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To explore the association between lipids, polygenic profile scores (PPS) for biomarkers of lipid metabolism, markers of disease severity, and survival in patients with Amyotrophic Lateral Sclerosis (ALS).We meta-analyzed the current literature on the prognostic value of lipids in patients with ALS. Subsequently, we evaluated the relationship between lipid levels at diagnosis, clinical disease stage and survival in all consecutive patients diagnosed in The Netherlands. We determined the hazard ratio of each lipid for overall survival, defined as death from any cause. A subset of patients was matched to a previous Genome Wide Association Study (GWAS); data were used to calculate PPS for biomarkers of lipid metabolism, and to determine the association between observed lipid levels at diagnosis and survival.Meta-analysis of four studies indicated that none of the biomarkers of the lipid metabolism were statistically significantly associated with overall survival; there was, however, considerable heterogeneity between study results. Using individual patient data (N = 1,324), we found that increased HDL-cholesterol was associated with poorer survival (HR of 1.33 (95% CI 1.14 to 1.55,Lipids may contain valuable information about disease severity and prognosis, but their main value may be driven as a consequence of disease progression. Our results underscore that gaining further insight into lipid metabolism and longitudinal data on serum concentrations of the lipid profile could improve the monitoring of patients and potentially further disentangle ALS pathogenesis.

BackgroundWhile safety in US coal mining has improved over the past two decades, general occupational health research shows that risk of injury varies across individual worksites and is influenced by worksite safety cultures and practices.MethodsIn this longitudinal study, we evaluated whether mine-level characteristics reflecting poor adherence to health and safety regulations in underground coal mines are associated with higher acute injury rates. We aggregated Mine Safety and Health Administration (MSHA) data by year for each underground coal mine for the period 2000–2019. Data included part-50 injuries, mine characteristics, employment and production, dust sampling, noise sampling, and violations. Multivariable hierarchical generalised estimating equations (GEE) models were developed.ResultsBased on the final GEE model, despite an average annual decline in injury rates by 5.5%, the following indicators of inadequate adherence to health and safety regulations were associated with increased average annual injury rates: +2.9% for each 10% increase in dust samples exceeding the permissible exposure limit; +0.6% for each 10% increase of permitted 90 dBA 8-hour noise exposure dose; +2.0% for every 10 substantial-significant MSHA violations in a year; +1.8% for each rescue/recovery procedure violation; +2.6% for each safeguard violation. If a fatality occurred in a mine, injury rates increased by 11.9% in the same year, but declined by 10.4% in the following year. The presence of safety committees was associated with a 14.5% decline in injury rates.DiscussionIn US underground coal mines, injury rates are associated with poor adherence to dust, noise and safety regulations.

In pediatric cancer care, medication non-adherence is a significant driver of avoidable suffering and death. There is a lack of interventions designed for families of young children, where patient medication refusal/avoidance is a common barrier to adherence. We developed the

Purpose: Treatment options for recurrent or refractory Ewing's sarcoma (ES) are limited. Vigil is a novel autologous tumor cell therapy expressing bi-shRNA furin/GMCSF plasmid, which previously demonstrated monotherapy activity in advanced ES. Herein we report safety and evidence of benefit to Vigil for ES as potential treatment. Patients and Methods: In this pilot trial, eligible patients with recurrent or refractory ES who failed initial standard-of-care therapy received treatment with temozolomide (TEM) 100 mg/m2/day oral and irinotecan (IRI) 50 mg/m2/day oral, Days 1 to 5, in combination with Vigil (1 × 106–107 cells/mL/day intradermal, Day 15), every 21 days (Vigil/TEM/IRI). Objective response rate (ORR) by RECIST v1.1, progression-free survival (PFS), and overall survival (OS) were assessed. Circulating tumor (ct) DNA analysis was done by patient-specific droplet digital PCR on baseline and serially collected on-treatment samples. Results: Eight of 10 enrolled patients were evaluable for safety and efficacy (mean age 24.6; 12.6–46.1 years old); 2 did not receive Vigil. Seven of 8 patients previously received TEM/IRI. No Vigil-related adverse events were reported. Common ≥Grade 3 chemotherapy-related toxicity included neutropenia (50%) and thrombocytopenia (38%). We observed two partial response patients by RECIST; both showed histologic complete response without additional cancer therapy. Median PFS was 8.2 months (95% confidence interval, 4.3–NA). Five patients showed stable disease or better for ≥6 months. Patient-specific EWS/FLI1 ctDNA was detectable in all 8 evaluable patients at baseline. Changes in ctDNA levels corresponded to changes in disease burden. Conclusions: Results demonstrated safety of combination Vigil/TEM/IRI.

Background Actigraphy has been proposed as a measure for tracking functional decline and disease progression in patients with Motor Neuron Disease (MND). There is, however, little evidence to show that wrist-based actigraphy measures correlate with functional decline, and no consensus on how best to implement actigraphy. We report on the use of wrist actigraphy to show decreased activity in patients compared to controls, and compared the utility of wrist- and hip-based actigraphy for assessing functional decline in patients with MND. Methods In this multi-cohort, multi-centre, natural history study, wrist- and hip-based actigraphy were assessed in 139 patients with MND (wrist, n = 97; hip, n = 42) and 56 non-neurological control participants (wrist, n = 56). For patients with MND, longitudinal measures were contrasted with clinical outcomes commonly used to define functional decline. Results Patients with MND have reduced wrist-based actigraphy scores when compared to controls (median differences: prop. active = − 0.053 [− 0.075, − 0.026], variation axis 1 = − 0.073 [− 0.112, − 0.021]). When comparing wrist- and hip-based measures, hip-based accelerometery had stronger correlations with disease progression (prop. active: τ = 0.20 vs 0.12; variation axis 1: τ = 0.33 vs 0.23), whereas baseline wrist-based accelerometery was better related with future decline in fine-motor function (τ = 0.14–0.23 vs 0.06–0.16). Conclusions Actigraphy outcomes measured from the wrist are more variable than from the hip and present differing sensitivity to specific functional outcomes. Outcomes and analysis should be carefully constructed to maximise benefit, should wrist-worn devices be used for at-home monitoring of disease progression in patients with MND.

ObjectivesCoal miners suffer increased mortality from non-malignant respiratory diseases (NMRD), including pneumoconioses and chronic obstructive pulmonary disease, compared with the US population. We characterised mortality trends from NMRD, lung cancer and ischaemic heart disease (IHD) using data from the Federal Black Lung Program, National Coal Workers’ Health Surveillance Program and the National Death Index.MethodsWe compared mortality ORs (MORs) for NMRD, lung cancer and IHD in former US coal miners to US white males. MORs were computed for the study period 1979–2017 by birth cohort (ResultsThe study population totalled 235 550 deceased miners, aged >45 years. Odds of death from NMRD and lung cancer across all miner birth cohorts averaged twice those of US males. In Central Appalachia, MORs significantly increased across birth cohorts. There was an eightfold increase in odds of death from NMRD among miners born after 1940 (MORBC≥19408.25; 95% CI 7.67 to 8.87). Miners with progressive massive fibrosis (PMF) were younger at death than those without PMF (74 vs 78 years; pConclusionUS coal miners have excess mortality from NMRD and lung cancer compared with total US and Appalachian populations. Mortality is highest in the most recent birth cohorts, perhaps reflecting increased rates of severe pneumoconiosis.

Food reinforcement or one's motivation to eat may be established early in life; it might not be the food reinforcement per se that drives weight gain, but rather the imbalance between food and nonfood reinforcement.We implemented a 2-y randomized, controlled trial to assess the effects of a music enrichment program (music, n = 45) compared with an active play date control (control, n = 45) in 9- to 15-mo-old healthy infants who were strongly motivated to eat.The 12-mo intensive intervention phase included 4 semesters of Music Together® or a play date program (Winter, Spring, Summer, and Fall), comprised of once per week group meetings, followed by a 12-mo maintenance phase with monthly meetings. Parents were encouraged to listen to the Music Together® program CD or play with the play date group's toy with their infants at home, respectively. We performed a modified intention-to-treat analysis using all randomly assigned, non-excluded subjects for the outcome measures [relative reinforcing value of food (RRVfood), food reinforcement, music reinforcement, and weight-for-length z-score (WLZ)].There were significant differential group changes across time for RRVfood (group × month; P = 0.016; Cohen's f2 = 0.034). The music group had significantly greater RRVfood decreases than the control group from baseline to the end of the intensive intervention phase (music change = -0.211; control change = -0.015; P = 0.002; Cohen's D = 0.379). However, these differences were not maintained during months 12-24 (music change = -0.187; control change = -0.143; P = 0.448; D = 0.087). We observed an overall moderation effect by sex for food reinforcement and WLZ. Boys in the music group had a significant attenuation in food reinforcement and WLZ compared with boys in the control group.This study extends our knowledge in infant eating behavior by providing insight into the role of nonfood alternatives in altering one's motivation to eat. There may be sex differences in altering one's motivation to eat.This trial was registered at clinicaltrials.gov as NCT02936284.

We report our experience of performing an extra, earlier physics plan check as recommended by the American Association of Physicists in Medicine Task Group 100 and Task Group 275 reports. We assessed utilization and timing of the extra check as well as the time required in a medium-sized clinic.We retrospectively extracted and analyzed timestamp data from the record and verify system for the quality checklist (QCL) items related to treatment planning and physics "prechecks" for 3487 patients treated at our institution from February 2017 to February 2021. The dosimetry staff was interviewed for their perception of the value and efficacy of the practice.Physics prechecks were requested for 19.0% of plans. The number of requests declined from 43.9% of cases in 2017 to 18.4% in 2018. The introduction of automated plan-check tools and a dosimetrist checklist further contributed to a drop in number of precheck requests to 3.5% in 2019. For patients who received a physics precheck, the treatment planning process was a median 3.6 hours longer compared with those without (P.001). A total of 12.9% of the precheck requests were canceled by the dosimetrist after waiting a median time of 5.3 hours. There was a strong positive correlation (0.899) between a precheck being requested and the time remaining until treatment start. Higher complexity plans and plans with a specific concern (eg, possible collision) were more likely to have a precheck requested.Physics prechecks have become standard practice for certain cases in our clinic. However, the perception in the department was that, as a universal practice, waiting for a precheck was not worth the time saved redoing work on the few cases in which an error was caught. Dosimetrist access to automated checking tools and checklists, which were motivated by the precheck process, contributed to this perception.

Patients with immune-mediated inflammatory diseases (IMIDs) receiving B cell-depleting therapy (BCDT) are among the most vulnerable to severe COVID-19, as well as the most likely to suboptimally respond to SARS-CoV-2 vaccines. However, little is known about the frequency or severity of breakthrough infection in this population. We retrospectively analyzed a large group of vaccinated IMID patients undergoing BCDT in order to identify breakthrough COVID-19 infections and assess their outcomes.In this retrospective cohort study, the pharmacy records and COVID-19 registry at the Cleveland Clinic were searched using specific International Statistical Classification of Diseases and Related Health Problems, Tenth Revision codes to identify IMIDs patients who 1) received treatment with BCDT, 2) were vaccinated against SARS-CoV-2, and 3) experienced breakthrough infections. Each electronic medical record was reviewed to extract clinical data and outcomes. Univariate and multivariable logistic/proportional odds regression models were used to examine the risk factors for severe outcomes.Of 1,696 IMID patients receiving BCDT, 74 developed breakthrough COVID-19 prior to December 16, 2021. Outcomes were severe, with 29 patients hospitalized (39.2%), 11 patients requiring critical care (14.9%), and 6 deaths (8.1%). Outpatient anti-SARS-CoV-2 monoclonal antibodies were used to treat 21 patients, with 1 hospitalization and no deaths. A comparator analysis examining 1,437 unvaccinated IMID patients receiving BCDT over the same time period identified 57 COVID-19 cases (4.0%), with 28 requiring hospitalization (49.1%), including 7 deaths (12.3%).IMID patients receiving BCDT regardless of vaccine status appear to be vulnerable to infection with SARS-CoV-2, and use of BCDT is frequently associated with severe outcomes. Outpatient use of anti-SARS-CoV-2 monoclonal antibody therapy appears to be associated with enhanced clinical outcomes.

Rationale: The reasons for resurgent coal workers' pneumoconiosis and its most severe forms, rapidly progressive pneumoconiosis and progressive massive fibrosis (PMF), in the United States are not yet fully understood. Objectives: To compare the pathologic and mineralogic features of contemporary coal miners with severe pneumoconiosis with those of their historical counterparts. Methods: Lung pathology specimens from 85 coal miners with PMF were included for evaluation and analysis. We compared the proportion of cases with pathologic and mineralogic findings in miners born between 1910 and 1930 (historical) with those in miners born in or after 1930 (contemporary). Results: We found a significantly higher proportion of silica-type PMF (57% vs. 18%; P < 0.001) among contemporary miners compared with their historical counterparts. Mineral dust alveolar proteinosis was also more common in contemporary miners compared with their historical counterparts (70% vs. 37%; P < 0.01). In situ mineralogic analysis showed that the percentage (26.1% vs. 17.8%; P < 0.01) and concentration (47.3310(8) vs. 25.8310(8) particles/cm(3); P = 0.036) of silica particles were significantly greater in specimens from contemporary miners compared with their historical counterparts. The concentration of silica particles was significantly greater when silica-type PMF, mineral dust alveolar proteinosis, silicotic nodules, or immature silicotic nodules were present (P < 0.05). Conclusions: Exposure to respirable crystalline silica appears causal in the unexpected surge of severe disease in contemporary miners. Our findings underscore the importance of controlling workplace silica exposure to prevent the disabling and untreatable adverse health effects afflicting U.S. coal miners. Alpha Foundation for the Improvement of Mine Safety and Health [AFC417-1] Published version Supported by Alpha Foundation for the Improvement of Mine Safety and Health grant AFC417-1. The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention, or Alpha Foundation for the Improvement of Mine Safety and Health, Inc. Any use of trade, firm, or product names is for descriptive purposes only and does not imply endorsement by the U.S. government.

© The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/., There is a strong genetic contribution to Amyotrophic lateral sclerosis (ALS) risk, with heritability estimates of up to 60%. Both Mendelian and small effect variants have been identified, but in common with other conditions, such variants only explain a little of the heritability. Genomic structural variation might account for some of this otherwise unexplained heritability. We therefore investigated association between structural variation in a set of 25 ALS genes, and ALS risk and phenotype. As expected, the repeat expansion in the C9orf72 gene was identified as associated with ALS. Two other ALS-associated structural variants were identified: inversion in the VCP gene and insertion in the ERBB4 gene. All three variants were associated both with increased risk of ALS and specific phenotypic patterns of disease expression. More than 70% of people with respiratory onset ALS harboured ERBB4 insertion compared with 25% of the general population, suggesting respiratory onset ALS may be a distinct genetic subtype., This research was funded in whole, or in part, by the Wellcome Trust [Grant number]. For the purpose of open access, the author has applied a CC BY public copyright licence to any Author Accepted Manuscript version arising from this submission. The authors acknowledge use of the research computing facility at King’s College London, Rosalind (https://rosalind.kcl.ac.uk), which is delivered in partnership with the National Institute for Health Research (NIHR) Biomedical Research Centres at South London & Maudsley and Guy’s & St. Thomas’ NHS Foundation Trusts, and part-funded by capital equipment grants from the Maudsley Charity (award 980) and Guy’s & St. Thomas’ Charity (TR130505). The views expressed are those of the author (s) and not necessarily those of the NHS, the NIHR, King’s College London, or the Department of Health and Social Care. National. The authors acknowledge Institute for Health Research (NIHR) Biomedical Research Centre at South London and Maudsley NHS Foundation Trust and King’s College London. The authors acknowledge Health Data Research UK, which is funded by the UK Medical Research Council, Engineering and Physical Sciences Research Council, Economic and Social Research Council, Department of Health and Social Care (England), Chief Scientist Office of the Scottish Government Health and Social Care Directorates, Health and Social Care Research and Development Division (Welsh Government), Public Health Agency (Northern Ireland), British Heart Foundation and Wellcome Trust. A.A.K. is funded by The Motor Neurone Disease Association (MNDA), NIHR Maudsley Biomedical Research Centre and and ALS Association Milton Safenowitz Research Fellowship. This project was funded by the MND Association and the Wellcome Trust. This is an EU Joint Programme-Neurodegenerative Disease Research (JPND) project. The project is supported through the following funding organisations under the aegis of JPND - www.jpnd.eu (United Kingdom, Medical Research Council (MR/L501529/1 and MR/R024804/1) and Economic and Social Research Council (ES/L008238/1)). A.A.C. is an NIHR Senior Investigator. C.E.S. and A.A.C. receive salary support from the National Institute for Health Research (NIHR) Dementia Biomedical Research Unit at South London and Maudsley NHS Foundation Trust and King’s College London. The views expressed are those of the authors and not necessarily those of the NHS, the NIHR or the Department of Health. The work leading up to this publication was funded by the European Community’s Health Seventh Framework Program (FP7/2007–2013; grant agreement number 259867) and Horizon 2020 Program (H2020-PHC-2014-two-stage; grant agreement number 633413). This project has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement no. 772376 - EScORIAL. The collaboration project is co-funded by the PPP Allowance made available by Health~Holland, Top Sector Life Sciences & Health, to stimulate public-private partnerships. Project MinE Belgium was supported by a grant from IWT, the Belgian ALS Liga and a grant from Opening the Future Fund (KU Leuven). PVD holds a senior clinical investigatorship of FWO-Vlaanderen and is supported by E. von Behring Chair for Neuromuscular and Neurodegenerative Disorders, the ALS Liga België and the KU Leuven funds “Een Hart voor ALS”, “Laeversfonds voor ALS Onderzoek” and the “Valéry Perrier Race against ALS Fund”. R.L.McL. is supported by Science Foundation Ireland (17/CDA/4737). MinE USA is funded by the US ALS Association. We thank the patients and families from the Emory ALS Clinic participating in this research. Funding was provided by US National Institutes of Health (NIH)/National Institute of Neurological Disorders and Stroke (NINDS) (R01NS073873, J.E.L.) and the American ALS Association (J.E.L.).

Rhabdomyosarcoma (RMS) is a common childhood cancer that shares features with developing skeletal muscle. Yet, the conservation of cellular hierarchy with human muscle development and the identification of molecularly defined tumor-propagating cells has not been reported. Using single-cell RNA-sequencing, DNA-barcode cell fate mapping and functional stem cell assays, we uncovered shared tumor cell hierarchies in RMS and human muscle development. We also identified common developmental stages at which tumor cells become arrested. Fusion-negative RMS cells resemble early myogenic cells found in embryonic and fetal development, while fusion-positive RMS cells express a highly specific gene program found in muscle cells transiting from embryonic to fetal development at 7-7.75 weeks of age. Fusion-positive RMS cells also have neural pathway-enriched states, suggesting less-rigid adherence to muscle-lineage hierarchies. Finally, we identified a molecularly defined tumor-propagating subpopulation in fusion-negative RMS that shares remarkable similarity to bi-potent, muscle mesenchyme progenitors that can make both muscle and osteogenic cells.

OBJECTIVE: Correlated longitudinal and time-to-event outcomes, such as the rate of cognitive decline and the onset of Alzheimer’s disease, are frequent (co-)primary and key secondary endpoints in randomized clinical trials (RCTs). Despite their biological associations, these types of data are often analyzed separately leading to loss of information and increases in bias. In this paper, we set out how joint modelling of longitudinal and time-to-event endpoints can be used in RCTs to answer various research questions. STUDY DESIGN AND SETTING: The key concepts of joint models are introduced and illustrated for a completed trial in amyotrophic lateral sclerosis. RESULTS: The output of a joint model can be used to answer different clinically relevant research questions, where the interpretation of effect estimates and those obtained from conventional methods are similar. Albeit joint models have the potential to overcome the limitations of commonly used alternatives, they require additional assumptions regarding the distributions as well as the associations between two endpoints. CONCLUSION: Improving the uptake of joint models in RCTs may start by outlining the exact research question one seeks to answer, thereby determining how best to prespecify the model and defining which parameter should be of primary interest.

Background and objectives Primary lateral sclerosis (PLS) is a motor neuron disease characterised by loss of the upper motor neurons. Most patients present with slowly progressive spasticity of the legs, which may also spread to the arms or bulbar regions. It is challenging to distinguish between PLS, early-stage amyotrophic lateral sclerosis (ALS) and hereditary spastic paraplegia (HSP). The current diagnostic criteria advise against extensive genetic testing. This recommendation is, however, based on limited data. Methods We aim to genetically characterize a PLS cohort using whole exome sequencing (WES) for genes associated with ALS, HSP, ataxia and movement disorders (364 genes) and C9orf72 repeat expansions. Patients fulfilling the definite PLS criteria by Turner et al. and with available DNA samples of sufficient quality were recruited from an on-going, population-based epidemiological study. Genetic variants were classified according to the ACMG criteria and assigned to groups based on disease association. Results WES was performed in 139 patients and the presence of repeat expansions in C9orf72 was analysed separately in 129 patients. This resulted in 31 variants of which 11 were (likely) pathogenic. (Likely) pathogenic variants resulted in 3 groups based on disease association: ALS-FTD (C9orf72, TBK1), pure HSP (SPAST, SPG7), “ALS-HSP-CMT overlap” (FIG4, NEFL, SPG11). Discussion In a cohort of 139 PLS patients, genetic analyses resulted in 31 variants (22%) of which 10 (7%) (likely) pathogenic associated with different diseases (predominantly ALS and HSP). Based on these results and the literature, we advise to consider genetic analyses in the diagnostic work-up for PLS.

Background:Treatment options for recurrent or refractory Ewing's sarcoma (ES) are limited. Vigil is a novel autologous tumor cell therapy expressing bi-shRNA furin/GMCSF plasmid, which previously demonstrated monotherapy activity in advanced ES. Herein we report safety and evidence of benefit to Vigil for ES as potential treatment.Patients and Methods:In this pilot trial, eligible patients with recurrent or refractory ES who failed initial standard-of-care therapy received treatment with temozolomide (TEM) 100 mg/m2/day oral and irinotecan (IRI) 50 mg/m2/day oral, Days 1 to 5, in combination with Vigil (1 × 106–107 cells/mL/day intradermal, Day 15), every 21 days (Vigil/TEM/IRI). Objective response rate (ORR) by RECIST v1.1, progression-free survival (PFS), and overall survival (OS) were assessed. Circulating tumor (ct) DNA analysis was done by patient-specific droplet digital PCR on baseline and serially collected on-treatment samples.Results:Eight of 10 enrolled patients were evaluable for safety and efficacy (mean age 24.6; 12.6–46.1 years old); 2 did not receive Vigil. Seven of 8 patients previously received TEM/IRI. No Vigil-related adverse events were reported. Common ≥Grade 3 chemotherapy-related toxicity included neutropenia (50%) and thrombocytopenia (38%). We observed two partial response patients by RECIST; both showed histologic complete response without additional cancer therapy. Median PFS was 8.2 months (95% confidence interval, 4.3–NA). Five patients showed stable disease or better for ≥6 months. Patient-specific EWS/FLI1 ctDNA was detectable in all 8 evaluable patients at baseline. Changes in ctDNA levels corresponded to changes in disease burden.Conclusions:Results demonstrated safety of combination Vigil/TEM/IRI.

Notch signaling determines cell fates in mouse intestine. Notch receptors contain multiple epidermal growth factor-like (EGF) repeats modified by O-glycans that regulate Notch signaling. Conditional deletion of protein O-fucosyltransferase 1 (Pofut1) substantially reduces Notch signaling and markedly perturbs lineage development in mouse intestine. However, mice with inactivatedPofut1are viable, whereas complete elimination of Notch signaling in intestine is lethal. Here we investigate whether residual Notch signaling enabled by EOGT permits mice lackingPofut1in intestine to survive. Mice globally lackingEogtalone were grossly unaffected in intestinal development. In contrast, mice lacking bothEogtandPofut1died at ∼28 days after birth with greater loss of body weight, a greater increase in the numbers of goblet and Paneth cells, and greater downregulation of Notch target genes, compared toPofut1deletion alone. These data establish that both O-fucose and O-GlcNAc glycans are fundamental to Notch signaling in the intestine and provide new insights into roles for O-glycans in regulating Notch ligand binding. Finally, EOGT and O-GlcNAc glycans provide residual Notch signaling and support viability in mice lackingPofut1in the intestine.

With the advent of gene therapies for amyotrophic lateral sclerosis, there is a surge in gene testing for ALS. Although there is ample experience with gene testing for C9orf72, SOD1, FUS and TARDBP in familial ALS, large studies exploring genetic variation in all ALS-associated genes in sporadic ALS (sALS) are still scarce. Gene testing in a diagnostic setting is challenging given the complex genetic architecture of sALS with genetic variants with large and small effect sizes. Guidelines for interpretation of genetic variants in gene panels and for counselling of patients are lacking. We aimed to provide a thorough characterization of genetic variability in ALS genes by applying the ACMG criteria on whole genome sequencing data from a large cohort of 6013 sporadic ALS patients and 2411 matched controls from Project MinE. We studied genetic variation in 90 ALS-associated genes and applied customized ACMG-criteria to identify pathogenic and likely pathogenic variants. Variants of unknown significance were collected as well. In addition, we determined the length of repeat expansions in C9orf72, ATXN1, ATXN2 and NIPA1 using the ExpansionHunter tool. We found C9orf72 repeat expansions in 5.21% of sALS patients. In 50 ALS-associated genes, we did not identify any pathogenic or likely pathogenic variants. In 5.89%, a pathogenic or likely pathogenic variant was found, most commonly in SOD1, TARDBP, FUS, NEK1, OPTN or TBK1. Significantly more cases carried at least one pathogenic or likely pathogenic variant compared to controls (OR 1.75; p-value 1.64 × 10−5). Isolated risk factors in ATXN1, ATXN2, NIPA1 and/or UNC13A were detected in 17.33% of cases. In 71.83%, we did not find any genetic clues. A combination of variants was found in 2.88%. This study provides an inventory of pathogenic and likely pathogenic genetic variation in a large cohort of sALS. Overall, we identified pathogenic and likely pathogenic variants in 11.13% of ALS patients in 38 known ALS genes. In line with the oligogenic hypothesis, we found significantly more combinations of variants in cases compared to controls. Many variants of unknown significance may contribute to ALS risk, but diagnostic algorithms to reliably identify and weigh them are lacking. This work can serve as a resource for counselling and for the assembly of gene panels for ALS. Further characterization of the genetic architecture of sALS is necessary given the growing interest in gene testing in ALS.

Supplementary Figure 1 depicts copy number alterations in the described cohort

The short-chain fatty acid sodium butyrate (NaB), produced in the colonic lumen, induces cell cycle arrest, differentiation, and/or apoptosis in colorectal carcinoma cells in vitro, establishing a potential role for NaB in colon cancer prevention. We have previously shown that butyrate decreases cyclin D1 and c-myc expression, each essential for intestinal tumor development, by transcriptional attenuation. Here, we determined that butyrate-induced transcriptional attenuation of the cyclin D1 and c-myc genes in SW837 human colorectal adenocarcinoma cells occurs at ∼100 nucleotides downstream of the transcription start site, with a similar positioning in Caco-2 cells. A concomitant decrease in RNA polymerase II occupancy at the 5′ end of each gene was observed. Because transcriptional regulation is associated with chromatin remodeling, we investigated by chromatin immunoprecipitation whether the histone deacetylase inhibitory activity of butyrate altered chromatin structure at the attenuated loci. Although the distributions of histone H3 trimethylated on K4 and K36 along the cyclin D1 and c-myc genes were consistent with current models, butyrate induced only modest decreases in these modifications, with a similar effect on acetylated H3 and a modest increase in histone H3 trimethylated on K27. Finally, transcriptome analysis using novel microarrays showed that butyrate-induced attenuation is widespread throughout the genome, likely independent of transcriptional initiation. We identified 42 loci potentially paused by butyrate and showed that the transcription patterns are gene specific. The biological functions of these loci encompass a number of effects of butyrate on the physiology of intestinal epithelial cells. Cancer Prev Res; 3(10); 1292–302. ©2010 AACR.

Supplementary Figure 1 from Transcriptional Attenuation in Colon Carcinoma Cells in Response to Butyrate

Supplementary Figure 2 cont. from Transcriptional Attenuation in Colon Carcinoma Cells in Response to Butyrate

Supplementary Table 1 shows point mutations found in DSRCT samples

Supplementary Figure 3 provides a breakdown of the structural variant types found via whole genome sequencing in DSRCT samples

Perspective on This Article from Abdominal Obesity, Independent from Caloric Intake, Accounts for the Development of Intestinal Tumors in Apc1638N/+ Female Mice

Supplementary Table 2 from Transcriptional Attenuation in Colon Carcinoma Cells in Response to Butyrate

Supplementary Figure 2 depicts whole genome sequencing findings

Desmoplastic small round cell tumor (DSRCT) is characterized by the EWSR1–WT1 t(11;22) (p13:q12) translocation. Few additional putative drivers have been identified, and research has suffered from a lack of model systems. Next-generation sequencing (NGS) data from 68 matched tumor-normal samples, whole-genome sequencing data from 10 samples, transcriptomic and affymetrix array data, and a bank of DSRCT patient-derived xenograft (PDX) are presented. EWSR1–WT1 fusions were noted to be simple, balanced events. Recurrent mutations were uncommon, but were noted in TERT (3%), ARID1A (6%), HRAS (5%), and TP53 (3%), and recurrent loss of heterozygosity (LOH) at 11p, 11q, and 16q was identified in 18%, 22%, and 34% of samples, respectively. Comparison of tumor-normal matched versus unmatched analysis suggests overcalling of somatic mutations in prior publications of DSRCT NGS data. Alterations in fibroblast growth factor receptor 4 (FGFR4) were identified in 5 of 68 (7%) of tumor samples, whereas differential overexpression of FGFR4 was confirmed orthogonally using 2 platforms. PDX models harbored the pathognomic EWSR1–WT1 fusion and were highly representative of corresponding tumors. Our analyses confirm DSRCT as a genomically quiet cancer defined by the balanced translocation, t(11;22)(p13:q12), characterized by a paucity of secondary mutations but a significant number of copy number alterations. Against this genomically quiet background, recurrent activating alterations of FGFR4 stood out, and suggest that this receptor tyrosine kinase, also noted to be highly expressed in DSRCT, should be further investigated. Future studies of DSRCT biology and preclinical therapeutic strategies should benefit from the PDX models characterized in this study.Implications:These data describe the general quiescence of the desmoplastic small round cell tumor (DSRCT) genome, present the first available bank of DSRCT model systems, and nominate FGFR4 as a key receptor tyrosine kinase in DSRCT, based on high expression, recurrent amplification, and recurrent activating mutations.

Supplementary Figure 4 shows data related to FGFR4 inhibition in DSRCT and control models

Background People with human immunodeficiency virus (PWH) are at increased risk for comorbidities, and plasma interleukin 6 (IL-6) levels are among the most robust predictors of these outcomes. Tocilizumab (TCZ) blocks the receptor for IL-6, inhibiting functions of this cytokine. Methods This was a 40-week, placebo-controlled, crossover trial (NCT02049437) where PWH on stable antiretroviral therapy (ART) were randomized to receive 3 monthly doses of TCZ or matching placebo intravenously. Following a 10-week treatment period and a 12-week washout, participants were switched to the opposite treatment. The primary endpoints were safety and posttreatment levels of C-reactive protein (CRP) and CD4+ T-cell cycling. Secondary endpoints included changes in inflammatory indices and lipid levels. Results There were 9 treatment-related toxicities of grade 2 or greater during TCZ administration (mostly neutropenia) and 2 during placebo administration. Thirty-one of 34 participants completed the study and were included in a modified intent-to-treat analysis. TCZ reduced levels of CRP (median decrease, 1819.9 ng/mL, P < .0001; effect size, 0.87) and reduced inflammatory markers in PWH, including D-dimer, soluble CD14, and tumor necrosis factor receptors. T-cell cycling tended to decrease in all maturation subsets after TCZ administration, but was only significant among naive CD4 T cells. Lipid levels, including lipid classes that have been related to cardiovascular disease risk, increased during TCZ treatment. Conclusions TCZ is safe and decreases inflammation in PWH; IL-6 is a key driver of the inflammatory environment that predicts morbidity and mortality in ART-treated PWH. The clinical significance of lipid elevations during TCZ treatment requires further study.

To determine whether visceral fat (VF), independent of other confounders, is causally linked to intestinal tumorigenesis, we surgically removed visceral fat in Apc1638/N+ mice. At 15 weeks of age, male and female Apc1638/N+ mice were randomized to one of three groups: ad libitum, visceral fat removal (VF-) and ad libitum fed, or caloric restriction, and were studied for effects on tumorigenesis and survival. As compared with ad libitum, VF− and caloric restriction reduced macroadenomas to a similar extent (P < 0.05), but only caloric restriction significantly improved survival (P < 0.05). Given that a significant group × gender interaction was observed, we next examined males and females separately. In females, macroadenomas were markedly attenuated by VF− (1.33 ± 0.23 mean ± SE; P < 0.05), but not by caloric restriction (2.35 ± 0.25; P = 0.71), as compared with ad libitum (2.50 ± 0.34). In males, however, caloric restriction (1.71 ± 0.26; P < 0.01), but not VF− (2.94 ± 0.42; P = 0.29), reduced macroadenomas, as compared with ad libitum males (3.47 ± 0.30). In females, both VF− (P = 0.05) and caloric restriction (P < 0.01) improved survival, but not in male mice (P = 0.15). The benefits observed with caloric restriction were consistent with favorable metabolic adaptations, but protection conferred in VF− females was despite lower adiponectin levels (P < 0.05), and failure to reduce body mass, total adiposity, glucose, insulin, leptin, and chemokine (C–X–C motif) ligand 1 (CXCL-1) levels. In conclusion, these data provide the first causal evidence linking visceral fat to intestinal cancer risk, and suggest that factors, other than known metabolic mediators, may impact tumor development. Furthermore, these data emphasize that strategies designed to deplete visceral fat stores in humans should be considered in the prevention of intestinal cancer. Cancer Prev Res; 6(3); 177–87. ©2012 AACR.

Supplementary Figure 2 from Transcriptional Attenuation in Colon Carcinoma Cells in Response to Butyrate

Supplementary Table 2 shows the raw data further described in Figure 3

supplementary table

Absolute numbers of CD3+ and CD4+ HLA-DR+ activated T cells increase at day 21 and day 42 following ipilimumab infusion.

Tumor mutational age (TMB) by age (p=0.58 for fusion-negative patients, Pearson coefficient = -0.07, R2 = 0.005; and p=0.04 for fusion-positive patients, Pearson coefficient = 0.44, R2 = 0.20)

Purpose:Increased availability of next-generation sequencing has allowed for the genomic characterization of a variety of pediatric tumors, although genomic determinants of response to treatment remain largely unknown. We sought to evaluate the genomic landscape and genomic determinants of clinical outcomes in rhabdomyosarcoma (RMS).Experimental Design:Of 29,067 patients who underwent genomic profiling at our institution using a 468-gene oncopanel with complete records, 87 had RMS, of whom 22 were fusion positive. The 10 most common genetic alterations were associated with locoregional control (LC), disease-free survival (DFS), and overall survival (OS). Tumor mutational burden (TMB), defined as the total number of somatic nonsynonymous mutations normalized to the number of sequenced megabases, was also associated with clinical outcomes.Results:Median age at diagnosis was 16.4 years and median follow-up, 2.1 years. Patients with fusion-negative RMS had more genomic alterations and a higher TMB than those with fusion-positive RMS (mean number of genomic alterations, 6.0 vs. 2.9; P = 0.007 and mean TMB, 2.6 vs. 1.0; P = 0.01). Genetic alterations in TP53 were associated with worse OS (P = 0.03). High TMB (defined as the top quartile ≥ 2.8) was associated with worse LC (P = 0.05), DFS (P = 0.04), and OS (P = 0.01), with significance retained on multivariable analysis after controlling for risk group, fusion status, and receipt of chemotherapy as per pediatric protocols.Conclusions:High TMB was associated with worse clinical outcomes in patients with RMS. With further validation, TMB and other genomic classifiers may be combined with traditional clinicopathologic risk factors to guide risk stratification and ultimately treatment decisions.

Purpose: Ipilimumab is a first-in-class immune checkpoint inhibitor approved for treatment of metastatic melanoma but not studied in children until this phase I protocol.Experimental Design: This study examined safety, pharmacokinetics, and immunogenicity, and immune correlates of ipilimumab administered to subjects ≤21 years old with recurrent or progressive solid tumors. Dose escalation cohorts received 1, 3, 5, or 10 mg/m2 intravenously every 3 weeks in a 3 + 3 design. Response was assessed after 6 weeks and 12 weeks, and then every 3 months. Treatment was continued until disease progression or unacceptable toxicity.Results: Thirty-three patients received 72 doses of ipilimumab. Patients enrolled had melanoma (n = 12), sarcoma (n = 17), or other refractory solid tumors (n = 4). Immune-related adverse events included pancreatitis, pneumonitis, colitis, endocrinopathies, and transaminitis with dose-limiting toxicities observed at 5 and 10 mg/kg dose levels. Pharmacokinetics revealed a half-life of 8 to 15 days. At day 21, subjects had increased levels of cycling T cells, but no change in regulatory T-cell populations. Six subjects had confirmed stable disease for 4 to 10 cycles (melanoma, osteosarcoma, clear cell sarcoma, and synovial sarcoma).Conclusions: Ipilimumab was safely administered to pediatric patients using management algorithms for immune-related toxicities. The spectrum of immune-related adverse events is similar to those described in adults; however, many of the pediatric toxicities were evident after a single dose. Although no objective tumor regressions were observed with ipilimumab as a single agent, subjects with immune-related toxicities had an increased overall survival compared with those who showed no evidence of breaking tolerance. Clin Cancer Res; 22(6); 1364–70. ©2015 AACR.

Supplementary Figure Legends

TP53 mutational status by age (p=0.05)

Supplementary Legends for Figures 1-5, Table 1 from An A13 Repeat within the 3′-Untranslated Region of Epidermal Growth Factor Receptor (EGFR) Is Frequently Mutated in Microsatellite Instability Colon Cancers and Is Associated with Increased EGFR Expression