Veterinary Clinical Pathology ISSN 0275-6382

CASE REPORT

Hypercalcemia and parathyroid hormone-related peptide expression in a dog with thyroid carcinoma and histiocytic sarcoma Jennifer L. Scruggs1, Michelle Nobrega-Lee1, Michael M. Fry1, Rory Applegate2 Departments of 1Biomedical and Diagnostic Sciences, and 2 Small Animal Clinical Sciences, College of Veterinary Medicine, University of Tennessee, Knoxville, TN, USA

Key Words Calcium, canine, paraneoplastic Correspondence J.L. Scruggs, Department of Biomedical and Diagnostic Sciences, College of Veterinary Medicine, University of Tennessee, 2407 River Drive, Room A205, Knoxville, TN 37996-4543, USA E-mail: [email protected] DOI:10.1111/vcp.12244

Abstract: A 9.5-year-old, male castrated Walker Hound was presented for evaluation of progressive weakness, anorexia, and weight loss. Imaging revealed multiple abdominal and thoracic masses and ascites; fine-needle aspirates of mesenteric and splenic masses confirmed malignancy, most likely histiocytic sarcoma. Laboratory analyses revealed increased ionized calcium and parathyroid hormone-related peptide (PTH-rP) concentrations, and concurrent low–normal parathyroid hormone concentration, consistent with humoral hypercalcemia of malignancy. Necropsy was performed after euthanasia. The dog had disseminated histiocytic sarcoma, including sarcomatosis, as well as bilateral thyroid carcinoma. PTH-rP immunostaining was positive in the thyroid carcinoma but negative in the histiocytic neoplasm. These results suggest that thyroid carcinomaassociated hypercalcemia can be caused by tumor secretion of PTH-rP.

Case Presentation A 9.5-year-old, male castrated Walker Hound was referred to the University of Tennessee Veterinary Medical Center for a recent history of progressive weakness, anorexia, and weight loss. At presentation, the dog was recumbent, 8–10% dehydrated, and had an abdominal fluid wave and palpable organomegaly. A CBC (Table 1), biochemical panel (Table 2), thoracic radiographs, and abdominal radiographs and ultrasound examination were performed. Results from the CBC were consistent with marked inflammation, although paraneoplastic leukocytosis could not be excluded. Clinical dehydration probably masked mild anemia. Pertinent biochemical results included marked hypercalcemia, mild hypoalbuminemia, moderate azotemia, and moderate hyperphosphatemia (Table 2). Urinalysis was not performed. Imaging analyses demonstrated abdominal effusion and masses in the mediastinum, lungs, spleen, and mesentery. Fine-needle aspirates from the splenic and mesenteric masses, and abdominal fluid samples were obtained for cytologic evaluation using Wright’s stain (Wescor Aerospray Pro Hematology; Elitech Biomedical Systems, Logan, UT,

USA). The cytologic interpretation was malignant neoplasia for all samples, most consistent with histiocytic sarcoma. Histopathology and possible special staining were recommended to more definitively determine the cell of origin. Due to the poor prognosis, the owners elected euthanasia of the dog. Prior to euthanasia, plasma was collected for endocrinology testing at the Michigan State University Animal Health Diagnostic Laboratory. Test results (Table 3) showed increased parathyroid hormone-related peptide (PTH-rP) and ionized calcium concentrations, and low–normal parathyroid hormone (PTH) concentration, consistent with humoral hypercalcemia of malignancy. At necropsy, 1.6 L of opaque, red peritoneal effusion were present with disseminated pinpoint to small (most ≤ 0.5 cm in diameter), soft to firm, white, raised, cystic nodules on the visceral and parietal peritoneum. Small, firm, white nodules with a central red area were present throughout all liver lobes. Multiple peripheral and visceral lymph nodes were markedly enlarged, white-tan, and contained central cavitations. The thyroid glands were bilaterally enlarged (right 3.0 9 2.2 9 0.6 cm; left 2.5 9 1.8 9 1.5 cm), dark green to black, and contained red fluid. Segmental duodenal and jejunal mucosal hemorrhage was

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Table 1. Select CBC data of a dog with histiocytic sarcoma and thyroid carcinoma. Variable HCT (%) WBC (9103/lL) Segmented neutrophils (9103/lL) Band neutrophils (9103/lL) Lymphocytes(9103/lL) Monocytes (9103/lL) Platelet (9103/lL) Total protein (g/dL)

Patient Value

Reference Interval

41.3 90.5 80.55 5.43 0.91 3.6 Clumped, adequate 6.8

41.0–60.0 5.1–14.0 2.65–9.8 0–0.3 1.1–4.6 0.165–0.85 147.0–423.0 5.7–7.9

Bold values indicate out-of reference interval data.

Table 2. Select biochemical results of a dog with histiocytic sarcoma and thyroid carcinoma. Variable Urea (mg/dL) Creatinine (mg/dL) Total Protein (g/dL) Albumin (g/dL) Globulins (g/dL) Calcium (mg/dL) Phosphorous (mg/dL) Sodium (mEq/L) Potassium (mEq/L) Chloride (mEq/L) Bicarbonate (mmol/L) Anion Gap Magnesium (mmol/L)

Patient Value

Reference Interval

85.0 3.2 5.8 2.9 2.9 18.6 10.8 149.0 4.4 111.0 13.0 29.4 1.5

8.0–32.0 0.4–1.2 5.4–6.8 3.2–4.1 2.0–3.2 10.0–11.9 2.6–5.8 142.0–149.0 3.1–4.8 109.0–117.0 14.0–22.0 13.0–22.0 0.7–0.9

Bold values indicate out-of reference interval data

present. The kidneys were mottled red and tan, and there was a focal acute infarct in the left cortex. The anal sacs were grossly unremarkable. Tissue samples were fixed in 10% buffered formalin, paraffin embedded, and routinely processed for histologic examination. Multiple organs, including the diaphragm, liver, tracheobronchial lymph nodes, and lung, were infiltrated by highly pleomorphic, neoplastic, polygonal to elongated cells, arranged in interlacing streams and occasional whorls. Neoplastic cells had variably distinct cellular borders, abundant lightly eosinophilic, occasionally vacuolated cytoplasm, and polygonal to oval nuclei with vesicular chromatin. There was marked anisokaryosis with frequent multi-nucleation (up to 12 nuclei), bizarre nuclei, and 46 mitotic figures per 10 4009 fields. Multifocal vascular and lymphatic invasion were observed. The right thyroid gland was expanded and effaced by neoplastic cuboidal to polygonal cells forming solid nests, follicles, and tubules separated by variably thick collagenous septa (Figure 1A). The left thyroid gland

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was replaced by a focal cystic structure lined by dense collagen and a single layer of cuboidal to flattened epithelium. Within the cystic structure was a mass composed of neoplastic cuboidal to polygonal cells forming follicles, tubules, and trabeculae. Neoplastic cells in both thyroid glands contained abundant eosinophilic cytoplasm, round nuclei, and coarse to dense chromatin, and exhibited moderate anisocytosis and anisokaryosis; mitoses were not apparent. Multifocal to coalescing areas of necrosis with hemorrhage and fibrin were also noted. The kidneys had microscopic changes, including marked, chronic multifocal segmental membranous glomerulonephritis and a unilateral, cortical infarct. There was no evidence of lymphoma or apocrine gland adenocarcinoma of the anal sacs. Based on the histologic findings, the animal was diagnosed with bilateral thyroid carcinoma and presumptive histiocytic sarcoma with peritoneal sarcomatosis. Immunohistochemistry for CD18, cytokeratin, and vimentin was performed (University of Tennessee, College of Veterinary Medicine) to confirm the diagnosis of histiocytic sarcoma. Histologic sections (5 lm) were deparaffinized and rehydrated, and antigen retrieval was performed with Proteinase K-induced Epitope Retrieval (Dako, Carpenteria, CA, USA) for CD18 and cytokeratin, and with heat-induced Epitope Retrieval (Dako) for vimentin. Endogenous peroxidase block with 3% hydrogen peroxide was performed for CD18. Serum-free protein block (Dako) was used for cytokeratin and vimentin. Primary antibodies were against CD18 (Peter Moore, University of California, Davis, CA, USA, clone Ca16.3c10, diluted 1:10), cytokeratin (Dako, clone AE1/AE3, diluted 1:800), and vimentin (Biogenix, Freemont, CA, USA, clone V9, diluted 1:8000). The secondary reagent for all primary antibodies was a labeled polymer system (EnVision+System HRP Anti-mouse, Dako). The chromogen for all 3 markers was 3,30 -diam

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Table 3. Endocrinology results in a dog with histiocytic sarcoma and thyroid carcinoma. Variable Ionized Calcium (pmol/L) Parathyroid hormone (pmol/L) Parathyroid hormone-related protein (pmol/L) 25-Hydroxyvitamin D (nmol/L)

Patient Value

Reference Interval

1.89 0.6 21.0 33.0

1.25–1.45 0.5–5.80 0.0–1.0 60.0–215.0

Bold values indicate out-of reference interval data.

A

B

Figure 1. (A) Histologic section of a thyroid carcinoma from a dog. Neoplastic cuboidal cells form follicles containing scant to moderate amounts of colloid (amorphous eosinophilic material). H&E. 9100 objective. (B) Histologic section of a thyroid carcinoma from a dog. There is mild, widespread cytoplasmic staining for parathyroid hormone-related peptide immunohistochemistry, 3,30 - diaminobenzidine tetrahydrochloride chromogen. 9100 objective.

inobenzidine tetrahydrochloride (DAB, Dako), and slides were counterstained with hematoxylin. Neoplastic cells from the presumptive histiocytic sarcoma were immunoreactive for CD18 and vimentin, and negative for cytokeratin, confirming the initial diagnosis of histiocytic sarcoma (not shown). To determine which tumor was secreting PTH-rP, immunohistochemistry for PTH-rP was performed at The Ohio State University College of Veterinary Medicine. Histologic sections (5 lm) were deparaffinized, hydrated, and pretreated using target antigen retrieval solution (Dako). Sections were incubated with antiPTH-rP antibody (Dako, diluted 1:100), rinsed, and incubated with biotinylated rabbit anti-goat antibody (Vector Laboratories, Burlingame, CA, USA, diluted 1:200). Following rinsing, sections were incubated with VECTASTAIN Elite ABC complex (Vector Laboratories). The chromogen was 3,30 diaminobenzidin tetrahydrochloride (DAB) (Dako); sections were counterstained with hematoxylin. Both thyroid carcinomas had mild, diffuse cytoplasmic immunoreactivity (Figure 1B), while the histiocytic sarcoma exhibited minimal, nonspecific, background staining (image not shown). These results suggest that the increased serum levels of PTH-rP and resulting hypercalcemia were caused by PTH-rP secretion by the bilateral thyroid carcinoma.

Discussion Paraneoplastic hypercalcemia can be caused by a number of mechanisms, including secretion of PTH-rP, cytokines, or vitamin D by the tumor, and primary or metastatic, tumor-induced, boney lysis.1,2 Previous reports (1992) observed increased PTH-rP and total calcium serum concentrations in a dog with thyroid carcinoma, suggesting a cause and effect. However, necropsy data were not reported, and up to a third of dogs with thyroid carcinoma may have a second malignancy, introducing some doubt as to the strength of the association.3 A more recent case report describes a dog with unilateral thyroid carcinoma and hypercalcemia.4 The hypercalcemia resolved after removal of the carcinoma, suggesting an association, but PTH-rP was not measured. In human medicine, there has been at least one case report linking thyroid carcinoma with PTH-rP and hypercalcemia using both immunohistochemistry and plasma tests.5 This current case report provides similar information for dogs. The data are unique in that PTH-rP immunoreactivity was demonstrated within neoplastic thyroid cells, and concurrently measured serum concentrations of PTH-rP and ionized calcium were increased. A study examining PTH-rP expression in nondiseased tissues from dogs found that thyroid follicular epithelial cells do not normally express PTH-rP, providing additional support for atypical protein expression by the thyroid carcinoma in this case.6 The dog in this study was diagnosed with histiocytic sarcoma in addition to thyroid carcinoma. A shortcoming of this report is that the patient’s serum vitamin D (1, 25-dihydroxyvitamin D) concentration was not measured. Some cells of monocyte lineage express 1-a-hydroxylase, permitting conversion of 25hydroxyvitamin D to 1,25-dihydroxyvitamin D.7 As granulomatous inflammation can cause increased total and ionized calcium concentrations, it seems plausible that a histiocytic malignancy could also produce vitamin D, resulting in hypercalcemia.8 Hypercalcemia has been noted in at least one dog with malignant histiocytosis, although the mechanism was not pursued.9

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Serum 25-hydroxyvitamin D concentration was low (33.0 nmol/L [reference interval 60–215 nmol/L]) in the dog described here suggesting, but not confirming, hypovitaminosis D. The dog had both hypercalcemia and hyperphosphatemia. Increased PTH-rP was considered the most likely cause of hypercalcemia. Renal failure can also result in hypercalcemia and may have been a contributing factor. However, a urine specific gravity was not obtained, limiting assessment of tubular function. Blood gas information was also not obtained and the extent to which the dog’s acid–base status contributed to the increased ionized calcium concentration is not clear. Decreased renal excretion of phosphorous was considered the most likely cause of hyperphosphatemia. Clinical dehydration and azotemia were consistent with decreased glomerular filtration rate, and the dog had histologic evidence of renal pathology. Increased PTH-rP typically is associated with hypophosphatemia or normophosphatemia.10 Hypervitaminosis D causes hypercalcemia and hyperphosphatemia, and cannot be excluded as a cause of these biochemical abnormalities in this dog, but direct evidence is lacking.11 This report is the first in the veterinary literature to provide immunohistochemical data demonstrating PTH-rP protein expression within neoplastic thyroid follicular cells and concurrent increased serum concentrations of ionized calcium and PTH-rP. Data provided here indicate that thyroid carcinomas can cause humoral hypercalcemia of malignancy via production and secretion of PTH-rP.

Acknowledgements The authors are grateful to Dr. Thomas J. Rosol, DVM, PhD, DACVP, and his laboratory staff, College of Veterinary Medicine, The Ohio State University, for performing and interpreting the PTH-rP immunohistochemistry. Dr. Shelley Newman, DVM, DVSc, DACVP and Dr. Danielle Reel, DVM, DACVP, College of Veterinary Medicine, the University of Tennessee, are appreciated for review of the immunohisto-

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chemistry methods and acknowledged as the attending pathologist on this case, respectively.

References 1. Rosol TJ, Nagode LA, Couto CG, et al. Parathyroid hormone (PTH)-related protein, PTH, and 1,25-dihydroxyvitamin D in dogs with cancer-associated hypercalcemia. Endocrinology. 1992;131:1157–1164. 2. Bergman P. Topical review: paraneoplastic hypercalcemia. Top Companion Anim Med. 2012;27:156–158. 3. Rebhun RB, Thamm DH. Multiple distinct malignancies in dogs: 53 Cases. J Am Anim Hosp Assoc. 2010;46:20–30. 4. Lane AE, Wyatt KM. Paraneoplastic hypercalcemia in a dog with thyroid carcinoma. Can Vet J. 2012;53:1101– 1104. 5. Hiroshi I, Yasuhro O, Norihiko A. Anaplastic thyroid carcinoma with humoral hypercalcemia of malignancy (HHM): an autopsy case report. Endocr J. 2004;51:303– 310. 6. Gr€ one A, Werkmeister JR, Steinmeyer CL, Capen CC, Rosol TJ. Parathyroid hormone-related protein in normal and neoplastic canine tissues: immunohistochemical localization and biochemical extraction. Vet Pathol. 1994;31:308–315. 7. Chun RF, Peercy BE, Orwoll ES, Nielson CM, Adams JS, Hewison M. Vitamin D and DBP: the free hormone hypothesis revisited. J Steroid Biochem Mol Biol. 2014;144:132–137. Available at: http://www.sciencedirect.com/science/article/pii/S0960076013001866. 8. LeBlanc CJ, Echandi RL, Moore RR, Souza C, Grooters AM. Hypercalcemia associated with gastric pythiosis in a dog. Vet Clin Pathol. 2008;37:115–120. 9. Uehlinger P, Glaus T, Hauser B, Reusch C. Differential diagnosis for hypercalcemia – A retrospective study of 46 dogs. Schweiz Arch Tierheilkd. 1998;140:188–197. 10. Law F, Ferrari S, Rizzoli R, Bonjour JP. Parathyroid hormone-related protein and calcium phosphate metabolism. Pediatr Nephrol. 1993;7:827–833. 11. Alshahrani F, Aljohani N. Vitamin D: deficiency, sufficiency and toxicity. Nutrients. 2013;5:3605–3616.

Vet Clin Pathol 44/2 (2015) 249–252 ©2015 American Society for Veterinary Clinical Pathology

Hypercalcemia and parathyroid hormone-related peptide expression in a dog with thyroid carcinoma and histiocytic sarcoma.

A 9.5-year-old, male castrated Walker Hound was presented for evaluation of progressive weakness, anorexia, and weight loss. Imaging revealed multiple...
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