Immunochemical Localization of Parathyroid Hormone in Cancer Tissue from Patients with Ectopic Hyperparathyroidism
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Immunochemical Localization of Parathyroid Hormone in Cancer Tissue from Patients with Ectopic Hyperparathyroidism Genaro M. A. Palmieri, … , Robert E. Nordquist, Gilbert S. Omenn J Clin Invest. 1974;53(6):1726-1735. https://doi.org/10.1172/JCI107724. Research Article Immunoreactive parathyroid hormone (PTH) in nonparathyroid malignant tumors associated with hypercalcemia and hypophosphatemia in the absence of demonstrable bone metastases was determined by radioimmunoassay and immunofluorescent techniques. Six of seven tumors contained material with immunological cross-reactivity to bovine PTH by radioimmunoassay and immunofluorescence. The intensity of the immunofluorescent stain varied considerably in the different tumors. From 15 to 90% of neoplastic cells were stained specifically with fluorescein-labeled anti-PTH. In contrast, normal parathyroid glands and parathyroid adenomas showed uniform distribution of immunofluorescence in all parenchymal cells. In one malignant tumor, PTH was localized also by immunoautoradiography. In every case PTH was detected only in the cytoplasm of parenchymal cells. One patient lacked detectable PTH in his tumor, yet showed regression of the hypercalcemia to normal values after removal of large masses of neoplastic tissue and recurrence of hypercalcemia when new growth occurred. Dilutional radioimmunoassay curves of nonparathyroid malignant tumors were in most cases different from those obtained with extracts of normal parathyroid glands and parathyroid adenomas. Although both nonparathyroid neoplasmas and parathyroid extracts demonstrated immunoheterogeneity by gel filtration, greater heterogeneity was found in nonparathyroid malignant tumors. In those tumors in which immunological cross-reactivity to PTH was detected, the capability of secreting PTH may be restricted to derepressed cell clones amidst other neoplastic cells, whereas the greater heterogeneity of ectopic PTH may […] Find the latest version: https://jci.me/107724/pdf Immunochemical Localization of Parathyroid Hormone in Cancer Tissue from Patients with Ectopic Hyperparathyroidism GENARO M. A. PALMIERI, ROBERT E. NORDQUIST, and GILBERT S. OMENN From the Veterans Administration Hospital, Oklahoma Medical Research Foundation, Departments of Medicine and Pathology, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104; and the Department of Medicine, University of Washington Medical School, Seattle, Washington 98105 A B S T R A C T Immunoreactive parathyroid hormone from those obtained with extracts of normal parathyroid (PTH) in nonparathyroid malignant tumors associated glands and parathyroid adenomas. Although both non- with hypercalcemia and hypophosphatemia in the ab- parathyroid neoplasms and parathyroid extracts demon- sence of demonstrable bone metastases was determined strated immunoheterogeneity by gel filtration, greater by radioimmunoassay and immunofluorescent techniques. heterogeneity was found in nonparathyroid malignant Six of seven tumors contained material with immuno- tumors. logical cross-reactivity to bovine PTH by radioimmuno- In those tumors in which immunological cross-reactiv- assay and immunofluorescence. The intensity of the ity to PTH was detected, the capability of secreting immunofluorescent stain varied considerably in the dif- PTH may be restricted to derepressed cell clones amidst ferent tumors. From 15 to 90% of neoplastic cells were other neoplastic cells, whereas the greater heterogeneity stained specifically with fluorescein-labeled anti-PTH. of ectopic PTH may reflect hormone cleavage by proteo- In contrast, normal parathyroid glands and parathyroid lytic enzymes in the tumor that is less specific than the adenomas showed uniform distribution of immunofluo- Pro-PTH cleaving enzyme in the parathyroids. rescence in all parenchymal cells. In one malignant tu- mor, PTH was localized also by immunoautoradiog- raphy. In every case PTH was detected only in the INTRODUCTION cytoplasm of parenchymal cells. One patient lacked de- Nonparathyroid malignant tumors in patients without tectable PTH in his tumor, yet showed regression of skeletal metastasis may be associated with hypercalcemia, the hypercalcemia to normal values after removal of hypophosphatemia, and clinical complications indis- large masses of neoplastic tissue and recurrence of hy- tinguishable from the syndrome of primary hyperpara- percalcemia when new growth occurred. thyroidism (1, 2). Various immunologic techniques have Dilutional radioimmunoassay curves of nonpara- demonstrated parathyroid hormone (PTH)1 in extracts thyroid malignant tumors were in most cases different of kidney, lung, liver, adrenal, parotid, spleen, and This work was presented in part at the 4th International breast tumors (3-6). A venous-arterial gradient for Congress of Endocrinology, Washington, D. C., 18-24 June PTH was found in cases due to hepatoma (5) and hyper- 1972. nephroma (7). Some malignancies may produce hu- Dr. Palmieri is a Clinical Investigator at the Veterans moral factors other than PTH capable of inducing hyper- Administration Hospital, Oklahoma City, Okla., and Dr. since is known that bone is stim- Omenn is the recipient of Career Development Award 43122 calcemia, it resorption from the National Institutes of Health. Address reprint ulated by a variety of biological agents, including 1,25- requests to Dr. Palmieri, Section of Endocrinology, De- hydroxycholecalciferol, vitamin A, and prostaglandin El partment of Medicine, University of Tennessee, Memphis, Tenn. 38163. 1Abbreviations used in this paper: HPTH, human para- Received for publication 26 July 1973 and in revised form thyroid tissue extract; PBS, phosphate-buffered saline; 16 November 1973. PTH, parathyroid hormone. 1726 The Journal of Clinical Investigation Volume 53 June 1974-1726-1735 (8). Gordan, Cantino, Erhardt, Hansen, and Lubich PTH (TCA-PTH, Wilson Labs., Chicago, Ill.). PTH in (9) related hypercalcemia to the production of a sterol plasma of normal subjects, patients with primary hyper- by some breast carcinomas, while Powell, Singer, Mur- parathyroidism, and patients with ectopic PTH syndrome ray, Minkin, and Potts (10) found no radioimmuno- was determined using an identical procedure in 500-pl samples of plasma. Antiserum GP 456-5-62 in a 1: 50,000 assayable PTH in extracts of 11 malignant tumors asso- dilution was also used in determinations of PTH in plasma. ciated with hypercalcemia and hypophosphatemia, five This antiserum is more sensitive than GP 012-6-23 for de- of which produced bone resorption in vitro. tection of circulating PTH. In malignant tumors secreting PTH, the Chromatographic fractionation. Lyophilized tissue ex- concentra- tracts resuspended in 3 ml of 0.2 M ammonium acetate tion of the hormone, based on dry weight measurements, buffer pH 4.6 were applied to a 2.0 X 90 cm Bio-Gel P-10 is 30-300 times lower than in parathyroid adenomas (4). column (Bio-Rad Laboratories, Richmond, Calif.) equili- The lower concentration of PTH in these nonpara- brated at 40C with 0.2 M ammonium acetate buffer pH thyroid neoplasms may be due to a slower rate of syn- 4.7. The flow rate was approximately 0.18 ml/min. 50-100- IAI samples of 1-ml fractions were diluted in 500 /Al of thesis or more rapid turnover in most of the cells or to hypoparathyroid plasma for radioimmunoassay. high levels of production by only a small fraction of the Morphological studies. Immunofluorescent studies were neoplastic cells. performed on normal parathyroid tissue, parathyroid ade- We present here studies of the content, intracellular nomas, ectopic hormone-secreting tumors, and in other localization, and tumors and tissues which contained no detectable para- chromatographic characteristics of thormone by radioimmunoassay. PTH in normal parathyroid glands, parathyroid ade- The tissues obtained were divided and cryostat sectioned nomas, and nonparathyroid malignant tumors associated on the face adjacent to the tissue taken for determination with ectopic hyperparathyroidism. of PTH. 7-/Am sections were fixed in acetone which was made basic by the addition of enough saturated NaOH in METHODS absolute ethyl alcohol to bring the pH to 7.5. After 10 min fixation in this solution the slides were air dried and Subjects. Ectopic production of PTH was investigated then washed with phosphate-buffered saline (PBS) at in patients with malignancies accompanied by hypercal- pH 7.2 (0.15 M NaCl, 0.01 M phosphate). Whole guinea cemia with no evidence of bone metastasis by X ray or at pig antiserum against bovine parathormone (GP 012-6-23) autopsy. In those cases in which a careful exploration of was diluted 1:10 with PBS, applied to the sections, and in- the parathyroid glands was performed at autopsy, the para- cubated in a humidified chamber at room temperature for thyroid glands were found to be normal (Table I). Normal 30 min. Increasing dilutions of the anti-PTH serum were parathyroid glands were obtained at autopsies of patients applied to sections of all tissues. No detectable immuno- without a history of metabolic or endocrine diseases. Para- fluorescence was observed with dilutions greater than 1: 100. thyroid adenomas were obtained during surgical explora- After incubation, the tissues were rinsed in three changes tion of patients with a diagnosis of primary hyperpara- of PBS for a total of 30 min and postcoupled with a 1: 30 thyroidism. dilution of fluorescein-labeled rabbit anti-guinea pig IgG Preparation of tissue extracts. Normal parathyroid glands, for 30 min. After