Quantitative determination of telomerase activity in breast cancer and benign breast diseases.

Telomerase plays an important role in maintaining the stability of chromosomes. This ribonucleoprotein prevents chromosome ends (telomeres) from gradual loss with each cell division. It enables tumor cells to maintain telomere length, allowing indefinite replicative capacity. Telomerase activity has been detected in the majority of tumor and germ cells and in immortalized cell lines. Quantitative telomerase PCR-ELISA (TeloTAGGG Telomerase PCR ELISA(PLUS)) was evaluated for distinguishing benign and malignant breast tissue. Activity of telomerase was determined in 27 samples of fibrocystic and dysplastic tissues, 28 fibroadenomas and phylloid tumors, and 154 breast cancer tissues; 59 specimens were analyzed retrospectively. Analytical precision and linearity of the assay was tested using breast carcinoma cell line ZR-75-1 and breast tumor tissue extracts. About 4% of tumor samples were excluded from analysis due to interferences in the PCR reaction. Relative telomerase activity differed significantly in the groups of dysplastic tissues, fibroadenomas and carcinomas. The highest activity was found in breast cancer tissue. This method can identify breast cancer tissue with 73% clinical sensitivity and 93% specificity as compared to benign breast tumors. We did not find a correlation between telomerase activity and the tissue levels of estrogen and progesterone receptors, HER-2/neu oncoprotein concentration, tumor size, and lymph node positivity. Probability of disease-free survival was significantly lower for patients with telomerase activity higher than median value. As the assay for telomerase activity has very high analytical sensitivity and high specificity for cancer cells, this routinely used method may prove useful for distinguishing malignant phenotype of breast tissues.

Phylloid breast tumors and three steroid hormone receptors.

The concentrations of three steroid hormones (estrogen, progesteron and 1,25-dihydroxycholecalciferol) receptors (ER, PgR, DR) in tissue cytosol were analyzed in a group of 17 breast phylloid tumors. Comparison with breast carcinoma tissue samples (n = 37) did not reveal significant differences in average values of ER, PgR, and DR. Comparison with another control set of 30 samples of dysplastic tissue of the mammary gland showed significant differences only in PgR values. Only 18% of phylloid tumor samples contained levels above cut-of-line of all three receptors (ER, PgR, DR-5,10,10 resp. fmol/ mg protein). The most frequent combination was ER+PgR+DR-(41%). As far as we know, DR in phylloid breast tumors have never been examined before. In approximately 60% of our samples we found the expression of DR, in 36% the estimated values were above 10 fmol/mg protein. Cells of the tissue not expressing DR seem to belong to a special phenotype. We found no ER+PgR- or ER-PgR-combinations in them. The group which expresses DR is characterized by a higher dispersion of PgR values.

Biochemical analysis of breast cyst fluid as a possible predictor of breast carcinoma development.

The occurrence of breast cancer in patients with gross cystic disease is 2-5 times higher as compared to control group of women. During 3 years, 183 cyst fluid samples were analyzed in 129 females, in 30 patients of them the samples were analysed repeatedly. The distribution of the Na+/K+ ratio, considered as the measure of cancer risk, was found to be bimodal. In repeated analyses the type I cyst fluid markedly predominated (Na+/K+ < or = 4.0). A direct dependence on this ratio was found in the concentration of glucose, albumin, carcinoembryonic antigen (CEA), tissue polypeptide antigen (TPA) and its specific form, TPS; an indirect dependence was found for the level of uric acid, phosphates, alkaline phosphatase (ALP) and alpha-amylase (AMS). The predominance of apocrine metaplasia cells released into the cyst fluid is characteristic of type I cysts. A definitive assessment of significance of these parameters will be enabled by a long-term follow-up of the disease in the respective patients.

Early diagnosis of breast cancer dissemination by tumor markers follow-up and method of prediction.

A mathematical model of prediction of progression was tested in patients with breast cancer employing long-term monitoring of tumor markers CEA, CA 15-3, MCA and TPA, erythrocyte sedimentation rate (FW), and the enzymes gamma-glutamyl transferase (GGT), alkaline phosphatase (ALP) and lactate dehydrogenase (LD) in serum. At the same time, specificity, sensitivity, lead time and positive predictive value were evaluated along with false positivity for all these parameters and their combinations. A model was proposed for the follow-up of patients with breast cancer after the completion of basic therapy.

Carcinoembryonic antigen and alpha-lactalbumin in phylloid tumor tissues.

The concentration of carcinoembryonic antigen and alpha-lactalbumin in tumor tissue cytosol were analyzed in a group of 19 tumors of cystosarcoma phyllodes type. Both antigens were also localized in the tissue of identical tumors by means of immunohistochemical procedure. The cytosol levels of both proteins were found to be higher in the histologically defined malignant type of phylloid tumors. This group was also characterized by the simultaneous occurrence of both antigens. We did not manage to prove any relationship between the presence of alpha-lactalbumin and the steroid hormone receptor positivity in tumor tissue.

Immunohistochemical localization of alpha-lactalbumin in human breast cancer tissue.

One hundred and eleven formalin-fixed breast cancer tissue samples were examined for the presence of alpha-lactalbumin using our polyclonal antibodies to this specific milk protein. Alpha-lactalbumin was shown to be present in 67 tumor samples (60%), in 15% of cases the occurrence of alpha-lactalbumin was questionable. No relationship was found between alpha-lactalbumin positivity and the histological type of the tumor, differentiation grading, type of invasivity, or stromal reactivity. Disease progression occurred in an equal number of patients with tumors either alpha-lactalbumin positive or negative. However, negativity was often connected with a disease-free interval shorter than 24 months after primary operation.

Complex biochemical analysis of human breast tumor tissue.

The quantitative biochemical analysis of tissue specimens from 76 human breast carcinomas consisted of examination for cytosolic estrogen receptors (cER), nuclear estrogen receptors (nER), progesterone receptors (PgR), 1,25-dihydroxycholecalciferol receptors (DR), carcinoembryonic antigen (CEA), alpha-lactalbumin (aLA), and gamma-glutamyl transferase (gGT). The highest incidence was found in CEA (76%), DR (70%), and aLA (62%). There was a high percentage of tumors containing only DR, in contrast to the tumors containing only cER or PgR. The simultaneous occurrence of DR and CEA was considerably high (61%). No statistically significant differences were observed in these biochemical parameters in relation to the grade of differentiation of the tumors. The values of aLA in tumors that invaded lymphatic or blood vessels were lower as compared to those tumors that invaded adipose or connective tissues. The level of statistical significance of this difference was close to 5%, the differences in other parameters were statistically insignificant. For prognosis assessed at the time of surgery, after a 2-3-year follow-up of 36 patients the level of gGT in the tumor seems to be the most promising prognostic factor. The values of gGT were significantly lower in those patients whose tumors were in progression during this time. The significance of nER and aLA was also taken into consideration.

Cathepsin B in human breast tumor tissue and cancer cells.

The cysteine proteinase cathepsin B (EC 3.4.22.1) has been proposed to play an important role in the proteolytic mechanism of the ability of breast cancer cells to invade into and through normal tissues during metastasis. In this study, activity of cathepsin B was measured with a fluorometric microtiter plate assay in human breast tumors as well as in mammary gland dysplasias and in four human breast cancer cell lines (BT-20, MDA-MB-231, PMC42 and T47D). It was found that primary breast carcinomas and cystosarcomas phyllodes contain significantly higher levels of cathepsin B activity than mammary dysplasias; the activity of cathepsin B in cystosarcomas phyllodes was comparable with that in breast carcinomas. The enzyme from breast carcinoma tissue exhibited properties of a mature form of cathepsin B. All investigated breast cancer cell lines display positive cytochemical staining for cathepsin B activity with granular pattern of distribution of the final reaction product. Biochemically, the breast cancer cell lines differed significantly from each other in the level of cathepsin B activity decreasing in the following order: T47D, PMC42, MDA-MB-231 and BT-20.

Partial purification of gamma-glutamyltransferase from human brain microvessels.

Two forms of gamma-glutamyltransferase from human brain cortex microvessels were partially purified by gel permeation and ion-exchange and group-affinity chromatography. The specific activity of the purified preparations was 320-fold (detergent form) and 830-fold (proteolytic form) higher than that of the enzyme in the brain cortex homogenate. The relative molecular mass of the proteolytic form of the enzyme was about 90,000 as determined by gel permeation chromatography. The major part of the enzyme (about 80%) was absorbed on Con A-Sepharose 4B. The pH optima for transfer reactions with gamma-glutamyl-4-nitroanilide as donor and glycylglycine and L-cystine as acceptors were in the range of 8.2 to 9.0. The studied enzyme was inhibited by a mixture of L-serine and borate and by bromcresol green.

Solubilization and some properties of gamma-glutamyltransferase from human brain microvessels.

Detergents Triton X-100, sodium deoxycholate, and octyl-beta-D-glucopyranoside, and proteinase papain proved to be excellent agents solubilizing the gamma-glutamyltransferase (gamma-GT) from human brain cortex microvessels. Ficin also solubilized gamma-GT but to a lesser extent than papain. The relative molecular mass of the detergent-solubilized enzyme form was greater than 200,000 (in the presence of Triton X-100). The relative molecular mass of the proteinase-solubilized form was slightly greater than that of albumine. gamma-GTs of microvessels from five human brain regions and from the choroid plexus were tested for their specificity toward acceptors. The best acceptors were found to be (in decreasing order of activity) L-cystine, glycylglycine, L-glutamine, L-methionine, and L-alanine. The findings suggest that the main features of gamma-GT of the human blood-brain barrier are very similar to those of gamma-GTs from other human tissues.