Influence of cytokines and autologous lymphokine-activated killer cells on leukemic bone marrow cells and colonies in AML.

We have already shown that cytokine cocktails (IL-1beta, IL-3, IL-6, SCF, GM-CSF) and/or lymphokine-activated killer (LAK) cells can reduce the amounts of clonal, CD34-positive mononuclear bone marrow cells (BM-MNC) in acute myeloid leukemia (AML). In addition, the influence of those cocktails and/or LAK cells on the clonogenic potential of AML BM-MNC was investigated. BM colonies cultured in agar during different stages of the disease were immunophenotyped in situ: 17 patients at diagnosis, 14 patients in complete remission, 8 patients at relapse, 8 healthy donors. A significant reduction in leukemic cells and colonies positive for CD34 after in vitro culture of BM-MNC with cytokine cocktails was achieved with all samples obtained at diagnosis (n = 8, p < 0.01), in 6 of 8 cases in complete remission but only in 2 of 6 cases at relapse. Cytokine cocktails stimulated granulopoiesis as well as B and T lymphopoiesis. Colonies with leukemic phenotype could never be detected in healthy BM. A significant reduction in leukemic colonies was achieved by coculture of BM-MNC (uncultured or cytokine precultured) with autologous LAK cells in all 4 cases at diagnosis and in 1 case at relapse. An additive effect of in vitro cytokine preincubation of BM samples on the leukemia-reducing effect of LAK cells could be demonstrated in all samples studied (p < 0.001; diagnosis: n = 10, relapse: n = 3, complete remission: n = 7). Patients had a better prognosis if CD34-positive colonies in AML could be reduced by cytokine incubation (p = 0.03) or coculture with autologous LAK cells in vitro (p = 0.04). Our data show that cytokines as well as LAK cells alone and in combination can reduce, however not eliminate clonogenic AML cells. Such mechanisms might be responsible for maintaining stable remissions in AML.

Cytokines can reduce clonal, CD34-positive cells in acute myeloid leukemia in vitro.

We studied the influence of cytokine mixes on the survival of acute myeloid leukemia (AML) bone-marrow (BM) cells in a 14-day culture assay in vitro. Southern-blot analysis using a panel of different probes in combination with densitometry and flow cytometry were used to detect and compare the amount of clonal or CD34-positive BM cells before and after the culturing procedure. A significant reduction of CD34-positive cells after incubation with a cytokine mix [interleukin (IL)-1beta, IL-3, IL-6, stem cell factor (SCF), erythropoietin (EP) with granulocyte macrophage/colony-stimulating factor (GM-CSF, Cytok1) could be achieved in all 16 cases with a CD34-positive blast phenotype studied at diagnosis (P<0.001), in 3 of 10 cases at relapse, and in 8 of 18 cases in complete remission. In healthy donors, an increase of CD34-positive cells was demonstrated in 5 of 5 samples. A reduction of clonal DNA through incubation with Cytok1 was achieved in 5 of 5 (100%) cases studied at diagnosis, in 1 of 4 (25%) cases at relapse, and in 7 of 9 cases (78%) in complete remission. Cytokine cocktails with GM-CSF (Cytok1) were more efficient in reducing (clonal) CD34-positive cells than cocktails without GM-CSF (Cytok2). AML patients at diagnosis and in complete remission had a better survival probability if their CD34-positive or clonal cells could be reduced in vitro by cytokine cultivation (P<0.05). Vitality of BM cells was not influenced by 14-day cytokine treatment; however, the total cell count could be increased by Cytok1 and Cytok2 by 55-174%, but not by the control medium. Our data show that: (1) clonal cell populations can be regularly detected at diagnosis, during complete remission, and at relapse; (2) CD34-positive cells in AML can be demonstrated to be clonal, gene-rearranged cells; (3) incubation of AML BM-cells with Cytokl leads to a reduction of the CD34-positive, clonal cell load in all cases at diagnosis and in 78% of the cases in complete remission of AML, but in only 25% of the cases at relapse; (4) in all healthy BM samples, proportions of 'healthy' CD34-positive cells were increased. Moreover, absolute cell counts were increased by cytokine incubation of cells obtained at diagnosis, relapse, or complete remission of AML and from healthy donors indicating a selective stimulation of healthy, but not of leukemic CD34-positive cells; (5) cytokine cocktails containing GM-CSF are more efficient in reducing leukemic cells than cocktails without GM-CSF; and (6) in vitro reactivity of clonal or CD34-positive BM cells against Cytokl has clinical relevance. We conclude, that Southern-blot analysis and flow cytometry are suitable methods to detect and quantify leukemic disease and to distinguish between clonal or non-clonal CD34-positive cells. The ex vivo or clinical application of specific combinations of cytokines might be a feasible and successful application of immunotherapy in AML that merits further investigations.

Gene rearrangements in bone marrow cells of patients with acute myelogenous leukemia.

At diagnosis, clonal gene rearrangement probes [retinoic acid receptor (RAR)-alpha, major breakpoint cluster region (M-bcr), immunoglobulin (Ig)-JH, T cell receptor (TcR)-beta, myeloid lymphoid leukemia (MLL) or cytokine genes (GM-CSF, G-CSF, IL-3)] were detected in bone marrow samples from 71 of 153 patients with acute myelogenous leukemia (AML) (46%): in 41 patients with primary AML (pAML) (58%) and in 30 patients with secondary AML (42%). In all cases with promyelocytic leukemia (AML-M3) RAR-alpha gene rearrangements were detected (n = 9). Gene rearrangements in the Ig-JH or the TcR-beta or GM-CSF or IL-3 or MLL gene were detected in 12, 10, 16 and 12% of the cases, respectively, whereas only few cases showed gene rearrangements in the M-bcr (6%) or G-CSF gene (3%). Survival of pAML patients with TcR-beta gene rearrangements was longer and survival of pAML patients with IL-3 or GM-CSF gene rearrangement was shorter than in patients without those rearrangements. No worse survival outcome was seen in patients with rearrangements in the MLL, Ig-JH or M-bcr gene. In remission of AML (CR), clonal gene rearrangements were detected in 23 of 48 cases (48%) if samples were taken once in CR, in 23 of 26 cases (88%) if samples were taken twice in CR and in 23 of 23 cases (100%) if samples were studied three times in CR. All cases with gene rearrangements at diagnosis showed the same kind of rearrangement at relapse of the disease (n = 12). Our data show that (1) populations with clonal gene rearrangements can be regularly detected at diagnosis, in CR and at relapse of AML. (2) Certain gene rearrangements that are detectable at diagnosis have a prognostic significance for the patients' outcome. Our results point out the significance of gene rearrangement analyses at diagnosis of AML in order to identify 'poor risk' patients - independently of the karyotype. Moreover, the persistence of clonal cells in the further course of AML can be studied by gene rearrangement analysis.

Cytogenetic and Southern blot analysis to demonstrate clonality and to estimate prognosis in patients with myelodysplastic syndromes.

We examined the bone marrow of 109 patients with myelodysplastic syndrome (MDS) at the time of diagnosis and during the course of the disease by means of Southern blot analysis and/or cytogenetic studies to detect and evaluate clonal markers, their implications for the prognosis of the disease, and the response to treatment. The patients either were enrolled in an EORTC study and received low-dose Ara-C with (n=31) or without (n=21) growth factors, according to the study protocol, or were treated supportively (only one patient received regular chemotherapy for concomitant lymphoma). Full or at least partial remission was achieved by 34% of the treated patients (n=54). In 57% (53 of 93) of all patients a clonal marker of either kind was detected by Southern blot analysis and/or cytogenetic examination. Clonal chromosomal aberrations were found in 45% (35 of 77) of the cases examined at diagnosis, with solitary del(5q) aberrations occurring in 10% of the cases and complex aberrations in 18%, trisomy 8 or monosomy 7 being a frequent finding. Of all patients, 49% (28 of 57) were characterized by one or more gene rearrangements (e.g., Ig-JH, TcR-beta, M-bcr, GM-CSF, G-CSF, or IL-3) at diagnosis. In five of 21 cases (24%) studied in hematological remission of the disease chromosomal aberrations were still detectable, and in seven of 23 (30%) a gene rearrangement persisted. We also found six cases with multiple clones exhibiting different susceptibilities to treatment and thereby indicating the oligoclonal character of this disease. Clinical evaluation revealed that the prognosis of the respective patients was directly related to the particular clonal markers detected at diagnosis: Risk groups were subdivided according to the karyotypes, with a solitary del(5q) aberration meaning a favorable, a normal karyotype an intermediate, solitary aberrations without del(5q) a poor, and complex karyotypes a very poor prognosis. We showed that densitometry helps to increase the sensitivity of Southern blot analysis by quantifying the amount of altered DNA, which often increases shortly before or at progression of MDS. Overall, there was a high level of concordance of both clonality examinations with the clinical course of the disease and the response rate. Therefore, we recommend cytogenetic studies and Southern blot analysis to detect clonal markers at diagnosis of MDS, to detect oligoclonality and clonal evolution, or to quantify the amount of clonal DNA, which appears to be a sensitive tool for evaluating the prognosis and response to therapy in MDS.

Lymphokine-activated killer (LAK) cells and cytokines synergize to kill clonal cells in acute myeloid leukemia (AML) in vitro.

We studied the influence of autologous lymphokine-activated-killer (LAK) cells on the survival of clonal and CD34-positive bone marrow (BM) cells from patients with acute myeloid leukemia (AML) in a coculture assay in vitro. (1) LAK cells were grown in the presence of IL-2, in some cases additionally with IL-6. (2) These cytotoxic cells were cocultured with (untreated or cytokine pretreated) AML-BM cells obtained at different stages of the disease. Therefore BM cells were (a) either frozen in liquid nitrogen or (b) precultured for 14 days with cytokines: IL-1beta, IL-3, IL-6, erythropoietin (EPO), stem cell factor (SCF) with ('Cytok1') or without granulocyte macrophage colony stimulating factor (GM-CSF) ('Cytok2') or with no added cytokines ('ISC/FCS') as a control. (3) Southern blot analysis was used to detect clonal BM cells. At diagnosis, 76 of 151 cases (50%) studied showed clonal gene rearrangements in marker genes. (4) Southern blot analysis and flow cytometry were used to compare the amount of clonal and CD34 positive BM cells before and after coculture procedures. Coculture experiments with untreated BM and autologous LAK cells led to a reduction of clonal cells in 2 of 5 cases at diagnosis, in 11 of 17 BM samples in complete remission but not in the one case studied at relapse. Similar results were found if precultured AML cells (with or without cytokines) were cocultivated with LAK cells. However the cytotoxic effect of LAK cells was more pronounced if cytokines (especially GM-CSF and SCF) were comprised. Our data indicate, that (1) clonality in AML can be demonstrated by Southern blot analysis; (2) CD34 positive cells in AML are clonal, gene rearranged cells; (3) clonal cell populations persist in BM during complete remission and relapse in most of the patients; (4) incubation of AML-BM cells with LAK cells lead to a reduction of clonal, rearranged cells in 11 of 17 AML cases in complete remission, but only in 2 of 6 cases at diagnosis or relapse; (5) AML cells can be sensitized to theLAK cell treatment by preincubation of AML-BM cells with cytokines (IL-1beta, IL-3, IL-6, SCF, EPO and GM-CSF) or by adding SCF to the coculture conditions. Southern blot analysis and flow cytometry are appropriate methods to detect and quantify leukemic disease. Cytokines and LAK cells synergize to kill AML blasts in vitro. This is a feasible approach to immunotherapy of AML and merits further investigations.

GM-CSF stimulates proliferation of clonal leukemic bone marrow cells in acute myeloid leukemia (AML) in vitro.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is known to stimulate granulocytes, monocytes, and macrophages. We studied the effect of GM-CSF on (clonal) bone marrow (BM) cells obtained from AML patients after 7 days of culture in vitro: BM samples were obtained from 19 AML patients at diagnosis (DIA), from two patients with persisting disease (PERS), from eight patients in complete remission (CR), and from 12 healthy donors. Flow-cytometric comparison of differentiated, CD 15-positive cells or of CD34-positive blast cells before and after cultivation showed that the proportion of CD15-positive cells was increased in nine of 12 healthy BM samples, in 14 of 19 cases at DIA, in one of three cases during PERS, and in five of six cases in CR of AML. The proportion of CD34-positive cells was increased in one of 12 healthy BM samples, in seven of 19 cases at DIA, in one of two cases during PERS, and in three of seven cases in CR of AML. Southern blot analysis (SBA) performed in six cases during the course of AML, before and after cell culture, showed that clonal DNA increased after GM-CSF treatment in three of five cases studied at DIA, in six of nine cases studied in CR, in the one case studied at PERS, and in the one studied at relapse (REL). In one case of trisomy 8 at DIA a normal karyotype was demonstrated in CR. However, after 7 days of cultivation of the cells in GM-CSF the trisomy 8 was detected in two of 17 metaphases isolated from colony-cells from methylcellulose cultures. Our data show that a 7-day treatment of BM cells with GM-CSF induced a differentiation of healthy and leukemic BM cells in the great majority of cases. An enrichment of CD34-positive cells was not achieved in healthy BM samples. However, in 70% of the cases in CR and in 30% of the cases at DIA of AML, clonal CD34-positive cells were enriched. This means that GM-CSF stimulates ('primes') leukemic cell growth in vitro.

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.

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.

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.

Subclassification of diffuse large B-cell lymphomas according to the Kiel classification: distinction of centroblastic and immunoblastic lymphomas is a significant prognostic risk factor.

Among high-grade malignant non-Hodgkin's lymphomas the updated Kiel classification identifies three major B-cell entities: centroblastic (CB), B-immunoblastic (B-IB), and B-large cell anaplastic (Ki-1+) (now termed anaplastic large cell [CD30+], [B-ALC]). The clinical prognostic relevance of this distinction was evaluated in a randomized prospective treatment trial (COP-BLAM/IMVP-16 regimen randomly combined +/- radiotherapy in complete responders) conducted in adult (age 15 to 75) patients with Ann Arbor stage II-IV disease (n = 219) diagnosed by optimal histomorphology (Giemsa staining) and by immunohistochemistry. Overall survival was significantly better in CB lymphoma as compared to B-IB (P = .0002) or B-ALC (P = .046). Relapse-free survival was worse for B-IB (P = .0003) as compared to CB lymphomas. The prognostic differences between CB and B-IB were confirmed by multivariate analyses including the risk factors of the International Index. Overall survival was significantly determined by performance status (P = .0003), serum-LDH (P = .036), and B-IB histology subtype (P = .036). Relapse-free survival was influenced by age (P = .007) and histological subtype (P = .007). Thus, the diagnosis of the CB and B-IB lymphomas by the histological criteria of the Kiel classification was identified as an independent prognostic factor in diffuse large B-cell lymphomas.

Effect of GM-CSF, 1, 25-Dihydroxycholecalciferol (Vit.D) and All-Trans-Retinoic Acid (ATRA) on the Proliferation and Differentiation of MDS-Bone Marrow (BM)-Cells In Vitro.

Myelodysplastic syndromes (MDS) are a heterogenous group of stem cell disorders characterized by an impaired differentiation of the pluripotent stem cell resulting in dysplastic changes of all three hemopoietic lineages. We studied the effect of vitamin D (Vit.D) or all-trans retinoic acid (ATRA; 10(-6) and 10(-8)M) in combination with GM-CSF on the proliferation and differentiation of mononuclear bone marrow-cells (MNC) of 48 MDS-patients as compared to 9 normal bone marrow (BM)-controls in a special colony assay: 30,000 MNC were cultured in agar plugs for 7 days (d) and the resulting colonies immunophenotyped in situ by a panel of monoclonal antibodies. In 14 of 48 cultured MDS-BM-samples hemopoietic clones could be grown which expressed myelomonocytic antigens (CD14 (21%*), CD15 (35%*)) as well as blast antigens (CD20 (9%*), CD34 (10%*), Glycophorin A (Glyco A, 18%*)) whereas all normal BM-colonies were negative for blast markers. Vit.D or ATRA in combination with GM-CSF could not induce an (immunologically measurable) increased differentiation (5 higher percentage of differentiated clones) as compared to GM-CSF alone. We conclude that Vit.D and ATRA have no sufficient differentiation effect on MDS-cells. Our colony assay in combination with immunophenotyping enables an in vitro measurement of differentiation and proliferation in MDS. We suggest the use of this technique to measure effects of therapy in the course of the disease.

Interleukin-3 plus low-dose cytosine arabinoside for advanced myelodysplasia: a pilot study. EORTC Leukemia Group.

In an attempt to reestablish normal hematopoiesis in symptomatic myelodysplasia (MDS) and to show the tolerability of a combination treatment of low-dose cytosine arabinoside (LD AraC) and interleukin-3 (IL-3), we treated 31 patients (pts., median age 65 years) who had more than 10% blasts in the bone marrow (BM) and hematopoietic failure with LD AraC (2 x 10 mg/m2 sc, day 1-14) plus IL-3 (once daily sc, day 8-21) at different dose steps (1.0, 2.5, 5.0, and 10.0 micrograms/kilogram body weight). The numbers of each 21-day cycle varied between 1 (3 pts.), 2 (6 pts.), 3 (8 pts.), 4 (1 pt.), 5 (5 pts.), and 6 (8 pts.), in total 116 cycles on an outpatient basis. Subjective tolerability was good in 20 cases (65%). Toxicities were fever (29 pts.), flu-like symptoms (17 pts.), infections (15 pts.), hepatic toxicity (10 pts.), and skin reactions (8 pts.). Overall response was seen in 13 cases (42%) and 5 complete responses (CR), while 10 pts. had stable disease (SD), 5 progressed (2 to acute leukemia), 2 were considered toxic deaths, and 1 died due to the disease. Median survival is 18 months, progression-free survival is 12.5 months (18.0 months in responding pts.), with an actuarial follow-up of 31 months. The data from this phase I/II study show that a combination of LD-AraC and IL-3 is well tolerated and that stable responses can be achieved in MDS by means of an easy outpatient therapy.

Detection of acute myeloid leukemic cells in complete remission and in extramedullary sites by clonal analyses.

We report the case of a 49-year-old woman with acute myeloid leukemia (AML, M5b). The leukemic cells expressed blast as well as myelomonocytic antigens and were characterized by a clonal gene rearrangement of the immunoglobulin (Ig) JH gene. During the course of the disease in clinical/cytological complete remission (CR) the persistence of leukemic cells was shown by surface marker analyses on bone marrow (BM) cells or BM clones grown in agar. Moreover, clonal leukemic cells could be detected by Southern blot analyses indicating the persistence of blasts. Occasionally BM cells obtained in CR showed a cytologically, immunologically differentiated phenotype, but nevertheless DNA-rearranged cells indicated differentiated blasts ('clonal remission'). Five years after diagnosis the patient presented with an isolated pleural effusion. Southern blot analysis identified the original leukemic clone because pleural cells showed the same clonal Ig gene rearrangement as the cells at diagnosis. The patient died 6 years after diagnosis during her fourth relapse of AML. This shows the value of Southern blot analyses, immunophenotyping and culture techniques to monitor the course of leukemic tumor burden even if it is not cytologically identifiable.

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.

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.

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.

Randomized, double-blind, placebo-controlled, phase III study of recombinant human granulocyte-macrophage colony-stimulating factor as adjunct to induction treatment of high-grade malignant non-Hodgkin's lymphomas.

We evaluated recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF; Sandoz Pharma [Basel, Switzerland]/Schering-Plough [Kenilworth, NJ]) as an adjunct to a modified (mainly cyclophosphamide and doxorubicin increased 1.5-fold) COP-BLAM regimen in the primary treatment of high-grade malignant non-Hodgkin's lymphomas (NHL). Patients (n = 182; stage II-IV; age, 15 to 73 years) were randomized to rhGM-CSF (400 micrograms) or placebo for 7 days subcutaneously after chemotherapy. Efficacy was analyzed for patients receiving at least 70% of study medication (n = 125). The frequency of clinically relevant infection was reduced by rhGM-CSF (28 v 69 infections, 16 v 30 patients, P = .02) with a cumulative probability of remaining infection free in 70% versus 48% (P = .05 log rank test at 190 days). Periods of neutropenia (P = .01 in 5 of 6 courses), days with fever (2.1 v 4.0, P = .04) and days of hospitalization for infection (3.5 v 8.0 days, P = .01) were significantly reduced. Complete response (CR) rates, assessed by prognostic risk, were 15 of 19 (79%) in treated versus 20 of 21 (95%) in controls in the low-risk group (P = .12). In the high-risk group, 31 of 45 (69%) treated patients achieved CR versus 25 of 52 (48%) of controls (P = .04). No difference in survival has been seen after 1 year. Only injection site reactions (45% treated v 7% controls) and rash (26% v 2%) occurred more frequently in treated patients (n = 176). These data show that rhGM-CSF is well tolerated in most patients with NHL, significantly reduces infection, and improves response.