The Role of Surface Immunoglobulin Isotype in Chronic Lymphocytic Leukemia Disease Biology and Clinical Outcome

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2850-2850
Author(s):  
Danielle M. Brander ◽  
Sallie D. Allgood ◽  
Karen M. Bond ◽  
J. Brice Weinberg ◽  
Mark C Lanasa
Keyword(s):  
Flow Cytometry ◽  
B Cells ◽  
Conflicts Of Interest ◽  
Fold Increase ◽  
Surface Expression ◽  
Lymphocytic Leukemia ◽  
Mutation Status ◽  
Immunoglobulin Isotype ◽  
Surface Immunoglobulin

Abstract Abstract 2850 Background: Upon encountering cognate antigen, naïve B cells may undergo germinal center reaction. This results in DNA modification including somatic hypermutations (HSM) of the immunoglobulin heavy chain variable region gene (IGHV) and immunoglobulin class switch recombination (CSR). HSM and CSR are unlinked, but share initiating and mechanistic factors. Prior evidence indicates CLL B cells are antigen experienced; however, half of all CLL patients have an unmutated IGHV and follow more aggressive clinical courses. The biologic importance of the CLL immunoglobulin isotype and CSR in B cell receptor (BCR)-mediated signaling is not well understood. We hypothesized the immunoglobulin isotype and expression density of surface immunoglobulin would correlate with IGHV mutation status and other clinical parameters, and predict BCR responsiveness in vitro. Methods: 195 samples from our CLL specimen repository with detailed clinical and biologic characterization were evaluated. Surface expression of IgD, IgM and IgG was determined using multi-parameter flow cytometry and samples were classified as either IgG+ or IgM+IgD+ co-expressing. An ROC analysis was performed to identify the most discriminate value for dichotomization of IgM surface expression as a function of IGHV mutation status. IgMhiIgD+, and IgMlowIgD+ patients were then analyzed for associations with clinical and biologic parameters. To investigate the biological mechanisms of potential associations, BCR-mediated signaling after isotype specific crosslinking in vitro was measured by quantification of downstream phospho-proteins (SYK, AKT, ERK, MEK1, NFκB) by flow cytometry (phospho-flow). Samples were crosslinked with IgM (or IgG if IgG+), IgD, and isotype control and incubated for 0, 15, 30, 60, and 120 minutes. The extent of phosphorylation at each timepoint was expressed as mean fluorointensity (MFI) of the examined proteins. Crosslinked samples were also analyzed using oligonucleotide microarray gene analysis (GEP) to assess differential transcription. Results: A correlation was identified between the percentage of CLL cells expressing surface IgM and IGHV mutation status (r2=0.33); higher surface expression of IgM correlated with unmutated IGHV. The most discriminate value of IgM expression for prediction of IGHV mutation was 47%, with an area under ROC curve of 0.72. Of the 195 patient samples analyzed, 91 (47%) were IgMhiIgD+; 85 (43%) were IgMlowIgD+; and 19 (10%) were IgG+. IgMhiIgD+ samples showed increased expression of CD38 (p<0.01) and ZAP70 (p<0.05) compared to IgMlowIgD+ samples. Only 1 of 19 IgG+ samples was IGHV unmutated. Neither immunoglobulin isotype nor density correlated with gender, lymphocyte doubling time, age, or Rai stage at diagnosis. There was a trend toward shorter time to first treatment by IgM expression (IgMhiIgD+ 7.9yrs vs. IgMlowIgD+ 9.4yrs) but this was not significant (p=0.09). To date, we have tested the biologic relevance of surface immunoglobulin (sIg) density and isotype in 13 patients using phospho-flow analysis. Crosslinking in the IgMlow patients (sIgM <30%; n=5) demonstrated no change in MFI of tested proteins. Among IgMmod patients (sIg 31–60%; n=2), a maximal 3 fold increase in p-SYK MFI with IgM crosslinking and 3.8 fold increase with IgD crosslinking was observed. In IgMhi samples (sIgM >60%; n=3), there was a maximal 3.5 fold increase in MFI with IgM but no change with IgD crosslinking. In the IgG+ samples (n=3), a maximum 5 fold increase in MFI was observed. Analysis of GEP data is ongoing. Conclusion: Surface expression of IgM in CLL varies considerably between patients. This difference in density of surface IgM expression correlates with markers of clinical risk. Although we do not propose immunoglobulin isotype or expression density as a novel prognostic factor, the findings that high surface expression of IgM in CLL predicts BCR mediated signaling after crosslinking provides insight into CLL immunobiology and disease responsiveness to antigens in the microenvironment. In addition, the observation of significant BCR activation within the IgG+ population (which was predominantly IGHV mutated) suggests that the responsiveness of the BCR to crosslinking may be related to the immunoglobulin isotype and density, and not solely determined by the IGHV mutation status. Disclosures: No relevant conflicts of interest to declare.

Adaphostin-Induced Apoptosis in CLL B-Cells Is Associated with Induction of Oxidative Stress and Exhibits Synergy with Fludarabine.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3482-3482 ◽  
Author(s):  
Tait D. Shanafelt ◽  
Y. Kit Lee ◽  
Nancy D. Bone ◽  
Ann K. Strege ◽  
Scott H. Kaufmann ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Death ◽  
B Cells ◽  
Alkylating Agents ◽  
Single Agent ◽  
Lymphocytic Leukemia ◽  
Parp Cleavage ◽  
Ros Generation ◽  
Mutation Status

Abstract Background : B-Chronic Lymphocytic leukemia (CLL) is the most common leukemia in North America. The standard current treatments use purine nucleoside analogues as single agent therapy or in combination with rituximab, steroids, and alkylating agents. Overall response rates with these treatments in previously untreated patients may reach 90 % with CR rates of 50–60%, however, most patients will relapse. Thus, despite refinements in therapy, CLL remains an incurable malignancy and there remains significant and urgent need to identify and develop new agents with novel mechanisms of action for the treatment of CLL. In line with this we have explored the mechanism and in vitro activity for a tyrphostin, adaphostin. Methods: We evaluated the in vitro efficacy of adaphostin to induce apoptosis in CLL B-cells. Peripheral blood was collected from patients with CLL (n=57). Highly purified CLL B-cells ((minimum 80% CD19+, mean 92.2% CD19+ and 94.4% CD19+/CD5+ positive)) were cultured with freshly prepared adaphostin for 24 – 120 hours. Cell death was analyzed by flow cytometry using Anexin V/Propidium iodide (PI). PARP cleavage and anti-apoptotic protein levels were measured by immunoblot techniques. The effect of combination treatment on CLL B cells with adaphostin and fludarabine on CLL B-cells was also assessed. Results: Analysis by Annexin V/PI staining revealed that the mean IC50 for adaphostin at 24 h was 4.2 uM (range 1.10 uM-11.25 uM; median = 4.25; n = 29) for CLL isolates and >10 uM for B and T-cells isolated from normal donors. Median IC50 levels for Adaphostin were not significantly different based on IgVH mutation status, level of CD38 expression, or cytogenetic abnormalities by FISH testing (Table 1). Immunoblots demonstrated adaphostin-induced PARP cleavage and cleavage of caspase 3 substrates, suggesting that adaphostin induces cell death through apoptosis. Adaphostin increased the intra-cellular level of reactive oxygen species (ROS) in CLL B-cells; and the antioxidant N-acetylcysteine blocked both adaphostin-induced ROS generation and apoptosis (mean reduction cell death=60%; range 23–99% reduction) suggesting generation of ROS is critical to adaphostin’s induction of apoptosis. Adaphostin also caused a decrease in the level of the anti-apoptotic proteins Bcl-2 in a majority of patients on both flow cytometry and immunoblots. When adaphostin was combined with fludarabine (F-ARA-ATP), a synergistic effect on cell death was observed in all 10 CLL samples when analyzed by mathematical modeling software. Summary: These findings indicate that adaphostin induces selective apoptosis in CLL B-cells from all risk categories through a mechanism that involves ROS generation. Importantly, we also demonstrate its ability to augment the effects of fludarabine. We continue to explore the preclinical development of adaphostin as a novel agent for the treatment of CLL. IC50 Adaphostin Dose Levels by Prognostic Groups FACTOR N Median IC 50 Adaphostin Range p-Value     Rai Stage Group Low/Int (0–II) 19 4.7 1.8 – 11.25 0.018 High (III – IV) 10 3.05 1.1 – 5.4 - IgVH Mutation Status Mutated 11 4.45 1.1 – 11.25 0.72 Nonmutated 10 3.93 1.8 – 5.8 - CD38 Status Negative 20 4.55 1.1 – 11.25 0.74 Positive 9 4.25 1.8 – 6.6 -     FISH defects 13q- 8 4.70 1.75 – 11.25 Normal 6 3.53 1.1 – 5.8 0.83 12+ 4 3.93 2.25 – 6.6 17p/11q 5 4.20 2.0 – 5.4

CD23 Receptor Positivity Has No Prognostic Implication in Chronic Lymphocytic Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4397-4397
Author(s):  
Jahan Aghalar ◽  
Charles Chu ◽  
Rajendra N Damle ◽  
Che-Kai Tsao ◽  
Nina Kohn ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Overall Survival ◽  
Chronic Lymphocytic Leukemia ◽  
Prognostic Markers ◽  
Conflicts Of Interest ◽  
Prognostic Significance ◽  
Lymphocytic Leukemia ◽  
Mutation Status ◽  
Percent Positivity ◽  
Cd23 Expression

Abstract Abstract 4397 BACKGROUND Chronic Lymphocytic Leukemia (CLL) phenotypically expresses CD23, although the percentage of positive cells measured by flow cytometry is variable. We sought to analyze whether the percent of CD23 positive cells in the CLL clone correlates with time to treat (TTT), overall survival (OS) and prognostic markers CD38, ZAP-70, and IGHV mutation status. METHODS We retrospectively analyzed the flow cytometry data of 332 CLL patients on the gated population of cells that were CD5 and CD19 positive. Percentage positivity for CD23, CD38, and ZAP-70 was noted. CD38 and ZAP-70 were considered positive at cut-offs of >= 30% and >=20%, respectively. CD23 was considered negative at <30% and positive at >= 30%. IGHV sequence was determined from cDNA and then compared to germline to assess mutation status using IMGT/V-QUEST. The distributions of time from diagnosis until start of treatment and overall survival were stratified by CD23 positivity, estimated using the product limit method, and compared using the log rank test. Those who had expired without treatment or were alive and not treated at this time point were censored in the TTT analysis. Those who were still alive were censored in the OS analysis. Associations of CD23 positivity with IGHV mutation status, ZAP-70, and CD38 positivity were examined using the chi-square test. RESULTS Out of 332 patients, 25 had diminished CD23 expression (<30%) whereas 307 had normal CD23 expression (>30%). There was no difference in time until start of treatment or overall survival based on CD23 %positivity. CD23 %positivity showed no associations with IGHV mutation status, ZAP-70 or CD38 positivity. CONCLUSION CD23 percent positivity has no prognostic significance in CLL. There is no correlation between CD23 percent positivity and poor prognostic markers such as CD38, ZAP-70, or IGHV mutation status. Disclosures: No relevant conflicts of interest to declare.

Serum IgM Activates BCR Signaling Pathways and Increases Survival of Chronic Lymphocytic Leukemia B Cells

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4131-4131
Author(s):  
Stefania Gobessi ◽  
Binu K Sasi ◽  
Luca Laurenti ◽  
Dimitar G Efremov
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Signaling Pathways ◽  
Target Genes ◽  
Conflicts Of Interest ◽  
Direct Interaction ◽  
Leukemic Cell ◽  
Fold Increase ◽  
Lymphocytic Leukemia ◽  
Bcr Signaling

Abstract Serum IgM would be expected to bind chronic lymphocytic leukemia B cells through two different mechanisms. The first mechanism is via interactions between the immunoglobulin heavy chain CDR3 of the leukemic B cell receptors (BCRs) and internal epitopes located in the FR2 and FR3 regions of serum IgM molecules, analogous to the recently identified cell-autonomous BCR-BCR interaction. The latter interaction represents a general feature of human CLL BCRs and was recently shown to be positively selected during leukemia development in the Eμ-TCL1 transgenic murine model. The second mechanism is by binding of serum IgM to the recently identified Fc receptor for IgM (FcμR), which is overexpressed on CLL B cells. In the present study we investigated the consequences of the interaction between serum IgM and CLL cells. Incubation of CLL cells with Alexa488-conjugated human IgM resulted in strong cell surface labeling, confirming that IgM binds to CLL cells. Binding was substantially inhibited by preculture of CLL cells with Fcμ, suggesting that IgM interacts with CLL B cells primarily through the FcμR. To investigate whether IgM also binds to the leukemic BCRs, we analyzed activation of downstream BCR signaling pathways and expression of a well-defined set of BCR-target genes (Herishanu Y et al, Blood. 2011;117:563-74) in CLL cells cultured in the presence or absence of purified IgM. After three hours in culture with polyclonal or monoclonal human IgM, 5 of the 7 investigated BCR target genes (OAS3, RGS1, GFI1, CCND2 and KLF4) showed a 2- to 9-fold increase with respect to unstimulated CLL cells, whereas the remaining two genes (EGR1 and EGR2) were not induced. The induced BCR target genes were also upregulated to an equal or even greater extent by Fcμ, suggesting that these effects are primarily or exclusively caused by binding of IgM to the FcμR. Analysis of downstream signaling events, such as SYK and ERK phosphorylation, also showed similar induction by IgM and Fcμ. However, intracellular Ca2+ flux was induced to a substantially greater extent with IgM, suggesting that certain effects are mediated by a direct interaction between serum IgM and the leukemic cell BCRs. Since co-ligation of the FcμR was recently shown to enhance the survival of anti-IgM-stimulated murine B lymphocytes (Ouchida R et al, J Immunol. 2015;194:3096-101), we investigated the consequences of IgM binding on CLL cell survival. CLL cells from 18 patients were cultured with or without purified human IgM for 72 hours and then analyzed by Annexin V/PI staining. A modest but significant increase in the percentage of viable CLL cells was observed in the presence of IgM (percentage of viable CLL cells without IgM: 40.5±17.8; with IgM: 43.8±18.4; P =0.016), which was replicated in a smaller series of samples cultured with Fcμ (n=12, percentage of viable CLL cells without Fcμ: 41.1±17.8; with Fcμ: 49.5±15.6; P =0.019). Altogether, these data suggest that binding of serum IgM results in activation of prosurvival pathways in CLL cells and that this effect is most likely mediated by co-triggering the FcμR and BCR. Disclosures No relevant conflicts of interest to declare.

scholarly journals High Peripheral Blood Circulating BDNF Levels Are Associated with Good Prognosis in CLL Patients; A CXCR-4 Dependent Effect?

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 5562-5562
Author(s):  
David Azoulay ◽  
Yair Herishanu ◽  
Mika Shapiro ◽  
Yarden Brenshaft ◽  
Celia Suriu ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
B Cells ◽  
Serum Levels ◽  
Surface Expression ◽  
Independent Effect ◽  
Total Serum ◽  
Healthy Controls ◽  
Disease Prognosis ◽  
And Migration

Abstract Introduction: BDNF is a neuronal growth factor that previously showed to exert survival effect in B cells. The role of BDNF in CLL is unknown. Objectives: To study the circulating levels of BDNF in CLL patients and their association with disease prognosis. We also looked for CXCR-4 as a possible mechanisms underlying BDNF effect in CLL. Design and methods: The total BDNF levels and the levels of the BDNF precursor; proBDNF were quantified in the serum of 36 CLL patients and 5 healthy controls by commercial ELISA kits (DY248 and DY3175 DuoSet, respectively, from R&D Systems, Minneapolis, MN, USA). The patients' BDNF levels were correlated to the disease characteristics and clinical course. mRNA and protein expression of the high affinity receptor for BDNF; TrkB were evaluated by real time PCR and flow cytometry respectively. The effect of BDNF on CXCR4 surface expression and migration of CLL cells towards SDF-1 were studied in-vitro. Results: The total serum levels of BDNF in CLL patients was not statistically different compared to healthy controls (mean±SD; 19.9±15.1ng/ml vs. 10.5±9.5ng/ml respectively, p=0.19). Serum proBDNF levels in both CLL patients and healthy controls were under the detection limit of the kit. Within the CLL group we found higher total BDNF levels in patients with mutated immunoglobulin variable heavy-chain (IGHV) status than in patients with unmutated IGHV (25.2±14.6ng/ml vs. 14.1±13.0 in mutated and unmutated status, respectively, p=0.028). We also found higher serum levels of total BDNF in CLL patients in Binet stage A compared to those with a more advance clinical stage (mean±SD ; 24.5±14.6ng/ml vs. 17.0±16.9 and 10.2±9.6 in A,B and C, respectively, p=0.013). Accordantly, higher BDNF levels were detected in patients who had a clinically stable disease compared to patients who had disease progression and required treatment (23.1±14.7ng/ml vs. 13.4±14.5, respectively, p=0.013). The mRNA levels of the BDNF receptor; TrkB, were twofold lower in CLL cells compared to normal B cells. However, the overall TrkB mRNA transcripts were very low in CLL cells and normal B cells compared to the normalized housekeeping genes of Beta-2-microglobulin and CD19. Likewise, surface expression of TrkB in CLL was undetectable using several commercial monoclonal Abs by flow cytometry. In vitro culture of CLL cells in serum free media for 24h resulted in an increased CXCR4 surface expression and greater apoptosis. Culture of CLL cells in the presence of recombinant BDNF (50ng/ml) resulted in downregulation CXCR-4 surface levels and protection from spontaneous apoptosis, irrespectively of the IGHV mutational status. The effect of BDNF on CXCR4 downregulation was also confirmed at 1h culture as it shows to induce similar effect to SDF-1 (50ng/ml) and additive effect when combined with SDF-1. Finally, possible competition between BDNF and SDF-1 was indicated as pretreatment of CLL cells with BDNF inhibit their migration toward SDF-1. Discussion: Our findings show association between high BDNF levels and favorable disease prognosis in CLL. Undetected TrkB expression in CLL cells is compatible with previous reports showing sequestration of TrkB in normal and malignant B cells and suggests TrkB independent effect of BDNF in CLL. The effect of BDNF on the survival and migration of CLL cells via CXCR-4 needs to be further investigated. Disclosures No relevant conflicts of interest to declare.

scholarly journals Impact of BCR Stimulation on Mir-181b in Chronic Lymphocityc Leukemia

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2026-2026
Author(s):  
Amerigo Mirco Di Marco ◽  
Francesco Autore ◽  
Paola Lanuti ◽  
Idanna Innocenti ◽  
Giuseppe Leone ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Immediate Release ◽  
Cell Receptor ◽  
Surface Expression ◽  
Lymphocytic Leukemia ◽  
Lymphoid Tissues ◽  
Bcr Signaling ◽  
Enhanced Expression ◽  
Signaling Inhibitors

Abstract B cell receptor (BCR) signaling plays an important pathogenic role in chronic lymphocytic leukemia (CLL) enhancing homing and homeostasis of B-CLL cells and favoring the interaction with the pro-survival stromal lymph node microenvironment. Surface expression of CD69 is required to block the cells in lymphoid compartment while Sfingosine-1-phosphate receptor-1 (S1PR1) is necessary for egress of cells to blood. BCR signaling inhibitors such as Ibrutinib and Acalabrutinib, (anti-bruton tyrosine kinase) or Idelalisib (anti-Phosphatidylinositol 3-kinases ) represent a significant therapeutic advance in CLL. At least some of these drugs are distinctive to lead to an initial lymphocitosis due to rapid release of cells from lymphoid organs to blood, where they die. Changes in microRNAs expression characterize clinical progression of CLL with a strong decrease of miR-181b associated with the more aggressive phase of the disease. In this study we demonstrate that miR-181b is part of the BCR pathway in that it influences the anatomical redistribution of CLL cells from lymph nodes into the blood by balancing the expression of CD69 and S1PR1. We co-cultured MEC_01 and pure B-CLL cells in medium supplemented or not with IgM F(ab')2, specific antibodies for BCR. We observed a significant decrease of miR-181b after 24 hrs of BCR stimulation compared with the same cells cultured in medium without IgM F(ab')2. To establish that this effect was due to the stimulation of the BCR, studies were performed on MEC_01 and in pure B-CLL cells treated separately with 1μM Ibrutinib or 1 μM Idelalisib, which are a potent BCR signaling inhibitors. After 1 hours of treatment the relative expression of miR-181b was assessed noting an increase of the transcription of miR, suggesting that the activation of BCR down regulate the expression of miR-181b. To confirm what we observed in vitro, RNA was isolated from B cells of the CLL patients at different time-point before and after treatment with a BTK inhibitor, Acalabrutinib (ACP-196). We observed a marked increase of miR-181b in the patients in therapy with this drugs compared with respective baseline. Since BTK inhibitors lead to the immediate release of CLL cells from lymphoid tissues to circulation, we evaluated whether this miRNA could be involved in this process. To test this hypothesis MEC01 cells were infected with either LV-miR-181b_coGFP or the LV-CTRL_coGFP after which migration was assessed in transwell assays. The Cells (5 × 105 cells/well) were seeded on top of the transwells and allowed to migrate for 4 h. S1P (100 nM) was added to the lower chamber as a chemo attractant. Transwell assay was performed to determine the migratory capacity of MEC-01 GFP+ cells, mimicking spleen (S1P-) vs blood (S1P+) compartments. Our data indicate that enhanced expression of miR-181b accelerate the migration of B-CLL cells. On the same cells we also evaluated the expression of S1PR1 and CD69. We observed that the restoration of miR-181b down regulated CD69 expression and increased the S1PR1. We also demonstrated that both proteins are direct targets and that the regulation on SIPR1 by the miR-181b occurs through a not canonical way. In conclusion, our findings indicate miR-181b is down regulated by BCR stimulation in CLL cells and that its enhanced expression reduced CD69 while increased S1PR1 by direct targeting. This could facilitate the egress from lymphoid tissue to blood and in turn their death. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

The Shaving Reaction. Rituximab/CD20 Complexes Are Removed from Mantle Cell Lymphoma Cells (Z138) and Chronic Lymphocytic Leukemia (CLL) Cells by Monocyte/Macrophages: In Vitro Studies and a SCID Mouse Model.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2968-2968
Author(s):  
Ronald P. Taylor ◽  
Paul V. Beum ◽  
Yongli Li ◽  
Margaret A. Lindorfer ◽  
Michael E. Williams
Keyword(s):  
Flow Cytometry ◽  
Monoclonal Antibodies ◽  
B Cells ◽  
Mouse Model ◽  
Plasma Concentrations ◽  
Lymphocytic Leukemia ◽  
Fcγ Receptors ◽  
Antigenic Modulation ◽  
Substantial Loss

Abstract Clinical investigations have revealed that infusion of immunotherapeutic monoclonal antibodies directed to normal or tumor cells can lead to loss of targeted epitopes, a phenomenon called antigenic modulation. Recently we reported that Rituximab (RTX) treatment of CLL patients induced substantial loss of CD20 on B cells found in the circulation after RTX infusion, when RTX plasma concentrations were high. Antigenic modulation can severely compromise therapeutic efficacy, and we postulated that B cells had been “shaved” of RTX/CD20 complexes by monocytes or macrophages of the mononuclear phagocytic system (MPS) in a reaction mediated by Fcγ receptors. Our in vitro model is based on reacting RTX-opsonized CD20+ cells with acceptor THP-1 monocytes differentiated to a macrophage-like phenotype. After 45 min at 37°C, ~50–80% of both RTX and CD20 are removed from opsonized cells, and both proteins are demonstrable on acceptor THP-1 cells, as ascertained by flow cytometry, fluorescence microscopy, and immunoblotting. Tests with inhibitors and use of F(ab’)2 fragments of RTX indicate shaving is mediated by Fcγ receptors, and shaving occurs equally well in the presence and absence of complement. We developed a pre-clinical xenograft mouse model to replicate shaving: 5 X 106 Z138 cells were infused iv or sc, into SCID/CB17 mice, and ~25 days later animals were treated with varying doses of RTX. Z138 cells recovered from lungs (iv model) or tumors (sc model) of treated and control mice 30 min to 3 days after RTX infusion were analyzed by flow cytometry. Substantial loss of CD20 was demonstrable on RTX-targeted cells, and this loss was more pronounced at longer times after RTX infusion. Moreover, Z138 cells in untreated mice had moderate to high levels of CD20 and few cell-associated C3 fragments, but in RTX-treated mice the situation was reversed: the Z138 cells were CD20 poor (&lt;20% of the CD20 levels compared to untreated mice) but had significant amounts of deposited C3dg. These findings replicate our earlier studies which demonstrated that treatment of CLL patients with RTX promotes loss of CD20 as well as complement activation and C3 fragment deposition on circulating B cells. It is likely that previously described antigenic modulation, usually reported for conditions of high circulating malignant cell burdens targeted by monoclonal antibodies, was also mediated by cells of the MPS via the shaving reaction. Our findings may have profound implications for the use of monoclonal antibodies in the immunotherapy of cancer.

Expression of CD38 and High Levels of CD5 Identifies Members of CLL Clones That Have Progressed beyond G1 and Proliferated.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1114-1114
Author(s):  
Carlo Calissano ◽  
Rajendra N. Damle ◽  
Marc Hellerstein ◽  
Elisabeth J. Murphy ◽  
Gregory M. Hayes ◽  
...  
Keyword(s):  
Cell Cycle ◽  
B Cells ◽  
Similar Pattern ◽  
Great Majority ◽  
Surface Expression ◽  
Cyclin B1 ◽  
Lymphocytic Leukemia ◽  
Cellular Activation ◽  
Deuterium Enrichment

Abstract Chronic lymphocytic leukemia (CLL) is characterized by the monoclonal expansion of CD5+ B cells with the morphology of small mature lymphocytes. Although the great majority of circulating CLL B cells appears as quiescent/non-proliferating lymphocytes, a small percentage of cells in peripheral blood appear to be cycling. Furthermore, within these clones, the CD38+ subset is enriched in those cells that entered G1, as evidenced by a larger number of Ki67-expressing cells. In an attempt to further identify submembers of CLL clones with different proliferative capacities, we measured, by flow cytometry, expression of Ki67 along with mcm6 and Cyclin D1, expressed from G1 to M, and Cyclin B1, expressed in G2-M. Since the expression of CD5 is characteristic of CLL cells and increases in vitro upon cellular activation, the intensity of CD5 expression was used to further divide CD38+ and CD38− cells into CD5bright and CD5dim fractions. The leukemic clones of eighteen IgVH unmutated CLL (U-CLL) and 16 IgVH mutated CLL (M-CLL) patients were analyzed. A similar pattern of expression for each cell cycle related marker studied was found. The presence of CD38 and high expression of CD5 was correlated with increased percent of cells expressing the various cell cycle markers (CD38−CD5dim<CD38−CD5bright; CD38+CD5dim<CD38+CD5bright). Strikingly, significant differences characterized the intraclonal comparisons between the 4 subpopulations (Table). To confirm these findings, the cells of 4 CLL patients involved in a deuterated “heavy” water protocol were sorted using CD38 and CD5 expression. In two of these cases, because of limiting cell numbers, CD5bright and CD5dim cells could be obtained only among CD38− cells. CD38−CD5bright cells contained more deuterium in their DNA than CD38−CD5dim cells indicating that more CD38−CD5bright cells had divided than CD38−CD5dim cells. A similar pattern was found in two patients for whom all four subfractions could be sorted. Thus, lowest deuterium enrichment in DNA was found in CD38−CD5dim cells and highest in CD38+CD5bright cells. The combined use of surface and intracellular markers, together with the 2H labeling, effectively further subdivides the proliferative leukemic compartment in CLL. Expression of Cell Cycle related markers in CLL B cells defined by surface expression of CD38 and CD5 N=34 mcm6 % CyclinD1 % Ki67 % CyclinB1 % For every marker studied: all intraclonal comparisons (1 vs. 2, 1 vs. 3 etc.) are statistically significant (P<0.05) with the exception of 2 vs. 3 and, for cyclinB1, 3 vs. 4 CD38−CD5dim (1) 4.0±0.8 9.9±2.6 3.8±0.8 0.8±0.2 CD38−CD5bright (2) 9.8±1.9 17±4.2 10.2±1.9 2.1±0.5 CD38+CD5dim (3) 8.5±1.4 13.7±2.8 7.8±1.1 2.3±0.6 CD38+CD5bright (4) 19.5±2.3 24.3±4.0 15.2±1.8 2.8±0.6

Family-Associated Monoclonal B Lymphocytosis Shows Differences From CLL That Suggest An Indolent Biology.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1241-1241
Author(s):  
Mark C Lanasa ◽  
Sallie D Allgood ◽  
Susan L Slager ◽  
Nicola J Camp ◽  
Neil E. Kay ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Peripheral Blood ◽  
Conflicts Of Interest ◽  
Family Members ◽  
Surface Expression ◽  
Cellular Activation ◽  
Immunoglobulin Isotype ◽  
Cell Counts ◽  
Intracellular Expression ◽  
Trisomy 12

Abstract Abstract 1241 Poster Board I-263 Background and Significance Monoclonal B lymphocytosis (MBL) is a hematologic syndrome characterized by accumulations of clonal B lymphocytes in the peripheral blood. Most MBL have a CLL-like immunophenotype, though other, less common phenotypes are observed. Although some MBL, particularly those with MBL cell counts >1.9 × 109 / L, progress to CLL, “low count” MBL (<0.2 × 109 MBL / μL) have little potential to progress to MBL and may have different biologic characteristics from MBL with lymphocytosis or Rai Stage 0 CLL. Our hypothesis was that detailed characterization of family-associated MBL may also provide biologic insights into the pathogenesis of CLL. Methods Individuals with MBL were identified by flow cytometry screening of fresh and cyropreserved peripheral blood from unaffected members of CLL kindreds ascertained by Genetic Epidemiology of CLL Consortium (GEC) sites. We defined MBL as populations of CD5+, CD19+, CD20lo, CD23+ B cells that comprised at least 0.02% of the PBMC and did not exceed 5.0 × 109 MBL cells / L. Flow cytometry was used to determine the surface immunophenotype including prognostic parameters of CD38, intracellular ZAP-70, immunoglobulin isotype, CD49d, and the ratio of CD69:71. mRNA and genomic DNA from single MBL cells isolated by flow cytometric sorting were analyzed using PCR to determine immunoglobulin heavy and light chain sequences (IGVH status). MBL cells were sorted in bulk for FISH determination of genetic loci associated with clinical CLL. Results Fifty-four unaffected family members were found to have MBL, of these 48 (89%) showed a typical CLL immunophenotype (CLL-like MBL). We observed significant variability in the size of the MBL clone as a percentage of the CD19+ B cell compartment (mean 30%, median 16%; range 2% - 97%). CD38 positive (defined as CD38 surface expression in ≥ 30% of MBL cells) was observed in 6 of 38 (16%) subjects tested. ZAP-70 positive (defined as intracellular expression in ≥ 20% of MBL cells) was expressed in 5 of 28 (18%) participants. CD49d positive (defined as surface expression in ≥ 45% of MBL cells) was observed in 4 of 17 (24%) subjects. The ratio of surface expression of CD69:CD71, a measure of cellular activation that also correlates with an unmutated IGVH, was ≥2.0 in 3 of 34 (9%) subjects tested. Among 36 subjects tested, 9 (25%) MBL expressed both surface IgD and IgM (defined as surface expression ≥ 40% for both IgM and IgD), 12 (33%) expressed IgD only, 2 (6%) expressed IgM only, and 13 did not express IgD or IgM (36%). Analysis of IGVH status has been completed in 10 individuals. Both immunoglobulin heavy chain variable (IGVH) region mutated (n = 14) and unmutated (n = 5) sequences were observed. Six of 10 individuals had 2 or more unrelated MBL clones (range 2 - 4), including two individuals with both unmutated and mutated clones. Among the 19 MBL clones identified in these 8 subjects, VH3 or VH4 rearrangements were observed in all MBL clones. The most commonly rearranged IGVH genes were 3-07 (3 MBL clones), 3-15 (3), and 4-34 (3). No VH1 family gene rearrangements were observed. MBL cells were bulk sorted for FISH from 14 subjects. Mono or biallelic deletion of 13q14.3 was observed in 9 subjects, 4 were normal, and one showed trisomy 12. Conclusions Our data affirms that CLL-like MBL are commonly observed among the unaffected family members from CLL kindreds. We found that family associated MBL clones (most of which were small clones) express ZAP-70, CD38, and CD49d at an apparently lower frequency than observed in CLL. Unlike in CLL, the surface immunoglobulin isotype showed co-expression of IgM and IgD in only one third of cases. Moreover, small MBL clones are commonly oligoclonal and predominantly express mutated IGVH genes with an IGVH usage that also appears different from CLL. Taken together, these findings suggest that small MBL clones, though phenotypically similar to CLL, have important biologic differences from CLL that may explain the limited potential of these clones to progress to CLL. The clinical outcome of these MBL clones in relation to our baseline prognostic characterization will be of interest. In addition the further investigation of family associated MBL is being conducted and may clarify the genetics and immunobiology of familial CLL. Disclosures No relevant conflicts of interest to declare.

Surface IgM of Chronic Lymphocytic Leukemia Cells Displays Unusual Glycans Indicative of Antigen Engagement In Vivo.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 55-55
Author(s):  
Graham Packham ◽  
Serge Krysov ◽  
Christopher Ian Mockridge ◽  
Kathy N Potter ◽  
Freda K Stevenson
Keyword(s):  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Conflicts Of Interest ◽  
Variable Region ◽  
Immunoglobulin M ◽  
Lymphocytic Leukemia ◽  
Glycosylation Pattern ◽  
Mature Form

Abstract Abstract 55 Several lines of evidence support the idea that surface immunoglobulin M (sIgM) plays a key role in determining the clinical behavior of chronic lymphocytic leukemia (CLL). For example, the presence of somatic mutations in immunoglobulin variable region genes is a strong prognostic marker with unmutated CLL (U-CLL) associated with a poor outcome relative to mutated CLL (M-CLL). U-CLL also generally express higher levels of sIgM and retain the ability to signal via this receptor. In this study, we used surface biotinylation to analyse sIgM in CLL and discovered that it exists in two forms with differing mobility on SDS-PAGE. Treatment with glycosidases revealed that these forms were due to different N-glycosylation patterns in the μ constant region. One form is similar to that of normal B cells in bearing mature complex glycans common to most cell surface glycoproteins. The other is an immature mannosylated form more characteristic of endoplasmic reticulum (ER)-located μ chains. CLL cells expressed variable proportions of the immature mannosylated form and quantitative analysis demonstrated that, on average, the proportion of mannosylated sIgM was approximately 2-fold higher (p=0.006) in U-CLL compared to M-CLL. Although normal B cells isolated from blood expressed only the mature form of sIgM, in vitro treatment with anti-μ resulted in upregulation of the immature form, suggesting that glycan modification is a consequence of antigen exposure. Consistent with this, in vitro incubation of CLL cells was associated with increased expression of the mature form of sIgM. Phosphotyrosine analysis demonstrated that both forms of sIgM were able to signal following sIgM engagement in vitro. Taken together, these findings support the concept that CLL cells are continuously exposed to antigen in vivo. This process leads to a change in the N-glycosylation pattern of the re-expressed sIgM so that a mannosylated form predominates, especially in U-CLL. Strikingly, expression of mannosylated sIgM is also characteristic of follicular lymphoma, where it is constitutively displayed via N-glycosylation sites in the Ig variable region (Radcliffe et al. J Biol Chem. 2007; 282, 7405-15). Persistent mannosylation of sIgM appears to be a feature common to several B-cell malignancies, suggesting a role in pathogenesis. Disclosures: No relevant conflicts of interest to declare.

The Sesquiterpene Oil α-Bisabolol Induces Apoptosis of B-Chronic Lymphocytic Leukemia Primary Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1319-1319
Author(s):  
Massimiliano Bonifacio ◽  
Antonella Rigo ◽  
Angela Bonalumi ◽  
Emanuele Guardalben ◽  
Ilaria Nichele ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Lipid Rafts ◽  
Conflicts Of Interest ◽  
Membrane Integrity ◽  
Leukemia Cell ◽  
Lymphocytic Leukemia ◽  
Primary Cells

Abstract Abstract 1319 We have recently demonstrated that the sesquiterpene oil α-bisabolol is cytotoxic against primary acute leukemia cells ex vivo and in chronic myeloid leukemia cell lines. It enters cells via lipid rafts and activates the mitochondrial-dependent intrinsic pathway of apoptosis, exerting a preferential toxicity against malignant vs normal cells probably due to their higher content in lipid rafts. Here we investigated the in vitro activity of α-bisabolol in primary cells from patients with B-Chronic Lymphocytic Leukemia (B-CLL). Twenty-six patients with newly diagnosed B-CLL gave their informed consent to the study. Cells were collected before any treatment, purified and cultured for 24 hours with serial dilutions of α-bisabolol. Citotoxicity was quantified in flow cytometry by the BD Trucount™ technology to allow comparison between neoplastic and normal residual lymphocytes. B-CLL cells (IC50 42±15 μM) were significantly more sensitive towards α-bisabolol than normal B- (IC50 82±34 μM, p=.005) and T-cells (IC50 120±35 μM, p<.001). Citotoxicity was similar between the IgVH mutated (n=11) and the IgVH unmutated samples (n=7), as well as between the Binet stage A (n=20) and B-C (n=6) patients. To investigate the mechanisms of α-bisabolol-induced toxicity we treated B-CLL cells with 40 μM α-bisabolol for up to 3 hours. We observed a time-dependent increase in fluorescence of cells treated with the membrane-impermeant nucleic acid stain TO-PRO-3, already detactable after 30 minutes. When cells were loaded with the Ca2+ indicator Fluo-4 AM, an increase of Ca2+ influx was revealed already after 15 minutes. These early events indicate that α-bisabolol induces the loss of cellular membrane integrity, so triggering the apoptotic cascade. Then we assessed the mitochondrial transmembrane potential (ΔΨm) with the fluorochrome JC-1 to confirm that a mitochondrial damage is a concurrent mechanism in the apoptotic process induced by α-bisabolol. By flow cytometry we demonstrated that, after 3-hour incubation with 40 μM α-bisabolol, ΔΨm dissipation was already detectable in leukemic cells, while T-lymphocytes, evaluated as internal control in the same samples, stayed vital. To investigate the mitochondrial target of α-bisabolol we examined the function of the mitochondrial permeability transition pore (mPTP). After 5-hour incubation with 40 μM α-bisabolol we loaded cells with the calcein AM dye and added CoCl2 to distinguish between intact and damaged mitochondria, confirming that the function of mPTP was compromised in B-CLL cells but not in normal controls. Finally, to determine whether α-bisabolol affects the oxydative state of treated cells, we evaluated the intracellular concentration of reactive oxygen species (ROS) by measuring the fluorescent signal of CM-H2DCFDA loaded cells. When B-CLL cells were exposed to 40 μM a-bisabolol for 3 hours, they exhibited a clear fluorescence increase, indicating the striking generation of ROS: this was completely abrogated by the addition of N-acetylcysteine, a scavenger of intracellular ROS. Clues about the molecular mechanistics of α-bisabolol have also emerged from in vitro models based on treating cells previously transfected with BH3-only molecules. In this setting, α-bisabolol exposed cells seem to undergo detrimental, non-selective autophagy-like phenomena. Our data indicate that α-bisabolol exerts a level of cytotoxicity against B-CLL cells at concentrations that only partially affect normal B- and T-cells. Moreover, a brief exposure (3–5 hours) to α-bisabolol is sufficient to elicit multiple pro-apoptotic signals independently of the patients' mutational status. Disclosures: No relevant conflicts of interest to declare.

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