Chromogranin A in a Cohort of Pheochromocytomas and Paragangliomas: Usefulness at Diagnosis and as an Early Biomarker of Recurrence.

OBJECTIVE: To evaluate the usefulness of chromogranin A (CgA) in the management of patients with pheochromocytomas (PHEOs) and paragangliomas (PGLs). METHODS: We retrospectively reviewed the charts of 132 patients with confirmed PHEOs/PGLs (PPGLs) followed at our medical center. CgA was measured in 80 patients at diagnosis. The exclusion criteria removed 19 of these patients. Five patients with relapses were also analyzed. RESULTS: Our cohort of 61 patients included 34 PHEOs, 14 head and neck PGLs, and 13 thoracoabdominal PGLs. CgA levels were elevated in 53 of 61 patients (86.9%) at diagnosis: 33 of 34 (97.1%) PHEOs, 9 of 14 (64.3%) head and neck paragangliomas, and 11 of 13 (84.6%) thoracoabdominal paragangliomas. For 8 of 13 (61.5%) nonfunctional PPGLs (5 head and neck paragangliomas and 3 thoracoabdominal paragangliomas), increased CgA levels showed potential as a tumor marker during follow-up. Of 10 patients with malignant PPGLs, only 1 had normal CgA levels (10.0%). Among 54 patients with PPGLs who underwent genetic testing, elevated CgA levels were positive in 73.7% of patients carrying a germline genetic variant (pathogenic and of unknown significance) versus 91.4% of patients without a known germline variant. We also report 5 PPGL cases with increased CgA levels as the first detectable marker of tumoral recurrence or progression preceding other biochemical markers or imaging. CONCLUSION: CgA is a sensitive marker for the diagnosis of PHEO (97.1%) and thoracoabdominal paraganglioma (84.6%). CgA may be useful in the follow-up of nonfunctional PGLs and may also play a complementary role in the early detection of recurrence in secreting PPGLs.

The PRONTO study: Clinical performance of ID NOW in individuals with compatible SARS-CoV-2 symptoms in walk-in centres-accelerated turnaround time for contact tracing.

Background: This PRONTO study investigated the clinical performance of the Abbott ID NOWTM (IDN) COVID-19 diagnostic assay used at point of care and its impact on turnaround time for divulgation of test results. Methods: Prospective study conducted from December 2020 to February 2021 in acute symptomatic participants presenting in three walk-in centres in the province of Québec. Results: Valid paired samples were obtained from 2,372 participants. A positive result on either the IDN or the standard-of-care nucleic acid amplification test (SOC-NAAT) was obtained in 423 participants (prevalence of 17.8%). Overall sensitivity of IDN and SOC-NAAT were 96.4% (95% CI: 94.2-98.0%) and 99.1% (95% CI: 97.6-99.8), respectively; negative predictive values were 99.2% (95% CI: 98.7-99.6%) and 99.8% (95% CI: 99.5-100%), respectively. Turnaround time for positive results was significantly faster on IDN. Conclusion: In our experience, IDN use in symptomatic individuals in walk-in centres is a reliable sensitive alternative to SOC-NAAT without the need for subsequent confirmation of negative results. Such deployment can accelerate contact tracing, reduce the burden on laboratories and increase access to testing.

Quality assurance practices for point of care testing programs: Recommendations by the Canadian society of clinical chemists point of care testing interest group.

Point of Care Testing (POCT) refers to clinical laboratory testing performed outside the central laboratory, nearer to the patient and sometimes at the patient bedside. The testing is usually performed by clinical staff, such as physicians or nurses, who are not laboratory trained. This document was developed by the POCT Interest group of the Canadian Society of Clinical Chemists (CSCC) as practical guidance for quality assurance practices related to POCT performed in hospital and outside hospital environments. The aspects of quality assurance addressed in this document include: (1) device selection, (2) initial device verification, (3) ongoing device verification, (4) ongoing quality assurance including reagent and quality control (QC) lot changes, and (5) quality management including operator and document management.

Correlation of cord blood pH, base excess, and lactate concentration measured with a portable device for identifying fetal acidosis.

OBJECTIVE: To determine the effectiveness of portable lactate analyzers in identifying fetal acidosis by correlating arterial and venous lactate values from umbilical cord blood with lactate, pH, and base excess measurements from central laboratory analyzers. METHODS: We performed a prospective study using arterial and venous cord blood from 52 women with a singleton fetus delivered at term. We evaluated the correlation between the cord blood lactate concentration measured using two of the same portable devices (Lactate Plus, Nova Biomedical) with the result from a central laboratory analyzer. Analyses of the correlation between arterial lactate concentration measured on the portable device with arterial pH and base excess were then performed. RESULTS: We observed a median arterial pH of 7.24 (range 7.05 to 7.35) and a median arterial lactate concentration of 3.7 mmol/L (range 1.7 to 8.8 mmol/L). An excellent correlation was observed between lactate concentrations measured by the two portable devices (arterial R² = 0.98 and venous R² = 0.98), and between the portable device and the central laboratory analyzer (arterial R² = 0.94 and venous R² = 0.95). In our population, the optimal cut-offs to predict a pH < 7.20 or a base excess > -8.0 mmol/L were a lactate concentration of 4.9 mmol/L and 5.3 mmol/L, respectively, according to receiver operator characteristic analysis. With a lactate concentration > 4.9 mmol/L, the portable device had a sensitivity of 82% and a specificity of 90% to identify samples with an arterial pH < 7.20. CONCLUSION: Cord blood lactate concentration measured with a portable device is a good predictor of cord blood base excess and pH. Future studies should be designed to correlate scalp blood lactate measurements with clinical outcomes.

Objectif : Déterminer l'efficacité des analyseurs de lactate portatifs, pour ce qui est de l'identification de l'acidose fœtale, en mettant en corrélation les valeurs artérielle et veineuse du lactate constatées dans le sang de cordon ombilical et les mesures du lactate, du pH et de l'excès de bases révélées par les analyseurs du laboratoire central. Méthodes : Nous avons mené une étude prospective en utilisant le sang de cordon artériel et veineux prélevé chez 52 femmes qui ont connu une grossesse monofœtale s'étant soldée en un accouchement à terme. Nous avons évalué la corrélation entre la concentration en lactate du sang de cordon mesurée au moyen de deux exemplaires du même appareil portatif (Lactate Plus, Nova Biomedical) et le résultat obtenu au moyen d'un analyseur du laboratoire central. Nous avons par la suite procédé à des analyses de la corrélation entre la concentration artérielle en lactate mesurée au moyen de l'appareil portatif et les valeurs artérielles du pH et de l'excès de bases. Résultats : Nous avons constaté un pH artériel médian de 7,24 (plage : 7,05 - 7,35) et une concentration artérielle en lactate médiane de 3,7 mmol/l (plage : 1,7 - 8,8 mmol/l). Une excellente corrélation a été constatée entre les concentrations en lactate mesurées par les deux appareils portatifs (R2 artériel = 0,98 et R2 veineux = 0,98) et entre les concentrations mesurées par l'appareil portatif et par l'analyseur du laboratoire central (R2 artériel = 0,94 et R2 veineux = 0,95). Au sein de notre population, les seuils optimaux permettant de prédire un pH < 7,20 ou un excès de bases > −8,0 mmol/l ont été des concentrations en lactate de 4,9 mmol/l et de 5,3 mmol/l, respectivement, selon l'analyse de la fonction d'efficacité du récepteur. En présence d'une concentration en lactate > 4,9 mmol/l, l'appareil portatif comptait une sensibilité de 82 % et une spécificité de 90 % pour ce qui est de l'identification des prélèvements présentant un pH artériel < 7,20. Conclusion : La concentration en lactate du sang de cordon qui est mesurée au moyen d'un appareil portatif constitue un bon facteur prédictif pour ce qui est du pH et de l'excès de bases du sang de cordon. De futures études devraient être conçues de façon à pouvoir mettre en corrélation les concentrations en lactate dans le sang prélevé sur le cuir chevelu et les résultats cliniques.

Tissue factor mediates the HGF/Met-induced anti-apoptotic pathway in DAOY medulloblastoma cells.

The classical treatment scheme for medulloblastoma (MB) is based on a tri-therapy approach consisting of surgical tumor resection, craniospinal axis radiation and chemotherapy. With current treatments relying mainly on non-specific cytotoxic therapy, a better understanding of the mechanisms underlying resistance to these treatments is important in order to improve their effectiveness. In this study, we report that stimulation of DAOY with HGF resulted in the protection of these cells against etoposide-induced apoptosis, this anti-apoptotic effect being correlated with an increase in the expression of tissue factor (TF), the initiator of the extrinsic pathway of coagulation. HGF-mediated protection from apoptosis was abolished by a c-Met inhibitor as well as by siRNA-mediated reduction of TF levels, implying a central role of Met-dependent induction of TF expression in this process. Accordingly, stimulation of DAOY with FVIIa, the physiological ligand of TF, also resulted in a significant protection from etoposide-mediated cytotoxicity. Overall, our results suggest the participation of the haemostatic system to drug resistance in MB and may thus provide novel therapeutic approaches for the treatment of these tumors.

c-Met activation in medulloblastoma induces tissue factor expression and activity: effects on cell migration.

Met, the receptor for hepatocyte growth factor (HGF), is a receptor tyrosine kinase that has recently emerged as an important contributor to human neoplasia. In physiological and pathological conditions, Met triggers various cellular functions related to cell proliferation, cell migration and the inhibition of apoptosis, and also regulates a genetic program leading to coagulation. Since medulloblastomas (MBs) express high levels of tissue factor (TF), the main initiator of blood coagulation, we therefore examined the link between Met and TF expression in these pediatric tumors. We observed that stimulation of the MB cell line DAOY with HGF led to a marked increase of TF expression and procoagulant activity, in agreement with analysis of clinical MB tumor specimens, in which tumors expressing high levels of Met also showed high levels of TF. The HGF-dependent increase in TF expression and activity required Src family kinases and led to the translocation of TF to actin-rich structures at the cell periphery, suggesting a role of the protein in cell migration. Accordingly, addition of physiological concentrations of the TF activator factor VIIa (FVII) to HGF-stimulated DAOY cells promoted a marked increase in the migratory potential of these cells. Overall, these results suggest that HGF-induced activation of the Met receptor results in TF expression by MB cells and that this event probably contribute to tumor proliferation by enabling the formation of a provisional fibrin matrix. In addition, TF-mediated non-hemostatic functions, such as migration toward FVIIa, may also play a central role in MB aggressiveness.

The flavonols quercetin, kaempferol, and myricetin inhibit hepatocyte growth factor-induced medulloblastoma cell migration.

Medulloblastoma, the most common malignant brain tumor in children, is a highly metastatic disease, with up to 30% of children having evidence of disseminated disease at presentation. Recently, the hepatocyte growth factor (HGF) and its receptor, the tyrosine kinase Met, have emerged as key components of human medulloblastoma growth and metastasis, suggesting that inhibition of this pathway may represent an attractive target for the prevention and treatment of this disease. Using immunoblotting procedures, we observed that the dietary-derived flavonols quercetin, kaempferol, and myricetin inhibited HGF/Met signaling in a medulloblastoma cell line (DAOY), preventing the formation of actin-rich membrane ruffles and resulting in the inhibition of Met-induced cell migration in Boyden chambers. Furthermore, quercetin and kaempferol also strongly diminished HGF-mediated Akt activation. Interestingly, the inhibitory effects of quercetin on the tyrosine kinase receptor Met [half-maximal inhibitory effect (IC(50)) of 12 micromol/L] or on the Met-induced activation of Akt (IC(50) of 2.5 micromol/L) occurred at concentrations achievable through dietary approaches. These results highlight quercetin, kaempferol, and myricetin as dietary-derived inhibitors of Met activity and suggest that this inhibitory effect may contribute to the chemopreventive properties of these molecules.

Tissue factor pathway inhibitor (TFPI) interferes with endothelial cell migration by inhibition of both the Erk pathway and focal adhesion proteins.

Tissue factor pathway inhibitor (TFPI) is a plasma Kunitz-type serine protease inhibitor that is mainly known for its inhibition of tissue factor-mediated coagulation. In addition to its anticoagulant properties, emerging data show that TFPI may also regulate endothelial cell functions via a non-haemostatic pathway. In this work we demonstrate that at concentrations within the physiological range, TFPI inhibits both endothelial cell migration and their differentiation into capillary-like structures in vitro. These effects were specific to endothelial cells since no inhibitory effect was observed on the migration of tumor (glioblastoma) cells. Inhibition of endothelial cell migration was correlated with a concomitant loss in cell adhesion, suggesting an alteration of focal adhesion complex integrity. Accordingly, we observed that TFPI inhibited the phosphorylation of focal adhesion kinase and paxillin, two key proteins involved in the scaffolding of these complexes, and that this effect was specific to endothelial cells. These results suggest that TFPI influences the angiogenic process via a non-haemostatic pathway, by downregulating the migratory mechanisms of endothelial cells.

Direct-acting fibrinolytic enzymes in shark cartilage extract: potential therapeutic role in vascular disorders.

Fibrinogen and fibrin are molecules with overlapping roles in blood clotting, fibrinolysis, wound healing, inflammation, matrix and cellular interactions and neoplasia. There is currently much interest in the possible use of fibrinolytic agents in human therapeutics. In this study, we report the presence of fibrinolytic activities in shark cartilage extract (SCE). In vitro, SCE at 100 microg/ml completely degraded fibrin gel in an aprotinin-insensitive manner, suggesting a non-plasmin molecular nature. SCE was able to cleave all chains of fibrinogen and fibrin and the cleavage was completely inhibited by 1,10-phenanthroline, suggesting an essential role for metalloprotease(s) in this process. Using fibrinogen zymography, we show that SCE contains two plasmin-independent fibrinolytic activities and that these activities are correlated with the presence of 58 and 62 kDa proteases in the extract. SCE-fibrinolytic activities are inhibited by dithiothreitol, suggesting that disulfide bonds are necessary for the protease structure. Finally, using thromboelastography, SCE markedly induced retraction of human platelet-rich plasma (PRP) clot, this process being completely abolished by 1,10-phenanthroline. These data suggest the presence of novel non-plasmin fibrinolytic activities within SCE. This extract may thus represent a potential source of new therapeutic molecules to prevent and treat vaso-occlusive and thromboembolic disorders.

Purification and characterization of a stimulator of plasmin generation from the antiangiogenic agent Neovastat: identification as immunoglobulin kappa light chain.

We have recently shown that Neovastat, an antiangiogenic extract from shark cartilage, stimulates the in vitro activation of plasminogen by facilitating the tissue-type plasminogen activator (tPA)-dependent conversion of plasminogen to plasmin. In this report, we describe the purification and characterization of the stimulatory molecules. Neovastat was subjected to a three-step purification procedure including gel filtration, preparative isoelectric focusing, and preparative SDS-PAGE. Two 28-kDa proteins with pIs of approximately 4.5 and 6.5 were purified to apparent homogeneity and identified as immunoglobulin (Ig) kappa light chains by N-terminal microsequencing. Ig light chains do not directly stimulate the activity of tPA or plasmin, suggesting a mechanism of action involving an interaction with plasminogen. Kinetic analysis showed that both Ig light chains accelerate the in vitro tPA-dependent conversion of plasminogen in plasmin by increasing the affinity of tPA for plasminogen by 32- and 38-fold (Km decrease from 456 nM to 12-14 nM). Shark Ig light chains also stimulated the degradation of fibrin by the tPA/plasminogen system in an in vitro assay. A direct interaction between Ig light chains and plasminogen (KA=4.0-5.5 x 10(7) M(-1); KD=18-25 nM) and with tPA (KA=2.8 x 10(7) M(-1); KD=36 nM) was demonstrated using real time binding measured by surface plasmon resonance. Ig light chain is the first molecule associated with the antiangiogenic activity of Neovastat to be purified and identified.