Concurrent chronic lymphocytic leukemia and primary hyperparathyroidism in a mule

Abstract A 26‐year‐old mule gelding was evaluated for chronic weight loss and decreased appetite. The mule had been losing weight and intermittently hypophagic for approximately 7 months. Laboratory analysis of whole blood and plasma identified severe total hypercalcemia, marked hypophosphatemia, markedly increased parathyroid hormone concentration, and marked lymphocytosis. A sestimibi scan intended to identify parathyroid gland tissue was nondiagnostic. Results of flow cytometry and PCR for antigen receptor rearrangement (PARR) were consistent with a B cell lymphoproliferative disorder, likely chronic lymphocytic leukemia (CLL). Although not previously described concurrently, these conditions may sometimes arise together, complicating definition of the underlying mechanism for weight loss and hypercalcemia in aged equids.


| Second presentation
The gelding was bright and alert. Rectal temperature (36.7 C) was normal but heart (56 bpm) and respiratory (40 breaths per minute) rates were increased. The mule was still thin (BCS, 3/9) and weight had slightly decreased (408 kg). The EOTRH was unchanged. Symmetrical lymph node enlargements were evident, especially in the mandibular and inguinal regions. Bilaterally symmetrical, firm, $10 cm spherical masses were palpated in the pre-scapular region. Further testing for lymphoproliferative neoplasia included abdominothoracic and cervical ultrasonography, peritoneal fluid analysis, and fine needle aspiration of cervical lymph nodes. Ultrasonography disclosed increased peritoneal fluid and mesenteric lymphadenomegaly.
Peritoneal fluid nucleated cell count and protein concentration were normal; presence of lymphocytes similar to those seen in blood suggested that they likely resulted from lymphoproliferative neoplasia.
Enlarged cervical lymph nodes were ultrasonographically characterized by a mixed echogenic pattern. Aspirates of the right mandibular node and the lymph node at the right thoracic inlet were interpreted as reactive lymphoid hyperplasia. However, small cell lymphoma with leukemia was not entirely discounted.
wA flow cytometric immunophenotyping panel (Table 1)      Hyperparathyroidism is uncommon in equids and can be primary or secondary. [8][9][10][11][12] Whereas secondary hyperparathyroidism results from dietary factors that interfere with the oral bioavailability of calcium, renal secondary hyperparathyroidism have not been reported in this species. 13,14 Primary hyperparathyroidism results from parathyroid gland hyperplasia or adenoma, with the chief cells of ≥1 affected parathyroid glands secreting excessive PTH and dysregulating calcium and phosphorus homeostasis. 8,9,11 Increased PTH concentration results in increased osteoclast activity, osteoid resorption (osteopenia), increased renal calcium resorption, and urinary phosphorus wasting.
Diagnosis of PHPT in horses should be suspected if hypercalcemia and hypophosphatemia are identified in the absence of renal disease and cancer. As with this mule, affected equids commonly develop weight loss and inappetence. In other cases, lameness and headshaking may arise because of stimulated bone resorption. Diagnostic affirmation is obtained using endocrine testing (increased PTH concentration in the absence of increases in PTHrP, vitamin D, and creatinine concentrations). Hyperparathyroidism may be discovered incidentally because some affected equids do not exhibit clinical signs over many years. 15 This mule likely was affected for >2 years with only mild weight loss as the principal concern. Moreover, CLL, EOTRH, advancing age, and PPID probably contributed to the weight loss.
If a solitary adenomatous parathyroid gland is identified, surgical excision (parathyroidectomy) can be curative. 9,11 Recently, percutaneous ultrasound-guided ethanol ablation was described as an alternative treatment. 16 However, treatment success is limited by the fact that positive identification of an affected parathyroid gland is challenging in horses. As with humans and dogs, horses have 2 pairs of parathyroid glands. Being located anywhere along the length of the carotid arteries from the cranial cervical region to the thymus makes detection of a parathyroid adenoma affecting caudal parathyroid glands challenging using ultrasonography alone. 8,9 The sensitivity of 99m Tc-sestamibi nuclear medicine scans for detecting parathyroid adenomas is unknown in horses but is reported to be $90% in humans 17 and 17% in dogs. 18 Successful employment of 99m Tc-sestamibi scintigraphic scanning to detect parathyroid adenomas in affected horses has been reported in 13 of 16 (81%) published cases. [8][9][10][11]16 As reported in other equine cases, 99m Tc-sestamibi nuclear medicine scanning failed to identify an abnormal parathyroid gland in this mule. 8 Explanations for failure of 99m Tc-sestamibi to identify a cause for high PTH concentration in human patients include patient motion, ectopy, small glands (<500 mg), radionuclide uptake interference by comorbid thyroid gland neoplasia, and parathyroid carcinoma. 19,20 Even on necropsy, an enlarged parathyroid gland could not be identified in this mule; it is likely that extensive lymphadenopathy in the area complicated the examination.
Chronic lymphocytic leukemia and small lymphocytic lymphoma (SLL) are grouped as a single entity in the WHO classification schemes of hematopoietic neoplasms for both humans and horses. 21,22 This is partly because, morphologically, the neoplastic cells are identical. However, oncologists in human medicine classify the disease as CLL when a clonal peripheral blood lymphocytosis >5 Â 10 3 cells/μL is present for at least 3 months. 23 The same is true in veterinary oncology, and for these reasons, this mule was diagnosed with B-cell CLL. As in our case, lymphadenomegaly is commonly observed in patients with CLL.
Chronic lymphocytic leukemia is rare in equids and has never been reported in a mule. improved our ability to subcategorize lymphomas and leukemias. 32,33 Lymphocytes in this mule were characterized by flow cytometry and expressed CD44 (91%) and CD21 (66%), most consistent with B cell CLL. Polymerase chain reaction for antigen receptor rearrangement also was performed to confirm clonal expansion of B cells in lymph nodes. 32 With concurrent hypercalcemia and CLL in this case, there was concern for paraneoplastic hypercalcemia, but PTHrP concentration was normal whereas PTH concentration was increased. 34 Parathyroid hormone may be secreted by neoplastic tissue, and thus immunohistochemical staining for PTH was performed on neoplastic lymph nodes. [34][35][36][37] No stain uptake was noted. It therefore was concluded that ectopic parathyroid tissue was present, albeit not identified in this case.
A limitation of our case report is that the antibodies used for flow cytometry and immunohistochemistry have been validated in the horse, but their performance in a mule is unknown. Ideally, tissues from a control healthy mule would have been evaluated concurrently, but such samples were not available. However, the concordant data obtained from flow cytometry, immunohistochemistry (using different B cell markers and antibody clones) and PARR is compelling evidence that the assays were reliable for the diagnosis of a B cell neoplasm in this mule.

| SUMMARY
Ours is the first reported case of CLL in a mule, and concurrent PHPT was particularly interesting. The B cell CLL in this case progressed slowly, which is contrary to previous reports of the same disease in horses but similar to the clinical course in dogs and humans. Hyperparathyroidism can present concurrently with neoplasia and should be ruled out as a differential diagnosis in equids with cancer.

ACKNOWLEDGMENT
University funded clinical case. No external funding was provided.