Novel, non-invasive markers for detecting therapy induced neuroendocrine differentiation in castration-resistant prostate cancer patients.

Neuroendocrine prostate cancer (NEPC), a highly aggressive variant of castration-resistant prostate cancer (CRPC), often emerges upon treatment with androgen pathway inhibitors, via neuroendocrine differentiation. Currently, NEPC diagnosis is challenging as available markers are not sufficiently specific. Our objective was to identify novel, extracellular vesicles (EV)-based biomarkers for diagnosing NEPC. Towards this, we performed small RNA next generation sequencing in serum EVs isolated from a cohort of CRPC patients with adenocarcinoma characteristics (CRPC-Adeno) vs CRPC-NE and identified significant dysregulation of 182 known and 4 novel miRNAs. We employed machine learning algorithms to develop an 'EV-miRNA classifier' that could robustly stratify 'CRPC-NE' from 'CRPC-Adeno'. Examination of protein repertoire of exosomes from NEPC cellular models by mass spectrometry identified thrombospondin 1 (TSP1) as a specific biomarker. In view of our results, we propose that a miRNA panel and TSP1 can be used as novel, non-invasive tools to identify NEPC and guide treatment decisions. In conclusion, our study identifies for the first time, novel non-invasive exosomal/extracellular vesicle based biomarkers for detecting neuroendocrine differentiation in advanced castration resistant prostate cancer patients with important translational implications in clinical management of these patients that is currently extremely challenging.

A three-platelet mRNA set: MAX, MTURN and HLA-B as biomarker for lung cancer.

BACKGROUND: During the development of tumors, tumors "educate" platelets causing changes in their mRNAs expression profiles and phenotypes, thereby, tumor-educated platelet (TEP) mRNA profile has the potential to diagnose lung cancer. The current study aimed to examine whether TEPs might be a potential biomarker for lung cancer diagnostics. METHODS: Platelet precipitation was obtained by low-speed centrifugation and subjected to Trizol for total RNA extraction. Platelet MAX, MTURN, and HLA-B mRNA were selected by microarray, validated by qPCR, and analyzed combined with related clinical factors. RESULTS: Our results showed that a three-platelet mRNA set: MAX, MTURN, and HLA-B was significantly up-regulated in lung cancer patients as well as in early-stage lung cancer patients compared with those from healthy donors, the area under the curve (AUC) was 0.734, 0.787, respectively, among which platelet MTURN mRNA processed a dramatically high diagnostic efficiency in female patients with lung cancer, its AUC for female was 0.825. More importantly, the three-platelet mRNA set: MAX, MTURN, and HLA-B was associated with chemotherapeutic effect, low mRNA expression of this three-platelet set was correlated with "favorable" first chemotherapy response. CONCLUSIONS: A three-platelet mRNA set: MAX, MTURN and HLA-B enables blood-based lung cancer diagnosis and chemotherapy response prediction.

Thrombospondin-I concentrations behavior in plasma of patients with ovarian cancer.

PURPOSE: To determine whether thrombospondin-1 might be used as a prognostic factor in ovarian cancer patients. METHOD: Ninety-six female subjects hospitalized in years 2011-2014 was included in the study. Transvaginal ultrasound examination was performed in all patients. In 39 cases of suspected ovarian cancer, CT scans were also performed. Each patient had been subjected to collection of a 5-mL blood sample before the laparoscopic procedure. Thrombospondin-1 concentrations were quantified in serum by multiplex fluorescent bead-based immunoassays (Luminex) at the Laboratory of the Department of General Pathology. RESULTS: Statistical analysis performed using the Kaplan-Meier survival curves and log-rank test revealed no statistically significant correlations between the median, 75th percentile and 95th percentile thrombospondin-1 levels with progression-free survival of patients (p= 0.47). In the univariate OS model, median thrombospondin-1 level was a significant variable. Correlation was demonstrated between baseline thrombospondin-1 levels and overall survival of patients (p= 0.04, HR = 0.99). The higher the baseline TSP1 level, the longer the overall survival of patients. CONCLUSION: In our study, we were the first to demonstrate correlation between the levels of thrombospondin-1 and overall survival of patients. Therefore, it appears that thrombospondin-1 may be used as a prognostic factor in ovarian cancer patients.

Genetic and Environmental Biomarkers Associated with Triglyceride Levels in Two Groups of Slovak Women.

OBJECTIVE: This study analyzed the association between the MLXIPL gene polymorphism (rs3812316) and triglyceride (TG) levels and selected environmental biomarkers in Slovak women at risk for cardiovascular disease compared to a reference sample. MATERIALS AND METHODS: The studied sample consisted of 200 women at cardiovascular risk (mean age 52.96 ± 6.01 years) and 244 healthy women (mean age 47.52 ± 5.34 years). Participants gave details of their health and lifestyle during their medical examination, and peripheral blood samples were used for biochemical analyses and DNA genotyping. A nested polymerase chain reaction-restriction fragment length polymorphism assay was used to detect the rs 3812316 SNP. RESULTS: We determined that there were significantly different genotype distributions in two TG categories: (1) subjects with normal TG values had a significantly higher G allele frequency than those with elevated TG levels (χ2 = 6.1556, df = 2, p = 0.046); and (2) the rare G allele frequency was 0.11 in the cardiovascular risk group and 0.15 in the reference group. Binary regression analysis showed that women with at least one G allele had a significantly lower relative risk of hypertriglyceridemia than women with the CC genotype (OR = 0.399, p = 0.022, 95% CI = 0.182-0.876). CONCLUSION: This cross-sectional study suggests that MLXIPL rs3812316 genotypes may be associated with TG levels. However, further analysis is advisable because of study limitations.

Platelet-specific markers are associated with monocyte-platelet aggregate formation and thrombin generation potential in advanced atherosclerosis.

Platelet activation and thrombin generation are crucial steps in primary and secondary haemostasis. However, both also play major roles in intravascular thrombus formation and therefore in the development of adverse cardiovascular events. In the current study, we first sought to investigate the associations of the platelet biomarkers platelet factor (PF)-4, thrombospondin (TSP)-1, soluble CD40 ligand (sCD40L), and soluble P-selectin (sP-selectin) with each other and with monocyte-platelet aggregate (MPA) formation in 316 patients undergoing angioplasty and stenting. To better understand the interplay between platelet activation and thrombin generation, we subsequently investigated the associations of the platelet biomarkers with thrombin generation potential. The mostly platelet-specific markers PF-4, TSP-1 and sCD40L correlated strongly with each other (all p < 0.001), and the best correlation was observed between PF-4 and TSP-1 (r=0.91). In contrast, sP-selectin, which derives from platelets and endothelial cells, correlated rather poorly with TSP-1 (r=0.12, p=0.04), and did not correlate with PF-4 and sCD40L. While PF-4, TSP-1 and sP-selectin correlated significantly with in vivo MPA formation (all p< 0.001), no such association was found between sCD40L and MPA formation. PF-4, TSP-1 and sCD40L correlated strongly with peak thrombin generation (all p< 0.001) with the best correlation between PF-4 and peak thrombin generation (r=0.55), whereas sP-selectin did not correlate with peak thrombin generation. Likewise, PF-4, TSP-1 and sCD40L correlated significantly with the area under the thrombin generation curve (AUC; all p< 0.01), whereas sP-selectin did not correlate with the AUC. In conclusion, platelet-specific markers are associated with MPA formation and thrombin generation potential in patients with advanced atherosclerosis.

Altered angiogenesis in low birth weight individuals: a role for anti-angiogenic circulating factors.

OBJECTIVE: Low birth weight (LBW) is a risk factor for hypertension at adulthood. Endothelial progenitor cells (EPCs) dysfunction has been characterized in LBW neonates. We hypothesized that changes in soluble, plasma pro- or anti-angiogenic factors are associated with EPCs dysfunction and impaired angiogenesis in LBW neonates. METHOD: Venous umbilical cord blood was collected from 42 normal, term neonates and 75 LBW neonates. Cord blood endothelial colony forming cells (ECFC) from control patients were cultured in the presence of 10% of serum obtained from both groups. RESULTS: The proliferation and the migration of ECFC were significantly reduced when cultured with 10% of serum of LBW neonates compared to serum of control neonates. Matrigel invasion assay was not significantly altered. Umbilical vein plasma VEGF concentration was significantly reduced in LBW neonates while that of sVEGFR and PF4 were significantly higher. Addition of VEGF corrected the inhibitory effect of LBW serum on normal ECFC proliferation. CONCLUSIONS: Serum obtained from LBW babies contains factors that exhibit an antiangiogenic effect on ECFC proliferation and migration. VEGF/sVEGF/PF4 pathway seems to be involved in the EPCs dysfunction in LBW neonates.

VEGF and TSP1 levels correlate with prognosis in advanced non-small cell lung cancer.

PURPOSE: There is a need for biomarkers that may help in selecting the most effective anticancer treatments for each patient. We have investigated the prognostic value of a set of angiogenesis, inflammation and coagulation markers in patients treated for advanced non-small cell lung cancer. PATIENTS AND METHODS: Peripheral blood samples were obtained from 60 patients before first line platinum-based chemotherapy ± bevacizumab, and after the third cycle of treatment. Blood samples from 60 healthy volunteers were also obtained as controls. Angiogenesis, inflammation and coagulation markers vascular endothelial growth factor (VEGF), their soluble receptors 1 (VEGFR1) and 2 (VEGFR2), thrombospondin-1 (TSP-1), interleukin-6 (IL6), sialic acid (SA) and tissue factor (TF) were quantified by ELISA. RESULTS: Except for TSP-1, pre- and post-treatment levels of all markers were higher in patients than in controls (p < 0.05). There was a positive and significant correlation between VEGF and VEGFR2 before treatment. VEGF also correlated with inflammatory markers IL-6 and SA. Moreover, there was a positive and significant correlation between levels of VEGFR1 and TF. Decreased levels of TSP-1 and increased levels of VEGF were associated with shorter survival. Bevacizumab significantly modified angiogenesis parameters and caused a decrease of VEGF and an increase of TSP-1. CONCLUSION: Angiogenesis, inflammation and coagulation markers were increased in NSCLC patients. Increased levels of VEGF and low levels of TSP-1 correlated with a poor prognosis.

Somatic mosaicism in a mother of two children with Pitt-Hopkins syndrome.

Pitt-Hopkins syndrome (PTHS) is a neurodevelopmental disorder characterized by intellectual disability, unusual face and breathing abnormalities and can be caused by haploinsufficiency of TCF4. The majority of cases are sporadic. Somatic mosaicism was reported infrequently. We report on a proband with typical manifestations of PTHS and his younger brother with a less striking phenotype. In both, a heterozygous frameshift mutation (c.1901_1909delinsA, p.Ala634AspfsX67) was found in exon 19 of TCF4. The same mutation was found at low levels in DNA extracted from the mother's blood, urine and saliva. This report of familial recurrence with somatic mosaicism in a healthy mother has important consequences for genetic counseling. We suggest careful studies in parents of other patients with PTHS to determine the frequency of germline and somatic mosaicism for TCF4 mutations.

Decreased TSP-1 following percutaneous coronary intervention is associated with major adverse cardiac events in ST-elevation myocardial infarction.

BACKGROUND: TSP-1 is a vasoconstrictive protein, which is released from both endothelium and cardiomyocytes during ischemia and promotes platelet aggregation and adhesion to subendothelial layers in atherosclerotic lesions. During myocardial ischemia and reperfusion, TSP-1 disturbs local microcirculation by disrupting both NO-signaling as well as VEGF-pathways by activation of CD47 and CD36. Furthermore, activation of TGF-ß might induce excessive fibrosis after infarction. It was assumed that TSP-1 is washed out after successful coronary reperfusion. In this study, we examined circulating TSP-1 post emergency PCI as a risk factor for major adverse cardiac events after STEMI with and without ventricular fibrillation. METHODS: TSP-1 levels in platelet poor plasma were measured in 54 patients after ST-elevation myocardial infarction. Major adverse cardiac events were monitored for 426 days. RESULTS: Patients with decreased TSP levels after coronary stenting showed a significantly higher risk for MACE than patient with higher TSP levels (TSP-1[d0]: n = 46, no MACE = 16.38 ± 1.98 ug/mL vs. MACE 7.11 ± 1.54 ug/mL; p = 0.003). Kaplan-Meyer-analysis for MACE showed a better outcome above 10 ug/mL (p = 0.02). For MACE later than 3 months post-STEMI, the corresponding Kaplan-Meier-analysis yielded a p-value of 0.01. The number needed to diagnose for late MACE was 2.158. CONCLUSION: Low plasma levels of TSP1 after PCI are associated with MACE. Due to its procoagulant effects and dysregulation of microvascular tone, adequately powered prospective studies are warranted to test the impact of TSP-1 on cardiac microcirculation, endothelial function and remodeling. TSP-1 might serve as a new diagnostic and therapeutic approach in cardiovascular disease.

Platelet-stored angiogenesis factors: clinical monitoring is prone to artifacts.

BACKGROUND: The analysis of angiogenesis factors in the blood of tumor patients has given diverse results on their prognostic or predictive value. Since mediators of angiogenesis are stored in platelets, their measurement in plasma is sensitive to inadvertent platelet activation during blood processing. METHODS: Variants of blood withdrawal and plasma preparation were evaluated by ELISA for the detection of TSP-1, PF-4, VEGF and PD-ECGF. A total of 22 pancreatic cancer patients and 29 healthy volunteers were evaluated. RESULTS: Plasma preparation with the anticoagulant mix of citrate, theophylline, adenosine, dipyridamole (CTAD) and immediate blood processing at 4°C was required for reproducible measurements of TSP-1, PF-4 and VEGF. Blood collection by venflon or inadvertent hemolysis during blood withdrawal caused significantly elevated TSP-1 and PF-4 values. When optimized plasma preparation was applied, a significant increase of TSP-1 and VEGF in cancer patients was detected (P=0.006; P< 0.001). CONCLUSION: The reliable plasma analysis of circulating platelet-stored angiogenesis factors requires preparation with CTAD at 4°C and blood collection by butterfly needle. Suboptimal procedures of plasma preparation are commonly applied in clinical monitoring of angiogenesis parameters which may account for the differences in reported plasma values and may have masked their predictive or prognostic marker potential.

[A clinical study of congenital thrombotic thrombocytopenic purpura].

OBJECTIVE: To study on the first Chinese congenital thrombotic thrombocytopenic purpura (TTP) patient with respect to peripheral blood platelet count and ADAMTS13 activity, ADAMTS13 antigen, anti-endothelial cell antibody (ACEA), and thrombospondin1 (TSP1) etc. to compared it with idiopathic TTP and to explore something special and meaningful in the pathogenesis of congenital TTP. METHODS: A total of 30 volunteers served as controls. The congenital TTP patient and 10 carriers in her pedigree as well as 7 idiopathic TTP patients were included in this study. Residue collagen binding assay and a newly developed sandwich ELISA were used for determination of ADAMTS13 activity and antigen respectively. An ELISA for ACEA and a commercial kit for TSP1 were use in the study. RESULTS: After a "3-4 weeks regular relapse" of the congenital TTP, which is similar to that in other reports, the Chinese patient moved into a new relapse cycle. The average of the antigen levels of this patient before plasma exchange (PE) and at the interval between relapses was (22.79 +/- 14.61) U/L (P < 0.01), while that of Chinese normal control (NC) turned out to be (600.93 +/- 145.36) U/L. TSP1 detected at the same time (4.67 +/- 1.62) mg/L in the congenital TTP patient was much lower than that in NC (18.34 +/- 7.24) mg/L (P < 0.01). A value in ACEA assay was 0.58 +/- 0.06 (P < 0.01) for congenital TTP, which was a little higher than that in NC (0.40 +/- 0.13). CONCLUSIONS: Congenital TTP may have changeable relapse cycles during episodes. ADAMTS13 antigen and TSP1 and ACEA in congenital TTP showed significant difference as compared with those in NC. ADAMTS13 and ACEA in congenital TTP were also markedly lower than those in idiopathic TTP.

[A study on the significance of plasma thrombospondin1 in thrombotic thrombocytopenic purpura and the relationship between thrombospondin1 and von Willebrand factor cleaving protease (ADAMTS13)].

OBJECTIVE: To measure plasma thrombospondin1 (TSP1) in thrombotic thrombocytopenic purpura (TTP) and other diseases such as idiopathic thrombocytopenic purpura (ITP), systemic lupus erythematosus (SLE), myocardial infarction, brain infarction, and malignant tumor et al. and 8 patients after bone marrow transplantation (BMT) were also investigated. Then to study on the relationship between TSP1 and von Willebrand factor cleaving protease (ADAMTS13); and to identify the significance of plasma TSP1 in TTP. METHODS: TSP1 was measured by a commercial kit and the activity of ADAMTS13 was evaluated by residue collagen binding assay. RESULTS: TSP1 in TTP plasma before plasma exchange or plasma infusion was 6.49 mg/L, and stepping up to 13.02 mg/L after therapy, but still significantly lower than 18.34 mg/L in normal control. While the decrease in different degree of ADAMTS13 activity was observed from 0%-52%, and there were 8 samples whose activity of ADAMTS13 were no more than 10%; the different extent of increase in those patients after therapy was demonstrated to be 2.9%-93.4%, only one patient's ADAMTS13 activity was below 10%. The activity of ADAMTS13 in some ITP and SLE patients were mildly decreased (63% +/- 16% and 70% +/- 14% respectively), TSP1 were also decreased (16 mg/L +/- 8 mg/L). TSP1 in patients of myocardial infarction and brain infarction were increased (24.0 mg/L +/- 2.9 mg/L), while ADAMTS13 activity had no significant change (72% +/- 16%). Same things happened in BMT patients. CONCLUSION: There was some concordance between the decrease of ADAMTS13 activity and TSP1 in plasma of TTP patients. And the change of TSP1 was restricted to TTP. TSP1 may contribute to the episode of TTP in a still unclear fashions.