Defining the neoplasm

These are neoplasms of lymphoblasts that produce leukemia or lymphoma (Figure 7.1A,B).^1–4 Immunophenotyping is required to determine if they are of B‐ or T‐cell type as the cells look identical via cytology.^5–8 B‐cell acute lymphoblastic leukemia (B‐ALL) is a malignant proliferation of lymphoblasts that arise in the bone marrow, there are numerous neoplastic cells in circulation, cytopenias are expected and it has a rapidly progressive course with a fatal outcome. B‐ALL is reported to be more common than T‐ALL in dogs but the data are not uniform.^9–12 ALL and CLL are the most common lymphoid leukemias in dogs and they have very different prognoses. Their distinction is important. ALL is rare in cats and CLL is uncommon.¹³ B‐cell lymphoblastic lymphoma (B‐LBL) is also a tumor of lymphoblasts. It is uncommon to rare: it was not recognized in 600 dogs with lymphoma¹⁴ and was less than 2% in a report of 1000 dogs.¹⁵ T‐cell lymphoblastic tumors are more common than B‐cell ones in dogs.^13–15

LBL arises in a peripheral lymph node and does not involve the bone marrow or blood, at least not initially. These are defining lines between the tumors, but as each disease progresses their morphologic features start to overlap. ALL (B‐ or T‐cell) will enter lymph nodes and other organs, and LBL (B‐ or T‐cell) will infiltrate organs and bone marrow and leukemia can develop. The latter is referred to as secondary leukemia, leukemic phase of lymphoma, or stage V lymphoma, if it was certain the origin was in solid lymphoid tissues such as lymph nodes.

Some classifications rely on where the greatest tumor burden is suspected to be – bone marrow, nodal, or extranodal. In some cases the distinction is difficult or impossible. Regardless of definitions, at these stages when the tumors are advanced and neoplastic cells can be found in nodes, bone marrow, blood, and organs, the prognosis is poor and survival is measured in days to weeks. B‐ALL and B‐LBL are tumors of lymphoblasts and they cannot be differentiated in advanced disease states.

The lines also become blurred between B‐cell and T‐cell ALL and therefore throughout this section many times only the abbreviation ALL is used. One study reported that the majority of ALL in dogs were B‐cell in origin (>90%),⁹ another study that T‐cell types were more common (>90%),¹⁰ and two other studies reported approximately 60–70% B‐ALL and 30–40% T‐ALL, but a few had atypical phenotypes.^11,12 Differences this wide make summarizing data problematic. Level of expertise, antibodies used, and how tumors are defined influence final conclusions. Until we have standardized techniques, antibodies, and definitions there will be conflicting data which makes how to use that data, especially for non‐hematopathologists, difficult in diagnostic settings. Regardless, ALL of B‐cell or T‐cell origin is a lethal disease with short survival times.^11,15,16

Also, confounding diagnoses is the use of the term “lymphoblast.” Lymphoblasts can be defined by morphologic, immunologic, or functional parameters. The term lymphoblastic is used in human and veterinary histology to describe a relatively small cell fully capable of cell division. In contrast, in veterinary pathology large dividing lymphoid cells, especially if nucleoli are seen, are loosely called “blasts,” which renders the cytologic recognition of this cell type less specific. True stem cell lymphomas are rare or not recognized in veterinary pathology. Lymphoblastic is a term we have used imprecisely in veterinary pathology, probably most frequently by non‐hematology specialists, for a large immature cell with large nuclei and nucleoli. Many of these were likely large cell lymphomas and not lymphoblastic tumors. The designation lymphoblastic based on morphology should be reserved for cells that are intermediate in size, larger than a mature lymphocyte. They have minimal cytoplasm, a high nucleus‐to‐cytoplasmic ratio, and nuclei that are approximately 1.5–2 red blood cells in diameter and that have uniformly fine dispersed chromatin with no or inconspicuous nucleoli. If evaluated they will have a high proliferative rate. They could be confused with CLL or AML. Lymphoblasts are not large lymphoid cells with open chromatin and prominent nucleoli, these are large cell lymphomas. B‐LBL is uncommon but DLBCL is the most common lymphoma in dogs and common in cats. B‐LBL and DLBCL are different tumors but there are no antibodies or clear morphologic criteria to distinguish these two diagnoses in veterinary medicine. A neoplasm that is positive for CD34 favors ALL over lymphoma but exceptions will exist. CLL are considered CD34 negative. Until markers are developed that recognize B‐LBL it is likely that intermediate to large B‐cell lymphomas will be classified as DLBCL which is the most common lymphoma of dogs.

Reports of “high‐grade lymphoma” of B‐cell type also do not clearly define or separate B‐LBL from DLBCL.⁹ Therefore to distinguish B‐LBL from DLBCL we rely on subtle morphologic differences in nuclear size and chromatin. Canine B‐cell (<2%) and T‐cell (3%) lymphoblastic lymphomas are uncommon lymphomas.^7,9,14,15 They are also uncommon in cats and horses, less than 1%. Large B‐cell lymphomas are common in dogs, cats and horses but these are not lymphoblastic.

Epidemiology, occurrence, and clinical presentation

Lymphoblastic leukemias and lymphomas occur in all species^4,17 and are most often seen in cattle¹⁷ and dogs.^3,9,10,17 ALL can be identified as T‐ or B‐cell, but cases reported in the older literature were not separated into T‐ or B‐cell phenotypes. The typing of acute leukemias in animals needs continued investigations as it is likely that many undifferentiated leukemic cells have been assumed to be lymphoid and actually are of myeloid origin. Broad batteries of antibodies have not been available until recently to verify this distinction (see Myeloid section of this chapter). The morphology of poorly differentiated neoplasms can look similar and should not be relied on to identify cell of origin.

Lymphoblastic leukemia and lymphoma can occur in calves less than 6 months of age.¹⁷ Rarely, calves may develop the tumor in utero and it causes dystocia due to marked hepatosplenomegaly. Cases of ALL in the calf are not associated with BLV. These calves are in poor body condition and infarcts in bones are present. Dairy cows with ALL, in contrast to lymphoma, present in good body condition and are first noticed because of a rapid reduction in milk production.¹⁷ Dairy bulls also maintain good body condition and the diagnosis is sometimes made from blood taken during an annual examination. The laboratory result of a leukemia is a paradox compared to the apparent excellent health of the bull. In contrast to most other animals, calves that present with ALL are in poor body condition with a rough dull hair coat and enlarged peripheral lymph nodes, up to 10 cm in diameter.¹⁷ Calves are often recumbent, likely due to bone pain from multiple areas of marrow infarction (see Figure 10.44).

Horses are rarely identified with ALL. The few cases reported were horses in racing or other disciplines and were examined because of a rapid decline in performance.¹⁷

Dogs with LBL are also usually in good condition and anorexia or physical exam abnormalities are recent developments as the disease has an acute onset and progresses rapidly. Only one or two lymph nodes may be enlarged at presentation but multiple nodes quickly become enlarged. Dogs with ALL present from 4 to 12 years of age.^3,9,12,17 In dogs the disease seems to have a bimodal age distribution, appearing most often in dogs less than 5 years of age and in old dogs.¹⁷ In a study of 210 dogs with hematopoietic neoplasia 51 (24%) were ALL and 61 (29%) were CLL, 92% of ALL were B‐cell and 89% of CLL were T‐cell.⁹ The average age of dogs with ALL was 7.4 years, there was no sex or specific breed predominance but larger breeds were overrepresented.⁹ Younger dogs that develop B‐cell ALL or CLL seem to have a worse prognosis than older dogs.¹⁸ In one study the golden retriever was overrepresented.¹²

The incidence of ALL in cats has decreased with increased vaccination for FeLV. ALL occurs in cats but is a rare neoplasm compared to other leukemias and lymphomas.¹³

Pathology

Cellular morphology

Cytologically, lymphoblastic tumors are intermediate in size with nuclei about 1.5 red cells in diameter. However, some nuclei are long and oval‐shaped and could be called large cells in any other subtype of lymphoma. The chromatin does not have coarse aggregations; it is dispersed and this pattern makes visualization of the multiple small nucleoli that are present difficult (Figure 7.1A). In histologic preparations this feature should be examined at the periphery where fixation is best. Cytoplasm is minimal and lightly basophilic. Nuclei reside close together and there is a high mitotic count with 10 or more mitoses/400× field. The mitotic figures are unique and are not the characteristic metaphase type that is easy to recognize. There will be numerous apoptotic cells in each field and these should not be counted as mitotic figures. Having provided these features, it is a reality that many cases of ALL will look identical to AML or even CLL. Morphology can be used but should not be the sole diagnostic criterion. All the case data need to be interpreted, including onset of disease (rapid or indolent), phenotype, broad batteries of antibodies, and which cytopenias are present if any.

Cytochemistry and immunohistochemistry

Immunohistochemistry or flow cytometry are needed to identify B‐ versus T‐cell immunophenotype. B‐ALL in tissues is routinely positive with the B‐cell markers CD79a, CD20, and CD21 after antigen retrieval is carried out on FFPE samples.² The cells in blood can also be effectively phenotyped by flow cytometry or fixing unstained blood films for up to a minute in formalin. This adheres the cells to the glass slide and permits a wide range of stains to be used after heat‐activated antigen retrieval, including CD79a, CD3, and CD45.⁷ If only stained films are available it is possible to decolorize Wright–Giemsa‐stained slides with acetone before or after formalin fixation.^1,7 A large battery of antibodies can be used in flow cytometry and these may be needed to distinguish B‐ versus T‐cell ALL and to distinguish both from AML and CLL. Detection of cytoplasmic antigens by flow cytometry requires permeabilization of cell membranes, which makes the technique prone to artifact. Therefore, flow cytometry data that use antibodies directed at cytoplasmic antigens should be interpreted cautiously. Flow cytometry and IHC identify phenotypes but do not differentiate neoplastic cells from reactive cells. However, the homogeneity of patterns in tissue sections or in flow cytometry help to identify clonal versus heterogeneous cellular proliferations.

A 2015 report used alkaline phosphatase (ALP) to distinguish lymphoma and CLL (all were negative) from myeloid and myelomonocytic leukemias (all positive).¹⁰ ALP did not completely distinguish ALL from AML as it was weakly positive in about one‐third of the cases of ALL.¹⁰ AML is usually CD34 positive, ALL is variable but CLL is CD34 negative. Several genetic changes have been identified in humans but in animals the diagnosis rests on morphologic recognition of lymphoblastic cells in cytology and histology combined with immunophenotyping and histochemistry.

See T‐cell lymphoblastic leukemia/lymphoma in this chapter for details on IHC. If the neoplasm is positive to CD79a in histology or cytology it is considered B‐cell. However, some AMLs may be positive to CD79a and some B‐ALL may be CD79a negative. Therefore consider using more than one B‐cell antibody and technique. A dog with tumor cells in circulation that are immunoreactive to CD79a, CD20, CD21, and CD34 and negative to CD3 via flow cytometry is considered B‐ALL. B‐CLL is possible but it is uncommon (80% are T‐CLL), cells are CD34 negative and the onset and progression of CLL should be indolent and neutropenia absent. Another potential problem is that CD79a may bind with the nucleus and this cannot easily be differentiated from cytoplasmic binding and therefore could cause a false‐positive. If this is a concern use IHC or immunocytochemistry (ICC) to determine the staining pattern of CD79a (membrane staining is a positive reaction) and/or consider other antibodies.

Differential diagnosis

The diagnosis of lymphoma is usually easy with cytology or histopathology as neoplastic cells are numerous in the blood or have effaced or at least filled lymph nodes and/or bone marrow. Categorizing as lymphoblastic versus large cell is subjective and is based on size of cell, nuclei, and prominence of nucleoli. The larger the cells and nuclei and the more prominent the nucleoli the more likely the diagnosis is a large cell lymphoma and not lymphoblastic. Most large cell lymphomas in dogs and cats are DLBCL. The high‐grade B‐cell lymphomas and Burkitt’s‐like lymphoma will have numerous tingible body macrophages, which are low or absent in LBL. The node appears more deeply stained than usual because of the prominent and dispersed nuclear chromatin. ALL has an immature lymphocytosis and CLL has mature lymphocytosis, ALL is usually CD34 positive and CLL is CD34 negative. Neutropenia and other cytopenias are expected with ALL and not CLL. The onset of disease and the duration of survival are entirely different. B‐cell ALL is differentiated from T‐cell ALL by immunophenotyping techniques, but techniques and antibodies can influence results. Both B‐ and T‐cell ALL can look similar to myeloid leukemia especially if the cells in AML have few or no granules. If an AML does not demonstrate maturation, that further confounds the distinction. See the Myeloid neoplasms section of this chapter for complete antibody profiles that could be used.

If leukemic cells react with T‐ or B‐cell markers it is considered lymphoid. Both B‐ and T‐cell ALL should be negative for mature myeloid markers. If a tumor was CD34 positive and CD45 positive, but negative for B‐cell and T‐cell markers then the diagnosis from immunophenotyping is AML. AML should also be positive for myeloid markers CD11b, CD11c, and/or CD14 on flow cytometry, and/or histochemistry positive with myeloperoxidase, chloroacetate esterase, or Sudan black B. Histochemistry for ALP will be negative for lymphoma and CLL and positive for monocytic myeloid leukemias and about one‐third of ALL.¹⁰ If a leukemia was CD34 positive, ALP positive, and had morphologic features of myeloid lines then AML is favored over ALL. See T‐ALL in this chapter for further use of histochemistry and IHC to differentiate these neoplasms.

Prognosis

B‐ALL and T‐ALL are malignant tumors and some of the most aggressive cancers in veterinary medicine. Dogs with ALL and AML have short survival times measured in days to weeks.^11,15,16,18 Although the numbers of dogs in the different groups were small, those with B‐ALL had median survival time (MST) of 8 days, range 5–46 days; dogs with T‐ALL had MST of 10 days, range 4–120; dogs with AML had MST of 10 days, range 3–73 days.¹¹ The clinical features of advanced disease include lymphocytosis >30,000/μL, cytopenias, neoplastic cells 50–100% in the marrow, systemic involvement of liver, spleen and other organs and signs of hepatic or renal failure. CD34 is expressed by early precursors, dogs with CD34‐positive leukemic cells have short survival times (MST of 16 days).^11,16 Dogs with a neutrophil count in the reference interval have longer survival times and perhaps the absence of anemia is a more favorable indicator.¹¹ Regardless, ALL is a lethal disease.

Dogs with B‐cell lymphocytosis (CD21 positive) were separated by the size of the neoplastic cells.¹⁶ Dogs with large cell B‐lymphocytosis had an MST of 130 days and the MST was not reached for B‐lymphocytosis of small cells. Cell size was determined by flow cytometry on the peripheral blood. This study did not try to distinguish leukemias from stage V lymphoma or to classify the neoplasms with the REAL/WHO system. Therefore there were likely dogs with different diseases in each group (e.g. B‐ALL, B‐LBL with leukemia, B‐CLL, DLBCL, etc.). This is not a criticism; in fact it makes identifying groups of dogs easier as there are fewer parameters evaluated and histology is not needed for prognostic purposes. See Precursor T‐cell lymphoblastic leukemia/lymphoma in this chapter for additional information.

With advancing ALL an FNA of any area of the liver will provide tumor cells similar to those in the blood. With LBL, more favorable signs at diagnosis are probable if the tumor is limited to a single node, and there is an absence of systemic disease and retention of appetite and activity. With advancing disease multiple nodes are enlarged, liver and spleen is involved, leukemia can be present, the animal is lethargic, and there are signs of systemic disease.

Defining the neoplasm

These are neoplasms of small lymphocytes, either B‐ or T‐cell types that result in leukemia or lymphoma (Figure 7.2). Immunophenotyping is needed to distinguish B versus T, but if cytoplasmic granules are present then the tumor is of T‐cell type. In humans, CLL is a B‐cell tumor (>95%),^1,2 but in dogs only 7 of 61 cases (11%),³ 26% of 73 cases,⁴ or 17 of 43⁵ were of B‐cell type and 1 of 18 cats was B‐cell.⁶ Approximately 80% (75–90%) of canine CLL cases are T‐cell,^3,4 95% (17/18) of cats are T‐cell, and 94% of these were T‐helper CD3+/CD4+/CD8− phenotype.⁶ Aberrant phenotypes have been reported for dogs.^4,5 Horses have been inadequately studied. Cows with CLL are usually of B‐cell type. If tumors are not phenotyped then the diagnosis is CLL.

B‐cell CLL is a neoplasm of the bone marrow and is characterized by marked lymphocytosis (leukemia) of small mature B lymphocytes. Animals often have few or no clinical signs and the disease has a slow but persistent progression. Spleen and liver are usually enlarged due to tumor cells but lymph nodes may be normal size. B‐cell small lymphocytic lymphoma (SLL) is a tumor of small mature B lymphocytes involving lymph nodes, spleen, and solid organs but leukemia is not present or neoplastic cells in blood are few. SLL is an uncommon lymphoma in dogs and is much less common than CLL. It has a slow rate of progression.

B‐cell prolymphocytic leukemia (PLL) is related but is an earlier stage of maturation than mature lymphocytes and is more aggressive. In terms of organ distribution it is a mirror image of CLL. Morphologic identification is accomplished better with cytology in which the nuclei appear more “immature,” are larger, the chromatin is in aggregates joined by fine chromatin bands, and the nucleolus is prominent (Figure 7.3). Since the prognosis and therapy differ from those in CLL the distinction is clinically important.

Epidemiology and occurrence

Lymphoma and leukemia of small mature lymphocytes are indolent, B‐cell and T‐cell. They are slowly progressive neoplasms that may involve bone marrow or peripheral tissues or both. They are considered neoplasms of accumulation rather than proliferation. Aggressive variants have been identified, usually associated with atypical phenotypes.^4,5 B‐cell CLL is uncommon in domestic animals, although reported in dogs, cats, and other species.^4,7,8–12 In the American College of Veterinary Pathologists (ACVP) study of 1000 cases of canine lymphoma there were 8 cases of B‐cell SLL. In humans there is a strong male predominance but there is no sex or no breed predominance in animals. In animals, as in humans, the tumor is found most often on routine examination of blood. Cells of PLL type are seen in humans,¹³ mature cows, and dogs, but are relatively rare.⁹

Clinical presentation

Most cases are asymptomatic and the disease is discovered from a CBC taken at an annual exam that reveals lymphocytosis. Weight loss is reported in approximately 50% of the cases. Cats and dogs with CLL and SLL are usually over 5 years of age, the mean age for 134 dogs with CLL (B and T) was 10 years, the range was 1.5–19 years^3,4 and 17 cats with T‐cell CLL the mean age was 12.5 years.⁶ Only one cat in this series had B‐cell CLL. Animals in an accelerated phase of the disease occasionally present with epistaxis, likely due to thrombocytopenia; some will have diarrhea and vomiting. In dairy cattle the most common clinical problem is a drop in milk production and feed consumption.

Pathology

Cytochemistry and immunohistochemistry

B‐cell CLL and SLL are differentiated from the more common T’cell phenotype by IHC or flow cytometry (Figure 7.2). The B‐cell CLL and SLL cells are positive in tissues with CD79 alpha, CD20, or CD21 and are negative with CD3. B‐cell CLL cells are negative with CD5 (unlike humans) and should express CD1c (95%) and CD1a (78%).⁴ Cells from animals and humans with CLL are negative for CD34, which is expected to be positive in ALL and AML. CD34 is a surface glycoprotein expressed on hematopoietic stem cells (and other cells) and is useful to distinguish acute and chronic leukemia. It should be negative in CLL and most lymphomas and positive in ALL and AML.

All of these neoplastic cells can have stages or types in which the morphology of the cells are identical or so similar that phenotyping, especially with flow cytometry and multiple antibodies, may be necessary to correctly identify the neoplasm. Neutropenia is not present in CLL and is a feature of acute leukemias. Acute leukemias are symptomatic and have a rapid course with a fatal outcome, while CLL is often asymptomatic and has a clinical course of months to years. Differential diagnoses and how to distinguish them is discussed in the section on T‐cell chronic lymphocytic leukemia.

Tumor cell transformation and prognosis

These are indolent lymphomas. Animals may live for years without treatments. Since these tumors are slowly progressive and clinically occult they are usually not diagnosed until the tumor has been present for an extended period, perhaps 1–2 years. Despite the slow progression a diagnosis of leukemia is considered to be more serious than a lymphoma. Humans with SLL have a 2‐year survival of 60–70% and a 5‐year survival of 50–70%.

A monoclonal antibody has been used for treatment in humans.^14,15 Dogs are treated with a variety of chemotherapies that may include prednisolone. Some are treated with only prednisolone and others are not treated.

In animals, CLL and SLL can undergo an accelerated phase similar to these diseases in humans. When this occurs in the tumor’s life is not known, but the cells gain in mitotic rate and the cells enlarge to prolymphocytic type (Figure 7.3). The next stage is similar to Richter’s syndrome, in which the small cells evolve into large cell lymphoma.^11,13,16 Further progression in humans is classified as blastic transformation, plasmacytoid transformation, and, finally, immunoblastic transformation.^9–11,15 The diagnostic features taken to predict the progression of human CLL are the clinical stage, lymphocyte count and doubling time, pattern of marrow involvement, cytogenetic abnormalities, and atypical immunophenotype.^1,16 These methods are not available on a diagnostic basis in veterinary medicine.

The distinction of blast transformation in CLL or SLL from a large cell lymphoma is not clear unless there was documentation of a prior small cell CLL or SLL. Most canine large cell lymphomas are B‐cell, multicentric, and some may have concurrent leukemia so the distinction may be difficult when either disease is seen at one point in time. Dogs with CD21‐positive lymphocytosis have different survival times that can be separated by size of the neoplastic cells as determined from flow cytometry.¹⁷ Dogs with large cell B‐lymphocytosis had MSTs of 130 days versus an MST that was not reached for small cell lymphocytosis (>1–2 years) as determined by flow cytometry.¹⁷ See T‐cell chronic lymphocytic leukemia section in this chapter and Appendix of this book for more information on survival and prognosis.

Clinical presentation and occurrence

Plasmacytomas are common in dogs, are seen in cats, and are unusual in horses and cattle.^{1,2,5,6,8–11} They are described in more detail in Chapter 5. Plasmacytomas are usually solitary, benign tumors in the skin, subcutis, or gastrointestinal tract and most do not recur following excision. Solitary tumors have been identified in the liver of dogs and cats and rarely have been associated with paraneoplastic syndromes of hypoglycemia, hypercalcemia, or paraproteinemia. Paraproteinemia is a feature of myeloma, MRB, and LPL. Plasmacytomas are also found in spleen, kidney, and intestines, but in these locations consider LPL, plasmablastic lymphoma, and MRD. Plasmacytomas are well described in cats. They are divided into cutaneous and extracutaneous and compared to MRD.^5,6,8 In cats the data suggest they originate in soft tissues but later may be present in bones, which is a different pathogenesis from that proposed for human MRD.⁵ Cats with MRD are FeLV and FIV negative. Some of the classifications and categories in MRD are similar to other diagnoses that use different nomenclature.

Regardless of names, several important conclusions from these investigations in cats are that cutaneous plasmacytomas respond well to excision, well‐differentiated tumors are associated with survival times of approximately 250 versus 14 days for poorly differentiated tumors, there is good concordance between cytology and histopathology, many tumors will not label with CD79a, mitotic figures are not frequent, and giant cells and giant nuclei may be seen in more aggressive tumors, as they are in dogs and humans.⁵

Most plasmacytomas are benign but infrequently cutaneous plasmacytomas behave aggressively and shorten the life of the dog or cat. When plasmacytomas are present in the aerodigestive system of dogs they are almost always solitary and benign. However, those that occur in the stomach or rectum may be clonal and aggressive and may cause vomiting and weight loss. When multiple tumors with plasma cell features are present in the intestines, and especially if they are also found in liver, spleen, or lymph nodes, the diagnosis in dogs is more likely plasmablastic lymphoma or LPL. Plasmacytomas in the rectal area usually present with some history of difficulty in defecation and are accompanied by recurrent bloody stool.¹⁰ The potential for plasmacytomas in the gastric and rectal areas to be aggressive necessitates wide surgical excision of tumors in these areas.^9,10,12 Plasmacytomas in dogs are rarely associated with monoclonal gammopathy. If a monoclonal gammopathy and hypercalcemia are present the dog is more likely to have multiple myeloma.

Cats with MRD are reported to have paraproteinemia and a few have had hypercalcemia.^5,6 Degree of differentiation is useful to predict survival times in cats. Well‐differentiated tumors had MSTs of 254 days versus 14 days for poorly differentiated tumors.⁵ Well‐differentiated tumors are more common.

LPLs are rare tumors in animals and humans. In humans this lymphoma as well as other lymphoproliferative diseases are associated with Waldenstrom‐type macroglobulinemia (IgM) that in about 20% of cases may have cryoglobulinema.^1,2 Clinically this protein causes hyperviscosity that results in cold extremities and may require plasma exchange to reverse the syndrome. In animals this syndrome is exceedingly rare but has been associated with multiple myelomas that produced IgM. A category of MRD includes this syndrome.

Plasmablastic lymphomas are uncommon tumors seen in mature dogs and cats. The animals usually present with severe systemic disease and have profound weight loss and a poor hair coat. On clinical examination multiple nodes are enlarged. Leukemic manifestations are rare but focal lesions in the spleen and perivascular cuffs in the liver occur. The tumor tends to be aggressive. Others may conceivably classify these as plasmablastic MRD or plasmablastic large cell lymphoma.

Light microscopic features

The features common to plasmacytoma, LPL, plasmablastic lymphoma, and MRD are that they are composed of tumors with plasma cell features and they are derived from B lymphocytes. Histologic and cytologic features of plasmacytomas range from well‐differentiated tumors that are easily recognized to poorly differentiated neoplasms that require IHC to confirm the diagnosis (Figure 7.4).^5,13 The majority have characteristic plasma cell morphology: uniform round cells, moderate amount of basophilic cytoplasm, perinuclear semi‐clear Golgi zone, single or binucleate eccentric uniform nuclei, chromatin that forms aggregates in the centers of nuclei or along the nuclear membrane, and a low mitotic count. The diagnosis on cytology or histopathology of well‐differentiated types is straightforward, IHC is not needed and these types of plasmacytomas are benign. Some tumor cells may contain intracytoplasmic inclusions that range from semi‐clear vacuoles to distinct eosinophilic globules or rhomboid‐shaped packets of immunoglobulin (Figure 7.4B,C). These are best appreciated in cytologic preparations and are referred to as Mott cells. The tumors are well delineated and surgical excision is usually curative. Unfortunately, other types have a wider range of cellular and nuclear pleomorphism such that IHC may be required to identify them correctly. These types of plamacytomas will have more binucleated cells, multinucleated cells, marked anisokaryosis, no Golgi, no cytoplasmic inclusions and may resemble amelanotic melanoma and histiocytic tumors. Plasmacytomas with these features are aggressive and considered malignant.

Differentiation of cells was a good means to identify cats with longer survival times.⁵ Giant multinucleated plasma cells have been described in MRD of cats. Mitotic activity is low in cats with MRD. Only 5 of 26 cases had mitoses seen and when they were found they were <10/400× field.⁵ Plasmacytomas in animals are infrequently (10%) associated with deposition of amyloid, but when present in the tumor amyloid is a helpful diagnostic feature.^5,9

LPL and plasmablastic lymphomas are much less common than plasmacytoma. They look similar to plasmacytoma but they arise in a lymph node rather than skin or oral cavity. The node may be focally involved or diffusely. Both may cause generalized lymphadenomegaly, whereas plasmacytomas do not. They may also be in spleen or liver or gastrointestinal tract, whereas plasmacytomas do not have this widespread organ distribution unless malignant. LPLs have a plasmacytic morphology. They have well‐delineated cell borders, the cytoplasm is usually intensely pink, and nuclei are eccentric and may have a distinct shallow nuclear indentation (Figures 7.5 and 7.6). Nuclei are approximately 1.5 RBC in diameter. The mitotic count is low, 0–2 mitoses/400× field. They are low‐grade, indolent, mature B‐cell lymphomas. Plasmablastic lymphomas also look plasmacytoid but have larger nuclei, 2.0–2.5 RBC in diameter, and prominent multiple central nucleoli and a coarsely aggregated chromatin that is hyperchromatic (Figure 7.7). Cytoplasm is usually abundant and deeply stained. Neoplastic cells have a high mitotic count with several in each field at 400× magnification. In lymph nodes they can form clusters or sheets that may be mistaken for benign plasmactyic hyperplasia or plasmacytoma. They will expand from these regions and can be diffuse in nodes or spleen. They are high‐grade mature B‐cell lymphomas and some classify these as a plasmablastic variant of DLBCL.

Cytochemistry and immunohistochemistry

Canine plasmacytomas stain more reliably with MUM1 than with CD79a or CD20. In one study 101 of 109 plasmacytomas (93.5%) were positive for MUM1, 59/105 (56%) were positive for CD79, and only 21/108 (19%) were positive for CD20.¹³ In this same study the authors applied MUM1 on 139 other tumors, including tumors that can resemble plasmacytomas: 13 melanomas, 7 mast cell tumors, 17 histiocytomas, 2 cutaneous and 3 systemic histiocytoses, 7 histiocytic sarcomas, 17 epitheliotropic lymphomas, and 17 T‐cell lymphomas, and the only other positive tumors were 10 B‐cell lymphomas and one anaplastic lymphoma.¹³

MUM1 can be used on FFPE tissue sections. It primarily stains nuclei and there may be slight staining of the cytoplasm. MUM1 is in the family of interferon regulatory factors. It is required for immunoglobulin light‐chain rearrangement and is expressed in B cells, plasma cells, activated T cells and a subset of macrophages and dendritic cells in humans. Not all nuclei of a plasmacytoma mark with the MUM1, therefore interpretation is needed when only a proportion of tumor cells are labeled. In general if 20% or more of the nuclei are labeled the immunoreactivity is considered positive and if less than 10% are labeled it is considered negative. Tumors in cats with MRD do not stain reliably with CD79a: 12 cases were negative for CD79a and CD3, one case was positive for both and 6 cases stained well with CD79a.⁵

Plasmablastic lymphomas mark poorly with CD79a (Figure 7.7B) and strongly with CD20. LPLs generally stain strongly with CD79a and reliably with CD20. Both tumors are negative with CD3 (see Figures 7.23–25). If the tumor is in a lymph node, it looks like a plasma cell tumor and stains strongly with CD79a or CD20 it is likely a plasmacytoid lymphoma or LPL. Twelve of 19 MRD cases did not label with CD79a and 18 of 19 had Ig immunolabeling.⁵

In animals (exception is swine) the “normal” ratio of immunoglobulin light chains is approximately 90:10 lambda to kappa.¹¹ Therefore light‐chain restriction is a part of normal plasma cells and should not be relied on to identify a clonal expansion. If the predominant light chain was kappa, a rare occurrence, then it should represent clonality. See the section on Myeloma in this chapter. Use of lambda light chain to identify neoplastic proliferations of plasmacytic tumors in animals should be done with other diagnostic tests and should not be relied on alone.

Differential diagnosis

Tumors with plasma cell morphology include plasmacytoma, LPL, plasmablastic lymphoma, myeloma, MRD, and even some osteosarcomas that have numerous osteoblasts. The more differentiated the tumor and the more fully developed the associated syndromes, the easier the distinction. Generalizations follow and exceptions are numerous. Myeloma is in bone marrow, multiple osteolytic lesions and paraproteinemia are present. Plasmacytoma is a singular soft tissue tumor without paraproteinemia. LPL and plasmablastic lymphoma are in nodes and/or organs, LPL is more differentiated and paraproteinemia may be present.

Osteosarcomas produce osteoid. They have a characteristic clinical appearance and are the only type listed that are not of B‐cell origin. Osteosarcomas can be stained reliably with ALP and are negative to lymphoid markers.

Well‐differentiated plasmacytomas are not a diagnostic challenge but less‐differentiated tumors may be confused with other round cell tumors, melanoma, histiocytic sarcoma, or poorly differentiated mast cell tumors. The presence of binucleated and occasionally tri‐ or multinucleated cell types as well as scattered large nuclei are suggestive of a plasmacytoma or histiocytic tumor.¹⁴ The more bizarre the nuclear pleomorphism the more likely the tumor is of histiocytic origin. Cytophagia favors histiocytic, but plasma cell tumors can be added to the growing list of tumors that are capable of cytophagia.¹⁵ Differentiation of these two tumors in FFPE sections can be accomplished by IHC using MUM1, which recognizes plasma cell tumors, and CD18, which favors histiocytic tumors. Many of the antibodies used for histiocytic lineage work better on frozen sections rather than FFPE (see Chapter 3 for IHC details and Chapter 8 or a review article for details about histiocytic neoplasms and how best to recognize them).¹⁴

If the tumor is in nodes and organs it is more likely a histiocytic tumor or one of the other lymphomas, plasmablastic lymphoma, or LPL. Plasmablastic lymphoma and LPL will immunostain well with CD20, whereas plasmacytomas stain weakly or are negative with CD20 but stain very well with MUM1. If the tumor is in the lungs it is much more likely to be histiocytic than lymphoid.

Plasmablastic lymphoma and LPL look like plasmacytoma but are in lymph nodes, focally or diffusely, and may also be in intestine, liver, spleen, or bone marrow. The more widespread the neoplasm the more likely it is plasmablastic lymphoma or LPL and not plasmacytoma. LPL is more indolent and mature. Plasmablastic lymphoma is less differentiated and the prominent nucleoli and high mitotic activity differentiate it from LPL. LPL is associated with macroglobulinemia. These same generalizations can be used for cats but manuscripts describing MRD should be read for the details provided.^5,6 The authors also discuss the different pathogenesis between cats and humans with MRD.

Defining the neoplasm

Myeloma is a clonal proliferation of malignant plasma cells that originates in bone marrow and is associated with monoclonal gammopathy and multiple osteolytic bone lesions. It is considered a primary tumor of bone and is also described in Chapter 10. Bone pain and radiographically visible bone lesions are present in approximately 50–70% of dogs and humans with myeloma. The diagnosis is established by visualization of tumor cells in an aspirational cytology or a bone marrow core biopsy from an osteolytic lesion (Figure 7.8; see also Figure 10.43A–D). In most cases the diagnosis is straightforward as there are multiple, discrete osteolytic foci (see Figure 10.43) seen on radiographs and the tumor yields numerous cells with FNA. If needed, an electrophorectic examination of serum demonstrating a monoclonal gammopathy offers further confirmation.

The classification of human monoclonal gammopathies according to the International Working Group defines three categories of gammopathies based on the concentration of serum M‐protein and percent of clonal plasma cells in the bone marrow.^1–3 Plasmacytoma may progress to myeloma. In humans, altered expression of the c‐myc oncogene is associated with multiple myeloma and the cell cycle protein cyclin D is associated with myeloma in humans and dogs.^4,5

Occurrence and clinical presentation

Myeloma is seen primarily in dogs, and less frequently in cats and has been reported in the cow, horse, and pig.^3,6–9 In cats it accounts for approximately 1% of all feline malignancies; it is not associated with FeLV or FIV and osteolytic lesions are rare.^6–8 In cats the myeloma‐related diseases (MRD) are a group of about seven disorders, one of which is myeloma. Most cases of MRD are extramedullary and are described in detail in several publications.⁸ The majority of the cats with MRD present with extramedullary tumors (see section on Plasmacytoma, plasmablastic lymphoma, lymphoplasmacytic lymphoma, and myeloma‐related disorders) and most will have organ involvement and paraproteinemia. About half will have bone marrow involvement, but bone lesions are not a prominent feature of MRD.⁸

In the dog, multiple myeloma accounts for approximately 0.5% of all malignant tumors, with no apparent sex or breed predisposition. No cases were found in the ACVP study of 1000 cases of canine lymphoma, likely because the disease was diagnosed from characteristic clinical presentations including a high serum globulin and monoclonal gammopathy. There is no known cause, but chronic immune stimulation is suspected to be involved in humans, where dendritic cells infected with Herpesvirus 8 secrete high levels of IL‐6 that drives plasma cell differentiation and possible malignant transformation.

A common finding in dogs is lameness and radiographs demonstrate multiple lytic lesions (multiple myeloma) in bones.⁸ The lesions can be found in any bone of the axial or appendicular skeleton but are seen more commonly in areas of active hematopoiesis. Animals in later stages of the disease may present with a history of weight loss, decreased vigor, and signs of renal malfunction. A characteristic feature of myeloma is a monoclonal or less commonly a biclonal gammopathy (paraprotein). Serum total protein values over 9.0 g/dL are a clinical aid to suspect myeloma and suggest electrophoresis to characterize the serum proteins or search for bone lesions. Theoretically, any of the immunoglobulins could be produced but IgG is the most common, followed by IgA and IgM. Cats also have paraproteinemia, usually IgG. A small percent of myelomas will be nonsecretory with no gammopathy present. IgM‐secreting tumors can cause a hyperviscosity syndrome. Most myelomas with gammopathy are accompanied by overproduction of light‐chain immunoglobulins small enough to pass the kidney and be detected in the urine as Bence‐Jones protein. SSA (sulfosalicylic acid) is a more sensitive technique to recognize these proteins than are colorimetric dipsticks used for routine urinalyses. These light‐chain immunoglobulins may also be deposited in the basement membranes of glomeruli, resulting in glomerulonephritis. The light‐chain immunoglobulins may also form amyloid, which is deposited in glomeruli, resulting in the nephrotic syndrome: proteinuria, hypoalbuminemia, ascites, and increased serum cholesterol. Patients with the nephrotic syndrome are not necessarily azotemic. Amyloid may also be deposited subendothelially in other organs, most notably spleen and liver.

Monoclonal gammopathy is highly suggestive of myeloma but can also be seen in canine ehrlichiosis, leishmaniasis, pyoderma, B‐CLL, B‐cell lymphoma, plasmacytoma, and feline infectious peritonitis.¹⁰ Hypercalcemia occurs in approximately 10% of canine myelomas as a paraneoplastic syndrome and is less common in cats.⁸ The mechanism of hypercalcemia is attributed to the paracrine effects of cytokines produced by the tumor cells, which induce local osteoclastic osteolysis (see Figure 10.43D). In addition there may be humoral substances (parathyroid hormone‐related protein (PTHrP)) that act on renal tubules to reabsorb calcium from the glomerular filtrate and block the absorption of phosphorus, resulting in hypercalcemia and hypophosphatemia. Concurrent hypercalcemia in dogs with myeloma suggests a worse prognosis.³

Although the tumor originates in bone marrow, leukemia is rarely detected. If present the prognosis is worse. At the time of diagnosis there is usually a mild or moderate non‐regenerative normocytic normochrocmic anemia, most likely due to cytokine production associated with the anemia of chronic inflammatory disease. Petechiae and bleeding problems are fairly common and are associated with thrombocytopenia and/or adherence of paraproteins to platelet membranes that alter platelet function.¹⁰

Pathology

Cytochemistry and immunohistochemistry

Immune staining is usually not needed for a diagnosis. If performed, the results may vary but most canine cases stain positively with CD79a in both cytological and histological preparations. CD20 is not reliable in myeloma but the nuclei will stain very well with MUM1.¹¹ CD38 and CD138 are used in humans and myelomas are negative with CD3.

Homogeneity of light‐chain restriction can be used in human plasma cell tumors to differentiate neoplasia versus inflammation. This is based on the principle that there is a balanced distribution of lambda and kappa light chains.¹² However, in animals (exception is swine) the “normal” ratio is approximately 90:10 lambda to kappa.¹² Therefore light‐chain restriction is a part of normal plasma cells and should not be relied on to identify a clonal expansion. If the predominant light chain was kappa, a rare occurrence, then it should represent clonality.

Staging and survival

Treatment of multiple myeloma in animals, as in humans, has not been curative, with most animals succumbing to the tumor. Chemotherapy is used but most cases survive less than 2 years. Factors associated with a poor prognosis are: hypercalcemia, leukemia, azotemia, cytopenias, and Bence‐Jones proteinuria. One parameter that may be useful to predict prognosis is the relation of the rate of increase in serum M‐globulin to the number of surviving neoplastic cells secreting protein. New treatments in humans have greatly improved the survival of younger patients. A staging system has been developed for humans based on the levels of β₂‐microglobulin and albumin.

Defining the neoplasm

Marginal zone lymphoma (MZL) originates in lymph nodes or spleen and it is the most common type of lymphoma in the spleen of dogs and humans.^1–14 Although it is common in the spleen of humans, it makes up <1% of lymphomas. It is one of the more common lymphomas in dogs, at 5–15%.^5,7,9 MZL is considered a low‐grade B‐cell lymphoma that has an indolent course but more cases need to be followed and morphology and molecular studies integrated.^10–14 It is one of the lymphomas that forms discrete nodules which become diffuse in only late stages. Histologic sections are required to make this diagnosis as the architectural arrangement in a node or spleen is critical and this cannot be appreciated in cytological preparations. MZL has a characteristic arrangement of neoplastic lymphocytes surrounding fading germinal centers which results in a nodular pattern (Figure 7.9). IHC reveals neoplastic lymphocytes are B‐cell type. There is no specific marker for MZL in humans or animals. The diagnosis is dependent on their histologic organization combined with anatomical location in body, cytologic features, and immunophenotyping. MZL was one of the more common lymphomas in the studies that total approximately 1700 canine lymphomas.^4,7,8

The marginal cell zone of lymph nodes is only seen in nodes that are hyperplastic (reactive) or neoplastic (Figures 7.10 and 7.11). The nodular architecture in the cortex of nodes is due to a central germinal center, B‐cell rich, surrounded by a distinct mantle cell zone that is composed of approximately 90% B lymphocytes and 10% T lymphocytes. When a node is antigenically stimulated, the marginal zone layer proliferates and becomes visible, forming a polar pattern in which the marginal zone is thinner toward the paracortex and thicker toward the subcapsular sinus (Figure 7.10). The lighter pole has the proliferating cells (centroblasts).

Marginal zone hyperplasia (MZH) is an important differential for MZL, as both originate in the marginal zone of lymphoid follicles. MZH is a common finding in the spleen of aged dogs and can be observed concurrently with faded germinal centers. Hyperplastic marginal zones can have a quite monomorphic appearance with intermediate‐sized lymphocytes that often have a single prominent nucleolus. In MZH this change is generally widespread within the white pulp of the spleen; however, coalescence of proliferating marginal zones into nodules of varying sizes is a feature of MZL not MZH.

Epidemiology and clinical presentation

MZL is seen in mature dogs and cats and occurs in horses and likely in all domestic animals. In the ACVP study of 1000 cases of canine lymphoma 62 cases of MZL included 13 in the spleen, 2 cases in both node and spleen, and 1 case of mucosal‐associated lymphoid tissue (MALT) type in a salivary gland.⁴ The gender was about even and the mean age of the cases of MZL in nodes was 9.7 years and those with MZL in the spleen had a mean age of 11.4 years.⁴ In the review of 600 canine cases there were 66 MZL; most were in lymph nodes and only a few were extranodal.⁷ In some reviews MZL represents approximately 15% of selected canine lymphomas.⁷ MZL may be found in other organs, including the central nervous system in humans⁶ and in the spleen of p53‐deficient mice.¹⁵

MZL occurs in mature dogs, typically large breeds, with a single enlarged node that remains fully mobile. Dogs with MZL usually have no other clinical signs and leukemia is not present; however, leukemia is seen in humans if MZL is in the spleen. A series of 75 dogs with indolent lymphomas detailed the clinical characteristics and survival data;⁹ two other studies reported on approximately 45 dogs.^13,14 MZL in lymph nodes becomes slowly generalized and even with multiple large nodes the dogs generally feel well, perhaps due to the low mitotic count and slow growth of the tumor. The tumor may also start in the spleen and abdominal ultrasound of these dogs reveals focal or multifocal splenic enlargement. Spread from the spleen is slow and likely first to the splenic hilar nodes and then to mesenteric nodes. In human patients MZL cells may be found in the peripheral blood and in darkfield examination with a wet mount these cells have villous cytoplasm. This aspect has not been reported in dogs. In humans MZL is rarely associated with a macroglobulinemia like that of the Waldenstrom type.

Other organs

The only other organs reported to be involved with MZL of dogs are the salivary glands, tonsil, and nictating membrane.^4,7 MZL invades the salivary tissue, producing a “lymphoepithelial‐like lesion” characteristic of MALT lymphoma (Figure 7.12). Occasionally the same lesion may be present in lymphomas of the intestine in dogs and, much less commonly, in cats. Concurrent involvement of a node is likely but the tumor may only be found in the salivary gland if a thorough autopsy is performed. Rarely a MALT lesion may be found in the thyroid gland, and in humans the lung and breast may also be involved. The key to diagnose this subtype of lymphoma is invasion of the salivary epithelium by neoplastic lymphocytes, producing a lymphoepithelial lesion.

Immunohistochemistry and clonality

MZL in all sites, in all animals, is strongly positive with CD79a and CD20 antibodies and negative with CD3 (Figures 7.9–7.12). There is no specific marker for MZL and the diagnosis is dependent on anatomical location, histologic organization, cytologic features and B‐cell immunophenotype.

Clonality was detected in 28 of 35 cases (80%) of canine MZL.⁵ In a separate study, 24/33 dogs had clonal rearrangement of Ig loci, 6 polyclonal and 3 pseudoclonal rearrangements.¹⁴ Absence of detectable clonality in 20% of cases is consistent with results from PARR that may have false‐negative results up to 30%. False negatives can be attributed to multiple possibilities, including gene rearrangements which the primers used do not recognize, mediocre quality of sample, or partial rearrangements of IGH. It is also noteworthy that 6 of 28 cases with a clonal IGH gene rearrangement had a concurrent TCRG gene rearrangement.⁵ Multiple possibilities exist, including reactive but clonal T cells within the lymphoma, which can happen with other neoplasms. Material for clonality was from FFPE material; fresh material might have revealed different results.⁵ Regardless, clonality must be interpreted as only one part of the case data and, depending on the results, it is important to consult with the laboratory that ran the tests.

Defining the neoplasm

Mantle cell lymphoma (MCL) is an uncommon indolent lymphoma of B lymphocytes seen in animals and humans. It starts focally in the spleen in mantle cell regions. Most cases in dogs occur as a primary splenic lymphoma and lymph nodes are not involved. Two types of MCL are seen in the spleen, a focal mass, which is the more common type, and a generalized or “blastoid” type. MCL was not recognized as an entity in prior classifications because it was included with the small B‐cell non‐Hodgkin’s lymphomas.

In humans, MCL has a slow progressive course that is unresponsive to treatment and therefore the prognosis is not good. In humans MCL has a unique genetic signature arising from a translocation t(11;14) and is evident by the overexpression of cyclin D1. Immunoreactivity for this protein has not been demonstrated in the dog and it is a difficult stain to perform accurately on human tissues.¹

Epidemiology, occurrence, and clinical presentation

In one study, 16 MCLs were seen in 1000 cases of canine lymphoma, suggesting that approximately 1–2% of canine lymphomas are of MCL type.² Fifteen of the 16 cases were in the spleen. Dogs with blastoid type of MCL may have a low‐level gammopathy and recurrent fever that resolve with splenectomy. The latter changes could be related to the multiple foci of necrosis in hemorrhagic neoplastic nodules. Nine dogs were female, 5 were male, and in 2 cases the sex was not indicated. The ages ranged from 6 to 12 years and the mean age was 9.5 years.² Only four cases were classified as MCL in the 600‐dog study. MCL occurs rarely in the cat and pig. MCL likely occurs in most mammals. In humans it constitutes about 5% of lymphomas, males predominate 3:1, and the disease is seen in elderly patients with a median age of 65–75 years.^3–7

Spleen

Most cases in dogs are in the spleen, where it is a primary neoplasm that forms a locally expansile mass. Localized masses are obvious because of compression of surrounding tissue. The splenic tumor may have other concurrent lesions, including myelolipoma, plasmacytoma, hemorrhagic infarction, nodular hyperplasia, and the fibrotic reaction previously termed fibrohistiocytic nodules (Figures 7.13–7.15). Adjacent lymph nodes are usually not affected early in the development of the tumor. In early cases the proliferating neoplastic B lymphocytes will be in separate colonies with focal areas of coalescence. In advanced splenic MCL the infiltrations become contiguous and may be diffuse, resulting in wide separation and thinning of the splenic smooth muscle trabeculae. In the blastoid type of MCL the entire spleen is involved. The lymphoid nodules are about twice the size of a normal germinal center. These nodules enlarge until the pressure of the proliferation causes the central area to undergo ischemic necrosis that is followed by hemorrhage (Figure 7.15A). This type is described in humans^3–6 and in dogs.^1,2,8 In both types of splenic MCL other areas of the spleen have numerous but non‐neoplastic lymphocytes and plasma cells.

MCL and MZL occur as primary splenic lymphomas and both are characterized by fading germinal centers surrounded by an enlarging envelope of neoplastic B lymphocytes that create a distinctive nodular pattern (Figure 7.13A,B). In the focal form of MCL there is usually a locally extensive, multifocal proliferation of lymphocytes around end arterioles (Figure 7.13) with several foci starting to coalesce. Nuclei are situated close together as the cells have relatively little cytoplasm. The nuclei have coarsely aggregated chromatin with small nucleoli that are not readily apparent. Nuclei are small, the largest nuclei are about 1.5 RBC in diameter (Figure 7.13). The mitotic count is low. Cytology of FNA reveals the neoplastic lymphoid cells have more cytoplasm and larger nuclei than appreciated in histologic sections (Figure 7.14D). Although a cytologic diagnosis of lymphoma is readily apparent, the classification of MCL requires knowledge of location (spleen), histologic organization (fading germinal centers surrounded by neoplastic lymphocytes), and B‐cell immunophenotype. Neoplastic cells of the blastoid type are larger, cytoplasm is fairly abundant and deeply stained. Nuclei are nearly 2.0 RBC in diameter. Some nuclei are irregularly shaped and have sharp shallow nuclear indentations and prominent nucleoli (Figure 7.15B). There can be 4–6 mitoses/400× field. In the blastoid type the nuclei are larger, more irregular, nucleoli are more prominent and mitoses are more numerous than in the focal type of MCL. In the blastoid type MCL neoplastic cells tend to colonize the endothelial lining of the large muscular veins.

Cytochemistry and immunohistochemistry

The cells of MCL are uniformly positive with CD79a and CD20 and are negative with CD3.^2,8 The IHC staining will highlight the fading germinal centers and nodular pattern better than H&E. In humans, the cells of MCL are strongly positive with cyclin D1.¹

Differential diagnosis

Because of its histologic association with the end arterioles in the spleen and fading germinal centers the most likely differential is MZL, which is also an indolent B‐cell lymphoma with a nodular organization. The major difference is that the nucleoli in MZL are large and very prominent and in the focal type of MCL they are small and inconspicuous. In the blastoid type of MCL the nucleoli are larger and centrally located (Figure 7.15). Tumors in lymph nodes favor MZL. MCL should have a light center in the nodules. This is from the remaining germinal center, which is surrounded by the tumor. MZL should have darker staining mantle cells left as remnants and they are surrounded by neoplastic B cells. TZL can look similar via histology and cytology but is readily distinguished with IHC for CD3. Hyperplasia is also a differential. The larger the mass, the more coalescence of nodules that is seen, and the more uniform the cells the more likely the diagnosis is lymphoma. Hyperplasia or a “reactive process” has a mixture of lymphocytes and other cells in the proliferation and this can be appreciated in H&E‐ or IHC‐stained sections. Clonality testing is an option to help distinguish hyperplasia.

Survival time

Too few cases have been followed with accurate clinical outcomes to predict therapies and reliable survival times. A recent study reported 9 cases of MCL treated with splenectomy alone had a 1‐year survival rate of 89%.⁹ In the 16 cases seen in the ACVP study the range of survival was 126–155 days and the mean survival was 140 days.² Despite these observed survival times this is considered an indolent lymphoma. All dogs were assumed to have been treated. Survival times for all tumors are affected to some degree by the owner of the dog or cat and their willingness to continue treating versus electing euthanasia. In humans the median survival of patients with MCL is 3 years and the most prognostic factors are patient age, leukemia, and splenomegaly.^4–6 Genetic studies have been done in humans to determine changes associated with survival.^5,6

Defining the neoplasm

Follicular lymphoma is a tumor of B lymphocytes that forms characteristic follicles in affected lymph nodes and spleen. It is a very uncommon lymphoma in animals but is one of the most common lymphomas in humans; more than 30% of all lymphomas in humans are follicular lymphoma and all are B‐cell phenotype.^1–4 Follicular lymphoma is one of the nodular indolent lymphomas; others are MZL and MCL. MZL appears to arise in regions with lymphoid hyperplasia.

Follicular lymphoma needs to be distinguished from follicular hyperplasia, which is a common lesion in animals, but interestingly also has an unfavorable outcome, at least in dogs.⁵ These two lesions will be described and compared in this section as they have similar morphology. Follicular lymphoma, MZL, and MCL all have such a characteristic nodular histologic organization that their B‐cell phenotype can be predicted in H&E‐stained sections. In the review of the REAL/WHO classification of hematopoietic neoplasms the MD pathologists proposed that phenotyping was essential for all human lymphomas except for those with a follicular architecture, as these will be follicular lymphoma and they are all B‐cell.

Epidemiology, occurrence, and clinical presentation

Follicular lymphoma is recognized in most domestic animals but is very uncommon.^6,7 In a review of lymphoma in cats there were 6 cases in a group of 602⁸ and in 1198 cases of bovine lymphoma there were 4 cases of follicular lymphoma.⁹ One case was found in over 200 cases of equine lymphoma.¹⁰ In 600 canine lymphomas there were 4 follicular lymphoma and in 1000 canine cases 0.3% were diagnosed as follicular lymphoma and approximately 4% follicular hyperplasia.⁵ In 300 canine cases there were only 3 cases of follicular lymphoma and 11 (3.7%) diagnoses of lymphoid hyperplasia, 2 of which were benign follicular hyperplasia (BFH) and 9 benign atypical follicular hyperplasia (BAFH).¹¹ However, dogs with benign hyperplasia had an MST of approximately only 350 days.^5,11 Perhaps there were misdiagnoses or follicular hyperplasia is associated with occult but potentially lethal disease processes.⁵

Animals with follicular lymphoma present with one or more enlarged lymph nodes but they have a normal appetite and level of activity.¹² Dogs with follicular lymphoma, MCL, and MZL are often not ill, or only mildly so. Any peripheral node(s) may be involved as well as thoracic nodes, but oddly, follicular lymphoma has not been described in mesenteric nodes. The nodes are mobile and not fixed. Like many other types of lymphoma those in the head and neck region are most likely to be involved.

Lymph node

In both follicular lymphoma and follicular hyperplasia the diagnostic feature is the presence of distinct follicles (Figures 7.16–7.18). Both can have infiltration of perinodal fat by lymphocytes and both may have areas where the follicles are coalescing and are separated by narrow bands of connective tissue. The more infiltrate that is extranodal, especially if the cells form extranodal follicles, and the more coalescence of nodules, the more follicular lymphoma is favored. The distinction between follicular lymphoma and follicular hyperplasia is interpretative and the following features are used. Try to evaluate the cortical region of the node as this region has germinal centers and within these evaluate for polarity (follicular hyperplasia) or its absence (follicular lymphoma).

The following features all favor follicular hyperplasia: retention of nodal architecture, antigen polarity in the follicles with larger lighter stained cells to one side of the follicle usually facing the capsule, the mantle cell cuffs remain and form a dark rim around the germinal center, any perinodal lymphocytes are scattered and do not form follicles, there is a mixture of cell types in the perinodal and interfollicular regions, and there should be numerous plasma cells in medullary cords and interfollicular (Figure 7.16). Tumor and follicles may form in the perinodal fat of follicular lymphoma. Depending on how advanced the follicular lymphoma is the overall cellular proliferation will be greater and the mantle cell cuffs and polarity in the follicle are lost (Figure 7.17). The mantle cell cuffs may be obliterated by tumor cells and their absence (Figure 7.17B) or presence (Figure 7.16B) is highlighted in IHC‐stained sections. Intrafollicular tingible body macrophages should be more prominent in follicular hyperplasia and there are few or none with follicular lymphoma, which seems a contradiction but there is inactivation of the apoptotic gene by the neoplastic cells.

At highest magnifications another feature to look for is the proportion of small cells (centrocytes) and large cells capable of division (centroblasts).¹² In follicular lymphoma there is the same proportion of centrocytes and centroblasts across individual follicles and between follicles, imparting a uniform color pattern (Figure 7.17A). In follicular hyperplasia there should be no centrocytes between follicles, only within the germinal centers.¹² IHC should identify heterogenity of lymphocytes in follicular hyperplasia and homogeneity in follicular lymphoma. Numerous plasma cells is a feature of follicular hyperplasia.

Most nodes with follicular lymphoma are not diffusely involved, but if nodes are sampled late in the disease then neoplastic follicles occupy the entire node (Figure 7.17). The capsule is thinned and the peripheral sinus is compressed and may be focally invaded. Intrafollicular tingible macrophages, common in many lymphomas, are not prominent in follicular lymphoma, even in nodes heavily laden with tumor, and may be more prominent in germinal centers of follicular hyperplasia. The likely explanation is inactivation of the apoptotic gene by the neoplastic cells. Inactivating the apoptotic caspase enzyme system results in few or no cells dying and therefore the need for tingible body macrophages is reduced or absent.^1,2,4

Cytochemistry and immunohistochemistry

The neoplastic cells in follicles are strongly positive with CD79 alpha and CD20 but are negative with CD3 (Figures 7.17 and 7.18). There may be a few CD3‐positive T cells scattered in neoplastic follicles and in interfollicular regions. IHC will reveal more intrafollicular T lymphocytes in follicular hyperplasia than in follicular lymphoma.

CD79a will label cells in germinal centers of follicular hyperplasia, but in contrast to follicular lymphoma the mantle cell cuffs are present in follicular hyperplasia and are well stained (Figure 7.16B). CD3 for T cells leaves the germinal centers relatively unstained but the lymphocytes in the outer region of each follicle and in the interfollicular region are strongly positive in follicular hyperplasia. This staining pattern may impart a distinct polar cap to each follicle (Figure 7.16C).

In humans there are follicular dendritic cells in the follicles of follicular lymphoma that consistently stain positive with CD21 and CD23 and these are presumably present in follicular lymphoma of animals. Since the neoplastic follicles have produced the BCL2 protein and inactivated their apoptotic system, the cells of follicular lymphoma will have staining for the BCL2 protein not seen in benign follicles.

Differential diagnosis

The light microscopic criteria to differentiate follicular hyperplasia and follicular lymphoma are described in the section under lymph nodes and under IHC. If the differentiation is not clear then consider PARR to determine B‐cell clonality and all the other data in the case. In gray zone cases do not rely on only one test. In addition, benign follicles do not express the BCL2 protein while those that are malignant will be positive to some extent.

Staging

Human follicular lymphoma is graded on the proportions of centroblasts to centrocytes present in each follicle. The larger cells (centroblasts) capable of division increase in numbers as the disease progresses. The WHO grading system for human follicular lymphoma may apply to animals. If there are 0–5 centroblasts/400× field the tumor is graded 1. If there are 6–15 centroblasts/400× field, the tumor is graded 2. If there are over 15 centroblasts/400× field the tumor is graded as 3. These criteria have been altered slightly as the original description was made on oculars with field number (FN) of 20 mm diameter and most oculars now have an FN of 22–25 mm diameter.¹² As the FN increases so does the total field‐of‐view area.

Typical survival time

Too few canine follicular lymphomas have been followed to predict survival times with or without treatment, but if they behave in dogs as they do in people then survival times are likely less than 1 year.⁵ More cases of follicular hyperplasia need to be followed but it appears that dogs with follicular hyperplasia also have survival times of approximately 1 year, suggesting that follicular hyperplasia is an indicator of an underlying and potentially lethal disease process or that it leads to lymphoma or there may be misdiagnoses.¹²

Defining the neoplasm

Burkitt’s lymphoma is a B‐cell lymphoma which is a well‐characterized disease in humans but not animals.^1–3 In veterinary pathology there is no uniform agreement on this diagnosis. The distinction from DLBCL in dogs is accomplished by subjective features of cell size and nuclear and nucleolar features. In humans it is associated with the Epstein–Barr virus (EBV) and dysregulation of the c‐myc gene by one of several chromosomal translocations located at 8q24.^3,4 Chronic infection with EBV in humans is usually asymptomatic but a portion of infected individuals co‐infected with malaria will develop lymphomas, most notably Burkitt’s lymphoma.

Serologic evidence indicates that privately owned dogs with and without lymphoma are exposed to a gammaherpesvirus, and further studies implicated its presence in B‐cell lymphomas of dogs and suggested it may be associated with lymphomagenesis in a subset of canine lymphomas.⁵

Burkitt’s lymphoma is a unique tumor with interesting epidemiologic associations that brought some historical firsts to human hematopathology.² It was the first tumor curable by chemotherapy, it was the first human tumor to be associated with a virus, and it was one of the first tumors in which epidemiology helped define the tumor and its pathogenesis.

Several types of Burkitt’s lymphoma are recognized, one is the type seen in regions of the world where EBV and malaria are endemic. It is thought that chronic malaria reduces the resistance to EBV, allowing the virus to proliferate and the incidence of lymphoma to increase. An interesting aspect of Burkitt’s lymphoma is the tendency for the tumor to arise in an area of benign lymphoid cell proliferation, as occurs in the mouths of young children and in the breast tissue of girls reaching menarche. This accounts for the tumor arising in the jaws of young Africans and later in the breasts of teenage girls.¹ In endemic areas of EBV and malaria Burkitt’s lymphoma characteristically involves the mouth, teeth, jaw bones, and other facial bones, resulting in large tumors and disfigurement.

The descriptions for Burkitt’s lymphoma use some of the classical descriptors to characterize lymphoma: sheets of monotonous neoplastic lymphoid cells with a high mitotic count and a “starry sky” appearance due to numerous tingible body macrophages. In veterinary pathology similar descriptors are used for DLBCL. The classical form of Burkitt’s lymphoma is composed of cells of uniform, intermediate size, described as like “peas in a pod.” In histological preparations the mild separation of the cytoplasm of each cell tends to form a geometric pattern of cell interfaces known as a “squaring off” of cytoplasmic surfaces. This form of the human Burkitt’s lymphoma is associated with prominent cytoplasmic vacuoles. “Burkitt’s‐like” lymphoma is a provisional entity in the WHO classification and is the type recognized in animals. This is a high’grade B‐cell lymphoma that has numerous tingible body macrophages and looks similar to DLBCL.¹

Occurrence and clinical presentation

In the ACVP study of 1000 cases of lymphoma in dogs there were 21 cases classified as Burkitt’s‐like lymphoma. The dogs had a mean age at diagnosis of 8.8 years. This lymphoma is seen most often in dogs and cats and likely occurs in the pig, cow, horse, and mouse.⁶ In humans there is often leukemia and marrow involvement but this is not seen or not recognized in animals. Of all the domestic animals it is described most frequently in cats.⁷ In the series of 600 feline lymphomas 79 were considered to be Burkitt’s lymphoma or Burkitt’s‐like.⁷ A neurotrophic form was described in detail in a cat with clinical signs and lesions of peripheral neuropathy.⁸ The tumor was only found in peripheral nerves even after an autopsy. The tumor and cat were negative for FeLV and multiple human (EBV) and animal herpesviruses. The tumor was of B‐cell phenotype, had intermediate to large cells, and was similar to human diffuse neurolymphomatosis.⁸

Pathology

Lymph nodes

Nodes are filled with tumor that consists of sheets of monotonous medium‐sized lymphocytes with a high mitotic count (Figure 7.19). A “starry sky” appearance is expected at low magnification and is due to numerous tingible body macrophages (phagocytic cells containing dead apoptotic tumor cells). Macrophages will be fewer where the mitotic count is lower. An old descriptive term of “small non‐cleaved cell” is misleading. The tumor cells are intermediate in size. Tumor cells possess small amount of basophilic cytoplasm. Nuclei have mild anisokaryosis and are up to 1.5 RBC in diameter. Chromatin is aggregated such that it is open and nucleoli are prominent and numerous; often 3–5 nucleoli/nucleus. Cytologic preparations of DLBCL look similar. The mitotic count is high, at up to 10/400× field.

The capsule of nodes is usually thickened and at least focally invaded by the lymphoid cells. The nodal tissue is friable and ischemic necrosis is not a feature.

Cytochemistry and immunohistochemistry

The neoplastic cells are uniformly labeled with CD79a and CD20 and are negative with CD3. In humans, the cells are positive with CD10 and BCL6 and are negative with BCL2. The Burkitt’s lymphoma cells have light‐chain restriction and in humans they may have cytoplasmic IgM.

Defining the neoplasm

Diffuse large B‐cell lymphoma (DLBCL) is the most common type of lymphoma in dogs. Almost half of nearly 1700 lymphomas in dogs were of this subtype.^1–3 It is also one of the most common lymphomas in all species and is the “classical” form of lymphoma seen frequently in cytologic or histologic specimens, appearing as enlarged lymph nodes filled or effaced by a homogeneous population of large immature lymphoid cells, large nuclei, small to moderate amount of basophilic cytoplasm, a high mitotic count, high‐grade lymphoma, and numerous tingible body macrophages. The diagnosis of lymphoma is made easily, but other immature large cell lymphomas look almost identical in routine specimens and the distinction between the different types of large cell lymphomas requires immunophenotyping, cytology, and histologic organization of the tumor within lymph nodes.

DLBCL can be divided into centroblastic or immunoblastic (single, central nucleolus) based on examination of nuclei and nucleoli. The majority are high‐grade lymphomas. T‐cell‐rich B’cell lymphoma is a subtype of DLBCL, it is common in cats and horses and rare in dogs.^4–8 Two other rare variants of DLBCL are mediastinal and intravascular. However, most mediastinal and intravascular lymphomas in dogs are of T‐cell phenotype.⁹ DLBCL is associated with BLV and FeLV.¹⁰ As more parameters, and careful follow‐up information on biologic behavior are collected and correlated with molecular techniques it is likely that DLBCL will be subdivided in animals as it is in humans. Human DLBCL can be subdivided into two subtypes: tumors that are believed to arise from the germinal center B cell are called GCB lymphomas and those arising from cells exiting the germinal center, referred to as activated B cells, are called ABC lymphomas. Expression of different NF‐kB pathway–related genes is part of the molecular classification of human DLBCL: GCB subtype originates from centroblasts and expresses BCL6 and ABC subtype originates from plasmablasts and expresses XBP1, PRDM1, and IRF4.¹¹ This may be the case for canine B‐cell lymphomas, but the data for this separation in canine studies is not uniform.^12,13 At least the shared signaling pathways between the two species make the dog an attractive model.¹¹

Epidemiology and occurrence

DLBCL is the most common lymphoma in most domestic species, birds, and many wild animals.¹⁴ It is a common type of lymphoma in cats using the REAL/WHO system. Locations may be mediastinal, in URT, in segments of the bowel, or multicentric. By anatomic location, enteric lymphoma has emerged as the most common lymphoma in cats, and most are T‐cell.^4–6,15 In dogs, DLBCL accounts for almost 50% of all lymphomas.^1,2,16,17 TCRLBCL, a variant of DLBCL, is the most common lymphoma in horses (40–50%).^7,8 DLBCL makes up another 8–10% of the equine cases.⁷

In cattle the large cell lymphomas comprised almost 70% of 1200 cases, but immunophenotyping was not performed.¹⁸ Large cell lymphoma, particularly those with cleft nuclei, are usually related to BLV.¹⁴ The less common types that occur in calves as leukemia and in 2‐year‐olds in the thymus or skin (mycosis fungoides) are more likely to be T‐cell lymphomas and are not BLV related. In swine, approximately 60% of 136 cases of lymphoma were of large cell type.¹⁴ In the cat, DLBCL has been associated with FeLV but there also are DLBCLs that are not virally induced.^4,5,14 One study in cats reported that all cats infected with FeLV or FIV had high‐grade lymphomas.⁵ Six of 7 cats with FIV had B‐cell lymphomas and the phenoptype was split fairly evenly in the 9 cats infected with FeLV.⁵ In dogs no association has been found between lymphoma of any type and viral infection. In birds there is a constant association of large T‐cell lymphoma affecting peripheral nerves with herpes virus (Marek’s disease).¹⁴ HIV lymphomas in humans are also T‐cell lymphomas.

Types of diffuse large cell lymphoma

Clinical presentation

Most dogs with DLBCL have multicentric lymphoma with a generalized lymphadenopathy, but approximately 10% are diagnosed when only a single peripheral node is enlarged.^1,2,14 Up to 20% will involve abdominal nodes or viscera and 10% will be in the skin.¹ Large cell lymphomas are common in cats; they may be T‐cell or B‐cell and are found in different anatomic locations.^{4–6,10,19–21} Nasal lymphoma in cats are almost all large B‐cell lymphomas.^4,5,10,19 Large B‐cell lymphomas are common in the stomach and ileal–cecal region of cats.¹⁵ The number of cases of mediastinal lymphomas in cats has decreased since the implementation of testing and vaccination for FeLV but it still represents a frequent anatomic site.

Horses may present with multiple subcutaneous tumors (TCRLBCL) or with enlarged abdominal nodes that cause colic.^7,8 Reports indicate that multicentric lymphoma with numerous enlarged lymph nodes is the most common lymphoma in horses and the great majority are DLBCL,^7,8 but cutaneous lymphoma is also common in horses and unique. The most common problems were weight loss (25 of 37 horses) and ventral edema (21 horses, 19 of which had hypoalbuminemia).⁷ Immunophenotyping revealed 26 T‐cell and 7 B‐cell lymphomas; 4 could not be classified. Morphology of tumors was heterogeneous (n = 17) and it was difficult to assign definite classifications. Eight tumors had a marked histiocytic component with giant cells.

Multiple tumors were considered TCRLBCL, which is a common lymphoma in horses. Horses can have hemolytic anemia and a huge splenic lymphoma that may weigh several hundred pounds, but these cases are rare.¹⁴ In the era before ultrasound, splenic lymphoma was difficult to diagnose because the spleen was not palpable on rectal examination and an important differential for the hemolytic anemia was equine infectious anemia virus.

Pathology

Lymph nodes

One or more peripheral lymph nodes are enlarged, some markedly so. Often the entire node is effaced by a diffuse infiltration. This is the prototype lymphoma that completely fills and effaces lymph nodes with uniform large immature lymphocytes. If seen early, the tumor seems to start in the cortex. The subcapsular sinus is compressed or filled with tumor, the capsule is thin and tumor extends through the capsule into the perinodal tissues. The interior architecture is diffusely involved and medullary cords are replaced by neoplastic cells. Nodes that are not completely effaced will have remnants of normal structures remaining: small darkly stained regions of germinal centers in the outer cortex, outlines of the medullary cords may be evident, subcapsular sinus focally compressed and fibrovascular supporting structures present. The rate at which the tumor progressed can be deduced from the absence or prominence of these normal histologic structures. In rapidly advancing lymphomas there is little medullary structure present, while in slower developing cases the medullary supporting structures will be hyperplastic and more apparent. DLBCL neoplasms are not sclerotic and ischemic necrosis is not present. On gross inspection the nodes are white and soft and cut surfaces bulge. Touch imprints from cut surfaces yield many large, high‐grade type immature lymphocytes (Figure 7.20).

Enlarged nodes are diffusely filled with neoplastic cells in monotonous sheets (Figures 7.20–7.22). In histologic sections the cells are best interpreted near the tissue margins where fixation has been rapid. DLBCL is composed of large cells, most of the nuclei are 2.0 or more red blood cells in diameter. Nuclei are round to oval and almost never elongated as seen in lymphoblastic lymphoma of B‐ or T‐cell types. Nuclei are rarely cleaved or indented. Nuclei tend to fill the entire cell such that only a small amount of basophilic cytoplasm is seen. Mitotic counts vary considerably from less than 1/400× field to 20 or more. As the mitotic count increases so do macrophages. Tumors with high mitotic counts will have numerous tingible body macrophages (Figure 7.19), seen easily at low magnification as a moth‐eaten appearance. Tumors with a lower mitotic count will have a paucity of these phagocytic macrophages.

DLBCL can be divided into centroblastic or immunoblastic based on examination of nuclei and nucleoli. Neoplastic cells with a single prominent central nucleolus are termed immunoblastic (Figure 7.21A,B). Centroblastic types have multiple nucleoli. These are not as obvious as in immunoblastic types and they are often situated near the nuclear margin (Figure 7.20A,B). Approximately 25% of canine DLBCLs are immunoblastic and 75% centroblastic. Many tumors will have a mixture of each type, and the designation immunoblastic was only used when 90% or more of the nuclei were of this type.¹

T‐cell‐rich large B‐cell lymphoma (TCRLBCL) is a type of large B‐cell lymphoma composed predominantly of small, non‐neoplastic T cells with variable numbers of large neoplastic B cells scattered through the tumor. They usually have abundant fine stroma.

Cytochemistry and immunohistochemistry

The cells of DLBCL label strongly with CD79 alpha and CD20 and are negative with CD3. In horses, CD20 is a preferable B‐cell marker. Neoplastic cells can readily be stained in cytological preparations after fixation in formalin and with heat‐activated antigen retrieval. Phenotyping can also be performed on slides stained with Wright–Giemsa. These slides need to be decolorized in the antigen retrieval process and then stained via IHC. In the dog, most of the tumors of DLBCL type have surface or cytoplasmic Ig and are positive for light chains.^1,2,14 It is likely that in dogs, as in people, there are biologic and morphologic variations in DLBCL that are not presently detectable by our limited immunophenotypic analyses.

Differential diagnosis

The diagnosis of lymphoma is straightforward at gross examination in an autopsy; multiple nodes are enlarged and there may be hepatospenomegaly. The diagnosis of lymphoma from cytology or histopathology is easy and the neoplasm is identified as a large cell lymphoma, likely DLBCL. Differentials for DLBCL include peripheral T‐cell lymphoma of large cell type, T‐cell lymphoma not otherwise specified, LBL, MZL, and Burkitt’s‐type lymphoma; even benign lymphoid proliferations can be considered in some cases, especially those in the gastrointestinal tract. DLBCL and large T’cell lymphoma look identical and are distinguished by immunophenotype. LBL has smaller nuclei. They are intermediate in size with dispersed chromatin that may obscure nuclear detail, while the nuclei of DLBCL are larger, with aggregated chromatin, and prominent nucleoli. MZL is of B‐cell type and the characteristic arrangement of neoplastic cells around fading follicles combined with smaller nuclei, a low mitotic count, and more abundant cytoplasm help to identify it. However, stages of both MZL and DLBCL are morphologically similar and molecular studies indicate the two diseases may represent a continuum rather than separate tumors.^12,13 Molecular separation of the two tumors was not distinct.¹² Follicular lymphoma is B‐cell but the tumor should form distinct follicles as opposed to diffuse sheets of neoplastic cells as seen in DLBCL. Follicular lymphoma is rare in animals. DLBCL and Burkitt’s lymphoma can look similar morphologically and both are B‐cell lymphomas. Burkitt’s lymphoma has intermediate‐sized cells and DLBCL has large cells. Nucleoli in Burkitt’s lymphoma are numerous, small, and always central. However, in DLBCL the nucleoli are single, and larger in immunoblastic types. In centroblastic DLBCL the multiple nucleoli should impinge on the nuclear membrane, which is not seen in Burkitt’s lymphoma.

DLBCL is a common type of lymphoma in animals and Burkitt’s lymphoma is rare. In the few dogs studied, Burkitt’s lymphoma had a translocation t(8;13) at the IGH locus of canine chromososome 8 (CFA 8) and the MYC locus in CFA 13.¹² If confirmed on larger series, having molecular signatures to complement morphology will obviously improve our ability to distinguish between tumors that look similar.

Tumor cell transformation and progression

No cytologic changes are associated with progression of DLBCL. In human large B‐cell lymphoma the hypermutation of the Ig gene is detectable by PCR and the subtype of lymphoma can be related to gene expression profiling.

Survival time

See sections on B‐ and T‐cell CLL and the appendix on lymphoma (p. 961) for additional prognostic information. The majority of DLBCLs are high‐grade lymphomas that eventually cause the death of dogs, cats and horses, usually in less than one year. As a generalization, B‐cell lymphomas have a better prognosis than T‐cell lymphomas, but tumors within each of these two groups are heterogeneous and subdividing them has prognostic implications. Size of tumor cells has been used to subdivide B‐cell tumors. Small cell tumors (SLL, CLL) had MST that was not reached and dogs with large cell B‐lymphocytosis had MST of 130 days.^23,24 Medium to large B‐cell lymphomas can be subdivided to help predict outcome via histopathology, survivin expression, or MHC expression. Using histopathology, the MST of B‐cell lymphoma ranged from 15 days for Burkitt’s‐like lymphoma to 510 days for other subtypes.²⁵ Using survivin as a marker, the time to first remission was 171 days for high levels and 321 days for low levels of survivin expression.²⁶ Approximately 160 dogs with B‐cell lymphoma could be divided with flow cytometry by class II MHC expression or cell size to help predict outcome when treated with a multi‐agent chemotherapy protocol. Dogs with low level of expression of class II MHC had decreased adjusted survival times of 120 days versus 314 days for dogs with high expression of class II MHC.²³ The subset of dogs with low expression of class II MHC was small. Most dogs had high expression and therefore shorter survival.

The poorer prognosis identified is similar to that observed in humans. The explanation may be decreased presentation of tumor antigen to CD4 T cells, resulting in decreased host immune response. The data from both species suggests that the biology and the immune system of the host, not just the type of neoplasm present, is an important or more important component of the prognosis. Dogs with large B‐cell lymphoma (via flow cytometry) had adjusted MST of 120 days versus 275 days for medium‐sized tumor cells. Surprisingly, a CD34 (or CD5 or CD21)‐positive phenotype was not predictive of prognosis for dogs.²³ Previous studies indicated that dogs with large B‐cell leukemia (some of which were likely stage V lymphoma) and CD34‐positive cells had a very poor prognosis, with an MST of 16 days.²⁴ Only about 10% of dogs with B‐cell lymphoma express CD34. These authors concluded that in dogs CD34‐positive lymphoma and CD34‐positive leukemia are different types of lymphoproliferative disease.²³ This same study indicated that young age was a negative prognostic factor.

An older and large study found an overall survival time of 330 days in dogs with B‐cell lymphoma treated with multi‐agent chemotherapy.²⁷ A study of 63 dogs with lymphoma treated with a CHOP‐based chemotherapy protocol reported on the stage of lymphoma, histologic types, morphologic groups, and clinical parameters.²⁸ The majority of the dogs had DLBCL and the results indicated that aggressive B‐cell lymphomas had an MST of 256 days. The study correlated remissions with different histologic types as well as indolent and aggressive B‐ and T‐cell lymphomas.²⁸ In the ACVP study the DLBCL cases were divided into centroblastic and immunoblastic types.² Each of these were subdivided into three groups based on mitotic count as low (0–5 mitoses/400× field), intermediate (6–10), and high (>10) grade. Most dogs received chemotherapy, however some cases were terminated shortly after the diagnosis. Treatments varied from practice to practice and the stage of the lymphoma was not provided. From the available information the MST of dogs treated for DLBCL was less than 1 year. A few cases survived longer and none survived 2 years or longer.

Therefore within the classification of B‐cell lymphoma, most of which will be DLBCL, there are subsets with different prognoses and there are multiple methods that can be used to identify these subsets and predict approximate survival times. In humans, morphologically similar DLBCL can be subdivided by gene expression profiles into GCB and ABC patients that have different survival times.^12,13,29 GCB patients have a 5‐year survival of 75% and ABC patients 15% at 5 years. It appears that canine DLBCL or at least large B‐cell lymphomas have germinal center and post‐germinal center subtypes with modestly different survivals; however, the distinction is not as definitive as in humans and needs clarification.^12,13 Molecular classifications of human DLBCL are predictive of biologic behavior and treatment outcomes but are based on hundreds to thousands of analyses. Present data from dogs are based on a low number of cases in which the data are promising.

One 2015 study on high‐grade multicentric lymphomas in dogs did not use phenotyping but used cytology to provide prognostic information.³ Presumably these were a mixture of different lymphomas but certainly DLBCL were in the study group. Subjective cytologic features were evaluated in FNA preparations from 20 dogs with high‐grade multicentric lymphoma. In addition, a numerical scoring system was created and morphometry was performed. At diagnosis, multinucleation in the neoplastic cells was associated with decreased survival time and binucleation was associated with shorter remission. None of the other cytologic features evaluated at diagnosis or the scoring system or morphometry were predictive of survival. At relapse, the number of mitoses in lymph node aspirates and the total cytologic scores assigned were greater than those obtained at the time of diagnosis.³ However, the increased total score was due to the increased mitoses and not increased morphologic abnormalities. Mitotic counts as determined in this study did not correlate with survival time or other time points assessed. The overall MST of the dogs followed was 236 days.³ Despite relatively low numbers of cases, no immunophenotyping data, and no staging data, the results suggest that cytologic evaluation for bi‐ and multinucleation at the time of diagnosis has predictive value for survival. Cytology of good‐quality preparations can diagnose 80–90% of lymphomas. Incorporating assessment of bi‐ and multinucleation may provide additional information to help oncologists and owners make decisions. The original paper should be read for the many details and comparisons that were studied.³

Lymphomas can be classified by pathologists as outlined in Box 7.1 and DLBCL in animals can be divided into centroblastic and immunoblastic types. Molecular techniques are being used to subdivide these and other lymphomas to determine their utility as models for similar diseases in humans.^12,13,29,30 For future studies we need standardized classification of lymphomas between pathologists and for clinicians to standardize treatment protocols. These data will then need to be correlated with accurate clinical follow‐up, including autopsies on a large number of cases, if we are to determine reliable survival times, prognoses, and effective therapeutic protocols. It is unlikely there will be a sufficient number of cases in which no treatment is received and the dogs are allowed to live out their lives to compare survival times between treated and nontreated dogs. A confounder in all types of clinical studies will be the level of commitment to continue therapy and how different clinicians and owners will use the option of euthanasia.

Definition of the neoplasm

T‐cell‐rich large B‐cell lymphoma (TCRLBCL) is a B‐cell lymphoma but the diagnostic characteristic is a mixture of lymphocytes and other cells. Early TCRLBCL may have 80–90% small to intermediate‐sized non‐neoplastic T cells, with the rest of the cells being neoplastic large B cells, histiocytes, and connective tissue cells. The neoplastic large B cells gradually increase in proportion to the other cells. TCRLBCL is the or one of the most common lymphomas in horses.^1–4 It is fairly common in cats⁵ but is uncommon in dogs (1% of all lymphomas).⁶

In cats this lymphoma has been called “feline Hodgkin’s disease” because of the way the tumor spreads and the presence of large, atypical, binucleated B‐cell lymphocytes that vaguely resemble the Reed–Sternberg cells of human Hodgkin’s lymphoma.⁵ The characteristic that most resembles the human tumor in cats is that the disease spreads only to contiguous lymph nodes and does not skip to nodes in other anatomic regions.⁵

Epidemiology, occurrence, and clinical presentation

TCRLBCL constitutes about 10% of all feline lymphomas. Most cats are mature, they are in good body condition, typically without loss of weight or appetite.⁵ Oddly, the presentation is almost always for a single enlarged node in the neck area, usually in a submandibular node and more often on the right side⁵ (Figure 7.23). Approximately 25% of cats will have multicentric tumors in almost any organ. In the dog it is seen in a peripheral node but may appear in any area of the body, even in the spinal canal.

In horses, TCRLBCL is the most common lymphoma, accounting for approximately 40% of equine lymphomas.^1–4 The mean age is 10 years and horses appear in good health with minimal loss of weight or appetite.¹ This lymphoma shares the slow progression seen in cats but has very different clinical presentations.^1–5,7,8 In horses this lymphoma is unique in that the most common problem is subcutaneous nodules (see Figure 7.25). There may be a few or up to 100 or more discoid shaped tumors irregularly distributed over the entire body but most are concentrated at the lower area of the neck.¹ These lesions are not in the skin but appear to arise along lymphatics in the subcutis. The subcutaneous tumors are typically 2–3 cm in diameter but some may become large, ulcerate the skin and form a mass weighing up to 30 pounds. The skin lesions have regressed during pregnancy and recurred following parturition attributed to progesterone receptors on the neoplastic cells.^7,8 The other presentation is multicentric lymphoma with tumors in the gastrointestinal tract and many other organs. If only a few nodules are found and they are excised fully this has proven an effective treatment in some cases.⁴ Those cases in which the tumor does not recur post excision have survival times of up to 10 years.⁴

Pathology

Lymph nodes or skin nodules

The diagnosis rests on the recognition of a mixed population of cells with a few characteristic large binucleated cells with large central nucleoli and abundant cytoplasm (Figure 7.23). Unlike most lymphomas, TCRLBCL is not a homogeneous population of uniform lymphoid cells. Early lesions have more small cells and are T’cell‐rich. As the tumor progresses the larger B cells increase disproportionately (Figures 7.23–7.27). B cells may predominate in some regions of the tumor. The increase of B cells is usually accompanied by increased numbers of macrophages. Histiocytic cells are very obvious in some of the equine subcutaneous tumors and they will be accompanied by multinucleated giant cells (Figure 7.25). Some giant cells may have thin, crystalline‐like cytoplasmic inclusions.

An unusual, yet characteristic part of this lymphoma are the large B cells that can have marked anisokaryosis (Figures 7.23, 7.27, and 7.28). The nuclei in these cells will range from 2 RBC in diameter to 3–5. The chromatin of the large B cells is peripheralized, which accentuates a single large central nucleolus. The large cells are often binucleated and a few cells will have 3–4 nuclei. Some large cells are necrotic and have contracted brightly pink cytoplasm and a pyknotic nucleus. Necrosis of individual tumor cells should be looked for as this feature is always present (Figure 7.23).

In all species there is fine sclerosis which renders the tumor cohesive (Figure 7.23). The nodes are firm to palpation and cut surfaces are solid, the typical soft white appearance of lymphoma is not present.

Cytochemistry and immunohistochemistry

The patterns seen with IHC are unusual because of the heterogeneity of cells within a tumor and different tumors from the same animal may vary in the proportion of B‐ versus T lymphocytes. The large B cells mark consistently with CD20 and less frequently with CD79 alpha (Figures 7.24, 7.26, and 7.28). CD20 is preferred for horses. The smaller T cells are positive with CD3 and in some cases, or at some stages, CD3‐positive cells will be predominant (Figure 7.24). IHC for the small T cells accentuates the large nuclei of the unstained B cells. IHC is useful to identify cell types but it does not distinguish neoplastic and non‐neoplastic cells. If histochemical stains are done for reticulin there are diffuse fine fibrillar fibers in all areas of nodal and subcutaneous tumors.

Differential diagnosis

The main differentials for TCRLBCL are hyperplasia or an infectious agent. Some cases, especially in horses will have so many multinucleated giant cells and histiocytic cells that stains for acid‐fast organisms or fungi are performed. These will be negative. Tumors with foci of ischemic necrosis can look inflammatory. Awareness that TCRLBCL is a mixture of cells combined with the large nuclei and anisokaryosis that is present in the neoplastic large cell population helps focus the diagnosis on lymphoma. A high index of suspicion for this lymphoma in cats and horses is helpful, especially if the lesion is a subcutaneous nodule from a horse. The large nuclei, binucleation, and prominent nucleoli in large cells are further indications of neoplasia. Clonality detection by PCR should be able to identify clonal antigen receptor genes for B cells if the clonal expansion is greater than 1%. However, the sensitivity and specificity of these assays vary with the expertise of the laboratory. This is a tumor in which expertise with reagents and interpretation is needed.

Evaluation of treatment and survival

Because of the slow rate of progression and the fact that in the cat and dog most of the early cases involve a single node, excision is a consideration. However tumors tend to recur in the same area as the excision. If there is only one or a few subcutaneous tumors then excision may be effective in horses. As stated earlier, some horses respond well to surgical excision and will have long survival times.⁴ The tumors may be so numerous that excision is not possible. Clinical oncology texts and references provide treatment recommendations. In the ACVP study of canine lymphoma there were 9 cases of TCRLBCL.⁶ These cases had a surprisingly short survival with a range of 89–105 days and a mean of 97 days; however, the follow‐up data in this study need to be confirmed.