An Integrated Approach for Analyzing Clinical Genomic Variant Data from Next-Generation Sequencing

Next-generation sequencing (NGS) technologies provide the potential for developing high-throughput and low-cost platforms for clinical diagnostics. A limiting factor to clinical applications of genomic NGS is downstream bioinformatics analysis for data interpretation. We have developed an integrated approach for end-to-end clinical NGS data analysis from variant detection to functional profiling. Robust bioinformatics pipelines were implemented for genome alignment, single nucleotide polymorphism (SNP), small insertion/deletion (InDel), and copy number variation (CNV) detection of whole exome sequencing (WES) data from the Illumina platform. Quality-control metrics were analyzed at each step of the pipeline by use of a validated training dataset to ensure data integrity for clinical applications. We annotate the variants with data regarding the disease population and variant impact. Custom algorithms were developed to filter variants based on criteria, such as quality of variant, inheritance pattern, and impact of variant on protein function. The developed clinical variant pipeline links the identified rare variants to Integrated Genome Viewer for visualization in a genomic context and to the Protein Information Resource’s iProXpress for rich protein and disease information. With the application of our system of annotations, prioritizations, inheritance filters, and functional profiling and analysis, we have created a unique methodology for downstream variant filtering that empowers clinicians and researchers to interpret more effectively the relevance of genomic alterations within a rare genetic disease.     

Miniature Swine as a Clinically Relevant Model of Graft-Versus-Host Disease

Miniature swine provide a preclinical model of hematopoietic cell transplantation (HCT) for studies of graft-versus-host disease. HCT between MHC-matched or ‑mismatched pigs can be performed to mimic clinical scenarios with outcomes that closely resemble those observed in human HCT recipients. With myeloablative conditioning, HCT across MHC barriers is typically fatal, with pigs developing severe (grade III or IV) GVHD involving the gastrointestinal tract, liver, and skin. Unlike rodent models, miniature swine provide an opportunity to perform extended longitudinal studies on individual animals, because multiple tissue biopsies can be harvested without the need for euthanasia. In addition, we have developed a swine GVHD scoring system that parallels that used in the human clinical setting. Given the similarities of GVHD in pigs and humans, we hope that the use of this scoring system facilitates clinical and scientific discourse between the laboratory and the clinic. We anticipate that results of swine studies will support the development of new strategies to improve the identification and treatment of GVHD in clinical HCT scenarios.Abbreviations: BMT, bone marrow transplantation; GVHD, graft-versus-host disease; GVL, graft-versus-leukemia; HCT, hematopoietic cell transplantation; TBI, total-body irradiation     

Surgical Stress Abrogates Pre-Existing Protective T Cell Mediated Anti-Tumor Immunity Leading to Postoperative Cancer Recurrence

Anti-tumor CD8⁺ T cells are a key determinant for overall survival in patients following surgical resection for solid malignancies. Using a mouse model of cancer vaccination (adenovirus expressing melanoma tumor-associated antigen (TAA)—dopachrome tautomerase (AdDCT) and resection resulting in major surgical stress (abdominal nephrectomy), we demonstrate that surgical stress results in a reduction in the number of CD8⁺ T cell that produce cytokines (IFNγ, TNFα, Granzyme B) in response to TAA. This effect is secondary to both reduced proliferation and impaired T cell function following antigen binding. In a prophylactic model, surgical stress completely abrogates tumor protection conferred by vaccination in the immediate postoperative period. In a clinically relevant surgical resection model, vaccinated mice undergoing a positive margin resection with surgical stress had decreased survival compared to mice with positive margin resection alone. Preoperative immunotherapy with IFNα significantly extends survival in surgically stressed mice. Importantly, myeloid derived suppressor cell (MDSC) population numbers and functional impairment of TAA-specific CD8⁺ T cell were altered in surgically stressed mice. Our observations suggest that cancer progression may result from surgery-induced suppression of tumor-specific CD8⁺ T cells. Preoperative immunotherapies aimed at targeting the prometastatic effects of cancer surgery will reduce recurrence and improve survival in cancer surgery patients.     

Process‐based functions for seed retention on animals: a test of improved descriptions of dispersal using multiple data sets

Studies of external seed transport on animals usually assume that the probability of detachment is constant, so that seed retention should show a simple exponential relationship with time. This assumption has not been tested explicitly, and may lead to inaccurate representation of long distance seed dispersal by animals. We test the assumption by comparing the fit to empirical data of simple, two‐parameter functions. Fifty‐two data sets were obtained from five published studies, describing seed retention of 32 plant species on sheep, cattle, deer, goats and mice. Model selection suggested a simple exponential function was adequate for data sets in which seed retention was followed for short periods ( <48 h). The data gathered over longer periods (49–219 days) were best described by the power exponential function, a form of the stretched exponential which allows a changing dropping rate. In these cases the power exponential showed that seed dropping rate decreased with time, suggesting that seeds vary in attachment, with some seeds becoming deeply buried or wound up in the animal's coat. Comparison of fitted parameters across all the data sets also confirmed that seeds with adhesive structures have lower dropping rates than those without. We conclude that the seed dropping rate often changes with time during external transport on animals and that the power exponential is an effective function to describe this change. We advise that, to analyse seed dropping rates adequately, retention should be measured over reasonable time periods – until most seeds are dropped – and both the simple and power exponential functions should be fitted to the resulting data. To increase its utility, we provide functions describing the seed dropping rate and the dispersal kernel resulting from the power exponential relationship.     

Selective estrogen receptor degraders with novel structural motifs induce regression in a tamoxifen-resistant breast cancer xenograft

Potent estrogen receptor ligands typically contain a phenolic hydrogen-bond donor. The indazole of the selective estrogen receptor degrader (SERD) ARN-810 is believed to mimic this. Disclosed herein is the discovery of ARN-810 analogs which lack this hydrogen-bond donor. These SERDs induced tumor regression in a tamoxifen-resistant breast cancer xenograft, demonstrating that the indazole NH is not necessary for robust ER-modulation and anti-tumor activity.     

Severe Obesity Is Associated With Impaired Arterial Smooth Muscle Function in Young Adults

The degree of arterial dilatation induced by exogenous nitrates (nitrate‐mediated dilatation, NMD) has been similar in obese and normal‐weight adults after single high‐dose glyceryl trinitrate (GTN). We examined whether NMD is impaired in obesity by performing a GTN dose‐response study, as this is a potentially more sensitive measure of arterial smooth muscle function. In this cross‐sectional study, subjects were 19 obese (age 31.0 ± 1.2 years, 10 male, BMI 44.1 ± 2.1) and 19 age‐ and sex‐matched normal‐weight (BMI 22.4 ± 0.4) young adults. Blood pressure (BP), triglycerides, high‐density lipoprotein (HDL), and low‐density lipoprotein (LDL)‐cholesterol, glucose, insulin, high‐sensitivity C‐reactive protein (hs‐CRP), carotid intima‐media thickness (CIMT), and flow‐mediated dilatation (FMD) were measured. After incremental doses of GTN, brachial artery maximal percent dilatation (maximal NMD) and the area under the dose‐response curve (NMD AUC) were calculated. Maximal NMD (13.4 ± 0.9% vs. 18.3 ± 1.1%, P = 0.002) and NMD AUC (54,316 ± 362 vs. 55,613 ± 375, P = 0.018) were lower in obese subjects. The obese had significantly higher hs‐CRP, insulin, and CIMT and lower HDL‐cholesterol. Significant bivariate associations existed between maximal NMD or NMD AUC and BMI‐group (r = ?0.492, P = 0.001 or r = ?0.383, P = 0.009), hs‐CRP (r = ?0.419, P = 0.004 or r = ?0.351, P = 0.015), and HDL‐cholesterol (r = 0.374, P = 0.01 or r = 0.270, P = 0.05). On multivariate analysis, higher BMI‐group remained as the only significant determinant of maximal NMD (r² = 0.242, β = ?0.492, P = 0.002) and NMD AUC (r² = 0.147, β = ?0.383, P = 0.023). In conclusion, arterial smooth muscle function is significantly impaired in the obese. This may be important in their increased cardiovascular risk.     

Molecular analysis of a novel family of complex glycoinositolphosphoryl ceramides from Cryptococcus neoformans: structural differences between encapsulated and acapsular yeast forms

Complex glycoinositolphosphoryl ceramides (GIPCs) have been purified from a pathogenic encapsulated wild-type (WT) strain of Cryptococcus neoformans var. neoformans and from an acapsular mutant (Cap67). The structures of the GIPCs were determined by a combination of tandem mass spectrometry, nuclear magnetic resonance spectroscopy, methylation analysis, gas chromatography-mass spectrometry, and chemical degradation. The main GIPC from the WT strain had the structure Manp(alpha1-3)[Xylp(beta1-2)] Manp(alpha1-4)Galp(beta1-6)Manp(alpha1-2)Ins-1-phosphoryl ceramide (GIPC A), whereas the compounds from the acapsular mutant were more heterogeneous in their glycan chains, and variants with Manp(alpha1-6) (GIPC B), Manp(alpha1-6) Manp(alpha1-6) (GIPC C), and Manp(alpha1-2)Manp(alpha1-6)Manp(alpha1-6) (GIPC D) substituents linked to the nonreducing terminal mannose residue found in the WT GIPC A were abundant. The ceramide moieties of C. neoformans GIPCs were composed of a C(18) phytosphingosine long-chain base mainly N-acylated with 2-hydroxy-tetracosanoic acid in the WT GIPC while in the acapsular Cap67 mutant GIPCs, as well as 2-hydroxy-tetracosanoic acid, the unusual 2,3-dihydroxy-tetracosanoic acid was characterized. In addition, structural analysis revealed that the amount of GIPC in the WT cells was fourfold less of that in the acapsular mutant.