Changes of Megakaryocyte Gene Expression When Essential Thrombocythemia (ET) Transform Into Myelofibrosis (Post-ET MF) by Microarray Analysis,

Abstract 3845

Patients with ET eventually transform into Post- ET MF. The underlying mechanism of the transformation is still unknown. Megakaryocytes play an important role in the pathogenesis of bone marrow fibrosis in MF; ET and Post-ET MF also are characterized by abnormal megakaryocytic proliferation. Therefore we have performed microarray analysis of gene expression profiles to compare megakaryocytes derived from patients with Post-ET MF to ET. CD 34+ cells were obtained from peripheral blood, then cultured with Serum-Free Medium of stem cell factor (100 ng/mL) and thrombopoietin (TPO 100 ng/mL). Megakaryocytes (MK) were obtained by MACS selection of CD 61+ cells after 10 days culture. MK from two pooled ET-MF and a pooled from eight ET patients were extracted into RNA then converted to cDNA and amplified by PCR, followed by hybridization to an oligonucleotide microchip in PhalanxBio Inc. (Palo Alto, Ca). Gene expression levels in MK cells from ET-MF patients expressed in threshold cycle (CT) values were validated by statistical analysis of replicates and normalized to that from pooled ET patients to generate fold-change and statistical relevance. The resulting signal log ratios (SLRs) were used to classify the expressed genes into various groups according to its graded values. The results of post-ET MF compared with ET were as follows: 1) with analysis of 4544 genes, SLR <-1 were 6.58 %, and 10.12 % were >1, 2) in apoptosis genes, over expressed genes were IGF-1R, CFLAR, NFKBA,(GLR between 1–1.5) down-regulated genes were BAX, LGALSI, PPPIR 15A, CASP3 (GLR between -0.5 to -0.1), 3) in cell cycle controlling genes, over expressed were IL8, INSIG1, IGF-1R, BCL3(GLR between 1 to 1.5), down-regulated were PPPIR15A (GLR= -0.7). 4) in megakaryocyte-important development genes, growth factor genes including PDGF A, C were up-regulated (GLR=1.8), and VEG B was down-regulated (GLR= -1. 4):; growth factor receptor genes including IL1R 1and 2, CXCR4, IL8, PF4 were up-regulated (GLR between 1–2), IL3RA, KIT were down-regulated (GLR between -1.6 to -3); cell cycle regulator genes including CCND1, 3, RASGRP3 were up-regulated (GLR between 1 to 2.5), CDK4 was down-regulated (GLR = -1); transcription regulators including CEBPE, CEBPG were up-regulated (GLR-1.5); all Signal transducers including all STAT 1–5, MAK14 were up-regulated (GLR between 1.5 to 2.4). There are 20 up-regulated genes and 20 down-regulated genes which need further analysis for their function. These analyses of MK genes comparison between Post ET-MF and ET suggest that many anti-apoptosis, cell cycle regulator genes, growth factor genes, growth factor receptor genes and all of the signal transducer genes for the MK development genes were up-regulated while many of the pro-apoptosis genes were down-regulated. These gene changes found in the present study, when transformation of ET into post-ET MF occurs will be important for understanding this disease process and target therapy may be emerged.