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Hodgkin lymphoma (HL) and diffuse large B cell lymphoma (DLBCL) represent 15% and 20%, respectively, of all lymphoma types. The aim of this study was to identify and compare circulating serum miRNA (c-miRNA) and peripheral whole blood miRNA (wb-miRNA) profiles in patients with these lymphomas. Serum samples (20 HL, 21 DLBCL, and 30 healthy controls) and whole blood samples (21 HL, 17 DLBCL patients, and 30 healthy controls) were collected at the time of diagnosis. Serum and whole blood were also collected from 18 HL/17 DLBCL and eight HL/nine DLBCL patients, respectively, after treatment. Pairwise comparisons identified 125 c-miRNAs (adjusted P value < 0.05) showing significant dysregulation between 30 healthy controls and patients; of these, 47 and 55 differentiated controls from pretherapeutic HL and DLBCL patients, respectively. In addition, 60 and 16 c-miRNAs differentiated controls from posttherapeutic HL and DLBCL, respectively. Pairwise comparisons identified 292 wb-miRNAs (adjusted P value < 0.05) showing significant dysregulation between 30 controls and patients; of these, 103 and 169 differentiated controls from pretherapeutic HL and DLBCL, respectively, and 142 and 151 wb-miRNAs differentiated controls from posttherapeutic HL and DLBCL, respectively. Thus, lymphoma-associated miRNAs may be a better source of noninvasive candidate biomarkers than miRNAs in serum. It is unclear whether miRNA alterations in lymphoma cells are similar to those observed in white blood cells.

Purpose. Nonfunctioning gonadotropic pituitary neuroendocrine tumors (PitNETs) are among the most frequent neoplasms of pituitary gland. Although PitNETs are commonly considered benign, a notable part of patients suffer from tumor recurrence after treatment. Invasive growth of pituitary tumor is among the most important prognostic factors. Since molecular features of invasiveness are of potential clinical usefulness, this study was aimed to verify whether invasive and noninvasive nonfunctioning gonadotropic PitNETs differ in the miRNA expression profile and whether the differences could provide a possible molecular classifier. Methods. miRNA profiles were determined in 20 patients (11 invasive and 9 noninvasive tumors) using next-generation sequencing. The expression of selected miRNAs was assessed in the independent cohort of 80 patients with qRT-PCR. Results. When miRNA profiles of invasive and noninvasive tumors were compared, 29 miRNAs were found differentially expressed. Hsa-miR-184, hsa-miR-181a-2-3p, hsa-miR-93-3p, hsa-miR-574-5p, hsa-miR-185-5p, and hsa-miR-3200-5p showed a potential clinical value according to ROC curve analysis. Unfortunately, differential expression of only hsa-miR-185-5p was confirmed in the validation cohort, with AUG at 0.654. Conclusion. Differences in miRNAs expression profiles in invasive and noninvasive gonadotropic PitNETs are slight and the level of miRNA expression seems not to be applicable as useful classifier of tumor invasiveness.

Background Inflammatory bowel diseases are classic polygenic disorders, with genetic loads that reflect immunopathological processes in response to the intestinal microbiota. Herein we performed the multiomics analysis by combining the large scale surveys of gut bacterial community, stool microRNA (miRNA) and short chain fatty acid (SCFA) signatures to correlate their association with the activity of Crohn’s disease (CD). Methods DNA, miRNA, and metabolites were extracted from stool samples of 15 CD patients, eight with active disease and seven in remission, and nine healthy individuals. Microbial, miRNA and SCFA profiles were assessed using datasets from 16S rRNA sequencing, Nanostring miRNA and GC-MS targeted analysis, respectively. Results Pairwise comparisons showed that 9 and 23 taxa differed between controls and CD patients with active and inactive disease, respectively. Six taxa were common to both comparisons, whereas four taxa differed in CD patients. α-Diversity was lower in both CD groups than in controls. The levels of 13 miRNAs differed (p-value 1.5) in CD patients and controls before FDR correction and 4 after. Of six SCFAs, the levels of two differed significantly (p-value 1.5) in CD patients and controls, and the levels of four differed in patients with active and inactive CD. PLS-DA revealed models with smallest error rate for controls in bacterial component and inactive disease in metabolites. Conclusion A complex interrelationship may exist between gut dysbiosis, miRNA profiling and SCFA level in response to intestinal inflammation.

Background: Sarcomas are rare malignant tumors of mesenchymal origin. The discovery of circulating biomarkers with high diagnostic value could supplement diagnosis of this heterogenous group of tumors. The aim of this study was to identify the profiles of circulating miRNA (c-miRNAs) in four groups of common bone and soft tissue sarcomas. Methods: At the time of diagnosis, blood samples were collected from 86 patients: 36 with locally advanced/unresectable/metastatic gastrointestinal stromal tumor (GIST) who received first-line treatment with imatinib; 16 with locally advanced osteosarcoma (OS); 26 with locally advanced synovial sarcoma (SS); and eight with locally advanced Ewing sarcoma (ES). In addition, samples were collected from 30 healthy controls. C-miRNAs were isolated using a miRCURY RNA Isolation Kit, followed by preparation of cDNA libraries and sequencing on the Ion Proton platform. Results: Pair-wise comparisons identified 156 unique c-miRNAs (adjusted P-value < 0.05) showing significant dysregulation between controls and patients; of these, 24, 36, 42, and 99 differentiated controls from pretherapeutic OS, SS, ES, and GIST, respectively. Ten c-miRNAs were commonly altered in at least three sarcoma types. Receiver operating characteristic curves and area under the curve (ROC-AUC) analyses revealed that a four-miRNA diagnostic classifier was able to differentiate controls from ES, GIST, OS, and SS, with AUC-ROC values of 1, 0.97, 0.95, and 0.94, respectively. Conclusions: Aberrant miRNA expression signatures were identified in serum from patients with four different sarcoma subtypes. Differences in miRNA expression profiles between sarcoma patients and healthy volunteers suggest that miRNAs may play a role in sarcoma development.

Sepsis is the leading cause of death in intensive care units worldwide. Current treatments of sepsis are largely supportive and clinical trials using specific pharmacotherapy for sepsis have failed to improve outcomes. Here, we used the lipopolysaccharide (LPS)-stimulated mouse RAW264.7 cell line and AlphaLisa assay for TNFa as a readout to perform a supervised drug repurposing screen for sepsis treatment with compounds targeting epigenetic enzymes, including kinases. We identified the SCH772984 compound, an extracellular signal-regulated kinase (ERK) 1/2 inhibitor, as an effective blocker of TNFa production in vitro. RNA-Seq of the SCH772984-treated RAW264.7 cells at 1, 4, and 24 h time points of LPS challenge followed by functional annotation of differentially expressed genes highlighted the suppression of cellular pathways related to the immune system. SCH772984 treatment improved survival in the LPS-induced lethal endotoxemia and cecal ligation and puncture (CLP) mouse models of sepsis, and reduced plasma levels of Ccl2/Mcp1. Functional analyses of RNA-seq datasets for kidney, lung, liver, and heart tissues from SCH772984-treated animals collected at 6 h and 12 h post-CLP revealed a significant downregulation of pathways related to the immune response and platelets activation but upregulation of the extracellular matrix organization and retinoic acid signaling pathways. Thus, this study defined transcriptome signatures of SCH772984 action in vitro and in vivo, an agent that has the potential to improve sepsis outcome.

The data demonstrating a correlation between sonographic markers of malignancy of thyroid cancer (TC) and its genetic status are scarce. This study aimed to assess whether the addition of genetic analysis at the preoperative step of TC patients’ stratification could aid their clinical management. The material consisted of formalin-fixed paraffin-embedded tumor fragments of 49 patients who underwent thyroidectomy during the early stages of papillary TC (PTC). Tumor DNA and RNA were subjected to next-generation sequencing (NGS) on Ion Proton using the Oncomine™ Comprehensive Assay panel. We observed a significant correlation between BRAF V600E and a higher EU-TIRADS score (p-value = 0.02) with a correlation between hypoechogenicity and taller-than-wide tumor shape in analysed patients. There were no other significant associations between the identified genetic variants and other clinicopathological features. For TC patient’s stratification, a strong suspicion of BRAF V600E negativity in preoperative management of TC patients could limit the over-treatment of asymptomatic, very low-risk, indolent disease and leave room for active surveillance.

Crohn's disease is an inflammatory disorder that can affect the entire gastrointestinal tract but typically involves the ileocecal region. Before endoscopy was widely used, involvement of the esophagus, stomach, and duodenum was thought to be rare. Recent publications demonstrated that not only are upper gastrointestinal lesions common in Crohn's disease (affecting up to 75% of the patients), but they also present characteristic endoscopic findings with potential clinical significance. It was suggested that lesions in the stomach with a bamboo joint-like appearance might be an endoscopic biomarker for Crohn's disease. It was also found that this occurrence is related to a more severe disease course. Our review summarizes the literature, as well as our own observations and considerations, concerning the issue of upper gastrointestinal involvement in Crohn's disease and its clinical meaning.

Aims Mesenchymal stromal cells isolated from different tissues are claimed to demonstrate similar therapeutic potential and are often incorrectly named mesenchymal stem cells. However, through comparison of such cells is lacking. This study aimed to compare the transcriptome of mesenchymal cells of the same phenotype isolated from the heart muscle and epicardial fat of the same patient, before and after culture. Methods and results Cells were isolated from biopsies of the right ventricle and epicardial fat collected from five patients (three men and two women, mean age 59.4 ± 2.6) who underwent heart transplantation due to ischaemic cardiomyopathy. In both tissues, immunophenotyping revealed three distinct populations: (i)CD31- CD45- CD90+ CD34+ CD146- , (ii) CD31- CD45- CD90+ CD34- CD146+ , and (iii) CD31- CD45- CD90- CD34- CD146+ , of which only the first one could be grown after sorting. Material for RNA-seq was collected from these cells before culture (250 cells) and at passage 6 (5000 cells). Transcriptomic analysis revealed that cells of the same phenotype (CD31- CD45- CD90+ CD34+ CD146- ) upon isolation preferentially clustered according to the tissue of origin, not to the patient from whom they were isolated. Genes up-regulated in the right ventricle-derived cells were related to muscle physiology while down-regulated genes included those encoding proteins with transmembrane signalling receptor activity. After six passages, heart-derived and fat-derived cells did not acquire similar transcriptome. Cells isolated from the right ventricle in comparison with their epicardial fat-derived counterparts demonstrated higher level of transcripts related, among others, to RNA processing and muscle development. The down-regulated genes were involved in the nucleosome assembly, DNA packaging and replication, and interleukin-7-mediated signalling pathway. Cells from epicardial fat demonstrated higher heterogeneity both before and after culture. Cell culture significantly changed gene expression profile within both tissues. Conclusions This study is an essential indication that mesenchymal cells isolated from different tissues do not demonstrate similar properties. Phenotypic identification and ease of isolation cannot be considered as a criterion in any therapeutic utilization of such cells.

Background Fetuses whose mothers have produced antibodies to red blood cell (RBC) or platelet antigens are at risk of being affected by hemolytic disease or alloimmune thrombocytopenia, respectively, only if they inherit the incompatible antigen. Noninvasive diagnosis of the fetal antigen is employed for management of immunized pregnancies, but the specific detection of SNPs, encoding the majority of antigens, in maternal plasma is still a challenge. We applied targeted next-generation sequencing (NGS) to predict the fetal antigen based on the detection of fetomaternal chimerism. Methods and materials The DNA of 13 pregnant women (with anti-K [3] anti-k [1], anti-Fya [1], anti-D + C + Jka [1], anti-D + E + K [1], anti-HPA-1a [1], anti-HPA-3b [1], anti-HPA-5b [1], and nonimmunized [3]) was sequenced using primers for regions encoding RhD, RhC, Rhc, RhE/e, K/k, Fya/b, Jka/b, MN, Ss, and HPA-1, 2, 3, 5, 15, 4 X-polymorphisms on the Ion Torrent Personal Genome Machine (PGM) System (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Results NGS results were in agreement with the phenotype/genotype of women and their neonates (except for the unsuccessful detection of MN and RhC). NGS determined fetal allele chimerism for K, k, Fya, Fyb, Jka, Jkb, S, RhE (from 0.42% to 6.08%); RhD, Rhc (100%); HPA-1a, -2b, -3a, 3b, -5b, -15a, 15b (from 0.23% to 4.11%). NGS revealed fetal chimerism for incompatible antigens (from 0.7% to 4.8%) in 7 immunized cases, excluded in 3 (with anti-K, anti-Fya , anti-HPA-3b). Conclusion The designed NGS predicts the fetal RBC and platelet antigen status universally in cases with various clinically significant antibodies as well as providing confirmation of the presence of fetal DNA. However, some improvement of the unsuccessful primers is required.

Background and Aims The study investigates the practical utility of whole-blood gene expression profiling to diagnose inflammatory bowel diseases [IBDs]. Methods The discovery cohorts included 102 and 51 paediatric IBD patients and controls, and 95 and 46 adult IBD patients and controls, respectively. The replication cohorts included 447 and 76 paediatric IBD patients and controls, and 271 and 108 adult IBD patients and controls, respectively. In the discovery phase, RNA samples extracted from whole peripheral blood were analysed using RNA-Seq, and the predictive values of selected biomarkers were validated using quantitative polymerase chain reaction [qPCR]. Results In all, 15 differentially expressed transcripts [adjusted p ≤0.05] were selected from the discovery sequencing datasets. The receiver operating characteristic curves and area under the curve [ROC-AUC] in replication analyses showed high discriminative power [AUC range, 0.91–0.98] for 11 mRNAs in paediatric patients with active IBD. By contrast, the AUC-ROC values ranged from 0.63 to 0.75 in comparison among inactive paediatric IBDs and active/inactive adult IBDs, indicating a lack of discriminative power. The best multi-mRNA diagnostic classifier showed moderate discriminative power [AUC = 0.81] for paediatric inactive IBD, but was not able to discriminate active or inactive adult IBD patients from controls. The AUC-ROC values did not confirm an ability of the mRNAs abundances to discriminate between active ulcerative colitis and active Crohn’s disease in paediatric or adult populations. Conclusions This study identifies and validates blood transcriptional biomarkers that could be used in clinical settings as diagnostic predictors of IBD clinical activity in paediatric, but not adult, IBD patients.

Background: USP8 mutations are the most common driver changes in corticotroph pituitary tumors. They have direct effect on cells&rsquo, proteome through disturbance of ubiquitination process and also influence gene expression. The aim of this study was to compare microRNA profiles in USP8-mutated and wild-type tumors and determine the probable role of differential microRNA expression by integrative microRNA and mRNA analysis. Methods: Patients with Cushing&rsquo, s disease (n = 28) and silent corticotroph tumors (n = 20) were included. USP8 mutations were identified with Sanger sequencing. MicroRNA and gene expression was determined with next-generation sequencing. Results: USP8-mutated patients with Cushing&rsquo, s disease showed higher rate of clinical remission and trend towards lower tumor volume than wild-type patients. Comparison of microRNA profiles of USP8-mutated and wild-type tumors revealed 68 differentially expressed microRNAs. Their target genes were determined by in silico prediction and microRNA/mRNA correlation analysis. GeneSet Enrichment analysis of putative targets showed that the most significantly overrepresented genes are involved in protein ubiquitination-related processes. Only few microRNAs influence the expression of genes differentially expressed between USP8-mutated and wild-type tumors. Conclusions: Differences in microRNA expression in corticotropinomas stratified according to USP8 status reflect disturbed ubiquitination processes, but do not correspond to differences in gene expression between these tumors.

Recently, liquid biopsy has emerged as a tool to monitor oncologic disease progression and the effects of treatment. In this study we aimed to determine the clinical utility of liquid biopsy relative to conventional oncological post-treatment surveillance. Plasma cell-free (cf) DNA was collected from six healthy women and 37 patients with breast cancer (18 and 19 with stage III and IV tumors, respectively). CfDNA was assessed using the Oncomine Pan-Cancer Cell-Free Assay. In cfDNA samples from patients with BC, 1112 variants were identified, with only a few recurrent or hotspot mutations within specific regions of cancer genes. Of 65 potentially pathogenic variants detected in tumors, only 19 were also discovered in at least one blood sample. The allele frequencies of detected variants (VAFs) were &lt, 1% in cfDNA from all controls and patients with stage III BC, and 24/85 (28.2%) variants had VAFs &gt, 1% in only 8 of 25 (32%) patients with stage IV BC. Copy number variations (CNVs) spanning CDK4, MET, FGFR1, FGFR2, ERBB2, MYC, and CCND3 were found in 1 of 12 (8%) and 8 of 25 (32%) patients with stage III and IV tumors, respectively. In healthy controls and patients without BC progression after treatment, VAFs were &lt, 1%, while in patients with metastatic disease and/or more advanced genomic alterations, VAFs &gt, 1% and/or CNV were detected in approximately 30%. Therefore, most patients with stage IV BC could not be distinguished from those with stage III disease following therapy, based on liquid biopsy results.

The severity of Duchenne muscular dystrophy (DMD), an incurable disease caused by the lack of dystrophin, might be modulated by different factors, including miRNAs. Among them, miR-378 is considered of high importance for muscle biology, but intriguingly, its role in DMD and its murine model (mdx mice) has not been thoroughly addressed so far. Here, we demonstrate that dystrophic mice additionally globally lacking miR-378 (double-KO [dKO] animals) exhibited better physical performance and improved absolute muscle force compared with mdx mice. Accordingly, markers of muscle damage in serum were significantly decreased in dKO mice, accompanied by diminished inflammation, fibrosis, and reduced abundance of regenerating fibers within muscles. The lack of miR-378 also normalized the aggravated fusion of dystrophin-deficient muscle satellite cells (mSCs). RNA sequencing of gastrocnemius muscle transcriptome revealed fibroblast growth factor 1 (Fgf1) as one of the most significantly downregulated genes in mice devoid of miR-378, indicating FGF1 as one of the mediators of changes driven by the lack of miR-378. In conclusion, we suggest that targeting miR-378 has the potential to ameliorate DMD pathology., miR-378 deficiency improves the dystrophic phenotype in mdx mice, suggesting that modulation of miR-378 level may be a potential approach to attenuate DMD severity.

Bladder cancer (BC) remains the most common malignancy of urinary tract. Sex-related differences in BC epidemiology, diagnosis, therapy, and outcomes have been reported. Throughout the recent years, extensive research has been devoted to genetic and molecular alterations in BC. Apart from the molecular background, another related concept which has been speculated to contribute to gender diversities in BC is the role of urinary pathogens in bladder carcinogenesis. Microbiome studies, fueled by the availability of high-throughput DNA-based techniques, have shown that perturbation in the microbiome is associated with various human diseases. The aim of this review is to comprehensively analyze the current literature according to sex-related differences in the microbiome composition in BC.

Background Anti-HPA-1a alloantibodies in HPA-1a negative mothers can lead to fetal/neonatal alloimmune thrombocytopenia (FNAIT). Noninvasive prenatal testing (NIPT) of HPA-1a determines fetuses at risk and the course of maternal antenatal treatment. Study design and methods The aim was to develop and validate HPA-1a NIPT by real-time polymerase chain reaction (PCR) or next-generation sequencing (NGS) for a high-throughput screening setting. DNA from 328 plasma samples of 299 HPA-1a negative pregnant women was examined for HPA-1a by real-time PCR and in two cases also by NGS (Ion Torrent). The results were compared with neonatal HPA-1a genotyping in 281 cases. Results HPA-1a NIPT was negative in 44 of 51 HPA-1a negative fetuses, inconclusive in five, and false positive in two. In 228 of 229 HPA-1a positive fetuses, the NIPT results were positive (mean threshold cycle 36.0 ± 1.7) and inconclusive in one. In 22 cases with HPA-1a positive fetuses analyzed twice, the sensitivity of HPA-1a detection was significantly higher at 28 weeks compared with 16 to 20 weeks. NGS efficiently detected the ITGB3 coding HPA-1a/b (1% and 5% fetal HPA-1a reads). Conclusion Real-time PCR is reliable to predict the fetal HPA-1a positive genotype in a screening study, but false-positive results are reported in 4%, with unnecessary prenatal treatment if anti-HPA-1a is detected.

// Tomasz Rzymski 1, * , Michal Mikula 2, * , Eliza Żylkiewicz 1 , Agnieszka Dreas 1 , Katarzyna Wiklik 1 , Aniela Golas 1 , Katarzyna Wojcik 1 , Magdalena Masiejczyk 1 , Anna Wrobel 1 , Izabela Dolata 1 , Agata Kitlinska 1 , Malgorzata Statkiewicz 2 , Urszula Kuklinska 2 , Krzysztof Goryca 2 , Łukasz Sapala 1 , Aleksandra Grochowska 3 , Aleksandra Cabaj 2, 4 , Malgorzata Szajewska-Skuta 1 , Ewelina Gabor-Worwa 1 , Katarzyna Kucwaj 1 , Arkadiusz Bialas 1 , Adam Radzimierski 1 , Michal Combik 1 , Jakub Woyciechowski 1 , Maciej Mikulski 1 , Renata Windak 1 , Jerzy Ostrowski 2, 3 and Krzysztof Brzozka 1 1 R&D Department, Selvita S.A., Krakow, Poland 2 Department of Genetics, Maria Sklodowska-Curie Memorial Cancer Center, Warsaw, Poland 3 Department of Gastroenterology, Hepatology and Clinical Oncology, Medical Center for Postgraduate Education, Warsaw, Poland 4 Laboratory of Bioinformatics, Nencki Institute of Experimental Biology, Warsaw, Poland * These authors have contributed equally to this work Correspondence to: Krzysztof Brzozka, email: krzysztof.brzozka@selvita.com Keywords: CDK8 mediator, kinase inhibitor, STAT5, AML, leukemia stem cells Received: August 04, 2016 Accepted: March 09, 2017 Published: April 04, 2017 ABSTRACT Inhibition of oncogenic transcriptional programs is a promising therapeutic strategy. A substituted tricyclic benzimidazole, SEL120-34A, is a novel inhibitor of Cyclin-dependent kinase 8 (CDK8), which regulates transcription by associating with the Mediator complex. X-ray crystallography has shown SEL120-34A to be a type I inhibitor forming halogen bonds with the protein’s hinge region and hydrophobic complementarities within its front pocket. SEL120-34A inhibits phosphorylation of STAT1 S727 and STAT5 S726 in cancer cells in vitro . Consistently, regulation of STATs- and NUP98-HOXA9- dependent transcription has been observed as a dominant mechanism of action in vivo . Treatment with the compound resulted in a differential efficacy on AML cells with elevated STAT5 S726 levels and stem cell characteristics. In contrast, resistant cells were negative for activated STAT5 and revealed lineage commitment. In vivo efficacy in xenotransplanted AML models correlated with significant repression of STAT5 S726. Favorable pharmacokinetics, confirmed safety and in vivo efficacy provide a rationale for the further clinical development of SEL120-34A as a personalized therapeutic approach in AML.

This article is a summary of the opinions of the authors, as presented at the annual conference of the Nowotwory Journal of Oncology , ‘Oncological Debates’, held in Warszawa, 8–9th April 2016. This work is not a typical review, in that it contains only the viewpoints of the authors, as opposed to any critical review of the literature.

A certain minimum plasma concentration of (Z)-endoxifen is presumably required for breast cancer patients to benefit from tamoxifen therapy. In this study, we searched for DNA variants that could aid in the prediction of risk for insufficient (Z)-endoxifen exposure. A metabolic ratio (MR) corresponding to the (Z)-endoxifen efficacy threshold level was adopted as a cutoff value for a genome-wide association study comprised of 287 breast cancer patients. Multivariate regression was used to preselect variables exhibiting an independent impact on the MR and develop models to predict below-threshold MR values. In total, 15 single-nucleotide polymorphisms (SNPs) were significantly associated with below-threshold MR values. The strongest association was with rs8138080 (WBP2NL). Two alternative models for MR prediction were developed. The predictive accuracy of Model 1, including rs7245, rs6950784, rs1320308, and the CYP2D6 genotype, was considerably higher than that of the CYP2D6 genotype alone (AUC 0.879 vs 0.758). Model 2, which was developed using the same three SNPs as for Model 1 plus rs8138080, appeared as an interesting alternative to the full CYP2D6 genotype testing. In conclusion, the four novel SNPs, tested alone or in combination with the CYP2D6 genotype, improved the prediction of impaired tamoxifen-to-endoxifen metabolism, potentially allowing for treatment optimization.

Background Acute myeloid leukemia (AML) is characterized by rapid proliferation of myeloid blood cells. Due to its heterogeneity and the high rate of acquired drug resistance, new treatment modalities are needed. SEL120 is a specific type I selective inhibitor of Cyclin-dependent kinase 8 (CDK8) and Cyclin-dependent kinase 19 (CDK19). A first- in- human phase Ib clinical trial with SEL120 in patients with AML or HR-MDS was initiated in June 2019. Preclinical studies demonstrated high efficacy of SEL120 in experimental AML models via mechanisms involving differentiation and induction of apoptosis. Transcriptomic analysis of hematological cell lines demonstrated that SEL120 treatment upregulated expression of an apoptotic activator BIM from BCL-2 family of proteins. Methods Efficacy of the compound alone or in combination was tested in viability assays in a broad panel of cancer cell lines. Activity and mechanism of action of CDK8 inhibitor - SEL120 alone and in combination was investigated by flow cytometry, western blotting, co-immunoprecipitation, differential gene expression and ChIPseq analysis. In vivo efficacy was tested in mice injected with MV4-11 cell line both subcutaneously and intravenously. Results Here we provide a strong rationale for combination of SEL120 and BCL-2-selective inhibitor Venetoclax (ABT-199). We found that SEL120 synergistically induced apoptosis with Venetoclax in AML cells. Combination of both compounds significantly reduced levels of prosurvival protein MCL-1 and induced hallmarks of apoptosis including Caspase-3 activation and PARP cleavage. Previous studies associated Venetoclax resistance with increased sequestration of proapoptotic BIM by high levels of MCL-1. While a SEL120 treatment alone had no effects on MCL-1 levels, combination of both compounds resulted in sensitization of Venetoclax-resistant AML cells. We demonstrated that mechanism of Venetoclax resistance can be abrogated by the cotreatment with SEL120 leading to changes in proportions of BIM and MCL-1 levels. Synergistic interaction between SEL120 and Venetoclax resulted in apoptotic cell death in established cell lines and patient derived AML cells. Finally, using murine models of subcutaneous or disseminated AML, we found complete remissions of AML and associated recovery of normal cells in bone marrow of animals treated with both SEL120 and Venetoclax. Conclusion Taken together, these data provided rationale for a novel clinical strategy that may lead to durable responses in AML patients. Citation Format: Tomasz Rzymski, Marta Obacz, Milena Mazan, Marion Chappelier, Marcus Järås, Michal Mikula, Elżbieta Adamczyk, Katarzyna Wiklik, Michal Combik, Aniela Golas, Magdalena Masiejczyk, Przemyslaw Juszczynski, Jerzy Ostrowski, Krzysztof Brzózka. Synergistic effect of CDK8 and BCL-2 inhibition in AML through regulation of MCL-1 and BIM balance [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6217.

Primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC), and inflammatory bowel diseases (IBDs), including Crohn’s disease (CD) and ulcerative colitis (UC), are heterogeneous chronic autoimmune diseases that may share underlying pathogenic mechanisms. Herein, we compared simultaneously analyzed blood transcriptomes from patients with PBC, PSC, and IBD. Microarray-based measurements were conducted using RNA isolated from whole blood samples from 90, 45, 95 and 93 patients with PBC, PSC, CD, and UC, respectively, and 47 healthy controls. Expression levels of selected transcripts were analyzed by quantitative reverse-transcribed PCR using an independent cohort of 292, 71 and 727 patients with PBC, PSC, and IBD, respectively. Of 4026, 2650 and 4967 probe sets differentially expressed (adjusted p-value

Deregulated transcription, one of the key features of acute myelogenous leukemia (AML), remains largely unactionable by recently approved therapies. Preclinical studies indicate that targeting Cyclin Dependent Kinase 8 (CDK8) may be a novel therapeutic strategy for AML. CDK8 and its paralog CDK19, restrain activation of super-enhancer-associated tumor suppressors and lineage commitment genes in AML cells. SEL120 is a first-in-class, specific and selective inhibitor of CDK8/CDK19 and has shown activity in preclinical AML models. Preclinical characterization of SEL120 demonstrated a unique mechanism of action, related to known functions of CDK8 in the regulation of transcription. Cells sensitive to SEL120 show enrichment for leukemia stem cells (LSCs) signatures and higher signal transducer and activator of transcription 5 (STAT5) levels. Treatment with SEL120 reduced STAT5 phosphorylation in sensitive cell lines both in vitro and in vivo. Transcriptomic analysis of AML cells revealed that SEL120 regulated genes involved in lineage controlling functions. Specifically, trimethylation of lysine 27 on histone H3 (H3K27me3) by the polycomb repressive complex 2 (PRC2), was proposed to maintain the stemness of LSCs by preventing differentiation. Derepression of lineage commitment genes marked with H3K27me3, was one of the earliest transcriptional events in sensitive cells treated with SEL120. Consistent with its transcriptional effects, SEL120 induced differentiation of AML cells. Additionally SEL120 significantly repressed the MYC Proto-Oncogene-dependent transcriptomic signatures. Efficacy of SEL120 in AML was confirmed in models with high translational potential, including patient-derived AML cells (PDC) in vitro and in vivo. PDCs treated with SEL120 showed reduced viability, induction of apoptotic cell death and differentiation commitment. Administration of SEL120 in orthotopic AML patient-derived xenograft models reduced tumor burden to the level undetectable by flow cytometry, decreased splenomegaly and resulted in partial bone marrow recovery. CLI120-001 is a first-in-human, open-label, multi-center, modified 3+3 dose escalation phase Ib study of SEL120 with a dose-escalation cohort (DC) followed by an enrichment cohort (EC) in adult patients with AML or high-risk myelodysplastic syndrome who have relapsed or refractory disease and have received no more than 3 prior lines of therapy. Other key inclusion criteria are Eastern Cooperative Oncology Group performance status of 0-2, white blood cell (WBC) count 10000/µL, adequate organ function defined as: aspartate aminotransferase and alanine aminotransferase ≤3x the upper limit of normal (ULN), total bilirubin ≤1.5x ULN, creatinine clearance ≥60 ml/min, left ventricular ejection fraction ≥40%. Major exclusion criteria include Q to T wave interval corrected for heart rate (QTc) ≥450 ms, taking concomitant medications that are known to be strong inhibitors or inducers of cytochrome P450 1A2 or that can prolong QTc and/or cause torsade de pointes. The primary objective of the study is to assess safety and tolerability of SEL120 and to establish the recommended dose (RD) for further clinical development. Secondary objectives include evaluation of preliminary anti-leukemic activity and characterization of the pharmacokinetic profile of SEL120. The exploratory objective is to assess pharmacodynamics of SEL120 by using relevant biomarkers, including STAT5 pS726, transcriptional profiling by RNAseq and immunophenotypic changes related to stemness and differentiation of AML cells. SEL120 is administered as a single oral dose every other day for a total of 7 doses i.e. on days 1, 3, 5, 7, 9, 11 and 13, in a 21-day treatment cycle. Patients receive SEL120 until disease progression, unacceptable toxicity, or withdrawal of consent. The DC is now enrolling patients who are treated at dose levels defined by a modified Fibonacci sequence, and will end with selection of the RD based on all available study data. In the EC, additional patients will be treated at the RD to further support the evaluation of the RD of SEL120 monotherapy. The study is currently running in the United States and is planned to be completed in 2020. The ClinicalTrials.gov Identifier: NCT04021368. Disclosures Borthakur: Eli Lilly and Co.: Research Funding; FTC Therapeutics: Membership on an entity's Board of Directors or advisory committees; Agensys: Research Funding; Eisai: Research Funding; Oncoceutics: Research Funding; Oncoceutics, Inc.: Research Funding; BioTheryX: Membership on an entity's Board of Directors or advisory committees; GSK: Research Funding; NKarta: Consultancy; Cyclacel: Research Funding; Argenx: Membership on an entity's Board of Directors or advisory committees; Janssen: Research Funding; BioLine Rx: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Research Funding; Bayer Healthcare AG: Research Funding; Novartis: Research Funding; Cantargia AB: Research Funding; Strategia Therapeutics: Research Funding; BMS: Research Funding; PTC Therapeutics: Consultancy; Xbiotech USA: Research Funding; AbbVie: Research Funding; Arvinas: Research Funding; Polaris: Research Funding; Merck: Research Funding; AstraZeneca: Research Funding; Tetralogic Pharmaceuticals: Research Funding. Abboud:Jazz Pharma: Speakers Bureau; Novartis: Other: Member on an entity's Board of Directors or advisory committees (Ended 12/30/2017), Research Funding; Agios: Other: Member on an entity's Board of Directors or advisory committees (Ended 12/30/2017); Tetraphase Pharmaceuticals: Consultancy, Honoraria; Seattle Genetics: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; NKarta: Consultancy, Honoraria; Incyte: Consultancy, Honoraria; Bayer: Consultancy, Honoraria. Nazha:Abbvie: Consultancy; Incyte: Speakers Bureau; Daiichi Sankyo: Consultancy; Jazz Pharmacutical: Research Funding; Novartis: Speakers Bureau; MEI: Other: Data monitoring Committee; Tolero, Karyopharma: Honoraria. Mazan:Selvita S.A.: Employment. Majewska:Selvita S.A.: Employment. Wiklik:Selvita S.A.: Employment. Golas:Selvita S.A.: Employment. Bialas:Selvita S.A.: Employment. Windak:Selvita S.A.: Employment. Juszczynski:Selvita S.A.: Consultancy, Membership on an entity's Board of Directors or advisory committees. Chrom:Selvita S.A.: Employment. Rzymski:Selvita S.A.: Employment, Equity Ownership. Brzózka:Selvita S.A.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Background. The proper use of new medical tests in clinical practice requires the establishment of their value and range of diagnostic usefulness. While whole-exome sequencing (WES) has already entered the medical practice, recognizing its diagnostic usefulness in multifactorial diseases has not yet been achieved. Aims. The objective of this study was to establish usability of WES in determining genetic background of chronic cholestatic liver disease (CLD) in young patients. Methods. WES was performed on six young patients (between 17 and 22 years old) with advanced fibrosis or cirrhosis due to CLD and their immediate families. Sequencing was performed on an Ion Proton sequencer. Results. On average, 19,673 variants were identified, of which from 7 to 14 variants of an individual were nonsynonymous, homozygous, recessively inherited, and considered in silico as pathogenic. Although monogenic cause of CLD has not been determined, several heterozygous rare variants and polymorphisms were uncovered in genes previously known to be associated with CLD, including ATP8B1, ABCB11, RXRA, and ABCC4, indicative of multifactorial genetic background. Conclusions. WES is a potentially useful diagnostic tool in determining genetic background of multifactorial diseases, but its main limitation results from the lack of opportunities for direct linkage between the uncovered genetic variants and molecular mechanisms of disease.

Most inflammatory bowel diseases (IBDs) are classic complex disorders represented by common alleles. Here we aimed to define the genetic architecture of pediatric and adult-onset IBDs for the Polish population. A total of 1495 patients were recruited, including 761 patients with Crohn’s disease (CD; 424 pediatric), 734 patients with ulcerative colitis (UC; 390 pediatric), and 934 healthy controls. Allelotyping employed a pooled-DNA genome-wide association study (GWAS) and was validated by individual genotyping. Whole exome sequencing (WES) was performed on 44 IBD patients diagnosed before 6 years of age, 45 patients diagnosed after 40 years of age, and 18 healthy controls. Altogether, out of 88 selected SNPs, 31 SNPs were replicated for association with IBD. A novel BRD2 (rs1049526) association reached significance of P = 5.2 × 10−11 and odds ratio (OR) = 2.43. Twenty SNPs were shared between pediatric and adult patients; 1 and 7 were unique to adult-onset and pediatric-onset IBD, respectively. WES identified numerous rare and potentially deleterious variants in IBD-associated or innate immunity-associated genes. Deleterious alleles in both groups were over-represented among rare variants in affected children. Our GWAS revealed differences in the polygenic architecture of pediatric- and adult-onset IBD. A significant accumulation of rare and deleterious variants in affected children suggests a contribution by yet unexplained genetic components.

Cyclin-dependent kinase 8 inhibitors (CDK8i) have anti-cancer activity in human acute myeloid leukaemia (AML) cell lines both in vitro and in vivo. Activity of CDK8i often involves deregulation of super-enhancer-associated genes in AML cell lines. Previous studies established SEL120 as a specific CDK8 inhibitor active in AML cells with increased STAT1/5 signalling pathways. Differential gene expression analysis demonstrated high enrichment of leukaemia stem cell (LSC) signatures in responding cells, linked to resistance to standard therapies and relapsed disease. Cells sensitive to SEL120 treatment were positive for CD34 and negative for lineage commitment surface markers. SEL120 markedly reduced STAT5 phosphorylation on serine 726 (STAT5 pS726) in sensitive cell lines. Prolonged SEL120 treatment led to significant downregulation of CD34 and induction of lineage commitment markers. Transcriptomic analysis revealed that SEL120 regulated many genes involved in differentiation and apoptosis. We observed synergistic effects of SEL120 with standard of care cytotoxic drugs such as cytarabine. Treatment of AML cells with cytarabine spared many CD34+ cells, which could be effectively eradicated by subsequent treatment with SEL120. Many cell lines which were resistant to SEL120 treatment could be sensitized by concomitant treatment with BH3 mimetic agent ABT-199. Combination of both compounds resulted in potent induction of apoptosis in AML cells in vitro and in vivo. Treatment of mice bearing subcutaneously implanted human leukaemia cell lines resulted in significant tumour growth inhibition, whereas cotreatment with ABT-199 led to complete regressions at doses which were vey well tolerated by animals. Next we have selected patient derived primary AML cells using gene expression signatures identifying SEL120 -responder cell lines. In these cells SEL120 significantly reduced viability, induced apoptosis and lineage commitment. Further, the same cells were implanted into NOD scid gamma mice. Animals succumbed to AML, diagnosed by a significant presence of human CD45/CD34 positive leukaemia cells in a peripheral blood and splenomegaly. Stand-alone treatment with SEL120 resulted in the complete remission of AML cells in a peripheral blood and bone marrow, and reduced spleen weight, without symptoms of compound-related toxicity. These results validate SEL120 as a promising agent in the treatment of AML. Citation Format: Milena Mazan, Eliza Majewska, Michal Mikula, Katarzyna Wiklik, Michal Combik, Aniela Golas, Magdalena Masiejczyk, Elzbieta Fiedor, Anna Polak, Magdalena Cybulska, Aleksandra Grochowska, Michal Kopczynski, Urszula Kuklinska, Zuzanna Sandowska-Markiewicz, Malgorzata Statkiewicz, Agnieszka Paziewska, Michalina Dabrowska, Arkadiusz Bialas, Maciej Mikulski, Renata Windak, Jerzy Ostrowski, Przemyslaw Juszczynski, Krzysztof Brzozka, Tomasz Rzymski. SEL120, a potent and specific inhibitor of CDK8 induces complete remission in human patient derived xenograft models of acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1306.

International audience; Background: Prognosis of localised gastrointestinal stromal tumour (GIST) is heterogeneous, notably for patients with AFIP intermediate or high risk of relapse, who are candidates to adjuvant imatinib. We hypothesised that gene expression profiles might improve the prognostication and help to refine the indications for imatinib.Methods: We collected gene expression and histoclinical data of 146 pre-treatment localised GIST samples treated with surgery alone. We searched for a gene expression signature (GES) predictive for relapse-free survival (RFS) and compared its performances to that of three published prognostic proliferation-based GES (Genomic Grade Index (GGI), 16-Kinase, and CINSARC) and AFIP classification. We also analysed a data set from 28 patients with advanced GIST treated with neo-adjuvant imatinib.Results: We identified a 275-gene GES (gene expression signature) predictive of RFS in a learning set and validated its robustness in an independent set. However, the GGI outperformed its prognostic performances, and those of the two other signatures and the AFIP intermediate-risk classification in two independent tests sets in uni- and multivariate analyses. Importantly, GGI could split the AFIP intermediate/high-risk samples into two groups with different RFS. Genomic Grade Index 'high-risk' tumours were more proliferative and genetically unstable than 'low-risk' tumours, and more sensitive to imatinib.Conclusion: GGI refines the prediction of RFS in localised GIST and might help tailor adjuvant imatinib.

Possession of a BRCA1/2 mutation increases risk of contralateral breast and ovarian cancer recurrence and may have an impact on health management decisions, such as imaging screening, preventive surgical interventions and systemic therapies. A hospital-based study was conducted to assess the frequency and spectrum of pathogenic germline BRCA1 and BRCA2 mutations in Polish women with familial and nonfamilial breast cancer. Genomic DNA was extracted from 1581 women with breast cancer and from 2225 healthy individuals. For genotyping BRCA1 (5382insC, T300G, 3819del5, 185delAG, C5370T, 3875del4, 3896delT, 4153delA, 4184del4, 4160delAG, G5332A) mutations and BRCA2 (G1408T, 5467insT, 6174delT, 6192delAT, 6675delTA, 8138del5, 9152delT, C9610T, 9630delC) mutations, a Custom TaqMan (Applied Biosystems) PCR-based technology was adopted. A BRCA1 mutation was found in 26 and 12.5 % of women with familial breast cancer and in 13 and 8.3 % nonfamilial (sporadic) breast cancer, diagnosed before or after 50 years of age, respectively. A much lower frequency of BRCA2 mutation was observed. The predominance of seven BRCA1 mutations (5382insC, T300G, 3819del5, 185delAG, C5370T, 3875del4, 4153delA) studied in the Masovian voivodeship population confirmed a strong founder effect for BRCA1 mutations in the Polish population, and the results of BRCA2 testing confirmed a high diversity in the studied pathogenic mutations in BRCA2 gene. We propose offering inexpensive testing for the presence of BRCA1 founder mutations to all Polish women at the time of initial breast cancer diagnosis, regardless of the patient's family history or age of disease onset.

Purpose Aggressive intervention against the bladder wall during transurethral resection of bladder tumors (TURBT) causes damage and leakage from blood vessels to the bladder lumen. The aim of this study was to determine whether TURBT could increase the level of circulating urothelial cells. Methods Expression of tumor markers, discriminative for nucleated blood cells and urothelium, was evaluated by quantitative (q) RT-PCR on RNA isolated from peripheral blood samples of 51 patients who underwent TURBT for ≥cT1c bladder tumors. Results Four of 14 studied genes, epidermal growth factor receptor (EGFR), Collagen α-1(I) chain, Mast/stem cell growth factor receptor (KIT) and CD47, exhibited significant differences in gene expression between controls and cancer patients. While TURBT did not significantly increase the number of PCR-positive results of any transcripts, positive RT-PCR detection for EGFR was significantly less frequent on day 30 compared to results obtained before surgery. Conclusions Although the results of our study do not provide evidence for increased tumor cell release into the peripheral blood after TURBT, they seem to indicate that EGFR mRNA measurement in the blood may provide useful information for urologists. Electronic supplementary material The online version of this article (doi:10.1007/s11255-011-0102-z) contains supplementary material, which is available to authorized users.

Proteomic mass spectrometry is gaining an increasing role in diagnostics and in studies on protein complexes and biological systems. This experimental technology is producing high-throughput data which is inherently noisy and may contain various errors. Mathematical processing can help in removing them.In this paper we focus on the peak alignment problem in LC-MS spectra. As an alternative to heuristic approaches to the problem, we propose a mathematically sound method which exploits a model-based clustering. In this framework experiment errors are modeled as deviations from real values and mass spectra are regarded as finite Gaussian mixtures. The advantage of such an approach is that it provides convenient techniques for adjusting parameters and selecting solutions of best quality. The method can be parameterized by assuming various constraints. In this paper we investigate and compare different classes of models. We analyze the results in terms of statistically significant biomarkers that can be identified after the alignment of spectra. The study was conducted on a dataset of plasma samples of colorectal cancer patients and healthy donors.

Gastroesophageal reflux disease is regarded as a spectrum of diseases: non-erosive reflux disease (NERD), erosive reflux disease (ERD), and the far end of the spectrum represented by patients with Barrett's esophagus. Among predisposing factors, both risk and protective polymorphic variants of several genes may influence the clinical outcomes of reflux disease. Consequently, different molecular mechanisms are likely to underlie the development of clinical variants of reflux disease. Ninety six patients with reflux disease were screened for polymorphisms of CARD15, SLC22A4 (OCTN1), SLC22A5 (OCTN2), DLG5, ATG16L1 and IL23R genes which had previously been found to associate with immune-mediated chronic inflammatory disorders. While none of the polymorphisms were associated with NERD or ERD, the 1142G/A variant of the IL23R gene was found to be a risk variant in Barrett's esophagus patients. The IL23/IL23R pathway may modulate STAT3 transcriptional activity which is an essential regulator not only of immune-mediated inflammation, but also of inflammatory-associated apoptosis resistance. Although the mechanisms of metaplastic transition of inflamed squamous epithelium are undetermined as yet, our findings suggest potential involvement of alternations in the IL23/IL23R pathway as a molecular background of Barrett's esophagus development.

Here we develop a fully automated procedure for the analysis of liquid chromatography–mass spectrometry (LC–MS) datasets collected during the analysis of complex peptide mixtures. We present the underlying algorithm and outcomes of several experiments justifying its applicability. The novelty of our approach is to exploit the multidimensional character of the datasets. It is common knowledge that highly complex peptide mixtures can be analyzed by liquid chromatography coupled with mass spectrometry, but we are not aware of any existing automated MS spectra interpretation procedure designed to take into account the multidimensional character of the data. Our work fills this gap by providing an effective algorithm for this task, allowing for automated conversion of raw data to the list of masses of peptides.

Background: Although Helicobacter pylori is a significant etiologic factor of peptic ulcer disease, it remains unknown why ulcers develop only in the minority of infected individuals. Aim: The aim of this cross-sectional study was to evaluate the association between the presence of duodenal ulcer in H. pylori-infected patients and different risk factors. Methods: A total of 122 H. pylori-infected patients were enrolled; 79 had duodenal ulcer and 43 gastritis. Univariate analysis was conducted using either Fisher’s exact test or exact Cochrane-Armitage trend test. In multivariate analysis the logistic model was used. Results: Univariate analysis indicated six factors (male sex, smoking, antral H. pylori density, cagA presence in antrum, and vacA s1a presence in antrum and corpus). Four factors (sex, smoking-alcohol index, H. pylori density index, and cagA index) were found to be significant in multivariate analysis. The best model predicting duodenal ulcer included male sex, smoking, presence of H. pylori on histopathology in antrum and cagA presence in corpus. Conclusion: Although several risk factors were significantly associated with duodenal ulcer, we failed in the identification of either a single risk factor or a set of factors that can unequivocally differentiate patients with ulcer from those with gastritis.

Colorectal cancer (CRC) is an epidemiological problem of a great importance in Poland; each year approximately 14,600 new cases of the disease are diagnosed. Mortality associated with CRC reaches approximately 10,400 cases per year (according to the National Cancer Registry). The 5-year survival rate is approximately 25 %, which is one of the lowest rates in Europe. The etiology of sporadic colorectal cancer (CRC) is multifactorial and has been attributed to an interplay between both environmental and genetic risk factors. In addition, there is a general consensus that genetic factors may modulate the influence of environmental insults. Following these assumptions, we performed a study on widely described polymorphisms in xenobiotic-metabolizing enzymes and DNA repair genes which may influence individual susceptibility to cancer. We selected five candidate polymorphisms in following genes: ERCC1 Asp118Asn (rs11615), XPC i11C/A (rs2279017), XRCC3 Met241Thr (rs861539) CYP1A1 Ile462Val (rs1048943) and NAT2 A803G (rs1208) and assessed the importance of chosen SNPs on groups consisting of 478 CRC patients and 404 controls. Only CYP1A1 Ile462Val was statistically significant in CRC patients over 50 years old: OR 2.05 (1.29–3.28); p = 1.25E−02 and this association was more pronounced in the female group of CRC patients after the age of 50: OR 2.72 (1.43–5.14); p = 1.14E−02. Electronic supplementary material The online version of this article (doi:10.1007/s12032-014-0072-y) contains supplementary material, which is available to authorized users.

A total proctocolectomy with ileal pouch-anal anastomosis (IPAA) is considered the surgery of choice for definitive management of familial adenomatous polyposis (FAP) and some patients with ulcerative colitis (UC). However, this surgical treatment is often associated with pouchitis, a long-term complication that occurs mostly in UC patients. The purpose of this study was to better define the molecular background of pouchitis. A microarray-based survey was performed using pouch mucosal samples collected from 28 and 8 patients undergoing surgery for UC and FAP, respectively. There were 4,770 genes that significantly differentiated uninflamed from inflamed mucosal samples, and their functional features were represented mostly by metabolic and cell proliferation pathways. In contrast, functional analyses of aberrantly expressed genes between UC and FAP samples, irrespective of mucosal inflammation status, revealed multiple pathways and terms that were linked to changes in immune response. Interestingly, the comparison of uninflamed UC and FAP samples identified a set of 29 altered probe sets, including an inflammation-related transcript encoding a Charcot-Leyden crystal (CLC) protein. The most distinct changes in gene expression profiles differentiating uninflamed UC and FAP pouch mucosal samples were attributed to the Gene Ontology category innate immune response. Our study confirmed that alterations in immune responses can be found between patients who underwent surgery for UC and FAP, independent of the pouch inflammation status. This observation may be important when managing IPAA patients.

Background: We analysed critically the potential usefulness of RNA- and DNA-based biomarkers in supporting conventional histological diagnostic tests for prostate carcinoma (PCa) detection. Methods: Microarray profiling of gene expression and DNA methylation was performed on 16 benign prostatic hyperplasia (BPH) and 32 cancerous and non-cancerous prostate samples extracted by radical prostatectomy. The predictive value of the selected biomarkers was validated by qPCR-based methods using tissue samples extracted from the 58 prostates and, separately, using 227 prostate core biopsies. Results: HOXC6, AMACR and PCA3 expression showed the best discrimination between PCa and BPH. All three genes were previously reported as the most promising mRNA-based markers for distinguishing cancerous lesions from benign prostate lesions; however, none were sufficiently sensitive and specific to meet the criteria for a PCa diagnostic biomarker. By contrast, DNA methylation levels of the APC, TACC2, RARB, DGKZ and HES5 promoter regions achieved high discriminating sensitivity and specificity, with area under the curve (AUCs) reaching 0.95−1.0. Only a small overlap was detected between the DNA methylation levels of PCa-positive and PCa-negative needle biopsies, with AUCs ranging between 0.854 and 0.899. Conclusions: DNA methylation-based biomarkers reflect the prostate malignancy and might be useful in supporting clinical decisions for suspected PCa following an initial negative prostate biopsy.

Background: Prevention of Helicobacter pylori infection may help to control related gastritis, peptic ulcer and cancer. Of the possible preventive measures, immunization was successfully employed in various animal studies. However, no immunization protocol has been accepted for humans. A better characterization of the immune response against the pathogen may be required before a human vaccine is developed. Aim: To identify bacterial proteins which induce an immune response in infected humans or H. pylori-immunized rabbits. Methods: An expression library of H. pylori genes was screened with sera from infected humans and from immunized rabbits. Positive clones were partially sequenced and identified on the basis of a homology search of a H. pylori genome database. Encoded proteins were expressed directly from positive clones and analyzed by SDS-PAGE/Western blot techniques. Results: 114 positive clones were isolated: 79 by screening with human sera and 35 by screening with rabbit sera. Western blot analysis demonstrated that selected clones encoded one or more strongly immunoreactive proteins. 64 clones selected with human sera had no counterparts among clones from screening with rabbit serum. 13 of these clones encoded a total of 21 unknown H. pylori proteins. 17 clones selected with rabbit sera were not immunostained with human sera. They represent 2 various regions of the H. pylori genome which encoded 3 bacterial proteins of unknown function. Conclusions: Screening of H. pylori expression library identified immunogenic proteins – potential vaccine antigens.

Background: A novel 1-h topical method eradicated Helicobacter pylori in 96% of dyspeptic patients. The eradication rate of amoxycillin/omeprazole therapy varies from 0 to 93%. Aim: To compare both methods in patients with endoscopically proven duodenal ulcer. Methods: Eighty patients (59 males, 21 females; median age 43 years) were randomized into two therapeutic groups. The first group (group A) was treated with a 6-week course of ranitidine 300 mg/day, then omeprazole 20 mg b.d. with pronase 36 000 units/day for 2 days, followed by 1-h topical therapy with a solution of bismuth, metronidazole, amoxycillin and pronase. The second group (group B) consisted of patients treated with omeprazole 20 mg b.d. and amoxycillin 2 g/day for 2 weeks, followed by a 4-week course of ranitidine 300 mg/day. Eradication of H.␣pylori was assessed by urease test, histology, a polymerase chain reaction and a 13C-urea breath test, all of which were performed 4 weeks after discontinuation of the antibacterial treatment. Results: Eradication rates in groups A and B were 2.5% and 35% in an intention-to-treat analysis, respectively. Side-effects were encountered in 40.5% and 12.5% of subjects in groups A and B, respectively. Treatment tolerance was rated as poor by 54% of patients in group A and 2.5% of patients in group B. Conclusions: Both treatment regimens, the 1-h topical method and amoxycillin with omeprazole, have low eradication rates in patients with duodenal ulcer. In addition, the topical treatment is characterized by a high rate of side-effects and poor tolerance. Based on the results of our study, neither method can be recommended for eradication of H. pylori in patients with duodenal ulcer.

11024Background: Recent studies have shown that isocitrate dehydrogenase 1/2 (IDH1/2) activating mutations occur in a variety of cancers, including acute myeloid leukemia, gliomas, and chondrosarco...

BACKGROUND: Clinical progression of colorectal cancers (CRC) may occur in parallel with distinctive signaling alterations. We designed multidirectional analyses integrating microarray-based data with biostatistics and bioinformatics to elucidate the signaling and metabolic alterations underlying CRC development in the adenoma-carcinoma sequence. METHODOLOGY/PRINCIPAL FINDINGS: Studies were performed on normal mucosa, adenoma, and carcinoma samples obtained during surgery or colonoscopy. Collections of cryostat sections prepared from the tissue samples were evaluated by a pathologist to control the relative cell type content. The measurements were done using Affymetrix GeneChip HG-U133plus2, and probe set data was generated using two normalization algorithms: MAS5.0 and GCRMA with least-variant set (LVS). The data was evaluated using pair-wise comparisons and data decomposition into singular value decomposition (SVD) modes. The method selected for the functional analysis used the Kolmogorov-Smirnov test. Expressional profiles obtained in 105 samples of whole tissue sections were used to establish oncogenic signaling alterations in progression of CRC, while those representing 40 microdissected specimens were used to select differences in KEGG pathways between epithelium and mucosa. Based on a consensus of the results obtained by two normalization algorithms, and two probe set sorting criteria, we identified 14 and 17 KEGG signaling and metabolic pathways that are significantly altered between normal and tumor samples and between benign and malignant tumors, respectively. Several of them were also selected from the raw microarray data of 2 recently published studies (GSE4183 and GSE8671). CONCLUSION/SIGNIFICANCE: Although the proposed strategy is computationally complex and labor-intensive, it may reduce the number of false results.

Anastomosis leakage is one of the most serious complications of colorectal surgery. A role for extracellular matrix remodelling in the healing process of the colon wall has been recently postulated. Changes in matrix metalloproteinase (MMP) activity in the intestinal wall occurring prior to elective resection and primary anastomosis appear to be responsible for dehiscence leading to anastomosis. Thrombophylaxis using low-molecular-weight heparins is routinely administered to all patients during the perioperative period. However, adverse antiproliferative and proapoptotic effects such as limitation of bioavailability of growth factors and angiogenesis inhibition have been characterized in various cell types as a result of heparin administration. It is also likely that relationships exist between extracellular matrix homeostasis and the coagulation/fibrinolysis system. We hypothesize that subcutaneous administration of LMWHs (low-molecular-weight heparins) may influence matrix metalloproteinase activity in the colon wall and increase the risk of postoperative leakage.

Background: The vacA genotypes and the cagA gene status were investigated in 80 Helicobacter pylori-infected patients with duodenal ulcer (DU) and 49 with gastritis only. Methods: Lysates of gastric biopsy specimens were used directly for polymerase chain reaction-based detection. Results: The m1 subtype was found in 36% and 31% and the m2 in 36% and 46% of specimens from patients with DU and gastritis, respectively (P > 0.05). In 15% of samples the midregion remained unclassified. The prevalence rate of s1 subtypes was higher in cases of DU (69%) than in gastritis (43%) (P < 0.0001); the opposite correlation was observed for s2. The cagA gene was detected in 80% of patients with DU and in 52% of those with gastritis (P < 0.0001). Infections with multiple H. pylori strains exceeded 50% in both groups. Conclusions: These results suggest that vacA s1 genotype and cagA + status are associated with higher DU prevalence and that mixed H. pylori infections are very common in our geographic region.

Some aspects of the design of SAW acceleration sensors are investigated. The principles of operation are briefly reassumed. Our studies reveal that the cantilever beam is the optimum mechanical structure for the device. The calculations and experiments allow to determine the limits of applicability of lithium niobate as the substratum for the SAW acceleration sensor.© (1997) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

The results of theoretical and experimental studies on the construction of refractive index phase transducer for the range below 1.45 are presented. This transducer may be applied in liquid refractometer. Presented results concern the technology of measurement head based on fiberoptic taper and measurements ofthis device. 1. 1TRODUCTION Biconical tapered fiber..optic has been Studied for some time past from the point of view of application in fiber-opticrefractometry. For instance, Kumar and co-workers ' presented the intensity refractometer based on the unilaterally tap-ered multi-mode optic fiber. On this base L. Loyd, C. Bobb and H. D. Krumbaltz described a new refractometer construc-tion with tapered core of multi-mode optic fiber as an active element2 order to ensure an access of studied liquid to thecore, an optic fiber coating should be removed in this construction. An analogous refractometer, which does not requirecoating removal, however, has been presented by authors of this work.3 A considerable sensitivity increase, but for de-creased measurement range, may be obtained with application of bi-conically tapered single-mode optic fiber as it hasbeen suggested by, e.g., S. Lacroix and co-workers.4 The base of action of such a refractometric head is an inter-mode in-terference obtained in a "sensitive" area. In this work a scheme of technological set-up for the preparation of bi-conicaltapered single-mode optic fiber is presented. The example of refractometric characteristic of obtained transducer is alsopresented.

Use of optical fiber sensors and gyroscopes require a number of in-line components. One important device is a polarizing element. The design of a fiber polarizer using a biconical taper is reported. The model performances for the operating wavelength of 1.3 micrometers are characterized by an extinction ratio of 40 dB or more, with a loss of the desired polarization (throughput loss) of about 2 dB.

The technological aspects and researches results of single - mode fiber polarizer are described. Single - mode fiber has been polished and covered with metalic layer for undiserable polarization mode attenuation. The extinction ratio range from 10 to 28 dB was obtained at λ = 0.63 and 0.85 µm. The general optimlization analysis of this kind of polarizers is presented, too.

The aminopyrine breath test and measurement of postprandial serum bile acids were performed in 67 patients with well-documented hereditary hepatic porphyria. Elevated postprandial serum bile acids levels were found in 75% and the aminopyrine breath test was abnormal in 42% of the patients studied. These function tests exhibited a statistically significant correlation with the excretion of porphyrin precursors in patients with acute intermittent porphyria. Furthermore, all 12 patients with acute intermittent porphyria who had a liver biopsy because of abnormalities in the aminopyrine breath test or postprandial serum bile acids measurement, or both, were found to have diffuse, moderately severe, but nonspecific ultrastructural changes of hepatocytes, indicative of significant subcellular damage. While a direct relationship between functional and morphologic abnormalities is speculative, it is possible that the defective heme biosynthesis, perhaps related to the reduced aminopyrine demethylation, could form the basis for the ultrastructural hepatic changes in porphyria. This, in turn, could be the cause of the elevated postprandial serum bile acids levels.

Serum retinol levels were determined by a fluorometric method in patients with colorectal cancer or polyps and those with inflammatory bowel disease. Serum retinol levels in patients with benign or malignant colorectal polyps and stage B cancer (modified Dukes' classification) were similar to those found in controls. By contrast, serum retinol levels were significantly lower in patients with Dukes' stage C or D. Among cancer patients that were followed after surgical treatment serum retinol levels did not differ significantly from those found in controls. Patients who died of metastases during follow-up possessed very low serum retinol levels. These findings suggest that a decreased serum retinol level in cancer patients is a consequence rather than a precursor of the neoplastic process. Furthermore, this study suggests that the marked decrease in serum retinol level might be an indicator of poor prognosis in colorectal cancer patients after surgery.