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.  

Supplementary Figure Legends

Figure S1. Pre-operative PTH levels are lower in vitamin D-deficient and vitamin D-insufficient PHPT patients.  Serum PTH levels determined at the time of referral for parathyroidectomy were compared in patients with vitamin D-deficiency (<20 ng/ml; n=2), vitamin D-insufficiency (20-30 ng/ml; n=8), or vitamin D replete status (>30 ng/ml; n=24). *p = 0.0034.  **p = 0.0025.

Figure S2.  RGS5 immunofluorescence scoring.  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.

Figure S3.  RCAN1 immunofluorescence scoring.  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).  

Figure S4.  MEN1 relative abundance is variable in PHPT adenomas.  Immunofluorescence images were acquired as described above.  Green = MEN1; red = WGA 594; blue = DAPI.

Figure S5.  Gcmb localization and abundance is variable in PHPT adenomas. Immunofluorescence images were acquired as described above.  Green = Gcmb.  

Figure S6. RET expression in PHPT tumors. Immunofluorescence images were acquired as described above.  Green = RET; red = WGA 594; blue = DAPI.  

Figure S7. CDKN2B expression in PHPT tumors. Immunofluorescence images were acquired as described above.  Green = CDKN2B; red = WGA 594; blue = DAPI.

Figure S8.  CDC73 expression in PHPT tumors.  Immunofluorescence images were acquired as described above.  Green = CDC73; red = WGA 594; blue = DAPI.  Scale bar = 50 microns.