1 Dept. Oncology and Hematology, BMT-Unit,
2 Dept. of TransfusionMedicine.
3 Dept. Pediactric Hematology, University Hospital Eppendorf, Hamburg, Germany

Introduction

The hematopoietic growth factors G-CSF and GM-CSF are able to stimulate granulopoiesis after autologous transplantation of bone marrow or blood stem cells (PBSC) and can also mobilize peripheral blood progenitor cells [1, 2, 3, 4]. Rapid recovery of platelets has been observed following addition of G-CSF mobilized PBSC to autologous bone marrow transplants [3]. The kinetics of engraftment are mainly dependent on the number and qualitiy of progenitor/stem cells in the graft [5, 6, 7, 8]. We have studied the impact of higher doses of G-CSF (Filgrastim) on the yield of CD34 positive cells, CFU-GM and mononucelar cells in leukapheresis products in patients with Non Hodgkin Lymphomas, Hodgkins disease, testicular cancer, AML and neuroblastoma as well as in normal donors for allogeneic progenitor cell transplantation. We report our experience with mobilization of GCSF in two different doses: 10 µg/kg per day versus 24 (2 x 12) µg/kg per day for stem cell collection, separation and transplantation.

Materials and methods

Mobilization: G-CSF (Filgrastim) was administered subcutaneously for 5- 6 days at a daily dose of 10 µg/kg (group A) or 2x12 µg/kg (group B) to 45 patients. Beyond this a total of 18 mobilizations followed by 1 to 4 leukapheresis procedures, was carried out in normal donors. 15 transplants, involving mobilized progenitor cells either alone or in conjunction with bone marrow, were carried out. The characteristics of the autologous patients are shown in table 1.

Table 1. Patient characteristics

15 patients with the following diagnosis: AML: 4, ALL: 5, Multiple Myeloma: 1, Severe Aplastic Anaemia: 3, CML blastic crisis: 1, Myeloproliferative Syndromes 1 received an allogeneic stem cell transplant. The donor-recipient relationships were the following: matched siblings: 8, one mismatch: 2, parent to child: 3, child to parent: 1, matched unrelated donor: 1. Conditioning regimens: without conditioning as a rescue graft for rescue from insufficient bone marrow function: 5; TBICyclophosphamide-VP16: 5; TBI-Cyclophosphamide: 1, Busulfan, Cyclophosphamid +- Etoposid: 4.

Colony forming assay: Colony forming units granulocyte-monocyte (CFU-GM) were assayed in Iscove's methylcellulose. Colonies were enumerated after 14 days of culture in 37° C, 5 % CO2 and 100 % humidity by using an inverted phase microscope (9).

Flow cytometric analysis: Surface antigen expression was evaluated using the monoclonal antibody HPCA-2, directly conjugated to FITC (Becton-Dickinson). Analysis was performed on a FACScan (Becton-Dickinson) using Lysis II research software [5,10,11].

Statistics: Data are shown as median (range), statistical significance was determined by the Student's t-test.

Results

Autologous: The percentage of CD34+ mononuclear cells (MNC) in the first leukapheresis product increased significantly (p=0.024) from 0.68 (0.03-1.96) (group A) to 1.23 (0.4-4.28) (group B) (Fig. 1 ).

Fig.1 Leukapheresis products 10µg vs. 2x12µg G-CSF

Statistically significant differences were also found with respect to total CD34+ MNC per leukapheresis product (LP1): 10.08x10high 7 (0.4-44.0) (group A) versus 42.51 x 10 high 7 (3.46-86.3) (group B) (p=0.012) (Fig. 2, Tabl. 2).

Fig. 2 Leukapheresis products 10µg vs. 2x12µg G-CSF

Table 2. Graft Data

CFU-GM increased from 37.05 (1.6- 95.1) x 105 (group A) to 106.5 (8.7- 390.5) (group B) (p = 0.004). These results confirm, that higher doses of G-CSF can significantly increase the number of CD34 positive cells and CFU-GM in leukapheresis products.

Allogeneic: An average of 3 leukapheresis (range 1 to 4) were carried out, which yielded 13 x 10 high8 (range 2.9 -29) mononuclear cells/kg BW of the recipient, 5 x 10 high 6 CD34 positive cells/kg BW of recipient (range 1.2- 39), CFC 3 x 10 high 5 /kg BW (0,88 60,4). The mobilization with G-CSF was generally well tolerated with the exception of moderate bone pain in 2/3 of the donors.

Engraftment in 9 evaluable patients revealed neutrophil engraftment of more than 0,5/nl by day 14 (range 9 -22), neutrophils more than 1.0/nl day 15 (range 10- 24), thrombocytes more than 20/nl day 16 (range 8 -32), more than 50/nl 24 (range 13 to more than 100 days).

GvH grade 0 -1: 3 patients, grade 2: 4 patients, grade 4: 2 patients and moderate chronic GvH in 1 patient. Total of 12 of the 15 patients are alive in continuous complete remission, 3 have died of GvH and infection (aspergillus 1, candida crusei 1 ).

Discussion

We could show that the mobilization with a higher than usual dosis of G-CSF is well tolerated in normal donors as well as in patients and yields grafts which allow engraftment after myeloablative therapy. GvH occured in 2/3 of evaluable patients and led in conjunction with infection to death in 3 patients. 1 patient received an allogeneic graft from a matched unrelated donor, showed engraftment but unfortunately died of a preexisting fungus disease.

Based on our experience in the autologous setting, where we found statistically significant differences in the number of CD34+ cells and CFU-GM in patients mobilized with 10 µg/kg G-CSF compared with 24 µg/kg and based on the observation that the side effects in both dose levels are comparable, we would recommend 24 µg/kg BW G-CSF per day, given in 2 divided doses or continuously intravenous infusion for mobilization of normal donors.

References

1. Socinski MA, Cannistra SA, Elias A, Antman KH, Schnipper L, Griffin JD: Granulocyte-macrophage colony stimulating factor expands the circulating haemopoietic progenitor cell compartment in man. Lancet 1: 1194, 1988

2. Dührsen U, Villeval JL, Boyd J, Kannourakis G, Morstyn G, Metcalf D: Effects of recombinant human granulocyte colony-stimulating factor on hematopoietic progenitor cells in cancer patients. Blood 72: 2074, 1988

3. Sheridan WP, Begley CG, Jutner CA, Szer J, To LB, Maher D, McGrath KM, et al. : Effect of peripheral-blood progenitor cells mobilised by filgrastim (GCSF) on platelet recovery after high-dose chemotherapy. Lancet 339: 640, 1992

4. Gianni AM, Siena S, Bregni M, Tarella C, Stern AC, Pileri A, Bonnadonna G: Granulocyte-macrophage colony-stimulating factor to harvest circulating haemopoietic stem cells for autotransplantation. Lancet 2: 580, 1989

5. Siena S, Bregni M, Brando B, Belli N, Ravagnani F, Gandola L, et al.: Flow cytometry for clinical estimation of circulating hematopoietic progenitors for autologous transplantation in cancer patients. Blood 77: 400, 1991

6. Weaver CH, Buckner CD, Longin F, Appelbaum FR, Rowley S, Lilleby K, et al. : Syngenic transplantation with peripheral blood mononucelar cells collected after administration of recombinant human granulocyte colonystimulating factor. Blood 82: 1981, 1993

7. Chao NJ, Schriber JR, Grimes K, Long GD, Negrin RS, Raimondi CM, et al.: Granulocyte colony-stimulating factor "mobilized" peripheral blood progenitor cells accelerate granulocyte and platelet recovery after highdose chemotherapy. Blood 81: 2031, 1993

8. Bensinger W, Singer J, Appelbaum F, Lilleby K, Longin K, Rowley S, et al.: Autologous transplantation with peripheral blood mononuclear cells collected after administration of recombinant granulocyte stimulating factor. Blood 81: 3158, 1993

9. Messner HA, Curtis JE, Minden MD, Tritchler D, Lockwood G, Takahashi T, Lepine J, Jamal N, Tweeddale M, Wandl U: Clonogenic hemopoietic precursors in bone marrow transplantation. Blood 70: 1425, 1987

10. Berenson RJ, Andrews RG, Bensinger WI, et al.: Selection of CD34+ marrow cells for autologous marrow transplantation. Autologous Bone Marrow Transplantation: Proceedings of the 4th International Symposium, Dicke KA, Spitzer G, Jagannath S, et al. (eds.). Houston, Univ. of Texas, 1989,55

11 . Shpall EJ, Jones RB, Bearman SI, Franklin WA, Archer PG, Curiel T, Bitter M, et. al.: Transplantation of enriched CD34-positive autologous marrow into breast cancer patients following high-dose chemotherapy: influence of CD34-positive peripheral-blood progenitors and growth factors on engraftment. Journal of Clinical Oncology 12: 1994, 28