Rituximab therapy of patients with B-cell chronic lymphocytic leukemia

Rituximab (IDEC-C2B8) is a chimeric antibody that binds to the B-cell surface antigen CD20. Rituximab has significant activity in follicular non-Hodgkin lymphomas. Much less is known about the effects in chronic lymphocytic leukemia (CLL). We have initiated a phase II trial to evaluate the efficacy and safety of rituximab in patients with CD20+ pretreated CLL. To avoid the rituximab-associated toxicity, we restricted the tumor cell load, as measured by the number of circulating lymphocytes and the spleen size, in the first 2 cohorts of patients included in the study. Patients received 4 intravenous infusions of 375 mg/m2 once a week over a period of 1 month. Of the 28 patients evaluable for response, 7 patients showed a partial remission (National Cancer Institute criteria) lasting for a median of 20 weeks, with 1 patient still in remission after 71 weeks. Based on lymphocyte counts only, we found at least a 50% reduction of lymphocyte counts lasting for at least 4 weeks in 13 (45%) of 29 patients. Fifteen patients from 3 institutions were monitored for the immunophenotype profile of lymphocyte subsets. The number of CD5+CD20+ cells decreased significantly and remained low until day 28 after therapy. T-cell counts were not affected. With the exception of one rituximab-related death, adverse events in the remaining patients were mild. The results suggest that rituximab has clinical activity in pretreated patients with B-CLL. Toxicity is tolerable. Response duration after withdrawal of rituximab is rather short. Therefore, other modes of application and the combination with other agents need to be tested.     

Interleukin 2 stimulates chronic lymphocytic leukemia colony formation in vitro

The requirements of clonogenic cells of B cell-type chronic lymphocytic leukemia (B CLL) for interleukin 2 (IL 2) were analyzed. Using the cells of five patients, we measured IL 2 receptor expression on the cell surface and the colony-forming abilities of the cells in response to IL 2. In four of the cases, significant percentages of the CLL cells expressed IL 2 membrane receptors (as assessed with the monoclonal antibody anti-Tac), indicative of their potential sensitivity to IL 2. Pure recombinant interleukin 2 (r-IL2) was added to colony cultures that also contained the lectin phytohemagglutinin (PHA) or the phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA) to activate the CLL cells. Colony formation completely depended on the presence of r-IL 2 and PHA or TPA in culture, with the exception of one case, in which the addition of IL 2 was not required for colony growth in TPA-supplemented cultures. Twenty-five to fifty units of r-IL 2 per milliliter of culture medium provided optimal stimulation. Under these conditions, a linear relationship was observed between plated cell numbers and colony numbers formed. Morphological and immunologic analysis of colony cells indicated that these were monoclonal CLL cells that had matured toward plasmacellular lymphocytes and plasma cells.     

Immunoregulation of B lymphocyte colony formation by T cell subsets in patients with chronic lymphocytic leukemia

Normal B lymphocytes are activated, proliferate, and then differentiate into plasma cells and secrete immunoglobulin (Ig). We have reported that chronic lymphocytic leukemia (CLL) T4 cells help and CLL T8 cells lack suppressor effects on Ig synthesis by normal B cells (Blood 62:767, 1983). We have now explored the earlier phase, proliferation, using B cell colony formation; in semisolid media. B lymphocyte colonies from normal individuals and from patients with CLL were grown in 0.3% agarose overlayed with T cells or T cell subsets and the B cell mitogen staphylococcal protein A. Enriched T cells, OKT4 or OKT8, were obtained either by sheep erythrocyte rosettes or depletion of OKT8 or OKT4 cells by monoclonal antibody or complement, respectively. Twenty thousand B cells from normal subjects yielded 65 +/- 9, 64 +/- 7, and 19 +/- 6 colonies with autologous unfractionated T-, OKT4-, or OKT8- positive cells, respectively. This compared to 29 +/- 11, 81 +/- 11, and 15 +/- 4 colonies from patients with CLL with added autologous unfractionated T-, OKT4-, or OKT8-positive cells. To determine whether the fewer number of colonies in both normal subjects and patients with CLL with OKT8-positive cells was due to suppression or lack of help, the number of OKT4-positive cells was held constant, and OKT8-positive cells were added in increasing numbers. No suppression of colony formation could be demonstrated. Furthermore, the addition of increasing numbers of concanavalin A (Con A)-activated OKT8-positive cells did not suppress colony formation. These results suggest that the CLL T cell subsets behave in a functionally similar manner to normal T cell subsets, namely, (1) that normal and CLL B cell colony growth is helped by OKT4 cells; and (2) in contrast to immunoglobulin secretion by B cells, neither normal nor CLL OKT8 cells, unstimulated or activated by Con A, suppress B cell colony growth.     

TRAIL receptors in the serum of patients with B-cell chronic lymphocytic leukemia

Great importance in the course of chronic B-cell lymphocytic leukemia (B-CLL) has been ascribed to cytokines belonging to the superfamily of the tumor necrosis factor (TNF), including TRAIL (TNF-related apoptosis inducing ligand) and its specific receptors: TRAIL receptor 1 (TRAIL-R1), TRAIL receptor 2 (TRAIL-R2), TRAIL receptor 3 (TRAIL-R3), TRAIL receptor 4 (TRAIL-R4) and osteoprotegerin (OPG). Both the molecule and the receptors may occur in membrane and soluble forms, except for OPG which has only a soluble form. The aim of the study was to assess the levels of sTRAIL molecule and soluble TRAIL receptors - sTRAIL-R2 and OPG in the serum of patients with B-CLL. The findings revealed reduced concentrations of sTRAIL both before and after treatment and elevated levels of sTRAIL-R2 and OPG in patients before treatment. After treatment with CC (2CdA/Cladrybin and Cyklofosfamid) and FC (Fludarabin and Cyklofosfamid) we observed an increase in sTRAIL and a decrease in sTRAIL-R2. OPG levels were found to increase after treatment with CHOP (Vincristini, Cyklofosfamid, Adriamycin and Prednisol) and they decreased after administration of Leukeran (Chlorambucyl) and CMC (2CdA/Cladrybin, Mitoxanton and Cyklofosfamid). The relationships between TRAIL and its natural regulators in the serum of BCLL patients prior to treatment may impair apoptosis of leukemic B cells. Changes in these relationships after treatment with CC and FC seem to promote enhancement of apoptosis in these cells.     

B-cell receptor signaling in chronic lymphocytic leukemia

The B-cell receptor (BCR) is a key survival molecule for normal B cells and for most B-cell malignancies. Recombinatorial and mutational patterns in the clonal immunoglobulin (Ig) of chronic lymphocytic leukemia (CLL) have revealed 2 major IgMD-expressing subsets and an isotype-switched variant, each developing from distinct B-cell populations. Tracking of conserved stereotypic features of Ig variable regions characteristic of U-CLL indicate circulating naive B cells as the likely cells of origin. In CLL, engagement of the BCR by antigen occurs in vivo, leading to down-regulated expression and to an unanticipated modulation of glycosylation of surface IgM, visible in blood cells, especially in U-CLL. Modulated glycoforms of sIgM are signal competent and could bind to environmental lectins. U-CLL cases express more sIgM and have increased signal competence, linking differential signaling responses to clinical behavior. Mapping of BCR signaling pathways identifies targets for blockade, aimed to deprive CLL cells of survival and proliferative signals. New inhibitors of BCR signaling appear to have clinical activity. In this Perspective, we discuss the functional significance of the BCR in CLL, and we describe strategies to target BCR signaling as an emerging therapeutic approach.     

CD43 in B-cell chronic lymphocytic leukemia

CD43 (other names: sialophorin, leukosialin, sialoglycoprotein of white blood cells) is an integral cell membrane mucin. In population of peripheral B cells CD43 occurs only on activated B cells and CD5 positive B cells. These last cells create neoplasm population in patients with B-cell chronic lymphocytic leukemia (B-CLL). Anti-CD43 monoclonal antibodies are used routinely in investigations of tissue fragments in cases of non-Hodgkin's lymphoma, whereas we did not find publication on theme of CD43 expression on peripheral blood B cells in patients with B-cell chronic lymphocytic leukemia. Wherefore advisable appeared estimation CD43 expression on B-CLL cells and comparison it with expression of typical B-CLL markers--such as CD5 and CD6. Immunological phenotype of peripheral blood and bone marrow lymphocytes has been evaluated using flow cytometry (Cytoron Absolute Ortho-Diagnostic Systems) and two-color staining. Twenty six untreated patients with B-CLL were studied. Because on well-known correlations between CD43 expression and metastasis potential of tumor, patients were divided on two groups differing score of total tumor mass (score TTM). Score TTM was evaluated according to criterion of Jaksic and Vitale. Twelve patients whose TTM score was equal or lower than 9 and median lymphocytosis was 24.6 x 10(9) in microliter were included in group I. 14 patients whose TTM score was higher than 9 were included in group II. Median lymphocytosis in these patients was 152.6 x 10(9) in microliter. The median percentage of CD43+/CD19+ cells in peripheral blood was 62.6% in the group I, and 75% in the group II (p < 0.05). Median fluorescence intensity (MFI) of CD43 antigen was 87.7 in the I group comparing to 77.4 in the group II. So one observed tendency to lowering MFI during tumor growing but the difference was not significant (p = 0.25). In peripheral blood during progression of disease more clearly than CD43+ cells increased percentage of CD5+ and CD6+ cells. The median percentage of CD19+/CD5+ cells was 62.7% in the group I, 82.4% in the group II and the difference was significant (p < 0.002). The difference in the median percentages CD6+/CD19+ cell 71.8% in group I and 84.3% in the II one were also significant (p < 0.03). MFI of CD5 and also CD6 antigens did not change in course of disease. Moreover, examination of CD43 and CD5 expression in marrow additionally to blood study were performed in 12 cases (6 from group I, 2 from group II and 4 new not included). The median percentage of CD43+/CD19+ cell was 35.1% in blood and 43.7% In marrow, in contrast to these results was the median percentage of CD19+/CD5+ cell, which was higher in peripheral blood (70.4%) than in bone marrow (60.9%). The results of this study indicate that CD43 is present on peripheral blood B-CLL cells. Moreover, percentage of these cell increases during progression of disease however more weakly than percentage of CD5 and CD6 positive cells. Expression of CD43 is independent from expression CD5 and CD6 and diminishes during tumor mass increasing, what can depended from releases exocellular domains of CD43. CD43+ cell from B-CLL patients have a tendency to accumulation in tissues what is illustrated by higher percentage of CD43+ cell in bone marrow than in peripheral blood.     

Cytotoxic lymphocytes in B-cell chronic lymphocytic leukemia

Summary The occurrence of cytotoxic lymphocyte subpopulations (i.e., CD 16⁺, CD57⁺ and cytotoxic CD 8⁺) was studied in the peripheral blood of 18 B-cell chronic lymphocytic leukemia (B-CLL) patients. The absolute numbers of CD 57⁺, CD 16⁺ and cytotoxic CD 8⁺ lymphocytes were increased in the peripheral blood of untreated patients as compared with healthy donors, suggesting a causal relation with the accumulation of malignant B-cells. For 5 B-CLL patients and 5 hematological normal donors, the lymphocyte subpopulations in peripheral blood, lymph nodes and bone marrow were determined. A significant immune response was observed in the lymph nodes of the patients, as reflected by the CD 3⁺ lymphocytes, which were 1.7–27 times larger in the patients lymph nodes than in their peripheral blood and bone marrow. In contrast, with peripheral blood this was mainly caused by an increase in CD 4⁺ lymphocytes. The CD 57 lymphocytes in the lymph nodes of the patients had abnormal orthogonal light-scattering signals and an abnormal density of CD 57⁺ receptors in comparison with their peripheral blood CD 57⁺ lymphocytes or the CD57⁺ lymphocytes in the peripheral blood, bone marrow and tonsils of the hematological normal donors. This study shows that although a significant increase of cytotoxic lymphocytes in the peripheral blood of B-CLL patients is observed, the actual distributions of the non-malignant lymphocytes can be quite different at the actual tumor sites, i.e., bone marrow and lymph nodes.     

Rheumatoid arthritis and B-cell chronic lymphocytic leukemia

The association between lymphoproliferate malignancies, especially lymphoma, and rheumatoid arthritis (RA) has been confirmed by several studies. However; there are few reports of RA patients who developed B-cell chronic lymphocytic leukemia (B-CLL) and vice versa. We report a patient with B-CLL who developed RA and another with RA who presented with B-CLL during follow-up. We discuss the incidence of B-CLL among the RA population and the possible interaction of the pathogenetic mechanisms of these two entities.     

Fludarabine uptake mechanisms in B-cell chronic lymphocytic leukemia

Nucleoside derivatives are currently used in the treatment of hematologic malignancies. Although intracellular events involved in the pharmacologic action of these compounds have been extensively studied, the role of plasma membrane transporters in nucleoside-derived drug bioavailability and action in leukemia cells has not been comprehensively addressed. We have monitored the amounts of mRNA for the 5 nucleoside transporter isoforms cloned so far (CNT1, CNT2, CNT3, ENT1, and ENT2) in several human cell types and in normal human leukocytes. We then examined the expression patterns of these plasma membrane proteins in patients with chronic lymphocytic leukemia (CLL) and correlated them with in vitro fludarabine cytotoxicity. Despite a huge individual variability in the mRNA amounts for every transporter gene expressed in CLL cells (CNT2, CNT3, ENT1, and ENT2), no relationship between mRNA levels and in vitro fludarabine cytotoxicity was observed. Fludarabine accumulation in CLL cells was mostly, if not exclusively, mediated by ENT-type transporters whose biologic activity was clearly correlated with fludarabine cytotoxicity, which reveals a role of ENT-mediated uptake in drug responsiveness in patients with CLL.     

B-cell acute lymphocytic leukemia in HIV-antibody-positive patients

B-cell non-Hodgkin lymphoma (NHL) has been well described in association with human immunodeficiency virus (HIV) and the acquired immunodeficiency syndrome (AIDS). Many of these lymphomas are of the diffuse, aggressive, subtype B-cell NHL, including Burkitt and Burkitt-like lymphoma. Recently, there have been reports of B-cell acute lymphocytic leukemia (ALL), Burkitt type, in patients who were either HIV antibody-positive or at high risk for AIDS. We have seen three cases of B-cell ALL, Burkitt type, and herein describe their clinical and laboratory characteristics. All patients were HIV antibody-positive. Since stage IV Burkitt lymphoma in blood phase and B-cell ALL, Burkitt type, represent a continuum of the same disease, and since it is also an aggressive B-cell malignancy, we suggest that B-cell ALL, Burkitt type, in HIV antibody-positive patients should support the diagnosis of AIDS.     

Prognostic factors in chronic lymphocytic leukemia

The Rai staging system is widely used and is easy to apply in clinical practice. The system introduced by Binet et al offers an advantage that it consists only of three stages and, therefore, is more practical for initiation of controlled therapeutic trials than the five-stage Rai system. However, when we take into consideration the survival curves, it is important to note that the Rai system has three groups (and not five), low risk (stage O), intermediate risk (stages I and II), and high risk (stages III and IV). These groups are roughly equivalent to Binet's A, B, and C stages, respectively. Although the IWCLL has recommended a unified, integrated method of combining Binet and Rai systems, in practice, however, such a combination is cumbersome and unusable. Both systems, however, suffer from an important limitation--they cannot predict for the large majority of patients in the low and intermediate risk groups (Binet's stages A and B and Rai's O, I, and II) who will have an indolent course and good prognosis in contrast to those in the same stages whose disease will progress rapidly. It is necessary, therefore, to combine the bone marrow histology findings, the lymphocyte doubling time and the threshold values of blood lymphocyte count with the clinical staging systems to distinguish between indolent disease course and aggressive disease on a prospective basis within a given clinical stage.     

Molecular cytogenetic analysis of B-cell chronic lymphocytic leukemia

 The genetic alterations underlying the pathogenesis of B-cell chronic lymphocytic leukemia (B-CLL) are difficult to assess. Cytogenetic studies are hindered by the low in vitro mitotic activity of the tumor cells and the limited resolution of chromosome banding. Molecular genetic analyses are hampered by nonclonal cells contained in the specimens and by the limited knowledge of candidate genes involved. As a complement to cytogenetic and molecular genetic techniques, fluorescence in situ hybridization (FISH) has proven powerful in the molecular cytogenetic analysis of B-CLL. FISH allows the detection of aberrations such as trisomies, deletions, and translocation breakpoints on the single cell level in dividing as well as nondividing cells without the prerequisite of detailed physical maps or knowledge of involved genes. As detected by the interphase cytogenetic FISH approach, the most common chromosome abnormalities of B-CLL are deletions in band 13q14, followed by deletions in 11q22-q23, trisomy 12, deletions in 17p13, and deletions in 6q21. Abnormalities in 17p13 seem to involve the TP53 tumor suppressor gene, but as yet no candidate genes have been identified for the other frequent aberrations. Toward the identification of such genes by positional cloning, FISH can be applied for detailed aberration mapping at the molecular level. Furthermore, the accurate detection of chromosome aberrations in B-CLL by FISH provides a valid basis for the evaluation of their prognostic significance. Inactivation of TP53 in 17p13 and deletions in 11q22-q23 have already been shown to be among the most important independent prognostic factors. Genetic abnormalities may eventually provide biological parameters, allowing a risk assessment for individual patients at the time of diagnosis of this clinically heterogeneous disease. Received: 23 January 1998 / Accepted: 3 February 1998