Exploring the tumor genomic landscape of aggressive prostate cancer by whole-genome sequencing of tissue or liquid biopsies.

Treatment resistance remains a major issue in aggressive prostate cancer (PC), and novel genomic biomarkers may guide better treatment selection. Circulating tumor DNA (ctDNA) can provide minimally invasive information about tumor genomes, but the genomic landscape of aggressive PC based on whole-genome sequencing (WGS) of ctDNA remains incompletely characterized. Thus, we here performed WGS of tumor tissue (n = 31) or plasma ctDNA (n = 10) from a total of 41 aggressive PC patients, including 11 hormone-naïve, 15 hormone-sensitive, and 15 castration-resistant patients. Across all variant types, we found progressively more altered tumor genomic profiles in later stages of aggressive PC. The potential driver genes most frequently affected by single-nucleotide variants or insertions/deletions included the known PC-related genes TP53, CDK12, and PTEN and the novel genes COL13A1, KCNH3, and SENP3. Etiologically, aggressive PC was associated with age-related and DNA repair-related mutational signatures. Copy number variants most frequently affected 14q11.2 and 8p21.2, where no well-recognized PC-related genes are located, and also frequently affected regions near the known PC-related genes MYC, AR, TP53, PTEN, and BRCA1. Structural variants most frequently involved not only the known PC-related genes TMPRSS2 and ERG but also the less extensively studied gene in this context, PTPRD. Finally, clinically actionable variants were detected throughout all stages of aggressive PC and in both plasma and tissue samples, emphasizing the potential clinical applicability of WGS of minimally invasive plasma samples. Overall, our study highlights the feasibility of using liquid biopsies for comprehensive genomic characterization as an alternative to tissue biopsies in advanced/aggressive PC.

Noninvasive prenatal screening for cystic fibrosis using circulating trophoblasts: Detection of the 50 most common disease-causing variants.

OBJECTIVES: Cystic fibrosis (CF) is one of the most common severe autosomal recessive disorders. Prenatal or preconception CF screening is offered in some countries. A maternal blood sample in early pregnancy can provide circulating trophoblasts and offers a DNA source for genetic analysis of both the mother and the fetus. This study aimed to develop a cell-based noninvasive prenatal test (NIPT) to screen for the 50 most common CF variants. METHODS: Blood samples were collected from 30 pregnancies undergoing invasive diagnostics and circulating trophoblasts were harvested in 27. Cystic fibrosis testing was conducted using two different methods: by fragment length analysis and by our newly developed NGS-based CF analysis. RESULTS: In all 27 cases, cell-based NIPT provided a result using both methods in agreement with the invasive test result. CONCLUSION: This study shows that cell-based NIPT for CF screening provides a reliable result without the need for partner- and proband samples.

Circulating tumor DNA for prognosis assessment and postoperative management after curative-intent resection of colorectal liver metastases.

The recurrence rate of colorectal liver metastases (CRLM) patients treated with curative intent is above 50%. Standard of care surveillance includes intensive computed tomographic (CT) imaging as well as carcinoembryonic antigen (CEA) measurements. Nonetheless, relapse detection often happens too late to resume curative treatment. This longitudinal cohort study enrolled 115 patients with plasma samples (N = 439) prospectively collected before surgery, postoperatively at day 30 and every third month for up to 3 years. Droplet digital PCR (ddPCR) was used to monitor serial plasma samples for somatic mutations. Assessment of ctDNA status either immediately after surgery, or serially during surveillance, stratified the patients into groups of high and low recurrence risk (hazard ratio [HR], 7.6; 95% CI, 3.0-19.7; P < .0001; and HR, 4.3; 95% CI, 2.3-8.1; P < .0001, respectively). The positive predictive value (PPV) of ctDNA was 100% in all postoperative analyses. In multivariable analyses, postoperative ctDNA status was the only consistently significant risk marker associated with relapse (P < .0001). Indeterminate CT findings were observed for 30.8% (21/68) of patients. All patients (9/21) that were ctDNA positive at the time of the indeterminate CT scan later relapsed, contrasting 42.6% (5/12) of those ctDNA negative (P = .0046). Recurrence diagnoses in patients with indeterminate CT findings were delayed (median 2.8 months, P < .0001). ctDNA status is strongly associated with detection of minimal residual disease and early detection of relapse. Furthermore, ctDNA status can potentially contribute to clinical decision-making in case of indeterminate CT findings, reducing time-to-intervention.

Early career opportunities and practice characteristics of recent graduates of shoulder and elbow fellowship programs.

BACKGROUND: There exists anecdotal evidence that influential faculty members actively dissuade orthopedic surgery residents from pursuing a fellowship in shoulder and elbow due to a perceived lack of availability of jobs following graduation. The aims of the present study are to evaluate whether this perception is true by characterizing the early career opportunities and practice characteristics of recent graduates of shoulder and elbow fellowship programs through a survey of graduates of the past 5 years. METHODS: An anonymous 39-question survey was designed and approved by the leadership of the American Shoulder and Elbow Surgeons (ASES). The survey was distributed via electronic mail to the fellowship directors and coordinators of the 31 ASES-recognized shoulder and elbow fellowship programs. The fellowship directors and coordinators then sent the survey to the shoulder and elbow fellowship graduates of the last 5 years (2016-2020). Following a total of 4 emails, the survey closed after 8 weeks, and descriptive statistical analysis was performed. RESULTS: Seventy-six of 171 (44.4%) fellows responded. In total, 89.5% were very or somewhat satisfied with their fellowship experience, and 90.8% would pursue a shoulder and elbow fellowship again. Notably, 100% of shoulder and elbow fellowship graduates surveyed in the past 5 years have secured employment. The average number of total orthopedic and shoulder and elbow cases performed annually was 296.7 (SD 141.3) and 173.9 (SD 120.2), respectively. In addition, 62.7% and 89% of respondents were very or somewhat satisfied with the volume and surgical complexity of shoulder and elbow cases, respectively. For the fellows that believed their case experience to be inadequate, the most common reasons were competition from other practices (66.0%) and inadequate patient volume (59.6%). CONCLUSION: Recent graduates of the ASES-recognized shoulder and elbow fellowship programs demonstrate a high level of satisfaction with the fellowship and their subspeciality selection. The majority of fellows believed that completing a shoulder and elbow fellowship enhanced their job opportunities. Altogether, there are ample job opportunities and high satisfaction with the volume and complexity of cases as an early career shoulder and elbow surgeon.

Validation of computational determination of microsatellite status using whole exome sequencing data from colorectal cancer patients.

BACKGROUND: Microsatellite instability (MSI), resulting from a defective mismatch repair system, occurs in approximately 15% of sporadic colorectal cancers (CRC). Since MSI is associated with a poor response to 5-fluorouracile based chemotherapy and is a positive predictive marker of immunotherapy, it is routine practice to evaluate the MSI status of resected tumors in CRC patients. MSIsensor is a novel computational tool for determining MSI status using Next Generation Sequencing. However, it is not widely used in the clinic and has not been independently validated in exome data from CRC. To facilitate clinical implementation of computational determination of MSI status, we compared MSIsensor to current gold standard methods for MSI testing. METHODS: MSI status was determined for 130 CRC patients (UICC stage I-IV) using immunohistochemistry, PCR based microsatellite stability testing and by applying MSIsensor to exome sequenced tumors and paired germline DNA. Furthermore, we investigated correlation between MSI status, mutational load and mutational signatures. RESULTS: Eighteen out of 130 (13.8%) patients were microsatellite instable. We found a 100% agreement between MSIsensor and gold standard methods for MSI testing. All MSI tumors were hypermutated. In addition, two microsatellite stable (MSS) tumors were hypermutated, which was explained by a dominant POLE signature and pathogenic POLE mutations (p.Pro286Arg and p.Ser459Phe). CONCLUSION: MSIsensor is a robust tool, which can be used to determine MSI status of tumor samples from exome sequenced CRC patients.

Arthroscopic anatomy medial to the coracoid: an anatomic study of the axillary and musculocutaneous nerves.

PURPOSE: The purpose of this study was to provide arthroscopic measurements and orientations of the axillary and musculocutaneous nerves medial to the coracoid. METHODS: A retrospective chart review of 29 patients undergoing arthroscopic subscapularis repair and arthroscopic cadaveric dissection of 23 shoulders was used to analyze neuroanatomical distances to arthroscopic landmarks and to document the orientations of the axillary and musculocutaneous nerves using a clock face analogy. The clock face data was analyzed by separating the clock face into four quadrants and the frequency of any crossing nerve within each of the four quadrants was then determined. RESULTS: In vivo, the axillary nerve was found 1.5 ± 0.5 cm medial to the coracoid tip and the musculocutaneous nerve was found 1.6 ± 0.6 cm medial to the coracoid tip. In cadavera, the axillary nerve was found 2.0 ± 0.6 cm medial to the coracoid tip and the musculocutaneous nerve was found 1.5 ± 0.5 cm medial to the coracoid tip. The posterosuperior quadrant of the subcoracoid space contained a crossing nerve in 4 of 29 (13.8%) patients undergoing arthroscopic rotator cuff repair medial to the coracoid, compared to 9 of 23 (39.1%) cadavera undergoing arthroscopic dissection medial to the coracoid. The posteroinferior quadrant contained a crossing nerve in 16 of 29 (55.2%) patients compared to 17 of 23 (73.9%) cadavera. CONCLUSIONS: The axillary and musculocutaneous nerves run in close proximity to the coracoid tip and coracoid arch, most consistently within 1-2 cm medial to these structures, which is closer than has been previously documented in the literature. Crossing nerves are least frequently encountered within the posterosuperior quadrant of the subcoracoid space medial to the coracoid, followed by the posteroinferior quadrant. Arthroscopic dissection of this space should begin in the posterosuperior quadrant and carefully progress to the posteroinferior quadrant to decrease the risk of intraoperative nerve injury. Given the close proximity and frequently encountered nerves in this area, extreme caution must be exercised when working arthroscopically within the subcoracoid space.

A bimodular mechanism of calcium control in eukaryotes.

Calcium ions (Ca(2+)) have an important role as secondary messengers in numerous signal transduction processes, and cells invest much energy in controlling and maintaining a steep gradient between intracellular (∼0.1-micromolar) and extracellular (∼2-millimolar) Ca(2+) concentrations. Calmodulin-stimulated calcium pumps, which include the plasma-membrane Ca(2+)-ATPases (PMCAs), are key regulators of intracellular Ca(2+) in eukaryotes. They contain a unique amino- or carboxy-terminal regulatory domain responsible for autoinhibition, and binding of calcium-loaded calmodulin to this domain releases autoinhibition and activates the pump. However, the structural basis for the activation mechanism is unknown and a key remaining question is how calmodulin-mediated PMCA regulation can cover both basal Ca(2+) levels in the nanomolar range as well as micromolar-range Ca(2+) transients generated by cell stimulation. Here we present an integrated study combining the determination of the high-resolution crystal structure of a PMCA regulatory-domain/calmodulin complex with in vivo characterization and biochemical, biophysical and bioinformatics data that provide mechanistic insights into a two-step PMCA activation mechanism mediated by calcium-loaded calmodulin. The structure shows the entire PMCA regulatory domain and reveals an unexpected 2:1 stoichiometry with two calcium-loaded calmodulin molecules binding to different sites on a long helix. A multifaceted characterization of the role of both sites leads to a general structural model for calmodulin-mediated regulation of PMCAs that allows stringent, highly responsive control of intracellular calcium in eukaryotes, making it possible to maintain a stable, basal level at a threshold Ca(2+) concentration, where steep activation occurs.

Computational discovery of specificity-conferring sites in non-ribosomal peptide synthetases.

MOTIVATION: By using a class of large modular enzymes known as Non-Ribosomal Peptide Synthetases (NRPS), bacteria and fungi are capable of synthesizing a large variety of secondary metabolites, many of which are bioactive and have potential, pharmaceutical applications as e.g. antibiotics. There is thus an interest in predicting the compound synthesized by an NRPS from its primary structure (amino acid sequence) alone, as this would enable an in silico search of whole genomes for NRPS enzymes capable of synthesizing potentially useful compounds. RESULTS: NRPS synthesis happens in a conveyor belt-like fashion where each individual NRPS module is responsible for incorporating a specific substrate (typically an amino acid) into the final product. Here, we present a new method for predicting substrate specificities of individual NRPS modules based on occurrences of motifs in their primary structures. We compare our classifier with existing methods and discuss possible biological explanations of how the motifs might relate to substrate specificity. AVAILABILITY AND IMPLEMENTATION: SEQL-NRPS is available as a web service implemented in Python with Flask at http://services.birc.au.dk/seql-nrps and source code available at https://bitbucket.org/dansondergaard/seql-nrps/. CONTACT: micknudsen@gmail.com or cstorm@birc.au.dk SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Anatomic landmarks for arthroscopic suprascapular nerve decompression.

PURPOSE: Arthroscopic suprascapular nerve (SSN) decompression has become a more frequently utilized procedure in the treatment of SSN entrapment and has gained popularity over recent years. Despite increasing technical notes and outcomes information regarding this technique, there remains a paucity of data with respect to clear anatomic guidelines for teaching this procedure. The purpose of this study was to provide guidelines that are visible arthroscopically and palpable externally to allow safer and more efficient surgery for arthroscopic decompression by analysing the superior scapular anatomy with respect to local landmarks. METHODS: A cadaveric study was used to examine neurovascular structural measurements obtained in twelve cadavera with 23 usable shoulders. Arthroscopic dissection of the pertinent anatomy as determined by previously described approaches was followed by meticulous open regional dissection and measurements of the local landmarks. RESULTS: Measurements of the pertinent arthroscopic anatomy with respect to local landmarks of the superior shoulder were recorded in 23 shoulders and are included herein. Measurements taken arthroscopically on 22 shoulders revealed that the lateral insertion of the transverse suprascapular ligament to the acromioclavicular joint was 3.6 cm (SD 0.5 cm). One of the anatomic measurements on open dissection had a significant correlation with our subject's demographics and was found between cadaveric height and the linear distance from the lateral acromion to the suprascapular notch (mean distance = 66.53 ± 5.30 mm; Pearson's correlation = 0.739; p = 0.006). CONCLUSIONS: This cadaveric study describes meaningful landmarks and their measurements, which are identifiable arthroscopically and enable safer surgery in this area. Using these numbers, surgeons can know that it is safe to bluntly dissect to 2.5 cm medial to the acromioclavicular joint (and 5 cm medial to the palpable lateral acromion) before dissection is likely to encounter the SSN or artery. This knowledge will allow surgeons to learn this surgical technique, and for surgical educators to safely teach dissection and release in this uncommonly accessed anatomic region.

The low-anterolateral portal for arthroscopic biceps tenodesis: description of technique and cadaveric study.

PURPOSE: Arthroscopic biceps tenodesis surgery is an important procedure for the correction of biceps tendonitis or in conjunction with rotator cuff repair with biceps symptoms. Recent trends have developed in placing the biceps tendon lower in the bicipital groove for a tenodesis. However, a more distal biceps tenodesis location is technically challenging when carried out arthroscopically with standard posterior and lateral portals. We aimed to establish the safety of a low-anterolateral portal location for direct access to the lowest aspect of the bicipital groove. METHODS: An anatomical study design was used to examine portal to neurovascular structural measurements in 23 cadaveric shoulders. These shoulders had undergone low-anterolateral portal placement over the inferior most aspect of the bicipital groove as determined by palpation and direct arthroscopic visualization. No arthroscopic irrigation was performed. Following this, the shoulders underwent open dissection with the cannula in place to evaluate for any potential damage to any portion of the axillary nerve. RESULTS: All of the resultant portals in this study provided direct access to the inferior most aspect of the bicipital groove, and the dissection revealed that the portal was touching a small distal axillary nerve branch on the undersurface of the anterior deltoid in nearly half of the shoulders. CONCLUSIONS: The placement of a low-anterolateral portal for arthroscopic biceps tenodesis at the distal bicipital groove does not produce significant neurovascular damage; the portal trajectory comes close to distal anterior branches of the axillary nerve. Given these findings, this portal should be placed bluntly to best protect these underlying neurovascular structures.

"Trend" Statement Use in the Orthopaedic Literature.

INTRODUCTION: For research to effectively guide clinical decision making, appropriate interpretation of data is paramount. The P-value is a useful tool for guiding the interpretation of data. However, despite its utility, the P-value is not without limitations. Of particular concern is the use of "trend statements" to describe non-statistically significant findings, a practice which introduces subjectivity and variability into data interpretation and can lead to the drawing of undue conclusions. METHODS: An audit of original research articles published from January 2022 to December 2022 in four high-impact orthopaedic journals was conducted. The selected journals were queried to identify instances in which a non-statistically significant result was labeled as a "trend." The use of trend statements and associated information was recorded and analyzed. RESULTS: One thousand two hundred sixty articles were included in the analysis. 81 articles (6.4%) included a trend statement to describe a non-statistically significant result. Only two articles (2.5%) formally defined what constituted a trend. In 28.8% of cases, the associated P-value was > 0.10. DISCUSSION: Trend statements are used to describe non-statistically significant findings with moderate frequency in the orthopaedic literature. Given the potentially misleading effects of trend statements, efforts should be made to mitigate their use. If trend statements are to be used, attention should be paid to defining what constitutes a "trend", explicitly acknowledging the lack of statistical significance of the finding to which the trend statement refers, and avoiding drawing undue conclusions from non-statistically significant data.

Graft Resorption After Posterior Distal Tibial Allograft Augmentation for Posterior Shoulder Instability: A Case Report.

CASE: A 19-year-old man underwent arthroscopic posterior glenoid reconstruction with a distal tibia allograft (DTA) after failing 2 posterior, soft-tissue instability surgeries. Although he experienced near-complete resolution of symptoms and return to sport, graft resorption was noted 7 months postoperatively. The patient underwent revision surgery for screw removal. CONCLUSION: Graft resorption has not previously been reported in the setting of arthroscopic DTA use for posterior instability. It is believed that stress shielding contributed to resorption. In such situations, screw removal may be warranted. Consideration of alternative fixation techniques and additional investigation into the causes, clinical significance, and optimal management of posterior DTA resorption are warranted.

Exploring the molecular landscape of cancer of unknown primary: A comparative analysis with other metastatic cancers.

Cancer of unknown primary (CUP) tumors are biologically very heterogeneous, which complicates stratification of patients for treatment. Consequently, these patients face limited treatment options and a poor prognosis. With this study, we aim to expand on the current knowledge of CUP biology by analyzing two cohorts: a well-characterized cohort of 44 CUP patients, and 213 metastatic patients with known primary. These cohorts were treated at the same institution and characterized by identical molecular assessments. Through comparative analysis of genomic and transcriptomic data, we found that CUP tumors were characterized by high expression of immune-related genes and pathways compared to other metastatic tumors. Moreover, CUP tumors uniformly demonstrated high levels of tumor-infiltrating leukocytes and circulating T cells, indicating a strong immune response. Finally, the genetic landscape of CUP tumors resembled that of other metastatic cancers and demonstrated mutations in established cancer genes. In conclusion, CUP tumors possess a distinct immunophenotype that distinguishes them from other metastatic cancers. These results may suggest an immune response in CUP that facilitates metastatic tumor growth while limiting growth of the primary tumor.

Field Cancerization Is Associated with Tumor Development, T-cell Exhaustion, and Clinical Outcomes in Bladder Cancer.

BACKGROUND: Field cancerization is characterized by areas of normal tissue affected by mutated clones. Bladder field cancerization may explain the development and recurrence of bladder cancer and may be associated with treatment outcomes. OBJECTIVE: To investigate the predictive and prognostic roles of field cancerization in patients with high-risk non-muscle-invasive bladder cancer (NMIBC) treated with bacillus Calmette-Guérin (BCG). DESIGN, SETTING, AND PARTICIPANTS: We conducted comprehensive genomic and proteomic analyses for 751 bladder biopsies and 234 urine samples from 136 patients with NMIBC. The samples were collected at multiple time points during the disease course. Field cancerization in normal-appearing bladder biopsies was measured using deep-targeted sequencing and error correction models. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Endpoints included the rates of recurrence and progression. Cox regression and Wilcoxon rank-sum and Fisher's exact tests were used. RESULTS AND LIMITATIONS: A high level of field cancerization was associated with high tumor mutational burden (p = 0.007), high tumor neoantigen load (p = 0.029), and high tumor-associated CD8 T-cell exhaustion (p = 0.017). In addition, high field cancerization was associated with worse short-term outcomes (p = 0.029). Nonsynonymous mutations in bladder cancer-associated genes such as KDM6A, ARID1A, and TP53 were identified as early disease drivers already found in normal-appearing bladder biopsies. Urinary tumor DNA (utDNA) levels reflected the bladder tumor burden and originated from tumors and field cancerization. High levels of utDNA after BCG were associated with worse clinical outcomes (p = 0.027) and with disease progression (p = 0.003). High field cancerization resulted in high urinary levels of proteins associated with angiogenesis and proliferation. Limitations include variation in the number of biopsies and time points analyzed. CONCLUSIONS: Field cancerization levels are associated with tumor development, immune responses, and clinical outcomes. utDNA measurements can be used to monitor disease status and treatment response. PATIENT SUMMARY: Molecular changes in the tissue lining the bladder result in tumor recurrence. Urinary measurements may be used to monitor bladder cancer status and treatment responses.

Whole-genome Mutational Analysis for Tumor-informed Detection of Circulating Tumor DNA in Patients with Urothelial Carcinoma.

BACKGROUND AND OBJECTIVE: Circulating tumor DNA (ctDNA) can be used for sensitive detection of minimal residual disease (MRD). However, the probability of detecting ctDNA in settings of low tumor burden is limited by the number of mutations analyzed and the plasma volume available. We used a whole-genome sequencing (WGS) approach for ctDNA detection in patients with urothelial carcinoma. METHODS: We used a tumor-informed WGS approach for ctDNA-based detection of MRD and evaluation of treatment responses. We analyzed 916 longitudinally collected plasma samples from 112 patients with localized muscle-invasive bladder cancer who received neoadjuvant chemotherapy (NAC) before radical cystectomy. Recurrence-free survival (primary endpoint), overall survival, and ctDNA dynamics during NAC were assessed. KEY FINDINGS AND LIMITATIONS: We found that WGS-based ctDNA detection is prognostic for patient outcomes with a median lead time of 131 d over radiographic imaging. WGS-based ctDNA assessment after radical cystectomy identified recurrence with sensitivity of 91% and specificity of 92%. In addition, genomic characterization of post-treatment plasma samples with a high ctDNA level revealed acquisition of platinum therapy-associated mutational signatures and copy number variations not present in the primary tumors. The sequencing depth is a limitation for studying tumor evolution. CONCLUSIONS AND CLINICAL IMPLICATIONS: Our results support the use of WGS for ultrasensitive ctDNA detection and highlight the possibility of plasma-based tracking of tumor evolution. WGS-based ctDNA detection represents a promising option for clinical use owing to the low volume of plasma needed and the ease of performing WGS, eliminating the need for personalized assay design. PATIENT SUMMARY: Detection of tumor DNA in blood samples from patients with cancer of the urinary tract is associated with poorer outcomes. Disease recurrence after surgery can be identified by the presence of tumor DNA in blood before it can be detected on radiography scans.

Improved protocol for single-nucleus RNA-sequencing of frozen human bladder tumor biopsies.

This paper provides a laboratory workflow for single-nucleus RNA-sequencing (snRNA-seq) including a protocol for gentle nuclei isolation from fresh frozen tumor biopsies, making it possible to analyze biobanked material. To develop this protocol, we used non-frozen and frozen human bladder tumors and cell lines. We tested different lysis buffers (IgePal and Nuclei EZ) and incubation times in combination with different approaches for tissue and cell dissection: sectioning, semi-automated dissociation, manual dissociation with pestles, and semi-automated dissociation combined with manual dissociation with pestles. Our results showed that a combination of IgePal lysis buffer, tissue dissection by sectioning, and short incubation time was the best conditions for gentle nuclei isolation applicable for snRNA-seq, and we found limited confounding transcriptomic changes based on the isolation procedure. This protocol makes it possible to analyze biobanked material from patients with well-described clinical and histopathological information and known clinical outcomes with snRNA-seq.

Single-nucleus and Spatially Resolved Intratumor Subtype Heterogeneity in Bladder Cancer.

Background: Current bulk transcriptomic classification systems for bladder cancer do not consider the level of intratumor subtype heterogeneity. Objective: To investigate the extent and possible clinical impact of intratumor subtype heterogeneity across early and more advanced stages of bladder cancer. Design setting and participants: We performed single-nucleus RNA sequencing (RNA-seq) of 48 bladder tumors and additional spatial transcriptomics for four of these tumors. Total bulk RNA-seq and spatial proteomics data were available from the same tumors for comparison, along with detailed clinical follow-up of the patients. Outcome measurements and statistical analysis: The primary outcome was progression-free survival for non-muscle-invasive bladder cancer. Cox regression analysis, log-rank tests, Wilcoxon rank-sum tests, Spearman correlation, and Pearson correlation were used for statistical analysis. Results and limitations: We found that the tumors exhibited varying levels of intratumor subtype heterogeneity and that the level of subtype heterogeneity can be estimated from both single-nucleus and bulk RNA-seq data, with high concordance between the two. We found that a higher class 2a weight estimated from bulk RNA-seq data is associated with worse outcome for patients with molecular high-risk class 2a tumors. The sparsity of the data generated using the DroNc-seq sequencing protocol is a limitation. Conclusions: Our results indicate that discrete subtype assignments from bulk RNA-seq data may lack biological granularity and that continuous class scores may improve clinical risk stratification of patients with bladder cancer. Patient summary: We found that several molecular subtypes can exist within a single bladder tumor and that continuous subtype scores can be used to identify a subgroup of patients with poor outcomes. Use of these subtype scores may improve risk stratification for patients with bladder cancer, which can help in making decisions on treatment.

Post-operative Baseplate Radiographic Evaluation Using Routine pre-Operative CT.

Background: There is limited data evaluating post-operative component position and fixation in reverse shoulder arthroplasty (RSA). Therefore, the purpose of this study was to evaluate baseplate position and fixation using routine pre-operative CT and post-operative radiographs. Methods: A retrospective analysis of a series consecutive patient who underwent primary RSA was performed. Pre-operative and post-operative glenoid retroversion and inclination were measured using radiographs aligned with projection silhouettes of 3D scapula models in Mimics software. Baseplate retroversion and inclination were measured followed by evaluating for the presence of radiolucent lines (RLLs). Results: Twenty-four patients met inclusion criteria. The average age was 73.4 ± 10.7 years (range, 45-89 years). Radiographic follow-up was 3.4 ± 1.3 years. Post-operative glenoid baseplate retroversion was 2 ± 10 degrees (range, 30 to -9). Post-operative glenoid baseplate inclination was 3.8 ± 9.1 (range, -13 to 19). Five (21%) RSAs had baseplate retroversion >10 degrees. Follow-up radiographs revealed no RLLs around the baseplate, central post, or peripheral screws in any patient. Conclusions: Pre-operative CT imaging enabled evaluation of baseplate component placement and fixation on post-operative radiographs. Baseplate version was within 10 degrees of neutral in 79% (19/24) of patients. No RLLs or loss of fixation were found in any cases. Level of Evidence: Level IV: Diagnostic Study.

Meniscal Injuries Are Decreasing but Are Increasingly Being Treated Surgically With Excellent Return to Play Rates in Professional Baseball Players.

Purpose: The purposes of this study were to determine the incidence and key characteristics of meniscus injuries in professional baseball players, assess current treatment strategies, determine the return to play rates at any level (RTP) and at the same level (RSL), and identify prognostic factors that predict injury severity. Methods: After approval from the Major League Baseball (MLB) Research Committee and our institutional review board, the MLB Health and Injury Tracking System was used to identify meniscus injuries occurring across MLB and Minor League Baseball (MiLB) from 2011 to 2017. Analyzed injuries occurred during normal baseball activity in a player who was active on an MLB or MiLB roster and resulted in at least 1 day missed. Results: A total of 293 professional baseball players sustained 314 meniscus injuries from 2011 to 2017 (7 years) for a mean of 44.9 injuries/y. Pitchers were the most injured position (31.8%), followed by infielders (26.4%). Catchers and infielders missed the most median number of days (50 days). When comparing injuries to landing leg vs push-off leg in pitchers, injury to the push-off leg resulted in significantly more days missed per injury compared to the lead leg (59.6 vs 39.9 days, P = .048). Overall, RTP was 93.0%, while RSL was 84.4%. Conclusions: Over 7 professional baseball seasons, 314 meniscus injuries occurred in 293 players. Pitchers and catchers were most injured, and overall, the number of meniscal injuries per year declined while the percentage of injuries that required surgery increased over time. High rates of RTP were observed. Level of Evidence: Level IV, therapeutic case series.

Circular stable intronic RNAs possess distinct biological features and are deregulated in bladder cancer.

Until recently, intronic lariats were regarded as short-lasting splicing byproducts with no apparent function; however, increasing evidence of stable derivatives suggests regulatory roles. Yet little is known about their characteristics, functions, distribution, and expression in healthy and tumor tissue. Here, we profiled and characterized circular stable intronic sequence RNAs (sisRNAs) using total RNA-Seq data from bladder cancer (BC; n = 457, UROMOL cohort), healthy tissue (n = 46), and fractionated cell lines (n = 5). We found that the recently-discovered full-length intronic circles and the stable lariats formed distinct subclasses, with a surprisingly high intronic circle fraction in BC (∼45%) compared to healthy tissues (0-20%). The stable lariats and their host introns were characterized by small transcript sizes, highly conserved BP regions, enriched BP motifs, and localization in multiple cell fractions. Additionally, circular sisRNAs showed tissue-specific expression patterns. We found nine circular sisRNAs as differentially expressed across early-stage BC patients with different prognoses, and sisHNRNPK expression correlated with progression-free survival. In conclusion, we identify distinguishing biological features of circular sisRNAs and point to specific candidates (incl. sisHNRNPK, sisWDR13 and sisMBNL1) that were highly expressed, had evolutionary conserved sequences, or had clinical correlations, which may facilitate future studies and further insights into their functional roles.