Chronic inflammation has arised as a major underlying cause of atherosclerosis, obesity and diabetes. It is mediated by cells of innate immune system like macrophages but also by their antecedents, ...circulating monocytes. Roles of monocyte subsets and different markers of monocyte activation in the context of metabolic disorders have been reviewed. Applying cell based approach through flow cytometry in this field has resulted with new understanding of pathophysiologic mechanisms. Possible implications of these insights in diagnosis, prognosis and revealing of therapeutic targets in metabolic disorders remain a challenge for future.
Flow cytometry (FC) immunophenotyping is an important tool in the evaluation of lymphadenopathy and is widely used in the diagnosis of non-Hodgkin's lymphomas (NHLs) on fine-needle aspirates of lymph ...nodes and extranodal sites. Because at least 80% of NHLs are of B-cell type, detection of immunoglobulin (Ig) light-chain-restriction is the most commonly used method for confirmation of monoclonality. The aim of our study was to evaluate usefulness of endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) for FC analysis from deep-seated lymph nodes and to compare results of FC clonality analysis to cytomorphologic diagnosis of sampled lymph nodes. For cytological diagnosis direct smears were made, selected slide was stained for rapid-on site evaluation procedure. Sixteen patients with suspected NHL of deep-seated lymph nodes obtained by EUS-FNA were submitted for FC clonality analysis using four-color multiparameter flow cytometry stained with kappa/lambda/CD19/CD45. Clonality analysis was performed on 11 samples. Monoclonality was demonstrated in seven of 11 cases cytologically diagnosed as NHL and four of 11 cases cytologically diagnosed as benign were polyclonal. Our results show that EUS-FNAC with FC is a sensitive and specific tool in the diagnosis of deep-seated B-NHL. Cytologic diagnosis combined with FC clonality analysis can be performed in majority of cases and may eliminate need for open biopsy in some cases.
Today lymphomas are defined according to a combination of morphology, immunophenotype, genetic features and clinical presentation, so beside the pure cytomorphologic analysis in diagnosis of lymphoma ...ancillary techniques such as cytochemistry, immunocytochemistry, molecular diagnosis and flow cytometry (FC) are often used. Our goal was to determinate how is information given by fine-needle aspiration cytology (FNAC) and FC correlated with pathohistologic diagnosis and to evaluate ability to diagnose and subclassify malignant lymphomas by FNAC and FC. This study is a retrospective chart review of patients with suspicion of lymphoma processed at University Hospital Dubrava in Zagreb. After analysis 50 patients fulfilled inclusion criteria for this study (FNAC diagnosis with or without FC and consecutive confirmatory pathohistological diagnosis). When analyzing accuracy of FNAC according to suspicion of lymphoma or NHL and differential diagnosis lymphoma sensitivity was 97.7%, specificity 85.7% and the diagnostic accuracy was 96%. When analyzing accuracy of FNAC according to the subclassification of lymphoma, sensitivity was 74.4%, specificity 85.7% and the diagnostic accuracy 76%. Combined FNAC and FC improved sensitivity, positive predictive value, negative predictive value and diagnostic accuracy. Sensitivity was 79.1% and the diagnostic accuracy 80%. We have shown that these methods can distinguish benign lymphadenopaties from lymphomas and also subclassify lymphomas and quickly provide clinicians with that information.
Human CD38, an ecto-enzyme and a receptor, performs as an independent negative prognostic marker for patients with chronic lymphocytic leukemia (CLL), a hematological malignancy characterized by the ...accumulation of a population of mature B lymphocytes expressing CD5. Patients with a CD38⁺ CLL clone display a more aggressive form of the disease with earlier treatment requirements and ultimately shorter overall survival than patients with a CD38⁻ clone. Several lines of evidence indicate that CD38 is not only a diagnostic marker but also a key element in the molecular network regulating disease maintenance and progression. First, CD38 is a receptor that induces proliferation and increases survival of CLL cells. Second, CD38 signals facilitate access of CLL cells to growth-favorable districts. This is achieved by enhancing i) chemotaxis towards CXCL12, ii) integrin-mediated adhesion and iii) matrix metalloprotease synthesis and secretion. Third, blocking monoclonal antibodies targeting CD38 impair CLL homing to spleen and bone marrow in xenograft models. These functions appear to be modulated by frontal interactions with CD31 as well as by lateral associations on the CLL membrane to form a large supramolecular complex similar to the invadosomes of epithelial cells. Our understanding has evolved from considering CD38 as a marker of unfavorable prognosis to recognizing its function as a disease modifier. Studies in the next few years will likely determine whether the molecule can also serve as a target for new therapies, using monoclonal antibodies, inhibitors of the enzymatic activity or both.
The inflammatory phenotype of neck adipose tissue (NAT) might reflect its involvement in the pathogenesis of carotid atherosclerosis. We investigated inflammatory gene expression in the subcutaneous ...and the perivascular (pericarotid) adipose tissue from patients with carotid stenosis (CS) undergoing endarterectomy and a control group of patients without significant carotid atherosclerosis undergoing thyroid surgery. Only male patients were included (n = 13 in each study group). Clinical and biochemical data along with serum leptin, adiponectin, and monocyte chemoattractant protein 1 (MCP-1) were collected. Adipose tissue samples were obtained from both the subcutaneous and pericarotid compartments. Real-time polymerase chain reaction was used to measure gene expression of macrophage markers and adipokines. The CS group had higher subcutaneous and pericarotid visfatin gene expression and higher pericarotid expression of MCP-1 and CD68 genes. The ratio between pericarotid CD206 and CD68 gene expression was similar between study groups. Adiponectin gene expression in both NAT compartments did not differ between groups, but it was negatively associated with body weight. These observations suggest that NAT, and especially the pericarotid compartment, express enhanced inflammatory properties in patients with CS, but the proportion of anti-inflammatory macrophages in advanced atherosclerosis seems to be maintained.
BACKGROUND.NOTCH1 PEST domain mutations were among the first to be identified using whole exome sequencing and their prevalence and prognostic power for CLL patients has since been extensively ...studied. Nevertheless, the functional contribution of NOTCH1 and the impact of its mutations in CLL remain poorly understood. This is mainly due to the intrinsic limits in using primary CLL cells and to the rapid inactivation of the NOTCH1 pathway in vitro.
AIM. The aim of the study was to highlight the NOTCH1-dependent mechanisms contributing to CLL pathogenesis and progression and the effects of PEST mutations in this disease context.
RESULTS. By using the CRISPR/Cas9 technology, we first generated a cellular model either lacking NOTCH1 (Mec-1/KO) or expressing it in its wild-type or mutated forms. RNA-seq analysis of the transcriptome of Mec-1/WT and /KO cells, highlighted signaling and migration as the most prominently down-regulated pathways in KO cells. These findings were confirmed by showing that Mec-1/KO cells migrated significantly less towards CCL19 than their WT counterparts, as the result of the down-modulation of CCR7, the CCL19 receptor. Mechanistically, Mec-1/KO were characterized by increased expression of the tumor suppressor gene DUSP22, a phosphatase that negatively regulates MAPK and STAT3 signaling, in turn responsible for CCR7 gene transcription. Up-regulation of DUSP22 in Mec-1/KO was caused by decreased methylation of the gene promoter, as shown using a methylation specific PCR. Re-expression of the NOTCH1 Intracellular Domain (NICD) rescued the phenotype, increasing methylation of the DUSP22 promoter, which in turn led to decreased gene and protein expression. Mec-1 cells with reconstituted NICD expression showed increased STAT3 phosphorylation, CCR7 expression and, ultimately, migrated more actively in response to CCL19. Importantly, reconstitution with an NICD carrying the PEST domain mutation most commonly found in patients (ΔCT_7541-7542), significantly enhanced all these events, including DUSP22methylation, STAT3 phosphorylation, CCR7 expression, generating cells with the highest chemotactic responses to CCL19.
NOTCH1 directly participates to the epigenetic regulation of DUSP22, by conditioning the activity of the DNA methyltransferase 3A (DNMT3A) on the gene promoter. By binding RBPJk, the NICD displaces HDAC1 from the repressor complex and initiates NOTCH1-dependent signaling. Consequently, free HDAC1 binds to and stabilizes DNMT3A, promoting its activity on DUSP22promoter.
These results were first corroborated in a large cohort of CLL (n=113) carrying mutations in NOTCH1 PEST domain (ΔCT_7541-7542)at the clonal (>12%) or subclonal (≤12%) level. The clonally mutated subset showed significant downregulation of DUSP22expression and gene promoter hypermethylation compared to subclonal samples. Consistently, the clonal subset displayed higher constitutive STAT3 phosphorylation, expressed higher levels of CCR7 and migrated more efficiently to CCL19 than subclonally mutated CLL.
The second confirmation was obtained by studying a Mec-1 variant with PEST domain mutation (Mec-1/PEST), generated using the CRISPR/Cas9 system. Like primary cells, Mec-1/PEST needed the ligand to initiate signaling. As predicted, the mutant NICD was more stable and more transcriptionally active. Consistently, these cells had the lowest DUSP22 levels and the highest CCR7 expression. When Mec-1/PEST were xenografted in immunocompromised mice, activation of the NOTCH1 pathway was more pronounced than that of Mec-1/WT cells. In keeping with their minimal expression of DUSP22, Mec-1/PEST cells were characterized by markedly increased metastatic properties, with extensive colonization of the liver, the spleen, and the brain, at variance with the other cell variants.
CONCLUSIONS. Considered together, these results show that PEST mutations increase NICD stability, in turn affecting a complex nuclear balance. The final outcome for the CLL cell is decreased expression of the tumor suppressor gene DUSP22 and increased chemotaxis towards CCL19. As this chemokine regulates homing to secondary organs, conceivably NOTCH1 mutations might favor CLL recirculation to lymph node and spleen, where the local environment triggers proliferation and protects from apoptosis, two conditions that are associated with a more aggressive disease and an unfavorable prognosis.
Coscia:Karyopharm: Research Funding; Janssen: Honoraria; Mundipharma: Honoraria; Gilead: Honoraria; ROCHE: Honoraria, Other: Advisory board. Furman:Pharmacyclics, LLC, an AbbVie Company: Consultancy, Honoraria, Speakers Bureau. Gaidano:Roche: Consultancy, Honoraria, Speakers Bureau; Gilead: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau; Morphosys: Consultancy, Honoraria; Karyopharm: Consultancy, Honoraria.
Abstract 3877
B-cell chronic lymphocytic leukemia (B-CLL) is characterized by variable clinical presentation with different involvement of various lymphoid compartments i.e. peripheral blood, bone ...marrow and lymphoid organs such as lymph nodes and spleen. Also there is a well documented intraclonal and interclonal variability of B-CLL cells in different microenvironments regarding a number of surface and intracellular molecules (for example CD38 and ZAP-70). This variable distribution of tumor mass has strong association with prognosis and a well documented influence on response to antibodies like rituximab and alemtuzumab. There is a well documented efficacy of rituximab in cases with with 11q deletion (associated with significant lymphadenopathy), and known resistance to alemtuzumab in pateints with bulky lymphadenopaty (>5cm).
Aim of this study was to evaluate level of expression of CD20 and CD52 along with key chemokine receptor CXCR-4 and proliferation marker Ki-67 on B-CLL lymphocytes and intra and interclonal differences dependent on different microenvironment, ie peripheral blood (PB), bone marrow (BM) and lymph nodes (LN).
PB, BM and LN samples were taken by conventional techniques (venepuncture and fine needle aspiration) on the same day. The expression levels of CD20, CD52, CXCR-4 and Ki-67 molecules on CD19+CD5+ B-CLL cells were analyzed by flow cytometry. Results were expressed as mean fluorescence intensity (MFI) and analyzed by paired tests.
We have analyzed samples taken from 25 typical B-CLL patients with median age of 72 years. There were 14 males and 11 females. Mean beta-2 microglobulin was 4.3mg/l, mean TTM size was 9.8 and mean TD was 0.74. There were 13, 5 and 7 patients in Binet stage A, B and C, respectively. There were 4 previously treated patients (patients were not treated 3 months before sampling) of whom one patient was previously treated with both rituximab and alemtuzumab. Among included patients there were patients with 11q deletion and with 17p deletion. Median expression level (MFI) of CD52 on B-CLL cells was 171, 193, and 352 for PB, BM and LN respectively (p<0.05) and on T cells was 224, 171 and 137 (p<0.05). Median expression level (MFI) of CD20 on B-CLL cells was 7.5, 5.7 and 4.7 for PB, BM and LN respectively (p<0.05). These results were very consistent in this clinically and cytogenetically heterogenous group of B-CLL patients showing the same pattern in almost all patients. Median expression level (MFI) of CXCR-4 was 9.6, 5.9 and 2.6 (p<0.05) and Ki-67 was 1.08, 1.27 and 1.64 (p<0.05) for PB, BM and LN respectively. There was no correlation of CXCR-4 with CD20 and C52 expression in any compartment and there is positive correlation of Ki-67 with both CD20 and CD52 in PB but not in other compartments.
Relatively unexpected results demonstrating the lowest level of expression of CD20 on B-CLL cells in lymph nodes compared to PB and BM and the highest expression of CD52 on B-CLL cells in LN compared to PB and BM (but with the opposite pattern on T cells) is inversely related to known efficacy of agents (ie rituximab and alemtuzumab) targeting these molecules in these lymphoid compartments. These results indicate that other factors in selected microenvironment (beside number of molecules on cell surface) regulate sensitivity of B-CLL cells on rituximab and alemtuzumab in vivo. These may include other cells (like T cells) and soluble factors. Also in this study levels of expression were not clearly related to molecules involved in recirculation (CXCR-4) and proliferation (Ki-67) indicating that other factors in microenvironment may be important for the observed expression levels. These results warrant further studies to indentify these factors which may eventually uncover novel therapeutic targets. Display omitted
No relevant conflicts of interest to declare.
Chronic lymphocytic leukemia (CLL), is characterized by the expansion of mature B lymphocytes present in blood, bone marrow and lymphoid organs. Clinical behavior is highly heterogeneous, thus ...requiring timely identification of high-risk patients.
NOTCH1 encodes a trans-membrane receptor acting as a ligand-activated transcription factor. NOTCH signaling initiates when the ligand, from either the Jagged or Delta families, binds to the receptor and induces successive proteolytic cleavages, resulting in the release and nuclear translocation of the NOTCH intra-cellular domain (NICD). Signaling is terminated by phosphorylation of the PEST domain of NOTCH1, triggering its ubiquitination and proteasomal degradation.
Whole exome sequencing approaches have revealed NOTCH1 mutations in 5-10% newly diagnosed CLL cases, with their prevalence increasing to 15-20% in progressive or relapsed patients. The most frequent mutation is 7544-7545delCT frameshift deletion in exon 34, resulting in disruption of the C-terminal PEST domain. Truncation of the PEST domain is predicted to result in NOTCH1 impaired degradation, stabilization of the active NOTCH1, and deregulated signaling.
The present study was undertaken with the aim to compare NOTCH1 expression and functional role in CLL patients harboring wild type (WT) or mutated (M) NOTCH1 gene.
NOCTH1 mRNA and surface protein were expressed at comparable high levels in peripheral blood (PB) CLL cells obtained from NOTCH1 M and WT patients, consistent with a more general requirement of NOTCH1 signaling in this leukemia. However, at a variance of NOTCH1 WT cases, NOCTH1 M patients displayed remarkable accumulation of both the intermediate molecular species of the activated NOCTH1 receptor, as well as of the active NICD. Consistently, by gene expression profiling NOCTH1 M patients displayed significantly higher levels of HES1 and DTX1, the main NOTCH1 target genes. Overall, these data suggest a more active signaling pathway in NOTCH1 M CLL than in NOTCH1 WT cases.
Expression of NOTCH1 and of its target gene (DTX1) varied across disease compartments, being higher in CLL cells obtained from the lymph nodes (LN), as compared to paired samples derived from the PB or the bone marrow (BM). By immunohistochemical analyses of primary LN tissue samples, NOTCH1 M CLL showed an intense nuclear staining as opposed to the more cytoplasmic distribution observed in NOTCH1 WT samples. These data suggest a more active NOTCH1 signaling in CLL residing in the LN microenvironment and confirm the functional effect of NOTCH1 mutations in vivo.
When PB CLL cells were cultured in vitro in the absence of any supporting layer or stimulation, they showed a rapid down regulation of the NOTCH pathway, with complete loss of NICD after 24 hours paralleled by a sharp decrease in HES1 and DTX1 transcription. Consistently, levels of presenilin-1 (PSEN1), the catalytic subunit of the g-secretase complex, were also down-regulated offering a partial mechanistic explanation for the NICD loss. NOTCH1 mRNA levels remained unchanged, with accumulation of the receptor at the plasma membrane. These effects were independent of NOTCH1 mutation status and suggested the dependence of NOTCH1 signaling activation upon in vivo microenvironmental interaction, even in NOTCH1 M CLL.
Within primary LN biopsies from CLL patients, the NOTCH1 ligand, was highly expressed on CD68+ elements of myeloid origin. This observation prompted the in vitro recreation of a lymphoid niche by co-culturing Jagged1+ nurse-like cells (NLC) with autologous CLL cells. Under these conditions, NOTCH1 activity in CLL cells was sustained over time, as shown by Q-PCR analyses of DTX1 and PSEN1. Moreover, NLCs protect NOTCH1 M CLL cells from fludarabine-induced apoptosis. This microenvironment-induced chemoresistance was prevented by pre-treatment of NOTCH1 M CLL cells with specific g-secretase inhibitors, to block NOTCH1 activation.
Taken together, these results show that the 7544-7545delCT mutation in the PEST domain of NOTCH1 has a stabilizing effect on NOTCH1 signaling pathway. They also show that micro-environmental interactions are critical in activating NOTCH1 pathway both in the M and WT patients. Lastly, these results show that NOTCH1 signals micht create local conditions that favour drug resistance, thus making NOTCH1 a potential molecular target in CLL.
No relevant conflicts of interest to declare.
Monocytes actively participate in inflammatory mechanisms that contribute to the development of adipose tissue dysfunction and atherogenesis. The aim of this study was to evaluate the association of ...monocyte CCR2 chemokine receptor expression and intracellular oxidative burst with the metabolic and inflammatory factors related to body weight.
The study was performed in 67 postmenopausal women with normal, overweight and obese body mass index. Monocyte CCR2 surface expression and intracellular oxidative burst activity (determined using 2', 7'-dichlorofluorescin diacetate) were analyzed by flow cytometry. Serum levels of HMW adoponectin, monocyte chemoattractant protein-1 (MCP-1), insulin, glucose, lipids and C-reactive protein were determined.
Subjects with homeostasis model assessment-estimated insulin resistance (HOMA-IR) above the median had significantly higher proportion of CCR2+ monocytes and higher mean fluorescence intensity (MFI) of CCR2 and oxidative burst. The proportion of CCR2+ monocytes and CCR2 MFI were correlated with body weight, body mass index, fat mass, insulin and HOMA-IR. Oxidative burst also correlated with anthropometric measures, fat mass and expression of CCR2. No correlations were found between these markers of monocyte activation and HMW adiponectin or monocyte chemoattractant protein-1. The absolute number of monocytes was associated with insulin and this association remained significant after adjusting for C-reactive protein. In the multiple regression model the monocyte number was determined to be an independent predictor of insulin level.
These results provide support for significant associations of monocyte number and markers involved in monocyte activation with obesity and insulin resistance.
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