Oncologist use and perception of large panel next-generation tumor sequencing.

BACKGROUND: Genomic profiling is increasingly incorporated into oncology research and the clinical care of cancer patients. We sought to determine physician perception and use of enterprise-scale clinical sequencing at our center, including whether testing changed management and the reasoning behind this decision-making. PATIENTS AND METHODS: All physicians who consented patients to MSK-IMPACT, a next-generation hybridization capture assay, in tumor types where molecular profiling is not routinely performed were asked to complete a questionnaire for each patient. Physician determination of genomic 'actionability' was compared to an expertly curated knowledgebase of somatic variants. Reported management decisions were compared to chart review. RESULTS: Responses were received from 146 physicians pertaining to 1932 patients diagnosed with 1 of 49 cancer types. Physicians indicated that sequencing altered management in 21% (331/1593) of patients in need of a treatment change. Among those in whom treatment was not altered, physicians indicated the presence of an actionable alteration in 55% (805/1474), however, only 45% (362/805) of these cases had a genomic variant annotated as actionable by expert curators. Further evaluation of these patients revealed that 66% (291/443) had a variant in a gene associated with biologic but not clinical evidence of actionability or a variant of unknown significance in a gene with at least one known actionable alteration. Of the cases annotated as actionable by experts, physicians identified an actionable alteration in 81% (362/445). In total, 13% (245/1932) of patients were enrolled to a genomically matched trial. CONCLUSION: Although physician and expert assessment differed, clinicians demonstrate substantial awareness of the genes associated with potential actionability and report using this knowledge to inform management in one in five patients. CLINICAL TRIAL NUMBER: NCT01775072.

[Choosing wisely recommendations in nephrology]. / Klug-entscheiden-Empfehlungen in der Nephrologie.

The German Society of Internal Medicine ("Deutsche Gesellschaft für Innere Medizin", DGIM) founded the Choosing wisely initiative in order to address diagnostic and therapeutic procedures that are frequently inappropriately applied, whether this be in terms of over-, under-, or misuse of health services. The German Society of Nephrology ("Deutsche Gesellschaft für Nephrologie," DGfN) strongly supports the initiative and has contributed five positive and five negative recommendations. These ten recommendations are discussed in the current publication. The positive recommendations reflect the importance of early recognition of renal disease via simple blood and urine tests, the use of radiocontrast media in cases of impaired renal function, as well as the problems associated with low vaccination rates. Three of the negative recommendations are focused on hydration and diuretics. The remaining two negative recommendations concern angioplasty in cases of renal artery stenosis and the unconsidered use of nonsteroidal anti-inflammatory drugs.

Different in vivo and in vitro transformation of intestinal stem cells in mismatch repair deficiency.

Mutations in mismatch repair (MMR) genes result in microsatellite instability (MSI) and early onset of colorectal cancer. To get mechanistic insights into the time scale, sequence and frequency of intestinal stem cell (ISC) transformation, we quantified MSI and growth characteristics of organoids of Msh2-deficient and control mice from birth until tumor formation and related them to tissue gene expression. Although in Msh2-deficient organoids MSI continuously increased from birth, growth characteristics remained stable at first. Months before tumor onset, normal Msh2-deficient tissue contained tumor precursor cells forming organoids with higher MSI, cystic growth and growth rates resembling temporarily those of tumor organoids. Consistently, Msh2-deficient tissue exhibited a tumor-like gene signature. Normal Msh2-deficient organoids showed increased inheritable transient cyst-like growth, which became independent of R-spondin. ISC transformation proceeded faster in vitro than in vivo independent of the underlying genotype but more under MMR deficiency. Transient cyst-like growth but not MSI was suppressed by aspirin. In summary, as highlighted by organoids, molecular alterations continuously proceeded long before tumor onset in MMR-deficient intestine, thus increasing its susceptibility for ISC transformation.

["Choosing wisely" in infectious diseases : Overuse of antibiotics - too few vaccinations]. / "Klug entscheiden" bei Infektionskrankheiten : Zu häufig Antibiotika - zu wenig Impfungen.

The "choosing wisely" recommendations of the German Society of Internal Medicine (DGIM) and its specialist societies address diagnostic and therapeutic procedures, which are of particular medical importance but applied too often or too rarely in clinical practice. The aim is to further improve treatment of patients. Important topics of overuse and insufficient treatment related to the diagnostics, therapy, prevention and exclusion of infectious diseases could be identified. These topics not only play an important role in the discipline of infectious diseases but are also relevant for other internal medical disciplines. These topics related to infectious diseases have also been integrated into the recommendations of the German Society of Gastroenterology, Digestive and Metabolic Diseases as well as the German Societies for Internal Intensive Care and Emergency Medicine, for Pneumology, for Nephrology and for Rheumatology. The pivotal issues of the recommendations are the inappropriate use of antibiotics and insufficient vaccination rates.

[Incidental finding of proteinuria]. / Zufallsbefund Proteinurie.

A positive signal when testing urine for proteinuria is a frequent finding, either in the context of a routine medical check-up or when searching for a specific renal disorder. This brief overview aims to provide assistance in the classification of proteinuria and to provide guidance to the next diagnostic and therapeutic steps. The normal urine protein loss of a healthy adult is less then 150 mg/day. Higher rates of proteinuria should be confirmed as this is often a sign of glomerular or tubular damage. In addition, proteinuria is a strong prognostic factor for cardiovascular and total mortality. Principally, proteinuria is 1) a symptom of renal diseases, 2) a progression factor for renal diseases and 3) a risk factor for cardiovascular diseases and total mortality. In this article proteinuria is defined, the correlation to various renal diseases is described and the relevance for progression of renal diseases and total mortality is shown. Finally, diagnostic procedures are described and a perspective on therapeutic measures is provided.

Micromechanical testing with microstrain resolution.

Simple test equipment has been developed for studying the elastic limit and plastic deformation of thin metal wires and thin foils (down to 10 µm) under torsion, tension, and bending. Using load-unload methods and gauge lengths up to 1 m, plastic strain as low as 10(-6) can be measured accurately.

Tumors in the lung--morphologic features and the challenge of integrating biomarker signatures into diagnostics.

In the last decade, pathologic approaches concerning diagnosis and treatment of lung carcinomas have increasingly moved towards the implementation of molecular methods into the process of decision. In this study, an overview is given referring to the variety of tumors in the lung including common primary lung neoplasms and secondary tumors, and a modus operandi is presented which integrates immunology as well as molecular pathology within the process of finding correct diagnoses. Besides the conventional and approved methods and techniques leading to appropriate treatment including so-called targeted therapies, pathologist's work meanwhile depends on both histologic and molecular results. Since molecular techniques have increasingly entered the field of routine diagnostics, challenges and possibilities have changed and are still rapidly developing. The proceeding integration of molecular-biologic investigations into the process of diagnosing has changed the nature of diagnostics and will continuously grow in the near future. Only by obtaining a proper diagnosis, the optimal treatment of a patient can be assured, whereupon the knowledge of gene mutations and/or altered protein expression is crucial. By identifying those novel molecular target structures, the therapeutic spectrum is tremendously enlarged and will finally improve the patient's prognosis by personalized targeted therapies.

Mesenchymal stem cells in cartilage repair: state of the art and methods to monitor cell growth, differentiation and cartilage regeneration.

Degenerative joint diseases caused by rheumatism, joint dysplasia or traumata are particularly widespread in countries with high life expectation. Although there is no absolutely convincing cure available so far, hyaline cartilage and bone defects resulting from joint destruction can be treated today by appropriate transplantations. Recently, procedures were developed based on autologous chondrocytes from intact joint areas. The chondrocytes are expanded in cell culture and subsequently transplanted into the defect areas of the affected joints. However, these autologous chondrocytes are characterized by low expansion capacity and the synthesis of extracellular matrix of poor functionality and quality. An alternative approach is the use of adult mesenchymal stem cells (MSCs). These cells effectively expand in 2D culture and have the potential to differentiate into various cell types, including chondrocytes. Furthermore, they have the ability to synthesize extracellular matrix with properties mimicking closely the healthy hyaline joint cartilage. Beside a more general survey of the architecture of hyaline cartilage, its composition and the pathological processes of joint diseases, we will describe here which advances were achieved recently regarding the development of closed, aseptic bioreactors for the production of autologous grafts for cartilage regeneration based on MSCs. Additionally, a novel mathematical model will be presented that supports the understanding of the growth and differentiation of MSCs. It will be particularly emphasized that such models are helpful to explain the well-known fact that MSCs exhibit improved growth properties under reduced oxygen pressure and limited supply with nutrients. Finally, it will be comprehensively shown how different analytical methods can be used to characterize MSCs on different levels. Besides discussing methods for non-invasive monitoring and tracking of the cells and the determination of their elastic properties, mass spectrometric methods to evaluate the lipid compositions of cells will be highlighted.

Impact of oxygen environment on mesenchymal stem cell expansion and chondrogenic differentiation.

INTRODUCTION: In vitro expansion and differentiation of mesenchymal stem cells (MSC) rely on specific environmental conditions, and investigations have demonstrated that one crucial factor is oxygen environment. OBJECTIVES: In order to understand the impact of oxygen tension on MSC culture and chondrogenic differentiation in vitro, we developed a mathematical model of these processes and applied it in predicting optimal assays. METHODS AND RESULTS: We compared ovine MSCs under physiologically low and atmospheric oxygen tension. Low oxygen tension improved their in vitro population growth as demonstrated by monoclonal expansion and colony forming assays. Moreover, it accelerated induction of the chondrogenic phenotype in subsequent three-dimensional differentiation cultures. We introduced a hybrid stochastic multiscale model of MSC organization in vitro. The model assumes that cell adaptation to non-physiological high oxygen tension reversibly changes the structure of MSC populations with respect to differentiation. In simulation series, we demonstrated that these changes profoundly affect chondrogenic potential of the populations. Our mathematical model provides a consistent explanation of our experimental findings. CONCLUSIONS: Our approach provides new insights into organization of MSC populations in vitro. The results suggest that MSC differentiation is largely reversible and that lineage plasticity is restricted to stem cells and early progenitors. The model predicts a significant impact of short-term low oxygen treatment on MSC differentiation and optimal chondrogenic differentiation at 10-11% pO(2).

Low oxygen expansion improves subsequent chondrogenesis of ovine bone-marrow-derived mesenchymal stem cells in collagen type I hydrogel.

BACKGROUND/OBJECTIVE: A crucial factor when investigating cartilage tissue engineering using mesenchymal stem cells (MSCs) is their application in large-animal models and preclinical trials. However, in vitro studies using cells of these model organisms must proceed. Considering that oxygen tension is an important parameter for stem cell culture, we investigated the effect of low oxygen tension during the expansion of ovine MSCs on colony-forming unit-fibroblast (CFU-F) formation, senescence and subsequent chondrogenesis in pellet culture and a collagen I hydrogel which is in clinical use for matrix-associated autologous chondrocyte transplantation (MACT). MATERIALS AND METHODS: Ovine MSCs were isolated from bone marrow aspirates and cultured at 5 and 20% O(2) in monolayer. CFU-F formation was detected by Giemsa staining. Senescence was analyzed by detection of senescence-associated beta-galactosidase and flow cytometry. Chondrogenic differentiation was carried out in pellet and collagen I hydrogel culture and assessed by gene expression, immunohistochemistry and measurement of sulfated glycosaminoglycans (sGAG). RESULTS: MSCs expanded at 5% O(2) revealed a 2-fold higher CFU-F potential and diminished senescence compared to those expanded at 20% O(2). Most notably, our results show enhanced chondrogenic differentiation in both pellet culture and the MACT-approved collagen I hydrogel. CONCLUSION: The findings demonstrate that physiologically low oxygen tension during monolayer expansion of ovine MSCs is advantageous in order to improve cartilage tissue engineering in a sheep model. The ovine system is shown to represent an appropriate basis for large-animal studies and preclinical trials on MSC-based cartilage repair.

From single cells to tissue architecture-a bottom-up approach to modelling the spatio-temporal organisation of complex multi-cellular systems.

Collective phenomena in multi-cellular assemblies can be approached on different levels of complexity. Here, we discuss a number of mathematical models which consider the dynamics of each individual cell, so-called agent-based or individual-based models (IBMs). As a special feature, these models allow to account for intracellular decision processes which are triggered by biomechanical cell-cell or cell-matrix interactions. We discuss their impact on the growth and homeostasis of multi-cellular systems as simulated by lattice-free models. Our results demonstrate that cell polarisation subsequent to cell-cell contact formation can be a source of stability in epithelial monolayers. Stroma contact-dependent regulation of tumour cell proliferation and migration is shown to result in invasion dynamics in accordance with the migrating cancer stem cell hypothesis. However, we demonstrate that different regulation mechanisms can equally well comply with present experimental results. Thus, we suggest a panel of experimental studies for the in-depth validation of the model assumptions.

D1 receptor antagonist-induced long-term depression in the medial prefrontal cortex of rat, in vivo: an animal model of psychiatric hypofrontality.

The objective of the following experiment was to induce a pathogenic hypofrontal condition by administering a dopamine-1 receptor (D(1)R) antagonist to rats. The pathophysiological effect of this manipulation upon glutamate-based long-term potentiation (LTP) in the medial prefrontal cortex (mPFC) was examined in vivo. Subjects were surgically implanted with stimulating electrodes into the corpus callosum and recording electrodes into the mPFC. High-frequency stimulation (HFS) was combined with the administration of the selective D(1)R family agonist A68930 hydrochloride (0.4 mg/kg/mL) and the selective D(1)R family antagonist SKF 83566 (0.15 mg/kg/mL). The administration of SKF 83566 hydrobromide prevented mPFC LTP, and resulted in HFS-induced long-term depression. This indicates that D(1)R activation is necessary for the induction of mPFC glutamate-based LTP. This is supported by our finding that the administration of A68930 hydrochloride combined with HFS induced LTP comparable with saline control levels, suggesting that D(1)R activation is necessary for the induction of baseline levels of mPFC LTP. Given that the mPFC governs executive behaviours that are subserved by LTP, such as working memory, these findings are relevant for the study of psychopathological conditions in which hypodopaminergic conditions exist in the mPFC and are correlated with psychiatric symptomotology, such as drug addiction and schizophrenia.

Secondary bronchial botryomycosis due to foreign body aspiration.

Botryomycosis is recognised mainly as a visceral disorder with rare cases of pulmonary manifestation. The most frequent cause of pulmonary Botryomycosis is aspiration of a foreign body which induces bacteria to group together instead of spreading out forming conglomerates resembling the granules of Actinomyces. Here we report on the clinical and pathologic findings of a 38-year-old patient without any further predisposing factors. It should be mentioned that the disease was cured following the extraction of a foreign body without the need for any surgery or antibiotic therapy. Factors influencing the course of the disease are discussed below.

Downregulation of organic anion transporters OAT1 and OAT3 correlates with impaired secretion of para-aminohippurate after ischemic acute renal failure in rats.

Ischemic acute renal failure (iARF) was described to reduce renal extraction of the organic anion para-aminohippurate (PAH) in humans. The rate-limiting step of renal organic anion secretion is its basolateral uptake into proximal tubular cells. This process is mediated by the organic anion transporters OAT1 and OAT3, which both have a broad spectrum of substrates including a variety of pharmaceutics and toxins. Using a rat model of iARF, we investigated whether impairing the secretion of the organic anion PAH might be associated with downregulation of OAT1 or OAT3. Inulin and PAH clearance was determined starting from 6 up to 336 h after ischemia-reperfusion (I/R) injury. Net secretion of PAH was calculated and OAT1 as well as OAT3 expression was analyzed by RT-PCR and Western blotting. Inulin and PAH clearance along with PAH net secretion were initially diminished after I/R injury with a gradual recovery during follow-up. This initial impairment after iARF was accompanied by decreased mRNA and protein levels of OAT1 and OAT3 in clamped animals compared with sham-operated controls. In correlation to the improvement of kidney function, both mRNA and protein levels of OAT1 and OAT3 were upregulated during the follow-up. Thus decreased expression of OAT1 and OAT3 is sufficient to explain the decline of PAH secretion after iARF. As a result, this may have substantial impact on excretion kinetics and half-life of organic anions. As a consequence, the biological effects of a variety of organic anions may be affected after iARF.

The HOPE technique opens up a multitude of new possibilities in pathology.

Fixation of tissues with formalin results in well-preserved morphology but to a high degree leads to degradation of nucleic acids, which substantially constricts the spectrum of applicable molecular techniques. The novel HOPE-fixative with subsequent paraffin embedding, as an alternative to formalin, has been shown to result in a morphological preservation comparable to formalin-fixed, paraffin-embedded specimens. Due to a similar workflow like in formalin-fixation and paraffin embedding, the HOPE technique can be successfully established within any pathological institute. We have shown that DNA, RNA and proteins are protected in HOPE-fixed, paraffin-embedded tissues for at least eight years. Moreover, we described procedures which permit successful application of all common molecular techniques such as in situ hybridization targeting either DNA or RNA, immunohistochemistry without antigen retrieval and for formalin-refractory antigens, PCR, RT-PCR, Western blot, Northern blot, and transcription microarrays to HOPE-fixed, paraffin-embedded tissues. Furthermore, HOPE-fixed tissues can be used for the construction of tissue microarrays for enhanced high-throughput analyses on the molecular level. Using the HOPE technique as its crucial methodological base, ex vivo model systems could be established, e.g. for the simulation of early events in human infections and detection of chemotherapy resistances in human cancer. In addition to tissues, cell-culture preparations have been prepared utilizing the HOPE technique, which were then successfully applied to in situ hybridization targeting mRNA or immunocytochemistry with excellent preservation of morphological details. Taken together, the HOPE technique to date represents an alternative fixation that is, in contrary to other procedures, scientifically broadly analyzed. Therefore new possibilities are opened up especially within the rapidly growing field of molecular pathology.

Individual cell-based models of the spatial-temporal organization of multicellular systems--achievements and limitations.

Computational approaches of multicellular assemblies have reached a stage where they may contribute to unveil the processes that underlie the organization of tissues and multicellular aggregates. In this article, we briefly review and present some new results on a number of 3D lattice free individual cell-based mathematical models of epithelial cell populations. The models we consider here are parameterized by bio-physical and cell-biological quantities on the level of an individual cell. Eventually, they aim at predicting the dynamics of the biological processes on the tissue level. We focus on a number of systems, the growth of cell populations in vitro, and the spatial-temporal organization of regenerative tissues. For selected examples we compare different model approaches and show that the qualitative results are robust with respect to many model details. Hence, for the qualitative features and largely for the quantitative features many model details do not matter as long as characteristic biological features and mechanisms are correctly represented. For a quantitative prediction, the control of the bio-physical and cell-biological parameters on the molecular scale has to be known. At this point, slide-based cytometry may contribute. It permits to track the fate of cells and other tissue subunits in time and validated the organization processes predicted by the mathematical models.