Dissecting mechanisms of chromosomemicrotubule interaction in oocytes by new imaging tools

Chromosome alignment and orientation within the spindle in mitosis and meiosis are determined by chromosome-microtubule interaction. Evidence suggests that within the acentrosomal spindle the mechanism of chromosome positioning is different from in mitotic spindle but its molecular bases are not well understood. I investigated how chromosome-microtubule interactions position the chromosomes within the spindle using Drosophila oocytes. I addressed the role and molecular mechanisms of kinetochore and chromosome interaction with microtubules in this process. I developed new live imaging reagents to observe dynamic chromosome-microtubule interaction. Live imaging combined with inactivation of kinetochores in oocytes revealed that kinetochore-microtubule attachment is required for three-step chromosome positioning in Drosophila oocytes: de-congression, change of orientation and re-congression. Augmin, a γ-tubulin recruiting complex, has been previously shown to be important for chromosome congression specifically in oocytes. Live imaging further showed that Augmin facilitates chromosome congression particularly in early stages of spindle assembly. Study of Augmin dynamics revealed that Augmin stably associates with spindle polar regions, specifically in oocytes. This meiotic regulation of Augmin function may contribute to generation of force pushing chromosomes toward spindle equator. Sentin protein has been shown to be important for microtubule plus end dynamics in mitosis. In meiosis, sentin mutant results in reduced distance between centromeres of homologous chromosomes. However, its meiotic role is unknown. Live imaging of the sentin mutant showed that in oocytes Sentin is required for preventing premature stabilization of kinetochore-microtubule attachments. As conclusion, I have used live imaging to reveal molecular basis of the interaction between chromosomes and microtubules particularly important for oocytes

Tumor Digital Masking Allows Precise Patient Triaging: A Study Based on Ki-67 Scoring in Gastrointestinal Stromal Tumors

Background. Technological advances constantly provide cutting-edge tools that enhance the progress of diagnostic capabilities. Gastrointestinal stromal tumors belong to a family of mesenchymal tumors where patient triaging is still based on traditional criteria such as mitotic count, tumor size, and tumor location. Limitations of the human eye and randomness in choice of area for mitotic figure counting compel us to seek more objective solutions such as digital image analysis. Presently, the labelling of proliferative activity is becoming a routine task amidst many cancers. The purpose of the present study was to compare the traditional method of prediction based on mitotic ratio with digital image analysis of cell cycle-dependent proteins. Methods. Fifty-seven eligible cases were enrolled. Furthermore, a digital analysis of previously performed whole tissue section immunohistochemical assays was executed. Digital labelling covered both hotspots and not-hotspots equally. Results. We noted a significant diversity of proliferative activities, and consequently, the results pointed to 6.5% of Ki-67, counted in hotspots, as the optimal cut-off for low–high-grade GIST. ROC analysis (AUC = 0.913; 95% CI: 0.828–0.997, p<0.00001) and odds ratio (OR = 40.0, 95% CI: 6.7–237.3, p<0.0001) pointed to Ki-67 16% as the cut-off for very high-grade (groups 5–6) cases. With help of a tumor digital map, we revealed possible errors resulting from a wrong choice of field for analysis. We confirmed that Ki-67 scores are in line with the level of intracellular metabolism that could be used as the additional biomarker. Conclusions. Tumor digital masking is very promising solution for repeatable and objective labelling. Software adjustments of nuclear shape, outlines, size, etc. are helpful to omit other Ki-67-positive cells especially small lymphocytes. Our results pointed to Ki-67 as a good biomarker in GIST, but concurrently, we noted significant differences in used digital approaches which could lead to unequivocal results

CD63 and GLUT-1 Overexpression Could Predict a Poor Clinical Outcome in GIST: A Study of 54 Cases with Follow-Up

Background and Goals. In light of current knowledge, it seems that alternations underlying GISTs are well explained, although all that is enhanced by various aspects on a daily basis. More recently, attention has been pointed towards exosomes as important particles able to modify healthy and also diseased tissues including cancer. The goal of the present study was an analysis of CD9, CD63, and GLUT-1 as a marker of hypoxia status within 54 cases of GIST and evaluation of their predictive value. Methods. 54 cases of patients suffering from GIST were enrolled into the study, predominantly in the gastric location. All operated cases had no Imatinib and other chemotherapies up to the day of operation. Expression of targeted proteins was performed by immunohistochemistry and, after that, the results with tabulated clinical data were compared by Kaplan-Meier method and multivariate Cox proportional hazard model of statistical analysis. Results. Our results presented a marked dependence of worsening clinical outcome with high expression CD63 (p=0.008) as well as with GLUT-1 (p=0.014). We noted a strict correlation of GLUT-1 expression with CD63 expression (p=0.03), which could confirm the thesis about the contribution of exosomes in intratumoural hypoxia status. The collected material did not confirm CD9 contribution. Conclusions. As presented here, CD63 and GLUT-1 have a prognostic value in GIST cases. The results confirm the other studies in this scope and can be used in future as an additional prognostic factor

Role of immunohistochemical and histochemical profiling in H&E-based diagnosis of scrotal leiomyosarcoma of dartos muscle

Abstract: Histochemical and immunohistochemical methods should often but not always complement standardized histopathologic procedures. Here, we illustrate use of these ancillary techniques in a report of scrotal leiomyosarcoma. 62-year-old male patient presented with a palpable, subcutaneous 2,5 cm wide tumor arising from dartos muscle. The tumor was diagnosed leiomyosarcoma G2 pT1b. Interestingly, the sarcomatous mass was focally strictly attached to convoluted, benign bundles of smooth muscles that were intermingled with tumor mass at peripheral, lateral and superior sides of the lesion. We have used immune- and histochemical methods to confirm histopathological findings based on H&amp;E staining. As expected, in tumor cells smooth muscle actin and desmin were strongly immunopositive similarly as Masson trichrome staining, while S100 and CD34 antigens were immunonegative except for sustained positivity for CD34 in vessels. The auxiliary staining methods can provide additional information on the tumorigenesis of leiomyosarcoma. They can also serve to determine additional features of prognostic significance, since e.g. immunoreactivity of CD34 accurately maps vascular density of tumor and enables a careful assessment of vascular invasion in course of leiomyosarcoma as well

Conventional colon adenomas harbor various disturbances in microsatellite stability and contain micro-serrated foci with microsatellite instability

<div><p>Introduction</p><p>Colorectal cancer belongs to the most frequent occurring malignancies. A prediction of the clinical outcome and appropriate choice of neoadjuvant chemotherapy needs personalized insight to the main driving pathways. Because most CRCs have polyps as progenitor lesions, studying the pathways driving to adenomagenesis is no less important.</p><p>Goals</p><p>Our purpose was the evaluation of microsatellite stability status within conventional colon adenomas and also β-catenin, BRAFV600E and p53 contribution.</p><p>Material and methods</p><p>The cohort included 101 cases of typical colon adenomas with high grade epithelial dysplasia according to WHO. An immunohistochemistry method was used for the depiction of the expression of targeted proteins, as also their heterogeneity.</p><p>Results</p><p>Generally, we noted a 10% frequency of MSI events where MSI-H reached a 5% share occurred within the left colon and rectal polyps. β-catenin nuclear overexpression was noted with a 70% frequency and p53 with close to a 24% frequency. In addition, we found a presence of micro-serration foci more often within tubular adenomas, where focal MSI took place more often. Our results indicate that MSI events occur more often than had been theorized earlier. It results in tumour heterogeneity, more complex underlying pathways and finally ontogenetic molecular-diversity of tumours besides similar occurring histopathological features.</p></div

The characteristics of used antibodies.

<p>The characteristics of used antibodies.</p