In vitro characterization of the hematopoietic system in pediatric patients with acute lymphoblastic leukemia

Abstract

Acute lymphoblastic leukemia (ALL) has been recognized as a hematologic neoplasia that originates at the level of a primitive lymphoid stem/progenitor cell. To date, however, the biology of the hematopoietic system in this disorder is still not fully understood. In the present study, we have determined the progenitor cell content (including myeloid, erythroid and multipotent progenitors) in 14 children with ALL and followed the proliferation kinetics of these cells in Dexter-type long-term marrow cultures. We have also characterized some aspects related to the composition and function of the hematopoietic microenvironment developed in vitro. All patients included in this study showed extremely reduced levels of progenitor cells (median of 6.2% of the levels found in normal marrow). Proliferation of these cells in long-term cultures was markedly deficient, since they showed very low numbers — compared to normal cultures — and reached undetectable levels after only a few weeks. Regarding the microenvironment developed in vitro, whereas normal marrow samples contained a median of 8 fibroblastic progenitors/10 5 marrow cells and the stromal cell layers developed in culture contained a median of 341 000 adherent cells per well, ALL marrow samples showed no fibroblastic progenitors and the numbers of adherent cells were 21% of those in normal cultures. Interestingly, the levels of TNFα and IL-6 in ALL culture supernatants were significantly increased, compared to normal cultures. Bone marrow samples from all 14 children were also analyzed once they reached a complete clinical and hematological remission. Myeloid, erythroid and multipotent progenitor cell levels were significantly increased, compared to patients at diagnosis, and proliferation of myeloid progenitors in long-term cultures was also improved. In contrast, proliferation of erythroid progenitors showed no difference to that in cultures from patients at diagnosis. The numbers of fibroblastic progenitors and adherent cells were significantly increased, compared to patients at diagnosis, and TNFα and IL-6 levels returned to normal. In summary, in the present study, we have demonstrated significant in vitro alterations of the hematopoietic system, both in terms of its composition and function, in pediatric patients with ALL. Importantly, most of these alterations are corrected, at least partially, after chemotherapy.