CD49d Expression Identifies a Chronic Lymphocytic Leukemia (CLL) Subset with High Levels of Circulating CD34 +Cells Co-Expressing Endothelial Cell Markers.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2329-2329
Author(s):  
Francesca Maria Rossi ◽  
Davide Rossi ◽  
Antonella Zucchetto ◽  
Riccardo Bomben ◽  
Michele Dal Bo ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Chronic Lymphocytic Leukemia ◽  
Lymphocytic Leukemia ◽  
Bone Marrow Biopsies ◽  
Endothelial Markers ◽  
Cd38 Expression ◽  
Cell Counts ◽  
Cd34 Cells ◽  
Stromal Component ◽  
The Absolute

Abstract Abstract 2329 Poster Board II-306 Introduction: In chronic lymphocytic leukemia (CLL), CD49d, often in association with CD38, has been shown to mark a disease subset with poor prognosis. Functionally, both molecules act as counter-receptors for surface structures (i.e. VCAM-1/CD106 and CD31) usually expressed by the endothelial/stromal component of tumor micro-environment. We have recently identified a micro-environmental circuitry which involves CD38 triggering, and eventually determines an enrichment of the VCAM-1/CD106-expressing endothelial component detected in the context of CLL infiltrates found in bone marrow biopsies. Data was also provided that CD49d/VCAM-1 interactions are active in delivering pro-survival signals to CD49d-expressing CLL cells (Zucchetto et al, Cancer Res, 69, 4001, 2009). In this study, we investigated the amount of circulating progenitors with endothelial phenotype in CLL samples with different CD49d and CD38 expression levels. Methods: Peripheral blood (PB) samples from 91 CLL cases purposely selected with WBC>25,000/μl (B cells absolute lymphocyte count >10,000/μl) were evaluated by multiparametric flow cytometry for the absolute count of circulating CD34+ cells (ISHAGE protocol in single platform). Whenever possible (i.e. if a cluster of at least 100 CD34+ cells was detectable), a further characterization was performed (4-6 colours flow cytometry) for circulating endothelial cells (CEC), identified as a CD34+CD45low cell population co-expressing one of the following endothelial markers: CD309/VEGFR-2, CD144/VE-cadherin, CD106/VCAM-1 and CD146/Muc-18. CD49d and CD38 expression by CLL cells was considered positive if exceeding the standard cut-off value of 30% of positive cells. Results: PB absolute CD34+ cell counts were 7.5±7.5/μl in CD49d+ CLL (32 cases), vs. 3.3±2.7/μl in CD49d− CLL (59 cases; p=2.6×10−4), or 9.4±8.7/μl in CD49d+ CLL (30 cases) vs. 4.6±2.9/μl in CD49d− CLL (18 cases; p=0.004) when only cases phenotyped for CEC were considered. Furthermore, when samples were stratified also for CD38 expression, values of circulating CD34+ cells increased to 10.6±10.1/μl in CD38+CD49d+ CLL (11 cases) vs. 3.1±2.4/μl in CD38−CD49d− (51cases; p=1×10−5). Regarding the absolute quantification of CEC, a CD49d+ phenotype again marked the CLL subset with the highest CEC count, as identified by the expression of either the CD309/VEGFR-2 (CEC counts 1.7±2.3/μl in CD49d+ CLL vs. 0.5±0.5/μl CD49d− CLL; p=0.009) or the CD144/VE-cadherin (CEC counts 0.8±1/μl in CD49d+ CLL vs. 0.3±0.5/μl in CD49d− CLL; p=0.057) endothelial markers on CD34+CD45low cells. Notably, CEC from CD49d+ CLL expressed CD106/VCAM-1 in virtually all cells (1.6±2.4/μl), while the other marker of endothelial activation CD146/Muc-18 was detected in a fraction of CEC only (0.4±0.9/μl). Conclusions: CD49d and CD38 expression by CLL cells identify a disease subset with significantly higher number of both circulating CD34+ cells and CEC. This phenomenon could be explained considering several aspects: i) the sharing of common phenotypic markers between CLL cells and CD34+ progenitors, including CD38 and CD49d, which could be responsible for a displacement of CD34+ progenitors in the context of micro-environmental niches; ii) the known capacity of CLL cells, especially with a unmutated IGHV gene status and/or a CD38+CD49d+ phenotype to produce pro-angiogenic factors including Ang-2; iii) the rare PB cells expressing CD34 and CEC markers may represent CLL cell precursors with tumor-initiating cell features. Studies are currently ongoing to dissect among these hypotheses Disclosures: No relevant conflicts of interest to declare.

CD38 expression as an important prognostic factor in B-cell chronic lymphocytic leukemia

Blood ◽  
2001 ◽  
Vol 98 (1) ◽  
pp. 181-186 ◽  
Author(s):  
Sherif Ibrahim ◽  
Michael Keating ◽  
Kim-Anh Do ◽  
Susan O'Brien ◽  
Yang O. Huh ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Prognostic Factor ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Aggressive Disease ◽  
Cd38 Expression ◽  
Important Prognostic Factor ◽  
Cell Chronic Lymphocytic Leukemia

Abstract CD38 is a transmembrane glycoprotein expressed on the surface of leukemic cells in a significant percentage of patients with B-cell chronic lymphocytic leukemia (B-CLL). A recent study suggested that CD38 expression has prognostic value in CLL. Peripheral blood samples from 218 patients with B-CLL were analyzed by flow cytometry for CD38 expression on CD5/19+ leukemic cells. Various patient characteristics were studied including age, sex, Rai and Binet stages, splenomegaly, hepatomegaly, hemoglobin (Hgb) level, β-2 microglobulin (β2M) level in the serum, number of nodal sites involved with disease, and length of survival. The Kaplan-Meier method was used to construct survival curves, and the log-rank statistic was used to compare these curves. CD38 was expressed in 20% or more of leukemic cells in 43% of the patients. Patients with high CD38 expression (20% or more) had significantly shorter survival times (P =.00005). Multivariate analyses showed that CD38 expression is an important prognostic factor associated with high incidence of lymph node involvement (P = .004), lower hemoglobin level (P = .001), hepatomegaly (P = .05), and high β2M level (P = .00005). CD38 expression identified a group of patients with aggressive disease that was considered by Rai staging to be early-stage disease (Rai stages 0-II). Patients with CD38+ samples have significantly aggressive disease regardless of their clinical stage. Measurement of CD38 expression by flow cytometry should become a routine test in the evaluation of patients with CLL.

scholarly journals Next-Generation IgVH Sequencing of Monoclonal B-Cell Lymphocytosis Reveals Clonal Heterogeneity, Ongoing Mutation, and Repertoire Differences Relative to Chronic Lymphocytic Leukemia

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1960-1960
Author(s):  
Mark Klinger ◽  
Malek Faham ◽  
Jianbiao Zheng ◽  
Kojo S.J. Elenitoba-Johnson ◽  
Sherrie L. Perkins ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
B Cells ◽  
Chronic Lymphocytic Leukemia ◽  
B Cell ◽  
Lymphocytic Leukemia ◽  
Cell Repertoire ◽  
Clonal Heterogeneity ◽  
Cell Counts ◽  
Lambda Expression ◽  
Antigen Selection

Abstract Background: Chronic lymphocytic leukemia (CLL) usually develops from asymptomatic monoclonal expansions of CD5 positive B-cells termed monoclonal B-cell lymphocytosis (MBL), present in the peripheral blood (PB) of approximately 5% of otherwise healthy older individuals. Although MBL only occasionally progresses to CLL, cases that do progress typically have higher MBL cell counts in the 1500-4000/µL range. Although antigen selection appears to play a central role in the development CLL, it is unclear whether this occurs at an early MBL stage or primarily during the progression of MBL to CLL. One prior study has reported clonal heterogeneity in MBL finding it in 4 of 6 low count MBL cases from familial CLL kindreds using a single cell PCR technique (Leukemia 2010,24:133-140). In this study, we assessed the VH repertoire and degree of clonal heterogeneity in sporadic MBL cases using next-generation sequencing (NGS) of the rearranged immunoglobulin heavy chain (IgH) locus. Methods: The 35 cases selected for sequencing represented residual, cryopreserved material from PB specimens submitted to ARUP for clinical phenotyping studies. All contained polytypic CD5 negative B-cells in addition to MBL/CLL phenotype cells, and had 2 or more vials for analysis. The majority (80%) had counts of MBL cells below 1000/µL (mean 294/, range 795-30 cells/µL). FACS purification of MBL cells (CD20+CD5+) and CD5 negative B-cells was performed on all samples. The IgH repertoire from the unsorted and two sorted populations was determined by NGS using the LymphoSIGHT method. Results: Five cases could not be analyzed due to insufficient numbers of MBL cells. Clonal VDJ rearrangements or clonotypes were identified in the remaining 30 based on their high frequency within the B-cell repertoire of the unsorted sample, and having a higher frequency in the sorted MBL cells relative to the sorted CD5 negative B-cells. Functional clonotypes were identified in 29 of these 30 cases. Interestingly, 5 cases had 2 functional unrelated clonotypes using different D and/or J segments that also employed different V segments. Of the 5 cases with 2 unrelated clonotypes, 3 had MBL cell counts below 1000/µL (32, 275, and 865) and 2 above (1640, 2600). Moreover, 1 of the clones in the case with 865 cells/µL represented only 25% of the MBL cells or 220 cells/µL, while 1 clone in the case with 2600 MBL cells/µL represented 18% of the MBL cells or 470 cells/µL. By flow cytometry, the CD5+ CD20+ cells in 2 of the cases with 2 functional clonotypes showed polytypic kappa/lambda expression (ratios near 1), 2 cases had uniform dim monotypic kappa expression, and 1 case showed 90% dim kappa and 10% dim lambda expression. The most frequently used VH segments were V4-34 in 6/34 or 18% of functional clonotypes, followed by V3-23 (11%), and V3-21 (9%). The V1-69 segment was used by only 1/34 (3%) functional clonotypes. The VH segments in 72% of cases with functional clonotypes were mutated (homology to germline < 98%), with 6 cases showing clear evidence of ongoing mutation by having 2 or more related clones. Conclusions: We demonstrate that MBL exhibits considerable clonal heterogeneity, with 2 distinct unrelated clones identified in 17% of 30 analyzed cases. Finding 2 distinct clones cannot be explained by a lack of allelic exclusion or the presence of 1 cell with 2 productive IgH rearrangements since each clone had different frequencies within the sorted MBL cell repertoire. This is further supported by finding the ratios of the two MBL clones in 2 cases being different in the unsorted compared to the MBL sorted cells. Clonal heterogeneity appears to occur at an early stage since the majority of clones (6/10) had cell counts below 500 cells/µL. We also found that clonal heterogeneity of MBL may not be detectable by flow cytometry or may appear as polytypic CD5+CD20+ B-cells. To our knowledge, this represents the first report of clonal heterogeneity in sporadic MBL. Our identification of infrequent use of V1-69 (1/34) supports prior studies indicating the VH repertoire of MBL is different than CLL which frequently employs V1-69. Finding evidence of ongoing VH mutation suggests antigen selection may occur in early MBL. Overall, our findings are consistent with recent observations (Cancer Cell 2011, 20;246-259) suggesting that hematopoietic stem cells from CLL patients can generate mono-or oligoclonal MBL phenotype cells that can then be selected through antigen binding for expansion. Disclosures Faham: Sequenta, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Flow Cytometric Detection and Phenotype of Circulating CD34+ Cells in Patients Affected by Chronic Lymphocytic Leukemia.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4329-4329
Author(s):  
Fabio Stagno ◽  
Nunziata Laura Parrinello ◽  
Giovannella Fargione ◽  
Anna Triolo ◽  
Antonella Privitera ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Peripheral Blood ◽  
Flow Cytometric Analysis ◽  
Absolute Number ◽  
Lymphocytic Leukemia ◽  
Unexpected Finding ◽  
Advanced Stage ◽  
Flow Cytometric ◽  
Cd34 Cells ◽  
The Absolute

Abstract Flow cytometric determination of peripheral blood CD34+ cells provides reliable measurements of circulating hemopoietic progenitors. Since the detection of the absolute number of circulating CD34+ cells has been found of clinical utility in the setting of chronic myeloproliferative disorders, we investigated whether peripheral CD34+ cells could play any role in the clinical work-up of B-cell chronic lymphocytic leukemia (B-CLL). In this view, we determined by flow cytometry the absolute number of circulating CD34+ cells in the peripheral blood of 28 patients (16 males and 12 females, median age 67 years) affected by typical B-CLL (Matutes score 5,4,3) and in different Rai stages of the disease (19 early stage: Rai 0, I, II; 9 advanced stage: Rai III, IV). Conventional and multiparameter flow cytometric analysis was performed utilizing a FACSCalibur cytometer (Becton Dickinson). Our data showed a significant increase in the number of circulating CD34+ cells in the peripheral blood of patients with B-CLL (median CD34+ cells:7.8mL) as compared to controls (median CD34+ cells 0.1mL) (p=0.008). No statistical difference between B-CLL patients in early versus advanced stage (p=0.5) and between untreated versus treated (p=0.7) was found, as well as there was no correlation with some of the clinical characteristics of B-CLL (WBC-count, LDH levels, Beta-2M). In 10 out of 28 B-CLL affected patients, circulating CD34+ cells were correlated with ZAP-70 and CD38 antigen but no correlation was found. In addition, we detected in the peripheral blood of 22 out of 28 patients small numbers of circulating CD34+ cells displaying the CD19+/CD5+ phenotype (median CD34+/CD19+/CD5+ cells:5.7mL) whereas these cells were absent in normal controls. This unexpected finding, whose significance remains to be clarified and still restricted to a small number of cases, could be directly correlated to the underlying lymphproliferative disease and might represent a pool of leukemic stem cells. However, further studies are warranted.

Phase IIa Study of the Anti-CXCL12/SDF-1 Spiegelmer® Nox-A12 Alone and Combined with Bendamustine/Rituximab in Patients with Relapsed Chronic Lymphocytic Leukemia (CLL)

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4593-4593
Author(s):  
Marco Gobbi ◽  
Federico Caligaris-Cappio ◽  
Marco Montillo ◽  
Stephanie Vauléon ◽  
Stefan Zöllner ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Plasma Concentration ◽  
Chronic Lymphocytic Leukemia ◽  
Healthy Volunteers ◽  
Peripheral Blood ◽  
Combined Treatment ◽  
Single Agent ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Cd34 Cells

Abstract Abstract 4593 Background NOX-A12 is a novel, potent, L-aptamer inhibitor of CXCL12/SDF-1, a chemokine which attracts and activates immune- and non-immune cells. The signaling of CXCL12 has been shown to play an important role in the pathophysiology of chronic lymphocytic leukemia (CLL), especially in the interaction of leukemic cells with their tissue microenvironment. The therapeutic concept of NOX-A12 is to inhibit such tumor-supporting pathways and thereby sensitizing the CLL cells towards chemotherapy. Methods The purpose of this phase IIa study is to evaluate the safety and efficacy of NOX-A12 in combination with background chemo-immunotherapy of bendamustine and rituximab (BR) in patients with relapsed CLL. The described study is being performed in compliance with ethical principles based on the Declaration of Helsinki and ICH-GCP guidelines. The study population was split into a pilot and expansion group. In the pilot group, 3 cohorts of 3 patients each received escalating doses of single agent NOX-A12 two weeks prior to the combined treatment of NOX-A12 and BR. Interim data from these patients are reported. Based on previous Phase I studies in healthy volunteers, pilot patients received a dose of 1, 2 or 4 mg/kg body weight (BW) single agent NOX-A12 on day -14, followed by a 2-weeks period of safety, PK and PD assessments prior to the combined treatment with NOX-A12 and BR. To date, the first cohort of the pilot group already progressed to the 2nd cycle of combined treatment. Evaluation criteria included adverse events according to CTCAE V4, flow cytometry of peripheral blood CD34+ cells and CLL cells, pharmacokinetics of NOX-A12, plasma concentration of CXCL12 and tumor response (NCI-WG 1996 criteria, updated 2008). Results To date 3 patients (age range: 58 – 65 years) have been enrolled in the pilot group of this study. They had received 1 or 2 prior therapies, but no bendamustine. Single i.v. doses of 1 mg/kg BW NOX-A12 had no clinically relevant effects on vital signs, 12-lead ECG parameters and laboratory parameters. One patient reported grade 1 pain in the lower limbs two days after treatment with NOX-A12. This event was not dose-limiting and resolved spontaneously on the same day. Flow cytometry of CD34+ cells and CLL cells (CD19+/CD5+high) showed a rapid mobilization of these cells into the peripheral blood on day 1. Interestingly, return to baseline was not complete at the last assessment on day 3 (for details see Figure 1). The NOX-A12 pharmacokinetics in these 3 patients (for concentration-time profile see Figure 2) is very comparable to healthy volunteers receiving i.v. NOX-A12, with a maximum plasma concentration of 1.52 ± 0.14 μM after 1 h (tmax) and a plasma elimination half-life of about 50 h. As seen in healthy volunteers the plasma concentration of CXCL12 increased upon NOX-A12 treatment and reached a maximum of 0.434 ± 0.076 μM at 24 to 72 h p.a. without ever approaching the plasma concentration of NOX-A12 (Figure 2). Conclusion Single i.v. doses of NOX-A12 at 1 mg/kg BW were safe and well tolerated; the maximum tolerated dose was not reached. NOX-A12 induced a long-lasting mobilization of CD34+ cells and leukemic cells in patients with relapsed CLL, consistent with a mechanism of action based on CXCL12 inhibition. Patient accrual and identification of an optimal chemosensitization regimen of NOX-A12 combined with BR is being continued. Disclosures: Vauléon: NOXXON Pharma AG: Employment. Zöllner:NOXXON Pharma AG: Employment. Dümmler:NOXXON Pharma AG: Employment. Kruschinski:NOXXON Pharma AG: Employment. Fliegert:NOXXON Pharma AG: Employment.

A Single Tube, Four-Color Flow Cytometry Assay for Evaluation of ZAP-70 and CD38 Expression in Chronic Lymphocytic Leukemia

2010 ◽  
Vol 133 (5) ◽  
pp. 708-717 ◽  
Author(s):  
Nagwa M. Hassanein ◽  
Kathryn R. Perkinson ◽  
Felisa Alcancia ◽  
Barbara K. Goodman ◽  
J. Brice Weinberg ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Chronic Lymphocytic Leukemia ◽  
Lymphocytic Leukemia ◽  
Flow Cytometry Assay ◽  
Color Flow ◽  
Single Tube ◽  
Cd38 Expression

Correlative Study of ZAP-70 Gene Expression by Quantitative PCR and CD-38 Surface Marker in Patients with Chronic Lymphocytic Leukemia (CLL).

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4796-4796
Author(s):  
Melissa L. Larson ◽  
Jamile M. Shammo ◽  
Sari H. Enschede ◽  
Parameswaran Venugopal ◽  
Daniel Russell ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Prognostic Factors ◽  
Chronic Lymphocytic Leukemia ◽  
Median Number ◽  
Lymphocytic Leukemia ◽  
Stage I ◽  
Cd38 Expression ◽  
Mutational Status ◽  
Multiple Treatments

Abstract Patients with chronic lymphocytic leukemia have heterogeneous clinical courses. Many possible prognostic factors have been evaluated as a way to predict each patient’s future course. Two such prognostic factors are IgVH gene mutational status and CD38 expression. Recently, ZAP-70 expression has been found to correlate with IgVH gene mutation, but little is known about the correlation between ZAP-70 and CD 38 expression. We sought to evaluate the concordance between the two. METHODS: A procedure was developed at our institution to evaluate ZAP-70 transcripts by PCR. The results were then confirmed by flow cytometry. The blood or bone marrow samples of 14 patients diagnosed with CLL were analyzed by PCR for ZAP-70 transcripts and flow cytometry for ZAP-70 protein expression and CD38 expression. The results of the ZAP-70 transcripts and CD38 expression were then correlated to the clinical courses of the patients. RESULTS: Of the 14 patients analyzed, the majority had Rai Stage I disease at diagnosis (4 Stage 0, 8 Stage I, and 2 Stage II). There was a median follow-up of 28 months. The median number of treatments for these patients was one (range 0–6). Overall, the results for ZAP-70 and CD38 expression were concordant in eight with CD38 data unavailable for 2 patients. In the four patients with discordant results, 3 of them had high CD38 expression and low ZAP-70. However, patients with high ZAP-70 were more likely to require multiple treatments once treatment was indicated. Patients with high CD38 were also more likely to be treated. Cytogenetic data was available for 9 patients with the following results: 5 with normal cytogenetics, 1 with deletion of 11q, 1 with deletion of 13q, and 2 with deletion of 17p. Five of the nine patients had high ZAP-70 expression. CONCLUSIONS: In our institution, the results of ZAP-70 and CD38 expression were concordant 67% of the time. Longer follow-up is necessary and may further delineate the roles of cytogenetics, ZAP-70 expression, and CD38 expression on prognosis in CLL.

SYK Inhibition Targets CD38−Mediated Survival and Proliferation Signals in Chronic Lymphocytic Leukemia Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 800-800
Author(s):  
Maike Buchner ◽  
Natalie Stickel ◽  
Arlette Dörffel ◽  
Ariane Ott ◽  
Marcus Duehren von Minden ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Chronic Lymphocytic Leukemia ◽  
Signaling Pathway ◽  
Conflicts Of Interest ◽  
Lymphocytic Leukemia ◽  
Concomitant Treatment ◽  
Ki 67 ◽  
Direct Involvement ◽  
Cd38 Expression ◽  
Syk Inhibitor

Abstract Abstract 800 Chronic Lymphocytic Leukemia (CLL) is characterized by an accumulation of mature monoclonal B cells in the blood, secondary lymphoid tissue, and marrow. The highly variable prognosis of the disease may be predicted using a number of biomarkers, including the expression level of CD38. Human CD38 is a surface glycoprotein with enzymatic activity and the ability to mediate cell-cell interactions by binding the non-substrate ligand CD31. CD31/CD38 interactions drive CLL proliferation and chemotaxis, and increase CXCL-12-mediated signals and homing of CLL cells towards lymphoid organs. We and others have previously identified SYK, a key component of the BCR signalling pathway, as a candidate for targeted therapy in CLL due to its enhanced expression and activity and the apoptotic effects of pharmacological SYK inhibition even in the context of the microenvironment. In this study, we demonstrate direct involvement of SYK in the CD38 signaling pathway and that SYK inhibition may prevent CD38−mediated CLL cell proliferation and migration. CD38 stimulation of primary CLL cells by its ligand CD31 resulted in SYK activation as indicated by phospho-protein analysis by flow cytometry (n=18, p=0.0002) and immunoblotting. Analysis of CD38 expression on CLL cells by flow cytometry with SYK inhibition using the pharmacological SYK inhibitor R406 (Sellek) revealed a significant decrease of CD38 by 31% (n=25, p<0.0001). Particularly, CLL cells with high CD38 expression showed a substantial downregulation following SYK inhibition. Since CD38 expression is associated with poor clinical outcome, the proliferative signals delivered by its ligation might be critical for this association. Therefore, we tested the influence of SYK inhibition on proliferative signals as assessed by the expression of the proliferation marker Ki-67. 12 hours of stimulation with CD31 resulted in up-regulation of Ki-67 in CLL cells, an effect completely abrogated by concomitant treatment with the SYK inhibitor R406 (n=8). Since the expression of CD38 is regulated by different pathways including CD40 ligation and SYK involvement has been implicated in CD40 signaling, we evaluated whether R406 is capable to inhibit CD38 upregulation induced by CD40L. Indeed, in vitro stimulation with 3 μg/ml recombinant CD40L for 30 hours resulted in a significant increase of CD38 expression (p=0.0059) and was reversed by concomitant SYK inhibition (p=0.0142, n=9). As positive control, we treated CLL cells with ATRA that upregulated CD38 in myeloid cells. ATRA is directly translocated to the nucleus without requirement of signal transduction. Hence, SYK inhibition did not influence ATRA-mediated CD38 upregulation. In order to functionally characterize the role of SYK in the CD38/CD31 interaction in CLL cells, we analyzed the expression of the anti-apoptotic molecule MCL-1 in the presence and absence of SYK inhibition with and without CD38 stimulation. CD31 ligation enhanced MCL-1 expression, an effect reversed when CLL cells were coincubated with R406 (n=8). Taken together, our data demonstrate direct involvement of SYK in the CD38 signaling pathway. Beyond the known effects of SYK inhibition on chemokine- and integrin mediated CLL-stroma interactions, these data establish interference with the CD31/CD38 axis as novel therapeutic mode of action of SYK inhibition in CLL. Since CD38 expression identifies CLL cells with high proliferative and migratory function and clinically associates with poor responsiveness to chemotherapy SYK inhibition may be a particularly promising therapeutic option in CLL patients expressing CD38. Disclosures: No relevant conflicts of interest to declare.

Additional value of quantitative expression of ZAP-70 and CD38 for prognostic factors in chronic lymphocytic leukemia.

2017 ◽  
Vol 35 (15_suppl) ◽  
pp. e19006-e19006 ◽  
Author(s):  
Martin Klabusay ◽  
Viera Hrabcakova ◽  
Petr Coupek ◽  
Martin Trbusek ◽  
Michaela Pevna
Keyword(s):  
Flow Cytometry ◽  
Prognostic Factors ◽  
Chronic Lymphocytic Leukemia ◽  
Lymphocytic Leukemia ◽  
Patients Âgés ◽  
Quantitative Expression ◽  
Cd38 Expression ◽  
Kaplan Meier ◽  
Trisomy 12 ◽  
Additional Value

e19006 Background: Chronic lymphocytic leukemia (CLL) has heterogeneous clinical course. Identification of prognostic markers is important for proper management of disease. Cytogenetics, IgVH mutation status, TP53 functional status, CD38, ZAP-70 and CD49d expression are accepted prognostic factors in CLL. ζ-chain-associated protein kinase 70 (ZAP-70) is associated with disease progression. However, measurement of ZAP-70 and CD38 expression relies on qualitative flow cytometry. Authors tested hypothesis whether quantitative analysis of markers can improve information about prognosis of CLL. Methods: 217 CLL patients were included; diagnosis was made from peripheral blood by morphology and immunophenotyping by flow cytometry with common antibodies. Expressions of intracellular ZAP-70 and surface CD38 were measured using quantitative fluorescence cytometry. Standardized fluorescent microparticles were used to quantify molecules of equivalent soluble fluorochrome (MESF units). Analysis of rearrangement of the IgVH gene was performed according to the European recommendation. Deletions at 11q22-q23 ( ATM), 17p13 ( TP53), 13q34 ( RB1) loci and trisomy of chromosome 12 were detected by I-FISH. Results: Patients’ ages ranged from 33 to 86 years and included all Rai stages. Expression of ZAP-70 ranged from 546 to 5,955 MESF, CD38 from 1,886 to 31,619 MESF. 48% patients embodied mutated and 52% unmutated IgVH status. In cytogenetics, del(13q) was detected in 59%, trisomy 12 in 13%, del(11q) in 21%, and del(p53) in 8% of patients. IgVH (in %) was plotted against ZAP-70 (in MESF) and patients were divided into high (HR) and low risk (LR) groups by cluster analysis based on Kaplan-Meier overall survival curves. Patients in HR group had significantly poorer prognoses (p = 0.0001) than those in LR group. When patients from HR group were clustered according to CD38 expression, very high risk (VHR) group with high CD38 and significantly worse prognosis (p = 0.035) was identified. Conclusions: We have demonstrated that quantitative ZAP-70 and CD38 expressions cannot replace IgVH analysis, but, contrary to that, addition of quantitative data can further specify prognoses of patients with CLL.

Rapid Mobilization of CD34+ Cells Following Administration of the CXCR4 Antagonist AMD3100 to Patients With Multiple Myeloma and Non-Hodgkin's Lymphoma

2004 ◽  
Vol 22 (6) ◽  
pp. 1095-1102 ◽  
Author(s):  
Steven M. Devine ◽  
Neal Flomenberg ◽  
David H. Vesole ◽  
Jane Liesveld ◽  
Daniel Weisdorf ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Hodgkin’S Lymphoma ◽  
Hodgkin's Lymphoma ◽  
Clinical Effects ◽  
Cxcr4 Antagonist ◽  
Cell Counts ◽  
Non Hodgkin’S Lymphoma ◽  
Non Hodgkin's Lymphoma ◽  
Cd34 Cells ◽  
The Absolute

PurposeInteractions between the chemokine receptor CXCR4 and its ligand stromal derived factor-1 regulate hematopoietic stem-cell trafficking. AMD3100 is a CXCR4 antagonist that induces rapid mobilization of CD34+ cells in healthy volunteers. We performed a phase I study assessing the safety and clinical effects of AMD3100 in patients with multiple myeloma (MM) and non-Hodgkin's lymphoma (NHL).Patients and MethodsThirteen patients (MM, n = 7; NHL, n = 6) received AMD3100 at a dose of either 160 μg/kg (n = 6) or 240 μg/kg (n = 7). WBC and peripheral blood (PB) CD34+ cell counts were analyzed at 4 and 6 hours following injection.ResultsAMD3100 caused a rapid and statistically significant increase in the total WBC and PB CD34+ counts at both 4 and 6 hours following a single injection. The absolute CD34+ cell count increased from a baseline of 2.6 ± 0.7/μL (mean ± SE) to 15.6 ± 3.9/μL and 16.2 ± 4.3/μL at 4 hours (P = .002) and 6 hours after injection (P = .003), respectively. The absolute CD34+ cell counts observed at 4 and 6 hours following AMD3100 were higher in the 240 μg/kg group (19.3 ± 6.9/μL and 20.4 ± 7.6/μL, respectively) compared with the 160 μg/kg group (11.3 ± 2.7/μL and 11.3 ± 2.5/μL, respectively). The drug was well tolerated and only grade 1 toxicities were encountered.ConclusionAMD3100 appears to be a safe and effective agent for the rapid mobilization of CD34+ cells in patients who have received prior chemotherapy. Further studies in combination with granulocyte colony-stimuating factor in patients with lymphoid malignancies are warranted.

Sign in / Sign up

Export Citation Format

Share Document