Search

Cell fate of hematopoietic progenitors is regulated by interaction between Notch proteins on progenitors and Notch ligands such as Jagged1 on stromal cells. Since acute myeloid leukemia (AML) originates from dysregulated hematopoietic progenitors, some abnormalities in the Notch-Jagged system may exist in AML cells. As the first step to clarify this, we examined the expression of Notch1 and Jagged1 proteins in eight AML cell lines and 15 fresh AML samples by immunoblotting. In the Notch1 protein, two bands, a 300 kDa band and a 120 kDa band, which appeared to be a full-length protein and a transmembrane fragment, respectively, were recognized in five AML cell lines and six fresh samples. In addition, three of the five cell lines showed a 110 kDa fragment, which appeared to be from an intracellular domain, namely an active form. One cell line showed aberrant sized fragments, which suggested a structural abnormality. Jagged1 protein was recognized in six cell lines and six fresh samples. In four cell lines and four fresh samples, both Notch1 and Jagged1 proteins were observed. In these cells, Notch1 and Jagged1 proteins may interact among themselves. We showed that Notch1 and Jagged1 proteins are widely expressed in AML cells. We hypothesize that some abnormalities in the Notch-Jagged system which cause the excessive self-renewal and the block of differentiation, may be involved in the abnormal proliferation of AML cells.

Molecular diagnostic tests are widely performed in managing hematologic malignancies such as leukemia and lymphoma. In this article, we review the present application and problems of the tests. Karyotyping is performed at diagnosis of all kinds of hematologic malignancies. This method needs dividing cells as samples and skilled experts. Fluorescence in situ hybridization(FISH) analysis using cells in interphase is performed, for example, to monitor the effect of interferon on chronic myelogenous leukemia patients. The weak point of this method is that approximately 2% of false-positive cells are inevitable. Southern blot method is used for clonal analysis in some disease, for example, adult T-cell leukemia/lymphoma. Polymerase chain reaction(PCR) method using genomic DNA is performed for limited types of diseases such as lymphoma with bcl-2/IgH fusion gene. Reverse transcription(RT)-PCR method can detect fusion gene transcripts with high sensitivity. This method is useful for detecting minimal residual diseases after chemotherapy or bone marrow transplantation. To perform quantitative analysis, real-time PCR or competitive PCR must be done. In the near future, new technology such as gene expression profiling analysis using DNA microarrays or spectral karyotyping(SKY) method will be used in clinical practice.

Using DNA extracted from plasma samples of B-cell non-Hodgkin's lymphoma (B-NHL) patients, we attempted to detect the monoclonal rearrangement of immunoglobulin heavy chain gene by amplifying complementarity-determining region 3 (CDR3) by semi-nested polymerase chain reaction (PCR) method (plasma PCR). In 19 of 37 (51%) cases, clonal DNA was detected. With the same PCR method using DNA extracted from peripheral blood mononuclear cells, clonal DNA was detected in 8 of the 37 cases (22%). These 8 were in advanced stages with bone marrow (BM) invasion mostly. On the other hand, the 19 positive cases by plasma PCR included those in early stages without BM invasion or with normal soluble interleukin-2 receptor (sIL-2R) and lactate dehydrogenase (LDH) values. In 15 healthy volunteers, plasma PCR showed no clonal DNA. In cases in which tumor biopsy was difficult to perform, plasma PCR was helpful for determining whether or not the tumor was B-NHL. Plasma PCR is simple and has high specificity, although its sensitivity is insufficient.

The recent advances in molecular biology and gene engineering have contributed considerably to the diagnosis and treatment of hematopoietic malignancies, such as leukemia and malignant lymphoma. These advances also made possible precise determination of the clonal origin of malignant cells, the subtype of leukemia or lymphoma, and the clinical prognosis in each patient. Furthermore, minimal residual malignant cells in leukemia or lymphoma patients after achieving complete remission could be detected by DNA analysis. Based on these analyses, treatment can theoretically be tailored for each patient. We discuss in the present paper the usefulness of DNA or gene analyses of immunoglobulin heavy chain gene in clonal assessment of lymphocytic malignancies and in detecting minimal residual disease in the patient.

We studied the effects of vesnarinone, a quinolinone derivative used clinically for the treatment of chronic congestive heart failure, on the leukemic blast progenitors in acute myelogenous leukemia (AML) patients and on the normal hematopoietic precursors, colony-forming unit in culture (CFU-C), and colony-forming unit erythroid (CFU-E). Leukemic blast progenitors made blast colonies in the presence of granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF), or interleukin-3 (IL-3). Blast colony-formation was suppressed by vesnarinone in a dose-responsive manner regardless of growth factor added. Vesnarinone suppressed the primary (PE1) and secondary (PE2) colony-formation of leukemic blast progenitors in six AML patients tested. The suppression by vesnarinone did not significantly differ between PE1 and PE2. This finding suggests that vesnarinone exerts an almost equivalent effects on the terminal divisions and the self-renewal of leukemic blast progenitors. Furthermore, this drug suppressed the growth of clonogenic cells of five AML cell lines, OCI/AML1a, OCI/AML2, OCI/AML3, OCI/AML5, and OCI/AML6. Normal hematopoietic precursors CFU-C and CFU-E were also suppressed by vesnarinone, although vesnarinone was less toxic to normal hematopoietic than to leukemic blast progenitors. The possible usefulness of vesnarinone as a new approach to the treatment of AML patients is discussed.

A 49-year-old man was admitted with swelling in the left lower extremity, and a mass in the left lower abdomen. Laboratory findings showed an increased WBC of 15,000/microliter with 41% plasma cells, and immunoglobulin (Ig) A of 2,557mg/dl with a monoclonal component. A roentgenogram and computed tomograph of the abdomen revealed that a 5 x 10 cm mass with calcification located in the iliopsoas muscle. Plasma cell leukemia with extramedullary plasmacytoma was diagnosed, and the patient was treated with high-dose dexamethasone (40 mg/day for 4 days), resulting in a good response with the disappearance of plasma cells in peripheral blood and a marked decrease in serum Ig A. However, the patient's condition deteriorated in spite of various treatments, and he died of heart failure 5 months after admission. With informed consent from relatives, a necropsy was performed and infiltration of plasma cells in the mass in the iliopsoas muscle was noted. We reported this case because plasma cell leukemia with amyloid deposition and osteogenesis at the site of extramedullary plasmacytoma is very rare.

We report three cases of drug-induced hemolytic uremic syndrome (HUS). Three patients with advanced gastrointestinal cancer underwent a curative operation and adjuvant chemotherapy with Mitomycin C (MMC), 5FU and Ara-C. Later, progressive anemia, thrombocytopenia, renal dysfunction and elevation of serum LDH were recognized. A diagnosis of HUS was made. As they had no symptoms of infectious diseases or relapse of cancer, the cause of HUS was thought to be MMC. Treatment with antiplatelet drugs and fresh frozen plasma was effective for two patients. However, one patient died of pulmonary edema.

The effect of basic and acidic fibroblast growth factors on leukemic blast progenitors was studied in 14 patients with acute myelogenous leukemia and in one patient with chronic myelocytic leukemia in myeloid crisis. bFGF and aFGF stimulated blast-colony formation by leukemic blast progenitors cultured in methylcellulose in two patients. In the other 13 patients, no significant effect of either FGF on blast-colony formation was noted. The combination of bFGF or a FGF and G-CSF, GM-CSF, interleukin-3, or stem cell factor (SCF) had a synergistic effect on blast-colony formation in three patients. In the other patients, however, synergism between FGF and CSF was not detected. In fact, bFGF was found to suppress the stimulation of blast-colony formation due to GM-CSF in one of 10 patients and that due to SCF in four of eight patients. aFGF suppressed the stimulation of blast-colony formation due to GM-CSF in two of 11 patients and that due to SCF in four of eight patients. The results show that bFGF and aFGF do not directly play a major role in leukemic hematopoiesis but that they may modulate the cytokine network affecting leukemic cell growth.

Diversity and similarity in signaling pathways of granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and stem cell factor (SCF) in five human factor-responsive leukemia cell lines were investigated by immunoblotting to detect tyrosine phosphorylation of intracellular proteins. G-CSF induced tyrosine phosphorylation of a set of proteins with few different components according to the cell lines. IL-3 also induced phosphorylation of several proteins. In a lymphoid cell line, phosphorylation patterns induced by IL-3 were somewhat different from that in myeloid cell lines. Phosphorylation patterns by G-CSF and those by IL-3 were similar in myeloid cell lines. In a cell line which responded to both IL-3 and SCF, almost similar sets of proteins were phosphorylated by each, although phosphorylation of a 92-KDa protein was specific to IL-3 and that of a 140-200-KDa protein was specific to SCF. Taken together, proliferative growth factors induced tyrosine phosphorylation of similar sets of proteins with little difference according to each growth factor and each target cell line.

For the optimal growth of clonogenic cells in acute myeloblastic leukemia (AML), several cytokines such as tumor necrosis factor alpha (TNF-alpha), interleukin-1 (IL-1) and IL-6 are required in addition to colony-stimulating factor (CSF), which may be produced by blast cells themselves. In the present study, we addressed the potential role of endogenous production of TNF-alpha and/or IL-1 in the in vitro growth of AML clonogenic cells supported by IL-3. Addition of a specific neutralizing antibody against TNF-alpha (anti-TNF-alpha) to the culture significantly reduced the growth-stimulating effect of IL-3 on the cells in 11 of 14 patients. Simultaneous addition of anti-IL-1 alpha and anti-IL-1 beta also partly affected the growth, although to a much lesser extent when compared to the effect observed with anti-TNF-alpha. In 3 patients, the growth-stimulating effect of IL-3 was completely abrogated by anti-TNF-alpha or a combination of all three antibodies. Constitutive TNF-alpha transcript was observed in 5 patients and TNF-alpha protein was present in culture supernatant. Following in vitro culture, a transient but profound increase in c-fos, c-jun, TNF-alpha and IL-1 beta mRNA levels was observed. Anti-TNF-alpha inhibited the accumulation of TNF-alpha transcript, suggesting that membrane-integrated TNF-alpha may be partly responsible for the induction of TNF-alpha mRNA. It seems likely that the accumulation of these genes occurs through a protein kinase C-independent signaling pathway.

The in vitro growth activities and drug sensitivities of leukemic blast progenitors were compared among the subgroups of acute myelocytic leukemia (AML) classified according to the French-American-British (FAB) cooperative group. Leukemic cells separated from the peripheral bloods of AML patients were cultured in methylcellulose media, and the plating efficiencies of primary colonies (PE1) and secondary colonies after replating (PE2) were determined. PE1 and PE2 have been considered to reflect the capacities of terminal divisions and self-renewal of leukemic blast progenitors, respectively. PE1 and PE2 were variable among AML patients; these findings suggest that AML is a heterogeneous disease in terms of the proliferative activities of leukemic cells. No significant correlation was noted between PE1 or PE2 and the AML subtype. The sensitivities to cytosine arabinoside (Ara-C) of leukemic blast progenitors were studied in methylcellulose and suspension cultures. Ara-C sensitivity was not significantly correlated with the AML subtype, either. In contrast, there was statistically significant correlation between PE2 and the remission outcome of the patients, whereas PE1 was not significantly associated with the clinical outcome. The results in the present study indicate that the proliferative activity, especially self-renewal capacity, of leukemic blast progenitors is highly predictive of the prognosis of AML patients but is not significantly correlated with the AML subtype classified by the blast morphology.

A 49 years-old man presented with dry cough, low grade fever, and abnormal shadow on a chest X-ray. He had suffered from follicular lymphoma of the liver 5 years previously. He received irradiation therapy in combination with chemotherapy for approximately three years and had been in complete remission. Physical and radiological examination revealed pleural effusion and softly dense masses in the right lung. The laboratory data were within normal limits. He was diagnosed as having lymphomatoid granulomatosis (LYG) by open lung biopsy. The lung lesion was mainly infiltrated with T cells. The patient received prednisolone and the lung lesions disappeared. However, when a lung mass was noted two months later, he started to receive combination chemotherapy consisting of cyclophosphamide, adriamycin, vincristine, and prednisolone every three months. He has not shown relapse of LYG so far. To investigate the association between the preceding follicular lymphoma and subsequent LYG at this time, DNA analysis using the PCR technique was carried out. The LYG lesion did not show a rearranged band for the JH probe, while the paraffin-embedded specimen of the preceding follicular lymphoma had shown rearranged band for the JH band. Southern blot analysis of the LYG lesion, showed no rearrangement for TCR beta, gamma or JH probe. These findings indicate that the LYG was different from the preceding follicular lymphoma in terms of origin. LYG is considered to be induced in the immunosuppressive state due to lymphoma.

We report here a rare case of pure red cell aplasia (PRCA) following autoimmune haemolytic anaemia (AIHA). After 4 years of AIHA, the patient developed anaemia with severe erythroid hypoplasia and was diagnosed as having PRCA. At this time, Coombs' test was negative and parvovirus infection was not recognized. The patient received azathioprine, and PRCA improved. To determine the pathogenesis of PRCA, in vitro culture studies of erythropoietic and granulopoietic precursors were performed. The patient's serum or IgG did not suppress the colony formation of bone marrow colony-forming units-erythroid (CFU-E) of normal subjects and the patient. In contrast, mononuclear cells in the peripheral blood of the patient significantly suppressed CFU-E of normal subjects. Media conditioned by the patient's mononuclear cells did not significantly suppress CFU-E. The significant production of suppressive cytokine such as tumour necrosis factor or gamma-interferon by the patient's mononuclear cells was not recognized. These findings suggest that the cytotoxic mononuclear cells affected directly the erythropoietic precursors and caused PRCA in this patient. The pathogenesis of PRCA was, therefore, considered to be different from that of the preceding AIHA.

The responses to retinoic acid (RA) of acute myeloblastic leukemia (AML) blasts and normal hemopoietic progenitors was examined under defined growth factor conditions. For the leukemic cells marked patient to patient variation was seen; blast colony formation by cells from some patients was stimulated by RA without growth factors or in the presence of recombinant granulocyte colony-simulating factor (rG-CSF), recombinant granulocyte-macrophage-CSF rGM-CSF and recombinant interleukin-3 (rIL-3); for other populations inhibition was observed under the same conditions. Some blast cells were stimulated by RA in the presence of rGM-CSF and rIL-3 and inhibited when cultured with RA and rG-CSF. Supernatants prepared from blasts cultured with RA and growth factors did not show activities that were not readily explained by the carry-over of growth factors; this result did not provide evidence that RA and growth factors interact to produce factors. Titrations of RA showed that activity was first observed at concentrations of 10(-9) M and was maximum at concentrations of 10(-7) M. Different effects of RA in combination with rG-CSF compared with rGM-CSF or IL-3 were not seen when the cells were tested in suspension culture rather than in methylcellulose, a finding that may be interpreted to mean that the interaction between RA and factors affects terminally-dividing blast cells. Three normal bone marrow samples were cultured with RA and growth factors. Colony formation was stimulated by RA in the presence of rGM-CSF or rIL-3 but inhibited by RA with rG-CSF. Thus a differential effect of RA in combination with growth factors occurs in normal hemopoietic cells and persists in some AML populations.

The effects of the inhibitor for protein kinase A or C, or tyrosine kinase (H-8, staurosporine, or genistein, respectively) on the proliferation of leukemic and normal bone marrow cells stimulated by granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), or interleukin-3 (IL-3) were studied using the MTT assay. These inhibitors suppressed the proliferation of leukemic and normal bone marrow cells in a dose-dependent manner. Although the suppressive effect of each inhibitor on cell proliferation was varied in each instance, the effects were almost similar whichever CSF was added. A significant difference was not recognized between leukemic and normal bone marrow cells in terms of sensitivity to these inhibitors. The data indicate that protein kinase inhibitors have an inhibitory effect on leukemic and normal hematopoietic cell proliferation and that further studies are required to determine if this effect is due to the inhibition of protein kinases acting as the second messenger of CSFs.

A 54-year-old man was admitted to our hospital for precise examination of pancytopenia in October 1988. He had been cut off his left femur and irradiated because of osteosarcoma in 1954. After 30 years, he was diagnosed as Waldenström's macroglobulinemia. Melphalan had been given 2 mg daily for 19 months until August 1988, when it was discontinued due to pancytopenia. Peripheral blood showed Hb 6.6 g/dl, platelet 40 x 10(3)/microliters, and WBC 2000/microliters with 33% blasts. Bone marrow showed normocellularity with 36% blasts. Although blasts were negative for peroxidase staining, surface marker analysis revealed myeloid (CD 13, CD 33) phenotypes. Chromosome analysis showed 45, XY, -7, inv (3). A CT scan of the liver showed a mass, 10 by 10 cm, compatible with hepatocellular carcinoma. He was treated with very low dose Ara-C without noticeable effect. Hepatic tumor gradually enlarged, and he died of hepatic failure. This is a rare case of quadruplicate malignancies. The chromosomal abnormality suggests that AML was secondary leukemia which might be associated with immunosuppression due to macroglobulinemia and/or melphalan therapy.

We tested the effect of interleukin 1 (IL-1) on the growth of leukemic blast progenitors from patients with acute myeloblastic leukemia (AML). A purified blast cell fraction depleted of both T cells and phagocytic cells was tested at different cell densities. Addition of 1 ng/ml of IL-1 alpha alone enhanced blast colony formation in 10 of 13 cases tested, and the enhancement was prominent when plated cell densities were lowered. The conditioned media (CM) from AML patients contained varied levels of IL-1 activity, and following depletion of phagocytic cells, the levels decreased markedly in all cases tested. Addition of either antiserum against IL-1 alpha or IL-1 beta reduced the IL-1 activity in CM, suggesting that AML blasts produce both IL-1 alpha and IL-1 beta. Addition of IL-1 alpha or IL-1 beta antiserum inhibited blast colony formation in a dose-dependent manner, and a combination of both antisera showed the most marked inhibition. However, the augmentation of blast colony formation was almost completely inhibited by addition of anti-granulocyte-macrophage colony-stimulating factor (GM-CSF) serum in all three cases tested. IL-1 is also devoid of this activity when tested in the presence of a combination of granulocyte CSF (G-CSF), GM-CSF, and interleukin 3 (IL-3) at an optimal concentration. These results suggest that blast cells could produce and secrete CSF(s) and/or IL-1, and that the growth-enhancing effect of IL-1 on AML blasts is indirect, via production of CSFs by leukemic cells.

To determine the mechanism by which haematopoiesis is suppressed in aplastic anaemia, the effect of the sera from 6 patients on the granulopoietic precursors (colony-forming units in culture; CFU-C) was studied in vitro. Addition of the sera from 2 patients significantly suppressed CFU-C. The suppressive effect of the sera on CFU-C was inhibited by the addition of 1.5 NU/ml of anti-gamma-IFN antibody. In another patient, anti-gamma-IFN antibody increased autologous CFU-C although the serum of the patient did not suppress CFU-C. Serum gamma-IFN levels of all patients were under 2 IU/ml. The above findings suggest that humoral factors inhibit haematopoiesis in some patients with aplastic anaemia, and that gamma-IFN plays a role as an inhibitor even at a low concentration.

The blasts obtained from three freshly diagnosed acute myeloblastic leukemia (AML) patients were cultured in suspension to determine whether leukemic blast progenitors can indeed differentiate to form mature granulocytes. One patient was AML M2. The other two patients were bilineal and biphenotypic leukemia, respectively. Media conditioned by human bladder carcinoma line 5637 (5637-CM) or recombinant human granulocyte colony-stimulating factor (rhG-CSF) was added to stimulate growth. In suspension, clonogenic cells grew for 1-3 weeks in two patients, while they did not increase in one patient. After repeated subculture, cells of blast morphology decreased in percentage and polymorphonuclear neutrophils, eosinophils, and monocyte-macrophages appeared. Lymphoid cell component of the patient 2, who was diagnosed as bilineal leukemia by dual-color immunofluorescence analysis, decreased in number after suspension culture and cells of myeloid phenotype became dominant. The findings show that clonogenic blast progenitors can renew themselves and can also undergo terminal differentiation to mature end cells.

To determine the growth requirement of leukemic blast progenitors in acute myeloblastic leukemia (AML), leukemic cells from the peripheral blood of eight AML patients were cultured in the serum-free culture system. Blast progenitors made colonies in methylcellulose culture and showed exponential growth in suspension culture, although the growth of blast progenitors in the absence of fetal calf serum (FCS) in some patients was inferior to that in the FCS-enhanced culture system. Recombinant human granulocyte colony-stimulating factor (rhG-CSF) stimulated the growth of blast progenitors in a dose-responsive manner. When cells were cultured at high cell density, blast colonies were formed even in the absence of CSF. Irradiated blasts also supported the growth of intact blast progenitors. These results confirm the finding noted in the FCS-enhanced culture studies that granulopoietic factor, G-CSF, plays an important role on the leukemic growth. The importance of cell to cell interaction for the growth of blast progenitors was also confirmed.

A 73-year-old male was admitted because of slight fever. Systemic lymphadenopathy, polyclonal hypergammaglobulinemia, swelling of pancreas and submaxillary gland and progressing pancytopenia probably by immune mechanism were observed. Histological examination of lymph node showed no destruction of the structure and mild capillary proliferation in the follicle centers. Between follicles, many immunoblasts and mature plasma cells which were polyclonal and mild capillary proliferation were recognized. He was diagnosed as having multicentric Castleman's lymphoma. Administration of prednisolone 10 mg per day improved his symptoms and laboratory data. Then he has been followed up. Referring to the literatures, we discussed the relation between multicentric Castleman's lymphoma and the related diseases.

The effects of human recombinant tumor necrosis factor type alpha (rTNF alpha) on the blast progenitors from 14 acute myeloblastic leukemia (AML) patients and 1 chronic myelogenous leukemia patient in blastic crisis were studied in methylcellulose and suspension cultures. Blast progenitors renew themselves and/or undergo terminal divisions. Plating efficiency of primary colony formation (PE1) in methylcellulose, which is considered to reflect the terminal divisions of blast progenitors, was suppressed by rTFN alpha in a dose-dependent manner in all cases. Plating efficiency of secondary colony formation (PE2) and the recovery of clonogenic cells in suspension culture, which are considered to reflect the self-renewal capacity of blast progenitors, were also suppressed by rTNF alpha in a dose-dependent manner in almost all cases. rTNF alpha also inhibited both PE2 and clonogenic cells in suspension culture, even in relapsed AML patients who were very refractory to intensive chemotherapies. The results demonstrate that rTNF alpha inhibits not only terminal divisions but also the self-renewal capacity of leukemic blast progenitors. The finding that rTNF alpha suppressed the self-renewal capacity of leukemic blast progenitors proposes the utility of rTNF alpha to AML therapy.

We describe here the presence of a single class of interleukin 1 beta (IL-1 beta) receptors on the surface of blast cells freshly obtained from eight acute myeloblastic leukemia patients and one chronic myelocytic leukemia patient in blast crisis. Blast cells possessed a low number of high-affinity receptors (range, less than 10-173 receptors/cell) with a Kd of 1.8-12.8 x 10(-10) M. At the same time, we have investigated the effects of IL-1 on the growth of leukemic blast progenitors, and a significant heterogeneity of responsiveness was observed. IL-1 beta (1 ng/ml) enhanced blast colony formation in six patients. No significant effect was observed by addition of up to 100 ng/ml of IL-1 beta in the remaining three patients. No significant correlation was observed between the receptor number, receptor affinity, and the cellular responsiveness to IL-1; in some acute myeloblastic leukemia cases with apparent IL-1 receptors, no proliferation response to added IL-1 was observed. Our data show that IL-1 alone can enhance blast colony formation and that lack of responsiveness to IL-1 in some acute myeloblastic leukemia patients is not related to the absence of IL-1 receptors on blast cells.

The effects of transforming growth factor-beta (TGF-beta) on the blast progenitors from nine acute myeloblastic leukemia patients were studied in methylcellulose and suspension cultures. Leukemic blast progenitors undergo terminal divisions with a limited differentiation in methylcellulose culture, making blast colonies. Blast progenitors can renew themselves. The self-renewal can be reflected by secondary colony formation after replating primary blast colonies in fresh methylcellulose media and by the exponential growth of clonogenic cells in suspension culture. TGF-beta suppressed primary and secondary colony-formation in methylcellulose culture. Furthermore, TGF-beta suppressed the recovery of clonogenic cells in suspension. The results indicate that TGF-beta is effective in inhibiting not only terminal divisions but also self-renewal of leukemic blast progenitors.