A quantitative study of peripheral blood stem cell contamination in diffuse large-cell non-Hodgkin's lymphoma: one-half of patients significantly mobilize malignant cells.

Autologous transplantation using peripheral blood stem cells (PBSCs) collected after chemotherapy, followed by growth factor administration (ASCT), is increasingly used in the treatment of non-Hodgkin's lymphoma (NHL). However, quantitative data regarding contaminating malignant cells in the harvests are still scarce. We prospectively investigated 37 diffuse large-cell lymphomas (DLCLs) in complete remission (CR) that were treated according to multicentric protocols at our centre. DNA was extracted from the diagnostic lymph node. The complementarity-determining region (CDR) III was sequenced and a patient-specific oligomer synthesized. Contamination was evaluated semiquantitatively by polymerase chain reaction (PCR) and was confirmed by a limiting dilution analysis. PBSCs collected at regeneration after administration of granulocyte colony-stimulating factor (G-CSF), steady-state bone marrow (BM) and peripheral blood samples at CR were compared. DNA was available in 37 patients, from which 22 rearrangements could be sequenced. Patients (n = 15) who had both the required follow-up samples and a suitable clonal marker were investigated. In two cases, the patient-specific PCR assay set up at diagnosis later gave false-negative results in samples in which clonal DNA was still detectable by other sets of primers. PBSC contamination was highly variable: 7 out of 15 patients showed a PBSC/BM ratio of NHL cells greater than 1 log, whereas 8 out of 15 patients showed no difference and could vary from one apheresis to another. Eight ASCTs were performed, five of which used highly contaminated PBSCs: four patients relapsed early, three with disseminated lymphoma. Thus, 50% of DLCLs in CR seem to mobilize significantly malignant cells at regeneration under G-CSF. Considering the higher numbers of cells reinfused, this translates into a much higher number of lymphoma cells reinfused when compared with autologous bone marrow transplantation (ABMT). However, their clonogenic potential remains unknown and, despite concerning observations in certain cases, it is still unclear whether this has an impact upon the outcome of ASCT.

Peripheral blood stem cell contamination in mantle cell non-Hodgkin lymphoma: the case for purging?

Intensification using peripheral blood stem cells collected after chemotherapy followed by growth factors is being increasingly investigated as an alternative to conventional chemotherapy for mantle cell non-Hodgkin lymphoma. We investigated 14 grades III-IV, t(11;14)-positive cases for contamination of PBSC collected after a polychemotherapy regimen followed by G-CSF. Patients were first treated with a polychemotherapy regimen. There were four CR, seven PR, two refractory and one early death. Seven patients have been transplanted, in whom PBSC were mobilized, using either cyclophosphamide/VP16 or Dexa-BEAM followed by G-CSF. For all patients, whether actually autografted or not, PB cells were tested at the time of regeneration on G-CSF after the first polychemotherapy or after the mobilizing regimen. PCR evaluation of contamination was performed first by a semi-quantitative approach, using serial dilutions of initial DNA, then confirmed using a limiting-dilution analysis. Two patients were not informative (one early death and one without an available molecular marker). PB cells collected at regeneration contained at least one log more lymphoma cells than steady-state blood or marrow, apart from in two cases. Moreover, where a mobilizing treatment diminished tumor burden in the patient, at the same time it increased PB contamination in most cases. We conclude that advanced mantle cell NHL appears to be largely resistant to significant in vivo purging by conventional chemotherapy. Where treatment brings benefits by reducing tumor load, it may at the same time negate it by mobilizing malignant cells into the collections used to intensify. Although the clonogenic potential of this massive infiltration is unknown (only gene marking studies could provide a definitive answer regarding the source of relapses), strategies aimed at reducing the level of contamination in the graft should be considered when designing future protocols.