Pavel Dvorak

Energy and dose rate dependence of BANG-2 polymer-gel dosimeter.

The purpose of this study was to evaluate dependence of BANG-2 polymer-gel dosimeter sensitivity on different photon and electron energies as well as on different mean dose rates expressed as repetition rates for a standard clinically used linear accelerator. The sensitivity of the dosimeter was represented by the slope of calibration curve in the linear region measured for each modality. A calibration curve (in the linear region) based on five dosimeters (four irradiated and one background) was obtained for each photon and electron energy and different repetition rates. Dosimeter sensitivity dependence on energy was studied for 4, 6, and 18 MV x-ray photons and for nominal electron energies 9, 12, 16, and 20 MeV. Dosimeter sensitivity dependence on mean dose rate was separately studied for electron and photon beams with the use of repetition rates 80, 160, 240, 320, and 400 MU min(-1). Evaluation of dosimeters was performed on Siemens MAGNETOM EXPERT 1T scanner in the head coil. A multiecho sequence with 16 equidistant echoes was used for the evaluation of irradiated polymer-gel dosimeters. The parameters of the sequence were as follows: TR 2000 ms, TE 22.5-360.0 ms, slice thickness 5 mm, FOV 255 mm, one acquisition. There was observed a trend in polymer-gel dosimeter sensitivity dependence on the quality index of high energy x-ray beams used and on mean electron energy absorbed in the center of the dosimeter. Polymer-gel dosimeter sensitivity was decreasing with increasing photon or electron energy. There was observed no trend in polymer-gel dosimeter sensitivity dependence on mean dose rate expressed as a repetition rate for both photon and electron beams.

Independent coexistence of clones with 13q14 deletion at reciprocal translocation breakpoint and 13q14 interstitial deletion in chronic lymphocytic leukemia

13q14 deletion is the most frequent chromosomal aberration in chronic lymphocytic leukemia (CLL), found by interphase fl uorescence in situ hybridization (FISH) in more than 50% of cases of CLL [1]. As a sole aberration it is associated with a favorable prognosis; however, it (13q14 deletion) does not improve the impaired prognosis of complex karyotype [1,2]. A minimal deleted region (MDR) of approximately 900 kbp in size was established by genomic profi ling [3], containing several genes including DLEU7 and miR15a and miR16 1, recognized as tumor suppressor genes [4 – 6]. Deletion larger than the MDR was repeatedly associated with an increased risk of disease progression [3,7]. 13q14 deletion is found predominantly as an interstitial deletion, but it has previously been described as a form of reciprocal translocation with deletion at the 13q14 breakpoint [8 – 12]. In most cases the translocation was reported to bring about heterozygous loss of the 13q14 region, although in one study it was reported as a cause of homozygous loss [9] and in another as a source of second allele loss along with loss of the fi rst by interstitial deletion [10]. Th e parallel presence of two clones with monoallelic 13q14 region loss, in one clone via the mechanism of interstitial deletion and in the second via reciprocal translocation with deletion at the breakpoint, has been noted only once to date [10]. No relevant information about the mechanism of formation of the 13q14 translocation with deletion has been reported so far. Th e coexistence of clones with interstitial deletion and with deletion at the translocation breakpoint provides two possibilities for the formation of translocation with deletion: either by consecutive clonal evolution from the clone with interstitial deletion, or as a separate event, independent from the clone with interstitial deletion. In contrast with 13q14 deletion, the presence of even one translocation aff ecting any chromosome in CLL is associated with impaired prognosis [13,14]. However, an unfavorable infl uence of the translocation form of 13q14 deletion on prognosis has not been proven [10]. Th e 13q14 translocation has been described with various translocation partners, but nevertheless activation of any oncogene has not yet been revealed. Th us, the leukemic eff ect of the 13q14 reciprocal translocation with deletion is presumed to occur only through the loss of tumor suppressor genes caused by the concomitant 13q14 deletion. In this report we present fi ve cases of CLL with the coexistence of clones with interstitial deletion and with deletion at the reciprocal translocation breakpoint. In one of the cases a subsequent clonal analysis proved an independent origin of both clones, providing evidence for their purely coincidental presence. 13q14 deletion was found by interphase FISH performed on bone marrow or peripheral blood cells in 62% of our cohort of unselected patients with CLL (146/235). Detailed metaphase analysis (G-banding, FISH) could be accomplished in 135 patients, and revealed the translocation form of 13q14 deletion in 10% of them (13/135). Th e coexistence of a clone with deletion at the reciprocal translocation breakpoint and another clone with interstitial deletion was found in 38% of the translocation cases (5/13). Th is study focused on the fi ve patients with coexistence of clones with both forms of 13q14 deletion: interstitial deletion and deletion at the translocation breakpoint. In three patients (numbers 2, 3 and 5), cytogenetic analyses were accomplished at the time of diagnosis, and in the two other cases (numbers 1 and 4) at disease progression. Only patient 4 had received previous chemotherapy, including fl udarabine with cyclophosphamide. In three patients (numbers 2, 4 and 5) elevated expression of CD38 was found, and ZAP70 expression was high in two patients (numbers 1 and 2); immunoglobulin heavy chain variable gene (IGHV) mutational status was not analyzed. Cytogenetic analysis was performed on bone marrow (patients 1 – 4) or peripheral blood (patient 5) cells stimulated L eu k L ym ph om a D ow nl oa de d fr om in fo rm ah ea lth ca re .c om b y D uk e U ni ve rs ity o n 12 /2 8/ 14

The translocation t(2;11)(p21;q23) without MLL gene rearrangement—a possible marker of good prognosis in myelodysplastic syndrome patients

The translocation t(2;11)(p21;q23) is associated with de novo myelodysplastic syndromes (MDS) and has an overall frequency of approximately 1%. The outcome of MDS patients with this translocation is not clear until now, because most of the clinical data addressing the t(2;11)(p21;q23) has been collected without investigating the status of the mixed lineage leukemia (MLL) gene. In this report, we present seven new patients with MDS diagnosis and the t(2;11)(p21;q23) in bone marrow cells; all of them without MLL gene rearrangement. They were found in two databases consisting of 1185 patients of two Czech institutions. These patients tended to be younger and showed a strong male predominance. A cytological and histological assessment of bone marrow at diagnosis revealed only mild MDS with marked dysplasia in megakaryopoiesis. Similar to other primary abnormalities in MDS (e.g. deletion of 11q), the t(2;11)(p21;q23) was frequently associated with deletion of 5q. Our results stress the common clinicopathological features of this entity and indicate that the t(2;11)(p21;q23) may be associated with a good prognosis for MDS patients (median survival 72 months). Copyright

ABC gene expression profiles have clinical importance and possibly form a new hallmark of cancer

Adenosine triphosphate–binding cassette proteins constitute a large family of active transporters through extracellular and intracellular membranes. Increased drug efflux based on adenosine triphosphate–binding cassette protein activity is related to the development of cancer cell chemoresistance. Several articles have focused on adenosine triphosphate–binding cassette gene expression profiles (signatures), based on the expression of all 49 human adenosine triphosphate–binding cassette genes, in individual tumor types and reported connections to established clinicopathological features. The aim of this study was to test our theory about the existence of adenosine triphosphate–binding cassette gene expression profiles common to multiple types of tumors, which may modify tumor progression and provide clinically relevant information. Such general adenosine triphosphate–binding cassette profiles could constitute a new attribute of carcinogenesis. Our combined cohort consisted of tissues from 151 cancer patients—breast, colorectal, and pancreatic carcinomas. Standard protocols for RNA isolation and quantitative real-time polymerase chain reaction were followed. Gene expression data from individual tumor types as well as a merged tumor dataset were analyzed by bioinformatics tools. Several general adenosine triphosphate–binding cassette profiles, with differences in gene functions, were established and shown to have significant relations to clinicopathological features such as tumor size, histological grade, or clinical stage. Genes ABCC7, A3, A8, A12, and C8 prevailed among the most upregulated or downregulated ones. In conclusion, the results supported our theory about general adenosine triphosphate–binding cassette gene expression profiles and their importance for cancer on clinical as well as research levels. The presence of ABCC7 (official symbol CFTR) among the genes with key roles in the profiles supports the emerging evidence about its crucial role in various cancers. Graphical abstract

Downregulation of ABC Transporters in Non-neoplastic Tissues Confers Better Prognosis for Pancreatic and Colorectal Cancer Patients

Transport of a wide variety of substrates, including xenobiotics, is one of the main functions attributed to human ATP-binding cassette (ABC) proteins. Overexpression of ABC genes is considered to be an important mechanism facilitating the development of chemoresistance. Relationships between the expression levels of ABC genes in tumor tissues and established clinicopathological features were extensively studied previously. The current study tested our hypothesis that the expression levels of ABC genes in non-neoplastic (control) tissues also provide important information in relation to the relevant tumor progression. Expression levels of all human ABC genes (48 protein coding and one pseudogene), measured by qRT-PCR, were bioinformatically analyzed. The data originated from four independently collected cohorts covering three types of tumors - breast, colorectal and pancreatic carcinomas. ABC gene expression profiles (signatures) in non-neoplastic tissues (matched to tumor samples from three different tumor types) were characteristically clustered into three main types - those with the vast majority of the genes downregulated, upregulated or heterogeneously regulated. The clusters with mostly downregulated and upregulated genes were shown to possess significant relations to good and poor prognostic markers, respectively, in pancreatic and colorectal cancers. The present findings support the theory that the expression of ABC genes in non-neoplastic tissues can significantly contribute to tumor pathogenesis. Suggested multi-gene panels, consisting of the reduced number of ABC genes, have the potential to be implemented as new prognostic markers, which are especially urgent in pancreatic cancer. The results can also stimulate further primary research in carcinogenesis.

Protective Effect of Aspirin Against Oligomeric Aβ42 Induced Mitochondrial Alterations and Neurotoxicity in Differentiated EC P19 Neuronal Cells

BACKGROUND Amyloid-beta (Aβ) induced mitochondrial dysfunction is one of the major causes of neuronal toxicity in Alzheimers disease. A number of recent reports suggest involvement of mitochondrial alterations through intracellular accumulation of oligomeric Aβ. These mitochondrial alterations include increased Reactive Oxygen Species (ROS), mt-DNA depletion, decreased oxidative phosphorylation and ATP production, membrane depolarization, reduced number of mitochondria etc. All these defects cumulatively caused neural toxicity and alterations in cellular energy homeostasis. On the other hand, anti-inflammatory drug aspirin is reported to promote both mitochondrial biogenesis and improvement in cellular energy status. METHODS Taking altogether the mentioned clues, we evaluated protective effect of aspirin, if any on oligomeric Aβ42 induced toxicity and mitochondrial alterations in differentiated neuronal cells. RESULTS A significant reduction in neuronal viability and increased apoptosis was observed in Aβ42 treated cells, as evident by MTT assay, apoptosis ELISA and immunofluorescence from β-III tubulin antibody staining of neuronal cells. A concomitant decrease was also observed in the intensity of mitotracker red FM staining and mt-DNA to nDNA ratio, suggesting mitochondrial membrane depolarization and/or reduced number of mitochondria along with depletion in mt-DNA. However, simultaneous treatment of 5 μM aspirin to oligomeric Aβ42 treated cells protected them from mitochondrial dysfunction and neurotoxicity. CONCLUSION We suggest mitochondrial biogenesis, changes in mitochondrial membrane potential and / or inhibition of Aβ42 aggregation by aspirin as possible underlying mechanism(s).

Paying for Banking Services: What Determines the Fees?

We analyze a unique dataset to test an empirical model of retail bank fee determinants in five Central European countries. Due to the data structure we can cope with heterogeneity and cross-subsidization by employing a representative fee index instead of using variables associated with individual fees. We find support for the Structure-Conduct-Performance hypothesis about the effect of industry concentration, the importance of differences in reliance on cashless payments, and differences in the labor intensity and technology level of bank operations. We also show that cross-country differences in retail bank fees can be explained by fundamental economic factors.

Trends in gene expression changes during adipogenesis in human adipose derived mesenchymal stem cells under dichlorodiphenyldichloroethylene exposure

BackgroundsExposure to lipophilic environmental pollutants has been explored as a risk factor of development of diabetes mellitus in obese. Adipose tissue is a reservoir of bioaccumulative lipophilic contaminants including p,pʹ-dichlorodiphenyldichloroethylene (DDE). Our aim was to analyze the effect of DDE (in concentrations 0.1 μM, 1 μM, and 10 μM) on adipocyte differentiation and insulin signalling pathway on in vitro adipogenic model of human adipose derived mesenchymal stem cells (hADMSC).MethodsThe effect of DDE was monitored by analysis of expression (RT qPCR, Western blotting) of genes involved in adipocyte differentiation and insulin signalling pathway including lipid metabolism on days 0, 4, 10, 21, 28 of differentiation.ResultsThe main observation was significant increase of INSR, LIPE, FASN, SREBP1, OCT4 and AKT2 expression under influence of DDE. We did not record any increase of the active form of Akt.ConclusionOur findings suggest that DDE exposure changes the differentiation of adipocytes, enhances the lipid metabolism and so may play a role in the development of obesity and metabolic diseases by affecting the insulin signalling pathway. The influence of DDE seems to be similar to the effect of insulin itself. However, further studies to elucidate the mechanism of action of DDE are necessary.

PO-163 Identification of candidate genes underlying soft tissue sarcoma progression using a progression series of murine fibrosarcoma cell lines

Introduction Soft tissue sarcomas are known for their great variability in clinical behaviour, ranging from almost indolent lesions to rapidly metastasing tumours. Genes responsible for sarcoma progression have been poorly characterised by now. Towards this end, we established a unique single-background progression series of murine sarcoma cell lines, consisting of the slowly proliferating nonmotile and noninvasive cell line JUN-2, rapidly proliferating, motile and invasive cell line JUN-3, and the cell line JUN-2fos-3 that exhibits a unique transformation pattern, with little deregulation of cell growth and proliferation, but pronounced motility and invasiveness. Material and methods This unique distribution of transformation related-traits made us possible to identify two separate groups of genes tentatively involved in sarcoma progression in a single transcriptomic analysis – on the one hand, proliferation-related genes could be identified by their differential expression in JUN-3 compared to both JUN-2 and JUN-2fos3, and, on the other hand, motility and invasiveness-related genes could be identified by their common expression pattern in JUN-2fos3 and JUN-3 cells compared to JUN-2. The high-throughput gene expression analysis has been performed using the GeneChip Mouse Genome 430 2.0 Array (ThermoFisher Scientific). Results and discussions In total, we identified 277 upregulated and 212 downregulated unique transcripts in JUN-2 and JUN-2fos3 compared to the JUN3 cells (adjustP <10-4). Simultaneously, we showed 29 upregulated and 112 downregulated unique transcripts in JUN3 and JUN2fos3 compared to JUN2 cell cultures (adjustP <10-4). The chemokine Ccl-8 was identified as a possible druggable target responsible for sarcoma motility, as it is overexpressed in both motile cell lines and its pharmacological inhibition significantly downregulated JUN-3 cell motility. Interestingly, the scrutiny of differentially expressed genes in the motile cell lines JUN-2fos3 and JUN-3 revealed a significant downregulation of genes that are put into context with stem cells in general (Abcg2 – 21x reduction) or specifically in sarcoma (Connective Tissue Growth Factor – 10x reduction). This is reminiscent of the phenomenon ‘go or grow’ independently described for brain tumours. Conclusion Our sarcoma cell lines could thus provide a valuable model to identify key genes responsible for sarcoma progression as well as to decipher a relationship between stemness and cancer invasion. Supported by the Czech Grant Agency project No 17–17636S.

Dysregulation of KRAS signaling in pancreatic cancer is not associated with KRAS mutations and outcome

Pancreatic ductal adenocarcinoma (PDAC) is a tumor with a poor prognosis, and no targeted therapy is currently available. The aim of the present study was to investigate the prognostic significance of the expression of V-Ki-ras2 Κirsten rat sarcoma viral oncogene homolog (KRAS), downstream signaling pathway genes and the association with clinical characteristics in PDAC patients undergoing radical surgery. Tumors and adjacent non-neoplastic pancreatic tissues were examined in 45 patients with histologically verified PDAC. KRAS and B-Raf proto-oncogene, serine/threonine kinase (BRAF) gene mutation analysis was performed using the KRAS/BRAF/phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α array. The transcript profile of 52 KRAS downstream signaling pathway genes was assessed using quantitative-polymerase chain reaction. KRAS mutation was detected in 80% of cases. The genes of four signaling pathways downstream of KRAS, including the phosphoinositide 3-kinase/3-phosphoinositide-dependent protein kinase 1/V-akt murine thymoma viral oncogene homolog 1, RAL guanine nucleotide exchange factor, Ras and Rab interactor 1/ABL proto-oncogene-1, non-receptor tyrosine kinase, and RAF proto-oncogene serine/threonine-protein kinase/mitogen-activated protein kinase pathways, exhibited differential expression in PDAC compared with that in the adjacent normal tissues. However, no significant differences in expression were evident between patients with KRAS-mutated and wild-type tumors. The expression of KRAS downstream signaling pathways genes did not correlate with angioinvasion, perineural invasion, grade or presence of lymph node metastasis. Additionally, the presence of KRAS mutations was not associated with overall survival. Among the KRAS downstream effective signaling pathways molecules investigated, only v-raf-1 murine leukemia viral oncogene homolog 1 expression was predictive of prognosis. Overall, KRAS mutation is present in the majority of cases of PDAC, but is not associated with changes in the expression of KRAS downstream signaling pathways and the clinical outcome. This may partly explain the failure of KRAS-targeted therapies in PDAC.