Discovery of TYRA-300: First Oral Selective FGFR3 Inhibitor for the Treatment of Urothelial Cancers and Achondroplasia.

Activating FGFR3 alterations have been identified in up to 15-20% of muscle-invasive bladder cancer and metastatic urothelial carcinoma (mUC), and as high as 80% in nonmuscle invasive bladder cancers. FGFR3 germline mutations have also been associated with a variety of skeletal dysplasias. Achondroplasia, the most common form of dwarfism in humans, results from a G380R mutation in FGFR3. The pan-FGFR inhibitor erdafitinib was approved for the treatment of mUC with FGFR3 alterations but is limited due to FGFR isoform off-target toxicities and the development of on-target gatekeeper resistance mutations. TYRA-300 (22) was conceived using a structure-based approach as a potent FGFR3-selective inhibitor to avoid the toxicities associated with inhibition of FGFR1, FGFR2, and FGFR4, and to be agnostic for the FGFR3 gatekeeper mutations. TYRA-300 is being evaluated in a Phase 1 clinical trial in urothelial cancers and solid tumors, with intention to initiate Phase 2 studies in urothelial cancers and achondroplasia.

Advances, limitations and perspectives in the use of celecoxib-loaded nanocarriers in therapeutics of cancer.

Cancer is highlighted as a major global health challenge in the XXI century. The cyclooxygenase-2 (COX-2) enzyme rises as a widespread tumor progression marker. Celecoxib (CXB) is a selective COX-2 inhibitor used in adjuvant cancer therapy, but high concentrations are required in humans. In this sense, the development of nanocarriers has been proposed once they can improve the biopharmaceutical, pharmacokinetic and pharmacological properties of drugs. In this context, this article reviews the progress in the development of CXB-loaded nanocarriers over the past decade and their prospects. Recent advances in the field of CXB-loaded nanocarriers demonstrate the use of complex formulations and the increasing importance of in vivo studies. The types of CXB-loaded nanocarriers that have been developed are heterogeneous and based on polymers and lipids together or separately. It was found that the work on CXB-loaded nanocarriers is carried out using established techniques and raw materials, such as poly (lactic-co-glicolic acid), cholesterol, phospholipids and poly(ethyleneglycol). The main improvements that have been achieved are the use of cell surface ligands, the simultaneous delivery of different synergistic agents, and the presence of materials that can provide imaging properties and other advanced features. The combination of CXB with other anti-inflammatory drugs and/or apoptosis inducers appears to hold effective pharmacological promise. The greatest advance to date from a clinical perspective is the ability of CXB to enhance the cytotoxic effects of established chemotherapeutic agents.

Hospital and Surgeon Surgical Valvar Volume and Survival after Multi-Valve Cardiac Surgery in Medicare Beneficiaries.

OBJECTIVE: Long-term outcomes after multi-valve cardiac surgery remain under-evaluated. METHODS: Medicare administrative claims from 2008-2019 identified beneficiaries undergoing multi-valve surgery. Operative characteristics were doubly-adjudicated using International Classification of Diseases and Current Procedural Technology codes. A multivariable flexible parametric model evaluated predictors of survival; regression standardization was performed to predict standardized survival probabilities (SSP) at varying percentiles of annual valvar volume. RESULTS: Of 476,092 cardiac surgeries involving the aortic (AVS), mitral (MVS), or tricuspid (TVS) valve, 63,083 (13.3%) were identified as involving multi-valve surgery: 22,884 MVS+TVS, 30,697 AVS+MVS, 3,443 AVS+TVS and 6,059 AVS+MVS+TVS. Surgery occurred at 1,157 hospitals by 2,922 surgeons. Annual valvar volume (total AVS+MVS+TVS) was tallied for surgeons and hospitals. Median survival varied substantially by type of multi-valve surgery: 8.09 [7.90-8.24] years in MVS/TVS, 6.65 [6.49-6.81] years in AVS/MVS, 5.77 [5.37-6.13] in AVS/TVS, and 6.02 [5.64-6.38] in AVS/MVS/TVS. SSPs were calculated across combined hospital/surgeon volume percentiles; the median SSP increased with increasing percentile of combined hospital/surgeon volume: 5%tile: 5.77 [5.58,5.98], 25%tile: 6.18 [6.07,6.28], 50%tile: 6.56 [6.44,6.68], 75%tile: 6.86 [6.75,6.97], and 95%tile: 7.58 [7.34,7.83] years, respectively. CONCLUSIONS: Survival varied significantly by type of multi-valve surgery, worsened with addition of concomitant interventions and improved substantially with increasing annual hospital and surgeon volume. Hospital volume was associated with an improved early hazard for death that abated beyond 3 months post-surgery), while surgeon volume was associated with an improved hazard for death that persisted even beyond the first post-operative year. Consideration should be given to referring multi-valve cases to high-volume hospitals and surgeons.

Impact of post-operative transoral robotic surgery hemorrhage on adjuvant treatment delays in patients with oropharyngeal squamous cell carcinoma.

OBJECTIVES: Transoral robotic surgery (TORS) for the treatment for oropharyngeal squamous cell carcinoma (SCC) carries a risk of post-operative hemorrhage. Increased time from surgery to completion of adjuvant therapy has been associated with decreased survival. Our objective was to assess for adjuvant treatments delays in patients with post-operative bleeding. Secondarily, to assess post-operative swallowing outcomes. MATERIALS AND METHODS: Retrospective chart review of all patients who underwent TORS from 2014 to 2021 at a tertiary care center. Patient demographics, adjuvant therapy course, treatment-related dysphagia outcomes, incidence and severity of post-operative bleeding were reviewed. RESULTS: 221 patients underwent TORS, 160 (72%) of which were recommended to undergo adjuvant treatment. 33 patients developed post-operative bleeding, of which 22 patients underwent at least partial radiation therapy (RT) where there was an average of 53.0 ± 12 days elapsed from surgery to the initiation of RT. In the control group, 124 completed at least partial adjuvant treatment and there was an average of 55.3 ± 23 days from surgery to start of adjuvant RT. Time to start of RT was not significantly different between the cohorts (p=0.47). 9.1% of patients with bleeding and 23.7% of those without bleeding started radiation therapy within 6 weeks. The odds ratio of requiring a feeding tube during treatment in patients with post-operative bleeding compared to those without was 1.3 (95% C.I. 0.54-3.13). CONCLUSION: Patients with post-operative bleeding following TORS with TAL were not found to have a significantly higher risk of treatment delays or dysphagia burden, independent of hemorrhage severity.

Sex and Age-Specific Analysis of Basketball-Related Shoulder Dislocations in the United States: A National Injury Data Review.

HYPOTHESIS/PURPOSE: Basketball-related shoulder dislocations frequently present to emergency departments (EDs) in the US. This study aimed to identify the primary mechanisms, distributions, and trends of these injuries. METHODS: All data was extracted from the National Electronic Injury Surveillance System (NEISS), a public database representing approximately 100 US EDs. NEISS was queried for all basketball-related injuries and shoulder dislocations from January 1, 2013 to December 31, 2022. Clinical narratives were used to assign injury mechanisms and the presence of player contact. RESULTS: Between 2013 and 2022, 52,417 basketball-related shoulder dislocations were reported. 30.9% of all basketball-related shoulder injuries were dislocations, and 30.5% of all joint dislocations occurred at the shoulder. Basketball-related shoulder dislocations decreased significantly from 2013-2022 (p<.001). From 2019 to 2020, a 31.0% decrease was identified. The most common mechanism of shoulder dislocation was falling (36.9%). Males accounted for 92.5% of all shoulder dislocations. However, females were significantly more likely than males to dislocate their shoulders from player contact (15.5% of female dislocations v. 10.0% of male dislocations, p<.001). Only 0.2% of all dislocations resulted in hospitalization. 10.4% of dislocations resulted from contact with another player. Compared to other age groups, young adults (43.3%) and adolescents (42.7%) presented with the majority of shoulder dislocations. Children were more likely to dislocate their shoulder from sustaining a direct blow (25.5%), while all other age groups were more likely to have fallen. Children were also the most likely to sustain a dislocation involving player contact (23.9%). CONCLUSION: Basketball-related shoulder dislocations decreased significantly from 2013 to 2022. Females and children were significantly more likely to present with a dislocation by sustaining player contact. Across all demographics, teaching athletes how to break their falls safely may decrease rates of dislocation by minimizing impacts on a posteriorly outstretched arm.

Early drivers of clonal hematopoiesis shape the evolutionary trajectories of de novo acute myeloid leukemia.

Mutations commonly found in AML such as DNMT3A, TET2 and ASXL1 can be found in the peripheral blood of otherwise healthy adults - a phenomenon referred to as clonal hematopoiesis (CH). These mutations are thought to represent the earliest genetic events in the evolution of AML. Genomic studies on samples acquired at diagnosis, remission, and at relapse have demonstrated significant stability of CH mutations following induction chemotherapy. Meanwhile, later mutations in genes such as NPM1 and FLT3, have been shown to contract at remission and in the case of FLT3 often are absent at relapse. We sought to understand how early CH mutations influence subsequent evolutionary trajectories throughout remission and relapse in response to induction chemotherapy. Here, we assembled a retrospective cohort of patients diagnosed with de novo AML at our institution that underwent genomic sequencing at diagnosis as well as at the time of remission and/or relapse (total n = 182 patients). Corroborating prior studies, FLT3 and NPM1 mutations were generally eliminated at the time of cytologic complete remission but subsequently reemerged upon relapse, whereas DNMT3A, TET2 and ASXL1 mutations often persisted through remission. Early CH-related mutations exhibited distinct constellations of co-occurring genetic alterations, with NPM1 and FLT3 mutations enriched in DNMT3A mut AML, while CBL and SRSF2 mutations were enriched in TET2 mut and ASXL1 mut AML, respectively. In the case of NPM1 and FLT3 mutations, these differences vanished at the time of complete remission yet readily reemerged upon relapse, indicating the reproducible nature of these genetic interactions. Thus, early CH-associated mutations that precede malignant transformation subsequently shape the evolutionary trajectories of AML through diagnosis, therapy, and relapse.

Small extracellular vesicles as biomarkers of response in recurrent/metastatic HNSCC patients treated with immunotherapy.

Background: Biomarkers that effectively predict response to anti-PD-1 mAb therapy in cancer patients are an unmet need. We evaluated the utility of small extracellular vesicles (sEV) as biomarkers of response to immunotherapy in recurrent/metastatic (R/M) head and neck squamous cell carcinoma (HNSCC) patients. Methods: Plasma sEV were isolated from 24 R/M HNSCC patients prior to immunotherapy initiation. sEV were separated by immune capture into T cell-derived CD3(+) and tumor-enriched CD3(-) subsets. Stimulatory and suppressive profiles of CD3(-) sEV were determined by on-bead flow cytometry. Differences were assessed using nonparametric tests. Multivariable Cox regression was used to evaluate the relationship with overall (OS) and progression free survival (PFS). Results: CD3(-)CD44v3(+) sEV represented the majority of plasma sEV; the T-cell-derived CD3(+) fraction was significantly smaller. High CD3(+) sEV was associated with better OS and PFS. Total CD3(-)CD44v3(+) sEV was not associated with outcome. However, suppressive and stimulatory profiles were associated with OS; the suppressive/stimulatory ratio was associated with best response. Exploration of individual proteins on CD3(-) sEV showed that high PD-L1 and high CTLA-4 were associated with better outcomes. Conclusions: Evaluation of the T cell-derived-CD3(+) and tumor-enriched CD3(-) plasma sEV subsets indicated their potential utility as biomarkers of response to immunotherapy.

Artificial Intelligence Models Are Limited in Predicting Clinical Outcomes Following Hip Arthroscopy: A Systematic Review.

BACKGROUND: Hip arthroscopy has seen a significant surge in utilization, but complications remain, and optimal functional outcomes are not guaranteed. Artificial intelligence (AI) has emerged as an effective supportive decision-making tool for surgeons. The purpose of this systematic review was to characterize the outcomes, performance, and validity (generalizability) of AI-based prediction models for hip arthroscopy in current literature. METHODS: Two reviewers independently completed structured searches using PubMed/MEDLINE and Embase databases on August 10, 2022. The search query used the terms as follows: (artificial intelligence OR machine learning OR deep learning) AND (hip arthroscopy). Studies that investigated AI-based risk prediction models in hip arthroscopy were included. The primary outcomes of interest were the variable(s) predicted by the models, best model performance achieved (primarily based on area under the curve, but also accuracy, etc), and whether the model(s) had been externally validated (generalizable). RESULTS: Seventy-seven studies were identified from the primary search. Thirteen studies were included in the final analysis. Six studies (n = 6,568) applied AI for predicting the achievement of minimal clinically important difference for various patient-reported outcome measures such as the visual analog scale and the International Hip Outcome Tool 12-Item Questionnaire, with area under a receiver-operating characteristic curve (AUC) values ranging from 0.572 to 0.94. Three studies used AI for predicting repeat hip surgery with AUC values between 0.67 and 0.848. Four studies focused on predicting other risks, such as prolonged postoperative opioid use, with AUC values ranging from 0.71 to 0.76. None of the 13 studies assessed the generalizability of their models through external validation. CONCLUSION: AI is being investigated for predicting clinical outcomes after hip arthroscopy. However, the performance of AI models varies widely, with AUC values ranging from 0.572 to 0.94. Critically, none of the models have undergone external validation, limiting their clinical applicability. Further research is needed to improve model performance and ensure generalizability before these tools can be reliably integrated into patient care. LEVEL OF EVIDENCE: Level IV. See Instructions for Authors for a complete description of levels of evidence.

Association of Patient and Tumor Characteristics With Outcomes in Young Head and Neck Squamous Cell Carcinoma Patients.

INTRODUCTION: We retrospectively studied young patients with head and neck squamous cell carcinoma (HNSCC) to identify factors associated with disease-specific survival (DSS). METHODS: Patient and tumor characteristics of patients aged ≤45 who received treatments for non-metastatic HNSCC were collected to identify factors associated with DSS. Proportional hazards regression was applied separately for surgical and non-surgical patients. RESULTS: 230 patients were included. Surgical and non-surgical patients had similar DSS. Higher pathologic stages, positive margins, perineural invasion (PNI), extranodal extension and negative HPV status were associated with worse DSS for surgical patients and negative HPV status for non-surgical patients. In the multivariate analysis, pathologic stages, positive margins, and PNI were associated with worse DSS in surgical patients. CONCLUSION: Pathologic stages, positive margins, and PNI are independently associated with worse DSS in young surgical HNSCC patients. PNI is a uniquely strong prognostic factor for young patients.

Moderately hypofractionated, preoperative radiotherapy in patients with soft tissue sarcomas (HYPORT-STS): Updated local control, late toxicities, and patient-reported outcomes.

BACKGROUND: Moderately hypofractionated, preoperative radiotherapy in patients with soft tissue sarcomas (HYPORT-STS; ClinicalTrials.gov identifier NCT03819985) investigated a radiobiologically equivalent, moderately hypofractionated course of preoperative radiotherapy (RT) 15 × 2.85 Gy in patients with soft tissue sarcoma (STS). Here, the authors report longer term follow-up to update local control and report late toxicities, as well as functional and patient-reported outcomes. METHODS: HYPORT-STS was a single-center, open-label, single-arm, prospective phase 2 clinical trial that enrolled 120 eligible adult patients with localized STS of the extremities or superficial trunk between 2018 and 2021. Patients received a 3-week course of preoperative RT followed by surgery 4-8 weeks later. End points and follow-up were analyzed from the date of surgery. RESULTS: The median follow-up was 43 months (interquartile range, 37-52 months), and the 4-year local recurrence-free survival rate was 93%. Overall RT-related late toxicities improved with time from local therapy (p < .001), and few patients had grade ≥2 toxicities (9%; n = 8 of 88) at 2 years. These included: 2% grade ≥2 skin toxicity, 2% fibrosis, 3% lymphedema, and 1% joint stiffness. Four patients (3%) had bone fractures. Both functional outcomes, as measured by the Musculoskeletal Tumor Society Rating Scale (p < .001), and quality of life, as measured by the Functional Assessment of Cancer Therapy-General (p < .001), improved with time from treatment, and both measures were better in follow-up at 2 years compared with baseline. CONCLUSIONS: Long-term follow up suggests that moderately hypofractionated preoperative RT for patients with STS is safe and effective. Higher grade late toxicities affect a minority of patients. Late toxicities decrease over time, whereas functional outcomes and health-related quality of life seem to improve with more time from combined modality treatment.

Use of Low-Dose Platelets in Actively Bleeding Patients: A Retrospective Analysis of a Cardiac Surgery Cohort.

CONTEXT.­: During platelet shortages, many hospitals produce low-dose platelets by splitting a standard platelet unit (>3 × 1011 platelets in the United States) in 2, then providing these low-dose units to patients. While low-dose units were previously found to be effective for prophylactic purposes in patients undergoing chemotherapy in the Prophylactic Platelet Dose (PLADO) trial, their use in actively bleeding patients has not yet been assessed. OBJECTIVE.­: To assess the use and safety of low-dose platelets in actively bleeding patients. DESIGN.­: We performed a retrospective review of cardiac surgery cases receiving platelet units for 18 months at 1 hospital. Two cohorts, those receiving only whole-dose platelets (37 cases) and those receiving only low-dose platelets (38 cases), were compared during the intraoperative and the 24-hour perioperative period. Mean number of platelet transfusions, dose of other blood products, estimated blood loss, bleeding complications in index cases, and all-cause mortality within 30 days of discharge were compared. RESULTS.­: There was no significant difference in mean number of intraoperative platelet transfusions between the cohorts (1.61 versus 1.53, P = .57). There was no significant increase in the transfusion of other blood products, estimated blood loss, bleeding complications in index cases, or all-cause mortality within 30 days of discharge in the low-dose platelet cohort, apart from a small increase in requirement for fresh frozen plasma in the perioperative period. CONCLUSIONS.­: These results suggest that low-dose platelets are tentatively equivalent to whole-dose platelets in cardiac surgery during shortages, with similar transfusion requirements and clinical outcomes between groups. Future multicenter studies are needed to confirm these findings.

Towards Preanalytical Best Practices for Liquid Biopsy Studies: A BLOODPAC Landscape Analysis.

BLOODPAC is a public-private consortium that develops best practices, coordinates clinical and translational research, and manages the BLOODPAC Data Commons to broadly support the liquid biopsy community and accelerate regulatory review to aid patient accessibility. BLOODPAC previously recommended 11 preanalytical minimal technical data elements (MTDEs) for BLOODPAC-sponsored studies and data submitted to BLOODPAC Data Commons. The current landscape analysis evaluates the overlap of the BLOODPAC MTDEs with current best practices, guidelines, and standards documents related to clinical and research liquid biopsy applications. Our findings indicate an existing high degree of concordance among these documents. Where differences exist, the BLOODPAC preanalytical MTDEs can be considered a minimal practicable set for organizations to utilize. These MTDEs were developed following extensive examination of best practices and iterative conversations with the U.S. FDA. BLOODPAC recommends the use of these MTDEs in submissions to data commons and to support liquid biopsy clinical trials and research globally.

Association between a Recalled Positive Airway Pressure (PAP) Device and Incident Cancer: A Population-Based Study.

BACKGROUND: The real-world consequences of a Philips/Respironics recall for positive airway pressure (PAP) devices distributed between 2009 and 2021 are unknown. METHODS: We conducted a retrospective population-based study using health administrative databases (Ontario, Canada) on all new adult PAP users identified through the provincial funding system, free of cancer at baseline, who initiated (claimed) PAP treatment between 2012 and 2018. Everyone was followed from the PAP claim date to the earliest of incident cancer diagnosis, death, or the end of the follow-up (March 2022). We used inverse probability of treatment weighting to balance baseline characteristics between individuals on recalled devices and those on devices from other manufacturers. Weighted hazard ratios of incident cancer were compared between groups. RESULTS: Of 231 692 individuals identified, 58 204 (25.1%) claimed recalled devices, and 173 488 (74.9%) from other manufacturers. A meaningful baseline difference between groups (standardised difference≥0.10) was noted only by location-relevant covariates; other variables were mostly equally distributed (standardised differences≤0.06). Over a median follow-up of 6.3 years (IQR: 4.9-8.0), 11 166 (4.8%) developed cancer: unadjusted rates per 10 000 Person-Year (95 CI%) of 78.8 (76.0-81.7) in the recall group versus74.0 (72.4-75.6) in others (p=0.0034). Propensity score weighting achieved excellent balance in baseline characteristics between groups (standardised differences≤0.07). On a weighted sample, there was no statistical difference in the hazard of incident cancer between groups: cause-specific hazard ratio (recalled versus others) of 0.97, 95% CI: 0.89-1.06. CONCLUSION: In our real-world population study, compared to other manufacturers and adjusting for confounders, recalled devices do not appear to be independently associated with developing cancer.

The Lack of Reporting Social Determinants of Health in Pediatric Orthopaedic Randomized Controlled Trials.

BACKGROUND: Social determinants of health (SDOHs) affect health outcomes outside the hospital, and understanding them can enhance postoperative outcomes in orthopaedic surgery patients. This study aimed to describe the prevalence of randomized controlled trials (RCTs) in pediatric orthopaedic journals reporting on the SDOHs of their patient cohorts. We hypothesize that many SDOHs will be underreported in RCTs investigating pediatric orthopaedic surgery. METHODS: Utilizing the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines, the authors queried the PubMed database to examine SDOHs from 2 pediatric orthopaedic journals: Journal of Pediatric Orthopaedics and Journal of Pediatric Orthopaedics: Part B. The inclusion criteria incorporated RCTs published between 2005 and April 2024. The exclusion criteria included any articles that were not RCTs published in this period. RESULTS: One hundred thirteen articles met the search criteria, with 31 excluded because they did not fall from 2005 to 2024. Eighty-two were published from 2005 to 2024, but 6 RCTs were excluded, as 3 were non-RCTs, and 3 examined cadavers. Seventy-six RCTs were included for analysis, with 65 articles from the Journal of Pediatric Orthopaedics and 11 articles from the Journal of Pediatric Orthopaedics: Part B. Articles originated from 17 countries, with the United States producing 61.8% (47) of the included studies. Of all 76 included studies, 96.1% (73) reported age, 88.2% (67) reported sex/gender, 30.3% (23) reported BMI, 21.1% (16) reported race/ethnicity, 5.3% (4) reported educational level, 2.6% (2) reported stress, and 2.6% (2) reported insurance. Smoking status, socioeconomic status, income levels, and employment status were each reported by only 1 study. CONCLUSIONS: The RCTs examining pediatric orthopaedic surgery tend to scarcely report SDOHs. Future RCTs should expand beyond demographic characteristics such as age, race/ethnicity, sex/gender, and BMI and incorporate other relevant SDOHs. This will allow us to develop a more comprehensive understanding of health outcomes in the pediatric orthopaedic population. LEVEL OF EVIDENCE: I; Therapeutic Studies.

Saliva-derived DNA is suitable for the detection of clonal haematopoiesis of indeterminate potential.

Clonal haematopoiesis of indeterminate potential (CHIP) has been associated with many adverse health outcomes. However, further research is required to understand the critical genes and pathways relevant to CHIP subtypes, evaluate how CHIP clones evolve with time, and further advance functional characterisation and therapeutic studies. Large epidemiological studies are well placed to address these questions but often collect saliva rather than blood from participants. Paired saliva- and blood-derived DNA samples from 94 study participants were sequenced using a targeted CHIP-gene panel. The ten genes most frequently identified to carry CHIP-associated variants were analysed. Fourteen unique variants associated with CHIP, ten in DNMT3A, two in TP53 and two in TET2, were identified with a variant allele fraction (VAF) between 0.02 and 0.2 and variant depth ≥ 5 reads. Eleven of these CHIP-associated variants were detected in both the blood- and saliva-derived DNA sample. Three variants were detected in blood with a VAF > 0.02 but fell below this threshold in the paired saliva sample (VAF 0.008-0.013). Saliva-derived DNA is suitable for detecting CHIP-associated variants. Saliva can offer a cost-effective biospecimen that could both advance CHIP research and facilitate clinical translation into settings such as risk prediction, precision prevention, and treatment monitoring.

PURE-seq identifies Egr1 as a Potential Master Regulator in Murine Aging by Sequencing Long-Term Hematopoietic Stem Cells.

Single-cell transcriptomics is valuable for uncovering individual cell properties, particularly in highly heterogeneous systems. However, this technique often results in the analysis of many well-characterized cells, increasing costs and diluting rare cell populations. To address this, we developed PURE-seq (PIP-seq for Rare-cell Enrichment and Sequencing) for scalable sequencing of rare cells. PURE-seq allows direct cell loading from FACS into PIP-seq reactions, minimizing handling and reducing cell loss. PURE-seq reliably captures rare cells, with 60 minutes of sorting capturing tens of cells at a rarity of 1 in 1,000,000. Using PURE-seq, we investigated murine long-term hematopoietic stem cells and their transcriptomes in the context of hematopoietic aging, identifying Egr1 as a potential master regulator of hematopoiesis in the aging context. PURE-seq offers an accessible and reliable method for isolating and sequencing cells that are currently too rare to capture successfully with existing methods.

Comparison of EV characterization by commercial high-sensitivity flow cytometers and a custom single-molecule flow cytometer.

High-sensitivity flow cytometers have been developed for multi-parameter characterization of single extracellular vesicles (EVs), but performance varies among instruments and calibration methods. Here we compare the characterization of identical (split) EV samples derived from human colorectal cancer (DiFi) cells by three high-sensitivity flow cytometers, two commercial instruments, CytoFLEX/CellStream, and a custom single-molecule flow cytometer (SMFC). DiFi EVs were stained with the membrane dye di-8-ANEPPS and with PE-conjugated anti-EGFR or anti-tetraspanin (CD9/CD63/CD81) antibodies for estimation of EV size and surface protein copy numbers. The limits of detection (LODs) for immunofluorescence and vesicle size based on calibration using cross-calibrated, hard-dyed beads were ∼10 PE/∼80 nm EV diameter for CytoFLEX and ∼10 PEs/∼67 nm for CellStream. For the SMFC, the LOD for immunofluorescence was 1 PE and ≤ 35 nm for size. The population of EVs detected by each system (di-8-ANEPPS+/PE+ particles) differed widely depending on the LOD of the system; for example, CellStream/CytoFLEX detected only 5.7% and 1.5% of the tetraspanin-labelled EVs detected by SMFC, respectively, and median EV diameter and antibody copy numbers were much larger for CellStream/CytoFLEX than for SMFC as measured and validated using super-resolution/single-molecule TIRF microscopy. To obtain a dataset representing a common EV population analysed by all three platforms, we filtered out SMFC and CellStream measurements for EVs below the CytoFLEX LODs as determined by bead calibration (10 PE/80 nm). The inter-platform agreement using this filtered dataset was significantly better than for the unfiltered dataset, but even better concordance between results was obtained by applying higher cutoffs (21 PE/120 nm) determined by threshold analysis using the SMFC data. The results demonstrate the impact of specifying LODs to define the EV population analysed on inter-instrument reproducibility in EV flow cytometry studies, and the utility of threshold analysis of SMFC data for providing semi-quantitative LOD values for other flow cytometers.

Premalignant Progression in the Lung: Knowledge Gaps and Novel Opportunities for Interception of Non-Small Cell Lung Cancer. An Official American Thoracic Society Research Statement.

Rationale: Despite significant advances in precision treatments and immunotherapy, lung cancer is the most common cause of cancer death worldwide. To reduce incidence and improve survival rates, a deeper understanding of lung premalignancy and the multistep process of tumorigenesis is essential, allowing timely and effective intervention before cancer development. Objectives: To summarize existing information, identify knowledge gaps, formulate research questions, prioritize potential research topics, and propose strategies for future investigations into the premalignant progression in the lung. Methods: An international multidisciplinary team of basic, translational, and clinical scientists reviewed available data to develop and refine research questions pertaining to the transformation of premalignant lung lesions to advanced lung cancer. Results: This research statement identifies significant gaps in knowledge and proposes potential research questions aimed at expanding our understanding of the mechanisms underlying the progression of premalignant lung lesions to lung cancer in an effort to explore potential innovative modalities to intercept lung cancer at its nascent stages. Conclusions: The identified gaps in knowledge about the biological mechanisms of premalignant progression in the lung, together with ongoing challenges in screening, detection, and early intervention, highlight the critical need to prioritize research in this domain. Such focused investigations are essential to devise effective preventive strategies that may ultimately decrease lung cancer incidence and improve patient outcomes.

The IL-6 hypothesis in COVID-19: A phase 2, randomised, double-blind, placebo-controlled study to evaluate the efficacy and safety of free IL-6 sequestration by the monoclonal antibody sirukumab in severe and critical COVID-19.

BACKGROUND: Upregulation of IL-6 has been associated with worse prognosis in COVID-19 patients. Impact on IL-6 signalling has mostly been limited to clinical outcomes in IL-6 receptor antagonist trials. METHODS: We performed a phase 2, randomised, double-blind, placebo-controlled trial (NCT04380961) of US-based hospitalised adults (<85 years) with laboratory-confirmed SARS-CoV-2 infection and severe (low levels of supplemental oxygen) or critical disease (high levels of oxygen supplementation). Patients received sirukumab 5 mg/kg or placebo single dose IV on Day 1 plus standard of care. The primary endpoint was time to sustained clinical improvement up to Day 28 based on an ordinal scale. Secondary endpoints included clinical improvement, all-cause mortality, and safety. Following an interim analysis, the protocol was amended to only recruit patients with critical COVID-19. FINDINGS: From May 2020 to March 2021, 209 patients were randomised; 112 had critical disease (72 sirukumab, 40 placebo) at baseline. Median time to sustained clinical improvement in critical patients was 17 and 23 days in the sirukumab and placebo groups (HR, 1∙1; 95% CI, 0∙66-1∙88; p > 0∙05). At Day 28, 59∙4% versus 55∙0% of patients achieved clinical improvement with sirukumab versus placebo and rates of all-cause mortality were 24∙6% versus 30∙0%, respectively. Rates of grade ≥3 adverse events were comparable between the sirukumab and placebo groups (25∙9% vs 32∙9%; all patients). INTERPRETATION: In critical COVID-19 patients who received sirukumab, there was no statistically significant difference in time to sustained clinical improvement versus placebo despite objective sequestration of circulating IL-6, questioning IL-6 as a key therapeutic target in COVID-19.