Comparison of Chromogenic In Situ Hybridisation with Fluorescence In Situ Hybridisation and Immunohistochemistry for the Assessment of Her-2/neu Oncogene in Archival Material of Breast Carcinoma

The successful treatment of breast cancer is dependent upon a number of complex factors. Her-2/neu gene amplification is known to be one of the most common genetic alterations associated with breast cancer and its accurate determination has become necessary for the selection of patients for trastuzumab therapy

NUCKS overexpression in breast cancer

<p>Abstract</p> <p>Background</p> <p>NUCKS (Nuclear, Casein Kinase and Cyclin-dependent Kinase Substrate) is a nuclear, DNA-binding and highly phosphorylated protein. A number of reports show that <it>NUCKS </it>is highly expressed on the level of mRNA in several human cancers, including breast cancer. In this work, NUCKS expression on both RNA and protein levels was studied in breast tissue biopsies consisted of invasive carcinomas, intraductal proliferative lesions, benign epithelial proliferations and fibroadenomas, as well as in primary cultures derived from the above biopsies. Specifically, in order to evaluate the level of NUCKS protein in correlation with the histopathological features of breast disease, immunohistochemistry was employed on paraffin sections of breast biopsies of the above types. In addition, NUCKS expression was studied by means of Reverse Transcription PCR (RT-PCR), real-time PCR (qRT-PCR) and Western immunoblot analyses in the primary cell cultures developed from the same biopsies.</p> <p>Results</p> <p>The immunohistochemical Results showed intense NUCKS staining mostly in grade I and II breast carcinomas compared to normal tissues. Furthermore, NUCKS was moderate expressed in benign epithelial proliferations, such as adenosis and sclerosing adenosis, and highly expressed in intraductal lesions, specifically in ductal carcinomas <it>in situ </it>(DCIS). It is worth noting that all the fibroadenoma tissues examined were negative for NUCKS staining. RT-PCR and qRT-PCR showed an increase of <it>NUCKS </it>expression in cells derived from primary cultures of proliferative lesions and cancerous tissues compared to the ones derived from normal breast tissues and fibroadenomas. This increase was also confirmed by Western immunoblot analysis. Although NUCKS is a cell cycle related protein, its expression does not correlate with Ki67 expression, neither in tissue sections nor in primary cell cultures.</p> <p>Conclusion</p> <p>The results show overexpression of the NUCKS protein in a number of non malignant breast lesions and cancerous tissues. In particular, the NUCKS overexpression in ADH and DCIS indicates a significant role of this protein in neoplastic progression.</p

Altered expression pattern of integrin alphavbeta3 correlates with actin cytoskeleton in primary cultures of human breast cancer

Background: Integrins are transmembrane adhesion receptors that provide the physical link between the actin cytoskeleton and the extracellular matrix. It has been well established that integrins play a major role in various cancer stages, such as tumor growth, progression, invasion and metastasis. In breast cancer, integrin alphavbeta3 has been associated with high malignant potential in cancer cells, signaling the onset of widespread metastasis. Many preclinical breast cancer studies are based on established cell lines, which may not represent the cell behavior and phenotype of the primary tumor of origin, due to undergone genotypic and phenotypic changes. In the present study, short-term primary breast cancer cell cultures were developed. Integrin alphavbeta3 localization was studied in correlation with F-actin cytoskeleton by means of immunofluorescence and immunogold ultrastructural localization. Integrin fluorescence intensities were semi-quantitatively assessed by means of computerized image analysis, while integrin and actin expression was evaluated by Western immunoblotting. Results: In the primary breast cancer epithelial cells integrin alphavbeta3 immunofluorescence was observed in the marginal cytoplasmic area, whereas in the primary normal breast epithelial cells it was observed in the main cell body, i.e. in the ventrally located perinuclear area. In the former, F-actin cytoskeleton appeared well-formed, consisting of numerous and thicker stress fibers, compared to normal epithelial cells. Furthermore, electron microscopy showed increased integrin alphavbeta3 immunogold localization in epithelial breast cancer cells over the area of stress fibers at the basal cell surface. These findings were verified with Western immunoblotting by the higher expression of integrin beta3 subunit and actin in primary breast cancer cells, revealing their reciprocal relation, in response to the higher motility requirements, determined by the malignant potential of the breast cancer cells. Conclusion: A model system of primary breast cancer cell cultures was developed, in an effort to maintain the closest resembling environment to the tumor of origin. Using the above system model as an experimental tool the study of breast tumor cell behavior is possible concerning the adhesion capacity and the migrating potential of these cells, as defined by the integrin alphavbeta3 distribution in correlation with F-actin cytoskeleton

HER2 and TOP2A in high-risk early breast cancer patients treated with adjuvant epirubicin-based dose-dense sequential chemotherapy

<p>Abstract</p> <p>Background</p> <p>HER2 and TOP2A parameters (gene status, mRNA and protein expression) have individually been associated with the outcome of patients treated with anthracyclines. The aim of this study was to comprehensively evaluate the prognostic/predictive significance of the above parameters in early, high-risk breast cancer patients treated with epirubicin-based, dose-dense sequential adjuvant chemotherapy.</p> <p>Methods</p> <p>In a series of 352 breast carcinoma tissues from patients that had been post-operatively treated with epirubicin-CMF with or without paclitaxel, we assessed HER2 and TOP2A gene status (chromogenic in situ hybridization), mRNA expression (quantitative reverse transcription PCR), as well as HER2 and TopoIIa protein expression (immunohistochemistry).</p> <p>Results</p> <p>HER2 and TOP2A amplification did not share the same effects on their downstream molecules, with consistent patterns observed in HER2 mRNA and protein expression according to HER2 amplification (all parameters strongly inter-related, p values < 0.001), but inconsistent patterns in the case of TOP2A. TOP2A gene amplification (7% of all cases) was not related to TOP2A mRNA and TopoIIa protein expression, while TOP2A mRNA and TopoIIa protein were strongly related to each other (p < 0.001). Hence, TOP2A amplified tumors did not correspond to tumors with high TOP2A mRNA or TopoIIa protein expression, while the latter were characterized by high Ki67 scores (p = 0.003 and p < 0.001, respectively). Multivariate analysis adjusted for nodal involvement, hormone receptor status, Ki67 score and HER2/TOP2A parameters revealed HER2/TOP2A co-amplification (21.2% of HER2 amplified tumors) as an independent favorable prognostic factor for DFS (HR = 0.13, 95% CI: 0.02-0.96, p = 0.046); in contrast, increased HER2/TOP2A mRNA co-expression was identified as an independent adverse prognostic factor for both DFS (HR = 2.41, 95% CI: 1.31-4.42, p = 0.005) and OS (HR = 2.83, 95% CI: 1.42-5.63, p = 0.003), while high TOP2A mRNA expression was an independent adverse prognostic factor for OS (HR = 2.06, 95% CI: 1.23-3.46, p = 0.006). None of the parameters tested was associated with response to paclitaxel.</p> <p>Conclusions</p> <p>This study confirms the favorable prognostic value of HER2/TOP2A co-amplification and the adverse prognostic value of high TOP2A mRNA expression extending it to the adjuvant treatment setting in early high-risk breast cancer. The strong adverse prognostic impact of high HER2/TOP2A mRNA co-expression needs further validation in studies designed to evaluate markers predictive for anthracyclines.</p> <p>Trial registration</p> <p>Australian New Zealand Clinical Trials Registry <a href="http://www.anzctr.org.au/ACTRN12611000506998">ACTRN12611000506998</a>.</p

Nitrosative and Oxidative Stresses Contribute to Post-Ischemic Liver Injury Following Severe Hemorrhagic Shock: The Role of Hypoxemic Resuscitation

Purpose: Hemorrhagic shock and resuscitation is frequently associated with liver ischemia-reperfusion injury. The aim of the study was to investigate whether hypoxemic resuscitation attenuates liver injury. Methods: Anesthetized, mechanically ventilated New Zealand white rabbits were exsanguinated to a mean arterial pressure of 30 mmHg for 60 minutes. Resuscitation under normoxemia (Normox-Res group, n = 16, PaO2 = 95–105 mmHg) or hypoxemia (Hypox-Res group, n = 15, PaO 2 = 35–40 mmHg) followed, modifying the FiO 2. Animals not subjected to shock constituted the sham group (n = 11, PaO 2 = 95–105 mmHg). Indices of the inflammatory, oxidative and nitrosative response were measured and histopathological and immunohistochemical studies of the liver were performed. Results: Normox-Res group animals exhibited increased serum alanine aminotransferase, tumor necrosis factor- alpha, interleukin (IL)-1b and IL-6 levels compared with Hypox-Res and sham groups. Reactive oxygen species generation, malondialdehyde formation and myeloperoxidase activity were all elevated in Normox-Res rabbits compared with Hypox-Res and sham groups. Similarly, endothelial NO synthase and inducible NO synthase mRNA expression was up-regulated and nitrotyrosine immunostaining increased in animals resuscitated normoxemically, indicating a more intense nitrosative stress. Hypox-Res animals demonstrated a less prominent histopathologic injury which was similar to sham animals. Conclusions: Hypoxemic resuscitation prevents liver reperfusion injury through attenuation of the inflammatory respons

Η συμπεριφορά ευαίσθητων και ανθεκτικών στελεχών candida albicans έναντι της αμφοτερικίνης Β ως προς την προσκολλητικότητα, την φαγοκυττάρωση και την βακτηριοκτόνο δράση

The adherence of the bacteria and yeasts to human epithelial cells and their resistance against the phagocytosis and the bactericidal action of the human polymorphonuclear leucocytes (PMN), play a significant role in the evolution of the disease. It has been proved that strains of C. albicans sensitive to Amphotericin B (AMB) have different behavior than the resistant ones. In the present study we cross-examined three strains of C. albicans, one sensitive (E), one resistant in vivo (KA) and one resistant in vitro (EA), in relation of their adherence to human epithelial cells of healthy individuals and their phagocytosis of human PMN. It was found that the adherence of C. albicans of the strain E was 21±5,5 , of the strain EA was 11,2±4,1 and of the strain KA was 14±4,5 (method of Ellen and Gibbons, 1972).[121] The average of phagocytosis of the E strain was 123±17,3 , of the KA was 86±14,2 and of the EA was 76,5±13,7. The bactericidal action of the E strain was 34,5±8,3 , of the KA strain was 11,8±2,51 and of EA strain was even lower 8,4±2,1 (method of Smith and Rommel, 1977). [122] Results are compatible with the hypothesis that resistance of C. albicans is related to the conversion of the cell membrane (pili) and also with internal factors (e.g. katalase).Η προσκόλληση των μικροβίων σε ανθρώπινα επιθηλιακά κύτταρα και η αντίστασή του στην φαγοκυττάρωση και τη βακτηριοκτόνο δράση των ουδετερόφιλων πολυμορφοπύρηνων (ΠΜΠ) διαδραματίζουν σημαντικό ρόλο στην ανάπτυξη της λοίμωξης. Έχει αποδειχθεί ότι στελέχη της C. albicans ευαίσθητα στην Αμφοτερικίνη B (ΑΜΒ) έχουν διαφορετική παθογένεια. Στην παρούσα εργασία μελετήσαμε τρία στελέχη, ένα ευαίσθητο (Ε) , ένα ανθεκτικό in vivo (ΚΑ) και ένα ανθεκτικό in vitro (ΕΑ), όσον αφορά την προσκολλητικότητά τους σε ανθρώπινα επιθηλιακά κύτταρα υγιών ατόμων και την φαγοκυττάρωσή τους από ανθρώπινα ΠΜΠ. Βρέθηκε ότι ο αριθμός μυκήτων C. albicans τού στελέχους Ε που προσκολλήθηκαν σε κάθε επιθηλιακό κύτταρο ήταν 21±5,5, για το στέλεχος ΕΑ ο αριθμός μυκήτων που προσκολλήθηκαν σε κάθε επιθηλιακό κύτταρο ήταν 11,2±4,1 ενώ για το στέλεχος ΚΑ ο αριθμός αυτός ήταν 14±4,5. 0 μέσος όρος φαγοκυτταρωμένων μυκήτων του Ε στελέχους ανά ΠΜΠ ήταν 123±17,3. Για το ΚΑ στέλεχος ο αριθμός αυτός ήταν 86±14,2 και για το ΕΑ στέλεχος ήταν 76,5±13,7. Η μικροβιοκτόνος δράση για το Ε στέλεχος ήταν 34,5±8,3%. Για το ΚΑ στέλεχος η μικροβιοκτόνος δράση ήταν 11,8±2,5%, ενώ για το ΕΑ στέλεχος ήταν ακόμα χαμηλότερη 8,4±2,1%. Από τα παραπάνω συνάγεται ότι η ανθεκτικότητα της C. albicans στην ΑΜΒ σχετίζεται με τροποποίηση τόσο της κυτταρικής μεμβράνης (υποδοχείς), όσο και με τροποποίηση ενδογενών παραγόντων (καταλάση)