Canine Lymphoma

• Lymphoma is a diverse, heterogeneous disease that results from the uncontrolled clonal expansion of malignant lymphocytes. 
• The B-cell phenotype is predominant; the remainder consists of T- or rarely NK-cell phenotypes.¹

• Lymphoma is generally considered a systemic disease. 
• Sites of origin include lymphoid-rich tissues (ie, lymph nodes, spleen, thymus, bone marrow). 
• Extranodal sites affected may consist of epithelium, intestinal tract, and CNS. 
• Because of the systemic nature of this disease, any tissue may be involved. 

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• Lymphoma comprises 7% to 24% of all canine cancers; it is the most common hematopoietic cancer in dogs.^2,3
• Incidence is estimated at 13-24 per 100 000 dogs at risk.^2,3
• Incidence rate for dogs <1 year of age is 1.5 per 100 000; for dogs 10-11 years of age, incidence is 84 per 100 000.⁴

• Lymphoma is diagnosed worldwide in the canine population.

• There is no known cause; the disease is likely multifactorial.⁵
• Hypothesized but unproven causes include retroviral infection with Epstein-Barr virus–like viruses, environmental contamination with phenoxyacetic acid herbicides (2,4-dichlorophenoxyacetic acid [2,4-D]), magnetic field exposure, and immune dysfunction.⁵

• Multiple genetic and molecular pathway aberrations have been noted, but none of these factors have translated into clinically relevant information. 
• Chromosomal aberrations reported include gain of chromosome 13 and 31, as well as loss of chromosome 14.⁶
• Germline and somatic mutations, altered oncogene/tumor-suppressor gene expression, and epigenetic changes have been reported.^7,8
• Immunophenotypic differences among different breeds suggest heritable risks.⁹

• Middle-aged dogs are most commonly affected, but young dogs can be affected.⁵
• No sex predisposition has been reported consistently.⁵
• Boxers, golden retrievers, basset hounds, Saint Bernard dogs, Scottish terriers, and mastiffs are overrepresented.⁵

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• Dogs with impaired immune function are at increased risk for lymphoma.¹⁰
• Dogs with immune-mediated diseases are at increased risk independent of age and sex.¹⁰
• A case report of a dog that developed lymphoma following cyclosporine treatment has been reported.¹¹
• Infectious factors such as retroviral or Helicobacter infection may be involved though not definitive.⁵
• Environmental factors include herbicides (eg, 2,4-D)^12,13 and a weak association with magnetic fields.¹⁴

• Uncontrolled, clonal, neoplastic transformation and expansion of lymphocytes not restricted to specific anatomic sites 
• Disease progression in the lymph nodes, soft tissue, or extranodal sites leads to development of clinical signs. 
• Other signs may be related to paraneoplastic syndromes; the most common of these are anemia,¹⁵ hypercalcemia,¹⁶ and immune-mediated thrombocytopenia.¹⁷ 
• Hypercalcemia, most commonly with T-cell variant of the disease¹⁸ 

• Classification is based on anatomic location, staging, histologic criteria, and immunophenotype.^5,19 
• The most common form of the disease is the multicentric form; this most commonly involves the peripheral nodes but may also include liver, spleen, and bone marrow. 
• Other forms of the disease include GI (small or large bowel), mediastinal, cutaneous, and extranodal forms such as CNS, ocular, nasal, cardiac, lung, bladder, and bone. 
• World Health Organization’s Clinical Staging System for Lymphoma⁵:
• Stage I: 1 Lymph node involved or lymphoid tissue in a single organ (excluding bone marrow).
• Stage II: Involvement of many lymph nodes in a regional area.
• Stage III: Generalized lymphadenopathy.
• Stage IV: Liver and spleen involved ± stage III.
• Stage V: Bone marrow involvement or extranodal disease.
• Substage^{5, 20-24}
• a: without systemic signs.
• b: with systemic signs.

• Patients typically are presented with a history of rapidly progressive, nonpainful, generalized lymphadenopathy ± hepatosplenomegaly.
• Most are presented without signs of systemic illness (substage a).
• Clinical signs may be nonspecific (eg, mild lethargy, weight loss) or may represent the organ system that is infiltrated.
• Dogs that are clinically ill (substage b) have profound inappetence, lethargy, weight loss, vomiting, and/or diarrhea.^5,20-24
• Polyuria and polydipsia may be present in dogs with hypercalcemia of malignancy, secondary to other underlying disease or UTI.
• Other clinical signs related to the anatomic location of the disease include:
• Uveitis
• Cranial abdominal enlargement
• Dyspnea
• Mediastinal involvement or pleural fluid
• Regional lymph edema
• Dermal or subcutaneous masses
• Vomiting or diarrhea
• Bruising
• Pallor
• Stridor and stertor if retropharyngeal nodes are involved

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• Complete physical examination, including rectal examination, should be performed. 
• Complete staging before initiation of therapy is always recommended; however, in select cases, staging may not be performed in entirety because of owner financial constraints.
• Minimum database includes CBC, serum chemistry panel, urinalysis ± urine culture if clinically indicated.
• CBC may indicate infection that requires treatment prior to chemotherapy or cytopenia consistent with myelophthisic or paraneoplastic syndromes. 
• Serum chemistry panel often reveals changes consistent with infiltrative disease such as:
• Elevated ALP, ALT
• Hyperbilirubinemia
• Hypercalcemia
• Azotemia
• Urinalysis may indicate infectious cystitis that should be treated prior to initiation of chemotherapy. 
• Three-view thoracic radiographs are recommended.
• Mediastinal lymph node enlargement or tracheobronchial lymph node enlargement may be noted.
• Occult pneumonia may be present.
• Rarely, infiltrative disease (diffuse interstitial pattern) will be seen.
• Abdominal ultrasonography is recommended if GI signs are present or if no peripheral lymphadenopathy is appreciated.
• Echocardiography prior to doxorubicin may be recommended if patient is an at-risk breed for cardiomyopathy or if murmur or arrhythmias are noted.^5,24,25 
• Lymphoma findings may be present on examination of a bone marrow aspirate.
• Involvement conveys a worse overall prognosis.
• Determine if marrow reserves are sufficient for chemotherapy. 
• Fine-needle aspiration of affected node(s) or organs should be performed.
• Results are often adequate to obtain diagnosis, as most cases are large-cell variant; monomorphic population of small or intermediate cells may require histopathology or molecular diagnostics to further characterize.
• The author recommends avoiding areas of high reactivity if possible (eg, mandibular nodes).
• Cells are generally >2 times the diameter of a red blood cell or larger than neutrophils and appear as a monomorphic population.
• Histopathology is considered the gold standard and allows for evaluation of tissue architecture.
• It also allows for classification into low-, intermediate-, and high-grade variants, which may affect treatment and prognosis.¹⁹
• Immunohistochemistry is provided through commercial laboratories.²⁶
• Molecular diagnostics may be recommended when cytology and histopathology are suggestive but confirmation or immunophenotyping is indicated.
• PARR (PCR for Antigen Receptor Rearrangement) can determine if the majority of cells in the sample are derived from the same original clone vs multiple clones.²⁶
• PARR assay is 94% specific for lymphoid neoplasia; sensitivity is 75%.
• It can be performed on blood, lymph node, bone marrow, cavity fluid, and CSF.^5,26
• Flow cytometry involves staining live cells with labeled antibodies that bind proteins expressed on the cell surface. 
• Flow cytometry is an interpretive test and can provide additional useful information regarding prognosis and treatment.^27,28
• T-cells express CD3 (CD4 and CD8)
• B-cells express CD21 (CD20, CD79a)
• This can be performed on blood, lymph node, bone marrow, cavity fluid, and CSF.

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• Reactive lymphoid hyperplasia
• Systemic infection
• Bacterial (eg, Rickettsial disease)
• Fungal
• Parasitic
• Viral
• Immune-mediated disease

• The prognostic factors with the most significance regarding overall survival time are phenotyping and clinical substaging.
• Median survival times for dogs without treatment is 4-6 weeks^29,30; with gold standard therapy, survival times are 1 year with 25% chance for 2-year survival.⁵
• The most significant negative prognostic factors are substage b, T-cell phenotype, mediastinal location, and hypercalcemia.
• Other negative factors include the presence of anemia, gastrointestinal location, and stage V disease (bone marrow involvement).
• Small cell/low grade/indolent lymphomas are associated with longer survival times because of slow progression of disease but are considered less chemoresponsive.
• Clinical staging is controversial; in general, I=II>III=IV>V.
• Dogs with a longer initial remission generally have a better long-term outcome and often respond favorably to re-induction therapy when relapse is noted. 

• Because of the systemic nature of disease, chemotherapy is considered the mainstay of therapy for large-cell lymphoma.
• There are many protocols available, and the individual protocol should be tailored to patient and owner. 
• However, current standard of care consists of CHOP-based protocols.
• The following protocols are recommended as a starting point when discussing therapeutic options with owners:
• CHOP-based therapy ^5,22,29-31
• 80%-90% of patients experience remission within the first 4 weeks of therapy
• Median survival time of 12 months with 25% survival at 2 years⁵
• 70% response and 6-8 month survival for stage V or T-cell disease
• Single-agent doxorubicin therapy ^32,33
• 50%-75% response rate
• Median survival of 6-9 months
• Single-agent CCNU therapy³⁴
• ≈40%-50% response rate
• Median survival of ≈4 months
• Frontline therapy for canine cutaneous lymphoma³⁵
• Prednisone monotherapy³⁰
• ≈50% response rate
• 1-3 month survival time
• The author strongly recommends reviewing the necessary personal protective equipment, administration techniques, and chemotherapy side effects/adverse events before consideration of any chemotherapeutic agent.
• Consultation with a board-certified medical oncologist is recommended.
• There is not a one-size-fits-all protocol, and new protocols, treatment options, and clinical trials may be available.

• Monoclonal antibody therapy for large T-cell lymphoma³⁶
• This is being used on a clinical trial basis under a conditional USDA license in combination with chemotherapy.
• AT005, first T-cell biological therapeutic
• Caninized monoclonal antibody³⁷
• Targeted immunotherapy that specifically recognizes CD-52 expressed on T-Cell
• No known contraindications for use in T-cell lymphoma.
• Standard hypersensitivity reactions (eg, vomiting, nausea, cutaneous erythema  swelling, pruritus) are uncommon.
• Efficacy data is pending trial results.
• Monoclonal antibody therapy for large B-cell lymphoma.^38-40
• Canine Lymphoma Vaccine, DNA (merial.com; conditional licensure by USDA)
• Targeted immunotherapy that specifically recognizes CD20.
• Therapeutic immunization to be used on achieving remission with chemotherapy. 
• Survival time of vaccinates after completion of 25-week CHOP protocol is >734 days, median not reached, which represents a significant improvement over previously reported historical survival times of 1 year.³¹

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• The role of radiation therapy in the management of lymphoma remains under investigation, and no protocols are well-established in clinical practice.^41-42
• Discussion with a board-certified radiation oncologist is strongly recommended prior to referral for this modality. 
• Radiation therapy may be used in select cases: 
• Localized (nasal, CNS) or stage I disease (1 node involved)
• Palliation of local disease (solitary location or refractory node)
• Whole-body radiation with bone-marrow transplant
• Staged half-body irradiation after chemotherapy

• Staging test costs are dependent on decision-making between owner and clinician, depending on the degree of work up: $$-$$$$
• Diagnosis, cytology $$
• Diagnosis, histopathology $$$
• Diagnosis, IHC-Flow cytometry $$$
• Treatment
• Multi-agent protocol $$$$$
• Monoclonal therapy $$$$$
• Single-agent doxorubicin $$$$
• Radiation therapy $$$-$$$$
• Single-agent CCNU $$$
• Prednisone therapy $

2,4=D = 2,4-dichlorophenoxyacetic acid, ALP = alkaline phosphatase, ALT = alanine aminotransferase, CNS = central nervous system, CSF = cerebrospinal fluid, PARR = PCR for Antigen Receptor Rearrangement, PCR = polymerase chain reaction