Molecular Pathways of Response to Recombinant Human Erythropoietin (rHuEpo) in Myelodysplastic patients Assayed with Macroarrays.

Recently, among myelodysplastic syndromes with IPSS low/intermediate 1 risk classes, promising results have been obtained in the treatment of anemia by high dosage recombinant human erythropoietin (rHuEPO) treatment, in the cases with low endogenous erythropoietin (EPO). We have reproduced these clinical results in the patients treated at our Institution. Based on these premises, in order to evaluate the molecular basis of the rHuEPO response in MDS patients, we investigated the differential gene expression in the bone marrow erythroid cells expressing glycophorin (Gly+) of EPO-responders (ER) and EPO-non responders (ENR) MDS patients, using commercially available macroarrays (Clontech), comparing gene level of expression with a baseline represented by a pool of erythoid cells isolated from normal bone marrow donors. We analysed 4 ER and 4 ENR MDS patients (RA and RARS). BM erythroid cells, after Ficoll density gradient, were separated by positive selection using an immunomagnetic procedure (MACS, glycophorin isolation kit; Miltenyi Biotech, Auburn, CA). The purity of Gly+ cells after magnetic immunosorting was tested by flow-cytometry and was superior to 97%, in all the cases. RNA extraction, cDNA synthesis and macroarrays hybridization and analysis were performed following manufacturer instructions (Clontech Laboratories, Palo Alto, CA). The data were validated on 4 differentially expressed genes by real time RT-PCR TaqMan technology, obtaining results that were superimposable to the macroarrays ones. Using a cut off of an at least two fold difference in gene expression, we obtained a differential pattern of expression in ER and ENR patients, BM Gly+ erythroid cells, with respect to normal donors. In particular, ER patients presented the up-regulation of interleukin-1 beta, apolipoprotein E precursor, CREB2, high mobility group protein (HMG-I), transducin beta-1 subunit and the down regulation of protein kinase MLK-3, cAMP-dependent phosphodiesterase (PDE43), IL-5R-alpha, caspase-9. On the other hand, in ENR group, we found a down regulation of proliferating cyclic nuclear antigen (PCNA), B-myb, prothymosin alpha, phenylethanolamine N-methyltransferase, thymosin beta-10 and an up regulation of homeobox protein HOX-A5, monocarboxylate transporter 1, interleukin-6, deoxyribonuclease II, sterol regulatory element-binding protein, metalloproteinase inhibitor 1 precursor, rap1 GTPase activating protein 1 (RAP1GAP). In conclusion, ER and ENR MDS patients erythroid cells present a different pattern of gene expression with respect to normal. These results may provide the basis for early testing of the genes differentially expressed in ER and ENR patients in order to elucidate their role in the prognostication of rHuEPO response in MDS patients with low endogenous EPO levels.