The clinical course of acquired aplastic anemia in childhood; a retrospective study.

We reviewed the clinical courses of 38 children with acquired aplastic anemia (AA). The patients were classified according to the severity criteria by the Japanese Ministry of Health and Welfare (JMHW) Study Group (22 severe, 15 moderate, 1 mild). Early death was observed only in severe cases. Eight of the non-severe cases progressed to severe in 0.5-125 months, and the long-term survival rate of non-severe AA did not differ from that of severe AA. The frequency of lymphocytes in the bone marrow was significantly higher, and the peripheral blood neutrophil count was lower in patients who died within a year, and these patients should be treated as very severe. These findings suggest that the JMHW Study Group criteria are useful for identifying AA patients with a poor prognosis, but even non-severe cases should be repeatedly evaluated. Sixteen of the 33 patients treated with corticosteroids and/or anabolic steroids (AS) showed hematological recovery. Bolus methylprednisolone (mPSL) therapy was effective in one of the 8 patients. Allogenic marrow transplant (BMT) was performed on 3 patients. One died from sepsis and engraftment was not achieved in the other two. Trilineage recovery was obtained in 3 of 6 patients treated with rhG-CSF and rhEPO with or without AS, and hemopoiesis has been maintained 6-12 months after discontinuation in 2 cases. In the other 3 patients, the neutrophil count showed transient increase. Therefore, the treatment for severe AA patients, who have no sibling donor for BMT, should be started with the combination therapy including these cytokines.

[Treatment and prognosis after late relapse in childhood acute lymphoblastic leukemia].

We evaluated the clinical courses and laboratory features in 13 late-relapse cases of 55 children with acute lymphoblastic leukemia who had been in complete remission for longer than their years. In 8 of 13 cases, leukemia relapsed in bone marrow; 2 with testicular, 1 with central nervous system and one with ovarian involvement. Further, extramedullary relapse not involving bone marrow occurred in 5 cases (4 testicular and 1 CNS). Late-relapse was more frequently observed in boys (37.5%) than in girls (4.4%). Initial age and leukocyte counts were of no value in predicting late-relapse. The relapse rate in cases initially treated by the VPL regimen was twice that of those by a multi-drug regimen. A second prolonged remission was achieved in 5 of 10 cases by combinations of intensive chemotherapy (modified HEX) and irradiation to the testes or CNS. On the contrary, all late relapse patients initially treated by the multi-drug chemotherapy had a poor outcome. More intensive chemotherapy, including high-dose chemoradiotherapy and bone marrow transplantation, should be employed in this group of patients.

[High-dose busulfan, VP-16 and ACNU therapy with stem cell transplantation for the treatment of children with acute leukemia].

In order to strengthen the anti-leukemia effect, we developed a new conditioning regimen with high dose busulfan, VP-16 and ACNU (BVA) for cytoreduction before stem cell transplantation. Fourteen patients with refractory acute leukemia received allogeneic bone marrow transplantation (BMT) or peripheral blood stem cell transplantation (PBSCT) after conditioning with the BVA regimen, 7; allogeneic BMT, 1; syngeneic BMT, 6; PBSCT. # Seven patients were transplanted in the first complete remission, and 8 patients were in their second or third remission. Although total body irradiation or cyclophosphamide was not included in this regimen, engraftment was obtained in all cases. Two patients suffered relapse, and one patient died of cytomegalovirus interstitial pneumonitis (IP) 64 days after PBSCT. The other 11 patients are alive and free of disease at a median follow up time of 647 days (98-1235 days). Major regimen-related toxicity was pulmonary complications such as IP (3 cases) and pulmonary edema (2 cases). However, all patients recovered rapidly following steroid therapy. The results indicate that this conditioning regimen is highly effective for the treatment of childhood acute leukemia.

[Serum-free culture for leukemia cells].

Several human and murine acute myeloid leukemia cell lines growing in serum-free and protein-free culture medium are known to be established. In this paper, recent development of serum-free and protein-free culture methods for leukemia cells and the characteristics of the established cell lines are reviewed. Chemically defined culture medium is preferable as compared with serum-containing culture medium because the latter contains various, undefined factors which make it difficult to investigate the interactions between cell proliferation or differentiation and various growth factors. Investigations using leukemia cell lines which are maintained in a serum-free and protein-free chemically defined medium will provide the important information with respect to cell biology of leukemia cells and leukemia therapy.

[Cell differentiation action of 4'-epi-adriamycin in WEHI-3B myelomonocytic leukemia cells].

We studied the effect of 4'-epi-adriamycin on the differentiation of WEHI-3B (D+) murine myelomonocytic leukemia cell line (parent line) and WEHI-3B-MIT-R subline resistant to induction of differentiation by mitoxantrone. WEHI-3B(D+) cells were differentiated into mature granulocytes in suspension culture when exposed to 4'-epi-adriamycin regardless of the cell densities. The maximum differentiation rate was produced by 50 nM 4'-epi-adriamycin; at this concentration, approximately 40% of the cell population expressed a differentiated phenotype, as assessed by their ability to reduce nitroblue tetrazolium Induction of differentiation was observed by 4'-epi-adriamycin even at low cell densities (less than 10(5)/ml) that excluded the effects of autoinduction of differentiation. WEHI-3B-MIT-R cell line, which have a capacity of G-CSF-induced differentiation, was cross-resistance to 4'-epi-adriamycin. These results suggest that the mechanism of action differs from that of the induction of differentiation by G-CSF (which is known to show the activity by the secondary autoinduction of differentiation) and the differentiation induction is due to the direct action of 4'-epi-adriamycin.

Establishment and characterization of a murine megakaryoblastic cell line growing in protein-free culture (L8057Y5).

A murine megakaryoblastic cell line growing in protein-free culture (L8057Y5) was established from an experimentally induced murine leukemia (MK8057). Most of the Y5 cells were small and blast-like, with 2-4N in DNA content. Also, large cells possessing a lobulated nucleus characteristic of megakaryocytes, which showed polyploidization to more than 4N up to 16N, were occasionally seen. Nearly 5% of the total number of Y5 cells were positive for acetylcholinesterase reaction. The survival time of C3H/He mice after injection with Y5 cells was longer than that of mice injected with the original MK8057 cells. The colony-forming ability of Y5 cells in the spleen of the lethally irradiated mouse was much lower, whereas the number of in vitro colonies derived from Y5 was greater than that of MK8057. The plating efficiency of colony formation in serum-free methylcellulose culture was higher at a low O2 tension. Conditioned medium of Y5 cells enhanced colony formation as well as 3H-TdR uptake by Y5 cells, which implies that Y5 cells may produce autocrine growth factor(s). mRNAs for IL-6, LIF, and INF-gamma were expressed in Y5 cells; these cytokines may have roles in the growth mechanisms of the cell line.

Growth and differentiation of a murine interleukin-3-producing myelomonocytic leukemia cell line in a protein-free chemically defined medium.

We established the continuous growth of WEHI-3B D+ cells in protein-free chemically defined F-12 medium by stepwise decreases in the concentration of fetal calf serum. This cell line, designated as WEHI-3B-Y1, has now been propagated in protein-free F-12 medium for 3 years. The population-doubling time of the cells in culture is about 24 hr. WEHI-3B-Y1 cells are immature undifferentiated cells which show positive staining for naphthol ASD chloroacetate esterase and alpha-naphthyl butyrate esterase and spontaneously exhibit a low level of differentiation to mature granulocytes and macrophages. Medium conditioned by WEHI-3B-Y1 cells stimulated the proliferation of an interleukin-3 (IL-3)-dependent FDCP-2 cell line. This conditioned medium was shown to have erythroid burst-promoting activity when assayed using normal murine bone marrow. The colony formation of WEHI-3B-Y1 cells in semi-solid agar culture was not stimulated by purified recombinant human granulocyte colony-stimulating factor (rhG-CSF). However, in the presence of human transferrin, rhG-CSF enhanced the number of colonies of WEHI-3B-Y1 cells but did not induce their differentiation. These results suggest that WEHI-3B-Y1 cells cultured in protein-free medium produced murine IL-3. In addition, human G-CSF enhanced the clonal growth but did not induce the differentiation of WEHI-3B-Y1 cells cultured in serum-free medium.

[Induction of differentiation of myelomonocytic leukemia cells by mitoxantrone].

We studied the effect of mitoxantrone (MIT) on the proliferation and differentiation of murine myelomonocytic leukemia cell line WEHI-3B (D+), human myelocytic leukemia cell line HL-60 and cells from patients with newly diagnosed acute nonlymphocytic leukemia (ANLL). In a liquid culture, growth inhibition and differentiation of WEHI-3B (D+) cells to mature myeloid cells and a reinforcement tendency of the induction of nitroblue tetrazolium reducing capacity as well as of ASD chroloacetate esterase staining was observed by the treatment with MIT. Fresh ANLL cells classified as M4 were induced by MIT to undergo terminal differentiation to macrophage-like cells. Since at concentrations of WEHI-3B (D+) cells of less than 1 x 10(5)/ml induction of differentiation was observed due to MIT, it is suggested that its mechanism of action differs from that of the induction of differentiation by granulocyte colony-stimulating factor and is due to the direct action of MIT.

[Autoinduction of differentiation in human myelocytic leukemia cells (HL-60-Y3)].

We conducted a study on autoinduction of differentiation in human myelocytic leukemia cells (HL-60-Y3) in which the effects of serum cytodifferentiation were excluded by the use of a serum-free semisolid culture. In the culture dish the HL-60-Y3 colony count per dish was kept at 100 or below, and only the formation of clumping-type colonies, which consisted of blastoid cells, was observed. The formation of spreading-type colonies increased with the colony count and when the colony count reached 500 per dish, more than 90% of the colonies formed were spreading-type colonies. The main component cells of the spreading-type colonies were mature monocytoid cells, which were positive for alpha-naphthyl butyrate esterase. Moreover, a marked reduction in the recloning ability was observed in differentiated colonies compared to undifferentiated colonies. These results indicate the autoinduction of differentiation in human myelocytic leukemia cells. Furthermore, a single cell study that excluded the effect of colony to colony interactions suggested the presence of a differentiation autoinducing factor in the medium.

Three quantitative assays for human erythroid burst-promoting activity of recombinant growth factors and of omentum-conditioned medium.

Human erythroid burst-promoting activity (BPA) of recombinant growth factors and crude materials, of media conditioned by omentum tissue (OMCM), and of media conditioned by the bladder carcinoma cell line (HTB9CM) was measured by three different culture methods. Using the two-stage culture method, significant activity was shown in OMCM (137%-329% of the control), HTB9CM (102%-333%), recombinant human (rh) granulocyte-macrophage colony-stimulating factor (rhGM-CSF) (179%-220%), rh interleukin 3 (rhIL-3) (232%-676%), and rh insulin-like growth factor 1 (rh IGF-1) (106%-175%), whereas there was no significant increase in the number of erythroid bursts by the same additives when the one-stage culture or the delayed erythropoietin method was employed. Linear dose-response curves were observed in the tested range of rhIL-3 and rhGM-CSF. We also observed that 1) a larger amount of rhGM-CSF was required for the optimal stimulation of erythroid burst-forming units (BFU-E) than for the optimal stimulation of granulocyte-macrophage colony-forming units (CFU-GM), and 2) even the maximum dose of rhGM-CSF increased erythroid bursts to a lesser extent than was possible by the addition of rhIL-3. The former results implies that BPA is not the major activity of GM-CSF, and the latter result, although it is not conclusive, suggests that the GM-CSF-responsive BFU-E represent only a subset population of BFU-E responsive to IL-3. The two-stage culture is a useful assay method for screening BPA in biological materials with respect to accuracy, dose responsiveness, and reproducibility.

[Clonal response of murine myelomonocytic leukemia cells (WEHI-3B-Y1) to purified recombinant human hemopoietic factors in serum-free culture].

We tested purified recombinant hemopoietic factors for their effects on the proliferation and differentiation of murine myelomonocytic leukemia cells (WEHI-3B-Y1) in serum-free agar culture. We found that purified recombinant human G colony-stimulating factor (CSF) markedly increased the colony number of WEHI-3B-Y1 cells and differentiation-inducing activity. However, at low colony densities [( 100/dish), G-CSF did not induce the differentiation of WEHI-3B-Y1 cells. We conclude that G-CSF does not induce the differentiation of WEHI-3B-Y1 cells directly, but induce the differentiation as a result of the secondary autoinduction of differentiation. Interleukin 3 (IL-3) slightly enhanced but erythropoietin (Epo) did not alter the colony number of WEHI-3B-Y1 cells. GM-CSF or M-CSF decreased the colony number of WEHI-3B-Y1 cells. Such purified recombinant human IL-3, Epo, GM-CSF and M-CSF did not induce morphologically the distinct differentiation of WEHI-3B-Y1 cells.

The production of differentiation autoinducing activity by WEHI-3B D+ leukemia cells.

We studied the differentiation autoinducing activity in WEHI-3B D+ cell-conditioned medium (WCM). After culturing 10(6)/ml WEHI-3B D+ cells in RPMI-1640 medium without fetal calf serum (FCS) for 4 days, the supernatant was collected. The medium, concentrated 50-fold by YM-5 membrane filtration, was fractionated by gel exclusion on Ultrogel AcA44. We evaluated the effect of each of the four fractions on differentiation in WEHI-3B D+ cells by morphological, functional, and cytochemical criteria after adding the fractions to liquid or soft-agar cultures of 10(3) cells in 1 ml RPMI-1640 medium containing 10% FCS; the experimental cultures contained 10% of the fractions, with a control for each without the fraction. The growth of WEHI-3B D- cells in culture was inhibited by the addition of fraction P only (mol. wt. 10,000-20,000 daltons). In these same cultures, the cells were granulocyte-like, strongly positive for naphthol ASD chloroacetate esterase, and had phagocytic activity. Colonies grown in agar culture with fraction P also exhibited a peripheral halo of loosely dispersed cells around a central aggregate. Fraction P contained neither granulocyte colony-stimulating activity nor burst-promoting activity. These results suggest that fraction P contains differentiation autoinducing factor that is different from granulocyte colony-stimulating factor or interleukin 3.

[Congenital stomatocytosis associated with aplastic anemia].

A seven year-old boy with hereditary stomatocytosis complicated with aplastic anemia was reported. He was admitted to our hospital because of pale and general fatigue. On physical examination, he had severe anemia, petechiae, but no hepatosplenomegaly. Peripheral blood cell count revealed pancytopenia; RBC 103 X 10(4)/microliters, Hb 3.5 g/dl, Ret 21%, WBC 1,200/microliters, Pl 1.3 X 10(4)/microliters, and bone marrow revealed markedly hypocellular marrow. Red cell morphology demonstrated stomatocytosis. Red cell life span (51Cr T1/2) was 12 days, Coombs' test and Ham's test were negative. Indirect bilirubin was 1.1 mg/dl and marked decrease of haptoglobin was found. Family studies showed that his father and sister had stomatocytosis on peripheral blood examination, but no anemia. The patient had severe anemia because of complicated aplastic anemia. Congenital stomatocytosis with aplastic anemia is extremely rare. The authors are interested in a possible relationship between hereditary stomatocytosis and aplastic anemia although the precise mechanism remains to be elucidated.

[Autostimulation of growth by murine myelomonocytic leukemia cells (WEHI-3B-Y1)].

We studied the effects of medium conditioned by WEHI-3B-Y1 (WCM) on the growth of the cells using serum-free culture. The growth of WEHI-3B-Y1 cells was stimulated by the addition of WCM in serum-free culture. Furthermore, WCM stimulated the proliferation of interleukin-3 (IL-3) dependent FDCP-2 cell line and contained burst-promoting activity when assayed on normal murine bone marrow. Recombinant murine IL-3 also stimulated the growth of WEHI-3B-Y1 cells in serum-free culture. These results indicate that WEHI-3B-Y1 cells cultured in a protein-free medium produce IL-3, which autostimulates the growth of the cells.