Poster Board III-282
CB transplantation (CBT) is often complicated by delayed or failed engraftment. We conducted a study of ex vivo co-culture of CB mononuclear cells with either third party family member ≥ 2/6 HLA matched marrow derived MSCs (N=8) or off-the-shelf mesenchymal progenitor cells (MPCs) from Angioblast Systems Ltd. (N=9). MSCs provide a microenvironment for the generation of complex molecular cues that direct proliferation and regulate the differentiation and maturation of hematopoietic progeny. Patients had two CB units matched in at least 4/6 HLA antigens, with a minimum of 1×107 TNC/Kg per unit.
Diagnoses were AML/MDS (N=10), ALL (N=3), NHL (n=1), MM (n=1), and CLL (N=2). Five patients (29%) were in CR (CR1, n=1) and 12 (71%) had active disease at CBT. Preparative regimen: myeloablative fludarabine, melphalan, thiotepa and ATG (n=17), with rituximab in the 3 ALL pts. GVHD prophylaxis: tacrolimus and MMF. Median weight was 79.7 Kg (range, 15-102) and median age was 36 years (2-55 years). Donor-recipient HLA matching was 5 of 6 in 29% of the cases and 4 of 6 in 71%.
Ex-vivo EXP: 100 ml of marrow was aspirated from the family donor and MSCs generated in ten T175 flasks, which took ∼21 days (n=8) or one vial of Angioblast MPCs was thawed and expanded to confluence in 10 flasks within 4 days (n=9). The CB unit with the lowest TNC dose was then thawed, divided into 10 fractions, and each placed into 1 flask containing the confluent layers of MSCs/MPCs in expansion media with SCF, FLT3, G-CSF and TPO. After 7 days at 37°C, the non-adherent cells were removed from each flask, placed into each of ten one-liter Teflon-coated culture bags (American Fluoroseal) and cultured for an additional 7 days (14 days total), while 50 ml of media/growth factors was added to the flasks to culture the remaining adherent layer during that time period. On day 14 the cells from the bags and the flasks were combined, washed and infused along with a second unmanipulated CB unit.
The median number of infused total nucleated cell (TNC) and CD34+ cells per kg in unmanipulated CB was 2.3 × 107 (range, 1.9-8.2) and 1.6 × 105 (range, 0.3-1.26). There were no toxicities attributable to the EXP cells. The median TNC fold-EXP for recipients of family-MSCs was 11.7 (0.7-28) and for Angioblast-MPCs was 15.5 (3.1-22.5); Fold-EXP of CD34+ cells was 11.7 (1.7-50.1) for family-MSCs and 46.6 (3.85-71.9) for Angioblast-MPCs. The median number of infused TNC and CD34+ cells per kg after EXP for all pts was 5.8 × 107 (range, 0.1-1.43) and 6.05 × 105 (range, 0.18-30). Median time to neutrophil and platelet engraftment was 15 days (9-25 days) and 37.5 days (13-56). Sixteen (94%) and 14 (82%) of all patients engrafted neutrophils and platelets, respectively. One patient died before engraftment. All evaluable patients became complete donor(s) chimeras. One CB unit dominated in all patients; on transplant day +21, EXP unit contributed with a mean of 10% of T cell and 24% of myeloid chimerism; on day +40, corresponding proportions were 2% and 4%, and on day +70, 0% and 3%, respectively. Acute grade II-IV and III-IV GVHD rate was 38% and 12%, while 50% of at-risk patients developed chronic GVHD. Ten patients (59%) are alive (3-14 months after CBT), while 7 patients have died due to infections (n=4), relapse (n=2) and GVHD (n=1). Actuarial 6 and 12 month survival is 70% and 50%, respectively.
The decreased logistical demands and expansion results demonstrate that off-the-shelf Angioblast-MPCs are the preferred stroma for this CB EXP procedure. MSC/MPC-CB EXP is feasible and may provide rapid engraftment of neutrophils and platelets. Platelet engraftment occurred in a high proportion of patients (80%) in this cohort of high-risk patients with a median weight of 80 Kg.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.