Mature lymphocytosis (ie, 7,000/ L) in the blood of

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1 J Vet Intern Med 2005;19: Differentiating Benign and Malignant Causes of Lymphocytosis in Feline Bone Marrow Douglas J. Weiss Differentiation of benign and malignant causes of lymphocytosis in blood or bone marrow can be problematic. In the present study, reports of examinations of bone marrow from cats, submitted over an 8-year period, were reviewed to identify cats with increased numbers of small lymphocytes. Of 203 reports reviewed, 12 (5.9%) indicated increased numbers of small lymphocytes. Diagnoses for these cats included chronic lymphocytic leukemia (CLL; n 2), pure red cell aplasia (PRCA; n 4), immune-mediated hemolytic anemia (IMHA; n 3), thymoma (n 1), cholangiohepatitis (n 1), and fever of unknown origin (n 1). Several factors were identified that could be used to differentiate reactive lymphocytosis from CLL. Cats with CLL tended to be older, and lymphocytes were slightly larger and had cleaved or lobulated nuclei. Reactive lymphocytosis was associated with immunemediated anemias and inflammatory diseases. In reactive lymphocytosis, the proliferating lymphocytes were organized into lymphoid aggregates in bone marrow and were predominately B cells. Alternatively, in CLL and thymoma, the proliferating lymphocytes were diffusely distributed and were predominately T cells. Therefore, differentiation of the causes of lymphocytosis should include evaluation of signalment, concurrent disease conditions, lymphocyte morphology, lymphocyte distribution in bone marrow, and immunophenotype. Cat age, presence of severe anemia, and evidence of inflammatory disease also should be considered. Key words: Chronic lymphocytic leukemia; Immune-mediated hemolytic anemia; Pure red cell aplasia; Reactive lymphocytosis; Thymoma. Mature lymphocytosis (ie, 7,000/ L) in the blood of cats is a relatively frequent finding that presents a diagnostic challenge. 1 A variety of associated conditions, including physiologic causes, chronic antigenic stimulation, hypoadrenocorticism, hyperthyroidism, and chronic lymphocytic leukemia, have been identified. 1 4 Physiologic lymphocytosis is thought to result from redistribution of lymphocytes as a result of epinephrine release. 1 Lymphocyte numbers in the blood are usually less than 20,000/ L but have been reported to exceed 36,000/ L. 5 Lymphocytosis was observed in 2 of 10 cats with hypoadrenocorticism and 9% of cats with hyperthyroidism. 2,3 In cats with hypoadrenocorticism, lymphocytosis is probably the result of decreased cortisol production. Lymphocytosis can be associated with antigenic stimulation secondary to feline leukemia virus infection, feline herpesvirus type 1 infection, and recent vaccination. 1,4 The associated lymphocytosis is usually mild but in some cases can exceed 40,000/ L. 1,4 This type of lymphocytosis may be associated with the presence of reactive or blast-transformed lymphocytes. Lymphocytosis in bone marrow has been less well studied. Mean values for the percentage of lymphocytes in bone marrow of cats vary between 5% and 9%. 6 9 The highest values for healthy cats in several studies varied between 13% and 16%. 7 9 However, in a study of 15 healthy cats, 1 cat had 28% lymphocytes. 6 Discrete clusters of lymphocytes (ie, lymphoid aggregates) have been reported in cats with feline immunodeficiency virus infection. 10 Chronic lymphocytic leukemia (CLL) is a rare disease in cats. 11 Lymphocyte numbers in the blood are frequently From the Department of Veterinary Biomedical Sciences, College of Veterinary Medicine, University of Minnesota, St. Paul, MN. Reprint requests: Douglas J. Weiss, University of Minnesota, 1971 Commonwealth Avenue, St. Paul, MN 55108; weiss005@ umn.edu. Submitted January 5, 2005; Revised April 13, 2005; Accepted June 3, Copyright 2005 by the American College of Veterinary Internal Medicine /05/ /$3.00/0 high (25, ,000/ L), and lymphocytes are reported to be slightly larger and more homogeneous than normal small lymphocytes. 11 It has been suggested that bone marrow lymphocyte percentages exceeding 15% or 20% of total nucleated cells in bone marrow are suggestive of CLL. 11 In the present study, I reviewed bone marrow reports from cats evaluated over an 8-year period to identify cats with lymphocytosis. The case records and blood and bone marrow morphology were evaluated to differentiate associated benign and malignant conditions. Materials and Methods Bone marrow cytology reports from cats, evaluated at the University of Minnesota Veterinary Medical Center between July 1, 1996, and June 30, 2004, were reviewed. Of 203 bone marrow reports reviewed, 12 (5.9%) indicated the presence of increased numbers of small lymphocytes either in the aspiration smears or core biopsy specimens. Blood and bone marrow smears were reviewed for all cats, and core biopsy specimens were reviewed for 9 cats. A 1,000-cell differential cell count was performed on each marrow aspiration smear. Case records were also evaluated. Data extracted included signalment, clinical signs, all laboratory and other diagnostic tests, clinical diagnosis, pathologic diagnosis (when available), and outcome. Inclusion criteria for diagnosis of lymphocytosis were based on previously published reference values for cat bone marrow. 6 9 Inclusion criteria included the presence of greater than 16% small lymphocytes in cats with a normocellular or hypercellular bone marrow. Evaluating only cats with cellular bone marrow eliminated relative lymphocytosis resulting from erythroid or myeloid hypoplasia. Bone marrow cellularity was assessed by determining the cellularity of unit particles in aspiration smears and by determining the percentage of the marrow space occupied by hematopoietic tissue in core biopsy specimens. In each sample, a hemic cell-to-fat ratio of greater than 50% was defined as a cellular sample Results All 12 cats met the criteria for inclusion in the study. Clinical diagnoses for these cats included CLL (n 2), pure red cell aplasia (PRCA; n 4), immune-mediated hemolytic anemia (IMHA; n 3), thymoma (n 1), cholangiohepatitis (n 1), and fever of unknown origin (n

2 856 Weiss Table 1. Characteristics of cats with lymphocytosis in bone marrow. a Clinical Diagnosis No. of Cats Age (years) (x, range) Hematocrit (%) Blood Lymphocytes (/ L) Lymphocyte Morphology Bone Marrow Lymphocytes (%) Lymphoid Aggregates CLL ( ) 38 32,913 Cleaved or lobulated 40 No nuclei PRCA ( ) 6.3 5,950 Normal 36 Yes IMHA (1.5 12) ,879 Normal 23 Yes Thymoma ,990 Cleaved or lobulated 33 NE nuclei Cholangio-hepatitis Normal 39 NE Fever of unknown origin ,150 Normal 54 Yes CLL, chronic lymphocytic leukemia; PRCA, pure red cell aplasia; IMHA, immune-mediated hemolytic anemia; NE, not evaluated. a Values listed as values for an individual or a mean for 2 or more cats. b Two cats were less than 3 years old and one cat was 12 years old. 1). CLL was listed as a possible diagnosis in all 12 cats. None of the cats had a history of recent vaccination. CLL Fig 1. Blood smear from a cat with a diagnosis of chronic lymphocytic leukemia. Notice lymphocytes that are slightly larger than typical small lymphocytes and have cleaved or lobulated nuclei (arrows). Wright-Giemsa stain; Bar 10 m. Two cats with a diagnosis of CLL were 10 and 11.5 years old, respectively (Table 1). Both cats had a history of chronic vomiting and diarrhea, and 1 cat had repeated episodes of constipation. At the time of initial evaluation, neither cat was anemic, and lymphocyte counts in the blood were 23,256/ L and 42,570/ L, respectively. Lymphocyte morphology was similar in both cats. Lymphocytes were m in diameter, and 80% of cells had nuclear cleaving or lobulation (Fig 1). Bone marrow had normal or increased cellularity, and lymphocytes comprised 38% and 42% of total nucleated cells, respectively. Lymphocyte morphology was similar to that in peripheral blood. Bone marrow lymphocytes were immunophenotyped by labeling cells with anti-cd21 (B-cell marker) and anti-cd3 (T-cell marker). The immunophenotype of the lymphoid population indicated that the cells were of T-cell origin. A test for clonality was done on the peripheral blood of 1 cat. A clonal population was not detected. Both cats tested negative for feline leukemia virus and feline immunodeficiency virus. The total lymphocyte count continued to increase in both cats, reaching 53,200/ L and 120,000/ L, respectively. Both cats were treated with chlorambucil and prednisone and were alive at 8 months and 1.3 years after diagnosis, respectively. PRCA Four cats had a diagnosis of PRCA (Table 1). The cats were 1, 1.8, 2, and 2.3 years old, respectively. All cats had a history of severe weakness and pale mucous membranes. All cats were severely anemic, with hematocrits ranging between 5% and 9%. Lymphocyte counts in the blood were within the reference interval for cats, with the exception of 1 cat (lymphocyte count 14,200/ L). Lymphocytes in the blood consisted mostly of small lymphocytes with round nuclei and minimal cytoplasm. One of the cats had a moderate number of reactive lymphocytes. Tests for feline leukemia virus and feline immunodeficiency virus were negative. The bone marrow was characterized by normal or increased cellularity, with a marked increase in myeloid-toerythroid (M : E) ratio. No erythroid cells were seen in 3 cats, and only rare rubriblasts and prorubricytes were seen in the other cat. Lymphocyte percentages were 25, 30, 41, and 47%, respectively. Core biopsy specimens were characterized by multiple small aggregates of basophilic cells consistent with small lymphocytes (Fig 2). Immunohistochemistry was performed on core biopsy specimens of 2 cats with anti-cd19 as a B-cell marker. Labeling of cell aggregates with anti-cd19 confirmed that they were B cells. Immunophenotyping of marrow cells was performed on 2 cats. In both cats, lymphocytes were 95% B cells. Clonal analysis was performed on 2 cats. A clonal population was not detected in either cat. One cat was euthanized without treatment, and 3 cats were treated with immunosuppressive doses of prednisone. One cat died 7 days after initiation of treatment. The hematocrit of the other 2 cats returned to normal within 4 months, and the cats were alive at last contact (1.2 and 1.5 years after diagnosis, respectively).

3 Feline Lymphocytosis 857 a normal M : E ratio. Thirty-three percent of nucleated cells were lymphocytes. Most lymphocytes were small but had cleaved or lobulated nuclei similar to those in the peripheral blood. Immunophenotyping of blood and bone marrow indicated that 95% of lymphocytes were T cells. Lymphocyte numbers in the peripheral blood were 8,140/ L and 8,840/ L at 1 and 2 months after surgery, respectively. Fig 2. Bone marrow core biopsy specimen from a cat with pure red cell aplasia. Notice the presence of a lymphoid aggregate (arrows). Wright-Giemsa stain; Bar 20 m. IMHA Three cats had a diagnosis of immune-mediated anemia (Table 1). These cats were 1.5, 3, and 12 years of age. One cat had a severe regenerative anemia, and 2 cats had moderate regenerative anemias. All 3 cats had autoagglutination, and 1 of 2 cats tested had a positive direct Coombs test. Lymphocyte counts in the blood ranged between 11,300/ L and 20,380/ L. The lymphocytes were predominately small lymphocytes. One cat had moderate numbers of blast-transformed lymphocytes, consistent with immunologically stimulated lymphocytes. Feline leukemia virus and feline immunodeficiency virus tests were negative. All cats had hypercellular bone marrow. Lymphocyte numbers were 18, 21, and 30% of all nucleated cells, respectively. One cat had all small lymphocytes in bone marrow, 1 cat had 3% lymphoblasts, and 1 cat had 2% lymphoblasts. Core biopsy specimens were available for all cats. One or more lymphoid aggregates were present in 2 core biopsy specimens. Immunophenotyping was performed on bone marrow of all cats. One cat had 76% B cells and 24% T cells, another cat had 95% B cells, and the third cat had 88% B cells. Two cats were treated with prednisone and 1 was lost to follow-up. One cat had no hematologic response and died 6 months after diagnosis. The hematocrit of the other cat returned to normal within 4 months, and the cat was alive 1.5 years after diagnosis. Peripheral blood lymphocyte counts were within the reference interval in the last complete blood count obtained from all 3 cats. Thymoma A 13-year-old cat was referred for evaluation of chronic weight loss (Table 1). A mass was identified in the anterior mediastinum. The mass was surgically removed and histopathologic examination revealed a thymoma. A complete blood count was characterized by a total lymphocyte count of 21,990/ L. The majority of lymphocytes were small but had cleaved or lobulated nuclei. Tests for feline leukemia virus and feline immunodeficiency virus were negative. Bone marrow was characterized by normal cellularity and Cholangiohepatitis An 8-year-old cat had a history of lethargy, anorexia, ataxia, and vomiting (Table 1). A complete blood count showed neutropenia, left shift, lymphopenia, and toxic neutrophils consistent with bacterial sepsis. A serum chemistry profile revealed a marked increase in alanine transaminase and a liver biopsy indicated chronic cholangiohepatitis. Bone marrow had normal cellularity, 37% small lymphocytes, and 2% lymphoblasts. A core biopsy was not obtained. The cat was euthanized 5 days after admission to the hospital. Postmortem examination revealed chronic cholangiohepatitis, cardiomyopathy, and peritoneal hemorrhage. Fever of Unknown Origin An 8-month-old cat was referred for evaluation of persistent fever and lymphocytosis. A complete blood count was within reference intervals except for a total lymphocyte count of 11,150/ L (Table 1). Lymphocytes were small and had round nuclei. Tests for feline leukemia virus and feline immunodeficiency virus were negative. Bone marrow cellularity was normal and M : E was decreased. Fifty-four percent of nucleated cells were lymphocytes. Most lymphocytes were small, but 4% were lymphoblasts. A core biopsy revealed diffuse distribution of lymphoid cells with no lymphoid aggregates. Immunophenotyping indicated that 95% of the lymphocytes were B cells. Clonal analysis of bone marrow failed to identify a clonal population. The cat was reevaluated 8 times over the next 13 months. The cat appeared to be healthy throughout this period. Lymphocyte numbers in the peripheral blood varied between 4,400/ L and 11,250/ L, with no abnormal lymphoid morphology noted. Discussion Results of this study indicate that differentiation of the causes of lymphocytosis in cats can be problematic. Chronic lymphocytic leukemia was listed as a differential diagnosis for all 12 cats in this study. However, definitive evidence to support a diagnosis of CLL was present in only 2 cats. Neither the total number of lymphocytes in the blood nor the percentage of lymphocytes in bone marrow clearly differentiated CLL from reactive lymphocytosis. Several factors were identified that may aid in differentiating CLL from other causes of lymphocytosis. First, cat age may be useful. Six of 7 cats with evidence of immunemediated diseases were less than 3 years old. In contrast, the average age of 2 cats with a diagnosis of CLL was 10.8 years. Second, blood and bone marrow lymphocyte morphology may be useful. In the 2 cats with a diagnosis of CLL that did not have evidence of immune-mediated disease, lymphocytes were slightly larger than typical small

4 858 Weiss lymphocytes and had cleaved or lobulated nuclei. Although cleaved nuclei have been reported to be a normal finding in cats, large numbers of lobulated nuclei are not a normal finding for either B cells or T cells in the blood. 5,11 Third, lymphoid aggregates were observed in the bone marrow of 4 of 5 cats with evidence of immune-mediated anemia. In the cat in which lymphoid aggregates were not observed, the core biopsy specimen was very small, and, therefore, lymphoid aggregates may have been missed. Lymphoid aggregates were not seen in cats with CLL. Although the number of cats studied was small, these data indicate that chronic antigenic stimulation associated with immune-mediated diseases may result in lymphoid aggregate formation in bone marrow of cats. 10,12 Fourth, immunophenotyping may be helpful in differentiating the cause of lymphocytosis. Immunophenotyping was performed on 5 cats with evidence of immune-mediated anemias and 1 cat with fever of unknown origin. All cats had a predominance of B cells in the bone marrow. Of 2 cats with CLL and 1 cat with thymoma, all had exclusively T cells in the bone marrow. Recent studies have revealed that 20 of 20 CLL cats evaluated had a T-cell phenotype. a Fifth, clonality testing has been recommended for differentiating benign and malignant lymphoid proliferative disorders. 13,14 However, this test has not been adequately evaluated for use in cats. The sensitivity of the test is low (approximately 60%; Avery, personal communication). Therefore, a negative test result cannot be used to rule out malignancy. Two cats had strong support for a diagnosis of CLL. This included old age, absence of concurrent disease, progressive increase in lymphocyte count (eventually exceeding 50,000/ L), and response to chemotherapy. As noted previously, the negative test for clonality in 1 cat did not rule out CLL because of the low sensitivity of the test. Additionally, unlike the other cats, CLL was associated with a T-cell phenotype. Seven cats had severe anemia with evidence to indicate that it was immune-mediated. It is unlikely that any of these cats had concurrent CLL. Six of the cats were less than 3 years of age, 6 had less than 10,000 lymphocytes/ L in the blood, all 7 had normal lymphocyte morphology in blood and bone marrow, 4 of 5 cats had lymphoid aggregates in bone marrow, and 4 of 4 cats had mostly B cells in the bone marrow. Furthermore, in 3 cats for which long-term follow-up was available, the lymphocyte count returned to normal as the anemia resolved. All of these findings differ from that for 2 cats with a selective diagnosis of CLL. Therefore, the most likely conclusion is that B-cell hyperplasia in blood or bone marrow is an outcome of immune dysregulation associated with immune-mediated anemias. The hematologic data for the cats with cholangiohepatitis and fever of unknown origin were very similar to those with immune-mediated diseases. This included normal lymphocyte count or mild lymphocytosis in the blood and small cell lymphocytosis in bone marrow, consisting mostly of B cells. This indicates that the bone marrow lymphocytosis was the result of chronic disease-associated antigenic stimulation. The cause of the lymphocytosis associated with thymoma was not determined. Because the lymphocyte count decreased after thymectomy, concurrent CLL is unlikely. Thymoma is thought to be a primary epithelial tumor with a secondary benign proliferation/infiltration of lymphocytes. 15 However, it is possible that the T cells were a neoplastic population in this tumor or, alternatively, that growth factors released by the neoplastic epithelial cells stimulated T- cell proliferation. In conclusion, differentiation of the causes of persistent lymphocytosis in cats is difficult. Immune-mediated anemias and chronic inflammatory diseases appear to be major causes of reactive lymphocytosis. These lymphocytes generally have normal morphology, but reactive or blast-transformed lymphocytes may be seen. The proliferating lymphocytes tend to be organized into lymphoid aggregates in bone marrow and are mostly B cells. Alternatively, in cats with CLL, the proliferating lymphocytes are more likely to be T cells that have subtle morphologic abnormalities and are diffusely distributed in bone marrow. Differentiation of the causes of lymphocytosis should include careful evaluation of lymphocyte morphology, lymphocyte distribution in bone marrow, and immunophenotyping. Other considerations should include cat age, presence of severe anemia, and evidence of inflammatory disease. Footnotes a Workman HC, Vernau W, Schmidt PS, et al. Chronic lymphocytic leukemia in cats is primarily a T-helper cell disease. Vet Clin Pathol 2004;33:184 References 1. Cowell RC, Decker LS. 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5 Feline Lymphocytosis Weiss DJ. Nonmalignant histopathology of bone marrow. Vet Clin Pathol 1986;15: Keller RL, Avery AC, Burnett RC, et al. Detection of neoplastic lymphocytes in peripheral blood of dogs with lymphoma by polymerase chain reaction for antigen receptor gene rearrangement. Vet Clin Pathol 2004;33: Burnett RC, Vernau W, Modiano JF, et al. Diagnosis of canine lymphoid neoplasia using clonal rearrangements of antigen receptor genes. Vet Pathol 2003;40: Carpenter JL, Holzworth J. Thymoma in 11 cats. J Am Vet Med Assoc 1982;181: