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Methods to analyze the clonality of an adverse event in preclinical or clinical retroviral stem cell gene therapy protocols are needed. We analyzed the progeny of retrovirally transduced human peripheral blood progenitor cells (PBPCs) after transplantation and engraftment in immune-deficient mice. The integration site of the provirus serves as a unique tag of the individual transduced PBPC. A plasmid library of junctions between proviral and human genomic DNA was generated. We were able to detect individual transduced cell clones that amounted to 0.14%-0.0001% of chimeric bone marrow cells. This is the first report in which the contribution of individual marrow-repopulating cells to human hematopoiesis is directly quantified.

It would be a clinical and economical advantage if the optimal time point of peripheral blood stem cell (PBSC) mobilization following G-CSF-supported chemotherapy (CT) was known in advance. Therefore, we retrospectively analyzed mobilization parameters in 113 adult tumor patients treated in our institution within 1 year. The start of apheresis was guided by CD34(+) cell measurements in the PB and occurred on or after day 11 after start of mobilization CT in 97% of patients. The median peak (p)CD34(+) cell count in PB uniformly occurred on day 14-15 (range: 6-32 days) after the start of CT, irrespective of the diagnosis (multiple myeloma n = 76, other histology n = 37), the type, but not the amount, of premobilization CT or radiotherapy (RT), the mobilization regimen, or the G-CSF dosage administered. Among more heavily pretreated patients (>six cycles of prior CT or RT), a higher proportion mobilized late (pCD34(+) cell count later than day 20 in 12% and 13%, respectively, versus 2%-5% in the other groups). Therefore, we propose to start measuring CD34(+) cells in the PB on day 11 after the start of mobilization therapy. The wide range of optimal mobilization time points argues for an individualized rather than a preset start of apheresis.