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In the Circulating Cell-free Genome Atlas (NCT02889978) substudy 1, we evaluate several approaches for a circulating cell-free DNA (cfDNA)-based multi-cancer early detection (MCED) test by defining clinical limit of detection (LOD) based on circulating tumor allele fraction (cTAF), enabling performance comparisons. Among 10 machine-learning classifiers trained on the same samples and independently validated, when evaluated at 98% specificity, those using whole-genome (WG) methylation, single nucleotide variants with paired white blood cell background removal, and combined scores from classifiers evaluated in this study show the highest cancer signal detection sensitivities. Compared with clinical stage and tumor type, cTAF is a more significant predictor of classifier performance and may more closely reflect tumor biology. Clinical LODs mirror relative sensitivities for all approaches. The WG methylation feature best predicts cancer signal origin. WG methylation is the most promising technology for MCED and informs development of a targeted methylation MCED test., Competing Interests: Declaration of interests A.J., O.V., E.H., J.F.B., S.G., Q.L., N.Z., E.T.F., K.N.K., H.A., C.B., D.C., K.D., S.F., C.H., R.J., B.J., S.T., C.M., V.D., J.N., O.S., E.S., A.S., S.S., K.K.S., V.N., A.P.F., T.C.C., S.B., N.H., M.D., Z.D., and M.P.H. are employees of GRAIL, LLC, with equity in Illumina, Inc. C.M. also holds stock in Novartis, Clovis, Cara, Gilead, and Bluebird. M.C.L. is an uncompensated consultant for GRAIL, LLC. The Mayo Clinic was compensated for M.C.L.’s and D.D.T.’s advisory board activities for GRAIL, LLC. E.A.K. is a consultant for GRAIL, LLC. D.A.R. is a consultant for Ipsen. M.A.S. is a consultant for Celgene, Millennium, and Syros Pharmaceuticals. A.M.A. was previously employed by GRAIL, LLC; has equity in Illumina, Inc.; is currently employed by Illumina, Inc.; and is an advisor to and an equity holder in Foresite Labs and Myst Therapeutics. M.V.S. is an employee of and holds stock in McKesson Corporation, and is a clinical adviser for GRAIL, LLC. D.A.B. is a co-owner of Berry Consultants, LLC. A.H.B. is a consultant for Pfizer, Merck, Bayer, and Astellas Pharmaceuticals. C.S. holds stock in Illumina, Inc., Epic Biosciences, and Apogen Biotech; receives grants from Pfizer and AstraZeneca; receives honoraria or consultant fees from Roche Ventana, Celgene, Pfizer, Novartis, Genentech, and BMS; and is a co-founder of Achilles Therapeutics. S.G., O.V., A.P.F., A.J., K.D., V.N., J.F.B., C.M., E.H., Q.L., N.Z., P.F., and O.S. are inventors on pending patent applications related to this work, for which GRAIL, LLC, has ownership rights. GRAIL, LLC, a subsidiary of Illumina, Inc., is currently held separate from Illumina, Inc., under the terms of the Interim Measures Order of the European Commission dated October 29, 2021. C.S. recieved grant support from AstraZeneca, Boehringer-Ingelheim, Bristol Myers Squibb, Pfizer, Roche-Ventana, Invitae (previously Archer Dx Inc), and Ono Pharmaceutical; is an AstraZeneca Advisory Board member and Chief Investigator for the AZ MeRmaiD 1 and 2 clinical trials and is also Co-Chief Investigator of the NHS Galleri trial funded by GRAIL and a paid member of GRAIL’s Scientific Advisory Board (SAB); received consultant fees from Achilles Therapeutics (also SAB member), Bicycle Therapeutics (also a SAB member), Genentech, Medicxi, Roche Innovation Centre – Shanghai, Metabomed, and the Sarah Cannon Research Institute; received honoraria from Amgen, AstraZeneca, Pfizer, Novartis, GlaxoSmithKline, MSD, Bristol Myers Squibb, Illumina, and Roche-Ventana; had stock options in Apogen Biotechnologies and GRAIL until June 2021, and currently has stock options in Epic Bioscience, Bicycle Therapeutics, and Achilles Therapeutics; and is a co-founder of Achilles Therapeutics; holds patents relating to assay technology to detect tumour recurrence (PCT/GB2017/053289), targeting neoantigens (PCT/EP2016/059401), identifying patent response to immune checkpoint blockade (PCT/EP2016/071471), determining HLA LOH (PCT/GB2018/052004), predicting survival rates of patients with cancer (PCT/GB2020/050221), and identifying patients who respond to cancer treatment (PCT/GB2018/051912); holds US patent relating to detecting tumour mutations (PCT/US2017/28013), methods for lung cancer detection (US20190106751A1); and holds both a European and US patent related to identifying insertion/deletion mutation targets (PCT/GB2018/051892). C.S. is a Royal Society Napier Research Professor (RSRP\R\210001) and has received funding from the Francis Crick Institute that receives its core funding from Cancer Research UK (CC2041), the UK Medical Research Council (CC2041), and the Wellcome Trust (CC2041); Cancer Research UK (TRACERx [C11496/A17786], PEACE [C416/A21999], and CRUK Cancer Immunotherapy Catalyst Network); Cancer Research UK Lung Cancer Centre of Excellence (C11496/A30025); the Rosetrees Trust, Butterfield and Stoneygate Trusts; NovoNordisk Foundation (ID16584); Royal Society Professorship Enhancement Award (RP/EA/180007); National Institute for Health Research (NIHR) University College London Hospitals Biomedical Research Centre; the Cancer Research UK-University College London Centre; Experimental Cancer Medicine Centre; the Breast Cancer Research Foundation (US) (BCRF-22-157); Cancer Research UK Early Detection and Diagnosis Primer Award (Grant EDDPMA-Nov21/100034); The Mark Foundation for Cancer Research Aspire Award (Grant 21-029-ASP); Stand Up To Cancer-LUNGevity American Lung Association Lung Cancer Interception Dream Team Translational Research Grant (Grant Number: SU2C-AACR-DT23-17); and an ERC Advanced Grant (PROTEUS) from the European Research Council under the European Union’s Horizon 2020 research and innovation programme (grant agreement no. 835297)., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

We investigate whether the heart rate can be treated as a semi-random source with the aim of amplification by quantum devices. We use a semi-random source model called ε-Santha-Vazirani source, which can be amplified via quantum protocols to obtain a fully private random sequence. We analyze time intervals between consecutive heartbeats obtained from Holter electrocardiogram (ECG) recordings of people of different sex and age. We propose several transformations of the original time series into binary sequences. We have performed different statistical randomness tests and estimated quality parameters. We find that the heart can be treated as a good enough, and private by its nature, source of randomness that every human possesses. As such, in principle, it can be used as input to quantum device-independent randomness amplification protocols. The properly interpreted ε parameter can potentially serve as a new characteristic of the human heart from the perspective of medicine.

Objective: Heart failure (HF) is a clinical syndrome resulting from structural or functional damages. Although clinical trials have shown that the plasma renin-angiotensin system (RAS) activation decreases HF functional status and increases hospitalization for HF patients, the effect of intrarenal RAS activity is still unknown. In this study, we investigated the relationship between the New York Heart Association (NYHA) class, duration, and number of hospitalizations in the previous year and urinary angiotensinogen (UAGT) in patients with HF with reduced ejection fraction (HFrEF)., Methods: This study included 85 patients who had an ejection fraction of <40% and were receiving optimal medical treatment. Among these, 22 were excluded from the study for various reasons. Demographically and biochemically, the remaining 63 patients were compared according to the NYHA functional classes and re-hospitalization status., Results: When the groups were compared in terms of N-terminal pro-B-type natriuretic peptide (NT-proBNP), UAGT, and high-sensitivity C-reactive protein (Hs-CRP), it was found that these parameters were significantly higher in patients who were hospitalized more than two times in the previous year [p<0.001; p=0.007; p<0.001, respectively]. There was a significant correlation between number of hospitalizations and NT-proBNP (r=0.507, p<0.001), Hs-CRP (r=0.511, p<0.001), hemoglobin (r=-0.419, p=0.001), serum sodium (r=-0.26, p=0.04), and systolic blood pressure (r=-0.283, p=0.02). When the independence of multiple correlations was assessed using multiple linear regression analysis, NT-proBNP, Hs-CRP, and hemoglobin levels were independent predictors of re-hospitalization, but this was not the same for UAGT., Conclusion: Although UAGT levels are high in patients with poor NYHA functional class and repeated hospitalizations, this marker is not valuable for predicting repeated hospitalization in patients with HFrEF.

Horizontal gene transfer (HGT) is common between prokaryotes and phagotrophic eukaryotes. In metazoans, the scale and significance of HGT remains largely unexplored but is usually linked to a close association with parasites and endosymbionts. Marine sponges (Porifera), which host many microorganisms in their tissues and lack an isolated germ line, are potential carriers of genes transferred from prokaryotes. In this study, we identified a number of potential horizontally transferred genes within the genome of the sponge, Amphimedon queenslandica. We further identified homologs of some of these genes in other sponges. The transferred genes, most of which possess catalytic activity for carbohydrate or protein metabolism, have assimilated host genome characteristics and are actively expressed. The diversity of functions contributed by the horizontally transferred genes is likely an important factor in the adaptation and evolution of A. queenslandica. These findings highlight the potential importance of HGT on the success of sponges in diverse ecological niches.

Triple-negative breast cancers (TNBCs) are a heterogeneous set of tumors defined by an absence of actionable therapeutic targets (ER, PR, and HER-2). Microdissected normal ductal epithelium from healthy volunteers represents a novel comparator to reveal insights into TNBC heterogeneity and to inform drug development. Using RNA-sequencing data from our institution and The Cancer Genome Atlas (TCGA) we compared the transcriptomes of 94 TNBCs, 20 microdissected normal breast tissues from healthy volunteers from the Susan G. Komen for the Cure Tissue Bank, and 10 histologically normal tissues adjacent to tumor. Pathway analysis comparing TNBCs to optimized normal controls of microdissected normal epithelium versus classic controls composed of adjacent normal tissue revealed distinct molecular signatures. Differential gene expression of TNBC compared with normal comparators demonstrated important findings for TNBC-specific clinical trials testing targeted agents; lack of over-expression for negative studies and over-expression in studies with drug activity. Next, by comparing each individual TNBC to the set of microdissected normals, we demonstrate that TNBC heterogeneity is attributable to transcriptional chaos, is associated with non-silent DNA mutational load, and explains transcriptional heterogeneity in addition to known molecular subtypes. Finally, chaos analysis identified 146 core genes dysregulated in >90 % of TNBCs revealing an over-expressed central network. In conclusion, use of microdissected normal ductal epithelium from healthy volunteers enables an optimized approach for studying TNBC and uncovers biological heterogeneity mediated by transcriptional chaos.

Triple-negative breast cancer (TNBC) is characterized by the absence of expression of estrogen receptor, progesterone receptor, and HER-2. Thirty percent of patients recur after first-line treatment, and metastatic TNBC (mTNBC) has a poor prognosis with median survival of one year. Here, we present initial analyses of whole genome and transcriptome sequencing data from 14 prospective mTNBC. We have cataloged the collection of somatic genomic alterations in these advanced tumors, particularly those that may inform targeted therapies. Genes mutated in multiple tumors included TP53, LRP1B, HERC1, CDH5, RB1, and NF1. Notable genes involved in focal structural events were CTNNA1, PTEN, FBXW7, BRCA2, WT1, FGFR1, KRAS, HRAS, ARAF, BRAF, and PGCP. Homozygous deletion of CTNNA1 was detected in 2 of 6 African Americans. RNA sequencing revealed consistent overexpression of the FOXM1 gene when tumor gene expression was compared with nonmalignant breast samples. Using an outlier analysis of gene expression comparing one cancer with all the others, we detected expression patterns unique to each patient's tumor. Integrative DNA/RNA analysis provided evidence for deregulation of mutated genes, including the monoallelic expression of TP53 mutations. Finally, molecular alterations in several cancers supported targeted therapeutic intervention on clinical trials with known inhibitors, particularly for alterations in the RAS/RAF/MEK/ERK and PI3K/AKT/mTOR pathways. In conclusion, whole genome and transcriptome profiling of mTNBC have provided insights into somatic events occurring in this difficult to treat cancer. These genomic data have guided patients to investigational treatment trials and provide hypotheses for future trials in this irremediable cancer.

High-throughput RNA sequencing enables quantification of transcripts (both known and novel), exon/exon junctions and fusions of exons from different genes. Discovery of gene fusions-particularly those expressed with low abundance- is a challenge with short- and medium-length sequencing reads. To address this challenge, we implemented an RNA-Seq mapping pipeline within the LifeScope software. We introduced new features including filter and junction mapping, annotation-aided pairing rescue and accurate mapping quality values. We combined this pipeline with a Suffix Array Spliced Read (SASR) aligner to detect chimeric transcripts. Performing paired-end RNA-Seq of the breast cancer cell line MCF-7 using the SOLiD system, we called 40 gene fusions among over 120,000 splicing junctions. We validated 36 of these 40 fusions with TaqMan assays, of which 25 were expressed in MCF-7 but not the Human Brain Reference. An intra-chromosomal gene fusion involving the estrogen receptor alpha gene ESR1, and another involving the RPS6KB1 (Ribosomal protein S6 kinase beta-1) were recurrently expressed in a number of breast tumor cell lines and a clinical tumor sample.

Transient bone marrow edema syndrome (TBMES) is a disorder with unknown etiology and must be included in the differential diagnosis of hip pain. TBMES generally has a good prognosis but, it can be result in avascular necrosis (AVN). We present two cases diagnosed with TBMES, one of whom experienced full remission and the other who developed AVN.

Ion channels establish and regulate membrane potentials in excitable and non-excitable cells. How functional diversification of ion channels contributed to the evolution of nervous systems may be understood by studying organisms at key positions in the evolution of animal multicellularity. We have carried out the first analysis of ion channels cloned from a marine sponge, Amphimedon queenslandica. Phylogenetic comparison of sequences encoding for poriferan inward-rectifier K(+) (Kir) channels suggests that Kir channels from sponges, cnidarians and triploblastic metazoans each arose from a single channel and that duplications arose independently in the different groups. In Xenopus oocytes, AmqKirA and AmqKirB produced K(+) currents with strong inward rectification, as seen in the mammalian Kir2 channels, which are found in excitable cells. The pore properties of AmqKir channels demonstrated strong K(+) selectivity and block by Cs(+) and Ba(2+). We present an original analysis of sponge ion channel physiology and an examination of the phylogenetic relationships of this channel with other cloned Kir channels.

Motivation: Gene duplications and losses (GDLs) are important events in genome evolution. They result in expansion or contraction of gene families, with a likely role in phenotypic evolution. As more genomes become available and their annotations are improved, software programs capable of rapidly and accurately identifying the content of ancestral genomes and the timings of GDLs become necessary to understand the unique evolution of each lineage., Results: We report EvolMAP, a new algorithm and software that utilizes a species tree-based gene clustering method to join all-to-all symmetrical similarity comparisons of multiple gene sets in order to infer the gene composition of multiple ancestral genomes. The algorithm further uses Dollo parsimony-based comparison of the inferred ancestral genes to pinpoint the timings of GDLs onto evolutionary intervals marked by speciation events. Using EvolMAP, first we analyzed the expansion of four families of G-protein coupled receptors (GPCRs) within animal lineages. Additional to demonstrating the unique expansion tree for each family, results also show that the ancestral eumetazoan genome contained many fewer GPCRs than modern animals, and these families expanded through concurrent lineage-specific duplications. Second, we analyzed the history of GDLs in mammalian genomes by comparing seven proteomes. In agreement with previous studies, we report that the mammalian gene family sizes have changed drastically through their evolution. Interestingly, although we identified a potential source of duplication for 75% of the gained genes, remaining 25% did not have clear-cut sources, revealing thousands of genes that have likely gained their distinct sequence identities within the descent of mammals., Availability: Query server, source code and executable are available at http://kosik-web.mcdb.ucsb.edu/evolmap/index.htm .

Background: The evolution of complex sub-cellular structures such as the synapse requires the assembly of multiple proteins, each conferring added functionality to the integrated structure. Tracking the early evolution of synapses has not been possible without genomic information from the earliest branching animals. As the closest extant relatives to the Eumetazoa, Porifera (sponges) represent a pivotal group for understanding the evolution of nervous systems, because sponges lack neurons with clearly recognizable synapses, in contrast to eumetazoan animals., Methodology/principal Findings: We show that the genome of the demosponge Amphimedon queenslandica possesses a nearly complete set of post-synaptic protein homologs whose conserved interaction motifs suggest assembly into a complex structure. In the critical synaptic scaffold gene, dlg, residues that make hydrogen bonds and van der Waals interactions with the PDZ ligand are 100% conserved between sponge and human, as is the motif organization of the scaffolds. Expression in Amphimedon of multiple post-synaptic gene homologs in larval flask cells further supports the existence of an assembled structure. Among the few post-synaptic genes absent from Amphimedon, but present in Eumetazoa, are receptor genes including the entire ionotropic glutamate receptor family., Conclusions/significance: Highly conserved protein interaction motifs and co-expression in sponges of multiple proteins whose homologs interact in eumetazoan synapses indicate that a complex protein scaffold was present at the origin of animals, perhaps predating nervous systems. A relatively small number of crucial innovations to this pre-existing structure may represent the founding changes that led to a post-synaptic element.

Spondylometaphyseal dysplasia is a type of bone dysplasia characterized by vertebral and metaphyseal changes of varying severity. Diagnosis of the disease is difficult because the severity of bone involvement differs and symptoms change according to the age of the patient. In this study, radiographic findings of a 16 month-old male patient diagnosed as Kozlowski type spondylometaphyseal dysplasia is reported.