Prognostic value of circulating T-lymphocyte subsets in advanced pancreatic cancer patients treated with mFOLFIRINOX or gemcitabine.

Advanced pancreatic ductal adenocarcinoma (PDAC) is commonly treated with a chemotherapy combination of mFOLFIRINOX or gemcitabine. However, predictive and prognostic factors for choosing a more appropriate treatment strategy are still lacking. This study aimed to evaluate how chemotherapy changes immune system parameters and whether these changes influence survival outcomes. We sought to identify an easily accessible marker to help choose the appropriate treatment. Patients with PDAC who were suitable for systemic chemotherapy were eligible for the study. Peripheral blood samples were obtained at baseline and after two months of treatment. Lymphocyte subsets were measured using flow cytometry. Correlation with clinical features and survival analyses were performed. In total, 124 patients were enrolled in this study. Seventy patients were treated with mFOLFIRINOX and 50 with gemcitabine monotherapy. Four patients could not be treated because of rapid deterioration. During overall survival analysis (OS), significant factors included age, Eastern Cooperative Oncology Group (ECOG) performance status, differentiation grade G3, carcinoma antigen (CA) 19-9 more than 100 kU/L, absolute white blood cell count, CD3 + CD8+, and CD8 + CD57-T lymphocytes. Natural killer CD3-CD56 + CD16 + and CD3-CD56 + CD16- and T regulatory CD4 + FOXP3 + and CD3 + CD56 + cells differed during treatment, but these differences did not influence the survival results. At baseline, CD8 + CD57- T lymphocyte count demonstrated a clear independent impact on progression-free survival and OS. Gemcitabine showed better survival in patients with extremely low baseline CD8 + CD57- levels. Therefore, circulating CD3 + CD8 + and CD8 + CD57- cells measured before treatment in PDAC may be considered prognostic and predictive biomarkers.

Multiple Myeloma Immunophenotype Related to Chromosomal Abnormalities Used in Risk Assessment.

(1) Background: At diagnosis, multiplemyeloma risk estimation includes disease burden, end-organ damage, and biomarkers, with increasing emphasis on genetic abnormalities. Multicolor flow cytometry (MFC) is not always considered in risk estimation. We demonstrate associations found between genetic abnormalities and antigen expression of plasma cells measured by MFC. (2) Methods: Single nucleotide polymorphism microarray (SNP-A) karyotyping as well as MFC using standardized next-generation flow (NGF) panels and instrument settings were performed from bone marrow aspirates at the time of diagnosis. (3) Results: We uncovered specific immunophenotype features related to different genetic risk factors. Specifically, we found higher malignant/normal plasma cell ratio and lower expression of CD27, CD38, CD45, CD56, CD117 and CD138 in higher-risk genetic groups or risk categories.

The Effects of Treatment on Peripheral Blood Immune Cell Profile in Pancreatic Ductal Adenocarcinoma (PDAC).

BACKGROUND/AIM: This study evaluated whether circulating lymphocytes, assessed by flow cytometry, is a prognostic biomarker in pancreatic ductal adenocarcinoma (PDAC). PATIENTS AND METHODS: We studied T cell subsets in blood samples from a cohort of 41 patients diagnosed with PDAC. Patients underwent surgery of the primary site and adjuvant chemotherapy or were treated with 1st line chemotherapy (mFOLFIRINOX regimen or gemcitabine alone). The changes in T cell subpopulations during treatment were evaluated at the initial diagnosis before surgery, and after 2 and 4 months. Friedman test was used for statistical analysis. RESULTS: A decline in CD19+ B lymphocytes, natural killer (NK) cells CD3-CD56+CD16+, and T regulatory cells CD4+FOXP3+ during treatment was observed. NKT-like cells CD3+CD56+ and cytotoxic T cells CD3+CD8+ tended to increase after two months and decrease after that. CONCLUSION: Statistically significant changes in lymphocyte counts in peripheral blood were detected in patients with PDAC during treatment.

Oxidative Properties of Blood-Derived Extracellular Vesicles in 15 Patients After Myocardial Infarction.

BACKGROUND In this study, we investigated the yield and composition of extracellular vesicles (EVs) derived from 40- to 60-year-old healthy male controls and post-myocardial infarction (post-MI) patients' blood samples and assessed their pro-inflammatory and oxidative-related properties. Our study aimed to determine the EV yield and composition differences between both groups and to find out if there were differences between EV-mediated oxidative stress reactions. MATERIAL AND METHODS Fifteen post-MI patients and 25 healthy individuals were included. EVs were isolated by ultracentrifugation and analyzed using nanotracking analysis (NTA), western blotting and fluorescent flow cytometry (FFC). Oxidative stress (OS) in blood samples was identified by measuring malondialdehyde concentration from serum, while EVs-induced OS was measured in the human vein endothelium cells (HUVEC) using H2DCFDA (2',7'-dichlorodihydrofluorescein diacetate) fluorescence as a marker. RESULTS We found higher EVs concentration in healthy controls than in the post-MI group (7.07±3.1 E+10 ml vs 3.1±1.9 E+10 ml, P<0.001) and a higher level of CD9-positive exosomes (MFI 275±39.5 vs 252±13, P<0.001). Post-MI patients' EVs carry pro-oxidative nicotinamide adenine dinucleotide phosphate (NADPH) oxidases isoforms NOX1 (NADPH oxidase 1), NOX5 (NADPH oxidase 5) and NOX2 (NADPH oxidase 2) and anti-oxidative thioredoxin, extracellular signal-regulated kinases 1/2 (ERK1/2), and protein kinase B (Akt B). In the post-MI EVs, there was a higher predominance of enzymes with anti-oxidative effects, leading to weaker OS-inducing properties in the HUVEC cells. CONCLUSIONS We conclude that post-MI patient blood sample EVs have stronger anti- than pro-oxidative properties and these could help fight against post-MI consequences.

Evaluation of multiple myeloma measurable residual disease by high sensitivity flow cytometry: An international harmonized approach for data analysis.

BACKGROUND: Multiple myeloma (MM) measurable residual disease (MRD) evaluated by flow cytometry is a surrogate for progression-free and overall survival in clinical trials. However, analysis and reporting between centers lack uniformity. We designed and evaluated a consensus protocol for MM MRD analysis to reduce inter-laboratory variation in MM MRD reporting. METHODS: Seventeen participants from 13 countries performed blinded analysis of the same eight de-identified flow cytometry files from patients with/without MRD using their own method (Stage 1). A consensus gating protocol was then designed following survey and discussions, and the data re-analyzed for MRD and other bone marrow cells (Stage 2). Inter-laboratory variation using the consensus strategy was reassessed for another 10 cases and compared with earlier results (Stage 3). RESULTS: In Stage 1, participants agreed on MRD+/MRD- status 89% and 68% of the time respectively. Inter-observer variation was high for total numbers of analyzed cells, total and normal plasma cells (PCs), limit of detection, lower limit of quantification, and enumeration of cell populations that determine sample adequacy. The identification of abnormal PCs remained relatively consistent. By consensus method, average agreement on MRD- status improved to 74%. Better consistency enumerating all parameters among operators resulted in near-unanimous agreement on sample adequacy. CONCLUSION: Uniform flow cytometry data analysis substantially reduced inter-laboratory variation in reporting multiple components of the MM MRD assay. Adoption of a harmonized approach would meet an important need for conformity in reporting MM MRD for clinical trials, and wider acceptance of MM MRD as a surrogate clinical endpoint.

Adhesion molecule immunophenotype of bone marrow multiple myeloma plasma cells impacts the presence of malignant circulating plasma cells in peripheral blood.

INTRODUCTION: Multiple myeloma (MM) patients with malignant plasma cells (MMPCs) in their bone marrow (BM) and malignant circulating plasma cells (MMCPCs) in the peripheral blood (PB) are an independent marker of a clinically aggressive disease, and it reflects a poor prognosis defined by a short time to progression and overall survival. We hypothesized that changes in ADM expression on BM MMPCs might contribute to MMCPC presence in the PB of relapsed/refractory multiple myeloma (RRMM) patients. METHODS: We assessed the difference in expression of adhesion molecules and receptors related to cell-cell interaction: integrins, hyaluronic acid receptors, chemokine receptors and other proteins on healthy donor PCs, RRMM BM and PB MMPCs. RESULTS: Adhesion immunophenotype showed a significant loss of many adhesion molecules when comparing BM MMPCs of MMCPC- and MMCPC+ MM patients (CD49d, CD49e, CD56, CD138). Further decrease of adhesion molecules was shown in MMCPCs (CD49d, CD49e, CD56, CD138, CD58), suggesting that loss of these molecules may allow cells to leave the BM. CONCLUSIONS: Loss of adhesion molecule expression enables MMPCs to leave the BM milieu and enter the PB. These changes can be seen in both the PB and BM of MMCPC+ MM patient.

Changes in circulating endothelial microvesicles in men after myocardial infarction.

PURPOSE: The objective of the study was to determine the differences in the numbers of endothelial microvesicles (EMV) after myocardial infarction (MI) and their association with oxidative stress. MATERIALS AND METHODS: We included 15 post MI patients and 28 healthy controls. Samples were analysed by flow cytometry. We examined four EMV populations: 1) CD144+, CD42a-, CD61-, 2) CD144+, CD42a+, CD61-, 3) CD105+, CD42a-, CD61-and 4) CD31+, CD42a-, CD61-and determined a percentage of CD62e + EMV. Malondialdehyde concentration was determined by ultra-high performance liquid chromatography. RESULTS: The median of EMV counts differed between controls and patients in: CD105+ (10.91 microvesicles/µl vs. 33.68 microvesicles/µl, P = 0.006), CD144+, CD42a+ (312.87 microvesicles/µl vs. 73.29 microvesicles/µl, P < 0.001) and CD31+ (2 microvesicles/µl vs. 1.38 microvesicles/µl, P = 0.021). The median of percentage of CD62e expression differed between controls and patients in: CD105+ (1.35% vs. 14.8%, P < 0.001), CD144+, CD42a+ (56.45% vs. 98.99%, P < 0.001) and CD144+, CD42a- (173.03% vs. 215.56%) EMV. In patients, EMV counts correlated with low-density lipoprotein cholesterol (LDL-C), total cholesterol (TC) and high-density lipoprotein cholesterol (HDL-C) concentrations: CD105+: R = -0.69, P = 0.004 (LDL-C), R = -0.64, P = 0.01 (TC); CD144+, CD42a-: R = -0.68, P = 0.005 (LDL-C), R = -0.63, P = 0.011 (TC); CD144+: R = -0.54, P = 0.038 (HDL-C) and CD144+, CD42a-, CD62e+: R = 0.78, P = 0.001 (HDL-C). In controls, HDL-C concentration correlated with CD105+ (R = -0.395, P = 0.038) and CD105+, CD62e+ (R = -0.716, P < 0.001) counts. Malondialdehyde concentration correlated with CD144+, CD42a- (P = 0.01, R = 0.48) and CD105+, CD62e+ (P = 0.012, R = 0.47) counts. CONCLUSIONS: Changes in EMV levels after the MI period were observed. Counts of EMV and their CD62e expression correlated with dyslipidaemia and oxidative stress.

Association of Platelet Membrane Fatty Acid Composition with Markers of Oxidative Stress in Healthy Men.

BACKGROUND Platelet membranes are extremely susceptible to peroxidation, forming a variety of lipid peroxides, including malondialdehyde (MDA), which has been implicated in the etiology of cardiovascular diseases. Moreover, platelet-leukocyte aggregates (PLAs) are known to contribute to advanced endothelial injury and atherogenesis. MATERIAL AND METHODS Fatty acid (FA) methyl esters of the platelet membranes of 79 apparently healthy men without any acute clinical condition at the time of the study were identified by GC/MS. MDA was measured by HPLC in blood serum, and PLAs were analyzed by whole-blood flow cytometry. Individuals were divided into quartiles according to MDA concentration and percentage of PLAs formation. The composition of platelet membrane FAs was compared to MDA concentration and the percentage of PLAs formation in apparently healthy individuals. RESULTS In quartiles (Q) with higher MDA concentration, percentage of C 16: 1ω7 (Q1 vs. Q3, p=0.021), C 20: 1ω9 (Q2 vs. Q4, p=0.028) and C 20: 5ω3 (Q2 vs. Q4, p=0.046) was lower. However, C 22: 5ω3 (Q1 vs. Q4, p=0.038) and total ω3 (Q1 vs. Q2, p=0.024) were higher. CONCLUSIONS MDA and the formation of platelet-monocyte aggregates stimulate the incorporation of monounsaturated fatty acids and polyunsaturated fatty acids in platelet phospholipid membranes, which may be a hallmark for a changed level of biologically active compounds required for the activation of future platelets.

Association of Hair Cortisol Concentration with Prevalence of Major Cardiovascular Risk Factors and Allostatic Load.

BACKGROUND The high prevalence of cardiovascular diseases cannot be explained completely by conventional risk factors such as older age, smoking, diabetes mellitus, hypertension, obesity, and dyslipidemia. Results of recent studies indicate that chronic stress may be an independent risk factor for cardiovascular morbidity and mortality. Thus, the aim of our study was to investigate the associations between the hair cortisol concentration (HCC), which is considered as a potential biomarker of long-term psychosocial stress, and traditional cardiovascular risk factors, including smoking, dyslipidemia, hypertension, and obesity. MATERIAL AND METHODS Fasting blood samples and anthropometric and lifestyle data were collected from 163 apparently healthy men. HCC was determined using high-performance liquid chromatography. Allostatic load (AL) index, defined as an integrated score of multiple interacting systems involved in the adaptation to adverse physical or psychosocial situations, was also calculated. RESULTS We found that many prevalent cardiovascular risk factors, including hypertension, smoking, higher than recommended waist circumference (WC), and low-density lipoprotein cholesterol (LDL-C) median values, are associated with higher HCC. Hair cortisol level was also positively associated with the manifestation of individual cardiovascular risk factors such as higher-than-recommended total cholesterol, LDL-C, non-high-density lipoprotein cholesterol, body mass index, and WC median values. Moreover, a significant positive relationship between HCC and AL index was observed. CONCLUSIONS The results of this study suggest that increased prevalence of traditional cardiovascular risk factors is associated with higher HCC. Also, both HCC and AL index might be appropriate markers for the evaluation of chronic stress level.

Chronic and oxidative stress association with total count of endothelial microvesicles in healthy young male plasma.

BACKGROUND: Chronic and oxidative stress promotes injury to the endothelium. This happens early in the disease and novel biomarkers describing the rate of the damage may be important in early diagnostics and prevention. Microvesicles are shed from endothelial cells in response to oxidative stress, inflammation, coagulation, and angiogenesis. Their increased level in plasma could reflect the state of the endothelium. OBJECTIVES: The objective of this study was to test the association between oxidative and chronic stress markers, atherosclerosis risk factors and endothelial microvesicle (EMV) count in peripheral blood. MATERIAL AND METHODS: The study included 81 males, aged 25-55 years and apparently healthy. Venous blood samples were labeled with anti-CD144-FITC, anti-CD105-BV421, anti-CD42a-PerCP, anti-CD62e-PE, anti-CD31-APCy7, and anti-CD61-APC (BD Biosciences, San Jose, USA), and tested using a BD LSR Fortessa cytometer (BD Biosciences). Events were gated on forward and side-scattered light parameters. Malondialdehyde (MDA) and cortisol concentrations were measured using high-performance liquid chromatography (HPLC). RESULTS: Four populations of EMV expressing a combination of CD105+, CD31+, CD144+, and CD62e with CD42aor CD42a+ markers were examined. We found correlations between MDA concentration and hair cortisol and a total count of CD144+ microvesicles, and weak correlations with diastolic blood pressure (DBP) (p = 0.003, r = 0.324) and systolic blood pressure (SBP) (p = 0.016, r = 0.267), especially with the microvesicles carrying CD62e. There was a median difference of CD105+ microvesicle count between smoking (n = 13) and non-smoking (n = 68) individuals. A predictive model showed an association between CD144+ microvesicle counts with cortisol and MDA concentrations and waist circumference. CONCLUSIONS: In conclusion, our data and predictive model showed that the total counts of microvesicle populations were associated with stress-related parameters - cortisol and MDA concentrations; expression of CD62e in various populations of EMV and the ratio of CD144+ to CD105+/CD62e+ were associated with increased DBP and SBP, and also with total cholesterol concentration in healthy young male population.