Mobilisation and collection of Peripheral Blood Stem Cells N Milpied University and Hospital Bordeaux

Principes Intensification-autogreffe Masse Intensification Chimio conventionnelle 10 3 Rémission clinique Rechute Seuil Clinique TEP ? Bio Mol ?

Auto-SCT: EBMT standard indications AllogeneicAutologous Sibling well-matchedmm unrelated Disease Disease status donor unrelated >1 ag mm related__________ Diffuse large B-cell lymphomaCR1 (intermediate/high IPI at dx) GNR/IIIGNR/III GNR/IIICO/I Chemosensitive relapse; ≥CR2 CO/IICO/II GNR/IIIS/I Refractory D/II D/II GNR/IIIGNR/II Mantle cell lymphomaCR1 D/IIID/IIIGNR/IIIS/II Chemosensitive relapse; ≥CR2 D/IID/IIGNR/IIIS/II Refractory D/IID/IIGNR/IIIGNR/II Lymphoblastic lymphomaCR1 CO/IICO/IIGNR/IIICO/II and Burkitt’s lymphoma Chemosensitive relapse; ≥CR2 CO/IICO/IIGNR/IIICO/II Refractory D/IIID/IIIGNR/IIIGNR/II Follicular B-cell NHLCR1 (intermediate/high IPI at dx) GNR/IIIGNR/IIIGNR/IIICO/I Chemosensitive relapse; ≥CR2 CO/IICO/IID/IIIS/I Refractory CO/IICO/IID/IIGNR/II T-cell NHLCR1 D/IID/II GNR/IIID/II Chemosensitive relapse; ≥CR2 CO/IICO/II GNR/IIID/II Refractory D/II D/II GNR/IIIGNR/II Hodgkin lymphoma CR1 GNR/IIIGNR/IIIGNR/IIIGNR/I Chemosensitive relapse; ≥CR2 CO/IICO/II CO/IIS/I Refractory D/IID/IIGNR/IICO/II Lymphocyte predominant nodular HLCR1 GNR/IIIGNR/IIIGNR/IIIGNR/III Chemosensitive relapse; ≥CR2 GNR/IIIGNR/III GNR/IIICO/III Refractory GNR/III GNR/III GNR/IIICO/III

EBMT Activity survey on HSCT in 2009: main indication - AUTOLOGOUS - Baldomero H et al. BMT 2011 Feb 28. [Epub]

Caractéristiques des greffons

Randomized trial of filgrastim-mobilized peripheral blood progenitor cell transportation versus autologous bone-marrow transplantation in lymphoma patients. N Schmitz, DC Linch, P Dreger, A H Goldstone, M A Boogaerts, A Ferrant, H M S Demuynck, H Link, A Zander, A Barge, K Borkett THE LANCET Lancet 1996; 347:

58 Pts* * Hodgkins or High grade NHL 27 G-CSF (10 µg/Kg/day) x 6 days 27 G-CSF (10 µg/Kg/day) x 6 days 31 Bone Marrow CHEMOTHERAPYCHEMOTHERAPY CHEMOTHERAPYCHEMOTHERAPY Harvest (Day 5-7) Harvest (Day 5-7) Reinfusion+G-CSFReinfusion+G-CSF 5 µg/Kg/day Schmitz et al. Lancet 1996; 347:

Results PBSC BMT p value Days to plt recovery Days to neutrophil recovery Plt Tf days 6 10 <0.001 N° RBC Tf Days in the hospital

Marrow vs. PBSCT

10 Déroulement 1- Chimiothérapies initiales 2- Mobilisation et collecte CSP 3- Conditionnement ( 3- Conditionnement (Effet dose-intensité (BEAM, Mel200)) 4 - Greffe = 4 - Greffe = Transfusion des CSP 5 - Reconstitution hématologique: 5 - Reconstitution hématologique: Aplasie 10 à 15 jours

PBSC Mobilization Regimens G-CSF only G-CSF + chemotherapy G-CSF side effects: –Headache: 75% –Bone pain: 63% –Swelling: %

How do we mobilize stem cells ? Growth factor + post chemo (Cy+ G-CSF) (Cy+ G-CSF) G-CSF CD-34 CY G-CSF CD Chemo +Growth factor Growth factor only

G-CSF Stimulation: How does it work ? Stem Cells G-CSF

G-CSF Stimulation: One Theory

VLA-4VLA-4 VCAMVCAM CD34 + Cell CD34 + Cell

Elastase CD34 + Cell CD34 + Cell G-CSF stimulates production of Neutrophils Neutrophils Release Elastase

Elastase CD34 + Cell CD34 + Cell Elastase Digests VCAM molecule

VLA-4VLA-4 CD34 + Cell CD34 + Cell VCAMVCAM CD-34 Cells break free and circulate in PB

What is CD 34? From: www. beckmancoulter.com Probably an adhesion molecule. Present in early hematopoietic cell precursors Present in early hematopoietic cell precursors Present in 0.1% of peripheral mononuclear cells Present in 0.1% of peripheral mononuclear cells 1-4% human bone marrow cells 1-4% human bone marrow cells kDa transmembrane Glycoprotein kDa transmembrane Glycoprotein

When to collect ?

Journal of Hematotherapy 7:45-52 (1998) Mary Ann Liepert, Inc. Evaluation of Mobilized CD-34+ Cell Counts to Guide Timing And Yield of Large-Scale Collection by Leukopheresis LENE MELDGAARD KNUDSEN, EVA GAARSDAL, LINDA JENSEN KRISTEN NIKOLAISEN and HANS JOHNSEN 130 patients (10µg/kg/day) 38G-CSF (10µg/kg/day) 52G-CSF + HDCY (chemo) 12G-CSF + CEF (chemo) 22G-CSF + other chemo 3 None 3 None 3 No data 3 No data PBSC (10 L) began when PB CD-34+ Cells  20 x10 3 /ml

From Meldgard et al,;Journal of Hematotherapy 7:45-52 (1998) CD34+ Cells in Peripheral Blood and Product collected on Day 1 CD34+ Cells in Peripheral Blood on day before and Product collected on Day 1 R=0.62 CD34 x 10 6 /Kg CD34 x 10 3 /ml blood R=0.87 CD34 x 10 3 /ml blood CD34+ cells: Peripheral counts vs product collected

Correlation between WBC count and CD34+ cells harvested on day 1

How many CD 34+ cells to collect and for what ?

What is your preferred (target) number of CD34+ cells (x10 6 /kg) for a single auto-SCT at your center? NHLMyeloma PREDICT Investigator’s Meeting Amsterdam, 13 November 2008

CD34 Cells Number of cells correlates with engraftment Number or cells correlates with speed of engraftment  2 x 10 6 / Kg (ideal body weight) is considered “sufficient”  4 to 5 x 10 6 / Kg ( more acceptable dose for engraftment)  >5 x 10 6 / Kg ( gives more rapid engraftment and lower incidence of graft failure Further increases, decrease the time to platelet engraftment

How often are these numbers harvested ?

High variability in published lymphoma mobilisation failure rates (11-53%) AuthorCountrynDiseaseMobilization regimenFailure rate* Stiff et al.US 48NHL, HDSCF + G-CSF30% 54NHL, HDG-CSF46% Pusic et al.US467NHLG-CSF27% Flomenberg et al.US15NHLG-CSF53% Hosing et al.US149NHL, HD G-CSF alone G-CSF plus chemotherapy 20% Russell et al. EU + Australia 29NHLICE + pegfilgrastim (6mg)31% 29NHLICE + pegfilgrastim (12mg)41% 32NHLICE + filgrastim (5 mcg/kg/day)28% Watts et al.UK 78NHL, HDESHAP15% 78NHL, HD1.5g Cy29% Pavone et al.Italy 38NHLDHAP15% 34NHL5g Cy11% Bashey et al.US94NHL1.5g Cy18% * Proportion of patients collecting < 2 x 10 6 cells/kg Stiff et al. BMT 2000; 26: ; Pusic et al. Biol BMT 2008;14(9):1045–56; Flomenberg et al. Blood 2005; 106(5): ; Hosing C et al. An analysis of the costs associated with peripheral blood hematopoietic stem cell mobilization collection and cryopreservation in patients with lymphomas undergoing autologous HSCT; Russell et al. Haematologica 2008; 93(3): ; Watts et al. Br J Cancer (2): 278–82; Pavone et al. BMT 2002;29(4): 285–90; Bashey A et al. Transfus 2007;47(11): 2153–60

Variations in defining “mobilisation failure” Significant variation both in definition of “mobilization failure” and mobilization practice lead to large variations in reported failure rates –Patients with a peripheral blood (PB) CD34+ count below 10 cells/µl usually do not go to apheresis and are often not counted as failures –“Successful” mobilisation may include patients transplanted with pooled cells from prior mobilizations –Target cell numbers may be defined differently (e.g. optimal numbers vs. minimal, as well as numerical differences)

Differences in clinical practice affect failure rates –G-CSF doses and schedules –Doses and regimens of chemotherapy during chemo- mobilisation –Blood volumes processed –Maximal numbers of apheresis sessions allowed –Extent of disease at time of mobilisation –Hematology parameters used as surrogate markers to initiate apheresis (e.g. some centres use CD34+ cell count, some use WBC)

Number of mobilization attempts by histological categories %

Number of mobilization attempts by age (n=3972) %

Who will be “poor” or will fail mobilization? Pre-treatment Age Radiotherapy/Mel/ Nitrosureas, Fludarabine lenalidomide anti-CD20? Marrow involvement Disease Many issues unknown Failed Mobilizers Frontline with G-CSF + Chemotherapy or Replace Chemo Predicted Poor Mobilizers Slow Mobilizers Frontline with G-CSF Alone

Solutions for poor mobilisers?

Endoxan + G-CSF G-CSF + SCF Bone Marrow harvest G-CSF + Plerixafor

Plerixafor = Mozobil ® = AMD3100 First in class hematopoietic stem cell mobilisation agent Unlike G-CSF, Mozobil is not a growth factor Reversibly binds the CXCR4 receptor and blocks SDF-1 interaction

DiPersio, J. F. et al. J Clin Oncol; 27: Fig 1. Study treatment

DiPersio, J. F. et al. J Clin Oncol; 27: Fig 3. (A) Kaplan-Meier estimate of proportion of patients reaching >= 5 x 106 CD34+ cells/kg

Criteria for optimal mobilization regimen G-CSF alone G-CSF plus chemo. Higher probability of collecting optimal number of cells Manageable tolerability Predictable time to apheresis Fewer apheresis days Prompt and durable engraftment Minimal toxicity and inconvenience for the patient Practical, logistical, and resource benefits Plerixafor as part of an ideal stem cell mobilization regimen Plerixafor + G-CSF

Collecte de CSP par cytaphérèses Thrombopénies Hypocalcémies / Hypomagnésémies Hypotensions (très rares) Allergies Problèmes mécaniques de CEC Incidents de voie d’abord Hématomes Importance de l’accès veineux +++

Manipulation du greffon 1) Congélation: obligatoire (DMSO 10%) Protègent les membranes et évite la cristallisation Ralentissent les échanges d’eau Réduisent la concentration intracellulaire des électrolytes 2) Stockage en cuve azote surveillée 3) Décongélation du greffon Lavage du DMSO (sinon troubles rythme cardiaque, malaises,céphalées, épilepsie, HTA, nausées-vomissements) Prémédication lors de la réinfusion / surveillance

Concluding remarks G-CSF +/- Chimio most often efficient. Close monitoring of circulating CD34+ cells allows for precise time to harvest. 2x10e6 CD 34+ cells/kg injected is enough to achieve a good engraftment. Poor mobilisation cannot be completely predicted Use of Perixafor with G-CSF either systematically after a 1st failure or upon low PB CD 34+ cells count on scheduled apheresis day may overcome poor mobilisation in # 60% of the cases

Thank you