Placental and umbilical cord blood, as an alternative course of haematopoietic stem cells for bone marrow reconstitution, have recently been showed to yield successful sibling-donor cord blood grafts children. The advantages of using cord blood are related to the high number of haematopoietic progenitors in circulation at birth. In our study there was a remarkable heterogeneity of volume, number of nucleated cells and the number of progenitors from sample to sample. Seven out of 40 samples of more than 61 ml blood volume contained 1.8 (+/- 1.0) x 10(5) CFU-GM or 6.0 (+/- 4.8) x 10(8) nucleated cells. 10 ml of whole blood was necessary for laboratory tests including ABO blood type, screening of infectious markers, HLA typing, as well as frozen sera and cells for the possible future tests. Collected cord blood of more than 70ml in volume may have sufficient numbers of CFU-GM for engraftment to pediatric patients weighing about 20 kg. A cord blood bank project is now going on by collecting blood with informed consent of the mother in cooperation with obstetrics and gynecology staff.

The effects of vesnarinone (3,4-dihydro-6-[4(3,4-dimethoxybenzoyl)-1-piperanizyl]-2 (1H)-quinolinone) on the hematopoietic precursors in 5 healthy volunteers and leukemic blast progenitors in 11 acute myeloid leukemia (AML) patients, 1 chronic myelocytic leukemia patient (CML) in blast crisis, and 3 leukemic cell lines were studied in methylcellulose and suspension cultures. Normal erythroid precursors (colony-forming unit erythroid: CFU-E and burst-forming unit erythroid: BFU-E) and granulopoietic precursors (colony-forming unit granulocyte/macrophage: CFU-GM) were suppressed in methylcellulose culture by vesnarinone in a dose-dependent manner. Leukemic blast progenitors may replicate themselves and/or undergo terminal divisions with limited differentiation. The plating efficiency of primary blast colony formation (PE1) in methylcellulose, which is considered to reflect the terminal divisions of leukemic blast progenitors, was suppressed by vesnarinone in a dose-dependent manner in all cases tested. The plating efficiency of secondary blast colony-formation (PE2) in methylcellulose culture and the recovery of clonogenic cells in the suspension culture, which are considered to reflect the self-replication function of leukemic blast progenitors, were also suppressed by vesnarinone in a dose-dependent manner in all cases tested. The results suggest that vesnarinone inhibits the growth of normal and leukemic hematopoietic progenitors. To determine the mechanism by which vesnarinone inhibits hematopoiesis, the effect of the agent on apoptosis (programmed cell death) of leukemic cells was studied. DNA ladder formation was recognized in OCI/AML 1 a cells after exposure to 100 micrograms/ml vesnarinone for 18 hours; this means that vesnarinone induced apoptosis in this cell line. Therefore, vesnarinone is considered to be the cause of apoptosis of granulopoietic precursors.

The strategies of gene therapy for cancer can be classified as: 1. regulation of oncogenes and anti-oncogenes, 2. immunogenetherapy, 3. support of chemotherapy, 4. suicide gene therapy, and 5. gene marking. The first one is the strategy to inhibit the expression of oncogenes by their antisenses or rhybozymes, or to introduce anti-oncogenes into those tumor cells with the inactivate effector cells (lymphocytes) by transducing cytokine genes, etc., followed by retransfering the gene-modified effector cells to patients (adoptive immunotherapy). The other one is to augment antigenicity of tumor cells. The immunogenetherapy method has been widely used for 70% of gene therapy of human cancer, because cells can be transduced ex vivo. The anti-tumor effects of human gene therapy using a GM-CSF gene by Muligan et al. or an IL-2 gene by Tahara and Lotze are expected. The third is the strategy to protect bone marrows from large dose of anti-cancer drug by transducing a multidrug resistance gene into those bone marrow cells or periphen blood stem cells, overcoming dose limiting of the drug. The fourth strategy is to transduce the herpes simplex virus thymidine kinase (KSV-TK) gene for activating the cytocydal prodrug (ganciclovir: GCV) into tumor cells in order to kill the tumor cells themselves following administration of GCV. At present, vectors most widely used for gene transduction are retroviruses and adenoviruses. However, the transduction using these vectors are primarily conducted ex vivo. The direct in vivo gene delivery method to target tumor cells are required.

The number of hematopoietic stem cells circulating in peripheral blood increases remarkably during the recovery of marrow function after myelosuppressive chemotherapy. In peripheral blood stem cell transplantation, these stem cells are collected and cryopreserved, and then used to restore marrow function after myelodisruptive (high-dose) anticancer therapy, Marrow recovery is faster with this procedure than with autologous bone marrow transplantation. Recently, this procedure has been used after high-dose chemotherapy for chemosensitive solid tumors such as breast cancer. We used high-dose chemotherapy with etoposide and carboplatin, followed by peripheral blood stem cell transplantation, to treat 5 patients with intrathoracic malignant tumors, including small cell lung cancer Neutrophils recovered (> 500 microliters) with 9 to 11 days and platelets recovered (> 5,000 microliters) within 8 to 13 days after the transplantation. No other serious complication was seen. Current topics regarding this procedure, problems to be solved, and prospects for further development are discussed.

Gaucher disease is a lysosomal storage disease which is characterized by deficient activity of lysosomal enzyme, known as glucocerebrosidase. This resulted in progressive accumulation of glucocerebroside only in bone marrow derived macrophages. This unique pathophysiology makes Gaucher disease an excellent candidate of gene therapy based on transferring therapeutic gene to hematopoietic stem cell. The extensive study based on transferring therapeutic gene to hematopoietic stem cell. The extensive study was already done using mouse in vivo system and human in vitro system. In mouse system, all of the macrophages from long term reconstituted mice were transduced. In human system, 40 approximately 60% of bone marrow progenitor cells were transduced. Because of this success, the clinical protocol for gene therapy for Gaucher disease was approved by RAC and FDA. The clinical trial was started in this year. The brief history of development and current limitation of gene therapy for Gaucher disease were discussed.

Gaucher disease is an inherited metabolic disease characterized by deficient activity of lysosomal enzyme, known as a glucocerebrosidase. Three clinical phenotype were documented depends on the onset of disease and neuronal involvement. Deficient activity of glucocerebrosidase results in progressive accumulation of glucocerebroside mainly in bone marrow derived macrophages. Diagnosis was made based on enzymatic activity in various tissue including WBC and fibroblasts. Molecular diagnosis was also possible. However, it is difficult to differentiate the three phenotypes. Although bone marrow transplantation and enzyme infusion therapy are both effective, the inherent problems limits their application. Gene therapy based on transfer of the therapeutic gene to hematopoietic stem cells were started in this year in USA.

Trisomy 13, as a sole karyotypic abnormality in acute leukemia, has been reported in several cases. However, in chronic myelogenous leukemia (CML), only two cases with this abnormality were reported so far. We describe herein a 68-year-old case with Philadelphia chromosome-negative CML and trisomy 13. Leukocytosis was pointed out during the treatment for other diseases. After 7 months, abrupt increase in leukocyte count (108,000/microliters) and splenomegaly developed. Decreased neutrophil alkaline phosphatase activity and morphological features fulfilled the diagnostic terms for CML. However, the karyotypic analysis revealed trisomy 13 instead of Philadelphia chromosome, and the BCR gene rearrangement was not detected. In cases with acute leukemia accompanied by trisomy 13, malignant transformation of an immature hematopoietic precursor cell has been suggested by the expression of antigens characteristic of both the myeloid and lymphoid lineage. In a few cases with myelodysplastic syndrome, a multipotent stem cell disorder, trisomy 13 has also been reported. From these standpoints, there might be a possibility that trisomy 13 as a sole abnormality in hematologic disorders would be related to tumorigenesis in the levels of multipotent stem cells.

A 45-year-old man was admitted with high fever and leukocytosis in August 1993. The diagnosis of acute myelogenous leukemia (AML; M2) was made on the basis of morphological, cytochemical and immunological characteristics of the blasts in the bone marrow. The induction therapy with BHAC, daunorubicin, 6-MP was unsuccessful in achieving remission; the bone marrow biopsy specimen revealed the proliferation of the remaining leukemic cells and massive fibrosis accompanied with unusual megakaryocyte-like giant bizarre cells. These megakaryocyte-like giant cells were positive for myeloperoxidase and CD34, but not GPIIIa and factor VIII, indicating that those were derived from myelogenous stem cells. Following the low-dose Ara-C therapy, improvement of fibrosis and disappearance of these giant cells were observed in the bone marrow. After the reinduction therapy with high-dose Ara-C and MIT against markedly increased blasts, the patient died of systemic fungal infection. The presence of myelofibrosis and giant atypical blasts might allow resistance to therapy and poor prognosis.

The X-linked form of hyper-IgM syndrome (HIGM1) is a rare disorder characterized by the inability of B cells to undergo isotype switch by a deficiency of CD40 ligand (CD40L) on activated T lymphocytes. The patients suffer from recurrent infections not only due to a lack of B lymphocyte activation but also due to defect of T lymphocyte functions. In addition, neutropenia is frequently accompanied by these symptoms. A patient with HIGM1, we experienced, suffered from recurrent infections and neutropenia. But he had a normal number of hematopoietic stem cell by the in vitro colony forming assay. CD34+ myeloid stem cell has been known to express CD40. We speculated by these facts that myeloid cell numbers are regulated by CD40-CD40L interaction.

We report a 3-year-old boy with acute myelogenous leukemia, who relapsed very early after peripheral blood stem cell transplantation (PBSCT). He was admitted with a tumor in maxillar sinus and hemorrhagic diathesis and was diagnosed as having acute myelogenous leukemia with t(8;21). He achieved complete remission with etoposide, cytosine arabinoside and mitoxantrone. After 8 courses of consolidation therapy and marrow ablative chemotherapy, he received PBSCT. G-CSF was given from day 0 because of severe infection. WBC and platelete counts rapidly increased, however, from day 20 platelet count spontaneously decreased. Concomitantly bone marrow examination revealed the presence of blastic cells. RT-PCR showed that the presence of AML 1/MTG 8 chimera mRNA in the cryopreserved PBSC samples. In vitro analysis also revealed that leukemic cells had G-CSF receptors and increased 3H-thymidine uptake in the presence of G-CSF. These findings strongly suggest that the reinfusion of leukemic cells in PBSC and the administration of G-CSF after PBSCT might be relevant to early relapse in this patient.

MX2, a new lipophilic morpholino anthracycline, has been reported to have superior chemotherapeutic effects to adriamycin against murine and human tumor cells. In this study the chemotherapeutic effect of MX2 against C6 glioma cells was examined as well as the photocytotoxicity of MX2 and the combination effect of MX2 and photodynamic therapy (PDT) in vitro. Colony formation is inhibited even with only 2 hour treatment with MX2 in a dose-dependent manner. In this colony forming efficiency assay the drug concentration required for 50% inhibition of colony formation for C6 glioma cells was 24.0 +/- 4.5 ng/ml. Mild photocytotoxicity of MX2 against C6 glioma cells was observed at a high concentration (100 ng/ml) of MX2 following exposure to white light but not red light. In combination, MX2 and the photosensitizer haematoporphyrin derivative (HpD) exhibited an additive cytotoxic effect against C6 glioma cells when the cells were treated with MX2 either immediately after red light illumination following incubation with HpD or at an interval of 24 hours before incubation with HpD. We conclude that MX2 may be clinically useful against malignant glioma alone, and in combination with other therapies such as PDT.

Autologous bone marrow transplantation (ABMT) and peripheral blood stem cell transplantation (PBSCT) are increasingly used to support high-dose chemotherapy for solid tumors of childhood. In this review we described practical aspects of myeloablative chemotherapy rescued by ABMT, PBSCT or combination of ABMT and PBSCT for the treatment of children with high-risk solid tumor, involving our experiences in 15 cases. Indication, method of harvesting bone marrow and peripheral blood stem cells, cryopreservation, transplantation, selection of anti-neoplastic agents for preconditioning, nutritional and G-CSF support, engraftment and outcomes for prognosis were discussed. In comparing the engraftment of stem cells between ABMT and PBSCT, the acceleration of platelet and erythrocyte recovery is less impressive, although there is a tendency to more rapid recovery of granulocyte in PBSCT group. The outcomes are distinctly improved only in patients who showed complete remission after induction chemotherapy, radiation and surgical excision. A better prognosis will be conferred especially in neuroblastoma and entities of small round cell tumor. It is noteworthy that relapses can occur as distant metastasis considerable years after complete clinical remission. This may be largely contributed by contaminated malignant cells in both harvested bone marrow and peripheral blood stem cells. There is no significant difference between the relapse rates after ABMT and PBSCT.

Germ cell tumor (GCT) is chemotherapy-sensitive, and with the development of a cisplatin combined regimen, the majority of patients can now be cured. Seventy percent to 80% of patients with advanced GCT have a lasting complete response to a cisplatin combined regimen, while 20 to 30% of these patients are refractory to the therapy. Refractory GCT patients need more effective and intense therapy. In order to overcome the conventional dose chemotherapy results, high-dose chemotherapy backed by autologous hematopoietic stem cell transplantation (AHSCT), has been applied to patients with GCT who have relapsed after achieving a complete response by first-line chemotherapy or to those in whom disease progression or the failure to achieve CR had occurred after initial and/or ifosfamide combined salvage therapy. The therapy with a high-dose combination of CBDCA (Carboplatin), VP-16 (Etoposide) and CPM (Cyclophosphamide) or IFM (Ifosfamide), supported with AHSCT is administered to the advanced GCT patients. The response rate (CR+PR) ranges from 40 to 78% by reported groups averaging about 50%, and disease-free survival is possible in about half the survivors thus far. Treatment related morbidity and mortality are observed, but are tolerable with the above dosage. Thanks to the technical development of collecting hematopoietic stem cells using either bone marrow or peripheral blood, and hematopoietic growth factor to lessen hematologic toxicities and obtain early hematological recovery, this method can be performed more safely. High-dose chemotherapy with AHSCT may well become more effective in patients with resistant GCT by a new combination of synergistic drugs or multicycle transplants.

In vitro chemosensitivity of established cell lines from human prostate cancer (8 PC 93, 19 PC 93, DU 145 and PC 3) to various anticancer drugs was examined by clonogenic assay. The drugs used were aclarubicin (ACR), adriamycin (ADM), carboquone (CQ), vincristine (VCR), ifosfamide (IFM), peplomycin (PEP) and cis-platinum (CDDP). To compare antitumor activity of different drugs, the predicted anticancer activity (PAA) was calculated from the 50% inhibition doses and the peak plasma concentration of the clinically used dose. High antitumor activity of drug was considered if PAA > or = 1 was observed. The chemosensitivities were: CQ > ADM > CDDP > VCR > PEP > IFM > ACR in the clonogenic assay. 19 PC 93 and DU 145 were sensitive to CQ, ADM and CDDP, but 8 PC 93 and PC 3 were sensitive to CQ, ADM, CDDP and VCR. Thus, a new combination chemotherapy with CDDP, ADM and CQ (PAQ therapy) was used clinically. PAQ therapy was given to sixteen patients with stage D advanced prostate cancer. Of these patients, 14 were undergoing relapse from antiandrogen therapy and 2 had hormone-resistant prostate cancer. The mean interval from the start of the prior treatment to relapse of the cancer was 18 months. The effectiveness of the new therapy was judged according to the response criteria for prostate cancer treatment of Japan. Three patients showed partial response (PR), 9 were stable disease (Stable) and 4 showed progressive disease (PD). The mean response duration in the patients with PR and with Stable was 11.6 months. The survival length of the responders (PR + Stable) was significantly longer than that of the nonresponders (PD) (p < 0.001). The side effect of PAQ therapy was lower than the moderate degree. Therefore, we considered PAQ therapy to be one of the clinical trials for the treatment of advanced prostate cancer.

The administration of perfluorotributylamine/pluronic F-68 stem-emulsion (FC43se), a fluorine compound emulsion, to a rodent discordant xeno-transplantation (dXT) model led to the discovery of the inhibitory effect of this compound against hyperacute rejection (HAR). When a guinea pig heart was transplanted to a rat administered with FC43se (10 microliters/g body weight), rhythmic beating was maintained for 1110 +/- 111.1 min (mean +/- SD; n = 8) whereas the untreated heart continued to beat for 15.5 +/- 6.6 min (mean +/- SD; n = 8). After 720 min of re-beating; pathologic changes observed in the untreated group, such as multiple thrombosis in the coronary vessels, were not observed in the FC43se-administered group by light and electron microscopic examination, and endothelial cells were well preserved. On the other hand, scattered necroses of the myocardium were observed and lymphocytic infiltration was demonstrated in the interstitium. We concluded that FC43se possessed a HAR inhibitory effect by inhibiting thrombus formation in the xeno-graft heart. Using this model, we speculated that action in the vessels of the graft heart (intravascular HAR) caused thrombus formation in the vessels. On the other hand, extravascular HAR causes myocardial necrosis more slowly, not resulting in cardiac arrest in the short term.