Stem Cell Mobilization Standards of Care for HSCT Novel Applications Richard Champlin, M.D.

Why is the bone marrow in the bone marrow?  Hematopoietic stem cells home to a “niche” in the marrow  Marrow microenvironment provides critical interactions controlling the growth and differentiation of hematopoietic cells  Maturing cells naturally egress the bone marrow into the blood and later into the tissues  Stem cells traffic into and out of the bone marrow physiologically

HSC Stem Cell Niche VLA-4 VCAM-1 Bone Marrow Stromal Microenvironment CXCR4 Osteoblasts SDF1 Pamela S, et al. ASH Abstract #: 858; Shivtiel et al. J Exp Med. 2008;205:2381.

Mechanisms Governing Stem Cell Mobilization with G-CSF Adhesive interactions between HSC and matrix components in the BM G-CSF Mobilization Cathepsin G (CG), chemokine receptor-4 (CXCR4), hematopoieic stem cell (HSC), hyaluronic acid (HA), interleukin 8 (IL-8), kit ligand (KL), matrix metalloproteinase- 9 (MMP-9), neutrophil elastase (NE), stromal cell derived factor-1 (SDF-1), vascular cell adhesion molecule-1 (VCAM-1), very late antigen-4 (VLA-4), P-selectin glycoprotein ligand-1 (PSGL). Source: Nervi B, et al. J Cell Biochem. 2006;99:

Considerations for Mobilization Regimen Reliable Collect sufficient number of HSCs and progenitors Predictable Able to predict day of collection Low failure rate Limited toxicity Cost Effective Limited number of days of apheresis required Low resource utilization Low tumor contamination

HSC Agents for Stem Cell Mobilization VLA-4 VCAM-1 G-CSF SDF1 Bone Marrow Stromal Microenvironment Fibronectin CXCR4 Osteoblasts D Plerixafor SDF1 MM9 G-CSF Scr kinase PP2 scr inhibitor Pamela S, et al. ASH Abstract #: 858; Shivtiel et al. J Exp Med. 2008;205:2381.

CD34 + Cells Are Heterogenous CD34 + CD34 + /CD38 - CD34 + /CD133 + CD34 + /CD133 - CD34 + /HLA-DR + CD34 + /HLA-DR - CD34 + /CD61 - CD34 + /CD61 + CD34 + /CD38 - = Most Primitive Stem Cells Hock H. J Exp Med. 2010;207:

When to Collect? Armitage S, et al. Bone Marrow Transplant. 1997;20: Correlation between PB CD34+ cells/µL and CD34+ cells/kg collection

How Many HSCT do you need for AutoSCT? Richard Champlin, MD

Platelet Engraftment Kinetics As A Function Of CD34 + Cell Dose Glaspy JA, et al. Blood. 1997;90(8): N = 212

Importance of CD34 + Cell Dose Stiff PJ, et al. Blood. 2008;112: Abstract Percent Patient Platelet Count > 150,000/  L

Defining a Target?  Generally accepted that ≥ 2 x 10 6 CD34 + cells/kg is ensures a threshold effect for a rapid hematopoietic engraftment 1-2  95% of patients receiving > 2.5 x 10 6 CD34 + cells/kg experience durable neutrophil engraftment by day 18  5 x 10 6 /kg may be threshold for rapid platelet engraftment 3-4  Unclear if > 5 x 10 6 /kg will result in any better engraftment, may be associated with improved outcome 5 1 To LB, et al. Blood. 1997;89: ; 2 Schiller G, et al. Blood. 1995;86:390-7; 3 Kiss JE, et al. Bone Marrow Transplant. 1997;19:303–10; 4 Weaver CH, et al. Blood 1995;86:3961–9; 5 Dercksen MW, et al. J Clin Oncol. 1995;13:1922–32.

Higher Cell Dose: Impact on Cost  Costs of transplant-related care in patients who experience “good” versus “poor” mobilization  Retrospective analysis of 172 NHL patients treated with HDT and autologous PBSC transplantation  Mobilizations categorized as “poor” (<2 ×10 6 CD34+ cells/kg) or “good” (≥2 ×10 6 CD34+ cells/kg)  Cyclophosphamide + G-CSF used for mobilization  Cost data in a subset of patients (n=57) Stockerl-Goldstein KE, et al. Biol Blood Marrow Transplant. 2000;6(5): a Includes cost of apheresis and bone marrow harvest, if performed.

How to collect HSCT  Chemo-Mobilization  Integrates collection into disease management  Improves CD34 yield  Cost, complications, can’t predict date of collection  We use this for aggressive lymphoma  Growth Factor Mobilization  Simple  Efficient, can schedule  Provides adequate CD34 yield in many categories of patients  Less Costly, few complications, can predict date of collection  May interrupt/delay chemotherapy  We use this for myeloma

Factors Affecting CD34 + Cell Yield Patient-related  Age  Mobilization regimen chosen  Generally higher CD34 yields with chemo-based mobilization  Amount and type of prior therapy  Alkylators, lenalidomide, radiation  Platelet count at the time of mobilization

G-CSF vs Chemotherapy + G-CSF Chemo = various chemotherapeutic agents; Cy = cyclophosphamide; HD = Hodgkin's disease; MM = multiple myeloma; NHL = non-Hodgkin's lymphoma; NR = not reported. 1 Alegre A, et al. Bone Marrow Transplant. 1997;20:211–217; 2 Desikan KR, et al. J Clin Oncol. 1998;16:1547–1553; 3 Dazzi C, et al. Leuk Lymphoma. 2000;39:301–310; 4 Narayanasami U, et al. Blood. 2001;98:2059–2064; 5 Pusic I, et al. Biol Blood Marrow Transplant 2008;14:1045–1056.

Rituximab and HSC Mobilization- MDACC Experience  Rituximab reduces circulating lymphoma cells  No impact on HSC mobilization, particularly with chemomobilization  Improvement in results of autoSCT using Rituximab in mobilization and transplant.

ASCT for Aggressive NHL Impact of Rituximab on DFS Months Post Transplant Cumulative Proportion Surviving Rituximab (N=67) No Rituximab (N=30) p = 0.004

Adhesion Molecules And HSC Mobilization Natalizumab BIO5192 Plerixafor Mobilization Nervi B, et al. J Cell Biochem. 2006;99:

20 Plerixafor  A sustained elevation of peripheral blood CD34+ cell levels was noted between 4 and 18 hours 1 G-CSF, granulocyte-colony stimulating factor. 1. Mozobil ™ [prescribing information]. Cambridge, MA: Genzyme Corp; Adapted from Liles WC, et al. Transfusion. 2005; 45: n = 3 healthy volunteers Time (hours) calculated after 4 days of G-CSF therapy and addition of G- CSF + Mozobil ™ (plerixafor injection) on day 5

Mobilization Using G-CSF With Plerixafor  Efficacy as single agent  Synergistic with G-CSF  Increases likelihood of successful CD34+ cell mobilization  QUESTIONS:  Are there important functional differences in the grafts collected?  Are the improvements in CD34 yield worth the added cost?  Effect on mobilization of malignant cells?

Gene Expression of Mobilized CD34 + Cells and Leukocytes Donahue RE, et al. Blood. 2009;114: Gene expression differs among CD34 cells mobilized plerixafor, G-CSF, and plerixafor + G-CSF 1.Composition of mobilized CD34 + cells is dependent on the mobilization protocol 2.Composition of CD34 + cells mobilized by the combination is not simply a mixture of cells mobilized by each agent separately

Functional Differences Noted With Plerixafor vs G-CSF-Mobilized HSPCs  Higher proportion of cells in G1 phase of the cell cycle  Higher proportion of more ‘primitive’ CD34 + CD38 - cells  More cells expressing CXCR4 and VLA-4 on the cell surface  Grafts contain more T, B and NK cells Larochelle A, et al. Blood. 2006; 107:3772–3778; Hess DA, et al. Biol Blood Marrow Transplant. 2007; 13:398–411; Fruehauf S, et al. Cytotherapy. 2009; 11:992–1001; Donahue RE, et al. Blood. 2009; 114:2530–2541;

Plerixafor vs G-CSF-Based Stem Cell Mobilization in HLA-Identical Donors: Allograft Composition *Mean. † Median. ‡ Includes 8 donors mobilized by both plerixafor and G-CSF. Devine S, et al. Blood. 2008;112(4):

Cancer Cell Mobilization in Autologous Donors??? CXCR4 / SDF-1 ? Release of Tumor Cells CXCR4 Antagonist Gazitt Y. Leukemia. 2004;18;1-10.

Apheresis Costs Treatment PhaseCost Pre-apheresisClinic visit Lab evaluation Insertion of CVC Chest –x-ray $1,800 ApheresisApheresis procedure (2) G-CSF treatment (2) CD34+ analysis $5,161 ($2,580.7 per day) Post-apheresisCryopreservation Supplies Storage Sterility testing $2,493 ($1,246 per bag) Total Cost*$9,454 Cost Savings of Eliminating One Day of Apheresis*,# $3,826 Hosing et al

MDACC Policy PBPC Collection  For autos and allos- goal 5 million, accept minimum 2 million CD34/kg,  Day 1 or 2 stop >4M  Day 3 stop >3M  Day >4 stop >2M  Collect If CD34 > 10/mcl  If collection is ≤ 0.3 million/kg/d x 2 consecutive days despite use of plerixafor or stop apheresis

Myeloma- plan for 2 transplants  Target doses: Goal 6-8 million/kg for 2 transplants (minimum acceptable 4 million/kg)  If after 1 or 2 collections CD34 collected is > 8 million/kg stop  If after 3 collections CD34 collected is > 6 million/kg stop  If after 4 collections CD34 collected > 4 million/kg stop  If after 5 collections CD34 collected > 2 million/kg stop, do one transplant

Just In Time Strategy for Cost Effectiveness  G-CSF alone successfully mobilizes many patients  Plerixafor is synergistic with G-CSF for stem cell mobilization  An approach to improving cost effectiveness is reserving plerixafor for patients with suboptimal mobilization  Use circulating CD34 on day 4 or first day’s collection to determine who needs addition of plerixafor.

Factors Associated With Poor Mobilization  Increasing cycles / duration of prior chemotherapy  Female gender  Prior radiation to bone marrow  Low pre-mobilization platelet count  Bone marrow positivity  Indolent lymphoma histology  Exposure to fludarabine, platinum-based chemotherapy, alkylating agents, lenalidomide  Low PB CD34 count during mobilization

Outcome of Mobilization by Disease 27% 33%14% Gertz M, et al. Bone Marrow Transplant Jan 11. epub.

Percent ≥ 2 Million CD34+ Cells/kg Phase III NHL Study Plerixafor +G-CSF Placebo + G-CSF HR=2.50, 95%CI (1.86, 3.36), p< Kaplan-Meier estimate of proportion of patients reaching ≥ 2 x 10 6 CD34+ cells/kg DiPersio JF, et al. J Clin Oncol. 2009;27:

Conclusions  Hematopoietic stem and progenitor cells are mobilized by G-CSF and plerixafor a CXCR4 inhibitor  Plerixafor mobilizes PBPC by inhibition of SDF-1 and CXCR4 interaction  Plerixafor and G-CSF are synergistic  The combination of Plerixafor and G-CSF will reduce the number of aphereses required for PBPC collection and enhance to ability to perform autologous HSCT in “hard to mobilize” patients  Chemotherapy plus growth factor enhances mobilization and is warranted when the chemotherapy is indicated for treatment of the malignancy