Total cell division in cancer patients measured through a new thymidine kinase assay based on BrdU incorporation into DNA. Basic features of the assay and serum TK, and clinical relevance illustrated in breast, renal, lung and prostate cancer

Konference: 2011 XXXV. Brněnské onkologické dny a XXV. Konference pro sestry a laboranty

Kategorie: Nádorová biologie/imunologie/genetika a buněčná terapie

Téma: Pokroky v biologii nádorů

Číslo abstraktu: 138

Autoři: J. Simon Gronowitz


Theory: Cytosolic thymidine kinase (TK1) is the key enzyme for dTTP (deoxy-thymidine-tri-phosphate) synthesis via the salvage pathway. It catalyzes the transfer of the terminal phosphate of ATP to the 5´ hydroxy l group to form dTMP which is then phosphorylated to TTP by thymidylate kinase and TDP-kinase. The synthesis of TTP is highly regulated to be in consort with the replication of the nuclear DNA during the G1-S stage and it is assumed that the TTP pool regulates the pools of the other three deoxynucleotides in order to obtain fidelity in the DNA synthesis (Munch-Petersen et al 1995, Hu, 2007). After DNA replication is completed, dTTP formation is no longer in great demand in G2/M phase, where TK1 becomes phosphorylated with a low catalytic efficiency as a result. In mitotic exit, TK1 is identified by its KEN-box and degraded via the anaphase-promoting complex/cyclosome (APC/C) pathway, i.e. the ubiquitin ligase, to limit dTTP production (Hu 2007, Kraft 2003, Ke 2004, 2007, Pfleger and Kirschner 2000). However, a recent study showed that TK is re-expressed during G2 repair of DNA in tumour cells, but not in normal cells (Chen et al 2010). The TK1 gene codes for a 24 kD protein, while the s-TK found in sera has an apparent molecular weight of 730kD (Karlström et al 1990). The nature of this complex has not yet been elucidated, albeit earlier reports indicate that TK1 may act in close contact with other replication associated enzymes in order to speed up the catalytic processes (Wickremasinge et al, 1983, li et al 2003). The TK1 gene is located on chromosome 17q25, while c-erbB2 and BRCA1 are located at 17q21. Inherited mutated BRCA1 gene occurs in breast and ovarian carcinoma but also in prostate carcinoma and its gene product seems to be an E3 ubiquitin ligase that has an impact among other on DNA repair and cell-cycle progression (Boulton 2006). Deletions in the KEN-box of TK1 lead to a less processed, and thus a more stable, enzyme which has been shown by a prolonged half-life (Zhu et al 2006). Further, the processing of the TK protein is affected both by phosphorylation (Chang and Huang 1993, Chang et al 1994) and presence of substrate thymidine/ATP (Li et al 2003, Ke et al 2007). Our recent results show that healthy women with BRCA mutations have in addition to the high risk of developing breast carcinoma also significantly higher s-TK levels than healthy women without BRCA mutation – These results are in analogy with what was earlier found in myelodysblastic syndrome (Musto 1995).

Clinical: Measurements of s-TK activity for whole body cell proliferation have been possible since the 1980´s. The original method based on conversion of 125I-deoxyUridine into its monophosphate is still used, but has a limited sensitivity and has thus been mainly applied to hematological malignancies (Gronowitz et al 1983, 1984, Källander et al 1984, Morgan et al 1985, Poley et al 1997). In addition, presence of pathological levels in metastasized semi-solid and solid cancers (Gronowitz et al 1986, Topolcan et al, 2006, Svobodova et al 2007) can be detected and have been shown to be highly prognostic (Gronowitz et al 1990; Mitzutani et al 2002).
Another example of how cell proliferation determination by s-TK activity measurements can be used in clinical practice is a study of children with premalignant lesions, for instance myelodysblastic syndrome. Only those children that at the same time expressed high s-TK were those who developed leukemia (Musto et al 1995). An alternative non radioactive assay for serum TK activity based on phosphorylation of AZT was recently launched by Diasorin, however, this assays is not more sensitive than the old radioactive assay. In addition, clinical results have been published for different cancers during the years using experimental Elisa assays for measuring the TK protein in sera. However, these assays give a smaller TK range for a given tumour type and do not correlate very well to assays measuring the enzymatic assay.
There is ample documentation that s-TK is an important cell proliferation marker, and that elevated levels are highly indicative of metastases, poor prognoses and that they are useful for the monitoring of therapy intervention. However, as a biomarker of cell division it should be noted that both transient and chronic s-TK elevations can be caused by non malignant diseases. I.e. trauma due to e.g. surgery, acute allergy attack or other cell killing infections. In these cases a transient increased s-TK is observed which normalizes rapidly upon the healing of the trauma. Chronic increase has been reported for autoimmune disease with a high cell turnover e.g. rheumatoid arthritis. These are facts which should be taken into consideration when using s-TK measrements on a routine basis within the clinic.

Scope: To present the DiviTum assay with its 100 times higher analytical sensitivity, based on incorporation of the thymidine analog BrdU into a growing DNA-strand (Gronowitz 2006) and its features and application to characterization of serum TK expressed upon different disease both malignant and non-malignant. In addition, reference levels for healthy donors and recently published data(Nisman et al 2010, 2011) comparing serum TK to other clinical parameters that address both prediction and therapy follow-up of cancer patients, will be detailed and presented.

Results: From the research based invention of 2005-2006 a standardized ATP dependent CE-labeled TK assay kit was developed as well as a CTP dependent variant for measurement of mitochondrial TK. Comparison of the novel assay to old radioactive technique and the AZT dependent technique showed a more than hundredfold higher detection sensitivity of TK activity using this novel assay. For serum analyses, normally having a reference level, a tenfold increase in sensitivity was unexpectedly found. This increase, which allows for the early detection of solid tumour growth seems to reside in much lower signals from healthy donors, i.e. approximately ten times less cell division can be significantly detected. Applying the increased sensitivity to basic studies showed that the s-TK activity induced in cancer is mainly of TK1 type and contained in a 700kD complex. Only minor increases in the CTP active mitochondrial s-TK with an apparent Mw less than 60kD were detected, leading to the conclusion that the TK released to the bloodstream is not due to cell death but rather to the mitotic exit. This was in part verified in another study using cell culture showing that S-TK measurement was different in cell proliferation contrary to apoptosis. Further, novel data indicates that the difference in sensitivity to detect tumour cell division between different assays may reside in capacity to measure G2 re-expressed TK.
Analysis of a blind coded cohort of sera from blood donors equally distributed with reference to sex and age in the span between 18-80 years of age showed no differences in s-TK activity. Follow-up of a limited number of healthy, as well as of cured cancer patients, show that the s-TK level over time is very constant for each individual. It should however be noted that a fraction of healthy women carrying BRCA 1 and 2 mutations at risk for developing breast carcinoma were found to have significantly higher S-TK levels than other women.
Analyses of the test, for the prediction of tumour recurrence in patients with localized breast- or renal- carcinoma, showed that the pre-surgery value efficiently predicted recurrence using a s-TK cut off at least 2-3 times higher than the upper normal limit. Further, s-TK was found to be the strongest predictor of recurrence in breast carcinoma when compared with current markers such as staging, histopathology, ER, PR and CA15-3. In the multivariate model only the size of the primary tumour gave additional information to s-TK. Considering the fact that these patients were defined to have local disease after costly imaging it is deduced that the s-TK level upon detection of solid cancer can be used for stratifying patients to extra imaging or not. The relation between high s-TK and spread of solid cancer is further illustrated by results for patient with prostatic carcinoma, i.e. where positivity in bone scintillography correlated to s-TK levels 2-3 times that of the upper normal.
Monitoring of patients, judged to have localized breast carcinoma post surgery, showed increasing s-TK levels for those with recurrence, while low constant levels were present for those without. Lead time between increase in s-TK and clinical symptoms varied between patients with more or less dedifferentiated tumour and so did the s-TK level which could be more than 40 times upper normal level when symptoms were presented. Spread cancers e.g. non-small lung cancer often presented with 10-50 fold increase in s-TK making this analyses very useful for following therapy intervention and for judgement of residual disease activity when terminating treatment. The response upon efficient therapy is fast in s-TK levels, as the half life of s-TK in serum is less than two days Results from lung cancer studies show that the decrease in e.g. tumour marker CA-125 comes many weeks later than the decrease in s-TK. This is probably due to the fact that CA-125 relates to tumour volume rather than cell division, in the same way as CA 15-3 relates to volume of tumour in breast carcinoma. Similar delay in answer about therapy response is naturally present for imaging with judging reduction in tumor volume. From monitoring both breast and lung cancer patients it was found that certain therapies induce high s-TK levels, i.e. folate/thymidylate interfering therapy such as pemetrexed, gemcitabine, 5-fluorouracil and methotrexate dependent on administration. Normalization in s-TK occurred rapidly after setting out such therapy. Further, transient increase in s-TK in connection with trauma caused by surgery will be illustrated and discussed as well as non-malignant diseases causing increased cell division,
Conclusions. It is concluded based on current and earlier results regarding thymidine kinase that the novel TK assay fills a gap within clinical chemistry being the first assay sensitive enough for early detection of pathological cell division. Such increased cell division may be transient after trauma, during wound healing, after trauma caused of various reasons, or chronic. A chronic increase may either be due to a nonmalignant cell consuming disease like rheumatoid arthritis or cancer. Cell proliferation being the hallmark of cancer and the major target for treatment, when a tumour cannot be excised, is an optimal biomarker. S-TK can be used for measuring disease activity, disease aggressivity and to enable personalized medicine, which is becoming more important in view of additional treatment options. Being a broad marker of cell division it is concluded that DiviTum complements current ´tumour markers´ which are differentiation and volume related and DiviTum should always be interpreted in context with other clinical findings. Finally it is concluded that the measurement of total cell division adds a new dimension to clinical cancer research and can in a cost efficient manner contribute upon clinical trials of new drugs and combinations thereof.

  1. Boulton J. Cellular functions of the BRCA tumour-suppressor proteins. Biochem.Soc.Trans. 2006:34(5):633-45.
  2. Chen YL, Eriksson S and ZF Chang. The regulation and functional contribution of thymidine kinase 1 in repair of DNA damage. J. Biol.Chem. 2010:285:27327-335.
  3. Chang ZF and DY Huang The regulation of thymidine kinase in HL-60 human promyeloleukemia cells. JBC 268:1266-71, 1993.
  4. Chang ZF, Huang DY, Hsue NC. Differential phosphorylation of human thymidine kinase in proliferating an M phase-arrested cells. JBC 269:21249-21254, 1994.
  5. Gronowitz J.S., Hagberg H., Källander C.F.R. and B. Simonsson. The use of serum deoxythymidine kinase as a prognostic marker, and in the monitoring of patients with non-Hodgkins´ lymphoma. Brit.J.Cancer 47:487-495, 1983.
  6. Gronowitz J.S., Källander C.F.R.,Diderholm H., Hagberg H. and U. Petterson. Application of an in vitro assay for serum thymidine kinase: results on viral disease and malignancies in humans. Int.J.Cancer, 33:5-12, 1984.
  7. Gronowitz J.S., Steinholtz L., Källander C.F.R., Hagberg H. and J. Bergh. Serum deoxythymidine kinase in small-cell carcinoma of the lung; relation to clinical features, prognosis, and other biochemical markers. Cancer 58:111-118, 1986.
  8. Gronowitz J.S., Bergström R, Nou E, Pahlman S, Brodin O, Nilsson S, Källander C.F.R. Clinical and serological markers of stage and prognoses in kinase in small-cell carcinoma of the lung; A multivariate analysis. Cancer 66:722-732, 1990.
  9. Gronowitz JS. A method and kit for determination of thymidine kinase activity and use thereof. European patent EP 1 856 275 B1 Patent filed 24.02.2006, certificate 12.08.2009.
  10. Hu CM, Chang ZF. Mitotic control of dTTP pool: a necessity or coincidence? J. Biomed.Sci 2007:14(4):491-7. E-pub 2007 May 25.
  11. Karlström A.R. Neumüller M., Gronowitz J.S. and C.F.R. Källander. Molecular forms in human serum of enzymes synthetizing DNA precursors and DNA. Mol.Cell.Biochem. 92:23-35, 1990.
  12. Ke PY, Hu CM, Chang YC, Chang CF. Hiding human thymidine kinase 1 from APC/C mediated destruction by thymidine binding. FASEB J. 21(4):1276-84, 2007.
  13. Ke PY, Chang ZF. Mitotic degradation of human thymidine kinase 1 is dependent on the anaphase-promoting complex/cytosome-CDH1-mediated patway. Mol.Cell.Biol. 2004:24(2):514-26.
  14. Ke PY, Hu CM, Chang YC, Chang ZF. Hiding human thymidine kinase from APC/C-mediated destruction by thymidine binding. FASEB: 2007:21(4):1276-84.
  15. Kraft C, Herzog F, Gieffers C, Mechtler K, Hagting A, Pines J, Peters JM. Mitotic regulation of the human anaphase-promoting complex by phosphorylation. Embo J. 15:22(24):6598-6609, 2003.
  16. Källander C.F.R, Simonsson B., Hagberg H., and J.S. Gronowitz. Serum deoxythymidine kinase gives prognostic information in chronic lymphocytic leukemia. Cancer 54:2450-2455, 1984.
  17. Li CL, Lu CY, Ke PY, Chang ZF. Perturbation of ATP-induced tetramerization of human cytosolic thymidine kinase by substitution of serine-13 with aspartic acid at the mitotic phosphorylation site. Biochem.Biophys.Res. Commun 313:587-593, 2003
  18. Morgan M. A. M., Cooper E.H., Bailey C.C., Gronowitz J.S. and C.F.R. Källander. Serum deoxythymidine kinase in acute lymphoblastic leukaemia in children. TumourDiagnostik & Therapie 6:155-158, 1985.
  19. Munch-Petersen B, Cloos L. Jensen HK, Tyrstedt G. Human thymidine kinase 1. Regulation in normal and malignant cells. Adv.Enzyme.Regul. 35:69-87, 1995.
  20. Musto P, Bodenizza C, Falcone A, D´Arena G, Scalzulli P, Perla G, Modoni S, Parlatore L, Valvano MR, Carotenuto M. Prognostic relevance of serum thymidine kinase in primary myelodysplastic syndromes: relationship to development of acute myeloid leukaemia. Br J Haematol. 1995 May;90(1):125-30.
  21. M2 pyruvate kinase and thymidine kinase 1 are potential predictors for disease recurrence in renal cell carcinoma after nefrectomy. Urology 2010:76(3):513-18.
  22. Nisman B, Tanir Allweis, Luna Kaduri, Bella Maly, Simon Gronowitz, Tamar Hamburger, Tamar Peretz1. Serum thymidine kinase 1 activity in breast cancer. Cancer Biomarkers 2010:7: 65-72.
  23. Nisman B, Tanir Allweis, Luna Kaduri, Simon Gronowitz, Tamar Peretz. Serum thymidine kinase 1 activity is elevated in healthy BRCA1 and BRCA2 mutation carriers. Manuscript 2011
  24. Petterson I, Shao X, Gronowitz S, Källander C. (2001) A method for measuring DNA polymerisation and applications of the method. US-provisonal 60/297,773. June 2001,
  25. PCT/SE02/01155 (WO 02/103039 A1) designation December 2003
  26. Pfleger, C.M. and Kirschner, M. W. (2000) The KEN box: an APC recognition signal distinct from the D box targeted by Cdh1. Genes & Development 14: 655-665.
  27. Poley S, Stieber P, Nussler V, Pahl H, Fateh-Moghadam A. Serum thymidine kinase in non-Hodgkin lymphomas with special regard to multiple myeloma. Anticancer Res; 17(4B):3025-29, 1997
  28. Svobodova S, Topolcan O, Holubec L, Treska V, Sutnar A, Rupert K, Kormunda S, Rousarova M, and Finek J Prognostic importance of thymidine kinase in colorectal and breast cancer. Anticancer research 27(4A):1907-9, 2007
  29. Topolcan O, L Holubec, S Svoboda, V Treska, OJ Wolfe, Finek J. The diagnostic and prognostic significance of thymidine kinas in tumor diseases. J. Clin. Ligand. Assay 2006:29(4), 190-193.
  30. Wickremasinghe R, Gitendra et al. Gel filtration of a complex of DNA polymerase and DNA precursor synthesizing enzymes from a human lymphoblastoid cell line. Gen structure and expression. Biochem.Biophys. Acta, 740:243-48, 1983
  31. Zhu C, Harlow LS, Berenstein D, Munch-Petersen S. Munch-Petersen B. Effect of C-terminal of human cytosolic thymidine kinase (TK1) on in vitro stability and enzymatic properties. Nucleosides, Nucleotides and Nucleic Acids, 25:1185-1188, 2006

Datum přednesení příspěvku: 22. 4. 2011