Predicting Metastatic Potential in Pheochromocytoma and Paraganglioma: A Comparison of PASS and GAPP Scoring Systems.

PURPOSE: The Pheochromocytoma of the Adrenal Gland Scaled Score (PASS) and the Grading System for Adrenal Pheochromocytoma and Paraganglioma (GAPP) are scoring systems to predict metastatic potential in pheochromocytomas (PCC) and paragangliomas (PGLs). The goal of this study is to assess PASS and GAPP as metastatic predictors and to correlate with survival outcomes. METHODS: The cohort included PCC/PGL with ≥5 years of follow-up or known metastases. Surgical pathology slides were rereviewed. PASS and GAPP scores were assigned. Univariable and multivariable logistic regression, Kaplan-Meier survival analysis, and Cox proportional hazards were performed to assess recurrence-free survival (RFS) and disease-specific survival (DSS). RESULTS: From 143 subjects, 106 tumors were PCC and 37 were PGL. Metastases developed in 24%. The median PASS score was 6.5 (interquartile range [IQR]: 4.0-8.0) and median GAPP score was 3.0 (IQR: 2.0-4.0). Interrater reliability was low-moderate for PASS (intraclass correlation coefficient [ICC]: 0.6082) and good for GAPP (ICC 0.7921). Older age (OR: 0.969, P = .0170) was associated with longer RFS. SDHB germline pathogenic variant (OR: 8.205, P = .0049), extra-adrenal tumor (OR: 6.357, P < .0001), Ki-67 index 1% to 3% (OR: 4.810, P = .0477), and higher GAPP score (OR: 1.537, P = .0047) were associated with shorter RFS. PASS score was not associated with RFS (P = .1779). On Cox regression, a GAPP score in the moderately differentiated range was significantly associated with disease recurrence (HR: 3.367, P = .0184) compared with well-differentiated score. CONCLUSION: Higher GAPP scores were associated with aggressive PCC/PGL. PASS score was not associated with metastases and demonstrated significant interobserver variability. Scoring systems for predicting metastatic PCC/PGL may be improved by incorporation of histopathology, clinical data, and germline and somatic tumor markers.

EphA4 Receptor Forward Signaling Inhibits Glucagon Secretion From α-Cells.

The loss of inhibition of glucagon secretion exacerbates hyperglycemia in type 1 and 2 diabetes. However, the molecular mechanisms that regulate glucagon secretion in unaffected and diabetic states remain relatively unexplained. We present evidence supporting a new model of juxtacrine-mediated regulation of glucagon secretion where neighboring islet cells negatively regulate glucagon secretion through tonic stimulation of α-cell EphA receptors. Primarily through EphA4 receptors, this stimulation correlates with maintenance of a dense F-actin network. In islets, additional stimulation and inhibition of endogenous EphA forward signaling result in inhibition and enhancement, respectively, of glucagon secretion, accompanied by an increase and decrease, respectively, in α-cell F-actin density. Sorted α-cells lack endogenous stimulation of EphA forward signaling from neighboring cells, resulting in enhanced basal glucagon secretion as compared with islets and the elimination of glucose inhibition of glucagon secretion. Restoration of EphA forward signaling in sorted α-cells recapitulates both normal basal glucagon secretion and glucose inhibition of glucagon secretion. Additionally, α-cell-specific EphA4(-/-) mice exhibit abnormal glucagon dynamics, and EphA4(-/-) α-cells contain less dense F-actin networks than EphA4(+/+) α-cells. This juxtacrine-mediated model provides insight into the functional and dysfunctional regulation of glucagon secretion and opens up new therapeutic strategies for the clinical management of diabetes.

Intrinsic islet heterogeneity and gap junction coupling determine spatiotemporal Ca²âº wave dynamics.

Insulin is released from the islets of Langerhans in discrete pulses that are linked to synchronized oscillations of intracellular free calcium ([Ca(2+)]i). Associated with each synchronized oscillation is a propagating calcium wave mediated by Connexin36 (Cx36) gap junctions. A computational islet model predicted that waves emerge due to heterogeneity in ß-cell function throughout the islet. To test this, we applied defined patterns of glucose stimulation across the islet using a microfluidic device and measured how these perturbations affect calcium wave propagation. We further investigated how gap junction coupling regulates spatiotemporal [Ca(2+)]i dynamics in the face of heterogeneous glucose stimulation. Calcium waves were found to originate in regions of the islet having elevated excitability, and this heterogeneity is an intrinsic property of islet ß-cells. The extent of [Ca(2+)]i elevation across the islet in the presence of heterogeneity is gap-junction dependent, which reveals a glucose dependence of gap junction coupling. To better describe these observations, we had to modify the computational islet model to consider the electrochemical gradient between neighboring ß-cells. These results reveal how the spatiotemporal [Ca(2+)]i dynamics of the islet depend on ß-cell heterogeneity and cell-cell coupling, and are important for understanding the regulation of coordinated insulin release across the islet.

Alpha,beta-methylene-2'-deoxynucleoside 5'-triphosphates as noncleavable substrates for DNA polymerases: isolation, characterization, and stability studies of novel 2'-deoxycyclonucleosides, 3,5'-cyclo-dG, and 2,5'-cyclo-dT.

We report synthesis and characterization of a complete set of alpha,beta-methylene-2'-dNTPs (alpha,beta-m-dNTP; N = A, C, T, G, 12-15) in which the alpha,beta-oxygen linkage of natural dNTP was replaced by a methylene group. These nucleotides were designed to be noncleavable substrates for DNA polymerases. Synthesis entails preparation of 2'-deoxynucleoside 5'-diphosphate precursors, followed by an enzymatic gamma-phosphorylation. All four synthesized alpha,beta-m-dNTPs were found to be potent inhibitors of polymerase beta, with K i values ranging 1-5 microM. During preparation of the dG and dT derivatives of alpha,beta-methylene diphosphate, we also isolated significant amounts of 3,5'-cyclo-dG (16) and 2,5'-cyclo-dT (17), respectively. These novel 2'-deoxycyclonucleosides were formed via a base-catalyzed intramolecular cyclization (N3 --> C5' and O2 --> C5', respectively). In acidic solution, both 16 and 17 underwent glycolysis, followed by complete depurination. When exposed to alkaline conditions, 16 underwent an oxidative deamination to produce 3,5'- cyclo-2'-deoxyxanthosine (19), whereas 17 was hydrolyzed exclusively to dT.

Rapid identification of the pharmacophore in a peptoid inhibitor of the proteasome regulatory particle.

Here we report a simple and effective method to identify the minimal pharmacophore in the first peptoid inhibitor of the 19S proteasome regulatory particle, which has led to the development of a derivative that exhibits improved cellular activity, presumably due to a reduction in mass of about two-fold and the elimination of positively charged lysine-like residues.