Identifying tumor type and cell type-specific gene expression alterations in pediatric central nervous system tumors.

Central nervous system (CNS) tumors are the leading cause of pediatric cancer death, and these patients have an increased risk for developing secondary neoplasms. Due to the low prevalence of pediatric CNS tumors, major advances in targeted therapies have been lagging compared to other adult tumors. We collect single nuclei RNA-seq data from 84,700 nuclei of 35 pediatric CNS tumors and three non-tumoral pediatric brain tissues and characterize tumor heterogeneity and transcriptomic alterations. We distinguish cell subpopulations associated with specific tumor types including radial glial cells in ependymomas and oligodendrocyte precursor cells in astrocytomas. In tumors, we observe pathways important in neural stem cell-like populations, a cell type previously associated with therapy resistance. Lastly, we identify transcriptomic alterations among pediatric CNS tumor types compared to non-tumor tissues, while accounting for cell type effects on gene expression. Our results suggest potential tumor type and cell type-specific targets for pediatric CNS tumor treatment. Here we address current gaps in understanding single nuclei gene expression profiles of previously under-investigated tumor types and enhance current knowledge of gene expression profiles of single cells of various pediatric CNS tumors.

Associations in cell type-specific hydroxymethylation and transcriptional alterations of pediatric central nervous system tumors.

Although intratumoral heterogeneity has been established in pediatric central nervous system tumors, epigenomic alterations at the cell type level have largely remained unresolved. To identify cell type-specific alterations to cytosine modifications in pediatric central nervous system tumors, we utilize a multi-omic approach that integrated bulk DNA cytosine modification data (methylation and hydroxymethylation) with both bulk and single-cell RNA-sequencing data. We demonstrate a large reduction in the scope of significantly differentially modified cytosines in tumors when accounting for tumor cell type composition. In the progenitor-like cell types of tumors, we identify a preponderance differential Cytosine-phosphate-Guanine site hydroxymethylation rather than methylation. Genes with differential hydroxymethylation, like histone deacetylase 4 and insulin-like growth factor 1 receptor, are associated with cell type-specific changes in gene expression in tumors. Our results highlight the importance of epigenomic alterations in the progenitor-like cell types and its role in cell type-specific transcriptional regulation in pediatric central nervous system tumors.

Treatment options for isolated aortic valve insufficiency: a review.

Aortic insufficiency (AI) is a valvular disease with increasing prevalence in older patients. The modern era provides numerous options for the management of AI which is explored here. Traditional interventions included aortic valve replacement with either mechanical or bioprosthetic aortic valves. While the former is known for its durability, it has grown out of favor due to the potential complications of anticoagulation. The preference for bioprosthetic valves is thus on the rise, especially with the advancements of transcatheter technology and the use of valve-in-valve therapy. Surgical options are also no longer limited to replacement but include complex techniques such as those required for aortic valve repair, Ozaki neocuspidization, Ross procedure and valve-sparring aortic root repair. Transcatheter options for the management of AI are not commercially available currently; however, preliminary data from ad-hoc trials, showed promising results and potential use of transcatheter technology in a variety of patients with pure AI.

Sixteen-Year Institutional Review of Magnetic Resonance Imaging-Guided Breast Biopsies: Trends in Histologic Diagnoses With Radiologic Correlation.

Background: Breast magnetic resonance imaging (MRI) is an important imaging tool for the management of breast cancer patients and for screening women at high risk for breast cancer. Objectives: To examine long-term trends in the distribution of histologic diagnoses obtained from MRI-guided breast biopsies. Design: Retrospective analysis. Methods: We retrospectively reviewed the distribution of histologic diagnoses of MRI-guided breast biopsies from 2004 to 2019. All cases underwent central pathology review and lesions were classified based on the most prominent histologic finding present. Magnetic resonance imaging features were extracted from radiology reports when available and correlated with pathology diagnoses. Results: Four hundred ninety-four MRI-guided biopsies were performed on 440 patients; overall, 73% of biopsies were benign and 27% were malignant. The annual percentages of benign and malignant diagnoses remained similar throughout the 16-year period. Of the benign entities commonly identified, the percentage of benign papillary and sclerosing lesions detected in the benign biopsies increased significantly (13% in 2004-2011 vs 31% in 2012-2019, P = .03). The mean size of malignant lesions was larger than benign lesions (30.1 mm compared with 14.2 mm, P = .045); otherwise, there were no distinguishing radiologic features between benign and malignant lesions. Conclusion: The specificity of breast MRI remained constant over a 16-year period; however, there was a shift in the distribution of benign diagnoses with increased detection and biopsy of benign papillary and sclerosing lesions. Monitoring the distribution of breast MRI biopsy diagnoses over time with radiology-pathology correlation might improve the suboptimal specificity of breast MRI.

Magnesium increases numbers of Foxp3+ Treg cells and reduces arthritis severity and joint damage in an IL-10-dependent manner mediated by the intestinal microbiome.

BACKGROUND: Rheumatoid arthritis (RA) is a common autoimmune disease with emerging environmental and microbiome risk factors. The western diet is typically deficient in magnesium (Mg), and there is some evidence suggesting that Mg may have anti-inflammatory properties. But the actual role of Mg supplementation in arthritis or in T cell subsets has not been explored. METHODS: We investigated the role of a high Mg diet in two different mouse models of RA induced with the KRN serum, and collagen-induced arthritis. We also characterized the phenotypes of splenocytes, gene expression, and an extensive intestinal microbiome analyses including fecal material transplantation (FMT). FINDINGS: The high Mg diet group was significantly protected with reduced arthritis severity and joint damage, and reduced expression of IL-1ß, IL-6, and TNFα. The high Mg group also had increased numbers of Foxp3+ Treg cells and IL-10-producing T cells. The high Mg protective effect disappeared in IL-10 knockout mice. FMT from the high Mg diet mice recreated the phenotypes seen in the diet-treated mice, with reduced arthritis severity, increased Foxp3+ Treg, and increased IL-10-producing T cells. Intestinal microbiome analyses using 16S rDNA sequencing revealed diet-specific changes, including reduced levels of RA-associated Prevotella in the high Mg group, while increasing levels of Bacteroides and other bacteria associated with increased production of short-chain fatty acids. Metagenomic analyses implicated additional pathways including L-tryptophan biosynthesis and arginine deiminase. INTERPRETATION: We describe a new role for Mg in suppressing arthritis, in expanding Foxp3+ T reg cells and in the production of IL-10, and show that these effects are mediated by the intestinal microbiome. Our discoveries suggest a novel strategy for modifying the intestinal microbiome to treat RA and other autoimmune and inflammatory diseases. FUNDING: None.

Tumor type and cell type-specific gene expression alterations in diverse pediatric central nervous system tumors identified using single nuclei RNA-seq.

Central nervous system (CNS) tumors are the leading cause of pediatric cancer death, and these patients have an increased risk for developing secondary neoplasms. Due to the low prevalence of pediatric CNS tumors, major advances in targeted therapies have been lagging compared to other adult tumors. We collected single nuclei RNA-seq data from 35 pediatric CNS tumors and three non-tumoral pediatric brain tissues (84,700 nuclei) and characterized tumor heterogeneity and transcriptomic alterations. We distinguished cell subpopulations associated with specific tumor types including radial glial cells in ependymomas and oligodendrocyte precursor cells in astrocytomas. In tumors, we observed pathways important in neural stem cell-like populations, a cell type previously associated with therapy resistance. Lastly, we identified transcriptomic alterations among pediatric CNS tumor types compared to non-tumor tissues, while accounting for cell type effects on gene expression. Our results suggest potential tumor type and cell type-specific targets for pediatric CNS tumor treatment. In this study, we address current gaps in understanding single nuclei gene expression profiles of previously uninvestigated tumor types and enhance current knowledge of gene expression profiles of single cells of various pediatric CNS tumors.

Hydroxymethylation alterations in progenitor-like cell types of pediatric central nervous system tumors are associated with cell type-specific transcriptional changes.

Although intratumoral heterogeneity has been established in pediatric central nervous system tumors, epigenomic alterations at the cell type level have largely remained unresolved. To identify cell type-specific alterations to cytosine modifications in pediatric central nervous system tumors we utilized a multi-omic approach that integrated bulk DNA cytosine modification data (methylation and hydroxymethylation) with both bulk and single-cell RNA-sequencing data. We demonstrate a large reduction in the scope of significantly differentially modified cytosines in tumors when accounting for tumor cell type composition. In the progenitor-like cell types of tumors, we identified a preponderance differential CpG hydroxymethylation rather than methylation. Genes with differential hydroxymethylation, like HDAC4 and IGF1R, were associated with cell type-specific changes in gene expression in tumors. Our results highlight the importance of epigenomic alterations in the progenitor-like cell types and its role in cell type-specific transcriptional regulation in pediatric CNS tumors.

DNA 5-hydroxymethylcytosine in pediatric central nervous system tumors may impact tumor classification and is a positive prognostic marker.

BACKGROUND: Nucleotide-specific 5-hydroxymethylcytosine (5hmC) remains understudied in pediatric central nervous system (CNS) tumors. 5hmC is abundant in the brain, and alterations to 5hmC in adult CNS tumors have been reported. However, traditional approaches to measure DNA methylation do not distinguish between 5-methylcytosine (5mC) and its oxidized counterpart 5hmC, including those used to build CNS tumor DNA methylation classification systems. We measured 5hmC and 5mC epigenome-wide at nucleotide resolution in glioma, ependymoma, and embryonal tumors from children, as well as control pediatric brain tissues using tandem bisulfite and oxidative bisulfite treatments followed by hybridization to the Illumina Methylation EPIC Array that interrogates over 860,000 CpG loci. RESULTS: Linear mixed effects models adjusted for age and sex tested the CpG-specific differences in 5hmC between tumor and non-tumor samples, as well as between tumor subtypes. Results from model-based clustering of tumors was used to test the relation of cluster membership with patient survival through multivariable Cox proportional hazards regression. We also assessed the robustness of multiple epigenetic CNS tumor classification methods to 5mC-specific data in both pediatric and adult CNS tumors. Compared to non-tumor samples, tumors were hypohydroxymethylated across the epigenome and tumor 5hmC localized to regulatory elements crucial to cell identity, including transcription factor binding sites and super-enhancers. Differentially hydroxymethylated loci among tumor subtypes tended to be hypermethylated and disproportionally found in CTCF binding sites and genes related to posttranscriptional RNA regulation, such as DICER1. Model-based clustering results indicated that patients with low 5hmC patterns have poorer overall survival and increased risk of recurrence. Our results suggest 5mC-specific data from OxBS-treated samples impacts methylation-based tumor classification systems giving new opportunities for further refinement of classifiers for both pediatric and adult tumors. CONCLUSIONS: We identified that 5hmC localizes to super-enhancers, and genes commonly implicated in pediatric CNS tumors were differentially hypohydroxymethylated. We demonstrated that distinguishing methylation and hydroxymethylation is critical in identifying tumor-related epigenetic changes. These results have implications for patient prognostication, considerations of epigenetic therapy in CNS tumors, and for emerging molecular neuropathology classification approaches.

MethylSPWNet and MethylCapsNet: Biologically Motivated Organization of DNAm Neural Networks, Inspired by Capsule Networks.

DNA methylation (DNAm) alterations have been heavily implicated in carcinogenesis and the pathophysiology of diseases through upstream regulation of gene expression. DNAm deep-learning approaches are able to capture features associated with aging, cell type, and disease progression, but lack incorporation of prior biological knowledge. Here, we present modular, user-friendly deep-learning methodology and software, MethylCapsNet and MethylSPWNet, that group CpGs into biologically relevant capsules-such as gene promoter context, CpG island relationship, or user-defined groupings-and relate them to diagnostic and prognostic outcomes. We demonstrate these models' utility on 3,897 individuals in the classification of central nervous system (CNS) tumors. MethylCapsNet and MethylSPWNet provide an opportunity to increase DNAm deep-learning analyses' interpretability by enabling a flexible organization of DNAm data into biologically relevant capsules.

A large-scale internal validation study of unsupervised virtual trichrome staining technologies on nonalcoholic steatohepatitis liver biopsies.

Non-alcoholic steatohepatitis (NASH) is a fatty liver disease characterized by accumulation of fat in hepatocytes with concurrent inflammation and is associated with morbidity, cirrhosis and liver failure. After extraction of a liver core biopsy, tissue sections are stained with hematoxylin and eosin (H&E) to grade NASH activity, and stained with trichrome to stage fibrosis. Methods to computationally transform one stain into another on digital whole slide images (WSI) can lessen the need for additional physical staining besides H&E, reducing personnel, equipment, and time costs. Generative adversarial networks (GAN) have shown promise for virtual staining of tissue. We conducted a large-scale validation study of the viability of GANs for H&E to trichrome conversion on WSI (n = 574). Pathologists were largely unable to distinguish real images from virtual/synthetic images given a set of twelve Turing Tests. We report high correlation between staging of real and virtual stains ([Formula: see text]; 95% CI: 0.84-0.88). Stages assigned to both virtual and real stains correlated similarly with a number of clinical biomarkers and progression to End Stage Liver Disease (Hazard Ratio HR = 2.06, 95% CI: 1.36-3.12, p < 0.001 for real stains; HR = 2.02, 95% CI: 1.40-2.92, p < 0.001 for virtual stains). Our results demonstrate that virtual trichrome technologies may offer a software solution that can be employed in the clinical setting as a diagnostic decision aid.

Preoperative Type and Screen is Unnecessary in Elective Anatomic Lung Resection and Esophagectomy.

BACKGROUND: Preoperative type and screen (TS) is routinely performed before elective thoracic surgery. We sought to evaluate the utility of this practice by examining our institutional data related to intraoperative and postoperative transfusions for two common, complex procedures. MATERIALS AND METHODS: A single-center, retrospective review of a prospective thoracic surgery database was performed. Patients who underwent consecutive elective anatomic lung resection (ALR) and esophagectomy from January 2015 to April 2018 were included. Perioperative characteristics between patients who received transfusion of packed red blood cells and those who did not were compared. The rates of emergent and nonemergent transfusions were evaluated. Cost data were derived from institutional charges and Centers for Medicare & Medicaid Services fee schedules. RESULTS: Of 370 patients, 16 (4.3%) received a transfusion and four (1.1%) were deemed emergent by the surgeons and 0 (0%) by blood bank criteria. For ALR (n = 321), 13 (4.0%) received a transfusion, and four (1.2%) were emergent. For esophagectomies (n = 49), three (6.1%) received a transfusion, and none were emergent. Patients who underwent ALR requiring a transfusion had a lower preoperative hemoglobin (11.7 versus 13.4 gm/dL, P = 0.001), higher estimated blood loss (1325 versus 196 mL, P < 0.001), and longer operative time (291 versus 217 min, P = 0.003) than nontransfused patients. Based on current volumes, eliminating TS in these patients would save at least an estimated $60,100 per year. CONCLUSIONS: Emergent transfusion in ALR and esophagectomy is rare. Routine preoperative TS is most likely unnecessary for these cases. These results will be used in a quality improvement initiative to change practice at our institution.