25P A Novel Serum Thymidine Kinase Activity (S-TK) Assay for Quantification of Total Cell Division in Man, with Special Reference to Breast Carcinoma

Abstract

ABSTRACT Scope To presents a clinical chemistry test (DiviTum) for analyses of total cell division in human and to show the biochemical character of the s-TK expressed in breast carcinoma (BRCa), and to relate the degree of pathological increase found in pretreatment sera from women with localized BRCa to other clinical parameters as well as to outcome of disease. Methods and patients A standardised assay for s-TK is presented. In principle, the s-TK converts BrdU to its monophosphate which immidiatly is phosphorylated by yeast enzymes to triphosphate, which instantly is immobilised by polymerisation to a DNA-strand by reverse transcriptase using a prA-template bound to the bottom of the 96 well plate (i.e thymidine incorporation). The amount of produced DNA proportional to the s-TK is determined by ELISA using colorimetri. For comparison the Liason TK test was used. Molecular size fractioning of sera used a Superose 6 column. Serum cohorts included healthy women, healthy women with BRCA1/BRCA2 mutations in different loci, women with breast lumps judged as benign, and women with non-metastatic invasive BRCa. Results The pathological s-TK used only ATP as phosphate donor and was confined for all sera to a complex of approx. 700kD. With DiviTum, contrary to Liason, significantly higher s-TK levels, compared to healthy blood donors, were found in patients having benign lumps with atypical hyperplasia and in healthy persons with BRCA1/2 mutations in certain loci. Further, pretreatment s-TK correlated to all parameters indicating aggressivity such as absence of ER/PR receptor, vascularisation, tumor grade and size, triple negativity and BRCA mutation, but not to age, menopausal status, histopathology, HER/neu or nodal status. Univariate analyses showed that pretreatment s-TK (>134Du/L, p 80Du/L p Conclusion The degree of cell division in the BRCA patients, measured as s-TK by DiviTum assay, is an important biomarker of survival and thus useful for staging, prognoses, and therapy monitoring. Disclosure S. Gronowitz: I, Simon Gronowitz am the founder of Biovica International and the inventor of the DiviTum assay for seru thymidine kinase and my family has still shares in the company. All other authors have declared no conflicts of interest.