The investigation of some microenvironmental markers in canine mast cell tumors

Mast cells are sentinel cells reside in the tissues. Early differentiation of mast cell takes place from hematopoietic stem cells. The alternative and late differentiation of mast cells takes place from the monocyte granulocyte progenitor. Mast cells are found around blood vessels in many different tumors and also at the edges of tumors. Mast cells play proinflammatory and antitumorigenic role in the tumor microenvironment. After activation and degranulation, they recruit neutrophils, eosinophils and macrophages and the cells of acquired immune system to manage antitumoral role. However, mast cells also release angiogenic compounds that facilitate tumor vascularization and invasiveness. They also produce matrix metallopeptidase 9 (MMP-9) that degrade extracellular matrix and helps in the metastasis of tumor cells. Mast cells are the origin of mast cell tumors (MCTs). In animals, MCTs are found quite commonly in dogs, less commonly in cats and are rarely found in horses, cattle, goats and pigs, similar to that in humans. Canine MCTs are the most common tumors in domestic dogs accounting for almost 20% of all skin tumors in dogs. Location of MCTs is commonly cutaneous, less commonly subcutaneous and rarely extracutaneous. Although no live animals were included, ethics committee approval was obtained from Afyon Kocatepe University Animal Experiments Local Ethics Committee with the number AKUHADYEK-22-21 for the confirmation of this study. Sixty samples of MCTs from the archive of Department of Pathology were selected for this study. The MCT samples were taken from 33 female and 27 male dogs. Eighteen different dog breeds were presented in this study. Twenty-three Golden Retrievers, 5 Dogo Argentinos, 3 Labradors, 3 Pugs, 2 French Bulldogs, 2 Siberian Huskies, 2 Cane Corso, 2 Jack Russel Terrier, 2 Dachshund, 1 Samoyed, 1 German Shepherd, 1 American Pitbull Terrier, 1 Cocker Spaniel, English Pointer 1, Terrier 1, American Bulldog 1, Boxer 1 and 8 dogs of mix breeds were also included in this study. Tumor samples arrived at the laboratory in 10% buffered formalin solution. After the routine procedures of histopathological laboratory, the tissues were stained with hematoxylin and eosin (HE) for the diagnosis via grading, evaluation of necrosis and counting of mitotic figures (MC). For the evaluation of microenvironmental markers, the MCTs were stained with C-kit, Ki67, VEGF, OPN, Oct-3/4, and TNF-α markers by immunohistochemical method. The correlation between markers and other histopathological variables (grades of MCTs, presence of necrosis and MC) was also investigated with the use of Chi-square test. The mean age of dogs was 9.25 years for the development of canine cutaneous MCTs. The Golden Retriever and mixed breeds of dogs showed high incidence of cutaneous MCTs. All the tumors were diagnosed and graded according to the Patnaik and Kiupel grading systems for the cutaneous MCTs. According to the Patnaik system, 17 dogs were diagnosed as grade 1, 33 dogs as grade 2 and 10 dogs as grade 3 MCTs. The percentages of grades were as follows, grade 1 (28.33%), grade 2 (55%) and grade 3 (16.67%). After that, the classification of tumors was done according to the Kiupel grading system. All the grade 3 tumors were high grade tumors and all the grade 1 tumors were low grade tumors. Twenty-five tumors from the grade 2 MCTs were graded as low grade and 8 tumors were graded as high grade MCTs. According to the Kiupel grading system, 70% tumors were graded as low grade and 30% were graded as high-grade canine cutaneous MCTs. Then, the comparison of 3-tier and 2-tier grading system was done and it was found that 2-tier grading system has more inter-observer consistency for the diagnosis and prognostication of canine cutaneous MCTs. The percentage of clean margins of canine cutaneous MCTs was 70% and 20% MCTs showed dirty margins. Other 10% MCTs were not clear because of the incomplete information provided by the clinicians. The depth of all the tumors was checked to confirm the cutaneous MCTs. Tumors were present in three areas including epidermis, superficial dermis and deep dermis. Thirty-two (53.33%) MCTs revealed membranous immunopositivity, 22 (36.67%) MCTs revealed stippled cytoplasmic immunopositivity and 6 (10%) MCTs revealed diffused cytoplasmic immunopositivity via KIT antibody. The relation of KIT immunopositivity with the other histopathological variables was investigated with Chi-square test and significant (P<0.05) correlations were evaluated between KIT and grades of MCTs, mitotic count (MC) and the presence of necrosis in this study. Intensity of OPN, TNF-α, VEGF, Ki67, and Oct-3/4 antibody expressions was graded as nonspecific positivity (-), mild positivity (+), moderate positivity (++), diffuse positivity (+++). Eighteen MCTs showed (+), 17 MCTs showed (++) and 25 MCTs showed (+++) immunopositivity with OPN. Thirty-three MCTs showed (+), 17 MCTs showed (++) and 10 MCTs showed (+++) immunopositivity with TNF-α. Twenty-six MCTs showed (+), 22 MCTs showed (++) and 12 MCTs showed (+++) immunopositivity with VEGF. Twenty-eight MCTs showed (+), 16 MCTs showed (++) and 16 MCTs showed (+++) immunopositivity with Ki67. Eighteen MCTs showed (+), 16 MCTs showed (++) and 26 MCTs showed (+++) immunopositivity with Oct-3/4. The correlation between the markers (KIT, Ki67, VEGF, OPN, Oct-3/4 and TNF-α) and other histopathological variables (presence of necrosis and MC) was considered significant. A significant correlation was found between KIT and all other markers (Ki67, VEGF, OPN, Oct-3/4 and TNF-α). High expression of proliferation markers and expression of KIT pattern II and III were the indicators of less survival time in dogs. Tyrosine kinase inhibitors therapy was preferred in the MCTs showing expression of KIT pattern II and III. Margin evaluation plays important role in the diagnosis and prognostication of canine cutaneous MCTs. KIT, Ki67, and VEGF were found to be reliable markers for differentiation, diagnosis, prognostication, and selection of chemotherapy in canine cutaneous MCTs, as identified in previous studies and confirmed in our study. In our study, after evaluating the immunohistochemical and statistical results, it was concluded that OPN, Oct-3/4 and TNF-α, which we used for the first time in this study, may be good microenvironmental markers for the differentiation, diagnosis and prognostication of canine cutaneous MCTs.