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Journal articles on the topic 'B-CLL'

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Published: 4 June 2021

Last updated: 15 June 2025

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Parker, Anton, Shilu Amin, Tricia Zwiefelhofer, et al. "Epigenetic Regulation of ZAP70 in Chronic Lymphocytic Leukemia." Blood 112, no. 11 (2008): 2246. http://dx.doi.org/10.1182/blood.v112.11.2246.2246.

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Abstract ZAP70 expression has been shown to be involved in enhanced signalling and more aggressive disease in a subset of CLL. Mechanisms regulating ZAP70 expression are unknown. We have shown previously that despite the absence of a 5’ CpG island, the methylation status of a small region of CpG dinucleotides (CpGs) correlates with the transcriptional state of the gene in both normal lymphocytes and B cell leukemias. Quantitative methylation analysis of 605 CpGs across the 28kb genomic region spanning ZAP70 was performed by MassARRAY on a panel of 17 CLL tumor cell samples, 4 lymphoid cell lines and B cell, T cell and myeloid cell samples pooled from 3 normal individuals. All samples showed hypermethylation of the gene body and of the gene’s two 3’ CpG islands. However, there was variability between samples in the methylation of 12 consecutive CpGs within a 1kb predicted promoter region (PPR), spanning the transcription start site (TSS) and in the methylation of 24 consecutive CpGs in an adjacent 1kb differentially methylated region (DMR), downstream of the TSS. The methylation of the PPR and DMR, together with the expression status of the samples, suggested four different states for the gene (Table 1). Table 1 - ZAP70 gene states defined by ZAP70 expression status and methylation of the PPR and DMR. MEAN CpG METHYLATION (%) SAMPLE ZAP70 EXPRESSION STATUS PPR DMR GENE STATE NAMALWA − 65 82 I B CELLS − 48 82 I MYELOID − 53 80 I CLL6 − 4 86 II CLL7 − 5 75 II CLL8 − 12 78 II CLL10 − 12 62 II CLL11 − 4 62 II CLL12 − 21 77 II HBL2 − 18 60 II CLL13 + 4 40 III CLL14 + 5 46 III CLL15 + 7 45 III CLL16 + 9 43 III CLL17 + 5 56 III NALM6 + 8 52 III CLL1 + 3 4 IV CLL2 + 3 4 IV CLL9 + 4 6 IV CLL4 + 3 8 IV CLL5 + 4 16 IV CLL3 + 4 17 IV JURKAT + 3 4 IV T CELLS + 9 13 IV Bisulphite cloning and sequencing of a PCR amplicon spanning an exon1 C/A SNP (rs2276645) and the PPR/DMR junction was performed together with cDNA pyrosequencing of rs2276645 on the five CLL tumor samples identified with gene state III. All samples showed allele specific methylation (ASM) of the A allele within the DMR and almost complete restriction of ZAP70 expression to the hypomethylated C allele. Bisulphite pyrosequencing of two DMR CpGs in purified leukocyte populations from these cases showed that ASM appears restricted to CLL cells, with hypermethylation and hypomethylation of the myeloid and T cells respectively (Table2). This suggests that while methylation of the DMR is sufficient for allele restriction, ASM does not result from imprinting and may be restricted to CLL tumor cells. Table 2 – Mean methylation of 2 DMR CpGs in leukocyte populations from CLL patients with known ASM of the DMR in their tumor cells. MYELOID CELLS CLL CELLS T CELLS PATIENT CD15 (%) METHYLATION(%) CD19 (%) METHYLATION (%) CD2 (%) METHYLATION (%) CLL13 83 90 98 59 71 21 CLL14 98 99 98 50 88 10 CLL15 88 84 93 45 85 24 CLL16 99 96 99 52 82 18 CLL17 92 89 98 49 90 22 Native chromatin immunoprecipitation (N-ChIP) using anti-AcH3, H3K14Ac and H3K14Me2 antibodies was performed on the 4 cell lines and tumor cells from CLLs 1, 2, 6, 7, 13 and 14 from the MassARRAY series. PCR for amplicons across the PPR and DMR showed the presence of all 3 histone modifications in ZAP70 expressing JURKAT and NALM6 cells but these modifications were absent in the ZAP70 negative NAMALWA and HBL2 cells. In contrast, all 6 CLL samples showed enrichment for all 3 modifications, regardless of gene state, suggesting an open, active/permissive chromatin structure, despite clear differences in methylation of the DMR. Further bisulphite pyrosequencing and N-ChIP of NAMALWA and HBL2 cells cultured for 6 days in the presence of 0.5μM Decitibine showed concomitant DMR demethyaltion, increased AcH3 within the DMR and up regulation of ZAP70 expression, all of which were reversed when the drug stimulus was removed. Taken together this data suggests that ZAP70 is regulated by epigenetic mechanisms, with the methylation status of a small DMR playing a key role, sufficient to differentiate the transcriptional activity of two alleles within a single cell. It is apparent that the gene is primed for expression in all CLL cells and that methylation of the DMR is part of the key switching process between active transcription and silencing. The differences in DMR methylation between an individual’s expressing T cells and CLL cells, suggests that differences may exist in the mechanism of regulation between T and B cells and raises the possibility that such differences could be exploited as targets for therapy.

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Silber, R., CM Farber, E. Papadopoulos, et al. "Glutathione depletion in chronic lymphocytic leukemia B lymphocytes." Blood 80, no. 8 (1992): 2038–43. http://dx.doi.org/10.1182/blood.v80.8.2038.2038.

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Abstract Glutathione (GSH) content may be the major determinant of a cell's sensitivity to cytotoxic alkylating agents. In the present study, the GSH concentration was determined in lymphocytes isolated from the blood of normal subjects and patients with chronic lymphocytic leukemia (CLL). Comparable levels were found in both types of cells. Incubation for 20 hours led to a decrease in GSH to 51% of baseline values in CLL B cells. Under the same conditions, normal B- or T-lymphocyte GSH content remained constant. GSH depletion was shown to be a characteristic of the B-CLL B lymphocyte. It was not found in the T cells of patients with B-CLL or in cells from patients with T-CLL. Chlorambucil (CLB) contributes to the decrease in GSH in B-CLL lymphocytes; after incubation with the drug, lower levels of GSH were found than in the normal B or T lymphocytes, B-CLL T cells, or T-CLL (CD4 or CD8) cells. GSH depletion of CLL B lymphocytes may be related to the greater therapeutic efficacy of CLB in B-CLL than in T-CLL.

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Glutathione (GSH) content may be the major determinant of a cell's sensitivity to cytotoxic alkylating agents. In the present study, the GSH concentration was determined in lymphocytes isolated from the blood of normal subjects and patients with chronic lymphocytic leukemia (CLL). Comparable levels were found in both types of cells. Incubation for 20 hours led to a decrease in GSH to 51% of baseline values in CLL B cells. Under the same conditions, normal B- or T-lymphocyte GSH content remained constant. GSH depletion was shown to be a characteristic of the B-CLL B lymphocyte. It was not found in the T cells of patients with B-CLL or in cells from patients with T-CLL. Chlorambucil (CLB) contributes to the decrease in GSH in B-CLL lymphocytes; after incubation with the drug, lower levels of GSH were found than in the normal B or T lymphocytes, B-CLL T cells, or T-CLL (CD4 or CD8) cells. GSH depletion of CLL B lymphocytes may be related to the greater therapeutic efficacy of CLB in B-CLL than in T-CLL.

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Goodman, Alice. "B-CLL." Oncology Times &NA;, Supplement (2006): 21. http://dx.doi.org/10.1097/01.cot.0000316106.04463.a9.

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Silber, R., B. Degar, D. Costin, et al. "Chemosensitivity of lymphocytes from patients with B-cell chronic lymphocytic leukemia to chlorambucil, fludarabine, and camptothecin analogs." Blood 84, no. 10 (1994): 3440–46. http://dx.doi.org/10.1182/blood.v84.10.3440.3440.

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Abstract Chemosensitivity of B lymphocytes, obtained from 65 patients with B- cell chronic lymphocytic leukemia (B-CLL), Rai stages 0 through IV, was determined using the MTT assay. The results were expressed by the drug concentration required for 50% inhibition of cell viability (IC50). The cytotoxicity of chlorambucil (CLB) was compared with that of fludarabine and the DNA topoisomerase I inhibitors, camptothecin, 9- aminocamptothecin, 10,11-methylenedioxy-20(S)-camptothecin (10,11-MDC) and 9-amino-10,11-methylenedioxy-20(S)-campthothecin (9-A-10,11-MDC), and topotecan. Considerable heterogeneity in sensitivity to CLB was observed, with a median IC50 of 40.5 mumol/L in untreated patients. B- CLL cells from patients treated with CLB had a significantly higher median IC50 of 86.0 mumol/L (P < .01). Untreated as well as CLB-treated patients were divided into two subsets. For the purpose of this study, B-CLL lymphocytes with an IC50 CLB of less than 61.0 mumol/L were designated as “sensitive” and those with an IC50 CLB of > or = 61.0 mumol/L were designated as “0resistant.” After baseline assays, 15 untreated patients received CLB; after treatment, the IC50 increased in B-CLL lymphocytes from 13 of 15 patients. The response to CLB treatment, determined by its effect on the absolute lymphocyte count and by the Eastern Cooperative Oncology Group clinical criteria, was significantly better in patients whose lymphocytes had an IC50 CLB of less than 61.0 mumol/L before therapy (P < .01). B-CLL lymphocytes also had a variable degree of sensitivity in vitro to each of the other drugs. There was significant cross-resistance between CLB and fludarabine (P < 0.01). Whereas only 29% of CLB-resistant B-lymphocyte specimens obtained from individual patients were sensitive to fludarabine in vitro, 52% and 67% of CLB-resistant lymphocyte samples were sensitive to 10,11-MDC and 9-A-10,11-MDC, respectively. We have previously reported that p53 gene mutations were associated with aggressive B-CLL and a poor prognosis. B lymphocytes from seven patients with these mutations were resistant to CLB, and five of six were resistant to fludarabine. Lymphocytes from four of seven were resistant to 10,11-MDC, and three of four were resistant to 9-A-10,11- MDC. This study implies that the MTT assay may be useful in identifying subsets of CLL patients resistant to conventional chemotherapy. However, definitive conclusions can not be drawn in view of the small number of patients studied prospectively. In addition, these results suggest the potential of camptothecin-based therapy for patients unresponsive to standard treatment.

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Chemosensitivity of B lymphocytes, obtained from 65 patients with B- cell chronic lymphocytic leukemia (B-CLL), Rai stages 0 through IV, was determined using the MTT assay. The results were expressed by the drug concentration required for 50% inhibition of cell viability (IC50). The cytotoxicity of chlorambucil (CLB) was compared with that of fludarabine and the DNA topoisomerase I inhibitors, camptothecin, 9- aminocamptothecin, 10,11-methylenedioxy-20(S)-camptothecin (10,11-MDC) and 9-amino-10,11-methylenedioxy-20(S)-campthothecin (9-A-10,11-MDC), and topotecan. Considerable heterogeneity in sensitivity to CLB was observed, with a median IC50 of 40.5 mumol/L in untreated patients. B- CLL cells from patients treated with CLB had a significantly higher median IC50 of 86.0 mumol/L (P < .01). Untreated as well as CLB-treated patients were divided into two subsets. For the purpose of this study, B-CLL lymphocytes with an IC50 CLB of less than 61.0 mumol/L were designated as “sensitive” and those with an IC50 CLB of > or = 61.0 mumol/L were designated as “0resistant.” After baseline assays, 15 untreated patients received CLB; after treatment, the IC50 increased in B-CLL lymphocytes from 13 of 15 patients. The response to CLB treatment, determined by its effect on the absolute lymphocyte count and by the Eastern Cooperative Oncology Group clinical criteria, was significantly better in patients whose lymphocytes had an IC50 CLB of less than 61.0 mumol/L before therapy (P < .01). B-CLL lymphocytes also had a variable degree of sensitivity in vitro to each of the other drugs. There was significant cross-resistance between CLB and fludarabine (P < 0.01). Whereas only 29% of CLB-resistant B-lymphocyte specimens obtained from individual patients were sensitive to fludarabine in vitro, 52% and 67% of CLB-resistant lymphocyte samples were sensitive to 10,11-MDC and 9-A-10,11-MDC, respectively. We have previously reported that p53 gene mutations were associated with aggressive B-CLL and a poor prognosis. B lymphocytes from seven patients with these mutations were resistant to CLB, and five of six were resistant to fludarabine. Lymphocytes from four of seven were resistant to 10,11-MDC, and three of four were resistant to 9-A-10,11- MDC. This study implies that the MTT assay may be useful in identifying subsets of CLL patients resistant to conventional chemotherapy. However, definitive conclusions can not be drawn in view of the small number of patients studied prospectively. In addition, these results suggest the potential of camptothecin-based therapy for patients unresponsive to standard treatment.

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Matvieieva, A. S., L. M. Kovalevska, and E. V. Kashuba. "The SMAD4 transcription factor shows cytoplasmic retention in B-cells of patients with chronic lymphocytic leukemia (CLL)." Faktori eksperimental'noi evolucii organizmiv 22 (September 9, 2018): 144–48. http://dx.doi.org/10.7124/feeo.v22.939.

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Aim. To illuminate the reason of inactivity of the TGFB-SMAD2/3 pathways in CLL cells. Methods. CLL cells were isolated from peripheral blood of CLL patients, using gradient centrifugation at the ficoll. Expression and cellular localization of SMAD2, 3 and 4 proteins were analyzed by fluorescence microscopy, using specific antibodies. Results. The SMAD2 protein was basically not expressed in CLL cells, in contrast to B cells, isolated from the peripheral blood of a healthy donor. Moreover, the SMAD3 and SMAD4 proteins were localized exclusively in the cytoplasm (a proportion of SMAD3 was detected in the membrane) of CLL cells. Conclusions. The TGFB-SMAD2/3 signaling pathway is not active in CLL cells. We have found that SMAD2 is not expressed. Also, the nuclear heterodimers that consisted of SMAD3 and SMAD4 proteins, were not detected. Keywords: chronic lymphocytic leukemia (CLA), acute myeloid leukemia (AML), peripheral blood B-cells, SMAD, the TGFB-SMAD2/3 signaling pathway.

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Santiago-Schwarz, F., C. Panagiotopoulos, A. Sawitsky, and KR Rai. "Distinct characteristics of lymphokine-activated killer (LAK) cells derived from patients with B-cell chronic lymphocytic leukemia (B-CLL). A factor in B-CLL serum promotes natural killer cell-like LAK cell growth." Blood 76, no. 7 (1990): 1355–60. http://dx.doi.org/10.1182/blood.v76.7.1355.1355.

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Abstract We show that lymphokine-activated killer (LAK) cell precursors derived from patients with B-cell chronic lymphocytic leukemia (B-CLL) and cultured in the presence of recombinant interleukin-2 and normal human serum (NHS), develop into primarily NK cell-like (CD 57+) LAK cells, whereas identically prepared LAK cell precursors from normal subjects develop into mainly T cell-like (CD 3+, CD 8+) LAK cells. B-CLL LAK cells exhibited greater proliferative capacity than did normal LAK cells. When normal LAK cells were grown in B-CLL serum instead of NHS, their proliferation increased; NK cell levels also increased to those found in B-CLL LAK cells, suggesting that B-CLL serum contains a factor that promotes NK cell-like growth, LAK cells derived from normal or B- CLL patients demonstrated similar lytic activity toward K562 and Raji cells. Growth in B-CLL serum did not reduce their lytic potential. Thus, the altered phenotype and growth exhibited by B-CLL LAK cells and normal LAK cells grown in B-CLL serum does not lead to abnormalities in their cytolytic functions. We propose instead that the predominance of NK-like cells in B-CLL LAK cell populations and the presence of an NK cell-like growth factor in B-CLL serum reflect abnormalities related to NK cell-mediated B-cell regulation; ie, either inhibition of normal B- cell growth and/or growth stimulation of the leukemic clone in B-CLL.

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We show that lymphokine-activated killer (LAK) cell precursors derived from patients with B-cell chronic lymphocytic leukemia (B-CLL) and cultured in the presence of recombinant interleukin-2 and normal human serum (NHS), develop into primarily NK cell-like (CD 57+) LAK cells, whereas identically prepared LAK cell precursors from normal subjects develop into mainly T cell-like (CD 3+, CD 8+) LAK cells. B-CLL LAK cells exhibited greater proliferative capacity than did normal LAK cells. When normal LAK cells were grown in B-CLL serum instead of NHS, their proliferation increased; NK cell levels also increased to those found in B-CLL LAK cells, suggesting that B-CLL serum contains a factor that promotes NK cell-like growth, LAK cells derived from normal or B- CLL patients demonstrated similar lytic activity toward K562 and Raji cells. Growth in B-CLL serum did not reduce their lytic potential. Thus, the altered phenotype and growth exhibited by B-CLL LAK cells and normal LAK cells grown in B-CLL serum does not lead to abnormalities in their cytolytic functions. We propose instead that the predominance of NK-like cells in B-CLL LAK cell populations and the presence of an NK cell-like growth factor in B-CLL serum reflect abnormalities related to NK cell-mediated B-cell regulation; ie, either inhibition of normal B- cell growth and/or growth stimulation of the leukemic clone in B-CLL.

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Tummala, Hemanth, Alexey Goltsov, Hilal S. Khalil, et al. "Advocating the need of a systems biology approach for personalised prognosis and treatment of B-CLL patients." BioDiscovery 6 (December 30, 2012): e8939. https://doi.org/10.7750/BioDiscovery.2012.6.4.

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The clinical course of B-CLL is heterogeneous. This heterogeneity leads to a clinical dilemma: can we identify those patients who will benefit from early treatment and predict the survival? In recent years, mathematical modelling has contributed significantly in understanding the complexity of diseases. In order to build a mathematical model for determining prognosis of B-CLL one has to identify, characterise and quantify key molecules involved in the disease. Here we discuss the need and role of mathematical modelling in predicting B-CLL disease pathogenesis and suggest a new systems biology approach for a personalised therapy of B-CLL patients.

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Buhmann, Raymund, Annette Nolte, Doreen Westhaus, Bertold Emmerich, and Michael Hallek. "CD40-Activated B-Cell Chronic Lymphocytic Leukemia Cells for Tumor Immunotherapy: Stimulation of Allogeneic Versus Autologous T Cells Generates Different Types of Effector Cells." Blood 93, no. 6 (1999): 1992–2002. http://dx.doi.org/10.1182/blood.v93.6.1992.406k23_1992_2002.

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Although spontaneous remissions may rarely occur in B-cell chronic lymphocytic leukemia (B-CLL), T cells do generally not develop a clinically significant response against B-CLL cells. Because this T-cell anergy against B-CLL cells may be caused by the inability of B-CLL cells to present tumor-antigens efficiently, we examined the possibility of upregulating critical costimulatory (B7-1 and B7-2) and adhesion molecules (ICAM-1 and LFA-3) on B-CLL cells to improve antigen presentation. The stimulation of B-CLL cells via CD40 by culture on CD40L expressing feeder cells induced a strong upregulation of costimulatory and adhesion molecules and turned the B-CLL cells into efficient antigen-presenting cells (APCs). CD40-activated B-CLL (CD40-CLL) cells stimulated the proliferation of both CD4+ and CD8+ T cells. Interestingly, stimulation of allogeneic versus autologous T cells resulted in the expansion of different effector populations. Allogeneic CD40-CLL cells allowed for the expansion of specific CD8+cytolytic T cells (CTL). In marked contrast, autologous CD40-CLL cells did not induce a relevant CTL response, but rather stimulated a CD4+, Th1-like T-cell population that expressed high levels of CD40L and released interferon-γ in response to stimulation by CD40-CLL cells. Together, these results support the view that CD40 activation of B-CLL cells might reverse T-cell anergy against the neoplastic cell clone, although the character of the immune response depends on the major histocompatibility complex (MHC) background on which the CLL or tumor antigens are presented. These findings may have important implications for the design of cellular immunotherapies for B-CLL.

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Kay, Neil E., Ann K. Strege, Tait D. Shanafelt, Nancy D. Bone, and Yean K. Lee. "CLL B Cell Interaction with Bone Biopsy Generated Marrow Stromal Elements Enhances Their Apoptosis Resistance in Association with an Angiogenic Switch." Blood 104, no. 11 (2004): 1914. http://dx.doi.org/10.1182/blood.v104.11.1914.1914.

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Abstract CLL B cells have significant opportunity to interact with multiple tissue sites that are critical to the leukemic cell’s ability to survive. The marrow stromal elements (MSE) in B-chronic lymphocytic leukemia (B-CLL) are very likely one of the most frequently involved in this important cell-cell interaction. We have utilized a novel bone marrow biopsy technique (Alvi S, Leuk Res, 2001) that consistently generates robust and long-lived marrow stromal elements from B-CLL patients in order to study CLL B cell apoptosis status in relation to angiogenic factors. The latter angiogenic parameters were studied as we have previously detected both a VEGF based autocrine pathway for CLL B cells and found an extensive neovascularization in CLL marrows. Bone biopsy material from B-CLL patients (N=25) yielded active cell growth in vitro which expresses the features of marrow stromal constituents. Importantly, these marrow constituents were able to be sustained for several months (range, 3 – 52 weeks) and also could be transferred repetitively to new culture dishes. CLL B cells were able to spontaneously release VEGF and TSP-1 as we have previously published (Kay N, Leukemia, 2002). MSE from CLL marrow (N=5) were found to spontaneously secrete basic fibroblast growth factor (bFGF) 5.2 ± 0.9 pg/ml (mean ± one SEM), vascular endothelial growth factor (VEGF) 162 ± 48 pg/ml and thrombospondin-1 (TSP-1) 90 ± 26 ng/ml into the culture medium. Significant reduction in CLL B cell spontaneous apoptosis,measured by Annexin/PI incorporation (mean ± one SEM), was seen after exposure to the CLL MSE (N=6) both after 24 hours (i.e., Non exposed CLL B cells vs. MSE co-cultured CLL B cells was 70% ± 9 vs. 39% ± 10 respectively for 24 hours, p < 0.01) and 72 hrs (data not shown). Exposure to MSE also enhanced the in vitro drug resistance of CLL B cells to chlorambucil (C). Thus, for CLL B cells (n=5) cultured with CLL MSE and C (1μM) for 24 hours, apoptosis levels were reduced from 58.8% ± 7.8 for B cells without MSE compared to 39.8% ± 3.8 for B cells exposed to MSE. Apoptosis resistance enhancement for CLL B cells was found to be similar with normal marrow stromal elements and a stromal cell line (HS-5). Anti-apoptotic proteins detected by immunoblot were found to increase for CLL B cells exposed to stromal elements from bone marrow biopsies and included Mcl-1, Bcl-2, Survivin and XIAP. While Mcl-1 was found to increase, there was a decrease in Bcl-2 and Survivin detected by immunoblot when CLL B cells were exposed to the HS-5 cell line implying a different mechanism for apoptosis enhancement. When CLL B cells were added to CLL MSE for 24 hours, there was an increase in VEGF released into the culture medium (283 pg ± 114 for the co-culture vs. 14 pg ± 1.7 and 162 pg ± 48.8 for CLL B cells and MSE cultured alone respectively). In contrast co-culture of CLL B cells with MSE (24 hours) resulted in less TSP-1 in the culture medium than expected (106 ng ± 28.8 for co-cultured CLL and MSE cells vs. 89.5 ng ± 27.5 and 90.9 ng ± 26.4 for CLL B cells and MSE cultured alone respectively). This change in the ratio of pro- to anti-angiogenic factors favoring VEGF strongly suggests that CLL B cell interaction with stroma can facilitate an angiogenic switch and may be linked to disease progression. In addition, we have found that bone biopsies from B-CLL patients can generate long-term marrow stromal elements that are able to reliably enhance CLL B cell apoptosis resistance.

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Wojdacz, Tomasz K., Harindra E. Amarasinghe, Latha Kadalayil, et al. "Clinical significance of DNA methylation in chronic lymphocytic leukemia patients: results from 3 UK clinical trials." Blood Advances 3, no. 16 (2019): 2474–81. http://dx.doi.org/10.1182/bloodadvances.2019000237.

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Abstract Chronic lymphocytic leukemia patients with mutated immunoglobulin heavy-chain genes (IGHV-M), particularly those lacking poor-risk genomic lesions, often respond well to chemoimmunotherapy (CIT). DNA methylation profiling can subdivide early-stage patients into naive B-cell–like CLL (n-CLL), memory B-cell–like CLL (m-CLL), and intermediate CLL (i-CLL), with differing times to first treatment and overall survival. However, whether DNA methylation can identify patients destined to respond favorably to CIT has not been ascertained. We classified treatment-naive patients (n = 605) from 3 UK chemo and CIT clinical trials into the 3 epigenetic subgroups, using pyrosequencing and microarray analysis, and performed expansive survival analysis. The n-CLL, i-CLL, and m-CLL signatures were found in 80% (n = 245/305), 17% (53/305), and 2% (7/305) of IGHV-unmutated (IGHV-U) cases, respectively, and in 9%, (19/216), 50% (108/216), and 41% (89/216) of IGHV-M cases, respectively. Multivariate Cox proportional analysis identified m-CLL as an independent prognostic factor for overall survival (hazard ratio [HR], 0.46; 95% confidence interval [CI], 0.24-0.87; P = .018) in CLL4, and for progression-free survival (HR, 0.25; 95% CI, 0.10-0.57; P = .002) in ARCTIC and ADMIRE patients. The analysis of epigenetic subgroups in patients entered into 3 first-line UK CLL trials identifies m-CLL as an independent marker of prolonged survival and may aid in the identification of patients destined to demonstrate prolonged survival after CIT.

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Paul, Santanu, Steven L. Allen, Kanti R. Rai, and Nicholas Chiorazzi. "Expression of Angiogenin in Normal B Lymphocytes and B-CLL Cells." Blood 106, no. 11 (2005): 1188. http://dx.doi.org/10.1182/blood.v106.11.1188.1188.

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Abstract Angiogenesis is critical for the clinical progression of hematopoietic malignancies and depends on a series of angiogenic factors. Angiogenin is a potent angiogenic factor produced by the host microenvironment and certain neoplastic cells. The association between angiogenin, cancer progression and poor outcome in solid tumors has been documented, but its significance in B-CLL has not been defined. A previous study suggests that B-CLL patients in Rai stage O with higher angiogenin levels have a better clinical outcome. This is surprising in light of the association of angiogenin levels with progression in solid tumors. Therefore we analyzed angiogenin mRNA and protein expression by the leukemic cells of B-CLL patients in order to correlate the production of this molecule with disease progression in B-CLL. Angiogenin was expressed in B-CLL cells as well as in B cells of normal donors, although the expression in the former was much higher than in the latter. Q PCR revealed a 7-fold induction in angiogenin-specific mRNA transcription in B-CLL cells (n=10) in comparison to normal B cells (n=13). In addition, angiogenin protein was identified by confocal microscopy both within and on the cell surface of B-CLL cells. All B-CLL cases, regardless of IgV gene mutation status, express angiogenin as defined by FACS. Approximately 85% of the cells that comprise B-CLL clones (n=28) display angiogenin on their surface membranes (range: 55-98%). Furthermore approximately 13% of polyclonal B cells from normal subjects (n=12) also produce angiogenin (range: 1–33%). Using enzyme immunoassay, we also measured serum angiogenin levels and detected significant differences in 66 B-CLL patients (median: 381 ng/mL; range: 185–875) versus 24 age- and sex-matched healthy controls (median: 301 ng/mL; range: 192–536) (P = 0.0056). Surprisingly, there was no correlation between surface and serum angiogenin levels and the different V gene-defined B-CLL subgroups. Our results show for the first time that a small fraction of normal B cells and all cases of B-CLL express angiogenin transcripts as well as intracellular and surface membrane protein. Angiogenin levels do not correlate with IgV gene mutations status or expression of surface membrane CD38. The role of angiogenin in the pathogenesis and progression of B-CLL needs further study.

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Douglas, Raymond S., Renold J. Capocasale, Roberta J. Lamb, Peter C. Nowell та Jonni S. Moore. "Chronic Lymphocytic Leukemia B Cells Are Resistant to the Apoptotic Effects of Transforming Growth Factor-β". Blood 89, № 3 (1997): 941–47. http://dx.doi.org/10.1182/blood.v89.3.941.

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Abstract Chronic lymphocytic leukemia (CLL) is the most common leukemia of the western world and is characterized by a slowly progressing accumulation of clonal CD5+ B cells. Our laboratory has investigated the role of transforming growth factor-β (TGF-β) and interleukin-4 (IL-4) in the pathogenesis of B-cell expansion in CLL. In vitro addition of TGF-β did not increase spontaneous apoptosis of B cells from most CLL patients, as determined using the TUNEL method, compared with a twofold increase observed in cultures of normal B cells. There was similar expression of TGF-β type II receptors on both CLL B cells and normal B cells. In contrast to apoptosis, CLL B-cell proliferation was variably inhibited with addition of TGF-β. In vitro addition of IL-4, previously reported to promote CLL B-cell survival, dramatically reduced spontaneous apoptosis of CLL B cells compared with normal B cells. CLL B-cell expression of IL-4 receptors was increased compared to normal B cells. Thus, our results show aberrant apoptotic responses of CLL B cells to TGF-β and IL-4, perhaps contributing to the relative expansion of the neoplastic clone.

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Palena, Claudia, Loni McIntosh, Jeffrey Schlom, Kenneth Foon, Dennis Panicali, and Kwong Y. Tsang. "Modification of B-CLL Cells Via Infection with a Replication-Defective MVA Virus Encoding Three Costimulatory Molecules: A Potential Approach to Tumor Cell Immunotherapy of B-CLL." Blood 104, no. 11 (2004): 2516. http://dx.doi.org/10.1182/blood.v104.11.2516.2516.

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Abstract Chronic lymphocytic leukemia of B-cell origin (B-CLL) is a lymphoproliferative disorder characterized by the accumulation of slowly dividing, apoptosis-defective, clonal CD5+ B cells that typically involve the bone marrow, lymph nodes, spleen and blood of affected individuals. New therapeutic approaches are necessary to be investigated for the treatment of B-CLL. Several characteristics of the neoplastic B cells make B-CLL a good candidate for immunotherapy; evidence exists that B-CLL cells can be recognized by autologous anti-tumor T cells in an MHC-restricted manner. B-CLL cells, however, are themselves ineffective antigen-presenting cells mainly because of their inadequate costimulatory capacity. We have investigated the ability of in vitro manipulated B-CLL cells that express a triad of costimulatory molecules, designated TRICOM, to stimulate effective anti-tumor T-cell responses and their potential as cancer vaccines. Delivery of transgenes to the B-CLL cells could be accomplished through the use of a modified vaccinia virus strain Ankara designated MVA. This is a highly attenuated, non-replicative virus that has been demonstrated to be safe and well tolerated in both animals and humans. Here we report the use of a recombinant MVA vector to deliver the simultaneous expression of three human costimulatory molecules (B7-1, ICAM-1, LFA-3) on the surface of B-CLL cells. Infection was conducted using various multiplicity-of-infection (MOI) of MVA-TRICOM and the efficiency of the vector to enhance the expression of the encoded transgenes was evaluated by FACS analysis. The infection of B-CLL cells with MVA-TRICOM markedly increased the expression of B7-1, ICAM-1 and LFA-3 on the cell surface. The ability of the MVA-TRICOM-infected B-CLL cells to activate specific T cells was subsequently analyzed in proliferation assays, cytotoxic, and cytokine production assays. Proliferation of both autologous and allogeneic T cells was observed when MVA-TRICOM-infected B-CLL cells were used as stimulators in mixed lymphocyte reactions. Autologous CTLs were then generated in vitro using MVA-TRICOM-infected B-CLL cells as APC; these CTLs were capable of lysing uninfected autologous as well as allogeneic B-CLL cells. From our results we conclude that MVA-TRICOM-infected B-CLL cells can be used to induce B-CLL-specific CTLs that, in turn, can mediate the lysis of unmodified B-CLL cells. Our findings have implications for a potential use of MVA-TRICOM-infected B-CLL cells as tumor vaccines in the immunotherapy of B-CLL.

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Sonia Jaramillo, Andreas Agathangelidis, Christof Schneider, et al. "Prognostic impact of prevalent chronic lymphocytic leukemia stereotyped subsets: analysis within prospective clinical trials of the German CLL Study Group (GCLLSG)." Haematologica 105, no. 11 (2019): 2598–607. http://dx.doi.org/10.3324/haematol.2019.231027.

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Almost one-third of all patients with chronic lymphocytic leukemia (CLL) express stereotyped B cell receptor immunoglobulins (BcR IG) and can be assigned to distinct subsets, each with a particular BcR IG. The largest stereotyped subsets are #1, #2, #4 and #8, associated with specific clinicobiological characteristics and outcomes in retrospective studies. We assessed the associations and prognostic value of these BcR IG in prospective multicenter clinical trials reflective of two different clinical situations: i) early-stage patients (watch-and-wait arm of the CLL1 trial) (n=592); ii) patients in need of treatment, enrolled in 3 phase III trials (CLL8, CLL10, CLL11), treated with different chemo-immunotherapies (n=1861). Subset #1 was associated with del(11q), higher CLL international prognostic index (CLL-IPI) scores and similar clinical course to CLL with unmutated immunoglobulin heavy variable (IGHV) genes (U-CLL) in both early and advanced stage groups. IGHV-mutated (M-CLL) subset #2 cases had shorter time-to-first-treatment (TTFT) versus other M-CLL cases in the early-stage cohort (HR: 4.2, CI: 2-8.6, p<0.001), and shorter time-to-next-treatment (TTNT) in the advanced-stage cohort (HR: 2, CI: 1.2-3.3, p=0.005). M-CLL subset #4 was associated with lower CLL-IPI scores and younger age at diagnosis; in both cohorts, these patients showed a trend towards better outcomes versus other M-CLL. U-CLL subset #8 was associated with trisomy 12. Overall, this study shows that major stereotyped subsets have distinctive characteristics. For the first time in prospective multicenter clinical trials, subset # 2 appeared as an independent prognostic factor for earlier TTFT and TTNT and should be proposed for risk stratification of patients.

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Lotz, M., E. Ranheim, and T. J. Kipps. "Transforming growth factor beta as endogenous growth inhibitor of chronic lymphocytic leukemia B cells." Journal of Experimental Medicine 179, no. 3 (1994): 999–1004. http://dx.doi.org/10.1084/jem.179.3.999.

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Chronic lymphocytic leukemia (CLL) B cells are hyporesponsive or refractory to mitogens and growth factors in vitro. This study examined whether transforming growth factor beta (TGF-beta), a potent inhibitor of lymphocyte proliferation may play a role in the growth regulation of CLL B cells. CLL B cells from all donors treated expressed detectable TGF-beta 1 mRNA. In vitro release of TGF-beta by unstimulated cultures, or cultures stimulated by antibody to cell surface immunoglobulin (anti-mu) plus phorbol 12-myristate 13-acetate (PMA) was higher in CLL than in normal B cells. High levels of TGF-beta activity were also detected in plasma samples of CLL patients. The role of TGF-beta in growth regulation of CLL B cells was tested in assays using different B cell activators. Purified neoplastic B cells from most CLL patients proliferated in response to anti-mu, or the combination of anti-mu plus PMA. Levels of CLL B cell proliferation were lower than observed in normal B cells. Some CLL were refractory to these stimuli. Antibody to CD40 induced proliferation of CLL B cells from all donors tested when presented on Fc gamma RII (CDw32)-expressing L cells. Neutralizing antibodies to TGF-beta increased CLL B cell proliferation in the absence or presence of additional stimuli. These effects were dose dependent and specific. Exogenous TGF-beta completely inhibited CLL B cell proliferation induced by anti-mu, PMA, and anti-TGF-beta. CLL B cell proliferation induced by anti-CD40 was reduced by exogenous TGF-beta. However, even at high doses, TGF-beta did not completely inhibit the anti-CD40 effect. In summary, TGF-beta is overexpressed in CLL. CLL B cells are sensitive to TGF-beta and this cytokine functions as an autocrine growth inhibitor accounting at least in part for reduced proliferative responses of these leukemic cells and for the slow progression of the malignant process in vivo.

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Seifert, Marc, Ludger Sellmann, Johannes Bloehdorn, et al. "Cellular origin and pathophysiology of chronic lymphocytic leukemia." Journal of Experimental Medicine 209, no. 12 (2012): 2183–98. http://dx.doi.org/10.1084/jem.20120833.

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The cellular origin of chronic lymphocytic leukemia (CLL) is still debated, although this information is critical to understanding its pathogenesis. Transcriptome analyses of CLL and the main normal B cell subsets from human blood and spleen revealed that immunoglobulin variable region (IgV) gene unmutated CLL derives from unmutated mature CD5+ B cells and mutated CLL derives from a distinct, previously unrecognized CD5+CD27+ post–germinal center B cell subset. Stereotyped V gene rearrangements are enriched among CD5+ B cells, providing independent evidence for a CD5+ B cell derivation of CLL. Notably, these CD5+ B cell populations include oligoclonal expansions already found in young healthy adults, putatively representing an early phase in CLL development before the CLL precursor lesion monoclonal B cell lymphocytosis. Finally, we identified deregulated proteins, including EBF1 and KLF transcription factors, that were not detected in previous comparisons of CLL and conventional B cells.

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Sinha, Sutapa, Charla R. Secreto, Justin C. Boysen, et al. "Enhanced Expression of Beta-Catenin and Axl Receptor Tyrosine Kinase (RTK) in Chronic Lymphocytic Leukemia (CLL) B-Cells with Co-Culture on Marrow Stromal Cells: Implications for Leukemic Cell Drug Resistance." Blood 132, Supplement 1 (2018): 3125. http://dx.doi.org/10.1182/blood-2018-99-110981.

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Abstract Introduction CLL remains an incurable disease and represents a significant health problem in the western world. Increasing evidence highlights that the impact of marrow stromal cells is a key component influencing CLL B-cell survival. We have utilized an in vitro bone marrow stromal cell (BMSC) model system and found unique alterations in CLL B-cells with BMSC co-culture that point to previously unidentified biologic changes in the CLL B-cells that may influence CLL B-cell signaling and drug resistance. Methods Purified primary CLL B-cells (n= 39) from previously untreated CLL patients were cultured alone or co-cultured with primary BMSCs from either normal individuals (n=26) or CLL patients (n=17) at a 50:1 ratio in AIMV medium. After 48 hours, separated CLL B-cells or BMSCs were examined by immunoprecipitation/Western blot analyses and where needed real time PCR was done to assess the presence of intracellular proteins. In separate experiments to assess CLL B-cell killing, purified CLL B-cells were treated with TP-0903, fludarabine, chlorambucil and ibrutinib as single agents with or without BMSC co-culture. Results We observed significant increases in expression of Axl for both mRNA and protein levels in CLL B-cells co-cultured with BMSCs compared to CLL B-cells cultured alone. We also detected significantly increased expression of β-catenin at the protein level in CLL B-cells co-cultured with BMSC. But, we did not see any significant change in β-catenin or Axl protein expression in BMSCs co-cultured with CLL B-cells. Co-culturing of CLL B-cells with BMSCs using transwells confirmed that the upregulation of both Axl and β-catenin is dependent on the direct contact of CLL B-cells with BMSCs. The CLL B-cells from co-culture also had upregulation in phosphorylated (P)-ERK-1/2 but no change in P-AKT(Ser473). High nuclear β-catenin and P-ERK-1/2 levels were also detected in co-cultured CLL B-cells. ERK associates with and inactivates GSK-3β resulting in the up-regulation of β-catenin. We next checked for P-GSK-3β (Ser9) in co-cultured CLL B-cells. Upregulation in P-GSK-3β (Ser9) detected in co-cultured CLL B-cells suggests inactivation of GSK-3β and increasing β-catenin accumulation in co-cultured CLL B-cells. Moreover, inhibition of P-ERK-1/2 with inhibitor PD98059 in CLL B-cells cultured with BMSCs inhibited β-catenin as well as Axl expression levels. We also determined the phosphorylation status of Axl in CLL B-cells in co-culture with BMSC but found no change either at Y702 (Axl kinase domain) or total tyrosine phosphorylation levels for Axl in CLL B-cells. Thus, we assume that the role of Axl in co-cultured leukemic B-cells is independent of its kinase activity. Next we determined the effect of the highly specific Axl inhibitor TP-0903 on CLL B-cell status of Axl and b-catenin while in BMSC co-culture. Interestingly, both Axl and β-catenin protein expression levels were found to be further upregulated in CLL B-cells exposed to sub-lethal doses of TP-0903 in co-culture with BMSC. Treatment with chemotherapeutic or targeted therapy drugs, (i.e. fludarabine, chlorambucil or ibrutinib) also led to increase in expression levels of both β-catenin and Axl CLL B-cells co-cultured with BMSC. Of interest CLL B-cells were less sensitive to the chemotherapy drugs in presence of BMSCs, suggesting a role for both Axl and β-catenin in stromal mediated CLL B-cell drug resistance to these agents. This was not true for the Axl inhibitor as TP-0903 was able to induce robust cell death by targeting P-Axl and overcome BMSC mediated protection even in the presence of increased Axl and b-catenin. We also found that TP-0903 decreased P-Axl as well as the Axl downstream mediator, P-Akt(S473) and reduced Mcl-1 expression in CLL B-cells in BMSC co-culture. Conclusions Here we show that marrow stromal cell mediated increased expression in both β-catenin and Axl in CLL B-cells is associated with leukemic B cell survival and drug resistance. The mechanism for this may in part be via activated ERK levels that also occur when CLL B-cells contact BMSC. The BMSC resistance appears to be more profound for chemotherapeutic agents since Axl inhibitor can still induce CLL B-cell killing with BMSC co-culture. These studies suggest that a further understanding of the roles of Axl and β-catenin in the resistance status of CLL B-cells mediated by contact with BMSC are warranted. Disclosures Warner: Tolero Pharmaceuticals: Employment. Bearss:Tolero Pharmaceuticals, Inc: Employment. Kay:Morpho-sys: Membership on an entity's Board of Directors or advisory committees; Cytomx Therapeutics: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Infinity Pharm: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Acerta: Research Funding; Tolero Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Agios Pharm: Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees.

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Liang, Xueqing, Veronika Bachanova, and Wei Chen. "Deletion and mutation of IL10RA gene on chromosome 11q23 to avert CpG-B oligodeoxynucleotides-induced apoptosis of human chronic lymphocytic leukemia B cells." Journal of Clinical Oncology 31, no. 15_suppl (2013): 3023. http://dx.doi.org/10.1200/jco.2013.31.15_suppl.3023.

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3023 Background: Targeting TLR9 expressed on human CLL cells with CpG-B oligodeoxynucleotides leads to IL-10-induced tyrosine phosphorylation of STATs and apoptosis of B-CLL cells. However, B-CLL cells from a small subset of patients were resistant to CpG-B ODN treatment. Here, we investigated the molecular mechanism by which B-CLL cells are sensitive or resistant to CpG-B ODN treatment. Methods: Purified CD19+CD23+CD5+primary B-CLL cells were cultured for 5 days with/without CpG-B ODN (CpG 2006, CpG 685) or rh-IL-10. B-CLL cell apoptosis were determined by viable cell counts, Annexin V/PI and TMRE staining, Western blot and intracellular staining of the activation/cleavage of caspases, PARP, Bax translocation and cytochrome c release, IL-10R1 expression and IL-10 binding by flow cytometry, IL10RA gene deletion by FISH, IL10R1 S138G mutation by Bi-PASA, The tyrosine or serine phosphorylation of STATs by Western blot. Results: Fifteen CpG-sensitive and 11 CpG-resistant primary CLL samples were comparatively studied. B-CLL cells from 15/15 CpG-sensitive samples were induced into apoptosis by either CpG-B ODNs or IL-10 in a treatment time and dose-dependent manner. Both CpG-B ODNs and IL-10 significantly increased pTyr701-STAT1/pTyr705-STAT3 expression and induced apoptosis via the mitochondrial apoptotic pathway in 15 primary B-CLL cells. No IL-10RA gene deletions were detected, 13/15 patients were IL10R1 S138G AA wildtypes and 2/15 patients were AG heterozygotes. In contrast, CpG-B ODNs or IL-10 failed to induce apoptosis in 11/11 CpG-resistant B-CLL cells. Interesting, 2/11 CLL samples had IL10RA genes deletion and 7/11 had IL10R1 S138G GG homozygote mutation. IL10RA gene deletion significantly decreased the IL10R1 expression; both IL10RA gene deletion and mutation significantly decreased the IL-10 binding, abolished or reduced CpG-B ODNs or IL10-induced pTyr701-STAT1/pTyr705-STAT3 expression in B-CLL cells. Conclusion: Deletion and mutation of IL10RA gene on chromosome 11q23 averts CpG-B ODN-induced apoptosis of human B-CLL cells, which may serve as biomarker to predict sensitivity or resistance of CLL to CpG-B ODN treatment.

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Hwang, Kwan-Ki, Xi Chen, Daniel M. Kozink, et al. "Enhanced outgrowth of EBV-transformed chronic lymphocytic leukemia B cells mediated by coculture with macrophage feeder cells." Blood 119, no. 7 (2012): e35-e44. http://dx.doi.org/10.1182/blood-2011-08-371203.

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Abstract B-cell chronic lymphocytic leukemia (B-CLL) is characterized by the clonal expansion of CD5-expressing B lymphocytes that produce mAbs often reactive with microbial or autoantigens. Long-term culture of B-CLL clones would permit the collection and characterization of B-CLL mAbs to study antigen specificity and of B-CLL DNA to investigate molecular mechanisms promoting the disease. However, the derivation of long-term cell lines (eg, by EBV), has not been efficient. We have improved the efficiency of EBV B-CLL transformation of CpG oligonucleotide-stimulated cells by incubating patient peripheral blood mononuclear cells in the presence of an irradiated mouse macrophage cell line, J774A.1. Using this approach, peripheral blood mononuclear cells isolated from 13 of 21 B-CLL patients were transformed as documented by IGHV-D-J sequencing. Four clones grew and retained CD5 expression in culture for 2 to 4 months. However, despite documentation of EBV infection by expression of EBNA2 and LMP1, B-CLL cells died after removal of macrophage feeder cells. Nevertheless, using electrofusion technology, we generated 6 stable hetero-hybridoma cell lines from EBV-transformed B-CLL cells, and these hetero-hybridomas produced immunoglobulin. Thus, we have established enhanced methods of B-CLL culture that will enable broader interrogation of B-CLL cells at the genetic and protein levels.

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Baskar, Sivasubramanian, Jessica M. Suschak, Ivan Samija, et al. "A human monoclonal antibody drug and target discovery platform for B-cell chronic lymphocytic leukemia based on allogeneic hematopoietic stem cell transplantation and phage display." Blood 114, no. 20 (2009): 4494–502. http://dx.doi.org/10.1182/blood-2009-05-222786.

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Abstract Allogeneic hematopoietic stem cell transplantation (alloHSCT) is the only potentially curative treatment available for patients with B-cell chronic lymphocytic leukemia (B-CLL). Here, we show that post-alloHSCT antibody repertoires can be mined for the discovery of fully human monoclonal antibodies to B-CLL cell-surface antigens. Sera collected from B-CLL patients at defined times after alloHSCT showed selective binding to primary B-CLL cells. Pre-alloHSCT sera, donor sera, and control sera were negative. To identify post-alloHSCT serum antibodies and subsequently B-CLL cell-surface antigens they recognize, we generated a human antibody-binding fragment (Fab) library from post-alloHSCT peripheral blood mononuclear cells and selected it on primary B-CLL cells by phage display. A panel of Fab with B-CLL cell-surface reactivity was strongly enriched. Selection was dominated by highly homologous Fab predicted to bind the same antigen. One Fab was converted to immunoglobulin G1 and analyzed for reactivity with peripheral blood mononuclear cells from B-CLL patients and healthy volunteers. Cell-surface antigen expression was restricted to primary B cells and up-regulated in primary B-CLL cells. Mining post-alloHSCT antibody repertoires offers a novel route to discover fully human monoclonal antibodies and identify antigens of potential therapeutic relevance to B-CLL and possibly other cancers. Trials described herein were registered at www.clinicaltrials.gov as nos. NCT00055744 and NCT00003838.

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Maiti, Guru P., Sutapa Sinha, Justin C. Boysen, Neil E. Kay, and Asish K. Ghosh. "Epigenetic Silencing of Catalase Induces Accumulation of Reactive Oxygen Species in Chronic Lymphocytic Leukemia B Cells Leading to Activation of Axl: An Escape Strategy?" Blood 128, no. 22 (2016): 4363. http://dx.doi.org/10.1182/blood.v128.22.4363.4363.

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Abstract Background: A challenge for novel therapeutic strategies will be the fine tuning of intracellular reactive oxygen species (ROS) signaling in order to effectively deprive cells from ROS-induced tumor promoting events and subsequent tipping the balance to ROS-induced apoptotic signaling. ROS plays a critical role in regulation of the pro-survival receptor tyrosine kinase (RTK) signaling pathways in human cancers. Studies have identified mitochondrial metabolism as the key source for abundant ROS in chronic lymphocytic leukemia (CLL). Unlike in other malignant cells, increased oxidative phosphorylation but not increased aerobic glycolysis has been found in CLL B-cells. While we have detected constitutively phosphorylated/active RTKs like Axl in CLL B-cells, the mechanism of activation remains largely undefined. Here we report that (i) elevated ROS activates the Axl signaling pathway in CLL B-cells and, (ii) mechanism of increased ROS accumulation in the leukemic B-cells. Methods: CLL B- and normal B-cells were purified from peripheral blood of previously untreated CLL patients and normal healthy individuals, respectively using a RossetteSep B-Cell enrichment kit. CLL B-cells were exposed to H2O2 for 5 min and cell lysates were analyzed for activation of RTKs including Axl by immunoprecipitation/Western blots. We also examined expression status of SIRT3, acetylated-superoxide dismutase (SOD)2 and catalase in CLL B-cells by Western blots. Catalase mRNA levels in CLL B-/normal B-cells were determined by qRT-PCR. In some experiments, genomic DNA was isolated from CLL B-/normal B-cells for catalase promoter methylation studies. Finally, accumulation of O2‾ and H2O2 in CLL B- and normal B-cells were measured by flow cytometry after treating the cells withdihydroethidium (DHE) and dichlorohydrofluorescein diacetate (DCFDA), respectively. Results: We found that enforced induction of ROS significantly increases tyrosine phosphorylation levels on multiple cellular proteins in CLL B-cells (Fig. 1). Further analysis finds that increased ROS activates Axl and its downstream targets AKT/Erk1/2 MAPK and the fibroblast growth factor receptor (FGFR) which we recently defined as a downstream target of Axl; while ROS accumulation did not show any significant effect on other RTKs, cMET or IGFR1, in CLL B-cells. Interestingly, the histone deacetylase SIRT3 which activates mitochondrial SOD2 via deacetylation, we found, was overexpressed in CLL B-cells as compared to normal B-cells indicating more efficient conversion of O2‾ into H2O2 in the leukemic B-cells (Fig. 2). However expression of catalase, which converts H2O2 into O2 and H2O, was reduced significantly in CLL B-cells as compared to normal B-cells both at mRNA and protein levels. To delineate the mechanism of reduced expression of catalase in CLL B-cells we studied the human catalase promoter region for potential methylation sites using the method of HpaII restriction enzyme digestion where HpaII is unable to cut DNA (CCGG site) when the internal cytosine is methylated. Initial findings from this approach suggest that while the catalase gene promoter in CLL B-cells may contain methylated cytosines, the catalase gene in normal B-cells may not confer methylation in the promoter region. Next, genomic DNA from CLL B-cells (n=10) and normal B-cells (n=6) was subjected to bisulfite treatment, followed by PCR amplification and sequencing. This approach revealed no significant promoter methylation of the catalase gene in normal B-cells, while moderate to heavily methylated promoter of the catalase gene was detected in CLL B-cells from majority of CLL patients (9 of 10). Further studies are in progress to examine catalase promoter methylation status in a larger cohort of CLL patients and its association with H2O2 accumulation, RTK phosphorylation and risk-factors. Indeed, flow cytometric analysis finds lower levels of O2‾ but accumulation of H2O2 in CLL B-cells as compared to normal B-cells. Taken together, these findings suggest that although SOD2 remains highly active, increased accumulation of H2O2 may occur in CLL B-cells due to epigenetic silencing of the catalase gene. Conclusion: These observations may explain, at least in part, the expression of constitutively active Axl signaling pathway in CLL B-cells that plays a role in CLL B cell survival and may account for the prolonged survival and/or apoptotic resistance of the leukemic B-cells. Disclosures No relevant conflicts of interest to declare.

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Sinha, Sutapa, Zhiquan Wang, Weiguo Han, et al. "Abstract 1252: Role of purine and pyrimidine metabolism in CLL B cell survival and drug resistance mediated via interaction with bone marrow microenvironment." Cancer Research 83, no. 7_Supplement (2023): 1252. http://dx.doi.org/10.1158/1538-7445.am2023-1252.

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Abstract Multiple studies including ours have shown that the interaction of chronic lymphocytic leukemia (CLL) B cells with the bone marrow (BM) microenvironment promotes cell survival and protects these cells from the cytotoxic effects of therapy. A detailed understanding of this interaction will help better understand CLL leukemic cell survival and drug resistance mechanisms. We performed RNA-seq in paired CLL B cells isolated from untreated blood and BM (n=6) and in untreated CLL B cells (n=4) cultured alone or co-cultured with bone marrow stromal cells (BMSCs) derived from either normal individuals (n=2) or CLL patients (n=2). We found upregulation of 232 and 917 genes in CLL B cells from BM and in co-cultured CLL B cells compared to the CLL B cells from the same patient’s blood and CLL B cells cultured alone respectively (p&lt;0.05, fold change &gt;1.5). However we found only 13 genes overlapped between BM CLL B cells and co-cultured CLL B cells. To explore the functional importance of these 13 genes, we analyzed the expression of these genes in CLL blood B cells from an additional 169 untreated patients by RNA-seq and found a positive relationship between 5 of these 13 genes (MKI67, PNP, C16orf54, MOB3A, CDK2AP2) with CLL patient’s clinical outcome including overall survival (OS), progression free survival (PFS), and time to first treatment (TTFT) [univariate Cox regressions analysis (UCRA) p&lt;0.05]. Out of 5 genes, purine nucleotide phosphorylase (PNP) which is an enzyme in the purine salvage pathway, showed the significant association for patient’s OS, PFS, and TTFT using UCRA [hazard ratio (HR) {95% CI}: 2.8 {1.8-4.3}, 2.7 {1.9-3.8}, 2.9 {2.0-4.2}, respectively, p&lt;.0001], suggesting purine metabolism plays an important role in BM induced CLL B cell survival. We also observed an increase in PNP protein levels in CLL B cells co-cultured with BMSCs or from BM using Western blot analysis. Untargeted metabolomic profiling (LC-MS+GC-MS) showed an increased level of purine salvage pathway metabolites adenosine, inosine, and hypoxanthine in co-cultured CLL B cells. Consistent with this altered nucleotide metabolism data, we also found increased pyrimidine pathway enzyme thymidylate synthase (TYMS) at RNA and protein levels in co-cultured CLL B cells and in BM CLL B cells. We further analyzed the RNA-seq data generated from the 169 CLL patients and it also showed a positive association of TYMS gene with their OS, PFS and TTFT using UCRA [HR (95% CI): 1.35 (1.0-1.8), 1.3 (1.0-1.6), 1.63 (1.3-2.1) respectively, p&lt;0.05]. In conclusion, these results indicate that active purine/pyrimidine metabolism in CLL B cells may be augmented by the BM environment to support CLL B cell survival. Future studies to understand the exact mechanism of PNP and TYMS mediated CLL B cell survival for both untreated and treated CLL B cells will uncover unique approaches for therapy. Citation Format: Sutapa Sinha, Zhiquan Wang, Weiguo Han, Kari G. Rabe, Susan L. Slager, Chantal E. McCabe, Daniel R. O'Brien, Sameer A. Parikh, Esteban Braggio, Neil E. Kay. Role of purine and pyrimidine metabolism in CLL B cell survival and drug resistance mediated via interaction with bone marrow microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1252.

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Yasukawa, M., T. Shiroguchi, A. Inatsuki, and Y. Kobayashi. "Antigen presentation in an HLA-DR-restricted fashion by B-cell chronic lymphocytic leukemia cells." Blood 72, no. 1 (1988): 102–8. http://dx.doi.org/10.1182/blood.v72.1.102.102.

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Abstract The ability of B-cell chronic lymphocytic leukemia (B-CLL) cells to present antigen to antigen-specific T cells was investigated. B-CLL cells present herpes simplex virus (HSV) antigen and purified protein derivative (PPD) to HSV- and PPD-specific, interleukin-2-dependent T- cell lines in an antigen-specific manner. Treatment of B-CLL cells with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) induced markedly increased levels of HLA-DR expression. TPA-treated B-CLL cells showed substantially more effective presentation, especially at low antigen concentrations, than did untreated B-CLL cells. By coculturing different allogeneic combinations of B-CLL cells and T cells and by adding anti-HLA-DR monoclonal antibody to cultures, it was found that antigen presentation by B-CLL cells was restricted by HLA-DR in the same way as for macrophages. We concluded from these experiments that B- CLL cells have a capacity to serve as antigen-presenting cells in an HLA class II-restricted fashion and that increasing the amount of HLA class II antigen and activation of B-CLL cells resulted in effective antigen presentation.

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The ability of B-cell chronic lymphocytic leukemia (B-CLL) cells to present antigen to antigen-specific T cells was investigated. B-CLL cells present herpes simplex virus (HSV) antigen and purified protein derivative (PPD) to HSV- and PPD-specific, interleukin-2-dependent T- cell lines in an antigen-specific manner. Treatment of B-CLL cells with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) induced markedly increased levels of HLA-DR expression. TPA-treated B-CLL cells showed substantially more effective presentation, especially at low antigen concentrations, than did untreated B-CLL cells. By coculturing different allogeneic combinations of B-CLL cells and T cells and by adding anti-HLA-DR monoclonal antibody to cultures, it was found that antigen presentation by B-CLL cells was restricted by HLA-DR in the same way as for macrophages. We concluded from these experiments that B- CLL cells have a capacity to serve as antigen-presenting cells in an HLA class II-restricted fashion and that increasing the amount of HLA class II antigen and activation of B-CLL cells resulted in effective antigen presentation.

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Jurlander, Jesper, Chun-Fai Lai, Jimmy Tan, et al. "Characterization of Interleukin-10 Receptor Expression on B-Cell Chronic Lymphocytic Leukemia Cells." Blood 89, no. 11 (1997): 4146–52. http://dx.doi.org/10.1182/blood.v89.11.4146.

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Abstract B-cell chronic lymphocytic leukemia (B-CLL) cells accumulate in vivo in the G0/G1 phase of the cell cycle, suggesting that their malignant expansion is due, at least in part, to a delay in cell death. However, the cellular or molecular factors responsible for a delay in B-CLL cell death are unknown. B-CLL cells do express receptors for interferon-α (IFN-α) and IFN-γ, and activation of both has been shown to promote B-CLL survival in vitro by preventing apoptosis. The interleukin-10 (IL-10) receptor is another member of the IFN receptor family, but its ligand, IL-10, has been reported to induce apoptosis in B-CLL cells. In the current study, we undertook a biochemical analysis of IL-10 receptor expression on freshly isolated B-CLL cells and characterized the functional responsiveness of IL-10 binding to its constitutively expressed receptor. We show that B-CLL cells bind IL-10 with significant specificity and express between 47 and 127 IL-10 receptor sites per cell, with a dissociation constant in the range of 168 to 426 × 10−12 mol/L. Ligand binding and activation of the IL-10 receptor expressed on B-CLL cells results in the phosphorylation of signal transducer and activator of transcription 1 (STAT1) and STAT3 proteins. This pattern of STAT protein phosphorylation is identical to IL-10 receptor activation on normal cells and similar to IFN-α (STAT1 and STAT3) and IFN-γ (STAT1) receptor activation in CLL. Further, in consecutive samples of fresh blood obtained from patients with B-CLL cells, the addition of IL-10 inhibited B-CLL proliferation, enhanced B-CLL differentiation, but did not induce apoptosis. Indeed, IL-10, like IFN-γ, was able to significantly reduce the amount of B-CLL cell death caused by hydrocortisone-induced apoptosis. We conclude that cytokines, which signal through the interferon family of receptors, have comparable functional effects on B-CLL cells.

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Xing, Dongxia, Alan G. Ramsay, Simon Robinson, et al. "Lenalidomide Treatment Enhances Immunological Synapse Formation of Cord Blood Natural Killer Cells with B Cells Derived From Chronic Lymphocytic Leukemia." Blood 118, no. 21 (2011): 1794. http://dx.doi.org/10.1182/blood.v118.21.1794.1794.

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Abstract Abstract 1794 Immune dysfunction is a hallmark of chronic lymphocytic leukemia (CLL) including suppressed humoral and cell-mediated immune responses. The immunomodulatory agent lenalidomide has shown effective clinical activity against CLL, but its mechanism of action is poorly understood. Previous work has demonstrated that the T cell immunological synapse and functional defects in CLL can be reversed following lenalidomide treatment (J Clin Invest. 2008; 118). Polymerization of F-actin at the NK cell immunological synapse with tumor cells is required for signaling molecules to assemble and regulate NK cell activation and effector function. Confocal microscopy was used to visualize and analyze F-actin polymerization at the immune synapse between NK cells and CLL cells. The impaired immune synapse defect identified in CLL could result from not only the defects of CLL B cells but also defects in the CLL NK cells or a combination of both factors. To investigate the contribution of each factor, we examined synapse formation in experiments using CLL B cells with autologous CLL NK cells or healthy allogeneic NK cells. Conjugates formed with healthy NK cells and CLL B cells exhibited a strong band of F-actin at the immune synapse. In contrast, significantly less actin polymerization at the synapse was observed in autologous CLL NK cells and CLL B cells (P < 0.01). These results indicate CLL B cells, together with CLL NK cells contributed to the immune dysfunction in CLL. As autologous NK cell function in CLL is suppressed, we investigated the utility of CB as a potential functional source of NK cells for CLL immunotherapy. We examined the effect of lenalidomide on NK cell immune synapse function with CLL B cells acting as APCs. We demonstrated that ex vivo treatment of CLL cells with lenalidomide (500 ng/ml) for 48 hours caused a significant increase in the ability of autologous CLL NK cells to form F-actin immune synapses with CLL B cells. The same treatment of CLL B cells also significantly increased the ability of CB-NK cells to form F-actin immunological synapses with these treated CLL B cells compared to untreated CLL B cells (33.6% to 67.3%, P < 0.01, n=6). Our results also show that lenalidomide treatment of autologous NK cells from CLL patients enhanced synapse formation with treated CLL cells compared to experiments using untreated NK cells, but with reduced function compared to CB NK cells. Of note, lenalidomide treatment was shown to increase the recruitment of the signaling molecule Lck to NK cell:CLL cell synapse site, that is known to regulate lytic synapse function. Importantly, lenalidomide treatment significantly increased CB-NK killing of CLL B cells compared to untreated CLL B cells (20.5% versus 48.2%, E:T ratio of 10:1, n = 6, p < 0.001). These results provide insight into the potential mechanism of action of lenalidomide's anti-leukemic function – priming CLL tumor cells for enhanced NK cell lytic synapse formation and effector function. In addition, the data suggests that immunotherapeutic strategies utilizing a combination of CB-NK cells and lenalidomide has an enhanced clinical efficacy in CLL. Disclosures: Gribben: Roche: Honoraria; Celgene: Honoraria; GSK: Honoraria; Mundipharma: Honoraria; Gilead: Honoraria; Pharmacyclics: Honoraria.

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Lankester, AC, GM van Schijndel, CE van der Schoot, MH van Oers, CJ van Noesel, and RA van Lier. "Antigen receptor nonresponsiveness in chronic lymphocytic leukemia B cells." Blood 86, no. 3 (1995): 1090–71. http://dx.doi.org/10.1182/blood.v86.3.1090.1090.

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Abstract B chronic lymphocytic leukemia (B-CLL) are clonal populations of mIgM+ or mIgM+/mIgD+ CD5+ B cells that appear to be arrested in the follicular mantle-zone B-cell stage. Functional analyses have shown two groups of B-CLL that can be distinguished based on their capacity to proliferate in response to B-cell antigen receptor complex (BCR) cross- linking. To investigate the molecular basis for this phenomenon, we have analyzed both architecture and functional properties of BCR complexes on these two groups of B-CLL. Both groups were found to express structurally similar BCR. However, protein tyrosine kinase (PTK) activity associated with and specific for BCR constituents was strongly diminished in nonresponsive B-CLL. Moreover, the PTK-dependent assembly of Shc/Grb2 complexes, which may couple the BCR to p21ras, was absent in these B-CLL. Finally, of all PTKs tested, the expression of PTK syk was found to be considerably lower in nonresponsive B-CLL. Thus, absence of mitogenic responses upon BCR cross-linking in particular B-CLL was found to be strictly correlated with diminished induction of BCR-associated PTK activity and lower levels of PTK syk. Because nonresponsive B-CLL closely resembles tolerant autoreactive B cells both functionally and biochemically, distinction between B-CLL with respect to functional properties in vitro may be determined by differences in antigen encounter in vivo.

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B chronic lymphocytic leukemia (B-CLL) are clonal populations of mIgM+ or mIgM+/mIgD+ CD5+ B cells that appear to be arrested in the follicular mantle-zone B-cell stage. Functional analyses have shown two groups of B-CLL that can be distinguished based on their capacity to proliferate in response to B-cell antigen receptor complex (BCR) cross- linking. To investigate the molecular basis for this phenomenon, we have analyzed both architecture and functional properties of BCR complexes on these two groups of B-CLL. Both groups were found to express structurally similar BCR. However, protein tyrosine kinase (PTK) activity associated with and specific for BCR constituents was strongly diminished in nonresponsive B-CLL. Moreover, the PTK-dependent assembly of Shc/Grb2 complexes, which may couple the BCR to p21ras, was absent in these B-CLL. Finally, of all PTKs tested, the expression of PTK syk was found to be considerably lower in nonresponsive B-CLL. Thus, absence of mitogenic responses upon BCR cross-linking in particular B-CLL was found to be strictly correlated with diminished induction of BCR-associated PTK activity and lower levels of PTK syk. Because nonresponsive B-CLL closely resembles tolerant autoreactive B cells both functionally and biochemically, distinction between B-CLL with respect to functional properties in vitro may be determined by differences in antigen encounter in vivo.

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Hegde, Ganapati V., Katie J. Peterson, Katy Emanuel, et al. "GLI1 Transcription Factor: A Potential Therapeutic Target in B-CLL." Blood 110, no. 11 (2007): 3100. http://dx.doi.org/10.1182/blood.v110.11.3100.3100.

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Abstract B-cell Chronic Lymphocytic Leukemia (B-CLL) is characterized by accumulation of clonal lymphocytes resistant to apoptosis. Therefore, identification of molecules responsible for the increased resistance to apoptosis is warranted. The identified molecules could be further targeted to develop effective therapy. Emerging evidence on the mechanism of resistant to apoptosis in several cancers suggests that GLI1 transcription factor, target of hedgehog signaling, may have a potential role in the increased resistance to apoptosis in B-CLL. However such studies have not been reported for B-CLL. Therefore, we investigated the role of sonic hedgehog (Shh)-GLI signaling in the pathogenesis B-CLL using 38 different patient samples and different parameters. The results were analyzed based on good and poor clinical outcome B-CLL subgroups identified on the basis of cytogenetic abnormality and/or CD38 expression levels. Microarray and RT-PCR analyses demonstrated active Shh-GLI signaling molecules in B-CLL cells including GLI1. Our results demonstrate that GLI1 transcripts were significantly (p = 0.003) over-expressed in B-CLL cells of poor clinical outcome patient subgroups (17pdel, 11qdel, trisomy12; N = 20) compared to good clinical outcome (13qdel, normal karyotype; N = 18) as observed in microarray analysis and confirmation by real time PCR. In addition, similar observation was also made with poor prognosis (high CD38 expression) versus good prognosis (low CD38 expression) B-CLL subgroups. Furthermore, higher expression of GLI1 was associated with significantly (p = 0.02) shorter time to treatment compared to lower GLI1 expressing B-CLL patients as determined by Kaplan-Meier survival analysis using log rank test, suggesting the increased expression of GLI1 and its association with the disease progression in poor clinical outcome B-CLL patient sub-groups. Addition of exogenous Shh on the significantly (p<0.01) increased cell survival and up-regulation of GLI1 transcripts, and decreased cell survival and downregulation of GLI1 transcripts in the presence of cyclopamine, known inhibitor of signaling, as determined by MTT assay and RT-PCR in vitro, suggesting the role of GLI1 in the survival of B-CLL. Downregulation of GLI1 expression in B-CLL cells using antisense oligonucleotides resulted in the significantly (p<0.01) increased susceptibility of the B-CLL cells to fludarabine mediated cytotoxicity compare to fludarabine alone or antisense alone or B-CLL cells treated with irrelevant oligonucleotides as determined by MTT assay and Annexin V method for identifying apoptotic cells. There was a significant decrease in BCL2 expression in GLI1-downregulated B-CLL cells suggesting that GLI1 may regulates BCL2 and thereby regulate cell survival/apoptosis in B-CLL. Together, these results demonstrate a role for GLI1 transcription factor for the increased resistance to apoptosis and thereby disease progression in B-CLL, and hence may be a potential therapeutic target to further improve treatment for B-CLL.

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Lee, Yean K., Ann K. Strege, Nancy D. Bone та ін. "Hypoxia Inducible Factor-1α Is over Expressed in CLL B Cells Because of an Impaired Proteasome Pathway Associated with Defective Interaction with von Hippel-Landau Protein." Blood 106, № 11 (2005): 2115. http://dx.doi.org/10.1182/blood.v106.11.2115.2115.

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Abstract We have found that CLL B cells spontaneously secrete vascular endothelial growth factor (VEGF) and that a VEGF autocrine pathway can induce apoptosis resistance in these cells. Recently, we also found that hypoxia-inducible factor-1 alpha (HIF-1α) is highly expressed in CLL B cells. Since this protein is a potent transcription factor for the induction of VEGF, we were interested in further definition of HIF-1α regulation and its function in CLL B cells. CLL blood B cells overexpress HIF-1α protein but not mRNA for HIF-1α compared to normal blood and splenic B cells. Immunohistochemistry (IHC) showed that circulating blood CLL B cells and a subset of CLL marrow cells uniformly express HIF. Hypoxic conditions (i.e., 1% O2) did not increase the protein levels of HIF-1α nor mRNA for HIF-1α in CLL B cells, indicating that the high HIF-1α protein level is due to post-translation modification. Blockade of signaling pathways known to increase HIF-1α levels also did not alter the high levels of HIF-1α in CLL B cells. IHC and nuclear extraction assay demonstrated that HIF-1α was predominantly located in the CLL B cell nucleus. In addition, the nuclear extract when immunoprecipitated for HIF-1α was shown to be complexed with the co-activator p300, indicating that HIF-1α is transcriptionally active. Co-immunoprecipitation assay showed that HIF-1α from CLL B cells does not associate and form a complex with von Hippel-Landau protein tumor suppressor (pVHL), indicating that the proteasome dependent degradation pathway for HIF-1α protein in CLL B cells is dysfunctional. Using immunoblot or IHC methods, we were unable to detect pVHL protein in CLL B cells; however, we were able to use immunoprecipitation of CLL B cell lysates to demonstrate there is pVHL in CLL B cells. Prolyl hydroxylases (PHD 1, 2, and 3) are negative regulators for HIF-1α via hydroxylation of amino acid prolines in the oxygen degradation domain (ODD) which permits interaction with pVHL. RT-PCR results revealed that there is a subset of CLL patients who had ≥ 50% reduction of PHD 1 and 3 mRNA levels. However using a hydroxylation specific polyclonal antibody we found that HIF-1α from CLL B cells is indeed hydroxylated. Finally, silencing of HIF-1α by RNA interference in CLL B cells was associated with a selective decrease in VEGF mRNA levels but not VEGF-R1, Mcl-1 and prolyl hydroxylases (PHD 1–3) other downstream target genes of HIF-1α. These data show that the high endogenous HIF-1α levels in CLL B cells are due to a defect in HIF-1α degradation via the proteosomal pathway. We believe that this abnormality is linked to the autocrine VEGF pathway in CLL B cells and ultimately results in increases in their apoptotic resistance. Inhibition of HIF-1α levels may be of therapeutic benefit to CLL patients.

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Cro, Lilla, Luca Baldini, Lucia Nobili, et al. "The Cytofluorimetric/FISH Diagnostic Approach Define a B-Cell Variant-CLL with Peculiar Clinico-Biological Features." Blood 110, no. 11 (2007): 2077. http://dx.doi.org/10.1182/blood.v110.11.2077.2077.

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Abstract In the present study, we describe the clinico-biological features of 63 cases of variant B-CLL (v-B-CLL) defined according to a previously described diagnostic algorithm for CD5+ mature B-cell leukemias, based on the combined use of Cytofluorimetric (CFM) analysis and t(11;14)(q13;q32) detection by means of fluorescence in situ hybridisation (FISH). These leukemias were characterized by SIgbright/CD23+ or CD23+/− , CD79b/CD20 bright, and negativity for t(11;14)(q13;q32). A historical series of 112 classical B-CLL was used as comparison. The mean age was 61 years (33–85) and M/F ratio 1.9. The v-B-CLL cases were significantly different from the B-CLL cases in terms of the following clinico-hematological variables: age <70 yrs, lymphocytosis <20 ×109/L (P <.0001), lymphocyte doubling time ≤ 12 months (P=.04), high serum ß2-microglobulin levels (P=.003) and presence of splenomegaly (P=.007). No differences were found in terms of sex, Hb levels, platelets count, presence of serum monoclonal component, serum LDH levels and HCV-Ab positivity. In v-B-CLL and B-CLL pts the median follow-up was respectively 56 and 84 months. The median overall survival was 80 months in v-B-CLL pts while it was not reached in B-CLL pts; 37 pts (58.7%) in the first group started chemotherapy vs 84 pts (56.4%) in the latter. The analysis of leukemic cell biological characteristics related to the prognosis was also made in part of the cases. A classic or mixed-CLL cytomorphology was seen only in 22/61 pts (36.1%). The percentage of CD38 positive cases was higher in v-B-CLL (60.3) than in B-CLL (44.6; P=.028); the IgVH mutation status, evaluated in 30/63 cases, was more frequently hypermutated in v-B-CLL group (23/30) than B-CLL (P=.03); the percentage of CFM Zap-70 positivity, evaluated in 42/63 cases, was similar in two groups (47.6 vs 57.4%, respectively). Interestingly, significant differences were found about the frequence of the recurrent chromosome alterations, evaluated in 48/63 cases, by means of FISH analysis: trisomy 12 was more frequent in v-B-CLL (37.5 vs 18.4%, p=0.02), while del13q14 was more frequent in B-CLL (14.5 vs 37.8%, P=0.007). In both groups, del 11q22.3 and del 17p13.1 were relatively rare (4 vs 8%, and 2 vs 7.7%, respectively). In conclusion, our study identifies, on the basis of a defined CFM-FISH diagnostic approach, a variant form of B-CLL that shows significant differences in terms of genetic and clinical features.

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Kikushige, Yoshikane, Fumihiko Ishikawa, Toshihiro Miyamoto, and Koichi Akashi. "Hematopoietic Stem Cells Are Primarily Involved In Pathogenesis of Chronic Lymphocytic Leukemia." Blood 116, no. 21 (2010): 4604. http://dx.doi.org/10.1182/blood.v116.21.4604.4604.

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Abstract Abstract 4604 Chronic lymphocytic leukemia (CLL) is characterized by consistent expansion of B cells in peripheral lymphoid organs. CLL B cells express CD5 antigen, and have clonal rearrangement of immunoglobulin heavy chain (IGH) genes with the restricted usage of VH genes. CLL has thus been believed to represent retention or proliferation of abnormal B cell clones presumably with anti-apoptotic potential, or with deregulated response to auto-antigens. In this study, we extensively search for CLL-initiating cells by utilizing the xenogeneic transplantation system, in which human hematopoietic stem cells (HSCs) can normally develop multi-lineage cells including polyclonal B cells. In the xenotransplantation system, neither human (h)CD34-hCD19+ circulating B cells, nor hCD34+hCD38+ bone marrow (BM) progenitor populations from CLL patients engrafted after injection of >106 cells. We then transplanted hCD34+hCD38- BM HSC population from 12 CLL patients. Mice transplanted with as few as 3×103 cells of the hCD34+hCD38- BM population from CLL patients exhibited multi-lineage human hematopoietic reconstitution. Surprisingly, monoclonal or oligoclonal hCD5+ mature CLL-like B cells were reconstituted in the mice transplanted with HSCs from 11 out of 12 CLL patients. IGH gene rearrangement analyses demonstrated that these clonal abnormal B cells derived from hCD34+hCD38- HSCs of CLL patients (CLL-HSCs) were completely independent from the original leukemia. Interestingly, these clonal B cells showed highly biased usage of VH genes for their IGH rearrangement like primary CLL cells. We then checked the chromosomal abnormalities including del13q14 and del11q22 in CLL-HSCs and the clonal B cells reconstituted in the recipient mice. In 3 cases examined, both of them were negative for the chromosomal abnormalities detected in the original CLL cells, suggesting that the chromosomal abnormalities were acquired as a 2nd leukemogeneic events during CLL progression in the patients. The fact that CD34+CD38- HSCs from CLL patients but not those from normal individuals reconstitute monoclonal B cell lymphocytosis (MBL)-like human hematopoiesis in our xenograft model strongly suggests that some genetic abnormalities for mature monoclonal B cell development are acquired already at the HSC level in CLL patients. HSCs in CLL patients are multipotent, but once they commit to the B cell lineage, they use preferentially the VH1, VH3 and VH4 regions for IGH recombination and MBL-like clonal B cell expansion occur. Our hypothesis is that such B cell clones may already be abnormal in that they clonally expand in response, for example, to auto-antigens (xeno-antigens in mice), and these preleukemic B cell clone may possibly sequentially receive additional genetic events including chromosomal abnormalities during development of CLL and finally progress to clinical CLL. Although this xenograft model may not recapitulate full picture of CLL progression, our data clearly show that primary leukemogenic event occurs at the multipotent HSC stage in human CLL. Disclosures: No relevant conflicts of interest to declare.

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Hallaert, Delfine Y. H., Laura A. Smit, Rene Spijker, et al. "Increased Expression Level of Noxa in Peripheral Versus Lymph Node Chronic Lymphocytic Leukemia Cells Is Correlated with Survival Capacity." Blood 108, no. 11 (2006): 4972. http://dx.doi.org/10.1182/blood.v108.11.4972.4972.

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Abstract Background: The gradual accumulation of chronic lymphocytic leukemia (B-CLL) cells is presumed to derive from proliferation centers in lymph nodes and bone marrow. To what extent these cells possess the purported anti-apoptotic phenotype of peripheral B-CLL cells is unknown. Recently, we have described that in B-CLL samples from peripheral blood, aberrant apoptosis gene expression was not limited to protective changes but also included increased levels of pro-apoptotic BH3-only member Noxa. The functional consequence of this finding is not known, nor whether this aberrant apoptosis gene profile is also present in CLL proliferation centers. Aim: To perform a functional comparison of apoptosis gene profiles from peripheral blood (PB) B-CLL versus lymph node (LN) proliferation centers. Methods: Immunofluorescence microscopy, RT-Multiplex-Ligation-dependent Probe Amplification (RT-MLPA), Western Blot, Transduction, RNA interference. Results: PB samples (>90% CD5/CD19/CD23+ B-CLL cells) from 16 B-CLL patients and LN samples from 9 B-CLL patients were included in this study. LN samples contained over 90% CD5/CD19/CD23+ lymphocytes with Ki67+ cells either scattered throughout the LN or in follicle-like structures. RNA samples were subjected to the RT-MLPA procedure which monitors expression of 34 apoptosis genes. Apart from expected differences in survivin and Bcl-xL, the most prominent distinction with PB B-CLL cells was the generally low levels of Noxa in LN samples. Figure 1: Profiling of apoptosis genes in peripheral blood and lymph node samples of B-CLL patients Figure 1:. Profiling of apoptosis genes in peripheral blood and lymph node samples of B-CLL patients A reduction in Noxa RNA and protein levels could also be obtained by in vitro stimulation of PB B-CLL with CD40. Direct manipulation of Noxa protein levels was achieved by proteasome inhibition in B-CLL cells and via RNAi in model cell lines. In all these instances the viability of the cells was inversely correlated with Noxa levels. Conclusions: These data indicate that spontaneous apoptosis of PB B-CLL cells in vitro is linked with high Noxa levels. We propose that suppression of Noxa in the LN contributes to the persistence of B-CLL, and that therapeutic targeting of Noxa might be beneficial.

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Tinhofer, Inge, Ingrid Marschitz, Marion Kos, et al. "Differential Sensitivity of CD4+ and CD8+T Lymphocytes to the Killing Efficacy of Fas (Apo-1/CD95) Ligand+ Tumor Cells in B Chronic Lymphocytic Leukemia." Blood 91, no. 11 (1998): 4273–81. http://dx.doi.org/10.1182/blood.v91.11.4273.411k25_4273_4281.

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B-chronic lymphocytic leukemia (B-CLL) is characterized by cellular and humoral immune defects resulting in increased rates of infection and disturbed immune surveillance against cancer cells as well as by the expansion of slowly proliferating tumor cells. We found increased Fas receptor (FasR) expression in peripheral blood CD4+and CD8+ cells of B-CLL patients compared with the equivalent cells of healthy donors. Although increased Fas receptor expression was significant in both T-lymphocytic subsets, only CD4+ cells from B-CLL patients underwent apoptosis after treatment with the agonistic Fas antibody CH11. In CD4+cells of B-CLL patients, the Fas-sensitivity also correlated with a CD4+/CD8+ ratio below the lower threshold of healthy individuals (<1.0). By contrast, FasR expression in the CD19+ fraction of B-CLL patients was downregulated compared with normal controls, and this was associated with an insensitivity to CH11-induced apoptosis. The B-CLL cell line EHEB as well as CD19+ cells from B-CLL patients constitutively expressed Fas ligand (FasL). The FasL was functionally active, as the B-CLL cell line as well as T-cell–depleted CD19+ B-CLL fractions were able to kill target T-acute lymphatic leukemia (T-ALL) cells in vitro. This effect was inhibited by the antagonistic FasR-antibody ZB4, the neutralizing anti-FasL monoclonal antibody (MoAb) NOK-2 or by transfection of the caspase inhibitor crmA. These data point to the fact that expression of FasL on CD19+B-CLL cells, together with enhanced susceptibility of CD4+ T cells toward FasL-bearing effector cells, are causally linked to the relative reduction of CD4+ cells occurring during B-CLL progression. These findings could explain the inversion of the ratio of CD4+/CD8+ cell numbers, which may be causally linked to the immune deficiency observed in these patients and to the expansion of the neoplastic clone in B-CLL.

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Sinha, Sutapa, Charla R. Secreto, Justin C. Boysen та ін. "Β-Catenin and Axl Receptor Tyrosine Kinase Modulation in CLL B-Cells with Co-Culture on Marrow Stromal Cells: Implications for Drug Resistance". Blood 134, Supplement_1 (2019): 1739. http://dx.doi.org/10.1182/blood-2019-121772.

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Introduction: Bone marrow stromal cells (BMSCs), a major component of CLL microenvironment influences CLL B-cell survival. We have utilized an in vitro BMSC model system and found unique alterations in CLL B-cells with short term BMSC co-culture that point to previously unidentified biologic changes in the CLL B-cells that may influence CLL B-cell signaling and their drug resistance. Methods: Purified primary CLL B-cells (n=41) from previously untreated CLL patients were cultured alone or co-cultured with primary BMSCs from either normal individuals (n=24) or CLL patients (n=18) at a 50:1 ratio in AIMV medium. After 48 hours, separated CLL B-cells or BMSCs were examined by immunoprecipitation/Western blot analyses to assess the presence of intracellular signal proteins and real time PCR to determine the RNA level expression. In separate experiments to assess CLL B-cell response in co-culture with BMSC, purified CLL B-cells were treated with the following agents at sub lethal doses; fludarabine, chlorambucil, ibrutinib and venetoclax as single agents. Results: Consistent and significant increase in expression of Axl receptor tyrosine kinase for both mRNA and protein levels was observed in CLL B-cells co-cultured with BMSCs compared to CLL B-cells cultured alone. We also detected significantly increased expression of β-catenin at the protein level in CLL B-cells co-cultured with BMSCs. In contrast there was no significant change in β-catenin or Axl protein expression in the co-cultured BMSCs. Co-culturing of CLL B-cells with BMSCs using transwells confirmed that the upregulation of both Axl and β-catenin is dependent on the direct contact of CLL B-cells with BMSCs. Experiments showed that the increased Axl level in co-cultured CLL B-cells was independent of Axl kinase activity. Furthermore, the CLL B-cells from co-culture also had clear upregulation of downstream P-ERK-1/2 but no change in P-AKT(Ser473). Next we reasoned that if increases in Axl and β-catenin are related to drug resistance then CLL B-cells should have increases in those proteins when cultured with drugs and in presence of BMSCs. Treatment with chemotherapeutic or targeted therapy drugs, (i.e. fludarabine, chlorambucil, ibrutinib, venetoclax) with sublethal doses was found to lead to increase in expression levels of both β-catenin and Axl in co-cultured CLL B-cells. However these increases were significantly over that seen with simple co-culture of CLL B-cells with BMSCs. Since CLL B-cells were less sensitive to these latter chemotherapy drugs in presence of BMSCs, this suggests to us a role for both Axl and β-catenin in stromal mediated CLL B-cell drug resistance to these agents. Moreover, high nuclear β-catenin and P-ERK-1/2 levels were also detected in co-cultured CLL B-cells. It is also known that ERK associates with and inactivates GSK-3β resulting in the up-regulation of β-catenin. We found upregulation in P-GSK-3β (Ser9) an inactive molecule which can result in increasing accumulation of β-catenin. Inhibition of P-ERK-1/2 using the ERK inhibitor PD98059 in co-cultured CLL B-cells inhibited β-catenin as well as Axl expression levels. Furthermore, it has been shown that Axl expression can be regulated by c-Jun activity. In that regard we have observed upregulation of P-c-Jun(Ser73) which can enhance Axl level in co-cultured CLL B-cells and inhibition of c-Jun activity using SP600125 (c-jun upstream JNK inhibitor), as well inhibited the Axl expression. Finally, we studied 5 CLL patients before and while being treated with ibrutinib/chemo-immuno therapy for the expression of CLL B-cell Axl, β-catenin, P-ERK-1/2, P-c-Jun(Ser73) levels. We found that 2 patients had increase in Axl expression, 4 patients had increased β-catenin and P-ERK-1/2 levels and 3 patients showed increase in P-c-Jun(S73) level after the therapy. Conclusion: Here we show that marrow stromal cell contact with CLL B-cells consistently mediate increased expression in both β-catenin and Axl in CLL B-cells (Figure1). The mechanism for this may, in part, via activated ERK and c-Jun levels (Figure1). We believe that these changes in both molecules are associated with leukemic B-cell survival and drug resistance (Figure1). These studies suggest that a further understanding of the roles of Axl and β-catenin in the CLL B-cells mediated by contact with BMSC will help to develop potential strategies for the management of CLL disease resistant to various drugs. Disclosures Warner: Tolero Pharmaceuticals: Employment. Bearss:Tolero Pharmaceuticals: Employment. Kay:Agios: Other: DSMB; Infinity Pharmaceuticals: Other: DSMB; Celgene: Other: Data Safety Monitoring Board; MorphoSys: Other: Data Safety Monitoring Board.

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Tiribelli, Mario, Elisa Barbarotto, Claudio Celeghini, et al. "Aberrant Expression of Trail in B Chronic Lymphocytic Leukemia (B-CLL) Cells." Blood 106, no. 11 (2005): 4976. http://dx.doi.org/10.1182/blood.v106.11.4976.4976.

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Abstract Chronic lymphocytic leukemia (CLL) is a quintessential example of human malignancies that are caused primarily by defect in apoptosis. Defects in apoptotic pathways contribute also to chemoresistance and can promote resistance to cellular immune responses. TNF-related apoptosis inducing ligand (TRAIL), interacts with four high affinity membrane receptors (TRAIL-R1 – R4): R1 and R2 are thought to transducer apoptotic signals, while R3 and R4 may act as “decoy” receptors, protecting cells from apoptosis. Despite its potential anti–cancer activity, TRAIL alone has a low cytotoxic activity on B-CLL, and no data are available on the expression of TRAIL and its biological potential function in B-CLL. We examined the expression of TRAIL in peripheral blood CD19+/CD5+ B lymphocytes from 44 patients affected by CLL at diagnosis, the susceptibility of B-CLL cells to recombinant TRAIL and the role of endogenous membrane-bound TRAIL on autologous cell survival. Each B-CLL patient had a follow up time of 12 to 24 months from diagnosis and was clinically stable. None of the patients received chemotherapy. Lymphocytes from normal PBMC revealed an absent or dim expression of TRAIL. Surface TRAIL was detected in all 44 B-CLL examined, at variable intensity from patient to patient, but with an overall significantly greater MFI with respect to normal lymphocytes. Higher levels of TRAIL mRNA and protein were documented in purified CLL CD19+ B lymphocytes, confirming that TRAIL was overexpressed at the transcriptional level. The addition in culture of a TRAIL-R1-Fc chimera, which binds at high affinity to surface TRAIL, significantly decreased the percentage of viable cells with respect to untreated cultures. No effects were noted when TRAIL-R1-Fc chimera was added to normal CD19+ B cells. Preincubation of TRAIL-R1-Fc chimera with recombinant TRAIL significantly counteracted the decrease of viability induced by TRAIL-R1-Fc chimera. These data indicate that surface TRAIL might be involved in a promoting a prosurvival activity, at least in some B-CLL samples. We then investigated the response in terms of cell viability to exogenously added recombinant TRAIL (1 μg/ml) of all 44 B-CLL samples examined in this study. On the basis of their response to recombinant TRAIL, B-CLL samples could be subdivided in 3 groups. In 11 patients (group 1), B-CLL cells showed a faster decline in the number of viable cells upon treatment with recombinant TRAIL. In 19 samples (group 2),no significant variation in terms of viable cell number in TRAIL-treated cultures was observed. In 14 patients (group 3), TRAIL-treatment results in a significantly higher numbers of viable cells compared to untreated cultures. At flow cytometry, B-CLL lymphocytes expressed at variable levels TRAIL-R1, TRAIL-R2 and TRAIL-R4, while TRAIL-R3 was never detected. TRAIL-R1 was expressed at low levels in a subset of B-CLL samples, while TRAIL-R2 was expressed in almost the totality of the B-CLL samples. TRAIL-R4 was detected in the majority of B-CLL lymphocytes, but at lower levels than in normal lymphocytes. Of note, the different apoptotic/survival response to recombinant TRAIL among the three groups of B-CLL samples described above, apparently did not depend on differences in death and/or decoy receptor patterns of expression. A progressive increase of TRAIL MFI was noticed from group 1 to group 3.

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Knauf, Wolfgang Ulrich, Toshko Lissichkov, Ali Aldaoud, et al. "Bendamustine Versus Chlorambucil in Treatment-Naive Patients with B-Cell Chronic Lymphocytic Leukemia (B-CLL): Results of an International Phase III Study." Blood 110, no. 11 (2007): 2043. http://dx.doi.org/10.1182/blood.v110.11.2043.2043.

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Abstract Introduction: Bendamustine (BEN) is a purine analog / alkylator hybrid agent with a particular mechanisms of action that provides effective treatment for a number of hematologic and non-hematologic malignancies. It is used primarily for chemo-naïve, relapsed or refractory B-CLL as well as for other types of non-Hodgkin’s lymphomas. The aim of this randomized phase III, open-label, multicenter study was to compare the efficacy and safety of BEN versus chlorambucil (CLB) in treatment-naïve patients (pts) with B-CLL Binet stage B/C. Patients and Methods: Pts with untreated B-CLL were randomized to receive BEN (100 mg/m2 on days 1+2) or CLB (0.8 mg/kg on days 1+15) for up to 6 treatment cycles. Primary endpoints were overall remission rate (ORR), defined as complete response (CR), nodular partial response (nPR) and partial response (PR), confirmed after 8 weeks, and progression-free survival (PFS). Secondary endpoints were duration of remission, overall survival (OS), safety, and quality of life (QoL). Follow-up was for ≥12 months after completion of treatment of the last patient, or until progression for pts with CR, nPR or PR and stable disease, or until death or lost to follow-up. A 5-stage, adaptive-group, sequential procedure was used with planned interim analyses to adjust the number of pts. Safety and efficacy were assessed by an Independent Data Monitoring Committee. Results: 305 pts were randomized to receive BEN (n=156) or CLB (n=149). As 7 pts did not receive study medication, 298 pts were included in the safety analysis. At the time of this analysis, 264 pts (139 BEN; 125 CLB) were available for the efficacy analysis. For both treatment groups: median age was 64 years; 70% had Binet stage B and 30% Binet stage C disease; median number of cycles/patient was 6; median follow-up was 18.5 months. ORR was significantly higher with BEN than with CLB (68% vs 39%; p&lt;0.0001), with a CR of 30% vs 2%, respectively. Among the subgroups with Binet stage B and C disease, ORR was 70% and 61%, respectively, with BEN, vs 47% and 22%, respectively, with CLB. Median PFS (Kaplan-Meier estimate) was 21.7 months with BEN and 9.3 months with CLB (p&lt;0.0001), and median duration of remission was 18.9 months with BEN and 6.1 months with CLB (p&lt;0.0001). No difference in OS was seen between groups. Toxicity of BEN was manageable and did not impair QoL when compared with CLB. Infection rates (common toxicity criteria grades III+IV) were low in both groups (5.8% BEN; 3.5% CLB). Conclusions: BEN was significantly more effective than CLB in achieving remissions in treatment-naïve pts with B-CLL Binet stage B/C; median PFS and duration of remission were also significantly longer. Furthermore, safety data indicate that BEN toxicities are manageable and the drug is well tolerated. On the basis of these results, BEN should be considered as first-line chemotherapy for patients with B-CLL Binet stage B or C.

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Ghosh, Asish K., Debabrata Mukhopadhyay, and Neil E. Kay. "Bosutinib, a Src/Abl Kinase Inhibitor Induces Apoptosis in CLL B Cells by Targeting Multiple Tyrosine Kinases and Overcomes Stroma Protection." Blood 114, no. 22 (2009): 2368. http://dx.doi.org/10.1182/blood.v114.22.2368.2368.

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Abstract Abstract 2368 Poster Board II-345 Background: B-cell chronic lymphocytic leukemia (CLL) is characterized by the progressive accumulation of CD5+ B lymphocytes in the peripheral blood, lymphoid organs and bone marrow. Despite aggressive therapy, CLL is still incurable partly because of intrinsic defect in apoptosis induction. A novel therapeutic agent, Bosutinib, was initially developed as an inhibitor of Src and Abl kinases and is currently in phase II clinical trials for the treatment of several human malignancies. Recently, Bosutinib has been shown to inhibit phosphorylation of a novel receptor tyrosine kinase, Axl which has been reported to be overexpressed in several types of human cancers including colon, prostatic, thyroid, breast, gastric, renal and lung. Previously, Dasatinib, another Src/Abl kinase inhibitor, showed cytotoxic effects on CLL B cells by decreasing levels of activated, phosphorylated forms of Akt, Erk1/2 and p38 and reducing expression of anti-apoptotic proteins Mcl-1 and Bcl-xL in CLL B cells. Here, we wished to examine receptor or non-receptor tyrosine kinases active in primary CLL B-cells and determine their status after exposure to Bosutinib as well as the latter drug's effect on CLL B-cell viability. Methods: We used freshly isolated CLL B cells after obtaining written consent from patients. Bosutinib was used at various doses (2.5, 5.0, 10.0 and 20.0 μM) to treat CLL B cells in vitro for 24/48 hrs. Induction of apoptosis was assessed by annexin/propidium staining. To examine the impact of stroma on Bosutinib induced CLL B-cell death, primary CLL-bone marrow stromal cells (BMSC) were cocultured with CLL B cells at a cell density ratio of 1:20 and treated with various doses of Bosutinib for 24 hrs. Expression status of various kinases and downstream targets were analyzed in CLL B cell lysates with or without Bosutinib-treatment by Western blot using specific antibodies. Results: Treatment of CLL B-cells with Bosutinib induces a massive apoptotic cell death in a dose- and time-dependent manner (IC50 for 24 h; ∼10 μM and IC50 for 48 h: 5-10 μM) which involves PARP cleavage as demonstrated by Western blot analysis. Moreover, Bosutinib-treatment reduced expression of several key anti-apoptotic proteins, Mcl-1, XIAP and Bcl-2 reported to be overexpressed in CLL B cells. Interestingly, we detected that the majority of CLL B-cells express constitutively active Axl. Importantly, Bosutinib treatment inhibited phosphorylation of Axl in CLL B cells resulting in inhibition of AKT-activation, one of its downstream signaling pathways. Previous studies have suggested a possible physical association between Axl and Src kinase. We observed that expression of constitutively active Axl was associated with the presence of highly phosphorylated Src kinase when compared with that in CLL B-cells with low or unphosphorylated Axl. These observations suggest that phosphorylation of Axl may be an upstream event for Src activation in CLL. We found inhibition of constitutively active Axl resulted in subsequent inhibition of Src kinase activation in CLL B-cells following Bosutinib-treatment. We also detected inhibition of ZAP70/Syk-phosphorylation in CLL B cells upon Bosutinib-treatment. Finally, we found Bosutinib was able to overcome stomal protection of CLL B cells at a dose of 10 μM in an in vitro coculture system suggesting its potential as a therapeutic agent against CLL. Conclusion: Together, these observations suggest that Bosutinib induces apoptosis in CLL B-cells, even in the presence of stromal cells, in association with the down regulation of multiple kinases including the novel receptor-tyrosine kinase, Axl, and reduces expression of the anti-apoptotic proteins critical to CLL B-cell survival. In total, these findings for the first time indicate that Bosutinib has the potential to be a very potent therapeutic agent for CLL patients. Disclosures: Kay: Biogenc-Idec, Celgene, Genentech, genmab: Membership on an entity's Board of Directors or advisory committees; Genentech, Celgene, Hospira, Polyphenon Pharma, Sanofi-Aventis: Research Funding.

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Ponzoni, Maurilio, and Claudio Doglioni. "Prognostic factors in B-CLL." Advances in Anatomic Pathology 11, no. 3 (2004): 172–73. http://dx.doi.org/10.1097/00125480-200405000-00006.

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Farren, Timothy W., Feng-Ting Liu, Claire Stephens, et al. "CD160 Expression in B-Cell Lymphoproliferative Disorders: Derivation and Validation of a New Three-Antigen Scoring System Improving Diagnostic Precision in B-Cell Malignancies." Blood 114, no. 22 (2009): 4382. http://dx.doi.org/10.1182/blood.v114.22.4382.4382.

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Abstract Abstract 4382 The diagnosis of many chronic B-cell lymphoproliferative disorders (B-LPD) is based on the integration of characteristic morphology, immunophenotype and cytogenetic analysis. Atypical B-cell chronic lymphocytic leukaemia (CLL), mantle cell lymphoma (MCL) and monoclonal B cell lymphocytosis (MBL) share many features. which can lead to diagnostic confusion. Throughout the past decade, considerable progress has been achieved in defining new prognostic markers and treatment options in B-cell malignancies. However, over the same period, there has been only limited work performed on the diagnostic aspects of distinguishing CLL from other B-cell malignancies. On a cohort of 748 consecutive B-LPDs, we established the three most robust diagnostic markers of CLL were CD5, CD23 and CD160. From this data set a 3-antigen 'mini' CLL score was derived, with each scoring one point. In this validation study, we compare the 'mini CLL score' against the 5-antigen Royal Marsden CLL score. This validation study included 163 consecutive cases of B-cell malignancies (CLL n=113; CD5-negative B-LPD n=13; CD5-positive B-LPD n=10; MCL n=8; hairy cell leukaemia [HCL] variant n=5; HCL n=3; diffuse large B-cell lymphoma [DLBCL] n=3; splenic marginal zone lymphoma [SMZL] n=3; and lymphoplasmacytic lymphoma [LPC/WM] n=3; follicular lymphoma [FL] n=2). The prevalence of B-CLL in our validation group was 0.69 (0.61 – 0.76). Samples were analyzed using multicolour flow cytometry and sequential gating strategies. Same day analysis for all antigens was performed. 95% (107/113) of CLL cases were positive for the CD160 antigen (p=<0.0001). 7% of the confirmed CLL cases had an atypical phenotype and a Marsden CLL score of <3, suggestive of an alternative diagnosis to CLL. However, 75% (6/8) of these cases were CD160 positive. Table 1 (above): Mini CLL score results by final diagnosis. 'mini CLL score' 0 1 2 3 TOTAL CLL 0 0 7 106 113 MCL 0 6 2 0 8 HCL 0 1 2 0 3 SMZL/LPC/WM 5 5 1 0 11 Other BLPD 7 15 6 0 28 TOTAL 163 Table 2 (above): Global performance of the 5 point CLL Royal Marsden Scoring system against the 'mini CLL score'. CLL Score Mini CLL Score Sensitivity 0.93 (0.87 - 0.97) 0.92 (0.85 – 0.96) False positive rate 0 (0 – 0.07) 0 (0 – 0.07) Diagnostic odds ratio ° (129 - °) ° (116 - °) A mini CLL score of 3 was diagnostic of CLL and excluded all other B-cell malignancies (including MCL) in this cohort of 163 cases (p<0.0001). However, 7 CLL cases with a mini CLL score of 2 could not be distinguished from other B-cell malignancies, especially CD23+ MCL. This led to the approach of defining the ratio of CD23 to CD5 expression on malignant B-cells. The CD23r proved of value with a ratio of >0.94 in 80% of patients (0.7104-0.8664), ie, a sensitivity and a specificity of 0.7965 and 1.000 (0.9598-1.000), respectively. A CD23r cut-off of 0.51 between the two diagnostic groups increased the sensitivity to 0.9912 (0.9517-0.9998) with an odds ratio of having CLL of 1275 (48.15-33765). In summary, the near universal expression of CD160 in CLL allows a simplified and accurate immunophenotypic diagnosis of B-LPD. CD160 expression in CLL had a sensitivity and specificity of 0.9496 (0.8880-0.9803) and 0.8400 (0.7089-0.9283), respectively. Despite the Royal Marsden Score and the new 'mini' score giving the same overall global performance (table 2), by combining the 'mini-score' with the CD23:CD5 ratio provides a complete discrimination between CLL and MCL, including cases of CD23+ MCL (p<0.0001). This study validates the previously reported expression of CD160 antigen in CLL and further justifies its use in the diagnosis of B-cell malignancies. Disclosures: No relevant conflicts of interest to declare.

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Tiwari, Sanjay, Kyriacos Felekkis, Eun-Yi Moon, Amanda Flies, David H. Sherr, and Adam Lerner. "Among circulating hematopoietic cells, B-CLL uniquely expresses functional EPAC1, but EPAC1-mediated Rap1 activation does not account for PDE4 inhibitor-induced apoptosis." Blood 103, no. 7 (2004): 2661–67. http://dx.doi.org/10.1182/blood-2003-06-2154.

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Abstract Type 4 cyclic adenosine monophosphate (cAMP) phosphodiesterase (PDE4) inhibitors and other agents that raise intracellular cAMP levels induce apoptosis in B-cell chronic lymphocytic leukemia (B-CLL) but not in T-CLL or peripheral blood T cells. Two principal effector proteins for cAMP are protein kinase A (PKA) and EPAC (exchange protein directly activated by cAMP), a Rap guanosine 5′-diphosphate (GDP) exchange factor. We here examine whether varying expression of EPAC accounts for the discrepant sensitivity of B-CLL and T cells to PDE4 inhibitor-induced apoptosis. B-CLL and peripheral blood B cells express EPAC1 transcript, whereas T-CLL, peripheral blood T cells, monocytes, and neutrophils do not. Treatment with the PDE4 inhibitor rolipram induces Rap1 activation in B-CLL cells but not in peripheral blood B cells, T-CLL, or any of the normal hematopoietic lineages examined. The EPAC-specific cAMP analog 8CPT-2Me-cAMP (8-(4-chloro-phenylthio)-2′-O-methyladenosine-3′,5′-cAMP) activates Rap1 in B-CLL cells, but, unlike rolipram/forskolin or 8-Bromo-cAMP, it does not induce PKA activation, as judged by phosphorylation of the transcription factor cAMP-response element binding protein (CREB). Unexpectedly, whereas rolipram/forskolin and 8-Bromo-cAMP induce apoptosis in B-CLL cells, 8CPT-2Me-cAMP decreased basal apoptosis in B-CLL cells by an average of 25% (P &lt; .002). Our results demonstrate that B-CLL cells uniquely activate Rap1 in response to PDE4 inhibitors and suggest that physiologic stimuli that activate EPAC may transmit an antiapoptotic signal. (Blood. 2004;103:2661-2667)

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Ghosh, Asish K., Charla Secreto, Justin Boysen, et al. "The novel receptor tyrosine kinase Axl is constitutively active in B-cell chronic lymphocytic leukemia and acts as a docking site of nonreceptor kinases: implications for therapy." Blood 117, no. 6 (2011): 1928–37. http://dx.doi.org/10.1182/blood-2010-09-305649.

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Abstract Recently, we detected that chronic lymphocytic leukemia (CLL) B-cell–derived microvesicles in CLL plasma carry a constitutively phosphorylated novel receptor tyrosine kinase (RTK), Axl, indicating that Axl was acquired from the leukemic B cells. To examine Axl status in CLL, we determined the expression of phosphorylated-Axl (P-Axl) in freshly isolated CLL B cells by Western blot analysis. We detected differential levels of P-Axl in CLL B cells, and further analysis showed that expression of P-Axl was correlated with the other constitutively phosphorylated kinases, including Lyn, phosphoinositide-3 kinase, SyK/ζ-associated protein of 70 kDa, phospholipase C γ2 in CLL B cells. We found that these intracellular signaling molecules were complexed with P-Axl in primary CLL B cells. When Axl and Src kinases were targeted by a Src/Abl kinase inhibitor, bosutinib (SKI-606), or a specific-inhibitor of Axl (R428), robust induction of CLL B-cell apoptosis was observed in both a dose- and time-dependent manner. Therefore, we have identified a novel RTK in CLL B cells which appears to work as a docking site for multiple non-RTKs and drives leukemic cell survival signals. These findings highlight a unique target for CLL treatment.

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Jung, Byeongho, Anita Ng, Pui Yan Chiu, Barbara Sherry та Nicholas Chiorazzi. "Skewing Towards Effector Memory Cells in CLL Is Associated with IGHV -Mutation Status and IFNγ and IL-4". Blood 142, Supplement 1 (2023): 4625. http://dx.doi.org/10.1182/blood-2023-190595.

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Memory T cells are a small, heterogenous population of T cells that remain following pathogen clearance and provide long-lasting protection. They are generally characterized as either central memory (T CM) or effector memory (T EM) based on differences in their surface and intracellular markers, anatomic location, effector function, and cytokine production. It is well known that patients with chronic lymphocytic leukemia (CLL) display differences in both T cell function and population compared to healthy individuals. Previous studies have shown that CLL patients skew towards the CD4 + memory compartment and among these, a skewing toward T EM is especially associated with unmutated IGHV and more progressive disease. Moreover, T EM cells appear to protect CLL cells, possibly via IFNγ and IL-4 mediated pathways. To understand this crosstalk between memory T cells and CLL B cells, we co-cultured FACS-purified T CM (CD3 +CD4 +CD19 -CCR7 +CD45RA -CD45RO +CD62L +) and T EM (CD3 +CD4 +CD19 -CCR7 -CD45RA -CD45RO +CD62L -) cells with CLL B cells and assessed shifts in the memory population as well as cytokines produced. Furthermore, as leukemic B-cell subpopulations differing in time since cell birth/division display distinct functional abilities to present antigen, we also investigated the effect of B-cell activation on this crosstalk. FACS-purified peripheral blood memory T cells and CLL B cells were obtained from treatment-naïve patient samples and co-cultured for 7 days to determine if the presence of CLL B cells, either resting (B rest) or pre-activated with CpG-ODN2006 and IL-15 (B act), would alter the T-cell population distribution. In the presence of either B rest or unmanipulated PBMC, overall, there were no significant changes in population of T EM or T CM. However, the distribution of T EM with B rest was bimodal and when grouped based on IGHV mutation status, we found significantly reduced counts of T EM in co-cultures with M-CLL (14.50% T EM, 11,019 cells) as compared to U-CLL (26.68% T EM, 20,648 cells) based on relative percentages and absolute cell counts ( P&lt;0.001). Conversely, we saw an increase of T CM in M-CLL B rest co-cultures that correlated with decrease of T EM ( P&lt;0.01). We then analyzed supernatants taken at the end of the 7-day co-culture period for IFNγ, IL-4, IL-17A, and IL-17F. We found elevated concentrations of IFNγ in co-cultures with both mutated-CLL and M- CLL, but significantly higher concentrations ( P&lt;0.01) were found with U-CLL (312pg/ml) than M-CLL (173pg/ml). Similarly, IL-4 was elevated but more so (p&lt;0.001) in U-CLL (88pg/ml) than M-CLL (44pg/ml). Of note, IFNγ and IL-4 have been implicated in progression of CLL with literature suggesting their role in prolonging CLL B cell survival. IL-17A and IL-17F were not elevated above baseline and no differences were found. Consistent with literature, the relative fraction of T EM in our patients negatively correlated with TTFT. We then repeated the experiments with pre-activated CLL B cells. When B act were co-cultured with memory T cells, we found significantly ( P&lt;0.001) elevated absolute number of T EM and T CM. This indicated heightened proliferation of the memory population, with a greater degree of increase in the T EM population, resulting in a skewing toward T EM. Subsequent experiments found that co-culture with B act led to significantly elevated IL-4 and IFNγ levels. Interestingly, the effects mediated by B act, on both memory cell proliferation and cytokine production were markedly greater than with B rest. Moreover, unlike B rest, the effects of B act did not depend on IGHV mutation status but rather showed uniform increase in both T EM proliferation and IFNγ and IL-4 levels. Lastly, we analyzed the survival of CLL B cells at the end of the co-cultures. Overall, the addition of memory T cells significantly ( P&lt;0.001) enhanced survival of CLL B cells and this effect was more prominent in B act (U-CLL: 51.1%, M-CLL: 40.3% survival) than B rest (U-CLL: 34.8%, M-CLL: 23.5% survival). Together, our in vitro results suggest crosstalk between memory T cells and CLL B cells in which CLL B cells, especially U-CLL, promote the expansion of T EM which is associated with shorter TTFT and more progressive disease. T EM, in turn, promote the survival of CLL B cells possibly through increased production of IFNγ and IL-4.

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Burger, Jan A., Meike Burger, and Thomas J. Kipps. "Chronic Lymphocytic Leukemia B Cells Express Functional CXCR4 Chemokine Receptors That Mediate Spontaneous Migration Beneath Bone Marrow Stromal Cells." Blood 94, no. 11 (1999): 3658–67. http://dx.doi.org/10.1182/blood.v94.11.3658.

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Abstract Chemokines play a central role for lymphocyte trafficking and homing. The mechanisms that direct the tissue localization of B cells from patients with chronic lymphocytic leukemia (B-CLL) are unknown. We found that CLL B cells express functional CXCR4 receptors for the chemokine stromal cell-derived factor-1 (SDF-1), as demonstrated by receptor endocytosis, calcium mobilization, and actin polymerization assays. Moreover, CLL B cells displayed chemotaxis to this chemokine that could be inhibited by monoclonal antibodies (MoAbs) against CXCR4, pertussis toxin, or Wortmannin, a phosphatidylinositol 3-kinase inhibitor. That this chemotaxis may be involved in the homing of CLL cells is argued by studies in which CLL B cells were cocultured with a murine marrow stromal cell line that secretes SDF-1. Within 2 hours, CLL B cells spontaneously migrated beneath such stromal cells in vitro (pseudoemperipolesis). This migration could be inhibited by pretreatment of CLL B cells with anti-CXCR4 MoAbs, SDF-1, or pertussis-toxin. Furthermore, we noted strong downmodulation of CXCR4 on CLL B cells that migrated into the stromal cell layer. These findings demonstrate that the chemokine receptor CXCR4 on CLL B cells plays a critical role for heterotypic adherence to marrow stromal cells and provide a new mechanism to account for the marrow infiltration by neoplastic B cells.

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Chemokines play a central role for lymphocyte trafficking and homing. The mechanisms that direct the tissue localization of B cells from patients with chronic lymphocytic leukemia (B-CLL) are unknown. We found that CLL B cells express functional CXCR4 receptors for the chemokine stromal cell-derived factor-1 (SDF-1), as demonstrated by receptor endocytosis, calcium mobilization, and actin polymerization assays. Moreover, CLL B cells displayed chemotaxis to this chemokine that could be inhibited by monoclonal antibodies (MoAbs) against CXCR4, pertussis toxin, or Wortmannin, a phosphatidylinositol 3-kinase inhibitor. That this chemotaxis may be involved in the homing of CLL cells is argued by studies in which CLL B cells were cocultured with a murine marrow stromal cell line that secretes SDF-1. Within 2 hours, CLL B cells spontaneously migrated beneath such stromal cells in vitro (pseudoemperipolesis). This migration could be inhibited by pretreatment of CLL B cells with anti-CXCR4 MoAbs, SDF-1, or pertussis-toxin. Furthermore, we noted strong downmodulation of CXCR4 on CLL B cells that migrated into the stromal cell layer. These findings demonstrate that the chemokine receptor CXCR4 on CLL B cells plays a critical role for heterotypic adherence to marrow stromal cells and provide a new mechanism to account for the marrow infiltration by neoplastic B cells.

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Perri, RT. "Impaired expression of cell surface receptors for B cell growth factor by chronic lymphocytic leukemia B cells." Blood 67, no. 4 (1986): 943–48. http://dx.doi.org/10.1182/blood.v67.4.943.943.

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Abstract Normal human B cell proliferation is controlled by various immunoregulatory signals including the T cell-derived lymphokine B cell growth factor (BCGF). The role of BCGF in the regulation of malignant B cell proliferation is unclear. Therefore, we studied the proliferative response of purified chronic lymphocytic leukemia (CLL) B cells to BCGF. For all CLL patients studied, CLL B cells showed a decreased proliferative response as compared with control B cells for BCGF- induced B cell proliferation (patient 291 +/- 59 cpm v control 3,942 +/- 622, mean +/- SEM). This impaired proliferative response appeared to be intrinsic to CLL B cells since it was not corrected by incubation with increasing concentrations of BCGF. Attainment of normal B cell responsiveness to BCGF requires the processing of an initial activation signal which results in the expression of cell surface receptors for BCGF. Increasing concentrations of the B cell activation signal (the F(ab')2 fragment of goat anti-human mu chain) did not improve CLL B cell responsiveness to BCGF. Three-day activated CLL B cells compared with activated control B cells demonstrated a marked impairment in their ability to absorb out the BCGF activity present in the BCGF preparation (BCGF activity absorbed out, patient 12.8% v control 53%). Pretreatment of CLL B cells with neuraminidase failed to improve either the proliferative response to BCGF or the expression of cell surface receptors for BCGF by the CLL B cells. This study suggests that the impaired responsiveness to BCGF by CLL B cells is the result of impaired expression of cell surface receptors for BCGF when CLL B cells are exposed to activation signals.

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Normal human B cell proliferation is controlled by various immunoregulatory signals including the T cell-derived lymphokine B cell growth factor (BCGF). The role of BCGF in the regulation of malignant B cell proliferation is unclear. Therefore, we studied the proliferative response of purified chronic lymphocytic leukemia (CLL) B cells to BCGF. For all CLL patients studied, CLL B cells showed a decreased proliferative response as compared with control B cells for BCGF- induced B cell proliferation (patient 291 +/- 59 cpm v control 3,942 +/- 622, mean +/- SEM). This impaired proliferative response appeared to be intrinsic to CLL B cells since it was not corrected by incubation with increasing concentrations of BCGF. Attainment of normal B cell responsiveness to BCGF requires the processing of an initial activation signal which results in the expression of cell surface receptors for BCGF. Increasing concentrations of the B cell activation signal (the F(ab')2 fragment of goat anti-human mu chain) did not improve CLL B cell responsiveness to BCGF. Three-day activated CLL B cells compared with activated control B cells demonstrated a marked impairment in their ability to absorb out the BCGF activity present in the BCGF preparation (BCGF activity absorbed out, patient 12.8% v control 53%). Pretreatment of CLL B cells with neuraminidase failed to improve either the proliferative response to BCGF or the expression of cell surface receptors for BCGF by the CLL B cells. This study suggests that the impaired responsiveness to BCGF by CLL B cells is the result of impaired expression of cell surface receptors for BCGF when CLL B cells are exposed to activation signals.

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