EORTC receptor and biomarker study group report analytical and technical evaluation of a thymidine kinase radio-enzymatic assay in breast cancer cytosols.
BACKGROUND
High thymidine kinase (TK) activity in cancer cells could counteract adjuvant chemotherapy directed at the inhibition of de novo DNA synthesis. TK is an independent prognostic factor in breast cancer patients receiving adjuvant chemotherapy.


METHODS
In this paper, we describe the effects of extraction and dilution buffer composition on TK enzymatic activity values obtained in breast cancer cytosols with the Prolifigen serum TK-REA kit (Sangtec Medical, Sweden).


RESULTS
The addition of MgCl2 and ATP early in the assay, preferably during the extraction of tumor tissue, seems critical to stabilise the enzyme. Furthermore, the use of normal calf serum to dilute both standards and samples is necessary to obtain satisfactory parallelism between TK values in serial dilutions of breast cancer cytosols.


CONCLUSIONS
Based on the data reported here, the manufacturer has changed the cytosol diluent composition and is adding a specific cytosol assay insert to the Prolifigen TK-REA kit. As evidenced by the laboratory reproducibility, these modifications to the serum assay led to an adequate, standardized protocol for analyzing TK activity in breast tumor cytosols.
Span
P
P
Department of Chemical Endocrinology, University Hospital Nijmegen, St Radboud, The Netherlands. p.span@ace.azn.nl
Heuvel
J
J
Romain
S
S
Piffanelli
A
A
Martin
P M
PM
Geurts-Moespot
A
A
Sweep
F
F
D014408:Biomarkers, Tumor / Q000032:analysis*; D001943:Breast Neoplasms / Q000737:chemistry* / Q000201:enzymology / Q000473:pathology; D003600:Cytosol / Q000737:chemistry; D004795:Enzyme Stability; D005260:Female; D006801:Humans; D007065:Idoxuridine; D007457:Iodine Radioisotopes; D000091402:Prohibitins; D011865:Radioisotope Dilution Technique; D011933:Reagent Kits, Diagnostic; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013937:Thymidine Kinase / Q000032:analysis*
D014408:Biomarkers, Tumor; D007457:Iodine Radioisotopes; C000717331:PHB2 protein, human; D000091402:Prohibitins; D011933:Reagent Kits, Diagnostic; D013937:Thymidine Kinase; D007065:Idoxuridine