DIAGNOSI E TERAPIA DELLE LEUCEMIE LINFOBLASTICHE ACUTE e

DIAGNOSI E TERAPIA DELLE LEUCEMIELINFOBLASTICHE ACUTE e DELLE SINDROMI LINFOPROLIFERATIVE CRONICHE, DEI LINFOMI E DEL MIELOMA MULTIPLOGiovanni Martinelli, MD

Istituto di Ematologia e Oncologia MedicaL. & A. Sergnoli, Universit degli Studi di Bologna

DANNO GENETICOALLANLL oAML

Lanalisi molecolare:Identifica gruppi di pazienti con diversa prognosiAnomalie genetiche non citogeneticamente identificate o caratterizzateSignificato clinico diverso della persistenza di MMRLivelli di malattia minima residua assai piccoli.Suggerisce la risposta alla chemioterapiaMeccanismi molecolari sconosciuti.

Geni non di FusioneSi definiscono geni non di fusione gli oncogeni associati a traslocazioni cromosomiche specifiche, generati dalla ricombinazione tra due geni.Danno origine a m-RNA neoplastici, il cui prodotto e spesso over espresso ed e oncogeno anche se solo uno dei due geni e funzionante.Un tipico esempio e loncogene Myc associato alla traslocazione t(8;14)Istituto di Ematologia ed Oncologia Medica Sergnoli, Bologna

GENI NON DI FUSIONECRCRCRAB

Geni di FusioneSi definiscono geni di fusione gli oncogeni associati a traslocazioni cromosomiche specifiche, generati dalla ricombinazione tra due geni.Danno origine a m-RNA chimerici il cui prodotto e espresso ed e neoplastico se entrambi i due oncogeni sono funzionanti.Un tipico esempio e loncogene BCR-ABL associato alla traslocazione t(9;22) ed alla Leucemia Mieloide Cronica, oppure loncogene PML-RAR alfa associato alla t(15;17) ed alla Leucemia Acuta a promielociti.Istituto di Ematologia ed Oncologia Medica Sergnoli

GENI DI FUSIONEC3RCRCRC5

Acute Lymphoid Leukemia

FREQUENCY OF PRINCIPAL TRANSLOCATION AND FUSION GENEs IN ALL

none 19%random 25%BCR-ABL4%TEL-AML1 28%MLL fusions 6%11q23 (AMLL1/Htrx/Hrx/ALL-1) 13 (1% ) E2A-PBX1%Principali alterazioni molecolari e fusion gene e loro frequenza nelle ALLIstituto di Ematologia ed Oncologia Medica Seragnoli, Bologna

Estimated Frequency of Specific Genotypes of ALL in Children and Adults.

Transformation of Hematopoietic Cells in the Pathogenesis of ALL.

Mechanism of Transcriptional Repression by TEL-AML1.

The Retinoblastoma (RB) and p53 Tumor-Suppressor Network.

KaplanMeier Analysis of Event-free Survival According to the Subtype of Leukemia in 467 Children with ALL Who Were Enrolled in Three Consecutive Treatment Protocols at St. Jude Children's Research Hospital from 1991 to 1999.

Influence of Host Germ-Line and ALL Blast Genotypes on the Probability of Cure and of Adverse Events.

Analisi molecolare: Microarray

random 25%BCR-ABL4%TEL-AML1 28%MLL fusions 6%11q23 (AMLL1/Htrx/Hrx/ALL-1) 13 (1% ) E2A-PBX1%none 19%Principali alterazioni molecolari e fusion gene e loro frequenza nelle ALLIstituto di Ematologia ed Oncologia Medica Seragnoli, Bologna

Is ALL Ph+ homogenous? INDIVIDUAL VARIABILITY?

Deletions on the derivative chromosome 9q+

Struttura dei geni ABL e BCR e sonde utilizzate in FISH a 3 colori e in D-FISH(Oncor)Sinclair et al., Blood 2000; 95:738-744

Prostaglandin E synthase

LMX2 NEK6PSMB7NR5A1NR6A1RPL35GOLGA1PPP6CHSPA5SIN1_HUMANPBX3ANGPTL2SLCA8RPL12STXBP1SH2D3CCDK9FPGSENGAK1SIAT7DDPM2ZNF297BLMX1BC9orf15C9orf16LCN2CIZ1_HUMANDNM1GOLGA2SLC27A4ODF2GLE1LSPTAN1SETSH3GLB2CRATPPP2R4CCBL1ENDOGPMX2_HUMANPTGESDYT1USP2OFREQASSFUBP3PRDM12RRP4_HUMANchr.9centel

LMX2 NEK6PSMB7NR5A1NR6A1RPL35GOLGA1PPP6CHSPA5SIN1_HUMANPBX3ANGPTL2SLCA8RPL12STXBP1SH2D3CCDK9FPGSENGAK1SIAT7DDPM2ZNF297BLMX1BC9orf15C9orf16LCN2CIZ1_HUMANDNM1GOLGA2SLC27A4ODF2GLE1LSPTAN1SETSH3GLB2CRATPPP2R4CCBL1ENDOGPMX2_HUMANPTGESDYT1USP2OFREQASSFUBP3PRDM12RRP4_HUMANABL

LMX2 NEK6PSMB7NR5A1NR6A1RPL35GOLGA1PPP6CHSPA5SIN1_HUMANPBX3ANGPTL2SLCA8RPL12STXBP1SH2D3CCDK9FPGSENGAK1SIAT7DDPM2ZNF297BLMX1BC9orf15C9orf16LCN2CIZ1_HUMANDNM1GOLGA2SLC27A4ODF2GLE1LSPTAN1SETSH3GLB2CRATPPP2R4CCBL1ENDOGPMX2_HUMANPTGESDYT1USP2OFREQASSFUBP3PRDM12RRP4_HUMANtel

LMX2 NEK6PSMB7NR5A1NR6A1RPL35GOLGA1PPP6CHSPA5SIN1_HUMANPBX3ANGPTL2SLCA8RPL12STXBP1SH2D3CCDK9FPGSENGAK1SIAT7DDPM2ZNF297BLMX1BC9orf15C9orf16LCN2CIZ1_HUMANDNM1GOLGA2SLC27A4ODF2GLE1LSPTAN1SETSH3GLB2CRATPPP2R4CCBL1ENDOGPMX2_HUMANPTGESDYT1USP2OFREQASSFUBP3PRDM12RRP4_HUMAN

LMX2 NEK6PSMB7NR5A1NR6A1RPL35GOLGA1PPP6CHSPA5SIN1_HUMANPBX3ANGPTL2SLCA8RPL12STXBP1SH2D3CCDK9FPGSENGAK1SIAT7DDPM2ZNF297BLMX1BC9orf15C9orf16LCN2CIZ1_HUMANDNM1GOLGA2SLC27A4ODF2GLE1LSPTAN1SETSH3GLB2CRATPPP2R4CCBL1ENDOGPMX2_HUMANPTGESDYT1USP2OFREQASSFUBP3PRDM12RRP4_HUMAN

SMARCB1: SWI/SNF related, actin dependent regulator of chromatin subfamily B member 1GSTT1:Glutathione S-transferase theta 1

FBXW3IGLL1ZNF70VPREB3MMP11SMARCB1SLC2A11MIFGSTT2DDTGSTT1CABI_HUMANGGTLA1

chr.22centel

random 25%BCR-ABL4%TEL-AML1 28%MLL fusions 6%11q23 (AMLL1/Htrx/Hrx/ALL-1) 1% E2A-PBX1%none 19%Principali alterazioni molecolari e fusion gene e loro frequenza nelle ALLIstituto di Ematologia ed Oncologia Medica Seragnoli, Bologna

11q23 and MLL

MLL t(11;19) t(11;22) t(X;11) (q13;q23) t(X;11)(q24;q23)del 11 (q11q23)del 11 (q21q23)t(1;11) t(4;11)t(6;11)t(9;11)t(10;11) t(11;17)centromeretelomereBreakpoints in MLL5678910111213MLLA-T hooksMTZn fingersENLMLLENLder 11 chromosomefusion pointMLL positive leukemias

MLLENLAFAAF6q21ELLAF19Eps15MSFABIENNAF3q21AFXAF5q31AF17AF10CALMAMLL1/Htrx/Hrx/ALL-1ATMTasePHDBP??MLL and Fusion Patners in Leukemia

Chr. 8 and t(8;14) and BURKITT LUKEMIA

TRANSLOCATIONS OF c-Myc LocusReciprocal chromosomal translocations in Burkitt's lymphoma, a solid tumour of B lymphocytes involves chr.14 and chr 8 chr.2 chr 22.

The genes for making the heavy chains of antibodies (CH) are located on chromosomes 14, whereas those for making the light chains are on chromosomes 2 and 22.

Struttura dellanticorpo

Patologia dell sintesi Ig

Struttura dellanticorpoLe Ig possono essere suddivise in classi o sottoclassi sulla base delle catene pesante ISOTIPIIgMmIgAa1 a2IgGg1, g2, g3, g4IgDdIgEe

Configurazione geni Ig germinaleIgH: Cromosoma 14Igk: Cromosoma 2Igl: Cromosoma 22

Riarrangiamento catena leggera

Mapping of cloned germline and rearranged antigen receptor gene segments

Genomic structure of selected antigen receptor loci

Cloning of the V(D)J recombinase genes.

The RAG proteins and the RAG cleavage reaction(A) Schematic of RAG-1 and RAG-2, illustrating known mofits and the putative RAG-1 DDE active site(B) Summary of the RAG cleavage reaction.

V(D)J recombination and the non-homologous end-joining pathway

Memory-BPlasma cellMaturazione del B linfocitaVKJK

Antigene interection with naive B cellSecrezione di Ig a bassa affinitPartecipazione reazione CGSelezione Ag-dipendenteProliferazioneIpermutazione somaticaSelezione per: > affinit vs < affinit e/o autoreattivit Switch isotipico

CD40 on B cellCD40L: on T cell (CD154)ProliferazioneSurvivallMemory differentiationT-cell dependent B-cell immune response

Activation-Induced Deaminasi (AID)

Somatic hypermutations (SHM)IgV Gene conversion (IGC)Class switch recombination (CSR) Deaminazione G to U:G to URepair with Uracil-DNA-glycosylase (UNG)

Comparazione delle caratteristiche cliniche e della sopravvivenza fra i gruppi mutato e germline.

BASI MOLECOLARI DELLE SINDROMI IMMUNOPROLIFERATIVE: MM

MM: UN MODELLO DI PATOGENESINormal Plasma CellMGUSIntra-medullary MyelomaExtra-medullary MyelomaMyeloma Cell LinePrimary Ig translocationsKaryotypic instability, 13q/13q14 deletions/monosomyN-Ras, K-Ras, or FGFR3 mutations Secondary (Ig) translocations p53 mutationsSmouldering Myeloma p16 methylation/ p18 deletionIstituto Sergnoli - Bologna

IgH14q32Istituto Sergnoli - Bologna

IgH14q3211q13BCL-1/ CCND1/ PRAD1, myeovIstituto Sergnoli - Bologna

IgH14q324p1611q13BCL-1/ CCND1/ PRAD1, myeovMMSET, FGFR3Istituto Sergnoli - Bologna

IgH14q324p1611q13BCL-1/ CCND1/ PRAD1, myeovMMSET, FGFR316q23c-mafIstituto Sergnoli - Bologna

IgH14q324p166p2111q13CCND3BCL-1/ CCND1/ PRAD1, myeovMMSET, FGFR316q23c-mafIstituto Sergnoli - Bologna

IgH14q324p166p2111q13CCND3~ 20% of pts with 14q translocationsMMSET, FGFR316q23c-mafIstituto Sergnoli - Bologna

IgH14q324p166p2111q13CCND3~ 20% of pts with 14q translocations~ 10-15% of pts with 14q translocations16q23c-mafIstituto Sergnoli - Bologna

IgH14q324p166p2111q13CCND3~ 20% of pts with 14q translocations~ 10-15% of pts with 14q translocations16q23~ 8% of pts with 14q translocationsIstituto Sergnoli - Bologna

IgH14q324p166p2111q13~ 20% of pts with 14q translocations~ 10-15% of pts with 14q translocations16q23~ 8% of pts with 14q translocations~ 4% of pts with 14q translocationsIstituto Sergnoli - Bologna

regcodingregcodingTRANSLOCATIONregcodingTRANSCRIPTIONAL UPREGULATIONIgH locusoncogeneIgH locusoncogeneIstituto Sergnoli - Bologna

MM: UN MODELLO DI PATOGENESINormal Plasma CellMGUSIntra-medullary MyelomaExtra-medullary MyelomaMyeloma Cell LinePrimary Ig translocationsKaryotypic instability, 13q/13q14 deletions/monosomyN-Ras, K-Ras, or FGFR3 mutations Secondary (Ig) translocations p53 mutationsSmouldering Myeloma p16 methylation/ p18 deletionIstituto Sergnoli - Bologna

Istituto Sergnoli - BolognaMICROARRAY ANALYSIS

Tutti i pz affetti da MM di nuova diagnosi overesprimono una delle cicline DIstituto Sergnoli - BolognaMICROARRAY ANALYSIS

Cyc D2Cyc D3Cyc D1t(11;14)? (unknown)t(6;14)t(4;14)t(14;16)MECCANISMI DI UP-REGOLAZIONE DELLE CICLINE D NEL MMMultiple MyelomaIstituto Sergnoli - Bologna???

CCND1 CCND2 CCND3Genomic location 11q13 12p13 6p21Protein size, kD 33 34 33Expression in none high low hematopoietic tissue LA FAMIGLIA DELLE CICLINE DIstituto Sergnoli - Bologna

pRB/E2F-mediated transcriptional repression of genes required for G1/S transitionHDACpRBE2FDPIstituto Sergnoli - Bolognaearly G1late G1SPPCell cycle progression:growth factors

pRB/E2F-mediated transcriptional repression of genes required for G1/S transitionHDACpRBE2FDPIstituto Sergnoli - Bolognaearly G1late G1SPPCell cycle progression:cyc Dgrowth factors

pRB/E2F-mediated transcriptional repression of genes required for G1/S transitionHDACpRBE2FDPIstituto Sergnoli - Bolognaearly G1late G1SPPCell cycle progression:CDK4/6cyc Dgrowth factors

pRB/E2F-mediated transcriptional repression of genes required for G1/S transitionHDACpRBE2FDPIstituto Sergnoli - BolognapRBearly G1late G1SPPPPPPPCell cycle progression:CDK4/6cyc Dphosphorilation by cdk4/cyc D and cdk2/cyc E relieves pRB repression of E2F transcription factors E2FDPgrowth factorsP

Cyclin D1 and CDK inhibitors

CCND1 CCND2 CCND3Genomic location 11q13 12p13 6p21Protein size, kD 33 34 33Expression in none low high hematopoietic tissue Cyclin D gene family membersIstituto di Ematologia e Oncologia Medica Sergnoli - Bologna

Istituto di Ematologia e Oncologia Medica Sergnoli - BolognaTyr15Thr161Thr14cdk4/6cyclin D1CDC25CAK2. dephosphorylation3. phosphorylation1. cyclin D1 sinthesis and associationGROWTH FACTORS

pRB/E2F-mediated transcriptional repression of genes required for G1/S transitionHDACpRBE2FDPIstituto di Ematologia e Oncologia Medica Sergnoli - BolognapRBearly G1late G1SPPPPPPPPassage of the Restriction point:CDK4DCDK2Ephosphorilation by cdk4/cyc D and cdk2/cyc E relieves pRB repression of E2F transcription factors E2FDP

CYCLIN D1RASRaf 1Ral-GDSPI3KAkt/PKBGSK3bWntb-catenin + TCFGSK3bMEK/MAPKERK/MAPintegrinsFAKIstituto di Ematologia e Oncologia Medica Sergnoli - Bologna-++++++++-+

A/G polymorphism at the splice donor region (nt.870) Ex.1 Ex.2 Ex.3 Ex.4Ex.5Intron 4if nt.870 is G: preferentially this splicing patternif nt.870 is A: preferentially this splicing patternCCND1/BCL-1/PRAD1 gene: alternative splicingIstituto di Ematologia e Oncologia Medica Sergnoli - Bologna

Ex.1 Ex.2 Ex.3 Ex.4 Ex.5 3 UTR Ex.1 Ex.2 Ex.3 Ex.4 In.4 3 UTRTranscript aTranscript bcyclin box (nt.312-630)destruction box (PEST- rich region)Cyclin D1 isoformsIstituto di Ematologia e Oncologia Medica Sergnoli - Bologna

Cromosoma 11Cromosoma 14TRASLOCAZIONE t(11;14)(q13;q32) q p q pBCL-1/CCND1/PRAD1IgHEaswitch regionBCL-1IgH locusChr.11Chr.14tel.cen.+Istituto Sergnoli - Bologna

t(11;14), OVERESPRESSIONE CICLINA D1: SIGNIFICATO PROGNOSTICO?Istituto Sergnoli - Bologna

Fonseca et al. Multiple myeloma and the translocation t(11;14)(q13;q32): a report on 13 cases Br J Haematol, 1998Sonoki et al. Expression of PRAD1/cyclin D1 in plasma cell malignancy: incidence and prognostic aspects Br J Haematol, 1998Hoechtlen-Vollmar et al. Amplification of cyclin D1 in multiple myeloma: clinical and prognostic relevance Br J Haematol, 1998Pruneri et al. Immunohistochemical analysis of cyclin D1 shows deregulated expression in multiple myeloma with the t(11;14) Am. J. Pathol. 2000FATTORE PROGNOSTICO SFAVOREVOLE??

Fonseca et al. Myeloma and the t(11;14)(q13;q32); evidence for a biologically defined unique subset of patients Blood. 2002;99:3735-41 DISEGNO DELLO STUDIO:336 pz. (ECOG 9486/9487) trattati con chemioterapia convenzionale, analizzati in FISH per la presenza della traslocazione t(11;14)(q13;q32)Istituto Sergnoli - Bologna RISULTATI:- 53/336 pz. (16%) con evidenza di t(11;14)- pts positivi per t(11;14): sopravvivenza pi lunga OS: 49.6 vs. 38.7 mesi (log-rank P > .2) PFS: 33.0 vs. 27.1 mesi (log-rank P > .2)

Istituto Sergnoli - BolognaMoreau et al. Recurrent 14q32 translocations determine the prognosis of multiple myeloma, especially in patients receiving intensive chemotherapy Blood. 2002;100:1579-83 DISEGNO DELLO STUDIO:168 pz. (IFM) trattati con diversi protocolli di chemioterapia ad alte dosi, analizzati in FISH per le principali traslocazioni 14q32RISULTATI:- 26/168 pz. (15.5%) con evidenza di t(11;14)(q13;q32)- OS attesa a 80 mesi significativamente pi lunga per i pazienti t(11;14)-positivi (87.5 vs. 55.4 mesi; P=0.055)

SCOPO DELLA RICERCAAnalizzare loverespressione della ciclina D1 in termini di:- frequenza; - correlazione con alterazioni chr.11; - associazione con delezione/monosomia chr.13 (D13);- significato clinico e prognostico.Soverini et al. Cyclin D1 overexpression is a favorable prognostic variable for newly diagnosed multiple myeloma patients treated with high-dose chemotherapy and single or double autologous transplantation Blood, 2003;102:1588-94Istituto Sergnoli - Bologna

BM da 74 pazienti affetti da MM di nuova diagnosi ed arruolati nel protocollo Bologna 96quantificazione dellmRNA per ciclina D1 mediante real-time RT-PCR (74 pts)citogenetica convenzionale (CC) ed eventualmente FISH per chr. 11 e 13 (46/74 pts)Istituto Sergnoli - Bologna PAZIENTI E METODI

No 11q abn.(n = 28)(n = 9)t(11;14) +11 (n = 9)(n = 10)NormalIstituto Sergnoli - BolognaCORRELAZIONE TRA LIVELLI DI CICLINA D1 E ALTERAZIONI DEL CHR.11

Foglio1

PATIENTCt #1Ct #2MEAN CtCt #1Ct #2MEAN CtCt #1Ct #2MEAN CtQUANT.#2TYPE ATYPE B(FISH)CYC D1 TYPE ACYC D1 TYPE B

CYCLIN D1 TYPE ACYCLIN D1 TYPE BABL (CONTROL GENE)DDCtDDCtCITOGEN.norm. (X10^-9)norm. (X10^-9)

66ALLEGRA29.9429.9529.9531.1430.9231.0329.0430.5129.7820.4617.38693.315883.07

72ARNAO28.8828.9928.9431.3531.2831.3228.1128.0428.0821.1519.36429.751486.14

86ARNO31.2930.831.0532.2732.1332.228.7728.9728.8722.4719.45172.731396.26

81BARONIO28.9829.0629.0230.3730.7530.5627.0727.4227.2522.0719.44227.921410.85

54BARTOLI27.1727.4827.3328.2328.2928.2630.6430.8730.7616.8613.638406.8779152.80

6BELLI31.0931.1931.1433.0532.7032.8829.228.7328.9722.4720.03neg172.73934.05

73BERTOLI32.9732.2632.6235.2135.8135.5131.4932.0131.7521.1619.88428.261036.39

40BIAGIOTTI22.5722.5122.5424.6524.5524.630.2530.3230.2912.5510.44pos: t(11;14)167331.89722357.18

15BIANCONI25.0825.425.2429.1829.0929.1429.7229.5329.6315.9115.63neg16297.3819719.74

89BONAVITA31.0330.2730.6535.2935.3335.3128.4828.1428.3122.6323.12154.06109.69

85BORGHI34.1233.233.6634.0534.0434.04531.531.6731.5922.3718.58185.122551.89

24BORGHINI23.1223.2823.2030.6230.5230.5728.5328.5228.5314.9718.17pos31266.993402.44

83BOSCO29.7729.929.8433.3033.1333.21527.8827.8527.8722.2621.47199.10344.26

71BOSI29.2629.3129.2933.1732.7432.9628.428.5628.4821.1020.60446.45631.37

27BOZZOLINI26.8226.7826.8030.4430.5630.5028.2228.2728.2518.8518.38pos2123.682941.53

91BRIGO31.3330.9531.1434.6534.3134.4828.1928.1828.1923.2522.42100.59178.82

67CAMPANA27.8928.0127.9533.3133.6933.527.1327.6427.3920.8622.24527.25202.58

61CAMPANINI29.0529.0129.0333.5834.0833.8329.729.8729.7919.5420.171316.37850.61

38CANTO27.6427.5227.5831.7031.431.5530.930.9430.9216.9516.75pos: t(11;14)7898.449072.93

68CANTONI29.7729.929.8434.2934.0234.15529.2929.1629.2320.9021.05511.06460.59

8CAPPELLI32.1332.3832.2633.1333.2833.2127.0226.8826.9525.6022.38neg19.73183.85

58CASSANI25.6425.6225.6330.7430.6130.6827.2727.2027.2418.6919.562372.761293.76

5CASTRO29.5529.3629.4631.9831.9931.98529.2529.8429.5520.2018.56neg830.222587.52

21CAVALIERI30.4630.6730.5732.8533.5433.2025.2325.6425.4425.4223.88neg22.2764.77

63CONTE28.9928.9828.9931.2531.1631.2129.1229.1529.1420.1418.19865.483343.99

80CORAZZARI30.6430.7430.6930.9330.8630.9029.0128.9528.9822.0018.04238.423723.27

1CORVINO29.5329.3829.4631.7732.2732.0229.0629.0729.0720.6819.08neg595.251810.73

34DAL MONTE29.5829.3929.4933.4433.7633.6031.6931.7131.7018.0818.02pos3621.453762.18

2D'AMICO23.523.3623.4328.5028.2728.3926.2226.1126.1717.5618.34neg5193.003013.77

4DE BON29.7430.0529.9032.5832.832.6928.0729.1928.6321.5620.18neg324.56841.81

11DI PAOLA30.5731.2430.9131.2631.8231.5428.6928.7128.7022.5018.96neg169.171960.97

92FABIANI32.0432.4432.2432.5032.2432.3729.4129.1129.2623.2719.2398.861626.27

37FANTUZZI25.0425.1225.0828.2528.2328.2430.1830.9930.5914.7913.78pos: t(11;14)35421.8771336.50

55FEDRIGA24.4124.5524.4827.9328.3228.12527.427.3827.3917.3816.865862.718436.06

84FRANCESCHI32.4832.3532.4233.6133.7933.730.4330.4630.4522.2619.38199.101470.77

33GALLERANI26.6326.5726.6030.5930.2230.4128.5528.5628.5618.3417.97pos3024.233894.85

69GERMANO30.1930.4430.3233.7733.8833.8329.6429.5229.5821.0320.37468.65740.50

9GIARDI31.5432.7332.1433.9034.3734.1427.8628.0827.9724.4622.29neg43.48195.68

82GIOVANNINI31.1331.3131.2234.7134.9734.8429.4329.2729.3522.1621.61213.39312.42

39GOFFREDI22.0422.0322.0426.4926.2526.3731.0731.2431.1611.1711.34pos: t(11;14)434005.22387101.63

3GUIDI30.4530.4830.4733.2533.1133.1828.0628.0928.0822.6821.23neg148.81407.98

49KMS12 (linea)20.7720.720.7425.3225.2425.2840.7741.3941.08-0.050.32pos: t(11;14)1.040.80

30LAURITO21.3821.4221.4025.5725.8525.7129.5629.5529.5612.1412.28pos222331.50201770.10

87LUCA32.0732.5432.3134.9935.1935.0929.9630.2830.1222.4821.09171.53448.00

93MAIOLI33.9733.5733.7735.3035.0535.1830.0329.8629.9524.1221.3555.04374.12

42MANZI26.8426.6926.7730.5131.0630.78531.1130.9531.0316.0315.88pos: t(11;14)14996.6516639.83

16MARRAS29.7229.5829.6531.5831.9331.75528.3528.3828.3721.5819.51neg320.091339.38

18MARTININI29.4929.4429.4732.7533.0932.9227.5627.6527.6122.1521.44neg214.87352.71

32MARZOLA27.1226.5126.8230.2330.4130.3232.7832.7132.7514.3613.70pos47556.4375403.97

41MASSA29.1729.1229.1534.7634.2934.52534.4634.4734.4714.9716.18pos: t(11;14)31158.8213468.98

60MENIN29.3829.3529.3733.3133.1233.21530.1830.3730.2819.3819.061465.681829.65

45MM1424.8224.8924.8628.8928.6928.7933.3833.433.3911.7611.52pos: t(11;14)289329.29340513.59

46MM3022.2422.2922.2724.4324.0424.2430.0529.9830.0212.5410.34pos: t(11;14)167912.82771524.72

47MM3223.3723.523.4426.5826.4926.5431.2731.3431.3112.4211.35pos: t(11;14)182476.71383097.70

12MM4229.5729.5929.5832.7232.5332.6329.5229.6429.5820.2919.17neg780.011701.22

14MM6533.4433.1233.2833.5433.133.3230.0930.1630.1323.4519.32neg87.571533.22

13MM8029.3129.2929.3032.3432.7732.5628.8128.9228.8720.7319.81neg576.971087.92

90MOHAMMAD33.1532.4732.8134.5334.4834.5130.0629.829.9323.1720.70105.96589.09

65MUSSIDA31.7232.4332.0837.0237.1937.1132.0231.9732.0020.3721.23737.94406.57

56NAPPO26.352726.6829.4129.4729.4429.7129.0829.4017.5716.175139.2913609.75

36NEGRI20.7821.4121.1024.8725.0124.9429.6929.4929.5911.8011.47pos: t(11;14)281417.57352521.78

62PALANDRA32.2932.4832.3934.5634.7134.6432.8733.0832.9819.7017.781174.114443.11

52PATRONE25.5025.6925.6030.2130.4830.34529.6729.5829.6316.2616.8412742.438524.22

94PIEGARI38.6437.6738.1638.8138.8838.84532.9932.5732.7825.6722.1918.80209.72

31PIETROLINI27.1627.0827.1229.8130.0129.9131.3331.5731.4515.9614.58pos15687.7740830.31

25POLI26.3526.3531.0730.9531.0128.7328.6528.6917.9518.44pos3949.222811.95

10POLIDORI30.5430.7430.6434.2334.4634.3528.428.5628.4822.4521.99neg174.53240.91

70POTENTE29.3629.3629.3633.0833.0633.0728.6228.5828.6021.0520.59460.59633.57

74PRESOTTO34.1535.3634.7636.0336.6736.3533.8533.8533.8521.2018.62416.552482.11

76RAVAGNAN29.1830.1229.6528.7528.5628.6628.3928.4228.4121.5416.37329.0911806.98

20RICCI34.2834.8534.5733.9533.9133.9330.5130.5130.5124.3519.54neg46.931311.82

35ROMAGNOLI G.26.326.1426.2230.9231.0530.9927.8827.9827.9318.5819.18pos2551.891689.47

64ROMAGNOLI R.29.9330.0329.9832.0231.9932.00530.0230.2230.1220.1518.01859.503801.50

50ROSA24.4324.4224.4326.9926.9826.9932.5732.8332.7012.0210.41241615.39737535.44

19SCAFORA'28.5628.6628.6130.5430.6730.6128.2728.428.3420.5718.39neg644.642911.11

29SCINARDO28.8728.5228.7029.0429.0729.05533.0932.7632.9316.0612.25pos14637.21205296.98

79SENNI30.9330.2530.5934.4034.7234.5629.022929.0121.8721.67260.90299.70

22SILVESTRI30.7331.2230.9832.0732.0632.0726.4126.426.4124.8621.78neg32.84277.69

44STAGLIANO24.4224.5224.4730.6430.7330.6930.8730.8130.8413.9215.97pos: t(11;14)64515.2615633.50

59STECCANELLA28.3728.3228.3531.9931.8831.9429.5729.6929.6319.0118.431900.752841.33

57SUPPA29.9629.7629.8631.3431.5231.4332.1532.1132.1318.0215.423762.1822809.59

43TEMPRATI21.6122.1121.8626.3726.3726.3730.7131.0330.8711.2811.62pos: t(11;14)402144.05317710.41

26TENEBRUSO29.429.229.3033.8334.4834.15534.4434.2834.3615.2315.92pos26020.3516184.81

23TENTOR32.1132.2332.1734.1234.1534.1430.0130.2130.1122.3520.15neg187.06862.48

75TONI32.132.2932.2034.3434.6634.5031.2431.0131.1321.3619.50371.531353.38

53TORRESI25.2925.0125.1527.9227.7527.8429.0328.9128.9716.4714.9911016.3130836.53

78TOSCA29.5629.9129.7433.4833.3533.4228.4828.3728.4321.6021.11314.60441.83

48U266 (linea)20.1820.0620.1224.3024.2824.2940.0040.8140.410.000.00pos: t(11;14)1.001.00

7VANDELLI V.28.0128.6828.3532.6632.5432.6028.4428.4128.4320.2120.30neg824.49777.31

88VECCHIETTINI31.3831.931.6434.4034.3534.37529.3129.5929.4522.4821.05170.94462.19

77VENTURI31.431.2331.3235.1234.6634.8930.0330.1130.0721.5420.94329.09497.09

28VERLECCHI26.0525.725.8832.2932.1132.2030.7630.6630.7115.4617.61pos22262.884998.75

51VIETRI23.2922.9923.1427.1127.0727.0930.4530.4430.4512.9912.77123346.12143665.37

17VIGHI27.0527.2327.1431.0831.0231.0527.0027.0527.0320.4120.15neg720.25862.48

CYCLIN D1 TYPE ACYCLIN D1 TYPE BABL (CONTROL GENE)DDCtDDCtCYC D1 TYPE ACYC D1 TYPE B

NPATIENTCt #1Ct #2MEAN CtCt #1Ct #2MEAN CtCt #1Ct #2MEAN CtQUANT.#2TYPE ATYPE Bnorm. (X10^-9)norm. (X10^-9)

1NORMALE 332.9232.6732.8029.3429.4729.4129.329.2329.2723.8216.2667.5312742.43

2NORMALE 532.8133.6633.2432.3032.2932.3029.3629.4929.4324.1018.9955.611920.62

3NORMALE 734.1834.2234.2035.7635.4135.5931.9331.531.7222.7819.99139.33960.31

4NORMALE 831.5131.8631.6931.5131.5631.5428.0928.1328.1123.8719.5565.451307.28

5NORMALE 933.3232.8833.1034.0634.1834.1232.6832.3932.5420.8617.71527.254680.19

6NORMALE 1232.9132.4332.6733.8833.7433.8132.2632.2432.2520.7117.68583.004762.00

7NORMALE 1534.0133.8833.9534.5534.2234.3930.8530.5530.7023.5419.8182.271091.69

media =22.8118.57

mediana=23.5418.99

dev.st. =1.451.39

Foglio2

NPATIENTCt #1Ct #2MEAN CtCt #1Ct #2MEAN CtCt #1Ct #2MEAN CtQUANT.#2TYPE ATYPE Bnormalizednormalized(FISH)

CYCLIN D1 TYPE ACYCLIN D1 TYPE BABL (CONTROL GENE)DDCtDDCtTYPE ATYPE BCITOGEN.

1CORVINO29.5329.3829.4631.7732.2732.0229.0629.0729.07-2.130.544.380.69neg

2VIGHI27.0527.2327.1431.0831.0231.0527.0027.0527.03-2.411.615.300.33neg

3D'AMICO28.528.2728.3931.2131.2531.2326.2226.1126.17-0.302.651.230.16neg

4GUIDI30.4530.4830.4733.2533.1133.1828.0628.0928.08-0.132.691.090.16neg

5DE BON29.7430.0529.9032.5832.832.6928.0729.1928.63-1.261.642.390.32neg

6CASTRO29.5529.3629.4631.9831.9931.9929.2529.8429.55-2.610.026.110.99neg

7BELLI31.0931.1931.1433.0532.7032.8829.228.7328.97-0.341.491.270.36neg

8VANDELLI V.28.0128.6828.3532.6632.5432.6028.4428.4128.43-2.601.756.060.30neg

9CAPPELLI32.1332.3832.2633.1333.2833.2127.0226.8826.952.793.840.150.07neg

10GIARDI31.5432.7332.1433.9034.3734.1427.8628.0827.971.653.750.320.07neg

11POLIDORI30.5430.7430.6434.2334.4634.3528.428.5628.48-0.363.451.280.09neg

12DI PAOLA30.5731.2430.9131.2631.8231.5428.6928.7128.70-0.320.421.240.75neg

13MM4229.5729.5929.5832.7232.5332.6329.5229.6429.58-2.520.635.740.65neg

14MM8029.3129.2929.3032.3432.7732.5628.8128.9228.87-2.091.274.240.41neg

15MM6533.4433.1233.2833.5433.133.3230.0930.1630.130.640.780.640.58neg

16CAVALIERI30.4630.6730.5732.8533.5433.2025.2325.6425.442.615.340.160.02neg

17SILVESTRI30.7331.2230.9832.0732.0632.06526.4126.4026.412.053.240.240.11neg

18ROMAGNOLI R.29.9330.0329.9832.0231.9932.00530.0230.2230.12-2.66-0.536.321.45neg

19BIANCONI25.0825.425.2429.1829.0929.1429.7229.5329.63-6.91-2.91119.847.52neg

20MARRAS29.7229.5829.6531.5831.9331.75528.3528.3828.37-1.240.972.350.51neg

21MARTININI29.4929.4429.4732.7533.0932.9227.5627.6527.61-0.662.901.580.13neg

22SCAFORA'28.5628.6628.6130.5430.6730.6128.2728.428.34-2.25-0.154.741.11neg

23RICCI34.2834.8534.5733.9533.9133.9330.5130.5130.511.541.000.350.50neg

24TENTOR32.1132.2332.1734.1234.1534.1430.0130.2130.11-0.461.611.380.33neg

25BORGHINI23.1223.2823.2030.6230.5230.5728.5328.5228.53-7.85-0.37229.921.30pos

26POLI26.3526.3531.0730.9531.0128.7328.6528.69-4.86-0.1029.041.07pos

27BOZZOLINI26.8226.7826.8030.4430.5630.5028.2228.2728.25-3.97-0.1615.621.12pos

28VERLECCHI26.0525.725.8832.2932.1132.2030.7630.6630.71-7.36-0.93163.711.91pos

29LAURITO21.3821.4221.4025.5725.8525.7129.5629.5529.56-10.68-6.271634.9276.90pos

30PIETROLINI27.1627.0827.1229.8130.0129.9131.3331.5731.45-6.85-3.96115.3615.56pos

31MARZOLA27.1226.5126.8230.2330.4130.3232.7832.7132.75-8.45-4.85349.7128.74pos

32GALLERANI26.6326.5726.6030.5930.2230.4128.5528.5628.56-4.48-0.5722.241.48pos

33DAL MONTE29.5829.3929.4933.4433.7633.6031.6931.7131.70-4.74-0.5226.631.43pos

34TENEBRUSO29.429.229.3033.8334.4834.15534.4434.2834.36-7.58-2.63191.346.17pos

35ROMAGNOLI G.26.326.1426.2230.9231.0530.9927.8827.9827.93-4.230.6418.770.64pos

80SCINARDO28.8728.5228.7029.0429.0729.05533.0932.7632.93-6.75-6.29107.6378.25pos

36NEGRI20.7821.4121.1024.8725.0124.9429.6929.4929.59-11.02-7.072069.40134.36pos: t(11;14)

37FANTUZZI25.0425.1225.0828.2528.2328.2430.1830.9930.59-8.03-4.77260.4727.19pos: t(11;14)

38CANTO27.6427.5227.5831.7031.431.5530.930.9430.92-5.86-1.7958.083.46pos: t(11;14)

39GOFFREDI22.0422.0322.0426.4926.2526.3731.0731.2431.16-11.64-7.213191.46147.54pos: t(11;14)

40BIAGIOTTI22.5722.5122.5424.6524.5524.630.2530.3230.29-10.27-8.111230.48275.33pos: t(11;14)

41MASSA29.1729.1229.1534.7634.2934.52534.4634.4734.47-7.84-2.36229.135.13pos: t(11;14)

42MANZI26.8426.6926.7730.5131.0630.78531.1130.9531.03-6.79-2.67110.286.34pos: t(11;14)

43TEMPRATI21.6122.1121.8626.3726.3726.3730.7131.0330.87-11.53-6.922957.17121.10pos: t(11;14)

44STAGLIANO24.4224.5224.4730.6430.7330.6930.8730.8130.84-8.89-2.58474.415.96pos: t(11;14)

45MM1424.8224.8924.8628.8928.6928.7933.3833.433.39-11.06-7.022127.58129.79pos: t(11;14)

46MM3022.2422.2922.2724.4324.0424.2430.0529.9830.02-10.27-8.201234.75294.07pos: t(11;14)

47MM3223.3723.523.4426.5826.4926.5431.2731.3431.31-10.39-7.191341.84146.02pos: t(11;14)

48U266 (linea)20.1820.0620.1224.3024.2824.2940.0040.8140.41-22.81-18.547328060.58379831.49pos: t(11;14)

49KMS12 (linea)20.7720.720.7425.3225.2425.2840.7741.3941.08-22.87-18.227639252.25305327.92pos: t(11;14)

50CONTE28.9928.9828.9931.2531.1631.2129.1229.5229.32-2.86-0.547.241.45

51GERMANO30.1930.4430.3233.7733.8833.8329.6429.5229.58-1.791.833.450.28

52MUSSIDA31.7232.4332.0837.0237.1937.1132.0231.9732.00-2.442.695.430.15

53ROSA24.4324.4224.4326.9926.9826.9932.5732.8332.70-10.80-8.141776.72281.11

54BORGHI34.1233.233.6634.0534.0434.04531.531.6731.59-0.450.041.360.97

55FEDRIGA24.4124.5524.4827.9328.3228.12527.427.3827.39-5.43-1.6943.113.22

56RAVAGNAN29.1830.1229.6528.7528.5628.6628.3928.4228.41-1.28-2.172.424.50

57PRESOTTO34.1535.3634.7636.0336.6736.3533.8533.8533.85-1.620.083.060.95

58ALLEGRA29.9429.9529.9531.1430.9231.0329.0430.5129.78-2.35-1.175.102.24

59ARNAO28.8828.9928.9431.3531.2831.3228.1128.0428.08-1.660.823.160.57

60ARNO31.2930.831.0532.2732.1332.228.7728.9728.87-0.340.911.270.53

61BARONIO28.9829.0629.0230.3730.7530.5627.0727.4227.25-0.750.901.680.54

62BONAVITA31.0330.2730.6535.2935.3335.3128.4828.1428.31-0.184.581.130.04

63CAMPANA27.8928.0127.9533.3133.6933.527.1327.6427.39-1.963.703.880.08

64CASSANI25.6425.6225.6330.7430.6130.6827.2727.2027.24-4.131.0217.450.49

65PALANDRA32.2932.4832.3934.5634.7134.6432.8733.0832.98-3.11-0.768.631.69

66MENIN29.3829.3529.3733.3133.1233.21530.1830.3730.28-3.430.5210.780.70

67NAPPO26.352726.6829.4129.4729.4429.7129.0829.40-5.24-2.3837.795.19

68TONI32.132.2932.2034.3434.6634.5031.2431.0131.13-1.450.962.730.52

69VIETRI23.2922.9923.1427.1127.0727.0930.4530.4430.45-9.83-5.78907.0354.76

70CANTONI29.7729.929.8434.2934.0234.15529.2929.1629.23-1.912.513.760.18

71POTENTE29.3629.3629.3633.0833.0633.0728.6228.5828.60-1.762.053.390.24

72FABIANI32.0432.4432.2432.5032.2432.3729.4129.1129.260.460.690.730.62

73BOSCO29.7729.929.8433.3033.1333.21527.8827.8527.87-0.552.931.460.13

74BRIGO31.3330.9531.1434.6534.3134.4828.1928.1828.190.433.880.740.07

75MAIOLI33.9733.5733.7735.3035.0535.1830.0329.8629.951.312.810.400.14

76VENTURI31.431.2331.3235.1234.6634.8930.0330.1130.07-1.282.402.420.19

77LUCA32.0732.5432.3134.9935.1935.0929.9630.2830.12-0.342.551.260.17

78FRANCESCHI32.4832.3532.4233.6133.7933.730.4330.4630.45-0.550.841.460.56

79GIOVANNINI31.1331.3131.2234.7134.9734.8429.4329.2729.35-0.653.071.570.12

81SENNI30.9330.2530.5934.4034.7234.5629.022929.01-0.943.131.920.11

82STECCANELLA28.3728.3228.3531.9931.8831.9429.5729.6929.63-3.81-0.1213.981.08

83VECCHIETTINI31.3831.931.6434.4034.3534.37529.3129.5929.45-0.332.511.260.18

84PIEGARI38.6437.6738.1638.8138.8838.84532.9932.5732.782.863.650.140.08

85BOSI29.2629.3129.2933.1732.7432.9628.428.5628.48-1.722.063.280.24

86CORAZZARI30.6430.7430.6930.9330.8630.9029.0128.9528.98-0.81-0.511.751.42

87PATRONE25.5025.6925.6030.2130.4830.34529.6729.5829.63-6.55-1.7093.703.25

88CAMPANINI29.0529.0129.0333.5834.0833.8329.729.8729.79-3.281.639.680.32

89BARTOLI27.1727.4827.3328.2328.2928.2630.6430.8730.76-5.95-4.9261.8230.17

90BERTOLI32.9732.2632.6235.2135.8135.5131.4932.0131.75-1.661.343.150.40

91MOHAMMAD33.1532.4732.8134.5334.4834.5130.0629.829.930.362.160.780.22

92TORRESI25.2925.0125.1527.9227.7527.8429.0328.9128.97-6.34-3.5681.0111.75

93TOSCA29.5629.9129.7433.4833.3533.4228.4828.3728.43-1.212.572.310.17

94SUPPA29.5629.7629.6631.3431.5231.4332.1532.1132.13-4.99-3.1231.788.69

CYCLIN D1 TYPE ACYCLIN D1 TYPE BABL (CONTROL GENE)DDCtDDCtTYPE ATYPE B

NPATIENTCt #1Ct #2MEAN CtCt #1Ct #2MEAN CtCt #1Ct #2MEAN CtQUANT.#2TYPE ATYPE Bnormalizednormalized

1NORMALE 332.9232.6732.8029.3429.4729.4129.329.2329.271.51-2.150.354.44

2NORMALE 532.8133.6633.2432.3032.2932.3029.3629.4929.431.790.580.290.67

3NORMALE 734.1834.2234.2035.7635.4135.5931.9331.531.720.471.580.720.33

4NORMALE 831.5131.8631.6931.5131.5631.5428.0928.1328.111.561.140.340.46

5NORMALE 933.3232.8833.1034.0634.1834.1232.6832.3932.54-1.46-0.702.741.63

6NORMALE 1232.9132.4332.6733.8833.7433.8132.2632.2432.25-1.60-0.733.031.66

7NORMALE 1534.0133.8833.9534.1234.2234.1730.8530.5530.701.231.180.430.44

33.0932.9930.57

media=0.500.13

mediana=1.230.58

dev. st. =1.451.36

Foglio6

Pz. con overespressioneDDCtCITOGEN.

cyc D1 trascritto B(B - ABL)(FISH)

17.74pos

15.92pos

18.38pos

17.61pos

12.25pos

12.28pos

14.58pos

13.70pos

17.97pos

18.02pos

11.47pos

13.78pos

16.75pos

11.34pos

10.44pos

16.18pos

15.88pos

11.62pos

15.97pos

11.52pos

10.34pos

11.35pos

&A

Pagina &P

Foglio5

Pz. con livelli "normali"DDCtCITOGEN.

cyc D1 trascritto A(A - ABL)(FISH)

1Corvino20.68neg

2D'amico22.51neg

3Guidi22.68neg

4De Bon21.56neg

5Castro20.20neg

6Belli22.47neg

7Vandelli V.20.21neg

8Cappelli25.60neg

9Giardi24.46neg

10Polidori22.45neg

11Di Paola22.50neg

12Marras21.58neg

13Vighi20.41neg

14Martinini22.15neg

15Scafor20.57neg

16Ricci24.35neg

17Tentor22.35neg

18Silvestri24.86neg

19Cavalieri25.42neg

20MM42 (Neri)20.29neg



23Conte20.14n.v

24Romagnoli R.20.15in attesa

25Mussida20.37n.v

26Allegra20.46n.v

27Campana20.86n.v

28Cantoni20.90n.v

29Germano21.03n.v

30Potente21.05n.v

31Bosi21.10n.v

32Arnao21.15n.v

33Bertoli21.16n.v

34Presotto21.20n.v

35Toni21.36n.v

36Ravagnan21.54n.v

37Venturi21.54n.v

38Tosca21.60n.v

39Senni21.87n.v

40Corazzari22.00n.v

41Baronio22.07n.v

42Giovannini22.16n.v

43Bosco22.26n.v

44Franceschi22.26n.v

45Borghi22.37n.v

46Arno22.47n.v

47Luca22.48n.v

48Vecchiettini22.48n.v

49Bonavita22.63n.v

50Mohammad23.17n.v

51Brigo23.25n.v

52Fabiani23.27n.v

53Maioli24.12n.v

54Piegari25.67n.v

&A

Pagina &P

Grafico2 (2)

14.96517.9522.5123.82

14.3615.2321.55524.1

11.79518.84522.46522.775

16.9515.45520.2123.865

12.1415.9622.4520.855

11.7617.5622.49520.71

13.9218.07520.2923.535

16.0318.5820.72521.776

11.2816.02523.44522.02

15.9122.99

21.575

20.405

22.15

24.345

22.35

23.56

24.02

22.22

23.02

22.1

24

22

21.25

20.98

21.84

23.21

22.56

23.78

DDCt

Grafico I (2)

20.6814.96523.820

22.5117.9524.10.6

22.6815.2322.775

21.55518.84523.865

20.215.45520.855

22.46516.0620.71

20.2112.13523.535

2515.9621.776

24.45514.3622.02

22.4518.33522.99

22.49518.075

20.2918.58

20.72511.795

23.44514.785

15.9116.95

21.57511.17

20.40512.545

22.1514.97

20.56516.025

24.34511.28

22.3513.92

11.755

12.54

12.42

CC/FISH -

CC/FISH +

n = 18 pts

n = 23 pts

NORMAL

n = 10

CELL LINES

DDCt

Grafico I

20.6814.96523.82

22.5117.9524.1

22.6815.2322.775

21.55518.84523.865

20.215.45520.855

22.46516.0620.71

20.2112.13523.535

2515.9621.776

24.45514.3622.02

22.4518.33522.99

22.49518.075

20.2918.58

20.72511.795

23.44514.785

15.9116.95

21.57511.17

20.40512.545

22.1514.97

20.56516.025

24.34511.28

22.3513.92

23.5611.755

24.0212.54

21.2212.42

22

CC/FISH -

CC/FISH +

n = 24 pts

n = 20 pts

NORMAL

n = 10

DDCt

Grafico2

14.96517.9522.5123.821

14.3615.2321.55524.1

11.79518.84522.46522.775

16.9515.45520.2123.865

12.1415.9622.4520.855

11.7617.5622.49520.71

13.9218.07520.2923.535

16.0318.5820.72521.776

11.2816.02523.44522.02

15.9122.99

21.575

20.405

22.15

24.345

22.35

23.56

24.02

22.22

23.02

22.1

24

22

21.25

20.98

21.84

DDCt

Foglio3

1-2.13120.68214.97323.8240-1.952114.97114.97217.95322.51423.8214.97

1-2.41122.51217.95324.140.6-2.232217.95114.36215.23321.56424.114.36

1-0.30122.68215.23322.775-0.122215.23111.80218.85322.47422.77511.80

1-0.13121.56218.85323.8650.052218.85116.95215.46320.21423.86516.95

1-1.26120.20215.46320.855-1.082215.46112.14215.96322.45420.85512.14

1-2.61122.47216.06320.71-2.432116.06111.76217.56322.50420.7111.76

1-0.34120.21212.14323.535-0.162113.92218.08320.29423.53513.92

1-2.60125.00215.96321.776-2.422215.96116.03218.58320.73421.77616.03

12.79124.46214.36322.022.972114.36111.28216.03323.45422.0211.28

11.65122.45218.34322.991.832218.34315.91422.9917.95

1-0.36122.50218.08-0.182218.08321.5815.23

1-0.32120.29218.58-0.142218.58320.4118.85

1-2.52120.73211.80023.82-2.342111.8013.9217.56322.1515.46

1-2.09123.45214.79024.1-1.912324.3515.96

10.64115.91216.95022.7750.822116.95322.3517.56

12.61121.58211.17023.8652.792323.5618.08

12.05120.41212.55020.8552.232112.55324.0218.58

1-2.66122.15214.97020.71-2.482114.97322.2216.03

1-1.24120.57216.03023.535-7.672216.03323.02

1-0.66124.35211.28021.776-4.682111.28322.1

1-2.25122.35213.92022.02-4.222113.9232415.71

11.54123.56211.76022.99-7.182111.76322

1-0.46124.02212.54-10.502112.54321.25

2-7.85121.22212.42-6.672320.98

2-4.86122-8.27321.84

2-3.97-4.30323.21

2-7.36-4.56322.56

2-10.68-10.84323.78

2-6.85-7.85

2-8.45-5.6822.29

2-4.48-11.46

2-4.74-10.09

2-7.58-7.66

2-4.23-6.61

2-6.75-7.69

2-11.02-8.71

2-8.03-10.88

2-5.86-10.09

2-11.64-10.21

2-10.27

2-7.84

2-6.79

2-11.53

2-8.89

2-11.06

2-10.27

2-10.39

Foglio4

Pz. con overespressioneDDCtCITOGEN.FISH +

cyc D1 trascritto A(A - ABL)(FISH)

1Poli17.95pos17.95

2Tenebruso15.23pos15.23

3Bozzolini18.85pos18.85

4Verlecchi15.46pos15.46

5Scinardo16.06pos16.06

6Laurito12.14pos12.14

7Pietrolini15.96pos15.96

8Marzola14.36pos14.36

9Gallerani18.34pos18.34

10Dalmonte18.08pos18.08

11Negri11.80pos11.80

12Fantuzzi14.79pos14.79

13Canto16.95pos16.95

14Goffredi11.17pos11.17

15Biagiotti12.55pos12.55

16Massa14.97pos14.97

17Manzi16.03pos16.03

18Temprati11.28pos11.28

19Stagliano13.92pos13.92

20Borghini14.97pos14.97

21Romagnoli G.18.58pos18.58

22Rosa12.02n.v

23Fedriga17.38n.vmedia:15.21

24Nappo17.57n.vmediana:15.23

25Vietri12.99n.vdev.st.:2.43

26Patrone16.26n.vrange:11,17-18,85

27Bartoli16.86n.v

28Torresi16.47n.v

29Suppa18.02n.v

30Cassani18.69n.v

31Bianconi15.91neg!

32MM14 (Neri)11.76pos



Totale: 34 su 92 (36%)

"Borderline"

Campanini19.54

Palandra19.70

Menin19.38

Steccanella19.01

&A

Pagina &P

CORRELAZIONE TRA OVERESPRESSIONE DELLA CICLINA D1 E D13Istituto Sergnoli - Bologna38% dei pazienti ciclina D1-pos41% dei pazienti ciclina D1-negD13nessuna associazione

Median ageSex (M:F)5551.531:1121:11n.s.Istituto Sergnoli - BolognapCyc D1 posCyc D1 neg32 (43%)42No. patientsDurie and Salmon stage: I II III 342511823CORRELAZIONE TRA OVERESPRESSIONE DELLA CICLINA D1 E CARATTERISTICHE CLINICO-LABORATORISTICHE ALLESORDIOn.s.n.s.

Istituto Sergnoli - BolognaM component:IgGBJlkLight chain:8614720101228IgA1726pCyc D1 posCyc D1 neg32 (43%)42No. patientsMedian M component concentration:IgG (g/dL)(range)BJ (g/24hrs)(range)3.80 (2.20-4.20)4.25 (1.90-18.60)3.40 (1.90-6.40)3.65 (2.56-7.80)IgA (g/dL)(range)3.90 (1.00-11.17)4.20 (2.55-8.40)n.s.n.s.n.s.

Istituto Sergnoli - Bolognap50 (10-100%)40 (10-100%)Cyc D1 posCyc D1 negmedian BMPC (%)(range)32 (43%)42No. patientsmedian b2-m (mg/L)(range)median CRP (mg/L)(range)median creat (mg/dL)(range)2.80 (1.20-16.00)3.10 (0.10-70.20)0.90 (0.70-1.40)3.60 (0.10-62.50)1.10 (0.70-3.00)2.20 (1.10-6.90)n.s.n.s.n.s.n.s.

OVERALL SURVIVAL0.10.50.40.30.20.60.70.80.91.0612182430364248540monthsProbability of survival (%)Cyc D1 negCyc D1 pospnot reached after 48430.6Cyc D1 posCyc D1 negMedian OSIstituto Sergnoli - Bologna

TIME TO DISEASE PROGRESSIONmonths0.10.50.40.30.20.60.70.80.91.0Cyc D1 posCyc D1 negProbability of progression (%)612182430364248540p41260.02Cyc D1 posCyc D1 negMedian TTPIstituto Sergnoli - Bologna

EVENT-FREE SURVIVALmonthsCyc D1 posProbability of survival (%)Cyc D1 neg540.10.50.40.30.20.60.70.80.91.06121824303642480p33240.055Cyc D1 posCyc D1 negMedian EFSIstituto Sergnoli - Bologna

nei 18 pts con D13:4126D13 pos/Cyc D1 pos (n=9)D13 pos/Cyc D1 neg (n=9)Median TTP4123D13 pos/Cyc D1 pos (n=9)D13 pos/Cyc D1 neg (n=9)Median EFSIstituto Sergnoli - BolognaCICLINA D1 vs. D13

CONCLUSIONI (I): unalterazione frequente (43%) nei pazienti affetti da MM alla diagnosi; strettamente correlata ad alterazioni del cromosoma 11: - t(11;14), - trisomia 11.Loverespressione della ciclina D1: Istituto Sergnoli - Bologna

Istituto Sergnoli - BolognaCONCLUSIONI (II):E un fattore prognostico favorevole: identifica un sottogruppo di pazienti che presentano TTP ed EFS pi lunghi in seguito a chemioterapia ad alte dosi ed autotrapianto.Loverespressione della ciclina D1:

Istituto Sergnoli - BolognaQUESITI APERTI:Loverespressione della ciclina D1 in grado di controbilanciare la monosomia/delezione del chr.13?

- cariotipicamente silente- descritta solo nel MM e MGUS- frequenza nel MM = 20%ca.t(4;14)(p16;q32)Istituto Sergnoli - Bologna

IgH LOCUS

Promoter (V)D J EPromoter Switch 3-Enhancer

regions

Chr. 14q32

Telomere Centromere

MMSET

Prom. Prom.

Chr. 4p16

der(4)

der(14)

I

C

11

4

1

24

10

12

FGFR3

I

24

12

11

10

4

C

1

FGFR3

t(4;14)

100 kb

t(4;14) translocation in Multiple Myeloma (MM)Like many tumors of the B-cell lineage, multiple myeloma (MM) shows recurrent rearrangements of the immunoglobulin heavy-chain (IGH) locus at 14q32 and molecular studies have identified FGFR3, CCND1, CCND3, and MAF genes as targets of primary translocations in this malignancy.Region 14q32 = locus IgHMMS = Class Switch Recombination (CSR)

unico caso, nel MM, in cui si forma un gene di fusione (IgH-MMSET), che rende la t(4;14) rilevabile mediante RT-PCRIstituto Sergnoli - Bologna

Translocations involving locus IgHare observed in 60-80% of patients with MMcr.14q3211q134p1616q236p256p21 In patients with MM a number of recurrent translocations involving chromosome 14 at band q32 have been recently identified, the most common being the t(11;14) and the t(4;14). Both these chromosomal abnormalities may help to identify patients at different risk of death; The t(4;14) has been recently reported to be associated with an unfavorable clinical outcome.

4p16 Region The t(4;14) affects at least two potential oncogenes, MMSET on der(4) and Fibroblast Growth Factor Receptor 3 (FGFR3) on der (14); the role of both these genes in the pathogenesis of MM has not bee fully elucidated.

MMSET = Multiple Myeloma SET domain protein3 4 5 6 7 8 9 10 115UTORF 1911bp = 647aa5UT3 4 5 6 7 8 9 10 12 13 14 15 16 17 18 19 20 21 22 23 24ORF 4094bp = 1365aaType IType IIPolyadenilation Segnals NCM.Chesi et al., Blood (1998)

MMSET/IgH = fusion gene and alternative spicingIgH 3 4 5 6 MMSET IgH 4 5 6 MMSET IgH 5 6 MMSET

Region 14q32 = locus IgHMMS = sequenze di DNA ripetitivo, non codificante, associate alla Class Switch Recombination (CSR)TRANSLOCATION = CSR abherrant OR mechanismo involving regions near S

4p16 Region = genic clustercr.4p16

MMSET = Multiple Myeloma SET domain protein3 4 5 6 7 8 9 10 115UTORF 1911bp = 647aa5UT3 4 5 6 7 8 9 10 12 13 14 15 16 17 18 19 20 21 22 23 24ORF 4094bp = 1365aaType IType IIPolyadenilation Segnals NCM.Chesi et al., Blood (1998)

MMSET: FUNCTIONALS DOMAIN NC

QUINDI1. domini tipici di proteine nucleari coinvolte in- rimodellamento della cromatina- meccanismo regolatorio epigenetico2. deleto nella Wolf-Hirshhorn Syndrome (severi difetti di crescita, ritardo mentale, difetti scheletrici etc.)3. overespresso nel MM per effetto della t(4;14)MMSET: fattore trascrizionale?

Derivatives t(4;14)der(14):der(4):

AIM- frequency and prognostic signifiance t(4;14) in our casistic

molecular variability of t(4;14)

For this purpose we analyzed:the presence of t(4;14) by RT-PCR of the hybrid transcript between MMSET and the IgH locus; the overexpression of FGFR3 by Real-time RT-PCR; the frequency of potentially activating point mutations in the FGFR3 translocated coding region, by direct sequencing of RT-PCR products.The frequency of del 13 and t(4;14) In the present study we investigated the frequency and the prognostic relevance of the t(4;14) in a series of 63 patients with de novo MM, who were randomized to receive either a single autotransplant (Tx-1) or double autotransplants (Tx-2) as primary therapy for their disease.

PATIENTS & METHOD RT-nested PCR per IgH/MMSET (diagnosis + LFU) Real-Time PCR per FGFR3 (diagnosis) RT-PCR & direct sequencing for FGFR3 point mutation (diagnosis + LFU)

Sex (M/F)12/528/18Age (years, median, range) 51 (42-61)55 (40-62)Durie-Salmon stage (no, %)I4 (24%)8 (17%)II4 (24%)5 (11%)III9 (52%)33 (72%)Isotyp (no, %)IgG11 (65%)26 (65%)IgA6 (35%)10 (22%)Bence Jones010 (22%)High chain (no., %)k7 (41%)32 (70%)l10 (59%)14 (30%)Lytic bone disease (no, %)07 (41%)9 (20%)12 (12%)4 (9%)22 (12%)3 (6%)>36 (35%)30 (65%)Calcium (mg/dl)9.1 (8.2-12-3)9.1 (5-13)Hb (g/dl)11.4 (6.4-14.5)12.3 (5.6-15-5)Plts211 (157-300)212 (59-416)B2 mycroglobulin (mg/L) 2.1 (1.2-6.9)2.4 (1.1-16)C reactive protein (mg/L) 0.38 (0.01-6.6)0.31 (0.01-70)Creatinine (mg/dl) 1.1 (0.3-3.0)1.0 (0.6-1.7)Plasmacells (%) 50 (10-90)50 (10-100)

THERAPY RECEIVEDNo Tx03 (7%)Tx111 (65%)26 (56%)Tx26 (35%)17 (37%)

RESPONSE TO TRANSPLANTN. Evaluables 17/1743/46Progression 01 (2%)No response 2 (12%)5 (12%)Partial response 11 (65%)15 (35%)Complete response (IF+) 3 (17%)7 (16%)Complete response (IF-) (?) (6%)15 (35%)

GLOBAL SURVIVALMedian Follow-up (months):MMSET/IgH+: 36 (16-50)MMSET/IgH-: 45 (13-72)n.s

EVENT FREE SURVIVALMedian, range (months):MMSET/IgH+: 23 (6-29)MMSET/IgH-: 32 (6-72)MMSET/IgH-MMSET/IgH+p= 0.01

TIME TO DISEASE PROGRESSION in MMSET+ patients Median, range (months):MMSET/IgH+: 23 (6-29)MMSET/IgH-: 32 (6-72)

t (4;14): PROGNOSTIC SIGNIFICANCE t(4;14) ASSOCIATED TO BAD THERAPY- Moreau et al. (Blood, 2002): FISH - CD138+168 pz. (high dose chemotherapy )13% t(4;14)+ OS & EFS less than (p

t(4;14) & del(13)del (13)5 (42%)7 (29%)NO del (13)7 (54%)17 (71%)

t(4;14) & del (13)* = by 3 years (number; percentual)

mortality*

relapse*

del(13)

3/38 patients

0

1/3 (33%)

t(4;14)

7/38 patients

0

4/7 (57%)

del(13) + t(4;14)

5/38 patients

3/5 (60%)

4/5 (80%)

CONCLUSIONS-1 27% of patients studied has t(4;14)

t(4;14) = worst rensponse to transplant

EFS & TTP shorter in t(4;14) carring patients

t(4;14): longitudinal study: MMSET type of transcript is conserved through the therapy

IgH/ex3

D LFU

IgH/ex4

D LFU

IgH/ex5

D LFU

alter.splic.

D LFU

ov. FGFR3

mut. FGFR3

D LFU

FU

1

X X

X

X

no no

+43m

2

X X

X

no no

+37m

3

X X

X

X

no X

+37m

4

X nv

X

X X

+40m

5

X X

X

no no

+41m

6

X nv

X

X nv

+16m

7

X X

X X

X

no no

+25m

8

X X

X X

X

no no

+42m

9

X X

X X

X X

X

no no

+45m

10

X X

X X

X X

X

no no

+55m

11

X X

X

X X

X X

X

X X

+49m

12

X X

X

no no

+58m

13

X

X X

X

no X

+28m

14

X X

no

no no

+52m

15

X X

X X

no

no no

+40m

16

X X

X

no

no no

+34m

17

X X

X

no

no no

+50m

tot

7 8

12 8

3 3

3 5

13

3 4

t(4;14): longitudinal study: alternative spiced MMSET transcript are associated with longest clinical outcome

IgH/ex3

D LFU

IgH/ex4

D LFU

IgH/ex5

D LFU

alter.splic.

D LFU

ov. FGFR3

mut. FGFR3

D LFU

FU

1

X X

X

X

no no

+43m

2

X X

X

no no

+37m

3

X X

X

X

no X

+37m

4

X nv

X

X X

+40m

5

X X

X

no no

+41m

6

X nv

X

X nv

+16m

7

X X

X X

X

no no

+25m

8

X X

X X

X

no no

+42m

9

X X

X X

X X

X

no no

+45m

10

X X

X X

X X

X

no no

+55m

11

X X

X

X X

X X

X

X X

+49m

12

X X

X

no no

+58m

13

X

X X

X

no X

+28m

14

X X

no

no no

+52m

15

X X

X X

no

no no

+40m

16

X X

X

no

no no

+34m

17

X X

X

no

no no

+50m

tot

7 8

12 8

3 3

3 5

13

3 4

t(4;14):

IgH/ex3

D LFU

IgH/ex4

D LFU

IgH/ex5

D LFU

splic. alter.

D LFU

ov. FGFR3

mut. FGFR3

D LFU

FU

1

X X

X

X

no no

+43m

2

X X

X

no no

+37m

3

X X

X

X

no X

+37m

4

X nv

X

X X

+40m

5

X X

X

no no

+41m

6

X nv

X

X nv

+16m

7

X X

X X

X

no no

+25m

8

X X

X X

X

no no

+42m

9

X X

X X

X X

X

no no

+45m

10

X X

X X

X X

X

no no

+55m

11

X X

X

X X

X X

X

X X

+49m

12

X X

X

no no

+58m

13

X

X X

X

no X

+28m

14

X X

no

no no

+52m

15

X X

X X

no

no no

+40m

16

X X

X

no

no no

+34m

17

X X

X

no

no no

+50m

tot

7 8

12 8

3 3

3 5

13

3 4

t(4;14): alternative splicing & survivalX = 83% alive 47m (28-55m.)X = 45% alive 40m (16-58m.)

IgH/ex3

D LFU

IgH/ex4

D LFU

IgH/ex5

D LFU

vari

D LFU

ov. FGFR3

mut. FGFR3

D LFU

FU

status

1

X X

X

X

no no

+43m

V

2

X X

X

no no

+37m

M

3

X X

X

X

no X

+37m

M

4

X nv

X

X X

+40m

M

5

X X

X

no no

+41m

M

6

X nv

X

X nv

+16m

M

7

X X

X X

X

no no

+25m

M

8

X X

X X

X

no no

+42m

V

9

X X

X X

X X

X

no no

+45m

RC

10

X X

X X

X X

X

no no

+55m

V

11

X X

X

X X

X X

X

X X

+49m

V

12

X X

X

no no

+58m

V

13

X

X X

X

no X

+28m

M

14

X X

no

no no

+52m

V

15

X X

X X

no

no no

+40m

V

16

X X

X

no

no no

+34m

V

17

X X

X X

no

no no

+50m

V

tot

7 8

12 8

3 3

3 5

13

3 4

FGFR3: overexpression & point mutations

IgH/ex3

D LFU

IgH/ex4

D LFU

IgH/ex5

D LFU

vari

D LFU

ov. FGFR3

mut. FGFR3

D LFU

FU

status

1

X X

X

X

no no

+43m

V

2

X X

X

no no

+37m

M

3

X X

X

X

no X

+37m

M

4

X nv

X

X X

+40m

M

5

X X

X

no no

+41m

M

6

X nv

X

X nv

+16m

M

7

X X

X X

X

no no

+25m

M

8

X X

X X

X

no no

+42m

V

9

X X

X X

X X

X

no no

+45m

RC

10

X X

X X

X X

X

no no

+55m

V

11

X X

X

X X

X X

X

X X

+49m

V

12

X X

X

no no

+58m

V

13

X

X X

X

no X

+28m

M

14

X X

no

no no

+52m

V

15

X X

X X

no

no no

+40m

V

16

X X

X

no

no no

+34m

V

17

X X

X X

no

no no

+50m

V

tot

7 8

12 7

3 3

3 5

13

3 4

CONCLUSIONS-2 t(4;14) and MMSET was not lost during disease progression

which is the role of aberant transcripts?

not all the t (4;14) positive patients over-express FGFR3

CONCLUSIONS-3prognostic significance

therapeutic implications

PROTOCOL MM02- patients enrolled: 126 (at 15/07/03)

- samples for molecular byology:126 (at diagnosis) 91 (follow up)

- CD138+:95 (at diagnosis)24(post 4 month)

PROTOCOL MM02: t(4;14)25 patients at diagnosis9 patients: t(4;14)+(36%)16 patients: t(4;14)-6 patients: del132 patients: NO1 patients: n.v.2 patients: del1314 patients: NO

PROTOCOL MM02:genic expression profileStudiare contemporaneamentelespressione genica di migliaia di geni

GENIC EXPRESSION PROFILE IN MM- Zahn et al: Global gene expression profiling of multiple myeloma, monoclonal gammopathy of undetermined significance, and normal bone marrow plasma cells. Blood (2002) - Claudio et al. Molecular compendium of gene expressed in multiple myeloma Blood (2002)- De Vos et al. Comparison of gene expression profiling between mulitple myeloma and normal plasmacells Oncogene (2002)- Underhill et al. Gene expression profiling reveals a highly specialized genetic program of plasma cells Blood (2003)- Shaughnessy JD and Barlogie B Interpreting the molecular biology and clinical behavior of multiple myeloma in the context of global gene expression profiling Immunol. Rev. (2003)

PROTOCOL MM02:genic expression profile- CD138+: selezione magnetica (95 patients at diagnosis; 24 patients post 4 months)- RNA extraction - vetri MWG 10K

AbstractIn patients with MM a number of recurrent translocations involving chromosome 14 at band q32 have been recently identified, the most common being the t(11;14) and the t(4;14). Both these chromosomal abnormalities may help to identify patients at different risk of death; in particular, the t(11;14) predicts for good prognosis, whereas the t(4;14) has been recently reported to be associated with an unfavorable clinical outcome. The t(4;14) affects at least two potential oncogenes, MMSET on der(4) and Fibroblast Growth Factor Receptor 3 (FGFR3) on der (14); the role of both these genes in the pathogenesis of MM has not bee fully elucidated.

In the present study we investigated the frequency and the prognostic relevance of the t(4;14) in a series of 63 patients with de novo MM, who were randomized to receive either a single autotransplant (Tx-1) or double autotransplants (Tx-2) as primary therapy for their disease.

For this purpose we analyzed (1) the presence of t(4;14) by RT-PCR of the hybrid transcript between MMSET and the IgH locus; (2) the overexpression of FGFR3 by Real-time RT-PCR; (3) the frequency of potentially activating point mutations in the FGFR3 translocated coding region, by direct sequencing of RT-PCR products.

Overall, the t(4;14) was detected by RT-PCR in 17/63 patients (27%); of these 17 patients, 13 had both MMSET/IgH fusion gene and FGFR3 overexpression, while 4 patients had MMSET/IgH but did not overexpress FGFR3. This finding further confirms the possible discrepancy between MMSET/IgH positivity and FGFR3 overexpression. Comparison between t(4;14) positive and t(4;14) negative patients revealed that both groups were well balanced with respect to the most common presenting features of MM. In 36 patients, for whom material was available, FISH analysis for the detection of 13q deletion and/or monosomy was performed.

Results showed that t(4;14) positive patients were more likely to carry del(13) than t(4;14) negative patients (46% vs. 29%, respectively). On an intention-to-treat basis, the probability of attaining stringently defined complete remission following either Tx-1 or Tx-2 was significantly lower for t(4;14) positive patients in comparison with t(4;14) negative patients (6% vs. 35%, respectively; p = 0.05). With a median follow-up of 40 months, no difference in overall (OS) was detected between the two groups. At the opposite, in comparison with the t(4;14) negative subgroup, patients carrying the t(4;14) had significatly lower event free survival (EFS) (23 vs 32 months, respectively; p=0.01) and duration of remission (23 vs 32 months, respectively; p=0.01). In summary, 27% of our MM patients carried the t(4;14). In this cohort of homogeneously treated patients, the t(4;14) predicted for lower response to high-dose therapy. Longer follow-up is required to assess the influence of this abnormalities on OS. In a subgroup of six patients carrying t(4;14), point mutations were detected in the FGFR3 coding region, thus suggesting a possible constitutive FGFR3 activation.

t(4;14)(p16;q32)- kariotipicamente silente- described only in MM e MGUS- frequency in MM = 20%ca.

A subset of multiple myeloma harboring the t(4;14)(p16;q32) translocation lacks FGFR3 expression but maintains an IGH/MMSET fusion transcript Madhumita Santra, Fenghuang Zhan, Erming Tian, Bart Barlogie, and John Shaughnessy Jr From the Donna and Donald Lambert Laboratory of Myeloma Genetics at the Myeloma Institute for Research and Therapy, University of Arkansas for Medical Sciences, Little Rock, ARLike many tumors of the B-cell lineage, multiple myeloma (MM) shows recurrent rearrangements of the immunoglobulin heavy-chain (IGH) locus at 14q32 and molecular studies have identified FGFR3, CCND1, CCND3, and MAF genes as targets of primary translocations in this malignancy. The multiple myeloma (MM) specific t(4;14)(p16;q32) not only results in the activation of FGFR3 but also the creation of a chimeric fusion transcript between IGH and MMSET. Given the transcription-activating nature of 14q32 translocations, microarray profiling of global gene expression has emerged as a powerful method for identifying IGH-associated rearrangements in B-cell malignancies. We have previously used this strategy to identify a novel 14q32 translocation involving the cyclin D3 gene (CCND3) as well as all known 14q32 translocations in MM. Using a combination of gene expression profiling reverse transcriptase-polymerase chain reaction (RT-PCR) and interphase fluorescence in situ hybridization (FISH) we present evidence that nearly 20% of newly diagnosed cases of MM harbor the t(4;14)(p16;q32), and approximately 32% of these cases, while expressing the IGH/MMSET fusion transcript, lack FGFR3 expression. Blood, 2003, Vol.101 pp. 2374-2376

t(4;14): PROGNOSTIC SIGNIFIANCE La disponibilit di fattori prognostici genetici fondamentale per:- identificare sottogruppi di pazienti che potrebbero condividere il meccanismo patogenetico proporre eventuali terapie mirate

Aim- studio molecolare t(4;14)

- frequenza e significato prognostico t(4;14) nella nostra casistica

famiglia di recettori ad attivit tirosin-chinasica coinvolti in una grande variet di processi mitogenici e morfogenici: sviluppo embrionale, angiogenesi, riparazione tissutale.. FIBROBLAST GROWTH FACTOR RECEPTORS (FGFR):Istituto Sergnoli - Bologna

4 membri: FGFR1, FGFR2, FGFR3, FGFR4, con distribuzione (almeno in parte) tessuto specifica, in grado di riconoscere con affinit variabile almeno 9 diversi FGFs.FIBROBLAST GROWTH FACTOR RECEPTORS (FGFR):Istituto Sergnoli - Bologna

FGFR3: glicoproteina di 125 kDa, espressa principalmente a livello del SNC, del polmone, del rene e delle cartilagini; il gene che la codifica stato isolato nel 1990 da una libreria di cDNA ottenuta dalla linea cellulare K562 (Keegan et al, Proc. Natl. Acad. Sci., 1991).Istituto Sergnoli - Bologna

FGFR3 = Fibroblast Growth Factor Receptor 3 is a tyrosin-kinase receptor mutazioni nella linea germinale causano una grave forma di nanismo espressione assente nel tessuto ematopoietico delladulto sano

in MM: could be over-expressed in alcuni casi sono presenti le stesse mutazioni che causano il nanismo

FGFR3 GENE AT 4p16.3: 1 2 6 7 (IIIa) 8 (IIIb) 9 (IIIc) 10 18 1916,5 kbalternative splicingcoding regionIIIa: secreted formIIIb: epithelial formIIIc: mesenchimal formIstituto Sergnoli - Bologna

s-ss-ss-ss-ss-ss-sSplit Tyrosine Kinase DomainTrans-Membrane DomainIg-like DomainsIIIIIIIIIIIIOUTINcell surfaceIstituto Sergnoli - Bologna

Istituto Sergnoli - Bologna

FGF + heparan-sulfate

GDP GTP

Shc exchange

dimerization and

transphosphorilation

* o MEK

o ERK

P

P

FGFR3

Ras

GRB22

Raf-1

P

Sos

P

P

MAPK*K *

MAPK

80K-H

kk

GRB2

Sos

P

DNA

NUCLEUS

Jun

Fos

1. Achondroplasia(Gly375Cys; Gly380Arg)5. Hypochondroplasia(Asn540Lys)3. Crouzon syndrome with acantosis nigricans (Ala391Glu)4. Muenke coronalcraniosynostosis(Pro250Arg)FGFR3 mutations2. Thanatophoric Dysplasia type I and II (Tyr373Cys; Arg248Cys; Lys650Glu; Thr807Cys)Istituto Sergnoli - Bologna

Ex.7 Ex.10 Ex.13 Ex.15 Ex.19cod.248 TM domain cod.540 cod.650 cod.807 mutations constitutively activated FGFR3SUBSEQUENT SELECTION OF SOMATIC MUTATIONS:Istituto Sergnoli - Bologna

EFFECTS OF FGFR3 ECTOPIC EXPRESSION ON MURINE B9 MM CELLS: (Plowright EE et al, Blood 2000; 95:992-997)FGFR3STAT3Bcl-xLPphosphorilationDECREASED APOPTOSIS?ENHANCED PROLIFERATIVE RESPONSE TO IL-6Istituto Sergnoli - Bologna

In multiple myeloma, t(4;14)(p16;q32) is an adverse prognostic factor irrespective of FGFR3 expression.Keats et al. Blood 2003;101:1520-29


Clinical and biologic implications of recurrent genomic aberrations in myeloma Fonseca, et al Blood 2003;101:4569-4575

Recurrent 14q32 translocations determine the prognosis of multiple myeloma, especially in patients receiving intensive chemotherapy Moreau et al. Blood 2002;100:1579-1583

Chr. 12 and t(12;21)Tel-AML1

Chr. 1 and t(1;19)E2a- PBX

The genes for making the heavy chains of antibodies (CH) are located on chromosomes 14, whereas those for making the light chains are on chromosomes 2 and 22. These genes are expressed exclusively in B lymphocytes, because only these cells have the necessary transcription factors to switch on their expression. In most (over 90%) of Burkitt's lymphoma cases, a reciprocal translocation moves the proto-oncogene c-myc from its normal position on chromosome 8 to a location close to the antibody heavy-chain genes on chromosome 14. In other cases, c-myc is translocated close to the antibody genes on chromosome 2 or 22. In every case, c-myc now finds itself in a region of active gene transcription, and it may simply be the overproduction of the c-myc product (a transcription factor essential for cell division) that propels the lymphocyte down the pathway towards cancer.

Virus and Lymphoma

Figure 1|Schematic representation of the HCV genome and encoded viral proteins. The boxed area corresponds to the single open reading frame of the hepatitis C virus (HCV) genome. The stemloop structures represent the 5' and 3' non-translated (NTR) regions, including the internal ribosome-entry site (IRES) and 3'X regions. The function and molecular mass (in kDa) of the gene products after polyprotein processing are shown. Core (C)E1, E1E2, E2p7 and p7non-structural protein 2 (NS2) junctions are cleaved by a cellular signal peptidase(s) to yield structural proteins. The NS2NS3 metalloproteinase undergoes autocatalytic cleavage, which releases the mature NS3 serine protease. NS3 cleaves the remainder of the NS polypeptide. The two regions that have extreme sequence variability in E2, known as hypervariable regions 1 and 2 (HVR1 and HVR2), are indicated. A region in NS5A, known as the interferon (IFN)-sensitivity-determining region (ISDR), has been linked to the response to IFN- therapy in some strains of HCV. Both NS5A and E2 have been implicated as antagonists of IFN (for review, see Ref. 34). ARFP/F, alternative reading-frame protein/frameshift protein; LDLR, low-density lipoprotein receptor; RdRp, RNA-dependent RNA polymerase.

Cenni di terapia delle ALL: nuovi approcci

CICLO A1 PREDNISONE 60 mg/sqm p os dal +1 al +5ENDOXAN 200 mg/sqm e.v. dal +1 al +5Giorno +6: PAUSARITUXIMAB 375 mg/sqm e.v. g +7 (CD20 > 20%)DEX

MTX

IFOSFAMIDE

ARA-C

VM-2610 mg/sqm p. os. dal +8 al +12

500 mg/sqm p.c. g +8

400 mg/sqm ev dal +8 al +12

120 mg/sqm ev gg +11 e +12

60 mg/sqm ev gg +11 e +12Rachicentesi :gg +8(MTX 12 mg)

G-CSF 5 mcgr/Kg sc dal +14 a PMN > 1000/mmc

CICLO B1 (DAL +28) RITUXIMAB 375 mg/sqm e.v. g +28 (CD20> 20%)DEX

VCR

MTX

ENDOXAN

ADRIAMICINA10 mg/sqm p. os. dal +29 al +33

1 mg ev g +29

500 mg/sqm p.c. g +29

200 mg/sqm ev dal +29 al +33

25 mg/sqm ev gg +32 e +33Rachicentesi :gg +29( MTX 12 mg)

G-CSF 5 mcgr/Kg sc dal +35 a PMN > 1000/mmc

CICLO A2 (DAL +49) RITUXIMAB 375 mg/sqm e.v. g +49 (CD20> 20%)DEX

MTX

IFOSFAMIDE

ARA-C


500 mg/sqm p.c. g +50

400 mg/sqm ev dal g +50 al g +54

120 mg/sqm ev gg +53 e +54

60 mg/sqm ev gg +81 e +82G-CSF 5 mcgr/Kg sc dal +56 a PMN > 1000/mmc

Rachicentesi :gg +50(MTX 12 mg)

CICLO B2 (DAL +77) RITUXIMAB 375 mg/sqm e.v. g +77 (CD20> 20%)DEX

VCR

MTX

ENDOXAN


1 mg ev g +78

500 mg/sqm p.c. g +78




CICLO A3 (DAL +98) RITUXIMAB 375 mg/sqm e.v. g +98 (CD20> 20%)DEX

MTX

IFOSFAMIDE

ARA-C


500 mg/sqm p.c. g +99


120 mg/sqm ev gg +102 e +103



CICLO B3 (DAL +119) RITUXIMAB 375 mg/sqm e.v. g +119 (CD20 >20%)DEX

VCR

MTX

ENDOXAN


1 mg ev g +120

500 mg/sqm p.c. g +120




CONSOLIDAMENTO RITUXIMAB 375 mg/sqm e.v. SETTIMANA 21 (G +147) E 24 (G+ 168)RADIOTERAPIA (dopo la fine della chemioterapia) SNC+: 24 Gy

Bulky mediastinico (> 7.5 cm): 36 Gy

Malattia residua dopo CHT: 36 Gy

Localizzazioni extranodali: decisione libera

RAS inhibitors

Extracellular signalsRAS GDP (inactive)RAS GTP (active)RAS effectors: PI3K, MAPK...Effects: cellular growth, proliferation, differentiationInactivationGTPase activating proteins (GAPs)ActivationExchange factors (GEFs)Istituto di Ematologia e Oncologia Medica Sergnoli - Bologna

RasCAAXSHFarnesyl transferase1. IsoprenylationRasC AAXS2. ProteolysisIstituto di Ematologia e Oncologia Medica Sergnoli - Bolognafarnesyl-PPfarnesylRasC Sfarnesyl3. Methylation4. PalmitoylationRasC SfarnesylCH3RasscPost-translational RAS modifications:palmitoyl-PPCAAX-proteasefully activated RAS

Posttranscriptional modification of RasFTase I orGGTaseRascell membrane1. Prenylation2.Proteolysis3. Methylation4. PalmytolationCaaXFPPGGPPRasC-S-F(or GG)a a XRasC-S-F(or GG)microsomal membraneaaXRasSAMPPMTaseRasC-SF(or GG)C-S-F(or GG)MePPSCH 66336 PHASE IIR 115777PHASE II

RASRalGDSPI 3- kinasePKB/Akt?GSK3bBadCaspase-9PDKForkhead?Fas ligandRafMekErkEtsp21Cyclin D1FosIstituto di Ematologia e Oncologia Medica Sergnoli - BolognaRacRho?

mTOR inhibitors

TORProtein Kinase Csignalingstationaryphase (G0)autophagynutrient transporterturnovertranslationtranscriptionactin organization tRNA andribosomebiogenesisTOR controls a Large and Diverse Set of Growth-related Readout Green arrows indicate activation; red bars indicate repressionShown is a composite of yeast and mammalian readouts.

RADICICOLRAPAMICIN

TRAIL inhibitors

Targeting the :

Tyrosine kinase activity of BCR-ABL

FTI (Farnesyl-transferase activity)

Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL or Apo-2L)

Src pathway TK pathway

Proteasome activity

Se, da un lato, sembra ormai essere un dato di fatto che queste traslocazioni rappresentino levento oncogenetico primario nalla patogenesi del mieloma multiplo, dallaltro viene spontaneo domandarsi quale possa essere, in un quadro cos eterogeneo di alterazioni ed oncogeni coinvolti, il minimo comune denominatore responsabile del fenotipo neoplastico.

Per diverso tempo stato suggerito che la t(11;14) e la conseguente overespressione della ciclina D1 potessero rappresentare un fattore prognostico sfavorevole per i pazienti affetti da MM. Tuttavia, le segnalazioni comparse in letteratura a questo proposito, e che qui vedete elencate, derivavano tutte da studi condotti su casistiche estremamente limitate. E dellaprile di questanno inveceil lavoro di Fonseca su 336 pazienti arruolati nellECOG; questi pazienti sono stati analizzati in FISH per la presenza della t(11;14) e 53, ossia il 16%, sono risultati positivi. Qui elencate potete vedere le uniche correlazioni emerse in questo studio, tra presenza della traslocazione e caratteristiche cliniche e biologiche allesordio: componente monoclonale nel siero inferiore ai 10g/L e cariotipo raramente iperdiploide. I pz. con t(11;14) presentavano una risposta pressoch uguale alla chemioterapia convenzionale, ed un overall survival ed un progression-free survival lievemente migliori; le differenze sono pero lontane dalla significativit statistica.Cyclin D1 mRNA levels were quantified in the bone marrow of 85 MM patients at diagnosis. These patients were subsequently randomized to receive either a single or a double autologous PBSC transplantation after remission induction chemotherapy with VAD and high-dose cyclophosphamide (HD-CTX). In 46 cases with sufficient material, FISH analysis was also done to evaluate the presence of chromosome 11 abnormalities so as to validate our real-time assay.

Up-regulation of cyclin D1 correlated only with higher bone marrow plasma cell infiltration and more advanced clinical stage at diagnosis.No statististical differences were found between cyclin D1 positive and negative pts. with respect to age, gender, immunoglobulin subtype, stage, serum beta-2-microglobulin, C-reactive protein or creatinine levels.

DIAGNOSI E TERAPIA DELLE LEUCEMIE LINFOBLASTICHE ACUTE e

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