B‐cell lymphoblastic lymphoma of the nictitating membrane as the first presenting sign in a 2‐year‐old Springer Spaniel

Key Clinical Message B‐cell lymphoblastic lymphoma is an aggressive malignant disease. Necropsy and microscopical examination revealed widespread disease with a high proliferation index. This is the first reported case of B‐cell lymphoblastic lymphoma presenting in the ocular region and only the second reported lymphoma of the nictitating membrane.

finely distributed chromatin, indistinct nucleoli, and scant cytoplasm. 3 The architecture of the cells was diffuse, and there was a high mitotic rate. 3 Canine lymphoma can be staged according to the World Health Organization staging system: stage I with involvement of a single node or a single organ (excluding bone marrow); stage II with regional involvement of multiple lymph nodes (± tonsils); stage III with generalized lymph node involvement; stage IV being stages I-III with involvement of liver and/or spleen; and stage V being stages I-IV with involvement of blood or bone marrow. Furthermore, canine lymphoma can be substaged with A (absence of systemic signs) or B (presence of systemic signs: fever, >10% weight loss, hypercalcemia). 6 Periocular and intraocular presentations in lymphomas can be seen in dogs. However, these tumors have received limited attention in the literature. [7][8][9][10][11][12][13][14][15][16] To the best of our knowledge, this is the first case in the literature to report B-LBL as the first presenting sign in the ocular region in a dog.

| CASE REPORT
A 2-year-and-7-month-old, intact, female Springer Spaniel presented to a veterinary ophthalmology referral clinic with a 1-month history of a unilateral problem of the nictitating membrane. Sixteen days prior to referral, the patient was treated with an injection of amoxicillin trihydrate 150 mg/ mL (Curamox Prolongatum ® , Boehringer Ingelheim A/S, Copenhagen, Denmark), amoxicillin trihydrate and clavulanic acid 250 mg/12.5 mg (Clavubactin ® , Dechra Veterinary Products A/S, Uldum, Denmark) a half tablet twice a day, and fusidic acid (Isathal®, Dechra Veterinary Products A/S) eye drops 10 mg/g in viscous vehicle one drop twice a day.
After 9 days, no improvement was observed and fusidic acid was discontinued. Topical dexamethasone sodium phosphate and chloramphenicol 1 mg/mL/5 mg/mL (Spersadex Comp ® , Laboratoires THEA, Clermont-Ferrand, France) was instilled one drop twice a day.
On presentation, a protrusion of the right side nictitating membrane (NM) was evident. On the bulbar aspect of the NM, the tumor area was thickened to approximately 5 mm, flabby, and mildly hyperemic. Slit-lamp biomicroscopy (SL-17, Kowa Ltd., Nagoya, Japan) of the cornea, anterior chamber, iris, and lens was unremarkable. Indirect ophthalmoscopy was not performed. Schirmer tear testing (STT, Mark Blu Optitech Eyecare, Allahabad, India) was 20 mm/ min OD and 19 mm/min OS. Intraocular pressure measured with applanation tonometry (Tonopen Vet Medtronic Solan, Reichert Technologies, Munich, Germany) was 20 mm Hg OD and 17 mm Hg OS. Direct and indirect pupillary light reflex, menace response, and palpebral reflexes were normal. Examination of the oral cavity showed no abnormal signs. The weight was 17.6 kg and, apart from the eye problem, the patient was agile and in a good health condition. No laboratory tests were performed at this time. Local treatment from the referring veterinarian continued in this period.
A transpalpebral ultrasound scan with linear probe SL 1543 (Esaote MyLab Gamma, Genova, Italy) revealed no bulbar or retrobulbar involvement. A small amount of fluid with a high number of neutrophils was retrieved with fine needle aspiration from the NM swelling.
On suspicion of an abscess or intramembranal foreign body, the membrane was bluntly opened caudal to the Tshaped cartilage. An amount of 0.5 to 1 mL pus-like fluid with two or three small foreign bodies resembling plant material escaped. The cavity was flushed through a contralateral opening with a 0.9% NaCl solution. Openings were left open for secondary intention healing.
Topical chloramphenicol (Kloramfenikol Viskouse DAK ® , Takeda Pharma A/S, Taastrup, Denmark) and carprofen 50 mg 4 mg/kg per oral (Norodyl Vet ® , ScanVet Animal Health) continued postoperatively. Due to the initial suspicion of an abscess and the fact that the patient was young, no staging for lymphoma was done at this point.
Another 6 days later, the NM protruded even more, but the patient still showed no discomfort. The swelling had become more firm and multinodular with no content of pus. A small sample of tissue of the NM was harvested for histopathology under general anesthesia. Postoperative medication continued unchanged.
The NM continued to enlarge for 2 weeks and started to cause the patient discomfort ( Figure 1A). A decision to remove the NM was made. After standard pre-surgical procedure, the NM was lifted and a full resection performed with the openings left for secondary intention healing. Postsurgical treatment with chloramphenicol and carprofen continued. The tissue was submitted for histopathological investigation.
At follow-up 37 days after initial presentation, the eye was comfortable and the wound in the conjunctiva was healing properly. However, the general condition had deteriorated and the patient was now in poor condition. During the last days, the patient developed inappetence and depression, with moderate weight loss, a high temperature of 39.3°C, and generalized lymphadenopathy. While awaiting the result of the histopathology, a treatment with subcutaneously administered steroids against a suspected lymphoma was initiated with dexamethasone sodium phosphate 0.1 mg/kg (Rapidexon ® 2 mg/mL, Dechra Veterinary Products A/S). The diagnosis of a malignant lymphoma in the NM was reported. Due to the initial suspicion of an abscess and the fact the patient initially presented with no other signs of affection, no staging was done initially. We could not determine whether this was a primary lymphoma disseminating or a secondary lymphoma disseminated from elsewhere in the body, because of the lacking initial staging.
The owner had decided not to continue treatment in case of a malignant disease, and thus, no further staging was done after the suspicion of lymphoma arose. The patient was euthanized and, in accordance with the owner's wish, samples from the patient could be used for scientific purposes.

| NECROPSY
Necropsy showed a generalized condition with tumor growth in the liver, spleen, and left ovary. Lymphadenopathy was pronounced throughout the whole body. Carcass weight was 16.5 kg, showing a mild weight reduction of 1.1 kg since initial presentation. In the spleen, disseminated growth of firm, round tumors ranging from a few millimeters to 3-4 cm in diameter were found (Figure 2A). In one liver lobe, there was a horseshoe to umbilicated-shaped process of 8 × 6 cm with central invagination and purulent secretion, suggesting tumor growth with severe necrosis in the center ( Figure 2C). The left ovary measured 10 × 6 × 4 cm containing one large, firm homogenous tumor. Tonsillar lymph nodes were moderately enlarged ( Figure 2E). The cranial sternal lymph node measured 4 × 3 × 2 cm. Especially in the abdomen, intestinal lymph nodes were conspicuous, with considerable thickening of the small intestinal walls.

| Materials and methods
All specimens were processed routinely, paraffin-embedded, sectioned, and stained with hematoxylin-eosin. Further staining with Giemsa-AR was performed. Immunohistochemical procedures were performed on a Ventana Benchmark ULTRA platform (Ventana Medical Systems Inc., Tucson, AZ, USA) as previously described 17  . For each of the above-mentioned antibodies, we ran both internal and external controls to validate our stainings. The external controls were human tissue, whereas we used a normal lymph node from the same patient as internal control. The positive external controls expressed the proteins of interest, whereas the negative external controls did not express the proteins of interest, indicating that the procedure was working. The lymph node demonstrated CD3+, CD4+, and CD8+ T cells in the parafollicular cortex (positive internal control), whereas no cells stained positive for these T-cell markers in the follicles of the lymph node (negative internal control). Furthermore, a non-specific negative control for each antibody was performed. These internal controls indicate that the stainings could be used in this particular patient. The specimens with tumor infiltration were also stained with Ki-67 (clone MIB1, code M724001, mouse anti-human, 1:100, Dako, Glostrup, Denmark). blast morphology. They were densely stained round cells of moderate size with intermediate-sized nuclei. The cell borders were distinct. The chromatin was finely distributed with almost indistinct nucleoli. The cytoplasm was scant ( Figure 1B,C). A starry-sky configuration could be observed. There were 13 mitotic figures per high power field (×400 magnification). The tumor cells were infiltrating diffusely, and there was no necrosis. The tumor cells stained positive for PAX5 ( Figure 1D) and negative for CD3 ( Figure 1E) and CD34. The tumor cells did not stain for TdT or the T-cell markers CD4 or CD8; nor did they stain for the B-cell markers CD20 and CD79α. The Ki-67 index was 90% ( Figure 1F). The morphology and immunohistochemistry were consistent with the diagnosis of B-LBL.

| Spleen
The white and red pulp was almost completely substituted by B-LBL tumor cells ( Figure 2B). These cells were positive for PAX5 and negative for CD3 and CD34. The Ki-67 proliferation index was 40%-50%.

| Tonsils
The crypts of the tonsil were heavily and diffusely infiltrated with tumor cells of B-LBL, making it difficult to see the normal tonsil structure of the specimen ( Figure 2F). The tumor cells were positive for PAX5 and negative for CD3 and CD34. The Ki-67 proliferation index was 50%-60%.

| Popliteal lymph node
In the cortex of the lymph node, B cells were found in follicles. In the parafollicular cortex, CD3+ T cells, CD4+ T cells, and CD8+ T cells were found. The medulla contained lines of B cells in variable stages as well as macrophages. This specimen showed no signs of infiltration of tumor cells.

| Bone marrow
Blood vessels, hematopoietic cells, and adipocytes were diffusely infiltrated by B-LBL tumor cells. The tumor cells demonstrated positivity for PAX5 and negativity for CD3 and CD34. The Ki-67 proliferation index was 40%-50%.

| Staging
As described above, heavy tumor infiltration of several organs was present at necropsy. The tumor stage of our case was thereby stage VB with involvement of bone marrow and a systemic sign, fever.

| DISCUSSION
We herein present a case of B-LBL in a 2-year-and-7-monthold, intact, female Springer Spaniel with the first presenting sign in the NM of a systemic disease. Lymphoblastic lymphomas are the most aggressive lymphoma subtypes encountered commonly in veterinary practice. 18 Likewise, the present case showed a very aggressive behavior. LBL typically presents with the dog being visibly ill but in good condition, indicating a rapid onset of the lymphoma. 18 Primary unicentric canine periocular and intraocular lymphomas are rare. However, ocular manifestations in multicentric lymphomas are quite common. Some reports suggest that one-third of multicentric lymphomas have ocular involvement, making it the second most common clinical sign. 19 Canine B-LBL is less common than the T-cell counterpart T-LBL 3 ; however, it seems that LBL is more frequent in younger individuals, as in the present case. 20 When clinical suspicion of a lymphoma is raised, a thorough diagnostic procedure should begin. This includes clinical evaluation, computed tomography (CT) scan, and a bone marrow biopsy in order to determine the stage. Due to prognosis as well as treatment options, it is of great importance to know the specific subtype of lymphoma. By applying immunohistochemistry in canine lymphoma, it has become possible to make the specific diagnosis. In the present case, we saw diffuse infiltration of lymphoblastic cells; however, to further classify the lymphoma, we stained with the T-cell markers CD3, CD4, CD8, and the B-cell markers CD20, CD79α, as well as PAX5. We also stained for CD34, which can be used as a marker for the leukemic B-cell subtype, B-cell acute lymphoblastic leukemia. We used internal as well as external controls for each specimen and each stain. The positive internal controls indicate that our immunostainings did work in this particular patient and that the tumor cells were positive for PAX5 and negative for CD3, CD4, and CD8, and thus, the diagnosis of B-LBL was confirmed.
The high Ki-67 proliferation index of the tumor of the NM suggests a very aggressive behavior, and the rapid growth of the tumor likewise suggests aggressive behavior.
Lymphoblastic lymphoma is a highly aggressive subtype of lymphoma with a rapid clinical course. The B-cell variant is less common than the T-cell counterpart in canines, and little information about the clinical course is known for both subtypes. In a study of 13 cases of T-LBL, the longest duration of clinical signs was 4 weeks and all patients were either stage III with generalized lymph node enlargement in both the front half and the back half of the body or stage IV with involvement of the liver and/or spleen, suggesting a very aggressive behavior. 20 Due to the aggressive nature of B-LBL and the widespread lymphoma, chemotherapy is the therapy of choice for lymphoblastic lymphoma, regardless of B-cell or T-cell linage.
To better understand lymphomas and their subtypes in the canine family, further research is needed. With the use of immunohistochemistry, it has become possible to diagnose lymphomas more specifically, and in the future, this can provide valuable information regarding lymphoma subtypes in the eye region. This will lead to more differentiated information regarding prognosis and available therapeutic modalities.