Background: In about half of patients with lower-risk (LR) myelodysplastic syndromes (MDS), thrombocytopenia is present at the time of diagnosis, being associated with shortened survival and a higher risk of progression to acute myeloid leukemia (AML). Romiplostim (ROM), a thrombopoietin receptor agonist (TPO-RA), has shown safety and clinical efficacy in prospective trials in LR-MDS. Post-hoc analyses have demonstrated hematologic improvement of platelets (HI-P) after ROM treatment contingent on endogenous thrombopoietin (TPO) levels and platelet transfusion events (PTE) (Sekeres et al. BJH 2014). Aims: The prospective EUROPE multicenter phase 2 trial (NCT02335268) investigated the predictive value of biomarkers like endogenous TPO levels, PTE and molecular markers on the clinical efficacy of single-agent ROM treatment within the `European Myelodysplastic Syndromes Cooperative Group` (EMSCO) network. Patients with IPSS low or intermediate 1 risk were eligible if baseline bone marrow blast count was <5% (central morphology) and platelet count was ≤30 Gpt/L or ≤50 Gpt/L in case of a bleeding history. Methods: According to a previously published model of response to TPO-RA (Sekeres et al. BJH 2014), patients were assigned into two different cohorts at the time of screening based on previous PTE and centrally assessed TPO serum levels (cohort A: TPO<500 ng/l and PTE<6 units/past year; cohort B: TPO>500 ng/l, and/or PTE36 units/past year). The primary efficacy endpoint was the rate of HI-P according to IWG 2006 criteria lasting for 38 weeks. ROM was initiated at a dose of 750 μg weekly by subcutaneous injection, the dose was adjusted based on the patient`s platelet counts. Results: From 2015 to 2019, a total of 77 patients were included at 29 different trial sites in Germany, France and the Czech Republic. Regarding the primary endpoint, 32 out of 77 (42%) responded (HI-P) with a numerically higher response rate in cohort A (47%, n=24) vs. cohort B (31%, n=8) (p=0.2953). At 16 weeks of ROM treatment, three (4%) and seven (9%) patients had additional neutrophil (HI-N) and erythroid (HI-E) responses, respectively. None of the patients achieved trilineage responses (HI-P, HI-E and HI-N). Median duration of response was significantly longer for patients in cohort A (351 days) compared to cohort B (315 days) (p=0.006, log-rank-test). Mutated SRSF2 was significantly more frequent in responders (41%) compared to non-responders (16%) (p=0.018, Fisher’s exact test). In patients with an SRSF2 mutation, the probability to achieve HI-P was 65% compared to 33% inpatients with SRSF2 wildtype (Figure 1A). Comparing responders vs. non-responders, we found no significant changes of variant allelic burden of variants detected pre- and post-ROM (Figure 1B). Finally, we developed a response prediction model to ROM therapy with the aim to improve personalized patient stratification in the future. The percentage of correctly predicted HI-P was highest for the model, which included the variables platelet count, SRSF2 mutation status and the hemoglobin level using the threshold of 11.4 g/dl and resulted in an overall accuracy of 70 % for a correct ROM response prediction (Figure 1A). Image:Summary/Conclusion: In conclusion, this prospective study confirms the efficacy and overall safety of ROM in this subgroup of LR-MDS patients with thrombocytopenia. To avoid overfitting of variables and to confirm our results, the here presented response prediction model needs to be validated in an external independent cohort. * U.P. and L.A. contributed equally to this study as senior authors

Deregulated apoptosis is an identifying feature of myelodysplastic syndromes (MDS). Whereas apoptosis is increased in the bone marrow (BM) of low-risk MDS patients, progression to high-risk MDS correlates with an acquired resistance to apoptosis and an aberrant expression of BCL-2 proteins. To overcome the acquired apoptotic resistance in high-risk MDS, we investigated the induction of apoptosis by inhibition of pro-survival BCL-2 proteins using the BCL-2/-X L /-W inhibitor ABT-737 or the BCL-2-selective inhibitor ABT-199. We characterized a cohort of 124 primary human BM samples from MDS/secondary acute myeloid leukemia (sAML) patients and 57 healthy, age-matched controls. Inhibition of anti-apoptotic BCL-2 proteins was specifically toxic for BM cells from high-risk MDS and sAML patients, whereas low-risk MDS or healthy controls remained unaffected. Notably, ABT-737 or ABT-199 treatment was capable of targeting the MDS stem/progenitor compartment in high-risk MDS/sAML samples as shown by the reduction in CD34 + cells and the decreased colony-forming capacity. Elevated expression of MCL-1 conveyed resistance against both compounds. Protection by stromal cells only partially inhibited induction of apoptosis. Collectively, our data show that the apoptotic resistance observed in high-risk MDS/sAML cells can be overcome by the ABT-737 or ABT-199 treatment and implies that BH3 mimetics might delay disease progression in higher-risk MDS or sAML patients.

Myelodysplastic syndromes (MDS) are a group of common bone marrow disorders characterized by ineffective hematopoiesis, peripheral cytopenias, and a propensity for transformation to acute myeloid leukemia (AML). For many years, the main treatment option for MDS was best supportive care which alleviates symptoms but has no effect on the natural course of the disease. The recent approval of the demethylating agent azacitidine represents a significant advance in the treatment of MDS. The results of two randomized trials with azacitidine have shown an overall response rate between 40% and 60%, an improved quality of life, a reduced risk of transformation to AML and a definite survival advantage compared to best supportive care or low-dose chemotherapy. Current data on azacitidine and its place in the treatment of MDS are reviewed.

Mobilization and recruitment of endothelial progenitor cells (EPC) contributes to vasculogenesis in vivo. So far, applications for cell therapy are limited by the number of available cells. Expansion of EPC or their progeny may, therefore, facilitate its therapeutic use in ischemic disease. The aim of this study was to expand CD34+ EPC-derived progeny from different sources, characterize them, and investigate their potential for use in therapeutic vasculogenesis. CD34+ cells from G-CSF-mobilized peripheral blood (PB) and cord blood (CB) were isolated using immunomagnetic beads and cultured in endothelial cell medium. Cells were expanded up to 16 (PB) and up to 46 (CB) population doublings, respectively. Immunophenotypic and mRNA expression analyses showed a high degree of similarity between the cultured cells and human umbilical vein endothelial cells (HUVEC). By day 14 after transplantation, transplanted human CD31-positive EPC-derived cells were detected. These cells expressed the proliferation marker Ki67 and formed vessel-like structures in ischemic myocardium. Most strikingly, transplantation of EPC-derived cells improved left ventricular function after experimental ischemia, as shown by echocardiography. In conclusion, cells cultured from CD34+ EPC can be expanded in vitro to clinically relevant numbers. In vivo, these cells proliferate, form vascular structures, and improve left ventricular function after experimental myocardial infarction. Therefore, in vitro expanded EPC-derived endothelial cells may be beneficial in the treatment of ischemic disease.

CD34 is a sialomucin expressed on hematopoietic cells, endothelial cells and muscle satellite cells. Within the hematopoietic system, CD34 expression has been associated with very immature progenitor cells as well as hematopoietic stem cells (HSC), and it is widely used to assess stem cell activity in clinical protocols. In the past, HSC activity was thought to be retained exclusively in the subset of cells expressing CD34. This view has been challenged by recent observations in mice in which HSC activity was also found in the CD34-negative fraction. These findings have since been reproduced using human marrow and cord blood cells. However, the exact relationship between CD34+ and CD34− stem cells remains unclear. We investigated the regulation of CD34 expression as dependent on cell division history. To follow cell division, human cord blood cells were labeled with the fluorescent dye CFSE. Lin-CD34−CD133+CFSE+ (CD34−) and CD34+ populations were almost indistinguishable in their ability to produce CAFCweek6 content. After three days of serum-free culture with stem cell factor, Flt3 ligand and thrombopoietin, almost all initially CD34− cells had acquired expression of CD34, including all undivided cells. We found that, in cultures initiated from CD34− cells, virtually all CAFCweek6 were produced from the divided, now CD34+ cells, indicating these cells had self-renewed. In contrast, similar cultures from initially CD34+ cells demonstrated that hematopoietic activity associated with the undivided cell fraction. We did not find any hematopoietic activity in the cell fraction that remained CD34− or the fraction that lost CD34 after division. Analysis of mRNA expression showed that CD34− and CD34+ cells expressed almost equal levels of CD34, AC133, Flt1, Flk1 and Flt4, while CD34− cells expressed significantly lower levels of Tie1 and Tie2 than CD34+ cells. The expression of CD34 message in CD34− cells was explained by our observation that these cells contained intracellular CD34, indicating that they are “primed” to express the antigen on their cell surface. In conclusion, Lin−CD34−CD133+ cells acquire expression of CD34, even in the absence of cell divisions. These CD34− cells self-renew more rapidly in vitro than cells initially expressing CD34, and self-renewal is preceded by acquisition of CD34 antigen.

Chronic myeloid leukemia (CML) and a subset of acute lymphoblastic leukemias arise from the genetic reciprocal translocation t(9;22), forming the BCR-ABL fusion gene. These lead to the expression of the constitutively active tyrosine kinase BCR-ABL, which is the causative oncogene for these leukemias. Allogeneic bone marrow transplantation (BMT) or stem cell transplantation (SCT) is currently considered the only curative treatment for chronic myeloid leukemia (CML). Recently, the selective tyrosine kinase inhibitor imatinib mesylate (Glivec, formerly STI-571) has been shown to induce durable hematologic and major cytogenetic responses in a high percentage of patients with chronic phase CML. In patients with advanced disease remissions are transient and most patients relapse despite continued imatinib treatment. Some of these patients go on to receive allogeneic BMT or SCT, during which administration of imatinib is usually discontinued as it is believed to interfere with bone marrow engraftment. In this study, we examined the effect of imatinib on hematopoietic engraftment in a syngeneic mouse model. We found that imatinib has no significant influence on hematopoietic recovery in lethally irradiated mice in vivo. Thus, our results suggest that continued administration of imatinib in the course of BMT or SCT may be a feasible therapeutic regimen.

Expansion of primitive hematopoietic progenitor cells (HPC) is a major challenge in stem cell biology. Stimulation by growth factors (GF) is essential for proliferation of HPC, while the role of stromal cell coculture for maintenance of progenitor/stem cell potential is unclear. We evaluated the potential of a murine stromal cell layer providing hematopoietic GF to support expansion of human CD34 + cells. Murine MS-5 cells were transfected with the cDNA encoding huFlt3 ligand and the interleukin6/sinterleukin-6R fusion protein hyper-IL-6. Expansion of CFC and week6 CAFC was at least as efficient in transfected clones compared to control cocultures supported with exogenous GF. Cell numbers reached 17.5- to 62.3- (day 14) and 17.4- to 92.4-fold (day 21) of input cells. Expansion of CFU-GM/Mix was 4.0- to 12.8-fold (day 14) and 4.9- to 11.7-fold (day 21). Primitive week6 CAFC were expanded up to 6.5-fold (day 14) and 6.2-fold (day 21) without exogenous GF. When direct contact of HPC and stromal cells was inhibited, a loss of CFC and much more of CAFC potential was observed with unaffected overall cell proliferation. Here, we show the generation of GF producing murine stromal cells which efficiently support early hematopoiesis without exogenous GF. Direct stromal cell–HPC contact is advantageous for maintenance of differentiation potential.

ObjectiveExperimental strategies for ex vivo expansion of human hematopoietic stem/progenitor cells involve the use of exogenous cytokines as well as direct interaction with stromal elements. We examined the use of the interleukin-6/soluble interleukin-6 receptor (IL-6/sIL-6R) fusion protein Hyper-IL-6 (H-IL-6), which interacts directly with gp130, in conjunction with stromal support for expansion of human progenitors.Materials and MethodsPeripheral blood CD34+ cells were cultured on the murine stromal cell line FBMD1 or in suspension for up to 28 days with different cytokines. Cells were evaluated at various time points for phenotype, proliferative and clonogenic capacity, and long-term hematopoietic activity.ResultsThe combination of Flt3 ligand and H-IL-6 was markedly more effective than Flt3 ligand and IL-6/sIL-6R for expansion of CD34+ cells in suspension culture and on FBMD1. Addition of kit ligand but not thrombopoietin to Flt3 ligand and H-IL-6 significantly augmented proliferation and enhanced colony formation three-fold. However, long-term cobblestone area-forming cell assays indicated that although multipotent progenitors were maintained up to 21 days on FBMD1 in the presence of Flt3 ligand alone and were amplified three-fold by addition of H-IL-6, CD34+ cells cultured in the absence of stromal support rapidly lost their cobblestone area-forming cell potential. Immunophenotyping revealed that stromal support prevented up-regulation of IL-6R on CD34+ cells, which was induced within 3 days in stroma-free cultures and was enhanced in the presence of kit ligand. Delayed addition of H-IL-6 to the cultures resulted in reduced proliferation and colony-forming unit potential.ConclusionH-IL-6 synergizes with stromal elements to effectively enhance proliferation and maintenance of primitive hematopoietic progenitors under prolonged ex vivo culture conditions.

Human herpes virus-8 (HHV8) encodes a cytokine named viral interleukin-6 (vIL-6) that shares 25% amino-acid identity with its human homologue. Human IL-6 is known to be a growth and differentiation factor of lymphatic cells and plays a potential role in the pathophysiology of various lymphoproliferative diseases. vIL-6 is expressed in HHV8-associated-diseases including Kaposi's sarcoma, Body-cavity-based-lymphoma and Castleman's disease, suggesting a pathogenetic involvement in the malignant growth of B-cell associated diseases and other malignant tumours. We expressed vIL-6 in Escherichia coli as a fusion protein with recombinant periplasmic maltose binding protein. After cleavage from the maltose binding protein moiety and purification, vIL-6 was shown to be correctly folded using circular dichroism spectroscopy. A rabbit antiserum was raised against the recombinant vIL-6 protein. vIL-6 turned out to be active on cells that expressed gp130 but no IL-6 receptor (IL-6-R) suggesting that, in contrast to human IL-6, vIL-6 stimulated gp130 directly. Accordingly, vIL-6 activity could be inhibited by a soluble gp130 Fc Fusion protein. vIL-6 was shown to induce neuronal differentiation of rat pheochromocytoma cells and to stimulate colony formation of human hematopoietic progenitor cells. Thus, vIL-6 exhibits biologic activity that has only been observed for the IL-6/soluble IL-6-R complex but not for IL-6 alone. These properties are important for the evaluation of the pathophysiological potential of vIL-6.