Quantitative Assessment of Preanalytic Variables on Clinical Evaluation of PI3/AKT/mTOR Signaling Activity in Diffuse Glioma.

Biomarker-driven therapeutic clinical trials require the implementation of standardized, evidence-based practices for sample collection. In diffuse glioma, phosphatidylinositol 3 (PI3)-kinase/AKT/mTOR (PI3/AKT/mTOR) signaling is an attractive therapeutic target for which window-of-opportunity clinical trials could facilitate the identification of promising new agents. Yet, the relevant preanalytic variables and optimal tumor sampling methods necessary to measure pathway activity are unknown. To address this, we used a murine model for isocitrate dehydrogenase (IDH)-wildtype glioblastoma (GBM) and human tumor tissue, including IDH-wildtype GBM and IDH-mutant diffuse glioma. First, we determined the impact of delayed time-to-formalin fixation, or cold ischemia time (CIT), on the quantitative assessment of cellular expression of 6 phosphoproteins that are readouts of PI3K/AK/mTOR activity (phosphorylated-proline-rich Akt substrate of 40 kDa (p-PRAS40, T246), -mechanistic target of rapamycin (p-mTOR; S2448); -AKT (p-AKT, S473); -ribosomal protein S6 (p-RPS6, S240/244 and S235/236), and -eukaryotic initiation factor 4E-binding protein 1 (p-4EBP1, T37/46). With CITs ≥ 2 hours, typical of routine clinical handling, all had reduced or altered expression with p-RPS6 (S240/244) exhibiting relatively greater stability. A similar pattern was observed using patient tumor samples from the operating room with p-4EBP1 more sensitive to delayed fixation than p-RPS6 (S240/244). Many clinical trials utilize unstained slides for biomarker evaluation. Thus, we evaluated the impact of slide storage conditions on the detection of p-RPS6 (S240/244), p-4EBP1, and p-AKT. After 5 months, storage at -80°C was required to preserve the expression of p-4EBP1 and p-AKT, whereas p-RPS6 (240/244) expression was not stable regardless of storage temperature. Biomarker heterogeneity impacts optimal tumor sampling. Quantification of p-RPS6 (240/244) expression in multiple regionally distinct human tumor samples from 8 patients revealed significant intratumoral heterogeneity. Thus, the accurate assessment of PI3K/AKT/mTOR signaling in diffuse glioma must overcome intratumoral heterogeneity and multiple preanalytic factors, including time-to-formalin fixation, slide storage conditions, and phosphoprotein of interest.

Challenges in the discovery of tumor-specific alternative splicing-derived cell-surface antigens in glioma.

Despite advancements in cancer immunotherapy, solid tumors remain formidable challenges. In glioma, profound inter- and intra-tumoral heterogeneity of antigen landscape hampers therapeutic development. Therefore, it is critical to consider alternative sources to expand the repertoire of targetable (neo-)antigens and improve therapeutic outcomes. Accumulating evidence suggests that tumor-specific alternative splicing (AS) could be an untapped reservoir of antigens. In this study, we investigated tumor-specific AS events in glioma, focusing on those predicted to generate major histocompatibility complex (MHC)-presentation-independent, cell-surface antigens that could be targeted by antibodies and chimeric antigen receptor-T cells. We systematically analyzed bulk RNA-sequencing datasets comparing 429 tumor samples (from The Cancer Genome Atlas) and 9166 normal tissue samples (from the Genotype-Tissue Expression project), and identified 13 AS events in 7 genes predicted to be expressed in more than 10% of the patients, including PTPRZ1 and BCAN, which were corroborated by an external RNA-sequencing dataset. Subsequently, we validated our predictions and elucidated the complexity of the isoforms using full-length transcript amplicon sequencing on patient-derived glioblastoma cells. However, analyses of the RNA-sequencing datasets of spatially mapped and longitudinally collected clinical tumor samples unveiled remarkable spatiotemporal heterogeneity of the candidate AS events. Furthermore, proteomics analysis did not reveal any peptide spectra matching the putative antigens. Our investigation illustrated the diverse characteristics of the tumor-specific AS events and the challenges of antigen exploration due to their notable spatiotemporal heterogeneity and elusive nature at the protein levels. Redirecting future efforts toward intracellular, MHC-presented antigens could offer a more viable avenue.

The Surgical Management of NCAA Division 1 College Football Injuries Post COVID-19: A Single Institution Retrospective Review.

ABSTRACT: Cohen, JL, Cade, WH, Harrah, TC, Costello II, JP, and Kaplan, LD. The surgical management of NCAA Division 1 college football injuries post COVID-19: A single institution retrospective review. J Strength Cond Res 38(5): 906-911, 2024-The unprecedented COVID-19 pandemic had a significant impact on college football operations, including athletes' training regimens. As a result of these changes, concern for increased injury susceptibility post COVID-19 regulations has become a point of discussion. The current study sought to evaluate the incidence of surgical injury among NCAA Division 1 college football players at the authors' institution during the first full season after start of the COVID-19 pandemic compared with previous years. Retrospective chart review was performed for all players who sustained injuries requiring surgery while a member of the NCAA Division 1 football program during the 2009-2021 seasons. A p -value of ≤0.05 was used to determine significance. A total of 23 surgical injuries occurred in 22 players during the 2021 season compared with 121 in 118 players in the 12 previous seasons combined ( p = 0.0178; RR = 1.47). There was a significant increase in shoulder injuries ( n = 13 vs. n = 31; p = <0.0001; RR = 3.05) and specifically a significant increase in labral tears ( n = 10 vs. n = 30; p = 0.0003; RR = 2.74). No difference was seen in knee injuries ( n = 10 vs. n = 77; p = 0.27; RR = 1.35) and specifically no difference in anterior cruciate ligament injuries ( n = 3 vs. n = 31; p = 0.77; RR = 1.17). This phenomenon is multifactorial in nature, but alterations to players' training and preparations because of the COVID-19 pandemic likely resulted in suboptimal conditioning, leading to the increased incidence of surgical injuries emphasizing the importance of adequate strength training and conditioning.

Glioblastoma evolution and heterogeneity from a 3D whole-tumor perspective.

Treatment failure for the lethal brain tumor glioblastoma (GBM) is attributed to intratumoral heterogeneity and tumor evolution. We utilized 3D neuronavigation during surgical resection to acquire samples representing the whole tumor mapped by 3D spatial coordinates. Integrative tissue and single-cell analysis revealed sources of genomic, epigenomic, and microenvironmental intratumoral heterogeneity and their spatial patterning. By distinguishing tumor-wide molecular features from those with regional specificity, we inferred GBM evolutionary trajectories from neurodevelopmental lineage origins and initiating events such as chromothripsis to emergence of genetic subclones and spatially restricted activation of differential tumor and microenvironmental programs in the core, periphery, and contrast-enhancing regions. Our work depicts GBM evolution and heterogeneity from a 3D whole-tumor perspective, highlights potential therapeutic targets that might circumvent heterogeneity-related failures, and establishes an interactive platform enabling 360° visualization and analysis of 3D spatial patterns for user-selected genes, programs, and other features across whole GBM tumors.

Deconstructing intratumoral heterogeneity through multiomic and multiscale analysis of serial sections.

Tumors may contain billions of cells including distinct malignant clones and nonmalignant cell types. Clarifying the evolutionary histories, prevalence, and defining molecular features of these cells is essential for improving clinical outcomes, since intratumoral heterogeneity provides fuel for acquired resistance to targeted therapies. Here we present a statistically motivated strategy for deconstructing intratumoral heterogeneity through multiomic and multiscale analysis of serial tumor sections (MOMA). By combining deep sampling of IDH-mutant astrocytomas with integrative analysis of single-nucleotide variants, copy-number variants, and gene expression, we reconstruct and validate the phylogenies, spatial distributions, and transcriptional profiles of distinct malignant clones. By genotyping nuclei analyzed by single-nucleus RNA-seq for truncal mutations, we further show that commonly used algorithms for identifying cancer cells from single-cell transcriptomes may be inaccurate. We also demonstrate that correlating gene expression with tumor purity in bulk samples can reveal optimal markers of malignant cells and use this approach to identify a core set of genes that is consistently expressed by astrocytoma truncal clones, including AKR1C3, whose expression is associated with poor outcomes in several types of cancer. In summary, MOMA provides a robust and flexible strategy for precisely deconstructing intratumoral heterogeneity and clarifying the core molecular properties of distinct cellular populations in solid tumors.

Lower Perioperative Complication Rates and Shorter Lengths of Hospital Stay Associated With Technology-Assisted Total Knee Arthroplasty Versus Conventional Instrumentation in Primary Total Knee Arthroplasty.

BACKGROUND: The use of technology allows increased precision in component positioning in total knee arthroplasty (TKA). The objectives of this study were to compare (1) perioperative complications and (2) resource utilization between robotic-assisted (RA) and computer-navigated (CN) versus conventional (CI) TKA. METHODS: A retrospective cohort study was performed using a national database to identify patients undergoing unilateral, primary elective TKA from January 2016 to December 2019. A total of 2,174,685 patients were identified and included RA (69,445), CN (112,225), or CI (1,993,015) TKA. Demographics, complications, lengths of stay, dispositions, and costs were compared between the cohorts. Binary logistic regression analysis was performed. RESULTS: The RA TKA cohort had lower rates of intraoperative fracture (0.05 versus 0.08%, P < .05), respiratory complications (0.6 versus 1.1%, P < .05), renal failure (1.3 versus 1.7%, P < .05), delirium (0.1 versus 0.2%, P < .05), gastrointestinal complications (0.04 versus 0.09%, P < .05), postoperative anemia (8.9 versus 13.9%, P < .05), blood transfusion (0.4 versus 0.9%, P < .05), pulmonary embolism, and deep vein thrombosis (0.1 versus 0.2%, P < .05), and mortality (0.01 versus 0.02%, P < .05) compared to conventional TKA, though the cohort did have higher rates of myocardial infarction (0.09 versus 0.07%, P < .05). The CN cohort had lower rates of myocardial infarction (0.02 versus 0.07%, P < .05), respiratory complications (0.8 versus 1.1%, P < .05), renal failure (1.5 versus 1.7%, P < .05), blood transfusion (0.8 versus 0.9%, P < .05), pulmonary embolism (0.08 versus 0.2%, P < .05), and deep vein thrombosis (0.2 versus 0.2%, P < .05) over CI TKA. Total cost was increased in RA (16,190 versus $15,133, P < .05) and CN (17,448 versus $15,133, P < .05). However, the length of hospital stay was decreased in both RA (1.8 versus 2.2 days, P < .05) and CN (2.1 versus 2.2 days, P < .05). CONCLUSIONS: Technology-assisted TKA was associated with lower perioperative complication rates and faster recovery.

Whole tumor analysis reveals early origin of the TERT promoter mutation and intercellular heterogeneity in TERT expression.

BACKGROUND: The TERT promoter mutation (TPM) is acquired in most IDH-wildtype glioblastomas (GBM) and IDH-mutant oligodendrogliomas (OD) enabling tumor cell immortality. Previous studies on TPM clonality show conflicting results. This study was performed to determine whether TPM is clonal on a tumor-wide scale. METHODS: We investigated TPM clonality in relation to presumed early events in 19 IDH-wildtype GBM and 10 IDH-mutant OD using 3-dimensional comprehensive tumor sampling. We performed Sanger sequencing on 264 tumor samples and deep amplicon sequencing on 187 tumor samples. We obtained tumor purity and copy number estimates from whole exome sequencing. TERT expression was assessed by RNA-seq and RNAscope. RESULTS: We detected TPM in 100% of tumor samples with quantifiable tumor purity (219 samples). Variant allele frequencies (VAF) of TPM correlate positively with chromosome 10 loss in GBM (R = 0.85), IDH1 mutation in OD (R = 0.87), and with tumor purity (R = 0.91 for GBM; R = 0.90 for OD). In comparison, oncogene amplification was tumor-wide for MDM4- and most EGFR-amplified cases but heterogeneous for MYCN and PDGFRA, and strikingly high in low-purity samples. TPM VAF was moderately correlated with TERT expression (R = 0.52 for GBM; R = 0.65 for OD). TERT expression was detected in a subset of cells, solely in TPM-positive samples, including samples equivocal for tumor. CONCLUSIONS: On a tumor-wide scale, TPM is among the earliest events in glioma evolution. Intercellular heterogeneity of TERT expression, however, suggests dynamic regulation during tumor growth. TERT expression may be a tumor cell-specific biomarker.

"De novo replication repair deficient glioblastoma, IDH-wildtype" is a distinct glioblastoma subtype in adults that may benefit from immune checkpoint blockade.

Glioblastoma is a clinically and molecularly heterogeneous disease, and new predictive biomarkers are needed to identify those patients most likely to respond to specific treatments. Through prospective genomic profiling of 459 consecutive primary treatment-naïve IDH-wildtype glioblastomas in adults, we identified a unique subgroup (2%, 9/459) defined by somatic hypermutation and DNA replication repair deficiency due to biallelic inactivation of a canonical mismatch repair gene. The deleterious mutations in mismatch repair genes were often present in the germline in the heterozygous state with somatic inactivation of the remaining allele, consistent with glioblastomas arising due to underlying Lynch syndrome. A subset of tumors had accompanying proofreading domain mutations in the DNA polymerase POLE and resultant "ultrahypermutation". The median age at diagnosis was 50 years (range 27-78), compared with 63 years for the other 450 patients with conventional glioblastoma (p < 0.01). All tumors had histologic features of the giant cell variant of glioblastoma. They lacked EGFR amplification, lacked combined trisomy of chromosome 7 plus monosomy of chromosome 10, and only rarely had TERT promoter mutation or CDKN2A homozygous deletion, which are hallmarks of conventional IDH-wildtype glioblastoma. Instead, they harbored frequent inactivating mutations in TP53, NF1, PTEN, ATRX, and SETD2 and recurrent activating mutations in PDGFRA. DNA methylation profiling revealed they did not align with known reference adult glioblastoma methylation classes, but instead had unique globally hypomethylated epigenomes and mostly classified as "Diffuse pediatric-type high grade glioma, RTK1 subtype, subclass A". Five patients were treated with immune checkpoint blockade, four of whom survived greater than 3 years. The median overall survival was 36.8 months, compared to 15.5 months for the other 450 patients (p < 0.001). We conclude that "De novo replication repair deficient glioblastoma, IDH-wildtype" represents a biologically distinct subtype in the adult population that may benefit from prospective identification and treatment with immune checkpoint blockade.

Challenges in the discovery of tumor-specific alternative splicing-derived cell-surface antigens in glioma.

Background: Despite advancements in cancer immunotherapy, solid tumors remain formidable challenges. In glioma, profound inter-and intra-tumoral heterogeneity of antigen landscape hampers therapeutic development. Therefore, it is critical to consider alternative sources to expand the repertoire of targetable (neo-)antigens and improve therapeutic outcomes. Accumulating evidence suggests that tumor-specific alternative splicing (AS) could be an untapped reservoir of neoantigens. Results: In this study, we investigated tumor-specific AS events in glioma, focusing on those predicted to generate major histocompatibility complex (MHC)-presentation-independent, cell-surface neoantigens that could be targeted by antibodies and chimeric antigen receptor (CAR)-T cells. We systematically analyzed bulk RNA-sequencing datasets comparing 429 tumor samples (from The Cancer Genome Atlas [TCGA]) and 9,166 normal tissue samples (from the Genotype-Tissue Expression project [GTEx]), and identified 13 AS events in 7 genes predicted to be expressed in more than 10% of the patients, including PTPRZ1 and BCAN , which were corroborated by an external RNA-sequencing dataset. Subsequently, we validated our predictions and elucidated the complexity of the isoforms using full-length transcript amplicon sequencing on patient-derived glioblastoma cells. However, analyses of the RNA-sequencing datasets of spatially mapped and longitudinally collected clinical tumor samples unveiled remarkable spatiotemporal heterogeneity of the candidate AS events. Furthermore, proteomics analysis did not reveal any peptide spectra matching the putative neoantigens. Conclusions: Our investigation illustrated the diverse characteristics of the tumor-specific AS events and the challenges of antigen exploration due to their notable spatiotemporal heterogeneity and elusive nature at the protein levels. Redirecting future efforts toward intracellular, MHC-presented antigens could offer a more viable avenue.

3D Printing Applications for Craniomaxillofacial Reconstruction: A Sweeping Review.

The field of craniomaxillofacial (CMF) surgery is rich in pathological diversity and broad in the ages that it treats. Moreover, the CMF skeleton is a complex confluence of sensory organs and hard and soft tissue with load-bearing demands that can change within millimeters. Computer-aided design (CAD) and additive manufacturing (AM) create extraordinary opportunities to repair the infinite array of craniomaxillofacial defects that exist because of the aforementioned circumstances. 3D printed scaffolds have the potential to serve as a comparable if not superior alternative to the "gold standard" autologous graft. In vitro and in vivo studies continue to investigate the optimal 3D printed scaffold design and composition to foster bone regeneration that is suited to the unique biological and mechanical environment of each CMF defect. Furthermore, 3D printed fixation devices serve as a patient-specific alternative to those that are available off-the-shelf with an opportunity to reduce operative time and optimize fit. Similar benefits have been found to apply to 3D printed anatomical models and surgical guides for preoperative or intraoperative use. Creation and implementation of these devices requires extensive preclinical and clinical research, novel manufacturing capabilities, and strict regulatory oversight. Researchers, manufacturers, CMF surgeons, and the United States Food and Drug Administration (FDA) are working in tandem to further the development of such technology within their respective domains, all with a mutual goal to deliver safe, effective, cost-efficient, and patient-specific CMF care. This manuscript reviews FDA regulatory status, 3D printing techniques, biomaterials, and sterilization procedures suitable for 3D printed devices of the craniomaxillofacial skeleton. It also seeks to discuss recent clinical applications, economic feasibility, and future directions of this novel technology. By reviewing the current state of 3D printing in CMF surgery, we hope to gain a better understanding of its impact and in turn identify opportunities to further the development of patient-specific surgical care.

Tumor-wide RNA splicing aberrations generate immunogenic public neoantigens.

T-cell-mediated immunotherapies are limited by the extent to which cancer-specific antigens are homogenously expressed throughout a tumor. We reasoned that recurrent splicing aberrations in cancer represent a potential source of tumor-wide and public neoantigens, and to test this possibility, we developed a novel pipeline for identifying neojunctions expressed uniformly within a tumor across diverse cancer types. Our analyses revealed multiple neojunctions that recur across patients and either exhibited intratumor heterogeneity or, in some cases, were tumor-wide. We identified CD8+ T-cell clones specific for neoantigens derived from tumor-wide and conserved neojunctions in GNAS and RPL22 , respectively. TCR-engineered CD8 + T-cells targeting these mutations conferred neoantigen-specific tumor cell eradication. Furthermore, we revealed that cancer-specific dysregulation in splicing factor expression leads to recurrent neojunction expression. Together, these data reveal that a subset of neojunctions are both intratumorally conserved and public, providing the molecular basis for novel T-cell-based immunotherapies that address intratumoral heterogeneity.

Augmented Reality for Perioperative Anxiety in Patients Undergoing Surgery: A Randomized Clinical Trial.

Importance: Both augmented reality (AR) and virtual reality (VR) have had increasing applications in medicine, including medical training, psychology, physical medicine, rehabilitation, and surgical specialties, such as neurosurgery and orthopedic surgery. There are little data on AR's effect on patients' anxiety and experiences. Objective: To determine whether the use of an AR walkthrough effects patient perioperative anxiety. Design, Setting, and Participants: This randomized clinical trial was conducted at an outpatient surgery center in 2021 to 2022. All patients undergoing elective orthopedic surgery with the senior author were randomized to the treatment or control group. Analyses were conducted per protocol. Data analysis was performed in November 2022. Intervention: AR experience explaining to patients what to expect on their day of surgery and walking them through the surgery space. The control group received the standard educational packet. Main Outcomes and Measures: The main outcome was change in State-Trait Anxiety Inventory (STAI) from the screening survey to the preoperative survey. Results: A total of 140 patients were eligible, and 45 patients either declined or were excluded. Therefore, 95 patients (63 [66.3%] male; mean [SD] age, 38 [16] years) were recruited for the study and included in the final analysis; 46 patients received the AR intervention, and 49 patients received standard instructions. The AR group experienced a decrease in anxiety from the screening to preoperative survey (mean score change, -2.4 [95% CI, -4.6 to -0.3]), while the standard care group experienced an increase (mean score change, 2.6 [95% CI, 0.2 to 4.9]; P = .01). All patients postoperatively experienced a mean decrease in anxiety score compared with both the screening survey (mean change: AR, -5.4 [95% CI, -7.9 to -2.9]; standard care, -6.9 [95% CI, -11.5 to -2.2]; P = .32) and preoperative survey (mean change: AR, -8.0 [95% CI, -10.3 to -5.7]; standard care, -4.2 [95% CI, -8.6 to 0.2]; P = .19). Of 42 patients in the AR group who completed the postoperative follow-up survey, 30 (71.4%) agreed or strongly agreed that they enjoyed the experience, 29 (69.0%) agreed or strongly agreed that they would recommend the experience, and 28 (66.7%) agreed or strongly agreed that they would use the experience again. No differences were observed in postoperative pain levels or narcotic use. Conclusions and Relevance: In this randomized clinical trial, the use of AR decreased preoperative anxiety compared with traditional perioperative education and handouts, but there was no significant effect on postoperative anxiety, pain levels, or narcotic use. These findings suggest that AR may serve as an effective means of decreasing preoperative patient anxiety. Trial Registration: ClinicalTrials.gov Identifier: NCT04727697.

Novel SOX10 indel mutations drive schwannomas through impaired transactivation of myelination gene programs.

BACKGROUND: Schwannomas are common peripheral nerve sheath tumors that can cause severe morbidity given their stereotypic intracranial and paraspinal locations. Similar to many solid tumors, schwannomas and other nerve sheath tumors are primarily thought to arise due to aberrant hyperactivation of the RAS growth factor signaling pathway. Here, we sought to further define the molecular pathogenesis of schwannomas. METHODS: We performed comprehensive genomic profiling on a cohort of 96 human schwannomas, as well as DNA methylation profiling on a subset. Functional studies including RNA sequencing, chromatin immunoprecipitation-DNA sequencing, electrophoretic mobility shift assay, and luciferase reporter assays were performed in a fetal glial cell model following transduction with wildtype and tumor-derived mutant isoforms of SOX10. RESULTS: We identified that nearly one-third of sporadic schwannomas lack alterations in known nerve sheath tumor genes and instead harbor novel recurrent in-frame insertion/deletion mutations in SOX10, which encodes a transcription factor responsible for controlling Schwann cell differentiation and myelination. SOX10 indel mutations were highly enriched in schwannomas arising from nonvestibular cranial nerves (eg facial, trigeminal, vagus) and were absent from vestibular nerve schwannomas driven by NF2 mutation. Functional studies revealed these SOX10 indel mutations have retained DNA binding capacity but impaired transactivation of glial differentiation and myelination gene programs. CONCLUSIONS: We thus speculate that SOX10 indel mutations drive a unique subtype of schwannomas by impeding proper differentiation of immature Schwann cells.

The Rate of Medial Ulnar Collateral Ligament Repair Is Increasing While Reconstruction Remains the Most Common Procedure Overall Among Early-Career Orthopaedic Surgeons.

Purpose: To determine the practice patterns and complication rates in medial ulnar collateral ligament (MUCL) repair versus reconstruction procedures performed by early-career orthopaedic surgeons each year between 2010 and 2020, stratified by fellowship training and concomitant procedures performed, during their 6-month American Board of Orthopaedic Surgery (ABOS) Case List collection period. Methods: The ABOS database was queried for MUCL reconstruction and MUCL repair procedures reported by ABOS Part II Oral Examination examinees from 2010 to 2020. Surgeon fellowship training background, patient demographics, procedural diagnosis codes, complications, and concomitant procedures were recorded for each case. Differences between overall procedure rates and the associated complications reported were examined. Data regarding the specific injury pathology and other patient-specific characteristics for each case were not available. Results: In total, 187 primary procedures performed to address isolated MUCL injuries were reported. Of those, 83% (n = 155) were reconstructions and 17% (n = 32) were repairs. The annual percentage of MUCL repair increased from 10% (1/10) in 2010 to 38% (8/21) in 2020 (linear regression; R2 = 0.56, P < .05). The cumulative complication rate for MUCL reconstruction (11.6%) was significantly lower than for MUCL repair (25%) from 2010 to 2020 (P < .05). This remained true among subsets of cases from Orthopaedic Sports Medicine, Shoulder & Elbow, and or Hand Surgery fellowship-trained examinees, although only statistically significant in the Hand Surgery subset. Reported complication rates were not significantly different among cases in which concurrent ulnar nerve neuroplasty and/or transposition or concurrent elbow arthroscopy were performed. Conclusions: Among cases reported by ABOS Part II Oral Examination examinees from 2010 to 2020, there was an increasing rate of MUCL repair whereas MUCL reconstruction remained more common overall. Interestingly, the overall complication rates were significantly lower for MUCL reconstruction than for MUCL repair both in isolation and when concurrent procedures were performed. Level of Evidence: Level III, retrospective cohort study.

Single-cell RNA sequencing and spatial transcriptomics reveal cancer-associated fibroblasts in glioblastoma with protumoral effects.

Cancer-associated fibroblasts (CAFs) were presumed absent in glioblastoma given the lack of brain fibroblasts. Serial trypsinization of glioblastoma specimens yielded cells with CAF morphology and single-cell transcriptomic profiles based on their lack of copy number variations (CNVs) and elevated individual cell CAF probability scores derived from the expression of 9 CAF markers and absence of 5 markers from non-CAF stromal cells sharing features with CAFs. Cells without CNVs and with high CAF probability scores were identified in single-cell RNA-Seq of 12 patient glioblastomas. Pseudotime reconstruction revealed that immature CAFs evolved into subtypes, with mature CAFs expressing actin alpha 2, smooth muscle (ACTA2). Spatial transcriptomics from 16 patient glioblastomas confirmed CAF proximity to mesenchymal glioblastoma stem cells (GSCs), endothelial cells, and M2 macrophages. CAFs were chemotactically attracted to GSCs, and CAFs enriched GSCs. We created a resource of inferred crosstalk by mapping expression of receptors to their cognate ligands, identifying PDGF and TGF-ß as mediators of GSC effects on CAFs and osteopontin and HGF as mediators of CAF-induced GSC enrichment. CAFs induced M2 macrophage polarization by producing the extra domain A (EDA) fibronectin variant that binds macrophage TLR4. Supplementing GSC-derived xenografts with CAFs enhanced in vivo tumor growth. These findings are among the first to identify glioblastoma CAFs and their GSC interactions, making them an intriguing target.

Hyperpolarized δ-[1- 13C]gluconolactone imaging visualizes response to TERT or GABPB1 targeting therapy for glioblastoma.

TERT promoter mutations are a hallmark of glioblastoma (GBM). Accordingly, TERT and GABPB1, a subunit of the upstream mutant TERT promoter transcription factor GABP, are being considered as promising therapeutic targets in GBM. We recently reported that the expression of TERT or GABP1 modulates flux via the pentose phosphate pathway (PPP). Here, we investigated whether 13C magnetic resonance spectroscopy (MRS) of hyperpolarized (HP) δ- [1-13C]gluconolactone can serve to image the reduction in PPP flux following TERT or GABPB1 silencing. We investigated two different human GBM cell lines stably expressing shRNAs targeting TERT or GABPB1, as well as doxycycline-inducible shTERT or shGABPB1cells. MRS studies were performed on live cells and in vivo tumors, and dynamic sets of 13C MR spectra were acquired following injection of HP δ-[1-13C]gluconolactone. HP 6-phosphogluconolactone (6PG), the product of δ-[1-13C]gluconolactone via the PPP, was significantly reduced in TERT or GABPB1-silenced cells or tumors compared to controls in all our models. Furthermore, a positive correlation between TERT expression and 6PG levels was observed. Our data indicate that HP δ-[1-13C]gluconolactone, an imaging tool with translational potential, could serve to monitor TERT expression and its silencing with therapies that target either TERT or GABPB1 in mutant TERT promoter GBM patients.

Zinc Finger MYND-Type Containing 8 (ZMYND8) Is Epigenetically Regulated in Mutant Isocitrate Dehydrogenase 1 (IDH1) Glioma to Promote Radioresistance.

PURPOSE: Mutant isocitrate dehydrogenase 1 (mIDH1) alters the epigenetic regulation of chromatin, leading to a hypermethylation phenotype in adult glioma. This work focuses on identifying gene targets epigenetically dysregulated by mIDH1 to confer therapeutic resistance to ionizing radiation (IR). EXPERIMENTAL DESIGN: We evaluated changes in the transcriptome and epigenome in a radioresistant mIDH1 patient-derived glioma cell culture (GCC) following treatment with an mIDH1-specific inhibitor, AGI-5198. We identified Zinc Finger MYND-Type Containing 8 (ZMYND8) as a potential target of mIDH1 reprogramming. We suppressed ZMYND8 expression by shRNA knockdown and genetic knockout (KO) in mIDH1 glioma cells and then assessed cellular viability to IR. We assessed the sensitivity of mIDH1 GCCS to pharmacologic inhibition of ZMYND8-interacting partners: HDAC, BRD4, and PARP. RESULTS: Inhibition of mIDH1 leads to an upregulation of gene networks involved in replication stress. We found that the expression of ZMYND8, a regulator of DNA damage response, was decreased in three patient-derived mIDH1 GCCs after treatment with AGI-5198. Knockdown of ZMYND8 expression sensitized mIDH1 GCCs to radiotherapy marked by decreased cellular viability. Following IR, mIDH1 glioma cells with ZMYND8 KO exhibit significant phosphorylation of ATM and sustained γH2AX activation. ZMYND8 KO mIDH1 GCCs were further responsive to IR when treated with either BRD4 or HDAC inhibitors. PARP inhibition further enhanced the efficacy of radiotherapy in ZMYND8 KO mIDH1 glioma cells. CONCLUSIONS: These findings indicate the impact of ZMYND8 in the maintenance of genomic integrity and repair of IR-induced DNA damage in mIDH1 glioma. See related commentary by Sachdev et al., p. 1648.

Isocitrate dehydrogenase (IDH) mutant gliomas: A Society for Neuro-Oncology (SNO) consensus review on diagnosis, management, and future directions.

Isocitrate dehydrogenase (IDH) mutant gliomas are the most common adult, malignant primary brain tumors diagnosed in patients younger than 50, constituting an important cause of morbidity and mortality. In recent years, there has been significant progress in understanding the molecular pathogenesis and biology of these tumors, sparking multiple efforts to improve their diagnosis and treatment. In this consensus review from the Society for Neuro-Oncology (SNO), the current diagnosis and management of IDH-mutant gliomas will be discussed. In addition, novel therapies, such as targeted molecular therapies and immunotherapies, will be reviewed. Current challenges and future directions for research will be discussed.

Characteristics and Trends of the Most Cited Arthroplasty Articles in the Journal of Bone and Joint Surgery.

Introduction: The Journal of Bone and Joint Surgery (JBJS) is one of most influential orthopaedic journals, with Total Joint Replacements (TJR) being a frequent topic. The importance of TJR research parallels it's high prevalence in American society. Objective: To compile and analyze the top 50 most frequently cited articles published in the Journal of Bone and Joint Surgery regarding total joint reconstruction or arthroplasty. Methods: Guidelines set by the Preferred Reporting Items for Systematic Reviews were used as the foundation for data collection and analysis. Scopus database was used to acquire the metric analyzed in the study. Data was then exported to an excel sheet for analysis. Results: The top 50 TJR publications analyzed for this study were cited a total of 35,850 times (including self-citations), with an average number of citations per article of 717. Kurtz and Neer II were the only authors contributing more than one. 38 of 50 articles analyzed met the criteria for Level II or III in terms of Level of Evidence (LOE). The United States contributed the most publications with a total of 34. Harvard University and Massachusetts General Hospital, with eight publications each, were the highest contributing institutions. Conclusion: The Journal of Bone and Joint Surgery has published very influential research papers as noted by the number of citations amassed by its most popular articles. JBJS's top cited publications hail largely from major institutions in the United States and are composed of high-quality reports of mostly Level 2 and Level 3 evidence classifications. Level of Evidence: 3.