The Prognostic Value of Plasma Small Extracellular Vesicles' Phenotype in Patients With Gastrointestinal Stromal Tumor.

BACKGROUND/AIM: For patients with local gastrointestinal stromal tumor (GIST), risk stratification is used to assess the prognosis and identify patients to offer adjuvant treatment. For patients with advanced or metastatic GIST, no such risk stratification exists. This study aimed to investigate the prognostic value of 31 different plasma small extracellular vesicles' (SEVs) surface proteins in GIST patients. MATERIALS AND METHODS: GIST patients from the two sarcoma centers in Denmark were included. Patients were divided into three groups; group 1: patients undergoing radical surgery; group 2: patients with local, locally advanced, or metastatic GIST; and group 3: patients without evidence of disease after radical surgery. Protein microarray technology was used for the analysis of plasma SEVs. The median plasma SEV marker level was used when comparing groups of patients. The primary endpoint was the progression of GIST. Iterative statistical modeling was used to identify a SEV marker profile/model with a prognostic value. RESULTS: A total of 157 patients were included, with a median follow-up time of 2.05 years. In group 2, a high level of carcinoembryonic antigen (CEA) and a low level of glucose transporter 1 (GLUT-1) were found to be poor prognostic factors [univariate analysis; GLUT-1: hazard ratio (HR)=0.47, 95% confidence interval (CI)=0.22-0.98; CEA: HR=2.12, 95%CI=1.02-4.44]. Composing a model consisting of CEA and GLUT-1 adjusted for age at inclusion was found to have a prognostic value (HR=4.93, 95%CI=2.30-10.57, p<0.0001). CONCLUSION: Plasma SEVs in GIST showed that CEA and GLUT-1 might be of prognostic value. However, external validation is needed.

Identification of potential autoantigens in anti-CCP-positive and anti-CCP-negative rheumatoid arthritis using citrulline-specific protein arrays.

The presence or absence of autoantibodies against citrullinated proteins (ACPAs) distinguishes two main groups of rheumatoid arthritis (RA) patients with different etiologies, prognoses, disease severities, and, presumably, disease pathogenesis. The heterogeneous responses of RA patients to various biologics, even among ACPA-positive patients, emphasize the need for further stratification of the patients. We used high-density protein array technology for fingerprinting of ACPA reactivity. Identification of the proteome recognized by ACPAs may be a step to stratify RA patients according to immune reactivity. Pooled plasma samples from 10 anti-CCP-negative and 15 anti-CCP-positive RA patients were assessed for ACPA content using a modified protein microarray containing 1631 different natively folded proteins citrullinated in situ by protein arginine deiminases (PADs) 2 and PAD4. IgG antibodies from anti-CCP-positive RA plasma showed high-intensity binding to 87 proteins citrullinated by PAD2 and 99 proteins citrullinated by PAD4 without binding significantly to the corresponding native proteins. Curiously, the binding of IgG antibodies in anti-CCP-negative plasma was also enhanced by PAD2- and PAD4-mediated citrullination of 29 and 26 proteins, respectively. For only four proteins, significantly more ACPA binding occurred after citrullination with PAD2 compared to citrullination with PAD4, while the opposite was true for one protein. We demonstrate that PAD2 and PAD4 are equally efficient in generating citrullinated autoantigens recognized by ACPAs. Patterns of proteins recognized by ACPAs may serve as a future diagnostic tool for further subtyping of RA patients.

Extracellular Vesicles: An Important Biomarker in Recurrent Pregnancy Loss?

Recurrent pregnancy loss (RPL) has an estimated incidence of 1-3% of all couples. The etiology is considered to be multifactorial. Extracellular vesicles (EVs) take part in numerous different physiological processes and their contents show the originating cell and pathophysiological states in different diseases. In pregnancy disorders, changes can be seen in the composition, bioactivity and concentration of placental and non-placental EVs. RPL patients have an increased risk of pregnancy complications. The aim of this prospective study was to examine whether measuring different specific EV markers in plasma before and during pregnancy could be used as predictors of pregnancy loss (PL) in women with RPL. Thirty-one RPL patients were included in this study; 25 had a live birth (LB group) and six had a new PL (PL group). Five blood samples were obtained, one before achieved pregnancy and the others in gestational week 6, 8, 10 and 16. Moreover, some of the patients received intravenous immunoglobulin (IVIG) infusions as part of treatment, and it was also examined whether this treatment influenced the EV levels. Seventeen EV markers specific for the immune system, coagulation, placenta and hypoxia were analyzed in the samples with EV Array, a method able to capture small EVs by using an antibody panel targeting membrane proteins. Comparing the LB and PL groups, one EV marker, CD9, showed a significant increase from before pregnancy to gestational week 6 in the PL group. The changes in the other 16 markers were nonsignificant. One case of late-onset PL showed steeply increasing levels, with sudden decrease after gestational week 10 in nine of 17 markers. Moreover, there was an overall increase of all 17 markers after IVIG treatment in the LB group, which was significant in 15 of the markers. Whether increases in EVs positive for CD9 characterize RPL patients who subsequently miscarry should be investigated in future larger studies.

Elevated blood plasma levels of tissue factor-bearing extracellular vesicles in patients with atrial fibrillation.

BACKGROUND: The risk of thrombus formation in the left atrial appendage (LAA) in patients with atrial fibrillation (AF) may result from blood stasis, local endocardial changes, and/or changed blood composition. Extracellular vesicles (EVs), especially subtypes exposing tissue factor (TF), have procoagulant capacity. We hypothesized that blood concentrations of TF-bearing EVs and other procoagulant biomarkers are elevated in AF patients, particularly in the LAA lumen. METHODS: From 13 AF patients and 12 controls a venous blood sample was drawn prior to cardiac surgery. Intraoperatively, venous blood and blood directly from the LAA was drawn. Plasma levels of EVs, including TF- and cell type specific antigen-bearing EVs, were measured using a protein microarray platform. Plasma levels of TF, von Willebrand factor (vWF), cell free deoxyribonucleic acid (cf-DNA), procoagulant phospholipids (PPLs), and total submicron particles were also evaluated. RESULTS: Significantly higher EV levels, including a several-fold higher median level of TF-bearing EVs were measured in AF patients compared with controls. Median concentrations of TF and vWF were approximately 40% and 30% higher, respectively, in the AF group than in the control group, while no significant differences in levels of cf-DNA, PPLs, or total submicron particles were observed. No significant differences in levels of any of the measured analytes were observed between intraoperative venous and LAA samples. CONCLUSIONS: Increased plasma concentrations of TF in AF patients are accompanied and probably at least partly explained by increased levels of TF-bearing EVs, which may be mechanistically involved in increased thrombogenicity in AF patients.

Postprandial Increase in Blood Plasma Levels of Tissue Factor-Bearing (and Other) Microvesicles Measured by Flow Cytometry: Fact or Artifact?

Tissue factor (TF)-bearing microvesicles (MVs) and exosomes may play a role in hemostasis and thrombosis. MVs may be quantified by flow cytometry (FC)-based detection of phosphatidylserine (PS)-positive submicron particles carrying specific antigens, although interference from lipoproteins complicates this approach. In this study, we evaluated the effect of food intake on blood levels of TF-bearing particles measured by FC and small extracellular vesicles (EVs) measured by a protein microarray-based test termed EV Array. Platelet-free plasma (PFP) was obtained from 20 healthy persons in the fasting state and 75 minutes after consumption of a meal. Postprandial changes in the concentration of PS-positive particles, including subgroups binding labeled antibodies against TF, CD41, CD146, and CD62E, respectively (FC), small EVs (EV Array), and TF antigen and procoagulant phospholipids (PPLs) were measured. Furthermore, we tested the effect on FC results of in vitro addition of lipoproteins to fasting PFP. We found significantly increased plasma concentrations of PS-positive particles and all examined subgroups postprandially, while no changes in small EVs, PPL, or TF antigen levels were found. Levels of all types of particles measured by FC were also elevated by lipoprotein spiking. In conclusion, meal consumption as well as in vitro addition of lipoproteins to fasting plasma induces increased levels of PS-positive particles as measured by FC, including TF-positive subtypes and subtypes exposing other antigens. While the observed postprandial increase may to some extent reflect elevated MV levels, our results indicate a substantial interference from lipoproteins.

Prospects and limitations of antibody-mediated clearing of lipoproteins from blood plasma prior to nanoparticle tracking analysis of extracellular vesicles.

Introduction: Nanoparticle tracking analysis (NTA) enables measurement of extracellular vesicles (EVs) but lacks the ability to distinct between EVs and lipoproteins which are abundantly present in blood plasma. Limitations in ultracentrifugation and size exclusion chromatography applied for EV isolation may result in inadequate EV purification and preservation. In this proof of concept study, we aimed to evaluate the potential of antibody-mediated removal of lipoproteins from plasma prior to extracellular vesicle (EV) analysis by nanoparticle tracking analysis (NTA). Methods: Ten platelet-free plasma (PFP) samples from healthy fasting subjects were incubated with magnetic beads coated with antibodies against apolipoprotein B-48 and B-100 (ApoB). Plasma samples were analysed with NTA before and after application of the bead procedure. Four fasting PFP samples were analysed with an ELISA specific for human ApoB to estimate the degree of removal of lipoproteins and EV array analysis was used for identification of possible EV loss. Results: The magnetic bead separation procedure resulted in a median reduction of the particle concentration in plasma by 62% (interquartile range 32-72%). The mean size of the remaining particles generally increased. ApoB concentration was reduced to a level close to the background signal, whereas a median reduction of the EV content by 21% (range 8-43%) was observed. Conclusion: Anti-ApoB antibody coated magnetic beads may hold potential for removal of lipoproteins from human PFP prior to EV measurement by NTA but some artefactual effect and EV loss may have to be endured.

Age-Related Changes in Plasma Extracellular Vesicle Characteristics and Internalization by Leukocytes.

Cells release lipid-bound extracellular vesicles (EVs; exosomes, microvesicles and apoptotic bodies) containing proteins, lipids and RNAs into the circulation. Vesicles mediate intercellular communication between both neighboring and distant cells. There is substantial interest in using EVs as biomarkers for age-related diseases including cancer, and neurodegenerative, metabolic and cardiovascular diseases. The majority of research focuses on identifying differences in EVs when comparing disease states and matched controls. Here, we analyzed circulating plasma EVs in a cross-sectional and longitudinal study in order to address age-related changes in community-dwelling individuals. We found that EV concentration decreases with advancing age. Furthermore, EVs from older individuals were more readily internalized by B cells and increased MHC-II expression on monocytes compared with EVs from younger individuals, indicating that the decreased concentration of EVs with age may be due in part to increased internalization. EVs activated both monocytes and B cells, and activation of B cells by LPS enhanced EV internalization. We also report a relative stability of EV concentration and protein amount in individual subjects over time. Our data provide important information towards establishing a profile of EVs with human age, which will further aid in the development of EV-based diagnostics for aging and age-related diseases.

The impact of various preanalytical treatments on the phenotype of small extracellular vesicles in blood analyzed by protein microarray.

The research field of extracellular vesicles (EVs) is increasing immensely and the potential uses of EVs seem endless. They are found in large numbers in various body fluids, and blood samples may well serve as liquid biopsies. However, these small membrane-derived entities of cellular origin are not straightforward to work with in regard to isolation and characterization. A broad range of relevant preanalytical issues was tested, with a focus on the phenotypic impact of smaller EVs. The influences of the i) blood collection tube used, ii) incubation time before the initial centrifugation, iii) transportation/physical stress, iv) storage temperature and time (short term and long term), v) choice of centrifugation protocol, vi) freeze-thaw cycles, and vii) exosome isolation procedure (ExoQuick™) were examined. To identify the impact of the preanalytical treatments, the relative amounts (detected signal intensities of CD9-, CD63- and/or CD81-positive) and phenotypes of small EVs were analyzed using the multiplexed antibody-based microarray technology, termed the EV Array. The analysis encompassed 15 surface- or surface-related markers, including CD9, CD63, CD81, CD142, and Annexin V. This study revealed that samples collected in different blood collection tubes suffered to varying degrees from the preanalytical treatments tested here. There is no unequivocal answer to the questions asked. However, in general, the period of time and prospective transportation before the initial centrifugation, choice of centrifugation protocol, and storage temperature were observed to have major impacts on the samples. On the contrary, long-term storage and freeze-thawing seemed to not have a critical influence. Hence, there are pros and cons of any choice regarding sample collection and preparation and may very well be analysis dependent. However, to compare samples and results, it is important to ensure that all samples are of the same type and have been handled similarly.

Exosomal proteins as potential diagnostic markers in advanced non-small cell lung carcinoma.

BACKGROUND: Lung cancer is one of the leading causes of cancer-related death. At the time of diagnosis, more than half of the patients will have disseminated disease and, yet, diagnosing can be challenging. New methods are desired to improve the diagnostic work-up. Exosomes are cell-derived vesicles displaying various proteins on their membrane surfaces. In addition, they are readily available in blood samples where they constitute potential biomarkers of human diseases, such as cancer. Here, we examine the potential of distinguishing non-small cell lung carcinoma (NSCLC) patients from control subjects based on the differential display of exosomal protein markers. METHODS: Plasma was isolated from 109 NSCLC patients with advanced stage (IIIa-IV) disease and 110 matched control subjects initially suspected of having cancer, but diagnosed to be cancer free. The Extracellular Vesicle Array (EV Array) was used to phenotype exosomes directly from the plasma samples. The array contained 37 antibodies targeting lung cancer-related proteins and was used to capture exosomes, which were visualised with a cocktail of biotin-conjugated CD9, CD63 and CD81 antibodies. RESULTS: The EV Array analysis was capable of detecting and phenotyping exosomes in all samples from only 10 µL of unpurified plasma. Multivariate analysis using the Random Forests method produced a combined 30-marker model separating the two patient groups with an area under the curve of 0.83, CI: 0.77-0.90. The 30-marker model has a sensitivity of 0.75 and a specificity of 0.76, and it classifies patients with 75.3% accuracy. CONCLUSION: The EV Array technique is a simple, minimal-invasive tool with potential to identify lung cancer patients.