No abstract

Our study indicates that DCC of 30 to 60 seconds has a small negative impact on collection of high-TNC-count CBUs. However, increasing BTC to more than 60 seconds decreases significantly both TNC content and volume, reducing drastically the chances of obtaining clinically useful CBUs.

Adoptive immunotherapy has shown efficacy in patients with relapsed/refractory acute myelogenous leukemia (AML). We conducted a prospective evaluation of cord blood (CB)-based adoptive cell therapy following salvage chemotherapy in patients with AML or myelodysplastic syndrome (MDS) and describe the safety and early outcomes of this approach. To enhance the antileukemic effect, we selected CB units (CBUs) with a shared inherited paternal antigen (IPA) and/or noninherited maternal antigen (NIMA) match with the recipients. Furthermore, the CBUs had total nucleated cell (TNC) dose <2.5 £ 10 7 /kg and were at least 4/6 HLA-matched with the patients; a higher allele-level match was preferred. Heavily pretreated adult patients with AML/MDS were enrolled. CBU searches were performed for 50 patients. CBUs with shared IPA targets were identified for all, and CBUs with NIMA matches were found for 80%. Twenty-one patients underwent treatment (AML, primary induction failure, n = 8; refractory relapse, n = 10, including 7 recipients of previous allogeneic HSCT; blast crisis chronic myelogenous leukemia, n = 1; MDS, n = 2). Most received combination chemotherapy; those not fit for intensive treatment received a hypomethylating agent. Response was defined as <10% residual blasts in hypocellular bone marrow at approximately 2 weeks after treatment. Ten of the 19 evaluable patients responded, including 5 of the 7 recipients of previous transplant. Response was seen in 4 of 4 patients with full CBU-derived chimerism, 2 of 2 of those with partial, low-level chimerism and 4 of 12 of the recipients with no detectable CBU chimerism. The most common adverse events were infections (bacterial, n = 5; viral, n = 2; fungal, n = 5). Grade IV acute graft-versus-host disease (GVHD) developed in 2 patients with full CBU chimerism; 2 other patients had grade 1 skin GVHD. A total of 11 patients died, 7 from disease recurrence and 4 from infections (1 early death; the other 3 in remission at the time of death). Overall, 12 patients proceeded to allogeneic HSCT; of those, 7 had responded to treatment, 3 had not (and had received additional therapy), and 2 had persistent minimal residual disease. In conclusion, the use of CB as adoptive immunotherapy in combination with salvage chemotherapy for patients with refractory AML/MDS is feasible, can induce disease control, can serve as a bridge to allogeneic HSCT, and has an acceptable incidence of adverse events. Alloreactivity was enhanced through the selection of CBUs targeting a shared IPA and/or NIMA match with the patients. CBUs with lower cell doses, already available in the CB bank and unlikely to be adequate grafts for adult transplants, can be used for cell therapy within a short time frame.

No abstract

485 CFU content of a cord blood (CB) graft is a potency indicator that evaluates the number, viability, functionality and repopulation capability of its hematopoietic progenitor cells (HPC). However, the CFU assay has not yet been amenable to standardization necessary for potency comparisons between CB units from different CB banks. As a result, this key graft attribute cannot be used widely in CB unit selection. One of the critical steps of the CFU assay is CFU counting, which, together with dish plating, contributes to the 14–36% of variability in CFU counts, as evaluated in the NCBP laboratory in a set of 64 CB samples that were plated and 240 CFU dishes independently red and counted by three operators. Our goal is to establish a method to determine HPC potency able to replace the manual CFU counting, that can be standardized, proving accurate high-throughput performance. Our technology includes traditional, manual CFU assay and High Resolution Digital Imaging of stained colonies (CFU-HRDI) [work in progress presented at ASH 2008 and 2009; abstracts 2306 and 2160]. After 14 days of CB culture in Stem Cell Technologies media, culture dishes contents were stained with MTT (3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide). A high resolution image of each dish was captured which allowed a clear view of HPC colonies in a color different for CFU-M/GM than for CFU-E. The image also included the dish's barcoded ID. CFU manual counting on the HRDI images showed good correlation with microscope counts (R2 = 0.95; p<0.01; n=151). To improve consistency, eliminate the variability of manual CFU counting and allow high-throughput performance that can be applied widely with acceptable ruggedness, we developed and validated a system that performs automated counting of CFU [ACC] imaged with HRDI. Algorithms were designed to count objects within a specific region of the dish, defined as a “CFU” based on size, optical density and separateness from other objects and also, to read a barcode ID label on the culture dish. The ACC required 20 seconds per image to complete the capture and counting of CFU. The system produces a batch report with CFU counts/dish including the CB unit ID, date and other desired data such as technologist ID, time or lot numbers. A total of N= 12,263 CB samples were tested in duplicate CFU assays. CFU were counted manually and using HRDI-ACC over a period of 24 months. Thus, N= 24,486 CFU culture dishes were evaluated. Assays and manual counting were performed by 8 operators. ACC and manual CFU counts correlated well (R2 = 0.85, slope 1.14, intercept 2.0). An average of n=490 CB samples were evaluated per month. Monthly correlations between manual CFU counts and ACC were consistent (range R2 =0.81–0.89; SD=0.024). In addition, CFU counts by operator and ACC correlated, with R2= between 0.8–0.9 (mean=0.84 and slope 1.11; range 0.92–1.21). ACC was highly reproducible: CFU counts of the same dish were within 1–2%, with n=50 culture dishes each read 10 times. To evaluate the inter-laboratory reproducibility of results and their stability despite operational and environmental variables the ruggedness of the CFU-HRDI-ACC system was evaluated by the NCBP and MDA teams in their respective laboratories. Aliquots of the same unprocessed CB units (n=173) were tested for CFU in duplicate and in parallel by two laboratories following a jointly-developed protocol. Automated CFU counts (ACC) per culture dish were comparable between laboratories. The CB units evaluated had CFU counts in the range of 3 to 93 and 4 to 88 CFU/culture dish in each lab respectively with a highly significant linear correlation (R2 = 0.62, slope 0.8, intercept 6). Manual CFU counts of the same culture dishes using HRDI images were also comparable between both labs (R2=0.67, slope 0.8, intercept 3). Conclusion: CFU is a potency assay that should evaluate the potency of the HPC in a CB graft more completely than the TNC or CD34 counts. The introduction of automated counting (ACC) with the CFU-HRDI system improves the reliability, reproducibility and ruggedness by eliminating the variability of manual CFU counting with contrast phase microscopy and importantly, supports high-throughput implementation and standardization as shown by the consistency achieved in different laboratories. Thus, the CFU content can be used for clinical CB unit selection. Although not described, CFU assessments can be performed before and after cryopreservation and thaw. Disclosures: No relevant conflicts of interest to declare.