Evaluation of Cyclin D1 expression by western blotting methods and immunohistochemistry in breast cancer patients.

PURPOSE: Considering that cyclin D1 had a prognostic and clinical value for breast cancer patients, adequate measurement of cyclin D1 is necessary. METHODS: In this investigation, we detect cyclin D1 expression in tumour and peritumoral tissue of breast cancer patients by Western blotting method and by immunohistochemistry. RESULTS: Cyclin D1 expression decreased significantly with each advanced clinical stage of disease and tumour size. Also, patients without lymph node involvement, with positive hormone receptors and Luminal A type of tumours had significantly increased the expression of cyclin D1. We show that cyclin D1 expression correlates with longer RFS in the entire group of patients, in the group of ER-positive and in the group of HER2-negative patients. Patients who were both ER and cyclin D1 positive had a better prognosis. CONCLUSION: Taken together, our results, showing correlation of cyclin D1 with clinical stage, tumour size and lymph nodes, suggest that cyclin D1 expression, detected by Western blotting, could be considered as an additional marker for the staging of breast cancer, as well as a marker for longer RFS and survival in ER-positive breast cancer patients.

pSTAT3 expression associated with survival and mammographic density of breast cancer patients.

BACKGROUND: Constitutive activation of STAT3 have been shown in several tumor types including breast cancer. We investigate STAT3 expresion as possible molecular marker for breast cancer early detection, as well as prognostic factor for determination of tumor agressiveness. METHODS: In this study we measure p(Y705)STAT3 expression in tumor and adjacent tissue of breast cancer patients by Western blot. For relapse-free survival (RFS) and overall survival (OS) we used Log-Rank test. RESULTS: We show that average expression of p (Y705) STAT3 in tumor tissue is higher compared to adjacent tissue. Moreover, we found that patients with HER2 positive receptors had significantly higher pSTAT3 expression compared to HER2 negative patients. We showed that patients with high mammographic density had significantly higher tumor expression of pSTAT3 compared to patients with low mammographic density. Also, we show that pSTAT3 expression correlates with longer RFS in the entire group of patients, as well as in the group of ER positive, in lymph node positive and in older group of breast cancer patients (with age over 50). Furthermore, in the entire group of patients, in ER positive, in lymph node positive and in older group of patient, high expression of pSTAT3 showed a better survival than low expression of pSTAT3. CONCLUSION: Considering that the expression of pSTAT3 is associated with longer RFS and survival, it can be used as prognostic tools for determination of group of breast cancer patients with low-risk.

Differentiation between progression and pseudoprogresion by arterial spin labeling MRI in patients with glioblastoma multiforme.

PURPOSE: To compare arterial spin labeling (ASL) perfusion technique with the clinically established dynamic susceptibility contrast-enhanced (DSC) perfusion weighted-imaging (PWI), and to determine its value in routine MRI evaluation of disease progression in patients with glioblastoma multiforme (GBM). METHODS: A prospective intraindividual study was performed in 31 patients with histologically proven GBM who had clinical and/or radiological deterioration after treatment, including surgery, radiotherapy and therapy with temozolomide. Conventional brain protocol with ASL and DSC techniques was performed on 3T MRI unit. Cerebral blood flow (CBF) and cerebral blood volume (CBV) maps were analyzed by means of regions of interest (ROI). Each ROI average value was normalized to the contralateral normal brain parenchyma ROI value. Neuroradiologists analyzed CBF and CBV maps separately, and classified patients into progression or pseudoprogression group. Radiological diagnosis was confirmed by clinical-radiological follow-up for at least three months after patient deterioration. RESULTS: High linear correlation existed between DSC-PWI and ASL in the tumor ROI (r=0.733; p<0.001). 92% of ASL CBF maps were informative. ASL detected all lesions as well as DSC MRI. Both techniques provided perfusion values closely correlated. CONCLUSION: ASL allows distinction between GBM progression and pseudoprogression, and it can be used as reliable alternative to DSC-PWI.

The Actual Role of LDH as Tumor Marker, Biochemical and Clinical Aspects.

Lactate dehydrogenase (LDH) among many biochemical parameters represents a very valuable enzyme in patients with cancer with possibility for easy routine measurement in many clinical laboratories. Previous studies where mostly based on investigated LDH in serum of patients with cancer with aims to estimate their clinical significance. The new directions in investigation of LDH where based on the principle that tumor cells release intracellular enzymes trough damaged cell membrane, that is mostly consequence in intracellular mitochondrial machinery alteration, and apoptosis deregulation. This consideration can be used not only in-vitro assays, but also in respect to clinical characteristics of tumor patients. Based on new techniques of molecular biology it is shown that intracellular characteristics of LDH enzyme are very sensitive indicators of the cellular metabolic state, aerobic or anaerobic direction of glycolysis, activation status and malignant transformation. Using different molecular analyses it is very useful to analyzed intracellular LDH activity in different cell line and tumor tissues obtained from patients, not only to understanding complexity in cancer biochemistry but also in early clinical diagnosis. Based on understandings of the LDH altered metabolism, new therapy option is created with aims to blocking certain metabolic pathways and stop tumors growth.

HER2-positive breast cancer patients: correlation between mammographic and pathological findings.

Human epidermal growth factor receptor 2 (HER2)-positive breast cancers represent a highly aggressive breast cancer subtype and are associated with a worse prognosis. This study was designed to investigate the mammography finding of HER2-positive breast cancer and to compare the results with the characteristics of HER2-negative breast cancer patients. From January 2010 to October 2011, mammography findings of 65 patients with pathologically confirmed HER2-positive breast cancers (n = 22) or HER2-negative breast cancers (n = 43) were retrospectively reviewed. The authors also reviewed pathological reports for information on the histological type and differentiation grade. Among the two types of breast cancer patients, estrogen receptor-negative/PR-negative/HER2-positive breast cancer patients most commonly had associated calcifications (18 of 22) on mammography. On mammography, cases with a cluster of calcifications usually were presented as pleomorphic calcifications (12 of 20) and branching calcifications (4 of 20). Patients with HER2-positive breast cancers showed a histological grade II. HER2-positive breast cancer patients usually had ductal invasive carcinoma (17 of 22). Moreover, postmenopausal patients showed a significantly higher frequency of HER2-positive tumours. Our results suggest that the imaging findings might be useful in diagnosing HER2-positive breast cancer patients.

Values of MMP-2 and MMP-9 in tumor tissue of basal-like breast cancer patients.

Gelatinase A (MMP-2) and gelatinase B (MMP-9) are proteolytic enzymes involved in process of tumor invasion, and they are considered as possible tumor markers in breast cancer patients. In this study, we measured activity of latent and active form of MMP-2 and MMP-9 in tumor and adjacent tissue of 60 breast cancer patients by SDS-PAGE zymography. The activity of both form of gelatinases significantly increased with each advancing clinical stage of disease. ProMMP-9 and aMMP-9 activity in tumor tissue shows a positive association with tumor size. Patients with lymph node involvement have higher proMMP-2, aMMP-2 and aMMP-9 activity than node negative patients. Steroid receptor-negative tumors had enhanced aMMP-2 and aMMP-9 activity. Patients with basal-like cancers had higher proMMP-2 tumor activity and aMMP-2 adjacent tissue activity compared to patients with luminal A tumors. Patients with negative hormone receptors are associated with increased activity of both form of gelatinases in adjacent tissue. Reported increased activity of MMP-2 in tumor and adjacent tissue of basal-like tumors implicates that MMP-2 might have a role in aggressive biology of basal-like cancers. Additional investigations regarding molecular pathways in adjacent tissue could give better insight into aggressive nature of basal-like carcinomas.

Lactate dehydrogenase, catalase, and superoxide dismutase in tumor tissue of breast cancer patients in respect to mammographic findings.

Lactate dehydrogenase (LDH), marker of anaerobic metabolism, is associated with highly invasive and metastatic breast cancer. Novel studies show that increased anaerobic metabolism (LDH), as well as activity of antioxidative enzymes (superoxide dismutase (SOD) and catalase (CAT)), is correlated with higher mammographic density, as known predictor of breast cancer risk. In this study, we measured LDH, MDH, and SOD activity in tumor and adjacent tissues of breast cancer patients by spectrophotometric assay. Mammograms were evaluated according to the American College of Radiology Breast Imaging Reporting and Data system. Mammographically dense breast tissue is associated with higher activity of LDH in tumor tissue of breast cancer patients. Moreover, patients with masses have significantly higher activity of LDH compared to patients with focal asymmetries or architectural distortion. Patients with spiculated mass margin had higher activity of LDH compared to patients with focal asymmetries or architectural distortion. Activity of LDH in patients significantly increases, while activity of CAT significantly decreases with the increase of BIRADS category. These results suggest that the association of activity of LDH and CAT in tumor tissue with mammographic characteristics could help in defining aggressive breast cancers.

Investigation of NK cell function and their modulation in different malignancies.

NK cells have become a subject of investigation not only in the field of tumor immunology and infectious diseases, but also within all aspects of immunology, such as transplantation, autoimmunity, and hypersensitivity. Our early studies aside from investigating NK cell activity in experimental animals and humans included studies of perforin expression and modulation in this lymphocyte subset. As NK cell activity is modified by their environment, we showed clinical stage-dependent impairment of their activity and in vitro effect of different sera, Th1 cytokines, and their combination in breast cancer, Hodgkin's disease, and non-Hodgkin's lymphoma patients, especially with respect to metabolic and cell membrane changes of peripheral blood lymphocytes evaluated by spontaneous release of the enzyme lactate dehydrogenase (LDH) that led to the correction of the LDH enzyme release assay for natural cytotoxicity. By long-term immuno-monitoring of patients with malignancies, we also showed the kinetics of NK cell modulation during chemo-immunotherapy. In our more recent studies, we give data of NK function and novel families of NK cell receptor expression in healthy individuals that may be of help in NK cell profiling, by giving referent values of basic and cytokine-induced expression of some NK cell receptors either in evaluation of disease or in immuno-monitoring during cytokine therapy of patients with malignancies. Moreover, we give novel aspects of modulation of NK cell activity by cytokines approved for immunotherapy, IFN and IL-2, in melanoma and other malignancies with respect to alterations in new activating (NKG2D and CD161) and inhibitory (CD158a and CD158b) receptor characteristics and signaling molecules in CD16- and CD56-defined NK cells and their small immunoregulatory and large cytotoxic subsets in peripheral blood and lymph nodes, as NK cell-mediated killing of tumor cells depends on the balance between stimulatory and inhibitory signaling.

Matrix metalloproteinases and membrane damage markers in sera of patients with acute myocardial infarction.

Coronary artery disease is a multifunctional disease and represents one of the leading causes of death worldwide. Oxidative stress appears as an etiological factor for myocardial damage during acute myocardial infarction. Some data suggest that acute coronary syndromes may also be influenced by matrix metalloproteinases through degradation of the fibrous cap of vulnerable atherosclerotic lesions. It has been indicated that gelatinases A and B play a key role in acute myocardial infarction and deoxyribonuclease I has been postulated to be a novel early phase marker of disease. The aim was to study activity of gelatinases A and B in acute myocardial infarction and its association with some membrane damage markers. Seventy-five patients with disease and seventy-five healthy controls were enrolled. Activities of lactate dehydrogenase, malate dehydrogenase, and deoxyribonuclease I were estimated using standard spectrophotometric assay and isoforms of lactate and malate dehydrogenases were determined using direct zymography. Activity of dehydrogenases was significantly higher in patients, while deoxyribonuclease I was lower. Isoform 2 of lactate dehydrogenase was significantly higher in the patient group. Gelatinases A and B were detected only in patients group. The results suggest determination of serum malate dehydrogenase activity to be used as an additional parameter for acute myocardial infarction diagnosis. Those findings suggest important role of gelatinases A and B as biomarkers of early stage of acute myocardial infarction together with membrane damage parameters.

[Gelatinases A and B activities in the serum of patients with various coronary artery disease stages].

BACKGROUND/AIM: The main characteristic of matrix metalloproteinases (MMPs) is the degradation of extracellular matrix. Synthesis of MMPs has been reported in coronary atherosclerotic lesions in patients with coronary disease (CD) suggesting a pathogenic role of MMPs in its development. Recently there is increasing evidence that gelatinase A (pro MMP-2) and gelatinase B (proMMP-9) play a pathogenic role in the development of the atherosclerotic plaques. The aim of the study was to determine, by the use of a gel image system, a possible presence of active gelatinases in the serum of the patients with CD, as well as if their activity is higher in these patients than in healthy people. METHODS: By gelatin zymography we analyzed the activity of proMMP-2 and proMMP-9 in the serum of 50 patients with various coronary artery disease stages and in the serum of 15 healthy controls. The activity was measured by using a gel image system (Kodak Image 1D 3.6.). RESULTS: ProMMP-2 and proMMP-9 activity was significantly higher in the serum of patients with CD compared to controls. There was higher activity of MMP-2 and MMP-9 in the serum of patients with acute myocardial infarction (AMI) compared to patients with stable angina pectoris, as well as higher proMMP-9 activity in patients with unstable angina pectoris compared to patients with stable angina pectoris. CONCLUSION: ProMMP-2 and proMMP-9 participate in processes associated with destabilizing plaques and understanding the processes of MMPs activation and regulation may have significant benefits in clinical interpretation. The reported higher proMMP-2 and proMMP-9 activity in the serum of patients with CD suggests a role of proMMP-2 and proMMP-9 in prognostic stratification of these patients and in designing new drugs.