DOI: 10.7272/q6891431
title: Supplemental figures: Calcium response behaviors and biomarker expression in human parathyroid tumors

description: Context. The biochemical basis for clinical variability in
 primary hyperparathyroidism (PHPT) is poorly
 understood.   Objectives. To define parathyroid
 tumor biochemical properties associated with calcium sensing failure in
 PHPT patients, and to relate differences in these profiles to variations
 in clinical presentation.  
 Design. Pre-operative clinical data were evaluated for
 correlation to parathyroid tumor biochemical behavior. 
 Setting. An endocrine surgery referral center at a large, public
 university hospital. Patients and Other Participants.  A
 sequential series of 39 patients undergoing surgery for
 PHPT.   Main Outcome Measures. An intact
 tissue, ex vivo interrogative assay was employed to evaluate the
 calcium-sensing capacity of parathyroid adenomas relative to normal donor
 glands.  Tumors were functionally classified based on
 calcium dose-response curve profiles, and clinical parameters were
 compared among the respective classes.  Changes in the
 relative expression of CASR, RGS5, and RCAN1, three key components in the
 calcium/PTH signaling axis were evaluated as potential mechanisms for
 calcium-sensing failure. Results. Parathyroid adenomas grouped
 into three distinct functional classes.  Tumors with
 diminished calcium sensitivity were the most common (18 of 39) and were
 strongly associated with reduced bone mineral density
 (p=0.0009).  Tumors with no calcium sensing deficit (11
 of 39) were associated with higher pre-operative PTH (p =
 0.036).  A third group (6/39) displayed a non-sigmoid
 calcium/PTH response curve; four of these six tumors expressed elevated
 RCAN1.   Conclusions. Calcium-sensing capacity
 varies among parathyroid tumors but down-regulation of the calcium sensing
 receptor (CASR) is not an obligate underlying
 mechanism.  Differences in tumor calcium responsiveness
 may contribute to variations in PHPT clinical
 presentation.  
description: <b>Supplementary Figure Legends</b> <b>Figure S1.</b> <i>Pre-operative PTH levels are lower in vitamin D-deficient and vitamin D-insufficient PHPT patients.</i>  Serum PTH levels determined at the time of referral for parathyroidectomy were compared in patients with vitamin D-deficiency (&lt;20 ng/ml; n=2), vitamin D-insufficiency (20-30 ng/ml; n=8), or vitamin D replete status (&gt;30 ng/ml; n=24). *p = 0.0034.  **p = 0.0025. <b>Figure S2</b>.  <i>RGS5 immunofluorescence scoring</i>.  Representative examples of RGS5 expression in adenomas relative to normal tissue, scored using the same index system.  Blue = DAPI; green = anti-RGS5; red = wheat germ agglutinin 594. <b>Figure S3</b>.  <i>RCAN1 immunofluorescence scoring.</i>  RCAN1 expression in tumors relative to normal tissue.   Representative image fields are shown, scored as described above. Blue = DAPI; green = anti-RCAN1; red = wheat germ agglutinin 594.  The graph indicates the distribution of tumors with elevated RCAN1 expression (scored 2+ and 3+) among non-sigmoid response curve tumors (n=6) compared to all others in the sample set (n=29).   <b>Figure S4</b>.  <i>MEN1 relative abundance is variable in PHPT adenomas</i>.  Immunofluorescence images were acquired as described above.  Green = MEN1; red = WGA 594; blue = DAPI. <b>Figure S5</b>.  <i>Gcmb localization and abundance is variable in PHPT adenomas</i>. Immunofluorescence images were acquired as described above.  Green = Gcmb.   <b>Figure S6</b>. <i>RET expression in PHPT tumors</i>. Immunofluorescence images were acquired as described above.  Green = RET; red = WGA 594; blue = DAPI.   <b>Figure S7</b>. <i>CDKN2B expression in PHPT tumors</i>. Immunofluorescence images were acquired as described above.  Green = CDKN2B; red = WGA 594; blue = DAPI. <b>Figure S8</b>.  <i>CDC73 expression in PHPT tumors</i>.  Immunofluorescence images were acquired as described above.  Green = CDC73; red = WGA 594; blue = DAPI.  Scale bar = 50 microns.