A New Sandwich ELISA for Quantification of Thymidine Kinase 1 Protein Levels in Sera from Dogs with Different Malignancies Can Aid in Disease Management.

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 (rs = 0.6, p<0.0001). Thus, the new TK1-ELISA has sufficient sensitivity and specificity for routine clinical use in veterinary oncology.

Properties of cellular and serum forms of thymidine kinase 1 (TK1) in dogs with acute lymphocytic leukemia (ALL) and canine mammary tumors (CMTs): implications for TK1 as a proliferation biomarker.

BACKGROUND: 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. RESULTS: Serum samples from 20 healthy dogs and 27 dogs with mammary tumors were analyzed for sTK1 activity, using an [(3)H]-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. CONCLUSIONS: 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.

Treatment efficacy and immune stimulation by AdCD40L gene therapy of spontaneous canine malignant melanoma.

Malignant melanoma is a serious disease in both humans and dogs, and the high metastatic potential results in poor prognosis for many patients. Its similarities with human melanoma make spontaneous canine melanoma an excellent model for comparative studies of novel therapies and tumor biology. We report a pilot study of local adenovector CD40L (AdCD40L) immunogene treatment in 19 cases of canine melanoma (14 oral, 4 cutaneous, and 1 conjunctival). Three patients were World Health Organization stage I, 2 were stage II, 10 stage III, and 4 stage IV. One to 6 intratumoral injections of AdCD40L were given every 7 days, followed by cytoreductive surgery in 9 cases and only immunotherapy in 10 cases. Tumor tissue was infiltrated with T and B lymphocytes after treatment, suggesting immune stimulation. The best overall response included 5 complete responses, 8 partial responses, and 4 stable and 2 progressive disease statuses according to the World Health Organization response criteria. Median survival was 160 days (range, 20-1141 d), with 3 dogs still alive at submission. Our results suggest that local AdCD40L therapy is safe and could have beneficial effects in dogs, supporting further treatment development. Clinical translation to human patients is in progress.

Homogeneous assay for real-time and simultaneous detection of thymidine kinase 1 and deoxycytidine kinase activities.

Measurement of thymidine kinase-1 (TK1) and deoxycytidine kinase (dCK) activity may be useful in cancer disease management. Therefore, a one-step homogeneous assay for real-time determination of TK1 and dCK was developed by combining enzyme complementation with fluorescent signal generation using primer extension and a quenched probe oligodeoxyribonucleotide system at 37 °C. Complementation, for producing dCTP and TTP from nucleoside substrates, was carried out by dTMP kinase and/or UMP/CMP kinase and nucleoside diphosphate kinase. dNTP was continuously incorporated into a fixed oligodeoxyribonucleotide primer, template, and probe system, and the fluorescent signal was generated by using the combined actions of primer extension and 5' exonuclease activity of Thermophilus aquaticus (Taq) DNA polymerase for specific relief of fluorescent quenching. Fluorescence was captured at 1-min intervals using a real-time polymerase chain reaction (PCR) instrument. A horizontal threshold line, crossing all sample relative fluorescent units (RFU) values at the level of the RFU of the blank sample at the end of the assay (i.e., 90 min), was drawn, obtaining RFU measurement data in minutes for each sample. Duplex proof of principle was demonstrated by the independent determination of different amounts of dCK and TK1 in combination. R(2) values of 0.90 were demonstrated with Prolifigen TK-REA U/L reference values obtained from pathological canine and human serum samples.

Serum thymidine kinase activity in clinically healthy and diseased cats: a potential biomarker for lymphoma.

The thymidine kinases are enzymes that convert deoxythymidine to deoxythymidine monophosphate and have a function in DNA synthesis. Rapidly proliferating cells will have higher levels of thymidine kinase. Serum thymidine kinase activity (sTK) is a useful tumour marker in humans and dogs, with utility as a prognostic indicator in lymphoma. In the current study serum samples were collected from 49 clinically healthy cats, 33 with lymphoma, 55 with inflammatory disease and 34 with non-haematopoietic neoplasia (NHPN). sTK was measured using a radioenzyme assay and a reference interval (1.96 × SD) was established from the clinically healthy cats (<5.5 U/l). Mean sTK activity for healthy cats was 2.2 U/l (range 0.8-8.4, ± SD 1.7). Mean sTK activity for cats with lymphoma was 17.5 U/l (range 1.0-100.0 SD ± 27.4). Mean sTK activity for cats with NHPN was 4.2 U/l (range 1.0-45.0, SD ± 8.6). Mean sTK activity for the inflammatory group was 3.4 U/l (range 1.0-19.6, SD 3.9). Cats with lymphoma had significantly higher sTK activity than healthy cats or cats with inflammatory disease (P <0.0001) and cats with NHPN (P <0.0002). sTK activity is a potentially useful biomarker for feline lymphoma and further study is required to assess its utility as a prognostic indicator.

Nonfatal suicide attempts and other self-inflicted harm. Beltrami County youths, 2002-2006.

Following a highly publicized murder-suicide by a high school student in Beltrami County, Minnesota, the Minnesota Department of Health initiated an investigation into suicide attempts and self-inflicted harm (SA/SIH) among youths in the county between 2002 and 2006. This article summarizes the results of that effort, which found an annualized rate of 356 hospital-treated SA/SIH per 100,000 population among Beltrami County residents ages 10 to 24 years--a rate more than 50% higher than that of other youths in Minnesota or the United States. In addition, the rate of SA/SIH for American Indian youths in Beltrami County was 2.5 times higher than that for white youths. An examination of medical history found 52% of the youths in this study had previously attempted suicide. This article also discusses several modifiable risk factors that were identified and potential interventions.