Ingen lysbildetittel

1
Høydosebehandling med autolog stamcellestøtte-
HMAS
2
Hematopoietic stemcell research
3
BONE MARROW HARVESTING
4
(No Transcript)
5
(No Transcript)
6
(No Transcript)
7
Department of Cellular Therapy,
Rikshospitalet-Radiumhospitalet Leucapheresis
unit 125-150 patients per year
8
Autotransplant of lymphomas at DNR Bone marrow
compared to PBPC

• BM PBPC
• (n83)
  (n50)
• CD34 x106/kg n.d.
  5,7(1,8-20,0)
• CFUx105/kg() 6.2(0.7-22.0) 14,1(0,4-62.5)
• Days neutgt0,5 24(11-140) 12(8-24)
• Days trcgt20 26(8-566) 11(7-43)

9
Hematopoietic stemcell research today
10
Large volume leukapheresis (LVL) processing at
least 3 blood volumes/harvest session
11
LVL in children-challenges

• Venous access
• Volume shifts
• Anticoagulants
• Patient cooperation

12
Venous access

• Dubbel lumen chatheter required to maintain a
  high inlet flow rate ( 2ml/kg bodyweight per
  minute) during processing

13

• In children red cell priming with irradiated
  blood in the Cobe Spectra prior to
  collection(adjust the hematocrit gt27)

14
ACD-A apheresis

• Rate of citrate return is the strongest limiting
  factor to LVL
• gtACD-A rate ltCa2symptoms

15
Citrate toxicity

• Greatest concern Cardiac arrhythmias
• Paresthesias
• Muscle cramping
• Hypotension

16
Prevention of citrate toxicity during LVL

• ACD-A/whole blood ratio (130)
• Prophylactic Calcium administration
• Calcium Sandoz 9mg/ml iv 4-5 ml/ 10 kg after
• 60 minutes
• 120 minutes

17
Patient cooperation

• Experienced nurse
• One family member
• Discuss prior to collection if special
  requirements is needed (food, entertainments,
  pants)

Staff at DCT
18
PURGING OF TUMOUR CELLS IN AUTOGRAFTS?
19
Positive/Negative Selection on Isolex
Isolex Magnet
20
Efficacy of T-cell removal from PBPC by CD34
cell enrichment alone(A) or in combination with
anti-CD2 T-cell depletion(B)
PBPC
CD34
A
B
21
Real time PCR of T(1418)-mcr in BM before and
after high dose therapy with B-cell purged CD34
cells . Relapse in spite of infusion of tumour
cell free graft
No of mcr copies per 5x105 cells
22
High-dose therapy in high-grade NHL, comparison
between purged BM and unmanipulated PBPC
Purged BM (n30)
Purged BM (n30)
Blystad et al.
23
No of absolute lymphocytes (ALC) at day 14 after
high dose therapy in patients with diffuse large
B-cell lymphoma and follicular lymphoma grade 3

• Blystad et al

24
Poor mobilizers

• 5-20 of the cancer patients

25
Poor mobilizers definitions

• lt 2x106 CD34 cells/kg following
• 3 consecutive apheresis?
• 5 consecutive apheresis?

26
Lymphoma patients characteristics assosiated with
mobilizing efficacy of CD34cells

• Previous chemotherapy
• Previous irradiation
• Patients with Hodgkin's disease
• Hass et al. 1994

27
Platelet engraftment correlateswith PBSC dose
of patients with gt28 days to recovery of PLT
gt20 x 103 /µL

PBSC dose (x 106 CD34 /kg)

Outcome
gt5
Optimum
1
25
Acceptable
10
12
Minimum
20
lt1
Sub-therapeutic
2070
Glaspy J, et al. Blood. 1997902939-2951.Perez-S
imon J, et al. Transfusion. 199838385-391.
28
PBPC collection experiences in lymphoma patients
at DNR
29

• 141 Lymphoma patients
• 33 Hodgkins lymphomas
• 89 High grade non Hodgkins lymphomas
• 19 low grade or transformed high grade non
  Hodgkins lymphomas

30

• 141 Lymphoma patients
• Median age 39(14-64) years
• Chemotherapy before mobilization Median 11
  (3-40) cycles
• (53 patients gt12 cycles)
• Previous radiotherapy 59 patients, 31 with
  extended field radiation

31
Schedule for chemotherapy with G-CSF mobilisation
in patient with lymphoma

• Day 1 2 3 4 5
• Mitoguazon 500 mg/m2
• Ifosfamide 1000 mg/m2
• (Mesna 300 mg/m2 x 3)
• Methotrexate 30 mg/m2
• Etoposide 100 mg/m2
• G-CSF from day 7

G-CSF granulocyte colony-stimulating factor
32
Myelo-suppression and MIME/G-CSF mobilization
efficacy

• Nadir leucocytes values
• G-CSF 5ug/kg 1.95(0,2-8,0) x 109/l
• G-CSF 10ug/kg 1,8(0,1-10,6) x 109/l
• Nadir platelets values
• G-CSF 5ug/kg 77(5-199) x 109/l
• G-CSF 10ug/kg 70(12-231) x 109/l
• No correlation between low nadir cell counts and
  yield of CD34 cells

33

• Cyclophosphamide 1,5 g/m2 G-CSF( Watts et al.
  J.Clin. Oncol 1997)
• 50 mobilised gt 3,5 x 106 CD34 cells/kg
• Higher doses of CY improve yield of CD34 cells,
  but increase toxicity
• MIME G-CSF
• 58 mobilised gt 5 x 106 CD34 cells/kg

34
(No Transcript)
35
(No Transcript)
36
Total yields of CD34 cells
Total number of harvested CD34 cells x 106 /kg
Number () of patients
Diagnosis
0
0 lt2.0
?2.0lt5.0
?5.0lt10.0
?10
Total
HD
33 (100)
9 (27)
8 (24)
16 (49)


H-NHL
89 (100)
6 (7)
9 (10)
24 (27)
29 (33)
21 (23)
L-Tra-NHL
2 (11)
1 (5)
8 (42)
4 (21)
4 (21)
19 (100)
HD Hodgkins disease H-NHL high-grade
non-Hodgkins lymphoma L-Tra-NHL low-grade
transformed non-Hodgkins lymphoma
37
Inadequate PBSC cell dose a clinical issue for
patients

• 70 with lt2 x 106 CD34 /kg have slow
  engraftment of platelets
• Current alternatives
• do not proceed to high-dose therapy
• full large volume BM harvest
• remobilise with multiple apheresis

Weaver C, et al. Blood. 19958639613969.Perez-
Simon J, et al. Bone Marrow Transplant.
19992412791283
38
Inadequate PBSC cell dose a clinical issue for
patients

• 70 with lt2 x 106 CD34 /kg have slow
  engraftment of platelets
• Current alternatives
• do not proceed to high-dose therapy
• full large volume BM harvest
• remobilise with multiple apheresis

Weaver C, et al. Blood. 19958639613969.Perez-
Simon J, et al. Bone Marrow Transplant.
19992412791283
39
Inadequate PBSC cell dose a clinical issue for
patients

• 70 with lt2 x 106 CD34 /kg have slow
  engraftment of platelets
• Current alternatives
• do not proceed to high-dose therapy
• full large volume BM harvest
• remobilise with multiple apheresis

Weaver C, et al. Blood. 19958639613969.Perez-
Simon J, et al. Bone Marrow Transplant.
19992412791283
40
Remobilize with multiple apheresis

• Treatment free interval until new mobilization -
  how long?
• G-CSF alone or combined with chemo?
• Other combinations?

41
AMD3100

• Chemokine receptor antagonist
• Reversibly blocks SDF-1/CXCR4 interaction
• Stem cell mobilizer

42
CD26
MMP-9, MMP-2
G-CSFR
C-kit
CD34
NE CG
VLA-4
CXCR4
Stromal Cell
VCAM-1
SDF-1
43
CD26
MMP-9, MMP-2
G-CSFR
CD34
C-kit
NE CG
VLA-4
CXCR4
AMD3100
Stromal Cell
VCAM-1
SDF-1
44
Phase I Study Liles, et al.

• 26 healthy volunteers, 13 male, 13 female
• Normal peripheral blood counts
• Group 1 (n10) AMD3100 80 mcg/kg subq x 1
• Group 2 (n13) 40-240 mcg sq x 1
• Group 3 (n3) AMD3100 80 mcg sq daily x 3
  consecutive days

Blood 2003 1028, 2728-2730
45
Results

• Single 80 mcg injection caused 4-fold increase in
  circulating CD34 cells, peak 6 hours
• Dose escalation study showed dose-dependent
  effect, peak 10-fold increase in PB CD34 cells
  at 9 hours after 240 mcg injection
• Daily 80 mcg injection caused increase in PB
  CD34 cells of similar magnitude each day
• Adverse effects injection site reaction,
  nausea, abdominal distension, not dose dependent
• Effects were temporary

Blood 2003 1028, 2728-2730
46
Phase I Study Devine, et al.

• 7 patients with MM, 6 with NHL
• Last dose of chemo between 4 8 weeks of study
  entry
• Normal peripheral blood cell counts
• 4 with prior RT
• Median age 53 (39-67)
• Median prior chemo regimens 1
• Median prior cycles 6

JCO 2004 221095
47
Phase I Study Devine, et al.

• 160 mcg/kg in 6
• 240 mcg/kg in 7
• WBC, PB CD34 cell counts analyzed at 4 and 6
  hours

JCO 2004 221095
48
Phase I Study Devine, et al.

• Results
• All patients mobilized successfully
• Rise in PB WBC and CD34 cells at 4 and 6 hours
• PB CD34 counts higher in 240 mcg group
• No greater than grade I toxicity

49
Fig 1. Total WBC count and peripheral blood CD34
cell count observed in patients receiving AMD3100
at 160 microg/kg or 240 microg/kg at baseline
and 4, 6, and 24 hours (WBC count only at 24
hours) after the dose
Devine, S. M. et al. J Clin Oncol 221095-1102
2004
50
Compassionate use study in patients who have
failed prior mobilization

• Inclusion criteria
• Age gt 18
• Advanced hematologic malignancy
• Failed prior attempt at mobilization
• Candidate for high dose chemotherapy with stem
  cell support

51
Patient Characteristics
52
Mobilization Schema

• G-CSF 10 mcg/kg daily x 4 days
• Evening of day 4 pts received AMD3100 240 mcg/kg
• Day 5 pts received G-CSF, apheresis commenced
• Apheresis continued until pt collected sufficient
  CD34 cells/kg for auto transplant

53
Conclusions

• AMD3100 plus G-CSF is an effective salvage
  regimen for pts failing mobilization due to prior
  chemo
• No significant increased toxicity compared with
  G-CSF alone
• Engraftment kinetics similar to cytokine or
  chemomobilization

54
(No Transcript)
55
Niche cells for hematopoietic stem cells
56
AastromReplicell System cell production platform

• gt9 x 108 BMMNC
• IMDM 10 FCS 10 horse serum
• pIXY321 5 ng/mL Flt-3 25 ng/mLEpo 0.1
  IU/mL
• 12 days incubation

IMDM Iscove's modified Dulbecco's medium FCS
fetal calf serum Epo erythropoietin
57
Engraftment study design

• non-randomised
• 19 Breast cancer patients
• Small volume marrow collection (75 ml)
• STAMP V chemotherapy
• BMT G-CSF post.transpl
• Endpoints and day 60 follow-up
• time to ANC gt500 /µL
• time to platelets gt10, 15 or 20 x 103 /µL
• incidence of febrile neutropenia

STAMP V carboplatin, thiotepa and
cyclophosphamide ANC absolute neutrophil count
58
SC-I Engraftment Study Data
Stiff et al.
59
SC-I Engraftment Study Data
Stiff et al.
60
Expansion capacity of bone marrow progenitor
cells using Aastrom Replicell bioreactor with
PIXY321, Flt3, SC-1
61
MSCs from bone marrow stroma
62
Clonogenic assays for mesenchymal stem cells

• Fibroblast colony-forming unit (CFU-F) assay
• 1 CFU-F per 104105 bone marrow mononuclear cells
  (BMMNC)
• Contains a mixture of colonies with both
  restricted lineage potential and full mesenchymal
  differentiation potential

63
Study design
Patients undergoing PBSC collection
lt2.0 x 106 CD34 /kg PBSC
gt2.0 x 106 CD34 /kg PBSC
Meet entrance criteria
Small volumeBM collection
lt18 x 108TNC
Off study
Expansion of small volume BM in the
AastromReplicell
Off study
HDC with PBSC and ex vivoexpanded BM cells
HDC high-dose chemotherapy
64
Patient characteristics

• Non Hodgkins lymphomas (no 7)
• Myeloma (no2)
• Median age 55 (48-62)
• Neuroblastoma (no1)
• Mobilisation regimen G-CSF(no3)
• G-CSF chem(no7)
• High dose therapy BEAM(no8)
• Melphalan(no2)

65
Cells infused and time to engraftment median
PBPC CD34 x106 /kg 1.3 (0.9-1.8) median
bioreactorCD34x106/kg 0.2
(0.02-0.52) medianPBPC CFU-GM x105/kg 2.8
(2.4-3.9) median bioreactor CFU-GM x105/kg 3.9
(0.02-35) median CFU-F x103/kg 1.5
(0.06-725) median days to ANCgt 0.5 11
(10-28) median days to PLTgt 20 21 (9-76) (?
n10)
66

• Conclusions
• Small volume ex vivo expanded bone marrow
  combined with subtherapeutic doses of PBPC
• 1. Give no infusion related toxicity.
• 2. Give a sustained engraftment of both
  granulocytes and platelets.
• 3. Make high dose therapy possible in poor
  mobilizers as well.
• Our results suggest the existence of a
  synergistic effect on
• haematopoietic reconstitution.
• The results showed a wide variation in the
  clonogenic capacity
• of the expanded BM cells.
• No correlation was found between haematopoietic
  reconstitution and the number of CFU-GM, CFU-F
  and LTC-ic in the ex vivo expanded BM products

67
Dynamic observation of MSCs proliferation and
migration in vivo with Luciferase reporter gene
IV in animals treated with TBI 5GY
68
FCS and GTP
69
hBM-MSC grown in LPO2 serum free medium
supplemented with platelet rich AB plasma
Cell cultured for 7 days in 25ml culture flasks
70
ADAS grown in LPO2 serum free medium supplemented
with platelet rich AB plasma
Cell cultured for 7 days in 25ml culture flasks
71
Phenotypic comparison of types of MSCs and growth
conditions
72
CYTOKINE PROFILE OF MSCs (Bioplex)
IL-2, IL-5, IL-9, IL-13, IL-17, MIP-1a,
MIP-1b,PDGF-BB not detectable
73
(No Transcript)