The use of CD38 expression by monoclonal B lymphocytes as a prognostic factor in B-cell chronic lymphocytic leukemia

In B-cell chronic lymphocytic leukemia (B-CLL) the Rai and Binet staging criteria are not always able to accurately predict the prognosis of each patient. Rapidly evolving, violent disease is often seen in the so-called "good-prognosis" group, which highlights the need of additional and more refined prognostic markers. Several of these markers are described in the literature, with varying abilities to predict patient survival. Among the promising prognostic markers is flowcytometric analysis of CD38 on the monoclonal B cells in CLL. Several studies have shown that expression of CD38 is associated with a decreased overall-, or progression free survival. CD38 expression may be analyzed as percentage positive cells or as antibodies bound per cell. Addition of CD38 to the flow cytometry antibody panel for B-CLL analysis is a relatively easy way to obtain important prognostic information.     

Reactivity of B leukemic lymphocytes of patients with chronic lymphocytic leukemia to PWM and PHA

The ability of leukemic B lymphocytes to proliferate after in vitro stimulation with PWM and PHA was studied in 15 patients with chronic lymphocytic leukemia. Peripheral blood lymphocytes of five healthy subjects as well as purified normal B lymphocytes were used as controls. Leukemic lymphocytes of all donors expressed the same membrane phenotype, M receptor, and B7 and Ia antigens. The lymphocyte populations investigated were not completely free from myelomonocytic cells and contained small numbers of T lymphocytes. DNA synthesis was determined on days 3, 5, and 7 of culture by measuring the incorporation of tritiated thymidine. PWM-induced proliferation of leukemic B lymphocytes of nine patients was within normal limits, while the response of leukemic cells of six patients was very low. On the other hand, all CLL donors responded very well to PHA. Moreover, the response of leukemic B lymphocytes was significantly higher than the response of normal B cells. It was concluded that leukemic B lymphocytes of CLL patients are capable of proliferation after stimulation with PWM and PHA. The mechanisms underlying these responses to PWM and PHA are likely to be different.     

Genetic features of B-cell chronic lymphocytic leukemia

The genetic features of B-cell chronic lymphocytic leukemia (CLL) are currently being reassessed by molecular cytogenetic techniques such as fluorescence in situ hybridization (FISH). Conventional cytogenetic studies by chromosome banding are difficult in CLL mainly because of the low in vitro mitotic activity of the tumor cells, which leads to poor quantity and quality of metaphase spreads. Molecular genetic analyses are limited because candidate genes are known for only a few chromosomal aberrations that are observed in CLL. FISH was found to be a powerful tool for the genetic analysis of CLL as it overcomes both the low mitotic activity of the CLL cells and the lack of suitable candidate genes for analysis. Using FISH, the detection of chromosomal aberrations can be performed at the single cell level in both dividing and non-dividing cells, thus circumventing the need of metaphase preparations from tumor cells. Probes for the detection of trisomies, deletions and translocation breakpoints can be applied to the regions of interest with the growing number of clones available from genome-wide libraries. Using the interphase cytogenetic FISH approach with a disease specific set of probes, chromosome aberrations can be found in more than 80% of CLL cases. The most frequently observed abnormalities are losses of chromosomal material, with deletions in band 13q14 being the most common, followed by deletions in 11q22-q23, deletions in 17p13 and deletions in 6q21. The most common gains of chromosomal material are trisomies 12q, 8q and 3q. Translocation breakpoints, in particular involving the immunoglobulin heavy chain locus at 14q32, which are frequently observed in other types of non-Hodgkin's lymphoma, are rare events in CLL. Genes affected by common chromosome aberrations in CLL appear to be p53 in cases with 17p deletion and ataxia telangiectasia mutated (ATM), which is mutated in a subset of cases with 11q22-q23 aberrations. However, for the other frequently affected genomic regions, the search for candidate genes is ongoing. In parallel, the accurate evaluation of the incidence of chromosome aberrations in CLL by FISH allows the correlation of genetic abnormalities with clinical disease manifestations and outcome. In particular, 17p abnormalities and deletions in 11q22-q23 have already been shown to be among the most important independent prognostic factors identifying subgroups of patients with rapid disease progression and short survival. In addition, deletion 17p has been associated with resistance to treatment with purine analogs. Therefore, genetic abnormalities may allow a risk assessment for individual patients at the time of diagnosis, thus giving the opportunity for a risk-adapted management.     

Functional proteomic insights in B-cell chronic lymphocytic leukemia

Introduction: B cell chronic lymphocytic leukemia (B-CLL) is a hematological malignancy considered as the most common leukemia in the Western world. The understanding of B cell differentiation is crucial for the diagnosis, prognosis, and treatment of the disease.

Areas covered: In this review, B-cell ontogeny and its relation with the CLL development, in combination with the proteomic approaches which could provide a deep characterization of the disease through the characterization of the cellular signaling pathways involved in the pathological cells is described.

Expert commentary: Although conventional strategies (genome sequencing, morphology assays, and immunophenotyping by flow cytometry and/or immunochemistry) have allowed the establishment of the disease stage based on different parameters, it is still necessary to utilize novel approaches (e.g., proteomics) that have the potential to simultaneously analyze thousands of molecules to improve understanding of CLL.     

Menstruation protects women from B-cell chronic lymphocytic leukemia

B-cell chronic lymphocytic leukemia (B-CLL) affects women and men with different frequency: men show a more than double as high risk to acquire this disease than women. The reason for this sex-related difference is unknown. It is proposed here that menstruation confers advantages to women in two ways: i) early stage B-CLL cells and/or their potential precursors are partially removed from the body with menstrual bleeding which includes shedding of endometrial tissue; and ii) during degradation of the remaining endometrial tissue an immune response against B-CLL is triggered. The regular reduction of potential B-CLL cells throughout pre-menopausal life as well as the immunization against B-CLL would enable the female organism to better control outbreak and course of the disease. Both processes depend on specific binding of the leukemic cells to the endometrial tissue. CD23 expressed on the surface of B-CLL cells is suggested to mediate binding to the vitronectin receptor/CD47 expressed on endometrium. The menstrual inflammatory process includes danger signals that might facilitate initiation of an anti-leukemia immune response. Menstrual immunization might explain sex-related differences in clinical features of other malignancies as well and might therefore have broad implications for the development of individualized therapies.     

Some oncogene and tumour suppressor gene protein products expression in B-cell chronic lymphocytic leukaemia

The expression of Bcl-2, P53 proteins and known markers of proliferation, namely proliferating cell nuclear antigen (PCNA) and Ki67, in 29 patients with B-cell chronic lymphocytic leukaemia (B-CLL) was investigated. All leukaemic patients were classified, and immunophenotyped by the two-colour immunofluorescence method with the use of fluorocytometry. B-CLL was heterogeneous in the range of biological parameters of tumour cells. B-CLL patients manifested 34% positive Ki67 and 61% PCNA expression, whereas Bcl-2 and P53 positivity was 81% and 42%, respectively. The level of intracellular expression of Bcl-2 and P53 proteins did not depend on the stage of disease estimated by routine methods. Ki67 and PCNA expression was significantly higher in B-CLL patients with more advanced stages of the disease. A statistically significant correlation was established between their mutual expression.     

Apoptotic effect of fludarabine is independent of expression of IAPs in B-cell chronic lymphocytic leukemia

Despite the efficiency of fludarabine in the induction of clinical responses in B-cell chronic lymphocytic leukemia (B-CLL) patients, resistance to this drug has been documented. The present study tested whether resistance to fludarabine is related to the expression of inhibitor of apoptosis proteins (IAPs) family members. We analyzed the expression of c-IAP1, c-IAP2 and XIAP, by immunocytochemistry, in 30 blood samples from B-CLL patients and correlated protein expression to fludarabine-induced apoptosis estimated by an annexin-V assay. Expression of c-IAP1, c-IAP2 and XIAP were found predominantly in the cytoplasm, and a wide range of staining intensities was observed among distinct samples. No correlation was found between the levels of IAPs expression and prognostic factors such as age, gender, lymphocyte doubling time, white blood cell count or previous treatment. The expression of IAPs also failed to predict the sensitivity to fludarabine-induced apoptosis. Alternative pathways of cell death may explain the independence of fludarabine-induced apoptosis from the high expression of IAPs.     

Bone marrow angiogenesis and proliferation in B-cell chronic lymphocytic leukemia

OBJECTIVE: To assess angiogenesis and the proliferative activity of bone marrow in patients with chronic lymphocytic leukemia (CLL) in relation to the bone marrow infiltration pattern. STUDY DESIGN: Bone marrow samples were obtained by trephine biopsy from 46 patients with B-cell CLL (B-CLL). Infiltration pattern was diffuse in 20 patients and nondiffuse--i.e., nodular, interstitial or mixed--in the remaining 26 patients. Ten normal bone marrow samples were used as a control group. Studies were carried out by immunohistochemical staining of paraffin-embedded bone marrow samples. Angiogenesis was assessed in the zones of highest vascular density (hot spots), visualized by the expression of endothelial antigen CD34 and expressed as a number of microvessels per high-powerfield (hpf) (final magnification, 400x). Proliferative activity was estimated by the expression of nuclear protein Ki-67, cyclin A and mean number of nucleolar organizer regions (AgNORs). RESULTS: Microvessel density was higher in B-CLL marrow than in normal marrow (30.1 and 16.44 per hpf, respectively) and was higher in the diffuse than nondiffuse pattern (33.6 and 27.5 per hpf, respectively). B-CLL bone marrow also showed higher proliferative activity, as assessed by mean number of AgNORs, than did normal marrow (1.52 and 1.25, respectively) and a higher mean percentage of cyclin A-positive cells (7.5 and 6.8, respectively). In contrast, mean Ki-67 expression was similar in B-CLL and the control group. Mean AgNORs number, Ki-67 and cyclin A-positive cell percentage were significantly higher in B-CLL marrow with a diffuse as compared to nondiffuse involvement pattern (AgNORs, 1.75 and 1.35; cyclin A, 9.27% and 3.95%; Ki-67, 34.9% and 23.3%, respectively). CONCLUSION: Our results indicate enhancement of bone marrow angiogenesis in B-CLL and a relationship between microvessel density and the bone marrow infiltration pattern. The study points also to a possible relationship between the bone marrow infiltration pattern and proliferative activity of bone marrow cells.     

Modulation of apoptosis by cytokines in B-cell chronic lymphocytic leukemia.

B-cell chronic lymphocytic leukemia (B-CLL) is characterized by the slow and progressive accumulation of monoclonal apparently mature, CD5(+) B lymphocytes. The majority of circulating cells appear to be nondividing, and it has been suggested that a prolonged life span is mainly responsible for the accumulation of the leukemic cells. However, spontaneous programmed cell death by apoptosis occurs when B chronic lymphocytic leukemia cells are cultured in vitro. This may be because of the lack of an unidentified essential cytokine present in vivo. Thus, we investigate interleukin-2 (IL-2), IL-4, IL-6 and IL-10 in vitro effects on apoptosis of B cells from 32 previously untreated patients with B-CLL in initial clinical stages. B cells were isolated from peripheral blood, and apoptosis was measured in these cells immediately after isolation and following incubation in vitro, without and with the different cytokines, for 24 and 48 h. Distribution of cellular DNA content and quantitative analysis of apoptosis were determined by standard propidium iodide staining and flow cytometry. Spontaneous apoptosis occurred in B-CLL cells incubated in vitro in the absence of cytokines. Our results indicate that both IL-2 and IL-4, but not IL-6, inhibit in vitro apoptosis in a large percentage of B-CLL patients. IL-10 increases in vitro apoptotic cell number in stage 0 patients, but not in stage I and II. These data support the hypothesis that IL-2 or IL-4, may be cell survival factors in vivo and that IL-10 might be a candidate for immune therapy of early B-CLL.     

Different maturation capabilities of chronic lymphocytic leukaemia lymphocytes in vitro

Peripheral blood lymphocytes from five patients with B-derived chronic lymphocytic leukaemia were stimulated by Staphylococcus aureus strain Cowan together with T cell mitogen phytohaemagglutinin in 5-9 days suspension cultures. The responses of B lymphocytes were studied on a T cell depleted subpopulation, obtained from harvested lymphocyte cultures using the sheep red blood cell rosette technique. Proliferation tests were performed using a 3H-TdR blast cell index. The maturation process of B-lymphocytes was examined with cytoplasmic Ig studied by FITC-conjugated antisera. Results analysed indicate various degrees of maturation of B cells in different patients.     

Cortisol catabolism by lymphocytes of patients with chronic lymphocytic leukemia

A low rate of catabolism of cortisol by lymphocytes correlates with high sensitivity of the cells to the steroid and causes them to die at a greater rate than control samples. Since lymphocytes of patients with chronic lymphocytic leukemia respond to treatment with glucocorticosteroids and are cortisol sensitive, we attempted to see whether their capability to catabolize cortisol differs from that of normal lymphocytes. No difference was found between the two groups of cells with regard to the pattern of cortisol metabolites. However, the lymphocytes of the chronic lymphocytic leukemia groups showed a total cortisol catabolism per cell that was significantly lower than that of the control group. Patients with low lymphocyte count in peripheral blood showed a relatively higher cortisol metabolism by lymphocytes per cell than those with high counts.     

Cytogenetic and molecular cytogenetic investigations in relapse of B-cell chronic lymphocytic leukemia

Сhromosomal abnormalities have been analyzed in bone marrow cells of 61 patients with relapse of B-cell chronic lymphocytic leukemia. The cytogenetic results have allowed the structural stratification of the obtained karyotypes into ten groups of clones: normal, normal/near tetraploid, abnormal/normal, abnormal/ near tetraploid/normal, evolution of clonal chromosome abnormalities; evolution of clonal chromosome abnormalities/normal, evolution of clonal chromosome abnormalities/near tetraploid/normal, independent clones, independent/normal clones; and independent/near tetraploid/normal clones. The identified structural rearrangements included translocations, deletions, insertions, and duplications; however, deletions with the involvement of bands 17p12, 13q12–q14, 11q14, and 11q23 dominated (63.8%). The application of i-FISH helped to show the presence of one to four abnormalities per karyotype. The identified cytogenetic and molecular cytogenetic rearrangements may signify a multilevel nature of the process underlying the development of resistant karyotypes. The results obtained under both methods have revealed the presence of a heterogenic cell population with possibly different levels of chemotherapy resistance.     

Comparison of different markers on blood lymphocytes of chronic lymphocytic leukemia

Mononuclear cells (MNC) of 17 patients suffering from B chronic lymphocytic leukemia (B-CLL) were analysed by various immunological methods. The B cell nature of CLL cell was determined by classical tests (MRBC-rosette-test, immunofluorescence test for detection of membrane bound immunoglobulins). The cytochemical detection of the new T-cell marker dipeptidyl peptidase IV (DP IV) was found to be suitable for the characterization of B-CLL. The B-CLL cells showed granular pattern of alpha-naphthylacetate esterase (ANAE) reaction and binding of the monoclonal pan T antibody BL-T2. These non typical reactions for normal B lymphocytes can be used for differential diagnosis of B-CLL in combination with other reliable T cell markers. Avoiding the separation of T cells, the mixed rosette assay was used to enumerate Fc-IgG-receptor bearing T(TG) and non T cells. Both cell populations were found to be significantly elevated in MNC of B-CLL.     

Cytotoxic lymphocytes in B-cell chronic lymphocytic leukemia. A flow cytometric study of peripheral blood, lymph nodes and bone marrow.

The occurrence of cytotoxic lymphocyte subpopulations (i.e., CD 16+, CD 57+ and cytotoxic CD 8+) wa 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 CD 57+ 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.     

ICOS gene polymorphisms in B-cell chronic lymphocytic leukemia in the Polish population

There is strong evidence that altered immunological function entails an increased risk of B-cell chronic lymphocytic leukemia (B-CLL). The main mechanism of an anti-tumor response depends on T-cell activation. Unlike the constitutively expressed CD28, inducible costimulatory molecule (ICOS) is expressed on the T-cell surface after activation. ICOS enhances all the basic T-cell responses to a foreign antigen, namely proliferation, secretion of lymphokines, the upregulation of molecules that mediate cell-cell interaction, and effective help for antibody secretion by B cells. ICOS is essential for both efficient interaction between T and B cells and normal antibody responses to T cell-dependent antigens. It does not upregulate the production of interleukin-2, but superinduces the synthesis of interleukin-10. Our previous results indicated the ICOS gene has a role as a susceptibility locus to B-CLL. Therefore an extended study was undertaken to evaluate the association between four ICOS polymorphisms (which were recently described as functional ones) and susceptibility to B-CLL in the Polish population. A case-control study of 296 individuals, including 146 B-CLL patients, was conducted on four polymorphisms in the ICOS gene. Genotyping of the polymorphisms ICOS ISV1+173T>C (rs10932029), ICOSc.1624C>T (rs10932037), ICOSc.2373G>C (rs4675379), and ICOSc.602A>C (rs10183087) was carried out using allelic discrimination methods with the TaqMan SNP Genotyping Assay. There were no statistically significant differences in the allele, genotype, or haplotype distributions between B-CLL patients and healthy controls for any of the investigated polymorphic markers in the ICOS gene. However, we noted that patients carrying genotype ICOS ISV1+173T>C [TT], ICOSc.602A>C [AA], ICOSc.1624C>T [CC], and ICOSc.2373G>C [GG] have a decreased frequency of progression to a higher Rai stage during 60-month follow-up (21.35% vs. 40.8%, p = 0.013) compared to other individuals. This indicates that the investigated polymorphisms do not modulate the risk of B-CLL in the Polish population, but are associated with disease dynamics, in particular with the time to Rai stage progression.     

Acid phosphatase cytochemistry of mitogen-transformed normal and chronic lymphocytic leukemia lymphocytes

Acid phosphatase cytochemistry was performed on lymphocytes stimulated in vitro with phytohemagglutinin, pokeweed mitogen, or concanavalin A. These electron microscopic studies demonstrated that activated lymphocytes from both normals and patients with chronic lymphocytic leukemia (CLL) had an increased number of lysosomes relative to resting cells. At the time of maximum thymidine incorporation, a reduced number of lysosomes was present in many transformed CLL lymphocytes, mainly medium-sized blast cells, in comparison to transformed normal cells. The findings demonstrate a lysosomal abnormality in phytomitogen transformed CLL lymphocytes which may be related to functional defects of these cells or to an incomplete transformation of a residual population of normal lymphocytes.     

Bone marrow histopathologic patterns and immunologic phenotype in B-cell chronic lymphocytic leukaemia

The present work analyzes the clinicobiological and immunological characteristics - the latter hitherto unexplored - of the different bone marrow histopathological patterns of the B-cell chronic lymphocytic leukaemia (B-CLL). In addition, we studied whether any or some of these parameters were able to predict the probability of a particular pattern of bone marrow involvement appearing. Of the 100 B-CLL cases studied 41 had a diffuse pattern and 59 were non-diffuse - interstitial 27, nodular 11 and mixed 21 -. Neither clinical nor immunological differences were observed among the distinct non-diffuse patterns. The patients in the diffuse group displayed an increased incidence of mu+ isotype and a higher proportion of HLA-DR and HAN-PC 1 positive cells while, conversely, reactivity with the FMC 8 McAb was lower. In addition, patients with a diffuse pattern of BM involvement displayed features of a more extensive disease: a higher incidence of adenopathies (p less than 0.05), hepatomegaly (p less than 0.01), splenomegaly (p less than 0.01), anaemia (p less than 0.01) and thrombopenia (p less than 0.01) as well as higher levels of peripheral blood lymphocytosis (p less than 0.05) and a higher percentage of BM lymphocytic infiltration (p less than 0.001). Multiple regression analysis showed that thrombopenia and splenomegaly were the two most important features in predicting the probability of a diffuse pattern.     

Induced differentiation of subpopulations of leukemic blood cells in patients with chronic B-cell lympholeukemia

M+-cell subpopulation forming M-rosettes and M(-)-cell subpopulation not forming M-rosettes were revealed in the peripheral blood of patients with B-cell chronic lympholeukemia (B-CLL) by means of mouse red blood rosette formation test. M+-subpopulation contained a larger percentage of cells expressing Ia-like antigens, as compared to M- subpopulation. On the other hand, the latter contained a significantly higher amount of cytoplasmatic immunoglobulin-containing cells. M+ appeared to be less mature than M- cells. Cells of B-CLL patients had a heterogeneous response to 12-0-tetradecanoylphorbol-13-acetate (TPA). Less mature cells with surface immunoglobulin expression did differentiate, while more mature cells containing cytoplasmatic immunoglobulins did not. Differentiation was accompanied by the acquisition of characteristics peculiar to more mature cells, i. e. cytoplasmatic immunoglobulin accumulation. Subpopulations of M+ and M- cells from each patient also had a different pattern of response to TPA: less mature M+ cells did differentiate, while more mature M- cells did not. Maturation of less mature leukemia cells, as the disease progresses, is suggested to result in a heterogeneous pattern of immunological B-CLL phenotype.