532339 research-article2014
VDIXXX10.1177/1040638714532339Chronic T-cell leukemia in a macaqueCazzini et al.
Case Report
Spontaneous chronic T-cell leukemia in a male rhesus macaque (Macaca mulatta)
Journal of Veterinary Diagnostic Investigation 2014, Vol. 26(4) 571–574 © 2014 The Author(s) Reprints and permissions: sagepub.com/journalsPermissions.nav DOI: 10.1177/1040638714532339 jvdi.sagepub.com
Paola Cazzini, Paula M. Krimer, Misty J. Williams-Fritze, Abigail M. Butler, and Uriel Blas-Machado1
Abstract. Blood smears from a 24-year-old male rhesus macaque (Macaca mulatta) used for cognitive function studies were evaluated. The macaque had an 8-month history of gradual weight loss and increasing lymphocytosis. Most of the lymphocytes present were small to medium and had a mature morphology. Based on the degree and duration of the lymphocytosis, and the appearance of the lymphocytes, a diagnosis of chronic lymphocytic leukemia was made. The animal tested negative for 4 viral diseases that are commonly associated with lymphoproliferative disorders in Old World monkeys. Over the course of 12 months, the lymphocytosis progressed from 18.4 to 384 × 103 lymphocytes/µl (reference range: 0.8–17 × 103 cells/µl), and euthanasia was elected. On histologic examination, cluster of differentiation (CD)3- and CD8-positive, CD79-negative neoplastic cells comprised 40–60% of the bone marrow, diffusely obscured the normal splenic architecture, and were present in the vascular channels in other organs. Findings were characteristic of T-cell lymphocytic leukemia. Naturally occurring T-cell lymphocytic leukemia has been rarely reported in rhesus macaques and, to the authors’ knowledge, never in males. A persistent lymphocytosis characterized by a monomorphic population of CD3- and CD8-positive cytotoxic T-lymphocytes and the presence of neoplastic cells in the bone marrow led to a diagnosis in the current case. Key words: Chronic lymphocytic leukemia; immunohistochemical staining; lymphoproliferative diseases; neoplasia; rhesus macaques. The rhesus macaque (Macaca mulatta) genome was unraveled in 2007 and demonstrated that human beings and macaques share approximately 93% of their nucleotide sequence.2 Because of their genetic and physiological similarity to human beings, rhesus monkeys (Macaca mulatta) are the best studied10 and most widely used nonhuman primate (NHP) in medical research.2 Rhesus macaques are also excellent models in studies related to the immune system10 because several phenotypic markers that define innate and adaptive immune cells are highly conserved between human beings and Old World NHPs.7 Lymphoid leukemias are characterized by the clonal proliferation of malignant lymphoid progenitor cells in the bone marrow. In veterinary medicine, lymphoid leukemias are divided into 2 broad categories, acute and chronic, based on clinical presentation and the proliferative rate of the neoplastic cell clone.4,6 Acute leukemias are clonal proliferations of a progenitor cell4 with limited differentiation; identification of cellular markers is often necessary for a definitive identification of the cell of origin. In chronic leukemias, neoplastic cells are terminally differentiated, and the cellular origin is more readily identifiable. Chronic lymphocytic leukemia (CLL) is a neoplastic clonal proliferation of small and morphologically matureappearing lymphocytes that present as a persistent (>3 months), often marked, peripheral lymphocytosis.4 Frequently, as in the case reported herein, the initial suspicion of
CLL is based on an accidental finding of lymphocytosis found during a routine blood work or a geriatric wellness examination. In fact, the affected subjects are usually asymptomatic during the initial phases of the disease. During later phases, the clinical signs are generic and nonspecific and include lethargy, anorexia, and slowly progressive weight loss. If the disease has advanced with effacement of the bone marrow, cytopenias can be observed.4 In human medicine, leukemias of mature lymphocytes are classified in a more complex way. What in human literature is labeled as CLL generally refers to a neoplasm of B-cell origin. Lymphoid neoplasms in general are most commonly of B-cell origin, with most of the remaining being of T-cell and only rarely of natural killer (NK) cell origin.5 Among the T-cell leukemias, the ones characterized by cells with mature morphology and phenotype can be divided into T-cell large granular leukemia (T-LGL), T-prolymphocytic leukemia, and adult T-cell leukemia/lymphoma. T-cell large granular From the Department of Pathology (Cazzini, Krimer, Butler, BlasMachado) and the Athens Veterinary Diagnostic Laboratory (Krimer, BlasMachado), College of Veterinary Medicine, University of Georgia, Athens, GA; and the Laboratory Animal Services, Georgia Regents University, Augusta, GA (Williams-Fritze). 1 Corresponding Author: Uriel Blas-Machado, College of Veterinary Medicine, University of Georgia, 501 DW Brooks Drive, Athens, GA 30602. [email protected]
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Figure 1. Peripheral blood count chart; rhesus macaque (Macaca mulatta). The chart illustrates the variation in the number of lymphocytes (green bars) compared with other white blood cells (WBC; red bars) per microliter (µl) of blood at different time points. Note how the numbers go from 2.4 × 103 in May 2010 to 384 × 103 in November 2011.
leukemia accounts for 2–3% of all small lymphocytic leukemia and is generally found in older people.6 Normal T-LGL cells are positive for cluster of differentiation (CD)8, CD3, CD2, CD5, and CD7 and are negative for CD4, CD16, and CD56 markers.6 Expression of NK-related antigens CD16 and CD57 are usual in T-LGL while the T-cell lymphoma 1 (TCL-1) marker is not seen in T-LGL.6 On blood smear examination, T-LGLs are characterized by intermediate to large size (15–18 µm), with increased amount of pale cytoplasm and variable amounts of randomly distributed azurophilic granules and a rounded nucleus with mature chromatin. Several lymphoproliferative disorders have been reported in rhesus macaques and, to the authors’ knowledge, among those naturally occurring, CLL has only been reported in a female macaque.3,4 Blood smears from a 24-year-old male rhesus macaque used in cognitive function studies and presenting with an 8-month history of poor body condition score were evaluated. The rhesus macaque had a persistent and gradually increasing lymphocytosis (Fig. 1) over a 1-year period. Smears were stained with a modified Romanowsky stain and analyzed by light microscopy (Fig. 2). The majority of the lymphocytes present were small to medium in size, and had an abundant granular basophilic cytoplasm that occasionally contained a scant dusting of eosinophilic granules. The nucleus was round to oval with a clumped chromatin and was occasionally cleaved with an inconspicuous nucleolus. Rare binucleated lymphocytes were also present. Based on the degree and the persistence of the lymphocytosis and on the morphologic appearance of the lymphocytes, a diagnosis of CLL was made. The animal was seronegative for Macacine herpesvirus 1 (formerly Herpes simian B virus), Primate T-lymphotropic
Figure 2. Peripheral blood smear; rhesus macaque (Macaca mulatta). The smear had small- to medium-sized lymphocytes (arrows), with abundant granular basophilic cytoplasm with scant dusting of eosinophilic granules, round to oval to cleaved nucleus with a clumped chromatin with an inconspicuous nucleolus. The arrowhead points to a neutrophil. Modified Romanowsky stain; original magnification 1,000×.
virus 1, Simian immunodeficiency virus (SIV), and Simian retrovirus (SRV). The lymphocytosis progressed to the remarkable number of 384 × 103 lymphocytes/µl (reference range: 0.8–17 × 103 cells/µl; Fig. 1). Because of deteriorating health conditions, the macaque was euthanized; necropsy and bone marrow cytology were performed. Formalin-fixed tissue samples of femoral head with bone marrow, liver, spleen, kidney, and adrenal gland were submitted along with unstained bone marrow cytology smears. Tissue samples were fixed in neutral buffered, 10% formalin solution. Trimmed tissues were routinely processed, embedded in paraffin, and sectioned at approximately 4 μm. All tissue sections were stained with hematoxylin and eosin. Sections from the spleen, liver, and bone marrow tissues were subjected to CD3, CD79, CD18, CD4, and CD8 immunohistochemical staining. Briefly, paraffin-embedded tissues were sectioned at approximately 4 µm, deparaffinized, and rehydrated. The following primary antibodies were used: polyclonal rabbit anti-human CD3 antibody,a monoclonal mouse anti-human CD79αcy,b mouse monoclonal antiCD18,c monoclonal mouse anti-human CD4,d and monoclonal mouse anti-human CD8.e Antibody dilutions have previously been carried out on positive controls to find the greatest dilution of the antibody, which yielded optimal staining. The following optimal antibody dilutions were determined: CD3, 1:800 with staining time of 60 min; CD79, 1:100 with staining time of 60 min; CD18, 1:50 with staining time of 60 min; CD4, 1:25 with
Downloaded from vdi.sagepub.com at University of Tokyo on June 6, 2015
Chronic T-cell leukemia in a macaque staining time of 90 min; and CD8, 1:25 with staining time of 90 min. All primary antibodies were diluted,f and antigen retrieval was performed. Heat-induced epitope retrieval (HIER) using citrate buffer with a pH of 6.0g was used for CD3 and CD18. For CD79, CD4, and CD8, HIER was also used but with target retrieval solution (pH 9.0; 10×).h For CD3, CD79, and CD18, endogenous peroxidase was quenched using 3% hydrogen peroxide.i For all antibodies, blocking was completed with a universal blocking reagent.j For CD3-positive tissue, controls consisted of formalinfixed, paraffin-embedded (FFPE) canine lymph node; for CD79-, CD4-, CD8-positive tissue, controls consisted of FFPE canine tonsil; and for CD18-positive tissue, controls consisted of FFPE equine lymphoid tissue. As a negative reagent control, the primary antibodies (CD79, CD18, CD4, and CD8) were eliminated and substituted with purified mouse immunoglobulins in bufferk; for CD3, the buffer contained purified rabbit immunoglobulins.l For CD3, the secondary antibody used was biotinylated anti-rabbit IgG, made in goat.m For CD79 and CD18, the secondary antibody used was biotinylated anti-mouse IgG, rat absorbed, made in horse.n For CD3, CD79, and CD18, streptavidin conjugated to horseradish peroxidase in phosphate buffered saline was used as detection.o For CD4 and CD8, detection was completed using a commercial kitp; both kit reagents were used with a staining time of 10 min each. For CD3, CD79, and CD18, the substrate chromogen system used was 3,3’-diaminobenzidine (DAB)q with an incubation time of 12 min. For CD4 and CD8, the substrate chromogen system used was Warp Red,r with an incubation time of 10 min. For all, the tissue sections were counterstained with Gill II hematoxylin and bluing; dehydrated in alcohol levels of 70%, 95%, and 100%; cleared in xylene; and mounted with xylene-based mounting medium. Bone marrow cytology preparations, which were routinely stained with a modified Romanowsky stain, revealed mild to moderate lymphocytosis. On histologic evaluation, the epiphysis and metaphysis of the femur were diffusely hypercellular, with little or no adipose tissue between the bony trabeculae (Fig. 3). The marrow consisted of a heterogeneous population of CD3- and CD8-positive lymphocytes and hematopoietic cell precursors, including myeloid and erythroid cells and scattered megakaryocytes. In the spleen, sheets of moderately pleomorphic round cells, with discrete foci of hemorrhage and fibrin strands, replaced most of the red pulp, displaced adjacent muscle trabeculae, and markedly attenuated the white pulp. Round cells were CD3 and CD8 positive and CD79α and CD4 negative, and had mildly abundant to scant pale eosinophilic to clear cytoplasm with distinct cell borders. The hyperchromatic nuclei varied from round to oval to indent to lobed, with mild anisokaryosis and indistinct nucleoli. Mitoses in 10 random fields at 400× were zero. Neoplastic cells extended into many vascular channels. The splenic capsule was intact.
573
Figure 3. Bone marrow, femoral head; rhesus macaque (Macaca mulatta). The marrow is diffusely hypercellular. A homogenous population of cells has replaced most of the fatty marrow and osseous trabeculae. Bar = 0.5 cm. Upper inset: immunostaining for cluster of differentiation (CD)3. Neoplastic cells with cell membrane brown pigment are positive. Lower inset: immunostaining for CD8. Neoplastic cells with cytoplasmic red pigment are positive.
In the liver, widely scattered collections of pleomorphic CD3- and CD8-positive lymphocytes expanded the sinusoids and extended into many vascular channels. In the kidney and adrenal gland, vascular channels contained CD3- and CD8positive lymphoblasts. The kidney had mild tubular degeneration and necrosis. History and findings were characteristic of a T-cell lymphocytic leukemia, with origin in the marrow and dissemination into many organs, including the spleen and liver. In Old World NHPs, lymphoproliferative disorders are often associated with oncogenic herpes viruses, such as PTLV-1, SIV, and SRV1,8,9; however, these viruses were not detected in the animal described in the current report. Naturally occurring T-cell CLL has been rarely reported in rhesus macaques,3 and seems to be underreported in male macaques. Patients with CLL are often asymptomatic, but, as in the current case, patients may develop nonspecific signs such as lethargy and anorexia. The diagnosis was made by serial blood analysis revealing persistent lymphocytosis characterized by a monomorphic population of T-lymphocytes (Fig. 1), which is characteristic for CLL, later confirmed by histology and immunohistochemical staining. A limitation of the current case report is the absence of clonality studies on the neoplastic cells. Clonality studies on T-lymphocytes are based on the rearrangements of the T-cell receptor and are often performed in lymphoproliferative disorders to support the exclusion of a reactive expansion of lymphocytes. Given the extraordinary numbers of lymphocytes
Downloaded from vdi.sagepub.com at University of Tokyo on June 6, 2015
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present in circulation, there was no doubt regarding their neoplastic nature; however, it would have been interesting to detect and identify a clonal rearrangement of the T-cell receptor. Furthermore, immunophenotyping with markers such as TCL-1 and CD56, commonly used in human medicine but less frequently used in veterinary medicine, would have been interesting in the current case to better compare this neoplasm with those occurring in human beings. In conclusion, the current case presents a case of CLL in a male rhesus macaque. More reports of such uncommon lymphoproliferative disorders and better characterization of the neoplastic cells phenotype are needed to further assess this neoplastic disease in rhesus macaques. Acknowledgements The authors would like to thank Dr. Alvin Terry for the care and maintenance of the macaque as well as the case referral, and Dr. Kristen Weishaar for clinical advice on this case.
Sources and manufacturers a. Polyclonal rabbit anti-human CD3 antibody, A0452; Dako North America Inc., Carpinteria, CA. b. Monoclonal mouse anti-human CD79αcy clone HM57, M7051; Dako North America Inc., Carpinteria, CA. c. Monoclonal mouse anti-human CD4, clone BC/1F6, CM153; Biocare Medical, Concord, CA. d. Monoclonal mouse anti-human CD18, clone H20A; VMRD Inc., Pullman, WA. e. Monoclonal mouse anti-human CD8, clone BC/1A5, CM154; Biocare Medical, Concord, CA. f. Antibody diluent, S0809; Dako North America Inc., Carpinteria, CA. g. Citrate buffer with a pH of 6.0, HK086-9K; BioGenex Laboratories Inc., San Ramon, CA. h. Target retrieval solution pH 9.0, S2367; Dako North America Inc., Carpinteria, CA. i. Hydrogen peroxide, H312-500; Fisher Scientific, Fair Lawn, NJ. j. Power block, HK085-5K; BioGenex Laboratories Inc., San Ramon, CA. k. Purified mouse immunoglobulins in buffer, NC494H; Biocare Medical, Concord, CA. l. Purified rabbit immunoglobulins in buffer, NC495H; Biocare Medical, Concord, CA. m. Biotinylated anti-rabbit IgG, made in goat, BA-1000; Vector Laboratories Inc., Burlingame, CA. n. Biotinylated anti-mouse IgG, rat absorbed, made in horse, BA-2001; Vector Laboratories Inc., Burlingame, CA.
o. LSAB 2 streptavidin conjugated to horseradish peroxidase in PBS, K1016; Dako North America Inc., Carpinteria, CA. p. Universal AP detection kit, AP506; Biocare Medical, Concord, CA. q. DAB, K3466; Dako North America Inc., Carpinteria, CA. r. Warp Red, WR806; Biocare Medical, Concord, CA.
Declaration of conflicting interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding The Athens Veterinary Diagnostic Laboratory (Athens, GA) provided funds for the preparation and publication of the current article.
References 1. Fortgang IS, Didier PJ, Levy LS: 2000, B-cell leukemia in a rhesus macaque (Macaca mulatta) infected with simian immunodeficiency virus. Leuk Lymphoma 37:657–662. 2. Gibbs RA, Rogers J, Katze MG, et al.: 2007, Evolutionary and biomedical insights from the rhesus macaque genome. Science 316:222–234. 3. Gilardi KV, Spinner A, Canfield DR, et al.: 2000, T-cell lymphoproliferative disorder in an aged rhesus macaque. J Am Vet Med Assoc 217:384–387, 341. 4. Helfand SC, Kisseberth WC: 2010, General features of leukemia and lymphoma. In: Schalm’s veterinary hematology, ed. Weiss DJ, Wardrop KJ, pp. 455–466. Wiley-Blackwell, Ames, IA. 5. Kumar V, Abbas AK, Fausto N, Aster JC: 2010, Diseases of the immune system. In: Robbins and Cotran’s pathologic basis of disease, ed. Kumar V, Abbas AK, Fausto N, Aster JC, 8th ed., pp. 183–258. Saunders Elsevier, Philadelphia, PA. 6. O’Malley DP: 2007, T-cell large granular leukemia and related proliferations. Am J Clin Pathol 127:850–859. 7. Pitcher CJ, Hagen SI, Walker JM, et al.: 2002, Development and homeostasis of T cell memory in rhesus macaque. J Immunol 168:29–43. 8. Rosenwirth B, Kondova I, Niphuis H, et al.: 2011, Herpesvirus saimiri infection of rhesus macaques: a model for acute rhadinovirus-induced T-cell transformation and oncogenesis. J Med Virol 83:1938–1950. 9. Seggewiss R, Pittaluga S, Adler RL, et al.: 2006, Acute myeloid leukemia is associated with retroviral gene transfer to hematopoietic progenitor cells in a rhesus macaque. Blood 107:3865–3867. 10. Vaccari M, Franchini G: 2010, Memory T cells in Rhesus macaques. Adv Exp Med Biol 684:126–144.
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VDIXXX10.1177/1040638714532339Chronic T-cell leukemia in a macaqueCazzini et al.
Case Report
Spontaneous chronic T-cell leukemia in a male rhesus macaque (Macaca mulatta)
Journal of Veterinary Diagnostic Investigation 2014, Vol. 26(4) 571–574 © 2014 The Author(s) Reprints and permissions: sagepub.com/journalsPermissions.nav DOI: 10.1177/1040638714532339 jvdi.sagepub.com
Paola Cazzini, Paula M. Krimer, Misty J. Williams-Fritze, Abigail M. Butler, and Uriel Blas-Machado1
Abstract. Blood smears from a 24-year-old male rhesus macaque (Macaca mulatta) used for cognitive function studies were evaluated. The macaque had an 8-month history of gradual weight loss and increasing lymphocytosis. Most of the lymphocytes present were small to medium and had a mature morphology. Based on the degree and duration of the lymphocytosis, and the appearance of the lymphocytes, a diagnosis of chronic lymphocytic leukemia was made. The animal tested negative for 4 viral diseases that are commonly associated with lymphoproliferative disorders in Old World monkeys. Over the course of 12 months, the lymphocytosis progressed from 18.4 to 384 × 103 lymphocytes/µl (reference range: 0.8–17 × 103 cells/µl), and euthanasia was elected. On histologic examination, cluster of differentiation (CD)3- and CD8-positive, CD79-negative neoplastic cells comprised 40–60% of the bone marrow, diffusely obscured the normal splenic architecture, and were present in the vascular channels in other organs. Findings were characteristic of T-cell lymphocytic leukemia. Naturally occurring T-cell lymphocytic leukemia has been rarely reported in rhesus macaques and, to the authors’ knowledge, never in males. A persistent lymphocytosis characterized by a monomorphic population of CD3- and CD8-positive cytotoxic T-lymphocytes and the presence of neoplastic cells in the bone marrow led to a diagnosis in the current case. Key words: Chronic lymphocytic leukemia; immunohistochemical staining; lymphoproliferative diseases; neoplasia; rhesus macaques. The rhesus macaque (Macaca mulatta) genome was unraveled in 2007 and demonstrated that human beings and macaques share approximately 93% of their nucleotide sequence.2 Because of their genetic and physiological similarity to human beings, rhesus monkeys (Macaca mulatta) are the best studied10 and most widely used nonhuman primate (NHP) in medical research.2 Rhesus macaques are also excellent models in studies related to the immune system10 because several phenotypic markers that define innate and adaptive immune cells are highly conserved between human beings and Old World NHPs.7 Lymphoid leukemias are characterized by the clonal proliferation of malignant lymphoid progenitor cells in the bone marrow. In veterinary medicine, lymphoid leukemias are divided into 2 broad categories, acute and chronic, based on clinical presentation and the proliferative rate of the neoplastic cell clone.4,6 Acute leukemias are clonal proliferations of a progenitor cell4 with limited differentiation; identification of cellular markers is often necessary for a definitive identification of the cell of origin. In chronic leukemias, neoplastic cells are terminally differentiated, and the cellular origin is more readily identifiable. Chronic lymphocytic leukemia (CLL) is a neoplastic clonal proliferation of small and morphologically matureappearing lymphocytes that present as a persistent (>3 months), often marked, peripheral lymphocytosis.4 Frequently, as in the case reported herein, the initial suspicion of
CLL is based on an accidental finding of lymphocytosis found during a routine blood work or a geriatric wellness examination. In fact, the affected subjects are usually asymptomatic during the initial phases of the disease. During later phases, the clinical signs are generic and nonspecific and include lethargy, anorexia, and slowly progressive weight loss. If the disease has advanced with effacement of the bone marrow, cytopenias can be observed.4 In human medicine, leukemias of mature lymphocytes are classified in a more complex way. What in human literature is labeled as CLL generally refers to a neoplasm of B-cell origin. Lymphoid neoplasms in general are most commonly of B-cell origin, with most of the remaining being of T-cell and only rarely of natural killer (NK) cell origin.5 Among the T-cell leukemias, the ones characterized by cells with mature morphology and phenotype can be divided into T-cell large granular leukemia (T-LGL), T-prolymphocytic leukemia, and adult T-cell leukemia/lymphoma. T-cell large granular From the Department of Pathology (Cazzini, Krimer, Butler, BlasMachado) and the Athens Veterinary Diagnostic Laboratory (Krimer, BlasMachado), College of Veterinary Medicine, University of Georgia, Athens, GA; and the Laboratory Animal Services, Georgia Regents University, Augusta, GA (Williams-Fritze). 1 Corresponding Author: Uriel Blas-Machado, College of Veterinary Medicine, University of Georgia, 501 DW Brooks Drive, Athens, GA 30602. [email protected]
Downloaded from vdi.sagepub.com at University of Tokyo on June 6, 2015
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Figure 1. Peripheral blood count chart; rhesus macaque (Macaca mulatta). The chart illustrates the variation in the number of lymphocytes (green bars) compared with other white blood cells (WBC; red bars) per microliter (µl) of blood at different time points. Note how the numbers go from 2.4 × 103 in May 2010 to 384 × 103 in November 2011.
leukemia accounts for 2–3% of all small lymphocytic leukemia and is generally found in older people.6 Normal T-LGL cells are positive for cluster of differentiation (CD)8, CD3, CD2, CD5, and CD7 and are negative for CD4, CD16, and CD56 markers.6 Expression of NK-related antigens CD16 and CD57 are usual in T-LGL while the T-cell lymphoma 1 (TCL-1) marker is not seen in T-LGL.6 On blood smear examination, T-LGLs are characterized by intermediate to large size (15–18 µm), with increased amount of pale cytoplasm and variable amounts of randomly distributed azurophilic granules and a rounded nucleus with mature chromatin. Several lymphoproliferative disorders have been reported in rhesus macaques and, to the authors’ knowledge, among those naturally occurring, CLL has only been reported in a female macaque.3,4 Blood smears from a 24-year-old male rhesus macaque used in cognitive function studies and presenting with an 8-month history of poor body condition score were evaluated. The rhesus macaque had a persistent and gradually increasing lymphocytosis (Fig. 1) over a 1-year period. Smears were stained with a modified Romanowsky stain and analyzed by light microscopy (Fig. 2). The majority of the lymphocytes present were small to medium in size, and had an abundant granular basophilic cytoplasm that occasionally contained a scant dusting of eosinophilic granules. The nucleus was round to oval with a clumped chromatin and was occasionally cleaved with an inconspicuous nucleolus. Rare binucleated lymphocytes were also present. Based on the degree and the persistence of the lymphocytosis and on the morphologic appearance of the lymphocytes, a diagnosis of CLL was made. The animal was seronegative for Macacine herpesvirus 1 (formerly Herpes simian B virus), Primate T-lymphotropic
Figure 2. Peripheral blood smear; rhesus macaque (Macaca mulatta). The smear had small- to medium-sized lymphocytes (arrows), with abundant granular basophilic cytoplasm with scant dusting of eosinophilic granules, round to oval to cleaved nucleus with a clumped chromatin with an inconspicuous nucleolus. The arrowhead points to a neutrophil. Modified Romanowsky stain; original magnification 1,000×.
virus 1, Simian immunodeficiency virus (SIV), and Simian retrovirus (SRV). The lymphocytosis progressed to the remarkable number of 384 × 103 lymphocytes/µl (reference range: 0.8–17 × 103 cells/µl; Fig. 1). Because of deteriorating health conditions, the macaque was euthanized; necropsy and bone marrow cytology were performed. Formalin-fixed tissue samples of femoral head with bone marrow, liver, spleen, kidney, and adrenal gland were submitted along with unstained bone marrow cytology smears. Tissue samples were fixed in neutral buffered, 10% formalin solution. Trimmed tissues were routinely processed, embedded in paraffin, and sectioned at approximately 4 μm. All tissue sections were stained with hematoxylin and eosin. Sections from the spleen, liver, and bone marrow tissues were subjected to CD3, CD79, CD18, CD4, and CD8 immunohistochemical staining. Briefly, paraffin-embedded tissues were sectioned at approximately 4 µm, deparaffinized, and rehydrated. The following primary antibodies were used: polyclonal rabbit anti-human CD3 antibody,a monoclonal mouse anti-human CD79αcy,b mouse monoclonal antiCD18,c monoclonal mouse anti-human CD4,d and monoclonal mouse anti-human CD8.e Antibody dilutions have previously been carried out on positive controls to find the greatest dilution of the antibody, which yielded optimal staining. The following optimal antibody dilutions were determined: CD3, 1:800 with staining time of 60 min; CD79, 1:100 with staining time of 60 min; CD18, 1:50 with staining time of 60 min; CD4, 1:25 with
Downloaded from vdi.sagepub.com at University of Tokyo on June 6, 2015
Chronic T-cell leukemia in a macaque staining time of 90 min; and CD8, 1:25 with staining time of 90 min. All primary antibodies were diluted,f and antigen retrieval was performed. Heat-induced epitope retrieval (HIER) using citrate buffer with a pH of 6.0g was used for CD3 and CD18. For CD79, CD4, and CD8, HIER was also used but with target retrieval solution (pH 9.0; 10×).h For CD3, CD79, and CD18, endogenous peroxidase was quenched using 3% hydrogen peroxide.i For all antibodies, blocking was completed with a universal blocking reagent.j For CD3-positive tissue, controls consisted of formalinfixed, paraffin-embedded (FFPE) canine lymph node; for CD79-, CD4-, CD8-positive tissue, controls consisted of FFPE canine tonsil; and for CD18-positive tissue, controls consisted of FFPE equine lymphoid tissue. As a negative reagent control, the primary antibodies (CD79, CD18, CD4, and CD8) were eliminated and substituted with purified mouse immunoglobulins in bufferk; for CD3, the buffer contained purified rabbit immunoglobulins.l For CD3, the secondary antibody used was biotinylated anti-rabbit IgG, made in goat.m For CD79 and CD18, the secondary antibody used was biotinylated anti-mouse IgG, rat absorbed, made in horse.n For CD3, CD79, and CD18, streptavidin conjugated to horseradish peroxidase in phosphate buffered saline was used as detection.o For CD4 and CD8, detection was completed using a commercial kitp; both kit reagents were used with a staining time of 10 min each. For CD3, CD79, and CD18, the substrate chromogen system used was 3,3’-diaminobenzidine (DAB)q with an incubation time of 12 min. For CD4 and CD8, the substrate chromogen system used was Warp Red,r with an incubation time of 10 min. For all, the tissue sections were counterstained with Gill II hematoxylin and bluing; dehydrated in alcohol levels of 70%, 95%, and 100%; cleared in xylene; and mounted with xylene-based mounting medium. Bone marrow cytology preparations, which were routinely stained with a modified Romanowsky stain, revealed mild to moderate lymphocytosis. On histologic evaluation, the epiphysis and metaphysis of the femur were diffusely hypercellular, with little or no adipose tissue between the bony trabeculae (Fig. 3). The marrow consisted of a heterogeneous population of CD3- and CD8-positive lymphocytes and hematopoietic cell precursors, including myeloid and erythroid cells and scattered megakaryocytes. In the spleen, sheets of moderately pleomorphic round cells, with discrete foci of hemorrhage and fibrin strands, replaced most of the red pulp, displaced adjacent muscle trabeculae, and markedly attenuated the white pulp. Round cells were CD3 and CD8 positive and CD79α and CD4 negative, and had mildly abundant to scant pale eosinophilic to clear cytoplasm with distinct cell borders. The hyperchromatic nuclei varied from round to oval to indent to lobed, with mild anisokaryosis and indistinct nucleoli. Mitoses in 10 random fields at 400× were zero. Neoplastic cells extended into many vascular channels. The splenic capsule was intact.
573
Figure 3. Bone marrow, femoral head; rhesus macaque (Macaca mulatta). The marrow is diffusely hypercellular. A homogenous population of cells has replaced most of the fatty marrow and osseous trabeculae. Bar = 0.5 cm. Upper inset: immunostaining for cluster of differentiation (CD)3. Neoplastic cells with cell membrane brown pigment are positive. Lower inset: immunostaining for CD8. Neoplastic cells with cytoplasmic red pigment are positive.
In the liver, widely scattered collections of pleomorphic CD3- and CD8-positive lymphocytes expanded the sinusoids and extended into many vascular channels. In the kidney and adrenal gland, vascular channels contained CD3- and CD8positive lymphoblasts. The kidney had mild tubular degeneration and necrosis. History and findings were characteristic of a T-cell lymphocytic leukemia, with origin in the marrow and dissemination into many organs, including the spleen and liver. In Old World NHPs, lymphoproliferative disorders are often associated with oncogenic herpes viruses, such as PTLV-1, SIV, and SRV1,8,9; however, these viruses were not detected in the animal described in the current report. Naturally occurring T-cell CLL has been rarely reported in rhesus macaques,3 and seems to be underreported in male macaques. Patients with CLL are often asymptomatic, but, as in the current case, patients may develop nonspecific signs such as lethargy and anorexia. The diagnosis was made by serial blood analysis revealing persistent lymphocytosis characterized by a monomorphic population of T-lymphocytes (Fig. 1), which is characteristic for CLL, later confirmed by histology and immunohistochemical staining. A limitation of the current case report is the absence of clonality studies on the neoplastic cells. Clonality studies on T-lymphocytes are based on the rearrangements of the T-cell receptor and are often performed in lymphoproliferative disorders to support the exclusion of a reactive expansion of lymphocytes. Given the extraordinary numbers of lymphocytes
Downloaded from vdi.sagepub.com at University of Tokyo on June 6, 2015
574
Cazzini et al.
present in circulation, there was no doubt regarding their neoplastic nature; however, it would have been interesting to detect and identify a clonal rearrangement of the T-cell receptor. Furthermore, immunophenotyping with markers such as TCL-1 and CD56, commonly used in human medicine but less frequently used in veterinary medicine, would have been interesting in the current case to better compare this neoplasm with those occurring in human beings. In conclusion, the current case presents a case of CLL in a male rhesus macaque. More reports of such uncommon lymphoproliferative disorders and better characterization of the neoplastic cells phenotype are needed to further assess this neoplastic disease in rhesus macaques. Acknowledgements The authors would like to thank Dr. Alvin Terry for the care and maintenance of the macaque as well as the case referral, and Dr. Kristen Weishaar for clinical advice on this case.
Sources and manufacturers a. Polyclonal rabbit anti-human CD3 antibody, A0452; Dako North America Inc., Carpinteria, CA. b. Monoclonal mouse anti-human CD79αcy clone HM57, M7051; Dako North America Inc., Carpinteria, CA. c. Monoclonal mouse anti-human CD4, clone BC/1F6, CM153; Biocare Medical, Concord, CA. d. Monoclonal mouse anti-human CD18, clone H20A; VMRD Inc., Pullman, WA. e. Monoclonal mouse anti-human CD8, clone BC/1A5, CM154; Biocare Medical, Concord, CA. f. Antibody diluent, S0809; Dako North America Inc., Carpinteria, CA. g. Citrate buffer with a pH of 6.0, HK086-9K; BioGenex Laboratories Inc., San Ramon, CA. h. Target retrieval solution pH 9.0, S2367; Dako North America Inc., Carpinteria, CA. i. Hydrogen peroxide, H312-500; Fisher Scientific, Fair Lawn, NJ. j. Power block, HK085-5K; BioGenex Laboratories Inc., San Ramon, CA. k. Purified mouse immunoglobulins in buffer, NC494H; Biocare Medical, Concord, CA. l. Purified rabbit immunoglobulins in buffer, NC495H; Biocare Medical, Concord, CA. m. Biotinylated anti-rabbit IgG, made in goat, BA-1000; Vector Laboratories Inc., Burlingame, CA. n. Biotinylated anti-mouse IgG, rat absorbed, made in horse, BA-2001; Vector Laboratories Inc., Burlingame, CA.
o. LSAB 2 streptavidin conjugated to horseradish peroxidase in PBS, K1016; Dako North America Inc., Carpinteria, CA. p. Universal AP detection kit, AP506; Biocare Medical, Concord, CA. q. DAB, K3466; Dako North America Inc., Carpinteria, CA. r. Warp Red, WR806; Biocare Medical, Concord, CA.
Declaration of conflicting interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding The Athens Veterinary Diagnostic Laboratory (Athens, GA) provided funds for the preparation and publication of the current article.
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