Recovery of circulating haemopoietic progenitor cells in the early phase of haemopoietic reconstitution after autologous and allogeneic bone marrow transplantation

The recovery of circulating haemopoietic progenitor cells was evaluated serially in seven patients for 3-4 weeks after bone marrow transplantation (two autologous and five allogeneic) as treatment for leukaemia. Eight normal healthy volunteers were used as controls. CFU-G (colony forming unit-granulocyte) was found to be the earliest progenitor cell to recover at a mean interval of 16 +/- 1 (SE) days post-transplantation. A lag of 7 days was found before circulating CFU-GM (colony forming unit-granulocyte, monocyte) reappeared, while BFU-E (burst forming unit-erythroid) were detectable in only two patients in the first 4 weeks. The peak level of circulating progenitors was very low, 28 +/- 8/ml, compared with a mean level of 619 +/- 235/ml in eight normal individuals. This pattern of circulating progenitor cell recovery post-transplantation was consistently seen in all patients. CFU-G reappeared significantly earlier than CFU-GM suggesting that early granulocytic recovery after bone marrow transplantation is mediated by proliferation of mature progenitors committed to the granulocytic lineage, whereas later reconstitution is accompanied by the emergence of CFU-GM.     

Effect of stem cell factor with and without granulocyte colony-stimulating factor on neonatal hematopoiesis: in vivo induction of newborn myelopoiesis and reduction of mortality during experimental group B streptococcal sepsis.

Neonatal hematopoiesis and host defense are developmentally immature and under states of increased demand predispose the newborn to peripheral cytopenias and depletion of bone marrow storage pool reserves. We have previously demonstrated that recombinant human granulocyte colony-stimulating factor (rhG-CSF) can significantly modulate neonatal rat granulopoiesis and act synergistically with antibiotic therapy to reduce the mortality rate during experimental group B streptococcal sepsis. Stem cell factor (SCF) has been shown to stimulate early hematopoietic progenitor cells and, in the presence of lineage-specific CSFs, enhance committed progenitor cell proliferation. In the present study we examined the in vivo neonatal hematologic effects of recombinant rat (rr) SCF (14 days), simultaneous rrSCF + rhG-CSF (14 days), and sequential combination of rrSCF (7 days) + rhG-CSF (7 days). Sprague-Dawley newborn rats (less than or equal to 24 hours) were injected intraperitoneal (IP) x 14 days with the above combinations. rrSCF (0 to 200 micrograms/kg/d) had a negligible effect on the peripheral platelet count and absolute neutrophil count (ANC) but the diminution in the hematocrit during the first 10 days of treatment was less pronounced (P = .0001). However, the simultaneous use of rrSCF + rhG-CSF synergistically increased the circulating day 6 to 13 ANC (P = .001). Similarly, sequential rrSCF + rhG-SCF also had a synergistic significant effect during the second week of therapy on the circulating ANC (P = .01). The bone marrow neutrophil storage and proliferative pools were also significantly increased in newborn rats treated with rrSCF + rhG-CSF versus rhG-CSF (P = .02). The bone marrow and liver/spleen CFU-GM pool was unchanged; however, the CFU-GM proliferative rates were significantly increased in the rrSCF + rhG-CSF group (P = .04). rrSCF also induced a significant increase in the bone marrow and liver/spleen mast cell pool (P = .002). Lastly, rrSCF x 14 days +/- rhG-CSF significantly reduced the mortality rate at 48 and 120 hours after experimental group B streptococcus sepsis (P = .03 and .05, respectively). These data suggest that combination SCF + G-CSF therapy compared with G-CSF alone significantly increases the neonatal rat peripheral neutrophil count, bone marrow myeloid pools and proliferative rates, and induces a reduction in the mortality rate during experimental bacterial sepsis. SCF therapy may have future potential applications in the modulation of human neonatal hematopoiesis and host defense.     

Hematopoietic damage prior to PBSCT and its influence on hematopoietic recovery.

BACKGROUND AND OBJECTIVE: Patients with malignancies receive chemotherapy to induce tumor remission which could damage hematopoiesis and adversely influence hematopoietic reconstitution after transplantation. In the present study we used a long-term culture (LTBMC) system and clonogenic assays to evaluate the marrow damage in patients selected to receive peripheral blood stem cell transplantation (PBSCT). DESIGN AND METHODS: Thirty-five patients - 20 with breast cancer (BC), 9 with non-Hodgkin's lymphoma (NHL) and 6 with Hodgkin's disease (HD) - were included. Bone marrow aspiration was performed one day prior to the initiation of the conditioning therapy. CFU-GM were cultured in methylcellulose with PHA-LCM. Delta assays of plastic adherent progenitor cells (PD) were performed according to Gordon's method. LTBMC were established for 5 weeks. RESULTS: There were fewer CFU-GM from all patient groups than from normal BM (p<0.05). In contrast, the number of immature progenitor cells (PD) was not decreased. The total number of CFU-GM produced by LTBMC patients was significantly reduced (p<0.05). The adherent layer from patients was often qualitatively different. In order to know whether the hematopoietic damage could affect hematopoietic reconstitution, we correlated culture data with time taken to reach peripheral cell counts. A negative correlation (r= - 0.71) was found between percentage of stromal layer and time taken to reach 20x10(9) platelets/L (tplat= 20x3-0.08% stromal layer). INTERPRETATION AND CONCLUSIONS: We can conclude that prior to PBSCT, hematopoietic function is impaired at both the level of committed progenitor cells and that of BM stroma. This damage could influence platelet recovery.     

The recovery of circulating progenitor cells after chemotherapy in AML and ALL and its relation to the rate of bone marrow regeneration after aplasia

Peripheral blood levels of BFU-e, CFU-GM and CFU-mix were studied serially in nine patients with acute leukaemia in remission during the period of recovery that followed induction or consolidation chemotherapy. Following 23 courses of treatment in the nine patients, the values for all three classes of progenitor were found to be higher in ALL than in AML (mean peak CFU-GM levels 5.8 x 10(3)/ml and 0.8 x 10(3)/ml respectively) and the highest levels were observed in patients recovering the most rapidly from bone marrow aplasia. Peak levels of these progenitors correlated best with the rate and extent of platelet recovery and all patients achieving blood levels of greater than 1.0 x 10(3) CFU-GM/ml had recovered greater than 100 x 10(9)/l platelets by 20 d and had peak platelet counts of greater than 400 x 10(9)/l within 40 d following chemotherapy. The peak values for circulating progenitors fell markedly after repeated courses of treatment in three of the five AML patients studied and this is likely to limit useful harvesting of such cells during later consolidation courses in this disease.     

Expansion of peripheral blood progenitors by recombinant human granulocyte colony-stimulating factor]

The levels of peripheral progenitor cells was measured serially after cancer chemotherapy in 4 patients with non-Hodgkin's lymphoma and one patient with rhabdomyosarcoma who received recombinant human granulocyte colony-stimulating factor (rG-CSF). This study was composed of two independent phases: in the first phase, patients received a course of cytotoxic chemotherapy only, and in the second phase, they received the same chemotherapy followed by subcutaneous injection of rG-CSF (2 micrograms/kg/day) for 10-14 days. In the control phase, the levels of granulocyte-macrophage colony-forming units (CFU-GM) and erythroid burst-forming units (BFU-E) per milliliter increased during the early recovery phase, but rG-CSF treatment increased the number of CFU-GM 3 to 18-folds, and the number of BFU-E increased 1.3 to 4.6-folds. An overshoot in the blood progenitor levels occurred at the day 8-10 of rG-CSF administration. And then, the peak of neutrophil count followed 3-5 days later. After the discontinuation of rG-CSF, the number of blood CFU-GM and BFU-E fell rapidly. This results suggest that in vivo expansion of circulating hemopoietic progenitors can be achieved by the administration of rG-CSF, and this approach might be clinically applicable to cancer patients who are a candidate of peripheral blood stem cell autotransplantation.     

Prolonged exposure to cytosine arabinoside in the presence of hematopoietic growth factors preferentially kills leukemic versus normal clonogenic cells

We investigated the cytotoxic effect of the cell cycle-specific agent cytosine arabinoside (Ara-C) on clonogenic leukemic and normal bone marrow cells. To overcome kinetic resistance and to increase cytotoxicity, the cells were exposed to Ara-C in liquid culture medium for extended time periods, that is, 5 and 10 days. Subsequently the number of surviving clonogenic cells was determined in a semi-solid assay. All cultures were stimulated with the combination of recombinant human interleukin 3 (rhIL-3), granulocyte-macrophage colony-stimulating factor (rhGM-CSF), and granulocyte colony-stimulating factor (rhG-CSF) to induce optimal cell proliferation. In comparison to normal clonogenic bone marrow cells (granulocyte-macrophage colony-forming units, CFU-GM) 5-day Ara-C exposure resulted in an equal to a slightly more effective kill of leukemic colony-forming cells (CFU-L). The Ara-C dose resulting in 50% inhibition (ID50) was 1.6 +/- 1.6 x 10(-8) M for CFU-L (n = 9) and 6.7 +/- 4.3 x 10(-8) M for CFU-GM (n = 4, p = 0.096). Prolongation of the Ara-C exposure time from 5 to 10 days increased the cytotoxicity towards the majority of the leukemic clonogenic cells (ID50: 0.8 +/- 0.6 x 10(-8) M) but not towards CFU-GM (ID50: 5.7 +/- 2.8 x 10(-8) M). Overall, significantly more leukemic clonogenic cells than normal CFU-GM were killed after 10 days of exposure to Ara-C (p = 0.039). These results indicate that leukemic clonogenic cells can be eradicated preferentially by prolonged exposure to low dosages of Ara-C in the presence of hematopoietic growth factors with relative preservation of the normal hematopoietic progenitor cells.     

Flow cytometry for clinical estimation of circulating hematopoietic progenitors for autologous transplantation in cancer patients.

Optimum methods of harvesting circulating hematopoietic progenitors for autologous transplantation to support myeloablative cancer therapy are still uncertain, mostly because of the lack of an assay for marrow-repopulating stem cells. The CFU-GM assay, the commonly used indirect indicator of the quality of the graft, is poorly standardized and provides results evaluable only retrospectively. Based on the knowledge that hematopoietic progenitors express CD34 and CD33 differentiation antigens, we developed a dual-color direct immunofluorescence flow cytometry assay with the aim of replacing the CFU-GM assay advantageously. For this purpose, we applied both assays to 157 blood samples obtained daily throughout 20 different recoveries from pancytopenia induced by high-dose cyclophosphamide or etoposide cancer therapy with or without recombinant human GM colony-stimulating factor (rhGM-CSF). The appearance of CD34+ cells in the circulation indicated that hematopoietic progenitors had increased to more than 500 CFU-GM/mL, a level clinically adequate for large-scale harvest by leukapheresis. Total CD34+ cells correlated well with CFU-GM (r = .89), and data could be fitted by a linear regression line described by the equation y = 388.3 + 64.0x, where y = CFU-GM/mL and x = CD34+ cells per microliter. Moreover, in a series of six patients treated with myeloablative chemoradiotherapy, early hematopoietic recovery of marrow functions was predicted more accurately by the number of transplanted CD34+/CD33+ cells than by either total nucleated cells, CFU-GM, CD34+/CD33- cells, or CD34-/CD33+ cells. Data presented in this article favor clinical use of the CD34/CD33 flow cytometry assay to guide harvesting of circulating hematopoietic progenitors for autologous transplantation and contribute to better understanding of the role played by circulating hematopoietic progenitor cell subsets in marrow recovery after myeloablative cancer therapy.     

Factors influencing hematopoietic recovery after autologous blood stem cell transplantation in patients with acute myeloblastic leukemia and with non-myeloid malignancies

Factors influencing hematopoietic recovery (HR) after autologous blood stem cell transplantation (ABSCT) were analyzed in 73 patients with various non-myeloid malignancies (NMM), and in 58 patients with acute myeloblastic leukemia (AML). Peripheral blood stem cells were collected following mobilization with chemotherapy, granulocyte colony-stimulating factor (G-CSF), or chemotherapy plus G-CSF. The conditioning regimen used consisted of either chemotherapy alone (112 cases) or chemotherapy plus total body irradiation (19 cases). The median number of colony-forming units granulocyte-macrophage (CFU-GM) was similar in both groups of patients, with the median number of CD34(+) cells infused being higher in the AML group (5.4 vs 4 x 10(6)/kg; P = 0.03). Median time neutrophils >0.5 x 10(9)/l was 13 days in both groups, and median time to a platelet count >20 x 10(9)/l was longer in AML patients (14 vs 12 days; P = 0.01). In multivariate analysis, the only factors affecting neutrophil recovery in the NMM group were the CD34+ cell number (continuous model) and the CFU-GM dose (categorized model) infused, whereas for platelet recovery, previous chemotherapy also remained significant. In the AML group, the only factors significantly affecting the speed of neutrophil recovery were dose of CD34+ cells administered and the patient's age. As for platelet recovery, only the progenitor dose administered remained significant. In the NMM group, the most discriminating cut-off values for a rapid neutrophil and platelet recovery were 1.5 x 10(6) and 2.5 x 10(6) CD34+ cells/kg, respectively, and for AML patients these figures were 1.5 x 10(6) and 4 x 10(6) CD34+ cells/kg, respectively. Our results confirm the slower HR after ABSCT in AML, and highlight the importance of progenitor cell dose in accelerating HR after ABSCT.     

In vitro and in vivo hematopoietic effect of mutant human granulocyte colony-stimulating factor.

About 100 derivatives of human recombinant granulocyte colony-stimulating factor (rhG-CSF) were created by various gene-mutagenic techniques, and KW-2228, in which amino acids were replaced at five positions of N-terminal region of intact rhG-CSF, was picked up and evaluated for its biologic and physicochemical properties in comparison with intact rhG-CSF. KW-2228 showed two to four times higher specific activity than that of intact rhG-CSF in mouse and/or human bone marrow progenitor cells by colony-forming unit assay in soft agar, and by cell-proliferation assay in liquid culture. KW-2228 showed a potency to increase peripheral neutrophil counts when it was administered to normal C3H/He mice by single intravenous injection. Increase of total leukocyte count and neutrophils was observed, with peak level at 8 to 12 hours at low doses (0.5 to 1.0 micrograms/mouse), and the highest level was maintained for 24 to 30 hours at high doses (5 to 10 micrograms/mouse). The granulopoietic effect of KW-2228 was examined by several doses of single course (once daily for 10 days) or multiple courses (twice daily injection for 5 days followed by cessation for 9 days on one cycle, 3 cycles in total) of treatment. KW-2228 showed higher activity than that of rhG-CSF, especially at sub-optimal doses of multiple courses of treatment. Furthermore, KW-2228 was found to be more stable physicochemically and biologically than intact rhG-CSF, especially under thermal conditions at 56 degrees C and in the human plasma at 37 degrees C, suggesting a protease resistancy. Pharmacokinetic study showed that plasma concentration of KW-2228 assayed for its bioactivity maintained a higher level than that of intact rhG-CSF for 60 minutes after intravenous injection of this protein to normal mice. Those results suggest that KW-2228 might show a superior in vivo hematopoietic effect to intact rhG-CSF due to its high specific activity to progenitor cells, and also due to its improved physicochemical, biologic, and pharmacokinetic stability in host animals.     

Hematopoietic progenitor cells from allogeneic bone marrow transplant donors circulate in the very early post-transplant period

Despite the therapeutic efficacy of allogeneic bone marrow transplantation (allo-BMT), circulating hematopoietic progenitor cells after bone marrow transplantation have not been well characterized. In the present study, we focused on these 'post-transplant circulating progenitor cells (PTCPC)' which may be on their way to bone marrow. We analyzed the number of myeloid progenitor cells (CFU-GM) per 10 ml of peripheral blood (PB) on days 0 (just before transplantation), 1 (8-15 h after the completion of transplantation), 2, 3, 5, 7, 10, 14, 17, 21, 28 and 35 after allo-BMT in five transplant patients using a standard methylcellulose assay. In addition, high proliferative potential colony-forming cells (HPP-CFC) of the harvested donor bone marrow (BM) and day 1 PB of recipients were assayed in five patients. The origin of HPP-CFC from day 1 PB was analyzed by polymerase chain reaction of a DNA region containing a variable number of tandem repeats. The replating potential of these HPP-CFC was evaluated by a secondary colony assay. The proportion of CD38negative cells among CD34+ cells in the harvested BM and day 1 PB was evaluated by two-color flow cytometric analysis. The number of CFU-GM on day 1 ranged from 6 to 73/10 ml PB, and became undetectable on day 5. The reappearance of PTCPC was observed on day 14, along with hematopoietic recovery. The proportion of HPP-CFC among myeloid colonies from day 1 PB was significantly higher than that from harvested BM (44.3+/-10.4% vs 11.3+/-2.1%, respectively, n=5, P=0.0030). These HPP-CFC from day 1 PB were confirmed to be of donor origin. More than 90% of these HPP-CFC had replating potential. Two-color flow cytometric analysis revealed that the proportion of CD34+CD38negative cells was significantly higher in day 1 PB than in the harvested BM (61.0+/-16.5% vs 9.3+/-3.5%, respectively, n=7, P=0.0002). These observations suggest that both primitive and committed transplanted myeloid progenitor cells may circulate in the very early period following allo-BMT.     

Analysis of circulating hematopoietic progenitor cells after peripheral blood stem cell transplantation

We investigated the kinetics of posttransplant circulating progenitor cells (PTCPC) in the early phase after autologous (auto-) and allogeneic (allo-) peripheral blood stem cell transplantation (PBSCT). We analyzed the number of myeloid progenitor cells (CFU-GM) per 10 ml of peripheral blood (PB) on days 0 (just prior to transplantation), 1 (12-15 hours after completion of first transplantation), 2, 3, 5, 7, 10, 14, 17, 21 and 28 (after auto-PBSCT), and also additionally on day 35 after allo-PBSCT. A standard methylcellulose colony assay was used for analysing the number of CFU-GGM and BFU-E on all of the days. In addition, high proliferative potential-colony forming cells (HPP-CFC) of the harvested PBSC from donors and day 1 PB from recipients were assayed in 5 allo-PBSCT patients. Furthermore, a proportion of CD38- cells among CD34+ cells in the harvested PBSC and day 1 PB was evaluated by two-color flow cytometric analysis in 5 allo-PBSCT patients. The number of CFU-GM on day 1 ranged from 7 to 119 per 10 ml PB after auto-PBSCT, and from 15 to 61 per 10 ml PB after allo-PBSCT. After these transient increases, PTCPC diminished rapidly. Then, PTCPC emerged again on day 7 after auto-PBSCT and on day 10 or 14 after allo-PBSCT along with neutrophil recovery. A proportion of HPP-CFC among myeloid colonies from day 1 PB of recipients was significantly higher than that from the harvested PBSC from donors (65.6 +/- 12.7% vs. 17.4 +/- 13.0%, respectively, n = 5, P = 0.0013). In addition, two-color flow cytometric analysis revealed that the proportion of CD34+CD38- cells was significantly higher in day 1 PB of recipients than in the harvested PBSC from donors (57.5 +/- 17.6% vs. 11.7 +/- 4.9%, n = 5, P = 0.005). These observations suggest that both primitive and committed transplanted myeloid progenitor cells may circulate in the very early period following PBSCT.     

In vivo synergy between recombinant human stem cell factor and recombinant human granulocyte colony-stimulating factor in baboons enhanced circulation of progenitor cells

Recombinant human stem cell factor (rhSCF) and recombinant human granulocyte colony-stimulating factor (rhG-CSF) are synergistic in vitro in stimulating the proliferation of hematopoietic progenitor cells and their precursors. We examined the in vivo synergy of rhSCF with rhG-CSF for stimulating hematopoiesis in vivo in baboons. Administration of low-dose (LD) rhSCF (25 micrograms/kg) alone did not stimulate changes in circulating WBCs. In comparison, administration of LD rhSCF in combination with rhG-CSF at 10 micrograms/kg or 100 micrograms/kg stimulated increases in circulating WBCs of multiple types up to twofold higher than was stimulated by administration of the same dose of rhG-CSF alone. When the dose of rhG-CSF is increased to 250 micrograms/kg, the administration of LD rhSCF does not further increase the circulating WBC counts. Administration of LD rhSCF in combination with rhG-CSF also stimulated increased circulation of hematopoietic progenitors. LD rhSCF alone stimulated less of an increase in circulating progenitors, per milliliter of blood, than did administration of rhG-CSF alone at 100 micrograms/kg. Baboons administered LD rhSCF together with rhG-CSF at 10, 100, or 250 micrograms/kg had 3.5- to 16-fold higher numbers per milliliter of blood of progenitors cells of multiple types, including colony-forming units granulocyte/macrophage (CFU-GM), burst-forming unit-erythroid (BFU-E), and colony-forming and burst-forming units-megakaryocyte (CFU- MK and BFU-MK) compared with animals given the same dose of rhG-CSF without rhSCF, regardless of the rhG-CSF dose. The increased circulation of progenitor cells stimulated by the combination of rhSCF plus rhG-CSF was not necessarily directly related to the increase in WBCs, as this effect on peripheral blood progenitors was observed even at an rhG-CSF dose of 250 micrograms/kg, where coadministration of LD rhSCF did not further increase WBC counts. Administration of very-low- dose rhSCF (2.5 micrograms/kg) with rhG-CSF, 10 micrograms/kg, did not stimulate increases in circulating WBCs, but did increase the number of megakaryocyte progenitor cells in blood compared with rhG-CSF alone. LD rhSCF administered alone for 7 days before rhG-CSF did not result in increased levels of circulating WBCs or progenitors compared with rhG- CSF alone. Thus, the synergistic effects of rhSCF with rhG-CSF were both dose- and time-dependent. The doses of rhSCF used in these studies have been tolerated in vivo in humans.(ABSTRACT TRUNCATED AT 400 WORDS)     

Increased efficiency of gene transfer with retroviral vectors in neonatal hematopoietic progenitor cells

The retroviral vector N2, which is derived from the Moloney murine leukemia retrovirus, was used to transfer the bacterial NeoR gene (conferring resistance to the neomycin analogue G418) into hematopoietic progenitor cells from fetal, neonatal, and adult dogs and cats. Infection of canine and feline bone marrow cells with the N2 vector resulted in resistance of granulocyte-macrophage colony-forming units (CFU-GM) to G418. Approximately 2%-4% of fetal liver, fetal bone marrow, and adult bone marrow day-7 CFU-GM were resistant to 1.75 mg/ml G418, a dose toxic to cells not expressing the NeoR gene, after infection with the N2 retrovirus. In sharp contrast to the low rate of infectivity of both fetal and adult marrow samples, the mean +/- SD of G418-resistant CFU-GM was 11.7% +/- 14.1% and 14.0% +/- 18.1% for neonatal dog and cat marrow samples, respectively. The neomycin phosphotransferase enzyme activity was detected in G418-resistant CFU-GM, confirming that G418-resistant CFU-GM expressed the NeoR gene. The increased efficiency of retroviral vector-mediated gene transfer into neonatal hematopoietic progenitor cells was not due to an increased fraction of actively dividing cells, as determined by tritiated thymidine suicide. Understanding the basis for increased gene transfer into neonatal hematopoietic progenitor cells may be helpful in designing effective retroviral vectors/gene transfer protocols for gene therapy.     

Kinetics of circulating haematopoietic progenitors during chemotherapy-induced mobilization with or without granulocyte colony-stimulating factor

Summary. Kinetics of circulating haematopoietic progenitors was analysed during chemotherapy- or chemotherapy plus granulocyte colony-stimulating factor (G-CSF)-induced mobilization of peripheral blood stem cells. Circulating progenitors including colony-forming unit granulocyte/macrophage (CFU-GM), burst forming-unit erythroid (BFU-E) and multilineage colony forming unit (CFU-Mix) were studied serially on alternate days during a recovery phase from chemotherapy for consolidation of complete remission. In 18 patients with acute leukaemia, 27 courses of consolidation chemotherapy were performed with a combination of an intermediate-dose cytosine arabinoside with etoposide (Ara-C/Etop) or mitoxantron (Ara-C/Mit). G-CSF (5 μg/kg) was administered during the recovery phase in 6/14 courses with Ara-C/Etop and in 4/13 courses with Ara-C/Mit. G-CSF induced a significant and synchronized increase of circulating CFU-GM, BFU-E and CFU-Mix by more than 4-fold at their peaks. The peak of CFU-GM was significantly correlated with that of both BFU-E and CFU-Mix, irrespective of additional G-CSF mobilization. G-CSF also produced a significant increase of monocytes in a synchronized fashion with an increase of circulating CFU-GM. Interestingly, peripheral blood monocytes spontaneously produced high concentrations of IL-6; a significant correlation was observed between absolute monocyte counts and plasma levels of IL-6 or peak levels of CFU-GM.
These observations indicate that the addition of G-CSF to chemotherapy-induced mobilization can facilitate further expansion of a blood progenitor pool during the haematopoietic recovery, probably through the stimulation of monocytes to proliferate and to induce their monokine production such as IL-6. The data also suggest that absolute monocyte counts may be a useful indicator to predict the peak of circulating progenitors for collecting autologous blood stem cells.     

Rapid and complete hematopoietic reconstitution following peripheral blood stem cell autotransplantation in a child with T cell acute lymphoblastic leukemia

Rapid and complete hematopoietic reconstitution was achieved in a child with T cell acute lymphoblastic leukemia who was autografted with peripheral blood stem cells (PBSC). A large number of PBSC was collected by two courses of 3-4 hour-lasting lymphopheresis during early remission induced by the second-line chemotherapy and then cryopreserved in liquid nitrogen. A myeloid progenitor cell dose of 203 X 10(4) CFU-GM/kg body weight was reinfused to the patient following marrow-ablative chemotherapy (MCNU 600 mg/m2, cytosine arabinoside 6 g/m2, etoposide 300 mg/m2, cyclophosphamide 160 mg/kg). Neutrophil count reached 0.5 X 10(9)/l by day + 7 and platelet count reached 20 X 10(9)/l by day + 9. Thereafter, white blood cell count continued to increase and reached a maximum of 38 X 10(9)/l on day + 14. Thus, the rapid recovery of hematopoiesis minimised marrow aplasia-related risks. This approach of stem cell rescue operation can be applied to the treatment of children with cancer, who otherwise have no hope to be cured, as an alternative to bone marrow transplantation.     

Very large amounts of peripheral blood progenitor cells eliminate severe thrombocytopenia after high-dose melphalan in advanced breast cancer patients

We analyzed the relationship between the reinfusion of large or very large amounts of peripheral blood progenitor cells (PBPC) and hematologic toxicity in twenty-one advanced breast cancer patients subjected to a myeloablative dose of melphalan at the end of a high-dose sequential chemotherapy (HDSC) program. We also evaluated the influence of the white blood cell (WBC) count to predict an optimal PBPC harvest after high-dose chemotherapy and growth factor priming. Twenty-one patients with high-risk or metastatic breast cancer sequentially received: high-dose cyclophosphamide (HD-Cy) and G-CSF followed by PBPC harvest, HD-methotrexate plus vincristine, HD-doxorubicin, cisplatin and finally HD-melphalan 200 mg/m2 (HD-L-PAM) followed by PBPC reinfusion. No growth factor was administered after HD-L-PAM. CD34+ cytofluorimetric analysis, WBC count and clonogenic assays were employed to monitor circulating cells and to analyze the PBPC harvest. Correlation between different PBPC doses and hematologic toxicity as well as leukocyte and platelet recovery time was attempted. Patients received a median number of 16 (4-25.1) x 10(6)/kg CD34+ cells, 81.3 (30.8-228) x 10(4)/kg CFU-GM and 4.2 (1.3-7.3) x 10(8)/kg nucleated cells (NC) after HD-L-PAM. The number of days with fewer than 1 x 10(9)/l leukocytes and 20 x 10(9)/l platelets were 6 (range 4-9) and 0 (range 0-3), respectively. The CD34+ cell dose significantly correlated with both platelet count nadir (r = 0.73) and time to 50 x 10(9)/l platelets (r = 0.7), but did not correlate with time to reach more than 1 x 10(9)/l WBC count (r = 0.2). In particular, we found that in 12 patients given very large amounts of CD34+ cells, ranging between 15.8 and 25. 1 x 10(6)/kg (V-LA-CD34+), the platelet nadir count never fell below 20 x 10(9)/l and platelet transfusions were not required. Conversely, nine patients who received only large amounts of CD34+ cells, ranging between 4 and 12 x 10(6)/kg (LA-CD34+), experienced a platelet nadir lower than 20 x 10(9)/l and required 2 days (range 1-4) to achieve independence from platelet transfusions (P = 0.001 and P = 0. 0005). The requirement for packed red blood cells (RBC) was 1.5 vs 3 units in the V-LA-CD34+ and LA-CD34+ groups respectively (P = 0.063). The analysis of 44 PBPC collections demonstrated that 29 aphereses performed with a WBC count <20 x 10(9)/l yielded a mean of 312 +/- 43 x 10(6) CD34+ cells and 1831 +/- 201 x 10(4) CFU-GM, whereas 15 collections performed with WBC count >20 x 10(9)/l yielded 553 +/- 64 x 10(6) CD34+ cells and 3190 +/- 432 x 10(4) CFU-GM (P = 0.004). In conclusion, our data suggests that V-LA-CD34+ eliminates severe thrombocytopenia and platelet transfusion requirements in breast cancer patients subjected to HD-L-PAM, and higher PBPC collections seems to coincide with WBC count higher than 20 x 10(9)/l after HD-Cy and G-CSF mobilization. These results justify a prospective study to establish whether large doses of CD34+ cells result in significant clinical benefits.     

Clinical significance of recombinant human granulocyte colony-stimulating factor (rG-CSF) in the chemotherapy of patients with malignant lymphoma]

We examined the effects of recombinant human granulocyte colony-stimulating factor (rG-CSF) on neutropenia induced by chemotherapy in 10 patients with non-Hodgkin's lymphoma (NHL). The numbers of peripheral blood hematopoietic progenitors were also evaluated before and after administration of rG-CSF. Six patients received an administration of 2 micrograms/kg/body weight of rG-CSF subcutaneously for 14 days after 2nd chemotherapy. Four patients received intravenous infusion of rG-CSF (300 micrograms/body/day) for 4 days from nadir state after chemotherapy. Administration of rG-CSF from the termination of chemotherapy, markedly shortend the period of bone marrow hypoplasia induced by chemotherapy. On the other hand, administration of rhG-CSF from nadir state after chemotherapy have accelerated the recovery of neutrophil counts. In addition, this type of therapy induced 26 to 60 folds increase of peripheral blood hematopoietic progenitors. These results demonstrate the validity of administration of rhG-CSF not only in the chemotherapy of NHL, but also in peripheral blood stem cell transplantation (PBSCT).     

Comparative study of peripheral blood progenitor cell collection in patients with multiple myeloma after single-dose cyclophosphamide combined with rhGM-CSF or rhG-CSF

Summary. Patients suffering from high-risk multiple myeloma (MM) were randomized to receive single high-dose cyclophosphamide followed by either rhGM-CSF or rhG-CSF in order to harvest circulating peripheral blood progenitor cells. The safety of the procedure, the mobilization kinetics, the relative efficacy of rhGM-CSF and rhG-CSF to mobilize progenitor cells and their relative toxicity were studied. Special attention was paid to the antigenic profile of CD34⁺ progenitor cells.
Group I patients (n=ll) were treated with cyclophosphamide 4 g/m² i.v. followed by rhGM-CSF at 10 /ig/kg/d by subcutaneous administration. Group II (n=ll) patients received rhG-CSF s.c. at 10/zg/kg/d after the same dose cyclophosphamide. Both mobilization regimens appeared to be equally effective. No significant differences in absolute numbers of circulating progenitors, determined by CD34 expression or in yields of MNC, CFU-GM, BFU-E and CD34 subsets were observed. rhGM-CSF administration resulted however in delayed haemopoietic recovery and an increased complication rate.
We conclude that rhG-CSF may be preferred because of its markedly lower toxicity and lower in-hospital costs.     

Range and clinical significance of the number of myeloid-committed stem cells in the blood of patients with acute leukaemia in remission

Circulating granulocyte/macrophage progenitor cells (CFU-GM) were assayed serially during remission in 17 patients with acute leukaemia (9 ALL, 8 AML). In patients with ALL receiving cyclophosphamide, 6-mercaptopurine and methotrexate, CFU-GM numbers were significantly lower than in normal individuals; cycles of 'reinduction' chemotherapy (vincristine, prednisolone) caused a 10-fold increase in CFU-GM per ml of blood. In 2 ALL patients a substantial increase in CFU-GM numbers preceded the morphologically detectable relapse. In patients with AML receiving repeated courses of cytosine-arabinoside and 6-mercaptopurine, circulating CFU-GM were likewise reduced. In 6 patients who relapsed, a further reduction of CFU-GM was seen. A complete absence of circulating CFU-GM was observed in 10 of the 23 investigations performed within 6 weeks prior to the morphologically detectable relapse, while such a 'zero'-growth occurred in only 1 of 54 experiments performed during stable remission. In summary, in patients with ALL in remission, circulating CFU-GM are increased following treatment with vincristine and prednisolone. In patients with AML, declining numbers of circulating CFU-GM may predict an imminent relapse.     

Ex-vivo expansion of bone marrow progenitor cells for hematopoietic reconstitution following high-dose chemotherapy for breast cancer

The use of hematopoietic growth factors, stromal monolayers, and frequent medium exchange allows the expansion of hematopoietic progenitors ex-vivo. We evaluated the use of ex-vivo expanded progenitor cells for hematopoietic reconstitution following high dose chemotherapy (HDC) in breast cancer patients. Patients with high-risk Stage II or metastatic breast carcinoma underwent bone marrow aspirations using general anesthesia. A total of 675-1125 x 10(6) mononuclear cells (MNC) were seeded for ex-vivo expansion for 12 days in controlled perfusion bioreactors (Aastrom Biosciences, Inc.). The bone marrow cultures, which included the stromal cells collected with the aspirate, were supplemented with erythropoietin, granulocyte-macrophage-colony stimulating factor (GM-CSF)/IL-3 fusion protein (PIXY 321), and flt3 ligand. Stem cell transplant was performed with expanded cells after HDC. A median bone marrow volume of 52.9 mL (range 42-187 mL) was needed to inoculate the bioreactors. Median fold expansion of nucleated cells (NC) and colony forming unit granulocyte-macrophage (CFU-GM) was 4.9 and 9.5, respectively. The median fold expansion of CD34+lin- and long-term culture-initiating culture (LTC-IC) was 0.42 and 0.32, respectively. Five patients were transplanted with ex-vivo expanded NC. Median days to an absolute neutrophil count > 500/microL was 18 (range 15-22). Median days to a platelet count > 20,000/microl was 23 (range 19-39). All patients had sustained engraftment of both neutrophils and platelets. Immune reconstitution was similar to that seen after HDC and conventional stem cell transplantation. We conclude that ex-vivo expansion of progenitor cells from perfusion cultures of small volume bone marrow aspirates, allows hematopoietic reconstitution after HDC.