Document Detail

Elevated levels of thymidine kinase 1 peptide in serum from patients with breast cancer.
Jump to Full Text
MedLine Citation:
PMID:  19396699     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
OBJECTIVES: Thymidine kinase (TK) has an important role in DNA synthesis and is thus related to cell proliferation and turn-over. Traditionally, TK has been measured by enzymatic activity or radioimmunoassays. These assays are difficult to adapt to random access instruments. The aim of this study was to evaluate a new immunological sandwich assay for detection of TK peptides in serum from breast cancer patients.
MATERIAL AND METHODS: Serum samples were collected from patients with breast cancer and stored frozen at -70 degrees C. The samples were collected after surgery, after metastatic tumor recurrence and after chemotherapy due to tumour recurrence. Patients' serum samples were analysed by the TK enzyme-linked immunosorbent assay (ELISA).
RESULTS: In receiver operating characteristics (ROC) analyses of TK1 for diagnosis of breast cancer, the area under the curve (AUC) collected four weeks after surgery was 0.56 (95% CI 0.47-0.65), for samples collected postsurgically after tumour recurrence 0.73 (95% CI 0.65-0.80), and after chemotherapy 0.64 (95% CI 0.56-0.72).
CONCLUSIONS: This study indicates that the tumour proliferation marker TK has a potential as a serum marker in breast cancer. Further studies are warranted to verify this observation.
Authors:
Lena Carlsson; Anders Larsson; Henrik Lindman
Related Documents :
12691319 - The contribution of the cea marker to ca 15.3 in the follow-up of breast cancer.
8322709 - Metastatic adenocarcinoma of an unknown primary site. a comparison of the relative cont...
1780689 - Tumor-associated trypsin inhibitor (tati) in patients with colorectal carcinoma. a crit...
15373709 - Variation of tumor markers in 277 breast cancer cases.
11381189 - Clinical significance and prognostic value of ca72-4 compared with cea and ca19-9 in pa...
3163149 - A clinical evaluation of monoclonal (ca19-9, ca50, ca12-5) and polyclonal (cea, tpa) an...
18592369 - Lobular involution: localized phenomenon or field effect?
17311529 - Genetics of preneoplasia: lessons from lung cancer.
14568079 - Diterpenes from alomia myriadenia (asteraceae) with cytotoxic and trypanocidal activity.
Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Upsala journal of medical sciences     Volume:  114     ISSN:  2000-1967     ISO Abbreviation:  Ups. J. Med. Sci.     Publication Date:  2009  
Date Detail:
Created Date:  2009-04-27     Completed Date:  2009-07-20     Revised Date:  2013-06-02    
Medline Journal Info:
Nlm Unique ID:  0332203     Medline TA:  Ups J Med Sci     Country:  Sweden    
Other Details:
Languages:  eng     Pagination:  116-20     Citation Subset:  IM    
Affiliation:
Department of Medical Sciences, Clinical Chemistry, University Hospital, Uppsala, Sweden. Lena.carlsson@medsci.uu.se
Export Citation:
APA/MLA Format     Download EndNote     Download BibTex
MeSH Terms
Descriptor/Qualifier:
Breast Neoplasms / blood,  enzymology*
Enzyme-Linked Immunosorbent Assay
Female
Humans
Peptide Fragments / blood*
ROC Curve
Sensitivity and Specificity
Thymidine Kinase / blood*,  chemistry
Tumor Markers, Biological / blood*
Chemical
Reg. No./Substance:
0/Peptide Fragments; 0/Tumor Markers, Biological; EC 2.7.1.21/Thymidine Kinase; EC 2.7.1.21/thymidine kinase 1
Comments/Corrections

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine

Full Text
Journal Information
Journal ID (nlm-ta): Ups J Med Sci
Journal ID (publisher-id): UPS
ISSN: 0300-9734
ISSN: 2000-1967
Publisher: Informa Healthcare
Article Information
Download PDF
? Upsala Medical Society
open-access:
Received Day: 05 Month: 12 Year: 2008
Accepted Day: 10 Month: 12 Year: 2008
Print publication date: Month: 6 Year: 2009
Electronic publication date: Day: 24 Month: 4 Year: 2009
Volume: 114 Issue: 2
First Page: 116 Last Page: 120
PubMed Id: 19396699
Publisher Id: UPS_A_369053_O
DOI: 10.1080/03009730802688835

Elevated levels of thymidine kinase 1 peptide in serum from patients with breast cancer
Lena Carlsson1
Anders Larsson1
Henrik Lindman2
1Department of Medical Sciences, Clinical Chemistry, University HospitalUppsalaSweden
2Department of Oncology, University HospitalUppsalaSweden
Correspondence: Correspondence: Lena Carlsson, Department of Clinical Sciences, Clinical Chemistry, University Hospital, SE-751 85 Uppsala, Sweden. E-mail: Lena.carlsson@medsci.uu.se

Introduction

Thymidine kinase (TK) (EC.2.7.1.21) is an enzyme in the pyrimidine salvage pathway and catalyses the phosphorylation of thymidine to thymidine monophosphate (1). There are two forms of TK: a cytosolic (thymidine kinase 1 (TK1)) and a mitochondrial one (TK2) (2). The level of TK1 is very low in non-proliferating cells but increases dramatically at late G1 to late S-phase/early G2 phase during the cell cycle in proliferating cells and tumour cells. This makes TK1 an interesting marker for cell proliferation and tumour growth. In patients with malignancies more than 95% of the TK1 activity in serum is derived from the malignant cells (3). Thus, serum TK1 should be a good marker for tumour cell proliferation. Serum TK1 activity has been used to monitor the extent of tumour metastasis and prognosis in patients with acute leukaemia, chronic leukaemia, Hodgkin's and non-Hodgkin's lymphoma, bladder carcinoma, and cervical carcinoma (4?10). The most widely used TK1 assays are radioimmunoassay techniques, discouraging their routine clinical use. It is therefore important to find non-radioactive alternatives to these assays. Both monoclonal and polyclonal antibodies are available against TK1. One of these antibodies is a chicken anti-TK1 polyclonal antibody raised against a 31-amino acid synthetic peptide (GQPAG PDNKE NCPVP GKPGE AVAAR KLFAPQ, residues 194?225) corresponding to the C-terminal part of human TK1 (3). This antibody has been used to develop a sandwich enzyme-linked immunosorbent assay (ELISA) for TK1.

Breast cancer incidence has approximately doubled compared with 1960. The number of new breast cancer cases in Sweden in the year 2001 was 6,569, resulting in an age-adjusted incidence of 138 per 100,000 women (11). The number of deaths from breast cancer was 1,487, giving a mortality rate of 30 per 100,000. Breast cancer represented 29% of all new cancers in women, and for a Swedish woman the lifetime risk of developing breast cancer is now above 12% (11). Most new breast cancers are detected in women aged 55?59 years. The incidence of and mortality from breast cancer is markedly higher in Western countries than in Asian or African countries (12). The average annual age-standardized breast cancer rate increase in Sweden has been 1.8% during the last decade, but the mortality figures have slowly decreased and are, despite the high incidence, one of the lowest in Europe (13). The annually increasing cure rate, since the late 1980s also observed in the United States and the United Kingdom, could have many explanations, but earlier diagnosis and more efficient adjuvant therapies are corner-stones that cannot be neglected (14).

A proliferation marker such as TK1 could theoretically be used to distinguish between those cancers that proliferate slowly and more malignant forms that show rapid growth. The present report is the first clinical evaluation of a new sensitive and specific immunoassay (enzyme-linked immunosorbent assay, ELISA) for determination of thymidine kinase 1 in serum from breast cancer patients.


Material and methods
Patients? sera and control sera

Blood samples were collected from patients with breast cancer treated at the Department of Oncology, Uppsala University Hospital. Twenty-four samples were collected four weeks after surgery, 39 samples were collected after postsurgical tumour recurrence, and 41 samples were collected after chemotherapy. A group of 100 healthy blood donors from Uppsala University Hospital Blood Bank were used as controls. This study was approved by the local Ethics Committees.

TK1 enzyme-linked immunosorbent assay (ELISA)

Samples were analysed using an ELISA kit for thymidine kinase (TK1) (Arocell, Uppsala, Sweden). Briefly, the microtitre plates had been coated with affinity-purified polyclonal chicken antibodies specific for TK1 peptide, and the first step was to add standards and samples to the wells. During the subsequent incubation period TK1 present in standards and samples was bound to the immobilized antibody.

The plate was washed four times, and a biotinylated polyclonal antibody specific for TK1 was added. After incubation and four washes, enzyme-labelled streptavidin was pipetted into the wells, and, following an incubation step and four washes, a substrate solution was added and colour developed in proportion to the amount of TK1 bound. The colour development was subsequently stopped, and the intensity of colour was measured by spectrophotometry. Calculation of results was performed according to manufacturer's recommendations, and TK1 concentration was expressed in U/L.

Statistical analyses

Receiver operating characteristics (ROC) curves were plotted, and areas under the ROC curves were compared for specificity and sensitivity in the three breast cancer groups. Sensitivity (the ratio of patients with breast cancer who were positive for the variable) and specificity (the ratio of patients without breast cancer who were negative for the variable), negative predictive value (probability that the disease is not present when the test is negative), and positive likelihood ratio (ratio between the probability of a positive test result given the presence of the disease and the probability of a positive test result given the absence of the disease) were also calculated. These analyses were performed on MedCalc Software (Mariakerke, Belgium). Statistical significance was considered at P < 0.05.


Results
Clinicopathological characteristics of the patients

Median value for the samples collected four weeks after surgery was 7.05?U/L (range 0.8?130.8?U/L), the median value for samples collected after postsurgical tumour recurrence was 13.7?U/L (range 0.8?170.1?U/L), and median value for samples collected after chemotherapy was 9.1?U/L (range 0.4?199.8?U/L).

The mean value for the controls was 5.78?U/L (range 1.5?159.4?U/L).

Thymidine kinase 1 ELISA titres

Twenty-four samples were collected four weeks after surgery. The accuracy of the TK1 ELISA to detect breast cancer prior to surgery according to receiver operating characteristics is given in Figure 1. The area under the curve (AUC) for this patient group was 0.56 (95% CI 0.47?0.65, P = 0.34). The sensitivity of the assay was 33.3% and the specificity 89% using a cut-off of 12.3.

Thirty-nine samples were collected upon postsurgical tumour recurrence. The accuracy of the TK1 ELISA to detect breast cancer in this patient group according to receiver operating characteristics is given in Figure 2. The AUC for this patient group was 0.73 (95% CI 0.65?0.80, P < 0.0001). The sensitivity of the assay in this patient group was 64% and the specificity 86% using a cut-off of 11.0.

Forty-one samples were collected after chemotherapy. The AUC after chemotherapy was 0.64 (95% CI 0.56?0.72, P = 0.008) (Figure 3). The sensitivity of the assay in this patient group was 53.7% and the specificity 79.0% using a cut-off of 8.9.


Discussion

TK1 activity is low or absent in resting cells, starts to occur in late G1 cells, increases in S- phase, and disappears during mitosis. The cell cycle regulation makes TK1 a highly interesting marker for cell proliferation and as a marker for tumour growth. Also, TK1-deficient mice have been shown to have increased mutation rates (15). The role of TK1 in tumour growth has led to the use of TK1 as a cancer serum marker.

The present study shows that TK1 can be a potential tumour marker in breast cancer patients. This result could reasonably be explained by the fact that the level and activity of TK1 are highly dependent on the growth state and cell cycle phase and that the transcription of the TK1 gene is strongly induced in response to growth stimulation in the S-phase (16?19).

The assay used in the present study is a non-radioactive immunological alternative to the traditional TK1 radioimmunoassay. The assay could also be adopted to fit the format of high throughput immunoanalysers currently used in clinical laboratories. This would facilitate the use of TK1 as a tumour marker. The anti-TK1 Immunoglobulin Y (IgY) antibody used in the ELISA has previously been characterized (3). Antibodies derived from egg-yolk offer interesting advantages over mammalian antibodies in several aspects and may sometimes be a more suitable choice in designing solid-phase immunometric assays than mammalian antibodies. Chicken antibodies do not activate the human complement system which is a well known source of interference in sandwich immunoassays (20). Capture antibodies bound to a solid surface are potent complement activators, and the activated complement components will react with the assay antibodies thus partly blocking the antigen-binding sites (21). Another advantage is that rheumatoid factors (RF) do not bind to IgY. RF is a major source of interference in many immunoassays, reacting with the Fc portion of mammalian IgG (22). The disease usually associated with RF is rheumatoid arthritis, but RF is also present in blood samples from patients with many other diseases and also healthy individuals (6,7). Most immunoassays use mammalian polyclonal or monoclonal antibodies, which are subjected to RF binding, thus potentially giving false positive results. As RF is not able to bind to IgY, chicken antibodies can be useful in assays (e.g. nephelometry, turbidimetry, or ELISA) where RF could interfere (22). Another interfering factor is human anti-mouse IgG antibody (HAMA). An increasing number of patients are in vivo treated with monoclonal mouse antibodies, and this often provokes an antibody response in the patient resulting in HAMA production. Chicken antibodies do not react with HAMA, so they can be used to eliminate such interference (23,24). Thus, chicken antibodies should theoretically have advantages over mammalian antibodies in immunoassays especially for tumour marker assays as mouse monoclonal antibodies are used for tumour treatment and this treatment increases the prevalence of HAMA (24).

In conclusion, the current study has demonstrated that the serum concentration of TK1 is increased in patients with breast cancer. These findings could provide the basis for future studies of the clinical utility of the assay.


Acknowledgements

This study was financially supported by the Swedish Cancer Society.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.


References
1. Hu CM,Chang ZF. Mitotic control of dTTP pool: a necessity or coincidence?J Biomed Sci.Year: 200714491717525869
2. Al-Madhoun AS,Tjarks W,Eriksson S. The role of thymidine kinases in the activation of pyrimidine nucleoside analoguesMini Rev Med Chem.Year: 200443415015134537
3. Wu C,Yang R,Zhou J,Bao S,Zou L,Zhang P,et al. Production and characterisation of a novel chicken IgY antibody raised against C-terminal peptide from human thymidine kinase 1J Immunol Methods.Year: 20032771576912799048
4. Gowda A,Byrd JC. Use of prognostic factors in risk stratification at diagnosis and time of treatment of patients with chronic lymphocytic leukemiaCurr Opin Hematol.Year: 2006132667216755224
5. Madewell BR. Serum thymidine kinase activity: an alternative to histologic markers of cellular proliferation in canine lymphomaJ Vet Intern Med.Year: 200418595615515571
6. Di RF,Giustolisi R,Lerner S,Cacciola E,O'Brien S,Kantarjian H,et al. Retrospective study of the prognostic role of serum thymidine kinase level in CLL patients with active disease treated with fludarabineAnn Oncol.Year: 200112621511432619
7. Schwartz MK. Enzymes as prognostic markers and therapeutic indicators in patients with cancerClin Chim Acta.Year: 199220677821572080
8. O'Neill KL,Zhang F,Li H,Fuja DG,Murray BK. Thymidine kinase 1?a prognostic and diagnostic indicator in ALL and AML patientsLeukemia.Year: 200721560317252017
9. Zhang J,Jia Q,Zou S,Zhang P,Zhang X,Skog S,et al. Thymidine kinase 1: a proliferation marker for determining prognosis and monitoring the surgical outcome of primary bladder carcinoma patientsOncol Rep.Year: 2006154556116391869
10. Fujiwaki R,Hata K,Moriyama M,Iwanari O,Katabuchi H,Okamura H,et al. Clinical value of thymidine kinase in patients with cervical carcinomaOncology.Year: 200161475411474248
11. Socialstyrelsen. Statistics Health and Diseases 2003:6, Cancer incidence in Sweden. http://www.socialstyrelsen.se/NR/rdonlyres/8D37532A-BC53-4B38-9F06-73CD8BC5AD62/967/2003426.pdf
12. Cancer incidence in five continentsVIIIIARC Sci PublYear: 20021781
13. Ros?n M,Lundin A,Nystr?m L,Rutqvist L,Stenbeck M,Talb?ck M. Incidens och d?dlighet i br?stcancer under 25 ?r. Internationella och regionala j?mf?relserL?kartidningen.Year: 20009729499
14. Peto R,Boreham J,Clarke M,Davies C,Beral V. UK and USA breast cancer deaths down 25% in year 2000 at ages 20?69 yearsLancet.Year: 2000355182210832853
15. Dobrovolsky VN,Bucci T,Heflich RH,Desjardins J,Richardson FC. Mice deficient for cytosolic thymidine kinase gene develop fatal kidney diseaseMol Genet Metab.Year: 20037811012559842
16. Bello LJ. Regulation of thymidine kinase synthesis in human cellsExp Cell Res.Year: 197489263744457349
17. Chang ZF,Huang DY,Lai TC. Different regulation of the human thymidine kinase promotor in normal human diploid IMR-90 fibroblasts and HeLa cellsJ Biol Chem.Year: 19952702737497593001
18. Munch-Petersen B,Cloos L,Jensen HK,Tyrsted G,Human thymidine kinase 1Regulation in normal and malignant cellsAdv Enzyme RegulYear: 19953569897572355
19. Roehl HH,Conrad SE. Identification of a G1-S-phase-regulated region in the human thymidine kinase gene promotorMol Cell Biol.Year: 199010383472355925
20. K?pyaho K,Tanner P,Weber T. Effect of complement binding on a solid-phase immunometric TSH assayScand J Clin Lab Invest.Year: 19894921152662381
21. Carlander D,Larsson A. Avian antibodies can eliminate interference due to complement activation in ELISAUps J Med Sci.Year: 20011061899512166511
22. Larsson A,Karlsson-Parra A,Sjoquist J. Use of chicken antibodies in enzyme immunoassays to avoid interference by rheumatoid factorsClin Chem.Year: 19913741142004449
23. Larsson A,Sjoquist J. Chicken IgY: utilizing the evolutionary differenceComp Immunol Microbiol Infect Dis.Year: 1990131992012076606
24. Larsson A,Mellstedt H. Chicken antibodies: a tool to avoid interference by human anti-mouse antibodies in ELISA after in vivo treatment with murine monoclonal antibodiesHybridoma.Year: 1992113391737638

Figures

[Figure ID: F0001]
Figure 1. 

Receiver operating characteristics (ROC) curves for thymidine kinase 1 (TK1) analysed in serum samples from patients with breast cancer (n=24) collected four weeks after surgery in comparison with healthy controls (n=100). Area under the curve (AUC) = 0.56, 95% confidence interval (CI) 0.47?0.65, P=0.34.



[Figure ID: F0002]
Figure 2. 

Receiver operating characteristics (ROC) curves for thymidine kinase 1 (TK1) analysed in serum samples from patients with breast cancer (n=39) collected after metastatic tumour recurrence after surgery in comparison with healthy controls (n=100). Area under the curve (AUC) = 0.73, 95% confidence interval (CI) 0.65?0.80, P<0.0001.



[Figure ID: F0003]
Figure 3. 

Receiver operating characteristics (ROC) curves for thymidine kinase 1 (TK1) analysed in serum samples from patients with breast cancer (n=41) collected after chemotherapy in comparison with healthy controls (n=100). AUC = 0.64, 95% CI 0.56?0.72, P = 0.0076.



Article Categories:
  • Original Article

Keywords: Antibodies, breast cancer, immunological sandwich assay, thymidine kinase.

Previous Document:  Thrombospondin 1 expression and angiogenesis in breast carcinoma and their relation with platelet ac...
Next Document:  Langerhans cell histiocytosis of the sternum.