We assessed the ability of granulocyte colony-stimulating factor (G-CSF) to mobilize peripheral blood stem cells (PBSC), the efficacy of PBSC infusion in the rescue of hematopoiesis after high-dose chemotherapy in 22 adult patients with hematological malignancies, and the long-term quality of engraftment. Bolus subcutaneous injections of G-CSF (12 micrograms/kg/day) were administered for 6 days. A median of three leukaphereses resulted in the collection of a median of 5.45 X 10(8) mononuclear cells/kg, 4.52 X 10(6) CD34+ cells/kg, and 3.97 X 10(4) CFU-GM/kg. G-CSF (5 micrograms/kg daily) was administered after PBSC infusion until granulocyte recovery. The median time to attain a neutrophil level > 0.5 X 10(9)/L and a platelet count > 20 X 10(9)/ L was 11 days. The median follow-up in 17 survivors was 23.8 months. These patients have maintained a complete and stable graft and some of them with neoplastic recurrence tolerated further chemotherapy. These results confirm that mobilization of PBSC by G-CSF can be performed on an outpatient setting and used in heavily pretreated patients. G-CSF mobilized PBSC transplantation provides early and complete engraftment in most patients.

Twenty-one previously untreated multiple myeloma (MM) patients and 10 previously treated patients with refractory or relapsed disease received two or three cycles of intermediate-dose melphalan (70 mg/m2) (IDM), administered intravenously every 6 weeks. Seven previously untreated patients received three and all other patients received two courses of IDM. The objective of the study was to reduce the toxicity of high-dose melphalan (140 mg/m2) (HDM) while maintaining its cytotoxic efficacy and secondly to ensure the possibility of collecting sufficient numbers of peripheral blood stem cells (PBSC) for transplantation. 18 (85%) previously untreated patients responded, of whom four achieved CR (18%). In addition five out of 10 previously treated patients with refractory or relapsed disease responded although bone marrow toxicity in this category was a major drawback. Toxicity was moderate, consisting of alopecia and moderate bone marrow suppression: the granulocyte count dropped below 0.5 x 10(9)/l and platelets below 25 x 10(9)/l for a median of 8 and 6 d, respectively. No serious infections occurred and the majority of patients attended the out-patient clinic. In 12/14 previously untreated patients sufficient peripheral blood CD34+ cells for harvest were present in the repopulation phase after the first IDM. In nine patients peripheral blood stem cells were collected and eight patients have undergone successful transplantation. Repeated IDM followed by filgrastim is highly effective in untreated MM and may be safely administered to reduce tumour load prior to PBSC collection. Autologous stem cells harvested after repeated IDM have a full long-term repopulating capacity.

To study the toxicity and efficacy of combined granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) administration for mobilization of hematopoietic progenitor cells (HPCs).

The effectiveness of arabinosylcytosine (ara-C) for the treatment of acute myelogenous leukemia (AML) depends on the formation of its active metabolite, the triphosphate of ara-C (ara-CTP). Using biochemical modulation strategies to increase the accumulation of ara-CTP in leukemia blasts, a clinical protocol was designed combining 2-chlorodeoxyadenosine (CdA), an inhibitor of ribonucleotide reductase, and ara-C for adults with AML. The protocol stipulated an infusion of 1 g/m2 of ara-C over 2 hours on day 1. A continuous infusion of CdA (12 mg/m2/d) begun 24 hours later and continued for 5 days. Identical doses of ara-C were administered on days 3, 4, 5, and 6. Pharmacokinetic and pharmacodynamic interactions between CdA and ara-C during therapy were investigated. To complement these studies, molecular actions of the triphosphate of ara-C and CdA on DNA extension by human DNA polymerase alpha in an in vitro model system was conducted. In the circulating leukemia blasts of 7 of the 9 patients studied, ara-CTP pharmacokinetics showed a median 40% increase in the rate of ara-CTP accumulation after 24 hours of CdA infusion. The ex vivo effect of CdA on accumulation of ara-CTP in AML blasts was similar to that during therapy except that the enhancement was less. The DNA synthetic capacity of the circulating blasts was inhibited to a greater extent by administration of CdA and ara-C in combination than by either one alone. Additionally the lowered level of DNA synthesis was maintained until the next infusion of ara-C. Endogenous levels of deoxynucleotides increased 24 hours after ara-C infusion. Administration of CdA in general lowered the concentrations of all dNTPs. DNA pol alpha incorporated CdATP and ara-CTP with high affinity in a DNA primer extending over an oligonucleotide template of defined sequence. Human DNA polymerase alpha extended DNA primers terminated by CdA monophosphate (CdAMP) at its 3'-end by incorporating ara-C monophosphate (ara-CMP). The tandem incorporation of CdAMP and ara-CMP resulted in nearly complete inhibition of DNA primer extension. The insertion of two analogs in sequence, inhibition of ribonucleotide reductase, and the metabolic potentiation of ara-CTP by CdA infusion may be responsible for sustained inhibition of DNA synthesis in the circulating leukemia blasts during therapy with this combination regimen.

The response of acute promyelocytic leukemia (APL) peripheral blood and bone marrow cells to trans-retinoic acid (RA) was cytogenetically characterized during RA treatment using the techniques of premature chromosome condensation (PCC) and fluorescence in situ hybridization (FISH). Before treatment, the predominant immature bone marrow cells were found to have t(15;17), whereas the residual mature granulocytes were diploid and lacked evidence of the translocation. In response to RA treatment, an increase in the leukocyte count was noted. The majority of these cells exhibited a t(15;17). Subsequently (eg, between days 6 and 23), 32% to 91% of the maturing myeloid cells still exhibited t(15;17). The appearance of t(15;17) in gradually maturing elements suggests that RA contributed to a release of the maturation block of the leukemic elements. As responding patients obtained complete remission, diploid elements without evidence of the translocation prevailed in the blood and bone marrow. In 16 patients studied after 1 month in complete remission, all but 2 showed all diploid cells. The residual t(15;17) cells disappeared 18 days later in 1 patient, whereas the second patient exhibited clinical evidence of relapse 20 days later. These results suggest that response of patients with APL to RA is associated with maturation, subsequent loss of the mature leukemic elements, and preferential regeneration of normal diploid hematopoietic elements.

Fourteen poor risk acute myeloid leukemia (AML) patients were treated with G-CSF prior (from day 0) and during chemotherapy with fludarabine and Ara-C from day 1 to day 5 (the FLAG regimen). Several biological parameters were monitored on the blast population: multidrug resistance (MDR) functional expression by rhodamine-123 efflux (Rhd-E), cell cycle changes, induction of apoptosis and leukemic clonogenic cell growth (CFU-L). The mean basal Rhd-E value was 14.4% (range 0-51.2), and 12/14 patients exhibited a dye efflux > 4%, efficiently blocked by the MDR-reversal agent cyclosporin A. After 24 h of G-CSF administration, cell cycle studies showed in bone marrow (BM) samples a significant mean increase in S phase (p = 0.04) and in RNA content of G1 cells (p = 0.01), coupled to a significant increase in apoptosis (p = 0.02). Clonogenic cell growth analysis showed a twofold increase in BM CFU-L in 6 of the 14 cases tested. When G-CSF activity was assessed without the addition of exogenous growth factors (autonomous proliferation), a significant increase (p = 0.02) in CFU-L was found only in patients who achieved a complete remission (CR); these patients were also characterized by lower S-phase values at diagnosis. Eight of the 14 patients treated achieved CR, but the median response duration was three months, and only two cases are still in CR. The FLAG regimen can thus induce remission in poor risk AML patients. The responses, however, are short, suggesting that resistant cells are not efficiently affected by either the use of agents not involved in the MDR-efflux mechanism or by the G-CSF priming strategy. Other post-induction therapies need to be considered in further approaches.

Thalassemia is widely distributed throughout the world and is one of the major public health problems. The use of bone marrow transplantation, the only curative therapy for thalassemia, is limited because less than 30% of the patients have unaffected and HLA-identical siblings as donors. Cord blood stem cells, an alternative source of stem cells for transplantation, have been successfully transplanted into patients with several diseases after myeloablative therapy. Twenty cord blood samples from unaffected neonates whose siblings had severe thalassemia were collected. The median volume was 80 ml. The median number of cells and colony forming units-granulocyte-macrophage in cord blood was 9.2 x 10(8) and 3.4 x 10(5), respectively. Four of 20 cord blood samples had HLA-matched to the affected siblings. One patient underwent cord blood transplantation with success; one patient is waiting for transplantation.

In June 1992, we started a dose-escalated cytotoxic therapy with peripheral blood progenitor cell (PBPC) transplantation in patients with chemosensitive multiple myeloma (MM). At the time of best response to conventional treatment, 70 patients received high-dose cyclophosphamide (HD-CY) or, in case of pre-existing heart disease, dose-escalated ifosfamide/mitoxantrone followed by filgrastim (R-metHuG-CSF, 300 micrograms/day). PBPC collection was commenced when CD34+ cells were detectable using direct immunofluorescence analysis. Fifty-four out of 70 patients were successfully harvested (> or = 2.5 x 10(6) CD34+ cells/kg body weight [BW]) after the first cycle of HD chemotherapy. Conditioning therapy consisted of 140 mg/m2 melphalan plus TBI (14.4 Gy hyper-fractionated) or 200 mg/m2 melphalan in patients not eligible for TBI because of previous radiotherapy. To date, 56 patients have been transplanted. Autografts contained a median of 3.4 x 10(6) CD34+ cells/kg BW. Following reinfusion of PBPC, rapid engraftment was achieved in 54 out of 56 patients with a median of 14 days (range 9-23) to reach 0.5 x 10(9)/l neutrophils and 10 days (range 5-22) for an unsubstituted platelet count of > 20 x 10(9)/l. One patient died of transplantation-related complications. Sequential HD treatment improved the remission status (European Bone Marrow Transplantation criteria) in 19 out of 46 patients (9 patients too early). Of note, in 11 patients the immunofixation became negative and a polyclonal immunoglobulin reconstitution was achieved. Our protocol provides an effective treatment strategy for patients with advanced MM combined with low treatment-related toxicity.

Between September 1991 and April 1995, high-dose therapy with peripheral blood progenitor cell (PBPC) support was administered to 105 patients with non-Hodgkin's lymphoma (NHL). Thirty-three patients had high-grade NHL, while 72 patients had different forms of low- or intermediate-grade NHL. Except for three patients who received G-CSF during steady-state hematopoiesis, PBPCs were collected following cytokine-supported cytotoxic chemotherapy. This included G-CSF or the sequential administration of interleukin 3 (IL-3) and GM-CSF. Assessing bone marrow (BM) samples before the start of chemotherapy and leukapheresis (LP) products collected during cytokine-enhanced marrow recovery, a 2.3-fold greater mean concentration of CD34- cells was found in peripheral blood (p < 0.005). The blood-derived progenitor cells were enriched with a particular subset of more primitive progenitors, as the mean proportion of CD34+/Thy-1+ cells in LP products was three-fold greater in comparison to premobilization BM samples, respectively (p < 0.001). In contrast, the mean proportion of CD34+/CD19+ and CD19+ cells in LP products was 8.8- and 80-fold smaller compared to BM samples, respectively (p < 0.001). Following high-dose conditioning therapy including TBI in 74 patients, reinfusion of PBPC resulted in rapid and sustained engraftment in the majority of patients, while in seven patients an unsubstituted platelet count of greater than 20 x 10(9)/l was reached between 31 and 51 days. Five patients died of treatment-related complications between 13 and 188 days following transplantation. The probability of long-term disease-free survival at 30 months in patients autografted while they were in first remission was 70% in high-grade and 83% in low-grade NHL, respectively. The data may provide the rationale for the use of PBPC-supported high-dose regimens as first-line treatment for patients at high risk of treatment failure.

Autologous peripheral stem cell transplantation was initiated at the University of Nebraska Medical Center to provide hematopoietic rescue for patients who were candidates for high-dose therapy but had marrows that were unfit for autografting. From 1984 until 1991, the cells were collected during steady state without mobilization. These cells restored hematopoietic function at a rate similar to that of autologous marrow in patients not treated with total body irradiation. Patients who received total body irradiation experienced slower hematopoietic recovery. When growth factors became generally available in the United States in 1991, mobilization with cytokines became standard at Nebraska. In recent years, the function of cells other than hematopoietic progenitors contained in a peripheral stem cell apheresis product has been studied. Detection of tumor cells using cell culture, immunocytochemical and polymerase chain reaction techniques has revealed that such collections are less likely to contain, or contain fewer, tumor cells than autologous bone marrow harvests. More antitumor cytotoxic activity was found in cells in peripheral stem cell collections than in marrow cell collections. The immunocompetent lymphocyte population is larger in peripheral stem cell collection than in marrow harvests, and immunologic recovery after peripheral stem cell transplant has differed compared to recovery following bone marrow transplantation. The future of peripheral stem cell transplantation is likely to include engineering of the graft products specific for the patient and disease being treated. Determining the function of accessory cells in a peripheral stem cell collection will be important to provide the best engineered product for the patient.

Since our initial report of successful peripheral blood stem cell transplantation (PBSCT) in a patient with acute myeloid leukemia (AML), we have performed more than 300 PBSCTs; 49 of them were done for AML patients. PBSC mobilization (and collection) was influenced by the number of previous courses of chemotherapy and significantly increased when G-CSF was combined with chemotherapy for mobilization. Hematopoietic recovery (HR) was complete in every patient. The HR rate was influenced by the number of cells transplanted. Platelet recovery was significantly quicker for patients given G-CSF for mobilization. The outcome of patients transplanted in first or second remission was similar to that usually observed after bone marrow transplantation.

Expression of the multidrug resistance (MDR-1) gene product, P-glycoprotein (P-170), and the stem cell antigen, CD34, at diagnosis were determined using monoclonal antibodies (MoAbs) MRK-16 and 12.8 respectively, in 130 pediatric acute myeloid leukemia (AML) patients entered onto Childrens Cancer Group (CCG) study CCG-2891. Fluorescein isothiocyanate (FITC) as a second step reagent was employed for the measurement of P-170 expression since it is commonly used in clinical laboratories. Nine of 30 (30%) infant ( < 1 year of age) de novo specimens expressed P-170 at levels > or = 20% of control cells. In contrast, eight of 100 (8%) AML samples from older children ( > or = 1 year of age) expressed the multidrug resistance surface protein at diagnosis. With the exception of one infant, all de novo samples that expressed P-170 also expressed CD34. Pediatric patients of any age with positive P-170 expression using MoAb MRK-16 with a FITC-conjugated second step reagent fared no worse than remaining patients treated on the same treatment with regard to induction failure, incidence of relapse, event-free survival, or overall survival. Further investigation is necessary to determine whether P-170 assay systems with greater sensitivity will distinguish pediatric AML patients with poor prognosis.

To improve the management of chronic myeloid leukemia (CML) in a single center, we used interferon alpha (IFN alpha) to treat newly diagnosed CML patients and investigated the factors predictive of a major cytogenetic response. Fifty-two patients (pts) with a median age of 51.5 years (16-68), were given interferon alpha (IFN alpha) (5 millions/m2/day, subcutaneously). The median interval between diagnosis and IFN alpha was 41.5 days (0-160). The doses of INF alpha were adjusted to maintain the white blood cell (WBC) count between 1.5 and 5 x 10(9)/l and the platelet count between 50 and 100 x 10(9)/l. At diagnosis, Sokal's criteria were used to classify patients into three groups: low (n = 24), intermediate (n = 19) and high risk (n = 9). A complete hematological response (CHR) was achieved in 42 cases (80.7%). A partial response was present in nine; only one patient did not respond. By multivariate logistic regression analysis, only the age at diagnosis was found to influence the CHR rate (P = 0.06). Cytogenetic response was evaluated in 46 responder patients. Twenty-three patients achieved a major cytogenetic response (MCR) which was either partial ( > or = 65% pH negative cells) (n = 3) or complete (CCR) (n = 20). By univariate analysis, two disease-related variables were found to influence the MCR rate in 40 evaluable CHR patients: spleen size at diagnosis and peripheral blood blast percentage. However, using either univariate or multivariate analysis, the most significant factor was the achievement of CHR within 3 months (P < 0.0004 and P < 0.0002, respectively). These results show that IFN alpha can induce high rates of hematological and cytogenetic responses when administered in doses leading to myelosuppression. The achievement of CHR within 3 months could be useful to identify early, those patients who will not respond to IFN alpha and who need alternative treatments such as allogeneic or autologous stem cell transplantation.

As clinical trials for gene therapy in Gaucher disease (GD) begin, questions regarding the biology of the hematopoietic stem cell in this disease remain unanswered. This study demonstrates the ability to mobilize and collect CD34+ cells in three patients with the disorder. Our RAC/FDA-approved clinical trial utilizes mobilized peripheral blood stem cells (PBSC) as the target cells for gene transfer. In this approach, a white blood cell fraction is collected by apheresis, enriched for CD34+ cells, and transduced with a retroviral vector carrying the glucocerebrosidase (GC) gene. Transduced cells from the patient with activity corrected to at least normal levels will be returned to the patient without myelosuppressive therapy. We report here the effect of cytokines in mobilizing PBSC in three patients with GD. Two (patients 1 and 2) were given granulocyte colony-stimulating factor (G-CSF) at a dose of 5 micrograms/kg/d and one (patient 3) was given 10 micrograms/kg/d for 10 days. Leukaphereses were done daily for 5 days and the products enriched for CD34+ cells using the clinical Ceprate (CellPro) column. The CD34+ cells in all fractions were monitored daily during mobilization and leukaphereses. Subset analysis for the expression of Thy-1, CD38, HLA-DR, and CD33 on the CD34+ cells was performed. An increase in CD34+ cells in the peripheral blood was noted from day 5 onward (up to a six-fold increase). Up to a 625-fold enrichment in CD34+ cells in the apheresis product was noted using the clinical Ceprate column. Totals of 1.2, 3.5, and 2.1 x 10(6) CD34+ cells/kg were collected in the three patients. A diminution in the percent of CD34+/Thy-1+ cells was noted with enrichment. In vitro retroviral transduction of the CD34-enriched cells using centrifugation promoted transduction protocol previously described (Bahnson AB et al., Centrifugal enhancement of retroviral-mediated gene transfer. Journal of Virology Methods 54:131, 1995) and modified for clinical use, demonstrated a mean transduction efficiency of 37% (range 8.3-87.1%) in clonogenic cells and up to 50% in long-term culture-initiating cells (LTC-IC) at week 6. Significantly, we have been able to achieve up to a 50-fold increase in the level of GC above deficient levels in the patients' CD34+ enriched cells when maintained in vitro in culture. The study demonstrates that up to a six-fold increase in CD34+ cells in the PB can be achieved with cytokines in patients with GD. CD34+ cells can be collected in numbers sufficient for conventional transplantation and transduced efficiently in vitro. In gene therapy trials for genetic disorders to date, myelosuppressive therapy is not advocated. The clinical trial will demonstrate whether this number of transduced CD34+ cells will be adequate for competitive engraftment of genetically corrected PBSC.

Remission marrow from patients with BCR-ABL+ acute lymphoblastic leukemia (ALL) achieving clinical remission (CR) after induction or consolidation chemotherapy according to the German multicenter adult ALL (GMALL) protocol showed high titers of residual BCR-ABL+ cells. Therefore, we initiated a pilot study to monitor circulating BCR-ABL+ cells and to collect, purge, and autograft peripheral blood stem cells (PBSC) in these patients. After GMALL 05/93 high-risk phase II of induction chemotherapy (high-dose AraC 3 g/m2 x 8 does and mitoxantrone 10 mg/m2 x 3 doses), patients received 5-10 micrograms/kg subcutaneous recombinant human granulocyte colony-stimulating factor (rhG-CSF) daily. Mobilized CD34+ cells peaked between 20 and 26 days after starting chemotherapy at 4.8-75.6 (median 10.8) x 10(4)/mL peripheral blood (PB) (n = 5). Patients treated with additional chemotherapy cycles failed to mobilize adequate numbers of CD34+ cells. PB stem cells (PBSC) were purged using a cocktail of CD10, CD19, and AB4 monoclonal antibodies (mAbs) coupled to immunomagnetic beads (IMB). The median recoveries of total nucleated cells (TNC) and CD34+ cells after mAb/IMB purging were 84 and 81%. The peak numbers of CD34+ cells collected in a single leukapheresis were median 8.6 x 10(6)/kg pre- and 5.2 x 10(6)/kg postpurge (n = 4). The absolute prepurge CD19+ cells were as low as median 2.7 (range 1.4-19) x 10(6) per leukapheresis. Residual BCR-ABL+ cells in unpurged leukapheresis products were assessed by limiting-log10-dilution nested reverse-transcriptase polymerase chain reaction (RT-PCR) as one in 10(5) to one in 10(6) normal cells and were consistently undetectable in all purged PBSC autografts. We conclude that sufficient numbers of CD34+ cells for PBSCT can be collected after phase II but not at later stages of the GMALL 05/93 high risk protocol; PBSC grafts are 3 log less contaminated with residual BCR-ABL+ cells compared to an historical series of 13 autologous BM grafts; and purging of PBSC with mAb/IMB is feasible with minor loss of CD34+ cells and abolished BCR-ABL signals in the grafts.

The more mature erythroid progenitor assayable in vitro, the colony-forming unit-erythroid (CFU-E), is normally found in the bone marrow (BM) but is virtually absent from peripheral blood (PB), unlike the more immature progenitor, the burst-forming unit-erythroid (BFU-E). We report on the detection of CFU-E in the PB of six of 18 patients during hematopoietic recovery following allogeneic bone marrow transplantation (BMT); three of six patients with PB CFU-E were under treatment with recombinant human erythropoietin (rhEpo) as well as six of 12 who did not present with PB CFU-E. PB CFU-E were found as early as day 14 following BMT, reached a peak on day 28, and were still detectable on day 60. The presence of PB CFU-E was associated with signs of stimulated erythroid engraftment--an accelerated reticulocyte recovery, an increased number of reticulocytes, higher levels of serum transferrin receptor, and a reduction in transfusional requirements were found in these patients compared to those without PB CFU-E. The numbers of PB and BM BFU-E were similar in the two groups, as well as the numbers of PB and BM CFU-granulocyte/macrophage (CFU-GM) and multipotential CFU (CFU-GEMM); on the other hand, the percentage of BM BFU-E in S phase of the cell cycle was higher in the group of patients with PB CFU-E. While there was no difference between the two groups in serum Epo levels assayed on days 14 and 28 after BMT, patients with PB CFU-E had higher Epo levels in serum samples collected before starting the BMT procedure. These data suggest that the appearance of circulating CFU-E early after BMT is characteristic of a group of patients with an accelerated erythroid engraftment, although the mechanisms leading to the circulation of CFU-E after BMT remain unclear.

Bone-marrow (BM) hematopoietic precursors are recruited into proliferative activity when colony-stimulating factors (CSF) are sequenced with chemotherapy (CT). Previous studies suggested that further CT can be safely administered only when the increased proliferative activity of these cells has subsided, because most cytostatic drugs selectively damage cycling cells. The safest interval between CSF discontinuation and the start of the next CT course needs to be ascertained in vivo. Thirty patients with advanced breast cancer were treated with an intensified FEC regimen, planned at 21-day intervals, sequenced with granulocyte-macrophage (GM)-CSF (15 patients) or granulocyte (G)-CSF (15 patients). Using flow cytometry (FCM) we evaluated the proliferation kinetics of CD34+ BM hematopoietic progenitors before CT+CSF and at different times after CSF administration was stopped. FEC+GM- and FEC+G-CSF sequences both induced a rapid and sustained increase in the percentage of BM myeloid precursors (BMMP%) and in the cycling status of CD34+BM cells. However, while the BMMP% remained elevated in both cases after CSF were stopped, the enhanced proliferative activity of CD34+ cells decreased more rapidly after GM- than after G-CSF. Using FCM, CD34+ BM-derived hematopoietic presursor cell kinetics is readily evaluated in the clinical setting. The administration of CSF following CT increases both the proliferative activity of CD34+ BM cells and the BMMP%. After CSF were discontinued a kinetic refractoriness of hematopoietic progenitors was more evident after GM-CSF than after G-CSF. These data may be of value in designing clinical trials to avoid cytostatic damage to the BM hematopoietic stem-cell compartment.

The CD34 antigen is expressed by committed and uncommitted hematopoietic progenitor cells and is increasingly used to assess stem cell content of peripheral blood progenitor cell (PBPC) collections. Quantitative CD34 expression in PBPC collections has been suggested to correlate with engraftment kinetics of PBPCs infused after myeloablative therapy. We analyzed the engraftment kinetics as a function of CD34 content in 692 patients treated with high-dose chemotherapy (HDC). Patients had PBPCs collected after cyclophosphamide based mobilization chemotherapy with or without recombinant human granulocyte colony-stimulating factor (rhG-CSF) until > or = 2.5 x 10(6) CD34+ cells/kg were harvested. Measurement of the CD34 content of PBPC collections was performed daily by a central reference laboratory using a single technique of CD34 analysis. Forty-five patients required a second mobilization procedure to achieve > or = 2.5 x 10(6) CD34+ cells/kg and 15 patients with less than 2.5 x 10(6) CD34+ cells/kg available for infusion received HDC. A median of 9.94 x 10(6) CD34+ cells/kg (range, 0.5 to 112.6 x 10(6) CD34+ cells/kg) contained in the PBPC collections was subsequently infused into patients after the administration of HDC. Engraftment was rapid with patients requiring a median of 9 days (range, 5 to 38 days) to achieve a neutrophil count of 0.5 x 10(9)/L and a median of 9 days (range, 4 to 53+ days) to achieve a platelet count of > or = 20 x 10(9)/L. A clear dose-response relationship was evident between the number of CD34+ cells per kilogram infused between the number of CD34+ cells per kilogram infused and neutrophil and platelet engraftment kinetics. Factors potentially influencing the engraftment kinetics of neutrophil and platelet recovery were examined using a Cox regression model. The single most powerful mediator of both platelet (P = .0001) and neutrophil (P = .0001) recovery was the CD34 content of the PBPC product. Administration of a post-PBPC infusion myeloid growth factor was also highly correlated with neutrophil recovery (P = .0001). Patients receiving high-dose cyclophosphamide, thiotepa, and carboplatin had more rapid platelet recovery than patients receiving other regimens (P = .006), and patients requiring 2 mobilization procedures versus 1 mobilization procedure to achieve > or = 2.5 x 10(6) CD34+ cells/kg experienced slower platelet recovery (P = .005). Although a minimal threshold CD34 dose could not be defined, > or = 5.0 x 10(6) CD34+ cells/kg appears to be optimal for ensuring rapid neutrophil and platelet recovery.

The aim of this study was to evaluate the efficacy, safety and toxicity of short-term priming with recombinant human granulocyte colony-stimulating factor (rhG-CSF) immediately after diagnosis but before combination chemotherapy (CHOP) for non-Hodgkin's lymphomas. Of fourteen patients entering the study, seven received five days subcutaneous injection of rhG-CSF (5 micrograms/kg/day) before CHOP (CSF-group), and seven were treated with CHOP alone (control group). Blood samples were studied before and on days 1-5 during rhG-CSF priming as well as twice weekly after treatment. The number of blood and bone marrow progenitors was identified by clonogenic growth day 7, 14 and 21 of GM-CFU in semisolid medium. Blood absolute neutrophil counts increased in all rhG-CSF primed patients. The expansion of marrow myelopoiesis resulted in increased myeloid:erythroid ratios, increased bone marrow cellularity and increased numbers of myeloid progenitors both in the blood as well as the marrow. Chemotherapy induced neutropenia developed on day 9-12 in all patients independent of myeloid growth factor priming. However, neutropenia appeared earlier in the cytokine primed group (P = .0038). Five patients in the CSF-group and three patients in the control group were hospitalized with neutropenic fever, and septicemia was documented in three patients in the CSF-group. RhG-CSF induced expansion of myelopoiesis immediately before combination chemotherapy mobilized sufficient number of blood progenitors for apheresis but did not result in reduction of duration and degree of neutropenia in patients with newly diagnosed non-Hodgkin's lymphoma. Although the small number of patients prevents drawing definite conclusions, this time schedule for priming should be used with caution in the future due to an increased risk of hematologic toxicity.