Thymidine kinase 1 (TK1) is a cellular enzyme involved in DNA precursor synthesis, and its activity has been used as a proliferation marker for monitoring malignant diseases. Here, for the first time, we evaluated both TK1 activity and protein levels in sera from patients with different malignancies. Serum samples from patients with myelodysplastic syndrome (MDS, n = 22), breast cancer (n = 42), prostate cancer (n = 47) and blood donors (n = 30) were analyzed for TK1 protein and activity levels, using a serum TK1 (STK1) protein assay based on antibodies and an activity assay that measured [³H]-deoxythymidine (dThd) phosphorylation. The molecular forms of TK1 in sera from some of these patients were analyzed using size-exclusion chromatography. Mean STK1 activities in sera from MDS, breast and prostate cancer were 11 ± 17.5, 6.7 ± 19 and 1.8 ± 1.4 pmol/min/mL, differing significantly from blood donors (mean ± standard deviation (SD) = 1.1 ± 0.9 pmol/min/mL). Serum TK1 protein (25 kDa polypeptide) levels were also significantly higher in MDS, breast, prostate cancer compared to blood donors (mean ± SD = 19 ± 9, 22 ± 11, 20 ± 12, and 5 ± 3.5 ng/mL, respectively). The STK1 specific activities of sera from patients with MDS and blood donors were significantly higher when compared with activities in sera from breast and prostate cancer patients. Size-exclusion analysis of sera from breast and prostate cancer showed that the detected active TK1 was primarily a high molecular weight complex, similar to the forms found in sera from MDS patients and blood donors. However, Western blotting demonstrated high TK1 25 kDa protein levels in fractions lacking TK1 activity in sera from cases with breast and prostate cancer. These results demonstrate that there are differences in the specific activities and the subunit compositions of STK1 in hematological malignancies compared with breast and prostate cancer. This fact has several important implications for the use of STK1 as a tumor biomarker. One is that STK1 protein assays may differentiate early-stage tumor development in breast and prostate cancer more effectively than STK1 activity assays.

Thymidine kinase (TK1) is an enzyme involved in DNA synthesis that leaks into the blood as a result of high cell turnover, particularly in the case of cancer. Serum TK1 activity has been used for prognosis and monitoring of leukemia and lymphoma patients for many years. Here, we describe the first clinical results with the newly developed TK 210 ELISA from AroCell AB. Sera from 124 breast cancer patients with known TNM classification along with sera from 53 healthy females were analyzed by TK 210 ELISA for TK1 protein and TK1 activity levels by the ³[H]-deoxythymidine (dThd) phosphorylation assay. The limit of detection for the TK 210 ELISA was 0.17 ng/ml, and 60 % of the sera from female blood donors were below this value. The median TK1 levels found in sera from breast cancer patients with T1 to T4 stage disease were 0.31, 0.46, 0.47, and 0.55 ng/ml, and these levels significantly differed from healthy controls. The median values of the biomarker CA 15-3 were also increased in patient sera from T1 to T4 patients (16, 34, 36, 40 U/ml, respectively). TK 210 ELISA showed significantly higher sensitivity for the T1 and T2 breast cancer patients compared to the TK activity assay. The combination of the TK1 ELISA and CA 15-3 biomarkers demonstrated a significant increase in sensitivity up to 15 % compared to each marker alone. This evaluation of the TK 210 ELISA strongly suggests that it can provide independent and complementary information for patients with breast cancer.

Background: The early detection of ovarian cancer is presently not effective, and it is crucial to establish biomarkers for the early diagnosis of ovarian cancer to improve the survival of patients. Materials and methods: The aim of this study was to investigate the role of thymidine kinase 1 (TK1) in combination with CA 125 or HE4 to serve as a potential diagnostic biomarkers for ovarian cancer. In this study, a set of 198 serum samples consisting of 134 ovarian tumor patients and 64 healthy age-matched controls were analyzed. The TK1 protein levels in serum samples were determined using the AroCell TK 210 ELISA. Results: A combination of TK1 protein with CA 125 or HE4 showed better performance than either of them alone in the differentiation of early stage ovarian cancer from the healthy control group, but also a significantly better performance than the ROMA index. However, this was not observed using a TK1 activity test in combination with the other markers. Furthermore, the combination of TK1 protein and CA 125 or HE4 could differentiate early stage disease (stage I, II) more efficiently from advanced-stage (stage III, IV) disease (p < 0.0001). Conclusions: The combination of TK1 protein with CA 125 or HE4 increased the potential of detecting ovarian cancer at early stages.

Thymidine kinase 1 (TK1) is a salvage enzyme involved in DNA precursor synthesis, and its expression is proliferation dependent. A serum form of TK1 has been used as a biomarker in human medicine for many years and more recently to monitor canine lymphoma. Canine TK1 has not been cloned and studied. Therefore, dog and human TK1 cDNA were cloned and expressed, and the recombinant enzymes characterized. The serum and cellular forms of canine and human TK1 were studied by size-exclusion chromatography and the level of TK1 protein was determined using polyclonal and monoclonal anti-TK1 antibodies.

Thymidine kinase 1 (TK1) is a deoxyribonucleic acid (DNA) precursor enzyme and a proliferation biomarker used for prognosis and treatment monitoring of breast cancer in humans. The aim was to determine if serum thymidine kinase 1 (sTK1) activity and sTK1 protein levels in dogs with mammary tumors could be useful in veterinary medicine. Serum samples from 20 healthy dogs and 27 dogs with mammary tumors were analyzed for sTK1 activity, using an [3H]-deoxythymidine (dThd) phosphorylation assay, and for sTK1 protein levels by immune affinity/Western blot assay. The molecular forms of sTK1 in acute lymphocytic leukemia (ALL), canine mammary tumor (CMT), and healthy sera were determined by size exclusion chromatography. Mean sTK1 activities in CMT were 1.0 ± 0.36 pmol/min/mL, differing significantly from healthy dogs (mean ± SD = 0.73 ± 0.26 pmol/min/mL). Serum TK1 protein (26 kDa polypeptide) levels were also significantly higher in CMTs compared to healthy dogs (mean ± SD = 28.5 ± 11.4, and 8.5 ± 4 ng/mL, respectively). Cellular TK1 isolated from ALL tumor cells was predominantly a dimer, while the serum TK1 activity eluted as a high molecular weight (MW) oligomer. In analyses of CMT tissue extracts, TK1 activity eluted in two peaks, a minor peak with a high MW oligomer and a major tetramer peak. Western blot analysis of chromatographic fractions showed that cellular TK1 protein in both ALL and CMT dogs, and to some extent serum TK1 from ALL dogs, correlated with activity profiles, but a large fraction of inactive TK1 protein was detected in CMT. Serum TK1 protein and activity levels were significantly higher in CMT than in healthy dogs. Size exclusion chromatography demonstrated major differences in the molecular forms of sTK1 in ALL, healthy, and CMT dogs, with a large fraction of inactive TK1 protein in CMT. Our results showed that the sTK1 protein assay can differentiate benign tumors (early stage tumors) from healthy more efficiently than sTK1 activity assay. This preliminary data supports that sTK1 protein assay is clinically useful. Further studies are needed to evaluate the diagnostic or prognostic role of serum TK1 protein in CMTs.

Thymidine kinase 1 (TK1) is a DNA precursor enzyme whose expression is closely correlated with cell proliferation and cell turnover. Sensitive serum TK1 activity assays have been used for monitoring and prognosis of hematological malignancies in both humans and dogs. Here we describe the development of a specific sandwich TK1-ELISA for the quantification of TK1 protein levels in sera from dogs with different malignancies. A combination of rabbit polyclonal anti-dog TK1 antibody and a mouse monoclonal anti-human TK1 antibody was used. Different concentrations of recombinant canine TK1 was used as standard. Clinical evaluation of the ELISA was done by using sera from 42 healthy dogs, 43 dogs with hematological tumors and 55 with solid tumors. An established [³H]-dThd phosphorylation assay was used to determine the TK1 activity levels in the same sera. The mean TK1 activities in dogs with hematological tumors were significantly higher than those found in healthy dogs. In agreement with earlier studies, no significant difference was observed in serum TK1 activities between healthy dogs and dogs with solid tumors. However, the mean TK1 protein levels determined by new TK1-ELISA were significantly higher not only in hematological tumors but also in solid tumors compared to healthy dogs (mean ± SD = 1.30 ± 1.16, 0.67 ± 0.55 and 0.27± 0.10 ng/mL, respectively). Moreover, TK1-ELISA had significantly higher ability to distinguish lymphoma cases from healthy based on receiver operating characteristic analyses (area under the curve, AUC, of 0.96) to that of the activity assay (AUC, 0.84). Furthermore, fluctuations in TK1 protein levels during the course of chemotherapy in dogs with lymphoma closely associated with clinical outcome. Overall, the TK1-ELISA showed significant linear correlation with the TK1 activity assay (r_s = 0.6, p<0.0001). Thus, the new TK1-ELISA has sufficient sensitivity and specificity for routine clinical use in veterinary oncology.