Phenotypic heterogeneity of B cell chronic lymphocytic leukaemia

The peripheral blood lymphocytes from 39 patients from the Latvian S.S.R.T., U.S.S.R. with chronic lymphocytic leukaemia (CLL) have been phenotyped with various monoclonal antibodies representing the major clusters of differentiation (CD) used for phenotyping B cells. A clear delineation of two groups of patients was evidenced. The major group (33/39) possessed leukaemic cells bearing surface immunoglobulins (SIg) at a low density, Class II HLA, and CD5, CD24 and CD37 molecules but not CD21, CD22 and CD35. CD23 antigen was seen only once under microscope examination, but could be visualized by flow cytometry. CD6 antibody reacted with cells from about 1/3 of this group of patients. In the six patients of the second group the leukaemic phenotype was SIg+, Class II HLA+, CD5+, 24+, 37+, 21+, 22+, 35+, 23+ and 6-. The main finding is the concomitant expression of CD22, CD21 (CR2) and CD35 (CR1) molecules, all involved in B cell activation. It is not yet known whether these observations correlate with different clinical evolutions of the disease.     

Impact of cryopreservation on B cell chronic lymphocytic leukaemia phenotype.

BACKGROUND AND OBJECTIVES: Freezing is a practical approach for cell preservation for retrospective studies. The aim of this work was to check the cryopreservation impact on B cell chronic lymphocytic leukaemia phenotype. MATERIAL AND METHODS: Blood samples from 15 CLL patients were analyzed freshly and after freezing at -196 degrees C, without separation, and thawing. Results were compared by Student's paired t-test. RESULTS: The phenotype of fresh CLL cells was as follows: CD19+, CD5+, faint CD20, CD23+/-, weak CD22 and sIg, CD37+, HLA-DR+, FMC7-. After cryopreservation, the percentage of CD5 and CD23 positive cells decreased, whereas HLA-DR positive cells increased moderately. The CLL Matutes's score was modified in 6 cases out of 15 (40%). CONCLUSION: Cryopreservation modifies B cell chronic lymphocytic leukaemia phenotype, by decreasing CD5 and CD23 expression.     

Further evidence for T cell abnormalities in chronic lymphocytic leukaemia of the B cell type.

Functional properties were studied in the purified T cell fraction of patients with chronic lymphocytic leukaemia of the B cell type (B-CLL). This analysis included the evaluation of T suppressor activity when investigated patients' T cells were co-cultured together with allogenic normal B and OKT4 enriched T cells in the presence of pokeweed mitogen (PWM). The Ig secreting cells (ISC) were assessed in a reverse haemolytic plaque assay (RHPA). Antibody-dependent cytotoxicity (ADCC) and natural killer activity (NK) were determined in a 51Cr release assay. Furthermore, purified T cells reactive with the monoclonal antibody HNK1, known to recognize most effector cells in ADCC and NK, were enumerated using an indirect immunofluorescence. Our results revealed increased T suppressor cell activity and markedly deficient NK activity in peripheral blood lymphocytes (PBL), T cell and T gamma cell fractions from B-CLL patients, whereas ADCC potential was only increased in T cells and T gamma cells. Accordingly, T cells were recognized by HNK1 in greater numbers in B-CLL patients than in healthy subjects. Our data suggest that there may be a link between our findings and the hypogammaglobulinaemia as well as the increased incidence of second neoplasias reported in CLL.     

Imbalance of T cell subpopulations in patients with chronic lymphocytic leukaemia of the B cell type.

T cell enriched mononuclear cells from the peripheral blood of 20 patients with histologically and immunologically defined chronic lymphocytic leukaemia of the B cell type (B-CLL) and 20 healthy individuals of various ages were investigated with T cell-specific monoclonal antibodies (OKT 4 and OKT 8) with regard to their subpopulation distribution. In B-CLL, a significant increase of lymphocytes reacting with OKT 8 could be demonstrated. Whereas there was a ratio of OKT 4 to OKT 8 of 1 X 72 in the control group, an OKT 4 to OKT 8 ratio of 0 X 67 was found in the B-CLL as a whole. With increasing clinical stage in accordance with the Rai scheme (Rai et al., 1975), a further displacement of this ratio in favour of OKT 8 positive cells was found. These results clearly show that, in peripheral blood of patients with B-CLL, an abnormal distribution pattern of circulating T cell subpopulations is present and that this also has prognostic relevance.     

Expression of immunoglobin G on blood lymphocytes in chronic lymphocytic leukaemia.

A sensitive rosette test utilizing antibody-coated red cells has been applied to the study of the immunoglobulins on the surface of blood lymphocytes in chronic lymphocytic leukaemia (CLL). Contrary to other reports, IgG has been found to be a common surface membrane immunoglobulin (SmIg) on CLL cells. The reasons for this variation are discussed. Evidence is presented to show that (a) the anti-IgG-coated erythrocytes really are detecting IgG and not a cross-reacting substance, (b) the IgG is intrinsic to the cell and not cytophilically bound and (c) Fc-binding and other artefacts have been excluded. Expression of Ig by individual cells was studied by using mixtures of fluorescein- and rhodamine-labelled red cells coated with various anti-Ig. Fifty-one cases of untreated CLL were tested and the lymphocytes of thirty-eight of these cases bore Ig of a single light chain type. These cases could be classified on the basis of their lymphocyte SmIg as follows: eighteen expressing M and D and G, eleven expressing D and G but not M, six expressing M and D but not G, and three expressing G alone.     

Effect of interleukins on the proliferation and survival of B cell chronic lymphocytic leukaemia cells.

AIMS--To investigate the effects of interleukin (IL) 1, 2, 4, and 5 on the proliferation and survival of peripheral blood B cells from patients with B chronic lymphocytic leukaemia (B-CLL) and compare them with the effects on normal peripheral blood B cells. METHODS--The proliferation and survival of pokeweed mitogen (PWM) activated B cells from B-CLL (n = 12) and normal peripheral blood (n = 5) were studied in vitro in response to IL-1, IL-2 IL-4, and IL-5. Survival of cells in cultures with or without added interleukins was studied by microscopic examination of cells and DNA agarose gel electrophoresis. RESULTS--Proliferation was observed in both B-CLL and normal peripheral blood cells on culture with IL-2 alone and also in some, but not all, B-CLL and normal peripheral blood cells with IL-1 and IL-4. However, there was greater variability in B-CLL cell responses than in normal peripheral blood cells. Il-5 did not affect normal peripheral blood cell proliferation but it increased proliferation in two B-CLL cases. Synergistic effects of these cytokines were not detected. IL-4 inhibited normal peripheral blood and B-CLL cell proliferation after the addition of IL-2. Inhibition of B-CLL cell responses to IL-2 was also observed with IL-5 and Il-1. Survival of B-CLL cells in cultures was enhanced with IL-4 not by an increase in proliferation but by reduced apoptosis. No such effect was seen in normal peripheral blood cells. IL-2 had a less noticeable antiapoptotic effect; IL-5 enhanced apoptosis in B-CLL cells. CONCLUSIONS--B-CLL and normal peripheral blood cells proliferated equally well in response to IL-2. IL-4 had a much lower effect on B-CLL cell proliferation, but had noticeable antiapoptotic activity. IL-5 enhanced cell death by apoptosis.     

Direct effect of interleukin 2 on chronic lymphocytic leukaemia B cell functions and morphology.

The functional and morphological changes induced by recombinant interleukin 2 (IL-2) were studied in purified B cells from patients with untreated B chronic lymphocytic leukaemia (B-CLL). In eight of nine patients, purified B-CLL cells increased their DNA synthesis in response to IL-2 without preactivation in vitro. This response, studied in detail in three patients, was dose dependent and reached a maximum on day 5 or 6. IL-2 induced or increased IgM secretion in cultures from five of the nine patients studied. Two of this responsive group were particularly interesting as IL-2 not only stimulated IgM secretion but also induced the secretion of IgG. Immunoglobulin production was invariably monoclonal. B CLL cells incubated with IL-2 showed distinct morphological changes including an increase in the size of cytoplasm and enlargement of nuclei together with the appearance of nucleoli. These changes were present in all IL-2 treated cultures but were more pronounced in those containing immunoglobulin secreting cells. None of the IL-2 induced changes appeared to correlate with the clinical stage of the disease or the level of Tac antigen expression on the freshly isolated CLL B cells.     

Activation of B chronic lymphocytic leukaemia cells by Branhamella catarrhalis.

Cells from the blood of patients with chronic lymphocytic leukaemia were cultured in the presence of two polyclonal activators of human B cells, the bacteria Branhamella catarrhalis (Bc) and Staphylococcus aureus Cowan 1 (SAC). Although the magnitude of the responses varied, cells from seven of the eight patients studied were induced to proliferate in response to Bc. In contrast, the response to SAC was low or negligible in seven of the eight patients, and only one patient responded well to this mitogen. Bc was also effective in inducing secretion of IgM in cells from seven of the eight patients, and this was unaffected by removal of T cells. Fractionation of CLL cells on density gradients showed that the highest level of IgM production was induced in cells with a low buoyant density, whilst cells with a high buoyant density secreted little or no immunoglobulin in response to Bc. Together, these results demonstrate that Bc is an effective, T-independent activator of both DNA synthesis and immunoglobulin production in CLL cells.     

Bone marrow aplasia in B cell chronic lymphocytic leukaemia: successful treatment with antithymocyte globulin.

Pure red cell aplasia is a rare but well known association of chronic lymphocytic leukaemia (CLL). Pancytopenia due to bone marrow aplasia has not been previously described in CLL. A 42 year old man with B cell CLL became severely pancytopenic with bone marrow aplasia. Bone marrow culture resulted in a greatly reduced colony formation. High dose corticosteroids and intravenous immunoglobulin treatment were unsuccessful. Prompt and complete marrow recovery ensued after administration of antithymocyte globulin.     

Lycurim (R-74) in the therapy of chronic lymphocytic leukaemia

Lycurim was used in the therapy of 41 CLL patients, in 34 a positive clinical effect was found. The effect was marked in cases of CLL associated with autoimmune haemolytic anaemia. The immunodepressive property of Lycurim was confirmed by immunological investigation. It has been shown that the levels of IgG, IgA and IgM fall after Lycurim treatment of CLL patients. At the same time the T-lymphocyte count decreases and a tendency for EAC rosette-forming B-lymphocyte decrease is marked.     

Bax gene G(-248)A promoter polymorphism is associated with increased lifespan of the neutrophils of patients with osteomyelitis.

BACKGROUND: Patients with osteomyelitis have a decreased rate of spontaneous apoptosis of their peripheral blood neutrophils. The G(-248)A polymorphism in the promoter region of the bax gene is associated with prolonged peripheral blood neutrophil survival in leukemic patients and may play some role in osteomyelitis. METHODS: Bax G(-248)A promoter polymorphism was detected by DNA amplification using polymerase chain reaction, followed by restriction fragment length polymorphism analysis. Spontaneous apoptosis of peripheral blood neutrophils was measured by propidium iodide, annexin V, and flow cytometry, and Bax was quantified by Western blotting. RESULTS: The bax promoter polymorphism A allele was significantly more frequent in 80 patients with osteomyelitis than in 220 healthy donors (18.1% vs. 10.6%, chi=4.84, odds ratio=1.81, 95% confidence interval=1.06-3.07, P=.028). Carriers of the A allele had a lower apoptotic rate of their peripheral blood neutrophils compared with noncarriers (33.3+/-16.7 vs. 43.1+/-3.1, P=.036). Patients with the AA genotype showed a lower expression of the Bax protein compared with carriers of other genotypes (P=.038). CONCLUSIONS: Substitution of a nucleotide G-->A at position -248 in the bax gene was more frequent in patients with osteomyelitis and was associated with a longer lifespan of their peripheral blood neutrophils and lower Bax protein expression. These findings may play a role in the pathogenesis of osteomyelitis.     

Impaired production of mononuclear cell procoagulant activity in chronic lymphocytic leukaemia.

Chronic lymphocytic leukaemia (CLL) is associated with a low incidence of thrombotic complications, or disseminated intravascular coagulation (DIC), or both, despite the frequent occurrence of severe infections. We have investigated the capacity of blood mononuclear cells to produce procoagulant activity when stimulated with bacterial endotoxin in 16 patients with untreated chronic lymphocytic leukaemia (CLL). Procoagulant activity generated by patients' cells after prolonged incubation with endotoxin was significantly lower than that produced by cells from a matched control group (p less than 0.001). The defect could not be attributed to an inhibitory effect of leukaemic lymphocytes. It is suggested that in CLL the monocyte has an intrinsic functional abnormality of the procoagulant response to endotoxin and possibly to other stimuli. These findings help explain why CLL patients do not develop thrombotic complications despite the high incidence of severe infectious diseases.     

T cells in B cell chronic lymphocytic leukaemia. I. Decreased frequency of T lymphocytes secreting suppressor factor.

A reverse T cell plaque assay was employed to study the ability of purified T cells isolated from the blood of seven patients with B cell chronic lymphocytic leukaemia (B-CLL) to secrete antigen-specific helper or suppressor factors (ThF and TsF respectively) after activation in vitro. It was found that in spite of the phenotypical presence of CD8+ cells, the frequency of TsF-secreting cells was strongly decreased as compared to normal values. T cells secreting ThF could be generated in all B-CLL patients tested in about normal frequencies. These results may indicate a tumour induced change in the distribution of cellular subsets within the CD8+ cell compartment.     

Leukaemic mantle cell lymphoma with t(11;14) and trisomy 12 showing clinical features of state A0 B cell chronic lymphocytic leukaemia

The precise diagnosis of lymphoma usually requires the histological examination of lymph nodes or involved tissues. Mantle cell lymphoma is a form of intermediate grade non-Hodgkin's lymphoma in which typical morphological immunophenotypic and cytogenetic features have been recognised. A case of leukaemic mantle cell lymphoma with the characteristic reciprocal translocation t(11;14) together with trisomy 12, a chromosomal abnormality usually associated with B cell chronic lymphocytic leukaemia (CLL), is presented. This combination of cytogenetic abnormalities has not been reported previously. The lack of lymphadenopathy and hepatosplenomegaly in this patient is more in keeping with stage A0 CLL. This case demonstrates the close clinical and biological relationship between mantle cell lymphoma and CLL.     

Soluble forms of CD21 and CD23 antigens in the serum in B cell chronic lymphocytic leukaemia

By using pairs of monoclonal antibodies (MAbs) to different epitopes on CD21 and CD23 antigens, it has been shown that both antigens are readily detectable in cell-free supernates of cultures of B cells expressing these antigens on the cell surface. The antigens remained in the soluble fraction after high speed centrifugation. Sera from normal individuals contained significant amounts of CD21 antigen, whereas little CD23 antigen was detectable. By contrast CD23 but not CD21 antigen was present in urine. Sera from patients with B cell chronic lymphocytic leukaemia (B-CLL) contained increased amounts of both antigens. The levels were related to the surface expression of antigen on the leukaemic cells and the number of cells in the blood. The possible functional role of soluble forms of B cell antigens and the diagnostic potential of their detection in body fluids are discussed.     

Decreased expression of complement receptor type 2 (CR2) on neoplastic B cells of chronic lymphocytic leukaemia.

Neoplastic cells from 49 patients with B cell chronic lymphocytic leukaemia (B-CLL) were studied and compared with normal peripheral and tonsillar B cells using CD21 monoclonal antibodies. Membrane expression of CR2 was quantified by calibrated flow cytometry and by binding analysis with radiolabelled antibody. Both assays indicate that B-CLL cells express only 30% of the CR2 found on normal B cells. These findings are further evidence of the aberrant phenotype of B-CLL cells.     

Surface glycoproteins as markers of the cellular status of B chronic lymphocytic leukaemia lymphocytes.

In order to indirectly assess T and B cell function in vivo in spontaneously autoimmune MRL mice, IgM plaque forming cell (PFC) responses to the thymus-independent antigens type III pneumococcal polysaccharide (S3) and polyvinylpyrrolidone (PVP) were determined. Both MRL/Mp-lpr/lpr (MRL/l) and MRL/Mp+/+ (MRL/n) mice responded well to S3 and, in fact, low doses of S3 which are not immunogenic in normal strains of mice elicited good responses in MRL mice. PVP was less immunogenic than S3, however, doses of PVP which are considered sub-immunogenic in normal mice did elicit responses in MRL mice. The effect of ageing on S3 and PVP responsiveness in MRL mice was also determined. Responses to S3 and PVP declined minimally in MRL/l mice and were unchanged in MRL/n mice. Amplifier T cell (TA) activity in MRL mice was indirectly assessed by determining the effect on concanavalin A or anti-lymphocyte serum on PFC responses to S3 and PVP. Whereas significant enhancement of the S3 and PVP IgM PFC responses occurred in MRL/n mice, neither method elicited remarkable enhancement in MRL/l mice. The lack of IgM enhancement was not due to altered kinetics of activation nor to a switch to IgG PFC responses. Possible reasons for the apparent dysfunction of TA in MRL/l mice are discussed.     

Mouse red cell rosette formation and the colchicine sensitivity test: relative usefulness in the differential diagnosis of chronic lymphocytic leukaemia and B lymphocytic lymphoma.

Mouse erythrocyte (M) rosette formation and colchicine sensitivity were compared for their ability to differentiate chronic lymphocytic leukaemia (CLL) from B cell non-Hodgkin's lymphoma (NHL) with overspill. Twenty-two cases of CLL and eight of NHL were studied along with 31 normal adults. Results from the patients in both tests differed significantly from the controls but colchicine sensitivity failed to differentiate them further. M Rosettes, on the other hand, while increased in some patients with NHL were, without overlap, much more numerous in those with CLL, and clearly distinguished the two conditions. A significant autolymphocytotoxic effect of plasma from both study groups was also noted which was not found in the controls.     

B cell chronic lymphocytic leukaemia/small lymphocytic lymphoma: role of ZAP70 determination on bone marrow biopsy specimens

BACKGROUND: The course of chronic lymphocytic leukaemia/small lymphocytic lymphoma (CLL/SLL) partly depends on the mutational status of the variable region of immunoglobulin heavy chain genes (IgV(H)), which defines two subgroups of tumours: mutated and unmutated. The expression of zeta-associated protein 70 (ZAP70) is significantly associated with the more aggressive unmutated forms. AIMS: To assess the feasibility of the ZAP70 immunohistochemical test on bone-marrow biopsy (BMB) specimens and to compare the results with those of western blotting (WB) and IgV(H) mutational status assessed on neoplastic cells from peripheral blood. METHODS: 26 patients with CLL/SLL detected on BMB and with known IgV(H) mutational status were selected. ZAP70 was determined by immunohistochemistry (IHC) comparing three antibodies from different sources (Upstate, Cell Signaling, Santa Cruz, California, USA) and two different methods (APAAP and EnVision(+)). In 23 cases, ZAP70 WB results were also available. RESULTS: ZAP70 determination on BMB specimens turned out to be easily feasible with routine procedures with reagents from Upstate and Cell Signaling. The results were concordant with those obtained with WB and mutational status analysis in >80% of the cases with both reagents. Three of four discordant cases were mutated/ZAP70 positive, with two staining weakly for ZAP70 on both WB and IHC. CONCLUSIONS: The study confirms the role of ZAP70 as a possible surrogate of mutational status and emphasises its application in routine diagnostics; it discloses a small subset of discordant cases (mutated/ZAP70 weakly positive) that clinically cluster with the more favourable forms.