Ifosfamide, carboplatin and etoposide in children with poor-risk relapsed Wilms' tumor: a Children's Cancer Group report.

BACKGROUND: The outcome of children with relapsed Wilms' tumor is poor, especially with poor-risk factors such as unfavorable histology, early recurrence, previous three-drug therapy, relapse not confined to lungs and abdominal relapse following abdominal radiotherapy. We report the overall response rate, progression-free survival and overall survival of 11 children with relapsed and poor-risk Wilms' tumor following ifosfamide/carboplatin/etoposide (ICE) chemotherapy. PATIENTS AND METHODS: ICE therapy consisted of ifosfamide 1800 mg/m2/day (on day 0-4), carboplatin 400 mg/m2/day (on day 0-1) and etoposide 100 mg/m2/day (on day 0-4). The median age at diagnosis was 39 months (range from 13 months to 16 years) and the median time to relapse after initial diagnosis was 9 months (range 4-72 months). All but one patient had at least one poor prognostic feature, with eight patients showing three or four. RESULTS: After ICE chemotherapy the number of patients showing a complete response (CR) was three (27%) and a partial response (PR) was six (55%). The overall response rate (CR+PR) was 82%. Five of the six patients with a PR subsequently achieved a CR with further therapy. The 3-year event-free survival and overall survival were 63.6 +/- 14.5%. CONCLUSIONS: The response rate in children with relapsed and poor-risk Wilms' tumor is >80% with ICE re-induction chemotherapy followed by post-ICE therapy. The optimal approach for post-ICE consolidation therapy has yet to be determined.

Results of a randomized phase III trial in children and adolescents with advanced stage diffuse large cell non Hodgkin's lymphoma: a Pediatric Oncology Group study.

The Pediatric Oncology Group (POG) adopted a histology-based approach to the management of pediatric non-Hodgkin's lymphomas (NHL) utilizing the National Cancer Institute Working Formulation for Clinical Usage. Patients with diffuse large cell lymphoma (DLCL) were treated on a separate protocol from small cell diffuse undifferentiated or lymphoblastic lymphomas. This study assessed the overall and event free survival of children with DLCL and determined the effects of cyclophosphamide upon these end-points in a prospective randomized trial. One hundred and twenty eligible stage III or IV NHL patients with the confirmed diagnosis of diffuse large cell or immunoblastic histology were enrolled on study between October 1986 and November 1991. Patients were randomized to receive or not receive cyclophosphamide; 58 received cyclophosphamide, doxorubicin, vincristine, 6-mercaptopurine (6-MP), and prednisone (ACOP+) and 62 were treated with doxorubicin, vincristine, 6-MP, and prednisone (APO). In both treatment programs methotrexate was substituted when the doxorubicin cumulative dose reached 450 mg/m2. Radiation was administered to bulky disease if progression or no response were observed after induction therapy. Planned duration of therapy was 12 months. The 5-year event free survival (EFS) rates of patients treated with ACOP+ versus APO were 62% +/- 7% and 72% +/- 6%, respectively. While there was no statistically significant difference between the two treatment arms (p = 0.28), we can only say that we are 95% confident that the difference in 5-year EFS falls in the wide range from 28% in favor of APO to 8% favoring ACOP+. Marrow suppression was the main toxicity with one fatal infection. There were three other deaths on study due to respiratory failure in patients with mediastinal masses. Only one patient experienced cardiotoxicity requiring discontinuation of doxorubicin. Ten patients received radiation therapy to achieve. In conclusion the efficacy of elimination of cyclophosphamide from the treatment program of children and adolescents with advanced stage diffuse large cell lymphoma was inconclusive as to its effect on EFS. Furthermore, the majority of the patients (92%) did not require any radiation therapy to bulky disease indicating that the chemotherapy regimens are quite efficient for achievement of complete remission.

CD34 expression by murine hematopoietic stem cells. Developmental changes and kinetic alterations.

For more than a decade it was believed that hematopoietic stem cells express CD34. However, this dogma was recently challenged by the observation that stem cells of normal adult mice are CD34-. In order to clarify the controversy, we carried out systematic examination of stem cells by using C57BL/6 mice that are congenic for Ly-5. As reported previously, stem cells in the normal adult mice were CD34-. However, stem cells stimulated in vivo by 5-fluorouracil injection or in vitro by a combination of interleukin-11 and steel factor were CD34+. The activated CD34+ stem cells reverted to CD34- when the recipients' marrow achieved steady state. The majority of G-CSF-mobilized stem cells also were CD34+ and reverted to CD34- under steady-state conditions. Most recently, we examined the developmental changes of stem cell CD34 expression. In order to gain information on the total population of stem cells we prepared CD34+ and CD34- populations of mononuclear cells without prior enrichment and studied their engrafting potentials. All stem cells from perinatal to 5-week-old mice were CD34+. In 7-week-old mice CD34- stem cells began to emerge, and the majority of the stem cells were CD34- in the 10- and 20-week-old mice. An estimated 20% of the adult stem cells expressed CD34. These observations provide insight into the current controversy regarding CD34 expression by adult hematopoietic stem cells.

Reciprocal expression of CD38 and CD34 by adult murine hematopoietic stem cells.

The effects of activation of adult murine stem cells on their expression of CD38 were studied using a murine transplantation model. First, the published finding that the majority of long-term engrafting cells from normal adult steady-state marrow are CD38(+) was confirmed. Next, it was determined that the majority of stem cells activated in vivo by injection of 5-fluorouracil (5-FU) or mobilized by granulocyte colony-stimulating factor are CD38(-). Stem cells that were activated in culture with interleukin-11 and steel factor were also CD38(-). Previous studies have shown that expression of CD34 by adult stem cells is also modulated by in vivo or in vitro activation. To determine whether there is reciprocal expression of CD38 and CD34, 4 populations of post-5-FU marrow cells were analyzed. The majority of the stem cells were in the CD38(-)CD34(+) fraction. However, secondary transplantation experiments indicated that when the bone marrow reaches steady state, the majority of the stem cells become CD38(+)CD34(-). In addition, the minority populations of CD34(+) stem cells that occur in steady-state bone marrow are CD38(-). This reversible and reciprocal expression of CD38 and CD34 by murine stem cells may have implications for the phenotypes of human stem cells.

Assessment of barriers to bone marrow donation by unrelated African-American potential donors.

African Americans have a lower registration rate for becoming potential bone marrow and stem cell donors. The same attitudes and behaviors are exhibited in regard to solid organ and blood donations, causing a serious under-representation of the African-American population in the donor pool. In our efforts to increase donor availability for African Americans through a project funded by the Medical University of South Carolina, we used a survey to determine the reasons African Americans do not participate as donors for bone marrow. We surveyed 589 African Americans, a great majority of whom were women. Our survey identified major barriers to donation to be the lack of awareness that transplantation can save lives, the cost of donation, and the lack of opportunities to donate. The most effective interventions in increasing donation have been to provide both educational programs preceding marrow drives and the opportunity to donate. Through these efforts, the number of potential African-American donors has increased from 768 (accrued over a period of 12 years) to 1977 in less than 2 years. We conclude that a minority recruitment program targeting African-American volunteers for the National Marrow Donor Program (NMDP) should include an education component addressing the most common barriers before drives.

CD34 expression by murine hematopoietic stem cells mobilized by granulocyte colony-stimulating factor.

Controversy has existed about CD34 expression by hematopoietic stem cells. We recently reported that CD34 expression reflects the activation state of stem cells by using a murine transplantation model. It has been generally held that mobilized blood stem cells express CD34.However, it has also been reported that mobilized stem cells and progenitors are in G0/G1 phases of the cell cycle. To address the state of CD34 expression by the mobilized stem cells, we again used the mouse transplantation model. We prepared CD34(-) and CD34(+) populations of nucleated blood cells from granulocyte colony-stimulating factor-treated Ly-5.1 mice and assayed each population for long-term engrafting cells in lethally irradiated Ly-5.2 mice. The majority of the stem cells were in the CD34(+) population. The CD34 expression by mobilized stem cells was reversible because re-transplantation of Ly-5.1 CD34(-) marrow cells harvested from the Ly-5.2 recipients of CD34(+)-mobilized stem cells 8 months posttransplantation revealed long-term engraftment. These results may support the use of total CD34(+) cells in mobilized blood as a predictor for engraftment and CD34 selection for enrichment of human stem cells. (Blood. 2000;96:1989-1993)

Cost analyses of adjunct colony stimulating factors for acute leukemia: can they improve clinical decision making.

Colony stimulating factors reduce the duration of neutropenia following intensive chemotherapy in a variety of settings, but the advantages in the management of leukemia are inconclusive. The variations in clinical results and the high costs of granulocyte colony-stimulating factor (G-CSF) and granulocyte macrophage colony-stimulating factor (GM-CSF) have led to confusion over appropriate use for leukemia patients. In this paper, we reviewed published information on costs and cost-effectiveness of growth factors for childhood and adult leukemia patients. Medline and Healthstar databases were searched for original research articles that contain cost or cost-effectiveness analyses of G-CSF (filgrastim) and GM-SCF (sargramostim) in oncology cooperative group trials. Published manuscripts and abstracts presented at national or international oncology conferences were included. The cost of adjunct treatment was evaluated in two studies of pediatric ALL, one study of adult AML, and two studies of AML in older adults (>55 years). The use of G-CSF for children with ALL was associated with reductions in days to ANC recovery, fewer documented infections, a shorter duration of hospitalization, and small (but not significant) additional costs. In adult AML patients, benefits included a shortening of the duration of neutropenia and hospital stays, a lower incidence of infection and febrile episodes, less use of antibiotics, and cost savings of $2,230 and $2,310 in two studies and an increase if $120 in the third study. This summary suggests that economic analyses can provide useful information to assist clinical decision-making. For pediatric ALL patients, this information indicates that G-CSF use is unlikely to have significant cost implications, and its use should be based on clinical considerations. In studies of adult and older adult AML patients, both GM-CSF and G-CSF have clinical benefits and can be expected to lead to a decrease in overall costs.

Effects of cranial radiation in children with high risk T cell acute lymphoblastic leukemia: a Pediatric Oncology Group report.

Contemporary chemotherapy has significantly improved event-free survival among patients with T cell-lineage acute lymphoblastic leukemia (T-ALL). Unlike B-precursor ALL, most investigators are still using cranial radiation (CRT) and are hesitant to rely solely on intrathecal therapy for T-ALL. In this study we assessed the effects of CRT upon event-free survival and central nervous system (CNS) relapses in a cohort of children with high risk features of T cell leukemia. In a series of six consecutive studies (1987-1995) patients were non-randomly assigned their CNS prophylaxis per individual protocol. These protocols were based on POG 8704 which relied on rotating drug combinations (cytarabine/cyclophosphamide, teniposide/Ara-C, and vincristine/doxorubicin/6-MP/prednisone) postinduction. Modifications such as high-dose cytarabine, intermediate-dose methotrexate, and the addition of G-CSF, were designed to give higher CNS drug levels (decreasing the need for CRT), to eliminate epidophyllotoxin (decreasing the risk of secondary leukemia), and to reduce therapy-related neutropenia (pilot studies POG 9086, 9295, 9296, 9297, 9398). All patients included in this analysis qualified for POG high risk criteria, WBC >50000/mm3 and/or CNS leukemia. Patients without CNS involvement received 16 doses of age-adjusted triple intra-thecal therapy (TIT = hydrocortisone, MTX, and cytarabine) whereas patients with CNS disease received three more doses of TIT during induction and consolidation. Patients who received CRT were treated with 2400 cGy (POG 8704) or 1800 cGy (POG 9086 and 9295). CNS therapy included CRT in 144 patients while the remaining 78 patients received no radiation by original protocol design. There were 155 males and 57 females with a median age of 8.2 years. The median WBC for the CRT+ and CRT- patients were 186000/mm3 and 200000/mm3, respectively. CNS involvement at diagnosis was seen in 16% of the CRT+ and 23% of the CRT- groups. The complete continuous remission rate (CCR) was not significantly different for the irradiated vs. non-irradiated groups (P = 0.46). The 3-year event-free survival was 65% (s.e. 6%) and 63% (s.e. 4%) for the non-irradiated vs. the radiated group. However, the 3-year CNS relapse rate was significantly higher amongst patients who did not receive CRT; 18% (s.e. 5%) vs. 7% (s.e. 3%) in the irradiated group (P = 0.012). Our analysis in a non-randomized setting, suggests that CRT did not significantly correlate with event-free survival but omitting it had an adverse effect on the CNS involvement at the time of relapse.

Cost analysis of filgrastim for the prevention of neutropenia in pediatric T-cell leukemia and advanced lymphoblastic lymphoma: a case for prospective economic analysis in cooperative group trials.

BACKGROUND: Growth factor use has been shown to ameliorate chemotherapy-induced neutropenia, leading to shorter hospital stays and lower use of parenteral antibiotics, two costly areas of cancer treatment. Prior reports on pediatric patients have shown evidence of cost savings in some studies, but no such evidence in others. In this study a retrospective analysis compared the costs of inpatient supportive care for pediatric patients with T-cell leukemia and advanced lymphoblastic lymphoma enrolled in a Pediatric Oncology Group trial. PROCEDURE: Patients 1-22 years of age were randomized to receive either granulocyte colony-stimulating factor (G-CSF; n = 45) or no G-CSF (n = 43) following induction and two cycles of maintenance therapy. There were no significant differences in neutropenia-related outcomes during the induction phase. During maintenance therapy, G-CSF patients had significantly fewer days to an ANC >500 cells/microl and a trend towards fewer days of hospitalization. Data on resource utilization were tabulated from case report forms. Costs were imputed from national data on hospitalization costs, average wholesale prices of pharmaceuticals, and patient billing information from a single institution. RESULTS: Total median costs of supportive care were $34,190 for patients receiving G-CSF and $28,653 for patients not receiving G-CSF (P > 0. 05 for the cost difference). Sensitivity analyses demonstrated that the total cost difference was not statistically significant, even in scenarios that included reasonable variations in estimates of the range of the length of stay, antibiotic regimen, and dosage and cost of G-CSF. CONCLUSIONS: In the setting of pediatric leukemia, the cost of growth factor may offset potential savings from shorter hospital stays or lower antibiotic use, a finding consistent with that from the Children's Cancer Study Group.

Interleukin 8 in middle ear fluid during acute otitis media: correlation with aetiology and bacterial eradication.

OBJECTIVES: To study the concentration of interleukin 8 (IL-8) in the middle ear fluid of children with acute otitis media and the association between IL-8 concentrations, aetiology of acute otitis media, and bacteriological sterilisation. STUDY DESIGN: Middle ear fluid was obtained by tympanocentesis at enrollment (day 1) and on day 4-5 in 81 children aged 3-36 months with acute otitis media who received antibiotic treatment. IL-8 concentrations were measured by enzyme linked immunosorbent assay. RESULTS: 101 samples were obtained on day 1 and 47 samples on day 4-5. 94 pathogens were isolated in 79 of 101 samples obtained on day 1: 56 Haemophilus influenzae, 35 Streptococcus pneumoniae, 2 Moraxella catarrhalis, and 1 Streptococcus pyogenes. Among 40 paired, initially culture positive samples, sterilisation was achieved on day 4-5 in 22 but not in 18 (13 H influenzae, 2 S pneumoniae, and 3 H influenzae and S pneumoniae concomitantly). IL-8 was detected in 96 of 101 and 46 of 47 samples obtained on days 1 and 4-5, respectively. Mean (SD) IL-8 concentration on day 1 was significantly higher in culture positive than in negative samples (12,636 (23,317) v 5,920 (7,080) pg/ml). In paired samples, IL-8 concentration fell in 12 of 22 ears in which sterilisation was achieved and in 9 of 21 ears with persistent or new infection. Mean (SD) IL-8 concentrations on day 4-5 were significantly higher in culture positive than in negative samples (15,420 (15,418) v 6,695 (5,092) pg/ml). CONCLUSIONS: Higher IL-8 concentrations are found in culture positive middle ear fluid in acute otitis media. Bacterial eradication is associated with a fall in these concentrations.

Reversible expression of CD34 by murine hematopoietic stem cells.

We used a mouse transplantation model to address the recent controversy about CD34 expression by hematopoietic stem cells. Cells from Ly-5.1 C57BL/6 mice were used as donor cells and Ly-5.2 mice were the recipients. The test cells were transplanted together with compromised marrow cells of Ly-5.2 mice. First, we confirmed that the majority of the stem cells with long-term engraftment capabilities of normal adult mice are CD34(-). We then observed that, after the injection of 150 mg/kg 5-fluorouracil (5-FU), stem cells may be found in both CD34(-) and CD34(+) cell populations. These results indicated that activated stem cells express CD34. We tested this hypothesis also by using in vitro expansion with interleukin-11 and steel factor of lineage(-) c-kit(+) Sca-1(+) CD34(-) bone marrow cells of normal mice. When the cells expanded for 1 week were separated into CD34(-) and CD34(+) cell populations and tested for their engraftment capabilities, only CD34(+) cells were capable of 2 to 5 months of engraftment. Finally, we tested reversion of CD34(+) stem cells to CD34(-) state. We transplanted Ly-5.1 CD34(+) post-5-FU marrow cells into Ly-5.2 primary recipients and, after the marrow achieved steady state, tested the Ly-5.1 cells of the primary recipients for their engraftment capabilities in Ly-5.2 secondary recipients. The majority of the Ly-5.1 stem cells with long-term engraftment capability were in the CD34(-) cell fraction, indicating the reversion of CD34(+) to CD34(-) stem cells. These observations clearly demonstrated that CD34 expression reflects the activation state of hematopoietic stem cells and that this is reversible.

Dynamics of interleukin-1 production in middle ear fluid during acute otitis media treated with antibiotics.

In an ongoing prospective study, IL-1 concentrations were measured in 78 children (aged 3-36 months) with acute otitis media receiving antibiotics. Middle ear fluid IL-1 concentrations were determined using ELISA kits. Ninety-eight middle ear fluid samples were obtained by tympanocentesis at enrollment (day 1) and 43 samples were collected on days 4-5. Ninety-two pathogens were isolated in 77/98 samples obtained on day 1: 55 Haemophilus influenzae, 34 Streptococcus pneumoniae, 2 Moraxella catarrhalis and 1 Streptococcus pyogenes. Among 37 paired samples initially culture-positive, eradication of the pathogen was achieved on day 4-5 in 20 while pathogens were still present in 17. On day 1, IL-1 was detected in 61/77 (79%) culture-positive samples vs 9/21 (43%) culture-negative ones (P = 0.003). The mean +/- SD middle ear fluid concentration of IL-1 on day 1 was significantly higher in culture-positive (316 +/- 508 pg/ml) than in culture-negative samples (111 +/- 245 pg/ml) (P = 0.01). When paired samples were evaluated, IL-1 decreased on days 4-5 in 13/20 (65%) ears where bacterial eradication was achieved, but also in 11/19 (58%) with persistent or new infection. The mean IL-1 concentrations decreased on days 4-5 in the 20 samples from ears where bacterial eradication was achieved (330 +/- 460 vs 118 +/- 294 pg/ml, P = 0.1) but also in the 17 samples where it was not (465 +/- 660 vs 232 +/- 289 pg/ml, P = 0.02). No significant differences were found between day 1 and days 4-5 in the mean IL-1 concentrations measured in patients with H. influenzae vs S. pneumoniae or concomitant H. influenzae and S. pneumoniae. It was concluded that: 1) IL-1 was detected in the middle ear fluid of most patients with acute otitis media; 2) significantly higher IL-1 concentrations were found in patients with culture-positive than in those with culture-negative acute otits media; 3) IL-1 concentrations decreased on days 4-5 of antibiotic therapy, whether the pathogen was eradicated or not.

NK cell colony formation from human fetal thymocytes.

We established a clonal cell culture system for human natural killer (NK) cells from fetal thymocytes. Thymocytes of 16 to 22 gestational weeks were cultured in methylcellulose in the presence of interleukin (IL)-7, IL-15, and steel factor (SF). After 14 days in incubation, large, diffuse colonies consisting of small cells were identified. Cells in the colonies were medium- to large-sized granular lymphocytes, expressing CD56 but not CD3, and revealed lytic activity against K562 cells. Colony-forming units (CFU)-NK were enriched in lineage negative (Lin- ) CD34++ subpopulations of fetal thymocytes, whereas a smaller number of CFU-NK also existed in Lin-CD34+ and Lin-CD34- subpopulations. Cytokine requirement for the NK cell colony formation was examined under serum-free conditions. As a single agent, only IL-15, but not IL-2, IL-7, or SF, supported NK cell colony formation. IL-15 had synergy with IL-7 and SF independently, and the maximal number of colonies were obtained when the three cytokines were present. IL-2 also supported NK cell colony formation in the presence of SF. When IL-2 was added to cultures containing IL-15 alone, IL-15 plus SF, or IL-15, SF, and IL-7, the numbers of NK cell colonies were reduced relative to those without IL-2. These results indicate that IL-2 may regulate IL-15-responsive NK cell progenitors. This clonal culture system will be a useful tool in the investigation of NK cell ontogeny.

Postdural puncture headache in paediatric oncology patients.

PURPOSE: Previous studies have not determined the correlation between dural puncture and postural headache in paediatric patients. Furthermore, no studies have evaluated the correlation between atypical headache and dural puncture in the paediatric population. Therefore, we prospectively analyzed the incidence of typical postdural puncture headache (PDPHA) and atypical headache in paediatric oncology patients following dural puncture. METHODS: The study population consisted of 66 paediatric patients undergoing 128 consecutive procedures, including 99 lumbar punctures and 29 bone marrow aspirations without concomitant lumbar puncture. Patients were prospectively randomized into four groups: Group I, preteens (< 13 yr) undergoing lumbar puncture, Group II, adolescents (13-21 yr) undergoing lumbar puncture, Group III, preteens undergoing bone marrow aspiration, and Group IV, adolescents undergoing bone marrow aspiration. The presence and description of headache was documented immediately after dural puncture or bone marrow aspiration, and on post-procedure days # 1, 3 and 5 by personnel blinded to the type of procedure. RESULTS: There was an increase in the incidence of headache (9.1%) after lumbar puncture in patients < 21 yr relative to patients undergoing bone marrow aspiration (P < 0.05). No difference was found between the incidence of typical PDPHA after dural puncture in preteens and adolescents. There was also no difference in the incidence of atypical headache after dural puncture or after bone marrow aspiration among preteens and adolescents. CONCLUSIONS: Paediatric patients experience an increased incidence of typical postdural puncture headache after dural puncture compared with age-matched patients undergoing bone marrow aspiration only. Atypical headache is relatively common in the paediatric population after dural puncture or bone marrow aspiration.

Therapy of patients with T-cell lymphomas and leukemias using an anti-CD7 monoclonal antibody-ricin A chain immunotoxin.

Anti-CD7-dgA, DA7, consists of deglycosylated ricin A chain coupled to a mouse monoclonal anti-human CD7 antibody. This study determined the maximally tolerated dose (MTD) of this immunotoxin administered as a one hour infusion over five days to 11 patients with T-cell lymphoma (>30% CD7+ malignant cells). The MTD was 0.2 mg/kg/day or 1 mg/kg/120 hours (maximal toxicity grade 3) with vascular leak syndrome (VLS) as dose-limiting toxicity (DLT). Predictors of severe VLS included age and absence of circulating lymphoma cells. Two partial responses and one minimal response were seen. Patients with minimal lymphoma burden or T-cell large granular lymphocyte (LGL) leukemia showed the best responses. The mean maximal serum concentration of immunotoxin at the MTD was 2.5 ug/ml. The mean alpha-phase half-life was 1.5 hours and the mean beta-phase half-life was 8 hours. Repeated dosing had minimal effects on either peak serum immunotoxin concentrations or serum half-lives. While human antimouse antibodies were observed, they were low in concentration (<55 ng/ml). Human anti-ricin antibody was elevated in one patient (190 ng/ml). VLS presented with hypoalbuminemia, dyspnea, pulmonary edema, aphasia, and peripheral edema and cleared over a two week period. Serum fibronectin levels were measured in three patients and were very low in one patient who developed VLS. No specific binding of DA7 immunotoxin was seen with vascular endothelium in various human tissues.

Transient therapy-related myelodysplastic syndrome associated with monosomy 7 and 11q23 translocation.

Secondary acute myelocytic leukemia (AML) and myelodysplastic syndromes (MDS) are known to develop in patients previously treated with different chemotherapeutic regimens. Nonrandom chromosomal abnormalities have been demonstrated in these therapy-related myeloid disorders which often evolve into refractory AML. The prognosis of these patients with conventional chemotherapy has been dismal and only allogeneic bone marrow transplantation offers a potential cure. We describe two patients who developed MDS after chemo/radiotherapy and had a spontaneous recovery. One patient was treated with MOPP-ABVD hybrid therapy for Hodgkin's disease, developed pancytopenia, marrow hypoplasia and dyserythropoiesis associated with monosomy 7. The other was treated with a combination of chemotherapy including VP-16 for Ewing's sarcoma, developed thrombocytopenia, marrow hypoplasia and dyserythropoiesis associated with an 11q23 translocation. Both patients received rhG-CSF after their cycles of chemotherapy and were considered for a bone marrow transplant. Marrow aspirates at frequent intervals showed gradual disappearance of the abnormal clone with parallel normalization of the peripheral count. In both patients G-CSF might have played a role in the development of the abnormal clone. We suggest that patients with therapy-related MDS without excess of blasts could be closely monitored for karyotypic and hematological improvement rather than transplanted immediately.

Thrombopoietin, steel factor and the ligand for flt3/flk2 interact to stimulate the proliferation of human hematopoietic progenitors in culture.

We have tested the effects of steel factor (SF) the ligand for flt3/flk2 (FL) and thrombopoietin (TPO, Mpl ligand), on the proliferation of primitive human bone marrow progenitors in serum-deprived culture. Varying combinations of SF, FL and TPO supported formation of only few colonies from CD34+/c-Kit(low)/CD38neg/low cells. However, the addition of interleukin 3 (IL-3) to the three cytokines significantly increased the number of colonies. When this population of cells was tested in suspension culture for one week for production of colony-forming cells there was synergism among SF, FL and TPO. Addition of IL-3 to the three cytokines further increased the number of erythroid colony-forming cells. The effects of these four factors on CD34+/c-Kit(low)/CD38high cells were merely additive. Studies of individual CD34+/c-Kit(low)/CD38neg/low cells demonstrated the direct effects of SF, FL and TPO. In the presence of SF, FL and TPO, approximately half of the individual CD34+/c-Kit(low)/CD38neg/low cells proliferated in seven day suspension culture. Addition of IL-3 to the combination of SF, FL and TPO did not increase the frequencies of proliferating clones, but increased the size of individual clones. These observations suggest that SF, FL and TPO play important roles in survival and proliferation of primitive human hematopoietic progenitors.

Thrombopoietin supports proliferation of human primitive hematopoietic cells in synergy with steel factor and/or interleukin-3.

We have studied the effects of recombinant human thrombopoietin (TPO; mpl ligand) on the proliferation of human primitive hematopoietic progenitors in vitro. CD34+ cells were enriched for cell-cycle-dormant primitive progenitors by separation on the basis of expression of c-kit and CD38. In the presence of varying combinations of TPO, Steel factor (SF), and interleukin-3 (IL-3), CD34+/c-kit(low)/CD38neg/low cells produced fewer colonies than CD34+/c-kit(low)/CD38high cells. However, when cultured in suspension for 7 days and replated in methylcellulose culture for measurement of colony-forming cells, the former population generated more colony-forming cells than the latter. In suspension culture of CD34+/c-kit(low)/CD38neg/low cells, TPO acted synergistically with SF and/or IL-3 in support of the production of colony-forming cells for granulocyte/macrophage colonies, erythroid colonies, and mixed colonies. Culture studies of individual CD34+/c-kit(low)/CD38neg/low cells provided the evidence for the direct nature of the effects of TPO. When combined with SF, TPO showed stronger stimulation of production of progenitors in suspension culture than other early-acting factors, such as IL-6, IL-11, and granulocyte colony-stimulating factor (G-CSF). TPO may be an important cytokine for in vitro manipulation of human hematopoietic stem cells.

Characterization of c-kit expression by primitive hematopoietic progenitors in umbilical cord blood.

Human umbilical cord blood (CB) appears to be an exciting new source of transplantable stem cells for a variety of clinical conditions. In this study, we have attempted to further characterize the primitive progenitors in CB. First we analyzed the effects of early-acting growth factors on blast cell colony formation from CD34+ progenitors. Addition of Steel factor (SF), interleukin-6 (IL-6), or granulocyte colony-stimulating factor (G-CSF) to cultures containing interleukin-3 enhanced blast cell colony formation. These results indicated that cell cycle-dormant progenitors are present in CB. Next, based on results obtained in the murine system, we tested whether c-kit expression could separate the CB progenitors into cycle-dormant vs. cycle-active progenitors. Cells were separated into CD34+ c-kit-, c-kitlow, and c-kithigh. The results suggested that the c-kitlow population contains the majority of cycle-dormant progenitors and the c-kithigh population contains most of the forming cells were in the c-kitlow population, while the opposite is true for other colony-forming cells. Expression of c-kit may be useful in identifying CB progenitors with long-term engraftment capability.

Recombinant human thrombopoietin (Mpl ligand) enhances proliferation of erythroid progenitors.

We have studied the effects of recombinant human thrombopoietin (TPO, Mpl ligand) on human hematopoiesis in vitro. TPO alone did not support erythroid burst formation but, in the presence of erythropoietin, it enhanced erythroid burst formation from CD34+ bone marrow and cord blood cells. The burst-promoting activity (BPA) was stronger under 5% serum than 30% serum conditions. The direct nature of BPA effects was documented by replating studies of early erythroid bursts. The BPA of TPO was less than that of interleukin-3 but was comparable with that of granulocyte/macrophage colony-stimulating factor and steel factor. The soluble form of Mpl receptor inhibited burst enhancing effects of TPO, suggesting that the BPA effects of TPO are mediated through the Mpl receptor. These results further delineate the physiologic roles of TPO and may aid in the determination of the clinical usages of TPO.