Chronic lymphocytic leukemia modeled in mouse by targeted miR-29 expression

B-cell chronic lymphocytic leukemia (B-CLL), the most common leukemia in the Western world, occurs in two forms, aggressive (showing for the most part high ZAP-70 expression and unmutated IgH V_H) and indolent (showing low ZAP-70 expression and mutated IgH V_H). We found that miR-29a is up-regulated in indolent human B-CLL as compared with aggressive B-CLL and normal CD19⁺ B cells. To study the role of miR-29 in B-CLL, we generated Eμ-miR-29 transgenic mice overexpressing miR-29 in mouse B cells. Flow cytometric analysis revealed a markedly expanded CD5⁺ population in the spleen of these mice starting at 2 mo of age, with 85% (34/40) of miR-29 transgenic mice exhibiting expanded CD5⁺ B-cell populations, a characteristic of B-CLL. On average, 50% of B cells in these transgenic mice were CD5 positive. At 2 y of age the mice showed significantly enlarged spleens and an increase in the CD5⁺ B-cell population to ∼100%. Of 20 Eμ-miR-29 transgenic mice followed to 24–26 mo of age, 4 (20%) developed frank leukemia and died of the disease. These results suggest that dysregulation of miR-29 can contribute to the pathogenesis of indolent B-CLL.     

E-selectin and very late activation antigen-4 mediate adhesion of hematopoietic progenitor cells to bone marrow endothelium

 Adhesion of CD34⁺ hematopoietic progenitor cells (HPCs) to sinusoidal endothelium probably plays a key role in homing of transplanted CD34⁺ HPCs to the bone marrow (BM). We have investigated the role of various adhesion molecules in the interaction of purified CD34⁺ HPCs derived from BM or peripheral blood (PB) and a human BM-derived endothelial cell line. Adhesion of CD34⁺ HPCs to endothelial cells was measured with the use of a double-color flow microfluorimetric adhesion assay. In this assay, adhesion is measured under stirring conditions, simulating blood flow in sinusoidal marrow vessels. Adhesion of PB CD34⁺ cells to human BM endothelial cells (HBMECs) was observed only after interleukin (IL)-1β prestimulation of the endothelial cells. This adhesion was strongly increased after addition of phorbol-myristate acetate (PMA). Adhesion of PB CD34⁺ cells to IL-1β-prestimulated HBMECs was inhibited by blocking monoclonal antibodies (mAbs) against E-selectin and by neuraminidase treatment of the PB CD34⁺ cells. mAbs against very late activation antigen (VLA)-4 inhibited adhesion only when the E-selectin-mediated interaction was prevented. No clear inhibiting effect was found with blocking mAbs against β₂-integrins. Stimulation with the β₁-integrin-activating mAb, 8A2, induced adhesion of CD34⁺ cells to endothelial cells. In conclusion, stimulation of both endothelial cells and CD34⁺ HPCs is necessary for adhesion of CD34⁺ HPCs to endothelial cells. We furthermore demonstrated that E-selectin and VLA-4 mediated this adhesion. Received: 26 April 1999 / Accepted: 8 February 2000     

Blood-derived macrophage layers in the presence of hydrocortisone support myeloid progenitors in long-term cultures of CD34+ cord blood and bone marrow cells

 Monocytes/macrophages secrete various cytokines that induce proliferation of colony-forming unit granulocyte-macrophage (CFU-GM) in short-term assays. To determine whether macrophages also support proliferation of more primitive progenitors, i.e., cells that give rise to colony forming cells in a 5-week long-term culture (LTC), we established plastic-adherent macrophage layers from human peripheral blood (PB) and filgrastim (G-CSF)-mobilized progenitor cell collections in the presence of hydrocortisone, and compared these layers with bone marrow (BM) stroma regarding their suitability to support proliferation and differentiation of CD34⁺ BM and cord blood (CB) cells in 5-week LTCs. CD34⁺ cells were seeded onto irradiated macrophage and BM stromal layers, as well as without any preformed layer. After 5 weeks, colony formation (CFU-GM, BFU-E/CFU-E) and cell expansion were determined. CD34⁺ cells from BM and CB yielded more CFU-GM and total nucleated cells at 5 weeks in the presence of both types of adherent layer compared with cultures without a layer (p<0.05). For CD34⁺ BM cells, macrophage layers were superior to BM stroma in enhancing CFU-GM and CFU-E/BFU-E output (p<0.05). In contrast, BM stroma was favorable compared with macrophages concerning nucleated cell expansion from CD34⁺ CB cells (p=0.027). The macrophage nature of PB-derived adherent cells was confirmed immunocytochemically by positive staining for CD68, Ki-M1p, CD31, CD54, inconstant staining for CD14, and negative staining for CD1a, CD3, CD15, CD34, and CD62E. Cytochemical reactions were positive for α-naphthyl acetate esterase and negative for peroxidase and periodic acid-Schiff, consistent with the immunophenotype. In conclusion, the results show that blood-derived macrophages support CFU-GM generation from CD34⁺ CB and BM progenitors for 5 weeks in vitro. Differential effects on proliferation and maturation of BM versus CB progenitors are discussed. Received: February 16, 1999 / Accepted: June 21, 1999     

Natural killer expansion,human leukocyte antigens‐E expression and CD14+ CD56+ monocytes in a myelodysplastic syndrome patient

Myelodysplastic syndromes (MDS) are clonal disorders characterized by ineffective hematopoiesis and possible evolution to acute leukemia. Occurrence of stem cell defects and of immune‐mediated mechanisms was evidenced as relevant for pathophysiology of MDS. Here, we described one case of MDS patient carrying CD14⁺CD56⁺ monocytes in bone marrow (BM), in the presence of a defective human leukocyte antigen (HLA)‐E expression on peripheral blood (PB) cells and of natural killer (NK) cell expansion in PB and BM. The defective HLA‐E expression and the NK expansion are proposed to be relevant for the pathogenesis of myelodysplasia in those patients showing CD14⁺CD56⁺ monocytes in BM.     

GM-CSF stimulates proliferation of clonal leukemic bone marrow cells in acute myeloid leukemia (AML) in vitro

 Granulocyte-macrophage colony-stimulating factor (GM-CSF) is known to stimulate granulocytes, monocytes, and macrophages. We studied the effect of GM-CSF on (clonal) bone marrow (BM) cells obtained from AML patients after 7 days of culture in vitro: BM samples were obtained from 19 AML patients at diagnosis (DIA), from two patients with persisting disease (PERS), from eight patients in complete remission (CR), and from 12 healthy donors. Flow-cytometric comparison of differentiated, CD 15-positive cells or of CD34-positive blast cells before and after cultivation showed that the proportion of CD15-positive cells was increased in nine of 12 healthy BM samples, in 14 of 19 cases at DIA, in one of three cases during PERS, and in five of six cases in CR of AML. The proportion of CD34-positive cells was increased in one of 12 healthy BM samples, in seven of 19 cases at DIA, in one of two cases during PERS, and in three of seven cases in CR of AML. Southern blot analysis (SBA) performed in six cases during the course of AML, before and after cell culture, showed that clonal DNA increased after GM-CSF treatment in three of five cases studied at DIA, in six of nine cases studied in CR, in the one case studied at PERS, and in the one studied at relapse (REL). In one case of trisomy 8 at DIA a normal karyotype was demonstrated in CR. However, after 7 days of cultivation of the cells in GM-CSF the trisomy 8 was detected in two of 17 metaphases isolated from colony-cells from methylcellulose cultures. Our data show that a 7-day treatment of BM cells with GM-CSF induced a differentiation of healthy and leukemic BM cells in the great majority of cases. An enrichment of CD34-positive cells was not achieved in healthy BM samples. However, in 70% of the cases in CR and in 30% of the cases at DIA of AML, clonal CD34-positive cells were enriched. This means that GM-CSF stimulates ('primes') leukemic cell growth in vitro. Received: August 17, 1998 / Accepted: April 28, 1999     

Variability in responsiveness to lovastatin of the primitive CD34+ AML subfraction compared to normal CD34+ cells

In the present study, we questioned whether the cholesterol synthesis inhibitor lovastatin potentiates the cytotoxicity of chemotherapeutic agents in the primitive CD34⁺ subpopulation of acute myeloid leukemia (AML) cells. AML mononuclear cells (n = 17) were sorted in CD34⁺ and CD34⁻ fractions and compared to normal CD34^+/− cells (n = 7). The percentage of surviving cells upon exposure to lovastatin (25–100 μM) and/or chemotherapeutics (cytarabin or daunorubicin) was determined with a luminescent cell viability assay. The results demonstrate that the primitive CD34⁺ subpopulation of normal and AML cells displayed a higher sensitivity to lovastatin than the more mature CD34⁻ subpopulation. The combination of lovastatin and chemotherapeutics resulted in a more pronounced inhibitory effect on both subpopulations. In contrast to the homogeneous results in normal CD34⁺ cells, a distinct heterogeneity in lovastatin sensitivity was found in AML samples. Therefore, a group of normal (n = 11) and abnormal (n = 6) responders were identified based on a reduced or increased cell survival compared to normal CD34⁺ cells. This distinction was not only observed in the CD34⁺ AML subfraction but also in CD34⁺CD38⁻AML cells. In the abnormal responder group, 50% of patients presented with unfavorable cytogenetics and significant higher peripheral blast cell counts, which coincided with poor treatment results. In summary, the findings indicate that the primitive subfraction of CD34⁺ AML cells is in the majority of cases affected by lovastatin treatment, which is potentiated when combined with chemotherapeutics. Heterogeneity of the response observed in AML patients allowed identification of a subgroup with poor prognosis.     

Phenotypic and functional analysis of bone marrow progenitor cell compartment in bone marrow failure

SUMMARY. Many laboratory findings have demonstrated that the haemopoietic stem cell compartment is defective in aplastic anaemia (AA). AA bone marrow (BM) and peripheral blood (PB) are profoundly deficient in colonyforming cells, and AA progenitors fail to proliferate in longterm assays even in the presence of an intact stroma. Our study was designed to characterize some quantitative and qualitative aspects of the progenitor cell defect in AA. Using flow cytometric analysis of BM from new AA patients and from those recovering after immunosuppressive therapy, we determined that the numbers of CD34⁺ and CD33⁺ cells were markedly decreased in AA. Although PB neutrophil counts did not correlate with BM CD34⁺ cell numbers in acute disease, there was an association between the overall severity of the disease and the degree of CD34⁺ cell reduction. A decrease in BM CD33⁺ cells was a common finding in MDS patients, but reduction in CD34⁺ cells was found only in some hypoplastic MDS cases. Sorting experiments demonstrated lower plating efficiency for purged CD34⁺ cells from AA BM in comparison to controls. Thus, diminished colony formation of total BM appeared to result from both quantitative and qualitative defects. Based on the association between increased cycling and c-kit receptor expression on CD34⁺ cells, we found that the mitotically active CD34⁺ cells bearing the c-kit antigen were reduced in AA. With clinical improvement, CD34⁺ and CD33⁺ cells increased in correlation with PB parameters, but they did not return to normal values. Sorted CD34⁺ cells from recovered patents showed improved plating efficiency. In patients with aplastic anaemia, use of CD34 antigen as a phenotypic marker of progenitor cells may be helpful for the analysis of the early haemopoietic cell compartment and BM recovery.     

Alloantibody from a patient with severe von Willebrand disease inhibits von Willebrand factor-FVIII interaction

 Older patients with RAEB-T or AML are extremely difficult to treat. They are at high risk of infection and/or bleeding complications and have a low probability of cure and short overall survival with conventional treatments. We treated 12 patients with an outpatient low-dose chemotherapy regimen consisting of Ara-C 100 mg subcutaneously on day 1, and 6-thioguanine 80 mg orally on days 2–5, repeated every week. Nine patients had MDS, six RAEB-T, and three RAEB (median age 57 years) and three had de novo AML (median age 73 years). All patients were transfusion dependent. The mean peripheral blast count at the beginning of treatment was 29% (4–51%). The median follow-up is 13 months (2–34 months) for all the patients and 14 months (2–34 months) for those with RAEB-T. Nine of the 12 patients are alive, including seven RAEB-T patients with a median of 18 months (range 6–34+ months). During treatment, the peripheral blast count was markedly reduced to a mean of 5% (0–23%). The mean pre-therapy platelet count, with transfusion support, was 24.0×10⁹/l, while the mean post-therapy platelet count without transfusion support is 95.0×10⁹/l. All patients except two became transfusion independent at some time. Treatment for 6–10 weeks was required to show reduction of blast number and increase in hemoglobin, platelet, and WBC counts. Initial cytopenias were the only side effects of this regimen. One patient had granulocytopenic fever. In conclusion, this low-dose regimen is effective and well tolerated for outpatient palliation in high-risk or elderly patients with RAEB-T or AML. Received: 11 September 1996 / Accepted: 8 January 1997     

Acute myeloid leukemia does not deplete normal hematopoietic stem cells but induces cytopenias by impeding their differentiation

Acute myeloid leukemia (AML) induces bone marrow (BM) failure in patients, predisposing them to life-threatening infections and bleeding. The mechanism by which AML mediates this complication is unknown but one widely accepted explanation is that AML depletes the BM of hematopoietic stem cells (HSCs) through displacement. We sought to investigate how AML affects hematopoiesis by quantifying residual normal hematopoietic subpopulations in the BM of immunodeficient mice transplanted with human AML cells with a range of genetic lesions. The numbers of normal mouse HSCs were preserved whereas normal progenitors and other downstream hematopoietic cells were reduced following transplantation of primary AMLs, findings consistent with a differentiation block at the HSC–progenitor transition, rather than displacement. Once removed from the leukemic environment, residual normal hematopoietic cells differentiated normally and outcompeted steady-state hematopoietic cells, indicating that this effect is reversible. We confirmed the clinical significance of this by ex vivo analysis of normal hematopoietic subpopulations from BM of 16 patients with AML. This analysis demonstrated that the numbers of normal CD34⁺CD38⁻ stem-progenitor cells were similar in the BM of AML patients and controls, whereas normal CD34⁺CD38⁺ progenitors were reduced. Residual normal CD34⁺ cells from patients with AML were enriched in long-term culture, initiating cells and repopulating cells compared with controls. In conclusion the data do not support the idea that BM failure in AML is due to HSC depletion. Rather, AML inhibits production of downstream hematopoietic cells by impeding differentiation at the HSC–progenitor transition.     

Intracellular tumor necrosis factor production by T- and B-cells in B-cell chronic lymphocytic leukemia

BACKGROUND AND OBJECTIVES: The pathogenesis of B-cell chronic lymphocytic leukemia (B-CLL) has been linked with the production and activity of certain growth factors. Tumor necrosis factor (TNF-alpha) is important for the growth and survival of B-CLL cells. TNF-alpha promotes the proliferation of the malignant cell clone and is believed to play a role in the progression of B-CLL. The aim of our study was to examine the level of production and intracellular expression of TNF-alpha by T- and B-cells in B-CLL in correlation with stage of disease and clinical parameters. DESIGN AND METHODS: Using a three-color flow cytometry technique we analyzed intracellular TNF-alpha expression by B-cells (CD19+) and by T-cell subsets (CD3+/CD4+ and CD3+/CD8+) in peripheral blood (PB) and bone marrow (BM) from 40 patients with B-CLL and 24 healthy controls. RESULTS: A higher number of TNF-a-positive B-cells were found in PB and BM in B-CLL patients than in normal controls. In BM this difference was statistically significant (p<0.007). Likewise, in PB and BM the percentage of T-cells expressing TNF-alpha was significantly higher in B-CLL patients than in normal controls (PB: p<0.00001; BM: p<0.007). CD3+/CD4+ cells from patients displayed a lower level of intracellular TNF-alpha expression than did CD3+/CD8+ cells (PB: p<0.0001; BM: p<0.04). The number of T-cells expressing TNF-alpha in B-CLL patients was higher in those with stages III-IV than in patients with early stage disease (PB: p<0.007; BM: p<0.01). Additionally, PB and BM T-cell subsets from patients in stages III-IV had a statistically significant higher level of cytoplasmic TNF-a expression than the corresponding cells from healthy controls (PB: p<0.02; BM: p<0.05). In PB the percentage of CD4+ and CD8+ T-cells expressing cytoplasmic TNF-alpha positively correlated with the stage of disease, total tumor mass (TTM) score and lymphocytosis. In BM only the percentage of CD8 T-cells positively correlated with TTM score and lymphocytosis. The expression of TNF-alpha in leukemic B-cells did not correlate with any progression parameters of disease. INTERPRETATION AND CONCLUSIONS: The results obtained suggest that malignant B-cells are exposed to large numbers of T-cells able to synthesize and secrete TNF-alpha. Moreover, T-cells even though fewer than B-cells may be an important source of TNF-a in advanced stages of disease. This indicates that the TNF-alpha can be associated with progression of B-CLL and may be implicated in some side-effects associated with this disease.     

Cerebellar myeloblastoma formation in CD7-positive, neural cell adhesion molecule (CD56)-positive acute myelogenous leukemia (M1)

 We present a first report of a CD7⁺ acute myelogenous leukemia patient who developed intracranial myeloblastomas. The patient was neurologically normal on physical examination at presentation. The peripheral leukocyte count was extremely high (203.6×10⁹/l). The blasts expressed CD7 and CD56 (neural cell adhesion molecule) in addition to CD13, CD33, CD34, and HLA-DR. The karyotype of bone marrow cells was normal. The patient was diagnosed as having acute myelogenous leukemia (AML, M1). Following a short period of complete remission, bone marrow relapse and meningeal leukemia occurred, and the patient died of respiratory failure. Autopsy revealed that blasts had invaded the subarachnoid space and cerebellum, and two myeloblastomas were found in the cerebellar hemisphere. Both CD7⁺ and CD56⁺ AML have been reported to have a high incidence of central nervous system involvement. CD7⁺ CD56⁺ AML calls for prophylaxis of central nervous system leukemia. Received: 2 May 1997 / Accepted: 17 July 1997     

Bone marrow CD34+ progenitor cells from rheumatoid arthritis patients support spontaneous transformation of peripheral blood B cells from healthy individuals

We show that bone marrow (BM) CD34⁺ progenitor cells from rheumatoid arthritis (RA) patients have the capacity to support spontaneous transformation of peripheral blood B cells. CD34⁺ cells purified from BM blood from eight RA patients and eight osteoarthritis (OA) patients were expanded with granulocyte/macrophage colony stimulating factor (GM-CSF) for 4–6 weeks. GM-CSF-stimulated BM CD34⁺ cells from three of eight RA patients, but none from seven OA patients, gave rise to spontaneous transformation of highly purified B cells of Epstein-Barr virus (EBV)- seronegative healthy donors. GM-CSF-stimulated BM CD34⁺ cells from four of six RA patients and from one of four OA patients also supported the spontaneous transformation of peripheral blood B cells from EBV-seropositive healthy donors. All the transformed B cell lines were positive for EBV-DNA as determined by PCR. Neither GM-CSF-stimulated BM CD34⁺ cells alone nor highly purified B cells alone gave rise to spontaneously transformed B cell lines. These results suggest that the capacity of BM CD34⁺ cells to support survival of B cells might contribute to the pathogenesis of RA by sustaining abnormal B cell responses. Received: 8 November 1999 / Accepted: 10 January 2000     

Flow-cytometric detection of minimal residual disease with atypical antigen combinations in patients with de novo acute myeloid leukemia

 The immunophenotypic features in adult de novo acute myeloid leukemia (AML) patients at diagnosis using flow cytometry double marker analysis and the detection of minimal residual disease with atypical leukemia-associated antigen combinations during remission were investigated. Fifty adult patients with de novo AML at diagnosis were studied. Bone marrow samples from 21 patients with AML were analyzed upon achievement of complete remission and during continuous complete remission. Ten bone marrow samples of normal donors were also studied. CD34/CD13, CD34/CD33, CD33/CD7, CD33/CD10, CD33/CD19 and CD33/TdT are the leukemia-associated antigen combinations used for the detection of minimal residual disease. The outcome of 19 patients has been evaluated. Of these 19 patients, 10 were found to be in immunophenotypic remission (median follow-up after the study: 837 days, range 620–1343 days). Only one patient in this group has relapsed so far. In the other nine patients residual disease was detected. Seven of these patients developed systemic relapse following a median follow-up time of 86 days after the study (range 34–273 days), one received allogeneic bone marrow transplantation 70 days after the study, and another has been in complete remission and off chemotherapy for 36 months. The presence of cells with atypical antigen combinations identified at diagnosis in certain patients is valuable for monitoring the disease in remission. The persistence of such a population in remission has indicated the impending relapse in this study. Received: 11 October 1999 / Accepted: 11 February 2000     

Molecular and clinical follow-up after stem cell transplantation for multiple myeloma

 Molecular follow-up has been carried out using immunoglobulin heavy-chain (IgH) gene fingerprinting, a polymerase chain reaction (PCR)-based technique with a sensitivity of 0.1–0.01% (10^–3–10^–4), in 22 patients affected by multiple myeloma and submitted to stem cell transplantation (SCT). Twelve patients were submitted to either single or double autologous unselected peripheral blood progenitor cell transplantation, eight patients were submitted to autologous CD34+ immunoselected transplantation and two patients were submitted to allogeneic bone marrow (one patient) or peripheral blood CD34+ stem cell (one patient) transplantation. At diagnosis, all patients showed clonal CDIII rearrangement. The molecular analysis performed on leukapheresis products and CD34+ purified fractions proved to be contaminated by myeloma cells. During follow-up after autografting, all but one patient retained clonal rearrangement despite clinical complete remission (CR) in ten of them. These ten patients either relapsed (Rel) or showed progressive disease (PD) after transplantation; four of them died. Only one patient did not retain clonal rearrangement after autologous transplantation; she is currently alive in CR after a follow-up of 100 months. One patient submitted to allogeneic transplantation is currently alive with no evidence of the disease, but still retains clonal rearrangement after a follow-up of 47 months. Another patient died 4 months after transplantation after succumbing to fatal pneumonia showing myeloma progression. Received: 28 February 2000 / Accepted: 1 August 2000     

B-lymphocyte suppression in multiple myeloma is a reversible phenomenon specific to normal B-cell progenitors and plasma cell precursors

The reduced levels of normal immunoglobulin in patients with myeloma may be due to suppression of normal B-cell differentiation. However, reports on the numbers of B cells vary, with some finding decreases consistent with immunoparesis, and others reporting expansions of phenotypically aberrant cells. We have therefore assessed the phenotype and levels of B lymphocytes in patients at presentation (n = 23), in plateau or complete remission (PB n = 42, BM n = 18), and in relapse (PB n = 17, BM n = 14), in comparison to normal individuals (n = 10). Phenotypic analysis was performed using five-parameter flow cytometry, with CD14 used to exclude monocytes where necessary. We found no evidence of a phenotypically distinctive blood or marrow B-cell population in patients with myeloma, nor of an increase in the levels of any B-cell subset. Numbers of blood CD19⁺38⁺ normal plasma cell precursors were significantly reduced in presentation/relapse patients, but not in patients in plateau/remission. Total CD19⁺ cells were significantly reduced only in patients with circulating myeloma cells, detected by IgH-PCR. In the marrow, CD19⁺ B cells expressing CD5, CD10, CD34, CD38, CD45^low and Syndecan-1 were significantly decreased at presentation/relapse, but not in patients in plateau/remission. The majority of these antigens are expressed by normal B-cell progenitors, indicating that myeloma also affects the early stages of B-cell development. The suppression of progenitor cells was not restricted to B-lymphoid differentiation, as total CD34⁺ cells were also significantly reduced in the marrow of myeloma patients at presentation. These results indicate that, if neoplastic B cells are present in myeloma, they are low in number and have a phenotype similar to their normal counterparts. Furthermore, there is a reversible suppression of CD19⁺ B lymphocytes that correlates inversely with disease stage, and specifically affects the early and late stages of normal B-cell differentiation.     

Cytokines can reduce clonal, CD34-positive cells in acute myeloid leukemia in vitro

We studied the influence of cytokine mixes on the survival of acute myeloid leukemia (AML) bone-marrow (BM) cells in a 14-day culture assay in vitro. Southern-blot analysis using a panel of different probes in combination with densitometry and flow cytometry were used to detect and compare the amount of clonal or CD34-positive BM cells before and after the culturing procedure. A significant reduction of CD34-positive cells after incubation with a cytokine mix [interleukin (IL)-1β, IL-3, IL-6, stem cell factor (SCF), erythropoietin (EP) with granulocyte macrophage/colony-stimulating factor (GM-CSF, Cytok1] could be achieved in all 16 cases with a CD34-positive blast phenotype studied at diagnosis (P<0.001), in 3 of 10 cases at relapse, and in 8 of 18 cases in complete remission. In healthy donors, an increase of CD34-positive cells was demonstrated in 5 of 5 samples. A reduction of clonal DNA through incubation with Cytok1 was achieved in 5 of 5 (100 %) cases studied at diagnosis, in 1 of 4 (25%) cases at relapse, and in 7 of 9 cases (78%) in complete remission. Cytokine cocktails with GM-CSF (Cytok1) were more efficient in reducing (clonal) CD34-positive cells than cocktails without GM-CSF (Cytok2). AML patients at diagnosis and in complete remission had a better survival probability if their CD34-positive or clonal cells could be reduced in vitro by cytokine cultivation (P<0.05). Vitality of BM cells was not influenced by 14-day cytokine treatment; however, the total cell count could be increased by Cytok1 and Cytok2 by 55–174%, but not by the control medium. Our data show that: (1) clonal cell populations can be regularly detected at diagnosis, during complete remission, and at relapse; (2) CD34-positive cells in AML can be demonstrated to be clonal, gene-rearranged cells; (3) incubation of AML BM-cells with Cytok1 leads to a reduction of the CD34-positive, clonal cell load in all cases at diagnosis and in 78% of the cases in complete remission of AML, but in only 25% of the cases at relapse; (4) in all healthy BM samples, proportions of ‘healthy’ CD34-positive cells were increased. Moreover, absolute cell counts were increased by cytokine incubation of cells obtained at diagnosis, relapse, or complete remission of AML and from healthy donors indicating a selective stimulation of healthy, but not of leukemic CD34-positive cells; (5) cytokine cocktails containing GM-CSF are more efficient in reducing leukemic cells than cocktails without GM-CSF; and (6) in vitro reactivity of clonal or CD34-positive BM cells against Cytok1 has clinical relevance. We conclude, that Southern-blot analysis and flow cytometry are suitable methods to detect and quantify leukemic disease and to distinguish between clonal or non-clonal CD34-positive cells. The ex vivo or clinical application of specific combinations of cytokines might be a feasible and successful application of immunotherapy in AML that merits further investigations. Received: 19 April 1999 / Accepted: 13 January 2000     

Antigen modulation followed by quantitative flow cytometry of B-chronic lymphocytic leukemia cells after treatment

Presented study analyzed the immunophenotypic characteristics and antigen density of peripheral blood (PB) and bone marrow (BM) cells of 23 patients with B-chronic lymphocytic leukemia (B-CLL) and 10 control subjects using flow cytometry. The patients were subclassified into two groups. Group I formed 13 patients with B-CLL at the time of diagnosis and group II 10 patients with B-CLL after the therapy but not in remission. For definition of B-CLL cells we used immunological marker analysis of surface markers characteristic for B-CLL pattern: CD5, CD19, CD20, CD23 and HLA DR and enumeration of fluorescence intensity of these markers given by molecular equivalent of soluble fluorochrome--MESF. In group II of B-CLL patients, who were already treated, in PB and BM somehow lower MESF values for CD19, CD20 and CD23 markers and higher MESF values for CD5 marker (in PB and BM) than in group I patients have been detected. The MESF level of HLA DR marker was little higher in group II than in group I B-CLL patients. However in PB and BM the percentage expression of HLA DR and CD19 markers in both patients groups was approximately the same. The values of HLA DR, CD19, CD20, CD23 and CD5 markers (% expression and MESF values) in both patients groups with B-CLL were significantly higher versus controls (p<0.001 resp. p<0.01) even in PB and BM. In conclusion, in our study we observed that the patients with B-CLL after therapy (group II) had similar or a little smaller (except CD5) but nonsignificantly decreased expression level of markers characteristic for B-CLL, but the MESF values of some of them (CD19, CD23) were significantly (p<0.05) decreased when compared with untreated B-CLL patients (group I). The determination of antigen density (MESF values) may be an important marker to characterize the leukemic cells. Our results showed that chemotherapy did not influence in a significant level the antigen modulation of B-CLL cells, however, could influence MESF values of some characteristic markers. Quantitative analysis of some markers in B-CLL cells seems to offer valuable information concerning possible influence of some chemotherapeutics on antigen equipment of leukemic cells.     

Chronic lymphocytic leukemia with two cellular populations: a biphenotypic or biclonal disease

 A case of CLL with two different cellular populations is reported. A 50-year-old man was evaluated for persistent absolute lymphocytosis. A peripheral blood smear revealed numerous small lymphocytes (83% of white blood cells counted). Frequent Grumpecht shadows were present, too. On bone marrow aspirate smears lymphocytes comprised 85% of the total cells counted, and the bone marrow biopsy showed a mixed nodular-interstitial infiltration pattern. The immunophenotypic study showed two different leukemic populations. The first one (comprising 79% leukemic cells) was CD5+, CD19+, CD10–, CD20+, CD18–, CD22–, CD23++, lambda dim, and FMC7–. The second population (comprising 21% leukemic cells) was CD5+, CD19+, CD10–, CD20+, CD18+, CD22+, CD23+, lambda++, and FMC7+. Gene rearrangement studies detected the germline and one rearranged band in Jk blot with each restriction endonuclease. In the Jh blot the germline and two rearranged bands were detected with EcoRI and BamHI and three rearranged bands with HindIII. The JBI/JBII blot detected only the germline band. The detection of three rearranged bands was interpreted as evidence of the presence of at least two monoclonal populations of cells with the same light chain restriction. Received: 10 September 1996 / Accepted: 17 February 1997