Serum Thymidine Kinase 1 Activity Research Articles

Serum thymidine kinase activity in patients with HR-positive/HER2-negative advanced breast cancer treated with ribociclib plus letrozole: Results from the prospective BioItaLEE trial

BackgroundThymidine kinase 1 (TK1) is an enzyme downstream of the CDK4/6 pathway, with a critical role in DNA synthesis; serum TK1 activity (sTKa) is a novel liquid biopsy biomarker of tumour cell proliferation. MethodsThe phase IIIb, BioItaLEE trial (NCT03439046) collected sera from postmenopausal patients with hormone receptor–positive (HR+), HER2-negative (HER2–) advanced breast cancer (ABC) treated with first-line ribociclib plus letrozole at baseline, day 15 of cycle 1 (C1D15), day 1 of cycle 2 (C2D1), and at first imaging. Associations between sTKa assessed at different time points or sTKa dynamic patterns, and progression-free survival (PFS) were evaluated using multivariate Cox models. ResultsOverall, 287 patients were enroled. Median follow-up was 26.9 months. High sTKa (>median) at baseline was associated with higher risk of progression (hazard ratio [HR], 2.21; 95% confidence interval [95% CI], 1.45, 3.37; P = 0.0002); similar results were observed for patients with high sTKa levels at C1D15 and C2D1. Early sTKa dynamic patterns were strongly predictive of PFS. The pattern with high sTKa levels at C2D1 following initial decrease at C1D15 was associated with higher risk of progression versus the pattern with low sTKa levels at both time points (HR, 2.89; 95% CI, 1.57, 5.31; P = 0.0006), while the pattern with high sTKa levels at C1D15 was associated with the shortest PFS (HR, 5.65; CI: 2.84, 11.2; P < 0.0001). Baseline and dynamic sTKa changes provided independent information. ConclusionssTKa appears to be a new promising prognostic and pharmacodynamic biomarker in patients with HR+/HER2– ABC treated with ribociclib plus letrozole as first-line therapy.

Serum thymidine kinase 1 activity as a prognostic biomarker in dogs with chemotherapy-treated diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is frequently treated with chemotherapy incorporating cyclophosphamide, doxorubicin, vincristine and prednisone (CHOP), which induces remission in 80% to 95% of cases. However, not all dogs derive meaningful benefit from CHOP, and prognostic factors for dogs with DLBCL are poorly defined. Serum thymidine kinase 1 (TK1) activity, a marker of tumour cell proliferation, has shown promising initial results as a prognostic biomarker in dogs with multicentric lymphomas. The purpose of this study was to determine if baseline serum TK1 activity is associated with clinical outcome in dogs with CHOP-treated DLBCL. Baseline serum TK1 activity was measured in banked sera from 98 dogs with CHOP-treated DLBCL using a commercially available ELISA kit. Data on other potential prognostic factors were abstracted retrospectively from electronic medical records. Multivariable statistical methods were used to identify associations between TK1 and other potential prognostic factors with progression-free survival (PFS) and attainment of complete remission. TK1 activity at baseline was not associated with PFS (p=.299) or attainment of complete remission (p=.910) following CHOP chemotherapy. Of the other prognostic factors analysed, only purebred (vs. mixed breed) status (HR 8.81, 95% CI 1.68-46.30, p=.010), attainment of complete (vs. partial) remission (HR 0.09, 95% CI 0.02-0.49, p=.006), and baseline serum C-reactive protein concentration (HR 1.19, 95% CI 1.07-1.32, p=.001) were independently associated with PFS. Based on these findings, baseline serum TK1 activity does not appear to be a useful prognostic biomarker in dogs with CHOP-treated DLBCL.

Analytical and clinical characterization of an optimized dual monoclonal sandwich ELISA for the quantification of thymidine kinase 1 (TK1) protein in human blood samples.

Thymidine Kinase 1 (TK1) plays an important role in DNA precursor synthesis and serum TK1 activity has been used as a biomarker for prognosis and therapy monitoring of different malignancies. AroCell has developed a dual monoclonal antibody ELISA for determination of TK1 protein in clinical samples. The purpose of the study is to validate the ELISA analytically in relation to the gold standard, [3H]-deoxythymidine (dThd) phosphorylation assay for TK1 activity using sera from patients with different malignancies. The colorimetric TK 210 ELISA was validated analytically by assessment of precision, linearity, interfering substances, and stability. For the clinical validation, serum samples from patients with hematological malignancies (n = 100), breast cancer (n = 56), prostate cancer (n = 70) and blood donors (n = 159) were analyzed using TK 210 ELISA and TK1 activity by [3H]-deoxythymidine (dThd) phosphorylation assay. The sandwich TK 210 ELISA was highly specific for TK1 protein having a detection limit of 0.12 ng/mL, with a functional sensitivity of 0.25 ng/mL. Within-run CVs ranged from 5.5% to 10% and between-run CVs ranged from 5% to 15%. The ratio of observed to expected dilutional parallelism of 5 serum samples was in the range of 80–120%. Samples exhibited stability through four freeze/thaw cycles and 5 days at 4°C. Further, the ROC curve analysis showed that TK 210 ELISA and [3H]-dThd phosphorylation assay had similar sensitivity (62% vs 59%) in hematological malignancies. However, in the case of breast and prostate cancer sera, TK 210 ELISA had higher sensitivity (59% and 44%) compared to [3H]-dThd phosphorylation assay (47% and 25%) at a specificity of 98%. These data demonstrate that the dual monoclonal antibody based AroCell TK 210 ELISA is a robust, accurate and precise tool for measuring TK1 protein in different malignancies that can improve the clinical applications of TK1 as a biomarker in cancer management.

Abstract 1268: Early proliferative response via [18]FLT-PET/CT and serum TK1 activity are predictive biomarkers for synergistic effect of sequential cell cycle targeted therapy in sarcoma

Abstract Introduction: Sarcomas are rare, aggressive tumors that make up a higher percentage of overall cancer mortality in children and young adults. Primary management of sarcoma patients includes surgical resection with adjuvant or neoadjuvant radiation/chemotherapy. Even with aggressive multimodal treatment regimens, patients with high-grade sarcomas experience suboptimal prognosis and advanced disease is almost universally fatal. Innovative biomarker driven targeted therapies are in urgent need to improve survival in these patients. Discoveries in molecular and genetic basis of sarcoma have revealed new targets that deserve consideration. Inactivation of retinoblastoma (Rb) pathway, which causes unabated cell proliferation, through overexpression of cyclin D1 and/or CDK4/6 has been detected in the majority of sarcomas even when wild-type Rb protein is expressed. Hence, inhibition of cell cycle progression through reactivation of the tumor suppressor function of Rb by inhibiting CDK4/6 activity poses a potential avenue for new therapeutic options. Methods: A panel of 4 sarcoma cell lines and 4 patient derived xenograft (PDX) models were used: 2 with an intact Rb pathway (Rb+ve, p16-ve) and 2 with an altered Rb pathway (Rb low/-ve, p16+ve). CDK4/6 inhibitor, abemaciclib, was used to reactivate the Rb pathway followed by treatment with gemcitabine or WEE1-kinase-inhibitor, to target the enriched S and G2/M phase cells post-abemaciclib removal. 18F-FLT PET imaging and serum thymidine kinase 1 (TK1) were used to monitor changes in cell proliferation and treatment response in both in vitro and in vivo. Changes in cell cycle biomarkers in response to treatment were also interrogated. Results: Rb+ve sarcoma cell lines were sensitive to the anti-proliferative activity of abemaciclib leading to their reversible G1 arrest, while Rb null cells did not arrest in any phase of cell cycle in response to CDK4/6 inhibition. Following abemciclib removal, Rb+ve models (but not Rb-ve) showed significant increase in 18F-FLT biotracer uptake concomitant with elevated serum TK1 and enrichment of cells in S phase. The enrichment of cells in S phase maximized therapeutic efficacy of S-phase or G2/M targeting agents leading to synergistic cell killing in Rb+ve cell lines and PDX models. Conclusion: Our study presents an innovative treatment strategy inhibiting the Rb pathway followed by S or G2/M pathway sequentially resulting in synergistic cell killing in those cells/tumors with wild-type Rb. We also incorporate real-time PET imaging and serum TK1 as non-invasive indicators of response to the treatment strategy. The study with PDX models also identified biomarkers of cell proliferation to be used in conjunction with PET imaging to provide the pre-clinical rationale to translate our findings into clinical trials for this aggressive disease. Citation Format: Tuyen Duong Nguyen, Marc A. Pina, Yan Wang, Cansu Karakas, Tuyen N. Bui, Osama Mawlawi, Alexander J. Lazar, Davis R. Ingram, Peter J. Hougton, Kelly K. Hunt, Khandan Keyomarsi. Early proliferative response via [18]FLT-PET/CT and serum TK1 activity are predictive biomarkers for synergistic effect of sequential cell cycle targeted therapy in sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1268.

Serum thymidine kinase 1 and its kinetics in HER2-positive breast cancer: Results from the Swedish phase II PREDIX HER2 trial.

e12598 Background: Thymidine kinase 1 (TK1) plays a pivotal role in DNA synthesis and cellular proliferation. TK1 has been studied as a prognostic marker and as an early indicator of treatment response in HER2-negative early and metastatic breast cancer (BC). Here we present the first report of TK1 in HER2-postitive BC. Methods: In the PREDIX HER2 trial, 202 patients with HER2-positive BC were randomized to 6 cycles of neoadjuvant trastuzumab, pertuzumab and docetaxel or trastuzumab emtansine every three weeks followed by surgery and adjuvant epirubicin and cyclophosphamide. Serum was prospectively collected from all participants at multiple timepoints: At baseline, after cycles 2, 4 and 6, at end of adjuvant therapy and then annually for 5 years and in case of recurrence. TK1 activity was measured by DiviTum assay (Biovica, Sweden), blinded to treatment allocation, patient characteristics and outcome. TK1 activity was correlated with baseline characteristics, pathologic complete response (pCR) and event-free survival (EFS). Results: Baseline TK1 activity as categorical variable was not associated with tumor size or hormone receptor expression. An increase of TK1 activity was seen in the majority of cases after treatment (mean TK1 at baseline = 111.7, after cycle 2 = 1257.0, after cycle 4 = 1157.0, after cycle 6 = 1178.0). Neither baseline TK1, on-treatment TK1 nor its change from baseline to cycle 2 were significantly associated with pCR in multivariable logistic regression analysis (Table). Baseline TK1 activity was not predictive for differential benefit to the study treatments ( pinteraction = 0.19). After a median follow-up of 52.5 months, 21 patients had EFS events. There was no significant correlation between baseline TK1 activity and EFS in multivariable Cox regression analysis. Conclusions: Serum TK1 activity in HER2-positive BC increased following treatment with neoadjuvant therapy but was not correlated to pCR rates or EFS. [Table: see text]

Serum TK1 protein and C-reactive protein correlate to treatment response and predict survival in dogs with hematologic malignancies

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 [3H]–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.

Molecular characterization of equine thymidine kinase 1 and preliminary evaluation of its suitability as a serum biomarker for equine lymphoma

BackgroundThymidine kinase 1 (TK1) plays a key role in the synthesis of deoxythymidine triphosphate (dTTP) and is thus important for DNA replication and cell proliferation. The expression of TK1 is highest during S-phase, and it is rapidly degraded after mitosis. In cancer cells, TK1 is upregulated, resulting in leakage of excess TK1 into the blood. Consequently, serum TK1 has been used as a diagnostic and prognostic cancer biomarker, mainly in human medicine. The aims of this work were to characterize equine TK1 and to evaluate its suitability as a serum biomarker for equine lymphoma.ResultsEquine TK1 was cloned, expressed in E. coli and affinity purified. The purified recombinant horse TK1 showed broad substrate specificity, phosphorylating pyrimidine deoxyribo- and ribonucleosides and, to some extent, purine deoxynucleosides, including anticancer and antiviral nucleoside analogues. ATP was the preferred phosphate donor. Serum TK1 activity was measured in samples collected from horses with confirmed or suspected lymphoma and control horses with and without concurrent diseases. Serum TK1 activity levels were significantly higher in horses with lymphoma (p < 0.0005) and suspected lymphoma (p < 0.02) and in tumour-free groups with diverse diseases (p < 0.03) than in controls without concurrent diseases. There was a significant difference between the lymphoma group and the tumour-free group with diverse diseases (p < 0.0006). Furthermore, receiver operating characteristic analysis revealed a sensitivity of 0.86, a specificity of 0.95 and an AUC (area under the curve) of 0.92 compared to the controls without concurrent diseases, with a sensitivity of 0.97, a specificity of 0.71 and an AUC of 0.88 when compared with the tumour-free group with diverse diseases.ConclusionEquine TK1 showed high specific activity and broader substrate specificity than human TK1. Anticancer and antiviral thymidine analogues were efficiently phosphorylated by horse TK1, suggesting that these analogues might be good candidates for chemotherapy in horses. Serum TK1 activity was significantly higher in horses with lymphoma than in controls. ROC analysis indicated that serum TK1 could serve as a promising cancer biomarker in horses.

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

BackgroundThymidine 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.ResultsFeline 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.ConclusionFeline 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.

292P Serum thymidine kinase 1 activity in patients with hormone receptor positive (HR+)/HER2 negative (HER2-) advanced breast cancer (aBC) treated in first-line with ribociclib (R) and letrozole (L) in the BioItaLEE trial

Thymidine kinase 1 (TK1) is an enzyme downstream of the CDK4/6 pathway, with a critical role in DNA synthesis and cell proliferation. We assessed serum TK1 activity (TKa) as a prognostic and predictive biomarker of response to R+L. 287 postmenopausal patients (pts) were enrolled in the BioItaLEE trial (NCT03439046). Sera were collected at baseline (D0; n=263), day 15 of cycle 1 (D15; n=245), day 1 of cycle 2 (C2D1; n=241), and at first imaging (FI; n=203). TKa was measured with DiviTum®, an ELISA-based assay, with a limit of detection (LOD) of 20 DiviTum units (Du)/L. The predictive role of TKa dynamics was explored by defining three patterns (P): P1, TKa <LOD at D15 and C2D1; P2, TKa <LOD at D15 and >LOD at C2D1; P3, TKa >LOD at D15 and C2D1. Multivariate Cox models assessed the association between biomarkers and progression-free survival (PFS). Median follow-up was 26.9 months. High TKa (>median) at D0 was associated with poor prognosis (HR for disease progression 2.21; 95% CI 1.45,3.37; p=0.0002), whereas only 4% of pts with low TKa at FI had concomitant disease progression. Robust and complex TKa dynamics were observed, with a decrease below LOD seen in 85%, 29% and 40% of pts at D15, C2D1 and FI, respectively; 65% of pts with TKa <LOD at D15 had TKa >LOD at C2D1 (P2). TKa dynamics were strongly predictive of PFS: P2 indicated a worse outcome vs P1 (HR 2.89; 95% CI 1.57,5.31; p=0.0006), while P3 was associated with shortest PFS (HR 5.65; CI 2.84,11.2; p<0.0001). For pts in P2, low TKa (<median) at D0 correlated with a better PFS vs high TKa (HR=3.47; 95% CI 1.64,7.36; p=0.001). TKa appears to be a new promising prognostic, predictive and monitoring biomarker in pts with HR+/HER2- aBC treated with R+L as first-line therapy. Baseline and dynamic TKa changes provided independent information. Lack of TKa suppression at D15, as seen in P3, may indicate enrichment for primary resistance and poor prognosis. TKa rebound at C2D1, as seen in P2, may indicate early adaptation to R+L, while persistent suppression as observed in P1 may identify pts with sustained inhibition and excellent prognosis.

Clinical performance of a commercially available thymidine kinase 1 assay for diagnosis of lymphoma in 42 hospitalized horses (2017-2020).

BackgroundAntemortem definitive diagnosis of lymphoma in horses is often difficult. Thymidine kinase 1 (TK1) assay is a potentially useful biomarker for lymphoma in horses.Hypothesis/objectivesTo report the clinical performance of a commercially available TK1 assay for diagnosis of lymphoma in horses. We hypothesized that there would be no association between serum TK1 activity and a diagnosis of lymphoma in horses.AnimalsForty‐two hospitalized horses, 14 with a definitive diagnosis of lymphoma, 4 with other neoplasia, and 24 with inflammatory disease.MethodsRetrospective medical record review, groups were compared via Kruskal‐Wallis and Mann‐Whitney tests, and logistic regression was performed.ResultsMedian (range) TK1 was 3 U/L (0.4‐17.7 U/L) in horses with lymphoma and 3.9 U/L (0.8‐94 U/L) in horses without lymphoma (P = .59). There was no significant difference in total protein between horses with and without lymphoma (6.6 g/dL [5.5‐8.3 g/dL] vs 6.6 g/dL [4.7‐10.4 g/dL]; P = .83). There was no significant difference in fibrinogen between horses with and without lymphoma (447 [100‐1364] mg/dL vs 433 [291‐2004] mg/dL; P = .47). On logistic regression, serum TK1 activity was not associated with a diagnosis of lymphoma (odds ratio, 0.97; 95% confidence interval, 0.9‐1.05, P = .48).Conclusion and Clinical ImportanceSerum TK1 values were not predictive of lymphoma diagnosis in this cohort of horses.

Prognostic role of serum thymidine kinase 1 kinetics during neoadjuvant chemotherapy for early breast cancer

BackgroundEmerging data support the use of thymidine kinase 1 (TK1) activity as a prognostic marker and for monitoring of response in breast cancer (BC). The long-term prognostic value of TK1 kinetics during neoadjuvant chemotherapy is unclear, which this study aimed to elucidate.MethodsMaterial from patients enrolled to the single-arm prospective PROMIX trial of neoadjuvant epirubicin, docetaxel and bevacizumab for early BC was used. Ki67 in baseline biopsies was assessed both centrally and by automated digital imaging analysis. TK1 activity was measured from blood samples obtained at baseline and following two cycles of chemotherapy. The associations of TK1 and its kinetics as well as Ki67 with event-free survival and overall survival (OS) were evaluated using multivariable Cox regression models.ResultsCentral Ki67 counting had excellent correlation with the results of digital image analysis (r = 0.814), but not with the diagnostic samples (r = 0.234), while it was independently prognostic for worse OS [adjusted hazard ratio (HRadj) = 2.72, 95% confidence interval (CI) 1.19-6.21, P = 0.02]. Greater increase in TK1 activity after two cycles of chemotherapy resulted in improved event-free survival (HRadj = 0.50, 95% CI 0.26-0.97, P = 0.04) and OS (HRadj = 0.46, 95% CI 0.95, P = 0.04). There was significant interaction between the prognostic value of TK1 kinetics and Ki67 (pinteraction 0.04).ConclusionSerial measurement of serum TK1 activity during neoadjuvant chemotherapy provides long-term prognostic information in BC patients. The ease of obtaining serial samples for TK1 assessment motivates further evaluation in larger studies.

Elevated Free Triiodothyronine Is Associated With Increased Proliferative Activity in Triple-negative Breast Cancer.

Thyroid hormones (THs) stimulate breast cancer (BC) cell proliferation. We hypothesized that these hormones and the proliferative marker thymidine kinase 1 (TK1) represent the initial and final steps of the proliferative pathway, respectively. We measured the serum levels of thyroid-stimulating hormone (TSH), free triiodothyronine (FT3), and free thyroxine (FT4), along with serum TK1 activity, in 144 newly diagnosed BC patients, and examined the associations between THs and proliferation in different BC receptor profiles. TK1 activity did not correlate with TSH (r=0.06, p=0.473) or FT4 levels (r=0.04, p=0.665), but did correlate with FT3 levels (r=0.28, p=0.001). Elevated FT3 (>6.0 pmol/l) predicted increased TK1 activity (>140 Du/l) after adjusting for age (odds ratio 4.1, p=0.014). We also found a significant association of the combined elevation of FT3 and TK1, assumed as a surrogate marker of accomplished proliferative signal, with triple-negative (TN) profile (p=0.003). The rates of combined FT3 and TK1 elevation in TN and ER-positive profiles were 20.0% and 1.8%, respectively (p=0.005). FT3 may be involved in proliferative signaling, as measured by TK1 activity, predominately in TN breast cancer.

Serial evaluation of serum thymidine kinase activity is prognostic in women with newly diagnosed metastatic breast cancer

The rapid development of new therapies in metastatic breast cancer (MBC), entails a need for improved prognostic and monitoring tools. Thymidine kinase 1 (TK1) is involved in DNA synthesis and its activity correlates to outcome in cancer patients. The aim of this study was to evaluate serum TK1 activity (sTK1) levels in MBC patients as a tool for prognostication and treatment monitoring. 142 women with MBC scheduled for 1st line systemic treatment were included in a prospective observational study. sTK1 was measured at baseline (BL) and at 1, 3 and 6 months and correlations to progression-free and overall survival (PFS, OS) evaluated. High sTK1 levels (above median) correlated to worse PFS and OS at BL, also after adjusting for other prognostic factors. sTK1 levels were significantly associated with PFS and OS measured from follow-up time points during therapy. Changes from 3 to 6 months during therapy significantly correlated to PFS and OS, whereas early changes did not. We could demonstrate sTK1 level as an independent prognostic factor in patients with newly diagnosed MBC. Changes in sTK1 levels from 3 to 6 months correlated to PFS and OS. Future studies of sTK1 are warranted to further define its clinical utility.

Abstract P5-06-11: Serum thymidine kinase-1 activity (TKa) as a prognostic marker in premenopausal women with hormone receptor positive (HR+) operable breast cancer (BC)

Abstract Introduction: Thymidine kinase is an established marker of cancer cell proliferation and its activity can be measured in blood. We and others have recently shown that baseline and dynamic evaluation of circulating thymidine kinase activity (TKa) during treatment gives prognostic and predictive information in patients with HR+, HER2-negative metastatic BC treated with endocrine therapy alone, as well as in the setting of CDK4/6 inhibition. However, there is limited data regarding the role of TKa as a prognostic biomarker in operable BC. Here we present a retrospective analysis of TKa in serum samples collected in a cohort of premenopausal women with operable BC enrolled in a phase III adjuvant multicenter clinical trial (NCT00201851). Materials and methods: Serum samples were available for 644 (87%) of participants prospectively enrolled in a randomized trial between 2003 and 2009 in South East Asia. All women were premenopausal, had stage II-IIIB HR+ operable BC and uniformly received bilateral surgical oophorectomy concurrent with mastectomy followed by tamoxifen alone for five years. Patients did not receive chemotherapy or targeted therapy pre- or post-operatively. Participants were randomized in the study according to the timing of surgery with respect to the phase of the menstrual cycle. Serum samples were collected preoperatively on the day of surgery. Serum TKa was measured using the ELISA-based DiviTum™ assay (Biovica, Sweden). TKa analysis was performed at a central laboratory, blind to clinical data. Baseline TKa values were correlated with clinico-pathological characteristics and clinical outcome. Clinical outcome was estimated using the Kaplan-Meier method. Results: The majority of patients had both estrogen and progesterone receptor positive tumors (94% and 92% respectively), 65% were HER2 negative (18% positive; 17% unknown). Most had pT2 or pT3 disease (60% and 27% respectively), and more than half were node-positive (pN0 42%, pN1 27%, pN2 19%, pN3 11%, pNx 1%). The overall median TKa value was 65.4 Du/L. At five years, patients with a baseline TKa value below the median had a disease-free survival (DFS) rate of 75% versus 61% in those with a baseline over the median (HR 1.82, 95% CI 1.37-2.4, p&amp;lt;0.001). Similar results were observed when women with HER2+ disease were excluded from analysis (HR 1.71, 95% CI 1.21-2.42, p=0.0025). Further prognostic precision was achieved when TKa values were divided by quartiles, with a 5 year DFS rate of 81%, 69%, 63% and 58% observed in the 1st, 2nd, 3rd and 4th quartiles respectively. After adjusting for major prognostic factors and randomization arm, TKa remained an independent marker. Conclusions: This study shows pre-operative TKa measured in serum is a strong prognostic marker in a large cohort of women with HR+ operable BC uniformly treated within a clinical trial. The notable rate of recurrence seen within this cohort of patients derived from non-high income countries may be mainly attributed to the relative degree of disease burden at diagnosis. TKa may be seen as a potential circulating marker of proliferation akin to tumor Ki67, which may provide useful prognostic information to guide adjuvant therapies. Citation Format: Luca Malorni, Ilenia Migliaccio, Chiara Biagioni, Lorenzo Rossi, Irene De Santo, Richard L Love, Amelia McCartney, Mattias Bergqvist, Martina Bonechi, Francesca De Luca, Francesca Galardi, Matteo Benelli, Dario Romagnoli, Emanuela Risi, Laura Biganzoli, Adriano Laudico, Nguyen Van Dinh, Angelo Di Leo. Serum thymidine kinase-1 activity (TKa) as a prognostic marker in premenopausal women with hormone receptor positive (HR+) operable breast cancer (BC) [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-06-11.

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.

Evaluation of serum thymidine kinase 1 activity as a biomarker for treatment effectiveness and prediction of relapse in dogs with non-Hodgkin lymphoma.

BackgroundSerum thymidine kinase 1 (sTK1) activity is closely correlated with DNA synthesis.ObjectivesEvaluate sTK1 activity as a biomarker for treatment response and early detection of relapse in dogs with lymphoma.AnimalsNinety‐seven client‐owned dogs with naive or relapsed lymphoma and 23 healthy dogs.MethodsProspective study. Serum TK1 activity measured by refined ELISA‐based method (DiviTum assay, Biovica International) before treatment, at clinical response, and every 4 weeks until relapse or last follow‐up.ResultsSerum TK1 activity was ≤20 Du/L in 96% (22/23) of healthy dogs. Pretreatment sTK1 activity was >20 Du/L in 88% (85/97) dogs with lymphoma. At clinical response, sTK1 activity was significantly lower in dogs with complete (CR, n = 36) versus partial (PR, n = 29) response (P < .0001). Sensitivity (Se) and specificity (Sp) of sTK1 activity for detecting nonfully responders were 76% and 100%, respectively, with cutoff of 119.5 Du/L (AUC, 0.90; 95%‐CI, 0.81‐0.98; P < .0001). In dogs with CR, a 5‐fold increase in sTK1 activity at a 4‐week interval predicted relapse at the subsequent 4‐week assessment with a Se 50% and Sp 94% (AUC, 0.72; 95%‐CI, 0.55‐0.90; P = .02). An increase of sTK1 activity (>2.7‐fold value measured at clinical response) predicted relapse at subsequent 4‐week assessment with a Se 61% and Sp 88% (AUC, 0.79; 95%‐CI, 0.64‐0.95; P = .004).Conclusions and Clinical ImportanceMonitoring sTK1 activity could help to detect complete responders and early disease progression in dogs with lymphoma.

Prognostic role of serum thymidine kinase 1 activity in patients with hormone receptor–positive metastatic breast cancer: Analysis of the randomised phase III Evaluation of Faslodex versus Exemestane Clinical Trial (EFECT)

BackgroundThymidine kinase 1 (TK1) plays a critical role in DNA synthesis and cell proliferation. Recent studies have shown potential for serum TK1 activity (sTKa) as a prognostic marker and indicator of early response to endocrine therapy in advanced breast cancer. The aim of this study is to assess the correlation between sTKa and patient outcome. Patients and methodsThe Evaluation of Faslodex versus Exemestane Clinical Trial (EFECT) was a double-blind, double-dummy, randomised trial of fulvestrant versus exemestane after progression on non-steroidal aromatase inhibitor therapy, in postmenopausal women with advanced breast cancer. Retrospective analyses of serum archived from EFECT were conducted. sTKa was assessed using the DiviTum® assay on samples collected at baseline, after three and six months of endocrine therapy, and at disease progression. ResultsThe median time to progression (mTTP) for patients with low baseline sTKa levels was 5.03 months (95% confidence interval [CI]: 3.91–5.89) versus 2.57 months (95% CI: 2.04–3.52) in patients with high sTKa baseline levels (P < 0.0001). On treatment, patients whose sTKa increased from baseline had a significantly shorter mTTP (3.39 months, 95% CI: 2.14–4.11) than those without an sTKa increase (5.39 months, 95% CI: 4.01–6.68) (P = 0.0045). Similar results were observed in the separate EFECT treatment arms. After adjusting for major prognostic factors, sTKa remained an independent marker. ConclusionsTKa is a potential circulating prognostic marker in patients with advanced breast cancer treated with endocrine therapy. It may also represent a tool for upfront identification of endocrine therapy resistance and early positive response to therapy. Independent validation of these results is warranted.

Role of serum thymidine kinase-1 (TK1) activity in patients (pts) with hormone receptor positive (HR+) advanced breast cancer (ABC) treated with endocrine therapy (ET) in the EFECT trial.

12031Background: TK1 plays a critical role in DNA synthesis and cell proliferation. The DiviTum assay measures serum TK1 activity (sTKa), reflecting cancer cell proliferation. Recent studies sugges...

Abstract P3-08-13: Serum thymidine kinase activity is an independent prognostic factor for progression-free and overall survival in women with metastatic breast cancer

Abstract Introduction: Although prognosis and treatment of metastatic breast cancer (MBC) have improved over the last years, there is still an unmet clinical need for more precise prognostic and treatment monitoring tools. Liquid-based markers are preferred since they reflect real-time tumor progression and are not dependent on repeated invasive tissue biopsies. Thymidine kinase 1 (TK1) is an enzyme involved in nucleotide metabolism and has a fundamental role in the DNA synthesis. It can be used as a marker of cell proliferation rate and the TK1 activity has demonstrated correlations to prognosis and usefulness for treatment monitoring in different malignancies. The aim of this study was to determine serum TK1 activity (sTK1) levels measured with the DiviTum assay (Biovica, Sweden), in women with MBC scheduled for 1st line systemic therapy and to evaluate its potential for prediction of outcome and treatment monitoring. Methods: 142 women with MBC scheduled for 1st line systemic treatment and included in a prospective monitoring trial (CTC-MBC, NCT01322893) were evaluated for sTK1 at baseline (BL) and during treatment at 1, 3 and 6 months. 132 patients had at least one follow-up sample. sTK1 activity levels were measured and correlations to important clinicopathological variables and prognosis (PFS and OS) at BL and during treatment were evaluated. Results: The median sTK1 level at BL was 391 u/L (range 10-35520 u/L). When comparing patients with high (above median) versus low (below median) sTK1 levels at BL, high sTK1 levels were found to be associated to worse performance status (p=0.001) and high number of metastatic sites (p=0.03). There was also a statistically significant association between high sTK1 levels and high Ki67 expression in biopsies from metastatic lesions (p=0.038). In univariable analyses high sTK1 levels correlated to worse PFS and OS (HRPFS-BL 2.32, p&amp;lt;0.001; HROS-BL 2.54, p&amp;lt;0.001) at BL. In multivariable analysis adjusted for clinically used prognostic factors, sTK1 was an independent prognostic factor for PFS and OS (HRPFS-BL 2.4, p&amp;lt;0.001; HROS-BL 2.0, p=0.01). During treatment, sTK1 was significantly associated with OS from each of the four time points and onwards (BL, 1, 3, 6 months) (HROS-1m 1.93, p=0.01; HROS-3m 2.35, p=0.02; HROS-6m 2.78, p=0.002) in univariable analysis. High sTK1 levels were also associated with impaired PFS (HRPFS-1m 1.48, p=0.06; HRPFS-3m 1.52, p=0.07; HRPFS-6m 2.03, p=0.009) and these associations were significant at BL and 6 months. Discussion: sTK1 activity level is an independent prognostic factor for PFS and OS in patients with MBC scheduled for 1st line systemic therapy. During treatment, sTK1 is prognostic for OS evaluated from all time-points up to 6 months. The sTK1 effects observed for PFS are slightly weaker, but still propose potential usefulness for treatment monitoring. Further, sTK1 levels correlate to Ki67 expression in metastatic lesions suggesting that it can be useful as a liquid-based real-time proliferation marker. In conclusion, these results are clinically relevant for prognostication and treatment monitoring in patients with MBC. Future studies of sTK1 are justified to further elucidate in what settings this marker is most useful. Citation Format: Larsson A-M, Jansson S, Bendahl P-O, Baker S, Bergqvist M, Aaltonen K, Rydén L. Serum thymidine kinase activity is an independent prognostic factor for progression-free and overall survival in women with metastatic breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P3-08-13.

Serum thymidine kinase 1 activity as a pharmacodynamic marker of cyclin-dependent kinase 4/6 inhibition in patients with early-stage breast cancer receiving neoadjuvant palbociclib

BackgroundThymidine kinase 1 (TK1) is a cell cycle-regulated enzyme with peak expression in the S phase during DNA synthesis, and it is an attractive biomarker of cell proliferation. Serum TK1 activity has demonstrated prognostic value in patients with early-stage breast cancer. Because cyclin-dependent kinase 4/6 (CDK4/6) inhibitors prevent G1/S transition, we hypothesized that serum TK1 could be a biomarker for CDK4/6 inhibitors. We examined the drug-induced change in serum TK1 as well as its correlation with change in tumor Ki-67 levels in patients enrolled in the NeoPalAna trial (ClinicalTrials.gov identifier NCT01723774).MethodsPatients with clinical stage II/III estrogen receptor-positive (ER+)/HER2-negative breast cancer enrolled in the NeoPalAna trial received an initial 4 weeks of anastrozole, followed by palbociclib on cycle 1, day 1 (C1D1) for four 28-day cycles, unless C1D15 tumor Ki-67 was > 10%, in which case patients went off study owing to inadequate response. Surgery occurred following 3–5 weeks of washout from the last dose of palbociclib, except in eight patients who received palbociclib (cycle 5) continuously until surgery. Serum TK1 activity was determined at baseline, C1D1, C1D15, and time of surgery, and we found that it was correlated with tumor Ki-67 and TK1 messenger RNA (mRNA) levels.ResultsDespite a significant drop in tumor Ki-67 with anastrozole monotherapy, there was no statistically significant change in TK1 activity. However, a striking reduction in TK1 activity was observed 2 weeks after initiation of palbociclib (C1D15), which then rose significantly with palbociclib washout. At C1D15, TK1 activity was below the detection limit (<20 DiviTum units per liter Du/L) in 92% of patients, indicating a profound effect of palbociclib. There was high concordance, at 89.8% (95% CI: 79.2% - 96.2%), between changes in serum TK1 and tumor Ki-67 in the same direction from C1D1 to C1D15 and from C1D15 to surgery time points. The sensitivity and specificity for the tumor Ki-67-based response by palbociclib-induced decrease in serum TK1 were 94.1% (95% CI 86.2% - 100%) and 84% (95% CI 69.6% -98.4%), respectively. The κ-statistic was 0.76 (p < 0.001) between TK1 and Ki-67, indicating substantial agreement.ConclusionsSerum TK1 activity is a promising pharmacodynamic marker of palbociclib in ER+ breast cancer, and its value in predicting response to CDK4/6 inhibitors warrants further investigation.Trial registrationClinicalTrials.gov, NCT01723774. Registered on 6 November 2012.