Proteinedellafaseacutadell’infiammazionenellemala4emieloprolifera6vecroniche

SilviaSalmoiraghi

Firenze,31Marzo2011

Anac&va&ngmuta&oninthepseudokinasedomainofJAK2knownasV617Fpresentin•>95%ofPolycythemiaVera•~50%ofEssen&alThrombocythemia•~50%ofPrimaryMyelofibrosis

MolecularPathogenesisofPolycythemiaVera,Essen&alThrombocythemiaandMyelofibrosis

CytokinePlasmaLevelsinPa&entswithMF

Verstovsek,Setal.:NEnglJMed2010;363:1117‐27

Pa&entswithMyelofibrosisvsHealthycontrols

Primary Pro-Inflammatory Cytokines (eg, IL-1, TNF-α)

IL-6“Messenger” Cytokine

ICAM-1Selectins, HSPs, etc.

LiverEndotheliumand other cells

CRPSAA

Adapted from Libby P et al.Circulation. 1999;100:1148–1150.

InflammatoryPathwaysinAtherogenesis

Circulation

Pro-inflammatory RiskFactors

CytokinePlasmaLevelsinPa&entswithMF

Verstovsek,Setal.:NEnglJMed2010;363:1117‐27

Pa&entswithMyelofibrosisvsHealthycontrols

RiskFactorsforFutureCardiovascularEvents:WHS

0 1.0 2.0 4.0 6.0

Lipoprotein(a)

Homocysteine

IL-6

TC

LDLC

sICAM-1

SAA

Apo B

TC: HDLC

hs-CRP

hs-CRP + TC: HDLC

Relative Risk of Future Cardiovascular EventsRidker et al, N Engl J Med. 2000;342:836-43

MATRIXDEPOSITION

andANGIOGENESIS

Interaction withTSG-6 and IαI.

Interaction with FGF2

Mø myeloidDC

FibroblastsDecidual

stromal cells

adipocytes mesangialcellsSMC

EndotheliumEpithelium

Granulosacells

FERTILITYCumulus oophorus

maturationFertilization

Decidualizationand placentation

Implantation

PTX3

TLR AGONISTS(LPS. OmpA, CpG..)

PRIMARY INFLAMMATORYCYTOKINES (IL-1, TNF) IL-10 oxLDL

HDL

GDF9FSH, cAMP, EGF

(+ oocyte)

INNATEIMMUNITY

Resistance to A.fumigatus,

P. brasiliensis,P. aeruginosa,

K. pneumoniae,CMV, Influenza virus...

PMN

INFLAMMATIONModulation of

Complement activity(C1q and Factor H).

Interaction withapoptotic cells.

P-selectin.

CELLULARSOURCESandFUNCTIONSOFPTX3

Norata,GDetal.:TrendsCardiovascMed20:35–40,2010

TheLongPentraxinPTX3:AModulatoroftheImmunoinflammatoryResponseinAtherosclerosisand

CardiovascularDiseases

Norata,GDetal.:TrendsCardiovascMed20:35–40,2010

PTX3inhumans,likeCRP,isamarkerofatherosclerosisandcorrelateswiththeriskofdevelopingvascularevents

DeficiencyofPTX3isassociatedwithincreasedheartdamagewithagreaterno‐reflowareaandincreasedinflammatoryrespons

Differentobserva&onssuggesttheincreasedlevelofPTX3insubjectswithcardiovasculardiseasemaybeprotec&veandcorrelatewiththeseverityofthedisease.

Regulation of leukocyte recruitment andRegulation of leukocyte recruitment andinflammation by PTX3inflammation by PTX3

Under conditions of massive leukocyte activation, release of PTX3acts locally as a negative feedback mediator dampening neutrophilrecruitment. (Deban et al. Nat Imm. 2010)

Leukocytosisisariskfactorforthrombosisinessen&althrombocythemia:interac&onwithtreatment,standard

riskfactors,andJak2muta&onstatus

CarobbioCarobbioetaletalBlood,2007CarobbioCarobbioetaletalJClinOncol,2008

CarobbioCarobbioetaletalBlood,2008CarobbioCarobbioetaletalExpHematol,2009.

BarbuietalBlood,2009.BarbuietalBlood,2009CarobbioetalBlood2010

RQ‐PCRanalysisofPTX3innormalandPV,ETandMFWBC

Ctgene

PTX3

PTX3expressionbyRQ‐PCRinPV,ETandMF

DataexpressedasfoldincreasecomparedtonormalWBC

1

3

5

7

9

11

13

MF PV TE

PTX3expressionbyRQ‐PCRinPV,ETandMF

DataexpressedasfoldincreasecomparedtonormalWBC

1

3

5

7

9

11

13

15

17

MF JAK2V617FPOS MF JAK2V617FNEG

MF

1

3

5

7

9

11

13

15

17

PV JAK2V617F POS >50% PV JAK2V617F POS <50%

PV

1

3

5

7

9

11

13

15

17

ET JAK2V617FPOS ET JAK2V617FNEG

ET

AimofAimofthestudystudy

VerifywhetherhsCRPandPTX3plsmalevelscanbecorrelatedwiththrombo&ccomplica&onsinpa&entswithETandPV

Exploretherela&onshipwithJAK2V617Fmuta&oninordertorecognizenewfactorstoassessthevascularriskinMPNs

Inflamma&onandthrombosisinPVandET:adifferentroleofhsCRPandPTX3?

Copyright ©2011 Ferrata Storti Foundation

Barbui, T. et al. Haematologica 2011;96:315-318

Characteristics of patients participating in the study

PlasmalevelsofhsCRPinETandPVpa&ents

Normal ET PV controls

02

46

8

CONTROLLO ET PVexcludes outside values

PCR distribution

N3217371

Pentraxin3(PTX3)

05

1015

20

Normal controls ET PV PMFexcludes outside values

N3217371

ng/m

L

Copyright ©2011 Ferrata Storti Foundation

Barbui, T. et al. Haematologica 2011;96:315-318

Meaningful variables associated with values of hs-CRP and PTX3

Copyright ©2011 Ferrata Storti Foundation

Barbui, T. et al. Haematologica 2011;96:315-318

Unadjusted and sequentially multivariable adjusted risk of thrombosis associated to different hs-CRPand PTX3 levels

INFLAMMATIONANDTHROMBOSISINESSENTIALTHROMBOCYTHEMIAANDPOLYCYTHEMIAVERA:

DIFFERENTROLEOFC‐REACTIVEPROTEINANDPENTRAXIN3

TizianoBarbuietal.:Haematologica96(2):315,2011

Majorthrombosisratewashigherinthehighesths‐CRPter6le(p=0.01)andloweratthehighestPTX3levels(p=0.045)

Theseassocia6onsremainedsignificantinmul6variateanalysesandindicatethatbloodlevelsofhs‐CRPandPTX3independentlyandinanoppositemannermodulatetheintrinsicriskofCVinpa6entswithMPN.

theeffectofGivinostatandotherJAK2inhibitorsonPTX3andhsCRPmRNA/proteinlevel

TheroleofnewdrugsonhsCRPandPTX3plasmalevels

Givinostat: a novel HDAC inhibitor

At nanomolar concentrations,Givinostat inhibits

Gene expression and synthesis ofTNFα and IFNγ by mononuclear cellsLeoni, Mol Med. 2005

Secretion of IL-1β by preventing theexocytosis of IL-1β-containingsecretory lysosomesCarta, Blood 2006

Secretion of IL-6, VEGF and IFN-γby mesenchymal stromal cellsGolay, Leukemia 2007

Guerini V, Leukemia 2008

0.25

µM

0,1 µ

M

0,01

µM

0.5 µ

M

0,00

1µM

ITF2357

Con

trol

Col

onie

s (%

of c

ontr

ol)

20

40

60

80

100

120140

160

0

Inhibition of EEC assay in JAK2 V617F

TE (n=7) and PV (n=6)

µM ITF2357 0 0.01 0.25 0 0.01 0.25

HEL K562

p-JAK2

JAK2

p-STAT5

STAT5

p-STAT3

STAT3

Actin

EPO-R

Down-modulation of JAK2 V617F, pSTAT5and pSTAT3

Inhibition of EEC assay and JAK2 signal transduction by Givinostat

0 20 40 60 80 100 120 140 160 1800

20

40

60

80

100

Singlecolon

ygeno

type

(%oftotal)

Time(days)

323127201823

%V617FPMN

WT

Homo

Hetero

Singlecolonygenotype

Rambaldi,Aetal.:BJH2010

HCT normalization during the pre-study time

HU non responders(maximum tolerated dose)

Randomization (1 : 1)

HU MTD+

ITF2357 50 mg o.d.

Primary endpoint assessment:Overall response rate

Response No Response

continue 50 mg dose escalation

12 w

ks12

wks

HU MTD+

ITF2357 50 mg b.i.d.

baseline W12 W24

0

10

20

30

40hC

RP(m

g/L)

hsCRP before and after Givinostat+HU in PV patients

BASELINE WEEK12 WEEK24

4

6

8

10

12

14

16

18

20P

TX-3

BASELINE WEEK12 WEEK24

PTX3 before and after Givinostat+HU in PV patients

(ng/

ml)

CytokinePlasmaLevelsinPa&entswithMFbeforeandanertreatmentwithINCB018424,aJAK1andJAK2Inhibitor

Verstovsek,Setal.:NEnglJMed2010;363:1117‐27

PTX3 and hsCRP can represent a novel marker of disease inchronicmyeloprolifera6veneoplasms(ET,PVandMF)

PTX3andhsCRPplasmalevelsmayprovidenewinsightsinthepathogenesisandprognosisofthesediseases

PTX3 and hsCRP plasma levels may represent an innova6vesurrogate marker to evaluate the efficacy of novel therapeu6capproachesof thesedisease including JAK2andHDAC inhibitorsaswellaspegIFNa

PTX3andhsCRPasamarkerofcancer‐relatedinflamma6on

hshs‐CRPasaRiskFactorForFutureCVD:‐CRPasaRiskFactorForFutureCVD:PrimaryPreven&onCohortsPrimaryPreven&onCohorts

0 1.0 2.0 3.0 4.0 5.0 6.0

Kuller MRFIT 1996 CHD DeathRidker PHS 1997 MIRidker PHS 1997 StrokeTracy CHS/RHPP 1997 CHDRidker PHS 1998,2001 PADRidker WHS 1998,2000,2002 CVDKoenig MONICA 1999CHDRoivainen HELSINKI 2000 CHDMendall CAERPHILLY 2000 CHDDanesh BRHS 2000 CHDGussekloo LEIDEN 2001 Fatal StrokeLowe SPEEDWELL 2001 CHDPackard WOSCOPS 2001 CV Events*Ridker AFCAPS 2001 CV Events*Rost FHS 2001 StrokePradhan WHI 2002 MI,CVD deathAlbert PHS 2002 Sudden DeathSakkinen HHS 2002 MI

Relative Risk (upper vs lower quartile)Ridker PM. Circulation 2003;107:363-9

Cadiovascularprotec&veeffectsofPTX‐3

Norata,GDetal.:TrendsCardiovascMed20:35–40,2010