OR07-04 A Novel Ex Vivo Live-Cell Interrogative Assay of Human Parathyroid Tissue Reveals Distinct Mechanisms of Calcium Sensing Failure in Primary, Secondary, and Tertiary Hyperparathyroidism
Authors: Zhang Jie , Zhang Run , Foft Jessica , Starks Catherine , Gosnell Jessica Erin , Roman Sanziana , Sosa Julie Ann , Koh James 
Published: May 2020

Abstract
Disruption of calcium homeostasis is common to all forms of hyperparathyroidism (HPT), but the underlying biochemical mechanisms that distinguish the various forms of HPT pathology remain poorly characterized. We previously have observed that the kinetics and amplitude of CASR-mediated signaling vary significantly among parathyroid (PT) adenomas and found specific functional and gene expression profiles preferentially associated with increased risk of bone density loss. While these data established a clear connection between CASR activity and clinical phenotype, a direct comparison of the kinetics of PTH secretory behavior between normal and neoplastic intact human PT tissue has yet to be performed. Utilizing eucalcemic normal human organ donor tissues (n=3) as a reference standard, we examined a series of cryopreserved live PT tissue specimens obtained from patients with primary (n=9), secondary (n=12) and tertiary (n=5) HPT. PT tissue fragments matched for viability, mass, and cellular content were placed on permeable membranes and exposed to a series of extracellular calcium concentrations over equivalent time intervals of challenge and normocalcemic recovery to interrogate dynamic PTH secretory induction or suppression. As expected, normal tissue exhibited a sigmoid response curve indicative of allosteric calcium-mediated inhibition, with a mean EC50 of 0.95 mM (95% CI: 0.859–1.254). In contrast, the majority of primary HPT adenomas (n=6) displayed a concave response curve indicative of non-competitive inhibition, consistent with a primary sensing deficit, such as loss of CASR expression. Two distinct PTH secretory behaviors were observed in secondary HPT specimens. One subset (secondary type 1, n=4) retained a sigmoid response curve but with a modest EC50 increase (mean EC50=1.50 mM, 95% CI: 1.41–1.61) and maximal suppression similar to normal tissue, features reflective of competitive inhibition in response to elevated calcium. This pattern could indicate enhanced CASR antagonist activity relative to normal tissue. A second subset, (secondary type 2, n=8) demonstrated a large EC50 shift (mean EC50=2.46 mM; 95% CI: 1.844–2.621), a sigmoid response curve, and an elevated threshold of persistent PTH secretion at high calcium conditions. These parameters are suggestive of non-competitive inhibitory behavior, consistent with loss of a CASR-dependent downstream effector. Three of the primary HPT adenomas shared this response phenotype. Of the tertiary specimens, four matched the primary HPT adenoma pattern, while one exhibited secondary type 2 behavior. These results reveal a series of progressively attenuated dynamic response patterns, where PTH secretion becomes increasingly uncoupled from extracellular calcium sensing. These findings suggest that primary, secondary, and tertiary HPT arise through distinct mechanisms of calcium sensing failure.