Plasma free amino acid profiles of canine mammary gland tumors

The purpose of this study was to elucidate the relationship between plasma free amino acid (PFAA) levels and the clinical stages of mammary gland tumors (MGT) in dogs. PFAA levels in canines with malignant mammary tumors were decreased compared to those of healthy animals. The levels of aspartate and ornithine, in the dogs with tumor metastasis were significantly decreased when compared to those of dogs that did not have metastases. Results of this study indicate that PFAA levels could be a risk factor or biomarker for canine MGT metastasis.     

Expression of vascular endothelial growth factor in tumors and plasma from dogs with primary intracranial neoplasms

OBJECTIVE: To quantitatively evaluate expression of vascular endothelial growth factor (VEGF) in intracranial tumors in dogs and determine whether relationships exist between circulating and intratumoral VEGF concentrations and tumor type and grade. ANIMALS: 27 dogs with primary intracranial neoplasms and 4 unaffected control dogs. PROCEDURES: Plasma and brain tumor samples were obtained from each dog, and plasma and intratumoral concentrations of VEGF were measured by use of an ELISA. RESULTS: Dogs with meningiomas (n = 11) were significantly older than dogs with oligodendrogliomas (7) or astrocytomas (9). Measurable VEGF was detected in all tumors, and a significant negative correlation between age and intratumoral VEGF concentration was detected. Age-adjusted comparisons identified significant differences in intratumoral VEGF concentrations among all tumor types; the highest VEGF concentrations were associated with astrocytomas. Within each tumor type, increasing tumor grade was significantly associated with increasing VEGF expression. Plasma VEGF concentrations were detectable in 9 of 27 dogs; the proportion of dogs with astrocytomas and a detectable circulating VEGF concentration (7/9 dogs) was significantly higher than the proportion of dogs with meningiomas (1/11 dogs) or oligodendrogliomas (1/7 dogs) with a detectable circulating VEGF concentration. CONCLUSIONS AND CLINICAL RELEVANCE: Overexpression of VEGF appears common in canine astrocytomas, oligodendrogliomas, and meningiomas. In the neoplasms examined, intratumoral VEGF concentrations correlated well with tumor malignancy. The VEGF expression patterns paralleled those of analogous human tumors, providing evidence that dogs are a suitable species in which to study angiogenesis and intracranial neoplasia for human application.     

The Prognostic Significance of Angiogenesis in Canine Mammary Tumors

The purpose of the study was to determine if neovascularization, a measure of angiogenesis, is correlated with metastasis of mammary tumors in dogs. Forty-six paraffin-embedded tissue blocks of benign and malignant canine mammary tumors obtained from 42 clinical cases at the Iowa State University Veterinary Teaching Hospital were retrieved from the archives of the Department of Veterinary Pathology. Of the dogs with malignant tumors, cases with and without lymph node metastasis were chosen. Neovascularization was quantified by light microscopy on formalin-fixed, paraffin-embedded sections of canine mammary tumors using an avidin biotin immunoperoxidase assay for factor VIII-related antigen. Mean microvessel counts for each group were statistically evaluated using analysis of variance. The mean number of microvessels was highest in the malignant tumors of dogs with lymph node metastasis (44). This number was significantly different from the mean number of microvessels in the benign tumors (28; P = .03) and a trend occurred toward higher microvessel counts in malignant tumors with lymph node metastasis versus malignant tumors of dogs without metastasis (32; P = .1). No significant difference was found between the number of microvessels found in malignant tumors without metastasis versus benign tumors. The trend toward higher microvessel counts in mammary tumors that have metastasized supports the premise that angiogenesis may be an independent and significant prognostic indicator in dogs with malignant mammary tumors, as it is in women with breast cancer.     

Evaluation of circulating IGF-I and IGFBP-3 as biomarkers for tumors in dogs

BackgroundSerum-based parameters are considered non-invasive biomarkers for cancer detection. In human studies, insulin-like growth factor-I and II (IGF-I and IGF-II) and insulin-like growth factor binding protein-3 (IGFBP-3) are useful as diagnostic or prognostic markers and potential therapeutic targets.ObjectivesThis study examined the diagnostic utility of circulating IGF-I, IGF-II, and IGFBP-3 levels in healthy dogs and dogs with tumors.MethodsThe serum concentrations of these biomarkers in 86 dogs with tumors were compared with those in 30 healthy dogs using an enzyme-linked immunosorbent assay (ELISA).ResultsThe ELISA results showed no difference between healthy dogs and dogs with tumors in the serum IGF-II concentrations. On the other hand, there was a significant difference in the circulating IGF-I and IGFBP-3 levels between healthy dogs and dogs with tumors. The concentrations of serum IGF-I (median [interquartile range], 103.4 [59.5–175] ng/mL) in dogs with epithelial tumors were higher than those (58.4 ng/mL [43.5–79.9]) in healthy dogs. Thus, the concentrations of serum IGFBP-3 (43.4 ng/mL [33.2–57.2]) in dogs with malignant mesenchymal tumors were lower than those (60.8 ng/mL [47.6–70.5]) in healthy dogs.ConclusionsThe serum IGF-I and IGFBP-3 levels can be used as diagnostic biomarkers in dogs with tumors.     

Serum vascular endothelial growth factor in dogs with soft tissue sarcomas

This work aimed to evaluate serum vascular endothelial growth factor (VEGF) in 25 dogs with soft tissue sarcoma, and in 30 healthy dogs. Blood was collected once time from the control animals and three times, in the same way, from animals with sarcoma. Blood count was performed in the blood collected, and serum VEGF was measured by enzyme‐linked immunosorbent assay quantitative method. Serum VEGF in control animals was similar to patients with soft tissue sarcoma. There was a reduction in serum VEGF after the sarcoma resection. There was positive correlation between serum VEGF and neutrophil counts, and negative between VEGF and hemoglobin content in animals with sarcoma. Animals with hemangiopericytoma showed higher serum VEGF levels compared to the patients with malignant peripheral nerve sheath. Circulating blood cells can contribute to elevate VEGF serum concentrations in dogs with soft tissue sarcomas and a possible role of VEGF in the angiogenesis of these tumors.     

Investigation of serum survivin in dogs suffering from cancer: a multicenter study

BackgroundIn contrast to human medicine, only a small number of serum tumor markers are established in veterinary medicine even though they are a non-invasive diagnostic tool.ObjectivesThis study examined whether survivin could be suitable as a potential canine serum tumor marker.MethodsThis study measured the serum survivin concentrations of dogs with mammary tumors (n = 33), squamous cell carcinoma (n = 9), soft-tissue sarcoma (n = 18) and multicentric lymphoma (n = 22), using a commercially available, competitive immunoassay kit (BlueGene). The serum survivin concentrations were compared with those of a healthy control group (n = 20) and a control group of dogs with non-neoplastic diseases (n = 17).ResultsDogs with malignant tumors had serum survivin concentrations between 15 and 5,906 pg/mL (median, 72 pg/mL), those in the healthy group ranged from 7 to 99 pg/mL (median, 21 pg/mL) and those in the group of dogs suffering from non-neoplastic diseases from 15 to 93 pg/mL (median, 42 pg/mL). The differences in the survivin concentrations between the healthy dogs and dogs with malignant tumors and between the dogs with non-neoplastic diseases and those with malignant tumors were significant (p < 0.001 and p = 0.006, respectively).ConclusionsThe serum survivin concentrations in dogs with malignant tumors, with some exceptions, are higher than in dogs with benign tumors and dogs that do not suffer from a malignancy. Therefore, survivin can provide information on the presence of malignant tumors and be used as a tumor marker in dogs.     

Signs of metastatic disease on thoracic radiographs of dogs suffering from mammary gland tumours: a retrospective study (1990-1998)

A mammary gland tumour (MGT) was clinically diagnosed in 136 dogs. Histologically 71% were malignant and 29% benign. Intrathoracic metastatic disease was noted or suspected radiographically in 13.5% of the dogs with malignant and in 2.5% of the dogs with benign MGT. Six dogs with malignant MGT were necropsied, 5 had pulmonary metastases but only 1 had radiographic signs of intrathoracic metastatic disease. We conclude that radiographs are not very sensitive for detection of early intrathoracic metastatic disease of MGT.     

Vascular endothelial growth factor in diabetic and nondiabetic canine cataract patients

Objective To measure vascular endothelial growth factor (VEGF) levels in aqueous humor, serum, and plasma in diabetic and nondiabetic cataractous dogs. Methods Canine VEGF was assayed in the plasma and serum of 32 dogs (20 diabetics; 12 nondiabetics) and aqueous humor in 57 eyes of those dogs (39 diabetic; 18 nondiabetic) undergoing phacoemulsification, using a commercial canine VEGF assay. Statistical analysis was performed using Fisher’s PLSD, t‐test, and regression analysis to compare values by diabetic status, duration of diabetes, age, weight, gender, left vs. right eye, and blood clarity. Results Plasma, but not serum or aqueous humor VEGF values of diabetics were significantly greater than nondiabetics (P = 0.019). Older nondiabetics (10–15 years) had higher plasma VEGF values than younger (0–5 and 5–10 years) dogs (P = 0.0002 and 0.0001, respectively). There was no significant difference in aqueous humor VEGF between left and right eyes in all patients. Serum and plasma, but not aqueous humor, VEGF values in females were significantly higher than males in both groups. Conclusion Similar to human diabetic patients, VEGF aqueous humor values in all dogs are significantly higher than blood values. Aqueous humor VEGF values in human diabetics are elevated and correlate with the severity of diabetic retinopathy. However, aqueous humor values of VEGF in diabetic dogs are not greater than nondiabetics and may serve to protect the dog against development of diabetic retinopathy.     

Serum and Plasma Vascular Endothelial Growth Factor Levels in Various Cat Populations

Introduction:  Vascular endothelial growth factor (VEGF) is one of the most important mediators of angiogenesis. Elevated levels within tumors, and in serum, plasma, and tumor effusion have been correlated with the development of metastatic disease, recurrence, and poor prognosis in many tumors in humans. Canine VEGF has been sequenced as homologous with the human form, and elevated serum and plasma VEGF have been found in dogs with hemangiosarcoma. Feline VEGF has also been sequenced, and shares homology with the human and canine forms.
Materials and Methods:  Stored serum and plasma samples from normal cats, cats with various neoplasms, and cats with non‐neoplastic disease were evaluated with a commercial ELISA kit (R&D Systems, Minneapolis MN). Samples were run in duplicate, and a standard curve was performed for each plate. The data were analyzed for differences between populations, and between serum and plasma levels in the same patient to determine the optimal sample for evaluating VEGF in cats.
Results:  In seven apparently healthy cats mean plasma VEGF was 95.6 pg/mL. In non‐neoplastic disease (7 cases), mean plasma VEGF was 117.3 pg/mL and mean serum VEGF level was 219.7 pg/mL. In ten tumor‐bearing patients mean plasma VEGF was 247.1 pg/mL, and mean serum VEGF was 322.3 pg/mL. Statistical analysis showed no significant difference between mean serum and plasma VEGF concentrations within each group or between groups (p > 0.05).
Discussion:  Serum and plasma VEGF levels could not be used to distinguish between healthy cats and cats with neoplastic or non‐neoplastic disease.     

Immunohistochemical detection of HER-2/neu expression in spontaneous feline mammary tumours

Mammary gland tumours (MGT) are the third most common tumours in the cat. At least 85% are malignant and metastasis is common. The HER‐2/neu protooncogene encodes a 185‐kDa transmembrane tyrosine receptor kinase protein. Approximately 25–30% of human MGT demonstrate HER‐2/neu protein overexpression in the malignant cells, and overexpression has been associated with an increased metastatic propensity and a decreased prognosis. No reports have been published, to date, investigating the expression of Her‐2/neu in cats or cats with spontaneous mammary tumours. Based on the increased percentage of malignant mammary cancers in cats, compared to that in dogs, and the correlation of an increased malignancy and a decreased prognosis with Her‐2 overexpression in human mammary cancer, we hypothesized that cats with spontaneous malignant mammary adenocarcinoma overexpress Her‐2/neu in the neoplastic mammary epithelial cells. Thirty cats with MGT were assayed for Her‐2/neu immunohistochemical expression. The median percentage of cells from feline MGT expressing Her‐2/neu by utilizing the Dako polyclonal and CB11 monoclonal antibodies was 85 and 92.5, respectively. Her‐2/neu expression intensity grades 2 and 3 consistent with the overexpression by utilizing the Dako polyclonal and CB11 monoclonal antibodies were observed in 90 and 76.7% of cats with MGT, respectively. The level of overexpression concordance across the two antibodies was 70%. The results from this study suggest that Her‐2/neu overexpression is common in cats with spontaneous MGT, and therefore appears to represent an excellent model for Her‐2/neu‐overexpressing human breast cancer.     

Alteration of somatostatin receptor 2 expression in canine mammary gland tumor

Somatostatin receptor 2 (SSTR2) is a negative regulator of cell proliferation in human breast cancer. Since there is little information about SSTR2 in canine mammary gland tumor (MGT), we clarified its distribution and expression level in normal mammary gland, benign MGT and malignant MGT. SSTR2 expression determined by immunohistochemical staining was observed in the cytoplasm of luminal epithelial cells. The intensity was negatively correlated with malignancy: normal tissues and some of the benign tumors had the highest levels, while the malignant tumors had little or no SSTR2 expression. As for the Western blotting, SSTR2 protein level in benign tumors was significantly lower than the normal mammary gland. On the other hand, SSTR2 protein levels in two of three malignant tumors were higher than the other groups. These results suggest that SSTR2 expression alters according to the malignancy of canine MGT.     

Preliminary study of plasma vascular endothelial growth factor (VEGF) during low- and high-dose radiation therapy of dogs with spontaneous tumors

High plasma vascular endothelial growth factor (VEGF) concentrations are associated with radiation resistance and poor prognosis. After an exposure to ionizing radiation in cell culture an early phase and a late phase of increased VEGF have been documented. The activation was dependent on the radiation dose. Therefore, the purpose of this study was to measure baseline plasma VEGF and changes in VEGF over the course of fractionated radiation therapy in dogs with spontaneous tumors. Dogs with tumors had a significantly higher pretreatment plasma VEGF than did dogs without tumors. Immediately after irradiation no increased plasma VEGF was observed. Over the course of radiation therapy there was an increased plasma VEGF in dogs treated with low doses per fraction/high total dose, whereas plasma VEGF remained stable in dogs irradiated with high doses per fraction/low total dose. The regulatory mechanisms are very complex, and therefore the value of plasma VEGF measurements as an indirect marker of angiogenesis induced by radiotherapy is limited.     

Expression of vascular endothelial growth factor in canine oral malignant melanoma

Serum, plasma and tissue expression of vascular endothelial growth factor (VEGF) was measured in 20 dogs previously diagnosed histologically with oral melanoma. The concentrations of VEGF in serum and plasma were significantly higher in dogs with melanoma than in a control population (P ≤ 0.002). Concentrations of VEGF in the serum and plasma of dogs with melanoma were highly correlated (r = 0.867). Ninety‐five per cent of melanoma tissues expressed VEGF. Two staining patterns were detected: diffuse and granular cytoplasmic staining. High blood concentrations of VEGF were correlated to a shorter survival time in dogs receiving definitive therapy (P = 0.002). Survival times were significantly longer in dogs receiving definitive therapy versus palliative therapy (median 496 versus 97 days, P = 0.007). Blood concentrations of VEGF were associated with stage (P < 0.05). Dogs with oral melanoma have increased serum, plasma and tissue concentrations of VEGF. Increased expression of VEGF may be a reasonable target for future therapy of canine oral melanoma.     

Serum vascular endothelial growth factor concentrations and postsurgical outcome in dogs with osteosarcoma

Vascular endothelial growth factor (VEGF) is a key angiogenic growth factor, playing putative roles in both tumour growth and metastasis. The purpose of this study was to correlate pretreatment serum concentrations of VEGF in dogs with osteosarcoma (OSA) with disease‐free interval (DFI) and overall survival (OS). Additionally, the effect of serum from dogs with OSA on ex vivo canine endothelial cell (EC) growth was determined. Pretreatment platelet‐corrected serum VEGF levels correlated significantly with DFI. No other examined variable predicted outcome. The ability of sera from dogs with OSA to stimulate canine EC proliferation did not correlate with VEGF concentration or outcome. These data support a role for VEGF in the development or progression of metastatic disease in dogs with OSA. The VEGF concentration in tested sera was not a major determinant of ex vivo canine EC proliferation in this study.     

Serum alkaline phosphatase isoenzyme activities in canine malignant mammary neoplasms with and without osseous transformation

BACKGROUND: Increased serum activity of total alkaline phosphatase (TALP) has been found in dogs with mammary neoplasms, especially malignant mixed tumors. We hypothesized that the bone isoenzyme of alkaline phosphatase (BALP), a specific indicator of osteoblastic activity and bone formation, may contribute to increased TALP in dogs with mammary neoplasms with osseous transformation. OBJECTIVE: The purpose of this study was to compare serum TALP, BALP, and other ALP isoenzyme activities in dogs with mammary malignant neoplasms with and without osseous transformation. METHODS: Twenty-one female dogs with malignant mammary neoplasms were compared with 21 clinically healthy, age-matched female control dogs. Physical, clinicopathologic (including preprandial and postprandial serum bile acids, ACTH stimulation, and low-dose dexamethasone suppression tests), radiographic, and ultrasonographic examinations were performed on all dogs with tumors to assess coexisting conditions. On the basis of histologic examination of excised tumors, dogs were further classified as having epithelial (n = 11) or mesenchymal/mixed (epithelial-mesenchymal) (n = 10) neoplasms, the latter of which had histologic and radiologic evidence of bone formation. Serum TALP, BALP, liver alkaline phosphatase (LALP), and corticosteroid-induced alkaline phosphatase (CALP) activities were measured using biochemical methods. RESULTS: Dogs with malignant mammary tumors had significantly higher (P < .05) median serum TALP (170 U/L), BALP (59 U/L), LALP (49 U/L), and CALP (24 U/L) activities, compared with control dogs (81, 32, 37, and 5 U/L, respectively). Significantly higher activities of BALP and LALP were found in dogs with epithelial neoplasms; whereas, only CALP activity was higher in dogs with mesenchymal/mixed neoplasms. There was no significant difference in TALP or isoenzyme activitities between epithelial and mesenchymal/mixed groups. CONCLUSION: BALP activity is increased in some dogs with malignant mammary tumors but does not account for the increase in TALP in dogs with neoplasms that have osseous transformation.     

Endostatin and vascular endothelial growth factor concentrations in healthy dogs, dogs with selected neoplasia, and dogs with nonneoplastic diseases

To evaluate the relationship between endostatin and vascular endothelial growth factor (VEGF) in cancers of dogs, circulating concentrations of these 2 tumor-associated markers were measured prospectively in healthy dogs (n = 44), dogs with tumors (n = 54), and dogs with nonneoplastic diseases (n = 42 for endostatin; n = 16 for VEGF). A canine-directed enzyme-linked immunosorbent assay kit was used for determination of endostatin, and a human-directed kit was validated for detection of canine VEGF. Concentrations of endostatin for all dogs were 28-408 ng/mL. Increasing serum endostatin concentration was associated with increasing age (P = .0396). Concentrations of endostatin were not different among groups of dogs (P = .1989) when adjusted for age. Mean endostatin concentrations for all dogs were higher in dogs (P = .0124) with detectable VEGF concentrations. Endostatin concentrations, when corrected for age, were related to decreasing PCV (P = .032) but not white blood cell count (P = .225) or platelet count (P = .1990). Measurable VEGF (> or = 2.5 pg/mL) was detected in 3 (7.0%) of 43 healthy dogs. Dogs with tumors had detectable VEGF in 24 (44%) of 54 dogs, with concentrations ranging from 2.5-274 pg/mL; only 1 dog with a nonneoplastic disease process had detectable VEGF. VEGF concentrations for all dogs after correcting for age, endostatin, and disease categories were associated with increased white blood cell count (P = .0032) and platelet counts (P = .0064) and decreased PCV (P = .0017). Linkage between increased endostatin and VEGF concentrations suggests that similar factors may influence concentrations of these markers. Further evaluation of endostatin and VEGF associations in dogs with tumors may provide information on the extent and progression of the disease.     

Evaluation of circulating PD-1 and PD-L1 as diagnostic biomarkers in dogs with tumors

BackgroundProgrammed cell death protein-1 (PD-1) and programmed cell death ligand-1 (PD-L1) have important roles in tumor evasion of the immune system.ObjectivesThis study aimed to assess the diagnostic utility of circulating PD-1 and PD-L1 levels in healthy dogs and dogs with tumors.MethodsCirculating PD-1 and PD-L1 levels in the serum of 71 dogs with tumors were compared with those of 52 healthy dogs by performing enzyme-linked immunosorbent assay (ELISA).ResultsThe ELISA results revealed higher circulating PD-1 and PD-L1 levels in dogs with tumors (2.9 [2.2–3.7] ng/mL; median [IQR] and 2.4 [1.4–4.4] ng/mL, respectively) than in healthy dogs (2.4 [1.9–3.0] ng/mL; p = 0.012 and 1.4 [0.9–2.1] ng/mL; p < 0.001, respectively). Especially, there was a significant difference in circulating PD-1 levels between healthy dogs and dogs with malignant epithelial tumors (2.4 [1.9–3.0] ng/mL and 3.1 [2.6–4.4] ng/mL, respectively; p < 0.01). In addition, there was a significant difference in circulating PD-L1 levels between healthy dogs and dogs with lymphomas (1.4 [0.9–2.1] ng/mL and 2.7 [1.6–5.8] ng/mL, respectively; p < 0.001).ConclusionThis study indicates that circulating PD-1 and PD-L1 have potential as tumor diagnostic biomarkers in dogs with tumors.     

Vascular endothelial growth factor concentrations in body cavity effusions in dogs

Vascular endothelial growth factor (VEGF) has potent angiogenic, mitogenic, and vascular permeability enhancing properties specific for endothelial cells. VEGF is present in high concentrations in inflammatory and neoplastic body cavity effusions and has been implicated in the pathogenesis of neoplastic and inflammatory effusion formation. In this study, VEGF was quantitated by solid-phase enzyme-linked immunoadsorbent assay (ELISA) in samples of pericardial, pleural, and peritoneal effusions (N = 38) from dogs (N = 35) with neoplastic and non-neoplastic diseases. VEGF was detected in 37 of 38 effusions (median, 754; range, 18-3,669 pg/mL) and was present in much higher concentrations than in previously established normal concentrations for canine plasma (median, < 1 pg/mL; range, < 1-18 pg/mL) or in those previously noted in the plasma of dogs with hemangiosarcoma (HSA; median, 17 pg/mL; range, < 1-67 pg/mL). In 4 dogs with HSA, the concurrent plasma VEGF concentration was much lower than in the abdominal effusion (P = .029). No significant correlation was demonstrated between VEGF effusion concentration and effusion total protein content or nucleated cell count. Mean VEGF concentrations were significantly higher in pericardial (median, 3,533; range, 709-3,669 pg/mL) and pleural effusions (median, 3,144; range, 0-3,663 pg/mL) compared to peritoneal effusions (median, 288; range, 18-2,607 pg/mL; P < .05). There was no marked difference demonstrated between effusions associated with malignant and nonmalignant diseases. Further studies are necessary to elucidate the role of VEGF in body cavity effusion formation in dogs.     

Rapid visualization of mammary gland tumor lesions of dogs using the enzyme-activated fluorogenic probe; γ-glutamyl hydroxymethyl rhodamine green

Since gamma-glutamyl transpeptidase (GGT) is highly and locally expressed in human breast cancer, a GGT-enzymatically activatable fluorescent probe, gamma-glutamyl hydroxymethyl rhodamine green (gGlu-HMRG), has been developed to detect the human breast cancer area with high performance. In this study, GGT expression and the efficacy of gGlu-HMRG on visualization were investigated in canine mammary gland tumors (MGT). Seventeen non-fixed fresh-frozen MGT specimens and each peritumoral control tissue were utilized. The GGT mRNA levels were highly observed in the tumor specimens compared with the control. GGT immunostaining was mostly observed on the cell membrane and cytosol of the alveolar and duct mammary epithelium of MGT tissues. These signals were strongly positive in several cases while they were mild to not observed in other cases. When gGlu-HMRG solution was dropped to the non-fixed tissue pieces of MGT or control tissues, the fluorescence intensities (FIs) were measured using Maestro in-vivo imaging device. FIs in MGT tissues were significantly higher than each control tissue 20 min after treatment. Based on Youden index method said that the maximum sensitivity and specificity of FI was 82.4% and 82.4%. These findings suggest that GGT is highly expressed in several MGTs in dogs and gGlu-HMRG could visualize at least a part of MGT tissues in dogs. Nevertheless, it should be needed to assess the false-negative areas more carefully in canine than human cases.