Molecular profiling reveals prognostically significant subtypes of canine lymphoma.

We performed genomewide gene expression analysis of 35 samples representing 6 common histologic subtypes of canine lymphoma and bioinformatics analyses to define their molecular characteristics. Three major groups were defined on the basis of gene expression profiles: (1) low-grade T-cell lymphoma, composed entirely by T-zone lymphoma; (2) high-grade T-cell lymphoma, consisting of lymphoblastic T-cell lymphoma and peripheral T-cell lymphoma not otherwise specified; and (3) B-cell lymphoma, consisting of marginal B-cell lymphoma, diffuse large B-cell lymphoma, and Burkitt lymphoma. Interspecies comparative analyses of gene expression profiles also showed that marginal B-cell lymphoma and diffuse large B-cell lymphoma in dogs and humans might represent a continuum of disease with similar drivers. The classification of these diverse tumors into 3 subgroups was prognostically significant, as the groups were directly correlated with event-free survival. Finally, we developed a benchtop diagnostic test based on expression of 4 genes that can robustly classify canine lymphomas into one of these 3 subgroups, enabling a direct clinical application for our results.

Immunohistochemical detection of retinoid receptors in tumors from 30 dogs diagnosed with cutaneous lymphoma.

BACKGROUND: Retinoids exert their effects by binding to retinoid receptors. Two types of retinoid receptors have been described: retinoic acid receptor (RAR) and retinoid X receptor (RXR), and their subtypes α, ß, and γ. The expression of subtypes varies depending on the disease process. This study intended to detect the pattern of retinoid receptor expression in cutaneous lymphomas in dogs. HYPOTHESIS: Cutaneous lymphomas in dogs have variable expression of retinoid and retinoid X receptors. ANIMALS: Biopsy specimens from 30 dogs with cutaneous lymphoma. METHODS: Tissues of dogs with cutaneous lymphoma were evaluated by immunohistochemistry for expression of retinoid receptors. The tissues were tested for the presence of 3 RAR and RXR subtypes (α, ß, and γ). Lymphoma immunophenotype was determined by the use of the immunohistochemical markers CD79a (B-cell) and CD3 (T-cell) in all cases. RESULTS: Twenty-nine of 30 dogs were CD3 positive. The retinoid receptors expressed with the greatest frequency were RARß (87% of cases), and RXRα and RXRγ (77% of cases). The expression of RARγ was not observed. CONCLUSIONS AND CLINICAL RELEVANCE: Retinoid and rexinoid receptor binding drugs may have an impact on the treatment of dogs with cutaneous lymphoma.

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.

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.

CD20 expression in normal canine B cells and in canine non-Hodgkin lymphoma.

We examined the expression of CD20 in normal canine peripheral blood mononuclear cells, normal canine spleen, and canine non-Hodgkin lymphoma (NHL) to determine the feasibility of using this antigen as a diagnostic aid and as a possible target for therapy. An antibody generated against a C-terminal (intracytoplasmic) epitope of human CD20 recognized proteins of 32-36 kd in normal and malignant canine lymphocytes. This antibody showed restricted membrane binding in a subset of lymphocytes in peripheral blood, in the B-cell regions from a normal canine spleen and lymph node, and in malignant cells from 19 dogs with B-cell NHL, but not from 15 dogs with T-cell NHL. The patterns of CD20 reactivity in these samples overlapped those seen using an antibody that recognizes canine CD79a. This anti-CD20 antibody is therefore suitable as an aid to phenotype canine NHL. In contrast, normal canine B cells were not recognized by any of 28 antibodies directed against the extracellular domains of human CD20 (including the chimeric mouse-human antibody Rituximab) or by any of 12 antibodies directed against the extracellular domains of mouse CD20. Thus, the use of CD20 as a therapeutic target will require the generation of specific antibodies against the extracellular domains of canine CD20.