Acute myeloid leukemia (AML) can be oligoclonal.

Acute myeloid leukemia (AML) is usually thought to represent a monoclonal disease. Among 45 cases of newly diagnosed AML we found rearranged bands in 14 cases using the Southern blot methodology and immunoglobulin (Ig) joining region (JH) or T-cell receptor (TCR beta) probes. In three patients, the findings indicated an oligoclonal disease. One case was characterized by several bands in the JH blot, some of which reappeared at different time points during remission. A second case had monoclonally rearranged Ig-JH sequences in the bone marrow but exclusively germline configuration in DNA from peripheral blood cells despite the presence of 84% blasts. A third case was characterized by two different, Ig-JH and c mu gene rearranged cell populations at diagnosis but relapsed with a germline pattern without reappearance of the previous clones. These data indicate that AML may differentiate along different lineages with predominant appearance of one or the other subclone in the course of the disease.

Detection of minimal residual disease in acute myeloid leukemia.

The distinction of clonogenic leukemic cells (CFU-L) and normal myeloid progenitors (GM-CFU) is a problem because both types of cells respond to the same growth factors and their clones resemble each other morphologically in culture. We investigated by means of an indirect enzyme-immunoassay the expression of "early" and "late" differentiation markers on bone marrow cell suspensions, as well as on agar clones in 18 cases of newly diagnosed acute myeloid leukemia (AML) as compared with 13 normal controls. Uncultured AML cells carried only low amounts of "late" myeloid differentiation antigen (CD15) but expressed nearly normal levels when cultured in agar with colony-stimulating factor (CSF). In contrast to normal bone marrow, AML cells were strongly reactive with "early" differentiation markers (CD10, CD20, CD34) and remained so during culture. Normal and leukemic agar clones could be specifically distinguished by CD20- and CD34 antibodies. By means of a double marker technique, it could be shown that "late" myeloid differentiation markers (CD15) and "early" markers (CD10, CD20, CD34) were coexpressed on the same cells only in AML but not in normal bone marrow. Leukemic clones were identified by phenotyping of agar clones in 17 of 19 cases investigated during complete clinical remission (CR) of the disease. A formal proof of the leukemic origin of CD20/CD34 positive clones grown in CR was made possible in four cases either by Southern blot analysis or by a cytogenetic marker. These results demonstrate that AML cells can partially differentiate in vitro in the presence of CSF. A distinction of AML from normal clones, however, is possible by their reactivity with "early" differentiation markers, because this is maintained under the differentiating influence of CSF. The technique described here identifies residual leukemic clones in the majority of AML in CR, which persist at a constant rate and increase 6 months before cytological relapse.

Clonality analysis as a tool to study the biology and response to therapy in myelodysplastic syndromes.

We examined the bone marrow of 45 patients with MDS at the time of diagnosis and in the course of the disease by means of Southern blot analysis and cytogenetic studies to detect and evaluate clonal markers and their implication on the prognosis of the disease and the response to treatment. All patients were enrolled in an EORTC study and received low-dose Ara-C with or without growth factors according to the study protocol. Thirty patients (67%) were characterized by different clonal markers, such as various gene rearrangements (eg Ig-JH, tcR-beta, bcr, GM-CSF, G-CSF or IL-3) and/or chromosomal markers at the time of diagnosis or early in the course of the disease. In 23 of 30 cases that could be studied in the course of the disease, a statement about the clonal situation was possible: in three cases (8%) the clonal situation did not change, in nine cases (39%) at least a transient reduction of clonal cells could be demonstrated, suggesting partial or complete response to therapy. In eight cases (35%) a change for the worse could be seen. In four cases (17%) involvement of multiple clones could be demonstrated with the clones exhibiting different susceptibilities to treatment. Clinical evaluation showed that patients without clonal markers at diagnosis had a better prognosis as compared to patients who presented with clonal markers. We suggest that clonality analysis at diagnosis and in the course of the disease will be a useful tool to study the biology and response to treatment in MDS.

A randomized phase II study of low-dose cytosine arabinoside (LD-AraC) plus granulocyte-macrophage colony-stimulating factor (rhGM-CSF) in myelodysplastic syndromes (MDS) with a high risk of developing leukemia. EORTC Leukemia Cooperative Group.

In a randomized phase II study, patients with myelodysplastic syndromes (MDS) with 10-30% blasts in the bone marrow and hematopoietic failure were treated with low-dose Ara C (2 x 10 mg/m2 subcutaneously (s.c.) days 1-14) and rhGM-CSF (fully glycosylated, Sandoz/Schering-Plough, 2 x 150 micrograms protein/day s.c.) given either following Ara C (days 15-21) or simultaneously (days 8-14) for 1-5 cycles. 108 patients with a median age of 65 years, range 17-80 years and refractory anemia with an excess of blasts (RAEB, n = 54), RAEB with transformation (RAEBt, n = 50) or with chronic myelomonocytic leukemia (CMML, n = 4) were evaluable. Complete remission was achieved in 15 cases (14%), 11 had a partial response (10%), and 16 a minor response (15%). Stable disease was reached in 35 cases (32%). There were 16 cases of toxic death (15%), progression occurred in 15 patients (14%). No differences existed between the two treatment arms with respect to response and duration of response. Prognostic factors for poor response included the presence of cytogenetic abnormalities and a history of previous blood transfusions. Major adverse events during treatment were hemorrhage (55%), infections (54%), and fever associated with GM-CSF administration (40%). The overall response rate ws 39%, median duration was 12.5 months from start of treatment which allowed responding patients to lead good quality life without further therapy. The question whether the combination is indeed superior to LD-Ara C alone is not settled but will be evaluated in an ongoing clinical trial.

Immunological phenotyping in situ of myeloid colonies in agar cultures.

A technique to immunologically stain hematopoietic colonies in agar cultures would make the analysis of clonal aggregates in situ possible without destroying their architecture and avoid the necessity of picking and washing the cells from semi-solid media. A method is described employing mild glutaraldehyde fixation of agar cultures grown directly on polysiloxane-coated slides. This procedure provided agar plugs which were thin enough to allow the complete penetration of reagents without any loss in plating efficiency. Incubation with primary antibodies for 1.5 h and final development in an indirect enzyme immunoassay using an alkaline phosphatase-conjugated secondary antibody gave excellent morphological and immunological results. As an example, the immunological characterization of normal myeloid colonies with a number of monoclonal antibodies is demonstrated.

Immunological classification of chronic myeloid leukemia distinguishes chronic phase, imminent blastic transformation, and acute lymphoblastic leukemia.

The clinical course of chronic myeloid leukemia (CML) is highly variable and therefore it is difficult to predict the duration of the chronic phase. We studied the immunological expression of maturation patterns in 62 cases of CML (30 cases in clinical/cytological blast crisis (BC), 32 cases in clinical/cytological chronic phase (CP) by means of a double marker enzyme immuno assay (DM-EIA). Immunological findings were supplemented by Southern blots using Ig-JH-, TCRbeta- and bcr-probes. Patients in BC (n = 30) expressed high proportions of CD10, CD20, CD33, CD34 and low degrees of a mature myeloid marker (CD15). Myeloid BC bone marrow (BM) cells showed a high degree of coexpression of unusual, lineage restricted markers: 25% of CD15-positive cells also expressed markers like CD10, CD20 or CD34. In contrast, BM cells in lymphoid BC did not show this coexpression. In CP two groups were distinguished immunologically: concordant cases which were immunologically normal (n = 14) and discordant cases (n = 18) which showed increased proportions of unusual, lineage restricted markers and double labelled cells (e.g. CD15/CD34). The latter group developed clinical BC earlier during further follow up (p = 0.009). Cases of lymphoid BC (n = 11)--in contrast to acute lymphoblastic leukemia (ALL) patients (n = 21)--did not show coexpression of CD15/CD10, CD20, CD34. These data show that blast clones can be detected in CML-CP by characteristic immunological maturation defects several months before the clinical onset of BC. Moreover, the lymphoid "blasts" of CML-BC represent a relatively differentiated lymphoid population of cells which can be distinguished from ALL by their lack of coexpression of unusual, lineage restricted markers.

Velocity sedimentation and cell cycle characteristics of granulopoietic progenitor cells (CFUc) in canine blood and bone marrow: influence of mobilization and CFUc depletion.

Populations of granulopoietic progenitor cells, as measured by the colony formation technic (CFUc), of canine bone marrow and blood were compared by velocity sedimentation and tritiated thymidine (3H-TdR) induced cell death rates. Blood CFUc were smaller than those of the bone marrow and had a lower proportion in S-phase, as reflected by these methods. CFUc which were mobilized into the peripheral blood by dextran sulfate had unaltered velocity sedimentation and cell death fraction rates. Removal of CFUc from the blood by 24-hour leukapheresis resulted in doubling of 3H-TdR suicide of bone marrow CFUc, whereas that of blood CFUc was not affected. Depending on the degree of CFUc depletion, granulopoietic progenitor cells which were larger than normal appeared in the blood and bone marrow. It is concluded that there is a subpopulation of CFUc within the bone marrow which is ready for discharge into the circulation. These cells are characterized by small size and a lower proportion in S-phase. Some bone marrow/blood barrier retains large CFUc as well as those in active proliferation within the bone marrow. One might speculate that enlarged CFUc in the bone marrow are a subpopulation which normally provides only a small influx to the total CFUc pool.

Allogeneic transplantation of blood stem cells concentrated by density gradients.

Discontinuous albumin density gradients were used to physically separate hemopoietic cells from immunocompetent lymphocytes in peripheral blood mononuclear cells of dogs. Transplantation of these stem cell concentrates into lethally irradiated allogeneic recipients restored hemopoiesis in nine of 12 animals. Tolerance and long-term survival, however, were achieved only in pairs matched for major histocompatibility antigens. These animals showed stable chimerism in bone marrow cells but transiently regenerated autochthonous cells demonstrable only in the peripheral blood. As compared with earlier studies using grafts of unseparated peripheral blood mononuclear cells, the numbers of CFUc transplanted were similar; the number of mononuclear cells, however, was reduced by a factor of 60. It thus appears that the granulocyte-macrophage colony-forming assay (GM-CFUc) is able to predict within limits the hemopoietic potential of a graft. Although very low numbers of blood-derived CFUc were effective (33,000-42,000/kg body weight), the superiority of blood stem cells over bone marrow grafts remains to be established.

Reduction of infection rates in cancer patients associated with the use of haematopoietic growth factors.

As the risk of infection associated with chemotherapy is related to the depth of the fall in neutrophil counts, protection from neutropenia has been used as an endpoint for growth factors in this setting. However, the functional status of these and other myeloid cells are also important. Therefore, more direct measurements of clinical improvement will also be useful. Several studies have suggested that the use of granulocyte-macrophage colony-stimulating factor (GM-CSF) can result in improvements in hospital stay, days of fever, antibiotic use and thrombocytopenia. Similar findings have been confirmed by our own work which indicates that GM-CSF not only shortens the period of leukopenia, but also reduces the complications of infection. More sensitive and appropriate endpoints should be included in future trials, including rate of and survival from infection as well as overall and disease-free survival.

The use of dose-intensified chemotherapy in the treatment of metastatic nonseminomatous testicular germ cell tumors. German Testicular Cancer Study Group.

With the use of a cisplatin-based chemotherapy, metastatic testicular cancer has become a model for a highly curable malignant disease. Current data show that 70% to 80% of patients with this disease will achieve long-term survival following cisplatin/etoposide/bleomycin therapy. The role of high-dose chemotherapy with autologous stem cell support is being investigated in metastatic germ cell cancer in attempts to improve outcome for patients whose disease relapses after standard-dose chemotherapy and for those who present initially with advanced metastatic disease. Prognostic categories for patients receiving high-dose salvage chemotherapy have recently been developed: cisplatin-refractory disease, beta-human chorionic gonadotropin values greater than 1,000 U/L, and primary mediastinal germ cell tumors are factors characterizing patients who will derive less benefit from high-dose chemotherapy than those with chemosensitive disease at relapse. While standard-dose salvage chemotherapy achieves only a 20% long-term survival rate, high-dose salvage chemotherapy may yield a cure rate of approximately 40%. A randomized study comparing high-dose therapy with conventional-dose therapy (IT94 coordinated by the European Group for Blood and Marrow Transplantation) in patients with relapsed disease is ongoing to substantiate this observation. The use of dose-intensive therapy as first-line treatment is currently being studied by several institutions. High-dose therapy may be better tolerated when used first line compared with its use in the salvage situation, and may also achieve a rapid initial cell kill before cytostatic drug resistance develops. The German Testicular Cancer Study Group has developed a sequential high-dose combination regimen of cisplatin/etoposide/ifosfamide given with granulocyte colony-stimulating factor and peripheral blood stem cell support for four cycles every 3 weeks. This ongoing study, started in 1990, had accrued 218 patients with advanced testicular germ cell tumors as of June 1997. Of 141 evaluable patients receiving dose levels 1 through 5, 82 (58%) have achieved complete remission with no evidence of disease and 32 (23%) have achieved partial remission with marker normalization. The early death rate was 8%. Overall and event-free survival rates at 2 years are 78% and 73%, respectively, with a projected 5-year overall survival rate of 74%. Despite favorable preliminary results, this approach cannot be considered standard treatment. Currently, high-dose chemotherapy with peripheral blood stem cell transplantation should be administered to patients with testicular cancer only within controlled clinical trials to allow long-term cure rates and treatment-related late side effects to be evaluated.

Intervention treatment of established neutropenia with human recombinant granulocyte-macrophage colony-stimulating factor (rhGM-CSF) in patients undergoing cancer chemotherapy.

Recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) was given to 60 patients, in a double-blind, non-prophylactic study of already established chemotherapy-induced leucopenia, for 5 days by continuous intravenous infusion and twice or once daily by subcutaneous injection. Four patients were randomized to rhGM-CSF (3) or placebo (1) at each dose (1.3, 1.7, 5.5, 11, or 22 micrograms of protein/kg). Leucocyte recovery was significantly enhanced compared with controls, in a dose-dependent manner except for 22 micrograms/kg which was ineffective with a worse experience of side effects in some patients. Most adverse events occurred in equal proportions in the treated and placebo cases. Fourteen patients developed infection and were treated with antibiotics in addition to rhGM-CSF. They were joined by a further 18 febrile patients and treated with rhGM-CSF in a subsequent open-label trial. The survival from infection was related to white blood cell (WBC) count: 19 of 32 responded with increased numbers of leucocytes (WBC count above 1.5 x 10(9)/1) after 5 days of GM-CSF. Sixteen of the 19 leucocyte 'responders' recovered from infection, two died from the underlying disease and one from persistent infection. Six of the 13 patients who did not have a leucocyte response died with persistent infection. These data indicate that rhGM-CSF enhances the leucocyte count following chemotherapy and in this way saves critically ill neutropenic patients from fatal infections.

Clonogenic assays and engraftment in allogeneic bone marrow transplantation.

The significance of clonogenic assays for determining the hematopoietic potential of bone marrow grafts is still a matter of controversy. We determined the number of myeloid (GM-CFU), early erythroid (BFUe) and mixed (CFU-GEM) clones in 23 consecutive allogeneic bone marrow grafts. The growth of GM-CFU was stimulated by placental-conditioned medium, whereas both phytohemagglutinin-stimulated leucocyte-conditioned medium (PHA-LCM) and 'pluripoietin' from the 5637 cell line served as equally efficient stimulators of BFUe and CFU-GEM growth. Plating efficiency (e.o.p.) of GM-CFU and numbers of myeloid and mixed progenitors transplanted per kg body weight were significantly lower in those patients who died in the aplastic phase 2-6 weeks postgrafting (n = 4). These data show that low numbers of clonogenic cells, in particular GM-CFU, indicate a higher risk of death from infection following bone marrow transplantation (BMT) and argue for a contribution of GM-CFU in the seeding of an aplastic bone marrow.

Myeloablative conditioning for marrow transplantation in myelodysplastic syndromes and paroxysmal nocturnal haemoglobinuria.

Paroxysmal nocturnal haemoglobinuria (PNH) and myelodysplastic syndromes (MDS) are disorders of pluripotent stem cells resulting in haematopoietic insufficiency which can be cured by marrow transplantation. The extent of myeloablative conditioning necessary for elimination of the non-malignant and premalignant clones is not known. We report our results of marrow transplantation with and without myeloablative conditioning in two patients with PNH and seven patients with MDS. Conditioning was not used in a patient with PNH and a monozygotic twin as donor. In this patient the disease remained unchanged. Myeloablative treatment with busulphan (BUS) in addition to immunosuppression with cyclophosphamide (CY) was used for conditioning in a patient with PNH and a 2-year-old boy with chronic myelomonocytic leukaemia (CMML). Fractionated total body irradiation (FTBI) and CY was used in six patients with refractory anaemia with excess of blasts (RAEB) and RAEB in leukaemic transformation (RAEB-T). Haematopoiesis was fully restored in all patients conditioned with myeloablative treatment except for a patient in leukaemic transformation with myelofibrosis and a HLA-DR-incompatible donor. Chimerism was complete in all patients except for the 2-year-old boy conditioned with BUS and CY. Our results and those reviewed in the literature indicate that myeloablative conditioning with either BUS or FTBI is advantageous for marrow transplantation in PNH and MDS.

Cyclophosphamide-resistant Yoshida ascites tumor cells and their cross resistance to some alkylating agents.

A cyclophosphamide-resistant subline of a Yoshida ascites tumor was developed by giving increasing doses of the drug after transplantation. The effect of several alkylating agents on the cell proliferation of both the sensitive and resistance cell line was compared establishind dose response curves and D50 values. The developed subline revealed a 260 fold resistance to cyclophosphamide. It was completely cross-resistant to hydroperoxycyclophosphamide, whereas for triaziquone, N-oxide-mustard, and N-methyl-mustard only a partial cross resistance existed. These results give further evidence that cyclophosphamide and hydropeoxycyclophosphamide have the same mechanism of action. Regarding the other alkylating agents the results demonstrate differences concerning either the molecular mode of action or protecting effects. A decreased activation or uptake of substance is probably not the base for resistance.

Perspective of rhGM-CSF in the treatment of neutropenic infections and aggressive lymphomas.

Non-Hodgkin lymphomas (NHL) of intermediate and high-grade malignancy respond well to doxorubicin-containing regimens, but long-term survival does not exceed 30% in large studies with long-term follow-up. Any attempt to improve this somehow disappointing result by adding more drugs, increasing doses or shortening time intervals of chemotherapy have so far failed in randomized settings. Even autologous bone marrow transplantation (ABMT) could not improve long-term survival when applied in first remission of the disease. Prophylactic use of hematopoietic growth factors in the chemotherapy of aggressive NHL did prevent neutropenia and positively influenced the occurrence of infectious complications, and also led to an increase of dose intensity (DI) by 15% but this did not affect survival. In contrast, a retrospective analysis of an NHL study showed that a high DI may in fact be deleterious rather than beneficial. Thus the prophylactic use of hematopoietic growth factors still has to be considered experimental in the chemotherapy of NHL and should be studied in controlled settings like the one proposed here.

Myeloid progenitors in acute non-lymphocytic leukemia: prognostic value in remission.

The prognostic value of in vitro cloning of bone marrow and blood cells was tested in 32 patients with acute non-lymphocytic leukemia (ANLL) at various stages of disease. Using placental conditioned medium (PCM) as a stimulus and panoptical staining methods for the analysis of colony morphology, four different growth types could be distinguished at presentation. Poor response to colony-stimulating activity (GM-CSF) in vitro seemed to be associated with poor survival. Probably due to low patient numbers, this association could not be proven statistically. During remission a progressive decrease of morphologically normal colonies was recognized in the bone marrow and peripheral blood. Thus by in vitro cloning methods the quality of remission could be substantiated.

Cloning of early erythroid and mixed myeloid/erythroid human bone marrow progenitor cells: comparison of different sources of burst-promoting activity (BPA).

The ability of conditioned media from the 5637 cell line and human placenta (HPCM) to stimulate the in vitro growth of human early erythroid and mixed myeloid/erythroid clones was tested and compared to phytohemagglutinin-stimulated leukocyte supernatants (PHA-LCM). Both 5637 supernatant and PHA-LCM were equally effective with a linear dose-response relationship. HPCM at various concentrations did not exhibit burst-promoting activity (BPA). Thus, "pluripoietin"-containing media provide a large-scale source of BPA similar in its biological activity to standard sources used for studying human hematopoiesis in vitro.