Minimal residual disease in leukemia: studies in an animal model for acute myelocytic leukemia (BNML)

The possibilities for studying minimal residual disease (MRD) in human acute myelocytic leukemia (AML) are limited. Animal models are, therefore, indispensable for gaining insight into the characteristics of leukemia growth during the MRD phase. Studies were done to compare AML to acute myelocytic leukemia in the Brown Norway rat (BNML). The BNML model exhibited a high degree of similarity to human AML with regard to its general growth characteristics, its cell kinetic parameters, its biophysical parameters and its response to chemotherapy. This implied that studies of the BNML model have predictive value for clinical application. In the BNML model a number of independent methods are available to quantify the number of leukemic cells, i.e., indirectly by means of various bioassays or directly by using monoclonal antibody labeling and flow cytometry. Studies of the BNML model in relation to the understanding of various aspects of MRD in leukemia are discussed in this concise review. Insight has been obtained with regard to the kinetics of MRD; the efficacy of certain treatment modalities, e.g., cytostatic drug treatment with or without total body irradiation to eradicate MRD; the efficacy of various methods for eliminating residual leukemic cells from autologous marrow grafts; the emergence of drug resistance during MRD; and the progression of residual disease during the remission phase ultimately leading to a relapse and the implications of these observations for staging leukemia patients during the phase of MRD.     

Bivariate flow karyotyping of acute myelocytic leukemia in the BNML rat model

Univariate as well as bivariate flow karyotyping has been performed on chromosome suspensions obtained from the Brown Norway myelocytic leukemia (BNML), a rat model for human acute myelocytic leukemia (AML). Flow karyograms were obtained from both the in vivo transplantable parent line and from an in vitro established cell line. Density gradient centrifugation performed on cells arrested in mitosis resulted in an enrichment of mitotic cells. Furthermore, with this procedure leukemic and nonleukemic cells could be separated. Univariate analysis with propididum iodide (PI) as a DNA stain revealed the position of the several tumor-specific marker chromosomes in the in vitro cell line. Estimations of the peak position of the various chromosomes was done by comparing the univariate flow karyogram with a computer-simulated karyogram from the BNML that was derived from the mean length of the individual chromosomes in conventionally prepared metaphase slides. By comparing the bivariate flow karyogram of the in vivo BNML cells with the flow karyogram of normal BN cells, it was clearly demonstrated which peaks are involved in the altered chromosomal pattern of the BNML. No differences were found between the flow karyograms of the in vitro- and the ex vivo-derived chromosome suspensions in this rat leukemia model.     

A unique antigen expressed on myeloid cells and acute leukemia blast cells defined by a monoclonal antibody

A murine hybridoma-derived monoclonal antibody, PM-81, was obtained from a fusion of cells of the NS-1 myeloma cell line with cells from a mouse immunized with the HL-60 promyelocytic leukemia cell line. This cytotoxic IgM monoclonal antibody was specific for myeloid cells. Employing indirect immunofluorescence and flow cytometry, we determined that this antibody reacts strongly with normal human granulocytes, eosinophils, and monocytes but not lymphocytes (including phytohemagglutinin-activated lymphocytes), null cells, red blood cells, or platelets. Moreover, the PM-81 antibody reacts with leukemia cells from 19 of 22 patients with acute myelocytic leukemia of all FAB subclasses, three of three patients with common acute lymphocytic leukemia, four of four patients with chronic myelocytic leukemia (CML) in myeloid blast crisis (terminal transferase (TdT)-negative) but did not react with cells from two patients with CML in lymphoid blast crisis (TdT-positive) or five patients with chronic lymphocytic leukemia. The myeloid cell lines HL-60, K562, KG-1, and U937 were all reactive with PM-81. The lymphoid lines CCRF-CEM and Daudi did not express PM-81 but HSB-2 was positive. The PM-81 antigen was absent on myeloid and erythroid progenitor cells as determined by their insusceptibility to complement-dependent lysis. In addition, only PM-81-unreactive cells were capable of colony formation. Furthermore, the PM-81 antibody does not appear to induce modulation of the antigen to which it binds. Thus, this monoclonal antibody appears to fulfill several criteria for clinical utility in the diagnosis and treatment of both acute myelocytic and acute lymphocytic leukemia.     

Plasma and intracellular levels of lactate dehydrogenase, phosphohexose isomerase and lysozyme activity in acute leukemia

Plasma and intracellular levels of lactate dehydrogenase (LDH), phosphohexose isomerase (PHI) and lysozyme activities were investigated in 20 patients with acute myelocytic leukemia (AML), 18 patients with acute lymphatic leukemia (ALL) and 10 patients with chronic myelocytic leukemia in blast transformation (CML/BT). Though the plasma levels of LDH and PHI in all patients with acute leukemia were elevated as compared to control persons there was no distinctive pattern which could be of use in the classification of acute leukemia. On the other hand the intracellular levels of these enzymes could be of value in classifying acute leukemia. The leukemic lymphoblasts were characterized by low levels of PHI and lysozyme as compared to leukemic myeloblasts or to normal lymphocytes (p less than 0.01). The LDH/PHI ratio is also significantly higher in leukemic lymphoblasts than in leukemic myeloblasts or in normal lymphocytes (p always less than 0.01). These characteristics might also be made use of in identifying the blasts of CML/BT als "lymphoid" or "myeloid" in corresponding cases.     

Double labeling and in vitro versus in vivo incorporation of bromodeoxyuridine in patients with acute nonlymphocytic leukemia

A monoclonal antibody against bromodeoxyuridine (BrdUrd) was produced, and a rapid slide technique (RPMB technique) was developed for the estimation of S-phase cells in a population using this antibody. Bone marrow cells from patients with acute nonlymphocytic leukemia (ANLL) were studied by both the RPMB technique and tritiated thymidine (3HdThd) labeling index studies. The percentage of S-phase cells obtained by each method was compared in 50 samples, and the correlation coefficient was r = 0.89. A "double label" method is also described in which cells were simultaneously incubated with either BrdUrd and 3HdThd or BrdUrd and tritiated cytosine arabinoside (3HAra-C). The samples were first processed by the RPMB technique and then by autoradiography. Results showed only black grains overlying the nuclei of fluorescent cells in each group. An automated microphotometer was used to quantitate grains and fluorescence from each cell. This demonstrated an almost direct relationship between grains and fluorescence from BrdUrd + 3HdThd slides, whereas different patterns of relationship were noted from BrdU + 3HAra-C slides of leukemic patients. Their implications are discussed in the text. Finally, intravenous infusions of BrdUrd was given to five leukemic patients. S-phase cells were recognized distinctly within 5 min of starting the infusion. The percentage of S-phase cells was almost identical from in vivo and in vitro samples. Various possibilities of studying the biological behavior of acute leukemias and analyzing cell cycle characteristics are discussed.     

Blast cell and granulocyte production in human leukemia: pathophysiological concepts based on computer simulation using discrete modeling techniques

The granulocyte production of two patients suffering from leukemia was studied extensively by means of the tritiated thymidine method of cellular kinetics. The data obtained (1-h labeling index, pattern of cell labeling, labeling intensity, as well as other conventional parameters of bone marrow and blood) were used to develop a computer model (GPSS-language) to fit the observations. From these models, it was concluded that patients with leukemia may have an abnormal granulopoiesis, characterized by a high degree of inefficiency (premature cell death, skipping of divisions with undisturbed maturation). However, the underlying mechanisms may be quite different. While it cannot be excluded that in acute myelocytic leukemia there is a stem and/or progenitor cell pool that is highly ineffective but still capable of feeding some cells into the granulocytic pathway, it is nevertheless possible, as shown in plasma cell leukemia, that the ineffective granulopoiesis may be the result of direct or indirect interaction between the "leukemic" and the "normal" cell clone.     

TIM-3 is a promising target to selectively kill acute myeloid leukemia stem cells

Acute myeloid leukemia (AML) originates from self-renewing leukemic stem cells (LSCs), an ultimate therapeutic target for AML. Here we identified T cell immunoglobulin mucin-3 (TIM-3) as a surface molecule expressed on LSCs in most types of AML except for acute promyelocytic leukemia, but not on normal hematopoietic stem cells (HSCs). TIM-3(+) but not TIM-3? AML cells reconstituted human AML in immunodeficient mice, suggesting that the TIM-3(+) population contains most, if not all, of functional LSCs. We established an anti-human TIM-3 mouse IgG2a antibody having complement-dependent and antibody-dependent cellular cytotoxic activities. This antibody did not harm reconstitution of normal human HSCs, but blocked engraftment of AML after xenotransplantation. Furthermore, when it is administered into mice grafted with human AML, this treatment dramatically diminished their leukemic burden and eliminated LSCs capable of reconstituting human AML in secondary recipients. These data suggest that TIM-3 is one of the promising targets to eradicate AML LSCs.     

Targeting of a developmentally regulated epitope of CD43 for the treatment of acute leukemia

Previously, we developed a JL1 mouse monoclonal antibody that specifically recognizes the leukemic cells of T, B, and myeloid lineages, but not the peripheral blood cells and pluripotent hematopoietic stem cells. Here, we identified that JL1 mAb recognized a specific epitope of human CD43 and validated its potential as an anti-leukemic targeting agent. After the comprehensive screening of JL1 Ag in the human thymocyte cDNA library, multiple fusion gene constructs encoding human CD43 were generated to identify its specific epitope to JL1 antibody. JL1 antibody interacted with a developmentally regulated and non-glycosylated epitope of the human CD43 extracellular domain (AA 73-81, EGSPLWTSI). In an in vivo leukemia model using NOD/SCID mice injected with CCRF-CEM7 cells, JL1 antibody induced effective cytotoxicity in tumor cells and prolonged survival (p < 0.05). Saporin conjugation to JL1 antibody effectively depleted tumor cells in in vitro cytotoxic assays and also prolonged survival in a leukemic mouse model (p < 0.001). These preclinical results further support the therapeutic potential of the JL1 antibody in the management of acute leukemia.     

Effect of thymic humoral factor in vitro on human bone marrow and blood cells from B-, T- and null-cell acute lymphatic leukemia.

The nature of null-cell acute lymphatic leukemia (ALL) was investigated with the aid of a thymic humoral factor (THF), bone marrow cells, and a local xenogeneic graft-versus-host reaction (GVHR). Lymphocytes obtained from the blood and bone marrow of six children with T-cell ALL, five with null-cell ALL, one with perinatal B-cell ALL, one with acute myelocytic leukemia, and one with erythroleukemia were tested for membrane surface markers (E, EAC, and SM Ig); functional activity of T cells was tested by a local GVHR. All of the specimens obtained at the initial presentation showed a lack of functional activity of the lymphocytes. Incubation of null cell and acute myelocytic leukemia (AML) bone marrow with THF led to the acquisition of the characteristics of functional, immunocompetent T cells. No such effect was seen when the bone marrow of T-cell ALL and peripheral blood lymphocytes of B-cell perinatal ALL were incubated with THF. This study demonstrates that the null cell in ALL bone marrow can be differentiated into a T cell whereas the stem cell in AML bone marrow constitutes a pluripotential undifferentiated cell which also can mature into a T cell.     

Characterization of an acute T lymphoblastic leukemia expressing the gamma/delta T-cell receptor

Leukemic cells of a 20 year old patient, suffering from acute lymphoblastic leukemia, were characterized by surface marker and functional analysis. A significant cell population within this type of leukemia expresses concomitantly the CD4 and CD8 antigen on the same cell and might represent a new differentiation stage of T-cells with the gamma/delta receptor. The leukemic cells show a distinct pattern of growth response to mitogens and lymphokines, which might correlate to their differentiation stage. Moreover, a "natural killer"-like activity can be induced in these cells by IL-2.     

Increased cellular hypoxia and reduced proliferation of both normal and leukaemic cells during progression of acute myeloid leukaemia in rats

The microenvironmental changes in the bone marrow, spleen and liver during progression of the transplantable promyelocytic leukaemia in the Brown Norwegian rat (BNML) have been studied. We used flow cytometry to estimate cellular hypoxia and proliferation based on in vivo pulse-labelling with a mixture of 2-nitroimidazole linked to theophylline (NITP) and bromodeoxyuridine (BrdUrd). The leukaemic cells were identified with the RM124 antibody. In rats inoculated with leukaemic cells the fraction of RM124+ cells was significantly increased from day 20 onwards in the spleen and from day 27 in the bone marrow and liver, reaching a level of 65-87% in these organs at day 32. At day 32, the NITP+ fraction of RM124+ cells had increased significantly in the bone marrow and spleen to 88% and 90%, respectively. The corresponding fractions of NITP+ normal cells reached 63% and 65%, respectively. From day 13 to day 32, the DNA-synthesizing (BrdUrd+) fraction of RM124+ cells in the bone marrow decreased significantly from 52% to 25%, and of normal cells from about 20% to 6%. In the bone marrow and spleen at day 27 and 32, the S-phase and G2/M-phase fractions according to DNA content were higher for the NITP+ than for the NITP- cells. This could partly be explained by an impaired cell cycle progression due to hypoxia. Nevertheless, we found indications of leukaemic cells that were simultaneously labelled with NITP and BrdUrd, in the bone marrow and spleen. These latter findings suggest that in contrast to normal cells some of the leukaemic cells can proliferate even during hypoxia, and this subpopulation may consequently renew and expand the leukaemic cell load.     

Deficient expression of class-I HLA in some cases of acute leukemia

Summary Leukemic cells from the blood and marrow of 25 cases of newly diagnosed acute leukemia were presented as target cells to alloreactive effector cells from unrelated normal donors in cell-mediated cytotoxicity assays. In three cases the leukemic targets were poorly killed relative to nonleukemic, HLA-identical target cells. The poor killing of the leukemic cells from one of these cases was shown by competitive inhibition to be due to deficient expression of normal class-I HLA antigens rather than resistance to lysis. Furthermore, the leukemic cells from these three patients were also deficient in binding monoclonal antibodies to nonpolymorphic determinants of class-I HLA and B2 microglobulin. Two additional cases were identified as having a less extensive deficit of HLA, and may be representative of a group with relatively subtle changes in these cell surface antigens. The possible significance of reduced expression of HLA in leukemic progression and in susceptibility to graft-vs-leukemia reactions after bone marrow transplantation is discussed.     

A comparison of the rate of DNA synthesis in myeloblasts from peripheral blood and bone marrows of patients with acute nonlymphocytic leukemia

Durations of S-phase (Ts) and total cell cycle times (Tc) were measured from the peripheral blood (PB) and bone marrow aspirates (BM) of five patients with acute nonlymphocytic leukemia (ANLL). Intravenous bromodeoxyuridine (BrdU) was used as the first label for S-phase cells and a monoclonal anti-BrdU antibody was used to detect the positive cells. Tritiated thymidine [( 3H]Tdr) was used as a second label in vitro, and the Ts was calculated by counting the number of cells labeled either by BrdU or by [3H]Tdr or by both. Our data demonstrate that the duration of S-phase in myeloblasts obtained from BM is quite similar to that of circulating leukemic cells. Finally, the most accurate assessment of percentage of myeloblasts actively engaged in DNA synthesis can be obtained only from bone marrow biopsies following in vivo labeling.     

A new human T-cell differentiation antigen: unexpected expression on chronic lymphocytic leukemia cells

Monoclonal antibody 10.2 reacts with a monomorphic antigen expressed on the surface of virtually all thymocytes, as well as thymus-dependent lymphocytes in the peripheral blood and bone marrow. In contrast, antibody 10.2 did not react with normal peripheral blood B cells, monocytes, or the non-T-cell fraction of bone marrow. This complement fixing IgG2a antibody also reacted with established leukemic T-cell lines, but not with cell lines of either normal or malignant B-cell origin. Similarly, when tested against acute leukemia blasts, the 10.2 antibody reacted with those from patients with T-cell acute leukemia, but not with those from patients with acute null cell or non-lymphocytic leukemia. An unexpected exception to this pattern was the reaction of 10.2 antibody with leukemic cells from patients with B-cell type chronic lymphocytic leukemia. Immune precipitates formed with 10.2 antibody and detergent lysates of radiolabeled T-cells contained three polypeptides with molecular weights of 65 000, 55 000, and 50 000 daltons. It has not been determined whether all three of these polypeptides contain the 10.2 antigenic determinant, or whether these proteins represent a multimeric antigen complex.     

A new human T-cell differentiation antigen: Unexpected expression on chronic lymphocytic leukemia cells

Monoclonal antibody 10.2 reacts with a monomorphic antigen expressed on the surface of virtually all thymocytes, as well as thymus-dependent lymphocytes in the peripheral blood and bone marrow. In contrast, antibody 10.2 did not react with normal peripheral blood B cells, monocytes, or the non-T-cell fraction of bone marrow. This complement fixing IgG2a antibody also reacted with extablished leukemic T-cell lines, but not with cell lines of either normal or malignant B-cell origin. Similarly, when tested against acute leukemia blasts, the 10.2 antibody reacted with those from patients with T-cell acute leukemia, but not with those from patients with acute null cell or non-lymphocytic leukemia. An unexpected exception to this pattern was the reaction of 10.2 antibody with leukemic cells from patients with B-cell type chronic lymphocytic leukemia. Immune precipitates formed with 10.2 antibody and detergent lysates of radiolabeled T-cells contained three polypeptides with molecular weights of 65 000, 55 000, and 50000 daltons. It has not been determined whether all three of these polypeptides contain the 10.2 antigenic determinant, or whether these proteins represent a multimeric antigen complex.PJM is a Junior Faculty Clinical Fellow of the American Cancer Society.     

Growth of normal versus leukemic bone marrow cells in long term culture from acute lymphoblastic and myeloblastic leukemias

The ability of the in vitro long-term bone marrow culture (LTBMC) system to impair the survival of leukemic cells and to enhance the growth of normal progenitors has been studied. Bone marrow cells from 19 acute lymphoblastic leukemia (ALL) and 30 acute myeloid leukemia (AML) patients at diagnosis were grown in LTBMC for 4-10 weeks. In half of the cases the leukemic population declined down to undetectable levels and was replaced by putative normal hemopoietic precursors, both in ALL and in AML. In the remaining cases, leukemic cells persisted throughout the culture time and few if any normal hemopoietic cells were detected. These data led us to extend to the lymphoid compartment the previous observation of decreasing leukemic myeloid blasts in LTBMC. The potential of such cultures as an in vitro purging system for autologous bone marrow transplantation in selected poor-prognosis lymphoid malignancies should be explored, as has been done for acute and chronic myeloid leukemias.     

Immunotoxin studies in a model of human T-cell acute lymphoblastic leukemia developed in severe combined immune-deficient mice

The transplantation of the human T-cell acute lymphoblastic leukemia (T-ALL) cell line HSB-2 into severe combined immune-deficient (SCID) mice was found to produce a disseminated pattern of leukemia similar to that seen in humans. The iv injection of 10⁷ HSB-2 cells was associated with a universally fatal leukemia. Histopathological examination of animals revealed the spread of leukemia initially from bone marrow to involve all major organs including the meninges. An immunotoxin (HB2-Sap) was constructed by conjugating the anti-CD7 monoclonal antibody (MAb) HB2 to the ribosome inactivating protein (RIP) saporin. An in vitro protein synthesis inhibition assay revealed specific delivery of HB2-Sap immunotoxin (IT) to CD7⁺ HSB-2 target cells with an IC₅₀ of 4.5 pM. In an in vivo study, the IT was shown to significantly prolong the survival of SCID mice injected with HSB-2 cells compared to untreated control animals. This therapeutic effect was seen both with a single injection of 10 μg of IT given 7 d after the injection of HSB-2 cells, and was even more effective when IT was administered as three daily injections of 10 μg on d 7, 8, and 9. These results demonstrate the useful application of human leukemia xenografts in SCID mice and the potential therapeutic effect of an anti-CD7 IT in human T-ALL.     

High frequency of CD34+CD38-/low immature leukemia cells is correlated with unfavorable prognosis in acute myeloid leukemia

AIM To evaluate the importance of the CD34+CD38-cell population when compared to the CD34+CD38+/low and CD34+CD38+/high leukemic cell sub-populations and to determine its correlations with leukemia characteristics and known prognostic factors, as well as with response to therapy and survival.METHODS Two hundred bone marrow samples were obtained at diagnosis from 200 consecutive patients with newly diagnosed acute myeloid leukemia(AML) were studied between September 2008 and December 2010 at our Institution(Hematology Department, Lyon, France). The CD34/CD38 cell profile was analyzed by multiparameter flowcytometry approach using 8 C panels and FACS CANTO and Diva software(BD Bioscience).RESULTS We analyzed CD34 and CD38 expression in bone marrow samples of 200 AML patients at diagnosis, and investigated the prognostic value of the most immature CD34+CD38-population. Using a cut-off value of 1% of CD34+CD38-from total "bulk leukemic cells" we found that a high( 1%) level of CD34+CD38-blasts at diagnosis was correlated with advanced age, adverse cytogenetics as well as with a lower rate of complete response after induction and shorter disease-free survival. In a multivariate analysis considering age, leukocytosis, the % of CD34+ blasts cells and the standardized cytogenetic and molecular risk subgroups, a percentage of CD34+CD38-leukemic cells 1% was an independent predictor of DFS [HR = 2.8(1.02-7.73), P = 0.04] and OS [HR = 2.65(1.09-6.43), P = 0.03].CONCLUSION Taken together, these results show that a CD34/CD38 "backbone" for leukemic cell analysis by multicolour flowcytometry at diagnosis provides useful prognostic information.     

Identification of a human endothelial cell antigen with monoclonal antibody 44G4 produced against a pre-B leukemic cell line

Staining of a variety of human tissue sections (lymph node, tonsil, spleen, thymus, kidney, lung, and liver) by the indirect immunoperoxidase method indicated that mAb 44G4, produced against a human pre-B leukemic cell line, was strongly reactive with vascular endothelium. All other cell types observed in these tissues were unreactive. Immunofluorescence staining of endothelial cells isolated from umbilical cord vein and grown in culture confirmed that mAb 44G4 recognized a surface membrane component of vascular endothelium. Granulocytes, monocytes, B and T lymphocytes, and T lymphocytes cultured in the presence of PHA for 72 h did not express the 44G4 Ag. mAb 44G4 reacted weakly with leukemic cells from 28 of 41 patients with non-T cell acute lymphocytic leukemia and 4 of 7 patients with acute myelocytic leukemia, whereas 8 of 10 cases of T cell acute lymphocytic leukemia were negative. Moderate reactivity with leukemic cell lines of pre-B and myelomonocytic origin was also observed. The level of 44G4 Ag on umbilical endothelial cells was three to five times that of leukemic cell lines and 25 times the average levels observed on leukemic cells isolated from patients. Immunoprecipitation of lysates prepared from surface-iodinated endothelial cells and the immunizing pre-B leukemic cell line revealed that the 44G4 Ag from both cell types was composed of two subunits of apparent m.w. 95,000 linked by disulfide bond(s). Comparison of the cellular localization and subunit structure of 44G4 to that of known Ag suggests that it represents a previously undescribed marker of endothelial cells.