Matrix metalloproteinase-2 and -9 involvement in canine tumors

Matrix metalloproteinases (MMPs) are a family of enzymes implicated in the degradation and remodeling of extracellular matrix and in vascularization. They are also involved in pathologic processes such as tumor invasion and metastasis in experimental cancer models and in human malignancies. We used gelatin zymography and immunohistochemistry to determine whether MMP-2 and MMP-9 are present in canine tumors and normal tissues and whether MMP production correlates with clinicopathologic parameters of prognostic importance. High levels of pro-MMP-9, pro-MMP-2, and active MMP-2 were detected in most canine tumors. Significantly higher MMP levels were measured in canine tumors than in nontumors, malignancies had higher MMP levels than benign tumors, and sarcomas had higher active MMP-2 than carcinomas. Cartilaginous tumors produced higher MMP levels than did nonsarcomatous malignancies, benign tumors, and normal tissues, and significantly greater MMP-2 than osteosarcomas and fibrosarcomas. Pro-MMP-9 production correlated with the histologic grade of osteosarcomas. The 62-kd form of active MMP-2 was detected only in high-grade, p53-positive, metastatic malignancies. Zymography proved to be a sensitive and quantitative technique for the assessment of MMP presence but has the limitation of requiring fresh tissue; immunohistochemistry is qualitative and comparatively insensitive but could be of value in archival studies. MMP presence was shown in a range of canine tumors, and their link to tumor type and grade was demonstrated for the first time. This study will allow a substantially improved evaluation of veterinary cancer patients and provides baseline information necessary for the design of clinical trials targeting MMPs.     

Identification of matrix metalloproteinases in canine neoplastic tissue

OBJECTIVE: To identify matrix metalloproteinases (MMP) 2 and 9 in canine tumor tissue and to compare the amount of activity to that in unaffected stromal tissue. ANIMALS: 30 dogs with spontaneously developing, high-grade osteosarcoma. PROCEDURE: Tumor and nearby stromal tissue (muscle) were obtained at the time of surgery. Specimens were homogenized, and supernatants were assayed, using gelatin zymography. Human derived standards were run concurrently. Densitometry was done to obtain a semiquantitative arbitrary unit value for each specimen. The amount of activity in tumor tissue was compared with the amount in stromal tissue. RESULTS: Gelatinolytic bands were observed from the analysis of all tumor tissues and in most stromal tissues. These bands migrated in the same molecular weight area as the human MMP 2 and 9 standards. Gelatinolytic activity could be quenched by the addition of 50 mM EDTA and 1 microg of synthetic tissue inhibitor of metalloproteinase (TIMP) 2 per 100 ml. There was significantly more gelatinolytic activity in tumor tissue than in stromal tissue. CONCLUSIONS AND CLINICAL RELEVANCE: MMP 2 and 9 are detectable in canine neoplastic tissue. Matrix metalloproteinases activity in tumor tissue is higher than in unaffected stromal tissue, indicating that canine MMP may be involved in the pathogenesis of tumor growth and metastasis.     

Increased expression of tissue inhibitor of metalloproteinase‐1 correlates with improved outcome in canine cutaneous mast cell tumours

Canine mast cell tumour (MCT) is a biologically heterogeneous disease. The extracellular matrix degradation promoted by matrix metalloproteinases (MMPs) has been studied in an attempt to elucidate the mechanisms involved in the biological behaviour of tumours. The aim of this study was to characterize the expression of MMP‐2 and ‐9 and tissue inhibitors of metalloproteinase (TIMP)‐1 and ‐2 in canine cutaneous MCTs and to evaluate their prognostic values. Immunohistochemical staining for MMP‐2, MMP‐9, TIMP‐2 and TIMP‐1 was performed in 46 canine cases of MCTs. TIMP‐1 expression showed an independent prognostic value for post‐surgical survival and disease‐related mortality. Dogs with MCTs showing less than 22.9% mast cell TIMP‐1 positivity were more prone to die because of the disease and had a shorter post‐surgical survival. This article suggests the involvement of TIMP‐1 in MCT progression, by contributing to a good outcome in patients with MCTs.     

Immunohistochemical detection of p53 tumor-suppressor protein is a poor indicator of prognosis for canine cutaneous mast cell tumors

Eighty-three canine cutaneous mast cell tumors were graded histologically and evaluated immunohistochemically for p53 tumor-suppressor protein expression. An avidin-biotin immunohistochemical protocol incorporated a rabbit polyclonal antibody (CM-1) directed against normal and mutant p53 protein. Positive staining was observed in 44.6% (37/83) of tumors and included 50% (12/24) of grade I (well differentiated) tumors, 46.9% (23/49) of grade II (intermediate differentiation) tumors, and 20% (2/10) of grade III (poorly differentiated) tumors. A statistically significantly higher proportion (P < 0.019) of tumors from the head and neck (83.3%, 10/12), stained positive for p53 than tumors from the thorax, back, abdomen, and axilla (39.4%, 13/33), legs (35.7%, 10/28), or prepuce, scrotal, or inguinal areas (44.4%, 4/9). No statistically significant difference between p53 labeling and histologic grade, breed, or tumor size was present. Survival data were available for 53/83 (63.9%) of dogs. Positive reactivity for p53 was observed in 47% (25/53) of tumors within this group, with 57.9% (11/19) of grade I, 43.3% (13/30) of grade II, and 25% (1/4) of grade III tumors labeled. Mean survival time for the 53 dogs was 12.1 months. The median survival time for dogs with grade III tumors or tumors >5 cm was statistically significantly shorter (P < 0.0001) than for dogs with grades I and II or smaller tumors. Although p53 protein abnormalities may play a role in tumor development or behavior in some canine cutaneous mast cell tumors, immunoreactivity was not associated with lack of tumor differentiation, tumor locations previously shown to demonstrate aggressive biological behavior, breed predisposition, or survival times.     

Matrix metalloproteinase activity in tumor, stromal tissue, and serum from cats with malignancies

Matrix metalloproteinases (MMPs) are enzymes that play key roles in angiogenesis, tumor invasion, and metastasis in a wide variety of species. The purpose of this study was to evaluate pro and active MMP 2 and 9 concentrations in tumor, normal stromal tissue, and serum from tumor-bearing cats. We hypothesized that serum concentrations of pro and active forms of MMPs 2 and 9 would be predictive of MMP concentrations in tumor tissue and that these MMP concentrations would correlate with the histopathologic grade of the malignancies. Pro and active forms of MMPs 2 and 9 were determined by gelatin zymography and subsequent computerized densitometry from tumor and nearby stromal tissue and serum from 49 cats with various malignancies. The serum concentrations of MMPs from these tumor-bearing cats were compared with serum concentrations of MMPs from 44 normal cats of similar age and gender. Measurable concentrations of MMPs 2 and 9 were found within tumor, stromal, and serum samples. Mean concentrations of total pro and active MMPs 2 and 9 within tumor tissue were significantly higher (P values <.0001, .0031, <.001, and .0064, respectively) when compared with stromal tissue from the same animals. Serum MMP concentrations from tumor-bearing cats were higher than those from normal cats. Poor correlation was found between serum MMP concentrations and tissue MMP concentrations of increasing histologic grades of malignancies.     

Mutations in the juxtamembrane domain of c-KIT are associated with higher grade mast cell tumors in dogs

Mast cell tumors are among the most commonly seen tumors of the skin in dogs and are more highly aggressive than mast cell tumors of other species. Some breeds display a markedly higher incidence of mast cell tumor development than others and appear to have some genetic predisposition. Recently, mutations have been found in canine mast cell tumor tissues and cell lines within the juxtamembrane domain of the protooncogene c-KIT In previous studies utilizing a small number of cases, no association between the presence of a mutation and the breed of dog or grade of the tumor could be identified. An expanded study with a larger sample set was performed to explore this possibility. The juxtamembrane domain of c-KIT was amplified using the polymerase chain reaction from genomic DNA preparations of 88 paraffin-embedded mast cell tumors from selected breeds. Mutations, consisting of duplications and deletions, were found in 12 of the tumors. A significant association was found between the presence of a mutation and a higher grade of tumor but not between breed and grade or between breed and the presence of a mutation.     

Immunohistochemical and histochemical stains for differentiating canine cutaneous round cell tumors

Immunohistochemical and histochemical stains are useful adjunct techniques in the diagnosis of canine cutaneous round cell tumors, which can appear histologically similar. We applied a panel of monoclonal antibodies (recognizing tryptase, chymase, serotonin for mast cells; CD1a, CD18, MHC class II for histiocytes; CD3 for T lymphocytes; CD79a for B lymphocytes and plasma cells) and one histochemical stain (naphthol AS-D chloroacetate for chymase activity) to formalin-fixed, paraffin-embedded sections of canine cutaneous mast cell tumors, histiocytomas, lymphosarcomas, plasmacytomas, and unidentified round cell tumors. Of 21 tumors with a histologic diagnosis of mast cell tumor, 7/7 (100%) grade I, 6/7 (85.7%) grade II, and 3/7 (42.9%) grade III tumors were diagnosed as mast cell tumors based on positive staining for tryptase antigen and chymase activity. Mast cells were positive for both tryptase antigen and chymase activity, indicating equal efficacy of tryptase immunohistochemistry and chymase histochemistry. Chymase was detected immunohistochemically in both tumor and nontumor cells, while serotonin was not detected in most mast cell tumors, and thus, neither was useful in the diagnosis of mast cell tumors. Immunohistochemistry to detect CD18 and MHC class II was equally effective in staining histiocytomas, although lymphosarcoma must be ruled out through the use of CD3 and CD79a immunohistochemistry. Immunohistochemistry using three different monoclonal antibodies to human CD1a showed no cross-reactivity in canine histiocytomas and was not useful. A final diagnosis was obtained for 4/5 (80%) of the unidentified tumors, indicating the usefulness of multiple stains in poorly differentiated round cell tumors.     

Variation among pathologists in histologic grading of canine cutaneous mast cell tumors.

Ten veterinary pathologists at 1 veterinary institution independently assigned histologic grades to the same 60 canine cutaneous mast cell tumors (MCTs). There was significant variation among pathologists in grading the MCTs (P < 0.001). The probability of assigning a low grade was significantly higher for the pathologists in this study who use a published reference for histologic grading of canine cutaneous MCTs that allows subcutaneous MCTs or MCTs with mitotic figures to be included in the low-grade category (P < 0.0001 and P < 0.0001, respectively).     

Cytology of canine cutaneous round cell tumors. Mast cell tumor, histiocytoma, lymphosarcoma and transmissible venereal tumor.

Sixty-four canine cutaneous round cell tumors were divided into 25 mast cell tumors, 15 histiocytomas, nine cutaneous lymphosarcomas and 15 transmissible venereal tumors. The final diagnosis was made from cytologic, clinical and histologic findings. Cytologic features were significantly distinctive in mast cell tumor, transmissible venereal tumor, and most cases of histiocytoma and lymphosarcoma to allow a diagnostic opinion. This opinion was supported by subsequent histologic examination. In some instances cytology was considered essential in rendering a diagnostic opinion even though histology was available.     

Immunohistochemical expression of vascular endothelial growth factor and vascular endothelial growth factor receptor associated with tumor cell proliferation in canine cutaneous squamous cell carcinomas and trichoepitheliomas

The expression of 5 markers associated with angiogenesis was studied in canine squamous cell carcinomas (SCCs) (n = 19) and canine trichoepitheliomas (TCPs) (n = 24). SCCs were assigned histologic grades, and tissue sections from both tumor types were immunohistochemially stained for the expression of vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor-2 (VEGFR-2), as well as intratumoral microvessel density (iMVD), tumor proliferation index (PI), and tumor apoptotic index (AI), using antibodies against VEGF, VEGFR-2, von Willebrand's factor, Ki-67 antigen, and the terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate end-labeling method (TUNEL), respectively. VEGF and VEGFR-2 were detected in 17/19 (89.4%) and 19/19 (100%) SCCs and in 17/24 (70.8%) and 20/24 (83.3%) TCPs, respectively. In SCCs, there was substantial correlation between histologic grade and PI (r = 0.51); and moderate correlation between VEGF and histologic grade (r = 0.43), VEGFR-2 and histologic grade (r = 0.47), VEGF and PI (r = 0.47), and VEGFR-2 and PI (r = 0.47) (Spearman rank correlation coefficient). In TCPs, there was substantial correlation between VEGF and PI (r = 0.51) and a moderate correlation between VEGFR-2 and iMVD (r = 0.36). The median iMVD of SCCs (15.5) was significantly higher than the median iMVD of TCPs (9.05) (P value < .05). It was concluded that VEGF and VEGFR-2 may promote tumor cell proliferation in TCPs and SCCs. An autocrine pathway for VEGF probably operates in canine SCCs and TCPs, as VEGF and VEGFR-2 expression was found in most tumors and was associated with evidence for tumor cell proliferation.     

Correlation of DNA ploidy to tumor histologic grade, clinical variables, and survival in dogs with mast cell tumors.

By using flow cytometry, a retrospective analysis of the DNA content of 40 primary canine mast cell tumors and seven lymph nodes that contained metastatic mast cell tumor from 44 dogs of various breed, sex, and age was performed on formalin-fixed, paraffin-embedded samples of the tumors and nodes. These samples were chosen according to the following criteria: samples contained sufficient well-preserved tumor tissue in the paraffin block for processing, sufficient patient history data were available, clean and homogeneous cell suspensions were obtained after processing, and interpretable DNA histograms were produced on analysis. The ploidy data obtained were compared with the histopathologic grade, the anatomical site of occurrence, the clinical stage of the tumors, and the survival of the dogs. Over 70% (29/40) of the mast cell tumors were diploid. Three metastatic mast cell tumors in lymph nodes had the same ploidy status as their corresponding primary tumors. In five dogs, mast cell tumors from multiple sites in each dog displayed similar ploidy status. Of 26 dogs evaluated for survival times, 69% (18/26) had diploid tumors and 31% (8/26) had aneuploid tumors. When numbers of diploid versus aneuploid tumors were compared, no significant difference was found between any two grades, clinical stages, or anatomic sites. A significant difference (P = 0.02) was found, however, between aneuploid and diploid tumors when comparing Stage I and non-Stage I disease. The Kaplan-Meier survival plot indicated a tendency towards an increased survival within the first year in dogs with diploid versus aneuploid tumors (P = 0.06).     

Expression of the KIT protein (CD117) in primary cutaneous mast cell tumors of the dog.

Thirty-one canine cutaneous masses, diagnosed as mast cell tumors (MCT) by histopathologic analysis, were used to evaluate the immunohistochemical pattern of expression of KIT protein (CD117), a type III tyrosine kinase protein involved in mast cell growth and differentiation. Lesions were graded as I (well differentiated), II (intermediate differentiation), or III (poorly differentiated) according to the following morphologic features: invasiveness, cellularity and cellular morphology, mitotic index, and stromal reaction. Immunohistochemical KIT expression was compared with histologic grade and some histomorphologic features (cell differentiation and nuclear grade) evaluated separately. A possible predictive role of biologic behavior in MCTs for KIT expression was also investigated. Immunohistochemical analysis revealed three different patterns of KIT expression: a cytoplasmic diffuse pattern, a membranous pattern with immunostaining located on the cell surface, and a cytoplasmic perinuclear pattern, where KIT expression was detected in the cytoplasm of the neoplastic mast cells, close to the nucleus. Statistical analysis showed a close relationship between different KIT immunohistochemical patterns and histologic grade (P < 0.00000), cell differentiation (P < 0.00000), and nuclear grade (P < 0.0024). According to Kaplan-Meier-estimated survival curves compared by survival analysis, KIT expression was significantly associated with survival time (P = 0.037) but not cancer-free interval (P = 0.50). Similar to other well-known histomorphological features, KIT expression is a useful parameter of malignancy in cutaneous MCTs. KIT expression also predicted the biological behavior of the tumors in this study.     

Prognostic value of histologic stage and proliferative activity in canine malignant mammary tumors.

The aim of this study was to investigate the correlation between the histologic invasiveness (histologic stage) and various cell proliferation activity assays (quantity of argyrophil proteins associated with nucleolar organizer regions [AgNORs], mitotic activity, MIB1 [Ki67] immunohistochemical detection) for predicting the biologic behavior of malignant canine mammary tumors. Sixty specimens from malignant canine mammary tumors with no distant metastases (M0) at surgery were selected, and follow-up data were collected over a 2-year period. The histologic invasiveness was graded by histologic stage (stage 0 = tumors without stromal invasion; stage I = tumors with stromal invasion; stage II = tumors with neoplastic emboli in vessels), and the proliferative indices were expressed as MIB1 index (the percentage of nuclear area immunohistochemically stained by MIB1 antibody), mitotic index (the number of mitoses per 1,000 neoplastic cells), and AgNOR index (the ratio between mean AgNOR area of tumor cells and the mean AgNOR area of fibroblasts/lymphocytes). The measures of proliferative activity were compared among groups with different histologic stages, and the influence of different prognostic variables (histologic stage, AgNOR index, mitotic index, MIB1 index) on survival time was evaluated. A significant difference in the proliferation patterns was recorded between the different histologic stages for the mitotic index (P = 0.0006) and MIB1 index (0.0013). Among the different parameters considered, histologic stage (P < 0.05), AgNOR index (P = 0.0291), and MIB1 index (P = 0.014) revealed a significant association with prognosis in univariate analysis. AgNOR index for 1-year survival and histologic stage for 2-year survival were the most significant parameters influencing survival, as determined by multiple nonlinear logistic regression.     

Immunohistochemical and clinical evaluation of p53 in canine cutaneous mast cell tumors

One hundred twenty-six cutaneous mast cell tumors obtained by excisional biopsy from 106 dogs were evaluated using immunohistochemical staining for the presence of p53 protein. A standard avidin-biotin immunohistochemical protocol was used incorporating a polyclonal antibody of rabbit origin (CM-1) as the primary antibody. Histopathologic grading of tumors was performed on hemotoxylin and eosin-stained samples. There was a significant difference in the percentage of cells staining positive for p53 for the histopathologic grades (P = 0.0005). Grade III tumors had a significantly greater p53 content than did grade I or II tumors (P < 0.05). Clinical data obtained retrospectively was available for 54 dogs. Tumor recurred in 19 of 54 (35.2%) dogs. Twenty-nine dogs died by the end of the study; 9 of 29 (31.0%) died of mast cell tumor disease. Histopathologic grade showed a significant negative association with survival time. Both clinical stage and histopathologic grade showed a significant negative association with time to recurrence. The percentage of cells staining positive for p53 did not significantly improve the forward analysis. Immunohistochemical detection of p53 did not appear useful in characterizing the clinical association between cutaneous mast cell tumor cellular features and survival time or time to tumor recurrence in dogs.     

Pilot study utilizing Fluorine‐18 fluorodeoxyglucose‐positron emission tomography/computed tomography for glycolytic phenotyping of canine mast cell tumors

The goal of this prospective pilot study was to use naturally occurring canine mast cell tumors of various grades and stages as a model for attempting to determine how glucose uptake and markers of biologic behavior are correlated. It was hypothesized that enhanced glucose uptake, as measured by 2‐[fluorine‐18]fluoro‐d ‐glucose‐positron emission tomography/computed tomography (F18 FDG PET‐CT), would correlate with histologic grade. Dogs were recruited for this study from a population referred for treatment of cytologically or histologically confirmed mast cell tumors. Patients were staged utilizing standard of care methods (abdominal ultrasound and three view thoracic radiographs), followed by a whole body F18 FDG PET‐CT. Results of the F18 FDG PET‐CT were analyzed for possible metastasis and standard uptake value maximum (SUV_max) of identified lesions. Incisional or excisional biopsies of the accessible mast cell tumors were obtained and histology performed. Results were then analyzed to look for a possible correlation between the grade of mast cell tumors and SUV_max. A total of nine animals were included in the sample. Findings indicated that there was a correlation between grade of mast cell tumors and SUV_max as determined by F18 FDG PET‐CT (p‐value = 0.073, significance ≤ 0.1). Based on the limited power of this study, it is felt that further research to examine the relationship between glucose utilization and biologic aggressiveness in canine mast cell tumors is warranted. This study was unable to show that F18 FDG PET‐CT was a better staging tool than standard of care methods.     

The activity of matrix metalloproteinases (MMPS) and tissue inhibitors of metalloproteinases (TIMPs) in mammary tumors of dogs and rats

We conducted zymography for detecting the activity of matrix metalloproteinases (MMPs) and reverse zymography for the activity of tissue inhibitors of metalloproteinases (TIMPs) in canine spontaneous and rat 7, 12-dimethylbenz(a)anthracene (DMBA)-induced mammary tumor tissues. The activities of MMPs of canine mammary tumors were quite higher than those of the rat chemically induced tumors. The activities of MMPs were significantly higher in malignant tissues than in benign ones of canine tumors, whereas the activity of only MMP-2 was higher in both benign and malignant rat tumors compared to normal tissues. There were no differences of MMPs activities between benign and malignant rat tumors. The results of reverse zymography indicated that the activities of TIMP-1, -2 and -3 were strikingly higher in rat tumors than in canine tumors. The activities were higher in malignant tissues than in benign ones of dogs, and higher in tumor tissues than in normal mammary tissues of rats. The results of film in situ zymography for tissue localization of gelatinolytic activity showed that the digested area was more extended in malignant tumors than in benign ones of dogs. However, the area was similarly extended in both benign and malignant rat tumors. These results may indicate that the canine spontaneous malignant mammary tumors possess more aggressive nature than the rat chemically induced counterpart, resulting from the high level of MMPs and low level of TIMPs activities of the tumor tissues.     

Variation among pathologists in the histologic grading of canine cutaneous mast cell tumors with uniform use of a single grading reference.

Ten veterinary pathologists independently assigned histologic grades to the same 60 canine cutaneous mast cell tumors using the Patnaik classifications. The degree of agreement in grading among the pathologists was compared with the degree of agreement among the same pathologists in a previous study, in which each pathologist used the reference for grading that he/she uses routinely. Mean agreement improved significantly from 50.3% to 62.1% with uniform use of the Patnaik classifications (P = 0.00001), suggesting that there is value in uniform application of a single grading scheme for canine cutaneous mast cell tumors. Agreement among pathologists was still not 100%, suggesting that a more objective grading scheme should be developed and that other histologic indicators of prognosis should be investigated.     

Immunohistochemical assay for detecting estrogen receptors in canine mammary tumors.

OBJECTIVE: To determine the estrogen receptor (ER) content of canine mammary gland tumors by use of immunohistochemical (IHC) examination of formalin-fixed sections. SAMPLE POPULATION: 21 mammary gland tumors from 20 adult dogs. PROCEDURE: ER were detected in formalin-fixed tissues, using an avidin-biotin alkaline phosphatase IHC assay and were quantified on fresh-frozen tumor samples, using a modified dextran-coated charcoal (DCC) assay. RESULTS: 7 of 21 tumors had visually detectable nuclear ER by use of IHC staining, whereas 8 of 21 tumors were positive for ER by use of the DCC assay. The ER-positive cells in 5 IHC-positive tumors were epithelial cells with histologic criteria of early malignancy. The remaining 2 ER-positive tumors detected by use of IHC had ER-positive mast cells within areas of connective tissue around the tumor. CONCLUSIONS: Immunohistochemistry is an additional method for detection of ER in canine mammary tumors. The major advantage of this type of assay is that it may be performed on formalin-fixed tissues, and individual ER-positive cells may be identified. Discovery of ER-positive mast cells by use of IHC is of concern, particularly if the ER status of a tumor is based on DCC results alone. CLINICAL RELEVANCE: Because most canine mammary tumors are fixed in formalin prior to histologic evaluation, an IHC assay that identifies ER-positive cells is desirable. Adjunctive antiestrogen therapy could be administered to dogs with ER-positive tumors.     

Imatinib mesylate treatment in a dog with gastrointestinal stromal tumors with a c-kit mutation

A 13-year-old spayed mixed-breed dog was diagnosed with a gastrointestinal stromal tumor (GIST) after histopathological examination of an abdominal mass. Five months after surgical resection of the tumor, we detected the recurrence of GIST with multiple disseminated abdominal lesions. A sequence analysis of cDNA obtained from a biopsy of the recurrent tumors revealed a mutation within exon 9 of the c-kit gene (1523A>T, Asn⁵⁰⁸Ile), which has been shown to cause ligand-independent phosphorylation of the KIT protein in GISTs and canine mast cell tumors (MCTs). Upon detection of the recurrent tumors, we initiated treatment with imatinib mesylate (10 mg/kg, q 24 hr). After 2 months, the dog achieved complete remission. Our findings indicate that canine GIST, and possibly MCT, may be responsive to molecular-targeted therapy.