Antigen receptor gene rearrangements are applied for the PCRbased minimal residual disease (MRD) detection in acute lymphoblastic leukemia (ALL). It is known that ongoing rearrangements result in subclone formation, and that the relapsing subclone(s) can contain antigen receptor rearrangement(s) that differ from the rearrangements found in the major clone(s) at diagnosis. However, the mechanism leading to this so-called clonal evolution is not known, particularly at which time point in the disease the relapsing subclone obtains its (relative) therapy resistance. To obtain insight in clonal evolution, we followed the kinetics of several subclones in three oligoclonal ALL patients during induction therapy. Clone-specific nested PCR for immunoglobulin heavy chain or T cell receptor ␦ gene rearrangements were performed in limiting dilution assays on bone marrow samples taken at diagnosis, at the end of induction therapy and at possible relapse in three children with oligoclonal B-precursor ALL. We demonstrated that in all three patients the subclones were behaving differently in response to therapy. Moreover, in the two patients who relapsed, the clones that grew out during relapse showed the slowest regression or even evoluated during induction therapy and the clones that were not present at relapse showed good response to induction therapy. These results support the hypothesis that at least in some patients already at diagnosis or in the very first weeks, subclones have important differences in respect to resistance. Hence, these data give experimental evidence for the need to develop, during the first months after diagnosis, quantitative PCR assays for at least two different Ig/TCR gene rearrangement targets for every ALL patient. Leukemia (2001) 15, 134-140.

Reinfusion of ex vivo-expanded autologous megakaryocytes together with a stem cell transplantation may be useful to prevent or reduce the period of chemotherapy-induced thrombocytopenia. In this study, we analyzed several serum-containing and serum-free media to identify the most suitable medium for megakaryocyte expansion. Moreover, two thrombopoietin (Tpo)-mimetic peptides were tested to evaluate whether they could replace Tpo in an expansion protocol. To analyze the effects of different media on megakaryocyte expansion, we used an in vitro liquid culture system. For this purpose, CD34(+) cells were isolated from peripheral blood and cultured for 8 days in the presence of Tpo and interleukin-3 (IL-3). The presence of megakaryocytes was analyzed by flow cytometric analysis after staining for CD41 expression. For our standard culture procedure, megakaryocyte medium (MK medium) supplemented with 10% AB plasma was used. Addition of 5% or 2.5% AB plasma yielded higher numbers of megakaryocytes, implying the presence of inhibitory factors in plasma. However, some plasma components are required for optimal megakaryocyte expansion because addition of less than 1% AB plasma or addition of human serum albumin instead of AB plasma resulted in the formation of lower numbers of megakaryocytes. Two commercially available serum-free media were also tested: Cellgro and Stemspan. If CD34(+) cells were cultured in Cellgro medium similar numbers of megakaryocytes were obtained as when CD34(+) cells were cultured in MK medium supplemented with 10% AB plasma. In MK medium with 2.5% AB plasma, higher numbers of megakaryocytes were cultured than in MK medium supplemented with 10% AB plasma. Therefore, Cellgro medium is not the best alternative medium. In cultures with Stemspan medium, higher numbers of megakaryocytes were obtained compared to MK medium with 10% AB plasma. Stemspan is thus a good alternative for MK medium. Two Tpo-mimetic peptides, AF13948 and PK1M, were tested for their ability to replace Tpo. In cultures with AF13948, comparable numbers of megakaryocytes were obtained as in the presence of Tpo, but in cultures with PK1M the number of megakaryocytes was lower. This study shows that high concentrations of plasma in medium inhibits megakaryocyte formation, but some plasma components are required for optimal megakaryocyte expansion. For an ex vivo expansion protocol, it is worthwhile to test several media, because the number of megakaryocytes differs widely with the medium used.