Comparison of in vitro inhibition of etoposide (VP16) on leukemic and normal myeloid, erythroid clonogenic cells

We have compared in various clonogenic assays the in vitro sensitivity to etoposide (VP16) of 1) human leukemic precursors (leukemia colony-forming units; L-CFU), 2) normal erythroid progenitors (erythroid burst-forming units; BFU-E, and 3) normal committed myeloid progenitors (granulocyte-macrophage colony-forming units; CFU-GM and more primitive hemopoietic precursors (PPC) that adhere to cultured marrow stromal cells. Bone marrow samples were obtained from 15 normal subjects and 16 leukemic patients: 9 in the acute phase of acute nonlymphoblastic leukemia (ANLL) and 7 in complete remission. VP16 was tested at concentrations ranging from 10(-8) to 10(-3) M. The median recoveries at 10(-3) M VP16 were respectively 0%, 0.5%, 0%, and 0% for leukemic progenitors, CFU-GM from leukemic patients in complete remission, normal CFU-GM, and BFU-E, and 23% for PPC. This indicates that CFU-GM, BFU-E, and L-CFU are highly sensitive to VP16, whereas PPC, more primitive myeloid precursors, are spared. These results suggest that VP16 may be used as an "ex vivo" purging agent for autologous bone marrow.     

Secretion of tumor necrosis factor-alpha by fresh human acute nonlymphoblastic leukemic cells: role in the disappearance of normal CFU-GM progenitors.

The disappearance of normal hematopoiesis during acute nonlymphoblastic leukemia (ANLL) is poorly understood. Several reports indicate that conditioned medium obtained from leukemic cells might inhibit the formation of normal hematopoietic progenitors. However, these blast-conditioned medium (BCM) inhibitory activities are not well characterized. In order to evaluate whether BCM might contain an activity inhibiting the growth of normal marrow progenitors, BCM from 13 consecutive patients with ANLL were tested on normal bone marrow in methylcellulose assays. In all the cases, a significant inhibition of the growth of granulocyte-macrophage colony-forming unit (CFU-GM) progenitors was observed, whereas erythroid burst-forming unit (BFU-E) progenitors were not affected. Further characterization of the BCM inhibitory activity showed using both a biological assay and RIA, the presence of tumor necrosis factor-alpha (TNF-alpha) in 10 out of 13 BCM. Northern blot analysis performed in six patients showed a correlation between the expression of TNF-alpha mRNA by leukemic cells and the presence of TNF-alpha in BCM. Moreover, the BCM inhibitory activity could be neutralized with an anti-TNF-alpha antiserum. These data indicate that leukemic cells express and release frequently TNF-alpha, which may therefore play an important role in the inhibition of granulopoiesis during leukemia.     

Effect of recombinant interferons on colony formation of blast progenitors in acute myeloblastic leukemia

The effect of recombinant interferons (rIFNs) on primary and secondary colony formation by blast progenitors (leukemic colony-forming units, L-CFU) from the peripheral blood of patients with acute myeloblastic leukemia (AML) was examined. rIFN-alpha, rIFN-beta, and rIFN-gamma inhibited L-CFU in a dose-dependent manner. When 3000 U/ml rIFN-alpha, -beta, and -gamma were added, L-CFU were suppressed to 20%, 12%, and 40% of the control cultures, respectively. The concentrations of rIFN-alpha, -beta, and -gamma required for 50% inhibition of colony formation were 63, 29, and 250 U/ml, respectively. The self-renewal capacity of L-CFU was inhibited by rIFNs in a dose-dependent manner, the degree of inhibition being the same for all three rIFNs. These rIFNs also inhibited normal granulocyte-macrophage progenitors to a similar degree as L-CFU. Taken together, these in vitro studies indicate that these rIFNs may be efficacious in the treatment of AML, though development of granulocytopenia may be observed as a complication of IFN therapy.     

The effect of a prolonged in vitro exposure to 1-beta-D arabinofuranosylcytosine and deoxycytidine on the survival of normal (CFU-GM) and leukemic (L-CFU) human myeloid progenitor cells in suspension culture

We examined the effect of a 96-h exposure to 1-beta-D arabinofuranosylcytosine (Ara-C) and deoxycytidine (dCyd) in medium lacking an exogenous source of colony-stimulating activity (phytohemagglutinin-stimulated leukocyte-conditioned medium, PHA-LCM) on the survival of normal human committed myeloid progenitor cells (day-7 and day-14 granulocyte-macrophage colony-forming units [CFU-GM] as well as leukemic progenitors (leukemic colony-forming units, L-CFU) derived from myeloblasts obtained from 13 patients with acute nonlymphocytic leukemia (ANLL). Coadministration of Ara-C (20-50 microM) in conjunction with dCyd (50-100 microM) permitted the survival of an average of 9%-57% of day-7 CFU-GM and 32%-65% day-14 CFU-GM, depending upon the relative concentrations of dCyd and Ara-C. In contrast, exposure of leukemic myeloblasts to identical regimens resulted in considerably greater reductions in L-CFU survival, which in general exceeded 90% and in some cases was 100%. In addition, exposure of leukemic myeloblasts to Ara-C and dCyd for 96 h in culture medium lacking PHA-LCM eliminated the secondary plating efficiency (PE2) of leukemic colonies in 11 of 13 samples assayed and reduced values dramatically in the remaining 2. Substantial preservation of CFU-GM formation was also noted when normal bone marrow samples depleted of T cells and marrows obtained from two patients with ANLL in remission were assayed. These studies suggest that in contrast to certain normal committed myeloid progenitor cells, leukemic progenitors, particularly those with self-renewal capacity, are highly vulnerable to a prolonged exposure to Ara-C and dCyd in the absence of an exogenous source of colony-stimulating activity. They also raise the possibility that a combined regimen utilizing chemotherapeutic agents in conjunction with modifications of long-term culture techniques may represent a novel approach to the ex vivo purging of leukemic cells from bone marrow in conjunction with autologous bone marrow transplantation.     

In vitro sensitivity of normal and leukemic myeloid clonogenic cells to hyperthermia: absence of selective effect

The in vitro heat sensitivity of myeloid clonogenic cells was tested in 22 normal marrows (granulo-monocytic progenitors) and in 40 marrows of patients with acute myelogenous leukemia (leukemic progenitors). Cells were treated for 1 h at 42 degrees, 43 degrees, 44 degrees, or 45 degrees C prior to plating. A temperature-dependent inhibition of growth was seen without a selective effect on the two kinds of progenitors. Because these two kinds of progenitors have the same heat sensitivity, hyperthermia should not be used alone as a technique for in vitro depletion of residual myeloid leukemic cells.     

The tetrapeptide AcSDKP, an inhibitor of the cell-cycle status for normal human hematopoietic progenitors, has no effect on leukemic cells.

The tetrapeptide acetyl-N-Ser-Asp-Lys-Pro (AcSDKP) inhibits the entry into DNA synthesis of murine spleen colony-forming units (CFU-S) and protects these cells during chemotherapy. This synthetic peptide also inhibits the growth of normal human marrow progenitors granulocyte-macrophage colony-forming units (CFU-GM) and erythroid burst-forming units (BFU-E) and decreases their percentage in DNA synthesis at nanomolar concentration. In view of its clinical application as a marrow protector, we have investigated its effects on malignant cells. Studies were carried out on HL-60 cells and on fresh leukemic cells from patients with either chronic myeloid leukemia (CML) or acute myeloid leukemia (AML). Results showed that AcSDKP, whatever the doses used, did not modify the proliferation of both HL-60 cells and AML cells even when enhanced by stimulating factors such as interleukin 3 or granulocyte-macrophage colony-stimulating factor (GM-CSF). In addition, no change in the number and the percentage in S-phase of both HL-60 clonogenic cells and CML progenitors was observed. Our data clearly demonstrate that the tetrapeptide AcSDKP was ineffective on leukemic cells and therefore by acting selectively on normal progenitors represents a potent therapeutical agent for the protection of normal bone marrow progenitors during chemotherapy.     

Antibody-dependent cellular cytotoxicity of leukemic cells from the patients with acute myeloid leukemia to human erythrocytes coated with anti-D antiserum

We examined the antibody-dependent cellular cytotoxicity (ADCC) of normal leukocytes from healthy volunteers, immature neutrophils from 4 patients with chronic myelocytic leukemia (CML) and leukemic cells from 22 patients with acute myeloid leukemia, employing human erythrocyte targets. Ability to mediate ADCC was demonstrated only in monocytes among normal leukocytes and in leukemic cells from all the 6 patients with acute myelomonocytic or monocytic leukemia (AMMoL, AMoL). CML immature neutrophils and leukemic cells from 14 patients with acute myeloblastic or promyeloblastic leukemia displayed very low levels of ADCC. In the remaining 2 patients with acute myeloblastic leukemia, the ADCC levels were high. We described the usefulness of this ADCC assay for distinguishing AMMoL or AMoL from other types of acute myeloid leukemia.     

Unresponsiveness of primitive chronic myeloid leukemia cells to macrophage inflammatory protein 1 alpha, an inhibitor of primitive normal hematopoietic cells.

Most primitive hematopoietic cells appear to be normally quiescent in vivo, whereas their leukemic counterparts in patients with chronic myeloid leukemia (CML) are maintained in a state of rapid turnover. This difference is also seen in the long-term culture system, where control of primitive hematopoietic progenitor proliferation is mediated by interactions of these cells with marrow-derived mesenchymal cells of the fibroblast lineage. We now show that exogenous addition of macrophage inflammatory protein 1 alpha (MIP-1 alpha) to normal long-term cultures can reversibly and specifically block the activation of "primitive" (high proliferative potential), but not "mature" (lower proliferative potential), progenitors in the adherent layer of these cultures. Moreover, addition of MIP-1 beta after primitive-progenitor activation can prevent the subsequent return of these cells to a quiescent state a few days later as shown previously in similar experiments using antibodies to transforming growth factor beta. This suggests that the level of MIP-1 alpha (or a related MIP-1 alpha agonist) produced in LTCs, like the level of transforming growth factor beta, may be necessary, but is not on its own sufficient, to mediate the inhibitory activity of the regulatory cells in the adherent layer. Addition of MIP-1 alpha to similar long-term cultures containing normal marrow adherent layers but supporting exclusively neoplastic (CML) hematopoiesis did not block the cycling of primitive neoplastic progenitors. A defect in the responsiveness of CML cells to MIP-1 alpha (or a similarly acting chemokine) would explain their deregulated proliferative behavior in this model and, by extrapolation to the in vivo setting, suggests a molecular mechanism whereby the leukemic clone may become amplified at the stem-cell level. In addition, these findings suggest approaches to the therapy of CML, using inhibitors such as MIP-1 alpha for the protection of primitive normal cells.     

CD34-positive cell selection by immunomagnetic beads and chymopapain.

BACKGROUND. Pluripotent hemopoietic stem cells, progenitors of all hemolymphopoietic lineages, and clonogenic cells from many patients with acute nonlymphocytic leukemia (ANLL) and chronic myeloid leukemia (CML) express the CD34 antigen on their surface. Isolation of these cell populations is of primary experimental and clinical importance. METHODS. Six bone marrow (BM) and 10 peripheral blood (PB) samples were obtained from 2 normal individuals, 3 patients with CML and 9 with ANLL. The CD34+ cell fraction was isolated using MY10 antibody, sheep anti-mouse immunomagnetic beads and the enzyme chymopapain. Indirect immunofluorescence and semisolid culture were employed to evaluate the percentage of CD34+ cells and that of clonogenic cells in each cell fraction. RESULTS. The frequency of CD34+ cells in the original unseparated populations was (mean +/- SE) 24.3 +/- 7.3%, and reached 85.0 +/- 2.7% in the isolated CD34-positive fractions; in the negative fractions it was only 2.7 +/- 1.7%. According to these results, the great majority of clonogenic cells was separated in the CD34-positive fractions and depleted in those CD34-negative. Moreover, chymopapain was shown to be non-toxic to the clonogenic cells. CONCLUSIONS. Positive immunoselection using My10 Ab, immunomagnetic beads and chymopapain is a method for isolating almost pure progenitors from the BM and PB of normal individuals and patients with myeloid leukemias.     

Fas ligand is highly expressed in acute leukemia and during the transformation of chronic myeloid leukemia to blast crisis.

Fas ligand (FasL) induces apoptosis in susceptible Fas-bearing cells and is critically involved in regulating T-cell immune responses. It is highly expressed in several human malignancies, and a role in the suppression of antitumor immune responses has been suggested. We evaluated FasL expression in leukemia and normal hematopoietic cells. By Western blotting, all acute leukemic cell lines (n = 9) and primary samples of acute leukemic marrow (n = 4) revealed high levels of FasL. In contrast, much weaker signals were observed in samples of normal marrow (n = 5), and either weak or intermediate expression was seen in chronic myeloid leukemia (CML) in chronic phase (n = 7). Additional leukemic samples were examined by immunohistochemistry. Staining for FasL was negative in 7 of 9 cases of chronic-phase CML, whereas all cases of CML in blast crisis (n = 6), acute lymphoblastic leukemia (n = 6), and acute myeloid leukemia (n = 11) stained strongly in 60 to 100% of nucleated cells. FasL+ leukemic cell lines did not trigger Fas-mediated apoptosis in either Jurkat cells or activated human T lymphocytes, possibly related to the intracellular location of the ligand. Western analysis of normal marrow subpopulations revealed that most FasL in marrow mononuclear cells was expressed by CD7+ lymphocytes. FasL also was strongly expressed in CD34+ hematopoietic progenitor cells from both normal and chronic-phase CML marrow, suggesting a correlation with primitive maturation stage. In summary, high levels of FasL expression were associated with aggressive biologic behavior in leukemia, including transformation of CML to blast crisis. This could potentially represent a response to loss of proapoptotic Fas signaling, which is known to occur in acute leukemic blasts.     

Blocking the APRIL circuit enhances acute myeloid leukemia cell chemosensitivity

Resistance to chemotherapy-induced cell death represents a major obstacle in the treatment of acute myeloid leukemia. APRIL (A Proliferation Inducing Ligand) is a member of the tumor necrosis factor superfamily that plays a key role in normal B-cell development, while promoting survival and proliferation of malignant B cells. We investigated APRIL expression and activity in acute myeloid leukemia. We found that APRIL mRNA and protein, including the secreted form, are expressed in leukemic cells of patients with M0, M2 and M4 acute myeloid leukemia subtypes but not in normal hematopoietic progenitors. Retrovirus-mediated APRIL expression in normal hematopoietic progenitors confers resistance to chemotherapeutic drugs-induced apoptosis. Conversely, blocking APRIL function by recombinant soluble APRIL receptors increased chemotherapeutic drugs-induced cell adeath in acute myeloid leukemia cells. These results indicate that APRIL acts in an autocrine fashion to protect acute myeloid leukemia cells from drug-induced death and foresee a therapeutic potential of APRIL antagonists in the treatment of acute myeloid leukemia.     

Anti‐leukemic activity of valproic acid and imatinib mesylate on human Ph+ ALL and CML cells in vitro

The armamentarium of anti‐leukemic drugs has increased substantially since anti‐leukemic activities were recently found for a variety of non‐classical cytostatic drugs, among them the histone deacetylase (HDAC) inhibitor valproic acid (VPA). This study investigated the effect of VPA on proliferation and apoptosis of human Philadelphia chromosome‐positive (Ph+) acute lymphatic (ALL) and chronic myeloid leukemia (CML) cells and on colony formation of human chronic‐phase CML progenitor cells. Strong anti‐proliferative and pro‐apoptotic effects of VPA were observed on human ALL and CML cell lines at concentrations achievable in vivo. These effects were most pronounced in ALL cell lines as well as in primary ALL cells. Notably, VPA revealed enhanced activity with imatinib mesylate, nilotinib, the farnesyl transferase inhibitor SCH66336, interferon‐alpha and cytosine arabinoside. VPA inhibited the growth of colony‐forming cells from 12 Ph+ chronic‐phase CML patients but also of those from normal healthy controls in a dose‐dependent fashion. HDAC‐inhibiting activity of VPA was confirmed on ALL and CML cells. In conclusion, VPA, whether alone or in combination with other non‐classical anti‐leukemic compounds, exerts significant anti‐leukemic effects on human ALL and CML cells.     

Effect of alpha interferon on growth of leukemic and normal hematopoietic progenitors. Synergism with H2 histamine receptor antagonists

We studied whether the histamine H2 receptor antagonist, cimetidine, potentiates antiproliferative effects of recombinant alpha interferon (IFN alpha) on in vitro clonal growth of certain normal and malignant hematopoietic cells. Cimetidine alone, at therapeutic serum concentrations, was not inhibitory to the cells studied. IFN alpha alone inhibited the growth of HL-60 leukemic cells only at concentrations greater than 1000 U/ml, whereas with cimetidine, inhibition was seen at greater than 1 U/ml of IFN alpha. The enhancing effect of cimetidine on IFN alpha inhibition of clonal growth was neutralized by histamine and was not seen with histamine H1 receptor antagonist. In HL-60 cells, cimetidine also increased the enzymatic activity of (2'-5')-oligoadenylate synthetase, induced by IFN alpha. The combination of cimetidine and IFN alpha had a synergistic inhibitory effect on the growth of leukemic granulocyte-macrophage colony-forming units (CFU-GM) from chronic myeloid leukemia patients, normal CFU-GM, and normal erythroid burst-forming unit (BFU-E) progenitors. These data suggest that cimetidine may play a role in overcoming resistance to IFN alpha therapy in leukemia, but may also increase IFN alpha hematopoietic toxicity.     

Imatinib and plasmacytoid dendritic cell function in patients with chronic myeloid leukemia

Plasmacytoid dendritic cells (PDCs) are crucial effectors in innate immunity. In this study, we show that imatinib, a potent inhibitor of BCR/ABL tyrosine kinase activity, in the presence of Flt3-Ligand, could induce CD34+ progenitors from chronic myeloid leukemia (CML) to give rise in vitro to typical BDCA-2+ type I interferon-producing PDCs. The effect of imatinib on PDC generation was related to up-regulation of Flt3 on leukemic CD34+ progenitors. Moreover, patients with chronic myeloid leukemia (CML) who were in complete cytogenetic or molecular response after imatinib treatment restored their blood PDCs both quantitatively and functionally comparable to healthy donors, in contrast to patients not responding to imatinib, further confirming that disease response to imatinib is accompanied by restoration of PDC function in vivo. These findings provide evidence that response to imatinib is capable to restore some DC-related immune functions in CML that might be beneficial for long-term disease control.     

Inhibition of apoptosis by BCR-ABL in chronic myeloid leukemia

BCR-ABL expression is presumed to effect clonal expansion in chronic myeloid leukemia (CML) by deregulation of cell proliferation. However, most studies have found that relative rates of cell proliferation are not increased in CML. Moreover, we found that CML progenitors display a normal proliferative response to growth factors and do not manifest greater proliferative potential than normal progenitors. Growth of malignancies depends on an imbalance between the rate of cell production and the rate of cell death. We found that BCR-ABL expression inappropriately prolongs the growth factor-independent survival of CML myeloid progenitors and granulocytes by inhibiting apoptosis, a genetically programmed process of active cell death; inhibition of BCR- ABL expression by antisense oligonucleotides reversed the suppression of apoptosis as well as the enhancement of survival. The decreased rate of programmed cell death appears to be the primary mechanism by which BCR-ABL effects expansion of the leukemic clone in CML.     

Heavy chain immunoglobulin gene rearrangement in acute nonlymphocytic leukemia

Samples of leukemic cell DNA from 14 children with acute nonlymphocytic leukemia (ANLL) and 4 human myeloid leukemia cell lines were analyzed for rearrangement in the heavy chain region of the immunoglobulin gene. The diagnosis of ANLL was confirmed in all patients by morphological, cytochemical, and immunologic studies. By restriction endonuclease digestion and hybridization with cloned heavy chain immunoglobulin gene probes for the constant (Cmu) and joining (JH) regions, the DNA of 2 patients and 1 cell line (ML-1) was found to contain rearrangements. The DNA from the remaining 12 patients and 3 cell lines was not rearranged (germline configuration). Both patients with apparent immunoglobulin gene rearrangement achieved complete remission on therapy for ANLL. Immunoglobulin gene rearrangement in phenotypically defined ANLL suggests (1) that such changes may not be limited to lymphoid leukemia of B cell lineage, or (2) that, in some patients, the leukemic transforming event may involve stem cells capable of both B cell and myeloid differentiation.     

Bmi1 reprograms CML B-lymphoid progenitors to become B-ALL-initiating cells

The characterization and targeting of Philadelphia chromosome positive (Ph(+)) acute lymphoblastic leukemia (ALL)-initiating cells remains unresolved. Expression of the polycomb protein Bmi1 is up-regulated in patients with advanced stages of chronic myelogenous leukemia (CML). We report that Bmi1 transforms and reprograms CML B-lymphoid progenitors into stem cell leukemia (Scl) promoter-driven, self-renewing, leukemia-initiating cells to result in B-lymphoid leukemia (B-ALL) in vivo. In vitro, highly proliferating and serially replatable myeloid and lymphoid colony-forming cultures could be established from BCR-ABL and Bmi1 coexpressing progenitors. However, unlike in vivo expanded CML B-lymphoid progenitors, hematopoietic stem cells, or multipotent progenitors, coexpressing BCR-ABL and Bmi1 did not initiate or propagate leukemia in a limiting dilution assay. Inducible genetic attenuation of BCR-ABL reversed Bmi1-driven B-ALL development, which was accompanied by induction of apoptosis of leukemic B-lymphoid progenitors and by long-term animal survival, suggesting that BCR-ABL is required to maintain B-ALL and that BCR-ABL and Bmi1 cooperate toward blast transformation in vivo. Our data indicate that BCR-ABL targeting itself is required to eradicate Ph(+)/Bmi1(+) B-ALL-initiating cells and confirm their addiction to BCR-ABL signaling.     

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

Summary 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.     

Malignant progenitors from patients with CD87+ acute myelogenous leukemia are sensitive to a diphtheria toxin-urokinase fusion protein

In previous studies, we demonstrated that the diphtheria toxin-urokinase fusion protein DTAT was selectively toxic to acute myeloid leukemia (AML) cell lines overexpressing the CD87 urokinase receptor. In the present study, we analyzed the sensitivity of patient leukemic progenitors to DTAT and correlated the sensitivity with CD87 expression.We isolated leukemic blasts by density gradient centrifugation and performed immunophenotyping by flow cytometry and blast sensitivity measurements by inhibition of cell proliferation and colony formation in semisolid media.We found CD87 overexpression in 18 (25%) of 71 patient leukemic blast samples, including 18 (28%) of 64 myeloid malignancies and 0 (0%) of 7 lymphoid malignancies. DTAT was toxic to patient leukemic blasts by both proliferation inhibition (IC50 85% inhibition by 10 nM DTAT in 11/41 evaluable samples). Only AML and chronic myeloid leukemia (CML) blast crisis blasts (18/61 [30%]) were sensitive to DTAT by the proliferation inhibition assay. Lymphoid leukemia and chronic phase CML/chronic myelomonocytic leukemia (CMML) progenitors were insensitive to DTAT by the proliferation inhibition assay (n = 7 and n = 3, respectively). Similarly, normal marrow progenitors were insensitive to DTAT by both proliferation inhibition (n = 2) and colony inhibition (n = 5) assays. The DTAT toxicity measured by both proliferation inhibition assay and colony inhibition assay correlated with CD87 density (p < 0.0001 and p = 0.001, respectively). DTAT toxicity results were similar for leukemic blasts measured by either of the two assays (p = 0.0002).This study provides the first evidence that a urokinase receptor targeted diphtheria fusion protein is toxic to patient AML blasts. The work also suggests that blast proliferation assays yield similar responses to leukemia colony-forming cell colony assays.     

Adenosine deaminase (ADA) in leukemia: clinical value of plasma ADA activity and characterization of leukemic cell ADA

Adenosine deaminase (ADA) activity was measured in plasma, erythrocytes, and mononuclear cells from 18 patients with acute and chronic leukemia. High levels of ADA activities were found in plasma, erythrocytes, and mononuclear cells from patients with acute leukemia, especially acute lymphoblastic leukemia, and blastic crisis of chronic myeloid leukemia. Serial determination of plasma ADA activities was done in 9 patients with acute leukemia. All patients untreated or in relapse had an elevation of plasma ADA activity, which decreased to normal or subnormal levels during complete remission. On starch gel electrophoresis, plasma ADA in leukemic patients separated into two bands. The major band showed a mobility identical to that of normal red cells and mononuclear cells, and the minor band corresponded to that of normal plasma ADA. Enzymatic and immunological studies were performed on ADA from leukemic cells of acute myeloid and lymphoblastic leukemia. There were no differences in Michaelis constant for adenosine, thermostability, electrophoretic mobility, immunological reactivity, and specific activity between ADA of leukemic cells and normal mononuclear cells. These results strongly suggest that the increased ADA activity in leukemic cells is caused by an increased synthesis of a structurally normal enzyme and that increased plasma ADA activity in leukemic patients reflects an increment of leukemic cells in bone marrow. Therefore, serial determination of plasma ADA activities seems to provide a good indicator of the total mass of leukemic cells in bone marrow.