Thymidine kinase in clinical chemistry

History

The incorporation of thymidine in DNA was demonstrated around 1950. Somewhat later, it was shown that this incorporation was preceded by phosphorylation and around 1960, the enzyme responsible was purified and characterized. The potential use as a tumor marker was suggested by Gronowitz et al.

Biochemistry

Mammals have two isoenzymes that are chemically very different, TK1 and TK2. The former was first found in fetal tissue, the second was found to be more abundant in adult tissue, and initially they were termed fetal and adult thymidine kinase. Soon it was shown that TK1 is present in the cytoplasm only in anticipation of cell division (cell cycle-dependent) whereas TK2 which is located in mitochondria is cell cycle-independent. TK1 is synthesized by the cell during the S phase of cell division. After cell division is completed, TK1 is degraded intracellularly, so that it does not pass to body fluids after normal cell division. The enzyme suggested as a tumor marker in malignant disease is the cytosolic cell cycle dependent TK1 that is present during cell division in quantities much larger than TK2 and is therefore set free to the circulation in quantities that completely dominate the activity of thymidine kinase in blood and other body fluids.

In addition to cellular TK:s, virus specific thymidine kinases have been identified in Herpes simplex virus, Varicella zoster virus and Epstein-Barr virus. They differ biochemically from thymidine kinase from mammalian cells and are inhibited by specific inhibitors that do not influence the activity of mammalian thymidine kinases. Determination of viral thymidine kinase has been suggested for confirmation of diagnosis and for control of treatment of viral infections.

+ ATP ---> + ADP

Thymidine reacts with ATP to give thymidine monophosphate and ADP.

Physiological context

Thymidine monophosphate, the product of the reaction catalyzed by thymidine kinase, is in turn phosphorylated to thymidine diphosphate by the enzyme thymidylate kinase and further to thymidine triphosphate by the enzyme nucleoside diphosphate kinase. The triphosphate is included in a DNA molecule, a reaction catalyzed by a DNA polymerase and a complementary DNA molecule (or an RNA molecule in the case of reverse transcriptase, an enzyme present in retrovirus). Thymidine monophosphate is produced by the cell in two different reactions - either by phosphorylation of thymidine as described above or by methylation of deoxyuridine monophosphate, a product of other metabolic pathways unrelated to thymidine, by the enzyme thymidylate synthase. The second route is used by the cell under normal conditions, and it is sufficient to supply thymidine monophosphate for DNA repair. When a cell prepares to divide, a complete new set-up of DNA is required, and the requirement for building blocks, including thymidine triphosphate, increases. Cells prepare for cell division by making some of the enzymes required during the division. They are not normally present in the cells and are downregulated and degraded afterwards. Such enzymes are called salvage enzymes. Thymidine kinase 1 is such a salvage enzyme, whereas thymidine kinase 2 is not cell cycle-dependent.

Background

Thymidine kinase is a salvage enzyme that is only present in anticipation of cell division. The enzyme is not set free from cells undergoing normal division where the cells have a special mechanism to degrade the proteins no longer needed after the cell division. In normal subjects, the amount of thymidine kinase in serum or plasma is therefore very low. Tumor cells release enzyme to the circulation, probably in connection with the disruption of dead or dying tumor cells. The thymidine kinase level in serum therefore serves as a measure of malignant proliferation, indirectly as a measure of the aggressivity of the tumor. It is interesting to note that the form of enzyme present in the circulation does not correspond to the protein as encoded by the gene: the gene corresponds to a protein with molecular weight around 25 kD. It is a dimer with a molecular weight of around 50 kD, if activated by ATP a tetramer with molecular weight around 100 kD. The main fraction of the active enzyme in the circulation has a molecular weight of 730 kD and is probably bound in a complex to other proteins.

Measurement

The level of thymidine kinase in serum or plasma is so low that the measurement is best based on the enzymatic activity. In commercial assays, this is done by incubation of a serum sample with a substrate analogue. The oldest commercially available technique uses iodo-deoxyuridine (idoxuridine) wherein a methyl group in thymidine has been replaced with radioactive iodine. This substrate is well accepted by the enzyme. The monophosphate of iodo deoxyuridine is adsorbed on aluminum oxide that is suspended in the incubation medium. After decantation and washing the radioactivity of the aluminum oxide gives a measure of the amount of thymidine kinase in the sample. Kits using this principle are commercially available from the companies Immunotech/Beckman and DiaSorin.

A non-radioactive assay method has been developed by the company Dia-Sorin. In this technique 3'-azido-2',3'-deoxythymidine (Zidovudine, AZT) is first phosphorylated to AZT 5'-monophosphate (AZTMP) by TK1 in the sample. AZTMP is measured in an immunoassay with anti-AZTMP antibodies and AZTMP-labeled peroxidase. The assay runs in a closed system on the laboratory robot from DiaSorin. The DiviTum assay from Biovica International uses another thymidine analogue, bromodeoxyuridine, as substrate to the enzyme. The product of the reaction is further phospholylated to tri-phosphate and incorporated in DNA-strings of polythymidine. The polythymidine binds to strings of polyadenine coupled to the bottom of the wells in microtiter plates. There it is detected with ELISA technique: The wells are filled with a solution of a monoclonal antibody to bromo-deoxyuridine. The monoclonal antibody has been bound (conjugated) to the enzyme alkaline phosphatase. After the unbound antibody with attached alkaline phosphatase has been washed away, a solution of a substrate to the alkaline phosphatase, para-nitrophenylphosphate, is added. The product of the reaction, para-nitrophenol, is yellow at alkaline pH and can be measured by photometry. This method has been evaluated against the previous radioactive technique. It gives a considerably more sensitive determination than the previous methods and is therefore suitable for use with solid tumors that give less elevation of the thymidine kinase in body fluids. Comparisons of the methods have been published. A variation of the technique is using homogeneous ELISA for the measurement. Direct determination of the thymidine kinase protein by immunoassay has also been used. This method does not correlate well with the methods measuring the enzymatic activity. One explanation is that the method also measures protein that is enzymatically inactive and that the degree of inactivation varies for different sources of thymidine kinase in the body.

A microchip electrophoresis immunoaffinity assay for determination of serum thymidine kinase concentration has been described. Its function was demonstrated using recombinant TK1. It is claimed to be fast and simple to perform.

Hematologic malignancies

The most dramatic increases of serum thymidine kinase are seen in hematologic malignancies. The differences between the increases in hematologic malignancies and solid tumors are so large that they have been classified as different by kind, not only by degree.

Non-Hodgkin lymphoma

The main use of serum thymidine kinase activity assay now is in non-Hodgkin lymphoma. This disease has a wide range of aggressivity, from slow-growing indolent disease that hardly requires treatment to highly aggressive, rapidly growing forms that should be treated urgently. This is reflected in the values of serum thymidine kinase activity, that range from close to the normal range for slow-growing tumors to very high levels for rapidly growing forms.

Leukemias

Leukemias normally do not present major diagnostic difficulties, as the microscopic analysis of the cells in blood mostly give unequivocal results. Thymidine kinase, however, may give supplementary information about the aggressivity and the risk for progression.

Myeloma

Also myelomas often constitute a diagnostic challenge. The malignant cells are often not available for microscopic analysis, and the prognosis is often uncertain. Therefore, information on the prognosis may be essential in the decision of the treatment. Several studies verify the close connection between prognosis and thymidine kinase activity in myelomas.

Myelodysplastic syndrome

A very interesting case is the myelodysplastic syndrome: Some rapidly change to acute leukemia, whereas others remain indolent for very long time. Identification of those tending to change to overt leukemia is important for the treatment. The relationship between the prognosis and the thymidine kinase value in myelodysplastic syndrome has been demonstrated.

Solid tumors

Also solid tumors give increased values of thymidine kinase activity in serum. The increases for solid tumors are not as large as they are for hematologic malignancies. The first methods for determination of thymidine kinase activity in serum had a limited sensitivity. In the case of the methods employing radioactivity one reason was that the quantity of radioactivity allowed by law in normal radioimmunoassay laboratories has strict limitations. The experimental method first developed by Gronowitz et al. used quantities of radioisotope much above those used in commercial radioassays and therefore the sensitivity was sufficient to detect increases also from solid tumors. With commercial radioimmunoassays this was difficult, and the results were not very convincing. Later non-radioactive techniques give higher sensitivity allowing the lower increases from solid tumors to be measured accurately.

Lung cancer

Lung cancer is one of the major tumor localizations, both by incidence (about 15% for both men and women in USA and in Europe) and by mortality (25% for women and 30% for men). One major reason why the mortality lies higher than corresponding to the incidence is that lung cancer is mostly detected and diagnosed at a late stage. Early detection would reduce the mortality. Another reason is that lung cancer, particularly small cell lung cancer, is very aggressive with very low 5 year survival rates.

There are several reports of the utility of thymidine kinase activity measurements in serum in lung cancer.

Breast cancer

Breast cancer is the largest cancer form in women by incidence (about 25% of cancer cases in USA and Europe) and the second largest form by mortality (about 15%). The reason for this difference is the advances during the last decennia in the treatment of breast cancer cases and, above all, the public awareness that has allowed earlier diagnosis. One contributing factor is the widespread use of mammography for early detection, self-examination is another.

Many tumor markers including thymidine kinase are used for the follow-up and detection of recurrences in breast cancer patients.

Prostate cancer

Among men, prostate cancer is by far the most common cancer form, with an incidence that corresponds to about 25% of the total cancer incidence among men in USA and Europe. The mortality is much lower than would be expected from the incidence, around 10% of the total cancer mortality of men in USA and Europe. A major reason for the lower mortality is that many prostate cancers grow slowly so that the patients do not die from this cancer but from other unrelated reasons.

In the management of prostate cancer, it is therefore very important to be able to discriminate between slowly and rapidly growing cancers. Thymidine kinase has been suggested as a supplement to PSA (Prostate Specific Antigen), the tumor marker most frequently used in prostate cancer. Whereas PSA is considered to give an indication of the tumor mass, thymidine kinase activity indicates the rate of proliferation and the markers thus supplement each other.

Other localizations

The use of TK has also been reported in the following localizations:

kidney cancer, bladder cancer, gastric cancer, liver cancer, neurological cancers and melanoma.

Non-malignant elevations

There are several non-malignant causes for elevation of thymidine kinase in serum including vitamin B12 deficiency, leading to pernicious anemia viral infections (particularly by virus from the herpes group) and wound healing after trauma and operation.

Thymidine kinase in domestic animals

There are also reports of the use of thymidine kinase as a tumor marker in domestic animals, in horse, in dogs in cats and in cows. Elevations in dogs with bacterial infections have also been reported.

Thymidine kinase in tissue

Thymidine kinase has been determined in tissue samples after extraction of the tissue and a relation between the results and disease progression has been shown. However, no standard method for the extraction or for the assay has been developed and TK determination in extracts from cells and tissues have not been validated in relation to any specific clinical question, see however Arnér et al. Romain et al. and Alegre et al.

In the studies referred to below the methods used and the way the results are reported are so different that comparisons between different studies are not possible.

The TK1 levels in fetal tissues during development are higher than those of the corresponding tissues later.

Certain non-malignant diseases also give rise to dramatic elevation of TK values in cells and tissue: in peripheral lymphocytes during monocytosis and in bone marrow during pernicious anemia. As TK1 is present in cells during cell division, it is reasonable to assume that the TK activity in malignant tissue should be higher than in corresponding normal tissue. This is also confirmed in most studies: a higher TK activity is found in neoplastic than in normal tissue, in brain tumors, in hematological malignancies, in cancer and polyps in colon, in breast cancer, in lung cancer, in gastric cancers, in ovarian cancer, in mesotheliomas, in melanomas, in thyroid tumors in leukemia and in breast cancer.

Therapy that influences the rate of cell proliferation influences the TK values correspondingly. Although most studies do not show this, it seems probable that differences between samples from healthy tissue and samples from tumor tissue primarily represents changes in the levels of TK1, since this enzyme is much more strongly coupled to cell proliferation than TK2.

A method has been developed for specific determination of TK2 in cell extracts using the substrate analogue 5-Bromovinyl 2'-deoxyuridine.

Uses of thymidine kinase determinations

Tumor markers may be used for the following purposes