ARCHITETTURA DELLA RICERCA

Lucio Luzzatto,Scientific Director,

Istituto Toscano Tumori,Firenze, ITALY

“Cosa stai cercando”17 novembre 2010

RICERCA BIOMEDICA

•Osservazionale•Sperimentale

MacroMacro--evolutionevolution

Problemipostidallapraticaclinica

Nuoviportatidallaricercadi base

PROGETTO DI RICERCA

• Delineare il progetto: obiettivi e criteri di importanza scientifica

• Elaborazione: letteratura, metodologie, piano sperimentale, considerazioni statistiche

• Preventivo economico• Principi etici e Comitato Etico• Realizzazione del progetto• Rendicontazione scientifica• Rendicontazione economica

HemoglobinuriaHemoglobinuria: : Sometimes Paroxysmal, Sometimes NocturnalSometimes Paroxysmal, Sometimes Nocturnal

WORLDWIDE PNH PATIENTSWORLDWIDE PNH PATIENTS

22

88

77

3838 132132

33

• Intravascular Hemolysis

• Thrombosis

• Cytopenias

Paroxysmal Nocturnal Hemoglobinuria(PNH) is an acquired disorder defined by a clinical triad:

Patient RBCPatient RBCControlControlRBCRBC

dAcdAc dSdS dAcdAc dHidHi pHipHi pAcpAc HH2200

Ham Test in a PNH Patient

QuickTime™ and aGIF decompressor

are needed to see this picture.

CLONAL ORIGIN OF PNH CELLS CLONAL ORIGIN OF PNH CELLS IN A G6PD A/B HETEROZYGOTEIN A G6PD A/B HETEROZYGOTE PATIENTPATIENT

G6PDG6PD--AA G6PDG6PD--BB Whole RBCWhole RBC PNH PNH RBCRBC

ControlsControls PatientPatient

University College University College HospitalHospitalIbadan NigeriaIbadan Nigeria

TThe Complement he Complement CCascaascadede

Cell ActivationCell ActivationLysisLysis

C6 C7 C8 C9C6 C7 C8 C9

Potent AnaphylatoxinPotent AnaphylatoxinChemotaxisChemotaxis

Cell ActivationCell Activation

C5bC5b--99

C3 ConvertaseC3 Convertase

C3C3

C1qC1q Activated C1Activated C1

C4+C2C4+C2

C5 ConvertaseC5 Convertase

C3bC3b

Classical PathwayClassical PathwayActivationActivation

Antibody/Antigen ComplexesAntibody/Antigen Complexes

C3aC3aWeakWeak

AnaphylatoxinAnaphylatoxin

C4b2aC4b2a C4b2a3bC4b2a3b

C5bC5b

C5aC5a

C5C5Immune ComplexImmune Complexand Microbialand MicrobialOpsonizationOpsonization

Factor B+DFactor B+DC3 ConvertaseC3 Convertase C5 ConvertaseC5 ConvertaseC3, C3HC3, C3H22OO C3bC3b

Alternative PathwayAlternative PathwayActivationActivation

Microbiological MembranesMicrobiological MembranesBacterial LPSBacterial LPS

Immune ComplexesImmune ComplexesMammalian Cell MembranesMammalian Cell Membranes

C3bBb3bC3bBb3bC3bBbC3bBb

LectinLectin PathwayPathwayActivationActivation

MBLMBL

adaptedadapted fromfrom: Meri: Meri etet al, al, 19901990

C5bC5b--8844ººCCC9 loosely boundC9 loosely bound(displaceable)(displaceable)

C9C9

C5bC5b--883737ººCCC9 tightly boundC9 tightly bound(not displaceable)(not displaceable)

C9C9

C5bC5b--88

PolyPoly--C9C9

C5bC5b--88

C9C9

C5bC5b--88

CD59CD59C9C9

Mechanism of Action of CD59Mechanism of Action of CD59

with CD59with CD59

without CD59without CD59QuickTime™ and aGIF decompressor

are needed to see this picture.

CD59, CD90, CD109

CD55CD58*CD59CD48CDw52PrPcCD16*

CD24 CD55CD58* CD59 CD48 PrPCCD73

CDw108

CD55CD58*CD59CD109PrPCGP500Gova/b

CD55CD58*CD59PrPCAChEJMH AgDombrochHG Ag

CD55 CD58*CD59 CD14CD16 CD24CD48 CD66bCD66c CD87CD109 CD157LAPNB1 PrPCp50-80 GPI-80ADP-RT

NA1/NA2

CD14 CD55CD58*

CD59 CD48 CDw52

CD87 CD109CD157

Group 8 PrPC GPI-80CD16*

CD55 CD58*CD59 CD48CDw52 CD87 CDw108 PrPcADP-RT CD73CD90

CD109CD16*

Proteins Deficient on PNH Blood Cells

Hematopoietic Stem Cell

Platelets

PMNMonocytes

NK cells

RBC

B cells

T cells

QuickTime™ and aGIF decompressor

are needed to see this picture.

all these proteins are GPIall these proteins are GPI--linkedlinked

[From Kinoshita et al., J Biochem (JPN) 144:287,2008]

THE BIOSYNTHETIC PATHWAY OF THE GPI ANCHOR

INVOLVES AT LEAST TEN DISCRETE STEPS

nucleus

ER

X chr.

PIG-A

+GPIprotein

Etn-P GlcN

Inositol-P

glycan

PIG-A

GPI anchornucleus

ER

X chr.

PIG-A

+GPIprotein

PIG-A

GPI anchor

Etn-P GlcN

Inositol-P

glycan

Cellula Staminale Ematopoietica

Normale NormaleEPN

Emoglobinuria Parossistica Notturna (EPN)Patogenesi

..

4

982 11898497161

1

1452Null mutations

3

AvaIpolymorphism

del 735 bp

Large deletions

***** *

****

***

**

50 bp

**

652

del exons 3-4-5

Non-null mutations

Mutations in the PIG-A Gene

A. Normal, steady state

C3b

C3

+

CD55

CD55 CD59

C3b

C3b

C5 convertase

Classicalpathway

Lectinpathway

Alternativepathway

PhysiologicalC3 tick-over

C6 C7 C8 C9 MAC

CD55

CD55 CD59

C5b

C5

A normal (CD55+, CD59+) red cell can cope with the threat of complement activation

(From Luzzatto, Risitano & Notaro, 2010)

C3b

C3

+ C5b

C5

C3b

C5 convertase

MAC-mediated intravascular hemolysis

Classicalpathway

Lectinpathway

Alternativepathway

C6 C7 C8 C9 MAC

PhysiologicalC3 tick-over

C3bC3b

B. PNH, steady state

An abnormal (CD55-, CD59-) red cell (PNH red cell) will be lysed sooner or later by activated complement

(From Luzzatto, Risitano & Notaro, 2010)

TARGETING THE COMPLEMENT TARGETING THE COMPLEMENT CCASCAASCADEDE

Cell ActivationCell ActivationLysisLysis

C6 C7 C8 C9C6 C7 C8 C9

Potent Potent AnaphylatoxinAnaphylatoxinChemotaxisChemotaxis

Cell ActivationCell Activation

C5bC5b--99

C3 C3 ConvertaseConvertase

C3C3

C1qC1q Activated C1Activated C1

C4+C2C4+C2

C5 C5 ConvertaseConvertase

C3bC3b

Classical PathwayClassical PathwayActivationActivation

Antibody/Antigen ComplexesAntibody/Antigen Complexes

C3aC3aWeakWeak

AnaphylatoxinAnaphylatoxin

C4b2aC4b2a C4b2a3bC4b2a3b

C5bC5b

C5aC5a

C5C5Immune ComplexImmune Complexand Microbialand MicrobialOpsonizationOpsonization

Factor B+DFactor B+DC3 C3 ConvertaseConvertase C5 C5 ConvertaseConvertase

C3, C3HC3, C3H22OO C3bC3b

Alternative PathwayAlternative PathwayActivationActivation

Microbiological MembranesMicrobiological MembranesBacterial LPSBacterial LPS

Immune ComplexesImmune ComplexesMammalian Cell MembranesMammalian Cell Membranes

C3bBb3bC3bBb3bC3bBbC3bBb

LectinLectin PathwayPathwayActivationActivation

MBLMBL

XX

AntiAnti--C5 C5 (ECULIZUMAB)(ECULIZUMAB)

CH1

CH

3C

H2

hinge

EculizumabEculizumab (anti(anti--C5 antibody)C5 antibody)

human IgG4 heavy chainconstant regions 2 and 3

complementarity determining regions(murine origin)

CL

human framework regions

human IgG2 heavy chainconstant region 1 and hinge

Levels of Lactate Dehydrogenase and PNH Type III Erythrocytes during Treatment with Eculizumab

Hillmen P et al. N Engl J Med 2006;355:1233-1243

ECULIZUMAB ARRESTS THE SELECTIVE INTRAVASCULAR

LYSIS OF PNH III RED CELLS

(Hillmen et al., New Eng J Med355:1233,2006)

Kaplan-Meier Curves for the Time to the First Transfusion during Treatment

Hillmen P et al. N Engl J Med 2006;355:1233-1243

ECULIZUMAB CAN ABROGATE THE NEED FOR BLOOD TRANSFUSION

(Hillmen et al., New Eng J Med 355:1233,2006)

Change in Fatigue Scores from Baseline to Week 26

Hillmen P et al. N Engl J Med 2006;355:1233-1243

ECULIZUMAB HAS AN IMPACT ON THE QUALITY OF LIFE

(Hillmen et al., New Eng J Med 355:1233,2006)

3D STRUCTUREOF THE EPIDERMALGROWTH FACTORRECEPTOR

THE IRESSA/LUNG CANCER STORY. I.

• Non-small cell lung cancer (NSCLC) is a major cause of death in many countries

• NSCLC over-express the epidermal growth factor receptor (EGFR) in 40-80% of cases

• Gefitinib (Iressa) is an inhibitor of the TyrKin activity of EGFR

(From Lynch et al., NEJM 350:2129,2004)

Response to Gefitinib in a Patient with Refractory Non–Small-Cell Lung Cancer

(From Lynch et al., NEJM 350:2129,2004)

THE IRESSA/LUNG CANCER STORY. II.

• In a study at Mass General Hospital 25 out of 275 patients had an impressive clinical response to gefitinib

• In 8 out of 9 responders in whom the EGFR gene was sequenced a somatic mutation was found within the kinase domain

• No mutations were found in 7 out of 7 non-responders(From Lynch et al., NEJM 350:2129,2004)

RICERCA NEL SETTORE SALUTE

• Geografia• Storia• Tipologie

– Epidemiologia– Prevenzione– Diagnosi– Cure– Procedure infermieristiche– Qualità di vita– Modalità somministrazione cure– Aspetti economici

PROGETTI ITT

• Ricerca sul CANCRO in TOSCANA– Cancer Research– Clinical Cancer Research– Epidemiology– Supportive care

• Università, Ospedali, CNR• Peer review in due stadi: referees

esterni, ISAB• Progetti approvati

– 2007: 25 su 85– 2009: 33 su 109

ITT INTERNATIONAL SCIENTIFIC ADVISORY BOARD

• Sydney BRENNER (Cambridge), Chair• Dino AMADORI, Forlì• Andrea ARDIZZONI, Parma• Paolo BRUZZI, Genova• Giannino DEL SAL, Trieste• Jean-Claude Horiot, Genolier• Pier Paolo PANDOLFI, Boston• Robert PINEDO, Amsterdam• Paolo VINEIS, London

NOMINA NUMINA(what’s in a name?)

• Grant• Principal investigator

(PI)• Post-doctoral Fellow

(post-doc)• Graduate Student• Resident• Fellow

• Finanziamento• Titolare della ricerca

(capo-cordata)• Borsista; Co.co.co.;

(precario)• Dottorando• Specializzando• Specializzando

CLINICAL QUALITY CONTROLSmade user friendly

MORTALITY AND MORBIDITY REVIEWS•Regular periodicity•Appropriate notice•Mix of flagged and random cases•Collegial•Analysis carried out by an independent

but internal colleague•Frank discussion•No attacks•Experienced sensible chairman essential

ETHICAL and REGULATORY ISSUES IN THE CLINICAL USE

OF STEM CELLS & GENE TRANSFER

• Compliance with Good Manufacturing Practice (GMP) standards

• Compliance with all rules and regulations on clinical trials

• Ethics Committee approval

ETHICAL and REGULATORY ISSUES IN THE CLINICAL USE OF GENE

TRANSFER

An investigator involved in a clinical trial should not have any financial interests in any aspect of the trial itself

American Society of Gene Therapy. 1999

STEPS IN CANCER MANAGEMENT

• Diagnosis• Staging• Radical treatment• Surveillance of remission• Treatment of relapse• Salvage treatment• Management of patient with

advanced cancer

LA COMUNICAZIONE CON I PAZIENTI CON TUMORI

• Dire/non dire• Quanto dire• Quando dire• Comunicazione con

familiari• Comunicazione con

paziente• Comunicazione e sostegno

psicologico

CAPIRE,CURARE.PREVENIRE IL CANCROAL MEGLIOPER TUTTI

www.ittumori.it