Clinical Significance of the TK1-Specific Activity in the Early Detection of Ovarian Cancer.

INTRODUCTION: Ovarian cancer is the eighth most common cause of cancer death in women. One of the major concerns is almost two-thirds of cases are typically diagnosed in the late stage as the symptoms are unspecific in the early stage of ovarian cancer. It is known that the combination of TK1 protein with CA 125 or HE4 showed better performance than either of them alone. That is why, the aim of the study was to investigate whether the TK1-specific activity (TK1 SA) could function as a complement marker for early-stage diagnosis of ovarian cancer. METHODS: The study included a set of 198 sera consisting of 134 patients with ovarian tumors (72 benign and 62 malignant) and 64 healthy age-matched controls. The TK1 SA was determined using TK1 activity by TK-Liaison and TK1 protein by AroCell TK 210 ELISA. Further, CA 125, HE4, as well as risk of ovarian malignancy algorithm index were also determined in the same set of clinical samples. RESULTS: The TK1 SA was significantly different between healthy compared to ovarian cancer patients (p < 0.0001). Strikingly, TK1 SA has higher sensitivity (55%) compared to other biomarkers in the detection of benign ovarian tumors. Further, the highest sensitivity was achieved by the combination of TK1 SA with CA 125 and HE4 for the detection of benign tumors as well as malignant ovarian tumors (72.2% and 88.7%). In addition, TK1 SA could significantly differentiate FIGO stage I/II from stage III/IV malignancies (p = 0.026). Follow-up of patients after surgery and chemotherapy showed a significant difference compared to TK1 SA at the time of diagnosis. CONCLUSIONS: These results indicate that TK1 SA is a promising blood-based biomarker that could complement CA 125 and HE4 for the detection of early stages of ovarian cancer.

Strand displacement-triggered FRET nanoprobe tracking TK1 mRNA in living cells for ratiometric fluorimetry of nucleic acid biomarker.

A precisely designed dual-color biosensor has realized a visual assessment of thymidine kinase 1 (TK1) mRNA in both living cells and cell lysates. The oligonucleotide probe is constructed by hybridizing the antisense strand of the target and two recognition sequences, in which FAM serves as the donor and TAMRA as the acceptor. Once interacting with the target, two recognition strands are replaced, and then the antisense complementary sequence forms a more stable double-stranded structure. Due to the increasing spatial distance between two dyes, the FRET is attenuated, leading to a rapid recovery of FAM fluorescence and a reduction of TAMRA fluorescence. A discernible color response from orange to green could be observed by the naked eye, with a limit of detection (LOD) of 0.38 nM and 5.22 nM for spectrometer- and smartphone-based assays, respectively. The proposed ratiometric method transcends previous reports in its capacities in visualizing TK1 expression toward reliable nucleic acid biomarker analysis, which might establish a general strategy for ratiometric biosensing via strand displacement.

Transcription factor Dp-1 knockdown downregulates thymidine kinase 1 expression to protect against proliferation and epithelial-mesenchymal transition in cervical cancer.

Thymidine kinase 1 (TK1) level is an independent survival prognostic factor for both premalignant and malignant cervical pathologies. Herein, this study sought to probe the impacts of TK1 on cervical cancer (CC) progression and its underlying mechanism. Transcription factor Dp-1 (TFDP1) and TK1 expression was assessed using qRT-PCR in CC cell lines. After ectopic expression and knockdown experiments, cell counting kit-8 and colony formation assays were adopted to measure cell proliferation, western blot to examine the expression of epithelial-mesenchymal transition (EMT)-related proteins, and Transwell assays to assess cell invasion and migration. The binding of TFDP1 to TK1 was predicted by bioinformatic sites and verified by chromatin immunoprecipitation and dual-luciferase reporter assays. Tumor xenograft experiments in nude mice were performed to validate the influence of TFDP1/TK1 on CC progression in vivo. CC cells had high TK1 and TFDP1 expression. TFDP1 or TK1 knockdown restrained CC cell EMT, invasion, migration, and proliferation. TFDP1 facilitated TK1 expression in CC via transcription. Overexpression of TK1 counteracted the suppressive impacts of TFDP1 knockdown on CC cell malignant behaviors. Moreover, TFDP1 knockdown depressed CC growth in vivo by downregulating TK1. TFDP1 knockdown restricted proliferation and EMT in CC by downregulating TK1 expression.

Prediction of Overall Survival by Thymidine Kinase 1 Combined with Prostate-Specific Antigen in Men with Prostate Cancer.

Thymidine kinase 1 (TK1) is an intracellular enzyme involved in DNA-precursor synthesis. Increased serum TK1 levels are used as a biomarker in various malignancies. We combined serum TK1 with PSA and evaluated its capacity to predict overall survival (OS) in 175 men with prostate cancer (PCa), detected by screening in 1988-1989 (n = 52) and during follow-up (median 22.6 years) (n = 123). TK1 was measured in frozen serum, age was stratified into four groups, and dates of PCa diagnosis and dates of death were obtained from Swedish population-based registries. The median concentration of TK1 and PSA was 0.25 and 3.8 ng/ml. TK1 was an independent variable of OS. In the multivariate analysis, PSA was not statistically significant in combination with age whereas the significance remained for TK1 + PSA. Measured once, TK1 + PSA predicted a difference of up to 10 years (depending on patient subgroup) in OS at a median of 9 years before PCa diagnosis. The TK1 concentration in 193 controls without malignancies did not differ from that of the PCa patients, hence TK1 was likely not released from incidental PCa. Thus, TK1 in the blood circulation may indicate the release of TK1 from sources other than cancers, nonetheless associated with OS.

Inhibitors of Anti-apoptotic Bcl-2 Family Proteins Exhibit Potent and Broad-Spectrum Anti-mammarenavirus Activity via Cell Cycle Arrest at G0/G1 Phase.

Targeting host factors is a promising strategy to develop broad-spectrum antiviral drugs. Drugs targeting anti-apoptotic Bcl-2 family proteins that were originally developed as tumor suppressors have been reported to inhibit multiplication of different types of viruses. However, the mechanisms whereby Bcl-2 inhibitors exert their antiviral activity remain poorly understood. In this study, we have investigated the mechanisms by which obatoclax (OLX) and ABT-737 Bcl-2 inhibitors exhibited a potent antiviral activity against the mammarenavirus lymphocytic choriomeningitis virus (LCMV). OLX and ABT-737 potent anti-LCMV activity was not associated with their proapoptotic properties but rather with their ability to induce cell arrest at the G0/G1 phase. OLX- and ABT-737-mediated inhibition of Bcl-2 correlated with reduced expression levels of thymidine kinase 1 (TK1), cyclin A2 (CCNA2), and cyclin B1 (CCNB1) cell cycle regulators. In addition, small interfering RNA (siRNA)-mediated knockdown of TK1, CCNA2, and CCNB1 resulted in reduced levels of LCMV multiplication. The antiviral activity exerted by Bcl-2 inhibitors correlated with reduced levels of viral RNA synthesis at early times of infection. Importantly, ABT-737 exhibited moderate efficacy in a mouse model of LCMV infection, and Bcl-2 inhibitors displayed broad-spectrum antiviral activities against different mammarenaviruses and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Our results suggest that Bcl-2 inhibitors, actively being explored as anticancer therapeutics, might be repositioned as broad-spectrum antivirals. IMPORTANCE Antiapoptotic Bcl-2 inhibitors have been shown to exert potent antiviral activities against various types of viruses via mechanisms that are currently poorly understood. This study has revealed that Bcl-2 inhibitors' mediation of cell cycle arrest at the G0/G1 phase, rather than their proapoptotic activity, plays a critical role in blocking mammarenavirus multiplication in cultured cells. In addition, we show that Bcl-2 inhibitor ABT-737 exhibited moderate antimammarenavirus activity in vivo and that Bcl-2 inhibitors displayed broad-spectrum antiviral activities against different mammarenaviruses and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Our results suggest that Bcl-2 inhibitors, actively being explored as anticancer therapeutics, might be repositioned as broad-spectrum antivirals.

Feline thymidine kinase 1: molecular characterization and evaluation of its serum form as a diagnostic biomarker.

BACKGROUND: Thymidine kinase 1 (TK1) catalyzes the initial phosphorylation of thymidine in the salvage pathway synthesis of dTTP, an essential building block of DNA. TK1 is a cytosolic enzyme with its highest level during the S-phase of the cell cycle. In cancer cells TK1 is upregulated and excess TK1 is leaked into the blood. Therefore, serum TK1 has been used as biomarker for cancer diagnosis and prognosis in human medicine. Feline TK1 shows high sequence similarity to TK1 from other species. The aim of this study was to characterize feline TK1 and evaluate if serum TK1 can be used as a diagnostic biomarker. RESULTS: Feline TK1 was cloned, expressed and affinity purified. The purified feline TK1 phosphorylated not only pyrimidine deoxyribonucleosides but also pyrimidine ribonucleosides and to some extent purine deoxynucleosides. A number of anticancer and antiviral nucleoside analogs also served as substrates with fairly high efficiency. ATP and dATP were the preferred phosphate donor. Serum TK1 activity in felines with malignant diseases was significantly higher than that in healthy individuals. ROC analysis revealed an area under the curve (AUC) of 0.98 with a sensitivity of 0.83 and a specificity of 0.95 for felines with lymphoma. Serum TK1 activity in felines with IBD or inflammatory disease was within the same range as healthy ones. Furthermore, in felines with lymphoma serum TK1 activity returned to normal levels in response to treatment. CONCLUSION: Feline TK1 has high specific activity and a broader substrate specificity in comparison with TK1 from other species. Serum TK1 activity in felines with malignant diseases is significantly higher than that in normal felines and in felines with inflammatory diseases. These results suggest that serum TK1 may be a promising biomarker for the diagnosis and monitoring of malignant diseases and for the differential diagnosis of certain inflammatory disease.

Plasma thymidine kinase 1 activity and outcome of ER+ HER2- metastatic breast cancer patients treated with palbociclib and endocrine therapy.

PURPOSE: Previous cohort studies have reported plasma TK1 activity (pTKa) as a potential prognostic biomarker in estrogen receptor-positive (ER+) HER2-negative (HER2-) metastatic breast cancer (MBC). In this prospective study, we report here the prognostic impact of pTKa in ER+/HER2- MBC patients treated with endocrine therapy and CDK4/6 inhibitor. EXPERIMENTAL DESIGN: Patients were included into the prospective, ethics committee-approved ALCINA study (NCT02866149). Eligibility criteria were patients with ER+/HER2- MBC treated at Institut Curie with endocrine therapy and palbociclib. Plasma samples were obtained at baseline and after 4 weeks of treatment. pTKa was quantified by the DiviTum® assay (Biovica, Sweden). RESULTS: From May 2016 to August 2018, 103 patients treated with endocrine therapy and palbociclib were included. Patients had received a median of two prior systemic therapies for MBC (range 0-14). Median follow-up was 13.8 months (range 6-31), with median PFS and OS of 9.6 months (95%CI [7.0-11.3]) and 28 months (95%CI [23-not reached]), respectively. Median baseline pTKa was 292 Du/L (range 20-27,312 Du/L, IQR [89-853]). After adjusting for other prognostic factors, baseline pTKa remained an independent prognostic factor for both PFS (HR = 1.3 95%CI [1.1-1.4], p = 0.0005) and OS (HR = 1.3 95%CI [1.2-1.6], p < 0.0001), and 4-week pTKa was associated with OS (HR = 1.6 95%CI [1.3-2], p < 0.0001). That survival prediction was significantly improved by the addition of baseline pTKa to clinicopathological characteristics. Adding pTKa changes at 4 weeks to baseline pTKa did not further increase survival prediction. CONCLUSION: This study demonstrates the clinical validity of pTKa as a new circulating prognostic marker in ER+/HER2- MBC patients treated with endocrine therapy and palbociclib.

Novel monoclonal antibodies against thymidine kinase 1 and their potential use for the immunotargeting of lung, breast and colon cancer cells.

BACKGROUND: Thymidine kinase 1 (TK1) is a pyrimidine salvage pathway enzyme that is up-regulated in malignant tissues and elevated in the serum of cancer patients. While TK1 has been well established as a tumor biomarker, little has been done to explore its potential as a tumor target. Recently, we reported the membrane expression of TK1 on malignant cells, but not on normal cells. This study explores the possible use of monoclonal antibodies for the targeting of membrane associated TK1 in lung, breast, colon and prostate cancer cells. METHODS: We generated and evaluated a panel of monoclonal antibodies against six different epitopes exposed in the tetrameric form of TK1. Antibodies were developed with hybridoma technology and validated with Western blot, siRNA TK1 knockdown, enzyme-linked immunosorbent assay (ELISA) and flow cytometry. The therapeutic potential of the antibodies was evaluated in vitro in antibody-dependent cell-mediated-cytotoxicity (ADCC) experiments. RESULTS: Binding of the antibodies to TK1 was confirmed by Western blot in purified recombinant protein, cancer serum, and cell lysate. After a TK1 knockdown was performed, a reduction of TK1 expression was observed with five antibodies. Using indirect ELISA, we identified 3B2E11, 9C10, 7H2, 3B4, 8G2 among the most sensitive antibodies (LOD = 10.73-66.9 pg/ml). Surface expression of TK1 on the membrane of various cancer cell lines was analyzed with flow cytometry. Antibodies 8G2, 3B4, 7HD and 5F7G11 detected TK1 on the membrane of various cancer cell lines, including lung, prostate, colon and breast. No significant binding was detected on normal lymphocytes. Increased cytolysis of lung (~ 70%. p = 0.0001), breast (~ 70%, p = 0.0461) and colon (~ 50% p = 0.0216) cancer cells by effector cells was observed when anti-TK1 antibodies were added during ADCC experiments. CONCLUSIONS: The antibodies developed showed potential to be used to detect and target TK1 on the membrane of various tumor cells. The targeting of TK1 in malignant cells using monoclonal antibodies may be a feasible approach for the elimination of high TK1 expressing tumor cells.

In vivo imaging of cell proliferation in meningioma using 3'-deoxy-3'-[18F]fluorothymidine PET/MRI.

PURPOSE: Positron emission tomography (PET) with 3'-deoxy-3'-[18F]fluorothymidine ([18F]FLT) provides a noninvasive assessment of tumour proliferation in vivo and could be a valuable imaging modality for assessing malignancy in meningiomas. We investigated a range of static and dynamic [18F]FLT metrics by correlating the findings with cellular biomarkers of proliferation and angiogenesis. METHODS: Seventeen prospectively recruited adult patients with intracranial meningiomas underwent a 60-min dynamic [18F]FLT PET following surgery. Maximum and mean standardized uptake values (SUVmax, SUVmean) with and without normalization to healthy brain tissue and blood radioactivity obtained from 40 to 60 min summed dynamic images (PET40-60) and ~ 60-min blood samples were calculated. Kinetic modelling using a two-tissue reversible compartmental model with a fractioned blood volume (VB) was performed to determine the total distribution volume (VT). Expressions of proliferation and angiogenesis with key parameters including Ki-67 index, phosphohistone-H3 (phh3), MKI67, thymidine kinase 1 (TK1), proliferating cell nuclear antigen (PCNA), Kirsten RAt Sarcoma viral oncogene homolog (KRAS), TIMP metallopeptidase inhibitor 3 (TIMP3), and vascular endothelial growth factor A (VEGFA) were determined by immunohistochemistry and/or quantitative polymerase chain reaction. RESULTS: Immunohistochemistry revealed 13 World Health Organization (WHO) grade I and four WHO grade II meningiomas. SUVmax and SUVmean normalized to blood radioactivity from PET40-60 and blood sampling, and VT were able to significantly differentiate between WHO grades with the best results for maximum and mean tumour-to-whole-blood ratios (sensitivity 100%, specificity 94-95%, accuracy 99%; P = 0.003). Static [18F]FLT metrics were significantly correlated with proliferative biomarkers, especially Ki-67 index, phh3, and TK1, while no correlations were found with VEGFA or VB. Using Ki-67 index with a threshold > 4%, the majority of [18F]FLT metrics showed a high ability to identify aggressive meningiomas with SUVmean demonstrating the best performance (sensitivity 80%, specificity 81%, accuracy 80%; P = 0.024). CONCLUSION: [18F]FLT PET could be a useful imaging modality for assessing cellular proliferation in meningiomas.

Early prediction of pathologic response to neoadjuvant treatment of breast cancer: use of a cell-loss metric based on serum thymidine kinase 1 and tumour volume.

BACKGROUND: After neoadjuvant chemotherapy of breast cancer pathologic complete response (pCR) indicates a favorable prognosis. Among non-selected patients, pCR is, however, achieved in only 10-30%. Early evaluation of tumour response to treatment would facilitate individualized therapy, with ineffective chemotherapy interrupted or changed. The methodology for this purpose is still limited. Tumour imaging and analysis of macromolecules, released from disrupted tumour cells, are principal alternatives. OBJECTIVE: To investigate whether a metric of cell-loss, defined as the ratio between serum concentration of thymidine kinase1 (sTK1, ng x ml- 1) and tumour volume, can be used for early prediction of pathologic response. METHODS: One hunred four women with localized breast cancer received neoadjuvant epirubicin/docetaxel in 6 cycles, supplemented with bevacizumab in cycles 3-6. The cell-loss metric was established at baseline (n = 104), 48 h after cycle 2 (n = 104) and prior to cycle 2 (n = 57). The performance of the metric was evaluated by association with pathologic tumour response at surgery 4 months later. RESULTS: Treatment caused a rise in sTK1, a reduction in tumour volume and a marked increase in the cell-loss metric. Patients were subdivided into quartiles according to the baseline cell-loss metric. For these groups, baseline means were 0.0016, 0.0042, 0.0062, 0.0178 units. After subtraction of baselines, means for the quartiles 48 h after treatment 2 were 0.002, 0.011, 0.030 and 0.357 units. pCR was achieved in 24/104, their distribution in the quartiles (11, 11, 23 and 46%) differed significantly (p = 0.01). In 80 patients with remaining tumour, tumour size was inversely related to the metric (p = 0.002). In 57 patients studied before treatment 2, positive and negative predictive values of the metric were 77.8 and 83.3%, compared to 40.5 and 88.7% 48 h after treatment 2. CONCLUSION: A cell-loss metric, based on serum levels of TK1, released from disrupted tumour cells, and tumour volume, reveal tumour response early during neoadjuvant treatment. The metric reflect tumour properties that differ greatly between patients and determine the sensitivity to cytotoxic treatment. The findings point to the significance of cell loss for tumour growth rate. The metric should be considered in personalized oncology and in the evaluation of new therapeutic modalities. TRIAL REGISTRATION: PROMIX (Clinical Trials.govNCT000957125).

Effect of hypoxia on human equilibrative nucleoside transporters hENT1 and hENT2 in breast cancer.

Elevated proliferation rates in cancer can be visualized with positron emission tomography (PET) using 3'-deoxy-3'-l-[18F]fluorothymidine ([18F]FLT). This study investigates whether [18F]FLT transport proteins are regulated through hypoxia. Expression and function of human equilibrative nucleoside transporter (hENT)-1, hENT2, and thymidine kinase 1 (TK1) were studied under normoxic and hypoxic conditions, and assessed with [18F]FLT-PET in estrogen receptor positive (ER+)-MCF7, triple-negative MDA-MB231 breast cancer (BC) cells, and MCF10A cells (human mammary epithelial cells). Functional involvement of hENT2 [18F]FLT transport was demonstrated in all cell lines. In vitro [18F]FLT uptake was higher in MDA-MB231 than in MCF7: 242 ± 9 vs. 147 ± 18% radioactivity/mg protein after 60 min under normoxia. Hypoxia showed no significant change in radiotracer uptake. Protein analysis revealed increased hENT1 (P < 0.0963) in MDA-MB231. Hypoxia did not change expression of either hENT1, hENT2, or TK1. In vitro inhibition experiments suggested involvement of hENT1, hENT2, and human concentrative nucleoside transporters during [18F]FLT uptake into all cell lines. In vivo PET imaging revealed comparable tumor uptake in MCF7 and MDA-MB231 tumors over 60 min, reaching standardized uptake values of 0.96 ± 0.05 vs. 0.89 ± 0.08 (n = 3). Higher hENT1 expression in MDA-MB231 seems to drive nucleoside transport, whereas TK1 expression in MCF7 seems responsible for comparable [18F]FLT retention in ER+ tumors. Our study demonstrates that hypoxia does not significantly affect nucleoside transport as tested with [18F]FLT in BC.-Krys, D., Hamann, I., Wuest, M., Wuest, F. Effect of hypoxia on human equilibrative nucleoside transporters hENT1 and hENT2 in breast cancer.

The combination of AroCell TK 210 ELISA with Prostate Health Index or prostate-specific antigen density can improve the ability to differentiate prostate cancer from noncancerous conditions.

BACKGROUND: Prostate-specific antigen (PSA) is an established tumour marker for prostate cancer (PCa). Serum thymidine kinase 1 is a possible new marker for the detection of PCa. The aim of the study was to investigate the diagnostic value of the AroCell TK 210 enzyme-linked immunosorbent assay (ELISA) together with free PSA, [-2]proPSA, and Prostate Health Index (PHI) in differentiating PCa from benign urological conditions. METHODS: Serum samples from 140 patients with PSA values in the range between 2 and 10 µg/L were collected at the Ljubljana University Medical Centre and the Maribor University Medical Centre. Thymidine kinase (TK1) protein levels were determined using the AroCell TK 210 ELISA and PSA-related parameters analysed with commercial assays. RESULTS: Serum TK1 protein, total and free PSA, proPSA, PSA density (PSAD), and PHI levels in patients with confirmed PCa were significantly higher than in patients with benign urological conditions (P < 0.05). Overall, the AroCell TK 210 ELISA results showed a significant correlation with PHI ( r = 0.25, P = 0.0031). Receiver-operating characteristic curve analyses were used to compare the area under the curve (AUC) of TK 210 ELISA, PHI, and PSA density. For PHI, the AUC was 0.73, comparable to those of TK 210 ELISA (0.67) and PSAD (0.66), with no significant differences in pairwise comparisons (PHI vs TK 210 ELISA P = 0.32, PHI vs PSAD P = 0.24, and TK 210 ELISA vs PSAD P = 0.95). The AUC for the combination of TK1 plus PSAD was significantly higher than those for the individual PSA-related biomarkers and marginally PHI, while the AUC for the combination of TK1 plus PHI was significantly higher than those for the individual PSA-related biomarkers except for PHI and marginally for PSAD. Total PSA concentration was the only marker, that was significantly higher in patients with an increasing Gleason grade. CONCLUSIONS: These results suggest that TK1 protein determinations together with PHI or PSAD could be a valuable additional tool in PCa management.

Thymidine kinase 1 concentration in pleural effusion is a diagnostic marker and survival predictor for malignant pleural effusion.

OBJECTIVE: Thymidine kinase 1 (TK1) is a key enzyme in the pyrimidine salvage pathway. Increased TK1 concentration correlates with cell division. TK1 is an emerging biomarker in cancer diagnosis; however, its effectiveness in diagnosis and management for malignant pleural effusion (MPE) is unclear. We evaluated the diagnostic efficiency and prognostic value of pleural effusion TK1 (pTK1) concentration for MPE. METHODS: From 2013 to 2017, 210 pleural effusion samples were collected from 160 patients diagnosed with MPE and 50 patients diagnosed with benign pleural effusion (BPE). TK1 concentrations in pleural effusion were measured by chemiluminescence dot blot assays. The median follow-up was 12 months. We constructed a receiver-operating characteristic (ROC) curve to find the optimal cutoff value for MPE diagnosis. The hazard ratios were estimated using a multivariable Cox proportional hazard model. A nomogram was drawn to illustrate the prognostic characteristics of MPE. RESULTS: The TK1 concentration in pleural effusion was significantly higher in MPE than BPE (P < 0.001), and patients with MPE could be distinguished by an optimal cutoff value of 3.10 pmol/L with a sensitivity of 0.894 and a specificity of 0.800. The multivariate analysis suggested that pTK1 concentration was an independent predictor of survival in patients with MPE. CONCLUSIONS: The diagnostic and prognostic prediction of MPE may be improved by measuring pTK1 concentration and utilizing a multivariate nomogram.

Membrane expression of thymidine kinase 1 and potential clinical relevance in lung, breast, and colorectal malignancies.

BACKGROUND: Lung, breast, and colorectal malignancies are the leading cause of cancer-related deaths in the world causing over 2.8 million cancer-related deaths yearly. Despite efforts to improve prevention methods, early detection, and treatments, survival rates for advanced stage lung, breast, and colon cancer remain low, indicating a critical need to identify cancer-specific biomarkers for early detection and treatment. Thymidine kinase 1 (TK1) is a nucleotide salvage pathway enzyme involved in cellular proliferation and considered an important tumor proliferation biomarker in the serum. In this study, we further characterized TK1's potential as a tumor biomarker and immunotherapeutic target and clinical relevance. METHODS: We assessed TK1 surface localization by flow cytometry and confocal microscopy in lung (NCI-H460, A549), breast (MDA-MB-231, MCF7), and colorectal (HT-29, SW620) cancer cell lines. We also isolated cell surface proteins from HT-29 cells and performed a western blot confirming the presence of TK1 on cell membrane protein fractions. To evaluate TK1's clinical relevance, we compared TK1 expression levels in normal and malignant tissue through flow cytometry and immunohistochemistry. We also analyzed RNA-Seq data from The Cancer Genome Atlas (TCGA) to assess differential expression of the TK1 gene in lung, breast, and colorectal cancer patients. RESULTS: We found significant expression of TK1 on the surface of NCI-H460, A549, MDA-MB-231, MCF7, and HT-29 cell lines and a strong association between TK1's localization with the membrane through confocal microscopy and Western blot. We found negligible TK1 surface expression in normal healthy tissue and significantly higher TK1 expression in malignant tissues. Patient data from TCGA revealed that the TK1 gene expression is upregulated in cancer patients compared to normal healthy patients. CONCLUSIONS: Our results show that TK1 localizes on the surface of lung, breast, and colorectal cell lines and is upregulated in malignant tissues and patients compared to healthy tissues and patients. We conclude that TK1 is a potential clinical biomarker for the treatment of lung, breast, and colorectal cancer.

Thymidine kinase 1 expression in ovarian serous adenocarcinoma is superior to Ki-67: A new prognostic biomarker.

Cancer is a disease with abnormally proliferating cells and therefore proliferation rate is an important index for assessing tumour growth. Ki-67 is a commonly used proliferation marker considered to be an unfavourable prognostic marker in some tumors, while Thymidine kinase 1 (TK1) is an interesting proliferation marker because its levels are highly dependent on the growth stage of cells. To define the immunohistochemistry (IHC) expression of the TK1 in patients with ovarian serous adenocarcinoma and establish its potential role as a new biomarker for progressive disease, we analyzed the expression patterns of TK1 and Ki-67 in 109 patients with ovarian serous adenocarcinoma. TK1 and Ki-67 expression both showed a statistically significant correlation to MD Anderson Cancer Center (MDACC) grade, but not to age, tumour size, lymph node metastasis or pathological TNM (pTNM) stages. TK1 expression, MDACC grades, pathological stages and lymph node metastasis correlate to relapse incident rate and overall survival, but Ki-67 does not. Although TK1 expression, MDACC grade, pTNM stage and lymph node metastasis significantly correlate to relapse in the Cox univariate analysis, in the multivariate Cox analysis only TK1 expression and lymph node metastasis were independent prognostic factors. The overall survival also correlated significantly to TK1 expression, MDACC grade, pTNM stage and lymph node metastasis in the Cox univariate analysis. However, only the pTNM stage was found to be an independent prognostic factor for survival in the Cox multivariate analysis. Therefore, though TK1 expression was an independent prognostic factor for relapse, but not for survival, TK1 is a more informative expression than Ki-67 for LI, relapse and overall survival rates. Thus, when TK1 is combined with MDACC grading, pTNM staging and lymph node metastasis, IHC determination of TK1 expression may improve the overall prediction of prognosis in patients with ovarian cancer.

A clinical evaluation of the TK 210 ELISA in sera from breast cancer patients demonstrates high sensitivity and specificity in all stages of disease.

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.

Serum thymidine kinase 1 protein concentrations and presence of its autoantibody as biomarkers for screening dogs with malignant tumors.

BACKGROUND: An accurate and easily accessible method for diagnosing malignancies in local veterinary clinics has not yet been established. OBJECTIVES: To investigate the usefulness of serum thymidine kinase 1 (TK1) protein and its autoantibody as tumor biomarkers in dogs. ANIMALS: Serum samples from 1702 dogs were collected from local animal hospitals and referral animal medical centers in South Korea. METHODS: TK1 protein OD value and TK1 autoantibody ratio (TK1 autoantibody OD/total IgG OD) in serum samples of dogs classified into healthy controls, group with nontumor disease, group with benign and group with malignant tumors were measured using lateral flow immunochromatographic assay methods. RESULTS: TK1 autoantibody levels were significantly higher in malignant tumor group (median 0.71) than in healthy controls (median 0.34), group with nontumor disease (median 0.34), and group with benign tumor (median 0.32, Welch t test, P < .0001). They were also significantly different among dogs with carcinomas (median 0.77), hematopoietic tumors (median 0.71), and sarcomas (median 0.56) than in healthy controls (median 0.34, post hoc Games-Howell test, P < .0001). In the receiver operating characteristic curve of TK1 protein, AUC was 0.633 (95% CI: 0.592-0.675, P < .0001). The AUC of TK1 autoantibody ratio was 0.758 (95% CI: 0.723-0.793, P < .0001). CONCLUSIONS AND CLINICAL IMPORTANCE: TK1 autoantibody is a potentially useful biomarker for differentiating between healthy and tumor-bearing dogs, better than TK1 protein measurement. However, both were inadequate when used as single biomarkers for screening dogs to discover occult malignant tumors.

Development of a gold nanoparticle-based lateral flow immunochromatographic assay for the rapid and quantitative detection of thymidine kinase 1 in human serum.

Thymidine kinase 1 (TK1) is a marker of cell proliferation that can be used for early screening, treatment monitoring, and evaluating the prognosis of patients with tumors. The main purpose of this study was to develop clinically applicable TK1 antibodies, establish an appropriate detection method, and provide material and technical support for the research and clinical application for different types of tumors. Experimental mice were immunized with the C-terminal 31 peptide of human TK1 to screen monoclonal cell lines capable of stably secreting specific antibodies. Monoclonal antibodies were then prepared, purified and screened for optimal pairing following the identification of purity and isotype. Finally, based on the principles adopted by the double-antibody sandwich detection method, we constructed a lateral flow immunochromatographic assay (LFIA) to quantify the concentration of TK1 in serum samples when using a gold nanoparticle-labeled anti-TK1 monoclonal antibody as a probe. The limit of detection for TK1 in serum was 0.31 pmol/L with a detection range of 0.31-50 pmol/L. The spiked recoveries ranged from 97.7% to 109.0% with an analytical precision of 5.7-8.2%; there was no cross-reactivity with common proteins in the serum. The established LFIA also exhibited good consistency with commercially available chemiluminescent immunoassay kits for the detection of clinical samples. The LFIA developed in this study has the advantages of high sensitivity, accuracy, reproducibility and strong specificity, and provides a new technical tool for the quantitative detection of TK1.

A stable thymidine kinase 1 tetramer for improved quality control of serum level quantification.

DNA synthesis is a critical process for cell growth and division. In cancer patients, an enzyme called thymidine kinase 1 (TK1) is often elevated in the blood, making it a valuable biomarker for cancer diagnosis and treatment. However, previous studies have shown that recombinant TK1 can exist in unstable mixtures of tetramers and dimers, leading to inconsistent results and potentially affecting accuracy. To address this issue, we hypothesized that incorporating tetrameric coiled-coil peptides could enhance TK1 self-assembly into stable tetramers without requiring additional adenosine triphosphate. In this study, we successfully expressed a recombinant TK1 tetramer protein in the Escherichia coli system. We optimized the induction conditions, significantly increasing protein expression levels, functionality, and solubility. Size exclusion chromatography confirmed the formation of a tetrameric structure in the expressed TK1 protein, with a molecular weight of 127.2 KDa, consistent with our expectations. We also found that the TK1 tetramer exhibited higher affinity with anti-TK1 IgY than wild-type TK1, as shown by enzyme-linked immunosorbent assay experiments. Moreover, the TK1 tetramer demonstrated good stability against heating, freeze-thawing and lyophilization with almost no immunoactivity lost. These findings suggest that recombinant TK1 tetramers have the potential to serve as calibrators in diagnostic assay kits, becoming promising candidates for quality control of clinical laboratory and in vitro diagnostic reagents.

Increased levels of thymidine kinase 1 in malignant cell-derived extracellular vesicles.

Extracellular vesicles (EVs), whose main subtypes are exosomes, microparticles, and apoptotic bodies, are secreted by all cells and harbor biomolecules such as DNA, RNA, and proteins. They function as intercellular messengers and, depending on their cargo, may have multiple roles in cancer development. Thymidine kinase 1 (TK1) is a cell cycle-dependent enzyme used as a biomarker for cell proliferation. TK1 is usually elevated in cancer patients' serum, making the enzyme a valuable tumor proliferation biomarker that strongly correlates with cancer stage and metastatic capabilities. Here, we investigated the presence of TK1 in EVs derived from three prostate cancer cell lines with various p53 mutation statuses (LNCaP, PC3, and DU145), EVs from the normal prostate epithelial cell line RWPE-1 and EVs isolated from human seminal fluid (prostasomes). We measured the TK1 activity by a real-time assay for these EVs. We demonstrated that the TK1 enzyme activity is higher in EVs derived from the malignant cell lines, with the highest activity from cells deriving from the most aggressive cancer, compared to the prostasomes and RWPE-1 EVs. The measurement of TK1 activity in EVs may be essential in future prostate cancer studies.