Tumor Cell Purging

All of these processing procedures either depend on negative selection (removal of the cell type that is unwanted) or positive selection (selection of stem/progenitor cells, leaving all other cells behind). CD34 selection is the primary positive selection technique available to stem cell labs. CD34 is a cellular antigen that is expressed on stem cells, as well as progenitor cells of all hematopoietic lineages. Automated processes that select the CD34+ cell population (Strauss et al. 1991) away from the 99% of PBSC that are irrelevant for engraftment are available, and one of these technologies, the Isolex 300i device, is FDA approved. An alternative device, the Miltenyi Clini-MACS device (Schumm et al. 1999), is approved in Europe and may soon become available in the United States. In general, CD34 selection will result in a product that is 60-95 % CD34+, removing more than 99% of T cells (Beelen et al. 2000) and tumor cells (Donovan et al. 2000; Klein et al. 2001; Mohr et al.

2001), providing that the tumor cells do not express the CD34 antigen. Hematopoietic tumors, such as acute leukemias, often express CD34 and are therefore not depleted by CD34 selection. CD34 selection has been used to purge stem cell products in patients with NB,but concerns have been raised that some NB cells or cell lines may express CD34 or express surface epitopes that cross-react with monoclonal antibodies (MoAbs) that recognize CD34 (Hafer et al. 1999; Voigt et al. 1997). Our data have not confirmed expression of CD34 on NB (Donovan et al. 2000), and we and others have shown purging of NB cells from PBSC products in the clinical setting (Kanold et al. 2000). These data suggest that CD34 selection may be a purging alternative for PBSC products obtained from NB patients.

Negative selection procedures, by contrast, are tumor- or cell-type specific. For example, many techniques have been developed to negatively select T cells or T-cell subsets away from stem and progenitor cells in bone marrow and PBSC (see Ho and Soiffer 2002 for a recent review). These include: (a) a variety of monoclonal antibodies directed against T cells; (b) counterflow centrifugal elutriation (Wagner et al. 1990), which separates out lymphocytes based on physical characteristics; (c) sheep red blood cell rosetting (Reisner et al. 1981); and (d) immunomag-netic removal of T cells. Some of these procedures allow for more specific graft engineering by removing specific T-cell subsets such as CD8+ T cells or T cells expressing activation markers such as CD25 or CD69 (Fehse et al. 2000).

It is also possible to deplete tumor cells using specific anti-tumor monoclonal antibodies, relying on complement (Stein et al. 1988) plus cell-mediated cyto-toxicity (Cheung et al. 2002), or more often an im-munomagnetic depletion method (Reynolds et al. 1986). This approach has been proven to purge tumor cells from stem cell products collected from patients with B-cell lymphomas (Freedman et al. 1999). That tumor-contaminating stem cell products may contribute to relapse was shown in gene-marking studies in NB patients undergoing autologous bone marrow transplant. In these studies, a bone marrow aliquot was transfected with a marker gene and infused after transplant. Tumor cells at sites of relapse were found to con tain the marker gene, suggesting that clonogenic tumor had been infused with the graft (Rill et al. 1994). In follicular lymphoma, inability to detect tumor cells in the stem cell product after purging is associated with improved outcome after autologous transplant (Freed-man et al. 1999), but no study has shown that purging itself improves outcome. To study this question, the Children's Oncology Group (COG) is conducting a randomized comparison (COG A3973) of purged vs un-purged PBSC given in support of HDC/SCR in NB. Compared with bone marrow, PBSC from a patient receiving HDC/SCR for a malignancy are less likely to contain tumor cells (Ladetto et al. 2002; Moss et al. 1990; Faulkner et al. 1998) and have a lower content of tumor cells if any are present, and are therefore more likely to be purged successfully of tumor cells (Faulkner et al. 1998,2000; Ladetto et al. 2001).

0 0

Post a comment