Identification of hypoxia-induced metabolism-associated genes in canine tumours.

Canine tumours including urothelial carcinoma, lung adenocarcinoma, mammary gland tumour, squamous cell carcinoma, and melanoma have been identified as causes of death, but effective therapies are limited due to insufficient knowledge of the molecular mechanisms involved. Within the tumour microenvironment, hypoxia activates hypoxia-inducible factor 1α (HIF1α) in tumour cells. High HIF1α expression correlates with enhanced glycolysis and poorer outcomes in human cancers. However, the molecular mechanisms underlying hypoxic tumour cells remain elusive in dogs. In our study, we investigated upregulated genes in a canine malignant melanoma cell line during hypoxia using RNA-sequencing analysis. Glycolysis and HIF1 signalling pathways were upregulated in hypoxic melanoma cells. HIF1α knockout melanoma cells revealed that the glycolysis marker MCT4 is regulated by HIF1α activation. Hypoxia induces high lactate secretion due to enhanced glycolysis in canine melanoma cells. Furthermore, we examined monocarboxylate transporter 4 (MCT4) expression in malignant melanoma and eight other types of canine tumour tissues using immunohistochemistry (IHC). Membrane-localized MCT4 protein was mostly detected in urothelial carcinoma and lung adenocarcinoma rather than malignant melanoma. We conclude that canine MCT4 protein plays a role in lactic acid efflux from glycolytic cells and may serve as a marker for hypoxia and glycolysis in canine tumours. These findings could inform future therapeutic strategies targeting MCT4.

Establishment of a novel canine soft tissue sarcoma cell line and comparison of its characteristics with other soft tissue sarcoma cell lines.

Soft tissue sarcoma (STS) is a relatively common tumor in dogs. However, very few canine STS cell lines are available. This study aimed to establish a new cell line, STS-YU1, derived from a recurrence of myxosarcoma in an 11-year-old mixed-breed dog. We examined STS-YU1 for in vitro cell proliferation, migration, anticancer drug sensitivity, transcriptome analysis using next-generation sequencing (RNA-seq), and in vivo tumorigenicity in mice and compared it with previously established STS cell lines, MUMA-G and A72. The cell proliferation and migration of STS-YU1 were higher than MUMA-G although MUMA-G only exhibited tumorigenicity in mice. STS-YU1 showed dose-dependent cytotoxicity to anticancer drugs, but with weak effects. RNA-seq analysis revealed the molecular phenotype of STS-YU1 was different from that of a previously reported cell line, A72. Hence, the use of STS-YU1 would help in efficient drug screening against canine STS in vitro.

Proof-of-concept study of the caninized anti-canine programmed death 1 antibody in dogs with advanced non-oral malignant melanoma solid tumors.

BACKGROUND: The anti-programmed death 1 (PD-1) antibody has led to durable clinical responses in a wide variety of human tumors. We have previously developed the caninized anti-canine PD-1 antibody (ca-4F12-E6) and evaluated its therapeutic properties in dogs with advance-staged oral malignant melanoma (OMM), however, their therapeutic effects on other types of canine tumors remain unclear. OBJECTIVE: The present clinical study was carried out to evaluate the safety profile and clinical efficacy of ca-4F12-E6 in dogs with advanced solid tumors except for OMM. METHODS: Thirty-eight dogs with non-OMM solid tumors were enrolled prospectively and treated with ca-4F12-E6 at 3 mg/kg every 2 weeks of each 10-week treatment cycle. Adverse events (AEs) and treatment efficacy were graded based on the criteria established by the Veterinary Cooperative Oncology Group. RESULTS: One dog was withdrawn, and thirty-seven dogs were evaluated for the safety and efficacy of ca-4F12-E6. Treatment-related AEs of any grade occurred in 13 out of 37 cases (35.1%). Two dogs with sterile nodular panniculitis and one with myasthenia gravis and hypothyroidism were suspected of immune-related AEs. In 30 out of 37 dogs that had target tumor lesions, the overall response and clinical benefit rates were 6.9% and 27.6%, respectively. The median progression-free survival and overall survival time were 70 days and 215 days, respectively. CONCLUSIONS: The present study demonstrated that ca-4F12-E6 was well-tolerated in non-OMM dogs, with a small number of cases showing objective responses. This provides evidence supporting large-scale clinical trials of anti-PD-1 antibody therapy in dogs.

Gene expression profiles associated with early relapse during first remission induction in canine multicentric high-grade B-cell lymphoma.

Although chemotherapy using CHOP-based protocol induces remission in most cases of canine multicentric high-grade B-cell lymphoma (mhBCL), some cases develop early relapse during the first induction protocol. In this study, we examined the gene expression profiles of canine mhBCL before chemotherapy and investigated their associations with early relapse during the first whole CHOP-based protocol. Twenty-five cases of mhBCL treated with CHOP-based protocol as first induction chemotherapy were included in this study. Sixteen cases completed the first whole CHOP-based protocol without relapse (S-group), and nine developed relapse during the chemotherapy (R-group). RNA-seq was performed on samples from neoplastic lymph nodes. Differentially expressed genes (DEGs) were extracted by the comparison of gene expression profiles between S- and R-groups, and the differences in the expression levels of these genes were validated by RT-qPCR. Extracted 179 DEGs included the genes related to chemokine CC motif ligand, T-cell receptor signaling pathway, and PD-L1 expression and PD-1 checkpoint pathway. We focused on chemokine CC motif ligand, and CCL4 was confirmed to be significantly downregulated in the R-group (P=0.039). We also focused on the genes related to T-cell signaling pathway, and CD3E (P=0.039), ITK (P=0.023), and LAT (P=0.023) genes were confirmed to be significantly upregulated in the R-group. The current results suggest that both changes in tumor cells and the interactions between tumor cells and immune cells are associated with the efficacy of the chemotherapy for first remission induction.

Activation of the Akt signalling pathway as a prognostic indicator in canine soft tissue sarcoma.

Canine soft tissue sarcoma (STS) is relatively common in dogs and is the generic term for tumours that originate from mesenchymal cells. While histopathological grade and immunolabelling with Ki-67 have been used for estimating prognosis, additional indicators are needed for predicting prognosis. Aberrant cell signalling pathways may contribute to disease activity and, therefore, prognostic markers. However, their role in canine STS remains poorly understood. The aim of this study was to investigate expression of phosphorylated Akt (phospho-Akt) and phosphorylated S6 (phospho-S6) as potential prognostic indicators. Immunohistochemical labelling was conducted on clinical samples of canine STS (n = 67). We found that phospho-Akt expression was positively correlated with histopathological grade (P = 0.001) and Ki-67 index (P <0.01). There was no apparent relationship between the type of STS and the expression of phospho-Akt. The number of cases that expressed phospho-S6, which is the downstream molecule of the Akt signalling pathway, was higher in immunopositive phospho-Akt cases than in immunonegative phospho-Akt cases (P <0.0001). Furthermore, phospho-Akt expression was significantly higher in recurrent and metastatic cases. We also confirmed that phosphorylation of Akt occurred in conjunction with S6 phosphorylation in three canine STS cell lines. These results suggest that immunolabelling for phospho-Akt, phospho-S6 and Ki-67 could potentially be used as a prognostic indicator and therapeutic target in canine STS.

Effect of tolvaptan on hyponatremia in a dog with syndrome of inappropriate secretion of antidiuretic hormone.

A 1-year-old spayed female Miniature Schnauzer had chronic hyponatremia, accompanied by polyuria and polydipsia. Blood tests and urinalysis revealed severe hyponatremia, low plasma osmolality with euvolemia, and increased sodium excretion in urine. Hypothyroidism and hypoadrenocorticism were ruled out as causes. These findings led to the diagnosis of syndrome of inappropriate secretion of antidiuretic hormone (SIADH). Magnetic resonance imaging (MRI) showed dilation of the lateral ventricles, indicating severe hydrocephalus. Tolvaptan, a vasopressin V2 receptor antagonist commonly used in human SIADH, was administered along with water restriction. This treatment resulted in a consistent increase in plasma sodium levels without any adverse effects. This case report represents the first documented evidence of the therapeutic efficacy of tolvaptan in treating SIADH in a dog.

Cancer-specific apoptosis induction in canine lymphoma cell lines by the endocytosis inhibitor dynasore.

Canine lymphoma is the most common cancer in dogs and has a poor prognosis. We recently found that the endocytosis inhibitor dynasore suppresses the viability of human cancer cell lines, especially hematopoietic cancers, by inducing apoptosis. In the present study, we examined the anticancer effects of dynasore on five previously established canine lymphoma cell lines (CLBL-1, Ema, Nody-1, CLC, and GL-1). Dynasore suppressed cell viability in these canine lymphoma cell lines more effectively than in human cancer cell lines. It also induced apoptosis in CLBL-1 and Ema cells but not in peripheral blood mononuclear cells in healthy dogs or in Madin-Darby canine kidney (MDCK) cells, suggesting that the ability of dynasore to induce apoptosis is cancer-specific. Furthermore, dynasore induced a DNA damage response in CLBL-1 and Ema cells, suggesting that it acts as a genotoxic agent in canine lymphoma cell lines. These findings suggest that endocytosis inhibitors may provide a new anticancer treatment for canine lymphoma.

Cross-reactivity of anti-human programmed cell death ligand 1 (PD-L1) monoclonal antibody, clone 28-8 against feline PD-L1.

Immunotherapy is a breakthrough in human cancer therapy and has become a major concern in veterinary oncology. However, in cats, many unclear points of the tumor microenvironment exist, including immune checkpoint molecules. A reason is that very few monoclonal antibodies have been proven to react with feline molecules. Therefore, this study investigated whether anti-human programmed cell death ligand 1 (PD-L1) monoclonal antibody, clone 28-8, which is currently commercially available, can also recognize feline PD-L1 by flow cytometry, immunoprecipitation, and immunohistochemical (IHC) staining. We confirmed that the antibody's specificity by flow cytometry and immunoprecipitation using NIH3T3 cells transfected with feline PD-L1. Additionally, we revealed that PD-L1 was expressed on the surface of some feline cell lines by flow cytometry and clone 28-8 antibody unbound to the cells where feline PD-L1 was knocked out. Furthermore, IHC analysis revealed that PD-L1 was expressed in macrophages in the spleen and lymph nodes from healthy cats and mast cell tumor cells. Therefore, we indicated that the clone 28-8 antibody is a valuable tool in detecting feline PD-L1, and further analysis of tumor tissues is expected in the future.

Development of anti-feline PD-1 antibody and its functional analysis.

Antibodies against immune checkpoint molecules restore T-cell function by inhibiting the binding of PD-1 and PD-L1 and have been shown to exert therapeutic effects in various human cancers. However, to date, no monoclonal antibody that recognizes feline PD-1 or PD-L1 has been reported, and there are many unknowns regarding the expression of immune checkpoint molecules and their potential as therapeutic targets in cats. Here we developed anti-feline PD-1 monoclonal antibody (1A1-2), and found that the monoclonal antibody against anti-canine PD-L1 (G11-6), which was previously developed in our laboratory, cross-reacted with feline PD-L1. Both antibodies inhibited the interaction of feline PD-1 and feline PD-L1 in vitro. These inhibitory monoclonal antibodies augmented the interferon-gamma (IFN-γ) production in activated feline peripheral blood lymphocytes (PBLs). Furthermore, for clinical application in cats, we generated a mouse-feline chimeric mAb by fusing the variable region of clone 1A1-2 with the constant region of feline IgG1 (ch-1A1-2). Ch-1A1-2 also augmented the IFN-γ production in activated feline PBLs. From this study, 1A1-2 is first anti-feline PD-1 monoclonal antibody with the ability to inhibit the interaction of feline PD-1 and PD-L1, and the chimeric antibody, ch-1A1-2 will be a beneficial therapeutic antibody for feline tumors.

Defucosylated Mouse-Dog Chimeric Anti-Human Epidermal Growth Factor Receptor 2 Monoclonal Antibody (H77Bf) Exerts Antitumor Activities in Mouse Xenograft Models of Canine Osteosarcoma.

Human epidermal growth factor receptor 2 (HER2) has been studied in many human cancer types, and its overexpression and/or gene mutation contribute to the poor prognosis. Therefore, HER2 is an important therapeutic target in various cancer types, including breast and gastric cancers. We previously developed an anti-HER2 monoclonal antibody (mAb), H2Mab-77 (mouse IgG1, kappa), which detects HER2 and dog HER2 (dHER2) with high sensitivity and specificity. In this study, we produced a defucosylated mouse-dog chimeric anti-HER2 mAb (H77Bf), and investigated the reactivity against canine osteosarcoma D-17 cells by flow cytometry. Furthermore, we showed that H77Bf exerted antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity against D-17 cells in vitro and exhibited the potent antitumor activity in vivo. These results suggest that H77Bf exerts antitumor effects against dHER2-expressing canine tumors and could be valuable as part of an antibody treatment regimen for them.

Antitumor Activities in Mouse Xenograft Models of Canine Fibroblastic Tumor by Defucosylated Mouse-Dog Chimeric Anti-HER2 Monoclonal Antibody (H77Bf).

Human epidermal growth factor receptor 2 (HER2) is a cell surface type I transmembrane glycoprotein that is overexpressed on a variety of solid tumors and transduces the oncogenic signaling upon homo- and heterodimerization with HER families. Anti-HER2 monoclonal antibodies (mAbs) including trastuzumab and its antibody-drug conjugate have been shown to improve patients' survival in HER2-positive breast, gastric, and lung cancers. Canine tumors have advantages as naturally occurring tumor models, and share biological and histological characteristics with human tumors. In this study, we generated a defucosylated version of mouse-dog chimeric anti-HER2 mAb (H77Bf) derived from H2Mab-77 (mouse IgG1, kappa). H77Bf possesses the high binding affinity (a dissociation constant: 8.7 × 10-10 M) for a dog HER2 (dHER2)-expressing canine fibroblastic tumor cell line (A-72). H77Bf exhibited antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity for A-72 cells. Moreover, intraperitoneal administration of H77Bf significantly suppressed the development of A-72 tumor compared with the control dog IgG in a mouse xenograft model. These results indicate that H77Bf exerts antitumor activities against dHER2-expressing canine cancers, which could provide a valuable information for canine cancer treatment.

Decreased sensitivity of cyclin-dependent kinase 4/6 inhibitors, palbociclib and abemaciclib to canine lymphoma cells with high p16 protein expression and low retinoblastoma protein phosphorylation.

Canine lymphoma/leukemia cell lines with p16 protein expressions: high (17-71 and GL-1) and low (CLBL-1, CLC, Nody-1, and UL-1) were treated in vitro with cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors, palbociclib or abemaciclib. Cell proliferation decreased as a result, with higher IC50 levels observed in the high p16 (17-71 and GL-1) and one low p16 (UL-1) cell lines compared with the low p16 cells (CLBL-1, CLC, and Nody-1). As expected, palbociclib and abemaciclib treatment reduced pRb phosphorylation in a dose-dependent manner, especially in cells with low p16. These results suggest that CDK4/6 inhibitors have potential as new chemotherapeutic agents for canine lymphoma and high p16 protein expression may be used as a biomarker for resistance to CDK4/6 inhibitor therapy.

Antitumor Activity of an Anti-EGFR/HER2 Bispecific Antibody in a Mouse Xenograft Model of Canine Osteosarcoma.

The overexpression of epidermal growth factor receptors (EGFRs) has been reported in various human tumors, including breast, gastric, lung, colorectal, and pancreatic cancers. Humanized anti-EGFR and anti-human epidermal growth factor receptor 2 (HER2) monoclonal antibodies (mAbs) have been shown to improve patients' survival. Canine tumors resemble human tumors in the initiation and progression. We previously established a defucosylated mouse-dog chimeric anti-EGFR mAb (E134Bf) and a mouse-dog chimeric anti-HER2 mAb (H77Bf), which exerted antitumor activities in canine tumor xenograft models. Here, we produced E134Bf antibody fused to H77Bf single chain Fv at the light chains (E134Bf-H77scFv). The bispecific E134Bf-H77scFv recognized dog EGFR (dEGFR) and dog HER2 (dHER2)-overexpressed Chinese hamster ovary-K1 cells by flow cytometry. E134Bf-H77scFv also reacted with dEGFR/dHER2-positive canine osteosarcoma D-17 cells, and possesses a high binding-affinity (KD: 1.3 × 10-9 M). Furthermore, E134Bf-H77scFv exerted antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity against D-17 cells in the presence of canine mononuclear cells and complement, respectively. Moreover, administration of E134Bf-H77scFv suppressed the development of D-17 xenograft tumor in mice early compared with the control dog IgG, E134Bf and H77Bf alone. These results indicate that E134Bf-H77scFv exerts antitumor activities against dEGFR/dHER2-positive canine tumors, and could be a valuable treatment regimen for canine tumors.

Intratumoral heterogeneity of c-KIT mutations in a feline splenic mast cell tumor and their functional effects on cell proliferation.

A cat was presented with mast cell tumors (MCTs) of the skin and spleen. During the initial diagnosis, the exon 8 mutation of c-KIT was detected in the masses from skin and spleen by a commercial laboratory test. Consequently, treatment with toceranib was started. After complete remission, because of recurrence on day 117, the spleen and skin tumors were removed, but the cat eventually died on day 191. The analysis of ten cDNA clones of the c-KIT gene cloned from the surgically removed spleen revealed that seven different cDNA patterns were included, indicating the heterogeneity of this gene in the splenic MCT. The seven cDNA nucleotide patterns can be classified into four protein sequence patterns. In addition to the previously known mutations in exon 8, we identified novel mutations in exons 9, 10, and 18; four amino acids deletion in exon 9, and a point mutation in exons 10 and 18. Mouse IL-3-dependent cell line, Ba/F3, was transduced with these mutant clones, and c-KIT phosphorylation and proliferation assays were performed. We found that certain mutations affected the c-KIT phosphorylation status and cell proliferation. This suggests that heterogeneity among the population of tumor cells exists in MCTs, and that the dominant clones of this heterogeneity may contribute to the subsequent tumor cell growth.

Intraepithelial cytotoxic lymphocytes are associated with a poor prognosis in feline intestinal T-cell lymphoma.

The expression of cytotoxic molecules in feline intestinal T-cell lymphoma cells was examined immunohistochemically using endoscopic samples of 50 cases. Cases included 14 large-cell lymphomas (LCLs) and 36 small-cell lymphomas (SCLs). Most LCL and some SCL exhibited marked erosion and villous atrophy. Clonal T-cell receptor (TCR) gene rearrangement was detected in 10/14 (71%) LCL cases and 33/36 (92%) SCL cases. No clonal immunoglobulin heavy chain (IgH) gene rearrangement was detected. Immunohistochemically, all cases were positive for CD3 and negative for CD79α, CD30, CD56, and Foxp3. LCLs were positive for CD8 in 13/14 cases (93%), T-cell intracellular antigen 1 (TIA1) in 14/14 cases (100%), and granzyme B in 6/14 cases (43%). SCLs were positive for CD8 in 28/36 cases (78%), TIA1 in 33/36 cases (92%), and granzyme B in 2/36 cases (6%). TIA1- and granzyme B-positive neoplastic lymphocytes were predominantly observed in the mucosal epithelium of 10/50 cases (20%) and 6/50 cases (12%), respectively. No significant differences in survival time were found based on cell size or epitheliotropism. However, cases with TIA1+ and/or granzyme B+ neoplastic lymphocytes predominantly in the mucosal epithelium had significantly shorter survival times (P < .05), suggesting that mucosal epithelium infiltration of neoplastic cells with a cytotoxic immunophenotype is a negative prognostic factor. Therefore, intraepithelial cytotoxic lymphocytes may be associated with mucosal injury and impaired intestinal function, leading to a poor prognosis in cats with intestinal T-cell lymphoma.

Relationship of microsatellite instability to mismatch repair deficiency in malignant tumors of dogs.

BACKGROUND: Microsatellite instability (MSI) is a type of genomic instability caused by mismatch repair deficiency (dMMR) in tumors. Studies on dMMR/MSI are limited, and the relationship between dMMR and MSI is unknown in tumors of dogs. OBJECTIVES: We aimed to identify the frequency of dMMR/MSI by tumor type and evaluate the relationship between dMMR and MSI in tumors of dogs. ANIMALS: In total, 101 dogs with 11 types of malignant tumors were included. METHODS: We extracted DNA from fresh normal and tumor tissues. Twelve microsatellite loci from both normal and tumor DNA were amplified by PCR and detected by capillary electrophoresis. Each microsatellite (MS) was defined as MSI if a difference in product size between the tumor and normal DNA was detected. The dMMR was evaluated by immunohistochemistry with formalin-fixed paraffin-embedded tumor tissues. Next, we confirmed whether dMMR induces MSI by serial passaging of MMR gene knockout cell lines for 3 months. RESULTS: Microsatellite instability was detected frequently in oral malignant melanoma. The number of MSI-positive markers was higher in cases with dMMR than in those with proficient MMR (P < .0001). Statistical analysis indicated that the occurrence of MSI in FH2305 might have relevance to dMMR. Furthermore, MSI occurred in dMMR cell lines 3 months after passaging. CONCLUSIONS AND CLINICAL IMPORTANCE: Microsatellite instability and dMMR more frequently were found in oral malignant melanoma than in other tumors, and dMMR has relevance to MSI in both clinical cases and cell lines.

The effect of 5-aminolevulinic acid on canine peripheral blood mononuclear cells.

5-aminolevulinic acid (ALA) is a natural amino acid and a product of the first heme synthesis pathway in mitochondria. Its immunomodulatory effects have garnered recent attention for their potential application to cancer, inflammation, and autoimmune diseases in humans. A supplement containing ALA is now available in Japan to enhance ATP synthesis via mitochondrial activity. However, how ALA affects canine immunity is unclear. Here we studied the effects of ALA on peripheral blood mononuclear cells (PBMCs) from healthy dogs in vitro. Heme oxygenase-1 (HO-1) protein was expressed in Madin-Darby canine kidney (MDCK) cells and PBMCs treated with ALA and ferrous sodium citrate (SFC), which showed that ALA works in dogs as well as humans. ALA also induced concanavalin A (ConA)-stimulated PBMCs to produce significantly more interferon-gamma (IFN-γ). Next-generation RNA sequencing (RNA-seq) revealed that ALA enhanced T cell immunity among Th1, Th2, and Th17 subsets, especially the IL-17 signaling pathway. We then confirmed that ALA promoted interleukin (IL)- 17A production in ConA-stimulated PBMCs. Together, these findings indicate that ALA promotes heme synthesis in mitochondria and enhances ConA-induced T cell immune responses in canine PBMCs.

Improvement of anemia in five dogs with nonregenerative anemia treated with allogeneic adipose-derived stem cells.

Background: Nonregenerative anemia is occasionally seen in dogs and can be caused by many factors, among which nonregenerative immune-mediated anemia (NRIMA) and pure red cell anemia are relatively common causes. These are thought to be caused by immune-mediated destruction of the erythroid lineage and are treated with immunosuppressive drugs, but some of them are refractory or recurrent, so new treatments are needed. Objectives: To examine the efficacy of allogeneic adipose-derived stem cells (ADSCs) for the treatment of nonregerative anemia in dogs. Methods: ADSCs were administered to total five nonregenerative anemia cases; two NRIMA cases and two suspected NRIMA cases that were refractory to immunosuppressive agents, and one NRIMA case that has not been treated with immunosuppressive agents. Results: In all cases, anemia was improved, and blood transfusion was no longer necessary. Conclusions: This study suggests that allogeneic ADSCs may be one of the rescue therapies for the refractory immune-mediated anemia in dogs.

Defucosylated mouse­dog chimeric anti­HER2 monoclonal antibody exerts antitumor activities in mouse xenograft models of canine tumors.

Human epidermal growth factor receptor 2 (HER2) overexpression has been reported in various types of cancer, including breast, gastric, lung, colorectal and pancreatic cancer. A humanized anti­HER2 monoclonal antibody (mAb), trastuzumab, has been shown to improve survival of patients in HER2­positive breast and gastric cancer. An anti­HER2 mAb, H2Mab­77 (mouse IgG1, kappa) was previously developed. In the present study, a defucosylated version of mouse­dog chimeric anti­HER2 mAb (H77Bf) was generated. H77Bf possesses a high binding­affinity [a dissociation constant (KD): 7.5x10­10 M, as determined by flow cytometric analysis] for dog HER2­overexpressed CHO­K1 (CHO/dHER2) cells. H77Bf highly exerted antibody­dependent cellular cytotoxicity (ADCC) and complement­dependent cytotoxicity (CDC) for CHO/dHER2 cells by canine mononuclear cells and complement, respectively. Moreover, administration of H77Bf significantly suppressed the development of CHO/dHER2 xenograft tumor in mice compared with the control dog IgG. H77Bf also possesses a high binding­affinity (KD: 7.2x10­10 M) for a canine mammary gland tumor cell line (SNP), and showed high ADCC and CDC activities for SNP cells. Intraperitoneal administration of H77Bf in mouse xenograft models of SNP significantly suppressed the development of SNP xenograft tumors compared with the control dog IgG. These results indicated that H77Bf exerts antitumor activities against dHER2­positive canine cancers, and could be valuable treatment regimen for canine cancers.

Nodal T-zone lymphoma and T-zone hyperplasia in dogs.

T-zone lymphoma (TZL) is an indolent, nodal lymphoma that has been clinically characterized in detail in dogs, and T-zone hyperplasia (TZH) is a hyperplastic change in lymph nodes associated with antigen processing. In some cases, histopathological features of TZL and TZH are similar, and are difficult to differentiate by morphology alone. Since there have been few publications characterizing their immunohistochemical profiles, histological, immunohistochemical, and clonality examinations were performed using formalin-fixed paraffin-embedded samples of canine lymph nodes with TZL (14 cases) and canine lymph nodes with TZH associated with nonlymphocytic tumors (10 cases). Immunohistochemically, small- to medium-sized lymphocytes of TZL were immunopositive for CD3, CD5, and HLA-DR, and negative for CD45, FOXP3, and granzyme B (GRB) in all cases. Among these 14 cases, 11 were immunopositive for CD8 and 1 was CD20 positive. Paracortical lymphocytes in TZH were diffusely immunopositive for CD3, CD5, and CD45, with scattered immunopositivity for CD8, HLA-DR, FOXP3, and GRB, and negative for CD20 in all cases. A clonal TCR gene rearrangement was detected in 13/14 TZL and none of the TZH cases. The present study revealed that TZL is a clonal proliferation of monomorphic CD8+CD45-GRB- T cells, while TZH consists of an immunophenotypically heterogenous population of CD45+ T cells that are variably positive for CD8 and FOXP3. These results suggest that canine TZL is a clonal proliferation of naïve or premature cytotoxic T cells. Regarding TZH, variable immunopositivity for cytotoxic and regulatory T-cell antigens may reflect immune responses to a variety of regional neoplastic lesions.