Lezioni sui linfomiLezioni sui linfomi

A cura di Stefano Aldo PileriA cura di Stefano Aldo PileriMD, MD, PhDPhD, Prof., Dr. h.c., Prof., Dr. h.c.

Dedicate ai miei Maestri Professori Antonio Maria Mancini ed Dedicate ai miei Maestri Professori Antonio Maria Mancini ed Alessandro PiccalugaAlessandro Piccaluga

Prof. Dr. Dr. h.c. Karl LennertProf. Dr. Dr. h.c. Karl Lennert

LINFOMI: NEOPLASIE LINFOIDILINFOMI: NEOPLASIE LINFOIDI•• I linfomi sono al terzo posto in ordine di incidenza fra I linfomi sono al terzo posto in ordine di incidenza fra

le neoplasie al Mondo.le neoplasie al Mondo.

•• Si distinguono in nonSi distinguono in non--Hodgkiniani e di Hodgkin.Hodgkiniani e di Hodgkin.

•• I primi hanno riconosciuto nel corso degli ultimi 20 I primi hanno riconosciuto nel corso degli ultimi 20 anni un aumento esponenziale della loro incidenza; anni un aumento esponenziale della loro incidenza; tale tendenza tale tendenza èè destinata a mantenersi o, pidestinata a mantenersi o, piùùprobabilmente, a subire unprobabilmente, a subire un’’ulteriore progressione nel ulteriore progressione nel prossimo mezzo secolo. I secondi, invece, sono prossimo mezzo secolo. I secondi, invece, sono rimasti stabili, con un tipico andamento stagionale.rimasti stabili, con un tipico andamento stagionale.

•• Al momento, il rapporto fra le forme nonAl momento, il rapporto fra le forme non--Hodgkiniane Hodgkiniane ed Hodgkiniane ed Hodgkiniane èè pari ad 10/1.pari ad 10/1.

BB

TT

TT

MOMO

TimoTimo

LNLN

MilzaMilza

OrganiOrganiperifericiperiferici

ClassificazioneClassificazione deidei linfomilinfomi

Rappaport Rappaport Classification, 1966Classification, 1966

NodularNodular DiffuseDiffuse

Lymphocytic, well differentiatedLymphocytic, well differentiatedLymphocytic, poorly differentiatedLymphocytic, poorly differentiated

Mixed, lymphocyticMixed, lymphocytic--histiocytichistiocyticHistiocyticHistiocytic

UndifferentiatedUndifferentiated

Lukes and Collins Lukes and Collins Classification, 1974Classification, 1974

BB--cellcell TT--cellcell

Small lymphocyte B (CLL, PLL, HCL) Small lymphocyte B (CLL, PLL, HCL) Small lymphocyte (CLL, PLL)Small lymphocyte (CLL, PLL)

Plasmacytic Plasmacytic –– lymphocyticlymphocytic Cerebriform T (MF/SS)Cerebriform T (MF/SS)

Small cleaved FCCSmall cleaved FCC IBLIBL--like Tlike T--cell lymphomacell lymphoma

Large cleaved FCCLarge cleaved FCC

Large nonLarge non--cleaved FCCcleaved FCC

Small nonSmall non--cleaved FCC (Burkitt, non Burkitt) T convolutedcleaved FCC (Burkitt, non Burkitt) T convoluted

BB--immunoblastic sarcomaimmunoblastic sarcoma TT--immunoblastic sarcomaimmunoblastic sarcoma

KielKiel Classification, 1974Classification, 1974

Working Formulation, 1982Working Formulation, 1982

No communication between Europe and USA No communication between Europe and USA with detriment for patients and sciencewith detriment for patients and science

•• Realizzata per consenso a Realizzata per consenso a due meeting organizzati a:due meeting organizzati a:Berlino, Aprile 1993,Berlino, Aprile 1993,Boston, Maggio 1994.Boston, Maggio 1994.

•• Pubblicata su: Blood Pubblicata su: Blood 1994, 84:13611994, 84:1361--92.92.

CampioneCampionetessutotessutocellulecellule

Informazioni Informazioni clinichecliniche

Quadri morfologiciQuadri morfologici•• ArchitetturaArchitettura•• CitologiaCitologia

FenotipoFenotipoDIAGNOSIDIAGNOSI

Specifica entitSpecifica entitààPrognosiPrognosiBersagli terapeuticiBersagli terapeutici

La diagnosi di un linfoma La diagnosi di un linfoma èè un processo integratoun processo integrato

CitogeneticaCitogenetica

Biologia molecolareBiologia molecolare

1010 1010 1010 1010 101000 11 22 33 44

CD43 PE CD43 PE -->>C

D19

TC

CD

19 T

C -- >>

ControparteContropartenormalenormale

Bethesda, March 1994Bethesda, March 1994

Blood, 89:3909-18, 1997

20012001

20082008

DateDate TaskTask DoneDone

Oct 1Oct 1--15, 200615, 2006 SHS to send out a completed list from beginning to end that inclSHS to send out a completed list from beginning to end that includes everyoneudes everyone’’s revised files and which will include a near s revised files and which will include a near final draft of table of contents, final draft of table of contents, chaptchapt titles, preferred 1titles, preferred 1stst authors, additional authors, suggested page numbers, suggestionauthors, additional authors, suggested page numbers, suggestions s for revising/writing each chapter. This will be used to create for revising/writing each chapter. This will be used to create the author letters so it is very important. the author letters so it is very important. (Also includes (Also includes ongoing issues regarding future meetings)ongoing issues regarding future meetings)

9/26/069/26/06

October 22, 2006October 22, 2006 Final comments back to SHS based on reviewing the entire set of Final comments back to SHS based on reviewing the entire set of attached documents. Editors submit contact information attached documents. Editors submit contact information for all authors in their sections (1for all authors in their sections (1stst and other authors). and other authors).

donedone

November 1, 2006November 1, 2006 SHS will notify IARC of all chapter titles, 1SHS will notify IARC of all chapter titles, 1stst authors, contact information and suggestions/other requests to authors, contact information and suggestions/other requests to first authors. first authors. donedone

December 1, 2006December 1, 2006 Reply to SHS Reply to SHS ““questionnairequestionnaire”” so letters to authors can be sent by IARC.so letters to authors can be sent by IARC. donedone

December 1, 2006December 1, 2006 Letter goes to first authors from IARC on behalf of the entire sLetter goes to first authors from IARC on behalf of the entire steering committee based on the template. The initial letter teering committee based on the template. The initial letter will include the will include the chaptchapt title and the deadline. title and the deadline.

donedone

Dec 15, 2007Dec 15, 2007 Deadline for first author to accept or decline the offer to writDeadline for first author to accept or decline the offer to write the chapter. IARC sends another letter with logistical e the chapter. IARC sends another letter with logistical information, comments from editors, version from last edition, einformation, comments from editors, version from last edition, entire outline. to authors who accept.ntire outline. to authors who accept.

donedone

December 22, 2007December 22, 2007 IARC notifies SHS of author acceptance/rejection of our offer. IARC notifies SHS of author acceptance/rejection of our offer. SHS will notify responsible editor if any problem chapters SHS will notify responsible editor if any problem chapters among those they are responsible for. among those they are responsible for.

DelayedDelayed

Jan 1, 2007Jan 1, 2007 Agreed upon secondary authors are confirmed (based on whether 1Agreed upon secondary authors are confirmed (based on whether 1stst author accepted offer) and then IARC notifies them.author accepted offer) and then IARC notifies them. donedone

Jan 15, 2007Jan 15, 2007 Templated invitation letters to be sent to other authors by IARCTemplated invitation letters to be sent to other authors by IARC. . donedone

February 9February 9--10, 200710, 2007 Myeloid Clinical Advisory Committee meetingMyeloid Clinical Advisory Committee meeting donedone

March 7March 7--9, 20079, 2007 Lymphoma Clinical Advisory Committee meetingLymphoma Clinical Advisory Committee meeting donedone

March 23, 2007March 23, 2007 Letter with detailed instructions to all authorsLetter with detailed instructions to all authors donedone

March, 2007March, 2007 USCAP miniUSCAP mini--meeting of available editorsmeeting of available editors donedone

July 15, 2007July 15, 2007 Chapters & images due to responsible editor (text and suggested Chapters & images due to responsible editor (text and suggested image changes/additions) image changes/additions) –– submitted through IARC submitted through IARC website. website. ChaptsChapts available for viewing by all authors/editors.available for viewing by all authors/editors.

donedone

Editor to author communications if necessary (and authors responEditor to author communications if necessary (and authors respond), other editors may send comments also to responsible d), other editors may send comments also to responsible editor regarding any concerns/suggestions for editor regarding any concerns/suggestions for chaptchapt or final consensus conference.or final consensus conference.

donedone

reviserevise All comments from other editors should have been sent by this daAll comments from other editors should have been sent by this date to the responsible editor. te to the responsible editor. donedone

reviserevise Responsible editor organizes & prioritizes topics for consensusResponsible editor organizes & prioritizes topics for consensus meeting for their meeting for their chaptschapts –– forwards organized topics to forwards organized topics to SHS. Dr. SHS. Dr. OhgakiOhgaki will also suggest specific topics for agenda. will also suggest specific topics for agenda.

donedone

September 1, 2007September 1, 2007Revise?Revise?

Final version of all chapters for Lyon meeting as OKFinal version of all chapters for Lyon meeting as OK’’d by editor available on web site for review by all authors and d by editor available on web site for review by all authors and editors editors to prepare for to prepare for ““consensusconsensus”” meeting. meeting.

donedone

Sept 15, 2007Sept 15, 2007 Agenda established for Agenda established for ““consensusconsensus”” meeting so all can prepare and take care of any additional taskmeeting so all can prepare and take care of any additional tasks that need to be s that need to be completed prior to the meeting.completed prior to the meeting.

donedone

October 25October 25--27, 200727, 2007 Meeting in Lyon (need to establish goals for this meeting)Meeting in Lyon (need to establish goals for this meeting) donedone

September, 2008September, 2008 Publication of monographPublication of monograph donedone

WHO CAC Meeting WHO CAC Meeting -- Airlie House Airlie House -- March 8March 8--9, 20079, 2007

Steering Committee, Airlie HouseSteering Committee, Airlie House

Editorial Meeting Editorial Meeting –– IARC, Lyon IARC, Lyon –– October 25October 25--27, 200727, 2007

10/200810/200801/200901/200909/200909/200909/201009/2010

15.000 copie15.000 copie20.000 copie20.000 copie15.000 copie15.000 copie20.000 copie20.000 copie

(

).

.

26/13926/139

La REAL/WHO Classification La REAL/WHO Classification non prevede alcuna indicazione non prevede alcuna indicazione

circa il grado di malignitcirca il grado di malignitàà..

Infatti, i criteri istologici (quali le Infatti, i criteri istologici (quali le dimensioni cellulari), utilizzati dalle usuali dimensioni cellulari), utilizzati dalle usuali classificazioni anatomoclassificazioni anatomo--patologiche, non patologiche, non

costituiscono dei reali indicatori costituiscono dei reali indicatori prognostici.prognostici.

CLLMALTFollicularMCLDLBCLBurkitt

Years

Prob

abilt

y

0,2

0,4

0,6

0,8

1,0

0 2 4 6 8 10 120,0

*

*

*

““Linfomi a basso grado!Linfomi a basso grado!

LinfomiLinfomi indolentiindolenti

CorrispondonoCorrispondono a a quellequelle neoplasieneoplasie linfoidi linfoidi la cui la cui sopravvivenzasopravvivenza èè valutabilevalutabile in in annianni, , indipendentementeindipendentemente daldal fattofatto cheche sianosiano o o menomeno trattatetrattate..

Longo DL. The REAL Classification of lymphoid neoplasms: one Longo DL. The REAL Classification of lymphoid neoplasms: one clinicianclinician’’s view. In Rosenberg S (ed): PPO Updates. Philadelphia: s view. In Rosenberg S (ed): PPO Updates. Philadelphia: Lippincott 1995; 9:1Lippincott 1995; 9:1--12.12.

Linfomi/leucemieLinfomi/leucemie disseminatedisseminateleucemialeucemia linfaticalinfatica cronicacronica B/B/linfomalinfoma a piccoli linfociti Ba piccoli linfociti Bimmunocitomaimmunocitomaleucemialeucemia a tricoleucociti a tricoleucociti linfomalinfoma splenicosplenico della della zonazona marginalemarginaleplasmocitoma/mielomaplasmocitoma/mieloma multiplomultiploleucemialeucemia a linfociti a linfociti ampiampi e e granulatigranulati (T)(T)

LinfomiLinfomi extranodaliextranodalilinfomalinfoma della della zonazona marginalemarginale di di tipotipo MALTMALT

LinfomiLinfomi nodalinodalilinfomalinfoma follicolarefollicolarelinfomalinfoma della della zonazona marginalemarginale

Un aspetto comune a molti tipi di linfoma Un aspetto comune a molti tipi di linfoma indolente indolente èè la tendenza a subire una trasla tendenza a subire una tras--formazione blastica, con accelerazione del formazione blastica, con accelerazione del quadro clinico.quadro clinico.

LinfomaLinfoma aggressivoaggressivo

•• TermineTermine utilizzatoutilizzato per per indicareindicare queiquei tumoritumorilinfoidi linfoidi cheche esordisconoesordiscono con con unauna sintomatosintomato--logia logia importanteimportante e e cheche -- non non trattatitrattati -- riconoricono--sconoscono unauna rapidarapida progressioneprogressione..

Longo DL. The REAL Classification of lymphoid neoplasms: Longo DL. The REAL Classification of lymphoid neoplasms: one clinicianone clinician’’s view. In Rosenberg S (ed): PPO Updates. s view. In Rosenberg S (ed): PPO Updates. Philadelphia: Lippincott 1995; 9:1Philadelphia: Lippincott 1995; 9:1--12.12.

NEOPLASIE DEI LINFOCITI BNEOPLASIE DEI LINFOCITI BDei Dei precursoriprecursori: : leucemia/linfomaleucemia/linfoma linfoblasticolinfoblasticoDelleDelle cellule mature/cellule mature/perifericheperiferiche:: linfomalinfoma mantellaremantellare

linfomalinfoma follicolarefollicolare di III di III gradogradolinfomalinfoma a a grandigrandi cellule B, cellule B, diffusodiffusolinfoma/leucemialinfoma/leucemia di Burkittdi Burkitt

NEOPLASIE DEI LINFOCITI T/NKNEOPLASIE DEI LINFOCITI T/NKDei Dei precursoriprecursori: : leucemia/linfomaleucemia/linfoma linfoblasticolinfoblasticoDelleDelle cellule mature/cellule mature/perifericheperiferiche:: leucemialeucemia prolinfocitica Tprolinfocitica T

leucemialeucemia a cellule NK, a cellule NK, aggressivaaggressivaleucemia/linfomaleucemia/linfoma T T delldell’’adultoadulto (HTLV(HTLV--11++))linfomalinfoma T/NK di T/NK di tipotipo nasalenasalelinfomalinfoma T T enteropaticoenteropaticolinfomalinfoma T epatosplenicoT epatosplenicolinfomalinfoma T similT simil--panniculiticopanniculiticolinfomalinfoma T, NAS T, NAS linfomalinfoma T angioimmunoblasticoT angioimmunoblasticolinfomalinfoma a a grandigrandi cellule anaplastiche cellule anaplastiche

ZINZANI (Blood, 1999)ZINZANI (Blood, 1999)

00 66 1212 1818 2424 3030 3636 4242 4848 5454 6060 6666 7272

MesiMesi

00

2020

4040

6060

8080

100100%%

VNCOPVNCOP--B: OSB: OS (1992(1992--1997)1997)

LNHLNH--AG ANZIANOAG ANZIANO

Istituto Istituto ““SerSerààgnolignoli”” -- BolognaBologna

PrincipiPrincipi didi patobiologiapatobiologia

ImprintsImprints

ElectronElectronmicroscopymicroscopy

CryoCryo--preservationpreservation CellCell

suspensionssuspensions

FACS analysis,FACS analysis,cytocyto--genetics,genetics,

molecular biologymolecular biology

CellCellculturescultures

Morphologic analysis,Morphologic analysis,immunohistochemistry,immunohistochemistry,

molecular biologymolecular biology

RoutineRoutinetechniquestechniques

IImmunohistochemistry,mmunohistochemistry,molecular biology, molecular biology,

genomics, proteomicsgenomics, proteomics

Wright, Wright, cytochemistrycytochemistry,,immunocytochemistryimmunocytochemistry

VaccineVaccine

FreshFresh

No FNANo FNA

((scopertascoperta in in rossorosso, , validazionevalidazione in in neronero,, provaprova funzionalefunzionale in in verdeverde))

Sequenziamento Sequenziamento massivomassivo del genomadel genomaProfiloProfilo didi espressioneespressione

genicagenica e  SNPse  SNPs

miRNAsmiRNAsepigeneticaepigenetica

QQ‐‐RTPCR RTPCR –– SS SS –– 454J454J

TMATMA

FISHFISH

ColtureColture cellularicellulari

ModelloModello murinomurino

GERMINAL CENTRE

MANTLE

MARGINAL ZONE

PLASMACELL

MEMORY B-CELL

VERGIN B-CELLS

BCL-2?

p53

Mantle cellLymphoma

BCL-1

Primary effusion

HHV-8

Lympho-plasmacytic lymphoma

PAX-5

MALT lymphoma

API2/MALT

c-MYCp53

BCL-6 Burkitt’s Lymphoma

FollicularLymphoma

Diffuse large B-cell lymphoma

LinfomaLinfoma follicolarefollicolare

CD10CD10 BarcusBarcus ME et al. ME et al. ApplAppl ImmunohistochemImmunohistochem MoleculMoleculMorpholMorphol 4:2634:263--6, 2000.6, 2000.

BclBcl--66 Flenghi L et al. Am J Pathol 148:1543Flenghi L et al. Am J Pathol 148:1543--55, 1996.55, 1996.

RaibleRaible MD et al. Am J Clin Pathol 112:101MD et al. Am J Clin Pathol 112:101--7, 1999.7, 1999.

CD20CD20

DrugDrug

CD20CD20

CC RituximabRituximab

RadioimmunoterapiaRadioimmunoterapia

IttrioIttrio--9090IttrioIttrio--9090 RadiazioneRadiazionebetabeta

RadiazioneRadiazionebetabeta

ChelanteChelanteTiuxetanoTiuxetanoChelanteChelante TiuxetanoTiuxetano

AnticorpoAnticorpomonoclonalemonoclonale

((MAbMAb))

ZevalinZevalin: : MAbMAb antianti--CD20 coniugato con ICD20 coniugato con I9090

MOMO

CD22CD22

Terapia combinata con MAb antiTerapia combinata con MAb anti--CD20 e CD22 in CD20 e CD22 in pazienti con linfoma ricaduto o refrattariopazienti con linfoma ricaduto o refrattario

J Clin J Clin OncolOncol 2005; 23: 50442005; 23: 5044--5051 5051

Genetica dei TumoriGenetica dei Tumori•• PerchPerchèè una cellula una cellula ““normalenormale”” diviene diviene

““tumoraletumorale””??•• PerchPerchèè diverse cause (spesso diverse cause (spesso

sconosciute) inducono un danno al sconosciute) inducono un danno al patrimonio genetico della cellula.patrimonio genetico della cellula.

Danno al DNADanno al DNAVirus, Tossici, RadiazioniVirus, Tossici, Radiazioni

IncapacitIncapacitàà di riparare il dannodi riparare il dannoCellula normaleCellula normale

Cellula NeoplasticaCellula Neoplastica

MeccanismiMeccanismi didi linfomagenesilinfomagenesi

EZIOLOGIAEZIOLOGIA PATOGENESIPATOGENESI FENOTIPOFENOTIPO

AttivazioneAttivazione didiprotoproto--oncogenioncogeni

InattivazioneInattivazione didigenigeni oncoonco--

soppressorisoppressori

proliferazioneproliferazione

apoptosiapoptosi

differenziazionedifferenziazione

??((~~75 %)75 %)

patogenipatogeni(15(15--20%)20%)

immunodeficitimmunodeficitodod autoimmunitautoimmunitàà

(~5 %)(~5 %)

pesticidipesticidi(0.5(0.5--1 %)1 %)

LinfomiLinfomi malignimaligni e e patogenipatogeni•• Helicobacter pyloriHelicobacter pylori MZL (MALT) MZL (MALT) dellodello stomacostomaco•• Campylobacter Campylobacter jujenijujeni IPSIDIPSID•• ClamydiaClamydia psittacipsittaci MZL (MALT) MZL (MALT) oculareoculare•• BorreliaBorrelia burgdorferiburgdorferi MZL (MALT) MZL (MALT) cutaneocutaneo

•• HCVHCV MLDUS, MZL MLDUS, MZL nodalenodale e e splenicosplenico,,•• HBVHBV DLBCLDLBCL•• HHV8HHV8 PEL, PEL, malattiamalattia didi Castleman, Castleman, •• EBVEBV BL, CHL, DLBCLBL, CHL, DLBCL--E, LYG, CIAL, E, LYG, CIAL,

TLPDTLPD-- C, N/KC, N/K--TCL NT, PTLD, TCL NT, PTLD, IDLD IDLD

•• HTLV1HTLV1 ATLLATLL•• HIVHIV

HTLV1HTLV1 HPHPHCVHCV BBBBEBVEBV CPCPHHVHHV--88 CJCJ

ModelloModello didi linfomagenesi linfomagenesi infezioneinfezione--correlatocorrelato

CCRRtraslocationetraslocatione

Espansione linfoide poli/Espansione linfoide poli/oligoclonaleoligoclonale LinfomaLinfoma

GG LLGG LLGG LL GG LL

Malattie autoMalattie auto--immuni e linfomagenesiimmuni e linfomagenesi

•• Sindrome di Sindrome di SjSjöögrengren•• Tiroidite di Tiroidite di HashimotoHashimoto•• Enteropatia da ipersensibilitEnteropatia da ipersensibilitàà al glutineal glutine•• Lupus erythematosusLupus erythematosus•• Artrite reumatoide Artrite reumatoide

LinfomiLinfomi malignimaligni e e farmacifarmaci

LinfomiLinfomi malignimaligni eded etetàà

•• Paediatric type FLPaediatric type FL•• Paediatric type NMZLPaediatric type NMZL•• Systemic EBV+ TSystemic EBV+ T--cell LPD of childhoodcell LPD of childhood•• Hydroa vaccineformeHydroa vaccineforme--like lymphomalike lymphoma

•• EBV+ DLBCL of the elderlyEBV+ DLBCL of the elderly

LinfomiLinfomi malignimaligni e e sedesede anatomicaanatomica

•• Splenic diffuse red pulp small BCL Splenic diffuse red pulp small BCL •• FL of the jejunumFL of the jejunum•• FL of the testisFL of the testis•• FL of the skin (EORTC)FL of the skin (EORTC)•• Primary cutaneous DLBCL, legPrimary cutaneous DLBCL, leg--type (EORTC)type (EORTC)

•• Primary cutaneous aggressive epidermotropic Primary cutaneous aggressive epidermotropic CD8+ cytotoxic PTCL (EORTC)CD8+ cytotoxic PTCL (EORTC)

•• Primary cutaneous gammaPrimary cutaneous gamma--delta PTCL (EORTC)delta PTCL (EORTC)•• Primary cutaneous small/medium CD4 positive Primary cutaneous small/medium CD4 positive

PTCL (EORTC)PTCL (EORTC)

MeccanismiMeccanismi didi linfomagenesilinfomagenesi

EZIOLOGIAEZIOLOGIA PATOGENESIPATOGENESI FENOTIPOFENOTIPO

AttivazioneAttivazione didiprotoproto--oncogenioncogeni

InattivazioneInattivazione didigenigeni oncoonco--

soppressorisoppressori

proliferazioneproliferazione

apoptosiapoptosi

differenziazionedifferenziazione

??((~~75 %)75 %)

patogenipatogeni(15(15--20%)20%)

immunodeficitimmunodeficitodod autoimmunitautoimmunitàà

(~5 %)(~5 %)

pesticidipesticidi(0.5(0.5--1 %)1 %)

Fattori genetici intrinseci congenitiFattori genetici intrinseci congeniti

Fattori genetici intrinseci acquisitiFattori genetici intrinseci acquisiti

Traslocazione Traslocazione didi un protoun proto--oncogene oncogene

CCRRCCRR

gene Iggene Igprotoproto--oncogeneoncogene

CCRR

Deregolazione Deregolazione trascrizionaletrascrizionaledel protodel proto--oncogeneoncogene

Traslocazione Traslocazione cromosomialecromosomiale

SenseSense primerprimer AntiAnti--sensesense primerprimer

55’’ 33’’

ChrChr. 18. 18 ChrChr. 14. 14

EXEX.1.1 EXEX22 EXEX.3.3

JJHH SSμμ CCμμ

MBR MBR breakpointbreakpoint

55’’ 33’’

ChrChr. 18. 18 ChrChr. 14. 14

EXEX.1.1 EXEX22 EXEX.3.3JJHH SSμμ CCμμ

mcrmcr breakpointbreakpoint

SenseSense primerprimer AntiAnti--sensesense primerprimer

BCLBCL--2 & LF2 & LF

t(11;14) > riarrangiamento del gene BCL1 > sovraespressione della Ciclina D1 nel linfoma mantellare

Centro germinativoresiduo

ProduzioneProduzione didi un gene un gene didi fusionefusione

CCRRCCRR

CCRR

SintesiSintesi didi unauna proteinaproteina ibridaibrida

CC

Traslocazione Traslocazione cromosomialecromosomiale

00

2020404060608080

100100

1212 2424 3636 4848 6060 7272 mesimesi

t(2;5)t(2;5)++

t(2;5)t(2;5)--

%% sopravvivenzasopravvivenza globaleglobale

ALCL: ALCL: linfomalinfoma a a grandigrandi cellule anaplastichecellule anaplasticheSopravvivenzaSopravvivenza in base in base allaalla presenzapresenza o o menomenodelladella t(2;5)(p23;q35) o t(2;5)(p23;q35) o didi sue sue variantivarianti..

NPMNPM

ALKALK

+404+404 +1144+1144BCL6BCL6 gene point mutationgene point mutation

1 1 2 3 4 5 2 3 4 5 6 7 8 9 106 7 8 9 10

MutazioniMutazioni didi genigeni regolatoriregolatori

SovraSovra--espressioneespressione didi p53 p53 dada mutazionimutazioni puntiformipuntiformi del del gene gene corrispondentecorrispondente

Eventi epigeneticiEventi epigenetici

•• MetilazioneMetilazione

•• Modificazione degli istoniModificazione degli istoni

, 2007

DroshaDrosha and Dicer: and Dicer: RNaseIIIRNaseIII--type nucleases; DGCR8 and TRBP/PACT: cotype nucleases; DGCR8 and TRBP/PACT: co--factors;factors;miRISCmiRISC: microRNA: microRNA--induced silencing complex.induced silencing complex.

Involvement in CLLInvolvement in CLLpredispositionpredisposition

Involvement inInvolvement inhuman leukaemiashuman leukaemias

PlasticitPlasticitàà e dinamica delle popolazioni linfoidie dinamica delle popolazioni linfoidiSono i linfomi entitSono i linfomi entitàà stabili?stabili?

TransTrans--differenziazionedifferenziazione di elementi linfoididi elementi linfoidi

TT--LBLLBL Langerhans Cell Langerhans Cell HystiocytosisHystiocytosis ((FeldmanFeldman et al, Lancet Oncol 2005; 6: 435)et al, Lancet Oncol 2005; 6: 435)

(Xie H et al Cell 2004)

Truth is rarely pure andTruth is rarely pure and

never simple.never simple.

Oscar Oscar Wilde, The importance of being EarnestWilde, The importance of being Earnest

The Human Genome ProjectThe Human Genome ProjectA Global Perspective in MedicineA Global Perspective in Medicine

MicroMicro--arrayarray

Le tecniche basate sul principio dei microLe tecniche basate sul principio dei micro--array costituiscono il fronte avanzato della array costituiscono il fronte avanzato della ricerca bioricerca bio--medica, specie in campo medica, specie in campo oncologico.oncologico.

Array: allineamento, disposizione a schiera.Array: allineamento, disposizione a schiera.

Tipo di organizzazione del materiale Tipo di organizzazione del materiale biologico da analizzare.biologico da analizzare.

•• Fino a 1000 campioni diversi Fino a 1000 campioni diversi sullo stessa vetrinosullo stessa vetrino

•• Analisi in situ ad altissima resaAnalisi in situ ad altissima resa

Kononen et al. Nat Med. 1998; 4:844Kononen et al. Nat Med. 1998; 4:844--77

MicroMicro--array tissutalearray tissutale((““tissue chiptissue chip””))

Tecnologie basate sui microarrayTecnologie basate sui microarray

12 TMAs 12 TMAs from 193 from 193

PCTL (148 U PCTL (148 U & 45 AILD)& 45 AILD)

ββF1F1

00

.2.2

.4.4

.6.6

.8.8

11

Cum

. Sur

viva

lC

um. S

urvi

val

00 2020 4040 6060 8080 100100 120120 140140 160160 180180TimeTime

p<0.0001p<0.0001

Score 1Score 1

Score 2Score 2

Score 3Score 3

Identification of a new prognostic Identification of a new prognostic clinicoclinico--pathologic score based on pathologic score based on

age, LDH, PS, and Kiage, LDH, PS, and Ki--67 index. 67 index.

Tecnologie basate sui microarrayTecnologie basate sui microarray

•• DNA microarrayDNA microarray–– Valutazione qualitativa e quantitativa Valutazione qualitativa e quantitativa

delldell’’espressione dello espressione dello mRNAmRNA–– Valutazione dellValutazione dell’’intero genoma su di unaintero genoma su di una

singola chipsingola chip–– Oligonucleotide microarrayOligonucleotide microarray–– DNA DNA microarraymicroarray–– SNP SNP arrayarray–– GCH GCH arrayarray

UnsupervisedUnsupervised analysisanalysis (ordinamento gerarchico)(ordinamento gerarchico)

BLBLGC GC cellscells

FLFL

DLCLDLCL

N/M N/M cellscells

CLLCLL

MCLMCL

HCLHCL

•• Classificazione dei Classificazione dei campioni sulla base campioni sulla base delle similitudini del delle similitudini del profilo di espressione profilo di espressione genica.genica.

Supervised analysisSupervised analysis

Supervised Supervised AnalysisAnalysis334 genes334 genes

GerminalGerminalcentercenter

NaiveNaiveMemoryMemory

•• Confronto fra Confronto fra categorie categorie predefinite.predefinite.

High resolution SNP array genomic profiling of peripheral T cellHigh resolution SNP array genomic profiling of peripheral T cell lymphomas, not lymphomas, not otherwise specified, identifies a subgroup with chromosomal aberotherwise specified, identifies a subgroup with chromosomal aberrations affecting the rations affecting the

RELREL locuslocus

Sylvia Hartmann, Stefan Sylvia Hartmann, Stefan GeskGesk, Ren, Renéé ScholtysikScholtysik, Markus , Markus KreuzKreuz, Stefanie Bug, Inga , Stefanie Bug, Inga VaterVater, , Claudia Claudia DDööringring, Sergio , Sergio CogliattiCogliatti, Marie , Marie ParrensParrens, Jean, Jean--Philippe Philippe MerlioMerlio, Anna , Anna KwiecinskaKwiecinska, Anna , Anna

PorwitPorwit, Pier Paolo Piccaluga, Stefano Pileri, Gerald , Pier Paolo Piccaluga, Stefano Pileri, Gerald HoeflerHoefler, Ralf , Ralf KKüüppersppers, Reiner Siebert, , Reiner Siebert, MartinMartin--Leo Leo HansmannHansmann

Br. J. Br. J. HaematolHaematol 2010 2010 FebFeb;148(3):402;148(3):402--12.12.

Genomic imbalances affected several regions containing members oGenomic imbalances affected several regions containing members of NFf NF--kappaBkappaB signalling and signalling and genes involved in cell cycle control. Gains of 2p15genes involved in cell cycle control. Gains of 2p15--16 were confirmed in three cases by 16 were confirmed in three cases by fluorescencefluorescence--in situin situ--hybridization (FISH) and were associated with breakpoints in thehybridization (FISH) and were associated with breakpoints in the RELREL locus in locus in two of these cases. Three additional cases with gains of two of these cases. Three additional cases with gains of RELREL were detected by FISH among 18 were detected by FISH among 18 further PTCL NOS. However no additional breakpoints in the further PTCL NOS. However no additional breakpoints in the RELREL locus were identified. Five of 27 locus were identified. Five of 27 PTCL NOS investigated showed nuclear expression of the REL protePTCL NOS investigated showed nuclear expression of the REL protein by immunohistochemistry, in by immunohistochemistry, partly associated with genomic gains of the partly associated with genomic gains of the RELREL locus.locus.

13 13 annianni –– 13 13 miliardimiliardi didi dollaridollari

2 2 giornigiorni –– 2000 Euro2000 Euro

PlatformsPlatformsInstrument Company Year Feature generation Sequencing by synthesis Read lenght Mappable

data /runAccuracy

454 Roche 2004 Emulsion PCR Polymerase (pyrosequencing) 450 bp 0.5Gb >99.5%

HiSeq Illumina 2010 Bridge PCR Polymerase (reversibile terminators) 100 bp 30-35 Gb 99.9%

SOLiD Applied Biosystems

2007 Emulsion PCR Ligase (octamers) 50 bp 25-30 Gb 99.9%

HelicosHelicos HeliscopeHeliscopeTMTM : recently available: recently availablePacific Pacific BioscienciesBiosciencies SMRT: launching 2010SMRT: launching 2010

ApplicationsApplicationsCategory ApplicationComplete genome resequencing Mutation , SNP…. discovery in individual genomes.

Detection of human genomic variationTargeted genomic resequencing Targeted mutation and SNP discovery. Population

genetics. Paired-end sequencing Discovery of inherited and acquired structural

variationsRNAseq (transcriptome seq) Quantification of gene expression and alternative

splicing. Discovery of SNP and somatic mutations. Discovery of structural variants.

De novo assembly Sequencing and assembly of unknown genomesSmall RNA sequencing miRNA profilingMetagenomics Discovery and characterization of complex

enviromental or human microbial communities. ChIP seq Genome-wide mapping of protein-DNA interactionsEpigenomics Pattern of methylation in genomic DNA

>40,000 genes / 1 tumor>40,000 genes / 1 tumor 1 gene / >1,000 1 gene / >1,000 tumorstumors

MicroMicro--array strategyarray strategy

PTCL: overall survivalPTCL: overall survival

0

20

40

60

80

100

120

0 8 16 24 32 40 48 56 64 72

Control Dauno 5mM Imatinib 1 mM

PTCL/NOS is related to activated PTCL/NOS is related to activated TT--cells, cells, mainly of the CD4+ subsetmainly of the CD4+ subset

DRDR-- PTCL/NOSPTCL/NOSDRDR++

Supervised analysisSupervised analysisgenes=185genes=185

Supervised analysisSupervised analysisgenes=70 genes=70

CD4CD4 CD8CD8 PTCL/NOSPTCL/NOS

Training setTraining set Test setTest set CD4CD4--CD8CD8--DR+DR+

00

PTCL/NOSPTCL/NOS

Normal lymphocytesNormal lymphocytes

Genes differentially expressed in PTCL/NOS and Genes differentially expressed in PTCL/NOS and normal Tnormal T--cells (training set + test set)cells (training set + test set)

Supervised analysis 155 genes: 91 downSupervised analysis 155 genes: 91 down--regulated and 64 upregulated and 64 up--regulatedregulated

Cellular programs de-regulated in PTCL/NOS

AdhesionApoptosis

Matrix

Proliferation

Signal transduction

Cytoskeleton TranscriptionPER1PER1CBX4CBX4CHD2CHD2COPEBCOPEBCREMCREMEPC1EPC1JMJD1CJMJD1CMAFMAFNR4A2NR4A2NR4A3NR4A3SERTAD1SERTAD1ZBTB10ZBTB10ZBTB24ZBTB24ZNF198ZNF198ZNF331ZNF331BCL10BCL10

PTCL/NOS CD4-CD8-DR+GJA1GJA1TNSTNSVCAM1VCAM1LIFRLIFR

PTCL/NOS CD4-CD8-DR+

CD69CD69DUSP2DUSP2DUSP8DUSP8GADD45AGADD45AGADD45BGADD45BING3ING3JUNDJUNDMOAP1MOAP1PPP1R15APPP1R15A

PTCL/NOS CD4-CD8-DR+

FN1FN1COL12A1COL12A1COL1A2COL1A2COL3A1COL3A1COL4A1COL4A1COL4A2COL4A2FBN1FBN1LAMB1LAMB1SPARCSPARCCDH11CDH11

AXUD1AXUD1FOXP1FOXP1RHOBTB3RHOBTB3CAV2CAV2PLEKHC1PLEKHC1BTG1BTG1CLK1CLK1HECAHECAJUNJUNRGC32RGC32TOB1TOB1

PTCL/NOS CD4-CD8-DR+

CALD1CALD1STK17BSTK17BMKNK2MKNK2HIPK1HIPK1PTP4A1PTP4A1PDE4DPDE4DMAP3K8MAP3K8ITPKBITPKBSEPT10SEPT10TJP1TJP1IRS2IRS2

PTCL/NOS CD4-CD8-DR+

TPM1TPM1Dlc2Dlc2MGAT4AMGAT4AMYLIPMYLIPNFIBNFIBWASPIPWASPIP

PTCL/NOS CD4-CD8-DR+

PTCL/NOS CD4-CD8-DR+

CaldesmonCaldesmon ((⇑⇑))

LIFR (LIFR (⇑⇑))

BCL10 (BCL10 (⇓⇓))

pp--PDGFRPDGFRαα

p27 (p27 (⇑⇑))

IGFBP7 (IGFBP7 (⇑⇑))

PDGFRPDGFRαα ((⇑⇑))

CYR61 (CYR61 (⇑⇑))

Piccaluga PP, et al The Lancet Oncology, 2005; 6: 440Piccaluga PP, et al The Lancet Oncology, 2005; 6: 440

Expression of plateletExpression of platelet--derived growth factor receptor in derived growth factor receptor in PTCL/NOS, AITL and ALCLPTCL/NOS, AITL and ALCL

Training setTraining set Test setTest set CD4CD4--CD8CD8--DR+DR+

PDGFRaPDGFRa

pPDGFRApPDGFRA

PTCL/NOSPTCL/NOS

pPDGFRApPDGFRA

ParacortexParacortexTK TK inhibitorsinhibitors

Alessandro Pileri, Claudio Agostinelli, Pier Paolo PiccalugaAlessandro Pileri, Claudio Agostinelli, Pier Paolo Piccaluga

PDGFRA inhibition determines cell viability reduction, PDGFRA inhibition determines cell viability reduction, proliferation arrest and apoptosis in proliferation arrest and apoptosis in PTCLnosPTCLnos primary cellsprimary cells

0

20

40

60

80

100

120

0 6 12 18 24 30 36 42 48 54 60 66 72

Control Sugen 0,3 mM Sugen 1 mM

0

20

40

60

80

100

120

0 6 12 18 24 30 36 42 48 54 60 66 72

Control Sugen 0,3 mM Sugen 1 mM

AnnexinAnnexin V: t48hV: t48h

MediumMedium

Medium +Medium +Imatinib 1 Imatinib 1 mMmM

BrdUrdBrdUrd proliferationproliferation assayassay; 48 h; 48 h

00101020203030404050506060707080809090

100100

ControlControl ImatinibImatinib

G1G1SSG2G2--MM

Dai Dai protocolliprotocolli rigidirigidi applicatiapplicatia a tuttitutti i i PazientiPazienti allaalla……. .

““Tailored Tailored therapytherapy””

La diagnosi di linfomaLa diagnosi di linfomaQuale approccioQuale approccio??

•• Profonda conoscenza delle singole entitProfonda conoscenza delle singole entitàà

•• Tecnologia multiTecnologia multi--disciplinaredisciplinare

•• Integrazione di tutti i dati da parte del patologo Integrazione di tutti i dati da parte del patologo

•• KeepKeep anan Open Mind Open Mind

A. A. LincolnLincoln

SalvadorSalvadorDalDalìì, 1975, 1975

PP Piccaluga, E Sabattini, C Agostinelli, F Bacci, C Sagramoso, PP Piccaluga, E Sabattini, C Agostinelli, F Bacci, C Sagramoso, M Rossi, S Righi, M Rossi, S Righi, A A GazzolaGazzola, T Sista, M Piccioli, MR Sapienza, C , T Sista, M Piccioli, MR Sapienza, C MannuMannu, L , L ChilliChilli, F , F SandriSandri, P , P

ArtioliArtioli, G De , G De BiaseBiase, G Da Pozzo, C , G Da Pozzo, C TigriniTigrini and I Bareseand I Barese