Eva Jäger

CD34+/CD38− stem cells in chronic myeloid leukemia express Siglec-3 (CD33) and are responsive to the CD33-targeting drug gemtuzumab/ozogamicin

Background CD33 is a well-known stem cell target in acute myeloid leukemia. So far, however, little is known about expression of CD33 on leukemic stem cells in chronic leukemias. Design and Methods We analyzed expression of CD33 in leukemic progenitors in chronic myeloid leukemia by multi-color flow cytometry and quantitative polymerase chain reaction. In addition, the effects of a CD33-targeting drug, gemtuzumab/ozogamicin, were examined. Results As assessed by flow cytometry, stem cell-enriched CD34+/CD38−/CD123+ leukemic cells expressed significantly higher levels of CD33 compared to normal CD34+/CD38− stem cells. Moreover, highly enriched leukemic CD34+/CD38− cells (>98% purity) displayed higher levels of CD33 mRNA. In chronic phase patients, CD33 was found to be expressed invariably on most or all stem cells, whereas in accelerated or blast phase of the disease, the levels of CD33 on stem cells varied from donor to donor. The MDR1 antigen, supposedly involved in resistance against ozogamicin, was not detectable on leukemic CD34+/CD38− cells. Correspondingly, gemtuzumab/ozogamicin produced growth inhibition in leukemic progenitor cells in all patients tested. The effects of gemtuzumab/ozogamicin were dose-dependent, occurred at low concentrations, and were accompanied by apoptosis in suspension culture. Moreover, the drug was found to inhibit growth of leukemic cells in a colony assay and long-term culture-initiating cell assay. Finally, gemtuzumab/ozogamicin was found to synergize with nilotinib and bosutinib in inducing growth inhibition in leukemic cells. Conclusions CD33 is expressed abundantly on immature CD34+/CD38− stem cells and may serve as a stem cell target in chronic myeloid leukemia.

In vitro and in vivo effects of JAK2 inhibition in chronic myelomonocytic leukemia

In chronic myelomonocytic leukemia (CMML), colony‐forming units granulocyte/macrophage (CFU‐GM), which grow in vitro in the absence of exogenous growth factors, arise from the abnormal clone that is responsible for the overproduction of granulomonocytic cells. Previous in vitro findings including ours suggest that divergent molecular aberrations in CMML seem to converge within the GM‐CSF signaling pathway. As JAK2 is a sentinel kinase in this pathway, JAK2 inhibition may be an attractive treatment approach in CMML. We investigated the in vitro effects of the specific JAK2 inhibitor TG101209 on the autonomous CFU‐GM formation from peripheral blood mononuclear cells of patients with CMML. TG101209 was found to either block or strongly inhibit spontaneous CFU‐GM growth in all 10 patients tested. This inhibitory effect was dose dependent and significantly more pronounced as compared to the inhibitory effect on stimulated CFU‐GM growth from normal individuals. In a CMML patient with splenomegaly, who was treated with the JAK1/2 inhibitor ruxolitinib off label, we can demonstrate a spleen response and the disappearance of constitutional symptoms which was associated with a decrease in autonomous CFU‐GM formation ex vivo. Pharmacological JAK2 inhibition may be an interesting approach to be systematically studied in patients with CMML.

Evaluation of treatment responses and colony-forming progenitor cells in 50 patients with aplastic anemia after immunosuppressive therapy or hematopoietic stem cell transplantation: a single-center experience

SummaryWe analyzed the clinical course and outcome in 50 patients (27 males, 23 females) suffering from aplastic anemia (AA), treated in our department between 1987 and 2007. The median age was 37 years (range: 14–70 years). A total of 42 patients received antithymocyte globulin and cyclosporine A (CSA). Seven patients were transplanted using a matched sibling donor upfront, and one patient was treated with CSA and growth factors only. A total of 34 patients (68 %) achieved a complete remission, and 7 (14 %), a partial remission. Eight patients (16 %) did not respond to treatment, and one died shortly after transplantation. Relapses of AA occurred in eight patients (20 %). No obvious correlations between clinical parameters, including age, karyotype, existence of paroxysmal nocturnal hemoglobinuria clones, pretreatment blood counts, progenitor cell counts, and the response to immunosuppressive therapy (IST), were found. We also examined the numbers of colony-forming progenitor cells (CFUs) before and after therapy. In most responding patients, CFU numbers increased substantially after successful therapy. However, even in patients without a substantial increase in CFU, stable remissions were observed. Together, both IST and stem cell transplantation are reasonable treatment options for patients with AA.ZusammenfassungIn der vorliegenden Studie wurden die Daten von 50 Patienten (27 Männer/23 Frauen) mit Aplastischer Anämie (AA), die von 1987–2007 an unserer Klink behandelt wurden, analysiert. Das mediane Alter betrug 37 Jahre (14–70 Jahre). Zweiundvierzig Patienten erhielten Antithymozytenglobulin/ CyclosporinA (CSA) und 7 wurden primär mit einem Geschwisterspender transplantiert. Ein Patient erhielt nur CSA und Wachstumsfaktoren. Vierunddreißig Patienten (68 %) erreichten eine komplette Remission und 7 (14 %) eine partielle Remission. Acht Patienten sprachen nicht auf die Therapie an und einer verstarb kurz nach der Transplantation. Rezidive traten bei 8 Patienten (20 %) auf. Es konnten keine offensichtlichen Korrelationen zwischen klinischen Parametern wie Alter, Karyotyp, Vorhandensein eines paroxysmal-nächtliche Hämoglobinurie (PNH) – Klons, Blutbilder vor Therapie, Vorläuferzellzahl und dem Ansprechen auf die immunsuppressive Therapie gefunden werden. Wir untersuchten auch die Vorläuferzellzahl vor und nach Therapie. In den meisten Patienten stieg die Vorläuferzellzahl nach erfolgreicher Therapie deutlich an. Allerdings erreichten sogar manche Patienten ohne einen substantiellen Anstieg der Vorläuferzellzahl stabile Remissionen. Insgesamt sind sowohl die immunsuppressive Therapie als auch die Stammzelltransplantation wirksame Therapieoptionen bei Patienten mit Aplastischer Anämie.

Recombinant human interleukin-10 in patients with chronic myelomonocytic leukemia

With the successful use of ruxolitinib in patients with myelofibrosis, cytokine inhibition as a therapeutic concept has regained increased attention. We report here our limited clinical experience in patients with chronic myelomonocytic leukemia (CMML) with the cytokine synthesis inhibiting molecule rhIL-10 (Schering-Plough Corp., Kenilworth, NJ, USA) that was suggested by us as a potential candidate for anticytokine treatment several years ago [1]. We have originally demonstrated that in vitro CMML cells can form CFUGM in semisolid cultures without the addition of exogenous growth factors [2]. Moreover, we have shown that spontaneous colony growth from CMML cells can be markedly reduced by addition of antigranulocyte/macrophage colonystimulating factor (GM-CSF) antibodies, but not by addition of antibodies against G-CSF, IL-3, or IL-6 suggesting that GM-CSF signaling may play a major role in the pathophysiology of this disease [3]. Because of its cytokine synthesisinhibiting activity [4], we have tested interleukin-10 (IL-10) in order to affect leukemic cell growth in CMML in vitro. The specific binding of IL-10 had a profound and dose-dependent inhibitory effect on autonomous in vitro growth of CMML cells [3]. IL-10-induced suppression of CMML cell growth was reversed by the addition of exogenous GM-CSF and correlated with a substantial decrease in GM-CSF production by leukemic cells, both at the mRNA and protein levels. These data indicated that IL-10 profoundly inhibits the autonomous growth of CMML cells in vitro most likely through suppression of endogenous GM-CSF release. This observation suggested therapeutic evaluation of rhIL-10 in patients with CMML. In a small pilot trial, we have treated five patients with CMML (four patients with CMML I and one patient with CMML II) with 4 μg/kg/day recombinant human IL-10 (rh IL-10) sc for 1 month and with 8 μg/kg/day for another month. The dosages of IL-10 used in this pilot trial were based on experiences from IL-10 trials in other indications including Crohn’s disease [5], rheumatoid arthritis [6], and psoriasis [7]. We did not observe meaningful effects of IL-10 treatment on the WBC counts in any of the five CMML patients. In one out of three patients, histologically confirmed skin infiltration markedly improved during IL-10 therapy (Fig. 1). In this patient, IL-10 therapy was associated with a clear drop of serum levels of lysozyme which has been shown to be released from peripheral blood monocytes in culture [8] and which is commonly increased in patients with CMML [9]. Moreover, CD86 expression which has been shown to be upregulated by GM-CSF and downregulated by IL-10 in vitro [10] gradually decreased in this patient. With regard to toxicity, one patient (CMML II) died at home with the symptoms of an acute heart attack in the third week of IL-10 treatment, another patient who had splenectomy because of immunothrombocytopenia 4 months prior to study entry developed thrombocytopenia which was reversible after the end E. Jäger Department of Laboratory Medicine, Medical University of Vienna, Vienna, Austria

Circulating hematopoietic progenitor cells in essential thrombocythemia versus prefibrotic/early primary myelofibrosis

2.7 (95% CI: 1.1–5.8, P5 0.032) and the age and HU-adjusted HR was 2.6 (95% CI: 1.1–6.4, P5 0.031) (Fig. 1C). Nephrin, a major component of the slit diaphragm that maintains normal podocyte foot process architecture, is a sensitive urinary biomarker for glomerular injury and is detected before albuminuria develops in animal models [4]. Kidney injury molecule-1 (KIM-1) is a transmembrane proximal tubular protein with increased expression after tubular injury [5]. We used ELISA to measure urine concentrations of nephrin (Exocell, Philadelphia, PA) and KIM-1 (R&D Systems, Minneapolis, MD) in 32 adult SCA patients, six from the follow-up cohort just described and 26 additional consecutive outpatients with eGFR >60mL/min/1.73 m, between March and May 2013. Urine nephrin concentration correlated significantly with eGFR (r5 0.36, P5 0.044) but urine KIM-1 concentration did not (r5 0.11, P5 0.6). Urine nephrin concentration also correlated strikingly with urine albumin concentration (r5 0.92, P< 0.0001) (Fig. 1D). Our findings are consistent with the possibility that a high eGFR causes damage to the glomerulus reflected by its correlation with a urine biomarker of glomerular injury but not with a urine biomarker of tubular injury and by the increased risk for developing microalbuminuria in SCA patients with hyperfiltration. Limitations to this study include that we did not directly measure the GFR, although the CKD-Epidemiology-based eGFR is reported to have good correlation with measured GFR in patients with SCA [6]. Another potential limitation is that nephrin is expressed in the pancreas and may be circulating in the plasma and filtered through the glomerulus with albumin which will need to be evaluated in future studies. Furthermore, our study may have been underpowered to detect associations between hydroxyurea therapy or BMI and the development of microalbuminuria. In conclusion, a higher degree of persistent hyperfiltration in adults may be an important risk factor for the development of CKD in addition to other factors such as joint polymorphisms in MYH9 and APOL1. Future research is needed to understand mechanisms of how hyperfiltration may damage the glomerulus and to identify interventions to protect the kidney in this context.

High spontaneous granulocyte/macrophage-colony formation in patients with myelofibrosis

Unstimulated methylcellulose cultures in 25 myelofibrosis (MF) patients were performed to better understand the role of cytokines in the proliferation of MF cells. Compared to controls MF patients show a variable but highly increased spontaneous CFU-GM formation (66 vs 4.8/10(5) PBMNC). There was a marked reduction of autonomous CFU-GM growth by the cytokine-synthesis-inhibiting molecule IL-10 as well as by antibodies against GM-CSF whereas antibodies against IL-3, G-CSF, M-CSF and IL-1β showed heterogeneous effects. Spontaneous CFU-GM growth >100/10(5) PBMNC predicted shorter survival. Constitutive release of GM-CSF seems to contribute to proliferation of MF cells in vitro and possibly in vivo.

Is ruxolitinib a potentially useful drug in hematological malignancies with RAS pathway hyperactivation

It was with great interest that we read this journal’s article by Sachs et al. in which, in a preclinical mouse model, the critical role of Stat5 in the development and maintenance of myeloproliferative neoplasms (NPM) initiated by Nf1-deficiency, has been nicely demonstrated.[1][1] Since

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.