Bone marrow mammaglobin expression as a marker of graft-versus-tumor effect after reduced-intensity allografting for advanced breast cancer.

We assessed mammaglobin (MMG) gene expression in bone marrow (BM) aspirates from patients with advanced breast cancer who had received a reduced-intensity conditioning and stem cell allografting, in order to detect a graft-versus-tumor effect on micrometastatic disease. Nine patients received a reduced-intensity conditioning with fludarabine, cyclophosphamide, and thiotepa, followed by peripheral blood allografting from HLA-identical sibling donors. Nested RT-PCR analysis with sequence-specific primers for MMG was carried out on a monthly basis on BM samples. Three patients had MMG-positive BM, four patients had MMG-negative BM before allografting, and two were undetermined. In two patients, a clinical response after allografting (partial remission) occurred concurrently with the clearance of MMG expression, at a median of 6 months after allografting, following immune manipulation. In two patients, a prolonged stable disease and negative MMG expression occurred after day +360 from allografting. In two patients, progression of the disease was associated with MMG RT-PCR changing from negative to positive. In one case, a disease response occurring after donor lymphocyte infusion and grade II acute GVHD was heralded by negativization of MMG expression. Although preliminary, these data suggest that a graft-versus-breast cancer effect is detectable on micrometastatic BM disease.

Optimisation of retroviral supernatant production conditions for the genetic modification of human CD34+ cells.

BACKGROUND: Clinically applicable protocols for ex vivo modification of human CD34+ hematopoietic stem/progenitor cells rely on incubation of the target cell with supernatant containing recombinant retroviral particles. Although components of the supernatant may have a profound impact on both preclinical and clinical outcome, to date supernatant production has not been properly addressed with regard to CD34+ cells. We wanted to investigate and optimise production conditions for this target using simple, reproducible and clinically applicable procedures and reagents. METHODS: Retroviral supernatant was obtained from producer cell GP+Am12 under various production conditions and tested for bulk transduction efficiency and endpoint titre on murine and human cell lines. Gene transfer efficiency into CD34+ cells from mobilised peripheral blood, after a single exposure to retroviral supernatant, was measured by transgene expression, colony forming assay and long-term culture colony forming assay. RESULTS: Bulk gene transfer or endpoint titre values obtained on cell lines for the different production conditions were not predictive of gene transfer efficiency into hematopoietic progenitors. Time of virus production appeared to have the greatest impact on gene transfer, peaking at 6 h and decreasing 2-3-fold at longer time points. Neither the culture vessel used nor the temperature for virus production had any significant effect on gene transfer into CD34+ cells. Supernatant could be produced under defined serum-free conditions as efficiently as serum containing conditions for CD34+ cell gene transfer. CONCLUSIONS: The present data provide important implications for the establishment of quality controls for small- and large-scale clinical grade supernatant production for gene transfer into human hematopoietic stem/progenitor cells.