Lymphocytic Leukemia Of B-cell Type Research Articles

Array-CGH Based Genomic Profiling in B-Cell Chronic Lymphocytic Leukemia Reveals Specific Correlations of Genomic Imbalances and Prognostic Subgroups and Underlines the Consistency of Chromosomal Aberration Patterns within This Disease.

Chronic lymphocytic leukemia of B-cell type (B-CLL) is characterized by a number of typical genomic aberrations. In comparison to patients with normal karyotypes or 13q deletions patients with high risk imbalances such as deletions of 11q, 17p or unmutated IgVH status have a higher risk for advanced disease and a significantly shorter survival.For a precise mapping of chromosomal imbalances as well as to verify the number of aberrations per case in different genetic subgroups of B-CLL (e.g. del11q, del13q, del17p, +12 or unmutated IgVH status) we performed high resolution genomic profiling using a genomic DNA-chip containing 2.800 probes. Target clones compriseda large genome-wide cluster of clones covering the genome at a distance of approx. 1.5Mb andclones mapping to genomic regions or genes of possible pathogenetic relevance in lymphoma.This chip covers approximately 10% of the human genome.In 93 (70%) of 133 analyzed B-CLL cases 171 genomic imbalances were identified (between 1–7 aberrations/case). Besides the confirmation of known recurrent chromosomal aberrations, previously unknown recurrent imbalances were detectable on 2p (8%), 4p (4%), 7p-q (5%), 10q (5%) and 20p (3%). Most of these imbalances were of larger extension (> 10 Mb) and therefore impeded a further delineation of minimal aberrant regions and the identification of possible candidate genes. The mean number of chromosomal aberrations per case (= genomic complexity) in IgVH unmutated CLLs was approx. 2 times higher than in mutated cases (0,77 vs. 1,58 per case). 84% of samples with > 2 aberrations showed an unmutated IgVH status. Moreover, most of the previously unknown imbalances were identified within this group. A higher genomic complexity was also shown for samples with gain on 2p vs. balanced 2p status (2.2 times higher; 2.5 vs. 1.2) and in samples with del17p vs. balanced 17p status (3.7 times higher; 3.52 vs. 0.95). 11q aberrations had no impact on the number of genomic aberrations per case (1.6 vs. 1.2). Moreover, we found a strong association of 2p gains and an unmutated IgVH status (100%).Array based genomic profiling confirmes the chromosomal aberration structure and underlines the consistency of chromosomal aberration patterns of B-CLL. The biological and prognostic relevance of 2p gains and unmutated IgVH mutational status have to be further investigated.

Vδ1 T Lymphocytes from B-CLL Patients Recognize ULBP3 Expressed on Leukemic B Cells and Up-Regulated by Trans -Retinoic Acid

We analyzed 38 untreated patients with chronic lymphocytic leukemia of B-cell type (B-CLL): 24 low-, 8 intermediate-, and 6 high-risk stage. In 15 patients (13 low risk and 2 intermediate risk), circulating Vdelta1 T lymphocytes were significantly increased (100 to 300 cells/muL) compared with most intermediate, all high-risk stage, and 15 healthy donors (50 to 100 cells/muL). We studied these Vdelta1 T lymphocytes and observed that they proliferated in vitro and produced tumor necrosis factor alpha or IFN-gamma in response to autologous leukemic B cells but not to normal lymphocytes. However, they were unable to kill resting autologous B cells, which lack the MHC-related MIC-A antigen and express low levels of the UL16-binding protein (ULBP) 3 and undetectable levels of ULBP1, ULBP2, and ULBP4. All these molecules are reported ligands for the NKG2D receptor, which is expressed by gammadelta T cells and activates their cytolytic function. The Vdelta1 T lymphocytes studied were able to lyse the ULBP3(+) C1R B-cell line upon transfection with MIC-A. More importantly, they also lysed autologous B-CLL cells when transcription and expression of MIC-A or up-regulation of ULBP3 were achieved either by activation or by exposure to trans-retinoic acid. The NKG2D receptor expressed on Vdelta1 T cells was involved in the recognition of B-CLL. Finally, in six patients with low numbers of circulating Vdelta1 T cells and undetectable ULBP3, the disease progressed over 1 year, whereas no progression occurred in patients with high Vdelta1 T lymphocytes and detectable/inducible ULBP3. These data suggest that Vdelta1 T lymphocytes may play a role in limiting the progression of B-CLL.

Expression of vascular endothelial growth factor in lymphomas and Castleman's disease.

Vascular endothelial growth factor (VEGF) is one of the main angiogenic cytokines in human solid tumours and inhibition of VEGF-induced angiogenesis suppresses tumour growth. Some groups of malignant lymphoma, including peripheral T-cell lymphomas and Hodgkin's disease, are characterized by a conspicuous proliferation of small vessels. To test the hypothesis that VEGF may also be involved in the angiogenesis in lymphomas and other lesions of the lymphoid system, VEGF expression was analysed in tissues, employing in situ hybridization with a 35S-labelled RNA probe specific for this cytokine. Significant expression of VEGF transcripts was observed in Hodgkin's disease and peripheral T-cell lymphomas, particularly of the angioimmunoblastic type. In contrast, expression of this cytokine was minimal or absent in follicle centre lymphoma and chronic lymphocytic leukemia of B-cell type. VEGF was mainly observed in reactive non-lymphoid CD68-negative cells, which probably represent fibroblasts or myofibroblasts. In normal and ulcerated tonsils, VEGF was expressed in the squamous epithelium but only rarely found in the lymphoid tissue. Although infectious mononucleosis tonsils contained high numbers of VEGF-positive cells in the interfollicular zone, expression of this cytokine was not found in Epstein-Barr virus (EBV)-infected cells, as determined by simultaneous in situ hybridization for VEGF and EBV-encoded small nuclear RNAs (EBER). In 5/8 cases of Castleman's disease, germinal centres containing small vessels also showed expression of VEGF, in contrast to normal tonsillar germinal centres which are devoid of both vessels and VEGF transcripts. It is concluded that VEGF may be involved in the induction of the angiogenesis of both peripheral T-cell lymphomas and Hodgkin's disease, but not in low-grade B-cell lymphomas. In contradistinction to solid tumours, in which this cytokine is commonly secreted by the tumour cells themselves, in malignant lymphoma VEGF is not a product of neoplastic cells. Vascularization of germinal centres in Castleman's disease may also be a consequence of abnormal local expression of VEGF.

Studies on the regulation and localization of 5-lipoxygenase in human B-lymphocytes.

Stimulated B-lymphocytes, isolated from patients with chronic lymphocytic leukemia of B-cell type (B-CLL cells) or from human tonsils, produced similar amounts of leukotriene (LT) B4 and 5-hydroxyeicosatetraenoic acid (5-HETE) as polymorphonuclear granulocytes. Unlike intact granulocytes or monocytes, human B-lymphocytes require calcium ionophore, exogenous arachidonic acid and an oxidative environment in order to produce 5-lipoxygenase products. Several thiol-reactive compounds such as N-ethylmaleimide, methyl methanethiosulfonate, azodicarboxylic acid bis[dimethylamide] (diamide) as well as hydrogen peroxide were all found to stimulate cellular leukotriene biosynthesis. Reverse transcriptase (RT)-PCR analysis demonstrated the expression of 5-lipoxygenase, 5-lipoxygenase-activating protein (FLAP) and LTA4 hydrolase mRNA in B-CLL cells. Western blot analysis demonstrated a band corresponding to the molecular size of FLAP in the B-CLL cell membrane. Furthermore, MK886, the FLAP-binding cellular leukotriene biosynthesis inhibitor, reduced both LTB4 and 5-HETE formation. Immunocytochemistry showed that 5-lipoxygenase was mainly localized in the nuclei of non-activated B-CLL cells, tonsillar B-lymphocytes and monoclonal B-cells. In contrast, neither human peripheral T-lymphocytes nor Jurkat cells were stained. These results suggest that 5-lipoxygenase and its products function in the nucleus of B-lymphocytes.

G Protein Subunit G αl6 Expression Is Restricted to Progenitor B CellsDuring Human B-Cell Differentiation

Recently G alpha 16, a new guanosine triphosphate (GTP) binding protein alpha subunit has been described to be specifically expressed in human hematopoietic cells. Expression of G alpha 16 was observed in human cell lines of myelomonocytic and T-lymphocytic origin, but not in human B-cell lines Raji and IM9. We studied the expression of G alpha 16 in human B cells corresponding to different stages of B-cell differentiation by means of reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting. The human Burkitt's lymphoma cell lines Raji, Ramos, BJAB, the lymphoblastoid cell line SKW6.4, and the plasmocytoma cell line U266 were devoid of G alpha 16. In contrast, G alpha 16 was detected in the human progenitor B cell lines Reh and Nalm-6. Using the mu+, k- cell line BLIN-1 (pre-B cell phenotype) and its derived subclone 1E8 (surface mu+, k+; B-cell phenotype) G alpha 16 expression was found to disappear on transition from pre-B to B-cell differentiation stage. The analysis of a broad panel of human neoplastic B lymphocytes ranging from progenitor B-acute lymphatic leukemia (pre-pre-B-ALL), common acute leukemias (cALL), pre-B-ALL, mature B-ALL to low grade B-cell lymphoma (chronic lymphocytic leukemia of B-cell type, leukemic centrocytic non-Hodgkins lymphoma [NHL], hairy cell leukemia) showed that G alpha 16 expression is limited to progenitor and pre-B-ALL cells. Therefore, we conclude that within B-cell differentiation, G alpha 16 is expressed solely during early B cell ontogeny and downregulated during differentiation. Thus, G alpha 16 might be an important regulator involved in signaling processes in progenitor B cells.

Expression of adhesion molecules CD11/CD18 (Leu-CAMs, beta 2-integrins), CD54 (ICAM-1) and CD58 (LFA-3) in B-chronic lymphocytic leukemia.

Cell adhesion molecules (CAMs) are cell surface proteins with unique specificities that allow intercellular adhesion. The importance of CAMs for normal lymphocyte growth and differentiation is underscored by the association between neoplastic disease states and abnormal CAM expression. In the present study we analysed the cell surface expression of several CAMs on peripheral blood lymphocytes from patients with progressive chronic lymphocytic leukemia of B-cell type (B-CLL) (n = 21) and stable monoclonal B-lymphocytosis of undetermined significance (B-MLUS) (n = 20). The CAM expression was analysed on the B-cell clone and on normal T- and NK-cell populations separately using monoclonal antibodies (MAbs). A phorbol ester-induced lymphocyte aggregation assay and blocking MAbs were also used. The B-cell clone in B-CLL expressed ICAM-1 (CD54) more frequently and at a higher density than in B-MLUS. The brightest CD54 expression was noted in patients with prominent lymphadenopathy and/or splenomegaly. The beta 2 integrin CD11a (Leu-CAMa, LFA-1) was detected on some B-cell clones and seemed to relate to tissue localization of the disease. T and NK cells showed a low expression of CD11a in B-CLL patients, while in B-MLUS a high proportion of non-clonal cells coexpressed CD11a with a high staining intensity. The relative numbers of both CD18+ as well as CD2+ cells showed a positive correlation with phorbol ester induced cell aggregation in B-MLUS patients (p < 0.05). The aggregation was blocked by adding MAbs against CD18 in most cases but to a greater extent in B-CLL. These results extend and corroborate our earlier findings on surface phenotypic characteristics of clonal and non-clonal lymphocytes in different clinical subtypes of B-CLL. CAM expression on the monoclonal lymphocytes may play a role in their interaction with regulatory immune cells and their tissue localization.

Production of B‐cell growth factor interleukin 2 and gamma interferon by peripheral blood lymphocytes from patients with chronic lymphocytic leukemia of B‐cell type

Chronic lymphocytic leukemia of B-cell type (B-CLL) is a malignant disease characterized by monoclonal proliferation of small lymphocytes of B-cell origin, usually associated with suppression of polyclonal B-cell activation (i.e., proliferation and differentiation). Normal human B-cell proliferation is controlled by different T-cell-derived lymphokines, including interleukin 2 (IL2) and gamma interferon (gamma-IFN), that account for the majority of the B-cell growth factor (BCGF) activity produced by mitogen-activated peripheral blood mononuclear cells (PBMCs). We have previously shown an increased and dysregulated secretion of IL2 in peripheral blood from patients with B-CLL. BCGF, IL2, and gamma-IFN productions by phytohemagglutinin (PHA)-stimulated PBMCs were investigated in 13 patients with active untreated B-CLL and 11 healthy donors. B-CLL PBMCs produced a significant amount of BCGF (6 U/ml) despite the low percentage of T cells (10%) associated with this disease as compared with that found in healthy donors (61%). BCGF production in normal controls and B-CLL patients was tripled after irradiation of PBMCs or addition of indomethacin. gamma-IFN secretion in B-CLL patients was decreased when compared with normal controls. Therefore, when gamma-IFN was calculated per fixed number of T cells, production was significantly higher in B-CLL patients than in normal controls, showing a dilution of the productive cells. This study suggests that T cells from B-CLL patients are functional in terms of BCGF production despite their decreased percentage and abnormalities in surface markers.

Epidemiological Features of B-Cell Lymphoma in Japan

Age-adjusted incidence rates for malignant lymphomas in eastern Asian countries except for the Kyushu district of Japan are lower than those in northern European, North and South American and Oceanian countries. Particularly, the incidence rates for Hodgkin's disease and follicular lymphoma are remarkably low in eastern Asian countries. Immunological and clinico-pathological analyses suggested that the estimated rate of incidence of extra-nodal B-cell lymphoma in Japan is not very different from that in the U.S.A. However, their primary sites differ as seen in gastrointestinal lymphomas, most of which are included in extranodal B-cell lymphoma. It is interesting epidemiologically that patients with colorectal lymphoma, whose distribution is closely correlated with that of colorectal cancer in both countries, is much rarer in Japan than in the U.S.A. From the epidemiological viewpoint, extranodal B-cell lymphoma in Japan could be classified by the difference in possible risk factors as follows; lymphoma of the alimentary tract, lymphoma of solid organs, lymphoma of the liver and spleen, medullary or extramedullary plasmacytoma, and Burkitt's lymphoma. In order to clarify the possible risk factors for each type of extranodal B-cell lymphoma including chronic lymphocytic leukemia of B-cell type, it seems necessary to conduct collaborative nationwide epidemiological studies in Japan.

Macroglobulinemia and Chronic B-Cell Type Lymphocytic Leukemia in Japan — Study of Autopsy Cases —

Autopsy cases of Waldenström's macroglobulinemia (WM) and chronic lymphocytic leukemia of B-cell type (B-CLL) were examined to determine the geographical distribution and histological characteristics of these diseases in Japan. Both diseases occurred primarily at 50 to 60 years of age with a predominance of males and were distributed throughout Japan with a population slightly lower than that of T-CLL in southwestern Japan. Histologically WM (31 cases) consisted mainly of small cell type malignant lymphoma with plasmacytic differentiation (27 cases) according to the Japanese Classification (LSG) and cytoplasmic immunoglobulins with a monoclonal pattern of immunoglobulin M (IgM) (lambda 12; kappa seven). The bone marrow involvement (27 cases) was follicular, mixed (follicular and diffuse) or diffuse. B-CLL (58 cases) also showed principally a small cell type histology (48 cases) and 16 of the cases showed plasmacytic differentiation as in WM. Cytoplasmic immunoglobulins with a monoclonal pattern were found in 10 (six of IgG, two of IgM, one of IgG and M, and one of IgA). Bone marrow involvement (56 cases) revealed the same histological pattern as WM. According to the growth pattern of the tumor cells in the bone marrow and lymph nodes, both diseases may be categorized as bone marrow-based lymphoma.

Immunologic markers in the differential diagnosis of non-Hodgkin's lymphomas.

To improve the classification of the phenotypes of the various types of non-Hodgkin's lymphoma (NHL), 250 cases of NHL were analyzed with immunologic and enzyme cytochemical techniques. The results confirmed previous findings. Chronic lymphocytic leukemia of B-cell type (B-CLL) is characterized by a small amount of surface immunoglobulin (SIg), a predominance of C3d receptors, a large number of mouse erythrocyte receptors, and a low T-cell content. Chronic lymphocytic leukemia of T-cell type (T-CLL) has at least two subtypes: one shows a dot-like reaction product in cells stained with acid nonspecific esterase and the other does not. In prolymphocytic leukemia, there is a constantly high percentage of SIg-positive cells and a large amount of SIg on each positive cell; C3b receptors usually preponderate over C3d receptors; and there is a large number of IgG-Fc receptors. The surface marker phenotype of hairy-cell leukemia is similar to that of prolymphocytic leukemia except that hairy cells are devoid of C3 receptors and usually show a high density of IgM-Fc receptors. T-zone lymphoma usually contains both T cells and B cells. The T cells are capable of binding sheep erythrocytes only at 4 °C and can be identified cytologically as the tumor cells. In contrast, the B cells stem from residual follicles, which are often present in T-zone lymphoma at the time of the first biopsy. Three types of lymphoplasmacytic/-cytoid lymphoma (LP immunocytoma) are distinguished on the basis of morphologic features. The marker constellation of the lymphoplasmacytic subtype resembles that of centroblastic-centrocytic lymphoma. The lymphoplasmacytoid subtype and borderline cases between this subtype and B-CLL show the same markers as does B-CLL. The third subtype of LP immunocytoma, the polymorphic subtype, differs in its marker profile from all other types of NHL. The three types of lymphoma derived from germinal center cells resemble each other in the expression of nearly equal numbers of C3b and C3d receptors and a low percentage of IgG-Fc receptors. Centrocytic lymphoma is distunguished from centroblastic-centrocytic lymphoma by a large proportion of cells bearing SIg and C3 receptors and by the absence, or small proportion, of T cells and cells rosetting with mouse erythrocytes. Centroblastic lymphoma shows a marker profile that is similar to that of centroblastic-centrocytic lymphoma. The Burkitt type of lymphoblastic lymphoma shows a unique marker profile, with a high percentage of SIg-positive cells and no other markers. Analysis of lymphoblastic lymphoma of the convoluted-cell type (including cases of acute lymphoblastic leukemia with a focal acid phosphatase reaction) revealed four phenotypes. Cases with the first phenotype show C3 receptors (usually both subtypes) and a lack of sheep erythrocyte receptors. In cases with the second phenotype, the cells express both C3 receptors and sheep erythrocyte receptors. Cases with the third phenotype lack C3 receptors but contain cells rich in receptors for sheep erythrocytes that bind at 37°C. Nearly all of the cases with these three phenotypes are devoid of acid nonspecific esterase. Cases with the fourth phenotype lack C3 receptors, exhibit sheep erythrocyte receptors that bind only at 4°C, and show a focal acid nonspecific esterase reaction. Eight of nine cases of immunoblastic lymphoma showed SIg and were thus identified as B-cell derived. The ninth case was of T-cell type, as indicated by the capacity of the tumor cells to form rosettes with sleep erythrocytes.

Enzyme polymorphism in the classification of human malignant lymphoma.

Recent studies on the polymorphism of lysosomal hydrolases have shown that all individual blood cell types in the human being possess their own isoenzyme pattern. In the present study acid phosphatase activity of normal human B-lymphocytes and of four different types of low-grade malignant non-Hodgkin's lymphomas according to the Kiel classification was estimated. In addition, the isoenzyme pattern of AcP was investigated by isoelectric focusing. Chronic lymphocytic leukemia of B-cell type (N = 9) and centroblastic centrocytic follicular lymphoma (N = 10) demonstrated significantly lower values than lymphoplasmacytic/lymphoplasmacytoid lymphomas (N = 28) and plasmacytomas (N = 8). The isoenzyme pattern of normal human B-lymphocytes comprised 12 bands between pH 6.3 and 3.85. This basical pattern was shared by all four lymphoma entities. Only lymphoplasmatic lymphoplasmacytoid lymphoma and plasmacytoma revealed additional bands, which probably account for the higher net enzyme activity in these cases.

Histologic and immunohistochemical findings in the differential diagnosis of chronic lymphocytic leukemia of B-Cell type and lymphoplasmacytic/lymphoplasmacytoid lymphoma

114 cases of malignant lymphoma consisting chiefly of lymphocytes were classified by histology as chronic lymphocytic leukemia of the B-cell type (B-CLL) or lymphoplasmacytic/lymphoplasmacytoid lymphoma (LP immunocytoma) and investigated with the immunoperoxidase-bridge (PAP) method for the presence of heavy and light immunoglobulin chains. Fifteen cases were excluded because they showed a completely negative reaction, which might have been an artifact. Of the remaining 99 cases, 46 revealed polyclonal immunoglobulin-positive plasma cells only and could be clearly classified as B-CLL. In 33 cases there were a moderate or large number of plasma cells or plasmacytoid cells with monoclonal intracytoplasmic positivity. Two heavy chain classes were demonstrated in three other cases, and both light chain types were detected in one case. These 37 cases were finally classified as LP immunocytoma. Ten cases contained only a few monoclonal plasmacytoid cells and were interpreted as borderline cases between B-CLL and LP immunocytoma. Six cases have not yet been clarified — there was an inexplicable discrepancy between their histology and immunostaining.