Anemia Associated with Chronic Disease in Patients with Chronic Lymphocytic Leukemia Is Not Attributed to Decreased Production or Impaired EPO Signaling of Erythroid Precursors.
Authors: Tsopra Olga , Ziros Panos , Lagadinou Eleni , Kouraklis Alexandra , Symeonidis Argiris , Thanopoulou Eleni , Zikos Panagiotis , Zoumbos Nicholas C. 
Published: April 2020

Abstract
Anemia is a common finding in Chronic Lymphocytic Leukemia (CLL) both at diagnosis and during disease progression and has been correlated with poorer outcome and shorter survival. Anemia associated with chronic disease (ACD) has been implicated, among other subjects, in the pathogenesis of anemia in CLL. The etiology of ACD, although multifactorial, still remains uncertain. To investigate the contribution of ACD to the development of anemia in CLL, we examined bone marrow (BM) mononuclear cells from 10 patients with CLL and anemia and 8 patients with CLL without anemia. Flow cytometric analysis did not reveal any significant difference in the percentage of BM CD34+ cells (hematopoietic stem cells), CD34+/CD71+ cells (erythroid progenitors) and CD36+/glycophorinA+ (GPA) cells (early erythroid precursors) of both groups studied. Moreover, CD34+ cells of anemic and non-anemic patients exhibited similar clonogenic capacity when cultured in semisolid culture media. To evaluate the ability of CLL patients with anemia to generate erythroid precursors (GPA+ cells), CD34+ cells were cultured in an one step liquid system in the presence of erythropoietin (EPO) (2IU/ml), stem cell factor (20ng/ml) and interleukin-3 (1ng/ml) for 8–10 days. Interestingly, CD34+ cells of anemic patients normally proliferated and differentiated into the erythroid lineage, as it was also observed in non anemic patients. Furthermore, CD34+ derived GPA+ cells from both groups studied normally responded to EPO, as EPO stimulation (10IU/ml for 10 minutes) activated the JAK2-STAT5 pathway, which represents the main intracellular pathway mediating EPO signaling. Tumor Necrosis Factor-a (TNF-a) has been related to the pathogenesis of ACD and elevated levels of this pro-inflammatory cytokine have been reported in the serum of CLL patients with anemia. To further investigate the role of TNF-a in CLL with anemia, various concentrations of TNF-a (1,10,100ng/ml) were added in liquid cultures. TNF-a decreased the production of GPA+ cells in all samples examined, whereas it increased the generation of GPA-cells. Nevertheless, TNF-a did not have an impact in EPO intracellular signaling pathway, as normal activation of JAK2-STAT5 pathway was detected when CD34+ derived GPA+ cells stimulated with EPO and TNF-a for 10 minutes after preincubation for 18hs with 10 and 100ng/ml of TNF-a. Our in vitro study suggests that there is no defect both in the capacity of BM hematopoietic stem cells of patients with CLL and anemia to generate erythroid cells and in the efficacy of erythroid precursors to respond normally to EPO stimulation. Additionally, TNF-a decreased the production of GPA+ cells but did not affect their response to EPO. More studies are needed to elucidate the pathogenetic mechanisms of anemia in CLL and the role of TNF-a in the process.