Ex Vivo Expansion Reduces Neutropenia Post Autologous Transplantation of Peripheral Blood Hematopoïetic Stem Cells in Patients with Follicular Lymphoma. A Randomized, Double Blind Trial,

Abstract 4009

The ex vivo culture of Hematopoietic Stem Cells (HSC) with various combinations of cytokines can increase the number of mature hematopoietic cells that are theoretically capable of rapidly release neutrophils and platelet and reduce recovery duration post transplantation. In patients, the infusion of such cells has been reported, but the short-term effect was not clear.

In a randomized, double blind study, we used expanded cells from 4×10⁶/kg peripheral blood hematopoietic selected CD34⁺cells in comparison to a non manipulated graft containing the same number of CD34⁺ cells; we designed an ex vivo expansion protocol based on a cocktail of early or late acting cytokines with different culture duration in order to obtain progenitors at various stages of differentiation 1) primitive progenitors obtained from selected CD34⁺cells cultured for 8 days in presence of fetal liver tyrosine kinase 3 ligand (FLT3-ligand), stem cell factor (SCF), interleukin-3 (IL-3) and thrombopoietin (TPO), 50 ng/ml, each 2) committed megakaryocyte progenitors (Mks) obtained after culture for 10 days in presence of TPO et SCF (50 ng/ml, each) or 3) committed granulocytes and megakaryocyte (GMks) progenitors obtained after culture for 10 days in presence of TPO, SCF and G-CSF (100 ng/ml).

Eighteen Non Hodgkin Lymphoma patients submitted to autologous transplantation after a myeloablative regimen consisting of AraC: Day (D)-6 to D-3: 200mg/m2/12h - VP16: 100mg/m2/12h: D-6 to D-3 - BCNU: 300mg/M2: D-6- Melphalan: 140mg/M2: D-2 could be evaluated. Patients in the Expansion Group received graft with 1×10⁶/kg non manipulated cells combined with primitive progenitors issues from 2×10⁶/kg CD34⁺ cells and Mks progenitors (Mks Expansion Group) or GMks progenitors (GMks Expansion Group) coming from 1×10⁶/kg CD34⁺cells; unmanipulated cells were used as a source of immunocompetent cells.

In the mean, patients of control group (n=10) received 1.3±0.9×10⁸Total Nucleated Cells (TNC)/Kg, 2.7±1.2×10⁶ CD34⁺/kg, 49±17×10⁴/kg CFU-GM and 17.7±.710⁴CFU-Mks/kg; in the Mks (n=4) and GMks (n=4) Expansion group, they received respectively in the mean: 61.5±18.5 and 90.1±21.3×10⁸TNC /Kg, 26.5±10.7 et 31± 11.2 x10⁶CD34⁺/kg, 451± 188 et 557±216×10⁴/kg CFU-GM, 358±212 et 39±18.1 x10⁴/kg CFU-Mk. No cytokines were administered after transplantation. No toxicity was observed after cell infusion. The mean times to reach white blood cell (WBC) recovery (WBC >1x 10⁹/l) was significantly shorter after administration of expanded cells, 14 (10–16), 12 (11–14) and 9 (9–10) days respectively in control, Mks and GMks Expansion Group (p=0.01). Median profound neutropenia (neutrophils<0,5×10⁹/l) duration was 4 (4–5) days in the GMk Expansion Group versus 9 (7–11) in the Mks Expansion Group and 9 (6–14) in the control group (p<0.05); no patient demonstrated abrogation of neutropenia. In contrast, ex vivo expansion did not reduce the time to platelet recovery (>25 or50×10⁹/L) despite numerous mature megakaryocytes and CFU-Mks in the Mks Expansion Group; that could be related to the colonies size, very small from expanded cells. No secondary hypoplasia was observed during the 12 months follow-up.

This study shows that in comparison with unmanipulated cells, peripheral blood haematopoietic cells expanded from similar doses of CD34⁺ cells accelerate neutrophil recovery without impairing long-term haematopoiesis and open interesting perspectives in the field of allogeneic cord blood cell transplantation.