Hematopoietic Tumors

Canine Lymphoma and Lymphoid Leukemia

David M. Vail , Karen M. Young

LYMPHOMA

The lymphomas (malignant lymphoma or lymphosarcoma) are a diverse group of neoplasms that have in common their origin from lymphoreticular cells. They usually arise in lymphoid tissues, such as lymph nodes, spleen, and bone marrow; however, they may arise in almost any tissue in the body. Lymphoma is one of the most common neoplasms in the dog. The annual incidence has been estimated at 13 to 24 cases per 100,000 dogs at risk. 1 , 2 The annual incidence rates at specific ages are estimated to be 1.5 per 100,000 for dogs less than 1 year old and 84 per 100,000 in the 10- to 11-year-old age group. 3 Lymphoma accounts for approximately 7% to 24% of all canine neoplasia and 83% of all canine hematopoietic malignancies. 4 , 5 In a review of the Veterinary Medical Data Base (VMDP) program, based at Purdue University, from 1987 to 1997, the frequency with which canine lymphoma patients were presented to 20 veterinary institutions increased from 0.75% of the total caseload to 2%, and the frequency apparently continues to increase. A similar trend is present in physician-based oncology. Non-Hodgkin's lymphoma accounts for 5% of all new cancer cases; it is the fifth leading cause of cancer death and the second fastest growing cancer in terms of mortality in humans. 6

Lymphoma affects primarily middle-aged to older dogs (median age, 6 to 9 years). 1 , 7 A lower risk has been reported for intact females, 8 but most reports show that gender is not an important risk factor. 7 , 8b Dog breeds reported to have a higher incidence of lymphoma include boxers, bull mastiffs, basset hounds, Saint Bernards, Scottish terriers, Airedales, and bulldogs; breeds with a lower risk include dachshunds and Pomeranians. 8 , 9

Etiology

The etiology of canine lymphoma is largely unknown and likely multifactorial; however, current investigations are shedding significant light on the subject.

Genetic and molecular factors

Recent advances in molecular cytogenetics (see Chapter 1, section A), including gene microarray techniques, have been and currently are being applied to investigations of chromosomal aberrations in dogs with lymphoma. 10 , 11 , 12 Breen's group has documented gain of canine chromosomes 13 and 31 and loss of chromosome 14 as the most common aberrations in a group of 25 cases analyzed. 11 The recent publication of the canine genome and the commercial availability of canine gene microarrays (e.g., GeneChip Canine Genome 2.0 array; Affymetrix Santa Clara, California) certainly will lead to advances in our understanding of the genetic events in lymphoma in the very near future. 13 Several genetic predispositions have been reported for a pedigree of bull mastiffs, 14 a group of related otter hounds, a family of rottweilers, and a breeding pair of unrelated Scottish terriers. 15 Germ line and somatic genetic mutation in the p53 tumor suppressor gene (see Chapter 2) and the N- ras gene have been documented in bull mastiffs and in a dog with lymphoma, respectively. 16 , 17 , 18

In addition, differences in the prevalence of immunophenotypic subtypes of lymphoma among different breeds have been shown to indicate heritable risks. 19 , 19b Epigenetic modifications also have been investigated in dogs with lymphoma; deoxyribonucleic acid (DNA) hypomethylation (see Chapter 1, section A) was a feature of neoplastic cells in most lymphoma cases and in one third of the leukemia cases investigated and likely is involved in malignant transformation of lymphoid cells. 20

In humans, characteristic chromosomal abnormalities are being described with increasing frequency as more precise banding and other high-resolution techniques are applied. Chromosomal aberrations are nonrandom in human lymphoma, and several aberrations serve as markers for various subtypes of lymphoma. In addition, several oncogenes that may play a role in the pathogenesis of lymphoma have been detected based on the identification of cytogenetic abnormalities. 21 , 22 , 23 Chromosomal aberrations also have been reported in canine lymphoma. 10 , 11 , 12 , 24 A study of 61 dogs with lymphoma demonstrated a treatment advantage in dogs with trisomy of chromosome 13 (25% of the dogs studied), as evidenced by an increase in duration of the first remission and overall survival time. 24

As our knowledge of molecular events and tumorigenesis has expanded, several molecular aberrations have been implicated in various canine tumor types, and some associated with lymphoma have been identified. Altered oncogene/tumor suppressor gene expression, epigenetic changes, signal transduction, and death-pathway alterations are common in human lymphomas and likely are also involved in the dog. As mentioned earlier, N- ras and p53 aberrations, although rare in dogs, have been implicated in some dogs with lymphoma. 16 , 17 , 18 , 25 , 26 , 27 Telomerase activity (see Chapter 14, section D) also has been documented in canine lymphoma tissues. 28 , 29 , 30 Alterations in cellular death-pathways, particularly the Bcl-2 family of proapoptotic and antiapoptotic governing molecules, have been implicated in human non-Hodgkin's lymphoma 31 and currently are under investigation in canine lymphoma.

Infectious factors

The hypothesis that a retrovirus may be involved in the pathogenesis of canine lymphoma has not been confirmed. 32 However, viral particles with properties similar to those of retroviruses have been identified in short-term cultures of canine lymphoma tissue. 33 , 34 , 35 , 36

In physician-based oncology, a direct association has been made between Helicobacter infections and the development of gastric lymphoma. 37 Although this has not been shown definitively in dogs, evidence indicates that Helicobacter infection in laboratory beagles results in gastric lymphoid follicle formation, which is considered a precursor of mucosa-associated lymphoid tissue (MALT) lymphoma in humans. 38

Environmental factors

Some evidence has accumulated that implicates phenoxyacetic acid herbicides, particularly 2,4-dichlorophenoxyacetic acid (2,4-D), in the development of human non-Hodgkin's lymphoma. 39 , 40 A population case control study of non-Hodgkin's lymphoma in Kansas farmers reported a twofold to sixfold higher risk in individuals who frequently mixed or applied herbicides (specifically 2,4-D). 41 A published, hospital-based case control study of dogs indicated that owners in households with dogs that developed malignant lymphoma applied 2,4-D herbicides to their lawn or employed commercial lawn care companies to treat their yard more frequently than owners of dogs without lymphoma. 42 The risk of canine lymphoma was reported to rise twofold (odds ratio, 1.3) with four or more yearly applications of 2,4-D. The results of this study since have drawn criticism, and three follow-up investigations have not validated the assertion of increased risk. 43 , 44 , 45

In another study, dogs exposed to lawn treatment within 7 days of application were more than 50 times more likely to have urine levels of 2,4-D of 50 μg/L or higher. 46 The highest concentration was noted 2 days after application. In a study of 28 dogs with lymphoma, the tumors of 20 dogs with known exposure to 2,4-D were analyzed using polymerase chain reaction (PCR) technology for cellular N- ras oncogene mutations. 47 The ras genes influence cell proliferation and may induce differentiation through signal transduction pathways. Mutation in the ras genes results in ras proteins that promote cell growth. One dog in the series showed a mutation of N- ras , indicating that such mutations are uncommon in canine lymphoma, a finding similar to that for humans with lymphoma. 47

In an environmental case control study performed in Europe, two variables, residency in industrial areas and use of chemicals (defined as paints or solvents) by owners, modestly increased the risk of lymphoma; however, no link was found between the use of pesticides and risk. 48

A weak association between lymphoma in dogs and exposure to strong magnetic fields was observed in a preliminary epidemiologic study. 49 In this hospital-based, case control study, dogs categorized as having high or very high exposure had an increased risk of lymphoma (odds ratio, 1.8). More thorough studies are necessary to evaluate this association further.

Immunosuppression

Impaired immune function has been identified in dogs with lymphoma. 50 , 51 Immune system alterations in the dog (e.g., immune-mediated thrombocytopenia), independent of age and gender, have been associated with a higher risk of subsequent development of lymphoma compared to the normal population. 52 , 53 Additional evidence for the role of the immune system in the development of lymphoma comes from observation in human transplantation patients. Individuals with immunosuppression have a higher risk of lymphoreticular cancer, 54 , 55 and organ transplant patients have a higher incidence of non-Hodgkin's lymphoma. 56 A case of lymphoma that developed in a dog after treatment with cyclosporine, although only one case, supports a link to immunosuppressive therapy in the species. 57

Pathology and Natural Behavior

The classification of malignant lymphoma in dogs can be distinguished on the basis of anatomic location, histologic criteria, and immunophenotypic characteristics. The most common anatomic forms of lymphoma, in order of decreasing prevalence, are the multicentric, craniomediastinal, gastrointestinal, and cutaneous forms. Primary extranodal forms, which can occur in any location outside the lymphatic system, include the eyes, central nervous system (CNS), bone, testes, bladder, heart, and nasal cavity.

Eighty percent of dogs with lymphoma develop the multicentric form, which is distinguished by the presence of superficial lymphadenopathy ( Figure 31-1 ). 58 Lymph node enlargement usually is painless, rubbery, and discrete and may be localized initially to the mandibular and prescapular nodes. Most animals are asymptomatic at the time of presentation, but approximately 20% to 40% have a history of weight loss, lethargy, anorexia, and febrile episodes. 59 , 60 Diffuse pulmonary infiltration also may be seen in 27% to 34% of affected dogs, as detected by radiographic changes ( Figure 31-2 ). 61 , 62 , 63 , 64 Based on bronchoalveolar lavage, the actual incidence of lung involvement may be higher. 62 , 65 Hepatosplenomegaly is the most common manifestation of abdominal involvement and usually is associated with an advanced stage of multicentric disease.

The alimentary form of lymphoma is much less common, usually accounting for 5% to 7% of all canine lymphomas. 58 This form is reported to be more common in male dogs than female dogs, 8 similar to observations in humans. 66 Dogs with infiltrative disease of the intestinal tract show weight loss, anorexia, panhypoproteinemia, and evidence of malabsorption. 67 , 68 Primary gastrointestinal (GI) lymphoma in dogs usually occurs multifocally and diffusely throughout the submucosa and lamina propria of the small intestine, with frequent superficial ulceration and occasional transmural infiltration of the serosa. Lymphocytic-plasmacytic inflammation can be seen adjacent to or distant from the primary tumor. 67 Pathologically, some of these neoplasms may resemble plasma cell tumors, and aberrant production of immunoglobulins may occur.

Histopathologically, distinguishing between gastrointestinal lymphoma and lymphocytic-plasmacytic enteritis (LPE) can be difficult. 67 Some have suggested that LPE may be a prelymphomatous change in the GI tract. A syndrome of immunoproliferative intestinal disease characterized by lymphocytic-plasmacytic enteritis has been described in basenjis, which subsequently develop gastrointestinal lymphoma. 69 In addition, plasma cell–rich areas with heterogeneous lymphomatous infiltration may resemble lesions of LPE.

Only a few reports specifically identify the immunophenotype of the lymphocyte subpopulations in alimentary lymphoma. Historically, it was presumed that they most likely originate from B cells; however, recent evidence suggests that most gastrointestinal lymphomas in dogs originate from T cells. 70 The boxer and Shar-pei breeds appear to be overrepresented in alimentary lymphoma and are reported to have morphologic features (epitheliotropism) consistent with T-cell disease. 71

The mediastinal form of lymphoma occurs in approximately 5% of cases. 58 This form is characterized by enlargement of the craniomediastinal lymph nodes or the thymus, or both ( Figure 31-3 ). However, as previously noted, 20% of dogs with multicentric lymphoma have radiographic evidence of craniomediastinal lymphadenopathy. 63 Hypercalcemia is reported to occur in 10% to 40% of dogs with lymphoma and is most common with the mediastinal form. 72 , 73 In a study of 37 dogs with lymphoma and hypercalcemia, 16 (43%) had mediastinal lymphoma. 74 The mediastinal form in dogs is most commonly associated with a T-cell phenotype. 60 , 75

Cutaneous lymphoma can be solitary or more generalized and usually is classified as epitheliotropic (mycosis fungoides) or nonepitheliotropic. 76 , 77 , 78 , 79 , 80 , 81 , 82 , 83 , 84 Cutaneous lymphoma may also involve the oral mucosa, 76 and extracutaneous involvement can occur, most often in the lymph nodes, spleen, liver, and bone marrow. Canine epitheliotropic cutaneous lymphoma is the most common form of cutaneous lymphoma and usually originates from T cells, 85 similar to its development in humans. In dogs the T cells are CD8+ cells, whereas in humans they are mostly CD4+ cells. 86 A rare form of cutaneous T-cell lymphoma is characterized by generalized skin involvement with evidence of circulating malignant T cells in the peripheral blood. These lymphocytes usually are large (15 to 20 μm in diameter) and have folded, grooved nuclei. In humans this is called Sézary syndrome , 87 which also has been reported in dogs and cats. 79 , 80 , 88 , 89 B-cell cutaneous lymphomas usually spare the epidermis and papillary dermis and affect the middle and deep portions of the dermis.

Hepatosplenic lymphoma is a relatively uncommon, distinct presentation in the dog marked by a lack of peripheral lymphadenopathy in the face of hepatic, splenic, and bone marrow infiltration with malignant lymphocytes, usually of T-cell origin. Biologically, this form of lymphoma is extremely aggressive and poorly responsive to therapy. In humans the tumor usually is composed of γδT cells (i.e., T cells that express the γδT-cell receptor), and this immunophenotype has been confirmed in at least one dog in the veterinary literature. 90 , 91 , 92

Intravascular (angiotrophic, angioendotheliomatosis) lymphoma is a distinct form of lymphoma defined as proliferations of neoplastic lymphocytes within the lumen and wall of blood vessels in the absence of a primary extravascular mass or leukemia. It has been reported several times in the veterinary literature, and in most cases it involves the central and peripheral nervous system (including the eye). 93 , 94 , 95 , 96 , 97 , 98 The B-cell immunophenotype is most common in humans; however, in most reported cases in dogs, the origin is either T cell or null cell (neither B nor T cell), although one case of a B-cell phenotype has been reported.

Histologic Classification Systems

Lymphomas arise from a clonal expansion of lymphoid cells with distinctive morphologic and immunophenotypic features. Many histologic systems have been used to classify non-Hodgkin's lymphoma (NHL) in humans, and some of these have been applied to lymphoma in the dog and other species. 99 , 100 , 101 , 102 , 103 , 104 , 105 , 106 , 107 The National Cancer Institute (NCI) of Working Formulation and the updated Kiel system have been adapted to canine tumors with some success ( TABLE 31-1 , TABLE 31-2 ). The World Health Organization (WHO) also publishes a histologic classification scheme, which uses the revised European American lymphoma (REAL) system as a basis for defining histologic categories of hematopoietic tumors in domestic animals. 107 This system incorporates both histologic and immunohistologic criteria (B- and T-cell immunophenotype). The clinical relevance of this system is likely to be high; however, it awaits further investigation. More recently, the Oncology Committee of the American College of Veterinary Pathologists (ACVP) has established a Lymphoma Subcommittee to formulate a classification system for lymphoma that has clinical relevance. The subcommittee has obtained information from an international group of veterinary oncologists and pathologists, and its report is expected to be completed by 2008.

The Working Formulation (WF) was developed to allow investigators to “translate” among the numerous classification systems so that clinical trials could be compared in humans. Most of the larger compilations agree that most canine lymphomas are intermediate or high grade; however, diffuse immunoblastic forms appear to predominate in the United States, whereas the follicular large cell variations predominate in Europe. A comparison of European and American classifications is warranted based on this discrepancy. The WF categorizes tumors according to pattern (diffuse or follicular) and cell type (e.g., small cleaved cell, large cell, immunoblastic), but it does not include information about the immunophenotype of the tumor. 103 The WF subtypes are related to the biology of the tumor and patient survival. The updated Kiel classification includes the architectural pattern, morphology (centroblastic, centrocytic, or immunoblastic), and immunophenotype (B cell or T cell) of the tumor cells. 102 In both systems, the tumors then can be categorized as low-grade, intermediate-grade, or high-grade malignancies. Low-grade lymphomas composed of small cells with a low mitotic rate typically progress slowly and are associated with long survival times but are incurable. High-grade lymphomas with a high mitotic rate progress rapidly but are more likely to respond to chemotherapy and, in humans, are potentially curable.

Several features of canine lymphomas become apparent when the WF or updated Kiel classification is applied. The most striking difference between canine and human lymphomas is the scarcity of follicular lymphomas in the dog. 108 , 109 , 110 , 111 Some diffuse lymphomas in the dog initially may be follicular, but these may progress to the more aggressive, diffuse form by the time of diagnostic biopsy. Only a small percentage of canine lymphomas (5.3% to 29%) are considered low-grade tumors. 60 , 104 , 105 , 112 Most low-grade small cell lymphomas are T cell in origin. 105

High-grade lymphomas occur frequently if the diffuse large cell lymphomas, classified as intermediate-grade in the WF, are considered high-grade, as in the updated Kiel classification (in which they are labeled diffuse centroblastic lymphomas ). Canine lymphoblastic lymphomas are uncommon. 72 , 104 Most high-grade lymphomas are of B-cell origin. 105 However, a documented difference exists in the prevalence of the various lymphoma immunophenotypes based on breed. 19 For example, cocker spaniels and Doberman pinschers are more likely to develop B-cell lymphoma, boxers are more likely to have T-cell lymphoma, and golden retrievers appear to have an equal likelihood of B- and T-cell tumors.

To be clinically useful, these classification systems in the end must yield information about response to therapy, maintenance of remission, and survival. Some studies suggest that the subtypes in the WF can be correlated with survival, and the Kiel system may be useful for predicting relapse. 113 , 114 In most studies, high-grade lymphomas show a complete response to chemotherapy significantly more often than low-grade tumors. However, dogs with low-grade tumors may live a long time without aggressive chemotherapy. Dogs with T-cell lymphomas have shown a lower rate of complete response to chemotherapy and shorter remission and survival times than dogs with B-cell tumors. 60 , 75 , 112 , 113 Furthermore, T-cell lymphomas tend to be associated with hypercalcemia. 115 , 116 , 117

In the veterinary literature, 60% to 80% of lymphoma cases in dogs are B-cell lymphoma; T-cell lymphomas account for 10% to 38%; mixed B- and T-cell disease accounts for as many as 22%; and null cell tumors (i.e., neither B-cell nor T-cell immunoreactive) represent fewer than 5%. * The development of monoclonal antibodies to detect specific markers on canine lymphocytes has made immunophenotyping of tumors in dogs routinely available in many commercial laboratories. Such techniques also can be performed on paraffin-embedded samples and on cytologic specimens obtained by fine-needle aspiration. 118 , 119 , 120 , 121 , 122

The Rappaport classification system, proposed in 1956 for human NHL, described the architectural pattern (follicular or diffuse) and the cytologic features (well differentiated, poorly differentiated, or histiocytic) of the tumors. 99 , 123 The term histiocytic was applied to tumors in which most of the lymphocytes had larger diameters, more vesicular nuclei, and more prominent nucleoli than those of lymphocytic or undifferentiated lymphomas; it also was used because the malignant cells have some morphologic features of benign histiocytes. However, immunophenotyping has failed to document a biologic relationship between these cells and true histiocytes, therefore the term now is largely considered a misnomer. Furthermore, this subgroup of histiocytic lymphomas included tumors with different morphologic and immunophenotypic features. The Rappaport classification has not been useful in providing prognostic information or in guiding therapy in dogs with lymphoma because of the low number of follicular tumors in dogs, the problematic “histiocytic” subgroup, and the failure to account for different morphologic and immunologic cell types. 100

One criticism of these classification systems is that they fail to include extranodal lymphomas as a separate category. Although differences between nodal and extranodal tumors in biologic behavior and prognosis are well recognized, comparative information about the histogenesis of these tumors has been lacking. For example, in humans, small cell lymphomas arising from MALT are composed of cells with a different immunophenotype from that of other small cell lymphomas (i.e., MALT lymphomas typically are negative for both CD5 and CD10). 124 , 125 , 126 Except for cutaneous lymphoid neoplasms, detailed characterization of extranodal lymphomas in dogs has not been done. Although cutaneous lymphoma is a heterogeneous group of neoplasms that includes an epitheliotropic form resembling mycosis fungoides and a nonepitheliotropic form, most cutaneous lymphomas have a T-cell phenotype. 85 , 127

To summarize, it is important to determine the histologic grade of canine lymphomas as low (small lymphocytic or centrocytic lymphomas) or intermediate to high (diffuse large cell, centroblastic, and immunoblastic lymphomas). Furthermore, determining the immunophenotype of the tumor provides useful information. Response rates to chemotherapy are better in animals with B-cell tumors and intermediate- to high-grade lymphomas. Dogs with low-grade lymphomas can have long survival times without aggressive therapy.

History and Clinical Signs

The clinical signs associated with canine lymphoma are variable and depend on the extent and location of the tumor. Multicentric lymphoma is the most common form (80%), and generalized painless lymphadenopathy (see Figure 31-1 ) is the most consistent finding. In addition, hepatosplenomegaly and bone marrow involvement are common. Most dogs with multicentric lymphoma do not have dramatic signs of systemic illness (WHO substage a ) ( Box 31-1 ), however, a large array of nonspecific signs can occur, such as anorexia, weight loss, vomiting, diarrhea, emaciation, ascites, dyspnea, polydipsia, polyuria, and fever (WHO substage b ). 59 , 60 , 128 , 129 , 130 Dogs with T-cell lymphoma are more likely to have constitutional signs (i.e., substage b ). 118 Polydipsia and polyuria are particularly evident in dogs with hypercalcemia of malignancy. Dogs also may have a history of or clinical signs related to blood dyscrasias secondary to marked tumor infiltration of the bone marrow (myelophthisis) or paraneoplastic anemia, thrombocytopenia, or neutropenia. These signs could include fever, sepsis, anemia, and hemorrhage. Dogs with gastrointestinal or alimentary lymphoma usually have nonspecific GI signs, such as vomiting, diarrhea, weight loss, and malabsorption. 66 , 67 , 68 The mesenteric lymph nodes, spleen, and liver may be involved.

World Health Organization Clinical Staging System for Lymphosarcoma in Domestic Animals

Anatomic site

Generalized

Alimentary

Thymic

Skin

Leukemia (true) *

Others (including solitary renal tumors)

Stage (includes anatomic site)

The mediastinal form of lymphoma is characterized by enlargement of the craniomediastinal structures or thymus or both (see Figure 31-3 , A ), and clinical signs are associated with the extent of disease or polydipsia and polyuria from hypercalcemia. These patients commonly have respiratory distress caused by a space-occupying mass and pleural effusion, exercise intolerance, and possibly regurgitation. Dogs with mediastinal lymphoma also may have precaval syndrome, characterized by pitting edema of the head, neck, and forelimbs secondary to tumor compression or invasion of the cranial vena cava ( Figure 31-4 ).

Signs in dogs with extranodal lymphoma depend on the specific organ involved. Cutaneous lymphoma usually is generalized or multifocal. 76 , 77 , 78 , 79 , 80 , 81 , 82 , 83 , 84 Tumors occur as nodules, plaques, ulcers, and erythremic or exfoliative dermatitis. Epitheliotropic T-cell lymphoma (e.g., mycosis fungoides) has a chronic clinical course with three apparent clinical stages. Initially, scaling, alopecia, and pruritus are seen ( Figure 31-5 , A ). As the disease progresses, the skin becomes more erythematous, thickened, ulcerated, and exudative. The final stage is characterized by proliferative plaques and nodules with progressive ulceration ( Figure 31-5 , B ). Oral involvement also may occur, which can appear as multicentric, erythematous, plaquelike lesions or nodules on the gums and lips ( Figure 31-5 , C ). 76

Dogs with primary CNS lymphoma may have either multifocal or solitary involvement. 131 , 132 , 133 Seizures, paralysis, and paresis may be noted. Ocular lymphoma is characterized by infiltration and thickening of the iris, uveitis, hypopyon, hyphema, posterior synechia, and glaucoma ( Figure 31-6 ). 134 In one study of 94 cases of canine multicentric lymphoma, 37% had ocular changes consistent with lymphoma, and in a series of 102 cases of uveitis in dogs, the condition occurred secondary to lymphoma in 17% of the cases. 135 , 136 Anterior uveitis was most often seen in advanced stage disease (stage V). Dogs with intravascular lymphoma usually have signs related to CNS, peripheral nervous system (PNS), or ocular involvement, 93 , 94 , 95 , 96 , 97 , 98 including paraparesis, ataxia, hyperesthesia, seizures, blindness, lethargy, anorexia, weight loss, diarrhea, polyuria, polydipsia, and intermittent fever. Dogs with pure hepatosplenic lymphoma usually have nonspecific signs such as lethargy, inappetence, and weakness.

The differential diagnosis of lymphadenopathy depends on the dog's travel history (i.e., relative to infectious disease) and the size, consistency, and location of the affected lymph nodes. Other causes of lymphadenopathy include bacterial and viral infections, parasites ( Toxoplasma and Leishmania spp.), rickettsial organisms (salmon poisoning, Ehrlichia sp.), and fungal agents ( Blastomyces and Histoplasma spp.). The potential for hypercalcemia to accompany systemic fungal diseases may further complicate differentiation from lymphoma. Discrete, hard, asymmetric lymph nodes, particularly if fixed to underlying tissues, may indicate metastatic tumors, such as mast cell tumors or carcinomas. Immune-mediated diseases (e.g., pemphigus, systemic lupus erythematosus) also may cause mild to moderately enlarged lymph nodes. The various diseases or conditions that can be misdiagnosed as canine lymphoma are listed in Box 31-2 .

Disorders That May be Misdiagnosed as Canine Lymphoma

Canine lymphoma also may be associated with paraneoplastic syndromes (see Chapter 5). Anemia is the most common lymphoma-related paraneoplastic syndrome. 137 , 138 Paraneoplastic hypercalcemia is also common in dogs with lymphoma. Hypercalcemia is characterized clinically by anorexia, weight loss, muscle weakness, lethargy, polyuria, polydipsia and, rarely, CNS depression and coma. 117 , 139 , 140 In most cases lymphoma-induced hypercalcemia results from a hormone-like substance, parathyroid hormone–related peptide (PTHrP), elaborated by neoplastic cells; however, it also can be related to the production of several other humoral factors, including interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF-α), transforming growth factor-beta (TGF-β), and vitamin D analogs (e.g., 1,25-dihydroxyvitamin D). 125 , 141 , 142 , 143 , 144 Several investigators have reported that hypercalcemia in dogs with lymphoma is most commonly associated with T-cell lymphoma. 60 , 113 , 117 , 118 Other paraneoplastic syndromes that may be encountered include monoclonal gammopathies, neuropathies, and cancer cachexia. 145 , 146

Diagnostics and Clinical Staging

For most animals suspected of having lymphoma, the diagnostic evaluation should include a thorough physical examination; complete blood count (CBC) with a differential cell count, including a platelet count; serum biochemistry profile; and urinalysis. Ultimately, obtaining tissue or cytologic specimens for a definitive diagnosis is essential.

Physical examination

A thorough physical examination should include palpation of all assessable lymph nodes, including those palpable by rectal examination; in the authors’ experience, a significant proportion of dogs have rectal polyps consisting of aggregates of neoplastic lymphocytes. The mucous membranes should be inspected for pallor, icterus, petechiae, and ulceration, because these signs may indicate anemia or thrombocytopenia secondary to myelophthisis or immune-mediated disease or may be evidence of major organ failure or uremia. Abdominal palpation may reveal organomegaly, intestinal wall thickening, or mesenteric lymphadenopathy. Thoracic auscultation may reveal the presence of a mediastinal mass or pleural effusion or both. An ocular examination that includes funduscopic assessment may reveal abnormalities such as uveitis, retinal hemorrhage, and ocular infiltration in approximately one third to one half of dogs with lymphoma. 135 , 136

Complete blood count, biochemistry profile, and urinalysis

Anemia, the most common lymphoma-related hematologic abnormality, 137 , 138 usually is normochromic and normocytic (nonregenerative), consistent with anemia of chronic disease. However, hemolytic anemia may also occur, and regenerative anemias may reflect concomitant blood loss or hemolysis. In addition, if significant myelophthisis is present, the anemia may be accompanied by thrombocytopenia and leukopenia. In animals with anemia or evidence of bleeding, a reticulocyte count, platelet count, and coagulation studies also may be indicated.

Thrombocytopenia may be seen in 30% to 50% of cases, but bleeding is seldom a clinical problem. 138 , 147 , 148 Neutrophilia can be seen in 25% to 40% of dogs with lymphoma. 138 , 147 Lymphocytosis is uncommon and occurs in approximately 20% of affected dogs. 138 Circulating atypical lymphocytes may indicate bone marrow involvement and leukemia. Differentiating multicentric lymphoma with bone marrow involvement (i.e., stage V disease) from primary lymphoblastic leukemia (discussed later) is important, because the prognoses are entirely different. Hypoproteinemia is observed more often in animals with alimentary lymphoma. If the dogs has a high total protein or evidence of an increased globulin fraction on a chemistry profile, serum proteins should be evaluated by serum electrophoresis. Monoclonal gammopathies have been reported to occur in approximately 6% of dogs with lymphoma. 145

Serum biochemical abnormalities often reflect the anatomic site involved. In addition, approximately 15% of dogs with lymphoma are hypercalcemic (30% to 40% of those with mediastinal involvement and approximately 35% of those with T-cell lymphomas). 74 , 118 , 147 In cases of hypercalcemia of unknown origin, lymphoma should always be considered high on the differential disease list, and diagnostics directed at this possibility should be undertaken (see Chapter 5). The presence of hypercalcemia also can serve as a marker for response to therapy. Elevations in serum urea nitrogen and creatinine can occur secondary to renal infiltration with tumor, hypercalcemic nephrosis, or prerenal azotemia from dehydration. Increases in liver-specific enzyme activity or bilirubin concentrations may result from hepatic parenchymal infiltration. Serum globulin elevations, usually monoclonal, occur infrequently with B-cell lymphoma.

A urinalysis is part of the minimum database used to assess renal function and the urinary tract. For example, isosthenuria and proteinuria in the absence of an active sediment may indicate renal disease, and hematuria may result from a hemostatic abnormality. It is important to remember that isosthenuria in azotemic dogs with hypercalcemia does not necessarily indicate renal disease, because the high calcium levels interfere with tubular concentration capabilities through disruption of antidiuretic hormone (ADH) control.

Abnormalities in serum levels of alpha fetoprotein, alpha-1 glycoprotein, zinc, chromium, iron, and endostatin also have been investigated in dogs with lymphoma. 149 , 150 , 151 , 152 The clinical and biologic significance of these alterations has yet to be elucidated.

Histologic and cytologic evaluation of lymph nodes

Morphologic examination of the tissue and cells that comprise the tumor is essential to the diagnosis of lymphoma. Care should be taken to avoid lymph nodes from reactive areas (e.g., mandibular lymph nodes); the prescapular or popliteal lymph nodes are preferable. Also, lymphoid cells are fragile, and in preparing smears of aspirated material, only gentle pressure should be applied in spreading the material on the slides. In most cases, a diagnosis of lymphoma can be made on evaluation of fine-needle aspirates of affected lymph nodes or other tissues. Typically, most of the cells are large lymphoid cells (larger than neutrophils), and they may have visible nucleoli and basophilic cytoplasm ( Figure 31-7 , A ) or fine chromatin with indistinct nucleoli. Because tissue architecture is not maintained in cytologic specimens, effacement of the lymph node or capsular disruption cannot be detected. Therefore, marked reactive hyperplasia characterized by increased numbers of large lymphoid cells may be difficult to distinguish from lymphoma, and small cell lymphomas may have few cytologic clues that point to their malignancy. Also, classification of lymphoma into the subcategories that comprise the low-, intermediate-, and high-grade forms, which has been attempted using the cytologic appearance and immunophenotypic analysis, 153 is performed most accurately on histologic sections.

For accurate histopathologic evaluation, an entire lymph node, including the capsule, should be removed, placed in buffered formalin, and submitted to a pathologist. Although needle core biopsies may be satisfactory, it is important to avoid crush artifact or inadequate sample size. Most pathologists prefer whole node biopsies because they provide the maximum amount of information. Effacement of the normal nodal architecture by neoplastic lymphocytes and capsular disruption are characteristic findings ( Figure 31-7 , C and D ). Diagnostic ultrasonography and ultrasound-guided fine-needle aspiration or needle biopsy have been very useful for evaluation of involvement of the liver, spleen, or abdominal lymph nodes. 154 , 155 , 156 , 157 , 158 If possible, the diagnosis should be made by sampling peripheral nodes, avoiding percutaneous biopsies of the liver and spleen. However, if there is no peripheral node involvement, it is appropriate to biopsy affected tissues in the abdominal cavity.

Histologic and cytologic evaluation of extranodal sites

With alimentary lymphoma, an open surgical wedge biopsy of the intestine must be obtained, ideally without entering the intestinal lumen, and adequate tissue must be obtained; this is important because of the difficulty involved in differentiating lymphoma from LPE. Endoscopic biopsies may be inadequate because only a superficial specimen is obtained; however, more aggressive endoscopic biopsy techniques combined with more accurate histopathologic assessments are improving the diagnostic yield of these less invasive techniques. 159 In many dogs with primary gastrointestinal lymphoma, an inflammatory, nonneoplastic infiltrate (e.g., LPE) may be misdiagnosed on biopsy specimens that are too superficial.

Cytologic examination of cerebrospinal fluid (CSF), thoracic fluid, or aspirates of an intracavitary mass is indicated in animals with CNS disease, pleural effusion, or an intrathoracic mass, respectively. In one study of dogs with CNS involvement, CSF analysis was diagnostic in seven of eight dogs. 131 The characteristics of the CSF included an elevated nucleated cell count in the seven dogs, and 95% to 100% of the cells were atypical lymphoid cells. The CSF protein concentration was higher in five of seven dogs, ranging from 34 to 310 mg/dl (the reference interval was less than 20 mg/dl).

For cutaneous lymphoma, punch biopsies (3 to 4 mm) should be taken from the most representative and infiltrative, but not infected, skin lesions. Staging procedures for cutaneous lymphoma vary, and the stage has shown no prognostic importance. 160

Molecular diagnostic techniques

Molecular techniques can be used to establish a diagnosis of lymphoma or to further characterize a tumor after the initial diagnosis has been made. Tissues and cells from peripheral blood, lymph nodes, or other sites can be analyzed by histochemical and cytochemical, immunohistochemical and immunocytochemical, flow cytometric, and PCR techniques. For example, the tumor's immunophenotype (B cell or T cell), proliferation rate (e.g., expression of Ki-67, proliferating cell nuclear antigen [PCNA], and argyrophilic nucleolar organizer regions [AgNORs]), and clonality (PCR for antigen receptor gene rearrangement [PARR]) can be determined. 60 , 105 , 113 , 119 , 120 , 121 , 122 , 161 , 162 , 163 , 164 , 165 , 166 , 167 , 168 , 169 , 170 The availability of such analyses is increasing, although currently only the immunophenotype consistently predicts the prognosis in dogs.

Immunophenotyping

Immunophenotyping typically is used to determine the type of cells that make up the lymphoma, but sometimes the technique is helpful for making the diagnosis of lymphoma. When a diverse population of lymphocytes is expected in a tissue, the presence of a homogeneous population of the same immunophenotype is supportive of a neoplastic process. The immunophenotype of a lymphocyte is identified by determining the expression of molecules specific for B cells (e.g., CD79a) and T cells (e.g., CD3). Although tumor cells sometimes have morphologic characteristics that typify a particular immunophenotype, exceptions occur, and morphology cannot be used as the sole determinant of the type of lymphocyte. For example, in a series of nine high-grade T-cell lymphomas and leukemias in dogs, the cells had a plasmacytoid appearance, typically associated with B-cell lymphoma. 171 Similarly, anatomic location does not always predict the immunophenotype. In a series of 44 cases of gastrointestinal lymphoma in dogs, often considered a neoplasm of B cells, the neoplastic cells were identified as T cells (CD3 positivity) in 75% of the cases. 70

For accurate determination of the immunophenotype, antibodies against lymphocyte markers are applied to tissue sections (immunohistochemistry), cytologic specimens (immunocytochemistry), or individual cells in a fluid medium (flow cytometry). Flow cytometric evaluation of cells from needle aspirates also is feasible. 172 For T cells, markers include CD3 (pan T), CD4 (helper T), and CD8 (cytotoxic T); for B cells, the markers are CD79a (see Figure 31-7 , B ) and CD21. 173 Interestingly, aberrant expression of CD molecules has been reported in canine lymphoma. In a study of 59 dogs with lymphoma, tumor cells from six dogs were positive for both T- and B-cell CD markers; however, a clonality assay (discussed later) revealed clonality for either the T-cell or the immunoglobulin receptor but not both. This indicates that in some cases of B- and T-cell lymphoma, the malignant cells may coexpress B- and T-cell CD markers. 119 Antibodies against these molecules are used to determine the immunophenotype; however, they also have a potential use as a therapeutic modality if tumor cells could be targeted using these antibodies.

Histologic assessment of markers of multidrug resistance and apoptotic pathways (e.g., P-glycoprotein, p53, and Bcl-2 proteins) currently are being evaluated in dogs with lymphoma. However, their significance requires further evaluation. 16 , 163 , 174 , 175 , 176 , 177

Clonality assay

Occasionally the diagnosis of lymphoma and the differentiation of malignant verses benign proliferation of lymphocytes is not possible based on standard histologic and cytologic criteria. In these cases, advanced molecular analyses are necessary to help confirm a diagnosis. Clonality is the hallmark of malignancy; that is, the malignant cell population theoretically should be derived from expansion of a single malignant clone characterized by a particular DNA region unique to that tumor. For example, in a dog with T-cell lymphoma, all the malignant cells should have the same DNA sequence for the variable region of the T-cell receptor (TCR) gene; likewise, in a dog with B-cell lymphoma, the tumor cells should have identical DNA sequences in the variable region of the immunoglobulin receptor gene. Conversely, in reactive lymphocytosis, the cells are polyclonal for their antigen receptors. Using this knowledge, investigators have used polymerase chain reaction (PCR) PCR techniques to amplify the variable regions of the T-cell and immunoglobulin receptor genes to detect clonal lymphocyte populations in dogs ( Figure 31-8 ). 167 , 168 , 169 , 170

In physician-based medicine, such assays of clonality are approximately 70% to 90% sensitive and have a false positive rate of approximately 5%, and recent studies report similar rates in the dog. 169 False negative and false positive results can occur with clonality assays. For example, cells from a dog with lymphoma may be negative for clonality if the clonal segment of DNA is not detected with the primers used, if the malignant cells are natural killer (NK) cells (rare), or if the malignant cells are present in too low a frequency to be detected. 169 False positive results occur rarely with some infectious diseases, such as ehrlichiosis and Lyme disease. In these cases a diagnosis should be made only after the results of all the diagnostic tests are considered, including histologic and cytologic evaluation, immunophenotyping, and clonality studies, in conjunction with the signalment and physical findings. Molecular techniques, in addition to aiding the diagnosis, could also be useful in determining early recurrence, more accurate clinical staging, and so-called molecular remission rates, because they are more sensitive than standard cytologic assessment for the peripheral blood, bone marrow, and lymph nodes.

Clinical Staging

After a diagnosis has been established, the extent of disease should be determined and correlated to the clinical stage of disease. The WHO staging system routinely used to stage dogs with lymphoma is presented in Box 31-1 . Most dogs (more than 80%) are presented in advanced stages (stage III or IV). Some type of imaging and an assessment of bone marrow involvement may be indicated for staging. The degree to which thorough staging is implemented depends on three factors: whether the result will alter the treatment plan; whether relevant prognostic information will be gleamed; and whether the client needs to know. In addition, when different protocols are compared with respect to efficacy, consistent and similar staging systems should be used to avoid so-called stage migration, which results when one staging methodology is more accurate than another. 178 The effect of stage migration currently is being evaluated in veterinary patients with lymphoma, and until the concept has been thoroughly explored, the impact on the prognosis should be considered when different published outcomes are compared.

Bone marrow evaluation

A bone marrow aspirate or biopsy (from the proximal humerus or iliac crest) is indicated for complete staging and in dogs with anemia, lymphocytosis, peripheral lymphocyte atypia, or other peripheral cytopenia. In one study of 53 dogs with lymphoma, 28% had circulating malignant cells and were considered leukemic, whereas bone marrow examination indicated involvement in 57% of the dogs. 179 The presence of a few prolymphocytes and large lymphocytes with nucleoli in the circulation of dogs with lymphoma may indicate bone marrow involvement. It is important to remember that these cells also can be seen with GI parasitism, immune-mediated hemolytic anemia, and other immune-mediated diseases. Recently, circulating tumor cells in dogs with stage III lymphoma were identified using a clonality assay (PARR). 168 The assay is more sensitive than routine microscopy in detecting malignant cells in circulation, but the correlation of these results with staging and prognosis has not been determined. Bone marrow evaluation offers prognostically valuable information, but if the client is committed to treatment regardless of the stage of disease, it is not necessary.

Imaging

Evaluation of thoracic and abdominal radiographs may be important in determining the extent of internal involvement. Approximately 60% to 75% of dogs with multicentric lymphoma have abnormalities on thoracic radiographs; one third have evidence of pulmonary infiltrates, and two thirds have thoracic lymphadenopathy (sternal and tracheobronchial lymph nodes) and widening of the cranial mediastinum (see Figure 31-2 , Figure 31-3 ). 61 , 62 , 63 , 64 Craniomediastinal lymphadenopathy is detected in 20% of dogs with lymphoma. 63 Abdominal radiographs reveal evidence of sublumbar (iliac) and/or mesenteric lymph node, spleen, or liver involvement in approximately 50% of cases. 63 , 64 In the authors’ practice, in typical cases of canine multicentric lymphoma, imaging is limited to thoracic radiographs because no prognostic difference exists between dogs with stage III disease and those with stage IV disease (i.e., liver or spleen involvement); however, the presence of craniomediastinal lymphadenopathy is prognostically significant (see Prognosis later in this section). If clinical signs attributable to abdominal disease are present, further imaging of the abdomen is warranted. In addition, as stated previously, abdominal ultrasonography can be important for obtaining ultrasound-guided intra-abdominal samples for diagnosis. It is also useful for the diagnosis of gastrointestinal and hepatosplenic lymphoma. 90 , 91 , 92 , 180 , 181 Advanced imaging modalities, including computed tomography (CT), magnetic resonance imaging (MRI), and positron emission/computed tomography (PET/CT), are becoming more commonplace in veterinary practice, and their usefulness is only now being fully determined. 182 , 183 , 184 , 185 , 186

Treatment of Multicentric Lymphoma

The therapeutic approach to a particular patient with lymphoma is determined by the stage and substage of disease, the presence or absence of paraneoplastic disease, the patient's overall physiologic status, the financial and time commitments of the client, and the client's level of comfort with regard to the likelihood of treatment-related side effects. Because most canine lymphomas are intermediate- to high-grade tumors, histopathologic characterization has played a less important role in determining the optimal treatment.

Without treatment, most dogs with lymphoma die of the disease in 4 to 6 weeks. 145 With few exceptions, canine lymphoma is considered a systemic disease and therefore requires systemic therapy to achieve remission and prolonged survival. Systemic chemotherapy continues to be the therapy of choice for canine lymphoma. In general, combination chemotherapy protocols are superior in efficacy to single agent protocols. Single agent protocols, except for doxorubicin, have a lower response rate that is not as durable as combination chemotherapy. In rare cases in which lymphoma is limited to one site (especially an extranodal site), the animal can be treated with a local modality, such as surgery or radiation therapy, as long as the caregiver and clinician are committed to diligent re-evaluation to document subsequent systemic involvement.

Multidrug combination protocols

Many chemotherapy protocols for dogs with lymphoma have been developed over the past 15 to 20 years ( Table 31-3 ). 59 , 61 , 72 , 128 , 187 , 188 , 189 , 190 , 191 , 192 , 193 , 194 , 195 , 196 , 197 , 198 , 199 , 200 , 201 Most complex combination protocols are modifications of CHOP protocols initially designed for human oncologic use. CHOP represents combinations of cyclophosphamide (C), doxorubicin (represented by the H , for hydroxydaunorubicin), vincristine (O, Oncovin) and prednisone (P). Conventional chemotherapy induces complete remission (CR) in approximately 60% to 90% of dogs, with median survival times of 6 to 12 months depending on the protocol used. Approximately 20% to 25% of dogs live 2 years or longer after initiation of these protocols ( Figure 31-9 ). Response rates and the duration of response vary according to the presence or absence of prognostic factors discussed in the Prognosis section. The cost to the owner depends on the drug or drugs selected, the size of the animal, the frequency of administration, and the laboratory tests needed to monitor toxicity.

Dogs that respond to chemotherapy and achieve complete remission usually are free of clinical signs associated with lymphoma and subsequently return to a very good quality of life. Treating dogs with lymphoma is gratifying, because a high percentage have a complete response. Most dogs tolerate chemotherapy well, and in our experience only a minority of dogs develop significant toxicity. Studies assessing clients’ perceptions of medical treatment for cancer in general and lymphoma in particular generally report a positive experience; most owners felt that the treatment was worthwhile, that it resulted in improvement in their pet's well-being, and that the animal's quality of life during treatment was good. 202 , 203 Very few clients expressed regret about having the lymphoma treated with a multidrug protocol.

With lymphoma, the fundamental goals of chemotherapy are to induce a complete and durable (longer than 6 months) first remission (induction) , to reinduce remission when the tumor recrudesces (or the patient relapses) after achievement of a remission (reinduction) , and finally to induce remissions when the cancer fails to respond to induction or reinduction using drugs not present in standard protocols (rescue).

A previously unanswered question in the treatment of lymphoma was whether long-term maintenance chemotherapy was useful after an initial course of aggressive induction chemotherapy that lasted 6 months or less. Long-term maintenance chemotherapy has been shown to be ineffective in humans with Hodgkin's disease, NHL, and multiple myeloma. However, the initial induction course of chemotherapy in humans is much more aggressive than that used in veterinary patients. Although no randomized studies have been performed to address the therapeutic benefit of long-term maintenance chemotherapy in dogs, comparisons of dogs treated with CHOP-based protocols in which all treatment was stopped after 6 months of induction therapy 129 were compared with sequentially treated, historical controls that received a nearly identical protocol that included long-term maintenance therapy. 60 The dogs that received the shortened, less expensive, no-maintenance protocol had comparable remission and survival durations and were more likely to achieve second remissions when they relapsed after completion of chemotherapy than their counterparts that received long-term maintenance therapy. Other studies, although not prospective randomized trials, suggest that aggressive induction or discontinuous therapy (i.e., induction without maintenance) is as good as or superior to protocols that use an extended maintenance phase. 190 , 193 , 194 , 200 These data, taken together, suggest that maintenance therapy is not necessary and indeed may be inappropriate for dogs with lymphoma that are treated with similar combination chemotherapy protocols.

Single agent chemotherapy with known activity for dogs with lymphoma

Currently, the most effective chemotherapeutic agents for lymphoma are doxorubicin, L-asparaginase, polyethylene glycol (PEG)-L-asparaginase, 129 , 130 vincristine, cyclophosphamide, and prednisone. * Other drugs that also have activity and could be considered second-line agents include vinblastine, actinomycin-D, mitoxantrone, chlorambucil, methotrexate, DTIC, 9-aminocamptothecin, ifosfamide, cytosine arabinoside, gemcitabine, lomustine, and dolastatin-10. 206 , 207 , 208 , 209 , 210 , 211 , 212 , 213 , 214 , 215 , 216 , 217 , 218 Of these, cytosine arabinoside, ifosfamide, dolastatin-10, and gemcitabine appear to have only minimal activity. Except for doxorubicin, induction with single agent chemotherapy does not result in durable remission durations compared with standard combination protocols. The efficacy of incorporating these newer drugs with single agent activity into standard combination protocols awaits further investigation.

Overall Chemotherapy Treatment Recommendations

Providing precise treatment recommendations for the wide variety of clinical settings of dogs with lymphoma is difficult, especially in light of the plethora of published combination drug protocols (see Table 31-3 ). Because of the large and ever increasing number of protocols available, several factors should be considered and discussed with caregivers on a case-by-case basis in making the choice of protocol. These factors include the cost, time commitment, efficacy, toxicity, and experience of the clinician with the protocols in question. With the increased availability of generic drugs, protocols are becoming affordable to a larger segment of veterinary clients. In general, more complex combination chemotherapy protocols are more expensive, more time-consuming (i.e., requiring repeated office visits and closer monitoring), and more likely to result in toxicity than simpler, single agent protocols. However, as a general rule, more complex combination protocols result in longer remission and survival durations than single agent protocols.

The earliest treatment protocol used in veterinary patients was a non-doxorubicin-based combination chemotherapy (i.e., cyclophosphamide, vincristine, and prednisone [CVP]), a relatively simple, easy protocol that is well-tolerated and results in a 60% to 70% CR rate and a median survival time of 6 to 7 months. 187 , 188 However, it has been clearly established that the standard of care combination protocols used in dogs with lymphoma include doxorubicin, and all are essentially variations on the CHOP protocols previously discussed and listed in Table 31-3 . Although many of these CHOP protocols include L-asparaginase, which is added either at initiation or at varying times throughout the protocol, several studies have confirmed that the addition of L-asparaginase in induction protocols does not result in clinically relevant increases in remission rate, speed of attaining remission, or first-remission duration; therefore its use is best reserved for rescue situations (discussed later). 128 , 190 , 219 , 220 Regardless of the veterinary CHOP-based combination protocol used, they all generally result in an 80% to 90% CR rate with median survival times of 12 months. About 25% of dogs are long-term survivors (longer than 2 years), and some are cured. The CHOP protocol used by the authors at the time of publication ( Box 31-3 ) generally is well tolerated by dogs. This protocol does not have a maintenance therapy arm, and all treatments stop at 19 weeks if the animal is in complete remission.

Current Canine Lymphoma Protocol (L-VCA–Short)

If client factors or other considerations preclude a CHOP-based protocol, single agent doxorubicin can be offered as an alternative, with the patient receiving five doxorubicin treatments (30 mg/m 2 given intravenously every 3 weeks). The expected complete response rate will range from 50% to 75% with an anticipated median survival time of 6 to 8 months. 128 , 188 , 189 , 197

If financial or other client concerns preclude the use of more aggressive systemic chemotherapy, prednisone therapy alone (2 mg/kg given orally daily) often results in a short-lived remission of approximately 1 to 2 months. In these cases, it is important to inform clients that should they decide to pursue more aggressive therapy later, dogs with previous prednisone therapy are more likely to develop multiple drug resistance (MDR) while receiving single agent prednisone and to have shorter remission and survival durations with subsequent combination protocols. This is especially true after long-term prednisone therapy and in dogs that have experienced a recurrence while receiving prednisone. 195 , 221 Therefore, the earlier a client opts for more aggressive therapy, the more likely it is that a durable response will result.

A CBC should be performed before each chemotherapy treatment. A neutrophil count of at least 2000/μl and a platelet count of 50,000/μl should be present before the chemotherapeutic drugs are administered. If the neutrophil count is below 2000/μl, it is best to wait 5 to 7 days and repeat the CBC. If the count has risen above 2000 cells/μl, the drug can be safely administered. A caveat to these restrictions: In dogs presented before initiation of chemotherapy that have low neutrophil and platelet counts because of bone marrow effacement, myelosuppressive chemotherapy is instituted in the face of cytopenias to “open” the bone marrow and allow counts to normalize.

With regard to breeds likely to have MDR-1 gene mutations (e.g., collies; see Chapter 11) and which therefore are at risk for serious, unexpected chemotherapy toxicity, the authors initiate a CHOP protocol out of sequence, beginning with non-MDR-1–associated drugs, such as cyclophosphamide. This ensures treatment of the lymphoma while allowing sufficient time for analysis of MDR-1 gene mutations (see Chapter 11) before MDR-1–associated drugs are initiated.

Reinduction and Rescue Chemotherapy

Eventually, most dogs that achieve a remission relapse or experience a recrudescence of lymphoma. This usually represents the emergence of tumor clones that are inherently more resistant to chemotherapy than the original tumor; so-called MDR clones that either were initially drug resistant or became so after exposure to selected chemotherapeutic agents. 175 , 222 , 223 , 224 Evidence suggests that in recurrent lymphoma in dogs, tumor cells are more likely to express the MDR-1 gene that encodes the protein transmembrane drug pump often associated with multiple drug resistance. 174 , 175 , 224 Other causes of relapse after chemotherapy include inadequate dosing and frequency of administration of chemotherapy and failure to achieve high concentrations of chemotherapeutic drugs in certain sites, such as the central nervous system.

At the first recurrence of lymphoma, reinduction should be attempted first by reintroducing the induction protocol that was successful initially. Special attention must be given to the cumulative dose of doxorubicin that will result from reinduction; also, a baseline cardiac assessment, the use of cardioprotectants, alternative drug choices, and client education should all be considered. In general, the likelihood of a response and the length of the reinduction are half those seen in the initial therapy; however, some animals enjoy long-term reinductions, especially if the patient had completed the initial induction regimen and was off chemotherapy when the relapse occurred. A reinduction rate of nearly 90% can be expected in dogs that have completed CHOP-based protocols and then relapse while off therapy. 129

If reinduction fails or the dog does not respond to the initial induction, use of so-called rescue agents or rescue protocols can be attempted. These are drugs or drug combinations that typically are not found in the standard CHOP protocol and are withheld for use in cases of drug resistance. The most common rescue protocols most commonly used in dogs include single agent or combination use of actinomycin D, mitoxantrone, doxorubicin (if doxorubicin was not part of the original induction protocol), a doxorubicin/dacarbazine combination, lomustine (CCNU), L-asparaginase, and the combination mechlorethamine, Vincristine (oncovin) procarbazine, and prednisone (MOPP). 176 , 206 , 209 , 210 , 211 , 225 , 226 , 227 , 228 , 229 Overall rescue response rates of 40% to 50% are reported, but these responses usually are not durable, and median response times of 1.5 to 2.5 months are typical. A small number of animals will enjoy longer rescue durations. Table 31-4 presents a summary of canine rescue protocols and published results.

Strategies to Enhance the Effectiveness of Chemotherapy in Lymphoma

Despite the plethora of published chemotherapy protocols for dogs with lymphoma, it appears that veterinary medicine has achieved about as much as it can from the currently available chemotherapeutic drugs in standard settings, because no dramatic improvement has been made in the 12-month median survival “wall” and the 25% 2-year survival rate. Advances in remission and survival durations await the development of new methods of delivering or targeting old chemotherapeutic drugs and the development of new generation chemotherapeutics or novel nonchemotherapeutic treatment modalities. Mechanisms of avoiding or abrogating MDR, enhancing tumor apoptosis (programmed cell death), and targeting treatments with immunoconjugates (i.e., antibody-directed therapies), as well as novel immunomodulatory therapies, are all active areas of investigation in both human and veterinary medicine.

Mechanisms of drug resistance

Drug resistance can develop in cancer patients after exposure to selected chemotherapeutic agents and often is associated with expression of P-glycoprotein (see Chapter 11). P-glycoprotein acts as a drug efflux pump that actively extrudes drugs from tumor cells, preventing a cytotoxic drug from reaching the cellular site of action. MDR and P-glycoprotein are controlled by the MDR-1 gene. 222 , 223 , 230 MDR has been reported in canine lymphoma after treatment with chemotherapy. 174 , 224 , 231 In one study, expression levels of messenger ribonucleic acid (mRNA) that encodes the canine MDR-1 gene was characterized in canine cell lines and lymphomas. 231 Although expression of MDR-1 mRNA correlated with in vitro drug sensitivity, it did not correlate with in vivo doxorubicin sensitivity in dogs with lymphoma in this study.

Altering drug pharmacokinetics

Methods of increasing the time that tumor cells are exposed to chemotherapeutic drugs theoretically should enhance tumor killing. These methods could include long-term continuous infusions (impractical in many veterinary situations), increasing the frequency of treatments, or enhancing the drugs’ circulation time. In one study, dogs with lymphoma received doxorubicin weekly rather than every 3 weeks in an attempt to reduce cardiac toxicity and increase the duration of drug exposure. 232 No benefit was noted, and in fact, remission rates were inferior. Some studies have evaluated pegylated long-circulating doxorubicin-containing liposome drug delivery systems in dogs with lymphoma. 233 , 234 Although efficacy was established, no enhancement of remission or survival durations over equivalent doses of native doxorubicin were observed.

Treatment Approaches Using Immunologic or Biologic Agents

Results to date for immunologic procedures and biologic agents have been mixed. A study using the chemical immunomodulator levamisole in combination with chemotherapy proved to be unrewarding in dogs with lymphoma. 235 Autologous vaccines have been shown to have some positive effects in dogs when combined with chemotherapy. A tumor vaccine extract using killed lymphoma cells combined with Freund's adjuvant was administered to dogs after remission induction with combination chemotherapy. The median survival time was 336 days (11 dogs) for the vaccine-treated group versus 196 days (nine dogs) for the group that received chemotherapy alone. 236 A subsequent study by the same investigators reported that the prolonged survival was due to the Freund's adjuvant. 237

Intralymphatic (IL) administration of an autologous killed lymphoma tumor cell vaccine has been done in dogs in which remission was achieved with a combination chemotherapy protocol. In a study that compared 28 dogs that received chemotherapy and then IL vaccination with 30 dogs that received chemotherapy alone, the median remission times were 98 and 28 days, respectively (p < 0.024). 238 Unfortunately, the survival times for the two groups were not significantly different (305 days for chemotherapy plus IL vaccine and 184 days for chemotherapy alone). Dogs that responded had significant increases in specific antibody to lymphoma antigens compared to those that did not respond.

Another immunotherapy approach involved MAb-231, a murine-derived anticanine monoclonal antibody (IgG2a). This antibody mediates antibody-dependent cellular cytotoxicity (ADDC) and complement-mediated cellular cytotoxicity (CMCC), 239 , 240 and it prevented outgrowth of canine lymphoma xenografts in nude mice. 241 In a noncontrolled clinical study, 215 dogs were treated with chemotherapy (L-asparaginase, vincristine, cyclophosphamide, and doxorubicin). 242 After two cycles of chemotherapy, 174 dogs had achieved complete remission and were treated with an intravenous infusion of MAb-231 daily for 5 days. The median survival time of the dogs treated with MAb-231 was 493 days. The 2-year survival rate was 15.6%. The median number of chemotherapy cycles in the first year was three, and the median number of MAb-231 cycles was 1.5. The MAb-231 antibody went off the commercial market in the mid-1990s. Definitive randomized trials to determine its effectiveness are still lacking.

Surgery

Most dogs with lymphoma have the multicentric form and need systemic chemotherapy for effective treatment of the disease. However, surgery has been used to treat solitary lymphoma (early stage I) or solitary extranodal disease. In such cases, careful staging is necessary to rule out multicentric involvement before the local disease is treated.

The benefit of surgical removal of the spleen in dogs with massive splenomegaly remains unclear. 243 , 244 In a published report, 16 dogs with lymphoma underwent splenectomy for a massive spleen and subsequently were treated with chemotherapy. Within 6 weeks of splenectomy, five of the 16 dogs died of disseminated intravascular coagulation (DIC) and sepsis. The remaining 11 dogs had a complete response rate of 66%, and seven of these, which were followed until their death, had a median survival time of 14 months. Splenectomy should be considered only if the lymphoma is in remission in other sites and if the splenic enlargement is caused by lymphoma that is not responsive to chemotherapy. In dogs with lymphoma, splenectomy also can be considered as a treatment for uncontrolled hemolytic anemia and persistent thrombocytopenia.

Radiation Therapy

The role of radiotherapy for the management of lymphoma in dogs currently is under investigation. The use of whole body irradiation without bone marrow transplantation has yielded poor results. However, radiation therapy may be indicated in selected cases. 245 , 246 , 247 , 248 The indications are:

Local stage I or stage II disease (i.e., nasal lymphoma, CNS lymphoma)

Palliation of local disease (e.g., mandibular lymphadenopathy, rectal lymphoma, mediastinal lymphoma accompanied by precaval syndrome, localized bone involvement)

Whole body irradiation combined with bone marrow or stem cell transplantation

Staged half-body irradiation after chemotherapy-induced remission

The use of staged half-body irradiation after achieving remission with induction chemotherapy has undergone preliminary investigation as a form of consolidation or maintenance. 247 , 248 In these investigations, radiation therapy is delivered either to the cranial or the caudal half of the dog's body in two consecutive 4 Gray daily fractions; then, after a rest of 3 or 4 weeks, the other half of the body is irradiated in a similar fashion. Although these preliminary investigations were not randomized, their results suggest that radiation therapy, either after completion of chemotherapy or sandwiched between chemotherapy sessions, when dogs are in either complete or partial remission, is safe and should be investigated more extensively to determine whether a significant therapeutic gain could be realized.

Nonmulticentric Lymphoma

In general, the veterinary literature offers little information on the treatment of the various extranodal forms of lymphoma in dogs, therefore the ability to predict outcome is limited. The authors recommend that, after extensive staging, local therapies (e.g., surgery, local radiation therapy) should be used in cases in which disease is localized to a solitary site. If multiple extranodal sites are involved or if they are part of a more generalized process, systemic chemotherapy should be given.

Alimentary lymphoma

Most dogs with alimentary lymphomas have diffuse involvement of the intestinal tract. Involvement of local lymph nodes and the liver is common. Chemotherapy in dogs with diffuse disease has been reported to be unrewarding for the most part. 67 However, in the authors’ experience, more aggressive, CHOP-based protocols (which are used extensively for multicentric lymphoma in dogs) have resulted in several cases of durable remission for alimentary lymphoma. Solitary alimentary lymphomas are rare in the dog, but if the tumor is localized and can be removed surgically, the results with or without follow-up chemotherapy can be encouraging.

Primary central nervous system lymphoma

Most CNS lymphoma in dogs results from metastasis of multicentric lymphoma. However, primary central nervous system lymphoma (PCNSL) has been reported. 131 , 132 If tumors are localized, local radiation therapy should be considered. Few studies have reported on the use of chemotherapy. In one study, cytosine arabinoside (Ara-C) at a dosage of 20 mg/m 2 was given intrathecally by bolus injection after withdrawal of an equal volume of CSF. 131 The dose was diluted in 2 to 4 ml of lactated Ringer's solution and was injected twice weekly for a total of six treatments. This treatment was combined with systemic chemotherapy and CNS irradiation. Overall, the response rates were low and of short duration (several weeks to months).

Cutaneous lymphoma

The treatment of cutaneous lymphoma depends on the extent of disease. Solitary lesions may be treated with surgical excision or radiation therapy. Fractionated radiation therapy (to a total dose of 30 to 45 Gy) has been associated with long-term control. 80 Diffuse non-T-cell lymphoma is best managed with combination chemotherapy, although the response rate is less than in multicentric lymphoma. In one study, investigators reported that combination chemotherapy with cyclophosphamide, vincristine (Oncovin), cytosine arabinoside, and prednisone (COAP) induced long-term remission in some cases. 249 Five of six dogs with diffuse non-T-cell cutaneous lymphoma attained a complete or partial remission, with a median remission duration of longer than 250 days and a median survival of longer than 399 days. 249

Retinoids, such as isotretinoin (Accutane) and etretinate (Tegison), have been used successfully in canine and human T-cell cutaneous lymphoma. 82 , 250 In one study, 12 dogs with cutaneous lymphoma were treated with isotretinoin (3 to 4 mg/kg given orally daily), and two were treated with etretinate (1.25 to 1.45 mg/kg given orally daily, continuously). Eleven of these 14 dogs had T-cell cutaneous lymphoma, and six of the 14 dogs achieved remission. In another study, four dogs with T-cell lymphoma were treated successfully with isotretinoin for 13, 11, 10, and 5 months. 82

In the authors’ experience, retinoid treatment must be given for at least 2 months to note a response. Polyethylene glycol (PEG)-L-asparaginase (30 mg/kg given intramuscularly weekly) induces responses in dogs with cutaneous T-cell lymphomas, although remissions are not durable, and no cures have been noted. 251 Prednisone may also be necessary to control pruritus. In the authors’ experience, pegylated-liposomal doxorubicin (Doxil) has produced remissions in approximately 40% of cases. Although most of these were short-lived responses, remissions of 1 year or longer occasionally have occurred.

A preliminary abstract reported activity for oral CCNU with cutaneous lymphoma in dogs. 252 All seven dogs (five with mycosis fungoides [MF] and two with nonepitheliotropic disease) that were treated with CCNU (50 mg/m 2 given orally every 3 weeks) achieved a complete response, and two of those responses were relatively durable (7 and 15 months). A larger cohort needs to be treated before definitive response rates and durations can be given.

Topical chemotherapy is another strategy for treating cutaneous T-cell lymphoma, although it is rarely used in veterinary medicine because of “patient compliance” problems. Mechlorethamine (Mustargen) can be applied topically as an aqueous solution or an ointment base. The aqueous solution is prepared by combining 10 mg of mechlorethamine with 50 ml of tap water. The ointment is prepared by mixing 90 mg of mechlorethamine with 10 ml of absolute alcohol and further combining enough xipamide (Aquaphor) to prepare 900 g of ointment. Hair must be removed before application. Gloves must be used when the drug is applied, because mechlorethamine is carcinogenic and can cause contact hypersensitivity in humans. The response to therapy varies, and the treatment often is only palliative. 253

Prognosis

The prognosis for canine lymphoma varies and depends on a number of factors, such as the location of disease, the extent of disease (the clinical stage), the presence or absence of clinical signs (the substage), the histologic grade, the immunophenotype (T cell or B cell), exposure to previous chemotherapy or corticosteroids and subsequent development of MDR (see Chapter 11), altered cell death processes (apoptosis), the proliferation rate of the tumor, the presence of concurrent medical problems or paraneoplastic conditions (e.g., hypercalcemia, weight loss, and liver insufficiency), and possibly gender. * Although canine lymphoma is rarely curable (fewer than 10% of cases), complete responses and a good quality of life during extended remissions and survival are typical.

Factors that have been shown to influence survival are shown in Table 31-5 . The two factors most consistently identified as being prognostically important in dogs with lymphoma are the immunophenotype and WHO substage (see Figure 31-9 ). Many reports have confirmed that dogs with CD3-immunoreactive tumors (i.e., T-cell derivation) are associated with significantly shorter remission and survival durations. † This holds true primarily for dogs with multicentric lymphoma, because the immunophenotype of solitary or extranodal forms of lymphoma has not been thoroughly investigated with respect to the prognosis. In addition, it has been shown that dogs with B-cell lymphomas that express lower than normal levels of B5 antigen (expressed in 95% of nonneoplastic lymphocytes) also have shorter remission and survival durations. 75 Dogs with WHO substage b disease (i.e., clinically ill) also do poorly compared to dogs with substage a disease (see Figure 31-9 ). ‡ Dogs with stage I or stage II disease have a better prognosis than dogs with more advanced disease (stage III, IV, or V). 187 , 188 In some studies, an elevated serum calcium (over 11.9 mg/dl) has been shown to be a negative prognostic factor 72 , 257 ; however, this rarely holds true with multivariate analysis because hypercalcemia is associated with the T-cell phenotype.

The histologic grade (subtype) has been found to influence the prognosis in some studies; however, our ability to predict outcome based on subtype is still quite limited. Dogs with lymphoma classified as intermediate- or high-grade (large cell, centroblastic, and immunoblastic) tend to respond to chemotherapy but can relapse early. Dogs with low-grade lymphomas (small lymphocytic or centrocytic) have a lower response rate to chemotherapy, yet have a survival advantage over dogs with intermediate- or high-grade lymphomas ( Figure 31-10 ) in that the course of disease may be more indolent. 113 Using the Working Formulation, dogs with low-grade lymphomas have a survival advantage compared to dogs with intermediate- or high-grade tumors. 78

Recently, proliferative assays (e.g., analysis of bromodeoxyuridine [BrdU] uptake, Ki-67 antibody reactivity, and AgNOR indices) to measure the proliferative activity of tumor cells have been shown to provide significant prognostic information in dogs treated with combination chemotherapy. 60 , 163 , 254 , 258 , 259 , 260 However, the results of different studies are contradictory. In two trials, dogs that had tumors with short doubling times, high AgNOR frequencies, or high Ki-67 immunoreactivity had a better prognosis than dogs with long doubling times or low AgNOR frequencies. 60 , 163 In other trials, the low-proliferating tumor groups were associated with a better prognosis. 254 , 259 In one trial, the proportion of tumor cells undergoing apoptosis was modestly predictive of remission duration. 163

The anatomic site of disease also has considerable prognostic importance. Primary diffuse cutaneous, diffuse gastrointestinal, hepatosplenic, and primary CNS lymphomas tend to be associated with a poor prognosis. Cutaneous lymphoma tends to progress slowly, and in our experience the responses to systemic chemotherapy are less durable. Localized lymphomas in the skin can be managed with radiation therapy or surgery or both, and these tumors have a better prognosis. In some dogs with lymphoma, significant involvement in the bone marrow may be present (i.e., tumor cells comprise more than 50% of all nucleated cells) and circulating malignant lymphocytes may be present in the peripheral blood. These dogs tend to have an overall poor prognosis. In some cases it is difficult to determine whether the disease arises from the bone marrow (e.g., acute lymphoblastic leukemia [ALL]) or is a diffuse lymphoma with extensive involvement in the marrow. Immunophenotyping is helpful in these cases, because the tumor cells in ALL typically are CD34 positive.

Gender has been shown to influence the prognosis in some studies. 59 , 191 Neutered females tend to have a better prognosis. Males may have a higher incidence of the T-cell phenotype, which may account for the poorer prognosis. 60

Other reported potential biomarkers of the prognosis include circulating levels of glutathione-S-transferase, thymidine kinase, and vascular endothelial growth factor (VEGF). 261 , 262 , 263 One report suggests that a history of chronic inflammatory disease of several types predicts a likelihood of early relapse. 264 These putative prognostic indicators require confirmation in larger trials.

LYMPHOID LEUKEMIA

Lymphoid leukemia is the proliferation of neoplastic lymphocytes, which usually originate in the bone marrow but occasionally may originate in the spleen. The neoplastic cells may or may not be circulating in the peripheral blood.

Incidence and Risk Factors

Lymphocytic leukemia is more common than nonlymphocytic leukemia and other myeloproliferative disorders (MPDs). The true incidence is not known. In a series of 30 cases of ALL, German shepherds accounted for 27% of the cases, and the male to female (M:F) ratio was 3:2. 265 In this study, the median age was 5.5 years (range, 1 to 12 years), and eight dogs were younger than 4 years of age. Recently, ALL was reported in a 12-week-old greyhound. 266 Well-differentiated or chronic lymphocytic leukemia (CLL) is seen less frequently than ALL but more commonly than MPD. The median age is 10 to 12 years. The M:F ratio has been reported as 1.8:1 (22 dogs) and approximately 2.3:1 in 15 dogs with granular lymphocytic (GL) T-cell CLL. 267 In a large study of 73 dogs with CLL, no gender predilection was found in dogs with B-cell or non-GL CLL; female dogs were overrepresented among dogs with GL CLL, (M:F ratio, 1:1.7). 268 , 269

Etiology

As with lymphoma, the etiology of lymphoid leukemia is unknown. Retroviruses have been implicated in diverse animal species such as cats, cattle, fish, snakes, birds, rodents, and nonhuman primates. A retroviral cause in dogs has not been proved. However, a retrovirus with morphology typical of lentiviruses has been isolated from mononuclear cells obtained from the peripheral blood of a dog with ALL. 270

In humans, acute leukemia has been associated with exposure to radiation, benzene, phenylbutazone, and antineoplastic agents. 271 , 272 , 273 , 274 , 275 , 276 The alkylating agents can cause chromosomal damage and are clearly carcinogenic. 276 Human T-lymphotropic virus type 1 (HTLV-1) is a proven cause of leukemia in a large cohort of human patients from the southern islands of Japan. 277 , 278 The etiology of CLL is less clear, but genetic factors likely are important. Extrapolation of predisposing factors across species is not warranted, and the etiologic factors for dogs may be quite different from those for humans, given the difference in the predominant immunophenotype of the neoplastic cells (discussed later).

Pathology and Natural Behavior

In ALL, the blast cells infiltrate the bone marrow, and the result is variable degrees of anemia, thrombocytopenia, and neutropenia. Infiltration of the spleen and liver is common, and extramedullary sites (e.g., the nervous system, bone, and GI tract) also may be involved. Some animals may have lymph node involvement and develop generalized lymphadenopathy. 265

The lymphocytes of CLL are virtually indistinguishable morphologically from normal small lymphocytes, and they have a low proliferation rate. The accumulation of lymphocytes likely results from their prolonged life span. In B-cell CLL, the marrow is infiltrated with mature lymphocytes, and the extent of infiltration is less than that seen with ALL or MPD. The neoplastic cells in GL T-cell CLL originate in the spleen, and bone marrow involvement may or may not occur. 173 Dogs with CLL tend to have a mild anemia, and granulocytes and platelets are only mildly reduced. Splenomegaly is common, and lymph nodes can be slightly to moderately enlarged. 269

Despite the well-differentiated appearance of the lymphocytes in CLL, these cells may function abnormally. Paraneoplastic syndromes include monoclonal gammopathies, immune-mediated hemolytic anemia, pure red cell aplasia and, rarely, hypercalcemia. 173 , 279 Hypercalcemia was reported in a giant schnauzer with B-cell CLL, which is highly unusual given that hypercalcemia is associated with T-cell lymphoproliferative disorders. In one study of 22 dogs with CLL, 68% had monoclonal gammopathies. 269 The immunophenotypes were not reported, but a monoclonal gammopathy is associated with production of immunoglobulins by the leukemic cells (B cells). The immunoglobulin is usually IgM or IgA. The term macrogammaglobulinemia is used to describe IgM gammopathy (see Chapter 31, section D ). Dogs with CLL and an IgM monoclonal gammopathy are said to have Waldenström's macroglobulinemia. Dogs can also develop hyperviscosity syndrome (see Chapter 31, section D ).

Reports of the immunophenotype of neoplastic cells in canine leukemias are increasing in frequency. One consistent finding in acute leukemias (either ALL or acute myeloid leukemia) is the presence of CD34 on the blast cells. This marker is useful for distinguishing ALL (CD34-positive) from CLL and lymphoma with bone marrow infiltration (CD34-negative). In one study of 12 cases of CLL, eight dogs were associated with a CD8-positive T-cell phenotype. 75 In a larger study of 73 cases, only 26% of the cases had a B-cell immunophenotype, whereas 73% were identified as T-cell CLL (CD3-positive). In 54% of these T-cell leukemias, the cells had the morphology of GLs, and most of them were CD8-positive. These findings are difficult to reconcile with the high frequency of monoclonal gammopathies reported in the earlier study. It also should be noted that this differs from human CLLs, which typically are lymphoproliferative diseases of B cells.

History and Clinical Signs

Dogs with ALL usually have a history of anorexia, weight loss, polyuria, polydipsia, and lethargy. Splenomegaly is typical, and other physical abnormalities may include hemorrhages, lymphadenopathy, and hepatomegaly. Anemia, thrombocytopenia, and an elevated white blood cell (WBC) count are common. The anemia may be severe and usually is characterized as normocytic and normochromic (nonregenerative). WBC counts usually are increased despite neutropenia because of an increased number of circulating lymphoblasts (more than 14,000 cells/μl). However, some dogs may be leukopenic. 265 Neoplastic lymphoblasts may infiltrate the bone marrow extensively, resulting in depression of normal hematopoietic elements.

Dogs with CLL often are asymptomatic, although some owners report lethargy and a decreased appetite. Mild lymphadenopathy and splenomegaly may be present. Most dogs tend to be mildly anemic (packed cell volume [PCV] less than 35%) and thrombocytopenic (110,000 to 190,000 platelets/μl). The WBC count usually exceeds 30,000 cells/μl but can vary from normal to more than 100,000 cells/μl, owing to an increase in circulating mature lymphocytes. 269 Lymphocytosis is persistent, and granulocytes usually are present in normal numbers. In some dogs the disease is identified incidentally when the patient undergoes evaluation for an unrelated problem.

Diagnostics and Clinical Staging

The clinician must consider the signalment, history, physical findings, cell morphology, and immunophenotype to diagnose the lymphoid leukemias accurately. A knowledge of the profile of lymphocyte subsets in the peripheral blood of normal dogs is helpful for determining whether a particular subset has expanded. Approximately 80% of circulating lymphocytes in dogs are T cells, and about 15% are B cells. NK cells and double-negative (CD4-CD8-) T cells account for the remaining fraction. In the T-cell fraction, helper T cells (CD4+) outnumber cytotoxic T cells (CD8+). 173

Lymphocytic leukemia should be a consideration if atypical lymphocytes are in circulation, if the immunophenotype of the lymphocytes in circulation is homogenous, or if a phenotype typically present in low numbers has increased. Differential diagnoses for lymphocytosis include certain infectious diseases (e.g., chronic ehrlichiosis), postvaccinal responses in young dogs, IL-2 administration, and transient physiologic or epinephrine-induced lymphocytosis. Reactive and neoplastic lymphocytoses sometimes are difficult to differentiate. In these cases, PCR assays are used to determine the clonality of the T cell or immunoglobulin receptor genes in a population of lymphocytes (see earlier discussion). 167 , 169 , 268

Infiltration of the bone marrow by neoplastic lymphoid cells is the hallmark of ALL and is seen in most cases of CLL; careful examination of peripheral blood and bone marrow by experienced cytopathologists is essential for establishing a diagnosis of lymphocytic leukemia. If adequate diagnostic bone marrow cannot be obtained by aspiration, a bone marrow core biopsy should be performed. In ALL, lymphoblasts predominate in the bone marrow and also are present in peripheral blood. Infiltration of bone marrow by lymphoblasts is accompanied by a decrease in the granulocytic, erythroid, and megakaryocytic cell lines.

Perhaps the distinguishing feature of lymphoblasts is the nuclear chromatin pattern, which is more condensed than the chromatin in myeloblasts. 280 Lymphoblasts are larger than neutrophils, have a high nucleus-to-cytoplasm ratio, and blue cytoplasm, which in some cases is intensely basophilic ( Figure 31-11 , A ). Nucleoli, although present, are less prominent in lymphoblasts than in myeloblasts. Nevertheless, these cells cannot be easily distinguished from blast cells of other hematopoietic lineages, and lineage-specific markers on the cells must be identified by immunocytochemistry or flow cytometry. 278 The most frequently used markers are CD3 for T cells and CD79a for B cells. As mentioned earlier, CD34 is found on blast cells of both lymphoid and myeloid lineages, and positivity for CD34 helps to confirm leukemia of immature cells (acute leukemia) and to distinguish ALL from stage V lymphoma. Immature and differentiating lymphocytes may stain strongly for alkaline phosphatase activity, which suggests that this cytochemical staining procedure is not specific for myeloid leukemia. 278 , 280 , 281

In B-cell and non-GL T-cell CLL, the lymphocytes are small mature cells ( Figure 31-11 , B ) that appear in excessive numbers in bone marrow (30% or more of all nucleated cells) early in the disease. 269 Infiltration becomes more extensive as the disease slowly progresses, and eventually the neoplastic cells replace normal marrow. The lymphoid lineage of the cells in CLL typically is easy to identify, and immunophenotyping is done to determine the lymphocyte subset. In dogs, most cases of CLL are T-cell (CD3+) proliferations. In GL CLL, the neoplastic cells originate in the spleen. 173

A separate clinical staging system has not been developed for lymphocytic leukemia. Currently, all dogs with leukemia are classified as stage V based on the WHO staging system for lymphoma (see Box 31-1 ). Although a specific clinical staging system for CLL has been used in humans, it has not been evaluated in the dog. 282

Treatment of the acute lymphoblastic leukemia

Similar to other infiltrative bone marrow malignancies, ALL causes morbidity by suppressing bone marrow function. Neutropenia, thrombocytopenia, and anemia may be severe. Therapy must be aggressive; to restore hematopoiesis, a 1.5 to 2 logarithmic reduction in leukemic cell numbers (to less than 100 million cells) must be achieved. Patients need supportive therapy, such as fresh whole blood, broad-spectrum antibiotics, fluid therapy, and nutritional support. Patients must be monitored carefully for bleeding and thrombosis, which may signal the development of DIC.

ALL requires aggressive chemotherapy. Consistently efficacious protocols for ALL have not been developed in veterinary medicine. CHOP-based protocols, similar to those used for lymphoma (see Box 31-3 ), tend to be used as treatment protocols for dogs with ALL. In one report on the use of vincristine and prednisone in dogs with ALL, 40% of the dogs responded to vincristine and prednisone, 20% with a complete remission and 20% with a partial remission. 265 With the addition of doxorubicin and L-asparaginase, it is anticipated that response rates will increase over those previously reported using vincristine and prednisone alone; however, the relative rarity of ALL limits the ability to identify effective protocols.

Treatment of chronic lymphocytic leukemia

Because of the indolent nature of CLL in many animals, the question of whether all dogs with the disease should be treated is controversial. 283 , 284 Most oncologists recommend observation over active therapy when the discovery of CLL is incidental, when no physical or clinical signs are present, and when no significant hematologic abnormalities are identified. Therapy should be instituted if the animal is anemic or thrombocytopenic, is showing evidence of significant lymphadenopathy or hepatosplenomegaly, or has an excessively high WBC count (over 60,000 lymphocytes/μl) ( Box 31-4 ). The most effective drug evaluated thus far is chlorambucil. 269 Chlorambucil is administered at a dosage of 0.2 mg/kg or 6 mg/m 2 given orally once daily for 7 to 14 days. The dosage then can be reduced to 0.1 mg/kg or 3 mg/m 2 given orally once daily. For long-term maintenance, a dosage of 2 mg/m 2 given orally every other day can be used. The dosage is adjusted according to the clinical response and bone marrow tolerance. Chlorambucil should be administered without food to increase the rate of absorption. 285

Treatment of Chronic Lymphocytic Leukemia

Regimen 1

Chlorambucil, 0.2 mg/kg, PO, days 7-14; then 0.1 mg/kg, PO, continuously or 2 mg/m 2 , PO, QOD *

Prednisone, 30 mg/m 2 , PO, SID, for 7 days; then 20 mg/m 2 , PO, SID, for 7 days; then 10 mg/m 2 , PO, QOD

Regimen 2

Vincristine, 0.02 mg/kg, IV, weeks 1-3

Chlorambucil, 0.2 mg/kg, PO, SID, for 7 days; then 0.1 mg/kg, PO, SID, continuously or 2 mg/m 2 , PO, QOD *

Prednisone, 30 mg/m 2 , PO, SID, for 7 days; then 20 mg/m 2 , PO, SID, for 7 days; then 10 mg/m 2 , PO, QOD, continuously

Corticosteroids are lymphocytolytic and lead to cell death by apoptosis. Studies in humans have shown that the antitumor activity of chlorambucil combined with prednisone is better than that of chlorambucil alone. 286 When the bone marrow is heavily infiltrated with CLL cells, and neutropenia, thrombocytopenia, and anemia occur, use of a more aggressive alkylating agent (usually cyclophosphamide [250 mg/m 2 given once]) when initiating oral chlorambucil and prednisone therapy can be considered to increase the speed of initial remission; however, this modification of the protocol has not been scrutinized in clinical trials. If chlorambucil or cyclophosphamide fails, the choice of treatment is combination chemotherapy similar to that presented in Box 31-3 . The treatment of CLL is primarily palliative, and complete remissions are rare. Because of the indolent nature of this disease, however, survival times have been in the range of 1 to 3 years with a good quality of life. 269 , 284

In humans, splenectomy has been shown to increase survival significantly in individuals with aggressive forms of CLL; however, splenectomy in dogs with CLL has not been evaluated. 287 The phenotypic expression of CLL usually is stable over months to years. However, the disease may evolve into an acute phase, and some dogs develop a rapidly progressive, pleomorphic (immunoblast) lymphoma. 269 In humans, this is called Richter's syndrome . 288 The prognosis for response to treatment is poor for this form.

Prognosis

In general, the prognosis for ALL in the dog is very poor. In a study of 21 dogs treated with vincristine and prednisone, the dogs achieving complete or partial remission (29%) had a median survival time of 120 days, and few dogs survived longer than 8 months with that protocol. 265 In one case report, a dog with ALL was treated with an infusion of a large volume of fresh canine plasma and whole blood, and a complete remission was maintained for 19 months without additional therapy. This is a very unusual response, which indicates that normal blood contains some antileukemic factor or factors. 289

As stated before, CLL is a slowly progressive disease, and some animals do not require therapy. One dog was observed for almost 2 years without treatment. 283 For dogs that are treated, normalization of WBC counts can be expected in 70% of cases. In one report of 17 dogs treated with vincristine, chlorambucil, and prednisone, the median survival time was approximately 12 months, with an expected 30% survival at 2 years. 269 In other studies, dogs treated intermittently with chlorambucil and prednisone have had remissions of 10 to 30 months. 284 , 290 , 291

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Feline Lymphoma and Leukemia

David M. Vail

LYMPHOMA

The lymphomas (malignant lymphoma and lymphosarcoma) are a diverse group of neoplasms that have in common their origin from lymphoreticular cells. They usually arise in lymphoid tissues, such as lymph nodes, spleen, and bone marrow; however, they may arise in almost any tissue in the body. Lymphoma is one of the most common neoplasms seen in the cat.

Incidence and Etiology

Viral factors

The feline leukemia virus (FeLV) was the most common cause of hematopoietic tumors in the cat during the so-called FeLV era of the 1960s through the 1980s, when 60% to 70% of lymphoma cases were associated with FeLV antigenemia. 1 , 2 , 3 , 4 , 5 , 6 , 7 Several studies have documented the potential molecular means by which FeLV can result in lymphoid neoplasia. 8 , 9 However, over the past 20 years in North America, a profound change has occurred in the viral status, presentation, signalment, and frequency of anatomic sites in cats with lymphoma, as documented by two large studies of feline lymphoma involving more than 700 cats. 10 , 11 The change in the epidemiology and characteristics of lymphoma in cats appears to coincide with the widespread integration of clinically relevant FeLV diagnostic assays (indirect immunofluorescence assay) and the affected animal elimination regimens of the late 1970s; it was further enhanced by the appearance of commercially available FeLV vaccines in the late 1980s.

The decline in FeLV-associated lymphoma was mirrored by a decline in the overall prevalence per year of FeLV positivity in cats tested, as characterized by reports from the Tufts Veterinary Diagnostic Laboratory from 1989 to 1997 10 , 12 and by Louwerens’ group, 11 which reported a decline in FeLV association in more than 500 cases of lymphoma in cats brought to the University of California–Davis Veterinary Teaching hospital. In these reports, FeLV antigenicity represented only 14% to 25% of the cats with lymphoma.

The incidence figures for feline lymphoma generated before the use of FeLV vaccination and testing became widespread suggest that lymphoma accounted for 50% to 90% of all hematopoietic tumors in the cat 13 , 14 ; because hematopoietic tumors (lymphoid and myeloid) represent approximately one third of all feline tumors, the estimated incidence of lymphoid neoplasia was 200 per 100,000 cats at risk. 15 In one series of 400 cats with hematopoietic tumors, 61% had lymphoma and 39% had leukemias and MPDs (21% of the MPDs were categorized as undifferentiated leukemias, most likely myeloid in origin). 16 An important fact that Louwerens’ study revealed is that despite a sharp drop in FeLV-associated lymphoma, the overall prevalence of lymphoma in cats is increasing. 11 This appears to be due to an increase in the number of affected cats and the relative frequency of the abdominal (particularly the intestinal) anatomic form of lymphoma in the species ( Figure 31-12 ).

As might be expected, along with a shift away from FeLV antigen–associated tumors came a shift away from the traditional signalment and relative frequency of anatomic sites. 10 , 11 This observation is supported outside North America by the similar signalment and anatomic frequency data obtained in Australia, where FeLV infection is quite rare. 17 , 18 , 19 , 20 The median age of approximately 11 years now reported in North America for lymphoma in cats is considerably higher than the median age of 4 to 6 years reported during the FeLV era. 1 , 3 , 4 , 5 , 6 , 10 , 11 , 21 The median age of cats within various anatomic tumor groupings has not changed, and anatomic forms traditionally associated with FeLV, such as the mediastinal form, still occur in younger, FeLV-antigenemic cats. Similarly, the alimentary form occurs most often in older, FeLV-negative cats. 10 , 11 , 18 , 19 , 20 Table 31-6 presents an overview of the characteristics of the various anatomic sites of lymphoma in cats. Younger cats with lymphoma tend to be FeLV antigenemic and are more likely to have mediastinal or multicentric lymphoma. Most cats with spinal lymphoma (85% to 90%) also are FeLV antigenemic. 22 , 23 Older cats usually are FeLV negative and develop alimentary lymphoma. 10 , 17 , 19 , 20 , 24 , 25 , 26 , 27 In a large compilation of Australian cases, male cats and the Siamese/oriental breeds were overrepresented. 17 Similar breed findings have been observed in North America, 11 but similar gender findings have not emerged. 28 , 29 The Siamese/oriental breeds appear to have a predisposition for the mediastinal form that affects a younger population (median age, 2 years). 11

Evidence also indicates that feline immunodeficiency virus (FIV) infection can increase the incidence of lymphoma in cats. 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 In contrast to FeLV, which plays a direct role in tumorigenesis, FIV appears to have an indirect role, likely secondary to the immunosuppressive affects of the virus. 35 Shelton and colleagues 30 determined that FIV infection alone in cats was associated with a fivefold increased risk for the development of lymphomas. Coinfection with FeLV further potentiates the development of lymphoproliferative disorders. Experimentally, cats infected with FIV have developed lymphoma in the kidney, alimentary tract, and liver and in multicentric sites. FIV-associated lymphoma is more likely to have a B-cell immunophenotype whereas T-cell type is predominantly associated with FeLV. 32 , 33 , 35 , 36 , 38 , 39 , 40 , 41 Some have suggested that FIV infection may be associated more often with alimentary lymphoma of B-cell origin, 40 , 41 which may be related to chronic dysregulation of the immune system or activation of oncogenic pathways. However, in another large compilation of cases, FIV antigenemia only rarely was associated with alimentary lymphoma. 10 , 24 , 25 , 26 , 27

Genetic and molecular factors

As discussed in Chapter 31, section A , recent advances in molecular cytogenetics (see Chapter 1, section A), including gene microarray techniques, are being used in investigations of chromosomal aberrations in veterinary species with lymphoma. The previously mentioned predisposition of the oriental cat breeds to the development of lymphoma suggests a genetic predisposition and indicates heritable risks. 11 , 17 As our knowledge of molecular events and tumorigenesis has expanded, several molecular aberrations have been implicated in various feline tumor types, and some associated with lymphoma have been identified. Altered oncogene/tumor suppressor gene expression, epigenetic changes, signal transduction, and death-pathway alterations are common in human lymphomas and likely are also involved in the cat. N- ras aberrations, although rare in cats, have been implicated. 42 Telomerase activity (see Chapter 14, section D) also has been documented in feline lymphoma tissues. 43 , 44 Other factors implicated in feline lymphoma include alterations in cellular proliferation and in cell cycle and death (apoptosis) pathways, particularly the cyclin-dependent kinase cell cycle regulators and the Bcl-2 family of proapoptotic and antiapoptotic governing molecules, which have been implicated in human NHL. 45 , 46

Environmental factors

Evidence that exposure to environmental tobacco smoke (ETS) is a risk factor for lymphoma in humans has prompted investigations in cats. In one report, cats with any exposure to ETS had a relative risk of developing lymphoma of 2.4; the relative risk for cats with 5 or more years of exposure was 3.2. 47

Immunosuppression

Immune system alterations in the cat, such as those brought on by FIV infection, have been implicated in the development of lymphoma. 30 , 31 , 32 , 33 , 35 , 36 , 37 In a report on 95 feline renal transplant recipients, nearly 10% developed de novo malignant lymphoma, which bolsters support for a link to immunosuppression in the species. These findings are similar to those seen in immunosuppressed human organ transplant patients. 48

Chronic inflammation

Although definitive proof is lacking, a growing body of indirect evidence suggests that lymphoma may be associated with a state of chronic inflammation, such as in intestinal and nasal lymphoma. In particular, an association has been suggested between intestinal lymphoma and inflammatory bowel disease 11 , 49 , 50 , 51 ; however, others have not found support for this concept. 52 Additional support for this association is provided by a recent report that suggests that cats with vaccine site–associated sarcoma, a syndrome directly linked to inflammation, are also at risk for the development of lymphoma. 53

Diet and intestinal lymphoma

Although no direct evidence exists, a link between diet and the development of intestinal lymphoma in cats has been suggested. 11 This association is supported by the relative and absolute increase in the intestinal form of lymphoma over the past 20 years and by the fact that several dietary modifications in cat food have occurred in a similar time frame in response to conditions such as urinary tract disease. Further investigation is warranted to prove or disprove such assertions.

Pathology and Natural Behavior

Lymphoma can be classified on the basis of anatomic location and histologic criteria. Several systems of anatomic classification exist for lymphoma in the cat. Some categorize the disease into mediastinal, alimentary, multicentric, nodal, leukemic, and individual extranodal forms. Others have combined various nodal and extranodal forms into categories of atypical, unclassified, and mixed, while still others have combined intestinal, splenic, hepatic, and mesenteric nodal forms into one category (intra-abdominal). For the purposes of this discussion, the “mixed” form is defined as cases involving multiple sites in which no primary site categorization was possible. Some discrepancies in the discussion of frequency inevitably will result from the variations in classification used in the literature. The relative frequency of anatomic forms and the immunophenotype may vary with geographic distribution and may be related to genetic and FeLV strain differences, as well as to the prevalence of FeLV vaccine use (see Table 31-6 ).

In most larger studies, most cases of lymphoma in cats (approximately 70% to 75%) are of the B-cell immunophenotype; however, the mediastinal, leukemic, and purely hepatic forms are more likely to be of T-cell derivation. 10 , 18 Cats with T cell–rich B-cell lymphoma (Hodgkin's-like lymphoma) and NK-like T-cell lymphoma (non-T, non-B-cell) have been described. 54 , 55 As with dogs, most lymphomas in cats have an intermediate-grade or a high-grade histology according to the working formulation (WF) criteria. 18 , 56

Alimentary/intestinal lymphoma

Alimentary/intestinal lymphoma can manifest as a purely intestinal infiltration or as a combination of intestinal, mesenteric lymph nodes, and liver involvement. 11 Some reports limit the alimentary form to GI involvement, with or without extension to the liver. Approximately two thirds to three fourths of reported cases are of the B-cell immunophenotype. Most patients are older cats, and very few cases appear to be associated with FeLV antigenemia. 10 , 11 , 18 , 24 , 25 , 26 , 57 , 58 , 59 However, some discordance on these points is seen in the literature. In one smaller report, most alimentary cases sampled were found to be of the T-cell immunophenotype. 24 The population size was relatively small in comparison and may represent a geographic variation. The epitheliotropic form of intestinal lymphoma also has been reported to be more commonly T-cell in origin. 49 , 57 A Canadian group investigated formalin-fixed archival lymphoma tissue in cats and found a nearly even distribution between the B-cell and T-cell immunophenotype; they also found that approximately two thirds of the samples were FeLV positive by PCR techniques. 2 , 60 , 61 , 62 Some FeLV-negative tumors may be derived from transformation of multipotent lymphoid or monocyte precursors 61 or FIV-transformed B lymphocytes. 32 , 33 , 38 , 39

The most common site of involvement in the alimentary tract is the small intestine (50% to 80% of cases), followed by the stomach (approximately 25%), ileocecocolic junction, and colon. 25 , 26 , 62 The tumor can be solitary or diffuse throughout the intestines ( Figure 31-13 ), muscle layers, and intestinal submucosa, resulting in annular thickening that leads to partial or complete intestinal obstruction. In a series of colonic neoplasias in cats, lymphoma was the second most common malignancy (41%), exceeded only by adenocarcinoma. 27 An abstract report has described chronic lymphocytic-plasmacytic enteritis in cats that progressed to overt lymphoma after 6 to 18 months of conservative therapy. 63

Mediastinal form

The mediastinal form can involve the thymus and the mediastinal and sternal lymph nodes. Pleural effusion is common. In two large compilations, 63% of cats with thymic disease and 17% of cats with pleural effusion were documented as having lymphoma. 64 , 65 Occasionally the tumor extends from the thoracic inlet and can be palpated in the ventral neck region. Hypercalcemia is common with mediastinal lymphoma in dogs but is rare in cats with lymphoma. 66 , 67 , 68 PTHrP has been detected through immunoradiometric assays in cats with hypercalcemia of malignancy, including one cat with lymphoma. 69 Most cats with mediastinal lymphoma are young and FeLV positive, and their tumors are of the T-cell immunophenotype. *

Nodal forms

Involvement of peripheral lymph nodes alone is very unusual in cats with lymphoma, representing approximately 4% to 10% of cases. 10 , 11 In contrast, approximately one fourth of all other anatomic forms of lymphoma have some degree of lymph node involvement. 11 One third of cats with nodal lymphoma are of the T-cell immunophenotype and are FeLV antigenemic. 10 , 18 As lymphoma progresses, bone marrow infiltration with malignant cells and hepatosplenomegaly may develop.

Recently an uncommon and distinct form of nodal lymphoma in cats, referred to as Hodgkin's-like lymphoma , has been reported. This form typically involves solitary or regional nodes of the head and neck and histologically resembles Hodgkin's lymphoma in humans. 17 , 54 , 70 These tumors generally manifest as an enlargement of a single mandibular or cervical node and immunophenotypically are classified as T cell–rich B-cell lymphoma by immunohistochemical analysis. 54 , 70 None have been associated with either FeLV or FIV.

Several reports of nonneoplastic peripheral lymphadenopathy in cats have been published. 71 , 72 , 73 , 74 , 75 This condition resembles lymphoma clinically, and it has histologic features that also may resemble those of lymphoma. 71 , 72 , 73 , 74 , 75 Affected lymph nodes may be two or three times normal size. In one report, the syndrome was called distinctive peripheral lymph node hyperplasia (DPLH) of young cats. 72 These cats tend to be young (2 to 4 years old), and many have had episodes of fever or previous viral infections, or they may have hypergammaglobulinemia (polyclonal gammopathy); most are FeLV negative. 71 Histopathologically, the nodal architecture is severely distorted, showing loss of subcapsular and medullary sinuses. The cell population shows an admixture of histiocytes, lymphocytes, plasma cells, and immunoblasts and occasionally effaced lymphoid follicles. The lymph nodes regress spontaneously in most of these cats. The histologic changes noted in these lymph nodes resemble the histologic features of acquired immunodeficiency syndrome (AIDS)-related lymphadenopathy in humans. 72 In another report, benign lymphadenopathy in cats was associated with argyrophilic intracellular bacteria. 75

Extranodal lymphoma

The most common extranodal sites for lymphoma are the kidneys, nasal cavity, eyes, retrobulbar space, CNS, and skin.

Renal lymphoma can be primary or associated with alimentary lymphoma. Based on several studies, the median age for cats with renal lymphoma is approximately 7.5 years. One fourth of cases are FeLV antigenemic, and most are of the B-cell immunophenotype. 10 , 11 , 18 , 76 The frequency of renal lymphoma is reported to be approximately 5% of all lymphomas. Extension to the CNS is a common sequela to renal lymphoma and occurs in 40% to 50% of treated cats. 76

Nasal/paranasal lymphoma usually is a localized disease; however, systemic extension occasionally is seen. 10 , 77 , 78 Neoplasia accounts for most nonviral nasal/paranasal disease in cats, and lymphoma has been reported to represent nearly one third to one half of these cases. 79 , 80 , 81 It occurs primarily in older, FeLV-negative cats (median age, 9 to 12 years). At least three fourths of cases are B cell in origin, and most are intermediate grade or high grade histologically. 10 , 79 , 80 , 81 A subset of epitheliotropic T-cell nasal lymphomas has been reported. 81 Cats that are concurrently FeLV positive are more likely to have concurrent systemic disease.

In the older literature, CNS lymphoma was most often seen extradurally in the spinal canal of FeLV-positive cats (85% to 90%). 20 , 21 , 82 However, in more recent reviews, both spinal cord and intracranial lymphoma occurs mostly in older, FeLV-negative cats, and spinal cord sites are more commonly intradural than extradural. 83 , 84 After meningioma, lymphoma is the second most common tumor involving the CNS in cats. 84 Feline CNS lymphoma may be primary or may occur secondary to multicentric involvement (especially of the renal system or bone marrow). 83 , 84 , 85 , 86 Bone involvement is rarely seen radiographically. 87 Multiple cord regions and the brain are involved in nearly 50% of cats with spinal lymphoma, and more than 80% of these patients have other organ (e.g., renal) and bone marrow involvement. 20 , 21 , 83 Only one third of cases of intracranial lymphoma are primary and confined to the CNS, and all cats tested have been FIV negative. 84

Cutaneous lymphoma generally is primary but can be seen secondary to multicentric involvement. It commonly is seen in older cats (median age, 10 to 12 years) and gender sex or breed predominance has not been found. 88 , 89 , 90 , 91 , 92 Although patients usually are FeLV negative, one case report using PCR techniques found evidence of FeLV provirus in tumor DNA. 92 Cutaneous lymphoma can be solitary or generalized. Two forms of cutaneous lymphoma have been distinguished histologically and immunohistochemically. 88 , 89 , 90 , 91 , 92 In most species, the epitheliotropic form, sometimes referred to as mycosis fungoides , is composed of T lymphocytes, whereas the nonepitheliotropic form usually is composed of B lymphocytes. In contrast, one report of nonepitheliotropic cutaneous lymphoma in cats found that five of six cases were of T-cell derivation. 93

Neoplastic T lymphocytes are large and have abundant cytoplasm and convoluted nuclei (mycosis cells). They usually form intraepidermal nests of five to 10 cells, which are separated from surrounding keratinocytes by a clear space (Pautrier's microabscesses). The B-cell lymphomas show lymphocytes deep in the epidermis, with sparing of the papillary dermis and epidermis. A recent report described 23 cases of cutaneous lymphocytosis, an uncommon disease histologically resembling well-differentiated lymphoma. 94 Solitary lesions were most common, and all were composed primarily of T-cells, although two thirds had some B-cell aggregates. Cutaneous lymphocytosis was characterized as a slowly progressive disorder, but internal organ infiltration developed in a few cases.

A cat with cutaneous T-cell lymphoma and circulating atypical lymphocytes has been reported. 91 The circulating cells were lymphocytes with large, hyperchromatic, grooved nuclei. In humans, cutaneous T-cell lymphoma with circulating malignant cells is called Sézary syndrome , 95 which also has been reported in dogs. 96 , 97 , 98 , 99

Histologic Grade Classification of Lymphomas

A number of histopathologic grading systems have been used to classify human NHL, including the WF (discussed earlier in the chapter). Most recently, the WF was used to classify more than 600 cases of feline lymphoma. 56 Low-grade lymphoma was found in 11% of the cases, intermediate-grade disease in 35%, and high-grade lymphoma in 54%. About 1.1% were plasmacytomas. More than one third of the tumors were the immunoblastic type. Lymphoblastic lymphoma, a subtype of the high-grade tumor, accounted for less than 3%. Similarly, in a large group of cases compiled in Australia (n = 118), 90% of the cases were medium- to high-grade disease as classified by the WF. 18

Also as discussed earlier in the chapter, WHO has published a histologic classification scheme that uses the revised European American lymphoma (REAL) system as a basis for defining histologic categories of hematopoietic tumors of domestic animals. 100 This system incorporates both histologic criteria and immunohistologic criteria (B- and T-cell immunophenotype). The clinical relevance of this system is likely to be high; however, it awaits further investigation and evaluation.

In a series of 28 cases of alimentary lymphoma, 89% were the high-grade lymphoblastic type. 25 However, in another report, many of the intestinal lymphomas had several characteristics of MALT, as described in humans; that is, a tendency to remain localized in the lamina propria, a relatively indolent clinical behavior, and an excess of low-grade tumors. 56 The age of affected animals varied considerably with various subtypes, but in general, low-grade tumors tended to develop in older cats (over 10 years of age), and high-grade tumors tended to develop in younger cats (under 6 years of age).

A less commonly reported, distinct form of alimentary lymphoma has been described and classified as large granular lymphoma . 101 , 102 , 103 , 104 , 105 , 106 , 107 , 108 , 109 , 110 These lesions are granulated, round cell tumors that also have been called either globule leukocyte tumors or large granular lymphocyte lymphoma , although they probably are variations of the same disease. Large granular lymphocytes are characterized by abundant cytoplasm with prominent azurophilic granules. This population of cells includes NK cells and cytotoxic T cells. Several reports have identified their immunophenotype as CD3, CD57-like, perforin positive, and CD20 negative; these cells also have been described as having a T-cell receptor gene rearrangement. These tumors commonly originate in the small intestine, especially the jejunum or mesenteric lymph nodes; however, most cases show widespread metastasis to the lung, myocardium, salivary gland, and spinal cord. Leukemia also has been reported with this disease. Affected cats generally are FeLV negative. Large granular lymphocytes must be differentiated from several other granular cell types that may be found in the small intestine, including enterochromaffin cells, mast cells, and eosinophils.

History and Clinical Signs

The clinical signs associated with feline lymphoma vary and depend on the location and extent of disease.

The alimentary form is most commonly associated with an abdominal mass that originates in the GI tract. The condition often is associated with enlarged mesenteric lymph nodes or other organ involvement. In 50% to 85% of cases, a palpable abdominal mass or thickened bowel loops are present. 24 , 25 , 27 , 62 , 111 Clinical signs may consist of weight loss, anorexia, diarrhea, and occasional vomiting. In approximately half of cases, the only historical finding is anorexia and weight loss. 25 Other reported presentations include abdominal distention, splenomegaly, persistent thrombocytopenia, and pica. Hematochezia and tenesmus may be present if the lymphoma involves the colon. 27 Polyuria and polydipsia have been reported in approximately 10% of cases. 62 In a small percentage of cases, the patient may have signs consistent with an acute abdomen as a result of intestinal perforation and concurrent peritonitis. 24

Clinical signs of the mediastinal form of lymphoma include dyspnea, tachypnea, and a noncompressible anterior mediastinum with dull heart and lung sounds. 20 , 111 , 112 , 113 , 114 In rare cases, Horner's syndrome may be present as a result of involvement of the sympathetic nerve as it ascends around the first rib, and edema of the head may be caused by pressure on the cranial vena cava. 111 Pleural effusion is common; the pleural fluid is characterized by serohemorrhagic to chylous effusion, and in most cases neoplastic cells (lymphoblasts) are identified. 65 , 114

Cats with the nodal form of lymphoma have variable clinical signs, depending on the location and extent of disease; however, these cats often are depressed and lethargic. Peripheral lymphadenopathy as the only physical finding is a very uncommon presentation for cats. As stated earlier, an uncommon and distinct form of nodal lymphoma in cats is Hodgkin's-like lymphoma. This form typically involves a single mandibular or cervical node, and the cat usually does not have any overt clinical signs. 54 , 70 In cats, unlike in dogs, peripheral lymphadenopathy without organomegaly is more often hyperplastic or reactive and does not represent lymphoma. 73

The extranodal sites include the kidneys, skin, eyes, nasal area, and CNS. Renal lymphoma is most consistently bilateral, even in cats that appear to have unilateral disease. 76 In general, the kidneys are uniformly enlarged; however, they may also feel lumpy and irregular on palpation. More than 50% cats have signs consistent with renal insufficiency. 76

Cats with CNS lymphoma most often have signs associated with thoracolumbar involvement. 22 , 23 The most common sites are between the second thoracic and fourth lumbar vertebrae. Signs include gradual or sudden onset of weakness, upper motor neuron paralysis to the bladder, tail flaccidity, hyperpathia in the region of the lesion, and progressing ataxia. The neurologic dysfunction may be insidious or may progress rapidly. 23 Cats with cervical spinal cord or nerve root involvement generally show peracute tetraparesis and diminished sensation in the thoracic limbs. Those with cervical root involvement may show root lesions (root signature), such as lameness and hyperesthesia upon shoulder extension. Common presenting signs for intracranial lymphoma, in decreasing order of frequency, include anorexia, ataxia, lethargy, altered consciousness, and aggression. 84

Cats with nasal lymphoma usually have signs localized to the nasal passage. In a large compilation of cases, the presenting complaints, in decreasing order of frequency, were unilateral nasal discharge (bilateral is less common), facial deformity, dyspnea, and epistaxis. 79 , 80 , 81 Stertor, anorexia, epiphora, exophthalmia, sneezing, and regional lymphadenopathy also can occur.

Cutaneous lymphoma may be solitary or diffuse and manifest with alopecia, erythema, and crusted papules. Minimal peripheral lymphadenopathy may also be present. In most cats the signs have a prolonged duration (i.e., several months). 88 , 89 , 90 , 91 , 92 , 94

Nonspecific signs

All cats with lymphoma, regardless of the site, may have secondary bone marrow infiltration that leads to anemia and a leukemic blood profile. Anemia is a common condition in cats with lymphoma, and at least 50% have moderate to severe nonregenerative anemia. Signs related to paraneoplastic hypercalcemia (PU/PD) can occur in cats, but the syndrome is much less common than in the dog. In one survey of hypercalcemia in cats, approximately 10% were diagnosed with lymphoma of various anatomic types. 115 One case of hypereosinophilic paraneoplastic syndrome and one case of symmetric cutaneous necrosis have been reported in cats with lymphoma. 116 , 117 A number of disease conditions can be confused with lymphoma in cats ( Table 31-7 ).

Diagnostics and Clinical Staging

For most cats suspected of having lymphoma or leukemia, the diagnostic evaluation should include a CBC with differential cell count, platelet count, serum chemistry profile, and a test for FeLV and FIV. Bone marrow aspiration or biopsy may be indicated to evaluate for possible involvement and complete staging of the extent of disease. Bone marrow evaluation is particularly indicated if anemia, cellular atypia, and leukopenia are present. Histopathologic evaluation of lymph nodes or involved organ tissue (procured by means of surgical incision, endoscopy, or needle core biopsy) is essential for a definitive diagnosis. In the cat, lymph node fine-needle aspiration alone is not sufficient in most cases because of the difficulty involved in distinguishing lymphoma from benign hyperplastic lymph node syndromes unique to the species; these include idiopathic peripheral lymphadenopathy, plexiform vascularization of lymph nodes, and peripheral lymph node hyperplasia of young cats. 71 , 72 , 73 , 74 , 75 Whole lymph node excision is preferred in these cases, because determination of the orientation, invasiveness, and architectural abnormalities may be necessary for diagnosis. Additional site-specific cytologic or histologic assessments may be warranted when extranodal sites are suspected.

Histologic and cytologic samples also can be analyzed by various histochemical and immunohistochemical techniques to determine the immunophenotype (B cell or T cell), tumor proliferation rate (e.g., Ki-67, PCNA, AgNORs), and telomerase activity (see Chapter 14, section D), and histologic subtype (low, intermediate, or high grade). 10 , 18 , 26 , 34 , 43 , 44 , 46 The availability of such analysis is increasing, but currently none has proved to be predictive of outcome in cats.

Serum chemistry profiles can help establish the overall health and clinical staging of cats, but the results are not specific for a diagnosis. Elevated liver enzymes may indicate hepatic infiltration with lymphoma, and elevated blood urea nitrogen (BUN) and creatinine levels may indicate renal lymphoma. For cats with alimentary lymphoma, hypoproteinemia and anemia are reported to occur in up to 23% and 76% of cases, respectively. 25 , 118 Hypercalcemia is rare in cats but has been reported in patients with lymphoma at various anatomic sites. 25 , 66 , 67 , 68 Elevated globulin levels may indicate the presence of a monoclonal gammopathy with or without serum hyperviscosity (this is a rarely reported paraneoplastic syndrome in cats with lymphoma). 119 , 120 Hypoglycemia was reported in approximately one third of cats with lymphoma in an Australian study. 118 Serum alpha 1–acid glycoprotein concentrations have been reported to be elevated in cats with lymphoma, but no clinical relevance has been associated with this finding. 121

Most cats with alimentary lymphoma develop a palpable abdominal mass or thickened intestinal loops. Approximately one third of patients have a mass that can be visualized on abdominal radiographs, and about 90% have ultrasound abnormalities. 24 , 25 The ultrasound abnormalities may include mesenteric lymphadenopathy in 33% to 50% of cases, an intestinal mass or thickening in approximately 40% of cases, and possibly splenomegaly (approximately 33% of cats with ultrasonographic changes in the spleen have lymphoma 122 ), hepatomegaly, or effusion. 24 , 25 In one compilation of 28 cases, the lesions appeared ultrasonographically to be localized in 70% of cases and diffuse in 30%; however, at surgery only 33% were resectable. 25 For alimentary lymphoma, especially if primary GI lymphoma is suspected, caution must be exercised when endoscopically obtained tissue is used because of the difficulty in differentiating lymphoplasmacytic gastroenteritis from primary, diffuse, intestinal lymphoma. 123 However, diagnosis of this disease by means of endoscopically derived biopsies is improving with more advanced techniques and instrumentation. A wedge biopsy through serosa and muscularis, avoiding the mucosa, may be necessary to establish a diagnosis in cases in which endoscopic samples are equivocal. As an alternative, because nearly half of alimentary lymphomas have secondary mesenteric lymph node involvement, ultrasound-guided biopsy specimens or fine-needle aspirates may be adequate for a diagnosis.

For cats with mediastinal lymphoma, diagnostic suspicion may begin with a noncompressible cranial thorax on physical examination and conformation of a mediastinal mass or pleural effusion on a thoracic radiograph ( Figure 31-14 ). Fine-needle aspiration of any suspected mass or cytologic evaluation of pleural fluid may be sufficient to establish a diagnosis. In most cats the finding of a monotonous population of lymphoblasts establishes a diagnosis. 65 , 124 , 125 However, definitive diagnosis of lymphoma in cats with a mediastinal mass and concurrent chylothorax can be challenging. 114 The CT appearance of these lesions has been evaluated in only a small number of cats with mediastinal masses, and CT generally does not contribute to a definitive diagnosis. 126 If lymphoblasts are not identified in the pleural chylous effusion, cholesterol and triglyceride concentrations can be measured. 127 In chylous effusions, the pleural fluid triglyceride concentration is greater than in the serum; however, anorectic cats have lower triglyceride levels in the pleural fluid. A major differential diagnosis for mediastinal lymphoma is thymoma. The cytologic features of thymoma were recently described, and although these were found to be distinct from lymphoma in many cases, the diagnosis was challenging because of a preponderance of small lymphocytes in thymoma. 128 Mast cells can also be seen in up to 50% of aspirate samples from thymomas.

Diagnostics for most extranodal forms of lymphoma are site specific. In cats suspected of having spinal lymphoma, survey radiographs of the spine rarely reveal osseous lesions. Myelograms, CT, or MRI is indicated, and in approximately 75% of cases, an extradural or intradural mass is detected. 22 , 23 , 83 , 84 Most lesions occur at a thoracolumbar or lumbosacral location. 83 Fluoroscopic-guided, fine-needle aspiration of epidural lesions may yield enough tissue for a cytologic diagnosis. In most of the cats evaluated in one study, CSF analysis revealed a clear, colorless fluid with a mixed pleocytosis (mean, 140 cells/μl; range, 0 to 1625 cells/Ul) and an elevated protein content (mean, 140.7 mg/dl; range, 12 to 405 mg/dl). 23 Malignant lymphocytes were identified in six of 17 cats evaluated with CSF analysis. In a report of intracranial neoplasia, one of two cats with lymphoma had lymphoblasts in the CSF fluid. 84

Bone marrow and renal involvement are common in cats suspected of having CNS lymphoma, and cytologic assessment of these organs generally is easier than in spinal sites. If nasal lymphoma is suspected, a biopsy specimen can be obtained either by intranasal procurement or by flushing one hemicavity with a bulb syringe and saline while occluding the contralateral cavity and collecting samples flushed out the nasopharynx ( Figure 31-15 ). Thorough staging to make sure that the disease is confined to the nasal passages is recommended, because this presentation can be treated locally with radiotherapy if systemic involvement is ruled out. A CT scan is indicated to determine local involvement and to plan radiotherapy if it is to be pursued. For cutaneous lymphoma, punch biopsies (4 to 8 mm) should be taken from the most representative and infiltrative sites, although overtly infected skin lesions should be avoided.

Complete staging to rule out systemic disease is also recommended for cats with cutaneous lymphoma, because local therapies can be applied in cases of solitary disease. In the case of renal lymphoma, physical examination findings of massive and often bilateral renomegaly raise the index of suspicion. The radiographic appearance is a smooth to irregular renomegaly ( Figure 31-16 , A ) that is most consistent with either renal lymphoma or polycystic kidney disease. The disease usually is diffuse throughout the renal cortex ( Figure 31-16 , B ), and transabdominal needle aspiration or core biopsy is diagnostic in most cases.

A WHO staging system exists for the cat that is similar to the one used in the dog (see Box 31-1 ). However, because of the high incidence of visceral involvement in the feline species, another staging system is used more often ( Box 31-5 ). 21 Because lymphoma in cats is more varied with respect to location and anatomic types, staging systems generally are less helpful for predicting response.

Clinical Staging System for Feline Lymphoma

Stage 1

Single tumor (extranodal) or single anatomic area (nodal)

Includes primary intrathoracic tumors

Stage 2

Single tumor (extranodal) with regional lymph node involvement

Two or more nodal areas on the same side of the diaphragm

Two single (extranodal) tumors with or without regional lymph node involvement on the same side of the diaphragm

Resectable primary gastrointestinal tract tumor, usually in the ileocecal area, with or without involvement of associated mesenteric nodes only

Stage 3

Two single tumors (extranodal) on opposite sides of the diaphragm

Two or more nodal areas above and below the diaphragm

All extensive, primary, unresectable, intra-abdominal disease

All paraspinal or epidural tumors, regardless of other tumor site or sites

Stage 4

Stage 1, 2, or 3 tumor or tumors with liver or spleen involvement (or both)

Stage 5

Stage 1, 2, 3, or 4 tumor or tumors with initial involvement of the central nervous system or bone marrow (or both)

Modified from Yunis JJ, Oken MM, Theologides A et al: Recurrent chromosomal defects found in most patients with non-Hodgkin's lymphoma, Cancer Genet Cytogenet 13:17-28, 1984.

Advanced molecular diagnostics

Occasionally the diagnosis of lymphoma and the differentiation of malignant and benign proliferation of lymphocytes is not possible based on standard histologic and cytologic criteria. In these cases, advanced molecular analyses may be helpful for confirming a diagnosis. Clonality is the hallmark of malignancy; that is, the malignant cell population theoretically is derived from expansion of a single malignant clone. This was discussed earlier in the chapter for lymphoma in the dog (see Section A and Figure 31-8 ), and the same assays can be applied for advanced diagnosis of lymphoma in cats. 85 Investigators have used PCR techniques to amplify the variable regions of the T-cell and immunoglobulin receptor genes to detect clonal lymphocyte populations in cats, and these techniques appear to be a useful adjunctive diagnostic tool, although they are undergoing a more thorough assessment. Molecular techniques may also prove useful for assessing early recurrence, for more accurate clinical staging, and for providing so-called molecular remission rates, because they are more sensitive than the standard cytologic assessment of peripheral blood, bone marrow, or lymph nodes.

Treatment of Malignant Lymphoma

Our knowledge base for treating cats with lymphoma is less well established and less predictable than that for dogs, primarily because of the greater variation in histologic type and anatomic locations observed in felines. The chemotherapeutic agents most often used to treat feline lymphoma are similar to those used for dogs and humans with lymphoma (see Section A ); they include doxorubicin, vincristine, cyclophosphamide, methotrexate, L-asparaginase, CCNU, and prednisone. * In general, cats tolerate chemotherapy quite well. Selected published protocols for the treatment of feline lymphoma are detailed in Table 31-8 .

Most current combination protocols in North America are modifications of CHOP protocols initially designed for human oncologic use. In Europe, COP (i.e., without the addition of doxorubicin) is used more often in cats with lymphoma, and one compilation reported results similar to those for CHOP. 133 Some studies with relatively few case entries have reported limited activity for doxorubicin as a single agent in cats with lymphoma 19 , 132 ; however, larger studies using combination protocols have more consistently reported that the addition of doxorubicin is necessary to attain more durable responses. 10 , 129 In European and Australian studies that reported less favorable doxorubicin responses, the patient populations consisted of a higher proportion of the mediastinal form in Siamese cats, a population less frequently included in North American reports. 19 , 133

The protocol the authors use in their practice is presented in Table 31-9 . This protocol has been used in many cats with various forms of lymphoma and is well tolerated. Currently, our canine lymphoma protocols (see Section A ) discontinue chemotherapy by week 25, and we have sufficient data exist to show that in dogs, this short, maintenance-free protocol is as good as if not superior to longer maintenance protocols. Because long-term maintenance protocols have not proved superior to maintenance-free protocols in dogs and humans with lymphoma, the same likely holds true in the cat, although no data exist to document this. Until such time as evidence to the contrary exists, we recommend discontinuation of chemotherapy at week 25 in cats that have attained complete remission.

Although doxorubicin appears to be the most effective agent for treating cats with lymphoma in North America, the species generally is less tolerant of doxorubicin than are dogs, and significant toxicity results when it is used at the dog dosage of 30 mg/m 2 given intravenously every 3 weeks. However, at lower dosages (e.g., 25 mg/m 2 or 1 mg/kg, given intravenously) doxorubicin can be used without significant toxicity. 10 , 129 The major toxicities noted with doxorubicin are anorexia, myelosuppression, renal toxicity and, if perivascular leakage occurs, severe tissue damage. Clinical doxorubicin-induced cardiac toxicity has not been documented in cats, but no information indicates that cats are resistant to myocardial damage. Renal toxicity has been produced experimentally in rats and rabbits and has been reported in cats. 136 , 137 , 138 In our experience, the incidence is significant enough that renal function in cats should be monitored closely (i.e., serial creatinine and urine specific gravity analysis) before each therapy session. A stealth liposomal form of doxorubicin has been shown to increase the likelihood of renal toxicity and therefore is limited in its use in this species. 138 A small number of cats with lymphoma have been treated with single agent CCNU at dosages ranging from 30 to 60 mg/m 2 given orally every 3 to 6 weeks. 130 , 131 Although activity was noted, only partial responses were reported.

Little is known about the treatment of solitary or regional nodes of the head and neck that closely resemble Hodgkin's lymphoma histologically. 1 , 54 One study described the clinical outcome in four cats with this condition; these researchers found that the course of disease was prolonged, but after surgical excision of the single affected node, only one cat showed recurrence 6 months after surgery. 54 A second surgery was performed in this cat, and a second recurrence was noted, again 6 months after excision.

Cats with granular cell lymphoma or globule leukocyte tumors tend to respond poorly to chemotherapy, although durable responses have been reported. 102 Currently, too few cases have been treated with aggressive chemotherapy to allow an assessment of response to therapy for this disease.

A distinct form of intestinal lymphoma in cats that is composed of small, mature lymphocytes has been referred to as lymphocytic lymphoma . This syndrome has been reported to respond well to oral prednisone (10 mg/cat/day) and chlorambucil (15 mg/m 2 given once daily for 4 consecutive days out of every 3 weeks). A complete response rate of 69% was reported, with median remission and survival durations of 16 and 17 months, respectively. 62

Radiation therapy has been used effectively to treat localized lymphoma, such as epidural, mediastinal, and nasal lymphoma. Total doses of 10 to 15 Gy usually result in a complete remission. 77 In one study, 10 cats with localized lymphoma were treated with radiation, alone or with chemotherapy, at doses of 8 to 40 Gy. 78 Eight of the 10 cats achieved complete remission, with a median remission duration of just over 2 years.

Radiation therapy also has been used effectively to treat nasal lymphoma. 78 Complete response is the norm (80% to 100%), and median remission durations exceeding 1.5 years can be expected in FeLV-negative cats with disease confined to the nasal and paranasal cavities. For nasal lymphoma, radiotherapy appears to be superior to chemotherapy, for which reported median remission durations are 151 to 380 days. 10

Very little has been published about the treatment of cutaneous lymphoma or mycosis fungoides in cats; however, a report of a complete response to CCNU exists. 135 Cats with a solitary mass should be treated with surgical excision, although clinical staging is necessary to rule out possible internal involvement. Combination chemotherapy can be considered if multiple sites are involved. If the disease is localized to a small region, radiation therapy usually is also effective. Mycosis fungoides may be treated effectively in dogs with retinoids, such as isotretinoin (Accutane) at a dosage of 3 to 4 mg/kg given orally daily or etretinate (Tegison) at a dosage of 1.25 mg/kg given orally daily; however, no clinical studies have been published demonstrating efficacy in cats.

General Treatment Recommendations

Providing precise treatment recommendations for the wide variety of clinical settings of cats with lymphoma is difficult. Our current recommendation is to treat cats with lymphoma using the CHOP- based protocols (see Table 31-9 ). Doxorubicin alone (25 mg/m 2 given IV every 3 weeks for five total treatments) or palliative prednisone therapy is offered if the client declines more aggressive therapy.

Nutritional support is especially important, particularly for cats with alimentary lymphoma. A feeding tube may need to be placed in cats undergoing chemotherapy for alimentary lymphoma, particularly if anorexia is a factor (see Chapter 16, section B.)

Ultimately, most cats with lymphoma that are successfully treated with chemotherapy have a relapse of the disease. This often represents a recrudescence of the tumor in a more drug-resistant form. At the first recurrence, reinduction first should be attempted by using the induction protocol that was successful initially. In general, the likelihood of a response and the length of the response are half those for the initial therapy; however, a subset of animals enjoy long-term reinduction.

If reinduction fails or if the cat does not respond to the initial induction, so-called rescue agents or rescue protocols can be attempted. These are drugs or drug combinations that typically are not found in the standard CHOP protocol and are withheld for use in drug-resistant cases. A number of rescue protocols have been reported in the veterinary literature and were reviewed previously. 139 , 140 The most common rescue protocols advocated for cats with resistant relapse include single agent use or combination use of mitoxantrone, doxorubicin (if doxorubicin was not part of the original induction protocol), CCNU, and MOPP. In general, overall rescue response rates of 40% to 50% are reported, but these responses usually are not durable; median responses of 1.5 to 2 months are the norm. A small subset of animals enjoy longer rescue durations.

Prognosis

In general, cats do not enjoy response rates as high or remission and survival durations as long as dogs with lymphoma. Complete response rates range from 50% to 70% after combination chemotherapy, and overall median remission and survival durations are approximately 4 and 6 months, respectively. 1 , 5 , 6 , 10 , 129 However, a significant proportion of cats (25% to 30%) that achieve a complete response with combination chemotherapy enjoy more durable overall remission and survival times (i.e., 1 year or longer).

The response rate and the length of response vary according to the presence or absence of several prognostic factors. The wide variation in frequency and the great heterogeneity of anatomic forms of lymphoma in cats make specific prognostications more difficult than in dogs with lymphoma. Most studies have lumped anatomic groups together to produce remission and survival data, and individual group numbers generally have been too small to apply statistical analysis with meaningful power. That being said, the factors that appear to be most strongly associated with a more positive prognosis in cats are a complete response to therapy ( Figure 31-17 ), which unfortunately cannot be determined before treatment; negative FeLV status ( Figure 31-18 ); early clinical stage ( Figure 31-19 ); anatomic location; and addition of doxorubicin to the treatment protocol. 5 , 10 , 24 , 129 Unlike in the dog, CD3 immunoreactivity has not been established as a negative prognostic factor in the cat. 10 Early reports may contradict more recent studies partly because of the decline in FeLV-associated lymphoma, and patients reported in the early literature may not equate to more recent populations studied. In general, FeLV-negative cats that achieve a complete response on CHOP-based protocols have a high likelihood of long-term survival, with approximately 30% alive at 1.5 years after diagnosis.

Location-specific prognosis

For cats with alimentary lymphoma, median survival times of 7 to 10 months are expected with chemotherapy that includes doxorubicin. 10 , 26 , 129 In one study of 28 cats with alimentary lymphoma in which the treatment protocol did not include doxorubicin, the median survival time was only 50 days. 25

Mediastinal lymphoma in FeLV-positive young cats is associated with the poorest prognosis, and survivals times of approximately 2 to 3 months are expected with chemotherapy. 6 , 10 , 19 In contrast, older, FeLV-negative Siamese cats with mediastinal lymphoma appear to experience remission rates approaching 90%, and responses tend to be quite durable.

Overall, cats with nasal lymphoma have the best prognosis, because local radiotherapy (or chemotherapy if radiotherapy is not available) generally results in excellent control, with median survival times approaching 1.5 years. 10 , 78 However, cats with nasal lymphoma and concurrent FeLV infection have much shorter survival durations.

Renal lymphoma is associated with a shorter survival time, with published medians ranging from 3 to 6 months. 1 , 6 , 10 , 76 In a study by Mooney and colleagues, 76 28 cats with primary renal lymphoma were treated with combination chemotherapy. A complete response (CR) was noted in 17 cats (61%), and nine (32%) had a partial response. The median survival times were 4 months for the cats that showed a complete response and 1 month for those that showed a partial response. The duration of response to chemotherapy did not correlate with the degree of renal insufficiency except in cats with a BUN higher than 150 mg/dl. Extension to the CNS occurred in 40% of these treated cats. The investigators revised their chemotherapy protocol to include cytosine arabinoside, which can penetrate the blood barrier, theoretically to prevent or reduce CNS metastasis; however, definitive improvement with this modification has not been documented.

Few studies have reported on the results of treatment of cats with spinal lymphoma. In one study of four cats treated with chemotherapy (L-asparaginase, vincristine, and prednisone) combined with spinal radiation (n = 3) or surgical cytoreduction (n = 1), most of the cats were euthanized by 5 months, although one cat survived 13 months. 23 In another study of nine cats treated with chemotherapy (vincristine, cyclophosphamide, and prednisone), three cats achieved a complete response with a duration of 14 weeks and three achieved a partial response with a duration of 6 weeks. 22 One cat treated with dorsal decompression laminectomy and chemotherapy survived 13 months. Although the numbers are small overall, the prognosis for spinal lymphoma is poor.

FELINE LEUKEMIAS, MYELOPROLIFERATIVE DISORDERS, AND MYELODYSPLASIA

Section C of this chapter presents a complete discussion of leukemias and MPDs, including a general discussion of hematopoiesis, etiologies, lineage classification, and descriptions.

The classification of leukemias in cats is difficult because of the similarity of clinical and pathologic features and the transition, overlap, or mixture of cell types involved. 110 , 141 , 142 , 143 , 144 , 145 , 146 , 147 , 148

Leukemia is a neoplastic proliferation of hematopoietic cells that originate in the bone marrow. Cell lineage includes myeloid cells, neutrophils, basophils, eosinophils, monocytes, lymphoid cells, megakaryocytes, and erythrocytes. Box 31-6 presents a classification scheme for the leukemias identified in the cat. Leukemias also must be classified according to their degree of differentiation. 110 Well-differentiated leukemia is referred to as chronic leukemia, and poorly differentiated leukemia usually is called acute leukemia; this distinction is very important in the therapeutic management and prognosis of leukemias. Cats with acute leukemia usually show signs of severe anemia (pale mucous membranes), splenomegaly, and febrile episodes. Cats with chronic leukemia may have a longer duration of signs and mild anemia with or without splenomegaly.

Classification of Leukemia in Cats

Myeloproliferative disorders

Granulocytes

Granulocytic (myeloid, neutrophilic) leukemia

Eosinophilic leukemia

Basophilic leukemia

Monocytes

Monocytic leukemia

Erythrocytes

Erythremic myelosis

Primary erythrocytosis

Megakaryocytes

Megakaryocytic leukemia (myelosis)

Primary thrombocythemia

Miscellaneous

Mast cell leukemia

Myelofibrosis

Malignant histiocytosis

Undifferentiated leukemia

Mixed cell lines

Myelomonocytic leukemia (neutrophils and monocytes)

Erythroleukemia (erythrocytes and granulocytes)

Myelodysplastic syndrome

Lymphoproliferative disorders

Lymphocytes

Acute lymphoblastic leukemia

Chronic lymphocytic leukemia

In cats suspected of having leukemia, bone marrow aspiration or biopsy usually is diagnostic. The preferred sites for bone marrow aspiration are the proximal humerus and the iliac crest. Cats with acute leukemia are likely to have malignant cellular infiltrates in organs other than the bone marrow. 144 The bone marrow aspirate must contain more than 30% abnormal blast cells to support a diagnosis of an acute leukemia. In cats suspected of having CLL, infiltration of the bone marrow with more than 20% mature lymphocytes helps confirm the diagnosis. All cats with leukemia should be tested for FeLV and FIV. Determining the lineage of some leukemias can be challenging; most can be distinguished from one another by histologic appearance, histochemical stains, immunohistochemical analysis of cell surface antigens, and flow cytometric analysis of the leukemic cells for cellular antigens that identify their lineage. 141 , 143 , 149 , 150 In addition, examination of blast cells by electron microscopy may reveal characteristic ultrastructural features. The French-American-British (FAB) classification system ( Box 31-7 ) is considered useful in cats with myelodysplastic syndromes, and almost all these patients are FeLV antigenemic. 151 , 152

French-American-British (FAB) Classification System for Subtypes of Leukemias and Dysplasias

Acute leukemias

Chronic myeloid leukemias

Hematopoietic dysplasia

Lymphoid Leukemia

Lymphoid leukemia is the most common leukemia in cats. Approximately 25% of cats with lymphoma also have a leukemic blood profile. 153 ALL is the most common lymphoid leukemia. 154 ALL is characterized by the presence of poorly differentiated lymphoblasts and prolymphocytes in the blood and bone marrow. 155 Most cats with ALL have normal to low WBC counts. A few cats have leucocytosis with circulating blasts. A moderate to marked anemia is common. Bone marrow aspiration usually reveals extensive infiltration with lymphoblasts. Approximately 60% to 80% of cats with ALL are FeLV positive, and most malignant cells have a T-cell phenotype. 156

CLL, which is rarely reported in cats, is characterized by the presence of well-differentiated, small, mature lymphocytes in the peripheral blood and bone marrow. 155 , 156 Although most of these cells are of the T-cell lineage (particularly T helper), B-cell CLL has been reported. 150 , 157 , 158 Most cats with CLL have an elevated WBC count (i.e., over 50,000/μl), and most are FeLV negative.

Granulocytic Leukemia (Myeloid, Neutrophilic) 141 , 142 , 143 , 144

The total leukocyte count varies and may range from leukopenia to marked leukocytosis. Chronic granulocytic leukemia (GL) must be distinguished from leukemoid reactions associated with infections. Acute GL is characterized by a large percentage of myeloblasts and/or progranulocytes in the blood and bone marrow. Myeloblasts can be difficult to distinguish from lymphoblasts, but the former have a finer chromatin pattern, a smaller nucleus-to-cytoplasm ratio, more prominent or multiple nucleoli, and sometimes cytoplasmic granules. It is not uncommon in cats with GL to have no recognizable neoplastic cells in the peripheral blood. The bone marrow is hypercellular as a result of granulocytic leukemia cells.

Myelomonocytic Leukemia 141 , 145 , 146 , 159

Myelomonocytic leukemia (MML) results from malignant transformation of both neutrophils and monocytes. This form of leukemia is one of the most common forms reported.

Monocytic Leukemia 143 , 146 , 147

Monocytic leukemia (ML) is a rarely reported leukemia. It generally is considered an acute leukemia regardless of the morphologic appearance of the cells.

Eosinophilic Leukemia

Eosinophilic leukemia (EL) is rarely diagnosed in cats and is considered a variant of chronic granulocytic or myeloid leukemia. 148 , 160 FeLV has induced EL experimentally. 160 Mature eosinophils outnumber immature stages, and anemia is uncommon in cats associated with EL. Cats usually have an eosinophil count of 15,000 cells/μl or higher with or without immature cells in the peripheral blood. The bone marrow shows hyperplasia of eosinophilic precursors, and the myeloid to erythroid ratio (M:E) is significantly increased. Organ infiltration, such as in lymph nodes, the spleen, and the liver, can be seen. 161 In establishing a diagnosis of EL, it is important to rule out eosinophilic enteritis, parasitism, eosinophilic granuloma complex, and allergic disorders. Diagnosing EL can be very difficult because of the hypereosinophilic syndrome (HES) seen with other disease conditions in cats. 161 , 162 , 163 HES is characterized by a marked increase in the eosinophil count, bone marrow hyperplasia of eosinophilic precursors, and multiple organ infiltration by mature eosinophils. Most cats have signs related to GI involvement.

Basophilic Leukemia 164

Basophilic leukemia (BL) is considered a variant of chronic granulocytic leukemia. Only one confirmed case of basophilic leukemia has been reported in the cat. It is important to differentiate BL from systemic mastocytosis with mast cell leukemia. Mast cells have numerous cytoplasmic granules and round nuclei. Basophils have segmented nuclei and cytoplasmic granules that superimpose on the nucleus, giving it a moth-eaten appearance.

Erythremic Myelosis 165 , 166 , 167 , 168

Erythremic myelosis (EM) is an MPD characterized by excessive proliferation of erythroid elements, which results in an increase in nucleated erythrocytes (rubriblasts to metarubricytes). Severe anemia is common, and the peripheral blood shows numerous nucleated erythrocytes, moderate to marked anisocytosis, and an increased erythrocyte mean cell volume. The bone marrow contains a preponderance of normal-appearing erythrocyte precursors. Some cats undergo blast transformation to myeloblastic, granulocytic, or a poorly differentiated leukemia (previously called reticuloendotheliosis). The transition from erythremic myelosis to erythroleukemia to acute granulocytic leukemia is well recognized in humans. 169

Erythroleukemia (Reticuloendotheliosis) 110 , 143 , 146

Erythroleukemia, or acute erythremic myelosis, can develop from blast transformation of erythremic myelosis. Primitive erythroid precursors in the blood and bone marrow predominant. Primitive cells resembling myeloblasts often are present in low numbers.

Primary Erythrocytosis 170 , 171 , 172 , 173

Primary erythrocytosis is rarely reported in cats, and the diagnosis is based on an elevated PCV (65% to 80%) with low to normal serum erythropoietin activity. Most clinical signs are associated with an increased red blood cell (RBC) volume, which increases blood volume and viscosity, causing impaired blood flow, stasis, and tissue hypoxia. Neurologic signs such as seizures, blindness, and mental depression are common. 172 The oral mucous membranes may appear brick red. It is important to differentiate this condition from secondary polycythemia caused by renal tumors, chronic hypoxia, and right-to-left cardiac shunts.

Megakaryocytic Leukemia 174 , 175 , 176

Megakaryocytic leukemia is characterized by abnormal megakaryocytic hyperplasia in the bone marrow. The megakaryocytes are morphologically abnormal, and some are small (dwarf megakaryocytes) and have few or no nuclear lobulations. Thrombocytopenia or thrombocytosis may be present. In humans this form of leukemia often is associated with extensive marrow fibrosis and an increase in reticulum or collagen. 169

Primary Thrombocythemia 177

Primary thrombocythemia is a very rare, chronic MPD characterized by proliferation of megakaryocytes and elevated platelet counts exceeding 1 million. Giant platelets and platelets with abnormal morphology may be seen in the peripheral blood. One case has been reported in the cat.

Malignant Histiocytosis 178 , 179 , 180 , 181 , 182

Malignant histiocytosis, a rare condition in cats, is characterized by systemic proliferation of malignant macrophages (histiocytes) and their precursors. A distinguishing characteristic of this disease is erythrophagocytosis. Hepatosplenomegaly with progressive anemia (sometimes Coombs positive) and thrombocytopenia are characteristic. The erythrophagocytosis may be confused with a possible immune-mediated anemia. Bone marrow biopsy, rather than aspiration, and splenic biopsy may be necessary to establish a diagnosis. Special stains using acid phosphatase and nonspecific esterase with fluoride inhibition (naphthol butyrate substrate) may be necessary to indicate macrophage origin.

Myelofibrosis and Myeloid Metaplasia 183 , 184 , 185 , 186

Myelofibrosis and myeloid hyperplasia are characterized by abnormal growth and differentiation of erythroid, myeloid, and megakaryocytic cell types with varying proliferation of fibroblasts in the marrow. Anemia, leukopenia, or thrombocytopenia or varying combinations are common. Myelofibrosis has been diagnosed in FeLV-positive cats and is directly associated with the virus rather than a consequence of myeloproliferative disorders. Myeloid metaplasia may terminate in acute leukemia and therefore may be considered a preleukemic event.

Treatment of Leukemias

Lymphoid leukemia

The use of chemotherapy to treat ALL has been disappointing. A 27% complete response rate has been reported with a COP regimen. 1 , 10 , 138 , 187 In 15 cats treated with COP for ALL, four achieved a CR and 6 cats had a partial response. 1 The median remission was 7 months (range, 1 to 24 months). One report described a short-term remission in a cat with lymphoid leukemia that was treated with a low dose of cytosine arabinoside (10 mg/m 2 given subcutaneously twice daily). 188

CLL can be treated with chlorambucil (0.2 mg/kg given orally or 2 mg/cat given every other day) and prednisone at a dose of 1 mg/kg given orally daily. Cats with CLL have a better prognosis and survive 1 to 2 years when treated with chlorambucil. As in humans and dogs, treatment can be withheld if the patient has no significant clinical signs and no profound cytopenias. In one study, a cat with CLL remained stable without chemotherapy for over a year. 158

Nonlymphoid leukemia

The prognoses for acute nonlymphoblastic leukemias generally are very poor, although some exceptions exist. A treatment regimen consisting of a combination of cytosine arabinoside and cyclophosphamide and multiple blood transfusions was effective at inducing a response for 3 months in a cat with acute megakaryocytic leukemia. 176 Hydroxyurea (Hydrea) can be used to treat chronic myeloid leukemia and primary erythrocytosis. Cats with primary erythrocytosis that go untreated are reported to survive 6 to 20+ weeks. 170 , 172 Phlebotomy alone every 2 to 3 months was used to treat one cat, which survived longer than 20 months. 171 Hydroxyurea treatment for primary erythrocytosis was used in eight cats, and all survived longer than 1 year. 172 Hydroxyurea is available in 500 mg capsules, and the dosage is 25 to 50 mg/kg given orally daily. Some cats have been given 500 mg every 5 to 7 days, but methemoglobulinemia and hemolytic anemia with Heinz bodies has been seen. 172 A better recommendation is to have the drug formulated into 125 mg capsules, which is a more appropriate dosage. However, care must be used in making these capsules because hydroxyurea is potentially carcinogenic. A recommended treatment schedule for hydroxyurea is 125 mg daily to every other day, depending on the type of leukemia under treatment. The drug is tolerated very well at this dosage. A case of acute ML in a cat undergoing treatment with cytarabine, doxorubicin, vincristine, and prednisolone has been reported; a partial remission was noted for approximately 2 months, and the cat survived for 3 months after diagnosis. 147

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Canine Acute Myeloid Leukemia, Chronic Myeloproliferative Diseases, and Myelodysplasia

Karen M. Young , David M. Vail

Myeloproliferative diseases are a group of neoplastic diseases of bone marrow marked by unregulated clonal proliferation of hematopoietic stem cells. 1 Excessive proliferation of cells with defective maturation and function leads to reduction of normal hematopoiesis and invasion of other tissues. MPDs have been classified based on the biologic behavior, the degree of cellular differentiation, and the lineage of the neoplastic cells (granulocytic, monocytic, erythroid, megakaryocytic, or mixed), and newer classification systems in humans have incorporated genetics. In 1991 the Animal Leukemia Study Group made recommendations for the classification of nonlymphoid leukemias in dogs and cats. 2 More recently, the Oncology Committee of the American College of Veterinary Pathologists (ACVP) established a Myeloid Neoplasm Subcommittee to re-examine criteria for a classification system and to spearhead large multi-institutional studies to validate the criteria.

Incidence and Risk Factors

MPDs are uncommon or rare in the dog; they occur 10 times less frequently than lymphoproliferative disorders. 3 Accurate information about the incidence and other epidemiologic information await consistent use of a uniform classification system (see later discussion). No known age, breed, or gender predisposition exists, although large breed dogs have been overrepresented in some retrospective studies. 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 In dogs, the etiology of spontaneously occurring leukemia is unknown. Genetic and environmental factors (including exposure to radiation, drugs, or toxic chemicals) probably play a role. In humans, acquired chromosomal derangements lead to clonal overgrowth with arrested development. 13 Chromosomal abnormalities have been reported in dogs with acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), and lymphoid leukemia. 14 , 15 However, because karyotyping is difficult to perform in dogs, owing to the large number and morphologic similarity of their chromosomes and their resistance to banding, definition of the genetic factors in canine MPDs awaits application of molecular techniques and use of the canine genome map. 14 , 16 , 17 Certain forms of leukemia in dogs have been produced experimentally by irradiation. 18 , 19 , 20 In contrast to MPDs in cats, no causative viral agent has been demonstrated in dogs, although retrovirus-like budding particles were observed in the neoplastic cells of a dog with granulocytic leukemia. 21

Pathology and Natural Behavior

A review of normal hematopoiesis can aid the clinician's understanding of the various manifestations of MPDs. Hematopoiesis is the process of proliferation, differentiation, and maturation of stem cells into terminally differentiated blood cells (a simplified schematic is presented in Figure 31-20 ). Pluripotent stem cells differentiate into either lymphopoietic or hematopoietic multipotent stem cells. 22 Under the influence of specific regulatory and microenvironmental factors, multipotent stem cells in bone marrow differentiate into progenitor cells committed to a specific hematopoietic cell line, such as erythroid, granulocytic-monocytic, or megakaryocytic cells. Maturation results in the production of terminally differentiated blood cells—erythrocytes, granulocytes, monocytes, and platelets—that are delivered to the circulation. In some cases, as in the maturation of reticulocytes to erythrocytes, final development may occur in the spleen.

The proliferation and differentiation of hematopoietic cells are controlled by a group of regulatory growth factors. 22 , 23 Of these, erythropoietin is the best characterized; it regulates erythroid proliferation and differentiation and is produced in the kidneys, where changes in oxygen tension are detected. The myeloid compartment depends on a group of factors, collectively referred to as colony-stimulating factors (CSFs) . These factors act at the level of the committed progenitor cells but also influence the functional capabilities of mature cells. Some of these factors have a broad spectrum of activity; others are more restricted in their target cells and actions. CSFs are produced in vitro by a multitude of cell types, including monocytes, macrophages, lymphocytes, and endothelial cells, and these cells likely play a role in the production and regulation of these factors in vivo . The gene for thrombopoietin also has been cloned, and this hormone alone apparently can induce differentiation of megakaryocytes and platelet production. 24 Recombinant forms of many of these hormones are becoming increasingly available.

The clonal disorders of bone marrow include myeloaplasia (usually referred to as aplastic anemia ), myelodysplasia, and myeloproliferation. A preleukemic syndrome, characterized by peripheral pancytopenia and bone marrow hyperplasia with maturation arrest, is more correctly called myelodysplasia because the syndrome does not always progress to overt leukemia. This syndrome has been described in cats, usually in association with FeLV infection, but it has only rarely been recognized in dogs. 25 , 26 , 27 , 28 MPDs may be manifested by abnormalities in any or all of the different cell lines, because hematopoietic cells share a common stem cell. In addition, transformation from one MPD to another may occur. 29

MPDs are classified in several ways. The terms acute and chronic refer to the degree of cellular differentiation of the leukemic cells, but these terms also correlate with the biologic behavior of the neoplasm. 30 Disorders resulting from uncontrolled proliferation of cells incapable of maturation lead to the accumulation of poorly differentiated, or blast, cells. These disorders have been called acute MPDs or acute nonlymphocytic leukemias, but they now are included under the umbrella term acute myeloid leukemia . Disorders resulting from unregulated proliferation of cells that exhibit progressive, albeit incomplete and defective, maturation lead to the accumulation of differentiated cells. These disorders are called chronic MPDs . They include polycythemia vera, CML and its variants, essential thrombocythemia, and possibly primary myelofibrosis. MPDs are further classified by the lineage of the dominant cell type or types, as defined by Romanowsky stains, special cytochemical stains, ultrastructural features, and immunologic cell markers, and they recently have been classified into subtypes (see later discussion).

Acute leukemias have a more sudden onset and are more aggressive. In both acute and chronic disorders, however, abnormalities in proliferation, maturation, and functional characteristics can occur in any hematopoietic cell line. 1 In addition, normal hematopoiesis is adversely affected. Animals with leukemia usually have decreased numbers of circulating normal cells. The pathogenesis of the cytopenias is complex and may result in part from production of inhibitory factors. Eventually, neoplastic cells displace normal hematopoietic cells, a process called myelophthisis . Anemia and thrombocytopenia are particularly common. Neutropenia and thrombocytopenia result in infection and hemorrhage, which may be more deleterious to the animal than the primary disease process. Despite the disseminated nature of the disease at the time of diagnosis, parenchymal organ dysfunction usually occurs only in very advanced cases of MPD.

ACUTE MYELOID LEUKEMIA

AML is rare and is characterized by aberrant proliferation of a clone of cells without maturation. This results in the accumulation of immature blast cells in the bone marrow and peripheral blood ( Figure 31-21 ). The WBC count varies, ranging from leukopenia to counts greater than 150,000/μl. The spleen, liver, and lymph nodes commonly are involved, and other tissues may be infiltrated as well, including the tonsils, kidneys, heart, and CNS. No characteristic age has been noted, and even very young dogs may be affected. 31 The clinical course of these disorders tends to be rapid. Production of normal peripheral blood cells usually is diminished or absent, and anemia, neutropenia, and thrombocytopenia are common. Infection and hemorrhage are common sequelae. Occasionally, malignant blasts are present in the bone marrow but not the peripheral blood; this is called aleukemic leukemia . Subleukemic leukemia suggests a normal or decreased WBC with some neoplastic cells in circulation.

In 1985 the Animal Leukemia Study Group was formed under the auspices of the American Society for Veterinary Clinical Pathology to develop specific morphologic and cytochemical criteria for classifying acute nonlymphocytic leukemias in dogs and cats. Recognition of specific subtypes of leukemia is necessary for the accumulation of accurate, useful information about prognosis and response to treatment and for comparison of studies from different sites. In 1991 this group proposed a classification system following adaptation of the French-American-British (FAB) system and criteria established by the National Cancer Institute Workshop. 2 The group members examined blood and bone marrow from 49 dogs and cats with MPDs. Romanowsky-stained specimens were examined first to identify blast cells and their percentages. Lineage specificity then was determined using cytochemical markers. The percentage of blasts and the information about lineage specificity were used in combination to classify disorders as acute undifferentiated leukemia (AUL), acute myeloid leukemia (AML, subtypes M1 to M5 and M7), and erythroleukemia with or without erythroid predominance (subtypes M6 and M6Er) (see Box 31-7 ).

Except for acute promyelocytic leukemia (subtype M3), all these subtypes have been described in dogs. However, because this modified FAB system has only recently been adopted, the names given to these disorders in the literature vary considerably. In addition, in the absence of special cytochemical staining, immunophenotyping, and/or electron microscopy, the specific subtype of leukemia often has been uncertain, making retrospective analysis of epidemiologic information, prognosis, and response to therapy confusing at best.

Although defining specific subtypes may seem to be an academic exercise owing to the uniformly poor prognosis of acute leukemias, this information is critical to improving the management of these diseases. Because of the low incidence of MPDs, national and international cooperative efforts are required to accumulate information on the pathogenesis of specific subtypes and their response to different treatment modalities; use of a uniform classification system is an essential first step.

Different forms of AML are shown in Figure 31-21 , A - E . The most frequently reported forms of AML in the dog are acute myeloblastic leukemia (M1 and M2) and acute myelomonocytic leukemia (M4). 3 , 4 , 5 , 6 , 7 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 Megakaryoblastic leukemia (M7) also is well recognized in dogs 8 , 40 , 41 , 42 , 43 , 44 , 45 , 46 and may be associated with platelet dysfunction. 44 Monocytic leukemias likely have included those with and without monocytic differentiation (M5a and M5b), 9 , 47 but in some cases the diagnosis may have been chronic myelomonocytic or chronic monocytic leukemia (see later discussion). There are few reports in dogs of spontaneously occurring erythroleukemia (M6) in which the leukemic cells comprise myeloblasts, monoblasts, and erythroid elements. 48 , 49 AULs have uncertain lineages, because they are negative for all cytochemical markers. These leukemias should be distinguished from lymphoid leukemias by flow cytometric analysis of the leukemic cells for cellular antigens that identify their lineage. 50 In addition, examination of blast cells by electron microscopy may reveal characteristic ultrastructural features.

CHRONIC MYELOPROLIFERATIVE DISEASES

Chronic MPDs are characterized by excessive production of differentiated bone marrow cells, which results in the accumulation of erythrocytes (polycythemia vera), granulocytes and/or monocytes (chronic myelogenous leukemia and its variants), or platelets (essential thrombocythemia). Primary myelofibrosis as a clonal disorder of marrow stromal cells, characterized by proliferation of fibroblasts with accumulation of collagen in bone marrow, is not recognized in animals. Myelofibrosis is considered a response to injury and may occur secondary to MPDs.

Polycythemia Vera

Polycythemia vera (PV) is a clonal disorder of stem cells, although whether the defect is in the pluripotent stem cell or the hematopoietic multipotent stem cell is still unclear. In humans, progenitor cells have an increased sensitivity to insulin-like growth factor 1 (IGF-1), which stimulates hematopoiesis. 51 Whether this hypersensitivity is the primary defect or occurs secondary to another gene mutation is unknown. In any case, the result is overproduction of RBCs. The disease is rare and must be distinguished from more common causes of polycythemia, including relative and secondary absolute polycythemia (see later discussion). In PV, neoplastic proliferation of the erythroid series with terminal differentiation to RBCs occurs. The disease has been reported mostly in middle-aged dogs, and no breed or gender predilection has been noted. 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 PV is characterized by an increased RBC mass, evidenced by an elevated PCV, RBC count, and hemoglobin concentration. The PCV typically is in the range of 65% to 85%. The bone marrow is hyperplastic, although the M:E tends to be normal. In contrast to the disease in humans, other cell lines do not appear to be involved, and transformation to other MPDs has not been reported. The disease in dogs may be more appropriately called primary erythrocytosis.

Chronic Myelogenous Leukemia

CML is a neoplastic proliferation of the neutrophil series, although concurrent eosinophilic and basophilic differentiation can occur. CML can occur in dogs of any age. 31 , 61 , 62 , 63 , 64 , 65 Neutrophils and neutrophilic precursors accumulate in the bone marrow and peripheral blood and invade other organs. The peripheral WBC count usually, but not always, is higher than 100,000/μl. Both immature and mature neutrophils are present (see Figure 31-21 , F ). Mature forms usually are more numerous, but sometimes an “uneven” left shift is present. Signs of dysplasia may be evident, including hypersegmentation, ringed nuclei, and giant forms. Eosinophils and basophils may also be increased. The bone marrow is characterized by granulocytic hyperplasia, and morphologic abnormalities may not be present. Erythroid and megakaryocytic lines may be affected, resulting in anemia, thrombocytopenia or, less commonly, thrombocytosis.

CML must be distinguished from severe neutrophilic leukocytosis and “leukemoid reactions” caused by inflammation or immune-mediated diseases. Leukemoid reactions also can occur as a paraneoplastic syndrome. In humans with CML, characteristic cytogenetic abnormalities are present in all bone marrow cells, signifying a lesion at the level of an early multipotent stem cell. Typically these individuals have a chromosomal translocation, resulting in the Philadelphia chromosome. 66 No consistent cytogenetic abnormalities have been demonstrated in spontaneously occurring CML in dogs. Variants of CML are chronic myelomonocytic leukemia (CMML) and chronic monocytic leukemia (CMoL). These diagnoses are made based on the percentage of monocytes in the leukemic cell population.

Besides accumulating in the bone marrow and peripheral blood, leukemic cells invade the red pulp of the spleen, the periportal and sinusoidal areas of the liver, and sometimes the lymph nodes. Other organs (e.g., the kidneys, heart, and lungs) are less commonly affected. In addition, extramedullary hematopoiesis may be present in the liver and spleen. Death usually results from complications of infection or hemorrhage secondary to neutrophil dysfunction and thrombocytopenia. In some cases, CML may terminate in “blast crisis,” in which a transformation occurs from a predominance of well-differentiated granulocytes to excessive numbers of poorly differentiated blast cells in the peripheral blood and bone marrow; this phenomenon is well documented in the dog. 61 , 62 , 64

Basophilic and Eosinophilic Leukemia

Basophilic leukemia, although rare, has been reported in dogs and is characterized by an elevated WBC count with a high proportion of basophils in the peripheral blood and bone marrow. 67 , 68 , 69 Hepatosplenomegaly, lymphadenopathy, and thrombocytosis may be present, and these dogs have all been anemic. Basophilic leukemia should be distinguished from mast cell leukemia (mastocytosis). Whether dogs develop eosinophilic leukemia remains in question. Reported cases have had high blood eosinophil counts and eosinophilic infiltrates in organs. 70 , 71 One dog responded well to treatment with corticosteroids. The distinction between neoplastic proliferation of eosinophils and idiopathic hypereosinophilic syndrome remains elusive. Disorders associated with eosinophilia (e.g., parasitism, skin diseases, and diseases of the respiratory and GI tracts) should be considered first in an animal with eosinophilia. One distinguishing feature should be clonality, with reactive eosinophilia comprising polyclonal cells and the neoplastic condition arising from a single clone. As clonality assays become more available, this discrepancy may be resolved.

Essential Thrombocythemia

In humans, essential thrombocythemia, or primary thrombocytosis, is characterized by platelet counts that are persistently higher than 600,000/μl. No blast cells are in circulation, and marked megakaryocytic hyperplasia of the bone marrow without myelofibrosis is present. Thrombosis and bleeding are the most common sequelae, and most patients have splenomegaly. Other MPDs, especially PV, should be ruled out, and the patient should have no primary disorders associated with reactive thrombocytosis, 72 such as inflammation, hemolytic anemia, iron deficiency anemia, malignancies, recovery from severe hemorrhage, rebound from immune-mediated thrombocytopenia, or abscence of a spleen. In addition, certain drugs, such as vincristine, can induce thrombocytosis.

Essential thrombocythemia has been recognized in dogs. 29 , 73 , 74 , 75 , 76 In one dog, the platelet count exceeded 4 million/μl, and bizarre giant forms with abnormal granulation were present. The bone marrow contained increased numbers of megakaryocytes and megakaryoblasts, but circulating blast cells were not seen. Other findings included splenomegaly, GI bleeding, and increased numbers of circulating basophils. Causes of secondary or reactive thrombocytosis were ruled out. 73

Basophilia also was reported in a more recent case. 75 In another dog, primary thrombocytosis was diagnosed and then progressed to CML. 29 In some cases reported in the literature as essential thrombocythemia, the dogs had microcytic hypochromic anemias. Because iron deficiency anemia is associated with reactive or secondary thrombocytosis, care must be taken to rule out this disorder. However, spurious microcytosis may be reported if a dog has many giant platelets that are counted by an analyzer as small RBCs. 76 Microscopic review of the blood film may be helpful in these cases.

OTHER BONE MARROW DISORDERS

Myelofibrosis

Primary myelofibrosis with clonal proliferation of marrow fibroblasts has not been reported in dogs. 77 In humans, myelofibrosis is characterized by collagen deposition in the bone marrow and increased numbers of megakaryocytes, many of which have morphologic abnormalities. In fact, breakdown of intramedullary megakaryocytes and subsequent release of factors that promote fibroblast proliferation or inhibit collagen breakdown may be the underlying pathogenesis of the fibrosis. 78 Focal osteosclerosis sometimes is present. Anemia, thrombocytopenia, splenomegaly, and myeloid metaplasia (production of hematopoietic cells outside the bone marrow) are consistent features. The extramedullary hematopoiesis is ineffective at maintaining or restoring normal peripheral blood counts.

In dogs, myelofibrosis occurs secondary to MPDs, radiation damage, and congenital hemolytic anemias. 79 , 80 , 81 , 82 In some cases the inciting cause is unknown (idiopathic myelofibrosis). Concurrent marrow necrosis may occur in cases of ehrlichiosis, septicemia, or drug toxicity (estrogens, cephalosporins), and some speculate that fibroblasts proliferate in response to release of inflammatory mediators associated with the necrosis. 77 Myeloid metaplasia has been reported to occur in the liver, spleen, and lungs. 82 Extramedullary hematopoiesis is ineffective at preventing or correcting the pancytopenia that eventually develops.

Myelodysplastic Syndrome

Dysfunction of the hematopoietic system can be manifested by a variety of abnormalities that comprise myelodysplastic syndromes (MDS). In dogs, in which the syndrome is rare, cytopenias usually are seen in two or three lines in the peripheral blood (anemia, neutropenia, and/or thrombocytopenia). Other blood abnormalities can include macrocytic erythrocytes and metarubricytosis. The bone marrow typically is normocellular or hypercellular, and dysplastic changes are evident in several cell lines. If blast cells are present, they account for fewer than 30% of all nucleated cells, 2 although this threshold may be altered to fewer than 20%. 16 Myelodysplasia sometimes is called preleukemia because in some cases it may progress to an acute leukemia. 25 , 26 , 27 In humans and in cats, MDS are clonal disorders and are considered neoplastic.

History and Clinical Signs

Dogs with MPDs have similar presentations regardless of the specific disease entity, although animals with acute MPDs have a more acute onset of illness and a more rapid clinical course. A history of lethargy, inappetence, and weight loss is common. Clinical signs include emaciation, persistent fever, pallor, petechiation, hepatosplenomegaly and, less commonly, lymphadenopathy and enlarged tonsils. Shifting leg lameness, ocular lesions, and recurrent infections also are seen. Vomiting, diarrhea, dyspnea, and neurologic signs are variable features. Serum chemistries may be within reference intervals but can change if significant organ infiltration occurs.

Animals with MSD may be lethargic and anorectic and may have pallor, fever, and hepatosplenomegaly. In PV, dogs often have erythema of mucous membranes because of the increased RBC mass. Some dogs are polydipsic. In addition, neurologic signs may be present, such as disorientation, ataxia, or seizures, and these are thought to be the result of hyperviscosity or hypervolemia. 56 Hepatosplenomegaly usually is absent.

Peripheral blood abnormalities are consistently found. In addition to the presence of neoplastic cells, other abnormalities may be present, including a decrease in the numbers of normal cells of any or all hematopoietic cell lines. Occasional nucleated RBCs are present in the blood of about half of dogs with acute nonlymphocytic leukemia. 2 Nonregenerative anemia and thrombocytopenia are present in most cases. The anemia usually is normocytic and normochromic, although macrocytic anemia sometimes is present. Pathogenic mechanisms include the effects of inhibitory factors, leading to ineffective hematopoiesis, myelophthisis, immune-mediated anemia secondary to neoplasia, and hemorrhage secondary to thrombocytopenia, platelet dysfunction, or DIC. Anemia is most severe in acute MPDs, although both anemia and thrombocytopenia may be milder in animals with the M5 subtype (acute monocytic leukemia). In myelofibrosis, the anemia is characterized by anisocytosis and poikilocytosis. In addition, pancytopenia and leukoerythroblastosis, in which immature erythroid and myeloid cells are in circulation may be present. These phenomena probably result from the replacement of marrow by fibrous tissue, with resultant shearing of red cells and escape of immature cells normally confined to bone marrow. In PV, the PCV is elevated, usually in the range of 65% to 85%. The bone marrow is hyperplastic, and the M:E usually is in the normal range.

The neoplastic cells often are defective functionally. Platelet dysfunction has been reported in a dog with acute megakaryoblastic leukemia (M7), 44 and in CML, the neutrophils have decreased phagocytic capacity and other abnormalities. One exception to this was a report of CML in a dog in which the neutrophils had enhanced phagocytic capacity and superoxide production. 83 The authors hypothesized that increased synthesis of granulocyte-macrophage colony-stimulating factor (GM-CSF) resulted from a lactoferrin deficiency in the neoplastic neutrophils and mediated the enhanced function of these cells.

Diagnostic Techniques and Workup

In all cases of MPD, the diagnosis depends on the examination of peripheral blood and bone marrow. Acute MPDs are diagnosed on the basis of finding blast cells with clearly visible nucleoli in blood and bone marrow. Most dogs with acute leukemia have circulating blasts. These cells may be present in low numbers in peripheral blood, and the smear should be searched carefully, especially at the feathered edge. Even if blasts are not detected in circulation, indications of bone marrow disease, such as nonregenerative anemia or thrombocytopenia, usually are present. Occasionally neoplastic cells can be found in CSF in animals with invasion of the CNS. Smears of aspirates from tissues such as the lymph nodes, spleen, or liver may contain blasts but usually contribute little to the diagnostic workup.

Examination of blasts stained with standard Romanowsky stains may give clues to the lineage of the cells (see Figure 31-21 , A to C and E ). In AML, in addition to myeloblasts, some progranulocytes with their characteristic azurophilic granules may be present. In myelomonocytic leukemia, the nuclei of the blasts usually are pleomorphic and have round to lobulated forms. In some cells the cytoplasm may contain large azurophilic granules or vacuoles. Blasts in megakaryocytic leukemia may contain vacuoles and may have cytoplasmic blebs. In addition, bizarre macroplatelets may be present. Although these distinguishing morphologic features may suggest a definitive diagnosis, cytochemical stains or immunophenotyping usually is required to define the lineage of the blasts. Several investigators have reported modification of diagnoses after cytochemical staining. 84 , 85 It is especially important to distinguish AML from lymphocytic leukemia to provide prognostic information to the owner.

The Animal Leukemia Group recommends the following diagnostic criteria ( Figure 31-22 ). 2 Using well-prepared Romanowsky-stained blood and bone marrow films, a minimum of 200 cells are counted to determine the leukocyte differential in blood and the percentage of blast cells in bone marrow or blood or both. In bone marrow, blast cells are calculated both as a percentage of all nucleated cells (ANC) and nonerythroid cells (NEC) and are further characterized using cytochemical markers. 84 , 85 , 86 Neutrophil differentiation is identified by positive staining of blasts for peroxidase, Sudan black B, and chloracetate esterase. Nonspecific esterases (alpha-naphthyl acetate esterase or alpha-naphthyl butyrate esterase), especially if inhibited by sodium fluoride, mark monocytes. Canine monocytes may also contain a few peroxidase-positive granules. Acetylcholinesterase is a marker for megakaryocytes in dogs and cats. In addition, positive immunostaining for von Willebrand's factor (factor VIII–related antigen) and platelet glycoproteins on the surface of blasts identifies them as megakaryocyte precursors. 8 , 42 , 43 , 44 , 45 , 46 Alkaline phosphatase (AP) only rarely marks normal cells in dogs and cats but is present in blast cells in acute myeloblastic and myelomonocytic leukemias. However, because of reports of AP activity in lymphoid leukemias in dogs, its specificity as a marker for myeloid cells is not certain. Omega-exonuclease is a specific marker for basophils, which are also positive for chloracetate esterase activity. 69

Blood and bone marrow differential counts and cytochemical staining should be performed and interpreted by experienced veterinary cytopathologists. If erythroid cells comprise less than 50% of ANC and the blast cells account for more than 30%, a diagnosis of AML or AUL is made. If erythroid cells are more than 50% of ANC and the blast cells are more than 30%, a diagnosis of erythroleukemia (M6) is made. If rubriblasts comprise a significant proportion of the blast cells, a diagnosis of M6Er, or erythroleukemia with erythroid predominance, can be made. (It should be noted that in the human AML classification system, the blast threshold has been lowered to 20%.)

In some cases, electron microscopy is required to identify the lineage of the blast cells. For example, megakaryocyte precursors are positive for platelet peroxidase activity and contain demarcation membranes and alpha granules. 42 , 46 Both these features are detected at the ultrastructural level. Immunophenotyping, used to identify cell lineages in human patients, awaits development of appropriate markers for animal species (see later discussion). Hematopoietic cells from humans with leukemia often have abnormal chromosome patterns. Cytogenetic abnormalities have been found in leukemic cells from a small number of dogs. 14 , 15 It is not clear whether chromosomal aberrations are primary (causative) or secondary (caused by the leukemia). If consistent karyotypic patterns can be identified and correlated with leukemic subtypes, cytogenetic analysis eventually may yield important diagnostic and prognostic information and become a valuable tool for evaluating remission and predicting relapse.

Although morphologic and cytochemical analyses have formed the mainstay of cell identification, newer technologies now are used routinely to classify leukemias by using monoclonal antibodies to detect antigens associated with certain cell types. Cells can be immunophenotyped using flow cytometric analysis or immunocytochemistry. Both lymphoid and nonlymphoid acute leukemias are positive for CD34. Many lymphocyte markers, including CD3, CD4, CD8, CD18, CD21, CD45, CD79, and IgG, are available for dogs and can be used to rule out lymphoblastic leukemia in dogs with acute leukemias. 50 , 87 Other markers include myeloperoxidase (MPO) and CD11b for myeloid cells and CD41 for megakaryoblasts. Some overlap in the expression of these cell antigens occurs. For example, canine (but not human) granulocytes express CD4. It is best to use a panel of antibodies (similar to using a battery of cytochemical stains), because antigens often are expressed on multiple lineages, and lineage infidelity can occur. These tests have become more valuable with the availability of canine reagents. Currently, the Myeloid Neoplasm Subcommittee of the ACVP Oncology Committee recommends that the following immunophenotyping panel be done on bone marrow and/or blood smears to characterize animal leukemias: for B lymphocytes, CD79a; for T lymphocytes, CD3; for myeloid cells, MPO and CD11b; for megakaryoblasts, CD41; for dendritic cells, CD1c; and for acute leukemias, CD34. 16

Owing to the degree of differentiation of cells in chronic MPDs, these disorders must be distinguished from nonneoplastic causes of increases in these cell types. To allow a diagnosis of PV, tests first must establish that the polycythemia is absolute rather than relative. In relative polycythemias, plasma volume is decreased from hemoconcentration, dehydration, or hypovolemia, and the absolute red cell mass is not increased. Splenic contraction also can result in relative polycythemia. Absolute polycythemia, in which the RBC mass is increased, usually occurs secondary to tissue hypoxia, causing appropriate increased production of erythropoietin. In rare cases, erythropoietin may be produced inappropriately by a tumor (e.g., renal cell carcinoma) or in renal disease (pyelonephritis) or localized renal hypoxia. 88 , 89 , 90 These causes of polycythemia should be eliminated by appropriate laboratory work, thoracic radiographs, arterial blood gas analysis, and renal ultrasonography. In humans with PV, plasma erythropoietin (Epo) levels are low. Epo levels in dogs with PV tend to be low or low-normal, whereas in animals with secondary absolute polycythemia, the levels are high. 91 , 92 Samples for determination of Epo concentrations should be taken before therapeutic phlebotomy to treat hyperviscosity and, owing to fluctuations in Epo levels, should be repeated if results are incongruous with other information.

CML in dogs has no pathognomonic features, and other common causes for marked leukocytosis with a left shift (leukemoid reaction) and granulocytic hyperplasia of bone marrow must be eliminated. These include infections, especially pyogenic ones; immune-mediated diseases; and other malignant neoplasms. In CML, maturation sometimes appears disorderly, and variation in the size and shape of neutrophils may occur at the same level of maturation. In addition, neoplastic leukocytes may disintegrate more rapidly and appear vacuolated. 31 Because of the invasive nature of CML, biopsy of the liver or spleen may also help distinguish true leukemia from a leukemoid reaction, assuming the animal can tolerate the procedure. If characteristic cytogenetic abnormalities can be found in dogs with CML, this analysis may be helpful.

Basophilic leukemia is diagnosed by finding excessive numbers of basophils in circulation and in bone marrow. Basophilic leukemia must be differentiated from mastocytosis based on the morphology of the cell type present. Basophils have a segmented nucleus and variably sized granules, whereas mast cells have a round to oval nucleus that may be partly or totally obscured by small, round, metachromatic-staining granules. This distinction usually is easy to make; however, in basophilic leukemia, changes in the morphology of the nucleus and granules make the distinction less clear. 68

Essential thrombocythemia has been diagnosed based on finding persistent and excessive thrombocytosis (more than 600,000/μl) without circulating blast cells and in the absence of another MPD (e.g., polycythemia vera), myelofibrosis, or disorders known to cause secondary thrombocytosis. 72 These include iron deficiency anemia, chronic inflammatory diseases, recovery from severe hemorrhage, rebound from immune-mediated thrombocytopenia, and absence of a spleen. Thrombocytosis is transient in these disorders or abates with resolution of the primary disease. In essential thrombocythemia, platelet morphology may be abnormal, with bizarre giant forms and abnormal granulation. 73 In the bone marrow, megakaryocytic hyperplasia is a consistent feature, and dysplastic changes may be evident in megakaryocytes. 76 Spurious hyperkalemia may be present in serum samples from dogs with thrombocytosis from any cause because of the release of potassium from platelets during clot formation. 93 Measurement of the potassium in plasma is recommended in these cases and usually demonstrates a potassium concentration within reference interval. Platelet aggregability has been variably reported as impaired 73 or enhanced. 76 In the one dog in which it was measured, the plasma thrombopoietin (Tpo) concentration was normal. 75 Whether Tpo plays a role in essential thrombocythemia or is suppressed by the high platelet mass is unclear. Elucidation of the pathogenesis of this disorder should be aided by the recent cloning of the genes for thrombopoietin and its receptor, the proto-oncogene mpl. 94

In MDS, abnormalities in two or three cell lines usually are manifested in the peripheral blood as neutropenia with or without a left shift, nonregenerative anemia, or thrombocytopenia. Other changes include macrocytosis and metarubricytosis. The bone marrow typically is normocellular or hypercellular with an increased M:E, and blasts cells, although increased, account for fewer than 30% of the nucleated cells. This blast threshold may be changed to fewer than 20%, and in a report of 13 dogs with primary or secondary MDS, in all but one dog the blast cell percentage was less than 20%. 95 Dysplastic changes can be detected in any cell line. Dyserythropoiesis is characterized by asynchronous maturation of erythroid cells, typified by large hemoglobinized cells with immature nuclei (megaloblastic change). If the erythroid component is dominant, the MDS is called MDS-Er (see Box 31-7 ). 2 , 28 In dysgranulopoiesis, giant neutrophil precursors and abnormalities in nuclear segmentation and cytoplasmic granulation can be seen. Finally, dysthrombopoiesis is characterized by giant platelets and micromegakaryocytes.

Myelofibrosis should be suspected in animals with nonregenerative anemia or pancytopenia; abnormalities in erythrocyte morphology, especially shape; and leukoerythroblastosis. Bone marrow aspiration usually is unsuccessful, resulting in a “dry tap”; this necessitates a bone marrow biopsy taken with a Jamshidi needle. 96 The specimen is processed for routine histopathologic examination and, if necessary, special stains for fibrous tissue can be used. Because myelofibrosis occurs secondary to other diseases of bone marrow (e.g., MPD), chronic hemolytic anemia, or bone marrow necrosis, the clinician should look for a primary disease process.

TREATMENT

Acute Leukemias

Because of the poor response of acute nonlymphocytic leukemias, treatment has been unrewarding to date. However, little information is available on the response of specific subtypes of leukemia to uniform chemotherapeutic protocols, partly because of the rarity of these disease processes and the paucity of cases in the literature. The clinician is advised to contact a veterinary oncologist for advice on new protocols and appropriate management of these cases.

The therapeutic goal is to eradicate leukemic cells and re-establish normal hematopoiesis. Currently, this is best accomplished by cytoreductive chemotherapy, and the agents most commonly used include a combination of cytosine arabinoside and an anthracycline (e.g., doxorubicin or cyclophosphamide), vincristine, and prednisone. * In humans, the introduction of cytosine arabinoside has been the single most important development in the therapy of acute nonlymphocytic leukemia. 99 In dogs, a regimen of 100 to 200 mg/m 2 of cytosine arabinoside, given by slow infusion (over 12 to 24 hours) daily for 3 days and repeated weekly, has been used. Doxorubicin (30 mg/m 2 given intravenously every 2 to 3 weeks) can be administered at intervals alternating with cytosine arabinoside. If remission is achieved, as evidenced by normalization of the hemogram, the COAP protocol (cyclophosphamide, vincristine, cytosine arabinoside, and prednisone), as described for canine lymphoma, could be used as maintenance therapy. 7 , 97 Another protocol that has been used to treat acute myeloblastic leukemia is presented in Table 31-10 .

Regardless of the chemotherapy protocol used, significant bone marrow suppression will develop, and intensive supportive care will be necessary. Transfusions of whole blood or platelet-rich plasma may be required to treat anemia and thrombocytopenia, and infection should be managed with aggressive antibiotic therapy. Because of the generally poor response, the major thrust of therapy may be to provide palliative supportive care.

Polycythemia Vera

For PV, therapy is directed at reducing the red cell mass. The PCV should be reduced to 50% to 60% or by one sixth of its starting value. Phlebotomies should be performed as needed, with administration of appropriate colloid and crystalloid solutions to replace lost electrolytes; 20 ml of whole blood per kilogram of body weight can be removed at regular intervals. 54 In humans, phlebotomy continues to be the therapeutic approach used most frequently.

Radiophosphorus ( 32 P) has been shown to provide long-term control but can be used only in specialized centers. 100 The chemotherapeutic drug of choice is hydroxyurea, an inhibitor of DNA synthesis. This drug should be administered at an initial dosage of 30 mg/kg for 10 days; the dosage then is reduced to 15 mg/kg given orally daily. 56 The major goal of treatment is to maintain the PCV as close to normal as possible.

Chronic Myelogenous Leukemia

CML is best managed with chemotherapy to control the proliferation of the abnormal cell line and improve the quality of life. Hydroxyurea is the most effective agent for treating CML during the chronic phase. 61 , 101 The initial dosage is 20 to 25 mg/kg given twice daily. Treatment with hydroxyurea should continue until the leukocyte count falls to 15,000 to 20,000 cells/μl. 61 , 65 , 67 The dosage of hydroxyurea then can be reduced by 50% on a daily basis or to 50 mg/kg given biweeky or triweekly. In humans, the alkylating agent busulfan can be used as an alternative. 102 An effective dosage has not been established in the dog, but following human protocols, 0.1 mg/kg/day given orally is administered until the leukocyte count is reduced to 15,000 to 20,000 cells/μl.

Despite response to chemotherapy and control for many months, most dogs with CML eventually enter a terminal phase of disease. In one study of seven dogs with CML, four underwent terminal phase blast crisis. 61 In humans, blast crisis may be lymphoid or myeloid. 103 In dogs, the lineage of blast cells often has not been determined. These dogs have a poor prognosis, and the best treatment to consider, if any, would be that listed in Table 31-10 .

Essential Thrombocythemia

Few cases have been reported, but one dog was treated successfully with a combination chemotherapy protocol that included vincristine, cytosine arabinoside, cyclophosphamide, and prednisone. 74 Treatment is controversial in humans because of the lack of evidence that asymptomatic patients benefit from chemotherapy. Patients with thrombosis or bleeding are given cytoreductive therapy. Hydroxyurea is the drug of choice for initially controlling the thrombocytosis. 72

Myelodysplastic Syndrome

No standard therapeutic regimen exists for MDS. Often, humans receive no treatment if the cytopenias do not cause clinical signs. Transfusions are given when necessary, and patients with fever are evaluated aggressively to detect infections. Growth factors, such as erythropoietin, GM-CSF, granulocyte colony-stimulating factor (G-CSF), and IL-3, are sometimes used in patients who require frequent transfusions to increase their blood cell counts and enhance neutrophil function. 104 , 105 In one case report, human Epo (100 U/kg given subcutaneously every 48 hours) was administered to a dog with MSD because of profound anemia. The rationale for the erythropoietin was to promote terminal differentiation of dysplastic erythrocytes. The PCV increased from 12% to 34% by day 19 of Epo treatment. This dog remained in remission for longer than 30 months. 28 Other factors that induce differentiation of hematopoietic cells include retinoic acid analogs, 106 1,25 dihydroxyvitamin D 3 , 107 interferon-alpha, and conventional chemotherapeutic agents, such as 6-thioguanine and cytosine arabinoside. 108 The propensity of these factors to enhance progression to leukemia is not known in many cases, but the potential risk exists.

PROGNOSIS

In general, the prognosis for animals with chronic MPDs is better than for dogs with acute MPDs, for which the prognosis is grave. The prognosis for PV and chronic myelogenous leukemia is guarded, but significant remissions have been achieved with certain therapeutic regimens and careful monitoring. Animals commonly survive a year or longer. 61 , 67 The development of blast crisis portends a grave prognosis.

COMPARATIVE ASPECTS

The pathophysiology and therapy of nonlymphocytic leukemia in humans is being studied intensively. The MPDs have been demonstrated to be clonal, with abnormalities evident in all hematopoietic cell lines. Leukemogenesis likely is caused by mutation or amplification of proto-oncogenes in a two-step process that initially involves a single cell and is followed by additional chromosomal alterations that may involve oncogenes. 1 , 13 These alterations are manifested as chromosomal abnormalities. Environmental factors known to cause leukemia are exposure to high-dose radiation, benzene (chronic exposure), and alkylating agents. 109 New classification systems have incorporated genetic mutations, more accurately reflect prognoses, and facilitate use of consistent categorization among institutions. 110

Therapeutic modalities under investigation or development include combination chemotherapy, immunotherapy, cytokine therapy, drug-resistance modulators, proapoptotic agents, antiangiogenic factors, signal transduction-active agents, and bone marrow transplantation. The prognosis for chronic MPDs is better than for acute MPDs. For acute nonlymphocytic leukemias, the prognosis is better for children than adults; only 10% of adults who receive chemotherapy maintain remissions for longer than 5 years. 109 The spontaneous canine diseases probably occur too infrequently to serve as useful models. MPDs have been induced experimentally in the dog by irradiation and transplantation in an attempt to create models for study. Many similarities between human and canine MPDs exist, and veterinary medicine may benefit from any therapeutic advances made in the human field.

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Plasma Cell Neoplasms

David M. Vail

Plasma cell neoplasms arise when a cell of the B-lymphocyte plasma cell lineage proliferates to form a malignant population of similar cells. This population is believed in most instances to be monoclonal (i.e., derived from a single cell), because the cells typically produce homogenous immunoglobulin, although some examples of biclonal and polyclonal plasma cell neoplasms exist. A wide variety of clinical syndromes are represented by plasma cell neoplasms, including multiple myeloma, IgM (Waldenstrom's) macroglobulinemia, and solitary plasmacytoma (including solitary osseous plasmacytoma and extramedullary plasmacytoma). Based on incidence and severity, multiple myeloma is the most clinically important plasma cell neoplasm.

MULTIPLE MYELOMA

Incidence and Etiology

Although multiple myeloma (MM) represents fewer than 1% of all malignant tumors in animals, it accounts for approximately 8% of all hematopoietic tumors and 3.6% of all primary and secondary tumors affecting bones in dogs. 1 , 2 Early studies indicated a male predisposition, 3 but subsequent reports do not suggest a gender predilection for the dog. 1 , 4 Older dogs are affected most often (average age, 8 to 9 years). 1 , 3 , 4 In one large case series, German shepherds were overrepresented based on the hospital population. 1

The true incidence of MM in the cat is unknown, although it is a much rarer diagnosis in that species than in the dog. MM accounted for only one of 395 and four of 3248 tumors in two large compilations of feline malignancies and 0.9% of all malignancies and 1.9% of hematologic malignancies in another report. 5 , 6 , 7 MM occurs in aged cats (median age, 12 to 14 years), most often in domestic shorthairs, and no gender predilection has been consistently reported, although a male preponderance may exist. 4 , 7 , 8 MM has not been associated with coronavirus, FeLV or FIV infection.

The etiology of MM for the most part is unknown. Genetic predispositions, molecular aberrations, viral infections, chronic immune stimulation, and exposure to carcinogen stimulation all have been suggested as contributing factors. 4 , 8 , 9 , 10 , 11 , 12 , 13 , 14 Support for a familial association in cats comes from cases reported among siblings. 8 Evidence exists that molecular mechanisms of cellular control, including overexpression of cell cycle control components such as cyclin D1(see Chapter 2) and receptor tyrosine kinase dysregulation, may be involved in canine myeloma and plasma cell tumors. 13 , 14 In rodent models, chronic immune stimulation and exposure to implanted silicone gel has been associated with development of MM, 9 , 10 as have chronic infections and prolonged hyposensitization therapy in humans. 11 Viral Aleutian disease of mink results in monoclonal gammopathies in a small percentage of cases. 12 Working in the agricultural industry, petroleum products, and irradiation are known risk factors for the development of MM in humans. 15 , 16 , 17 Progression of solitary plasma cell tumors to MM has been reported in both dogs and cats, and a single case of a B-cell lymphoma that progressed to MM has been reported in the dog. 18 , 19

Pathology and Natural Behavior

MM is a systemic proliferation of malignant plasma cells or their precursors, which arise as a clone of a single cell that usually involves multiple bone marrow sites. Malignant plasma cells can have a varied appearance on histologic sections and cytologic preparations. The degree of differentiation ranges from cells that resemble normal plasma cells in late stages of differentiation ( Figure 31-23 ) to very large, anaplastic round cells with a high mitotic index, representing early stages of differentiation. 3 , 4 , 7 , 20

In cats with MM, most plasma cells (83% in one report) are immature and have marked atypia, including increased size, multiple nuclei, clefted nuclei, anisocytosis, anisokaryosis, variable nucleus-to-cytoplasm ratios, decreased chromatin density, and variable nucleoli. Nearly one fourth of the cells have “flame cell” morphology, characterized by peripheral eosinophilic cytoplasmic processes. 7

Malignant plasma cells typically produce an overabundance of a single type or component of immunoglobulin, referred to as the M component ( Figure 31-24 ). The M component may be represented by any class of the entire immunoglobulin or by only a portion of the molecule, such as the light chain (Bence Jones protein) or the heavy chain (heavy chain disease). In the dog, the M component usually is represented by either IgG or IgA immunoglobulin types in nearly equal incidence, whereas the ratio of IgG to IgA in cats is approximately 5:1. * If the M component is the IgM type, the term macroglobulinemia (also Waldenstrom's macroglobulinemia) often is applied. Several cases of biclonal gammopathy in dogs and cats have been reported. 7 , 8 , 26 , 27 , 28 , 29 , 30 Two cases of nonsecretory MM have been reported in dogs. 31 Rarely, cryoglobulinemia has been reported in dogs with MM and IgM macroglobulinemia, and it has been reported in a cat with IgG myeloma. 4 , 32 , 33 , 34 Cryoglobulins are paraproteins that are insoluble at temperatures below 37° C. They require blood collection and clotting to be performed at 37° C before serum separation. If whole blood is allowed to clot at temperatures below this, the protein precipitates in the clot and is lost. Pure light-chain M component is rare but has been reported in dogs. 35

The pathology associated with MM is a result of high levels of circulating M component or of organ or bone infiltration with neoplastic cells, or both. Associated pathologic conditions include bone disease, bleeding diathesis, hyperviscosity syndrome, renal disease, hypercalcemia, immunodeficiency (and subsequent susceptibility to infections), cytopenias secondary to myelophthisis, and cardiac failure.

Bone lesions can be isolated, discrete osteolytic lesions (including pathologic fractures [ Figure 31-25 , A ]) or diffuse osteopenias ( Figure 31-26 ). Approximately one fourth to two thirds of dogs with MM have radiographic evidence of bony lysis or diffuse osteoporosis. 1 , 3 , 4 , 36

Skeletal lesions in cats with MM were rarely reported in the older literature, but more recent reports document lesions in 50% to 65% of cases. 6 , 7 , 8 , 22 Bones engaged in active hematopoiesis are more commonly affected, including the vertebrae, ribs, pelvis, skull, and proximal or distal long bones. 3

Skeletal lesions are rare with IgM (Waldenstrom's) macrogammaglobulinemia, in which malignant cells often infiltrate the spleen, liver, and lymph tissue rather than bone. 4 , 33 , 37 , 38 , 39

Bleeding diathesis can result from one or a combination of events. M components may interfere with coagulation by inhibiting platelet aggregation and the release of platelet factor 3; causing adsorption of minor clotting proteins; generating abnormal fibrin polymerization; and producing a functional decrease in calcium. 4 , 40 , 41 , 42 Approximately one third of dogs and one fourth of cats have clinical evidence of hemorrhage. 1 , 7 , 8 In dogs, nearly half have abnormal prothrombin (PT) and partial thromboplastin (PTT) times. Thrombocytopenia may also play a role if bone marrow infiltration is significant (i.e., myelophthisis).

Hyperviscosity syndrome (HVS) represents one or a constellation of clinicopathologic abnormalities resulting from greatly increased serum viscosity. The magnitude of viscosity changes depends on the type, size, shape, and concentration of the M component in the blood. HVS is more common with IgM macroglobulinemias because of the high molecular weight of this class of immunoglobulin. 37 IgA myelomas, usually present as a dimer in the dog, may undergo polymerization, resulting in increased serum viscosity. 1 , 4 , 40 IgG-associated HVS also can occur, albeit less frequently. High serum viscosity occurs in approximately 20% of dogs with MM and can result in bleeding diathesis, neurologic signs (e.g., dementia, depression, seizure activity, and coma), ophthalmic abnormalities (e.g., dilated and tortuous retinal vessels, retinal hemorrhage [ Figure 31-27 ], and retinal detachment), and increased cardiac workload with the potential for subsequent development of cardiomyopathy. * These consequences are thought to be a result of sludging of blood in small vessels, ineffective delivery of oxygen and nutrients, and coagulation abnormalities. HVS was reported less commonly in cats with MM in the older literature but has been reported in association with IgG-, IgA-, and IgM-secreting tumors. 4 , 6 , 21 , 23 , 24 , 25 In four of nine cats with MM, the relative serum viscosity was above control ranges. 8

Renal disease is present in approximately one third to one half of dogs with MM, and azotemia was observed in one third of cats in one report. 1 , 3 , 7 The pathogenesis of renal failure often is multifactorial. It can develop as a result of Bence Jones (light-chain) proteinuria, tumor infiltration into renal tissue, hypercalcemia, amyloidosis, diminished perfusion secondary to hyperviscosity syndrome, dehydration, or ascending urinary tract infection. 1 , 4 , 40 , 41 , 42 Normally, heavy-chain and light-chain synthesis is well balanced in nonneoplastic immunoglobulin production. In MM, an unbalanced excess of light-chain products may result. 42 Light chains have a low molecular weight and normally are filtered by the renal glomerulus; their presence can result in protein precipitates and subsequent renal tubular injury. The presence of light chains in urine without a concomitant monoclonal spike in serum, although rare, is indicative of pure light-chain disease. 35 Tubules become obstructed by large laminated casts containing albumin, immunoglobulin, and light chains. 4 , 35 , 40 , 41 , 42 Bence Jones proteinuria occurs in approximately 25% to 40% of dogs with MM, and in two recent surveys, it occurred in approximately 50% of cases. *

Hypercalcemia is reported in 15% to 20 % of dogs with MM and is thought to result primarily from the production of osteoclast-activating factor by neoplastic cells. 1 , 4 , 46 Other factors have been implicated in human MM, including elevated levels of various cytokines, TNF, IL-1, and IL-6. In two dogs with MM and hypercalcemia, serum elevations in circulating N-terminal PTHrP were noted. 47 Hypercalcemia may also be exacerbated by associated renal disease. Initially thought to be a rare event in cats with MM, hypercalcemia was noted in 25% of recently reported cases. 7 , 8 , 48

Susceptibility to infection and immunodeficiency long have been associated with MM and often are the ultimate cause of death in affected animals. 1 , 4 , 22 Infection rates in humans with MM are 15 times higher than normal and usually represent pneumonia or a urinary tract infection. 49 The response to vaccination also has been shown to be suppressed in humans with MM. 50 Normal immunoglobulin levels often are severely depressed in affected animals. 4 In addition, leukopenias may develop secondary to myelophthisis.

Variable cytopenias may be observed in association with MM. Approximately two thirds of dogs with MM have a normocytic, normochromic, nonregenerative anemia 1 , 3 , 4 ; this can result from marrow infiltration (myelophthisis), blood loss from coagulation disorders, anemia of chronic disease, or increased erythrocyte destruction secondary to high serum viscosity. In dogs with MM, similar factors lead to thrombocytopenia in nearly one third of the dogs and leukopenia in nearly one fourth. In cats, approximately two thirds are anemic, one half are thrombocytopenic, and one third are neutropenic. 7 , 8

Cardiac disease, if present, usually is a result of excessive cardiac workload and myocardial hypoxia secondary to hyperviscosity. 40 , 41 Myocardial infiltration with amyloid and anemia may be complicating factors. In one report nearly half of the cats with MM had a cardiac murmur, for which the etiology was not established. 7

History and Clinical Signs

Clinical signs of MM may be present up to a year before diagnosis in dogs (median, 1 month). In one cat, M-component elevations were detected 9 years before clinical presentation. 1 , 7 In the latter case, the M-component elevation was consistent with monoclonal gammopathy of unknown significance (MGUS). MGUS (i.e., “benign,” “essential,” or “idiopathic” monoclonal gammopathy) is a benign monoclonal gammopathy that is not associated with osteolysis, bone marrow infiltration, or Bence Jones proteinuria. MGUS also has been reported in dogs. 51 , 52

Signs of MM can vary, depending on the wide range of possible pathologic effects. TABLE 31-11 , TABLE 31-12 list the relative frequencies of clinical signs observed in the dog and cat, respectively, based on a compilation of several reports. 1 , 4 , 7 , 8 , 22 Bleeding diathesis usually is represented by epistaxis and gingival bleeding. Funduscopic abnormalities may include retinal hemorrhage (see Figure 31-27 ), venous dilation with sacculation and tortuosity, retinal detachment and blindness. 1 , 4 , 40 , 43 , 44 , 45 CNS signs may include dementia, seizure activity, and deficiencies in midbrain or brainstem localizing reflexes secondary to HVS or extreme hypercalcemia. Signs reflecting transverse myelopathies secondary to vertebral column infiltration, pathologic fracture, or extradural mass compression also can occur. 1 , 4 , 32 , 36 , 53 One case of ataxia and seizure activity in a dog with extramedullary plasmacytoma (EMP) secondary to tumor-associated hypoglycemia has been reported. 54 In addition, paraneoplastic polyneuropathy has been reported in a dog with MM. 55

A history of chronic respiratory infections and persistent fever may also be present in cats. Hepatosplenomegaly and renomegaly can occur as a result of organ infiltration. Bleeding diathesis due to HVS is less common in the cat, but epistaxis, pleural and peritoneal hemorrhagic effusions, retinal hemorrhage, and central neurologic signs have been reported. 4 , 6 , 7 , 8 , 21 , 22 , 23 , 24 , 25 Polydipsia and polyuria can occur secondary to renal disease or hypercalcemia, and dehydration may develop. Hind limb paresis secondary to osteolysis of lumbar vertebral bodies or extradural compression has been reported in cats. 8 , 56

Diagnostics and Staging

The diagnosis of MM usually follows the demonstration of bone marrow plasmacytosis (see Figure 31-23 ), the presence of osteolytic bone lesions (see Figure 31-26 ), and the demonstration of serum or urine myeloma proteins (M component; see Figure 31-24 ). In the absence of osteolytic bone lesions, a diagnosis can also be made if marrow plasmacytosis is associated with a progressive increase in the M component. In the cat, because the degree of bone marrow infiltration may not be as marked, some have suggested consideration of plasma cell morphology and visceral organ infiltration in cases with demonstrable M-component disease in the absence of marked marrow plasmacytosis (less than 20%). 7

All animals suspected of having plasma cell tumors should receive a minimal diagnostic evaluation, including a CBC, platelet count, serum biochemistry profile, and urinalysis. Particular attention should be paid to renal function and serum calcium levels. Serum electrophoresis and immunoelectrophoresis are performed to detect a monoclonal spike (see Figure 31-24 ) and to categorize the immunoglobulin class involved. Heat precipitation with electrophoresis of urine is performed to detect Bence Jones proteinuria, because commercial urine dipstick methods are not capable of this determination.

Definitive diagnosis usually is done by bone marrow aspiration. A bone marrow core biopsy or multiple aspirates may be necessary because of the possibility of clustering of plasma cells in the bone marrow. Normal marrow contains fewer than 5% plasma cells, whereas in myelomatoid marrow, this level often is greatly exceeded. Current recommendations require the presence of marrow plasmacytosis greater than 20%; however, a 10% cutoff in cats recently has been recommended, with special attention to cellular atypia. 7 Patel and colleagues 7 comment that even the 10% threshold is problematic, and cellular atypia and visceral organ involvement should be considered equally important.

Imaging

Routine thoracic and abdominal radiographs are recommended. Occasionally, bony lesions can be observed in skeletal areas on these standard films, and organomegaly (liver, spleen, and kidneys) is observed in most cats ( Figure 31-28 ). 7 Abdominal ultrasound scans are recommended in all cats suspected of having MM, because they reveal one or more abdominal tumors in nearly all such patients. 7 These include splenomegaly with or without nodules, diffuse hyperechoic hepatomegaly with or without nodules, renomegaly, and iliac lymph node enlargement. Skeletal survey radiographs are recommended to detect and determine the extent of osteolytic lesions, which may have diagnostic, prognostic, and therapeutic implications. Nuclear scintigraphy (bone scans) for clinical staging of dogs with MM has been performed; however, because of the predominant osteolytic activity with osteoblastic inactivity, these scans seldom give positive results and therefore are not useful for routine diagnosis. 57 In physician-based oncology, bone mineral density analysis (DEXA scan) to document osteoporosis and MRI scans of bone marrow are commonly used for staging; however, neither of these has been applied consistently in the veterinary literature.

In rare cases, biopsy of osteolytic lesions (i.e., Jamshidi core biopsy; see Chapter 23) is necessary for diagnosis. In one case of MM in a dog, splenic aspirates were diagnostically helpful. 58 If clinical hemorrhage is present, a coagulation assessment (e.g., platelet count, PT, and PTT) and serum viscosity measurements should be done. All animals should undergo a careful funduscopic examination. Table 31-13 shows the overall frequency of clinical diagnostic abnormalities in dogs and cats with MM, as compiled from published series involving at least five cases each.

A clinical staging system for canine MM has been suggested 1 ; however, currently no prognostic significance has been attributed to it.

Molecular diagnostic techniques for MM have had limited use thus far in veterinary oncology; however, PCR techniques have been used to determine the clonality of the immunoglobulin heavy-chain variable region gene in feline plasmacytomas and myelomas (see Section A of this chapter and Figure 31-8 ). 59 The use of this technology in cases in which the diagnosis is not straightforward awaits further investigation.

Differential diagnosis of multiple myeloma

Disease syndromes other than plasma cell tumors can be associated with monoclonal gammopathies and should be considered in any list of differentials. These include other lymphoreticular tumors (lymphoma, extramedullary plasmacytoma, chronic and acute lymphocytic leukemia), chronic infections (e.g., ehrlichiosis, leishmaniasis, FIP), and MGUS. *

Treatment

Initial therapy

Therapy for MM is directed at both the tumor cell mass and the secondary systemic effects. All diagnostic procedures should be completed before primary therapy is started to ensure a complete diagnosis and to procure baseline values for monitoring response.

In most dogs with MM, chemotherapy is effective at reducing the myeloma cell burden, relieving bone pain, allowing for skeletal healing, and reducing levels of serum immunoglobulins. It also can greatly extend both the quality and duration of most patients’ lives. 1 , 4 Complete elimination of neoplastic myeloma cells is rare, but the chemotherapeutic drugs currently available can make MM a gratifying disease to treat for both the clinician and the client. However, eventual relapse is to be expected.

Only one half of cats with MM respond to chemotherapy, and most responses are short-lived. However, several long-term responses (i.e., longer than 1 year) have been reported, and treatment should be attempted when educated clients decide on a therapeutic option. †

Melphalan, an alkylating agent, is the chemotherapeutic drug of choice for the treatment of MM. 1 , 4 In the dog, an initial starting dosage of 0.1 mg/kg is given orally once daily for 10 days and then reduced to 0.05 mg/kg given orally once daily continuously. Addition of prednisone therapy is thought to increase the efficacy of melphalan therapy. Prednisone is started at a dosage of 0.5 mg/kg given orally once daily for 10 days and is then reduced to 0.5 mg/kg given orally every other day until the drug is discontinued after 60 days of therapy. Melphalan, however, is continued at 0.05 mg/kg/day until clinical relapse occurs or myelosuppression necessitates a dose reduction. Most dogs treated with this melphalan/prednisone combination tolerate the regimen well. The most clinically significant toxicity of melphalan is myelosuppression, particularly a delayed thrombocytopenia. A CBC, including a platelet count, should be performed biweekly for 2 months of therapy and monthly thereafter. If significant myelosuppression occurs (usually thrombocytopenia or neutropenia), the dosage or the treatment frequency may need to be reduced.

The author has successfully used an alternative, pulse-dosing regimen for melphalan (7 mg/m 2 given orally daily for 5 consecutive days every 3 weeks) in a small number of cases in which myelosuppression was limiting the more conventional, continuous low-dose therapy. The author now uses this pulse-dose regimen as a first-line treatment with the caveat that long-term response data are lacking.

Melphalan/prednisone therapy also can be used in cats with MM; however, the protocol appears to be more myelosuppressive in cats than in dogs, and careful monitoring is required. In the cat, a dosing schedule similar to that for the dog has been reported 8 , 22 ; 0.1 mg/kg (approximately 0.5 mg, or ¼ of a 2 mg tablet) is given orally once daily for 10 to 14 days, then every other day until clinical improvement occurs or leukopenia develops. One report has advocated long-term continuous maintenance (0.1 mg/kg given orally once every 7 days). 8

Cyclophosphamide has been used as an alternative alkylating agent or in combination with melphalan in dogs with MM. 1 , 4 , 64 No evidence indicates that it is superior to melphalan therapy. In the author's practice, cyclophosphamide is limited to patients with severe hypercalcemia or widespread systemic involvement in which a faster acting alkylating agent may alleviate systemic effects more quickly. Cyclophosphamide is initiated at a dosage of 200 mg/m 2 given intravenously once, at the same time oral melphalan therapy is started. Because cyclophosphamide is less likely to affect thrombocytes, it may be substituted in patients in which thrombocytopenia has developed secondary to long-term melphalan use.

Chlorambucil, another alkylating agent, has been used successfully for the treatment of IgM macroglobulinemia in dogs at a dosage of 0.2 mg/kg given orally once daily. 4 , 37 Few or no clinical signs of toxicity result from this dosing schedule.

CCNU, yet another alkylating agent, has been used in a limited number of cats with MM, and a partial response has been reported with a dosing schedule of 50 mg/m 2 given orally every 21 days. 65

Evaluation of the response to therapy

Evaluation of the response to systemic therapy for MM is based on improvement in clinical signs and clinicopathologic parameters and radiographic improvement of skeletal lesions. 1 , 4 Subjective improvement in clinical signs of bone pain, lameness, lethargy, and anorexia should be evident within 3 to 4 weeks after initiation of therapy. Objective laboratory improvement, including reduction in serum immunoglobulin or Bence Jones proteinuria, usually is noted within 3 to 6 weeks. Radiographic improvement in osteolytic bone lesions may take months, and only partial resolution may occur. Ophthalmic complications (including longstanding retinal detachment) and paraneoplastic neuropathies can be expected to resolve along with the tumor mass. 45 , 55 In one report on cats that responded to melphalan and prednisone, clinical improvement was noted in 4 weeks and serum protein and radiographic bone abnormalities were greatly improved by 8 weeks. 8

As previously discussed, MM does not resolve completely, and a good response is defined as a reduction in measured M component (i.e., immunoglobulin or Bence Jones proteins) by at least 50% of pretreatment values. 4 Reduction in the serum immunoglobulin levels may lag behind reduction in Bence Jones proteinuria, because the half-lives are 15 to 20 days and 8 to 12 hours, respectively. 66 For routine follow-up, quantification of the elevated serum immunoglobulin or urine Bence Jones protein is performed monthly until a good response is noted and then every 2 to 3 months. Repeat bone marrow aspiration for evaluation of plasma cell infiltration occasionally may be necessary. This is particularly prudent when cytopenias develop during chemotherapy and drug toxicity must be differentiated from marrow recurrence.

Therapy directed at complications of multiple myeloma

Long-term control of complications such as hypercalcemia, HVS, bleeding diathesis, renal disease, immunosuppression, ophthalmic complications, and pathologic skeletal fractures is achieved by controlling the primary tumor mass. However, therapy directed more specifically at these complications may be indicated in the short term.

If hypercalcemia is marked and significant clinical signs are present, standard therapies, including fluid diureses with or without pharmacologic agents (e.g., calcitonin), may be indicated (see Chapter 5). Moderate hypercalcemia typically resolves within 2 to 3 days after initiation of melphalan/prednisone chemotherapy.

HVS is best treated in the short term by plasmapheresis. 4 , 40 , 62 , 67 , 68 Whole blood is collected from the patient and centrifuged to separate plasma from packed cells. Packed red cells are resuspended in normal saline and reinfused into the patient. Bleeding diathesis usually resolves along with HVS, but platelet-rich plasma transfusions may be necessary with thrombocytopenia.

Renal impairment may require aggressive fluid therapy in the short term and maintenance of adequate hydration in the long term. Careful attention to secondary urinary tract infections and appropriate antimicrobial therapy are indicated. Ensuring an adequate water intake at home is important, and in some cases owners must be taught home subcutaneous fluid administration. Continued monitoring of renal function is recommended, along with follow-up directed at tumor response.

Patients with MM can be thought of as immunologic cripples. Some have recommended prophylactic antibiotic therapy in dogs with MM, 4 but in humans, no benefit for this approach has been observed over diligent monitoring and aggressive antimicrobial management when indicated. 41 Cidal antimicrobials are preferred over static drugs, and nephrotoxic antimicrobials should not be used.

Pathologic fractures of weight-bearing long bones and of vertebrae, resulting in spinal cord compression, may require immediate intervention in conjunction with systemic chemotherapy. Orthopedic stabilization of fractures should be performed and may be followed with external beam radiotherapy (see Figure 31-25 , B ). Recently, inhibition of osteoclast activity by bisphosphonate drugs has been shown to reduce the incidence and severity of skeletal complications of MM in humans. 57 This class of drugs may hold promise for use in dogs and cats with various skeletal tumors. 69

Rescue therapy

Rescue therapy may be attempted when MM eventually relapses in dogs undergoing melphalan therapy or in the uncommon patient that initially is resistant to alkylating agents. The author has had success with the VAD protocol, a combination of doxorubicin (30 mg/m 2 given intravenously every 21 days), vincristine (0.7 mg/m 2 given intravenously on days 8 and 15), and dexamethasone sodium phosphate (1 mg/kg given intravenously once a week on days 1, 8, and 15); this regimen is used in 21-day cycles. Although most dogs initially respond to this rescue protocol, the duration of response tends to be short, lasting only a few months. High-dose cyclophosphamide (300 mg/m 2 given intravenously every 7 days) also been has used as a rescue agent, although with limited success. Liposomal doxorubicin produced a long-term remission in a dog with MM that had been resistant to native doxorubicin. 70

Investigational therapies

Because MM ultimately is a uniformly fatal disease in most species, including humans, significant effort is being put into investigational therapies for this disease. Currently, bone marrow ablative therapy and marrow or stem cell rescue, thalidomide (and other antiangiogenic therapies), bortezomib (a proteasome inhibitor), arsenic trioxide, the bisphosphonates, and several molecular targeting therapies are under investigation; however, their use in veterinary species is limited or completely absent at present. 57 Nonetheless, the promise of molecular-targeted therapies is foreshadowed by a case of a dog with MM that was resistant to melphalan, prednisone, and doxorubicin 14 ; this dog achieved a partial response to tyrosine kinase inhibitor therapy (SU11654; see Chapter 14, section B) that was maintained for 6 months.

Prognosis

The prognosis for dogs with MM is good for initial control of the tumor and a return to good quality of life. In a group of 60 dogs with MM, approximately 43% achieved a complete remission (i.e., normalization of serum immunoglobulins), 49% achieved a partial remission (i.e., immunoglobulin levels less than 50% pretreatment values), and only 8% did not respond to melphalan/prednisone chemotherapy. 1 Long-term survival is the norm, with a median of 540 days reported ( Figure 31-29 ). Hypercalcemia, Bence Jones proteinuria, and extensive bony lysis are known negative prognostic indices in the dog. 1 The long-term prognosis for dogs with MM is poor, because recurrence of the tumor mass and associated clinical signs is expected. Eventually, the tumor no longer responds to available chemotherapeutic drugs, and death follows from renal failure, sepsis, or euthanasia for intractable bone or spinal pain. 1 , 4

The prognosis for MM in the cat is not as favorable in the short term as it is in the dog. 4 , 7 , 8 , 22 Although most cats (approximately 60%) transiently respond to melphalan/prednisone- or cyclophosphamide-based protocols, most responses are partial and not durable. Typically, cats with MM succumb to the disease within 4 months. 6 , 7 , 8 , 22 , 24 , 68 However, long-term survivors (longer than 1 year) have been reported. 7 , 8 , 22 , 30 , 48 One investigator grouped MM in cats into two prognostic categories, based on criteria known to predict disease behavior in dogs ( Table 31-14 ). 8 Although no rigorous statistical analysis was performed on this small group of cats (nine), the median survival times were 5 days for cats with disease categorized as “aggressive” and 387 days for those with disease categorized as “nonaggressive.”

Experience in dogs with IgM macroglobulinemia is limited. 4 , 35 Response to chlorambucil is to be expected, and in nine treated dogs, 77% achieved remission, with a median survival time of 11 months. 4

SOLITARY AND EXTRAMEDULLARY PLASMACYTIC TUMORS

Solitary collections of monoclonal plasmacytic tumors can originate in soft tissues (extramedullary plasmacytoma) or bone (solitary osseous plasmacytoma [SOP]). A number of large case compilations of cutaneous plasmacytoma have been reported in the dog. 13 , 71 , 72 , 73 , 74 , 75 , 76 , 77 Plasmacytomas represented 2.4% of all canine tumor submissions in one large compilation of cases. 78 In this series of 751 cases, the most common locations in the dog were cutaneous sites (86%) ( Figure 31-30 ), the mucous membranes of the oral cavity and lips (9%) ( Figure 31-31 ), and the rectum and colon (4%). The skin of the limbs and head (including the ears) is the most frequently reported cutaneous site. 71 , 72 , 77 All other sites accounted for the remaining 1% of cases; these sites may include the stomach, spleen, genitalia, eyes, uterus, and liver. The American cocker spaniel, English cocker spaniel, and West Highland white terrier (and perhaps Yorkshire terriers, boxers, German shepherds, and Airedale terriers) have an increased risk of developing plasmacytomas, and the median age of affected dogs is 9 to 10 years. 77 , 78

Cutaneous and oral EMP in dogs typically manifests as benign tumors that are highly amenable to local therapy. The natural behavior of noncutaneous/nonoral EMP appears to be somewhat more aggressive in the dog. Gastrointestinal EMP has been reported in a number of sites in the veterinary literature, including the esophagus, 79 stomach ( Figure 31-32 ), 64 , 80 small intestine, 81 and large intestine. 78 , 80 , 81 , 82 , 83 Metastasis to associated lymph nodes is more common in these cases; however, bone marrow involvement and monoclonal gammopathies are less commonly encountered. In nine cases of colorectal EMP, only two dogs experienced recurrence after surgical excision, and two cases involved multiple lesions. 78 EMP of the trachea, liver, and uterus also have been reported in a dog, and all had a benign course after local resection. 84 , 85 , 86

Most cases of SOP eventually progress to systemic MM; however, the time course from local tumor development to systemic MM may be many months to years. 31 , 87 SOPs reported in the dog have involved the zygomatic arch and the ribs. 31

Plasmacytomas are less common in cats, and fewer reports exist in the literature. 18 , 88 , 89 , 90 , 91 , 92 , 93 , 94 They occur in older cats (mean age, 8.5 years) with no significant gender predilection. The skin is the most common site; other sites include the oral cavity, eye, GI tract, liver, subcutaneous tissues, and brain. Two reports exist of cutaneous EMP in cats that progressed to systemic disease; one cat developed lymph node and distant metastasis, the other progressed to MM. 18 , 91

Clinical Signs

Clinical signs associated with solitary plasmacytomas relate to the location of involvement; in the rare cases involving high levels of M component, HVS may occur. Most cutaneous plasmacytomas are solitary, smooth, raised pink nodules that measure 1 to 2 cm in diameter (see Figure 31-30 ), although tumors as large as 10 cm have been reported. Combination of data from large series shows that more than 95% of the tumors occur as solitary masses, and fewer than 1% occur as part of a systemic MM process. 13 , 71 , 72 , 73 , 77 The cutaneous and oral forms of EMP usually have a benign course and no related clinical signs. Gastrointestinal EMP, however, typically produces relatively nonspecific signs that may suggest alimentary involvement. Colorectal plasmacytomas usually cause rectal bleeding, hematochezia, tenesmus, and rectal prolapse. 78 One case of ataxia and seizure activity in a dog with EMP secondary to tumor-associated hypoglycemia has been reported. 54 SOP usually is associated with pain and lameness (if the appendicular skeleton is affected) or with neurologic signs (if vertebral bodies are involved).

Diagnosis

Diagnosis of SOPs and EMPs usually requires tissue biopsy or fine-needle aspiration. The cells that make up solitary plasmacytic tumors in both cats and dogs have been classified histologically as mature, hyaline, cleaved, asynchronous, monomorphous blastic, and polymorphous blastic cell types. No prognostic significance has been observed with this classification system, although it has been suggested that the polymorphous blastic type may act more aggressively in the dog. 13 , 77 , 88

With poorly differentiated solitary plasmacytic tumors, immunohistochemical studies directed at detecting immunoglobulin, light and heavy chains, and thioflavine T may be helpful for differentiating the lesions from other round cell tumors. 31 , 76 , 77 , 92 , 93 , 94 Immunoreactivity has been demonstrated for canine IgG F(ab) 2 and vimentin. 73 A variant characterized by an IgG-reactive amyloid interspersed with the neoplastic cells also has been described. 74 In addition, PCR techniques can be used to determine the clonality of the immunoglobulin heavy-chain variable region gene in plasmacytomas and myelomas, which may be diagnostically useful in difficult cases.

Thorough staging is important in dogs and cats with plasmacytomas that are at higher risk for systemic spread. Staging, which must be done before therapy is started, should include bone marrow aspiration, serum electrophoresis, and skeletal survey radiographs to ensure that the disease is confined to a local site. This is most important for SOP and gastrointestinal EMP because of these tumors’ relatively high metastatic rate; it is less important for cutaneous and oral plasmacytomas because of their typically more benign behavior. In addition, endoscopic evaluation of the entire GI tract is recommended with gastrointestinal EMP.

Treatment

Cutaneous plasma cell tumors in the dog are almost always benign and have an excellent prognosis after conservative surgical excision. Successful therapy with melphalan and prednisone has been rarely applied for a local recurrence or incomplete margins in dogs and cats. 53 , 92 Radiation therapy has been used infrequently for cases that are nonsurgical. Surgery is recommended in combination with radiotherapy for SOP when the lesion results in an unstable long bone fracture (see Figure 31-25 ) or the patient is nonambulatory because of neurologic compromise caused by a vertebral body SOP. In the latter case, spinal cord decompression, mass excision, and possibly spinal stabilization may be necessary. 53 Radiotherapy can be used without surgery, when fractures are stable, as a palliative measure for bone pain or, in the case of vertebral SOP, if the patient is ambulatory and stable. Good local control usually is achieved, but most patients eventually develop systemic MM. 31 , 53 , 87 SOP of the axial skeleton can be managed by excision or radiotherapy alone.

Whether systemic chemotherapy should be initiated at the time of local therapy for SOP when systemic involvement is not documented is the subject of debate. Systemic spread may not occur for many months or even years beyond the primary SOP diagnosis in humans and dogs, and studies in humans reveal no benefit from initiation of systemic chemotherapy before documentation of subsequent systemic spread. 42 , 53 Similarly, EMP of the GI tract in humans most often is treated with surgical excision and thorough staging of disease. Systemic therapy is not initiated unless systemic involvement is documented. Systemic chemotherapy has been used following excision of a gastric EMP in a cat, but the utility of adjuvant therapy in the species is unkown. 94

Long-term follow-up of patients with a solitary plasmacytoma is indicated so that recurrence of disease and systemic spread can be recognized. In patients with SOP, careful attention should be paid to the serum globulin levels, bone pain, and the radiographic appearance of bone healing. Restaging of the disease, including bone marrow evaluation, is indicated if systemic spread is suspected.

Prognosis

The prognosis for solitary plasma cell tumors generally is good. Cutaneous and mucocutaneous plasmacytomas usually are cured by surgical excision. 13 , 77 In large compilations of cases in dogs, the local recurrence rate was approximately 5%, and nodal or distant metastasis occurred in only seven of 349 cases (2%). 13 , 71 , 72 , 73 , 77 New cutaneous plasmacytomas at sites distant from the primary tumor developed in fewer than 2% of cases. One caveat: the author has encountered a handful of cases of aggressive multiple cutaneous plasmacytoma that eventual resulted in the death of the affected dogs. Neither the tumor cell proliferation rate (as measured by Ki-67 immunohistochemistry) in the dog nor histopathologic grading in dogs and cats was prognostic in large compilations of cases, although it has been suggested that the polymorphous blastic type may act more aggressively in the dog. 13 , 77 , 88 The presence of amyloid and overexpression of cyclin D1 (prognostic in human plasmacytomas) were not shown to be prognostic in dogs. 13

Most dogs with EMP of the alimentary tract and other abdominal organs (e.g., liver, uterus) that is treated by surgical excision alone or in combination with systemic chemotherapy (if metastasis is present) can enjoy long-term survival. 31 , 64 , 78 , 79 , 80 , 82 , 83 , 84 , 85 , 86 In a compilation of nine dogs with colorectal plasmacytoma, two had local recurrence at 5 and 8 months after surgery; after surgery alone, the overall median survival time was 15 months. 78 DNA ploidy and c-myc oncoprotein expression in biopsy samples were determined to be predictive for EMPs in dogs; however, tumors that were malignant all were from noncutaneous sites (i.e., lymph node, colon, and spleen), therefore location appears to be as predictive. 96 As previously discussed, most patients with SOP eventually develop systemic disease, but long, disease-free periods usually precede the event.

The prognosis in cats is less well defined, owing to the scarcity of reported cases. If disease is confined to a local site and/or regional nodes, surgical excision and chemotherapy can result in long-term control; however, early, widespread metastasis and progression to MM is also reported in cats. *

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Footnotes

If no response, substitute cyclophosphamide, 50 mg/m 2 , PO, SID, for 4 days; repeat weekly. PO, Orally; QOD, every other day; SID, once a day; IV, intravenously.

60 , 72 , 75 , 105 , 112 , 113 , 118 , 119 , 120 .

59 , 61 , 72 , 128 , 187 , 188 , 189 , 190 , 191 , 192 , 193 , 194 , 195 , 196 , 197 , 198 , 199 , 200 , 204 , 205 .

59 , 60 , 61 , 63 , 72 , 74 , 75 , 113 , 174 , 187 , 188 , 189 , 224 , 254 , 255 , 256 .

† 60 , 72 , 75 , 112 , 113 , 256 .

59 , 60 , 72 , 112 , 128 , 255 .

2 , 10 , 11 , 17 , 18 , 19 , 20 , 30 , 60 .

1 , 5 , 6 , 10 , 19 , 24 , 26 , 76 , 129 , 130 , 131 , 132 , 133 , 134 , 135 .

7 , 10 , 25 , 34 , 97 , 98 .

1 , 3 , 4 , 6 , 7 , 21 , 22 , 23 , 24 , 25 .

1 , 4 , 37 , 39 , 40 , 43 , 44 , 45 .

1 , 3 , 4 , 6 , 7 , 8 , 22 , 24 .

3 , 7 , 29 , 51 , 52 , 60 , 61 , 62 , 63 .

4 , 7 , 8 , 21 , 22 , 30 .

18 , 75 , 89 , 90 , 93 , 95 .