Immunophenotypic and cytomorphologic subclassification of T-cell lymphoma in the boxer breed

The boxer breed is at high risk for developing lymphoma and, in contrast to the general canine population, is predisposed to the T-cell variant of the disease. The purpose of this study was to more accurately classify lymphoma in this breed. Clinical, cytomorphologic and immunophenotypic data were examined in 43 boxers with lymphoma. Twenty-five cases were collected prospectively and a further 18 cases were obtained retrospectively. Lymphomas were classified as multicentric (n=29), mediastinal (n=6) and intestinal (n=8). Of the 40 immunophenotyped samples, 34 (85%) were T-cell, 5 (12.5%) were B-cell and 1 was a non-B-cell non-T-cell lymphoma. Immunophenotypic subtyping was done on prospectively collected T-cell lymphoma samples (n=22) to differentiate CD4 (helper) from CD8 (cytotoxic) T-cell origin as well as to determine the T-cell receptor (TCR) expression (TCRalphabeta vs. TCRdeltagamma). Phenotypic expression was CD4+ (n=12), double negative (DN) (n=6), double positive (DP) (n=1) and CD8+ (n=1), respectively, while two samples had no interpretable result. 20/22 samples were TCRalphabeta+ with only 1 sample being TCRdeltagamma+ and 1 with no interpretable result. Cytomorphologic analysis was done on the same 22 samples using the World Health Organization (WHO) classification scheme. According to this scheme, 17/22 samples were classified as lymphoblastic, 2/22 as large cell peripheral T-cell lymphoma-not otherwise specified (PTCL-NOS), 2/22 as large granular lymphoma (LGL) high-grade and 1/22 as small lymphocytic. The results of this study indicate that lymphoma in the boxer breed is a disease comprised predominantly of TCRalphabeta+, CD4+ (helper) T-cells with lymphoblastic (high-grade) morphology.     

Use of fine needle aspirates and flow cytometry for the diagnosis, classification, and immunophenotyping of canine lymphomas.

Fifty canine lymphomas were classified cytomorphologically using the updated Kiel classification scheme. Aspirates of lymph nodes from dogs with lymphoma were stained using 5 canine-specific antibodies and 3 human-specific antibodies that cross-react with canine lymphocytes. The antibody-stained aspirates were analyzed by flow cytometry. A total of 32 (64%) of the 50 lymphomas were characterized as B-cell origin and 18 (36%) were of T-cell origin. B-cell lymphomas were identified in 12 females and 20 males with a mean age of 8.35 years. T-cell lymphomas were identified in 8 females and 10 males with a mean age of 7.9 years. A minority of the lymphomas were low-grade B-cell and T-cell lymphomas (6/50, 12% and 4/50, 8%, respectively). The most common morphologic types were high-grade centroblastic and unclassifiable plasmacytoid for B- and T-cell lymphomas (18/50, 36% and 7/50, 14%, respectively).     

Aberrant phenotypes and quantitative antigen expression in different subtypes of canine lymphoma by flow cytometry

Flow cytometry may be a useful tool to analyze lymphoma samples that are obtained from fine needle aspirations (FNA). This study aimed to determine if flow cytometric analysis add more objective and standardized information on the cellularity and morphology of lymphoma cells to conventional cytology. The typical immunophenotype of different lymphoma subtypes was assessed and leukocyte marker expression was evaluated to determine which antigens were more frequently over- or under-expressed in these lymphoma subtypes. Fifty FNA lymph node samples were evaluated from canine lymphomas. Thirty-one samples were identified to be of B-cell origin, sixteen were identified to be of T/NK-cell origin and three cases were classified as acute lymphoblastic leukaemia with lymph nodes involvement. The most common B-cell lymphoma subtypes were centroblastic lymphomas, whereas three cases were atypical and classified as B-large cell pleomorphic lymphomas. Among the T/NK lymphomas, small clear cells, large and small pleomorphic mixed cells, large granular lymphocytic cells and small pleomorphic cells were identified. Aberrant phenotypes and/or antigen under/over regulation was identified in thirty out of forty-seven lymphoma cases (64%; 18/31 B-cell=58% and 12/16 T-cell=75%). In B-cell lymphomas the most frequent finding was the diminished expression of CD79a (45%). CD34 expression was also observed in four cases (13%). Among T-cell lymphomas the prevalent unusual phenotype was the under-expression or absence of CD45 (25%). These findings reveal flow cytometry may be useful in confirming the diagnosis of lymphoma, as the technique allows one to add useful information about morphology of the neoplastic cells and identify antigenic markers and aberrant phenotypes.     

Prognostic significance of morphological subtypes in canine malignant lymphomas during chemotherapy

The aim of this study was to determine the response of different morphological subtypes of canine lymphoma to a standardized therapeutic protocol. Diagnosis of lymphoma was based on cytohistological analysis and immunophenotyping with antibodies against CD3 and CD79a of an enlarged lymph node or an extranodal mass. Fifty-seven cases were classified according to the updated Kiel classification adapted to the canine species, into 24 B-cell lymphomas (20 centroblastic polymorphic and four Burkitt-type subtypes), and 33 T-cell lymphomas (10 pleomorphic mixed, 10 lymphoblastic, eight unclassifiable high grade plasmacytoid, and five small clear-cell subtypes). All dogs were clinically staged at diagnosis. The protocol used l-asparaginase, vincristine, cyclophosphamide, doxorubicin, and prednisone. First remission duration and overall survival time were evaluated. Although the T-cell phenotype was associated, on the whole, with a poor prognosis, as previously reported in veterinary and human medicine, the study showed significant prognostic differences between the B- and the T-cell subtypes of canine lymphoma and suggests that clinico-morphological characterization of the disease is justified in dogs, as in humans.     

Inactivation of the p16 cyclin-dependent kinase inhibitor in high-grade canine non-Hodgkin's T-cell lymphoma

The significance of p16/Rb tumor suppressor pathway inactivation in T-cell non-Hodgkin's lymphoma (NHL) remains incompletely understood. We used naturally occurring canine NHL to test the hypothesis that p16 inactivation has specific pathologic correlates. Forty-eight samples (22 T-cell NHL and 26 B-cell NHL) were included. As applicable, metaphase- or array-based comparative genomic hybridization, Southern blotting, promoter methylation, and Rb phosphorylation were used to determine the presence, expression, and activity of p16. Fisher's exact test was used to test for significance. Deletion of p16 (or loss of dog chromosome 11) was restricted to high-grade T-cell NHL (lymphoblastic T-cell lymphoma and peripheral T-cell lymphoma, not otherwise specified). These were characterized by a concomitant increase of tumor cells with Rb phosphorylation at canonical CDK4 sites. Rb phosphorylation also was seen in high-grade B-cell NHL (diffuse large B-cell lymphoma and Burkitt-type lymphoma), but in those cases, it appeared to be associated with c-Myc overexpression. The data show that p16 deletion or inactivation occurs almost exclusively in high-grade T-cell NHL; however, alternative pathways can generate functional phenotypes of Rb deficiency in low-grade T-cell NHL and in high-grade B-cell NHL. Both morphologic classification according to World Health Organization criteria and assessment of Rb phosphorylation are prognostically valuable parameters for canine NHL.     

Cytomorphological and immunohistochemical features of lymphoma in ferrets

Twenty ferrets with histopathologically diagnosed lymphoma were classified cytomorphologically and immunohistochemically. According to site of origin, multicentric, gastrointestinal, mediastinal and cutaneous lymphomas accounted for 8 (40%), 9 (45%), 2 (10%) and 1 case (5%), respectively. According to the National Cancer Institute Working Formulation (NCI-WF), low-, high- and intermediate-grade lymphomas accounted for 4 (20%), 4 (20%) and 12 cases (60%), respectively. The 4 low-grade lymphomas showed no mitotic figures, whereas all 4 high-grade lymphomas exhibited > or = 3 mitotic figures (median,6). Higher grade thus appears to be associated with a higher number of mitotic figures. Immunohistochemical examination of 18 specimens, excluding 2 insufficient specimens, showed that 16 (88.9%) and 2 (11.1%) lymphomas were of T-cell origin and B-cell origin, respectively. According to the combination of the NCI-WF and immunophenotypes, all 4 low-grade lymphomas (2 multicentric, 1 gastrointestinal, and 1 cutaneous lymphoma) were classified as diffuse small lymphocytic lymphoma of T-cell origin. Of the 12 intermediate-grade lymphomas (6 multicentric, 4 gastrointestinal, and 2 mediastinal lymphomas), 11 were classified as diffuse mixed-cell lymphoma, and 1 as diffuse large cell lymphoma. Of these 11 lymphomas, 2 (both multicentric) were of B-cell origin, 7 (3 multicentric, 3 gastrointestinal, 1 mediastinal) were of T-cell origin, and 2 (1 multicentric, 1 mediastinal) were of unknown cell origin. The remaining 1 lymphoma (gastrointestinal) was of T-cell origin. All 4 high-grade lymphomas (gastrointestinal) were classified as diffuse immunoblastic lymphoma of T-cell origin.     

Lineage differentiation of canine lymphoma/leukemias and aberrant expression of CD molecules

Multiparameter flow cytometry analysis and specific cluster differentiation (CD) molecules were used to determine the expression profiles of B- and T-cell antigens on lymph node preparations from 59 dogs with generalized or multisystemic lymphoma. Lymph node samples from 11 healthy dogs were labeled to validate the specificity of antibodies and to formulate guidelines for interpretation of the results obtained from lymphoma samples. In normal lymph nodes, T-lymphocytes expressing CD3, CD4, or CD8 beta represented 59+/-11%, 43+/-8%, or 16+/-5% of the total cells, whereas B-lymphocytes expressing either CD21 or surface IgM (IgM) represented 37+/-9% or 14+/-5%, respectively. Small lymphocytes could be distinguished from large lymphocytes by forward light scatter. Of the patient samples 29 different breeds were represented with Golden and Labrador retriever being the most common. The lymphoma samples segregated into three groups based on CD antigen expression. Thirty cases predominantly expressed one or more combinations of CD79a, IgM, and CD21 representing a B-cell lineage. Three B-cell cases also expressed the stem cell antigen, CD34. Sixteen cases expressed one or more combinations of CD3, CD4, and CD8 consistent with a T-cell lineage and CD3+CD4+CD8--phenotype was the most common. Thirteen cases showed a mixed expression profile for T- and B-cell antigens and in three cases CD14 was highly expressed. Clinical response was poorest for T-cell lymphomas. Leukemic states occurred in all three phenotypes; but mixed cell cases had the greatest proportion. Dual immunofluorescence staining confirmed co-expression of T-cell (CD3) and B-cell antigens (CD79a or CD21) on neoplastic lymphocytes of six mixed cell cases. In one mixed cell case, dual immunostaining identified lymphocyte populations that stained mutually exclusive for CD79a and CD3. Six mixed cell lymphomas tested by PCR showed clonality for rearranged antigen receptor. Four cases that were CD79a+CD3+ had TCRgamma chain gene rearrangements, whereas two cases that were CD3+CD8+CD21+ had Ig heavy chain rearrangement. One case expressing multiple CD molecules (CD3+CD8+CD21+CD14+) was PCR negative for both Ig and TCRgamma gene rearrangement and could not be classified into a B- or T-cell lineage. We show for the first time co-expression of B- and T-cell markers on lymphoma cells that had specific T- or B-cell gene rearrangements. These findings suggest that aberrant CD molecule expression is not an uncommon finding in canine lymphomas and is a useful diagnostic marker for malignancy.     

Lymphoid neoplasms in swine

Seventeen cases of lymphoid neoplasms in swine were investigated and divided into eight histological types. Cases 1-3 were precursor B lymphoblastic leukemias, which occurred in three piglets from the same dam. Cases 4 and 5 were diagnosed, respectively, as a precursor B lymphoblastic lymphoma and a thymic B cell lymphoma, because there were cytological differences between the lymphomas. These five cases of immature B cell malignancies expressed CD79a and terminal deoxynucleotidyl transferase (TdT). Mature B cell lymphomas were divisible into follicular (case 6), diffuse centroblastic (case 7) and intestinal large B cell (cases 8-11) lymphomas. Unlike in case 7, the neoplastic cells in cases 8-11 showed cytological features intermediate between centroblasts and immunoblasts. The mature lymphomas were characterized by positive immunolabeling for CD79a and cytoplasmic immunoglobulins. A case of thymic γδ T cell lymphoma (case 12) were positive for CD3, CD5, WC1 and TdT. Instead of TdT, perforin was expressed in γδ T cell lymphomas (cases 13-17), whose histological characteristics were epitheliotropism, homing into T cell zones of lymphatic tissues, and cytological atypia and pleomorphism. In the present study, lymphoid neoplasms could be classified into discrete histological types, some of which were considered to be specific for swine.     

Canine indolent nodular lymphoma

Sixty-six cases of indolent canine lymphoid proliferation were reviewed. Age ranged from 1.5 to 16 years (median 9.0 years). Dogs of 26 breeds, plus 13 of mixed breeding or unknown lineage, were represented. B-Cell lymphomas (CD79a+) predominated. Marginal zone lymphoma (MZL), the largest group, involved lymph node (33 cases) and spleen (13 cases), with both tissues involved in five of these cases. Follicular lymphoma (FL) involved lymph nodes (five cases), and mantle cell lymphoma (MCL) occurred as solitary splenic masses (three cases). Nodal CD3+ T-zone lymphomas (TZL) (10 cases), were included since they resembled late-stage MZL at the architectural level. Two cases of marginal zone hyperplasia (MZH) were included to aid in differentiation of early MZL. Clonality status was determined in 54 cases by analysis of immunoglobulin heavy chain (IGH) and T-cell antigen receptor gamma (TCRG) gene rearrangement. Clonal rearrangement of IGH was detected in 28 of 35 MZL cases (80%), four of four FL cases (100%) and three of three MCL cases (100%). Concurrent cross lineage rearrangement of TCRG was detected in six MZL and two FL cases. Clonal rearrangement of TCRG was documented in five of eight TZL cases (63%). Limited survival data obtained for 18 dogs indicated that the B-cell lymphomas (MZL, MCL, and FL) and the T-cell lymphoma (TZL) were associated with indolent behavior and long survival. Although to the authors' knowledge, the true incidence of canine indolent lymphomas is unknown, the tumors are not rare and may have been underrecognized. Recognition of their architectural features, routine application of immunophenotyping, and molecular clonality assessment should alleviate this.     

Canine angiosarcoma: cytologic, histologic, and immunohistochemical correlations

BACKGROUND: Angiosarcomas (AS) are malignant tumors that arise from vascular endothelial cells and are common in dogs. Histologically, AS are markedly heterogeneous neoplasms that make interpretation by cytology difficult. OBJECTIVE: Our objective was to evaluate the cytologic features of canine AS and look for additional diagnostic criteria. METHODS: Cytologic specimens from 19 histologically and immunohistochemically confirmed cases of canine AS were extensively reviewed for cytologic features. We compared cytologic, histopathologic, and immunohistochemical findings. RESULTS: Neoplastic cells in 14 cytology specimens had a high-grade sarcomatous appearance, whereas in 4 specimens the cells were extremely pleomorphic, ranging from sarcomatous to epithelioid. In the remaining case, the neoplastic cells were low grade, spindle shaped, and monomorphic. Other relevant cytologic findings were blood contamination (18/19 cases), cellular cohesiveness (16/19), punctate cytoplasmic vacuolation (19/19), background neutrophilia (11/19) and eosinophilia (5/19), erythrophagocytosis (8/19), extramedullary hematopoiesis (8/19), and apoptotic leukocytes (14/19). Vasoformative features (ie, pseudoacinar structures) were observed in 7 of 19 samples. Histologically, 16 neoplasms had a proliferative pattern typical of well-differentiated canine AS. Three tumors were atypical poorly differentiated AS; 2 of these had a striking epithelioid pattern and 1 was a poorly differentiated spindle cell tumor with focal vascular differentiation. Immunohistochemically, tumor cells in 16 cases were positive for both endothelial markers tested (Factor VIII-related antigen [FVIII-ra] and CD31 antigen), 2 were positive for CD31 only, and 1 was positive for FVIII-ra only. The epithelioid AS were negative for cytokeratins. CONCLUSIONS: The cytologic characteristics of canine AS are widely heterogeneous, but supplementary findings can provide clues that are useful for making a cytologic diagnosis. Histologic and immunohistochemical confirmation is nonetheless warranted in all cases.     

Diagnosis of mediastinal masses in dogs by flow cytometry

BACKGROUND: Biopsy of mediastinal masses can be invasive, but the procedure may be necessary if cytology of a mass aspirate is inconclusive. The 2 most common mediastinal masses, lymphoma and thymoma, may both be comprised of small lymphocytes. We investigated the ability of flow cytometry to distinguish between these 2 neoplasms. HYPOTHESIS: Flow cytometry of mediastinal mass aspirates may provide a definitive diagnosis of thymoma or lymphoma, reducing the need for biopsy. ANIMALS: Dogs with mediastinal masses presenting to the Veterinary Teaching Hospital/Animal Cancer Center were included in the study. METHODS: Aspirates obtained over 2 years that met the inclusion criteria (i.e. sufficient viable cells and a definitive diagnosis by means other than flow cytometry) were analyzed by flow cytometry to determine the percentage of cells expressing B- and T-cell markers, and co-expressing CD4 and CD8. RESULTS: All cases of thymoma (n = 6) consisted of > or = 10% lymphocytes coexpressing CD4 and CD8, a phenotype that is characteristic of thymocytes, whereas 6 of 7 lymphomas contained <2% CD4+CD8+ lymphocytes. The CD4+CD8+ lymphoma could be readily distinguished flow cytometrically from thymoma by light scatter properties. The phenotypes of the remaining lymphomas were CD4+ T cell (4), CD34+ (1) and B cell (1). CONCLUSIONS: Our studies demonstrate that flow cytometry is a useful tool for discriminating mediastinal masses. Lymphocyte-rich mediastinal masses could be unambiguously identified by flow cytometry in 13/13 cases.     

High-grade canine T-cell lymphoma/leukemia with plasmacytoid morphology: a clinical pathological study of nine cases.

The aim of this study is to determine the clinical, morphological, and immunophenotypical presentation of 9 cases of a particular type of canine T-cell lymphoma/leukemia. The morphological presentation was a diffuse infiltration of small, medium-sized, or large blast cells with eccentric nuclei, hyperbasophilic cytoplasm, and a juxtanuclear, pale cytoplasmic area, giving a plasmacytoid appearance and suggesting a B-cell morphology. Surprisingly, all 9 cases were of T-cell phenotype (CD3+). Among the 7 immunophenotyped cases, 4 were CD4-/CD8+, 2 CD8+/CD4+, and 1 CD4+/CD8-. The median Ki-67 index was 65.7%, which placed this lymphoma in the high-grade group. This type of lymphoma/leukemia was found in dogs between 1 and 11 years of age, with a median age of 5.8. The male-female ratio was 0.8 for a reference population of 1.04. The most significant clinical findings were lymphadenopathy either generalized or localized in all cases, a mediastinal mass in 4 cases, bone marrow involvement in 7 cases, hypercalcemia in 4 cases, along with an aggressive clinical course and a poor response to chemotherapy in all cases, with a median disease-free survival time of 3 months.     

Feline large granular lymphocyte (LGL) lymphoma with secondary leukemia: primary intestinal origin with predominance of a CD3/CD8(alpha)(alpha) phenotype

Clinicopathologic and immunophenotypic characteristics of large granular lymphocyte (LGL) neoplasia in 21 cats were examined. All cats were domestic short (19) or long hair (2) with a mean age of 9.3 years at diagnosis. Increased peripheral blood LGL counts were present in 18/21 cats. Neutrophilia (12/21 cats) and increased serum liver enzymes (7/12), total and direct bilirubin (7/13), BUN (5/14), and creatinine (2/14) were observed. Cats usually presented with advanced disease and none survived longer than 84 days (mean 18.8 days) postdiagnosis. Cytologically, LGLs had a mature (6/21), immature (13/21), or mixed (2/21) morphology. Necropsy lesions consisted of neoplastic lymphoid infiltrates in the jejunum, ileum, and duodenum in decreasing order of frequency. In the small intestine, mucosal ulceration (9/13) and epitheliotropism of neoplastic cells (9/13) were common. Neoplastic infiltrates were also present in the mesenteric lymph nodes (13/13), liver (12/13), spleen (8/13), kidneys (5/7), and bone marrow (5/7). A T cell phenotype (CD3epsilon+) characterized LGL neoplasia in 19/21 cases. A CD8alphaalpha+ cytotoxic/suppressor phenotype was present in 12/19 T cell tumors, 2 had a CD4+CD8alphaalpha phenotype, 3 had a CD4-CD8- phenotype, and 2 were CD4+ helper T cells. CD8beta chain expression was not detected in any instance. In two cats, a B or T cell origin could not be established. CD103 was expressed by 11 of 19 (58%) of the lymphomas tested. The immunophenotypic features shared by neoplastic LGLs in the cat and feline intestinal intraepithelial lymphocytes (IELs) support a small intestinal IEL origin for feline LGL lymphoma.     

Immunohistochemical study of expression of immunoglobulins in canine B-cell lymphomas

Nineteen canine lymphomas were included in this study. Tumors were classified according to the updated Kiel classification adapted for canine lymphomas by Fournel-Fleury et al. Immunoglobulin light chains (kappa and lambda) and IgM and IgG expression were determined by immunohistochemical method. In all examined cases neoplastic cells were positive for one of the immunoglobulin light chains. Expression of lambda light chains and kappa light chains was observed in 18/19 and 1/19 tumors, respectively. In the majority of neoplastic cells in each examined specimen this reaction had a membranous pattern (skappa/slambda). In all examined cases the presence of immunoglobulin light chains was also observed in the cytoplasm of some neoplastic cells (ckappa/clambda). These cells were usally rare and never constituted a dominant population. The expression of immunoglobulin was found in 13/19 cases. Most lymphomas were sIgM positive (11/13 cases). In one case expression of IgG was found, and in another lymphoma two populations of neoplastic cells with different expression of examined immunoglobulins (cells with IgM+ and IgG+ phenotypes) were observed. The reaction also had a membranous pattern. The cells containing cytoplasmic immunoglobulins were rare, and in most cases were of the same type as the surface immunoglobulins. Our study has confirmed that canine lymphomas are a monoclonal proliferation of B-cells usually expressing immunoglobulin lambda light chains and that the vast majority of tumors deriving from B-cells express IgM. Our study also indicates a possibility of occurence of biclonal lymphomas in canine species.     

Perforin expression can define CD8 positive lymphocyte subsets in pigs allowing phenotypic and functional analysis of natural killer, cytotoxic T, natural killer T and MHC un-restricted cytotoxic T-cells

In this study we have used the expression of perforin to characterize subsets of porcine cytotoxic lymphocytes. Perforin positive lymphocytes expressed both CD2 and CD8, most were small dense lymphocytes (SDL) and up to 90% were CD3 negative. However, the numbers of perforin positive T-cells increased with the age of the animal and their populations increased after specific antigen stimulation in vitro. The remaining perforin positive lymphocytes were large and granular and contained more CD3⁺CD5⁺CD6⁺ T-cells (−40%) of which a substantial proportion also co-expressed CD4. Perforin was expressed in subpopulations of both CD8 and CD8β lymphocytes, but was not expressed in γδ T-cells or monocyte/macrophages. The perforin positive CD3⁻ subset was phenotypically homogeneous and defined as CD5⁻CD6⁻CD8β⁻CD16⁺CD11b⁺. This population had NK activity and expressed mRNA for the NK receptor NKG2D, and adaptors DAP10 and DAP12. Perforin positive T-cells (CD3⁺) could be divided into at least three subsets. The first subset was CD4⁻CD5⁺CD6⁺CD11b⁻CD16⁻ most were small dense lymphocytes with cytotoxic T-cell activity but not all expressed CD8β. The second subset was mainly observed in the large granular lymphocytes. Their phenotype was CD4⁺CD5⁺CD6⁺CD8β⁺CD16⁻CD11b⁻ and also showed functional CTL activity. Thus not all of double positive T-cells are memory helper T-cells. The third subset did not express the T-cell co-receptor CD6, but up to half of them expressed another T-cell co-receptor CD5. The majority of this subset expressed CD11b and CD16, thus the third perforin positive T-cell subset was CD3⁺CD4⁻CD5⁺CD6⁻CD8β^±CD11b⁺CD16⁺, and possessed MHC-unrestricted cytotoxicity and LAK activity.     

Immunohistochemical detection of multiple myeloma 1/interferon regulatory factor 4 (MUM1/IRF-4) in canine plasmacytoma: comparison with CD79a and CD20

Multiple myeloma oncogene 1/interferon regulatory factor 4 (MUM1/IRF4) is involved in lymphoid cell differentiation, particularly in the production of plasma cells. We examined the immunoreactivity of mouse monoclonal antibody Mum-1p to MUM1/IRF4 and compared it with expression of CD79a and CD20 in 109 plasmacytomas in 107 dogs. Tissues had been fixed in formalin and embedded in paraffin. One hundred one of 109 (93.5%) tumors were positive for MUM1/IRF4. The staining was nuclear with weak cytoplasmic reaction. Fifty-nine of 105 (56.2%) plasmacytomas were positive for CD79a; only 21 of 108 (19.4%) cases were positive for CD20. MUM1/IRF4 staining was performed on 139 other tumors including B- and T-cell lymphomas, histiocytic proliferations, mast cell tumors, and melanocytic tumors. The only MUM1/IRF4-positive nonplasmacytic tumors were 10 B-cell lymphomas and 1 anaplastic lymphoma. We conclude the following: 1) Antibody Mum-1p is very specific for canine plasmacytomas, 2) antibody Mum-1p is superior in sensitivity and specificity to CD79a and CD20 for the identification of canine plasmacytomas in formalin-fixed, paraffin-embedded tissues, 3) canine lymphomas that express MUM1/IRF4 are few and usually of B-cell origin, 4) other canine leukocytic and melanocytic tumors do not express MUM1/IRF4, and 5) prospective studies are needed to determine whether the expression of MUM1/IRF4, particularly in lymphomas, has prognostic significance.     

Canine lymphoproliferative disease characterized by lymphocytosis: immunophenotypic markers of prognosis

BACKGROUND: Canine lymphoproliferative disease often presents with lymphocytosis and is immunophenotypically diverse. HYPOTHESIS: Immunophenotype predicts prognosis in canine lymphoproliferative disorders involving circulating lymphocytosis. ANIMALS: Dogs that had peripheral blood evaluation performed by flow cytometry by the Clinical Immunology Service at Colorado State University between 2003 and 2005. METHODS: Outcome data regarding treatment and survival were sought on patients with lymphocytosis comprising a single lymphocyte subset. Ninety-six patients that met the inclusion criteria had sufficient follow-up information to be included in the study. RESULTS: Four main phenotypic classifications were found: CD8+ T-cell, CD21+ B-cell, CD4-8-5+ (aberrant T-cell phenotype), and CD34+ (undifferentiated progenitor). Expression of CD34 predicted poor outcome with median survival of 16 days (P < .0001) compared with other phenotypes. Within the CD8+ phenotype, dogs presenting with a lymphocytosis >30,000 lymphocytes/muL had significantly shorter median survival (131 days) than those presenting with <30,000 lymphocytes/muL (1098 days, P < .0008). Within the T-cell leukemias, there was no difference in outcome between dogs with CD4-8-5+ leukemia and dogs with the CD8+ T-cell phenotype nor was the loss of expression of the pan-leukocyte marker CD45 associated with decreased survival time. A CD21+ lymphocytosis composed of large cells was associated with shorter survival time (129 days) than those with smaller circulating cells (median survival not reached, P < .01). CONCLUSIONS AND CLINICAL IMPORTANCE: Immunophenotyping provides an objective method for determining prognosis in lymphoproliferative disorders characterized by lymphocytosis.     

Bovine leukemia virus high tax molecular clone experimentally induces leukemia/lymphoma in sheep

Sheep were inoculated with high tax coded pBLV-IF (H group, Nos.1-5) of bovine leukemia virus (BLV), wild tax coded pBLV-IF (W group, Nos. 6-11), or control plasmid (C group, Nos. 12-14). During the observation period (4 to 46 months), 5 of 5 cases in H group and 3 of 6 cases (Nos. 6, 7, 9) in W group became positive for gp 51. Only 1 case in H group became leukemic, and one case each of H and W groups developed lymphoma. In No. 3, lesions were found in multiple organs including the lymph nodes, gastrointestinal tract following abomasum, and heart. In No. 6, lesions of lymphoma were found only in the jejunum and heart. Morphologically, small to middle-sized lymphocytic neoplastic (NP) cells were found in both cases, but lymphoblastic NP cells were found only in No. 3. By immunohistochemical examination, the phenotypes of NP cells were determined as CD1-, CD4-, CD5- -, CD8alpha-, sIgM+, lambda light chain+, B-B4+, MHC class II+ in both case. The results of this study indicate that inoculation of pBLV-IF can induce lymphocytic and lymphoblastic leukemia/lymphoma in sheep. Additionally, it is suggested that the expression rate of tax gene is not associated with the development of leukemia/lymphoma in sheep experimentally inoculated with pBLV-IF.     

Clinical, laboratory, and histopathologic features of equine lymphoma

Clinical, laboratory and tissue findings from 37 horses with lymphoma were investigated. Horses ranged in age from 0.3 to 20.5 years (median 5.0 years) and included 18 females and 19 males. Weight loss (n = 25) and ventral edema (n = 21) were the most common historical and physical abnormalities. The most common laboratory abnormalities were hyperfibrinogenemia (n = 26), hypoalbuminemia (n = 19), anemia (n = 19), leukemia (n = 14), hyperglobulinemia (n = 13), and thrombocytopenia (n = 13). Thirty-four tumors involved multiple lymphoid tissues and abdominal or thoracic organs, and 3 tumors were restricted to cutaneous and subcutaneous sites. Histopathologically, all tumors diffusely effaced normal lymph node architecture. Tumor cell morphology was heterogeneous in 17 tumors, and 8 tumors had marked histiocytic and multinucleated giant cell infiltrates. Extensive necrosis or focal fibrosis was present in 22 and 4 lymphomas, respectively. Staining of tumor sections with antibodies against CD3 and CD79alpha molecules resulted in classification of T-cell (n = 26) or B-cell (n = 7) origin. Four tumors could not be classified. Most T-cell tumors comprised small to medium CD3(+) lymphocytes, whereas 5 of 7 B-cell tumors were infiltrated by numerous small T lymphocytes and classified as T-cell-rich B-cell lymphoma. Neither estrogen nor progesterone receptor expression was consistently identified by immunochemical assessment of tumor tissues. Fresh tumor cells from 6 horses bound antibodies reactive with equine CD4, CD5, CD8, CD21, or major histocompatibility class II molecules, confirming T-cell (n = 5) or B-cell origin (n = 1). These findings suggest that T-cell lymphoma is more common than B-cell lymphoma in horses and that inflammation, possibly from tumor cytokine production, is frequent.     

bcl-2 and MIB-1 labeling indexes in cats with lymphoma

Immunohistochemistry was used to examine feline lymphoid tumors for bcl-2 and MIB-1 expression. Tumor tissues from 29 cats were selected to represent 2 groups--cats that did not respond to chemotherapy and cats that responded to therapy. Median bcl-2 immunoreactivity was 60%, and median MIB-1 reactivity was 47%. There was no significant difference in median survival time between cats with tumors with high levels of bcl-2 expression and those with low levels of expression. There was no significant difference in median survival time between cats with tumors with high levels of MIB-1 expression and those with low levels of expression. Mean bcl-2 immunoreactivity was significantly (P = .0004) higher in low-grade (73.2%) than in high-grade (16.9%) lymphomas, whereas the mean MIB-1 immunoreactivity was significantly (P = .0201) higher in high-grade (61.2%) lymphomas than in low-grade (35.0%) lymphomas. The mean bcl-2 immunoreactivity was significantly (P = .0042) greater in T-cell lymphomas (66.8%) than in B-cell lymphomas (22.8%), whereas the mean MIB-1 immunoreactivity was significantly (P = .0052) lower in T-cell lymphomas (36.4%) than in B-cell lymphomas (65.2%). Although expression of bcl-2 and MIB-1 did not appear to be linked to responses to chemotherapy in cats with lymphoma, the data suggest a possible role for these regulatory proteins in the biology of feline lymphomas.