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Eine mogliche klassische Komplikation des M. Crohn ist die Ausbildung einer Stenose, die wahrend des gesamten Krankheitsverlaufs auftreten und sich in Abhangigkeit von der Lumeneinengung klinisch unterschiedlich prasentieren kann. Berichtet wird uber einen 39-jahrigen Patienten mit einer stenosierenden Ileitis terminalis, die sich letztendlich erst nach Verlegung durch einen Fremdkorper manifestierte.

Acute megakaryoblastic leukemia (AMKL) represents ∼10% of pediatric acute myeloid leukemia cases and typically affects young children (

Leukemia is a complex genetic disease caused by errors in differentiation, growth, and apoptosis of hematopoietic cells in either lymphoid or myeloid lineages. Large-scale genomic characterization of thousands of leukemia patients has produced a tremendous amount of data that have enabled a better understanding of the differences between adult and pediatric patients. For instance, although phenotypically similar, pediatric and adult myeloid leukemia patients differ in their mutational profiles, typically involving either chromosomal translocations or recurrent single-base-pair mutations, respectively. To elucidate the molecular mechanisms underlying the biology of this cancer, continual efforts have been made to develop more contextually and biologically relevant experimental models. Leukemic cell lines, for example, provide an inexpensive and tractable model but often fail to recapitulate critical aspects of tumor biology. Likewise, murine leukemia models of leukemia have been highly informative but also do not entirely reproduce the human disease. More recent advances in the development of patient-derived xenografts (PDXs) or human models of leukemias are poised to provide a more comprehensive, and biologically relevant, approach to directly assess the impact of the in vivo environment on human samples. In this review, the advantages and limitations of the various current models used to functionally define the genetic requirements of leukemogenesis are discussed.

Infiltration of tumor-promoting immune cells is a strong driver of tumor progression. Especially the accumulation of macrophages in the tumor microenvironment is known to facilitate tumor growth and to correlate with poor prognosis in many tumor types. TAp73, a member of the p53/p63/p73 family, acts as a tumor suppressor and has been shown to suppress tumor angiogenesis. However, what role TAp73 has in regulating immune cell infiltration is unknown. Here, we report that low levels of TAp73 correlate with an increased NF-κB-regulated inflammatory signature in breast cancer. Furthermore, we show that loss of TAp73 results in NF-κB hyperactivation and secretion of Ccl2, a known NF-κB target and chemoattractant for monocytes and macrophages. Importantly, TAp73-deficient tumors display an increased accumulation of protumoral macrophages that express the mannose receptor (CD206) and scavenger receptor A (CD204) compared to controls. The relevance of TAp73 expression in human breast carcinoma was further accentuated by revealing that TAp73 expression correlates negatively with the accumulation of protumoral CD163

Over the last two decades, molecular biology has been changed by the introduction of high-throughput technologies. Data sharing requirements have prompted the establishment of persistent data archives. A standardized approach for recording and managing these data was first proposed in the Minimal Information About a Microarray Experiment (MIAME) guidelines. The Minimal Information about a high throughput nucleotide Sequencing Experiment (MINSEQE) proposal was introduced in 2008 as a logical extension of the guidelines to next-generation sequencing (NGS) technologies used for transcriptome analysis.We present a historical snapshot of the data-sharing situation focusing on transcriptomics data from both microarray and RNA-sequencing experiments published between 2009 and 2013, a period during which RNA-seq studies became increasingly popular for transcriptome analysis. We assess how much data from RNA-seq based experiments is actually available in persistent data archives, compared to data derived from microarray based experiments, and evaluate how these types of data differ. Based on this analysis, we provide recommendations to improve RNA-seq data availability, reusability, and reproducibility.

Chromosomal translocations involving the KMT2A gene are among the most common genetic alterations found in pediatric acute myeloid leukemias although the molecular mechanisms that initiate the disease remain incompletely defined. To elucidate these initiating events we used a human model system of acute myeloid leukemia driven by the KMT2A-MLLT3 (KM3) fusion. More specifically, we investigated changes in DNA methylation, histone modifications, and chromatin accessibility at each stage of our model system and correlated these with expression changes. We observed the development of a pronounced hypomethyl - ation phenotype in the early stages of leukemic transformation after KM3 addition along with loss of expression of stem-cell-associated genes and skewed expression of other genes, such as S100A8/9, implicated in leukemogenesis. In addition, early increases in the expression of the lysine demethylase KDM4B was functionally linked to these expression changes as well as other key transcription factors. Remarkably, our ATAC-sequencing data showed that there were relatively few leukemia-specific changes and that the vast majority corresponded to open chromatin regions and transcription factor clusters previously observed in other cell types. Integration of the gene expression and epigenetic changes revealed that the adenylate cyclase gene ADCY9 is an essential gene in KM3-acute myeloid leukemia, and suggested the potential for autocrine signaling through the chemokine receptor CCR1 and CCL23 ligand. Collectively, our results suggest that KM3 induces subtle changes in the epigenome while co-opting the normal transcriptional machinery to drive leukemogenesis.

A possible classical complication of Crohn's disease is the formation of a stenosis, which can occur throughout the course of the disease and can present differently depending on the narrowing of the lumen. This article reports the case of a 39-year-old male patient with a stenosing terminal ileitis, which was ultimately only manifested after obstruction by a foreign body.Eine mögliche klassische Komplikation des M. Crohn ist die Ausbildung einer Stenose, die während des gesamten Krankheitsverlaufs auftreten und sich in Abhängigkeit von der Lumeneinengung klinisch unterschiedlich präsentieren kann. Berichtet wird über einen 39-jährigen Patienten mit einer stenosierenden Ileitis terminalis, die sich letztendlich erst nach Verlegung durch einen Fremdkörper manifestierte.

Hematopoietic clones with leukemogenic mutations arise in healthy people as they age, but progression to acute myeloid leukemia (AML) is rare. Recent evidence suggests that the microenvironment may play an important role in modulating human AML population dynamics. To investigate this concept further, we examined the combined and separate effects of an oncogene (c-MYC) and exposure to interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), and stem cell factor (SCF) on the experimental genesis of a human AML in xenografted immunodeficient mice. Initial experiments showed that normal human CD34+ blood cells transduced with a lentiviral MYC vector and then transplanted into immunodeficient mice produced a hierarchically organized, rapidly fatal, and serially transplantable blast population, phenotypically and transcriptionally similar to human AML cells, but only in mice producing IL-3, GM-CSF, and SCF transgenically or in regular mice in which the cells were exposed to IL-3 or GM-CSF delivered using a cotransduction strategy. In their absence, the MYC+ human cells produced a normal repertoire of lymphoid and myeloid progeny in transplanted mice for many months, but, on transfer to secondary mice producing the human cytokines, the MYC+ cells rapidly generated AML. Indistinguishable diseases were also obtained efficiently from both primitive (CD34+CD38−) and late granulocyte-macrophage progenitor (GMP) cells. These findings underscore the critical role that these cytokines can play in activating a malignant state in normally differentiating human hematopoietic cells in which MYC expression has been deregulated. They also introduce a robust experimental model of human leukemogenesis to further elucidate key mechanisms involved and test strategies to suppress them.

Although the history of intraoperative neuromonitoring (IONM) dates back to the 19th century, the method did not evolve further than the mere differentiation of nerves until recently. Only the development of continuous IONM (cIONM) has allowed for non-stop analysis of excitation amplitude and latency during surgical procedures, which is nowadays integrated into the software of almost all commercially available neuromonitoring devices. The objective of cIONM is real-time monitoring of nerve status in order to recognize and prevent impending nerve injury and predict postoperative nerve function. Despite some drawbacks such as false-positive/negative alarms, technical artefacts, and rare adverse effects, cIONM remains a good instrument which is still under development. Active (acIONM) and passive (pcIONM) methods of cIONM are described in literature. The main fields of cIONM implementation are currently thyroid surgery (in which the vagal nerve is continuously stimulated) and surgery to the cerebellopontine angle (in which the facial nerve is either continuously stimulated or the discharge signal of the nerve is analyzed via pcIONM). In the latter surgery, continuous monitoring of the cochlear nerve is also established.Obwohl die Geschichte des intraoperativen Neuromonitorings (IONM) bereits in das 19. Jahrhundert zurückdatiert werden kann, hat sich diese Methode bis vor Kurzem nicht von der reinen Differenzierung des Nervs weiterentwickelt. Erst das kontinuierliche IONM (cIONM) ermöglichte die durchgehende Analyse der Reizamplituden und -latenzen, welche mittlerweile ebenfalls in die Software gängiger Monitoringsysteme integriert wurde. Zielsetzung des cIONM ist ein Real-Time-Monitoring des Nervenstatus während des Eingriffs, um so drohende Nervenverletzung erkennen und verhindern zu können und die postoperative Funktion des Nervs vorhersehbar zu erhalten. Trotz einiger Nachteile wie falsch-positiver oder -negativer Alarme, technischer Artefakte und seltener Nebenwirkungen bleibt das cIONM ein gutes Hilfsmittel, das noch weiterentwickelt wird. In der Literatur sind sowohl aktive (acIONM) als auch passive (pcIONM) Reiz- und Ableitmethoden des cIONM beschrieben. Derzeit gängige Anwendungsgebiete des cIONM umfassen die Schilddrüsenchirurgie mit der kontinuierlichen Stimulation des N. vagus sowie die Chirurgie des Kleinhirnbrückenwinkels (KHBW) mit dem Monitoring des N. facialis; hierbei werden neben kontinuierlicher Stimulation auch die Entladungsmuster des Nervs analysiert. Des Weiteren ist in die Chirurgie des KHBW das kontinuierliche Monitoring des Hörnervs etabliert.

Objective To present and review the vascular consequences of arteritis in neurosarcoidosis. Patient and methods neurosarcoidosis is typically an inflammatory disorder of the meninges surrounding the brain and spinal cord. Although inflammation of small and medium sized vessels is seen pathologically and vasculitis is occasionally described, a large intracerebral arteritis has not previously been reported. A few case reports exist, however, which describe the vascular consequences of large vessel compromise in the disorder. We review the literature and present a new case with novel MRI features which imply carotid arteritis. Results The case presented with a disorder of the carotid artery on one side leading to a series of TIAs. Inflammation of the wall of the carotid artery was seen adjacent to a granulomatous leptomeningitis. The disorder responded to immunosuppressive therapy without recurrence. Conclusions The imaging features suggest a granulomatous infiltration of the carotid artery wall leading to arteritis followed by disorganisation of the internal elastic lamina and fibrosis. The data provide further insight into the pathogenesis of neurological impairments in neurosarcoidosis. The MRI features of carotid arteritis in neurosarcoidosis have not previously been demonstrated.

The advent of large scale genomic sequencing technologies significantly improved the molecular classification of acute megakaryoblastic leukaemia (AMKL). AMKL represents a subset (∼10%) of high fatality pediatric acute myeloid leukemia (AML). Recurrent and mutually exclusive chimeric gene fusions associated with pediatric AMKL are found in 60%-70% of cases and include RBM15-MKL1, CBFA2T3-GLIS2, NUP98-KDM5A and MLL rearrangements. In addition, another 4% of AMKL harbor NUP98 rearrangements (NUP98r), with yet undetermined fusion partners. We report a novel NUP98-BPTF fusion in an infant presenting with primary refractory AMKL. In this NUP98r, the C-terminal chromatin recognition modules of BPTF, a core subunit of the NURF (nucleosome remodeling factor) ATP-dependent chromatin-remodeling complex, are fused to the N-terminal moiety of NUP98, creating an in frame NUP98-BPTF fusion, with structural homology to NUP98-KDM5A. The leukemic blasts expressed two NUP98-BPTF splicing variants, containing one or two tandemly spaced PHD chromatin reader domains. Our study also identified an unreported wild type BPTF splicing variant encoding for 2 PHD domains, detected both in normal cord blood CD34+ cells and in leukemic blasts, as with the fly BPTF homolog, Nurf301. Disease course was marked by rapid progression and primary chemoresistance, with ultimately significant tumor burden reduction following treatment with a clofarabine containing regimen. In sum, we report 2 novel NUP98-BPTF fusion isoforms that contribute to refine the NUP98r subgroup of pediatric AMKL. Multicenter clinical trials are critically required to determine the frequency of this fusion in AMKL patients and explore innovative treatment strategies for a disease still plagued with poor outcomes.

Acute myeloid leukemia (AML) is an aggressive form of blood cancer defined by the uncontrolled proliferation and clonal expansion of immature myeloblast cells in the blood and bone marrow, leading to hematopoietic failure. Despite the use of aggressive and cytotoxic standard-of-care drugs, patients often relapse and succumb to the disease partially due to the inability of medically unfit patients to withstand the cytotoxic treatments, regrowth from minimal residual disease and the chemo-resistant nature of leukemic stem cells (LSCs) which can remain in a quiescent state and reside in a protective bone marrow niche. Hence, novel therapies targeting unique leukemic stem cell biology are highly needed to eliminate and avoid reoccurrence. High-throughput screens of human AML LSCs are not performed due to technical issues such as low LSC frequency within primary samples, an inability to purify LSCs, and the difficulty maintaining and expanding primary patient samples and LSCs in vitro. We were able to optimize conditions for a 4-week in vitro large-scale expansion (>600 million bulk) of the primary human AML sample 8227 (OCI-AML-8227), functionally validated to be enriched for LSCs in long-term xenotransplant assays (Eppert et al., 2011). These optimized conditions enabled the isolation and maintenance of the LSC-containing fraction for a chemical screen. We isolated the CD34+ LSC-containing fraction (>90% purity) and performed a high-throughput screen of 11,166 chemical molecules using a CellTiter Glo assay followed by a counter screen against normal CD34+ cord blood (CB) hematopoietic stem and progenitor cells. From this HT screen, a total of 61 hits had >70% inhibition on CD34+ 8227 cells and We then performed dose response assays for each candidate compounds and confirmed 35 potent anti-LSC compounds with IC 50 < 1 μM. This refined the types of compounds to including anti-apoptotic inhibitors, GSK inhibitors, protease inhibitors, metabolism inhibitors, HDAC inhibitors, BET inhibitors, nucleic acid synthesis inhibitors, cell cycle inhibitors and Wnt/β-catenin inhibitors. This is interesting as some of the classes of these compounds (inhibitors of GSK, BET, nucleic acid synthesis, Wnt/β-catenin and metabolism) have been shown to target bulk and leukemic stem cells in AML in vitro and in vivo. We now aim to examine LSC eradication in a panel of genetically defined primary AMLs confirmed through in vitro and in vivo assays. Our goal is to be able to understand and establish the molecular mechanisms and biomarkers on primary functional LSCs. Disclosures No relevant conflicts of interest to declare.

Background:Type I Interferons (IFNs), and especially INF-alpha, play a crucial role in the pathogenesis of SLE. Interferon-stimulated genes (ISGs) are generally up-regulated in SLE patients. Their pattern of up-regulation is often termed as an IFN signature. Even though composite IFN-scores are already used to express the up-regulation of the IFN-system, e.g. in studies testing therapeutic anti-IFN-antibodies, we are still lacking an in-depth understanding of the IFN signature.Objectives:To summarize the available data on the expression of ISGs in peripheral blood of patients with SLE compared to healthy controls; to assess which ISGs are most up-regulated in SLE patients; and to analyse if up-regulations of 6 ISGs typically used in IFN-scores [1,2] are associated with SLE disease activity and ethnicity.Methods:The electronic databases PubMed and EMBASE were searched using the terms “interferon signature”, “SLE”, “interferon-stimulated genes”, “microarray” and “gene expression” with inception date until Jan. 28, 2020. Original case-control studies containing quantitative data of ISG expression were included. Exclusion criteria were studies with animal models, clinical trials of drug treatment and studies withexvivo stimulation of cells prior to gene-expression analysis. Fold changes (FCs) (ISG expression in SLE/ISG expression in healthy controls) for the ISGs analysed in each study were extracted. FCs were plotted gene-wise on a heatmap for studies analysing ≥ 7 genes and for genes that were analysed in ≥ 5 studies. Cluster analysis using principle component analysis (PCA) and association analyses using generalized linear modelling (GLM) were performed for 6 ISGs (IFI27, IFI44, IFI44L, RSAD2, PRKR and IFIT1) as well as disease activity and ethnicity (SPSS).Results:16 case-control studies comprising a total of 851 SLE patients were included in the analyses, see Tab. 1. 24 ISGs had an average FC of ≥ 4. IFI27, RSAD2, IFI44L, IFI44, HERC5 and IFIT5 had an average FC > 6. The heatmap showed great variation in the expression of ISGs within the individual studies but also gene-wise between the studies, see Fig. 1. The inter-study variation was statistically explored for the selected 6 ISGs. PCA showed that PRKR, RSAD2, IFIT1, IFI44 and IFI44L clustered with African ancestry, see Fig. 2. Subsequent GLM confirmed that RSAD2 and PRKR were positively associated with African ancestry. However, none of the 6 ISGs were positively correlated with disease activity.Figure 1.Heatmap of ISG up-regulation.Figure 2.Principal component analysis.Conclusion:The degree of up-regulation of ISGs in SLE patients shows considerable variation within and between the individual studies. However, a pattern of up-regulation clearly emerges. We find a clustering of 5 prominent genes of the IFN signature (PRKR, RSAD2, IFIT1, IFI44 and IFI44L) and a positive correlation of RSAD2 and PRKR with African ancestry, pointing to the need to take ethnicity into account when using the IFN signature. Our results do not support the use of the IFN signature as traditionally defined as a surrogate marker for disease activity.References:[1]Kirou, K.A. et. al. Arthritis Rheum. 52, 1491–1503 (2005)[2]Morand E.F. et. Al. N Engl J Med. (2019)Table 1.Demographics on the studies in the heatmap. Characteristics refer to SLE patients. HC = healthy controls, SLEDAI = SLE Disease Activity Index, BILAG = British Isles Lupus Assessment Group.PaperSLE (N)HC (N)Female (N)Age (Mean)SLEDAI (Mean)African (N)Caucasian (N)Asian (N)Panousis, N.I. 201914258120405.2138Alcorta, D.A., 200740284035BILAG: 6.72018Assassi, S., 20101721163926Zhu, H., 2016302542912Li, Q.Z.,20092711253912Chaussabel, D., 20082212201412.163Bouquet, J., 201711251151353Wither, J.E. 201817022149348.5408050Mackay, M., 2016112011341314Yao, Y., 20084124394041Lyons, P.A., 201013251347121Lambers, W.M., 201939223243351Baechler, E.C., 2003484245Han, G.M., 200310189296.110Ishii, T., 2005313030343.831Tang, J., 200814460132336.4144Ye, S., 20035039452950Disclosure of Interests:None declared

Infant acute lymphoblastic leukemias (ALL) are rare hematological malignancies occurring in children younger than 1 year of age, most frequently associated with KMT2A rearrangements (KMT2A-r). The smaller subset without KMT2A-r, which represents 20% of infant ALL cases, is poorly characterized. Here we report two cases of chemotherapy-sensitive non-KMT2A-r infant ALL. Transcriptome analyses revealed identical ACIN1-NUTM1 gene fusions in both cases, derived from cryptic chromosomal rearrangements undetected by standard cytogenetic approaches. Two isoforms of the gene fusion, joining exons 3 or 4 of ACIN1 to exon 3 of NUTM1, were identified. Both fusion transcripts contained the functional DNA-binding SAP (SAF-A/B, Acinus, and PIAS) domain of ACIN1 and most of NUTM1. The detection of the ACIN1-NUTM1 fusion by RT-PCR allowed the molecular monitoring of minimal residual disease in a clinical setting. Based on publicly available genomic datasets and literature review, we predict that NUTM1 gene fusions are recurrent events in infant ALL. As such, we propose two clinically relevant assays to screen for NUTM1 rearrangements in bone marrow cells, independent of the fusion partner: NUMT1 immunohistochemistry and NUTM1 RNA expression. In sum, our study identifies ACIN1-NUTM1 as a recurrent and possibly cryptic fusion in non-KMT2A-r infant ALL, provides clinical tools to screen for NUTM1-rearranged leukemia and contributes to the refinement of this new subgroup.

The development of biosensor arrays able to detect specific protein interactions is highly needed to precisely dissect fundamental biological systems and to broaden the scope of biomarker detection, especially to refine diagnostics of subtyped diseases such as cancer. Graphene-based field-effect transistor sensors (GFETs) are a promising emerging technology for such biomolecular sensing applications: their atomically-thin surface provides label-free sensitivity to biomolecules via direct electrostatic interactions, and their small footprint enables compact, rapid and parallelized assays. The selectivity of GFET sensors, however, requires functionalization of the graphene surface with a layer of bio-recognition species (“probe molecules”). In this presentation, we will describe our recent progress in immobilizing proteins as probe molecules on graphene field-effect transistors, specifically (1) monoclonal antibodies against a biomarker specific to MLL-translocated acute myeloid leukemia and (2) small Ras GTPase proteins regulated by various effector proteins. First, we will describe our GFET sensor design, based on on-chip arrays of GFETs made from CVD-grown graphene, mounted with a multi-channel delivery flow-cell enabling parallel assays on sub-ensembles of sensors. We will then present our investigation of protein immobilization on graphene. We found that antibodies and small proteins can spontaneously adhere to the graphene surface, but this adhesion is partially reversible under solution flow. To create stable anchor groups at the graphene surface which can then capture the protein probes, we propose to use covalent chemistry on the graphene surface. In particular, we developed a protocol based on electrochemically-driven aryldiazonium chemistry to increase the rate of formation and the density of anchor groups on the graphene surface. Using time-resolved electrical measurements, we observed a specific electrical signal associated with the irreversible immobilization of protein probes on the graphene surface. Finally, we will discuss the use of such protein-functionalized GFETs for the detection of specific probe-target interactions, for applications in fundamental biophysics and cancer diagnostics.

Acute myeloid leukemias (AMLs) result from a series of genetic events occurring in a stem or progenitor hematopoietic cell that gives rise to their clonal expansion and an impaired capacity to differentiate. To circumvent the genetic heterogeneity of AML patient cohorts, we have developed a model system, driven by the MLL-AF9 (MA9) oncogene, to generate multiple human leukemias using progenitor cells from a single healthy donor. Through stepwise RNA-sequencing data generated using this model and AML patients, we have identified consistent changes associated with MA9-driven leukemogenesis and demonstrate that no recurrent secondary mutations are required. We identify 39 biomarkers whose high expression level is specific to this genetic subtype of AML and validate that many of these have diagnostic utility. We further examined one biomarker, the receptor tyrosine kinase (RTK) RET, and show through shRNA knockdowns that its expression is essential for in vivo and in vitro growth of MA9-AML. These results highlight the value of novel human models of AML derived from single donors using specific oncogenic fusions to understand their biology and to uncover potential therapeutic targets.

Umbilical cord blood (CB)-derived hematopoietic stem cells (HSCs) are essential in many life saving regenerative therapies, but their low number in CB units has significantly restricted their clinical use despite the advantages they provide during transplantation1. Select small molecules that enhance hematopoietic stem and progenitor cell (HSPC) expansion in culture have been identified2,3, however, in many cases their mechanisms of action or the nature of the pathways they impinge on are poorly understood. A greater understanding of the molecular pathways that underpin the unique human HSC self-renewal program will facilitate the development of targeted strategies that expand these critical cell types for regenerative therapies. Whereas transcription factor networks have been shown to influence the self-renewal and lineage decisions of human HSCs4,5, the post-transcriptional mechanisms guiding HSC fate have not been closely investigated. Here we show that overexpression of the RNA-binding protein (RBP) Musashi-2 (MSI2) induces multiple pro-self-renewal phenotypes, including a 17-fold increase in short-term repopulating cells and a net 23-fold ex vivo expansion of long-term repopulating HSCs. By performing a global analysis of MSI2-RNA interactions, we determined that MSI2 directly attenuates aryl hydrocarbon receptor (AHR) signaling through post-transcriptional downregulation of canonical AHR pathway components in CB HSPCs. Our study provides new mechanistic insight into RBP-controlled RNA networks that underlie the self-renewal process and give evidence that manipulating such networks ex vivo can provide a novel means to enhance the regenerative potential of human HSCs.

Field-effect transistor devices based on functionalized graphene (G-FETs) are a promising technology for biomolecular sensing applications due to the several advantages they present, including the label-free detection of biomolecules with direct electrical read-out, real-time detection and multiplexing capability. The development of highly selective and sensitive sensors for protein biomarkers is especially desirable to open novel technological avenues for the early detection and monitoring of biomarkers associated with cancer diseases. In this presentation, I’ll describe our recent progress on the design and development of a label-free immunosensor based on antibody-modified graphene field-effect transistors for protein biomarker detection. Specifically, monoclonal antibodies were selected to target a protein biomarker specific to MLL translocated acute myeloid leukemia and we tested approaches for their immobilization on graphene. We found that the antibodies can spontaneously adhere to the graphene surface but this adhesion is partially reversible under solution flow. In order to stabilize the immobilization of antibodies, we developed a protocol based on electrochemically-driven chemistry to form stable covalent anchor groups at the graphene surface which can then capture antibodies. We optimized the rate of formation of anchor groups at the surface in order to maximize the density of anchor groups while maintaining high electrical currents in the graphene. We were then able to record a specific electrical signature showing irreversible immobilization of antibodies on the graphene surface, thus allowing further optimization of target detection. Employing a combination of microfluidics and real-time electrical measurements, we then investigated the response of the sensors to non-specific interactions with graphene as well as their response to specific antibody-antigen interactions in phosphate buffer solution. Finally, the sensors response to different concentrations of target protein was characterized to assess their performance parameters.

Acute myeloid leukemia (AML) is a disease that results from the uncontrolled growth of primitive myeloid progenitor cells that are unable to undergo terminal differentiation. To better understand the genetic and epigenetic changes involved in leukemogenesis, we use a human model leukemia system where cord blood donor cells from a single donor are transduced with the human KMT2A-MLLT3 gene fusion to produce leukemias. We have generated multiple model leukemias and compared these to genetically matched pediatric AML patient samples that has allowed us to characterize the step-wise epigenetic and splicing changes that accompany leukemogenesis, allowing us to build an integrated view of this process. Our analysis of these data have revealed a number of candidate biomarker genes that are expressed in KMT2A translocated AMLs but not normal blood cells, some of which are important for leukemic growth. These candidate genes, which have validated diagnostic value, have also been used as targets for monoclonal antibody generation in order to assess their value as potential immunotherapeutics. Having established the value of this approach, we are now expanding our models to include additional oncogenic drivers in order to elucidate the differences in patient outcomes associated with the presence of specific fusions. Moreover, we have recently leveraged these model leukemias and matched patient samples to perform a high-throughput small molecule screen to identify novel therapeutic vulnerabilities and deconvolute the role of secondary mutations in pediatric patients. The establishment of this chemogenomic pipeline to generate, characterize, and then screen model leukemias will provide not only insight into the molecular mechanisms involved in this disease, but also how they can be rationally targeted to improve patient outcomes.

Next generation DNA sequencing has provided significant insights into the genetic determinants of acute myeloid leukemia (AML). Large scale sequencing studies of AML patient cohorts have revealed a remarkable level of genetic heterogeneity between patients who nevertheless have the same disease phenotype. As a solution to the problem of extensive genetic diversity between patients, we have modified a previously published model system in order to generate multiple human leukemias from CD34+ cord blood cells from a single healthy donor. A human MLL-AF9 (MA9) fusion gene is retrovirally introduced into the donor cells which are then cultured for 30 days before being transplanted into immunocompromised (NSG) mice that subsequently develop either AML or acute B-cell lymphoblastic leukemia (B-ALL) after ~24 weeks. We have now generated 22 leukemias from 4 single donors and have performed RNA-seq on the samples during their step-wise leukemic transformation (i.e. CD34+ cells, CD34+ cells with MA9, and the resulting leukemias). We have compared these data to RNA-seq data we have generated for several pediatric AML patients with MA9 translocations, as well as normal blood cells and other tissues. This analysis has revealed 39 candidate genes with an expression pattern highly specific for MA9 AMLs. Interestingly, we can find no evidence for secondary mutations acquired by the human leukemias in our model system, suggesting the human MA9 translocation is sufficient to generate leukemias in this context. To understand the epigenetic impact of the MA9 fusion gene, we have examined the DNA methylation changes at each stage in our model system using a capture survey approach (e.g. Human Methyl-Seq; Agilent) and used this same approach for several primary patient samples with MLL translocations. These data have been correlated with gene expression changes within the model system and have revealed a number of specific changes with relevance for the process of transformation. Lastly, functional assessment of specific candidate genes through shRNA knock-down experiments has shown that at least some of these candidate genes, which are known oncogenes in other tumor types, are essential for MA9 AML. In summary, the combination of RNA-seq data from patient and single donor model AMLs has highlighted consistent genetic changes associated with this AML sub-group, and has also revealed novel potential therapeutic targets. Citation Format: Frederic Barabe, Magalie Celton, Audrey Forest, Anne Bergeron, Radia Johnson, Laurine Gil, Angélique Bellemare-Pelletier, Sonia Cellot, Josee Hebert, Etienne Gagnon, Brian T. Wilhelm. Integrated genetic and epigenetic analysis of model and patient acute myeloid leukemias. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A2-14.

Using next-generation sequencing of primary acute myeloid leukemia (AML) specimens, we identified to our knowledge the first unifying genetic network common to the two subgroups of KMT2A (MLL)-rearranged leukemia, namely having MLL fusions or partial tandem duplications. Within this network, we experimentally confirmed upregulation of the gene with the most subtype-specific increase in expression, LOC100289656, and identified cryptic MLL fusions, including a new MLL-ENAH fusion. We also identified a subset of MLL fusion specimens carrying mutations in SPI1 accompanied by inactivation of its transcriptional network, as well as frequent RAS pathway mutations, which sensitized the leukemias to synthetic lethal interactions between MEK and receptor tyrosine kinase inhibitors. This transcriptomics-based characterization and chemical interrogation of human MLL-rearranged AML was a valuable approach for identifying complementary features that define this disease.

A methodology for evaluating the science benefit of adding space weather sensor data from a modest number of small satellites to the Utah State University Global Assimilation of Ionospheric Measurements—Full Physics (GAIM-FP) model is presented. Three orbital scenarios are presented, two focusing on improved coverage of narrowly specified regions of interest, and one on global coverage of the ionosphere as a whole. An Observing System Simulation Experiment is used to obtain qualitative and quantitative results of the impact of the various orbital scenarios on the ionospheric specifications. A simulated “truth” run of the ionosphere is obtained from a first principle model of the ionosphere/plasmasphere model and used to generate global simulated Global Positioning Satellite total electron content (GPS-TEC) data as well as in situ plasma density observations. Initially, only GPS data were assimilated by GAIM-FP, and the results of this limited run were compared to the truth run. Next, the simulated in situ plasma densities corresponding to our three orbital scenarios were assimilated together with the GPS data, and the results were compared to both the truth run and the limited GPS-TEC only GAIM-FP run. These model simulations have shown that adding a constellation of small satellites/sensors in addition to global TEC inputs does indeed converge the GAIM-FP model closer to truth in the situations described.

Acute myeloid leukemias (AMLs) with translocations of the mixed lineage leukemia (MLL/KMT2A) gene are common in young patients and are generally associated with poor clinical outcomes. The molecular biology of MLL fusion genes remains incompletely characterized and is complicated by the fact that more than 100 different partner genes have been identified in fusions with MLL. The continuously growing list of MLL fusions also represents a clinical challenge with respect to identification of novel fusions and tracking of the fusions to monitor progression of the disease after treatment. Recently, we have developed a novel single-donor model leukemia system that permits the development of human AML from normal cord blood cells. Gene expression analysis of this model and of MLL-AML patient samples has identified a number of candidate biomarker genes with highly biased expression on leukemic cells. Here, we present data demonstrating the potential clinical utility of several of these candidate genes for identifying known and novel MLL fusions.

For many decades thermally sprayed corrosion protection systems on the basis of ZnAl or Al carry out their service for structures in coastal areas, the offshore sector, as CUI (Corrosion Protection under insulation) or anywhere where the properties of thermally sprayed corrosion protection systems bring important advantages in terms of durability. A thermally sprayed corrosion protection system is about to protect the structure 25 to 30 years against corrosion. During this time it may be damaged due to factors like construction work, improper handling or simple aging. There are many standards and regulations, which describe the initial design of thermal spray systems, however they remain silent regarding repair. In particular, a mending of partial regions is hardly described. Specific repair instructions are rare and if present, they differ from one another. Overall there is a lack of knowledge of the proper procedures for partially repairing thermal spraying systems. This project was concerned with tangible corrosion-technical issues of the coating repairs: How does the critical overlap area perform? Have organic coatings benefits? To what extent does a renewed damage affect the lifetime? The aim of the study was to develop practice-relevant instructions for the repair of thermally sprayed duplex systems.

The development of next-generation sequencing technologies has had a profound impact on the field of cancer genomics. With the enormous quantities of data being generated from tumor samples, researchers have had to rapidly adapt tools or develop new ones to analyse the raw data to maximize its value. While much of this effort has been focused on improving specific algorithms to get faster and more precise results, the accessibility of the final data for the research community remains a significant problem. Large amounts of data exist but are not easily available to researchers who lack the resources and experience to download and reanalyze them. In this article, we focus on RNA-seq analysis in the context of cancer genomics and discuss the bioinformatic tools available to explore these data. We also highlight the importance of developing new and more intuitive tools to provide easier access to public data and discuss the related issues of data sharing and patient privacy.

Background: Current evidence suggests that genetic and epigenetic abnormalities drive the development of human Acute Myeloid Leukemias (AMLs). However, whether these are sufficient to establish a permanent, self-sustaining AML population, and the potential role of shared perturbed downstream pathways is unknown. We hypothesized that a modest upregulated expression of MYC might play such a role given its commonly increased expression in many AML patients' cells. To test this hypothesis, we assessed the dynamics and types of cells produced in sublethally irradiated NOD-Rag1-/--IL2Rγc-/-(NRG) mice transgenically producing human IL3, GM-CSF and SCF (NRG-3GS mice) following their transplantation with freshly isolated subsets of normal CD34+ cord blood (CB) cells that were first lentivirally transduced with a human MYC cDNA. Results: FACS and Western blot analyses indicated this produced a 2 to 5-fold increase in MYC mRNA and protein levels in MYC-transduced CD34+ CB cells, and 21/22 NRG-3GS mice injected with ≥6,500 of these cells developed a fatal human AML population within 7 weeks. Histological analysis of their bone marrow and spleen cells showed both contained a prominent human CD123+CD33+CD15±CD34-CD14-CD19-CD3- blast population. Additional limiting dilution transplants showed that both the CD34+CD38- cells (enriched for hematopoietic stem cells) and the more differentiated CD34+ GMPs were similarly highly susceptible (at frequencies of 1/14 and 1/46, respectively) and, in both cases, generated progeny that could initiate serially transplantable leukemias with the same phenotypic and transcriptomic features. Comparison to normal CB cells indicated these most closely resembled GMPs, and comparison to pediatric AML patient samples indicated a similarity to myelomonocytic leukemias with enhanced MYC expression. Interestingly, 14 sublethally irradiated NRG mice (the parental strain not producing human 3GS) transplanted with matched aliquots of CD34+ MYC-transduced cells regenerated a normal spectrum of CD19+ lymphoid cells, CD14+ and CD15+ GM cells and readily detectable CD34+ cells for up to 32 weeks of follow-up with no evidence of leukemogenesis. However, transfer of these regenerated human cells into secondary NRG-3GS mice, even after this extended period, enabled their rapid production of a lethal human AML in all 5 mice tested. In contrast, matched aliquots transplanted into 5 NRG recipients produced declining grafts of normal cells. This finding was then exploited to determine which growth factors were responsible for activating the AML program by transplanting NRG mice with CD34+ CB cells transduced with MYC and just a single growth factor, or all 3 as a positive control. In this set of experiments, a lethal human AML was obtained when MYC was paired with human IL3 or GM-CSF (or all 3 together), but not with SCF (or no growth factors). Conclusion: We report here a new in vivo model of MYC-induced human myeloid leukemogenesis that produces a serially transplantable AML closely resembling human pediatric myelomonocytic leukemias with elevated MYC expression. The rapidity, consistency, and high frequency of this transformation process obtained by transducing late granulopoietic as well as early types of normal human CD34+ progenitor cells makes this system highly attractive for future mechanistic and therapeutic testing experiments. The discovery that MYC deregulation alone generates a stable "latent program" that can be rapidly activated by exposure to exogenous growth factors typical of inflammatory states also raises intriguing questions about the potential role of such events in the genesis of AML populations that arise in patients. Disclosures Beer: Karus therapeutics Ltd.: Employment.

Acute myeloid leukemias (AML) comprise a genetically diverse group of human hematologic malignancies with a generally poor prognosis and modes of pathogenesis that have been difficult to investigate. Historic evidence and more recent findings have identified hematopoietic stem cell programs as major targets of mutations that predispose to and/or initiate a multistep process of transformation. However, details of the changes involved and how they interact in emerging human leukemic cells have been particularly challenging to characterize. This is due to an emerging appreciation of the heterogeneity in events that impact the differentiation processes of normal human hematopoietic cells, as well as difficulties in recreating the full leukemogenic alterations in the actual human cells affected, to avoid discrepancies inherent in studying model organisms. Here we describe several models of de novo human leukemogenesis that illustrate their power and the novel results they can generate. These include our recent discovery of a novel “latent” leukemic state that can be obtained in MYC-transduced human cord blood cells regenerating a normal spectrum of lymphoid and myeloid progeny in transplanted immunodeficient mice, but that can then be rapidly activated to an aggressive form of AML by in vivo exposure to a single human growth factor. These findings portend the future utility of de novo human leukemogenesis models as new platforms for elucidating shared molecular mechanisms responsible for different stages of human leukemogenesis that may be initiated by different mutations or exposure to different microenvironmental conditions. The flexibility and consistency of these models also make them attractive for identifying and testing new treatment strategies targeting mechanisms required for disease manifestation.

Genome-wide transcriptome profiling has enabled non-supervised classification of tumours, revealing different sub-groups characterized by specific gene expression features. However, the biological significance of these subtypes remains for the most part unclear. We describe herein an interactive platform, Minimum Spanning Trees Inferred Clustering (MiSTIC), that integrates the direct visualization and comparison of the gene correlation structure between datasets, the analysis of the molecular causes underlying co-variations in gene expression in cancer samples, and the clinical annotation of tumour sets defined by the combined expression of selected biomarkers. We have used MiSTIC to highlight the roles of specific transcription factors in breast cancer subtype specification, to compare the aspects of tumour heterogeneity targeted by different prognostic signatures, and to highlight biomarker interactions in AML. A version of MiSTIC preloaded with datasets described herein can be accessed through a public web server (http://mistic.iric.ca); in addition, the MiSTIC software package can be obtained (github.com/iric-soft/MiSTIC) for local use with personalized datasets.

Purpose Dynamic volume perfusion CT (dVPCT) provides valuable information on tissue perfusion in patients with hepatocellular carcinoma (HCC) and pancreatic cancer. However, currently dVPCT is often performed in addition to conventional CT acquisitions due to the limited morphologic image quality of dose optimized dVPCT protocols. The aim of this study was to prospectively compare objective and subjective image quality, lesion detectability and radiation dose between mean temporal arterial (mTA) and mean temporal portal venous (mTPV) images calculated from low dose dynamic volume perfusion CT (dVPCT) datasets with linearly blended 120-kVp arterial and portal venous datasets in patients with HCC and pancreatic cancer. Materials and methods All patients gave written informed consent for this institutional review board–approved HIPAA compliant study. 27 consecutive patients (18 men, 9 women, mean age, 69.1 years ± 9.4) with histologically proven HCC or suspected pancreatic cancer were prospectively enrolled. The study CT protocol included a dVPCT protocol performed with 70 or 80 kVp tube voltage (18 spiral acquisitions, 71.2 s total acquisition times) and standard dual-energy (90/150 kVpSn) arterial and portal venous acquisition performed 25 min after the dVPCT. The mTA and mTPV images were manually reconstructed from the 3 to 5 best visually selected single arterial and 3 to 5 best single portal venous phases dVPCT dataset. The linearly blended 120-kVp images were calculated from dual-energy CT (DECT) raw data. Image noise, SNR, and CNR of the liver, abdominal aorta (AA) and main portal vein (PV) were compared between the mTA/mTPV and the linearly blended 120-kVp dual-energy arterial and portal venous datasets, respectively. Subjective image quality was evaluated by two radiologists regarding subjective image noise, sharpness and overall diagnostic image quality using a 5-point Likert Scale. In addition, liver lesion detectability was performed for each liver segment by the two radiologists using the linearly blended120-kVp arterial and portal venous datasets as the reference standard. Results Image noise, SNR and CNR values of the mTA and mTPV were significantly higher when compared to the corresponding linearly blended arterial and portal venous 120-kVp datasets (all p Objective image quality of mTA and mTPV were rated significantly better when compared to the linearly blended 120-kVp arterial and portal venous datasets. Both readers were able to detect all liver lesions found on the linearly blended 120-kVp arterial and portal venous datasets using the mTA and mTPV datasets. The effective radiation dose of the dVPCT was 27.6 mSv for the 80 kVp protocol and 14.5 mSv for the 70 kVp protocol. The mean effective radiation dose for the linearly blended 120-kVp arterial and portal venous CT protocol together of the upper abdomen was 5.60 mSv ± 1.48 mSv. Conclusion Our preliminary data suggest that subjective and objective image quality of mTA and mTPV datasets calculated from low-kVp dVPCT datasets is non-inferior when compared to linearly blended 120-kVp arterial and portal venous acquisitions in patients with HCC and pancreatic cancer. Thus, dVPCT could be used as a stand-alone imaging technique without additionally performed conventional arterial and portal venous CT acquisitions.

Background Cancer genomics projects are producing ever-increasing amounts of rich and diverse data from patient samples. The ability to easily visualize this data in an integrated an intuitive way is currently limited by the current software available. As a result, users typically must use several different tools to view the different data types for their cohort, making it difficult to have a simple unified view of their data. Results Here we present Cascade, a novel web based tool for the intuitive 3D visualization of RNA-seq data from cancer genomics experiments. The Cascade viewer allows multiple data types (e.g. mutation, gene expression, alternative splicing frequency) to be simultaneously displayed, allowing a simplified view of the data in a way that is tuneable based on user specified parameters. The main webpage of Cascade provides a primary view of user data which is overlaid onto known biological pathways that are either predefined or added by users. A space-saving menu for data selection and parameter adjustment allows users to access an underlying MySQL database and customize the features presented in the main view. Conclusions There is currently a pressing need for new software tools to allow researchers to easily explore large cancer genomics datasets and generate hypotheses. Cascade represents a simple yet intuitive interface for data visualization that is both scalable and customizable. Electronic supplementary material The online version of this article (doi:10.1186/s12864-016-2389-8) contains supplementary material, which is available to authorized users.

The proliferation and survival of hematopoietic stem cells (HSCs) has to be strictly coordinated to ensure the timely production of all blood cells. Here we report that the splice factor and RNA binding protein hnRNP L (heterogeneous nuclear ribonucleoprotein L) is required for hematopoiesis, since its genetic ablation in mice reduces almost all blood cell lineages and causes premature death of the animals. In agreement with this, we observed that hnRNP L deficient HSCs lack both the ability to self-renew and foster hematopoietic differentiation in transplanted hosts. They also display mitochondrial dysfunction, elevated levels of γH2AX, are Annexin V positive and incorporate propidium iodide indicating that they undergo cell death. Lin-c-Kit+ fetal liver cells from hnRNP L deficient mice show high p53 protein levels and up-regulation of p53 target genes. In addition, cells lacking hnRNP L up-regulated the expression of the death receptors TrailR2 and CD95/Fas and show Caspase-3, Caspase-8 and Parp cleavage. Treatment with the pan-caspase inhibitor Z-VAD-fmk, but not the deletion of p53, restored cell survival in hnRNP L deficient cells. Our data suggest that hnRNP L is critical for the survival and functional integrity of HSCs by restricting the activation of caspase-dependent death receptor pathways.

A-kinase anchor protein 12 (AKAP12) is a regulator of protein kinase A and protein kinase C signaling, acting downstream of RAS. Epigenetic silencing of AKAP12 has been demonstrated in different cancer entities and this has been linked to the process of tumorigenesis. Here, we used quantitative high-resolution DNA methylation measurement by MassARRAY to investigate epigenetic regulation of all three AKAP12 promoters (i.e., α, β, and γ) within a large cohort of juvenile myelomonocytic leukemia (JMML) patient samples. The AKAP12α promoter shows DNA hypermethylation in JMML samples, which is associated with decreased AKAP12α expression. Promoter methylation of AKAP12α correlates with older age at diagnosis, elevated levels of fetal hemoglobin and poor prognosis. In silico screening for transcription factor binding motifs around the sites of most pronounced methylation changes in the AKAP12α promoter revealed highly significant scores for GATA-2/-1 sequence motifs. Both transcription factors are known to be involved in the haematopoietic differentiation process. Methylation of a reporter construct containing this region resulted in strong suppression of AKAP12 promoter activity, suggesting that DNA methylation might be involved in the aberrant silencing of the AKAP12 promoter in JMML. Exposure to DNMT- and HDAC-inhibitors reactivates AKAP12α expression in vitro, which could potentially be a mechanism underlying clinical treatment responses upon demethylating therapy. Together, these data provide evidence for epigenetic silencing of AKAP12α in JMML and further emphasize the importance of dysregulated RAS signaling in JMML pathogenesis.

The receptor tyrosine kinase, c-kit (Steel Factor (SF) receptor) controls survival, proliferation, chemotaxis, and secretion of proinflammatory cytokines in mast cells (MCs). Activation of c-kit results, amongst others, in induction of the PI3K and MEK/Erk pathways. Comparison of two MEK inhibitors, the specific, widely used U0126 and the more selective PD0325901, in different MC models revealed severe differences on SF-induced expression of proinflammatory cytokines IL-6 and TNF-α as well as the transcription factor Krüppel-like factor 2 (KLF2). Expression of the latter in MCs was not investigated so far. Whereas SF-induced expression of IL-6, TNF-α, and KLF2 was unaltered by U0126, it was significantly augmented by PD0325901. The effect of PD0325901 was corroborated by a second selective MEK inhibitor, PD184352 (Cl-1040), indicating the presence of MEK/Erk-based negative feedback mechanism(s) downstream of c-kit activation. Further analysis of KLF2 production revealed a positive function of PI3K. Depending on additional stimuli (e.g. antigen, IGF-1, LPS, thapsigargin), SF-triggered KLF2 expression was differentially modified, most likely controlled by the respective ratio between MEK/Erk and PI3K pathway activation. Moreover, the statin, simvastatin, was demonstrated to upregulate expression of KLF2 in MCs. In conclusion, data obtained by solely using the MEK inhibitor U0126 have to be carefully corroborated by using more selective inhibitors, such as PD0325901 or PD184352. SF-induced expression of the transcription factor KLF2 and its regulation by the MEK/Erk and PI3K pathways could impact on physiological as well as pathophysiological MC functions.

High levels of glutathione peroxidase 3 (GPx3) expression correlate with adverse prognosis in acute myeloid leukemia, and enhance activity of long-term repopulating hematopoietic stem cells in mice., The determinants of normal and leukemic stem cell self-renewal remain poorly characterized. We report that expression of the reactive oxygen species (ROS) scavenger glutathione peroxidase 3 (GPx3) positively correlates with the frequency of leukemia stem cells (LSCs) in Hoxa9+Meis1-induced leukemias. Compared with a leukemia with a low frequency of LSCs, a leukemia with a high frequency of LSCs showed hypomethylation of the Gpx3 promoter region, and expressed high levels of Gpx3 and low levels of ROS. LSCs and normal hematopoietic stem cells (HSCs) engineered to express Gpx3 short hairpin RNA (shRNA) were much less competitive in vivo than control cells. However, progenitor cell proliferation and differentiation was not affected by Gpx3 shRNA. Consistent with this, HSCs overexpressing Gpx3 were significantly more competitive than control cells in long-term repopulation experiments, and overexpression of the self-renewal genes Prdm16 or Hoxb4 boosted Gpx3 expression. In human primary acute myeloid leukemia samples, GPX3 expression level directly correlated with adverse prognostic outcome, revealing a potential novel target for the eradication of LSCs.