Leopold Öhler

Effect of interleukin-3 pretreatment on granulocyte/macrophage colony-stimulating factor induced mobilization of circulating haemopoietic progenitor cells

Summary. Recombinant human colony stimulating factors (CSFs) as single agents are increasingly used for mobilizing peripheral blood progenitor cells (PBPCs) for stem cell transplantation. We have shown in rhesus monkeys that interleukin‐3 (IL‐3) pretreatment markedly potentiated the increase in PBPC numbers of subsequent administration of granulocyte/macrophage‐CSF (GM‐CSF). Here we studied the effect of IL‐3 pretreatment on GM‐CSF‐induced mobilization of PB progenitors in patients who were potential candidates for autologous stem cell transplantation (n=16). Patients were treated with GM‐CSF at a dose of 5 μg/kg/d for 5 d and after a treatment free interval received another cycle of GM‐CSF immediately following pretreatment with IL‐3 at different doses and duration: 2.5 μg/kg/d (n = 4), 5μg/kg/d (n = 3) and 10μg/kg/d (n = 3) for 3d, 5μg/kg/d for 7d (n = 4) and 5μg/kg/d for 14 d (n = 2), respectively. Only 7d pretreatment with IL‐3 showed consistent effects. Although IL‐3 did not mobilize by itself, pretreatment with 5μg/kg/d of IL‐3 for 7d significantly potentiated GM‐CSF‐induced mobilization of PB CFU‐GM numbers, leading to a mean increase in PB CFU‐GM numbers over baseline by 18.5 +5.2 (SEM) fold by IL‐3/ GM‐CSF as compared to a 4.7 + 1.7‐fold increase by GM‐CSF alone. A significant enhancement by the 7d IL‐3 pretreatment was also observed for erythroid (BFU‐E) and multi‐potential progenitor cells (CFU‐mix) which were 3.3 + 1.3‐and 3.4 + 0.9‐fold, respectively, mobilized by GM‐CSF alone, as compared to 8.5 + 2.3‐ and 19.2 + 3.4‐fold, respectively, by the IL‐3/GM‐CSF combination. Our results suggest that 7 d pretreatment with IL‐3 may be a useful mean to augment mobilization of circulating progenitors by more lineage‐restricted CSFs. These findings may be important for the design of mobilization strategies that use growth factors without preceding chemotherapy.

Cytogenetic risk groups in acute myeloblastic leukaemia differ greatly in their semi-solid colony growth

We have analysed the results of semi‐solid bone marrow cultures in 296 patients with de novo acute myeloblastic leukaemia (AML) and correlated them with the leukaemic karyotype. A favourable prognostic karyotype was found in 52 patients (group A, 18·3%), an intermediate karyotype in 163 patients (group B, 57·4%), and unfavourable cytogenetics were observed in 69 patients (group C, 24·3%). Median colony growth according to the three risk groups was 2 (range 0–344) in group A, 14·5 (range 0–5000) in group B and 50·0 (0–3000) in group C (A vs. B, P < 0·001; A vs. C, P < 0·001; B vs. C, P < 0·01). Among the patients treated with chemotherapy (n = 257), median colony growth was 10 (range 0–5000) in those who achieved complete remission (CR) compared with 56·5 (range 0–1000) in patients without remission (NR) (P = 0·002). The median colony growth of all patients [13/105 bone marrow mononuclear cells (BMMCs); range 0–5000] significantly discriminated between patients regarding survival (OS 11 vs. 7 months, P = 0·044). However, multiple Cox regression analysis revealed cytogenetic risk groups as the most important predictor for achieving CR, disease‐free and overall survival, with colony growth adding no additional prognostic information. In 64 patients, colony growth was also investigated without the addition of exogenous cytokines. Interestingly, none of the patients with a favourable karyotype exhibited autonomous growth, whereas 50% with an intermediate and 73% of patients with an unfavourable karyotype displayed either partial or full autonomous growth in vitro (P = 0·0004). Our data suggest that the growth potential of the leukaemic clone seems to be critically influenced by the molecular changes emerging from chromosomal abnormalities.

Granulocyte colony-stimulating factor (rh G-CSF) as an adjunct to interferon alpha therapy of neutropenic patients with hairy cell leukemia

SummarySix patients with hairy cell leukemia (HCL) and neutropenia (median neutrophil count 563/μl, range 30–1200) were treated with recombinant human granulocyte colony-stimulating factor (G-CSF) at a dose of 5μg/kg by daily subcutaneous injection as an adjunct to interferon-alpha (IFN-a) therapy, in order to ameliorate neutropenia. Five of six patients responded to G-CSF with normalization of neutrophil counts (>1800/μl) within 2–11 days and a median neutrophil count of 5211/μl (range 4312–10160) at the end of G-CSF therapy. In three of these patients, infections resolved when neutropoiesis recovered. In one patient with very severe neutropenia (30/μl), in whom myeloid progenitors were not detectable, G-CSF therapy failed to restore granulopoiesis. Cessation or interruption of G-CSF after 2–5 weeks of therapy resulted in a rapid decline of neutrophil counts to lower or subnormal levels (median value 1478/μl, range 770–2739) within 1 week, suggesting that the improvement of granulopoiesis was dependent on G-CSF and not due to IFN-a therapy. G-CSF may be a useful adjunct to IFN-a therapy in patients with HCL in order to manage or prevent neutropenic complications in the early phase of treatment.

Diagnostic and prognostic value of colony formation of hematopoietic progenitor cells in myeloid malignancies.

ZusammenfassungHämatopoetische Vorläuferzellen des Knochenmarkes und des peripheren Blutes können im semisoliden Medium unter dem Einfluss von spezifischen Wachstumsfaktoren Kolonien ausbilden, die aus terminal differenzierten Blutzellen bestehen. Diese sogenannten „colony assays” wurden in den vergangenen Jahrzehnten zur Erforschung der normalen und malignen Hämatopoese eingesetzt. So kann mit Hilfe der colony assays das Wachstum und die Differenzierung der hämatopoetischen Vorläuferzellen unter dem Einfluss positiver oder negativer regulatorischer Moleküle untersucht werden. Überdies leisten die colony assays aber auch einen wichtigen Beitrag zur Diagnostik von hämatologischen Systemerkrankungen wie der aplastischen Anämie, der myelodysplastischen Syndrome und der myeloproliferativen Syndrome. Der vorliegende Artikel umreißt unser aktuelles Wissen über die diagnostische und prognostische Bedeutung der hämatopoetischen Vorläuferzellen im peripheren Blut und Knochenmark in myeloischen Systemerkrankungen.SummaryHematopoietic progenitor cells are capable of forming colonies of mature blood cells in semisolid media in response to specific growth factors. Colony assays have been extensively used for many years to study normal and malignant hematopoiesis in vitro. In fact, these assays have provided an excellent research tool for investigating growth and differentiation of progenitor cells in response to positive and negative regulators of hematopoiesis. However, apart from their role in basic research, colony assays are also widely used in routine clinical practice in the diagnosis of various hematologic disorders, such as aplastic anemia, myelodysplastic syndromes and myeloproliferative disorders. This review summarizes our current knowledge on the diagnostic value and prognostic significance of the growth of progenitor cells in peripheral blood and bone marrow in patients with myeloid malignancies.

Numbers of colony-forming progenitors in patients with systemic mastocytosis: potential diagnostic implications and comparison with myeloproliferative disorders.

Background An increase in colony‐forming progenitor cells (CFU) is typically seen in myeloproliferative disorders (MPD). Systemic mastocytosis (SM) is a haemopoietic neoplasm involving myeloid progenitors similar to MPD. In the present study, we measured the levels of peripheral blood (pb) and bone marrow (bm) CFU in patients with different categories of SM, and compared them with those obtained in MPD patients and healthy controls.

Decrease of circulating hematopoietic progenitor cells During interleukin-2 treatment is associated with an increase of vascular cell adhesion molecule-1, a critical molecule for progenitor cell adhesion.

Administration of interleukin-2 (IL-2) to cancer patients has been shown to transiently decrease the number of circulating hematopoietic progenitor cells, but the mechanism of this phenomenon is unknown. Recently, the interaction of vascular adhesion molecule-I (VCAM-I) with leukocyte very late antigen-4 (VLA-4) has been demonstrated to play a crucial role in the adhesion of progenitor cells to bone marrow stromal elements. Cytokine induced upregulation of VCAM-1 leads to increased binding of progenitor cells to stromal cells in vitro and inhibition of this interaction by monoclonal antibodies is associated with marked progenitor cell mobilisation in vivo. In the present study we serially determined peripheral blood progenitor cell numbers during IL-2 treatment (10 courses) in 6 cancer patients and determined in parallel levels of soluble VCAM-1 as a surrogate marker for the in vivo activation of this molecule. Our data indicate that continuous intravenous administration of IL-2 for 5 days leads to a marked decrease of circulating progenitor cells associated with a substantial increase of circulating VCAM-1. Circulating myeloid progenitor cells (CFU-GM) dropped from a mean value of 167 ± 187/ml pre IL-2 to 16 ± 15/ml on day 3 (p < 0.01). Similarily, mean erythroid progenitors (BFU-E) decreased from 282 ± 204/ml before IL-2 administration to 86 ± 61/ml on day 3 (p < 0.005). In contrast, soluble VCAM-1 rose from a mean value of 1814 ± 451 ng/ml before to 4607 ± 736 ng/ml at the end of IL-2 therapy (p < 0.0001). Sera from IL-2 treated patients did not inhibit hematopoietic colony formation from normal bone marrow. These results suggest redistribution and increased adhesion of progenitor cells to stromal and/or endothelial elements during IL-2 via the VCAM-I/VLA-4 interaction as a possible mechanism for the decrease of circulating progenitor cells during IL-2 therapy.

Semi-solid Colony Growth in Acute Myeloid Leukemia and its Relation to Cytogenetic Risk Groups

The prognostic significance of in vitro growth characteristics of leukemic blast cells in acute myeloid leukemia (AML) is well established in the literature. This review focuses on semi-solid colony growth from bone marrow mononuclear cells (BMMCs) of 322 newly diagnosed patients with AML in relation to the three established cytogenetic risk groups. The median numbers of colonies derived from cytokine-stimulated BMMCs significantly differed between patients with favorable karyotypes (2/10 6 ), intermediate cytogenetics (12/10 6 ) and unfavorable abnormalities (51.5/10 6 ). Colony growth in the absence of stimulatory cytokines was assessed in 113 patients. Individuals with unfavorable karyotypes exhibited significantly more often partial or full autonomous growth in vitro when compared to the intermediate and favorable prognostic group. Median stimulated colony growth of patients who entered complete remission (CR) was 9/10 6 BMMCs compared with 63 in patients with no response. Both, cytokine-stimulated as well as autonomous colony formation, significantly discriminated between patients regarding survival. However, multiple regression analysis revealed cytogenetic risk groups as the most important predictor for achieving CR, with colony growth adding no additional prognostic information. In conclusion, semi-solid colony growth predicts response to treatment and survival in AML but is clearly related to karyotypic abnormalities.

Relation of In Vitro Growth Characteristics to Cytogenetics and Treatment Outcome in Acute Myeloid Leukemia: Prognostic Significance in Patients with a Normal Karyotype

We analyzed in vitro growth characteristics of bone marrow mononuclear cells (BMMCs) from 322 patients with acute myeloid leukemia (AML) in relation to cytogenetic abnormalities. Median colony growth was low in each of the cytogenetic changes associated with a favorable outcome. Most karyotypic abnormalities in the intermediate prognosis group were associated with low growth potential, but 11q23 abnormalities exhibited 8 times higher in vitro growth. Cytogenetic changes that included abn(3q) seemed to display the highest colony growth in the unfavorable prognosis group, whereas isolated -7 may have been associated with limited growth potential. In vitro growth behavior was predictive of neither rate of complete remission (CR) nor survival of AML patients within the 3 cytogenetic risk groups. In contrast, colony growth differed significantly in the subgroup of patients with a normal karyotype who achieved remission with induction treatment and those who had no remission (10 versus 81.5/105 BMMCs;P = .015). Significantly more patients with normal cytogenetics and colony growth below the 50th percentile went into CR than did patients with colony growth above the 50th percentile (82.8% versus 71.2%). Only 4 (6.8%) of the patients in the low growth group had no remission, compared with 12 (23.1%) of the patients with higher in vitro growth (P = .031, chi-square test). In conclusion, colony growth may prove useful as a prognostic factor for early treatment failure in AML patients with a normal karyotype.Int J Hematol. 2003;78:241-247.

Clinical meaning and implications of serum hemoglobin levels in patients with rheumatoid arthritis

OBJECTIVE Anemia is a common problem in rheumatoid arthritis (RA), associated with radiographic progression and disability. We explored the association of hemoglobin with a comprehensive set of variables in RA patients. METHODS We included RA outpatients in the routine setting. For each patient we performed measurements (clinical measures, blood tests including serology, markers of acute phase response and iron metabolism, including hepcidin, and circulating hematopoietic precursor levels) at baseline and 12 weeks thereafter, and analyzed their changes in patients with a treatment adaptation at baseline. We performed principal component analysis (PCA) to identify thematic groups hemoglobin was related to. Then we constructed multivariable linear models to assess the contribution of individual variables to the variability of hemoglobin. RESULTS In total, 88 patients were included (age: 58 ± 12; disease duration: 9.3 ± 9.6 years). Cross-sectionally (at baseline and week 12) hemoglobin levels were tied to iron metabolism and hematopoiesis, but not to clinical activity, based on thematic groups extracted from the PCA. In contrast, longitudinal changes in hemoglobin levels were closely linked to changes in clinical activity. Conversely, hepcidin reflected iron metabolism cross-sectionally, but changes in acute phase response longitudinally. In multivariable analysis variability components of hemoglobin were explainable by ferritin, ESR, evaluator global assessment (EGA), and iron levels, while components of hemoglobin changes were explained by changes in EGA mostly. Hepcidin was not independently associated with hemoglobin. CONCLUSION Besides its dependence on body iron status, changes in hemoglobin levels are strongly tied to disease activity, possibly revealing more about disease activity than other laboratory markers.

Interleukin-10 inhibits autonomous myelopoiesis in patients with myelofibrosis.

The spontaneous formation of colony‐forming units granulocyte/macrophage (CFU‐GM) in semisolid cultures has been shown to be due to the endogenous release of cytokines and/or to the hypersensitivity of cells against growth factors. We have reported that increased autonomous CFU‐GM growth is an in vitro characteristic of myelofibrosis (MF) which may reflect aberrant hematopoiesis in vivo. Because of its cytokine synthesis‐inhibiting action, we speculated that interleukin‐10 (IL‐10) may inhibit pathological overproduction of myeloid cells in MF by suppression of autonomous myelopoiesis. In this study, IL‐10 significantly inhibited autonomous CFU‐GM formation in vitro from peripheral blood mononuclear cells (PB MNC) in 10 of 11 patients with MF tested. In all patients, there was a mean inhibition of 69% ranging from 35% to 100%. Suppression of autonomous CFU‐GM formation by IL‐10 was dose dependent and reversible by the addition of anti‐IL‐10 antibodies. Our results indicate that IL‐10 is a potentially useful molecule to affect aberrant myelopoiesis in patients with MF.