Biologic response of B lymphoma cells to anti-CD20 monoclonal antibody rituximab in vitro: CD55 and CD59 regulate complement-mediated cell lysis.

The chimeric anti-CD20 MAb rituximab has recently become a treatment of choice for low-grade or follicular non-Hodgkin's lymphomas (FL) with a response rate of about 50%. In this report, we have investigated the mechanism of action of rituximab on 4 FL and 1 Burkitt's lymphoma (BL) cell lines, 3 fresh FL samples and normal B cells in vitro. Rituximab efficiently blocks the proliferation of normal B cells, but not that of the lymphoma lines. We did not detect significant apoptosis of the cell lines in response to rituximab alone. All cell lines were targets of antibody-dependent cellular cytotoxicity (ADCC). On the other hand, human complement-mediated lysis was highly variable between cell lines, ranging from 100% lysis to complete resistance. Investigation of the role of the complement inhibitors CD35, CD46, CD55, and CD59 showed that CD55, and to a lesser extent CD59, are important regulators of complement-mediated cytotoxicity (CDC) in FL cell lines as well as in fresh cases of FL: Blocking CD55 and/or CD59 function with specific antibodies significantly increased CDC in FL cells. We conclude that CDC and ADCC are major mechanisms of action of rituximab on B-cell lymphomas and that a heterogeneous susceptibility of different lymphoma cells to complement may be at least in part responsible for the heterogeneity of the response of different patients to rituximab in vivo. Furthermore, we suggest that the relative levels of CD55 and CD59 may become useful markers to predict the clinical response. (Blood. 2000;95:3900-3908)

Primary effusion lymphoma after heart transplantation: a new entity associated with human herpesvirus-8.

Deep immunosuppression and Epstein-Barr virus (EBV) infection promote the emergence of lymphoproliferative disorders in patients undergoing solid organ transplantation. In the last few years a new herpesvirus, named human herpesvirus-8 (HHV-8), has been identified in Kaposi's sarcoma and primary effusion lymphoma (PEL) developing in AIDS patients. Subsequently, the same viral DNA sequences have been identified in almost all cases of Kaposi's sarcoma emerged outside HIV infection, thus suggesting their possible pathogenetic role in this tumor. Similarly, the association between HHV-8 and PEL also emerged in cases without HIV infection, even though the total number of these patients is still limited. Here, we focus on the emergence of this unusual lymphoma in patients undergoing solid organ transplant and underline once again its association with the HHV-8. Moreover, despite the characteristic local growth of this peculiar type of lymphoma, we demonstrate at the molecular level, an early neoplastic spread to the bone marrow suggesting the need to investigate in more detail the origin of the disease, as well as the molecular mechanisms controlling its systemic dissemination.

Flow cytometry of leucocyte alkaline phosphatase in normal and pathologic leucocytes.

Leucocyte alkaline phosphatase (LAP) is an enzyme expressed on the external aspect of the neutrophilic granulocyte plasma membrane, and represents a specific marker for the fully differentiated granulocyte. In this report we characterize 1B12.1, a monoclonal antibody raised against human bone alkaline phosphatase, by its ability to recognize the LAP protein. As assessed by Western blot analysis, following electrophoresis under non-reducing conditions, the antibody specifically reacts with LAP upon forced expression of the protein in simian COS-7 fibroblasts. In addition, the 1B12.1 antibody recognizes partially purified LAP isolated from peripheral blood granulocytes. With this antibody we developed a quantitative flow-cytometry-based method for the determination of LAP. Double fluorescence flow cytometry demonstrated that the LAP protein was present in relatively high amounts in neutrophilic granulocytes, but not in monocytes, natural killer cells, or B and T lymphocytes of normal individuals. The protein was completely absent in granulocytes obtained from chronic myeloid leukaemia and paroxysmal nocturnal haemoglobinuria patients. Higher than normal levels of LAP protein were evident in neutrophilic granulocytes of patients suffering from polycythaemia vera, essential thrombocythaemia and severe aplastic anaemia. However, the highest amounts of LAP protein were present in the granulocytes of normal individuals treated with G-CSF for the isolation of peripheral blood stem cells.

Redundant functions of B-Myb and c-Myb in differentiating myeloid cells.

We show in this report that the human myeloid leukemia cell line GFD8 is a useful model to compare the biological function of the structurally related c-Myb and B-Myb proto-oncogenes and to investigate the c-myb domains required for this function. GFD8 cells are dependent for growth on granulocyte-macrophage colony-stimulating factor and differentiate in response to phorbol myristate acetate (PMA). We have stably transfected this cell line with constructs constitutively expressing c-Myb or B-Myb. Deregulated expression of both c-Myb and B-Myb inhibited the differentiation observed in response to PMA and, in particular, the induction of the CD11b and CD11c antigens on the cell surface, and the induction of adherence. Furthermore, c-Myb and B-Myb enhanced expression of CD13 upon PMA treatment. Although deregulated Myb expression did not alter the growth factor dependence of the cells, it led to an increase in G2 relative to G1 arrest in cells induced to differentiate in response to PMA, whereas control vector-transfected cells were blocked mostly in G1. This decrease in G1 block took place despite normal induction of the cyclin-dependent kinase inhibitor protein p21 (CIP1/WAF1). Thus, GFD8 cells stably expressing the human B-Myb protein behaved in a manner indistinguishable from those stably expressing C-Myb for both differentiation and cell cycle parameters. In agreement with these findings and differently from most previous reports, transactivation assays show that B-myb can indeed act as a strong activator of transcription. Finally, we demonstrated that although the DNA-binding domain of c-myb is required for both the differentiation block and the shift in cell cycle after PMA treatment, phosphorylation by casein kinase II and mitogen-activated protein kinase at positions 11 and 12 or 532 of c-myb, respectively, are not. We conclude that c-Myb and B-Myb may activate a common cellular program in the GFD8 cell line involved in both differentiation and cell cycle control.

The natural history of monoclonal villous lymphocytosis: a chronic lymphoproliferative disorder of CD11c+ B cells.

The long-term outcome of three asymptomatic subjects with isolated persistent lymphocytosis of monoclonal villous B-cells (MVL) is reviewed. After 7.5 years, evolution to a splenic lymphoma variant (SLVL) was documented in only one patient, accompanied by a loss of interleukin-1beta autocrine production, confirming that MVL can be an early form of a malignant disorder. The clinical course was uneventful in the other two cases; a progressive lowering of lymphocyte count being noted in one. While the strict relationship of MVL to SLVL is confirmed, time to progression is unpredictable and the mechanisms by which it occurs still remain to be elucidated.

Granulocyte colony-stimulating factor following peripheral-blood progenitor-cell transplant in non-Hodgkin's lymphoma.

PURPOSE: To compare the hematologic recovery after high-dose chemotherapy and circulating peripheral-blood progenitor-cell (PBPC) transplant between patients who received recombinant human granulocyte colony-stimulating factor (G-CSF) (treated group) and those who did not (control group). PATIENTS AND METHODS: From December 1992 through June 1994, two sequential and consecutive cohorts of 20 patients each with histologically proven non-Hodgkin's lymphoma (NHL) received high-dose chemotherapy (carmustine [BCNU], cytarabine [Ara-C], etoposide and melphalan [BEAM]) followed by PBPC transplant. The first 20 patients were treated with G-CSF (5 micrograms/kg/d) after PBPC administration. Since the time of platelet and leukocyte recovery in this group was short (< 15 days), with a narrow standard deviation from the mean value, the last 20 patients were not given G-CSF. Hematologic recovery, number of febrile days, rate of documented infections, number of hospital days, duration of gastrointestinal complications, platelet and RBC transfusions, and antibiotic requirements were compared in the two groups. RESULTS: The two groups of patients were comparable according to disease status, histology, stage, bulky disease bone marrow involvement, elevated lactate dehydrogenase (LDH) level, and median number of infused CD34+ cells and colony-forming units granulocyte-macrophage (CFU-GM). The median time to reach 0.5 x 10(9)/L and 1.0 x 10(9)/L neutrophils was 2 days shorter in G-CSF group, but this difference was not statistically significant. The median times to reach 20 x 10(9)/L and 50 x 10(9)/L platelets were, respectively, 10 and 14 days in the G-CSF group and 11 and 16 days in the control group, but again this was not statistically significant. Moreover, when considering clinically relevant end points including the number of documented infections and antibiotic requirements, platelet transfusions, gastrointestinal toxicity, and days of hospitalization, no differences were demonstrated between the two groups. CONCLUSIONS: Provided an optimal dose of circulating progenitors is infused, NHL patients transplanted with PBPC do not benefit by the administration of hematopoietic growth factors.

Establishment and characterization of a new granulocyte-macrophage colony-stimulating factor-dependent and interleukin-3-dependent human acute myeloid leukemia cell line (GF-D8).

A novel factor-dependent human myeloid leukemia cell line (GF-D8) was established from the peripheral blood of an 82-year-old man suffering from acute myeloblastic leukemia (AML). By morphology, cytochemical staining, and analysis of surface antigens, GF-D8 cells are myeloblasts of immature progenitor origin. The consensus karyotype is 45, XY, -5, 7q-, inv(7) (q31.2q36), 8q+, +8q+, 11q+, 12p-, -15, -17, + marker. The long-term survival and proliferation of GF-D8 cells is dependent on the presence of either granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-3 (IL-3). Weak colony growth was observed after exposure of GF-D8 cells to stem cell factor (SCF) but not after exposure to granulocyte-CSF (G-CSF), macrophage-CSF (M-CSF), IL-1 beta, IL-2, IL-5, or tumor necrosis factor-alpha (TNF-alpha). GM-CSF- and IL-3-induced proliferation is dose dependent, with significant growth observed at concentrations as low as 0.1 ng/mL, but the combination of both factors has no synergistic effect. A significant proliferation is induced by GM-CSF and IL-3 even in serum-deprived cultures, although with a slightly decreased efficiency. GF-D8 cells were shown to express specific messenger RNAs for the alpha chains of the GM-CSF and IL-3 receptors as well as for the beta chain, common to both receptors. Interestingly, despite the absence of biologic response to G-CSF, specific transcripts for the G-CSF receptor gene were similarly identified by reverse polymerase chain reaction analysis. GF-D8 cells represent a useful tool for studying chromosome abnormalities of human AML as well as the regulation of myeloid proliferation and differentiation in vitro.