Peripheral blood is a source of BCR-ABL-negative pre-progenitors in early chronic phase chronic myeloid leukemia.

Manipulation of autologous bone marrow cells (BM) for transplantation in chronic myeloid leukemia (CML) to enrich for normal cells is a novel approach that may improve survival for patients not suitable for allogeneic transplantation. Limitations of this technique include the reported low frequency of normal stem cells in CML and the difficulties in obtaining sufficient BM for manipulation. To address these problems we compared the apheresis product with the diagnostic bone marrow at diagnosis as a source of primitive BCR/ABL-negative progenitors. We analyzed the CD34+ HLA-DR- and CD34+CD38(-) populations in five CML patients to evaluate the frequency of BCR-ABL-negative progenitors and pre-progenitors in these populations. Progenitor analysis was performed by RT-PCR of individual hemopoietic colonies from a standard CFU-GM assay. Analysis of pre-progenitors involved RT-PCR of secondary colonies derived from a stroma-free pre-CFU assay. Our results show variable levels of BCR-ABL-negative progenitors in the 34+DR- population but very low levels of BCR-ABL-negative progenitors in the 34+38- population in blood. Analysis of pre-progenitors from the 34+DR- fraction of peripheral blood (PB) and BM showed 80-100% and 85-100% of colonies were BCR-ABL negative at days 14 and 28, respectively. Analysis of pre-progenitors from the 34+38- fraction of PB and BM showed 23-100% and 42-100% of colonies were BCR-ABL negative at days 14 and 28, respectively. In summary, pre-progenitors from the 34+DR- and 34+38- populations are predominantly BCR-ABL negative in both marrow and blood at diagnosis. Apheresis product collected at diagnosis is a more abundant sources of BCR-ABL-negative pre-progenitors than BM. Thus, apheresis product could potentially be utilized as a source of BCR-ABL-negative stem cells in CML.

CFU-mix are no better than CFU-GM in predicting hemopoietic reconstitutive capacity of peripheral blood stem cells collected in the very early remission phase of acute nonlymphoblastic leukemia.

High levels of circulating myeloid progenitor cells (CFU-GM) occur during the very early remission phase of acute nonlymphoblastic leukemia (ANLL). Autologous stem cell rescue using blood cells collected during this phase has shown that successful hemopoietic reconstitution can be achieved, but a higher CFU-GM dose appears to be required than when bone marrow cells are used. This suggests that during very early remission, the level of marrow repopulating pluripotent stem cells (PSC) in blood does not undergo the same amount of increase as does the CFU-GM. This study set out to determine whether the levels of the multilineage progenitor cell (CFU-Mix) would be better indicators of the PSC in these cells than the CFU-GM. Serial peripheral blood CFU-Mix and CFU-GM measurements were carried out in six ANLL patients during very early remission. The levels of peripheral blood CFU-Mix showed a mean 12-fold increase, as compared to a mean 20-fold increase in the CFU-GM. The timing of the increase in the CFU-Mix paralleled that of the CFU-GM. These findings suggest that the CFU-Mix is no better than the CFU-GM in predicting PSC levels during very early remission of ANLL, and is closer to the CFU-GM than to the PSC in ontogeny.

Monoclonal antibody BB9 raised against bone marrow stromal cells identifies a cell-surface glycoprotein expressed by primitive human hemopoietic progenitors.

OBJECTIVE: The identification of cell-surface antigens whose expression is limited to primitive hematopoietic progenitor cells (HPC) is of major value in the identification, isolation, and characterization of candidate stem cells in human hemopoietic tissues. Based on the observation that bone marrow stromal cells and primitive HPC share several cell-surface antigens, we sought to generate monoclonal antibodies to HPC by immunization with cultured human stromal cells. METHODS: BALB/c mouse were immunized with human bone marrow (BM)-derived stromal cells. Splenocytes isolated from immunized mice were fused with the NS-1 murine myeloma cell line and resulting hybridomas selected in HAT medium, then screened for reactivity against stromal cells, peripheral blood (PB), and BM cells. RESULTS: A monoclonal antibody (MAb), BB9, was identified based on its binding to stromal cells, a minor subpopulation of mononuclear cells in adult human BM, and corresponding lack of reactivity with leukocytes in PB. BB9 bound to a minor subpopulation of BM CD34(+) cells characterized by high-level CD34 antigen and Thy-1 expression, low-absent expression of CD38, low retention of Rhodamine 123, and quiescent cycle status as evidenced by lack of labeling with Ki67. CD34(+)BB9(+) cells, in contrast to CD34(+)BB9(-) cells, demonstrated a capacity to sustain hematopoiesis in pre-CFU culture stimulated by the combination of IL-3, IL-6, G-CSF, and SCF. BB9 also demonstrated binding to CD34(+) cells from mobilized PB. CONCLUSION: Collectively, these data therefore demonstrate that MAb BB9 identifies an antigen, which is selectively expressed by hierarchically primitive human HPC and also by stromal cells.

Single high doses of cyclophosphamide enable the collection of high numbers of hemopoietic stem cells from the peripheral blood.

We used single high doses of cyclophosphamide (4 g/m2) to produce rebound increases in peripheral blood (PB) stem cells (PBSC) during recovery from myelosuppression, enabling their collection by apheresis for later autotransplantation. Thirty-three courses of cyclophosphamide were given to 30 patients with malignant lymphoma, multiple myeloma, or solid tumors. The neutrophil count was less than 0.5 x 10(9)/liter for a mean of 6.9 days (median 7 days), and fever occurred in 17 of 33 courses. Positive blood cultures occurred in two patients, one of whom died. The mean peak level of PB granulocyte-macrophage colony-forming units (CFU-GM) was 1517 x 10(3)/liter (median 2447 x 10(3)/liter), a 14-fold increase above the mean in normal subjects. The peak occurred at a mean of 16.6 days (median 16 days) after cyclophosphamide, generally coinciding with the time to reach 1.0 x 10(9) neutrophils per liter. Normal or minimally involved bone marrow and a rapid rise in leukocyte count during recovery were independent variables correlated to the peak of the rebound increase in PB CFU-GM levels. Previous chemotherapy and the duration of neutropenia were additional independent variables in the group with peak PB CFU-GM levels of greater than 1000 x 10(3)/liter. The mean total CFU-GM collected after a mean of five aphereses was 43.8 x 10(4)/kg body weight (BW) (median 35.5 x 10(4)/kg BW), significantly correlated with the mononuclear cell yield. We conclude that single 4 g/m2 doses of cyclophosphamide effectively produce high levels of PBSC, particularly but not exclusively in patients with normal or minimally involved bone marrow and who have not had intensive recent chemotherapy.

Identification of homeobox genes expressed in human haemopoietic progenitor cells.

Homeodomain (HD)-containing proteins have been shown to regulate cellular commitment and differentiation in fungal, invertebrate and vertebrate systems. Bone marrow cells synthesizing the CD34 antigen are a complex mix of early, stem and progenitor cells at various stages of commitment to the many haemopoietic lineages. Here, we report the cloning and sequencing of 31 homeobox (HB) sequences, identified using degenerate oligodeoxyribonucleotide primers, in a polymerase chain reaction with cDNA derived from a purified CD34+ population of human haemopoietic cells. Of these sequences, 16 correspond to previously identified genes, and 13 are located within the HOX A, B and C clusters. Ten of the clones most likely represent human homologues of genes identified previously in other species. Five of the clones reported here represent novel HD sequences. The identification of five new genes using a subclass-specific 5' primer, designed from the engrailed and Xanf1 sequences, suggests that there still remain several uncharacterized HB genes in the human genome. Haemopoietic cells purified on the basis of CD34 antigen synthesis are a rich source of regulatory genes consistent with their ability to differentiate into diverse haemopoietic cell types.

A differential sensitivity to recombinant human interferon-alpha 2a between normal and chronic myeloid leukaemic peripheral blood granulocyte-macrophage colony-forming units.

The sensitivity to recombinant human interferon-alpha 2a (IFN) of peripheral blood granulocyte-macrophage colony-forming units (PB CFU-GM) from patients with chronic myeloid leukaemia (CML) was studied in a semi-solid clonogenic assay, and compared with normal PB CFU-GM. Like normal PB CFU-GM, the growth of CML PB CFU-GM in vitro was found to be dependent on the plating concentration used. The optimal CFU-GM growth occurred when CML PB mononuclear cells (MNC) were plated at low concentrations in the range of 0.01-0.1 x 10(5)/ml, compared to the range of 0.3-3.0 x 10(5)/ml optimal for CFU-GM growth in normal subjects. The optimal plating concentration for CML PB CFU-GM was similar to that observed in PB collected from patients with ovarian carcinoma during haematological recovery following chemotherapy-induced myelosuppression (recovery phase). The recovery phase PB was used as a source of non-leukaemic cells with a higher incidence of CFU-GM similar to that of CML. IFN produced a dose-related inhibition of CFU-GM growth in normal, recovery phase ovarian carcinoma and CML, PB MNC. The IFN concentration required to inhibit 50% of the CFU-GM in culture (LD50) was found to be significantly influenced by the plating concentration. When cells were cultured at 1.0 x 10(5) MNC/ml the mean LD50 for 7 CML patients was similar to that in normal (n = 5) or recovery phase (n = 5) peripheral blood, 273 i.u./ml, 1047 i.u./ml and 795 i.u./ml, respectively. In contrast when CML cells were cultured at 0.03 x 10(5) MNC/ml the concentration for optimal CML CFU-GM growth, the mean LD50 was significantly lower than that in normal PB and recovery phase PB, 4 i.u./ml, 251 i.u./ml and 78 i.u./ml, respectively (p less than 0.05). This is the first report of a differential sensitivity to IFN between CML and non-CML progenitors using an optimized PB CFU-GM assay system and proposes that further study of the in vitro culture of CML progenitors may increase our understanding of the clinical effects of IFN.

Evaluation of a monoclonal IgM antibody for purging of bone marrow for autologous transplantation.

A monoclonal antibody of the IgM class, reacting with the CD9 (p24) antigen is described. The antibody (FMC27) is cytotoxic against cells of the common type of acute lymphoblastic leukaemia (c-ALL), giving killing at higher dilutions than an IgG antibody (FMC8) against the same antigen. FMC27 and FMC8 recognise different epitopes, and FMC27 may thus be used in a cocktail together with FMC8 and an antibody against the c-ALL antigen, WM21. Furthermore, the IgM antibody can be coated directly onto magnetic microparticles for magnetic purging, unlike the IgG antibody which must be used in a two-layer procedure.

Peripheral blood stem cells collected in very early remission produce rapid and sustained autologous haemopoietic reconstitution in acute non-lymphoblastic leukaemia.

Haemopoietic reconstitution was achieved in a patient with acute non-lymphoblastic leukaemia (ANLL) in relapse who was autografted with blood-derived stem cells collected during very early remission. The patient received a myeloid progenitor cell dose of 230 x 10(4) CFU-GM/kg body weight. Engraftment was evident in the bone marrow 7 days post-graft. Normal neutrophil and platelet counts were attained by day 14 and blood counts remained normal thereafter. An overshoot in peripheral blood haemopoietic progenitor levels occurred at the end of the second week, presumably the progeny of a family of early progenitor cells. The completeness of haemopoietic reconstitution is further illustrated by the satisfactory nucleated cell and myeloid progenitor cell yield when a bone marrow harvest was performed 4 1/2 months post-graft. Seven months post-graft, the patient remained in complete remission with normal blood counts and bone marrow cellularity, although haemopoietic progenitor levels were slightly reduced. The rapid recovery minimises aplasia-related risks and suggests that such autografting can be carried out safely in first remission. We propose that autografting using very early remission blood cells is a new therapeutic option for patients with acute ANLL.

An unusual pattern of hemopoietic reconstitution in patients with acute myeloid leukemia transplanted with autologous recovery phase peripheral blood.

Fourteen patients with acute myeloid leukemia (AML) were autotransplanted with peripheral blood cells collected during early remission. Seven were autotransplanted in first relapse and seven in first remission. They received a median of 3.3 X 10(8) nucleated cells/kg body weight (BW) and 92 X 10(4) myeloid progenitor cell (CFU-GM) per kg BW. Rapid hemopoietic reconstitution (HR) occurred in all patients with median time to reach normal neutrophil and platelet counts 13 and 18 days post re-infusion respectively. However, in three patients neutrophil counts fell to less than 1.0 x 10(9)/l and in seven patients platelet counts fell to less than 25 x 10(9)/l between 26 and 40 days post-transplant (trough count). In all but two patients who received the lowest CFU-GM dose the counts returned to normal or near normal levels (steady count). There were significant correlations between the CFU-GM dose and the trough and the steady platelet counts (p = 0.04 and 0.01 respectively). Patients receiving more than 50 x 10(4) CFU-GM/kg BW had higher steady neutrophil and platelet counts (p = 0.011 and 0.033 respectively) although some patients receiving greater than 50 x 10(4) CFU-GM/kg still experienced thrombocytopenia during the second month post graft. There was no significant correlation between the nucleated cell dose and HR. The cause of the fall in platelet and neutrophil counts in the second month post graft is not clear but is probably a reflection of a proliferative defect in the recovery phase stem cells in AML.

The optimization of collection of peripheral blood stem cells for autotransplantation in acute myeloid leukaemia.

Between November 1982 and November 1986 31 patients with acute myeloid leukaemia underwent peripheral blood stem cell apheresis during haemopoietic regeneration following induction chemotherapy. A retrospective analysis of the factors affecting the efficacy of stem cell harvest and of the clinical outcome of these patients was performed. The mean number of myeloid progenitor cells (CFU-GM) collected was significantly higher in the complete remission group (n = 22) than in the partial remission group (n = 9). Fifty x 10(4) CFU-GM/kg body weight or more, which produced rapid, complete and sustained haemopoietic reconstitution after autografting in our patients, were collected from six of nine patients who underwent three or four 7-litre aphereses over 5-7 days using a lymphocyte collection procedure on the Fenwal CS3000 [Protocol B] but only from two of 12 patients who underwent three or four 5-litre aphereses over 3-5 days using the Aminco Celltrifuge [Protocol A] (p less than 0.05). No adverse effects on the rates of neutrophil, platelet and lymphocyte recovery after induction chemotherapy or on long-term disease-free survival for patients who achieved a complete remission could be attributed to apheresis when compared with a historical control group of 39 patients who achieved complete remission following the same induction chemotherapy but did not undergo apheresis. We conclude that sufficient numbers of peripheral blood stem cells to produce safe and rapid haemopoietic reconstitution can be collected from most patients who achieve complete remission using apheresis Protocol B without impairment of haemopoietic recovery or adversely affecting the length of complete remission.

Comparison of haematological recovery times and supportive care requirements of autologous recovery phase peripheral blood stem cell transplants, autologous bone marrow transplants and allogeneic bone marrow transplants.

The haematological recovery time, infection rate and supportive care requirements of patients receiving recovery phase autologous peripheral blood stem cell transplants (APBSCT) (n = 38), autologous bone marrow transplants (autoBMT) (n = 13) and allogeneic bone marrow transplants (alloBMT) (n = 14) were compared with respect to the time post-transplant to reach 0.1, 0.5 and 2.0 x 10(9) neutrophils/l and 50 and 150 x 10(9) platelets/l, the length of hospitalization, fever and antibiotic use, the incidence of documented infection and the number of red cell and platelet transfusions. The APBSCT group had a significantly more rapid recovery of neutrophils and platelets and their supportive care requirements were significantly less than the autoBMT and the alloBMT groups. There was no difference between the latter two groups. The most significant variables contributing to the differences in haematological recovery times were the granulocyte-macrophage progenitor (CFU-GM) dose infused and, to a lesser extent, patient age. The APBSCT group received a higher CFU-GM dose of 87 +/- 12 x 10(4)/kg BW compared with 12 +/- 5 and 17 +/- 3 x 10(4)/kg BW in the autoBMT and the alloBMT groups, respectively (p = 0.0001). Patient age showed a negative correlation with the rate of recovery because the APBSCT group, which recovered faster was also older (48 +/- 2 years, compared with 33 +/- 3 and 31 +/- 2, respectively, p = 0.0001). On multivariate analysis, CFU-GM dose was the only variable to show a significant correlation with all the haematological recovery endpoints studied in these 65 patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Mucin-like molecules as modulators of the survival and proliferation of primitive hematopoietic cells.

Current data suggest that interplay between two classes of molecules contributes to the regulation of hematopoiesis: hematopoietic growth factors, which regulate the survival, proliferation, and development of primitive hematopoietic cells and cell adhesion molecules (CAMs), which are responsible for the localization of hematopoiesis to the bone marrow (BM) and for mediating physical association between developing hematopoietic cells and marrow stromal tissue. A range of cell surface molecules representing several CAM superfamilies including integrins, selectins, the immunoglobulin gene superfamily and an emerging family of mucin-like molecules (the sialomucins) are involved in supporting cell-cell and cell-extracellular matrix (ECM) interactions between primitive hematopoietic cells and the stromal cell-mediated hematopoietic microenvironment (HM) of the bone marrow. There is abundant evidence in non-hematopoietic tissues that CAMs are signalling molecules which participate in a range of signal transduction events important not only for regulating cell adhesion and motility, but also for cell growth and survival. Although the signalling functions of CAMs have not been studied extensively in primitive hematopoietic progenitors (HPCs), extrapolation from burgeoning data in other systems is consistent with the hypothesis that hematopoiesis within the BM is regulated by interaction between signals generated locally by CAMs and those elicited by cytokines. Evidence in support of this notion was initially provided by studies on normal HPCs demonstrating cross-talk between members of the integrin superfamily and cytokine receptors. In this article we review recent reports that mucin-like molecules are also signalling molecules on primitive hematopoietic cells and that the signals they deliver potently inhibit hematopoiesis.

Detection of minimal residual disease in an AML patient with trisomy 8 using interphase fish.

Patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) often exhibit clonal chromosomal abnormalities. Using a probe for the centromeric region of chromosome 8, fluorescence in situ hybridization (FISH) on interphase cells was used to detect trisomy 8 in an AML patient whose leukemia was characterised by the karyotype 47, XY, +8, del(9) (q21.1q32). We have demonstrated using FISH the presence of the trisomy at all stages of the patient's disease course (including remission, peripheral blood cell harvest and relapse), whereas conventional karyoptypic analysis was only able to detect the trisomy at diagnosis and clinical relapse. We have also shown using immunophenotyping, cell sorting and FISH, that the trisomic cells in this patient were restricted to the CD34+ subset of blood and bone marrow and could not be found in the CD 34-, T or B cell compartment. Overall we have shown FISH to be a rapid, quantitative method for the detection of cells with numerical chromosome abnormalities. FISH analysis of interphase cells provides valuable information on the status of the whole population, rather than just cycling cells, and can be applied successfully to monitor the level of leukemic cells.

Standardization of monoclonal antibodies for use in autologous bone marrow transplantation for common acute lymphoblastic leukemia.

Two monoclonal antibodies suitable for leukemia cell purging of remission bone marrow from patients with common acute lymphoblastic leukemia (common-ALL) are described. WM-21, reacting with the gp 100 common-ALL associated antigen (CALLA), and FMC-8, reactive with a p24 surface antigen, both bind to the majority of leukemic blast cells from cases of common ALL, and promote complement-mediated lysis of CALLA+ leukemias and cell lines. After initial dye exclusion studies to standardize antibody and rabbit complement concentrations and incubation times, an in vitro plating assay using CALLA+ p24+ cell lines was used to investigate the lytic ability of monoclonal antibody treatment. Incubation with WM-21, FMC-8, and complement produced up to 5 logs inhibition of growth in this system. Under similar conditions, no inhibition of in vitro growth of normal bone marrow myeloid progenitor cells was seen. These antibodies therefore appear to be useful therapeutic reagents for removing residual common ALL blast cells from bone marrow prior to autologous marrow transplantation.

The use of the APAAP technique as a rapid indicator of peripheral blood progenitor cell levels.

Rapid and sustained engraftment following autotransplantation with peripheral blood stem cells (PBSC) depends on adequate numbers of stem cells and progenitor cells. In this study we have compared the number of myeloid progenitor cells quantitated using the colony forming units-granulocyte macrophage (CFU-GM) clonogenic assay with the number of CD34+ cells estimated both by flow cytometry and by the alkaline phosphatase anti-alkaline phosphatase (APAAP) technique. We have analysed 15 peripheral blood mononuclear cells (PBMNC) samples from 13 normal subjects and 179 PBMNC from 32 patients undergoing PBSC harvests during the recovery phase of high dose cyclophosphamide chemotheraphy. The number of CD34+ cells measured by the APAAP technique correlated well with the number of CD34+ cells measured by flow cytometry (r = 0.727, p = 0.0001), and also with the number of CFU-GM measured in the clonogenic assay (r = 0.721, p = 0.0001). The APAAP method provides a rapid, reliable measure of progenitor cell levels that can be used to monitor the optimal time to harvest peripheral blood stem cells (PBSC), and to estimate the marrow repopulating ability (MRA) of stem cell preparations used for transplantation.