Resumen de Diagnosis and treatment of chronic lymphocytic leukemia in a bat-eared fox (Otocyon megalotis)
Case Description—Severe lymphocytosis and leukocytosis were detected during examination of a 10-year-old sexually intact male bat-eared fox (Otocyon megalotis) with regionally extensive alopecia.
Clinical Findings—A CBC revealed severe leukocytosis (39,100 leukocytes/μL) and marked lymphocytosis (90%). A blood smear consisted predominantly of intermediate-sized lymphocytes and few large lymphocytes, with mild to moderate nuclear atypia. These findings were highly suggestive of chronic lymphocytic leukemia (CLL). Cytologic evaluation of bone marrow aspirates revealed no evidence of overt malignancy, with 10% of all cells identified as small to intermediate-sized mature lymphocytes.
Treatment and Outcome—Treatment with chlorambucil and prednisone administered orally over a 1.8-year period decreased the leukocyte and lymphocyte counts to within reference intervals with no adverse effects. Although repeated flow cytometry revealed evidence of residual disease, the fox remained free of clinical disease, and WBC counts were within reference intervals for this species. At 22 months after initial evaluation, the fox was euthanized because of debilitating arthritis. No evidence of CLL was detected grossly or histologically during necropsy.
Clinical Relevance—To the authors’ knowledge, this was the first report of CLL in a bat-eared fox and first successful treatment in a nondomestic carnivore. Treatment in accordance with a chemotherapeutic protocol successfully resolved the leukocytosis and lymphocytosis with no serious adverse effects. Description of this fox and the treatment protocol should provide a valuable reference for future cases in this and other nondomestic canine species.
A 10-year-old 3.96-kg (8.71-lb) sexually intact male bat-eared fox (Otocyon megalotis) was examined because of regionally extensive alopecia. The fox was housed indoors, and the alopecia was suspected to be attributable to seasonal overgrooming (self-inflicted or from a conspecific animal).
The fox was anesthetized, and a comprehensive examination was performed (day 1). Alopecia was evident on the muzzle and feet. Results of cytologic evaluation of skin scrapings obtained from alopecic areas were unremarkable, and examination of biopsy specimens of the affected skin revealed chronic nonspecific dermatitis compatible with self-trauma (psychogenic alopecia or excessive grooming). Radiography revealed evidence of degenerative joint disease in the left elbow joint and bridging spondylosis in the lumbar portion of the vertebral column.
A CBC revealed severe leukocytosis (39,100 leukocytes/μL; reference mean ± SD,a 6,705 ± 2,770 leukocytes/μL) and marked lymphocytosis (90%; absolute count, 35,190 lymphocytes/μL; reference mean,a 1,930 ± 1,359 lymphocytes/μL). Microscopic examination of a blood sample was performed by a veterinary clinical pathologist (who also reviewed all subsequent blood samples); that examination revealed predominantly a monomorphic population of lymphocytes of intermediate size and few large lymphocytes, with mild to moderate nuclear atypia. Some lymphocytes had a prominent nucleolus, and only a few lymphoblasts were detected. In combination, cell counts and microscopic appearance of the blood smear were highly suggestive of lymphocytic leukemia. Because of the differentiated nature and intermediate size of the lymphocytes, CLL was considered the most likely diagnosis. Stage V lymphoma was considered less likely because of a lack of clinical signs or lymphadenomegaly.1 Mild hypoalbuminemia (2.5 g/dL; reference mean ± SD,a 3.2 ± 0.4 g/dL) with a moderate increase in the globulin concentration (4.6 g/dL; reference mean,a 2.8 ± 0.7 g/dL) was also detected. Ehrlichia canis may also cause lymphocytosis, but results of an antibody test for E canis were negative.
To confirm persistence of leukocytosis and further define the cause of lymphocytosis, another hematologic evaluation was performed 3 weeks later (day 22). The fox remained free of clinical signs of illness, but severe leukocytosis (33,500 leukocytes/μL) and marked lymphocytosis (87%; absolute count, 29,150 lymphocytes/μL) persisted (Figure 1). Cell morphology of a blood smear was cytologically similar to that of the sample obtained on day 1. The mild protein abnormalities persisted. An 18-gauge Rosenthal bone marrow biopsy needleb was used to aseptically obtain a bone marrow aspirate from the proximal portion of the right humerus. Cytologic examination of the bone marrow aspirate revealed appropriate numbers of lymphocytes, and there were no abnormalities detected in the overall morphology. Mildly increased numbers of plasma cells and macrophages were supportive of a reactive immune-stimulating condition but were not definitive for CLL. However, peripheral leukocytosis without bone marrow involvement was consistent with CLL.
View larger version(26K) Figure 1— Lymphocyte (light gray bars) and WBC (dark gray bars) counts before and during administration of chemotherapeutics to a bat-eared fox (Otocyon megalotis) with CLL. The fox was initially examined because of regionally extensive alopecia and results of the first CBC, which was performed on day 1. Administration of chemotherapeutics, consisting of chlorambucil (0.26 mg/kg [0.12 mg/lb], PO, q 48 h) and prednisone (1.26 mg/kg [0.57 mg/lb], PO, q 48 h), was initiated on day 59 (arrow).
The fox continued to have no clinical signs of illness. A follow-up examination was performed on day 43 to reevaluate the fox's hematologic status and to perform flow cytometry to better define the cause of the lymphocytosis. Severe leukocytosis (43,000 leukocytes/μL) and marked lymphocytosis (89%; absolute count, 38,540 lymphocytes/μL) were still evident, and cell numbers were higher than in previous samples (Figure 1). Cytologic examination of a blood smear revealed small to intermediate-sized lymphocytes with mild nuclear atypia. No lymphoblasts were detected in the sample. Flow cytometry revealed that the majority of T cells expressed the pan–T-lymphocyte markers CD5 (96%) and CD3 (97%). Two percent of the cells coexpressed CD3 and CD4, which is consistent with the expression for T-helper lymphocytes. None of the cells expressed the cytotoxic T-lymphocyte marker CD8. One percent of the cells expressed the B-cell marker CD21. None of the cells expressed CD45, a pan-leukocyte marker, which is also highly species specific. Fewer than 5% of the cells expressed CD34 (a stem cell marker), CD14 (a monocyte marker), or CD18 (a marker of granulocytes and monocytes). Although flow cytometric techniques have not been validated for this species, the marked lymphocytosis, cell morphology, and homogeneous population of T cells were consistent with T-cell CLL. Because few or no cells expressed CD4 and CD8, further determination of the subtype of T-cell leukemia could not be performed.
Considering that IgM monoclonal gammopathies can be associated with CLL in domestic dogs, serum globulins concentrations of the fox were evaluated by means of serum protein electrophoresis.c The IgA concentration was low (29 mg/dL; reference interval, 35 to 270 mg/dL), but the IgM concentration (189 mg/dL) was within the laboratory reference interval for domestic dogs (100 to 400 mg/dL). The IgG concentration (3,600 mg/dL) was high, compared with the reference interval for domestic dogs (670 to 1,650 mg/dL).
Abdominal ultrasonography revealed that the spleen had a mixed lacy echotexture with multiple 3- to 8-mm hypoechoic nodules throughout the parenchyma. The liver appeared slightly mottled with small (2 to 4 mm), evenly distributed hyperechoic areas. Cytologic examination of ultrasound-guided fine-needle aspirates of the spleen revealed predominantly intermediate-sized lymphocytes (approx 90%) and fewer small lymphocytes (10%) amid a background of admixed proteinaceous material and blood. Lymphocytes had scant amounts of basophilic cytoplasm and large round nuclei with finely stippled chromatin and mild anisokaryosis. No mitotic figures were detected. Findings were considered similar to those of previous peripheral blood smears and were suspected to be representative of blood contamination.
The fox continued to have no signs of clinical illness and was reevaluated on day 55. Physical examination revealed that hair had regrown in all previously alopecic areas. Severe leukocytosis (49,100 leukocytes/μL) and lymphocytosis (77%; absolute count, 37,810 lymphocytes/μL) were still evident (Figure 1).
A veterinary oncologist was consulted. It was determined that the bat-eared fox should receive chemotherapy for the subclinical CLL because of persistent and progressive leukocytosis and lymphocytosis. On day 59, long-term treatment with chlorambucild (0.26 mg/kg [0.12 mg/lb], PO, q 48 h) and prednisonee (1.26 mg/kg [0.57 mg/lb], PO, q 48 h) was initiated.
Thirty-three days after starting treatment (day 92), the fox was again evaluated. The CBC revealed a decreased, but still high, total WBC count (19,100 WBCs/μL) and absolute lymphocyte count (74%; absolute count, 14,130 lymphocytes/μL; Figure 1). Analysis of a blood smear revealed lymphocytes were predominantly small to intermediate in size with no lymphoblasts. Albumin (3.1 g/dL) and globulin (3.5 g/dL) concentrations had returned to within reference intervals. However, ALT activity (638 U/L; reference mean ± SD,a 83 ± 55 U/L) was increased, which was attributed to corticosteroid administration as part of the chemotherapeutic protocol.1 Findings on splenic ultrasonography were unchanged. Ultrasound-guided fine-needle aspirates of the spleen and a bone marrow aspirate from the proximal portion of the right humerus were obtained. Cytologic examination of both samples revealed that they contained peripheral blood contamination and were nondiagnostic. Because of the improvement in the leukocytosis and lymphocytosis, it was decided that the chemotherapy should be continued.
The fox remained free of clinical signs of disease and was examined again on day 135. Total WBC count (23,900 WBCs/μL) and lymphocyte count (72%; absolute count, 17,210 lymphocytes/μL) were slightly increased from those of the preceding sample but were still markedly improved from the leukocytosis before initiation of treatment (Figure 1). The blood smear contained increased numbers of small lymphocytes, a few intermediate-sized lymphocytes with some nuclear atypia, and no lymphoblasts. Alanine aminotransferase activity (364 U/L) was still high but had decreased from the value for the preceding sample. Cytologic examination of a bone marrow aspirate revealed approximately 3% small lymphocytes and 3% to 4% plasma cells, which was suggestive of mild plasma cell hyperplasia with no overt evidence of lymphocytic leukemia. Despite a mild increase in WBC and lymphocyte counts, oral administration of chemotherapeutics was continued at the same dose and dosing frequency because the fox continued to have no clinical signs of disease.
Additional follow-up examinations were performed on days 281, 504, and 603. The CBCs performed on each of those days revealed steady decreases of total WBC and lymphocyte counts to within reference intervals (Figure 1). Blood smears were obtained on each of those days, and examination revealed results were within reference limits with no lymphoblasts. The ALT activity on those days ranged between 288 and 672 U/L. Cause of the variation in ALT activity was unclear but may have been related to corticosteroid treatment or liver involvement. Cytologic examination of a bone marrow preparation on day 281 revealed a few lymphocytes (approx 1% to 2% of all cells) that were small and morphologically normal. Ultrasonography findings for the spleen remained consistent on days 281 and 603, with rounded margins and a diffuse heterogeneous appearance, which was an improvement from the appearance on previous ultrasonographic examinations.
Flow cytometry on day 504 revealed that the majority of lymphocytes (91%) expressed the pan–T-lymphocyte markers CD5 and CD3, which was similar to results for a previous sample. A greater percentage (5%) expressed CD4. Similar to previous results, a low percentage of cells expressed CD8, and none of the cells expressed CD45; small numbers of cells expressed CD21, CD34, CD14, and CD18. Overall, flow cytometry percentages were similar to those for previous samples, but with a lower percentage of T cells and a much lower absolute T-cell count. Most lymphocytes were T cells, and thus findings were considered compatible with residual disease. Oral administration of chemotherapeutics was continued because of the sustained improvement of clinical values and because results of flow cytometry were consistent with residual disease.
At 22 months (660 days) after the initial diagnosis, the fox's quality of life had declined because of progressive debilitating osteoarthritis and spondylosis, which prompted a decision for euthanasia. The fox remained subclinically affected by CLL. Examination of a blood sample collected immediately before the fox was euthanized revealed mild leukopenia, with a total WBC count of 3,300 WBCs/μL and a lymphocyte count within the low portion of the reference range (19%; absolute count, 627 lymphocytes/μL). The decreased WBC and lymphocyte counts were likely related to chronic administration of chemotherapeutic drugs. Because of the low numbers of lymphocytes in the blood sample, flow cytometry results were interpreted with caution. A predominance of T lymphocytes remained in lower percentages than for previous samples, with 54% of the cells expressing CD5 and 65% expressing CD3, both pan–T-lymphocyte markers. This could have been evidence of residual disease, but it may also have been an unremarkable finding, considering that T cells predominate in most species. Because of the low lymphocyte counts and a lack of flow cytometric lymphocyte immunophenotyping for this species, it was not possible to differentiate between residual disease and concentrations of T-cell markers typical of healthy animals.
Postmortem examination revealed age-related lesions, but no gross lesions were detected that were associated with CLL. Histologic examination revealed mild hepatopathy attributable to chronic corticosteroid treatment. Results for examination of sections of spleen and bone marrow were within anticipated limits, and there was a lack of evidence of neoplastic disease. To further rule out causes for the chronically increased numbers of lymphocytes, serum samples that had been obtained at the time of the first and second examinations (days 1 and 22) and stored frozen were thawed and tested for antibodies against borreliosis (Lyme disease), E canis, Ehrlichia ewingii, Anaplasma platys, and Anaplasma phagocytophilum by use of an ELISAf; against Leishmania donovani and Leishmania infantum by use of an immunofluorescence assay; and against Rocky Mountain spotted fever by use of antibody dilution titer. All test results were negative.
