Effect of mafosfamide (ASTA-Z-7654) on the clonogenic cells in precursor-B acute lymphoblastic leukaemia: significance for ex vivo purging of bone marrow for autologous transplantation.

Lymphoblasts from 11 patients with acute lymphoblastic leukaemia (ALL) of precursor-B type were exposed to the cyclophosphamide derivative mafosfamide (ASTA-Z-7654), and examined for growth inhibition using an in vitro colony assay. Leukaemic clonogenic cells were significantly more resistant to this cytotoxic drug (mean IC50 29.2 micrograms/ml, IC90 64.8 micrograms/ml) compared with myeloid progenitors from seven normal bone marrow samples (mean IC50 9.0, IC90 19.9) (p less than 0.0001). This effect was most pronounced in the four previously treated cases examined (mean IC90 84.4 micrograms/ml). The implications of these findings for bone marrow purging with ASTA-Z in patients with ALL for autologous marrow transplantation are discussed.

Effects of recombinant human cytokines on precursor-B acute lymphoblastic leukemia cells.

An in vitro colony assay was used to examine the growth stimulatory effects of a variety of recombinant human lymphohemopoietic cytokines on human precursor-B acute lymphoblastic leukemia (ALL) cells. Of 23 cases evaluated, 16 formed significant numbers of colonies (mean 280, range 36-939) when cultured in 10% fetal calf serum in 0.8% methylcellulose containing 10% partially purified B-cell growth factor (BCGF). Immunoperoxidase staining of cells from cultures confirmed a precursor-B phenotype (HLA-DR+, CD-10+, CD-19+, CD-34+, Ig-, CD-3-, CD-11C-). When these cases were cultured with recombinant human cytokines (but without BCGF) only a minority showed colony formation, in all instances less than seen with BCGF. Three cases were stimulated both by interleukin 3 (IL-3) and the putative pre-B growth factor interleukin 7 (IL-7). One case was stimulated both by tumor necrosis factor alpha and by interleukin 6 (IL-6); these results were confirmed on highly purified CD-10+, CD-19+ cells prepared by fluorescence-activated cell sorting. A further case was stimulated by granulocyte-macrophage colony-stimulating factor (GM-CSF), including CD-10+, CD-19+ purified cells. All cases responding to recombinant cytokines were heavily pretreated patients at relapse, whereas none of the newly diagnosed untreated cases showed any response. These results confirm that activities present in BCGF are the major stimulant for precursor-B ALL proliferation in vitro. None of the recombinant cytokines examined, including IL-7, appeared to have consistent activity under these culture conditions. The molecules regulating growth of ALL remain to be more precisely defined.

Effects of interleukin 7 on the growth of clonogenic cells in T-cell acute lymphoblastic leukaemia.

The effects of the recombinant human cytokines interleukin 2 (IL-2) and IL-7 on the proliferation of T-acute lymphoblastic leukaemia (T-ALL) cells were tested in a clonogenic assay. Highly purified leukaemic cells were obtained by immunomagnetic depletion of mature T cells and fluorescence-activated cell sorting for immature leucocyte markers (CD1, CD10, CD34). Of 9 cases tested, only 3 showed evidence of stimulation by cytokines. One was stimulated by both IL-2 and IL-7, one by IL-2 only, and the third by IL-7 alone. A further case showed proliferation without addition of cytokines. The remaining 5 cases were completely unresponsive. While both IL-2 and IL-7 are capable of stimulating leukaemic cells from some cases of T-ALL, the molecules regulating the proliferation of T-ALL cells in vitro remain to be more fully elucidated.

Autologous bone marrow transplantation for acute myeloid leukaemia in remission: a preliminary report.

Autologous bone marrow transplantation, using unpurged cryopreserved autologous marrow, was performed on ten adult patients with acute myeloid leukaemia in remission. Seven patients were in first chemotherapy-induced remission of their disease, while three were in later remission. Patients ages ranged from 24 to 52 years, with a median of 38.5 years. Conditioning therapy consisted of oral busulphan 16 mg/kg over four days and intravenous cyclophosphamide 60 mg/kg on two days. Bone marrow cells were thawed and infused two days later. All patients showed signs of marrow engraftment, however this was delayed in comparison with patients receiving allogeneic transplants. All patients developed fever requiring antibiotic therapy and one patient died of overwhelming sepsis. Another patient died of hepatic veno-occlusive disease two months after transplant. Serious, but non-fatal, hepatic complications occurred in two other patients. One patient, transplanted in third remission, relapsed 16 months post-autograft. No other relapses have been seen, with one second remission patient remaining leukaemia-free at 24 months, and six first remission patients in continuing remission 11 to 23 (median 20) months post transplant. These encouraging results require confirmation in a randomised clinical trial comparing autologous marrow transplantation versus standard chemotherapy.

Lack of correlation between immunoglobulin and T cell receptor gene rearrangements and TdT expression in acute myeloid leukaemia.

Thirty-two cases of acute myeloid leukaemia (AML) were examined for expression of terminal deoxynucleotidyl transferase (TdT) and rearrangements of the genes coding for the immunoglobulin heavy chain and the beta chain of the T cell receptor, in order to establish whether these two forms of lineage infidelity are linked. In 17 cases of AML with greater than or equal to 10% TdT+ cells, three cases showed evidence of gene rearrangement, two having clonal rearrangements in the immunoglobulin gene and one with a rearranged T cell receptor gene. Among 15 AML cases without significant numbers of TdT-positive blasts, three cases had rearrangements in both immunoglobulin and T cell receptor genes, while a fourth case had an immunoglobulin gene rearrangement. No relationship was seen between lymphoid gene rearrangements and expression of the lymphoid surface antigens CD7 and CD10. The lack of association between TdT expression and gene rearrangements does not support the concept of an orderly activation of the recombinase machinery in those cases of AML with features of early lymphoid differentiation.

A variant of Glanzmann's thrombasthenia characterized by abnormal glycoprotein IIb/IIIa complex formation.

Glanzmann's thrombasthenia is a congenital bleeding abnormality characterized by absent platelet aggregation due to the failure of fibrinogen to bind to activated thrombasthenic platelets. In the majority of cases, this defect is caused by the absence or marked reduction of a specific fibrinogen-binding aggregation receptor, the GP IIb/IIIa complex. E.T., an 18-year-old female with a life-long history of bleeding and easy bruising, had the normal clinical features of Glanzmann's thrombasthenia. Surprisingly, sodium dodecyl sulphate-polyacrylamide gel electrophoresis of her platelets showed no apparent abnormality of the GP IIb/IIIa complex. Control platelets washed in the presence of 2 mM EDTA and control and patient platelets washed in the presence of 2 mM calcium ions showed normal reactivity with anti-GP IIb, anti-GP IIIa, and anti-GP IIb/IIIa complex specific monoclonal antibodies as evaluated by flow cytometry. In contrast, patient's platelets washed in the presence of 2 mM EDTA reacted with anti-GP IIb, anti-GP IIIa, but not with the complex-specific monoclonal antibodies. The increased susceptibility of the patient's GP IIb/IIIa complex to EDTA dissociation was confirmed by crossed immunoelectrophoresis (CIE). CIE analysis further indicated that the patient's GP IIb/IIIa complex did not bind fibrinogen. The combined results suggest that this patient has Glanzmann's thrombasthenia due to an abnormal association of the GP IIb/IIIa complex which results in the failure of the complex to bind fibrinogen.

Immunophenotypic analysis of clonogenic cells in acute lymphoblastic leukemia using an in vitro colony assay.

A culture system was used to analyze the expression of membrane differentiation antigens on the proliferative or clonogenic fraction of cells from cases of common (precursor B) acute lymphoblastic leukemia (c-ALL). Colonies of leukemic cells were obtained in 18 of 20 cases after 1 week in culture in a liquid layer containing recombinant interleukin-2 (IL-2), phytohemagglutinin (PHA), and B-cell growth factor over an agar feeder layer containing irradiated peripheral blood mononuclear cells plus fetal calf serum (FCS) and horse serum. Cultured cells expressed HLA-DR, CD-9, CD-10, CD-20, and CD-34 antigens, indicating conservation of precursor B phenotype. Differentiation antigen expression on the clonogenic subpopulation giving rise to leukemic colonies was assessed by treating cells prior to culture with selected cytotoxic monoclonal antibodies (MoAbs) and complement or by fluorescence-activated cell sorting. Lytic treatment with HLA-DR, CD-10, CD-9, and CD-20 antibodies produced median reductions in colony formation of 93%, 81%, 73%, and 58% respectively. Combined treatment with CD-9 and CD-10 antibodies produced complete inhibition in 11 of 13 cases. Cell-sorting experiments after CD-10 staining indicated a good correlation with complement lysis studies and also demonstrated that the CD-19 antigen is expressed on a high proportion of clonogenic cells. Colony formation was also significantly reduced in 13 of 17 cases by lytic treatment of cells with the CD-33 antibody MY-9, suggesting expression of this "myeloid" lineage antigen on ALL clonogenic cells. The substantial case-to-case variation in expression of individual differentiation antigens indicates that leukemic cellular heterogeneity is a major consideration in ex vivo bone marrow purging using MoAbs and emphasizes the need for more critical evaluation of purging techniques.

Unusual immunophenotypes in acute leukaemias: incidence and clinical correlations.

The incidence and clinical implications of unusual patterns of expression of leucocyte differentiation antigens in acute leukaemia were assessed on 568 newly diagnosed paediatric and adult cases undergoing immunophenotyping with a panel of monoclonal antibodies at a single centre. Among patients with the precursor B (common) form of acute lymphoblastic leukaemia (ALL), the major variant seen was the group of 15 cases with expression of myeloid surface antigens. 4.5% of ALL cases tested with antibody to CD-11b were positive, 5.1% were CD-13+, and 10.8% CD-33+. All 15 patients achieved a complete remission with chemotherapy, with six of eight children and four of seven adults remaining disease free. A smaller proportion (1.5%) of precursor B ALL patients showed expression of the T lineage marker, CD-7. The only significant variant seen in the precursor T-ALL group was expression of HLA-DR antigen, which was found in five of 35 cases; although all responded to treatment, only one remains a disease-free survivor. Among patients with acute myeloid leukaemia (AML), expression of the lymphoid markers terminal transferase (TdT) and CD-7 were commonly seen (22.2% and 28.4% respectively of cases tested). Other lymphoid markers detected on AML cases were CD2 (11.1%), CD-10 (1%) and CD-19 (4.4%). These results confirm that examples of lineage infidelity are regularly seen in large series of patients with acute leukaemia. Prospective studies using uniform treatment protocols are required to establish whether these patients have significantly different disease outcomes.

Expression of a novel human pan leucocyte differentiation antigen on leukaemic cells.

A murine monoclonal antibody has been produced which identifies a novel surface membrane antigen present on virtually all normal human leucocytes and leukaemic cells. The antibody, designated WM-65, reacted with over 95% of peripheral blood, tonsil and thymic lymphocytes, and with a similar proportion of monocytes and granulocytes. A majority of nucleated normal bone marrow cells were also reactive with WM-65; however, these included only a small proportion of myeloid progenitor cells. WM-65 reacted with a wide range of acute and chronic leukaemias of both myeloid and lymphoid types, and with corresponding cell lines, but did not react with non-haemopoietic cells. By immunoprecipitation and SDS-PAGE, WM-65 identifies a heavily glycosylated surface protein of molecular weight between 40 and 50 kD. This property, and the broad non-lineage-specific distribution of the antigen on haemopoietically-derived cells, indicates that WM-65 is different from other monoclonal antibodies with "leucocyte common" reactivity patterns. The extensive reactivity of WM-65 with leukaemic cells raises the possibility of therapeutic applications of the antibody in haematological malignancies.

Further characterization of human myeloid antigens (gp160,95; gp150; gp67): investigation of epitopic heterogeneity and non-haemopoietic distribution using panels of monoclonal antibodies belonging to CD-11b, CD-13 and CD-33.

We have investigated the binding of over 30 different monoclonal antibodies (MAB) belonging to three distinct clusters of differentiation (CD-11b; CD-13; CD-33; as defined by the Third International Workshop on Leucocyte Differentiation Antigens (ILWS), 1986), and which are reactive with three distinct myeloid restricted surface antigens ('gp160,95'; 'gp150'; 'gp67'). By investigating reactivity with non-haemopoietic cells, we have confirmed that CD-11b and CD-33 MAB reactivity is largely restricted to haemopoietic cells, whilst CD-13 MAB showed additional binding to a wide range of non-haemopoietic cells. Epitopic heterogeneity was also investigated within each cluster of differentiation. Tested anti-CR3 (CD-11b) MAB varied in their ability to block the binding of complement coated sheep red blood cells and zymosan particles. A more detailed analysis of MAB binding heterogeneity was performed by competitive inhibition assays. It was demonstrated that MAB from both CD-11b and CD-13 bind to several distinct epitopes (at least six and five respectively) on their respective antigen molecules. In contrast, CD-33 MAB appear to bind to only a single site on 'gp67'. These data may allow for a clearer appreciation of the disparate functional effects obtained using different MAB reagents to individual myeloid antigens, as reported by a number of workers.

An acquired Bernard-Soulier-like platelet defect associated with juvenile myelodysplastic syndrome.

Bernard-Soulier syndrome is an inherited bleeding abnormality characterized by thrombocytopenia with large platelets and deficiency of the platelet membrane glycoprotein (GP) Ib-IX complex. We have identified a young female with an acquired Bernard-Soulier-like platelet defect and a coexisting primary myelodysplastic disorder. Abnormal bruising had developed at age 5. A normal platelet count with some giant platelets was noted at age 7. At age 9 she developed a large haematoma following surgery. Laboratory investigation revealed thrombocytopenia and large platelets. Platelet membrane glycoprotein analysis showed a marked deficiency of the components of the GP Ib-IX complex (approximately equal to 25% of normal). Flow cytometry revealed two populations of platelets: a predominant population of large platelets lacking the GP Ib-IX complex and a minor population of normal-sized platelets with normal GP Ib-IX expression. The patient developed progressive anaemia, more severe thrombocytopenia and neutropenia, and circulating blast cells were seen. A bone marrow showed gross hypercellularity with marked dysplasia of all three lineages and increased blasts. Marrow cytogenetic studies showed the presence of monosomy 7 in all metaphases, with an additional trisomy 21 in 10%. Peripheral blood cells were normal 46XX. The above data are consistent with an acquired myelodysplastic syndrome associated with a Bernard-Soulier-like platelet defect.

Immunophenotype of clonogenic cells in myeloid leukaemia.

Leukaemic clonogenic cells, capable of forming colonies of blast cells in an in-vitro assay, were examined for surface antigen expression using a panel of monoclonal antibodies (Mabs) to stem cell and myeloid differentiation antigens in nine cases of acute myeloid leukaemia (AML) and four cases of chronic myeloid leukaemia in myeloid blast crisis (CML-MBC). Clonogenic cells were found to be most frequently positive with anti-HLA-DR (positive in 100% cases) and RFB-1 (71%) Mabs, with significant reactivity also being seen with CD-33 (69%) and CD-13 (61%) myeloid specific antibodies. CD-11b and CD-15 antigens, expressed predominantly on mature leucocytes, were not significantly expressed on the clonogenic population. Interestingly, the CD-34 antigen, detected by MY-10 Mab on normal myeloid progenitor cells, was demonstrated on the clonogenic fraction of only one of seven cases tested. A discrepancy between antigen expression of clonogenic cells and immunophenotype of the total leukaemic population was frequently seen, with "early" markers (CD-33, HLA-DR, RFB-1) expressed on a higher proportion of the clonogenic fraction than the overall population, while the converse was the case for the "later" marker, CD-11b. Based on the known normal distribution of differentiation antigens, particularly the CD-13 antigen, cases could be ranked according to clonogenic phenotype into immature (CD-13- HLA-DR+ CD-33+ or CD-33-; five cases), and mature (CD-13+ HLA-DR+ CD-33+; eight cases), levels. However, there was no correlation between these maturation levels and the morphology according to the FAB classification. Of note, the mature group included three CML-MBC, as well as two AML cases with a history of myelodysplasia or myeloproliferative disorder. These immunophenotypic findings indicate a heterogeneity in the level of maturation of the clonogenic population, not only in cases of de-novo AML, but also in AML thought to derive from multipotential stem cells.

Characterization of monoclonal antibodies to the human myeloid-differentiation antigen, 'gp67' (CD-33).

This report describes the production and characterization of two new monoclonal antibodies (MoAb), WM-53 and WM-54, reacting with a human myeloid differentiation antigen, which has recently been assigned by the Third International Leucocyte Workshop on Human Differentiation Antigens into cluster CD-33 ('gp67'). To date, only three other MoAb (MY-9, L1B2, L4F3) have been reported to react with this antigen. In peripheral blood, WM-53 and WM-54 were found to bind to monocytes, but failed to react with erythrocytes, platelets and lymphoid cells. WM-54 was also faintly reactive with granulocytes. Myeloid 'specificity' was also observed with leukaemias as both WM-53 and WM-54 were reactive with most cases of acute myeloid leukaemia (AML), but with only a minority of lymphoid leukaemias. Fluorescence activated cell sorting of normal bone marrow cells demonstrated that both MoAb bound to the majority of CFUgm and CFUmix progenitor cells. Immunoprecipitation studies confirmed that these MoAb, in parallel with MY-9, bound to a protein of approximately 70 KD molecular weight, together with a previously undescribed higher molecular weight component of approximately 140 KD on non-reduced gels, possibly representing a disulphide linked dimer of the lower molecular weight protein. Competitive binding assays, using MoAb WM-53 and WM-54 as well as MY-9, L1B2, and L4F3, demonstrated that all five CD-33 MoAb are capable of competing with each other for binding onto HL-60 cells, suggesting that all recognize a single epitopic site on 'gp67'.

Transplantation of monoclonal antibody-purged autologous bone marrow for treatment of poor risk common acute lymphoblastic leukemia.

Two murine monoclonal antibodies, FMC-8 and WM-21, reactive with the human leucocyte differentiation antigens CD-9 (p-24) and CD-10 (CALLA), respectively, have been used for purging leukemic cells from remission bone marrow. Nine patients with the common variant of acute lymphoblastic leukemia (c-ALL) in second or subsequent remission underwent bone marrow harvesting. Bone marrow mononuclear cells underwent lytic incubation in vitro with antibodies FMC-8 and WM-21, and rabbit serum as a source of complement, and were then cryopreserved. A mean of 0.90 +/- 0.50 x 10(8) nucleated cells per kilogram of recipient body weight remained after treatment, with 0.38 +/- 0.24 x 10(8) nucleated cells and 8.3 +/- 10.3 x 10(4) CFUGM per kg being recovered on thawing. Seven patients subsequently received marrow-ablative treatment with high dose cyclophosphamide (120 mg/kg) and fractionated total body irradiation (12 Gy), followed by infusion of antibody-purged autologous bone marrow. Three deaths due to sepsis occurred within the first 35 days, compounded in one patient by poor marrow engraftment. All other patients engrafted promptly, and four remain in continuous complete remission at 2, 6, 9, and 28 months after transplantation. The procedure carries a substantial risk of early toxicity, but offers a significant chance of prolonged unmaintained remission to selected patients with poor prognosis acute lymphoblastic leukemia.

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.

Coexpression of p165 myeloid surface antigen and terminal deoxynucleotidyl transferase: a comparison of acute myeloid leukaemia and normal bone marrow cells.

A double immunofluorescence technique, using antibodies to terminal transferase (TdT) and a 165-kilodalton myeloid differentiation antigen (p165), has been used to investigate the phenomenon of TdT expression in cases of acute myeloid leukaemia (AML). Five cases of AML were shown to have significant (18-90%) numbers of leukaemic cells that concurrently expressed both TdT and p165 myeloid surface antigen. Examination of nonleukaemic bone marrow cells showed that the vast majority of normal TdT+ cells are p165 negative. However, in 5 of the 11 samples analyzed, rare cells staining for both p165 and TdT were found. These results suggest that some cases of TdT+ AML may arise from the clonal expansion of rare "biphenotypic" precursor cells existing in normal bone marrow.

Monoclonal antibody analysis of canine hemopoietic cells. Role of Ia-like and Thy-1 antigens in bone marrow engraftment.

The expression of Ia-like (class II MHC) antigens on canine hemopoietic cells was investigated using a cytotoxic murine monoclonal antibody, WM-2, reactive with dog Ia-like antigens. Another monoclonal antibody, WMD-1, reactive with canine Thy-1 antigen, was used as a positive control. Depletion of Ia+ cells from dog bone marrow by complement-mediated lysis with WM-2 antibody failed to inhibit growth of granulocyte-macrophage progenitors (CFUGM) in vitro, while WMD-1 produced complete inhibition of CFUGM. Lethally irradiated dogs receiving bone marrow autografts depleted of Ia-positive cells ex vivo showed initial engraftment, followed by prolonged pancytopenia, and eventual complete recovery of marrow function in the majority of animals. In contrast, dogs receiving autografts treated with WMD-1 and complement all died of marrow failure. We interpret these results as indicating: (1) that Thy-1 antigen is present on hemopoietic stem cells essential for marrow engraftment; and (2) that the expression of Ia antigens on hemopoietic cells is heterogeneous and related to the level of stem cell maturation. While Ia appears to be present on a stem cell population at an earlier stage than CFUGM, as evidenced by the transient phase of graft failure seen in dogs receiving Ia-depleted marrow, the most primitive stem cell, responsible for long-term engraftment, is effectively Ia-negative.

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.