Co-expression of thymidine kinase and cathepsin D in 200 primary breast carcinomas
Introduction
The evaluation of tumour markers concentration in cytosol is an important tool because it offers the possibility to obtain direct information relating to tumour phenotype. One such marker is thymidine kinase (TK), which catalyses the conversion of deoxythymidine to deoxythymidinemonophosphate (dTMP), subsequently used in the DNA synthesis, after the transformation to dTTP.
TK exists as two different isoenzymes in eukaryotic cells. One isoenzyme (foetal, cytoplasmic, TK1) is associated with cell division, the activity being high in the G1/S phase, while the other (adult, mitochondrial, TK2) remains stable throughout cell division [1]. TK1 is not released by normal dividing cells, but only after cell membrane disruption, i.e. during cancer proliferation, cytostatic treatment or is produced by non-dividing cells infected by RNA or DNA viruses [2]. TK1 accounts for the 95% of the serum TK activity, thus the total TK concentration determined by commercial assay kits largely represents the TK1 activity [3].
TK activity is higher in malignancies with high proliferation rate measured by thymidine labelling index (TLI), such as blood and lymphoid malignancies. Several studies have shown that TK activity is a useful prognostic indicator, and treatment-effectiveness marker for chronic and acute leukaemia [4], multiple myeloma [5], Hodgkin's and non-Hodgkin's lymphomas [6]. In univariate analysis of 290 breast cancer patients, high levels of TK were associated with shorter overall survival and in multivariate analysis, TK status is an independent prognostic factor for recurrence-free survival with a similar impact as progesterone receptor [7]. TK concentration is related to tumour size [8], histological grade [7] and solid-tubular and scirrhous type compared with papillotubular carcinomas [9]. Data regarding the relationship between TK and steroid receptors are uncertain. Some authors have reported a correlation between TK activity and oestrogen receptor (ER) and progesterone receptor (PgR) negativity [7], while others showed high TK activity in steroid receptors positive tumours [10], [11].
Cathepsin D (Cath-D) is another prognostic indicator, whose increased level is associated with a poor relapse-free and overall survival [12]. The precursor form of the stable Cath-D, pro-Cath-D or 52K, is a lysosomal protease [13] secreted either by ER-positive [14] or ER-negative cell lines [15]. A significant association between Cath-D level and high grade infiltrating ductal carcinomas, tumour size, lymph node metastases and peritumoural vascular invasion has been shown [16], which is related to the action of Cath-D through extracellular matrix degradation and fibroblast growth factor (FGF)-like growth factor release [17].
Since few information regarding the relationship between TK and Cath-D are so far available in the cancerous breast, in this study we determined the levels of both molecules in the cytosol of 200 primary breast tumours. Since TK is a well accepted indicator of tumour cell proliferation and Cath-D is related to metastatic potential we investigated their relationship and showed that their co-expression may identify a tumour population of high proliferation activity and invasiveness potential.
Section snippets
Patients
ER, PgR, Cath-D concentrations and TK activity were measured in the cytosol of 200 primary breast carcinomas excised between January 1998 and September 1999. Sixty-five out of the 200 patients were premenopausal, while the others were postmenopausal. Histological classification divided tumours into pure invasive ductal (IDC) (n=157) carcinomas and lobular carcinomas (ILC) (n=43). According to the modified Scarff–Bloom–Richardson method described by Elston and Ellis [18], tumours were divided in
Steroid receptors status
ER (estrogen receptor) concentration ranged from 0 to 400 fmol/mg protein (mean value 58.1 fmol/mg protein, median 21.5 fmol/mg protein) and PgR (progesterone receptor) concentration ranged from 0 to 420 fmol/mg protein (mean value 88.2 fmol/mg protein, median 26.5 fmol/mg protein). Using 10 fmol/mg protein we found that 100 and 106 out of the 200 tumours were positive for ER and PgR, respectively.
Dividing tumour population in pre and post-menopausal, we found that 90 out of the 135
Discussion
The evaluation of cell proliferation is commonly performed by monitoring the incorporation of radioactive thymidine, which is proportional to the number of dividing cells. The availability of an alternative and faster method able to assay TK activity directly makes it possible to obtain information regarding tumour proliferation in the same specimen used for steroid receptor assay. The TK-REA measures TK activity and is usually applied to serum, but it has been shown that it can also be used
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