Allogeneic cord blood transplantation for adult acute lymphoblastic leukemia: retrospective survey involving 256 patients in Japan.

We investigated the efficacy of cord blood transplantation (CBT) for adult acute lymphoblastic leukemia (ALL) by reviewing medical records of 256 patients reported to the Japan Cord Blood Bank Network between June 1997 and August 2006. Cumulative incidence of neutrophil engraftment at day 100 was 78%. Infused CD34-positive cell dose (>1 × 10(5) cells/kg) was associated with successful neutrophil engraftment. Cumulative incidence of grade II-IV acute graft-versus-host disease (GVHD) at day 100 was 37%. A 2-year disease-free and overall survival (OS) rates were 36% and 42%, respectively. Multivariate analysis showed that age (51 or older vs younger than 50) (hazard ratio 1.9, 95% confidence interval (CI), 1.3-2.8, P=0.001), disease status (non-remission vs remission) (hazard ratio 2.2, 95% CI, 1.5-3.2, P<0.0001), grade III-IV acute GVHD (hazard ratio 2.0, 95% CI, 1.2-3.2, P=0.006) and absence of chronic GVHD (hazard ratio 2.4, 95% CI, 1.1-5.1, P=0.02) were negatively associated with OS. CBT is effective for some patients with advanced ALL. It is worth considering for further evaluation.

Transient hematopoietic stem cell rescue using umbilical cord blood for a lethally irradiated nuclear accident victim.

We performed stem cell rescue and allogeneic skin transplantation on a lethally neutron-irradiated nuclear accident victim. HLA-DRB1 mismatched unrelated umbilical cord blood cells (2.08 x 10(7)/kg recipient body weight) were transplanted to an 8-10 Gy equivalent neutron-irradiated patient because of a lack of a suitable bone marrow or peripheral blood donor. Pre-transplant conditioning consisted of anti-thymocyte gamma-globulin alone, and GVHD prophylaxis was a combination of cyclosporine (CYA) and methylprednisolone (mPSL). Granulocyte colony-stimulating factor (G-CSF), erythropoietin (EPO), and thrombopoietin (TPO) were concurrently administered after transplantation. The absolute neutrophil count reached 0.5 x 10(9)/l on day 15, the reticulocyte count rose above 1% on day 23, and the platelet count was over 50 x 10(9)/l on day 27, respectively. Cytogenetic studies of blood and marrow showed donor/recipient mixed chimerism. Rapid autologous hematopoietic recovery was recognized after withdrawal of CYA and mPSL. Repeated pathological examinations of the skin revealed no evidence of acute GVHD. Eighty-two days after the irradiation, skin transplantation was performed to treat radiation burns. Almost 90% of the transplanted skin engrafted. Immunological examination after autologous hematopoietic recovery revealed an almost normal T cell count. However, immune functions were severely impaired. The patient died from infectious complication 210 days after the accident.

[A preliminary analysis of unrelated marrow transplantations facilitated by the Japan Marrow Donor Program (JMDP)].

Between January 1993 and June 1994, the JMDP facilitated marrow donations from unrelated donors for 171 patients with malignant and non-malignant disorders. The median age of the patients was 21 years. All patients received marrow from phenotypically HLA -A, -B, -DR identical donors. About half of the patients wrer treated with total body irradiation (TBI)-containing regimens and about 80% of the patients received short-courses methotrexate and cyclosporine for graft-versus-host disease (GVHD) prophylaxis. Eight out of 171 patients, (4.7%) had graft failure and 63 out of 144 patients (44%), who survived for more than 30 days posttransplant, developed grade II to IV acute GVHD. The incidence of chronic GVHD was 47% (extensive form; 27%); most of them were progressive/quiscent type. The incidence of moderate to severe acute GVHD was higher than that observed in sibling transplants. On the other hand, the incidence of chronic GVHD was similar to that observed in sibling transplants. Overall survival at 1.5 years posttransplant was about 50% with no significant differences between diseases. The age correlated significantly with the survival in standard risk leukemia but not in high-risk leukemia. Despite the risk of graft failure and acute GVHD, this preliminary analysis demonstrates that transplantation of marrow from unrelated donors can be an effective treatment for certain hematologic disorders.

[Plasma concentration of cytosine arabinoside (Ara-C) in the elderly patients with hematological malignancy treated by Ara-C or cytarabine ocfosfate (SPAC)].

Plasma concentration of cytosine arabinoside (Ara-C) was determined in elderly patients with myelodysplastic syndromes or acute myelocytic leukemia who were treated with subcutaneous injection of Ara-C (Ara-C s.c.; 10 mg/m2/12 hr, 14-21 days), continuous drip infusion of Ara-C (Ara-C d.i.v.; 20 mg/m2/day, 24 hr 14 days) and/or oral administration of cytarabine ocfosfate (SPAC) (SPAC p.o.; 100 mg-300 mg/body/day, 14 days) by radioimmunoassay. In the Ara-C s.c. patients, the peak plasma level (Cmax) of Ara-C was 103 ng/ml and the time to reach Cmax was 15 min. The elimination half-like (t1/2) was 25 min and no accumulation was detected after 14 days of consecutive Ara-C s.c. administrations. In the SPAC p.o. patients, Cmax of Ara-C was 3-8 ng/ml and it took 3-5 days to reach Cmax. The plasma concentration level of Ara-C remains almost at the Cmax level during the SPAC p.o. administration and it remained higher than 0.32 ng/ml for as long as 15 days after the end of administration. In a Ara-C d.i.v. patient, plasma level of Ara-C was detected 4-7 ng/ml during the administration (day 7 through day 14). In all patients bone marrow suppression was observed after chemotherapy regardless of regimen, and there was no significant difference between nadir peripheral cell blood counts of Ara-C s.c. patients and SPAC p.o. patients.

[Four cases of Hodgkin's disease treated with MOPP/ABV hybrid regimen using nitrogen mustard-N-oxide hydrochloride].

Four patients with fresh Hodgkin's disease were treated with MOPP/ABV hybrid regimen using nitrogen mustard-N-oxide hydrochloride (NH2-O). All patients responded well to this therapy and achieved complete remission. Toxicity was minimum except for an older patient. He was 70 years old and developed arrhythmia after MOPP therapy but recovered without any treatment. The continuation of therapy was possible by dose reduction in this case. We conclude MOPP/ABV regimen using NH2-O is valuable for the treatment of Hodgkin's disease.

The alloantigen-specific immunosuppressive activity of estradiol-chlorambucil conjugate (KM2210) and its beneficial effect on allogeneic bone marrow transplantation in mice.

KM2210, a conjugate of estradiol and chlorambucil (CBL), which was originally developed as an anti-breast cancer agent, inhibits proliferative response of human mononuclear cells to alloantigens in mixed lymphocyte culture in a dose-dependent manner, but has no effect on their response to phytohemagglutinin. Neither estradiol benzoate nor CBL alone showed these unique actions. The suppressive effect of KM2210 on MLC was abrogated by adding of anti-transforming growth factor-beta (TGF-beta) antibody to the culture, but was not affected by the addition of interleukin-2, suggesting that KM2210, unlike CBL, displays its actions via TGF-beta. In experimental allogeneic bone marrow transplantation using mice, daily oral administration of KM2210 (2 mg/kg/day) for 30 days posttransplant significantly inhibited the alloantigen-specific immune reactions. Furthermore, the survival rate of the KM2210-treated mice was significantly higher than that of the cyclosporine-treated (2 mg/kg/day, p.o.) mice, and no adverse effect of KM2210 on hematopoietic recovery was found. These results strongly suggest possible clinical benefits of KM2210 as a new immunosuppressive agent for the prevention and treatment of graft-versus-host disease and other allospecific immune reactions.

Effect of interferon-alpha in patients with chronic myelogenous leukemia in the accelerated phase: cytogenetic and molecular studies.

Human interferon-alpha (IFN-alpha) has been shown to be effective in the treatment of Philadelphia chromosome (Ph1)-positive chronic myelogenous leukemia (CML) in the benign stable phase. The present study indicates that IFN-alpha may have a suppressive effect on Ph1-positive clones not only in the early stable phase but also in the accelerated phase with additional chromosomal abnormalities in some patients. In this study, in addition to 5 benign-phase patients, 3 patients with CML in the accelerated phase who had additional chromosomal abnormalities were treated with IFN-alpha. The presence of the Ph1-positive clone was estimated by chromosomal analysis and by Southern analysis at the DNA level using a 3' breakpoint cluster region (bcr) probe. Hematological remission and the suppression of proliferation of Ph1-positive clone to various extents were achieved by IFN-alpha treatment in 2 benign-phase patients and 3 patients with additional chromosomal abnormalities. Interestingly, in one of the latter three patients, Ph1-positive clones with or without additional chromosomal abnormalities were completely suppressed judging from chromosomal analysis and from the disappearance of bcr gene rearrangements.

[Clinical application of recombinant human colony-stimulating factor (rhG-CSF) in bone marrow transplantation].

Human granulocyte colony-stimulating factor (G-CSF) specifically stimulates granulocyte production and enhances functions of mature granulocytes. It also proliferates myeloid leukemic cells. The molecule was purified and molecularly cloned in 1986. In this study, 51 patients received single daily injections with recombinant human (rh) G-CSF (2-20 micrograms/kg) after bone marrow transplantation were compared with control 36 patients. Blood neurophilic recovery was accelerated remarkably by rhG-CSF administration without any adverse effects including delay in reticulocyte and platelet recoveries. Furthermore, the duration for the management in laminar airflow room and febrile days greater than or equal to 38 degrees C were shortened in rhGS-CSF treated group. In 6 patients with myeloid leukemia in relapse, we also tested the effects of the rhG-CSF-combined conditioning regimen. In all the patients tested, the leukemic cells responded to rhG-CSF. Clinically no relapse was observed and 3 patients are well on day 224-357 for the time being. These findings indicate that rhG-CSF is very useful in bone marrow transplantation.

Establishment and characterization of a non-T, non-B cell lymphoma cell line with T cell receptor beta- and gamma-chain gene rearrangement and possessing MRK 20 monoclonal antibody-defined 85KD protein.

A new non-T cell, non-B cell lymphoma cell line, designated IN-1, was established from the ascitic fluid of a patient with non-Hodgkin lymphoma. The IN -1 cells did not show any T cell and B cell immunophenotypes. There were rearrangements of T cell receptor beta- and gamma-chain gene, but no rearrangement of T cell receptor delta-chain gene and immunoglobulin JH gene. Electron microscopically, the cell had numerous pseudopods, mitochondria, vesicles, a conspicuous nucleolus, and scattered heterochromatin at the periphery of the nucleus. They reacted with only OKT9 monoclonal antibody. Molecular analysis revealed that cellular DNA from the IN-1 cells did not hybridize with Bam HI W fragment of EB virus DNA. Cytogenetic analysis showed that the chromosome number of the IN-1 was in the range of 61 -63 whose karyotype analysis demonstrated multiple numerical and structural chromosome changes. The IN-1 cells were resistant to etoposide in comparison with an IC50 of K562 (human chronic myelogenous leukemia). Interestingly, this IN-1 cell possessed 85 KD protein, but not P-glycoprotein, both of which are considered to be multidrug resistance-related proteins.

Granulocyte colony-stimulating factor stimulates human mature neutrophilic granulocytes to produce interferon-alpha.

Granulocyte colony-stimulating factor (G-CSF) is a glycoprotein hormone that specifically stimulates both production and functional activation of neutrophils, while interferon-alpha (IFN-alpha) is known to suppress myelopoiesis, including neutrophil production in vivo and in vitro. On a possibility that IFN-alpha may operate as one of the inhibitory feedback factors in neutropoiesis, we examined whether neutrophils produce IFN-alpha in response to G-CSF. Northern blot analysis showed that messenger RNA (mRNA) for human IFN-alpha 1 became detectable time-dependently in highly purified human neutrophils incubated with purified recombinant human G-CSF (rhG-CSF). But such transcription was not observed either in neutrophils incubated with other neutrophil activators, such as formyl-methionyl-leucyl-phenylalanine (fMLP) or lipopolysaccharides (LPS), or in blood mononuclear cells incubated with rhG-CSF. In addition, radioimmunoassay for human IFN-alpha showed that its levels in culture medium of the rhG-CSF-treated neutrophils rose markedly (up to approximately 100 IU/mL/1 x 10(7) cells) in a time-dependent way, compared with those of nonstimulated neutrophils. These findings suggest that the G-CSF/IFN-alpha system may participate in the feedback regulatory loop of neutropoiesis.

High-level expression of MRK 16 and MRK 20 murine monoclonal antibody-define proteins (170,000-180,000 P-glycoprotein and 85,000 protein) in leukaemias and malignant lymphomas.

Using flow cytometry and immunocytochemistry, we investigated the reactivities of two different murine monoclonal antibodies (MAbs), MRK 16 and MRK 20, specific to adriamycin-resistant K562 cells (K562/ADM) with peripheral human mononuclear cells (MNC) (mainly blastic cells and lymphocytes) from 31 patients with leukaemia or malignant lymphoma. Reactivity with MRK 16 MAb was observed in five cases and reactivity with MRK 20 MAb in 18 cases. The cases were divided into three groups according to their reactivity patterns: group I, only the proportion of MRK 16-positive cells was increased; group II, only the proportion of MRK 20-positive cells was increased; group III, both MRK 16-and MRK 20-positive cells were increased. Some cases reflected the prior administration of adriamycin, vincristine, vinblastine and VP-16, which are known to induce P-glycoprotein expression. Expression of Mr 85,000 protein was observed more frequently than that of P-glycoprotein in leukaemia and malignant lymphoma, and this was not associated with either the total dose or period of administration of anticancer drugs. The expression of Mr 85,000 protein recognised by MRK 20 was further confirmed by Western blot analysis.

[A case of refractory testicular cancer showing complete response to high-dose chemotherapy with autologous bone marrow transplantation].

A patient with stage III testicular cancer was treated by 3 courses of BEP therapy and a partial response was obtained. Afterwards he underwent resection of pulmonary residual tumors and was treated by 2 courses of PE therapy immediately after operation. However, a new pulmonary tumor appeared after the first course of PE therapy was completed. He was treated by high-dose chemotherapy (etoposide 1200 mg/m2, cyclophosphamide 120 mg/kg, CDDP 60 mg/m2) with autologous bone marrow transplantation. Three weeks after high-dose chemotherapy his metastatic pulmonary tumor showed cavity formation which disappeared after 3 months. Fifteen months after the high-dose chemotherapy, no evidence of disease has been seen even without maintenance therapy.

Erythrocyte enzyme activities in myelodysplastic syndromes: elevated pyruvate kinase activity.

The erythrocyte enzyme activities in twenty-six cases of myelodysplastic syndromes were determined. There were remarkably abnormal levels in seven cases; namely, four cases showed increased hexokinase activity, three cases showed increased pyruvate kinase activity, and two cases showed increased adenosine deaminase activity. Among these, one case with elevated pyruvate kinase activity showed the novel expression of M2-type pyruvate kinase activity, in addition to the R-type pyruvate kinase activity normally found in erythrocytes. Southern blotting of peripheral leucocyte DNA revealed only an amplified PK-LR genome, which derived from the chromosomal abnormality of a 1;7 translocation. The mechanism responsible for switching M2-type to R-type during erythroid maturation was considered to be partially disrupted in this case.

Serum granulocyte colony-stimulating factor levels in healthy volunteers and patients with various disorders as estimated by enzyme immunoassay.

In order to better understand the patho-physiologic role of granulocyte colony-stimulating factor (G-CSF), we estimated its serum levels in healthy persons and patients with various disorders, using a newly developed enzyme immunoassay (Motojima et al). In 49 of 56 normal healthy persons (88%), the levels were beneath the sensitivity of the assay (less than 30 pg/mL), while in the remaining seven healthy persons, the levels ranged from 33 to 163 pg/mL. On the other hand, nine of 11 patients (82%) with idiopathic aplastic anemia (AA), one patient with Fanconi's anemia, six of 12 patients (50%) with myelodysplastic syndrome (MDS), five of 12 patients (42%) with acute leukemia without any blast cells in the blood (M4: one, M5: one, L1: one, and L2: two), six of 18 patients (33%) with chronic myeloid leukemia (CML), one of two patients with chronic lymphoid leukemia (CLL), two of four patients with lung cancer, one patient with cyclic neutropenia, two of seven patients with malignant lymphoma, and four patients with acute infection had G-CSF levels ranging from 46 pg/mL to greater than 2,000 pg/mL. Interestingly, a reverse correlation between blood neutrophil count and serum G-CSF level was clearly demonstrated for aplastic anemia (r = -.8169, P less than .01). Moreover, it was found that the G-CSF level rose during the neutropenic phase of cyclic neutropenia and after chemotherapy or bone marrow transplantation (BMT) in three patients with leukemia; also high G-CSF levels were positively correlated to blood neutrophil counts in some cases of infectious disorders and lung cancer. The cellular sources and the mechanisms for production and secretion of circulating G-CSF were not investigated in this study, but the data presented here strongly indicate that G-CSF plays an important role as a circulating neutrophilopoietin.

Postoperative erythrodermia (POED), a type of graft-versus-host reaction (GVHR)?

Postoperative erythrodermia (POED) is a rare disease appearing several days after surgery for which blood transfusion has been required, characterized by erythrodermia, fever, pancytopenia, hepatic insufficiency or diarrhea. Most affected patients die within four weeks. The etiology remains unknown, but some assume it to be a type of graft-versus-host reaction (GVHR) caused by transfused lymphocytes. In this study, skin biopsies taken from 9 POED patients were examined to clarify whether POED is a type of GVHR. In seven samples, changes compatible with GVHR, such as lymphocyte infiltration in the basal layer and occasional eosinophilic or vacuolar degeneration of the epidermal cells, with occasional satellitosis, were noted. Immunopathologically, infiltrating lymphocytes mostly expressed suppressor/cytotoxic T cell markers like those in GVHR. Meanwhile, immunostaining with anti-HLA-A type specific antibodies failed to differentiate infiltrating lymphocytes from host cells. Thus, the present morphologic and in vivo marker study suggested POED to be a type of GVHR, while in vivo HLA-A typing showed two contradictory possibilities: first, that POED is a GVHR in which major histocompatibility antigen-matched donor lymphocytes attack host cells, and secondly that POED is not a GVHR, the infiltrating lymphocytes being of host origin.