Uptake of low density lipoproteins by human leukemic cells in vivo: relation to plasma lipoprotein levels and possible relevance for selective chemotherapy.

The success of cancer chemotherapy is dependent on the possibility to utilize biological differences between malignant and normal cells to selectively destroy the tumor cells. One such difference may be that of receptor-mediated cellular uptake of low density lipoproteins (LDLs). Previous studies have shown that leukemic cells from patients with acute myelogenous leukemia have elevated receptor-mediated uptake and degradation rates of plasma LDL in vitro compared to normal white blood and bone marrow cells, and that plasma cholesterol levels at diagnosis are inversely correlated with the LDL receptor activity of the malignant cells. An important question is whether the uptake of LDL by the leukemic cells is also increased in vivo. To evaluate the in vivo uptake of LDL, 11 adult patients with newly diagnosed acute myelogenous leukemia received an i.v. injection of [14C]-sucrose-labeled LDL. On degradation of [14C]sucrose-LDL, the radiolabeled sucrose moiety is known to remain trapped in the lysosomal compartment of the cells. After injection, radioactivity accumulated progressively for at least 12 hr in the leukemic cells. The uptake of radioactivity in vivo correlated with the rate of receptor-mediated degradation of 125I-labeled LDL by the leukemic cells assayed in vitro (r = +0.88, P less than 0.001). An inverse correlation between plasma LDL cholesterol concentrations and the in vivo cellular uptake of [14C]sucrose-LDL in whole blood (r = -0.76, P less than 0.01) indicates that the hypocholesterolemia is due to elevated LDL uptake by the leukemic cells. Postmortem biopsies from virtually all tissues were obtained from one patient, and the distribution of radioactivity revealed that the liver and bone marrow had accumulated most radioactivity; the adrenals had the highest uptake of label per gram of tissue weight. The results indicate that LDL may be used as a carrier targeting lipophilic cytotoxic drugs to leukemic cells.     

急性髓性白血病患者血清胆固醇水平变化与低密度脂蛋白受体活性的关系研究

测定了52例初发未急性髓性白血病（AML）患者血清胆固醇浓度及外周血白血病细胞对^125I-低密度脂蛋白（^125I-LDL)的降解率,结果显示两者呈负相关,白细胞及^125I-LDL降解率较高者血清胆固醇浓度较低,化疗后,随着外周血白血病细胞消失,血清胆固醇及LDL胆固醇水平升高。据此推测AML患者的低胆固醇血症可能归因子白血病细胞的LDL受体活性增高,白血病细胞对LDL的高摄取及降解为利用LDL作为化疗药物的载体靶向治疗白血病提供了新思路。     

Leukemic cell colony formation in soft agar by bone marrow cells and peripheral blood cells from untreated acute leukemia patients

An in vitro culture technique for colony formation of marrow cells and peripheral blood cells from untreated acute leukemia patients and from patients in relapse is described. The colonies from bone marrow cells of an untreated acute myelogenous leukemia (AML) patient were demonstrated to be of leukemic origin by cytogenetic analysis. Cells obtained from colonies of leukemic origin contained the human malignancy-associated nucleolar antigen (HMNA) as detected by indirect immunofluorescence. This nucleolar antigen was not present in marrow or peripheral blood cells or cells from colonies of marrow from hematologically normal individuals. Colonies could be grown from over 70% of the marrow and peripheral blood samples from untreated acute leukemia patients. The median number of colonies obtained was 75 per 10(5) marrow cells from patients with AML. In 1/3 of the cases an increased number of colonies could be grown from marrow cell suspensions kept in liquid culture for 5 days. This is indicative of the proliferative capacity of the colony forming cell population. This assay may be useful for detection of residual clonogenic leukemic cells in marrow and peripheral blood cell suspensions.     

Isolated relapse of acute myelogenous leukemia presenting as a gastric ulcer.

Isolated gastrointestinal (GI) infiltrate is unusual at presentation or relapse of acute myelogenous leukemia (AML). We report a case of acute myelogenous leukemia (FAB-M4) whose isolated relapse presented as a bleeding gastric ulcer. The patient was a 30-year-old male who had been diagnosed to have AML in June 1988. While in third complete remission, he underwent a sibling allogeneic HLA-matched bone marrow transplant. Five months after transplantation, he was readmitted for pneumonia. While in the hospital, he had an episode of upper GI bleeding. The endoscopy revealed a leukemic gastric ulcer, with morphology and immunophenotyping identical to his initial AML. There was no evidence of leukemia in the blood or bone marrow. Although different types of leukemic infiltrates have been recognized at post-mortem examination, our case is unique because AML presenting as an isolated malignant ulcer has not been described previously. We conclude that relapsing AML may present as an isolated gastric ulcer and suggest that any suspicious lesion on upper GI endoscopy should be biopsied after aggressive platelet support.     

Histone deacetylase inhibitor treatment downregulates VLA-4 adhesion in hematopoietic stem cells and acute myeloid leukemia blast cells

The alpha4beta1 integrin very late activation antigen-4 (VLA-4) is an alpha4 (CD49d)/beta1 (CD29) heterodimer. It plays a key role in the adhesion of both hematopoietic progenitor cells and leukemic blast cells to bone marrow stromal cells which express the vascular cell adhesion molecule-1 (VCAM-1) or produce fibronectin. VLA-4 expression has been associated with bone-marrow minimal residual disease, which causes relapse after chemotherapy in patients with acute myelogenous leukemia. Conversely, the absence of VLA-4 reduces bone marrow retention of both hematopoietic progenitor and leukemic blast cells. We report on the downregulation of VLA-4/CD49d for various acute myelogenous leukemia cells lines, on primary cells from patients with acute myelogenous leukemia, and on hematopoietic stem cells and peripheral blood mononuclear cells from healthy donors on treatment with the histone deacetylase inhibitors suberoylanilide hydroxamic acid and valproic acid, which is associated with decreased adhesion to mesenchymal stromal cells. These findings suggest that HDAC-inhibitor treatment may on the one hand impair stem cell homing, while on the other it may improve peripheral blood stem cell mobilization and significantly help to reduce minimal residual disease from acute myelogenous leukemia.     

Flow cytometric analysis of P-glycoprotein in normal and leukemic cells

Summary Classical multidrug resistance is characterized by overexpression of a membrane protein, P-glycoprotein, which acts like a drug-extruding pump, reducing accumulation of cytotoxic drugs inside malignant cells. We have developed a simple method for detecting an intracellular epitope of P-glycoprotein in normal and leukemic cells by the monoclonal antibody JSB-1 and fluorescence-activated flow cytometry. Permeabilization of blood and bone marrow cells in unprocessed samples is achieved by a commercially available red blood cell lysing solution which excellently preserves the light scatter properties of leukocytes. The method is suitable for analyzing samples in clinical routine. Lower than 1% reactivity was seen in the lymphoid gate of normal peripheral blood and bone marrow samples as compared with over 60% of reacting cells in some leukemic samples. Twelve patients with acute de novo leukemia were studied at presentation, 13 patients at a refractory stage, and 28 in remission. There was a positive correlation between the P-glycoprotein and the CD 34 expression in acute myelogenous leukemia and an association between the P-glycoprotein expression and the blast count in both acute myelogenous and lymphatic leukemias.     

Terminal Deoxyribonucleotidyl Transferase in Human Leukemia

Terminal deoxyribonucleotidyl transferase (EC 2.7.7.31; nucleoside triphosphate:DNA nucleotidylexotransferase) is usually found only in thymus, but has been reported in leukemic cells from children with acute lymphoblastic leukemia. In an unusual adult patient with acute myelomonocytic leukemia, terminal transferase was found at a level of 16 units per 10(8) bone marrow cells and 14 units per 10(8) circulating leukocytes (1 unit = 1 nmol of nucleotide per hr). This activity is comparable to that found in normal thymus. Assays of transferase in marrow and peripheral leukocytes from patients with typical acute and chronic myelogenous leukemias gave average values of 0.5 and 0.3 unit per 10(8) cells, respectively. Transferase activity is also found in normal bone marrow at about 0.07 unit per 10(8) cells. Terminal deoxyribonucleotidyl transferase in all samples of human marrow and peripheral blood had reaction characteristics, sedimentation, and chromatographic properties similar to the homogeneous enzyme from calf thymus.     

Impaired low-density lipoprotein receptor activity in chronic B-lymphocytic leukaemia cells

Cellular degradation of ¹²⁵I-labelled low-density lipoprotein (LDL) was analysed in freshly isolated blood mononuclear cells from 26 patients with chronic B-lymphocytic leukaemia (CLL) and 8 healthy subjects, and in cells following 1, 2 and 3 d of culture in medium containing 10% human lipoprotein-deficient serum (LPDS). Fresh CLL cells had lower LDL degradation rates than mononuclear cells from healthy subjects (p < 0.01). The LDL degradation rates increased during culture (p < 0.001), but to a lesser degree in CLL cells than in normal blood mononuclear cells (p < 0.001). The cellular degradation rate of ¹²⁵I-LDL was markedly inhibited by an excess of unlabelled LDL, indicating that most of the ¹²⁵I-LDL that was degraded had been internalized following binding to the high-affinity cell surface receptor. There was a positive correlation between the LPDS-induced LDL degradation of CLL cells and the thymidine uptake in CLL cell cultures with (r = 0.70, p < 0.001) and without (r = 0.59, p < 0.01) the B cell mitogen, Epstein-Barr virus. The results indicate that LDL receptors might be involved in the regulation of CLL cell proliferation.     

Absence of common ALL antigen on normal bipotent myeloid, erythroid, and granulocyte progenitors

The presence of the common acute lymphoblastic leukemia antigen (CALLA) on leukemic cells from the great majority of patients with non-T cell acute lymphoblastic leukemia and chronic myelogenous leukemia in blast crisis suggests that CALLA could be differentiation antigen expressed by normal lymphoid and myeloid stem cells. Treatment with a murine monoclonal anti-CALLA antibody and complement lysed CALLA-positive leukemic cells quantitatively, whereas similar treatment of nucleated cells from peripheral blood and bone marrow failed to affect the expression, in semisolid culture, of CFU-G/E, BFU-E, CFU-E, or CFU-C. These data suggest that CALLA is not a normal differentiation antigen of the myeloid bipotent cell or its committed progenitors.     

The biological characteristics of dendritic cells derived in vitro from myelogeneous leukemia cells and healthy donor cells

Successful adoptive immunotherapy for leukemia depends on the generation of T cells that can specifically react with malignant cells. Dendritic cells (DCs) are important antigen-presenting cells in the development of antileukemia T-cell responses. Mononuclear cells (MNC) were isolated from peripheral blood or bone marrow of patients with chronic myelogenous leukemia (CML), and acute myelogenous leukemia (AML). After incubation with granulocyte-macrophage colony-stimulating factor, interleukin (IL)-4, and tumor necrosis factor-alpha, MNC developed morphological characteristics of DCs in vitro, which were confirmed by phenotypic assay. Fluorescence in situ hybridization demonstrated the presence of fusion gene in the nuclei of representative CML or AML-M3 samples, indicating that the cells were leukemic in origin. IL-12 levels were significantly higher in AML-DCs and CML-DCs prestimulated with phorbol 12-myristate 13-acetate than in the corresponding leukemic cells, but were lower than that of healthy donors. These cells were potent stimulators of lymphocyte proliferation in specific in vitro assays for DC function. However, the stimulatory abilities of allogeneic T cells in a mixed lymphocyte reaction were impaired compared with those of mature DCs derived from healthy donors, although T-cell stimulatory effects were significantly increased in these differentiated leukemia-DCs. These results suggest that functional DCs may be derived from leukemic (AML, CML) blasts in a significant number of patients and may be capable of inducing leukemia-specific immune responses with potentially clinically beneficial effects.     

Insulin binding of acute lymphocytic leukemia cells.

Because of differences in insulin binding of cultured lymphoic cell lines, T- and B-cell surface receptor and 125I-insulin binding studies were performed on the bone marrow and peripheral blood leukocytes of 13 children with active acute lymphocytec leukemia. Based on surface receptors, nine patients had null-cell disease and four had T-cell disease. The mean per cent insulin binding of the bone marrow cells from the null-cell patients was 10.0% +/- 8.1 and from the T-cell patients was 0.18% +/- 0.13. The mean insulin binding of the cell suspensions of the peripheral blood from the null-cell patients was 7.3% +/- 7.5 and 0.07% +/- 0.06 from the T-cell patients. Displacement studies with nonradioactive insulin indicated that null leukemic cells bore specific binding sites. These results indicated that there may be metabolic as well as surface membrane heterogeneity among the acute lymphocytic leukemias of childhood.     

Acute Myelogenous Leukemia With PIG-A Gene Mutation Evolved from Aplastic Anemia—Paroxysmal Nocturnal Hemoglobinuria Syndrome

We report a patient with aplastic anemia (AA)-paroxysmal nocturnal hemoglobinuria (PNH) syndrome who developed acute myelogenous leukemia (AML). Flow cytometric analysis showed that the leukemic cells in the bone marrow lacked CD59 antigen on their surface and were positive for P-glycoprotein. Heteroduplex and single-strand conformation polymorphism analysis followed by sequencing of the leukemic cells in the bone marrow disclosed 1 frameshift-type mutation in exon 2 of the phosphatidylinositol glycan-class A (PIG-A) gene, which deductively produces truncated PIG-A protein. These findings provide direct evidence that the leukemic cells evolved from the affected PNH clone. Cytogenetic analysis in the bone marrow in each stage of AA-PNH, AML, and at relapse of AML showed normal, -7, and -7 plus -20, respectively, showing evidence of a clonal evolution. Because complete remission of AML was not achieved by intensive chemotherapies, allogeneic peripheral blood stem cell transplantation (PBSCT) from the patient's HLA-matched sister was performed successfully with recovery of CD59 antigen on bone marrow hematopoietic cells; however, leukemia relapsed 4 months after PBSCT. Leukemia derived from PNH may be resistant to intensive chemotherapy, and a highly myeloablative regimen may be required for stem cell transplantation to eradicate the PNH-derived leukemia clone.     

Trisomy of chromosome 11 in a case of common ALL antigen-positive acute myeloblastic leukemia (FAB-M1)

In February 1986, a 68-year-old woman was diagnosed as having acute myeloblastic leukemia (FAB-M1). At the time of diagnosis, 86.0% of the bone marrow cells were myeloblastoid, and 15% of these myeloblastoid cells were positive to myeloperoxidase. Surface marker analysis by flow cytometry disclosed granulocyte-associated antigen (MY7) and also lymphocyte-associated antigen (CALLA) on the leukemic cells. Chromosomal banding studies of bone marrow cells revealed trisomy 11 in 6 of 19 metaphases examined and normal karyotype in the others. Complete remission was attained after intensive combination chemotherapy, and has remained for 38 months. Only 19 patients with trisomy 11-associated acute nonlymphocytic leukemia (ANLL) including the present case have been reported. Morphologic analyses have revealed that the frequency of FAB-M1 is high. However, except for the present case, surface marker findings were apparent in only one M5a patient, in whom monocyte-macrophage-associated antigen was detected. Accordingly, careful surface marker studies will be needed to clarify the frequency of acute mixed lineage leukemia in such patients.     

Case series of pediatric acute leukemia without a peripheral blood abnormality, detected by magnetic resonance imaging

Although abnormal peripheral blood counts are a key diagnostic finding for acute leukemia in children, between 2003 and 2010 we observed seven pediatric cases without peripheral blood abnormalities and showing abnormal signals in the bone marrow by magnetic resonance imaging (MRI). The common chief complaint in these patients was bone pain and fever. Bone marrow tests revealed six out of the seven cases to be acute leukemia, whereas one patient was diagnosed with juvenile idiopathic arthritis (JIA). There was no evident difference in MRI findings between leukemia patients and JIA patient. In three cases of leukemia, initial bone marrow aspiration failed to show the presence of leukemic cells, and diagnosis was only made by repeated bone marrow examination. Our findings indicate that in some cases MRI detects leukemia at an earlier phase than does bone marrow aspiration, suggesting that MRI is useful for the diagnosis of acute leukemia.     

Expression of two natural killer cell antigens, H-25 and H-366, by human immature myeloid cells and by erythroid and granulocytic/monocytic colony-forming units

Two monoclonal antibodies (MoAbs), H-25 and H-366, shown previously to react with human peripheral blood large granular lymphocytes with natural killer (NK) cell activity and some peripheral blood monocytes, have now been shown to also react with a significant proportion of the myeloid and erythroid precursor cells in human bone marrow and peripheral blood. In FACS IV cell sorting and immune rosetting of bone marrow cells, the antigens recognized by H-25 and H-366 were found to be expressed on most blasts and promyelocytes but sequentially fewer of the more mature cells of the myeloid lineage. Both antigens were also found on most monocytes but only a minor proportion of lymphoid and nucleated red cells in the bone marrow. In vitro assays detecting hematopoietic colony-forming units revealed that these antigens are expressed by virtually all mature erythroid colony-forming units (day-7 CFU-E), and the majority of the more primitive erythroid burst forming units (day-14 BFU-E). H-25 but not H-366 was also found on a variable proportion of the day-7 and day-14 granulocytic/monocytic colony- forming units (CFU-GM) in the bone marrow. The same type of precursor cells are also found in the H-25 and H-366 positive cell populations isolated from peripheral blood. In preliminary testing of cells from acute leukemic patients, FACS analysis showed that both antigens are also expressed on leukemic cells from patients with T cell acute lymphocytic leukemia and with myeloid leukemias. These studies demonstrate that the H-25 and H-366 positive NK cells in the peripheral blood retain some of the cell surface properties of early hematopoietic precursor cells, thus providing further evidence supporting the bone marrow origin of NK cells.     

Transplantation of mobilized peripheral blood cells to HLA-identical siblings with standard-risk leukemia

Allogeneic mobilized peripheral blood progenitor cells instead of bone marrow are increasingly used to restore hematopoiesis after myeloablative therapy. Data supporting this important change of clinical practice are scarce. We therefore assigned patients with early leukemias to peripheral blood or bone marrow transplantation; the occurrence of acute and chronic graft versus host disease, survival, transplantation-related mortality, and relapse rates were compared. A total of 350 patients between 18 and 55 years of age with acute leukemias in remission or chronic myelogenous leukemia in first chronic phase were randomized to receive either filgrastim-mobilized peripheral blood progenitor cells or bone marrow cells from HLA-identical sibling donors after standard high-dose chemoradiotherapy. Neutrophil and platelet recovery occurred significantly faster after transplantation of peripheral blood progenitor cells than after bone marrow transplantation. Acute graft versus host disease of grades II-IV was significantly more frequent in recipients of peripheral blood progenitor cells than in recipients of marrow cells (52% vs 39%, odds ratio 1.74, 95% confidence interval 1.12-2.69, P =.013). The cumulative incidence of chronic graft versus host disease was 67% with peripheral blood progenitor cells and 54% with bone marrow cells (hazard ratio 1.67, 95% confidence interval 1.15-2.42, P =.0066). The estimated overall probability of survival at 2 years was 65% with either source of stem cells (hazard ratio 1.15, 95% confidence interval 0.79-1.67, P =.46). Disease-free survival, transplantation-related mortality at day 100, and relapse rates did not significantly differ between treatment arms. Peripheral blood is an equivalent source of hematopoietic stem cells compared with bone marrow if administered to patients with standard-risk leukemias. Long-term observation of patients with different diseases and stages of disease is necessary to ultimately define the role of both sources of stem cells.     

Regulation of low density lipoprotein receptors by plasma lipoproteins from patients with abetalipoproteinemia.

Despite an absence of low density lipoproteins (LDLs) and chylomicron remnants from plasma, the rates of cholesterol synthesis or the number of LDL receptors expressed on freshly isolated cells from patients with abetalipoproteinemia are not markedly increased. These observations suggest that other lipoprotein particles present in the plasma of patients with abetalipoproteinemia may regulate LDL receptor activity and the rates of cellular cholesterol synthesis in this disorder. In the present report we have studied the effects of lipoprotein fractions from the plasma of normal subjects, patients with abetalipoproteinemia, and a patient with dysbetalipoproteinemia on the binding, internalization, and degradation of 125I-labeled LDL (125I-LDL) by cultured human fibroblasts. LDL from normal subjects or the high density lipoprotein fraction HDL2 from the plasma of patients with abetalipoproteinemia effectively down-regulated LDL receptor activity (greater than 50% inhibition at 20 micrograms of protein per ml). HDL2 from the plasma of patients with abetalipoproteinemia also effectively reduced the binding, internalization, and degradation of 125I-LDL by cultured human fibroblasts. 125I-HDL2 from the plasma of patients with abetalipoproteinemia was bound, internalized, and degraded by cultured human fibroblasts; this process was competitively inhibited by unlabeled normal LDL or HDL2 from abetalipoproteinemic plasma and was 1/6th to 1/8th times as high when 125I-HDL2 was incubated with fibroblasts from a patient with receptor-negative homozygous familial hypercholesterolemia. We conclude that lipoproteins present in the HDL2 fraction of plasma from patients with abetalipoproteinemia (which are relatively rich in apoprotein E) are effective regulators of LDL receptor activity in normal human fibroblasts. These in vitro findings may explain why the in vivo rates of cholesterol synthesis and the number of LDL receptors expressed on freshly isolated cells from patients with abetalipoproteinemia are not markedly increased.     

Production of colony-stimulating factor by leukemic leukocytes.

The production of colony-stimulating factor (CSF) by the peripheral blood cells of untreated patients with acute myeloid leukemia (AML) was measured in the agar culture system using normal human bone marrow as the source of colony-forming units (CFUc). CSF production was found to be variable and was related to the morphologic subtype of AML--cells from patients with monocytic leukemia produced normal or large quantities of CSF, while (with one exception) those from patients with myeloblastic leukemia produced little or no CSF. There was a general relationship between CSF production and serum lysozyme levels. Attempts to demonstrate a consistent inhibitory effect exerted by leukemic peripheral blood cells on normal leukopoiesis in vitro were negative. Results instead suggested that the addition to the feeder layer of cells from patients with monocytic leukemia could raise CSF levels above those obtained with normal peripheral blood leukocytes alone, possibly by recruiting additional CFUc from normal marrow.     

Monoclonal antibody against a unique antigen on human acute promyelocytic leukemia cell line (HL-60)

An IgM kappa monoclonal antibody (WI-1) reacted against HL-60 cells in indirect immunofluorescence and microcytotoxicity tests. It failed to react against 19 other cell lines representing acute myelogenous leukemia, chronic myelogenous leukemia, lymphocytic leukemias, multiple myeloma, Burkitt's lymphoma, monocytoid cells and virus induced lymphoid cell lines. Normal peripheral blood nucleated cells and bone marrow cells derived from acute granulocytic leukemia, chronic granulocytic leukemia, acute lymphocytic leukemia, and chronic lymphocytic leukemia also failed to react with WI-1. Normal peripheral blood lymphocytes, transferred with mitogens, showed no reaction against the antibody. There was some decrease in the reactivity of the cells, against WI-1, following maturation with dimethyl sulfoxide. However, a large percentage of the cells remained positive after maturation. WI-1 reacted specifically against an antigen on the HL-60 cells which had a molecular weight of about 42,000 dalton. Peripheral blood cells from one patient with acute promyelocytic leukemia failed to react with the antibody. It is possible that acute promyelocytic leukemia is an antigenically heterogeneous disease. A large population of acute promyelocytic leukemia patients needs to be tested to see if the specific antigen on HL-60 cells, detected by WI-1, is demonstrable in other patients with acute promyelocytic leukemia.     

A monoclonal antibody to myelogenous leukemia: isolation and characterization

A purified antigen from human acute myelogenous leukemia (AML) cells has been used to produce a myelogenous leukemia-associated monoclonal antibody. In limited FACS-IV analyses the monoclonal antibody to leukemia (CAMAL-1) as well as a conventional rabbit antiserum have been used to positively identify AML or chronic granulocytic leukemia patient cell samples. Neither CAMAL-1 nor the rabbit antiserum bound appreciably to acute lymphocytic leukemia cells, normal bone marrow, or normal peripheral blood leukocytes. CAMAL-1 was shown to be specific for AML cell extracts in the ELISA and was successfully used as an immunoadsorbent for the purification of the AML antigen from cell extracts. No significant levels of equivalent antigen were found when cell extracts from normal cells, lymphocytic leukemia cells, and lymphoma cells were similarly absorbed. These findings indicate that CAMAL-1 shows considerable specificity for an antigen associated with cells from patients with myelogenous leukemia.