Disease specific urinary biomarkers in the central nervous system.

Urinary biomarkers can diagnose and monitor pathophysiologic conditions in the central nervous system (CNS). However, focus is often on single diseases, with limited data on discriminatory capability of this approach in a general setting. Here, we demonstrate that different classes of CNS disease exhibit distinct biomarker patterns, evidence of disease-specific "fingerprinting." Urine from 218 patients with pathology-confirmed tumors or cerebrovascular disease, controls (n = 33) were collected. ELISA and/or bead-based multiplexing quantified levels of 21 putative urinary biomarkers. Analysis identified biomarkers capable of distinguishing each disease from controls and other diseases. Mann-Whitney U tests identified biomarkers with differential expression between disease types and controls (P ≤ 0.001). Subsequent receiver-operating characteristic (ROC) analyses revealed distinguishing biomarkers with high sensitivity and specificity. Areas under the curve (AUCs) ranged 0.8563-1.000 (P values ≤ 0.0003), sensitivities ranged 80.00-100.00%, and specificities ranged 80.95-100.00%. These data demonstrate proof-of-principle evidence that disease-specific urinary biomarker signatures exist. In contrast to non-specific responses to ischemia or injury, these results suggest that urinary biomarkers accurately reflect unique biological processes distinct to different diseases. This work can be used to generate disease-specific panels for enhancing diagnosis, assisting less-invasive follow-up and herald utility by revealing putative disease-specific therapeutic targets.

Dysregulation of the EphrinB2-EphB4 ratio in pediatric cerebral arteriovenous malformations is associated with endothelial cell dysfunction in vitro and functions as a novel noninvasive biomarker in patients.

We investigated (1) EphrinB2 and EphB4 receptor expression in cerebral AVMs, (2) the impact of an altered EphrinB2:EphB4 ratio on brain endothelial cell function and (3) potential translational applications of these data. The following parameters were compared between AVM endothelial cells (AVMECs) and human brain microvascular endothelial cells (HBMVECs): quantified EphrinB2 and EphB4 expression, angiogenic potential, and responses to manipulation of the EphrinB2:EphB4 ratio via pharmacologic stimulation/inhibition. To investigate the clinical relevance of these in vitro data, Ephrin expression was assessed in AVM tissue (by immunohistochemistry) and urine (by ELISA) from pediatric patients with AVM (n = 30), other cerebrovascular disease (n = 14) and control patients (n = 29), and the data were subjected to univariate and multivariate statistical analyses. Compared to HBMVECs, AVMECs demonstrated increased invasion (p = 0.04) and migration (p = 0.08), impaired tube formation (p = 0.06) and increased EphrinB2:EphB4 ratios. Altering the EphrinB2:EphB4 ratio (by increasing EphrinB2 or blocking EphB4) in HBMVECs increased invasion (p = 0.03 and p < 0.05, respectively). EphrinB2 expression was increased in AVM tissue, which correlated with increased urinary EphrinB2 levels in AVM patients. Using the optimal urinary cutoff value (EphrinB2 > 25.7 pg/µg), AVMs were detected with high accuracy (80% vs. controls) and were distinguished from other cerebrovascular disease (75% accuracy). Post-treatment urinary EphrinB2 levels normalized in an index patient. In summary, AVMECs have an EphrinB2:EphB4 ratio that is increased compared to that of normal HBMVECs. Changing this ratio in HBMVECs induces AVMEC-like behavior. EphrinB2 is clinically relevant, and its levels are increased in AVM tissue and patient urine. This work suggests that dysregulation of the EphrinB2:EphB4 signaling cascade and increases in EphrinB2 may play a role in AVM development, with potential utility as a diagnostic and therapeutic target.

Using urinary bFGF and TIMP3 levels to predict the presence of juvenile pilocytic astrocytoma and establish a distinct biomarker signature.

OBJECTIVE The authors report the use of urinary biomarkers as a novel, noninvasive technique to detect juvenile pilocytic astrocytomas (JPAs), capable of distinguishing JPAs from other CNS diseases, including other brain tumors. Preliminary screening of an array of tumors implicated proteases (including matrix metalloproteinases [MMPs]) and their inhibitors (tissue inhibitors of metalloproteinase [TIMPs]) as well as growth factors (including basic fibroblast growth factor [bFGF]) as candidate biomarkers. These data led the authors to hypothesize that tissue inhibitor of metalloproteinase 3 (TIMP3) and bFGF would represent high-probability candidates as JPA-specific biomarkers. METHODS Urine was collected from 107 patients, which included children with JPA (n = 21), medulloblastoma (n = 17), glioblastoma (n = 9), arteriovenous malformations (n = 25), moyamoya (n = 14), and age- and sex-matched controls (n = 21). Biomarker levels were quantified with enzyme-linked immunosorbent assay, tumor tissue expression was confirmed with immunohistochemical analysis, and longitudinal biomarker expression was correlated with imaging. Results were subjected to univariate and multivariate statistical analyses. RESULTS Using optimal urinary cutoff values of bFGF > 1.0 pg/µg and TIMP3 > 3.5 pg/µg, multiplexing bFGF and TIMP3 predicts JPA presence with 98% accuracy. Multiplexing bFGF and MMP13 distinguishes JPA from other brain tumor subtypes with up to 98% accuracy. Urinary biomarker expression correlated with both tumor immunohistochemistry and in vitro tumor levels. Urinary bFGF and TIMP3 decrease following successful tumor treatment and correlate with changes in tumor size. CONCLUSIONS This study identifies 2 urinary biomarkers-bFGF and TIMP3-that successfully detect one of the most common pediatric brain tumors with high accuracy. These data highlight potential benefits of urinary biomarkers and support their utility as diagnostic tools in the treatment of children with JPA.