B-cell multicentric lymphoma in a creole mare - a case reportABSTRACT
Lymphomas are neoplasms of origin in lymphocytes, rarely reported in equines, and classified as multicentric, alimentary, mediastinal, cutaneous, and extranodal in this species. This paper aims to report the case of a Creole mare with multicentric lymphoma. The case of a mare with weight loss, lethargy, and lymphadenomegaly unresponsive to therapy is reported. Clinical examination showed tachycardia, tachypnoea, and dyspnoea, in addition to enlargement and stiffening of palpable lymph nodes, suggestive of hyperplasia by aspiration cytology. The animal showed progressive worsening, not responding to therapy, and death occurred during the second clinical evaluation. Necropsy showed an increase in the size of all palpable lymph nodes and internal lymph nodes. Marked thoracic and abdominal effusions were also observed. Microscopically, the neoplastic proliferation of large lymphocytes was noted, infiltrated in multiple organs. Immunohistochemical (IHC) evaluation of tumor showed positivity for PAX-5 and Ki-67. The association of histopathology and IHC results allowed classifying the neoplastic process as a multicentric large B-cell lymphoma. This type of lymphoma in equines tends to have a silent clinical behavior, with a progressive evolution and clinical condition usually culminating in death.
Keywords:
horse; lymphadenopathy; effusion; immunohistochemistry; lymphoid neoplasm
RESUMO
Os linfomas são neoplasias de origem nos linfócitos, relatadas em equinos e definidas como multicêntricas, alimentares, mediastinais, pertinentes e extranodais nessa espécie. Este artigo tem como objetivo relatar o caso de linfoma multicêntrico em uma égua Crioula. É relatado o caso de uma égua com perda de peso, letargia e linfadenomegalia, sem resposta à terapia. O exame clínico mostrou taquicardia, taquipneia e dispneia, além de aumento e resistência dos linfonodos palpáveis, sugestivo de hiperplasia pela citologia de aspiração. O animal apresentou piora progressiva, não respondendo à terapia, com evolução à morte durante a segunda avaliação clínica. A necropsia mostrou linfadenomegalia de todos os linfonodos palpáveis e linfonodos internos. Efusões torácicas e abdominais acentuadas também foram observadas. Microscopicamente, foi observado o espectro neoplásico de linfócitos grandes, infiltrados em múltiplos órgãos. Na avaliação imuno-histoquímica (IHC), o tumor mostrou positividade para PAX-5 e Ki-67. A associação da histopatologia e dos resultados da IHC permitiu classificar o processo neoplásico como linfoma multicêntrico de células B grandes. Esse tipo de linfoma em equinos tende a ter um comportamento clínico silencioso, com uma evolução progressiva e uma condição clínica geralmente culminando em morte.
Palavras-chave:
cavalo; linfadenopatia; efusão; imuno-histoquímica; neoplasia linfoide
INTRODUCTION
Lymphomas are neoplasms of origin in T or B lymphocytes, considered the main hematopoietic neoplastic entities of domestic animals (Valli et al., 2017). It is not a common neoplasm in horses, but studies have reported the occurrence of this neoplasm in the species (Siegers et al., 2017; Bacci et al., 2020; Camacho-Luna et al., 2021).
In horses, this neoplasm can be classified as multicentric or generalized, the most commonly observed form, in addition to the alimentary/intestinal, mediastinal, cutaneous, and extranodal forms (Taintor and Schleis, 2011; Durham et al., 2013). The clinical signs observed depend, in most cases, on the affected organ, duration of the process, and dimensions of proliferation (Muñoz et al., 2009). In general, the animals may present lethargy, anorexia, inappetence, weight loss, reluctance to exercise, and fever (Muñoz et al., 2009; Bacci et al., 2020).
The most affected organs tend to be lymph nodes, liver, spleen, intestine, kidneys, lungs, and bone marrow (Muñoz et al., 2009). Similar to the clinical signs, topographic presentation is also related to the site of origin of the neoplastic process (Valli et al., 2017). Usually, nodules, when formed, are diffusely white and soft to slightly firm (Taintor and Schleis, 2011; Valli et al., 2017). The main type in the immunophenotypic classification is T-cell-rich large B-cell lymphoma (TCRLBCL), followed by peripheral T-cell lymphomas (PTCL) and diffuse large B-cell lymphoma (DLBCL). TCRLBCL tends to predominate even in all topographic distributions (Durham et al., 2013).
This report aims to show a case of multicentric lymphoma in a mare, describing its macroscopic, histopathological, and immunophenotypic characteristics.
CASE REPORT
A 22-year-old female Creole equine with a Zaina coat had a history of weight loss, lethargy, and lymphadenopathy (Fig. 1). Initially, she was treated with an anti-inflammatory (Maxican® 2%)1 and antibiotics (Pencivet® Plus PPU)2, but without success. Physical examination showed a body score of 2 (1-5), prostration, tachycardia, tachypnoea, and dyspnea with norm phonetic sounds. The inspection showed oedema in the ventral cervical region and an increase in mandibular, prescapular, precrural, cranial deep cervical, caudal deep cervical, medial and lateral retropharyngeal, and parotid lymph nodes, firm to pressure, generating the suspicion of lymphoma development. Blood samples were collected in two aliquots, one for hematological evaluation, which showed no significant changes, and another for diagnosis of equine infectious anemia (EIA) and glanders, both with negative results. Also, a fine-needle aspiration biopsy (FNAB) of the mandibular, prescapular, and caudal deep cervical lymph nodes was performed, without a conclusive diagnosis, but suggesting reactive lymphoid hyperplasia.
B-cell multicentric lymphoma in a Creole mare. Note prescapular (black dashed), cervical (yellow dashed), and retropharyngeal (red dashed) lymphadenomegaly.
Material collection from an incisional biopsy of lymph nodes for histopathological examination was suggested to the tutor due to the ineffectiveness of the complementary tests performed until that moment in determining the diagnosis and the still persistent suspicion of lymphoma development. However, the tutor chose not to do so. Euthanasia was indicated due to the inefficient response to therapeutic measures, inconclusive diagnostic suspicion, and the impossibility of keeping the patient in perfect conditions of well-being, but the tutor chose not to perform it at that time. A new consultation was requested on the fifteenth day after the first one due to a complaint of worsening in the clinical condition, characterized by prostration, marked hyporexia, and intense dyspnea at rest. The patient presented progressive respiratory distress during care when walking for physical examination, culminating in death.
The animal was referred for necropsy and the macroscopic evaluation showed a marked increase in the size of all palpable lymph nodes, firm to pressure and discreetly adhered, and diffusely white and irregular on section. The thoracic cavity was filled with approximately four liters of translucent serosanguineous fluid (Fig. 2A), compressing the lungs dorsally. Mediastinal lymph nodes were moderately enlarged, whitish, and firm, with multiple nodules similar in appearance to the parenchyma on section.
The abdominal cavity was filled with about five liters of translucent yellow fluid, with mild bundles of fibrin. The splenic hilum lymph node chain was diffuse and markedly enlarged, with firm, multinodular nodes (Fig. 2B), but without infiltrating the splenic parenchyma. A nodule of approximately 13 cm in diameter, soft on section and with the same appearance and color as the parenchyma, at the ventral end of the gastric surface, could also be observed in the spleen. Mesenteric lymph nodes were also diffusely enlarged, and firm, yellowish-white, and with more evident nodules than the previous ones on the section (Fig. 2C).
The lymph node chain of the ascending colon was also slightly enlarged, with no nodules in the lymph nodes despite being firm and white. The left ovary and salpinx intensely adhered to the retroperitoneal wall from a soft, yellowish-white irregular mass of approximately 15×8×3 cm, which infiltrated the abdominal teat musculature. Samples from all organs, enlarged lymph nodes, and periovarian mass were collected and placed in 10% buffered formalin for subsequent routine histological processing. The prepared histological slides were stained with hematoxylin and eosin and evaluated under optical microscopy.
Histopathological evaluation showed the development of a non-delimited or encapsulated neoplastic process, replacing the typical lymph node parenchyma, consisting of round cells similar to lymphocytes, which were organized in a dense sheet interspersed with scarce fibrovascular stroma (Fig. 2D). The nucleus of neoplastic cells was slightly peripheral, round to oval, with dense chromatin, measuring between 2 and 2.5 erythrocytes, and 2 to 3 evident central nucleoli. The cytoplasm of these cells was sparse, pale eosinophilic, and poorly delimited. There was moderate anisocytosis and anisokaryosis and about 3 mitoses per 2.37mm2. The histological features were compatible with the morphological diagnosis of large cell lymphoma with multicentric topography.
Macrophages with stained corpuscles, randomly distributed and in small amounts, were observed during the neoplastic process. This same neoplasm was observed infiltrated around the renal tubules and glomeruli, bordering bronchi and bronchioles, and compressing peripheral alveoli, infiltrating between the layers of the tunica adventitia of the aorta artery, between the serosa and the external muscular layer of the stomach and distending the periportal and portal spaces of the liver. The mass that kept the ovary and salpinx adhered to the retroperitoneum also comprised a lymph node deformed by neoplastic proliferation. In addition to the neoplasm, moderate to marked multifocal to coalescing atelectasis followed by marked oedema was observed in the lungs. The nodulation observed in the spleen comprised a white pulp hyperplastic process.
Furthermore, histological sections of lymph nodes were sent to the Nucleus of Pathological Anatomy of the Adolfo Lutz Institute (IAL), São Paulo, SP, Brazil, for anti-CD3 (polyclonal, 1:400)3, PAX-5 (Paired Box 5; BC-24, 1:200)3, and Ki-67 immunohistochemistry (MIB-1, 1:100)3. The reaction signal was amplified using a polymer conjugated to the enzyme peroxidase (Novolink)4 and visualized under light microscopy with the chromogenic substrate diaminobenzidine (DAB)5. Immunostaining showed positivity for PAX-5 (Fig. 2E) and Ki-67 (Fig. 2F) in neoplastic cells and CD3 in non-neoplastic lymphocytes infiltrated in the neoplasm, allowing classifying the neoplastic process as a large B-cell multicentric lymphoma.
B-cell multicentric lymphoma in a Creole mare. A. Thorax filled with serosanguineous fluid. B. Diffusely enlarged, irregular, and whitish splenic lymph nodes and splenic nodule with the same appearance as the parenchyma on section (asterisk) (bar: 12cm). C. Markedly enlarged and irregular mesenteric lymph node losing cortical and medullary conformation (bar: 0.5cm). D. Photomicrograph of mediastinal lymph nodes demonstrating neoplastic lymphocytes with nuclei ranging in size from 1.5 (red arrowhead) to 3 erythrocytes (black arrowhead) (Haematoxylin and Eosin, x300). E. Moderate anti-PAX-5 immunostaining in neoplastic B lymphocytes (x400). F. Moderate anti-Ki-67 nuclear staining in neoplastic cells (x100) [peroxidase-linked polymer (MACH 4 HRP), DAB chromogen].
DISCUSSION
The clinical presentation of lymphoma in equines is variable and nonspecific (Taintor and Schleis, 2011), resulting from dysfunction of affected organs, and weight loss, fever, inappetence, lymphadenopathy, colic, and ventral and limb oedema can be observed. The course of the disease is rapid after the appearance of these signs (Camacho-Luna et al., 2021). Except for colic, all other signs were observed in this mare during an evolution period of approximately 30 days.
The cytological evaluation of the patient did not allow an exact diagnosis. Although this diagnostic technique is efficient for the diagnosis of lymphomas in most cases, numerous factors can contribute to the inefficiency of the diagnosis, including sample quality, the interval between collection and preparation and the artifacts in the slide preparation, and the biopsy technique (Villiers, 2020). The cytological result that indicates a condition of nodal reactivity in the patient can be considered a false negative, in which the cellularity obtained from the lymph node is mixed, with small, medium, and large reactive cells, and not monomorphic as expected in classic cases of lymphoma, as described in humans (Taintor and Schleis, 2011).
Thoracic and abdominal effusions can occur in cases of lymphomas in equines, consisting of relatively uncommon findings rarely reported in these animals (Camacho-Luna et al., 2021), as described in this case, in which both cavities had together about nine liters of serosanguineous effusion. Isolated cavitary effusion in the thorax in cases of mediastinal lymphoma is more frequent in horses (Muñoz et al., 2009). However, multicentric lymphomas occur when intrathoracic neoplastic masses lead to important changes at the vascular level (Siegers et al., 2017).
The occurrence of effusions in cases of lymphoma is not considered a paraneoplastic condition, but a consequence of the expansion and infiltration of the tumor mass in the tissues (Siegers et al., 2017), as observed in this patient, whose lymph nodes reached large proportions and high peripheral compression capacity. Paraneoplastic syndromes associated with lymphomas in equines include hypercalcemia, hyperfibrinogenemia, thrombocytopenia, amyloidosis, tumor lysis syndrome, and pruritic and alopecic skin diseases (Axiak and Johnson, 2012), but the patient showed no changes that would indicate the occurrence of these clinical or hematological conditions.
Reports of the occurrence of numerous hematological changes in humans, such as hemolytic anemia, coagulopathies, and hemophagocytic syndromes can be found in the literature (Axiak and Johnson, 2012). However, except for anemia, these changes are not well studied and described in equines, nor have they been observed in the mare of this report. No changes were observed during the necropsy that would suggest a coagulopathic condition, commonly indicated by multiple hemorrhages.
Histologically, the classification of lymphomas is based mainly on the morphological characteristics of the cells since the arrangement is a little variable, predominantly in a dense cellular mantle (Valli et al., 2017). Although there are several classifications available for lymphomas in domestic animals, all are derived from adaptations of human medicine, there are no established criteria with complete reliability for the classification of these neoplasms in equines. The lymphoma of this patient was determined as large cell lymphoma based on the postulates of the Working Formulation of the National Cancer Institute, or simply WF-NCI, which defines the types of lymphoma based on the intrinsic characteristics of the cells, and adaptations of this classification for other animal species (Vezzali et al., 2010). These neoplastic lymphocytes have rounded to oval nuclei, with evident folds and cleavages, measuring between 7.5 and 15µm in diameter, equivalent to 1.5 to 3 erythrocytes.
This WF-NCI classification has little information regarding the determination of the neoplastic cell phenotype, diverging from the most current studies, such as that of the World Health Organization. Therefore, it alone is not capable of providing a wealth of information regarding the prognosis and behavior of the neoplasm.
The samples of neoplastic lymph nodes from this patient sent for IHC evaluation allowed the determination of the clonal lymphocyte as type B from the positive anti-PAX-5 staining. Few reports can be found in the literature regarding the use of PAX-5 as a lymphocytic marker for animals of the equine species, with results ranging from weak to strong, but efficient for phenotypic differentiation (Durham et al., 2013). This marker is important for the differentiation of B lymphocytes in humans, expressing in young lymphocytes even at the stage of transformation into plasma cells, but its use is still small (Shukla et al., 2015). Studies have shown that labelling for PAX-5 and CD20, one of the main markers for B lymphocytes, appears to be very similar, making their use safe for lymphocyte phenotype differentiation (Shukla et al., 2015).
The anti-CD3 was another IHC marker used for the samples of this patient. This marker is historically used to identify T lymphocytes and can be identified in small amounts in B-cell lymphomas and in higher amounts in T-cell-rich large B-cell lymphomas (TCRLBCL), one of the most frequently diagnosed types in horses (Durham et al., 2013). In this patient, the existing CD3 label is believed to be expressed only in residual cells from the affected lymph nodes and in cells acting directly as an immune component against the neoplastic process.
The definitive diagnosis consisted of diffuse large B-cell lymphoma (DLBCL) with a multicentric presentation due to the low positivity for CD3 in the patient in this report and the fact that it was present only in typical lymphocytes. This specific type of lymphoma is relatively common in equines, as described in a large study by Durham et al. (2013), in which 26 out of 203 cases classified as lymphoma were DLBCL. This lymphoma usually has a highly malignant behavior and is difficult to treat (Durham et al., 2013). Additionally, the malignancy of this neoplasm was confirmed by the expressiveness of the anti-Ki-67 marker, which is believed to be the staining intensity is proportional to the degree of malignancy for cases of lymphoma (Huber et al., 2021).
CONCLUSION
Multicentric lymphomas in equines tend to have a silent clinical behavior, with a progressive evolution and clinical condition usually culminating in death. It is important to consider lymphoma as a differential for cases of two-cell effusion in equines. The histological and phenotypic classification of lymphomas is essential for determining the degree of malignancy and prognostic complexity. The use of IHC as a method of lymphocyte differentiation proved to be very efficient for classifying the neoplastic type, and the use of this tool is recommended whenever possible.
ACKNOWLEDGMENTS
To the Adolfo Lutz Institute for performing the immunohistochemical reactions on the histological sections and to the University of Santa Catarina State (UDESC) for academic support.
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