Expression of ice, bcl-2, c-myc and p53 in different bone marrow cell populations from patients with diffuse large B-cell lymphoma.

We have previously reported functional alterations in vitro in the hematopoietic compartment of patients with diffuse large B-cell lymphoma (DLBCL). In the present study, we assessed the presence of molecular alterations in hematopoietic cells derived from DLBCL marrow. Accordingly, the expression of four genes (i.e. ice, bcl-2, c-myc and p53) was assessed both, at the mRNA and protein levels, in three cell populations: (i) population I, consisting of morphologically recognizable precursor and mature cells; (ii) population II, enriched for CD34+ Lineage-negative (Lin-) cells; and (iii) population III, enriched for CD34+ CD38- Lin- cells. By using a multiplex reverse transcriptase-polymerase chain reaction system, we observed reduced expression of bcl-2 in population I, and c-myc in populations I and II from lymphoma marrow compared to their normal counterparts. On the other hand, expression of ice and p53 was not significantly different when comparing normal and DLBCL samples. At the protein level, all four molecules were expressed in a higher proportion of samples from DLBCL patients than in marrow samples from normal subjects. Interestingly, these proteins were expressed predominantly in primitive cells (population III), whereas the proportion of positive samples was reduced in population II, and even more in population I. Taken together, our results indicate that, in DLBCL, molecular alterations are present in hematopoietic cells from bone marrow, including morphologically recognizable precursor and mature cells, as well as primitive hematopoietic progenitors (CD34+ cells). To date, the physiological implications of these alterations are still unclear, and further studies should be undertaken to address this issue.

In vitro proliferation and expansion of hematopoietic progenitors from patients with diffuse large B-cell lymphoma before and after chemotherapy.

We have previously demonstrated that when cultured in Dexter-type Long-Term Marrow Cultures (LTMC), hematopoietic progenitor cells (HPC) from patients with Diffuse Large B-Cell Lymphoma (DLBCL) show a defective proliferation, as compared to HPC from normal marrow. In that study it was also demonstrated that functional alterations were present in the hematopoietic microenvironment developed in culture; thus, it was not clear whether such a defective proliferation in vitro was due to an intrinsic defect in the HPC compartment of DLBCL patients, or to an altered microenvironment, or both. In order to address this question, in the present study we have assessed the proliferation and expansion potentials of HPC present in bone marrow from patients with DLBCL, in cytokine-supplemented liquid cultures initiated with a cell population enriched for CD34+ Lin- cells, in the absence of stromal cells and in the presence of reduced numbers of accessory cells. Our results demonstrated that bone marrow-derived HPC from patients with DLBCL, both before and right after chemotherapy, possessed reduced proliferation and expansion potentials in vitro, as compared to their normal counterparts. Interestingly, in patients analyzed 18 months after treatment the proliferation and expansion levels were similar to those of normal HPC, indicating a complete restoration of the hematopoietic function. Although the reason for these observations is not clear, our results suggest the possibility that primitive CD34+ progenitor cells present in bone marrow, which show deficient proliferation and expansion potentials in vitro, are involved in the origin/progression of DLBCL.