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The etiology of necrotizing enterocolitis (NEC) is not known. Alterations in gut microbiome, mucosal barrier function, immune cell activation, and blood flow are characterized events in its development, with stress as a contributing factor. The hormone corticotropin-releasing factor (CRF) is a key mediator of stress responses and influences these aforementioned processes. CRF signaling is modulated by NEC's main risk factors of prematurity and formula feeding. Using an established neonatal rat model of NEC, we tested hypotheses that: (i) increased CRF levels-as seen during stress-promote NEC in formula-fed (FF) newborn rats, and (ii) antagonism of CRF action ameliorates NEC. Newborn pups were formula-fed to initiate gut inflammation and randomized to: no stress, no stress with subcutaneous CRF administration, stress (acute hypoxia followed by cold exposure-NEC model), or stress after pretreatment with the CRF peptide antagonist Astressin. Dam-fed unstressed and stressed littermates served as controls. NEC incidence and severity in the terminal ileum were determined using a histologic scoring system. Changes in CRF, CRF receptor (CRFRs), and toll-like receptor 4 (TLR4) expression levels were determined by immunofluorescence and immunoblotting, respectively. Stress exposure in FF neonates resulted in 40.0% NEC incidence, whereas exogenous CRF administration resulted in 51.7% NEC incidence compared to 8.7% in FF non-stressed neonates (p

Well-differentiated papillary mesothelioma (WDPM) is an uncommon mesothelial proliferation that is most commonly encountered as an incidental finding in the peritoneal cavity. There is controversy in the literature about whether WDPM is a neoplasm or a reactive process and, if neoplastic, whether it is a variant or precursor of epithelial malignant mesothelioma or is a different entity. Using whole exome sequencing of five WDPMs of the peritoneum, we have identified distinct mutations in EHD1, ATM, FBXO10, SH2D2A, CDH5, MAGED1, and TP73 shared by WDPM cases but not reported in malignant mesotheliomas. Furthermore, we show that WDPM is strongly enriched with C > A transversion substitution mutations, a pattern that is also not found in malignant mesotheliomas. The WDPMs lacked the alterations involving BAP1, SETD2, NF2, CDKN2A/B, LASTS1/2, PBRM1, and SMARCC1 that are frequently found in malignant mesotheliomas. We conclude that WDPMs are neoplasms that are genetically distinct from malignant mesotheliomas and, based on observed mutations, do not appear to be precursors of malignant mesotheliomas.

Clear-cell renal cell carcinoma (ccRCC) is a common therapy resistant disease with aberrant angiogenic and immunosuppressive features. Patients with metastatic disease are treated with targeted therapies based on clinical features: low-risk patients are usually treated with anti-angiogenic drugs and intermediate/high-risk patients with immune therapy. However, there are no biomarkers available to guide treatment choice for these patients. A recently published phase II clinical trial observed a correlation between ccRCC patients' clustering and their response to targeted therapy. However, the clustering of these groups was not distinct. Here, we analyzed the gene expression profile of 469 ccRCC patients, using featured selection technique, and have developed a refined 66-gene signature for improved sub-classification of patients. Moreover, we have identified a novel comprehensive expression profile to distinguish between migratory stromal and immune cells. Furthermore, the proposed 66-gene signature was validated using a different cohort of 64 ccRCC patients. These findings are foundational for the development of reliable biomarkers that may guide treatment decision-making and improve therapy response in ccRCC patients.

TERT promoter mutations reactivate telomerase, allowing for indefinite telomere maintenance and enabling cellular immortalization. These mutations specifically recruit the multimeric ETS factor GABP, which can form two functionally independent transcription factor species: a dimer or a tetramer. We show that genetic disruption of GABPβ1L (β1L), a tetramer-forming isoform of GABP that is dispensable for normal development, results in TERT silencing in a TERT promoter mutation-dependent manner. Reducing TERT expression by disrupting β1L culminates in telomere loss and cell death exclusively in TERT promoter mutant cells. Orthotopic xenografting of β1L-reduced, TERT promoter mutant glioblastoma cells rendered lower tumor burden and longer overall survival in mice. These results highlight the critical role of GABPβ1L in enabling immortality in TERT promoter mutant glioblastoma.

Background The signaling protein p38 mitogen-activated protein kinase (MAPK) regulates the tumor cell microenvironment, modulating cell survival, migration, and invasion. This phase 1 study evaluated the safety of p38 MAPK inhibitor LY3007113 in patients with advanced cancer to establish a recommended phase 2 dose. Methods In part A (dose escalation), LY3007113 was administered orally every 12 h (Q12H) at doses ranging from 20 mg to 200 mg daily on a 28-day cycle until the maximum tolerated dose (MTD) was reached. In part B (dose confirmation), patients received MTD. Safety, pharmacokinetics, pharmacodynamics, and tumor response data were evaluated. Results MTD was 30 mg Q12H. The most frequent treatment-related adverse events (>10%) were tremor, rash, stomatitis, increased blood creatine phosphokinase, and fatigue. Grade ≥ 3 treatment-related adverse events included upper gastrointestinal haemorrhage and increased hepatic enzyme, both occurring at 40 mg Q12H and considered dose-limiting toxicities. LY3007113 exhibited an approximately dose-proportional increase in exposure and time-independent pharmacokinetics after repeated dosing. Maximal inhibition (80%) of primary biomarker MAPK-activated protein kinase 2 in peripheral blood mononuclear cells was not reached, and sustained minimal inhibition (60%) was not maintained for 6 h after dosing to achieve a biologically effective dose (BED). The best overall response in part B was stable disease in 3 of 27 patients. Conclusions The recommended phase 2 dosage of LY3007113 was 30 mg Q12H. Three patients continued treatment after the first radiographic assessment, and the BED was not achieved. Further clinical development of this compound is not planned as toxicity precluded achieving a biologically effective dose.

Patients and physicians report that communication about chronic pain and opioids is often challenging, but there is little empirical research on whether patient-physician communication about pain affects patient and physician visit experience. This study video recorded 86 primary care visits involving 49 physicians and 86 patients taking long-term opioids for chronic musculoskeletal pain, systematically coded all pain-related utterances during these visits using a custom-designed coding system, and administered previsit and postvisit questionnaires. Multiple regression was used to identify communication behaviors and patient characteristics associated with patients' ratings of their visit experience, physicians' ratings of visit difficulty, or both. After adjusting for covariates, 2 communication variables-patient-physician disagreement and patient requests for opioid dose increases-were each significantly associated with both worse ratings of patient experience and greater physician-reported visit difficulty. Patient desire for increased pain medicine was also significantly positively associated with both worse ratings of patient experience and greater physician-reported visit difficulty. Greater pain severity and more patient questions were each significantly associated with greater physician-reported visit difficulty, but not with patient experience. The association between patient requests for opioids and patient experience ratings was wholly driven by 2 visits involving intense conflict with patients demanding opioids. Patient-physician communication during visits is associated with patient and physician ratings of visit experience. Training programs focused on imparting communication skills that assist physicians in negotiating disagreements about pain management, including responding to patient requests for more opioids, likely have potential to improve visit experience ratings for both patients and physicians.

ObjectiveAssess patient-physician agreement on management goals for chronic musculoskeletal pain and its associations with patient and physician visit experiences.Materials and methodsPre-visit and post-visit questionnaires for 87 primary care visits that involved patients taking opioids for chronic musculoskeletal pain and primary care resident physicians. After each visit, patients and physicians independently ranked 5 pain treatment goals from most to least important.ResultsIn total, 48% of patients ranked reducing pain intensity as their top priority, whereas 22% ranked finding a diagnosis as most important. Physicians ranked improving function as the top priority for 41% of patients, and ranked reducing medication side effects as most important for 26%. The greatest difference between patient and physician rankings was for reducing pain intensity. In regression analyses, neither overall agreement on goals (ie, the physician's first or second priority included the patient's top priority) nor difference in patient versus physician ranking of pain intensity was significantly associated with patient-reported visit experience (β for overall agreement, -0.08; 95% confidence interval [CI], -0.45 to 0.30; P=0.69; β for intensity, -0.06; 95% CI, -0.17 to 0.04; P=0.24) or physician-reported visit difficulty (β for overall agreement, 1.92; 95% CI, -2.70 to 6.55; P=0.41; β for intensity, 0.42; 95% CI, -0.87 to 1.71; P=0.53).DiscussionPatients and physicians prioritize substantially different goals for chronic pain management, but there is no evidence that agreement predicts patient experience or physician-reported visit difficulty. Primary care physicians may have adapted to new recommendations that emphasize functional goals and avoidance of long-term opioid therapy, whereas patients continue to focus on reducing pain intensity.

Mutations in the isocitrate dehydrogenase gene IDH1 are common in low-grade glioma, where they result in the production of 2-hydroxyglutarate (2HG), disrupted patterns of histone methylation, and gliomagenesis. IDH1 mutations also cosegregate with mutations in the ATRX gene and the TERT promoter, suggesting that IDH mutation may drive the creation or selection of telomere-stabilizing events as part of immortalization/transformation process. To determine whether and how this may occur, we investigated the phenotype of pRb-/p53-deficient human astrocytes engineered with IDH1 wild-type (WT) or R132H-mutant (IDH1mut) genes as they progressed through their lifespan. IDH1mut expression promoted 2HG production and altered histone methylation within 20 population doublings (PD) but had no effect on telomerase expression or telomere length. Accordingly, cells expressing either IDH1WT or IDH1mut entered a telomere-induced crisis at PD 70. In contrast, only IDH1mut cells emerged from crisis, grew indefinitely in culture, and formed colonies in soft agar and tumors in vivo Clonal populations of postcrisis IDH1mut cells displayed shared genetic alterations, but no mutations in ATRX or the TERT promoter were detected. Instead, these cells reactivated telomerase and stabilized their telomeres in association with increased histone lysine methylation (H3K4me3) and c-Myc/Max binding at the TERT promoter. Overall, these results show that although IDH1mut does not create or select for ATRX or TERT promoter mutations, it can indirectly reactivate TERT, and in doing so contribute to astrocytic immortalization and transformation. Cancer Res; 76(22); 6680-9. ©2016 AACR.

ObjectiveTo describe the development and initial application of the Chronic Pain Coding System.DesignSecondary analysis of data from a randomized clinical trial.SettingSix primary care clinics in northern California.SubjectsForty-five primary care visits involving 33 clinicians and 45 patients on opioids for chronic noncancer pain.MethodsThe authors developed a structured coding system to accurately and objectively characterize discussions about pain and opioids. Two coders applied the final system to visit transcripts. Intercoder agreement for major coding categories was moderate to substantial (kappa = 0.5-0.7). Mixed effects regression was used to test six hypotheses to assess preliminary construct validity.ResultsGreater baseline pain interference was associated with longer pain discussions (P = 0.007) and more patient requests for clinician action (P = 0.02) but not more frequent negative patient evaluations of pain (P = 0.15). Greater clinician-reported visit difficulty was associated with more frequent disagreements with clinician recommendations (P = 0.003) and longer discussions of opioid risks (P = 0.049) but not more frequent requests for clinician action (P = 0.11). Rates of agreement versus disagreement with patient requests and clinician recommendations were similar for opioid-related and non-opioid-related utterances.ConclusionsThis coding system appears to be a reliable and valid tool for characterizing patient-clinician communication about opioids and chronic pain during clinic visits. Objective data on how patients and clinicians discuss chronic pain and opioids are necessary to identify communication patterns and strategies for improving the quality and productivity of discussions about chronic pain that may lead to more effective pain management and reduce inappropriate opioid prescribing.

Telomerase (TERT) activation is a fundamental step in tumorigenesis. By maintaining telomere length, telomerase relieves a main barrier on cellular lifespan, enabling limitless proliferation driven by oncogenes. The recently discovered, highly recurrent mutations in the promoter of TERT are found in over 50 cancer types, and are the most common mutation in many cancers. Transcriptional activation of TERT, via promoter mutation or other mechanisms, is the rate-limiting step in production of active telomerase. Although TERT is expressed in stem cells, it is naturally silenced upon differentiation. Thus, the presence of TERT promoter mutations may shed light on whether a particular tumor arose from a stem cell or more differentiated cell type. It is becoming clear that TERT mutations occur early during cellular transformation, and activate the TERT promoter by recruiting transcription factors that do not normally regulate TERT gene expression. This review highlights the fundamental and widespread role of TERT promoter mutations in tumorigenesis, including recent progress on their mechanism of transcriptional activation. These somatic promoter mutations, along with germline variation in the TERT locus also appear to have significant value as biomarkers of patient outcome. Understanding the precise molecular mechanism of TERT activation by promoter mutation and germline variation may inspire novel cancer cell-specific targeted therapies for a large number of cancer patients.

The evolutionary history of tumor cell populations can be reconstructed from patterns of genetic alterations. In contrast to stable genetic events, epigenetic states are reversible and sensitive to the microenvironment, prompting the question whether epigenetic information can similarly be used to discover tumor phylogeny. We examined the spatial and temporal dynamics of DNA methylation in a cohort of low-grade gliomas and their patient-matched recurrences. Genes transcriptionally upregulated through promoter hypomethylation during malignant progression to high-grade glioblastoma were enriched in cell cycle function, evolving in parallel with genetic alterations that deregulate the G1/S cell cycle checkpoint. Moreover, phyloepigenetic relationships robustly recapitulated phylogenetic patterns inferred from somatic mutations. These findings highlight widespread co-dependency of genetic and epigenetic events throughout brain tumor evolution.

Reactivation of telomerase reverse transcriptase (TERT) expression enables cells to overcome replicative senescence and escape apoptosis, which are fundamental steps in the initiation of human cancer. Multiple cancer types, including up to 83% of glioblastomas (GBMs), harbor highly recurrent TERT promoter mutations of unknown function but specific to two nucleotide positions. We identified the functional consequence of these mutations in GBMs to be recruitment of the multimeric GA-binding protein (GABP) transcription factor specifically to the mutant promoter. Allelic recruitment of GABP is consistently observed across four cancer types, highlighting a shared mechanism underlying TERT reactivation. Tandem flanking native E26 transformation-specific motifs critically cooperate with these mutations to activate TERT, probably by facilitating GABP heterotetramer binding. GABP thus directly links TERT promoter mutations to aberrant expression in multiple cancers.

UnlabelledRegulation of mRNA splicing, a critical and tightly regulated cellular function, underlies the majority of proteomic diversity and is frequently disrupted in disease. Using an integrative genomics approach, we combined both genomic data and exon-level transcriptome data in two somatic tissues (cerebella and peripheral ganglia) from a transgenic mouse model of neuroblastoma, a tumor that arises from the peripheral neural crest. Here, we describe splicing quantitative trait loci associated with differential splicing across the genome that we use to identify genes with previously unknown functions within the splicing pathway and to define de novo intronic splicing motifs that influence splicing from hundreds of bases away. Our results show that these splicing motifs represent sites for functional recurrent mutations and highlight novel candidate genes in human cancers, including childhood neuroblastoma.SignificanceSomatic mutations with predictable downstream effects are largely relegated to coding regions, which comprise less than 2% of the human genome. Using an unbiased in vivo analysis of a mouse model of neuroblastoma, we have identified intronic splicing motifs that translate into sites for recurrent somatic mutations in human cancers.

ImportanceEncouraging primary care patients to address depression symptoms and care with clinicians could improve outcomes but may also result in unnecessary treatment.ObjectiveTo determine whether a depression engagement video (DEV) or a tailored interactive multimedia computer program (IMCP) improves initial depression care compared with a control without increasing unnecessary antidepressant prescribing.Design, setting, and participantsRandomized clinical trial comparing DEV, IMCP, and control among 925 adult patients treated by 135 primary care clinicians (603 patients with depression and 322 patients without depression, defined by Patient Health Questionnaire-9 [PHQ-9] score) conducted from June 2010 through March 2012 at 7 primary care clinical sites in California.InterventionsDEV targeted to sex and income, an IMCP tailored to individual patient characteristics, and a sleep hygiene video (control).Main outcomes and measuresAmong depressed patients, superiority assessment of the composite measure of patient-reported antidepressant drug recommendation, mental health referral, or both (primary outcome); depression at 12-week follow-up, measured by the PHQ-8 (secondary outcome). Among nondepressed patients, noninferiority assessment of clinician- and patient-reported antidepressant drug recommendation (primary outcomes) with a noninferiority margin of 3.5%. Analyses were cluster adjusted.ResultsOf the 925 eligible patients, 867 were included in the primary analysis (depressed, 559; nondepressed, 308). Among depressed patients, rates of achieving the primary outcome were 17.5% for DEV, 26% for IMCP, and 16.3% for control (DEV vs control, 1.1 [95% CI, -6.7 to 8.9], P = .79; IMCP vs control, 9.9 [95% CI, 1.6 to 18.2], P = .02). There were no effects on PHQ-8 measured depression score at the 12-week follow-up: DEV vs control, -0.2 (95% CI, -1.2 to 0.8); IMCP vs control,  0.9 (95% CI, -0.1 to 1.9). Among nondepressed patients, clinician-reported antidepressant prescribing in the DEV and IMCP groups was noninferior to control (mean percentage point difference [PPD]: DEV vs control, -2.2 [90% CI, -8.0 to 3.49], P = .0499 for noninferiority; IMCP vs control, -3.3 [90% CI, -9.1 to 2.4], P = .02 for noninferiority); patient-reported antidepressant recommendation did not achieve noninferiority (mean PPD: DEV vs control,  0.9 [90% CI, -4.9 to 6.7], P = .23 for noninferiority; IMCP vs control,  0.3 [90% CI, -5.1 to 5.7], P = .16 for noninferiority).Conclusions and relevanceA tailored IMCP increased clinician recommendations for antidepressant drugs, a mental health referral, or both among depressed patients but had no effect on mental health at the 12-week follow-up. The possibility that the IMCP and DEV increased patient-reported clinician recommendations for an antidepressant drug among nondepressed patients could not be excluded.Trial registrationclinicaltrials.gov Identifier: NCT01144104.

Accurate detection of epimutations in tumor cells is crucial for -understanding the molecular pathogenesis of cancer. Alterations in DNA methylation in cancer are functionally important and clinically relevant, but even this well-studied area is continually re-evaluated in light of unanticipated results, such as the strong association between aberrant DNA methylation in adult tumors and polycomb group profiles in embryonic stem cells, cancer-associated genetic mutations in epigenetic regulators such as DNMT3A and TET family genes, and the discovery of altered 5-hydroxymethylcytosine, a product of TET proteins acting on 5-methylcytosine, in human tumors with TET mutations. The abundance and distribution of covalent histone modifications in primary cancer tissues relative to normal cells is an important but largely uncharted area, although there is good evidence for a mechanistic role of cancer-specific alterations in histone modifications in tumor etiology, drug response, and tumor progression. Meanwhile, the discovery of new epigenetic marks continues, and there are many useful methods for epigenome analysis applicable to primary tumor samples, in addition to cancer cell lines. For DNA methylation and hydroxymethylation, next-generation sequencing allows increasingly inexpensive and quantitative whole-genome profiling. Similarly, the refinement and maturation of chromatin immunoprecipitation with next-generation sequencing (ChIP-seq) has made possible genome-wide mapping of histone modifications, open chromatin, and transcription factor binding sites. Computational tools have been developed apace with these epigenome methods to better enable accurate interpretation of the profiling data.

ObjectiveTo determine whether parental reports and electronic health record documentation of physician counseling on nutrition and physical activity reflect actual counseling provided.MethodsParticipants were parents of 198 children 2 to 12 years of age seen in a primary care pediatric clinic at an academic medical center for well child care and their 38 physicians. Parents completed a post-visit questionnaire to report discussions on weight, nutrition, and physical activity that occurred during the visit. Electronic health records were reviewed to measure documentation of these topics during the visit. Parental reports and records were compared with actual discussions on the basis of coded audiotapes. Counseling was coded as having occurred if specific topics were mentioned during the encounter, however brief this mention was.ResultsA total of 48% of the children were female, they were a mean age of 5.4 years, and 28% were overweight or obese. Sensitivity of parental report was high (63%-96%), but specificity was low (43%-77%) because of parents' tendency to overreport counseling. Sensitivity of electronic health record documentation was generally low (40%-53%) except for discussion of screen time (92%) and physical activity (88%); the specificity of these data was also poor (42% and 21%, respectively, for screen time and physical activity).ConclusionsElectronic health record documentation may not be the most valid method of measuring physician counseling on weight, nutrition, and physical activity in pediatric primary care. Parental report via the use of a questionnaire administered immediately after the visit is a better alternative in quality improvement or research studies when resources do not allow for direct observation, with the caveat that parents may overreport whether counseling was provided.

PurposeDepression is common in primary care but often under-treated. Personal experiences with depression can affect adherence to therapy, but the effect of vicarious experience is unstudied. We sought to evaluate the association between a patient's vicarious experiences with depression (those of friends or family) and treatment preferences for depressive symptoms.MethodsWe sampled 1054 English and/or Spanish speaking adult subjects from July through December 2008, randomly selected from the 2008 California Behavioral Risk Factor Survey System, regarding depressive symptoms and treatment preferences. We then constructed a unidimensional scale using item analysis that reflects attitudes about antidepressant pharmacotherapy. This became the dependent variable in linear regression analyses to examine the association between vicarious experiences and treatment preferences for depressive symptoms.ResultsOur sample was 68% female, 91% white, and 13% Hispanic. Age ranged from 18-94 years. Mean PHQ-9 score was 4.3; 14.5% of respondents had a PHQ-9 score >9.0, consistent with active depressive symptoms. Analyses controlling for current depression symptoms and socio-demographic factors found that in patients both with (coefficient 1.08, p = 0.03) and without (coefficient 0.77, p = 0.03) a personal history of depression, having a vicarious experience (family and friend, respectively) with depression is associated with a more favorable attitude towards antidepressant medications.ConclusionsPatients with vicarious experiences of depression express more acceptance of pharmacotherapy. Conversely, patients lacking vicarious experiences of depression have more negative attitudes towards antidepressants. When discussing treatment with patients, clinicians should inquire about vicarious experiences of depression. This information may identify patients at greater risk for non-adherence and lead to more tailored patient-specific education about treatment.