Recurrent cytogenetic findings in subsets of patients with chronic lymphocytic leukemia expressing IgG-switched stereotyped immunoglobulins

We report a remarkable association of recurrent (stereotyped) cytogenetic aberrations with two subsets of chronic lymphocytic leukemia (CLL) cases expressing IgG-switched, stereotyped B-cell receptors (BCRs). Comparison with cases with heterogeneous BCRs showed that these recurrent cytogenetic aberrations were subset-biased. These findings further support a role for antigen in CLL development.     

Molecular and clinical features of chronic lymphocytic leukaemia with stereotyped B cell receptors: results from an Italian multicentre study

A fraction of chronic lymphocytic leukaemia (CLL) cases carry highly homologous B-cell receptors (BCR), i.e. characterized by non-random combinations of immunoglobulin heavy-chain variable ( IGHV ) genes and heavy-chain complementarity determining region-3 (HCDR3), often associated with a restricted selection of IGVK/L light chains. Such 'stereotyped' BCR occur more frequently in CLL with unmutated (UM) than mutated (M) IGHV genes. We analysed 1426 IG rearrangements (from 1398 CLL cases) by a clustering driven by HCDR3 similarities. Molecular findings were correlated to time-to-treatment (TTT) and presence of known prognosticators. Sixty-nine clusters (319 IG-rearrangements, 22·4%) with stereotyped BCR were identified. Among 30 confirmed clusters (≥3 IG-rearrangements/cluster), we found 14 novel clusters, of which 11 had M IG rearrangements (M clusters) and predominantly (8/11) used IGHV3 subgroup genes. Recurrent cluster-biased amino acid changes were found throughout IGHV sequences of these 'M clusters'. Regarding clinical outcome: (i) UM CLL from the IGHV1-2/1-3/1-18/1-46/7-4-1/IGKV1-39 cluster had poorer prognosis than UM/M cases, or UM cases using the same IGHV genes but not in clusters; (ii) M CLL from the IGHV3-21/IGLV3-21 cluster had TTT similar to UM CLL, and shorter than M CLL expressing IGHV3-21 but not in cluster. Altogether, our analysis identified additional molecular and clinical features for CLL expressing stereotyped BCR.     

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.     

Transient down-modulation of CD20 by rituximab in patients with chronic lymphocytic leukemia

Lymphoid cells in most patients with chronic lymphocytic leukemia (CLL), when treated with rituximab, become CD20-. This is thought to be due to masking of CD20 by rituximab. We used specific antimouse immunoglobulin antibodies to detect rituximab on the surface of CLL lymphocytes and we demonstrate that rituximab is rarely detectable after therapy. Only 3 of 65 patients with CLL had rituximab detectable on their lymphocytes after rituximab therapy despite the fact that most had no detectable CD20 expression. In vitro mixing of CLL or Raji cells with rituximab demonstrated that rituximab was detectable on the surface of cells due to its binding to CD20. However, the addition of plasma led to the down-modulation of CD20 expression, and the rituximab became undetectable. This down-modulation of CD20 protein expression was associated with a down-modulation of CD20 mRNA. CLL cells that lost their CD20 expression regained CD20 expression after 24 hours in culture. These data suggest that rituximab therapy leads to a substantial but transient down-modulation of CD20 expression and that negativity for CD20 in cells from patients treated with rituximab is not necessarily due to CD20 masking. The importance of this down-modulation in the efficacy of current therapy with rituximab needs further investigation.     

Distinct gene expression profiles in subsets of chronic lymphocytic leukemia expressing stereotyped IGHV4-34 B-cell receptors

Background

Numerous subsets of patients with chronic lymphocytic leukemia display similar immunoglobulin gene usage with almost identical complementarity determining region 3 sequences. Among IGHV4-34 cases, two such subsets with “stereotyped” B-cell receptors were recently identified, i.e. subset #4 (IGHV4-34/IGKV2-30) and subset #16 (IGHV4-34/IGKV3-20). Subset #4 patients appear to share biological and clinical features, e.g. young age at diagnosis and indolent disease, whereas little is known about subset #16 at a clinical level.

Design and Methods

We investigated the global gene expression pattern in sorted chronic lymphocytic leukemia cells from 25 subset/non-subset IGHV4-34 patients using Affymetrix gene expression arrays.

Results

Although generally few differences were found when comparing subset to non-subset 4/16 IGHV4-34 cases, distinct gene expression profiles were revealed for subset #4 versus subset #16. The differentially expressed genes, predominantly with lower expression in subset #4 patients, are involved in important cell regulatory pathways including cell-cycle control, proliferation and immune response, which may partly explain the low-proliferative disease observed in subset #4 patients.

Conclusions

Our novel data demonstrate distinct gene expression profiles among patients with stereotyped IGHV4-34 B-cell receptors, providing further evidence for biological differences in the pathogenesis of these subsets and underscoring the functional relevance of subset assignment based on B-cell receptor sequence features.     

50例慢性淋巴细胞白血病患者临床分期和免疫表型的研究

目的探讨慢性淋巴细胞白血病的免疫表型,临床分期以及它们之间的相互关系。方法采用CD45／SSC双参数散点图设门,应用三色流式细胞术对50例慢性淋巴细胞白血病的骨髓或外周血标本进行免疫分型,并根据临床资料进行临床分期。结果50例中绝大部分慢性淋巴细胞白血病患者表达CD19及CD20阳性率分别为96％和98．0％,T系相关抗原CD,阳性率为71．3％,HLA-DR的阳性率为100％;CD38的表达与临床分期存在显著的统计学差异（P〈0．05）。FMC-7与CD30共表达（P〈0．05）。35例随访病人中,14例死亡。死亡患者与存活患者CD23表达与否存在明显差异（P〈0．05）。结论Binet分期比Rai分期简便易行。CD5,CD19,CD20是B-慢性淋巴细胞白血病特异的免疫表型,CD5、FMC-7、CD13作为预后标志缺乏特异性,CD23和CD38可作为评价病人预后的指标。     

Stereotyped B-cell receptors in one-third of chronic lymphocytic leukemia: a molecular classification with implications for targeted therapies

Mounting evidence indicates that grouping of chronic lymphocytic leukemia (CLL) into distinct subsets with stereotyped BCRs is functionally and prognostically relevant. However, several issues need revisiting, including the criteria for identification of BCR stereotypy and its actual frequency as well as the identification of "CLL-biased" features in BCR Ig stereotypes. To this end, we examined 7596 Ig VH (IGHV-IGHD-IGHJ) sequences from 7424 CLL patients, 3 times the size of the largest published series, with an updated version of our purpose-built clustering algorithm. We document that CLL may be subdivided into 2 distinct categories: one with stereotyped and the other with nonstereotyped BCRs, at an approximate ratio of 1:2, and provide evidence suggesting a different ontogeny for these 2 categories. We also show that subset-defining sequence patterns in CLL differ from those underlying BCR stereotypy in other B-cell malignancies. Notably, 19 major subsets contained from 20 to 213 sequences each, collectively accounting for 943 sequences or one-eighth of the cohort. Hence, this compartmentalized examination of VH sequences may pave the way toward a molecular classification of CLL with implications for targeted therapeutic interventions, applicable to a significant number of patients assigned to the same subset.     

The prognostic value of clinical and laboratory parameters in patients with chronic lymphocytic leukemia

PURPOSE: Chronic lymphocytic leukemia (CLL) is heterogeneous in its clinical course. The aim of the present study was to identify some clinical and laboratory parameters with prognostic value in relation to survival of patients with CLL. PATIENTS AND METHODS: One hundred thirty one patients with CLL, treated in the Clinic of Hematology, Plovdiv, between 1992 and 2003 were studied. The patients' survival was analyzed by using the Kaplan-Meier method. Log-rank test was used to compare cumulative survival functions between different groups of patients. The parameters studied were: age, sex, Rai clinical stage, ECOG Performance status (PS), FAB morphological type, mediastinal lymphadenopathy, abdominal nodal mass, extranodal localization, absolute lymphocyte count, autoimmune hemolytic anemia, and response to therapy. RESULTS: The mean survival of patients was 76 months (95% Confidence Interval, 64-87). The following factors were found to carry significant prognostic value in relation to survival: Rai clinical stage, ECOG PS, mediastinal lymphadenopathy, extranodal localization, FAB morphological type and response to initial standard treatment. CONCLUSION: The prognostic factors identified in the present study can be easily applied in the clinical practice and may be used as a basis for creating mathematical prognostic models.     

Lipoprotein lipase in chronic lymphocytic leukemia: function and prognostic implications

Chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease characterized by the accumulation of a clonal population of B cells in peripheral blood, bone marrow, and lymphoid organs. More than 10 years ago, lipoprotein lipase (LPL) mRNA was identified as being strongly expressed in patients experiencing a more aggressive phenotype, while CLL patients with an indolent disease course lack expression of this marker. Since then, several reports confirmed the capability of LPL to predict CLL disease evolution at the moment of diagnosis. In contrast, data on the functional implications of LPL in CLL are scarce. LPL exerts a central role in overall lipid metabolism and transport, but plays additional, non‐catalytic roles as well. Which of those is more important in the pathogenesis of CLL remains largely unclear. Here, we review the current knowledge on the prognostic and biological relevance of LPL in CLL.     

PD-1/PD-L1 expression and its implications in patients with chronic lymphocytic leukemia

正Objective The observe the expression levels of PD-1/PD-L1 costimulatory molecules and explore the clinical significance in patients with chronic lymphocytic leukemia(CLL).Methods The expression of PD-1/PD-L1 in peripheral blood CD8+T cells,CD4+T cells,CD19+B,and dendrites cells(DC)was detected by flow cytometry in 57 CLL patients and 20 healthy controls.The correla-     

Improving treatment for patients with chronic lymphocytic leukemia

The last two decades have been time of tremendous progress in treatment for patients with chronic lymphocytic lymphoma (CLL). Chemoimmunotherapy (CIT) combining anti-CD20 monoclonal antibodies with purine nucleoside analogs has been a substantial advance for patients with CLL and results in increased response rates, progression-free survival, and overall survival. Despite these improved outcomes, only ≈ 45% of patients achieve a complete remission with CIT and nearly all patients eventually relapse and their remains a need to improve efficacy. Although new combinations of traditional agents may lead to incremental progress, more substantive improvements are likely to result through therapeutic targeting of novel pathways critical to CLL B-cell survival including targeting: (1) leukemia cell apoptotic resistance; (2) survival signals mediated through the B-cell receptor; and (3) nurturing interactions with the microenvironment. In this mini-review, we summarize Mayo Clinic's recent efforts to improve CIT for patients with CLL.     

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.     

p55 and p75 tumor necrosis factor receptors in patients with chronic lymphocytic leukemia.

We studied the expression of the two tumor necrosis factor (TNF) receptors, p55 and p75, on B cells from patients with chronic lymphocytic leukemia (CLL), and the presence of soluble TNF receptors in serum. Expression of membrane-associated receptors was quantified by double labeling of peripheral blood mononuclear cells (PBMC) with monoclonal antibodies against CD19 and p55/p75 TNF receptors and flow cytometry. A high fraction of the CD19+ cells expressed the p55 receptor (44% +/- 34% [SD]) and p75 receptor (61% +/- 31%). In healthy controls, 0% to 1% of the CD19+ cells expressed the p55 receptor and 0% to 10% expressed the p75 receptor. Incubation of CD19+ cells with 10 ng/mL of TNF increased the incorporation of thymidine in 11 patients tested, and this was decreased to 65% (P < .05) by antibodies to the p55 receptor or the p75 receptor, and to 35% +/- 7% (P < .001) when both antibodies were combined. With an enzyme-linked immunoassay, we measured soluble TNF receptors in serum from CLL patients. The mean level of p55 receptors was increased to 12.9 +/- 8.9 ng/mL (P < .000001 v normal). The mean level of p75 receptors was increased to 13 +/- 24 ng/mL (P = .01 v normal). The membrane expression of the two receptors was positively correlated (r +/- 97, P < .01); however, there was no correlation between membrane expression and serum concentration of either receptor. Autologous serum containing high levels of soluble TNF receptors inhibited TNF-induced proliferation of CD19+ cells. In conclusion, we have demonstrated that neoplastic cells from patients with CLL have increased expression of p55 and p75 TNF receptors, and that both receptors mediate signal to proliferation. Furthermore, serum from CLL patients has elevated levels of soluble TNF receptors, which may counteract the proliferative effect of TNF.     

T-cell responses against chronic lymphocytic leukemia cells: implications for immunotherapy

Chronic lymphocytic leukemia (CLL) cells are ineffective antigen-presenting cells (APCs) although CD40-activated CLL cells can stimulate proliferation of autologous and allogeneic T cells. We examined the antigen-presenting capacity of CD40-activated CLL cells as well as dendritic cells pulsed with apoptotic bodies of CLL cells to generate autologous and allogeneic immune responses against CLL cells. Both APC types were capable of generating T-cell lines that proliferate specifically in response to unstimulated CLL cells. Whereas cytotoxic responses against stimulated and unstimulated CLL cells could be repeatedly generated by allogeneic healthy donors, autologous cytotoxic immune responses against CD40-activated and native CLL cells were rarely detected. However, T cells isolated from patients with CLL could recognize and lyse allogeneic stimulated and unstimulated CLL cells, demonstrating that cytotoxic T cells from these tumor-bearing patients are functionally intact.