Abstract
Background/Aim: The aim of this prospective study was to determine whether serum Thymidine kinase –1 (TK1) could serve as a tumor marker in soft tissue sarcomas (STS). Patients and Methods: A total of 48 patients diagnosed with localized STS were included. None had received preoperative oncological treatment. Samples were collected before and after surgery and TK1 levels measured with the AroCell TK210 ELISA. Results: Mean preoperative TK1 was 0.32 μg/l, range=0.11-1.47, and 18 cases (38%) had values above the reference limit (0.41 μg/l). Mean postoperative TK1 was 0.35 μg/l (0.06-0.86). In patients with preoperative values above the reference limit, TK1 decreased significantly after surgery (n=13, p=0.001). We found no association between increased preoperative TK1 and age, sex, tumor size, grade, and the presence of vascular invasion or necrosis. Conclusion: TK1 has limited use as a tumor marker in localized STS.
A solid supply of DNA building blocks is required for cell proliferation in normal tissues as well as the uncontrolled replication found in tumors. Thymidine kinase –1 (TK1) is a key enzyme involved in DNA precursor synthesis. It catalyzes the phosphorylation of deoxythymidine (dT) to deoxythymidine monophosphate (dTMP) in an ATP-dependent manner, after which dTMP is further phosphorylated into deoxythymidine triphosphate (dTTP) and incorporated into DNA (1). TK1 has a role in the salvage pathway that recycles deoxyribonucleosides from degraded DNA, as opposed to the de novo pathway where nucleotides are being synthesized from low molecular weight precursors. It is expressed in a cell cycle dependent manner. The levels of TK1 are low in quiescent cells, start to increase in late G1, peak in S phase earlier than Ki67 expression and decrease in late G2/M phase (2, 3).
In normal cells TK1 is localized in the cytoplasm. Immunohistochemical studies have indicated that it is upregulated already in precancerous lesions and that high levels of TK1 in malignant tumors may predict poor survival (4, 5). It is released from cells that die during proliferation. Serum TK1 has been studied extensively in cancer as a diagnostic and prognostic biomarker (6). It can be determined from serum samples using several different assays (6), which do not necessarily give similar results. In this article we restrict analyses of the literature to studies using AroCell TK 210 ELISA, TK-REA, TK1 Liaison, the Divitum assay and the dTdht phosphorylation assays, results of which have shown strong or at least moderate correlation to each other in earlier studies (7-10). In a recent comparative study, serum TK1 protein concentration was shown to have higher capacity to discriminate patients with prostatic or breast carcinoma from healthy blood donors than TK1 activity assay (7).
High serum TK1 levels associate with progressive disease and poor survival in hematologic malignancies (11-13). Compared to healthy controls or patients with benign diseases, serum TK1 levels have been shown to be higher in various solid tumors, including breast and prostate cancer, pancreatic cancer and renal cell carcinoma (7, 14, 15). Results in lung cancer have been more uncertain, as different TK1 analysis methods have given different results. In one study TK1 measured by ELISA was shown to be a sensitive and specific marker for the detection of early-stage lung cancer (16), but TK1 was not able to differentiate cancer from benign lung disease in another (17). In some solid tumors, serum TK1 levels have been reported to associate with tumor stage and predict response to therapy and outcome (14, 18, 19). Most studies relating TK1 levels to a malignant diagnosis or its outcome have included cases with both early and advanced tumors (7, 17, 20, 21). There is still a lack of results on TK1 serum levels in patients with localized cancer.
Soft tissue sarcomas (STS) are rare tumors of mesenchymal origin that most often occur in the extremities but can be detected throughout the body. They include various histological types and subtypes with distinct differences in behavior, propensity to metastasize to a certain location and outcome (22). At the time of diagnosis most STS are high grade and over 5 cm (T2) in size. Metastasis to lymph nodes occurs only rarely as the primary site of distant metastasis is the lung. The 5-year overall survival (OS) of patients with STS remains around 50-70%, and still a large number of patients die of metastatic disease (22-24). The 5-year cause-specific survival varies considerably depending on histology and has been published to be highest in dermatofibroma (99.2%) and the lowest in angiosarcomas (53.8%) and rhabdomyosarcomas (54.7%) (24). Prognostic factors routinely used in the clinic include tumor histology, grade, size and depth, the site of the primary lesion, the presence of necrosis or microvascular invasion in the tumor, operation margin and metastasis, of which tumor histology and grade and presence of metastasis have been considered most important (22-25). Staging in STS has only limited relevance (26).
Currently there are no known serologic markers routinely used for determination of prognosis, prediction of treatment response or follow-up in STS, although elevated systemic inflammatory markers, especially C-reactive protein (CRP) levels have been associated with worse disease-specific survival (27, 28).
In this study, we aimed to evaluate whether serum TK1 levels could serve as a serum marker in localized STS. In order to study the effect of the primary tumor on TK1 levels, we sampled patients both immediately before surgery, and for disease-free patients a few weeks or months following surgery, before any adjuvant therapy. The association between preoperative TK1 levels and tumors most important clinical and pathological characteristics was analyzed, and the impact of preoperative TK1 on outcome was also evaluated.
Patients and Methods
Patients. The study was approved by the Helsinki University Hospital Ethics Committee and the Ministry of Social and Health Affairs. All research was performed in accordance with relevant guidelines and regulations and in accordance with the Declaration of Helsinki. Patients referred to the Helsinki University Hospital Soft Tissue Sarcoma group with local primary soft tissue sarcoma were considered eligible. Patients younger than 18 years old, diagnosed with Grade 1 liposarcoma, dermatofibrosarcoma protuberans or cutaneous leiomyosarcoma, operated elsewhere than in Helsinki University Hospital, patients who had received preoperative oncological treatment or diagnosed with a primarily metastatic disease were excluded from the study. Informed consent was obtained from all subjects.
Serum sample collection. Serum samples were prospectively collected at the time of diagnosis during the preoperative visit to the operating hospital preferably 5 to 10 days before the surgery, on the operation day before the operation, and during the 1st postoperative control visit not less than 3 weeks after operation. All the serum samples were stored at –20°C until analysis.
Analysis of serum concentrations of TK1. The serum concentrations of TK1 were measured with AroCell TK 210 ELISA kit (AroCell AB, Uppsala, Sweden) as instructed by the manufacturer (29). All samples were analyzed in duplicates. Briefly, the samples were preincubated in sample dilution buffer for 1h to make the TK1 aggregates more accessible. Monoclonal anti-TK1-antibody coated 96-well plates were prewashed four times, 100 μl of the diluted samples were added and the plate was incubated at room temperature for two h. Then the plate was washed four times and biotinylated anti-TK1-antibody was added. After an incubation time of one hour, the non-binding molecules were washed away, streptavidin-HRP-solution was added, and the plate incubated for 30 min. Again, the plates were washed four times and 100 μl of TMB substrate was added. After 15 min the reaction was stopped using a stop solution, and the absorbance in each well was determined using a microplate reader at 450 nm (Sunrise absorbance reader, Tecan, Männedorf, Switzerland). The TK1 protein concentrations were calculated, and values given as μg/l. The upper normal reference value was defined as 0.41 μg/l (20, 30, 31).
Statistical analysis. Spearman’s correlation coefficient (rs) was used to test correlations between TK1 and interval scale clinical and pathological variables. The association between TK1 and dichotomous variables was tested with the two-sample t-test, and the difference between pre- and postoperative values with the paired t-test. The association between tumor histology and TK1 was tested in a one-way ANOVA model. The association between preoperative TK1 and local, distant recurrence-free and OS was tested using a Cox model with TK1 as a continuous variable. The OS was defined from the time of diagnosis to death or last follow-up date and disease-free survival (DFS) from the operation day to disease relapse or death. The IBM®SPSS® Statistics version 23/26 (SPSS, Chicago, IL, USA) was used for all analyses. p-Values <0.05 were considered statistically significant.
Results
From November 2016 to February 2019, 48 patients diagnosed with a local soft tissue sarcoma and treated in the Helsinki University Hospital by the soft tissue sarcoma group were included in the study. The clinical characteristics of the patients are summarized in Table I.
Clinical characteristics of patients.
Sixty percent of the patients were male and median age was 68.5 years. The most common histological subtypes were undifferentiated pleomorphic sarcoma (UPS), sarcoma not otherwise specified, and liposarcoma (17%, 12% and 7%, respectively). Mean tumor size was eight cm and 13 tumors measured at least 10 cm in diameter. Ninety-two percent were classified as high grade. The mean Ki-67 proliferation index was 42%. During the 4-year-follow-up period, four patients developed a local recurrence, 13 were diagnosed with distant metastasis and 15 died, five of which due to sarcoma.
Association of serum TK1 levels with known clinicopathological characteristics. The preoperative TK1 serum concentrations varied between 0.11-1.47 μg/l with a mean of 0.32 μg/l. Based on previous studies using AroCell TK 210 ELISA that included samples from healthy controls (20, 30, 31), serum TK1 levels below 0.41 μg/l were considered to be in the reference range. Eighteen patients (38%) had a TK1 value above the reference value (0.41 μg/l). The associations between preoperative TK1 and clinicopathological factors are shown in Table II. We found no association between TK1 and age, sex, tumor size, grade, the presence of vascular invasion or necrosis. There was a weak statistically significant negative correlation between TK1 and Ki-67 (r=–0.29, p=0.043). There was also a significant association between Ki-67 and postoperative TK1 (r=–0.35, p=0.04).
Association between preoperative TK1 serum concentrations and clinicopathologic variables.
Restricting the clinicopathological analyses to those 18 patients with abnormal TK1 showed a significant negative correlation between TK1 and age (r=–0.61, p=0.007) while the other associations remained nonsignificant (Table III).
Association between high preoperative TK1 serum concentrations and clinicopathologic variables in patients with TK1 above the reference limit before surgery.
Thirty-three patients had available postoperative TK1 values before any further therapy or relapse at 21 to 93 days after surgery (mean 37). Pre- and postoperative TK1 levels in individual patients are shown in Figure 1. The mean postoperative TK1 value was 0.35 μg/l (0.06-0.86) and the difference between pre and postoperative TK1 values was not statistically significant (p=0.12). Thirteen of these patients had preoperative TK1 value above the reference range (mean 0.79 μg/l, range=0.44-1.47). TK1 decreased after operation in 12 of these 13 patients and the decrease was statistically significant (p=0.001). Postoperative TK1 in these patients was 0.35 μg/l, with a range of 0.06 to 0.86. In six patients the TK1 levels increased from the normal range into pathological after the operation. All these six patients had recovered without any major complications in wound healing or infections, and none have developed tumor recurrence.
Pre- and postoperative TK1 values. The vertical and horizontal line depict the reference limit. The oblique line separates values increasing (above) and decreasing (below) after surgery. The red area depicts cases where a preoperative TK1 before surgery rose above the reference limit after surgery. The green area depicts preoperative values above the reference limit decreasing after surgery. TK1: Thymidine kinase 1.
Prediction of local recurrence, distant metastasis and overall survival. Pre-operative TK1 had no significant effect on local recurrence-free survival (HR=0.01, 95%CI=0.0-24.7, p=0.27), distant recurrence-free survival (HR=0.78, 95%CI=0.12-5.24, p=0.80) or OS (HR=1.3, 95%CI=0.29-5.6, p=0.74).
Discussion
TK1 has long been used as a prognostic and predictive marker in hematological malignancies (11-13) and to some extent, in various solid tumors (7, 14-16). Currently, there are no serologic tumor markers available for STS diagnosis or follow-up, and patients are regularly being monitored for local recurrence and metastasis by chest X-rays and MRI- or CT-imaging. A reliable tumor marker would be useful for simplifying follow-up and detection of recurrences not covered by routine imaging. To our knowledge, this is the first study reporting the use of serum TK1 levels as a proliferation and prognostic marker in STS patients.
Most of the patients in this study had relatively low preoperative TK1 values; only 38% were above the reference range. Generally, values did not decline significantly after surgery, except for those patients, where TK1 was above the reference range before surgery. A few patients had moderately increased TK1 values after surgery without any obvious explanation.
The predictive and prognostic role of pre- and postoperative serum TK1 levels has been studied in some solid tumors, however few of these studies have been conducted in exclusively local cancer. In pancreatic cancer, high preoperative serum TK1 levels detected by DiviTum TK1 activity assay associated with worse survival compared to patients with lower serum TK1 activity (14). Similar findings were identified in the subgroup of patients with early-stage pancreatic cancer (stages I and IIA), and also when patients who had received neoadjuvant treatment had been excluded from the analysis. In a recent study from Sweden (31), comprising 104 women with primary breast cancer treated with neoadjuvant chemotherapy, only 25% had serum TK1 values exceeding 0.44 μg/l (close to the upper normal value of 0.41 μg/l used in the present study). The proportion of primary breast cancer patients with serum TK1 values exceeding the 95% range of healthy blood donors was even lower in a study from Israel using two different activity assays, 16% and 17.5% for the Divitum and Liaison assays, respectively (10). These results indicate that serum TK1 may not have sufficient sensitivity in detecting localized solid tumors, particularly when using TK activity-based tests.
The proportion of abnormally high preoperative serum TK1 levels measured by AroCell TK 210 ELISA in our study was similar to what has been published on prostate and breast cancer (20, 30) despite the fact that primary tumors in STS tend to be on average much larger than breast or prostate carcinomas at the time of diagnosis. Analysis of clinical and pathological characteristics did not indicate any significant correlation with TK1 levels in these patients. The proliferation marker Ki-67, unexpectedly, had a negative correlation to circulating preoperative TK1. Since TK1 after surgery in relapse-free patients had at least an equally strong negative correlation to tumor proliferation, tumor derived TK1 could not have been responsible for this association. Other patient related factors, e.g., age might at least partially explain the correlation between Ki-67 and TK1, since TK1 values decline with age (32) and in the present study older patients tended to have both larger and more aggressive tumors with higher Ki-67 indices. There was no correlation between tumor size and TK1 levels is STS. Altogether our results indicate that the number of proliferating tumor cells does not appear to be the main cause of increased TK1 values in STS.
Previous studies have suggested that serum TK1 may reflect not only overall cell proliferation but also the death of proliferating cells, since TK1 values regularly increase during chemotherapy (18, 33), and a large increase has even been associated with improved therapeutic effect (31).
Altogether, the relatively low preoperative TK1 values and the lack of significant associations between TK1 and tumor characteristics indicate that TK1 has limited value in localized STS, despite the fact that many of our patients had large tumors, which were histologically aggressive. TK1 decreased in patients with TK1 above the reference level before surgery, but not in the whole patient cohort, and in a few patients TK1 rose marginally from the reference range into values above the reference limit after surgery without any obvious explanation.
STS are a heterogenous group of different tumor types and their incidence is low, which makes it understandable that our study was limited by a low number of patients in most histological subgroups. Nonetheless, we believe that the total number of patients in this study was sufficient to clarify whether TK1 has the robustness required for a clinical tumor marker to be used in STS.
Conclusion
In conclusion, we could not show any clear association between high preoperative TK1 serum concentration and known prognostic markers in STS, recurrence or survival. Only 38% of preoperative TK1 levels were above the reference range, and abnormal values were generally only moderately raised. While TK1 values above the reference limit decreased after surgery, the great majority of patients had no significant decrease of TK1 following surgery, indicating that factors not related to the tumor were responsible for most of the variation in TK1 levels. Our results indicate that TK1 has insufficient sensitivity as a tumor marker in localized STS.
Acknowledgements
The Authors would like to thank Dr Staffan Eriksson for the help with the preparation of the manuscript and Outi Malkavaara for her excellent work as the study nurse.
Footnotes
Authors’ Contributions
MS and CB designed the study. CB designed the database. CB, ET and RN recruited the patients. KJ analyzed TK1-samples. SJ collected the data. SJ, CB, MS and RN interpreted the results. SJ, MS and CB prepared the manuscript. All Authors approved the final version of the manuscript.
Conflicts of Interest
Carl Blomqvist was a board member and received consultation fees from Arocel during the years 2014-2017. Kiran Jagarlamudi is an employee and stock owner of Arocell.
The other Authors declare no conflicts of interest in relation to this study.
Funding
The study was supported by grants from AroCell and the Cancer Foundation Finland (CB ja MS).
- Received January 16, 2022.
- Revision received February 5, 2022.
- Accepted February 7, 2022.
- Copyright © 2022 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
References
In this issue
Jump to section
Related Articles
Cited By...
- No citing articles found.