PREMIO Giuseppe Del Console. tutti quelli che non ce l hanno fatta

Transcript

1 PREMIO 2009 a Giuseppe Del Console e. a tutti quelli che non ce l hanno fatta

2 via Piero Gobetti via Giuseppe Di Vittorio

3 Infatti, per realizzalo sono stati necessari : 1) finanziamenti extra-istituzionali (interamente ottenuti dal gruppo) 2) Immaginazione scientifica 3) Collaborazioni con altri centri 4) Capacita di cogliere informazioni da esperimenti fallimentari 5) Senza possibilità di fare Scuola ; cioè di poter Crescere giovani e di premiarli e/o poter loro promettere un prospettiva di carriera

4 Acquistare topi Imparare a lavorare coi topi Tempi dilatati fino a sera stipendio modesto o (precari veramente esiguo) mentre gli altri fanno studio e carriera

5 La cosa di cui oggi parlo NON è una nuova tecnica. È una nuova idea o insieme di idee. Abbiamo dovuto cominciare da zero

6 The origin of Intra-Bone Marrow Transplant Lorenzo Magrassi Marina Podestà Francesco Frassoni Vincenzo Menditto Sandra Castello

7

8 Il patrimonio di cellule ematopoietiche nel midollo osseo di uomo 10^12 1/ Stem Cells ematopoietiche

9 Quando facciamo un trapianto di midollo osseo nell uomo trasferiamo 200 x 10^8 cellule ematopoietiche 1/100 del patrimonio

10 Abbiamo un numero di cellule ematopoietiche (e di stem cells) ridondante

11 Quando facciamo un trapianto di midollo osseo nell uomo trasferiamo 20 x 10^9 cellule ematopoietiche 1/100 del patrimonio [200 x 10 10^2] Stem Cells ematopoietiche

12 Quando facciamo un trapianto di cellule ottenute da cordone ombelicale x 10^7 cellule ematopoietiche

13 Quando facciamo un trapianto di cellule ottenute da cordone ombelicale trasferiamo 1/10 di cellule (e di cellule staminali) rispetto a quando facciamo un trapianto di midollo osseo

14

15 Seeding Efficiency

16 How many cells seed when injected into the vein? 10^5 cells injected After 24 hours seeding into Hematopoietic system = 9% or less

17 n engl j med 351;22 november 25, 2004 MUD N 682 CBT N 98

18 Cells collected Cells infused Cumulative incidence Eurocord data (V Rocha) UCBT malignant disorders (n=929) Platelets recovery according to number of nucleated cells collected or infused (10 7 /kg) P= > P< >5.0 < 3.0 < Days Days

19 .ma noi avevamo ipotizzato e poi dimostrato una potenzialità staminale formidabile per le cellule di Cordone Ombelicale

20 La ipotesi della Cellula Egoista Cellule Staminali del Sangue Post-Trapianto CB BM Blood. 2003;102: Frassoni Podestà Locatelli

21 Intra-Bone Marrow versus Intra-Vein 10^6 cells injected 950 cgy C57BL (EGFP) into C57BL = 37 % 950 cgy 10^5 cells injected C57BL (EGFP) into C57BL = 53 % (as mesured in the other femur) The cells injected directly into the bone provided 10 times more engraftment than cells injected into the vein Exp Hematol ;32:782-7.

22 Double cord transplant (A) Neutrophil recovery. (B) Unsupported platelet recovery platelet recovery greater than 50 x 10 9 /L at 6 months Umbilical cord blood transplantation after nonmyeloablative conditioning: impact on transplantation outcomes in 110 adults with hematologic disease Claudio G. Brunstein et al BLOOD, 15 OCTOBER 2007

23 Outcomes of transplantation of unrelated donor umbilicalcord blood and bone marrow in children with acute leukaemia: a comparison study Mary Eapen, et al Lancet 2007; 369: CELL DOSE: Low ( 3 10^7/kg) High( >3 10^7/kg)

24 Two barriers to the success of CBT in adults : 1) Cell dose 2) HLA matching

25 Clinical Intra-Bone Marrow Transplant

26 79 Patients received INTRA-BONE CBT

27 Rationale (endpoints) migliorare Seeding efficiency Attecchiemto Velocità di attecchimento

28 Site of injection CB cells are resuspended in ml of dextran/albumin, and aliquoted in 4 syringes of 5-6 ml each.

29

30 CBT IBM Patients selection pazienti che non avevano trovato un MUD pazienti che erano in condizioni cliniche spesso terminali e non avevano tempo per nessun altro tipo di trapianto

31 CBT IBM Patient charcteristics EARLY 14= 17% INT 15 =19% ADV 50 = 64% AML 12, 7, MDS 1,0,8 9 ALL 1,8,12 21 CML 0,0,6 6 HD 0,0,4 4 NHL 0,0,6 6 Other 0,0,3 3

32 Intervallo Diagnosi Trapianto : Pazienti con Leucemia Acuta in "CR1" : 250 d ( )

33 CBT IBM Patient charcteristics Age 45 (18-63) Patient b.w. 75 (44-108) TNC x10^7/kg 2.7 ( ) CD34 x10^5/kg 1.15 ( ) N= N= N=30 >3 HLA match 12 = 5/6 66 = 4/6 1 = 3/6

34 Engraftment 3 Patients did not engraft all the other patients engrafted maximum within 70 days* *(Plt longest recovery) The follow-up is : 13 months (2-38)

35 Results PMN > (14-44) PLT> (16-70)

36 Cumulative incidence UCBT malignant disorders (n=925) Cumulative incidence of Platelets recovery (>20.000) 54.7% ICC at day patients 506 engraftments at Day Days

37 platelets x10^9/l Transplants in Genova UD CB UD BM UD PB

38 Competing event = dead Matched pair analysis- Intrabone vs Iv CBT Cumulative Incidence for Platelets recovery (>20.000/mm3) P< IBCB IVCB 72±6% 50±4% days

39 Platelet recovery after INTRABONE CB transplants ( )* TNC x 10^7/kg <2 (n=7) 2-3(n=34)# >3 (n=23)@ Day PMN > 0.5 x10^9/l Day PLT > 20 x10^9/l *Not including: 3 Pts not engrafted 12 pts dead before day +14 HLA matching 1= 5/6 6 = 4/6 # 5= 5/6 28= 4/6 1= 2= 5/6 21= 4/6

40 GVHD Acute GvHD At risk 57 0 n =30 I n= 17 II n= 11 III-IV n= 0 Chronic GVHD At risk 43 0= 23 1= 13 2= 5 3= 2

41 Umbilical cord blood transplantation after nonmyeloablative conditioning: impact on transplantation outcomes in 110 adults with hematologic disease Claudio G. Brunstein et al BLOOD, 15 OCTOBER 2007 Incidence of Acute GVHD Grade II-IV Grade III-IV Incidence of 3-year treatment-related mortality.

42 The outcomes at 2 years according to Kaplan-Meier are: OS = 46%+/-5 RI = 18%+/-2 NRM = 39%+/-5 Cumulative incidence was: PMN >500/10 9 /L = 84+/-4 at day 50 PLT > /L = 82+/-4 at day 70

43 Another way to look at the results AW Rel NRM 14 Early INT ADV

44

45

46 Intra-Bone Marrow versus Intra-Vein 10^6 cells injected Rat model 10^6 cells injected

47 % of dose (% of cells) Defining lung entrapment of hemopoietic stem cells directly injected into the bone marrow (Rat model) 50,00% lungs LUNG 45,00% 40,00% 35,00% 30,00% 25,00% intrabone intravenous 20,00% 15,00% 10,00% 5,00% 0,00% Time seconds

48 intrabone intravenous Blood clearance of HSC (Cardiac volumes/minute) Gian Mario Sambuceti et al. Nuclear Medicine University of Genoa

49 Intrarabone vs I.V. transplant of CD90+ cells in rats The Logan plot analysis liver spleen forelimb Stem cell homing intravenous injection Ratio between retained and released cells Stem cell homing Intrabone injection Stem cell homing intrabone 4,5 4 3,5 3 2,5 2 1,5 1 0,5 * Intravenous naive Intrabone naive Hepatoma Intravenous irradiated Intrabone irradiated 0 Forelimb Liver Spleen

50

51 Un cordone ombelicale per tutti

52 Simulation of a Search for a CBU Simulation of a Search for a CBU 183 patients in search 1/2007 to 3/2009 Pool of CBU with DRB1 high resolution

53 Simulation of a Search for a CBU

54 Simulation of a Search for a CBU

55 Simulation of a Search for a CBU Body weight in kg

56 Conclusion This approach increases the chance of offering a transplant to a greater number of adult patients (94%)

57 Cord Blood Banks Clinica Ematoloigica Università di Genova M Gobbi Infermieri della Unità Trapiantologica e della Sala Operatoria Centro Cellule Staminali e Terapia Cellulare Ospedale San Martino - Genova GL Ubezio R. Varaldo S. Pozzi F. Bertolotti S. Lucchetti C. Cascianelli Divisione Ematologia Ospedale San Martino Genova Tutta la Divisione Ematologia e Trapianto Institute for Diabetes Research HSR Lorenzo Piemonti Dipartimento Medicina Nucleare Università di Genova GM Sambuceti S Morbelli C Marini IBMDR Ospedali Galliera Nicoletta Sacchi

58 .il gruppo che lavora a questi progetti il Centro Cellule Staminali stesso è a rischio di Estinzione

59 .ma il nostro lavoro è andato oltre le aspettative

60 Intra-Bone Marrow Islet Transplant Lorenzo Piemonti et al.

61

62

63

64 The origin of Intra-Bone Marrow Transplant Shielded limb The niche The Selfish Stem Cell hypothesis The problem of Seeding Efficiency

65 Jon van Rood D van Bekkum Rijswijk 1965

66 Van Bekkum DW, Schotmen E. Protection from haemopoietic death by shielding versus grafting of bone-marrow.

67

68 Intra-Bone Marrow versus Intra-Vein 10^6 cells injected 950 cgy C57BL (EGFP) into C57BL = 37 % 950 cgy 10^5 cells injected C57BL (EGFP) into C57BL = 53 % (as mesured in the other femur) The cells injected directly into the bone provided 10 times more engraftment than cells injected into the vein Exp Hematol ;32:782-7.

69 axis axis endosteum endosteum HSC prefer to live in an area rich in MSC and osteoblast Non random distribution of HSC Lord BI. The relative spatial distribution of CFU-S and CFU-C in the normal mouse femur. Blood (1975) F Frassoni, NG Testa, BI Lord. The relative spatial distribution of erythroid progenitor cells (BFUe and CFUe) in the normal mouse femur. Cell and Tissue Kinet Jul;15(4):447-55

70 The case of Cord Blood Transplant The selfish stem cell Hypothesis

71 Total Content of Progenitors Cord Blood Bone Marrow ANC /kg 2 x10^7 2-4 x10^8 LTC-IC/kg Podestà et al

72 BM CB 3 x10^8/kg 2 x10^7/kg is the delayed engraftment due to an insufficient number of stem cells in cord blood units?..or.

73 Hypothesis it is a selfish stem cell the problem of cord blood transplant? Self-renewal CB stem cells maturation

74 Hypothesis it not the stemness which is lacking in Cord Blood units but it is its scarce attitude to produce mature cells

75 Methods 12 Children who had received CBT 12 Children who had received BMT Reconstitution of hematopoietic progenitors (LTC-IC and CFC) 1 year after transplant Blood. 2003;102: Frassoni & Locatelli

76 When we transplant CB cells we transplant 1/10 of cells (and stem cells) respect to when we transplant a standard bone marrow harvets

77 Patients characteristics and Results Cord Blood Bone Marrow p Age (months) 63(3-180) 88(30-152) NS Cells infused x10^7/kg 3.7(1.9-18) 50 (21-120) PMN >1000 days 22(16-39) 16 (9-23) Plt > days 48(30-100) 27(21-58) CFC x10^4 20 (7-56) 11(3-25) LTC-IC x 10^6 8( ) 0.2(0-3.7) CBT provides 1 log higher reconstitution of hematopoietic reservoir than BM or PB transplants Blood. 2003;102:

78 Acute graft-versus-host disease after unrelated donor umbilical cord blood transplantation: analysis of risk factors Margaret L. MacMillan, et al BLOOD, 12 MARCH 2009

79

80

81