[Trastuzumab in the breast cancer treatment: efficacy and resistance mechanisms]. / Trastuzumab v liecbe karcinómu prsníka: mechanizmy úcinku a rezistencie.

Trastuzumab, a humanized monoclonal antibody against the HER-2 receptor, was the first targeted therapy for HER-2 positive breast cancer. The treatment of HER-2 positive breast cancer in monotherapy, but mainly in combination with cytostatics trastuzumab, showed significant efficacy and increased the time to progression/relapse and overall survival. On the other hand, despite it being administered to only a selected patient population, the response rate varies in range and duration due to the primary or acquired tumor resistance to trastuzumab. Several mechanisms contributing to the drug resistance are presumed and overcoming strategies are in development. In connection with the drug resistance there are the mechanisms of trastuzumab's action on tumor cells and the search for feasible biomarkers in order to predict the response of trastuzumab-based targeted therapies. Furthermore, we present here the actual possibilities to overcome primary or acquired trastuzumab resistance.

Prediction of ovarian cancer prognosis and response to chemotherapy by a serum-based multiparametric biomarker panel.

Currently, there are no effective biomarkers for ovarian cancer prognosis or prediction of therapeutic response. The objective of this study was to examine a panel of 10 serum biochemical parameters for their ability to predict response to chemotherapy, progression and survival of ovarian cancer patients. Sera from ovarian cancer patients were collected prior and during chemotherapy and were analysed by enzyme-linked immunosorbent assay for CA125, kallikreins 5, 6, 7, 8, 10 and 11, B7-H4, regenerating protein IV and Spondin-2. The odds ratio and hazard ratio and their 95% confidence interval (95% CI) were calculated. Time-dependent receiver-operating characteristic (ROC) curves were utilised to evaluate the prognostic performance of the biomarkers. The levels of several markers at baseline (c(0)), or after the first chemotherapy cycle (rc(1)), predicted chemotherapy response and overall or progression-free survival in univariate analysis. A multiparametric model (c(0) of CA125, KLK5, KLK7 and rc(1) of CA125) provided predictive accuracy with area under the ROC curve (AUC) of 0.82 (0.62 after correction for overfitting). Another marker combination (c(0) of KLK7, KLK10, B7-H4, Spondin-2) was useful in predicting short-term (1-year) survival with an AUC of 0.89 (0.74 after correction for overfitting). All markers examined, except KLK7 and regenerating protein IV, were powerful predictors of time to progression (TTP) among chemotherapy responders. Individual and panels of biomarkers from the kallikrein family (and other families) can predict response to chemotherapy, overall survival, short-term (1-year) survival, progression-free survival and TTP of ovarian cancer patients treated with chemotherapy.

[Symptomatic Meckel's diverticulum in children]. / Symptomatický Meckelov divertikul u detí.

Meckel's diverticulum (MD) is the most frequent anomaly of the small intestine, usually asymptotic. Complications of MD in children are usually bleeding and inflammation. Signs are non specific and diagnostics is prolonged. Scintigraphy is the method for the verification of bleeding from MD. A positive finding is the indication for exploration using laparoscopic or classic approach. In patients with bleeding of the MD, the literature presents, that there is usually gastric mucosa. The authors present patients with bleeding and the microscopic picture of the MD was intestinal mucosa with bleeding.

Malignant melanoma associates with Th1/Th2 imbalance that coincides with disease progression and immunotherapy response.

The immunological dysfunction associated with human cancer is well known phenomenon. It comprises number of pathological changes within immune network including imbalance in cytokines of Th1/Th2 origin. The objectives of our study were (i) to evaluate the abnormalities in serum levels of selected cytokines in malignant melanoma patients with regional lymph node metastases as compared to normal values, (ii) to examine the relationship between postoperative cytokine levels and disease progression and (iii) to correlate cytokine profile changes during IFN-alpha therapy with the disease progression and potential therapeutical response. Nine Th1/Th2 related cytokines and sIL-2R were determined in 26 malignant melanoma patients at clinical stage III prior and during adjuvant immunotherapy. Control group consisted of 26 healthy persons. Patients were treated with rIFN-alpha according to EORTC Melanoma group protocol 18952. Cytokines were quantified in patients sera using commercial ELISA kits. Majority of melanoma patients showed significantly lower values of IL-2 and IFN-gamma and pathologically elevated levels of IL-4, IL-6, IL-10 as compared to healthy subjects what indicates disease associated Th1/Th2 imbalance. In addition increased IL-12 and IL-15 values were noted in some patients (54% and 27%, respectively). All patients who manifested early relapse during immunotherapy had significantly lower pretreatment levels of IL-2 and IL-12 than those remaining without progression and probably benefiting from the treatment. Cytokine changes during immunotherapy disclosed that decreases in IL-2 and IL-12 and raises in IL-6 and IL-10 values occurred at least one month prior to relapse. Moreover, the continuous elevation of TNF-alpha and sIL-2R could be observed in patients who remained without progression during 10 months lasting immunotherapy. Our data illustrate that malignant melanoma associates with Th1/Th2 perturbances which are directed towards extended Th2 responses and that measurement of selected cytokines of Th1/Th2 category may be used as an early signal of disease deterioration and for evaluation of immunotherapy response.

Tiptoeing to chromosome tips: facts, promises and perils of today's human telomere biology.

The past decade has witnessed an explosion of knowledge concerning the structure and function of chromosome terminal structures-telomeres. Today's telomere research has advanced from a pure descriptive approach of DNA and protein components to an elementary understanding of telomere metabolism, and now to promising applications in medicine. These applications include 'passive' ones, among which the use of analysis of telomeres and telomerase (a cellular reverse transcriptase that synthesizes telomeres) for cancer diagnostics is the best known. The 'active' applications involve targeted downregulation or upregulation of telomere synthesis, either to mortalize immortal cancer cells, or to rejuvenate mortal somatic cells and tissues for cellular transplantations, respectively. This article reviews the basic data on structure and function of human telomeres and telomerase, as well as both passive and active applications of human telomere biology.

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.

Homocysteine in breast cyst fluid.

BACKGROUND: Elevated plasma homocysteine concentrations have been reported in a variety of carcinoma, including those of the breast. The risk of breast cancer is higher in patients suffering from gross cystic disease. The breast cyst fluid contains unusual amounts of low- and high- molecular substances, including steroid hormones and their conjugates. The present study was undertaken to find out the presence of homocysteine in the fluid filling the cysts and have its concentration compared with other thiols, levels of Na+/K+ ratio and steroid hormones. Materials and methods Fourteen women suffering from gross cystic disease were enrolled in this study. Cystic concentrations of homocysteine (Hcy), cysteine (Cys), cysteinylglycine (Cys-Gly) and glutathione (GSH) were determined by high performance liquid chromatography, with fluorescence detection; estradiol (E2), progesterone, allopregnanolone and pregnenolone sulfate (PregS) by RIA methods. RESULTS: Mean levels of Hcy, Cys, Cys-Gly, Na+/K+, E2 and PregS in the fluid filling the breast cysts were significantly higher than the corresponding plasma concentrations. In addition, a negative correlation was found between cystic Hcy and the Na+/K+ ratio (Rs = -0.72, P = 0.003) and positive correlations between cyst Hcy and estradiol (Rs = 0.64, P = 0.018) and Hcy and PregS (Rs = 0.60, P = 0.025). Conclusion The study provides the first evidence of thiol concentrations in the breast cyst fluid. The finding of a negative correlation between homocysteine and the Na+/K+ ratio support the idea that the homocysteine concentration in breast cysts might be used clinically as a marker for the development of breast cancer disease.

Relation of prenephrectomy CD profiles and serum cytokines to the disease outcome and response to IFN-alpha/IL-2 therapy in renal cell carcinoma patients.

Immune parameters, including cytokine levels and CD profiles were determined in 78 renal cell carcinoma patients (RCC) prior to nephrectomy. The values were correlated with the outcome of disease and response to cytokine-based treatment during a 3-year follow-up. Significantly lower frequency of progressions and higher proportion of survivors were recorded in 24 treated patients compared to 43 untreated ones (22.9% vs. 53.5% and 82.9% vs. 55.8%) illustrating the beneficial effect of immunotherapy on the course of RCC at localized stage. RCC-related immune changes are demonstrated by reduced proportion of CD19+, CD28+, HLA-DR+, CD19+/80+ and CD8+/28+ subsets, by increased serum levels of IL-6, sIL-2R, CRP and by impaired production of IL-2 and TNF-alpha released by in vitro stimulated PBMC. Only increased CRP, IL-6 serum values, decreased CD8+ and increased CD122+ were significantly related to patients' prognosis. Comparisons of preoperative CD profiles and cytokine values with the response to IL-2/IFN-alpha based therapy disclosed significant correlation in only CD80+ and CD19+/80+ subsets. Treated patients who relapsed during the 3-year follow-up exhibited at the diagnosis significantly reduced proportion of CD80+ and CD19+/80+ cells (CD80+ means - 0.79 vs. 1.69 and CD19+/80+ means - 0.32 vs. 0.61) comparing to those surviving disease-free. In addition initial proportion of CD3+, CD8+ and CD19+ cells was reduced in treated patients who manifested progression but statistical difference from those remaining disease-free was not proved.

Renal cell carcinoma-associated immune impairment that may interfere with the response to cytokine therapy.

This prospective study was carried out to explore cytokine-related immune alterations in 69 renal cell carcinoma patients (RCC) and to look for changes which might potentially serve as a reliable predictors of response to cytokine-based therapy. Interleukin-2 (IL-2), its soluble receptor (sIL-2R) and tumor necrosis factor (TNF-alpha) levels produced in vitro by PHA activated and intact mononuclear cells (PBMC) were determined. Concentrations of IL-2, IL-4, IL-6, sIL-2R, TNF-alpha and CRP were measured in sera. Cytokine level was evaluated by enzyme-linked immunoadsorbent assay (ELISA) and CRP was determined by means of turbidimetric method. All measurements were performed in patients without any prior treatment. PHA activated PBMC of RCC patients were significantly defective in producing IL-2 and TNF-alpha comparing to controls (p < 0.03 and p < 0.001). The difference of sIL-2R was noted in metastatic stage only (p < 0.03). Unstimulated PBMC manifested decrease in IL-2 (p < 0.03) and increased level of TNF-alpha in advanced disease (p < 0.02). This impairment reflected tumor size and differentiation stage. Serum concentrations of IL-2, sIL-2R and TNF-alpha were within normal range. However, in relation to the clinical stage, significantly increased serum IL-2 was noted in combined Stage I and II as compared to controls (p = 0.012). IL-6 and CRP showed markedly elevated levels with a significancy which allowed to distinguish samples from metastatic patients. In conclusion careful comparisons of these data with clinical course of cytokine treated patients will disclose which of those tests may possess predictive power in the individual patients who are likely to respond to cytokine-based treatment.

Significance of pre-treatment immunological parameters in colorectal cancer patients with unresectable metastases to the liver.

BACKGROUND/AIMS: In this study, we have compared the profiles of peripheral blood lymphocyte (PBL) subsets and serum cytokine levels of healthy individuals with those of patients with unresectable liver metastases from colorectal carcinoma before starting regional chemoimmunotherapy. Since the therapeutic responses are limited only to a subset of patients, we hypothesize that the initial status of immunity and individual immune response to a tumor might be significant to the therapeutic outcome. METHODOLOGY: Cellular and humoral immunological parameters were compared between 10 patients with colorectal cancer metastases to the liver responding and non-responding to regional intra-arterial chemo-immunotherapy, and 5 healty individuals. Analyses included a flow cytometric immunophenotyping of peripheral blood mononuclear cells (CD3, CD4, CD8, CD19, CD25, CD28, CD56, CD57, CD80 and HLA.DR), estimation of serum cytokine levels of interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-alpha), and other immunological parameters are soluble IL-2 receptor (sIL-2), carcinoembryonic antigen (CEA), gastrointestinal cancer-associated antigen (CA 19-9), and C-reactive acute phase protein (CRP). A significantly lower proportion of CD8 lymphocytes and a trend for decreased CD19, CD28 and CD80 was detected among colorectal cancer patients before liver-directed chemotherapy compared to healthy controls. RESULTS: The cancer patients showed a significantly increased population of peripheral NK cells as detected by both CD56+ and CD57+ phenotypes. Elevated serum levels of CRP, IL-4 and TNF-alpha, sIL-2R, but not IL-2, were also demonstrated in cancer patients as compared to controls. Activated CD25+ lymphocytes correlated negatively with CD28+ lymphocytes (r = -0.68, p < 0.01) and less significantly with CD4+ lymphocytes (r = -0.56, p < 0.05). The CD8+ cytotoxic cell subset might be negatively influenced by serum IL-4 (r = -0.57, p < 0.05). Positive correlation was found between sIL-2R and CRP (r = -0.78, p < 0.01), and between sIL-2R and TNF-alpha (r = 0.64, p < 0.05) serum levels in patients with progressive disease during the course of therapy, the initial proportions of CD4+, CD19+ and CD28+ lymphocytes were significantly lower than those among responders. Among humoral parameters, only sIL-2R showed a marginal correlation with therapeutic response, being more elevated among non-responding patients. Pre-treatment serum levels of CEA and CA 19-9 showed correlation with neither therapeutic response nor with any of the cellular or humoral immunological parameters analyzed. CONCLUSIONS: The results may serve as an initial guideline to open a discussion on the rationale of such a panel of tests, hopefully leading to standardized laboratory pre-selection and monitoring of patients treated with regional chemoimmunotherapy.

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.

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.

Polyclonal antibodies to human milk caseins.

Preparations of total human casein and its individual fractions were isolated for production of specific polyclonal antibodies. Immunization procedures used differed in the schedule for antigen administration, antigen concentrations, degree of additional purification, and modification of the size of molecules. Immunoprecipitation techniques failed to provide an unambiguous proof of the presence of antibodies to human milk caseins in antisera even after immunization of animals phylogenetically considerably different from man. Immunoblotting, however, allowed antibodies to beta-casein to be identified and compared with available monoclonal antibodies.

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.

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.

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.

A non-competitive enzyme immunoassay of human alpha-lactalbumin in biological fluids and tissue extracts.

A sandwich non-competitive enzyme immunoassay for the measurement of human alpha-lactalbumin in serum, milk, and tumour tissue cytosol was developed using affinity-purified polyclonal antibody adsorbed to solid phase. The detection limits of this procedure in tubes (macromethod) and in wells of microtitre plates (micromethod) are 50 pg and 25 pg/sample, respectively, which means 2.5 micrograms/l of serum at appropriate dilution. The micromethod enables a reduction of the volumes used in the reaction with equal sensitivity, accuracy and reproducibility. A comparison of alpha-lactalbumin concentrations in serum of healthy, pregnant, and lactating women determined by our method with those measured formerly by radioimmunoassays proved the specificity of our procedure.

Biosynthesis of nuclear RNAs during 3'-methyl-4-dimethyl-aminoazobenzene hepatocarcinogenesis and in transplantable rat hepatomas.

The long-term administration of 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) to rats causes marked changes in concentrations and synthesis of nucleic acids in the liver. In the beginning, a decrease in total cellular RNA concentration can be seen. On the other hand, at the stage of tumor onset the concentration of nuclear RNAs in precancerous lesions and hepatomas is elevated. Incorporation of 3H-orotic acid into nuclear RNA after 45 min of in vivo labeling serving as a measure of biosynthesis of nuclear RNAs is markedly decreased when compared to control livers. This decrease takes place from the very beginning of the process and goes on until primary hepatomas arise in which its values are approximately at 10% of those observed in controls. In the present study the problem of the increase of proliferative activity at early stages of carcinogenesis is discussed which is not in correlation with the changes in nuclear RNA biosynthesis.