Spontaneous chronic T-cell leukemia in a male rhesus macaque ( Macaca mulatta )

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.

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 studied ¹⁰ and most widely used nonhuman primate (NHP) in medical research. ² Rhesus macaques are also excellent models in studies related to the immune system ¹⁰ 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 cell ⁴ 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 mature-appearing 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 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.

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.

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.

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×.

The animal was seronegative for Macacine herpesvirus 1 (formerly Herpes simian B virus), Primate T-lymphotropic 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 anti-CD18, ^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 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 6.0 ^g 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 formalin-fixed, 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 buffer ^k ; 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 kit ^p ; 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.

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 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.

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 CD8-positive 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 SRV^1,8,9; 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 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.