Lymphoma in Horses

Krista E. EsteU

Lymphoma is the most commonly occurring hematopoietic neoplasia in horses. Previously, the terms lymphoma and lymphosarcoma have been used interchangeably, with the suffix “-sarcoma” used to characterize all malignant mesenchymal neoplasias.

Lymphoma is one of the most common malignant neoplasms in the horse; in postmortem studies, its prevalence is estimated to be 2% to 5%.² The underlying etiology of lymphoma in horses is undetermined. In humans there is an association between lymphoma and infection with the γ-herpesvirus Epstein Barr.^3,4 There are two case reports of horses with concurrent equine herpesvirus-5 infection and lymphoma or leukemia, one of which entered clinical remission after treatment with antivirals.^5,6 Virus-like particles have been described in lymph nodes of neonatal foals with lymphoma, though a causal relationship has not been proven.^7,8

Lymphoma can occur at any age, with reports in an aborted fetus to horses older than 30 years.^8,9 A large-scale retrospective study reports a median age of onset of all types of lymphoma to be 10 years; a previous study reviewing intestinal lymphoma reported a mean age of onset to be 17 years.^9,10 There is no apparent sex predilection, though certain breeds are more likely to develop particular subtypes of lymphoma.^10,11 Clinical signs vary depending on tumor location, but the most common include nonspecific lethargy, weight loss, pyrexia, and lymphadenopathy.

Although the hematologic features of lymphoma vary, anemia is a relatively common finding and is thought to be due to suppression of erythropoiesis, neoplastic bone marrow infiltra­tion, blood loss, or IMHA. A modest neutrophilic leukocytosis with an elevated fibrinogen is often seen. Atypical lymphocytes with or without elevated lymphocyte counts may be noted in the blood smears of more than 30% of affected horses, especially late in the disease course.¹⁷ When frank leukemia is present, neoplastic cells are often, but not always, found in the bone marrow.^18,19 Serum biochemistry profiles may provide an indication of internal organ involvement. Serum protein alterations are frequently observed and include hypoalbumin- emia, hyperglobulinemia with polyclonal or monoclonal gammopathy, and decreased albumin/globulin ratio.¹⁹ Additional immunologic derangements that have been reported in horses with lymphoma include hypogammaglobulinemia, neutropenia, and lymphopenia.^15,20,21

Though several different classification schemes exist, the World Health Organization classification system has been adopted for the accurate description of equine lymphoma. Using this classification system, tumors are characterized on the basis of anatomic location and cell lineage. In a recent retrospective study classifying 203 cases of equine lymphoma, 14 subtypes of lymphoma were identified and 13 anatomic sites were used for characterization: multicentric (involving at least 2 organs excluding the regional lymph node), gastro­intestinal, cutaneous, bone marrow, nodal, splenic, hepatic, mediastinal, heart, ocular/orbital, central nervous system, oral cavity, and nasal.

The diagnostic sample of choice is a biopsy of the affected lymph node or organ. Lymphoma is variably exfoliative, and even if there is a considerable amount of effusion, cytology may be inconclusive. Likewise, lymph node needle aspirates may be difficult to positively diagnose. Morphologically, lymphocytes are classified as small-cell type if their nuclear diameters are 1.5 times the diameter of erythrocytes or less; cells are classified as large if their nuclei average 2 times or more the diameter of erythrocytes.^23,24 Small-cell lym­phoma consists of 60% or more small cells while large-cell lymphoma consists of 60% or more large cells. The grade of lymphoma may also be assessed by determining the amount of mitoses seen within a high-powered field.^9,24

Immunohistochemical classification to determine the cell lineage is performed using monoclonal antibodies such as CD79a and CD20 for B-cell lymphomas and CD3 and CD8 for T-cell lymphomas. All three—B-, T-, and mixed B- and T-cell lymphomas—have been reported in the horse.^9,23,25 Several retrospective studies have identified a total of 14 subtypes of lymphoma in horses, with T-cell-rich B-cell lymphoma (TCRBCL) the most common subtype of lymphoma in the horse.^9,17,26 T-cell-rich, large B-cell lymphoma is a neoplastic proliferation of large B cells associated with a prominent component of nonneoplastic T cells and a dense fibrovascular network that prevents tissue fragmentation during sectioning.¹ The presence of large numbers of nonneoplastic T cells within a B-cell lymphoma could result in erroneous diagnosis of a T-cell tumor or a benign reactive lymph node, particularly if a small sample is submitted. Discriminating between neoplasia and chronic inflammation with reactive lymphocytes can be difficult in some cases; molecular clonality assays can be used to make this determination.

In addition to immunophenotyping and tumor cell mor­phologic description, establishing tumor location and presence of metastasis is necessary to determine disease severity and recommend a course of treatment. A thorough diagnostic work-up includes complete blood count with cytologic review by a hematopathologist, serum biochemical assay, thoracic radiographs, and abdominal ultrasound.²⁷ Though tumor biomarkers are used routinely in human medicine, there is limited research on their significance in horses. A recent paper describes significant elevations in serum thymidine kinase in horses with lymphoma when compared with horses with inflammatory conditions and nonhematopoietic neoplasia, indicating that this biomarker may be a useful ancillary diagnostic in horses with suspected lymphoma.²⁸ Accurate lymphoma characterization and staging in human medicine have been critical to establishing effective therapeutic manage­ment plans and accurate prognoses, and as more therapeutic options are explored in horses, it is likely that accurate diagnoses will have a similar impact.

Multicentric Lymphoma

The definition of multicentric lymphoma is lymphoma that involves two organ systems, excluding the regional lymph nodes, or involves multiple lymph nodes at distant anatomic locations. In previous literature, the term generalized lymphoma has been used to describe multicentric lymphoma, though this likely represents a more chronic or severe stage of disease.

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Alimentary or Intestinal Lymphoma

Lymphoma is the most common neoplasia of the intestinal tract in horses, and though the entire intestinal tract may be affected by lymphoma, disease occurs most frequently in the small intestine.^10,29 Though a breed predilection has not been established for lymphoma generally, a study reviewing intestinal neoplasia showed that Arabian horses are at an increased risk of developing intestinal lymphoma as compared with other breeds.^10,30 Lymphoma arises from the lymphocytes of the lamina propria of the intestinal tract, resulting in diffuse or segmental thickening, focal masses, or scattered crater-like ulcers with raised margins on the intestinal wall.^31-33 Peripheral lymphadenopathy is generally not present, though metastases to the lungs, mesenteric lymph nodes, liver, kidney, and spleen have been reported.^10,30,31 Intestinal lymphoma is usually associated with hypoalbuminemia secondary to protein-losing enteropathy, weight loss, edema, abdominal effusion, mild recurrent colic, and diarrhea.^10,29 Affected horses frequently have abnormal glucose or xylose absorption tests, indicating small intestinal malabsorption.³² Transabdominal ultrasound is the imaging diagnostic of choice and typically reveals thickened (>5 mm) small intestine, large colon, and/or cecum.

Thoracic Lymphoma: Mediastinal/Thymic

Lymphoma is the most common thoracic neoplasia in horses, and it may involve mediastinal lymph nodes, pericardium, thymus, or lungs.^33,34 The prevalence of mediastinal lymphoma varies from 4.5% to 29% of all forms of lymphoma.⁹ Primary and secondary (metastatic) mediastinal lymphomas have been reported, with the mediastinum and thoracic cavity commonly affected in horses with multicentric lymphoma.^17,34 Primary mediastinal lymphoma is usually seen in adult horses; however, the disease has been reported in a 1-month-old foal with a 1-week history of respiratory distress.³⁵ In the early stages of the disease, minimal clinical signs may be observed. Once disease has progressed, common clinical signs include nasal discharge, adventitious lung sounds, pleural effusion, thoracic ventral and limb edema, and regional lymphadenopathy at the thoracic inlet.^31,32 Other signs may include congestive heart failure, cough, pyrexia, pleurodynia, and respiratory distress.²⁹ Dysphagia may be observed in cases with retropharyngeal lymphadenopathy. An elevated nucleated cell count of several thousand cells∕μL, with a large percentage of abnormal lymphocytes, may be evident in pleural fluid cytologic analysis. However, collection of multiple pleural fluid samples may be necessary to establish a definitive diagnosis.³⁶ Cytologic abnormalities include the presence of prolymphocytes, lymphoblasts, and lymphocytes with evidence of nuclear blebbing, indented and cleaved nuclei, amorphous nuclei, altered nuclei/cytoplasm ratio, or mitotic figures.^17,29 Thoracic lymphomas may be of B-, T-, or mixed-cell origin, but T-cell lymphomas are most common.^23,34

Cutaneous Lymphoma

Cutaneous lymphoma originates in the skin-associated lym­phoid tissue. Single or multiple subcutaneous firm, nonpainful nodules ranging from less than 1 cm to greater than 20 cm in diameter can develop rapidly or slowly, regionally or over most of the body surface.^11,37 The predominant subtype of cutane­ous lymphoma is T-cell-rich, large B cell (TCRBCL). One retrospective study revealed a potential breed predilection for cutaneous lymphoma subtypes; Quarter Horses were diagnosed almost exclusively with TCRBCLs, while Thoroughbreds had an increased likelihood of cutaneous T-cell lymphoma (CTCL). In general, TCRBCLs tended to present with multiple nodules in different anatomic areas, while CTCLs and diffuse large B-cell lymphomas tended to be single masses. In some cases, the nodules are reported to wax and wane spontaneously or in association with sex hormone levels and seasonality, which likely reflects the presence of progesterone or other hormone receptors present within the tumors.³⁸ There is one report of complete regression of cutaneous lymphoma following surgical removal of an ovarian tumor.³⁹ Depending on the anatomic location, cutaneous lymphomas may cause mechanical lameness and limb edema secondary to vascular and lymphatic obstruction. In most cases, there are no associated hematologic abnormalities and generalized lymphadenopathy and internal organ involvement are rare in the early stage of disease. If the disease is allowed to progress untreated, cutaneous lymphoma may metastasize to internal organs or result in paraneoplastic syndromes.^11,33 If only a single or few tumors are present, surgical removal with clean margins may be curative.¹¹ Like­wise, local injection with cisplatin in sesame oil, as previously described, has also resulted in regression of the injected tumor.⁴⁰

Angiotrophic Lymphoma

Angiotrophic lymphoma, also known as endotheliotropic lymphoma or intravascular lymphoma, is lymphoma of the small blood vessels that spares the surrounding tissues. It has been described in humans, dogs, and cats.^41-43 There is a single report of angiotrophic lymphoma in a 12-year-old pregnant Thorough­bred presenting with anorexia, tachycardia, and mild fever.⁴⁴ The significant laboratory abnormalities in this case were profound anemia (PCV 12%) with a negative Coomb's test, marked anisocytosis, mild lymphopenia, low IgM, and marked erythroid hyperplasia and erythrophagocytosis on bone marrow biopsy. Histologic examination revealed pulmonary and renal vessels crowded with neoplastic lymphoid cells with scattered mitoses. The neoplastic cells were determined to be of T-cell origin based on immunohistochemical staining. Angiotrophic lymphoma may represent an antemortem diagnostic challenge owing to the lack of circulating neoplastic lymphocytes and identifiable masses.

Treatment of Lymphoma

If possible, surgical removal of lymphoma is indicated before treatment with ancillary treatments including radiation and chemotherapy. Radiation therapy has been effective in the treatment of cutaneous and paranasal lymphoma, though access to equipment makes treatment logistically challenging.^45,46 There are limited retrospective studies and clinical trials describing chemotherapy in the horse. The largest clinical investigation is a pilot phase II study evaluating the efficacy of doxorubicin in horses. Tumor response rate for lymphoma was 100% in the study population.⁴⁷ Signs of toxicity including hyperthermia and colic were controlled by premedication with diphenhydramine and flunixin before doxorubicin administration and treatment, at the recommended dose of 70 mg/m².⁴⁸

There are several case reports describing the use of single or multidrug protocols for the treatment of lymphoma. A recent case report describes surgical removal of a TCRBCL followed by treatment with lomustine (CCNU; 60 mg/m²q 30d for 4 treatments), followed by prednisolone, resulting in clinical remission for 16 months at the time of publication.⁴⁹ Other published protocols include single or multidrug therapies including L-asparaginase (10,000 to 40,000 U/m², IM q14-21d), doxorubicin (50 mg/m² IV), vincristine (1.4 mg/m² IV), and rituximab (375 mg/m² IV). A horse with thoracic lymphoma was treated with cytarabine (170 mg/m² IM) 2 weeks apart, alternating with cyclophosphamide (142 mg/m² IV) 7 days after each dosage of cytarabine, in addition to prednisolone.⁵⁰

Palliative therapy of lymphoma has also been achieved with corticosteroids including dexamethasone, prednisolone, and betamethasone. Ideally, pulse-dose therapy or treatment with a relatively high dose of corticosteroid for a short period of time should be implemented until tumor regression has occurred, and then the steroid should be quickly tapered. This strategy may prevent the neoplasia from becoming resistant to corticosteroid therapy. If clinical signs recur during steroid tapering, then the lowest dose of steroid that controls clinical signs should be administered.

Complications associated with chemotherapeutic agents in horses depend on the drug used and should be considered before the institution of therapy. Though no large retrospec­tives on treatment of lymphoma in horses exist, periods of remission following treatment of 8 months to 5 years have been reported.^33,49,50 The long-term prognosis for horses with lymphoma when treatment is not elected is poor.

Leukemia in the Horse thrombocytopenia, hyperproteinemia, and hyperfibrinogemia. As the disease progresses (days to months), marked leukocytosis (counts may be >200,000 WBC∕μL) and hyperproteinemia will be observed in leukemic leukemias.¹⁶ Thrombocytopenia is a common hematologic feature.^1,15,16,19 Pancytopenia and high blast cell counts (up to 1.6 ? 10⁹ cell∕μL) were reported in a lactating mare with myeloblastic leukemia.¹⁹ Pancytopenia due to severe infiltration of leukemic cells in the bone marrow, ineffective hematopoiesis, and myelofibrosis has been reported.¹⁵ Horses with multicentric lymphoma, pancytopenia, severe leukocytosis (more than expected for severe inflammation), or presence of atypical or blast cells in peripheral blood should prompt the collection of a bone marrow aspirate or biopsy. Bone marrow aspirates may have normal to altered myeloid/erythroid ratio (reference range, 0.5 to 3.76) with a disproportion of atypical or poorly differentiated blast or mature cells of certain lineage. Decreased or absent megakaryocytes is a common observation.

Hyperproteinemia due to hyperglobulinemia is a consistent finding. Serum electrophoresis typically shows a polyclonal increase of β₂- and γ-globulin fractions and, in some cases, α-globulin fraction. Marked increases in serum IgG and IgA concentrations have been detected in horses with lymphocytic leukemias.¹ Deficiencies in IgA and IgM have been reported in a single horse with chronic lymphocytic leukemia.¹²

Necropsy findings may include mucosal ulceration, edema, thrombosis, and hemorrhages of various organs due to intravascular leukostasis (aggregates of leukemic cells in blood vessels), disseminated intravascular coagulopathy, and thrombocytopenia secondary to decreased production of megakaryocytes. Leukemic-induced cell lysis may occur as the result of mediators released by neoplastic cells. Peripheral and internal lymphadenopathy may also be observed.

The diagnosis is based on results of a complete blood cell count (CBC) and chemistry panel, with cytologic and histo­pathologic evaluation of bone marrow, immunocytochemical analysis, flow cytometry, and immunophenotyping. Previously, leukemic cells in peripheral blood and bone marrow were described on the basis of their morphology. However, this may result in leukemia misclassification, especially in acute leukemias where mononuclear blast cells comprise the leukemic clone, which may lack specific morphologic identifying features. Numerous cytologic and cytochemical stains (e.g., Wright, Giemsa, Sudan black B, peroxidase, periodic acid-Schiff, alkaline phosphatase, acid phosphatase, various esterases, and surface glycoproteins) have been used to determine leukemia type. Leukemic cells may lack specific cytochemical properties, however. Currently, the various types of leukemias are identified by specific cellular antigens, using a panel of monoclonal antibodies and flow cytometry to analyze peripheral blood and/or bone marrow. Examples are B-cell/IgM-positive, B-cell/ IgM-negative, CD3/CD4/CD5-positive T-cell, CD3-negative/ CD4-positive T-cell, and CD13-positive/myeloid leukemias.^1,12,16 The various monoclonal antibodies for specific equine leukocyte antigens are shown in Table 37.2.

The prognosis is grave, resulting in death within days to a few months after its diagnosis. Treatment of leukemia in horses has been attempted with cytosine arabinoside, cytarabine, and prednisolone, but without much success.⁸,¹²,¹⁸