Abstract
CASE DESCRIPTION
A 17-year-old Friesian gelding was examined at a referral hospital because of a 1-month history of mild exercise intolerance and marked lymphocytosis.
CLINICAL FINDINGS
Physical examination revealed no peripheral lymphadenopathy or other abnormalities. Results of an abdominal palpation examination per rectum and thoracic and abdominal ultrasonographic examinations were unremarkable. B-cell chronic lymphocytic leukemia (CLL) was diagnosed on the basis of severe lymphocytosis and positive expression of the B-cell marker CD20 by lymphocytes in the bone marrow and peripheral blood.
TREATMENT AND OUTCOME
Treatment with prednisolone (2 mg/kg [0.9 mg/lb], PO, every other day) and chlorambucil (20 mg/m2, PO, every 3 weeks for 2 doses, then every 2 weeks) was initially associated with improvement in clinical signs and a decrease in the lymphocyte count. However, 3 weeks after administration of the first dose of chlorambucil, the lymphocyte count began to increase. One week later, the horse developed episodes of recurrent fever and the lymphocyte count continued to increase. Despite continued administration of the prednisolone-chlorambucil protocol, the horse's clinical condition deteriorated rapidly, and it was euthanized 6 weeks after initial examination at the referral hospital because of a poor prognosis. A necropsy was not performed.
CLINICAL RELEVANCE
B-cell CLL has been infrequently described in horses. This report was the first to describe the use of chemotherapy, albeit unsuccessful, for the treatment of B-cell CLL in a horse. This information should be useful for guiding expectations for prognosis and management of other horses affected with the disease.
A 17-year-old Friesian gelding was examined at a referral hospital because of a 1-month history of mild exercise intolerance and persistent lymphocytosis. Results of a CBCa (with an automated cell differential count) and serum biochemical analysis performed by the referring veterinarian 3 weeks earlier revealed mild anemia (RBC count, 5.9 × 106 RBCs/μL [reference interval, 6.4 × 106 RBCs/μL to 10.4 × 106 RBCs/μL]; Hct, 29% [reference interval, 30% to 47%]) and leukocytosis (73.4 × 103 WBCs/μL; reference interval, 4.9 × 103 WBCs/μL to 11.1 × 103 WBCs/μL) characterized by lymphocytosis (64.8 × 103 lymphocytes/μL; reference interval, 1.5 × 103 lymphocytes/μL to 5.1 × 103 lymphocytes/μL) and a serum total protein concentration within reference limits (5.9 g/dL; reference interval, 5.8 to 7.8 g/dL). Results of a CBCa performed 2 weeks later indicated that the lymphocytosis (109.8 × 103 lymphocytes/μL) had worsened.
During initial examination at the referral hospital, the horse was bright and alert and the physical examination findings were unremarkable; there was no peripheral lymphadenopathy. A CBCa revealed that the mild anemia (RBC count, 5.8 × 106 RBCs/μL; Hct, 26%) persisted along with marked leukocytosis (133.0 × 103 WBCs/μL) and lymphocytosis (120.7 × 103 lymphocytes/μL) and hyperfibrinogenemia (850 mg/dL; reference interval, 100 to 400 mg/dL). The SAA concentrationb was also increased from reference limits (1,000 μg/mL; reference interval, 0 to 50 μg/mL). A rebreathing examination revealed mildly increased bronchovesicular sounds bilaterally and a slightly prolonged recovery. Results of an abdominal palpation examination per rectum and abdominal and thoracic ultrasonographic examinations were unremarkable. The spleen was considered to be of normal size during palpation per rectum, and both the spleen and liver had clinically normal echogenic appearances and were subjectively normal in size during the abdominal ultrasonographic examination.
Because of the clinical signs and evidence of worsening lymphocytosis, a lymphoid leukemia was suspected. A bone marrow aspirate was obtained from the sternum with the horse sedated but standing. The bone marrow aspirate and peripheral blood samples were submitted to the Animal Health Diagnostic Center at Cornell University, Ithaca, NY, for cytologic examination of the bone marrow, microscopic evaluation of a peripheral blood smear, and immunophenotyping of the leukemia if necessary. The horse was discharged from the hospital with no changes in management recommended pending diagnostic test results.
A peripheral blood smear was created and stained with a modified Wright stain and examined with a light microscope. Results indicated the presence of marked lymphocytosis (lymphocytes accounted for 97% of the total WBC count). Approximately 94% of the lymphocytes were of intermediate size (diameter, 10 to 12 μm) with clumped chromatin that was lighter in color than the chromatin of normal (healthy) lymphocytes (Figure 1). The remaining 6% of lymphocytes were large cells (diameter, 15 to 20 μm), with a low number of cells having deep blue cytoplasm and clumped chromatin indicative of reactive lymphocytes. Results of serum total protein electrophoresis revealed mild hypoalbuminemia (2.2 g/dL; reference interval, 2.3 to 3.5 g/dL) but no evidence of a monoclonal gammopathy (Figure 2). Examination of modified Wright-stained bone marrow aspirate smears revealed a marked increase in cellularity owing to infiltration of up to 45% intermediate-sized lymphocytes and a few large lymphocytes. Megakaryocytes were observed in adequate numbers. Myeloid and erythroid progenitors were identified, and maturation was complete and balanced in both cell lines (Figure 3). The cytologic results for the bone marrow aspirate samples in conjunction with those for peripheral blood smears were most consistent with a diagnosis of CLL with concurrent erythroid hypoplasia secondary to inflammatory disease. Flow cytometric analysis of the bone marrow aspirate sample indicated that the abnormal cells were negative for most T-cell (CD3, antibody clone F6G.3[G12]; CD4, clone HB61A; and CD8, clone HT14A) and B-cell (CD19, clone cz2.1; CD21, clone B-ly4; and IgM, clone CM7) lymphoid markers and myeloid markers (CD14, clone 105; CD172a [clone DH59B])1–4 but did express MHC class I (clone cz3.1) and MHC class II (clone cz11) markers.2,3 Immunocytochemical staining of peripheral blood and bone marrow aspirate smears revealed a strong positive reaction with a rabbit anti-CD20 polyclonal antibody, which was supportive of B-cell CLL.5
Representative photomicrographs obtained at low (20X; A) and high (100X; B) magnification of a peripheral blood smear from a 17-year-old Friesian gelding that was examined because of a 1-month history of mild exercise intolerance and persistent lymphocytosis. Notice that both smears contain an abundance of intermediate-sized (diameter, 10 to 12 μm) lymphocytes with clumped chromatin that was lighter in color than the chromatin of healthy (normal) lymphocytes (arrow). Lymphocytes accounted for approximately 97% of the total WBC count. The horse was determined to have B-cell CLL. Modified Wright stain; bar = 50 μm (A) and 10 μm (B).
Citation: Journal of the American Veterinary Medical Association 255, 6; 10.2460/javma.255.6.716
Qualitative serum total protein electrophoretogram (A) and corresponding histogram (B) for the horse of Figure 1. No evidence of a monoclonal gammopathy was present. For each constituent protein component, the relative concentration in the sample was positively associated with the intensity of the corresponding band in the electrophoretogram and the height of the peak in the histogram.
Citation: Journal of the American Veterinary Medical Association 255, 6; 10.2460/javma.255.6.716
Representative photomicrographs of bone marrow aspirate smears for the horse of Figure 1. A—Notice the extensive infiltration of small lymphocytes (arrows) and a small number of large lymphocytes (arrowhead) that resemble those seen in the peripheral blood smear. Myeloid and erythroid progenitors are also present, and maturation was complete and balanced in both cell lines. Modified Wright stain; bar = 20 μm. B—Lymphocytes had a diffuse positive reaction (brown pigment) with a rabbit polyclonal antibody against CD20 (a B-cell marker), which was supportive of a diagnosis of B-cell CLL. Nucleated RBCs (arrow) and myeloid precursors (arrowhead) served as internal negative controls. Immunocytochemical stain; bar = 10 μm.
Citation: Journal of the American Veterinary Medical Association 255, 6; 10.2460/javma.255.6.716
A chemotherapeutic protocol was developed on the basis of protocols used for dogs with B-cell CLL. Treatment with prednisolonec (2 mg/kg [0.9 mg/lb], PO, every other day) was initiated for 1 week before addition of chlorambucild (20 mg/m2, PO, every 3 weeks for 2 doses, then every 2 weeks). The owner reported that the horse appeared to have more energy after initiation of prednisolone administration. Results of a CBCe (with a manual 200-cell differential count) at the start of chlorambucil administration indicated mild improvement of the lymphocytosis (91.8 × 103 lymphocytes/μL; reference interval, 1.2 × 103 lymphocytes/μL to 4.9 × 103 lymphocytes/μL). Examination of a peripheral blood smear revealed small to intermediate-sized lymphocytes with stippled to clumped chromatin and no large cells; a low number of apoptotic cells were observed.
One week after initiation of chlorambucil administration, a CBCe and serum biochemical analysis revealed that the lymphocyte count (56.2 × 103 lymphocytes/μL) had decreased by 53% and the SAA concentration (200 μg/mL) had decreased by 80%, compared with those values at the time of the initial examination at the referral hospital. A mild increase in sorbitol dehydrogenase activity (16 U/L; reference interval, 0 to 11 U/L) and hypoalbuminemia (2.8 g/dL; reference interval, 3.0 to 3.7 g/dL) were also detected; the serum total protein concentration (6.3 g/dL) was within reference limits. The owner reported that the horse was doing well at home, and physical examination findings remained unremarkable.
Two weeks later (3 weeks after administration of the first chlorambucil dose [at the time of administration of the second dose of chlorambucil]), results of a CBCe and serum biochemical analysis indicated that both the lymphocyte count (80.5 × 103 lymphocytes/μL) and SAA concentration (> 1,000 μg/mL) had increased from previous results. The sorbitol dehydrogenase activity (17 U/L) remained mildly increased, mild hypoalbuminemia (2.8 g/dL) persisted, and serum total protein concentration (6.2 g/dL) remained stable.
The horse was evaluated 1 week after administration of the second dose of chlorambucil. The owner reported that the patient was doing well at home. On physical examination, the horse had a slight fever (rectal temperature, 39.1°C [102.4°F]; reference interval, 37.2° to 38.6°C [99.0° to 101.5°F]). Because of concerns that the fever was caused by a secondary infection, administration of sulfamethoxazole-trimethoprimf (30 mg/kg [13.6 mg/lb], PO, q 12 h) was initiated. Results of a CBCe revealed severe lymphocytosis (103.9 × 103 lymphocytes/μL) with concurrent neutrophilia (10.6 × 103 neutrophils/μL; reference interval, 2.6 × 103 neutrophils/μL to 6.5 × 103 neutrophils/μL) and monocytosis (3.5 × 103 monocytes/μL; reference interval, 0 × 103 monocytes/μL to 0.6 × 103 monocytes/μL), which were supportive of an inflammatory response. Serum biochemical analysis results indicated a mild increase in aspartate aminotransferase (499 U/L; reference interval, 0 to 441 U/L) and alkaline phosphatase (872 U/L; reference interval, 10 to 277 U/L) activities and a creatine kinase activity within reference limits, which were suggestive of hepatic injury. The serum total protein concentration was slightly increased (7.1 g/dL), hypoalbuminemia (2.7 g/dL) persisted, and serum globulin concentration (4.4 g/dL; reference interval, 2.8 to 4.9 g/dL) approached the upper limit of the reference interval. Sorbitol dehydrogenase activity was not measured at that time.
The horse continued to have recurrent fevers over the next 2 weeks. The highest rectal temperature (41.7°C [107°F]) was recorded during the physical examination performed 2 weeks after administration of the second dose of chlorambucil. At that time, the lymphocyte count (176.5 × 103 lymphocytes/μL) continued to be markedly increased; however, the horse had become neutropenic (1.8 × 103 neutrophils/μL) without a compensatory increase in band neutrophils. No toxic changes were observed during microscopic examination of a peripheral blood smear, and the lymphocytes were mostly small to intermediate in size (92%) with a low proportion (8%) of large cells, some of which were in mitosis. Owing to the poor prognosis associated with the deteriorating clinical condition and rapid development and progression of neutropenia, the horse was euthanized 6 weeks after initial examination at the referral hospital. A necropsy was not performed.
Discussion
The horse of the present report had rapid clinical progression of CLL. The leukemia was phenotyped as B cell in origin on the basis of the lack of expression of T-cell markers and positive immunocytochemical results for CD20 (a B-cell marker). The lack of expression of CD19, CD21, and IgM molecules in horses with B-cell leukemia has been previously reported.6 Some horses with lymphocytic leukemias have variable expression of T- and B-cell signature markers.1–4 The horse of this report was treated, albeit unsuccessfully, with a combination of prednisolone and chlorambucil.
Leukemia is defined as the neoplastic transformation of 1 or more hematopoietic cell lines within the bone marrow or other blood-forming tissues.7 In horses, as with other veterinary species, leukemia is characterized on the basis of the affected lymphoid and myeloid types. Lymphoid leukemias are further classified as mature cell or CLL and as precursor or ALL, both of which can be of either T-cell or B-cell lineage.7–9 Large immature (blast) lymphocytes are the dominant cell population in patients with acute leukemia, whereas mature lymphocytes dominate in patients with CLL. In human patients, CLL has 3 stages: an asymptomatic or indolent stage, a clinical stage during which treatment is required, and a malignant stage (blast crisis) during which immature cells increase and eventually predominate.8,9 Differentiation of primary lymphoid leukemia from the leukemic phase of advanced-stage lymphoma can be challenging.6 For the horse of the present report, the combination of marked lymphocytosis characterized by lymphocytes with mature morphological features, extensive bone marrow infiltration by tumor cells, and absence of evidence of extramedullary lymphoid tissue involvement was most compatible with CLL rather than a leukemic phase of lymphoma.6,8–10
Leukemia has been rarely reported in horses, with < 30 cases of primary lymphoid leukemia described in the published literature. Of the cases reported, no specific breed was overrepresented, and the ages of the affected horses ranged from 2 months to 23 years.5,11–19 Leukemia was not specifically classified as CLL or ALL for the horses of those reports,5,11–19 but on the basis of the clinical and laboratory findings provided, it appears that 13 horses had CLL and 13 horses had ALL. All horses with presumed CLL were ≥ 9 years old, whereas the horses with presumed ALL ranged in age from 2 months to 8 years. Of the 13 horses with presumed CLL, 9 were of T-cell origin, 2 were of B-cell origin, and 2 were not immunophenotyped. One of the 2 horses with B-cell CLL was a 20-year-old gelding that was referred to a tertiary hospital because of a 2-day history of clinical signs, thrombocytopenia, and lymphocytosis (70.2 × 103 lymphocytes/μL) characterized by slightly enlarged lymphocytes12; that horse was euthanized at the time of diagnosis. The other horse with B-cell CLL was a 20-year-old gelding that was referred to a tertiary hospital because of a 2-year-history of clinical signs, peripheral lymphadenopathy, thrombocytopenia, and lymphocytosis (67.4 × 103 lymphocytes/μL) characterized by intermediate-sized lymphoctyes14; that horse was treated with prednisolone and was euthanized after 3.5 months of treatment because of clinical deterioration.
The small number of horses with lymphoid leukemia described in the literature makes it difficult to identify any breed, sex, or age predisposition for the disease. The survival time for horses with presumed CLL that were not euthanized at the time of diagnosis ranged from 3 days to 5 years, whereas that for horses with presumed ALL that were not euthanized at the time of diagnosis ranged from 2 to 42 days.5,11–19 For the horse of the present report, the duration between initial diagnosis of lymphocytosis and euthanasia due to clinical deterioration was 2 months. The rapid disease progression observed in this horse was in contrast to the indolent nature of CLL observed in other species.
In human medicine, B-cell CLL is the second most common type of leukemia diagnosed in the United States and the most common hematopoietic malignancy diagnosed in developed countries.20,21 Chronic lymphocytic leukemia of B-cell origin occurs most commonly in males and the elderly. Staging of CLL in human patients is performed by use of both clinical and molecular scoring systems, which are applied to determine prognosis and guide treatment.22,23 The clinical course of CLL in human patients varies, and some patients with the indolent form of the disease have a life span similar to that of a healthy cohort,21 whereas others develop aggressive disease and survive for only a short period.20,24 Treatment for human patients with CLL is palliative rather than curative and is recommended only for patients with severe clinical disease at the time of diagnosis or that develop signs of rapidly progressive disease.25 Although chemotherapeutics such as chlorambucil have been the standard of treatment for human patients with CLL, they have been largely replaced by immunotherapeutics and chemoimmunotherapeutics in recent years.26
In dogs, chronic leukemias are less common than acute leukemias, and unlike in humans, CLL in dogs is more commonly of T-cell origin rather than B-cell origin.27,28 Results of 1 study29 suggest that B-cell CLL is more common in small-breed dogs, whereas other studies27,28,30 have failed to identify an obvious breed, sex, or age predilection for B-cell CLL. Immunophenotyping is available to help definitively classify lymphoproliferative disease in dogs and can be used to determine the prognosis for affected patients.31 In general, CLL in dogs is slowly progressive, and affected dogs typically have a good quality of life and mean survival time of 1 to 3 years. One dog with CLL reportedly survived without treatment for 23 months and was euthanized for reasons unrelated to the leukemia.32 Dogs with CLL can develop a blast crisis and, in rare instances, a form of high-grade lymphoma (analogous to Richter syndrome in human patients) that is rapidly progressive and poorly responsive to treatment.33 Dogs with CLL are typically treated with a combination of chlorambucil and prednisolone.30
The SAA concentration was serially monitored for the horse of the present report. The SAA concentration was markedly increased (1,000 μg/mL) at the time of the initial examination at the referral hospital, decreased by 80% (200 μg/mL) in conjunction with a decrease in the lymphocyte count after initiation of the prednisolone-chlorambucil protocol, and then became markedly increased (> 1,000 μg/mL) when the neoplastic cell count increased and large lymphocytes were once again observed in the peripheral blood. Serum amyloid A is a positive acute-phase protein that is present in only trace concentrations in healthy animals and increases in response to certain infectious and noninfectious processes in horses and other species.34 Descriptions of the SAA concentration in horses with confirmed neoplasia are limited. In 1 report,35 the SAA concentration was within the reference interval for a horse with a granulosa cell tumor and another horse with T-cell lymphoma of the liver. In a recent case series19 of 12 horses with acute leukemia, the measured SAA concentration exceeded the upper reference limit in all horses and was > 100 times the upper reference limit in some horses. Use of SAA as a biomarker for disease is limited by the inability to differentiate between infection and neoplasia as the cause of the inflammatory response. Factors that confounded interpretation of the SAA concentration for the horse of the present report were the administration of a corticosteroid (prednisolone), which likely suppressed the production of inflammatory proteins, and the potential for other nonneoplastic sources of inflammation as evidenced by the abnormal rebreathing examination findings and recurrent fever. Nevertheless, although more studies are necessary, serial measurement of SAA concentration might be clinically useful for monitoring response to chemotherapy or as an early indicator of progressive or aggressive neoplastic processes in horses.
To our knowledge, the present report was the first to describe the use of chemotherapy in a horse with CLL. The prednisolone and chlorambucil protocol used was analogous to standard-of-care protocols that provide a good chance of response in other species. The decision to initiate treatment in this horse was made on the basis of the fairly mild clinical signs, presence of anemia, and rapid progression of lymphocytosis.25,30,36 When treatment was attempted in other horses with B-cell CLL, it typically consisted of administration of a corticosteroid (dexamethasone or prednisolone) and supportive care.13–15 For the horse of the present report, the lymphocyte count initially decreased after the prednisolone-chlorambucil protocol was begun but then rapidly increased despite continued administration of the protocol. Approximately 1 month after initiation of chemotherapy, the disease began to rapidly progress, and the horse developed recurrent episodes of intractable fever and the reemergence of large lymphocytes in peripheral blood smears. Although the chemotherapy protocol was well tolerated by the horse, it was unsuccessful in controlling disease, perhaps because chlorambucil is ineffective against CLL in horses or the dose was inadequate or perhaps owing to other unknown factors. Use of the chemotherapy protocol in other horses with CLL will help elucidate the reasons it does or does not work in particular patients.
The horse of the present report was only 1 of 2 horses with B-cell CLL for which follow-up clinical information has been provided. Thus, this case report contributed to the understanding of the clinical course and management of B-cell CLL in horses. The disease was particularly aggressive in the horse of this report, which was unexpected given the typically indolent nature of CLL in other species such as dogs. A limitation of this report was the fact that a necropsy was not performed on the horse; therefore, it was not possible to rule out possible visceral involvement of the leukemia. Although none of the visceral organs were grossly enlarged on the basis of results of an abdominal palpation examination per rectum and abdominal ultrasonography, small or microscopic leukemic infiltrates into those tissues were possible. Regardless, this report provided information about the clinical progression of B-cell CLL in a horse, which can be used to inform expectations for prognosis and management of other horses affected with the disease.
Acknowledgments
The authors thank Dr. Paul Mountan for assistance with case management.
ABBREVIATIONS
| ALL | Acute lymphoblastic leukemia |
| CLL | Chronic lymphocytic leukemia |
| SAA | Serum amyloid A |
Footnotes
ProCyte Dx Hematology Analyzer, Idexx Laboratories, West-brook, Me.
Stablelab EQ-1 Handheld Reader, Epona Biotech Ltd, Sligo, Ireland.
Henry Schein Animal Health, Dublin, Ohio.
Wedgewood Pharmacy, Swedesboro, NJ.
Animal Health Diagnostic Center, College of Veterinary Medicine, Cornell University, Ithaca, NY.
Amneal Pharmaceuticals, Bridgewater, NJ.
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