Strathclyde minor groove binders (S-MGBs) with activity against Acanthamoeba castellanii.

BACKGROUND: Acanthamoeba spp. is the causative agent of Acanthamoeba keratitis and granulomatous amoebic encephalitis. Strathclyde minor groove binders (S-MGBs) are a promising new class of anti-infective agent that have been shown to be effective against many infectious organisms. OBJECTIVES: To synthesize and evaluate the anti-Acanthamoeba activity of a panel of S-MGBs, and therefore determine the potential of this class for further development. METHODS: A panel of 12 S-MGBs was synthesized and anti-Acanthamoeba activity was determined using an alamarBlue™-based trophocidal assay against Acanthamoeba castellanii. Cross-screening against Trypanosoma brucei brucei, Staphylococcus aureus and Escherichia coli was used to investigate selective potency. Cytotoxicity against HEK293 cells allowed for selective toxicity to be measured. DNA binding studies were carried out using native mass spectrometry and DNA thermal shift assays. RESULTS AND DISCUSSION: S-MGB-241 has an IC50 of 6.6 µM against A. castellanii, comparable to the clinically used miltefosine (5.6 µM) and negligible activity against the other organisms. It was also found to have an IC50 > 100 µM against HEK293 cells, demonstrating low cytotoxicity. S-MGB-241 binds to DNA as a dimer, albeit weakly compared to other S-MGBs previously studied. This was confirmed by DNA thermal shift assay with a ΔTm = 1 ±â€Š0.1°C. CONCLUSIONS: Together, these data provide confidence that S-MGBs can be further optimized to generate new, potent treatments for Acanthameoba spp. infections. In particular, S-MGB-241, has been identified as a 'hit' compound that is selectively active against A. castellanii, providing a starting point from which to begin optimization of DNA binding and potency.

Effect of suckler cow breed type and parity on the development of the cow-calf bond post-partum and calf passive immunity.

BACKGROUND: Development of the cow-calf bond post-partum and passive immunity of calves from spring-calving beef × beef (B×B) and beef × dairy (B×D) cow genotypes was determined using primiparous and multiparous (Experiment 1), and primiparous and second-parity (Experiment 2) animals. In Experiment 1, calves either suckled colostrum naturally ('natural-suckling') (n = 126), or were fed colostrum, using an oesophageal-tube ('artificially-fed') (n = 26), from their dam within 1-h post-partum. In Experiment 2, all calves (n = 60) were artificially-fed colostrum from their dam. Prior to colostrum suckling/feeding, colostrum was sampled for IgG analysis. The cow-calf bond was assessed using CCTV recordings during the first 4-h post-partum. Calves were blood sampled at 48-h post-partum to determine IgG and total protein (TP) concentrations, and zinc sulphate turbidity (ZST) units. RESULTS: There was no difference (P > 0.05) in cow licking behaviours and calf standing and suckling behaviours between the genotypes, except in Experiment 2 where B×D calves had more attempts to suckle before suckling occurred (P ≤ 0.05) compared to B×B calves. In Experiment 1, multiparous cows licked their calves sooner (P ≤ 0.05) and for longer (P < 0.01), and their calves had fewer attempts to stand (P < 0.001), stood for longer (P = 0.05), and had fewer attempts to suckle before suckling occurred (P < 0.001) than primiparous cows; there was no parity effect on cow-calf behaviour in Experiment 2. Colostrum IgG concentrations and measures of calf passive immunity did not differ (P > 0.05) between the genotypes in either Experiment. In Experiment 1, colostrum IgG concentrations were greater (P ≤ 0.05) in multiparous compared to primiparous cows and their calves had superior (P ≤ 0.05) passive immunity; no effect of parity was found in Experiment 2. Passive immunity did not differ (P > 0.05) between suckled and artificially-fed calves in Experiment 1. CONCLUSIONS: Cow genotype had little effect on cow-calf behaviours, but under 'natural-suckling' conditions primiparous cows expressed maternal inexperience and their calves were less vigorous than multiparous cows. Colostrum IgG concentration and calf passive immunity measures were unaffected by genotype, but under 'natural-suckling' conditions calves from primiparous cows had lower passive immunity.

sUPRa is a dual-color reporter for unbiased quantification of the unfolded protein response with cellular resolution.

The unfolded protein response (UPR) maintains proteostasis upon endoplasmic reticulum (ER) stress, and is initiated by a range of physiological and pathological processes. While there have been advances in developing fluorescent reporters for monitoring individual signaling pathways of the UPR, this approach may not capture a cell's overall UPR activity. Here we describe a novel sensor of UPR activity, sUPRa, which is designed to report the global UPR. sUPRa displays excellent response characteristics, outperforms reporters of individual UPR pathways in terms of sensitivity and kinetics, and responds to a range of different ER stress stimuli. Furthermore, sUPRa's dual promoter and fluorescent protein design ensures that both UPR-active and inactive cells are detected, and controls for reporter copy number. Using sUPRa, we reveal UPR activation in layer 2/3 pyramidal neurons of mouse cerebral cortex following a period of sleep deprivation. sUPRa affords new opportunities for quantifying physiological UPR activity with cellular resolution.

Partitioning of an Enzyme-Polymer Surfactant Nanocomplex into Lipid-Rich Cellular Compartments Drives In Situ Hydrolysis of Organophosphates.

Most organophosphates (OPs) are hydrophobic, and after exposure, can sequester into lipophilic regions within the body, such as adipose tissue, resulting in long term chronic effects. Consequently, there is an urgent need for therapeutic agents that can decontaminate OPs in these hydrophobic regions. Accordingly, an enzyme-polymer surfactant nanocomplex is designed and tested comprising chemically supercharged phosphotriesterase (Agrobacterium radiobacter; arPTE) electrostatically conjugated to amphiphilic polymer surfactant chains ([cat.arPTE][S-]). Experimentally-derived structural data are combined with molecular dynamics (MD) simulations to provide atomic level detail on conformational ensembles of the nanocomplex using dielectric constants relevant to aqueous and lipidic microenvironments. These show the formation of a compact admicelle pseudophase surfactant corona under aqueous conditions, which reconfigures to yield an extended conformation at a low dielectric constant, providing insight into the mechanism underpinning cell membrane binding. Significantly, it demonstrated that [cat.arPTE][S-] spontaneously binds to human mesenchymal stem cell membranes (hMSCs), resulting in on-cell OP hydrolysis. Moreover, the nanoconstruct can endocytose and partition into the intracellular fatty vacuoles of adipocytes and hydrolyze sequestered OP.

Clinical impact of novel CMR technology on patients with congenital heart disease. A scientific statement of the Association for European Pediatric and Congenital Cardiology (AEPC) and the European Association of Cardiovascular Imaging (EACVI) of the ESC.

Cardiovascular magnetic resonance (CMR) imaging is recommended in patients with congenital heart disease (CHD) in clinical practice guidelines as the imaging standard for a large variety of diseases. As CMR is evolving, novel techniques are becoming available. Some of them are already used clinically, whereas others still need further evaluation. In this statement the authors give an overview of relevant new CMR techniques for the assessment of CHD. Studies with reference values for these new techniques are listed in the supplement.

Structural color in the bacterial domain: The ecogenomics of a 2-dimensional optical phenotype.

Structural color is an optical phenomenon resulting from light interacting with nanostructured materials. Although structural color (SC) is widespread in the tree of life, the underlying genetics and genomics are not well understood. Here, we collected and sequenced a set of 87 structurally colored bacterial isolates and 30 related strains lacking SC. Optical analysis of colonies indicated that diverse bacteria from at least two different phyla (Bacteroidetes and Proteobacteria) can create two-dimensional packing of cells capable of producing SC. A pan-genome-wide association approach was used to identify genes associated with SC. The biosynthesis of uroporphyrin and pterins, as well as carbohydrate utilization and metabolism, was found to be involved. Using this information, we constructed a classifier to predict SC directly from bacterial genome sequences and validated it by cultivating and scoring 100 strains that were not part of the training set. We predicted that SCr is widely distributed within gram-negative bacteria. Analysis of over 13,000 assembled metagenomes suggested that SC is nearly absent from most habitats associated with multicellular organisms except macroalgae and is abundant in marine waters and surface/air interfaces. This work provides a large-scale ecogenomics view of SC in bacteria and identifies microbial pathways and evolutionary relationships that underlie this optical phenomenon.

Distinct epigenomic landscapes underlie tissue-specific memory T cell differentiation.

The memory CD8+ T cell pool contains phenotypically and transcriptionally heterogeneous subsets with specialized functions and recirculation patterns. Here, we examined the epigenetic landscape of CD8+ T cells isolated from seven non-lymphoid organs across four distinct infection models, alongside their circulating T cell counterparts. Using single-cell transposase-accessible chromatin sequencing (scATAC-seq), we found that tissue-resident memory T (TRM) cells and circulating memory T (TCIRC) cells develop along distinct epigenetic trajectories. We identified organ-specific transcriptional regulators of TRM cell development, including FOSB, FOS, FOSL1, and BACH2, and defined an epigenetic signature common to TRM cells across organs. Finally, we found that although terminal TEX cells share accessible regulatory elements with TRM cells, they are defined by TEX-specific epigenetic features absent from TRM cells. Together, this comprehensive data resource shows that TRM cell development is accompanied by dynamic transcriptome alterations and chromatin accessibility changes that direct tissue-adapted and functionally distinct T cell states.

Temporal changes in mouse hippocampus transcriptome after pilocarpine-induced seizures.

Introduction: Status epilepticus (SE) is a seizure lasting more than 5 min that can have lethal consequences or lead to various neurological disorders, including epilepsy. Using a pilocarpine-induced SE model in mice we investigated temporal changes in the hippocampal transcriptome. Methods: We performed mRNA-seq and microRNA-seq analyses at various times after drug treatment. Results: At 1 h after the start of seizures, hippocampal cells upregulated transcription of immediate early genes and genes involved in the IGF-1, ERK/MAPK and RNA-PolII/transcription pathways. At 8 h, we observed changes in the expression of genes associated with oxidative stress, overall transcription downregulation, particularly for genes related to mitochondrial structure and function, initiation of a stress response through regulation of ribosome and translation/EIF2 signaling, and upregulation of an inflammatory response. During the middle of the latent period, 36 h, we identified upregulation of membrane components, cholesterol synthesis enzymes, channels, and extracellular matrix (ECM), as well as an increased inflammatory response. At the end of the latent period, 120 h, most changes in expression were in genes involved in ion transport, membrane channels, and synapses. Notably, we also elucidated the involvement of novel pathways, such as cholesterol biosynthesis pathways, iron/BMP/ferroptosis pathways, and circadian rhythms signaling in SE and epileptogenesis. Discussion: These temporal changes in metabolic reactions indicate an immediate response to injury followed by recovery and regeneration. CREB was identified as the main upstream regulator. Overall, our data provide new insights into molecular functions and cellular processes involved at different stages of seizures and offer potential avenues for effective therapeutic strategies.

Heparin Dose Intensity and Organ Support-Free Days in Patients Hospitalized for COVID-19.

Background: Clinical trials suggest that therapeutic-dose heparin may prevent critical illness and vascular complications due to COVID-19, but knowledge gaps exist regarding the efficacy of therapeutic heparin including its comparative effect relative to intermediate-dose anticoagulation. Objectives: The authors performed 2 complementary secondary analyses of a completed randomized clinical trial: 1) a prespecified per-protocol analysis; and 2) an exploratory dose-based analysis to compare the effect of therapeutic-dose heparin with low- and intermediate-dose heparin. Methods: Patients who received initial anticoagulation dosed consistently with randomization were included. The primary outcome was organ support-free days (OSFDs), a combination of in-hospital death and days free of organ support through day 21. Results: Among 2,860 participants, 1,761 (92.8%) noncritically ill and 857 (89.1%) critically ill patients were treated per-protocol. Among noncritically ill per-protocol patients, the posterior probability that therapeutic-dose heparin improved OSFDs as compared with usual care was 99.3% (median adjusted OR: 1.36; 95% credible interval [CrI]: 1.07-1.74). Therapeutic heparin had a high posterior probability of efficacy relative to both low- (94.6%; adjusted OR: 1.26; 95% CrI: 0.95-1.64) and intermediate- (99.8%; adjusted OR: 1.80; 95% CrI: 1.22-2.62) dose thromboprophylaxis. Among critically ill per-protocol patients, the posterior probability that therapeutic heparin improved outcomes was low. Conclusions: Among noncritically ill patients hospitalized for COVID-19 who were randomized to and initially received therapeutic-dose anticoagulation, heparin, compared with usual care, was associated with improved OSFDs, a combination of in-hospital death and days free of organ support. Therapeutic heparin appeared superior to both low- and intermediate-dose thromboprophylaxis.

Accuracy and safety of freehand vs. end-on fluoroscopic guided drill-hole placement in canine cadaveric thoracic, lumbar and sacral vertebrae.

Objective: To develop and evaluate the safety and accuracy of an open, end-on fluoroscopic guided (EOFG) drill hole position technique in canine cadaveric spinal surgery, in comparison to a traditional free-hand (FH) drilling technique. Study design: Cadaveric comparison study. Animals: Canine cadaveric vertebral columns (n = 4). Methods: Computed tomography (CT) scans were performed for in-silico planning. Ideal implant purchase depth and angulations were determined from previously published data. Plans for end-on fluoroscopic guided drill holes included angled reconstructions in thick slab mode to mimic fluoroscopic images. Following surgical preparation of T8 to S2, holes were drilled by one of two experienced surgeons randomized evenly by operated side, surgeon, and technique. C-arm fluoroscopy was utilized for the end-on technique. CT was repeated after the procedures. Safety was determined categorically using a modified Zdichavsky classification and "optimal" placement was compared between techniques. Continuous data for drill-hole accuracy was calculated as angle and depth deviations from the planned trajectories. Data sets were analyzed at both univariable and multivariable levels with logistic regression analysis. Results: Drill hole safety was categorized as optimal (modified Zdichavsky classification 1) in 51/60 (85%) of drill holes using EOFG and 33/60 (55%) using FH (P < 0.001) techniques. There were no "unsafe" holes (modified Zdichavsky classification 3a). Optimal drill hole placement was significantly associated with the EOFG technique and use of the largest cadaver, and was significantly less likely within the thoracic region. Mean angle and depth deviations were significantly lower with the EOFG technique. Angle deviations were significantly lower for EOFG in the lumbar region, whereas bone purchase deviations were significantly lower for EOFG in both the thoracic and lumbar regions. The mean time taken to drill the hole was significantly longer for the EOFG technique. Conclusion: Optimal drill hole placement was significantly more likely with the EOFG technique and improved the accuracy of bone purchase in the thoracic region. Clinical significance: The EOFG technique shows promise for translation into a clinically setting, potentially improving implant purchase and therefore stabilizing construct strength, whilst potentially reducing the likelihood of neurovascular injury and need for surgical revision.

State Implementation of Congregate Care Reforms for Children in Foster Care.

OBJECTIVES: The Family First Prevention Services Act (FFPSA) allows states to use federal Title IV-E funds to provide time-limited, clinically appropriate use of congregate care, including Qualified Residential Treatment Programs (QRTPs), for youth in foster care. October 1, 2021 marked the deadline for states to begin implementing these FFPSA congregate care reforms. From June to September 2022, we conducted a mixed-methods study to obtain a baseline understanding of implementation barriers, successes, and recommendations to inform congregate care policy and practice. METHODS: We fielded a national survey with state child welfare agency directors and conducted focus groups with youth with QRTP experiences, child welfare agency administrators, and QRTP executive leaders. We integrated a descriptive analysis of survey data with focus group themes to summarize state implementation progress. RESULTS: A total of 47 states (90%) responded to the survey. Most states reported ongoing congregate care reforms aligned with FFPSA, reducing the use of congregate care and increasing kinship foster care. QRTPs have become the primary congregate care setting. Top implementation barriers concerned workforce resource and capacity constraints, funding, and access to therapeutic foster care models and foster families. Focus group themes converged on the lack of tailored treatment, quality staff, coordinated aftercare, and a need for QRTP outcome evidence. CONCLUSIONS: Early implementation lessons of FFPSA congregate care reforms call for additional funding and technical assistance, oversight of congregate care, professionalization and investment in QRTP staff, youth advisory boards to promote youth-driven treatment, and performance- and outcome-based monitoring of QRTPs.

Single-Molecule Cross-Plane Conductance of Polycyclic Aromatic Hydrocarbon Derivatives.

In the cross-plane single-molecule junctions, the correlation between molecular aromaticity and conductance remained puzzling. Cross-plane break junction (XPBJ) provides new insight into understanding the role of aromaticity and conjugation to molecules on charge transport through the planar molecules. In this work, we investigated the modulation of cross-plane charge transport in pyrene derivatives by hydrogenation and substituents based on the XPBJ method that differs from those used in-plane transport. We measured the electrical conductance of the hydrogenated derivatives of the pyrenes and found that hydrogenation reduces conductance, and the fully hydrogenated molecule has the lowest conductance. Conductance of pyrene derivatives increased after substitution by both electron-donating and electron-withdrawing groups. By calculating, the trend in decreased conductance of hydrogenated pyrene was found to be consistent with the change in aromaticity. Electron-withdrawing substituents reduce the aromaticity of the molecule and narrow the HOMO-LUMO gap, while electron-donating groups increase the aromaticity but also narrow the gap. Our work reveals the potential of fine-tuning the structure of the pyrene molecule to control the cross-plane charge transport through the single-molecule junctions.

From bench to bedside: the past, present and future of IL-21 immunotherapy.

As immunotherapy gains momentum as a promising approach for treating several types of cancer, IL-21 has emerged as the latest discovery within the γ chain cytokine family, known for its decisive effects on innate and adaptive immunity and immunopathology. Through the modulation of immune cells, IL-21 has demonstrated significant anti-tumor effects in preclinical studies. The potential of IL-21 in cancer treatment has been explored in phase I and II clinical trials, where it has been utilized both as monotherapy and in combination with other drug agents. Further investigation, alongside larger studies, is necessary before final evaluation and application of IL-21 as immunotherapy. This review aims to summarize these pre-clinical and clinical studies and to discuss the possible future directions of IL-21 immunotherapy development. Such a study may be helpful to accelerate the process of clinical application for IL21 immunotherapy.

Optimizing extraction methods by a comprehensive experimental approach and characterizing polyphenol compositions of Ecklonia radiata.

Brown seaweed Ecklonia radiata harbors valuable polyphenols, notably phlorotannins, prized for their health benefits. This study optimized phlorotannin extraction via conventional solvent extraction and ultrasound-assisted extraction methods, utilizing variable concentrations of ethanol. Employing fractional factorial designs, key variables were identified. Steepest ascent/descent method and central composite rotatable designs refined optimal conditions, enhancing phlorotannin and polyphenol yields, and antioxidant capacities. Under optimized conditions, phlorotannin contents reached 2.366 ± 0.01 and 2.596 ± 0.04 PGE mg/g, total polyphenol contents peaked at 10.223 ± 0.03 and 10.836 ± 0.02 GAE mg/g. Robust antioxidant activity was observed: DPPH and OH radical scavenging capacities measured 27.891 ± 0.06 and 17.441 ± 0.08 TE mg/g, and 37.498 ± 1.12 and 49.391 ± 0.82 TE mg/g, respectively. Reducing power capacities surged to 9.016 ± 0.02 and 28.110 ± 0.10 TE mg/g. Liquid chromatography-mass spectrometry (LC-MS) and high-performance liquid chromatography (HPLC) analyses revealed enriched antioxidant compounds. Variations in polyphenol profiles were noted, potentially influencing antioxidant capacity nuances. This study illuminated the potential of E. radiata potential as a polyphenol source and offers optimized extraction methods poised to benefit various industries.

The lived experience of military beneficiaries with amputations at the hip and pelvic level.

BACKGROUND: Hip- and pelvic-level amputations are devastating injuries that drastically alter patient function and quality of life. This study examined the experience of military beneficiaries with a hip- or pelvic-level amputation to better characterize their challenges and specific needs and to optimize treatment in the future. METHODS: We conducted a retrospective review of the Military Health System and identified 118 patients with a history of one or more amputation(s) at the hip or pelvic level between October 2001 and September 2017. Surviving participants (n = 97) were mailed a letter which explained the details of the study and requested participation in a telephonic interview. A total of six individuals (one female, five males) participated in structured interviews. RESULTS: The study group included four participants with hip disarticulations and two participants with hemipelvectomies (one internal, one external). All six participants reported significant challenges with activities related to prosthetic use, mobility, residual limb health, pain, gastrointestinal and genitourinary function, psychiatric health, and sexual function. CONCLUSIONS: These interviews highlight the unique needs of individuals with hip- and pelvic-level amputations and may improve access to higher echelons of care that would enhance the function and quality of life for these participants.

'I Do It All Alone': The Burdens and Benefits of Being Diagnosed With, and Treated for, Colorectal Cancer During the Covid-19 Pandemic.

INTRODUCTION: The Covid-19 pandemic dramatically altered the way cancer care services were accessed and delivered, including for colorectal cancer (CRC). In the United Kingdom, patients were discouraged from presenting in primary care, many consultations took place remotely, investigative procedures and screening programmes were temporarily suspended, and fewer operations and treatments were delivered. People had to face the practical consequences of having cancer during a pandemic and navigate never before seen pathways, often alone. We examined the experience of being diagnosed and treated for CRC during the pandemic, and the implications of this on people's cancer journeys. METHODS: Semi-structured interviews were undertaken with people diagnosed with CRC during the Covid-19 pandemic (January 2020-May 2021), in the North East of England. An iterative topic guide was used during interviews, which took place remotely (telephone or Zoom), were audio recorded, pseudo-anonymised and transcribed. Initial transcripts were independently coded by two researchers, and a code 'bank' developed for application across transcripts. Development of themes and overarching analytical constructs was undertaken collaboratively by the research team. RESULTS: Interviews were conducted with 19 participants, analysed and four key themes identified: (1) The relative threats of Covid-19 and Cancer were not comparable, with cancer seen as posing a far greater risk than Covid-19; (2) Remote consultations were problematic, affecting patients' abilities to build rapport and trust with clinicians, assess nonverbal communication, and feel able to disclose, comprehend and retain information; (3) Stoma follow-up care was seen to be lacking, with long wait times for stoma reversal experienced by some; Finally, (4) Being alone during consultations negatively impacted some peoples' abilities to absorb information, and left them without the support of loved ones at an emotionally vulnerable time. However, some participants preferred being alone at certain points in their pathways, including receiving a diagnosis, and most frequently when receiving in-patient treatment. CONCLUSION: Being alone brought unexpected benefits, absolving people from undertaking emotions work for others, and instead focus on their recovery, however, remote consultations negatively impacted patients' experiences. This study highlights the complex benefits and burdens of pandemic-located cancer journeys, including how these shifted at different points across cancer pathways. PATIENT OR PUBLIC CONTRIBUTION: Lorraine Angell, a cancer survivor, has been central to this study from idea conception, contributing to: development of study focus and design; securing funding; production of patient-facing materials; development of interview topic guides; analysis and interpretation of data; and drafting of key findings and manuscripts.

Standardisation in Paediatric Echocardiography Reporting and Critical Interpretation of Measurements, Functional Parameters and Prediction Scores. A Clinical Consensus Statement of the European Association of Cardiovascular Imaging (EACVI) of the ESC and the Association for European Paediatric and Congenital Cardiology (AEPC).

This document has been developed to provide a guide for basic and advanced reporting in pediatric echocardiography. Furthermore, it aims to help clinicians in the interpretation of echocardiographic measurements and functional data for estimating the severity of disease in different pediatric age groups. The following topics will be reviewed and discussed in the present document: i) the general principle in constructing a pediatric echocardiography report, ii) the basic elements to be included, iii) the potential and limitation of currently employed tools used for disease severity quantification during paediatric reporting. A guide for the interpretation of Z-scores will be provided. Use and interpretation of parameters employed for quantification of ventricular systolic function will be discussed. Difficulties in the adoption of adult parameters for the study of diastolic function and valve defects at different ages, pressure and loading conditions will be outlined, with pitfalls for the assessment listed. A guide for careful use of prediction scores for complex congenital heart disease will be provided. Examples of basic and advanced (disease specific) formats for reporting in paediatric echocardiography will be provided. This document should serve as a comprehensive guide to i) structure a comprehensive paediatric echocardiographic report, ii) identify the basic morphological details, measures, and functional parameters to be included during echocardiographic reporting, and iii) correctly interpret measurements and functional data for estimating disease severity.

Can a Liquid Biopsy Detect Circulating Tumor DNA With Low-passage Whole-genome Sequencing in Patients With a Sarcoma? A Pilot Evaluation.

BACKGROUND: A liquid biopsy is a test that evaluates the status of a disease by analyzing a sample of bodily fluid, most commonly blood. In recent years, there has been progress in the development and clinical application of liquid biopsy methods to identify blood-based, tumor-specific biomarkers for many cancer types. However, the implementation of these technologies to aid in the treatment of patients who have a sarcoma remains behind other fields of cancer medicine. For this study, we chose to evaluate a sarcoma liquid biopsy based on circulating tumor DNA (ctDNA). All human beings have normal cell-free DNA (cfDNA) circulating in the blood. In contrast with cfDNA, ctDNA is genetic material present in the blood stream that is derived from a tumor. ctDNA carries the unique genomic fingerprint of the tumor with changes that are not present in normal circulating cfDNA. A successful ctDNA liquid biopsy must be able to target these tumor-specific genetic alterations. For instance, epidermal growth factor receptor (EGFR) mutations are common in lung cancers, and ctDNA liquid biopsies are currently in clinical use to evaluate the status of disease in patients who have a lung cancer by detecting EGFR mutations in the blood. As opposed to many carcinomas, sarcomas do not have common recurrent mutations that could serve as the foundation to a ctDNA liquid biopsy. However, many sarcomas have structural changes to their chromosomes, including gains and losses of portions or entire chromosomes, known as copy number alterations (CNAs), that could serve as a target for a ctDNA liquid biopsy. Murine double minute 2 (MDM2) amplification in select lipomatous tumors or parosteal osteosarcoma is an example of a CNA due to the presence of extra copies of a segment of the long arm of chromosome 12. Since a majority of sarcomas demonstrate a complex karyotype with numerous CNAs, a blood-based liquid biopsy strategy that searches for these CNAs may be able to detect the presence of sarcoma ctDNA. Whole-genome sequencing (WGS) is a next-generation sequencing technique that evaluates the entire genome. The depth of coverage of WGS refers to how detailed the sequencing is, like higher versus lower power on a microscope. WGS can be performed with high-depth sequencing (that is, > 60×), which can detect individual point mutations, or low-depth sequencing (that is, 0.1× to 5×), referred to as low-passage whole-genome sequencing (LP-WGS), which may not detect individual mutations but can detect structural chromosomal changes including gains and losses (that is, CNAs). While similar strategies have shown favorable early results for specific sarcoma subtypes, LP-WGS has not been evaluated for applicability to the broader population of patients who have a sarcoma. QUESTIONS/PURPOSES: Does an LP-WGS liquid biopsy evaluating for CNAs detect ctDNA in plasma samples from patients who have sarcomas representing a variety of histologic subtypes? METHODS: This was a retrospective study conducted at a community-based, tertiary referral center. Nine paired (plasma and formalin-fixed paraffin-embedded [FFPE] tissue) and four unpaired (plasma) specimens from patients who had a sarcoma were obtained from a commercial biospecimen bank. Three control specimens from individuals who did not have cancer were also obtained. The paired and unpaired specimens from patients who had a sarcoma represented a variety of sarcoma histologic subtypes. cfDNA was extracted, amplified, and quantified. Libraries were prepared, and LP-WGS was performed using a NextSeq 500 next-generation sequencing machine at a low depth of sequencing coverage (∼1×). The ichorCNA bioinformatics algorithm, which was designed to detect CNAs from low-depth genomic sequencing data, was used to analyze the data. In contrast with the gold standard for diagnosis in the form of histopathologic analysis of a tissue sample, this test does not discriminate between sarcoma subtypes but detects the presence of tumor-derived CNAs within the ctDNA in the blood that should not be present in a patient who does not have cancer. The liquid biopsy was positive for the detection of cancer if the ichorCNA algorithm detected the presence of ctDNA. The algorithm was also used to quantitatively estimate the percent ctDNA within the cfDNA. The concentration of ctDNA was then calculated from the percent ctDNA relative to the total concentration of cfDNA. The CNAs of the paired FFPE tissue and plasma samples were graphically visualized using aCNViewer software. RESULTS: This LP-WGS liquid biopsy detected ctDNA in 9 of 13 of the plasma specimens from patients with a sarcoma. The other four samples from patients with a sarcoma and all serum specimens from patients without cancer had no detectable ctDNA. Of those 9 patients with positive liquid biopsy results, the percent ctDNA ranged from 6% to 11%, and calculated ctDNA quantities were 0.04 to 5.6 ng/mL, which are levels to be expected when ctDNA is detectable. CONCLUSION: In this small pilot study, we were able to detect sarcoma ctDNA with an LP-WGS liquid biopsy searching for CNAs in the plasma of most patients who had a sarcoma representing a variety of histologic subtypes. CLINICAL RELEVANCE: These results suggest that an LP-WGS liquid biopsy evaluating for CNAs to identify ctDNA may be more broadly applicable to the population of patients who have a sarcoma than previously reported in studies focusing on specific subtypes. Large prospective clinical trials that gather samples at multiple time points during the process of diagnosis, treatment, and surveillance will be needed to further assess whether this technique can be clinically useful. At our institution, we are in the process of developing a large prospective clinical trial for this purpose.