Robert Möhle

High expression of the chemokine receptor CXCR4 predicts extramedullary organ infiltration in childhood acute lymphoblastic leukaemia

Childhood acute lymphoblastic leukaemia (ALL) is a malignancy with the potential to infiltrate the liver, spleen, lymph nodes and brain. Such extramedullary presentation is important for understanding the biology of childhood ALL and also for developing new prognostic parameters. A potential mechanism in the trafficking of leukaemia cells is the interaction of the chemokine receptor CXCR4, which is expressed on ALL cells, and its ligand stromal cell‐derived factor‐1 (SDF‐1), produced by stromal cells in bone marrow and extramedullary organs. Functionality of CXCR4 was demonstrated by a high correlation between cell surface density of CXCR4 and transendothelial migration of leukaemia blasts towards a gradient of SDF‐1 (r = 0·73, P = 0·001). Inhibition of SDF‐1‐induced migration by an anti‐CXCR4 monoclonal antibody (78·33 ± 23·86% inhibition) evidenced the specificity of CXCR4 to SDF‐1. In order to evaluate clinical significance of CXCR4 expression, lymphoblasts from the bone marrow of 73 patients with and without extramedullary organ infiltration were compared. Multiparameter flow cytometry revealed that lymphoblasts from patients with high extramedullary organ infiltration, defined as ultrasonographically measured enlargement of liver or spleen, expressed the CXCR4 receptor at higher fluorescence intensity (median 66·12 ± 66·17) than patients without extramedullary organ infiltration (median 17·56 ± 19·29; P < 0·001). Consequently, high expression of CXCR4 was strongly predictive for extramedullary organ involvement, independently of the peripheral lymphoblast count. Highest CXCR4 expression was seen in mature B ALL (median 102·74 ± 92·13; P < 0·003), a disease characterized by a high incidence of extramedullary bulky disease. As high expression of the chemokine receptor CXCR4 predicts extramedullary organ infiltration in childhood ALL, we suggest that CXCR4 and its ligand play an essential role in extramedullary invasion.

Functional response of leukaemic blasts to stromal cell-derived factor-1 correlates with preferential expression of the chemokine receptor CXCR4 in acute myelomonocytic and lymphoblastic leukaemia

The chemokine stromal cell‐derived factor‐1 (SDF‐1) that is released by bone marrow (BM) stromal cells and contributes to stem cell homing may also play a role in the trafficking of leukaemic cells. We analysed SDF‐1‐induced intracellular calcium fluxes in leukaemic blasts from the peripheral blood of patients with newly diagnosed acute myeloid leukaemia (AML) and lymphoblastic leukaemia (B‐lineage ALL), determined the effect of BM stromal cell‐conditioned medium on in vitro transendothelial migration (TM) and measured expression of the SDF‐1 receptor, CXCR4, by flow cytometry. AML FAB M1/2 blasts did not show calcium fluxes and TM was not stimulated. In myelomonocytic AML (M4/5), however, SDF‐1 induced significant calcium fluxes and TM was increased twofold by the conditioned medium. M3 and M4 blasts with eosinophilia (M4eo) showed intermediate activity and M6 blasts showed no functional activity. In ALL, strong calcium fluxes and increased TM (2.5‐fold) were observed. Accordingly, expression of CXCR4 was low in undifferentiated (M0) AML, myeloid (M1/2) AML and erythroid (M6) AML, but high [mean fluorescence (MF) > 50] in promyelocytic (M3) AML, myelomonocytic (M4/5) AML and B‐lineage ALL. We conclude that, in AML, SDF‐1 is preferentially active in myelomonocytic blasts as a result of differentiation‐related expression of CXCR4. Functional activity of SDF‐1 and high expression of CXCR4 in B‐lineage ALL is in accordance with the previously described activity of SDF‐1 in early B cells. SDF‐1 may contribute to leukaemic marrow infiltration, as suggested by increased CXCR4 expression and migratory response in BM‐derived blasts compared with circulating cells.

Expression of vascular endothelial growth factor (VEGF) and its receptors in human neuroblastoma

Angiogenic factors may play a role in the biology of neuroblastoma, a well vascularised tumour, which frequently spreads haematogenously. Therefore, we analysed expression of vascular endothelial growth factor (VEGF) in six human neuroblastoma cell lines and five primary neuroblastomas. High VEGF levels (1-3 ng/10(6) cells/day) were found in the supernatant of all cell lines examined (SK-N-LO, SK-N-SH, LS, SH-SY5Y, IMR-32, Kelly). VEGF peptide was also detected in tissue homogenates from four of five primary tumours. Reverse transcription-polymerase chain reaction (RT-PCR) revealed that VEGF165 is the major isoform produced by neuroblastomas. In addition, all cell lines and primary tumours expressed the mitogenic VEGF receptor FLK-1, whilst the non-mitogenic receptor FLT-1 was less frequently positive, suggesting that the tyrosine kinase FLK-1 is involved in malignant transformation of neuroblastoma cells. However, neutralising antibodies to VEGF did not inhibit growth of neuroblastoma cell lines, which argues against a role of VEGF as an autocrine growth factor, at least for cell lines in vitro. We conclude that neuroblastoma cells produce VEGF, which may contribute to tumour vascularisation, growth and invasion.

Expression and secretion of vascular endothelial growth factor-A by cytokine-stimulated hematopoietic progenitor cells: Possible role in the hematopoietic microenvironment

In the hematopoietic microenvironment, bone marrow endothelial cells may play an important role in trafficking and maintenance of progenitor and stem cells due to adhesive interactions and paracrine secretion of hematopoietic growth factors. However, it is unknown whether progenitors in turn modulate endothelial proliferation and function. We analyzed mRNA expression (Northern blot) and release of vascular endothelial growth factor-A (VEGF-A), which specifically acts on endothelial cells, by cytokine-stimulated peripheral blood-derived CD34+ hematopoietic progenitor cells. While unstimulated CD34+ cells expressed VEGF-A mRNA weakly without cytokine release in vitro, incubation for 24 hours with a single cytokine (e.g., kit ligand [KL]) resulted in increased VEGF-A mRNA expression and significant secretion of VEGF-A into the supernatant. The amount of VEGF released was substantially augmented by incubation with a combination of cytokines (e.g., KL, IL-3, GM-CSF, G-CSF), or by exposure to hematopoietic cytokines for a longer time period. In addition, we show that VEGF induced the release of hematopoietic growth factors (GM-CSF) by bone marrow endothelial cells and that in vitro stromal cell-derived factor-1 (SDF-1) driven transendothelial progenitor cell migration was increased by the presence of VEGF, which might be due to pore formation (increased endothelial fenestration). In vivo, release of VEGF by progenitor cells may result in a paracrine loop supporting proliferation of both endothelium and progenitors and may facilitate transendothelial migration during cytokine-induced progenitor cell mobilization.

The Role of Sphingosine 1‐Phosphate Receptors in the Trafficking of Hematopoietic Progenitor Cells

Abstract: Sphingosine 1‐phosphate (S1P) is an ubiquitously present extracellular lipid mediator that is released by several cell types, particularly by activated platelets. The effects of S1P are mediated by a specific family of G protein‐coupled sphingosine 1‐phosphate receptors (S1P1‐S1P5). We demonstrate that S1P acts on hematopoietic progenitor cells as a chemotactic factor, attracting peripheral blood CD34+ cells in vitro. Furthermore, constant activation of S1P receptors augments CXCR4‐mediated signal transduction induced by stromal cell‐derived factor 1 (SDF‐1). These effects are most likely mediated by the S1P1 receptor consistently expressed in both primitive and committed CD34+ hematopoietic progenitor cells (HPCs). In vivo, sustained activation of S1P1 by a receptor agonist during the homing process resulted in increased engraftment. Given the fact that activated platelets represent a major source of extracellular S1P, SDF‐1‐mediated stem cell homing may occur at sites of tissue injury in addition to the bone marrow. This could explain the previously observed contribution of primary hematopoietic stem cells to tissue repair in myocardial infarction and other diseases.

Regulation of transendothelial migration of hematopoietic progenitor cells.

Abstract: Transendothelial migration of hematopoietic progenitor cells occurs in the bone marrow during mobilization and homing, and may therefore play a key role in the trafficking of hematopoietic stem cells. We hypothesize that adhesion molecules, chemokines, and paracrine cytokines are involved in this multifactorial process. As suggested in several studies, downregulation of adhesion molecules (e.g., integrins) may contribute to mobilization of progenitors due to a decreased avidity to bone morrow stromal and endothelial cells, which express the corresponding ligands. Using an in vitro model of transendothelial migration, we have shown that only a small number of more mature, committed progenitors migrates spontaneously under the control of adhesion molecules of the beta‐2‐integrin family and their corresponding endothelial/ stromal ligands. However, transendothelial migration of progenitors in vitro is substantially enhanced by the chemokine stromal cell‐derived factor‐1 (SDF‐1), which is constitutively produced by bone marrow stromal cells. More primitive progenitors also respond to this chemokine. In addition, the ligand for SDF‐1, the chemokine receptor CXCR‐4, is expressed in greater levels on bone marrow CD34+ cells as compared to mobilized progenitors, suggesting that downregulation of chemokine receptors occurs during progenitor mobilization. Indeed, bone marrow CD34+ cells migrate more avidly in response to SDF‐1 than mobilized progenitors. Paracrine cytokines may also play a role in hematopoietic stem cell trafficking, since growth factor‐stimulated hematopoietic cells produce cytokines that act on endothelial cells (e.g., vascular endothelial growth factor, VEGF), modifying their proliferation, motility, permeability, and fenestration. We conclude that transendothelial migration of hematopoietic progenitor cells is regulated by adhesion molecules, paracrine cytokines, and chemokines. Cytotoxic therapy as well as exogenously administered hematopoietic growth factors may affect adhesion molecule expression, the local cytokine and chemokine milieu, and chemokine receptor expression, which indirectly results in mobilization of hematopoietic stem cells.

In vitro myogenic differentiation of human bone marrow-derived mesenchymal stem cells as a potential treatment for urethral sphincter muscle repair.

To explore a new treatment strategy for urinary incontinence, human bone marrow mesenchymal stem cells (MSC) of the first in vitro passage were exposed to 5‐azacytidine (AZA) to induce myogenic differentiation, and cultured for a total of six passages. Expression of stem cell surface antigens and intracellular α‐actin was examined by flow cytometry at the end of each passage and compared to that of native MSC (not exposed to AZA) cultured in parallel. To analyze differentiation into striated muscle, expression of the transcription factor MyoD1 and myosin heavy chain (MyHC) was examined by RT‐PCR. Both native and AZA‐exposed MSC of all passages were negative for the progenitor/endothelial antigen CD34, leukocytic CD45, and endothelial/monocytic CD31. In contrast, the MSC markers CD73, CD90, CD105, and intracellular actin were detected in both groups of MSC throughout the culture period. After an initial increase, the expression level of MSC antigens decreased over time particularly in AZA‐exposed MSC. Expression of smooth muscle α‐actin also declined, but was greater in AZA‐exposed MSC throughout the culture period. Varying percentages of MSC cultures expressed MyoD1 and MyHC mRNA. In late passages, AZA‐exposed MSC tended to be more frequently positive than native MSC. In pilot experiments, transplantation of MSC into the bladder neck tissue of athymic rats was feasible; long‐term analyses are pending. We conclude that independent of AZA exposure, MSC express smooth and striated muscle antigens. Treatment with AZA slightly increases myogenic differentiation, but may not be necessary in future studies of MSC as a treatment modality for urinary incontinence.

Phase II study of central nervous system (CNS)-directed chemotherapy including high-dose chemotherapy with autologous stem cell transplantation for CNS relapse of aggressive lymphomas

The prognosis of patients with central nervous system relapse of aggressive lymphoma is very poor with no therapy established so far. In a prospective multicenter phase II study, we evaluated a potentially curative chemotherapy-only regimen in these patients. Adult immunocompetent patients 65 years of age or under received induction chemotherapy with MTX/IFO/DEP (methotrexate 4 g/m2 intravenously (i.v.) Day 1, ifosfamide 2 g/m2 i.v. Days 3– 5 and liposomal cytarabine 50 mg intrathecally (i.th) Day 6) and AraC/TT/DEP (cytarabine 3g/m2 i.v. Days 1–2, thiotepa 40 mg/m2 i.v. Day 2 and i.th. liposomal cytarabine 50 mg i.th. Day 3) followed by high-dose chemotherapy with carmustine 400 mg/m2 i.v. Day −5, thiotepa 2×5 mg/kg i.v. Days −4 to −3 and etoposide 150 mg/m2 i.v. Days −5 to −3, and autologous stem cell transplantation Day 0 (HD-ASCT). Thirty eligible patients (median age 58 years) were enrolled. After HD-ASCT (n=24), there was a complete remission in 15 (63%), partial remission in 2 (8%) and progressive disease in 7 (29%) patients. Myelotoxicity was the most adverse event with CTC grade 3/4 infections in 12% of MTX/IFO/DEP courses, 21% of AraC/TT/DEP courses and 46% of HD-ASCT courses. The 2-year time to treatment failure was 49%±19 for all patients and 58%±22 for patients completing HD-ASCT. The protocol assessed proved feasible and highly active with long-lasting remissions in a large proportion of patients. (ClinicalTrials.govIdentifier NCT01148173)

Transendothelial Migration of Hematopoietic Progenitor Cells

Abstract: There is increasing evidence that hematopoietic stem cell mobilization and homing is regulated not only by adhesion molecules and cytokines, but also by chemotactic factors that support transendothelial migration across the bone marrow sinusoidal endothelium. Many receptors for chemotactic mediators belong to the family of G protein‐coupled seven‐transmembrane receptors (7‐TMR). Signaling via G proteins, particularly Gi proteins, results in a chemotactic response of the cells towards a gradient of the corresponding ligand. Recent studies have provided evidence for expression of several 7‐TMR on immature hematopoietic progenitor cells, which potentially mediate chemotactic effects: chemokine receptors (e.g., CXCR4, receptor for stromal cell‐derived factor‐1), receptors for lipid mediators (e.g., the cysteinyl leukotriene receptor cysLT1 and the peripheral cannabinoid receptor cb2), and receptors for neuroendocrine hormones (e.g., the somatostatin receptor sst2). From these studies it can be concluded that migration of hematopoietic progenitor and stem cells is controlled by a variety of chemotactic factors rather than by a single chemokine (e.g., SDF‐1). Trafficking of immature hematopoietic cells may require combined and interactive regulatory functions of these mediators.

Outcome of elderly patients with primary CNS lymphoma in the G-PCNSL-SG-1 trial

Objective: To assess the outcome of elderly patients with primary CNS lymphoma (PCNSL) treated within the G-PCNSL-SG-1 trial. Methods: We reviewed response, toxicity, and survival of patients with PCNSL aged 70 or more enrolled in the G-PCNSL-SG-1 trial. Results: A total of 126 of the 526 eligible patients (24%) and 66 of 318 patients (21%) in the per protocol population were aged 70 or more. Among all eligible patients, the rate of complete and partial responses (CR+PR) to HD-MTX-based chemotherapy was 44% in the elderly vs 57% in the younger patients (p = 0.016). Toxicity was age-independent except for a higher rate of grade III/IV leukopenia in the elderly (34% vs 21%, p = 0.007). Death on therapy was more frequent (18% vs 11%; p = 0.027), and progression-free survival (PFS) (4.0 vs 7.7 months, p = 0.014) and overall survival (12.5 vs 26.2 months, p < 0.001) inferior, in the elderly. A striking difference between younger and elderly patients was the PFS of CR patients of 35.0 in the younger vs 16.1 in the elderly patients (p = 0.024). Elderly patients were treated less often and less aggressively at salvage. However, age was not associated with survival from salvage whole brain radiotherapy in patients progressing during primary HD-MTX-based chemotherapy (p = 0.633). Conclusions: Lower response rate and higher mortality on HD-MTX-based chemotherapy as well as lower PFS of CR patients and less salvage therapy contribute to the poor prognosis of elderly patients with PCNSL.