We studied the blasts from 795 children greater than 1 year of age with newly diagnosed, untreated B-precursor acute lymphoblastic leukemia (ALL) for expression of the hematopoietic stem cell-associated antigen CD34. All cases were confirmed as B-lineage lymphoblastic leukemia by virtue of expression of CD19 and/or CD22, lack of T-cell antigens, and lack of surface-membrane immunoglobulin (Ig). The CD34 antigen was present in at least 10% of blast cells in 587 (73.8%) of the patients. There was no significant difference in presenting clinical characteristics between CD34+ and CD34- patients save for an increased incidence of CNS involvement at diagnosis in the latter. Patients with CD34+ leukemia were more likely to have blasts expressing CD22, CD9, and CD13 antigens but were less likely to coexpress CD20. Patients with pre-B (cytoplasmic mu) ALL were significantly more likely to lack CD34 on their blasts, while children with hyperdiploid ALL were more likely to be CD34+. Although remission induction rates were not significantly different between patients with CD34+ and CD34-ALL (P = .23), event-free survival was shorter for patients with CD34- leukemia (P = .0014). Even though CD34 expression was associated with certain other known prognostically favorable variables including hyperdiploidy and lack of cytoplasmic Ig, it had an independent favorable effect on treatment outcome, even after adjusting for competing prognostic factors.

The prognostic significance of chromosomal translocations, particularly t(1;19) (q23;p13), was evaluated in children with pre-B and early pre-B acute lymphoblastic leukemia (ALL). Patients were treated on a risk-based protocol of the Pediatric Oncology Group (POG) between February 1986 and May 1989. An abnormal clone was detected in 46% (130 of 285) of pre-B cases and 56% (380 of 679) of early pre-B cases. Translocation of any type was associated with a worse treatment outcome than other karyotypic abnormalities: 15 of 66 versus 3 of 64 failed therapy in the pre-B group (P = .001), and 37 of 141 versus 23 of 239 failed in the early pre-B group (P less than .001). The t(1;19) (q23;p13) occurred significantly more often in cases of pre-B ALL with a clonal abnormality than in early pre-B ALL cases (29 of 130 v 5 of 380, P less than .001). Among the 285 pre-B cases in which bone marrow was studied cytogenetically, those with t(1;19) had a significantly worse treatment outcome than all others (11 of 29 v 27 of 256 have failed therapy, P less than .001). This difference is significant (P less than .001) after adjustment for leukocyte count, age, and other relevant features. Cases with the t(1;19) also had a worse prognosis than pre-B patients with other translocations (4 of 37 have failed, P less than .01) or with any other karyotypic abnormality (7 of 101 have failed, P less than .001). We conclude that chromosomal translocations confer a worse prognosis for non-T, non-B-cell childhood ALL, and that the t(1;19) is largely responsible for the poor prognosis of the pre-B subgroup.

Although committed progenitor cells have been harvested during the standard plateletpheresis, only few experiences were specifically carried out in normal donors. The major drawback is the very low number of circulating haemopoietic stem cells and, besides a donor manipulation, a good harvesting technology is a fundamental prerequisite. To verify the effectiveness of an automate system, twenty normal volunteer donors were randomly assigned to four protocols of simultaneous PBSC and platelet collection. A Baxter separator CS 3000 was employed and we compared the yields and the efficiency of the modified Program 1 plus isoradial chamber (Protocol A), the modified Program 1 plus granulo chamber (Protocol B), the standard Program 3 plus isoradial chamber (Protocol C) and the standard Program 3 plus granulo chamber (Protocol D). The absolute yields for WBC (x10(9)), MNC (x10(9)), platelets (x10(11)) and CFU-GM (x10(4)) were respectively: 10.4, 10.1, 6.5 and 29.5 for Protocol A; 10.1, 9.9, 5.2 and 14.7 for Protocol B; 10.0, 9.9, 5.2, 14.7 for Protocol C, and 12.7, 12.4, 6.8 and 57.4 for Protocol D. The best CFU-GM efficiency was achieved with Protocol D (33%) vs. Protocol A (27.5%), Protocol B (14.5%) and Protocol C (27%). The above differences, although remarkable, were not statistically significant. Though preliminary, this study demonstrates the feasibility of a combined platelet and PBSC collection in normal donors.

Twenty uremic patients on regular hemodialysis received recombinant human Erythropoietin (rhEPO) in a dosage of 50 U/kg body wt (N = 9) and 80 U/kg body wt (N = 11), respectively, three times weekly. The number of circulating hemopoietic progenitor cells colony-forming unit-granulocyte-erythrocyte-macrophage (CFU-mix), burst-forming unit-erythroid (BFU-E) and colony-forming-granulocyte-macrophage (CFU-GM) in peripheral blood were assayed weekly by means of a commonly applied in vitro clonal assay. A significant increase of peripheral CFU-mix, BFU-E and CFU-GM could be observed within one week of supplementation therapy in both groups. The increase of BFU-E was followed by a rise of hematocrit within four and three weeks, respectively. These results suggest that the stimulatory in vivo effect of rhEPO administered in therapeutical doses is not restricted to the erythroid lineage but also includes progenitor cells committed to the myeloid lineage (CFU-GM) as well as the multipotent progenitors CFU-mix. The increment of circulating progenitor cells was seen with a dosage of 80 U/kg body wt and 50 U/kg body wt as well.

The dissatisfying results achieved in the therapy of ovarian carcinoma with an unchanging low rate (between 10% and 30%) of five-year-survival were the reason for efforts to develop a new treatment scheme combining chemotherapy with hormone therapy for epithelial ovarian carcinoma in FIGO stages III and IV. Basic theoretical and experimental reflections: Although in many cases patients may show good response to standard chemotherapy containing cisplatin, a large percentage (70% to 90%) suffers a relapse due to the fact that single tumour cells are resistant to chemotherapy. In order to counteract this resistance we developed a method of therapy (in accordance with the ideas of Coldman and Goldie) based on the sequential application of various non cross-resistant cytostatic agents. This new regimen, comprised of Doxorubicin, Cisplatin, Vincristine, Cyclophosphamide and Methotrexate (AP-VC-MTX), was compared to two standard types of chemotherapy (Doxorubicin/Cyclophosphamide or Doxorubicin/Cisplatin) in a prospective, randomised study. The AP-VC-MTX regimen showed equal therapeutical results but had a significantly lower level of nephrotoxicity and gastrointestinal toxicity than the combined Doxorubicin/Cisplatin therapy. As a result of these studies we chose the AP-VC-MTX method as standard therapy for patients suffering from advanced stage ovarian carcinoma. Hormone therapy was tested in numerous phase II studies on patients who had previously received cytostatics and was found to be an effective alternative. One of the substances that was most closely studied was Medroxyprogesterone acetate (MPA). Its therapeutic value was established in in vitro tests which showed direct cytotoxicity in ovarian carcinoma and is based on the theory that, on the one hand, the MPA is attached to the progesterone receptor and impairs growth in a similar way to endometrium and breast carcinoma and, on the other hand, the MPA reduces the level of gonadotropin and estrogen, which may be factors, which stimulate tumour growth in ovarian cancer. Furthermore, the myeloprotective effect and the corticoid-like effect of the MPA usually result in lower bone marrow toxicity and an increase in weight. 81 in vitro chemosensitivity tests carried out on tumour-cell cultures of 25 patients suffering from ovarian carcinoma, showed sensitivity to Cisplatin in 38%, Doxorubicin in 44%, 4-OOH-Cyclophosphamide (the in vitro active metabolite of Cyclophosphamide) in 50%, Vincristine in 53%, MTX in 19% and MPA in 36%.(ABSTRACT TRUNCATED AT 400 WORDS)

Zygotes from in vitro fertilization patients (n = 116) were randomly allocated to culture in either conventional plastic petri dishes or coculture on a monolayer of fetal bovine uterine fibroblasts. Embryos (n = 288) remained 26 to 32 hours in these culture systems. Video tape recording for later morphological analysis (11 parameters) was performed on 117 conventionally cultured and 104 cocultured embryos, shortly before replacement, by an independent observer, unaware of the culture conditions for each embryo. A significantly greater number of cocultured embryos (52%) had "good" morphology (zero or only one abnormal characteristic) as compared with conventionally cultured embryos (30%). The most outstanding morphological characteristic of cocultured embryos was the expanded appearance of their blastomeres. The incidence of implantation per embryo increased from 13% to 19% when the coculture rather than conventional culture system was used, and the incidence of ongoing pregnancy per patient after coculture doubled to 35%.

Red cell production in vertebrates is controlled by a glycoprotein hormone known as erythropoietin (Ep), which is produced by the kidney in response to hypoxia and acts on the marrow to selectively stimulate erythropoiesis. The gene for Ep has recently been cloned, and highly pure recombinant human Ep (rHuEp) is now available in considerable quantity. This has led to a better understanding of many aspects of Ep biology and to clinical trials in humans. The amino acid sequence of Ep is now completely known, and the protein portion of the natural hormone and the recombinant product are identical. Both the natural hormone and rHuEp produced in Chinese hamster ovary cells are heavily glycosylated in a very similar manner. This glycosylation is not necessary for in vitro activity but is required for activity in vivo. Radioimmunoassays (RIAs), which use labeled rHuEp, have been developed and are sufficiently sensitive to measure normal plasma levels. However, since Ep exists in plasma in several forms that vary in their immunologic and biologic activities, the ability of a RIA to provide information on the pathogenesis of clinical disease may be limited and should be referenced to the polycythemic mouse assay. The kidney's role in the production of Ep has been greatly clarified. Studies using probes to Ep mRNA have shown that Ep is primarily made in the kidney and secreted as the intact hormone. Moreover, renal secretion appears to be regulated by the rate of synthesis of the hormone, which in turn is dependent on the rate of synthesis of Ep mRNA. The cells that produce Ep have been identified as peritubular interstitial cells that may be endothelial in origin. The initiating mechanism for hormone production appears primarily to involve recruitment of additional cells rather than increased production by individual cells. Ep primarily acts on the marrow to stimulate the growth and maturation of early cells in the erythroid lineage that are known as the burst-forming unit-erythroid (BFU-E) and colony-forming unit-erythroid (CFU-E). The BFU-E is a very early cell closely related to the pluripotent stem cell, while the CFU-E is a later cell close to the first recognizable erythroblast.(ABSTRACT TRUNCATED AT 400 WORDS)

We studied the risk of the development of acute myeloid leukemia (AML) during initial remission in 733 consecutive children with acute lymphoid leukemia (ALL) who were treated with intensive chemotherapy. This complication was identified according to standard morphologic and cytochemical criteria in 13 patients 1.2 to 6 years (median, 3.0) after the diagnosis of ALL. At three years of follow-up, the cumulative risk of secondary AML during the first bone marrow remission was 1.6 percent (95 percent confidence limits, 0.7 and 3.5 percent); at six years, it was 4.7 percent (2 and 10 percent). The development of secondary AML was much more likely among patients with a T-cell than a non-T-cell immunophenotype (cumulative risk, 19.1 percent [6 and 47 percent] at six years). Sequential cytogenetic studies in 10 patients revealed entirely different karyotypes in 9, suggesting the induction of a second neoplasm. In eight of these patients, the blast cells had abnormalities of the 11q23 chromosomal region, which has been associated with malignant transformation of a pluripotential stem cell. There was no evidence of loss of DNA from chromosome 5 or 7, a karyotypic change commonly observed in cases of AML secondary to treatment with alkylating agents, irradiation, or both. We conclude that there is a substantial risk of AML in patients who receive intensive treatment for ALL, especially in those with a T-cell immunophenotype, and that 11q23 chromosomal abnormalities may be important in the pathogenesis of this complication.

Recombinant human erythropoietin (r-HuEPO) was administered in two phases to 12 patients with chronic renal insufficiency (creatinine clearances of 0.17-0.51 ml/second [10-30 ml/minute]) and uremic anemia. In addition to the routine tests done as part of a multicenter clinical trial, our patients had serial red cell mass measurements, quantitation of bone marrow stem cells, and marrow cytogenetic analysis. During the first eight weeks (acute phase), an equal number of patients was randomized to placebo or one of three doses of r-HuEPO (50, 100 or 150 unit/kg intravenously three times weekly). All three patients receiving 150 unit/kg responded by increasing their packed cell volume (PCV) to the normal range within eight weeks. There were lesser responses in PCV at the two lower doses of r-HuEPO and no response in the placebo group. The 51Cr red cell mass also increased significantly in a dose-related manner in patients receiving r-HuEPO but did not change in the placebo group. Marrow studies revealed increases in erythroid, megakaryocyte, and granulocyte-monocyte progenitor cells in those patients on r-HuEPO, but no mutagenic effects were seen. Subsequently, ten patients received open label r-HuEPO. During this maintenance phase, all ten achieved or maintained a normal PCV. Several adverse events occurred, but none were definitely linked to r-HuEPO. Recombinant human erythropoietin is an effective and potent treatment of anemia caused by renal failure.

A total of 100 mature oocytes from 13 consecutive patients were randomly assigned from each patient to one of two treatment groups (n = 53 for group 1, n = 47 for group 2). Group 1 oocytes were incubated throughout the culture periods in medium supplemented with 7.5% homologous patient serum. Group 2 oocytes were treated similarly, except the serum supplement was of fetal cord origin. End points for examination included fertilization frequency, normality of fertilization, stage of embryonic development at two time periods, and quality of embryonic development at two time periods. None of the end points examined revealed significant differences between patient serum and fetal cord serum.

Experimental studies in animals and recent preliminary clinical evidence raised the possibility that hypertransfusion might be capable of producing a beneficial effect on granulopoiesis recovery following irradiation or chemotherapy. This prompted us to design a study to determine the effect of hypertransfusion on the blood and marrow CFU-c of leukemic children during remission induction. Nineteen children with acute lymphoblastic leukemia have been randomized in pairs to normotransfused (Hb: 12-14 g/dl) and hypertransfused (Hb: 16-18 g/dl) groups. Anti-leukemic chemotherapy (vincristine and adriamycin weekly during 4 weeks and prednisone daily) was identical in all children. As expected, suppression of erythropoiesis was observed in the hypertransfused group. During the first three courses of chemotherapy, the number of marrow CFU-c remained very low in both groups. One week after the third course of chemotherapy the number of bone marrow CFU-c began to increase in both groups. One week after course four the CFU-c value was significantly larger in the hypertransfused group. We also observed that circulating CFU-c were almost absent before induction chemotherapy, whereas their number increased after course three and was higher in the hypertransfused group and remained higher after course four. These results show the kinetics of bone marrow recovery after chemotherapy and suggest that hypertransfusion increases the rate of recovery of granulopoiesis.

Misonidazole is a 2-nitroimidazole compound currently being assessed as a radiosensitizing agent. The effects of misonidazole on human bone marrow hematopoiesis were assayed by culture of committed granulocyte-macrophage progenitor cells (CFU-C). Three groups of patients with nonhematologic malignancies were selected for study. The first group of patients received a single, large dose of misonidazole; the second group received smaller, multiple doses of misonidazole; and the third group who did not receive any misonidazole served as irradiation controls. In 14 of 16 patients who received single or multiple doses of misonidazole, there was a significant decrease in the number of CFU-C present in the bone marrow after misonidazole therapy. In five patients who received irradiation only, there was no difference in the number of pre- and post-treatment bone marrow CFU-C. In misonidazole treated patients, extensive washing of post-treatment bone marrow samples failed to return CFU-C growth to control values. Suppression of CFU-C growth persisted for 3 weeks and returned to control values by 8 weeks. This reduction in the proliferative capacity of human bone marrow progenitor cells suggests that misonidazole may add to the myelotoxicity already associated with radiotherapy, systemic chemotherapy, or as combination of the two.

The effect of Levamisole on the human granulopoiesis was studied in patients randomized to receive, in addition to adjuvant chemotherapy for primary breast cancer, either no other treatment or additional unspecific immune therapy with Levamisole. The reaction of granulopoiesis to the cytostatic drugs, as characterized by changes of peripheral blood polymorphonuclear neutrophils (PMN), functional bone marrow granulocyte reserve, serial bone marrow cytology, and granulopoietic stem cells (CFU-C) in marrow and blood, was not affected by administration of Levamisole. The data support the concept that Levamisole has no direct effect on human bone marrow granulopoiesis, but that an allergic mechanism is involved in the pathogenesis of Levamisole-induced agranulocytosis. The expectation that Levamisole exerts a beneficial effect by stimulation of the granulopoiesis, as previously suggested for BCG and Corynebacterium parvum, could not be substantiated in our studies.

In a prospective, controlled trial 26 anaemic, neutropenic children with newly diagnosed acute lymphocytic leukaemia were randomised in pairs to receive either transfusion to a haemoglobin of 10--12 g/dl where clinically indicated (group A) or hypertransfusion to a haemoglobin of 16--18 g/dl (group B). Compared with group A (11 of 13 transfused), group B (all transfused) had a significantly more rapid rise in neutrophils at 7 and 10 days post-transfusion, a lower incidence of infection, and less interruption to chemotherapy. Hypertransfusion restored the myeloid/erythroid ratio to normal in bone-marrow of 5 of 6 children and the proportion of early myeloid precursors was greater than in controls.

Thirty two patients with malignant lymphoma - mainly Hodgkin's disease - were randomized for simultaneous treatment by high doses of metenolone during MOPP chemotherapy, to reduce its hematological toxicity. The results have shown surprisingly an increased hemato-toxicity in patients receiving androgens, with significantly more marked anemia and thrombocytopenia, reducing the total doses of anti-cancer drugs. This side effect could be explained by a cycling of the hematopoietic stem-cells and call to some caution when androgens are used during cancer chemotherapy.

In a prospective, randomized controlled study, 30 children who were receiving chemotherapy for malignant disease and who were anaemic and neutropenic, were randomized: 18 to receive transfusion to a Hb of 10-12 g/dl (group A) and 12 to receive moderate hypertransfusion to a Hb of 14-16 g/dl (group B). Children in group B had a significantly more rapid rise in polymorph count, lower incidence of infection, and lower incidence of interruption to chemotherapy. The findings of this study provide evidence for the existence of a common stem cell in human marrow, at least for erythroid and myeloid cell lines, and demonstrate that the concept of "stem-cell competition" derived from animal experiments has a human counterpart which is clinically significant.