Search

Bone marrow examination has been the confirmatory test for congenital dyserythropoietic anemia type II (CDAII). Occasional spherocytes on peripheral blood smear can confound the diagnosis. Since a screening test is still unavailable, we explored the feasibility of using flow cytometry as a preliminary screening method.Thirteen monoclonal antibodies with specificities for eight erythrocyte membrane proteins were used in FACS analysis to probe the cellular features of red cells from CDAII, normal adults, hereditary spherocytosis (HS), and cord red cells. Confocal microscopy was performed on normal and CDAII to determine the overall distribution of CD44 and CD47. Their expression levels on cultured erythroblasts were also analyzed.The densely stained band 3 as seen in CDAII in gel electrophoresis was also obtained for Dantu phenotype. Likewise analysis of CDAII cases (n = 26) using the eosin-5'maleimide (EMA) binding test found 57% of patients giving results either positive or in the grey area for HS. Enhanced fluorescence of CD44 was detected in 96% of the CDAII patients, and anti-CD47 binding was also elevated to a lesser degree. Although RNA expressions of CD44 and CD47 in the cultured erythroblasts of normal controls and CDAII were similar, confocal microscopy revealed more CDAII red cells giving elevated fluorescence than normal red cells.A distinction between CDAII and HS can be made using the EMA Binding test and anti-CD44 binding. Confirmation of CDAII can subsequently be made based on clinical presentation together with either bone marrow examination or DNA sequencing of SEC23B. © 2016 International Clinical Cytometry Society.

Congenital dyserythropoietic anemias are rare hematological disorders leading to ineffective erythropoiesis with chronic anemia, complicated by iron overload. Here we present a remarkable clinical course of an infant with CDA type II who first presented as a severe fetal hydrops, requiring serial intrauterine red cell transfusions. While postnatal transfusion dependency persisted, the patient was successfully transplanted with a myeloablative conditioning regimen and peripheral blood stem cells of a matched donor. We believe that allogeneic HSCT is a reasonable therapeutic approach for patients with very severe CDA, even if only a matched unrelated donor is available.

Ninety patients with Philadelphia chromosome-positive chronic myelogenous leukemia in blast crisis were reviewed to identify significant prognostic associations. At diagnosis of blast crisis the main clinical, laboratory, and cytogenetic data were recorded and evaluated for prognostic significance. At the time of the analysis 89 patients had died, with a median survival of 11 weeks from diagnosis of blast crisis. Patient characteristics demonstrated in the univariate analysis to have significant association with shorter survival were: thrombocythemia, leukocyte count above 20 x 10(9), Karnofsky index50%, nonlymphoid blast cell morphology, cytogenetic clonal evolution, the presence of a double Philadelphia chromosome or trisomy 8, and no response to therapy. In 17 of 59 patients (29%) evaluable for response to therapy a complete or partial remission was achieved. These responders had a significantly longer median survival (25 weeks) as compared with nonresponders (9 weeks). Response to therapy was significantly better in lymphoid blast crisis and in patients without clonal evolution. In a multivariate analysis containing all significant variables of the univariate analysis two parameters retained their prognostic significance: response to therapy and trisomy 8. In spite of the short overall survival in blast crisis, the determination of prognostic factors may be a useful tool for the clinician planning therapy, especially new therapeutic approaches.

Clinical trials underestimate the age of chronic myeloid leukemia (CML) patients. Incidence and median age of Ph/BCR-ABL-positive CML and other chronic myeloproliferative disorders in a representative area in Germany

Ovarian cancer is chemosensitive, but most patients with advanced disease die from tumor progression. As 25% of the patients can be cured by chemotherapy, it is reasonable to evaluate high-dose chemotherapy (HDCT). Forty-eight patients with untreated ovarian cancer were entered in a multicenter phase I/II trial of multicycle HDCT. Median age was 46 (19–59 years); International Federation of Gynecology and Obstetrics-stage was III in 79% and IV in 21%; 31% had residual disease >1 cm after surgery. Two courses of induction/mobilization therapy with cyclophosphamide (250 mg/m2) and paclitaxel (250 mg/m2) were used to collect peripheral blood stem cells. HDCT consisted of two courses of carboplatin (area under curve (AUC) 18–22) and paclitaxel followed by one course of carboplatin and melphalan (140 mg/m2) with or without etoposide (1600 mg/m2). Main toxicity was gastrointestinal. Limiting carboplatin to AUC 20 and eliminating etoposide resulted in manageable toxicity (69% without grade 3/4 toxicity). One patient died from treatment-related pneumonitis. At 8 years median follow-up, median progression-free-survival (PFS) and overall survival (OS) is 13.3 and 37.0 months. Five-years PFS and OS is 18 and 33%. Multicycle HDCT is feasible in a multicenter setting. A European phase III trial based on this regimen is evaluating the efficacy of HDCT.

Objective. To determine the aetiology and clinical significance of an elevated platelet count (thrombocytosis) in a large cohort of patients. Design. A retrospective review of the medical records was performed on all patients, who had at least one platelet count ≥ 500 × 109 L–1. Setting. Departments of Medicine and Surgery, University of Ulm, Germany. Subjects. A total of 732 patients with thrombocytosis. Main outcome measures. Classification of thrombocytosis and thromboembolic complications, and evaluation of laboratory parameters distinguishing between primary and secondary thrombocytosis. Results. Of the total of 732 patients, 89 (12.3%) had primary and 643 (87.7%) had secondary thrombocytosis. Essential thrombocythaemia was observed in 40 of 89 patients (45%) with primary thrombocytosis. The most frequent causes of secondary thrombocytosis were tissue damage (42%), infection (24%), malignancy (13%) and chronic inflammation (10%). Primary thrombocytosis was significantly associated with a higher platelet count and an increased incidence of both arterial and venous thromboembolic complications. In secondary thrombocytosis, thromboembolic events were restricted to the venous system and occurred only in the presence of other risk factors. Mean values of leucocyte count, haematocrit, erythrocyte sedimentation rate, fibrinogen, serum potassium and lactate dehydrogenase were significantly different in primary and secondary thrombocytosis. Conclusions. The finding of an elevated platelet count on routine blood examination has diagnostic, prognostic and therapeutic implications. It is of clinical importance to distinguish between primary and secondary thrombocytosis, as thrombotic complications occur more frequently in primary thrombocytosis. Unless additional risk factors are present, secondary thrombocytosis is not associated with a significant risk for thromboembolic events.

Thrombopoietin (TPO) is the most important regulator of megakaryocyte development and platelet production. Platelet production is thought to be regulated by a negative regulatory feed back loop. In an attempt to evaluate the role of TPO in the pathobiology of essential thrombocythemia (ET), we have examined levels of TPO and other cytokines with thrombopoietic activity (interleukin-6 and interleukin-11) in sera obtained from 25 patients with ET (ten treated, 15 untreated) and 117 healthy control subjects. TPO serum levels were assessed using a sandwich-antibody ELISA that utilizes a polyclonal rabbit antiserum for both capture and signal. The mean serum TPO level in 25 ET patients was significantly elevated (545+/-853 pg/ml) as compared with that in healthy controls (95.3+/-54.0 pg/ml,p0.001). The difference in TPO serum levels between ten treated (781+/-1229 pg/ ml) and 15 untreated ET patients (388+/-458 pg/ml) did not reach statistical significance (p = 0.09). We conclude that either consumption or production of TPO is altered in ET. Failure of appropriate feedback regulation and continued megakaryocyte stimulation by an elevated TPO may play an important role in the pathobiology of ET.

The treatment results of 96 patients with Philadelphia-positive chronic myeloid leukemia (CML) in accelerated phase (AP) were reviewed retrospectively. Treatment of AP consisted of allogeneic bone marrow transplantation in 20 (14 related and 6 unrelated donors) or conventional chemotherapy (CC) in 76 patients. Three main treatment strategies were followed in the CC group: continuation (group A) or dose escalation (group B) of chronic phase therapy or change of chronic phase therapy to hydroxyurea (group C). Median survival was 7.0 months in group A (range 1.8–110), in group B 8.3 months (range 0.9–40) and in group C 9.6 months (range 1.5–47.6), p = 0.89. Survival in CC was dependent on response to therapy as the achievement of a second chronic phase was significantly associated (p < 0.001) with a longer median survival (21 months) compared with stable accelerated phase disease (11 months) or treatment failure (5 months). Median survival in the BMT group was 16.7 months (range 5–77), the 5-yr probability of relapse was 25% and the 5-yr disease-free survial was 36%. For patients < 55 yr median survival after BMT was significantly prolonged compared with median survival after CC (n = 45, 8.3 months, p = 0.008). After developing criteria of AP, median survival in our analysis has been less than 1 yr. The results of conventional chemotherapy in the treatment of accelerated phase CML are disappointing. If a suitable donor is available allogeneic BMT should be performed without delay in patients with AP.

In an attempt to evaluate the role of thrombopoietin (TPO) in the pathobiology of aplastic anaemia (AA), we have examined TPO levels in sera from 54 AA patients and 119 healthy controls. A total of 92 samples were collected from AA patients: 43 samples were harvested at diagnosis, 23 samples in the cytopenic period after treatment, and 26 samples when patients were in partial (n=10) or complete remission (n=16) following immunosuppressive treatment. TPO serum levels were assessed by a sandwich-antibody ELISA that utilized a polyclonal rabbit antiserum for both capture and signal. Serum samples from normal donors revealed a mean TPO level of 95.3 +/- 54.0 pg/ml (standard deviation). Mean TPO levels in AA sera collected at diagnosis and before onset of treatment were 2728 +/- 1074 pg/ml (P0.001 compared to normal controls: mean platelet count at that time: 27x10(9)/l). TPO serum levels of AA patients in partial or complete remission after immunosuppressive treatment were significantly lower than TPO levels at diagnosis (P0.001). However, despite normal platelet counts (mean 167x10(9)/l), TPO levels remained significantly elevated in complete remission (mean TPO 1009 +/- 590 pg/ml, P0.001 compared to normal controls). There was a significant inverse correlation between serum TPO levels and platelet counts in AA patients who were not transfused for at least 2 weeks prior to sample collection (coefficient of correlation (r) = -0.70, P0.0001). In summary, TPO levels were highly elevated in sera of patients with AA. Thus there is no evidence to suggest an impaired TPO response contributing to thrombocytopenia in AA. Thrombopoietin did not return to normal levels in remission, indicating a persisting haemopoietic defect in remission of AA. We hypothesize that elevated levels of TPO may be required to maintain normal or near normal platelet counts in remission of AA.

This case report shows for the first time the usefulness of positron emission tomography (PET) with 2-[18F]-fluoro-2-deoxy-D-glucose (FDG) in the diagnosis of primary non Hodgkin's lymphoma of the liver. Results of FDG-PET, which in contrast to other imaging techniques offers the advantage of screening the whole body, demonstrated a high glycolytic activity of a solitary mass in the liver with central necrosis (loss of glycolytic activity), but no spread of lymphoma to the body. These results were confirmed by ultrasound, computed tomography, magnetic resonance imaging and were biopsy proven. From our findings we conclude that in patients with liver masses with high uptake of FDG, lack of liver dysfunction and absence of signs indicating other malignancies, a primary lymphoma of the liver should be considered as a possible diagnosis.

Survival times in chronic myeloid leukaemia (CML) may vary widely depending on the risk profiles of patients. This fact is frequently not, or not sufficiently, considered in evaluating survival in CML, and some studies do not report risk profiles. Therefore we analysed the relative impact of risk profile and therapy on survival using the median survival times of therapy groups and of risk groups of the three-arm randomized German CML Study I (interferon alpha v hydroxyurea v busulphan; median survival times 65 v 56 v 45 months, n = 490, median observation time 70.4 months). The impact of risk profile (Sokal) on survival as determined by the survival difference between high and low risk patients (40 months) was twice the maximum survival difference between treatment groups (20 months). A similar ratio was obtained after stratification for therapy and for risk profile. Since Sokal's index has been reported to prognostically discriminate IFN-treated patients less well than chemotherapy-treated patients, a new score with better discrimination of IFN-treated patients was also used. The results were similar for both scores. We conclude that the risk profile at diagnosis is still more important for survival of CML patients than therapy. Therefore patients should be stratified according to risk profile for comparisons of survival times between studies and treatment arms.

We analysed 459 samples from 206 chronic myeloid leukaemia (CML) patients at diagnosis and during or after treatment with interferon-alpha (IFN-alpha) by quantitative Southern blot analysis for BCR rearrangement. In a minority (2%) of Ph-positive patients, no BCR rearrangement was detectable due to breakpoints outside the major breakpoint cluster region (M-bcr) or possibly due to M-bcr deletions. Results from 235 samples were compared with the proportion of Ph-positive metaphases found in contemporaneous bone marrow specimens analysed by conventional cytogenetics. The rank correlation between both methods was 0.82 (P0.001). The proportion of CML cells in samples determined by Southern blot analysis (BCR ratio) was significantly different between cytogenetically-defined minor, partial, and complete response groups (P0.001). Empirically-derived cut-off points in the BCR ratio were introduced in order to define molecular response groups for comparison to standard cytogenetic response groups: a BCR ratio of 0% was defined as complete molecular response and ratios of 1-24%, 25-50%, and50% were defined as partial, minor, and no molecular response, respectively. Using these cut-off points the concordance between both methods was 67% (P0.0001), a major cytogenetic response could be predicted or excluded in more than 90% of cases (P0.0001). Our findings demonstrated that quantitative Southern blot was as sensitive as cytogenetics and as peripheral blood samples are suitable for this technique it should be considered as the method of choice for routine monitoring IFN-alpha therapy in CML patients.

We have assessed in a phase I/II clinical study the tolerability and efficacy of long-term application of recombinant human interleukin-3 (rh-IL-3) in combination with antithymocyte globulin (ATG) and cyclosporin A (CSA) in 13 patients with aplastic anemia who were refractory to or relapsed after previous immunosuppressive treatment. Four cohorts of three patients were consecutively enrolled so that they received rh-IL-3 on days 9, 6, 3 and 1 after start of ATG/CSA treatment. Yeast-derived recombinant human IL-3 was administered by daily subcutaneous injection until day 90 at a dosage of 250 micrograms/m2. Long-term application of rh-IL-3 was well tolerated. The combination of rh-IL-3 with immunosuppression did not modify the known toxicities of ATG and CSA. Incidence and severity of rh-IL-3-related adverse events was less than in other phase I/II trials of rh-IL-3 as single-agent therapy. One might speculate that co-medication with CSA alleviates rh-IL-3-induced side effects. Three of eight patients with refractory AA and all four patients with relapsed AA responded to the combined treatment within four months. The median time to response was 91.5 days. There was evidence for an rh-IL-3-dependent response in two patients. Long-term rh-IL-3 did not cause stem cell exhaustion. One patient died early during the course of the study from EBV-driven lymphoproliferative disease. Two patients developed acute myeloid leukemia 4 and 22 months after cessation of rh-IL-3. In conclusion, long-term rh-IL-3 in combination with immunosuppression is well tolerated. The response rate to the combined treatment in refractory and relapsed AA was high. Recombinant human IL-3-dependent responses suggest efficacy. A prospective randomized trial comparing immunosuppression alone versus a combination with rh-IL-3 is warranted.

It is the long-term goal of the German CML Study Group and of the Süddeutsche Hämoblastosegruppe (SHG) to improve survival of patients with chronic myelogenous leukemia (CML). In a first randomized study (CML Study I) monotherapies with hydroxyurea or interferon alpha (IFN-alpha) were compared with a standard busulfan regimen with regard to duration of the chronic phase and survival. The main results of this study were published, 1-3 and a long-term follow up is planned. In a second randomized study the effect of the combination of IFN-alpha and hydroxyurea versus hydroxyurea monotherapy on survival is being investigated. This paper provides a first preliminary report on the study concept, patient recruitment, state of documentation and initial patients' characteristics 9 months after closure of the study.

Antibodies to IFN-alpha have been recognized as a novel type of autoantibody developing after allogeneic BMT. Ninety-six patients undergoing BMT for various hematologic disorders were followed for the presence of spontaneous IFN-alpha antibodies until 12 years after transplantation. Seven of them (7.3%) developed IFN-alpha antibodies occurred late after BMT (or = 15 months), rose to very high titers in some patients, and persisted for years despite combined immunosuppressive treatment. They were oligo- or polyclonal in nature, predominantly IgG with a broad IgG subclass distribution, and neutralized the antiviral and antiproliferative activity of various natural and recombinant IFN-alpha types including the patients' endogenous IFN-alpha in vitro. All antibody-positive recipients suffered from chronic GVHD (n = 5) or chronic viral hepatitis (n = 2), but the only significant association was with prior severe aplastic anemia (3/9, 33%; P = 0.022). There was no discernible HLA association of IFN antibody development. Although the clinical relevance of the IFN-alpha antibodies is uncertain they may interfere with cellular defence mechanisms and immune regulation after BMT.

In one centre (the medical department of the University of Ulm), over a period of 25 years, the diagnosis of paroxysmal nocturnal haemoglobinuria (PNH) had been made in 40 patients (27 women, 13 men). The data on these patients were analysed and the findings compared with those reported in the literature. Criteria for inclusion were a positive sucrose haemolysis and acid-serum test. Leading clinical symptoms were anaemia, tendency towards bleeding and infections, haemolysis, abdominal pain and thromboembolism. The age at which main symptoms first appeared was most often in the third and fourth decade of life. The sex ratio of 2:1 (female to male) was unusual. PNH was frequently (in 22 patients) associated with aplastic anaemia. If bone marrow transplantation, at present the only curative procedure, was not possible and there was PNH alone, treatment was supportive and directed at relief of symptoms. The course of the disease was highly variable. Lack fo prognostic criteria made it difficult to define indications for intensive therapeutic measures. While haemolytic crisis and thromboembolism are frequent complications in isolated PNH haematopoetic insufficiency defines clinical outcome in PNH combined with aplastic anaemia.

We have performed a non-randomised GVHD prophylaxis trial comparing cyclosporin/methotrexate with in vivo/ex vivo T cell depletion with the monoclonal antibodies Campath 1G/1M in patients with acute leukaemias in first complete remission. We observed significantly less acute and chronic GVHD, neutropenic fever and severe mucositis in the T cell depletion group. The incidence of graft rejection and relapses was no higher than in the cyclosporin/methotrexate group. There is a trend in favour of improved disease-free survival in the in vivo/ex vivo T cell depletion group (80% vs. 62%).

Combined in vivo/ex vivo T cell depletion is effective in the prophylaxis of graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation (BMT), but influences the occurrence of active cytomegalovirus (CMV) infection and disease. Twenty nine patients receiving a T cell-depleted marrow graft (Campath-1M) after intravenous application of the monoclonal antibody Campath-1G prior to conditioning were monitored for virus shedding and antigenaemia from day -7 to day +100. In seropositive patients in this group active CMV infection occurred more frequently (10 of 16) and much earlier (nine of 10 until day +21) than in 27 seropositive patients (10 of 27, P0.02) receiving cyclosporin A and methotrexate (CsA/MTX). Early active CMV infection after in vivo/ex vivo T cell depletion correlated strictly with an early increase in blood lymphocyte counts (P0.01), with predominance of NK cells and/or CD8+ T cells. Three cases of very early interstitial pneumonitis (IP) occurred after in vivo/ex vivo T cell depletion compared with none in the CsA/MTX group. IP was fatal in the only patient with early active CMV infection, who remained lymphocytopenic till death on day +31. This may indicate that an early immune response against CMV is possible and essential for favourable clinical outcome.

The clinical interrelationship between paroxysmal nocturnal hemoglobinuria (PNH) and aplastic anemia (AA) promoted a search for a pathogenetic link. Since the molecular defect in PNH is a failure to express phosphatidylinositol glycan-anchored proteins (PIG-AP), we investigated whether this defect could also be demonstrated on peripheral blood cells of patients with typical AA. Quantification of the expression of PIG-AP was performed by flow cytometry using the monoclonal antibodies (MAbs) CD16 and CD66b for granulocytes, CD14 and CD48 for monocytes, CD48 and CD52 for lymphocytes, and CD55 and CD59 for erythrocytes. We analyzed cells from 52 patients with acquired AA. A PIG-AP-defective population was identified in 27 of 52 patients (52%) in at least one cell lineage. Granulocytes were involved in 25 of 27, monocytes in 18 of 25, lymphocytes in seven of 27, and erythrocytes in seven of 27 AA patients who were affected by a PIG-AP deficiency. The response rate to standard immunosuppressive therapy was significantly higher in the group of patients without a PIG-AP-deficient population than in patients with a PIG-AP-deficient population in at least one cell lineage (85.7 vs. 30.4%; p0.0003). Our results demonstrate that on the basis of PIG-AP expression, the proportion of AA patients who show features of typical AA along with a PNH phenotype is substantially higher than previously recognized. The pattern of PIG-AP expression might identify subgroups of AA patients who differ in the underlying mechanism as well as in the course of their disease.

As curative bone marrow transplantation is available only to a minority of patients with chronic myelogenous leukemia (CML), drug therapy remains of central interest. Several nonrandomized studies have suggested that interferon-alpha (IFN) may prolong survival in CML. In a randomized multicenter study the influence of IFN versus busulfan or hydroxyurea (HU) on survival of Philadelphia-positive (Ph+) CML was examined. A total of 513 Ph+ patients were randomized for treatment as follows: 133 for IFN, 186 for busulfan, and 194 for HU. IFN-treated CML patients have a significant survival advantage over busulfan-treated (P = .008), but not over HU-treated patients (P = .44). The longer survival is due to slower progression to blast crisis. Median survival of IFN-treated patients is 5.5 years [5-year survival, 59%; 95% confidence interval (CI), 48%-70%], of busulfan-treated patients, 3.8 years (5-year survival, 32%; CI, 24%-40%), and of HU-treated patients, 4.7 years (5-year survival, 44%; CI, 36%-53%). Patients who continue on IFN survive longer than those in whom IFN is discontinued before blast crisis (P = .007). Complete hematologic IFN-responders have a survival advantage over partial responders or nonresponders (P = .02). Cytogenetic IFN-responders have no significant survival advantage over nonresponders (P = .2). Patients who attain white blood cell (WBC) counts of 10 x 10(9)/L or less have a survival advantage in the IFN (P = .007) and HU (P = .05) groups. Whereas toxicity in the IFN group was considerably higher than in the busulfan or HU groups, long-lasting cytopenias necessitating discontinuation of therapy as observed with busulfan have not been seen with IFN or HU. The problems of conventional prognostic scores (Sokal's score, Score 1) that we observed in IFN-treated patients support the idea that IFN changes the natural course of CML. We conclude that, with regard to survival of CML in the chronic phase, IFN is superior to busulfan and as effective as HU. Whether and to what extent IFN is superior to HU appears to depend, at least in part, on the degree of WBC suppression by HU-therapy and on the risk profile of the patients.

CD 54 (ICAM-1) expression was studied on blasts of 13 cases of de-novo AML prior to and after stimulation by interferon-gamma (IFN-gamma). Furthermore, its functional activity was studied by analysis of the influence of CD 54 on homologous interaction of the blasts. Prior to culture only a minority of the blasts displayed CD 54 positivity. Timed incubation of the blasts in RPMI-1640/FCS or serum-free medium 'spontaneously' increased the percentage of CD 54 positive cells in 11/13 cases with a main increase after 24 h. IFN-gamma (500 IU/ml) further enhanced CD 54 positivity in 6/11 cases. In 2/13 cases neither a 'spontaneous' nor an IFN-gamma induced CD 54 upregulation occurred. The homotypic aggregation of the AML blasts paralleled ICAM-1 expression in that (1) in all but the two CD 54 negative cases autologous cluster formation could be detected, (2) IFN-gamma enhanced cluster formation in 5/6 cases in which it had enhanced CD 54 upregulation, (3) incubation of the blasts in the presence of an anti-CD 54 MoAb (clone 84H10) reduced the 'spontaneous' and IFN-gamma induced cluster formation in a majority of the cases. Taken together, ICAM-1 expression on AML blasts is heterogenous with respect to (1) its 'constitutional' expression and (2) its 'spontaneous' and IFN-gamma-induced upregulation, while it seems to be functionally active once expressed on the surface membrane. The reason for the heterogenity, which did not correlate with the FAB subtype, and its importance remain unclear.

In a randomized multicenter study the influence of hydroxyurea versus busulfan on the duration of the chronic phase and on survival of chronic myelogenous leukemia (CML) was determined. In addition cross resistance and adverse reactions of the drugs were analyzed. From July 1983 to January 1991, 441 CML patients were randomized to receive hydroxyurea or busulfan. Of these, 90.7% were Philadelphia positive; 25.7% were low, 38.2% intermediate, and 36.2% high risk patients according to Sokal's score. The median survival of the busulfan treated Philadelphia-positive patients is 45.4 months and of the hydroxyurea group 58.2 months (P = .008). The survival advantage for the hydroxyurea treated patients is recognized in all risk groups. Sixty four patients reached therapy resistance before blast crisis and were crossed over to the alternative drug. The 23 patients with primary hydroxyurea had a median survival of 5.6 years, the 41 patients with primary busulfan therapy a median survival of 2.7 years (P = .02). Adverse reactions were less frequent with hydroxyurea with no severe adverse effects (lung fibrosis, long lasting bone marrow aplasia). The analysis of white blood cell counts in the course of treatment showed lower counts in the hydroxyurea patients. We conclude that hydroxyurea is superior to busulfan in therapy of CML in chronic phase and should be used as first line therapy. Busulfan may have a role as secondary therapy after hydroxyurea resistance or intolerance.

Grafted immunocompetent cells are considered responsible for GVHD as well as for the elimination of residual leukemic cells ('graft-versus-leukemia reactivity', GVLR) in leukemic patients after allogeneic BMT. Clinical and experimental investigations have given contradictory answers to the question whether GVHD and GVLR are two manifestations of the same process or separate immunologic processes. We have addressed this question by analysing the primary in vitro response of BM-derived proliferating and cytotoxic T lymphocyte precursors (PTLp and CTLp) in HLA identical relative pairs (n = 17). PTLp frequency estimation reveals strong responses (1 in 5000) on non-leukemic as well as leukemic stimulation in a majority of cases. CTLp amount variably to 10-100% of the proliferating precursor cells. Preliminary specificity analyses show that on non-leukemic stimulation about 90% of colonies exhibit exclusive lysis of the non-leukemic target. At the same time, on leukemic stimulation, about 75% of cytolytic colonies are exclusively reactive against leukemic targets without crossreactivity against nonleukemic targets from the same patient. Our data show that primary in vitro responses in HLA identical sibling pairs may be as strong as those against allo MHC antigens. In addition CTL specifically lysing leukemic or non-leukemic targets may represent an in vitro model of the immunologic non-identity of GVHD and GVLR.

From July 1983 to January 1991 a total of 622 patients were randomized (585 eligible) to compare the effects of hydroxyurea, interferon alpha (IFN), and busulfan on the duration of chronic phase, and survival. Further goals included the determination of prognostic parameters. 598 CML patients were documented and 575 evaluable. The Ph-status was known for 547 patients. 89.4% of the patients were Ph-positive (+). 11% had additional chromosome aberrations. The median survival of Ph+ patients by now is 4.2 years, that of Ph-patients 1.4 years. Ph-negative patients are older, tend to have lower cell counts and, as a group are more ill at diagnosis. A survival difference of about one year is expected between busulfan and hydroxyurea treated patients. Prospectively evaluated age, organomegaly related symptoms, Karnofsky index, extramedullary manifestations, number of erythroblasts and percent of circulating blasts proved to be of prognostic significance. A prognostic score (score 1) was determined which was superior to Sokal's score in the study population. 164 patients were randomized to receive IFN. In 54 patients (33%) IFN had to be terminated because of adverse effects, therapy resistance or other reasons. Clinically relevant neutralizing antibodies were detected in 9 cases. Most frequent adverse events were flu-like symptoms in 74%, gastrointestinal symptoms in 52%, and neurologic-psychiatric symptoms in 30% of patients. Reduction of the Ph-chromosome was observed in 13% of evaluable patients (10 of 75). In 4 patients complete cytogenetic remissions were observed, in three of these ongoing. Cytogenetic responders have a survival advantage. Interferon treated Philadelphia-negative CML patients have no survival disadvantage. The study is expected to allow statements as to the advantages or disadvantages of the use of busulfan, hydroxyurea and IFN in the treatment of CML as well as to the reliability of prognostic markers.

Uncertain gait and weakness on standing, progressing to flaccid paraparesis of both legs, developed in a 76-year-old woman. Proprioceptor reflexes of both legs were absent and there was pallaesthesia up to the pelvic rim. Electromyography pointed to polyneuritis. Administration of prednisone, 1 mg/kg daily for 10 days, was ineffective, as was one-time plasmapheresis. An incomplete tetraparesis developed. Blood white-cell count increased within 2 weeks by 10,400/microliters (52% lymphocytes) to 17,400/microliters. Blood smear, bone-marrow cytology and histology, as well as immunocytology, revealed lymphoplasmocytoid immunocytomas (chronic lymphocytic leukemia). Oral cytoreductive treatment was started with chlorambucil, 0.1 mg/kg daily, and prednisone, 100 mg daily. After 19 days the patient was able to walk with support, after 28 was walking unaided. Treatment was continued over eight cycles (14 days' treatment, 14 days' interval per cycle). 17 weeks after onset of treatment paraesthesias of the legs required renewed administration of chlorambucil, 0.1 mg/kg per day, for 3 months. The patient has now been free of symptoms during a follow-up period of 12 months.

In the past 10 years 201 HLA-identical bone marrow transplantations (BMT) were performed with major ABO incompatibility in 41 (20%) and minor ABO incompatibility in 35 (18%) patients. ABO compatibility between donor and recipient showed no influence on granulocyte and platelet recovery after BMT. Erythrocyte reconstitution was significantly (p0.01) delayed for about 1 week in major ABO-incompatible BMT. In addition, a pure red cell aplasia lasting for 2-5 months occurred in 6 out of 21 blood group 0 patients who received transplants of group A. The rate of graft rejection, incidence of graft-versus-host disease as well as the leukemic relapse rate were similar in ABO-compatible and ABO-incompatible BMT. The probability of a 10-year survival after BMT is independent of ABO compatibility between donor and recipient.

Recent developments in CML research are illustrated by the results of one large randomized multicenter study carried out by the German CML Study Group. From July 1983 to January 1991, a total of 703 CML patients were recruited; 624 patients were randomized to compare hydroxyurea and interferon alpha (IFN) with busulfan. The median survival of Ph+ patients by now is 3.95 years, that of Ph- patients 1.1 years. Some difference in survival is recognizable between the treatment arms, but this is not yet significant. Fewer adverse effects are being observed in the hydroxyurea group. Ph-negative patients tend to have lower white blood cell and platelet counts. Patients (164) were randomized to receive IFN. In 50 patients (30%) IFN had to be terminated because of adverse effects, therapy resistance, or other reasons. Reduction of the Ph-chromosome was observed in 20% of evaluable patients. In 3 patients complete cytogenetic remissions were observed. Clinically relevant neutralizing antibodies were detected in 9 cases. Prospectively evaluated age, organomegaly related symptoms, Karnofsky index, extramedullary manifestations, erythroblasts, and percent of circulating blasts proved to be of prognostic significance. A prognostic score (score 1) was determined and compared to Sokal's score. It is expected that the study results will allow statements as to the advantages or disadvantages of the use of busulfan, hydroxyurea and IFN in the treatment of CML as well as to the reliability of prognostic markers.

Immunosuppression is the most effective treatment for patients with aplastic anemia, except for bone marrow transplantation. The best results are achieved with antilymphocyte globulin or cyclosporine. Patients have been treated successfully with a combination of both agents, but there has been no controlled evaluation of its efficacy. We conducted a randomized, multicenter trial in 84 patients not eligible for bone marrow transplantation, comparing treatment with antilymphocyte globulin and methylprednisolone (41 patients--the control group) with antilymphocyte globulin, methylprednisolone, and cyclosporine (43 patients--the cyclosporine group).At three months significantly more patients in the cyclosporine group had a complete or partial remission in response to treatment than did patients in the control group (65 percent vs. 39 percent, P less than 0.03); this difference was confirmed at six months (70 percent vs. 46 percent, P less than 0.05). The superior results of the regimen including cyclosporine were most evident in the patients with severe or very severe aplastic anemia, whose response rate at six months was 65 percent, as compared with 31 percent of such patients in the control group (P less than 0.02). Granulocyte and hemoglobin levels became normal in most patients who responded, but platelet counts continued to be subnormal in 61 percent of the patients. Ten of 52 patients with responses (3 in the cyclosporine group and 7 in the control group) relapsed 4 to 37 months after treatment. The actuarial survival of all patients at 41 months is 64 percent in the cyclosporine group and 58 percent in the control group (P = 0.16); among the patients with severe or very severe disease, survival is 80 percent and 44 percent, respectively (P = 0.077). Cyclosporine had substantial but reversible side effects.Immunosuppressive treatment of aplastic anemia with antilymphocyte globulin, methylprednisolone, and cyclosporine appears to be more effective than a regimen of antilymphocyte globulin and methylprednisolone without cyclosporine and may thus represent a treatment of choice for patients who are not eligible for bone marrow transplantation.

In a multicenter study it is being determined, whether the use of hydroxyurea or of interferon alpha instead of busulfan prolongs the chronic phase of Philadelphia-positive CML. Additional goals are the examination, whether the types of disease evolution and the terminal phases differ between the treatment groups, and the prospective recognition of prognostic criteria for the duration of the chronic phase of CML. By September 5, 1990, 593 CML-patients had been randomized, 221 for busulfan, 228 for hydroxyurea and 144 for interferon alpha. The average age is about 51 years. By April 30, 1990, 106 patients had reached the end of the chronic phase, 126 had died. The median survival of Philadelphia-positive patients was 3.95 years, that of all patients 3.75 years. The median observation time of all patients was 1.34 years (mean 1.60 years). At present, no significant difference in survival is recognizable between the three treatment arms, although fewer adverse effects have been observed in the hydroxyurea arm.

From a population-based study on the incidence of potentially drug-associated blood dyscrasias 28 cases were identified with pancytopenia. Who recovered within 90 days after diagnosis. Early recovery occurred more frequently in patients showing normal or increased cellularity of the bone marrow than in patients with bone marrow hypoplasia. Median recovery times of leukocytes were 14 and 10 days and of platelets 21 and 9 days in patients with and without bone marrow hypoplasia, respectively. Age and sex distribution were similar in both groups. Of 28 patients, 11 reported a period of fever before onset of pancytopenia. Sixteen patients in whom information on drug use was available had taken a median of 4 drugs before the onset of symptoms that were related to pancytopenia. From these results we present the hypothesis that transient pancytopenia with or without marrow hypoplasia can be the expression of the same type of bone marrow injury and that drugs or viral infections should be considered as etiological factors.

We investigated the effect of recombinant tumor necrosis factor-alpha (rTNF-alpha) and recombinant lymphotoxin (rLT) in the growth modulation of purified hairy cell leukemia (HCL) cells. In response to rTNF-alpha, HCL cells from five of eight patients showed a 3 to 23-fold thymidine incorporation above their unstimulated controls. The effect was time and dose dependent with a maximum between 10 and 25 ng/ml rTNF-alpha after 120-hr incubation. rLT (1-50 ng/ml), however, could not enhance DNA synthesis in six of six cases. Cell number of rTNF-alpha stimulated cells ranged from 2-3 x 10(6)/ml from days 0-50 whereas cell number of unstimulated controls decreased from 3 x 10(6)/ml at day 0 to 0.01-0.02 x 10(6)/ml after 50 days in culture. rTNF-alpha induced proliferation could be suppressed in all HCL cell populations by 0.3 ng/ml recombinant interferon alpha (100 U/ml rIFN-alpha). TNF binding studies in two patients revealed that both TNF-sensitive HCL cells (1,990 +/- 148 receptors/cell) as well as TNF-insensitive HCL cells (1,261 +/- 101 receptors/cell) express specific receptors for TNF-alpha. These data show that rTNF-alpha and rLT have different effects on the growth of HCL cells. In addition there is a subgroup of patients who show no response to rLT or rTNF-alpha.

The clinical course of 103 patients (50 males, 53 females; median age 59 years) with idiopathic myelofibrosis (IMF) seen at our hospital between 1967 and 1986 was analyzed retrospectively. Common symptoms and signs at the time of diagnosis were: myelofibrosis (96%), splenomegaly (84%), anemia (81%), osteosclerosis (45%), malaise (41%) and leukocytosis (41%). It was possible to follow the majority of patients without treatment or with transfusion therapy only for prolonged periods of time. The use of cytostatic drugs and radiotherapy was restricted as much as possible. Probably due to this treatment strategy the incidence of acute leukemia was low (5%). Major thromboembolic complications were seen in 19% of the patients. Median survival of the patients was 4.3 years. The prognostic influence of several disease parameters determined at the time of diagnosis was tested: age, sex, leukocytes, platelets, hemoglobin, reticulocytes, LDH, ANP-score, spleen size and percentage of peripheral blood blasts + promyelocytes had no significant influence on the length of survival. Osteosclerosis, a presumed sign of advanced disease, was not correlated with survival either.

We report the rare occurrence of essential thrombocythemia (ET) in two sisters. In one patient, the clinical phenotype of the disease evolved from ET to polycythemia vera (PV) after 4 years of follow-up. Clonal hematopoiesis was established in both cases by X-chromosomal inactivation analysis using a DNA polymorphism of the phosphoglycerate- kinase (PGK) gene. Cell separation studies suggested a common ancestor for granulocytes, monocytes, and T lymphocytes in one patient; however, in her sister, monoclonality could only be demonstrated convincingly for the granulocyte fraction. Our data indicate that ET may originate from heterogenous stem cell levels.

Restriction fragment length polymorphisms of the X-chromosome genes phosphoglycerate kinase and hypoxanthine phosphoribosyl transferase were used to study clonality in peripheral blood leukocytes from 48 women with chronic myeloproliferative disorders (c-MPD). A total of 50% of patients were heterozygous for one or both of the polymorphic loci. These included 17 cases with polycythemia vera, four patients with essential thrombocythemia (ET), and three cases with idiopathic myelofibrosis (IMF). A clear-cut monoclonal X-inactivation pattern was observed in 17 of 24 cases including all IMF patients. Only one patient with PV exhibited a nonclonal composition of her leukocytes, while six cases demonstrated a predominantly clonal pattern in peripheral blood cells. Among the latter category reckoned three of four ET patients. Cell separation analyses were performed in one ET and three PV patients. In all four cases a monoclonal pattern of the granulocyte fraction could be established, while T lymphocytes of these patients were of nonclonal origin. These data suggest that the vast majority of c-MPDs arise from multipotent hematopoietic stem cells. Moreover, this type of clonal analysis might be of help in discriminating between primary MPD and reactive processes.

The clinical course of 196 patients with chronic myelocytic leukemia (CML) was studied. Prognostic factors were analyzed using a standard prognostic model. From a univariate analysis of patients with nonblastic Philadelphia chromosome-positive CML, splenomegaly, bone marrow fibrosis, percentage of blasts and promyelocytes in the peripheral blood and LDH activity were shown to be factors with a significant negative influence on survival. However, age and the platelet count did not influence survival. The standard prognostic model, generated with the 4 variables (1) percentage of blasts and promyelocytes, (2) spleen size, (3) platelet count and (4) age did not provide a useful representation of risk status in this heterogenous patient population. However, the addition of further variables (LDH, additional chromosomal aberrations, percentage of basophiles/eosinophiles and percentage of bone marrow blasts) to the standard model allowed a separation into 2 patient groups: one with low and the other with intermediate to high risk. Our data support the general validity of the prognostic model; however, the applicability of the model may be compromised in hematologic centers with a heterogenous CML population due to the selection of high-risk patients. In this situation additional risk factors may have to be added to the prognostic formula.

Summary. Four patients with combined immunocytopenia of unknown origin were investigated. Two patients with pancytopenia had allo- and autoantibodies against erythrocytes, granulocytes and thrombocytes. Two other patients with granulocytopenia and thrombocytopenia showed allo- and autoantibodies against granulocytes and thrombocytes. All patients went into a transient or persistent remission under immunosuppressive therapy. The normalization of peripheral blood correlated with the disappearance of antibodies suggesting that the cytopenia was caused by an antibody mediated autoimmune mechanism.

The differentiation inducing effect of low-dose Ara-C on human myeloid leukemic cells was studied in two patients with subacute myelocytic and subacute myelomonocytic leukemia in vivo and in vitro. By continuous i.v. administration of 10 mg Ara-C/m2 over 12 h daily for 12 or 20 days complete remissions were obtained in both patients with normalization of the incidence of the committed progenitor cells BFU-E and CFU-C in the marrow while the incidence of pluripotent CFU-GEMM remained subnormal. Parallel cultures of the patients' bone marrow cells in diffusion chambers (DC) implanted in mice demonstrated a clear cytotoxic effect of low-dose Ara-C. The greater increase of granulopoietic cells within DC in the Ara-C exposed group than in control mice after the end of drug administration is, in addition, an indication for differentiation induction by this kind of Ara-C therapy.

Two unrelated cases of congenital dyserythropoietic anaemia (CDA) are described. They show striking similarities which could not be attributed to one of the well-known types of CDA or any other congenital disease of the erythroid system. Both patients were followed for many years before and after splenectomy. There was a long-lasting, prominent post-splenectomy erythroblastosis, suggesting impairment of red cell denucleation. The type of heredity is unknown, and the enzymatic or molecular basis of the changes observed is not understood.

Our results show a marked acute hematotoxicity of oral benzo(a)pyrene (BaP) in D2 mice as well as the extreme resistance of BDF1 individuals to bone marrow toxicity induced by oral BaP. Continued oral BaP produced severe bone marrow depression in D2 mice affecting all myelopoietic lineages, but produced only moderate bone marrow depression in BDF1 mice affecting erythropoiesis only. Pluripotent hematopoietic stem cells were almost completely destroyed in D2 individuals, but only reduced to approximately 40% in BDF1 individuals after 7 days of BaP. D2 mice were killed by 13 days of continued oral BaP, but BDF1 mice were still alive and in good condition even after 19 days of continued oral BaP. Analysis of the bone marrow and peripheral blood changes showed that severe toxic chemical bone marrow depression in D2 mice by continued oral BaP cannot serve as an experimental model system of acute aplastic anemia.

The clinical course of 141 unselected patients (64 m, 77 f, median age 59) with polycythemia vera (PV), treated during the period 1967 to 1986 was analyzed to study prognostic factors and the correlation between treatment strategies and complication rates. Therapy was performed according to a prospectively defined treatment protocol. Primary control of the disease was achieved by phlebotomy. Marrow suppression by radioactive phosphorus or low dose busulphan was used only as a second-line therapy or to lower high platelet counts. The clinical course of the patients was characterized by a low rate of acute leukemia (4%) and a high rate of thromboembolic complications (40%). Myelofibrosis developed in 17 patients (12%). Median survival of the patents was 9.4 years. The prognostic influence of several parameters at the time of diagnosis was tested: age, sex, spleen size, percentage of blood blasts + promyelocytes, leucocyte count, platelet count, hemoglobin, hematocrit, reticulocyte count and the values of the lactate-dehydrogenase (LDH) and the alkaline neutrophil phosphatase (ANP) all had no significant influence on the length of survival. The prognosis of PV patients with atypical disease presentation at diagnosis was not different from patients with typical disease.

Twenty-three patients with aplastic anemia (18/23 with severe aplastic anemia) were treated with an immunosuppressive regimen consisting of cyclosporin A (CsA) and methylprednisolone (MP) (n = 7) or CsA, MP, and antithymocyte globulin (ATG; n = 16). Nineteen patients are alive with a follow-up of 4 to 25 months; three patients died of infections and one of a gastrointestinal hemorrhage. Within 3 months, improvement of hematopoiesis was seen in 14 patients (61%). First signs of a response after 23 to 88 days were followed by complete remission in eight patients, partial remission in three patients, and minimal improvement in three patients. Two of the patients with only minimal improvement were treated with a second course of immunosuppression and reached a complete remission and partial remission. Interestingly, remission proved to be dependent on the continued administration of CsA in four of five patients with partial or complete remission who could be evaluated up to now. Thus, CsA must have been effective in the induction and/or maintenance of remission in three patients. This observation is a very strong argument for the role of T cells in the pathogenesis of at least some cases of aplastic anemia and warrants further evaluation of the role of CsA in the treatment of aplastic anemia.

The case of an immunoblastic lymphadenopathy with a leukaemoid reaction has been observed. All the clinical, serological and histological features described by previous authors were present. In addition, there were more than 1000000/mul nucleated peripheral cells, consisting of mature and a few immature granulocytes, a small number of normoblasts and many lymphocytes, "immunoblast-like" lymphoid cells and plasma cells. Polyclonality of the latter was demonstrated by serological methods. Spontaneous remission occurred and was apparently enhanced by the application of corticosteroids.