Transplantation of ex vivo expanded cord blood cells using the copper chelator tetraethylenepentamine: a phase I/II clinical trial.

The copper chelator tetraethylenepentamine (TEPA; StemEx) was shown to attenuate the differentiation of ex vivo cultured hematopoietic cells resulting in preferential expansion of early progenitors. A phase I/II trial was performed to test the feasibility and safety of transplantation of CD133+ cord blood (CB) hematopoietic progenitors cultured in media containing stem cell factor, FLT-3 ligand, interleukin-6, thrombopoietin and TEPA. Ten patients with advanced hematological malignancies were transplanted with a CB unit originally frozen in two fractions. The smaller fraction was cultured ex vivo for 21 days and transplanted 24 h after infusion of the larger unmanipulated fraction. All but two units contained <2 x 10(7) total nucleated cells (TNCs) per kilogram pre-expansion. All donor-recipient pairs were mismatched for one or two HLA loci. Nine patients were beyond first remission; median age and weight were 21 years and 68.5 kg. The average TNCs fold expansion was 219 (range, 2-620). Mean increase of CD34+ cell count was 6 (over the CD34+ cell content in the entire unit). Despite the low TNCs per kilogram infused (median=1.8 x 10(7)/kg), nine patients engrafted. Median time to neutrophil and platelet engraftment was 30 (range, 16-46) and 48 (range, 35-105) days. There were no cases of grades 3-4 acute graft-versus-host disease (GVHD) and 100-day survival was 90%. This strategy is feasible.

Microbial contamination of hematopoietic progenitor cell products: clinical outcome.

We reviewed the results of routine microbiological assays of 3078 infused hematopoietic progenitor cell (HPC) products for autologous and allogeneic transplantation between January 2001 and December 2005. Thirty-seven (1.2%) contaminated products were found. All patients receiving contaminated infusions received empirical antibiotic prophylaxis according to the assay result. None of these patients developed a positive blood culture with the same agent, developed infections that could be attributable to the contaminated product or experienced any clinical sequelae. Coagulase-negative Staphylococcus was found in 32 (86.5%) products. Admission lengths and time to engraftment were within the expected time frame for autologous and allogeneic transplants. Microbial contamination of HPC products occurs at a low frequency; prophylactic use of antibiotics based on the microbiological assay appears to be effective in preventing clinical complications.

Ex vivo expansion of umbilical cord blood.

The efficacy of cord blood (CB) transplantation is limited by the low cell dose available. Low cell doses at transplant are correlated with delayed engraftment, prolonged neutropenia and thrombocytopenia and elevated risk of graft failure. To potentially improve the efficacy of CB transplantation, approaches have been taken to increase the cell dose available. One approach is the transplantation of multiple cord units, another the use of ex vivo expansion. Evidence for a functional and phenotypic heterogeneity exists within the HSC population and one concern associated with ex vivo expansion is that the expansion of lower 'quality' hematopoietic progenitor cells (HPC) occurs at the expense of higher 'quality' HPC, thereby impacting the reserve of the graft. There is evidence that this is a valid concern while other evidence suggests that higher quality HPC are preserved and not exhausted. Currently, ex vivo expansion processes include: (1) liquid expansion: CD34+ or CD133+ cells are selected and cultured in medium containing factors targeting the proliferation and self-renewal of primitive hematopoietic progenitors; (2) co-culture expansion: unmanipulated CB cells are cultured with stromal components of the hematopoietic microenvironment, specifically mesenchymal stem cells (MSC), in medium containing growth factors; and (3) continuous perfusion: CB HPC are cultured with growth factors in 'bioreactors' rather than in static cultures. These approaches are discussed. Ultimately, the goal of ex vivo expansion is to increase the available dose of the CB cells responsible for successful engraftment, thereby reducing the time to engraftment and reducing the risk of graft failure.

Nucleotide excision repair genes as determinants of cellular sensitivity to cyclophosphamide analogs.

UNLABELLED: The objective of this study was to determine the relative importance of the first six complementation groups of the nucleotide excision repair cross-complementing genes (ERCC1-ERCC6) and the first complementation group of the X-ray repair cross-complementing genes (XRCC1), in the repair of DNA damage induced by the in vitro active cyclophosphamide (CP) derivatives 4-hydroperoxycyclophosphamide (4HC) and phosphorodiamidic mustard (PM). We compared the sensitivity of the wild-type CHO cell line, AA8, with that of the CHO mutant cell lines UV4 and UV20 (ERCC1-), UV5 (ERCC2-), UV24 (ERCC3-), UV41 (ERCC4-), UV135 (ERCC5-), UV61 (ERCC6-), and EM9 (XRCC1-). Cell survival was determined using both growth inhibition and conventional clonogenic assays. The yield of DNA crosslinks in selected cell lines was determined using an ethidium bromide fluorescence assay. RESULTS: The rank ordering of sensitivity to both 4HC and PM, based on the combined survival data, was UV41/UV4/UV20 > > UV61/UV24/UV135/EM9 > or = UV5 approximately AA8. Thus mutations in the ERCC1 and ERCC4 genes impart a hypersensitivity to CP analogs. To confirm the importance of the ERCC1 gene for cellular resistance to 4HC and PM, UV20 cells were transfected with the human ERCC1 gene and subsequently exposed to 4HC and PM. The transfected cells displayed essentially wild-type resistance to both drugs. Furthermore, two interspecific hybrids derived from UV41, both of which retained the region of human chromosome 16 that harbors the ERCC4 gene, displayed essentially wild-type resistance to 4HC and PM, confirming the importance of ERCC4 for the repair of 4HC-induced DNA damage. When crosslinks were assayed after a 60-min treatment with 4HC or a 15-min treatment with PM, their yield paralleled the sensitivity of the cell lines to both drugs: UV41 cells showed markedly elevated levels of crosslinks, whereas AA8 and UV5 cells showed similar (low) levels of crosslinks. CONCLUSIONS: Our findings confirm the general pattern indicating that the ERCC1 and ERCC4 gene products are crucial for the repair of 4HC-induced DNA damage, while the other nucleotide excision repair genes examined are relatively unimportant. These data suggest that the hypersensitivity of ERCC1- and ERCC4- mutants to DNA crosslinking agents may reflect a defect in recombinational repair rather than nucleotide excision repair.

Liposome encapsulation circumvents the hepatic clearance mechanisms of all-trans-retinoic acid.

All-trans-retinoic acid (ATRA) has been proven active against a range of malignancies in isolated tissue culture systems and in human clinical trials, but the duration of its effects has been transient. Recent evidence indicates that the basis for the limited duration of ATRA's activity, at least in one form of leukemia, is a pharmacological adaptation that results in reduced serum concentration after prolonged treatment. This finding suggests that an i.v. formulation of ATRA may significantly improve the potency and duration of ATRA's activity in leukemia and, potentially, other malignancies as well. Liposomal ATRA (L-ATRA) was developed to provide a formulation of this retinoic acid isomer that can be administered intravenously to provide potential pharmacological advantages over the oral formulation. When L-ATRA was administered to rats over a prolonged period, the blood levels of the drug did not change over time. In vitro studies of isolated liver microsomes revealed that catabolism of the drug was not altered in rats that were repeatedly administered the L-ATRA formulation. Whereas microsomes isolated from animals that were orally administered free ATRA the same number of times with the same doses showed a significant increase in metabolism of the drug. These results suggest that an i.v. formulation of ATRA such as L-ATRA could be extremely useful in inducing long-term remissions in patients with APL.