The Neuronal Ceroid Lipofuscinoses (NCLs) are a family of autosomal recessive neurodegenerative disorders that annually affect 1:100,000 live births worldwide. This family of diseases results from mutations in one of 14 different genes that share common clinical and pathological etiologies. Clinically, the diseases are subcategorized into infantile, late-infantile, juvenile and adult forms based on their age of onset. Though the disease phenotypes may vary in their age and order of presentation, all typically include progressive visual deterioration and blindness, cognitive impairment, motor deficits and seizures. Pathological hallmarks of NCLs include the accumulation of storage material or ceroid in the lysosome, progressive neuronal degeneration and massive glial activation. Advances have been made in genetic diagnosis and counseling for families. However, comprehensive treatment programs that delay or halt disease progression have been elusive. Current disease management is primarily targeted at controlling the symptoms rather than “curing” the disease. Recognizing the growing need for transparency and synergistic efforts to move the field forward, this review will provide an overview of the therapeutic approaches currently being pursued in preclinical and clinical trials to treat different forms of NCL as well as provide insight to novel therapeutic approaches in development for the NCLs. Lysosomal storage disorders (LSDs), a group of rare disorders involving the accumulation of storage material in the lysosome, have been estimated to occur in approximately one in 7500 live births , 2. NeurAs previously mentioned NCLs can be caused by one of a number of genetic mutations and can onset at different ages. This genetic heterogeneity results in approximately 9 different forms of NCLs , 7) withhttp://www.ucl.ac.uk/ncl/mutation.shtml and )Ppt1−/−/NSCID mouse model following treatment with human CNS derived stem cells and these findings resulted in a clinical trial . The ou0337636; ) and thuOne source of stem cells yet to be explored as a treatment option for NCLs are induced pluripotent stem cells (iPSCs). Since being first described in 2006 by Takahashi and Yamanaka, iPSCs have been used for various research purposes , 93. A lRNA modulation therapies are a relatively new therapeutic approach for lysosomal storage diseases, especially the NCLs. There are a number of different RNA modulation therapies  that have been used effectively in preclinical and clinical trials for a number of different diseases –111. TheOne therapeutic strategy aimed at treating diseases caused by nonsense mutations involves the use of nonsense suppression compounds which promote the readthrough of nonsense or premature termination codons (PTCs). A proportion of PTC-containing transcripts escape NMD and these compounds induce the ribosome to incorporate a near cognate amino acid at the PTC, essentially “reading through” the termination codon. For these mRNAs, translation continues through the entire transcript, terminates at the natural termination codon and thus generates a full-length protein with a single amino acid substitution at the nonsense mutation. Read-through therapy, or nonsense suppression therapy, is currently in clinical trials , , 113).CLN1 are present in more than 50 % of INCL patients [Cln1R151X mouse model [Can these therapies be effectively applied to the NCLs? Nonsense suppression therapies have been proposed as treatments for a whole host of LSDs that result from nonsense mutations. In fact, nonsense mutations in patients . Multiplpatients , 108. Sppatients . Moreovepatients , 107. Thse model  – so patse model , broadenAnother RNA modulating therapeutic approach is the use of antisense oligonucleotides (ASOs). These short, modified nucleic acids are designed to bind a target RNA through complementary base-pairing . The binASOs may be useful in combination with nonsense suppression therapies to improve the protein production from genes with nonsense mutations. A limitation to the nonsense suppression approach is the activity of the naturally occurring process in eukaryotic cells called nonsense mediated decay . DuBased on the aforementioned compounds and their results, small molecules seem to be a potential therapeutic option for the different forms of NCL. In order to identify new small molecule therapies, high throughput drug screens (HTS) have been used with some success in a number of LSD studies to screen various compound libraries –179. OveCln1 knockout mice [Analysis of natural compound treatments  for NCLs began in the late 20th century. Naidu et al., presented these studies in their report discussing the use of selenium in three cases of NCL . Since tout mice –183. EveAs the various treatment options that we have reviewed are being explored, we must simultaneously ensure that we have a strong foundation on which to successfully accelerate these treatments into and through clinical trials. This includes making sure that we have the necessary tools in place to expedite these studies including comprehensive natural history studies of the NCLs, detailed and easy-to-use clinical rating scales, systems for early diagnosis , and reliable biomarkers for tracking disease progression. Our clinical and basic research teams must be well trained in the ethical and management issues involved in conducting clinical trials. Additionally, even at the earliest stages of pre-clinical work in animal models of the NCLS, the rigor of clinical research must be applied. Failure to maintain a high level of rigor can result in our wrongly advancing (or dropping) therapeutic targets, unnecessary higher costs of production, unknown risks/benefits, ethical concerns pertaining to risking patients with invalid candidate therapeutics, and/or late state trial failures that in turn prevent patients from being involved in other trials. Thus, a number of reports from the NIH and the pharmaceutical industry have stressed the growing importance of rigor in how preclinical studies are designed and executed in order to optimize the predictive value of preclinical studies . How do We must also all acknowledge that although this review focuses on a number of potential NCL therapies, it does not include every possibility. In addition to drawing on the current literature to identify areas of current and potential focus for therapy development, the authors of this review are also very active in the NCL research community and attend a number of research forums focused on these topics. In particular, a portion of the topics discussed here and opinions on these potential therapeutic approaches are highlights from a periodic forum held between member of the research community, patient advocates and public policy group entitled Batten Disease: Updates on Translational Research for Management of INCL/LINCL. However, the field of translational research is moving at an accelerated pace and we as research scientist, clinical and regulatory officials must work collectively to streamline efforts that effectively and efficiently allow new drugs and treatment strategies to move from the basic laboratory to the patient as quickly as possible.


Circulating tumor DNA (ctDNA) in the plasma or serum of cancer patients provides an opportunity for non-invasive sampling of tumor DNA. This ‘liquid biopsy’ allows for interrogations of DNA such as quantity, chromosomal alterations, sequence mutations and epigenetic changes, and can be used to guide and improve treatment throughout the course of the disease. This tremendous potential for real-time ‘tracking’ in a cancer patient has led to substantial research efforts in the ctDNA field. ctDNA can be distinguished from non-tumor DNA by the presence of tumor-specific mutations and copy number variations, and also by aberrant DNA methylation, with both DNA sequence and methylation changes corresponding to those found in the tumor. Aberrant methylation of specific promoter regions can be a very consistent feature of cancer, in contrast to mutations, which typically occur at a wide range of sites. This consistency makes ctDNA methylation amenable to the design of widely applicable clinical assays. In this review, we examine ctDNA methylation in the context of monitoring disease status, treatment response and determining the prognosis of cancer patients. Genomics is anticipated to bring major improvements in the treatment of cancer patients. The capacity to quickly and relatively inexpensively sequence tumor DNA on a genome-wide scale allows the identification of potential molecular targets, assignment of cancer subtype and determination of patient prognosis, while also providing insights into cancer biology that can form the basis of further research. However, there are certain limitations to obtaining sequence information from solid cancers: due to the clonal evolution and heterogeneity of a tumor, a single biopsy may not represent the diversity of DNA changes present; metastasized disease may be difficult to identify and access; and repeated biopsies from a patient are not practicable.Circulating tumor DNA (ctDNA) containing the same molecular aberrations as the solid tumor is detectable in the bloodstream of many cancer patients , and samMethylation of CpG sites at selected DNA sequences provides a level of regulation over gene expression over that which is specified by DNA sequence alone. Genome methylation undergoes coordinated changes at defined stages of development and in response to environmental stimuli such as diet, chemical toxins and pollutants, and temperature stresses  for prostate cancer ; hence DctDNA in the blood has a half-life of ∼2 h . One appKRAS, APC, TP53 and PIK3CA) from each patient's FFPE tumor sample, and then designing probe-based PCR BEAMing assays for detection of the identified mutations in patient plasma  of 35%. In contrast, the methylation index of eight samples collected postoperatively was 3.5%, reflecting the decrease in ctDNA following removal of the tumor  were found to be methylated in significantly more pre-surgery samples than post-surgery samples. RARb2 has also been previously identified as a potential biomarker for breast cancer detection  was chosen to consist of genes frequently methylated in ER-positive and ER-negative breast cancer, and rarely methylated in healthy control samples. Using the panel to track 29 breast cancer patients treated in clinical trials with docetaxel and imatinib or capecitabine, a decrease in serum methylation levels after 1–2 cycles of treatment was seen in patients having stable disease or partial response, but not in patients with progressive disease. Where sequential serum samples from patients undergoing treatment were available, the rise in methylated ctDNA was detectable prior to clinical disease progression being observed  and overall survival  in six consecutive serum samples from breast cancer patients undergoing chemotherapy treatment. A correlation between reduction in total gene methylation and reduction in tumor volume in the respondents group was observed. At the single gene level, the frequency of BRCA1 methylation was significantly correlated with response to chemotherapy  and 90% specificity (95% CI: 84–94%)  biomarker discovery and development pathway has attempted to standardize biomarker development and provide an efficient framework to move useful biomarkers into clinical practice. All large Phase III therapeutic studies should collect prospective samples that are rationally devised to test available biomarkers. While associated costs to cover sample collection, storage and testing are significant, appropriate biomarker studies are crucial for tailoring treatment and improving outcomes. In response to this need, the NCI has prioritized biomarker inclusion in large Phase II (≥100 patients) and III therapeutic trials by establishing the Biomarker, Imaging and Quality of Life Studies Funding Program , which actDNA is detectable in the blood of many cancer patients, with levels reflecting tumor load . Thus thGSTP1 in plasma was a better predictor of overall survival than PSA in prostate cancer patients (Clinical trials of cancer treatments rely on overall survival time and relapse-free survival time to assess the efficacy of new drugs. While survival time and relapse-free survival are the gold standards in establishing treatment benefits, these data take a long time to accrue, prolonging the time it takes for new drugs to reach the clinic and adding considerably to trial costs. Surrogate endpoints, that is, clinical parameters related to disease progression that become apparent prior to relapse or death, have the potential to streamline drug development. However, surrogate endpoints have to date proved unreliable in evaluating cancer treatments. PSA, the most studied biomarker in prostate cancer, although useful for tracking individual response, does not correlate sufficiently well with overall survival times to effectively predict the outcome of treatment (Cancer monitoring by measuring tumor DNA dynamics in blood is a new and developing area, poised to advance rapidly both through the application of existing technology, and as a result of novel techniques that are constantly being pursued in the burgeoning field of molecular biology. The benefits can be anticipated to improve patient management, reduce unnecessary drug toxicity and accelerate data acquisition from clinical trials. Cancer-specific circulating DNA methylation offers a range of promising targets that can help to advance these aims.


Bronchopulmonary dysplasia (BPD) is a chronic lung disease of prematurity that is only now beginning to be understood at the molecular level. The review by Chao et al.  elegantlDespite the differences in etiology, both BPD and chronic adult lung injury are characterized by elevated expression and activity of transforming growth factor (TGF)-β, perhaps the most well-known profibrotic cytokine. Implicated in epithelial–mesenchymal transition (EMT) , myofibrAn intriguing aspect of the review by Chao et al. is the supportive role of the lipofibroblasts (LIFs), the interstitial fibroblasts identified in rodent lungs containing cytoplasmic lipid droplets  thought Mesenchymal–epithelial crosstalk, as described above for LIFs and type II alveolar epithelial cells, is obviously important for lung development, as nicely illustrated by Chao and colleagues . HoweverWe have certainly learned much about BPD pathogenesis through elegant mouse modeling and human pathologic studies. However, there is still much to learn; murine studies, while informative, cannot take the place of knowledge generated in human samples of disease. Moreover, although mouse lung development occurs in a stereotypic fashion which has been well characterized, it is not proven that human lung development occurs entirely in the same way. Thus, better methods of studying human lung development need to be created and validated, with findings hopefully informing our understanding of chronic adult lung diseases as well.As alluded to by Chao and colleagues , the incThe author confirms being the sole contributor of this work and approved it for publication.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Over the past few years there has been considerable investment in genomics (DNA-based) research, including the Prime Minister’s announcement of £100 million to sequence 100 000 whole genomes.Chronic disease prevention in primary care aims to identify individuals at increased risk who can then be given tailored disease prevention advice including lifestyle modification, disease screening, or preventive treatment. Current approaches to chronic disease risk assessment use a combination of factors including demographics, family history, lifestyle, physiological measurements, and biomarkers. GPs in the UK are familiar with using electronic risk calculators to provide a personal risk score, such as the QRISK2 algorithm for primary prevention of cardiovascular disease.While epidemiological studies have elucidated the most important risk factors for disease over the past half century, this work largely considered the population as homogeneous, with risk factors acting uniformly across the population in relation to diseases identified. Recent genomic advances have provided new insights such as that the diseases themselves are heterogeneous.7In the light of these advances, DNA-based risk assessment is increasingly being proposed as a means of determining chronic disease risk. Recent work undertaken by the European Commission funded multicentre Collaborative Oncological Gene-environment Study (COGS) on genetic variation and breast cancer risk has provided proof of principle that testing for genetic variants could be used to stratify the population so that those at higher risk might receive earlier or more frequent breast screening, while those at lower risk might even be recommended to forgo screening.Given adequate resourcing, a range of DNA-based tests could be used to fine-tune the current approach to prevention, and provide more tailored chronic disease prevention in primary care. This may be further enabled with near-patient testing for a particular set of genetic variants, undertaken in the surgery. Applied in this way, genomics could then support tailored cancer screening, the integration of genomics data with family history, sociodemographic, behavioural, and environmental risk factors into risk assessment tools, and even pharmacogenetically-informed prescribing such as for nicotine replacement treatment. However, there are still a number of issues that remain important to address before the incorporation of any of these approaches into routine general practice.What preparation might GPs need? The answer, in our mind, is not very much, as most of the genomics-based approaches will be delivered using GPs’ current expertise in communicating with patients about disease prevention, lifestyle and behaviour change. A detailed understanding of genetic variants and their additional contribution to risk profiling may not be required, assuming the validity of the risk assessment. We will need to incorporate discussions about the impact of DNA testing, including that a person’s level of risk could be determined at an earlier age and before phenotypic markers of risk or disease are detectable by biomarkers. The importance of adopting appropriate lifestyle changes will still need to be stressed. Where prevention options include interventions such as screening or drug treatment we will need to help patients understand that such interventions are not universally beneficial; those whose genetic background puts them at lower risk may need to understand the potential for harm in these activities. DNA-based risk assessment also raises ethical issues for primary care, particularly with near-patient testing, such as whether the genetic samples and test results will be stored for future reference or discarded. If stored, consent and access issues will become important, particularly around employment, insurance, and research by academic or commercial organisations.in some participants the genetic test results would reinforce healthy behaviour while others were falsely reassured.’‘... As genomics-based approaches begin to be implemented both within health services and, increasingly, through direct-to-consumer testing, there is still much research to be undertaken. In a recent issue Middlemass and colleagues reported a qualitative study investigating the introduction of genetic testing for cardiovascular disease in primary care.They emphasised that it will be important to build on the patient’s motivation to undertake genetic testing both for themselves and their families; to understand how patients deal with conflicting findings between genetic and conventional cardiovascular risk assessment; to learn how to promote responses that reinforce healthy behaviour change and reduce false reassurance; and to look at the potential benefit of genetic testing for younger people who may have more to gain from risk reducing behaviour.Educational strategies will also be of value to primary care clinicians. The NHS National Genetics and Genomics Education Centre has developed an e-learning programme for the RCGP entitled ‘Genetics in Primary Care’.


Coronary artery dissection is an infrequent cause of acute coronary syndrome in the general population. There is, however, a greater incidence of spontaneous coronary artery dissection (SCAD) in young women, especially in the peripartum period. However, the majority of cases have favorable outcomes with medical management or percutaneous coronary intervention; coronary artery bypass grafting (CABG) and transplantation are utilized in severe cases. This case is a one of a 30-year-old postpartum female with multivessel SCAD requiring CABG with subsequent biventricular failure and inability to wean from bypass. We believe this is the first reported case in which venoarterial extracorporeal membrane oxygenation (VA-ECMO) was used in the management of biventricular heart failure in a postpartum patient with SCAD. Acute coronary syndrome (ACS) is typically encountered in patients with a history of coronary atherosclerosis or associated risk factors; spontaneous coronary artery dissection has also been described as a rare but identifiable cause of this disease process. Overall, the incidence of SCAD has been reported to be between 0.28 and 1.1% in the general population. Young women encompass up to 70% of these cases, 30% of which are in the peripartum period [Coronary artery dissection can be categorized as either primary or secondary in nature, with secondary dissections typically being iatrogenic and trauma related . The etiManagement options for this conditions range from conservative, medical management to invasive interventions, such as coronary artery bypass grafting (CABG) and even cardiac transplantation . Most reA 30-year-old critically ill female with past medical history significant for tobacco use presented to our institution from an outside hospital after failure to wean from cardiopulmonary bypass (CPB) after a two-vessel CABG for management of spontaneous coronary artery dissection. She initially presented nine days after an uncomplicated cesarean delivery to the outside facility with complaints of chest pain. Work-up at that time revealed an ST segment elevation myocardial infarction (STEMI) and the patient was emergently taken to the cardiac catheterization lab for left heart catheterization (LHC). She was found to have dissection of the Right Coronary Artery (RCA) and left anterior descending (LAD) coronary artery. Multiple drug-eluting stents were placed and she was discharged home on aspirin, Plavix, and metoprolol. The patient presented again 7 days later with complaints of recurrent chest pain. Repeat LHC revealed a large dissection from the mid left main (LM) to the ostial circumflex (LCX) arteries with patency of the prior stented vessels. At that time she was taken for emergent coronary artery bypass grafting (CABG) of saphenous vein to LAD and saphenous vein to circumflex.The intraoperative course was relatively uncomplicated until it was evident that she could not be weaned from CPB despite maximum vasopressor and inotropic support. Intraoperative transesophageal echocardiogram (TEE) demonstrated severe biventricular dysfunction. Subsequently, our institution was consulted to convert the patient to central VA-ECMO. This was completed via the preexisting aortic and a two-stage right atrial cannula placed for CPB. She was maintained on multiple inotropic agents and transported by the team to our facility for a higher level of care. An attempt to transition to peripherally cannulated VA-ECMO via the right femoral artery and vein was unsuccessful secondary to severely limited flow related to grossly visible dissection in the proximal common femoral artery. This defect was immediately addressed with the assistance of the vascular surgeon who reestablished antegrade flow with Fogarty thrombectomy and repaired the arteriotomy site utilizing a vein patch. These interventions resulted in restoration of excellent forward flow. It was thought that this incident was likely iatrogenic given the small nature of the peripheral arteries in combination with their underlying propensity for dissection.During this operative course, repeat TEE revealed that the patient had a large previously unrecognized atrial septal defect (ASD) in the fossa ovalis which was believed to have intermittent bidirectional flow, measuring 1.5 cm. This was subsequently repaired via an open approach while that patient was on CPB. It was also confirmed by the surgeon using a flow probe that there was interval development of significant thrombosis within the newly placed saphenous vein graft (SVG) to left anterior descending (LAD) artery. It was decided that no operative intervention would be taken to correct this issue and the patient was transported to the ICU in stable condition on VA-ECMO with an open chest through persistent aortic cannula and newly placed R femoral vein cannula.Repeat TEE on postoperative day 1 showed persistent postbypass left ventricular dysfunction with an ejection fraction of 25%, global hypokinesis, normal RV size, and mild-to-moderate RV dysfunction. Repeat cardiac catheterization was performed via left common femoral artery to evaluate the possible etiology of her continued dysfunction and new drug-eluting stents (DES) were placed in the native LAD, left main (LM), and the SVG  as a cause of hypotension and inability to separate from the bypass circuit.Lastly, the cause of difficulty to wean in this case is ventricular dysfunction . Causes ECMO has a variety of uses ranging from the management of patient with acute respiratory distress syndrome to postcardiac surgery patient with significant global hypokinesis. In either instance, the main indications for ECMO support are to maintain right ventricular and/or pulmonary function . VanzettWhile the use of VA-ECMO in this report seems to be unique, there have been many reports which describe the utility of ECMO in caring for peripartum patients. For example, Hansen et al. published a case report in 2012 describing the use of ECMO in a postpartum patient who developed severe, acute mitral regurgitation related to a ruptured posteromedial papillary muscle . EarlierOur case report appears to be the first published use of VA-ECMO in a postpartum patient with SCAD requiring emergent on pump CABG who was unable to be weaned from CPB. SCAD has been described in numerous case reports over the years as a potential cause of cardiovascular morbidity and mortality in the peripartum patient. Multiple papers have discussed possible management options for SCAD in the peripartum period; however, currently, there are no guidelines for the management of this specific disease process.


Indeed, the results of three experimental studies demonstrate that reasoners associate conjunction descriptions with perceptually-dependent stimuli and disjunction descriptions with perceptually-independent stimuli, where visual dependency status follows the key Gestalt principles of common fate, proximity, and similarity.Reasoning, solving mathematical equations, or planning written and spoken sentences all must factor in stimuli perceptual properties. Indeed, thinking processes are inspired by and subsequently fitted to concrete objects and situations. It is therefore reasonable to expect that the mental representations evoked when people solve these seemingly abstract tasks should interact with the properties of the manipulated stimuli. Here, we investigated the mental representations evoked by conjunction and disjunction expressions in language-picture matching tasks. We hypothesised that, if these representations have been derived using key Gestalt principles, reasoners should use perceptual compatibility to gauge the goodness of fit between conjunction/disjunction descriptions (e.g.,  People have many options available for solving complex tasks in real-life situations. One option is to apply the rules of propositional logic –4 and idThe present paper builds on previous findings  suggestiGestalt psychologists ever since Koehler , Koffka the purple and the green) and disjunctions  by drawing on their experience with previous conjunction and disjunction situations. We thereby use insights from embodied and grounded cognition theories ).The structure of a trial was as follows. First, the word “Ready” appeared in the centre of the screen for 1000 ms, followed by a 500 ms blank screen, then by a binary visual display. In the alternative condition, the display remained visible for 500 ms and was followed by successive 400 ms displays featuring the left disk, both disks, the right disks, and both disks again. The final frame remained visible until response. In the simultaneous condition, the display remained visible for 1000 ms and was followed by a 1000 ms blank display, and then by a binary display. The final frame remained visible until response. The experiment consisted of 6 practice trials and 96 experimental trials  presented in individually randomized orders in two equal blocks. The dependent variables were the percentage of ‘yes’ responses and the response times across trials.An important question arising at this point is whether our dynamic experimental setup might have affected language reference verification and hence RT values, especially that meaning and verification processes differ when understanding conjunctions and disjunctions. In particular, stimuli perceptual properties might have nudged participants to stop halfway during the meaning verification process of the two nouns mentioned in disjunction trials given that verifying the reference of the first noun already satisfies disjunction validation. Indeed, recent findings suggest that language comprehension and reference verification unfold incrementally  such thMoreover, evidence from the visual perception literature suggests that, if anything, dynamic displays intensify the reference verification process. In particular, abrupt visual stimuli onsets were shown to capture attention even when they were unrelated to the items that participants were searching for . IndeedExperiment 2 investigated the effects of stimuli proximity on conjunction and disjunction processing in terms of accuracy scores and response times. Gestalt principles predict that participants perceive stimuli that are placed close to each other as forming a single Gestalt. Therefore they should validate such proximal displays as successful matches for conjunction descriptions but not for disjunction descriptions. Conversely, Gestalt principles predict that participants perceive stimuli placed far from each other as forming two separate Gestalts. Therefore they should validate such distal displays as successful matches of disjunction descriptions but not of conjunction descriptions. In terms of RT values, global precedence principles should facilitate processing of one-Gestalt displays, where stimuli are proximally placed, but not of two-Gestalts displays, where stimuli are distally placed. Nevertheless, Gestalt-reasoning principles might weaken or even reverse these effects, hence induce slower processing of one-Gestalt displays and faster processing of two-Gestalts displays in disjunction trials than in conjunction trials.Twenty participants were selected, as in Experiment 1.Stimuli in the experimental condition of interest consisted of 36 visual displays, each of them containing two disks of different colors placed in a grid of grey disks, as seen in The experiment followed a 2 (Coordination: AND vs. OR) x 2 Visual Stimuli: Two-Gestalts vs. One-Gestalt) factorial design. For filler trials containing a single disk, we only contrasted the two connective conditions, as the concepts “close” and “far” cannot be defined with respect to a single stimulus. As in Experiment 1, participants were informed as to what the correct responses to filler trials should be. In particular, a visual display where one disk matched the first or the second color mentioned was compatible with disjunction but not with conjunction descriptions, whereas a visual display where the disk matched neither color mentioned was always incompatible with coordination descriptions. Participants were not given any further information about the correct answer in the conditions of interest, but were encouraged to follow their intuition.On a typical trial, the word “Ready” first appeared in the centre of the screen for 800 ms, followed by a 500 ms blank screen and by a visual display that remained visible until response. The experiment consisted of 6 practice trials and of 108 experimental trials  presented in individually randomized orders in two equal blocks. As in Experiment 1, the dependent variables were the percentage of ‘yes’ responses and the response times across trials.However, we expected a stronger interaction between the fixed factors for both validation scores and RT values based on previous findings –57) that that57])Experiment 3 investigated the effects of stimuli similarity on conjunction and disjunction processing in terms of accuracy scores and response times. Based on Gestalt principles, we predicted that participants would perceive stimuli having the same shape  as forming a single Gestalt. Therefore they should validate such displays as successful matches of conjunction descriptions but not of disjunction descriptions. Conversely, we predicted that participants would perceive stimuli having different shapes  as forming two separate Gestalts. Therefore they should validate such displays as successful matches of disjunction descriptions but not of conjunction descriptions. In terms of RT values, global precedence principles should facilitate processing of one-Gestalt displays, where stimuli are similar to each other but not of two-Gestalts displays, where stimuli are dissimilar from each other. As in the previous experiments, however, Gestalt-reasoning principles might slow down and even reverse the processing of one-Gestalt displays and instead facilitate the processing of two-Gestalts displays in disjunction trials compared to conjunction trials.Twenty participants were selected, as in the previous two experiments.Stimuli consisted of 24 visual displays containing two geometrical figures whose colors matched the colors mentioned in the accompanying conjunction or disjunction description, as seen in The experiment followed a 2 (Coordination: AND vs. OR) x 2  factorial design. For filler trials, the experiment only contrasted the two connectives in the ‘Coordination’ condition, hence trials included only displays where stimuli had the same shape. A previous pilot study, where we used a fully factorial design, proved to be excessively difficult . As in Experiment 1 and in Experiment 2, participants were informed as to what the correct responses in filler trials should be, but received no further details about the correct answer in the conditions of interest where stimuli colors matched both colors mentioned in conjunction and disjunction descriptions.The structure of a trial was as follows. First, the word “Ready” appeared in the centre of the screen for 800 ms, followed by a 500 ms blank screen and by a visual display that remained visible until response. The experiment consisted of 6 practice trials and 72 experimental trials  presented in individually randomized orders in two equal blocks. The dependent variables were the proportion of ‘yes’ responses and the response times across trials.In three experimental studies, we provided support for the hypothesis that reasoning with conjunctions and disjunctions observes key Gestalt principles, which generate perceptual compatibility effects. In particular, high validation scores obtained for conjunctions describing perceptually-dependent stimuli and for disjunctions describing perceptually-independent stimuli, where dependency status was determined as follows. In Experiment 1, dependent and independent stimuli were moving simultaneously, respectively alternatively, thus illustrating the principle of common fate. In Experiment 2, dependent and independent stimuli were placed close to each other, respectively far from each other, thus illustrating the principle of proximity. In Experiment 3, dependent and independent stimuli exhibited similar shapes, respectively dissimilar shapes, thus illustrating the principle of similarity. The analysis of response times strongly supports these findings and further suggests that conjunction and disjunction meaning can override global-precedence effects, which otherwise apply systematically in visual perception. In particular, differences in accuracy scores and in response times between conditions were slightly greater in conjunction trials than in disjunction trials, as predicted by the global-precedence principle, which holds that global shapes  are processed faster than local shapes . However, participants were often faster to process two-Gestalts displays and slower to process one-Gestalt displays in disjunction trials compared to conjunction trials. It is thus remarkable that response times in disjunction trials indicate a weakening of the global-precedence effect in Experiment 1 and even a slight reversal in Experiment 2 and in Experiment 3.We may surmise that conjunction simulations are easy to build because individuals are often willing to accommodate conjunction descriptions irrespective of stimuli perceptual properties. Indeed, the end result in conjunction contexts  is the same irrespective of the practical circumstances where they occur. For example, when people consume soup and souffleé in sequence, they are still willing to describe them using a conjunction expression as long as they can perceive the two dishes as part of the same Gestalt . In contrast, individuals are less willing to accommodate disjunction descriptions in imperfect contexts because dependency status impacts the outcome of the selection process. For example, individuals cannot use a disjunction expression to describe two dishes consumed in sequence unless they are able to represent the dishes as distinct Gestalts .A comparison of effect sizes in validation scores across our experiments reveals that Gestalt grouping was strongest for stimuli varying in motion type and that it was weaker for stimuli varying in spatial placement and for stimuli varying in shape, thereby confirming previous findings reported in the literature on the relationship between grouping principles  would require instant access to the most concrete concept . In the second scenario, understanding an abstract concept would require instant access to a more concrete concept . Hence we cannot confirm the existence of a hierarchical, level-based activation mechanism for abstract concept comprehension. Our results also fail to confirm the idea that an abstract concept must be understood in terms of a less abstract concept via the least abstract concept. In particular, our results fail to suggest that joint motion, proximity, and similarity afford a particular abstract representation. Whether and how image schemas combine into concept types is an outstanding issue that conceptual-metaphor theorists need to address.We settled the issue of representation formation within the Gestalt-theoretical framework by providing evidence for independent effects of motion type, proximity, and similarity. In particular, we determined that any of these dimensions is a cue to visual dependency hence each of them can define the number of Gestalts involved in conjunction and in disjunction representations. More importantly, because differences in strength between grouping principles  follow the rules of the human visual system, reasoners may be unaware of the differences and commonalities between these principles or indeed of the fact that conjunction and disjunction representations are Gestalt-like. Evidence from optical illusions supports the idea that people are unaware of the workings of the visual system. In particular, humans may unduly perceive contrasts between stimuli properties in certain contexts despite being unable to objectively measure them. Subsequent studies will shed more light on Gestalt awareness and on further characteristics of conjunction and disjunction simulations beyond the number of their constitutive elements. Part of our investigation will be to determine whether rich Gestalt simulations are instantly triggered as people process coordination descriptions. If so, we should be able to elicit perceptual compatibility effects not only in reasoning tasks but also in indirect tasks, where individuals are not asked to judge on the validity of conjunction and disjunction descriptions.


Classical swine fever (CSF) is among the most detrimental diseases for the swine industry worldwide. Infected wild boar populations can play a crucial role in CSF epidemiology and controlling wild reservoirs is of utmost importance for preventing domestic outbreaks. Oral mass vaccination (OMV) has been implemented to control CSF in wild boars and limit the spill over to domestic pigs. This retrospective overview of vaccination experiences illustrates the potential for that option. The C-strain live vaccine was confirmed to be highly efficacious and palatable baits were developed for oral delivery in free ranging wild boars. The first field trials were performed in Germany in the 1990’s and allowed deploying oral baits at a large scale. The delivery process was further improved during the 2000’s among different European countries. Optimal deployment has to be early regarding disease emergence and correctly designed regarding the landscape structure and the natural food sources that can compete with oral baits. OMV deployment is also highly dependent on a local veterinary support working closely with hunters, wildlife and forestry agencies. Vaccination has been the most efficient strategy for CSF control in free ranging wild boar when vaccination is wide spread and lasting for a sufficient period of time. Alternative disease control strategies such as intensified hunting or creating physical boundaries such as fences have been, in contrast, seldom satisfactory and reliable. However, monitoring outbreaks has been challenging during and after vaccination deployment since OMV results in a low probability to detect virus-positive animals and the live-vaccine currently available does not allow serological differentiation of infected from vaccinated animals. The development of a new marker vaccine and companion test is thus a promising option for better monitoring outbreaks during OMV deployment as well as help to better determine when to stop vaccination efforts. After rabies in red fox, the use of OMV against CSF in European wild boar can be considered as a second example of successful disease control in wildlife. The 30 years of disease control experience included in this review may provide options for improving future disease management within wild populations. Classical swine fever (CSF) is a major threat to commercial pig production worldwide . This muGenerally, control of wildlife reservoirs is a significant challenge . To combDifferent vaccination approaches for wild boar have been developed and tested, some of them directly under field conditions , others under experimental conditions . Under eHowever, upon implementation of large scale oral CSF vaccination, it was discovered that the vaccination process and design need further improvement and was subsequently revisited. As a consequence, adaptations were introduced in all areas of CSF control in wild boar including the baiting strategy, population management, and surveillance design . This reSeveral CSFV vaccines are available and have been used successfully to control the disease in multiple countries worldwide . The mosThese traditional live attenuated vaccines have been used worldwide in eradication campaigns both intramuscularly (IM) in domestic pigs and in oral bait formulations in wild boar . IM applpestivirus chimera “CP7_E2alf” and flc11, were then compared within an EU-funded research project to decide which would be followed up for licensing. Based on the comparative trial and pre-existing data on safety and efficacy, “CP7_E2alf” was chosen for further assessment and marketing , palatability (taste), and uptake must all be considered. Wild boars are omnivores and consume a wide range of foods, but can have very clear preferences for certain food items such as acorns . During vailable .® company. Once the strategy is officially adopted, many practical problems must be solved before deployment including prerequisite and exhaustive census of the vaccination grounds, organizing the logistics for frozen or cold transportation and storage of several thousands of vaccine-baits within isolated areas, delivering the technical information to hunters and controlling bait distribution and consumption in the field. Stop-hunting 1 week before and during bait distributions has been implemented to avoid animal disturbance and to limit the risk of false PCR-positive results (Table 1). However, many challenges have to be still addressed for improving the baits delivery process, monitoring and efficacy (see further sections).There is always a big gap between the development of vaccination tools by researchers and the deployment of vaccination in the field . First,  results . Vaccine results  (Table 12 in North-eastern France) but vaccination efficacy is strongly influenced by the season and year in relation to natural food competing with feed stations and baits  on feeding grounds. Attempts to distribute baits by aircraft were completed , but arend baits . Thus, iMonitoring CSF outbreaks when vaccinating with the C-strain vaccine has been challenging during the past deployment attempts, since the non-marker vaccine (C-strain) strongly impacts the performance and significance of the diagnostic tools. First, antibodies targeting the C-strain and the wild CSFV strains cannot be differentiated using serological tests . TherefoVaccination success was determined through retrospective studies based on field hunting data collected in Germany, Luxembourg, and France, which showed evidence of a significant increase of seroprevalence up to 60% and a quick decrease of viroprevalence under 1% within vaccinated areas within 1–6 years . Neverth2), which depends on forest extend and structure, has been the main factor influencing outbreaks duration from 1985 up to 2009 , When OMV was performed three times a year, the average outbreaks duration decreased , but at the same time, the average delay between the last viropositive result and the end of restrictions measures increased ; possibly as a result of the confusing effect of OMV on serological and PCR results . Thus, OMV has not reduced the cost of CSF management, but it has been the only strategy preventing outbreaks re-emergence in large connected forested areas in Europe  (Table 1).As previously discussed by rd (Fr)] ; Table 1The epidemiological modeling of wildlife diseases is a tool used to support disease control and mitigation measures. Mathematical models from population ecology demonstrate the principle of OMV in wildlife . The appClassical swine fever virus can be assigned to three genotypes with three to four sub-genotypes each. These genotypes do not translate into serotypes that pose a problem in diagnostics or vaccination. Over the last decade, mainly strains of genotype 2, especially 2.1 and 2.3 were circulating in Europe. In the wild boar, only subtype 2.3 was prevalent  in strains , (iii) h strains . It is n strains .The intrinsic spreading of CSF within natural wild boar populations relies mainly on the forest structure and the presence of physical barriers. Due to the forest habitat of the species, the main factor influencing CSF spread (and persistence) within wild boar populations is the connectivity (∼distance) between neighbor forest patches , 2010. P2) well delimited by physical barriers by establishing hunting restrictions to avoid disease spread . Obviously, CSF control is also dependant on the precautionary measures taken for carcasses control and pig farm biosecurity. An integrated strategy is preferred to a single one to maximize the chance of success and also combining other strategies with vaccination should be considered. It is interesting to note that intensified hunting, feeding bans and fencing were recently re-evaluated as possible management measures for controlling ASF in Europe and were not considered to be adequate since now, given past experience during CSF outbreaks  has to be considered for determination of the infected areas and development of a monitoring scheme. This enables quick and proactive deployment of OMV (24 km buffer area). Second, a multiple-year application of OMV is necessary to prevent CSF re-emergence. Furthermore, OMV does not generate genetic evolution of the virus strains.Oral mass vaccination is costly during and after OMV deployment; carcass monitoring and restrictions last for several years after vaccination due to the confounding effect of non-marker vaccine on surveillance hunting data. Diagnostic tools have to be reliable and adapted for this purpose. Future outbreaks could be addressed and controlled more rapidly using oral marker vaccine  and companion serological tools . Even if the youngest piglets cannot eat the baits, experience of the last years of vaccination showed that they were protected by maternally derived antibodies. Therefore, applying repeated vaccination for adult females could avoid this possible failure in vaccination programs.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Tumour heterogeneity was the topic of the ‘Oncology Days’ series held at the 2015 OECI conference in which experts within the field provided an update on tumour heterogeneity and its relevance in the clinical setting.Here we present a summary of the presentations from the two major sessions of the meeting: clonal heterogeneity and phenotypic heterogeneity. Tumour heterogeneity describes differences between tumours of apparently the same type but in different host backgrounds  and differences between cancer cells within the same tumour . In both cases, this heterogeneity can lead to different responses to therapy.Evidence for intratumoural heterogeneity was described back in the 1980s , 3, but Genetic and epigenetic differences between cancer cells within a tumour might explain why some tumour cells are more aggressive and difficult to eradicate in patients treated with chemotherapy. In fact intratumour heterogeneity may also explain why some patients who initially respond well to a particular drug often relapse and develop new tumours which do not respond to the therapy.Most tumours initiate from a single cell which has acquired driver mutations necessary for malignant transformation. However, additional mutations can be acquired during the neoplastic process leading to genetic diversity within the tumour population i.e. heterogeneity. This genetic diversity is the substrate for adaptation to changing environments and for the development of drug resistance through Darwinian evolution. Recent work from our group and others has shown that genetic intratumour heterogeneity and branched evolution is common in many solid tumour types. These are major hurdles for the development of better treatment strategies and prognostic and predictive biomarkers for personalised cancer medicine approaches.We are interested in the impact of genetic and non-genetic intratumour heterogeneity on disease progression and how systemic therapies reshape cancer genomic landscapes. This should reveal the principles governing cancer evolution and should eventually translate into novel therapeutic strategies which can influence evolutionary trajectories for e.g. to prevent or delay the development of drug resistance. The development of biomarkers which are applicable to heterogeneous cancers, for e.g. through quantification of circulating tumour DNA, is a further priority of the group.Heterogeneity is a problem for treatment as these different clones are usually spatially separated within the tumour and leads to drug resistance.In order to avoid giving patients drugs that have little likelihood of working, we need to think about tumours in a different way. Thinking of the tumour as a heterogeneic unit may improve biomarker accuracy.There are different types of tumour heterogeneity: macroscopic i.e. clinical and ethnic heterogeneity which is very well recognised and widely reported. On a microscopic level, pathologists now recognise different histologic patterns within a single tumour with specific morphological characteristics. These characteristics lead to intertumour heterogeneity, but the same features may also lead to intratumour differences, such as different histological and cytological patterns in the same tumour with different degrees of differentiation with prognostic significance. What is more, it is known that in the same tumour there are different functional areas such as the centre or the infiltrative border of the tumour, also with different expression patterns. However, today tumour heterogeneity is mostly related to molecular differences at the inter- and intratumour level. It is the latter, intratumour heterogeneity (ITH) that can explain acquired resistance to cytotoxic chemotherapy or to new targeted drugs, and it occurs so often in tumours that in most of the cases it is the cause of treatment failure.in-situ methods such as in situ hybridisation (ISH), or immunohistochemistry (IHC), or fluorescence in situ hybridisation (FISH). Also molecular heterogeneity can be homotypic or heterotypic, and in the latter case it important to recognise the histological components of the microenvironment. Hopefully, by comparing what is common and what is different in the various tumour types will lead to specific characteristics which are more frequent at the level of pathology.At the moment, we do not have any precise indication on how to standardise this kind of heterogeneity, but we know that this ITH can be studied by using specific methodologies like single-cell sequencing or next generation sequencing (NGS) analysis from different tumour areas or We have to consider two different situations in which heterogeneity may influence prognosis and patient treatment. The first one is related to the primary tumour acquired after surgery which can give us information about ITH. We know that sometimes the killer clone is a minor one with less aggressive behaviour. For this reason, a qualified analysis of primary tumour subclones would allow a rational treatment to avoid recurrences. The second one is related to patients with advanced cancer or recurrence in which liquid biopsies can be extremely important to try to understand which are the best targets for therapy. ITH indeed is evident in cancer spreading; metastatic lesions are different at different secondary sites resulting in intermetastatic heterogeneity, but it can also be heterogeneous within a single metastasis and it is called intrametastatic heterogeneity. The comparison of molecular characteristics between primary tumours and their corresponding metastatic sites, even synchronous local lymph node metastases, can help to understand heterogeneity and also at the same time the biology of the metastatic process.Variation between primary tumours and metastasis could probably be explained by many factors ranging from a clonal selection in the metastatisation process, to the phenotypic plasticity related to the new microenvironment which can interact with the tumour cells, in a process of ‘inverted’ mesenchymal/epithelial transition. Metastases are supposed to derive from a single cell or subclone, but recently also polyclonal seeding from one metastasis to another was detected.A big improvement in the knowledge of cancer spreading comes from so-called ‘liquid biopsies’. Circulating tumour cells (CTC) and circulating free DNA (cfDNA) are huge contributors in the biological and clinical understanding of cancer recurrences and acquired mechanisms of resistance. In this process, ITH has a central position because low frequency clones can be resistant to therapy and could be the basis of therapy failure. These clones can be detected in blood during the follow-up of treatment outcomes, and the therapy itself represents the selection pressure factor for clonal evolution. Secondary acquired resistance to treatment is related to defined genetic or phenotypic heterogeneous mechanisms that are similar in different tumour types. Today the limits of liquid biopsies are still uncertain and connected with tumour ITH. Cells and DNA in blood can come from different and heterogeneous metastatic sites; sensitivity, and the reproducibility in detecting tumour clonal range still has to be proved.Human tumours are lesions which almost always develop over several years, e.g. for breast cancer it was shown that it needs approximately ten years from the first malignant cell until the lesion is clinically detectable. During this period roughly 109–1012 cell divisions take place which can produce a plethora of genetic alterations resulting in a high number of different cell clones. This is one major mechanism underlying not only intratumour heterogeneity, but also differences among different tumours that originate in the same organ  or epigenome-wide association studies (EWAS), integration of whole proteome or sub-proteome profiles—mass spectrometry, protein arrays, reversed protein arrays (beads) etc. and functional assays. Detection, quantification, and characterisation of methylome patterns and long non-coding RNAs  will complete the approach. These methods will be applied to tissue but also to tumour derived nucleic acids (ctDNA) or proteins in plasma, urine, sputum, and spinal fluid or in CTCs. These approaches are summarised under the term liquid biopsies.Computer-based image analyses to quantify immunohistochemical stains. Because of the fact that the human eye is neither an ideal ‘instrument’ to quantify colour intensity nor to estimate percentage positivity of structures, e.g. nuclei or membrane staining, the inter- and intra-observer variability in the evaluation of therapy relevant structures is high. Digital scanning of slides together with virtual microscopy and intelligent algorithms, e.g. object-based image analyses , will beNGS is becoming a routinely applied method for genetic profiling of malignant tumours in diagnostic molecular pathology. Currently, this may be restricted to so called druggable mutations , i.e. muIntroduction of liquid biopsies for follow-up or the continuous ‘real time’ analysis of heterogeneity that develops under therapy. It is generally known that because of mutational instability of tumour cells and/or drug-induced selection, drug resistance almost always develops under therapy. This process is independent of whether conventional chemotherapy or novel targeted drugs are applied. The ability to monitor the cellular changes and/or clonal evolution of the disease is becoming a novel challenge and may fundamentally influence the course of drug applications.Based on the new possibilities the establishment of molecular staging, follow-up of disease, tracing resistance, predicting therapy will become feasible.However, because of technical and financial limitations it will not be possible to apply all the techniques mentioned above in the foreseeable future in routine diagnostics. In the opinion of the author, three novel approaches exist which will have to be introduced in daily routine procedures as soon as possible.leading to an incomplete histological diagnosis,an incomplete immunoprofiling of malignant tumours which could have consequences for treatment decisions, in particular drug selection, andby over—or underestimation of genetic alterations that in turn could have consequences for (targeted) treatmentIn the daily clinical work, tumour heterogeneity may influence cancer diagnostics byIn histological diagnostics as many specimen regions as economically justifiable  should be evaluated.As many loci as economically justifiable should be used for immunoprofiling and should be complemented by,image analysis to reach a higher degree of reproducibility in tissue feature quantification.Genetic alterations should be investigated in at least several (e.g. n = 5) loci.Molecular profiling by NGS and in future other techniques should become a routine procedure in the diagnostic process of malignant diseases.The most relevant question is how to cope with tumour heterogeneity in clinical practice:in silico.Colorectal cancer (CRC) is a heterogeneous disease, displaying variable molecular marks and clinical behaviour. Recent studies have used gene expression profiles to identify colorectal cancer transcriptional subtypes endowed with distinctive molecular, biological, and clinical features. Among the subtypes, the stem/serrated/mesenchymal (SSM) one is characterised by expression of stem/mesenchymal cell transcripts and poor prognosis. Although it has been suggested that this subtype is comprised of cancer cells that have undergone a switch from an epithelial to a mesenchymal/stem cell–like state, we noted that genes upregulated in this subtype are also prominently expressed by stromal cells. This led us to hypothesise that the SSM transcripts could derive from the tumour microenvironment rather than being intrinsic to cancer cells. To test this hypothesis, we analysed RNA sequencing data from patient-derived xenografts (PDXs) of CRC, where human cancer cells are supported by mouse stroma. By using the mouse read fraction as a proxy for the stromal contribution to the expression of a gene, we found that stromal (mouse) transcripts were much more abundant than CRC epithelial (human) transcripts for the vast majority of the SSM signature genes, indicating that tumour-associated stromal cells are the main driver of the SSM class. Considering only genes not detected as human transcripts in PDXs, we built three expression signatures specifically reporting the abundance of cancer-associated fibroblasts (CAFs), leucocytes, or endothelial cells. In human CRC samples, all stromal signatures were strongly associated with the SSM subtype. A high CAF signature was associated with poor prognosis in untreated CRC patients, while in rectal cancer high stromal signatures jointly predicted radioresistance. Our results show that the distinctive transcriptional and clinical features of the SSM subtype can be ascribed to its particularly abundant stroma. The ambiguity between stromal cells and mesenchymal tumour cells can be addressed through RNA sequencing of xenograft models, where the expression characteristics of the human tumour cells can be distinguished from those of the mouse microenvironment Prostate carcinoma (PCa) is a heterogeneous disease at several levels, of which some were addressed in this presentation, namely heterogeneity in hereditary predisposition, intertumour heterogeneity of gene fusions, intra-tumour heterogeneity of gene fusions, genetic markers of prognosis, and molecular subtyping and targeted therapy.Regarding heterogeneity in inherited predisposition, up to 20% of all prostate cancer patients reporting a positive family history and evidence of the genetic contribution to prostate cancer comes from the fact that the relative risk of prostate cancer increases markedly when the number of affected family members increases, or when the age at onset of the index case decreases. Nevertheless, the identification of highly penetrant genes responsible for the inherited predisposition to prostate cancer has been more difficult than for other types of common cancer, namely those of the breast and colon. We have collected a series of 462 probands with early-onset and/or familial/ hereditary prostate cancer and found a small proportion of the cases that are associated with germline mutations in known genes, namely HOXB13, BRCA2, and MSH2 , 8. OngoTo characterise the intertumour heterogeneity regarding gene fusions in prostate cancer, tumour samples from a cohort of 200 patients with clinically localised PCa consecutively diagnosed and treated with radical prostatectomy were studied. The relative expression level of ETS family genes previously found rearranged in PCa , Ewing sarcoma  or leukaemia (ERG and ETV6), and additional ETS genes found overexpressed (ELK4) or localised between two genes commonly rearranged (ETS2), was analysed with TaqMan low-density arrays, followed with FISH, reverse transcription polymerase chain reaction (RT-PCR), and sequencing analyses in selected cases . This apFinally, we showed evidence of how the molecular heterogeneity of PCa might be explored therapeutically. We recently have shown that gastrin-releasing peptide receptor (GRPR) is an ERG and ETV1 target gene in prostate cancer, using a genome-wide scale and exonlevel expression microarray platform . Our worOne of the most recently described mechanisms of intercellular communication is exosomes. Exosomes are extracellular membrane vesicles of a size range of 50 to 150 nm diameter, protected by a lipid bilayer. During their biogenesis, nucleic acids and proteins are encapsulated into exosomes. Additionally, we have recently demonstrated that exosomes are the carriers of circulating genomic DNA. Exosomes are released into the extracellular space and enter the circulation. All cell types described to date, such as immune cells, platelets or endothelial cells, release exosomes into the blood stream. Exosomes can transfer their cargo to various recipient cells. Recently, exosomes have emerged as putative biological mediators in cancer. This is attributed to the fact that cancer exosomes carry molecular signatures and effectors of the disease, such as mutant oncoproteins, oncogenic transcripts, non-coding RNAS  and double-stranded DNA. Intercellular trafficking of exosomes may contribute to horizontal cellular transformation, phenotypic reprograming, and functional re-education of recipient cells.Exosomes derived from tumours carry molecular and genetic signatures associated with the cells or origin. Melanoma-derived exosomes contain the tumour-associated antigen Mart-1 and tyrosinase-related protein-2 (TYRP2). Exosomes from gastric cancer, breast cancer, and pancreas cancer carry members of the human epidermal growth factor receptor (HER) family. Therefore, we hypothesise that exosomes released by a tumour are heterogeneous and reflect the subpopulations of cancer cells co-existing in the tumour. Their heterogeneity is reflected in the different genetic material they carry that could affect other subpopulations of cells in the tumour and most likely regulate tumour progression and its ability to relapse after treatment by inducing plastic changes into therapy-resistant cells.Routine screening of the general population for a cancer such as pancreatic adenocarcinoma using magnetic resonance imaging (MRI) or computed tomography (CT) would be prohibitively expensive and associated with a high false positive rate. We have recently demonstrated that glypican-1 protein on the surface of tumour-derived exosomes can be used as a biomarker for the early detection of pancreatic cancer. Using mass spectrometry analyses, we identify a cell surface proteoglycan, glypican-1 (GPC1), specifically enriched on cancer-cell-derived exosomes. GPC11 circulating exosomes (crExos) were monitored and isolated using flow cytometry from the serum of patients and mice with cancer. GPC1 crExos were detected in the serum of patients with pancreatic cancer with absolute specificity and sensitivity, distinguishing healthy subjects and patients with a benign pancreatic disease from patients with early- and late-stage pancreatic cancer. Levels of GPC1 crExos correlate with tumour burden and the survival of pre- and postsurgical patients. GPC1 crExos from patients and from mice with spontaneous pancreatic tumours carry specific KRAS mutations and reliably detect pancreatic intraepithelial lesions in mice despite negative signals by MRI. GPC1 crExos may serve as a potential non-invasive diagnostic and screening tool to detect early stages of pancreatic cancer to facilitate possible curative surgical therapy.These results suggest the utility of exosomes in circulation as a detection and monitoring tool for pancreatic cancer, with an emphasis on its application in early detection, is a clinical challenge that remains to be addressed. Therefore, we propose to demonstrate that circulating exosomes-genetic content can be used as a promising non-invasive diagnostic and screening tool to detect early stages of pancreatic cancer that could aid the prospect of curative surgical therapy.Genomic profiling in solid tumour is notoriously challenging because of the presence of contaminating stromal and other kinds of cells. Microdissection is often attempted to enrich for the tumour cell fraction, but is inherently labour intensive and purity is limited especially when the tumour cells are highly intermingled with stromal cells, e.g. inflammatory cells. Also alternative approaches like the fluorescence-based cytometric analysis using fluorescence activated cell sorting (FACS) sorters to enrich tumour cells from disaggregated formalin fixed paraffin-embedded (FFPE) tumour tissues yield low efficiency and purity requiring a large input of cells material for instrument set-up and colour control. However the small sample size, e.g. biopsies, generally available in modern pathology impedes the use of conventional FACS for sorting. We present the results of genetic analysis for cell populations sorted by dieletrophoretic technology with our DEPArray system. This innovative technology solves two pressing problems in preparation of FFPE samples for genomic analysis: small sample size and unwanted admixture of stromal cells and other cell types. The presented procedure leads to robust and reproducible results, making it possible to unambiguously detect the true-positive variants and reliably analyse quantitative trait such as loss of heterozygosity (LoH) and copy number variations (CNVs), which cannot be evaluated precisely in unsorted FFPE samples. On several loci, we detected somatic mutations with 100% variant frequency in sorted tumour cell populations clearly indicating LoH and confirming 100% purity of sorted cells. Moreover, we identified and recovered cell subpopulations of potential epithelial-mesenchymal transition (EMT)-phenotype  exhibiting somatic mutations, different from tumour cells majority, and undetectable in DNA extracted from unsorted FFPE samples.To analyse tumour heterogeneity, the laser capture dissection (LCM) technology has been used in order to extract homogenous tumour cells or areas and apply antibody based studies. In a similar manner, the TMA can be used to extract tumour homogeneous areas from a tissue mass and use the selected areas or cores not only for antibody analysis but also for nucleic acids studies. The advantage in using TMAs is the absence of a laser and the speed in the molecular analysis since a large number of protein targets can be analysed simultaneously.Over the years, the number of applications using the TMA technology has increased, it is being used not only in basic research studies but is slowly paving its way into diagnostics.Various aspects of the TMA platform will be presented and its impact in Cancer Research and Diagnostics Applications. Tissue banks certainly have a central role in the preparation, storage, and distribution of tissue arrays (blocks and slides), and in the management of whole slide HD and TMA spots image.Although several advances have been made to facilitate imaging and archiving of TMA specimens, automatic evaluation and quantitative analysis of TMAs still remains an important challenge for modern investigators.Virtual pathology IT platform, will also be discussed, since it is a fundamental infrastructure in tissue banks since will prepare TMA’s for research, diagnostics, and educational purposes.intertumour (different breast cancers among patients) and intratumour (multifaceted features within a given breast cancer) heterogeneity. Such heterogeneity can be appreciated at different levels, spanning from morphology to gene resolution. Although heterogeneity in breast cancer has been dissected by studies based on single cell analysis, morphology is often neglected. Morphological features define the histological type, which has been known to impact on breast cancer patient survival since the ‘90s [It is well known that breast cancer is characterised by the ‘90s . Breast the ‘90s .Heterogeneity may represent a major issue in the neoadjuvant setting where core biopsies of large lesions are studied to plan the pre-surgery therapy. In this scenario multiple biopsies should be common practice in order to avoid improper sampling and to properly assess not only morphological but most importantly immunophenotypical features . Among tHER-2 positivity is heterogeneous by definition, since we consider a 10% cut-off to assign samples to distinct score categories . Score 0spatial heterogeneity, temporal heterogeneity should also be taken into account, when comparing the immunoprofile of primary versus metastatic lesions: there is indeed evidence to demonstrate that tumours evolve over the course of the disease between the primary tumour and local or distant recurrences [On top of this urrences . For insurrences . Immunopurrences .Taken together the whole panorama of morphological and immunophenotypical features here described should be taken into account when high throughput molecular studies are performed to accurately assess the meaning and clinical impact of heterogeneity in breast cancer.The WHO 2015 Lung Cancer Classification defines the main histological types of pulmonary carcinomas: adenocarcinomas, epidermoid carcinomas, neuroendocrine tumours, large cell carcinomas, adenosquamous carcinomas, sarcomatoid carcinomas, and pleomorphic carcinomas, and other rarer subtypes. Non-small cell lung cance (NSCLC) must include IHC profiling to highlight genetic alterations. Small biopsies can identify CK7/TTF1, CK5.6/p40, and vim/neuroendocrine markers.Heterogeneity is often controlled by driver mutations present i.e. micropapillary (and cribiform/microglandular) pattern relates to metastatic potentiality and vimentin is usually positive in tumour cells; this antibody is also convenient to report sarcomatoid/pleomorphic carcinomas in biopsies.Biopsies are the most reliable tissue to classify and treat pulmonary carcinomas because of the high percentage (70%) of tumours being diagnosed in advanced conditions.Two repair/carcinogenic pools of adult stem cells have been recognised: the TRU –terminal respiratory unit concerning the respiratory bronchiole and adjacent alveolar duct/septae and the adult respiratory epithelium till the TRU.Human induced pluripotent stem cells which have the potential to form a whole embryo have led to a better understanding of repair/ adaptation, and the possibilities of the intermingling of meso/ecto/endoderm in one single cell. After this preliminary study, CK7, CK5.7, TTF1, VIM, CD56, and Ki67 were shown to be sufficient for molecular pathology classification. Similar results have been obtained with chromium treatment of both fibroblasts and normal bronchial cells in contact where epithelial–mesenchymal transition was revealed.Tobacco can cause cellular remodelling and adaptation of the basal cells in both TRU and respiratory epithelium, with consequent hyperplasia of vimentine positive cells, and TTF1 bronchial positive cells. This has been demonstrated in carcinomas arising after molecular transformation of less mature cells resulting in multi-patterned carcinomas and in pleomorphic carcinomas. This approach facilitated pulmonary carcinoma classification in biopsies but does not correlate directly with metastatic potential.the coexistence of TP53/KRAS/CTNNB1/SMAD4/MET mutations with mutated EGFR. This may explain the variable response of the EGFR mutated tumours to the respective inhibitors. Prognosis will soon be explored by liquid biopsy sequencing in parallel with tumour re-biopsing to understand adaption to therapy. Primary lymph node metastasis studies revealed different morphology with the same molecular pathology where signet ring cell adenocarcinoma correlates with ALK fusion gene by FISH.NGS demonstrated Co-existence of driver mutations has been demonstrated and NGS will allow more precise understanding of the clinical implications. When combining IHC and molecular pathology, the following cascades have been demonstrated in pulmonary carcinomas: epidermoid carcinoma–epidermal growth factor receptor (EGFR) and HER-2 polysomy, and CK7/vimentin for EMT non-pure epidermoid carcinomas; bronchial-pulmonary adenocarcinomas -non-smoking female—mut EGFR and excision repair cross complement group 1 (ERCC1) expression; micropapillary pattern with VIM/RB/ERCC1 expression; acinar/BA—lepidic/micropappilary patterns express TTF1, and mutated EGFR.Higher ki67 and APC/ERCC1 expression correlates with stages IIA/IIIA while lower TTF1expression relates with mutated KRAS, mainly in papillary and solid patterns.Endothelial marker expression, CD133, ALDH, VEGFR, PDGFR, and VASH are mainly associated with solid, acinar, and micropapillary patterns.in situ and is characterised by one of the highest load of genetic alterations and mutations of all tumour types. In this type of urothelial carcinomas different histopathological variants exist which are often intermixed and can be regarded as sign of the frequent stem cell character of these tumours. Examples for this are micropapillary, plasmocytoid, nested, or neuroendocrine differentiation. In the last year it has become evident that this type of high-grade urothelial carcinoma shows different intrinsic subtypes which reflect the hallmarks of breast cancer biology. These intrinsic molecular subtypes of urothelial carcinoma differentially express markers of urothelial differentiation.Urothelial carcinoma of the bladder and upper urinary tract is a clinically, histopathologically and molecular heterogeneous disease. There are two types of urothelial carcinoma with distinct clinical and histopathological features. Papillary frequently noninvasive urothelial carcinomas are characterised by frequent recurrences and a low rate of progression to invasive and metastasising disease. This type of urothelial carcinoma shows only very infrequent genetic alterations which are limited to deletions of chromosome 9 and oncogenic driver mutations, like FGFR3 or PIK3Ca inducing proliferation. In contrast the second type of urothelial carcinomas is often characterised by primarily invasive disease, solid growth, poor differentiation, and a rapid progression to muscle invasive and metastasising disease. This type of urothelial carcinoma is developing through dysplasias and carcinoma Basal cell markers like basal cytokeratins and CD44 are detected in the basal type urothelial carcinoma and markers of the luminal umbrella cells like cytokeratin 20 or uroplakin are found in the luminal type of urothelial carcinoma. Interestingly, basal type urothelial carcinoma shows a decreased disease specific and overall survival (OS) in comparison to luminal type of urothelial carcinoma. Both intrinsic subtypes are associated with distinct genomic alterations like FGFR3and TSC1 mutations in the luminal and retinoblastoma (RB) alteration in the basal subtype. Furthermore luminal tumours express high levels of GATA3 and ER similar to luminal breast cancers and basal like tumours display enhanced MYC and E2F3 pathway signatures. The characteristic newly identified luminal and basal urothelial carcinoma subtypes were found in five different studies overall and showed in all studies a stable stratification by gene expression analysis. In a Consensus Meeting in Madrid in March 2015 IHC and RNA expression markers where defined which could be used in clinical studies in the near future. Most interestingly the different tumour types including a third p53-like subtype with an activated wild type p53 gene expression signature show a different response to neoadjuvant chemotherapy with best response in basal-like tumour and very poor response in p53-like tumours.A second important and specific feature of urothelial carcinoma is a large degree of intrapatient heterogeneity. Urothelial carcinoma is frequently a panurothelial disease characterised by preneoplasias, multifocal tumours, and frequent recurrences. It is evident that the urothelium of patients with urothelial carcinoma is characterised by a field cancerisation with a multitude of molecular and histologically dysplastic urothelial changes. Many clonality studies could show that multifocal urothelial carcinomas are oligoclonal with a monoclonal disease because of intraluminal seeding and intraurothelial tumour spread in 80% of patients and the development of different tumour clones because of field cancerisation in 20% of patients. New whole genome sequencing studies identified genomic heterogeneity at a nucleotide and chromosome level in urothelial carcinoma. Two types of urothelial carcinoma with low and high genetic diversity and a high degree of mutational heterogeneity are found. Further studies should include assessment of histological normal but possibly genetically altered urothelial lessons to further define genetic heterogeneity in urothelial carcinomas. The high degree of genomic alterations could be used to develop immune therapies strategies.Biobanks are collections of biological samples and accompanying data which are provided for scientific research. Biobanks for medical research can contribute to heterogeneity in clinical research through introducing varieties in the quality of samples on different levels.Medical research can be very demanding and often uses a variety of very sensitive high throughput tests. The high test sensitivity demands high quality samples and data, where ideally the samples are treated completely the same during the pre-analytical phase. This way, variations in sample quality are excluded and study bias is avoided. Biobank guidelines as well as certification and accreditation help to standardise the biobanks where the samples and data are stored safely and the procedures are completely standardised. Although there is a harmonising effect it leaves the individual biobanks free to choose the methods for collecting the samples.Looking closer at the biobank procedures it is clear that not every step in the pre-analytical phase can be standardised. In case standardisation is not possible, avoidance of the procedure or avoidance by choosing a test insensitive to the introduced variation is recommended. The latter is very common for pathology departments, since FFPE samples have large quality variations, and pathology diagnostic tests are well known to be developed and become insensitive for these variations. If however avoidance is also not possible the variations need to be recorded. Tissues can be preserved in different ways resulting in different qualities and fit for purpose issues. The standard pathology archive stores FFPE tissues, which usually have poor DNA, protein, and RNA quality. On the other hand these samples have the best morphology for microscopic evaluation and are fit for purpose for many available techniques. However, the snap frozen tissues kept at ultra-low temperatures are seen as the golden standard.A good biobank has standard operating procedures and a quality assurance and quality control programme in place and the best have these under an ISO certification/accreditation. Tissue banks are completely integrated in the Pathology department embedded in the health care pathways. In case the department becomes accredited the pathology tissue bank needs to take part because of this integration. This in combination with implementation of guidelines, best practices as well as dedicated and trained personnel helps to rule out bad samples when the study results are poor. This way, better support can be given to help obtain a good study result, which actually starts with good study design involving a multidisciplinary team where needed. Quality variety again becomes an issue when biobanks need to exchange samples in a multi-centre research project. In addition, the variations in patients and treatment contribute largely to these unwanted effects. Benchmarking of biobanks in proficiency testing (PT) and external quality assessmet (EQA) programmes could also help comparison of methods used within the biobanks and the hospitals to treat the patients. Furthermore the stimulation of multidisciplinary research teams for the study design and analysis can be key to tackle this problem.Of course next to quality there are other important issues when managing a biobank. Total sample exchangeability also depends after quality issues on good access rules, taking care of the implementation of the proper legal, ethical and social issues, and data interconnectivity. Sustainability of a biobank is of major importance. Therefore it is important to realise there is an obligation towards the donors and stakeholders of the biobank to stay active and keep the samples available for a very long period. This does not mean a biobank only needs to store samples, ideally the samples need to be given out almost at the same rate as they come in. Best sustainability is reached when the biobank is financially covered by either an institutional budget or a cost recovery business plan. This can be a combination perhaps stimulated here and there with project money. Therefore a biobank must be efficient in its collection strategy. Last but not least, sustainability also knows a social factor where the biobank needs to take good care of its stakeholders (PI’s), donors, and the general public.The scope for molecular analyses in pathology and diagnostics is increasing rapidly: more classes of molecular targets are attracting interest, including epigenetic marks, exosomes, interacting proteins and rare mutant DNA in plasma. More and more examples of each of these categories are subject to study in research and later clinically, and these analyses are being performed on samples from more tissues and taken at more time points for each individual. This trend towards greatly increased molecular analyses has prompted the development of very high-throughput and low cost analytic techniques. The construction of suitable molecular tools for analysis of proteins and nucleic acids is a central aspect of work in my group, and in our spin-out company Olink.in situ in pathological specimens. The assays use antibodies with attached oligonucleotides. Upon binding by a pair of such antibodies to a protein or a pair of interacting proteins the conjugated oligonucleotides can participate in ligation or extension reactions to give rise to amplifiable reporter DNA strands for highly sensitive and specific detection.We have developed proximity ligation assays for high-performance, high-throughput analyses of proteins in solution-phase patient samples, or in situ form of the assay, referred to as in situ PLA or Duolink®, uses localised amplification via a rolling circle mechanism of the reporter DNA strands that form, and it can offer enhanced specificity and sensitivity of protein detection in tissue sections, and analyses of patterns of protein interactions and modifications. Around 1000 users around the world are now using the methods for e.g. to investigate cellular signalling or to develop novel markers of diagnostic utility, and Ola Söderberg with colleagues have applied the technique to define cellular signalling cascades one by one or in low multiplex, but higher throughput assays are still under development.The Another variant of the assay, PEA or Proseek®, is used for detecting proteins in solution phase in samples such as plasma and other body fluids, and in lysates of tissues and even of single cells. This assay is used to measure sets of 92 proteins and four controls in 1 μl sample aliquots with excellent specificity and sensitivity. So far assays for some 800 proteins have been developed. In Uppsala alone, plasma samples from more than 40 000 individuals have been analysed for 3.5 million proteins in only just over one year by Dr Masood Kamali-Moghaddam.Another local colleague, Ulf Gyllensten, used the point, evidence, analysis (PEA) method to measure the dependence of protein levels on genetic background as well as on age, sex, and several more factors. He demonstrated that it was possible to define more narrow normal ranges for many of the proteins, and thus more readily identify significant variation, using these sources of information. Another means to better evaluate protein levels is by investigating trends of increasing or decreasing protein levels in samples from the same individuals over time. In this regard we have found that sets of 96 proteins and controls can be conveniently measured from discs of paper punched out of dried blood spots that can be collected by the donors themselves, sent by mail, and stored at room temperature. This simple technique can form the basis for very inexpensive collections of consecutive samples from the same individuals, and provide a valuable means of identifying and validating protein markers for early detection of disease.Caroline Gallant and Spyros Darmanis have demonstrated in yet unpublished work that the PEA technique permits measurements of sets of 90 or so proteins and similar numbers of transcripts in the same individual cells, to better measure cellular heterogeneity, e.g. in responses to putative therapeutic agents.Finally, the ultimate level of measuring heterogeneity is at the level of single molecules. A newly developed super rolling circle amplication (RCA) technology allows individual detected DNA, RNA, or protein molecules to be finely distinguished in highly specific reactions and to be counted as digital objects for e.g. using regular flow cytometers, as developed by the two PhD students Lei Chen and Johan Björkesten. These techniques open the doors to highly precise quantitation, and they are suitable to identify rare DNA sequence variants in plasma that may reflect the presence of minimal residual malignant disease.This short meeting has posed some very important questions regarding the diagnosis and treatment of tumours which may seem initially to be the same but on further investigation reveal quite different characteristics. In the future it will be important to characterise better intertumoral and intratumoral differences to offer the most appropriate therapy.