: 535–546. | PubMed | ISI | ChemPort |
17. Siena S, Schiavo R, Pedrazzoli P, Carlo-Stella C. Therapeutic relevance of CD34 cell dose in blood cell transplantation for cancer therapy. J Clin Oncol 2000; 18: 1360–1377. | PubMed | ISI | ChemPort |