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The expression of certain cell cycle regulatory proteins: cdk1, cdk2, cdk4, cyclin A, cyclin B, cyclin E, Bcl2 and PCNA was examined in peripheral blood lymphocytes (PBL) from 25 cases of chronic lymphocytic leukemias (CLL) in order to analyze a possible cell cycle involvement of CLL lymphocytes. For comparison, we also studied the expression of these proteins in: 23 samples of non-Hodgkin's lymphoma (NHL) tissue of different histological types, 10 samples of non-neoplastic lymphoid tissue (NLT), non-stimulated PBL (NS-PBL) and PHA-stimulated PBL (PHA-PBL) from three healthy donors. Samples were lysed and proteins were resolved on polyacrylamide gel followed by Western blot. The expression of cdk4 and cyclin E, both known to act in early cell cycle stage, was approximately on the same level in all groups of lymphoid pathology examined. In particular, we found that that 19 out of 24 CLL cases were cyclin E positive and all but one were cdk4 positive, ie they expressed these markers over twice the level of non-stimulated healthy PBL. The cdk1 expression was above the level seen in NS-PBL in 14 (56%) cases, but the average expression was significantly lower than in the other tissues examined, including low-grade lymphomas. Cdk2 expression was comparable in CLL and in low malignancy grade NHL, but weaker than in other NHL and in NLT. Cyclins A and B, normally observed in advanced cell cycle phases, were not seen in any CLL case. The presence of cdk4 and cyclin E in the blood cells of the majority of CLL cases studied, as well as cdk1 and cdk2 in some cases, indicate that the CLL cells are not quiescent, but are blocked in an early stage of the G1 cell cycle phase, and/or that the expression of these proteins is pathologically deregulated.

Peripheral blood stem cells (PBSC) from 15 patients with advanced non-Hodgkin's lymphoma (NHL), two patients with chronic lymphocytic leukemia, and two patients with myeloma were collected by continuous-flow leukapheresis after chemotherapy with MIV (mitoxantrone, ifosfamide, and etoposide, five patients) or high-dose cyclophosphamide (14 patients), followed by administration of GM-CSF. Sixteen patients (84%) had persistent marrow involvement at time of inclusion. Results were compared to those obtained in a control group of similar age and disease status in whom collection had been performed after MIV chemotherapy alone. The number of mononuclear cells, granulocyte-macrophage colony-forming units (CFU-GM), CD34+ cells were higher in GM-CSF treated patients with a lower mean number of leukapheresis (3.5 versus 6.4). Among the 19 patients harvested after chemotherapy plus GM-CSF, more progenitor cells were obtained in the cyclophosphamide group than in the MIV group. In all these patients except one, the number of mononuclear cells was sufficient to realize a transplantation. Seventeen patients received intensification with BEAM regimen (8 patients) or cyclophosphamide plus etoposide and total body irradiation (9 patients). Two patients failed to reconstitute correct hematopoiesis and three early toxic deaths occurred for a total of five procedure-related deaths. Nine of these 17 patients are in persistent complete remission with a median post-transplant follow-up of 18 months. Time to reach granulocyte and platelet recovery was not correlated with the number of mononuclear cells, CFU-GM, granulocyte-erythroid-macrophage-megakaryocyte colony-forming units (CFU-GEMM), CD34+ cells, and CD34+ CD33- cells but with the number of previous chemotherapy regimens. PBSC harvesting is achievable after chemotherapy plus GM-CSF in heavily pretreated patients with persistent marrow involvement. Moreover, these cells are able to reconstitute correct hematopoiesis after intensive treatment in these patients.

We describe a patient in whom the concomitant diagnosis of refractory anemia with excess of blasts (16% on initial marrow examination) and chronic lymphocytic leukemia was made more than 9 years ago. The myeloid clone showed a complex karyotypic abnormality. Evolution has so far been remarkably stable, without transformation into acute leukemia. Clonogenic assays showed that patient's serum inhibited the patient's own granulocyte-macrophage colony-forming units (CFU-GM). This inhibition was also present for a control subject's CFU-GM and acute myeloid leukemia clonogenic cells. This raises the problem of a down-regulation of the myeloid clone by the malignant lymphoid clone in this patient, and the possible mechanisms for this are discussed.