0013.7227/92/1313-1157$03.00/0 Endocrinology Copyright 0 1992 by The Endocrine Society
Vol. 131, No. 3 Printed in U.S.A.
Parathyroid Hormone (PTH)-Related Protein, and 1,25=Dihydroxyvitamin D in Dogs with Cancer-Associated Hypercalcemia* T. J. ROSOL, L. A. NAGODE, R. D. AYL, C. L. STEINMEYER,
C. G. COUTO, A. S. HAMMER, AND C. C. CAPEN
D. J. CHEW,
PTH,
J. L. PETERSON,
Departments of Veterinary Pathobiology (T.J.R., L.A.N., C.L.S, C.C.C.) and Clinical Sciences (C.G.C., A.S.H., D.J.C., J.L.P., R.D.A.), Ohio State University College of Veterinary Medicine, Columbus, Ohio 43210
ABSTRACT
icant linear correlation with total serum calcium in dogs with anal sac adenocarcinomas and hypercalcemia, but not in dogs with lymphoma and hypercalcemia. Serum N-terminal PTH concentrations were usually in the normal range (12-34 pg/ml) for all groups of dogs except dogs with parathyroid adenomas (83 f 38 pg/ml). The serum PTH concentration increased after successful treatment of CAH. Serum 1,25-(OH)sD concentrations were decreased, normal, or increased in dogs with CAH, and 1,25-(OH)*D levels decreased after treatment of CAH. In summary, circulating concentrations of PTHrP are consistently increased in dogs with CAH, and PTHrP appears to play an important role in the induction of hypercalcemia. (Endocrinology 131: 1157-1164,1992)
Circulating N-terminal PTH-related protein (PTHrP), N-terminal PTH, and 1,2&dihydroxyvitamin D [1,25-(OH),D] concentrations were measured in normal dogs and dogs with cancer-associated hypercalcemia (CAH), parathyroid adenomas, and miscellaneous tumors. PTHrP was undetectable (Cl.8 PM) in normal dogs and increased in dogs with CAH due to adenocarcinomas derived from apocrine glands of the anal sac (44.9 f 27 PM), lymphoma (8.3 + 4.4 PM), and miscellaneous carcinomas (13.3 f 11.4 PM). The PTHrP concentration decreased in dogs with lymphoma and anal sac adenocarcinomas after successful treatment of CAH. The PTHrP concentration had a signif-
C
ANCER -associated hypercalcemia (CAH) is a common complication of malignant tumors in dogs (1). Hypercalcemia occurs in some dogs with lymphoma, adenocarcinomas derived from apocrine glands of the anal sac, and miscellaneous carcinomas. CAH associated with carcinomas in dogs is usually due to humoral hypercalcemia of malignancy (HHM), since normocalcemia occurs after surgical resection of the inciting tumors, and the carcinomas infrequently metastasize to bone (2). CAH associated with lymphoma in dogs may be due to humoral hypercalcemia of malignancy (3) or local resorption of bone induced by lymphoma that has metastasized to the bone marrow (4). However, it appears that most dogs with lymphoma have a humoral component to the hypercalcemia, since excessive bone resorption may occur distant to the site of tumor metastases in bone (3, 5). PTH-related protein (PTHrP) is an important mediator of CAH in humans and experimental models of HHM (6-8). Humans with various forms of CAH have increased circulating concentrations of PTHrP (9-13). In addition, antibodies to PTHrP are effective in lowering serum calcium levels and inhibiting bone resorption in nude mice with HHM and transplanted human squamous cell carcinoma (14). PTH is not an important mediator of hypercalcemia in human patients with HHM because PTH levels are usually normal or Received April 6, 1992. Address all correspondence and requests for reprints to: Thomas J. Rosol, D.V.M., Ph.D., Ohio State University, 1925 Coffey Road, Columbus, Ohio 43210. *This work was supported by the State of Ohio Canine Research Fund (611368).
low in HHM, and tumors associated with HHM do not produce mRNA for PTH (15, 16). 1,25-Dihydroxyvitamin D [1,25-(OH)2D] is a humoral factor infrequently associated with HHM. Most patients with HHM have normal or low serum levels of 1,25-(OH)zD (16, 17). A few human patients with lymphoma and hypercalcemia have been shown to have increased serum levels of 1,25-(OH)*D (18,19). In these patients it has been concluded that 1,25-(OH)zD contributed to the induction of hypercalcemia. The purpose of this investigation was to measure circulating concentrations of PTHrP, PTH, and 1,25-(OH)*D in normal dogs and dogs with naturally occurring CAH. In some dogs measurements were completed before and after appropriate medical or surgical therapy to determine the responses of calcium-regulating hormones to correction of hypercalcemia. Finally, linear regression analysis was performed to determine the correlation of PTHrP and 1,25(OH)*D concentrations to serum calcium, PTH, phosphorus, and creatinine concentrations. Materials
and Methods
Samples Serum and/or plasma samples were collected from dogs from 1985 through 1991. The samples were kept frozen at -20 C for short time periods (weeks) and at -80 C for longer time periods (years) until assayed. Plasma samples were collected in EDTA-containing tubes with or without aprotinin [50 rg (300 kallikrein inhibitor units); Boehringer Mannheim, Indianapolis, IN). Serum/plasma samples were collected from the following hypercalcemic dogs (total serum calcium, >12.0 mg/ dl): 8 dogs with adenocarcinomas derived from apocrine glands of the anal sac, 28 dogs with lymphoma (thymic, multicentric, or extranodal forms), 2 dogs with myeloma, 8 dogs with miscellaneous carcinomas
1157
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PTHrP IN DOGS WITH [pulmonary, nasal, mammary, squamous cell, thymoma, thyroid (n = 2), and invasive acanthomatous epulis], and 6 dogs with parathyroid adenomas. Serum/plasma samples were collected from the following normocalcemic dogs (total serum calcium, C12.0 mg/dl): 8 clinically normal adult mature dogs, 9 dogs with adenocarcinomas derived from apocrine glands of the anal sac, 11 dogs with lymphoma, 1 dog with myeloma, and 12 dogs with miscellaneous tumors [perianal gland adenoma (n = 2), perianal gland carcinoma, thymoma, squamous cell carcinoma, undifferentiated adenocarcinoma, nasal carcinoma, hemangiosarcoma (n = 3), mast cell tumor, and ocular carcinoma]. Samples were collected before any form of surgical, radiation, or chemotherapy of the respective tumors. In some cases, serum/plasma samples were collected after successful treatment of the neoplasms and return to normocalcemia.
Assays N-Terminal PTH-related protein was determined by RIA using a kit from Incstar Corp. (Stillwater, MN). The PTHrP RIA has a sensitivity of 1.8 PM human PTHrP (l-84 or l-40). There is less than 0.001% crbssreactivity with the C-terminal regions of PTHrP, less than 0.001% crossreactivitv with PTH-(l-84), and less than 1.0% cross-reactivity with PTH-(l-34). The assay results for this investigation are reported as picomolar concentrations of PTHrP (human equivalents). The inter- and intraassay coefficients of variation of this assay using canine samples were 6.0% and 6.5%, respectively. Assays were also performed to determine whether dilution of canine plasma samples that contained detectable amounts of PTHrP [samples from dogs with apocrine adenocarcinomas (n = 4) or lymphoma (n = 4)] paralleled the standard curve of the PTHrP RIA. The samples were diluted with dog plasma with an undetectable PTHrP concentration. Initial dilutions (1:l) of the canine samples maintained linearity to the human standard curve, as evidenced by close values of the calculated PTHrP concentrations (picomolar) in the complete (200 ~1) compared to the diluted samples. When samples were further diluted (1:3), there was a lack of close linearity, and all samples had calculated PTHrP concentrations lower @O-40%) than the full sample of 200 rl. There was no relative difference between plasma samples from dogs with apocrine adenocarcinomas or lymphoma. Therefore, when used according to manufacturer’s directions and when 200~1 samples were utilized, the assay measured canine PTHrP in parallel to human PTHrP. N-Terminal PTH was determined by RIA using the INS assay kit (Nichols Institute, San Juan Capistrano, CA). This assay has been compared, using dog sera, by linear regression to an immunoradiometric assay (IRMA) for intact dog PTH (correlation coefficient, 0.97) (20), for which extensive validation studies have been made (21, 22). The interand intraassay coefficients of variation for this assay are 7.9% and 4.0%, respectively, and the limit of detectability is 8 pg/ml canine serum. The standard range of PTH in canine serum was determined in 25 normocalcemic dogs (20). The mean for the 25 dogs was 23 + 5.5 pg/ml &SD). The normal range for PTH in dogs is 12-34 pg/ml (mean + 2 SD). 1,25-(0H)rD was determined by a nonequilibrium RRA (23). The standard range of 1,25-(OHhD in canine serum was determined in 25 dogs (20). The mean for the 25 dogs was 35 + 7.7 pg/ml. The normal range for 1,25-(0H)zD in dogs was 20-50 pg/ml (mean + 2 SD). The limit of detectability was 5.0 pg/ml. The inter- and intraassay coefficients of variation for this assay were 9.4% and 5.6%, respectively. Serum total calcium, phosphorus, and creatinine concentrations were determined using a Dakos automated analyzer (Coulter Electronics Co., Hialeah, FL). All assays were performed in duplicate.
Statistical
analysis
Means and SDS were determined for all groups of dogs (control; either hypercalcemic or normocalcemic anal sac adenocarcinoma, lymphoma, or miscellaneous tumor; and parathyroid adenoma). Circulating hormone concentrations [PTHrP, PTH, and 1,25-(OH)2D] were analyzed by one-way analysis of variance with Tukey’s mean separation test (Statgraphics 5.0, Statistical Graphics Corp., Gaithersburg, MD), and measire-ments before and after treatment were evaluated by paired Student’s f test (Instat 1.0, GraphPAD Software, San Diego, CA). For purposes of
HYPERCALCEMIA
Endo. 1992 Voll31. No 3
statistical analysis, PTHrP concentrations that were undetectable (12 mg/dl) and anal sac adenocarcinomas (CAC), lymphoma, or miscellaneous tumors (MISC TUMOR); and dogs with normocalcemia (
Vol. 131, No. 3 Printed in U.S.A.
Parathyroid Hormone (PTH)-Related Protein, and 1,25=Dihydroxyvitamin D in Dogs with Cancer-Associated Hypercalcemia* T. J. ROSOL, L. A. NAGODE, R. D. AYL, C. L. STEINMEYER,
C. G. COUTO, A. S. HAMMER, AND C. C. CAPEN
D. J. CHEW,
PTH,
J. L. PETERSON,
Departments of Veterinary Pathobiology (T.J.R., L.A.N., C.L.S, C.C.C.) and Clinical Sciences (C.G.C., A.S.H., D.J.C., J.L.P., R.D.A.), Ohio State University College of Veterinary Medicine, Columbus, Ohio 43210
ABSTRACT
icant linear correlation with total serum calcium in dogs with anal sac adenocarcinomas and hypercalcemia, but not in dogs with lymphoma and hypercalcemia. Serum N-terminal PTH concentrations were usually in the normal range (12-34 pg/ml) for all groups of dogs except dogs with parathyroid adenomas (83 f 38 pg/ml). The serum PTH concentration increased after successful treatment of CAH. Serum 1,25-(OH)sD concentrations were decreased, normal, or increased in dogs with CAH, and 1,25-(OH)*D levels decreased after treatment of CAH. In summary, circulating concentrations of PTHrP are consistently increased in dogs with CAH, and PTHrP appears to play an important role in the induction of hypercalcemia. (Endocrinology 131: 1157-1164,1992)
Circulating N-terminal PTH-related protein (PTHrP), N-terminal PTH, and 1,2&dihydroxyvitamin D [1,25-(OH),D] concentrations were measured in normal dogs and dogs with cancer-associated hypercalcemia (CAH), parathyroid adenomas, and miscellaneous tumors. PTHrP was undetectable (Cl.8 PM) in normal dogs and increased in dogs with CAH due to adenocarcinomas derived from apocrine glands of the anal sac (44.9 f 27 PM), lymphoma (8.3 + 4.4 PM), and miscellaneous carcinomas (13.3 f 11.4 PM). The PTHrP concentration decreased in dogs with lymphoma and anal sac adenocarcinomas after successful treatment of CAH. The PTHrP concentration had a signif-
C
ANCER -associated hypercalcemia (CAH) is a common complication of malignant tumors in dogs (1). Hypercalcemia occurs in some dogs with lymphoma, adenocarcinomas derived from apocrine glands of the anal sac, and miscellaneous carcinomas. CAH associated with carcinomas in dogs is usually due to humoral hypercalcemia of malignancy (HHM), since normocalcemia occurs after surgical resection of the inciting tumors, and the carcinomas infrequently metastasize to bone (2). CAH associated with lymphoma in dogs may be due to humoral hypercalcemia of malignancy (3) or local resorption of bone induced by lymphoma that has metastasized to the bone marrow (4). However, it appears that most dogs with lymphoma have a humoral component to the hypercalcemia, since excessive bone resorption may occur distant to the site of tumor metastases in bone (3, 5). PTH-related protein (PTHrP) is an important mediator of CAH in humans and experimental models of HHM (6-8). Humans with various forms of CAH have increased circulating concentrations of PTHrP (9-13). In addition, antibodies to PTHrP are effective in lowering serum calcium levels and inhibiting bone resorption in nude mice with HHM and transplanted human squamous cell carcinoma (14). PTH is not an important mediator of hypercalcemia in human patients with HHM because PTH levels are usually normal or Received April 6, 1992. Address all correspondence and requests for reprints to: Thomas J. Rosol, D.V.M., Ph.D., Ohio State University, 1925 Coffey Road, Columbus, Ohio 43210. *This work was supported by the State of Ohio Canine Research Fund (611368).
low in HHM, and tumors associated with HHM do not produce mRNA for PTH (15, 16). 1,25-Dihydroxyvitamin D [1,25-(OH)2D] is a humoral factor infrequently associated with HHM. Most patients with HHM have normal or low serum levels of 1,25-(OH)zD (16, 17). A few human patients with lymphoma and hypercalcemia have been shown to have increased serum levels of 1,25-(OH)*D (18,19). In these patients it has been concluded that 1,25-(OH)zD contributed to the induction of hypercalcemia. The purpose of this investigation was to measure circulating concentrations of PTHrP, PTH, and 1,25-(OH)*D in normal dogs and dogs with naturally occurring CAH. In some dogs measurements were completed before and after appropriate medical or surgical therapy to determine the responses of calcium-regulating hormones to correction of hypercalcemia. Finally, linear regression analysis was performed to determine the correlation of PTHrP and 1,25(OH)*D concentrations to serum calcium, PTH, phosphorus, and creatinine concentrations. Materials
and Methods
Samples Serum and/or plasma samples were collected from dogs from 1985 through 1991. The samples were kept frozen at -20 C for short time periods (weeks) and at -80 C for longer time periods (years) until assayed. Plasma samples were collected in EDTA-containing tubes with or without aprotinin [50 rg (300 kallikrein inhibitor units); Boehringer Mannheim, Indianapolis, IN). Serum/plasma samples were collected from the following hypercalcemic dogs (total serum calcium, >12.0 mg/ dl): 8 dogs with adenocarcinomas derived from apocrine glands of the anal sac, 28 dogs with lymphoma (thymic, multicentric, or extranodal forms), 2 dogs with myeloma, 8 dogs with miscellaneous carcinomas
1157
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 13 November 2015. at 02:12 For personal use only. No other uses without permission. . All rights reserved.
PTHrP IN DOGS WITH [pulmonary, nasal, mammary, squamous cell, thymoma, thyroid (n = 2), and invasive acanthomatous epulis], and 6 dogs with parathyroid adenomas. Serum/plasma samples were collected from the following normocalcemic dogs (total serum calcium, C12.0 mg/dl): 8 clinically normal adult mature dogs, 9 dogs with adenocarcinomas derived from apocrine glands of the anal sac, 11 dogs with lymphoma, 1 dog with myeloma, and 12 dogs with miscellaneous tumors [perianal gland adenoma (n = 2), perianal gland carcinoma, thymoma, squamous cell carcinoma, undifferentiated adenocarcinoma, nasal carcinoma, hemangiosarcoma (n = 3), mast cell tumor, and ocular carcinoma]. Samples were collected before any form of surgical, radiation, or chemotherapy of the respective tumors. In some cases, serum/plasma samples were collected after successful treatment of the neoplasms and return to normocalcemia.
Assays N-Terminal PTH-related protein was determined by RIA using a kit from Incstar Corp. (Stillwater, MN). The PTHrP RIA has a sensitivity of 1.8 PM human PTHrP (l-84 or l-40). There is less than 0.001% crbssreactivity with the C-terminal regions of PTHrP, less than 0.001% crossreactivitv with PTH-(l-84), and less than 1.0% cross-reactivity with PTH-(l-34). The assay results for this investigation are reported as picomolar concentrations of PTHrP (human equivalents). The inter- and intraassay coefficients of variation of this assay using canine samples were 6.0% and 6.5%, respectively. Assays were also performed to determine whether dilution of canine plasma samples that contained detectable amounts of PTHrP [samples from dogs with apocrine adenocarcinomas (n = 4) or lymphoma (n = 4)] paralleled the standard curve of the PTHrP RIA. The samples were diluted with dog plasma with an undetectable PTHrP concentration. Initial dilutions (1:l) of the canine samples maintained linearity to the human standard curve, as evidenced by close values of the calculated PTHrP concentrations (picomolar) in the complete (200 ~1) compared to the diluted samples. When samples were further diluted (1:3), there was a lack of close linearity, and all samples had calculated PTHrP concentrations lower @O-40%) than the full sample of 200 rl. There was no relative difference between plasma samples from dogs with apocrine adenocarcinomas or lymphoma. Therefore, when used according to manufacturer’s directions and when 200~1 samples were utilized, the assay measured canine PTHrP in parallel to human PTHrP. N-Terminal PTH was determined by RIA using the INS assay kit (Nichols Institute, San Juan Capistrano, CA). This assay has been compared, using dog sera, by linear regression to an immunoradiometric assay (IRMA) for intact dog PTH (correlation coefficient, 0.97) (20), for which extensive validation studies have been made (21, 22). The interand intraassay coefficients of variation for this assay are 7.9% and 4.0%, respectively, and the limit of detectability is 8 pg/ml canine serum. The standard range of PTH in canine serum was determined in 25 normocalcemic dogs (20). The mean for the 25 dogs was 23 + 5.5 pg/ml &SD). The normal range for PTH in dogs is 12-34 pg/ml (mean + 2 SD). 1,25-(0H)rD was determined by a nonequilibrium RRA (23). The standard range of 1,25-(OHhD in canine serum was determined in 25 dogs (20). The mean for the 25 dogs was 35 + 7.7 pg/ml. The normal range for 1,25-(0H)zD in dogs was 20-50 pg/ml (mean + 2 SD). The limit of detectability was 5.0 pg/ml. The inter- and intraassay coefficients of variation for this assay were 9.4% and 5.6%, respectively. Serum total calcium, phosphorus, and creatinine concentrations were determined using a Dakos automated analyzer (Coulter Electronics Co., Hialeah, FL). All assays were performed in duplicate.
Statistical
analysis
Means and SDS were determined for all groups of dogs (control; either hypercalcemic or normocalcemic anal sac adenocarcinoma, lymphoma, or miscellaneous tumor; and parathyroid adenoma). Circulating hormone concentrations [PTHrP, PTH, and 1,25-(OH)2D] were analyzed by one-way analysis of variance with Tukey’s mean separation test (Statgraphics 5.0, Statistical Graphics Corp., Gaithersburg, MD), and measire-ments before and after treatment were evaluated by paired Student’s f test (Instat 1.0, GraphPAD Software, San Diego, CA). For purposes of
HYPERCALCEMIA
Endo. 1992 Voll31. No 3
statistical analysis, PTHrP concentrations that were undetectable (12 mg/dl) and anal sac adenocarcinomas (CAC), lymphoma, or miscellaneous tumors (MISC TUMOR); and dogs with normocalcemia (
