The Transcripts Of Platelet-Endothelial Adhesion Molecules and Inflamatory Chemokine Activation Pathway Molecules Are Hyperexpressed In The Bone Marrow Of Acute Promyelocytic Leukemia Patients Presenting Severe Coagulopathy

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Abstract Although Acute Promyelocytic Leukemia (APL) is now considered one of the most curable forms of acute leukemia, it is an aggressive disease associated with a high frequency of bleeding and thrombosis. Indeed, the leading cause of treatment failure in APL is not disease resistance or recurrence but death in induction by hemorrhage caused by APL-associated coagulopathy in which blast-derived pro-coagulant and fibrinolytic factors (Annexin A2 and Tissue factor), as well as cytokines, microparticles and proteases were reported to participate of the pathogenesis. However, why some APL patients are more prone to develop bleeding diathesis than others is presently unknown. In order to address this issue, we performed microarray analysis of leukemic cells from 6 pairs of patients matched for age, sex, and WBC and platelet counts at diagnosis, but differed regarding the presence of clinical signs of APL-associated coagulopathy and fibrinogen levels (Table 1). All patients were enrolled in the IC-APL trial whose results have been reported {Blood;121(11):1935-43}.Table 1Clinical characteristics of the selected patients at the diagnosis of APL. Values represent the median and percentiles (25 – 75)Fibrinogen(mg/dL)Age(years)WBC(x109/L)Platelets(x109/L)nClinical signs of CoagulopathyYESGroup 164 (51.5-98.5)41.5 (26.6-55.2)15.9 (7.8-29.9)31 (19-41.1)6NOGroup 2236 (194-431)40.8 (29.8-60.9)17.3 (4.7-28.9)23(4.2-70.5)6 Bone marrow (BM) samples from the diagnosis, were submitted to red blood cell lysis (RBC Lysis solution, Qiagen), stabilized in TRIZOL (Invitrogen) at 1x106 cells/mL and stored at -800C. For RNA isolation we performed an extraction with chloroform and ethanol followed by a resin column method (where the DNAse treatment was made ”in column”) - “Pure Link RNA Mini Kit” (Life Technologies). After quantification (Nanovue, GE Healthcare) the RNA samples were submitted to integrity analysis using the Bioanalyser system (Agilent). All samples presented a RNA integrity number (RIN) above 8, which attest the quality for microarray analysis. We used the Whole Human Genome Oligo Microarray Kit (Agilent, G4112F), which is a mRNA microarray slide with a content of more than 44.000 transcripts. A total of 200ng of RNA from each sample was used and we followed the instructions of the manufacturer. The quality control for all hybridizations revealed a coefficient of variation of less than 10% and a dynamic range of 4 orders of magnitude. The identification of the differentially expressed transcripts was based on a modified T test that uses the fold change (FC) value as the variable and the experimental “n” and the variance to calculate the p value. Table 2 shows the main hyperexpressed genes in the BM samples from patients with severe APL coagulopathy.Table 2Main differently expressed genes (more expression in patients with coagulopathy) in bone marrow samples from the diagnosis of APL. The genes are ordered by fold change (FC).GeneDescriptionpFCSELPselectin P0.015707.27IFI16interferon, gamma-inducible protein 160.004707.24GP1BAplatelet glycoprotein Ib alpha chain0.013645.63CXCL5chemokine (C-X-C motif) ligand 50.025424.79IFNAR1interferon (alpha, beta and omega) receptor 10.000863.18NOD1nucleotide-binding oligomerization domain containing 10.018172.69TLR4toll-like receptor 40.013582.63RLN2relaxin 20.036552.57LMAN1lectin, mannose-binding, 10.034182.52MAP3K13mitogen-activated protein kinase kinase kinase 130.049072.49ATRXalpha thalassemia/mental retardation syndrome X-linked0.036092.36MAP2K4mitogen-activated protein kinase kinase 40.027512.19PRKCQprotein kinase C, theta0.048212.17 In patients with bleeding complications, molecules associated with endothelial activation or related to chemokines are on the top hyperexpressed genes ordered by FC. SELP works as an adhesion molecule of activated endothelial cells. GP1BA is a component of the receptor for von-Willebrand factor, which facilitate platelet adhesion to activated endothelium. IFI16, CXCL5 and IFNAR1 are related to inflammatory responses by chemokines, however there are no reports of their involvement in APL coagulopathy. Our results suggest that these factors are involved in the pathogenesis of APL coagulopathy. Additionally, further analysis using large and independent APL cohorts, may test the serum levels of interferon isoforms and CXCL5 as possible markers of risk. Disclosures: No relevant conflicts of interest to declare.

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