Advancing Evidence Generation for Circulating Tumor DNA: Lessons Learned from A Multi-Assay Study of Baseline Circulating Tumor DNA Levels across Cancer Types and Stages.

Circulating tumor DNA (ctDNA) holds promise as a biomarker for predicting clinical responses to therapy in solid tumors, and multiple ctDNA assays are in development. However, the heterogeneity in ctDNA levels prior to treatment (baseline) across different cancer types and stages and across ctDNA assays has not been widely studied. Friends of Cancer Research formed a collaboration across multiple commercial ctDNA assay developers to assess baseline ctDNA levels across five cancer types in early- and late-stage disease. This retrospective study included eight commercial ctDNA assay developers providing summary-level de-identified data for patients with non-small cell lung cancer (NSCLC), bladder, breast, prostate, and head and neck squamous cell carcinoma following a common analysis protocol. Baseline ctDNA levels across late-stage cancer types were similarly detected, highlighting the potential use of ctDNA as a biomarker in these cancer types. Variability was observed in ctDNA levels across assays in early-stage NSCLC, indicative of the contribution of assay analytical performance and methodology on variability. We identified key data elements, including assay characteristics and clinicopathological metadata, that need to be standardized for future meta-analyses across multiple assays. This work facilitates evidence generation opportunities to support the use of ctDNA as a biomarker for clinical response.

Deep brain stimulation of the amygdala for treatment-resistant combat post-traumatic stress disorder: Long-term results.

Deep brain stimulation (DBS) holds promise for neuropsychiatric conditions where imbalance in network activity contributes to symptoms. Treatment-resistant Combat post-traumatic stress disorder (TR-PTSD) is a highly morbid condition and 50% of PTSD sufferers fail to recover despite psychotherapy or pharmacotherapy. Reminder-triggered symptoms may arise from inadequate top-down ventromedial prefrontal cortex (vmPFC) control of amygdala reactivity. Here, we report long-term data on two TR-PTSD participants from an investigation utilizing high-frequency amygdala DBS. The two combat veterans were implanted bilaterally with quadripolar electrodes targeting the basolateral amygdala. Following a randomized staggered onset, patients received stimulation with adjustments based on PTSD symptom severity for four years while psychiatric and neuropsychiatric symptoms, neuropsychological performance, and electroencephalography were systematically monitored. Evaluation of vmPFC-Amygdala network engagement was assessed with 18FDG positron emission tomography (PET). CAPS-IV scores varied over time, but improved 55% from 119 at baseline to 53 at 4-year study endpoint in participant 1; and 44%, from 68 to 38 in participant 2. Thereafter, during 5 and 1.5 years of subsequent clinical care respectively, long-term bilateral amygdala DBS was associated with additional, clinically significant symptomatic and functional improvement. There were no serious stimulation-related adverse psychiatric, neuropsychiatric, neuropsychological, neurological, or neurosurgical effects. In one subject, symptomatic improvement was associated with an intensity-dependent reduction in amygdala theta frequency power. In our two participants, FDG-PET findings were inconclusive regarding the hypothesized mechanism of suppression of amygdala hyperactivity. Our findings encourage further research to confirm and extend our preliminary observations.

Immunological Signatures in Blood and Urine in 80 Individuals Hospitalized during the Initial Phase of COVID-19 Pandemic with Quantified Nicotine Exposure.

This research analyzes immunological response patterns to SARS-CoV-2 infection in blood and urine in individuals with serum cotinine-confirmed exposure to nicotine. Samples of blood and urine were obtained from a total of 80 patients admitted to hospital within 24 h of admission (tadm), 48 h later (t48h), and 7 days later (t7d) if patients remained hospitalized or at discharge. Serum cotinine above 3.75 ng/mL was deemed as biologically significant exposure to nicotine. Viral load was measured with serum SARS-CoV-2 S-spike protein. Titer of IgG, IgA, and IgM against S- and N-protein assessed specific antiviral responses. Cellular destruction was measured by high mobility group box protein-1 (HMGB-1) serum levels and heat shock protein 60 (Hsp-60). Serum interleukin 6 (IL-6), and ferritin gauged non-specific inflammation. The immunological profile was assessed with O-link. Serum titers of IgA were lower at tadm in smokers vs. nonsmokers (p = 0.0397). IgM at t48h was lower in cotinine-positive individuals (p = 0.0188). IgG did not differ between cotinine-positive and negative individuals. HMGB-1 at admission was elevated in cotinine positive individuals. Patients with positive cotinine did not exhibit increased markers of non-specific inflammation and tissue destruction. The blood immunological profile had distinctive differences at admission (MIC A/B↓), 48 h (CCL19↓, MCP-3↓, CD28↑, CD8↓, IFNγ↓, IL-12↓, GZNB↓, MIC A/B↓) or 7 days (CD28↓) in the cotinine-positive group. The urine immunological profile showed a profile with minimal overlap with blood as the following markers being affected at tadm (CCL20↑, CXCL5↑, CD8↑, IL-12↑, MIC A/B↑, GZNH↑, TNFRS14↑), t48h (CCL20↓, TRAIL↓) and t7d (EGF↑, ADA↑) in patients with a cotinine-positive test. Here, we showed a distinctive immunological profile in hospitalized COVID-19 patients with confirmed exposure to nicotine.

Identification of a proteolysis regulator for an essential enzyme in Mycobacterium tuberculosis.

In Mycobacterium tuberculosis proteins that are post-translationally modified with Pup, a prokaryotic ubiquitin-like protein, can be degraded by proteasomes. While pupylation is reversible, mechanisms regulating substrate specificity have not been identified. Here, we identify the first depupylation regulators: CoaX, a pseudokinase, and pantothenate, an essential, central metabolite. In a Δ coaX mutant, pantothenate synthesis enzymes were more abundant, including PanB, a substrate of the Pup-proteasome system. Media supplementation with pantothenate decreased PanB levels in a coaX and Pup-proteasome-dependent manner. In vitro , CoaX accelerated depupylation of Pup∼PanB, while addition of pantothenate inhibited this reaction. Collectively, we propose CoaX contributes to proteasomal degradation of PanB by modulating depupylation of Pup∼PanB in response to pantothenate levels. One Sentence Summary: A pseudo-pantothenate kinase regulates proteasomal degradation of a pantothenate synthesis enzyme in M. tuberculosis .

Anesthetic Considerations for Patients with Hereditary Neuropathy with Liability to Pressure Palsies: A Narrative Review.

Hereditary neuropathy with liability to pressure palsies (HNPP) is an autosomal dominant demyelinating neuropathy characterized by an increased susceptibility to peripheral nerve injury from trauma, compression, or shear forces. Patients with this condition are unique, necessitating distinct considerations for anesthesia and surgical teams. This review describes the etiology, prevalence, clinical presentation, and management of HNPP and presents contemporary evidence and recommendations for optimal care for HNPP patients in the perioperative period. While the incidence of HNPP is reported at 7-16:100,000, this figure may be an underestimation due to underdiagnosis, further complicating medicolegal issues. With the subtle nature of symptoms associated with HNPP, patients with this condition may remain unrecognized during the perioperative period, posing significant risks. Several aspects of caring for this population, including anesthetic choices, intraoperative positioning, and monitoring strategy, may deviate from standard practices. As such, a tailored approach to caring for this unique population, coupled with meticulous preoperative planning, is crucial and requires a multidisciplinary approach.

Association of pulmonary function test abnormalities and quality-of-life measures after COVID-19 infection.

BACKGROUND: Long-COVID is a multisystem disease that can lead to significant impairments in health-related quality of life (HRQoL). Following COVID-19 infection, abnormalities on pulmonary function tests (PFT) are common. The primary aim of this study is to evaluate for any correlation between PFT abnormalities and impairment in HRQoL scores following COVID-19 infection. METHODS: This is an analysis of a prospective cohort of patients in Louisville, KY who were infected with COVID-19. Data collected included demographics, past medical history, laboratory tests, PFTs, and several HRQoL questionnaires such as the EuroQol 5 Dimension HRQoL questionnaire (EQ-5D-5 L), Generalized Anxiety Disorder 7 (GAD-7), Patient Health Questionnaire (PHQ-9), and Posttraumatic stress disorder checklist for DSM-5 (PCL-5). Descriptive statistics were performed, comparing PFTs (normal vs abnormal) and time since COVID-19 infection (3- vs 6- vs ≥ 12 months). RESULTS: There were no significant differences in FEV1, FVC, or the percentage of patients with abnormal PFTs over time after COVID-19 infection. Following COVID-19, patients with normal PFTs had worse impairment in mobility HRQoL scores and change in GAD-7 scores over time. There were no differences over time in any of the HRQoL scores among patients with abnormal PFTs. CONCLUSIONS: Among patients with an abnormal PFT, there was no temporal association with HRQoL scores as measured by EQ-5D-5 L, GAD-7, PHQ-9, and PCL-5. Among patients with a normal PFT, mobility impairment and anxiety may be associated with COVID-19 infection. Following COVID-19 infection, impairment in HRQoL scores is not completely explained by the presence of abnormalities on spirometry.

Early intermediates in bacterial RNA polymerase promoter melting visualized by time-resolved cryo-electron microscopy.

During formation of the transcription-competent open complex (RPo) by bacterial RNA polymerases (RNAP), transient intermediates pile up before overcoming a rate-limiting step. Structural descriptions of these interconversions in real time are unavailable. To address this gap, time-resolved cryo-electron microscopy (cryo-EM) was used to capture four intermediates populated 120 or 500 milliseconds (ms) after mixing Escherichia coli σ70-RNAP and the λPR promoter. Cryo-EM snapshots revealed the upstream edge of the transcription bubble unpairs rapidly, followed by stepwise insertion of two conserved nontemplate strand (nt-strand) bases into RNAP pockets. As nt-strand "read-out" extends, the RNAP clamp closes, expelling an inhibitory σ70 domain from the active-site cleft. The template strand is fully unpaired by 120 ms but remains dynamic, indicating yet unknown conformational changes load it in subsequent steps. Because these events likely describe DNA opening at many bacterial promoters, this study provides needed insights into how DNA sequence regulates steps of RPo formation.

Surgical Modulation of Pulmonary Artery Shear Stress: A Patient-Specific CFD Analysis of the Norwood Procedure.

PURPOSR: This study created 3D CFD models of the Norwood procedure for hypoplastic left heart syndrome (HLHS) using standard angiography and echocardiogram data to investigate the impact of shunt characteristics on pulmonary artery (PA) hemodynamics. Leveraging routine clinical data offers advantages such as availability and cost-effectiveness without subjecting patients to additional invasive procedures. METHODS: Patient-specific geometries of the intrathoracic arteries of two Norwood patients were generated from biplane cineangiograms. "Virtual surgery" was then performed to simulate the hemodynamics of alternative PA shunt configurations, including shunt type (modified Blalock-Thomas-Taussig shunt (mBTTS) vs. right ventricle-to-pulmonary artery shunt (RVPAS)), shunt diameter, and pulmonary artery anastomosis angle. Left-right pulmonary flow differential, Qp/Qs, time-averaged wall shear stress (TAWSS), and oscillatory shear index (OSI) were evaluated. RESULTS: There was strong agreement between clinically measured data and CFD model output throughout the patient-specific models. Geometries with a RVPAS tended toward more balanced left-right pulmonary flow, lower Qp/Qs, and greater TAWSS and OSI than models with a mBTTS. For both shunt types, larger shunts resulted in a higher Qp/Qs and higher TAWSS, with minimal effect on OSI. Low TAWSS areas correlated with regions of low flow and changing the PA-shunt anastomosis angle to face toward low TAWSS regions increased TAWSS. CONCLUSION: Excellent correlation between clinically measured and CFD model data shows that 3D CFD models of HLHS Norwood can be developed using standard angiography and echocardiographic data. The CFD analysis also revealed consistent changes in PA TAWSS, flow differential, and OSI as a function of shunt characteristics.

Ubiquitin-specific proximity labeling for the identification of E3 ligase substrates.

Protein ubiquitylation controls diverse processes within eukaryotic cells, including protein degradation, and is often dysregulated in disease. Moreover, small-molecule degraders that redirect ubiquitylation activities toward disease targets are an emerging and promising therapeutic class. Over 600 E3 ubiquitin ligases are expressed in humans, but their substrates remain largely elusive, necessitating the development of new methods for their discovery. Here we report the development of E3-substrate tagging by ubiquitin biotinylation (E-STUB), a ubiquitin-specific proximity labeling method that biotinylates ubiquitylated substrates in proximity to an E3 ligase of interest. E-STUB accurately identifies the direct ubiquitylated targets of protein degraders, including collateral targets and ubiquitylation events that do not lead to substrate degradation. It also detects known substrates of E3 ligase CRBN and VHL with high specificity. With the ability to elucidate proximal ubiquitylation events, E-STUB may facilitate the development of proximity-inducing therapeutics and act as a generalizable method for E3-substrate mapping.

Joint Replacement Technology in the Ambulatory Surgery Center: Current and Future Applications.

Total joint replacement (TJR) surgery in the ambulatory surgery centers (ASCs) has grown significantly over the past several years, along with the ability to improve the value of care. Standardization of high-quality, perioperative care is pivotal to the success of a TJR ASC program. As surgeons are experiencing increasing overhead with decreasing reimbursement, technology integration can provide major advantages. In this article, we will therefore highlight several examples of technologies that are changing the field and improving care in the preoperative, intraoperative, and postoperative settings.

Atraumatic intra-articular, extra-synovial ganglion cyst of the lateral knee deep to the iliotibial band: A case report and review of the literature.

Key Clinical Message: We highlight the rare case of an atraumatic, intra-articular ganglion cyst of the lateral knee deep to the iliotibial band that was successfully treated nonoperatively, a pathology yet to be reported in orthopedic literature. Abstract: Ganglion cysts are mucin-filled synovial cysts commonly found on the dorsal surface of the hands and feet. Intra-articular ganglion cysts of the knee are rare, and when they present clinically, are typically treated operatively through arthroscopic surgery. We present the first reported case of an atraumatic intraarticular, extra-synovial ganglion cyst of the lateral knee located deep to the iliotibial band that was successfully treated without operative intervention through repeated intra-articular aspirations of the knee.

Malignant ureteral obstruction: comparison of metallic, 8 French and 6 French ureteric stents after failure of initial ureteric stent.

PURPOSE: Malignant ureteric obstruction is a significant management challenge. The failure of ureteric stents often leads to long-term nephrostomy tubes. This is delayed for as long as possible due to its' associated morbidity. Several types of ureteric stents are available, however there is little evidence demonstrating which stents are better for preventing progression to nephrostomy tubes. This study looked to determine whether a new 6 French (Fr) polymer stent, 8Fr polymer stent or metallic stent achieved a longer functional duration once the initial polymer ureteric stent failed. METHODS: A retrospective, longitudinal study was performed at a single tertiary institution. All patients who underwent ureteric stenting with a 6Fr polymer stent for malignancy between 2010 and 2020 were included. Patients were followed up until death with ureteric stent in situ or permanent nephrostomy tube insertion. RESULTS: A total of 46 patients (66 ureters) had ureteric stents inserted for malignancy. From initial ureteric stent failure, 10 stents were changed to a new 6Fr polymer stent, 42 were changed to an 8Fr polymer stent and 14 were changed to a Resonance® 6Fr metallic stent. The Resonance 6Fr metallic stent had the longest median functional duration of 14 months (p = 0.012). CONCLUSION: Resonance® 6Fr metallic stents appear to have a significantly longer functional duration than a new 6Fr polymer stent or 8Fr polymer stent, which may allow patients to enjoy a better quality of life and delay permanent nephrostomy tube insertion.

Tommy John Ligament Repair with Ulnar Collateral Ligament Internal Brace.

Ulnar collateral ligament (UCL) injuries commonly occur in overhead athletes as a result of excess valgus stress on the elbow and can be functionally debilitating, requiring surgical intervention. Since the advent of the first initial UCL reconstruction technique pioneered by Dr. Frank Jobe performed on professional baseball player Tommy John, UCL, or Tommy John Ligament reconstruction has successfully returned athletes to sport following injury and shown enhanced clinical outcomes with minimal complication rates. Tommy John surgery continues to evolve with the development of various techniques over recent years. This technical note describes a UCL repair with an internal brace using knotless suture anchors and aims to contribute to the current literature a technique that is efficacious and reproducible and offers satisfactory stability, functionality, and return to play.

Improved short-term outcomes for a novel, fluoroscopy-based robotic-assisted total hip arthroplasty system compared to manual technique with fluoroscopic assistance.

BACKGROUND: While robotic-assisted total hip arthroplasty (RA-THA) has been associated with improved accuracy of component placement, the perioperative and early postoperative outcomes of fluoroscopy-based RA-THA systems have yet to be elucidated. METHODS: This retrospective cohort analysis included a consecutive series of patients who received manual, fluoroscopy-assisted THA (mTHA) and fluoroscopy-based RA-THA at a single institution. We compared rates of complications within 90 days of surgery, length of hospital stay (LOS), and visual analog scale (VAS) pain scores. RESULTS: No differences existed between groups with respect to demographic data or perioperative recovery protocols. The RA-THA cohort had a significantly greater proportion of outpatient surgeries compared to the mTHA cohort (37.4% vs. 3.8%; p < 0.001) and significantly lower LOS (26.0 vs. 39.5 h; p < 0.001). The RA-THA cohort had a smaller 90-day postoperative complication rate compared to the mTHA cohort (0.9% vs. 6.7%; p = 0.029). The RA-THA cohort had significantly lower patient-reported VAS pain scores at 2-week follow-up visits (2.5 vs. 3.3; p = 0.048), but no difference was seen after 6-week follow visits (2.5 vs. 2.8; p = 0.468). CONCLUSION: Fluoroscopy-based RA-THA demonstrates low rates of postoperative complications, improved postoperative pain profiles, and shortened LOS when compared to manual, fluoroscopy-assisted THA.

Caries detection and characterization in pediatric patients using iTero 5D near-infrared technology.

INTRODUCTION: Near-infrared imaging (NIRI) has been proposed as an alternative to radiographs and uses nonionizing radiation in the near-infrared spectrum to differentially scatter light off tooth surfaces and generate images allowing interproximal caries detection. The new iTero 5D Element Scanner (Align Technology, Santa Clara, Calif) has integrated NIRI capture and viewing technology but has not been specifically studied in a pediatric population. Therefore, this study aimed to assess clinicians' abilities to detect and characterize caries in pediatric patients using this instrument. METHODS: Bitewing (BW) radiographs and an intraoral scan were captured on 17 pediatric patients (344 surfaces were analyzed). Data were randomized and graded by 5 calibrated clinicians individually with 2 different rounds of grading. RESULTS: The reliability of lesion characterization (ie, grade) among examiners was poor to fair in both systems, whereas the reliability of caries detection was moderate. Both systems had a high specificity and low sensitivity. The reliability of the characterization of the combined dataset was moderate to substantial, whereas, for detection, it was substantial. CONCLUSIONS: When using either BW or NIRI analysis, reliability is relatively poor, and clinicians are more likely to correctly identify a healthy tooth surface when compared with a carious surface. There is a small difference in error rate between BW and NIRI systems that is not likely to be clinically significant. When NIRI and BW data are combined, clinician agreement for both lesion characterization and detection increases significantly.

Immune Surveillance of Acute Myeloid Leukemia Is Mediated by HLA-Presented Antigens on Leukemia Progenitor Cells.

Therapy-resistant leukemia stem and progenitor cells (LSC) are a main cause of acute myeloid leukemia (AML) relapse. LSC-targeting therapies may thus improve outcome of patients with AML. Here we demonstrate that LSCs present HLA-restricted antigens that induce T-cell responses allowing for immune surveillance of AML. Using a mass spectrometry-based immunopeptidomics approach, we characterized the antigenic landscape of patient LSCs and identified AML- and AML/LSC-associated HLA-presented antigens absent from normal tissues comprising nonmutated peptides, cryptic neoepitopes, and neoepitopes of common AML driver mutations of NPM1 and IDH2. Functional relevance of shared AML/LSC antigens is illustrated by presence of their cognizant memory T cells in patients. Antigen-specific T-cell recognition and HLA class II immunopeptidome diversity correlated with clinical outcome. Together, these antigens shared among AML and LSCs represent prime targets for T cell-based therapies with potential of eliminating residual LSCs in patients with AML. SIGNIFICANCE: The elimination of therapy-resistant leukemia stem and progenitor cells (LSC) remains a major challenge in the treatment of AML. This study identifies and functionally validates LSC-associated HLA class I and HLA class II-presented antigens, paving the way to the development of LSC-directed T cell-based immunotherapeutic approaches for patients with AML. See related commentary by Ritz, p. 430 . This article is featured in Selected Articles from This Issue, p. 419.

The T Cell Immunoscore as a Reference for Biomarker Development Utilizing Real-World Data from Patients with Advanced Malignancies Treated with Immune Checkpoint Inhibitors.

BACKGROUND: We aimed to determine the prognostic value of an immunoscore reflecting CD3+ and CD8+ T cell density estimated from real-world transcriptomic data of a patient cohort with advanced malignancies treated with immune checkpoint inhibitors (ICIs) in an effort to validate a reference for future machine learning-based biomarker development. METHODS: Transcriptomic data was collected under the Total Cancer Care Protocol (NCT03977402) Avatar® project. The real-world immunoscore for each patient was calculated based on the estimated densities of tumor CD3+ and CD8+ T cells utilizing CIBERSORTx and the LM22 gene signature matrix. Then, the immunoscore association with overall survival (OS) was estimated using Cox regression and analyzed using Kaplan-Meier curves. The OS predictions were assessed using Harrell's concordance index (C-index). The Youden index was used to identify the optimal cut-off point. Statistical significance was assessed using the log-rank test. RESULTS: Our study encompassed 522 patients with four cancer types. The median duration to death was 10.5 months for the 275 participants who encountered an event. For the entire cohort, the results demonstrated that transcriptomics-based immunoscore could significantly predict patients at risk of death (p-value < 0.001). Notably, patients with an intermediate-high immunoscore achieved better OS than those with a low immunoscore. In subgroup analysis, the prediction of OS was significant for melanoma and head and neck cancer patients but did not reach significance in the non-small cell lung cancer or renal cell carcinoma cohorts. CONCLUSIONS: Calculating CD3+ and CD8+ T cell immunoscore using real-world transcriptomic data represents a promising signature for estimating OS with ICIs and can be used as a reference for future machine learning-based biomarker development.

Gene partners of the EWSR1 fusion may represent molecularly distinct entities.

EWSR1 fusions are highly promiscuous and are associated with unique malignancies, clinical phenotypes, and molecular subtypes. However, rare fusion partners (RFP) of EWSR1 has not been well described. Here, we conducted a cross-sectional, retrospective study of 1,140 unique tumors harboring EWSR1 fusions. We identified 64 unique fusion partners. RFPs were identified more often in adults than children. Alterations in cell cycle control and DNA damage response genes as driving the differences between fusion partners. Potentially clinically actionable genomic variants were more prevalent in tumors harboring RFP than common fusions. While the data presented here is limited, tumors harboring RFP of EWSR1 may represent molecularly distinct entities and may benefit from further molecular testing to identify targeted therapeutic options.