Immunohistochemical detection of thymidine kinase 1 in canine mammary tumors and lymphomas

Abstract: The dog has often served as a model animal for humans in scientific studies. This is due to the fact that dogs are susceptible to a wide range of diseases which also affect humans. One example is cancer, a condition that affects dogs as well as owners. Neoplastic diseases account for 18-23 % of all deaths in dogs (Bonnet et al., 2005 and Jagielski et al., 2002). Mammary tumor, or tumor of the breast, is the most common tumor form in intact dogs and women (Im et al., 2013). Mammary tumors originate in the udder, and are classified according to their histological characteristics. The dog has five pairs of mammary glands, and tumors are most commonly found in the most cranial and caudal of the glands. Lymphoma, or lymphosarcoma, is defined as a malignant tumor form originating from lymphocytes. It is the most common hematopoietic tumor in dogs. The incidence of lymphoma may be even higher in dogs than in humans (Jagielski et al., 2002). Thymidine kinase 1 (TK1) is a cytosolic enzyme expressed during the S-phase of the cell cycle. It catalyzes the phosphorylation of deoxythymidine into deoxythymidine monophosphate (dTMP). dTMP is then phosphorylated into deoxythymidine triphosphate (dTTP), which can be used as a substrate in the salvage pathway synthesis of DNA. The availability of dTTP is the rate limiting step in the DNA synthesis. Measurement of the activity of thymidine kinase in serum has been utilized to determine prognosis and result of treatment in animals and humans with hematopoietic malignancies. However, some patients with solid tumors do not express elevated levels of thymidine kinase in serum, and this has been a problem in past studies focused on serum assays. The aim of this study is to investigate the expression of TK1 in tissue samples of lymphomas and mammary tumors immunohistochemically. Immunohistochemical detection of the proliferation marker TK1 may theoretically aid routine examination of histological samples postsurgically. Analysis of TK1 in tissue samples may be of use to establish minimal residual disease (MRD), and an elevated expression of TK1 may be indicative of a non-successful surgical treatment result. 20 mammary tumors (40% benign lesions) and 17 lymphoma cases were analyzed immunohistochemically at the National Veterinary Institute in Uppsala, Sweden. Out of those 17 lymphoma cases, 9 were of B-cell origin and 8 were of T-cell origin. 4 samples of normal lymph node tissue and 3 samples of normal udder tissue were also analyzed. The tissue samples used in this study had been previously fixed in formalin and were available in paraffinized sections. Tissue sections of 2-3 μm were deparaffinized and labeled with an anti-TK1 antibody (XPA 161, clone 528-2, Arocell). A labelled streptavidin-biotin system was used for detection (LSABTM, Dako) and 3,3´-diaminobenzidine (DAB) was used as chromogen. An appreciation of the percentage of positive cells was made manually by light microscopy and a mitotic index simultaneously counted. The expression of thymidine kinase was plotted against the mitotic index to see if a correlation existed. The chosen method did not render positive results in mammary tumor cells. This was due to dissatisfactory signal intensity and background staining. Some staining was apparent in capsular lymphocytic infiltrations and in the stroma of two tumors. The median percentage of positive cells in lymphoma samples was 0,8% for B-cell lymphomas and 1,8% for T-cell lymphomas. The median value in normal lymph nodes was 1,3%. The expression of thymidine kinase was not correlated to mitotic index, and could not be statistically validated due to insufficient amount of samples. In conclusion, there are indications that this method could be of use on tissue samples from patients with lymphoma. If the method is refined, detection of thymidine kinase may be of value to detect minimal residual disease. However, future studies will require trials of new antibodies and perhaps the use of an alkaline phosphatase based system. It would also be advisable to work with live patients, in order to analyze serum samples simultaneously.