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.Electronic supplementary materialThe online version of this article (doi:10.1007/s13277-016-5024-z) contains supplementary material, which is available to authorized users.

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Background: Thymidine Kinase 1 (TK1) is a cytosolic enzyme that plays an important role in DNA precursor synthesis and TK1 enzyme activity in serum is an established biomarker for prognosis and treatment monitoring, particularly for haematological malignancies. The AroCell TK 210 ELISA determines TK1 protein levels, in contrast to TK1 activity, and it has demonstrated superior sensitivity in distinguishing between subjects with solid tumours and controls. Pro PSA, free PSA and the prostate health index (PHI) have been proposed as more discriminating tests than PSA in distinguishing between benign and cancerous prostate changes. The purpose of this study was to compare the AroCell TK 210 ELISA with PSA, free PSA, pro PSA and PHI in men with pre-cancerous conditions, and patients with confirmed prostate cancer. Experimental Procedures: Serum samples from 94 patients with known PSA values (in the range 2 to 10 µg/L) that were under investigation for prostate cancer were collected at the University Medical Centre, Ljubljana. TK1 protein levels were determined using the AroCell TK 210 ELISA. PSA, fPSA and pro PSA levels were analysed with commercial assays (Hybritech PSA, Hybritech Free PSA and Access 2pPSA - Beckman Coulter USA) analysed on the Access 2 Beckman Coulter analyzer. Results: Overall, 16/94 patients had serum TK1 protein levels above the estimated cut-off value for age-matched healthy men (0.45µg/L). 65/94 men had pre-cancerous prostate conditions, including benign prostate hyperplasia, prostatitis and high grade prostatic intraepithelial neoplasia, while 29 patients had confirmed prostate cancer (PCa). Only 7 /65 in the pre-cancerous group (11%) had TK1 protein levels above the cut-off, whereas in the prostate cancer group 9 out of 29 (31%) were positive. Moreover, the TK1 protein values in the PCa group (mean=0.41µg/L) differed significantly from those with pre-cancerous conditions (mean=0.31µg/L, P=0.01). Further analysis showed that pro PSA and PHI also differentiated the prostate cancer group from those with pre-cancerous conditions (P= 0.01 and P<0.001, respectively). This was not observed for fPSA and PSA. Overall, the AroCell TK 210 ELISA showed significant correlation with PHI (r=0.32, P=0.0017) and pro PSA (r=0.21 P=0.044) but not with fPSA or PSA. Conclusions: This preliminary study has demonstrated that serum TK1 assayed with the AroCell TK 210 ELISA can differentiate between pre-cancerous and prostate cancer patients with a similar accuracy to that of pro PSA. Further clinical studies will establish the capacity of the AroCell TK 210 ELISA to complement pro PSA and PHI in distinguishing between pre-cancerous and prostate cancer, potentially providing another tool in prostate cancer management. Citation Format: Jagarlamudi Kiran Kumar, Staffan Eriksson, Zupan Moca, Kumer Kristina, Josko Osredkar, Fabjan Teja, Hlebic Gregor, T Smrkolj. The AroCell TK 210 ELISA may complement Pro PSA and the Prostate Health Index in differentiating pre cancerous and cancerous conditions in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 715. doi:10.1158/1538-7445.AM2017-715

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TK activity measurements have been used for many years to monitor cancer disease activity. However, what the blood concentrations of TK1 protein reflect is still uncertain. A TK1 immunoassay, the AroCell TK 210 ELISA, has been developed based on specific monoclonal antibodies against the C-terminal region of TK1. We utilized this assay to monitor the blood concentrations of TK1 in a cohort of patients with Hodgkin lymphoma (HL) before and after conventional treatment. TK1 concentrations in serum or plasma were measured using the AroCell TK 210 ELISA. For comparison, a large number of other biomarkers and cells were measured in blood such as C-reactive protein (CRP), Lactate dehydrogenase (LDH), hemoglobin, WBC, platelet counts, erythrocyte sedimentation rates (ESR), albumin, liver enzymes, creatinine. Fifty-eight patients with HL were included before start of treatment. Seventy three percent had the nodular sclerosis subtype and 52% stages I-II and 48% stages III-IV disease. Blood was sampled before treatment, during treatment and twice after completion of treatment. The upper normal TK1 concentration of healthy subjects (n=250) was 0.45 μg/L with a median of 0.25 μg/L (range 0.15-0.66 μg/L) as compared to HL, which was 0.26 μg/L (range 0.03-17 μg/L). The differences in ranges were significant (p<0.0001, F-statistics). Nodular sclerosis patients had higher TK1 levels as compared to the other subtypes (p=0.03). TK1 increased after treatment (p=0.001) in patients with normal concentrations from start i.e. <0.45 μg/L, whereas the concentrations decreased in 5/6 patients with TK1 >0.45 μg/L. TK1 concentrations before start of treatment was correlated to high CRP (p=0.01), low Hemoglobin (p=0.03), high WBC (p<0.001) and high platelet counts (p<0.01), but not to albumin or ESR or any other of the measured biomarkers. In a multivariate regression analysis, TK1 was correlated to LDH as the only independent variable (p<0.0001). In Hodgkin lymphoma patients, serum or plasma concentrations of TK1 may reflect malignant cell death and cell disruption rather than cell proliferation during treatment. Our results and hypothesis may help to interpret TK1 protein measurements as clinical tools in cancer management. Citation Format: Jagarlamudi Kiran Kumar, Staffan Eriksson, Johan Mattsson Ulfstedt, Per Venge, Daniel Molin. The thymidine kinase 1 (TK1) protein concentration in sera from Hodgkin lymphoma patients: A marker for cell death and disruption of malignant cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2618.