Identification of BCR/ABL-negative primitive hematopoietic progenitor cells within chronic myeloid leukemia marrow

Chronic myeloid leukemia (CML) is a malignant disorder of the hematopoietic stem cell. It has been shown that normal stem cells coexist with malignant stem cells in the bone marrow of patients with chronic-phase CML. To characterize the primitive hematopoietic progenitor cells within CML marrow, CD34+DR- and CD34+DR+ cells were isolated using centrifugal elutriation, monoclonal antibody labeling, and flow cytometric cell sorting. Polymerase chain reaction analysis of RNA samples from these CD34+ subpopulations was used to detect the presence of the BCR/ABL translocation characteristic of CML. The CD34+DR+ subpopulation contained BCR/ABL(+) cells in 11 of 12 marrow samples studied, whereas the CD34+DR- subpopulation contained BCR/ABL(+) cells in 6 of 9 CML marrow specimens. These cell populations were assayed for hematopoietic progenitor cells, and individual hematopoietic colonies were analyzed by PCR for their BCR/ABL status. Results from six patients showed that nearly half of the myeloid colonies cloned from CD34+DR- cells were BCR/ABL(+), although the CD34+DR- subpopulation contained significantly fewer BCR/ABL(+) progenitor cells than either low-density bone marrow (LDBM) or the CD34+DR+ fraction. These CD34+ cells were also used to establish stromal cell-free long-term bone marrow cultures to assess the BCR/ABL status of hematopoietic stem cells within these CML marrow populations. After 28 days in culture, three of five cultures initiated with CD34+DR- cells produced BCR/ABL(-) cells. By contrast, only one of eight cultures initiated with CD34+DR+ cells were BCR/ABL(-) after 28 days. These results indicate that the CD34+DR- subpopulation of CML marrow still contains leukemic progenitor cells, although to a lesser extent than either LDBM or CD34+DR+ cells.     

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.     

Characterization of primitive subpopulations of normal and leukemic cells present in the blood of patients with newly diagnosed as well as established chronic myeloid leukemia

Elevated numbers of primitive Philadelphia chromosome-positive (Ph+) progenitors, including long-term culture-initiating cells (LTC-IC) as well as colony-forming cells (CFC), have been previously described in the blood of patients with chronic myeloid leukemia (CML) in chronic phase with high white blood cell counts. In the present study, which focused primarily on an analysis of circulating progenitors present in such patients at diagnosis, we discovered the frequent and occasionally exclusive presence of circulating normal (Ph-) LTC-IC, often at levels above those seen for LTC-IC in the blood of normal individuals. The presence of detectable numbers of circulating Ph- LTC-IC was independent of the fact that the same peripheral blood samples also contained elevated numbers of predominantly or exclusively Ph+ CFC. Interestingly, both the Ph+ and Ph- LTC-IC in these samples were CD34+CD71- and variably CD38- and Thy-1+, as previously documented for LTC-IC in normal marrow. Thus, neither CD38 nor Thy-1 expression was useful for discriminating between Ph+ and Ph- LTC-IC in mixed populations. Nevertheless, an association of these phenotypes with LTC- IC function did allow highly enriched (> 5% pure) suspensions of either Ph+ or Ph- LTC-IC to be obtained from selected samples of CML blood in which the initial LTC-IC population was either predominantly Ph+ or Ph- , respectively. These findings suggest that the mechanisms causing mobilization of leukemic stem cells in untreated CML patients may affect their normal counterparts. They also indicate a possible new source of autologous cells for the support of intensive therapy of CML patients. Finally, they provide a method for obtaining the most highly purified populations of Ph+ LTC-IC described to date. This method should be useful for further analyses of the molecular activities of these very primitive neoplastic cells.     

Outcome of hematopoietic stem cell transplantation in patients with atypical chronic myeloid leukemia

Atypical chronic myeloid leukemia (aCML) occurs rarely and is associated with a poor prognosis when treated with conventional chemotherapy. We evaluated the outcome of aCML after allogeneic hematopoietic stem cell transplantation (HSCT). Nine patients were transplanted from HLA-identical siblings (n = 4), HLA-compatible unrelated donors (n = 4) or twin brother (n = 1). Median follow-up was 55 months after transplant (range, 9.1-118.1 months). One patient who was transplanted in advanced disease with bone marrow from his twin brother relapsed 19 months post transplant. This patient was successfully retransplanted from the original donor. All patients remained in complete remission. Analysis of the leukocyte chimerism of peripheral white blood cells and bone marrow buffy coat cells by VNTR-polymerase chain reaction (PCR) and single-nucleotide polymorphism real-time PCR revealed complete chimerism in all patients who had received an allogeneic transplant. One patient suffering from cerebral toxoplasmosis died 9 months post transplant. All other patients were alive at the time of analysis. Our findings suggest that the outcome of allogeneic or syngeneic transplantation in patients with aCML may not be worse than the outcome of transplantation for BCR-ABL-positive CML.     

Detection,isolation, and stimulation of quiescent primitive leukemic progenitor cells from patients with acute myeloid leukemia (AML)

Although many acute myeloid leukemia (AML) colony-forming cells (CFCs) and long-term culture-initiating cells (LTC-ICs) directly isolated from patients are actively cycling, quiescent progenitors are present in most samples. In the current study, (3)H-thymidine ((3)H-Tdr) suicide assays demonstrated that most NOD/SCID mouse leukemia-initiating cells (NOD/SL-ICs) are quiescent in 6 of 7 AML samples. AML cells in G(0), G(1), and S/G(2)+M were isolated from 4 of these samples using Hoechst 33342/pyroninY staining and cell sorting. The progenitor content of each subpopulation was consistent with the (3)H-Tdr suicide results, with NOD/SL-ICs found almost exclusively among G(0) cells while the cycling status of AML CFCs and LTC-ICs was more heterogeneous. Interestingly, after 72 hours in serum-free culture with or without Steel factor (SF), Flt-3 ligand (FL), and interleukin-3 (IL-3), most G(0) AML cells entered active cell cycle (percentage of AML cells remaining in G(0) at 72 hours, 1.2% to 37%, and 0% to 7.6% in cultures without and with growth factors [GFs], respectively) while G(0) cells from normal lineage-depleted bone marrow remained quiescent in the absence of GF. All 4 AML samples showed evidence of autocrine production of 2 or more of SF, FL, IL-3, and granulocyte-macrophage colony-stimulating factor (GM-CSF). In addition, 3 of 4 samples contained an internal tandem duplication of the FLT3 gene. In summary, quiescent leukemic cells, including NOD/SL-ICs, are present in most AML patients. Their spontaneous entry into active cell cycle in short-term culture might be explained by the deregulated GF signaling present in many AMLs.     

Phenotypic quantitative features of patients with acute myeloid leukemia

The recent WHO classification for acute myeloid leukemias (AML) separates entities by recurrent cytogenetic abnormalities and immunophenotypic features presenting prognostic impact. We have examined the expression of several lineage and maturation linked antigens used in routine immunophenotyping of patients with de novo AML, using a 3-color two-step panel. Cases were diagnosed by peripheral blood counts, bone marrow cytology, cytochemistry, cytogenetics and immunophenotyping (CD2, CD3, CD7, CD19, CD13, CD33, myeloperoxydase -- MPO, CD14, CD15, HLA-DR, CD34, CD56 and CD45). Antigen expression was measured by mean fluorescence intensity (MFI) by flow cytometry (Paint-a-gate software). Thirty five patients were analyzed. Median age: 51 years (15-79). Predominant FAB types were M2 and M4. In 6 cases more than one phenotypically distinct blast subpopulation could be detected. Although our set was small, we tried to analyze the impact of MFI of the examined antigens on the overall survival of the patients. In Cox univariate analysis, age, peripheral leukocytes (WBC) at diagnosis, MFI of CD45, and MPO were significant for worse a survival. In the multivariate analysis only MFI of CD45 and WBC remained in the model (p=0.018 and p=0.014 respectively). After bootstrap resampling, MFI of CD45 entered the model in 69%, WBCin 60%, age in 42% and MFI of MPO in 35% of the sets. Analysis of antigen expression by MFI permitted to detect cases presenting phenotypically distinct blast subpopulations. This may represent a pitfall in studies of minimal residual disease by flow cytometry, as chemotherapy may select one of these subsets.     

Evidence for a positive role of SHIP in the BCR-ABL-mediated transformation of primitive murine hematopoietic cells and in human chronic myeloid leukemia

Previous studies suggested that the SH2-containing inositol-5-phosphatase (SHIP) may play a tumor suppressor-like function in BCR-ABL-mediated leukemogenesis. To investigate this possibility, we first developed a new assay for quantitating transplantable multilineage leukemia-initiating cells (L-ICs) in hematopoietic stem cell (HSC)-enriched mouse bone marrow (BM) cells transduced with a BCR-ABL-GFP (green fluorescent protein) retrovirus. The frequency of L-ICs (1 of 430 Sca-1+lin- cells) was 7-fold lower than the frequency of HSCs in the Sca-1+lin- subset transduced with a control virus (1 of 65 cells). Forced BCRABL expression was also accompanied by a loss of regular HSC activity consistent with the acquisition of an increased probability of differentiation. Interestingly, the frequency and in vivo behavior of wild-type (+/+) and SHIP-/- L-ICs were indistinguishable, and in vitro, Sca-1+lin- BCR-ABL-transduced SHIP-/- cells showed a modestly reduced factor independence. Comparison of different populations of cells from patients with chronic myeloid leukemia (CML) in chronic phase and normal human BM showed that the reduced expression of full-length SHIP proteins seen in the more mature (CD34-lin+) leukemic cells is not mirrored in the more primitive (CD34+lin-) leukemic cells. Thus, SHIP expression appears to be differently altered in the early and late stages of differentiation of BCR-ABL-transformed cells, underscoring the importance of the cellular context in which its mechanistic effects are analyzed.     

Unregulated proliferation of primitive chronic myeloid leukemia progenitors in the presence of normal marrow adherent cells.

Previous studies have shown that Philadelphia (Ph1) chromosome-positive chronic myeloid leukemia (CML) results from the abnormal expansion at the pluripotent stem cell level of a single clone of hemopoietic cells. Although it seems likely that this is related to the heightened proliferative activity characteristic of primitive CML progenitor cell types, the underlying mechanism is unknown. In this report we show that either normal or CML peripheral blood progenitors can be maintained on preestablished normal marrow adherent layers for periods of 1-2 months. Under these conditions numbers of both normal and neoplastic progenitors are usually higher in the adherent layer than in the nonadherent fraction. Moreover, the number of primitive progenitors of high proliferative potential present in the adherent layer is sufficient to allow their cycling status to be determined. Such measurements demonstrate that primitive normal progenitors of blood origin, when cultured in the presence of a preestablished adherent marrow feeder layer, go in and out of cycle after each medium change but in the absence of a feeder layer remain continuously in cycle. In contrast, primitive CML progenitors of either blood or marrow origin cycle continuously regardless of the presence or absence of an adherent feeder layer. We suggest that early expansion of the CML clone is related to an ability of the neoplastic stem cells to ignore or overcome a negative regulatory signal produced by nonneoplastic adherent marrow cells whose normal function is to maintain the stem cell reserve in a quiescent state.     

Polyclonal normal hematopoietic progenitors in patients with acute myeloid leukemia

OBJECTIVE: To study the clonality of cytogenetically normal progenitors detected in the peripheral blood (PB) of acute myeloid leukemia (AML) patients. METHODS: Five female patients with cytogenetically abnormal, newly-diagnosed AML who were heterozygous for informative alleles of the androgen receptor (AR) gene were studied using the human androgen receptor allele (HUMARA) assay. RESULTS: PB mononuclear cells and bone marrow (BM) fibroblasts from these patients were monoclonal and polyclonal, respectively. Both cytogenetically normal and abnormal colony-forming cells (CFC) were detected from 3 AML samples and the HUMARA assay determined that most of these CFC were part of the leukemic clone. The fourth sample generated colonies that were 100% normal by cytogenetics and polyclonal by HUMARA. In contrast, 5-week-old long-term culture (LTC)-derived colonies were 100% cytogenetically normal by FISH and polyclonal by HUMARA in 4 of the 5 samples. The fifth sample, which showed a small number of karyotypically abnormal LTC-derived colonies, nevertheless showed amplification of the "leukemia-associated" AR allele in 46/50 LTC-derived colonies as well as all 40 directly clonogenic cells tested.CONCLUSIONS: Thus in 4 of 5 AML samples tested, both cytogenetics and the HUMARA assay indicate that a substantial number of normal, polyclonal hematopoietic progenitors often persist in AML PB at diagnosis in spite of the predominance of malignant blasts and the severe cytopenias of normal mature blood cells that are typically seen clinically.     

Staging of leukemic progenitor cells in acute myeloid leukemia by phenotyping with myeloid monoclonal antibodies

We studied the immune phenotype of leukemic progenitor cells (AML-CFU-L) in 16 cases of acute myeloid leukemia (AML) using 12 myeloid monoclonal antibodies (McAbs) in a complement-mediated cytotoxicity assay. On the basis of their reactivities with normal day-14 (D14) and day-7 (D7) myeloid progenitor cells (CFU-GM), these McAbs could be classified into three groups: six McAbs reacted strongly with "early" antigens on D14-CFU-GM, two McAbs reacted only with "late" antigens on D7-CFU-GM, while four McAbs formed an "intermediate" group that reacted both with the "late" antigens on D7-CFU-GM and to some extent with the "early" antigens of D14-CFU-GM. The McAbs all reacted with antigens on AML-CFU-L:McAbs that reacted with "early" antigens reacted consistently strongly with AML-CFU-L, in contrast to McAbs in the two other groups, which displayed greater heterogeneity. On the basis of antigenic phenotype, the predominating CFU-L in AML could thus be placed in one of three "stages." This phenotypic "staging" of AML correlated with the French-American-British (FAB) morphological classification of AML. The AML-CFU-L of patients with FAB morphological types M2, M4, and M5 expressed more differentiated antigenic phenotypes than those of the three patients with M7. The latter reacted poorly with the "intermediate" and "late" McAbs. Our data and those of others suggest that classification based on AML-CFU-L antigenic profile may complement the FAB classification of AML.     

Autologous hematopoietic stem cell transplantation in chronic myeloid leukemia with different clinical stages.

Seven patients with Philadelphia (Ph) chromosome-positive chronic myeloid leukemia (CML) were treated with an ICE-based regimen plus G-CSF with the aim of mobilizing and collecting Ph-negative peripheral stem cells in the setting of an autologous transplant program. Five patients had CML in the first chronic phase and 2 in the accelerated phase. All patients had been previously treated with interferon-alpha. Median value and ranges for harvested mononuclear cells, CD34+ cells and CFU-GM, respectively: 5.65 x 10(8)/kg (2.61-11.38); 1.48 x 10(6)/kg (0.216-3.5), and 3.43 x 10(4)/kg (0.243-11.6). FISH was the only useful method for detection of minimal residual disease on apheresis product showing <5% t(9;22) positive cells in 2 cases and <10% positive cells in 4 other cases. Four of seven autologous grafts have been transplanted to date. Busulfan conditioning was used in 1 case and TBI/Cy conditioning in 3 other cases. All patients are alive and well following transplantation and are on interferon-alpha therapy.     

Phenotypic and functional analysis of leukemic cells from 16 patients with adult T-cell leukemia/lymphoma

Surface phenotypes of leukemic cells from 16 patients with adult T-cell leukemia/lymphoma (ATLL) were analyzed by using monoclonal antibodies (anti-Leu-1, anti-Leu-2a, anti-Leu-3a, anti-HLA-DR and MAS 036 c), and the effect of leukemic cells on PWM-induced normal B-cell differentiation was also studied. The majority of ATLL cells bear Leu-1 and Leu-3a antigen on cell surface but lack Leu-2a antigen and were unreactive with MAS 036 c. These results indicate that ATLL cells are of peripheral inducer/helper T-cell origin. However, contrary to the surface phenotype, ATLL cells from 10 patients showed potent suppressor activity on PWM-induced normal B-cell differentiation to immunoglobulin- producing cells (Ig-PC) and no case showed helper activity. The dissociation between surface phenotype and function of ATLL cells is discussed in this article.