research article proinflammatory cytokine il-6 and jak...

Research ArticleProinflammatory Cytokine IL-6 and JAK-STAT SignalingPathway in Myeloproliferative Neoplasms

Vladan P HokiT1 Olivera MitroviT-AjtiT1 Bojana B Beleslin-HokiT2

Dragana MarkoviT1 Marijana BuaI1 Miloš DikliT1 Nada Kraguljac-KurtoviT3

Svetozar DamjanoviT24 Pavle MilenkoviT1 Mirjana GotiT34 and Puri K Raj5

1 Institute for Medical Research University of Belgrade 11129 Belgrade Serbia2Clinic of Endocrinology Diabetes and Diseases of Metabolism Clinical Center of Serbia 11000 Belgrade Serbia3Clinic of Hematology Clinical Center of Serbia 11000 Belgrade Serbia4Medical Faculty University of Belgrade 11000 Belgrade Serbia5Division of Cellular and Gene Therapies Center for Biologics Evaluation and Research Food and Drug AdministrationSilver Spring MD 20993 USA

Correspondence should be addressed to Vladan P Cokic vlimibgacrs

Received 26 June 2015 Revised 2 September 2015 Accepted 9 September 2015

Academic Editor Hannes Neuwirt

Copyright copy 2015 Vladan P Cokic et alThis is an open access article distributed under the Creative CommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The recent JAK12 inhibitor trial in myeloproliferative neoplasms (MPNs) showed that reducing inflammation can be morebeneficial than targeting gene mutants We evaluated the proinflammatory IL-6 cytokine and JAK-STAT signaling pathway relatedgenes in circulating CD34+ cells of MPNs Regarding laboratory data leukocytosis has been observed in polycythemia vera (PV)and JAK2V617F mutation positive versus negative primary myelofibrosis (PMF) patients Moreover thrombocytosis was reducedby JAK2V617F allele burden in essential thrombocythemia (ET) and PMF 261 significantly changed genes have been detectedin PV 82 in ET and 94 genes in PMF The following JAK-STAT signaling pathway related genes had augmented expression inCD34+ cells of MPNs CCND3 and IL23A regardless of JAK2V617F allele burden CSF3R IL6ST and STAT12 in ET and PVwith JAK2V617F mutation and AKT2 IFNGR2 PIM1 PTPN11 and STAT3 only in PV STAT5A gene expression was generallyreduced in MPNs IL-6 cytokine levels were increased in plasma as well as IL-6 protein levels in bone marrow stroma of MPNsdependent on JAK2V617Fmutation presence in ET and PMF patientsTherefore the JAK2V617Fmutant allele burden participatedin inflammation biomarkers induction and related signaling pathways activation in MPNs

1 Introduction

It has been generally accepted that chronic inflammationmayincrease the risk of cancer by providing bioactive moleculesfrom the tumor microenvironment including cytokines andgrowth factors that promote continuous cell proliferationand malignant transformation [1] Interleukin-6 (IL-6) is amember of the IL-6-type cytokines family with pro- and anti-inflammatory properties They activate the Janus tyrosinekinase (JAK) family members (JAK1 JAK2 and TYK2)leading to the activation of transcription factors of the signaltransducer and activator of transcription (STAT) family All

IL-6-type cytokines potently activate STAT3 and to a minorextent STAT1 while SOCS3 is the primary inhibitor of IL-6 signaling [2 3] IL-6 is produced by a wide range ofcells including monocytesmacrophages T cells endothelialcells fibroblasts and hepatocytes IL-6 regulates a varietyof cell functions such as a cell proliferation activation anddifferentiation and has been proposed as a predictor ofmalignancy [4 5]

IL-6 has been implicated as a critical activator ofmyelopoiesis in response to chronic inflammatory disordersincludingmyeloproliferative neoplasm (MPN) [6] IL-6 levelsare increased in primarymyelofibrosis (PMF) with a positive

Hindawi Publishing CorporationMediators of InflammationVolume 2015 Article ID 453020 13 pageshttpdxdoiorg1011552015453020

2 Mediators of Inflammation

correlation between IL-6 and angiogenesis in bone marrowof MPN patients [7 8] The endogenous levels of IL-6 andsoluble IL-6 receptor are also elevated in essential thrombo-cythemia (ET) [9] It has been reported that although serumlevel of IL-6 is not increased in polycythemia vera (PV)the increased percentage of megakaryocytes secreting thiscytokine is observed in bonemarrow of patients with PV [10]

Activation of the JAK-STAT signaling pathway resulted inthe production of IL-6 [11] Moreover JAK2-specific inhibi-tion blocks STAT3 phosphorylation and IL-6 production incancer cells [12] STAT3 is predominantly activated by IL-6receptor via JAK12 in cancer cell lines in association withtranscriptional silencing of SOCS-1 by hypermethylation [13]Moreover JAK12 inhibition decreases a circulating level ofproinflammatory cytokine IL-6 in MPN mouse model [14]In a transgenic mouse model of Jak2V617F-mediated MPNJAK2 inhibitor normalizes the pathologically high plasmaconcentrations of IL-6 [15] Tumor necrosis factor-alpha(TNF-120572) stimulated IL-6 production is also suppressed byinhibition of JAK2 in multiple myeloma cells [16]

According to previous reports proinflammatory cytokineIL-6 demonstrates JAK-STAT signaling dependence andhas been augmented in MPNs Majority of MPN patientshave JAK2V617Fmutation that constitutively stimulates JAK-STAT pathway Using microarray analysis we explore bothJAK-STAT and IL-6 signaling related genes in MPNs accord-ing to JAK2V617F mutant allele burden IL-6 levels havebeen examined both in peripheral blood and bonemarrow ofpatients with MPN In addition genes related to other majorinflammatory pathways such as transforming growth factor-beta (TGF-120573) and nuclear factor-kappa B (NF-120581B) have beenevaluated in MPNs

2 Material and Methods

21 Isolation of CD34+ Cells from the Peripheral Blood ofMPNPatients Thestudywas approved by the Local ResearchEthics Committee (Medical School University of Belgrade)Informed consent was obtained from all patients included inthe study All study de novo patients were subject to 30mLof peripheral blood draw on one occasion collected in 10sodium citrate Each 30mL of diluted peripheral blood (1 12with Ca2+Mg2+-free phosphate buffer saline (PBS)) was lay-ered gently on top of 15mL lymphocyte separation medium(Capricorn Scientific GmbH Ebsdorfergrund Germany)After centrifugation (400 g 30min 20∘C) the interface ofcontaining mononuclear cells was collected and washedwith PBS The CD34+ cells were isolated from the collectedmononuclear cells by magnetic separation column (SuperMacs II Miltenyi Biotec Bergisch Gladbach Germany) andmixture of magnetic microbeads conjugated with antibodyagainst CD34 (Miltenyi Biotec) according to the manu-facturerrsquos instructions The viable CD34+ cell counts wereperformed with the use of a trypan-blue exclusion technique(BioWhittaker)Thehigh purity of recoveredCD34+ cells wasdetermined by flow cytometry using PE-anti-CD34mAb (BDBiosciences San Jose CA USA) Karyotype analysis did notshow any chromosome aberrations in samples formicroarrayanalysis

22 Isolation of Total RNA We used the RNeasy protocolfor isolation of total RNA from CD34+ cells according to themanufacturerrsquos instructions (Qiagen GmbH Hilden Germa-ny) Concentration and integrity of total RNA were assessedusing NanoDrop spectrophotometer (Thermo Fisher Scien-tific Inc Wilmington Delaware USA) and Agilent 2100Bioanalyzer Software (Agilent Technologies WaldbronnGermany) comparing the ratio of 28S and 18S RNA peaks toensure that there is minimal degradation of the RNA sample

23 DNA Sequencing Genomic DNA was extracted fromperipheral blood granulocytes of MPN patients by usingthe proteinase K and phenol-chloroform technique Singlenucleotide mutation of JAK2V617F gene was characterizedby DNA sequencing after PCR amplification PCR amplifi-cation was performed with wild-type JAK-2-specific forwardprimer 51015840-tggcagagagaattttctgaact-31015840 and reverse primer 51015840-ttcattgctttcctttttcaca-31015840 confirmed by electrophoresis on anethidium bromide-impregnated 1 agarose gel PCR ampli-fied samples were analyzed by sequencing on an automatedABI PRISM 3130 Genetic Analyzer (Applied Biosystems LifeTechnologies Carlsbad CAUSA)withABDNASequencingAnalysis Software (v52) by using the Big Dye Terminator v31Ready Reaction Cycle Sequencing Kit

24 Immunohistochemical Analysis Bone marrow tissue sec-tions were fixed in 10 neutral formalin solution for 24ndash36hours and then decalcified in EDTA buffer for 3 hours andembedded in paraffin The tissue sections were cut at 5 120583mheated at 56∘C for 60min and then deparaffinized and rehy-drated through a series of xylenes and alcohols followed byan epitope retrieval step Samples were treated with 3H

2O2

solution in PBS to block endogenous peroxidase activity Thenext step was incubation with anti-IL-6 antibody (ThermoScientific dilution 1 400) in a humidity chamber overnightat room temperature Immunostaining was performed usingthe streptavidin-biotin technique (LSAB+HRP Kit DAKO)Immunoreactivity was visualized with DAKO Liquid DAB+SubstrateChromogen System counterstained with Mayerrsquoshematoxylin (Merck Whitehouse Station NJ) For the neg-ative control samples normal serum and PBS buffer (1 500)were pipetted without primary antibodies Immunoreactivecells were analyzed and scored at five powered fields in eachsample using a computer-supported imaging system (analysisPro 31) connected to the light microscope (Olympus AX70Hamburg Germany) with an objective magnification of times40Immunohistochemical staining for IL-6 was performed inbone marrow of 34 subjects (10 PV per 5 heterozygous andhomozygous 9 ET 5 no mutation and 4 heterozygous and9 PMF 5 no mutation 4 heterozygous for JAK2V617F and 6healthy subjects)

25 ELISA Assay Peripheral blood samples were obtainedfrom 45 patients with newly diagnosed untreated MPNsThe study included 14 PV 14 ET and 17 PMF cases Patientswere subclassified according to JAK2V617F mutational sta-tus as JAK2 homozygous JAK2 heterozygous and patientswithout mutation We analyzed 7 PV homozygous and 7 PV

Mediators of Inflammation 3

heterozygous patients 7 ET heterozygous and 7 ET patientswithout JAK2V617F mutation and 3 PMF homozygous 7PMF heterozygous and 7 PMF patients without JAK2V617FmutationThe control group comprised 7 healthy volunteersBlood samples were collected with EDTA and plasma wasseparated by centrifugation at 2000 rpm per 15 minutes Thesamples were stored at minus80∘C until analysis Plasma IL-6level was measured using an ELISA kit (Thermo ScientificRockford IL USA) according to the manufacturerrsquos instruc-tions All samples were tested in duplicate and data wereexpressed as average of IL-6 levels in pgmL for each groupMeasurements were performed on an ELISA Multiscan Plusplate reader (Labsystems Finland) Data were expressed asmeanplusmn standard error of themean (SEM) of each groupTheywere analyzed by Studentrsquos t-test A 119901 value lt 005 was chosenas the level of statistical significance

26MicroarrayAnalysis Thehumanoligo probe set was pur-chased fromOperonHumanGenomeArray-ReadyOligo SetVersion 40 (Eurofins MWG Operon Huntsville AL USA)which contains 35035 oligonucleotide probes representingapproximately 25100 unique genes We have followed theMIAME (minimum information about a microarray exper-iment) guidelines for the data presentation Also our priorexperience with primary cell cultures included quantitativePCR with housekeeping genes (S16 and HPRT) to establishsimilar efficiency of cDNA synthesis and PCR (data notshown) In microarray studies for determination of geneexpression in CD34+ cells of MPNs we used 8 healthydonors and 9 ET (4 negative and 5 positive heterozygotes forJAK2V617F mutation) 7 PV (3 heterozygotes and 4 homozy-gotes for JAK2V617F) and 4 PMF (2 negative and 2 positivefor JAK2V617F) patientsWe isolated a lowquantity of CD34+cells that correspond to low mRNA levels insufficient formicroarray analysis so we performed the amplification oftotal RNA using the Amino Allyl MessageAmp II aRNAAmplification kit (Life Technologies Corp Carlsbad CAUS) according to manufacturer instruction We used 03120583gof total RNA from MPN patients for amplification Formicroarray analyses we used 3 120583g of amplified RNA Totalhuman universal RNA (HuURNA) isolated from a collectionof adult human tissues to represent a broad range of expressedgenes from both male and female donors (BD BiosciencesPalo Alto CA) served as a universal reference control inthe competitive hybridization All examined MPN sam-ples were hybridized against HuURNA For hybridizationthe hybridization mixture of cDNA probe and aRNA waspreheated at 100∘C for 2 minutes and centrifuged for 1minute at 10000 rpm 20120583L of preheated (42∘C) Ambionhybridization buffer (20x SSC in 10 SDS) was mixed withhybridization mixture Total volume of the hybridizationmixture was added on the array in slide and covered withcover slip Slideswere placed inMAUIhybridization chamber(BioMicro Systems Inc Salt Lake City UT USA) andincubated overnight at 42∘C Slides were then washed for4 minutes each in 1x SSC and 01x SSC and spin-driedMicroarray slides were scanned in both Cy3 (532 nm) andCy5 (635 nm) channels using Axon GenePix 4000B scanner(Axon Instruments Inc Foster City CA) with a 10-micron

resolution Scanned microarray images were exported asTIFF files to GenePix Pro 30 software for image analysisThe average of the total Cy3 and Cy5 signal gave a ratio thatwas used to normalize the signals Each microarray exper-iment was globally normalized to make the median valueof the log

2-ratio equal to zero For advanced data analysis

gpr and jpeg files were imported into microarray databaseand normalized by software tools provided by NIH Centerfor Information Technology (httpnciarrayncinihgov)Spots with confidence interval of 99 (ge2 fold) with atleast 150-fluorescence intensity for both channel and 30120583mspot size were considered good quality spots for analysisThe microarray data were available from Gene Expres-sion Omnibus (httpwwwncbinlmnihgovgeo accessionnumber GSE55976)

27 Statistical Analysis For microarray data managementand analysis we used NCICIT microarray database (mAdb)system The one-way ANOVA was applied using mAdbsoftware for measurement of statistical significance in geneexpression among MPNs For mAdb hierarchical clusteringwe used uncentered correlation that applied a modifiedPearson correlation equation which assumes that the meansare 0

3 Results

31 Analysis of Peripheral Blood Cells in MPN Patientsaccording to JAK2V617FMutant Allele Burden We examinedthe levels of peripheral blood cells from MPN patients incorrelation to JAK2V617Fmutant allele burdenWe observed63 patients with ET 92 patients with PV 50 patients withPMF 12 healthy subjects and 10 patients with secondary ery-throcytosis Level of thrombocytes was reduced while ery-throcyte and hemoglobin levels were elevated in ET patientsheterozygous for JAK2V617F mutation versus ET patientswithout JAK mutation (Table 1) PV patients heterozygousand homozygous for JAK2V617F mutation had significantlyincreased levels of CD34+ cells leukocytes thrombocytesand erythrocytes but reduced MCV compared to secondaryerythrocytosis CD34+ cells were generally increased inperipheral blood of PMF patients particularly in JAK2V617Fmutation positive patients The average number of CD34+cells (cells120583L) in peripheral blood was approximately 6 inPV and ET and 111 in PMF group increased in comparisonto mean value for healthy subjects (275 plusmn 1 cells120583L) Inaddition leukocytes and erythrocytes were increased bothin heterozygous and homozygous JAK2V617Fmutated formscomparing to PMF patients without mutation We observedlower thrombocyte levels and MCV in homozygous PMFpatients while hemoglobin levels were significantly increasedin heterozygous PMF patients compared to JAK2V617Funmutated PMF patients (Table 1)

32 Microarray Analysis of Total Gene Expression in CD34+Cells of MPNs according to JAK2V617F Mutant Allele BurdenUsing microarray analysis the total gene expression hasbeen examined separately in CD34+ cells of PV ET and


Table 1 Blood test results of examined MPN patients according to JAK2mutant allele burden

MPN 1198691198601198702V617P

CD34+(cells120583L)

Leukocytes(times109L)

Thrombocytes(times109L)

Erythrocytes(times1012L) MCV Hemoglobin

(gL)

f0 63 (plusmn34) 877 (plusmn24) 1110 (plusmn358) 46 (plusmn04) 887 (plusmn37) 1341 (plusmn14)lt50 62 (plusmn61) 91 (plusmn35) 815 (plusmn249)2 5 (plusmn053)2 87 (plusmn39) 142 (plusmn13)1

PVSec Er 25 (plusmn07) 725 (plusmn19) 203 (plusmn46) 534 (plusmn04) 898 (plusmn36) 1616 (plusmn13)lt50 63 (plusmn46)1 122 (plusmn39)2 807 (plusmn349)2 61 (plusmn08)2 82 (plusmn88)2 1584 (plusmn15)gt50 94 (plusmn82)1 173 (plusmn95)2 631 (plusmn270)2 65 (plusmn09)2 746 (plusmn88)2 1524 (plusmn17)

PMF0 298 (plusmn37) 88 (plusmn31) 905 (plusmn320) 44 (plusmn044) 871 (plusmn56) 1265 (plusmn17)lt50 99 (plusmn212) 129 (plusmn51)2 804 (plusmn364) 49 (plusmn08)1 84 (plusmn62) 138 (plusmn14)1

gt50 126 (plusmn158) 142 (plusmn55)2 509 (plusmn331)2 534 (plusmn13)2 777 (plusmn11)2 1291 (plusmn20)119905-test 1119901 lt 005 2119901 lt 001 versus MPNs without JAK2 mutation (0) and secondary erythrocytosis (Sec Er)

PMF groups A distribution of total gene expression of MPNCD34+ cells has been presented in comparison to controlsand according to JAK2V617Fmutant allele burden (Figure 1)The quantity of shared genes in CD34+ cells was presentedby Venn diagram in JAK2V617F heterozygous homozygousand mutation-free forms (Figure 1) Moreover the statisticalanalyses of total gene expression have been performed inPV ET and PMF according to JAK2V617F mutant alleleburden (see Supplemental Table 1 in Supplementary Materialavailable online at httpdxdoiorg1011552015453020)Weobserved 261 significantly changed genes (119901 lt 001) in PV82 significant genes (119901 lt 001) in ET and 94 genes (119901 lt005) in PMF comparing to controls and sporadically amongMPNs (Supplemental Table 1) ET and PMF shared 43 and26of their significantly changed geneswith PV respectivelyCHMP5 gene expression was generally increased in MPNsand further on augmented by JAK2V617F allele burdenSOD2 gene expression was also generally increased in MPNsbut only in PV augmented by JAK2V617F allele burdenWAC gene expression was generally increased in PV ET andheterozygous PMF patients CCND3 HN1 and LOC124685were only significantly upregulated genes simultaneously inET and PMF compared to control subjects (SupplementalTable 1)

33 JAK-STAT Signaling Pathway Related Genes in CD34+Cells of MPNs The examination of JAK2V617F mutantallele burden influence has been expanded toward JAK-STAT signaling pathway in CD34+ cells of MPNs Accordingto Table 2 several genes involved in JAK-STAT signalinghave been modified by JAK2V617F allele burden Colonystimulating factor 3 receptor (CSF3R) has been significantlyincreased in JAK2V617F positive ET and PV patients The v-akt murine thymoma viral oncogene homolog 2 (AKT2) hasbeen significantly increased in JAK2V617F homozygous PVand JAK2V617F positive PMF patients Cyclin D3 (CCND3)has been generally augmented in MPNs not influencedby JAK2V617F mutation Pim-1 oncogene (PIM1) has beenalso generally augmented in MPNs but reached statisticalsignificance only in PV similar to protein tyrosine phos-phatase nonreceptor type 11 (PTPN11) However PTPN6 hasbeen significantly increased only in JAK2V617F heterozy-gous ET patients STAT1 and STAT2 gene expression have

been increased in heterozygous ET and PV patients whileSTAT3 only in homozygous PV patients STAT5A has beensignificantly decreased in ET and PMF with no JAK2V617Fmutation as well as in homozygous PV patients (Table 2) Inaddition these JAK-STAT signaling related genes were alsoshown in hierarchical clustering analysis to illustrate theirassociations (Figure 2)The genes only and steadily expressedin homozygous PV were GRB2 IL15 LEPR and PIK3CA(Table 2)

34 IL-6 and Inflammatory Signaling Pathways Related GeneExpression in CD34+ Cells ofMPNs Regarding IL-6 signalingpathway related genes several of themwere already presentedin Table 2 such as PTPN11 STAT3 JAK1 JAK2 interleukin 6signal transducer (IL6ST) and growth factor receptor-boundprotein 2 (GRB2) IL6ST has been increased in CD34+ cells ofJAK2V617F mutation positive ET and PV patients (Table 2)MAP2K1 was significantly increased in CD34+ cells of PV(Table 3) RAF1 and FOS gene expression were increased inET and PV but do not reach statistical significance comparedto controls (Table 3) Other proinflammatory pathways suchas NF-120581B and TGF-120573 signaling demonstrated elevation ofTNFRSF1A and TRADD genes expression in CD34+ cells ofPMF and PV respectively as well as decrease of CDKN2Band increase of TFDP1 genes in PMF (Table 3) Anti-inflammatory IL-10 signaling pathway related genes BLVRAand HMOX1 demonstrated increased expression in CD34+cells of PV and ET respectively (Table 3)

35 IL-6 Protein Levels in MPNs according to JAK2V617FMutant Allele Burden We examined IL-6 protein levelsin peripheral blood (plasma) and bone marrow of MPNsin accordance with JAK2V617F mutant allele burden IL-6protein levels were generally increased in plasma of MPNsreaching statistical significance in majority of patients notinfluenced by JAK2V617F mutant allele burden (Figure 3(a))Furthermore there is a positive correlation between IL-6protein levels and erythrocytes (119903 = 0933 119901 lt 001)hemoglobin (119903 = 0733119901 lt 005) and hematocrit (119903 = 0818119901 lt 005) levels in PV patients heterozygous for JAK2V617Fmutation IL-6 protein levels were also commonly andsignificantly increased in bone marrow stromal cells of


220 123 315

250

3450

576

645

A B

C

ET

A ControlB No mutation

C HeterozygoteD Homozygote

CD34+

(a)

189 101 205

166

3587

1076

951

C

D

A

PV



CD34+

(b)

273 231 738

501

3038

417

321

A B

C

PMF



CD34+

(c)

Figure 1 Microarray study of gene expression in CD34+ cells from peripheral blood of MPN patientsThe Venn diagram shows similarity oftotal gene expression in controls (119899 = 8) and (a) JAK2V617F heterozygous and no mutation ET forms (119899 = 9) (b) JAK2V617F heterozygousand homozygous PV forms (119899 = 7) and (c) JAK2V617F heterozygous and no mutation PMF forms (119899 = 4)

MPNs (Figure 3(b)) This bone marrow augmentation of IL-6 expression was more prominent in JAK2V617F positive ETand PMF patients (119901 lt 001 Figure 3(b))

4 Discussion

Presence of JAK2V617F mutation correlated with a reducedlevel of thrombocytosis in ET and increased absolute number

of circulating CD34+ cells leukocytosis and erythrocyto-sis in patients with PV In addition increased JAK2V617Fallele burden augmented leukocytosis and erythrocytosis butreduced thrombocytosis and MCV in patients with PMFUsing Venn diagram a distribution of gene expression hasbeen presented with a list of significantly modified genes inCD34+ cells ofMPNs compared to controls and according toJAK2V617F mutant allele burden 261 significantly changed


Table 2 Statistically significant genes related to JAK-STAT pathway in CD34+ cells of MPNs according to JAK2V617F mutant allele burden

Control ET PV PMFJAK2 minus minus + Hetero Homo minus +Genes AV plusmn SD AV plusmn SD AV plusmn SD AV plusmn SD AV plusmn SD AV plusmn SD AV plusmn SDAKT2 094 plusmn 018 111 plusmn 058 080 plusmn 040 066 plusmn 037 13 plusmn 01lowastlowast 07 plusmn 030 11 plusmn 017lowast

CSF3R 283 plusmn 020 299 plusmn 037 38 plusmn 06lowastlowast 32 plusmn 06lowast 32 plusmn 02lowastlowast 292 plusmn 101 320 plusmn 050CBL 049 plusmn 045 minus01 plusmn 03lowast 028 plusmn 065 029 plusmn 006 061 plusmn 014 016 plusmn 085 079 plusmn 013CCND2 030 plusmn 055 001 plusmn 005 minus038 plusmn 06 056 plusmn 039 038 plusmn 038 002 plusmn 020 033 plusmn 086CCND3 021 plusmn 022 11 plusmn 05lowastlowast 09 plusmn 04lowastlowast 066 plusmn 057 09 plusmn 03lowastlowast 12 plusmn 04lowastlowast 076 plusmn 02lowast

CNTFR 052 plusmn 099 065 plusmn 083 097 plusmn 071 025 plusmn 047 minus103 plusmn 02 minus12 minus064 plusmn 13CREBBP 023 plusmn 060 minus035 plusmn 03 035 plusmn 034 045 plusmn 044 010 plusmn 051 minus041 plusmn 06 005 plusmn 058GRB2 165 205 191 plusmn 012 148IFNAR2 051 plusmn 113 141 plusmn 046 176 plusmn 049 138 plusmn 025 132 plusmn 038 123 plusmn 069 083IFNGR1 077 plusmn 022 092 114 plusmn 028 085 plusmn 012 118 plusmn 040 188IFNGR2 077 plusmn 013 206 158 plusmn 060 194 plusmn 048 20 plusmn 01lowastlowast 196 187IL10RB 096 plusmn 028 088 plusmn 055 152 plusmn 060 123 plusmn 049 099 plusmn 079 086 plusmn 101 080 plusmn 005IL11RA 169 plusmn 013 031 187 166 plusmn 015 192 plusmn 039 149IL15 11 214 224 plusmn 004IL15RA 076 plusmn 047 019 plusmn 009 050 plusmn 034 027 plusmn 019 041 plusmn 007 016 plusmn 032 060 plusmn 039IL23A minus118 plusmn 02 minus07 plusmn 04lowast minus111 plusmn 04 minus075 plusmn 04 minus05 plusmn 04lowast minus13 plusmn 02lowast minus17 plusmn 002lowastlowast

IL6ST minus110 plusmn 02 minus046 plusmn 04 01 plusmn 06lowastlowast 006 plusmn 04lowast minus01 plusmn 06lowast minus092 plusmn 07 minus084JAK1 088 plusmn 024 12 093 142 plusmn 003 17 plusmn 01lowastlowast 147 111JAK2 225 354 plusmn 081 407 plusmn 027 396 plusmn 057 301LEPR 018 092 077 081 039 plusmn 021 113 107MYC minus186 plusmn 03 minus228 plusmn 02 minus170 plusmn 04 minus127 plusmn 07 minus158 plusmn 05 minus170 plusmn 11 minus079PIAS1 048 plusmn 038 004 plusmn 049 110 plusmn 061 104 plusmn 037 097 plusmn 012 021 plusmn 069 061 plusmn 005PIAS4 188 plusmn 043 205 plusmn 020 224 plusmn 077 186 plusmn 042 184 plusmn 012 189 plusmn 012 206 plusmn 051PIK3CA 145 151 164 plusmn 013PIK3R1 072 104 241 245 plusmn 068 195 221PIM1 028 plusmn 02 090 plusmn 019 110 plusmn 048 137 plusmn 04lowast 142 plusmn 04lowast 103 plusmn 038 082 plusmn 028PTPN11 078 plusmn 053 110 plusmn 046 084 plusmn 038 109 plusmn 043 103 plusmn 04lowast 091 044 plusmn 016PTPN6 044 plusmn 043 070 plusmn 018 135 plusmn 04lowast 112 plusmn 060 041 plusmn 019 005 plusmn 023 047SOCS2 148 plusmn 061 112 plusmn 094 121 plusmn 087 171 plusmn 073 175 plusmn 109 206 plusmn 004 220 plusmn 090SPRED2 017 plusmn 034 minus045 plusmn 02 minus058 minus082 minus073SPRY4 minus013 plusmn 07 minus014 plusmn 07 minus035 plusmn 02 minus061 plusmn 02 minus114 plusmn 02 minus103 minus100 plusmn 07STAT1 360 plusmn 055 337 plusmn 080 468 plusmn 08lowast 44 plusmn 005lowast 51 plusmn 05lowastlowast 331 plusmn 144 418 plusmn 039STAT2 145 plusmn 051 157 plusmn 038 239 plusmn 06lowast 225 plusmn 02lowast 201 plusmn 042 158 plusmn 074 175 plusmn 035STAT3 113 plusmn 026 154 plusmn 007 132 plusmn 030 105 plusmn 081 18 plusmn 03lowastlowast 159 plusmn 013 162 plusmn 057STAT5A 139 plusmn 025 08 plusmn 001lowast 090 plusmn 053 099 plusmn 043 101 plusmn 02lowast 05 plusmn 02lowastlowast 193TYK2 036 plusmn 028 063 085 plusmn 043 075 plusmn 033 035 plusmn 032 058lowast119901 lt 005 lowastlowast119901 lt 001 and gene expression in MPNs versus control

genes in PV have been observed 82 in ET and 94 genes inPMF We demonstrated that majority of JAK-STAT signalingpathway related genes have been augmented in MPNs withincreased JAK2V617F allele burden except STAT5 geneProinflammatory IL-6 (IL6ST PTPN11) NF-120581B (TNFRSF1ATRADD) and TGF-120573 (TFDP1) signaling pathways demon-strated increase of the related genes as well as certain anti-inflammatory IL-10 signaling pathway related genes (BLVRAHMOX1) in CD34+ cells of MPNs IL-6 cytokine levels wereincreased in plasma of MPNs as well as IL-6 protein levelsin bone marrow stroma of MPNs where they demonstrated

dependence of JAK2V617F presence in ET and PMF pa-tients

According to a large study with 1545 PV patients medianhemoglobin level was 184 gL (in range 151ndash265) medianleukocyte count was 104times 109L (in range 3ndash171) andmedianplatelet count was 466 times 109L (in range 7ndash2370) Leukocyto-sis was present in 49 while thrombocytosis in 53 of PVpatients [20] In other study with 327 PV patients medianhemoglobin level was 176 gL median leukocyte count was13 times 109L and median platelet count was 515 times 109L [21]In study with 707 PMF patients median hemoglobin level


JAK2V617FControl ET PV PMF

SOCS4Interleukin 23 alpha

v-myc avian myelocytomatosis viral oncogene homolog Protein tyrosine phosphatase nonreceptor type 6

SPRY4 Sprouty RTK signaling antagonist 4IL3EPOIL6STCCND2 Cyclin D2IL12BSPRED2 Sprouty-related EVH1 domain containing 2

Suppressor of cytokine signaling 7STAT1IL15RA

Colony stimulating factor 2 receptor alphaCBLSOCS3SOCS2PIK3CBPTPN11IL10RAPIM1TYK2PIK3CDIFNAR2

IL6RSTAT3BCL2L1CSF3RPIK3CA

GRB2IL7R

IL11RA

PIAS4PIAS2SOCS1CSF3RIL2RBJAK2STAT1STAT2SOCS5JAK1IFNGR2 Interferon gamma receptor 2STAT5AEP300

AKT2CCND3 Cyclin D3IL10RBIL15PTPN6PIK3R1LIFPIK3R2PIAS1PTPN6PRLR Prolactin receptorPIAS3CREBBIL7CNTFR Ciliary neurotrophic factor receptor

LEPR Leptin receptorIFNGR1

IRF9

IL23ASuppressor of cytokine signaling 4

MYCPTPN6

Interleukin 3ErythropoietinInterleukin 6 signal transducer

Interleukin 12B

SOCS7Signal transducer and activator of transcriptionInterleukin 15 receptor alpha

CSF2RACbl protooncogene E3 ubiquitin protein ligase Suppressor of cytokine signaling 3Suppressor of cytokine signaling 2Phosphatidylinositol-45-bisphosphate 3-kinase catalytic subunit betaProtein tyrosine phosphatase nonreceptor type 11Interleukin 10 receptor alphaPim-1 protooncogene serinethreonine kinaseTyrosine 2Phosphatidylinositol-45kinase-bisphosphate 3-kinase catalytic subunit deltaInterferon (alpha beta and omega) receptor 2

Interferon gamma receptor 1Interleukin 6 receptorSignal transducer and activator of transcription 3BCL2-like 1Colony stimulating factor 3 receptorPhosphatidylinositol-45-bisphosphate 3-kinase catalytic subunit alphaInterleukin 11 receptor alphaGrowth factor receptor-bound protein 2Interleukin 7 receptorProtein inhibitor of activated STAT 4Protein inhibitor of activated STAT 2Suppressor of cytokine signaling 1Colony stimulating factor 3 receptorInterleukin 2 receptor betaJanus kinase 2 Signal transducer and activator of transcription 1Signal transducer and activator of transcription 2Suppressor of cytokine signaling 5Janus kinase 1

Signal transducer and activator of transcription 5AE1A binding protein p300Interferon regulatory factor 9v-akt murine thymoma viral oncogene homolog 2

Interleukin 10 receptor betaInterleukin 15Protein tyrosine phosphatase nonreceptor type 6Phosphoinositide-3-kinase regulatory subunit 1Leukemia inhibitory factorPhosphoinositide-3-kinase regulatory subunit 2Protein inhibitor of activated STAT 1Protein tyrosine phosphatase nonreceptor type 6

Protein inhibitor of activated STAT 3CREB binding proteinInterleukin 7

minus minus minus+ + +++

minus121 0 121

0432 1 231

Figure 2 Hierarchical clustering of JAK-STAT signaling pathway related genes expressed in CD34+ cells of MPNs The color indicates therelative fold expression of each gene red indicates increased expression green negative expression and black not changed expression whilegray stands for absent expression per each examined sample The gene correlations are uncentered

was 110 gL (in range 63ndash149) median leukocyte count was13 times 109L (in range 27ndash350) and median platelet countwas 515 times 109L (in range 44ndash990) [22] In next study with612 MPN patients mean hemoglobin level was 137 174 and119 gL mean leukocyte count was 102 124 and 133 times 109Land mean platelet count was 889 579 and 517 times 109L forET PV and PMF patients respectively [23] According to asmall study with 21 ET patients median red cell level was 446times 1012L (314ndash578) median hemoglobin level was 135 gL(105ndash166) median leukocyte count was 107 times 109L (51ndash198) and median platelet count was 872 times 109L (310ndash1443)In the same report with 17 PV patients median red celllevel was 615 times 1012L (509ndash795) median hemoglobin levelwas 165 gL (138ndash205) median leukocyte count was 100 times109L (66ndash272) and median platelet count was 501 times 109L(221ndash936) [24] Reported laboratory findings are in range ofour presented results In our study hemoglobin mean level158 gL for PVwas reduced 142 gL for ETwas similar 138 gL

for PMF was increased in comparison to other studies but isstill in range of the observed results [20 21 24] The levelsof red blood cells and leukocytes in our MPN patients weresimilar to previous reports The level of platelets in our ETpatients was similar but elevated in PV and PMF patientsin comparison to previous results However the increasedvalues were within the limit of the reported results

Median numbers and ranges of circulating CD34+ cellswere 23 times 106L (0ndash5) in control subjects 22 times 106L (0ndash14)in those with PV 24 times 106L (0ndash14) in those with ET and114 times 106L (6ndash2520) in PMF patients [25] Comparing to ourstudy CD34+ cell levels were reduced in ET and PV patientsbut were still within range while they were similar for PMFpatients (Table 1) More similar median levels of circulatingCD34+ cells in PV and ET have been observed in anotherstudy 89times 106L (0ndash31) in thosewith PV 41times 106L (0ndash18) inthose with ET and 60 times 106L (11ndash449) in PMF patients [17]A significant and positive correlation between the proportion


Table3Inflammationsig

nalin

grelatedgene

expressio

nin

CD34+

cells

ofMPN

s

Genes

Con

trol

ETPV

PMF

Mean

SDMean

SDMean

SDMean

SDIL-6

signalin

gpathway

related

gene

expressio

nCE

BPB

CCAAT

enh

ancerb

inding

protein120573

minus03

048

minus05

063

minus03

049

minus01

054

FOS

FBJm

urineo

steosarcomav

iraloncogeneh

omolog

246

087

311

104

374

095

235

138

MAP2

K1Mito

gen-activ

ated

proteinkinase

kinase

1049

031

07

015

106

026

019

052

RAF1

v-raf-1

murineleukemiaviraloncogeneh

omolog

116

6029

202

027

211

034

195

038

SHC1

Srch

omolog

y2do

maincont

transfo

rmingprotein1

minus07

0minus03

054

027

004

minus11

0CS

NK2

A1

Casein

kinase

21205721p

olypeptid

e023

006

023

005

035

051

048

052

NF-120581Bsig

nalin

gpathway

related

gene

expressio

nTN

FRSF1A

Tumor

necrosisfactor

receptor

superfam

ilym

ember1A

058

072

126

086

154

042

164

040

NFK

BIA

Nuclear

factor

ofkapp

alight

polypept

gene

enhancer

inB-cells

inhibitor120572

424

082

370

102

357

091

282

106

TRADD

TNFR

SF1A

-associatv

iadeathdo

main

130

042

167

022

179

031

138

032

NFK

B1Nuclear

factor

ofkapp

alight

polypept

gene

enhancer

inB-cells

1069

017

028

113

117

024

134

116

TGF-betasig

nalin

gpathway

related

gene

expressio

nCD

KN2B

Cyclin-dependent

kina

seinhibitor2

B064

105

minus01

115

minus07

111

minus12

114

ID3

Inhibitoro

fDNAbind

ing3do

minan

tnegativeh

elix-lo

op-helix

protein

minus21

048

minus24

065

minus31

014

minus31

037

TFDP1

Tran

scriptionfactor

Dp-1

minus01

049

011

04

061

04

096

069

BMPR

2Bo

nemorph

ogeneticprot

rectypeII

199

010

2089

201

016

TGFB

R1Transfo

rminggrow

thfactor120573

recep1

059

035

104

05

066

014

TGFB

R2Transfo

rminggrow

thfactor120573

recepII

167

018

155

029

161

0IL-10sig

nalin

gpathway

related

gene

expressio

nBL

VRA

Biliv

erdinredu

ctaseA

070

059

113

045

150

040

131

029

IL10RB

Interle

ukin

10receptor

096

031

130

070

115

073

083

075

HMOX1

Hem

eoxygena

se(decyclin

g)1

130

044

241

044

232

059

Bolded

genesrepresent

significantly

changedexpressio

nversus

controls(119901lt005)


PVControl

PMFET

IL-6

(pg

mL)

lowastlowastlowast

lowast

lowast

0

1

2

3

4

5

6

7

++ + + minus +minus minus

JAK2V617F

(a) Peripheral bloodPVControl

PMFET

lowastlowast

lowast

lowastlowast

lowastlowast

lowastlowast

0

5

10

15

20

25

IL-6

posit

ive c

ells

()

++ + +minus + minus minus

JAK2V617F

(b) Bone marrow

Figure 3 IL-6 levels in peripheral blood and bone marrow of MPNs in accordance with JAK2V617F mutant allele burden (a) IL-6 levelin peripheral blood of patients with MPN determined by ELISA (119899 = 7) (b) IL-6 level in bone marrow of patients with MPN determinedby immunohistochemistry (minus) no JAK2V617F (+) heterozygosity and (++) homozygosity for JAK2V617F mutation (119899 = 4-5 plus control119899 = 6) Values are mean plusmn SEM lowast119901 lt 005 lowastlowast119901 lt 001 regarding MPNs versus Control

of JAK2V716F mutant alleles and circulating CD34+ countsin MPNs has been found CD34+ cell counts were similar inhealthy subjects (median value 13 times 106L) and individualswith secondary erythrocytosis (median value 18 times 106L)However PVpatients with JAK2V617F had higher circulatingCD34+ counts than healthy individuals without the mutation[26] This is in accordance with our observation PV patientscarrying up to 50 JAK2 mutant alleles had slightly butsignificantly higher CD34+ cell counts (median value 32 times106L) than controls doubled in our results Moreoverpatients with PV and more than 50 JAK2V617F allele hadmarkedly higher CD34+ cell counts (median value 214 times106L) than those with 50 or less mutant alleles [26] Thisvalue for CD34+ cell level in PV was doubled in comparisonto our result (94 times 106L) for PV patients with more than50 JAK2V617F allele In addition according to our resultsET patients regardless of JAK2mutant alleles had higher cir-culating CD34+ counts than healthy individuals MoreoverPMF patients had also increased level of circulating CD34+cells than healthy individuals more elevated in patients withJAK2V617F allele

Chronic inflammation may be an initiator of clonalevolution in patients with MPNs The presence of elevatedleukocyte and thrombocyte counts inMPNsmay be the resultof both clonal myeloproliferation and chronic inflammationpresented by hypersensitivity to cytokine stimulation [27]We demonstrated increased expression of JAK-STAT relatedgenes and increased IL-6 levels in MPNs IL-6-JAK1-STAT3signal transduction pathway had an important role in theconversion of differentiated cancer cells into cancer stemcells [28] Inhibition of JAK blocked IL-6-induced phos-phorylation of STAT3 but failed to block the phosphory-lation of mitogen-activated protein kinase (MAPK) [29]Another major signaling pathway for IL-6-type cytokines

was the MAPK cascade [2] Soluble calreticulin inducedproinflammatory cytokine IL-6 in macrophages via MAPK-NF-120581B signaling pathway [30] SOCS1 expression was ele-vated in MPN granulocytes but the level was independentof JAK2V617F mutational status SOCS3 is an inhibitorof IL-6 and STAT3 regulated by DNA methylation Thetranscript levels of SOCS3 were increased in granulocytesfrom JAK2V617F-positive MPN patients Hypermethylationof the SOCS3 promoter was identified in 32 of patients withPMF but not in patients with ET and PV [31]

We presented expression of genes related to inflammatoryNF-120581B and TGF-120573 signaling in MPNs IL-6 transsignalingwas dependent on STAT3 and mediated through enhancedTGF-120573 signaling [4] A dysregulation of NF-120581B was alreadyaddressed as responsible for the MPNs [31] NF-120581B dysreg-ulation in microRNA-146a-deficient mice drove the devel-opment of myeloid malignancies MicroRNA-146a has beenimplicated as a negative feedback regulator of NF-120581B acti-vation The mice also exhibited chronic myeloproliferationin their bone marrow with increased transcription of NF-120581B-regulated genes [32] NF-120581B was a central mediator ofTGF-120573 induction in monocytes from patients with PMF[33] A spontaneous activation of NF-120581B was also detectedin proliferating megakaryocytes and circulating CD34+ cellsof patients with PMF involved in TGF-1205731 secretion [34]Patients withMPNhad higher peripheral blood plasma levelsof both bioactive and total TGF-1205731 compared to healthycontrols [35] TGFBR2 was significantly overexpressed inadvanced PMF stages produced by endothelial cells of theincreased microvessel network [36] Expression of membersof bone morphogenetic protein (BMP) family BMP1 BMP6BMP7 and BMP-receptor 2 was significantly increased inadvanced stages of PMF [36] Cytokine IL-10 had increasedlevels in plasma of PMF patients [8] In our analysis gene


Table 4 Comparison of gene expression profiling between former and present studies in CD34+ cells of MPN subjects

Studies Guglielmelli et al [17] Jones et al [18] Steidl et al [19] Cokic et alMPN PMF PMF PV PV ET PMF

Purified from PB-PMFBM-HC

PB-PMFBM-HC

BM-PMFBM-HC

PB-PV ET and PMFPB-HC

RNA Pooled Amplified 50 ngof total RNA Individual Amplified 300 ng

of total RNAExaminedMPN patients

Mixed de novotreatedpaused

Mixed de novotreatedpaused De novo De novo

Number of patients PMF 3 times 5HC 3 times 5

PMF 8HC 6

PV 4HC 10

PV 7 ET 9PMF 4 and HC 8

Microarraychips

Affymetrix HG-U133AGeneChip

Affymetrix HGU95Av2chip

Atlas Clontech Human12 I Operon Human Genome 40

Number of examinedgenes

16000 genes(22238 probes)

9670 genes(12625 probes) 1185 genes 25100 genes

(35035 probes)

JAK2V617F Mixture of 8 mutatedand 7 nonmutated Not analyzed Not analyzed Separated according to

mutant allele burden

Characterized genes 174 95 (48 upregulated and47 downregulated) 107 PV 261 ET 82

PMF 94HC healthy controls PB peripheral blood and BM bone marrow

expression of receptor for IL-10 (IL10RB) was increased inCD34+ cells of ET and PV and reduced in PMF with nostatistical significance

The recent JAK12 inhibitor trials in MPNs unexpectedlyshowed that reducing inflammation can be more beneficialto patients than targeting gene mutants [37] Increased levelof cytokine IL-6 was associated with shorter survival inpatients with PMF [8] The proportion of hematopoieticcells expressing the JAK2V617F mutation decreased afterin vitro differentiation of CD34+ cells in the presence ofoptimal concentration of IL-6 and other growth factors[38] Panobinostat a pan-deacetylase inhibitor that depletesJAK2V617F levels and JAKSTAT signaling in MPN cellsreduced IL-6 level in plasma of PMF patients [39] More-over IL-6 secreted by stromal cells protected JAK2V617Fmutated cells MPN clones from JAK2 inhibitor therapy[40] Siltuximab a chimeric anti-IL-6 monoclonal antibodydid not reduce RBC transfusions in transfusion-dependentpatients with low- and intermediate-1-risk myelodysplasticsyndrome [41] The addition of siltuximab to bortezomib didnot improve progression-free or overall survival regardlessof a numerical increase in response rate of patients withrelapsed or refractorymultiplemyeloma [42]The addition ofsiltuximab to bortezomib-melphalan-prednisone improvedthe complete response rate by 5 overall response rate by8 and partial response rate by 20 in multiple myeloma[43] Siltuximab in combination with dexamethasone yieldeda partial response rate of 23 in patients with relapsed orrefractory multiple myeloma [44]

Formicroarray analyses of CD34+ cells we used amplifiedRNA Polacek et al observed 94 overlap of the differentiallyexpressed genes when starting with 100 ng total RNA input inthe amplification procedure [45] Greater than 90 overlaphas been reported with oligo arrays when starting with250 ng total RNA input [46 47] In addition the quality

of the array data was superior to that obtained using totalRNA [48] Therefore we used 300 ng total RNA input foramplification and it is couple times higher than or similar tothe previous observations with more than 90 overlap Bythis approach we greatly reduced the possibility of biases inthe relative representations of unique RNAs Previous profilereports included various quantities of examined genes frompurified CD34+ cells of PMF and PV subjects (Table 4) [17ndash19] We performed microarray studies with 25100 genes inCD34+ cells from the peripheral blood of MPN subjectsAccording to different technical approaches as presented inTable 4 (we prepared amplifiedRNA fromperipheral blood ofindividualMPNpatients and controls rather than performingpools of RNA from patient groups and bone marrow derivedCD34+ cells) we found some minor overlap in differentiallyexpressed genes In microarray study of well characterizedand significantly different genes performed by Guglielmelliet al FGFR1 KLF4 and TGFBI levels were decreased whileIFITM1 level was increased in PMF CD34+ cells [17] Inour study FGFR4 KLF2 gene expression was increased inPV augmented by JAK2V617F mutant allele burden whileTGFBI level was increased in PV and ET patients IFITM1level was also increased in our PV and ET CD34+ cellsenhanced by JAK2V617Fmutant allele burden (SupplementalTable 1) In microarray study of Jones et al TNFRSF1A andTGIF2-C20ORF24 were downregulated while NCF2 geneexpression was upregulated in PMF CD34+ cells [18] Inour study NCF2 gene expression was upregulated in PVwhile TNFRSF1A was upregulated in JAK2V617F positiveET patients In gene expression profiling study by Steidl etal CAPNS1 CASP1 GPX1 and HSPA8 were downregulatedwhile FGFR4 TGFBI and IL-6 were upregulated in PVCD34+ cells [19] In our study CAPNS1 and HSPA8 weredownregulated in PMF and PV whereas CASP1 IL6ST andGPX1 were upregulated in PV and ET respectively FGFR4


was upregulated in PV patients augmented by JAK2V617Fallele burdenTherefore the increased levels of inflammationbiomarkers (TGFBI IL-6) were also detected in previousmicroarray studies

5 Conclusion

Besides determination of significantly changed genes inCD34+ cells of MPNs we confirmed activation of JAK-STATsignaling related genes such as CSF3R IL6ST PIM1 andSTAT12 Regarding parameters of inflammation leukocyto-sis was increased in PV and PMF while thrombocytosis wasreduced in ET and PMF with JAK2V617F mutation Proin-flammatory cytokine IL-6 levels were generally increased inperipheral blood and bone marrow of patients with MPNbut were more prominent in JAK2V617F mutation positiveET and PMF patients Therefore JAK2V617F mutant alleleburden participated in inflammation biomarkers inductionThe presented inflammation biomarkers will provide a betterunderstanding of molecular mechanisms implicated in thedevelopment and progression of MPNs

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Vladan P Cokic Olivera Mitrovic-Ajtic Bojana B Beleslin-Cokic Dragana Markovic Marijana Buac Milos Diklic andNada Kraguljac-Kurtovic performed the research VladanP Cokic Pavle Milenkovic and Puri K Raj designed theresearch Svetozar Damjanovic Mirjana Gotic Puri K Rajand Nada Kraguljac-Kurtovic contributed essential reagentsor tools Vladan P Cokic Olivera Mitrovic-Ajtic BojanaB Beleslin-Cokic Dragana Markovic and Marijana Buacanalyzed the data Vladan P Cokic wrote the paper and PavleMilenkovic Mirjana Gotic and Puri K Raj revised the paper

Acknowledgments

This researchwas supported by Intramural Research Programof Alan N Schechter at the National Institute of Diabetesand Digestive and Kidney Diseases NIH Bethesda and bya Grant from the Serbian Ministry of Education Science andTechnological Development (no 175053)

References

[1] G Landskron M De La Fuente P Thuwajit C Thuwajit andM A Hermoso ldquoChronic inflammation and cytokines in thetumor microenvironmentrdquo Journal of Immunology Researchvol 2014 Article ID 149185 19 pages 2014

[2] P C Heinrich I Behrmann S Haan H M Hermanns GMuller-Newen and F Schaper ldquoPrinciples of interleukin (IL)-6-type cytokine signalling and its regulationrdquo The BiochemicalJournal vol 374 no 1 pp 1ndash20 2003

[3] J J Babon L N Varghese and N A Nicola ldquoInhibition of IL-6family cytokines by SOCS3rdquo Seminars in Immunology vol 26no 1 pp 13ndash19 2014

[4] S OrsquoReilly M Ciechomska R Cant and J M Van LaarldquoInterleukin-6 (IL-6) trans signaling drives a STAT3-dependentpathway that leads to hyperactive transforming growth factor-120573 (TGF-120573) signaling promoting SMAD3 activation and fibrosisvia gremlin proteinrdquo The Journal of Biological Chemistry vol289 no 14 pp 9952ndash9960 2014

[5] K Heikkila S Ebrahim and D A Lawlor ldquoSystematic reviewof the association between circulating interleukin-6 (IL-6) andcancerrdquo European Journal of Cancer vol 44 no 7 pp 937ndash9452008

[6] C Mirantes E Passegue and E M Pietras ldquoPro-inflammatorycytokines emerging players regulatingHSC function in normaland diseased hematopoiesisrdquo Experimental Cell Research vol329 no 2 pp 248ndash254 2014

[7] K E Panteli E C Hatzimichael P K Bouranta et al ldquoSeruminterleukin (IL)-1 IL-2 sIL-2Ra IL-6 and thrombopoietinlevels in patients with chronic myeloproliferative diseasesrdquoBritish Journal ofHaematology vol 130 no 5 pp 709ndash715 2005

[8] A Tefferi R Vaidya D Caramazza C Finke T Lasho and APardanani ldquoCirculating interleukin (IL)-8 IL-2R IL-12 and IL-15 levels are independently prognostic in primarymyelofibrosisa comprehensive cytokine profiling studyrdquo Journal of ClinicalOncology vol 29 no 10 pp 1356ndash1363 2011

[9] H-C Hsu W-H Tsai M-L Jiang et al ldquoCirculating levels ofthrombopoietic and inflammatory cytokines in patients withclonal and reactive thrombocytosisrdquo Journal of Laboratory andClinical Medicine vol 134 no 4 pp 392ndash397 1999

[10] CWickenhauser JThiele J Lorenzen et al ldquoPolycythemia veramegakaryocytes but not megakaryocytes from normal controlsand patients with smokers polyglobuly spontaneously expressIL-6 and IL-6R and secrete IL-6rdquo Leukemia vol 13 no 3 pp327ndash334 1999

[11] C Lee H-K Lim J Sakong Y-S Lee J-R Kim and S-H Baek ldquoJanus kinase-signal transducer and activator oftranscription mediates phosphatidic acid-induced interleukin(IL)-1120573 and IL-6 productionrdquoMolecular Pharmacology vol 69no 3 pp 1041ndash1047 2006

[12] M Colomiere A C Ward C Riley et al ldquoCross talk ofsignals between EGFR and IL-6R through JAK2STAT3 medi-ate epithelial-mesenchymal transition in ovarian carcinomasrdquoBritish Journal of Cancer vol 100 no 1 pp 134ndash144 2009

[13] T L Lee J Yeh C Van Waes and Z Chen ldquoEpigenetic mod-ification of SOCS-1 differentially regulates STAT3 activation inresponse to interleukin-6 receptor and epidermal growth factorreceptor signaling through JAK andor MEK in head and necksquamous cell carcinomasrdquoMolecular CancerTherapeutics vol5 no 1 pp 8ndash19 2006

[14] A Quintas-Cardama K Vaddi P Liu et al ldquoPreclinicalcharacterization of the selective JAK12 inhibitor INCB018424therapeutic implications for the treatment of myeloproliferativeneoplasmsrdquo Blood vol 115 no 15 pp 3109ndash3117 2010

[15] A Kirabo S O Park A Majumder et al ldquoThe Jak2 inhibitorG6 alleviates Jak2-V617F-mediated myeloproliferative neopla-sia by providing significant therapeutic efficacy to the bonemarrowrdquo Neoplasia vol 13 no 11 pp 1058ndash1068 2011

[16] C Lee J-I Oh J Park et al ldquoTNF 120572 mediated IL-6 secretionis regulated by JAKSTAT pathway but not by MEK phospho-rylation and AKT phosphorylation in U266 multiple myeloma


cellsrdquo BioMed Research International vol 2013 Article ID580135 8 pages 2013

[17] P Guglielmelli R Zini C Bogani et al ldquoMolecular profilingof CD34+ cells in idiopathic myelofibrosis identifies a set ofdisease-associated genes and reveals the clinical significance ofWilmsrsquo tumor gene 1 (WT1)rdquo Stem Cells vol 25 no 1 pp 165ndash173 2007

[18] L C Jones A Tefferi P T Vuong J C Desmond W-K Hof-mann and H P Koeffler ldquoDetection of aberrant gene expres-sion in CD34+ hematopoietic stem cells from patients withagnogenic myeloid metaplasia using oligonucleotide microar-raysrdquo Stem Cells vol 23 no 5 pp 631ndash637 2005

[19] U Steidl T Schroeder C Steidl et al ldquoDistinct gene expressionpattern ofmalignant hematopoietic stem and progenitor cells inpolycythemia verardquoAnnals of theNewYorkAcademy of Sciencesvol 1044 pp 94ndash108 2005

[20] A Tefferi E Rumi G Finazzi et al ldquoSurvival and prognosisamong 1545 patients with contemporary polycythemia vera aninternational studyrdquo Leukemia vol 27 no 9 pp 1874ndash1881 2013

[21] G Bonicelli K Abdulkarim M Mounier et al ldquoLeucocytosisand thrombosis at diagnosis are associatedwith poor survival inpolycythaemia vera a population-based study of 327 patientsrdquoBritish Journal of Haematology vol 160 no 2 pp 251ndash254 2013

[22] T Barbui A Carobbio F Cervantes et al ldquoThrombosis inprimary myelofibrosis incidence and risk factorsrdquo Blood vol115 no 4 pp 778ndash782 2010

[23] A Enblom E Lindskog H Hasselbalch et al ldquoHigh rateof abnormal blood values and vascular complications beforediagnosis of myeloproliferative neoplasmsrdquo European Journal ofInternal Medicine vol 26 no 5 pp 344ndash347 2015

[24] E Pourcelot C Trocme J Mondet S Bailly B Toussaint and PMossuz ldquoCytokine profiles in polycythemia vera and essentialthrombocythemia patients clinical implicationsrdquo ExperimentalHematology vol 42 no 5 pp 360ndash368 2014

[25] F Passamonti L Vanelli L Malabarba et al ldquoClinical utilityof the absolute number of circulating CD34-positive cells inpatients with chronic myeloproliferative disordersrdquo Haemato-logica vol 88 no 10 pp 1123ndash1129 2003

[26] F Passamonti E Rumi D Pietra et al ldquoRelation betweenJAK2 (V617F) mutation status granulocyte activation andconstitutive mobilization of CD34+ cells into peripheral bloodin myeloproliferative disordersrdquo Blood vol 107 no 9 pp 3676ndash3682 2006

[27] H C Hasselbalch ldquoPerspectives on chronic inflammation inessential thrombocythemia polycythemia vera and myelofi-brosis is chronic inflammation a trigger and driver of clonalevolution and development of accelerated atherosclerosis andsecond cancerrdquo Blood vol 119 no 14 pp 3219ndash3225 2012

[28] S-Y Kim J W Kang X Song et al ldquoRole of the IL-6-JAK1-STAT3-Oct-4 pathway in the conversion of non-stem cancercells into cancer stem-like cellsrdquo Cellular Signalling vol 25 no4 pp 961ndash969 2013

[29] P Kothari R Pestana R Mesraoua et al ldquoIL-6-mediatedinduction of matrix metalloproteinase-9 is modulated by JAK-dependent IL-10 expression in macrophagesrdquo The Journal ofImmunology vol 192 no 1 pp 349ndash357 2013

[30] C-C Duo F-Y Gong X-Y He et al ldquoSoluble calreticulininduces tumor necrosis factor-120572 (TNF-120572) and interleukin (IL)-6production bymacrophages throughmitogen-activated proteinkinase (MAPK) and NF120581B signaling pathwaysrdquo InternationalJournal of Molecular Sciences vol 15 no 2 pp 2916ndash2928 2014

[31] N Fourouclas J Li D C Gilby et al ldquoMethylation of thesuppressor of cytokine signaling 3 gene (SOCS3) inmyeloprolif-erative disordersrdquoHaematologica vol 93 no 11 pp 1635ndash16442008

[32] J L Zhao D S Rao M P Boldin K D Taganov RM OrsquoConnell and D Baltimore ldquoNF-kappaB dysregulationin microRNA-146a-deficient mice drives the development ofmyeloid malignanciesrdquo Proceedings of the National Academy ofSciences of the United States of America vol 108 no 22 pp9184ndash9189 2011

[33] P Rameshwar R Narayanan J Qian T N Denny C Colonand P Gascon ldquoNF-120581B as a central mediator in the induc-tion of TGF-120573 in monocytes from patients with idiopathicmyelofibrosis an inflammatory response beyond the realm ofhomeostasisrdquo Journal of Immunology vol 165 no 4 pp 2271ndash2277 2000

[34] E Komura C Tonetti V Penard-Lacronique et al ldquoRole forthe nuclear factor kappaB pathway in transforming growthfactor-beta1 production in idiopathic myelofibrosis possiblerelationship with FK506 binding protein 51 overexpressionrdquoCancer Research vol 65 no 8 pp 3281ndash3289 2005

[35] R Campanelli V Rosti L Villani et al ldquoEvaluation of thebioactive and total transforming growth factor 1205731 levels inprimarymyelofibrosisrdquoCytokine vol 53 no 1 pp 100ndash106 2011

[36] O Bock J Hoftmann K Theophile et al ldquoBone morpho-genetic proteins are overexpressed in the bone marrow ofprimarymyelofibrosis and are apparently induced by fibrogeniccytokinesrdquoTheAmerican Journal of Pathology vol 172 no 4 pp951ndash960 2008

[37] F P S Santos and S Verstovsek ldquoJAK2 inhibitors for myelofi-brosis why are they effective in patients with and withoutJAK2V617FmutationrdquoAnti-Cancer Agents inMedicinal Chem-istry vol 12 no 9 pp 1098ndash1109 2012

[38] T Ishii E Bruno R Hoffman and M Xu ldquoInvolvementof various hematopoietic-cell lineages by the JAK2 V617Fmutation in polycythemia verardquo Blood vol 108 no 9 pp 3128ndash3134 2006

[39] D J Deangelo R A Mesa W Fiskus et al ldquoPhase II trialof panobinostat an oral pan-deacetylase inhibitor in patientswith primary myelofibrosis post-essential thrombocythaemiaand post-polycythaemia vera myelofibrosisrdquo British Journal ofHaematology vol 162 no 3 pp 326ndash335 2013

[40] TManshouri Z Estrov A Quintas-Cardama et al ldquoBonemar-row stroma-secreted cytokines protect JAK2V617F-mutatedcells from the effects of a JAK2 inhibitorrdquo Cancer Research vol71 no 11 pp 3831ndash3840 2011

[41] G Garcia-Manero G Gartenberg D P Steensma et al ldquoAphase 2 randomized double-blind multicenter study compar-ing siltuximab plus best supportive care (BSC)with placebo plusBSC in anemic patients with International Prognostic ScoringSystem low- or intermediate-1-risk myelodysplastic syndromerdquoAmerican Journal of Hematology vol 89 no 9 pp E156ndashE1622014

[42] R Z Orlowski L Gercheva C Williams et al ldquoA phase2 randomized double-blind placebo-controlled study of sil-tuximab (anti-IL-6 mAb) and bortezomib versus bortezomibalone in patients with relapsed or refractorymultiplemyelomardquoAmerican Journal of Hematology vol 90 no 1 pp 42ndash49 2015

[43] J San-Miguel J Blade O Shpilberg et al ldquoPhase 2 randomizedstudy of bortezomib-melphalan-prednisone with or withoutsiltuximab (anti-IL-6) in multiple myelomardquo Blood vol 123 no26 pp 4136ndash4142 2014


[44] P M Voorhees R F Manges P Sonneveld et al ldquoA phase 2multicentre study of siltuximab an anti-interleukin-6 mono-clonal antibody in patients with relapsed or refractory multiplemyelomardquo British Journal of Haematology vol 161 no 3 pp357ndash366 2013

[45] D C Polacek A G Passerini C Shi et al ldquoFidelity andenhanced sensitivity of differential transcription profiles follow-ing linear amplification of nanogram amounts of endothelialmRNArdquo Physiological Genomics vol 13 no 2 pp 147ndash156 2003

[46] M G Carter T Hamatani A A Sharov et al ldquoIn situ-synthesized novel microarray optimized for mouse stemcell and early developmental expression profilingrdquo GenomeResearch vol 13 no 5 pp 1011ndash1021 2003

[47] S Klur K Toy M P Williams and U Certa ldquoEvaluationof procedures for amplification of small-size samples forhybridization onmicroarraysrdquoGenomics vol 83 no 3 pp 508ndash517 2004

[48] A L Feldman N G Costouros E Wang et al ldquoAdvantagesof mRNA amplification formicroarray analysisrdquo BioTechniquesvol 33 no 4 pp 906ndash914 2002

Submit your manuscripts athttpwwwhindawicom

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014


MEDIATORSINFLAMMATION

of


Behavioural Neurology

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Immunology ResearchHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinsonrsquos Disease

Evidence-Based Complementary and Alternative Medicine

Volume 2014Hindawi Publishing Corporationhttpwwwhindawicom


correlation between IL-6 and angiogenesis in bone marrowof MPN patients [7 8] The endogenous levels of IL-6 andsoluble IL-6 receptor are also elevated in essential thrombo-cythemia (ET) [9] It has been reported that although serumlevel of IL-6 is not increased in polycythemia vera (PV)the increased percentage of megakaryocytes secreting thiscytokine is observed in bonemarrow of patients with PV [10]

Activation of the JAK-STAT signaling pathway resulted inthe production of IL-6 [11] Moreover JAK2-specific inhibi-tion blocks STAT3 phosphorylation and IL-6 production incancer cells [12] STAT3 is predominantly activated by IL-6receptor via JAK12 in cancer cell lines in association withtranscriptional silencing of SOCS-1 by hypermethylation [13]Moreover JAK12 inhibition decreases a circulating level ofproinflammatory cytokine IL-6 in MPN mouse model [14]In a transgenic mouse model of Jak2V617F-mediated MPNJAK2 inhibitor normalizes the pathologically high plasmaconcentrations of IL-6 [15] Tumor necrosis factor-alpha(TNF-120572) stimulated IL-6 production is also suppressed byinhibition of JAK2 in multiple myeloma cells [16]

According to previous reports proinflammatory cytokineIL-6 demonstrates JAK-STAT signaling dependence andhas been augmented in MPNs Majority of MPN patientshave JAK2V617Fmutation that constitutively stimulates JAK-STAT pathway Using microarray analysis we explore bothJAK-STAT and IL-6 signaling related genes in MPNs accord-ing to JAK2V617F mutant allele burden IL-6 levels havebeen examined both in peripheral blood and bonemarrow ofpatients with MPN In addition genes related to other majorinflammatory pathways such as transforming growth factor-beta (TGF-120573) and nuclear factor-kappa B (NF-120581B) have beenevaluated in MPNs

2 Material and Methods

21 Isolation of CD34+ Cells from the Peripheral Blood ofMPNPatients Thestudywas approved by the Local ResearchEthics Committee (Medical School University of Belgrade)Informed consent was obtained from all patients included inthe study All study de novo patients were subject to 30mLof peripheral blood draw on one occasion collected in 10sodium citrate Each 30mL of diluted peripheral blood (1 12with Ca2+Mg2+-free phosphate buffer saline (PBS)) was lay-ered gently on top of 15mL lymphocyte separation medium(Capricorn Scientific GmbH Ebsdorfergrund Germany)After centrifugation (400 g 30min 20∘C) the interface ofcontaining mononuclear cells was collected and washedwith PBS The CD34+ cells were isolated from the collectedmononuclear cells by magnetic separation column (SuperMacs II Miltenyi Biotec Bergisch Gladbach Germany) andmixture of magnetic microbeads conjugated with antibodyagainst CD34 (Miltenyi Biotec) according to the manu-facturerrsquos instructions The viable CD34+ cell counts wereperformed with the use of a trypan-blue exclusion technique(BioWhittaker)Thehigh purity of recoveredCD34+ cells wasdetermined by flow cytometry using PE-anti-CD34mAb (BDBiosciences San Jose CA USA) Karyotype analysis did notshow any chromosome aberrations in samples formicroarrayanalysis

22 Isolation of Total RNA We used the RNeasy protocolfor isolation of total RNA from CD34+ cells according to themanufacturerrsquos instructions (Qiagen GmbH Hilden Germa-ny) Concentration and integrity of total RNA were assessedusing NanoDrop spectrophotometer (Thermo Fisher Scien-tific Inc Wilmington Delaware USA) and Agilent 2100Bioanalyzer Software (Agilent Technologies WaldbronnGermany) comparing the ratio of 28S and 18S RNA peaks toensure that there is minimal degradation of the RNA sample

23 DNA Sequencing Genomic DNA was extracted fromperipheral blood granulocytes of MPN patients by usingthe proteinase K and phenol-chloroform technique Singlenucleotide mutation of JAK2V617F gene was characterizedby DNA sequencing after PCR amplification PCR amplifi-cation was performed with wild-type JAK-2-specific forwardprimer 51015840-tggcagagagaattttctgaact-31015840 and reverse primer 51015840-ttcattgctttcctttttcaca-31015840 confirmed by electrophoresis on anethidium bromide-impregnated 1 agarose gel PCR ampli-fied samples were analyzed by sequencing on an automatedABI PRISM 3130 Genetic Analyzer (Applied Biosystems LifeTechnologies Carlsbad CAUSA)withABDNASequencingAnalysis Software (v52) by using the Big Dye Terminator v31Ready Reaction Cycle Sequencing Kit

24 Immunohistochemical Analysis Bone marrow tissue sec-tions were fixed in 10 neutral formalin solution for 24ndash36hours and then decalcified in EDTA buffer for 3 hours andembedded in paraffin The tissue sections were cut at 5 120583mheated at 56∘C for 60min and then deparaffinized and rehy-drated through a series of xylenes and alcohols followed byan epitope retrieval step Samples were treated with 3H

2O2

solution in PBS to block endogenous peroxidase activity Thenext step was incubation with anti-IL-6 antibody (ThermoScientific dilution 1 400) in a humidity chamber overnightat room temperature Immunostaining was performed usingthe streptavidin-biotin technique (LSAB+HRP Kit DAKO)Immunoreactivity was visualized with DAKO Liquid DAB+SubstrateChromogen System counterstained with Mayerrsquoshematoxylin (Merck Whitehouse Station NJ) For the neg-ative control samples normal serum and PBS buffer (1 500)were pipetted without primary antibodies Immunoreactivecells were analyzed and scored at five powered fields in eachsample using a computer-supported imaging system (analysisPro 31) connected to the light microscope (Olympus AX70Hamburg Germany) with an objective magnification of times40Immunohistochemical staining for IL-6 was performed inbone marrow of 34 subjects (10 PV per 5 heterozygous andhomozygous 9 ET 5 no mutation and 4 heterozygous and9 PMF 5 no mutation 4 heterozygous for JAK2V617F and 6healthy subjects)

25 ELISA Assay Peripheral blood samples were obtainedfrom 45 patients with newly diagnosed untreated MPNsThe study included 14 PV 14 ET and 17 PMF cases Patientswere subclassified according to JAK2V617F mutational sta-tus as JAK2 homozygous JAK2 heterozygous and patientswithout mutation We analyzed 7 PV homozygous and 7 PV








3 Results





MPN 1198691198601198702V617P

CD34+(cells120583L)




(gL)











220 123 315

250

3450

576

645

A B

C

ET



CD34+

(a)

189 101 205

166

3587

1076

951

C

D

A

PV



CD34+

(b)

273 231 738

501

3038

417

321

A B

C

PMF



CD34+

(c)



4 Discussion




















GRB2IL7R

IL11RA




IRF9


MYCPTPN6


Interleukin 12B








minus121 0 121

0432 1 231







nalin

grelatedgene

expressio

nin

CD34+

cells

ofMPN

s

Genes

Con

trol

ETPV

PMF

Mean

SDMean

SDMean

SDMean

SDIL-6

signalin

gpathway

related

gene

expressio

nCE

BPB

CCAAT

enh

ancerb

inding

protein120573

minus03

048

minus05

063

minus03

049

minus01

054

FOS

FBJm

urineo

steosarcomav

iraloncogeneh

omolog

246

087

311

104

374

095

235

138

MAP2

K1Mito

gen-activ

ated

proteinkinase

kinase

1049

031

07

015

106

026

019

052

RAF1

v-raf-1


omolog

116

6029

202

027

211

034

195

038

SHC1

Srch

omolog

y2do

maincont

transfo

rmingprotein1

minus07

0minus03

054

027

004

minus11

0CS

NK2

A1

Casein

kinase

21205721p

olypeptid

e023

006

023

005

035

051

048

052

NF-120581Bsig

nalin

gpathway

related

gene

expressio

nTN

FRSF1A

Tumor

necrosisfactor

receptor

superfam

ilym

ember1A

058

072

126

086

154

042

164

040

NFK

BIA

Nuclear

factor

ofkapp

alight

polypept

gene

enhancer

inB-cells

inhibitor120572

424

082

370

102

357

091

282

106

TRADD

TNFR

SF1A

-associatv

iadeathdo

main

130

042

167

022

179

031

138

032

NFK

B1Nuclear

factor

ofkapp

alight

polypept

gene

enhancer

inB-cells

1069

017

028

113

117

024

134

116

TGF-betasig

nalin

gpathway

related

gene

expressio

nCD

KN2B

Cyclin-dependent

kina

seinhibitor2

B064

105

minus01

115

minus07

111

minus12

114

ID3

Inhibitoro

fDNAbind

ing3do

minan

tnegativeh

elix-lo

op-helix

protein

minus21

048

minus24

065

minus31

014

minus31

037

TFDP1

Tran

scriptionfactor

Dp-1

minus01

049

011

04

061

04

096

069

BMPR

2Bo

nemorph

ogeneticprot

rectypeII

199

010

2089

201

016

TGFB

R1Transfo

rminggrow

thfactor120573

recep1

059

035

104

05

066

014

TGFB

R2Transfo

rminggrow

thfactor120573

recepII

167

018

155

029

161

0IL-10sig

nalin

gpathway

related

gene

expressio

nBL

VRA

Biliv

erdinredu

ctaseA

070

059

113

045

150

040

131

029

IL10RB

Interle

ukin

10receptor

096

031

130

070

115

073

083

075

HMOX1

Hem

eoxygena

se(decyclin

g)1

130

044

241

044

232

059

Bolded

genesrepresent

significantly

changedexpressio

nversus



PVControl

PMFET

IL-6

(pg

mL)

lowastlowastlowast

lowast

lowast

0

1

2

3

4

5

6

7


JAK2V617F


PMFET

lowastlowast

lowast

lowastlowast

lowastlowast

lowastlowast

0

5

10

15

20

25

IL-6

posit

ive c

ells

()


JAK2V617F

(b) Bone marrow










PB-PMFBM-HC

BM-PMFBM-HC







PMF 8HC 6

PV 4HC 10


Microarraychips







(35035 probes)











5 Conclusion






Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of























3 Results





MPN 1198691198601198702V617P

CD34+(cells120583L)




(gL)











220 123 315

250

3450

576

645

A B

C

ET



CD34+

(a)

189 101 205

166

3587

1076

951

C

D

A

PV



CD34+

(b)

273 231 738

501

3038

417

321

A B

C

PMF



CD34+

(c)



4 Discussion




















GRB2IL7R

IL11RA




IRF9


MYCPTPN6


Interleukin 12B








minus121 0 121

0432 1 231







nalin

grelatedgene

expressio

nin

CD34+

cells

ofMPN

s

Genes

Con

trol

ETPV

PMF

Mean

SDMean

SDMean

SDMean

SDIL-6

signalin

gpathway

related

gene

expressio

nCE

BPB

CCAAT

enh

ancerb

inding

protein120573

minus03

048

minus05

063

minus03

049

minus01

054

FOS

FBJm

urineo

steosarcomav

iraloncogeneh

omolog

246

087

311

104

374

095

235

138

MAP2

K1Mito

gen-activ

ated

proteinkinase

kinase

1049

031

07

015

106

026

019

052

RAF1

v-raf-1


omolog

116

6029

202

027

211

034

195

038

SHC1

Srch

omolog

y2do

maincont

transfo

rmingprotein1

minus07

0minus03

054

027

004

minus11

0CS

NK2

A1

Casein

kinase

21205721p

olypeptid

e023

006

023

005

035

051

048

052

NF-120581Bsig

nalin

gpathway

related

gene

expressio

nTN

FRSF1A

Tumor

necrosisfactor

receptor

superfam

ilym

ember1A

058

072

126

086

154

042

164

040

NFK

BIA

Nuclear

factor

ofkapp

alight

polypept

gene

enhancer

inB-cells

inhibitor120572

424

082

370

102

357

091

282

106

TRADD

TNFR

SF1A

-associatv

iadeathdo

main

130

042

167

022

179

031

138

032

NFK

B1Nuclear

factor

ofkapp

alight

polypept

gene

enhancer

inB-cells

1069

017

028

113

117

024

134

116

TGF-betasig

nalin

gpathway

related

gene

expressio

nCD

KN2B

Cyclin-dependent

kina

seinhibitor2

B064

105

minus01

115

minus07

111

minus12

114

ID3

Inhibitoro

fDNAbind

ing3do

minan

tnegativeh

elix-lo

op-helix

protein

minus21

048

minus24

065

minus31

014

minus31

037

TFDP1

Tran

scriptionfactor

Dp-1

minus01

049

011

04

061

04

096

069

BMPR

2Bo

nemorph

ogeneticprot

rectypeII

199

010

2089

201

016

TGFB

R1Transfo

rminggrow

thfactor120573

recep1

059

035

104

05

066

014

TGFB

R2Transfo

rminggrow

thfactor120573

recepII

167

018

155

029

161

0IL-10sig

nalin

gpathway

related

gene

expressio

nBL

VRA

Biliv

erdinredu

ctaseA

070

059

113

045

150

040

131

029

IL10RB

Interle

ukin

10receptor

096

031

130

070

115

073

083

075

HMOX1

Hem

eoxygena

se(decyclin

g)1

130

044

241

044

232

059

Bolded

genesrepresent

significantly

changedexpressio

nversus



PVControl

PMFET

IL-6

(pg

mL)

lowastlowastlowast

lowast

lowast

0

1

2

3

4

5

6

7


JAK2V617F


PMFET

lowastlowast

lowast

lowastlowast

lowastlowast

lowastlowast

0

5

10

15

20

25

IL-6

posit

ive c

ells

()


JAK2V617F

(b) Bone marrow










PB-PMFBM-HC

BM-PMFBM-HC







PMF 8HC 6

PV 4HC 10


Microarraychips







(35035 probes)











5 Conclusion






Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















MPN 1198691198601198702V617P

CD34+(cells120583L)




(gL)











220 123 315

250

3450

576

645

A B

C

ET



CD34+

(a)

189 101 205

166

3587

1076

951

C

D

A

PV



CD34+

(b)

273 231 738

501

3038

417

321

A B

C

PMF



CD34+

(c)



4 Discussion




















GRB2IL7R

IL11RA




IRF9


MYCPTPN6


Interleukin 12B








minus121 0 121

0432 1 231







nalin

grelatedgene

expressio

nin

CD34+

cells

ofMPN

s

Genes

Con

trol

ETPV

PMF

Mean

SDMean

SDMean

SDMean

SDIL-6

signalin

gpathway

related

gene

expressio

nCE

BPB

CCAAT

enh

ancerb

inding

protein120573

minus03

048

minus05

063

minus03

049

minus01

054

FOS

FBJm

urineo

steosarcomav

iraloncogeneh

omolog

246

087

311

104

374

095

235

138

MAP2

K1Mito

gen-activ

ated

proteinkinase

kinase

1049

031

07

015

106

026

019

052

RAF1

v-raf-1


omolog

116

6029

202

027

211

034

195

038

SHC1

Srch

omolog

y2do

maincont

transfo

rmingprotein1

minus07

0minus03

054

027

004

minus11

0CS

NK2

A1

Casein

kinase

21205721p

olypeptid

e023

006

023

005

035

051

048

052

NF-120581Bsig

nalin

gpathway

related

gene

expressio

nTN

FRSF1A

Tumor

necrosisfactor

receptor

superfam

ilym

ember1A

058

072

126

086

154

042

164

040

NFK

BIA

Nuclear

factor

ofkapp

alight

polypept

gene

enhancer

inB-cells

inhibitor120572

424

082

370

102

357

091

282

106

TRADD

TNFR

SF1A

-associatv

iadeathdo

main

130

042

167

022

179

031

138

032

NFK

B1Nuclear

factor

ofkapp

alight

polypept

gene

enhancer

inB-cells

1069

017

028

113

117

024

134

116

TGF-betasig

nalin

gpathway

related

gene

expressio

nCD

KN2B

Cyclin-dependent

kina

seinhibitor2

B064

105

minus01

115

minus07

111

minus12

114

ID3

Inhibitoro

fDNAbind

ing3do

minan

tnegativeh

elix-lo

op-helix

protein

minus21

048

minus24

065

minus31

014

minus31

037

TFDP1

Tran

scriptionfactor

Dp-1

minus01

049

011

04

061

04

096

069

BMPR

2Bo

nemorph

ogeneticprot

rectypeII

199

010

2089

201

016

TGFB

R1Transfo

rminggrow

thfactor120573

recep1

059

035

104

05

066

014

TGFB

R2Transfo

rminggrow

thfactor120573

recepII

167

018

155

029

161

0IL-10sig

nalin

gpathway

related

gene

expressio

nBL

VRA

Biliv

erdinredu

ctaseA

070

059

113

045

150

040

131

029

IL10RB

Interle

ukin

10receptor

096

031

130

070

115

073

083

075

HMOX1

Hem

eoxygena

se(decyclin

g)1

130

044

241

044

232

059

Bolded

genesrepresent

significantly

changedexpressio

nversus



PVControl

PMFET

IL-6

(pg

mL)

lowastlowastlowast

lowast

lowast

0

1

2

3

4

5

6

7


JAK2V617F


PMFET

lowastlowast

lowast

lowastlowast

lowastlowast

lowastlowast

0

5

10

15

20

25

IL-6

posit

ive c

ells

()


JAK2V617F

(b) Bone marrow










PB-PMFBM-HC

BM-PMFBM-HC







PMF 8HC 6

PV 4HC 10


Microarraychips







(35035 probes)











5 Conclusion






Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















220 123 315

250

3450

576

645

A B

C

ET



CD34+

(a)

189 101 205

166

3587

1076

951

C

D

A

PV



CD34+

(b)

273 231 738

501

3038

417

321

A B

C

PMF



CD34+

(c)



4 Discussion




















GRB2IL7R

IL11RA




IRF9


MYCPTPN6


Interleukin 12B








minus121 0 121

0432 1 231







nalin

grelatedgene

expressio

nin

CD34+

cells

ofMPN

s

Genes

Con

trol

ETPV

PMF

Mean

SDMean

SDMean

SDMean

SDIL-6

signalin

gpathway

related

gene

expressio

nCE

BPB

CCAAT

enh

ancerb

inding

protein120573

minus03

048

minus05

063

minus03

049

minus01

054

FOS

FBJm

urineo

steosarcomav

iraloncogeneh

omolog

246

087

311

104

374

095

235

138

MAP2

K1Mito

gen-activ

ated

proteinkinase

kinase

1049

031

07

015

106

026

019

052

RAF1

v-raf-1


omolog

116

6029

202

027

211

034

195

038

SHC1

Srch

omolog

y2do

maincont

transfo

rmingprotein1

minus07

0minus03

054

027

004

minus11

0CS

NK2

A1

Casein

kinase

21205721p

olypeptid

e023

006

023

005

035

051

048

052

NF-120581Bsig

nalin

gpathway

related

gene

expressio

nTN

FRSF1A

Tumor

necrosisfactor

receptor

superfam

ilym

ember1A

058

072

126

086

154

042

164

040

NFK

BIA

Nuclear

factor

ofkapp

alight

polypept

gene

enhancer

inB-cells

inhibitor120572

424

082

370

102

357

091

282

106

TRADD

TNFR

SF1A

-associatv

iadeathdo

main

130

042

167

022

179

031

138

032

NFK

B1Nuclear

factor

ofkapp

alight

polypept

gene

enhancer

inB-cells

1069

017

028

113

117

024

134

116

TGF-betasig

nalin

gpathway

related

gene

expressio

nCD

KN2B

Cyclin-dependent

kina

seinhibitor2

B064

105

minus01

115

minus07

111

minus12

114

ID3

Inhibitoro

fDNAbind

ing3do

minan

tnegativeh

elix-lo

op-helix

protein

minus21

048

minus24

065

minus31

014

minus31

037

TFDP1

Tran

scriptionfactor

Dp-1

minus01

049

011

04

061

04

096

069

BMPR

2Bo

nemorph

ogeneticprot

rectypeII

199

010

2089

201

016

TGFB

R1Transfo

rminggrow

thfactor120573

recep1

059

035

104

05

066

014

TGFB

R2Transfo

rminggrow

thfactor120573

recepII

167

018

155

029

161

0IL-10sig

nalin

gpathway

related

gene

expressio

nBL

VRA

Biliv

erdinredu

ctaseA

070

059

113

045

150

040

131

029

IL10RB

Interle

ukin

10receptor

096

031

130

070

115

073

083

075

HMOX1

Hem

eoxygena

se(decyclin

g)1

130

044

241

044

232

059

Bolded

genesrepresent

significantly

changedexpressio

nversus



PVControl

PMFET

IL-6

(pg

mL)

lowastlowastlowast

lowast

lowast

0

1

2

3

4

5

6

7


JAK2V617F


PMFET

lowastlowast

lowast

lowastlowast

lowastlowast

lowastlowast

0

5

10

15

20

25

IL-6

posit

ive c

ells

()


JAK2V617F

(b) Bone marrow










PB-PMFBM-HC

BM-PMFBM-HC







PMF 8HC 6

PV 4HC 10


Microarraychips







(35035 probes)











5 Conclusion






Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

































GRB2IL7R

IL11RA




IRF9


MYCPTPN6


Interleukin 12B








minus121 0 121

0432 1 231







nalin

grelatedgene

expressio

nin

CD34+

cells

ofMPN

s

Genes

Con

trol

ETPV

PMF

Mean

SDMean

SDMean

SDMean

SDIL-6

signalin

gpathway

related

gene

expressio

nCE

BPB

CCAAT

enh

ancerb

inding

protein120573

minus03

048

minus05

063

minus03

049

minus01

054

FOS

FBJm

urineo

steosarcomav

iraloncogeneh

omolog

246

087

311

104

374

095

235

138

MAP2

K1Mito

gen-activ

ated

proteinkinase

kinase

1049

031

07

015

106

026

019

052

RAF1

v-raf-1


omolog

116

6029

202

027

211

034

195

038

SHC1

Srch

omolog

y2do

maincont

transfo

rmingprotein1

minus07

0minus03

054

027

004

minus11

0CS

NK2

A1

Casein

kinase

21205721p

olypeptid

e023

006

023

005

035

051

048

052

NF-120581Bsig

nalin

gpathway

related

gene

expressio

nTN

FRSF1A

Tumor

necrosisfactor

receptor

superfam

ilym

ember1A

058

072

126

086

154

042

164

040

NFK

BIA

Nuclear

factor

ofkapp

alight

polypept

gene

enhancer

inB-cells

inhibitor120572

424

082

370

102

357

091

282

106

TRADD

TNFR

SF1A

-associatv

iadeathdo

main

130

042

167

022

179

031

138

032

NFK

B1Nuclear

factor

ofkapp

alight

polypept

gene

enhancer

inB-cells

1069

017

028

113

117

024

134

116

TGF-betasig

nalin

gpathway

related

gene

expressio

nCD

KN2B

Cyclin-dependent

kina

seinhibitor2

B064

105

minus01

115

minus07

111

minus12

114

ID3

Inhibitoro

fDNAbind

ing3do

minan

tnegativeh

elix-lo

op-helix

protein

minus21

048

minus24

065

minus31

014

minus31

037

TFDP1

Tran

scriptionfactor

Dp-1

minus01

049

011

04

061

04

096

069

BMPR

2Bo

nemorph

ogeneticprot

rectypeII

199

010

2089

201

016

TGFB

R1Transfo

rminggrow

thfactor120573

recep1

059

035

104

05

066

014

TGFB

R2Transfo

rminggrow

thfactor120573

recepII

167

018

155

029

161

0IL-10sig

nalin

gpathway

related

gene

expressio

nBL

VRA

Biliv

erdinredu

ctaseA

070

059

113

045

150

040

131

029

IL10RB

Interle

ukin

10receptor

096

031

130

070

115

073

083

075

HMOX1

Hem

eoxygena

se(decyclin

g)1

130

044

241

044

232

059

Bolded

genesrepresent

significantly

changedexpressio

nversus



PVControl

PMFET

IL-6

(pg

mL)

lowastlowastlowast

lowast

lowast

0

1

2

3

4

5

6

7


JAK2V617F


PMFET

lowastlowast

lowast

lowastlowast

lowastlowast

lowastlowast

0

5

10

15

20

25

IL-6

posit

ive c

ells

()


JAK2V617F

(b) Bone marrow










PB-PMFBM-HC

BM-PMFBM-HC







PMF 8HC 6

PV 4HC 10


Microarraychips







(35035 probes)











5 Conclusion






Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















nalin

grelatedgene

expressio

nin

CD34+

cells

ofMPN

s

Genes

Con

trol

ETPV

PMF

Mean

SDMean

SDMean

SDMean

SDIL-6

signalin

gpathway

related

gene

expressio

nCE

BPB

CCAAT

enh

ancerb

inding

protein120573

minus03

048

minus05

063

minus03

049

minus01

054

FOS

FBJm

urineo

steosarcomav

iraloncogeneh

omolog

246

087

311

104

374

095

235

138

MAP2

K1Mito

gen-activ

ated

proteinkinase

kinase

1049

031

07

015

106

026

019

052

RAF1

v-raf-1


omolog

116

6029

202

027

211

034

195

038

SHC1

Srch

omolog

y2do

maincont

transfo

rmingprotein1

minus07

0minus03

054

027

004

minus11

0CS

NK2

A1

Casein

kinase

21205721p

olypeptid

e023

006

023

005

035

051

048

052

NF-120581Bsig

nalin

gpathway

related

gene

expressio

nTN

FRSF1A

Tumor

necrosisfactor

receptor

superfam

ilym

ember1A

058

072

126

086

154

042

164

040

NFK

BIA

Nuclear

factor

ofkapp

alight

polypept

gene

enhancer

inB-cells

inhibitor120572

424

082

370

102

357

091

282

106

TRADD

TNFR

SF1A

-associatv

iadeathdo

main

130

042

167

022

179

031

138

032

NFK

B1Nuclear

factor

ofkapp

alight

polypept

gene

enhancer

inB-cells

1069

017

028

113

117

024

134

116

TGF-betasig

nalin

gpathway

related

gene

expressio

nCD

KN2B

Cyclin-dependent

kina

seinhibitor2

B064

105

minus01

115

minus07

111

minus12

114

ID3

Inhibitoro

fDNAbind

ing3do

minan

tnegativeh

elix-lo

op-helix

protein

minus21

048

minus24

065

minus31

014

minus31

037

TFDP1

Tran

scriptionfactor

Dp-1

minus01

049

011

04

061

04

096

069

BMPR

2Bo

nemorph

ogeneticprot

rectypeII

199

010

2089

201

016

TGFB

R1Transfo

rminggrow

thfactor120573

recep1

059

035

104

05

066

014

TGFB

R2Transfo

rminggrow

thfactor120573

recepII

167

018

155

029

161

0IL-10sig

nalin

gpathway

related

gene

expressio

nBL

VRA

Biliv

erdinredu

ctaseA

070

059

113

045

150

040

131

029

IL10RB

Interle

ukin

10receptor

096

031

130

070

115

073

083

075

HMOX1

Hem

eoxygena

se(decyclin

g)1

130

044

241

044

232

059

Bolded

genesrepresent

significantly

changedexpressio

nversus



PVControl

PMFET

IL-6

(pg

mL)

lowastlowastlowast

lowast

lowast

0

1

2

3

4

5

6

7


JAK2V617F


PMFET

lowastlowast

lowast

lowastlowast

lowastlowast

lowastlowast

0

5

10

15

20

25

IL-6

posit

ive c

ells

()


JAK2V617F

(b) Bone marrow










PB-PMFBM-HC

BM-PMFBM-HC







PMF 8HC 6

PV 4HC 10


Microarraychips







(35035 probes)











5 Conclusion






Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of

















PVControl

PMFET

IL-6

(pg

mL)

lowastlowastlowast

lowast

lowast

0

1

2

3

4

5

6

7


JAK2V617F


PMFET

lowastlowast

lowast

lowastlowast

lowastlowast

lowastlowast

0

5

10

15

20

25

IL-6

posit

ive c

ells

()


JAK2V617F

(b) Bone marrow










PB-PMFBM-HC

BM-PMFBM-HC







PMF 8HC 6

PV 4HC 10


Microarraychips







(35035 probes)











5 Conclusion






Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of




















PB-PMFBM-HC

BM-PMFBM-HC







PMF 8HC 6

PV 4HC 10


Microarraychips







(35035 probes)











5 Conclusion






Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of


















5 Conclusion






Acknowledgments


References

























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
























































of






Disease Markers



OncologyJournal of





PPAR Research



Journal of

ObesityJournal of
















research article proinflammatory cytokine il-6 and jak...

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