Mucus-degrading Bacteroides link carbapenems to aggravated graft-versus-host disease.

The intestinal microbiota is an important modulator of graft-versus-host disease (GVHD), which often complicates allogeneic hematopoietic stem cell transplantation (allo-HSCT). Broad-spectrum antibiotics such as carbapenems increase the risk for intestinal GVHD, but mechanisms are not well understood. In this study, we found that treatment with meropenem, a commonly used carbapenem, aggravates colonic GVHD in mice via the expansion of Bacteroides thetaiotaomicron (BT). BT has a broad ability to degrade dietary polysaccharides and host mucin glycans. BT in meropenem-treated allogeneic mice demonstrated upregulated expression of enzymes involved in the degradation of mucin glycans. These mice also had thinning of the colonic mucus layer and decreased levels of xylose in colonic luminal contents. Interestingly, oral xylose supplementation significantly prevented thinning of the colonic mucus layer in meropenem-treated mice. Specific nutritional supplementation strategies, including xylose supplementation, may combat antibiotic-mediated microbiome injury to reduce the risk for intestinal GVHD in allo-HSCT patients.

COVID-19 and pregnancy: A European study on pre- and post-infection medication use.

PURPOSE: The COVID-19 pandemic has impacted medication needs and prescribing practices, including those affecting pregnant women. Our goal was to investigate patterns of medication use among pregnant women with COVID-19, focusing on variations by trimester of infection and location. METHODS: We conducted an observational study using six electronic healthcare databases from six European regions (Aragon/Spain; France; Norway; Tuscany, Italy; Valencia/Spain; and Wales/UK). The prevalence of primary care prescribing or dispensing was compared in the 30-day periods before and after a positive COVID-19 test or diagnosis. RESULTS: The study included 294,126 pregnant women, of whom 8943 (3.0%) tested positive for, or were diagnosed with, COVID-19 during their pregnancy. A significantly higher use of antithrombotic medications was observed particularly after COVID-19 infection in the second and third trimesters. The highest increase was observed in the Valencia region where use of antithrombotic medications in the third trimester increased from 3.8% before COVID-19 to 61.9% after the infection. Increases in other countries were lower; for example, in Norway, the prevalence of antithrombotic medication use changed from around 1-2% before to around 6% after COVID-19 in the third trimester. Smaller and less consistent increases were observed in the use of other drug classes, such as antimicrobials and systemic corticosteroids. CONCLUSION: Our findings highlight the substantial impact of COVID-19 on primary care medication use among pregnant women, with a marked increase in the use of antithrombotic medications post-COVID-19. These results underscore the need for further research to understand the broader implications of these patterns on maternal and neonatal/fetal health outcomes.

DNA methylation-based classification of glioneuronal tumours synergises with histology and radiology to refine accurate molecular stratification.

AIMS: Glioneuronal tumours (GNTs) are poorly distinguished by their histology and lack robust diagnostic indicators. Previously, we showed that common GNTs comprise two molecularly distinct groups, correlating poorly with histology. To refine diagnosis, we constructed a methylation-based model for GNT classification, subsequently evaluating standards for molecular stratification by methylation, histology and radiology. METHODS: We comprehensively analysed methylation, radiology and histology for 83 GNT samples: a training cohort of 49, previously classified into molecularly defined groups by genomic profiles, plus a validation cohort of 34. We identified histological and radiological correlates to molecular classification and constructed a methylation-based support vector machine (SVM) model for prediction. Subsequently, we contrasted methylation, radiological and histological classifications in validation GNTs. RESULTS: By methylation clustering, all training and 23/34 validation GNTs segregated into two groups, the remaining 11 clustering alongside control cortex. Histological review identified prominent astrocytic/oligodendrocyte-like components, dysplastic neurons and a specific glioneuronal element as discriminators between groups. However, these were present in only a subset of tumours. Radiological review identified location, margin definition, enhancement and T2 FLAIR-rim sign as discriminators. When validation GNTs were classified by SVM, 22/23 classified correctly, comparing favourably against histology and radiology that resolved 17/22 and 15/21, respectively, where data were available for comparison. CONCLUSIONS: Diagnostic criteria inadequately reflect glioneuronal tumour biology, leaving a proportion unresolvable. In the largest cohort of molecularly defined glioneuronal tumours, we develop molecular, histological and radiological approaches for biologically meaningful classification and demonstrate almost all cases are resolvable, emphasising the importance of an integrated diagnostic approach.

Porphyromonas gingivalis initiates a mesenchymal-like transition through ZEB1 in gingival epithelial cells.

The oral anaerobe Porphyromonas gingivalis is associated with the development of cancers including oral squamous cell carcinoma (OSCC). Here, we show that infection of gingival epithelial cells with P. gingivalis induces expression and nuclear localization of the ZEB1 transcription factor, which controls epithelial-mesenchymal transition. P. gingivalis also caused an increase in ZEB1 expression as a dual species community with Fusobacterium nucleatum or Streptococcus gordonii. Increased ZEB1 expression was associated with elevated ZEB1 promoter activity and did not require suppression of the miR-200 family of microRNAs. P. gingivalis strains lacking the FimA fimbrial protein were attenuated in their ability to induce ZEB1 expression. ZEB1 levels correlated with an increase in expression of mesenchymal markers, including vimentin and MMP-9, and with enhanced migration of epithelial cells into matrigel. Knockdown of ZEB1 with siRNA prevented the P. gingivalis-induced increase in mesenchymal markers and epithelial cell migration. Oral infection of mice by P. gingivalis increased ZEB1 levels in gingival tissues, and intracellular P. gingivalis were detected by antibody staining in biopsy samples from OSCC. These findings indicate that FimA-driven ZEB1 expression could provide a mechanistic basis for a P. gingivalis contribution to OSCC.

Maternal and Neonatal Outcomes of Adolescent Pregnancies in the Military Health System.

OBJECTIVE: To determine if universal access to care for military beneficiaries improves timing of presentation to prenatal care (PNC) in adolescent and young adult (AYA) pregnancies, improving maternal and neonatal outcomes. STUDY DESIGN: Retrospective descriptive cohort study, which assessed PNC initiation in eligible military beneficiaries: dependent daughters, active-duty women, and active-duty spouses aged 13 to 26 between January 2015 and December 2019, and subsequent adverse maternal and neonatal outcomes. RESULTS: The cohort included 4,557 eligible pregnancies and 4,044 mothers aged 13 to 26. Late entry to PNC was not associated with gestational diabetes, prolonged rupture of membranes, pregnancy loss, elective abortion, substance use, or premature labor. Younger age was significantly associated with substance use, elective abortion, and sexually transmitted infection. There were 2,107 eligible newborns. There was no significant difference in gestational age at birth, incidence of prematurity, birthweight percentile, or occurrence of a neonatal intensive care unit admission based on maternal age. In comparison to published national outcomes, there was a significantly smaller occurrence of preterm (5.3% vs. 9.57-10.23%, 95% CI, 4.4-6.4%), small for gestational age (5.2% vs. 10-13%, 95% CI, 4.3-6.2%), and large for gestational age (4.8% vs. 9%, 95% CI, 4.0-5.8%) births, but a higher occurrence of neonatal intensive care unit admissions (16.9% vs. 7.8-14.4%, 95% CI, 15.4-18.6%) in infants born to military beneficiaries. CONCLUSIONS: Our findings suggest that expanded universal access to health care may improve AYA pregnancy and delivery outcomes. Infants born to AYA military beneficiaries have improved neonatal outcomes compared to nationally published data. These results may correlate to improved maternal access within a free or low-cost healthcare system.

Tsyn-Seq: a T-cell Synapse-Based Antigen Identification Platform.

Tools for genome-wide rapid identification of peptide-major histocompatibility complex targets of T-cell receptors (TCR) are not yet universally available. We present a new antigen screening method, the T-synapse (Tsyn) reporter system, which includes antigen-presenting cells (APC) with a Fas-inducible NF-κB reporter and T cells with a nuclear factor of activated T cells (NFAT) reporter. To functionally screen for target antigens from a cDNA library, productively interacting T cell-APC aggregates were detected by dual-reporter activity and enriched by flow sorting followed by antigen identification quantified by deep sequencing (Tsyn-seq). When applied to a previously characterized TCR specific for the E7 antigen derived from human papillomavirus type 16 (HPV16), Tsyn-seq successfully enriched the correct cognate antigen from a cDNA library derived from an HPV16-positive cervical cancer cell line. Tsyn-seq provides a method for rapidly identifying antigens recognized by TCRs of interest from a tumor cDNA library. See related Spotlight by Makani and Joglekar, p. 515.

Bacteroides ovatus alleviates dysbiotic microbiota-induced intestinal graft-versus-host disease.

Acute gastrointestinal intestinal GVHD (aGI-GVHD) is a serious complication of allogeneic hematopoietic stem cell transplantation, and the intestinal microbiota is known to impact on its severity. However, an association between treatment response of aGI-GVHD and the intestinal microbiota has not been well-studied. In a cohort of patients with aGI-GVHD (n=37), we found that non-response to standard therapy with corticosteroids was associated with prior treatment with carbapenem antibiotics and loss of Bacteroides ovatus from the microbiome. In a mouse model of carbapenem-aggravated GVHD, introducing Bacteroides ovatus reduced severity of GVHD and improved survival. Bacteroides ovatus reduced degradation of colonic mucus by another intestinal commensal, Bacteroides thetaiotaomicron, via its ability to metabolize dietary polysaccharides into monosaccharides, which then inhibit mucus degradation by Bacteroides thetaiotaomicron and reduce GVHD-related mortality.

Examining the role of carbonation and temperature on water swallowing performance: a swallowing reaction-time study.

Various therapeutic approaches for dysphagia management are based on modifications of bolus properties to change swallowing biomechanics and increase swallowing safety. Limited evidence exists for the effects of carbonation and bolus temperature on swallowing behavior. Here, we investigated the effects of carbonation and temperature on swallowing behavior using a novel automated and complex swallowing reaction time task via pressure signal recordings in the hypopharynx. Healthy participants (n = 39, 27.7±5 years old) were randomized in two different experiments and asked to perform 10 normal-paced swallows, 10 fast-paced swallows, and 10 challenged swallows within a predetermined time-window of carbonated versus still water (experiment 1) and of cold (4 °C) versus hot (45 °C) versus room temperature (21 °C) water (experiment 2). Quantitative measurements of latencies and percentage of successful challenged swallows were collected and analyzed nonparametrically. An increase in successfully performed challenged swallowing task was observed with carbonated water versus still water (P = 0.021), whereas only cold water shortened the latencies of normally paced swallows compared with room (P = 0.001) and hot (P = 0.004) temperatures. Therefore, it appears that chemothermal stimulation with carbonation and cold are most effective at modulating water swallowing, which in part is likely to be driven by central swallowing afferent activity.

The environmental impact of industrialization and foreign direct investment: empirical evidence from Asia-Pacific region.

Environmental degradation has been the main distress in recent years due to the drastic effect of climate change. To determine the gone thorough impact of industrialization and foreign direct investment on environmental degradation, this study utilized panel data of 55 countries of the Asia-Pacific region from 1995 to 2020 and it applies an autoregressive distributed lag (ARDL) model. The results showed that FDI, in general, has a significant negative impact on the environment and causes to increase in methane and CO2 emissions. Moreover, industrialization has a positive and significant impact on the environment. However, the size of the impact is moderate. This study also concludes that in the Asia-Pacific region, the environment Kuznets curve (EKC) and pollution heaven (PH) hypothesis are accepted. Finally, this study suggests the strict implication of environmental guidelines or the adoption of a new policy would be the key to ensuring the quality of the environment. Furthermore, the results confirmed that most of the panel countries are developing countries and do not have strict environmental management guidelines.

Post-Injury Buprenorphine Administration Is Associated with Long-Term Region-Specific Glial Alterations in Rats.

Traumatic brain injury (TBI) is a major leading cause of death and disability. While previous studies regarding focal pathologies following TBI have been done, there is a lack of information concerning the role of analgesics and their influences on injury pathology. Buprenorphine (Bup), an opioid analgesic, is a commonly used analgesic in experimental TBI models. Our previous studies investigated the acute effects of Buprenorphine-sustained release-Lab (Bup-SR-Lab) on diffuse neuronal/glial pathology, neuroinflammation, cell damage, and systemic physiology. The current study investigated the longer-term chronic outcomes of Bup-SR-Lab treatment at 4 weeks following TBI utilizing a central fluid percussion injury (cFPI) model in adult male rats. Histological assessments of physiological changes, neuronal damage, cortical and thalamic cytokine expression, microglial and astrocyte morphological changes, and myelin alterations were done, as we had done in our acute study. In the current study the Whisker Nuisance Task (WNT) was also performed pre- and 4w post-injury to assess changes in somatosensory sensitivity following saline or Bup-SR-Lab treatment. Bup-SR-Lab treatment had no impact on overall physiology or neuronal damage at 4w post-injury regardless of region or injury, nor did it have any significant effects on somatosensory sensitivity. However, greater IL-4 cytokine expression with Bup-SR-Lab treatment was observed compared to saline treated animals. Microglia and astrocytes also demonstrated region-specific morphological alterations associated with Bup-SR-Lab treatment, in which cortical microglia and thalamic astrocytes were particularly vulnerable to Bup-mediated changes. There were discernable injury-specific and region-specific differences regarding myelin integrity and changes in specific myelin basic protein (MBP) isoform expression following Bup-SR-Lab treatment. This study indicates that use of Bup-SR-Lab could impact TBI-induced glial alterations in a region-specific manner 4w following diffuse brain injury.

Assessment of Surgeon Performance of Advanced Open Surgical Skills Using a Microskills-Based Novel Curriculum.

Importance: The increase in minimally invasive surgical procedures has eroded exposure of general surgery residents to open operations. High-fidelity simulation, together with deliberate instruction, is needed for advanced open surgical skill (AOSS) development. Objective: To collect validity evidence for AOSS tools to support a shared model for instruction. Design, Setting, and Participants: This prospective cohort study included postresidency surgeons (PRSs) and second-year general surgery residents (R2s) at a single academic medical center who completed simulated tasks taught within the AOSS curriculum between June 1 and October 31, 2021. Exposures: The AOSS curriculum includes 6 fine-suture and needle handling tasks, including deep suture tying (with and without needles) and continuous suturing using the pitch-and-catch and push-push-pull techniques (both superficial and deep). Teaching and assessment are based on specific microskills using a 3-dimensional printed iliac fossa model. Main Outcomes and Measures: The PRS group was timed and scored (5-point Likert scale) on 10 repetitions of each task. Six months after receiving instruction on the AOSS tasks, the R2 group was similarly timed and scored. Results: The PRS group included 14 surgeons (11 male [79%]; 8 [57%] attending surgeons) who completed the simulation; the R2 group, 9 surgeons (5 female [55%]) who completed the simulation. Score and time variability were greater for the R2s compared with the PRSs for all tasks. The R2s scored lower and took longer on (1) deep pitch-and-catch suturing (69% of maximum points for a mean [SD] of 142.0 [31.7] seconds vs 77% for a mean [SD] of 95.9 [29.4] seconds) and deep push-push-pull suturing (63% of maximum points for a mean [SD] of 284.0 [72.9] seconds vs 85% for a mean [SD] of 141.4 [29.1] seconds) relative to the corresponding superficial tasks; (2) suture tying with a needle vs suture tying without a needle (74% of maximum points for a mean [SD] of 64.6 [19.8] seconds vs 90% for a mean [SD] of 54.4 [15.6] seconds); and (3) the deep push-push-pull vs pitch-and-catch techniques (63% of maximum points for a mean [SD] of 284.0 [72.9] seconds vs 69% of maximum points for a mean [SD] of 142.0 [31.7] seconds). For the PRS group, time was negatively associated with score for the 3 hardest tasks: superficial push-push-pull (ρ = 0.60; P = .02), deep pitch-and-catch (ρ = 0.73; P = .003), and deep push-push-pull (ρ = 0.81; P < .001). For the R2 group, time was negatively associated with score for the 2 easiest tasks: suture tying without a needle (ρ = 0.78; P = .01) and superficial pitch-and-catch (ρ = 0.79; P = .01). Conclusions and Relevance: The findings of this cohort study offer validity evidence for a novel AOSS curriculum; reveal differential difficulty of tasks that can be attributed to specific microskills; and suggest that position on the surgical learning curve may dictate the association between competency and speed. Together these findings suggest specific, actionable opportunities to guide instruction of AOSS, including which microskills to focus on, when individual rehearsal vs guided instruction is more appropriate, and when to focus on speed.

Diet-derived metabolites and mucus link the gut microbiome to fever after cytotoxic cancer treatment.

Not all patients with cancer and severe neutropenia develop fever, and the fecal microbiome may play a role. In a single-center study of patients undergoing hematopoietic cell transplant (n = 119), the fecal microbiome was characterized at onset of severe neutropenia. A total of 63 patients (53%) developed a subsequent fever, and their fecal microbiome displayed increased relative abundances of Akkermansia muciniphila, a species of mucin-degrading bacteria (P = 0.006, corrected for multiple comparisons). Two therapies that induce neutropenia, irradiation and melphalan, similarly expanded A. muciniphila and additionally thinned the colonic mucus layer in mice. Caloric restriction of unirradiated mice also expanded A. muciniphila and thinned the colonic mucus layer. Antibiotic treatment to eradicate A. muciniphila before caloric restriction preserved colonic mucus, whereas A. muciniphila reintroduction restored mucus thinning. Caloric restriction of unirradiated mice raised colonic luminal pH and reduced acetate, propionate, and butyrate. Culturing A. muciniphila in vitro with propionate reduced utilization of mucin as well as of fucose. Treating irradiated mice with an antibiotic targeting A. muciniphila or propionate preserved the mucus layer, suppressed translocation of flagellin, reduced inflammatory cytokines in the colon, and improved thermoregulation. These results suggest that diet, metabolites, and colonic mucus link the microbiome to neutropenic fever and may guide future microbiome-based preventive strategies.

An Sp1/Sp3 binding polymorphism confers methylation protection.

Hundreds of genes show aberrant DNA hypermethylation in cancer, yet little is known about the causes of this hypermethylation. We identified RIL as a frequent methylation target in cancer. In search for factors that influence RIL hypermethylation, we found a 12-bp polymorphic sequence around its transcription start site that creates a long allele. Pyrosequencing of homozygous tumors revealed a 2.1-fold higher methylation for the short alleles (P<0.001). Bisulfite sequencing of cancers heterozygous for RIL showed that the short alleles are 3.1-fold more methylated than the long (P<0.001). The comparison of expression levels between unmethylated long and short EBV-transformed cell lines showed no difference in expression in vivo. Electrophorectic mobility shift assay showed that the inserted region of the long allele binds Sp1 and Sp3 transcription factors, a binding that is absent in the short allele. Transient transfection of RIL allele-specific transgenes showed no effects of the additional Sp1 site on transcription early on. However, stable transfection of methylation-seeded constructs showed gradually decreasing transcription levels from the short allele with eventual spreading of de novo methylation. In contrast, the long allele showed stable levels of expression over time as measured by luciferase and approximately 2-3-fold lower levels of methylation by bisulfite sequencing (P<0.001), suggesting that the polymorphic Sp1 site protects against time-dependent silencing. Our finding demonstrates that, in some genes, hypermethylation in cancer is dictated by protein-DNA interactions at the promoters and provides a novel mechanism by which genetic polymorphisms can influence an epigenetic state.

Sensitive and specific detection of early gastric cancer with DNA methylation analysis of gastric washes.

BACKGROUND & AIMS: Aberrant DNA methylation is an early and frequent process in gastric carcinogenesis and could be useful for detection of gastric neoplasia. We hypothesized that methylation analysis of DNA recovered from gastric washes could be used to detect gastric cancer. METHODS: We studied 51 candidate genes in 7 gastric cancer cell lines and 24 samples (training set) and identified 6 for further studies. We examined the methylation status of these genes in a test set consisting of 131 gastric neoplasias at various stages. Finally, we validated the 6 candidate genes in a different population of 40 primary gastric cancer samples and 113 nonneoplastic gastric mucosa samples. RESULTS: Six genes (MINT25, RORA, GDNF, ADAM23, PRDM5, MLF1) showed frequent differential methylation between gastric cancer and normal mucosa in the training, test, and validation sets. GDNF and MINT25 were most sensitive molecular markers of early stage gastric cancer, whereas PRDM5 and MLF1 were markers of a field defect. There was a close correlation (r = 0.5-0.9, P = .03-.001) between methylation levels in tumor biopsy and gastric washes. MINT25 methylation had the best sensitivity (90%), specificity (96%), and area under the receiver operating characteristic curve (0.961) in terms of tumor detection in gastric washes. CONCLUSIONS: These findings suggest MINT25 is a sensitive and specific marker for screening in gastric cancer. Additionally, we have developed a new method for gastric cancer detection by DNA methylation in gastric washes.

Genome-wide profiling of DNA methylation reveals a class of normally methylated CpG island promoters.

The role of CpG island methylation in normal development and cell differentiation is of keen interest, but remains poorly understood. We performed comprehensive DNA methylation profiling of promoter regions in normal peripheral blood by methylated CpG island amplification in combination with microarrays. This technique allowed us to simultaneously determine the methylation status of 6,177 genes, 92% of which include dense CpG islands. Among these 5,549 autosomal genes with dense CpG island promoters, we have identified 4.0% genes that are nearly completely methylated in normal blood, providing another exception to the general rule that CpG island methylation in normal tissue is limited to X inactivation and imprinted genes. We examined seven genes in detail, including ANKRD30A, FLJ40201, INSL6, SOHLH2, FTMT, C12orf12, and DPPA5. Dense promoter CpG island methylation and gene silencing were found in normal tissues studied except testis and sperm. In both tissues, bisulfite cloning and sequencing identified cells carrying unmethylated alleles. Interestingly, hypomethylation of several genes was associated with gene activation in cancer. Furthermore, reactivation of silenced genes could be induced after treatment with a DNA demethylating agent or in a cell line lacking DNMT1 and/or DNMT3b. Sequence analysis identified five motifs significantly enriched in this class of genes, suggesting that cis-regulatory elements may facilitate preferential methylation at these promoter CpG islands. We have identified a group of non-X-linked bona fide promoter CpG islands that are densely methylated in normal somatic tissues, escape methylation in germline cells, and for which DNA methylation is a primary mechanism of tissue-specific gene silencing.

Integrated genetic and epigenetic analysis identifies three different subclasses of colon cancer.

Colon cancer has been viewed as the result of progressive accumulation of genetic and epigenetic abnormalities. However, this view does not fully reflect the molecular heterogeneity of the disease. We have analyzed both genetic (mutations of BRAF, KRAS, and p53 and microsatellite instability) and epigenetic alterations (DNA methylation of 27 CpG island promoter regions) in 97 primary colorectal cancer patients. Two clustering analyses on the basis of either epigenetic profiling or a combination of genetic and epigenetic profiling were performed to identify subclasses with distinct molecular signatures. Unsupervised hierarchical clustering of the DNA methylation data identified three distinct groups of colon cancers named CpG island methylator phenotype (CIMP) 1, CIMP2, and CIMP negative. Genetically, these three groups correspond to very distinct profiles. CIMP1 are characterized by MSI (80%) and BRAF mutations (53%) and rare KRAS and p53 mutations (16% and 11%, respectively). CIMP2 is associated with 92% KRAS mutations and rare MSI, BRAF, or p53 mutations (0, 4, and 31% respectively). CIMP-negative cases have a high rate of p53 mutations (71%) and lower rates of MSI (12%) or mutations of BRAF (2%) or KRAS (33%). Clustering based on both genetic and epigenetic parameters also identifies three distinct (and homogeneous) groups that largely overlap with the previous classification. The three groups are independent of age, gender, or stage, but CIMP1 and 2 are more common in proximal tumors. Together, our integrated genetic and epigenetic analysis reveals that colon cancers correspond to three molecularly distinct subclasses of disease.

Impact of parental injury on adolescent sleep.

STUDY OBJECTIVES: The aim of this study was to determine the impact of serious parental injury on adolescent sleep disorder diagnoses, outpatient care, and medication use. METHODS: U.S. military personnel who sustained a serious injury and were parents of adolescents aged 10-18 years were identified. Included adolescents were enrolled in the Military Health System for 2 years before their parent's injury and 2 years after the injury. We used logistic regression clustered by adolescents to compare the odds of having a sleep diagnosis and negative binomial regression analysis clustered by adolescents to compare outpatient sleep disorder visits and sleep medication days before and after parental injury. RESULTS: There were 96,318 parents seriously injured during 2004-2014 who had 117,577 children aged 10-18 years in 2002-2016. Approximately 2% of adolescents had a sleep disorder diagnosis, both before and after their parent's injury or illness. Outpatient sleep disorder visits increased 36% after a parent's injury (incidence rate ratio 1.36 [1.24-1.50]), with a twofold increase in insomnia visits (incidence rate ratio 2.35 [2.08-2.64]). Increases in sleep visits were most pronounced in adolescents of parents with traumatic brain injury, comorbid traumatic brain injury and posttraumatic stress disorder, battle injury, and those who were medically discharged from the military. The number of adolescents using sleep medications increased, but sleep medication days did not increase. CONCLUSIONS: Adolescents in our study used more outpatient medical care for sleep disorders; sleep medication use increased after parental injury. Sleep disorders should be considered in the care of adolescents with injured parents.

DNA methylation-based profiling for paediatric CNS tumour diagnosis and treatment: a population-based study.

BACKGROUND: Marked variation exists in the use of genomic data in tumour diagnosis, and optimal integration with conventional diagnostic technology remains uncertain despite several studies reporting improved diagnostic accuracy, selection for targeted treatments, and stratification for trials. Our aim was to assess the added value of molecular profiling in routine clinical practice and the impact on conventional and experimental treatments. METHODS: This population-based study assessed the diagnostic and clinical use of DNA methylation-based profiling in childhood CNS tumours using two large national cohorts in the UK. In the diagnostic cohort-which included routinely diagnosed CNS tumours between Sept 1, 2016, and Sept 1, 2018-we assessed how the methylation profile altered or refined diagnosis in routine clinical practice and estimated how this would affect standard patient management. For the archival cohort of diagnostically difficult cases, we established how many cases could be solved using modern standard pathology, how many could only be solved using the methylation profile, and how many remained unsolvable. FINDINGS: Of 484 patients younger than 20 years with CNS tumours, 306 had DNA methylation arrays requested by the neuropathologist and were included in the diagnostic cohort. Molecular profiling added a unique contribution to clinical diagnosis in 107 (35%; 95% CI 30-40) of 306 cases in routine diagnostic practice-providing additional molecular subtyping data in 99 cases, amended the final diagnosis in five cases, and making potentially significant predictions in three cases. We estimated that it could change conventional management in 11 (4%; 95% CI 2-6) of 306 patients. Among 195 historically difficult-to-diagnose tumours in the archival cohort, 99 (51%) could be diagnosed using standard methods, with the addition of methylation profiling solving a further 34 (17%) cases. The remaining 62 (32%) cases were unresolved despite specialist pathology and methylation profiling. INTERPRETATION: Together, these data provide estimates of the impact that could be expected from routine implementation of genomic profiling into clinical practice, and indicate limitations where additional techniques will be required. We conclude that DNA methylation arrays are a useful diagnostic adjunct for childhood CNS tumours. FUNDING: The Brain Tumour Charity, Children with Cancer UK, Great Ormond Street Hospital Children's Charity, Olivia Hodson Cancer Fund, Cancer Research UK, and the National Institute of Health Research.

A tailored molecular profiling programme for children with cancer to identify clinically actionable genetic alterations.

BACKGROUND: For children with cancer, the clinical integration of precision medicine to enable predictive biomarker-based therapeutic stratification is urgently needed. METHODS: We have developed a hybrid-capture next-generation sequencing (NGS) panel, specifically designed to detect genetic alterations in paediatric solid tumours, which gives reliable results from as little as 50 ng of DNA extracted from formalin-fixed paraffin-embedded (FFPE) tissue. In this study, we offered an NGS panel, with clinical reporting via a molecular tumour board for children with solid tumours. Furthermore, for a cohort of 12 patients, we used a circulating tumour DNA (ctDNA)-specific panel to sequence ctDNA from matched plasma samples and compared plasma and tumour findings. RESULTS: A total of 255 samples were submitted from 223 patients for the NGS panel. Using FFPE tissue, 82% of all submitted samples passed quality control for clinical reporting. At least one genetic alteration was detected in 70% of sequenced samples. The overall detection rate of clinically actionable alterations, defined by modified OncoKB criteria, for all sequenced samples was 51%. A total of 8 patients were sequenced at different stages of treatment. In 6 of these, there were differences in the genetic alterations detected between time points. Sequencing of matched ctDNA in a cohort of extracranial paediatric solid tumours also identified a high detection rate of somatic alterations in plasma. CONCLUSION: We demonstrate that tailored clinical molecular profiling of both tumour DNA and plasma-derived ctDNA is feasible for children with solid tumours. Furthermore, we show that a targeted NGS panel-based approach can identify actionable genetic alterations in a high proportion of patients.

Understanding the development of human bladder cancer by using a whole-organ genomic mapping strategy.

The search for the genomic sequences involved in human cancers can be greatly facilitated by maps of genomic imbalances identifying the involved chromosomal regions, particularly those that participate in the development of occult preneoplastic conditions that progress to clinically aggressive invasive cancer. The integration of such regions with human genome sequence variation may provide valuable clues about their overall structure and gene content. By extension, such knowledge may help us understand the underlying genetic components involved in the initiation and progression of these cancers. We describe the development of a genome-wide map of human bladder cancer that tracks its progression from in situ precursor conditions to invasive disease. Testing for allelic losses using a genome-wide panel of 787 microsatellite markers was performed on multiple DNA samples, extracted from the entire mucosal surface of the bladder and corresponding to normal urothelium, in situ preneoplastic lesions, and invasive carcinoma. Using this approach, we matched the clonal allelic losses in distinct chromosomal regions to specific phases of bladder neoplasia and produced a detailed genetic map of bladder cancer development. These analyses revealed three major waves of genetic changes associated with growth advantages of successive clones and reflecting a stepwise conversion of normal urothelial cells into cancer cells. The genetic changes map to six regions at 3q22-q24, 5q22-q31, 9q21-q22, 10q26, 13q14, and 17p13, which may represent critical hits driving the development of bladder cancer. Finally, we performed high-resolution mapping using single nucleotide polymorphism markers within one region on chromosome 13q14, containing the model tumor suppressor gene RB1, and defined a minimal deleted region associated with clonal expansion of in situ neoplasia. These analyses provided new insights on the involvement of several non-coding sequences mapping to the region and identified novel target genes, termed forerunner (FR) genes, involved in early phases of cancer development.