Rosette-forming multicentric B-cell lymphoma in a giant panda

Abstract

Literature covering diseases of the giant panda (Ailuropoda melanoleuca) is either in Chinese or focuses on infectious agents. Here we describe the clinical signs, gross and microscopic findings, and immunohistochemistry results of a B-cell lymphoma in multiple organs of a 35-y-old captive male giant panda. The animal was euthanized because of prolonged anorexia and vomiting. Postmortem examination revealed ascites, generalized lymphadenopathy, splenomegaly, hepatomegaly, and thickened gall bladder mucosa. Histologically, the architecture of these organs was effaced by a densely cellular neoplasm composed of large, CD79a-positive neoplastic B lymphocytes supported by a fine fibrovascular stroma. Neoplastic cells occasionally palisaded around an eosinophilic fibrillary center, sometimes resembling Homer Wright rosettes. To our knowledge, rosette-forming lymphoma has not been reported previously in animals.

A 35-y-old, male giant panda (Ailuropoda melanoleuca) had resided in Ocean Park Hong Kong for >23 y. He had intermittent and progressive nausea, anorexia, weakness, and ascites in April 2022. Blood tests revealed mild persistent nonregenerative anemia and hyperproteinemia. An abdominal mass was palpated and later confirmed by computed tomography to be mesenteric masses. Other medical history of the animal included chronic hypertension, arthritis, bilateral cataracts and lens luxation, previous aspiration pneumonia, and perineal sebaceous gland adenoma. The animal’s condition deteriorated, and he was euthanized after 3 mo of palliative care.

At autopsy, generalized subcutaneous edema affected the mandibular and inguinal areas most severely. Further inspection revealed generalized lymphadenopathy with enlargement of all examined lymph nodes. The axillary (~100 × 40 × 60 mm bilaterally) and mesenteric (up to 90 mm long) lymph nodes were affected most severely, with extensive necrosis (Fig. 1A). In addition, >2 L of translucent, straw-yellow ascites distended the abdomen markedly. The liver was swollen, pale, with rounded edges, and contained generalized miliary white 2–3-mm foci (Fig. 1B). The spleen was enlarged uniformly and had a meaty texture. Sectioning of the spleen revealed crowded, pale-tan, 2–5-mm nodules, with some nodules protruding above the capsular surface (Fig. 1C). The mesentery adjacent to the spleen contained a 10 × 5 × 5-mm, dark-red nodule surrounded by hemorrhage. The mucosal surface of the gall bladder was diffusely thickened by prominent finger-like projections, and it was distended with thick bile and green-yellow gelatinous material (Fig. 1D). Throughout the length of the intestinal tract, there was a small amount of soft, orange-to-yellow digesta. There was perineal staining with soft orange stool.

Figure 1.

Gross features of a B-cell lymphoma in a giant panda. A. Mesenteric lymph nodes were enlarged. B. The liver was swollen and contained myriad 2–3-mm white foci. C. Cross-section of the formalin-fixed spleen with crowded pale-tan nodules. D. Thickened gall bladder mucosa, with intraluminal green-yellow gelatinous material.

Histologically, the splenic white pulp was markedly expanded by nodules consisting of sheets of round-to-oval neoplastic cells supported by a fine fibrovascular stroma and compressed against the adjacent red pulp. Neoplastic cells often appeared to palisade around an eosinophilic fibrillary center sometimes resembling Homer Wright rosettes. They had distinct cell borders, small-to-moderate amounts of eosinophilic cytoplasm, and a round, oval, or convoluted nucleus that was 2–3 times the size of a red blood cell. Nuclei were vesiculated with 1 or 2 prominent nucleoli. Anisocytosis and anisokaryosis were moderate. The mitotic count was 12 in 2.37 mm ² (digital whole-slide imaging). A few fibrin strands were present within some nodules.

The normal architecture of the mandibular, superficial cervical, axillary, popliteal, inguinal, sternal, and mesenteric lymph nodes was replaced by diffuse sheets of neoplastic cells resembling those noted in the spleen, with rosette-like structures (Fig. 2A). In some sections, mitotic count was up to 23 in 2.37 mm ² . Areas of necrosis and hemorrhage were frequent, and neoplastic cells extended to the adjacent adipose tissues with resultant edema and fibrosis. Occasionally, neoplastic cells were present within blood vessels, lymphatic vessels, or sinuses. Hemosiderin-laden macrophages were sometimes present.

Figure 2.

Histologic features of a B-cell lymphoma in a giant panda. A. Neoplastic cells were often arranged in a rosette-like pattern in the mesenteric lymph nodes. H&E. B. Neoplastic cells infiltrated and expanded the gall bladder mucosa. H&E.

The lamina propria of the gall bladder mucosa was diffusely expanded by sheets of neoplastic lymphocytes with a mitotic count similar to that noted within the spleen, except that the rosette-like structure was inapparent (Fig. 2B). Neoplastic cells sometimes infiltrated the overlying epithelium, which was segmentally attenuated, and multifocally extended into the muscle layer and serosa. The serosa was edematous with dilated lymphatic vessels containing fibrin thrombi admixed with cellular debris. Within the lumen, there was cellular debris, degenerate leukocytes, fibrin, proteinaceous fluid, fewer macrophages, lymphocytes, and occasional bacterial colonies.

Within the liver, nodules of neoplastic lymphoid cells sometimes tracked portal triads. Multifocally, there were areas of hepatocyte atrophy or loss, with edema, fibrin, fibrosis, increased bile duct profiles, and infiltrates of neutrophils and lymphocytes. Other pathology findings included mitral valve fibromyxomatous degeneration, mycotic esophagitis, renal interstitial fibrosis with tubular degeneration and necrosis, anthracosis, pulmonary bullae and fibrosis, adrenal degeneration, ectopic spleen with lymphoma, and goniodysgenesis.

Selected paraffin-embedded sections, along with positive and negative control tissue sections, were cut at 4 µm and attached to charged slides for immunohistochemistry (IHC) analysis. The antibodies used were anti-CD3, anti-CD79a, anti-vimentin, anti-cytokeratin AE1/AE3, anti-chromogranin A, and anti-synaptophysin (Table 1). Automated IHC staining and Bond polymer refine detection systems (Leica) were used for staining, according to the manufacturer’s instructions. Tissue sections with the primary antibody replaced by antibody diluent (Leica) served as negative controls. Normal canine or feline lymph node (CD3, CD79a), canine or feline skin (vimentin, cytokeratin AE1/AE3), and canine or feline adrenal gland (chromogranin A, synaptophysin) were used as positive controls (Table 1). Immunolabeling was positive for vimentin and CD79a on the cell membrane and in the cytoplasm of neoplastic cells (Fig. 3A). Results for CD3 and the remaining antibodies used were negative (Fig. 3B). Our IHC results indicated that the neoplastic cells were derived from B lymphocytes.

Table 1.

Antibodies used in our case of a B-cell lymphoma in a giant panda.

Antibody	Host	Source	Clone	Antigen retrieval	Dilution	Positive canine or feline control
CD3	Rabbit	Dako	A0452	ER2*	1:200	Lymph node
CD79a	Mouse	BioCare	HM47/A9	ER2*	1:100	Lymph node
Vimentin	Mouse	Dako	VIM 3B4	ER1†	1:100	Skin
Cytokeratin AE1/AE3	Mouse	Dako	AE1/AE3	ER2*	1:100	Skin
Chromogranin A	Mouse	Dako	DAK-A3	ER1†	1:200	Adrenal gland
Synaptophysin	Mouse	Dako	DAK-SYNAP	ER1‡	1:100	Adrenal gland

The chromogen used in all instances was diaminobenzidine, and the autostainer was the Bond-III slide staining system (Leica).

^*Heat retrieval for 20 min (ER2, Bond epitope retrieval solution 2; Leica).

^†Heat retrieval for 20 min (ER1, Bond epitope retrieval solution 1; Leica).

^‡Heat retrieval for 30 min (ER1, Bond epitope retrieval solution 1; Leica).

Figure 3.

Immunohistochemical features of a B-cell lymphoma in the spleen of a giant panda. A. Large numbers of neoplastic cells labeled for CD79a. Immunohistochemistry (IHC) for CD79a, diaminobenzidine (DAB). B. Small numbers of CD3-positive non-neoplastic T cells scattered within the mass and forming a narrow cuff at the periphery. IHC for CD79a. DAB.

Being an endangered species, limited studies have reported on natural diseases in the giant panda. There is only one case report of systemic lymphoma in a female, 30-y-old giant panda published 40 y ago. ⁶ Similar to our case, the mesenteric lymph node was severely affected. The article was written in Chinese without immunohistochemical details. ⁶ Lymphoma is among the most frequently diagnosed malignancies in the dog; most cases are multicentric, diffuse large B-cell lymphoma. ⁴ There is no standard classification of lymphoma for giant pandas. In our case, the neoplastic cells had a diffuse distribution in all of the lymph nodes and a nodular pattern in the spleen.

An unusual finding in our case was the pronounced rosette arrangement of neoplastic lymphoid cells. We retrieved no cases of rosette-forming lymphoma in animals in a search of Google, PubMed, CABI Direct, Web of Science, and Scopus, using the search terms “rosette” and “lymphoma”, suggesting that rosette-forming lymphoma has not been reported previously in the veterinary literature. Rosettes have significant histologic diagnostic value in neural and neuroectodermal tumors, such as primitive neuroectodermal tumors, ependymoma, and glioblastoma. The positive CD79a immunohistochemical reactions and negative results for neuroendocrine markers in our case confirmed a diagnosis of B-cell lymphoma. In humans, rosette-forming lymphomas are rarely reported, and can be seen in a variety of B-cell and T-cell lymphomas; ultrastructurally, the central fibrillary material is composed of cytoplasmic processes projecting from the lymphocytes and contains thin and intermediate filaments.^1–3,5 Based on our findings, lymphoma should be considered as one of the differential diagnoses if rosettes are seen in lymphoid tissues.