Initial Diagnosis

• Fine needle aspiration (FNA) and cytology: From peripheral lymph node, organ, blood/bone marrow, or other site. Pros: quick, non-invasive. Immunocytochemistry may be done for immunophenotyping (T versus B cell). Definitive cytological diagnosis of lymphoma is reliable, however, in situations where this is not possible additional diagnostics are required. Such situations include small cell/low grade lymphoma, reactive lymph nodes, splenic location and non-diagnostic samples due to cell rupture or blood contamination. In the latter situation, adjustments in technique such as non-aspiration, smaller needle gauge, and using gentle techniques in making slides can improve diagnostic quality.
• Biopsy and histopathology: Histopathology with immunohistochemistry (IHC) is gold standard for canine lymphoma diagnosis and subtyping. Excision of a whole lymph node is recommended, but punch or needle core biopsies may also be diagnostic. Histopathology is indicated when cytology is inconclusive, FNA is low yield (e.g., minimally thickened intestinal wall), or additional information about subtype is desirable. Pros: generally relatively quick turnaround time, (IHC may take longer). Can give significantly more information if performed by a pathologist experienced in lymphoma subtyping. Cons: More invasive and expensive than FNA and cytology.
• PCR for antigen receptor rearrangement (PARR): Test for clonality, aim is to distinguish lymphoma from reactive lymphocyte population. Used where a definitive diagnosis of lymphoma cannot be made on cytology or histology alone. Pros: Can be performed on cytology or histologic preparations, usually without the need for collection of additional samples. High specificity. Cons: Can be long turnaround time, often expensive, variable sensitivity (approximately 70-90%, can be affected by the tissue sampled and primers used). Best used for diagnosis rather than immunophenotyping of known lymphoma. Most commonly applied in suspected small cell GI lymphoma, in combination with histopathology and IHC.
• Flow cytometry: Assess cell surface markers using specific antibody stains. Pros: Superior to PARR for immunophenotyping, may be suggestive for diagnosis (cannot be used to definitively identify a clonal population), can diagnose some specific subtypes (e.g., loss of CD45 in T-zone lymphoma), expression of additional proteins can be assessed (e.g., Ki67 or MHCII) which may have prognostic implications. Relatively non-invasive (fine needle aspiration), quick turnaround. Cons: handling and shipping requirements may limit availability, as cells must be alive and in suspension.
• Newer biomarker tests (cRP, TK, haptoglobin) are available but their utility in routine clinical practice has not been demonstrated.

Additional diagnostic tests: In my practice, I recommend additional diagnostic tests for a dog with known lymphoma if they alter prognosis, treatment options (what I would recommend or what the owner would choose to do), or there are abnormalities present that cannot be easily attributed to lymphoma.

• CBC, serum chemistry, urinalysis: Minimum database required if chemotherapy is considered. Thrombocytopenia, lymphocytosis, and neoplastic lymphocytes on blood smears in dogs with multicentric large cell lymphoma are associated with bone marrow involvement.^1,2 Assess for abnormalities that may require alterations in chemotherapy protocol (e.g., liver function in dogs to receive vinca alkaloids), be directly associated with lymphoma (e.g., hypercalcemia implying likely T cell immunophenotype in large cell lymphoma) or other concerns. Proteinuria is common in dogs with cancer, including lymphoma,^3,4 and may require ongoing monitoring, further investigation or intervention.
• Staging:
1. Involving a single lymph node, or lymphoid tissue in a single organ (excluding bone marrow).
2. Involvement of multiple lymph nodes in a regional area.
3. Generalised lymph node involvement.
4. Liver and/or spleen involvement (+/- stage III).
5. Blood, bone marrow, and/or other systems.

Each stage is subclassified into:

• Substage a: With systemic signs
• Substage b: Without systemic signs

In order to assign stage, full evaluation would involve, in addition to the minimum database, thoracic and abdominal imaging (usually chest radiographs and abdominal ultrasound) and sampling for cytology or histopathology, bone marrow evaluation, and other evaluation depending on clinical presentation (e.g., MRI if neurological signs). The prognostic impact of stage beyond stage III in dogs with multicentric lymphoma has not been fully defined. Certainly, involvement of liver and spleen does not seem to have a significant impact on prognosis, and bone marrow may or may not. Therefore, if complete staging does not significantly impact prognosis or treatment options, for many oncologists and owners, it is preferable to spend money on treatment rather than additional diagnostic tests. However, if there are specific localising signs e.g., vomiting, coughing, further evaluation may be more warranted. As will be discussed later, large cell gastrointestinal (GI) lymphoma generally has a poorer prognosis and so identifying GI involvement may impact approach in cases with suggestive clinical signs.

• Immunophenotype: In large cell multicentric lymphoma, immunophenotype is one of the strongest prognostic factors and, therefore, immunophenotyping is always recommended. IHC is considered the gold standard, however, collection of biopsies solely for purposes of IHC in a dog with previously diagnosed lymphoma may be difficult to justify from the standpoint of invasiveness and cost, when less invasive options exist. In my opinion, flow cytometry would be considered the next preferred option, followed by immunocytochemistry and then PARR because of the limitations already discussed in turnaround time and sensitivity.

Canine lymphoma classification:

• Subtype: The WHO classification scheme is based on tissue architecture, cell size, mitotic rate, cellular features, and immunophenotype. There are approximately 40 different subtypes. Pathologist expertise is important when classifying lymphoma and there is good, but not perfect agreement between pathologists (approximately 90% when only the 6 most common types are considered). From a practical standpoint, the important considerations are: identifying B versus T cell in large cell lymphoma, identifying T zone lymphoma separately from other T cell lymphomas, and identifying low grade B cell lymphomas, especially splenic marginal zone lymphoma. As well as considering the WHO subtype, clinical progression must be considered. T zone lymphoma, marginal zone lymphoma, mantle cell lymphoma and follicular lymphoma are generally considered indolent in behaviour. They often present as peripheral lymphadenomegaly (+/- lymphocytosis) which is slowly progressive. Although they are generally poorly responsive to standard maximum tolerated dose chemotherapy, prolonged survivals are common. Treatment is often not initiated at the time of diagnosis unless there are clinical signs or evidence of more rapid or advanced clinical progression. When treated, the most common initial protocol is chlorambucil and prednisolone. Within these ‘indolent’ subtypes, those of B cell origin generally do not have as good a prognosis as T zone lymphoma. There are some cases where an indolent lymphoma can undergo transformation to acquire more malignant behaviour and rapid progression. These are generally associated with a poor outcome, and reinforce the need to consider the clinical behaviour as well as the subtype.
• Anatomic location is also part of WHO classification, but some specific sites bear separate consideration.
• Hepatosplenic and primary hepatic - generally aggressive and associated with lower likelihood of response to chemotherapy.
• Splenic - often marginal zone and can do quite well with splenectomy alone, however, case selection is important - i.e., confined to spleen, not large B cell lymphoma.
• Rectal - mostly B cell and tend to have a good prognosis with chemotherapy based on the small numbers of dogs published in the literature.⁵
• Gastrointestinal - High grade/large cell GI lymphoma in dogs is generally associated with a poor outcome. Recently, low-grade/small cell GI lymphoma has been recognised and seems to behave like the more common low-grade/small cell GI lymphoma in cats. Outcomes tend to be much better and recommended treatment is different (oral chlorambucil and prednisolone versus multi-agent CHOP or other chemotherapy protocol for high grade/large cell lymphoma).
• Epitheliotropic T cell lymphoma
• Cutaneous: Most are diffuse and respond well initially to chemotherapy but progress within a few months. Alternative treatments such as isotretinoin or safflower oil may help in some cases. Solitary lesions may be associated with a better prognosis (Chan).
• Oral/mucocutaneous: If lesions are localised to the oral cavity then outcomes with chemotherapy or radiation therapy can be very good (>1–2 years), and again solitary lesions may be associated with better prognosis.^6,7

Summary

Canine lymphoma encompasses a group of diseases with varying clinical presentations, need for diagnostic tests, treatment options and prognosis. In my clinical practice, to treat a dog with lymphoma, the things I need to know are:

• Subtype as much as is reasonable i.e., large cell/small cell, immunophenotype, and clinical behaviour at a minimum. Flow cytometry is my preferred option for immunophenotyping following a cytological diagnosis as it also enables diagnosis of T zone lymphoma. If a diagnosis has been made on histopathology, IHC is preferred for immunophenotyping.
• Minimum database of CBC/serum chemistry/urinalysis.
• Substage - assigned based on systemic signs.
• In a dog with a confirmed diagnosis of multicentric lymphoma, additional diagnostic tests are not uniformly recommended in order to assign the case to stages I-V, unless it is felt that prognosis or treatment options would be changed by the result of these tests.

References

1.  Vail DM, Withrow SJ, eds. Withrow and McEwen’s Small Animal Clinical Oncology, 5th ed. W.B. Saunders, Philadelphia, PA: 2012 is recommended as a general resource.

2.  Graff EC, Spangler EA, Smith A, Denhere M, Brauss M. Hematologic findings predictive of bone marrow disease in dogs with multi-centric large-cell lymphoma. Vet Clin Pathol. 2014;43:505–512.

3.  Martini V, Melzi E, Comazzi S, Gelain ME. Peripheral blood abnormalities and bone marrow infiltration in canine large B-cell lymphoma: is there a link? Vet Comp Oncol. 2015;13:117–123.

4.  Prudic RA, Saba CF, Lourenco BN, Bugbee AC. Prevalence of proteinuria in a canine oncology population. J Small Anim Pract. 2018. DOI: 10.1111/jsap.12840 Epub ahead of print.

5.  Di Bella A, Maurella C, Cauvin A, Schmidt JM, Tapia BB, North SM. Proteinuria in canine patients with lymphoma. J Small Anim Pract. 2013;54:28–32.

6.  Van den Steen N, Berlato D, Polton G, Dobson J, Stewart J, Maglennon G, Hayes AM, Murphy S. Rectal lymphoma in 11 dogs - a retrospective study. J Small Animal Pract. 2012;53:586–591.

7.  Berlato D, Schrempp D, Van den Steen N, Murphy S. Radiotherapy in the management of localised mucocutaneous oral lymphoma in dogs: 14 cases. Vet Compar Oncol. 2012;10:16–23.

8.  Chan CM, Frimberger AE, Moore AS. Clinical outcome and prognosis of dogs with histopathological features consistent with epitheliotropic lymphoma: a retrospective study of 148 cases (2003–2015) in Australia. Vet Dermatol. 2017. DOI: 10.1111/vde.12504 Epub ahead of print.