Interactions between chronic lymphocytic leukemia (CLL) B cells and the bone marrow (BM) microenvironment play a major function in the physiopathology of CLL. Extracellular vesicles (EVs), which are ...composed of exosomes and microparticles, play an important role in cell communication. However, little is known about their role in CLL / microenvironment interactions. In the present study, EVs purified by ultracentrifugation from BM mesenchymal stromal cell (BM-MSC) cultures were added to CLL B cells. After their integration into CLL B cells, we observed a decrease of leukemic cell spontaneous apoptosis and an increase in their chemoresistance to several drugs, including fludarabine, ibrutinib, idelalisib and venetoclax after 24 hours. Spontaneous (
=0.0078) and stromal cell-derived factor 1α -induced migration capacities of CLL B cells were also enhanced (
=0.0020). A microarray study highlighted 805 differentially expressed genes between leukemic cells cultured with or without EVs. Of these, genes involved in the B-cell receptor pathway such as CCL3/4, EGR1/2/3, and MYC were increased. Interestingly, this signature presents important overlaps with other microenvironment stimuli such as B-cell receptor stimulation, CLL/nurse-like cells co-culture or those provided by a lymph node microenvironment. Finally, we showed that EVs from MSCs of leukemic patients also rescue leukemic cells from spontaneous or drug-induced apoptosis. However, they induce a higher migration and also a stronger gene modification compared to EVs of healthy MSCs. In conclusion, we show that EVs play a crucial role in CLL B cells/BM microenvironment communication.
Histone deacetylases (HDACs) play a crucial role in chromatin structure and, consequently, gene expression. Their deregulation has been reported in various cancers. We performed a complete and ...comprehensive study of the expression of 18 HDACs (including Sirtuin; SIRT) by real-time PCR in a cohort of 200 chronic lymphocytic leukemia (CLL) patients with a median follow-up of 77 mo, and compared it with the results obtained from normal B cells. We also compared HDAC expression at diagnosis and after relapse. We observed significant deregulation (mostly upregulation) of HDACs in CLL. In terms of clinical significance, only HDAC6 was significantly correlated with treatment-free survival (TFS), whereas HDAC3 and SIRT2, 3 and 6 were correlated with overall survival (OS). A multivariate Cox regression stepwise analysis indicated that HDAC6, 7 and 10 and SIRT3 were TFS independent predictors. Interestingly, poor prognosis was associated with an overexpression of HDAC7 and 10 but an underexpression of HDAC6 and SIRT3. Therefore, these factors were combined in a TFS score: patients with a score of 0-1-2, 3 and 4 had a median TFS of 107, 57 and 26 mo, respectively (HR = 4.03, p < 0.0001). For OS, SIRT5 and 6 allowed stratification into 3 groups, with a median OS of > 360, 237 and 94 mo (HR = 6.38, p < 0.0001). However, we could not find statistical differences in HDAC expression after relapse. These results, validated by a 5-fold cross-validation, highlight the complex impact of HDAC expression in CLL clinical course.
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BFBNIB, GIS, IJS, KISLJ, NUK, PNG, UL, UM, UPUK
Microparticles (MPs), also called microvesicles (MVs) are plasma membrane-derived fragments with sizes ranging from 0.1 to 1μm. Characterization of these MPs is often performed by flow cytometry but ...there is no consensus on the appropriate negative control to use that can lead to false positive results.
We analyzed MPs from platelets, B-cells, T-cells, NK-cells, monocytes, and chronic lymphocytic leukemia (CLL) B-cells. Cells were purified by positive magnetic-separation and cultured for 48h. Cells and MPs were characterized using the following monoclonal antibodies (CD19,20 for B-cells, CD3,8,5,27 for T-cells, CD16,56 for NK-cells, CD14,11c for monocytes, CD41,61 for platelets). Isolated MPs were stained with annexin-V-FITC and gated between 300nm and 900nm. The latex bead technique was then performed for easy detection of MPs. Samples were analyzed by Transmission (TEM) and Scanning Electron microscopy (SEM).
Annexin-V positive events within a gate of 300-900nm were detected and defined as MPs. Our results confirmed that the characteristic antigens CD41/CD61 were found on platelet-derived-MPs validating our technique. However, for MPs derived from other cell types, we were unable to detect any antigen, although they were clearly expressed on the MP-producing cells in the contrary of several data published in the literature. Using the latex bead technique, we confirmed detection of CD41,61. However, the apparent expression of other antigens (already deemed positive in several studies) was determined to be false positive, indicated by negative controls (same labeling was used on MPs from different origins).
We observed that mother cell antigens were not always detected on corresponding MPs by direct flow cytometry or latex bead cytometry. Our data highlighted that false positive results could be generated due to antibody aspecificity and that phenotypic characterization of MPs is a difficult field requiring the use of several negative controls.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
An unusual progressive stridor Apraxine, Matveï; Mineur, Philippe; Huet, Jean ...
Breathe (Lausanne, Switzerland),
06/2020, Volume:
16, Issue:
2
Journal Article
Peer reviewed
Open access
A 71-year-old man came to our pulmonary consultation with 6 weeks of progressive dyspnoea, hoarseness and, more recently, stridor. His past medical history included colorectal cancer treated with ...surgery and adjuvant chemotherapy, and type 2 diabetes mellitus. Prior to our consultation, his general practitioner had already ordered a chest computed tomography (CT) scan. The patient had undergone this CT scan 3 days before the consultation and it did not reveal any abnormalities.
A flow–volume curve with a flattening of the inspiratory and expiratory limb suggests a proximal obstruction of the upper airways. Plasma cell neoplasms need to be considered in the differential diagnosis of an invasion of the upper respiratory tract.
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Interactions with the microenvironment, such as bone marrow mesenchymal stromal cells and nurse-like cells, protect chronic lymphocytic leukemia cells from spontaneous and drug-induced apoptosis. ...This protection is partially mediated by the chemokine SDF-1α (CXCL12) and its receptor CXCR4 (CD184) present on the chronic lymphocytic leukemia cell surface.
Here, we investigated the ability of AMD3100, a CXCR4 antagonist, to sensitize chronic lymphocytic leukemia cells to chemotherapy in a chronic lymphocytic leukemia/mesenchymal stromal cell based or nurse-like cell based microenvironment co-culture model.
AMD3100 decreased CXCR4 expression signal (n=15, P=0.0078) and inhibited actin polymerization/migration in response to SDF-1α (n=8, P<0.01) and pseudoemperipolesis (n=10, P=0.0010), suggesting that AMD3100 interferes with chronic lymphocytic leukemia cell trafficking. AMD3100 did not have a direct effect on apoptosis when chronic lymphocytic leukemia cells were cultured alone (n=10, P=0.8812). However, when they were cultured with SDF-1α, mesenchymal stromal cells or nurse-like cells (protecting them from apoptosis, P<0.001), chronic lymphocytic leukemia cell pre-treatment with AMD3100 significantly inhibited these protective effects (n=8, P<0.01) and decreased the expression of the anti-apoptotic proteins MCL-1 and FLIP. Furthermore, combining AMD3100 with various drugs (fludarabine, cladribine, valproïc acid, bortezomib, flavopiridol, methylprednisolone) in our mesenchymal stromal cell co-culture model enhanced drug-induced apoptosis (n=8, P<0.05) indicating that AMD3100 could mobilize chronic lymphocytic leukemia cells away from their protective microenvironment, making them more accessible to conventional therapies.
Taken together, these data demonstrate that interfering with the SDF-1α/CXCR4 axis by using AMD3100 inhibited chronic lymphocytic leukemia cell trafficking and microenvironment-mediated protective effects. Combining AMD3100 with other drugs may, therefore, represent a promising therapeutic approach to kill chronic lymphocytic leukemia cells.
The multicenter observational BiRD study investigated the real-world effectiveness and safety of ibrutinib in patients with chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL) and ...Waldenström’s macroglobulinemia (WM) in Belgium. This interim analysis reports results for patients with CLL, with a median follow-up of 34 months. Overall, patients had predominantly relapsed/refractory disease (73%) and were elderly (median age 72 years) with high-risk features such as del17p and/or
TP53
mutations (59%). Patients were included either prospectively or retrospectively, and the total patient population effectiveness results were adjusted with left truncation. In the effectiveness population (
N
= 221: prospective,
n
= 71; retrospective,
n
= 150), the overall response rate was 90.0%. Median progression-free survival was 38.3 months (prospective, not estimable; retrospective, 51.5 months) and median overall survival was not yet estimable in the total, prospective and retrospective groups. Treatment-emergent adverse events (TEAEs) for the prospective and retrospective groups are reported separately. Any-grade TEAEs of interest in the prospective/retrospective groups included infections (67.1%/60.1%), diarrhea (20.5%/10.5%), hypertension (16.4%/9.8%) and atrial fibrillation (12.3%/7.2%). Major bleeding was reported in 5.5%/3.3% of prospective/retrospective patients, with little difference observed between those receiving versus not receiving antithrombotic treatment. Discontinuations due to toxicity were reported in 10.5% of patients. Results from this interim analysis show treatment with ibrutinib to be effective and tolerable, with no new safety signals observed. Future analyses will report on longer-term follow-up.
Several markers have been proposed to predict the outcome of chronic lymphocytic leukemia (CLL) patients. However, discordances exist between the current prognostic factors, indicating that none of ...these factors are totally perfect.
Here, we compared the prognostic power of new RNA-based markers in order to construct a quantitative PCR (qPCR) score composed of the most powerful factors. ZAP70, LPL, CLLU1, microRNA-29c and microRNA-223 were measured by real time PCR in a cohort of 170 patients with a median follow-up of 64 months (range3-330). For each patient, cells were obtained at diagnosis and RNA was extracted from purified CD19 cells. The best markers were included in a qPCR score, which was thereafter compared to each individual factor. Statistical analysis showed that all five RNA-based markers can predict treatment-free survival (TFS), but only ZAP70, LPL and microRNA-29c could significantly predict overall survival (OS). These three markers were thus included in a simple qPCR score that was able to significantly predict TFS and OS by dividing patients into three groups (0/3, 1-2/3 and 3/3). Median TFS were >210, 61 and 24 months (P<0.0001) and median OS were >330, 242 and 137 months (P<0.0001), respectively. Interestingly, TFS results were also confirmed in Binet stage A patients (P<0.0001). When compared to other classical factors, this score displays the highest univariate Cox hazard ratio (TFS: HR=9.45 and OS: HR=13.88) but also provides additional prognostic information.
In our hands, this score is the most powerful tool for CLL risk stratification at the time of diagnosis.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract
The prevalence of monoclonal gammopathy of undetermined significance (MGUS) is generally estimated at 3.4% in the general population over 50 years, and its incidence increases with age. MGUS ...represents a preneoplastic entity that can transform into multiple myeloma or other lymphoproliferative disorders. The risk of malignant transformation is estimated at 1% per year and persists over time. Predictors of malignant transformation have been identified such as the heavy chain isotype, The level of monoclonal proteins, increasing levels of the monoclonal component during the first years off follow-up, the percentage of bone marrow plasmocytosis, the dosage of serum free light chains, the presence of immunophenotypically abnormal plasma cells, aneuploidy, and the presence of circulating plasma cells. Prognostic scores that combine certain of these factors have been proposed and allow the identification of high-risk patients. Their use could assist in tailoring the care for each patient, based on his/her risk profile.
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