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.

Modulation of fatty acid metabolism and immune suppression are features of in vitro tumour sphere formation in ontogenetically distinct dog cancers.

Non-adherent, 3-dimensional sphere formation is used as an in vitro surrogate to evaluate cellular potential for tumour initiation and self-renewal. To determine if a shared molecular program underlies the capacity for sphere formation by cells originating from diverse tumour types, we characterized molecular and functional properties of 10 independent cell lines derived from 3 ontogenetically distinct dog cancers: hemangiosarcoma, osteosarcoma and glial brain tumours. Genome-wide gene expression profiling identified tumour-of-origin-dependent patterns of adjustment to sphere formation in a uniform culture condition. However, expression of the stem/progenitor markers CD34 and CD117, resistance to cytotoxic drugs and dye efflux (side population assays) showed no association with these gene expression profiles. Instead, primary sphere-forming capacity was inversely correlated with the ability to reform secondary spheres, regardless of tumour ontogeny. Primary sphere formation seemed to be proportional to the number of pre-existing cells with sphere-forming capacity in the cell lines. Cell lines where secondary sphere formation was more proficient than primary sphere formation showed enrichment of genes involved in fatty acid synthesis and immunosuppressive cytokines. In contrast, cell lines where secondary sphere formation was approximately equivalent to or less proficient than primary sphere formation showed upregulation of CD40 and enrichment of genes involved in fatty acid oxidation. Our data suggest that in vitro sphere formation is associated with upregulation of gene clusters involved in metabolic and immunosuppressive functions, which might be necessary for self-renewal and for tumour initiation and/or tumour propagation in vivo.

Interactions between CXCR4 and CXCL12 promote cell migration and invasion of canine hemangiosarcoma.

The CXCR4/CXCL12 axis plays an important role in cell locomotion and metastasis in many cancers. In this study, we hypothesized that the CXCR4/CXCL12 axis promotes migration and invasion of canine hemangiosarcoma (HSA) cells. Transcriptomic analysis across 12 HSA cell lines and 58 HSA whole tumour tissues identified heterogeneous expression of CXCR4 and CXCL12, which was associated with cell movement. In vitro, CXCL12 promoted calcium mobilization, cell migration and invasion that were directly proportional to surface expression of CXCR4; furthermore, these responses proved sensitive to the CXCR4 antagonist, AMD3100, in HSA cell lines. These results indicate that CXCL12 potentiates migration and invasion of canine HSA cells through CXCR4 signalling. The direct relationship between these responses in HSA cells suggests that the CXCR4/CXCL12 axis contributes to HSA progression.

Establishment of a Patient-Derived Xenograft of Canine Enteropathy-Associated T-Cell Lymphoma, Large Cell Type.

The pathogenesis of canine T-cell lymphoma remains incompletely understood, partly because there are no well-established in-vivo models to study these malignancies. For this study, we generated a patient-derived tumour xenograft (PDTX) from a 10-year-old neutered male golden retriever dog with enteropathy-associated intestinal T-cell lymphoma, large cell type. One of two female, 15-week-old beige/nude/XID mice developed a visible tumour 7 weeks after sections of tumour material from the spleen were surgically implanted. The histological appearance, immunophenotype and clonal antigen receptor rearrangements of the tumour from the recipient mouse showed that it was derived from the primary canine tumour. Our results indicate that immunodeficient mice are receptive hosts to develop in-vivo PDTX models to study the pathogenesis and management of canine T-cell lymphomas.

Clues to immune function and oncogenesis provided by events that activate the cell cycle machinery in normal human T cells.

A common feature seen in states of decreased immune competence or immunosuppression and in diseases of the blood, such as lymphohematopoietic cancers, is the disruption of the normal pathways of cell cycle control. In lymphocytes a series of nonlinear biochemical cascades leads to cellular proliferation and also controls the production of cytokines that provide immunologic help (i.e., aid in B and T cell proliferation, maturation, and differentiation). These two distinct outcomes can be dissociated, as stimuli that incite production of cytokines need not lead to cell division, and conversely, exogenously provided cytokines may promote lymphocyte proliferation. The signals that induce production of cytokines, particularly interleukin-2, have been extensively characterized. It also is known that the fidelity of cell cycle progression is dependent on a regulatory network whose key components include cyclin-dependent kinases and cyclins. This review describes the current state of knowledge linking the antigen receptor response pathways and the activation of the cell cycle machinery in T cells.

Association of Sphingosine-1-phosphate (S1P)/S1P Receptor-1 Pathway with Cell Proliferation and Survival in Canine Hemangiosarcoma.

BACKGROUND: Sphingosine-1-phosphate (S1P) is a key biolipid signaling molecule that regulates cell growth and survival, but it has not been studied in tumors from dogs. HYPOTHESIS/OBJECTIVES: S1P/S1P1 signaling will contribute to the progression of hemangiosarcoma (HSA). ANIMALS: Thirteen spontaneous HSA tissues, 9 HSA cell lines, 8 nonmalignant tissues, including 6 splenic hematomas and 2 livers with vacuolar degeneration, and 1 endothelial cell line derived from a dog with splenic hematoma were used. METHODS: This was a retrospective case series and in vitro study. Samples were obtained as part of medically necessary diagnostic procedures. Microarray, qRT-PCR, immunohistochemistry, and immunoblotting were performed to examine S1P1 expression. S1P concentrations were measured by high-performance liquid chromatography/mass spectrometry. S1P signaling was evaluated by intracellular Ca(2+) mobilization; proliferation and survival were evaluated using the MTS assay and Annexin V staining. RESULTS: Canine HSA cells expressed higher levels of S1P1 mRNA than nonmalignant endothelial cells. S1P1 protein was present in HSA tissues and cell lines. HSA cells appeared to produce low levels of S1P, but they selectively consumed S1P from the culture media. Exogenous S1P induced an increase in intracellular calcium as well as increased proliferation and viability of HSA cells. Prolonged treatment with FTY720, an inhibitor of S1P1 , decreased S1P1 protein expression and induced apoptosis of HSA cells. CONCLUSIONS AND CLINICAL IMPORTANCE: S1P/S1P1 signaling pathway functions to maintain HSA cell viability and proliferation. The data suggest that S1P1 or the S1P pathway in general could be targets for therapeutic intervention for dogs with HSA.

Canine osteosarcoma cells exhibit resistance to aurora kinase inhibitors.

We evaluated the effect of Aurora kinase inhibitors AZD1152 and VX680 on canine osteosarcoma cells. Cytotoxicity was seen in all four cell lines; however, half-maximal inhibitory concentrations were significantly higher than in human leukaemia and canine lymphoma cells. AZD1152 reduced Aurora kinase B phosphorylation, indicating resistance was not because of failure of target recognition. Efflux mediated by ABCB1 and ABCG2 transporters is one known mechanism of resistance against these drugs and verapamil enhanced AZD1152-induced apoptosis; however, these transporters were only expressed by a small percentage of cells in each line and the effects of verapamil were modest, suggesting other mechanisms contribute to resistance. Our results indicate that canine osteosarcoma cells are resistant to Aurora kinase inhibitors and suggest that these compounds are unlikely to be useful as single agents for this disease. Further investigation of these resistance mechanisms and the potential utility of Aurora kinase inhibitors in multi-agent protocols is warranted.

Sustained nuclear localization of p21/WAF-1 upon growth arrest induced by contact inhibition.

We assessed the expression and distribution of p21/Waf-1 in TLM1 melanoma cells that exhibit contact inhibition and require serum for growth. The growth stage of cells stimulated to enter the mitotic cell cycle synchronously and grow to confluence was characterized by distinct, yet consistent levels and patterns of distribution of p21/Waf-1. Significantly, sustained accumulation of p21/Waf-1 in the nuclear compartment was seen only after 4 days in culture when cell-to-cell contacts were established, leading to a diminished rate of cell growth. Overexpression of wild-type waf-1 in melanoma cells reduced growth of subconfluent cells, decreased Cdk4 activity with a concomitant increase in hypophosphorylated Rb, and promoted cell death by apoptosis. The data support the premise that cell-to-cell contacts provide signals that mediate sustained nuclear localization of p21/Waf-1 leading to cell growth arrest; furthermore, an elevation in the activity of this protein can lead to apoptosis.

Growth arrest of melanoma cells is differentially regulated by contact inhibition and serum deprivation.

Both growth-factor deprivation and contact inhibition suppress cell growth; however, the mechanisms by which they inhibit cell proliferation may not be identical. The function of antiproliferative genes and the induction of programmed cell death are among the potential differences between these growth-arrest mechanisms. Specifically, an inverse relation between the expression of cyclin-dependent kinase inhibitors (CDKIs) and the susceptibility to apoptosis has been reported. To test this relation, we examined the features of growth arrest in a canine melanoma cell line, TLM1. Both contact inhibition and serum deprivation halted cell-cycle progression of TLM1 cells in the G1 phase. Prolonged growth arrest of the cells without restimulation resulted in apoptosis; conversely, the cells reentered the cell cycle after release from contact inhibition or on restimulation with serum. Cell-to-cell contact, but not serum deprivation, led to the expression of p53 and p21/Waf-1. The expression of p21/Waf-1 did not prevent apoptosis. Moreover, the ectopic overexpression of CDKIs increased apoptosis. These results support the premise that growth arrest induced by contact inhibition and serum deprivation are mediated through distinct mechanisms. Furthermore, CDKIs are not universal inhibitors of apoptosis, and in some cases, they may initiate or enhance the apoptotic program.

Symmetry of the activation of cyclin-dependent kinases in mitogen and growth factor-stimulated T lymphocytes.

The entry of resting T cells into the G1 phase of the cell cycle after stimulation by mitogens is controlled by a series of biochemical events that are independent of growth factors. These events follow the initial signals stimulated through the engagement of the T-cell receptor and include activation of the cyclin-dependent kinases Cdk6, Cdk4, and Cdk2, as well as a transient phosphorylation of the retinoblastoma gene product (p110Rb) by one or several of these proteins. A progression signal such as that delivered by interleukin-2 then induces a second phase of Cdk6, Cdk4, and Cdk2 activation, along with sustained phosphorylation of p110Rb in the activated T cells. This second signal is required to carry the cells into the S phase and beyond. Quantitative and qualitative differences in the expression and activity of these proteins may be critical to maintain the delicate balance that is necessary to ensure the normal progression of T cells through the cell cycle.

Immunophysiological studies of interleukin-2 and canine lymphocytes.

Interleukin-2-dependent pathways of lymphocyte activation were investigated in canine peripheral blood lymphocytes (PBL) following stimulation with T-cell mitogens including phytohemagglutinin, phorbol ester (TPA), calcium ionophore (ionomycin), and human recombinant interleukin-2 (hrIL-2). The ability of the stimulated cells to produce interleukin-2 (IL-2) was determined using murine indicator cell lines. IL-2 receptor expression by mitogen-stimulated canine PBL was confirmed by the binding of hrIL-2 with high affinity, and with characteristics comparable to those of the human and murine IL-2 receptor. Examination of serum and PBL from two dogs that were treated with hrIL-2 and human recombinant tumor necrosis factor for systemic mast cell tumors showed that in one dog, IL-2 could be measured in the serum. Concurrently, the in vitro mitogenic response of this dog's PBL to hrIL-2 occurred earlier, possibly reflecting an increase in the relative number of IL-2-responsive cells within the PBL population.

Retrovirus-like activity in an immunosuppressed dog: pathological and immunological findings.

A putative retrovirus was isolated from a dog with a severe, acquired immunodeficiency-like syndrome. The haematological abnormalities and immunological deficiencies included anaemia, leucopenia (lymphopenia and neutropenia), thrombocytopenia, decreased humoral immunity, and ineffective T-cell responses in-vitro. The necropsy findings included generalized lymphoid depletion, severe bone marrow hypoplasia, plasmacytic infiltrates in lymphoid and non-lymphoid organs, and severe secondary infections. Supernates of peripheral blood mononuclear cell cultures from the affected dog contained an agent with manganese-dependent reverse transcriptase (RT) activity that sedimented at a density of 1.122 g/ml. RT activity was also found post-mortem in extracts prepared from the bone marrow, lymph nodes, and small intestine. The lymph nodes and small intestine expressed a 3.8 kb mRNA that was recognized by a bovine leukaemia virus (BLV) pol DNA probe by Northern blotting. DNA isolated from the lymph nodes and small intestine from the affected dog showed distinct band patterns by Southern analysis, suggesting an exogenous retrovirus. The retrovirus could be propagated in normal canine peripheral blood mononuclear cells or short-term canine lymphocyte cell lines in-vitro, and was cytopathogenic for cells of canine, but not human, origin. These results suggest the existence of a pathogenic canine retrovirus capable of producing disease of the type associated with retroviruses in other species.

The molecular basis of canine melanoma: pathogenesis and trends in diagnosis and therapy.

Melanoma is a common neoplastic disease of dogs with variable presentation and biological behavior. Canine malignant melanoma is a rapidly metastatic disease that generally is incurable. The loss of function of cellular safeguards built into the genetic program and of immune surveillance systems that cooperate to prevent tumor formation and progression appear to be important underlying causes of canine malignant melanoma. In effect, many existing cancer treatments restore the function of 1 or the other of these mechanisms. For example, chemotherapy and radiotherapy often kill tumor cells by initiating a genetic suicide mechanism (apoptosis), and immunotherapy initiates or enhances a response by the body's immune cells to identify and destroy cancer cells by mechanisms that rely on direct cytotoxicity or apoptotic cell death. Nevertheless, standard therapeutic approaches have not proved effective in treatment of canine malignant melanoma, with only marginal improvement in the outcome of dogs with this disease. The advantages of an improved understanding of the molecular basis of canine cancer are underscored by recent promising advances in diagnosis and in immunologic and genetic therapies that may help reduce the mortality of dogs affected with malignant melanoma.

Bone marrow cytological findings in 4 dogs and a cat with hemophagocytic syndrome.

Hemophagocytic syndrome or hemophagic histiocytosis was diagnosed in 4 dogs and 1 cat by evaluation of bone marrow aspirate smears. One of the dogs had a suspected infection with canine parvovirus and a confirmed infection with Salmonella spp, 2 dogs had presumptive diagnoses of myeloproliferative and lymphoproliferative disease, respectively, and 1 dog died without a diagnosis. The cat had hepatic lipidosis and lesions compatible with feline calicivirus infection. All animals had cytopenias involving 2 or more cell lines, and fragmented erythrocytes in the blood, along with mild to moderate increases in the number of macrophages in the bone marrow. Numerous marrow macrophages contained phagocytized hematopoietic cells. Other cytological features of the bone marrow were variable in each patient, but the degree of response in the blood was inadequate, even in those with bone marrow hyperplasia. The phagocytosis of hematopoietic elements did not appear to be caused by a primary immune disorder, but rather by the inappropriate activation of normal macrophages secondary to infectious, neoplastic, or metabolic diseases. These findings suggest that hemophagocytic syndrome may be an important factor in the development of cytopenias; the data also support the cytological evaluation of bone marrow aspirates as an aid in the diagnosis of hemophagocytic syndrome.

Use of human recombinant erythropoietin and prednisone for treatment of myelodysplastic syndrome with erythroid predominance in a dog.

A 7-year-old German Shepherd dog was referred for evaluation of severe nonregenerative anemia (PCV, 10%; reticulocyte fraction, 0.2%). Cytologic examination of a bone marrow aspirate indicated erythroid predominance and dyserythropoiesis, and a diagnosis of myelodysplastic syndrome (MDS) with erythroid predominance was made. The dog was given a single blood transfusion and was treated with prednisone and recombinant human erythropoietin (EPO). Eight weeks later, anemia had resolved. The dog remained clinically normal 30 months after treatment, with a PCV of 45%. Results suggest that EPO may be useful in the treatment of dogs with MDS with erythroid predominance or erythroleukemia. Additional studies are required to confirm the benefit of EPO to manage MDS-associated anemia in dogs.

Immunotherapy with autologous tumour antigen-coated microbeads (large multivalent immunogen), IL-2 and GM-CSF in dogs with spontaneous B-cell lymphoma.

Cytotoxic T-lymphocyte responses to subcellular antigens are enhanced when antigens are presented on cell-sized silica microbeads called large multivalent immunogens (LMIs). LMIs prepared with tumour cell membrane fragments have induced partial remissions in humans with melanoma and renal cell carcinoma. The purpose of this phase I study was to evaluate the safety of LMIs, prepared with autologous lymphoma cell membranes, along with subcutaneous interleukin 2 (IL-2) and granulocyte-macrophage colony stimulating factor (GM-CSF) in dogs with untreated B-cell lymphoma. After lymph node excision and induction chemotherapy, five dogs were vaccinated with three weekly doses of LMI alone; five with LMI and subcutaneous IL-2 and five with LMI, IL-2 and GM-CSF. No significant toxicity was noted, treatment did not adversely affect disease-free interval and half of the dogs showed measurable delayed-type hypersensitivity reactions to intradermal challenge with LMI, suggesting specific cell-mediated immunity.

A tumor-related lymphoid progenitor population supports hierarchical tumor organization in canine B-cell lymphoma.

BACKGROUND: Tumors have heterogeneous properties, which could be explained by the existence of hierarchically and biologically distinct tumor cells such as tumor-initiating cells (TICs). This model is clinically important, as TICs are promising targets for cancer therapies. However, TICs in spontaneous B-cell lymphoma have not been conclusively identified. HYPOTHESIS/OBJECTIVES: Tumor cells with a progenitor phenotype exist in B-cell lymphoma, reflecting a hierarchical organization. ANIMALS: Twenty-eight client-owned dogs with previously untreated B-cell lymphoma and 6 healthy dogs. METHODS: This was a prospective study. Flow cytometry was used to identify lymphoid progenitor cells (LPCs) that coexpressed hematopoietic progenitor antigens CD34, CD117, and CD133, with lymphoid differentiation markers CD21 and/or CD22 in B-cell lymphoma. The polymerase chain reaction for antigen receptor rearrangements was used to analyze clonality and relatedness of tumor populations. A xenograft model with NOD/SCID/IL-2Rγ(-/-) mice was adapted to expand and serially transplant primary canine B-cell lymphoma. RESULTS: LPCs were expanded in lymph nodes from 28 dogs with B-cell lymphoma compared with 6 healthy dogs (P= .0022). LPCs contained a clonal antigen receptor gene rearrangement identical to that of the bulk of tumor cells. Canine B-cell lymphoma xenografts in recipient mice that maintained LPCs in the tumors were recurrently observed. CONCLUSIONS AND CLINICAL IMPORTANCE: These results suggest the presence of a hierarchy of tumor cells in B-cell lymphoma as has been demonstrated in other cancers. These findings have the potential to impact not only the understanding of lymphoma pathogenesis but also the development of lymphoma therapies by providing novel targets for therapy.

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.