Feasibility study of IL-11 and granulocyte colony-stimulating factor after myelosuppressive chemotherapy to mobilize peripheral blood stem cells from heavily pretreated patients.

PURPOSE: Pediatric patients with solid tumors treated with prolonged dose-intensive chemoradiotherapy are poor mobilizers of peripheral blood stem cells (PBSC). We have conducted a pilot study to mobilize PBSC in eight pediatric patients with relapsed solid tumors using ifosfamide, carboplatin, and etoposide (ICE) followed-up by IL-11 plus granulocyte colony-stimulating factor (G-CSF). PATIENTS AND METHODS: Patients received ifosfamide 1.8 g/m2 per day for 5 days, carboplatin 400 mg/m2 per day for 2 days, and etoposide 100 mg/m2 per day for 5 days. After completion of ICE chemotherapy, patients received daily subcutaneous injections of G-CSF (5 microg/kg per day) and IL-11 (50-100 microg/kg per day) until peripheral stem cell apheresis. RESULTS: The median age was 11 years. Diagnosis included three relapsed Hodgkin disease, three relapsed central nervous system tumors, one relapsed Wilms tumor, and one relapsed rhabdomyosarcoma. The median number of apheresis procedures required to obtain 5 x 10(6) CD34+ cells/kg was one. The mean +/- standard error of mean (SEM) total CD34+ cells collected was 14.0+/-2.7 x 10(6)/kg. The mean +/- SEM total CD34+/CD41+ cells collected was 4.6+/-1.9 x 10(6)/kg. Seven of the eight patients have subsequently undergone myeloablative chemotherapy with autologous PBSC transplantation and have reconstituted hematopoiesis with a median time to neutrophil recovery of 10 days and platelet recovery of 15.5 days. CONCLUSIONS: We conclude that the regimen of ICE/IL-11 plus G-CSF is successful in mobilizing large numbers of CD34+ PBSC cells with a limited number (one) of apheresis collections in patients that have previously been heavily pretreated with chemotherapy/radiotherapy.

Successful treatment of relapsed Burkitt's lymphoma using unrelated cord blood transplantation as consolidation therapy.

We report a 10-year-old male with widespread recurrent Burkitt's lymphoma who underwent successful mismatched unrelated cord blood transplantation (UCBT) following salvage chemotherapy. He was conditioned with TBI, antithymocyte globulin (ATG) and high-dose VP-16 and achieved full donor engraftment. He experienced grade II skin and grade I gastrointestinal acute GVHD with no chronic GVHD. He is alive with no evidence of disease 24 months following UCBT. Bone Marrow Transplantation (2000) 25, 1311-1313.

A Phase I study of granulocyte-macrophage-colony stimulating factor/interleukin-3 fusion protein (PIXY321) following ifosfamide, carboplatin, and etoposide therapy for children with recurrent or refractory solid tumors: a report of the Children's Cancer Group.

BACKGROUND: This Phase I trial was developed to determine the safety, biologic activity, and effects on hematopoietic recovery of PIXY321 following ifosfamide, carboplatin, and etoposide chemotherapy for children with recurrent or refractory solid tumors. METHODS: Children (age < 22 years at diagnosis) received ifosfamide 1800 mg/m2/day x 5 days, carboplatin 400 mg/m2/day x 2 days, and etoposide 100 mg/m2/day x 5 days, followed by daily subcutaneous administration of PIXY321. Dose-limiting toxicity was defined as Grade IV toxicity related to PIXY321. Pharmacokinetic and endogenous cytokine production studies were conducted during Course 1, and peripheral blood (PB) progenitor cell and receptor expression studies were conducted during Course 1 when the white blood cell count recovered to > or=1000/mm3. RESULTS: Twenty-four children received ifosfamide, carboplatin, and etoposide chemotherapy plus PIXY321, the latter at doses of 500 /g/m2/day (n=3), 750 microg/m2/day (n=6), 1000 microg/m2/day (n=9), or 500 microg/m2/twice a day (n=6). PIXY321 was well tolerated, with only 1 dose-limiting toxicity (chills, occurring at a dose of 750 microg/m2/day). The maximum tolerated dose was not reached in this study. The median days to absolute neutrophil count recovery (> or =1000/mm3) and platelet recovery (>100,000/mm3) during Course 1 following PIXY321 (1000 microg/ m2/day) were 22 days (range, 5-33 days) and 20 days (range, 5-31 days), respectively. There was a 2500, 5000, 3000, and 390% increase in PB granulocyte-macrophage colony-forming units, erythrocyte blast-forming units, granulocyte erythrocyte macrophage and megakaryocyte colony-forming units, and CD34+ cells, respectively. CONCLUSIONS: In summary, this pediatric Phase I trial demonstrated that PIXY321 was well tolerated by children and resulted in platelet recovery a median of 20 days after ICE chemotherapy and an increase in the number of PB progenitor cells above baseline. However, based on recent negative results with PIXY321 in randomized Phase II/III trials involving adult subjects, PIXY321 is not currently available for future trials involving children.

The role of cytokines following bone marrow transplantation: indications and controversies.

The use of cytokines following bone marrow transplantation has become an area of increasing controversy with specific regard to indication, efficacy and cost. This review will attempt to summarize cytokine data in both adult and pediatric transplantation. Indications for cytokines following transplantation will be presented, as well as suggestions for specific issues that should be addressed in future clinical trials.

Analysis of apheresis content of progenitor cell collections from normal donors to whom granulocyte-colony-stimulating factor is administered.

BACKGROUND: Allogeneic blood cell transplantation is a new alternative to bone marrow transplantation. Preliminary data suggest that granulocyte-colony-stimulating factor (G-CSF)-mobilized peripheral blood progenitor cells from normal donors can provide rapid hematopoietic engraftment without significant increases in transplant-related morbidity. Potential advantages to donors include the elimination of an operation under general anesthesia. STUDY DESIGN AND METHODS: Twenty-one normal donors underwent high-dose (16 pg/kg/day for 5 days) G-CSF mobilization. Apheresis was performed on the fifth and sixth days of G-CSF administration, and apheresis components were analyzed by flow cytometry. Donor characteristics in relationship to apheresis yields were also analyzed. RESULTS: Apheresis components were analyzed according to donor weight. The median total numbers of white cells per kg, CD34+ cells per kg, and CD3+ cells per kg were 10.8 x 10(6), 7.2 x 10(8), and 295 x 10(8), respectively. Day 5 collections had significantly higher nucleated cell content and median CD34+ percentages than did Day 6 collections (0.71% on Day 5 vs. 0.58% on Day 6, p < 0.01). CD34+ content and total white cells were not related to age or sex. No donors experienced toxic effects that required their removal from the study. CONCLUSION: Day 5 is the optimal day for the harvest of normal-donor peripheral blood progenitor cells after mobilization with high-dose G-CSF. Older individuals are acceptable donors of allogeneic progenitor cells.

Clonogenicity of circulating neuroblastoma cells: implications regarding peripheral blood stem cell transplantation.

Peripheral blood stem cells (PBSCs) are being used as an alternative to autologous marrow rescue for hematopoietic reconstitution after high-dose chemotherapy in patients with neuroblastoma and other solid malignancies. Use of PBSCs is preferred by some because of the belief that there is less risk of tumor contamination. Because tumor stem cell contamination is thought to be one contributing cause of relapse after myeloablative therapy and autologous reconstitution, we examined the potential risk of reinfusing circulating neuroblastoma cells by in vitro evaluation of their clonogenicity. Immunocytologic and tumor cell clonogenic analyses were performed on 74 blood samples obtained from 56 children with advanced-stage neuroblastoma. Concurrently drawn bone marrow specimens were evaluated in 30 instances. Circulating neoplastic cells were detected in 19 of 74 (26%) for all specimens and by immunologic techniques (26%). Using a clonogenic assay, 13 grew identifiable tumor colonies. Comparing results with the two techniques showed tumor colony growth in 10 of the 19 positive specimens by immunocytology. However, 3 of 53 samples (6%) that were negative by immunocytology were positive by the clonogenic assay. Of the 11 positive blood samples, 9 concurrent marrows contained neuroblastoma cells; of the 19 negative blood specimens, 3 concurrent marrows had metastatic disease. We conclude that circulating neuroblastoma cells are present in peripheral blood and have clonogenic properties in vitro. This supports the view that tumor cell contamination may well be one cause of relapse after autologous reconstitution. Consequently, PBSC collections should also undergo meticulous monitoring for tumor contamination before autologous reinfusion.

Successful Pneumocystis carinii pneumonia prophylaxis using aerosolized pentamidine in children with acute leukemia.

PURPOSE: We report our experience replacing trimethoprim-sulfamethoxazole (TMP/SMX) with aerosolized pentamidine (AP) in 22 children with acute leukemia who could not tolerate TMP/SMX. PATIENTS AND METHODS: Children (age 1 to 15 years) with acute leukemia during maintenance chemotherapy or post-bone marrow transplantation (BMT) receiving prophylaxis for Pneumocystis carinii pneumonia (PCP) with TMP/SMX received AP following prolonged neutropenia or allergy to TMP/SMX. Patients received 300 mg of AP monthly (children < 4 years received 150 mg) dissolved in 5 mL of distilled water over 20 to 30 minutes. RESULTS: Over a 3-year period, 358 courses of AP were administered over 10,124 observable days. AP was adequately tolerated on a monthly basis for prophylaxis against PCP in 22 children with acute leukemia. AP was demonstrated to be effective in preventing PCP. There were minimal side effects observed during this trial. The majority of children with acute lymphoblastic leukemia (ALL; 12 of 14 [86%]) undergoing maintenance chemotherapy were able to resume full-dose therapy. CONCLUSION: AP in children is well tolerated and shows high efficacy for PCP prophylaxis in children with leukemia. We conclude that AP should be considered as second-line PCP prophylactic therapy for children with acute leukemia in instances in which TMP/SMX cannot be tolerated. Phase III trials are required to determine its effect on dose intensification and event-free survival.

Decreased endogenous circulating Steel factor (SLF) levels following allogeneic and autologous BMT: lack of an inverse correlation with post-BMT myeloid engraftment.

We have previously demonstrated an inverse relationship between circulating endogenous G-CSF levels and myeloid engraftment post-BMT. A new early-acting hematopoietic growth factor, Steel factor (SLF), has recently been demonstrated to induce the proliferation of early hematopoietic progenitor cells and synergistically stimulate committed progenitor cells in the presence of lineage-specific CSFs. In this pilot study, we determined the temporal relationship between endogenous SLF levels and the circulating absolute neutrophil count (ANC) (myeloid engraftment) in both children and adults undergoing both allogeneic and autologous BMT. Pre-BMT SLF levels were 2600 +/- 100 pg/ml compared to significantly lower levels of G-CSF (30-50 pg/ml). The circulating SLF level was significantly decreased throughout the post-BMT period (ANC < or = 200 x 10(6)/l: 1500 +/- 600 pg/ml; ANC 200-500 x 10(6)/l: 1780 +/- 130 pg/ml; ANC > or = 500 x 10(6)/l: 1690 +/- 110 pg/ml) (p < 0.001). There was a lack of an inverse relationship between the circulating SLF level and the ANC (r = -0.43) (p = NS). For comparison, SLF levels from immune thrombocytopenia (platelet < = or 20 x 10(9)/l) and chemotherapy-induced neutropenia patients (ANC < or = 200 x 10(6)/l) were similar to pre-BMT levels but significantly higher than post-BMT levels (p < or = 0.02 and < or = 0.001, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of human glucocerebrosidase following retroviral vector-mediated transduction of murine hematopoietic stem cells.

The human glucocerebrosidase (GC) gene has been expressed in the progeny of murine hematopoietic stem cells following transduction of marrow with a retroviral vector (G2) containing the human GC cDNA. Murine marrow was transduced via co-cultivation following prestimulation in the presence or absence of recombinant IL-3 and IL-6. A high rate of gene transfer and expression (95%) was demonstrated in primary day 12 CFU-S foci following bone marrow transplantation (BMT) of G2-transduced marrow into lethally irradiated syngeneic recipient mice. Immunoreactive human GC protein was also documented in the CFU-S foci. Primary recipient mice were examined 4-6 months following BMT. A higher rate of gene transfer (87%) was seen in hematopoietic organs of recipients of prestimulated donor marrow compared with organs from initially unstimulated marrow (25%). A high rate of expression of human GC was also documented in the prestimulated organs (50%) when compared with the unstimulated group (25%). Secondary BMT was performed using marrow from the long-lived primary recipients. The human GC gene was present in 88% of secondary day 12 CFU-S foci examined in the prestimulated group versus 23% in the unstimulated group. Expression of the human GC gene was documented in secondary day 12 CFU-S foci, providing strong evidence of initial hematopoietic stem cell transduction.

Toward gene therapy for Gaucher disease.

We are studying the transfer and expression by retroviral vectors of the human glucocerebrosidase (GC) gene into bone marrow cells as a model of gene therapy for genetic diseases of hematopoietic cells. A simple retroviral vector (G2) was developed that contains a normal human GC cDNA under the control of the Moloney murine leukemia virus long-terminal repeat (LTR) enhancer/promoter. Murine bone marrow was transduced with the G2 vector and maintained in long-term bone marrow culture (LTBMC). Expression of the human GC gene in the transduced murine LTBMC cells exceeded the level of endogenous murine GC mRNA. Murine bone marrow cells were also transduced with G2 and transplanted into irradiated syngeneic recipients. High levels of GC gene transfer and expression were seen in day-12 CFU-S foci, and to a lesser extent in the hematopoietic organs 4 months after gene transfer/bone marrow transplant (BMT). Human bone marrow, from a patient with Gaucher disease, was also used in studies of GC gene transduction. Gene transfer into 35-40% of the Gaucher hematopoietic progenitor cells was achieved, following prestimulation of the marrow with recombinant hematopoietic growth factors. Equal rates of gene transfer were obtained using either total marrow mononuclear cells or progenitor cells enriched 100-fold by immunomagnetic bead separation. GC gene transduction corrected the enzymatic deficiency of the Gaucher marrow. Our results demonstrate the potential utility of retroviral vector-mediated gene transfer for gene therapy of Gaucher disease. Current efforts are aimed at achieving more consistent in vivo GC expression in the murine BMT model and demonstrating transduction of pluripotent human hematopoietic stem cells.(ABSTRACT TRUNCATED AT 250 WORDS)

An unusual transposon with long terminal inverted repeats in the sea urchin Strongylocentrotus purpuratus.

A 3-kilobase DNA segment characteristic of a transposable element was found within a histone H2B pseudogene in a higher eukaryote, the sea urchin Stronglyocentrotus purpuratus. The inserted segment (TU1) is flanked by 8-base pair (bp) direct repeats of the H2B sequence. TU1 has long terminal inverted repeats approximately 840 bp long with an outer domain of 15-bp tandem repeats and a non-repeating inner domain, and is a member of a heterogeneous family of transposable elements. TU1 differs from most previously characterized eukaryotic transposable elements with terminal direct repats, but resembles the foldback transposon family in Drosophila.