The Effects of a Cardiac Rehabilitation Program on Endothelial Progenitor Cells and Inflammatory Profile in Patients with Chronic Heart Failure of Different Severity.

Endothelial dysfunction and inflammation are common pathophysiological characteristics of chronic heart failure (CHF). Endothelial progenitor cells (EPCs) are recognized as useful markers of vascular damage and endothelial repair. The aim of this study was to investigate the effects of a cardiac rehabilitation program on EPCs and inflammatory profile in CHF patients of different severity. Forty-four patients with stable CHF underwent a 36-session cardiac rehabilitation program. They were separated into two different subgroups each time, according to the median peak VO2, predicted peak VO2, VE/VCO2 slope, and ejection fraction. EPCs, C-reactive protein (CRP), interleukin 6 (IL-6), interleukin 10 (IL-10), and vascular endothelial growth factor (VEGF) were measured. Flow cytometry was used for the quantification of EPCs. Mobilization of EPCs increased and the inflammatory profile improved within each severity group (p < 0.05) after the cardiac rehabilitation program, but there were no statistically significant differences between groups (p > 0.05). A 36-session cardiac rehabilitation program has similar beneficial effects on the mobilization of EPCs and on the inflammatory profile in patients with CHF of different severity.

MTH1 Inhibition Alleviates Immune Suppression and Enhances the Efficacy of Anti-PD-L1 Immunotherapy in Experimental Mesothelioma.

BACKGROUND: MTH1 protects tumor cells and their supporting endothelium from lethal DNA damage triggered by oxidative stress in the tumor microenvironment, thus promoting tumor growth. The impact of MTH1 on the tumor-related immune compartment remains unknown. We hypothesized that MTH1 regulates immune fitness and therefore enhances the activity of currently used immunotherapeutic regimens. METHODS: Our hypotheses were validated in two syngeneic murine mesothelioma models using the clinically relevant MTH1 inhibitor, karonudib. We also examined the effect of combined MTH1 and PD-L1 blockade in mesothelioma progression, focusing on the main immune players. RESULTS: Karonudib administration enhances M1 macrophage polarization, stimulates CD8 expansion and promotes the activation of DC and T cells. Combined administration of PD-L1 and MTH1 inhibitors impairs mesothelioma tumor growth and mesothelioma-associated pleural effusion accumulation more effectively compared to each monotherapy. CONCLUSIONS: Combined MTH1 and PD-L1 inhibition holds promise for the successful clinical management of mesothelioma.

Flow cytometric analysis of myelodysplasia: Pre-analytical and technical issues-Recommendations from the European LeukemiaNet.

BACKGROUND: Flow cytometry (FCM) aids the diagnosis and prognostic stratification of patients with suspected or confirmed myelodysplastic syndrome (MDS). Over the past few years, significant progress has been made in the FCM field concerning technical issues (including software and hardware) and pre-analytical procedures. METHODS: Recommendations are made based on the data and expert discussions generated from 13 yearly meetings of the European LeukemiaNet international MDS Flow working group. RESULTS: We report here on the experiences and recommendations concerning (1) the optimal methods of sample processing and handling, (2) antibody panels and fluorochromes, and (3) current hardware technologies. CONCLUSIONS: These recommendations will support and facilitate the appropriate application of FCM assays in the diagnostic workup of MDS patients. Further standardization and harmonization will be required to integrate FCM in MDS diagnostic evaluations in daily practice.

Clinical application of flow cytometry in patients with unexplained cytopenia and suspected myelodysplastic syndrome: A report of the European LeukemiaNet International MDS-Flow Cytometry Working Group.

This article discusses the rationale for inclusion of flow cytometry (FCM) in the diagnostic investigation and evaluation of cytopenias of uncertain origin and suspected myelodysplastic syndromes (MDS) by the European LeukemiaNet international MDS Flow Working Group (ELN iMDS Flow WG). The WHO 2016 classification recognizes that FCM contributes to the diagnosis of MDS and may be useful for prognostication, prediction, and evaluation of response to therapy and follow-up of MDS patients.

The flow cytometry myeloid progenitor count: A reproducible parameter for diagnosis and prognosis of myelodysplastic syndromes.

BACKGROUND: The bone marrow blast count is central to the diagnosis and monitoring of myelodysplastic syndromes (MDS). It is an independent risk factor for worse prognosis whether based on the morphology blast count or the flow cytometry (FC) myeloid progenitor (MyP) count. It is a principal population in FC MDS analysis also because once defined; it provides significant contributions to the overall FC MDS score. METHODS: We elected to investigate inter-analyst agreement for the most fundamental parameter of the FC MDS diagnostic score: the MyP count. A common gating strategy was agreed and used by seven cytometrists for blind analysis of 34 routine bone marrows sent for MDS work-up. Additionally, we compared the results with a computational approach. RESULTS: Concordance was excellent: Intraclass correlation was 0.993 whether measuring %MyP of total cells or CD45+ cells, and no significant difference was observed between files from different centers or for samples with abnormal MyP phenotypes. Computational and manual results were similar. Applying the common strategy to individual laboratories' control cohorts produced similar MyP reference ranges across centers. CONCLUSION: The FC MyP count offers a reliable diagnostic and prognostic measurement in MDS. The use of manual and computational approaches side by side may allow for optimizing both strategies. Considering its known prognostic power, the MyP count could be considered a useful and reliable addition to existing prognostic scoring systems.

ELN iMDS flow working group validation of the monocyte assay for chronic myelomonocytic leukemia diagnosis by flow cytometry.

BACKGROUND: It was proposed that peripheral blood (PB) monocyte profiles evaluated by flow cytometry, called "monocyte assay," could rapidly and efficiently distinguish chronic myelomonocytic leukemia (CMML) from other causes of monocytosis by highlighting an increase in the classical monocyte (cMo) fraction above 94%. However, the robustness of this assay requires a large multicenter validation and the assessment of its feasibility on bone marrow (BM) samples, as some centers may not have access to PB. METHODS: PB and/or BM samples from patients displaying monocytosis were assessed with the "monocyte assay" by 10 ELN iMDS Flow working group centers with harmonized protocols. The corresponding files were reanalyzed in a blind fashion and the cMo percentages obtained by both analyses were compared. Confirmed diagnoses were collected when available. RESULTS: The comparison between cMo percentages from 267 PB files showed a good global significant correlation (r = 0.88) with no bias. Confirmed diagnoses, available for 212 patients, achieved a 94% sensitivity and an 84% specificity. Hence, 95/101 CMML patients displayed cMo ≥94% while cMo <94% was observed in 83/99 patients with reactive monocytosis and in 10/12 patients with myeloproliferative neoplasms (MPN) with monocytosis. The established Receiver Operator Curve again provided a 94% cut-off value of cMo. The 117 BM files reanalysis led to an 87% sensitivity and an 80% specificity, with excellent correlation between the 43 paired samples to PB. CONCLUSIONS: This ELN multicenter study demonstrates the robustness of the monocyte assay with only limited variability of cMo percentages, validates the 94% cutoff value, confirms its high sensitivity and specificity in PB and finally, also confirms the possibility of its use in BM samples.

Multiparameter flow cytometry in the evaluation of myelodysplasia: Analytical issues: Recommendations from the European LeukemiaNet/International Myelodysplastic Syndrome Flow Cytometry Working Group.

Multiparameter flow cytometry (MFC) is one of the essential ancillary methods in bone marrow (BM) investigation of patients with cytopenia and suspected myelodysplastic syndrome (MDS). MFC can also be applied in the follow-up of MDS patients undergoing treatment. This document summarizes recommendations from the International/European Leukemia Net Working Group for Flow Cytometry in Myelodysplastic Syndromes (ELN iMDS Flow) on the analytical issues in MFC for the diagnostic work-up of MDS. Recommendations for the analysis of several BM cell subsets such as myeloid precursors, maturing granulocytic and monocytic components and erythropoiesis are given. A core set of 17 markers identified as independently related to a cytomorphologic diagnosis of myelodysplasia is suggested as mandatory for MFC evaluation of BM in a patient with cytopenia. A myeloid precursor cell (CD34+ CD19- ) count >3% should be considered immunophenotypically indicative of myelodysplasia. However, MFC results should always be evaluated as part of an integrated hematopathology work-up. Looking forward, several machine-learning-based analytical tools of interest should be applied in parallel to conventional analytical methods to investigate their usefulness in integrated diagnostics, risk stratification, and potentially even in the evaluation of response to therapy, based on MFC data. In addition, compiling large uniform datasets is desirable, as most of the machine-learning-based methods tend to perform better with larger numbers of investigated samples, especially in such a heterogeneous disease as MDS.

Multicenter prospective evaluation of diagnostic potential of flow cytometric aberrancies in myelodysplastic syndromes by the ELN iMDS flow working group.

BACKGROUND: Myelodysplastic syndromes (MDS) represent a diagnostic challenge. This prospective multicenter study was conducted to evaluate pre-defined flow cytometric markers in the diagnostic work-up of MDS and chronic myelomonocytic leukemia (CMML). METHODS: Thousand six hundred and eighty-two patients with suspected MDS/CMML were analyzed by both cytomorphology according to WHO 2016 criteria and flow cytometry according to ELN recommendations. Flow cytometric readout was categorized 'non-MDS' (i.e. no signs of MDS/CMML and limited signs of MDS/CMML) and 'in agreement with MDS' (i.e., in agreement with MDS/CMML). RESULTS: Flow cytometric readout categorized 60% of patients in agreement with MDS, 28% showed limited signs of MDS and 12% had no signs of MDS. In 81% of cases flow cytometric readouts and cytomorphologic diagnosis correlated. For high-risk MDS, the level of concordance was 92%. A total of 17 immunophenotypic aberrancies were found independently related to MDS/CMML in ≥1 of the subgroups of low-risk MDS, high-risk MDS, CMML. A cut-off of ≥3 of these aberrancies resulted in 80% agreement with cytomorphology (20% cases concordantly negative, 60% positive). Moreover, >3% myeloid progenitor cells were significantly associated with MDS (286/293 such cases, 98%). CONCLUSION: Data from this prospective multicenter study led to recognition of 17 immunophenotypic markers allowing to identify cases 'in agreement with MDS'. Moreover, data emphasizes the clinical utility of immunophenotyping in MDS diagnostics, given the high concordance between cytomorphology and the flow cytometric readout. Results from the current study challenge the application of the cytomorphologically defined cut-off of 5% blasts for flow cytometry and rather suggest a 3% cut-off for the latter.

A Cardiac Rehabilitation Program Increases the Acute Response of Endothelial Progenitor Cells to Maximal Exercise in Heart Failure Patients.

Purpose: The purpose of this study was to investigate the effect of a cardiac rehabilitation program on the acute response on endothelial progenitor cells and circulating endothelial cells after maximal exercise in patients with chronic heart failure of different severity. Methods: Forty-four chronic heart failure patients were enrolled in a 36-session cardiac rehabilitation program. All patients underwent an initial maximal cardiopulmonary exercise test before and a final maximal cardiopulmonary exercise test after the cardiac rehabilitation program. The patients were divided in two groups of severity according to the median value of peak VO2. Blood was collected at 4 time points; 2 time points at rest, and 2 time points after each cardiopulmonary exercise test. Five endothelial cellular populations were quantified by flow cytometry. Results: Although there was a higher increase in the mobilization of subgroups of endothelial progenitor cells and circulating endothelial cells after the final cardiopulmonary exercise test compared to the initial test within each severity group (p < 0.05), no significant differences between severity groups were observed (p > 0.05). Conclusions: A 36-session cardiac rehabilitation program had similar beneficial effects on the acute response of endothelial progenitor cells and circulating endothelial cells after maximal exercise in patients with chronic heart failure of different severity.

CSF1/CSF1R signaling mediates malignant pleural effusion formation.

Malignant pleural effusion (MPE) is an incurable common manifestation of many malignancies. Its formation is orchestrated by complex interactions among tumor cells, inflammatory cells, and the vasculature. Tumor-associated macrophages present the dominant inflammatory population of MPE, and M2 macrophage numbers account for dismal prognosis. M2 polarization is known to be triggered by CSF1/CSF1 receptor (CSF1R) signaling. We hypothesized that CSF1R+ M2 macrophages favor MPE formation and could be therapeutically targeted to limit MPE. We generated mice with CSF1R-deficient macrophages and induced lung and colon adenocarcinoma-associated MPE. We also examined the therapeutic potential of a clinically relevant CSF1R inhibitor (BLZ945) in lung and colon adenocarcinoma-induced experimental MPE. We showed that CSF1R+ macrophages promoted pleural fluid accumulation by enhancing vascular permeability, destabilizing tumor vessels, and favoring immune suppression. We also showed that CSF1R inhibition limited MPE in vivo by reducing vascular permeability and neoangiogenesis and impeding tumor progression. This was because apart from macrophages, CSF1R signals in cancer-associated fibroblasts leading to macrophage inflammatory protein 2 secretion triggered the manifestation of suppressive and angiogenic properties in macrophages upon CXCR2 paracrine activation. Pharmacological targeting of the CSF1/CSF1R axis can therefore be a vital strategy for limiting MPE.

Peripheral blood natural killer cells in sarcoidosis are associated with early cardiac involvement.

AIM: To evaluate the distribution of circulating immune cell subsets in peripheral blood of patients with sarcoidosis and investigate if there is an association with an underlying cardiac involvement. METHODS AND RESULTS: Eighty-five newly diagnosed treatment-naïve patients with sarcoidosis (50 women) were included in the study. All patients underwent a thorough cardiac investigation, including cardiac magnetic resonance imaging (CMR). Of all patients, 19 (23.53%) had myocardial involvement, and the NK subpopulation in these patients in peripheral blood was significantly decreased compared to patients without (n = 63, p = 0.001 and p = 0.003 respectively). The absolute number of NKT cells (CD3+CD16/56+ ) in patients with cardiac involvement was highly correlated with T2 map increased values in MRI (r = -686, p = 0.041) showing that low NKT cell count correlates with the inflammatory process of the heart. No difference in CD19, CD3, CD4, CD8 and CD3- NK cell counts was found between groups. Lung severity was not found to correlate with the number of NK cells. CONCLUSION: We found that low NK cell count in peripheral blood of patients with sarcoidosis is associated with cardiac involvement, and the number of NK-T cells correlates with CMR findings indicative of myocardial inflammation. This finding might have a potential clinical application in detecting clinically silent cardiac involvement in sarcoidosis and may also suggest potential targets for therapeutic interventions.

Kinetics of Immune Subsets in COVID-19 Patients Treated with Corticosteroids.

RATIONALE: Changes in anti-SARS-CoV-2 defense immune subsets in patients treated with dexamethasone (DXM) for severe COVID-19 and their relation to disease outcomes are poorly understood. METHODS: Blood-lymphocyte subsets of 110 hospitalized COVID-19 patients were prospectively examined. A first sample was taken at enrollment and a second one 7-10 days later. Total B-, T-lymphocytes, CD4+, CD8+, T-regulatory (Treg), Natural-Killer (NK) and NK T-cells were counted using flow cytometry. RESULTS: At enrollment, patients with respiratory failure, characterized by DXM failure (intubation/death) or DXM success (hospital discharge) exhibited significantly fewer CD3+, CD4+ and CD8+ cells and B-lymphocytes compared to the control group (no respiratory failure/no DXM). At the time of treatment completion, the DXM-failure group exhibited significantly fewer CD3+, CD4+ and CD8+ cells, memory CD4+ and CD8+ T-lymphocytes, compared to the control and the DXM-success groups and fewer activated CD4+ T-lymphocytes, Tregs and NK cells compared to the control group. At the time of treatment completion, the number of all investigated lymphocyte subsets increased in the DXM-success group and was similar to those of the control group. NK cells significantly decreased over time in the DXM-failure group. CONCLUSION: The lymphocyte kinetics differ between DXM-treated and control COVID-19 patients and are associated with clinical outcomes.

Combination of the CD8+:B-cell and Neutrophil-to-Lymphocyte Ratio as a Novel Prediction Model for Intubation Need and Disease Severity in COVID-19 Patients.

BACKGROUND: The study provides a novel prediction model for COVID-19 progression and outcome by the combination of the CD8+: B-cells ratio with neutrophil-to-lymphocyte ratio (NLR). PATIENTS AND METHODS: Immune phenotyping was performed in 120 COVID-19 patients. RESULTS: A decrease in CD8+:B-cell (p<0.0001) and in lymphocyte-to-CRP (LCR) ratio (p<0.0001) was observed in intubated patients versus non-intubated with an increase for CD4+:CD8+ (p<0.01), NLR (p<0.0001) and CRP: Albumin (p<0.001). Receiving operating curve (ROC) analysis predicting requirement for mechanical ventilation revealed the highest AUC for CD8+:B-cells, (AUC=0.795, p<0.001) versus NLR (AUC=0.783, p<0.001), LCR (AUC=0.779, p<0.001), Albumin:CRP (AUC=0.750, p<0.001) and CD4+:CD8+ (AUC=0.779, p<0.001). Combination of the CD8+: B-cell ratio with the NLR increased the AUC (AUC=0.845, p<0.001). The combined ratios correlated with outcome defined as duration of hospital (r=0.435, p<0.001) or ICU stay (r=0.596, p<0.001). CONCLUSION: Combination of the CD8+: B-cell ratio and NLR serves as a useful prognostic tool for COVID-19 patient progression.

CSF1/CSF1R Axis Blockade Limits Mesothelioma and Enhances Efficiency of Anti-PDL1 Immunotherapy.

Colony-Stimulating Factor 1 (CSF1)/Colony-Stimulating Factor Receptor 1 (CSF1R) signaling orchestrates tumor-associated macrophage (TAM) recruitment and polarization towards a pro-tumor M2 phenotype, the dominant phenotype of TAMs infiltrating mesothelioma tumors. We hypothesized that CSF1/CSF1R inhibition would halt mesothelioma growth by targeting immunosuppressive M2 macrophages and unleashing efficient T cell responses. We also hypothesized that CSF1/CSF1R blockade would enhance the efficacy of a PDL1 inhibitor which directly activates CD8+ cells. We tested a clinically relevant CSF1R inhibitor (BLZ945) in mesothelioma treatment using syngeneic murine models. We evaluated the role of CSF1/CSF1R axis blockade in tumor-infiltrating immune subsets. We examined the effect of combined anti-CSF1R and anti-PDL1 treatment in mesothelioma progression. CSF1R inhibition impedes mesothelioma progression, abrogates infiltration of TAMs, facilitates an M1 anti-tumor phenotype and activates tumor dendritic and CD8+ T cells. CSF1R inhibition triggers a compensatory PD-1/PDL1 upregulation in tumor and immune cells. Combined CSF1R inhibitor with an anti-PDL1 agent was more effective in retarding mesothelioma growth compared to each monotherapy. In experimental mesotheliomas, CSF1R inhibition abrogates tumor progression by limiting suppressive myeloid populations and enhancing CD8+ cell activation and acts synergistically with anti-PDL1.

A novel ratio of CD8+:B-cells as a prognostic marker of coronavirus disease 2019 patient progression and outcome.

Infection with SARS-COV-2 may result in severe pneumonia potentially leading to mechanical ventilation and intensive care treatment. The aim of the present study was to analyze the immune responses in critically ill coronavirus 2019 (COVID-19) patients requiring mechanical ventilation and assess their potential use as markers of clinical progression and outcome. Confirmed COVID-19 patients were grouped into those requiring mechanical ventilation (intubated) and non-intubated. Immune phenotyping was performed and cytokine levels were determined. A novel ratio of CD8+:B cells was significantly lower in intubated versus non-intubated (p = 0.015) and intubated non-survivors (NSV) versus survivors (SV) (p = 0.015). The same ratio correlated with outcome, CRP, IL-6 levels and neutrophil count. Receiving operating curve (ROC) analysis for prediction of requirement of mechanical ventilation by the CD8+:B cells ratio revealed an AUC of 0.747 and a p = 0.007. The ratio of CD8+:B cells may serve as a useful prognostic marker for disease severity and outcome.

Monitoring treatment with 5-Azacitidine by flow cytometry predicts duration of hematological response in patients with myelodysplastic syndrome.

5-Azacitidine (AZA) therapy is used in high-risk myelodysplastic syndrome (MDS) patients who often show abnormalities in their immunophenotype. We explored the potential impact of AZA on these immunophenotypic abnormalities in serial bone marrow studies performed in 81 patients from five centers. We compared the immunophenotypic features before and after therapy with AZA, established definitions consistent with flow cytometry immunophenotyping (FCI) improvement, and explored its clinical significance. After a median of 6 cycles of AZA, 41% of patients showed a FCI improvement and this finding associated with best possible clinical response (P < 0.001). FCI improvement also correlated with hematological improvement (HI) (53/78 patients; 68%), independently of their eligibility for stem cell transplantation. Among patients who achieved a HI after 6 cycles of AZA, the probability of maintaining this response at 12 cycles of AZA was twice as large (67%) for those patients who also achieved a FCI improvement after 6 cycles of AZA as compared to patients who did not (33%, P < 0.01). These findings support that monitoring of the immunophenotypic abnormalities during therapy with AZA may assist in redefining the quality of response in patients with MDS.

The acute and long-term effects of a cardiac rehabilitation program on endothelial progenitor cells in chronic heart failure patients: Comparing two different exercise training protocols.

BACKGROUND: Vascular endothelial dysfunction is an underlying pathophysiological feature of chronic heart failure (CHF). Endothelial progenitor cells (EPCs) are also impaired. The purpose of the study was to assess the effect of a cardiac rehabilitation (CR) program on the increase of EPCs at rest and on the acute response after maximal exercise in patients with CHF and investigate whether there were differences between two exercise training protocols and patients of NYHA II and III classes. METHODS: Forty-four patients with stable CHF enrolled in a 36-session CR program and were randomized in one training protocol; either high-intensity interval training (HIIT) or HIIT combined with muscle strength (COM). All patients underwent maximum cardiopulmonary exercise testing (CPET) before and after the CR program and venous blood was drawn before and after each CPET. Five endothelial cellular populations, expressed as cells/106 enucleated cells, were quantified by flow cytometry. RESULTS: An increase in all endothelial cellular populations at rest was observed after the CR program (p < 0.01). The acute response after maximum exercise increased in 4 out of 5 endothelial cellular populations after rehabilitation. Although there was increase in EPCs at rest and the acute response after rehabilitation in each exercise training group and each NYHA class, there were no differences between HIIT and COM groups or NYHA II and NYHA III classes (p > 0.05). CONCLUSIONS: A 36-session CR program increases the acute response after maximum CPET and stimulates the long-term mobilization of EPCs at rest in patients with CHF. These benefits seem to be similar between HIIT and COM exercise training protocols and between patients of different functional classes.

International guidelines for the flow cytometric evaluation of peripheral blood for suspected Sézary syndrome or mycosis fungoides: Assay development/optimization, validation, and ongoing quality monitors.

Introducing a sensitive and specific peripheral blood flow cytometric assay for Sézary syndrome and mycosis fungoides (SS/MF) requires careful selection of assay design characteristics, and translation into a laboratory developed assay through development/optimization, validation, and continual quality monitoring. As outlined in a previous article in this series, the recommended design characteristics of this assay include at a minimum, evaluation of CD7, CD3, CD4, CD8, CD26, and CD45, analyzed simultaneously, requiring at least a 6 color flow cytometry system, with both quantitative and qualitative components. This article provides guidance from an international group of cytometry specialists in implementing an assay to those design specifications, outlining specific considerations, and best practices. Key points presented in detail are: (a) Pre-analytic components (reagents, specimen processing, and acquisition) must be optimized to: (i) identify and characterize an abnormal population of T-cells (qualitative component) and (ii) quantitate the abnormal population (semi/quasi-quantitative component). (b)Analytic components (instrument set-up/acquisition/analysis strategy and interpretation) must be optimized for the identification of SS/MF populations, which can vary widely in phenotype. Comparison with expert laboratories is strongly encouraged in order to establish competency. (c) Assay performance must be validated and documented through a validation plan and report, which covers both qualitative and semi/quasi-quantitative assay components (example template provided). (d) Ongoing assay-specific quality monitoring should be performed to ensure consistency.