Thymidine Kinase 1 Protein Levels Research Articles

A monoclonal antibody-based sandwich ELISA for measuring canine Thymidine kinase 1 protein and its role as biomarker in canine lymphoma.

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. 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. 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. These results demonstrate that the Canine TK1 ELISA can serve as an efficient tool in the diagnosis and management of dogs with lymphomas.

A Dual Biomarker TK1 Protein and CA125 or HE4-Based Algorithm as a Better Diagnostic Tool than ROMA Index in Early Detection of Ovarian Cancer

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. 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. 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). The combination of TK1 protein with CA 125 or HE4 increased the potential of detecting ovarian cancer at early stages.

Abstract 3130: Evaluation of TK1 protein changes using the AroCell TK 210 ELISA in an acute myeloid leukemia (AML) xenograft model during 5-fluoruracil treatment

Abstract Background Thymidine kinase 1 (TK1) is a cytosolic enzyme involved in DNA precursor synthesis and it is up-regulated in proliferating cells. Serum TK1 is an established biomarker for prognosis and therapy monitoring of malignancies. However, commercial TK1 enzyme activity assays have limitations for their use in xenograft models as they cross-react with TK1 activity from the host. The AroCell TK 210 ELISA is specific for human TK1 and offers the opportunity to measure the release of TK1 from human xenografts free from the background of TK1 release from the host (mouse). Here we studied the changes in serum TK1 protein levels using the AroCell TK 210 ELISA in a xenograft model during 5-fluorouracil (5FU) treatment of Acute Myeloid Leukemia (AML). Experimental Procedures Human MV-4-11 leukemic cells from ATCC were cultured with 95% O2 and 5% CO2 at 37°C and in IMD medium (Sigma) supplemented with 10% FBS (HI media) containing 4mmol/L L-glutamine, 5 ng/mL GM-CSF and 1% penicillin and streptomycin. The cells were harvested during exponential growth and injected subcutaneously into the left flank of athymic BALB/c nude mice 8-10 weeks old. The mice were divided into controls that received no therapy (n=8) and an active group receiving 5FU (n=8). The treatment was initiated when the average tumor volume reached 50-100 mm3. Tumor bearing mice were treated with PBS (control) or 5-FU (active group 10 mg/kg, once daily) for three weeks. Serum samples (70-80 µL) were collected before treatment initiation, during treatment (once weekly for 3 weeks) and a final termination. Serum samples were stored at -80°C and TK1 protein levels were determined with the AroCell TK 210 ELISA. All the animal experiments were performed at TheraIndx life sciences Pvt Ltd, India. Results The tumor volumes were measured post-implantation of cells every 3 days throughout the study. There was a significant difference (p&amp;lt;0.05) in the mean tumor volume between 5-FU treated (5-FU) (108.75± 36.3 mm3) and untreated controls (294.63± 50.17 mm3) at the end of treatment. There was a significant reduction in the tumor volume from 2nd week onwards in 5-FU treated mice up to termination (4th week) compared to control mice. TK1 protein concentrations increased significantly from the 2nd week onwards in 5-FU treated mice (0.42± 0.19 µg/L) in comparison to controls (0.1± 0.02 µg/L) in spite of the decrease in tumor volume in response to treatment. No cross-reactivity of TK 210 ELISA with mouse TK1 was observed. Conclusions This study is the first evaluation of TK 210 ELISA as a biomarker for monitoring therapy with anticancer drugs in an AML xenograft model. The changes in TK1 protein levels were significant enough to monitor the response of the xenograft to 5FU therapy in this animal model. Furthermore, the use of the AroCell TK 210 ELISA in xenograft models may enable more accurate measurement of the anti-cancer drug effects on cell proliferation and these results encourage further applications of TK 210 ELISA in drug development projects. Citation Format: Kiran Kumar Jagarlamudi, Ramesh Jayaraman, Sundaresh Babu, Martin Shaw, Staffan Eriksson. Evaluation of TK1 protein changes using the AroCell TK 210 ELISA in an acute myeloid leukemia (AML) xenograft model during 5-fluoruracil treatment [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3130.

AroCell TK 210 ELISA for determination of TK1 protein: age-related reference ranges and comparison with other TK1 assays.

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.

Abstract 2232: The assay of thymidine kinase 1 in sera from subjects with hematological and solid tumors with AroCell TK 210 ELISA: Comparison with TK-liaison assay and its clinical implications

Abstract Background: Thymidine Kinase 1 (TK1) is an ATP dependent enzyme involved in DNA precursor synthesis. It is released into the blood from cells undergoing un-controlled proliferation where it forms stable aggregates. Serum TK1 activity has been used as a biomarker for diagnosis and monitoring of hematological malignancies. Several commercial assays are available for measuring serum TK1 activity but they require radioactive materials or special instruments. To overcome these, AroCell has developed the TK 210 ELISA for TK1 protein measurements. In this study, we compared the performance of the TK 210 ELISA to that of the TK-Liaison assay on sera from subjects with hematological and solid tumors Experimental Procedures Sera from patients with hematologic malignancies (N=51) were collected at the University Hospital, Uppsala. Samples from solid tumors [breast cancer (n=60); subjects with benign prostate hyperplasia or prostate carcinoma (n=60)] along with blood donors (N=102) were collected at the University Medical Centre, Ljubljana. TK1 protein levels were determined with AroCell TK 210 ELISA and TK1 activity levels by the TK-Liaison assay. Results In blood donors, the TK1 protein levels were in the range of 0.1 to 0.35 µg/L with a median value of 0.2. Men had a higher median value TK1 protein levels compared to women (0.21 vs 0.19) with no significant difference. Both the TK 210 ELISA and the TK-Liaison assays showed significantly higher TK1 levels in hematological and solid tumors compared to blood donors (P&amp;lt;0.0001). ROC curve analysis demonstrated that across all malignancies, at 96% specificity, TK 210 ELISA (cut-off value of 0.34 µg/L) gave a higher sensitivity of 0.43 compared to TK-Liaison assay (cut-off value of 10 U/L) of 0.36. The performance of the assays differed depending on the type of malignancy, TK 210 ELISA and TK-Liaison had a similar sensitivity (0.65) for hematological malignancies. However, in the case of solid tumors, TK 210 ELISA showed a higher sensitivity (0.35) compared to TK-Liaison (0.26) at a specificity of 0.96. A regression analysis of the TK 210 ELISA (y) and TK-Liaison (x) assays across all three groups gave an equation of y = 0.15 + 0.017x (rs = 0.80, n = 273). The correlation value in hematological malignancies was higher than in blood donors and solid tumors (0.95 vs. 0.67 and 0.50 respectively). Conclusions This study showed that the AroCell TK 210 ELISA has a similar sensitivity and specificity to the TKLiaison assay for hematological malignancies. However, the AroCell TK 210 ELISA demonstrated a higher sensitivity for TK1 in sera from subjects with solid tumors and this may facilitate the application of TK1 as a biomarker for solid tumors. Furthermore, the robustness and convenience of the ELISA not only overcomes the limitations of TK-Liaison but also widens the clinical applications of TK1 in cancer management. Citation Format: Jagarlamudi Kiran Kumar, Staffan Eriksson, Kerstin Hamberg Levedahl, Martin Höglund, Mojca Zupan, Joško Osredkar, Diana Cviič, Urska Furar. The assay of thymidine kinase 1 in sera from subjects with hematological and solid tumors with AroCell TK 210 ELISA: Comparison with TK-liaison assay and its clinical implications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2232.

Abstract 715: 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 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&amp;lt;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

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

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.

BackgroundThymidine 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.MethodsSerum 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.ResultsMean 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.ConclusionsThese 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1073-8) contains supplementary material, which is available to authorized users.

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.

BackgroundThymidine 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.ResultsSerum 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.ConclusionsSerum 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.

Prognostic and Therapeutic Impact of Argininosuccinate Synthetase 1 Control in Bladder Cancer as Monitored Longitudinally by PET Imaging

Targeted therapies have yet to have significant impact on the survival of patients with bladder cancer. In this study, we focused on the urea cycle enzyme argininosuccinate synthetase 1 (ASS1) as a therapeutic target in bladder cancer, based on our discovery of the prognostic and functional import of ASS1 in this setting. ASS1 expression status in bladder tumors from 183 Caucasian and 295 Asian patients was analyzed, along with its hypothesized prognostic impact and association with clinicopathologic features, including tumor size and invasion. Furthermore, the genetics, biology, and therapeutic implications of ASS1 loss were investigated in urothelial cancer cells. We detected ASS1 negativity in 40% of bladder cancers, in which multivariate analysis indicated worse disease-specific and metastasis-free survival. ASS1 loss secondary to epigenetic silencing was accompanied by increased tumor cell proliferation and invasion, consistent with a tumor-suppressor role for ASS1. In developing a treatment approach, we identified a novel targeted antimetabolite strategy to exploit arginine deprivation with pegylated arginine deiminase (ADI-PEG20) as a therapeutic. ADI-PEG20 was synthetically lethal in ASS1-methylated bladder cells and its exposure was associated with a marked reduction in intracellular levels of thymidine, due to suppression of both uptake and de novo synthesis. We found that thymidine uptake correlated with thymidine kinase-1 protein levels and that thymidine levels were imageable with [(18)F]-fluoro-L-thymidine (FLT)-positron emission tomography (PET). In contrast, inhibition of de novo synthesis was linked to decreased expression of thymidylate synthase and dihydrofolate reductase. Notably, inhibition of de novo synthesis was associated with potentiation of ADI-PEG20 activity by the antifolate drug pemetrexed. Taken together, our findings argue that arginine deprivation combined with antifolates warrants clinical investigation in ASS1-negative urothelial and related cancers, using FLT-PET as an early surrogate marker of response.

Limits of [18F]-FLT PET as a Biomarker of Proliferation in Oncology

BackgroundNon-invasive imaging biomarkers of cellular proliferation hold great promise for quantifying response to personalized medicine in oncology. An emerging approach to assess tumor proliferation utilizes the positron emission tomography (PET) tracer 3’-deoxy-3’[18F]-fluorothymidine, [18F]-FLT. Though several studies have associated serial changes in [18F]-FLT-PET with elements of therapeutic response, the degree to which [18F]-FLT-PET quantitatively reflects proliferative index has been continuously debated for more that a decade. The goal of this study was to elucidate quantitative relationships between [18F]-FLT-PET and cellular metrics of proliferation in treatment naïve human cell line xenografts commonly employed in cancer research.Methods and Findings[18F]-FLT-PET was conducted in human cancer xenograft-bearing mice. Quantitative relationships between PET, thymidine kinase 1 (TK1) protein levels and immunostaining for proliferation markers (Ki67, TK1, PCNA) were evaluated using imaging-matched tumor specimens. Overall, we determined that [18F]-FLT-PET reflects TK1 protein levels, yet the cell cycle specificity of TK1 expression and the extent to which tumors utilize thymidine salvage for DNA synthesis decouple [18F]-FLT-PET data from standard estimates of proliferative index.ConclusionsOur findings illustrate that [18F]-FLT-PET reflects tumor proliferation as a function of thymidine salvage pathway utilization. Unlike more general proliferation markers, such as Ki67, [18F]-FLT PET reflects proliferative indices to variable and potentially unreliable extents. [18F]-FLT-PET cannot discriminate moderately proliferative, thymidine salvage-driven tumors from those of high proliferative index that rely primarily upon de novo thymidine synthesis. Accordingly, the magnitude of [18F]-FLT uptake should not be considered a surrogate of proliferative index. These data rationalize the diversity of [18F]-FLT-PET correlative results previously reported and suggest future best-practices when [18F]-FLT-PET is employed in oncology.

Abstract 376: Limits of [18F]-FLT PET as a predictive imaging biomarker of proliferation in oncology

Abstract Background: Surrogate, non-invasive imaging biomarkers of cellular proliferation hold great promise for quantifying response to personalized medicine in oncology. An emerging imaging approach to assess tumor proliferation utilizes the positron emission tomography (PET) tracer 3′-deoxy-3′[18F]-fluorothymidine, [18F]-FLT. Though several studies have associated serial changes in [18F]-FLT-PET with elements of therapeutic response, the degree to which [18F]-FLT uptake quantitatively reflects proliferative index has been continuously debated for more that a decade. The goal of this preclinical study was to elucidate quantitative relationships between [18F]-FLT-PET and cellular, proteomic, and genomic metrics proliferation in treatment naive tumors. Methods and Findings: [18F]-FLT-PET imaging was conducted in mice bearing human colorectal or breast cancer cell line xenografts. Quantitative relationships between PET imaging, thymidine kinase 1 (TK1) protein and RNA levels, and immunostaining for proliferation markers (Ki67, TK1) were evaluated using imaging-matched tumor specimens. Overall, we found that [18F]-FLT-PET reflects TK1 protein levels in tumors. However, the cell cycle specificity of TK1 expression and the variable degree to which tumors utilize thymidine salvage for DNA synthesis tend to decouple [18F]-FLT-PET data from estimates of proliferation by standard, clinically employed means. Conclusions: Our findings illustrate that [18F]-FLT-PET reflects tumor proliferation as a function of thymidine salvage pathway utilization. Therefore, unlike more generalizable proliferation markers, such as Ki67 immunostaining, tumor uptake of [18F]-FLT reflects proliferative indices to variable and potentially unreliable extents. [18F]-FLT-PET can not discriminate moderately proliferative, thymidine salvage driven disease from highly proliferative tumors that rely primarily upon de novo thymidine synthesis. For those reasons, the magnitude of [18F]-FLT uptake in treatment naive tumors should not be considered a surrogate of proliferative index. These data provide rationale for the diversity of correlative results reported, both positive and discordant, comparing [18F]-FLT-PET and tumor proliferation across numerous single-center clinical studies and provide a basis for future clinical best-practices when this imaging biomarker is employed in oncology. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 376. doi:1538-7445.AM2012-376

Thymidine Kinase 1 is a Potential Marker for Prognosis and Monitoring the Response to Treatment of Patients with Breast, Lung, and Esophageal Cancer and Non-Hodgkin's Lymphoma

Thymidine kinase 1 (TK1) is converting thymidine to thymidine monophosphate, and is related to DNA replication and cell proliferation. The use of the TK1 protein levels as a proliferation marker in malignancies is here summarized. TK1 protein in serum (STK1p) and TK1 expression in tissues were determined by a chemoluminescent dot blot assay and by immunohistochemistry staining, respectively. The expression of TK1 in tumor tissues correlated to pathological stages and clinical grades of carcinomas (ca) of esophagus, lung and in premalignancy of breast ductal ca. STK1p could monitor the out-come of tumor therapy by being correlated to remission [breast ca, non-Hodgkin's lymphoma], relapse [breast ca] and to survival [non-Hodgkin's lymphoma] of patients. In a health screening study of 12,641 persons, STK1p seemed to predict the risk of development of neoplasia related diseases at early stage.

Human inter-individual variability in metabolism and genotoxic response to zidovudine

A mainstay of the antiretroviral drugs used for therapy of HIV-1, zidovudine (AZT) is genotoxic and becomes incorporated into DNA. Here we explored host inter-individual variability in AZT-DNA incorporation, by AZT radioimmunoassay (RIA), using 19 different strains of normal human mammary epithelial cells (NHMECs) exposed for 24 h to 200 μM AZT. Twelve of the 19 NHMEC strains showed detectable AZT-DNA incorporation levels (16 to 259 molecules of AZT/10 6 nucleotides), while 7 NHMEC strains did not show detectable AZT-DNA incorporation. In order to explore the basis for this variability, we compared the 2 NHMEC strains that showed the highest levels of AZT-DNA incorporation (H1 and H2) with 2 strains showing no detectable AZT-DNA incorporation (L1 and L2). All 4 strains had similar (≥ 80%) cell survival, low levels of accumulation of cells in S-phase, and no relevant differences in response to the direct-acting mutagen bleomycin (BLM). Finally, when levels of thymidine kinase 1 (TK1), the first enzyme in the pathway for incorporation of AZT into DNA, were determined by Western blot analysis in all 19 NHMEC strains at 24 h of AZT exposure, higher TK1 protein levels were found in the 12 strains showing AZT-DNA incorporation, compared to the 7 showing no incorporation ( p = 0.0005, Mann–Whitney test). Furthermore, strains L1 and L2, which did not show AZT-DNA incorporation at 24 h, did have measurable incorporation by 48 and 72 h. These data suggest that variability in AZT-DNA incorporation may be modulated by inter-individual differences in the rate of induction of TK1 in response to AZT exposure.

The uptake of 3?-deoxy-3?-[18F]fluorothymidine into L5178Y tumours in vivo is dependent on thymidine kinase 1 protein levels

The aim of this study was to investigate the role of thymidine kinase 1 (TK1) protein in 3'-deoxy-3'-[18F]fluorothymidine ([18F]FLT) positron emission tomography (PET) studies. We investigated the in vivo kinetics of [18F]FLT in TK1+/- and TK1-/- L5178Y mouse lymphoma tumours that express different levels of TK1 protein. [18F]FLT-derived radioactivity, measured by a dedicated small animal PET scanner, increased within the tumours over 60 min. The area under the normalised tumour time-activity curve were significantly higher for the TK1+/- compared with the -/- variant (0.89+/-0.02 vs 0.79+/-0.03 MBq ml(-1) min, P=0.043; n=5 for each tumour type). Ex vivo gamma counting of tissues excised at 60 min p.i. (n=8) also revealed significantly higher tumour [18F]FLT uptake for the TK1+/- variant (6.2+/-0.6 vs 4.6+/-0.4%ID g(-1), P=0.018). The observed differences between the cell lines with respect to [18F]FLT uptake were in keeping with a 48% higher TK1 protein in the TK1+/- tumours versus the -/- variant (P=0.043). On average, there were no differences in ATP levels between the two tumour variants (P=1.00). A positive correlation between [18F]FLT accumulation and TK1 protein levels (r=0.68, P=0.046) was seen. Normalisation of the data for ATP content further improved the correlation (r=0.86, P=0.003). This study shows that in vivo [18F]FLT kinetics depend on TK1 protein expression. ATP may be important in realising this effect. Thus, [18F]FLT-PET has the potential to yield specific information on tumour proliferation in diagnostic imaging and therapy monitoring.