The immunologic detection of minimal residual disease in acute leukemia

Certain combinations of differentiation antigens are expressed on leukemia blasts and are absent or extremely rare among normal progenitors in the fetal liver and fetal and regenerating bone marrow. These combinations include cCD3/TdT, a thymic feature retained on thymic-acute lymphoblastic leukemia (T-ALL) blasts outside the thymus, and the coexpression of TdT and myeloid markers (CD13, CD33) on a proportion of ALL and acute myeloid leukemia (AML). Thus, double marker immunofluorescence assays are operationally leukemia-specific and can be applied in 35% of acute leukemias for detecting minimal disease at a less than 10(-4) level; only rare cases, 2 of 35 in our study, switch these relevant features during relapse. The sensitivity and specificity of these assays was tested as follows. First, bone marrow samples taken from patients who had originally presented with blasts expressing the leukemia-associated combinations but were in full morphologic remission were studied, and varying numbers (less than 0.01% to 10% of the mononuclear fraction) of cells with aberrant features were identified in 11.6% of the cases. Second, the outcome of 19 patients with minimal disease identified immunologically while in complete morphologic remission was investigated: all 19 patients have developed systemic relapse within 4 to 25 (median 14.5) weeks. In contrast, 17 of 25 patients also morphologically in complete remission and without residual disease identifiable immunologically after repeated testing are still in morphologic and immunologic remission (follow-up 17 to 114 weeks, median 28 weeks). Only eight patients in this group have relapsed so far: in two patients the relapse was localized in the cerebrospinal fluid, while in six patients a systemic relapse was observed 6 to 51 (median 21.5) weeks after the last negative immunologic bone marrow examination. In conclusion, no false-positive results were detected with these sensitive assays, and the introduction of appropriately planned prospective studies, including the immunologic detection of residual leukemia, is justified on the basis of these observations.     

Monoclonal antibodies to myeloid differentiation antigens: in vivo studies of three patients with acute myelogenous leukemia

Three patients with acute myelogenous leukemia (AML) in relapse were treated with intravenous infusions of one or more purified murine monoclonal antibodies (MoAbs) specific for differentiation antigens on normal and malignant myeloid cells. Three of the MoAbs used were IgM immunoglobulins that react with glycolipids, while the fourth, an IgG2b, reacts with a protein antigen. Peripheral blood leukemia cell counts decreased significantly, but transiently, during treatment. Evidence of in vivo binding of each MoAb to leukemia cells was obtained, although two of the four MoAbs could not be detected in the plasma following infusion, perhaps due to circulating blocking factors. Antigenic modulation was not encountered in these studies. However, the induction of human antibody to murine MoAb was observed in one patient who was treated over a 70-day period. Toxicities encountered were minimal and included fever (3 patients), back pain (1 patient), and arthralgias and myalgias (1 patient). This is the first reported clinical trial of (1) IgM MoAbs, (2) MoAb therapy in patients with AML, (3) combinations of MoAbs directed toward different myeloid differentiation antigens, and (4) MoAbs directed to glycolipids. The relative lack of toxicity and the positive effects of MoAb treatment in the reduction of leukemia cell counts permit the continued study of more innovative approaches to the treatment of AML with MoAbs.     

Divergent patterns of marrow cell suspension culture growth in the myeloid leukemias: correlation of in vitro findings with clinical features.

Cellular recovery, maturation, and colony-forming cell (CFC) generation patterns of bone marrow cells from 23 patients with acute, subacute, and chronic myeloid leukemia (AML, SML, and CML) were studied using liquid and agar culture techniques. Increased recovery of proliferative myeloid cells from liquid culture was noted in 6 of 8 AML patients at diagnosis or relapse and 5 of 7 untreated SML patients. Patients with either AML or SML with rapid clinical progression exhibited greater recovery of cells in vitro with less maturation than patients with more stable disease. Studies from 3 patients with CML showed normal to increased cellular recovery with normal maturation. Three of 4 studies of AML patients followed sequentially in apparent remission, but with impending relapse, exhibited increased numbers of myeloblasts and promyelocytes, whereas 28 of 32 studies performed during stable remission were normal. The normally observed increase in CFC during liquid culture was absent in most leukemic marrow samples studied (3 of 4 AML, 4 of 6 SML, and 2 of 3 CML). Persistent low recovery of CFC during AML remission was associated in 3 patients with short remission duration. These studies indicated the potential utility of these techniques for the clinical evaluation of patients with myeloid leukemia and for studying factors involved in the progression of these diseases.     

Cytogenetic studies in bone marrow transplant recipients

Summary Chromosome studies were performed in 24 patients who underwent allogeneic bone marrow transplantation (BMT) for severe aplastic anaemia (8), chronic myeloid leukemia (5 in chronic, 2 in accelerated phase and 1 in lymphoid blast crisis), acute myeloid leukemia (6), acute lymphoblastic leukemia in relapse (1) and Hodgkin's disease (1). Donor-cell type engraftment was demonstrated in 21 patients: in all 17 sex-mismatched transplants and — as demonstrated by reconstitution with Ph-negative cell populations — in 4 CML patients with a sex-matched donor. Recipient-type mitoses were seen in the bone marrow of 5 cases (1 SAA, 3 CML, 1 AML) after transplantation. They were only observed on one occasion in patients with SAA (4 of 25 on day 33) and AML (44 of 50 on day 14). Despite the continued demonstration of some Ph-positive mitoses in 3 patients with CML up to day 28, 323 and 451 after BMT, respectively, all surviving CML patients are still in complete haematological and clinical remission. So far the significance of these cytogenetically abnormal persisting host cells remains unknown. Present address: Roswell Park Memorial Institute, Department of Genetics and Endocrinology, 666 Elm Street, Buffalo, NY 14 222, USA     

Autologous bone marrow transplantation for acute myeloid leukemia following in vitro treatment with neuraminidase and monoclonal antibodies

Although monoclonal antibodies (MoAbs) to CD15, especially PM-81, react with leukemic blasts from the majority of patients with acute myeloid leukemia (AML), a small subset of patients have cells that are CD15 negative or dim. We determined previously that neuraminidase will increase the reactivity of PM-81 with AML blasts, as well as blasts from many patients with acute lymphoblastic leukemia (ALL). In this report, we describe the laboratory results and clinical course of the first patient with AML whose harvested bone marrow was treated with neuraminidase prior to MoAbs and complement treatment. Neuraminidase increased the percentage of the patient's leukemia cells that reacted with PM-81 from 18% to 90% and more than doubled the percentage of AML blasts that were lysed by PM-81 and complement. The patient suffered no acute toxicity, engrafted rapidly, and was transfusion independent by day 21 post-ABMT. This report demonstrates the probable safety and efficacy of pretreatment of bone marrow with neuraminidase, and increases the number of patients with AML or ALL who may benefit from ABMT using marrow purging with MoAb to CD15.     

Successful immunotherapy in early relapse of acute myeloid leukemia after nonmyeloablative allogeneic stem cell transplantation

We report on a 35-year-old woman who underwent allogeneic stem cell transplantation (SCT) in second complete remission (CR) of acute myeloid leukemia (AML) after reduced-intensity conditioning with fludarabine and 2 Gy of total body irradiation. For graft-versus-host disease (GVHD) prophylaxis, cyclosporin A (CsA) and mycophenolate mofetil (MMF) were given. On day 27 after SCT complete hematological remission and donor chimerism was documented. However, in CD34(+) bone marrow cells 28% of recipient hematopoiesis persisted. On day +59 leukemic relapse occurred. After discontinuation of CsA and onset of GVHD, complete donor chimerism and hematological CR were achieved which has been maintained for 14 months.     

Differentiation induction of myeloid leukemia cells by glucocorticoid--the differentiation induction effect in vitro and in vivo

The effects of glucocorticoid on the differentiation of myeloid leukemia cells were examined. Dexamethasone at 10(-6)M or 10(-7)M revealed marked effects not only on leukemic blasts' survival but also on its' differentiation in vitro. In 10 of 17 cases of myeloid leukemia, the obvious morphological and functional differentiation induction effects were observed in vitro. The direction of differentiation were differed in leukemia cell lineage and in cases. Granulocytic differentiation in M2 cells, mono-macrophage differentiation in M5 cells and either granulocytic or monocytic differentiation in M4 cells were induced. A AML (M2), it's leukemia cells were induced into granulocytic pathway by dexamethasone in vitro, was treated with prednisolone (40 mg per day).Ara-C (15 mg per day). The increase in peripheral leukocyte count and the decrease in immature cells were observed simultaneously. The peripheral leukocytes mainly consisted of intermediate forms of granulocytes and Pelger-Hu?t like neutrophils probably originated from leukemia cells. After that course, abnormal clone was eliminated from bone marrow. A AMoL, it's leukemia cells were induced into macrophage like cells completely by dexamethasone in vitro, was treated with prednisolone (30 mg per day) and complete remission was obtained without passing through a hematological nadir. It is indicated that anti-tumor effects of glucocorticoid on myeloid leukemia cells are closely related to it's differentiation inducing effect and glucocorticoid can be used as the drug intending for the differentiation induction therapy of acute myeloid leukemia.     

Relapse after allogeneic bone marrow transplantation for acute leukaemia: a survey by the E.B.M.T. of 117 cases

A multi-centre retrospective analysis on 117 patients relapsing after bone marrow transplantation (BMT) for acute leukaemia was carried out by the Leukaemia Working Party of the European Group for Bone Marrow Transplantation (E.B.M.T.). Forty-one patients had acute myeloid leukaemia (AML) and 76 had acute lymphoblastic leukaemia (ALL). Relapse occurred between 3 and 30 months after BMT and where investigated the leukaemia was found to have relapsed in recipient cells. In 10 cases the relapse was associated with new cytogenetic abnormalities. 74 patients received further treatment for leukaemia. Of these 21 out of 50 with ALL and 11 out of 24 with AML achieved a complete remission and had a median survival of 12 months compared with a median survival of 4 months for untreated patients or patients not achieving complete remission (P less than 0.001). Factors predictive for successful remission induction were a long interval between bone marrow transplant and relapse in ALL patients; and isolated extramedullary relapse. Presenting blast count, karyotype and remission status and number at the time of BMT were not predictive. Donor bone marrow was shown to be responsible for haemopoietic recovery occurring in the 21 out of 31 patients tested who achieved remission using donor karyotype or red blood cell antigens as markers. Nine patients received a second bone marrow transplant but only one became a long-term survivor. The results show that chemotherapy can usually prolong survival in selected patients with acute leukaemia relapsing after BMT but further BMT has a poor outlook.     

Concomitant chronic lymphocytic leukemia and acute myeloid leukemia with an uncommon immunophenotype

We report a case of simultaneous diagnosis of chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML), in which the use of flow cytometry analysis allowed the demonstration of two different cell populations and the study of both immunophenotyping patterns with a large panel of monoclonal antibodies (MoAbs). CLL cells showed a typical immunophenotype with coexpression of B cell markers with CD5, CD23, CD43, and weak surface immunoglobulin light chain restriction expression, whereas the AML population had a very uncommon phenotype with expression of myeloid markers and CD56 and lack of expression of other natural killer (NK) antigens, CD34 and HLA-DR. After chemotherapeutic treatment of AML with two induction courses, the patient achieved complete remission of the AML with persistence of a CD19/CD5 positive population. After consolidation chemotherapy, this latter population was no longer detectable despite the presence of lymphoid nodules in a bone marrow biopsy. Six months after diagnosis, the patient relapsed with AML and died shortly afterwards. Am. J. Hematol. 56:281–287, 1997. © 1997 Wiley-Liss, Inc.     

Prognostic value of immunophenotyping in acute myeloid leukemia. Australian Leukaemia Study Group

The diagnostic and prognostic value of immunophenotyping with 18 murine monoclonal antibodies (MoAbs) to a variety of leukocyte differentiation antigens was assessed in 168 adults aged 15 to 60 years with acute myeloid leukemia (AML). Patients were entered on the multicentre Australian Leukaemia Study Group M4 protocol, and were randomized to receive either standard or high-dose Ara-C together with daunorubicin and etoposide as induction chemotherapy, followed by standard consolidation and maintenance therapy. Diagnostic bone marrow aspirate (152 cases) or peripheral blood samples (16) were analyzed by indirect immunofluorescence and flow cytometry. MoAbs used were directed at myeloid (CD11b, CD13, CD14, CD15, CD33, CD41), lymphoid (CD2, CD3, CD7, CD9, CD10, CD19), or stem cell (HLA-DR, CD34, c-kit receptor) antigens, as well as the leukocyte integrins CD18 and CD49e, and the transferrin receptor CD71. Of the myeloid markers, CD13 and CD33 were the most useful diagnostically (71% and 79% of cases positive, respectively), with CD11b, CD14, and CD15 less commonly positive. A minority of cases expressed lymphoid antigens, either T cell (CD2 16%, CD3 7%, CD7 28%) or B cell (CD10 2%, CD19 7%). CD34 was detected on 42% and c-kit receptor on 48%. When patients were analyzed for response to treatment, CD2, CD9, and CD14 were significantly associated with complete remission rate: cases expressing these antigens had a poorer response than negative cases. In univariate analysis, CD11b+ cases had shorter periods of remission (relative risk of relapse, 2.33; P = .003) and shorter survival (relative death rate, 1.91; P = .006). In multivariate analysis, adjusting for other prognostic factors, CD9 and CD11b were significantly predictive of shorter survival. No other marker had a significant predictive effect. We conclude that myeloid MoAbs are useful in confirming the diagnosis of AML, but their prognostic value may be limited to CD11b. Lymphoid antigen expression is a consistent phenomenon in a minority of cases of AML, but appears to have little clinical significance.     

Spontaneous remission of acute monocytic leukemia after infection with Clostridium septicum

Spontaneous remissions of acute myeloid leukemia (AML) have been reported in association with infection. Here, we report a case of spontaneous remission of AML in a 47-year-old Saudi Arabian male patient who presented with a few weeks history of recurrent abdominal pain, vomiting and fever. He was diagnosed with acute monocytic leukemia (AML, FAB M5b) and a perforated bowel. He also had Clostridium septicum bacteremia and thus chemotherapy was deferred. He received supportive therapy and intravenous antibiotics. Six weeks later, he achieved spontaneous and complete remission lasting for about 4 months. The remission and relapse were documented by bone marrow examination. Similarly, previous reports of spontaneous remission of AML were short lived and were followed by relapse and progression.     

Autologous bone marrow transplantation for acute myeloid leukemia using busulfan plus etoposide as a preparative regimen

We have studied the use of a new preparative regimen for the treatment of patients in remission of acute myeloid leukemia (AML) with autologous bone marrow transplantation. Chemotherapy consisted of busulfan 1 mg/kg every 6 hours for 4 days (total dose, 16 mg/kg) on days -7 through -4 followed by an intravenous infusion over 6 to 10 hours of etoposide 60 mg/kg on day -3. Autologous bone marrow, treated in vitro with 100 micrograms/mL of 4-hydroperoxycyclophosphamide, was infused on day 0. We have treated 58 patients up to the age of 60 years, 32 in first remission, 21 in second or third remission, and 5 with primary refractory AML unresponsive to high-dose Ara-C, but achieving remission with aggressive salvage regimens. Of the first remission patients, there has been 1 treatment related death and 5 relapses. With median follow-up of 22 months, the actuarial relapse rate is 22% +/- 9% and disease-free survival is 76% +/- 9% at 3 years. Patients with favorable French-American-British (FAB) subtypes (M3 or M4 EO) did especially well, with no relapses seen in 15 patients observed for a median of 30 months. Actuarial relapse rate at 3 years was 48% for first remission patients with less favorable FAB subtypes. Of patients in second or third remission, there were 5 treatment related deaths and 4 relapses. With median follow-up of 22 months, the actuarial relapse rate is 25% +/- 11% and disease-free survival is 56% +/- 11% at 3 years. Four of five primary refractory patients died during treatment and 1 remains in remission with short follow-up. These preliminary data are very encouraging and, if confirmed, support the use of autologous purged bone marrow transplantation using aggressive preparative regimens as one approach to improve the outcome of adults with AML.     

Bulky lymphadenopathy in acute myeloid leukemia

 Two cases of acute myeloid leukemia (AML) presenting with bulky adenopathy are reported. Both patients were febrile at admission and showed massive and diffuse lymph node involvement, hepatomegaly, and splenomegaly. Erythematopapular leukemic skin lesions were present in one case at the onset and developed in the other at the time of relapse. Anemia, thrombocytopenia, and moderate leukocytosis were present in both. The presence of immature cells in peripheral blood and bone marrow allowed a rapid diagnosis of AML, FAB M1, in one patient. In the other case, owing to the paucity of immature cells in peripheral blood and bone marrow, lymph node biopsy with histology, imprint cytology, and immunocytochemistry were essential for the diagnosis (AML, FAB M2, with trilineage dysplasia and basophilic involvement). Both patients achieved complete remission (CR), followed by an early relapse 3 months later. They underwent allogeneic bone marrow transplantation (BMT) from HLA identical siblings. One patient is actually alive and in CR at 6 months after BMT; the other patient showed a leukemic regrowth after transplantation and died 4 months later. Received: December 8, 1997 / Accepted: April 29, 1998     

Solitary expression of CD7 among T-cell antigens in acute myeloid leukemia: identification of a group of patients with similar T-cell receptor beta and delta rearrangements and course of disease suggestive of poor prognosis.

In a series of 100 acute myeloid leukemia (AML) patients defined by cytochemistry and immunophenotyping, 20 expressed T-lymphocyte associated antigens on the surface of their blasts. While 15 expressed two or more T-cell antigens, five were found to express only CD7. All patients belonged to the French-American-British type M4, and four were under the age of 40. Despite intensive chemotherapy, four never obtained a complete remission and the fifth died of relapse after an allogenic bone marrow transplantation. While 12 randomly selected T-cell antigen negative AML patients showed only few rearrangements in Ig- or T-cell receptor (TCR) genes, such genetic alterations were demonstrated in four of five patients for the TCR delta gene and in all patients for the TCR beta gene. Interestingly, DNA fragments of similar size were demonstrated in three of five patients for both the beta and delta genes. These data suggest that the solitary presence of CD7 among T-cell antigens in otherwise clearcut AML cases identifies a group of patients with similarities in antigen receptor gene configuration as well as outcome.     

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     

Testicular infiltration in acute myeloid leukemia with complex karyotype including t(8;21)

Testicular infiltration is a well-known complication in acute lymphoblastic leukemia. In acute myeloid leukemia (AML), it has rarely been described and preferably occurred in cases with myelomonocytic or monoblastic differentiation. We report on a patient with AML with complex karyotype including translocation t(8;21) who presented with testicular infiltration at the time of his third bone marrow relapse. Cytological analysis of the specimen showed infiltration with blasts displaying the typical morphology of AML with translocation t(8;21) and comparable to those detected in the bone marrow. Fine needle aspiration cytology might suffice in these cases and should be performed if testicular involvement is suspected.     

Prognostic impact of the CD34+/CD38− cell burden in patients with acute myeloid leukemia receiving allogeneic stem cell transplantation

In acute myeloid leukemia (AML), leukemia‐initiating cells exist within the CD34+/CD38? cell compartment. They are assumed to be more resistant to chemotherapy, enriched in minimal residual disease cell populations, and responsible for relapse. Here we evaluated clinical and biological associations and the prognostic impact of a high diagnostic CD34+/CD38? cell burden in 169 AML patients receiving an allogeneic stem cell transplantation in complete remission. Here, the therapeutic approach is mainly based on immunological graft‐versus‐leukemia effects. Percentage of bone marrow CD34+/CD38? cell burden at diagnosis was measured using flow cytometry and was highly variable (median 0.5%, range 0%–89% of all mononuclear cells). A high CD34+/CD38? cell burden at diagnosis associated with worse genetic risk and secondary AML. Patients with a high CD34+/CD38? cell burden had shorter relapse‐free and overall survival which may be mediated by residual leukemia‐initiating cells in the CD34+/CD38? cell population, escaping the graft‐versus‐leukemia effect after allogeneic transplantation. Evaluating the CD34+/CD38? cell burden at diagnosis may help to identify patients at high risk of relapse after allogeneic transplantation. Further studies to understand leukemia‐initiating cell biology and develop targeting therapies to improve outcomes of AML patients are needed.     

Five year leukaemia-free survival of 72% and 77% for early stage acute and chronic myeloid leukaemia treated by HLA-identical sibling bone marrow transplantation

Background: HLA-identical sibling bone marrow transplantation is an accepted treatment for patients with acute myeloid leukaemia (AML) and chronic myeloid leukaemia (CML). We have recently reported improving results in HLA-identical sibling transplant over the ten year period 1981-1990. In this report we described the outcome in patients transplanted at St Vincent's Hospital, Sydney between 1989 and 1993. Aims: To determine the leukaemia-free survival, transplant-related mortality rate, and relapse rate for patients with AML or CML given HLA-identical sibling marrow transplants between 1989 and 1993. Methods: Sixty-two patients with AML or CML received high dose busulphan/cyclophos-phamide chemotherapy followed by infusion of T replete, HLA-identical sibling bone marrow. Cyclosporin/short methotrexate was utilised as prophylaxis for graft-versus-host disease, ganciclovir as prophylaxis for cytomegalovirus disease and cotrimoxazole as prophylaxis for Pneumocystis carinii pneumonia. Low dose intravenous heparin was used as prophylaxis for hepatic veno-occlusive disease. Results: The five year disease-free survival for patients with AML transplanted in first complete remission was 72% and for those with CML transplanted in first chronic phase was 77%. The relapse rate for AML transplanted in first complete remission was 15% and for CML in first chronic phase 0%. The transplant-related mortality for AML transplanted in first complete remission was 16% and for CML transplanted in first chronic phase 23%. In contrast, the disease-free survival, relapse rate and transplant-related mortality for patients with AML transplanted outside first complete remission and for CML transplanted beyond first chronic phase was 17%, 57% and 57% respectively. Conclusions: The outcome for patients transplanted for early AML or early CML continues to improve and exceeds that obtainable by conventional therapy. The salvage rate is so low for patients transplanted in later stages of AML or CML that all patients less than 55 years of age with these diseases, who have a HLA-identical sibling donor, should be offered bone marrow transplantation early in their disease course.     

Increased myeloid precursors in regenerating bone marrow; implications for detection of minimal residual disease in acute myeloid leukemia

Presented study is focused on exact immunophenotypic definition of myeloid precursors and their following stages in regenerating bone marrow during treatment of ALL/AML for correct interpretation of the immunophenotype results and proper distinction from minimal residual disease (MRD) by multiparameter flow cytometry. This study includes bone marrow samples from 36 controls, 27 patients with AML, 39 patients with B-ALL undergoing therapy who remained in complete remission after treatment and also 30 B-ALL patients one year after the end of therapy. We observed substantial expansion of immature bone marrow populations in the regenerating bone marrows, which were identified by expression of CD34 and/or CD117 markers by 4-color flow cytometry. Myeloid precursors were significantly increased after cessation of induction therapy cycle of B-ALL (1.27+/-2.04%, p=0.0064) and also AML patients (0.87+/-0.77%, p=0.001), but also during follow-up of B-ALL patients (1.42+/-2.36%, p=0.0001) when compared with non-treated controls (0.38+/-0.29%). Some cases where their frequencies achieved up to 12% reflect the massive regeneration of myeloid lineage in bone marrow after chemotherapy cycles. Especially in these cases accurate interpretation of such a high frequency of immature myeloid cells as myeloid precursors was very important to exclude incoming relapse or secondary leukemia. The myeloid precursors represented by CD34+ in regenerating bone marrow expressed CD45 (94.8+/-5.5%), CD117 (38.3+/-26.2%), CD38 (91.4+/-5.7%), HLA-DR (90.6+/-7.6%), CD13 (73.0+/-20.8%) and CD33 (85.2+/-15.6%), while CD90 (2.7+/-2.5%), CD133 (10.0+/-8.2%) and T or B lymphocyte markers were negative. Comparing immunophenotypes with control bone marrows, only difference in expression of CD33 marker was found (85.2+/-15.6% versus 63.0+/-17.4% p=0.024). In addition, according to expression of these markers three different subsets of myeloid precursor cells were identified in regenerating bone marrow samples: CD34+ CD117- HLA-DR+, CD34+ CD117+ HLA-DR+ and CD34- CD117+ HLA-DR-/+ without aberrant marker expression. In conclusion for the correct discrimination of MRD in acute leukemia it is indispensable to define the range of normality in myeloid differentiation by extensive studies of bone marrows not only from healthy donors but also from regenerating bone marrow of patients undergoing therapy.