| 1. |
- Jagarlamudi, Kiran Kumar, et al.
(författare)
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A clinical evaluation of the TK 210 ELISA in sera from breast cancer patients demonstrates high sensitivity and specificity in all stages of disease
- 2016
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Ingår i: Tumor Biology. - : Springer Science and Business Media LLC. - 1010-4283 .- 1423-0380. ; 37, s. 11937-11945
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 2. |
- Jagarlamudi, Kiran Kumar, et al.
(författare)
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A New Sandwich ELISA for Quantification of Thymidine Kinase 1 Protein Levels in Sera from Dogs with Different Malignancies Can Aid in Disease Management : Dog TK1-ELISA
- 2015
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Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 3. |
- Jagarlamudi, Kiran Kumar, et al.
(författare)
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Breast and prostate cancer patients differ significantly in their serum Thymidine kinase 1 (TK1) specific activities compared with those hematological malignancies and blood donors: implications of using serum TK1 as a biomarker
- 2015
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Ingår i: BMC Cancer. - : Springer Science and Business Media LLC. - 1471-2407. ; 15
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Tidskriftsartikel (refereegranskat)abstract
- Background: 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.Methods: 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 [H-3]-deoxythymidine (dThd) phosphorylation. The molecular forms of TK1 in sera from some of these patients were analyzed using size-exclusion chromatography.Results: 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.Conclusions: 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.
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| 4. |
- Jagarlamudi, Kiran Kumar, et al.
(författare)
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Clinical significance of the TK1 specific activity in the early detection of ovarian cancer
- 2024
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Ingår i: Oncology. - 0030-2414 .- 1423-0232. ; 102, s. 17-29
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Tidskriftsartikel (refereegranskat)abstract
- 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's why, the aim of the study is 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 ROMA index was also determined in the same set of clinical samples. Results: The TK1 SA was significantly different between healthy compared ovarian cancer patients (p < 0.0001). Strikingly, TK1 SA has higher sensitivity (55%) compared to other biomarkers in the detection of benign ovarian tumor. Further, the highest sensitivity was achieved by the combination of TK1 SA with CA 125 and HE4 for the detection of the benign tumor as well as malignant ovarian cancers (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 CA125 and HE4 for the detection of early stages of ovarian cancer.
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| 5. |
- Jagarlamudi, Kiran Kumar
(författare)
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Diagnostic implications of the molecular forms and levels of serum thymidine kinase 1 in different canine malignancies
- 2015
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Thymidine kinase 1 (ATP: thymidine 5’- phosphotransferase, EC 2.1.7. 21, TK1) is a cellular enzyme involved in salvage pathway for DNA precursor synthesis. TK1 activity fluctuates during cell cycle, it reaches peak in S phase and absent in M phase. Because of its tight regulation with cell cycle, TK1 has been used as proliferation marker for diagnosis and treatment monitoring of various malignancies in human and veterinary medicine. TK1 levels can be measured by activity based or antibody based assays. The main aim of the research described in this thesis was to develop TK1 antibody based assays for determining serum TK1 protein levels in comparison with TK1 activity from dogs with different malignancies. Further analysis revealed a significant difference in the molecular forms of TK1 in sera from canine leukemia and mammary tumours. In Study I, serum TK1 protein levels in dogs with different solid tumours were determined by using an antibody based assay i.e. immunoaffinity assay. TK1 protein levels were significantly higher in dogs with solid tumours than expected from the TK1 activity values. In contrast, the specific activity of TK1 in sera from healthy dogs was 2.5 fold higher than that of solid tumours. The molecular forms of recombinant, cellular and serum TK1 were investigated in study II. Dog recombinant and serum TK1 existed as oligomers with a molecular weight (MW) of 720-300 kDa. Cellular TK1 from both dogs and humans were mainly as tetramers. Human recombinant and serum TK1 activity eluted in two peaks: one at high and one at low MW, corresponding to 720-300 kDa and 200-50 kDa, respectively. In study III, TK1 protein levels in sera from dogs with mammary tumours were determined by immunoaffinity assay. The TK1 protein assay differentiated mammary adenomas efficiently from healthy dogs, and adenomas from carcinomas, but this was not possible with the TK1 activity assay. In mammary tumour sera, active TK1 eluted as high MW oligomer similar to leukemia however, TK1 protein was detected not only as high MW form but also in the fractions where no TK1 activity was found. This indicates that serum TK1 exits in multiple forms in mammary tumours with a large fraction of inactive TK1 protein. Study IV describes the development of TK1-ELISA, based on a polyclonal and a monoclonal anti TK1 peptide antibodies, that was used to measure TK1 protein levels in dog sera. TK1 protein levels were significantly higher in sera from dogs with haematological tumours as well as solid tumours in comparison with healthy dogs. Overall, the results demonstrate that TK1 protein assays provide valuable diagnostic information in a variety of canine malignancies.
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| 6. |
- Jagarlamudi, Kiran Kumar, et al.
(författare)
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Doxorubicin effects on leukemia and breast cancer cells in culture on the TK1 protein levels using AroCell TK 210 ELISA: a tool for drug development
- 2018
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Ingår i: Nucleosides, Nucleotides and Nucleic Acids. - : Informa UK Limited. - 1525-7770 .- 1532-2335. ; 37, s. 679-686
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Tidskriftsartikel (refereegranskat)abstract
- In this study changes in thymidine kinase 1 (TK1) levels after 24 hours of Doxorubicin (Dox) exposure of CEM and MDA MB-231 cells were determined using the commercially available AroCell TK 210 ELISA test. In cell extracts, TK1 levels increased twofold with 1 mu M Dox in both cell lines, while the TK1 levels in the culture media increased with 5 and 10 mu M of Dox only in case of CEM cells. In conclusion, this study reveals that the TK 210 ELISA can measure changes in intra- and extracellular TK1 levels apparently related to the mechanism of cytotoxicity of anti-cancer agents.
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| 7. |
- Jagarlamudi, Kiran Kumar, et al.
(författare)
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High levels of inactive thymidine kinase 1 polypeptide detected in sera from dogs with solid tumours by immunoaffinity methods: Implications for in vitro diagnostics
- 2013
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Ingår i: Veterinary Journal. - : Elsevier BV. - 1090-0233 .- 1532-2971. ; 197, s. 854-860
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Tidskriftsartikel (refereegranskat)abstract
- STK1 activity in dogs with haematological tumours was significantly higher than in the solid tumour and healthy dog groups (mean +/- standard deviation [SD] = 65 +/- 79, 1.1 +/- 0.5, and 1.0 +/- 0.4 pmol/min/mL, respectively). Serum samples were analyzed after immunoaffinity isolation by western blot and the TK1 26 kDa band intensities quantified revealing that concentrations were significantly higher in dogs with haematological tumours and solid tumours compared to healthy dogs (mean +/- SD = 33 +/- 12, 30 +/- 13, and 10 +/- 5 ng/mL, respectively). Pre-incubation with the reducing agent dithioerythritol (DTE) showed a decrease in STK1 activity and protein levels in most samples, but an increase of about 20% in sera from healthy dogs and from those with haematological malignancies. Compared to animals with solid tumours, the specific STK1 activity (nmol [H-3]-deoxythymidine monophosphate (dTMP)/min/mg of TK1 protein of 26 kDa) was 30-fold higher in haematological malignancies and 2.5-fold higher in healthy dogs, respectively. The results demonstrate that there is a large fraction of inactive TK1 protein, particularly in sera from dogs with solid tumours. The findings are important in the use of STK1 as a biomarker. (C) 2013 Elsevier Ltd. All rights reserved.
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| 8. |
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| 9. |
- Jagarlamudi, Kiran Kumar, et al.
(författare)
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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
- 2019
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Ingår i: Prostate. - : Wiley. - 0270-4137 .- 1097-0045. ; 79, s. 856-863
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Tidskriftsartikel (refereegranskat)abstract
- 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 mu g/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.
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| 10. |
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| 11. |
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| 12. |
- Jagarlamudi, Kiran Kumar
(författare)
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Thymidine kinase 1 as a tumor biomarker: technical advances offer new potential to an old biomarker
- 2018
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Ingår i: Biomarkers in Medicine. - : Future Medicine Ltd. - 1752-0363 .- 1752-0371. ; 12, s. 1035-1048
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Forskningsöversikt (refereegranskat)abstract
- Thymidine kinase 1 (TK1) is a key enzyme in DNA precursor synthesis. It is upregulated during the S phase of the cell cycle and its presence in cells is an indicator of active cell proliferation. In studies since the 1980s, TK1 has been shown as a clinically valuable biomarker for the management of hematological malignancies. However, TK1 activity assays may underestimate serum TK1 in subjects with solid tumors limiting its sensitivity. The development of TK1 immunoassays has made the assay of TK1 more widely available and increased its applicability to solid tumor diseases. This paper will review TK1 as a tumor biomarker with emphasis on recent studies and technologies plus highlight its potential in drug discovery and as a therapeutic target.
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| 13. |
- Kumar, Jagarlamudi Kiran, et al.
(författare)
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AroCell TK 210 ELISA for determination of TK1 protein : age-related reference ranges and comparison with other TK1 assays
- 2020
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Ingår i: BioTechniques. - : FUTURE SCI LTD. - 0736-6205 .- 1940-9818. ; 68:6, s. 335-342
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Tidskriftsartikel (refereegranskat)abstract
- Thymidine kinase 1 (TK1) is an enzyme involved in DNA precursor synthesis that has been used as a biomarker for prognosis and monitoring of different malignancies. In this study, we compared two immunoassays for measuring TK1 protein concentrations: the TK 210 ELISA (AroCell AB) and TK1 ELISA from Abcam. Overall, the TK 210 ELISA showed higher sensitivity than the Abcam TK1 ELISA for differentiating hematological malignancies (sensitivity of 0.77 vs 0.45) as well as for distinguishing sera of patients with solid tumors from those of apparently healthy individuals (0.61 vs 0.20). There was no significant difference in the TK1 protein levels determined with the TK 210 ELISA between different age groups from apparently healthy individuals. These results strongly indicate that the AroCell TK 210 ELISA is accurate and sensitive enough to be a valuable tool in cancer management. METHOD SUMMARY Thymidine kinase 1 (TK1) is an enzyme that leaks from S phase cells as a result of high cell turnover. Commercially available TK activity assays have certain limitations; to overcome these, we developed a dual monoclonal antibody-based ELISA, the AroCell TK 210 ELISA, which is commercially available. The ELISA includes a preincubation procedure with a special buffer that reduces high molecular weight complexes of serum TK1 and exposes the TK1 epitope to facilitate antibody binding. This provides a robust and convenient assay for the determination of TK1 protein concentrations in sera from patients with different malignancies.
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| 14. |
- Saellström, Sara, et al.
(författare)
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Serum TK1 protein and C-reactive protein correlate to treatment response and predict survival in dogs with hematologic malignancies
- 2022
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Ingår i: Research in Veterinary Science. - : Elsevier BV. - 0034-5288 .- 1532-2661. ; 145, s. 213-221
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Tidskriftsartikel (refereegranskat)abstract
- Thymidine kinase 1 (TK1), involved in DNA precursor synthesis, is used as a serum biomarker in cancer diagnostics in both human and veterinary medicine. We investigated the utility of serum TK1 protein (TK1p) and TK1 activity (TK1a) determinations for prognosis and monitoring of canine hematological malignancies. The combination of TK1p or TK1a with canine C-reactive protein (CRP) determinations was also investigated.Serum samples from 51 client-owned dogs with naive hematological malignancies and from 149 healthy subjects were included. Serum TK1p levels were determined using a prototype TK1-ELISA, TK1a using the [H-3]- dThd phosphorylation assay, and CRP using an immunoturbidimetric assay.Mean TK1p in sera from dogs with tumors was significantly higher than from healthy dogs (mean +/- SD = 3.9 +/- 5.9 vs. 0.45 +/- 0.15 ng/mL). Similarly, TK1a in hematological malignancies was significantly higher than in healthy dogs (mean + SD = 15.1 +/- 31.3 vs. 0.96 +/- 0.33 pmol/min/mL). The receiver-operating characteristic indicated that a combination of TK1p or TK1a with CRP gave higher sensitivity than either biomarker alone for the prognosis of hematological malignancies. Median pretreatment TK1p and TK1a levels were significantly higher than in dogs in remission and correlated with clinical outcome. Kaplan-Meier curve analysis showed that naive dogs with high TK1p, TK1a, and CRP had significantly shorter survival.This study present two new polyclonal antibodies used in an ELISA system to determine TK1p. The study also show that combining TK1p or TK1a with CRP gave higher sensitivity than either biomarker alone. Monitoring patients in the study while undergoing chemotherapy, suggests that the TK1 + CRP combination could be useful in a biomarker panel, possibly aiding the prognosis and therapy monitoring of hematological malignancies in dogs.
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| 15. |
- Sharif, Hanan, et al.
(författare)
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A monoclonal antibody-based sandwich ELISA for measuring canine Thymidine kinase 1 protein and its role as biomarker in canine lymphoma
- 2023
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Ingår i: Frontiers in Veterinary Science. - : Frontiers Media SA. - 2297-1769. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- Introduction: Dogs play an important role in society, which increased during the covid epidemics. This has led to a much higher workload for the veterinarians. Therefore, there is a need for efficient diagnostic tools to identify risk of malignant diseases. Here the development of a new test that can solve some of these problems is presented. It is based on serum Thymidine Kinase 1 (TK1), which is a biomarker for cell proliferation and cell lysis.Methods: Anti-TK1 monoclonal antibodies were produced against two different epitopes, the active site of the TK1 protein and the C-terminal region of canine TK1. The antibodies were developed with hybridoma technology and validated using dot blot, Quartz Crystal Microbalance (QCM) technology, western blots, immunoprecipitation (IP), and enzyme-linked immunosorbent assay (ELISA). Clinical evaluation of Canine TK1 ELISA was done by using sera from 131 healthy dogs and 93 dogs with lymphoma. The two selected Anti-TK1 monoclonal antibodies have Kd values in the range of 10(-9) M and further analysis with dot and western blots confirmed the high affinity binding of these antibodies. A sandwich Canine TK1 ELISA was developed using the anti-TK1 antibodies, and TK1 concentrations in serum samples were determined using dog recombinant TK1 as a standard.Results: Serum TK1 protein levels were significantly higher in dogs with lymphoma compared to those in healthy dogs (p < 0.0001). Receiver operating curve analysis showed that the canine TK1-ELISA obtain a sensitivity of 0.80, at a specificity of 0.95. Moreover, the Canine TK1 ELISA has a positive predictive value (PPV) of 97%, and the negative predictive value (NPV) of 83%, reflecting the proportion of test results that are truly positive and negative. Furthermore, Canine TK1 ELISA had significantly higher capacity to differentiate dogs with T-cell lymphoma from those with B-cell lymphoma compared to earlier used TK1 activity assays.Discussion: These results demonstrate that the Canine TK1 ELISA can serve as an efficient tool in the diagnosis and management of dogs with lymphomas.
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