A large isoniazid-resistant tuberculosis outbreak centred on London, United Kingdom, has been ongoing since 1995. The aim of this study was to investigate the power and value of whole genome sequencing (WGS) to resolve the transmission network compared to current molecular strain typing approaches, including analysis of intra-host diversity within a specimen, across body sites, and over time, with identification of genetic factors underlying the epidemiological success of this cluster.rpoB mutation associated with rifampicin resistance. Eleven longitudinal pairs of patient pulmonary isolates collected up to 48 mo apart differed from each other by between zero and four SNPs. Extrapulmonary dissemination resulted in acquisition of a SNP in two of five cases. WGS analysis of 27 individual colonies cultured from a single patient specimen revealed ten loci differed amongst them, with a maximum distance between any pair of six SNPs. A limitation of this study, as in previous studies, is that indels and SNPs in repetitive regions were not assessed due to the difficulty in reliably determining this variation.We sequenced 344 outbreak isolates from individual patients collected over 14 y (2 February 1998–22 June 2012). This demonstrated that 96 (27.9%) were indistinguishable, and only one differed from this major clone by more than five single nucleotide polymorphisms (SNPs). The maximum number of SNPs between any pair of isolates was nine SNPs, and the modal distance between isolates was two SNPs. WGS was able to reveal the direction of transmission of tuberculosis in 16 cases within the outbreak (4.7%), including within a multidrug-resistant cluster that carried a rare Our study suggests that (1) certain paradigms need to be revised, such as the 12 SNP distance as the gold standard upper threshold to identify plausible transmissions; (2) WGS technology is helpful to rule out the possibility of direct transmission when isolates are separated by a substantial number of SNPs; (3) the concept of a transmission chain or network may not be useful in institutional or household settings; (4) the practice of isolating single colonies prior to sequencing is likely to lead to an overestimation of the number of SNPs between cases resulting from direct transmission; and (5) despite appreciable genomic diversity within a host, transmission of tuberculosis rarely results in minority variants becoming dominant. Thus, whilst WGS provided some increased resolution over variable number tandem repeat (VNTR)-based clustering, it was insufficient for inferring transmission in the majority of cases. In this retrospective observational study, Francis Drobniewski and colleagues assess the power and value of whole genome sequencing to resolve transmission network of a large tuberculosis outbreak. Whole genome sequencing (WGS) of pathogenic organisms, such as the bacterium that causes tuberculosis, is increasingly used to better understand bacterial diversity, evolution, and drug resistance, and transmission to humans.WGS is believed to be superior in defining the extent and direction of tuberculosis transmission compared to the main existing standard method .Interpretation rules have been proposed using a 12 single nucleotide polymorphisms  distance as the upper threshold to identify plausible transmissions and argue that isolates separated by five or fewer SNPs are likely to have resulted from recent transmission; but the extent of variation amongst different bacteria in the same patient (intra-host variation) and the real value of WGS in analysing larger VNTR-defined outbreaks has received little attention.The study focused on understanding the transmission of tuberculosis strains resistant to one of the main therapeutic drugs (isoniazid) in an outbreak centred on London, England, from 1998 to 2012.WGS was performed on 344 strains, with 96 shown to be indistinguishable, 105 differing by a single SNP, and only one isolate differing from the major clone by more than five SNPs. Overall, only 270 SNPs discriminated isolates within the cluster.WGS was used to investigate intra-host strain variation, demonstrating between zero and three SNPs difference in six pairs of patient isolates obtained from pulmonary sites 9 to 35 mo apart, with up to a seven SNP variation in multiple single bacterial colonies isolated from the same patient.WGS provides increased resolution over VNTR-based clustering, but it is insufficient to fully resolve chains of transmission, as multiple transmission events can occur with no detectable SNP acquisition, and even identical isolate pairs cannot be proven to have resulted from a recent transmission event without supporting epidemiological evidence.Centralized routine WGS of TB strains for routine surveillance/outbreak investigation may be misleading and misdirect public health action without additional epidemiological evidence. RFLP-based typing of isoniazid-monoresistant isolates and, later, routine 24-loci mycobacterial interspersed repetitive units-variable number tandem repeats (MIRU-VNTR) typing showed that by 2008, there were 343 culture-proven cases, 299 (87%) of which were diagnosed in London ..26].inhA promoter, whereas those outside the cluster did not.In order to compare WGS-based clustering with MIRU-VNTR–based clustering, 14 isolates that differed from the canonical E1244 profile at 1 or 2 loci or had incomplete typing data were sequenced. Comparison of these variant isolates with the outbreak sequences showed that the isolates with a polymorphic number of repeats at one locus differed from their closest outbreak sequence by zero to three SNPs and clustered within the outbreak phylogeny, whereas isolates that were polymorphic at two VNTR loci differed by 32–34 SNPs from the closest neighbouring E1244 sequence ; S5 Fig.Spoligotypes derived from sequencing data indicated that a number of isolates differed from the canonical type by the loss of spoligotype spacer 19. The derived profiles were confirmed by laboratory-based typing of representative isolates. Mapping the deletion onto the SNP-derived phylogeny suggests homoplasic loss. However, deletion prior to acquisition of SNPs is a more parsimonious explanation and provides a link between these patients .Outbreak-specific polymorphisms were defined as those present in all E1244 sequences and absent from the three isolates that were separated from the major clone by 32–43 SNPs . ThirteeOutbreak isolates shared a 1 bp deletion within Rv1364c, an anti-sigma factor antagonist , which rM. tuberculosis reported to date. Analysis of 344 isolates, sharing an identical 24-loci VNTR profile molecular type, collected from individual patients over 14 y, revealed that 96 (38%) were indistinguishable, and only one differed by more than five SNPs from this clone. Contrary to that observed in some recent studies, WGS was able to reveal the direction of transmission of tuberculosis in only a small proportion (16/344) of cases within the outbreak. We observed the acquisition and spread of a rare RpoB mutation conferring rifampicin resistance that supported the conclusions of a public health investigation into this MDR cluster [To our knowledge, this is the largest WGS-based study of an outbreak of The relative lack of between-patient diversity compared to within-specimen diversity might suggest that transmission may not involve a narrow evolutionary bottleneck, or that the population typically involves one very abundant clone and multiple rare or minor clones. However, we speculate that where low numbers of bacilli are transmitted, such as during casual contact, variants existing in the source donor population become fixed in the recipient due to the founder effect. Neely et al.  showed tBased on studies in the low burden settings of Oxfordshire and the Midlands in the UK, Walker et al. ,29 propoThe level of diversity observed in this outbreak, averaging 0.8 SNPs per case (270 SNPs/344 isolates) is slightly lower than that found in other similar studies of large community clusters. A Hamburg cluster had an average of 1.0 SNPs per case (86 SNPs/85 isolates) , a ToronlpdA (Rv3303c), encoding a flavoprotein disulfide reductase that has been shown to protect M. tuberculosis from oxidative stress and contribute to enhanced survival in a mouse model [lpdA expression [The VNTR 3690 locus is located upstream of lpdA Rv03c, encopression ; thus, ipression . Rv1364cpression , which ipression .It is remarkable that of the four acquisitions of rifampicin-resistant genotypes within the outbreak reported here  plus two reported previously  ,24, noneOne limitation of the study is that investigation of intra-specimen diversity was only possible for one isolate, and the results may not be more generalizable. However, the data suggest that the practice of isolating single colonies prior to sequencing is likely to lead to an overestimation of the number of SNPs between cases resulting from direct transmission. Our approach, in which the sequence of the patient’s dominant genotype is determined, is preferable. A further limitation is that variation in repetitive regions, amounting to almost 10% of the genome, was not assessed due to the difficulty in reliably calling SNPs within these regions using current technologies (as described in Methods). Similarly, unambiguous data were not obtained for indels. In the future, this problem will be ameliorated by the availability of longer high-quality reads. Development of analysis workflows that incorporate a systematic and reliable analysis of repeat regions, small insertions, and large sequence polymorphisms may reveal some further variation within the outbreak.M. tuberculosis have suggested that the variation in the PE-PPE genes is 3-fold higher than in non-repetitive genes [However, studies have cast doubt on the previously widely held belief that the PE-PPE genes contain a large amount of variation ,37. Althve genes ,39, thisM. tuberculosis lineages and sublineages [WGS studies of large tuberculosis outbreaks, including this one, have typically been directed by VNTR- or RFLP-based clustering of isolates –32. In tlineages , and thiWe conclude that whilst WGS provides increased resolution over VNTR-based clustering, without supporting epidemiological data, WGS is insufficient to resolve transmission networks in tuberculosis outbreaks. Furthermore, the data suggest that the concept of a transmission chain or network may not be useful in institutional or household settings. The practice of applying a fixed SNP distance alone to identify plausible transmissions needs to be considered within the context of the specific population. WGS technology remains useful in ruling out the possibility of direct transmission when isolates are separated by a substantial number of SNPs. Although the value of WGS in defining drug resistance and identifying compensatory mutations was supported in this study, the real value of wide-scale investment in WGS to understand tuberculosis transmission in the context of public health action needs to be very carefully considered in the light of the above findings.S1 FigMST of 344 isolates from individual patients. Colour coding of nodes indicates the year of isolation. Each isolate is represented by a separate segment except for the central major clone, for which the number of isolates from each time period is given. Branches coloured red highlight links where a case can be linked to a single source case. The inferred node, indicated by the red unfilled circle, is occupied by the second isolate of a patient series.(EPS)Click here for additional data file.S2 Fig(a) MST of 344 isolates from individual patients. Colour coding of nodes indicates the year of isolation. Each isolate is represented by a separate segment except for the central major clone, for which the number of isolates from each time period is given. (b) Histogram illustrating pairwise SNP distances between isolates. (c) Graph showing date of specimen collection against its SNP distance from the major clone.(EPS)Click here for additional data file.S3 Fig2 marks the case suspected to be a cross-contamination from case x1.Nodes represent the 60 sequenced isolates shown in (EPS)Click here for additional data file.S4 FigLine graphs, generated by the bam-viewer Artemis, show the number of sequencing reads mapping to the H37Rv reference genome, which has two copies of the 3690 repeat.(EPS)Click here for additional data file.S5 FigNodes of isolates that differ from the E1244 VNTR profile or the canonical LAM10 spoligotype are coloured. Branch lengths are logarithmically scaled.(EPS)Click here for additional data file.S1 Text(PDF)Click here for additional data file.


While several clinical prediction rules (CPRs) of survival exist for patients with symptomatic spinal metastasis (SSM), these have variable prognostic ability and there is no recognized CPR for health related quality of life (HRQoL). We undertook a critical appraisal of the literature to identify key preoperative prognostic factors of clinical outcomes in patients with SSM who were treated surgically. The results of this study could be used to modify existing or develop new CPRs.Seven electronic databases were searched (1990–2015), without language restriction, to identify studies that performed multivariate analysis of preoperative predictors of survival, neurological, functional and HRQoL outcomes in surgical patients with SSM. Individual studies were assessed for class of evidence. The strength of the overall body of evidence was evaluated using GRADE for each predictor.very low for 39 and low for 7 predictors. Due to considerable heterogeneity in patient samples and prognostic factors investigated as well as several methodological issues, our results had a moderately high risk of bias and were difficult to interpret.Among 4,818 unique citations, 17 were included; all were in English, rated Class III and focused on survival, revealing a total of 46 predictors. The strength of the overall body of evidence was low. We failed to identify studies that evaluated preoperative prognostic factors for neurological, functional, or HRQoL outcomes in surgical patients with SSM. We formulated methodological recommendations for prognostic studies to promote acquiring high-quality evidence to better estimate predictor effect sizes to improve patient education, surgical decision-making and development of CPRs.The quality of evidence for predictors of survival was, at best,  Symptomatic spinal metastasis (SSM) afflicts up to 10% of cancer patients, of whicSince the randomized controlled trial conducted by Patchell et al. showing Clinical prediction rules (CPRs), which combine various clinical factors from an individual with a given health state and provide an estimate of the risk of experiencing a specific endpoint within a certain period, may allThe majority of published series assessed preoperative predictors of survival rather than HRQoL. We conducted a systematic review to ascertain the preoperative prognostic factors for 1) survival, 2) neurologic status, 3) functional status, and 4) HRQoL in surgical SSM patients. We also appraised the methodology and reporting of prognostic studies that met our eligibility criteria. The results of this study could not only be used to modify existing CPRs of survival to improve their prognostic value, but also to improve the theoretical framework to develop new CPRs for survival and HRQoL outcomes specific to surgical SSM patients.th, 2015 and was last updated on July 12th, 2016 (registration number CRD42015023831).This systematic review and best-evidence synthesis was conducted and reported in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. In compIn adult patients who underwent surgery for SSM, we sought to answer the following four key questions (KQs): What are the preoperative clinical factors associated with postoperative (1) survival; (2) neurologic status, such as muscle power on the Medical Research Council (MRC) scale for testing muscle strength, neurologic outcome measures  or Frankel grade) or autonomic functions (bladder / bowel control); (3) functional status, in terms of ambulatory status, functional outcome measures, such as functional independence measure (FIM), Barthel index, Eastern Cooperative Oncology Group (ECOG) or Karnofsky performance status (KPS); and (4) HRQoL, in terms of score on any HRQoL measure, such as short form health survey (SF-36), EuroQol 5 dimensions (EQ-5D) or Oswestry Disability Index (ODI)?The electronic databases MEDLINE, MEDLINE in Process, Embase, Web of Science, CINAHL, Cochrane Central Register of Controlled Trials, and Scopus were systematically searched for studies performed in humans from January 1, 1990 to December 31, 2015 with no language restrictions applied. The search strategies were developed in consultation with information specialists at the University Health Network Health Sciences Libraries. a priori determined inclusion and exclusion criteria  independently (1) screened the titles and abstracts to identify potential eligible studies to undergo full-text assessment and then (2) reviewed the selected full-text articles for final inclusion. Discrepancies between the two reviewers were resolved by consensus agreement; persisting disagreements were settled by consulting the senior author (MGF).The following data were extracted by AN and then checked by ARM: 1) first author and publication date; 2) publication language; 3) study design; 4) purpose; 5) patient sample and characteristics, with relevant inclusion and exclusion criteria; 6) preoperative predictors 7) outcome assessed; 8) postoperative follow-up characteristics, including length, rate, and information about how missing data were handled; 9) methodology, including details related to predictors’ selection, type of univariate and multivariate analyses conducted, multivariate modeling process and assumption(s) testing; and 10) univariate and multivariate estimates, including reported odds / hazard ratios and confidence intervals. Unless otherwise specified, a p-value < 0.05 was considered statistically significant.We are not aware of any consensus regarding a standardized approach for assessing the quality of prognostic studies.AN and ARM independently assessed the risk of bias of individual articles (Class I to IV) using the method described by Skelly et al., 33 for baseline strength of the overall body of evidence was assigned “High” if the majority of the studies were Class I or II and “Low” if the majority of the studies were Class III or IV. The strength could then be downgraded by one or two levels based on the risk of bias, consistency, directness, precision and publication bias. Alternatively, the strength could be upgraded by one or two levels if the effect was large, there was evidence of a dose response gradient or all plausible confounders would either reduce a demonstrated effect or would suggest a spurious effect when the results showed no effect. The final strength of the overall body of evidence for each predictor was classified as High, Moderate, Low or Very Low and expresses our confidence that the evidence reflects the true effect and the likelihood of further research to change our confidence in the latter estimate of effect  Working Group. The basef effect . OverallThe search yielded 4,818 unique citations, of which the title and abstract were screened, leading to the selection of 152 articles for full-text review. Among these, a total of 135 studies were excluded for one of the following reasons: preoperative prognostic factors were not evaluated or were assessed as part of a scoring system and not evaluated individually; multivariate analysis was not conducted; multivariate analysis included surgical and/or postoperative factors as predictors; the journal was not peer-reviewed at the time of publication on Ulrichsweb; surgicaAll 17 articles meeting our eligibility criteria were published in English and addressed KQ1, i.e. the preoperative clinical factors associated with survival in surgical SSM patients. There were six additional studies that examined the clinical prognostic factors of functional status (KQ3) in terms of the ability to walk–41 or reAll 17 studies investigated the prediction of survival, pursuing one or more of the following aims: to evaluate (1) predictors of survival; (2) predictors of survival and propose a prognostic model or a scoring system to predict survival; (3) the prognostic value of parameters included in an existing scoring system that predict survival. Ten studies examined aim #1–52, inclProspective prognostic studies meeting the following criteria for a good-quality cohort study are considered Class I evidence: (1) patients were followed for sufficient periods in order that outcomes could occur, (2) follow-up rate was ≥ 80%, (3) patients were at similar point in the course of their disease, and (4) the study accounted for other prognostic factors . AlthougA summary of the 17 studies is presented in Prognostic factors for survival varied substantially according to primary tumor types, with negative relationship as follows: breast cancer with shorter time interval from cancer diagnosis to SSM surgery, emergency hospital admission, primary tumor with poor/undifferentiated histologic grade and negative progesterone receptors; HHC witn divided by 10. In addition, six studies[All 17 studies used the Cox proportional hazards (PH) regression method for their multivariate survival analysis. Five studies, 47, 49 x studies, 53–56 ix studies, 55, 56 x studies, 53–57 dLow for preoperative prognostic factors of survival in studies that considered all sites of primary tumors including or excluding MM. When MM was included, only radioresistant site of primary tumor had a final strength of evidence of Low. All other predictors related to the site of primary tumor were downgraded to Very low due to high risk of bias related to inconsistency of results. Non-ambulatory status and CCIS ≥ 2 maintained a final strength of evidence of Low while the remaining seven predictors were downgraded to Very low for at least high risk of bias. The final strength of evidence for the three predictors of survival for all primaries excluding MM was also Very low for at least high risk of bias . A total of 20 factors were identified, among which 11 were related to the site/histology of the primary tumor, 56, 57. of bias .Low strength of evidence at baseline and their respective final strength of evidence was Very low due to high risk of bias , neurologic (KQ2), functional (KQ3), and HRQoL (KQ4) outcomes in patients with SSM who underwent surgical treatment. This systematic review identified 17 studies related to our KQ1 that conducted multivariate analysis and reported a total of 46 preoperative prognostic factors of survival in surgical SSM patients. All 17 prognostic studies were rated as having a moderately high risk of bias (Class III evidence). The final strength of the overall body of evidence was graded In spite of performing a literature search designed to maximize sensitivity, this review was only able to identify studies addressing KQ1. Six studies examined the clinical prognostic factors of functional status (KQ3) and one study isolated predictors of HRQoL (KQ4), but these studies were excluded because they included surgical and/or postoperative predictors. Inclusion of such predictors in the multivariate analysis runs the risk of precluding relevant preoperative predictors from either being selected in the final model or showing statistical significance. Also, final models that retain surgical/postoperative predictors are not relevant in the preoperative period, which is the critical time-point for clinical decision-making. Therefore, while this review had limited success in establishing preoperative prognostic factors of survival, it also highlights the dearth of evidence related to predictors of neurologic, functional, and HRQoL outcomes in surgical SSM patients.Due to the nature of cohort studies, prognostic data from these will be biased and may not be generalizable to patients with spinal metastases. Cohort studies are often limited by cost and timescales, and significant losses to follow-up. Spinal centers may cover large geographical areas, and patients may be transferred elsewhere for subsequent oncological treatments. Failure to return for spinal clinic follow-up at prearranged appointment times may be due to travel constraints, the patient may be too unwell, undergoing other treatments, or they may prefer to be reviewed by local oncologists instead.In addition, survival analyses are inherently complex, and our attempt to synthesize this group of 17 such studies was challenging due to considerable heterogeneity in patient samples and prognostic factors investigated. Furthermore, the design and reporting of the statistical analyses were problematic in many studies, leading to a moderately high risk of bias and difficulty interpreting the results. Since multivariate techniques applied to systematically collected data from a specific patient population may improve clinical prediction by identifying key prognostic factors, 60 and Prognostic studies should be designed and conducted to minimize potential biases related to six domains. (1) StudWhile there is no well recognized CPR for HRQoL, the variable prognostic ability of current CPRs of survival–26 in tha priori eligibility criteria were relatively narrow in their requirement and may have excluded studies that produced pertinent findings.The systematic literature review was conducted in accordance with the PRISMA guidelines. We assessed the quality of the studies and evaluated the strength of the overall body of evidence for each preoperative predictor identified through our sensitive and rigorous literature review. However, this review aimed to identify predictors of a wide range of outcomes, combining the results of studies with substantial heterogeneity in the prognostic factors, outcome measures, and patient populations that were assessed, which may constitute a problem with internal validity. Furthermore, our In predicting future outcomes by using patient data available at presentation, there will always be a degree of randomness or “chaos” in the system affecting clinical outcomes and survival. AlthoughLife expectancy and HRQoL are cornerstones to clinical decision-making in surgical SSM patients. Based on the results of 17 pertinent studies, this systematic review found a low overall strength of evidence for seven preoperative predictors of survival and very low strength evidence for 39 additional predictors. Consequently, we have low confidence that the evidence reflects the true effect size of these predictors. Furthermore, no evidence was found for the prediction of neurologic, functional, and HRQoL outcomes. Further rigorously conducted prospective studies are needed to better understand what preoperative factors are prognostic of these various outcomes, for the purpose of surgical decision-making, development of CPRs, patient education and levering treatment expectations. Genetic analysis of tumor subtypes will also need to be included in future prediction models, since novel chemotherapies and immunotherapies are showing promising influence on survival and HRQoL.S1 Checklist(DOC)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.


Every tissue in the body critically depends on meeting its energetic demands with sufficient oxygen supply. Oxygen supply/demand imbalances underlie the diseases that inflict the greatest socio-economic burden globally. The purpose of this review is to examine how hyperpolarized contrast media, used in combination with MR data acquisition methods, may advance our ability to assess oxygen metabolism non-invasively and thus improve management of clinical disease. We first introduce the concept of hyperpolarization and how hyperpolarized contrast media have been practically implemented to achieve translational and clinical research. We will then analyse how incorporating hyperpolarized contrast media could enable realization of unmet technical needs in clinical practice. We will focus on imaging cardiac and renal oxygen metabolism, as both organs have unique physiological demands to satisfy their requirements for tissue oxygenation, their dysfunction plays a fundamental role in society’s most prevalent diseases, and each organ presents unique imaging challenges. It is our aim that this review attracts a multi-disciplinary audience and sparks collaborations that utilize an exciting, emergent technology to advance our ability to treat patients adversely affected by an oxygen supply/demand mismatch. Oxygen must be delivered in sufficient amounts to a given tissue via blood flow and perfusion (oxygen supply).Oxygen must be extracted efficiently from haemoglobin in the blood to the cells where it is required (oxygen availability).Oxygen must be utilized in combination with the biochemical machinery in place and energetic substrate to produce ATP (oxygen consumption).Oxygen metabolism – the delivery of oxygen to tissues for the purpose of facilitating efficient breakdown of energetic substrates into ATP – is fundamental for the healthy function of all tissues in the body. For efficient oxygen metabolism to take place, a number of steps must occur continuously :Oxygen The outcome of successful oxygen metabolism in an organ is ample energy to power all of that organ’s specific physiological functions.As the anatomy and energetic requirements of each organ in the body vary widely, the physiological processes driving oxygen metabolism also vary among organs. What is true throughout the body, however, is that a defect in oxygen metabolism that prevents an organ from generating sufficient ATP probably is a cause or consequence of clinical disease. Imbalances in oxygen supply and demand underlie the two clusters of diseases that inflict the greatest socio-economic burden globally: cardiovascular diseases (particularly ischaemic heart disease) and diabetes . For exa15O-water considered an ideal diffusible agent to measure perfusion pyruvate, [2-13C]pyruvate, 13C-urea and fumarate most frequently because they polarize efficiently, retain hyperpolarization for a relatively long time  and probe unique aspects of cellular uptake and metabolism that are disturbed in disease.Theoretically, the DNP technique can be applied to a wide range of molecules labelled with  and use . Overall13C]pyruvate for infusion into patients  using their SpinLabs to generate sterile hyperpolarized pyruvate to simultaneously achieve metabolic and perfusion imaging in the absence and presence of hyperaemic stress (18F-FDG for metabolism and 13N-ammonia for perfusion and scan times approaching 1 h) [13C]lactate production could emerge as a novel marker of the functional significance of a perfusion defect: if oxygen supply is reduced to a level low enough that it affects myocardial substrate metabolism by enhancing glycolysis, that would be the ultimate indication that reduced tissue perfusion has affected cardiac physiology. Measuring stress-inducible [1-13C]lactate production in the context of perfusion mapping is poised to offer a new strategy for guiding treatment decisions in atypical patients: angina in the absence of obstructive CAD and conditions in which balanced hypoperfusion and microvascular dysfunction are purported to play a role.In the rodent heart, Lau et al.  demonstring 1 h) . Further13C-labelled 2-methylpropan-2-ol was hyperpolarized using DNP cyclopropane-d8, an efficiently polarized exogenous compound with a long T1 of 95 s ex vivo, 32 s in vivo at 3 T) have been used to demonstrate perfusion in liver and kidney tissue in mice with high spatial resolution (0.038 cm3 voxel size) pyruvate to interrogate the balance among the major ATP-producing pathways: anaerobic glycolysis, glucose oxidation and fatty acid oxidation acetate acetate .in vivo, the resultant dehydroxyascorbate/ascorbate (DHA/VitC) ratio appears to depend on redox-coupled (via the NADPH oxidases) glutathione activity [13C]lactate labelling following infusion of hyperpolarized [1-13C]pyruvate itself has indexed availability of cytosolic reducing equivalents (i.e. NADH) [13C]lactate-to-[1-13C]pyruvate conversion is sensitive to modulation of intracellular ROS themselves pyruvate through the PDC to form 13CO2 and 13C-bicarbonate, the first non-invasive measurement which has been confirmed to demonstrate the in vivo operation of the glucose–fatty acid cycle pyruvate into [1-13C]lactate has been shown to reflect an increased reliance on glycolytic ATP production in anaerobic states (hypoxia and/or ischaemia) or diseases in which glycolysis and glucose oxidation are uncoupled [13C]lactate has shown promise in delineating damaged, yet viable tissue in infarction models [13C]lactate production may be a biomarker of chronic, low-grade hypoxia and/or inflammation that characterize immunometabolic disorders such as HFpEF. Modelling the uptake and metabolism of hyperpolarized glucose pyruvate is used as a metabolic tracer, the 13C label is retained within acetyl-CoA rather than being released as 13CO2, enabling downstream metabolic steps to be visualized  in diabetic kidneys inspiring reduced oxygen levels typical of high altitude [13C]lactate and 13C-bicarbonate production), suggesting that poor glycaemic control places an additional burden to maintain renal energy metabolism and oxygenation without affecting the metabolic phenotype [13C]lactate production was elevated 24 h after reperfusion to a degree correlating with reductions in kidney function, indicating that the hyperpolarized metric could offer an imaging biomarker of tubular injury in the renal cortex pyruvate and [2-13C]dehydroxyacetone, may also clarify the kidney’s role in gluconeogenesis in various pathophysiological states. The kidney is still often overlooked as an important gluconeogenic organ, with primary focus on the liver. However, awareness of renal mechanisms of glucose homoeostasis is likely to increase in the near future because novel glucose-lowering drugs are being developed that inhibit the renal sodium–glucose co-transporter 2 (SGLT2) [13C]dehydroxyacetone enters gluconeogenesis at the level of the trioses [13C]pyruvate in the fasted and diabetic kidneys and liver, increased production of the gluconeogenic precursor lactate has been observed. This occurred in concert with increased expression of phosphoenolpyruvate carboxykinase (PEPCK), a key regulator of gluconeogenesis [13C]lactate production following infusion of hyperpolarized [1-13C]pyruvate was detected in both heart and kidney. Expression of the genes encoding regulatory enzymes PEPCK (Pck1) and glucose-6-phosphatase (G6pc) were both increased in kidney whereas only Pck1 expression was increased in heart, a non-gluconeogenic organ. Both of these sets of results suggested that hyperpolarized 13C MR may be valuable in assessing systemic processes (exemplified by gluconeogenesis) in which various organs have distinct roles urea may have a key role in the development of this technique, due to the multiple order-of-magnitude increases in T2 conferred by the 15N label. The lengthened T2 translated into enhanced signal in an SSFP acquisition, with flip angles approaching the fully refocused regime lactate production with measurements of tissue oxygenation could clarify the existence of an oxygenation reserve to buffer myocardial tissue against temporary oxygen supply/demand imbalances.There is no question that BOLD-MRI measurements of oxygenation in kidney and heart, limited as they are, have made a significant impact on clinical research and raised fascinating technical and clinical questions. In the kidney, for example, BOLD-MRI has confirmed abnormal oxygen supply and/or oxygenation occurs in unilateral ureter obstruction ,122, tis13C]pyruvate, and to assess/confirm their sterility, are being performed on an ongoing basis and for the longer term. Projecting forward one year, it is reasonable to expect that >10 sites worldwide will be performing clinical hyperpolarized MR studies with [1-13C]pyruvate in cancer and oxygen metabolism applications combined, and the growth rate of the field is set to increase further as the technology required to produce sterile hyperpolarized agents becomes simpler, more robust and in turn more affordable. The major MR scanner vendors have adapted their products to facilitate the addition of hardware and software tailored to the 13C nucleus, and the researchers who have led the way in developing custom MRI and MRS pulse sequences for hyperpolarized applications have been extremely generous in assisting with their implementation across imaging platforms.The technical advancements to the clinical hyperpolarizer (SpinLab) hardware that will be required for high-throughput patient studies are actively underway. Improvements in the ability to consistently produce sterile doses of hyperpolarized pyruvate [13C-α-ketoglutarate to target aberrant accumulation of the oncometabolite 2-hydroxyglutarate in isocitrate dehydrogenase-1 mutant glioma [13C2]fumarate to detect necrosis in vivo . Th. Th13C]pFinally, the most compelling results to emerge from hyperpolarized MR may be on the horizon: for in oxidative tissues, many of the clinical questions for which the technology is best suited to address can only truly be evaluated in patients. Experimental models of myocardial infarction as well as acute kidney injury, for example, mimic ischaemia and reperfusion but cannot reproducibly take into account confounding factors that occur in patients: mechanical crushing and fragmentation of the culprit lesion upon percutaneous coronary intervention (PCI), no-reflow phenomenon, subtleties in arterial and structural anatomy and the occurrence of co-morbidities such as age, hypertension and metabolic syndrome. To our knowledge no animal models are widely accepted for chronic angina or angina in the absence of CAD, and HFpEF models that reproduce the diverse characteristics of the patient population are notoriously difficult to achieve. In HFpEF and other immunometabolic diseases that have become a worldwide epidemic, the unique ability of hyperpolarized MR to simultaneously measure metabolism among multiple organs and elucidate coupling of their functions could extract information currently unavailable . FurtherEvery tissue in the body critically depends on meeting its energetic demands with sufficient oxygen supply, and conversely, oxygen supply/demand imbalances underlie the diseases that inflict the greatest socio-economic burden globally. The maturation of hyperpolarized MR has provided the foundation for new non-invasive methods to measure tissue oxygen metabolism as part of a ‘one-stop shop’ evaluation of organ structure and function. Pre-clinical studies have indicated that by using hyperpolarized tracers with advanced MRI and MRS pulse sequences, it may be possible to measure blood supply, mechanisms of oxidative metabolism and tissue oxygenation rapidly and robustly, with high signal generated by safe contrast. We expect the impact to be greatest in assessing the heart and the kidney, organs in which oxygen requirements are particularly stringent and which present imaging challenges that have not been met by current technology. The recent translation of hyperpolarized MR methods into the clinic will hasten the technological refinements and patient research required to cement its role as a clinical tool for assessment of oxygen metabolism.


Esophageal cancer (EC) is the eighth most common cancer and is the sixth leading cause of death worldwide. The incidence of histologic subtypes of EC, esophageal adenocarcinoma (EAC) and esophageal squamous carcinoma (ESCC), display considerable geographic variation. EAC arises from metaplastic Barrett’s esophagus (BE) in the context of chronic inflammation secondary to exposure to acid and bile. The main risk factors for developing ESCC are cigarette smoking and alcohol consumption. The main somatic genetic abnormalities showed a different genetic landscape in EAC compared to ESCC. EAC is a heterogeneous cancer dominated by copy number alterations, a high mutational burden, co-amplification of receptor tyrosine kinase, frequent TP53 mutations. The cellular origins of BE and EAC are still not understood: animal models supported a cellular origin either from stem cells located in the basal layer of esophageal epithelium or from progenitors present in the cardia region. Many studies support the existence of cancer stem cells (CSCs) able to initiate and maintain EAC or ESCC. The exact identification of these CSCs, as well as their role in the pathogenesis of EAC and ESCC remain still to be demonstrated. The reviewed studies suggest that current molecular and cellular characterization of EAC and ESCC should serve as background for development of new treatment strategies. Esophageal cancer represents the sixth most common cause of cancer death and the eighth in incidence worldwide. In fact, it accounts for about 4% of cancer diagnoses and for 6% of cancer deaths. The prognosis for esophageal carcinoma is poor, with a 5-year survival rate of 19% and only 0.9% for advanced esophageal carcinoma. The incidence and histologic subtypes of esophageal cancer display a considerable geographic variation. Esophageal squamous cell carcinoma (ESCC) is the most frequent esophageal cancer in the world, with the highest incidence in eastern Asia and parts of Africa. The major risk factors for ESCC are represented by tobacco and alcohol consumption, but also other environmental factors play a role in the development of this cancer, such as the consumption of hot beverages, nutritional deficiencies and limited intake of fruits and vegetables ,3. In coMost patients with an esophageal cancer have a disease at an advanced stage and, therefore, are not eligible for curative therapy or develop tumor recurrence, in spite treated with a curative intent. The treatment of esophageal cancer is mainly based on surgery. Radiotherapy, chemotherapy or chemoradiotherapy in both EAC and ESCC are of limited efficacy; therefore, there is an absolute need for an improvement of medical treatment through the identification of new therapeutic targets ,3.Developments in high-throughput genomic technologies have led to a better understanding of the disease heterogeneity and of the molecular basis underlying the development of EAC and ESCC . These sIn spite of the efforts to molecularly characterize esophageal cancer have led to the identification of patient subgroups who may potentially benefit from target therapies, the therapeutic success so far obtained was very limited. Thus, only the targeting of HER2-positive tumors was associated with some clinical efficacy ,6, whileThe present review will analyze the recent developments in our understanding of the molecular  and cellular (tumor initiating cells) basis underlying EAC and ESCC. Only an integrated genomic and cellular characterization at the level of single esophageal cancer, considering the major driver mutations, the tumor heterogeneity and the major biochemical pathways sustaining tumor survival and proliferation, may led to the identification of clinically suitable biomarkers and to drive the development of new multitargeting therapeutic approaches.The molecular alterations occurring in esophageal cancer have recently been explored in detail. In these studies, emphasis was given on determining the differences existing in the two major esophageal subtypes ,3,4. TP5A very recent study provided a detailed whole genome sequencing analysis of EACs with the molecular characterization of 129 cases, showing that EAC is a heterogeneous cancer dominated by copy number alterations with frequent large-scale rearrangements . ImportaThe use of neo-adjuvant chemotherapy to shrink tumors before surgery offers the unique opportunity to compare the evolution of cancers that respond well and poorly to this treatment. Findlay and coworkers observed that the response of EAC genome to neo-adjuvant chemotherapy greatly varies: a group of poor responders EAC display only minor genomic changes following treatment; another group of patients displays multiple genetic driver mutations that variably increase or decrease in frequency following treatment, sometimes showing complete loss or gain; finally, a third group of patients was marked by clonal shifts, reminiscent of genetic bottlenecking . In thisIt is important to note that Ras mutations are rare in esophageal cancer. Initial studies have highlighted the low frequency (<5%) of K-Ras mutations in both esophageal adenocarcinomas and squamous carcinomas, while a high frequency (around 40%) was observed in colon cancer . These iHelicobacter pylori Cag A seronegativity account for the large majority (about 75%–80%) of esophageal adenocarcinomas. However, there is evidence that in addition to the factors, also genetic factors play an important role in the genesis of esophageal adenocarcinoma and of its precursor lesions. Familial studies have suggested the existence of a common genetic background when a relative is affected by either chronic gastro-esophageal reflux or Barrett’s esophagus or esophageal adenocarcinoma (a 2–4-fold increased risk when a relative is affected); furthermore, twin studies have indicated a moderate heritability of gastro-esophageal reflux disease. Segregation analyses of many pedigrees of familial Barrett’s esophagus supports an incompletely dominant inheritance model with a polygenic component. These observations have stimulated the genesis of wide association genetic studies on Barrett’s disease. These studies have led to the identification of some genetic loci, associated with an increased risk of developing Barrett’s disease. The first study identified two regions associated with disease risk: (a) chromosome 6p21 involving the major histocompatibility locus; (b) chromosome 16q24, involving FOXF1, a gene involved in esophageal development and structure [As stated above, observational studies have indicated that a number of factors, including chronic gastro-esophageal reflux, cigarette smoking, obesity and tructure . A more tructure . Genome tructure . The loctructure .The presence in some individuals who develop EAC of a premalignant lesion offers a unique opportunity for genetic studies aiming to elucidate the evolution of genetic alterations occurring during the development of esophageal cancer. Barrett’s esophagus is the premalignant condition associated with the development of EAC and its study and characterization at cellular and molecular level is essential for a better understanding of the mechanisms responsible for EAC development. At histological level, Barrett’s esophagus is characterized by the replacement of the normal squamous epithelium of distal esophagus with columnar epithelium. Barrett’s esophagus progresses to EAC through intermediate histological stages: Barrett’s esophagus, low-grade dysplasia (LGD), high-grade dysplasia (HGD), EAC. Three types of non-dysplastic Barrett’s esophagus have been reported: with gastric metaplasia and length <3 cm; with intestinal metaplasia and length <3 cm; with intestinal metaplasia and length >3 cm. Barrett’s esophagus confers an absolute risk of progression to EAC of about 0.5 per patient per year; LGD is associated with a progression risk to HGD or EAC of about 9%–13% per patient per year; finally, HGD has a 25% risk of progress to EAC .Studies on the transition of Barrett’s esophagus to EAC have initially focused on the alterations of p16 and TP53 genes. According to these results, two models were proposed. One model proposed by Maley and coworkers suggests that an initial mutation (most commonly inactivation of p16) confers a selective advantage to a cell population and this mutation is present in most of cells of Barrett’s esophagus; the acquisition of additional mutations  give rise to cell clones able to expand across the Barrett’s lesion . LeedhamIn this context, Agrawal and coworkers have performed exome sequencing on 11 EAC samples and 2 samples of Barrett’s esophagus adjacent to the cancer; surprisingly, most of mutations were found to be present even in the Barrett’s esophagus samples . More reThe recent development of more sensitive sequencing techniques performed on small pieces of tumoral tissue allowed a more detailed comparison of the mutational spectrum of paired Barrett’s esophagus-EAC samples showing that surprisingly a relatively low degree of overlapping (<20%) was observed in most of cases; furthermore, the Barrett’s esophagus samples that had the best overlap with their paired EAC samples are those histologically classified as dysplastic . These sAnother study published in parallel carried out on 25 pairs of Barrett’s esophagus/EAC confirmed that the number of focal deletions and amplifications clearly increased during progression from Barrett’s esophagus without dysplasia, to Barrett’s esophagus with dysplasia and then to EAC . InteresLi and coworkers have carried out a genetic analysis of a group of patients with Barrett’s esophagus studied in the time: the large majority (>95%) of these patients do not progress to EAC during their lifetimes . The genIn conclusion, the recent studies on the characterization of Barrett’s esophagus have provided evidence that this lesion is not simply a metaplastic tissue, but a pre-cancerous tissue, characterized by frequent somatic genetic alterations predisposing, in some cases, to cancer progression. Some of these molecular abnormalities can be used to predict the risk of Barrett’s esophagus progression to dysplasia first and then to cancer . The molVarious genetic mutations have been identified in esophageal squamous cell cancers and many of them are associated with specific cellular pathways, such as cell cycle, apoptosis, DNA repair mechanisms, growth factor receptors. Recent studies have suggested that a major impact in this area could derive from comparative studies allowing a comparison of the mutational profile of ESCC, compared to EAC. In this context, particularly interesting are the results reported by Agrawal and coworkers . These aMore recently, Chen and coworkers explored the occurrence and the possible functional implications of NOTCH 1 mutations and NOTCH pathway mutations in ESCC cancer development and progression . These aAn involvement of the NOTCH pathway in ESCC is also supported by recent studies showing a need for NOTCH signaling in esophageal epithelial homeostasis; the crosstalk of NOTCH1 and NOTCH3 is required for squamous differentiation . Other sEGFR is overexpressed at protein level in about 50% of ESCCs and in about 30% of cases this gene is amplified ; interesGenes involved in the control of cell-cycle are frequently altered in ESCCs. Thus, a loss of heterozigosity of the Rb gene, associated with low/absent Rb expression, was observed in >50% of ESCC samples . P16INK4In initial studies mutations of the PI3KCA gene, which encodes the p110alpha catalytic subunit of PI3K have been reported in 2%–12% of ESCC patients. Recently, the occurrence and the prognostic impact of PI3KCA mutations was analyzed in many ESCC patients, showing that PI3KCA mutations were detected in 21% of patients and, compared with wild-type PI3KCA patients, these patients displayed a better prognosis, as analyzed in disease-free survival and overall survival . PI3KCA Another frequent genetic abnormality observed in ESCC is represented by the overexpression of the transcription factor SOX2. A copy gain number of SOX2 gene was observed in 15% of ESCC patients and SOX2 protein was overexpressed in 70% of ESCC tumors . The incThree recent studies have provided a global characterization of the molecular abnormalities occurring is squamous esophageal cancer. A first study by Lin and coworkers provided evidence about the recurrent mutation of TP53, PIK3CA, NOTCH1, FAT1, FAT2, ZNF750 and KTM2D genes in Chinese ESCC primary tumor samples . These mDeletions and translocations are the dominant structural variation types observed in ESCCs, and 16% of these deletions were complex deletions. The structural variations frequently led to disruption of cancer-associated genes  with different mutational mechanisms. Moreover, Chromotripsis, kataegis, and breakage-fusion-bridge (BFB) were identified as contributing to locally miss-arranged chromosomes that occurred in 55% of ESCCs. These genomic catastrophes led to amplification of oncogene through chromotripsis-derived double-minute chromosome formation  or BFB-affected chromosomes , with approximately 30% of ESCCs harboring BFB-derived CCND1 amplification . FurtherThe large majority of molecular studies on the characterization of genomic abnormalities of ESCCs have been carried out in Chinese patients. A recent study reported a wide exome sequencing and SNP array-based copy number analysis on 144 Japanese patients . A high Recent studies of characterization of the genomic alterations occurring in ESCC have led to the identification of mutational signatures associated with peculiar pathogenic mechanisms. Thus, Chang and coworkers have performed a whole-genome sequencing analysis of DNA and RNA in 94 Chinese patients with ESCC . ThroughSurprisingly, tumor heterogeneity in ESCC is not well studied. Cao and coworkers profiled the mutations and copy number alterations that were identified in multiple regions within an ESCC from two patients . The aveIt is important to note that the incidence of ESCC is particularly high in China and it was estimated that approximately half of the world’s new ESCC cases each year occur in China. This finding has prompted specific studies aiming to identify some genetic factors present in this population that could facilitate the development of ESCC. The screening of large numbers of ESCC patients and of control healthy individuals allowed the identification of some polymorphic alleles as ESCC susceptibility alleles .The comparative analysis of somatic copy number alterations in the adenocarcinomas of the esophagus, stomach and colon provided interesting data about the differential molecular mechanisms responsible for the genesis of these tumors. Thus, Dulak and coworkers observed a higher number of focal amplifications in the upper gastrointestinal adenocarcinomas (esophagus and stomach), compared to colon/rectal cancers . To explSome recent studies have comparatively analyzed the genomic profiling of EAC and ESCC showing similarities, but also several remarkable differences. At the level of genetic alterations observed in single biochemical pathways, some pathways were similarly affected (cell cycle and epigenetic regulations); other pathways such as ERBB, RAS/RAF/MEK and TGF-β signaling were less mutated in ESCC than in EAC; finally, other pathways such as KEAP1/NRF2, NOTCH, FGF and PI3K/AKT/MTOR signaling were more mutated in ESCC than in EAC . At the The Cancer Genome Atlas Research Network recently reported an integrated genomic characterization of esophageal carcinoma. Independent and integrated analysis of SNCA, DNA methylation, mRNA and miRNA expression profile allowed a clear separation between EAC and ESCC, thus indicating that they are really two distinct tumor entities. At the gene expression level, EAC showed increased E-cadherin signaling and upregulation of ARF6 and FOXA pathways, regulating E-Cadherin; E SCCs were characterized by upregulation of Wnt, syndecan and p63 pathways required for squamous cell differentiation . SNCAs rIn parallel to studies aiming to define genetic alterations present in esophageal cancers, other studies attempted to identify gene expression profiling in EAC and ESCC allowing to identify patient’s subgroups with different prognostic significance and predictive of response to therapy. However, the numerous studies until now performed were heterogeneous in their methodology and in their results and have been unable to define a repeatedly identified gene signature with clinical relevance . In spitSome single nucleotide polymorphisms consistently increase the risk of developing ESCC. Genome-wide association studies have identified two SNPs, rs671 in ALDH2 on 4q23 and rs1229984 in ALDH1B on 12q24 that are clearly associated with the risk of developing ESCC in a manner related with alcohol drinking and tobacco smoking status ,75. ChanRecent gene expression studies have suggested new classification criteria for ESCC. Thus, Xiong and coworkers, using the specific 185-gene signature generated by unsupervised consensus clustering of gene expression data, defined four subtypes of ESCC: tumors with high metastasis, associated with EMT , poor differentiation, active MAPK4/JNK signaling pathway and poor prognosis; tumors with high chromosomal instability and high expression of MYC-regulated targets; well differentiated tumors, with less aggressive invasivity and better prognosis; a group of tumors with intermediate properties and moderate prognosis .Finally, a recent study compared the gene expression profiles of the two major histological subtypes of solid tumors (adenocarcinomas and squamous cell carcinomas) across organs, with a focus on esophagus, lung and uterine cervix, showing that histology-related differences accounted for a more consistent degree of inherent molecular variation in the tumors that did the of origin . InteresThe esophagus is derived from the anterior portion of the foregut, a structure giving rise also to trachea, lung and stomach. Multiple signaling systems  and transcription factors (SOX2) are required for esophagus development. These factors play an essential role in the induction of differentiation of primitive esophageal epithelium undergoing a transition from a simple columnar to stratified squamous epithelium .The normal esophageal epithelium exhibits three compartments: (i) a superficial squamous cell compartment; (ii) a differentiating suprabasal cell compartment, containing proliferating cells at various stages of differentiation; (iii) a proliferative basal cell compartment, in which stem cells and proliferating progenitors are in contact with the basement membrane. Basal cells progressively differentiate and migrate in an outward direction to the lumen and move from initial proliferative stages to terminal non-proliferating differentiation stages. It is unclear whether all basal cells have an equal stem cell capacity of self-renew or whether the basal cells are organized according to a hierarchical pattern, in a stem cell-transit amplifying cell population.It is important to recall that the esophagus possesses submucosal glands composed by a single mucus-producing acinus that continues into developed ducts opening onto the surface epithelium. Most of these ducts is composed by columnar epithelium and the last distal, near to the surface, by squamous epithelium. Barrett’s metaplasia consists in the replacement of the normal squamous epithelium with a columnar-lined, mucus-secreting, epithelium.−) [+ cells results in reduced proliferation and epithelial atrophy [Before analyzing the few data available about the cancer stem cells of esophageal cancer, it is important to briefly discuss the problem of the identification of normal esophageal stem cells. Esophageal epithelium consists of layers of keratinocytes and lacks structures such as crypts, which at the level of the intestinal epithelium form stem cell niches. In this epithelium, the proliferation is confined at the level of the basal layer. Various studies have provided evidence about the existence in the basal layer of murine esophageal epithelium of cells exhibiting properties of stem cells. Kalabis and coworkers, using Hoechst dye extrusion and BrdU label-retaining assays identified in mice a putative stem cell population localized at the level of the basal layer . The sel−) . The act−) . Genetic atrophy .NTR, the low-affinity neutrophin receptor: the cells positive for this receptor are slowly cycling both in vitro and in vivo, but when grown in vitro generate a population of highly proliferating beta1-integrin positive cells that give rise to all distinguishable keratinocyte subsets [The study of human esophageal stem cells remains problematic and very few reports have addressed this important issue. The epithelial basal layer of the human esophagus consists of two distinct zones, one overlying the papillae of the supporting connective tissue and the other covering the interpapillary zone. Proliferating cells are rare in the interpapillary zone, where rare putative stem cells undergo asymmetric divisions, giving rise to one basal daughter and one suprabasal, differentiated daughter . Okumura subsets . However subsets . The mai subsets . These L subsets .Using 3D imaging, coupled with staining for a range of cell lineage markers, Barbera and coworkers have investigated the patterns of proliferation and mitosis in human esophageal epithelium. Using this approach several interesting conclusions have been reached: (i) the most quiescent cells expressing putative stem cell markers are located at the tip of the papillae; (ii) asymmetric divisions, which are a typical hallmark of stem cells, are not restricted to a specific cell compartment; (iii) cells at various stages of differentiation sorted according to the expression of progenitor cell markers have equal capacity for self-renewal and ability to reconstitute a squamous 3D architecture in vitro .The factors controlling the proliferation and differentiation of esophageal stem cells are poorly known. In this context, a recent study provided evidence that NOTCH signaling is essential for the differentiation of these cells. In fact, the introduction of a NOTCH mutant causing inhibition of the NOTCH signaling at the level of the esophageal basal cells promotes a suppression of differentiative mitosis of these cells, thus inducing their expansion as undifferentiated cells and the formation of clones expanding and progressively replacing the entire epithelium . AnalysiThe frequency of progression of Barrett’s esophagus to EAC is low. Two recent studies have provided an accurate evaluation of the frequency of malignant progression of Barrett’s esophagus: in a first study, 11,028 patients with Barrett’s esophagus have been analyzed in the time for 5.2 years showing an incidence rate of adenocarcinoma of 12 cases per 1000 per year, which corresponds to a risk of developing EAC 11.3 higher than in the general population  ; in a seAs stated above, the large majority (>95%) of subjects with Barrett’s esophagus do not progress to esophageal adenocarcinoma during their lifetimes, while a minority of these patients develop esophageal adenocarcinoma. Using a longitudinal case-cohort study, somatic chromosomal alterations have been explored in two groups of Barrett’s esophagus patients, one not-progressing and the other progressing to EAC . The gen+ cells) correlated with a high tumor score [Animal models to study Barrett’s metaplasia were mainly based on a rat model involving esophago-jejunostomy that induces gastro-duodenal reflux. Reflux injury in the esophagus results from chronic inflammation, associated with production of cytokines, such as IL-1beta, IL-6 and IL-8, all contributing to metaplastic and dysplastic conversion. These observations have represented the basis for the development of a model of Barrett’s-like metaplasia based on the overexpression of IL-1beta in the mouse esophagus . IL-1betor score . In a suor score . Low levor score . These f+ cells, and their metaplastic descendants into the esophagus. The role of inflammatory cytokines in the development of Barrett’s esophagus and in its progression to EAC is supported also by studies on patients with Barrett’s esophagus. These studies were based on the use of non-steroid anti-inflammatory drugs (NSAIDs) as agents capable of reducing the risk of cancer development. The action mechanism of these agents is largely unknown, but it is reasonable to assume that these agents act by lowering the development of somatic genetic abnormalities. This hypothesis was directly evaluated by studying the effect of NSAIDs administration on the number of somatic genetic abnormalities in patients with Barrett’s esophagus: the evaluation in the time of these patients showed that those receiving NSAIDs developed significantly less somatic genetic abnormalities than those not treated with these drugs [The inflammatory cytokines revealed at the level of the inflammatory esophagus resulted in a stimulation for the migration of cardia progenitor cells, including LGR5se drugs .A recent study provided some evidence about the progenitor responsible for the development of metaplasia and for a novel mechanism responsible for the generation of metaplasia, apparently not necessarily involving genetic mutations. In fact, according to the studies carried out in a peculiar murine system it was provided evidence that the earliest events that lead to metaplasia development do not depend on genetic changes, but rather on competition between cell lineages for access to basement membrane, a site essential for cell proliferation . This coThus, according to these studies, it must be concluded that the conversion of normal esophageal epithelium to intestinal metaplasia is not well understood and it unclear whether the metaplastic epithelium is derived from the normal esophageal epithelium or arises from gastric cardia stem cells or from remnant embryonic epithelial cells that persist at the gastro-esophageal junction.Studies on the origin of human Barrett’s metaplasia have shown a more complex pattern of cellular origin compared to those described through the study of animal models. Leedham and coworkers have provided evidence, through the analysis of tumor suppressor loss of heterozygosity at the level of individual crypts, that the ducts could represent the source of the Barrett’s metaplasia, as supported by the finding that normal squamous ducts contained the same somatic mutation as the contiguous metaplastic epithelium . This anThe factors that favor the metaplastic change of esophageal epithelium into columnar epithelium are largely unknown. Several signaling pathways and transcription factors have been potentially involved in the pathogenesis of Barrett’s metaplasia, for their role in the development and homeostasis of the normal gastro-intestinal system. A squamous epithelium may be induced to develop a columnar phenotype through either the decreased expression of squamous cell transcription factors (such as p61 or SOX2) or increased expression of columnar cell transcription factors . This hypothesis is supported by the observation that decreased expression of squamous cell transcription factors and increased expression of columnar cell transcription factors is observed at the level of Barrett’s esophagus tissue . During The origin of Barrett’s esophagus was recently explored by a combination of molecular studies and by the development of a new technology of isolation of intestinal stem cells derived from various areas of the human intestine. This new methodology of cultivation of human intestinal stem cells supports the maintenance of human intestinal stem cells in a highly clonogenic, ground state form ; this meIn conclusion, the studies carried out to define the cellular origin of Barrett’s esophagus have led to various hypotheses, indicating different cellular sources: progenitor cells located in the esophagus , columnar progenitors located in the squamo-columnar junction or in gastric cardia, migrating in the esophagus. Whatever the cellular origin is, the cells involved in the formation of Barrett’s esophagus must undergo a complex process of molecular reprogramming to generate a specialized intestinal metaplastic tissue. Some transcription factors play an essential role in the transdifferentiation/transcommitment processes. NTR or CD271) [+ cells isolated from ESCC cell lines display properties of cancer stem cells greatly stimulated the study of CD44 expression at the level of both normal and tumor esophageal tissues showing that: (a) in normal esophageal tissue CD44 expression was limited at the level of the basal compartment containing putative stem cells; (b) 70% of ESCC specimens exhibited a pronounced expression of CD44, with various patterns of positivity [Few studies have characterized cancer stem cells at the level of esophageal cancers . In thisr CD271) ,106 dispsitivity . Interessitivity .+ cells isolated from these cell lines have increased tumorigenic activity and are more chemoresistant. Interestingly, knockdown of ICAM-1 expression in esophageal cancer cell lines determines a concomitant upmodulation of CD44 expression; the same applies for CD44 in that CD44 downmodulation induces a concomitant ICAM-2 upmodulation [A more recent study showed that ICAM-1 is a potential membrane stem cell marker in ERSCC cancer cell lines. ICAM-1dulation . Accordidulation .NTR as a marker of tumor-initiating cells it is important to note that initial studies have shown that the low-affinity neurotrophin receptor p75 (NTR) characterizes human keratinocyte stem cells in vitro [NTR was expressed at the level of cellular elements present in the basal layer of the normal esophageal tissue [NTR expression was increased in ECs and the pattern of positivity at tissue level varied according to the degree of tumor differentiation [Concerning the identification of p75in vitro . P75NTR l tissue ; p75NTR ntiation .+ cells were isolated from these tumor specimens and were shown to exhibit an enhanced capacity to initiate tumor formation into immunodeficient mice, self-renew, differentiate and are resistant to standard cytotoxic drugs [+ cells were shown to possess a high metastatic potential in vivo [A recent study showed that CD90 is commonly and moderately overexpressed in ESCC clinical specimens . These fic drugs ; further in vivo . The met in vivo .+ cells is significantly associated with poor prognosis, poor differentiation and lymph node metastasis [+ cells display enhanced stemness features, associated with enhanced self-renewal capacities and resistance to chemotherapy. At functional level, ITGA7 regulates cancer stem cell properties through FAK-mediated signaling pathways triggered by ligand-integrin interaction [A recent study showed that integrin α7 (ITGA7) is a functional marker of ESCC cancer stem cells. The frequency of ITGA7tastasis . ITGA7+ eraction .+-ATPase (playing a key role in the control of intracellular pH); both these transporters are over-expressed in ESCC and their expression was associated with pathological grade [Another cell surface marker potentially identifying esophageal cancer stem cells is ABCG2, a member of group G in the ATP-binding cassette (ABC) transporter family. ABCG2 is expressed in the apical membrane of esophageal epithelial cells. ABCG2 contributes to the efflux of many chemotherapeutic drugs and is expressed together with vacuolar-Hal grade . ABCG2 eal grade .Other studies have further attempted to characterize populations of cells enriched in CSCs isolated from esophageal cancer cell lines. In this context, Zhang and coworkers have isolated tumorspheres from the Eca109 human EC cell line and have shown that these cells are enriched in Aldehyde Dehydrogenase positive cells and expressed various stem cell markers, such as Nanog and Oct4 . A parti+ cells isolated from the EC109 cell line [+ cells display molecular properties indicating an epithelial-mesenchymal transition [Yang and coworkers confirmed the stem-like properties of ALDH1Aell line . Taking ell line . Finallyansition .+ CSCs [These conclusions were confirmed in additional studies performed in EAC patients. Thus, Ajani and coworkers showed that ALDH1 expression correlated with the presence of CSCs in EAC patients and predicted complete tumor responses (CTRs): thus, patients with CTRs exhibited very low ALDH1 levels, while patients resistant to chemo-radiotherapy treatment display high ALDH1 levels and had higher incidence of tumor relapse and cancer-related deaths . Further+ CSCs ; in fact+ CSCs .A recent study explored the expression of the stem cell-associated intermediate filament nestin in primary EC specimens and showed that positive tumors had significantly shorter median survival than those with nestin-negative tumors; furthermore, nestin phenotype correlated with the expression of proliferation-associated markers in tumor specimens .Esophageal cancer stem cells could be involved in the mechanism of tumor metastasis. In line with this hypothesis, Chen and coworkers observed higher levels of Placental Growth Factor (PLGF) and Metalloproteinase (MMP9) in metastatic than in non-metastatic esophageal cancer . Using a+/CD44+ cells was observed [Some studies have investigated the sensitivity of esophageal cancer stem cells to radiation therapy. In this context, in an initial study Zhang and coworkers have shown that some radiation-resistant tumor cells exhibited characteristics of cancer stem cells after fractionated irradiation of esophageal cancer cells . The radobserved .As above mentioned, NOTCH signaling is frequently activated in EACs. A recent study provided evidence that NOTCH activity is required for stemness and tumorigenicity of EAC. Several lines of evidence support this view: NOTCH signaling is greater in less differentiated EACs than in differentiated EACs; NOTCH activity is required for tumor growth in esophageal adenocarcinoma xenograft models; the growth and the expression of CSC markers is inhibited in esophageal adenocarcinoma cell spheres; esophageal adenocarcinoma CSC population is more sensitive to inhibition of NOTCH than bulk tumor cells .+esophageal cancer cells though TGF-β [The study of animal models of esophageal cancer helped also to identify and characterize cancer stem cells. Thus, studies aiming to define the oncogenic role of Acylglycerol Kinase (AGK) in squamous epithelium, provided evidence that AGK overexpression induced constitutive JAK2/STAT3 activation, promoting the expansion of a population of cells with stem cell features, including CD44 expression, SP and expression of pluripotency-associated markers such as SOX2, OCT4, NANOG, BMI1, ABCG2 . Anothergh TGF-β . Epithelgh TGF-β .highALDH1A+ cells exhibiting cancer stem-like properties [The Wnt/β-catenin pathway is an important regulator of esophageal CSCs. The aberrant β-catenin expression and consequent Wnt pathway activation are determinants of ESCC cancer progression, invasion and metastasis. Two recent studies support a role for Wnt/β-catenin pathway activation in the maintenance of CSCs in ESCC. Thus, in a first study it was reported that miR-942 expression promotes ESCC stem cell traits by activating the Wnt/β-catenin pathway through a targeting effect on the inhibitors of this pathway  . miR-942operties . In suppoperties .In parallel with the study and characterization of EC stem like cells, the development of clinically relevant xenograft models is of fundamental importance. However, very few studies have reported the successful xenotransplantation of primary tumor cells issued either from EAC or ESCC. Initial studies have reported the sporadic successful establishment of primary xenografts injecting primary tumor cells derived from EAC or ESCC into nude mice ,131,132.The progress in molecular characterization of esophageal cancer provided fundamental data supporting that the histological subtypes EAC and ESCC are two distinct entities for all the molecular features analyzed. Although many of the same pathways are somatically altered in EACs and ESCCs, the specific genes affected are different, indicating distinct physiopathologic mechanisms and suggesting the need for different therapeutic approaches. For their molecular features, EACs mostly resemble the chromosomal instability subtype of gastric cancer, while ESCCs resemble other squamous carcinomas. These observations indicate that in studies of neoadjuvant, adjuvant or systemic therapies, EACs and ESCCs must be considered separate entities and cannot be combined. According to these molecular findings, mouse models suggest that Barrett’s esophagus and EAC may originate from proximal gastric cells or embryonic remnant cells located at the gastro-esophageal junction.On the other hand, the molecular studies indicate also that the genetic alterations observed in esophageal cancers are multiple and complex and a targeting of a single druggable genetic abnormality in this tumor is not sufficient to achieve the disease control. An additional problem is related to intra-tumor heterogeneity of both EACs and ESCCs, which fosters tumor evolution, and is a key challenge in response to drug treatment.The cellular studies have suggested the existence of a rare population of immature cancer cells that initiate and maintain the neoplastic process. These cells display a high metastatic potential and resistance to conventional anti-cancer therapy.The actual standard of care for patients with stages II-III EC involves chemotherapy, or chemoradiation either in the neoadjuvant or adjuvant setting of surgical resections of esophagus. Targeted therapies (anti-receptor tyrosine-protein kinase Erb-2 (HER2) or anti-Epidermal growth factor receptor (EGFR)) are sometimes used in concert with chemoradiation. However, the success of this therapeutic approach is limited in that the average 5-year survival of EC is less than 18%. In this context, given the progress in the understanding of the molecular abnormalities of EACs and ESCCs, it is expected the rapid development of a novel arsenal of therapeutic options in esophageal cancers. In parallel, oncologists, surgeons, pathologists and scientists can contribute to optimize the first-line therapy of ECs to try to improve survival rates. A straight-forward solution to this problem would imply a utilization in clinical studies of molecular classification and diagnostics of ECs, with identification of new therapeutic targets.


There is increasing evidence that these presynaptic Gi/o-coupled GPCRs have important roles in the actions of drugs of abuse, as well as behaviors related to these drugs. This topic will be reviewed, with particular emphasis on receptors for three neurotransmitters, Dopamine , Endocannabinoids  and glutamate (group II metabotropic glutamate (mGlu) receptors). The focus is on recent evidence from laboratory animal models (and some evidence in humans) implicating these receptors in the acute and chronic effects of numerous abused drugs, as well as in the control of drug seeking and taking. The ability of drugs targeting these receptors to modify drug seeking behavior has raised the possibility of using compounds targeting these receptors for addiction pharmacotherapy. This topic is also discussed, with emphasis on development of mGlu2 positive allosteric modulators (PAMs).Drug abuse and addiction cause widespread social and public health problems, and the neurobiology underlying drug actions and drug use and abuse is an area of intensive research. Drugs of abuse alter synaptic transmission, and these actions contribute to acute intoxication as well as the chronic effects of abused substances. Transmission at most mammalian synapses involves neurotransmitter activation of two receptor subtypes, ligand-gated ion channels that mediate fast synaptic responses and G protein-coupled receptors (GPCRs) that have slower neuromodulatory actions. The GPCRs represent a large proportion of neurotransmitter receptors involved in almost all facets of nervous system function. In addition, these receptors are targets for many pharmacotherapeutic agents. Drugs of abuse directly or indirectly affect neuromodulation mediated by GPCRs, with important consequences for intoxication, drug taking and responses to prolonged drug exposure, withdrawal and addiction. Among the GPCRs are several subtypes involved in presynaptic inhibition, most of which are coupled to the G Drug and alcohol use disorders are prominent neuropsychiatric conditions that create substantial economic, health and societal costs. Substance abuse definitions have evolved over time, but key features include relapse to drug use even after prolonged abstinence, escalation of drug intake, tolerance to the effects of drugs, craving, and continued use despite adverse consequences to health, financial status and relationships  are a large class of metabotropic receptors for neurotransmitters and hormones that couple to heterotrimeric G proteins. G proteins mediate a wide variety of cellular functions, including, but not limited to, altering the production of second messengers such as cyclic adenosine monophosphate (cAMP), mobilizing internal calcium stores, modulating ion channel function, altering neurotransmitter release, and influencing gene expression. Upon activation of a GPCR, the heterotrimeric G protein dissociates into α and βγ subunits, which then modulate the function of a diverse array of effector proteins to simultaneously influence many cellular functions  and operant drug self-administration have been used to evaluate the multifaceted relationships between presynaptic GPCRs and drugs of abuse  with the D1 and −5 subtypes generally coupled to Gs/olf G proteins, and the D2, −3 and −4 coupled to Gi/o . All of these drugs increase extracellular DA in the dorsal and ventral striatum , the major psychoactive ingredient in preparations of Cannabis sativa, which acts as a partial agonist at these receptors. The two major eCBs with synaptic actions are 2-arachidonoyl glycerol (2-AG) and arachidonoyl ethanolamide  are lipid metabolites that act as juxtacrine and paracrine modulators throughout the nervous system and body. Within the brain, eCBs produce their actions predominantly through activation of CB1, a class A GPCR that is almost exclusively localized to presynaptic terminals. The eCB name is obviously derived from the term cannabis, and this is due to the fact that CB1 is also the primary CNS molecular target for ΔA “retrograde” signaling mechanism in which eCBs are released from postsynaptic elements and act on presynaptic CB1 receptors, appears to be the predominant means for this neuromodulatory intercellular signaling. Both short- and long-term synaptic depression result from eCB signaling  couple to Gq family G proteins and are predominantly located postsynaptically, whereas the group II (mGlu2 and mGlu3) and group III  mGlu receptors couple to Gi/o family G proteins and frequently act as presynaptic auto- and heteroreceptors. Although the group III mGlu receptors, particularly mGlu7, have received attention in the addiction field, the current review will focus on the preclinical evidence that group II mGlu receptors (mGlu2/3) modulate the actions of drugs of abuse and represent potential therapeutic targets for the treatment of drug use disorders. Group II mGlu receptors are widely expressed in brain regions relevant to drug-related behaviors. Although physiological actions of postsynaptic group II mGlu receptors have been identified  are a group of class C GPCRs comprised of eight subtypes that are divided into three subgroups based on sequence homology, G protein-coupling specificity, and ligand-binding profiles  was recently identified in multiple lines of rats that are commonly employed in alcohol studies. Selectively bred alcohol-preferring (P) rats, which consume large amounts of alcohol and display relapse-like behaviors, are homozygous for Grm2 * 407. Intercrossing studies suggest that this allele is directly linked to increased alcohol consumption and preference in P rats  in various addiction-related circuits regulate drug seeking elicited by different stimuli. However, this hypothesis has not been exhaustively tested and may also vary between drugs of abuse.Additional evidence implicates group II mGlu receptors in both the VTA and the CeA as targets for blocking reinstatement of drug seeking. Infusion of LY379268 into the rat VTA reduces context-induced reinstatement of heroin seeking  for the treatment of drug abuse in humans; however, the common finding that lower doses of agonist are required to reduce drug self-administration vs. food self-administration suggests that a sufficient therapeutic window may exist to avoid adverse effects. Moreover, clinical studies of the mGlu2/3 agonist prodrug LY2142003 in schizophrenic patients suggest that activation of mGlu2/3 is generally safe and well-tolerated  and/or inhibit reinstatement of extinguished food seeking in both rats and squirrel monkeys. However, this effect is typically less robust than reduction of drug self-administration and is observed with higher doses of agonist than the minimal effective dose required to reduce drug self-administration  binding site of mGlu receptors have produced amino acid analogs, which often do not possess pharmacokinetic properties suitable for human dosing . Thus, PAMs of mGlu receptors can increase the potency of glutamate, as well as increase the maximal efficacy of receptor activation in the context of low receptor expression levels. Because allosteric binding sites are less conserved among GPCR families, many drug discovery campaigns have successfully created subtype-selective ligands  and JNJ-40411813 , have been evaluated in small clinical trials (Cross, 2 PAMs into efficacy in human populations. Interestingly, clinical studies using NAC have shown a modest ability to promote abstinence from cocaine, cannabis, and nicotine (reviewed in McClure et al., 2 activity could reduce drug seeking in humans. Additional clinical trials examining the ability of drugs targeting mGlu2 to prevent relapse and reduce the use of a variety of drugs will be necessary to more thoroughly interrogate the specific human addictive behaviors that are most likely to be impacted by mGlu2 activation.Recent progress towards the development of clinically useful drugs targeting mGlus Cross, . To dates Cross, . The U.Si/o-coupled GPCRs as mediators of the effects of abused drugs, substrates for neuroadaptations that impact behavior, and promising therapeutic targets for reducing drug intake and preventing relapse. These receptors include the three examples discussed above; however, this list is by no means exhaustive. For example, mu opioid receptors are important mediators of the rewarding effects of opiates such as heroin, and also undergo adaptations in function in response to drug exposure (Atwood et al., 7 have been the subject of substantial preclinical investigation and may provide alternative targets for future drug discovery efforts (Li et al., Substantial research using preclinical models of drug abuse has demonstrated key roles for presynaptic GAdvances in the ability to assess neuroadaptations in human patient populations will provide exciting opportunities to test the translational potential of the preclinical work highlighted here. For example, the increasing availability of PET ligands suitable for human testing will allow interrogation of drug-induced changes in receptor availability that could confirm preclinical observations of reduced presynaptic GPCR expression following repeated drug exposure without relying on the use of postmortem brain samples. In addition, methods such as magnetic resonance spectroscopy that can be used to study neurotransmitter dynamics in humans create the opportunity to assess the impact of long-term drug abuse on glutamate transmission and to test the ability of clinical drug candidates that target presynaptic GPCRs to modulate glutamate transmission in the addicted brain (Hillmer et al., KAJ and DML both conceived of idea and plan for the manuscript, and both participated in manuscript writing and editing. KAJ created the figures and tables.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.