Lezioni sui linfomi. A cura di Stefano Aldo Pileri MD, PhD,, Prof., Dr. h.c.

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1 Lezioni sui linfomi A cura di Stefano Aldo Pileri MD, PhD,, Prof., Dr. h.c. Dedicate ai miei Maestri Professori Antonio Maria Mancini ed Alessandro Piccaluga

2 Prof. Dr. Dr. h.c. Karl Lennert

3 LINFOMI: NEOPLASIE LINFOIDI I linfomi sono al terzo posto in ordine di incidenza fra le neoplasie al Mondo. Si distinguono in non-hodgkiniani e di Hodgkin. I primi hanno riconosciuto nel corso degli ultimi 20 anni un aumento esponenziale della loro incidenza; tale tendenza è destinata a mantenersi o, più probabilmente, a subire un ulteriore ulteriore progressione nel prossimo mezzo secolo. I secondi, invece, sono rimasti stabili, con un tipico andamento stagionale. Al momento, il rapporto fra le forme non-hodgkiniane ed Hodgkiniane è pari ad 10/1.

4 MO B T LN Timo T Organi periferici Milza

5 Classificazione dei linfomi

6 Rappaport Classification, 1966 Nodular Diffuse Lymphocytic, well differentiated Lymphocytic, poorly differentiated Mixed, lymphocytic-histiocytic histiocytic Histiocytic Undifferentiated

7 Lukes and Collins Classification, 1974 B-cell Small lymphocyte B (CLL, PLL, HCL) Plasmacytic lymphocytic Small cleaved FCC T-cell Small lymphocyte (CLL, PLL) Cerebriform T (MF/SS) IBL-like T-cell T lymphoma Large cleaved FCC Large non-cleaved FCC Small non-cleaved FCC (Burkitt, non Burkitt) T convoluted B-immunoblastic sarcoma T-immunoblastic sarcoma

8 Kiel Classification, 1974

9 Working Formulation, 1982

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

11 Realizzata per consenso a due meeting organizzati a: Berlino, Aprile 1993, Boston, Maggio Pubblicata su: Blood 1994, 84:

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13 La diagnosi di un linfoma è un processo integrato Campione tessuto cellule Informazioni cliniche Controparte normale Quadri morfologici Architettura Citologia Fenotipo Citogenetica Biologia molecolare DIAGNOSI Specifica entità Prognosi Bersagli terapeutici CD19 TC-> CD43 PE ->

14 Bethesda, March 1994

15 Blood, 89: , 1997

16 2001

17 2008

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19 Date Oct 1-15, 1 15, 2006 SHS to send out a completed list from beginning to end that includes everyone s s revised files and which will include a near final draft of table of contents, chapt titles, preferred 1 st authors, additional authors, suggested page numbers, suggestions for revising/writing each chapter. This will be used to create the author letters so it is very important. (Also includes ongoing issues regarding future meetings) October 22, 2006 Task Final comments back to SHS based on reviewing the entire set of attached documents. Editors submit contact information for all authors in their sections (1 st and other authors). November 1, 2006 SHS will notify IARC of all chapter titles, 1 st authors, contact information and suggestions/other requests to first authors. December 1, 2006 December 1, 2006 Dec 15, 2007 December 22, 2007 Reply to SHS questionnaire so letters to authors can be sent by IARC. Letter goes to first authors from IARC on behalf of the entire steering s committee based on the template. The initial letter will include the chapt title and the deadline. Deadline for first author to accept or decline the offer to write e the chapter. IARC sends another letter with logistical information, comments from editors, version from last edition, entire e outline. to authors who accept. IARC notifies SHS of author acceptance/rejection of our offer. SHS will notify responsible editor if any problem chapters among those they are responsible for. Jan 1, 2007 Agreed upon secondary authors are confirmed (based on whether 1 st author accepted offer) and then IARC notifies them. Jan 15, 2007 Templated invitation letters to be sent to other authors by IARC. February 9-10, Myeloid Clinical Advisory Committee meeting March 7-9, Lymphoma Clinical Advisory Committee meeting March 23, 2007 March, 2007 July 15, 2007 revise revise September 1, 2007 Revise? Sept 15, 2007 Letter with detailed instructions to all authors USCAP mini-meeting meeting of available editors Chapters & images due to responsible editor (text and suggested image changes/additions) submitted through IARC website. Chapts available for viewing by all authors/editors. Editor to author communications if necessary (and authors respond), other editors may send comments also to responsible editor regarding any concerns/suggestions for chapt or final consensus conference. All comments from other editors should have been sent by this date to the responsible editor. Responsible editor organizes & prioritizes topics for consensus meeting for their chapts forwards organized topics to SHS. Dr. Ohgaki will also suggest specific topics for agenda. Final version of all chapters for Lyon meeting as OK d d by editor available on web site for review by all authors and editors to prepare for consensus meeting. Agenda established for consensus meeting so all can prepare and take care of any additional tasks s that need to be completed prior to the meeting. October 25-27, 27, 2007 Meeting in Lyon (need to establish goals for this meeting) September, 2008 Publication of monograph Done 9/26/06 done done done done done Delayed done done done done done done done done done done done done done done

20 WHO CAC Meeting - Airlie House - March 8-9,

21 Steering Committee, Airlie House

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23 Editorial Meeting IARC, Lyon October 25-27, 27, 2007

24 10/ / / / copie copie copie copie

25 (. ). 26/139

26 La REAL/WHO Classification non prevede alcuna indicazione circa il grado di malignità. Infatti, i criteri istologici (quali le dimensioni cellulari), utilizzati dalle usuali classificazioni anatomo-patologiche, non costituiscono dei reali indicatori prognostici.

27 Linfomi a basso grado! 1,0 0,8 * Probabilty 0,6 0,4 0,2 * * CLL MALT Follicular MCL DLBCL Burkitt 0, Years

28 Linfomi indolenti Corrispondono a quelle neoplasie linfoidi la cui sopravvivenza è valutabile in anni, indipendentemente dal fatto che siano o meno trattate. Longo DL. The REAL Classification of lymphoid neoplasms: one clinician s s view. In Rosenberg S (ed): PPO Updates. Philadelphia: Lippincott 1995; 9:

29 Linfomi/leucemie disseminate leucemia linfatica cronica B/linfoma a piccoli linfociti B immunocitoma leucemia a tricoleucociti linfoma splenico della zona marginale plasmocitoma/mieloma multiplo leucemia a linfociti ampi e granulati (T) Linfomi extranodali linfoma della zona marginale di tipo MALT Linfomi nodali linfoma follicolare linfoma della zona marginale

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

31 Linfoma aggressivo Termine utilizzato per indicare quei tumori linfoidi che esordiscono con una sintomato- logia importante e che - non trattati - ricono- scono una rapida progressione. Longo DL. The REAL Classification of lymphoid neoplasms: one clinician s s view. In Rosenberg S (ed): PPO Updates. Philadelphia: Lippincott 1995; 9:

32 NEOPLASIE DEI LINFOCITI B Dei precursori: Delle cellule mature/periferiche periferiche: NEOPLASIE DEI LINFOCITI T/NK Dei precursori: Delle cellule mature/periferiche periferiche: leucemia/linfoma linfoblastico linfoma mantellare linfoma follicolare di III grado linfoma a grandi cellule B, diffuso linfoma/leucemia di Burkitt leucemia/linfoma linfoblastico leucemia prolinfocitica T leucemia a cellule NK, aggressiva leucemia/linfoma T dell adulto (HTLV-1 + ) linfoma T/NK di tipo nasale linfoma T enteropatico linfoma T epatosplenico linfoma T simil-panniculitico linfoma T, NAS linfoma T angioimmunoblastico linfoma a grandi cellule anaplastiche

33 ZINZANI (Blood, 1999) LNH-AG ANZIANO VNCOP-B: OS ( ) 1997) 100 % Mesi Istituto Seràgnoli - Bologna

34 Principi di patobiologia

35 Imprints Wright, cytochemistry, immunocytochemistry Fresh No FNA Electron microscopy Cell cultures Cryo- preservation Routine techniques Vaccine Cell suspensions Immunohistochemistry, molecular biology, genomics, proteomics Morphologic analysis, immunohistochemistry, molecular biology FACS analysis, cyto-genetics, molecular biology

36 (scoperta in rosso, validazione in nero, prova funzionale in verde) Sequenziamento massivo del genoma Profilo di espressione genica e SNPs Modello murino Colture cellulari mirnas epigenetica Q RTPCR SS 454J TMA FISH

37 Mantle cell Lymphoma VERGIN B- CELLS BCL-1 MALT lymphoma API2/MALT MARGINAL ZONE GERMINAL CENTRE Lympho-plasmacytic lymphoma PAX-5 HHV-8 Primary effusion PLASMACELL MANTLE MEMORY B- CELL Follicular Lymphoma BCL-2 BCL-6 Diffuse large B-cell lymphoma p53? c-myc p53 Burkitt s Lymphoma

38 Linfoma follicolare

39 CD10 Barcus ME et al. Appl Immunohistochem Molecul Morphol 4:263-6, 6, 2000.

40 Bcl-6 Flenghi L et al. Am J Pathol 148: , 55, Raible MD et al. Am J Clin Pathol 112:101-7, 1999.

41 CD20 Drug CD20 C Rituximab

42 Zevalin: MAb anti-cd20 coniugato con I 90 Radioimmunoterapia Anticorpo monoclonale (MAb) ChelanteTiuxetano Ittrio-90 Radiazione beta

43 Terapia combinata con MAb anti-cd20 e CD22 in pazienti con linfoma ricaduto o refrattario MO CD22 J Clin Oncol 2005; 23:

44 Genetica dei Tumori Perchè una cellula normale diviene tumorale? Perchè diverse cause (spesso sconosciute) inducono un danno al patrimonio genetico della cellula. Danno al DNA Virus, Tossici, Radiazioni Cellula Neoplastica Cellula normale Incapacità di riparare il danno

45 Meccanismi di linfomagenesi EZIOLOGIA? (~75 %) patogeni (15-20%) immunodeficit od autoimmunità (~5 %) pesticidi ( %) PATOGENESI Attivazione di proto-oncogeni oncogeni Inattivazione di geni onco- soppressori FENOTIPO proliferazione apoptosi differenziazione

46 Linfomi maligni e patogeni Helicobacter pylori Campylobacter jujeni Clamydia psittaci Borrelia burgdorferi HCV HBV HHV8 EBV HTLV1 HIV MZL (MALT) dello stomaco IPSID MZL (MALT) oculare MZL (MALT) cutaneo MLDUS, MZL nodale e splenico, DLBCL PEL, malattia di Castleman, BL, CHL, DLBCL-E, LYG, CIAL, TLPD- C, N/K-TCL NT, PTLD, IDLD ATLL

47 Modello di linfomagenesi infezione-correlato HTLV1 HCV EBV HHV-8 HP BB CP CJ R C traslocatione Espansione linfoide poli/oligoclonale oligoclonale Linfoma G L G L G L G L

48 Malattie auto-immuni e linfomagenesi Sindrome di Sjögren Tiroidite di Hashimoto Enteropatia da ipersensibilità al glutine Lupus erythematosus Artrite reumatoide

49 Linfomi maligni e farmaci

50 Linfomi maligni ed età Paediatric type FL Paediatric type NMZL Systemic EBV+ T-cell T LPD of childhood Hydroa vaccineforme-like lymphoma EBV+ DLBCL of the elderly

51 Linfomi maligni e sede anatomica Splenic diffuse red pulp small BCL FL of the jejunum FL of the testis FL of the skin (EORTC) Primary cutaneous DLBCL, leg-type (EORTC) Primary cutaneous aggressive epidermotropic CD8+ cytotoxic PTCL (EORTC) Primary cutaneous gamma-delta PTCL (EORTC) Primary cutaneous small/medium CD4 positive PTCL (EORTC)

52 Meccanismi di linfomagenesi EZIOLOGIA? (~75 %) patogeni (15-20%) immunodeficit od autoimmunità (~5 %) pesticidi ( %) PATOGENESI Attivazione di proto-oncogeni oncogeni Inattivazione di geni onco- soppressori FENOTIPO proliferazione apoptosi differenziazione

53 Fattori genetici intrinseci congeniti

54 Fattori genetici intrinseci acquisiti

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56 Traslocazione di un proto-oncogene oncogene R C R C proto-oncogene oncogene gene Ig Traslocazione cromosomiale R C Deregolazione trascrizionale del proto-oncogene oncogene

57 MBR breakpoint Chr.. 18 Chr.. 14 EX.1 EX2 EX Sense primer J H S μ C μ Anti-sense primer mcr breakpoint Chr.. 18 Chr.. 14 EX.1 EX2 EX.3 J H S μ C μ 5 3 Sense primer Anti-sense primer

58 BCL-2 2 & LF

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

60 Produzione di un gene di fusione R C R C Traslocazione cromosomiale R C C Sintesi di una proteina ibrida

61 ALCL: linfoma a grandi cellule anaplastiche Sopravvivenza in base alla presenza o meno della t(2;5)(p23;q35) o di sue varianti. % sopravvivenza globale ALK NPM t(2;5) + t(2;5) mesi

62 Mutazioni di geni regolatori BCL6 gene point mutation +1144

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64 Sovra-espressione di p53 da mutazioni puntiformi del gene corrispondente

65 Eventi epigenetici Metilazione Modificazione degli istoni

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67 , 2007 Drosha and Dicer: RNaseIII-type nucleases; DGCR8 and TRBP/PACT: co-factors; mirisc: : microrna-induced induced silencing complex.

68 Involvement in CLL predisposition Involvement in human leukaemias

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70 Plasticità e dinamica delle popolazioni linfoidi Sono i linfomi entità stabili? Trans-differenziazione di elementi linfoidi T-LBL Langerhans Cell Hystiocytosis (Feldman et al, Lancet Oncol 2005; 6: 435) (Xie H et al Cell 2004)

71 Truth is rarely pure and never simple. Oscar Wilde, The importance of being Earnest

72 The Human Genome Project A Global Perspective in Medicine

73 Micro-array Le tecniche basate sul principio dei micro- array costituiscono il fronte avanzato della ricerca bio-medica, specie in campo oncologico. Array: allineamento, disposizione a schiera. Tipo di organizzazione del materiale biologico da analizzare.

74 Tecnologie basate sui microarray Micro-array tissutale ( tissue chip ) Fino a 1000 campioni diversi sullo stessa vetrino Analisi in situ ad altissima resa Kononen et al. Nat Med. 1998; 4:844-7

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77 12 TMAs from 193 PCTL (148 U & 45 AILD) βf1

78 Identification of a new prognostic clinico-pathologic score based on age, LDH, PS, and Ki-67 index. 1.8 Cum. Survival.6.4 Score 2 Score 1 Score 3.2 p< Time

79 Tecnologie basate sui microarray DNA microarray Valutazione qualitativa e quantitativa dell espressione espressione dello mrna Valutazione dell intero genoma su di una singola chip Oligonucleotide microarray DNA microarray SNP array GCH array

80 Unsupervised analysis (ordinamento gerarchico) Classificazione dei campioni sulla base delle similitudini del profilo di espressione genica. BL GC cells FL DLCL N/M cells CLL MCL HCL

81 Supervised analysis Confronto fra categorie predefinite. Germinal center Naive Memory Supervised Analysis 334 genes

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85 High resolution SNP array genomic profiling of peripheral T cell lymphomas, not otherwise specified, identifies a subgroup with chromosomal aberrations rations affecting the REL locus Sylvia Hartmann, Stefan Gesk,, René Scholtysik,, Markus Kreuz,, Stefanie Bug, Inga Vater, Claudia Döring,, Sergio Cogliatti,, Marie Parrens,, Jean-Philippe Merlio,, Anna Kwiecinska,, Anna Porwit,, Pier Paolo Piccaluga, Stefano Pileri, Gerald Hoefler,, Ralf Küppers,, Reiner Siebert, Martin-Leo Hansmann Br. J. Haematol 2010 Feb;148(3):402 ;148(3): Genomic imbalances affected several regions containing members of NF-kappaB signalling and genes involved in cell cycle control. Gains of 2p were confirmed in three cases by fluorescence-in situ-hybridization (FISH) and were associated with breakpoints in the REL locus in two of these cases. Three additional cases with gains of REL were detected by FISH among 18 further PTCL NOS. However no additional breakpoints in the REL locus were identified. Five of 27 PTCL NOS investigated showed nuclear expression of the REL protein by immunohistochemistry, partly associated with genomic gains of the REL locus.

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88 13 anni 13 miliardi di dollari 2 giorni 2000 Euro

89 Platforms Instrument Company Year Feature generation Sequencing by synthesis Read lenght Mappable Accuracy data /run 454 Roche 2004 Emulsion PCR Polymerase (pyrosequencing) 450 bp 0.5Gb >99.5% HiSeq Illumina 2010 Bridge PCR Polymerase (reversibile terminators) 100 bp Gb 99.9% SOLiD Applied Biosystems 2007 Emulsion PCR Ligase (octamers) 50 bp Gb 99.9% Helicos Heliscope TM : recently available Pacific Biosciencies SMRT: launching 2010

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91 Applications Category Complete genome resequencing Targeted genomic resequencing Paired-end sequencing RNAseq (transcriptome seq) De novo assembly Small RNA sequencing Metagenomics ChIP seq Epigenomics Application Mutation, SNP. discovery in individual genomes. Detection of human genomic variation Targeted mutation and SNP discovery. Population genetics. Discovery of inherited and acquired structural variations Quantification of gene expression and alternative splicing. Discovery of SNP and somatic mutations. Discovery of structural variants. Sequencing and assembly of unknown genomes mirna profiling Discovery and characterization of complex enviromental or human microbial communities. Genome-wide mapping of protein-dna interactions Pattern of methylation in genomic DNA

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94 >40,000 genes / 1 tumor 1 gene / >1,000 tumors Micro-array strategy

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96 PTCL: overall survival Control Dauno 5mM Imatinib 1 mm

97 PTCL/NOS is related to activated T-cells, mainly of the CD4+ subset DR + DR - PTCL/NOS CD4 CD8 PTCL/NOS Supervised analysis genes=185 Supervised analysis genes=70

98 Genes differentially expressed in PTCL/NOS and normal T-cells T (training set + test set) Training set Test set CD4-CD8 CD8-DR+ DR+ PTCL/NOS 0 Normal lymphocytes Supervised analysis 155 genes: 91 down-regulated and 64 up-regulated

99 Cellular programs de-regulated in PTCL/NOS Adhesion Apoptosis Matrix Proliferation Signal transduction Cytoskeleton Transcription PER1 PER1 CBX4 CBX4 CHD2 CHD2 COPEB COPEB CREM CREM EPC1 EPC1 JMJD1C JMJD1C MAF MAF NR4A2 NR4A2 NR4A3 NR4A3 SERTAD1 SERTAD1 ZBTB10 ZBTB10 ZBTB24 ZBTB24 ZNF198 ZNF198 ZNF331 ZNF331 BCL10 BCL10 PTCL/NOS CD4-CD8-DR+ GJA1 GJA1 TNS TNS VCAM1 VCAM1 LIFR LIFR PTCL/NOS CD4-CD8-DR+ CD69 CD69 DUSP2 DUSP2 DUSP8 DUSP8 GADD45A GADD45A GADD45B GADD45B ING3 ING3 JUND JUND MOAP1 MOAP1 PPP1R15A PPP1R15A PTCL/NOS CD4-CD8-DR+ FN1 FN1 COL12A1 COL12A1 COL1A2 COL1A2 COL3A1 COL3A1 COL4A1 COL4A1 COL4A2 COL4A2 FBN1 FBN1 LAMB1 LAMB1 SPARC SPARC CDH11 CDH11 AXUD1 AXUD1 FOXP1 FOXP1 RHOBTB3 RHOBTB3 CAV2 CAV2 PLEKHC1 PLEKHC1 BTG1 BTG1 CLK1 CLK1 HECA HECA JUN JUN RGC32 RGC32 TOB1 TOB1 PTCL/NOS CD4-CD8-DR+ CALD1 CALD1 STK17B STK17B MKNK2 MKNK2 HIPK1 HIPK1 PTP4A1 PTP4A1 PDE4D PDE4D MAP3K8 MAP3K8 ITPKB ITPKB SEPT10 SEPT10 TJP1 TJP1 IRS2 IRS2 PTCL/NOS CD4-CD8-DR+ TPM1 TPM1 Dlc2 Dlc2 MGAT4A MGAT4A MYLIP MYLIP NFIB NFIB WASPIP WASPIP PTCL/NOS CD4-CD8-DR+ PTCL/NOS CD4-CD8-DR+

100 PDGFRα ( ) p-pdgfrα CYR61 ( )( BCL10 ( )( IGFBP7 ( )( LIFR ( )( p27 ( )( Caldesmon ( )

101 Expression of platelet-derived growth factor receptor in PTCL/NOS, AITL and ALCL Training set Test set CD4-CD8 CD8-DR+ DR+ ppdgfra PDGFRa Paracortex TK inhibitors ppdgfra PTCL/NOS Piccaluga PP, et al The Lancet Oncology, 2005; 6: 440

102 Alessandro Pileri, Claudio Agostinelli, Pier Paolo Piccaluga

103 PDGFRA inhibition determines cell viability reduction, proliferation arrest and apoptosis in PTCLnos primary cells Control Sugen 0,3 mm Sugen 1 mm Medium Medium + Imatinib 1 mm Control Imatinib G1 S G2-M Annexin V: t48h BrdUrd proliferation assay; ; 48 h

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106 Dai protocolli rigidi applicati a tutti i Pazienti alla.

107 Tailored therapy

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109 La diagnosi di linfoma Quale approccio? Profonda conoscenza delle singole entità Tecnologia multi-disciplinare Integrazione di tutti i dati da parte del patologo Keep an Open Mind

110 A. Lincoln Salvador Dalì,, 1975

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112 PP Piccaluga, E Sabattini, C Agostinelli, F Bacci, C Sagramoso, M Rossi, S Righi, A Gazzola,, T Sista, M Piccioli, MR Sapienza, C Mannu,, L Chilli,, F Sandri,, P Artioli,, G De Biase,, G Da Pozzo, C Tigrini and I Barese